Synthesis and structure-activity correlations of the cytotoxic bifunctional 1,4-diamidoanthraquinone derivatives

Article abstract of DOI:10.1016/j.bmc.2004.09.001

Anthraquinone-based compounds are attractive target for the design of new anticancer drugs. We have previously described a series of 1,5- and 1,4-difunctionalized anthraquinones, which exhibit different spectra of potency, together with human telomerase evaluation. The present study details the preparation of further, distinct series of regioisomeric difunctionalized amidoanthraquinone and examines their in vitro cytotoxicity in C6, Hepa G2, and 2.2.15 cell lines. Two structurally related compounds, mitoxantrone and adriamycin, were tested in parallel as positive controls. The structure-activity relationships indicated amido substitution may lead to a different mechanism of cytotoxicity. Compounds, which have -(CH₂)_n- side chains terminating in basic groups such as aminoalkyl-substituted, showed cytotoxic activity in several cell lines. The exact mode of intercalative binding may be dictated by the positional placement of substituent side chains. Implications for amidoanthraquinone cytotoxicity as potential anticancer agents are discussed. In addition, we further delineate the nature of the pharmacophore for this class of compounds, which provides a rational basis for the structure-activity relationships.

Full text of DOI:10.1016/j.bmc.2004.09.001

Bioorganic & Medicinal Chemistry 12 (2004) 6163–6170
Synthesis and structure–activity correlations of the cytotoxic
bifunctional 1,4-diamidoanthraquinone derivatives
Hsu-Shan Huang,a,* Hui-Fen Chiu,b An-Long Lee,a Ching-Long Guoa and
Chun-Lung Yuana
aSchool of Pharmacy, National Defense Medical Center, PO Box 90048-508, Neihu114 Taipei, Taiwan, ROC
bDepartment of Pharmacology, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC
Received 1 July 2004; revised 1 September 2004; accepted 1 September 2004
Available online 28 September 2004
Abstract—Anthraquinone-based compounds are attractive target for the design of new anticancer drugs. We have previously
described a series of 1,5- and 1,4-difunctionalized anthraquinones, which exhibit different spectra of potency, together with human
telomerase evaluation. The present study details the preparation of further, distinct series of regioisomeric difunctionalized amido-
anthraquinone and examines their in vitro cytotoxicity in C6, Hepa G2, and 2.2.15 cell lines. Two structurally related compounds,
mitoxantrone and adriamycin, were tested in parallel as positive controls. The structure–activity relationships indicated amido sub-
stitution may lead to a different mechanism of cytotoxicity. Compounds, which have –(CH2)n– side chains terminating in basic
groups such as aminoalkyl-substituted, showed cytotoxic activity in several cell lines. The exact mode of intercalative binding
may be dictated by the positional placement of substituent side chains. Implications for amidoanthraquinone cytotoxicity as poten-
tial anticancer agents are discussed. In addition, we further delineate the nature of the pharmacophore for this class of compounds,
which provides a rational basis for the structure–activity relationships.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
medicinal chemists have turned toward analog develop-
ment involving certain anthraquinones.12
There is continuing interest in the development of new
agents in which the small-molecule tricyclic anthraqui-
none structural motif represents an attractive target for
the rational design of new anticancer agents due to its
central role in the control of cellular proliferation.1–6
An area of particular activity and promise is concerned
with aminoalkyl-substituted anthraquinone and the
demonstration of clinical activity for the two structurally
related 1,4-disubstituted compounds mitoxantrone and
ametantrone.7–10 Moreover, a reversal in the direction
of mitoxantrone dipole moment occurs in the amido con-
geners. This may explain the lack of recognition of cleav-
able topoisomerase II-DNA complex and the loss of
cleavage stimulation.11 However, a more urgent need is
apparent from the common experience of clinically limit-
ing toxicities of most anticancer drugs, that is, the neces-
sity to develop less toxic clinical drug candidates. Thus,
We recently described the human telomerase activation
properties of a series of disubstituted anthraquinones
(1,4- and 1,5-isomers)3 and proposed that their activity
may be due to their ability to bind to and stabilize G-
quadruplex structures.5,13 The inhibition of telomerase
by molecules such as disubstituted amidoanthraquin-
ones is believed to be due to their stabilization of gua-
nine-quadruplex complexes.14 Computer modeling and
energy calculations have shown that some amidoanthra-
quinones can bind intercalatively to DNA and that there
are significant differences in the additional non-bonded
and electrostatic interactions possible at the DNA bind-
ing site. Solution DNA binding and closed-circular
DNA unwinding studies confirmed intercalative inter-
actions, and the predicted differences in strength of
interactions between mono- and disubstituted com-
pounds have been found.8 This may be due to marked
differences in the nature of chromophore-base stacking
and groove accessibility for these series compounds.
The experimental model results are in accord with the
predicted behavior and confirm that the 1,4-series bind
preferentially to double- rather than triple-stranded
Keywords: Anthraquinone; Cytotoxic; Regioisomeric; Mitoxantrone;
Amidoanthraquinone.
*
Corresponding author. Tel.: +886
87923169; e-mail: huanghs@ndmctsgh.edu.tw
2 87926454; fax: +886 2
0968-0896/$ - see front matter ꢀ 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.001

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H.-S. Huang et al. / Bioorg. Med. Chem. 12 (2004) 6163–6170
O
H
N
O
O
Planar rings
H
N
Cl
O
HN
OH
OH
X
O
O
HN
HN
n
R
R
O
NH2
NH2
O
HN
n
1
1
4
Cl(CH2)n-COCl
N,N-Diethylacetamide
pyridine, rt
4
X
O
HN
O
O
HN
N
H
n N
H
n Cl
O
1
2-4
Mitoxantrone (X = OH)
Ametantrone (X = H)
1,4-isomer
R-NH2
N,N-Diethylacetamide
pyridine, rt
R-COCl
EtOH, reflux
Chart 1.
O
O
H
N
O
HN
n
R
R
O
HN
HN
R
R
DNA.15 Although the precise mode of action in vitro or
in vivo for these compounds has not been fully eluci-
dated, a number of studies have shown that DNA is a
major biological target for mitoxantrone.6,8,16–19 Triplex
stabilization could be achieved with a series of disubsti-
tuted amidoanthraquinones, which are able to discrimi-
nate between duplex and triplex DNA by virtue of their
structural characteristics.15 Earlier studies by Palumbo
et al.,20,21 Keppler et al.,19,22 and Collier and Neidle8
have shown direct and indirect relationships between
DNA-binding parameters and at least some biological
effects for a series of mono- and disubstituted amido-
anthraquinones. Hence, amido substitution may lead
to a different mechanism of cytotoxicity, not related to
classical protein or free radical mediated DNA damage,
which points to a novel type of anticancer pharmaco-
phore (Chart 1).
O
HN
O
O
10-38
n N
H
O
5-9
Scheme 1.
1,4-diaminoanthraquinone with chloro-acyl chloride,
in an inert N,N-diethylacetamide as a solvent, with a
catalytic quantity of pyridine, producing the 1,4-bis-
(x-chloroalkaneamido) side chain compounds 2–4 in
essentially quantitative yield. Further condensation of
these intermediates with the appropriate amines in etha-
nol gave the desired disubstituted anthraquinones 5–9.
Compounds 10–38 were synthesized by direct one-stage
acylation reaction. Muller and Prinz23 have shown that
¨
The in vitro cytotoxicity of these compounds have been
reported and compared with mitoxantrone and adriamy-
cin, which were tested in parallel as positive controls. We
used XTT method to measure the relative cytotoxicity of
1,4-disubstituted amidoanthraquinone compounds, and
have examined how the attached base-functionalized
substituents affects their cytotoxicity to several tumor
cell lines. Of the compounds studied, the 1,4-diamino-
alkyl-substituted amidoanthraquinone displayed better
in vitro cytotoxicity. These results have been confirmed
by previous molecular modeling studies,19 which provide
a rational basis for the structure–activity relationships.
These suggest that, although all of the compounds bind
through an intercalative mode, the 1,5-disubstituted iso-
mers bind with their two side groups occupying adjacent
triplex grooves, in contrast with the other isomer, which
is positioned with both side groups in the same triplex
groove. This inhibitory ability also provides an indica-
tion of selective tumor cell lines inhibition. A range of
side chains has been examined to establish SAR as a
basis for subsequent rational drug design.
electrophilic additions or substitutions and nucleophilic
additions occur at different oxidation states of anthra-
quinones and the reactions are complementary from a
preparative viewpoint. These compounds were obtained
in good yield and determined to be pure by mass
spectrometry, 1H NMR, and 13C NMR. The amide
group is coplanar with the anthraquinone ring, and
there is an intramolecular hydrogen bond between the
amide hydrogen atom and the quinoid oxygen atom
on the ring.8 These heteroatoms have a significant effect
on the anthraquinone moiety, influencing the electron
density and thus affecting p-electron interaction with
DNA bases and also electron-accepting ability of the
quinonoid system, essential for the metabolic activation
of the molecule. This amidation causes the strong dimin-
ishment of p-electron density in the quinone moiety24
(Scheme 1).
2.2. Biological activity tests
The cytotoxic behavior of the compounds were evalu-
ated against a panel of murine tumor cell line and
human hepatoma cell lines (heap G2 and 2.2.15) using
the XTT assay, as described previously.25,26 Results
are presented in Table 1 as the concentrations required
to inhibit cell growth by 50% (IC50 values). Prior to
the evaluation of 1,5-isomers in XTT assay and telom-
erase assay,3,25,26 the agents were tested for their cyto-
toxicity and dual telomerase activity. As part of work
in our laboratories on the use of anthraquinones as anti-
cancer agents, a number of bis-(amino)-substituted27
2. Results and discussion
2.1. Synthetic approach
A series of 1,4-difunctionalized amidoanthraquinones
have been prepared, with side chains having basic nitro-
gen atoms. Compounds 5–9 were synthesized by a two-
stage reaction. The first stage involved acylation of the

H.-S. Huang et al. / Bioorg. Med. Chem. 12 (2004) 6163–6170
6165
Table 1. Chemical yields of 1,4-diamidoanthraquinones (2–38), and their cytotoxicity against suspended murine and human tumor cell lines
Compound
R
Yield (%)
IC50 (lM)a
Hepa G2c
C6b
8.51 0.08
2.2.15d
51.51 2.21
2
CH2Cl
CH2CH2Cl
CHClCH3
77
78
76
50
40
75
63
63
92
76
80
82
87
79
63
75
83
82
83
75
88
82
85
27.41 1.32
3
20.62 0.96
3.81 0.07
0.60 0.10
1.06 0.12
1.42 0.33
5.24 0.12
0.40 0.09
32.27 0.7431.36 0.87
35.37 0.5471.17 1.92
1.43 0.24
4
5
CH2N(CH2CH3)2
CH2CH2N(CH2CH3)2
CHCH3N(CH2CH3)2
CHCH3NHCH2CH(CH2)2
CH2CH2 NHCH2CH(CH2)2
CH3
1.98 0.01
1.15 0.04
6
0.89 0.02
7
13.49 0.39
10.62 0.30
1.01 0.01
16.74 1.63
15.42 0.28
6.04 0.59
8
9
10
19.12 0.8457.36 3.24108.3 3.74
11
12
Cyclopropane
Cyclopentane
Cyclohexane
CH2CH2C5H9
2-Thiophenyl
2-Furyl
13.25 0.03
22.82 1.82
29.51 2.17
24.41 1.94
17.39 0.07
11.70 0.03
8.26 0.03
10.15 1.21
25.52 1.07
23.08 0.97
13.12 0.76
9.48 0.03
25.16 0.12
21.24 1.56
0.53 0.01
46.62 1.07
44.57 1.78
57.05 1.96
3.24 0.03
2.61 0.03
43.57 1.14
47.25 1.24
64.28 2.14
9.33 0.05
13
14
15
16
18.44 1.36
13.33 0.92
53.12 2.17
103.84 3.17
44.42 0.57
54.34 1.27
10.42 0.72
27.23 1.78
29.75 0.32
23.51 0.17
13.21 0.06
39.54 1.92
86.17 4.67
100.26 4.13
94.20 3.47
12.31 0.61
93.10 3.62
21.56 0.84
17
18
2-Thiopheneacetyl
C6H5
3-CH3C6H4
19
20
2-FC6H4
3-FC6H4
21
22
4-FC6H4
2-ClC6H4
23
24
3-ClC6H4
4-ClC6H4
25
26
.02 1.02
8424 31.3318.816.04 0.92
2-NO2C6H4
4-CF3C6H4
70
78
73.68 2.12
40.88 0.62
58.11 3.01
6.51 0.07
15.23 0.92
32.93 0.32
10.26 0.13
48.31 1.07
36.57 2.91
6.57 0.06
18.62 0.92
34.82 0.62
10.95 0.06
27
28
24.51 0.62
0.49 0.05
22.33 1.67
22.16 0.86
0.71 0.21
2,5-(CF3)2C6H3
2,4-F2C6H3
2,4-Cl2C6H3
87
66
29
30
80
60
31
32
2,4,6-Cl3C6H2
2,3,6-F3C6H2
2,4,5-F3C6H2
2,3-Cl2-5-FC6H2
trans-CH(CH2)CHC6H5
CH2SC6H5
8420.62 0.68
70
25.9225.802.84 0.66
17.91 1.13
33
34
17.41 0.37
19.43 1.02
77
68
103.58 0.0410.71 0.11
11.31 0.06 12.86 0.46
23.413.32 0.05
13.53 0.14
35
36
21.10 1.67
845.82 60.051.21
37
38
CH2C6H4F(p)
2,5-Dimethylfuryl
85
79
14.14 0.72
11.83 0.07
0.07 0.01
1.00 0.16
25.88 0.72
8.12 0.67
2.00 0.50
0.90 0.01
16.43 0.04
21.17 0.21
0.40 0.02
1.60 0.04
Mitoxantrone
Adriamycin
a IC50, drug concentration inhibiting 50% of cellular growth following 48h of drug exposure. Values are in micromolar and represent an average of
three experiments. The variance for the IC50 values was less than 20%. Inhibition of cell growth was significantly different with respect to that of
the control; n = 3 or more, P < 0.01. Inhibition was compared with that of the control and standard errors.
b Hep G2, human hepatoma G2 cells.
c C6 cells, rat glioma C6 cells.
d 2.2.15 cells, hepatitis B virus transfected hepatoma cell lines, HepG 2.2.15 cells.
and bis-(amido)-substituted derivatives have been syn-
thesized and also fully characterized as potential telom-
erase inhibitors.
toxic activity shown by the substituents of tricyclic
derivatives is at first surprising given their structural
similarities. However, compounds 5–7, 9, 28, and 31
exhibited potent cytotoxic activities on C6 cell lines; 5,
6, 9, and 11 exhibited potent cytotoxic activities on
hepaG2 cell lines; and 5, 6, and 11 exhibited potent
cytotoxic activities on 2.2.15 cell lines.
The amidoanthraquinones described here exhibit no
cytotoxicity except for 5–9, 11, 28, and 31, which IC50
values in the low-micromolar range compared with
mitoxantrone and adriamycin, against a panel of three
cell lines (Murine C6, human Hepa G2, and 2.2.15).
The regioisomeric alkylamino-amido derivatives 5–9
were found to be the most potent of the amidoanthra-
quinone compounds studied. The most potent com-
pounds described here show levels of cytotoxicity that
compare favorably with mitoxantrone and adriamycin.
Where direct comparisons with analogous 1,4-isomer
anthraquinones are possible (Table 1), the levels of cyto-
3. Conclusions
This study has shown that other tricyclic systems in
addition to anthraquinone-based molecules can selec-
tively inhibit the growth of tumor cell lines. Further-
more, we also have demonstrated that conventional
cytotoxicity may be moderated through rational design

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H.-S. Huang et al. / Bioorg. Med. Chem. 12 (2004) 6163–6170
according to the substituent on the small-molecule tri-
cyclic anthraquinone structural motif. However, the dif-
ference between the 1,4-isomer and 1,5-isomer27 has a
significant effect on the shape of the chromophore.
The 1,5-disubstituted derivative also shows some inter-
action with duplex DNA, and these results have been
confirmed by molecular modeling studies, which provide
a rational basis for the structure–activity relationships.19
This, in turn, affects the angular disposition of the sub-
stituents compared to the mitoxantrone anthraquinone,
so that a good fit of the disubstituted substituents into
the putative binding site can only be achieved with a
degree of distortion of the site and a resultant energy
cost. The exploitation of rationally derived structure–
activity relationships, such as have been found in this
study, is likely to be a key to future success.
NMR: Varian GEMINI-300 (300MHz) and Brucker
AM-500 (500MHz); d values are in parts per million rel-
ative to TMS as an internal standard. Fourier-transform
IR spectra (KBr): Perkin–Elmer 983G spectrometer.
The UV spectra were recorded on a Shimadzu UV-
160A. Mass spectra (EI, 70eV, unless otherwise stated):
Finnigan MAT TSQ-46, Finnigan MAT TSQ-700 (Uni-
versita¨t Regensburg, Germany) and Finnigan MAT
LCQ-MS (National Research Institute of Chinese Med-
icine, Taipei, Taiwan). Typical experiments illustrating
the general procedures for the preparation of the anthra-
quinones are described below.
4.2. General procedure for the preparation of 1,4-diamido-
anthraquinones
4.2.1. Method A (compounds 2–9). Chloroacetylchloride
(12mmol) was added dropwise at 0ꢁC under N2 to a
solution of 1,4-diaminoanthraquinone (4mmol) and
pyridine (0.5mL) in N,N-diethylacetamide (70mL).
The reaction mixture was stirred for 24h at room tem-
perature. The resulting precipitate was collected by fil-
tration, washed with diethyl ether, and purified by
crystallization from ethyl acetate/n-hexane. A solution
of an appropriate amines (20mmol) in ethanol was
added dropwise under N2 to a suspended solution of
2–4 (1mmol) in 70mL of ethanol. The reaction mixture
was refluxed for 18h. The solvent was removed under re-
duced pressure to yield dark brown solids and the result-
ing precipitate was collected by filtration, washed with
diethyl ether, and purified by crystallization from ethyl
acetate/n-hexane.
The aim of this study was to elucidate the structure–
activity relationships of simple disubstituted amido-
anthraquinones and aminoanthraquinones to further
delineate the nature of the requirements of the pharma-
cophore. A striking feature of the results given in Table
1 is the marked cytotoxicity between the three different
tumor cell lines. Anthraquinone derivatives are known
for their antitumor activities. Also, these effects are very
sensitive to slight modifications of anthraquinone sub-
stituents. In light of these findings it is suggested that
cytotoxicity in cell lines is not sufficient for potent anti-
proliferative action. Finally, the effects of anthraqui-
nones on the cytotoxicity in cancer cells are very
diverse, so a simple correlation between the modifica-
tions of anthraquinones with their biological effects only
can be achieved when concerned with aminoalkyl-sub-
stituted anthraquinone. Thus, it appears that the anthra-
quinone chromophore itself causes the nature of
minimum requirements of the pharmacophore of cyto-
toxicity. Whatever be the molecular mechanism of the
cytotoxicity of anthraquinone derivatives and wherever
be its locus, the results described herein indicate that it
is sensitive to the slightest modification in the structure
of anthraquinones. However, the molecular mechanisms
behind these diversified biological effects are unclear.
Thus, a early decision in our attempts to develop new
therapeutically effective anticancer drugs was to keep
the anthraquinone unchanged3,25,26 because these struc-
tural portions may be important for the pharmaco-
phore. It now appears that the proposed structural
pattern hypothesis has been substantiated by the exper-
imental results obtained from the present paper. This
unique property should definitely be noted in future
drug design; and the potential application of these com-
pounds in tumor biology, stem cell research, and tissue
engineering has made them a group of compounds that
are worth extensive study.
4.2.2. Method B. Acyl chlorides (12mmol) were added
dropwise at 0ꢁC under N2 blanketing to a solution of
1,4-diaminoanthraquinone (4mmol) and pyridine
(0.5mL) in N,N-diethylacetamide (70mL). The reaction
mixture was stirred for 24h at room temperature. The
resulting precipitate was collected by filtration, washed
with diethyl ether, and purified by crystallization from
ethyl acetate/n-hexane.
4.2.3. 1,4-Bis(chloroacetamido)anthraquinone (2). Yield:
1
77%; mp 285–287ꢁC (chloroform); H NMR (DMSO):
d 4.60 (4H, s, CH2), 7.97–8.00 (2H, m, H-6,7), 8.24–
8.27 (2H, m, H-5,8), 8.99 (2H, s, H-2,3), 12.80 (2H, s,
NH); IR (KBr): 1595, 1650cmÀ1; UV kmax (MeOH):
486.0 (1.52); MS m/z: 390 (100%), 341 (95%), 237 (61%).
4.2.4. 1,4-Bis(3-chloropropionamido)anthraquinone (3).8
1
Yield: 78%; mp 230–231ꢁC (EA/n-hexane); H NMR
(CDCl3): d 3.04–3.08 (4H, t, J = 6.6Hz, CH2), 3.96–
4.00 (4H, t, J = 6.5Hz, CH2), 7.87–7.97 (H, m, H-6,7),
8.32–8.35 (2H, m, H-5,8), 9.23 (2H, s, H-2,3), 12.72
(2H, s, NH); 13C NMR (CDCl3): d 39.3 (CH2), 41.52
(CH2), 116.95 (CH), 127.17 (C), 129.12 (CH), 133.23
(CH), 134.56 (C), 138.12 (C), 169.08 (C), 186.98 (C);
IR (KBr): 1600, 1650, 1710cmÀ1; UV kmax (MeOH):
465.0 (1.04); MS m/z: 418 (68%), 328 (92%), 238 (100%).
4. Experimental
4.1. Materials
Melting points were determined with a Buchi B-545
¨
melting point apparatus and are uncorrected. All reac-
tions were monitored by TLC (silica gel 60 F254). H
4.2.5. 1,4-Bis(2-chloropropionamido)anthraquinone (4).
Yield: 76%; mp 233–235ꢁC (EA/n-hexane); H NMR
(CDCl3): d 1.91–1.94(6H, d, J = 7.2Hz, CH3), 4.62–
1
1

H.-S. Huang et al. / Bioorg. Med. Chem. 12 (2004) 6163–6170
6167
4.69 (2H, q, J = 7.7Hz, CH), 7.86–7.89 (2H, m, H-6,7),
8.36–8.39 (2H, m, H-5,8), 9.21 (2H, s, H-2,3), 13.24(2H,
s); 13C NMR (CDCl3): d 22.44 (CH3), 55.09 (CH),
118.10 (CH), 127.30 (C), 128.71 (CH), 133.05 (CH),
134.55 (C), 137.83 (C), 169.67 (C), 186.76 (C); IR
(KBr): 3155, 1648cmÀ1; UV kmax (MeOH): 459.0
(0.51); MS m/z: 418 (63%), 355 (100%), 292 (36%).
(MeOH): 465.0 (0.25); MS m/z: 321 (92%), 280 (66%),
237 (70%).
4.2.12. 1,4-Bis(cyclopropylamido)anthraquinone (11).
Yield: 76%; mp 281–282ꢁC (EA/n-hexane); 1H NMR
(CDCl3): d 0.98–1.02 (8H, m), 1.79–1.84(2H, m),
7.85–7.88 (2H, m, H-6,7), 8.33–8.36 (2H, m, H-5,8),
9.19 (2H, s, H-2,3), 12.86 (2H, s, NH); 13C NMR
(CDCl3): d 8.51 (CH2), 17.00 (CH2), 116.26 (CH),
127.01 (C), 129.22 (CH), 133.48 (CH), 134.22 (C),
138.63 (C), 173.40 (C), 186.88 (C); IR (KBr): 3315,
1680cmÀ1; UV (MeOH): 490.0 (0.64); MS m/z: 374
(87%), 306 (51%), 238 (100%).
4.2.6. 1,4-Bis[2-(diethylamino)acetamido]anthraquinone
1
(5). Yield: 50%; mp 85–86ꢁC (EA/n-hexane); H NMR
(CDCl3): d 1.50–1.55 (12H, t, J = 8.4Hz, CH3), 3.05–
3.08 (8H, s, CH2), 4.29–4.32 (4H, s, COCH2), 7.87–
7.90 (2H, m, H-6,7), 8.36–8.39 (4H, m, H-5,8), 9.22
(2H, s, H-2,3), 13.28 (2H, s, NH); IR (KBr): 3365,
1688cmÀ1; UV kmax (MeOH): 525.0 (0.50); MS m/z:
463 (55%), 237 (31%).
4.2.13. 1,4-Bis(cyclopentylamido)anthraquinone (12).
Yield: 80%; mp 243–244ꢁC (EA/n-hexane); 1H NMR
(CDCl3): d 1.90–1.99 (8H, m), 2.01–2.17 (8H, m),
2.90–3.01 (2H, d, J = 8.1Hz), 7.82–7.88 (2H, m, H-
6,7), 8.29–8.34(2H, m, H-5,8), 9.22 (2H, s, H-2,3),
12.63 (2H, s, NH); 13C NMR (CDCl3): d 25.88 (CH2),
29.62 (CH2), 30.37 (CH2), 48.13 (CH), 116.52 (CH),
127.00 (C), 129.18 (CH), 133.42 (CH), 134.17 (C),
138.77 (C), 176.09 (C), 186.85 (C); IR (KBr): 3375,
1645cmÀ1; UV (MeOH): 474.0 (0.50); MS m/z: 4 30
(36%), 334(28%), 238 (100%).
4.2.7. 1,4-Bis[3-(diethylamino)propionamido]anthraqui-
none (6).27 Yield: 40%; mp 169–171ꢁC (EA/n-hexane);
1H NMR (CDCl3): d 2.76–2.80 (12H, t, J = 7.6Hz,
CH3), 3.04–3.12 (8H, m, CH2), 3.21–3.25 (8H, m,
CH2), 7.80–7.86 (2H, m, H-6,7), 8.23–8.31 (2H, m, H-
5,8), 9.01 (2H, s, H-2,3), 12.68 (2H, br, NH); IR
(KBr): 3160, 1636cmÀ1; UV (MeOH): 485.0 (0.57);
MS m/z: 491 (45%), 237 (58%).
4.2.8. 1,4-Bis[2-(diethylamino)propionamido]anthraqui-
none (7). Yield: 75%; mp 175–177ꢁC (EA/n-hexane);
1H NMR (CDCl3): d 1.49–1.53 (12H, t, J = 7.5Hz,
CH3), 1.90–1.94(8H, m, CH 2), 3.06–3.08 (6H, d,
J = 8.0Hz, CH3), 4.59–4.66 (2H, m, CH), 7.76–7.86
(2H, m, H-6,7), 8.32–8.37 (2H, m, H-5,8), 8.93–8.96
(2H, d, J = 8.0Hz, H-2,3), 13.23 (2H, br, NH); IR
(KBr): 3390, 1645cmÀ1; UV (MeOH): 546 (0.9); MS
m/z: 491 (48%), 237 (65%).
4.2.14. 1,4-Bis(cyclohexylamido)anthraquinone (13).
Yield: 82%; mp 309–310ꢁC (EA/n-hexane); 1H NMR
(CDCl3): d 1.91–1.95 (12H, m), 2.11–2.15 (8H, d,
J = 8.0Hz), 2.42–2.52 (2H, m), 7.83–7.89 (2H, m, H-
6,7), 8.33–8.36 (2H, m, H-5,8), 9.24(2H, s, H-2,3),
12.61 (2H, s, NH); 13C NMR (CDCl3): d 25.72 (CH2 ),
29.64(CH 2), 47.45 (C), 116.66 (CH), 127.04 (C),
129.27 (CH), 133.39 (CH), 134.22 (C), 138.79 (C),
176.08 (C), 186.89 (C); IR (KBr): 3365, 1649cmÀ1; UV
(MeOH): 470.0 (0.70); MS m/z: 458 (96%), 348 (47%),
238 (100%).
4.2.9. 1,4-Bis[2-(cyclopropylmethylamino)propionamido]-
anthraquinone (8). Yield: 63%; mp 172–174ꢁC (EA/n-
hexane); 1H NMR (CDCl3): d 1.90–1.94(10H, t,
J = 6.6Hz), 4.61–4.66 (2H, m, CH), 7.78–7.88 (2H, m,
H-6,7), 8.32–8.38 (2H, m, H-5,8), 8.93–8.96 (2H, d,
J = 8.0Hz, H-2,3), 13.24(2H, br, NH); IR (KBr):
3170, 1660cmÀ1; UV (MeOH): 489.0 (0.72); MS m/z:
487 (58%), 341 (45%), 307 (38%), 237 (55%).
4.2.15. 1,4-Bis(3-cyclopentanepropylamido)anthraquinone
(14). Yield: 87%; mp 166–167ꢁC (EA/n-hexane); 1H
NMR (CDCl3): d 1.62–1.72 (4H,d, J = 5.1Hz), 1.86–
1.91 (10H, m), 2.58–2.63 (2H, t, J = 7.5Hz, CH2),
7.85–7.88 (2H, m, H-6,7), 8.33–8.36 (2H, m, H-5,8),
9.24(2H, s, H-2,3), 12.60 (2H, s, NH); 13C NMR
(CDCl3): d 25.14(CH 2), 31.68 (CH2), 32.50 (CH2),
38.21 (CH2), 39.69 (CH2), 114.98 (CH), 127.03 (CH),
4.2.10. 1,4-Bis[3-(cyclopropylmethylamino)propionamido]-
anthraquinone (9). Yield: 63%; mp 208–210ꢁC (EA/n-
hexane); 1H NMR (CDCl3): d 0.90–0.92 (10H, t,
129.15 (CH), 133.38 (CH), 134.26 (CH), 138.60 (C),
À1
173.14(CH), 186.87 (C); IR (KBr): 3360, 1625cm
UV (MeOH): 469.0 (038); MS m/z: 486 (47%), 362
;
J = 7.5Hz, CH2), 1.90–1.94(H4 , t,
J = 7.5Hz, CH2),
(33%), 238 (100%).
4.59–4.66 (10H, m), 7.77–7.86 (2H, m, H-6,7), 8.33–
8.38 (2H, m, H-5,8), 8.94–8.97 (2H, d, J = 9.6Hz, H-
2,3), 13.23 (2H, br, NH); IR (KBr): 3325, 1650cmÀ1
UV (MeOH): 525.0 (0.29); MS m/z: 487 (41%), 237
(75%).
;
4.2.16. 1,4-Bis(2-thiophenylamido)anthraquinone (15).
Yield: 79%; mp 321–322ꢁC (EA/n-hexane); H NMR
1
(CDCl3): d 7.26–7.29 (2H, t, J = 3.1Hz), 7.67–7.69
(2H, d, J = 5.1Hz), 7.88–7.91 (2H, m, H-6,7), 8.02–
8.03 (2H, t, J = 2.4Hz), 8.39–8.42 (2H, m, H-5,8), 9.38
(2H, s, H-2,3), 13.61 (2H, s, NH); IR (KBr): 3380,
1605cmÀ1; UV (MeOH): 514.0 (0.49); MS m/z: 4 58
(60%), 111 (100%).
4.2.11. 1,4-Bis(acetamido)anthraquinone (10). Yield:
92%; mp 278–280ꢁC (EA/n-hexane); 1H NMR (CDCl3):
d 2.38 (6H, s, CH3), 7.85–7.88 (2H, m, H-6,7), 8.31–8.34
(2H, m, H-5,8), 9.20 (2H, s, H-2,3), 12.56 (2H, s); 13C
NMR (CDCl3): d 25.70 (CH3), 116.05 (CH), 127.02
(C), 129.05 (CH), 133.33 (CH), 134.36 (C), 138.49 (C),
169.71 (C), 186.88 (C); IR (KBr): 3370, 1610cmÀ1; UV
4.2.17. 1,4-Bis(2-furylamido)anthraquinone (16). Yield:
63%; mp 365–367ꢁC (EA/n-hexane); 1H NMR (CDCl3):

6168
H.-S. Huang et al. / Bioorg. Med. Chem. 12 (2004) 6163–6170
d 6.66–6.67 (2H, d, J = 3.6Hz), 7.39–7.40 (2H, d,
J = 3.6Hz), 7.76 (2H, s), 7.87–7.90 (2H, m, H-6,7),
8.41–8.43 (2H, m, H-5,8), 9.42 (2H, s, H-2,3), 13.55
(2H, s, NH); IR (KBr): 3366, 1658cmÀ1; UV (MeOH):
578.0 (0.39); MS m/z: 427 (100%), 343 (12%), 166 (11%).
(C), 135.23 (C), 138.85 (C), 162.88 (C), 165.05 (C),
187.25 (C); IR (KBr): 3370, 1655cmÀ1; UV (MeOH):
521.0 (1.08); MS m/z: 482 (79%), 123 (100%).
4.2.23. 1,4-Bis(4-fluorobenzamido)anthraquinone (22).
1
Yield: 88%; mp 309–311ꢁC (EA/n-hexane); H NMR
(CDCl3): d 7.28–7.34(H4 , m), 7.89–7.92 (2H, m, H-
6,7), 8.22–8.26 (4H, m), 8.39–8.42 (2H, m, H-5,8), 9.47
(2H, s, H-2,3), 13.59 (2H, s, NH), 13C NMR (CDCl3):
d 116.35 (CH), 118.39 (CH), 122.85 (CH), 124.64
(CH), 127.11 (C), 129.57 (CH), 131.70 (CH), 133.36
(CH), 133.59 (CH), 134.23 (C), 138.19 (C), 160.88 (C),
163.09 (C), 186.60 (C); IR (KBr): 3390, 1680cmÀ1; UV
(MeOH): 590.0 (0.09); MS m/z: 482 (100%), 343 (34%).
4.2.18. 1,4-Bis(2-thiopheneacetamido)anthraquinone (17).
Yield: 75%; mp 158–160ꢁC (EA/n-hexane); 1H NMR
(CDCl3): d 4.09 (4H, s, CH2), 7.10–7.13 (2H, t,
J = 4.4Hz), 7.17–7.18 (2H, d, J = 3.3Hz), 7.34–7.36
(2H, d, J = 5.7Hz), 7.81–7.84(2H, m, H-6,7), 8.22–
8.25 (2H, m, H-5,8), 9.19 (2H, s, H-2,3), 12.58 (H, s,
NH); 13C NMR (CDCl3): d 39.71 (CH2), 117.23 (CH),
125.60 (CH), 127.05 (C), 127.22 (CH), 127.68 (CH),
128.91 (CH), 133.14 (CH), 134.29 (C), 134.97 (C),
138.19 (C), 169.67 (C), 186.66 (C); IR (KBr): 3375,
1610cmÀ1; UV (MeOH): 486.56 (0.72); MS m/z: 4 86
(19%), 389 (33%), 292 (29%), 265 (40%), 97 (100%).
4.2.24. 1,4-Bis(2-chlorobenzamido)anthraquinone (23).
1
Yield: 82%; mp 324–326ꢁC (EA/n-hexane); H NMR
(CDCl3): d 7.46–7.54 (4H, m), 7.55–7.60 (4H, m),
7.78–7.86 (2H, m, H-6,7), 8.28–8.31 (2H, m, H-5,8),
9.46 (2H, s, H-2,3), 13.05 (2H, s, NH); 13C NMR
(CDCl3): d 117.17 (CH), 127.14(C), 127.17 (CH),
129.27 (CH), 129.45 (CH), 131.65 (C), 131.72 (CH),
134.46 (C), 138.37 (C), 166.10 (C), 186.10 (C); IR
(KBr): 3365, 1655cmÀ1; UV (MeOH): 365.0 (1.18);
MS m/z: 514(10%), 141 (31%), 139 (100%).
4.2.19. 1,4-Bis(benzamido)anthraquinone (18). Yield:
83%; mp 285–287ꢁC (EA/n-hexane); 1H NMR (CDCl3):
d 7.64–7.69 (6H, m), 7.89–7.92 (2H, m, H-6,7), 8.23–8.26
(4H, d, J = 2.1Hz), 8.41–8.44 (2H, m, H-5,8), 9.52 (2H,
s, H-2,3), 13.62 (2H, s, NH); 13C NMR (CDCl3): d
117.15 (CH), 127.22 (C), 127.66 (CH), 128.86 (CH),
129.37 (CH), 132.23 (CH), 133.36 (CH), 134.42 (C),
134.61 (C), 139.05 (C), 166.41 (C), 187.19 (C); IR
(KBr): 3340, 1635cmÀ1; UV (MeOH): 486.0 (1.76);
MS m/z: 446 (46%), 105 (100%).
4.2.25. 1,4-Bis(3-chlorobenzamido)anthraquinone (24).
1
Yield: 85%; mp 244–246ꢁC (EA/n-hexane); H NMR
(CDCl3): d 7.54–7.64 (4H, m), 7.87–7.90 (2H, m, H-
6,7), 8.16–8.17 (2H, s), 8.37–8.40 (2H, m, H-5,8), 9.41
(2H, s, H-2,3), 13.56 (2H, s, NH); 13C NMR (CDCl3):
d 117.28 (CH), 125.45 (CH), 127.34 (C), 128.18 (CH),
129.30 (CH), 130.11 (CH), 132.30 (CH), 133.23 (CH),
134.60 (C), 135.23 (C), 136.35 (C), 138.82 (C), 164.96
(C), 187.22 (C); IR (KBr): 3380, 1665cmÀ1; UV
(MeOH): 486.0 (1.60); MS m/z: 514(49%), 139 (100%).
4.2.20. 1,4-Bis(3-methylbenzamido)anthraquinone (19).
Yield: 82%; mp 242–244ꢁC (EA/n-hexane); 1H NMR
(CDCl3): d 2.57 (6H, s, CH3), 7.47–7.56 (6H, m), 7.87–
7.90 (2H, m, H-6,7), 8.00–8.02 (2H, s), 8.39–8.42 (2H,
m, H-5,8), 9.48 (2H, s, H-2,3), 13.54 (2H, s, NH); 13C
NMR (CDCl3): d 21.09 (CH3), 117.30 (CH), 124.20
(CH), 126.87 (CH), 128.18 (C), 128.38 (CH), 129.07
(CH), 132.67 (CH), 133.03 (CH), 134.02 (C), 134.27
(CH), 138.39 (C), 138.70 (C), 166.31 (C), 186.80 (C);
IR (KBr): 3335, 1620 cmÀ1; UV (MeOH): 352 (2.33);
MS m/z: 474 (50%), 328 (12%), 119 (100%).
4.2.26. 1,4-Bis(4-chlorobenzamido)anthraquinone (25).
1
Yield: 84%; mp 320–322ꢁC (EA/n-hexane); H NMR
(CDCl3): d 7.59–7.62 (4H, d, J = 8.7Hz), 7.89–7.92
(2H, m, H-6,7), 8.15–8.17 (4H, d, J = 8.1Hz), 8.39–
8.42 (2H, m, H-5,8), 9.48 (2H, s, H-2,3), 13.61 (2H, s,
NH); 13C NMR (CDCl3): d 116.35 (CH), 118.39 (CH),
122.85 (CH), 124.64 (CH), 127.11 (C), 129.57 (CH),
131.70 (CH), 133.36 (CH), 133.59 (C), 134.23 (C),
138.19 (C), 160.88 (C), 163.09 (C), 186.60 (C); IR
(KBr): 3400, 1690cmÀ1; UV (MeOH): 517.0 (0.34);
MS m/z: 514(55%), 139 (100%).
4.2.21. 1,4-Bis(2-fluorobenzamido)anthraquinone (20).
Yield: 83%; mp 238–239ꢁC (EA/n-hexane); 1H NMR
(CDCl3): d 7.29–7.40 (4H, m), 7.58–7.65 (2H, m),
7.83–7.87 (2H, m, H-6,7), 8.12–8.18 (2H, t, J = 3.5Hz),
8.34–8.37 (2H, m, H-5,8), 9.41 (2H, s, H-2,3), 13.31
(2H, d, J = 6.9Hz, NH); 13C NMR (CDCl3): d 116.35
(CH), 118.29 (CH), 122.85 (C), 124.64 (CH), 127.11
(C), 129.57 (CH), 131.70 (CH), 133.36 (CH), 133.59
(CH), 134.23 (C), 138.19 (C), 160.88 (C), 163.09 (C),
186.60 (C); IR (KBr): 3375, 1660cmÀ1; UV (MeOH):
466.0 (1.09); MS m/z: 482 (48%), 361 (12%), 123 (100%).
4.2.27. 1,4-Bis(2-nitrobenzamido)anthraquinone (26).
1
Yield: 70%; mp 349–351ꢁC (EA/n-hexane); H NMR
(CDCl3): d 7.87–7.93 (4H, m), 7.94–8.04 (4H, m),
8.06–8.24(4H, m), 9.00 (2H, s, H-2,3), 12.65 (2H, s,
NH); IR (KBr): 3355, 1645cmÀ1; UV (MeOH): 437.0
(1.42); MS m/z: 536 (6%), 386 (100%), 150 (95%).
4.2.22. 1,4-Bis(3-fluorobenzamido)anthraquinone (21).
Yield: 75%; mp 273–275ꢁC (EA/n-hexane); 1H NMR
(CDCl3): d 7.34–7.40 (2H, t, J = 3.8Hz), 7.59–7.66
(4H, m), 7.89–7.92 (2H, m, H-6,7), 8.00–8.02 (2H, d,
J = 7.5Hz), 8.40–8.43 (2H, m, H-5,8), 9.47 (2H, s, H-
2,3), 13.61 (2H, s, NH); 13C NMR (CDCl3): d 114.87
(CH), 115.17 (CH), 119.15 (CH), 123.07 (CH), 127.31
(C), 129.30 (CH), 130.44 (CH), 133.27 (CH), 134.58
4.2.28. 1,4-Bis(4-trifluorobenzamido)anthraquinone (27).
1
Yield: 78%; mp 331–333ꢁC (EA/n-hexane); H NMR
(CDCl3): d 7.60 (4H, s), 7.90–7.92 (2H, d, J = 8.1Hz,
H-6,7), 8.32–8.35 (2H, d, J = 7.8Hz, H-5,8), 8.40–8.42
(4H, m), 9.51 (2H, s, H-2,3), 13.70 (2H, s, NH); 13C

H.-S. Huang et al. / Bioorg. Med. Chem. 12 (2004) 6163–6170
6169
NMR (CDCl3): d 117.82 (CH), 120.08 (C), 125.57 (CH),
127.30 (C), 127.72 (CH), 129.25 (CH), 133.03 (CH),
134.29 (C), 134.79 (C), 136.88 (C), 138.12 (C), 165.26
(C), 187.01 (C); IR (KBr): 3335, 1645cmÀ1; UV
(MeOH): 518.0 (0.35); MS m/z: 581 (11%), 359 (15%),
111 (100%).
4.2.35. 1,4-Bis(2,3-dichloro-5-fluorobenzamido)anthraqui-
none (34). Yield: 77%; mp 321–322ꢁC (EA/n-hexane); 1H
NMR (CDCl3): d 7.62 (2H, d, J = 8.7Hz), 7.67 (2H, d,
J = 6.6Hz), 7.87–7.89 (2H, m, H-6,7), 8.31–8.34(2H,
m, H-5,8), 9.38 (2H, s, H-2,3), 13.13 (2H, s, NH); IR
(KBr): 3300, 1650cmÀ1; UV (MeOH): 487.0 (0.87);
MS m/z: 620 (55%), 191 (100%).
4.2.29.
1,4-Bis(2,5-trifluorobenzamido)anthraquinone
(28). Yield: 87%; mp 245–247ꢁC (EA/n-hexane); 1H
NMR (CDCl3): d 7.16–7.19 (2H, d, J = 9.6Hz), 7.84–
7.87 (2H, m, H-6,7), 7.96–8.01 (2H, t, J = 8.0Hz),
8.04–8.07 (2H, d, J = 9.0Hz), 8.25–8.28 (2H, d,
J = 7.8Hz, H-5,8), 9.38 (2H, s), 13.03 (2H, s); 13C
NMR (CDCl3): d 112.40 (C), 117.82 (CH), 120.64 (C),
124.86 (CH), 125.57 (CH), 127.30 (C), 127.72 (CH),
129.25 (CH), 130.44 (C), 133.03 (CH), 134.29 (CH),
134.79 (C), 136.88 (C), 138.12 (C), 165.26 (C), 187.01
(C); IR (KBr): 3355, 1655cmÀ1 ; UV (MeOH): 524.0
(0.58); MS m/z: 717 (100%), 359 (13%), 211 (14%).
4.2.36. 1,4–Bis(trans-2-phenyl-1-cyclopropanecarboxam-
ido)anthraquinone (35). Yield: 68%; mp 272–274ꢁC (EA/
n-hexane); 1H NMR (CDCl3): d 1.79–1.86 (2H, m),
2.03–2.09 (2H, m), 2.69–2.76 (4H, m), 7.22–7.40 (10H,
m), 7.83–7.86 (2H, m, H-6,7), 8.29–8.32 (2H, m, H-
5,8), 9.26 (2H, s, H-2,3), 12.93 (2H, s, NH); 13C NMR
(CDCl3): d 16.59 (CH2), 17.07 (CH), 28.59 (CH),
116.34(CH), 126.24(CH), 126.64 (CH), 127.02 (C),
128.47 (CH), 129.14 (CH), 133.34 (CH), 134.32 (C),
138.51 (C), 140.21 (C), 171.76 (C), 186.85 (C); IR
(KBr): 3320, 1635cmÀ1; UV (MeOH): 486.0 (0.74);
MS m/z: 526 (100%), 365 (25%).
4.2.30.
1,4-Bis(2,4-difluorobenzamido)anthraquinone
(29). Yield: 66%; mp 315–317ꢁC (EA/n-hexane); 1H
NMR (CDCl3): d 7.03–7.13 (2H, m), 7.85–7.88 (2H,
m, H-6,7), 8.00–8.02 (2H, t, J = 6.3Hz), 8.34–8.37 (2H,
m, H-5,8), 9.38 (2H, s, H-2,3), 13.30 (2H, d,
J = 7.0Hz); IR (KBr): 3325, 1615cmÀ1; UV (MeOH):
469.0 (1.31); MS m/z: 518 (63%), 141 (100%).
4.2.37. 1,4-Bis(phenylthioacetamido)anthraquinone (36).
Yield: 84%; mp 137–139ꢁC (EA/n-hexane); 1H NMR
(CDCl3): d 3.91 (4H, s, CH2), 7.19–7.33 (6H, t,
J = 5.5Hz), 7.48–7.51 (4H, d, J = 4.5Hz), 7.84–7.87
(2H, m, H-6,7), 8.30–8.33 (2H, m, H-5,8), 9.13 (2H, s,
H-2,3), 12.18 (2H, s, NH); 13C NMR (CDCl3): d 40.16
(CH2), 117.75 (CH), 126.98 (CH), 127.11 (C), 128.76
(CH), 129.12 (CH), 129.63 (CH), 133.22 (CH), 134.32
(C), 134.55 (C), 137.82 (C), 158.59 (C), 186.52 (C); IR
(KBr): 3345, 1670cmÀ1; UV (MeOH): 485.0 (0.69);
MS m/z: 538 (94%), 415 (59%), 388 (23%).
4.2.31.
1,4-Bis(2,4-dichlorobenzamido)anthraquinone
(30). Yield: 80%; mp 331–333ꢁC (EA/n-hexane); 1H
NMR (CDCl3): d 7.45–7.49 (2H, d, J = 9.3Hz), 7.60
(2H, s), 7.75 (2H, d, J = 8.5Hz), 7.85–7.87 (2H, m, H-
6,7), 8.28–8.31 (2H, m, H-5,8), 9.42 (2H, s, H-2,3),
13.09 (2H, s, NH); IR (KBr): 3340, 1635cmÀ1; UV
(MeOH): 519.0 (0.07); MS m/z: 582 (2%), 354(100%),
298 (41%), 238 (30%).
4.2.38. 1,4-Bis(4-fluorophenylacetamido)anthraquinone
(37). Yield: 85%; mp 228–230ꢁC (EA/n-hexane); 1H
NMR (CDCl3): d 3.85 (4H, s, CH2), 7.11–7.17 (4H, t,
J = 4.0Hz), 7.41–7.46 (4H, m), 7.82–7.85 (2H, m, H-
6,7), 8.21–8.24(2H, m, H-5,8), 9.16 (2H, s, H-2,3),
12.54(2H, s); 13C NMR (CDCl3): d 45.09 (CH2),
115.62 (CH), 117.01 (CH), 127.04(C), 128.88 (CH),
129.79 (CH), 131.09 (C), 133.13 (CH), 134.36 (C),
138.31 (C), 161.20 (C), 170.62 (C), 186.72 (C); IR
(KBr): 3350, 1655cmÀ1; UV (MeOH): 485.0 (0.64);
MS m/z: 511 (100%), 427 (8%), 333 (4%), 166 (6%).
4.2.32. 1,4-Bis(2,4,6-trichlorobenzamido)anthraquinone
(31). Yield: 60%; mp 361–363ꢁC (EA/n-hexane); 1H
NMR (CDCl3): d 7.33–7.41 (2H, m), 7.85–7.91 (2H,
m), 8.08 (2H, d, J = 7.8Hz, H-6,7), 8.19 (2H, d,
J = 7.5Hz, H-5,8), 10.33 (2H, s, H-2,3), IR (KBr):
3355, 1675cmÀ1; UV (MeOH): 479.0 (0.43); MS m/z:
652 (2%), 482 (67%), 123 (100%).
4.2.33. 1,4-Bis(2,3,6-trifluorobenzamido)anthraquinone
(32). Yield: 84%; mp 345–347ꢁC (EA/n-hexane); 1H
NMR (CDCl3): d 7.08 (2H, t, J = 8.9Hz), 7.32–7.42
(2H, m), 7.85–7.88 (2H, m, H-6,7), 8.29–8.32 (2H, m,
H-5,8), 9.43 (2H, s, H-2,3), 13.18 (2H, s, NH); 13C
NMR (CDCl3): d 109.11 (CH), 112.11 (CH), 117.73
(CH), 127.28 (C), 129.22 (CH), 133.09 (CH), 134.69
(C), 138.02 (C), 144.80 (C), 158.44 (C), 149.50 (C),
165.05 (C), 186.95 (C); IR (KBr): 3370, 1610cmÀ1; UV
(MeOH): 463.0 (1.40); MS m/z: 554(34%), 158 (100%).
4.2.39. 1,4-Bis(2,5-dimethylfuryl-3-carbonylamido)anthra-
quinone (38). Yield: 79%; mp 284–286ꢁC (EA/n-hexane);
1H NMR (CDCl3): d 2.40 (6H, s, CH3), 2.69 (6H, s,
CH3), 6.59 (2H, s), 7.84–7.87 (2H, m, H-6,7), 8.36–
8.39 (2H, m, H-5,8), 9.37 (2H, s, H-2,3), 13.05 (2H, s,
NH); IR (KBr): 310, 1645cmÀ1; UV (MeOH): 582.0
(0.31); MS m/z: 482 (100%), 343 (48%).
4.3. Cell culture
4.2.34. 1,4-Bis(2,4,5-trifluorobenzamido)anthraquinone
(33). Yield: 70%; mp 310–312ꢁC (EA/n-hexane); 1H
NMR (CDCl3): d 7.14–7.19 (2H, m), 7.86–7.89 (2H,
m, H-6,7), 8.02–8.04(2H, m), 8.33–8.36 (2H, m, H-
5,8), 9.36 (2H, s, H-2,3), 13.32 (2H, s, NH); IR (KBr):
3340, 1620cmÀ1; UV (MeOH): 486.0 (0.84); MS m/z:
554(71%), 159 (100%).
Various cancer cell lines (G2, 2.2.15 cells, and C6 cells)
were cultured in minimum essential medium (MEM),
supplemented with 10% fetal calf serum, 100units/mL
penicillin and 100mg/mL streptomycin in a humidified
atmosphere in 5% CO2 at 37ꢁC. Cell culture media were
renewed every three days, up to the confluence of the
monolayer. Cell culture was passaged when they had

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H.-S. Huang et al. / Bioorg. Med. Chem. 12 (2004) 6163–6170
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compounds were stored at À70ꢁC and solublized in
100% DMSO. All the drug solutions were prepared
immediately before the experiments and were diluted
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All data presented in this report are from at least three
independent experiments showing the same pattern of
expression.
4.4. XTT method
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Acknowledgements
This research was supported by National Science Coun-
cil Grants NSC93–2113-M-016–003. The authors are in-
debted to Dr. Klaus K. Mayer (Universita¨t Regensburg,
Germany) for the mass spectrometry and analytical
determinations
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