Synthesis, cytotoxicity, and DNA topoisomerase II inhibitory activity of benzofuroquinolinediones

Article abstract of DOI:10.1016/j.bmc.2006.12.012

Benzofuroquinolinediones (7c and 7d) were synthesized by base-catalyzed condensation of dichloroquinolinediones with phenolic derivatives. Their dialkylaminoalkoxy derivatives (8i-8p) were prepared by reaction with various dialkylaminoalkyl chlorides. The cytotoxicity of the synthesized compounds was evaluated against eight types of human cancer cell lines, and their topoisomerase II inhibition was assessed. In general, the cytotoxicity of benzofuroquinolinediones (8i-8p) was similar or superior to that of doxorubicin and showed more potent inhibitory activity than naphthofurandiones (8a-8h). Also, most of the compounds exhibited excellent topoisomerase II inhibitory activity at a concentration of 5 μM and two compounds, 8d and 8i, showed IC₅₀ values of 1.19 and 0.68 μM, respectively, and were much more potent than etoposide (IC₅₀ = 78.4 μM), but similar to doxorubicin (IC₅₀ = 2.67 μM). However their inhibitory activity on topoisomerase I was lower, and 8d and 8i showed IC₅₀ values of 42.0 and 64.3 μM, respectively.

Full text of DOI:10.1016/j.bmc.2006.12.012

Bioorganic & Medicinal Chemistry 15 (2007) 1651–1658
Synthesis, cytotoxicity, and DNA topoisomerase II inhibitory
activity of benzofuroquinolinediones
a
a
a
Hee-Kyung Rhee, Hyen Joo Park, Sang Kook Lee,
b
a,
*
Chong-Ock Lee and Hea-Young Park Choo
a
School of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
Pharmaceutical Screening Division, Korea Research Institute of Chemical Technology, Daejeon 305-606, Republic of Korea
b
Received 24 October 2006; revised 8 December 2006; accepted 9 December 2006
Available online 13 December 2006
Abstract—Benzofuroquinolinediones (7c and 7d) were synthesized by base-catalyzed condensation of dichloroquinolinediones with
phenolic derivatives. Their dialkylaminoalkoxy derivatives (8i–8p) were prepared by reaction with various dialkylaminoalkyl chlo-
rides. The cytotoxicity of the synthesized compounds was evaluated against eight types of human cancer cell lines, and their topo-
isomerase II inhibition was assessed. In general, the cytotoxicity of benzofuroquinolinediones (8i–8p) was similar or superior to that
of doxorubicin and showed more potent inhibitory activity than naphthofurandiones (8a–8h). Also, most of the compounds exhib-
ited excellent topoisomerase II inhibitory activity at a concentration of 5 lM and two compounds, 8d and 8i, showed IC50 values of
1
(
6
ꢀ
.19 and 0.68 lM, respectively, and were much more potent than etoposide (IC50 = 78.4 lM), but similar to doxorubicin
IC50 = 2.67 lM). However their inhibitory activity on topoisomerase I was lower, and 8d and 8i showed IC50 values of 42.0 and
4.3 lM, respectively.
2007 Elsevier Ltd. All rights reserved.
1. Introduction
OH O
H CO
3
O
CH3
O
Many of the important chemotherapeutic drugs present-
ly in clinical use have been recognized to exert their
activity by interfering with the DNA. Compounds con-
taining a planar chromophore with two to four fused
aromatic rings act as DNA intercalator, which causes
enzyme blockade and reading errors during the replica-
tion process. A tetracyclic structural pattern consisting
of a benzene ring attached to the 2-position of a naph-
thalene nucleus or heterocyclic ring moiety was observed
among these compounds, and known as 2-phenylnaph-
thalene-type structural pattern hypothesis. For example,
a pentacyclic alkaloid, camptothecin 1, is a potent anti-
N
N
O
O
O
OH
O
1
2
HO
CH3
O
O(CH ) N(C H ) HCl
N
2
2
2 5 2
N
H
O
CH3
O
1
4
3
tumor agent against numerous types of cancers. WS-
2
5
995A 2 is active against L1210 leukemic cells, and a
natural plant alkaloid, ellipticine 3, is an anticancer
Figure 1. The ‘2-phenylnaphthalene type’ structures as anticancer
agents.
3
drug. Compound 4 was reported to be active against
human leukemia cells (HL-60) and small-cell lung can-
cer (SCLC) (Fig. 1).
4
The cytotoxic mechanisms of coplanar annulated poly-
cyclic compounds have been shown to consist of interca-
5
lation in human DNA and further, modification of
topoisomerases. Topoisomerase I and II are essential en-
zymes in the regulation of DNA topology and supercoil-
ing, which are crucial for transcription and replication.
Keywords: Benzofuroquinolinediones; Human cancer cell lines; Cyto-
toxicity; Topoisomerase II.
*
Corresponding author. E-mail: hypark@ewha.ac.kr
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.012

1652
H.-K. Rhee et al. / Bioorg. Med. Chem. 15 (2007) 1651–1658
Topoisomerase I (topo I) breaks one of the DNA
strands and the DNA can then unwind by revolving
about the remaining phosphodiester bond to remove
supercoils. The enzyme then relegates the break to re-
store the DNA with an altered linking number. The only
topo I inhibitors to have undergone clinical trials as
antitumor drugs are camptothecin 1 and its structural
derivatives.
base-catalyzed condensation of the prepared com-
4
pounds 7a–7d with dialkylaminoalkyl chlorides. The
products were purified by recrystallization in co-solvent
of chloroform and methanol or flash column chroma-
tography (chloroform/methanol 2:1) with 13–89%
yields. The yields for compounds with bis(1-methyleth-
ylamino) group were higher than the others. Introduc-
tion of (dimethylamino)isopropyl group to compounds
7
a–7d gave two isomers for each compound (8c and
Topoisomerase II (topo II) is an enzyme that catalyzes
changes in the topology of DNA via a mechanism that
involves the transient double-strand breaking and
rejoining of phosphodiester bonds. This enzyme plays
several key roles in DNA metabolism and chromosome
structure, and it is the primary molecular target for a
8d, 8g and 8h, 8k and 8l, 8o and 8p, respectively). Two
isomers were isolated by column chromatography and
rearrangement of methyl group was identified on the
1
H NMR spectra. These configurations can be elucidat-
ed by aziridinium ion (9) pathway in the alkylation step
1
3
as proposed for methadone synthesis (Scheme 2).
6
number of potent anticancer drugs such as etoposide.
Topo II inhibitors display great structural diversity
and are widely used as antitumor agents, and six topo
II cleavable complex inhibitors (Etoposide, Teniposide,
Doxorubicin, Daunorubicin, Idarubicin, and Mitoxan-
trone) have been approved for clinical use. Recently,
topo II inhibitors have created a great deal of interest
2.2. Cytotoxic activity
The in vitro cytotoxic activities of the synthesized com-
pounds were evaluated against various human tumor cell
lines (lung; A549, stomach; SNU-638, colon; HCT116,
fibro sarcoma; HT1080, myeloid leukemic; HL-60, ovar-
ian; SK-OV-3, melanoma; SK-MEL-2 and CNS; XF-
7
–10
in the design of new antitumor agents.
1
4
4
98) by SRB assay method. The inhibitory activities
The derivatives of benzonaphthoquinolindione 4 have a
suitable structure possessing the coplanar conformation
and were reported to have cytotoxicity and topoisomer-
were presented as micromolar concentrations of the
compounds that cause 50% inhibition per unit of enzyme
(IC ) under the assay conditions and compared with
5
0
4
ase II inhibitory activity. In our continuous effort in
that of doxorubicin, clinically used agents for the treat-
ment of solid tumors (Table 1). In general, cytotoxicity
of benzofuroquinolinediones (8i–8p) was similar or supe-
rior to that of doxorubicin and exhibited more potent
inhibitory activity against all kinds of cancer cell lines
than the corresponding benzonaphthofurandiones (8a–
8h). The higher cytotoxic activity of benzofuroquinolin-
ediones demonstrates that the presence of the nitrogen
atom plays an important role in determining the activity
development of novel DNA intercalators based on
nitrogen-containing heterocyclic ring with a para-conju-
gated quinone structure, we synthesized the ben-
zofuroquinolinedione analogues and their cytotoxicity
against the eight human tumor cell lines and inhibitory
activity against topo I and II are reported in this study.
Also some benzonaphthofurandiones were prepared to
compare the cytotoxic activity.
1
2
of the compound. Especially compound 8l showed sig-
nificantly high cytotoxicity among all compounds. All
the methyl branch side-chain containing compounds
such as 8k, 8l, 8o, and 8p showed high activity. However,
the largest molecule 8f, containing bulky bis(1-methyl-
ethylamino) group and adjacent chlorine atom, showed
no cytotoxic activity against all the tested human cancer
cell lines. To investigate the cytotoxic mechanism of test-
ed compounds, inhibitory activity for topo I and II,
essential enzymes for DNA metabolism, was evaluated.
2
. Results and discussion
2
.1. Chemistry
Condensation of 5 (2,3-dichloronaphthalene-1,4-dione
a or 6,7-dichloroquinoline-5,8-dione 5b) and 6 (resor-
cinol 6a or 4-chlororesorcinol 6b) was achieved with
5
4
,11
modification of the reported reaction conditions.
Treatment of compounds containing the hydroxyl group
with dialkylaminoalkyl chloride in base and phase trans-
fer catalysis (PTC, benzyltriethylammonium chloride)
gave the corresponding dialkylaminoalkoxy derivatives,
as shown the Scheme 1. Benzonaphthofurandiones (7a
and 7b) were synthesized by base-catalyzed condensa-
tion of the compounds (5a and 5b) with phenolic
derivatives (6a and 6b) in alcohol. Also, benzofuroqui-
nolinediones (7c and 7d) were designed and synthesized
because the introduction of nitrogen could increase the
reductive potential of the quinolinediones and their
cytotoxicity. However, the preparation of the com-
pounds required elevated temperature in the presence
of toluene as a solvent and pyridine as a base-catalyst/
solvent. The purified compounds were obtained by
recrystallization in dimethylformamide. Their dial-
kylaminoalkoxy derivatives (8a–8p) were prepared by
2.3. Inhibitory activity for topo I and II
DNA topoisomerases are nuclear enzymes regulating the
topological state of DNA by breaking and rejoining of
DNA strands. While DNA-topo I catalyzes changes in
the topological state of duplex DNA by performing tran-
sient single-strand breakage-union cycles, topo II alters
the topology of DNA by causing a transient enzyme
bridged, double-strand break, followed by strand pass-
ing and resealing. They are extensively involved in
DNA metabolism including replication and transcrip-
tion. The antitumor activity is associated with the ability
of the topoisomerase poisons to stabilize the enzyme–
DNA cleavable complex. The precise mechanism of
ellipticine has not yet been fully understood but it is
suggested including DNA intercalation and inhibition
1
2

H.-K. Rhee et al. / Bioorg. Med. Chem. 15 (2007) 1651–1658
1653
Y
O
O
O
No
a
X
Y
Y
Cl
Cl
OH
a
CH
H
+
OH
b
c
d
CH Cl
X
O
X
N
N
H
Cl
HO
O
5
6
7a-d
a
b
X=CH
X=N
a
b
Y=H
Y=Cl
b
Y
O
O
OR
O
X
8
a-p
a. Na in EtOH or KOH in MeOH (X=CH) or pyridine (X=N)
b. RCl HCl, K
2
CO
3
, benzyltriethylammonium chloride in CHCl
3
2
and H O
No
X
Y
H
R
8
a
CH
CH
CH
CH
CH
CH
CH
CH
N
(CH
(CH
2
)
2
N(C
N{CH(CH
)CH N(CH
CH(CH )N(CH
2 5 2
H )
8
b
H
2
)
2
) }
3 2 2
8
c
H
CH(CH
CH
3
2
3
)
2
8
d
H
2
3
3
)
2
8
e
Cl
Cl
Cl
Cl
H
(CH
(CH
2
)
2
N(C
N{CH(CH
)CH N(CH
CH(CH )N(CH
2 5 2
H )
8
f
2
)
2
) }
3 2 2
8
8
g
h
CH(CH
CH
3
2
3
3
)
)
2
2
3
2
8
i
j
(CH
(CH
2
)
2
N(C
N{CH(CH
)CH N(CH
CH(CH )N(CH
2 5 2
H )
8
N
H
2
)
2
) }
3 2 2
8
k
N
H
CH(CH
CH
3
2
3
3
)
)
2
8
l
N
H
2
3
2
8
m
N
Cl
Cl
Cl
Cl
(CH
(CH
2
)
2
N(C
N{CH(CH
)CH N(CH
CH(CH )N(CH
2 5 2
H )
8
n
o
p
N
2
)
2
) }
3 2 2
8
N
CH(CH
CH
3
2
3
3
)
)
2
8
N
2
3
2
Scheme 1. Synthesis of benzonaphthofurandiones and benzofuroquinolinediones.
N
N
N
Cl
Cl
Cl
9
Scheme 2. Aziridinium ion pathway.
of topo II activity. Also, it is a general tendency that the
antitumor activity of anthracyclines such as doxorubicin
mainly results from an inhibition of mammalian topo
in agarose gels. Based on this information, effects of the
synthesized compounds on the catalytic activity of topo
II were evaluated. The decatenation of KDNA was in-
duced by topo II. The results of topo II inhibition of
1
5,16
17,18
II.
The decatenation assay
is specific for measur-
6
ing topo II activity because it is based on the conversion
of catenated DNA to its decatenated form, which re-
quires DNA double-strand breakage followed by strand
rotation and ligation activities uniquely done by topo II.
The removal of these KDNA by the enzyme can be seen
the tested compounds and the standard drug etoposide
(VP-16, 200 lM as a positive control) are shown in Table
2. Also, the electrophotogram on the inhibitory activity
of test compounds is presented in Figure 2. All synthe-
sized compounds were found to be capable of inhibiting

1654
H.-K. Rhee et al. / Bioorg. Med. Chem. 15 (2007) 1651–1658
Table 1. Cytotoxicity against human cancer cells of benzonaphthofurandiones and benzofuroquinolinediones
Compound IC50 (lM)
A549
SNU-638
HCT 116
HT 1080
HL-60
SK-OV-3
SK-MEL-2
XF498
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
1.09
3.11
2.57
0.88
0.84
2.77
>20.0
0.10
1.14
0.14
0.45
0.14
0.11
0.14
0.53
0.10
0.18
0.23
0.06
1.68
1.39
0.27
2.20
1.52
>20.0
0.42
0.34
0.06
0.18
0.08
0.07
0.13
0.41
0.13
0.14
1.87
0.62
2.19
1.02
0.27
0.34
2.73
0.65
1.11
0.25
0.39
2.27
>20.0
0.67
0.63
0.25
0.08
0.09
0.18
0.31
0.70
0.06
0.22
0.37
0.04
>20.0
3.55
2.26
19.03
>20.0
>20.0
0.55
0.65
0.27
1.02
0.15
0.08
0.25
0.75
0.15
0.15
4.28
0.29
>20.0
1.53
1.52
2.15
5.73
>20.0
0.34
0.50
0.16
0.94
0.15
0.09
0.24
0.73
0.15
0.14
5.72
0.07
>20.0
3.17
1.75
4.69
>20.0
>20.0
0.55
0.55
0.14
0.94
0.12
0.10
0.40
0.91
0.17
0.14
3.48
0.04
2.01
0.37
0.21
1.59
>20.0
>20.0
g
h
i
0.62
0.45
0.05
0.37
0.06
0.02
0.09
0.18
0.10
0.09
1.16
0.15
0.49
0.65
0.07
0.18
0.17
0.06
0.15
0.22
0.10
0.11
0.25
0.06
j
k
l
m
n
o
p
Etoposide
Doxorubicin
A549, human lung tumor cell line; SNU-638, human stomach tumor cell line; HCT116, human colon tumor cell line; HT1080, human fibrosarcoma
tumor cell line; HL-60, human myeloid leukemic tumor cell line; SK-OV-3, human ovarian tumor cell line; SK-MEL-2, human melanoma tumor cell
line; XF-498, human CNS tumor cell line.
Table 2. Inhibitory activity of naphthofurans and furoquinolines on topoisomerase
Compound
Decatenation activity (for Topo II) % inhibition
Relaxation activity (for Topo I) % inhibition
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
91
36
84
100
50
59
88
68
100
71
24
67
47
35
11
59
89
—
78
12
10
12
14
19
14
6
g
h
i
0
0
j
0
k
l
0
0
m
n
o
p
0
0
0
0
Etoposide (200 lM)
Camptothecin (100 lM)
Doxorubicin (5 lM)
—
77
—
Decatenation assay for topo II and relaxation assay for topo I were done at a concentration of 5 lM of samples, respectively.
topo II completely at an initial concentration of 200 lM.
When these compounds were tested at a concentration of
for five compounds (8a, 8c, 8d, 8g, and 8i) with over 80%
inhibition against the topo II was carried out at concen-
tration of 200 lM and the electrophotogram on the re-
sult is presented in Figure 3. IC50 values of 8c, 8d, and
8i among these compounds were 59.5, 42.0, and
64.3 lM, respectively. As a result of these tests, all tested
benzonaphthofurandiones (8a–8h) and benzofuroqui-
nolinediones (8i–8p) were considered as potent positive
topo II inhibitors. Although the correlation of the cyto-
toxicity and the inhibitory activity of DNA decatenation
by topo II is not perfectly clear, at least, these results sug-
gest a possible mechanism for the cytotoxicity against tu-
mor cells and provide advanced opportunities to design
and develop new chemotherapeutic agents because
DNA topoisomerase based on the information of
5
lM, most of the test compounds showed overmoderate
activity. Especially, two compounds 8d and 8i showed
the IC50 values to be 1.19 and 0.68 lM, respectively,
and were 66 and 115 times more potent, respectively,
than that of etoposide (IC50 = 78.4 lM), but similar to
doxorubicin (IC50 = 2.67 lM). To further study the
mechanism of the synthesized compounds, topo I-medi-
ated DNA relaxation activity was also evaluated by com-
parison with camptothecin inhibiting DNA synthesis via
strand scission and causing cell death during the S-phase
1
of the cell cycle. The tested samples did not show inhib-
itory activity at concentration of 5 lM, the same concen-
tration in topo II assay. (Table 2) Topo I inhibitory assay

H.-K. Rhee et al. / Bioorg. Med. Chem. 15 (2007) 1651–1658
1655
Figure 2. Effects of the prepared compounds on the decatenation of KDNA by topo II. Lane A, KDNA without enzyme (catenated form); lane B,
*
KDNA with 1 U of topo II (decatenated form); lane C, decatenated KDNA marker, lane D; linear KDNA marker; lane E , etoposide, 200lM; lane
F, doxorubicin, 5 lM; lanes 8a–8p, KDNA with 1 U of topo II in the presence of compounds 8a–8p at a concentration of 5 lM, respectively.
methylsilane as an internal standard. Samples were dis-
solved in CDCl3 or DMSO-d . Mass spectra were
6
obtained on the Mass spectrometer JMS-700 (Jeol, Ja-
pan) at the Korea Basic Science Institute (Seoul). Most
of the reagents were purchased from Sigma–Aldrich
Chemical Company.
4.1.2. Cytotoxicity and topoisomerase inhibitory assay.
Trichloroacetic acid (TCA) and sulforhodamine B
Figure 3. Effects of the selected compounds on the relaxation of
supercoiled DNA by topo I Plasmid DNA (pHOT1, 0.25 mg) was
incubated with topo I (1 U) in the presence of test agents and then seen
on an agarose gel. Lane A, supercoiled DNA without enzyme; lane B,
supercoiled DNA with topo I (relaxed form), lane C, relaxed DNA
marker; lane D, supercoiled DNA with topo I in the presence of
(
(
SRB) were purchased from Sigma Chemical Co.
St. Louis, MO). Minimal essential medium with Eagles’
salt (MEME), fetal bovine serum (FBS), non-essential
amino acid solution (10 mM, 100·), trypsin-EDTA
solution (1·), and antibiotic-antimycotic solution were
from Invitrogen (Grand Island, NY). Topo I, II and as-
say kits were purchased from Topogen, Inc. (Columbus,
OH, USA). Stock solutions of all test agents were dis-
solved in dimethylsulfoxide (DMSO) for topo I relaxa-
tion assay and 10% DMSO in acetone for topo II
decatenation assay.
100 lM camptothecin. Lanes 5–9 in the presence of the selected
compounds at a concentration of 200 lM, respectively.
DNA intercalating potential is considered as important
1
targets for cancer chemotherapy.
9
3. Conclusion
4.2. Methods
The benzonaphthofurandione and benzofuroquinolin-
edione derivatives were synthesized by base-catalyzed
condensation. The cytotoxicity of the benzofuroquinol-
inedione derivatives was as high as that of doxorubicin
and higher than that of the corresponding naphthoqui-
none analogues. The compounds with dimethylamino-
isopropoxy group especially showed excellent
inhibitory activity throughout the entire in vitro test.
Most of the test compounds showed high activity on
topo II at a concentration of 5 lM, especially, 8d and
4.2.1. Synthesis. General procedure for the preparation
of benzonaphthofurandiones followed Cheng et al.’s
method. To a stirred ethanolic NaOEt solution or solu-
4
tion of MeOH containing KOH was added portionwise
at rt powdered 2,3-dichloro-1,4-naphthoquinone. After
stirring for a minute, the alcoholic solutions of resorcin-
ol, phloroglucinol, and 4-chlororesorcinol were added
dropwise with continuous stirring, respectively. The
mixture was stirred at rt overnight. The reaction mixture
was acidified with 6 N and 0.2 N HCl at 0 ꢁC. The
resulting solid was collected by filtration, washed succes-
sively with water, methanol, and diethyl ether, and dried
in vacuo at 60 ꢁC. Benzofuroquinolinediones were pre-
pared by refluxing for 15 h the mixture of 6,7-dichloro-
quinoline-5,8-dione and resorcinol or 4-chlororesorcinol
in toluene/pyridine (1:1). The reaction mixture was then
cooled and the organic solvent was evaporated, and the
residue was mixed with water and filtered. The prepared
solid was dried in vacuo at 60 ꢁC. These were recrystal-
lized from dimethylformamide (DMF).
8
i showed IC50 values of 1.19 and 0.68 lM, respectively.
However, their inhibitory activity on topo I was low and
IC50 values of 8d and 8i were 42.0 and 64.3 lM,
respectively.
4. Experimental
4
4
.1. Materials
.1.1. Synthesis. All melting points were taken in Pyrex
capillaries using electrothermal digital melting point
apparatus (B u¨ chi). H NMR spectra were recorded on
a 400 MHz Varian FT-NMR spectrometer using tetra-
4.2.2. 3-Hydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione
(7a). Deep yellow solid (24%); mp >310 ꢁC; H NMR
(DMSO-d ) d 10.53 (s, 1H), 8.10–8.13 (m, 2H), 8.00
6
1
1

1656
H.-K. Rhee et al. / Bioorg. Med. Chem. 15 (2007) 1651–1658
(
(
(
d, J = 8.8 Hz, 1H), 7.88–7.90 (m, 1H), 7.19 (s, 1H), 7.08
d, J = 8.8 Hz, 1H); HR-FABMS calcd for C H O
4
M+H) : 265.0501, found: 265.0505.
J = 6.0 Hz, 2H), 3.77–3.86 (m, 2H), 3.46 (q,
J = 5.2 Hz, 2H), 1.61 (d, J = 6.4 Hz, 6H), 1.53 (d,
J = 6.8 Hz, 6H); HR-FABMS calcd for C H NO
4
1
6
8
+
24
25
+
(M+H) : 392.1862, found: 392.1883.
4
1
.2.3. 2-Chloro-3-hydroxybenzo[b]naphtho[2,3-d]furan-6,
1
1-dione (7b). Brick red solid (31%); mp 309–310 ꢁC; H
4.2.9. 3-[2-(Dimethylamino)isopropoxy]benzo[b]naphtho
[2,3-d]furan-6,11-dione (8c). Orange red solid (32.7%);
NMR (DMSO-d ) d 11.37 (s, 1H), 8.11–8.13 (m, 2H),
6
1
8
MS calcd for C H ClO (M+H) : 299.0111, found:
.08 (s, 1H), 7.89–7.92 (m, 2H), 7.41 (s, 1H); HR-FAB-
mp 259–260 ꢁC; H NMR (CDCl ) d 8.19–8.27 (m,
3
+
2H), 8.15 (d, J = 8.8 Hz, 1H), 7.75–7.79 (m, 2H), 7.20
(s, 1H), 7.12 (d, J = 8.8 Hz, 1H), 4.57–4.70 (m, 1H),
16
7
4
2
99.0112.
2.68–2.76 (m, 1H), 2.45–2.55 (m, 1H), 2.35 (s, 6H),
1.38 (d, J = 6.0 Hz, 3H); HR-FABMS calcd for
4
.2.4. 8-Hydroxybenzofuro[3,2-g]quinoline-5,11-dione (7c).
1
+
C H NO (M+H) : 350.1392, found: 350.1404.
Dark brown solid (28%); mp >310 ꢁC; H NMR (DMSO-
2
1
19
4
d ) d 10.59 (s, 1H), 9.03 (d, J = 4.8 Hz, 1H), 8.46 (d,
6
J = 7.6 Hz, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.86 (dd,
J = 4.8 Hz; J = 4.4 Hz, 1H), 7.21 (s, 1H), 7.09 (d,
4.2.10. 3-[2-(Dimethylamino)propoxy]benzo[b]naphtho
[2,3-d]furan-6,11-dione (8d). Orange solid (21.2%); mp
13
1
J = 8.8 Hz, 1H);
C NMR (DMSO-d ) d 180.44,
260–261 ꢁC; H NMR (CDCl ) d 8.19–8.27 (m, 2H),
6
3
1
1
72.20, 160.23, 157.60, 153.68, 152.91, 148.32, 134.00,
29.89, 127.58, 123.68, 123.43, 116.66, 113.71, 98.35;
8.16 (d, J = 9.2 Hz, 1H), 7.75–7.80 (m, 2H), 7.14–7.17
(m, 2H), 4.12–4.17 (m, 1H), 3.94–4.01 (m, 1H), 3.03–
+
HR-FABMS calcd for C H NO (M+H) : 266.0453,
4
3.14 (m, 1H), 2.42 (s, 6H), 1.21 (d, J = 6.8 Hz, 3H);
HR-FABMS calcd for C H NO
4
15
7
+
(M+H) :
found: 266.0449.
2
1
19
3
50.1392, found: 350.1397.
4
.2.5. 7-Chloro-8-hydroxybenzofuro[3,2-g]quinoline-5,11-
1
dione (7d). Dark brown solid (30%); mp >310 ꢁC; H
NMR (DMSO-d ) d 9.03 (d, J = 4.4 Hz, 1H), 8.49 (d,
4.2.11. 2-Chloro-3-[2-(diethylamino)ethoxy]benzo[b]naph-
tho[2,3-d]furan-6,11-dione (8e). Dark yellow pearl solid
6
1
J = 7.6 Hz, 1H), 8.10 (s, 1H), 7.95 (s, 1H), 7.88 (dd,
J = 4.8 Hz; J = 4.8 Hz, 1H), 7.35 (s, 1H); HR-FABMS
(14.2%); mp: 211–212 ꢁC; H NMR (CDCl ) d 8.32 (s,
3
1H), 8.22–8.27 (m, 2H), 7.79–7.81(m, 2H), 7.28 (s,
1H), 4.77 (t, J = 6.8 Hz, 2H), 3.59 (t, J = 6.8 Hz, 2H),
3.37 (q, J = 5.8 Hz, 4H), 1.52 (t, J = 7.6 Hz, 6H); HR-
+
calcd for C H ClNO (M+H) : 300.0064, found:
1
5
6
4
3
00.0065.
+
FABMS calcd for C H ClNO (M+H) : 398.1159,
4
2
2
20
4
.2.6. General procedure for the preparation of (dialkyla-
found: 398.1169.
mino)alkoxy-benzo[b]naphtho[2,3-d]furan-6,11-diones
and (dialkylamino)alkoxy-benzofuro[3,2-g]quinoline-5,11-
diones. To a suspension of 0.15 mmol of benzo[b]naph-
tho[2,3-d]furan-6,11-diones or benzofuro[3,2-g]quino-
4.2.12. 2-Chloro-3-[2-[bis(1-methylethylamino)]ethoxy]-
benzo[b]naphtho[2,3-d]furan-6,11-dione (8f). Orange
1
pearl solid (24.8%); mp 246–247 ꢁC; H NMR (CDCl )
3
line-5,11-diones in 10 mL CHCl3,
a
solution of
.28 mmol K CO and 0.02 mmol benzyltriethylammo-
d 8.30 (s, 1H), 8.20–8.27 (m, 2H), 7.77–7.81 (m, 2H),
7.38 (s, 1H), 4.92 (t, J = 5.2 Hz, 2H), 3.84–3.95 (m,
2H), 3.56 (q, J = 5.1 Hz, 2H), 1.62 (d, J = 6.8 Hz, 6H),
1.55 (d, J = 6.8 Hz, 6H); HR-FABMS calcd for
1
nium chloride in 2 mL H O was added followed by
2
3
2
0
in 2 mL H O. The mixture was stirred at rt for 1 h and
.3 mmol of (dialkylamino)alkyl chloride hydrochloride
+
C H ClNO (M+H) : 426.1472, found: 426.1444.
24 24
2
4
refluxed overnight with vigorous stirring. It was cooled
and the organic layer was separated. The aqueous por-
tion was extracted with CHCl (210 mL). The combined
4.2.13. 2-Chloro-3-[2-(dimethylamino)isopropoxy]benzo-
[b]naphtho[2,3-d]furan-6,11-dione (8g). Orange solid
3
1
organic phase was washed with brine and water, and
dried (Na SO ). The solids were acquired by evapora-
(25.9%); mp 211–212 ꢁC; H NMR (CDCl ) d 8.29 (s,
3
1H), 8.21–8.26 (m, 2H), 7.76–7.81 (m, 2H), 7.32 (s,
1H), 4.60–4.70 (m, 1H), 2.73–2.82 (m, 1H), 2.55–2.63
(m, 1H), 2.37 (s, 6H), 1.43 (d, J = 6.0 Hz, 3H); HR-
2
4
tion of the solvent, which gave pure products upon
recrystallization (from EtOAc) or flash column chroma-
tography (CHCl /MeOH 2:1).
+
FABMS calcd for C H ClNO (M+H) : 384.1003,
21 18
3
4
found: 384.1016.
4.2.7. 3-[2-(Diethylamino)ethoxy]benzo[b]naphtho[2,3-d]-
furan-6,11-dione (8a). Orange pearl solid (35.4%); mp
4.2.14. 2-Chloro-3-[2-(dimethylamino)propoxy]benzo-[b]-
naphtho[2,3-d]furan-6,11-dione (8h). Dark orange solid
1
2
8
1
3
12–213 ꢁC; H NMR (CDCl ) d 8.19–8.26 (m, 2H),
3
1
.20 (d, J = 7.2 Hz, 1H), 7.77–7.80 (m, 2H), 7.19 (s,
H), 7.12 (d, J = 8.4 Hz, 1H), 4.69 (t, J = 4.6 Hz, 2H),
.52 (t, J = 7.2 Hz, 2H), 3.29 (q, J = 7.0 Hz, 4H), 1.50
(15.0%); mp 196–197 ꢁC; H NMR (CDCl ) d 8.30 (s,
3
1H), 8.21–8.26 (m, 2H), 7.76–7.81 (m, 2H), 7.19 (s,
1H), 4.18–4.22 (m, 1H), 3.97–4.02 (m, 1H), 3.10–3.18
(m, 1H), 2.42 (s, 6H), 1.25 (d, J = 6.8 Hz, 3H); HR-
(
(
t, J = 7.4 Hz, 6H); HR-FABMS calcd for C H NO
22 21 4
+
M+H) : 364.1549, found: 364.1547.
+
FABMS calcd for C H ClNO (M+H) : 384.1003,
2
1
18
4
found: 384.1023.
4.2.8. 3-[2-[Bis(1-methylethylamino)]ethoxy]benzo[b]na-
phtho[2,3-d]furan-6,11-dione (8b). Orange red solid
4.2.15. 8-(2-(Diethylamino)ethoxy)benzofuro[3,2-g]quino-
line-5,11-dione (8i). Yellow brown solid (24.2%); mp
1
(
8
84.3%); mp 183–184 ꢁC; H NMR (CDCl ) d 8.20–
3
1
.25 (m, 2H), 8.17 (d, J = 8.8 Hz, 1H), 7.76–7.78 (m,
H), 7.22 (s, 1H), 7.12 (d, J = 8.8 Hz, 1H), 4.80 (t,
189–190 ꢁC; H NMR (CDCl ) d 9.08 (d, J = 4.4 Hz,
3
2
1H), 8.56 (d, J = 7.6 Hz, 1H), 8.21 (d, J = 8.8 Hz, 1H),

H.-K. Rhee et al. / Bioorg. Med. Chem. 15 (2007) 1651–1658
1657
7
1
3
.73 (t, J = 6.2 Hz, 1H), 7.20 (s, 1H), 7.17 (d, J = 8.0 Hz,
H), 4.72 (t, J = 7.2 Hz, 2H), 3.52 (t, J = 4.0 Hz, 2H),
.31 (q, J = 4.0 Hz, 4H), 1.51 (t, J = 7.2 Hz, 6H); HR-
4.2.22. 8-(2-(Dimethylamino)propoxy)-7-chlorobenzof-
uro[3,2-g]quinoline-5,11-dione (8p). Brick brown solid
1
(22.4%); mp: 189–190 ꢁC; H NMR (CDCl ) d 9.07 (d,
3
+
FABMS calcd for C H N O (M+H) : 365.1501,
2
found: 365.1504.
J = 4.4 Hz, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.27 (s, 1H),
7.73 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 4.20–4.24 (m,
1
20
2
4
1H), 3.99–4.09 (m, 1H), 3.13–3.23 (m, 1H), 2.45 (s,
6H), 1.28 (d, J = 6.4 Hz, 3H); HR-FABMS calcd for
4.2.16. 8-(2-(Diisopropylamino)ethoxy)benzofuro[3,2-g]-
quinoline-5,11-dione (8j). Red brown solid (55.9%); mp
+
C H ClN O (M+H) : 385.0955, found: 385.0937.
2
0
17
2
4
1
2
1
7
34–235 ꢁC; H NMR (CDCl ) d 9.07 (d, J = 4.8 Hz,
3
H), 8.55 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 9.2 Hz, 1H),
.72 (t, J = 6.4 Hz, 1H), 7.23 (s, 1H), 7.18 (d,
4.2.23. In vitro antitumor activity evaluation by SRB
1
4
assay . The in vitro cytotoxic activities were evaluated
by SRB method. Human tumor cell lines: lung (A549),
stomach (SNU-638), colon (HCT116), fibro sarcoma
(HT1080), myeloid leukemic (HL-60), ovarian (SK-
J = 8.8 Hz, 1H), 4.82 (t, J = 6.0 Hz, 2H), 3.75–3.85 (m,
2
1
H), 3.45 (t, J = 5.2 Hz, 2H), 1.62 (d, J = 6.8 Hz, 6H),
.53 (d, J = 6.8 Hz, 6H); HR-FABMS calcd for
+
C H N O (M+H) : 393.1814, found: 393.1818.
4
OV-3), melanoma (SK-MEL-2), and CNS (XF-498)
2
3
24
2
4
(
5 · 10 cells/mL) were treated with different concentra-
4
[
.2.17. 8-(1-(Dimethylamino)propan-2-yloxy)benzofuro-
3,2-g]quinoline-5,11-dione (8k). Orange red solid
tions of the test agents for 3 days. After treatment, cells
were fixed with TCA and cell viability was determined
with sulforhodamine B (SRB) protein staining method.
The result was expressed as a percentage, relative to sol-
vent-treated control incubations, and the IC50 values
were calculated using non-linear regression analysis
(percent survival versus concentration).
1
(
15.7%); mp 134–135 ꢁC; H NMR (CDCl ) d 9.06 (d,
3
J = 4.4 Hz, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.14 (d,
J = 8.8 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.22 (s, 1H),
7
.16 (d, J = 8.8 Hz, 1H), 4.70–4.81 (m, 1H), 2.77–2.86
m, 1H), 2.58–2.67 (m, 1H), 2.43 (s, 6H), 1.39 (d,
J = 6.0 Hz, 3H); HR-FABMS calcd for C H N O
4
(
2
0
18
2
+
17
(
M+H) : 351.1345, Found: 351.1351.
4.3. Decatenation assay for topoisomerase II activity
4
.2.18. 8-(2-(Dimethylamino)propoxy)benzofuro[3,2-g]-
quinoline-5,11-dione (8l). Brick brown solid (12.9%);
The assay was done according to the protocol provided
by TopoGen, Inc. The total reaction volume of the
topoisomerase II-mediated cleavage reaction was fixed
at 20 lL. Briefly, assay buffer (50 mM Tris–HCl, pH
1
mp 195–196 ꢁC;
J = 4.8 Hz, 1H), 8.55 (d, J = 8.0 Hz, 1H), 8.16 (d,
H NMR (CDCl3) d 9.07 (d,
J = 8.4 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.20 (s, 1H),
8, 120 mM KCl, 10 mM MgCl , 0.5 mM ATP, 0.5 mM
2
7
1
.16–7.18 (m, 1H), 4.14–4.19 (m, 1H), 3.97–4.06 (m,
H), 3.07–3.17 (m, 1H), 2.44 (s, 6H), 1.23 (d,
dithiothreitol, 30 lg/mL bovine serum albumin (BSA))
containing 200 ng KDNA (TopoGEN) and a solution
of the test drugs were added to one unit of the human
recombinant topoisomerase II (the amount of enzyme
which resulted in the complete decatenation of 200 ng
KDNA). After 10 min of incubation at 37 ꢁC, the reac-
tion was stopped by addition of 5 lL of stop buffer con-
taining the loading dye (1% sarkosyl, 0.025%
bromophenol blue, and 5% glycerol), and then the reac-
tion mixture was analyzed on a 1% agarose gel by run-
ning at 40 V for 3.5 h in TBE buffer (89 mM Tris,
89 mM borate, and 2 mM Na–EDTA, pH 8.3). Gels
were stained with SYBR Green I (Molecular Probes,
Eugene, OR) and observed under UV illumination. Gels
were photographed, and remaining KDNA from photo-
graphic negatives was scanned using AlphaImager 2200
(AlphaEase version 5.5). The inhibition of topo II was
calculated from the equation: % Inhibition = (Intensity
of sample-treated DNA/Intensity of vehicle-treated con-
trol DNA) · 100.
J = 6.8 Hz, 3H); HR-FABMS calcd for C H N O
4
2
0
18
2
+
M+H) : 351.1345, found: 351.1338.
(
4.2.19. 8-(2-(Diethylamino)ethoxy)-7-chlorobenzofuro[3,2-
g]quinoline-5,11-dione (8m). Yellow brown solid
(48.5%); mp 198–199 ꢁC; H NMR (CDCl ) d 9.09 (d,
J = 4.8 Hz, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.30 (s, 1H),
1
3
7.74 (t, J = 6.2 Hz, 1H), 7.31 (s, 1H), 4.76–4.84 (m,
2H), 3.56–3.64 (m, 2H), 3.27–3.48 (m, 4H), 1.51 (t,
J = 7.2 Hz, 6H); HR-FABMS calcd for C H ClN O
4
2
1
19
2
+
M+H) : 399.1112, found: 399.1148.
(
4.2.20. 8-(2-(Diisopropylamino)ethoxy)-7-chlorobenzof-
uro[3,2-g]quinoline-5,11-dione (8n). Red brown solid
1
(
89.2%); mp 215–216 ꢁC; H NMR (CDCl ) d 9.08 (d,
3
J = 4.0 Hz, 1H), 8.56 (d, J = 8.0 Hz, 1H), 8.26 (s, 1H),
.73 (t, J = 6.0 Hz, 1H), 7.40 (s, 1H), 4.96 (t,
J = 4.4 Hz, 2H), 3.85–3.96 (m, 2H), 3.56 (t, J = 5.2 Hz,
H), 1.63 (d, J = 6.4 Hz, 6H), 1.56 (d, J = 6.4 Hz, 6H);
7
2
+
17
HR-FABMS calcd for C H ClN O
4
(M+H) :
4.4. Relaxation assay for topoisomerase I activity
2
3
23
2
4
27.1425, Found: 427.1397.
For the measurement of topoisomerase I catalytic activ-
ity, an assay was done using supercoiled pHOT1 DNA
as a substrate according to the protocol provided by
TopoGEN, Inc. (Columbus, USA). Supercoiled (Form
I) plasmid substrate DNA was used in a reaction volume
of 20 lL containing the following: 10 mM Tris–HCl, pH
7.9, 1 mM ethylene diamine tetraacetic acid (EDTA),
150 mM NaCl, 0.1% bovine serum albumin (BSA),
0.1 mM spermidine, 5% glycerol, and 1 U of purified
4.2.21. 8-(1-(Dimethylamino)propan-2-yloxy)-7-chloro-
benzofuro[3,2-g]quinoline-5,11-dione (8o). Orange red
1
solid (31.0%); mp 194–195 ꢁC; H NMR (CDCl ) d
3
9
(
.07 (d, J = 4.4 Hz, 1H), 8.56 (d, J = 8.0 Hz, 1H), 8.28
s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.35 (s, 1H), 4.66–
4
2
.79 (m, 1H), 2.78–2.88 (m, 1H), 2.60–2.70 (m, 1H),
.41 (s, 6H), 1.44 (d, J = 6.0 Hz, 3H); HR-FABMS calcd
+
for C H ClN O (M+H) : 385.0955, found: 385.0984.
17
2
0
2
4

1658
H.-K. Rhee et al. / Bioorg. Med. Chem. 15 (2007) 1651–1658
human topoisomerase I (TopoGEN, Inc.). The appro-
priate inhibitor was added as indicated, and the reaction
was started by the addition of the enzyme. After 10 min
of incubation at 37 ꢁC, the reaction was stopped by
addition of 5 lL of stop buffer containing the loading
dye (1% sarkosyl, 0.025% bromophenol blue, and 5%
glycerol), and then the reaction mixture was analyzed
on a 1% agarose gel by running at 40 V for 3.5 h in
TBE buffer (89 mM Tris, 89 mM borate, and 2 mM
Na–EDTA, pH 8.3). Gels were stained with SYBR
Green I (Molecular Probes, Eugene, OR) and observed
under UV illumination. For the quantitative determina-
tion of topo I activity, photographic negatives were den-
sitometrically scanned using AlphaImager 2200
3. Garbett, N. C.; Graves, D. E. Curr. Med. Chem: Anti-
Cancer Agents 2004, 4, 149.
4
. Cheng, C.-C.; Dong, Q.; Liu, D.-F.; Luo, Y.-l.; Liu, L. F.;
Chen, A. Y.; Yu, C.; Savaraj, N.; Chou, T.-C. J. Med.
Chem. 1993, 36, 4108.
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. Pindur, U.; Haber, M.; Sattler, K. J. Chem. Educ. 1993,
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0, 263.
. Baldwin, E. L.; Osheroff, N. Curr. Med. Chem: Anti-
Cancer Agents 2005, 5, 363.
6
7. Ji, Z.; Wang, H.-K.; Bastow, K.-H.; Zhu, X.-K.; Cho, S.
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9
(
AlphaEase version 5.5). The inhibition of topo I was
1
1
1
0. Temiz-Arpaci, O.; Tekiner-Gulbas, B.; Yldiz, I.; Aki-
Sener, E.; Yalcin, I. Bioorg. Med. Chem. 2005, 13, 6354.
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of sample-treated DNA/Intensity of vehicle-treated con-
trol DNA) · 100.
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986, 29, 1329.
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Acknowledgment
7
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This work was supported from the Korea Research
Foundation (KRF-2005-005-J01501).
1
1
1
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