Synthesis and antiproliferative evaluation of 6-aryl-11-iminoindeno[1,2-c] quinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2011.10.014

A number of 6-aryl-11-iminoindeno[1,2-c]quinoline derivatives were synthesized and evaluated for their antiproliferative activities. Among them, (E)-6-{4-[3-(dimethylamino)propoxy]phenyl}-2-fluoro-9-hydroxy-11H-indeno[1,2-c] quinolin-11-one O-3-(dimethylamino)propyl oxime (23a) was the most active, exhibited GI₅₀ values of 0.64, 0.39, 0.55, 0.67, and 0.65 μM against the growth of Hep G2, Hep 3B, A549, H1299, and MDA-MB-231, respectively. Compound 23a inhibited the growth of hepatoma cell lines in a dose- and time-dependent manner. The proportion of cells was decreased in the G1 and accumulated in G2/M phase after 12 h treatment of 23a, while the hypodiploid (sub-G0/G1 phase) cells increased. Further investigations have shown that 23a induced cell cycle arrest at G2/M phase and induce apoptosis via activation of p53, Bax, and caspase-8 which consequently cause cell death.

Full text of DOI:10.1016/j.bmc.2011.10.014

Bioorganic & Medicinal Chemistry 19 (2011) 7653–7663
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiproliferative evaluation of 6-aryl-11-iminoindeno[1,2-c]
quinoline derivatives
Chih-Hua Tseng a, Cherng-Chyi Tzeng a, Kuin-Yu Chung a, Chai-Lin Kao a, Chih-Yao Hsu a,
Chih-Mei Cheng b, Keng-Shiang Huang c, Yeh-Long Chen a,
⇑
a Department of Medicinal and Applied Chemistry, College of Life Science, and Center of Excellence for Environmental Medicine, Kaohsiung Medical University,
Kaohsiung City 807, Taiwan
b Department of Biomedical Science and Environmental Biology, College of Life Science, and Center of Excellence for Environmental Medicine, Kaohsiung Medical University,
Kaohsiung, Taiwan
c The School of Chinese Medicine for Post-Baccalaureate, I-Shou University, 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 6-aryl-11-iminoindeno[1,2-c]quinoline derivatives were synthesized and evaluated for their
antiproliferative activities. Among them, (E)-6-{4-[3-(dimethylamino)propoxy]phenyl}-2-fluoro-9-
hydroxy-11H-indeno[1,2-c]quinolin-11-one O-3-(dimethylamino)propyl oxime (23a) was the most
Received 1 August 2011
Revised 5 October 2011
Accepted 6 October 2011
Available online 20 October 2011
active, exhibited GI50 values of 0.64, 0.39, 0.55, 0.67, and 0.65 lM against the growth of Hep G2, Hep
3B, A549, H1299, and MDA-MB-231, respectively. Compound 23a inhibited the growth of hepatoma cell
lines in a dose- and time-dependent manner. The proportion of cells was decreased in the G1 and accu-
mulated in G2/M phase after 12 h treatment of 23a, while the hypodiploid (sub-G0/G1 phase) cells
increased. Further investigations have shown that 23a induced cell cycle arrest at G2/M phase and induce
apoptosis via activation of p53, Bax, and caspase-8 which consequently cause cell death.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
6-Arylindeno[1,2-c]quinoline derivatives
Antiproliferative activity
Apoptotic induction
1. Introduction
the positive topotecan and irinotecan.12 (E)-6-Hydroxy-9-meth-
oxy-11H-indeno[1,2-c]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl
Quinoline moiety constitutes a number of potential anticancer
agents. For examples, camptothecin (CPT) which bears a quinoline
skeletone, is an anticancer alkaloid isolated from Camptotheca
oxime (4), which exhibited GI50 values of 0.84, 0.89, and
0.79 M against SAS, A549, and BT483, respectively, was more
l
active than CPT.13 In continuation of our study to explore more
potent anticancer drug candidates, we described herein the prep-
aration of certain 6-aryl-11-iminoindeno[1,2-c]quinoline deriva-
tives and their evaluation in vitro against a panel of six cancer
cell lines including two human hepatocelluar carcinoma cells
(Hep G2 and Hep 3B), two non-small cell lung cancer cells
(A549 and H1299), and two breast cancer cells (MCF-7 and
MDA-MB-231). These cancers are common malignancies in the
world, and especially are the leading cause of cancer deaths in
Asian countries including Taiwan.14–17
acuminate.1,2 Camptothecin is
a prototypical topoisomerase I
(Topo I) inhibitor with poor water solubility. Subsequent intro-
duction of hydrophilic side chains led to the discovery of topotec-
an which are currently used as an anticancer drug. TAS-103 (1)
(Fig. 1), one of the indeno[2,1-c]quinoline derivatives, has been
proved to be a novel Topo I and Topo II targeting agent that
stabilizes cleavable complexes of Topo-DNA at the cellular le-
vel.3–5 A number of furo[2,3-b]quinoline derivatives, such as
CIL-102 (2), was synthesized and demonstrated to possess signif-
icant anticancer activity.6–10 We have also prepared certain inde-
no[1,2-c]quinoline and 6-arylindeno[1,2-c]quinoline derivatives
which have shown good antiproliferative activity, DNA binding
affinity, and topo I and topo II inhibitory activities.11–13 For exam-
2. Chemistry
ples,
amino)propoxy )phenyl)-11H-indeno[1,2-c]-quinolin-11-one (3),
which exhibited GI50 values of 0.60 and 0.68 M against the
2,9-bis(3-(dimethylamino)propoxy)-6-(4-(3-(dimethyl-
Treatment of the known 9-methoxy-6-(4-methoxyphenyl)-
11H-indeno[1,2-c]quinolin-11-one (5)12 with NH2OH gave
exclusively (E)-9-methoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-
c]-quinolin-11-one oxime (8) as a sole product. Alkylation of 8 with
3-chloro-N,N-dimethylpropanamine afforded (E)-9-methoxy-6-(4-
methoxyphenyl)-11H-indeno[1,2-c]-quinolin-11-one O-3-(dimeth-
ylamino)propyl oxime (11a) as described in Scheme 1. Compounds
l
growth of Hep G2 and A549, respectively, was more active than
⇑
Corresponding author. Tel.: +886 7 3121101x2684; fax: +886 7 3125339.
E-mail address: yeloch@kmu.edu.tw (Y.-L. Chen).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.014

7654
C.-H. Tseng et al. / Bioorg. Med. Chem. 19 (2011) 7653–7663
O
R3
N
R2
R1
Me
O
N
O
HN
O
N
HO
N
N
H
O
O
N
OH
Camptothecin R1 = H; R2 = H; R3 = H
1
, TAS-103
2, CIL-102
Topotecan R1 = OH; R2 = CH2N(CH3)2, R3 = H
OR4
11
OR2
OR
9
N
N
O
O
2
R1
OMe
RO
N
6
N
N
OR3
OR
, R = CH2CH2CH2N(CH3)2
N
OH
Targeted compounds
3
4
Figure 1. Structures of Camptothecin, Topotecan, 6-[2-(dimethylamino)ethylamino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one (1, TAS-103), 1-[4-(Furo[2,3-b]quinolin-4-
ylamino)phenyl]-ethanone (2, CIL-102), Aminoalkyl substituted indenoquinolines 3 and 4, and targeted compounds.
OR1
OMe
OH
OMe
OMe
O
N
N
(i)
(ii)
R
R
R
N
N
N
5
6
7
R = H
OMe
8 R = H
OMe
OMe
11 R = H
R = F
9 R = F
12 R = F
R = OMe
10 R = OMe
13 R = OMe
N
a R 1=
b R1 =
c R1 =
N
N
Scheme 1. Reagents and conditions: (i) NH2OHꢀHCl, 2-EtOEtOH, 150 °C, 3 h; (ii) NaH, alkyl halides, DMF, 80 °C, 1 h.
11b and 11c were obtained by alkylation of 8 with N-(2-chloro-
Structure of 18b was assigned based on the downfield protons
of OCH2 at 4.68 ppm and two broad signals of OH groups at 9.88
and 9.86 ppm. Structures of dialkylated products 20b and 22b
were distinguished by the NOESY spectra. Compound 20b was as-
signed as 9,11-dialkylated derivative based on the correlation of
ethyl)pyrrolidine and N-(2-chloroethyl)piperidine, respectively,
under the same reaction conditions. Compounds 12a–12c
were prepared from their oxime precursor 9 which in turn was
obtained by the reaction of 612 with NH2OH. Accordingly, oximiza-
tion of compound 712 gave its oxime derivative 10 which was then
alkylated with various aminoalkyl halides to afford 13a and
13b.
OCH2 (dH = 4.15 ppm)/8-H (dH = 6.61 ppm) and OCH2 (dH
=
4.15 ppm)/10-H (dH = 7.93 ppm). Compound 22b was assigned as
6-aryl,11-dialkylated isomer based on the correlations between
OCH2 (dH = 3.93 ppm) and meta-H (dH = 6.23 ppm). The same
assignments were applied for the mixtures of 18a, 20a, and 22a;
19a, 21a, and 23a; and 19b, 21b, and 23b (Fig. 2).
Reaction of 1412 with NH2OH afforded 9-hydroxy-6-(4-hydroxy-
phenyl)-11H-indeno[1,2-c]quinolin-11-one oxime (16) which was
then alkylated with 3-chloro-N,N-dimethylpropanamine to give a
mixture of (E)-9-hydroxy-6-(4-hydroxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one O-3-(dimethylamino)propyl oxime (18a),
(E)-9-[3-(dimethylamino)propoxy]-6-(4-hydroxyphenyl)-11H-in-
deno[1,2-c]quinolin-11-one O-3-(dimethylamino)propyl oxime
(20a) and (E)-6-{4-[3-(dimethylamino)propoxy]phenyl}-9-hydro-
xy-11H-indeno[1,2-c]quinolin-11-one O-3-(dimethylamino)propyl
oxime (22a) in a yield of 27%, 26%, and 21%, respectively as de-
scribed in Scheme 2. Compounds 18b, 20b, and 22b were obtained
by alkylation of their oxime precursor 16 which in turn was pre-
pared from 14 and NH2OH. Accordingly, oximization of compound
1512 gave its oxime derivative 17 which was then alkylated with 3-
chloro-N,N-dimethylpropanamine to give a mixture of 19a, 21a,
and 23a. Alkylation compound 17 with N-(2-chloroethyl)pyrroli-
dine afforded a mixture of 19b, 21b, and 23b.
3. Biological results and discussion
All the synthesized 6-aryl-11-iminoindeno[1,2-c]quinoline
derivatives were evaluated in vitro against a panel of six cancer cell
lines including two human hepatocelluar carcinoma cells (Hep G2
and Hep 3B), two non-small cell lung cancer cells (A549 and
H1299), and two breast cancer cells (MCF-7 and MDA-MB-231)
using XTT (2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetra-
zolium-5-carboxanilide) assay.18 The concentration that inhibited
the growth of 50% of cells (GI50) was determined from the linear por-
tion of the curve by calculating the concentration of agent that
reduced absorbance in treated cells, compared to control cells, by

C.-H. Tseng et al. / Bioorg. Med. Chem. 19 (2011) 7653–7663
7655
OH
OH
OH
N
O
(ii)
R
(i)
R
R
N
N
OH
OR1
14
15
16 R = H
R = H
OH
OR1
17 R = F
R = F
OR1
N
OR1
OH
OH
N
N
R
R
N
N
N
OH
OH
OR1
22 R = H
23 R = F
18
19
20 R = H
21 R = F
R = H
R = F
N
a
b
R1 =
R1 =
N
Scheme 2. Reagents and conditions: (i) Hydroxyamine, 2-EtOEtOH, 150 °C, 3 h; (ii) NaH, alkyl halides, DMF, 80 °C, 25 min.
N
N
N
4.68 ppm
4.67 ppm
4.78 ppm
N
O
N
OH
O
N
OH
O
N
O
4.15 ppm
N
N
N
N
OH
O
18b
20b
OH
22b
3.93 ppm
N
N
N
4.67 ppm
4.70 ppm
4.66 ppm
N
O
N
OH
O
N
OH
O
N
O
4.05 ppm
F
F
F
N
N
N
N
OH
19b
21b
23b
O
OH
4.21 ppm
Figure 2. NOESY correlations of compounds 20b, 21b, 22b and 23b.
50%. The GI50 results of 6-arylindeno[1,2-c]quinoline-11-one deriv-
atives are summarized in Table 1. Three oxime precursors 8–10 were
inactive against all the cancer cells tested with GI50 of >10 lM in
C-11 oxime precursor 9 while 12c was inactive. With the same ami-
noalkyl side chain on C-11 oxime moiety, the antiproliferative activ-
ity of C-2 methoxy derivative 13a and its C-2 unsubstituted
counterpart 11a are comparable while C-2 fluoro derivative 12a
was the most active. Compound 12a exhibited GI50 values of 0.66,
0.91, 0.61, and 0.70 lM against the growth of Hep 3B, A549,
H1299, and MDA-MB-231, respectively, was more active than the
positive Irinotecan.
With an exception of the MDA-MB-231, the oxime precursor 16
was inactive. Introduction of C-2 fluoro group improved antiprolif-
erativeactivity in whichcompound17 was marginallyactive against
the growth of Hep 3B, MCF-7, and MDA-MB-231. However, antipro-
liferative activity was significantly enhanced by the introduction of
each case. For the C-2 unsubstituted 6-arylindeno[1,2-c]quinoline
derivatives, introduction of an acyclic dimethylaminopropyl group
or a pyrrolidin-1-yl ethyl side chain at the C-11 oxime moiety en-
hanced antiproliferative activity in which each of compounds 11a
and 11b was more active than compound 8. However, with an
exception of Hep 3B, the antiproliferative activity did not improve
by the introductionof piperidin-1-yl ethyl on the sameposition indi-
cated a bulky substituent at C-11 oxime moiety is unfavorable. The
same trend was observed for C-2 fluoro derivatives 12a–12c in
which each of compounds 12a and 12b was more active than their

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C.-H. Tseng et al. / Bioorg. Med. Chem. 19 (2011) 7653–7663
Table 1
Antiproliferative activity of 6-arylindeno[1,2-c]quinoline-11-one derivatives (GI50, lM)
OR4
OR2
9
N
11
R1
2
6
N
OR3
R1 = H, F, OMe.
R2, R3, R4 = H, Me, or aminoalkyl groups.
Compound
R1
R2
R3
R4
Cell line (GI50, lM)
Hep G2
Hep 3B
A549
H1299
MCF-7
MDA-MB-231
8
9
10
H
F
OMe
Me
Me
Me
Me
Me
Me
H
H
H
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
N
N
N
11a
11b
H
H
Me
Me
Me
Me
6.54 0.11
5.48 0.01
3.14 0.04
4.68 0.03
6.69 0.07
5.27 0.03
6.61 0.06
6.31 0.04
6.57 0.11
6.71 0.02
7.01 0.02
5.33 0.03
N
N
11c
H
Me
Me
>10
8.29 2.02
>10
>10
>10
>10
12a
12b
F
F
Me
Me
Me
Me
5.75 0.03
9.46 1.42
0.66 0.02
3.11 0.02
0.91 0.24
9.24 2.55
0.61 0.01
3.15 0.02
6.18 0.02
6.36 0.03
0.70 0.03
5.99 0.04
N
N
12c
F
Me
Me
>10
>10
>10
>10
>10
>10
13a
13b
OMe
OMe
Me
Me
Me
Me
5.95 0.03
6.03 0.02
6.49 0.03
6.17 0.03
6.62 0.03
6.42 0.02
5.70 0.02
6.54 0.72
6.30 0.07
6.25 0.09
6.13 0.06
5.15 0.11
N
16
17
H
F
H
H
H
H
H
H
>10
>10
>10
7.60 0.87
>10
>10
>10
>10
>10
8.77 0.75
8.47 0.82
8.41 0.62
N
N
N
N
N
N
18a
18b
19a
19b
20a
20b
21a
21b
22a
22b
23a
23b
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
1.55 0.01
5.74 0.04
6.24 0.04
5.04 0.05
4.96 0.02
4.09 0.05
0.66 0.03
5.71 0.05
3.77 0.04
4.11 0.01
0.64 0.01
1.08 0.01
0.67 0.10
0.52 0.03
0.69 0.06
0.64 0.04
5.41 0.02
0.78 0.13
0.56 0.01
0.23 0.01
0.61 0.42
0.92 0.39
0.39 0.02
0.47 0.01
0.50 0.01
0.88 0.02
6.46 0.04
0.84 0.04
2.10 0.04
0.79 0.10
0.42 0.02
0.53 0.01
2.03 0.19
0.54 0.01
0.55 0.05
0.31 0.01
6.94 0.06
6.06 0.06
6.30 0.02
6.48 0.02
5.30 0.05
6.51 0.17
0.84 0.08
7.09 0.04
0.69 0.12
0.57 0.02
0.67 0.02
0.70 0.06
6.06 0.03
7.83 0.24
6.28 0.05
6.26 0.04
>10
0.93 0.03
0.58 0.05
4.74 0.09
0.68 0.07
3.07 0.01
0.68 0.12
0.67 0.03
0.33 0.02
0.63 0.03
0.51 0.05
0.65 0.01
0.48 0.01
N
N
N
N
N
N
F
N
N
H
H
F
6.42 0.07
6.25 0.01
6.20 0.08
6.65 0.09
5.92 0.02
6.46 0.05
6.04 0.01
N
N
F
N
N
H
H
F
H
H
H
H
N
N
F
Topotecana
Irinotecana
3.83 0.15
5.94 0.75
0.22 0.01
4.73 0.21
5.98 0.26
>10
>10
>10
>10
>10
0.078 0.002
9.17 0.02
a
Topotecan and irinotecan were obtained from ScinoPharm Taiwan Ltd. (Tainan, Taiwan).

C.-H. Tseng et al. / Bioorg. Med. Chem. 19 (2011) 7653–7663
7657
Table 2
at least one aminoalkyl side chain in which each of compounds 18a,
Effects of 23a on hepatoma cell cycle progression.
20a, and 22a was much more active than 16. The same trend was ob-
served in which in which each of compounds 19a, 21a, and 23a was
much more active than its oxime precursor 17. In general, H-bond
donating OH group was more favorable than H-bond accepting
OMe group at both C-6 aryl and C-9 positions in which compound
18a was more active than 11a while 18b was more active than
11b, and 19b was more active than 12b. For the dialkylated 6-aryl-
11-iminoindeno[1,2-c]quinoline derivatives, aminoalkyl side chain
substituted at both C-6 aryl and C-11 positions is more favorable
than that of substitution at both C-9 and C-11 positions in which
compound 22a was more active than 20a.
Cell lines
Concentration (
l
M)
Cell cycle distribution (%)
Sub G1
G1
S
G2/M
Hep G2 cell
DMSO
1.0
3.6
4.9
67.5
56.5
38.1
31.3
13.8
12.4
18.5
21.7
18.7
31.1
23.6
17.9
5.0
10.0
19.8
29.1
Hep 3B cell
DMSO
1
2.1
5.3
71.6
58.9
42.7
37.2
12.2
10.4
17.4
15.2
16.2
30.7
18.1
14.1
5.0
10.0
21.8
33.5
These 6-aryl-11-iminoindeno[1,2-c]quinoline derivatives dem-
onstrated selectivity on subtype of cancer cells. For examples,
Hep 3B was more susceptible than Hep G2, A549 was more suscep-
tible than H1299, and MDA-MB-231 was more susceptible than
MCF-7. Among six cancer cells tested, MDA-MB-231 and Hep 3B
were two of the most sensitive while MCF-7 was the most
resistant. Among these 6-aryl-11-iminoindeno[1,2-c]quinoline
derivatives evaluated, compound 23a was the most active, exhib-
pound 23a induced cell cycle arrest in a concentration dependent
manner as shown in Table 2. The proportion of cells was decreased
in the G1 and accumulated in G2/M phase after 12 h treatment of
23a, while the hypodiploid (sub-G0/G1 phase) cells increased.
Apoptosis can be characterized by morphological and biochemical
changes in the cell nucleus, including chromatin condensation and
nuclear shrinking. Morphological changes of cells treated with 23a
can be visually observed with light microscopy (Fig. 4). We found
that the hepatoma cell lines (Hep G2 & Hep 3B) treated with 23a
ited GI50 values of 0.64, 0.39, 0.55, 0.67, and 0.65 lM against the
growth of Hep G2, Hep 3B, A549, H1299, and MDA-MB-231, respec-
tively, was more active than the positive Irinotecan.
To gain further into the effects, hepatoma cell lines (Hep G2 &
at 5 lM for 12 h became shrinked. Such morphological changes
Hep 3B) were treated with 0.1–5.0 lM of compound 23a for 12,
were not apparent in the control cells. There are many regulators
involved in apoptosis induction. The Bcl-2 family proteins are
known to modulate apoptosis and the tumor suppressor protein
p53 responds to cellular damage and induces apoptosis through
caspase family or death receptor apoptosis pathways.19–21 Our re-
sults have shown that 23a promotes apoptosis by an increase in
p53, Bax and activate caspase-8 proteins expression was observed
in 23a-treated Hep G2 cells (Fig. 5). However, Bcl-2 protein was not
detected in Hep G2 cells, including control and 23a treated cells.
Previous studies by Kuo et al. have also reported that Bcl-2 protein
was not detected in hepatoma cell lines (Hep G2 & Hep 3B).22
These findings indicate that apoptosis induction may be a mecha-
nism by which 23a kills the cancer cells. Thus, compound 23a
induces cell cycle arrest at G2/M phase which led to apoptosis.
24, 36 and 48 h and the number of viable cells was counted by
XTT method. Compound 23a inhibited the growth of hepatoma cell
lines (Hep G2 & Hep 3B) in a dose- and time-dependent manner as
shown in Figure 3. These data indicated that 23a almost sup-
pressed cell viability at high concentration (5 lM) after 12 h. Com-
pound 23a was also selected for further evaluation on its effects of
hepatoma cell cycle distribution by flow cytometric analysis. Com-
4. Conclusion
A number of 6-aryl-11-iminoindeno[1,2-c]quinoline deriva-
tives were synthesized and evaluated for antiproliferative activi-
ties against the growth of six cancer cell lines including two
human hepatocelluar carcinoma cells (Hep G2 and Hep 3B),
two non-small cell lung cancer cells (A549 and H1299), and
two breast cancer cells (MCF-7 and MDA-MB-231). Among these
cancer cells tested, MDA-MB-231 and Hep 3B were two of the
most sensitive while MCF-7 was the most resistant. Among these
6-aryl-11-iminoindeno[1,2-c]quinoline derivatives evaluated,
(E)-6-{4-[3-(dimethylamino)propoxy]phenyl}-2-fluoro-9-hydro-
xy-11H-indeno[1,2-c]quinolin-11-one O-3-(dimethylamino)pro-
pyl oxime (23a) was the most active, exhibited GI50 values of
0.64, 0.39, 0.55, 0.67, and 0.65 lM against the growth of Hep
G2, Hep 3B, A549, H1299, and MDA-MB-231, respectively, was
more active than the positive Irinotecan. Flow cytometric analy-
sis indicated 23a effectively induced G2/M arrest and progress to
apoptosis in hepatoma cells after staining with propidium iodide
(PI). This suggested that 23a might inhibit the growth of hepa-
toma cell lines by the induction of apoptosis. The present work
demonstrated that incorporating the flexible aminoalkyl side
chains on the pharmacophore of 6-aryl-11-imino-indeno
[1,2-c]quinoline led to the discovery of novel compounds with
Figure 3. Compound 23a suppresses the viability of Hep G2 (A) and Hep 3B (B)
cells. Hepatoma cells were treated with DMSO, 0.1, 0.5, 1.0 and 5.0
lM of 23a. At 12,
24, 36 and 48 h after treatment, 50 L XTT reaction solution was added to each well
l
for an additional 4 h of incubation. The absorbance at 492 nm was measured on a
microtiter plate reader. The results represent the mean SD (n = 4).

7658
C.-H. Tseng et al. / Bioorg. Med. Chem. 19 (2011) 7653–7663
Hep G2 cell
DMSO
1 µM
5 µM
10 µM
Hep 3B cell
DMSO
1 µM
5 µM
10 µM
Figure 4. Induction of morphological change in Hep G2 and Hep 3B cells. Cells were treated with DMSO or compound 23a (1–10 lM) for 12 h at 37 °C and photographed
under a microscope.
potential apoptosis-inducing and anticancer activities. Further
structural optimization and detailed biological studies on the
molecular mechanism of action are ongoing.
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. Experimental
5.1. General
5.2. General procedure for the preparation of oxime compounds
8–10, 16 and 17
A mixture of 2-substituted 6-aryllindeno[1,2-c]quinolin-11-one
5–7 (1.0 mmol), hydroxylamineꢀHCl (3.0 mmol), and 2-ethoxyeth-
anol (30 mL) was heated at reflux for 3–6 h (TLC monitoring).
The solvent was removed in vacuo and the residue was suspended
in H2O (20 mL). The resulting precipitate that separated was col-
lected, washed with H2O, and dried to give a crude solid. The crude
product was purified by crystallization from EtOH to afford com-
pounds 8–10. Accordingly, compounds 16 and 17 were prepared
from their carbonyl precursors 14 and 15, respectively.
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 stan-
dard. Thin-layer chromatography was performed on Silica Gel 60
F-254 plates purchased from E. Merck and Co. The elemental
analyses were performed in the Instrument Center of National

C.-H. Tseng et al. / Bioorg. Med. Chem. 19 (2011) 7653–7663
7659
1 h. Appropriate tertiary-aminoalkyl halide derivatives (3 mmol)
was added and heated at 80 °C for 1–2 h (TLC monitoring). The or-
ganic phase was washed with brine, dried over MgSO4, filtered and
concentrated under vacuo. Crude product was purified by flash
chromatography on silica gel (MeOH/CH2Cl2 1:10) and crystallized
from EtOH.
5.3.1. (E)-9-Methoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one O-3-(dimethyl-amino)propyl oxime (11a)
Yield 80% a yellow solid. Mp 122–123 °C (EtOH). 1H NMR
(400 MHz, DMSO-d6): 2.02–2.08 (m, 2H, OCH2CH2CH2), 2.25 (s,
6H, NMe2), 2.52–2.55 (m, 2H, CH2N), 3.78 and 3.89 (two s, 6H, 40-
and 9-OMe), 4.62 (t, 2H, J = 6.4 Hz, OCH2), 6.85–6.91 (m, 2H, 7-
and 8-H), 7.12–7.16 (m, 2H, Ar-H), 7.57–7.73 (m, 4H, 2-, 3- and
Ar-H), 7.84–7.85 (m, 1H, 10-H), 8.01 (dd, 1H, J = 8.4, 0.8 Hz, 4-H),
8.83–8.86 (m, 1H, 1-H). 13C NMR (100 MHz, DMSO-d6): 26.64,
44.87(2C), 55.29, 55.46, 55.50, 74.86, 113.90(2C), 115.47, 115.61,
122.10, 123.29, 125.06, 128.22, 129.19, 129.69, 130.10(2C),
130.83, 131.30, 131.46, 132.01, 136.62, 147.00, 153.41, 154.35,
159.82 (2C). Anal. Calcd for C29H29N3O3ꢀ0.2 H2O: C, 73.93; H,
6.29; N, 8.92. Found: C, 73.74; H, 6.33; N, 8.74.
Figure 5. Immunoblot analysis of the expression of p53, caspase-8, Bcl-2, and Bax
in Hep G2 cells. Cells were incubated with compound 23a (0–10 M) and the
expression of the apoptosis-related proteins p53, caspase-8, Bcl-2, and Bax was
determined at 12 h by western blot. Protein loading was normalized to the
expression of b-actin. A representative experiment is shown of three performed.
l
5.2.1. (E)-9-Methoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one oxime (8)
Yield 72% as a yellow solid. Mp 270 °C (Dec). 1H NMR (400 MHz,
DMSO-d6): 3.79 and 3.89 (two s, 6H, 40- and 9-OMe), 6.86–6.91 (m,
2H, Ar-H), 7.13–7.17 (m, 2H, Ar-H), 7.59–7.74 (m, 4H, Ar-H), 8.00–
8.03 (m, 2H, Ar-H), 8.90 (dd, 1H, J = 8.4, 1.2 Hz, 1-H), 13.56 (br s,
1H, NOH). 13C NMR (100 MHz, DMSO-d6): 55.31, 55.51, 113.93
(2C), 115.09, 115.26, 122.25, 123.09, 125.21, 128.01, 129.14,
129.52, 130.15 (2C), 130.97, 131.04, 131.08, 132.01, 137.56,
146.71, 153.52, 154.33, 159.84 (2C). Anal. Calcd for
5.3.2. (E)-9-Methoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl oxime (11b)
Yield 86% a yellow solid. Mp: 135–136 °C (EtOH). 1H NMR
(400 MHz, CDCl3): 1.81–1.84 (m, 4H, pyrrolidinyl-H), 2.69–2.71
(m, 4H, pyrrolidinyl-H), 3.09 (t, 2H, J = 6.0 Hz, CH2N), 3.82 and
3.92 (two s, 6H, 40- and 9-OMe), 4.76 (t, 2H, J = 6.0 Hz, OCH2),
6.71 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 6.91 (d, 1H, J = 8.4 Hz, 7-H),
7.06–7.09 (m, 2H, Ar-H), 7.53–7.65 (m, 4H, 2-, 3-, and Ar-H), 7.98
(d, 1H, J = 2.4 Hz, 10-H), 8.10 (d, 1H, J = 8.4 Hz, 4-H), 8.94 (dd, 1H,
J = 8.8, 1.6 Hz, 1-H). 13C NMR (100 MHz, CDCl3): 23.52 (2C), 54.84
(2C), 54.93, 55.41, 55.57, 76.03, 114.08 (2C), 115.28, 115.75,
122.98, 123.52, 125.63, 127.78, 128.04, 129.97, 130.11 (2C),
131.71, 132.07, 132.49, 132.76, 137.80, 147.59, 154.32, 154.74,
159.95, 160.14. Anal. Calcd for C30H29N3O3: C, 75.13; H, 6.10; N,
8.76. Found: C, 74.97; H, 6.13; N, 8.60.
C
24H18N2O3ꢀ0.3HCl: C, 73.28; H, 4.69; N, 7.12. Found: C, 73.05; H,
4.79; N, 6.87.
5.2.2. 2-Fluoro-9-methoxy-6-(4-methoxyphenyl)-11H-
indeno[1,2-c]quinolin-11-one oxime (9)
Yield 70% as a yellow solid. Mp: 299–300 °C (EtOH). 1H NMR
(400 MHz, DMSO-d6): 3.76 and 3.88 (two s, 6H, 40- and 9-OMe),
6.85 (s, 2H), 7.12–7.14 (m, 2H, Ar-H), 7.56–7.61 (m, 3H, Ar-H),
7.95 (s, 1H, 10-H), 8.06 (dd, 1H, J = 9.2, 5.6 Hz, 4-H), 8.49 (dd, 1H,
J = 10.4, 2.8 Hz, 1-H), 13.61 (s, 1H, NOH). 13C NMR (100 MHz,
DMSO-d6): 55.39, 55.60, 108.51 (J = 24.3 Hz), 114.04 (2C), 114.98,
115.50, 119.42 (J = 23.1 Hz), 122.70 (J = 9.1 Hz), 123.43, 130.25
(2C), 130.46, 131.11, 131.74, 132.08, 132.56 (J = 9.9 Hz), 135.81
(J = 5.4 Hz), 145.43, 153.08, 153.68, 160.06, 160.18, 161.12
(J = 239.3 Hz). Anal. Calcd for C24H17FN2O3ꢀ0.1HCl: C, 71.34; H,
4.27; N, 6.93. Found: C, 71.56; H, 4.29; N, 6.83.
5.3.3. (E)-9-Methoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one O-2-(piperidin-1-yl)ethyl oxime (11c)
Yield 78% a yellow solid. Mp 161–162 °C (EtOH). 1H NMR
(400 MHz, CDCl3): 1.43–1.49 (m, 2H, piperidinyl-H), 1.61–1.66
(m, 4H, piperidinyl-H), 2.59 (br s, 4H, piperidinyl-H), 2.95 (t, 2H,
J = 6.0 Hz, CH2N), 3.83, 3.92 (two s, 6H, 40- and 9-OMe), 4.75 (t,
2H, J = 6.0 Hz, OCH2), 6.71 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 6.91 (d,
1H, J = 8.4 Hz, 7-H), 7.06–7.09 (m, 2H, Ar-H), 7.53–7.65 (m, 4H, 2-
, 3-, and Ar-H), 7.97 (d, 1H, J = 2.4 Hz, 10-H), 8.08–8.11 (m, 1H, 4-
H), 8.92–8.95 (m, 1H, 1-H). 13C NMR (100 MHz, CDCl3): 24.14,
25.97 (2C), 55.03 (2C), 55.41, 55.56, 57.93, 74.94, 114.08 (2C),
115.28, 115.68, 122.99, 123.50, 125.66, 127.75, 128.82, 129.97,
130.11 (2C), 131.72, 132.03, 132.49, 132.80, 137.83, 147.60,
154.25, 154.74, 159.94, 160.14. Anal. Calcd for C31H31N3O3: C,
75.43; H, 6.33; N, 8.51. Found: C, 75.04; H, 6.28; N, 8.42.
5.2.3. 2,9-Dimethoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one oxime (10)
Yield 60% as a yellow solid. Mp: 273–274 °C (EtOH). 1H NMR
(400 MHz, DMSO-d6): 3.77, 3.87 and 3.92 (three s, 9H, 40-, 2-, and
9-OMe), 6.84–6.89 (m, 2H, Ar-H), 7.10–7.14 (m, 2H, Ar-H), 7.35
(ddd, 1H, J = 9.2, 2.8, 0.8 Hz, 3-H), 7.54–7.58 (m, 2H), 7.91 (d, 1H,
J = 9.2 Hz, 4-H), 8.02 (d, 1H, J = 2.4 Hz, 10-H), 8.25 (d, 1H,
J = 2.8 Hz, 1-H), 13.44 (s, 1H, NOH). 13C NMR (100 MHz, DMSO-
d6): 55.28, 55.44, 55.50, 103.24, 113.87 (2C), 115.05, 115.25,
121.46, 123.10, 123.28, 130.19 (2C), 131.05 (2C), 131.21, 131.25,
132.30, 135.98, 143.22, 151.72, 153.66, 158.66, 159.62, 159.80.
Anal. Calcd for C25H20N2O4ꢀ1.7HCl: C, 63.29; H, 4.61; N, 5.90.
Found: C, 63.04; H, 4.69; N, 5.51.
5.3.4. (E)-2-Fluoro-9-methoxy-6-(4-methoxyphenyl)-11H-
indeno[1,2-c]quin-olin-11-one O-3-(dimethylamino)propyl
oxime (12a)
Yield 82% a yellow solid. Mp 164–165 °C (EtOH). 1H NMR
(400 MHz, CDCl3): 2.23–2.30 (m, 2H, OCH2CH2CH2), 2.47 (s, 6H,
NMe2), 2.78 (t, 2H, J = 7.2 Hz, CH2N), 3.84 and 3.92 (two s, 6H, 40-
and 9-OMe), 4.67 (t, 2H, J = 6.4 Hz, OCH2), 6.70 (dd, 1H, J = 8.4,
2.8 Hz, 8-H), 6.92 (d, 1H, J = 8.8 Hz, 7-H), 7.06–7.10 (m, 2H, Ar-H),
7.38 (ddd, 1H, J = 9.2, 8.0, 2.8 Hz, 3-H), 7.58–7.61 (m, 2H, Ar-H),
7.89 (d, 1H, J = 2.4 Hz, 10-H), 8.07 (dd, 1H, J = 9.2, 5.6 Hz, 4-H),
8.51 (dd, 1H, J = 10.4, 2.8 Hz, 1-H). 13C NMR (100 MHz, CDCl3):
5.3. General procedure for preparation of aminoalkylated oxime
derivatives: 11a–c, 12a–c, and 13a–b
To a stirred solution of oxime derivatives 8–10 (1.0 mmol) in
dry DMF (20 mL) was added NaH (60% in oil, 0.50 g) at 0 °C for

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26.68, 44.62 (2C), 55.42, 55.62, 56.03, 74.60, 108.95 (J = 24.2 Hz),
114.13 (2C), 115.13, 115.96, 119.06 (J = 25.7 Hz), 123.42
(J = 11.4 Hz), 123.83, 130.09 (2C), 131.65, 132.05, 132.39
(J = 9.1 Hz), 132.44, 132.64, 137.16, 137.22, 144.77, 154.04 (2C),
160.22 (J = 3.1 Hz), 161.64 (J = 246.4 Hz). Anal. Calcd for
s, 9H, 2-, 40-, and 9-OMe)), 4.75 (br s, 2H, OCH2), 6.86–6.90 (m, 2H,
7- and 8-H), 7.11–7.13 (m, 2H, Ar-H), 7.35 (dd, 1H, J = 9.2, 2.8 Hz, 3-
H), 7.54–7.56 (m, 2H, Ar-H), 7.88–7.90 (m, 2H, 4- and 10-H), 8.18
(d, 1H, J = 2.8 Hz, 1-H). 13C NMR (100 MHz, DMSO-d6): 22.90 (2C),
53.99 (2C), 55.29 (2C), 55.39, 55.57, 76.23, 103.04, 113.89 (2C),
115.72, 121.75, 123.10, 123.36, 130.16 (2C), 130.96, 131.27,
131.68, 132.06, 135.08, 143.41, 151.64, 153.96, 158.85, 159.69,
159.88. Anal. Calcd for C31H31N3O4ꢀ0.7 H2O: C, 71.30; H, 6.25; N,
8.05. Found: C, 71.15; H, 6.23; N, 7.82.
C29H28FN3O3: C, 71.74; H, 5.81; N, 8.65. Found: C, 71.66; H, 5.89;
N, 8.61.
5.3.5. (E)-2-Fluoro-9-methoxy-6-(4-methoxyphenyl)-11H-
indeno[1,2-c]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl oxime
(12b)
5.4. General procedure for 16 and 17
Yield 95% a yellow solid. Mp 169–170 °C (EtOH). 1H NMR
(400 MHz, CDCl3): 1.81–1.86 (m, 4H, pyrrolidinyl-H), 2.69–2.72
(m, 4H, pyrrolidinyl-H), 3.08 (t, 2H, J = 6.4 Hz, CH2N), 3.83 and
3.92 (two s, 6H, 40- and 9-OMe), 4.76 (t, 2H, J = 6.0 Hz, OCH2),
6.72 (dd, 1H, J = 8.8, 2.8 Hz, 8-H), 6.93 (d, 1H, J = 8.8 Hz, 7-H),
7.06–7.09 (m, 2H, Ar-H), 7.38 (ddd, 1H, J = 9.6, 8.4, 2.8 Hz, 3-H),
7.58–7.61 (m, 2H, Ar-H), 7.97 (d, 1H, J = 2.8 Hz, 10-H), 8.07 (dd,
1H, J = 9.2, 5.6 Hz, 4-H), 8.55 (dd, 1H, J = 10.4, 2.8 Hz, 1-H). 13C
NMR (100 MHz, CDCl3): 23.55 (2C), 54.84 (2C), 54.96, 55.42,
55.56, 76.22, 109.08 (J = 23.5 Hz), 114.13 (2C), 115.45, 115.65,
119.02 (J = 26.5 Hz), 123.48 (J = 11.3 Hz), 123.76, 130.11 (2C),
131.75, 132.06, 132.36 (J = 9.1 Hz), 132.52, 132.59, 137.28,
137.34, 144.78, 153.92, 154.05, 160.20, 161.64 (J = 246.4 Hz). Anal.
Calcd for C30H28FN3O3ꢀ0.2 H2O: C, 71.90; H, 5.71; N, 8.38. Found: C,
71.95; H, 5.71; N, 8.33.
5.4.1. 9-Hydroxy-6-(4-hydroxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one oxime (16)
Yield 61% as a yellow solid. Mp 340 °C (Dec). 1H NMR (400 MHz,
DMSO-d6): 6.75–6.84 (m, 2H, 7- and 8-H), 7.11–7.14 (m, 2H, Ar-H),
7.67–7.69 (m, 1H, Ar-H), 7.86–7.99 (m, 3H, 2-, 3-, and 10-H), 8.28
(d, 1H, J = 8.4 Hz, 4-H), 9.03 (dd, 1H, J = 8.4, 0.8 Hz, 1-H), 10.47 (br s,
2H, OH), 14.34 (br s, 1H, NOH). 13C NMR (100 MHz, DMSO-d6):
115.86 (2C), 116.07, 117.51, 122.52, 123.66, 125.85, 127.15,
129.94, 131.06, 131.10 (2C), 132.30, 132.58, 139.25, 142.80,
150.99, 153.03, 159.44, 160.32. Anal. Calcd for C22H14N2O3ꢀ0.5
H2Oꢀ1.0 HCl: C, 66.09; H, 4.03; N, 7.01. Found: C, 66.27; H, 3.79;
N, 6.83.
5.4.2. 2-Fluoro-9-hydroxy-6-(4-hydroxyphenyl)-11H-
indeno[1,2-c]quinolin-11-one oxime (17)
Yield 65% as a red solid. Mp 320 °C (Dec). 1H NMR (400 MHz,
DMSO-d6): 6.71 (dd, 1H, J = 8.8, 2.8 Hz, 8-H), 6.87 (d, 1H,
J = 8.4 Hz, 7-H), 7.02–7.04 (m, 2H, Ar-H), 7.53–7.56 (m, 2H, Ar-H),
7.66–7.71 (m, 1H, 3-H), 7.93 (d, 1H, J = 2.4 Hz, 10-H), 8.12 (dd,
1H, J = 9.2, 5.6 Hz, 4-H), 8.55 (dd, 1H, J = 10.4, 2.8 Hz, 1-H), 10.11
(br s, 2H, OH), 13.81 (br s, 1H, NOH). 13C NMR (100 MHz, DMSO-
d6): 108.91 (J = 24.2 Hz), 115.61 (2C), 116.40, 117.13 (J = 9.1 Hz),
120.31 (J = 26.5 Hz), 123.09 (J = 11.3 Hz), 123.91, 128.51, 129.91,
129.99, 130.66 (2C), 131.34, 132.76, 152.86, 152.92, 153.24,
5.3.6. (E)-2-Fluoro-9-methoxy-6-(4-methoxyphenyl)-11H-
indeno[1,2-c]quin-olin-11-one O-2-(piperidin-1-yl)ethyl oxime
(12c)
Yield 81% a yellow solid. Mp 180–181 °C (EtOH). 1H NMR
(400 MHz, CDCl3): 1.44–1.49 (m, 2H, piperidinyl-H), 1.60–1.66
(m, 4H, piperidinyl-H), 2.58 (br s, 4H, piperidinyl-H), 2.93 (t, 2H,
J = 6.0 Hz, CH2N), 3.83 and 3.92 (two s, 6H, 40- and 9-OMe), 4.74
(t, 2H, J = 6.0 Hz, OCH2), 6.70 (dd, 1H, J = 8.4, 2.8 Hz, 8-H), 6.91 (d,
1H, J = 8.4 Hz, 7-H), 7.05–7.09 (m, 2H, Ar-H), 7.38 (ddd, 1H,
J = 9.2, 8.4, 2.8 Hz, 3-H), 7.58–7.61 (m, 2H, Ar-H), 7.95 (d, 1H,
J = 2.8 Hz, 10-H), 8.07 (dd, 1H, J = 9.2, 5.6 Hz, 4-H), 8.54 (dd, 1H,
J = 10.4, 2.8 Hz, 1-H). 13C NMR (100 MHz, CDCl3): 24.17, 26.00
(2C), 55.03 (2C), 55.42, 55.57, 57.96, 75.08, 109.10 (J = 24.2 Hz),
114.13 (2C), 115.45, 115.60, 119.02 (J = 25.7 Hz), 123.48
(J = 11.3 Hz), 123.74, 130.11 (2C), 131.75, 132.06, 132.35, 132.55,
137.29, 137.35, 144.78, 153.83, 154.02, 160.20, 161.63
(J = 246.4 Hz). Anal. Calcd for C31H30FN3O3: C, 72.78; H, 5.91; N,
8.21. Found: C, 72.61; H, 5.72; N, 7.97.
159.05, 161.45 (J = 246.3 Hz). Anal. Calcd for
C22H13FN2
O3ꢀ0.6H2Oꢀ1.0HCl: C, 62.97; H, 3.65; N, 6.68. Found: C, 62.71; H,
3.87; N, 6.51.
5.5. General procedure for preparation of aminoalkylated oxime
derivatives and disubstituted derivatives: 18–23
To a stirred solution of oxime derivatives 16 or 17 (1.0 mmol) in
dry DMF (20 mL) was added NaH (60% in oil, 0.50 g) at 0 °C for 1 h.
An appropriate tertiary-aminoalkyl halide (5 mmol) was added
and heated at 80 °C for 2–5 h (TLC monitoring). The organic phase
was washed with brine, dried over MgSO4, filtered and concen-
trated under vacuo. Crude product was purified by flash chroma-
tography on silica gel, using a gradient of MeOH/CH2Cl2 (1:30 to
1:10) and crystallized from EtOH to give the 18–23.
5.3.7. (E)-2,9-Dimethoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one O-3-(dimethylamino)propyl oxime (13a)
Yield 76% a yellow solid. Mp 143–144 °C (EtOH). 1H NMR
(400 MHz, DMSO-d6): 1.98–2.05 (m, 2H, OCH2CH2CH2), 2.16 (s,
6H, NMe2), 2.42 (t, 2H, J = 7.2 Hz, CH2N), 3.76, 3.87 and 3.90 (three
s, 9H, 2-, 40-, and 9-OMe), 4.57 (t, 2H, J = 6.4 Hz, OCH2), 6.85–6.87
(m, 2H, 7- and 8-H), 7.09–7.12 (m, 2H, Ar-H), 7.32 (dd, 1H,
J = 9.2, 2.8 Hz, 3-H), 7.53–7.55 (m, 2H, Ar-H), 7.81–7.88 (m, 2H,
4- and 10-H), 8.17 (d, 1H, J = 2.8 Hz, 1-H). 13C NMR (100 MHz,
DMSO-d6): 26.94, 45.21 (2C), 55.27, 55.29, 55.50, 55.72, 74.97,
102.99, 113.86 (2C), 115.47, 121.63, 123.10, 123.32, 130.19 (2C),
130.99, 131.25, 131.54, 132.15, 135.19, 143.35, 151.62, 153.30,
158.77, 159.66, 159.79. Anal. Calcd for C30H31N3O4ꢀ0.1H2O: C,
72.15; H, 6.30; N, 8.41. Found: C, 72.00; H, 6.40; N, 8.33.
5.5.1. (E)-9-Hydroxy-6-(4-hydroxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one O-3-(dimethylamino)propyl oxime (18a), (E)-
9-[3-(dimethylamino)propoxy]-6-(4-hydroxyphenyl)-11H-
indeno[1,2-c]quinolin-11-one O-3-(dimethylamino)propyl
oxime (20a) and (E)-6-{4-[3-(dimethylamino)propoxy]phenyl}-
9-hydroxy-11H-indeno[1,2-c]quinolin-11-one O-3-
(dimethylamino)propyl oxime (22a)
Compound 18a was obtained in 27% yield (0.12 g) as a yellow
solid. Mp 185–186 °C (Dec). 1H NMR (400 MHz, DMSO-d6): 2.01–
2.08 (m, 2H, OCH2CH2CH2), 2.23 (s, 6H, NMe2), 2.50 (t, 2H,
J = 7.2 Hz, CH2N), 4.61 (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), 6.94–6.98 (m,
2H, Ar-H), 7.45–7.49 (m, 2H, Ar-H), 7.60–7.70 (m, 2H, 2- and 3-
H), 7.81 (d, 1H, J = 2.4 Hz, 10-H), 7.97 (d, 1H, J = 7.6 Hz, 4-H), 8.84
5.3.8. (E)-2,9-Dimethoxy-6-(4-methoxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl oxime (13b)
Yield 98% a yellow solid. Mp 202–203 °C (EtOH). 1H NMR
(400 MHz, DMSO-d6): 1.77 (br s, 4H, pyrrolidinyl-H), 2.85 (br s,
4H, pyrrolidinyl-H), 3.25 (br s, 2H, CH2N), 3.78, 3.88 and 3.93 (three

C.-H. Tseng et al. / Bioorg. Med. Chem. 19 (2011) 7653–7663
7661
(dd, 1H, J = 8.4, 1.2 Hz, 1-H), 9.88 (br s, 2H, OH). 13C NMR (100 MHz,
DMSO-d6): 26.83, 45.01 (2C), 48.61, 55.46, 74.78, 115.23 (2C),
116.83, 117.15, 122.13, 123.54, 125.01, 128.02, 128.91, 129.64,
129.98, 130.10 (2C), 130.53, 131.00, 131.79, 136.23, 146.86,
J = 2.8 Hz, 10-H), 8.08 (d, 1H, J = 8.0 Hz, 4-H), 8.85 (dd, 1H, J = 8.4,
0.8 Hz, 1-H). 13C NMR (100 MHz, CDCl3): 23.28 (2C), 23.45 (2C),
54.54 (2C), 54.78 (2C), 54.85, 54.90, 66.35, 75.36, 115.94 (2C),
116.09 (2C), 122.91, 123.67, 125.60, 127.73, 128.89, 129.60,
130.09 (2C), 131.52, 132.17, 132.68, 137.77, 147.38, 154.50,
155.10, 158.02, 158.86. Anal. Calcd for C34H36N4O3ꢀ1.3H2Oꢀ1.0HCl:
C, 67.10; H, 6.56; N, 9.21. Found: C, 67.19; H, 6.32; N, 9.11.
153.70,
27H25N3O3ꢀ0.3H2Oꢀ0.3HCl: C, 71.14; H, 5.73; N, 9.22. Found: C,
71.12; H, 6.02; N, 8.95.
154.63,
158.10,
158.28.
Anal.
Calcd
for
C
Compound 20a was obtained in 26% yield (0.14 g) as a red solid.
Mp 187–188 °C (EtOH). 1H NMR (400 MHz, CDCl3): 1.98–2.04 (m,
2H, OCH2CH2CH2), 2.13–2.20 (m, 2H, OCH2CH2CH2), 2.31 and
2.37 (two s, 12H, NMe2), 2.54 (t, 2H, J = 7.2 Hz, CH2N), 2.66 (t,
2H, J = 7.2 Hz, CH2N), 4.06 (t, 2H, J = 6.4 Hz, OCH2), 4.49 (t, 2H,
J = 6.0 Hz, OCH2), 6.39–6.46 (m, 2H, 7- and 8-H), 6.91–6.94 (m,
2H, Ar-H), 7.36–7.39 (m, 2H, Ar-H), 7.41–7.55 (m, 2H, 2- and 3-
H), 7.69 (d, 1H, J = 2.4 Hz, 10-H), 8.02 (dd, 1H, J = 8.4, 0.8 Hz, 4-
H), 8.67 (dd, 1H, J = 8.4, 0.8 Hz, 1-H). 13C NMR (100 MHz, CDCl3):
27.12, 27.19, 44.88 (2C), 45.29 (2C), 55.75, 56.27, 66.12, 73.95,
114.42 (2C), 116.97, 117.36, 122.78, 123.73, 125.72, 127.34,
128.43, 129.61, 130.04 (2C), 130.59, 131.43, 132.13, 132.42,
137.53, 147.11, 154.31, 154.33, 157.91, 159.20. Anal. Calcd for
Compound 22b was obtained in 29% yield (0.16 g) as a red solid.
Mp 176–177 °C (EtOH). 1H NMR (400 MHz, CDCl3): 1.88–1.94 (m,
8H, pyrrolidinyl-H), 2.75 (br s, 4H, pyrrolidinyl-H), 2.88–2.94 (m,
6H, CH2N and pyrrolidinyl-H), 3.18–3.20 (m, 2H, CH2N), 3.93 (t,
2H, J = 5.6 Hz, OCH2), 4.65–4.68 (m, 2H, OCH2), 5.97 (d, 1H,
J = 8.4 Hz, 7-H), 6.12 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 6.22–6.24 (m,
2H, Ar-H), 6.68–6.70 (m, 2H, Ar-H), 7.53–7.67 (m, 3H, 2-, 3- and
10-H), 8.12 (d, 1H, J = 8.4 Hz, 4-H), 8.84 (d, 1H, J = 8.0 Hz, 1-H).
13C NMR (100 MHz, CDCl3): 23.08 (2C), 23.47 (2C), 54.02 (2C),
54.90 (2C), 54.99, 55.45, 66.69, 73.98, 114.23 (2C), 116.40,
118.84, 122.31, 123.32, 125.83, 127.45, 128.15, 129.29 (2C),
129.60, 130.22, 130.73, 132.41, 132.97, 136.84, 147.20, 154.82,
154.91, 158.41 (2C). Anal. Calcd for C34H36N4O3: C, 74.43; H,
6.61; N, 10.21. Found: C, 74.12; H, 6.74; N, 10.05.
C32H36N4O3ꢀ1.5H2Oꢀ1.0HCl: C, 65.35; H, 6.86; N, 9.53. Found: C,
65.46; H, 6.82; N, 9.18.
Compound 22a was obtained in 21% yield (0.11 g) as a red solid.
Mp 75–76 °C (EtOH). 1H NMR (400 MHz, CDCl3): 1.94–2.03 (m, 2H,
OCH2CH2CH2), 2.14–2.19 (m, 2H, OCH2CH2CH2), 2.31 and 2.33 (two
s, 12H, NMe2), 2.52 (t, 2H, J = 7.6 Hz, CH2N), 2.60 (t, 2H, J = 7.6 Hz,
CH2N), 3.95 (t, 2H, J = 6.0 Hz, OCH2), 4.62 (t, 2H, J = 6.4 Hz, OCH2),
6.58 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 6.82–6.89 (m, 3H, 7- and Ar-H),
7.42–7.44 (m, 2H, Ar-H), 7.49–7.62 (m, 2H, 2- and 3-H), 7.86 (d,
1H, J = 2.0 Hz, 10-H), 8.08 (d, 1H, J = 8.0 Hz, 4-H), 8.89 (dd, 1H,
J = 8.4, 0.8 Hz, 1-H). 13C NMR (100 MHz, CDCl3): 27.07, 27.46,
45.16 (2C), 45.23 (2C), 45.31, 45.35, 56.18, 56.26, 66.31, 74.93,
115.30, 116.05 (2C), 116.56, 122.98, 123.61, 125.69, 127.69,
128.89, 129.51, 130.18 (2C), 131.31, 131.59, 132.15, 132.33,
137.93, 147.32, 154.13, 155.00, 157.78, 159.22. Anal. Calcd for
5.5.3. (E)-2-Fluoro-9-hydroxy-6-(4-hydroxyphenyl)-11H-
indeno[1,2-c]-quinolin-11-one O-3-(dimethylamino)propyl
oxime (19a), (E)-9-[3-(dimethylamino)propoxy]-2-fluoro-6-(4-
hydroxyphenyl)-11H-indeno[1,2-c]quinolin-11-one O-3-
(dimethylamino)propyl oxime (21a) and (E)-6-{4-[3-
(dimethylamino)propoxy]phenyl}-2-fluoro-9-hydroxy-11H-
indeno[1,2-c] quinolin-11-one O-3-(dimethylamino)propyl
oxime (23a)
Compound 19a was obtained in 30% yield (0.14 g) as a red solid.
Mp 262 °C (Dec). 1H NMR (400 MHz, DMSO-d6): 2.01 (quin, 2H,
J = 6.8 Hz, OCH2CH2CH2), 2.19 (s, 6H, NMe2), 2.45 (t, 2H,
J = 6.8 Hz, CH2N), 4.59 (t, 2H, J = 6.8 Hz, OCH2), 6.67 (dd, 1H,
J = 8.4, 2.8 Hz, 8-H), 6.82 (d, 1H, J = 8.4 Hz, 7-H), 6.92–6.95 (m,
2H, Ar-H), 7.42–7.45 (m, 2H, Ar-H), 7.58 (ddd, 1H, J = 9.2, 8.4,
2.8 Hz, 3-H), 7.76 (d, 1H, J = 2.4 Hz, 10-H), 8.01 (dd, 1H, J = 9.2,
5.6 Hz, 4-H), 8.43 (dd, 1H, J = 10.4, 2.8 Hz, 1-H), 9.88 (br s, 2H,
OH). 13C NMR (100 MHz, DMSO-d6): 26.94, 45.13 (2C), 55.53,
74.96, 108.19 (J = 23.5 Hz), 115.25 (2C), 116.75, 117.26, 118.93
(J = 25.7 Hz), 122.49 (J = 11.3 Hz), 123.82, 129.65, 130.13 (2C),
130.28, 131.05, 132.40, 132.54 (J = 9.9 Hz), 135.77 (J = 6.1 Hz),
144.10, 153.24, 154.11, 158.15, 158.57, 160.99 (J = 244.8 Hz). Anal.
Calcd for C28H23FN2O3ꢀ0.6HCl: C, 67.65; H, 5.17; N, 8.77. Found: C,
67.65; H, 5.26; N, 8.63.
C
32H36N4O3ꢀ0.7H2Oꢀ3.0HCl: C, 59.44; H, 6.30; N, 8.66. Found: C,
59.48; H, 6.01; N, 8.29.
5.5.2. (E)-9-Hydroxy-6-(4-hydroxyphenyl)-11H-indeno[1,2-
c]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl oxime (18b), (E)-6-
(4-hydroxyphenyl)-9-[2-(pyrrolidin-1-yl)ethoxy]-11H-
indeno[1,2-c]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl oxime
(20b) and (E)-9-hydroxy-6-{4-[2-(pyrrolidin-1-
yl)ethoxy]phenyl}-11H-indeno[1,2-c]quinolin-11-one O-2-
(pyrrolidin-1-yl)ethyl oxime (22b)
Compound 18b was obtained in 24% yield (0.11 g) as a yellow
solid. Mp 145–146 °C (EtOH). 1H NMR (400 MHz, DMSO-d6):
1.69–1.72 (m, 4H, pyrrolindinyl-H), 2.62 (br s, 4H, pyrrolidinyl-
H), 3.00 (t, 2H, J = 6.0 Hz, CH2N), 4.68 (t, 2H, J = 5.6 Hz, OCH2),
6.68 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 6.84 (d, 1H, J = 8.4 Hz, 7-H),
6.94–6.97 (m, 2H, Ar-H), 7.45–7.48 (m, 2H, Ar-H), 7.61–7.70 (m,
2H, 2- and 3-H), 7.83 (d, 1H, J = 2.0 Hz, 10-H), 7.97 (d, 1H,
J = 8.0 Hz, 4-H), 8.84 (d, 1H, J = 8.4 Hz, 1-H), 9.88 (br s, 2H, OH).
13C NMR(100 MHz, DMSO-d6): 23.21 (2C), 54.11 (2C), 54.36,
75.77, 115.23 (2C), 116.94, 117.18, 122.13, 123.54, 125.04,
128.06, 128.93, 129.65, 130.12 (2C), 130.28, 130.54, 131.02,
131.81, 136.25, 146.87, 153.75, 154.64, 158.11, 158.28. Anal. Calcd
for C28H25N3O3ꢀ0.7H2Oꢀ0.4HCl: C, 70.25; H, 5.64; N, 8.78. Found: C,
70.63; H, 5.98; N, 8.40.
Compound 20b was obtained in 28% yield (0.15 g) as a red solid.
Mp 174–175 °C. 1H NMR (400 MHz, CDCl3): 1.5–1.91 (m, 8H, pyrro-
lidinyl-H), 2.76–2.80 (m, 8H, pyrrolidinyl-H), 3.00 (t, 2H, J = 5.6 Hz,
CH2N), 3.17 (t, 2H, J = 5.6 Hz, CH2N), 4.15 (t, 2H, J = 5.6 Hz, OCH2),
4.78 (t, 2H, J = 5.6 Hz, OCH2), 6.61 (dd, 1H, J = 8.4, 2.4 Hz, 8-H),
6.82 (d, 1H, J = 8.4 Hz, 7-H), 6.86–6.90 (m, 2H, Ar-H), 7.38–7.42
(m, 2H, Ar-H), 7.49–7.62 (m, 2H, 2- and 3-H), 7.93 (d, 1H,
Compound 21a was obtained in 24% yield (0.13 g) as a yellow
solid. Mp 193–194 °C (EtOH). 1H NMR (400 MHz, DMSO-d6):
1.80–2.03 (m, 4H, OCH2CH2CH2), 2.13 (s, 6H, NMe2), 2.18 (s, 6H,
NMe2), 2.33 (t, 2H, J = 7.2 Hz, CH2N), 2.42 (t, 2H, J = 7.2 Hz, CH2N),
3.98 (t, 2H, J = 6.4 Hz, OCH2), 4.59 (t, 2H, J = 6.4 Hz, OCH2), 6.85–
6.96 (m, 4H, 7-, 8- and Ar-H), 7.42–7.46 (m, 2H, Ar-H), 7.57 (ddd,
1H, J = 8.8, 8.8, 2.4 Hz, 3-H), 7.78 (d, 1H, J = 2.4 Hz, 10-H), 8.01
(dd, 1H, J = 9.2, 6.0 Hz, 4-H), 8.40 (dd, 1H, J = 10.0, 2.4 Hz, 1-H).
13C NMR (100 MHz, DMSO-d6): 26.80, 26.97, 45.18 (4C), 55.58,
55.71, 66.19, 75.04, 108.2 (J = 24.2 Hz), 115.23 (2C), 115.84,
116.01, 119.06 (J = 25.8 Hz), 122.36 (J = 11.4 Hz), 123.62, 130.02,
130.06 (2C), 130.80, 131.00, 131.90, 132.48 (J = 11.3 Hz), 136.03
(J = 6.1 Hz), 144.21, 152.95, 154.19, 158.30, 159.37, 160.92
(J = 244.9 Hz). Anal. Calcd for C32H35FN4O3ꢀ0.3HCl: C, 69.43; H,
6.43; N, 10.12. Found: C, 69.44; H, 6.34; N, 10.00.
Compound 23a was obtained in 22% yield (0.12 g) as a yellow
solid. Mp 189–190 °C (EtOH). 1H NMR (400 MHz, DMSO-d6):
1.88–2.05 (m, 4H, OCH2CH2CH2), 2.18 (s, 6H, NMe2), 2.19 (s, 6H,
NMe2), 2.39–2.46 (m, 4H, CH2N), 4.11 (t, 2H, J = 6.4 Hz, OCH2),
4.61 (t, 2H, J = 6.4 Hz, OCH2), 6.68 (dd, 1H, J = 8.4, 2.4 Hz, 8-H),
6.81 (d, 1H, J = 8.4 Hz, 7-H), 7.09–7.12 (m, 2H, Ar-H), 7.54–7.61

7662
C.-H. Tseng et al. / Bioorg. Med. Chem. 19 (2011) 7653–7663
(m, 3H, 3-H and Ar-H), 7.78 (d, 1H, J = 2.4 Hz, 10-H), 8.04 (dd, 1H,
J = 9.2, 6.0 Hz, 4-H), 8.44 (dd, 1H, J = 10.4, 2.8 Hz, 1-H), 10.21 (br
s, 1H, OH). 13C NMR (100 MHz, DMSO-d6): 26.92, 26.97, 45.16
(2C), 45.23 (2C), 55.53, 55.70, 65.97, 74.98, 108.16 (J = 23.5 Hz),
114.35 (2C), 116.76, 117.26, 118.95 (J = 25.8 Hz), 122.54
(J = 11.4 Hz), 123.72, 129.44, 130.10 (2C), 131.02, 131.74, 132.34,
132.56 (J = 9.9 Hz), 135.81 (J = 5.4 Hz), 144.07, 153.18, 153.70,
158.65, 159.23, 161.02 (J = 244.8 Hz). Anal. Calcd for
5.6. Pharmacological methods
5.6.1. Antiproliferative assay
Cancer cells (Hep G2, Hep 3B, A549, H1299, MCF-7 and MDA-
MB-231) 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
C32H35FN4O3ꢀ0.4HCl: C, 68.97; H, 6.40; N, 10.05. Found: C, 69.00;
H, 6.33; N, 9.90.
and 0.25 lg/mL amphotercin. Culture was maintained at 37 °C
with 5% CO2 in a humidified atmosphere.
5.5.4. (E)-2-Fluoro-9-hydroxy-6-(4-hydroxyphenyl)-11H-
indeno[1,2-c]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl oxime
(19b), (E)-2-fluoro-6-(4-hydroxyphenyl)-9-[2-(pyrrolidin-1-yl)
ethoxy]-11H-indeno[1,2-c]quinolin-11-one O-2-(pyrrolidin-1-
yl)ethyl oxime (21b) and (E)-2-fluoro-9-hydroxy-6-{4-[2-
(pyrrolidin-1-yl)ethoxy]phenyl}-11H-indeno[1,2-c]quinolin-11-
one O-2-(pyrrolidin-1-yl)ethyl oxime (23b)
Compound 19b was obtained in 25% yield (0.12 g) as a red solid.
Mp 255 °C (Dec). 1H NMR (400 MHz, DMSO-d6): 1.69–1.72 (m, 4H,
pyrrolidinyl-H), 2.61 (br s, 4H, pyrrolidinyl-H), 2.99 (t, 2H,
J = 5.6 Hz, CH2N), 4.67 (t, 2H, J = 5.6 Hz, OCH2), 6.68 (dd, 1H,
J = 8.4, 2.4 Hz, 8-H), 6.83 (d, 1H, J = 8.4 Hz, 7-H), 6.93–6.96 (m,
2H, Ar-H), 7.43–7.46 (m, 2H, Ar-H), 7.56 (ddd, 1H, J = 9.2, 8.8,
3.2 Hz, 3-H), 7.79 (d, 1H, J = 2.4 Hz, 10-H), 8.02 (dd, 1H, J = 9.2,
5.6 Hz, 4-H), 8.42 (dd, 1H, J = 10.8, 3.2 Hz, 1-H), 9.86 (br s, 2H,
OH). 13C NMR (100 MHz, DMSO-d6): 23.19 (2C), 54.07 (2C),
54.37, 75.83, 108.17 (J = 24.3 Hz), 115.21 (2C), 116.83, 117.24,
118.88 (J = 25.7 Hz), 122.46 (J = 11.4 Hz), 123.76, 129.62, 130.08
(2C), 130.24, 131.02, 132.37, 132.51 (J = 9.1 Hz), 135.73
(J = 6.1 Hz), 144.08, 153.26, 154.09, 158.12, 158.51, 160.96
(J = 244.8 Hz). Anal. Calcd for C28H23FN2O3ꢀ0.4HCl: C, 69.47; H,
5.08; N, 8.68. Found: C, 69.49; H, 5.15; N, 8.52.
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%.18
5.6.2. Cell cycle analysis
Hepatoma cells were treated with DMSO, 23a at different con-
centrations (1.0, 5.0, 10.0 lM) for 12 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 containing 20 lg/lL of PI,
Compound 21b was obtained in 20% yield (0.11 g) as a yellow
solid. Mp 159–160 °C (EtOH). 1H NMR (400 MHz, DMSO-d6):
1.66–1.71 (m, 8H, pyrrolidinyl-H), 2.57 (br s, 4H, pyrrolidinyl-
H), 2.78 (t, 2H, J = 5.6 Hz, CH2N), 2.94 (t, 2H, J = 5.6 Hz, CH2N),
4.05 (t, 2H, J = 5.6 Hz, OCH2), 4.66 (t, 2H, J = 5.6 Hz, OCH2), 6.86–
6.97 (m, 4H, 7-, 8-, and Ar-H), 7.43–7.46 (m, 2H, Ar-H), 7.55–
7.60 (m, 1H, 3-H), 7.85 (d, 1H, J = 2.0 Hz, 10-H), 8.02 (dd, 1H,
J = 9.2, 5.6 Hz, 4-H), 8.40 (dd, 1H, J = 10.4, 2.8 Hz, 1-H), 9.86 (br
s, 1H, OH). 13C NMR (100 MHz, DMSO-d6): 23.15 (2C), 23.19
(2C), 53.95 (2C), 54.05 (2C), 54.15, 54.41, 67.11, 75.94, 108.17
(J = 24.3 Hz), 115.22 (2C), 115.53, 115.70, 116.27, 118.98
(J = 25.7 Hz), 122.32 (J = 11.4 Hz), 123.59, 130.09 (2C), 130.83,
131.07, 131.91, 132.52 (J = 9.1 Hz), 136.03 (J = 6.1 Hz), 144.22,
152.99, 154.16, 158.17, 159.21, 160.94 (J = 244.8 Hz). Anal. Calcd
for C28H23FN2O3ꢀ0.7HCl: C, 68.96; H, 6.08; N, 9.46. Found: C,
68.88; H, 6.06; N, 9.36.
Compound 23b was obtained in 16% yield (0.09 g) as a yellow
solid. Mp 192 °C (Dec). 1H NMR (400 MHz, DMSO-d6): 1.72–1.74
(m, 8H, pyrrolidinyl-H), 2.64 (br s, 4H, pyrrolidinyl-H), 2.94, 3.02
(m, 4H, CH2N ꢁ 2), 4.21 (t, 2H, J = 5.6 Hz, OCH2), 4.70 (t, 2H,
J = 5.6 Hz, OCH2), 6.68 (dd, 1H, J = 8.8, 2.4 Hz, 8-H), 6.81 (d, 1H,
J = 8.8 Hz, 7-H), 7.12–7.15 (m, 2H, Ar-H), 7.56–7.63 (m, 3H, 3-H
and Ar-H), 7.82 (d, 1H, J = 2.0 Hz, 10-H), 8.04 (dd, 1H, J = 9.2,
5.6 Hz, 4-H), 8.46 (dd, 1H, J = 10.4, 2.8 Hz, 1-H), 10.04 (br s, 1H,
OH). 13C NMR (100 MHz, DMSO-d6): 23.10 (2C), 23.16 (2C), 54.03
(4C), 54.18, 54.30, 66.52, 75.73, 108.18 (J = 23.5 Hz), 114.41 (2C),
116.87, 117.28, 118.99 (J = 25.7 Hz), 122.55 (J = 11.3 Hz), 123.70,
129.49, 130.09 (2C), 131.02, 131.87, 132.35, 132.59 (J = 9.9 Hz),
135.81 (J = 6.1 Hz), 144.08, 153.27, 153.66, 158.59, 158.97, 161.03
(J = 244.9 Hz). Anal. Calcd for C28H23FN2O3ꢀ1.0HCl: C, 67.71; H,
6.02; N, 9.29. Found: C, 67.53; H, 6.28; N, 8.86.
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).
5.6.3. Immunoblot analysis
After treatment of compound 23a, 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. 20 lg pro-
tein 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 pri-
mary antibodies, followed by 30 min with secondary antibodies in
milk containing Tris-buffered saline (TBS) and 0.5% Tween. The
membrane was then exposed to X-ray film. Protein bands were de-
tected using the enhanced chemiluminescence blotting detection
system (Amersham, USA).
Acknowledgments
Financial support of this work by the National Science Council of
the Republic of China is gratefully acknowledged. We also thank the
National Center for High-Performance Computing for providing com-
puter resources and chemical database services.

C.-H. Tseng et al. / Bioorg. Med. Chem. 19 (2011) 7653–7663
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