Novel angular furoquinolinones bearing flexible chain as antitumor agent: Design, synthesis, cytotoxic evaluation, and DNA-binding studies

Article abstract of DOI:10.1016/j.bmc.2008.07.081

A series of novel N-substituted angular furoquinolinone derivatives were synthesized and evaluated for their antitumor activities against QGY, K562, HeLa, P388, and A549 cell lines in vitro. The derivatives bearing basic amino side chain showed an improved antitumor activity. Compound 5h N-(2-dimethylamino-ethyl)-2-(4,8,9-trimethyl-2-oxo-2H-furo[2,3-h]quinolin-1-yl)-acetamide exhibited the highest activities against P388 and A549 cell lines, which are evidenced by the IC₅₀ values that are four to five fold lower than that for unsubstituted parent compound. DNA-binding experiments suggested that these derivatives bind to DNA through intercalation.

Full text of DOI:10.1016/j.bmc.2008.07.081

Bioorganic & Medicinal Chemistry 16 (2008) 8713–8718
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel angular furoquinolinones bearing flexible chain as antitumor agent:
Design, synthesis, cytotoxic evaluation, and DNA-binding studies
a,
a
a
c
a
Lijuan Xie a, Xuhong Qian a,b, , Jingnan Cui , Yi Xiao , Kewei Wang , Peichun Wu , Liying Cong
*
*
a State Key Laboratory of Fine Chemicals, Dalian University of Technology, PO Box 89, Zhongshan Road 158, Dalian 116012, China
b Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, PO Box 544, 130 Meilong Road, Shanghai 200237, China
c Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel N-substituted angular furoquinolinone derivatives were synthesized and evaluated for
their antitumor activities against QGY, K562, HeLa, P388, and A549 cell lines in vitro. The derivatives
bearing basic amino side chain showed an improved antitumor activity. Compound 5h N-(2-dimethyl-
amino-ethyl)-2-(4,8,9-trimethyl-2-oxo-2H-furo[2,3-h]quinolin-1-yl)-acetamide exhibited the highest
activities against P388 and A549 cell lines, which are evidenced by the IC50 values that are four to five
fold lower than that for unsubstituted parent compound. DNA-binding experiments suggested that these
derivatives bind to DNA through intercalation.
Received 5 June 2008
Revised 27 July 2008
Accepted 29 July 2008
Available online 3 August 2008
Keywords:
DNA-Intercalator
Antitumor
Ó 2008 Elsevier Ltd. All rights reserved.
Furoquinolinone
N-substituted furoquinolinone
1. Introduction
NMe2
DNA-intercalators containing a linear or angular planar chro-
mophore with a polyaromatic ring can influence the structures
and physiological functions of DNA.1–7 Some intercalators such as
furocoumarin, acridines, anthraquinones, naphthalimides, and
phenanthridine are used in cancer treatment.2,5,7,8 Angelicin (see
Fig. 1), an angular furocoumarin, has been used to treat pain in
the loins and knees,9 and skin diseases in phototherapy.10 Guiotto
et al. have synthesized a series of furoquinolinones,11–16 in which
the O atom of furocoumarin has been substituted by NH (see
Fig. 1), and some of them showed strong antiproliferative activity
against tumor cell lines upon UVA (Ultraviolet Radiation A) irradi-
ation. However, these furoquinolinones also revealed evident skin
phototoxicity and marked clastogenic activity due to the formation
of covalent monoadducts (MA) with DNA base and covalent DNA–
protein cross-links (DPC) upon UVA activation. In the dark, these
furoquinolinones exhibited weak antiproliferative activity with a
mechanism of action related to topoisomerase II inhibition. In con-
trast to normal cell, many tumor cells show high expression levels
of topoisomerase II, making this enzyme an ideal drug target.17–19
To enhance antitumor activity, generally the DNA-intercalator
should carry one or two flexible basic side chains on
chromophore.1–7,20–23 The flexible basic side chains are highly
influential in directing the thermodynamic-binding mechanism,
O
N
O
N
X
O
O
Me2N
N
O
H
NH2
Angelicin : X = O
Furoquinolinone : X = NH
DACA
Amonafide
Figure 1.
geometry of the ligand–DNA complex, and sequence selectivi-
ties.2,4,5,7,20–23 For example, the acridine does not have antitumor
activity, but the DACA (see Fig. 1),19 an acridine derivative with
a N-dimethylamino acetamide side chain, shows high activity
against solid tumours and has been used in clinic. Presence of
the dimethylamine or pyrrolidine side chain is essential for cyto-
toxic activity of the naphthalimide derivatives,2,24 such as in the
Amonafide (see Fig. 1). In an attempt to further explore effect of
side chain in furoquinolinone, we herein wish to report the
design, synthesis, and biological evaluation of a series of N-
substituted angular furoquinolinone derivatives with flexible side
chains. The introducing flexible amino chain could achieve high
activity enough to be used as antitumor agents even under no
irradiation. Thus, the side effects of phototherapy can be
neglected.
* Corresponding authors. Tel.: +86 21 64253589; fax: +86 21 64252603.
E-mail addresses: xhqian@ecust.edu.cn (X. Qian), jncui@dlut.edu.cn (J. Cui).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.081

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L. Xie et al. / Bioorg. Med. Chem. 16 (2008) 8713–8718
man liver cancer cells), K562 (human leukemia cells), and HeLa
(human cervicalcarcinoma cells) (Table 1) under no UVA irradiation.
2. Results and discussion
The IC50 represents the drug concentration (
cell growth by 50%.
lM) required to inhibit
2.1. Synthesis
These compounds with basic amino chain (5f, 5g, 5h, and 5j)
exhibited improved cytotoxicity compared with parent compound
5a and other non-amino chain compounds, especially the com-
pound 5h showed the highest activities against P388, A549, and
HeLa cell lines with IC50 values being four to five fold lower than
those for the 5a under the same experimental conditions. There-
fore, it is suggested that the N position of furoquinolinone is a cru-
cial point, the introduction of appropriate group to the position can
obviously improve the antitumor activity. The cytotoxicity of 5j
was weaker than that of 5f against HeLa cell. The possible reason
is that 5j, as quaternized product of 5f, has more difficulties to pen-
etrate the cell membrane. Moreover, 5j can not form hydrogen
bonding with DNA bases. Compounds 5f, 5g, and 5h exhibited anti-
tumor activities, but 5e and 5i were inactive. The nitrogen atom at
amino chain of 5f, 5g, and 5h could form hydrogen bonding with
DNA bases, which may contribute to the biological activity.28,29
The synthetic route of the target compounds is described in
Scheme 1. According to the literature,25 the intermediate
3
(4-methyl-7-hydroxyquinolin-2-one) could be prepared from
m-phenylenediamine and ethyl acetoacetate by three steps
including Pechmann condensation reaction, diazotization, and
hydrolysis. Compound 3 reacted with 3-chloro-2-butanone to give
intermediate 4 (4-methyl-7-[20-(30-oxo)butyloxy]quinolin-2-one).
Compound 5a was obtained by the cyclization of 4 with PPA
(polyphosphoric acid). An excess of halogenoalkane was added
dropwise into a mixture of 5a, NaH, and KI in dry DMF in the ice bath
to yield the target products 5b–j. Noticeably, the application of PPA
toformfuran ringin thereactiongreatly reducedthereactiontimeto
4–6 h and increased the yield to 62% compared with 40–60 h and
47% reported before.14 In addition, the cyclization of furan ring using
PPA has been rarely reported.
2.2. Cytotoxic evaluation in vitro and in the dark
2.3. DNA-binding studies
The in vitro antitumor activities of these compounds were evalu-
ated by examining their cytotoxic effects using sulforhodamine B
(SRB) assay26 against A549 (human lung cancer cells) and MTT tetra-
zolium dye assay27 against P388 (murine leukemia cells), QGY (hu-
It has been pointed out that only a combination of selected
methods provides sufficient information to determine the binding
mode.3–7,30,31 In order to reveal the influence of introducing side
NH2
1) NaNO2,HCl, -5-0 oC
CH3COCH2COOC2H5
150 oC, 48 h
2) 10M H2SO4, reflux 10min
HO
N
O
H2N
N
O
NH2
H
H
1
2
3
O
O
CH3COCHClCH3, K2CO3
CH3COCH3, reflux, 40 h
PPA
100-120 oC, 4-6 h
NaH, RX
DMF, 50 oC
O
N
R
O
N
O
O
N
O
H
H
4
5a
5b-j
5a: R = H
5d: R = CH2C6H5
5e: R = CH2CO2C2H5
5f: R= C2H4NMe2
5g: R = C3H6NMe2
5j: R = C2H4NMe3+I-
5b: R = C2H5
5c: R = CH2CN
5h: R = CH2CONH(CH2)2NMe2
5i: R = CH2CO2H
Scheme 1.
Table 1
Cytotoxic evaluation against QGY, K562, HeLa, P388, and A549 cell lines under no irradiation and Scatchard-binding constants
Compound
Cytotoxicity (IC50
HeLaa
,
l
M)
Kb (105 Mꢀ1
c
)
QGYa
K562a
P388a
A549b
5a
5b
5c
5d
5e
5f
5g
5h
5i
98.94 8.52
>100
>100
>100
>100
>100
>100
>100
>100
89.25 9.31
>100
>100
>100
>100
39.09 6.04
35 1.67
20.7 2.72
>100
>100
>100
>100
>100
99.29 10.90
>100
>100
>100
>100
2.57 0.09
2.79 0.08
2.06 0.09
3.15 0.02
2.54 0.07
2.66 0.06
2.53 0.10
1.58 0.07
2.69 0.05
3.18 0.06
>100
>100
72.68 9.81
74.87 6.45
NDd
55.73 5.37
71.09 7.91
NDd
51.54 4.92
45.32 3.25
14.45 2.12
NDd
30.40 4.57
29.62 3.81
20.45 2.13
NDd
NDd
NDd
5j
NDd
NDd
49.52 7.06
NDd
NDd
a
CTX (cytotoxicity) against tumor cells (P388, QGY, K562, and HeLa) was measured by microculture tetrazolium–formazan method.
CTX against human lung cancer cell (A549) was measured by sulforhodamine B dye-staining method.
Kb: Scatchard-binding constants
ND, not determined.
b
c
d

L. Xie et al. / Bioorg. Med. Chem. 16 (2008) 8713–8718
8715
chain on mechanism of antitumor action of these furoquinolinone
0.20
0.15
0.10
0.05
0.00
derivatives, the DNA-binding studies were investigated by spectro-
scopic techniques, electrophoresis and viscosity measurement.
The Kb (Scatchard-binding constants) were calculated according
to the fluorescence quenching technique (Table 1).32 Typical-bind-
ing constant between organic compound and DNA usually ranges
from 104–106 Mꢀ1, and these compounds are actually moderate
DNA-intercalators. In most cases, compounds strongly binding to
DNA are high cytotoxic agent. However, the results (Table 1) indi-
cated that there is no obvious relationship between DNA-binding
and cytotoxicity. The fluorescence and UV–vis spectra of 5h are
shown in Figures 2 and 3, respectively. The hypochromicity
(300–340 nm) and isosbestic points were observed in the absorp-
tion titration spectrum of 5h, and no significant shift was observed
(maximum absorption peak at 301 nm). The hypochromism and
the appearance of isosbestic points are characteristics of intercala-
tive binding.33 It was found that the fluorescence of 5h was
quenched with CT-DNA (calf-thymus DNA) concentration increas-
ing as most of the intercalators did.
5h
5h:DNA=1:0.1
5h:DNA=1:0.3
5h:DNA=1:0.5
5h:DNA=1:1
5h:DNA=1:2
260 280 300 320 340 360 380 400
Wavelength (nm)
Figure 3. Absorption spectral changes of 5h (10 lM) in the presence and absence of
CT-DNA in Tris–HCl buffer (30 mM, pH 7.5).
CD (circular dichroism) is a very powerful technique to moni-
tor the conformational state of the DNA double helix in solution.
The CD spectrum of free CT-DNA exhibited a negative band at
247 nm due to the helicity, and a positive band at 279 nm due
to the base stacking, which is the characteristic of DNA in the
right-hand B form. The increases in the intensity both of the po-
sitive band and the negative band were observed (Fig. 4) when
the compounds were incubated with CT-DNA. The changes in
CD signals of DNA after adding 5f and 5h were consistent with
Scatchard-binding constants, 5f > 5h, which indicated that DNA-
binding ability of 5f is stronger than that of 5h. The appearance
of small negative ICD signal (300–310 nm) suggested that 5f
and 5h intercalated into DNA with its long axis parallel to the
base-pair long axis.34
Viscosity measurement is regarded as a reliable tool to deter-
mine the binding model in solution.3–7,30,31 Intercalation of a mol-
ecule into DNA results in a lengthening, unwinding, and stiffening
of the helix which increases the viscosity of the solution.30,31,33 The
increase in viscosity of DNA solution was observed versus the in-
crease in concentration of 5f and 5h (Fig. 5).
9
6
5f
5h
CT-DNA
3
0
-3
-6
-9
-12
-15
250
300
350
400
Wavelength (nm)
Figure 4. CD spectra of CT-DNA in the absence and presence of 5f and 5h at
concentration of DNA 100
buffer (pH 7.0).
l
M, the concentration of 5f or 5h is 10 lM, in Tris–HCl
The gel mobility assay is sensitive to changes in DNA length or
conformation, since the electrophoretic mobility of nucleic acid is
proportional to the length of the nucleic acid molecules.33 Because
furoquinolinone could induce damage to DNA upon irradiation, the
5h
5f
1.25
1.20
1.15
1.10
1.05
1.00
160
140
4
4-CT
3
120
2
3-CT
100
2-CT
1
1-CT
80
60
40
20
0
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
[compound]/[DNA]
Figure 5. Effect of increasing amounts of compounds 5f (j) and 5h (N) on the
relative viscosities of CT-DNA at 25 ( 0.1) °C. [DNA] = 100 lM in Tris–HCl (30 mM,
pH 7.5).
g .
is the viscosity of DNA in the presence of the compounds and g0
350
400
450
500
550
cleavages to DNA were investigated using furoquinolinone deriva-
tives under dark and irradiation. No DNA cleavages by compounds
5a, 5f, 5g, and 5h in the absence of irradiation (Fig. 6), indicated
that these compounds have no ability to modify tertiary DNA
structure in the dark. The compounds 5a, 5f, and 5h caused signif-
Wavelength (nm)
Figure 2. Fluorescence spectrum before and after interaction of 5h and CT-DNA in
Tris–HCl buffer (30 mM, pH 7.5). Numbers 1–4 indicat the concentration of 5h, 10,
25, 50, and 100 lM, respectively. DNA applied was 50 lM (bp).

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L. Xie et al. / Bioorg. Med. Chem. 16 (2008) 8713–8718
4.1. Synthesis
4.1.1. 4, 8, 9-Trimethylfuro [2,3-h]quinolin-2(1H)-one (5a)14
Compound 4 (4.9 g, 20 mmol) was mixed with PPA (20 mL) and
stirred at 120–140 °C for 5 h, then cooled to room temperature,
washed with water, filtered and dried. The crude product was puri-
fied by chromatography on silica gel (CH2Cl2/C2H5OH = 40:1, v/v)
to get 5a as a white solid in 62% yield, mp 230–232 °C (reported
233 °C). 1H NMR (400 MHz CDCl3) d (ppm) 8.94 (s, 1H) 7.49 (d,
1H, J = 8.8 Hz) 7.28–7.25 (m, 1H) 6.48 (s, 1H) 2.53 (s, 3H) 2.49 (s,
3H) 2.42 (s, 3H); 13C NMR (100 MHz CDCl3) d (ppm) 162.2, 155.0,
151.1, 150.3, 132.6, 120.2, 118.1, 115.7, 115.3, 108.5, 106.8, 20.2
11.6, 10.4; IR (KBr, cmꢀ1) 3255, 2952, 1653, 841.
Figure 6. The cleavage of plasmid pBR322 DNA (25 ng/lL) by compounds 5a, 5f, 5g,
and 5h at various concentration in the buffer of Tris–HCl (20 mM, pH 7.5) at 25 °C
for 12 h under no irradiation. Lane 1, DNA marker; lanes 2, DNA alone (no
irradiation), lanes 3–5, DNA + 5a; lanes 6–8, DNA + 5f; lanes 9–11, DNA + 5g; lanes
12–14, DNA + 5h.
4.1.2. General procedure for N-substituted furoquinolinone
derivatives (5b–5j)
Compound 5a (114 mg, 0.5 mmol), NaH (100 mg, 4.17 mmol)
and KI (100 mg, 0.6 mmol) in dry DMF (10 mL) were mixed in ice
bath and under nitrogen. After stirring for 15 min, a solution of
halogenoalkanes (3 mmol) in dry DMF (10 mL), was added
dropwise for over a period of 30 min, and the reaction mixture
was stirred for 2 h at 0 °C and for 48 h at 50–60 °C under nitrogen
(TLC detection). The solvent was removed under reduced pressure.
The residue was purified by chromatography on silica gel with a
solution of CH2Cl2 and EtOH as eluent to give desired product.
4.1.3. 1-Ethyl-4,8,9-trimethyl-1H-furo[2,3-h]quinolin-2-one (5b)
Compound 5a (114 mg, 0.5 mmol) and iodoethane (0.24 mL,
3.0 mmol) were reacted according to the general procedure and
gave 5b after chromatography (CH2Cl2/C2H5OH = 50:1, v/v), in
45% yield as a white solid, mp 190–191.8 °C; 1H NMR (400 MHz
CDCl3) d (ppm) 7.64 (d, 1H, J = 9.2 Hz) 7.43 (d, 1H, J = 9.2 Hz) 6.70
(s, 1H) 4.58–4.56 (m, 2H) 2.63 (d, 3H, J = 7.6 Hz) 2.45 (s, 3H) 2.18
(s, 3H) 1.47 (t, 3H, J = 6.8 Hz); 13C NMR (100 MHz CDCl3) d (ppm)
161.5, 153.9 149.7, 147.0, 142.5, 124.1, 121.0, 118.7, 112.3, 110.5,
108.8, 61.4, 19.6, 14.7, 11.7, 10.4; IR (KBr, cmꢀ1) 3239, 2978,
1648, 840, 785; TOF MS (EI+) calcd C16H17NO2 255.0926; found:
255.0929.
Figure 7. The photocleavage of pBR322 DNA (25 ng/lL) by compound 5a, 5f, and
5h at various concentration in the buffer of Tris–HCl (20 mM, pH 7.5) upon
irradiation for 2 h. Lane1, DNA marker; lane 2, DNA alone (no irradiation); lane 3,
DNA alone (irradiation); lanes 4,-5, DNA + 5a; lanes 6–8, DNA + 5f; lanes 9–11,
DNA + 5h.
icant cleavages to DNA (form II- and form III-linear) (Fig. 7) upon
irradiation, which showed that these compounds are photoactive
depending on concentration.
3. Conclusions
4.1.4. (4,8,9-Trimethyl-2-oxo-2H-furo[2,3-h]quinolin-1-yl)-
acetonitrile (5c)
In summary, we developed a series of novel DNA-intercalators
based on furoquinolinone system. The introduction of amino side
chain is an effective way to improve the antitumor activity of
DNA-intercalator containing planar chromophore. Further studies
on action mechanism of furoquinolinone and their structural mod-
ification are currently under way. This research may provide some
new suggestions for the design of novel antitumor agent based on
furoquinolinone.
Compound 5a (114 mg, 0.5 mmol) and bromoacetonitrile
(0.21 mL, 3 mmol) were reacted according to the general procedure
and gave 5c after chromatography (CH2Cl2/C2H5OH = 50:1, v/v), in
38% yield as a white solid, mp 193.2–194.8 °C; 1H NMR (400 MHz
CDCl3) d (ppm) 7.68 (d, 1H, J = 8.8 Hz) 7.27 (d, 1H, J = 8.8 Hz) 6.79
(s, 1H) 5.15 (s, 2H) 2.69 (s, 3H) 2.63 (s, 3H) 2.47 (s, 3H). 13C
NMR (100 MHz CDCl3) d (ppm) 158.7, 154.1, 150.4, 148.7, 141.4,
124.4, 121.9, 118.7, 116.2, 112.3, 110.2, 109.3, 49.9, 19.7, 11.8,
10.4; IR (KBr, cmꢀ1) 2952, 2361, 1617; TOF MS (EI+) calcd for
4. Experimental
C16H14N2O2 266.1055; found: 266.1054.
All the solvents are of analytic grade. 1H NMR and 13C NMR
spectra were measured on a Bruker AV-400 spectrometer with
chemical shifts reported in ppm (in CDCl3, TMS as internal stan-
dard). Melting points were determined by using an X-6 micro-
melting point apparatus and are uncorrected. Column
chromatography was performed using silica gel 200–300 mesh.
IR spectra were obtained using a Nicolet 470 FT-IR instrument.
High-resolution mass spectra were obtained on a Micromass com-
pany GCT CA156 (TOF MS) spectrometer and Micromass LCT (ESI)
spectrometer.
4.1.5. 1-Benzyl-4,8,9-trimethyl-1H-furo[2,3-h]quinolin-2-one
(5d)
Compound 5a (114 mg, 0.5 mmol) and benzyl bromide
(0.36 mL, 3 mmol) were reacted according to the general procedure
and gave 5d after chromatography (CH2Cl2/C2H5OH = 40:1, v/v) in
62% yield as
a
white solid, mp 192.3–194.1 °C; 1H NMR
(400 MHz CDCl3) d (ppm) 7.65 (d, 1H, J = 8.8 Hz) 7.51 (d, 2H,
J = 7.2 Hz) 7.45 (d, 1H, J = 8.8 Hz) 7.39 (t, 2H, J = 7.2 Hz) 7.33–7.31
(m, 1H) 6.79 (s, 1H) 5.60 (s, 2H) 2.65 (s, 3H) 2.62 (s, 3H) 2.45 (s,
3H); 13C NMR (100 MHz CDCl3) d (ppm) 161.2, 154.0, 149.8,
147.3, 142.3, 137.8, 128.4, 127.9, 127.7, 124.2, 121.3, 118.7,
112.3, 110.5, 109.1, 67.2, 19.6, 11.7, 10.5; IR (KBr, cmꢀ1) 3039,
2952, 2921, 1601; TOF MS (EI+) calcd for C21H19NO2 317.1416;
found: 317.1411.
The intermediate 3 was prepared according to the published
methods25 and the 4 was prepared according to the literature
method.13 The structures of these compounds were characterized
by 1H NMR and electron ionization mass spectra.

L. Xie et al. / Bioorg. Med. Chem. 16 (2008) 8713–8718
8717
4.1.6. (4,8,9-Trimethyl-2-oxo-2H-furo[2,3-h]quinolin-1-yl)-
acetic acid ethyl ester (5e)
(d, 1H, J = 8.8 Hz) 6.88 (s, 1H) 5.11 (s, 2H) 2.70 (s, 3H) 2.53 (s,
3H) 2.41 (s, 3H); 13C NMR (100 MHz CDCl3) d (ppm) 160.1, 154.1,
150.2, 148.6, 141.6, 124.1, 121.8, 118.8, 112.2, 109.9, 109.8, 62.6,
19.7, 11.6, 10.3; IR (KBr, cmꢀ1) 3409, 2998, 1607, 1339; TOF MS
(EI+) calcd for C16H15NO4 285.1001; found: 285.1001.
Compound 5a (114 mg, 0.5 mmol) and ethyl bromoacetate
(0.33 mL, 3 mmol) were reacted according to the general procedure
and gave 5e after chromatography (CH2Cl2/C2H5OH = 50:1, v/v), in
51.4% yield as a white solid, mp 92.5–93.4 °C; 1H NMR (400 MHz
CDCl3) d (ppm) 7.65 (d, 1H, J = 8.8 Hz) 7.46 (d, 1H, J = 8.8 Hz) 6.86
(s, 1H) 5.05 (s, 2H) 4.25–4.23 (m, 2H) 2.67 (s, 3H) 2.55 (s, 3H) 2.44
(s, 3H) 1.26 (t, 3H, J = 6.8 Hz); 13C NMR (100 MHz CDCl3) d (ppm)
169.6, 160.1, 154.0, 149.9, 147.9, 141.9, 124.1, 121.6, 118.8, 112.3,
110.0, 109.5, 62.4, 61.0, 19.7, 14.2, 11.7, 10.3; IR (KBr, cmꢀ1) 2973,
2755, 2612, 1463, 1350; TOF MS (EI+) calcd for C18H19NO4
313.1314; found: 313.1312.
4.1.11. Trimethyl-[2-(4,8,9-trimethyl-2-oxo-2H-furo[2,3-
h]quinolin-1-yl)-ethyl]ammonium iodide (5j)
Compound 5f (149 mg, 0.5 mmol) and excess of 8–10 times iodo-
methane (0.50–0.62 mL, 4–5 mmol) were dissolved in EtOH (10 mL)
and stirred at 30–40 °C under nitrogen for 24 h. The solvent was re-
moved under reduced pressure, the residue was purified by chroma-
tography on silica gel (CH2Cl2/C2H5OH = 15:1, v/v) to give target
product in 38% yield as a white solid, mp 239.1–240.5 °C; 1H NMR
(400 MHz CDCl3) d (ppm) 7.66 (d, 1H, J = 8.8 Hz) 7.51 (d, 1H,
J = 8.8 Hz) 6.79 (s, 1H) 5.06 (s, 2H) 4.30 (s, 2H) 3.64 (s, 9H) 2.69 (s,
3H) 2.60 (s, 3H) 2.46 (s, 3H); 13C NMR (100 MHz CDCl3) d (ppm)
159.2, 154.3, 150.7, 121.7, 118.7, 112.0, 110.3, 109.5, 55.2, 19.7,
11.8, 11.0; IR (KBr, cmꢀ1) 3378, 2921, 1735; TOF MS (EI+) calcd for
4.1.7. 1-(2-Dimethylamino-ethyl)-4,8,9-trimethyl-1H-furo[2,3-
h]quinolin-2-one (5f)
Compound 5a (114 mg, 0.5 mmol) and 2-chloro-N,N-dimethyle-
thanamine (323 mg, 3 mmol) were reacted according to the general
procedure and gave 5f after chromatography (CH2Cl2/C2H5OH =
30:1, v/v), in 67% yield as a white solid, mp 197.4–199.2 °C; 1H
NMR (400 MHz CDCl3) d (ppm) 7.67 (d, 1H, J = 8.8 Hz) 7.50 (d, 1H,
J = 8.8 Hz) 6.80 (s, 1H) 5.05 (t, 2H, J = 4.8 Hz) 3.61 (t, 2H, J = 4.8 Hz)
2.94 (s, 6H) 2.68 (s, 3H) 2.58 (s, 3H) 2.46 (s, 3H). 13C NMR
(100 MHz CDCl3) d (ppm) 159.6, 154.1, 150.4, 148.5, 141.8, 124.1,
121.6, 118.8, 112.0, 109.9, 59.3, 56.5, 43.6, 19.7, 11.8, 10.7; IR (KBr,
cmꢀ1) 3291, 3245, 2942, 1606; TOF MS (EI+) calcd for C18H22N2O2
298.1681; found: 298.1674.
C
19H25N2O2I 227.0946; found: 227.0946; HMRS (ESI) m/z (M+H)+
calcd for C19H25N2O2I 313.1916; found: 313.1911.
4.2. Cytotoxic evaluation in vitro
The prepared compounds were submitted to Shanghai Institute
of Materia Medica Chinese Academy of Sciences and Dalian insti-
tute of chemical physics Chinese Academy of Sciences to test their
cytotoxicities. Growth inhibitory effect on the cell lines (P388,
QGY, K562, and HeLa) was measured by using the MTT assay.27
For A-549 cell lines, the growth inhibition was tested by the sulfo-
rhodamine B (SRB) assay.26
4.1.8. 1-(3-Dimethylamino-propyl)-4,8,9-trimethyl-1H-
furo[2,3-h]quinolin-2-one (5g)
Compound 5a (114 mg, 0.5 mmol) and 3-chloro-N,N-dimethyl-
propan-1-amine (364.83 mg, 3 mmol) were reacted according to
the general procedure and gave 5g after chromatography (CH2Cl2/
C2H5OH = 30:1, v/v), in 62% yield as a white solid, mp 198.2–
200 °C; 1H NMR (400 MHz CDCl3) d (ppm) 7.64 (d, 1H, J = 8.8 Hz)
7.47 (d, 1H, J = 8.8 Hz) 6.70 (s, 1H) 4.64 (t, 2H, J = 5.6 Hz) 3.36 (t,
2H, J = 7.6 Hz) 2.86 (s, 6H) 2.66 (s, 3H) 2.59 (s, 3H) 2.53 (br, 2H)
2.45 (s, 3H). 13C NMR (100 MHz CDCl3) d (ppm) 160.6, 154.0, 150.2,
147.8, 142.1, 124.1, 121.3, 118.7, 112.1, 109.9, 109.5, 62.0, 56.2,
43.2, 43.1, 24.1, 19.7, 11.8, 10.6; IR (KBr, cmꢀ1) 3409, 3003, 2936,
1596; TOF MS (EI+) calcd for C19H24N2O2 312.1838; found: 312.1844.
4.3. DNA-binding studies
UV–vis absorption spectra were recorded on a PGENERAL TU-
1901 UV–vis spectrophotometer and fluorescent spectra were
measured on a Hitachi F-4500 luminescence spectrophotometer.
Calf-thymus DNA was purchased from the Sino-American Biotech-
nology Company. Solutions of CT-DNA in Tris–HCl buffer (30 mM,
pH 7.5) gave a ratio of UV absorbance at 260 and 280 nm of 1.8–
1.9:1, indicating that the DNA was sufficiently free from protein.
The concentration of calf-thymus DNA was determined by its
absorption intensity at 260 nm with a known molar absorption
4.1.9. N-(2-Dimethylamino-ethyl)-2-(4,8,9-trimethyl-2-oxo-2H-
furo[2,3-h]quinolin-1-yl)-acetamide (5h)
coefficient value of 6600 Mꢀ1 cmꢀ1
.
Compound 5e (156.5 mg, 0.5 mmol) was dissolved in N,N-di-
methyl ethylenediamine (10 mL), the mixture was refluxed under
nitrogen for 24 h. The solvent was removed under reduced pres-
sure, the residue was purified by chromatography on silica gel
(CH2Cl2/C2H5OH = 25:1, v/v) to give title product in 51% yield as a
white solid, mp 200.2–201 °C; 1H NMR (400 MHz CDCl3) d (ppm)
7.65 (d, 1H, J = 8.8 Hz) 7.48 (d, 1H, J = 8.8 Hz) 6.82 (s, 1H) 5.03 (s,
2H) 3.43–3.39 (m, 2H) 2.69 (s, 3H) 2.59 (s, 3H) 2.45 (s, 3H) 2.41
(t, 2H, J = 6.0 Hz) 2.15 (s, 6H); 13C NMR (100 MHz CDCl3) d (ppm)
169.1, 159.7, 154.0, 150.1, 148.0, 142.0, 125.0, 124.3, 121.5,
118.6, 112.5, 109.8, 109.7, 64.7, 57.8, 45.0, 36.3, 29.4, 19.7, 11.7,
10.5; IR (KBr, cmꢀ1) 3286, 3101, 1653, 1606; HMRS (ESI) m/z
(M + H)+ calcd for C20H25N3O3 356.1974; found: 356.1956.
4.3.1. Fluorescence spectrum study
The two groups of samples for experiments were prepared, one
at a constant DNA concentration of 50
lM and at compound con-
centration ranging from 1 to 10 M in Tris–HCl (30 mM, pH 7.5),
l
and the other having the same concentration of compound but ab-
sence of DNA as control. All the above solutions were ultrasonically
shaken for 1 day at 25 °C in the dark. Fluorescence wavelength and
intensity area of samples were measured.
4.3.2. UV–vis absorption spectra study
The titration absorption spectra studies were performed by
keeping constant the concentration of compound, while varying
the DNA concentration at room temperature. Initially, solutions
of the blank buffer were placed in the reference and sample cuv-
ettes (1 cm path length), respectively, and then the first spectrum
was recorded in the range 200–400 nm. During the titration, ali-
quots of buffered DNA solution were added and the solutions were
mixed by repeated inversion. After mixing for 10 min, the absorp-
tion spectra were recorded. The titration processes were repeated
until there was no change in the spectra for at least four titrations
indicating binding saturation had been achieved.
4.1.10. (4,8,9-Trimethyl-2-oxo-2H-furo[2,3-h]quinolin-1-yl)-
acetic acid (5i)
Compound 5e (156.5 mg, 0.5 mmol) was mixed with aqueous
NaOH (10 mL, 20%), the mixture was refluxed for 30 h. After con-
centration, cooling and acidification with concd HCl, the residue
was collected and crystallized from EtOH to give target compound
in 35% yield as a white solid, mp 208.7–209.6 °C; 1H NMR
(400 MHz CDCl3) d (ppm) 8.04 (s, 1H) 7.68 (d, 1H, J = 8.8 Hz) 7.50

8718
L. Xie et al. / Bioorg. Med. Chem. 16 (2008) 8713–8718
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spectrophotometer (Jasco, Japan) using a 1-cm path quartz cell and
subtracted from the spectrum of Tris–HCl buffer alone. The CD
spectra were recorded at the compound concentration of 10 lM
and DNA concentration of 100 lM, in the region 200–600 nm.
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4.3.4. Viscometry studies
Calf-thymus DNA was dissolved in Tris–HCl buffer (30 mM, pH
7.5) and left at 4 °C overnight. It was treated in an ultrasonic bath
for 10 min, and the solution was filtered through a PVDF mem-
brane filter (pore size of 0.45
the DNA samples concentration is 100
ments were carried out using an Ubbelodhe viscometer maintained
at a constant temperature at 25 ( 0.1) °C in a thermostated bath.
Flow times were measured with a digital stopwatch and each sam-
ple was measured three times and an average flow time was calcu-
lated. Data are presented as (
concentration of compounds to that of DNA, where
l
m) to remove insoluble material,
l
M.35 Viscosity measure-
g/
g0 1/3
)
versus the ratio of the
is the viscos-
0 is the viscosity
g
ity of DNA in the presence of the compound and
of DNA in the absence of the compound.33,36
g
23. Yin, H.; Xu, Y.; Qian, X. Bioorg. Med. Chem. 2007, 15, 1356.
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4.3.5. DNA cleavage studies
The solution containing pBR322 DNA (form I, 250 ng, 1
compound in DMSO (1 L) was diluted by Tris–HCl buffer (pH 7.5,
20 mM) to 10 L, then irradiated for 2 h with light (300 nm) using
lL) and
l
l
lamp (50 W High Pressure Mercury Lamp) placed at 20 cm from
the sample. The samples were analyzed by gel electrophoresis in
1% agarose gel, which was stained with ethidium bromide. Super-
coiled DNA runs at position I, nicked DNA at position II, and linear
DNA at position III. Experiments were repeated in triplicate.
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