Synthesis, DNA binding and cytotoxicity of new pyrazole emodin derivatives

Article abstract of DOI:10.1016/j.ejmech.2006.04.006

A series of new anthrapyrazoles were derived from emodin by attaching various cationic alkyl amino side chains onto a pyrazole ring which had been incorporated into the anthraquinone chromophore. Compared with emodin, the derivatives had significantly higher DNA binding affinity based on interaction with calf thymus DNA, and much more potent cytotoxicity against different tumor cells. The derivatives with a mono-cationic alkyl side chain exhibited the highest DNA binding affinity and cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2006.04.006

European Journal of Medicinal Chemistry 41 (2006) 1041–1047
http://france.elsevier.com/direct/ejmech
Original article
Synthesis, DNA binding and cytotoxicity of new pyrazole emodin derivatives
J.-H. Tan a, Q.-X. Zhang b, Z.-S. Huang a, Y. Chen a, X.-D. Wang a,b, L.-Q. Gu a,*, J.Y. Wu b,*
a School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510080, China
b Department of Applied Biology and Chemical Technology and the State Key Lab of Chinese Medicine and Molecular Pharmacology,
the Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong
Received in revised form 17 April 2006; accepted 20 April 2006
Available online 22 May 2006
Abstract
A series of new anthrapyrazoles were derived from emodin by attaching various cationic alkyl amino side chains onto a pyrazole ring which
had been incorporated into the anthraquinone chromophore. Compared with emodin, the derivatives had significantly higher DNA binding
affinity based on interaction with calf thymus DNA, and much more potent cytotoxicity against different tumor cells. The derivatives with a
mono-cationic alkyl side chain exhibited the highest DNA binding affinity and cytotoxicity.
© 2006 Elsevier SAS. All rights reserved.
Keywords: Emodin derivatives; Anthrapyrazole; Amino side chain; DNA binding; Cytotoxicity; Tumor cells
1. Introduction
strictly defined orientation to the chromophore. They interact
with DNA via intercalative association and binding locations
(minor groove, major groove, backbone, etc.), which may in-
duce conformational changes in the regular helical structure,
unwinding the DNA at the binding site and interfering with
the function of DNA binding enzymes such as DNA topoi-
somerases and DNA polymerases [9–11]. The inhibition of to-
poisomerase II, in particular, has been established as the chief
mechanism of action for the antitumor and cytotoxic effects of
daunorubicin, mitoxantrone and related anthraquinones. The
inhibition of topoisomerase II eventually leads to DNA dou-
ble-strand breaks in the tumor cells and possibly, cell death.
Although daunorubicin and mitoxantrone have excellent DNA
binding and antitumor activities, they have shown serious side
effects during cancer therapy, especially cardiotoxicity [8,12].
Various semi-synthetic approaches have been exercised to de-
rive anthraquinone analogues with enhanced antitumor activ-
ities and reduced side effects. Some anthrapyrazole derivatives
such as losoxantrone have shown potent antitumor activity on a
number of tumor cell lines (including solid tumors) and lower
cardiotoxicity [13–15].
Chemical modification of bioactive components of medic-
inal herbs is one of the most common approaches in drug dis-
covery for new drugs and improved therapeutic properties.
Emodin (Fig. 1) is the major bioactive compound of numerous
herb species such as Rheum and Polygonum (Polygonaceae),
Rhamnus (Rhamnaceae) and Senna (Cassieae) [1]. It exhibits
a variety of biological effects such as anti-inflammatory, anti-
biotic, antiviral and antineoplastic [2–5] activities. Moreover, it
has been shown to inhibit the growth of various cancer cells
[5–7], and induce the apoptosis of certain cancer cells [6,7].
Emodin belongs to the same class of coplanar anthraqui-
nones as daunorubicin and mitoxantrone, which have been in
clinical use over 30 years for the treatment of various tumors
[8]. Their function as noncovalent DNA binders is generally
believed to be essential for their activity. Most DNA binding
compounds share two common structural features which ap-
pear to contribute to their activity, the presence of planar poly-
cyclic aromatic systems and the occurrence of one or two side
chains containing polymethyleneamine or sugar fragments in a
Emodin itself has low DNA binding affinity, and low or
insignificant cytotoxicity against various cancer cells [16]. Be-
cause of its coplanar structure, the incorporation of a pyrazole
ring into its anthraquinone chromophore may increase the elec-
* Corresponding authors.
E-mail addresses: cedc42@zsu.edu.cn (L.-Q. Gu), bcjywu@polyu.edu.hk
(J.Y. Wu).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.04.006

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J.-H. Tan et al. / European Journal of Medicinal Chemistry 41 (2006) 1041–1047
2. Results and discussion
2.1. Synthesis of emodin derivatives
The synthetic steps of the target compounds are shown in
Fig. 2. The starting compound emodin 1 was methylated to
corresponding di-O-methyl emodin 2 at the specified condi-
tions. Methylation of 1 yielded 6,8-O-dimethyl emodin 2 as
the major product together with a very small amount of 3,8-
O-dimethyl isomer. Then, 6,8-O-dimethyl emodin was purified
by flash chromatography and crystallization. Compound 2 was
converted to the corresponding tosylate 3, and further treatment
of 3 with 2-hydroxyethylhydrazine gave compound 4, which
was purified by column chromatography [21–23], and its struc-
tural assignment was confirmed with the nuclear Overhauser
enhancement spectroscopy (NOESY) experiment. The methy-
lene side chain adjacent to the pyrazole ring possessed NOEs
with the 3-aromatic proton but not the 10-methoxy group.
Compound 4 was further converted to the corresponding me-
sylate 5. The target compounds 6–13 were attained by nucleo-
philic substitution of 5 with appropriate substituted amines
(Fig. 3).
The emodin derivatives can be divided into three groups
based on their amino side chains, a mono-cationic alkyl group
(6–8), a di-cationic alkyl group (9, 10) and a heterocycle sub-
stituent group (11–13). The free base forms of compounds 6–
13 were converted into hydrochloride salts by treatment with
hydrochloride acid in ethanol to enhance the chemical stability
and solubility for the following biochemical assays.
Fig. 1. Emodin, daunorubicin, mitoxantrone and losoxantrone molecular
structures.
tron density of the π system area, which makes the chromo-
phore more resistant to enzymatic reduction to radical species,
thus to lower the cardiotoxicity [17]. The chromophore alone is
not sufficient, however, the addition of side chains such as
polymethyleneamine, sugar or heterocyclic to the chromo-
phore, is usually effective to gain higher DNA binding affinity
and antitumor activities [18,19]. The cationic amino side
chains are most desirable as their distal amino group binds
electrostatically to the phosphate moieties of DNA [18,20].
Moreover, chains of varying length, polarity, rigidity, charge
and steric bulk may confer different DNA binding affinities.
This work aims to synthesize a group of new emodin deri-
vatives with improved DNA binding affinity and antitumor ac-
tivity by incorporating a pyrazole ring into the anthraquinone
chromophore, and then attaching various cationic amino side
chains to the pyrazole ring. The DNA binding capacity of the
new anthrapyrazoles derived from emodin was evaluated based
on interaction with calf thymus (CT) DNA, and their cytotoxi-
city was assessed on three types of tumor cell lines, mouse
melanoma B16, human hepatocellular carcinoma HepG2, and
Lewis lung carcinoma (LLC) cells. The molecular structure
and bioactivity relationships were discussed.
2.2. DNA binding properties
The DNA binding properties of compounds 6–13 were eval-
uated based on their affinity or interaction with CT DNA, mea-
sured with absorption and fluorescence spectrometric titration
[24,25]. Fig. 4 shows the representative absorption spectra of
compound 6 (30 μM) in the presence of increasing concentra-
tions of CT DNA (0–165 μM). Other compounds exhibited the
similar absorption spectra pertaining to the chromophore but
with the hypochromicity and red shifts dependent on the catio-
nic amino side chains. These results were consistent with the
Fig. 2. Synthesis of anthrapyrazole derivatives with carboxamide side chains from emodin. Reagents: (a) Me2SO4, K2CO3, Me2CO (room temperature); (b) p-
toluenesulfonyl chloride, K2CO3, Me2CO (reflux); (c) 2-hydroxyethylhydrazine, 2-ethoxyethanol (120 °C); (d) methanesulfonyl chloride, Et3N, CH2Cl2; (e) N,N-
dialkylaminoethylamine, ethanol (reflux).

J.-H. Tan et al. / European Journal of Medicinal Chemistry 41 (2006) 1041–1047
1043
Fig. 3. Structures of emodin derivatives 6–13 from the synthetic steps shown in Fig. 2.
Fig. 4. Absorbance titration of 6 at 30 μM in 20 mM sodium phosphate buffer
(pH 6.3) with 150 mM NaCl at increasing CT DNA concentration (arrow: 0–
165 μM).
previous reports on the absorption titration of anthrapyrazole
derivatives, and the hypochromicity of anthrapyrazoles in the
presence of DNA is believed to be a result of their interactions
with the DNA [26]. On the other hand, the red shift is asso-
ciated with a decrease in the energy gap between the highest
occupied molecular orbital (HOMO) and the lowest occupied
molecular orbital (LUMO) when the drug binds to DNA [27].
The selection of ionic strength (150 mM NaCl) in the ab-
sorption titration experiments was mainly based on the avoid-
ance of DNA deposition (or precipitation) in all drug solutions
(30 μM). In the fluorescence spectrometric titration, a much
lower drug concentration (3 μM) could be used at a lower
NaCl concentration (20 mM) to measure the appropriate bind-
ing constants. Fig. 5a shows the fluorescence spectrometric ti-
tration spectra of compound 6, and Fig. 5b the corresponding
peak fluorescence intensity at increasing CT DNA concentra-
tion. The titration spectra and peaks for other compounds ex-
hibited the similar trend of changes with the CT DNA concen-
tration. The large increase in the fluorescence intensity with CT
DNA concentration is suggestive of a strong association of the
compound molecules with the DNA molecules. The associa-
tion may shield the DNA molecules from the solvent mole-
cules, leading to an increase in the emission intensity [28].
Table 1 summarizes the DNA binding constants and related
properties of emodin and compounds 6–13 after interaction
Fig. 5. (a) Fluorescence spectra of compound 6 at 3 μM in 20 mM sodium
phosphate buffer (pH 6.3) with 20 mM NaCl at increasing CT DNA
concentration (arrow: 0–138 μM); (b) the peak fluorescence intensity in (a)
at 565 nm versus CT DNA concentration.
with CT DNA. The relative binding affinities as indicated by
the binding constants Ki are in the order of
8 > 13 > 6 > 7 > 11 > 9 > 10 > 12 > emodin. Therefore, all de-
rivatives had a higher DNA binding constant than their lead
compound. This result indicates that introduction of the pyra-
zole ring and cationic amino side chains can dramatically in-
crease the DNA binding capacity of emodin. Among the deri-

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J.-H. Tan et al. / European Journal of Medicinal Chemistry 41 (2006) 1041–1047
Table 1
Binding constants (Ki) and photometric properties of emodin and its derivatives 6–13 in contact with CT DNA
Compound
Ki
λmax
(nm)
446
456
457
459
463
465
458
464
467
Red shift
Hypochromicity
Isosbestic point
(nm)
458
484
485
(× 10−5 M−1 a,c
0.68
)
(nm)b,d
(%)b,d
3
Emodin
6
7
8
1
9
9
6
5
4
11
2
4
6.09
5.52
7.47
3.76
2.53
4.09
2.25
6.98
18
18
15
11
13
20
7
482
9
Unclear
Unclear
482
Unclear
496
10
11
12
13
a
13
Three micromolars drug in 20 mM NaH2PO4-Na2HPO4 pH 6.3 buffer containing 20 mM NaCl at room temperature.
Thirty micromolars drug in 20 mM NaH2PO4–Na2HPO4 pH 6.3 buffer containing 150 mM NaCl at room temperature.
Obtained from spectrofluorimetric titration at λex = 410 nm and λem = 565 nm.
b
c
d
Obtained at λmax
.
Table 2
vatives, those with stronger DNA binding affinities, 6, 7, 8, 11
and 13 exhibited hypochromicity, red shifts and clear isobestic
points. The spectral changes are suggestive of intercalative as-
sociation as a major binding mode for these derivatives with
CT DNA [29,30]. In contrast, compounds 9, 10, 12 exhibited
no clear isobestic point, suggesting a different or a mixture of
DNA binding modes for these low-affinity compounds.
Compound 8 which has the same side chain as mitoxantrone
and losoxantrone had the largest DNA binding constant, which
may be attributed to the strong binding of DNA molecules
though electrostatic interaction and hydrogen bonding by its
secondary amino and hydroxyl groups. The other derivatives
6, 7, 11 bearing mono-cationic side chains also exhibited sig-
nificant binding capacity with the relative binding affinities in
the order of 6 > 7 > 11. The more bulky group with distal
amine appears to have a lower DNA binding affinity. In the
di-cationic alkyl group, much longer and more flexible ligands
9, 10 showed weaker binding properties. Side chain flexibility
and the two adjacent cationic centers may obstruct the chromo-
phore from accessing the DNA base pairs, which may be re-
sponsible for the lower binding affinity compared with the
mono-cationic compounds [31]. However, a higher binding af-
finity was observed in the more rigid compound 13, which may
be explained by that the rigid piperazine group can attach onto
the major groove of DNA when the chromophore is inserted
into the DNA base pairs [10,31]. Finally, the lower binding
affinity of compound 12 may be attributed to the low pKa value
of its morpholine group.
IC50 cytotoxicity values (μM) of emodin derivatives against tumor cells (data
derived from the mean of three independent assays)
Compound
Emodin
6
7
8
9
B16
> 100
2.7
3.6
1.8
5.2
10.1
6.9
n.d.
20.1
HepG2
55.1
2.0
4.9
2.7
5.7
37.6
4.9
n.d.
7.2
LLC
> 100
1.7
4.5
3.8
13.1
12.8
12.1
n.d.
8.9
10
11
12a
13
a
Not determined (n.d.) because of its poor solubility in solvents suitable for
the assay.
tively large DNA binding constants (Table 1). Therefore, the
higher cytotoxic activity against the tumor cells may be attrib-
uted, at least in part, to their intercalative association and high
binding affinity with the DNA of tumor cells. As for the struc-
ture–activity relationship, compounds 6, 7, 8, 11 bearing the
mono-cationic side chains had a stronger cytotoxic activity
than compounds 9, 10, 13 bearing the di-cationic side chains.
An explanation for the lower activity of compounds 9, 10, 13
is that these three compounds, because of their higher electrical
charge, are less lipophilic and thus more difficult to diffuse
through the plasma membrane.
In conclusion, the new anthrapyrazoles derived from emo-
din with cationic amino side chains had strong inhibiting activ-
ity against various tumor cells, which may be associated with
their DNA binding capacity. In particular, the mono-cationic
amino side chains introduced into the lead compound appeared
to be the major contributors to the DNA binding and cytotoxi-
city enhancement.
2.3. Cytotoxicity against tumor cells
Table 2 shows the IC50 values (cytotoxicity potency in-
dexes) of compounds 6–13 against three types of tumor cells
in culture. The lead compound emodin exhibited low or insig-
nificant cytotoxicity with high IC50 values, 55 μM on HepG2
cell and more than 100 μM on the other two cell lines. In com-
parison, all the derivatives had much more potent cytotoxicity
with significantly lower IC50 values on most of the tumor cells.
Of all the derivatives, compounds 6, 7, 8 were most potent in
inhibiting the tumor cell proliferation with the lowest IC50 va-
lues of 1.7–5.0 μM. Likewise, these compounds also had rela-
3. Experimental
3.1. Chemistry
Absorbance measurements were performed on a Shimadzu
UV-2501 spectrophotometer, and fluorometric measurements
were on a Shimadzu RF-5301PC spectrofluorophotometer.
Melting points (m.p.) were determined using an X-6 micro-

J.-H. Tan et al. / European Journal of Medicinal Chemistry 41 (2006) 1041–1047
1045
scope melting point instrument without correction. FAB-MS
spectra were obtained using a VG ZAB-HS spectrometer, and
1H NMR spectra and NOESY experiment were performed on a
Varian INOVA 500NB with tetramethylsilane (TMS) as an in-
ternal standard. Elemental analysis was carried out on an Ele-
mentar Vario EL CHNS Elemental Analyzer.
Emodin (purity 98.5%) was extracted from Chinese herb
Polygonum cuspidatum Sieb.et Zucc according to the method
of Kelly et al. [32]. Silica gel F254 was used in analytical thin-
layer chromatography (TLC) and silica gel H was used in col-
umn chromatography.
purified by column chromatography with cyclohexane/EtOAc
(50:50–10:90) elution to afford an orange solid compound 4
(0.19 g, 56%): m.p. 226–228 °C; 1H NMR (DMSO-d6,
300 MHz): δ 2.59 (s, 3H), 3.85 (q, 2H, J = 5.1 Hz), 3.90 (s,
3H), 3.98 (s, 3H), 4.55 (t, 2H, J = 5.1 Hz), 4.90 (t, 1H,
J = 4.8 Hz), 7.01 (d, 1H, J = 2.4 Hz), 7.42 (d, 1H,
J = 2.4 Hz), 7.74 (s, 1H), 7.86 (s, 1H); FAB-MS m/z: 339 [M
+ H]+; Anal. Calcd. for C19H18N2O4: C, 67.44; H, 5.36; N,
8.28. Found: C, 67.28; H,5.43; N, 8.19.
3.1.4. 2-(2-[(Methylsulfonyl)oxy]ethyl)-4-methyl-8,10-
dimethoxy-anthra[1,9-cd]pyrazol-6(2H)-one (5)
3.1.1. 1-Hydroxy-6,8-dimethoxy-3-methylanthracene-9,10-
dione (2)
To a stirred suspension of 4 (0.42 g, 1.00 mmol) and
triethylamine (1.0 ml, 7.2 mmol) in dry dichloromethane
(75 ml), methanesulfonyl chloride (0.5 ml, 6.46 mmol) was
added with cooling at 0 °C. The mixture was then stirred at
room temperature for 4 h. The reaction mixture was partitioned
between dichloromethane (200 ml) and 1 N sodium hydroxide
(20 ml), and the organic phase was washed with HCl (10%,
20 ml), water (20 ml), and finally brine (20 ml), and then con-
centrated to dryness. The residue was purified by flash chro-
matography with cyclohexane/EtOAc (50:50–20:80) elution to
afford an orange solid compound 5 (0.45 g, 87%): m.p.
To a solution of emodin (1) (1.05 g, 3.89 mmol) in dry
acetone (105 ml), dimethyl sulfate (1.0 ml) and anhydrous
K2CO3 (5.0 g) were added and the mixture was stirred for
28 h at 30 °C. The reaction product was evaporated under va-
cuum and then mixed with water (50 ml). The precipitate was
separated from water by filtration and the residue was purified
by flash column chromatography with CHCl3 elution. The pro-
duct was crystallized in acetone, yielding an orange solid as
compound 2 (0.70 g, 60%): m.p. 209–212 °C; 1H NMR
(CDCl3, 300 MHz): δ 2.43 (s, 3H), 3.98 (s, 3H), 4.02 (s,
3H), 6.76 (d, 1H, J = 2.4 Hz), 7.06 (s, 1H), 7.44 (d, 1H,
J = 2.4 Hz), 7.55 (s, 1H), 13.07 (s, 1H); FAB-MS m/z: 299
[M + H]+; Anal. Calcd. for C17H14O5: C, 68.45; H, 4.73.
Found: C, 68.33; H, 4.76. Compound 2 is the same as that
reported previously in [33] as the spectral data are all identical.
1
242–243 °C; H NMR (DMSO-d6, 300 MHz): δ 2.61 (s, 3H),
3.08 (s, 3H), 3.92 (s, 3H), 4.00 (s, 3H), 4.68 (t, 2H,
J = 4.2 Hz), 4.88 (t, 2H, J = 4.2 Hz), 7.03 (d, 1H,
J = 2.4 Hz), 7.43 (d, 1H, J = 2.4 Hz), 7.78 (s, 1H), 7.90 (s,
1H); FAB-MS m/z: 417 [M + H]+; Anal. Calcd. for
C20H20N2O6S: C, 57.68; H, 4.84; N, 6.73. Found: C, 57.41;
H, 4.90; N, 6.65.
3.1.2. 1,3-Dimethoxy-6-methyl-8-[(4-methylphenyl)sulfonyl]
oxyanthracene-9,10-dione (3)
3.1.5. General procedure for the preparation of the amines 6–
13
To a solution of compound 2 (0.45 g, 1.5 mmol) in dry
acetone (200 ml), p-toluenesulfonyl chloride (0.57 g 3.0 mmol)
and anhydrous K2CO3 (4.0 g, 29.0 mmol) were added under
nitrogen, and the mixture was refluxed for 5 h. The reaction
product was evaporated under vacuum and then mixed with
water (100 ml). The precipitate was separated from water by
filtration, rinsed with ether (50 ml), and then purified by col-
umn chromatography with 70:30 cyclohexane/EtOAc elution
to afford a light yellow solid compound 3 (0.58 g, 85%): m.p.
217–219 °C; 1H NMR (CDCl3, 500 MHz): δ 2.38 (s, 3H), 2.46
(s, 3H), 3.94 (s, 3H), 3.95 (s, 3H), 6.74 (d, 1H, J = 2.5 Hz),
7.28 (d, 2H, J = 8.0 Hz), 7.30 (d, 1H, J = 2.5 Hz), 7.41 (d, 1H,
J = 1.0 Hz), 7.89 (d, 2H, J = 8.0 Hz), 7.95 (d, 1H, J = 1.0 Hz);
FAB-MS m/z: 453 [M + H]+; Anal. Calcd. for C24H20O7S: C,
63.71; H, 4.45. Found: C, 63.65; H, 4.46.
To a stirred solution of compound 5 in anhydrous ethanol,
appropriate N,N-dialkylaminoethylamine (20 eq) was added.
The mixture was stirred at the reflux temperature for 6–24 h,
and then evaporated under vacuum. The residue was purified
by column chromatography and elution with CHCl3/MeOH
(99:1–80:20). Furthermore, the purified compound was con-
verted into its hydrochloride salt by treatment with excess
HCl in ethanol. The precipitate was collected, washed with
ethanol, and dried.
3.1.5.1. 2-[2-(Dimethylamino)ethyl]-4-methyl-8,10-dimethoxy-
anthra[1,9-cd]pyrazol-6 (2H)-one hydrochloride (6). An or-
ange solid, yield 80%; m.p. 252–254 °C; 1H NMR (10%
D2O in DMSO-d6, 300 MHz): δ 2.61 (s, 3H), 2.89 (s, 6H),
3.66 (t, 2H, J = 6.6 Hz), 3.91 (s, 3H), 4.01 (s, 3H), 4.98 (t,
2H, J = 6.6 Hz), 7.02 (d, 1H, J = 2.4 Hz), 7.40 (d, 1H,
J = 2.4 Hz), 7.77 (s, 1H), 8.00 (s, 1H); FAB-MS m/z: 366 [M
+ H]+. Anal. Calcd. for C21H23N3O3·HCl·3.4H2O: C, 54.46; H,
6.70; N, 9.07. Found: C, 54.74; H, 6.51; N, 9.01.
3.1.3. 2-(2-Hydroxyethyl)-4-methyl-8,10-dimethoxy-anthra
[1,9-cd]pyrazol-6(2H)-one (4)
To a solution of compound 3 (0.45 g, 1.0 mmol) in anhy-
drous 2-ethoxyethanol (20 ml), 2-hydroxyethylhydrazine
(0.34 ml, 5.0 mmol) was added and then heated under argon at
120 °C for 4 h. Upon cooling, the mixture was mixed with
water (200 ml), and the precipitate was then separated by fil-
tration and washed again with water (50 ml). The residue was
3.1.5.2. 2-[2-(Diethylamino)ethyl]-4-methyl-8,10-dimethoxy-
anthra[1,9-cd]pyrazol- 6(2H)-one hydrochloride (7). An or-
ange solid, yield 95%; m.p. 230–232 °C; 1H NMR (10%

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J.-H. Tan et al. / European Journal of Medicinal Chemistry 41 (2006) 1041–1047
D2O in DMSO-d6, 300 MHz): δ 1.26 (t, 6H, J = 7.0 Hz), 2.62
(s, 3H), 3.29 (q, 4H, J = 7.0 Hz), 3.71 (t, 2H, J = 5.7 Hz), 3.92
(s, 3H), 4.01 (s, 3H), 4.95 (t, 2H, J = 5.7 Hz), 7.03 (d, 1H,
J = 2.4 Hz), 7.41 (d, 1H, J = 2.4 Hz), 7.79 (s, 1H), 7.99 (s,
1H); FAB-MS m/z: 394 [M + H]+. Anal. Calcd. for
C23H27N3O3·HCl·2.1H2O: C, 59.06; H, 6.94; N, 8.98. Found:
C, 58.88; H, 6.61; N, 8.62.
(s, 3H), 4.69 (t, 2H, J = 5.7 Hz), 6.84 (d, 1H, J = 2.3 Hz), 7.55
(s, 1H), 7.63 (d, 1H, J = 2.3 Hz), 7.88 (s, 1H); FAB-MS m/z:
408 [M + H]+. Anal. Calcd. for C23H25N3O4·HCl·1.8H2O: C,
57.99; H, 6.26; N, 8.82. Found: C, 58.03; H, 6.01; N, 8.62.
3.1.5.8. 2-[2-(4-Methylpiperazin-1-yl)ethyl]-4-methyl-8,10-di-
methoxy-anthra[1,9-cd] pyrazol-6(2H)-one hydrochloride (13).
1
An orange solid, yield 85%; m.p. 189–190 °C; H NMR (not
in hydrochloride form, CDCl3, 300 MHz): δ 2.31 (s, 3H), 2.48
(m, 4H), 2.63 (s, 3H), 2.64 (m, 4H), 2.98 (t, 2H, J = 7.0 Hz),
3.95 (s, 3H), 4.08 (s, 3H), 4.62 (t, 2H, J = 7.0 Hz), 6.82 (d, 1H,
J = 1.9 Hz), 7.52 (s, 1H), 7.60 (d, 1H, J = 1.9 Hz), 7.86 (s,
1H); FAB-MS m/z: 421 [M + H]+. Anal. Calcd. for
C24H28N4O3·2HCl·3H2O: C, 52.65; H, 6.63; N, 10.23. Found:
C, 52.33; H, 6.66; N, 10.21.
3.1.5.3. 2-[2-[(2-Hydroxyethyl)amino]ethyl]-4-methyl-8,10-di-
methoxy-anthra[1,9-cd] pyrazol-6(2H)-one hydrochloride (8).
1
An orange solid, yield 80%; m.p. 236–238 °C; H NMR (10%
D2O in DMSO-d6, 300 MHz): δ 2.60 (s, 3H), 3.12 (t, 2H,
J = 6.0 Hz), 3.55 (t, 2H, J = 6.0 Hz), 3.66 (t, 2H, J = 6.0 Hz),
3.89 (s, 3H), 3.99 (s, 3H), 4.84 (t, 2H, J = 6.0 Hz), 7.01 (d, 1H,
J = 2.4 Hz), 7.40 (d, 1H, J = 2.4 Hz), 7.77 (s, 1H), 7.89 (s,
1H); FAB-MS m/z: 382 [M + H]+. Anal. Calcd. for
C21H23N3O4·HCl·2.5H2O: C, 54.49; H, 6.31; N, 9.08. Found:
C, 55.54; H, 6.48; N, 8.80.
3.2. Spectrometric titration and DNA binding assay
All types of spectrometric titration were conducted in a
1 cm quartz cuvette at room temperature (~30 °C). The CT
DNA (Sigma, St. Louis, MO) was dissolved in double distilled
deionized water with 50 mM NaCl, and dialyzed against a buf-
fer solution for 2 days. Its concentration was determined by
absorption spectrometry at 260 nm using a molar extinction
coefficient 6600 M−1 cm−1. The ratio A260/A280 > 1.80 was
used as an indication of a protein-free DNA.
Absorption titration was performed at a fixed concentration of
drugs (30 μM) in a sodium phosphate buffer (20 mM sodium
phosphate, 150 mM NaCl, pH 6.3). Small aliquots of concen-
trated CT DNA (3.9 mM) were added into the solution at final
concentrations from 0 to 165 μM, and stirred for 5 min before
measurement. The parameters, λmax, red shift, hypochromicity
and isosbestic point were found from the adsorption spectra.
Fluorescence titration was performed at a fixed concentration of
drugs (3 μM) in sodium phosphate buffer (20 mM sodium phos-
phate, 20 mM NaCl, pH 6.3). Small aliquots of concentrated CT
DNA (3.9 mM) were added into the solution at final concentra-
tions from 0 to 150 μM, and stirred for 5 min. Fluorescence in-
tensity was measured at Ex 410 nm and Ex/Em 10/10 nm. Bind-
ing constants were derived from the modified Scatchard
equation, r/Cf = Ki(1 – Nr)[(1 – Nr)/[1 – (N – 1)r]]n−1, where r
is the molar ratio of bound ligand to DNA, Cf the free ligand
concentration, Ki the binding constant and n the binding size in
base pairs [34].
3.1.5.4. 2-[2-[(2-Dimethylaminoethyl)amino]ethyl]-4-methyl-
8,10-dimethoxy-anthra [1,9-cd] pyrazol-6(2H)-one hydrochlor-
ide (9). An orange solid, yield 81%; m.p. 242–244 °C; H
NMR (10% D2O in DMSO-d6, 300 MHz): δ 2.54 (s, 3H),
2.80 (s, 6H), 3.40 (m, 4H), 3.59 (t, 2H, J = 6.0 Hz), 3.85 (s,
3H), 3.95 (s, 3H), 4.78(t, 2H, J = 6.0 Hz), 6.92 (d, 1H,
J = 2.4 Hz), 7.30 (d, 1H, J = 2.4 Hz), 7.67 (s, 1H), 7.80 (s,
1H); FAB-MS m/z: 409 [M + H]+. Anal. Calcd. for
C23H28N4O3·2HCl·3H2O: C, 51.59; H, 6.78; N, 10.46. Found:
C, 51.40; H, 6.74; N, 10.42.
1
3.1.5.5. 2-[2-[(2-Diethylaminoethyl)amino]ethyl]-4-methyl-
8,10-dimethoxy-anthra [1,9-cd]pyrazol-6(2H)-one hydrochlor-
1
ide (10). An orange solid, yield 85%; m.p. 234–236 °C; H
NMR (10% D2O in DMSO-d6, 300 MHz): δ 1.24 (t, 6H,
J = 7.0 Hz), 2.64 (s, 3H), 3.18 (q, 4H, J = 7.0 Hz), 3.41 (m,
4H), 3.67 (t, 2H, J = 4.0 Hz), 3.93 (s, 3H), 4.04 (s, 3H), 4.89
(t, 2H, J = 4.0 Hz), 7.04 (d, 1H, J = 1.8 Hz), 7.44 (d, 1H,
J = 1.8 Hz), 7.81 (s, 1H), 7.97 (s, 1H); FAB-MS m/z: 437 [M
+ H]+. Anal. Calcd. for C25H32N4O3·2HCl·1.2H2O: C, 56.54;
H, 6.91; N, 10.55. Found: C, 56.49; H, 6.88; N, 10.51.
3.1.5.6. 2-(2-Piperidin-1-yl-ethyl)-4-methyl-8,10-dimethoxy-
anthra[1,9-cd]pyrazol-6(2H)-one hydrochloride (11). A yellow
1
solid, yield 90%; m.p. 260–262 °C; H NMR (10% D2O in
DMSO-d6, 300 MHz): δ 1.93 (m, 6H), 2.63 (s, 3H), 3.06 (t,
2H, J = 6.5 Hz), 3.68 (m, 4H), 3.92 (s, 3H), 4.02 (s, 3H), 4.89
(t, 2H, J = 6.5 Hz), 7.03 (d, 1H, J = 2.5 Hz), 7.42 (d, 1H,
J = 2.5 Hz), 7.78 (s, 1H), 7.96 (s, 1H); FAB-MS m/z: 406 [M
+ H]+. Anal. Calcd. for C24H27N3O3·HCl·H2O: C, 62.67; H,
6.57; N, 9.14. Found: C, 62.38; H, 6.40; N, 9.01.
3.3. Cell cultures and cytotoxicity assay
All tumor cell lines, mouse melanoma B16, human hepato-
cellular carcinoma HepG2 and LLC cell (from ATCC, Rock-
ville, MD) were cultured on RPMI-1640 medium supplemen-
ted with 10% fetal bovine serum, 100 U ml–1 penicillin and
100 μg ml–1 streptomycin in 25-cm2 culture flasks at 37 °C
in humidified atmosphere with 5% CO2.
For the cytotoxicity tests, cells in exponential growth stage
were harvested from culture by trypsin digestion and centrifu-
ging at 180 × g for 3 min, then resuspended in fresh medium at
3.1.5.7. 2-(2-Morpholinoethyl)-4-methyl-8,10-dimethoxy-an-
thra[1,9-cd]pyrazol-6(2H)- one hydrochloride (12). An orange
1
solid, yield 91%; m.p. 242–244 °C; H NMR (not in hydro-
chloride form, CDCl3, 300 MHz): δ 2.62 (m, 4H), 2.65 (s,
3H), 3.03 (t, 2H, J = 5.7 Hz), 3.73 (m, 4H),3.97 (s, 3H), 4.10

J.-H. Tan et al. / European Journal of Medicinal Chemistry 41 (2006) 1041–1047
1047
a cell density of 5 × 104 cells per ml. The cell suspension was
dispensed into a 96-well microplate at 100 μl per well, and
incubated in humidified atmosphere with 5% CO2 at 37 °C
for 24 h, and then treated with the compounds at various con-
centrations (0, 1, 10, 100 μM). After 48 h of treatment, 50 μl of
1 mg ml–1 MTT solution was added to each well, and further
incubated for 4 h. The cells in each well were then solubilized
with DMSO (100 μl for each well) and the optical density
(OD) was recorded at 570 nm. All drug doses were tested in
triplicate and the IC50 values were derived from the mean OD
values of the triplicate tests versus drug concentration curves.
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