Synthesis and antiproliferative evaluation of 6-arylindeno[1,2-c]quinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2009.09.021

A number of 6-arylindeno[1,2-c]quinoline derivatives were synthesized and evaluated for their antiproliferative activities against the growth of five cancer cell lines including human hepatocelluar carcinoma (Hep G2, Hep 3B and Hep2.2.1 ), non-small cell

Full text of DOI:10.1016/j.bmc.2009.09.021

Bioorganic & Medicinal Chemistry 17 (2009) 7465–7476
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiproliferative evaluation of 6-arylindeno[1,2-c]quinoline
derivatives
Chih-Hua Tseng a, Yeh-Long Chen a, Kuin-Yu Chung a, Chih-Mei Cheng b, Chi-Huei Wang c,
Cherng-Chyi Tzeng a,
*
a Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
b Department of Biomedical Science and Environmental Biology, College of Life Science, 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 6-arylindeno[1,2-c]quinoline derivatives were synthesized and evaluated for their antipro-
liferative activities against the growth of five cancer cell lines including human hepatocelluar carcinoma
(Hep G2, Hep 3B and Hep2.2.1), non-small cell lung cancer (A549 and H1299), and normal diploid embry-
onic lung cell line (MRC-5). The preliminary results indicated that 9-(3-(dimethylamino)propoxy)-6-(4-
(3-(dimethylamino)propoxy)phenyl)-2-fluoro-11H-indeno[1,2-c]quinolin-11-one (14c) was the most
Received 18 August 2009
Revised 10 September 2009
Accepted 11 September 2009
Available online 16 September 2009
potent with GI50 values of 0.61, 0.67, 0.59, and 0.72 lM against the growth of Hep G2, Hep 3B, Hep
2.2.1, and H1299 cells, respectively. Results have also shown that 2,9-bis(3-(dimethylamino)propoxy)-
6-(4-(3-(dimethylamino)propoxy)phenyl)-11H-indeno[1,2-c]quinolin-11-one (17), which exhibited GI50
of 0.60 and 0.68 lM against the growth of Hep G2 and A549, respectively, was more active than the posi-
Keywords:
6-Arylindeno[1,2-c]quinoline derivatives
Antiproliferative activity
DNA fragmentation
tive topotecan and irinotecan. Compound 17 was less toxic than topotecan against the growth of normal
cell (MRC-5) and therefore, was selected for further evaluation. Results indicated that compound 17
induce cell cycle arrest in G2/M phase, DNA fragmentation, and disrupt the microtubule network in
A549 cells. The apoptotic induction may through the cleavage of PARP.
Apoptotic induction
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
c]quinoline derivatives (Fig. 1) and their evaluation in vitro against
a panel of five cancer cell lines including three human hepatocell-
Quinoline ring is found in a wide variety of biologically active
compounds and is frequently condensed with various heterocy-
cles.1–12 For example, camptothecin which bears a quinoline moi-
ety, is an anticancer alkaloid isolated from Camptotheca
acuminate.1,2 Camptothecin is a prototypical topoisomerase I (top
I) inhibitor with poor water solubility. Subsequent introduction
of hydrophilic side chains led to the discovery of topotecan and iri-
notecan which are currently used as anticancer drugs. However,
several drawbacks such as easy opening of the lactone ring and
the development of drug resistance have caused an urgent need
in search of alternative top I inhibitors which are chemically more
stable.13,14 Recently, a number of condensed quinoline derivatives
have been synthesized and evaluated for their top I inhibitory
activities. We have also synthesized certain indeno[1,2-c]quinoline
derivatives (1a–d) which were found to be more potent than cam-
ptothecin against the growth of human stomach adenocarcinoma
(AGS) and non-small cell lung cancer (A549).12 In continuation of
our study to explore more potent anticancer drug candidates, we
described herein the preparation of certain 6-arylindeno[1,2-
uar carcinoma cells (Hep G2, Hep 3B and Hep2.2.1) and two non-
small cell lung cancer cells (A549 and H1299). Hepatocellular car-
cinoma (HCC) and lung cancer are common malignancies in the
world, and are the leading cause of cancer deaths in Asian coun-
tries including Taiwan.15,16 Chronic hepatitis B and C, especially
in cirrhotic stage, are associated with the vast majority of cases
of HCC.15,17 Because of the high prevalence of hepatitis B and C
virus infection, HCC has been the leading cause of death in Taiwan
since 1984.15,18
2. Chemistry
The Pfitzinger reaction of indolin-2,3-dione (isatin) and
1,2-bis(4-methoxyphenyl)ethanone under basic conditions gave
2,3-bis(4-methoxyphenyl)quinoline-4-carboxylic acid (2)19 as de-
scribed in Scheme 1. Treatment of 2 with POCl3 and AlCl3 afforded
9-methoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-c]quinolin-
11-one (5) which was then reacted with 48% HBr to give 6-(4-
hydroxyphenyl)-9-methoxy-11H-indeno[1,2-c]quinolin-11-one
(8) in a fairly good overall yield. 2-Fluoro-9-hydroxy-6-(4-hydroxy-
phenyl)-11H-indeno[1,2-c]quinolin-11-one (9) and its 2-methoxy
counterpart 10 were, respectively prepared under the same reaction
* Corresponding author. Tel.: +886 7 3121101x2123; fax: +886 7 3125339.
E-mail addresses: tzengch@kmu.edu.tw, tzengch@cc.kmu.edu.tw (C.-C. Tzeng).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.021

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C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
R3
N
R2
N
R1
O
N
N
O
O
N
O
O
N
O
O
OH
O
Camptothecin R1 = H; R2 = H; R3 = H
Topotecan R1 = OH; R2 = CH2N(CH3)2, R3 = H
OH
Irinotecan
OR
N
OMe
OR2
9
O
11
1
R1
2
6
N
N
N
NH
1
OR3
6-Aryl-indeno[1,2-c]quinoline
b: R =
d: R =
a: R =
c: R =
(CH2)3NH2
(CH2)2
(CH2)2NMe2
(CH2)3NMe2
N
Figure 1. Chemical structures of camptothecin and indeno[1,2-c]quinoline derivatives.
O
OH
OMe
OMe
OMe
O
R
R
(i)
O
N
O
N
H3CO
H
2 R=H
R = H, F, OMe
3 R = F
4 R = OMe
OH
OMe
O
O
R
R
(ii)
(iii)
N
N
OH
OMe
8 R = H
5 R=H
9 R = F
6 R = F
10 R = OH
7 R = OMe
Scheme 1. Reagents and conditions: (i) KOH, EtOH, 80 °C, 48 h; (ii) POCl3, AlCl3, 150 °C, 24 h; (iii) 48% HBr, HOAc, reflux, 48 h.
conditions from 6 and 7 which in turn were obtained from 5-fluoroi-
satin and 5-methoxyisatin, respectively.
(Fig. 2). Accordingly, the mixtures of 12a–c and 14a–c were obtained
by the alkylation of 9 with N-(2-chloroethyl)pyrrolidine, N-(2-chlo-
roethyl)piperidine, and 3-chloro-N,N-dimethylpropanamine, resp-
ectively.
Treatment of 2,9-dihydroxy-6-(4-hydroxyphenyl)-11H-inde-
no[1,2-c]quinolin-11-one (10) with N-(2-chloroethyl)pyrrolidine,
N-(2-chloroethyl)piperidine, and 3-chloro-N,N-dimethylpropan-
amine, respectively afforded trialkylated products 15–17 as
described in Scheme 3.
Alkylation of 8 with N-(2-chloroethyl)pyrrolidine gave a mixture of
monoalkylated 9-hydroxy-6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-
11H-indeno[1,2-c]quinolin-11-one (11a) and dialkylated 9-(2-(pyrr-
olidin-1-yl)ethoxy)-6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-11H-
indeno[1,2-c]quinolin-11-one (13a) in a yield of 33% and 41%,
respectively as depicted in Scheme 2. Structure of 11a was assigned
by the NOESY spectrum in which correlations between OCH2
(dH = 4.13 ppm) and meta-H (dH = 7.13 ppm) were observed (Fig. 2).
Under the same reaction conditions, the mixture of 11b and 13b was
obtained from 8 and N-(2-chloroethyl)piperidine; and the mixture of
11c and 13c was obtained from 8 and 3-chloro-N,N-dimethylpropan-
amine. Structure of dialkylated product 13b was confirmed by the
NOESY spectrum in which correlations of OCH2 (dH = 4.12 ppm)/
meta-H (dH = 7.08 ppm); OCH2 (dH = 4.24 ppm)/8-H (dH = 6.70
ppm); and OCH2 (dH = 4.24 ppm)/10-H (dH = 7.20 ppm) were
observed
3. Results and discussion
All the synthesized 6-arylindeno[1,2-c]quinoline derivatives
wereevaluatedinvitroagainstapanelof fivecancercelllinesinclud-
ing three human hepatocelluar carcinoma cells (Hep G2, Hep 3B and
Hep2.2.1) and two non-small cell lung cancer cells (A549 and
H1299) using XTT (2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-

C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
7467
OR2
OH
OH
O
O
O
R1
(i)
R1
R1
N
N
N
OR2
OR2
OH
8 R1=H
9 R1=F
11a-11c R1=H
12a-12c R1= F
13a-13c R1=H
14a-14c R1=F
c R2 =
N
N
N
b R2 =
a R2 =
Scheme 2. Reagents and conditions: (i) NaH, alkyl halides, DMF, 80 °C, 25 min.
N
O
OH
10
6
O
O
8
N
N
6
N
N
O
O
13b
11a
Figure 2. NOESY correlations of compounds 11a and 13b.
OH
OR
O
O
N
15 R =
16 R =
17 R =
HO
RO
(i)
N
N
N
OH
10
OR
N
Scheme 3. Reagents and conditions: (i) NaH, alkyl halides, DMF, 80 °C, 60 min.
2H-tetrazolium-5-carboxanilide) assay.20 Tetrazolium salts (e.g.,
MTT, MTS, XTT) have been used for many years to distinguish living
cells from dead ones. They are cleaved to form a formazan dye only
by metabolic active cells. XTT is reduced in the cells to formazan
by mitochondrial succinate dehydrogenase. The normal diploid
embryonic lung cell line (MRC-5) was also evaluated since a poten-
tial anticancer drug candidate should selectively affect only tumor
cells and not somatic cells. The concentration that inhibited the
growth of 50% of cells (GI50) 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%. The
GI50 results of 6-arylindeno[1,2-c]quinoline-11-one derivatives are
summarized in Table 1. 9-Methoxy-6-(4-methoxyphenyl)-11H-in-
deno[1,2-c]quinolin-11-one (5) was inactive against all the cancer
its trimethoxy counterpart 7. Therefore, the substituents of hydro-
gen-bond donating groups are more favorable than that of hydro-
gen-bond accepting groups for the 6-arylindeno[1,2-c]quinoline
pharmacophore. The same trend was observed in which dihydroxy
derivatives 8 and 9 are more active than their respective dimethoxy
counterparts 5 and 6. For the C-2 unsubstituted 6-arylindeno[1,2-
c]quinoline derivatives, introduction of an aminoalkyl side chain
on the 6-aryl moiety enhanced antiproliferative activity in which
each of compounds 11a–c was more active than compound 8. Com-
parable activities of 11a, 11b and 11c indicated no difference in the
antiproliferative activity for various aminoalkyl side chains such as
pyrrolidin-1-yl ethyl, piperidin-1-yl ethyl, and acyclic dimethylami-
nopropyl group. Further introduction of an aminoalkyl side chain on
the C-9 position did not enhanced antiproliferative activity in which
activities of monoaminoalkylated 6-arylindeno[1,2-c]quinolines
11a–c were comparable with their respective diaminoalkylated
derivatives 13a–c.
cells tested with GI50 of >10 lM in each case. Introduction of a fluoro
or a methoxy group on the C-2 position enhanced antiproliferative
activity against the growth of H1299 cancer cell in which com-
pounds 6 and 7 exhibited GI50 of 6.44 and 7.84
With an exception of the H1299 cancer cell, 2,9-dihydroxy-6-(4-
hydroxyphenyl)-11H-indeno[1,2-c]quinolin-11-one (10) which
l
M, respectively.
For the C-2 fluoro-substituted 6-arylindeno[1,2-c]quinoline
derivatives, introduction of an aminoalkyl side chain on the 6-aryl
moiety enhanced antiproliferative activity in which each of com-
pounds 12a–c was more active than compound 9. Further intro-
duction of a pyrrolidin-1-yl ethyl side chain on the C-9 position
exhibited GI50 values of 5.51, 6.61, and 4.98
lM against the growth
of Hep G2, Hep 2.2.1, and A549, respectively, was more active than

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C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
Table 1
Antiproliferative activity of 6-arylindeno[1,2\-c]quinoline-11-one derivatives (GI50, lM)
OR2
O
R1
N
OR3
Compd
R1
R2
R3
Cell line (GI50, lM)
Hep G2
Hep 3B
Hep 2.2.1
A549
H1299
MRC-5
5
6
7
8
9
10
H
F
OMe
H
F
Me
Me
Me
H
H
H
Me
Me
Me
H
H
H
>10
>10
>10
9.12 0.35
>10
>10
>10
12.53 3.58
>10
9.01 0.78
12.15 3.81
>10
>10
>10
>10
>10
6.61 0.19
>10
>10
>10
>10
>10
4.98 0.05
4.33 0.01
5.06 0.05
>10
>10
>10
10.24 3.09
>10
>10
6.44 0.06
7.84 0.26
>10
5.88 0.07
10.44 0.05
OH
5.51 0.20
10.33 3.55
11a
11b
H
H
H
H
4.28 0.02
4.30 0.04
3.40 0.01
5.40 0.01
6.06 0.06
6.40 0.01
7.05 0.01
6.55 0.02
3.16 0.01
3.10 0.02
N
N
11c
12a
H
F
H
H
5.59 0.05
6.10 0.03
5.34 0.05
6.34 0.02
6.82 1.16
5.69 0.01
5.87 0.01
6.63 0.02
6.39 0.10
5.25 0.02
4.61 0.05
6.02 0.01
N
N
N
12b
F
H
H
4.78 0.01
6.61 0.01
6.03 0.06
6.00 0.02
5.32 0.03
5.91 0.01
12c
13a
F
5.92 0.01
4.03 0.02
6.72 0.02
3.46 0.02
5.62 0.01
6.15 0.03
6.35 0.01
3.75 0.01
6.13 0.01
6.95 0.01
4.75 0.01
5.31 0.02
N
H
N
N
N
N
13b
H
4.32 0.03
1.35 0.01
6.24 0.04
5.68 0.03
6.38 0.06
4.34 0.04
13c
14a
H
F
4.35 0.01
4.92 0.01
3.05 0.01
6.24 0.03
5.39 0.06
6.11 0.59
3.07 0.01
6.23 0.03
5.35 0.01
4.30 0.02
3.68 0.02
5.15 0.02
N
N
N
N
N
N
14b
14c
F
F
2.82 0.02
0.61 0.01
0.66 0.02
0.67 0.01
3.00 0.01
0.59 0.04
6.24 0.03
3.67 0.02
0.79 0.20
0.72 0.01
4.52 0.01
1.86 0.02
N
N

C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
7469
Table 1 (continued)
Compd
R1
R2
R3
Cell line (GI50, lM)
Hep G2
Hep 3B
Hep 2.2.1
A549
H1299
MRC-5
15
0.62 0.01
1.73 0.01
3.44 0.04
0.67 0.01
0.81 0.01
0.68 0.03
0.37 0.07
0.71 0.07
4.64 0.01
6.20 0.06
N
N
N
N
N
N
O
16
0.70 0.01
0.60 0.01
5.02 0.24
4.44 0.01
6.07 0.01
4.25 0.02
6.26 0.01
5.18 0.09
O
O
N
O
N
O
N
17
Topotecan
Irinotecan
3.83 0.15
5.94 0.75
0.22 0.01
4.73 0.21
4.95 0.08
>10
5.98 0.26
>10
>10
>10
4.25 0.81
77.53 1.82
did not enhanced antiproliferative activity in which activity of
diaminoalkylated derivative 14a was comparable with its mono-
aminoalkylated derivative 12a. However, introduction of the sec-
ond piperidin-1-yl ethyl side chain on the C-9 position enhanced
antiproliferative activity against certain specific cancer cells in
which compound 14b was especially active against the growth of
DNA at the internucleosomal linker sites yielding DNA fragments
in multiple fragments (180–200 bp) was regarded as a biochemical
hallmark of apoptosis.22 The appearance of such fragments resulted
in a ladder formation evidentlywhen fragmentedDNA from17-trea-
ted cells was separated by agarose gel electrophoresis (Fig. 6). Cells
in sub-G0/G1 phase indicate they are undergoing the DNA fragmen-
tation and celldeath. To examinetheeffectof compound17onapop-
tosis related protein, cleavage of PARP was evaluated in 17-treated
A549 cells by western blotting. 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.23 Our results have shown that PARP was cleaved from
116 kDa intact form into 85 kDa fragment after the treatment of 17
in a concentration-dependent manner (Fig. 7). Thus, compound 17
induces cell cycle arrest at G2/M phase followed by DNA fragmenta-
tion via cleavage of PARP and consequently to cause the cell death.
Hep 3B and H1299 with GI50 of 0.66 and 0.79 lM, respectively.
Introduction of the second dimethylaminopropyl side chain on
the C-9 position significantly enhanced antiproliferative activity
against all cancer cells in which compound 14c was much more ac-
tive than 12c. Compound 14b was found to be more selective and
less cytotoxic than 14c against the growth of normal diploid
embryonic lung cell line (MRC-5).
For the trisubstituted 6-arylindeno[1,2-c]quinoline derivatives,
the substituted aminoalkyl side chains play important roles in the
antiproliferative activities in which activities against cancer cells de-
creased in an order pyrrolidin-1-yl ethyl 15 > acyclic dimethylami-
nopropyl 17 > piperidin-1-yl ethyl 16. Although compound 15 was
the most potent among these 6-arylindeno[1,2-c]quinoline deriva-
tives, it was non-selective and highly cytotoxic against the normal
4. Conclusion
A number of 6-arylindeno[1,2-c]quinoline derivatives were
synthesized and evaluated for antiproliferative activities against
the growth of cancer cell lines including Hep G2, Hep 3B, Hep
2.2.1, A549, H1299, and normal cell line MRC-5. Among them,
2,9-bis(3-(dimethylamino) propoxy)-6-(4-(3-(dimethylamino)pro-
poxy)phenyl)-11H-indeno[1,2-c]-quinolin-11-one (17), which
MRC-5. Compound 17, which exhibited GI50 of 0.60 and 0.68 lM
against the growth of Hep G2 and A549, respectively, was more ac-
tive than the positive topotecan and irinotecan. In addition, com-
pound 17 was found to be less toxic than 15 and topotecan against
the growth of MRC-5. Therefore, compound 17 and its trimethoxy
substituted 6-arylindeno[1,2-c]quinoline analog 7 (as a negative
control) were selected for further evaluation on their effects of
A549 cell cycle distribution by flow cytometric analysis. Compound
7 had no effect on cell cycle progression even at a concentration of
exhibited GI50 of 0.60 and 0.68 lM against the growth of Hep G2
and A549, respectively, was more active than the positive topotec-
an and irinotecan. Compound 17 was less toxic than topotecan
against the growth of normal cell (MRC-5) and therefore, was se-
lected for further evaluation. Results indicated that 17 induce cell
cycle arrest at G2/M phase, DNA fragmentation, and disrupt the
microtubule network in A549 cells. The apoptotic induction may
through the cleavage of PARP. In vivo studies on compound 17
are currently under investigation.
10.0 lM whilecompound17 inducedcellcycle arrest in a concentra-
tion-dependent manner as shown in Figure 3 and Table 2. The pro-
portion of cells was slightly decreased in the S and accumulated in
G2/M phase after 24 h treatment of 17, while the hypodiploid
(sub-G0/G1 phase) cells increased. The morphological changes of
apoptosis include membrane blebbing, cell shrinkage, chromatin
condensation, and formation of apoptotic bodies.21 Stain of 17-trea-
ted A549 cells with DAPI clearly showed apoptotic bodies as shown
in Figure 4. The images obtained by immunofluorescence micros-
copy evaluate the effect of compound 17 on microtubule network
indirectly. The microtubule network in control cells displayed intact
organization and arrangement. However, when cells were exposed
to various concentrations of 17 for 24 h, it exhibited filament-like
structure and reduced microtubule extent in the cytoplasm (Fig. 4).
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.
The microtubule network shrank significantly at 1.0
lM and was
disrupted thoroughly at 10.0 M. Significant morphological changes
l
were also observed by the incubation of A549 cells with different
concentrations of 17 for 24 h as shown in Figure 5. Cleavage of

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C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
Figure 3. Flow cytometric analysis of A549 cells. Cells were treated with DMSO (A), compound 7 at 1.0
lM (B), 5.0 lM (C) or 10.0 lM (D); and compound 17 at 0.1 lM (E),
1.0 M (F) or 5.0 M (G). After the treatment for 24 h, cells were harvested, fixed, and stained with propidium iodide as described in Section 5.2.2. The percentage of cells in
l
l
each cell cycle phase was summarized in Table 2.
3.76 (two s, 6H, OCH3 ꢀ 2), 6.82 (m, 2H, Ar-H), 6.90 (m, 2H, Ar-
H), 7.14 (m, 2H, Ar-H), 7.30 (m, 2H, Ar-H), 7.70 (m, 1H, 5-H),
7.81–7.86 (m, 2H, 6- and 7-H), 8.11 (d, 1H, 8-H). 13C NMR
(100 MHz, DMSO-d6): 55.08, 55.11, 113.09 (2C), 113.59 (2C),
121.91, 124.78, 127.61, 128.94, 129.23, 129.36, 130.17, 131.25
(2C), 131.33 (2C), 132.37, 141.80, 146.36, 158.01, 158.65, 159.07,
168.33. Anal. Calcd for C24H19NO4: C, 74.79; H, 4.97; N, 3.63.
Found: C, 74.41; H, 5.37; N, 3.41.
Table 2
Effects of compounds 7 and 17 on A549 cell cycle progression
Compound
Concentration (
l
M)
Cell cycle distribution (%)
Sub G1
G1
S
G2/M
DMSO
7
7
7
17
17
17
0.9
0.9
1.0
1.0
1.5
76.2
78.2
77.9
76.9
73.9
75.0
71.9
14.3
13.2
14.3
14.7
16.3
13.6
12.3
9.5
8.6
7.8
8.4
9.8
1.0
5.0
10.0
0.1
1.0
5.0
2.0
17.1
11.0
15.8
5.1.2. 6-Fluoro-2,3-bis(4-methoxyphenyl)quinoline-4-carb-
oxylic acid (3)
From 5-fluoroisatin and 1,2-bis(4-methoxyphenyl)ethanone as
described for 2: Yield: 83%. Mp 310–311 °C. 1H NMR (400 MHz,
DMSO-d6): 3.74 and 3.76 (two s, 6H, OCH3 ꢀ 2), 6.82 (m, 2H, Ar-H),
6.91 (m, 2H, Ar-H), 7.14 (m, 2H, Ar-H), 7.28 (m, 2H, Ar-H), 7.48 (dd,
1H, J = 9.6, 2.8 Hz, 5-H), 7.75–7.80 (ddd, 1H, J = 9.2, 9.2, 2.8 Hz, 7-
H), 8.22 (d, 1H, J = 9.2, 5.6 Hz, 8-H), 13.92 (br s, 1H, COOH). 13C
NMR (100 MHz, DMSO-d6): 55.06, 55.09, 107.94 (J = 23.5 Hz),
113.08 (2C), 113.61 (2C), 120.28 (J = 25.8 Hz), 122.61 (J = 9.8 Hz),
129.05, 129.99, 131.20 (2C), 131.26 (2C), 132.03, 132.33
(J = 9.9 Hz), 141.11 (J = 6.0 Hz), 143.63, 157.63, 158.74, 159.07,
160.30 (J = 243.3 Hz), 167.87. Anal. Calcd for C24H18FNO4ꢁ0.5H2O:
C, 66.96; H, 4.93; N, 3.25. Found: C, 66.61; H, 5.29; N, 2.88.
Merck and Co. 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.
5.1.1. 2,3-Bis(4-methoxyphenyl)quinoline-4-carboxylic acid (2)
A
mixture of isatin (2.94 g, 20 mmol), 1,2-bis(4-methoxy-
phenyl)ethanone (6.15 g, 24 mmol), and KOH (3.37 g, 60 mmol)
in EtOH was heated at 80 °C for 48 h (TLC monitoring). 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 ice-cold aqueous phase was acidified to pH 1 with 37% HCl,
and the precipitate was collected, washed with H2O, and recrystal-
lized from EtOH to give 2 (3.23 g, 84%) as a white solid. Mp 312–
313 °C (lit. 308 °C).19 1H NMR (400 MHz, DMSO-d6): 3.74 and
5.1.3. 6-Methoxy-2,3-bis(4-methoxyphenyl)quinoline-4-
carboxylic acid (4)
From 5-methoxyisatin and 1,2-bis(4-methoxyphenyl)ethanone
as described for 2: yield: 86%. Mp 305–306 °C. 1H NMR

C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
7471
Figure 4. Microtubule effect of compound 17. The confocal laser scanning micrograph shows a merged image double-labeled with DAPI and b-tubulin antibodies. A549 cells
were fixed after the treatment of 17 for 24 h followed by the immunofluorescence analysis using anti-b-tubulin antibody, FITC-conjugated secondary antibody, and DAPI
staining as described in Section 5.2.3.
(400 MHz, DMSO-d6): 3.75, 3.76, and 3.90 (three s, 9H, OCH3 ꢀ 3),
6.81 (m, 2H, Ar-H), 6.90 (m, 2H, Ar-H), 7.06 (d, 1H, J = 2.4 Hz, 5-H),
7.13 (m, 2H, Ar-H), 7.26 (m, 2H, Ar-H), 7.52 (dd, 1H, J = 9.2, 2.8 Hz,
7-H), 8.04 (d, 1H, J = 9.2 Hz, 8-H). 13C NMR (100 MHz, DMSO-d6):
55.06, 55.07, 55.54, 102.33, 113.03 (2C), 113.56 (2C), 122.49,
122.91, 129.32, 129.51, 130.96, 131.11 (2C), 131.27 (2C), 132.41,
140.48, 142.58, 155.39, 157.98, 158.61, 158.83, 168.39. Anal. Calcd
for C25H21NO5: C, 72.28; H, 5.10; N, 3.37. Found: C, 72.41; H, 5.48;
N, 3.11.
5.1.4. 9-Methoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-c]quin-
olin-11-one (5)
A solution of 2 (3.85 g, 10 mmol) in POCl3 (30 mL) was heated at
150 °C for 24 h. After cooling to room temperature, chlorobenzene
(30 mL) and AlCl3 (1.33 g, 10 mmol) were added and refluxed for
3 h (TLC monitoring). After cooling, the mixture was poured into
ice-water (150 mL). The resulting precipitate thus obtained was
collected and then suspended in 5% NaHCO3 aqueous (200 mL)
with vigorous stirring for 1 h. The crude solid was then collected,

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C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
Figure 5. Induction of morphological change in A549 cells. Cells were treated with DMSO (A), compound 17 at 1 lM (B), 5 lM (C) or 10 lM (D) for 24 h at 37 °C and
photographed under a microscope.
Figure 6. Agarose gel electrophoresis for detecting DNA fragmentation in A549
Figure 7. Expression of PARP in A549 cells by the treatment of compound 17.
Exponentially growing cells were treated with 17 for 24 h. Equal amounts of cell
lysate were resolved using SDS–PAGE and analyzed by Western blot using anti-
PARP antibodies. The blots were re-probed with anti-b-actin antibody to confirm
equal protein loading.
cells treated with compound 17 for 24 h. Lane M, DNA size marker; lane 1, DMSO;
lane 2, 1.0 M; lane 3, 5.0 M; lane 4, 10.0 M.
l
l
l
purified by column chromatography (MeOH/CH2Cl2 1/50), and
crystallized from EtOH to give 5 (2.09 g, 57%) as a red solid. Mp:
214–215 °C. 1H NMR (400 MHz, CDCl3): 3.83 and 3.93 (two s, 6H,
9- and 40-OCH3), 6.71 (dd, 1H, J = 8.0, 2.4 Hz, 8-H), 6.84 (d, 1H,
J = 8.0 Hz, 7-H), 7.08 (m, 2H, Ar-H), 7.21 (d, 1H, J = 2.4 H, 10-H),
7.56–7.67 (m, 4H, 2-, 3- and Ar-H), 8.04 (m, 1H, 4-H), 8.82 (m,
1H, 1-H). 13C NMR (100 MHz, CDCl3): 55.44, 55.72, 110.68,
114.17 (2C), 114.20, 119.10, 122.51, 123.79, 124.47, 129.20,
129.57, 129.76, 130.14 (2C), 134.32, 135.58, 135.61, 136.73,
149.22, 154.96, 160.45, 160.85, 195.27. Anal. Calcd for
C24H17NO3ꢁ0.5H2O: C, 76.57; H, 4.83; N, 3.72. Found: C, 76.64; H,
4.99; N, 3.58.
5.1.5. 2-Fluoro-9-methoxy-6-(4-methoxyphenyl)-11H-
indeno[1,2-c]quinolin-11-one (6)
From 3 as described for 5: yield: 83%. Mp 260–261 °C. 1H NMR
(400 MHz, CDCl3): 3.83 and 3.92 (two s, 6H, 9- and 40-OCH3), 6.72
(dd, 1H, J = 8.4, 2.4 Hz, 8-H), 6.87 (d, 1H, J = 8.4 Hz, 7-H), 7.10 (m,
2H, Ar-H), 7.21 (d, 1H, J = 2.4 Hz, 10-H), 7.38–7.43 (m, 1H, 3-H),
7.65 (m, 2H, Ar-H), 8.07 (m, 1H, 4-H), 8.44 (dd, 1H, J = 9.2, 2.8 Hz,
1-H). 13C NMR (100 MHz, CDCl3): 55.46, 55.76, 107.20
(J = 23.4 Hz), 108.07, 110.78, 111.24, 114.23 (2C), 119.25, 120.45

C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
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(J = 26.6 Hz), 124.75, 130.15 (2C), 132.00 (J = 9.8 Hz), 135.11,
5.1.10. 9-Hydroxy-6-{4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-
135.58, 137.45, 154.15, 160.56, 161.11, 162.60 (J = 250.0 Hz),
194.32. Anal. Calcd for C24H16FNO3ꢁ0.3H2O: C, 73.75; H, 4.29; N,
3.58. Found: C, 73.47; H, 4.19; N, 3.53.
11H-indeno[1,2-c]quinolin-11-one (11a) and 9-[2-(pyrrolidin-
1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-yl)ethoxy]phenyl}-11H-
indeno[1,2-c]quinolin-11-one (13a)
To a solution of 8 (0.34 g, 1 mmol) in dry DMF (20 mL) was
added NaH (60% in oil, 0.50 g) at 0 °C for 1 h. N-(2-Chloroethyl)pyr-
rolidineꢁHCl (0.51 g, 3 mmol) was added and the mixture was
heated at 80 °C for 25 min. The reaction mixture was partitioned
between H2O (50 mL) and CH2Cl2 (50 mL). The organic layer was
separated, dried over MgSO4, and evaporated. The resulting residue
was purified by column chromatography (MeOH/CH2Cl2 1/10) to
give two fractions. The first fraction contained the monosubsti-
tuted product 11a (0.15 g, 33%) as a red solid. Mp 245 °C (Dec).
1H NMR (400 MHz, DMSO-d6): 1.72 (m, 4H, pyrrolininyl-H), 2.59
(m, 4H, pyrrolininyl-H), 2.88 (t, 2H, J = 6.0 Hz, CH2N), 4.19 (t, 2H,
J = 6.0 Hz, OCH2), 6.67–6.73 (m, 2H, 7- and 8-H), 6.98 (d, 1H,
J = 2.0 Hz, 10-H), 7.13 (m, 2H, Ar-H), 7.56 (m, 2H, Ar-H), 7.63–
7.70 (m, 2H, 2- and 3-H), 7.94 (m, 1H, 4-H), 8.63 (m, 1H, 1-H),
10.21 (br s, 1H, OH). 13C NMR (100 MHz, DMSO-d6): 23.17 (2C),
54.08 (2C), 54.30, 66.79, 112.23, 114.45 (2C), 120.25, 121.72,
122.97, 124.43, 129.28, 129.43, 129.77, 130.15 (2C), 131.48,
133.15, 133.25, 135.12, 136.63, 148.34, 154.55, 159.09, 159.25,
194.85. Anal. Calcd for C28H24N2O3ꢁ0.6H2O: C, 75.17; H, 5.69; N,
6.26. Found: C, 74.88; H, 5.90; N, 6.52.
The second fraction contained disubstituted product 13a
(0.22 g, 41%) as a red solid. Mp 150–151 °C. 1H NMR (400 MHz,
DMSO-d6+TFA): 1.92–2.16 (m, 8H, pyrrolininyl-H), 3.14–3.28 (m,
4H, OCH2N ꢀ 2), 3.64–3.75 (m, 8H, pyrrolidinyl-H), 4.42 (t, 2H,
J = 5.2 Hz, OCH2), 4.49 (t, 2H, J = 5.2 Hz, OCH2), 6.90 (d, 1H,
J = 8.4 Hz, 7-H), 7.01 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 7.32 (m, 2H, Ar-
H), 7.40 (d, 1H, J = 2.4 Hz, 10-H), 7.78–7.89 (m, 4H, 2-, 3-, and Ar-
H), 8.11 (d, 1H, J = 8.4 Hz, 4-H), 8.82 (dd, 1H, J = 8.4, 1.2 Hz, 1-H).
13C NMR (100 MHz, DMSO-d6+TFA): 22.82 (2C), 22.86 (2C), 53.11,
53.33, 54.30 (2C), 54.38 (2C), 63.92, 64.33, 112.33, 115.33 (2C),
120.68, 122.51, 123.78, 124.66, 127.61, 130.30, 130.58, 130.95
(2C), 131.74, 135.40, 135.44, 135.78, 136.88, 146.88, 153.91,
159.46, 159.65, 193.99. Anal. Calcd for C34H35N3O3ꢁ0.4H2O: C,
75.49; H, 6.68; N, 7.77. Found: C, 75.43; H, 6.56; N, 7.45.
5.1.6. 2,9-Dimethoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-c]-
quinolin-11-one (7)
From 4 as described for 5: yield: 74%. Mp 203–204 °C. 1H NMR
(400 MHz, CDCl3): 3.82, 3.92, and 4.00 (three s, 9H, OCH3 ꢀ 3), 6.70
(dd, 1H, J = 8.4, 2.4 Hz, 8-H), 6.85 (d, 1H, J = 8.4 Hz, 7-H), 7.07 (m,
2H, Ar-H), 7.18 (d, 1H, J = 2.4 Hz, 10-H), 7.26 (dd, 1H, J = 9.2,
2.8 Hz, 3-H), 7.62 (m, 2H, Ar-H), 7.92 (d, 1H, J = 9.2 Hz, 4-H), 8.11
(d, 1H, J = 2.8 Hz, 1-H). 13C NMR (100 MHz, CDCl3): 55.42, 55.70,
55.76, 100.47, 110.49, 114.12 (2C), 118.89, 123.45, 123.97,
124.46, 130.18 (2C), 131.05, 132.10, 132.75, 135.59, 135.80,
136.54, 146.11, 151.94, 160.23, 160.32, 160.77, 195.44. Anal. Calcd
for C25H19NO4: C, 75.55; H, 4.82; N, 3.52. Found: C, 75.46; H, 4.98;
N, 3.41.
5.1.7. 9-Hydroxy-6-(4-hydroxyphenyl)-11H-indeno[1,2-c]quin-
olin-11-one (8)
A solution 5 (0.37 g, 1.0 mmol) in 5 mL of 48% HBr was heated at
reflux under a nitrogen atmosphere for 48 h (TLC monitoring). The
mixture was then cooled and evaporated in vacuo to give a residue
which was treated with H2O (50 mL). The crude product was col-
lected and recrystallized with EtOH to give 0.27 g (78%) of 8 as a
red solid. Mp 338–339 °C. 1H NMR (400 MHz, DMSO-d6): 6.74 (m,
2H, 7- and 8-H), 7.00–7.57 (m, 3H, 10- and Ar-H), 7.55 (m, 2H,
Ar-H), 7.68–7.78 (m, 2H, 2- and 3-H), 8.02 (d, 1H, J = 8.4 Hz, 4-H),
8.67 (m, 1H, 1-H), 10.18 (br s, 1H, OH). 13C NMR (100 MHz,
DMSO-d6): 112.31, 115.44 (2C), 120.54, 121.85, 123.11, 124.56,
127.45, 127.70, 129.76, 130.39 (2C), 130.67, 132.62, 134.43,
134.96, 137.07, 146.45, 154.06, 159.02, 159.35, 194.24. Anal. Calcd
for C22H13NO3ꢁ0.8H2Oꢁ0.7HBr: C, 63.40; H, 3.71; N, 3.36. Found: C,
63.03; H, 3.51; N, 3.29.
5.1.8. 2-Fluoro-9-hydroxy-6-(4-hydroxyphenyl)-11H-indeno-
[1,2-c]quinolin-11-one (9)
From 6 as described for 8: yield: 98%. Mp 324–325°C. 1H NMR
(400 MHz, DMSO-d6): 6.68–6.74 (m, 2H, 7- and 8-H), 6.94–6.98
(m, 3H, 10- and Ar-H), 7.48 (m, 2H, Ar-H), 7.58 (ddd, 1H, J = 8.8,
8.8, 3.2 Hz, 3-H), 8.00 (dd, 1H, J = 9.2, 5.6 Hz, 4-H), 8.18 (dd, 1H,
J = 9.6, 2.8 Hz, 1-H). 13C NMR (100 MHz, DMSO-d6): 106.13
(J = 23.5 Hz), 112.26, 115.34 (2C), 120.27 (J = 21.3 Hz), 120.38,
122.20 (J = 12.2 Hz), 124.75, 128.89, 130.21 (2C), 131.69
(J = 9.9 Hz), 132.55, 133.20 (J = 6.0 Hz), 135.02, 137.46, 145.01,
154.06 (J = 2.3 Hz), 159.62, 159.36, 161.78 (J = 247.8 Hz), 194.15.
Anal. Calcd for C22H12FNO3ꢁ1H2Oꢁ0.3HCl: C, 68.39; H, 3.74; N,
3.63. Found: C, 68.65; H, 4.04; N, 3.38.
5.1.11. 9-Hydroxy-6-{4-[2-(piperidin-1-yl)ethoxy]phenyl}-11H-
indeno[1,2-c]quinolin-11-one (11b) and 9-[2-(piperidin-1-yl)-
ethoxy]-6-{4-[2-(piperidin-1-yl)ethoxy]phenyl}-11H-indeno-
[1,2-c]-quinolin-11-one (13b)
From 7 and N-(2-chloroethyl)piperidineꢁHCl as described for
11a and 13a. Compound 11b was obtained in 30% yield (0.14 g)
as a red solid. Mp 249 °C (Dec).1H NMR (400 MHz, DMSO-d6):
1.37–1.42 (m, 2H, piperidinyl-H), 1.47–1.54 (m, 4H, piperidinyl-
H), 2.46 (m, 4H, piperidinyl-H), 2.70 (t, 2H, J = 5.6 Hz, CH2N), 4.17
(t, 2H, J = 6.0 Hz, OCH2), 6.66–6.72 (m, 2H, 7- and 8-H), 6.96 (d,
1H, J = 2.4 Hz, 10-H), 7.11 (m, 2H, Ar-H), 7.57–7.69 (m, 4H, 2-, 3-,
and Ar-H), 7.92 (m, 1H, 4-H), 8.61 (dd, 1H, J = 8.0, 1.6 Hz, 1-H),
10.21 (br s, 1H, OH). 13C NMR (100 MHz, DMSO-d6): 23.98, 25.63
(2C), 54.52 (2C), 57.45, 65.83, 112.26, 114.52 (2C), 120.27,
121.75, 123.00, 124.46, 129.29, 129.45, 129.80, 130.17 (2C),
131.46, 133.15, 133.27, 135.15, 136.66, 148.37, 154.57, 159.14,
159.34, 194.89. Anal. Calcd for C29H26N2O3ꢁ0.5H2Oꢁ0.3HCl: C,
74.02; H, 5.86; N, 5.95. Found: C, 74.17; H, 6.06; N, 5.55.
Compound 13b was obtained in 47% yield (0.26 g) as a red solid.
Mp 167–168 °C. 1H NMR (400 MHz, CDCl3): 1.42–1.51 (m, 4H,
piperidinyl-H), 1.59–1.69 (m, 8H, piperidinyl-H), 2.51–2.59 (m,
8H, piperidinyl-H), 2.77 (t, 2H, J = 5.6 Hz, CH2N), 2.88 (t, 2H,
J = 6.0 Hz, CH2N), 4.12 (t, 2H, J = 6.0 Hz, OCH2), 4.24 (t, 2H,
J = 6.0 Hz, OCH2), 6.70 (dd, 1H, J = 8.4, 2.8 Hz, 8-H), 6.83 (d, 1H,
J = 8.4 Hz, 7-H), 7.08 (m, 2H, Ar-H), 7.20 (d, 1H, J = 2.4 Hz, 10-H),
5.1.9. 2,9-Dihydroxy-6-(4-hydroxyphenyl)-11H-indeno[1,2-c]quin-
olin-11-one (10)
From 7 as described for 8: yield: 72%. Mp 326 °C (Dec). 1H NMR
(400 MHz, DMSO-d6): 6.70 (dd, 1H, J = 8.4, 2.0 Hz, 8-H), 6.74 (d, 1H,
J = 8.4 Hz, 7-H), 6.94 (m, 2H, Ar-H), 6.98 (d, 1H, J = 2.0 Hz, 10-H),
7.25 (dd, 1H, J = 9.2, 2.8 Hz, 3-H), 7.46 (m, 2H, Ar-H), 7.81 (d, 1H,
J = 9.2 Hz, 4-H), 7.97 (d, 1H, J = 2.8 Hz, 1-H), 9.84, 10.18, and
10.51 (three br s, 3H, OH). 13C NMR (100 MHz, DMSO-d6):
104.41, 111.91, 115.23 (2C), 120.06, 122.94, 123.45, 124.39,
125.78, 130.17 (2C), 131.12, 133.28, 135.39, 136.47, 144.66,
151.17, 158.04, 158.47, 158.94, 195.09. Anal. Calcd for
C22H13NO4ꢁ1.0H2Oꢁ0.1HCl: C, 70.08; H, 4.05; N, 3.72. Found: C,
69.79; H, 4.07; N, 3.66.

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C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
7.55–7.65 (m, 4H, 2-, 3-, and Ar-H), 8.03 (m, 1H, 4-H), 8.82 (m, 1H,
1-H). 13C NMR (100 MHz,CDCl3): 24.05 (2C), 25.77 (2C), 25.78 (2C),
54.98 (2C), 55.08 (2C), 57.64, 57.83, 66.03, 66.46, 111.48, 114.84
(2C), 119.63, 122.51, 123.79, 124.48, 129.15, 129.57, 129.72,
130.11 (2C), 132.09, 134.32, 135.54, 135.61, 136.71, 149.23,
154.96, 159.63, 160.06, 195.24. Anal. Calcd for C36H39N3O3ꢁ1.0H2O:
C, 74.57; H, 7.14; N, 7.25. Found: C, 74.50; H, 6.82; N, 7.23.
J = 9.2, 5.6 Hz, 4-H), 8.44 (dd, 1H, J = 9.6, 2.8 Hz, 1-H). 13C NMR
(100 MHz, CDCl3): 23.43 (2C), 23.46 (2C), 54.69 (2C), 54.78 (2C),
54.96, 66.97, 67.50, 107.16 (J = 23.5 Hz), 111.49, 114.84 (2C),
119.72, 120.33 (J = 26.5 Hz), 123.15 (J = 12.1 Hz), 124.74, 130.09
(2C), 131.82, 132.08 (J = 9.9 Hz), 133.91 (J = 6.9 Hz), 135.14,
135.50, 137.37, 146.49, 154.16, 159.63, 160.26, 162.55
(J = 250.0 Hz), 194.71. Anal. Calcd for C34H34FN3O3ꢁ0.6HCl: C,
71.19; H, 6.09; N, 7.33. Found: C, 71.21; H, 6.38; N, 7.09.
5.1.12. 6-{4-[3-(Dimethylamino)propoxy]phenyl}-9-hydroxy-
11H-indeno[1,2-c]quinolin-11-one (11c) and 9-[3-(dimethyl-
amino)propoxy]-6-{4-[3-(dimethylamino)propoxy] phenyl}-
11H-indeno[1,2-c]-quinolin-11-one (13c)
5.1.14. 2-Fluoro-9-hydroxy-6-{4-[2-(piperidin-1-yl)ethoxy]-
phenyl}-11H-indeno[1,2-c]quino-lin-11-one (12b) and 2-fluoro-
9-[2-(piperidin-1-yl)ethoxy]-6-{4-[2-(piperidin-1-yl) ethoxy]-
phenyl}-11H-indeno[1,2-c]quinolin-11-one (14b)
From 7 and 3-chloro-N,N-dimethylpropanamineꢁHCl as de-
scribed for 11a and 13a. Compound 11c was obtained in 30% yield
(0.13 g) as a red solid. Mp 245 °C (Dec). 1H NMR (400 MHz, DMSO-
d6): 1.94 (m, 2H, OCH2CH2CH2), 2.23 (s, 6H, NMe2), 2.48 (t, 2H,
J = 7.2 Hz, CH2N), 4.13 (t, 2H, J = 6.4 Hz, OCH2), 6.73 (m, 2H, 7-
and 8-H), 7.01 (d, 1H, J = 2.0 Hz, 10-H), 7.13 (m, 2H, Ar-H), 7.61–
7.73 (m, 4H, 2-, 3-, and Ar-H), 7.96 (dd, 1H, J = 7.2, 1.2 Hz, 4-H),
8.66 (m, 1H, 1-H), 10.21 (br s, 1H, OH). 13C NMR (100 MHz,
DMSO-d6): 26.69, 45.02 (2C), 55.59, 65.92, 112.25, 114.41 (2C),
120.29, 121.74, 122.98, 124.45, 129.30, 129.47, 129.81, 130.17
(2C), 131.40, 133.15, 133.28, 135.13, 136.66, 148.35, 154.58,
159.13, 159.42, 194.90. Anal. Calcd for C27H24N2O3ꢁ1.0H2Oꢁ0.5HCl:
C, 70.37; H, 5.81; N, 6.08. Found: C, 70.48; H, 5.97; N, 5.93.
Compound 13c was obtained in 46% yield (0.23 g) as a red solid;
Mp 154–155 °C. 1H NMR (400 MHz, DMSO-d6): 1.91–2.07 (m, 4H,
OCH2CH2CH2), 2.25, 2.29 (two s, 12H, NMe2), 2.45 (t, 2H,
J = 7.2 Hz, CH2N), 2.53 (t, 2H, J = 7.2 Hz, CH2N), 4.05 (t, 2H,
J = 6.4 Hz, OCH2), 4.15 (t, 2H, J = 6.4 Hz, OCH2), 6.69 (dd, 1H,
J = 8.0, 2.4 Hz, 8-H), 6.84 (d, 1H, J = 8.0 Hz, 7-H), 7.08 (m, 2H, Ar-
H), 7.20 (d, 1H, J = 2.4 Hz, 10-H), 7.55–7.65 (m, 4H, 2-, 3-, and Ar-
H), 8.05 (d, 1H, J = 8.4 Hz, 4-H), 8.82 (m, 1H, 1-H). 13C NMR
(100 MHz, DMSO-d6): 27.37, 27.54, 45.51 (2C), 45.56 (2C), 56.14,
56.37, 66.40, 66.74, 111.28, 114.72 (2C), 119.60, 122.50, 123.78,
124.50, 129.13, 129.57, 129.69, 130.11 (2C), 131.91, 134.30,
135.48, 135.53, 136.78, 149.22, 155.02, 159.89, 160.29, 195.33.
Anal. Calcd for C32H35N3O3ꢁ0.5H2Oꢁ0.1HCl: C, 73.57; H, 6.98; N,
8.05. Found: C, 73.79; H, 7.00; N, 7.76.
From 9 and N-(2-chloroethyl) piperidineꢁHCl as described for
11a and 13a. Compound 12b was obtained in 37% yield (0.17 g)
as a red solid. Mp 245–246 °C. 1H NMR (400 MHz, DMSO-d6):
1.43 (m, 2H, piperidinyl-H), 1.57 (m, 4H, piperidinyl-H), 2.59 (m,
4H, piperidinyl-H), 2.83 (m, 2H, CH2N), 4.23 (t, 2H, J = 6.4 Hz,
OCH2), 6.68–6.74 (m, 2H, 7- and 8-H), 6.97 (d, 1H, J = 2.0 Hz, 10-
H), 7.16 (m, 2H, Ar-H), 7.56–7.61 (m, 3H, 3- and Ar-H), 8.00 (dd,
1H, J = 9.2, 5.6 Hz, 4-H), 8.19 (dd, 1H, J = 9.6, 2.8 Hz, 1-H), 10.25
(br s, 1H, OH). 13C NMR (100 MHz, DMSO-d6): 23.58, 25.19,
30.67, 54.25 (2C), 57.07, 65.37, 106.05 (J = 23.5 Hz), 112.27,
114.51 (2C), 120.05 (J = 25.8 Hz), 120.28, 122.21 (J = 12.1 Hz),
124.64, 130.11 (2C), 131.23, 132.30 (J = 9.1 Hz), 132.62, 132.87,
135.09, 137.29, 145.63, 153.90, 159.21, 159.33, 161.81
(J = 247.9 Hz), 194.30. Anal. Calcd for C29H25FN2O3ꢁ0.4HCl: C,
72.08; H, 5.30; N, 5.79. Found: C, 72.29; H, 5.48; N, 5.49.
Compound 14b was obtained in 53% yield (0.31 g) as a red solid;
Mp 165–166 °C. 1H NMR (400 MHz, CDCl3): 1.47–1.54 (m, 4H,
piperidinyl-H), 1.63–1.75 (m, 8H, piperidinyl-H), 2.57–2.67 (m,
8H, piperidinyl-H), 2.82 (t, 2H, J = 5.6 Hz, CH2N), 2.95 (t, 2H,
J = 5.6 Hz, CH2N), 4.19 (t, 2H, J = 5.6 Hz, OCH2), 4.31 (t, 2H,
J = 5.6 Hz, OCH2), 6.73 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 6.87 (d, 1H,
J = 8.4 Hz, 7-H), 7.09 (m, 2H, Ar-H), 7.21 (d, 1H, J = 2.4 Hz, 10-H),
7.38–7.43 (ddd, 1H, J = 9.2, 8.4, 2.8 Hz, 3-H), 7.62 (m, 2H, Ar-H),
8.03 (m, 1H, 4-H), 8.43 (dd, 1H, J = 9.6, 2.8 Hz, 1-H). 13C NMR
(100 MHz, CDCl3): 23.82, 23.91, 25.44 (2C), 25.58 (2C), 54.95
(2C), 55.00, 57.34, 57.55, 57.69, 65.70, 66.28, 107.18 (J = 23.5 Hz),
111.62, 114.87 (2C), 119.66, 120.36 (J = 26.5 Hz), 123.17
(J = 11.4 Hz), 124.74, 129.12, 130.13 (2C), 131.91, 132.09
(J = 9.8 Hz), 133.78, 135.18, 135.54, 137.35, 146.51, 159.52,
160.19, 162.57 (J = 250.2 Hz), 194.67. Anal. Calcd for
C36H38FN3O3ꢁ0.1HCl: C, 74.10; H, 6.59; N, 7.20. Found: C, 73.88;
H, 6.63; N, 7.15.
5.1.13. 2-Fluoro-9-hydroxy-6-{4-[2-(pyrrolidin-1-yl)ethoxy]-
phenyl}-11H-indeno[1,2-c] quinolin-11-one (12a) and 2-fluoro-
9-[2-(pyrrolidin-1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-yl)ethoxy]-
phenyl}-11H-indeno-[1,2-c]quinolin-11-one (14a)
From 9 and N-(2-chloroethyl) pyrrolidineꢁHCl as described for
11a and 13a. Compound 12a was obtained in 32% yield (0.15 g) as
a red solid. Mp 203–204 °C. 1H NMR (400 MHz, DMSO-d6): 1.78 (m,
4H, pyrrolidinyl-H), 2.62 (m, 4H, pyrrolidinyl-H), 2.91 (t, 2H,
J = 6.0 Hz, CH2N), 4.20 (t, 2H, J = 6.0 Hz, OCH2), 6.66–6.72 (m, 2H, 7-
and 8-H), 6.95 (d, 1H, J = 2.4 Hz, 10-H), 7.12 (m, 2H, Ar-H), 7.54–
7.59 (m, 3H, 3- and Ar-H), 7.98 (dd, 1H, J = 9.2, 5.6 Hz, 4-H), 8.18
(dd, 1H, J = 9.6, 2.8 Hz, 1-H). 13C NMR (100 MHz, DMSO-d6): 23.14
(2C), 54.07 (2C), 54.25, 66.66, 106.06 (J = 23.5 Hz), 112.26, 114.44
(2C), 119.91, 120.05 (J = 26.6 Hz), 120.29, 122.20 (J = 12.1 Hz),
124.65, 130.14 (2C), 131.18, 132.29 (J = 9.9 Hz), 132.62, 132.88
(J = 6.8 Hz), 135.08, 137.28, 145.63, 153.89, 159.28 (J = 8.3 Hz),
161.80 (J = 247.0 Hz), 194.29. Anal. Calcd for C28H23FN2O3ꢁ0.5HCl:
C, 71.12; H, 5.02; N, 5.92. Found: C, 71.50; H, 5.42; N, 5.82.
Compound 14a was obtained in 50% yield (0.28 g) as a red solid;
mp 163–164 °C. 1H NMR (400 MHz, CDCl3): 1.78–1.90 (m, 8H, pyr-
rolidinyl-H), 2.65 (m, 4H, pyrrolindinyl-H), 2.74 (m, 4H, pyrrolindi-
nyl-H), 2.92 (t, 2H, J = 5.6 Hz, OCH2N), 3.02 (t, 2H, J = 5.6 Hz,
OCH2N), 4.14 (t, 2H, J = 5.6 Hz, OCH2), 4.26 (t, 2H, J = 5.6 Hz,
OCH2), 6.74 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 6.86 (d, 1H, J = 8.4 Hz, 7-
H), 7.08 (m, 2H, Ar-H), 7.21 (d, 1H, J = 2.4 Hz, 10-H), 7.39 (ddd,
1H, J = 9.2, 9.2, 2.8 Hz, 3-H), 7.61 (m, 2H, Ar-H), 8.04 (dd, 1H,
5.1.15. 6-{4-[3-(Dimethylamino)propoxy]phenyl}-2-fluoro-9-
hydroxy-11H-indeno[1,2-c] quinolin-11-one (12c) and 9-[3-
(dimethylamino)propoxy]-6-{4-[3-(dimethylamino) propoxy]-
phenyl}-2-fluoro-11H-indeno[1,2-c]quinolin-11-one (14c)
From 9 and 3-chloro-N,N-dimethylpropanamineꢁHCl as de-
scribed for 11a and 13a. Compound 12c was obtained in 35% yield
(0.15 g). Mp 279–280 °C. 1H NMR (400 MHz, DMSO-d6): 2.10 (m,
2H, OCH2CH2CH2), 2.60 (s, 6H, NMe2), 2.97 (t, 2H, J = 7.2 Hz,
CH2N), 4.16 (t, 2H, J = 6.4 Hz, OCH2), 6.72 (m, 2H, 7- and 8-H),
7.01 (m, 1H, 10-H), 7.15 (m, 2H, Ar-H), 7.59–7.63 (m, 3H, 3- and
Ar-H), 8.02 (dd, 1H, J = 9.2, 5.6 Hz, 4-H), 8.25 (dd, 1H, J = 9.6,
2.8 Hz, 1-H). 13C NMR(100 MHz, DMSO-d6): d 24.98, 43.28, 54.70,
54.94, 65.36, 106.12 (J = 23.5 Hz), 112.35, 114.49 (2C), 120.20
(J = 33.4 Hz), 120.36, 122.29 (J = 11.4 Hz), 124.67, 130.20 (2C),
131.35, 132.37 (J = 9.8 Hz), 132.66, 133.02, 135.15, 137.38,
145.69, 153.99, 159.21, 159.41, 161.88 (J = 247.8 Hz), 194.40. Anal.
Calcd for C27H23FN2O3ꢁ1.5H2Oꢁ2.5HCl: C, 57.82; H, 5.13; N, 5.00.
Found: C, 58.04; H, 5.12; N, 5.17.
Compound 14c was obtained in 41% yield (0.22 g) as a red solid.
Mp 164–165 °C. 1H NMR (400 MHz, CDCl3): 1.92–2.07 (m, 4H,

C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
7475
OCH2CH2CH2), 2.26 (s, 6H, NMe2), 2.30 (s, 6H, NMe2), 2.44 (t, 2H,
J = 7.2 Hz, OCH2N), 2.53 (t, 2H, J = 6.8 Hz, OCH2N), 4.04(t, 2H,
J = 6.4 Hz, OCH2), 4.15 (t, 2H, J = 6.4 Hz, OCH2), 6.71 (dd, 1H,
J = 8.4, 2.4 Hz, 8-H), 6.86 (d, 1H, J = 8.4 Hz, 7-H), 7.08 (m, 2H, Ar-
H), 7.21 (d, 1H, J = 2.4 Hz, 10-H), 7.39 (ddd, 1H, J = 9.2, 8.4, 2.8 Hz,
3-H), 7.61 (m, 2H, Ar-H), 8.02 (dd, 1H, J = 9.2, 5.6 Hz, 4-H), 8.45
(dd, 1H, J = 9.2, 2.8 Hz, 1-H). 13C NMR (100 MHz, CDCl3): 27.31,
27.49, 45.44 (2C), 45.81 (2C), 56.09, 56.33, 66.42, 66.78, 107.17
(J = 23.5 Hz), 111.38, 114.77 (2C), 119.69, 120.27 (J = 26.6 Hz),
123.16 (J = 12.2 Hz), 124.74, 130.09 (2C), 131.65, 132.10
(J = 9.9 Hz), 133.93 (J = 6.8 Hz), 135.03, 135.56, 137.45, 146.51,
154.27, 159.97, 160.54, 162.56 (J = 250.2 Hz), 194.78. Anal. Calcd
for C32H34FN3O3ꢁ1.0H2O: C, 70.70; H, 6.66; N, 7.70. Found: C,
70.49; H, 6.56; N, 7.62.
CH2N ꢀ 2), 4.05 (t, 2H, J = 6.4 Hz, OCH2), 4.15 (t, 2H, J = 6.4 Hz,
OCH2), 4.24 (t, 2H, J = 6.4 Hz, OCH2), 6.69 (dd, 1H, J = 8.4, 2.4 Hz,
8-H), 6.84 (d, 1H, J = 8.4 Hz, 7-H), 7.07 (m, 2H, Ar-H), 7.19 (d, 1H,
J = 2.4 Hz, 10-H), 7.27 (dd, 1H, J = 9.2, 2.8 Hz, 3-H), 7.60 (m, 2H,
Ar-H), 7.90 (d, 1H, J = 9.2 Hz, 4-H), 8.12 (d, 1H, J = 2.8 Hz, 1-H).
13C NMR (100 MHz, CDCl3): 27.40 (2C), 27.56, 45.50 (2C), 45.56
(4C), 56.16, 56.38 (2C), 66.40, 66.70, 66.74, 101.23, 111.12,
114.70 (2C), 119.40, 123.57, 124.01, 124.47, 130.15 (2C), 131.03,
132.08, 132.75, 135.51, 135.81, 136.60, 146.09, 151.93, 159.69,
159.72, 160.24, 165.46. Anal. Calcd for C37H46N4O4ꢁ1.0H2Oꢁ0.2HCl:
C, 69.85; H, 7.65; N, 8.81. Found: C, 69.69; H, 7.45; N, 8.65.
5.2. Pharmacological methods
5.2.1. Antiproliferative assay
5.1.16. 2,9-Bis[2-(pyrrolidin-1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-
yl)ethoxy]phenyl}-11H-indeno [1,2-c]quinolin-11-one (15)
To a solution of 10 (0.36 g, 1 mmol) in dry DMF (20 mL) was
added NaH (60% in oil, 0.50 g) at 0 °C for 1 h. N-(2-Chloroethyl)pyr-
rolidineꢁHCl (0.51 g, 3 mmol) was added and the mixture was
heated at 80 °C for 60 min. The reaction mixture was partitioned
between H2O (50 mL) and CH2Cl2 (50 mL). The organic layer was
separated, dried over MgSO4, and evaporated. The resulting residue
was purified by column chromatography (MeOH/CH2Cl2 1/10) to
give 15 (0.21 g, 57%) as a orange solid. Mp 157–158 °C. 1H NMR
(400 MHz, CDCl3): 1.81–1.88 (m, 12H, pyrrolidinyl-H), 2.64 (m,
4H, pyrrolidinyl-H), 2.71 (m, 8H, pyrrolidinyl-H), 2.91 (t, 2H,
CH2N), 2.98–3.04 (m, 4H, CH2N ꢀ 2), 4.13 (t, 2H, J = 5.6 Hz, OCH2),
4.24 (t, 2H, J = 5.6 Hz, OCH2), 4.33 (t, 2H, J = 5.6 Hz, OCH2), 6.73
(dd, 1H, J = 8.0, 2.8 Hz, 8-H), 6.84 (d, 1H, J = 8.0 Hz, 7-H), 7.08 (m,
2H, Ar-H), 7.20 (d, 1H, J = 2.8 Hz, 10-H), 7.31 (dd, 1H, J = 9.2,
2.8 Hz, 3-H), 7.61 (m, 2H, Ar-H), 7.90 (d, 1H, J = 9.2 Hz, 4-H), 8.13
(d, 1H, J = 2.8 Hz, 1-H). 13C NMR (100 MHz, CDCl3): 23.47 (2C),
23.51 (4C), 54.69 (2C), 54.73 (2C), 54.78 (2C), 54.85, 54.87, 55.03,
67.15, 67.43, 67.56, 101.2, 111.25, 114.81 (2C), 119.52, 123.74,
123.98, 124.48, 130.16 (2C), 131.03, 132.23, 132.81, 135.64,
135.79, 136.57, 146.13, 152.00, 159.47, 159.60, 160.05, 195.41.
Anal. Calcd for C40H46N4O4ꢁ1.5H2Oꢁ1.0HCl: C, 67.62; H, 7.11; N,
7.89. Found: C, 67.45; H, 6.76; N, 7.58.
Cancer cells (Hep G2, Hep 3B, Hep 2.2.1, A549, H1299) and nor-
mal cell (MRC-5) were purchased from Bioresources Collection and
Research Center, Taiwan. Cell line was maintained in the same
standard medium and grown as a monolayer in DMEM (Gibco,
USA) and supplemented with 10% fetal bovine serum (FBS) and
antibiotics that is, 100 IU/mL penicillin, 0.1 mg/mL streptomycin
and 0.25 lg/mL amphotercin. Culture was maintained at 37 °C
with 5% CO2 in a humidified atmosphere.
Cells (5 ꢀ 103 cells/well) were treated as indicated for 72 h in
medium containing 10% FBS. (The medium was then changed
(100
liters of serum-free medium containing XTT (1 mg/mL) and phen-
azine methosulfate (PMS) (10 M) was added to the cells which
lL), and the cells were incubated for another day. Fifty micro-
l
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 (GI50
)
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%.20
5.2.2. Cell cycle analysis
5.1.17. 2,9-Bis[2-(piperidin-1-yl)ethoxy]-6-{4-[2-(piperidin-1-
yl)ethoxy]phenyl}-11H-indeno [1,2-c]quinolin-11-one (16)
From 10 and N-(2-chloroethyl)piperidineꢁHCl as described for
15: yield: 53%. Mp 159–160 °C. 1H NMR (400 MHz, CDCl3): 1.49
(m, 6H, piperidinyl-H), 1.61–1.72 (m, 12H, piperidinyl-H), 2.54
(m, 4H, piperidinyl-H), 2.62 (m, 8H, piperidinyl-H), 2.80 (t, 2H,
J = 5.6 Hz, CH2N), 2.88–2.93 (m, 4H, CH2N ꢀ 2), 4.15 (t, 2H,
J = 5.6 Hz, OCH2), 4.26 (t, 2H, J = 5.6 Hz, OCH2), 4.33 (t, 2H,
J = 5.6 Hz, OCH2), 6.70 (dd, 1H, J = 8.4, 2.8 Hz, 8-H), 6.84 (d, 1H,
J = 8.4 Hz, 7-H), 7.08 (m, 2H, Ar-H), 7.18 (d, 1H, J = 2.8 Hz, 10-H),
7.28 (dd, 1H, J = 9.2, 2.8 Hz, 3-H), 7.61 (m, 2H, Ar-H), 7.89 (d, 1H,
J = 9.2 Hz, 4-H), 8.11 (d, 1H, J = 2.8 Hz, 1-H). 13C NMR (100 MHz,
CDCl3): 23.96, 23.99, 24.07, 25.63 (2C), 25.69 2C), 25.76 (2C),
54.99 (2C), 55.02 (2C), 55.05 (2C), 55.15, 57.64, 57.78, 65.85,
66.12, 66.30, 101.26, 111.29, 114.79 (2C), 119.43, 123.67, 123.94,
124.45, 130.17 (2C), 131.01, 132.23, 132.77, 135.58, 135.74,
136.52, 146.09, 151.94, 159.32, 159.48, 159.94, 195.33. Anal. Calcd
for C43H52N4O4ꢁ0.5H2O: C, 73.99l; H, 7.67; N, 8.03. Found: C, 73.74;
H, 7.48; N, 7.94.
A549 cells were treated with DMSO, 7 or 17 at different con-
centrations (0.1, 1.0, 5.0, 10.0 lM) for 24 h. Cells were harvested,
rinsed in PBS, resuspended, fixed in 70% ethanol, and stored at
ꢂ20 °C in fixation buffer until ready for analysis. The pellets
were suspended in 1 mL of propidium iodide (PI) solution con-
taining 20 lg/lL of PI, 0.2 mg/mL RNase, and 0.1% (v/v) 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
A549 Cells were seeded on cover glasses in 12-well plates
with DMSO or compound 17 for 24 h were used for DAPI stain-
ing. After incubation, cells were washed with 1ꢀ 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 were
stained with b-tubulin monoclonal antibody (Santa Cruza
1:1000) at 4 °C overnight. After primary antibody incubation,
cells were washed with PBS containing 0.2% TritonX-100 three
times, and stained with fluorescein isothiocyanate-conjugated
anti-mouse IgG antibody (Santa Cruze, 1:200 diluted) at room
temperature for 1 h. Finally, washed with PBS and stained with
5.1.18. 2,9-Bis[3-(dimethylamino)propoxy]-6-{4-[3-(dimethyl-
amino)propoxy]phenyl}-11H-indeno[1,2-c]quinolin-11-one (17)
From 10 and 3-chloro-N,N-dimethylpropanamineꢁHCl as de-
scribed for 15: yield: 50%. Mp 140–141 °C. 1H NMR (400 MHz,
CDCl3): 1.93–2.08 (m, 6H, OCH2CH2CH2), 2.25, 2.29, and 2.30 (three
s, 18H, NMe2 ꢀ 3), 2.45 (t, 2H, J = 6.8 Hz, CH2N), 2.49–2.54 (m, 4H,

7476
C.-H. Tseng et al. / Bioorg. Med. Chem. 17 (2009) 7465–7476
DAPI (0.1 mg/mL) for 5 min at room temperature in the dark. Re-
moved the excess DAPI solution and washed with PBS twice.
Samples were mounted before analyzing under a fluorescence
microscopy.
Acknowledgments
Financial support of this work by the National Science Council
of the Republic of China is gratefully acknowledged. We also thank
National Cancer Institute (NCI) of the United States for the antican-
cer screenings and the National Center for High-Performance Com-
puting for providing computer resources and chemical database
services.
5.2.4. DNA fragmentation assay
DNA fragmentation was determined by agarose gel electropho-
resis. Cells were treated with various concentrations of compound
17 (1.0, 5.0, 10.0 lM) for 24 h and then washed twice with PBS.
References and notes
Total DNA was isolated using a commercial kit (Genomic DNA
Purification Kit, Fermentas Life Sciences). DNA agarose electropho-
resis was executed at 100 V on a 2.0% agarose gel in 1ꢀ TAE buffer
(40 mmol/L of Tris, 2 mmol/L of EDTA, 20 mmol/L of acetic acid).
DNA ladder marker (0.2–14.0 kb; GeneMark) was added to gel as
a reference for the analysis of internucleosomal DNA fragmenta-
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5.2.5. Immunoblot analysis
After treatment of compound 17, cells were collected and
washed twice with cold PBS and then lysed in lysis buffer
(50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM
EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM DTT, 1 mM
PMSF, 25 lg/mL aprotinin, and 25 lg/mL leupeptin) and kept on
ice for 30 min. The lysates were centrifuged at 12,000g at 4 °C for
20 min and the supernatants were stored at ꢂ70 °C. The protein
concentration was determined by the Bradford method. Protein
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(20 lg) were separated by 10% SDS–PAGE and transferred onto a
PVDF membrane using a glycine transfer buffer (192 mM glycine,
25 mM Tris–HCl, 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 anti-
bodies in milk containing Tris-buffered saline (TBS) and 0.5%
Tween. Anti-human-PARP antibodies were used at a 1:1000 dilu-
tion as the primary antibodies, while horseradish peroxidase-con-
jugated horse anti-rabbit IgG (Vector Laboratories, 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 de-
tected using the enhanced chemiluminescence blotting detection
system (Amersham, USA).
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