Synthesis and antiproliferative evaluation of certain indeno[1,2-c]quinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2007.12.028

Although the quinoline ring is found in a wide variety of biologically active compounds and is frequently condensed with various heterocycles, synthesis and biological evaluation of the indenoquinoline skeleton attracts only very limited attention. We rep

Full text of DOI:10.1016/j.bmc.2007.12.028

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3153–3162
Synthesis and antiproliferative evaluation of certain
indeno[1,2-c]quinoline derivatives
Chih-Hua Tseng,a,b Yeh-Long Chen,a Pei-Jung Lu,c,*
Chia-Ning Yangd and Cherng-Chyi Tzenga,*
aFaculty of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
bGraduate Institute of Pharmaceutical Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
cInstitute of Clinical Medicine, National Cheng-Kung University, School of Medicine, 138 Sheng-Li Road, Tainan 704, Taiwan
dInstitute of Biotechnology, National University of Kaohsiung, 700 Kaohsiung University Rd, Kaohsiung, Taiwan
Received 7 November 2007; revised 11 December 2007; accepted 12 December 2007
Available online 23 December 2007
Abstract—Although the quinoline ring is found in a wide variety of biologically active compounds and is frequently condensed with
various heterocycles, synthesis and biological evaluation of the indenoquinoline skeleton attracts only very limited attention. We
report herein the synthesis and antiproliferative evaluation of certain indeno[1,2-c]quinoline derivatives against the growth of six
cancer cell lines including human cervical epithelioid carcinoma (HeLa), oral squamous cell carcinoma (SAS), hepatocellular car-
cinoma (SKHep), human stomach adenocarcinoma (AGS), prostate cancer (PC-3), and non-small cell lung cancer (A549). The
results indicated that 9-methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (17b) is more active than its C6-amino deriv-
ative 17a, C6-morpholine and C6-piperidine isomers, 17c and 17d, respectively. Treatment of 17b with NH2OH afforded its hydrox-
yimino derivative 20 which is more active than the carbonyl precursor 17b. More potent agents were obtained by further
derivatization of 20. Thus, antiproliferative activities decreased in an order of aminoalkoxyimino 22a–d > hydroxyimino 20 > alk-
oxyimino 21, 22e > carbonyl 17b. Both AGS and A549 were resistant to camptothecin with GI50 values of 23.76 and 2.80 lM,
respectively, while GI50 values for 22a–d were in the range of 5.93–7.11 lM and 0.38–0.87 lM, respectively. Among them, 22b
was the most potent with GI50 values of 0.52, 0.74, 6.76, and 0.64 lM against the growth of HeLa, SKHep, AGS, and A549 cells,
respectively. Flowcytometric analysis indicated 22c can induce cell cycle arrest in S phase, and DNA polyploidy (>4n) followed by
apoptosis.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
more, the fact that some cancer cells develop resistance
to camptothecins has caused an urgent need to search
for alternative Top I inhibitors which are chemically
more stable. Indenoisoquinoline 4 (NSC 314622) was
first identified as a novel Top I inhibitor with better
pharmacokinetic features than camptothecin.5,6 Since
then, a number of indenoisoquinolines especially inde-
no[1,2-c]isoquinoline derivatives have been synthesized
and proved to possess DNA Top I inhibitory activ-
ity.7–10 These compounds bind to a transient Top I-
DNA covalent complex and inhibit the resealing of a
single-strand nick that the enzyme creates to relieve
superhelical tension in duplex DNA.11–13
Camptothecin (1), an alkaloid isolated from Camptot-
heca acuminata,1,2 and its derivatives such as topotecan
(2, Hycamptin) and irinotecan (3, Camptosar) are pro-
totypical topoisomerase I (Top I) inhibitors which are
currently used as anticancer drugs. However, several
drawbacks, such as easy opening of the lactone ring to
a hydroxycarboxylate which has a high affinity to hu-
man serum albumin, and the rapid reversibility of ter-
nary DNA-enzyme-camptothecin complex after the
removal of drug, limited their clinical utility.3,4 Further-
Since the discovery of indenoisoquinolines as a novel
class of potential anticancer drug candidates, extensive
structural modifications have been explored by altering
the substituent of the tetracyclic pharmacophore. How-
ever, synthesis and biological evaluation of the isomeric
Keywords: Indeno[1,2-c]quinoline derivatives; Antiproliferative activ-
ity; Cytotoxicity; Anticancer agents.
*
Corresponding authors. Tel.: +886 6 2353535x4415 (P.-J.L.); tel.:
+886 3121101x6985; fax: +886 3125339 (C.-C.T.); e-mail
addresses: pjlu2190@mail.ncku.edu.tw; tzengch@kmu.edu.tw
7
7
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.028

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C.-H. Tseng et al. / Bioorg. Med. Chem. 16 (2008) 3153–3162
R3
N
R2
N
R1
O
N
N
O
O
N
O
O
N
O
O
OH
O
1, Camptothecin R1 = H; R2 = H; R3 = H
2, Topotecan R1 = OH; R2 = CH2N(CH3)2, R3 = H
OH
3, Irinotecan
O
O
O
Me
O
MeO
MeO
HN
O
N
Me
N
HO
N
N
O
O
H
N
4, NSC 314622
5, TAS-103
6, CIL-102
indenoquinoline skeleton attracts only very limited
attention.14–19 Heterocycles containing the quinoline
ring constitute a wide variety of biologically active com-
pounds. For example, certain indeno[1,2-c]quinolin-11-
one scaffolds have been shown to possess antitumor
activity.15 TAS-103 (5), one of the indeno[2,1-c]quino-
line derivatives, has been proved to be a novel Top I
and Top II targeting agent that stabilizes cleavable com-
plexes of Top-DNA at the cellular level.17–19 A number
of furo[2,3-b]quinoline derivatives, such as CIL-102 (6),
have also been synthesized and demonstrated to possess
significant anticancer activity.20–24 We have also synthe-
sized and evaluated antiproliferative activities of certain
indolo[2,3-b]quinoline derivatives25 on the ground that
these tetracyclic heterocycles may intercalate into the
DNA double helix resulting in the inhibition of DNA
replication and transcription. In continuation of our
study to explore more potent anticancer drug candi-
dates, we describe herein the preparation and antiprolif-
erative evaluation of certain indeno[1,2-c]quinoline
derivatives.
The preparation of 9-methoxy-6-(piperazin-1-yl)-11H-
indeno[1,2-c]quinolin-11-one oxime (20) and its alkyl-
ated derivatives 21 and 22a–f is described in Scheme 2.
Reaction of 17b with NH2OH or NH2OMe gave 20 or
21, respectively. Alkylation of 20 with various aminoal-
kyl chlorides afforded their respective aminoalkoxyimi-
no derivatives 22a–e.
3. Pharmacological results and discussion
All the synthesized indeno[1,2-c]quinoline derivatives
were evaluated in vitro against a panel of six cancer cell
lines including human cervical epithelioid carcinoma
(HeLa), oral squamous cell carcinoma (SAS), hepatocel-
lular carcinoma (SKHep), human stomach adenocarci-
noma (AGS), prostate cancer (PC-3), and non-small
cell lung cancer (A549) using MTT (3-[4,5-dimethylthia-
zol-2-yl]-2,5-diphenyltetrazolium bromide) assay. 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 re-
duced absorbance in treated cells, compared to control
cells, by 50%. The GI50 results of 11H-indeno[1,2-
c]quinoline-11-one derivatives are summarized in Table
1. For the 9-methoxy derivatives, the C6-piperazine
substituted derivative 17b is more active than its amino
derivative 17a with exceptions of HeLa and SKHep, in
which 17a is fairly active against the growth of HeLa cell
with a GI50 value of 9.48 lM. Inactiveness of 17c and
17d indicated that the replacement of C6-piperazine with
its isosteric moieties such as morpholine or piperidine is
unfavorable. Comparable activities were observed by
the replacement of C6-piperazine with N-4 methyl piper-
azine (17b vs 17e) while the C-3 methyl piperazine coun-
terpart 17f exhibited more potent antiproliferative
activities than 17b. Comparison of positional isomers
indicated that the 8-OMe derivative 18b is more active
than its 9-OMe isomer 17b. The fact that 17b is less ac-
tive than its unsubstituted parent 19b implies that the
methoxy group is not essential for antiproliferative
2. Chemistry
Reaction of isatin (7) and 4–methoxyphenylacetic acid
(8) gave 2-hydroxy-3-(4-methoxyphenyl)quinoline-
4-carboxylic acid (11)15 as described in Scheme 1.
Compounds 12 and 13 were prepared under the same
reaction conditions from 3–methoxyphenylacetic acid
(9) and phenylacetic acid (10), respectively, with isatin.
Treatment of 11 with POCl3 afforded 6-chloro-9-meth-
oxy-11H-indeno[1,2-c]quinolin-11-one (14), which was
reacted with NH4OH or cyclic amines to give 6-amino-
9-methoxy-11H-indeno[1,2-c]quinolin-11-one (17a) or
its cyclic aminoalkyl derivatives 17b–f. Compound 14
had also been prepared previously from thermal cycliza-
tion of 11 followed by chlorination with POCl3.15
Accordingly, 18b and 19b were obtained from 15 and
16, which in turn were prepared from 12 and 13, respec-
tively, in a fairly good overall yield.

C.-H. Tseng et al. / Bioorg. Med. Chem. 16 (2008) 3153–3162
3155
4 R1
3
O
CO2H
4
R1
i
ii
3
+
O
N
CO2H
8-10
H
N
OH
7
8, 11 R1 = 4-OMe
9, 12 R1 = 3-OMe
10, 13 R1 = H
11-13
9
R1
O
R1
O
8
7
iii
14, 17 R1 = 9-OMe
15, 18 R1 = 8-OMe
16, 19 R1 = H
N
Cl
R2
N
14-16
17-19
b: R2 =
a: R2 =
e: R2 =
c: R2 =
N
O
d: R2 =
N
NH2
N
NH
N
Me
f: R2 =
N
NMe
NH
Scheme 1. Reagents and conditions: (i) NaOAc, 200 ꢁC, 3h; (ii) POCl3, 150 ꢁC, 48 h; (iii) Nh4OH in sealed tube, 200 ꢁC, 48 h or cyclic amines in
ethoxyethanol.
OMe
N
OMe
OMe
O
ii
N
N
N
N
NH
NH
21
17b
i
OR
N
OH
N
OMe
OMe
iii
N
N
N
N
NH
NH
22
20
b: R =
c: R =
(CH2)3NMe2
a: R =
d: R =
(CH2)3NH2
CH2
(CH2)2NMe2
(CH2)2
N
e: R =
Scheme 2. Reagents and conditions: (i) NH2OH HCl, MW (100 W), 30 min; (ii) NH2OMe HCl, MW (100 W), 30 min; (iii) NaH, RCl.
activities. On the contrary, the C-6 piperazine or substi-
tuted piperazine is crucial because morpholine counter-
part 17c and piperidine counterpart 17d are inactive.
Although camptothecin exhibited more active antipro-
liferative activities than 11H-indeno[1,2-c]quinoline-11-
one derivatives against the growth of most cancer cell
lines tested, it was inactive against the growth of AGS
with a GI50 value of 23.76 lM, while 18b and 19b were
relatively active with GI50 values of 4.11 and 6.54 lM,
respectively.
The fact that both 20 and 21 are more active than 17b
implied that a hydroxyimino or methoxyimino deriva-
tive is more favorable than the carbonyl precursor as

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C.-H. Tseng et al. / Bioorg. Med. Chem. 16 (2008) 3153–3162
Table 1. Antiproliferative activity of 11H-indeno[1,2-c]quinoline-11-one derivatives (GI50, lM)
R1
O
R2
N
R1
R2
HeLa
SAS
>30
SKHep
AGS
>30
PC-3
>30
A549
>30
17a
17b
9-OMe
NH2
9.48 0.59
21.69 5.93
9-OMe
9-OMe
9-OMe
9-OMe
9-OMe
8-OMe
H
15.18 1.50
>30
>30
>30
>30
25.34 15.46
>30
10.13 1.19
>30
20.38 11.02
>30
16.78 2.18
>30
N
N
N
NH
O
17c
17d
17e
17f
18b
19b
>30
>30
>30
>30
>30
7.29 1.94
3.44 1.01
0.36 0.11
25.13 11.23
13.40 2.60
5.10 0.82
20.24 7.86
2.90 0.50
0.73 0.31
7.40 0.30
7.93 1.16
4.11 1.20
>30
16.00 6.50
7.83 1.31
8.60 4.72
N
N
NMe
NH
Me
16.25 5.00
10.63 2.08
N
N
NH
1.07 0.61
0.18 0.10
10.70 0.92
6.00 2.70
1.75 1.08
0.22 0.13
6.54 2.59
11.91 3.96
0.09 0.02
3.60 1.25
2.80 0.70
NH
Camptothecin
23.76 0.73
summarized in Table 2. The antiproliferative activity de-
creased by substitution of 20 with methyl or benzyl
group, 21 and 22e, respectively, but was significantly en-
hanced by the introduction of aminoalkyl side chains
22a–d. Among these aminoalkoxyimino derivatives
22a–d, 22b was the most potent with GI50 values of
0.52, 0.74, 6.76, and 0.64 lM against the growth of
HeLa, SKHep, AGS, and A549 cells, respectively.
Among these cancer cells tested, AGS was the most
resistant to camptothecin with
a
GI50 value of
Table 2. Antiproliferative activity of 9-methoxy 11H-indeno[1,2-c]quinoline-11-one derivatives (GI50, lM)
OR
N
OMe
N
N
NH
R
HeLa
SAS
6.10 0.90
SKHep
AGS
PC-3
A549
20
21
H
Me
2.35 0.25
10.63 4.38
0.92 0.38
0.52 0.17
0.37 0.06
3.75 1.28
9.31 2.90
3.01 2.18
0.74 0.21
2.95 2.10
1.74 0.47
5.18 0.48
5.93 1.24
6.76 2.02
6.84 1.75
4.86 2.03
13.82 4.30
4.61 1.18
2.98 2.16
5.28 0.67
3.80 0.85
6.16 0.12
0.71 0.12
0.64 0.01
0.38 0.21
10.30 0.51
6.33 0.86
2.41 1.16
4.93 0.57
22a
22b
22c
–(CH2)2NMe2
–(CH2)3NH2
–(CH2)3NMe2
22d
22e
0.78 0.22
5.81 0.48
1.57 0.42
7.11 0.84
5.62 0.48
0.87 0.12
(CH2)2
CH2
N
6.14 0.34
0.18 0.10
7.62 0.63
6.00 2.70
4.15 1.82
0.22 0.13
6.12 1.40
7.81 1.07
0.09 0.02
5.40 0.22
2.80 0.70
Camptothecin
23.76 0.73

C.-H. Tseng et al. / Bioorg. Med. Chem. 16 (2008) 3153–3162
3157
23.76 lM while GI50 values for 22a–d were in the range
of 5.93–7.11 lM. Results have also shown that 22b and
22c exhibited more active antiproliferative activities
than camptothecin against the growth of SAS, AGS,
and A549. Compound 22c was the most active against
the growth of HeLa with a GI50 value of 0.37 lM and,
therefore, was selected along with its hydroxyimino pre-
cursor 20 for further investigation of their effects on the
growth curves of HeLa cells. Total 48 h period of
growth time was monitored and the results showed that
both compounds can effectively suppress the cell prolif-
eration in dosage dependent manners (Fig. 1A and B).
Compound 22c inhibited about 50% and almost 100%
of the cell proliferation at 0.3 and 1.0 lM, respectively,
which was more effective than 20. These results were
also consistent with GI50 values obtained from MTT
viability assay (Table 2).
The indeno[1,2-c]quinoline treated cells with higher
DNA content indicated that the cells may be arrested
in S phase or in G2 phase of cell cycle. To examine
the association between indeno[1,2-c]quinoline-induced
growth inhibition and cell cycle arrest, the cell cycle dis-
tribution was analyzed by flow cytometry. The results in
Figure 2 and Table 3 shows that 22c can induce cell
accumulation in S phase in a dose dependent manner
after treatment of 22c for 24 h. Figure 2D showed that,
at the concentration of 1.0 lM, 22c can not only induce
A 0.5
B
0.5
0.4
0.3
0.2
0.1
0
Mock
Mock
DMSO
0.1 uM
0.3 uM
1.0 uM
0.4
0.3
0.2
0.1
0
DMSO
0.1 uM
0.3 uM
1.0 uM
0
8
16 24 32 40 48 56
Time (hr)
0
8
16 24 32 40 48 56
Time (hr)
Figure 1. Antiproliferative effects of 20 (A) and 22c (B) on HeLa cells. Adherent cells proliferated in 96-well plates (104 cells/well) were incubated
with different concentrations of 20 (1.0, 3.0, and 10 lM) and 22c (0.1, 0.3, and 1.0 lM), respectively, and determined by MTT assay at various time
intervals.
B
A
C
Untreated
DMSO
2n
4n
6n
2n
4n
6n
D
1.0µΜ
0.3µΜ
2n
4n
6n
2n
4n
6n
DNA contents
Figure 2. Flow cytometric analysis of HeLa cells. Untreated HeLa cells were taken as control (A). Cells were treated DMSO (B), 0.3 lM (C), or
1.0 lM (D) of 22c; 24 h later the cells were harvested, fixed, and stained with propidium iodide as described in Experimental Section prior to analysis
by flow cytometry. The percentage of cells in each cell cycle phase was quantified and is given in Table 3.

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C.-H. Tseng et al. / Bioorg. Med. Chem. 16 (2008) 3153–3162
Table 3. Effects of 22c on HeLa cell cycle progression
PI staining
Cell cycle distribution (%)
S
SubG1
G1
G2/M (4n)
>4n (6n+8n)
Untreated
DMSO
0.4 0.1
0.4 0.2
1.5 0.2
5.9 0.5
58.3 2.9
57.6 3.9
10.8 1.4
3.9 0.8
13.8 1.8
12.9 3.4
50.8 4.4
21.8 2.8
25.2 1.9
29.3 1.8
19.8 1.3
15.3 2.4
1.3 0.4
1.8 0.6
22c (0.3 lM)
22c (1.0 lM)
17.2 3.7
53.2 4.1
the cell accumulation in S phase (between 2n and 4n) but
also increase DNA polyploidy (>4n) and the subG1
phase after 24 h drug treatment. The sub G0/G1 phase
increased after 24 h drug treatment indicated that these
cells may cause the DNA fragmentation and apoptosis.
5.1.1.
2-Hydroxy-3-(4-methoxyphenyl)quinoline-4-car-
boxylic acid (11). A mixture of isatin (2.21 g, 15 mmol),
4-methoxyphenylacetic acid (4.36 g, 26.25 mmol), and
sodium acetate (0.3 g) was heated at 200 ꢁC for 3 h
(TLC monitoring). After cooling, the mixture was added
AcOH (100 mL), and the precipitate was collected,
washed with H2O, and then crystallized from EtOH to
give 11 (3.14 g, 71%). mp 338–339 ꢁC. 1H NMR
(400 MHz, CDCl3): 3.89 (s, 3H, OMe), 7.03 (m, 2H,
Ar-H), 7.33 (m, 2H, Ar-H), 7.70 (m, 1H, 6-H), 7.84
(m, 2H, 7, 8-H), 8.14 (m, 1H, 5-H). 13C NMR
(100 MHz, CDCl3): 55.31, 114.12 (2C), 119.50, 120.75,
123.96, 123.99, 128.80, 129.10, 129.32, 131.26, 131.49
(2C), 146.81, 150.73, 160.42, 167.44. Anal. calcd for
C17H13NO4 0.1 H2O: C 68.73, H 4.48, N 4.71; found:
C 68.65, H 4.49, N 4.69.
4. Conclusion
Based on the structure of indeno[1,2-c]quinolines, we
found that the features for optimum antiproliferative
activities are: (1) substituent at C-6 should be piperazine
or alkylated piperazine; (2) substituent at C-11 is crucial
in which the potency decreased in an order of aminoalk-
oxyimino 22a–d > hydroxyimino 20 > alkoxyimino 21,
22e > carbonyl 17b.
Among these aminoalkoxyimino derivatives 22a–d, 22b
exhibited GI50 values of 0.52, 0.74, and 0.64 lM
against the growth of HeLa, SKHep, and A549 cells,
respectively, which are approximately 30-fold increase
in potency compared to the parent core 17b. Com-
pound 22b was more active than camptothecin against
the growth of SAS, AGS, and A549 cells, with GI50
values of 2.41, 6.76, and 0.64 lM, respectively. Results
have also shown that 22b and 22c exhibited approxi-
mately 4-fold increase in potency compared to campto-
thecin against the growth of AGS and A549.
Flowcytometric analysis indicated that 22c can induce
cell cycle arrest in S phase, DNA polyploidy (>4n) fol-
lowed by apoptosis. Further studies on the structural
optimization and the antiproliferative mechanism are
ongoing.
5.1.2.
2-Hydroxy-3-(3-methoxyphenyl)quinoline-4-car-
boxylic acid (12). From isatin and 3-methoxyphenylace-
tic acid as described for 11: 63% yield. mp 327–329 ꢁC.
1H NMR (400 MHz, DMSO-d6): 3.77 (s, 3H, 30-OMe),
6.93–6.98 (m, 3H, Ar-H), 7.26 (m, 1H, 6-H), 7.34 (m,
1H, Ar-H), 7.39 (d, 1H, J = 8.0 Hz, 8-H), 7.48 (dd, 1H,
J = 0.8, 8.0 Hz, 5-H), 7.58 (m, 1H, 7-H), 12.15 (br s,
1H, COOH). 13C NMR (100 MHz, DMSO-d6): 55.04,
113.39, 115.46, 115.50, 115.58, 122.04, 122.47, 125.46,
128.54, 128.88, 130.89, 135.71, 138.51, 142.24, 158.62,
160.64, 167.48. Anal. calcd for C17H13NO4: C 69.15, H
4.44, N 4.74; found: C 69.10, H 4.36, N 4.74.
5.1.3. 2-Hydroxy-3-phenylquinoline-4-carboxylic acid
(13). From isatin and phenylacetic acid as described
for 11: 78% yield. mp 332–333 ꢁC. 1H NMR
(400 MHz, DMSO-d6): 7.26 (m, 1H, 6-H), 7.33–7.45
(m, 6H, Ar-H), 7.49 (dd, 1H, J = 1.2, 8.4 Hz, 5-H),
7.58 (m, 1H, 7-H), 12.17 (br s, 1H, COOH), 13.84 (br
s, 1H, OH). 13C NMR (100 MHz, DMSO-d6): 115.44,
115.61, 122.43, 125.43, 127.73 (2C), 128.01, 128.74,
129.64 (2C), 130.82, 134.44, 138.50, 142.17, 160.74,
167.45. Anal. calcd for C16H11NO3: C 72.45, H 4.14,
N 5.27; found: C 72.42, H 4.18, N 5.28.
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 Var-
ian-Unity-400 spectrometer. Chemical shifts are ex-
pressed in parts per million (d) with tetramethylsilane
(TMS) as an internal standard. Thin-layer chromatogra-
phy was performed on silica gel 60 F-254 plates pur-
chased from E. Merck and Co. The elemental analyses
were performed in the Instrument Center of National
Science Council at National Cheng-Kung University
and National Chung-Hsing University using Heraeus
CHN-O Rapid EA, and all values are within 0.4% of
the theoretical compositions.
5.1.4. 6-Chloro-9-methoxy-11H-indeno[1,2-c]quinolin-11-
one (14). A solution of 11 (2.95 g, 10 mmol) in POCl3
(30 mL) was heated at 150 ꢁC for 48 h (TLC monitor-
ing). After cooling, the mixture was poured into ice-
water (150 mL). The resulting precipitate that separated
was collected by filtration. The filtered cake was sus-
pended in 5% NaHCO3 solution (200 mL) with vigorous
stirring for 1 h. The resulting precipitate was collected,
washed with H2O, and crystallized from EtOH to give
1
14 (2.69 g, 91%). mp 227–228 ꢁC H NMR (400 MHz,
CDCl3): 3.89 (s, 3H, 9-OMe), 6.99 (dd, 1H, J = 2.4,

C.-H. Tseng et al. / Bioorg. Med. Chem. 16 (2008) 3153–3162
3159
8.4 Hz, 8-H), 7.24 (d, 1H, J = 2.4 Hz, 10-H), 7.61 (m,
1H, 2-H), 7.67 (m, 1H, 3-H), 7.95 (dd, 1H, J = 0.4,
8.8 Hz, 4-H), 8.02 (d, 1H, J = 8.4 Hz, 7-H), 8.76 (dd,
1H, J = 0.8, 8.4 Hz, 1-H). 13C NMR (100 MHz, CDCl3):
55.81, 111.21, 119.48, 122.80, 123.97, 125.12, 128.59,
129.64, 130.61, 133.55, 135.14, 136.27, 137.20, 144.68,
149.30, 161.45, 193.63. Anal. calcd for C17H10ClNO2:
C 69.05, H 3.41, N 4.74; found: C 68.66, H 3.57, N 4.64.
(d, 1H, J = 8.4 Hz, 4-H), 8.69 (dd, 1H, J = 1.2, 8.4 Hz,
1-H). 13C NMR (100 MHz, CDCl3): 50.13 (2C), 55.81,
66.85 (2C), 110.99, 118.87, 121.07, 123.78, 124.25,
127.04, 128.01, 129.55, 132.65, 135.03, 135.19, 136.11,
148.62, 156.79, 160.76, 195.11. Anal. calcd for
C21H18N2O3: C 72.82, H 5.24, N 8.09; found: C 72.77,
H 5.26, N 8.03.
5.1.9. 9-Methoxy-6-(piperidin-1-yl)-11H-indeno[1,2-c]-
quinolin-11-one (17d). From 14 and piperidine as de-
scribed for 17a: 76% yield. mp 147–148 ꢁC. H NMR
5.1.5. 6-Chloro-11H-indeno[1,2-c]quinolin-11-one (16).26.
Chlorination of 13 as described for 14: 88% yield. mp
149–150 ꢁC. H NMR (400 MHz, CDCl3): 3.89 (s, 3H,
1
1
(400 MHz, CDCl3): 1.70 (m, 2H, piperidinyl-H), 1.84
(m, 4H, piperidinyl-H), 3.32 (m, 4H, piperidinyl-H),
3.87 (s, 3H, 9-OMe), 6.95 (dd, 1H, J = 2.4, 8.0 Hz, 8-
H), 7.21 (d, 1H, J = 2.4 Hz, 10-H), 7.42 (m, 2H, 2-H),
7.54 (m, 1H, 3-H), 7.59 (d, 1H, J = 8.0 Hz, 7-H), 7.82
(d, 1H, J = 8.4 Hz, 4-H), 8.68 (dd, 1H, J = 1.6, 8.4 Hz,
1-H). 13C NMR (100 MHz, CDCl3): 24.27, 25.92 (2C),
50.98 (2C), 55.79, 110.68, 118.84, 120.86, 123.74,
124.29, 126.60, 127.87, 129.31, 133.28, 135.11, 135.63,
135.83, 148.72, 158.03, 160.64, 195.50. Anal. calcd for
C22H20N2O2Æ0.1 H2O: C 76.33, H 5.88, N 8.09; found:
C 76.14, H 5.90, N 7.99.
9-OMe), 7.35 (m, 1H, 9-H), 7.54 (m, 1H, 8-H), 7.61
(m, 1H, 2-H), 7.67–7.72 (m, 2H, 3, 7-H), 7.96 (d, 1H,
J = 8.8 Hz, 4-H), 8.14 (d, 1H, J = 7.6 Hz, 10-H), 8.79
(dd, 1H, J = 0.4, 8.4 Hz, 1-H). 13C NMR (100 MHz,
CDCl3): 122.63, 123.98, 124.26, 124.94, 128.60, 129.69,
129.95, 131.11, 133.08, 135.36, 136.30, 136.55, 141.52,
145.03, 149.83, 193.65. Anal. calcd for C16H8ClNOÆ
0.1 H2O: C 71.35, H 3.03, N 5.20; found: C 71.64, H
3.18, N 5.23.
5.1.6. 6-Amino-9-methoxy-11H-indeno[1,2-c]quinolin-11-
one (17a). A mixture of 14 (0.3 g, 1 mmol), ammonia
water (5 mL), and 2-ethoxyethanol (20 mL) was heated
in the sealed tube at 200 ꢁC for 48 h. The solvent was re-
moved in vacuo and the residue was suspended in H2O
(50 mL). The resulting precipitate that separated was
collected, washed with H2O, and dried to give a crude
solid, which was crystallized from MeOH to give 17a
(0.17 g, 62%). mp 148–149 ꢁC. 1H NMR (400 MHz,
DMSO-d6): 3.85 (s, 3H, 9-OMe), 6.70 (br s, 2H, NH2),
7.04 (dd, 1H, J = 2.4, 8.4 Hz, 8-H), 7.16 (d, 1H,
J = 2.4 Hz, 10-H), 7.29 (m, 1H, 2-H), 7.48–7.55 (m,
2H, 3, 4-H), 7.87 (d, 1H, J = 8.4 Hz, 7-H), 8.45 (d, 1H,
J = 8.4 Hz, 1-H). 13C NMR (100 MHz, DMSO-d6):
56.24, 111.70, 118.89, 123.42, 124.38, 124.53, 124.71,
126.12, 128.37, 129.89, 134.16, 134.27, 134.92, 149.38,
153.79, 160.63, 195.55. Anal. calcd for C17H12N2O2: C
73.91, H 4.38, N 10.14; found: C 73.82, H 4.42, N 9.76.
5.1.10. 9-Methoxy-6-(4-methylpiperazin-1-yl)-11H-inde-
no[1,2-c]quinolin-11-one (17e). From 14 and 4-methyl-
piperazine as described for 17a: 74% yield. mp 147–
148 ꢁC. 1H NMR (400 MHz, CDCl3): 2.42 (s, 3H,
NMe), 2.70 (m, 4H, piperazinyl-H), 3.43 (m, 4H, piper-
azinyl-H), 3.87 (s, 3H, 9-OMe), 6.95 (dd, 1H, J = 2.4,
8.0 Hz, 8-H), 7.21 (d, 1H, J = 2.4 Hz, 10-H), 7.43 (m,
1H, 2-H), 7.53–7.57 (m, 2H, 3-, 7-H), 7.83 (d, 1H,
J = 8.4 Hz, 4-H), 8.68 (d, 1H, J = 8.4 Hz, 1-H). 13C
NMR (100 MHz, CDCl3): 46.28, 49.56 (2C), 55.03
(2C), 55.77, 110.78, 118.80, 120.88, 123.69, 124.33,
126.76, 127.95, 129.39, 132.70, 135.11, 135.26, 135.92,
148.59, 156.85, 160.62, 195.27. Anal. calcd for
C22H21N3O2Æ0.3 H2O: C 72.43, H 5.97, N 11.52; found:
C 72.19, H 6.02, N 11.70.
5.1.11. 9-Methoxy-6-(3-methylpiperazin-1-yl)-11H-inde-
no[1,2-c]quinolin-11-one (17f). From 14 and 3-methylpip-
erazine as described for 17a: 55% yield as a gum. H
5.1.7. 9-Methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]-
quinolin-11-one (17b). From 14 and piperazine as de-
scribed for 17a: 83% yield. mp 150–151 ꢁC. H NMR
1
1
NMR (400 MHz, CDCl3): 1.17 (d, 3H, J = 6.4 Hz,
Me), 1.82 (br s, 1H, piperidinyl-NH), 1.68 (m, 1H, pip-
erazinyl-H), 3.01 (m, 1H, piperazinyl-H), 3.14–3.25 (m,
3H, piperazinyl-H), 3.64 (m, 2H, piperazinyl-H), 3.88
(s, 3H, 9-OMe), 6.95 (dd, 1H, J = 2.8, 8.4 Hz, 8-H),
7.21 (d, 1H, J = 2.4 Hz, 10-H), 7.43 (m, 1H, 2-H),
7.53–7.58 (m, 2H, 3-, 7-H), 7.83 (d, 1H, J = 8.4 Hz, 4-
H), 8.69 (dd, 1H, J = 1.2, 8.0 Hz, 1-H). 13C NMR
(100 MHz, CDCl3): 19.79, 45.74, 50.17, 50.53, 55.78,
57.19, 110.85, 118.81, 120.90, 123.72, 124.26, 126.79,
127.92, 129.41, 132.81, 135.14, 135.28, 136.00, 148.64,
(400 MHz, DMSO-d6): 3.29 (m, 4H, piperazinyl-H),
3.45 (m, 4H, piperazinyl-H), 3.85 (s, 3H, 9-OMe), 7.12
(dd, 1H, J = 2.0, 8.0 Hz, 8-H), 7.17 (d, 1H, J = 2.0 Hz,
10-H), 7.50-7.56 (m, 2H, 2-, 7-H), 7.64 (m, 1H, 3-H),
7.79 (d, 1H, J = 8.4 Hz, 4-H), 8.54 (d, 1H, J = 8.4 Hz,
1-H). 13C NMR (100 MHz, DMSO-d6): 42.80 (2C),
47.10 (2C), 55.84, 111.01, 119.27, 120.41, 122.98,
124.77, 127.38, 127.79, 129.83, 132.34, 133.81, 134.43,
135.37, 147.62, 155.93, 160.55, 194.35. Anal. calcd for
C21H19N3O2Æ0.7 H2OÆ0.8 HCl: C 65.15, H 5.52, N
10.85; found: C 65.38, H 5.45, N 10.59.
157.02,
160.68,
195.24.
Anal.
calcd
for
C22H21N3O2Æ0.75.H2O: C 70.84, H 6.09, N 11.27; found:
C 70.50, H 6.12, N 11.20.
5.1.8. 9-Methoxy-6-morpholino-11H-indeno[1,2-c]quino-
lin-11-one (17c). From 14 and morpholine as described
for 17a: 81% yield. mp 170–171 ꢁC. 1H NMR
(400 MHz, CDCl3): 3.40 (m, 4H, morpholinyl-H), 3.87
(s, 3H, 9-OMe), 3.98 (m, 4H, morpholinyl-H), 6.95
(dd, 1H, J = 2.4, 8.4 Hz, 8-H), 7.21 (d, 1H, J = 2.4 Hz,
10-H), 7.45 (m, 1H, 2-H), 7.55 (m, 2H, 3-, 7-H), 7.83
5.1.12. 8-Methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]-
quinolin-11-one (18b). A solution of 12 (1.48 g, 5 mmol)
in POCl3 (15 mL) was heated at 150 ꢁC for 36 h (TLC
monitoring). After cooling, the mixture was poured
into ice-water (80 mL). The resulting precipitate that

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C.-H. Tseng et al. / Bioorg. Med. Chem. 16 (2008) 3153–3162
1
separated was collected by filtration. The filtered cake
was suspended in 5% NaHCO3 solution (100 mL) with
vigorous stirring for 1 h. The resulting precipitate was
collected, washed with H2O, and dried to give 15 as
the crude intermediate which was used for the next step
without further purification.
150 ꢁC. H NMR (400 MHz, CDCl3): 3.35 (m, 8H, pip-
erazinyl-H), 3.85 (s, 3H, OMe), 4.36 (s, 3H, NOMe),
7.15 (d, 1H, J = 7.2 Hz, 8-H), 7.52 (m, 1H, 2-H), 7.65
(m, 1H, 3-H), 7.74 (d, 1H, J = 8.4 Hz, 4-H), 7.86 (m,
2H, 7-, 10-H), 8.74 (d, 1H, J = 8.0 Hz, 1-H). 13C NMR
(100 MHz, CDCl3): 42.36 (2C), 46.45 (2C), 55.62,
64.57, 115.79, 115.87, 121.06, 123.87, 124.96, 126.46,
126.69, 128.26, 129.14, 130.34, 130.53, 138.28, 146.22,
153.45, 155.54, 159.80. Anal. calcd for C22H22N4O2Æ1.4
HCl: C 62.11, H 5.54, N 13.17; found: C 62.20, H
5.66, N 13.01.
The mixture of above crude intermediate 15, 2-ethoxy-
ethanol (15 mL), and piperazine (2.15 g, 25 mmol) was
heated in the sealed tube at 200 ꢁC for 48 h. The solvent
was removed in vacuo and the residue was suspended in
H2O (50 mL). The resulting precipitate that separated
was collected, washed with H2O, and dried to give a
crude solid, which was purified by column chromatogra-
phy (MeOH:CH2Cl2 1/50) to give 18b (0.59 g, 34%). mp
5.1.16. 9-Methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]-
quinolin-11-one O-2-(dimethylamino)ethyl oxime (22a).
To a stirred solution of 20 (0.36 g, 1 mmol) in dry DMF
(20 mL) was added NaH (60% in oil, 0.50 g) at 0 ꢁC and
stirring was continued for 1h. After stirring at rt for 8 h,
2-chloro-N,N-dimethylethanamineÆHCl (0.42 g, 3 mmol)
was added and stirring was continued for 1 h. The reac-
tion mixture was partitioned between H2O (50 mL) and
CH2Cl2(50 mL). The organic layer was collected, dried
over MgSO4, and evaporated in vacuo to give the resi-
due which was purified by column chromatography
(MeOH:CH2Cl2 1/10) to give 22a (0.23 g, 53%). mp
84–86 ꢁC. 1H NMR (400 MHz, CDCl3): 2.39 (s, 6H,
NMe2), 2.89 (t, 2H, J = 6.0 Hz, NCH2), 3.19 (m, 4H,
piperazinyl-H), 3.47 (m, 4H, piperazinyl-H), 3.88 (s,
3H, OMe), 4.68 (t, 2H, J = 6.0 Hz, NCH2), 6.95 (dd,
1H, J = 2.4, 8.4 Hz, 8-H), 7.41 (m, 1H, 2-H), 7.55 (m,
1H, 3-H), 7.76 (d, 1H, J = 8.4 Hz, 7-H), 7.87 (d, 1H,
J = 8.4 Hz, 4-H), 7.98 (d, 1H, J = 2.4 Hz, 10-H), 8.79
(dd, 1H, J = 1.2, 8.4 Hz, 1-H). 13C NMR (100 MHz,
CDCl3): 45.73 (2C), 46.01 (2C), 50.78 (2C), 55.58,
58.20, 74.97, 115.23, 115.86, 121.67, 123.34, 125.48,
125.67, 127.40, 128.39, 128.53, 131.26, 132.12, 139.20,
147.18, 154.46, 157.09, 159.78. Anal. calcd for
C25H29N5O2 Æ 0.4 H2O: C 68.44, H 6.85, N 15.96; found:
C 68.67, H 6.91, N 15.60.
1
193–194 ꢁC. H NMR (400 MHz, CDCl3): 3.17 (m, 4H,
piperazinyl-H), 3.36 (m, 4H, piperazinyl-H), 3.93 (s, 3H,
8-OMe), 6.70 (dd, 1H, J = 2.4, 8.4 Hz, 9-H), 7.30 (d, 1H,
J = 2.4 Hz, 7-H), 7.45 (m, 1H, 2-H), 7.57–7.62 (m, 2H,
3-, 10-H), 7.85 (d, 1H, J = 8.4 Hz, 4-H), 8.76 (dd, 1H,
J = 0.8, 8.4 Hz, 1-H). 13C NMR (100 MHz, CDCl3):
46.03 (2C), 51.33 (2C), 55.85, 111.32, 111.54, 113.47,
120.94, 124.12, 125.95, 126.70, 127.93, 129.94, 136.88,
137.85, 145.81, 149.38, 157.49, 165.31, 193.84. Anal.
calcd for C21H19N3OÆ0.1 HCl: C 72.27, H 5.52, N
12.04; found: C 72.07, H 5.64, N 12.03.
5.1.13. 6-(Piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-
one (19b). From 16 and piperazine as described for 17a:
81% yield. mp 152–153 ꢁC. 1H NMR (400 MHz,
DMSO-d6): 3.39 (m, 4H, piperazinyl-H), 3.51 (m, 4H, pip-
erazinyl-H), 7.34 (m, 1H, 9-H), 7.47 (m, 1H, 8-H), 7.55–
7.63 (m, 4H, 2-, 3-, 7-, 10-H), 7.77 (d, 1H, J = 8.4 Hz, 4-
H), 8.54 (dd, 1H, J = 1.2, 8.0 Hz, 1-H). 13C NMR
(100 MHz, DMSO-d6): 42.70 (2C), 46.88 (2C), 120.58,
123.51, 123.79, 124.68, 127.84, 128.07, 129.82, 130.75,
131.68, 132.55, 136.06, 136.13, 142.15, 148.43, 156.23,
194.89. Anal. calcd for C20H17N3OÆ1.0 HCl: C 68.28, H
5.16, N 11.94; found: C 68.08, H 5.25, N 11.71.
5.1.17. 9-Methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]-
quinolin-11-one O-3-aminopropyl oxime (22b). From 20
and 3-chloropropylamine Æ HCl as described for 22a:
5.1.14. 9-Methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]-
quinolin-11-one oxime (20). To a suspension of 17b
(0.35 g, 1.0 mmol) in 2-ethoxyethanol (30 mL) was
added NH2OHÆHCl (0.20 g, 3.0 mmol). The reaction
mixture was heated with stirring under microwave irra-
diation (100 W) for 30 min (TLC monitoring). The sol-
vent was removed in vacuo and the residue suspended
in H2O (20 mL). The resulting precipitate that separated
was collected, washed with H2O, and crystallized from
MeOH to give 20 (0.29 g, 81%). mp 146–147 ꢁC. 1H
NMR (400 MHz, CDCl3): 2.06 (m, 4H, piperazinyl-H),
3.24 (m, 4H, piperazinyl-H), 3.84 (s, 3H, OMe), 7.13
(d, 1H, J = 7.2 Hz, 8-H), 7.46 (m, 1H, 2-H), 7.59 (m,
1H, 3-H), 7.80 (m, 2H, 4-, 7-H), 8.02 (s, 1H, 10-H),
8.76 (d, 1H, J = 8.0 Hz, 1-H). 13C NMR (100 MHz,
CDCl3): 44.91 (2C), 50.15 (2C), 55.52, 115.04, 115.32,
120.99, 123.56, 125.03, 125.66, 126.39, 128.14, 128.70,
130.18, 130.52, 138.82, 146.37, 153.66, 156.94, 159.60.
ESIMS [M+H]+: 361.
1
41% yield. mp 89–90 ꢁC. H NMR (400 MHz, DMSO-
d6): 2.10 (quin, 2H, J = 6.4 Hz, NCH2CH2CH2N), 3.00
(t, 2H, J = 6.4 Hz, NCH2), 3.15 (m, 4H, piperazinyl-
H), 3.32 (m, 4H, piperazinyl-H), 3.87 (s, 3H, OMe),
4.67 (t, 2H, J = 6.4 Hz, NCH2), 7.18 (dd, 1H, J = 2.4,
8.4 Hz, 8-H), 7.51 (m, 1H, 2-H), 7.63 (m, 1H, 3-H),
7.78 (d, 1H, J = 8.4 Hz, 7-H), 7.83 (d, 1H, J = 8.4 Hz,
4-H), 7.87 (d, 1H, J = 2.4 Hz, 10-H), 8.73 (dd, 1H,
J = 1.2, 7.6 Hz, 1-H). 13C NMR (100 MHz, DMSO-
d6): 27.45, 36.07 (2C), 44.08, 49.14 (2C), 55.68, 73.47,
115.69, 115.90, 120.67, 123.93, 124.99, 126.19, 126.89,
128.24, 129.05, 130.28, 130.91, 138.09, 146.46, 153.92,
156.55, 159.74. ESIMS [M+H]+: 418.
5.1.18. 9-Methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]-
quinolin-11-one O-3-(dimethylamino)propyl oxime (22c).
From 20 and 3-chloro-N,N-dimethylpropanamineÆHCl
as described for 22a: 52% yield. mp 145–146 ꢁC. 1H
NMR (400 MHz, CDCl3): 2.14 (quin, 2H, J = 6.4 Hz,
NCH2CH2CH2N), 2.33 (s, 6H, NMe2), 2.59 (t, 2H,
J = 6.4 Hz, NCH2), 3.29 (m, 4H, piperazinyl-H), 3.44
5.1.15. 9-Methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]-
quinolin-11-one O-methyl oxime (21). From 17b and
NH2OMeÆHCl as described for 20: 83% yield. mp 149–

C.-H. Tseng et al. / Bioorg. Med. Chem. 16 (2008) 3153–3162
3161
(m, 4H, piperazinyl-H), 3.88 (s, 3H, OMe), 4.62 (t, 2H,
J = 6.4 Hz, NCH2), 6.95 (dd, 1H, J = 2.8, 8.4 Hz, 8-H),
7.41 (m, 1H, 2-H), 7.55 (m, 1H, 3-H), 7.80 (d, 1H,
J = 8.4 Hz, 7-H), 7.86 (d, 1H, J = 8.0 Hz, 4-H), 7.92 (d,
1H, J = 2.8 Hz, 10-H), 8.78 (dd, 1H, J = 1.2, 8.4 Hz, 1-
H). 13C NMR (100 MHz, CDCl3): 27.28, 44.68 (2C),
45.49 (2C), 50.41 (2C), 55.58, 56.24, 74.81, 115.01,
115.97, 121.69, 123.15, 125.45, 125.67, 127.09, 128.30,
128.64, 131.16, 131.73, 139.37, 146.96, 154.11, 156.30,
159.82. ESIMS [M+H]+: 446.
mide, (2 mg/mL) (MTT, 20 mL) was added to the cultures
and incubated during the final 1.5 h. The resultant tetra-
zolium salt was then dissolved by the addition of dimeth-
ylsulfoxide. Color was measured spectrophotometrically
in a microtiter plate reader at 570 nm and used as a rela-
tive measure of viable cell number. The number of viable
cells following treatment was compared to solvent and
untreated control cells and used to determine the percent
of control growth as (Abtreated/ Abcontrol) · 100, where Ab
represents the mean absorbance (n = 3). The concentra-
tion that killed 50% of cells (GI50) was determined from
the linear portion of the curve by calculating the concen-
tration of agent that reduced absorbance in treated cells,
compared to control cells, by 50%.
5.1.19.
9-Methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-
c]quinolin-11-one O-2-(piperidin-1-yl)ethyl oxime (22d).
From 20 and N-(2-chloroethyl)piperidineÆHCl as de-
scribed for 22a: 54% yield. mp 95–96 ꢁC. 1H NMR
(400 MHz, DMSO-d6): 1.38 (m, 2H, piperidinyl-H),
1.51 (m, 4H, piperidinyl-H), 2.51 (m, 4H, piperidinyl-
H), 2.81 (t, 2H, J = 5.6 Hz, NCH2), 3.28 (m, 4H, piper-
azinyl-H), 3.46 (m, 4H, piperazinyl-H), 3.85 (s, 3H,
OMe), 4.66 (t, 2H, J = 5.6 Hz, NCH2), 7.15 (dd, 1H,
J = 2.8, 8.4 Hz, 8-H), 7.48 (m, 1H, 2-H), 7.61 (m, 1H,
3-H), 7.76 (d, 1H, J = 8.4 Hz, 7-H), 7.81 (d, 1H,
J = 8.4 Hz, 4-H), 7.92 (d, 1H, J = 2.8 Hz, 10-H), 8.72
(dd, 1H, J = 0.8, 8.4 Hz, 1-H). 13C NMR (100 MHz,
DMSO-d6): 23.92, 25.62 (2C), 44.60 (2C), 49.70 (2C),
54.31 (2C), 55.54, 57.41, 74.44, 115.55, 115.85, 120.87,
123.79, 124.98, 126.03, 126.81, 128.18, 128.95, 130.34,
130.91, 138.10, 146.47, 153.58, 156.70, 159.68. Anal.
calcd for C28H33N5O2Æ0.1 H2O: C 71.04, H 7.07, N
14.79; found: C 70.76, H 7.40, N 14.45.
5.2.3. Immunofluorescence analysis.27,28. HeLa cells
grown on cover glasses in 12-well plates with the drug
treatment for 24 h were used for DAPI staining. At dif-
ferent time points, the cells were washed twice with 1X
HBSS and with 1X PBS three times. Then, the cells were
fixed with 0.4% paraformaldehyde for 5 min, followed
by three times washing by 1X PBS. In order to permea-
bilize cells, cells were incubated in 2% FBS and 0.4%
Triton X-100 in 1X PBS for 15 min at room temperature
followed by three times washing with 0.2% Triton X-100
in PBS. To stain DNA, DAPI (0.06 lg/mL) was added
onto cells and incubated at room temperature for
5 min. After washing several times with 0.2% Triton
X-100 in PBS, the cells with DAPI staining were exam-
ined by fluorescence microscopy.
5.1.20. 9-Methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]-
quinolin-11-one O-benzyl oxime (22e). From 20 and ben-
zyl chloride as described for 22a: 83% yield. mp 211–
212 ꢁC. H NMR (400 MHz, DMSO-d6): 3.16 (m, 4H,
5.2.4. Flow cytometric analysis.29. HeLa cells treated with
DMSO or 22c at a concentration of 0.3 or 1.0 lM for 24 h
were harvested, rinsed in PBS, resuspended and fixed in
80% ethanol, and stored at ꢀ20 ꢁC in fixation buffer until
ready for analysis. Then the pellets were suspended in
1 mL of propidium iodide (PI) solution containing
20 lg/lL of PI, 0.2 mg/mL RNase, and 0.1% (v/v) Triton
X-100. Cell samples were incubated at room temperature
in the dark for at least 30 min and analyzed by a FACScan
flow cytometer (Becton Dickinson, Mountain View, CA).
Data recording was made using CELLQuest software
(Becton Dickinson, Mountain View, CA) and cell cycle
data were analyzed using ModFitLT software (Veruty
Software House, USA).
1
piperazinyl-H), 3.49 (m, 4H, piperazinyl-H), 3.80 (s,
3H, OMe), 5.61 (s, 3H, OCH2), 7.13 (dd, 1H, J = 2.4,
8.4 Hz, 8-H), 7.36–7.51 (m, 4H), 7.58–7.63 (m, 3H),
7.74 (d, 1H, J = 8.4 Hz, 7-H), 7.81 (d, 1H, J = 8.4 Hz,
4-H), 7.84 (d, 1H, J = 2.4 Hz, 10-H), 8.70 (d, 1H,
J = 8.4 Hz, 1-H). 13C NMR (100 MHz, DMSO-d6):
40.13 (2C), 48.81 (2C), 55.49, 78.18, 115.56, 115.90,
120.87, 123.86, 124.97, 126.15, 126.86, 128.20, 128.39,
128.57 (2C), 128.60 (2C), 129.00, 130.28, 130.86,
136.80, 138.10, 146.41, 153.91, 156.36, 159.63. Anal.
calcd for C17H15N3OÆ1.0.H2O: C 71.76, H 6.03, N
11.96; found: C 71.53, H 5.81, N 11.70.
Acknowledgments
5.2. Antiproliferative activity
Financial support of this work by the National Science
Council of the Republic of China is gratefully acknowl-
edged. We also thank National Cancer Institute (NCI)
of the United States for the anticancer screenings and
the National Center for High-Performance Computing
for providing computer resources and chemical database
services.
5.2.1. Cell culture. Cancer cells were purchased from
Bioresources Collection and Research Center, Taiwan.
Each cell line was maintained in the same standard med-
ium 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 amphotericin. Cul-
ture was maintained at 37 ꢁC with 5% CO2 in a humid-
ified atmosphere.
References and notes
5.2.2. Antiproliferative assay. Cancer cells were treated as
indicated for 48 h in medium containing 10% FBS. 3-[4,5-
Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
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