Synthesis, antiproliferative activities and telomerase inhibition evaluation of novel asymmetrical 1,2-disubstituted amidoanthraquinone derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.10.059

A series of diversely asymmetrical mono- or disubstituted 1,2-diamidoanthraquinone derivatives were synthesized and evaluated for drug-induced cytotoxicity by SRB assay, telomerase inhibitory activity by TRAP assay, and hTERT expression by SEAP assay. Int

Full text of DOI:10.1016/j.ejmech.2011.10.059

European Journal of Medicinal Chemistry 47 (2012) 323e336
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiproliferative activities and telomerase inhibition evaluation
of novel asymmetrical 1,2-disubstituted amidoanthraquinone derivatives
Chia-Chung Lee a, Kuo-Feng Huang b, Pen-Yuan Lin c, Fong-Chun Huang d, Chun-Liang Chen a,
**
,
a,f,
*
Tsung-Chih Chen a, Jih-Hwa Guh e, Jing-Jer Lin d , Hsu-Shan Huang
a Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan
b Chi-Mei Medical Center, Tainan 710, Taiwan
c College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan
d Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan
e School of Pharmacy, National Taiwan University, Taipei 100, Taiwan
f School of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of diversely asymmetrical mono- or disubstituted 1,2-diamidoanthraquinone derivatives were
synthesized and evaluated for drug-induced cytotoxicity by SRB assay, telomerase inhibitory activity by
TRAP assay, and hTERT expression by SEAP assay. Interestingly, compounds 4, 11, 21, 32 and 36 exhibited
selective potent antiproliferative activities by NCI with IC50 values in the micromolar range. Of these, only
Received 2 September 2011
Received in revised form
26 October 2011
Accepted 31 October 2011
Available online 7 November 2011
compound 8 showed an IC50 value of 0.95 mM against PC-3 cell lines (human prostate cancer) by SRB
assay. All the synthesized compounds exhibited a poor or modest telomerase inhibitory activity by TRAP
assay suggesting another mode of action for these compounds. Compound 11 showed broad inhibition
against different types of cancer cell lines in the micromolar and submicromolar range.
Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
Keywords:
Anthraquinone
Sulforhodamine B (SRB) assay
NCI 60-cell panel assay
TRAP assay
Telomerase activity
1. Introduction
limited their applicability [4]. Doxorubicin and its analogs function
as topoisomerase poisons through stabilization of the cleavable
Anthraquinone-containing extracts from different plant sources
such as senna, cascara, aloe, frangula, and rhubarb have been found
to have wide variety of pharmacological activities such as antiin-
flammatory, wound healing, analgesic, antipyretic, antimicrobial,
and antitumor activities [1,2]. The main bioactive constituents
of rhubarb are anthraquinone derivatives, including emodin,
aloe-emodin, rhein, chrysophanol, physcion, and danthron [3].
Anthraquinone-based drugs have been shown to process strong
antiperliferative properties, such as mitoxantrone, ametantrone,
and anthracycline antibiotics (daunorubicin and doxorubicin), are
used for treatment of varied oncology (Scheme 1). However, these
clinically used drugs also suffered from frequent cardiotoxicity that
complex that forms as part of the catalytic cycle of the enzyme [5].
The detailed mechanism of how these compounds, which contain
an anthraquinone core that forms between the DNA base pairs, the
drug, and the enzyme in the minor groove has remained elusive.
Anthracyclines and their anthraquinone derivatives remain
‘evergreen’ drugs with broad clinical indications but have an
improvable pharmacological index [6,7]. In an attempt to identify
new targets for anticancer drugs, our attention has been focused on
cytotoxicity and telomerase. Nevertheless, because of their broad
bioactivities, anthraquinone-based analogs and their coplanar stru-
ctural motif still attract attentions for rational design anticancer
drugs. In addition, DNA appears to be the major cellular target for
most anthraquinone-based structures which is generally accepted by
DNA-interactive drugs [8]. It has been reported that daunorubicin
and its analogs are DNA-intercalating agent, and their cytotoxicity is
due to stabilization of the topoisomerase IIeDNA cleavage complexes
[9,10]. Furthermore, the bi-functionalized side-chains of regioiso-
meric amidoanthraquinones may simultaneously occupy both
DNA major and minor grooves, and mono- or disubstituted of
* Corresponding author. School of Pharmacy, National Defense Medical Center,
161, Minchuan E. Road, Sec. 6, Taipei 114, Taiwan. Tel.: þ886 2 87923100x18179;
fax: þ886 2 87923169.
** Corresponding author. Tel.: þ886 2 28267000x5603; fax: þ886 2 28267258.
E-mail addresses: jjlin@ym.edu.tw (J.-J. Lin), huanghs@ndmctsgh.edu.tw
(H.-S. Huang).
0223-5234/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.059

324
C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
O
OH
OH
O
O
HN
H
N
R
OH
OMe O
O
O
1-4
O
H3C
NH2
OH
O
O
NH2
O
NH2
H
N
Doxorubicin (R = OH)
Daunorubicin (R = H)
NH2
Cl
O
O
O
H
N
5
X
X
O
O
HN
HN
OH
OH
O
HN
H
N
N
H
O
Mitoxantrone (R = OH)
Ametantrone (R = H)
6-40
Scheme 1. Scaffold-based of bioactive pharmacophoric anthraquinones for drug design.
anthraquinone derivatives can potentially increase DNA binding
affinity [11].
sequential introduction of the 1,2-diamidoanthraquinone moiety
and the substituted aromatic or bicyclic aromatic system. The
acylated compound 5 with various acyl chlorides afforded the
asymmetrical compounds 6e28. The nucleophilic substitution
between the compound 11 and the appropriated amines under
catalysis conditions in the presence of DIPEA as well as in dry THF
resulted in the formation of asymmetrical compounds 29e40.
Based upon our desired structures obtained asymmetrical side-
chains, if we changed the order of synthetic route form acylation
Telomerase is a ribonucleoprotein for telomere maintenance
which utilizes its RNA component as the template to extend telo-
meric DNA length [12]. The activity of telomerase could be detected
in about 85e90% of tumor cells, whereas it is low or not present in
most somatic cells [13]. Thus, the maintenance of telomere length is
considered as a biological marker for determining the proliferation
of cancer cells. In previous studies [2,7,14e18], our groups have
reported a series of amidoanthraquinones at 1,4-, 1,5-, 2,6- and 2,7-
position that showed diversely in vitro antitumor activity and
telomerase inhibitory activities. Here we report the design and
synthesis of a series of novel asymmetrical mono- or disubstituted
1,2-diamidoanthraquinone derivatives. These compounds were
evaluated for cytotoxicity and telomerase inhibitory activity using
SRB assay [19e22], and repressing hTERT-expression, respectively.
Among these derivatives, compounds 1, 5, 6, 7, 8, 23, 24, 30 and 37
showed the highest potency against PC-3 (prostate cancer) with
Table 1
Drug screened for cytotoxicity of compounds 1e40 against the growth of human
prostate cancer cell lines (PC-3) by SRB assay.
Cell type/PC-3 (inhibition
m
M)a
IC50
1.19
Compound
Compound
IC50
1
22
>20
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
>20
3.94
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
2.44
IC50 from 0.95 mM to 2.64 mM (Table 1). Compounds 4 (NSC747246),
2.64
3.78
4.31
>20
5.39
3.92
1.46
6.68
17.56
>20
>20
19.06
3.46
2.07
>20
>20
>20
11 (NSC749232), 21 (NSC749233), 32 (NSC749757), and 36
(NSC749670) were selected by the NCI for one dose screening
program (Table 2) and further studies on 11 (NSC749232) and 36
(NSC749670) (Table 3) where the curves cross these lines represent
the interpolated values to cause 50% growth inhibition (GI50), total
growth inhibition (TGI) and 50% cell killing (LC50), respectively.
3.16
1.65
1.23
1.12
0.95
3.67
3.79
3.87
>20
2. Chemistry
3.48
3.14
3.09
3.51
There has been continuing interest in the synthesis of
anthraquinone-based derivatives and their systematic dissection
largely on account of their biological activities [16]. Herein, we
reported on the discovery of an efficiently method for synthesis of
asymmetrical mono- or disubstituted skeleton of amidoan-
thraquinones that involved simply synthetic route with appropriate
yields. The synthesis of target compounds is illustrated and out-
lined in schemes. Our synthetic strategies involved both the
>20
3.42
5.68
6.67
39
40
mitoxantrone
doxorubicin
0.29
0.25
>20
a
IC50 is the concentraion of drug (
mM) required to inhibit cell growth by 50%; all
experiments were independently performed at least three times (n ¼ 3).

Table 2
Cytotoxicity of selected compounds in the NCI in vitro 60-cell drug screen program.
Panel/Cell lines
Compound/Growth percenta
4
11
21
32
36
NSC747246
NSC749232
NSC749233
NSC749757
NSC749670
Leukemia
CCRF-CEM
HL-60(TB)
MOLT-4
SR
K562
RPMI-8226
Non-small cell lung cancer
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
A549/ATCC
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
79.02
73.11
89.63
87.69
107.54
101.77
e
33.97
41.36
11.40
ꢀ31.25
e
39.61
96.11
48.20
e
61.06
ꢀ8.54
26.04
ꢀ43.70
ꢀ4.52
e
ꢀ46.88
ꢀ23.17
ꢀ19.84
e
40.86
e
e
e
e
88.22
ꢀ28.62
ꢀ68.77
ꢀ25.30
ꢀ85.89
95.05
102.08
115.67
73.63
80.49
64.75
104.38
104.50
43.37
e
65.93
113.43
59.33
90.22
73.29
82.50
61.05
103.02
84.88
78.07
75.21
90.68
93.87
89.52
72.60
37.47
88.21
83.89
105.23
91.21
91.14
90.57
97.74
88.62
72.05
90.46
95.65
57.88
ꢀ81.71
108.57
84.68
78.55
102.44
99.39
94.21
93.79
ꢀ53.43
e
e
65.60
84.60
58.46
73.22
31.34
51.05
82.29
2.29
28.97
8.37
27.47
3.69
94.74
67.55
68.55
88.84
80.59
83.41
ꢀ54.27
ꢀ50.22
3.37
ꢀ19.53
44.05
39.17
ꢀ43.27
98.91
110.81
97.26
103.91
102.26
91.73
14.63
79.70
80.14
98.48
107.09
124.27
109.14
66.17
86.17
68.97
81.29
108.22
66.03
82.17
59.11
89.86
62.68
68.75
82.17
22.38
SF-295
SF-539
SNB-19
SNB-75
ꢀ31.42
88.40
6.31
U251
ꢀ44.02
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-62
UACC-257
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786e0
84.41
127.16
91.57
e
ꢀ57.05
ꢀ22.41
ꢀ22.68
4.82
36.29
47.07
87.10
80.40
ꢀ6.49
88.68
51.32
59.67
e
80.62
82.24
88.01
79.14
102.17
92.18
61.74
76.82
92.83
1.24
10.84
ꢀ60.04
11.15
100.65
99.46
94.04
87.50
98.46
52.00
123.64
ꢀ57.56
41.30
ꢀ31.94
ꢀ98.72
ꢀ67.80
e
ꢀ20.65
ꢀ50.95
78.71
97.90
104.32
110.18
85.43
e
51.41
ꢀ92.86
34.52
109.55
8.72
41.03
86.38
80.40
119.04
e
106.07
77.82
47.06
102.31
65.02
50.86
82.13
112.20
ꢀ6.15
57.11
106.66
46.43
72.17
86.87
e
66.74
108.91
109.62
106.24
116.17
124.08
112.43
98.59
124.91
66.54
11.30
123.78
21.92
ꢀ81.02
130.52
42.91
e
97.26
91.79
79.64
79.93
120.81
58.19
e
65.15
68.12
82.23
74.82
86.85
69.24
55.64
63.80
11.90
67.36
49.87
63.31
101.93
70.66
69.76
68.84
A489
ACHN
SN12C
TK-10
UO-31
CAKI-1
RXF 393
94.89
88.25
e
e
Prostate cancer
PC-3
DU145
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578-T
BT-549
82.91
95.93
6.81
ꢀ97.56
e
75.00
91.52
84.75
82.12
85.83
71.71
93.29
105.96
95.39
100.00
104.17
ꢀ79.28
24.67
e
51.00
82.04
66.65
79.78
e
70.79
73.34
69.39
81.58
44.33
69.27
31.79
37.00
87.29
91.88
52.62
7.43
28.19
ꢀ49.46
ꢀ83.42
T-47D
MDA-MB-468
51.02
Mean
Delta
Range
96.19
29.65
76.37
ꢀ1.96
73.78
105.03
155.52
74.53
43.19
82.09
46.71
106.75
183.68
96.76
229.24
a
Data obtained from NCI in vitro 60-cell lines drug screen program at 10 mM.

326
C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
Table 3
In vitro anticancer activity of compounds 11 (NSC749232) and 36 (NSC749670) in the NCI’s 60 human cancer cell lines.
Panel/Cell lines (
mM)
11 (NSC749232)
36 (NSC749670)
GI50
TGI
LC50
GI50
TGI
LC50
Leukemia
CCRF-CEM
HL-60(TB)
MOLT-4
SR
2.54
6.14
2.29
1.59
1.87
1.35
>50
>50
>50
>50
>50
>50
>50
e
e
e
e
e
24.6
2.95
>50
4.04
>50
e
e
e
e
e
4.65
>100
e
K562
RPMI-8226
2.09
3.25
e
>100
>100
Non-small cell lung cancer
EKVX
HOP-62
1.98
2.97
9.73
8.81
1.78
3.76
5.81
0.88
12.3
14.9
9.49
31.7
20.8
8.28
13.7
14.1
>50
e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
24.4
>50
49.1
31.4
45.4
34.1
>100
0.87
>100
>100
>100
3.36
e
HOP-92
9.19
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
A549/ATCC
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
>100
>100
>100
>100
>100
>100
2.03
29.2
e
>50
e
2.95
8.61
1.05
6.28
3.36
5.56
1.87
9.65
23.7
29.4
9.32
28.4
>50
28.3
>50
2.22
3.00
3.08
3.36
3.09
e
4.08
>100
>100
>100
>100
>100
>100
>100
1.59
2.46
1.34
2.41
12.6
9.31
>100
>100
>100
>100
>100
>100
HT29
KM12
SW-620
e
CNS cancer
SF-268
SF-295
SF-539
SNB-19
5.74
5.46
2.92
8.38
7.66
1.80
2.12
1.52
1.01
1.52
1.53
6.83
>50
42.2
25.1
27.6
30.5
30.2
e
>100
>100
e
>100
>100
e
>100
e
e
e
>100
>100
>100
SNB-75
U251
>100
3.64
>100
>100
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786e0
0.78
5.89
4.72
5.73
1.34
7.63
0.83
9.92
1.61
1.84
15.6
12.4
19.5
4.40
15.2
1.53
17.9
6.27
4.29
41.1
31.4
>50
>50
30.1
2.83
32.2
24.3
e
>100
e
>100
>100
e
e
2.22
3.13
>100
1.87
1.94
2.52
2.23
4.65
>100
>100
e
>100
>100
e
e
>100
>100
>100
e
5.81
5.60
1.35
5.52
11.3
3.32
9.46
13.2
17.0
3.54
>50
>100
2.43
e
>100
e
>100
>100
>100
>100
>100
>100
>100
15.2
25.9
32.7
>50
>50
>50
12.0
19.2
28.5
30.7
35.9
>100
>100
>100
>100
>100
>100
>100
e
>100
9.48
9.87
7.18
6.87
7.84
1.22
2.34
7.21
17.2
22.5
14.3
15.2
16.5
3.66
4.94
1.73
31.2
>50
28.6
33.7
34.6
>50
29.2
41.3
5.61
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
A489
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
>100
e
e
e
e
>100
e
Prostate cancer
PC-3
DU145
6.54
2.69
15.4
9.07
36.5
23.0
e
>100
>100
>100
>100
>100
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578-T
BT-549
1.12
6.51
1.64
2.58
1.19
0.83
4.95
>50
3.13
3.30
> 100
> 100
2.62
>100
>100
>100
>100
e
>100
>100
>100
>100
>100
>100
>50
9.12
16.3
4.04
1.82
>50
>50
>50
46.3
3.98
T-47D
MDA-MB-468
2.58
>100

C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
327
to amination, it should be noted for initially beginning amination of
compound 5. The reaction was due to the mono-substituted side
chain of intermediate 5 bearing an electrophilic carbon atom would
be possibly cyclized to imidazole group by heating in miniclave at
60 ꢁC [7] and some undesired byproducts before further subjected
to nucleophilic displacement with chlorine atom to a series of
amines. Notably, there was an interesting reaction to mentioned
that the starting material 1,2-diaminoanthraquinone, which was
treated with three kind of conditions but different equivalents of
chloroacetyl chloride and corresponding of catalyst in the presence
of pyridine, as well as control of reaction temperature and time that
could generate at least three kind of products and byproducts.
Consequently, the reactivity of compounds 1 and 5 has been
explored in a simply nucleophilic substitution via the regioselective
of 1,2-diaminoanthraquinone. The symmetrically side-chains of
compound 1 needed longer reaction time than compound 5. This
resulted in the intramolecular hydrogen bonding interaction
between first-positional aromatic amine (hydrogen bond donors)
and the neighboring quinone carbonyl group (hydrogen bond
acceptors) of the l,2-diaminoanthraquinone [23]. By contrast with
compound 5, the course of compound 1 through the intermediate
of intramolecular hydrogen bond existing could probably affect the
difference of both reactive orientation (slight steric hindrance) and
rate. According to the ratio of the reaction mixture indicated two
spots (compounds 1 and 5) on TLC which allowed us to isolate them
as being either major or minor. Besides, it was not only compounds
1 and 5, but also all the final products of these reactions were
monitored by TLC and the quantities of the byproducts were
separated from their physical chemistry properties and purification
by tedious recrystallization and chromatography. The chemical
structures of the compounds were established by FT-IR, high
resolution mass (HRMS), 1H NMR and 13C NMR spectra and the
results are presented in the experimental part Schemes 2 and 3.
3. Biological activity
All synthesized compounds (1e40) were tested against human
prostate cancer cell lines (PC-3) using SRB assay. Moreover, five of
these compounds (4, 11, 21, 32, and 36) were chosen by NCI and
tested against a panel of 60 human tumor cell lines. In addition,
compounds 11 and 36 were selected for further cell growth inhi-
bition analyses to determine the values of GI50, TGI, and LC50. We
also evaluated the effects of compounds (1e40) on telomerase
inhibition by PCR-based telomeric repeat amplification protocol
(TRAP) assay [2,24]. Finally, their effects on the transcriptional
activity of human telomerase reverse transcriptase (hTERT) was
O
Cl
n
O
O
HN
H
N
n
Cl
R2
i
O
O
O
O
HN
O
NH2
H
N
iii
NH2
1-3, (n=1; 56%; n=2: 51%; n=3: 45%)
Cl
O
O
O
HN
O
+
H
N
iii
21 (R2=H, 34%);
22 (R2=F, 37%)
O
O
O
X
Cl
O
Cl
Y
O
O
HN
4 (62%)
n
H
N
iii
Cl
O
HN
Cl
ii
H
N
O
i
Cl
O
O
O
O
NH2
23 (X=O, Y=C, 63%);
24 (X=S, Y=C, 55%);
25 (X=O, Y=N, 52%)
O
H
N
6 ( n=1: 33%) ; 7 (n=2: 38%)
Cl
R1
O
O
O
O
HN
O
H
N
O
HN
H
N
iii
5 (65%)
Cl
Cl
Cl
iii
O
O
O
O
No. R1
26 (63%)
O
8 (R1=H, 67%);
15 (R1=2-Cl, 46%);
16 (R1=3-Cl, 51%);
17 (R1=4-Cl, 67%);
18 (R1=2-CF3,32%);
19 (R1=3-CF3,48%);
20 (R1=4-CF3,57%)
9 (R =2-CH , 47%);
X
10 (R1=3-CH3, 56%);
O
HN
11 (R1=4-CH3, 66%);
H
N
12 (R11=2-F, 31%);
13 (R1=3-F, 47%);
14 (R1=4-F, 62%);
3
iii
O
O
27 (X=O, 55%);
28 (X=S, 59%)
Scheme 2. Synthesis of disubstituted anthraquinone derivatives 1e28. Reagents and conditions: (i) acyl chloride, anhydrous DMF, pyridine, rt, 24 h; (ii) anhydrous DMF, rt, 2 h;
(iii) acyl chloride, pyridine, anhydrous THF, miniclave, 3e4 h.

328
C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
O
O
O
O
O
HN
O
HN
O
HN
H
N
H
N
H
N
N
N
N
O
O
O
O
O
N
31 (32%)
33 (43%)
32 (39%)
O
O
O
O
O
HN
O
O
HN
O
HN
H
H
N
H
N
N
N
Cl
N
O
X
O
O
O
O
O
34 (X= O, 47%);
35 (X= S, 51%)
11 (66%)
30 (43%)
O
O
O
O
O
HN
O
HN
O
O
HN
H
N
H
N
H
N
N
N
N
O
N
O
N
O
R1
R2
O
No.
R2
29 (54%)
36 (R1 = CH3, 55%);
37 (R1 = CH2CH2OH, 48%)
N
N
38
; 39
; 40
N
(41%)
(63%)
(53%)
Scheme 3. Synthesis of disubstituted anthraquinone derivatives 29e40. Reagents and conditions: secondary amine, DIPEA, anhydrous THF, reflux, 14e16 h.
evaluated using the secreted alkaline phosphatase (SEAP) as the
reporter system [25].
the reaction mixture, and accelerated the reactivity of acylation in
SN2 reaction.
4. Results and discussion
4.2. In vitro anti-proliferative activities
4.1. Chemistry
The SRB assay provides a rapid, sensitive method for measuring
the cell growth inhibition in 96 well microtiter plates [21]. The
results were summarized in Table 1 and expressed as the
Based upon the 1H NMR spectrum, there were obviously two
major differences between compounds 1 and 5: (i) two singlet
concentration of drug inhibiting cell growth by 50% (IC50). As
a result of SAR studies showed that the amide-side chains obtained
one carbon atom as well as bearing chlorine atom (1 and 5e28)
were much potential antitumor activities then followed by ami-
nation of amido-side chains (compounds 29e40). However, the
peaks at
d 4.43 and d 4.45 ppm were present in compound 1 and
two protons of alkyl group were each for symmetrical moiety
(eCH2e and eCH2e) of double side chain than only one singlet
peak in the presence of
d 4.38 ppm for alkyl group (eCH2e),
respectively; (ii) compound 5 emerged from the broad peak con-
taining two protons where the first-positional amine of aromatic
amination compounds 36 (IC50 ¼ 3.46
m
M) and 37 (IC50 ¼ 2.07
mM)
were 10-folds more active than others. Both symmetrical and
asymmetrical side-chains results indicated that the length of
carbon side chain between amido group and chlorine atom at
secondary position of 1,2-diaminoanthraquinone scaffold played
an important role in enhancing their cytotoxic activies of synthe-
sized compounds. Compounds 4, 11, 21, 32, and 36 were choosed
and studied for their antiproliferative activity against human
cancer cell lines in NCI Drug Screen Program. As shown in Table 2,
compounds 11 and 36 showed strong growth-inhibitory effects
ring (AreNH2) was located at
9.80 ppm for second-positional amido group (AreNHeCO) of
aromatic ring. Compound 1 appeared as two siglets at 9.83 and
10.29 ppm due to the double amine of amido group. There were
two multiplets at 7.90e7.93 ppm and 8.12e8.18 ppm and
a triple-doublet appeared at
7.88 ppm. In addition to 1H NMR
data, the 13C NMR showed a couple of peaks at
165.61 and
166.02 ppm for the symmetrically double-side chains of amido
carbon (eNCO). Moreover, both peaks at 37.71 and 43.25 ppm
d 7.96 ppm, and one singlet at
d
d
d
d
d
d
d
that several cancer cell lines showed <10% growth upon 10 mM
were also elucidated on the two carbon of alkyl group (eCH2e)
than only one peak at 165.72 ppm. Compared with 5, compound 1
was making the condition more basic by addition of pyridine into
compound-treatments. Compounds 4, 21, and 32 did not show
significant antiproliferative activities for tumor-cell lines.
Compounds 11 and 36 were further analyzed by NCI to determine

C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
329
the values of GI50, TGI, and LC50. As shown in Table 3, both
compounds 11 (NSC749232) and 36 (NSC749670) showed potent
activity with GI50 ranging from 0.78 to 13.2 (mean
M) and 2.09 to > 100 M),
respectively. It is also apparent that compound 11 exhibited
stronger cancer-inhibitory effects than compound 36 with LC50
4.3. Telomerase activities
m
M
Anthraquinone analogs are one of the main structures that
targets telomere and affects telomerase activity [26e28]. Since
telomerase is an important component of cancers, we are inter-
ested in examining the effects of our synthesized compounds on
telomerase activities in the cell free extracts prepared from H1299
cells. However, none of the synthesized compounds showed telo-
value ¼ 3.63
m
m
M (mean value ¼ 12.88
m
and TGI as low as 2.83 and 1.01
It exhibited the best activity against melanoma cell lines with GI50
ranging from 0.78 to 9.92 M. The breast cancer subpanel was
ranked second sensitive with GI50 ranging from 0.83 to 6.51 M. It
mM in tested cell lines, respectively.
m
merase inhibitory effects at 10 mM concentration by TRAP assay. We
m
next analyzeds the hTERT repressing activity of these compounds.
The expression of SEAP in H1299 cells harboring PhTERT-SEAP was
used as the criteria to evaluate that anthraquinone derivatives
inhibited the expression of hTERT in cancer cells [29]. Results of all
tested compounds which showed MTT and SEAP inhibition activity
is also interesting to note that both compounds 11 and 36 dis-
played relatively differential cytotoxic activity profile against NCI
60-cell lines. Although the mechanism of how these compounds
showed differential growth-inhibitory effects toward cancer cells,
asymmetrical side chain within compound 11 might have a role on
the activity Fig. 1.
at 1,10 and 100
mM were summarized in Table 4. We found all of the
synthesized compounds showed parallel inhibitory effects toward
Fig. 1. Compounds 1e40 were tested the inhibition of telomerase activity by TRAP assay under concentration of 10 mM. [P: positive control (no inhibitor); N: negative control
(RNase A-treated cell extract, no inhibitor); IC: internal control].

330
C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
Table 4
lines screen, we computed structure activity relationships between
the five compounds (NSC747246), 11 (NSC749232), 21
(NSC749233), 32 (NSC749757), and 36 (NSC749670), respectively. A
variable pattern of sensitivity against some individual cell lines was
observed and the most potent compound 8 revealed a high activity
Effects of compounds 1e40 on activating or repressing hTERT expression and telo-
merase activity.
4
Cell type
H1299 (inhibition
MTT
m
M ꢂ SD)a
Compound
SEAP
100
toward a prostate cancer cell lines (IC50 ¼ 0.95
mM, PC-3). They also
exhibited dose-dependent inhibition of proliferation in all 60 cancer
100
0 ꢂ 4
10
1
10
1
1
2
3
4
5
6
7
8
0 ꢂ 3
8 ꢂ 3
13 ꢂ 1
15 ꢂ 1
15 ꢂ 1
30 ꢂ 2
13 ꢂ 1
15 ꢂ 1
30 ꢂ 2
15 ꢂ 1
0 ꢂ 2
0 ꢂ 0
0 ꢂ 0
2 ꢂ 0
0 ꢂ 0
0 ꢂ 2
1 ꢂ 2
3 ꢂ 0
4 ꢂ 0
2 ꢂ 0
0 ꢂ 1
6 ꢂ 0
7 ꢂ 0
1 ꢂ 0
2 ꢂ 0
2 ꢂ 0
1 ꢂ 0
0 ꢂ 0
4 ꢂ 0
0 ꢂ 0
9 ꢂ 1
10 ꢂ 0
22 ꢂ 2
3 ꢂ 0
8 ꢂ 0
3 ꢂ 0
12 ꢂ 1
8 ꢂ 4
0 ꢂ 0
15 ꢂ 1
9 ꢂ 2
19 ꢂ 5
13 ꢂ 1
94 ꢂ 5
92 ꢂ 6
97 ꢂ 4
13 ꢂ 1
92 ꢂ 6
97 ꢂ 4
94 ꢂ 5
0 ꢂ 2
15 ꢂ 1
96 ꢂ 3
cell lines. As shown in Table 3 for compound 11 (NSC 749232), the
5 ꢂ 1
19 ꢂ 5
62 ꢂ 6
0 ꢂ 4
19 ꢂ 5
62 ꢂ 5
5 ꢂ 1
0 ꢂ 2
0 ꢂ 1
0 ꢂ 1
0 ꢂ 1
1 ꢂ 0
0 ꢂ 0
0 ꢂ 0
0 ꢂ 0
4 ꢂ 0
0 ꢂ 0
3 ꢂ 1
0 ꢂ 0
3 ꢂ 1
6 ꢂ 0
2 ꢂ 0
0 ꢂ 0
0 ꢂ 1
0 ꢂ 0
4 ꢂ 0
0 ꢂ 0
0 ꢂ 0
3 ꢂ 0
1 ꢂ 1
5 ꢂ 1
10 ꢂ 2
15 ꢂ 3
20 ꢂ 2
3 ꢂ 3
0 ꢂ 1
23 ꢂ 3
6 ꢂ 9
59 ꢂ 13
83 ꢂ 6
79 ꢂ 6
116 ꢂ 4
0 ꢂ 3
101 ꢂ 4
103 ꢂ 7
100 ꢂ 4
8 ꢂ 3
average concentration required to inhibit GI50 was 3.63
a range of 0.78 M (Melanoma: LOX IMVI) to 9.92 M (Melanoma:
UACC-257). As shown in Table 3 for compound 36 (NSC749670), the
average concentration requiredto inhibit GI50 witha range of0.87
(Non-small celllungcancer: HOP-92) to >100 M. All these datawere
summarized in Tables 2 and 3 and used for further analysis.
mM with
113 ꢂ 6
108 ꢂ 9
15 ꢂ 1
113 ꢂ 6
108 ꢂ 9
96 ꢂ 3
98 ꢂ 7
116 ꢂ 1
109 ꢂ 2
105 ꢂ 1
26 ꢂ 1
11 ꢂ 9
81 ꢂ 5
80 ꢂ 3
98 ꢂ 5
93 ꢂ 6
99 ꢂ 4
85 ꢂ 8
84 ꢂ 3
93 ꢂ 5
84 ꢂ 2
5 ꢂ 2
m
m
79 ꢂ 6
116 ꢂ 4
83 ꢂ 6
0 ꢂ 3
103 ꢂ 7
100 ꢂ 4
101 ꢂ 4
73 ꢂ 7
72 ꢂ 3
103 ꢂ 7
78 ꢂ 7
18 ꢂ 1
10 ꢂ 2
101 ꢂ 4
89 ꢂ 2
92 ꢂ 7
93 ꢂ 2
94 ꢂ 3
86 ꢂ 2
81 ꢂ 1
88 ꢂ 5
80 ꢂ 3
0 ꢂ 0
mM
m
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
0 ꢂ 1
0 ꢂ 0
Although the mechanisms of how compounds 11 and 36
rendered it cytotoxic activity are still unclear, there is still no
significant correlation between telomerase activity and cytotox-
icity. Moreover, we anticipate that analysis of anthraquinone-based
small molecules with various asymmetrical amido side chains
might yield further insight into designing better lead compounds
for anticancer agents. The physical, chemical and biological prop-
erties of anthraquinone-base derivatives are greatly affected by its
various substituents of the planar ring system which considered as
aglycon analogs of anthracycline antibiotics. We are still interested
in examining the effects of these compounds on telomerase activity
in the cell free extracts prepared from H1299 cells. As shown in
Table 4, none of the synthesized compounds exhibited telomerase
inhibitory activity on SEAP expression, a tentative explanation
suggested could be a steric hindrance induced by substituents or by
other interactions due to the presence of functional groups on these
compounds. Although the mechanism of preferential inhibition of
compounds is still unclear to us, these results suggested that the
cell proliferation inhibition activity of our compounds could be
attributed to another mechanism of action. Therefore, more bio-
logical experiments are underway to determine a possible mech-
anism, which would give some information on how to improve the
efficiency of our leads by chemical structure modulation.
2 ꢂ 0
0 ꢂ 0
2 ꢂ 0
5 ꢂ 0
8 ꢂ 1
1 ꢂ 0
4 ꢂ 0
2 ꢂ 3
6 ꢂ 1
5 ꢂ 2
5 ꢂ 0
6 ꢂ 0
5 ꢂ 1
6 ꢂ 0
3 ꢂ 1
4 ꢂ 1
5 ꢂ 1
4 ꢂ 0
6 ꢂ 0
8 ꢂ 0
5 ꢂ 1
9 ꢂ 0
7 ꢂ 0
8 ꢂ 0
0 ꢂ 0
2 ꢂ 0
2 ꢂ 0
3 ꢂ 0
3 ꢂ 0
105 ꢂ 7
96 ꢂ 2
101 ꢂ 4
88 ꢂ 2
106 ꢂ 2
104 ꢂ 2
90 ꢂ 1
88 ꢂ 4
86 ꢂ 6
81 ꢂ 6
85 ꢂ 7
77 ꢂ 1
71 ꢂ 5
80 ꢂ 6
105 ꢂ 3
102 ꢂ 5
4 ꢂ 0
92 ꢂ 0
81 ꢂ 1
82 ꢂ 1
80 ꢂ 4
99 ꢂ 3
100 ꢂ 3
94 ꢂ 6
81 ꢂ 4
85 ꢂ 7
98 ꢂ 0
88 ꢂ 1
78 ꢂ 3
76 ꢂ 9
93 ꢂ 4
97 ꢂ 7
87 ꢂ 7
2 ꢂ 0
2 ꢂ 0
66 ꢂ 1
5 ꢂ 0
72 ꢂ 2
3 ꢂ 0
88 ꢂ 5
88 ꢂ 7
46 ꢂ 1
69 ꢂ 3
70 ꢂ 2
56 ꢂ 5
69 ꢂ 3
13 ꢂ 4
44 ꢂ 4
76 ꢂ 7
100 ꢂ 7
91 ꢂ 5
78 ꢂ 3
76 ꢂ 7
11 ꢂ 2
59 ꢂ 2
70 ꢂ 3
54 ꢂ 3
72 ꢂ 3
29 ꢂ 7
8 ꢂ 3
88 ꢂ 9
64 ꢂ 6
79 ꢂ 3
6. Materials and methods
a
SD, standard derivation, all experiments were independently performed at least
6.1. Chemistry
three times (n ¼ 3).
Melting points were determined by melting point apparatus
(Büchi 545). All reactions were monitored by TLC (silica gel 60
F254). 1H NMR spectra were recorded with GEMINI-300 MHz
both MTT and SEAP assays, indicating that none of these
compounds showed specific hTERT repression activity.
(Varian) and AM-500 MHz (Bruker). Chemical shift (d) values were
5. Conclusion
in ppm relative to TMS as an internal standard. Mass spectra were
obtained on Finnigan MAT 95 XL HRMS and Finnigan/Thermo
Quest MAT HRMS. FT-IR spectra were taken on PerkineElmer FTIR-
1615 spectrometer. The starting material of 1,2-diaminoanthra-
quinone was purchased form Aldrich Chemicals. Reagents and
solvents were purchased from Aldrich and Merck used without
further purification.
In the course of our continuing search for new antitumor agents
from anthraquinone moiety, we described a method of synthesizing
diversely symmetrical or asymmetrical substituted 1,2-
diamidoanthraquinone derivatives and comparing to their cytotox-
icity and telomerase activity. In this investigation, we focused our
attention on the role of our systematic synthesized tricyclic phar-
macophore bearing the symmetrical or asymmetrical side chain
linked to the planar anthraquinone moiety and to understand the
basis of tricyclic system selectivity. Forty new compounds have been
prepared among which twenty-seven displayed a broad spectrum of
6.2. General synthetic methods
All anthraquinone-based derivatives were synthesized by using
a simply acylation or two-stage reaction in miniclave (Büchi). These
compounds were obtained in good yield and their purity was
determined using FT-IR, 1H NMR, 13C NMR and high resolution mass
(HRMS) spectrometry.
antitumor activity below 10 mM range. As shownin the Tables 2 and 3,
the cytotoxicity elicited by the five analogs upon human-derived
carcinoma cells, is largely clustered within the Leukemia, Non-
small cell lung cancer, Colon cancer, CNS cancer, Melanoma,
Ovarian cancer, Renal cancer, Prostate cancer and Breast cancer
panels of the NCI 60 cell line screen. To understand the unique
antiproliferative activity pattern of test compounds in the NCI-60 cell
6.2.1. General procedure A: preparation of compound (1e3)
1,2-diaminoanthraquinone (0.92 g, 4 mmol) was dissolved in
anhydrous DMF (30 mL) in ice-bath and acyl chloride (12 mmol)

C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
331
and pyridine (0.5 mL) were added under nitrogen for 24 h. Ice was
added to precipitate out the crude product. The resulting precipi-
tate was collected and purified by crystallization from hot ethanol
to afford desired compounds 1e3.
134.35, 134.42, 134.63, 139.77, 165.61, 166.02, 181.60, 183.25.
HRMS (EI) m/z: calcd [M]þ, 390.0174 (C18H12Cl2N2Oþ4 ); found,
390.0170.
6.2.8. 1, 2-Bis-(3-chloropropionamido)-anthraquinone (2)
6.2.2. General procedure B: preparation of compound (4)
The pure compound was obtained as a yellow powder (yield
1,2-diaminoanthraquinone (0.92 g, 4 mmol) was dissolved in
anhydrous THF (30 mL) and 4-toluoyl chloride (1.6 mL, 12 mmol),
pyridine (0.5 mL) were added dropwise under nitrogen. The reac-
tion mixture was heated and refluxed at 70 ꢁC in miniclave for
3e4 h. After removal of THF, the reaction mixture was washed with
ethyl acetate/hexane and the crude product was extracted in ethyl
acetate. The organic layer was collect and dried over anhydrous
Na2SO4, and then the solvent was evaporated. The crude compound
was washed and purified by crystallization from hot ethanol to
afford desired compound 4.
51%). Mp: 179e180 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1704 (CO),
3301 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 2.94e3.03 (m,
4H), 3.86e3.94 (m, 4H), 7.88e7.90 (m, 2H), 8.10e8.15 (m, 2H), 8.08
(d, J ¼ 8.1 Hz, 1H), 8.42 (d, J ¼ 8.7 Hz, 1H), 9.50 (s, 1H), 10.02 (s, 1H).
13C NMR (75 MHz, DMSO-d6):
d ppm 39.58, 40.20, 40.26, 43.33,
125.94, 126.29, 126.87, 127.61, 127.79, 128.33, 129.79, 132.27,
134.26, 134.36, 134.52, 140.28, 168.86, 169.43, 181.63, 183.22.
HRMS (EI) m/z: calcd [M]þ, 418.0487 (C20H16Cl2N2Oþ4 ); found,
418.0494.
6.2.9. 1, 2-Bis-(4-chlorobutyramido)-anthraquinone (3)
6.2.3. General procedure C: preparation of compound (5)
1,2-diaminoanthraquinone (1.19 g, 5 mmol) was dissolved in
anhydrous DMF (30 mL) and chloroacetyl chloride (0.5 mL,
6.0 mmol) was added under nitrogen for 2 h. Ice was added to
precipitate out the crude product. The resulting precipitate was
collected and purified by crystallization from hot ethanol to afford
desired compound 5.
The pure compound was obtained as a yellow powder (yield
45%). Mp: 154e155 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1666 (CO),
3313 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 2.04e2.12 (m, 4H),
2.57e2.66 (m, 4H), 3.71 (t, J ¼ 6.6 Hz, 2H), 3.78 (t, J ¼ 6.6 Hz, 2H),
7.88e7.91 (m, 2H), 8.08e8.16 (m, 3H), 8.36 (d, J ¼ 8.7 Hz, 1H), 9.52
(s, 1H), 9.82 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d ppm 27.68,
27.76, 33.11, 33.37, 44.75, 44.99, 125.55, 126.28, 126.85, 127.88,
128.21 128.40, 129.57, 132.28, 134.23, 134.47, 134.53, 140.45, 171.12,
171.60, 181.71, 183.46. HRMS (EI) m/z: calcd [M]þ, 446.0800
(C22H20Cl2N2Oþ4 ); found, 446.0793.
6.2.4. General procedure D: preparation of compound (6e7)
A solution of compound 5 (1.28 g, 4 mmol) was dissolved in
anhydrous DMF (30 mL) and acyl chloride (12 mmol) was added
under nitrogen for 1 h. Ice was added to precipitate out the crude
product. The resulting precipitate was collected and purified by
recrystallization from hot ethanol to afford desired compounds
6e7.
6.2.10. 1, 2-Bis-(4-methylbenzamido)-anthraquinone (4)
The pure compound was obtained as a brown powder (yield
62%). Mp: 227e229 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1673 (CO),
3330 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 2.36 (s, 3H), 2.42
(s, 3H), 7.33 (d, J ¼ 8.4 Hz, 2H), 7.42 (d, J ¼ 8.4 Hz, 2H), 7.79 (d,
J ¼ 8.1 Hz, 2H), 7.90e7.93 (m, 2H), 8.01 (d, J ¼ 8.1 Hz, 2H), 8.13e8.16
(m, 1H), 8.18e8.20 (m, 1H), 8.27 (d, J ¼ 8.4 Hz, 1H), 8.42 (d,
J ¼ 8.7 Hz, 1H), 10.06 (s, 1H), 10.90 (s, 1H). 13C NMR (75 MHz, DMSO-
6.2.5. General procedure E: preparation of compound (8e28)
A solution of compound 5 (1.28 g, 4 mmol) was dissolved in
anhydrous THF (30 mL), and acyl chloride (12 mmol), pyridine
(0.5 mL) were added dropwise under nitrogen. The reaction
mixture was heated and refluxed at 70 ꢁC in miniclave for 3e4 h.
After removal of THF, the reaction mixture was washed with ethyl
acetate/hexane and the crude product was extracted in ethyl
acetate. The organic layer was collect and dried over anhydrous
Na2SO4, and then the solvent was evaporated. The crude compound
was washed and purified by crystallization from hot ethanol to
afford desired compounds 8e28.
d6):
d ppm 20.37, 20.48, 125.02, 125.90, 126.61, 126.90, 127.45,
128.88, 128.92, 129.28, 129.70,129.99, 130.39, 130.57, 131.79, 133.85,
134.00, 134.14, 138.94, 139.10, 142.12, 142.20, 164.50, 165.85, 181.21,
183.84. HRMS (EI) m/z: calcd [M]þ, 474.1580 (C30H22N2O4þ); found,
474.1572.
6.2.11. 1-(Amino)-2-(chloroacetamido)-anthraquinone (5)
The pure compound was obtained as a red powder (yield 65%).
Mp: 193e194 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1678 (CO), 3237
6.2.6. General procedure F: preparation of compound (29e40)
A solution of compound 11 (0.83 g, 2 mmol) was dissolved in
anhydrous THF (30 mL). The mixture was stirred for 10 min before
the addition of appropriate amines (8 mmol) and DIPEA (1 mL,
6 mmol), after which the reaction mixture was refluxed for 16 h.
After removal of THF, the reaction mixture was washed with ethyl
acetate/hexane and the crude product was extracted in ethyl
acetate. The organic layer was collected and dried over anhydrous
Na2SO4, and then the solvent was evaporated. The crude compound
was washed and purified by crystallization from hot ethanol to
afford desired compounds (28e40).
(NH), 3294, 3416 (NH2). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.38
(s, 2H), 7.48 (d, J ¼ 7.8 Hz, 1H), 7.74 (d, J ¼ 8.1 Hz, 1H), 7.88 (td,
J ¼ 12 Hz, J ¼ 1.8 Hz, 2H), 7.96 (br, 2H), 8.14 (dd, J ¼ 7.2 Hz, 1.8 Hz,
1H), 8.22 (dd, J ¼ 7.8 Hz, 1.5 Hz, 1H), 9.80 (s, 1H). 13C NMR (75 MHz,
DMSO-d6):
d ppm 43.25, 112.98, 115.62, 126.69, 126.55, 128.88,
128.92, 130.25, 131.01, 132.64, 133.65, 134.37, 146.11, 165.72, 182.28,
184.38. HRMS (EI) m/z: calcd [M]þ, 314.0458 (C16H11N2O3þ); found,
314.0455.
6.2.12. 1-[(3-Chloropropanyl)amido]-2-(chloroacetamido)-
anthraquinone (6)
The pure compound was obtained as a dark yellow powder
6.2.7. 1, 2-Bis-(chloroacetamido)-anthraquinone (1)
(yield 33%). Mp: 182e183 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1670
The pure compound was obtained as a yellowish brown
powder (yield 56％). Mp: 254e255 ꢁC (EtOH). FT-IR (KBr,
nmaxcmꢀ1): 1718 (CO), 3273 (NH). 1H NMR (300 MHz, DMSO-d6):
(CO), 3271 (NH). 1H NMR (300 MHz, CDCl3):
d ppm 3.13 (t,
J ¼ 6.3 Hz, 2H), 3.96 (t, J ¼ 6.6 Hz, 2H), 4.21 (s, 2H), 7.81e7.84 (m,
2H), 8.25e8.34 (m, 4H), 9.73 (s, 1H), 11.51 (s, 1H). 13C NMR
d
ppm 4.43 (s, 2H), 4.45 (s, 2H), 7.90e7.93 (m, 2H), 8.12e8.18 (m,
2H), 8.22 (d, J ¼ 8.1 Hz, 1H), 8.38 (d, J ¼ 9.0 Hz, 1H), 9.83 (s, 1H),
10.29 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
ppm 37.71, 43.25,
126.20, 126.93, 127.90, 128.04, 128.14, 130.33, 132.28, 134.25,
(125 MHz, DMSO-d6): d ppm 40.12, 40.36, 43.33, 125.92, 126.28,
126.82, 127.90, 128.00, 128.92, 130.28, 132.18, 134.19, 134.28, 134.52,
139.56, 164.50, 165.85, 181.45, 183.09. HRMS (EI) m/z: calcd [M]þ,
404.0331 (C19H14Cl2N2Oþ4 ); found, 404.0334.
d

332
C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
6.2.13. 1-[(4-Chlorobutanyl)amido]-2-(chloroacetamido)-
anthraquinone (7)
7.40e7.47 (m, 2H), 7.65e7.72 (m, 1H), 7.86e7.93 (m, 2H), 8.03e8.19
(m, 3H), 8.24 (d, J ¼ 8.4 Hz, 1H), 8.37 (d, J ¼ 8.7 Hz, 1H), 9.83 (s, 1H),
The pure compound was obtained as a yellow brown powder
10.48 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d ppm 43.10, 116.36,
(yield 38%). Mp: 163e164 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1673
116.65, 124.71, 125.86, 126.29, 126.94, 127.83, 128.50, 128.96, 130.43,
130.98, 132.23, 133.80, 133.92, 134.24, 134.35, 134.51, 139.27, 162.97,
165.41, 181.55, 183.82. HRMS (EI) m/z: calcd [M]þ, 436.0626
(C23H14ClFN2Oþ4 ); found, 436.0630.
(CO), 3290 (NH). 1H NMR (300 MHz, CDCl3):
d ppm 2.28e2.34 (m,
2H), 2.88 (t, J ¼ 7.2 Hz, 2H), 3.73 (t, J ¼ 6.3 Hz, 2H), 4.22 (s, 2H),
7.81e7.83 (m, 2H), 8.26e8.34 (m, 4H), 9.84 (s, 1H), 11.46 (s, 1H). 13C
NMR (125 MHz, DMSO-d6): d ppm 27.76, 33.05, 39.92, 43.34, 44.99,
125.67, 126.26, 126.79, 127.91, 128.01, 128.73, 130.21, 132.16, 134.23,
134.26, 134.52, 165.34, 171.81, 181.49, 183.21. HRMS (EI) m/z: calcd
[M]þ, 418.0487 (C20H16Cl2N2Oþ4 ); found, 418.0494.
6.2.19. 1-(3-Fluorobenzamido)-2-(chloroacetamido)-anthraquinone
(13)
The pure compound was obtained as a yellow powder (yield
47%). Mp: 197e198 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1671 (CO),
6.2.14. 1-(Benzamido)-2-(chloroacetamido)-anthraquinone (8)
The pure compound was obtained as a yellow powder (yield
67%). Mp: 203e204 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1689 (CO),
3211 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.43 (s, 2H),
7.48e7.55 (m, 1H), 7.63e7.70 (m, 1H), 7.86e7.96 (m, 4H), 8.10e8.13
(m, 1H), 8.15e8.19 (m, 1H), 8.26 (d, J ¼ 8.7 Hz, 1H), 8.44 (d,
J ¼ 8.7 Hz, 1H), 9.85 (s, 1H), 10.49 (s, 1H). 13C NMR (75 MHz, DMSO-
3318 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.42 (s, 2H),
7.58e7.67 (m, 3H), 7.88e7.92 (m, 2H), 8.09e8.18 (m, 4H), 8.24 (d,
d6): d ppm 43.21,114.61,114.91,118.63,118.90,124.18,126.12,126.30,
J ¼ 8.7 Hz, 1H), 8.40 (d, J ¼ 8.7 Hz, 1H), 9.86 (s, 1H), 10.58 (s, 1H). 13C
126.96, 128.14, 128.24, 128.84, 130.37, 130.64, 130.74, 132.23, 134.35,
136.55, 139.96, 162.43, 165.54, 181.55, 183.41. HRMS (EI) m/z: calcd
[M]þ, 436.0626 (C23H14ClFN2Oþ4 ); found, 436.0623.
NMR (75 MHz, DMSO-d6):
d ppm 43.19, 125.76, 126.28, 126.95,
127.79, 127.95, 128.40, 128.51, 129.56, 130.43, 132.00, 132.22, 133.97,
134.23, 134.34, 134.50, 139.44, 165.42, 166.35, 181.55, 183.77. HRMS
(EI) m/z: calcd [M]þ, 418.0720 (C23H15ClN2Oþ4 ); found, 418.0716.
6.2.20. 1-(4-Fluorobenzamido)-2-(chloroacetamido)-anthraquinone
(14)
6.2.15. 1-(2-Methylbenzamido)-2-(chloroacetamido)-anthraquinone
(9)
The pure compound was obtained as a greenish brown powder
(yield 62%). Mp: 184e185 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1672
The pure compound was obtained as a yellow powder (yield
(CO), 3213 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.42 (s, 2H),
47%). Mp: 179e180 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1664 (CO),
7.44 (t, J ¼ 9.0 Hz, 2H), 7.88e7.91 (m, 2H), 8.09e8.18 (m, 4H), 8.25
3277 (NH). 1H NMR (300 MHz, CDCl3):
d ppm 2.52 (s, 3H), 4.20 (s,
(d, J ¼ 8.7 Hz, 1H), 8.42 (d, J ¼ 8.7 Hz, 1H), 9.84 (s, 1H), 10.51 (s, 1H).
2H), 7.48e7.50 (m, 2H), 7.79e7.83 (m, 2H), 7.99e8.01 (m, 2H),
13C NMR (75 MHz, DMSO-d6):
d ppm 43.23, 115.33, 115.62, 125.97,
8.26e8.33 (m, 4H), 10.08 (s, 1H), 12.35 (s, 1H). 13C NMR (75 MHz,
126.33, 126.98, 128.07, 128.25, 129.24, 130.40, 130.64, 130.82,
132.25,134.25,134.38,134.56,139.79,165.44,165.52,181.58,183.58.
HRMS (EI) m/z: calcd [M]þ, 436.0626 (C23H14ClFN2Oþ4 ); found,
436.0625.
CDCl3):
d ppm 21.39, 43.12, 124.08, 125.16, 125.60, 127.24, 127.63,
128.91, 129.06, 131.71, 132.07, 132.91, 132.22, 133.32, 133.81, 134.29,
134.35, 134.83, 136.53, 139.15 165.44, 168.03, 181.68, 187.58. HRMS
(EI) m/z: calcd [M]þ, 432.0877 (C24H17ClN2Oþ4 ); found, 432.0877.
6.2.21. 1-(2-Chlorobenzamido)-2-(chloroacetamido)-anthraquinone
(15)
6.2.16. 1-(3-Methylbenzamido)-2-(chloroacetamido)-anthraquinone
(10)
The pure compound was obtained as a yellowish green powder
The pure compound was obtained as a brown powder (yield
(yield 46%). Mp: 193e194 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1671
56%). Mp: 202e203 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1671 (CO),
(CO), 3227 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.43 (s, 2H),
3233 (NH). 1H NMR (300 MHz, CDCl3):
d
ppm 2.52 (s, 3H), 4.20 (s,
7.57e7.60 (m, 3H), 7.89e7.94 (m, 2H), 8.08e8.19 (m, 3H), 8.26 (d,
2H), 7.48e7.50 (m, 2H), 7.80e7.83 (m, 2H), 7.99e8.01 (m, 2H),
J ¼ 8.7 Hz, 1H), 8.37 (d, J ¼ 8.7 Hz, 1H), 9.76 (s, 1H), 10.44 (s, 1H). 13C
8.26e8.32 (m, 4H), 10.08 (s, 1H), 12.36 (s, 1H). 13C NMR (75 MHz,
NMR (75 MHz, DMSO-d6):
d ppm 43.16, 126.16, 126.32, 126.93,
CDCl3):
d
ppm 21.39, 43.12, 124.07, 125.15, 125.60, 126.30, 127.24,
127.19, 127.87, 128.07, 128.37, 128.53, 129.49, 130.14, 130.63, 130.76,
131.73, 134.24, 134.34, 134.56, 135.29, 139.44, 165.36, 165.54, 181.55,
183.44. HRMS (EI) m/z: calcd [M]þ, 452.0331 (C23H14Cl2N2O4þ);
found, 436.0325.
127.63, 128.91, 129.06, 131.70, 132.08, 132.91, 133.30, 133.81, 134.29,
134.36, 136.84, 136.51, 139.15, 165.45, 168.03, 181.69, 187.58. HRMS
(EI) m/z calcd [M]þ, 432.0877 (C24H17ClN2Oþ4 ); found, 432.0873.
6.2.17. 1-(4-Methylbenzamido)-2-(chloroacetamido)-anthraquinone
(11)
6.2.22. 1-(3-chlorobenzamido)-2-(chloroacetamido)-anthraquinone
(16)
The pure compound obtained as a brown powder (yield 66%).
The pure compound was obtained as a yellow powder (yield
Mp: 198e199 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1666 (CO), 3230
51%). Mp: 188e189 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1678 (CO),
(NH). 1H NMR (300 MHz, DMSO-d6):
d
ppm 2.43 (s, 3H), 4.43 (s, 2H),
3235 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.43 (s, 2H),
7.41 (d, J ¼ 7.8 Hz, 2H), 7.88e7.91 (m, 2H), 8.01 (d, J ¼ 8. 1 Hz, 2H),
8.10e8.13 (m,1H), 8.16e8.19 (m,1H), 8.24 (d, J ¼ 8.7 Hz,1H), 8.39 (d,
J ¼ 9.0 Hz, 1H), 9.84 (s, 1H), 10.58 (s, 1H). 13C NMR (75 MHz, CDCl3):
7.72e7.64 (m, 2H), 7.87e7.91 (m, 2H), 8.02e8.19 (m, 4H), 8.26 (d,
J ¼ 8.4 Hz, 1H), 8.45 (d, J ¼ 8.4 Hz, 1H), 9.79 (s, 1H), 10.49 (s, 1H). 13C
NMR (75 MHz, DMSO-d6):
d ppm 43.28, 126.11, 126.24, 126.36,
d
ppm 20.90, 43.53, 123.39, 124.92, 126.62, 127.00, 127.64, 129.26,
126.79, 127.01, 127.88, 128.08, 128.37, 128.74, 130.36, 130.53, 131.73,
132.27, 133.33, 134.26, 134.60, 136.28, 140.17, 165.23, 165.64, 181.61,
183.36. HRMS (EI) m/z: calcd [M]þ, 452.0331 (C23H14Cl2N2O4þ);
found, 436.0330.
129.91, 131.06, 131.42, 132.33, 132.41, 133.01, 133.73, 134.20, 135.90,
143.22, 164.83, 167.15, 181.07, 187.00. HRMS (EI) m/z: calcd [M]þ,
432.0877 (C24H17ClN2Oþ4 ); found, 432.0881.
6.2.18. 1-(2-Fluorobenzamido)-2-(chloroacetamido)-anthraquinone
(12)
6.2.23. 1-(4-chlorobenzamido)-2-(chloroacetamido)-anthraquinone
(17)
The pure compound was obtained as a yellow brown powder
The pure compound was obtained as a yellow powder (yield
(yield 31%). Mp: 245e246 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1666
67%). Mp: 224e225 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1678 (CO),
(CO), 3261 (NH). 1H NMR (300 MHz, DMSO-d6):
d
ppm 4.41 (s, 2H),
3235 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.42 (s, 2H),

C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
333
7.68e7.71 (m, 2H), 7.88e7.91 (m, 2H), 8.08e8.18 (m, 4H), 8.25 (d,
J ¼ 8.4 Hz, 1H), 8.43 (d, J ¼ 8.7 Hz, 1H), 9.86 (s, 1H), 10.52 (s, 1H). 13C
114.97, 125.67, 126.26, 126.79, 127.79, 128.15, 128.28, 129.14, 130.45,
131.42, 131.53, 132.23, 134.27, 134.50, 139.18, 165.23, 170.70, 181.54,
183.46. HRMS (EI) m/z: calcd [M]þ, 450.0783 (C24H16ClFN2O4þ);
found, 450.0782.
NMR (75 MHz, DMSO-d6):
d ppm 43.23, 126.06, 126.33, 126.96,
128.21, 128.61, 129.06, 129.93, 130.40, 131.71, 132.25, 132.92, 134.25,
134.38, 134.56, 136.91, 139.87, 165.50, 165.53, 181.58, 183.51. HRMS
(EI) m/z: calcd [M]þ, 452.0331 (C23H14Cl2N2Oþ4 ); found, 436.0333.
6.2.29. 1-[(2-Furoyl)amido]-2-(chloroacetamido)-anthraquinone
(23)
6.2.24. 1-[2-(Trifluoromethyl)benzamido]-2-(chloroacetamido)-
anthraquinone (18)
The pure compound was obtained as a brown powder (yield
63%). Mp: 211e212 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1667 (CO),
The pure compound was obtained as a yellowlish green powder
1710 (C]C), 3260 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.43
(yield 32%). Mp: 248e250 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1671
(s, 2H), 6.77e6.79 (m, 1H), 7.38 (d, J ¼ 3.0 Hz, 1H), 7.88e7.92 (m,
2H), 8.06 (d, J ¼ 0.9 Hz, 1H), 8.11e8.18 (m, 2H), 8.22 (d, J ¼ 8.7 Hz,
1H), 8.39 (d, J ¼ 8.7 Hz, 1H), 9.93 (s, 1H), 10.60 (s, 1H). 13C NMR
(CO), 3282 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.38 (s, 2H),
7.80 (t, J ¼ 7.4 Hz,1H), 7.90e7.97 (m, 4H), 8.13e8.29 (m, 4H), 8.37 (d,
J ¼ 8.4 Hz, 1H), 9.72 (s, 1H), 10.49 (s, 1H). 13C NMR (75 MHz, DMSO-
(75 MHz, DMSO-d6):
d ppm 43.26, 112.51, 115.71, 125.74, 126.37,
d6):
d
ppm 43.32, 126.27, 126.33, 126.61, 126.63, 126.87, 128.07,
127.05, 127.56, 128.59, 128.82, 130.37, 132.27, 134.26, 134.47, 134.61,
139.34, 157.15, 165.60, 181.60, 184.03. HRMS (EI) m/z: calcd [M]þ,
408.0513 (C21H13ClN2Oþ5 ); found, 408.0518.
128.13, 128.53, 128.64, 128.71, 129.01, 130.66, 130.76, 132.18, 132.59,
134.23, 134.35, 134.58, 139.39, 165.31, 166.00, 181.45, 183.24. HRMS
(EI) m/z: calcd [M]þ, 486.0594 (C24H14ClF3N2Oþ4 ); found, 486.0587.
6.2.30. 1-[(2-Thiophenecarbonyl)amido]-2-(chloroacetamido)-
anthraquinone (24)
6.2.25. 1-[3-(Trifluoromethyl)benzamido]-2-(chloroacetamido)-
anthraquinone (19)
The pure compound was obtained as a brown powder (yield
The pure compound was obtained as a yellow powder (yield
55%). Mp: 190e191 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1673 (CO),
48%). Mp: 190e191 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1670 (CO),
1728 (C]C), 3288 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.45
3288 (NH). 1H NMR (300 MHz, DMSO-d6):
d
ppm 4.38 (s, 2H),
(s, 2H), 7.30e7.33 (m, 1H), 7.88e7.94 (m, 4H), 8.10e8.20 (m, 2H),
8.24 (d, J ¼ 8.4 Hz, 1H), 8.42 (d, J ¼ 6.9 Hz, 1H), 9.45 (s, 1H), 10.53 (s,
7.84e7.93 (m, 3H), 8.04 (d, J ¼ 7.5 Hz, 1H), 8.08e8.11 (m, 1H),
8.16e8.19 (m, 1H), 8.27 (d, J ¼ 9.0 Hz, 1H), 8.38e8.49 (m, 3H), 9.90
1H). 13C NMR (75 MHz, DMSO-d6):
d ppm 43.25, 125.95, 126.36,
(s, 1H), 10.58 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d
ppm 43.25,
127.01, 128.04, 128.24, 128.37, 128.88, 130.36, 132.26, 134.26, 134.42,
134.60, 139.08, 139.79, 161.17, 165.60, 181.61, 183.59. HRMS (EI) m/z:
calcd [M]þ, 424.0285 (C21H13ClN2O4Sþ); found, 424.0288.
124.55,124.66,126.16,126.32,126.63,126.96,127.98,128.39,128.48,
128.55, 129.82, 130.34, 132.07, 132.25, 134.22, 134.37, 134.55, 135.27,
140.30, 165.24, 165.63, 181.56, 183.25. HRMS (EI) m/z: calcd [M]þ,
486.0594 (C24H14ClF3N2Oþ4 ); found, 486.0596.
6.2.31. 1-[(5-Isoxazolecarbonyl)amido]-2-(chloroacetamido)-
anthraquinone (25)
6.2.26. 1-[4-(Trifluoromethyl)benzamido]-2-(chloroacetamido)-
anthraquinone (20)
The pure compound was obtained as a yellowish brown powder
(yield 52%). Mp: 212e213 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1598
The pure compound was obtained as a brown powder (yield
(C]N), 1673 (CO), 3268 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm
57%). Mp: 203e204 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1666 (CO),
4.43 (s, 2H), 7.31 (d, J ¼ 1.8 Hz,1H), 7.88e7.92 (m, 2H), 8.08e8.18 (m,
2H), 8.27 (d, J ¼ 8.7 Hz, 1H), 8.49 (d, J ¼ 8.7 Hz, 1H), 8.88 (d,
J ¼ 1.8 Hz, 1H), 10.03 (s, 1H), 10.69 (s, 1H). 13C NMR (75 MHz, DMSO-
3285 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.43 (s, 2H),
7.88e7.91 (m, 2H), 8.01 (d, J ¼ 8.4 Hz, 2H), 8.08e8.11 (m, 1H),
8.16e8.19 (m, 1H), 8.25e8.32 (m, 3H), 8.45 (d, J ¼ 8.3 Hz, 1H), 9.90
d6):
d ppm 43.25, 107.12, 126.35, 126.66, 126.82, 126.98, 127.84,
(s, 1H), 10.60 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d
ppm 43.25,
128.56, 130.17, 132.24, 134.18, 134.42, 134.59, 140.46, 151.99, 155.50,
162.77, 165.79, 181.50, 183.06. HRMS (EI) m/z: calcd [M]þ, 409.0465
(C20H12ClN3Oþ5 ); found, 409.0472.
125.53, 125.77, 126.27, 126.95, 128.11, 128.39, 128.66, 128.93, 130.37,
131.56, 131.99, 132.26, 134.24, 134.39, 134.57, 138.02, 140.12, 165.42,
165.61, 181.57, 183.35. HRMS (EI) m/z: calcd [M]þ, 486.0594
(C24H14ClF3N2Oþ4 ); found, 486.0597.
6.2.32. 1-[(2,5-Dimethyl-3-furoyl)amido]-2-(chloroacetamido)-
anthraquinone (26)
6.2.27. 1-[2-(Phenyl)acetylamido]-2-(chloroacetamido)-
anthraquinone (21)
The pure compound was obtained as a yellow powder (yield
63%). Mp: 207e208 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1676 (CO),
The pure compound was obtained as a brown powder (yield
3167 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 2.33 (s, 3H), 2.52 (s,
34%). Mp: 184e185 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1667 (CO),
3H), 4.44 (s, 2H), 6.70 (s, 1H), 7.89e7.92 (m, 2H), 8.13e8.19 (m, 2H),
8.21 (d, J ¼ 8.7 Hz, 1H), 8.34 (d, J ¼ 8.7 Hz, 1H), 9.83 (s, 1H), 10.29 (s,
3282 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 3.86 (s, 2H), 4.36
(s, 2H), 7.23e7.93 (m, 5H), 7.89e7.93 (m, 2H), 8.11e8.20 (m, 3H),
1H). 13C NMR (75 MHz, DMSO-d6):
d ppm 12.74, 13.04, 43.14, 105.21,
8.31 (d, J ¼ 8.7 Hz, 1H), 9.66 (s, 1H), 10.25 (s, 1H). 13C NMR (75 MHz,
116.20, 125.29, 126.29, 126.98, 128.82, 129.93, 130.50, 132.23, 134.23,
134.39, 134.52, 138.67, 149.74, 156.05, 162.99, 165.30, 181.55, 184.22.
HRMS (EI) m/z: calcd [M]þ, 436.0826 (C23H17ClN2Oþ5 ); found,
436.0832.
DMSO-d6):
d ppm 42.51, 43.34, 125.59, 126.26, 126.53, 126.83,
128.15, 128.86, 129.30, 129.66, 130.30, 132.16, 134.22, 134.29, 134.52,
135.35, 142.78, 145.07, 165.25, 170.57, 181.51, 183.25. HRMS (EI) m/z:
calcd [M]þ, 432.0877 (C24H17ClN2Oþ4 ); found, 432.0885.
6.2.33. 1-[2-(Phenoxy)acetylamido]-2-(chloroacetamido)-
anthraquinone (27)
6.2.28. 1-[2-(4-Fluorophenyl)acetylamido]-2-(chloroacetamido)-
anthraquinone (22)
The pure compound was obtained as a yellowish green powder
The pure compound was obtained as a yellowish brown powder
(yield 55%). Mp: 217e218 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1681
(yield 37%). Mp: 213e214 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1667
(CO), 3182 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.40 (s, 2H),
(CO), 3279 (NH). 1H NMR (300 MHz, DMSO-d6):
d
ppm 3.86 (s, 2H),
4.82 (s, 2H), 7.02 (t, J ¼ 6.0 Hz, 1H), 7.16 (d, J ¼ 7.5 Hz, 2H), 7.35e7.40
(m, 2H), 7.90e7.93 (m, 2H), 8.13e8.18 (m, 2H), 8.20 (d, J ¼ 8.7 Hz,
1H), 8.33 (d, J ¼ 8.4 Hz, 1H), 9.86 (s, 1H), 10.58 (s, 1H). 13C NMR
4.37 (s, 2H), 7.17 (t, J ¼ 9.0 Hz, 2H), 7.42e7.47 (m, 2H), 7.90e7.93 (m,
2H), 8.11e8.23 (m, 3H), 8.32 (d, J ¼ 8.7 Hz, 1H), 9.64 (s, 1H), 10.20 (s,
1H). 13C NMR (75 MHz, DMSO-d6):
d
ppm 41.48, 43.03, 114.69,
(75 MHz, DMSO-d6): d ppm 43.12, 67.24, 114.94, 118.49, 121.48,

334
C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
125.60, 126.29, 126.89, 127.44, 128.64, 129.54, 132.22, 134.21, 134.37,
134.52, 135.11, 138.87, 157.66, 165.41, 168.03, 181.50, 183.94. HRMS
(EI) m/z: calcd [M]þ, 448.0826 (C24H17ClN2Oþ5 ); found, 448.0824.
(CO), 3287 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 1.32 (br,
8H), 2.43 (s, 3H), 3.27 (s, 2H), 7.42 (d, J ¼ 8.1 Hz, 2H), 7.87e7.91 (m,
2H), 8.05 (d, J ¼ 8.1 Hz, 2H), 8.09e8.19 (m, 2H), 8.27 (d, J ¼ 8.7 Hz,
1H), 8.73 (d, J ¼ 8.4 Hz, 1H), 10.12 (s, 1H), 10.54 (s, 1H). 13C NMR
6.2.34. 1-[2-(Phenylsulfanyl)acetylamido]-2-(chloroacetamido)-
anthraquinone (28)
(75 MHz, DMSO-d6): d ppm 20.86, 23.20, 53.65, 59.09, 126.30,
126.54, 126.88, 127.80, 127.88, 127.92, 129.05, 129.25, 130.59,
132.33, 134.12, 134.38, 134.42, 136.24, 140.44, 142.43, 166.26,
169.56, 181.42, 183.68. HRMS (EI) m/z: calcd [M]þ, 467.1845
(C28H25N3Oþ4 ); found, 467.1840.
The pure compound was obtained as a brown powder (yield
59%). Mp: 161e162 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1671 (CO),
3260 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 4.10 (s, 2H), 4.43
(s, 2H), 7.18e7.22 (m, 1H),7.33e7.35 (m, 2H), 7.45 (d, J ¼ 7.2 Hz, 2H),
7.86e7.93 (m, 2H), 8.08e8.16 (m, 2H), 8.21 (d, J ¼ 8.7 Hz, 1H), 8.30
(d, J ¼ 8.7 Hz, 1H), 9.71 (s, 1H), 10.46 (s, 1H). 13C NMR (75 MHz,
6.2.39. 1-(4-Methylbenzamido)-2-[(2-piperidinoacetyl)amido]-
anthraquinone (33)
DMSO-d6):
d
ppm 43.82, 120.98, 126.43, 126.58, 127.00, 127.51,
The pure compound was obtained as a yellowish green powder
128.56, 129.20, 129.52, 129.66, 131.16, 132.93, 134.81, 134.96, 135.01,
135.23, 136.69, 139.82, 166.05, 168.92, 182.23, 184.16. HRMS (EI) m/
z: calcd [M]þ, 464.0598 (C24H17ClN2O4Sþ); found, 464.0602.
(yield 43%). Mp: 213e214 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1673
(CO), 3300 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 1.08 (d,
J ¼ 3.6 Hz, 2H), 1.19 (br, 4H), 2.33 (br, 4H), 2.43 (s, 3H), 3.07 (s, 2H),
7.43 (d, J ¼ 7.8 Hz, 2H), 7.87e7.91 (m, 2H), 8.07e8.19 (m, 4H), 8.27
(d, J ¼ 8.7 Hz, 1H), 8.78 (d, J ¼ 8.7 Hz, 1H), 10.13 (s, 1H), 10.52 (s, 1H).
6.2.35. 1-(4-Methylbenzamido)-2-[2-(dimethylamino)
acetylamido]-anthraquinone (29)
13C NMR (75 MHz, DMSO-d6):
d ppm 20.85, 22.80, 24.79, 54.13,
The pure compound was obtained as a yellowish brown powder
62.39, 125.19, 126.27, 126.53, 126.89, 127.84, 127.92, 128.02, 129.00,
129.22, 130.57, 132.31, 134.13, 134.36, 134.42, 140.63, 142.40, 166.25,
169.55, 181.42, 183.67. HRMS (EI) m/z: calcd [M]þ, 481.2002
(C29H27N3Oþ4 ); found, 481.2000.
(yield 54%). Mp: 190e191 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1685
(CO), 3299 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 2.11 (s, 6H),
2.44 (s, 3H), 3.09 (s, 2H), 7.43 (d, J ¼ 8.1 Hz, 2H), 7.88e7.91 (m, 2H),
8.00e8.04 (m, 2H), 8.11e8.19 (m, 2H), 8.26 (d, J ¼ 8.7 Hz, 1H), 8.64
(d, J ¼ 8.4 Hz, 1H), 10.16 (s, 1H), 10.54 (s, 1H). 13C NMR (75 MHz,
6.2.40. 1-(4-Methylbenzamido)-2-[(2-morpholinoacetyl)amido]-
anthraquinone (34)
DMSO-d6): d ppm 20.86, 45.16, 62.82, 125.73, 126.30, 126.47, 127.79,
127.99, 128.12, 128.89, 129.09, 129.39, 130.93, 132.32, 134.13, 134.36,
134.43, 140.34, 142.23, 166.35, 169.14, 181.45, 183.63. HRMS (EI) m/
z: calcd [M]þ, 441.1689 (C26H23N3Oþ4 ); found, 441.1689.
The pure compound was obtained as a yellow powder (yield
47%). Mp: 225e226 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1672 (CO),
3213 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 2.39 (br, 4H),
2.45 (s, 3H), 3.15 (s, 2H), 3.22 (br, 4H), 7.45 (d, J ¼ 7.8 Hz, 2H),
7.88e7.91 (m, 2H), 8.07e8.12 (m, 4H), 8.27 (d, J ¼ 8.7 Hz, 1H), 8.73
(d, J ¼ 8.7 Hz, 1H), 9.99 (s, 1H), 10.61 (s, 1H). 13C NMR (75 MHz,
6.2.36. 1-(4-Methylbenzamido)-2-{2-[(1,3-dioxolan-
2ylmethyl)(mehyl)amino]acetylamido}- anthraquinone (30)
The pure compound was obtained as a yellowish powder (yield
43%). Mp: 156e157 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1672 (CO),
DMSO-d6):
d ppm 20.95, 53.17, 61.94, 65.59, 125.73, 126.37, 126.53,
126.98, 127.82, 128.05, 128.54, 129.28, 129.46, 130.55, 132.35,
134.16, 134.42, 134.53, 140.31, 142.66, 166.32, 168.97, 181.50,
183.84. HRMS (EI) m/z: calcd [M]þ, 483.1794 (C28H25N3O5þ); found,
483.1793.
3282 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 2.18 (s, 3H), 2.44
(s, 3H), 2.54 (d, J ¼ 8.7 Hz, 2H), 3.26 (s, 2H), 3.52e3.55 (m, 2H),
3.72e3.77 (m, 2H), 4.58 (t, J ¼ 4.5 Hz, 1H), 7.43 (d, J ¼ 7.8 Hz, 2H),
7.88e7.92 (m, 2H), 8.04 (d, J ¼ 8.1 Hz, 2H), 8.10e8.19 (m, 2H), 8.27
(d, J ¼ 8.4 Hz, 1H), 8.70 (d, J ¼ 8.7 Hz, 1H), 10.15 (s, 1H), 10.53 (s, 1H).
6.2.41. 1-(4-Methylbenzamido)-2-[2-(1,4-thiazinan-4-yl)
acetylamido]-anthraquinone (35)
13C NMR (75 MHz, DMSO-d6):
d ppm 20.87, 43.49, 59.43, 61.73,
63.91, 102.61, 125.50, 126.31, 126.48, 126.90, 127.89, 127.91, 128.06,
129.06, 129.31, 130.75, 132.33, 134.16, 134.38, 134.45, 140.42, 142.29,
166.25, 169.59, 181.46, 183.70. HRMS (EI) m/z: calcd [M]þ, 513.1900
(C29H27N3Oþ6 ); found, 513.1902.
The pure compound was obtained as a yellowish green powder
(yield 51%). Mp: 210e211 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1677
(CO), 3234 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 2.39 (br,
4H), 2.45 (s, 3H), 3.15 (s, 2H), 3.23 (br, 4H), 7.45 (d, J ¼ 8.1 Hz, 2H),
7.88e7.91 (m, 2H), 8.07e8.19 (m, 4H), 8.27 (d, J ¼ 8.4 Hz, 1H), 8.73
(d, J ¼ 8.4 Hz, 1H), 10.00 (s, 1H), 10.61 (s, 1H). 13C NMR (75 MHz,
6.2.37. 1-(4-Methylbenzamido)-2-{{2-{methy[2-(2-pyridyl)ethyl]
amino}acetylamido}}eanthraquinone (31)
DMSO-d6):
d ppm 20.89, 53.13, 61.92, 65.54, 127.72, 126.30, 126.40,
The pure compound was obtained as a pale yellow powder
126.92, 127.69, 127.92, 127.98, 129.22, 129.43, 130.53, 132.30,
134.12, 134.41, 134.45, 140.21, 142.61, 166.27, 168.89, 181.42, 183.84.
HRMS (EI) m/z: calcd [M]þ, 499.1566 (C28H25N3O4Sþ); found,
499.1570.
(yield 32%). Mp: 166e167 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1666
(CO), 3263 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 2.15 (s, 3H),
2.40 (s, 3H), 2.68 (d, J ¼ 7.2 Hz, 4H), 3.20 (s, 2H), 7.03 (d, J ¼ 7.0 Hz,
1H), 7.13e7.18 (m, 1H), 7.39 (d, J ¼ 8.1 Hz, 2H), 7.60e7.65 (m, 1H),
7.89e7.93 (m, 2H), 8.03 (d, J ¼ 8.1 Hz, 2H), 8.11e8.20 (m, 2H), 8.28
(d, J ¼ 8.7 Hz, 1H), 8.40e8.42 (m, 1H), 8.69 (d, J ¼ 8.7 Hz, 1H), 10.18
6.2.42. 1-(4-Methylbenzamido)-2-[2-(4-methylpiperazino)
acetylamido]-anthraquinone (36)
(s, 1H), 10.54 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d
ppm 20.81,
The pure compound was obtained as a yellowish brown powder
42.45, 56.92, 61.03, 63.91, 121.62, 122.81, 125.50, 126.29, 126.44,
126.88, 127.83, 127.88, 128.07, 129.08, 129.30, 130.72, 132.33, 134.13,
134.36, 134.42, 136.24, 140.37, 142.30, 148.86, 159.40, 166.19, 169.59,
181.43, 183.74. HRMS (EI) m/z: calcd [M]þ, 532.2111 (C32H28N4O4þ);
found, 532.2119.
(yield 55%). Mp: 214e215 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1678
(CO), 3252 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 1.76 (s, 3H),
1.96 (br, 4H), 2.38 (br, 4H), 2.43 (s, 3H), 3.13 (s, 2H), 7.45 (d,
J ¼ 7.8 Hz, 2H), 7.87e7.93 (m, 2H), 8.08e8.16 (m, 4H), 8.27 (d,
J ¼ 9.0 Hz, 1H), 8.81 (d, J ¼ 8.7 Hz, 1H), 10.01 (s, 1H), 10.50 (s, 1H). 13C
NMR (75 MHz, DMSO-d6):
d ppm 20.82, 44.73, 52.79, 53.73, 61.60,
6.2.38. 1-(4-Methylbenzamido)-2-[2-(tetrahydro-1H-1-pyrrolyl)
acetylamido]-anthraquinone (32)
125.02, 126.28, 126.58, 126.89, 127.77, 127.91, 128.28, 129.14, 129.21,
130.61, 132.31, 134.12, 134.38, 134.42, 140.59, 142.53, 166.29, 169.20,
181.42, 183.67. HRMS (EI) m/z: calcd [M]þ, 496.2111 (C29H28N4O4þ);
found, 496.2115.
The pure compound was obtained as a dark brown powder
(yield 39%). Mp: 193e194 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1667

C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
335
6.2.43. 1-(4-Methylbenzamido)-2-{2-[4-(2-hydroxyethyl)
piperazino]acetylamido}- anthraquinone (37)
and penicillin (100 units/mL)/streptomycin (100
m
g/mL). Cultures
were maintained in a humidified incubator at 37 ꢁC in 5% CO2. Cells
were seeded in 96-well plates in medium with 5% FBS. After 24 h,
cells were fixed with 10% trichloroacetic acid (TCA) to represent cell
population at the time of compound addition (T0). After additional
incubation of vehicle (0.1% DMSO) or the indicated compound for
48 h, cells were fixed with 10% TCA and SRB at 0.4% (w/v) in 1%
acetic acid was added to stain cells. Unbound SRB was washed out
by 1% acetic acid and SRB bound cells were solubilized with 10 mM
Trizma base. The absorbance was read at a wavelength of 515 nm.
Using the following absorbance measurements, such as time zero
(T0), control growth (C), and cell growth in the presence of
compound (Tx), the percentage growth was calculated at each of
the compound concentrations levels. Percentage growth inhibition
was calculated as: 100ꢀ[(TxꢀT0)/(CꢀT0)] ꢃ 100 for concentrations
for which Tx ꢄ T0. Growth inhibition of 50% (IC50) was determined
at the drug concentration which resulted in 50% reduction of total
protein increased in control cells during the compound incubation.
The pure compound was obtained as a yellowish brown powder
(yield 48%). Mp: 210e211 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1677
(CO), 3264 (NH), 3314 (OH). 1H NMR (300 MHz, DMSO-d6):
d ppm
1.92 (t, J ¼ 6.0 Hz, 2H), 2.03 (br, 4H), 2.39 (br, 4H), 2.43 (s, 3H), 3.01
(s, 2H), 3.26e3.30 (m, 2H), 4.29 (t, J ¼ 5.4 Hz, 1H), 7.43 (d, J ¼ 8.1 Hz,
2H), 7.87e7.91 (m, 2H), 8.07e8.19 (m, 4H), 8.27 (d, J ¼ 8.7 Hz, 1H),
8.79 (d, J ¼ 8.7 Hz,1H), 10.00 (s, 1H),10.50 (s, 1H). 13C NMR (75 MHz,
DMSO-d6):
d ppm 20.93, 52.32, 52.93, 58.21, 59.10, 61.69, 125.11,
126.37, 126.72, 126.98, 127.81, 128.04, 128.33, 129.24, 129.41, 130.61,
132.36, 134.17, 134.48, 134.53, 140.68, 142.70, 166.37, 169.28, 181.50,
183.67. HRMS (EI) m/z: calcd [M]þ, 526.2216 (C30H30N4O5þ); found,
526.2219.
6.2.44. 1-(4-Methylbenzamido)-2-[2-(4-phenylpiperazino)
acetylamido]-anthraquinone (38)
The pure compound was obtained as a yellowish green powder
(yield 53%). Mp: 270e271 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1673
(CO), 3274 (NH). 1H NMR (300 MHz, DMSO-d6):
d
ppm 2.21 (s, 3H),
6.4. Cell cultures and assessment of hTERT
2.55 (br, 4H), 2.73 (br, 4H), 3.22 (s, 2H), 6.64 (d, J ¼ 7.5 Hz, 2H), 6.79
(d, J ¼ 7.2 Hz, 1H), 7.12e7.21 (m, 4H), 7.87e7.92 (m, 2H), 7.98 (d,
J ¼ 8.1 Hz, 2H), 8.08e8.19 (m, 2H), 8.29 (d, J ¼ 8.7 Hz, 1H), 8.80 (d,
J ¼ 8.7 Hz, 1H), 10.07 (s, 1H), 10.52 (s, 1H). 13C NMR (75 MHz, DMSO-
Non-small lung cancer cells H1299 [25] were grown in
RPMI1640 media supplemented with 10% fetal bovine serum,
100 units/mL penicillin and 100 mg/mL streptomycin in a humidi-
fied atmosphere with 5% CO2 at 37 ꢁC. Culture media were changed
every 3 days. To establish stable cell lines so that the expression of
hTERT could be monitored by a reporter system, a w3.3 kbp DNA
fragment ranging from ꢀ3338 to þ1 bp of the hTERT gene was
subcloned upstream to a secreted alkaline phosphatase gene
(SEAP) and transfectectd into H1299 by electroporation. The stable
clones were selected using G418. The stable clones derived from
H1299 was cultured using conditions that are similar to their
parental cells.
d6):
d ppm 20.76, 47.66, 52.81, 61.52, 115.40, 118.74, 125.16, 126.28,
126.57, 126.88, 127.84, 127.95, 128.16, 128.58, 129.05, 129.27,
130.28 132.31, 134.11, 134.42, 135.80, 140.51, 142.46, 150.77, 166.21,
169.05, 181.42, 183.65. HRMS (EI) m/z: calcd [M]þ, 558.2267
(C34H30N4Oþ4 ); found, 558.2271.
6.2.45. 1-(4-Methylbenzamido)-2-{2-[4-(2-pyridyl)piperazino]
acetylamido}-anthraquinone (39)
The pure compound was obtained as a yellowish brown powder
(yield 63%). Mp: 258e259 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1660
(CO), 3263 (NH). 1H NMR (300 MHz, DMSO-d6):
d
ppm 2.16 (s, 3H),
6.5. Secreted alkaine phosphatase (SEAP) assay
3.05 (br, 8H), 3.22 (s, 2H), 6.54 (d, J ¼ 8.4 Hz, 1H), 6.66 (t, J ¼ 6.3 Hz,
1H), 7.08 (d, J ¼ 7.8 Hz, 2H), 7.49e7.54 (m, 1H), 7.87e7.91 (m, 2H),
7.97 (d, J ¼ 8.1 Hz, 2H), 8.07e8.19 (m, 3H), 8.29 (d, J ¼ 8.7 Hz, 1H),
8.82 (d, J ¼ 9.0 Hz, 1H), 10.16 (s, 1H), 10.51 (s, 1H). 13C NMR (75 MHz,
Secreted alkaine phosphatase was used as the reporting system
to monitor the transcriptional activity of hTERT [31]. Here, about
2 ꢃ 103 cells each were grown in 96-well plates and incubated at
37 ꢁC for 24 h and changed with fresh media. Varying amounts of
drugs were added and cells were incubated for another 24 h.
Culture media were collected and heated at 65 ꢁC for 10 min to
inactivate heat-labile phosphatases. An equal amount of SEAP
DMSO-d6):
d ppm 20.88, 44.21, 52.56, 61.54, 107.00, 113.11, 124.91,
126.42, 126.89, 126.99, 127.63, 128.03, 128.21, 129.01, 129.29, 132.26,
133.39, 134.19, 134.52, 134.58, 137.30, 140.73, 142.51, 147.49, 159.15,
166.26, 166.31, 181.54, 183.63. HRMS (EI) m/z: calcd [M]þ, 559.2220
(C33H29N5Oþ4 ); found, 559.2224.
buffer (2 M diethanolamine, 1 mM MgCl2, and 20 mM L-homo-
arginine) was added to the media and p-nitrophenyl phosphate was
added to a final concentration of 12 mM. Absorptions at 405 nm
were taken, and the rate of absorption increase was determined.
6.2.46. 1-(4-Methylbenzamido)-2-{2-[4-(2-pyrimidinyl)piperazino]
acetylamido}-anthraq uinone (40)
The pure compound was obtained as a yellow powder (yield
41%). Mp: 267e268 ꢁC (EtOH). FT-IR (KBr, nmaxcmꢀ1): 1683 (CO),
6.6. MTT assay for cell viability
3236 (NH). 1H NMR (300 MHz, DMSO-d6):
d ppm 2.20 (s, 3H), 3.11
(s, 2H), 3.22 (br, 8H), 6.66 (d, J ¼ 4.8 Hz, 1H), 7.16 (d, J ¼ 7.8 Hz, 2H),
7.87e7.91 (m, 2H), 8.01 (d, J ¼ 6.0 Hz, 2H), 8.08e8.19 (m, 2H),
8.28e8.34 (m, 2H), 8.32 (d, J ¼ 7.2 Hz, 1H), 8.82 (d, J ¼ 8.7 Hz, 1H),
The tetrazolium reagent (MTT; 3-(4,5-di-methylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide, USB) was designed to yield
a colored formazan upon metabolic reduction by viable cells
[32,33]. Approximately 2 ꢃ 103 cells were plated onto each well of
a 96-well plate and incubated in 5% CO2 at 37 ꢁC for 24 h. To assess
the in vitro cytotoxicity, each compound was dissolved in DMSO
and prepared immediately before the experiments and was diluted
into the complete medium before addition to cell cultures. Test
compounds were then added to the culture medium for designated
10.19 (s, 1H), 10.54 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d ppm
20.98, 42.53, 43.01, 52.46, 61.44, 110.28, 111.11, 124.77, 126.32,
126.89, 127.44, 127.92, 128.14, 128.90, 129.18, 130.18, 132.26, 134.06,
134.48, 142.39, 157.65, 158.07, 161.24, 166.12, 169.08, 181.35, 183.39.
HRMS (EI) m/z: calcd [M]þ, 560.2172 (C32H28N6Oþ4 ); found,
560.2170.
various concentrations. After 48 h, an amount of 25
mL of MTT was
6.3. Cell cultures and sulforhodamine B (SRB) assay
added to each well, and the samples were incubated at 37 ꢁC for 4 h.
A 100 mL solution of lysis buffer containing 20% SDS and 50% N,N-
Human hormone-refractory prostate cancer cell lines (PC-3)
were from American Type Culture Collection (Rockville, MD) [30].
The cells were cultured in RPMI1640 medium with 10% FBS (v/v)
dimethylformamide was added to each well and incubated at 37 ꢁC
for another 16 h. The absorbency at 550 nm was measured using an
ELISA reader.

336
C.-C. Lee et al. / European Journal of Medicinal Chemistry 47 (2012) 323e336
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polyacrylamide gel electrophoresis and visualized by staining with
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derived from lung cancer cell lines (H1299) were used. Protein
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assay kit using BSA standards.
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The present study was supported by National Science Council
Grants NSC 100-2113-M-016, 99-2113-M-016, 99-3112-B-010-001,
and 98-2113-M-016-001, respectively. We are grateful to thank
NIH-NCI for their supports.
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