Synthesis, cytotoxicity and human telomerase inhibition activities of a series of 1,2-heteroannelated anthraquinones and anthra[ 1,2-d]imidazole-6,11 -dione homologues

Article abstract of DOI:10.1016/j.bmc.2009.09.033

A series of 1,2-heteroannelated anthraquinones and anthra[1,2-d]imidazole- 6,11-dione tetracyclic analogues with different side chain were prepared using an various synthetic route via acylation, cyclization, condensation, and intramolecular heterocyclization. Tetracyclic system containing alkyl and aryl, aromatic and heterocyclic, linear and cyclic, polar and apolar, and basic and acids residues were incorporated. They were evaluated for their effects on telomerase activity, hTERT expression, cell proliferations, and in vitro cytotoxicity against NCI's 60 cell line human tumor screen. Compounds 4, 11, 12, 14, 15, 16, 17, 19, 20, 23, 25, and 26 were selected by the NCI for one dose screening program and further studies on 4, 23 and 25 where the curves cross these lines represent the interpolated values to cause 50% growth inhibition (GI₅₀), total growth inhibition (TGI) and 50% cell killing (LC ₅₀). respectively. Further studies did not reveal any compound that showed potent and significant on telomerase inhibitory activity and hTERT repressing ability. Comparative testing of these compounds in the NCI's screen revealed varying levels of potency and differential cytotoxicity, apparently related to the unsaturation levels in and substitution patterns on the core ring system. It appeared that addition of a fourth planar aromatic system to a tricyclic chromophore might enhances potent cytotoxic agents, at a level equivalent to a second side chain in one of the tricyclic series. Although the exact mechanism of how this pharmacophore contributes to its activity is still unclear, however, the group in the extended arm of the tetracyclic system might contribute to proper binding to the residues within the grove of G-quadruplex structure.

Full text of DOI:10.1016/j.bmc.2009.09.033

Bioorganic & Medicinal Chemistry 17 (2009) 7418–7428
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, cytotoxicity and human telomerase inhibition activities
of a series of 1,2-heteroannelated anthraquinones and
anthra[1,2-d]imidazole-6,11-dione homologues
a
b
c
d
Hsu-Shan Huang a, , Tsung-Chih Chen , Ruei-Huei Chen , Kuo-Feng Huang , Fong-Chun Huang ,
*
Jing-Ru Jhan d, Chun-Liang Chen a, Chia-Chung Lee a, Yang Lo a, Jing-Jer Lin d,
*
a School of Pharmacy, National Defense Medical Center, No. 161, MinChuan E. Rd., Neihu, Taipei 11490, Taiwan, ROC
b Department of Basic Medical Science, Hungkuang University, Taichung 443, Taiwan
c Chi-Mei Medical Center, Tainan 710, Taiwan
d Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, 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 1,2-heteroannelated anthraquinones and anthra[1,2-d]imidazole-6,11-dione tetracyclic ana-
logues with different side chain were prepared using an various synthetic route via acylation, cyclization,
condensation, and intramolecular heterocyclization. Tetracyclic system containing alkyl and aryl, aro-
matic and heterocyclic, linear and cyclic, polar and apolar, and basic and acids residues were incorpo-
rated. They were evaluated for their effects on telomerase activity, hTERT expression, cell
proliferations, and in vitro cytotoxicity against NCI’s 60 cell line human tumor screen. Compounds 4,
11, 12, 14, 15, 16, 17, 19, 20, 23, 25, and 26 were selected by the NCI for one dose screening program
and further studies on 4, 23 and 25 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), respec-
tively. Further studies did not reveal any compound that showed potent and significant on telomerase
inhibitory activity and hTERT repressing ability. Comparative testing of these compounds in the NCI’s
screen revealed varying levels of potency and differential cytotoxicity, apparently related to the unsatu-
ration levels in and substitution patterns on the core ring system. It appeared that addition of a fourth
planar aromatic system to a tricyclic chromophore might enhances potent cytotoxic agents, at a level
equivalent to a second side chain in one of the tricyclic series. Although the exact mechanism of how this
pharmacophore contributes to its activity is still unclear, however, the group in the extended arm of the
tetracyclic system might contribute to proper binding to the residues within the grove of G-quadruplex
structure.
Received 22 August 2009
Revised 15 September 2009
Accepted 17 September 2009
Available online 23 September 2009
Keywords:
Heteroannelated anthraquinones
Anthra[1,2-d]imidazole-6,11-dione
Cytotoxicity
Telomerase
NCI’s 60 cell line human tumor screen
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
the DNA base pairs, the drug, and the enzyme in the minor groove
has remained elusive. Anthracyclines and their anthraquinone
The mechanism of the cytotoxicity of anthracyclines is pleiotro-
pic and its significance in cell growth inhibition seems to be highly
specific and dependent on cell type and anthracycline drug.1 The
anthracycline antibiotics daunorubicin, and doxorubicin (Scheme
1) have been used widely as anticancer drugs for more than
30 years, but their cardiotoxicity limits their clinical use.2 The clin-
ical use of anthracyclines like doxorubicin and daunorubicin can be
viewed as a sort of double-edged sword.3 Doxorubicin and its ana-
logues function as topoisomerase poisons through stabilization of
the cleavable complex that forms as part of the catalytic cycle of
the enzyme.4 The detailed mechanism of how these compounds,
all of which contain an anthraquinone core that forms between
derivatives remain ‘evergreen’ drugs with broad clinical indica-
tions but have an improvable pharmacological index. In an attempt
to identify new molecular or pharmacophore targets for anticancer
drugs, our attention has been focused on cytotoxicity and
telomerase.
Telomerase activity is thought to be required for the develop-
ment of cellular immortality and oncogenesis. Thus, considerable
interest is focused on telomerase because of its potential uses in
assays for cancer diagnosis, research into cell biology and for
anti-telomerase drugs as a strategy for cancer chemotherapy.5,6
At present, among different strategies pursued telomerase inhibi-
tors in chemotherapy, the development of G-quadruplex stabiliz-
ers has emerged as a highly promising approach.7,8 Herein, we
report the design and synthesis of a series of non-nucleoside
telomerase inhibitors, in an attempt to identify potent enzyme
* Corresponding authors. Tel.: +886 2 87926454; fax: +886 2 87923169 (H.-S.H.).
E-mail address: huanghs@mail.ndmctsgh.edu.tw (H.-S. Huang).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.033

H.-S. Huang et al. / Bioorg. Med. Chem. 17 (2009) 7418–7428
7419
O
O
O
HN
HN
N
NH
O
C
OH
B
O
O
R
4 (NSC745795)
20 (NSC745886)
OH
D
A
O
N
S
O
N
OMe O
OH
O
N
N
O
Me
NH2
O
O
OH
25 (NSC745887)
23 (NSC745885)
Doxorubicin (R = OH)
Daunorubicin (R = H)
O
O
O
HN
NH
O
26 (NSC745888)
Scheme 1.
inhibitors and worked towards its structural modifications to de-
velop some potential anti-cancer therapeutic leads compared to
parent molecules.
dione homologues can be synthesized and the preparation in-
volved various synthetic route with appropriate yields (overall
23–89% in all steps) (Scheme 2): (i) acylation reaction of 1,2-diami-
noanthraquinone 1 yielded the corresponding side chain com-
pounds 2 and 3; (ii) reaction of 1,2-diaminoanthraquinone 1 with
various aldehydes and concd sulfuric acid yielded the correspond-
ing side chain compounds 4–19, respectively; (iii) reaction of 1,2-
diaminoanthraquinone 1 with dry acetone and acetophenone and
concd sulfuric acid yielded the corresponding side chain com-
pounds 20 and 21, respectively; (iv) reaction of 1,2-diaminoanthra-
quinone 1 with carbon disulfide and triethylamine yielded the
corresponding compounds 22; (v) reaction of 1,2-diaminoanthra-
quinone 1 with thionyl chloride and triethylamine yielded the
corresponding compounds 23; (vi) reaction of 1,2-diaminoanthra-
quinone 1 with methyl vinyl ketone and concd sulfuric acid yielded
the corresponding compounds 24; (vii) reaction of 1,2-diaminoan-
thraquinone 1 with glyoxal in ethanol yielded the corresponding
compounds 25; (viii) reaction of 1,2-diaminoanthraquinone 1 with
oxalic acid and concd sulfuric acid yielded the corresponding com-
pounds 26. Compounds 4–19 were synthesized by direct one-step
imidazole cyclization reaction using aldehydes to obtain various
side chain substituted derivatives. However, under these reaction
conditions significant amounts of the reaction products were iso-
lated along with the desired reaction products. The quantity of
the byproducts isolated was substrate dependant and purification
of the reaction mixtures required tedious recrystallization or chro-
matography. The molecular formulas of 2–26 were all determined
by HRMS. The protons from thiadiazole and imidazole structure
were also obvious from the 1H NMR spectra.
As both benzimidazole and anthraquinone derivatives are
known topoisomerase inhibitors and telomerase inhibitors,9–13
the search for additional enzyme inhibitor is justified by the
remarkable anticancer activity of imidazole-fused and heteroann-
elated anthraquinone derivatives and by the possibility that differ-
ent enzyme poisons would exhibit different activity in cancer
chemotherapy. Non-nucleoside (small-molecule) inhibitors that
stabilize folded G-quadruplex structures might inhibit telomerase.
Inhibitory agents from many chemical classes have been identified,
many through screening, but their specificity of action is still in
doubt. A specific inhibitor is expected to immediately inhibit activ-
ity but not cell division, produce telomerase shortening over multi-
ple generations, and ultimately produce end-to-end chromosomal
fusion and growth arrest.14 In the course of our continuing search
for new antitumor agents from anthraquinone moiety, we found
that imidazole-fused and heteroannelated anthraquinone deriva-
tives exhibited very similar and striking differential cytotoxicity
profiles in the National Cancer Institute’s (NCI) 60 cell line primary
screen. Supposedly, an additional heterocyclic moiety or tetracyclic
system might substantially increase the binding with their intra-
cellular targets. We report here the synthesis methods, identifica-
tion, and high cytotoxic activity of novel compounds for a variety
of tumor cell lines. The synthesized compounds (4, 11, 12, 14, 15,
16, 17, 19, 20, 23, 25, 26) were screened for in vitro cytotoxicity,
besides; an attempt was made to understand their possible molec-
ular mode of action and reportedly possesses diverse telomerase
activity.
2.2. Antiproliferative activity
2. Results and discussion
2.1. Chemistry
The 1,2-heteroannelated anthraquinones and anthra[1,2-d]imid-
azole-6,11-dione derivatives were studies for antiproliferative
activity against human cancer cell lines in NCI Drug Screen Program.
Out of the synthesized derivatives, compounds 4, 11, 12, 14, 15, 16,
17, 19, 20, 23, 25, and 26 have been selected by NCI as prototypes
(Table 1). Those potential compounds were evaluated in the full pa-
nel of human tumor cell lines derived from nine cancer cell types
The compounds were synthesized, starting from 1,2-diamino-
anthraquinone. After considerable effort we were able to develop
a process of fused tetracyclic system by which a series of 1,2-het-
eroannelated anthraquinones and anthra[1,2-d]imidazole-6,11-

7420
H.-S. Huang et al. / Bioorg. Med. Chem. 17 (2009) 7418–7428
O
R
R1
N
O
NH
O
HN
O
O
HN
HN
N
O
O
O
2: R= H; 3: R=Cl
4-19
26
O
OH
O
ii
HO
vii
O
i
R
viii
O
Cl
H
R1
H
R2
H
R3
O
O
N
O
NH2
O
O
HN
NH
N
NH2
iii
O
O
H
O
H
R2
R3
O
vi
1
20: R2=R3= CH3
21: R2= CH3; R3= C6H5
25: R= H
iv
O
O
CS2
v
S
Cl
Cl
S
S
N
O
N
O
O
HN
N
NH
N
O
O
O
24
23
22
Scheme 2. Reagents and conditions: (i) 2: DMF, 60 °C, 10 h; 3: DMF, reflux, 10 h; (ii) R1CHO, H2SO4 stir, rt; (4: R1 = C2H5; 5: R1 = CH(CH3)2; 6: R1 = (CH2)3CH3; 7:
R1 = CH(CH3)C2H5; 8: R1 = C(CH3)3; 9: R1 = (CH2)6CH3; 10: R1 = CH=CHC2H5; 11: R1 = C6H5; 12: R1 = C6H4N(CH3)2 (p); 13: R1 = C6H4NO2 (p); 14: R1 = C6H3OH(p)(OCH3) (m); 15:
R1 = C6H4(CH3) (p); 16: R1 = C6H4(Br) (p); 17: R1 = C6H4(CN) (p); 18: R1 = C6H3(OCH3)2 (2,5); 19: R1 = C6H3OCH2O (iii) 20: dry acetone, H2SO4 stir, rt, 48 h, R2 = R3 = CH3; 21:
acetophenone, DMF, H2SO4 stir, rt, 72 h, R2 = CH3, R3 = C6H5; (iv) CS2, DMF, triethylamine, reflux, 10 h; (v) thionyl chloride, THF, triethylamine, stir, rt, 1 h; (vi) CH3COCH@CH2,
DMF, H2SO4, stir, rt, 72 h; (vii) 40% glyoxal in EtOH, DMF, reflux, 16 h, Dean–Stark trap; (viii) oxalic acid, DMF, H2SO4, reflux, 16 h, Dean–Stark trap.
(leukemia, non-small cell lung cancer, colon cancer, CNS cancer,
prostate cancer, melanoma, ovarian cancer, renal cancer and breast
cancer). Each subpanel uses several cell lines and the dose–response
curve is created by against the log10 of the corresponding drug con-
centration for each cell line by subpanel group. They were also eval-
uated in the 60 cell line panel and anticancer assay was performed in
accordance with the protocol of developmental therapeutics pro-
gram (one dose mean graph). Results for each compound were re-
ported as the growth percentage of the treated cells when
compared to that of the untreated control cells. The growth percent
values at 10À5 M for 60 cancer cell lines are shown in Table 1. For
each of these parameters the averaged values of mean graph mid-
point (MG_MID) were calculated. All of compounds, which reduced
the growth of any one of the cell lines to 32% or less, were selected for
further evaluation in the full panel of 60 human tumor cell lines. As
shown in Table 2, three compounds (4, 23, and 25) of the 12 tested
compoundsshowed significant cytotoxic activityhave been selected
extend the initial in vitro observation reported in the data above
and confirm the importance of anticancer activity and telomerase
inhibition.
The cancer panel, leukemia, melanoma and ovarian cancer lines
were quite sensitive to compound 23, while the non-small cell lung
cancer lines, colon, CNS, renal, prostate and breast panels were less
sensitive. It is surprising since compound 23 with heterocyclic
thiadiazole tetracyclic-system has differences in cytotoxicity
which might have a significant effect on their interaction with
DNA. Compound 23 exhibited an GI50 as little as 0.16
LC50 with as little as 0.99 mol/L and TGI with as little as
0.40 mol/L for leukemia HL-60(TB).
lmol/L,
l
l
It is especially notable that compound 4 with side chain –CH2CH3
displayed relatively potent and differential cytotoxic activity which
had both cytotoxicity and telomerase activity whereas for cytocidal
effects this difference was significant. However, it had significant
influence on the spectrum of antiproliferative activity. Compounds
4 and 25 had also selective cytotoxicity for nine cancer cell types.
It is especially notable that compound 25 have the best potent activ-
for a 60-cell panel assay, along with GI50, TGI and IC50
As result, they were found that (NSC745795), 23
(NSC745885) and 25 (NSC745887) have potent activity with GI50
conferred by 1.17–>100 mol/L (11.5 mol/L mean); 0.16–
17.4 mol/L (2.04 mol/L mean); 0.07–29.3 mol/L (5.24 mol/L
mean), respectively. Sensitive cell lines exhibit TGI and LC50 to 4
(NSC745795), all exhibited LC50 with >100 mol/L and TGI with
mol/L; 23 (NSC745885), exhibited an LC50 with
mol/L and TGI with as little as 0.40 mol/L; 25
mol/L and
mol/L, respectively. Results of the study
.
a
4
ity against leukemia K-562 with GI50 as little as 0.07
lmol/L, but LC50
l
l
and TGI with >100 mol/L, respectively. The overall dose–response
l
l
l
l
l
curves revealed that the leukemia subpanel was most sensitive for
23 and 25, with GI50 of six leukemia cell lines ranging from 0.07 to
l
3.65
l
mol/L. The ovarian cancer subpanel was ranked second most
mol/L. Compounds 11,
as little as 14.8
as little as 0.99
l
l
sensitive with GI50 between 0.55 and 29.3 l
l
12, 14, 15, 16, 17, 19, 20 and 26 are lack of significant antiprolifera-
tive activity for cytotoxic activity. NCI 60 cell line panel assays from
some selective compounds give only limited information and
(NSC745887), exhibited an LC50 with as little as 44.4
l
TGI with as little as 11.5
l

H.-S. Huang et al. / Bioorg. Med. Chem. 17 (2009) 7418–7428
7421
Table 1
Cytotoxicity of selected compounds in the NCI in vitro 60-cell Drug Screen Program
Panel/cell line
Compounds/growth percenta
4
11
745796
12
745794
14
745797
15
745798
16
745799
17
745883
19
745884
23
745885
20
745886
25
745887
26
745888
745795
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
68.24
52.31
97.73
41.43
75.94
71.39
31.42
59.82
41.48
78.97
77.08
108.51
50.68
80.48
91.66
77.44
86.21
80.36
113.27
91.86
102.47
89.88
89.30
93.83
94.79
101.38
87.08
97.86
89.10
55.73
91.20
110.24
77.27
100.62
98.79
84.56
93.63
84.67
94.98
82.70
105.67
99.84
—
56.20
103.05
90.86
126.73
105.15
141.18
101.67
94.18
114.06
65.33
97.05
104.18
103.18
À100.00
—
97.80
16.07
125.82
77.41
—
94.62
63.06
—
47.13
82.57
—
106.10
86.73
—
À15.08
95.83
97.09
159.05
101.45
100.37
116.18
120.18
100.41
103.05
107.02
90.13
104.06
83.53
102.60
93.14
167.19
96.24
76.50
HOP-92
5.10
—
NCl-H226
NCI-H23
NCl-H322 M
NCl-H460
NCl-H522
89.68
87.00
79.66
71.46
38.90
86.72
65.13
96.35
31.69
24.09
94.30
149.65
108.78
83.27
71.47
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
69.65
67.67
47.63
42.18
64.46
72.94
73.12
92.05
105.43
82.41
86.34
83.16
75.53
82.75
208.28
108.67
100.43
106.53
104.64
97.95
100.71
103.09
90.37
102.65
101.15
113.20
102.47
107.64
104.06
99.81
100.32
105.54
106.39
113.19
109.96
98.14
99.59
96.58
98.76
101.82
95.01
112.74
74.71
79.84
100.63
86.41
98.58
89.38
126.21
85.66
116.27
105.78
95.79
97.78
91.92
111.54
À0.74
À50.00
1.48
116.95
98.60
7.30
90.57
136.96
54.72
64.64
103.48
51.55
91.66
71.56
À7.15
53.27
52.15
81.90
61.00
39.19
101.14
77.59
91.14
99.34
114.87
97.82
97.00
103.02
Breast cancer
BT-549
HS 578T
—
—
—
—
94.14
138.21
41.92
97.59
105.10
—
—
—
—
—
—
—
—
À18.58
69.82
78.01
84.39
—
30.42
51.75
81.43
100.59
—
109.02
67.36
94.44
105.81
—
166.51
57.55
87.94
102.71
—
181.36
75.31
92.27
109.92
—
102.38
62.66
87.12
113.02
94.95
101.88
85.09
106.08
46.49
107.23
109.72
96.41
104.74
106.53
À7.40
À50.74
À17.33
À87.88
À73.17
À2.53
À45.52
68.42
83.58
72.90
124.56
77.25
102.64
82.96
76.75
39.68
81.39
102.03
75.28
67.96
85.19
79.00
85.17
92.54
144.42
37.35
98.23
87.10
MCF7
MDA-MB-231/ATCC
MDA-MB-435
MDA-MB-468
NCI/ADR-RES
T-47D
74.44
63.05
90.19
65.06
108.06
119.31
102.96
83.92
103.93
75.98
93.74
97.28
Ovarian cancer
IGROV1
—
—
—
—
—
—
66.31
110.54
101.11
98.10
98.86
103.78
93.33
106.64
100.52
111.14
96.47
À88.34
À15.47
À94.43
À20.07
À10.07
100.70
À2.80
78.22
80.14
96.48
90.23
35.91
3.44
71.65
94.22
106.59
47.54
78.16
13.96
93.18
103.31
110.43
104.07
99.07
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
78.22
48.81
102.30
72.94
25.77
69.18
73.43
108.78
91.00
114.69
111.00
98.31
117.44
111.08
201.85
124.78
83.02
97.12
93.42
113.22
96.14
96.83
105.69
96.39
84.50
100.32
94.06
93.21
96.34
105.41
138.75
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
45.36
41.75
64.43
64.17
56.17
52.55
86.32
99.05
76.04
88.35
61.11
95.53
63.86
89.68
96.09
101.48
78.39
118.40
94.69
122.73
95.49
96.36
81.02
73.92
100.60
124.51
102.37
105.87
91.52
98.71
100.33
108.81
101.29
95.18
117.25
145.27
54.91
87.09
113.71
84.08
99.64
127.01
80.62
104.77
96.21
À30.81
À21.77
49.61
À40.09
À27.97
4.96
74.01
128.84
101.39
116.41
99.93
—
90.48
125.76
105.90
121.52
108.01
108.60
102.34
37.23
22.91
—
113.66
92.44
91.74
72.44
82.92
Renal cancer
786-0
A498
76.60
28.88
24.97
—
111.09
76.95
71.86
51.87
—
87.49
81.97
57.59
109.24
86.10
96.24
108.25
—
94.51
115.78
75.53
109.05
121.53
100.47
134.19
—
102.48
174.50
80.74
112.66
175.54
101.41
110.01
—
103.28
153.53
75.19
111.34
117.25
91.37
86.56
—
111.51
280.47
66.20
124.28
96.21
107.42
91.71
104.19
113.75
119.55
101.03
—
À25.98
130.09
À94.31
1.97
86.18
80.60
50.17
61.72
85.14
91.98
62.39
30.65
28.45
98.83
46.52
87.92
104.88
64.65
126.89
51.45
131.54
99.78
81.33
123.62
110.90
94.07
274.15
61.43
ACHN
110.97
110.42
116.62
104.85
180.05
80.37
CAK1-1
RXF 393
SN12C
TK-10
—
20.11
68.53
33.82
23.94
À73.27
96.99
UO-31
73.36
À79.30
Melanoma
LOX IMVI
M14
MALME-3 M
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
39.98
60.14
56.94
40.07
86.80
60.75
39.69
51.35
79.66
100.92
74.47
62.56
60.44
65.18
—
90.55
112.55
109.58
73.15
96.83
93.80
109.22
114.00
106.72
82.23
131.70
88.06
—
103.97
106.09
97.63
79.59
154.12
87.97
—
102.36
101.55
224.05
72.25
130.82
87.08
—
101.18
243.01
200.54
22.47
114.61
102.76
104.79
104.25
99.59
120.39
—
41.52
116.26
98.73
116.58
98.10
À50.63
74.54
À71.54
À73.16
15.47
61.88
83.70
155.47
8.04
128.51
86.45
118.22
67.58
44.18
59.81
180.88
6.66
97.49
66.57
91.06
90.47
96.94
122.54
149.83
21.96
114.92
98.99
8.52
103.22
85.78
À83.07
118.21
95.25
70.40
92.24
90.01
104.94
À82.32
Prostate cancer
DU-145
PC-3
61.60
48.45
84.27
52.46
99.89
82.39
88.12
88.14
98.85
87.62
112.86
88.71
97.74
109.32
106.77
106.98
40.76
—
76.05
—
93.32
—
102.68
92.39
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
80.37
75.82
83.16
74.86
55.09
91.93
73.58
96.17
101.51
75.07
95.43
158.75
111.18
127.24
93.08
106.83
127.21
101.80
97.38
106.35
94.19
91.69
97.82
107.21
106.47
119.23
102.86
100.60
87.32
99.17
120.04
103.92
97.83
100.23
114.67
107.60
120.61
101.90
16.29
126.17
À47.11
78.98
90.16
97.99
93.56
94.28
78.03
67.98
80.81
40.72
87.30
104.52
98.26
140.32
101.16
93.94
85.95
63.17
79.03
109.34
(continued on next page)

7422
H.-S. Huang et al. / Bioorg. Med. Chem. 17 (2009) 7418–7428
Table 1 (continued)
Panel/cell line
Compounds/growth percenta
4
11
745796
12
745794
14
745797
15
745798
16
745799
17
745883
19
745884
23
745885
20
745886
25
745887
26
745888
745795
U251
Mean
Delta
Range
60.13
58.42
77.00
97.10
80.85
50.43
84.27
101.87
103.68
39.82
102.57
102.39
44.84
103.86
100.84
58.92
104.71
106.31
40.11
111.79
103.78
81.31
111.46
102.18
60.66
À89.26
2.68
102.68
230.09
87.84
80.41
83.21
63.47
69.36
76.51
95.92
100.83
115.91
289.23
120.88
144.42
116.95
133.62
214.27
220.54
108.13
158.27
188.03
a
Data obtained from NCI in vitro 60-cell Drug Screen program at 10À5 M concentration.
observations on mechanisms of drug action; for these DNA-affinic
tetracyclic ring systems more precise physicochemical measure-
ments are required which DNA polymorphic form is the target.
although it is not apparent whether the telomerase repression ef-
fects of these compounds are caused by their cytotoxicity, it is con-
ceivable that inhibition of telomerase expression by these
compounds inhibit cell proliferation. Although the mechanism of
preferential inhibition of compounds is still unclear to us, the com-
pounds might have the potential to be developed into a drug for
anti-cancer therapies.
2.3. Telomerase activity
Binding of low-molecular weight ligands to quadruplexes was
first studied using the classic DNA intercalator ethidium and the
telomere sequence T4G4 from Oxytricha.15 It is apparent that
G-quadruplexes are a targets for drug action, with unique struc-
tural and electronic features, compared with the more established
duplex or triplex DNA targets.16 However, the G-quadruplex inhib-
itors studied have at least some affinity for duplex DNA, and thus,
tend to exhibit conventional cytotoxicity, as well as particular
telomerase-associated properties.17 We evaluate the effects of
these test derivatives on telomerase inhibition using PCR-based
telomerase assay, and TRAP (telomeric repeat amplification proto-
col) assay. We also designed and expressed SEAP reporter gene
under the control of hTERT promoter to monitor the expression
of hTERT. Given the striking correlations between telomerase
activity and antiproliferation capacity in tumor cells, we antici-
pated that analysis of tetracyclic chromophore telomerase might
yield further insight into this relationship. We screened this series
of derivatives, searching for compounds that exhibit inhibition of
telomerase and/or SEAP assay with less cytotoxicity in MTT assay.
To understand the unique antiproliferative activity pattern of
test compounds in the NCI-60 cell line screen, we computed struc-
ture–activity relationships between the three compounds
4
(NSC745795), 23 (NSC745885) and 25 (NSC745887), respectively.
They exhibited dose-dependent inhibition of proliferation in all
60 cancer cell lines. As shown in Tables 1 and 2, the cytotoxicity
elicited by the three analogues 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. They may be appreciated from these representation of
the data that the three analogues represent the most potent deriv-
ative from this series of tetracyclic derivatives. As shown in Table 2
for compound 4 (NSC 745795), the average concentration required
to inhibit GI50 was 11.48
cancer: CAKI-1) to >100
l
mol/L with a range of 1.17
lmol/L (renal
lmol/L (leukemia: K-562; melanoma: SK-
MEL-28; renal cancer: A498; breast cancer: HS 578T). As shown in
Table 2 for compound 23 (NSC 745885), the average concentration
Under the concentration of 100
lm, none of the synthesized
required to inhibit GI50 was 2.04
lmol/L with a range of 0.16
lmol/
compounds gave profound telomerase inhibitory activity (Fig. 1).
L (leukemia cancer HL-60 TB) to 17.4
lmol/L (non-small cell lung
Results with all test compounds showed MTT and SEAP inhibition
cancer, NCI-H322 M). As shown in Table 2 for compound 25
activity in 1, 10 and 100
lM range are summarized in Table 3. We
(NSC745887), the average concentration required to inhibit GI50
did not found any of the synthesized compounds showed specific
repressive effects toward hTERT gene.
was 5.24
29.3
lmol/L with a range of 0.07 lmol/L (leukemia, K562) to
l
mol/L (ovarian cancer, OVCAR-5). In addition, >1 lmol/L
(10À6 mol/L) was required to achieve LC50 in the majority of human
tumor cell lines, cell line sensitive to compound 4 (NSC 745795),
3. Conclusion
exhibited an LC50 with >1
tle as 14.8 mol/L (ovarian cancer, IGROV1). Cell line sensitive to
compound 23 (NSC 745885), exhibited an LC50 with as little as
0.99 mol/L and also TGI with as little as 0.40 mol/L (leukemia,
HL-60 TB). Cell line sensitive to compound 25 (NSC 745887), exhib-
ited an LC50 with as little as 44.4 mol/L (melanoma, SK-MEL-5)
and TGI with as little as 11.5 mol/L (melanoma, HL-60 TB). Com-
l
mol/L (10À6 mol/L) and TGI with as lit-
l
At present, we described a method of synthesizing 1,2-hetero-
annelated anthraquinones and anthra[1,2-d]imidazole-6,11-dione
derivatives and comparing to their cytotoxicity and telomerase
activity. A structure–activity relationship study was not conducted
on a number of tetracyclic-system derivatives. In this investigation,
we continue to focus our attention on the role of our systematic
synthesized tetracyclic pharmacophore bearing the heterocycle
and side chain linked to the planar anthraquinone moiety and to
understand the basis of tetracyclic system selectivity. The physical,
chemical and biological properties of anthraquinone-base deriva-
tives are greatly affected by its various substituents of the planar
ring system which considered as aglycon analogues of anthracy-
cline antibiotics. Since telomerase is an important component of
cancers, we are interested in examining the effects of these com-
pounds on telomerase activity in the cell free extracts prepared
from H1299 cells. The telomerase activity is regulated mostly at
the transcriptional level for its catalytic subunit, hTERT, and partly
at the post-translational level.18 As shown in Table 3, most of the
compounds did show various effects on SEAP expression. However,
all of them also affect the proliferation of the treated cells. Thus,
l
l
l
l
pounds 4 (NSC 745795), 23 (NSC 745885) and 25 (NSC 745887)
exhibited dose-dependent inhibition of proliferation in all 60
cancer cell lines. Of the three, they were the most potent
compounds during the all selected evaluation, with an average
GI50 of 11.48 lmol/L for 4 (NSC 745795); with an average GI50 of
2.04
lmol/L for 23 (NSC 745885); with an average GI50 of
5.50
lmol/L for 25 (NSC 745887), respectively. All these data were
summarized in Tables 1 and 2 and used for further analysis. Given
the striking correlations between telomerase activity and prolifer-
ation capacity in tumor cells, we expected that analysis of tetracy-
clic analogues chromophore might yield further insight into
designing better lead compounds for anti-cancer therapies. How-
ever, there is still no significant correlation between telomerase
activity and cytotoxicity. The telomerase inhibitory data available

H.-S. Huang et al. / Bioorg. Med. Chem. 17 (2009) 7418–7428
7423
Table 2
In vitro anticancer activity by selected compounds 4 (NSC745795), 23 (NSC745885) and 25 (NSC745887) in the NCI’s 60 human cancer cell lines
Panel/cell line (
lM)
4 (NSC 745795)
23 (NSC 745885)
25 (NSC 745887)
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
16.5
2.72
>100
4.22
4.16
6.71
>100
64.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.08
0.16
1.92
0.49
1.18
—
4.1
0.4
15.9
1.84
3.46
—
>100
0.99
41.2
4.71
>100
—
2.46
0.68
0.07
3.65
1.61
—
>100
11.5
>100
>100
>100
—
>100
83.7
>100
>100
>100
—
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-460
28.5
4.6
>100
>100
>100
7.58
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
14.7
1.55
1.28
2.52
4.48
1.44
17.4
3.27
1.66
36.6
3.45
3.31
6.64
21.6
6.04
47.8
14.3
3.93
91
4.53
3.45
5.64
22.5
16.6
12
16.3
2.01
4.64
>100
72.1
>100
>100
95.2
70.3
96.1
26.9
32.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
7.66
8.53
>100
63.7
>100
>100
47.3
9.31
32.2
9.84
2.46
12.1
4.87
5.27
28.3
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
8.95
18.8
5.9
3.39
16.1
7.69
19.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
13.4
16.4
1.93
1.71
13.4
1.96
1.79
28.9
33.1
5.4
3.72
50.4
5.54
3.5
62.4
66.6
>100
8.12
>100
59.7
6.84
3.4
1.4
28.4
83.5
>100
84.3
>100
49.5
30.8
>100
>100
>100
>100
>100
>100
>100
12.3
4.63
7.01
5.26
1.71
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
20.3
4.15
11.7
18.1
62.5
16.3
>1000
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.17
1.9
1.46
2.28
2.03
1.61
5.77
4.04
2.94
5.82
5.2
27.7
8.56
5.91
26
18.6
5.44
4.98
4.35
2.18
13.4
11.9
5.38
>100
32.3
53.7
>100
>100
42.9
>100
>100
>100
>100
>100
>100
2.96
Melanoma
LOX IMV1
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
4.56
8.55
7.42
30.3
—
>100
12.7
47.2
8.09
>100
>100
>100
>100
—
>100
59.1
>100
>100
>100
>100
>100
>100
—
>100
>100
>100
>100
0.82
1.66
2.15
1.91
1.68
1.85
1.58
1.74
1.45
2.13
3.15
4.85
3.31
4.22
3.38
2.92
3.51
2.9
4.86
5.96
>100
5.76
22.5
6.18
5.41
7.09
5.79
4.2
38.9
20.7
>100
23.7
55.9
91.1
17.1
68.3
56.1
>100
70.8
>100
50.4
>100
>100
44.4
>100
>100
4.48
5.14
11.1
12.7
21.0
3.93
13
13.3
Ovarian cancer
IGROV1
1.54
16.5
25.7
77.4
23.2
18.9
2.59
14.8
>100
>100
>100
>100
>100
>100
>100
1.36
0.55
0.65
1.91
1.66
1.86
7.71
2.94
2.46
2.17
4.11
4.68
12.6
22.5
6.35
11.1
5.57
8.84
59.8
>100
53.4
2.55
2.34
10.5
29.3
5.07
5.68
6.95
35.9
12.5
>100
>100
>100
>100
>100
>100
>100
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Renal cancer
786-0
A498
43.6
>100
3.29
1.17
6.95
13.6
12.1
1.33
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6.17
12.4
1.46
1.61
1.75
1.33
2.85
1.62
19.3
25.4
2.77
2.96
4.4
3.17
10.9
3.12
50.4
52
8.87
9.31
3.85
5.83
6.43
13.2
5
>100
50.3
>100
>100
>100
92.7
>100
85.9
>100
>100
>100
>100
>100
>100
>100
>100
ACHN
5.27
5.44
14.6
7.56
50.4
6.01
CAKI-1
RXF 393
SN12C
TK-10
UO-31
3.02
Prostate cancer
PC-3
DU-145
28.2
9.43
>100
>100
>100
>100
3.23
1.67
>100
3.24
>100
6.27
—
11.4
>100
34.3
>100
>100
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
13
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.53
1.75
1.41
1.63
1.39
—
3.34
5.9
5.02
3.13
3.39
—
7.29
>100
56
6.01
8.27
—
0.72
19.1
14.1
12.2
12.6
—
29.8
>100
91.4
93.5
75.1
—
>100
>100
>100
>100
>100
—
8.86
>100
9.3
3.3
8.29
MDA-MB-468

7424
H.-S. Huang et al. / Bioorg. Med. Chem. 17 (2009) 7418–7428
Figure 1. Inhibition of telomerase activity by 1,2-heteroannelated anthraquinones and anthra[1,2-d]imidazole-6,11-dione homologues which tested concentration was
100 M. [P—positive control (no inhibitor); N—negative control (RNase A-treated cell extract, no inhibitor)].
l
here for tetracyclic anthraquinone system have demonstrated that
activity at the micromolar level can be achieved with pharmaco-
logically acceptable compounds. Their telomerase selectivity is rel-
atively modest, and this, combined with cytotoxicity at
comparable levels, suggests that these types of tetracyclic com-
pounds may produce potential candidates suitable for in vivo pre-
clinical evaluation.
otherwise stated): Finnigan MAT TSQ-46, Finnigan MAT TSQ-700
(Universität Regensburg, Germany) and Finnigan MAT LCQ-MS
(National Research Institute of Chinese Medicine, Taipei, Taiwan).
Typical experiments illustrating the general procedures for the
preparation of the anthraquinones are described below.
4.1. 2-Methyl-1(3)H-anthrasimidazole-6,11-dione (2)
4. Experimental
1,2-Diaminoanthraquinone (1.19 g, 5 mmol) and acetyl chloride
(0.5 mL, 6 mmol) were dissolved in DMF (30 mL) and stirred at
130 °C for 10 h. Once the starting materials were consumed, as
indicated by TLC, the reaction mixture was cooled to room temper-
ature. Water (200 mL) was added to precipitate out the crude prod-
uct. The resulting precipitate was collected by filtration, washed
with ethanol and purified by crystallization from ethanol to afford
desired compounds (Rf = 0.79 at ethyl acetate/dichlorometh-
Melting points were determined with a Büchi B-545 melting
point apparatus and are uncorrected. All reactions were monitored
by TLC (Silica Gel 60 F254). 1H NMR: Varian GEMINI-300 (300 MHz)
and Brucker AM-500 (500 MHz); d values are in ppm relative to
TMS as an internal standard. Fourier-transform IR spectra (KBr):
Perkin–Elmer 983G spectrometer. Mass spectra (EI, 70 eV, unless
Table 3
Effects of 1,2-heteroannelated anthraquinones and anthra[1,2-d]imidazole-6,11-dione homologues on activating or repressing hTERT expression and telomerase activity
Compound
Cell type (H1299) (inhibition
l
M
SD)
MTT
10 ( M)
SEAP
10 ( M)
1 (lM)
l
100 (l
M)
1 (lM)
l
100 (lM)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
64
7
8
7
5
8
9
5
6
5
44
79
82
8
7
9
33
26
37
6
6
4
101
94
70
83
91
67
88
89
63
81
92
85
85
86
89
91
77
61
96
99
97
89
95
3
4
7
5
4
9
4
5
1
6
5
7
6
6
4
5
8
4
8
2
4
9
6
82
81
59
76
84
5
5
1
8
3
19 12
80
88
95
85
77
83
85
69
5
7
25 12
73 15
34 24
13
36
8
9
70
83
81
69
48
72 10
62 12
52
70
8
2
7
5
4
20
43
56
40
14
34
22 16
33
38 11
16 15
20 17
19 11
13
40
31
29
54
17
9
5
7
5
5
4
58 12
11 14
87
88
34
78
73 10
62
50
82
71
83
76
61
73
97
100
6
3
1
8
29
32
13
10
7
6
5
7
79 11
88 10
21 10
84
71
75
96
80
91 12
79
98
99
101
40
78
98
9
9
9
9
2
4
9
9
5
8
2
9
9
4
4
7
1
5
6
8
7
6
6
4
7
4
6
4
6
8
13
72 13
85
77 10
29
36
30
41
34
9
29
31
9
8
39
67
93
99
99
16
69
13
65
1
9
3
6
2
1
8
9
9
3
4
5
9
5
7
4
6
2
4
7
3
4
5
10 13
25 14
6
25 14
88
26
89
8
8
8
26 10
4
105 10
101
24
20
7
8
85
4

H.-S. Huang et al. / Bioorg. Med. Chem. 17 (2009) 7418–7428
7425
ane = 1:4). The solid material was isolated in 67% yield. Mp >400 °C
(EtOH).17 FT-IR (KBr; cmÀ1) 1667 (CO). 1H NMR (300 MHz,
1.00 (t, J = 7.2 Hz, 3H), 1.50 (sx, J = 7.5 Hz, 2H), 1.93 (qt, J = 7.8 Hz,
2H), 3.04 (t, J = 7.5 Hz, 2H), 7.62–7.83 (m, 2H), 8.03 (d, J = 8.4 Hz,
1H), 8.20 (d, J = 8.1 Hz, 1H), 8.24–8.35 (m, 2H), 10.83 (s, 1H). 13C
NMR (75 MHz, CDCl3): d ppm 12.98, 21.78, 28.60, 29.27, 117.32,
121.07, 124.64, 125.98, 127.08, 127.83, 132.17, 132.84, 133.20,
133.61, 133.86, 148.25, 160.29, 182.31 (CO), 184.78 (CO). HR-MS
(ESI-TOF) m/z calcd, [M+H]+ 305.1276; found, 305.1282. Calcd,
[MÀH]À: 303.1131; found, 303.1135.
m
DMSO-d6): d ppm 2.72 (s, 3H), 7.75–7.82 (m, 2H), 7.93 (d,
J = 8.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 8.19–8.23 (m, 1H), 11.01
(br, 1H). 13C NMR (75 MHz, DMSO-d6): d ppm 23.89, 120.23,
121.22, 125.29, 126.19, 126.75, 127.19, 128.17, 128.87, 132.98,
134.18, 134.42, 148.22, 158.09, 182.43 (CO), 185.13 (CO). EI-MS
m/z: 262 (M+, 100).
4.2. 2-Chloroacetyl-1(3)H-anthra[1,2-d]imidazole-6,11-dione (3)
4.3.4. 2-sec-Butyl-1(3)H-anthra[1,2-d]imidazole-6,11-dione (7)
Product 7 was obtained as yellow powder (yield 40%): mp 117–
119 °C (EtOH) (Rf = 0.57 at ethyl acetate/dichloromethane = 1:4).
1,2-Diaminoanthraquinone (1.19 g, 5 mmol) and chloroacetyl
chloride (0.5 mL, 6 mmol) were dissolved in DMF (30 mL) and stir-
red at 50–60 °C for 10 h. Water (200 mL) was added to precipitate
out the crude product. The resulting precipitate was collected by
filtration, washed with ethanol and purified by crystallization from
ethanol to afford desired compounds (Rf = 0.5 at ethyl acetate/
dichloromethane = 1:4). The solid material was isolated in 80%
FT-IR (KBr;
m
cmÀ1) 1665 (CO). 1H NMR (300 MHz, CDCl3): d ppm
1.00 (t, J = 7.2 Hz, 3H), 1.52 (d, J = 6.9 Hz, 3H), 1.82–2.02 (m, 2H),
3.04 (sx, J = 7.2 Hz, 1H), 7.62–7.83 (m, 2H), 8.03 (d, J = 8.4 Hz,
1H), 8.20 (d, J = 8.1 Hz, 1H), 8.24–8.35 (m, 2H), 10.83 (s, 1H). 13C
NMR (75 MHz, CDCl3): d ppm 11.09, 18.09, 28.40, 35.71, 117.39,
121.07, 124.75, 125.95, 127.09, 127.84, 131.92, 132.83, 133.22,
133.59, 133.87, 148.06, 164.30, 182.31 (CO), 184.82 (CO). HR-MS
(ESI-TOF) m/z calcd, [M+H]+ 305.1276; found, 305.1280. Calcd,
[MÀH]À: 303.1131; found, 303.1137.
yield. Mp 272–273 °C (EtOH).19 FT-IR (KBr;
m
cmÀ1) 3359 (NH),
1660 (CO). 1H NMR (300 MHz, CDCl3): d (ppm) 4.92 (s, 2H), 7.80–
7.83 (m, 2H), 8.08 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H),
8.26–8.35 (m, 2H), 11.21 (br, 1H). 13C NMR (75 MHz, DMSO-d6):
d (ppm) 37.80, 119.35, 121.27, 125.95, 126.83, 127.40, 129.06,
132.35, 133.47, 133.64, 134.88, 135.10, 148.89, 156.93, 183.04
(CO), 183.83 (CO). HR-MS (ESI-TOF) m/z calcd, [M+H]+ 297.0425;
found, 297.0426.
4.3.5. 2-tert-Butyl-1(3)H-anthra[1,2-d]imidazole-6,11-dione (8)
Product 8 was obtained as yellow powder (yield 37%): mp 209–
210 °C (EtOH) (Rf = 0.80 at ethyl acetate/dichloromethane = 1:4).
FT-IR (KBr;
m
cmÀ1) 3568 (NH), 1664 (CO). 1H NMR (300 MHz,
CDCl3): d ppm 1.58 (s, 9H), 7.77–7.84 (m, 2H), 8.08 (d, J = 8.4 Hz,
1H), 8.21 (d, J = 8.4 Hz, 1H), 8.25–8.28 (m, 1H), 8.33–8.36 (m,
1H), 10.83 (s, 1H). 13C NMR (75 MHz, CDCl3): d ppm 29.24,
117.79, 121.47, 125.41, 126.39, 127.56, 128.17, 132.70, 133.23,
133.74, 133.96, 134.37, 148.73, 168.00, 182.77 (CO), 185.26 (CO).
HR-MS (ESI-TOF) m/z calcd, [M+H]+ 305.1276; found, 305.1283.
Calcd, [MÀH]À: 303.1131; found, 303.1136.
4.3. General procedure for the synthesis of compounds (4–19)
Various aldehyde (5 mmol) was added dropwise to a solution of
1,2-diaminoanthraquinone (1.19 g, 5 mmol) and concd sulfuric
acid (0.1 mL) in DMF (30 mL). The reaction mixture was stirred
for 1 h at room temperature. The mixture was treated with crushed
ice and the product was extracted into dichloromethane. After dry-
ing the organic layer over anhydrous Na2SO4, solvent was removed
and the residue was purified by crystallization from EtOH to afford
desired compounds.
4.3.6. 2-Heptyl-1(3)H-anthra[1,2-d]imidazole-6,11-dione (9)
Product 9 was obtained as brown powder (yield 38%): mp 85–
87 °C (EtOH) (Rf = 0.85 at ethyl acetate/dichloromethane = 1:4).
FT-IR (KBr;
m
cmÀ1) 3447 (NH), 1664 (CO). 1H NMR (300 MHz,
4.3.1. 2-Ethyl-1(3)H-anthra[1,2-d]imidazole-6,11-dione (4)
Product 4 was obtained as brown powder (yield 39%): mp 193–
195 °C (EtOH) (Rf = 0.75 at ethyl acetate/dichloromethane = 1:4).
CDCl3): d ppm 0.87–0.91 (m, 3H), 1.26–1.35 (m, 6H), 1.56 (sx,
J = 7.0 Hz, 2H), 2.36 (q, J = 7.0 Hz, 2H), 2.71 (t, J = 7.0 Hz, 2H),
7.75–7.81 (m, 2H), 8.04 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H),
8.23–8.25 (m, 1H), 8.31–8.33 (m, 1H), 10.93 (s, 1H). 13C NMR
(75 MHz, CDCl3): d ppm 14.08, 22.63, 27.99, 28.79, 29.24, 29.46,
31.79, 117.49, 121.66, 125.28, 126.37, 127.54, 130.56, 133.27,
133.67, 134.06, 134.31, 137.37, 149.40, 158.89, 182.69 (CO),
185.25 (CO). HR-MS (ESI-TOF) m/z calcd, [M+H]+ 347.1754; found,
347.1752.
FT-IR (KBr;
m
cmÀ1) 1669 (CO). 1H NMR (300 MHz, CDCl3): d
(ppm) 1.51 (t, J = 7.5 Hz, 3H), 3.05 (q, J = 7.5 Hz, 2H), 7.73–7.81
(m, 2H), 7.99 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 8.21–8.23
(m, 1H), 8.27–8.31 (m, 1H), 10.85 (br, 1H). 13C NMR (75 MHz,
CDCl3): d (ppm) 11.87, 22.89, 117.74, 121.50, 125.21, 126.47,
127.55, 128.21, 132.72, 133.24, 133.72, 133.99, 134.37, 148.90,
161.64, 182.81 (CO), 185.15 (CO). HR-MS (ESI-TOF) m/z calcd,
[M+H]+ 277.0971; found, 277.0975; calcd for C17H12N2O2Na+
[M+Na]+: 299.0971; found: 299.0794.
4.3.7. (E)-2-(But-1-enyl)-1(3)H-anthra[1,2-d]imidazole-6,11-
dione (10)
Product 10 was obtained as brown powder (yield 33%): mp
117–119 °C (EtOH) (Rf = 0.57 at ethyl acetate/dichlorometh-
ane = 1:4). FT-IR (KBr;
CDCl3): d ppm 0.98 (t, J = 6.9 Hz, 3H), 1.94–1.98 (m, 2H), 6.16–
6.29 (m, 1H), 6.51 (d, J = 18 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.82–
7.89 (m, 2H), 8.14 (d, J = 8.1 Hz, 1H), 8.27–8.35 (m, 2H), 10.74 (s,
1H). 13C NMR (75 MHz, CDCl3): d ppm 14.39, 27.40, 117.37,
120.03, 121.07, 124.75, 125.95, 127.09, 127.84, 131.92, 132.83,
133.22, 133.59, 133.87, 134.90, 135.37, 149.06, 18 2.73 (CO),
185.18 (CO). EI-MS m/z 302 (M+, 100).
4.3.2. 2-Isopropyl-1(3)H-anthra[1,2-d]imidazole-6,11-dione (5)
Product 5 was obtained as yellow powder (yield 41%): mp 117–
119 °C (EtOH) (Rf = 0.7 at ethyl acetate/dichloromethane = 1:4). FT-
m
cmÀ1) 1664 (CO). 1H NMR (300 MHz,
IR (KBr;
m
cmÀ1) 3445 (NH), 1662 (CO). 1H NMR (300 MHz, CDCl3):
d ppm 1.56 (d, J = 6.6 Hz, 6H), 3.40 (s, J = 6.6 Hz, 1H), 7.78–7.85 (m,
2H), 8.11 (d, J = 8.4 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.25–8.36 (m,
2H), 10.88 (s, 1H). 13C NMR (75 MHz, CDCl3): d ppm 21.15, 29.21,
117.66, 121.36, 125.21, 126.32, 127.42, 128.05, 132.49, 133.10,
133.61, 133.86, 134.24, 148.71, 165.35, 181.05 (CO), 182.73 (CO).
HR-MS (ESI-TOF) m/z calcd, [M+H]+ 291.1120; found, 291.1123.
4.3.8. 2-Phenyl-1(3)H-anthra[1,2-d]imidazole-6,11-dione (11)
Product 11 was obtained as yellowish brown powder (yield
74%): mp 232–233 °C (EtOH) (Rf = 0.55 at ethyl acetate/dichloro-
methane = 1:4). FT-IR (KBr;
NMR (300 MHz, CDCl3): d ppm 7.57 (t, J = 3 Hz, 3H), 7.89 (m, 2H),
4.3.3. 2-Butyl-1(3)H-anthra[1,2-d]imidazole-6,11-dione (6)
Product 6 was obtained as brown powder (yield 36%): mp 192–
193 °C (EtOH) (Rf = 0.65 at ethyl acetate/dichloromethane = 1:4).
m
cmÀ1) 3296 (NH),1660 (CO). 1H
FT-IR (KBr;
m
cmÀ1) 1669 (CO). 1H NMR (300 MHz, CDCl3): d ppm

7426
H.-S. Huang et al. / Bioorg. Med. Chem. 17 (2009) 7418–7428
8.03 (d, J = 8.4 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 8.16 (m, 2H), 8.40
(dd, J = 6.3 Hz, 2H). 13C NMR (75 MHz, CDCl3): d ppm 119.62,
121.72, 125.06, 126.85, 127.42, 128.79, 128.86, 129.41, 129.50,
131.72, 133.72, 133.77, 134.92, 135.07, 149.26, 158.25, 183.06
(CO), 183.79 (CO). HR-MS (ESI-TOF) m/z calcd, [M+H]+ 325.0971;
found, 325.0973. EI-MS m/z 324 (M+, 100), 325 (19).
(300 MHz, CDCl3): d ppm 7.72 (d, J = 8.7 Hz, 2H), 7.80–7.83 (m,
2H), 8.06 (d, J = 8.7 Hz, 2H), 8.13 (d, J = 8.4 Hz, 1H), 8.25 (d,
J = 8.4 Hz, 1H), 8.27–8.36 (m, 2H), 11.29 (s, 1H). 13C NMR
(75 MHz, CDCl3): d ppm 122.18, 125.86, 126.11, 126.57, 127.64,
127.69, 128.50, 128.89, 132.61, 133.20, 133.35, 133.87, 134.01,
134.57, 149.40, 155.62, 182.63 (CO), 185.25 (CO). HR-MS (ESI-
TOF) m/z calcd, [M+H]+ 403.0085; found, 403.0073. Calcd, [MÀH]À
400.9939; found, 400.9923. EI-MS m/z 402 (M+, 100), 404 (97).
4.3.9. 2-(4-N,N-Dimethylamino)phenyl-1(3)H-anthra[1,2-d
]imidazole-6,11-dione (12)
Product 12 was obtained as dark brown powder (yield 79%): mp
4.3.14. 2-(4-Cyanophenyl)-1H-anthra[1,2-d]imidazole-6,11-
dione (17)
239–240 °C (EtOH) (Rf = 0.60 at ethyl acetate/dichlorometh-
ane = 1:4). FT-IR (KBr;
m
cmÀ1) 3404 (NH),1659 (CO). 1H NMR
Product 17 was obtained as yellowish brown powder (yield
(300 MHz, CDCl3): d ppm 3.09 (s, 6H), 6.81 (d, 2H), 7.79–7.82 (m,
3H), 8.03–8.22 (m, 3H), 8.27–8.36 (m, 2H), 11.10 (br, 1H). 13C
NMR (75 MHz, CDCl3): d ppm 39.95, 111.65, 115.23, 117.13,
121.83, 124.24, 126.33, 127.37, 127.44, 128.27, 133.27, 133.45,
133.54, 134.12, 150.11, 152.10, 157.59, 182.47 (CO), 185.09 (CO).
HR-MS (ESI-TOF) m/z calcd, [M+H]+ 368.1393; found, 368.1393.
EI-MS m/z 366 (27), 367 (M+, 100), 368 (20).
77%): mp 353–354 °C (EtOH) (Rf = 0.65 at ethyl acetate/dichloro-
methane = 1:4). FT-IR (KBr;
m
cmÀ1) 3341 (NH), 2229 (CN), 1667
(CO). 1H NMR (300 MHz, CDCl3): d ppm 7.80–7.85 (m, 2H), 8.06
(d, J = 8.1 Hz, 2H), 8.18 (d, J = 8.4 Hz, 1H), 8.27–8.32 (m, 4H),
8.35–8.38 (m, 1H), 11.46 (s, 1H). 13C NMR (75 MHz, CDCl3): d
ppm 114.71, 118.04, 118.52, 122.39, 126.39, 126.63, 127.57,
127.75, 129.45, 132.76, 133.04, 133.11, 133.34, 133.93, 133.99,
134.70, 149.17, 154.25, 182.56 (CO), 185.21 (CO). HR-MS (ESI-
TOF) m/z calcd, [M+H]+ 350.0924; found, 350.0925.
4.3.10. 2-(4-Nitrophenyl)-1(3)H-anthra[1,2-d]imidazole-6,11-
dione (13)
Product 13 was obtained as red brown powder (yield 89%): mp
4.3.15. 2-(2,5-Dimethoxyphenyl)-1H-anthra[1,2-d]imidazole-
6,11-dione (18)
342–343 °C (EtOH) (Rf = 0.60 at ethyl acetate/dichlorometh-
ane = 1:4). FT-IR (KBr;
m
cmÀ1) 3460 (NH),1657 (CO), 1517, 1345
Product 18 was obtained as red brown powder (yield 74%): mp
(NO2). 1H NMR (300 MHz, DMSO-d6): d ppm 7.79–7.82 (m, 3H),
7.14 (d, J = 8.1 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 8.23–8.32 (m, 2H),
8.39 (d, J = 8.1 Hz, 2H), 8.58 (d, J = 8.1 Hz, 2H), 10.15 (br, 1H). 13C
NMR (75 MHz, DMSO-d6): d ppm 117.81, 122.43, 123.62, 125.24,
125.88, 126.10, 127.92, 133.22, 133.36, 134.53, 143.08, 146.39,
146.77, 155.89, 172.18, 178.35, 179.40, 183.20 (CO), 185.56 (CO).
HR-MS (ESI-TOF) m/z calcd, [M+H]+ 370.0822; found, 370.0823.
EI-MS m/z 249 (100), 369 (M+, 35).
251–252 °C (EtOH) (Rf = 0.40 at ethyl acetate/dichlorometh-
ane = 1:4). FT-IR (KBr;
m
cmÀ1) 3417 (NH), 1660 (C@O), 1226 (C–
O). 1H NMR (300 MHz, CDCl3): d ppm 3.93 (s, 3H), 4.21 (s, H),
7.09 (d, J = 1.2 Hz, 2H), 7.79–7.82 (m, 2H), 8.13 (d, J = 8.1 Hz, 1H),
8.13 (s, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.29–8.36 (m, 2H), 12.37 (s,
1H). 13C NMR (75 MHz, CDCl3): d ppm 56.08, 56.69, 113.13,
113.36, 116.86, 118.14, 119.98, 121.92, 124.92, 126.46, 127.54,
129.92, 132.57, 133.43, 133.70, 134.06, 134.24, 135.39, 152.20,
154.23, 155.18, 182.82 (CO), 184.88 (CO). HR-MS (ESI-TOF) m/z
calcd, [M+H]+ 385.1183; found, 385.1181.
4.3.11. 2-(4-Hydroxy-3-methoxyphenyl)-1H-anthra[1,2-
d]imidazole-6,11-dione (14)
Product 14 was obtained as brown powder (yield 47%): mp
4.3.16. 2-(Benzo[d][1,3]dioxol-5-yl)-1H-anthra[1,2-
d]imidazole-6,11-dione (19)
230–231 °C (EtOH) (Rf = 0.20 at ethyl acetate/dichlorometh-
ane = 1:4). FT-IR (KBr;
m
cmÀ1) 3411 (NH), 1664 (CO). 1H NMR
Product 19 was obtained as red brown powder (yield 81%): mp
(300 MHz, DMSO-d6): d ppm 3.91 (s, 3H), 6.90 (d, J = 8.4 Hz, 1H),
7.81–7.88 (m, 3H), 7.92–7.96 (m, 3H), 7.99 (s, 1H), 8.11 (d,
J = 8.4 Hz, 2H), 9.78 (br, 1H). 13C NMR (75 MHz, DMSO-d6): d ppm
56.57, 112.72, 116.37, 119.21, 119.65, 122.05, 122.95, 123.88,
126.81, 127.41, 128.42, 133.50, 133.64, 134.87, 135.09, 148.48,
150.87, 158.33, 182.85 (CO), 183.79 (CO). HR-MS (ESI-TOF) m/z
calcd, [M+H]+ 370.1026; found, 370.1025. EI-MS m/z 369 (57),
370 (M+, 100).
300–301 °C (EtOH) (Rf = 0.45 at ethyl acetate/dichlorometh-
ane = 1:4). FT-IR (KBr;
m
cmÀ1) 3444 (NH), 1670 (C@O), 1257 (C–
O), 1210 (C–O). 1H NMR (300 MHz, CDCl3): d ppm 6.11 (s, 2H),
7.00 (d, J = 7.8 Hz, 1H), 7.67 (s, 1H), 7.79–7.82 (m, 2H), 8.13 (d,
J = 8.1 Hz, 1H), 8.24 (d, J = 7.8 Hz, 1H), 8.25 (d, J = 8.1 Hz, 1H),
8.29–8.36 (m, 2H), 11.18 (s, 1H). 13C NMR (75 MHz, CDCl3): d
ppm 101.93, 107.37, 108.96, 117.86, 121.83, 122.05, 122.78,
125.35, 126.52, 127.62, 128.40, 133.27, 133.43, 133.76, 134.08,
134.44, 148.71, 149.62, 150.56, 156.55, 182.66 (CO), 185.27 (CO).
HR-MS (ESI-TOF) m/z calcd, [M+H]+ 369.0867; found, 369.0887.
4.3.12. 2-p-Tolyl-1H-anthra[1,2-d]imidazole-6,11-dione (15)
Product 15 was obtained as yellowish brown powder (yield
76%): mp 256–257 °C (EtOH) (Rf = 0.65 at ethyl acetate/dichloro-
4.3.17. 2,2-Dimethyl-2,3-dihydro-1H-anthra[1,2-d]imidazole-
6,11-dione (20)
methane = 1:4). FT-IR (KBr;
m
cmÀ1) 3397 (NH), 1659 (CO). 1H
NMR (300 MHz, CDCl3): d ppm 2.46 (s, 3H), 7.37 (d, J = 8.1 Hz,
2H), 7.79 (t, J = 3.6 Hz, 2H), 8.03 (d, J = 7.8 Hz, 2H), 8.08 (d,
J = 8.4 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.24–8.34 (m, 2H), 11.21
(s, 1H). 13C NMR (75 MHz, CDCl3): d ppm 21.58, 117.89, 121.96,
125.44, 125.75, 126.46, 127.00, 127.58, 128.43, 130.00, 133.20,
133.26, 133.72, 133.99, 134.38, 142.05, 149.50, 156.86, 182.60
(CO), 185.16 (CO). HR-MS (ESI-TOF) m/z calcd, [M+H]+ 339.1128;
found, 339.1128. EI-MS m/z 338 (M+, 100), 339 (24).
1,2-Diaminoanthraquinone (1.19 g, 5 mmol) was dissolved in
dry acetone (100 mL) and concd sulfuric acid (0.1 mL) was added
dropwise. The reaction mixture was stirred for 48 h at room tem-
perature. The mixture was purified by column chromatography
using potassium carbonate and crystallization from methanol to
yield the pure compound as a purple solid in 31% yield (Rf = 0.5
at ethyl acetate/dichloromethane = 1:4). Mp 235–237 °C (MeOH)
(lit: 234–237 °C).19 FT-IR (KBr;
m ) 3419 (NH), 3239
cmÀ1
(NH),1639 (CO). 1H NMR (300 MHz, DMSO-d6): d (ppm) 1.48 (s,
6H), 6.26 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.73–7.76 (m,
2H), 8.05 (1H, s), 8.08–8.12 (m, 2H), 8.79 (s, 1H). 13C NMR
4.3.13. 2-(4-Bromophenyl)-1H-anthra[1,2-d]imidazole-6,11-
dione (16)
Product 16 was obtained as red brown powder (yield 75%): mp
(75 MHz, DMSO-d6): d (ppm) 30.18, 81.70, 104.02, 108.04,
302–303 °C (EtOH) (Rf = 0.40 at ethyl acetate/dichlorometh-
120.99, 123.54, 126.32, 127.07, 133.41, 133.54, 134.79, 135.46,
143.05, 148.12, 179.89 (CO), 182.47 (CO). HR-MS (ESI-TOF) m/z
ane = 1:4). FT-IR (KBr;
m
cmÀ1) 3391 (NH), 1658 (CO). 1H NMR

H.-S. Huang et al. / Bioorg. Med. Chem. 17 (2009) 7418–7428
7427
calcd, [M+H]+ 279.1128; found, 279.1133. EI-MS m/z 278 (M+, 8.6),
EtOH to afford desired compound as a black solid in 25% yield
263 (100).
(Rf = 0.6 at ethyl acetate/dichloromethane = 1:4). Mp >400 °C
(EtOH). FT-IR (KBr;
m
cmÀ1) 1671 (CO). 1H NMR (300 MHz,
4.3.18. 2-Methyl-2-phenyl-2,3-dihydro-1H-anthra[1,2-
d]imidazole-6,11-dione (21)
DMSO-d6): d (ppm) 2.72 (s, 3H), 2.88 (s, 3H), 7.91–7.94 (m, 2H),
8.07 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 8.19–8.21 (m, 2H).
13C NMR (75 MHz, DMSO-d6): d (ppm) 14.91, 30.74, 120.19,
125.46, 126.21, 126.26, 127.16, 128.18, 128.87, 133.01, 133.10,
134.19, 134.27, 134.42, 158.87, 162.28, 182.49 (CO), 183.37
(CO). HR-MS (ESI-TOF) m/z calcd, [M+H]+ 289.0988; found,
289.0970.
Acetophenone (0.5 mL, 6 mmol) was added dropwise to a solu-
tion of 1,2-diaminoanthraquinone (1.19 g, 5 mmol) and concd sul-
furic acid (0.1 mL) in DMF (30 mL). The reaction mixture was
stirred for 72 h at room temperature. The mixture was treated with
crushed ice and the resulting precipitate was collected by filtration,
washed with diethyl ether and purified by crystallization from
EtOH to afford desired compound as a black solid in 28% yield
(Rf = 0.8 at ethyl acetate/dichloromethane = 1:4). Mp 368–371 °C
4.3.22. Naphtho[2,3-f]quinoxaline-7,12-dione (25)
Glyoxal (40%, 0.8 g, 5 mmol) in EtOH (50 mL) was added drop-
wise to a solution of 1,2-diaminoanthraquinone (1.19 g, 5 mmol)
in DMF (30 mL). The reaction mixture was refluxed for 16 h until
water was removed. The mixture was treated with crushed ice
and the resulting precipitate was collected by filtration, washed
with diethyl ether and purified by from EtOH and dichloromethane
to afford desired compound as a black solid in 23% yield (Rf = 0.45
at ethyl acetate/dichloromethane = 1:4). Mp 270–272 °C (EtOH).
(EtOH). FT-IR (KBr;
m
cmÀ1) 3348 (NH), 1671 (CO). 1H NMR
(300 MHz, DMSO-d6): d (ppm) 1.22 (s, 3H), 7.56–7.62 (m, 3H),
7.90–7.94 (m, 2H), 8.08 (d, J = 8.1 Hz, 1H), 8.22 (d, J = 8.1 Hz, 1H),
8.18–8.22 (m, 2H), 8.40–8.42 (m, 2H). 13C NMR (75 MHz, DMSO-
d6): d (ppm) 28.79, 83.56, 103.62, 109.74, 119.13, 121.35, 124.03,
126.20, 1267.76, 128.32, 128.77, 131.31, 132.99, 134.30, 134.45,
143.05, 157.25, 182.60 (CO), 182.89 (CO). HR-MS (ESI-TOF) m/z
calcd, [M+H]+ 341.1284; found, 341.1033.
FT-IR (KBr;
m
cmÀ1) 3413, 3365, 1626 (CO). 1H NMR (300 MHz,
CDCl3): d (ppm) 7.82–7.87 (m, 2H), 8.29–8.36 (m, 2H), 8.48 (d,
J = 8.7 Hz, 1H), 8.72 (d, J = 8.7 Hz, 1H), 8.99 (d, J = 1.5 Hz, 1H),
9.25 (d, J = 1.5 Hz, 1H). 13C NMR (75 MHz, CDCl3): d (ppm)
126.72, 127.05, 127.46, 130.05, 131.98, 133.77, 134.76, 135.18,
135.88, 135.93, 136.03, 145.42, 146.40, 147.77, 183.21 (CO),
183.61 (CO). HR-MS (ESI-TOF) m/z calcd, [M+H]+ 261.0659; found,
261.0663. EI-MS m/z 260 (M+, 100), 238 (73), 150 (54).
4.3.19. 2-Mercapto-1(3)H-anthra[1,2-d]imidazole-6,11-dione
(22)
1,2-Diaminoanthraquinone (1.19 g, 5 mmol) was dissolved in
DMF (30 mL) and carbon disulfide (0.4 g, 5 mm) and triethylamine
(3 mL) was added dropwise. The reaction mixture was continued
under refluxing condition for 10 h. The reaction mixture was
cooled to room temperature and treated with crushed ice and
the resulting precipitate was collected by filtration, washed with
diethyl ether and purified by crystallization from EtOH to afford
desired compound as a brown solid in 80% yield (Rf = 0.8 at ethyl
acetate/dichloromethane = 1:4). Mp 407–409 °C (EtOH). FT-IR
4.3.23. Naphtho[2,3-f]quinoxaline-2,3,7,12(1H,4H)-tetraone
(26)
Oxalic acid (0.46 g, 5 mmol) was added dropwise to a solution
of 1,2-diaminoanthraquinone (1.19 g, 5 mmol) and concd sulfuric
acid (0.1 mL) in DMF (30 mL). The reaction mixture was refluxed
for 16 h. The mixture was treated with crushed ice and the result-
ing precipitate was collected by filtration, washed with diethyl
ether and purified by crystallization from EtOH to afford desired
compound as a black solid in 30% yield (Rf = 0.25 at ethyl acetate/
(KBr;
m )
cmÀ1 3221 (NH), 3192 (NH),1665 (CO). 1H NMR
(300 MHz, DMSO-d6): d (ppm) 7.54 (d, J = 8.1 Hz, 1H), 8.02 (d,
J = 8.1 Hz, 1H), 7.91–7.94 (m, 2H), 8.18–8.22 (m, 2H), 12.73 (s,
1H), 13.29 (s, 1H). 13C NMR (75 MHz, DMSO-d6): d (ppm) 113.89,
115.27, 122.41, 126.26, 126.76, 126.88, 130.95, 132.89, 133.06,
134.25, 134.47, 138.19, 172.89, 181.79 (CO), 182.46 (CO). HR-MS
(ESI-TOF) m/z calcd, [M+H]+ 281.0379; found, 281.0389.
dichloromethane = 1:4). Mp dec. 370 °C (EtOH). FT-IR (KBr;
m
cmÀ1) 1710 (CO), 1671 (CONH). 1H NMR (300 MHz, DMSO-d6): d
(ppm) 7.71 (d, J = 8.0 Hz, 1H), 7.93–7.98 (m, 2H), 8.04 (d,
J = 8.0 Hz, 1H), 8.17–8.24 (m, 2H), 8.99 (d, J = 1.5 Hz, 1H), 9.25 (d,
J = 1.5 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): d (ppm) 118.08,
120.52, 122.87, 126.26, 126.34, 126.78, 127.71, 128.17, 129.58,
134.48, 134.55, 135.07, 154.64 (NHCO), 154.73 (NHCO), 180.08
(CO), 181.07 (CO). HR-MS (ESI-TOF) m/z calcd, [M+H]+ 293.0557;
found, 293.0568. EI-MS m/z 292 (M+), 248 (100).
4.3.20. Anthra[2,1-c][1,2,5]thiadiazole-6,11-dione (23)
1,2-Diaminoanthraquinone (1.19 g, 5 mmol) was dissolved in
dry THF (30 mL) and thionyl chloride (0.15 g, 20 mm) and triethyl-
amine (3 mL) was added dropwise. The reaction mixture was stir-
red for 1 h at room temperature. The mixture was treated with
crushed ice and the resulting precipitate was collected by filtration,
washed with diethyl ether and purified by crystallization from
EtOH to afford desired compound as a yellow solid in 74% yield
(Rf = 0.8 at ethyl acetate/dichloromethane = 1:4). Mp 227–228 °C
4.4. Cell culture and assessment of hTERT
(EtOH). FT-IR (KBr;
m
cmÀ1) 1671 (CO). 1H NMR (300 MHz, CDCl3):
Non-small lung cancer cells H129920 were grown in RPMI 1640
media supplemented with 10% fetal bovine serum, 100 units/mL
penicillin and 100 mg/mL streptomycin in a humidified atmo-
sphere with 5% CO2 at 37 °C. The hTERT immortalized hTERT-BJ1
(BD Biosciences Clontech)21 were grown in DMEM media supple-
mented with 10% fetal calf serum, 100 units/mL penicillin and
d (ppm) 7.84 (dd, J = 12.15, 6.9 Hz, 1H), 7.85 (dd, J = 13.2, 7.5 Hz,
1H), 8.33 (dd, J = 22.5, 7.2 Hz, 1H), 8.33 (dd, J = 22.5, 7.2 Hz, 1H),
8.41 (d, J = 9.3 Hz, 1H), 8.56 (d, J = 9.3 Hz, 1H). 13C NMR (75 MHz,
CDCl3): d (ppm) 125.07, 126.35, 126.99, 127.34, 127.61, 132.08,
133.47, 134.15, 134.75, 135.16, 150.93, 157.99, 181.97 (CO),
183.31 (CO). HR-MS (ESI-TOF) m/z calcd, [M+H]+ 267.0223; found,
267.0226. EI-MS m/z 266 (M+, 100), 238 (64), 210 (57).
100 mg/mL streptomycin, 1 mM sodium pyruvate, and 4 mM L-
arginine in a humidified atmosphere with 5% CO2 at 37 °C. Culture
media were changed every three days. To establish stable cell lines
that the expression of hTERT could be monitored by a reporter sys-
tem, a ꢀ3.3 kbp DNA fragment ranging from À3338 to +1 bp of the
hTERT gene was subcloned upstream to a secreted alkaline phos-
phatase gene (SEAP) and transfected into H1299 or hTERT-BJ1 by
electroporation. The stable clones were selected using G418. The
stable clones derived from H1299 or hTERT-BJ1 were cultured
using conditions that are similar to their parental cells.
4.3.21. 2,3-Dimethylnaphtho[2,3-f]quinoxaline-7,12-dione (24)
Methyl vinyl ketone (0.36 g, 5 mmol) was added dropwise to a
solution of 1,2-diaminoanthraquinone (1.19 g, 5 mmol) and concd
sulfuric acid (0.1 mL) in DMF (30 mL). The reaction mixture was
stirred for 72 h at room temperature. The mixture was treated with
crushed ice and the resulting precipitate was collected by filtration,
washed with diethyl ether and purified by crystallization from

7428
H.-S. Huang et al. / Bioorg. Med. Chem. 17 (2009) 7418–7428
4.5. Cytotoxicity assay
Acknowledgments
The tetrazolium reagent (MTT; 3-(4,5-di-methylthiazol)-2,5-
diphenyltetrazolium bromide, USB) was designed to yield a colored
formazan upon metabolic reduction by viable cells.22,23 Approxi-
mately 2 Â 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 com-
plete medium before addition to cell cultures. Test compounds
were then added to the culture medium for designated various
The present study was support by National Science Council
Grants NSC 96-2923-M-016-001-MY3, NSC 97-2113-M-016-002,
NSC 97-2311-B-010-005, and NSC 97-3112-B-010-013, and also
by National Health Research Institute Grant NHRI-EX97-9625SI.
The corresponding author cordially dedicated to Dr. Klaus K. Mayer
(Regensburg/Germany) on the occasion of his 70th birthday and
also thank NCI for collaborations.
References and notes
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