Synthesis of 6-substituted 9-methoxy-11H-indeno[1,2-c]quinoline-11-one derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bmc.2012.05.035

We have synthesized certain 6-substituted 9-methoxy-11H-indeno[1,2-c] quinolin-11-ones for antiproliferative evaluation. Results indicated that 6-alkylamine derivatives, 6-[2-(dimethylamino)ethylamino]-9-methoxy-11H- indeno[1,2-c]quinolin-11-one (5a) and its 6-[3-(dimethylamino)propylamino] derivative, 5b, were able to inhibit cells growth completely at a concentration of 100 μM while most of the 6-arylamine derivatives 6b-6h were inactive at the same concentration. Comparable mean GI₅₀ (drug molar concentration causing 50% cell growth inhibition) values for 5a (3.47 μM) and 5b (3.39 μM) indicated the cytotoxicity may not be affected by the length of alkyl substituents at C-6 position. Compound 5b, with a mean GI₅₀ value of 3.39 μM, was the most active and therefore was selected for further evaluation on its effects of H460 lung cancer cell cycle distribution. Results indicated that 5b induced cell cycle arrest in G2/M phase after 24 h treatment, while the hypodiploid (sub-G0/G1 phase) cells increased. We found that H460 cell became shrinked after the treatment of 5b, indicating that apoptosis may be a mechanism by which 5b kills the cancer cells. DNA unwinding assay indicated that 5b may bind to DNA through intercalation. Our results have also demonstrated that PARP was cleaved while caspase-3 and caspase-8 activities were induced after the treatment of 5b at 10 μM for 24 h. Thus, compound 5b intercalates DNA, induces cell cycle arrest at G2/M phase via cleavage of PARP, induces caspase-3 and caspase-8 activities, and consequently causes the cell death.

Full text of DOI:10.1016/j.bmc.2012.05.035

Bioorganic & Medicinal Chemistry 20 (2012) 4397–4404
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 6-substituted 9-methoxy-11H-indeno[1,2-c]quinoline-11-one
derivatives as potential anticancer agents
Chih-Hua Tseng a, Yeh-Long Chen a, Chiao-Li Yang a, Chih-Mei Cheng b, Chein-Hwa Han 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 Pharmacy, Chia Nan University of Pharmacy and Science, Tainan 717, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized certain 6-substituted 9-methoxy-11H-indeno[1,2-c]quinolin-11-ones for antiprolif-
erative evaluation. Results indicated that 6-alkylamine derivatives, 6-[2-(dimethylamino)ethylamino]-9-
methoxy-11H-indeno[1,2-c]quinolin-11-one (5a) and its 6-[3-(dimethylamino)propylamino] derivative,
Received 25 April 2012
Revised 15 May 2012
Accepted 15 May 2012
Available online 1 June 2012
5b, were able to inhibit cells growth completely at a concentration of 100
amine derivatives 6b–6h were inactive at the same concentration. Comparable mean GI50 (drug molar
concentration causing 50% cell growth inhibition) values for 5a (3.47 M) and 5b (3.39 M) indicated
the cytotoxicity may not be affected by the length of alkyl substituents at C-6 position. Compound 5b,
with a mean GI50 value of 3.39 M, was the most active and therefore was selected for further evaluation
lM while most of the 6-aryl-
l
l
Keywords:
Anticancer agents
Indeno[1,2-c]quinolinone derivatives
Cell cycle arrest
l
on its effects of H460 lung cancer cell cycle distribution. Results indicated that 5b induced cell cycle arrest
in G2/M phase after 24 h treatment, while the hypodiploid (sub-G0/G1 phase) cells increased. We found
that H460 cell became shrinked after the treatment of 5b, indicating that apoptosis may be a mechanism
by which 5b kills the cancer cells. DNA unwinding assay indicated that 5b may bind to DNA through
intercalation. Our results have also demonstrated that PARP was cleaved while caspase-3 and caspase-
Apoptosis
8 activities were induced after the treatment of 5b at 10 lM for 24 h. Thus, compound 5b intercalates
DNA, induces cell cycle arrest at G2/M phase via cleavage of PARP, induces caspase-3 and caspase-8
activities, and consequently causes the cell death.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
ethylamino)propoxy]phenyl}-11H-indeno[1,2-c]-quinolin-11-one
(3) exhibited IC50 value of 0.64, 0.89, and 0.68 lM, respectively
Quinoline skeleton is present in a large number of biologically
active compounds and is frequently condensed with various
heterocycles.1–7 For examples, camptothecin (CPT) is an anticancer
alkaloid isolated from Camptotheca acuminate which bears a con-
densed quinoline skeletone.1,2 The clinical use of CPT is hampered
due to its poor water solubility. Subsequent structural optimization
led to the discovery of highly water soluble topotecan which is cur-
rently used as an anticancer drug. TAS-103, which possesses the
tetracyclic indeno[2,1-c]quinoline pharmacophore, has been
proved to be a dual topoisomerases I and II targeting agent.8–10
Recently, we have synthesized certain isomeric indeno[1,2-c]quin-
oline derivatives of TAS-103 for anticancer evaluation.11–14 Among
them, 9-methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]quinolin-
11-one O-3-aminopropyl oxime (1), (E)-6-hydroxy-9-methoxy-
11H-indeno[1,2-c]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl
oxime (2), and 2,9-bis[3-(dimethylamino)propoxy]-6-{4-[3-(dim-
against the growth A549, which was more active than CPT and
topotecan (Fig. 1). The present report intends to establish the
antiproliferative structure–activity relationships (SAR) of indeno-
quinoline derivatives by the introduction of various substituents
at C-6 position and to identify more potent anticancer drug
candidates.
2. Chemistry
Reaction
of
2-hydroxy-3-(4-methoxyphenyl)quinoline-4-
carboxylic acid with POCl3 afforded 6-chloro-9-methoxy-11H-
indeno[1,2-c]quinolin-11-one (4),12 which was reacted with alkyl
amines to give 6-[2-(dimethylamino)ethylamino]-9-methoxy-
11H-indeno[1,2-c]quinolin-11-one (5a) and its 6-[3-(dimethyl-
amino)propylamino] derivative 5b. Treatment of
4 with the
respective arylamine in ethoxyethanol (50 mL) and was heated
with stirring under microwave irradiation (150 W) afforded
6-arylamino-9-methoxy-11H-indeno[1,2-c]quinolin-11-ones 6a–
6j as described in Scheme 1.
⇑
Corresponding author. Tel.: +886 7 3121101x2123; fax: +886 7 3125339.
E-mail address: tzengch@kmu.edu.tw (C.-C. Tzeng).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.035

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C.-H. Tseng et al. / Bioorg. Med. Chem. 20 (2012) 4397–4404
R3
N
R2
NH2
R1
O
N
O
N
O
OMe
NH
O
N
O
HO
N
N
OH
H
Camptothecin (CPT) R1 = H; R2 = H; R3 = H
TAS-103
Topotecan R1 = OH; R2 = CH2N(CH3)2, R3 = H
N
N
N
1
OR
O
OMe
O
O
9
OMe
RO
N
11
1
N
N
NHR
6
OR
3, R = CH2CH2CH2N(CH3)2
N
OH
2
Target compounds
Figure 1. Structures of Camptothecin, Topotecan, 6-[2-(dimethylamino)ethylamino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one (TAS-103), aminoalkyl substituted
indenoquinolines 1–3, and targeted compounds.
OMe
O
i
a R = CH2CH2NMe2
b
R = CH2CH2CH2NMe2
OMe
N
NHR
O
5
a R2 = R3 = H, R4 = Cl
b
R2 = R4 = F, R3 = H
OMe
c R2 = H, R3 = R4 = F
O
d R2 = H, R3 = Cl, R4 = F
N
Cl
e
R2 = R4 = H, R3 = OMe
R4
R3
4
f R2 = R3 = H, R4 = OMe
g R2 = R4 = OMe, R3 = H
ii
h
N
N
H
R2 = H, R3 = R4 = OMe
i R2 = R4 = H, R3 = COMe
j R2 = R3 = H, R4 = COMe
R2
6
Scheme 1. Reagents and conditions: (i) RNH2, reflux, 8 h (42% for 53a, 35% for 5b); (ii) ArNH2, MW, 1 h (32–87%).
The preparation of (E)-6-{{3-[(E)-1-(hydroxyimino)ethyl] phe-
nyl}amino}-9-methoxy-11H-indeno[1,2-c]quinolin-11-one oxime
(7a) and its positional isomer 8a are described in Scheme 2.
Reaction of 6-(3-acetylphenylamino)-9-methoxy-11H-indeno[1,2-
c]quinolin-11-one (6i) with NH2OH or NH2OMe gave exclusively
E-form isomer of oxime 7a or O-methyloxime 7b, respectively.6,13
Acetylation of 7a with acetic anhydride afforded (E)-6-{{3-[(E)-1-
(acetoxyimino)ethyl]phenyl}amino}-9-methoxy-11H-indeno[1,2-
c]quinolin-11-one O-acetyloxime (7c) in 86% yield. Accordingly,
compounds 8a–8c were prepared from 6j under similar reaction
conditions.
OR
OMe
OMe
O
N
i
O
N
N
N
3. Results and discussion
N
N
OR
H
H
Me
Me
iii
7a R = H
7b R = Me
7c R = COMe
6i
All compounds were evaluated in vitro against a 3-cell line
panel consisting of NCI-H460 (Lung), MCF7 (Breast), and SF-268
(CNS). In this protocol, each cell line is inoculated and preincubated
on a microtiter plate. Test agents are then added at a single concen-
OR
N
OMe
OMe
O
OR
tration (100 lM) and the culture incubated for 48 h. End-point
O
N
determinations are made with alamar blue.15 Results for each test
agent are reported as the percent of growth of the treated cells
when compared to the untreated control cells. Compounds which
reduced the growth percentage of any one of the cell lines to 32%
or less are passed on for evaluation in the full panel of 60 cell lines
over a 5-log dose range. Results from Table 1 indicated 6-alkyla-
mine derivatives 5a and 5b were able to inhibit cells growth
completely while most of the 6-arylamine derivatives 6b–6g were
inactive at the same concentration. Compounds 6a, 6i, and 6j
ii
Me
Me
N
N
N
N
H
H
8a R = H
R = Me
R = COMe
6j
iii
8b
8c
Scheme 2. Reagents and conditions: (i) NH2OH, MW, 0.5 h (67–69%); (ii) NH2OMe,
MW, 0.5 h (78–81%); (iii) Ac2O, pyridine, 1 h (82–86%).

C.-H. Tseng et al. / Bioorg. Med. Chem. 20 (2012) 4397–4404
4399
Table 1
Antiproliferative activity of 6-substituted 9-methoxyindeno[1,2-c]quinoline derivatives
OMe
X
N
R
Growth percentagesa
Mean GI50 (lM)
b
Compd
R
X
NCI-H460 (lung)
MCF7 (breast)
SF-268 (CNS)
5a
5b
6a
6b
6c
6d
6e
6f
NHCH2CH2NMe2
NHCH2CH2CH2NMe2
NHPh-4-Cl
NHPh-2,4-F
NHPh-3,4-F
NHPh-3-Cl-4-F
NHPh-3-OMe
NHPh-4-OMe
NHPh-2,4-OMe
NHPh-3,4-OMe
NHPh-3-COMe
NHPh-4-COMe
NHPh-3-C(@NOH)Me
NHPh-3-C(@NOMe)Me
NHPh-3-C(@NOCOMe)Me
NHPh-4-C(@NOH)Me
NHPh-4-C(@NOMe)Me
NHPh-3-C(@NOCOMe)Me
O
O
O
O
O
O
O
O
O
O
O
O
0
0
0
0
6
0
3.47
3.39
15.14
Ndc
Nd
Nd
Nd
Nd
Nd
Nd
12.88
39.81
28.18
Nd
53.70
4.17
Nd
10
96
96
Nd
83
69
110
68
0
18
11
82
96
0
96
99
106
Nd
79
74
103
90
17
61
84
80
32
1
42
68
69
Nd
87
65
107
41
7
11
66
92
43
2
6g
6h
6i
6j
7a
7b
7c
8a
8b
8c
NOH
NOMe
NOAc
NOH
NOMe
NOAc
112
37
89
24
108
36
52.48
a
Data obtained from NCI’s in vitro disease-oriented tumor cell screen. In this protocol, each cell line is inoculated and preincubated on a microtiter plate. Test agents are
added at a single concentration (100 M) and the culture is incubated for 48 h. End-point determinations are made with alamar blue. Results for each test agent are reported
l
as the percent of growth of the treated cells when compared to the untreated control cells. Compounds which reduced the growth percentage of any one of the cell lines to
32% or less are active.
b
Mean values over all cell lines tested. Theses cell lines are: leukemia (CCRF-CEM, HL-60 (TB), K-562, MOLT-4, PRMI-8226, and SR); non-small cell lung cancer (A549/ATCC,
EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, and NCI-H522); colon cancer (COLC 205, HCC-2998, HCT-116, HCT-15, HT29, KM12, and SW-620); CNS cancer (SF-
268, SF-295, SF-539, SNB-19, SNB-75, and U251); melanoma (LOX IMVI, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, and UACC-257); ovarian cancer (IGROV1,
OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, and SK-OV-3); renal cancer (786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, and UO-31); prostate cancer (PC-3 and DU-145);
and breast cancer (MCF7, MCF7/ADR-RES, MDA-MB-231/ATCC, HS 578T, MDA-MB-435, MDA-N and T-47D); GI50: Drug molar concentration causing 50% cell growth
inhibition.
c
Not determined.
exhibited only marginal antiproliferative activity. Therefore, an
alkylamino side chain at C-6 position is crucial for cytotoxicity.
Those active compounds were evaluated in the full panel of 60 hu-
man tumor cell lines derived from nine cancer cell types (leukemia,
non-small cell lung cancer, colon cancer, CNS cancer, melanoma,
ovarian cancer, renal cancer, prostate cancer, and breast cancer).
For each compound, dose–response curves for each cell line were
measured with five different drug concentration, the concentration
causing 50% cell growth inhibition (GI50) compared with the con-
trol were calculated.16 Comparable mean GI50 values for 5a (mean
Cytotoxicity of selected 9-methoxy-11H-indeno[1,2-c]quino-
line-11-one derivatives is summarized in Table 2. 6-[3-(Dimethyl-
amino)propylamino]-9-methoxy-11H-indeno[1,2-c]quinolin-11-
one (5b) was the most active with a mean GI50 value of 3.39
and inhibited the growth of SR, A549, H460, HCT-116, and U251
with GI50 values of less than 1.86 M in each case. Therefore,
lM
l
compound 5b was selected for further evaluation on its effects of
H460 cell cycle distribution by flow cytometric analysis. Com-
pound 5b induced cell cycle arrest in a concentration dependent
manner as shown in Figure 2 and Table 3. The proportion of cells
was decreased in the G1 and accumulated in G2/M phase after
GI50 = 3.47 lM) and 5b (mean GI50 = 3.39 lM) indicated the cyto-
toxicity may not be affected by the length of alkyl substituents at
C-6 position. However, the C-6 arylamine derivatives of these 9-
methoxy-11H-indeno[1,2-c]quinolin-11-ones demonstrated only
weakly active or inactive in which 6a, 6i, and 6j exhibited mean
Table 2
Cytotoxicity of selected 9-methoxy-11H-indeno[1,2-c]quinoline-11-one derivatives
[GI50 (l
M)]a
GI50 values of 15.14, 12.88, and 39.81 lM, respectively.
Cell lines
GI50 (lM)
The antiproliferative activity of 6,11-disubstituted 9-meth-
oxyindeno[1,2-c]quinoline derivatives is summarized in Table 1.
Compound 7a was weakly active with a mean GI50 value of
5b
6i
8a
1.48
1.45
29.50
1.48
2.29
3.80
15.14
<0.01
2.04
SR (Leukemia)
A549 (Lung)
H460 (Lung)
HCT-116 (colon)
U251 (CNS)
SK-MEL-5 (Melanoma)
IGROV1 (Ovarian)
RXF 393 (Renal)
DU-145 (Prostate)
HS 578t (Breast)
1.10
1.86
1.33
1.26
1.10
3.89
6.92
Ndb
21.38
17.70
23.50
2.63
3.09
5.25
3.63
9.77
11.75
3.16
28.18 lM while its methyl derivative 7b and acetoxy derivative
7c were inactive indicated that a hydrogen-donating group at C-
11 is essential for cytotoxicity. The same trend was observed for
its positional isomers in which (E)-6-{{4-[(E)-1-(hydroxyimi-
no)ethyl]phenyl}amino}-9-methoxy-11H-indeno[1,2-c]quinolin-
11-one oxime (8a) was active against the growth of all three cell
lines tested while its methyl derivative 8b and acetoxy derivative
8c were inactive. Compound 8a (with a mean GI50 value of
6.61
3.72
7.08
a
GI50: Drug molar concentration causing 50% cell growth inhibition.
Not determined.
4.17
lM) was more active than 7a implied the position of substitu-
b
tion is crucial for cytotoxicity.

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C.-H. Tseng et al. / Bioorg. Med. Chem. 20 (2012) 4397–4404
Figure 2. Flow cytometric analysis of H460 cells. Cells were treated with DMSO (A), compound 5b at 1.0 lM (B), 5.0 lM (C) or 10.0 lM (D) in H460 cells; 24 h later the cells
were harvested, fixed, and stained with propidium iodide as described in Section 5 prior to analysis by flow cytometry. The percentage of cells in each cell cycle phase was
quantified (Table 3).
24 h treatment of 5b, while the hypodiploid (sub-G0/G1 phase)
cells increased (Table 3). Apoptosis can be characterized by mor-
phological and biochemical changes in the cell nucleus, including
chromatin condensation and nuclear shrinking. Morphological
changes of cells treated with 5b can be visually observed with light
microscopy (Fig. 3). We found that the H460 cell became shrinked
Table 3
Effects of 5b on the cell cycle progression of lung cancer cell (H460)
Concentration (
l
M)
Cell cycle distributiona (%)
Sub G1
G1
S
G2/M
DMSO
1.0
5.0
0.9 0.3
3.7 1.2
8.2 1.6
25.3 3.7
66.2 2.1
58.1 3.5
49.2 1.7
41.4 2.2
14.3 1.9
10.1 1.5
9.1 2.5
18.6 1.7
28.1 2.2
33.5 2.3
21.5 1.8
after the treatment of 5b at 10 lM for 24 h. Such morphological
changes were not apparent in the control cells. These findings indi-
cated that apoptosis may be a mechanism by which 5b kills the
cancer cells. In order to evaluate if the antiproliferative effect of
10.0
11.8 1.4
a
Values representative mean SD from three experiments.
Figure 3. Induction of morphological change in H460 cells. Cells were treated with DMSO or compound 5b (1.0–10.0 lM) for 24 h at 37 °C and photographed under a
microscope.

C.-H. Tseng et al. / Bioorg. Med. Chem. 20 (2012) 4397–4404
4401
maintenance of cell viability.19 Our results have shown that PARP
was cleaved while caspase-3 and caspase-8 activities were induced
after the treatment of 5b for 24 h in a concentration-dependent
manner (Fig. 5). Thus, compound 5b may intercalate DNA, induces
cell cycle arrest at G2/M phase via cleavage of PARP, induces
caspase-3 and caspase-8 activities, and consequently causes the
cell death.
4. Conclusion
A number of 6-substituted 9-methoxy-11H-indeno[1,2-c]quin-
olin-11-ones were synthesized and evaluated for their antiprolifer-
ative activities. Among them, compound 5b was the most active
with a mean GI50 value of 3.39 lM and therefore was selected as
a new lead for further mechanism studies. Results indicated that
5b induced cell cycle arrest in G2/M phase after 24 h treatment,
while the hypodiploid (sub-G0/G1 phase) cells increased. DNA
unwinding assay indicated that 5b may bind to DNA through inter-
calation. In addition, PARP was cleaved while caspase-3 and
caspase-8 activities were induced after the treatment of 5b for
24 h in a concentration-dependent manner. Thus, compound 5b
may intercalate DNA, induces cell cycle arrest at G2/M phase via
cleavage of PARP, induces caspase-3 and caspase-8 activities and
consequently to cause the cell death. Further study on the struc-
ture optimization of 5b is ongoing.
Figure 4. Compound 5b unwind plasmid DNA. Increasing concentration of com-
pound 5b were incubated with negatively supercolied pBR322 (0.4 lg) for 12 h in
TE buffer and then run for 2.5 h on a 1% gel.
5b was a consequence of DNA intercalation, we performed an
in vitro DNA unwinding assay. DNA intercalators enhance the
unwinding of negatively supercoiled DNA (form I), which leads to
a decreased mobility in agarose gel during electrophoresis.17 As
shown in Figure 4, it seems that compound 5b between 0 and
5. Experimental
5.1. General
TLC: precoated (0.2 mm) Silica Gel 60 F254 plates from EM
Laboratories, Inc.; detection by UV light (254 nm). All chromato-
graphic separations were performed using silica gel (Merck 60
230–400 mesh). Mp: Yamato MP-21 melting-point apparatus;
uncorrected. 1H and 13C NMR spectra: Varian-Unity-400 spectrom-
eter at 400 and 100 MHz, chemical shifts in ppm with SiMe4 as an
internal standard (=0 ppm), coupling constants J in Hz. Mass spec-
tra (HRMS) were recorded on Finnigan/Thermo Quest MAT 95XL.
Elemental analyses were carried out on a Heraeus CHN-O-Rapid
elemental analyzer, and results were within 0.4% of calculated
values. Microwave reactions were conducted using a CEM Discover
Synthesis Unit (CEM Corp., Matthews, NC).
100 lM exhibited a dosage response, but not 1000 lM. The
plasmid DNA at this concentration cannot be clearly seen. Further,
the mechanism of shift in gel mobility can also be due to the
formation of huge complexes. These data indicate that 5b may bind
to DNA through intercalation. To determine whether 5b preferen-
tially kills cancer cells by apoptosis, procaspase-3, procaspase-8,
and PARP were evaluated in 5b-treated H460 cells by western
blotting. Caspases are the family of cysteine proteases and they
mediate apoptotic pathway in mammalian.18 PARP, a nuclear poly
(ADP-ribose) polymerase, is involved in DNA repair predominantly
in response to environmental stress, and is important for the
Figure 5. Expression of procapase-3, -8 and PARP in H460 cells by treatment with compound 5b for 24 h. Equal amounts of cell lysate were resolved using SDS–PAGE and
analyzed by western blot using anti-procaspase-3, -8, PARP and b-actin antibody to confirm equal protein loading.

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C.-H. Tseng et al. / Bioorg. Med. Chem. 20 (2012) 4397–4404
5.1.1. 6-[2-(Dimethylamino)ethylamino]-9-methoxy-11H-indeno
[1,2-c]quinolin-11-one (5a)
4-H), 8.33 (br s, 1H, NH), 8.52 (d, 1H, J = 8.4 Hz, 1-H). 13C NMR
(100 MHz, DMSO-d6): 55.80, 103.86, 104.12, 104.37, 110.76,
111.01, 111.19, 118.58, 119.31, 122.87, 124.72, 125.73, 126.79,
129.52, 129.64, 133.12, 134.13, 134.49, 147.82, 149.82, 160.43,
194.57. Anal. Calcd for C23H14F2N2O2: C, 71.13; H, 3.63; N, 7.21.
Found: C, 70.75; H, 3.93; N, 7.20.
A mixture of 6-chloro-9-methoxy-11H-indeno[1,2-c]quinolin-
11-one (4, 0.30 g, 1 mmol) and N,N-dimethylethylenediamine
(0.26 g, 3 mol) in 2-ethoxyethanol (30 mL) was refluxed for 8 h (by
TLC monitoring). The mixture was then cooled and evaporated in va-
cuo to give a residue which was treated with H2O (50 mL). The
resulting precipitate was filtered and washed with H2O. The crude
product was chromatographed on a column of silica gel using
CH2Cl2/MeOH = 10:1 to give 0.15 g (42%) of 5a as a red solid. Mp:
168–169 °C (dec). 1H NMR (400 MHz, CDCl3): 2.36 (s, 6H, N(CH3)2),
2.67 (t, 2H, J = 6.0 Hz, NHCH2CH2N), 3.71 (q, 2H, J = 6.0 Hz,
NHCH2CH2N), 3.85 (s, 3H, 9-OCH3), 5.84 (br s, 1H, NH), 6.88 (dd,
1H, J = 8.4, 2.4 Hz, 8-H), 7.21 (d, 1H, J = 2.4 Hz, 10-H), 7.26–7.33
(m, 2H, 2- and 7-H), 7.46–7.50 (m, 1H, 3-H), 7.67 (d, 1H, J = 8.4 Hz,
4-H), 8.59 (dd, 1H, J = 8.0, 1.2 Hz, 1-H). 13C NMR (100 MHz, CDCl3):
38.69, 45.19 (2C), 55.79, 57.66, 111.61, 118.18, 118.96, 122.20,
123.83, 124.37, 126.40, 129.17, 129.47, 134.13, 134.44, 135.41,
149.34, 152.62, 160.44, 195.49. HRMS (ESI) calcd for C21H22N3O2
[M+H]+ 348.1712; found 348.1709.
5.2.3. 6-(3,4-Difluorophenylamino)-9-methoxy-11H-indeno[1,2-
c]quinolin-11-one (6c)
Red solid (78%); mp: 240–241 °C. 1H NMR (400 MHz, DMSO-d6):
3.85 (s, 3H, 9-OCH3), 7.07 (dd, 1H, J = 8.4, 2.8 Hz, 8-H), 7.17 (d, 1H,
J = 2.8 Hz, 10-H), 7.36–7.45 (m, 2H, 2- and Ar-H), 7.51–7.59 (m, 2H,
3- and Ar-H), 7.64 (dd, 1H, J = 8.4, 2.0 Hz, 4-H), 7.82 (d, 1H,
J = 8.4 Hz, 7-H), 7.90 (ddd, 1H, J = 13.2, 7.8, 2.4 Hz, Ar-H), 8.50
(dd, 1H, J = 8.0, 1.2 Hz, 1-H), 8.67 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO-d6): 55.82, 109.19 (J = 21.2 Hz), 111.04, 116.49
(J = 9.1, 3.1 Hz), 116.90 (J = 17.4 Hz), 118.57, 119.65, 122.92,
125.07, 126.15, 126.91, 129.82, 130.23, 132.96, 134.43, 134.51,
137.97 (J = 8.3 Hz), 144.58 (J = 238.0, 12.9 Hz), 147.57, 149.05,
148.90 (J = 240.2, 12.8 Hz), 160.47, 194.46. Anal. Calcd for
C23H14F2N2O2: C, 71.13; H, 3.63; N, 7.21. Found: C, 70.77; H,
5.1.2. 6-[3-(Dimethylamino)propylamino]-9-methoxy-11H-indeno
[1,2-c]quinolin-11-one (5b)
3.87; N, 7.18.
Compound 5b was obtained from 4 and 3-dimethylaminopro-
pylamine as described for the preparation of 5a in 72% yield.
(0.13 g, 35%) as a red solid. Mp: 113–114 °C. 1H NMR (400 MHz,
CDCl3): 1.86 (m, 2H, NHCH2CH2CH2N) 2.33 (s, 6H, N(CH3)2), 2.57
(t, 2H, J = 5.6 Hz, NHCH2CH2CH2N), 3.77 (q, 2H, J = 5.6 Hz,
NHCH2CH2CH2N), 3.85 (s, 3H, 9-OCH3), 6.81 (dd, 1H, J = 8.4,
2.8 Hz, 8-H), 7.17 (br s, 1H, 6-NH), 7.19 (d, 1H, J = 2.8 Hz, 10-H),
7.23–7.27 (m, 1H, 2-H), 7.43–7.48 (m, 2H, 3-H and 7-H), 7.67 (d,
1H, J = 8.4 Hz, 4-H) 8.58 (dd, 1H, J = 8.0, 1.6 Hz, 1-H). 13C NMR
(100 MHz, CDCl3): 24.99, 42.98, 45.75 (2C), 55.69, 60.04, 111.09,
117.94, 118.69, 122.04, 123.71, 123.97, 126.26, 129.07, 129.26,
133.92, 134.67, 135.31, 149.39, 152.32, 160.24, 195.68. HRMS (EI)
calcd for C22H23N3O2 [M]+ 361.1790; found 361.1788.
5.2.4. 6-(3-Chloro-4-fluorophenylamino)-9-methoxy-11H-indeno
[1,2-c]quinolin-11-one (6d)
Red solid (40%); mp: 235–236 °C. 1H NMR (400 MHz, DMSO-d6):
3.85 (s, 3H, 9-OCH3), 7.06 (dd, 1H, J = 8.0, 2.4 Hz, 8-H), 7.16 (d, 1H,
J = 2.4 Hz, 10-H), 7.37 (d, 1H, J = 9.2 Hz, Ar-H), 7.40–7.44 (m, 1H, 2-
H), 7.54–7.62 (m, 2H, 3- and 4-H), 7.74 (ddd, 1H, J = 8.8, 4.0, 2.4 Hz,
Ar-H), 7.83 (d, 1H, J = 8.0 Hz, 7-H), 7.97 (dd, 1H, J = 6.8, 2.4 Hz, Ar-
H), 8.49 (dd, 1H, J = 8.4, 1.6 Hz, 1-H), 8.62 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO-d6): 55.84, 111.06, 116.41 (J = 22.0 Hz), 118.58,
118.71 (J = 24.2 Hz), 119.66, 120.75 (J = 6.8 Hz), 120.78, 121.82,
122.95, 125.09, 126.13, 126.86, 129.85, 130.20, 132.96, 134.47
(J = 8.3 Hz), 138.13, 147.56, 149.10, 152.50 (J = 239.5 Hz), 160.49,
194.48. Anal. Calcd for C23H14ClFN2O2: C, 68.24; H, 3.49; N, 6.92.
Found: C, 67.91; H, 3.68; N, 6.84.
5.2. General procedure for coupling of substituted-anilines
derivatives 6a–6j
5.2.5. 6-(3-Methoxyphenylamino)-9-methoxy-11H-indeno[1,2-
c]quinolin-11-one (6e)
A mixture of 4 (1.0 mmol), substituted aniline (3.0 mmol), and
K2CO3 (0.62 g, 4.5 mmol) in ethoxyethanol (50 mL) was heated
with stirring under microwave irradiation (150 W) for 1 h (TLC
monitoring). The mixture was cooled, concentrated, and triturated
with water. The resulting precipitate was collected and crystallized
from MeOH.
Purple solid (32%); mp: 113–114 °C. 1H NMR (400 MHz, DMSO-
d6): 3.79 (s, 3H, Ar-OCH3), 3.85 (s, 3H, 9-OCH3), 6.61 (dd, 1H, J = 8.0,
2.4 Hz, Ar-H), 7.07 (dd, 1H, J = 8.0, 2.4 Hz, 8-H), 7.18 (d, 1H,
J = 2.4 Hz, 10-H), 7.22–7.32 (m, 2H, Ar-H), 7.40–7.44 (m, 1H, 2-
H), 7.47–7.48 (m, 1H, Ar-H), 7.55–7.60 (m, 1H, 3-H), 7.655 (d, 1H,
J = 8.4 Hz, 4-H), 7.78 (d, 1H, J = 8.0 Hz, 7-H), 8.51 (d, 1H,
J = 8.0 Hz, 1-H), 8.54 (br s, 1H, NH). 13C NMR (100 MHz, DMSO-
d6): 54.97, 55.84, 105.73, 107.65, 111.03, 112.51, 118.58, 119.55,
122.96, 125.10, 126.00, 126.92, 129.19, 129.80, 130.57, 133.20,
134.34, 134.58, 142.19, 147.85, 149.45, 159.55, 160.43, 194.65.
Anal. Calcd for C24H18N2O3: C, 75.38; H, 4.74; N, 7.33. Found: C,
75.28; H, 4.74; N, 7.32.
5.2.1. 6-(4-Chlorophenylamino)-9-methoxy-11H-indeno[1,2-c]quin
olin-11-one (6a)
Red solid (54%); mp: 241–242 °C. 1H NMR (400 MHz, DMSO-d6):
3.83 (s, 3H, 9-OCH3), 7.04 (dd, 1H, J = 8.4, 2.8 Hz, 8-H), 7.14 (d, 1H,
J = 2.8 Hz, 10-H), 7.35–7.42 (m, 3H, 2- and Ar-H), 7.52–7.57 (m, 1H,
3-H), 7.62 (d, 1H, J = 8.4 Hz, 7-H), 7.73–7.76 (m, 2H, Ar-H), 7.78 (d,
1H, J = 8.4 Hz, 4-H), 8.48 (dd, 1H, J = 8.4, 1.6 Hz, 1-H), 8.62 (s, 1H,
NH). 13C NMR (100 MHz, CDCl3): 55.82, 111.00, 118.54, 119.63,
121.87 (2C), 122.94, 125.08, 125.44, 126.05, 126.89, 128.25 (2C),
129.76, 130.40, 133.07, 134.37, 134.52, 139.93, 147.70, 149.20,
160.43 (2C), 194.52. Anal. Calcd for C23H15ClN2O2Á0.1H2O: C,
71.06; H, 3.95; N, 7.21. Found: C, 70.82; H, 3.91; N, 7.09.
5.2.6. 6-(4-Methoxyphenylamino)-9-methoxy-11H-indeno[1,2-
c]quinolin-11-one (6f)
Purple solid (42%);mp: 225–226 °C. 1H NMR (400 MHz, DMSO-
d6): 3.78 (s, 3H, Ar-OCH3), 3.91 (s, 3H, 9-OCH3), 6.93–6.97 (m, 2H,
Ar-H), 7.18 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 7.18 (d, 1H, J = 2.4 Hz, 10-
H), 7.35–7.39 (m, 1H, 2-H), 7.50–7.57 (m, 2H, 3- and 4-H), 7.62–
7.66 (m, 2H, Ar-H), 7.87 (d, 1H, J = 8.4 Hz, 7-H), 8.30 (br s, 1H,
NH), 8.49 (d, 1H, J = 8.4 Hz, 1-H). 13C NMR (100 MHz, DMSO-d6):
55.22, 55.83, 111.08, 113.65 (2C), 118.43, 119.21, 122.91, 122.99
(2C), 124.92, 125.33, 126.63, 129.62, 129.99, 133.24, 133.68,
134.06, 134.56, 148.00, 150.08, 154.99, 160.36, 194.79. Anal. Calcd
for C24H18N2O3Á0.1H2O: C, 75.02; H, 4.78; N, 7.29. Found: C, 74.79;
H, 4.78; N, 7.20.
5.2.2. 6-(2,4-Diflourophenylamino)-9-methoxy-11H-indeno[1,2-
c]quinolin-11-one (6b)
Red solid (55%); mp: 225–226 °C. 1H NMR (400 MHz, DMSO-d6):
3.87 (s, 3H, 9-OCH3), 7.12–7.16 (m, 2H, 2- and 8-H), 7.21 (d, 1H,
J = 2.4 Hz, 10-H), 7.33–7.42 (m, 2H, 3- and Ar-H), 7.49–7.55 (m,
2H, 7- and Ar-H), 7.71–7.77 (m, 1H, Ar-H), 7.93 (d, 1H, J = 8.0 Hz,

C.-H. Tseng et al. / Bioorg. Med. Chem. 20 (2012) 4397–4404
4403
5.2.7. 6-(2,4-Dimethoxyphenylamino)-9-methoxy-11H-
indeno[1,2-c]quinolin-11-one (6g)
ing precipitate was collected, washed with H2O, and dried to give a
crude solid, which was purified by crystallization from MeOH to
give 7a (1.39 g, 67%). Mp: 239–240 °C. 1H NMR (400 MHz, DMSO-
d6): 2.19 (s, 3H, CH3), 3.85 (s, 3H, 9-OCH3), 7.10 (dd, 1H, J = 8.4,
2.4 Hz, 8-H), 7.29–7.42 (m, 4H), 7.54–7.7.58 (m, 1H, 3-H), 7.66 (d,
1H, J = 8.0 Hz), 7.73 (d, 1H, J = 7.6 Hz), 7.98 (d, 1H, J = 8.4 Hz, 7-
H), 8.06 (d, 1H, J = 2.0 Hz), 8.64 (s, 1H, NH), 8.75 (d, 1H, J = 8.4 Hz,
1-H), 11.18 (s, 1H, NOH), 13.54 (s, 1H, NOH). 13C NMR (100 MHz,
DMSO-d6): 11.81, 55.68, 115.14, 115.25, 117.60, 119.15, 120.45,
120.54, 124.03, 124.70, 124.91, 125.17, 127.27, 128.54, 129.09,
129.96, 130.71, 137.38, 138.34, 141.50, 146.35, 149.41, 153.20,
153.83, 159.72. HRMS (EI) calc. for C25H20N3O4 [M]+ 424.1535;
found 424.1538.
Purple solid (68%); mp: 240–241 °C. 1H NMR (400 MHz, DMSO-
d6 + TFA-d): 3.85 (s, 3H, 9-OCH3), 3.92 (s, 6H, Ar-OCH3), 6.79 (d, 1H,
J = 8.8 Hz, Ar-H), 6.88 (s, 1H, Ar-H), 7.16 (d, 1H, J = 6.8 Hz, 8-H),
7.35 (s, 1H, 10-H), 7.51 (d, 1H, J = 8.4 Hz, Ar-H), 7.57–7.60 (m,
1H, 2-H), 7.71–7.74 (m, 1H, 3-H), 8.09 (d, 1H, J = 8.0 Hz, 7-H),
8.26 (d, 1H, J = 8.0 Hz, 4-H), 8.75 (d, 1H, J = 7.8 Hz, 1-H). 13C NMR
(100 MHz, DMSO-d6 + TFA): 55.97, 56.37, 56.41, 106.48, 114.48,
114.33, 117.17, 119.31, 120.02, 124.87, 125.23, 124.87, 126.05,
127.67, 130.12, 131.21, 132.41, 133.09, 134.49, 138.28, 138.49,
156.72, 162.26, 162.33, 193.15. Anal. Calcd for C25H20N2O4: C,
72.80; H, 4.89; N, 6.79. Found: C, 72.59; H, 4.94; N, 6.73.
5.2.8. 6-(3,4-Dimethoxyphenylamino)-9-methoxy-11H-
indeno[1,2-c]quinolin-11-one (6h)
5.2.12. (E)-9-Methoxy-6-{{3-[(E)-1-(methoxyimino)ethyl]
phenyl}amino}-11H-indeno[1,2-c]quinolin-11-one O-meth
yloxime (7b)
Purple solid (32%). Mp: 166–167 °C. 1H NMR (400 MHz, DMSO-
d6): 3.78 (s, 3H, Ar-OCH3), 3.80 (s, 3H, Ar-OCH3), 3.88 (s, 3H, 9-
OCH3), 6.95 (d, 1H, J = 8.8 Hz, Ar-H), 7.07 (dd, 1H, J = 8.0, 2.4 Hz,
8-H), 7.19 (d, 1H, J = 2.4 Hz, 10-H), 7.28 (dd, 1H, J = 8.8, 2.4 Hz,
Ar-H), 7.37–7.41 (m, 1H, 2-H), 7.51 (d, 1H, J = 2.4 Hz, Ar-H), 7.53–
7.57 (m, 1H, 3-H), 7.61 (d, 1H, J = 8.0 Hz, 4-H), 7.88 (d, 1H,
J = 8.0 Hz, 7-H), 8.42 (br s, 1H, NH), 8.50 (d, 1H, J = 8.4 Hz, 1-H).
13C NMR (100 MHz, DMSO-d6): 55.43, 55.83, 55.84, 106.71,
111.06, 112.06, 113.15, 118.47 (2C), 125.23, 126.72, 127.16,
129.83 (2C), 130.58, 132.99, 133.59, 134.23, 134.46, 144.68,
148.55, 149.72, 160.40 (2C), 194.57. Anal. Calcd for
Compound 7b was obtained from 6i and 40% O-methylhydr-
oxylamine hydrochloride according to the preparation of 7a in
69% yield. Mp: 143–144 °C. 1H NMR (400 MHz, DMSO-d6): 2.21
(s, 3H, CH3), 3.84 (s, 3H, 9-OCH3), 3.92 (s, 3H, NOCH3), 4.35 (s,
3H, NOCH3), 7.09 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 7.29–7.41 (m, 5H),
7.53–7.57 (m, 1H, 3-H), 7.63 (d, 1H, J = 8.4 Hz), 7.76 (d, 1H,
J = 7.6 Hz), 7.87 (d, 1H, J = 2.4 Hz), 7.97 (d, 1H, J = 8.4 Hz), 8.10 (s,
1H), 8.65 (s, 1H, NH), 8.52 (d, 1H, J = 8.0 Hz, 1-H). 13C NMR
(100 MHz, DMSO-d6): 12.46, 55.63, 61.56, 64.54, 115.20, 115.88,
117.79, 119.34, 120.18, 121.13, 124.17, 124.96 (2C), 127.20,
128.42, 129.09, 130.27, 130.42, 136.18, 137.32, 141.37, 146.38,
149.19, 153.59, 154.18, 159.65. HRMS (EI) calc. for C27H24N3O4
[M]+ 452.1848; found 452.1850.
C
25H20N2O4Á0.4H2O: C, 71.54; H, 5.01; N, 6.67. Found: C, 71.30;
H, 4.94; N, 6.60.
5.2.9. 6-(3-Acetylphenylamino)-9-methoxy-11H-indeno[1,2-
c]quinolin-11-one (6i)
5.2.13. (E)-6-{{3-[(E)-1-(Acetoxyimino)ethyl]phenyl}amino}-9-
methoxy-11H-indeno[1,2-c]quinolin-11-one O-acetyloxime (7c)
To a suspension of 7a (0.42 g, 1.0 mmol) in acetic anhydride
(10 mL) was added pyridine (1.0 mL). The reaction mixture was
stirred at room temperature for 1 h. The solvent was removed
in vacuo, and the residue was triturated with H2O (20 mL), fil-
tered, and washed with H2O. The crude product was crystallized
from EtOH to give 7c (0.44 g, 86%). Mp: 214–215 °C. 1H NMR
(400 MHz, DMSO-d6): 2.25 (s, 3H, CH3), 2.41 (s, 3H, C(@O)CH3),
2.46 (s, 3H, C(@O)CH3), 3.86 (s, 3H, 9-OCH3), 7.13 (dd, 1H,
J = 8.4, 2.4 Hz, 8-H), 7.41–7.47 (m, 3H), 7.56–7.60 (m, 1H, 3-H),
7.66 (d, 1H, J = 8.4 Hz, 7-H), 7.86 (d, 1H, J = 2.4 Hz, 10-H), 7.88–
7.89 (m, 1H), 7.95 (d, 1H, J = 8.8 Hz, 4-H), 8.24 (br s, 1H), 8.69
(dd, 1H, J = 8.4, 0.8 Hz, 1-H), 8.73 (s, 1H, NH). 13C NMR
(100 MHz, DMSO-d6): 14.14, 19.34, 19.72, 55.64, 116.42,
117.44, 118.52, 120.05, 120.36, 122.51, 124.69, 124.92, 125.55,
126.60, 127.46, 128.71, 129.50, 130.02, 131.26, 134.82, 137.13,
141.41, 146.69, 148.97, 158.56, 159.74, 162.49, 167.63, 168.47.
Purple solid (82%); mp: 174–175 °C. 1H NMR (400 MHz,CDCl3):
2.66 (s, 3H, C(@O)CH3), 3.85 (s, 3H, 9-OCH3), 6.80 (br s, 1H, NH),
6.87 (dd, 1H, J = 8.0, 2.4 Hz, 8-H), 7.19 (d, 1H, J = 2.4 Hz 10-H),
7.28 (d, 1H, J = 8.4 Hz, Ar-H), 7.35–7.39 (m, 1H, 2-H), 7.46 (t, 1H,
J = 8.0 Hz, Ar-H), 7.51–7.55 (m, 1H, 3-H), 7.64–7.66 (m, 1H, Ar-H),
7.72 (d, 1H, J = 8.0 Hz, 7-H), 8.07 (dd, 1H, J = 8.0, 1.2 Hz, 4-H),
8.25–8.26 (m, 1H, Ar-H), 8.60 (d, 1H, J = 8.4 Hz, 1-H). 13C NMR
(100 MHz,CDCl3): 26.33, 55.82, 111.89, 118.47, 118.88, 120.20,
122.26, 122.68, 123.79, 123.97, 126.26, 127.26, 129.26, 129.97,
133.50, 135.19, 135.37, 137.83, 140.49, 148.35, 148.67, 148.75,
160.85, 194.49, 198.15. Anal. Calcd for C25H18N2O3Á0.1H2O: C,
75.77; H, 4.64; N, 7.07. Found: C, 75.66; H, 4.67; N, 7.06.
5.2.10. 6-(4-Acetylphenylamino)-9-methoxy-11H-indeno[1,2-
c]quinolin-11-one (6j)
Purple solid (84%); mp: 251–252 °C. 1H NMR (400 MHz, DMSO-
d6): 2.54 (s, 3H, C(@O)CH3), 3.84 (s, 3H, 9-OCH3), 7.07 (dd, 1H,
J = 8.4, 2.8 Hz, 8-H), 7.18 (d, 1H, J = 2.8 Hz, 10-H), 7.45–7.49 (m,
1H, 2-H),, 7.58–7.62 (m, 1H, 3-H), 7.69 (d, 1H, J = 8.0 Hz, 4-H),
7.73 (d, 1H, J = 8.4 Hz, 7-H), 7.79 (d, 2H, J = 8.8 Hz, Ar-H), 7.94 (d,
2H, J = 8.8 Hz, Ar-H), 8.52 (dd, 1H, J = 8.0, 0.8 Hz, 1H), 9.09 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6): 26.33, 55.82, 110.97, 118.25
(2C), 118.72, 120.05, 122.96, 125.23, 126.72, 127.16, 129.37 (2C),
129.90, 130.06, 131.04, 133.05, 134.56, 134.69, 145.90, 147.69,
148.56, 160.52, 194.39, 195.16. Anal. Calcd for C25H18N2O3Á0.7H2O:
C, 73.76; H, 4.81; N, 6.88. Found: C, 73.49; H, 4.86; N, 6.86.
HRMS (ESI) calc. for
509.1823.
C
29H25N4O5 [M+H]+ 509.1825; found
5.2.14. (E)-6-{{4-[(E)-1-(Hydroxyimino)ethyl]phenyl}amino}-9-
methoxy-11H-indeno[1,2-c]quinolin-11-one oxime (8a)
Compound 8a was obtained from 6j as described for 7a in 81%
yield. Mp: 165–166 °C. 1H NMR (400 MHz, DMSO-d6): 2.17 (s, 3H,
CH3), 3.86 (s, 3-H, 9-OCH3), 7.10 (dd, 1H, J = 8.4, 2.4 Hz, 8-H),
7.40–7.44 (m, 1H, 2-H), 7.56–7.77 (m, 6H), 7.97 (d, 1H, J = 8.4 Hz,
7-H), 8.07 (d, 1H, J = 2.4 Hz, 10-H), 8.73 (s, 1H, NH), 8.75 (dd, 1H,
J = 8.4, 0.8 Hz, 1-H), 11.00 (s, 1H, NOH), 13.52 (s, 1H, NOH). 13C
NMR (100 MHz, DMSO-d6): 11.46, 55.60, 115.05, 115.17, 119.44
(2C), 120.46, 123.96, 124.75, 124.90, 125.08, 125.82 (2C), 127.33,
128.96, 129.87, 130.61, 130.65, 138.28, 142.00, 146.36, 149.09,
152.69, 153.72, 159.66. HRMS (EI) calc. for C25H20N4O3 [M]+
424.1535; found 424.1532.
5.2.11. (E)-6-{{3-[(E)-1-(Hydroxyimino)ethyl]phenyl}amino}-9-
methoxy-11H-indeno[1,2-c]quinolin-11-one oxime (7a)
To a suspension of 6i (0.40 g, 1.0 mmol) in ethoxyethanol
(30 mL) was added NH2OHÁHCl (0.20 g, 3.0 mmol). The reaction
mixture was heated with stirring under microwave irradiation
(100 W) for 30 min (TLC monitoring). The solvent was removed
in vacuo and the residue was poured into H2O (20 mL). The result-

4404
C.-H. Tseng et al. / Bioorg. Med. Chem. 20 (2012) 4397–4404
5.2.15. (E)-9-Methoxy-6-{{4-[(E)-1-(methoxyimino)ethyl]phenyl}
amino}-11H-indeno[1,2-c]quinolin-11-one O-methyloxime (8b)
Compound 8b was obtained from 6j as described for 7b in 78%
yield. Mp: 108–109 °C. 1H NMR (400 MHz, DMSO-d6): 2.19 (s, 3H,
CH3), 3.85 (s, 3H, 9-OCH3), 3.92 (s, 3H, NOCH3), 4.37 (s, 3H, NOCH3),
7.10 (dd, 1H, J = 8.4, 2.8 Hz, 8-H), 7.41–7.45 (m, 1H, 2-H), 7.56–7.60
(m, 1H, 3-H), 7.65–7.77 (m, 5H), 7.87 (d, 1H, J = 2.8 Hz, 10-H), 7.93
(d, 1H, J = 8.4 Hz, 7-H), 8.71 (dd, 1H, J = 8.4, 0.8 Hz, 1-H), 8.81 (br s,
1H, NH). 13C NMR (100 MHz, DMSO-d6): 12.13, 55.64, 61.41, 64.58,
115.26, 115.86, 119.52 (2C), 120.22, 123.87, 124.17, 124.98,
125.23, 126.17 (2C), 127.04, 128.99, 129.20, 130.14, 130.40,
137.56, 142.28, 146.32, 148.78, 153.52, 153.72, 159.71. HRMS
(EI) calc. for C27H24N4O3 [M]+ 452.1848; found 452.1847.
samples was loaded onto a 1% agarose gel. The gel was run at 6 V/
cm for 2.5 h in TAE buffer (40 mM Tris, 20 mM sodium acetate,
1 mM EDTA-Na2, pH 8.5) and stained with ethidium bromide
and photographed under UV illumination using Polaroid instant
film.
5.3.4. Immunoblot analysis
After treatment of compound 5b, 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 À70 °C. The protein
concentration was determined by the Bradford method. Protein
5.2.16. (E)-6-{{4-[(E)-1-(Acetoxyimino)ethyl]phenyl}amino}-9-
methoxy-11H-indeno[1,2-c]quinolin-11-one O-acetyloxime (8c)
Compound 8c was obtained from 6j as described for 7c in 82%
yield. Mp: 188–189 °C. 1H NMR (400 MHz, DMSO-d6): 2.23 (s, 3H,
CH3), 2.36 (s, 3H, C(@O)CH3), 2.45 (s, 3H, C(@O)CH3), 3.86 (s, 3H,
9-OCH3), 7.09 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 7.40–7.44 (m, 1H, 2-
H), 7.56–7.60 (m, 1H, 3-H), 7.69–7.88 (m, 7H), 8.67 (br s, 1H,
NH), 8.69 (d, 1H, J = 8.4 Hz, 1-H). 13C NMR (100 MHz, DMSO-d6):
13.32, 18.94, 19.34, 55.38, 116.38, 117.15, 119.03 (2C), 119.99,
124.31, 124.68, 125.50, 126.66, 126.99 (2C), 127.27, 129.16,
129.84, 130.82, 131.06, 137.09, 143.48, 146.53, 148.28, 158.34,
159.63, 161.69, 167.30, 168.19. HRMS (ESI) calc. for C29H25N4O5
[M+H]+ 509.1825; found 509.1823.
(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. The membrane was then exposed to X-ray film. Protein
bands were detected using the enhanced chemiluminescence blot-
ting detection system (Amersham, USA).
Acknowledgements
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
Computing for providing computer resources and chemical data-
base services.
5.3. Pharmacological methods
5.3.1. Antiproliferative assay
The human tumor cell lines of the cancer screening panel are
grown in RPMI 1640 medium containing 5% fetal bovine serum
and 2 mM L-glutamine. For a typical screening experiment, cell
suspensions that were diluted according to the particular cell type
and the expected target cell density (5000–40,000 cells per well
based on cell growth characteristics) were added by pipet
(100 ml) into 96-well microtiter plates. Inoculates were allowed
a preincubation period of 24 h at 37 °C for stabilization. Dilution
at twice the intended test concentration was added at time zero
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5.3.3. DNA mobility assay (DNA unwinding)17
Negative supercolied pBR322 (400 ng) was incubated in TE buf-
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12 h at room temperature. Following the addition of 2
buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol), the
ll of loading