Novel acenaphthopyrazine derivatives as antitumor agents against MCF-7 cells: Molecular synthesis, optical properties, and DNA-binding studies

Article abstract of DOI:10.1007/s00706-011-0653-9

A series of novel acenaphthopyrazine derivatives was synthesized from acenaphthylene-1,2-dione via three steps, including bromination, cyclization, and SNAr^H reaction. These new compounds exhibited potential antiproliferative activity against MCF-7 cells in vitro, and 3-[2-(dimethylamino)ethylamino]acenaphtho[1,2-b]pyra-zine-8,9-dicarbonitrile exhibited the highest activity (IC50 = 4.60 μM). DNA-binding experiments suggested that these derivatives bind to DNA through intercalation with intrinsic binding constants K all above 10⁵ M^-1. Optical property studies indicated that these compounds have long emission wavelength (λ_em > 560 nm), high quantum yields in toluene (Φ_f = 0.59 for 3-(morpholin-4-yl)acenaphtho[1,2-b]pyrazine-8,9- dicarbonitrile), and large Stokes shift (ΔS > 130 nm).

Full text of DOI:10.1007/s00706-011-0653-9

Monatsh Chem (2012) 143:243–250
DOI 10.1007/s00706-011-0653-9
ORIGINAL PAPER
Novel acenaphthopyrazine derivatives as antitumor agents against
MCF-7 cells: molecular synthesis, optical properties,
and DNA-binding studies
•
Junling Xing Jingnan Cui Shuang Wang
•
•
•
Jin Gao Zhichao Zhang
Received: 19 March 2010 / Accepted: 3 September 2011 / Published online: 22 October 2011
Ó Springer-Verlag 2011
Abstract A series of novel acenaphthopyrazine deriva-
tives was synthesized from acenaphthylene-1,2-dione via
three steps, including bromination, cyclization, and SNArH
reaction. These new compounds exhibited potential
antiproliferative activity against MCF-7 cells in vitro, and
3-[2-(dimethylamino)ethylamino]acenaphtho[1,2-b]pyra-
Naphthalene, a famous chromophore, is often used to
design intercalators because of its various structural mod-
ifications. In 1973, Bran˜a et al. reported a series of
intercalators based on naphthalimide chromophore (Fig. 1,
A) [5]. Later, a great deal of work was done toward
modification of the chromophore in order to enhance the
cytotoxicity [6–17]. Recently, a series of new DNA target
molecules based on acenaphtho-heterocycle chromo-
phore (Fig. 1, B) was developed by our group [18]. The
biological evaluation of B indicated that 3-[2-(dimethyl-
amino)ethylamino]-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-
carboxylic acid was cytotoxic with an IC50 value of
16.5 lM against MCF-7 cells [18]. According to these
studies, it is considered that an ideal DNA-intercalator
having a planar and rigid chromophore, which consists of
three or four five- or six-membered rings, can easily
intercalate into DNA base pairs [19]. Because the degree of
electron deficiency in the chromophore affects the inter-
action between the intercalator and electron-rich bases in
DNA, the intensity and quantity of electron-withdrawing
groups on the chromophore could greatly influence the
intercalation ability. In addition, to enhance antitumor
activity, generally the DNA intercalator should carry one or
two flexible basic side chains on the chromophore [20–29].
Therefore, a series of new DNA intercalators was designed
on the basis of the above theories (Fig. 1, 1) and is
expected to have better antiproliferative ability against
MCF-7 cells.
zine-8,9-dicarbonitrile exhibited the highest activity (IC50
=
4.60 lM). DNA-binding experiments suggested that these
derivatives bind to DNA through intercalation with intrinsic
binding constants K all above 105 M-1. Optical property
studies indicated that these compounds have long emission
wavelength (kem [ 560 nm), high quantum yields in
toluene (Uf = 0.59 for 3-(morpholin-4-yl)acenaphtho[1,2-
b]pyrazine-8,9-dicarbonitrile), and large Stokes shift
(DS [ 130 nm).
Keywords Acenaphthopyrazine derivatives ꢀ
MCF-7 cells ꢀ Intercalator ꢀ DNA binding ꢀ Antitumor
Introduction
Breast cancer is the leading cancer affecting millions of
women worldwide and causes misery or death [1–3]. There
is a prevalent opinion that tumor is caused by an imbalance
between cell growth and death. Some of the antitumor
drugs, such as intercalators binding to DNA, result in
inhibition of cell growth or promotion of cell death [4].
However, the structure of fluorescent compounds having
both electron-withdrawing and electron-donating groups in
a conjugated planar chromophore is similar to that of the
DNA target molecule. Compounds A have large Stokes
shifts (about 75 nm) and emission wavelengths (about
530 nm) [30], whereas compounds B have smaller Stokes
shifts (about 10 nm) and longer emission wavelengths
J. Xing ꢀ J. Cui (&) ꢀ S. Wang ꢀ J. Gao ꢀ Z. Zhang
State Key Laboratory of Fine Chemicals,
Dalian University of Technology, PO Box 89,
Zhongshan Road 158, Dalian 116012, China
e-mail: jncui@dlut.edu.cn
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J. Xing et al.
contain terminal nitrogen in the substituted group; more-
over, 1a, 1b, and 1h showed potential activity, while 1d
showed no activity ([100 lM). The reasons could be that
the protonation of the terminal nitrogen in the experimental
situation greatly facilitates the interaction with the anionic
DNA polymer, which may contribute to the biological
activity. In addition, the terminal nitrogen of 1h linking to
benzene has a large steric hindrance to affect intercalation
ability.
N
NC
N
CN
N
O
CO2H
N
O
N
O
R
R
R
A
B
1
Fig. 1 Structures of some reported compounds (A and B) and novel
compounds 1
DNA-binding studies
(about 600 nm) because of their stronger electron-with-
drawing ability. Therefore, it is also expected that
compounds 1 might exhibit long wavelength emission
similar to compounds B and perhaps have potential appli-
cation as fluorescence sensors and photoelectricity
materials. Herein, we report the design, synthesis, cyto-
toxic evaluation against MCF-7 cells, DNA-binding
studies, and analysis of optical properties of the new ace-
naphthopyrazine derivatives, which contain two cyano
groups, four rigid planar rings, and aliphatic amino groups.
In order to confirm the DNA-binding properties of these
compounds, UV-vis absorption spectra, fluorescence
spectra, circular dichroism spectra (CD), and viscosity
titration were performed with calf-thymus DNA (CT-
DNA).
UV-vis absorption spectra studies
UV-vis absorption spectra are the most convenient and
common way to study how a compound binds to DNA.
Usually, hypochromicity and formation of isoabsorptive
point are symbols of a compound binding to DNA through
intercalation [22, 24, 25, 31, 32]. The intrinsic binding
constants K were calculated by the absorption spectrum
with the drippings of CT-DNA (Table 1) [33]. With
increasing concentration of CT-DNA, the hypochromicity
and isoabsorptive point could be found, indicating that
compounds 1 could bind to DNA (Fig. 2). Generally,
typical binding constants between organic compounds and
DNA range from 104 to 106 M-1, and these compounds are
actually moderate DNA intercalators. In most cases, com-
pounds strongly binding to DNA are highly cytotoxic
agents. Among compounds 1, the intrinsic binding constant
of 1a is the biggest (9.02 9 105), and the activity against
MCF-7 cells is the strongest. However, there is no obvious
relationship between cytotoxicity and DNA-binding ability
toward the others of compounds 1.
Results and discussion
Optical property studies
The optical properties were tested, and the UV-vis
absorption and fluorescence spectral data are listed in
Table 1. Although the maximal absorptions are similar to
those of compounds A, compounds 1 showed, as we
expected, long emission wavelength (kem [ 560 nm), large
Stokes shift (DS [ 130 nm), and high quantum yields in
toluene (Uf = 0.59 for 1 g). Because of fluorescence
quenching by PET (photo-induced electron transfer), the
quantum yields of 1a, 1b, 1d, and 1 h were lower than
those of others.
Cytotoxic evaluation
Fluorescence spectra studies
The in vitro cytotoxic potencies of the new compounds 1
against MCF-7 cells (human breast cancer cells) were
evaluated by MTT tetrazolium dye assay. The results are
summarized in Table 1 and compared with the activity of
B-a and B-h [18]. Compound B-h exhibited the strongest
cytotoxicities (15.03 lM) in the series of compounds B,
and compound 1h showed similar activity against the cells.
Among compounds 1, 1a showed the highest activity
(4.60 lM), which was 21-fold lower than that of B-a. The
results indicated that compounds with different chro-
mophores have different activities despite having the same
aliphatic amino groups. Compounds 1a, 1b, 1d, and 1h
Fluorescence spectra are an important method for studying
molecular binding to nucleic acid. Some compounds will
make the fluorescence enhance or quench after binding
DNA. In the absence of DNA, 1a can emit fluorescence in
Tris-HCl buffer at room temperature, with a maximum
emission wavelength at 640 nm. With the drippings of CT-
DNA, the emission intensities of 1a decreased, implying
that 1a can strongly interact with DNA (Fig. 3). The reason
for quenching might be energy or electron transfer between
1a and DNA base pairs to quench fluorescence [22, 24, 25,
34].
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Novel acenaphthopyrazine derivatives as antitumor agents against MCF-7 cells
245
Table 1 Spectral data, cytotoxicity (MCF-7), and intrinsic binding constants of compounds 1
Compound
R
kamax/nm
lg e
kbem/nm
Ucf/%
ICd50/lM
Kca/105 M-1
1a
432
3.84
562
0.29
4.60
9.02
NMe2
HN
1b
1c
433
431
4.01
4.40
564
560
0.26
0.55
25.1
1.09
2.26
NEt2
N
HN
HN
[100
1d
1e
429
433
4.21
4.15
565
579
0.23
0.45
[100
[100
1.76
0.53
NHPh
HN
N
1f
430
429
433
4.35
4.41
4.22
563
569
573
0.48
0.59
0.17
[100
6.46
3.96
1.75
3.11
N
N
S
1g
1h
O
13.33
N
NMe
B-a
B-h
98.08f
15.03f
NMe2
HN
N
NMe
a
The absorption was measured in dry toluene
b
c
d
e
f
The emission was measured in dry toluene and excitated at 430 nm
The quantum yield was measured with rhodamine B in ethanol as quantum yield standard (Uf = 0.97)
The cytotoxicity (CTX) against human breast cancer cells (MCF-7) was measured by the microculture tetrazolium-formazan method
The intrinsic binding constants K were calculated according to UV-vis absorption spectra
See [10]
Circular dichroism spectra studies
DNA in the right-hand B form. When the compounds
were incubated with CT-DNA, an increase in the intensity
of the positive band and a decrease in the intensity of the
negative band were observed. The increase of positive
bands is more obvious than that of negative ones (Fig. 4).
The increases in CD signal of DNA after adding 1a and
1g were consistent with the K value (1a [ 1g) (Fig. 5),
indicating that the DNA-binding ability of 1a is stronger
than 1g.
Circular dichroism spectra (CD) are a favorable technique
to monitor the conformational state of the DNA double
helix in solution, and could estimate whether the com-
pounds intercalate DNA or not [28]. The CD spectrum of
free CT-DNA exhibited a negative band at 247 nm
because of the helicity and a positive band at 279 nm
because of the base stacking, which is the characteristic of
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J. Xing et al.
Fig. 2 Absorption spectral changes of 1a (10 lM) in the presence of
CT-DNA whose respective ratio is 1:0.1, 1:0.3, 1:0.5, 1:1, and 1:2 in
Tris-HCl buffer (30 mM, pH 7.5)
Fig. 4 CD spectra of CT-DNA in the absence and presence of 1a at
65 lM DNA concentration, and varying the concentrations of 1a (0,
3, 5, 7, 10, 15 lM) in Tris-HCl buffer (30 mM, pH 7.5)
Fig. 3 Fluorescence spectrum before and after interaction of 1a and
CT-DNA in Tris-HCl buffer (30 mM, pH 7.5). Numbers 1–4 indicate
the concentration of 1a: 20, 25, 30, and 35 lM, respectively. DNA
applied was 50 lM
Fig. 5 CD spectra of CT-DNA in the absence and presence of 1a or
1g at 65 lM DNA concentration, the concentration of 1a or 1g is
15 lM, in Tris-HCl buffer (30 mM, pH 7.5)
and 1g on DNA conformation were found to be different,
although they could bind to DNA in the same way. Because
many factors such as DNA itself, the DNA-drug complex,
and environment could affect the UV-vis, fluorescence
spectra, CD spectra, and viscosity when compounds bind to
CT-DNA, the exact change of the conformation requires
further study.
Viscosity measurement
Viscosity measurement is regarded as a reliable tool to
determine the binding model in the absence of crystallo-
graphic structural data and NMR data [22]. Intercalation of
a molecule into DNA could result in a lengthening,
unwinding, and stiffening of the helix, and is usually
accompanied by an increase in solution viscosity [24, 25,
35]. The viscosities of DNA solution increased continually
with the concentration increase of 1a or 1g, implying the
lengthening and unwinding of CT-DNA (Fig. 6). The result
is contrary to the phenomenon of CD. Based on the UV-vis
absorption, fluorescence spectra, circular dichroism, and
viscosity measurement, the effects of both compounds 1a
In conclusion, we developed a novel series of ace-
naphthopyrazine derivatives that were potential inte-
rcalators endowed with antitumor activity against MCF-7
cells. The derivatives avoid the shortcoming of naphtha-
linamide and acenaphtho-heterocycle chromophores in
optical properties, and show not only long emission
wavelength, but also large Stokes shift.
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Novel acenaphthopyrazine derivatives as antitumor agents against MCF-7 cells
247
The mixture was refluxed under nitrogen until the reactions
were completed (TLC analysis) and the color of the solu-
tion changed from light yellow to black red. The mixture
was cooled and poured into ice water; the red solid was
collected by filtration and purified by silica gel to give
compounds 1a–1h in 50–60% yields. The structures of the
target products were identified by 1H NMR, 13C NMR, IR,
and HRMS.
3-[2-(Dimethylamino)ethylamino]acenaphtho[1,2-b]-
pyrazine-8,9-dicarbonitrile (1a, C20H16N6)
Yield 50%; red powder; m.p.: 252.1–253.8 °C; Rf = 0.50
(CH2Cl2/CH3OH = 50:1); 1H NMR (400 MHz, DMSO-
d6): d = 8.77 (d, J = 8.4 Hz, 1H), 8.53 (d, J = 7.2 Hz,
1H), 8.36 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.89 (t,
J = 8.0 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 3.65 (t,
J = 6.0 Hz, 2H), 2.83 (t, J = 5.6 Hz, 2H), 2.42 (s, 6H)
ppm; 13C NMR (100 MHz, DMSO-d6): d = 152.0, 151.0,
150.6, 136.5, 130.4, 129.1, 127.6, 126.4, 126.0, 125.7,
124.4, 119.0, 115.7, 115.4, 112.8, 106.9, 57.0, 46.3,
45.8 ppm; IR (KBr): vꢀ = 3,430, 3,074, 2,924, 2,228,
Fig. 6 Effect of increasing amounts of compounds 1a and 1g on the
relative viscosities of CT-DNA at 25 ( 0.1) °C. [DNA] = 160 lM in
Tris-HCl (30 mM, pH 7.5). g is the viscosity of DNA in the presence
of the compounds, and g0 is the viscosity of DNA in the absence of
the compounds
1,603, 1,534, 1,286 cm-1
340.1436, found 340.1444.
;
TOF MS (EI?): calcd
Experimental
All solvents were of analytic grade. 1H NMR and 13C NMR
spectra were measured on a Bruker AV-400 spectrometer
in DMSO-d6 or CDCl3 (TMS as an internal standard, with
chemical shifts reported as parts per million). IR spectra
were obtained using a Nicolet 470 FT-IR instrument, with
samples analyzed as KBr disks. High-resolution mass
spectra were obtained on a Micromass Co. GCT CA156
(TOF MS) spectrometer. Melting points were determined
by using an X-6 micro melting point apparatus. Column
chromatography was performed using silica gel 200–300
mesh. Compound 3 was prepared according to the litera-
3-[2-(Diethylamino)ethylamino]acenaphtho[1,2-b]-
pyrazine-8,9-dicarbonitrile (1b, C22H20N6)
Yield 40%; red powder; m.p.: 249.7–251.2°C; Rf = 0.43
(CH2Cl2/CH3OH = 50:1); 1H NMR (400 MHz, DMSO-
d6): d = 8.74 (d, J = 8.0 Hz, 1H), 8.55 (d, J = 6.8 Hz,
1H), 8.38 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.89 (t,
J = 7.8 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 3.56 (t, 2H),
2.78 (t, 2H), 2.59 (m, 4H), 1.00 (t, J = 6.4 Hz, 6H) ppm;
13C NMR (100 MHz, DMSO-d6): d = 152.2, 150.8, 150.4,
136.5, 130.4, 129.0, 127.3, 126.3, 125.9, 125.7, 124.2,
118.9, 115.4, 112.4, 106.8, 50.6, 46.6, 41.4, 11.8 ppm; IR
(KBr): vꢀ = 3,432, 3,074, 2,963, 2,925, 2,230, 1,603, 1,533,
1,284 cm-1; TOF MS (EI?): calcd 368.1749, found
368.1756.
1
ture; its structure was characterized by H NMR [36].
3-Bromoacenaphtho[1,2-b]pyrazine-8,9-dicarbonitrile
(4, C16H5BrN4)
Compound 3 (0.20 g, 0.77 mmol) and 0.11 g diaminoma-
leonitrile (1.07 mmol) were refluxed in 10 cm3 acetic acid 3-[(Pyridin-2-ylmethyl)amino]acenaphtho[1,2-b]pyrazine-
for 2 h. The solvent was evaporated, and the residue was 8,9-dicarbonitrile (1c, C22H12N6)
purified by chromatography (CH2Cl2/petroleum ether = Yield 50%; red powder; m.p.: 275.3–277.0 °C; Rf = 0.62
3:1) to afford compound 4. Yield 0.21 g (80%); Rf = 0.62; (CH2Cl2/CH3OH = 40:1); 1H NMR (400 MHz, DMSO-
1H NMR (400 MHz, CDCl3): d = 8.57 (d, J = 6.8 Hz, d6): d = 9.20 (s, 1H), 8.87 (d, J = 8.4 Hz, 1H), 8.59 (d,
1H), 8.49 (d, J = 8.4 Hz, 1H), 8.34 (d, J = 7.6 Hz, 1H), J = 6.4 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.94 (t, J =
8.19 (d, J = 7.2 Hz, 1H), 8.04 (t, J = 8.4 Hz, 1H) ppm; 7.6 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.47 (d, J = 8.0 Hz,
13C NMR (100 MHz, DMSO-d6): d = 152.6, 152.0, 1H), 7.32 (t, J = 6.0 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H),
137.1, 134.6, 131.8, 129.5, 128.3, 127.7, 127.3, 126.9, 4.84 (d, J = 6.0 Hz, 2H) ppm; 13C NMR (100 MHz,
126.4, 126.0, 125.7, 124.4, 115.9, 112.6 ppm.
DMSO-d6): d = 157.4, 152.0, 151.2, 150.7, 149.1, 136.9,
136.3, 130.2, 129.1, 127.5, 126.5, 126.1, 125.7, 124.6,
122.4, 121.2, 119.0, 115.6, 115.2, 113.2, 107.3, 48.1 ppm;
IR (KBr): vꢀ = 3,299, 3,053, 2,925, 2,226, 1,604, 1,532,
General synthesis procedure for 1a–1h
Compound 3 (0.10 g, 0.3 mmol) and aliphatic amine 1,288 cm-1; TOF MS (EI?): calcd 360.1123, found
(1.5 mmol) were mixed in 5 cm3 dry 2-methoxyethanol. 360.1115.
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J. Xing et al.
3-[(2-(Phenylamino)ethylamino]acenaphtho[1,2-b]-
pyrazine-8,9-dicarbonitrile (1d, C24H16N6)
127.5, 127.2, 125.7, 122.9, 119.9, 116.1, 115.2, 115.0,
66.0, 53.0 ppm; IR (KBr): vꢀ = 3,435, 3,075, 2,954, 2,234,
1,603, 1,540, 1,293, 1,211, 1,115, 1,002 cm-1; TOF MS
(EI?): calcd 339.1120, found 339.1118.
Yield 50%; red powder; m.p.: 230.1–232.0 °C; Rf = 0.56
1
(CH2Cl2); H NMR (400 MHz, DMSO-d6): d = 8.80 (d,
J = 8.0 Hz, 1H), 8.57 (d, J = 7.2 Hz, 1H), 8.52 (s, 1H),
8.31 (d, J = 8.0 Hz, 1H), 7.90 (t, J = 8.0 Hz, 1H), 7.10 (t,
J = 8.0 Hz, 2H), 6.95 (d, J = 8.4 Hz, 1H), 6.64 (d,
J = 7.6 Hz, 2H), 6.55 (t, J = 7.6 Hz, 1H), 5.82 (s, 1H),
3.68 (t, J = 6.0 Hz, 2H), 3.45 (t, J = 6.0 Hz, 2H) ppm;
13C NMR (100 MHz, DMSO-d6): d = 152.2, 150.9, 150.5,
148.3, 136.4, 130.3, 129.0, 128.9, 127.5, 126.3, 125.8,
125.6, 124.2, 119.0, 115.7, 115.5, 115.3, 112.6, 112.0,
106.7, 42.3, 41.3 ppm; IR (KBr): vꢀ = 3,405, 3,058, 2,924,
2,231, 1,602, 1,535, 1,283 cm-1; TOF MS (EI?): calcd
388.1436, found 388.1445.
3-(4-Methylpiperazin-1-yl)acenaphtho[1,2-b]pyrazine-8,9-
dicarbonitrile (1h, C21H16N6)
Yield 50%; red powder; m.p.: 253.3–255.5 °C; Rf = 0.68
(CH2Cl2/CH3OH = 30:1); 1H NMR (400 MHz, DMSO-
d6): d = 8.60 (d, J = 6.8 Hz, 1H), 8.50 (d, J = 8.4 Hz,
1H), 8.45 (d, J = 8.0 Hz, 1H), 7.96 (t, J = 7.6 Hz, 1H),
7.35 (d, J = 8.4 Hz, 1H), 3.55 (t, 4H), 2.67 (t, 4H), 2.32 (s,
3H) ppm; 13C NMR (100 MHz, DMSO-d6): d = 155.4,
152.2, 152.0, 136.3, 129.5, 129.0, 127.7, 127.6, 126.9,
126.6, 125.2, 122.7, 119.1, 115.8, 114.7, 114.6, 54.1, 52.1,
45.0 ppm; IR (KBr): vꢀ = 3,435, 3,055, 2,935, 2,234, 1,603,
1,538, 1,289, 1.245 cm-1; TOF MS (EI?): calcd 352.1436,
found 352.1432.
3-(Piperidin-1-yl)acenaphtho[1,2-b]pyrazine-8,9-
dicarbonitrile (1e, C21H15N5)
Yield 70%; red powder; m.p.: 246.7–249.2 °C; Rf = 0.45
(CH2Cl2/petroleum ether = 3:1); 1H NMR (400 MHz,
CDCl3): d = 8.53 (d, J = 8.4 Hz, 1H), 8.50 (d, J =
7.2 Hz, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.90 (t,
J = 7.6 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H), 3.58 (t,
J = 5.2 Hz, 4H), 2.04 (m, 4H), 1.82 (m, 2H) ppm; 13C
NMR (100 MHz, DMSO-d6): d = 156.3, 152.0, 151.8,
136.4, 129.5, 127.8, 127.4, 126.7, 126.3, 125.1, 122.7,
118.3, 115.5, 114.7, 114.6, 53.4, 25.2, 23.3 ppm; IR (KBr):
vꢀ = 3,435, 3,055, 2,935, 2,233, 1,602, 1,538, 1,242,
1,156 cm-1; TOF MS (EI?): calcd 337.1327, found
337.1303.
Cytotoxic evaluation
Cytotoxicity assays were performed on human breast
cancer cells (MCF-7), and the IC50 was evaluated by MTT
tetrazolium dye assay. Compounds were added into the
medium at concentrations from 0.1 to 100 lM for 24 h
after cells seeded into 96-well microculture plates and
allowed to adhere for 24 h.
DNA-binding studies
UV-vis absorption spectra were recorded on a PGENERAL
TU-1901 UV-vis spectrophotometer, and fluorescence
spectra were measured on a Hitachi F-4500 luminescence
spectrophotometer. The CD (circular dichroism) spectra
were scanned with a J-810. Calf-thymus DNA was
3-(Thiomorpholin-4-yl)acenaphtho[1,2-b]pyrazine-8,9-
dicarbonitrile (1f, C20H13N5S)
Yield 66%; red powder; m.p.: 255.2–256.8 °C; Rf = 0.43
(CH2Cl2/petroleum ether = 4:1); 1H NMR (400 MHz,
DMSO-d6): d = 8.62 (d, J = 7.2 Hz, 1H), 8.49 (d,
J = 7.6 Hz, 1H), 8.48 (d, J = 7.6 Hz, 1H), 7.99 (t,
J = 7.6 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 3.76 (t, 4H),
2.98 (t, 4H) ppm; 13C NMR (100 MHz, DMSO-d6):
d = 156.9, 153.2, 153.0, 136.9, 130.4, 130.1, 129.1,
128.6, 128.3, 128.0, 126.3, 123.9, 120.7, 117.6, 115.8,
115.6, 71.6, 55.7 ppm; IR (KBr): vꢀ = 3,437, 3,054, 2,921,
2,233, 1,602, 1,539, 1,289, 1,160, 1,064, 960 cm-1; TOF
MS (EI?): calcd 355.0892, found 355.0898.
3-(Morpholin-4-yl)acenaphtho[1,2-b]pyrazine-8,9-
dicarbonitrile (1g, C20H13N5O)
Yield 56%; red powder; m.p.: 249.0–251.0 °C; Rf = 0.57
(CH2Cl2/ethyl acetate = 25 :1); 1H NMR (400 MHz,
DMSO-d6): d = 8.61 (d, J = 7.2 Hz, 1H), 8.56 (d,
J = 8.0 Hz, 1H), 8.49 (d, J = 8.0 Hz, 1H), 7.98 (t,
J = 8.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 3.94 (t, 4H),
3.52 (t, 4H) ppm; 13C NMR (100 MHz, DMSO-d6):
d = 155.5, 152.5, 152.3, 136.3, 129.9, 128.1, 127.8,
Fig. 7 The CDNA/(ea – ef) vs CDNA plot for 1a as determined from
the absorption titration data
123

Novel acenaphthopyrazine derivatives as antitumor agents against MCF-7 cells
249
NC
N
CN
N
NC
N
CN
N
NMe2
NEt2
1a: R =
1b: R =
1e: R =
1f: R =
N
N
HN
O
O
O
O
NC
CN
S
HN
HN
RH
H2N
NH2
Br2
N
1c: R =
1d: R =
1g: R =
1h: R =
N
N
O
CH3OCH2CH2OH, reflux
60-70 o
C
acetic acid, reflux
Br
Br
R
NHPh
NMe
1
3
4
2
HN
Scheme 1
purchased from the Sino-American Biotechnology Co.
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 coefficient value of
compounds ranging from 20 to 35 lM in Tris-HCl (30 mM,
pH 7.5); the other set had the same composition, but in the
absence of DNA. All the above solutions were ultrasonically
shaken for 1 day at 25 °C in the dark. The fluorescence
wavelength and intensity of samples were measured.
Circular dichroism spectra studies
6,600 M-1 cm-1
.
The CD (circular dichroism) spectra were scanned with a
J-810 spectrophotometer (Jasco, Japan) using a 1-cm path
quartz cell and subtracted from the spectrum of Tris-HCl
buffer alone. Spectra of CT-DNA in Tris-HCl were
recorded in the range 200–400 nm. The appropriate con-
centration of compounds 1a and 1g in Tris-HCl was also
determined.
UV–vis absorption spectra studies
Spectroscopic titrations were carried out at room temper-
ature. Initially, solutions of the blank buffer were placed in
the reference and sample cuvettes (1 cm path length),
respectively, and then the blank spectrum was recorded in
the range 200–500 nm. During the titration, aliquots of
buffered DNA solution were added, and the solutions were
mixed by repeated inversion. After mixing for 10 min, the
absorption 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, then the spectrum of the compound in
buffer was recorded. The appropriate R ratio to get corre-
sponding spectra was achieved by keeping the con-
centration of the compound constant while varying the
DNA concentration at room temperature.
Viscosity experiments
Calf-thymus DNA was dissolved in Tris-HCl buffer
(30 mM, pH 7.5) and left at 4 °C overnight. The viscom-
etry was carried out in a thermostated bath maintained at
25 ( 0.1) °C using an Ubbelohde viscometer. The CT-
DNA solution was sucked into the upper bulb of the vis-
cometer. Flow times were measured with a digital
stopwatch, each sample was measured three times, and an
average flow time was calculated. Data are presented as
(g/g0)1/3 versus [complex]/[DNA], where g is the viscosity
of DNA in the presence of complex and g0 is the viscosity
of DNA alone [33, 37].
Based on the method of calculating intrinsic binding
constants K, the following Eq. 1 was used [33], where
CDNA is the the concentration of CT-DNA in base pairs, the
apparent absorption coefficients ea, ef, and eb correspond to
the absorbance for compounds, the free compounds, and
the compounds in the fully bound form. K is the equilib-
Acknowledgments We are grateful to the Natural Science Foun-
dation of China (90713030; 20876025) and the National Basic
Research Program of China (2009CB724706).
rium binding constant in M-1
.
À
Á
À
Á
À
Á
C
DNA= ea ꢁ ef ¼ CDNA= eb ꢁ ef þ 1=K eb ꢁ ef
ð1Þ
A plot of CDNA/(ea – ef) versus CDNA was calculated
using linear regression at the origin as shown in Fig. 7, and
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