Structure-based design, synthesis and evaluation of novel anthra[1,2-d]imidazole-6,11-dione derivatives as telomerase inhibitors and potential for cancer polypharmacology

Article abstract of DOI:10.1016/j.ejmech.2012.11.032

A series of anthra[1,2-d]imidazole-6,11-dione derivatives were synthesized and evaluated for telomerase inhibition, hTERT expression and suppression of cancer cell growth in vitro. All of the compounds tested, except for compounds 4, 7, 16, 24, 27 and 28

Full text of DOI:10.1016/j.ejmech.2012.11.032

European Journal of Medicinal Chemistry 60 (2013) 29e41
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure-based design, synthesis and evaluation of novel anthra[1,2-d]imidazole-
6,11-dione derivatives as telomerase inhibitors and potential for cancer
polypharmacology
,
Chun-Liang Chen a, Deh-Ming Chang a c, Tsung-Chih Chen a, Chia-Chung Lee a, Hsi-Hsien Hsieh e,
,
h
,
a,b,d,
*
Fong-Chun Huang e, Kuo-Feng Huang f, Jih-Hwa Guh g, Jing-Jer Lin e
, Hsu-Shan Huang
**
a Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan
b School of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan
c Rheumatology/Immunology/Allergy, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan
d Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan
e Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan
f Chi-Mei Medical Center, Tainan 710, Taiwan
g School of Pharmacy, National Taiwan University College of Medicine, Taipei 100, Taiwan
h Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei 100, 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 anthra[1,2-d]imidazole-6,11-dione derivatives were synthesized and evaluated for telomerase
inhibition, hTERT expression and suppression of cancer cell growth in vitro. All of the compounds tested,
except for compounds 4, 7, 16, 24, 27 and 28 were selected by the NCI screening system. Among them,
compounds 16, 39, and 40 repressed hTERT expression without greatly affecting cell growth, suggesting
for the selectivity toward hTERT expression. Taken together, our findings indicated that the analysis of
cytotoxicity and telomerase inhibition might provide information applicable for further developing
potential telomerase and polypharmacological targeting strategy.
Received 24 August 2012
Received in revised form
12 November 2012
Accepted 22 November 2012
Available online 29 November 2012
Keywords:
Telomerase
Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.
Imidazole-fused anthraquinones
TRAP assay
SEAP assay
Polypharmacology
1. Introduction
human cells [5]. Telomerase is a ribonucleoprotein that consists of
a catalytic subunit (human telomerase reverse transcriptase, hTERT)
The anthracycline antibiotics daunorubicin, doxorubicin,
mitoxantrone and ametantrone have been shown to process strong
antiproliferative properties and clinically used to treat various
cancers for many decades (Scheme 1) [1]. Recent studies illustrated
anthracycline antibiotics might interrupt telomere maintenance
through interfering the targeting of telomerase onto telomeres [2].
Telomeres are guanine-rich DNA sequences coated by specialized
proteins at the terminal ends of eukaryotic chromosomes [3,4]
which consists of hexameric TTAGGG repeats sequences in
and a template RNA (human telomerase RNA component, hTERC) to
add telomere repeats to the 30 single-strand overhang [6]. Exten-
sion of telomeres by telomerase is required for un-limited prolif-
eration of most of the immortal and cancer cells [7,8]. The
important roles of telomerase in cellular immortalization have
made telomerase as a potentially molecular target for cancer
therapeutic discovery which might have minimal side effects [9,10].
Moreover, the anthracycline antibiotics inhibit telomerase activities
through the stabilizing of G-quadruplex structure sequences. It is
also well accepted that human cancer cells achieve immortalization
in large part through the illegitimate activation of telomerase
expression [11,12]. Therefore, agents that suppress hTERT expres-
sion might further develop potential telomerase and poly-
pharmacological targeting strategy.
* Corresponding author. Department of Pharmacy Practice, Tri-Service General
Hospital, National Defense Medical Center, Taipei 114, Taiwan. Tel.: þ886
2
87923311x17327; fax: þ886 2 87923169.
** Corresponding author. Institute ofBiopharmaceutical Sciences, National Yang-Ming
University, Taipei 112, Taiwan. 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).
Based on our previous studies, some of the 1,2-heteroannelated
substitution on the anthraquinone and anthra[1,2-d]imidazole-
0223-5234/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.032

30
C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
Scheme 1. Design of new compounds through hybridization of bioactive chromophoric anthraquinones (anthracycline antibiotic anticancer agents) and benzimidazole.
6,11-dione scaffold homologs might be interesting for cytotoxicity
toward cancer cells or cancer polypharmacology [13]. Previous
SARs studies have also shown that both anthraquinone-based
compounds and bis-benzimidazole derivatives (Hoechst 33258)
are known telomerase inhibitors or G-quadruplex stabilizers [14e
16]. In the past, we also reported several results concerning the
discovery of new classes of anthraquinone-linked heterocyclic
derivatives [13,17e22]. Herein, we continue our efforts to develop
an efficient synthetic procedure for the preparation of a novel series
of N-substituent derivatives in the vacant 2-position of imidazole
ring of anthra[1,2-d]imidazole-6,11-diones. In the series of
designed analogs, we maintained benzimidazole as the central
structure moiety since it was considered as the primary pharma-
cophore for potential telomerase inhibitory activity. Lead optimi-
zation were then focused on varying of other substituent moiety at
the 2-position of the imidazole skeleton, by adding electron-
method for constructing the core structure of the compounds is
outlined in Scheme 2. The synthesis commences starting from 1,2-
diaminoanthraquinone, the cyclization with chloroacetyl chloride
formed compound 1. The preparation of compounds 2e33 involved
direct amination using various amines and TEA/DIPEA in THF to
obtain our desired derivatives. As shown in Scheme 2, compound
34 was prepared by amination between 1,2-diaminoanthraquinone
and 3-chloropropionyl chloride in the presence of pyridine. The
resulting compounds were further cyclization converted to
compound 35 that has a terminal chloroethyl group attached to the
imidazole-fused ring. Treating compound 35 with various amines
in DIPEA/THF gave the desired compounds 36e40 through nucle-
ophilic substitution of the chlorine atom by appropriately
substituted piperazine. The 13C NMR data showed that the signal
for carbonyl group on the core structure of anthraquinone was at
the range
d 182e185 ppm whereas the signal of imidazole ring was
withdrawing
or
electron-releasing
substituents.
These
at d 154e161 ppm, respectively. Besides, all the final products were
compounds were evaluated for cytotoxicity by MTT assay or sul-
forhodamine B assay (SRB assay) on non-small lung cancer cell,
H1299, prostate cancer cells PC-3 [23e26], as well as telomerase
inhibition using TRAP assay [27,28] and SEAP assay [29,30]. Finally,
the interplay between the cytotoxicity, hTERT repressing activities
and the drug explains the SARs of derivatives and the molecular
basis of drug-induced activities.
monitored by TLC; the quantities of the byproducts were separated
from their physical chemistry properties and purification by
tedious recrystallization and chromatography. All of the structural
compounds were determined by 1H NMR, 13C NMR and high
resolution mass (HRMS) spectra and the results are presented in
the experimental part.
3. In vitro anti-proliferative activities
2. Chemistry
As reported in previous sections, all of the newly synthesized
compounds were evaluated for their effects on cell viability in
H1299 cell (Table 1). We found that most of the tested compounds
showed cytotoxicity toward H1299 cells at micromolar ranges.
There has been continuing interest in the synthesis of anthra
[1,2-d]imidazole-6,11-dione derivatives and their systematic
dissection largely on account of their biological activities [13]. The

C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
31
aReagents and conditions: (i) chloroacetyl chloride, DMF, 80 oC, 8 hr. (ii) various
amine, TEA, THF, reflux, 8 hr. (iii) various amine, DIPEA, THF, reflux, 6 hr. (iv)
o
chloropropionyl chloride, pyridine, DMF, r.t., 24 hr. (v) 50% H2SO4 (aq), 110 C, 2 hr,
mini reactor. (vi) various amine, DIPEA, THF, reflux, 6 hr.
Scheme 2. aSynthesis of anthra[1,2-d]imidazole-6,11-dione derivatives 1e40.
Among them, compounds 1, 6, 10, 14, and 20 showed strong cyto-
less effective with IC50 > 10
mM. Since TRAP assay involves both
toxic effects toward H1299 cells. The anti-proliferation effects of
these compounds were further analyzed using a PC-3 cancer cell
line. The results were summarized in Table 2 and expressed as the
amount of compounds required to achieve 50% growth inhibition
(IC50). These compounds showed IC50 values ranging from several
telomerase extension and PCR amplification steps, the effect of
these compounds on Taq polymerase was also analyzed.
Compounds 20, 24 and 25 were added after the telomerase
extension step during the TRAP assays (Fig. 1, TRAP-m). All three
compounds did not show any detectable inhibitory activity in
TRAP-m assays, suggesting that the inhibitory effect observed for
these three compounds were due to the inhibition of telomerase
activity. Thus, our results show that all three compounds specifi-
cally inhibited telomerase activity. Compound 20 is the most potent
inhibitor among these compounds.
micromolars to weak inhibitory effect with the maximum 30 mM
concentration tested in our assays. It appeared that small side-
chain extension might have better cytotoxic effects toward PC-3
cell. Among them, compounds 1e10, 14e16, 18e26, 29e32, 35, 36
and 40 showed moderate potency against PC-3 with IC50 from
5.1
m
M to 27.9
m
M, respectively. With the exception of compounds
The catalytic subunit of telomerase hTERTexpression was shown
to be the key determining factor for telomerase activation in
telomerase-positive cancer cells [31,32]. Herein, we also evaluated
the hTERT repressing activity of our newly synthesized compounds
by using a reporter assay that fused a SEAP reporter gene under the
control of hTERT promoter. The expression of hTERTpromoter-driven
SEAP in H1299 cells was used as the criteria to determine the hTERT
repressing activities of our new anthraquinone derivatives. Results
of all tested compounds that showed SEAP inhibition activity at 1,10
22 and 35 that showed preferential cytotoxic effects toward PC-3
cells, most of these compounds showed similar cytotoxic profiles
toward H1299 and PC-3 cells. These compounds were also
submitted to National Cancer Institute for 60 cell line drug screen
program. Compounds
(NSC749236), 24 (NSC749235), 27 (NSC749237) and 28
4
(NSC749234),
7
(NSC749238), 16
(NSC749765) were selected to evaluate the cytotoxic effects toward
60 cell lines at a single dose of 10 mM (Table 3). These compounds
exhibited preferential growth inhibition effects toward leukemia
cell lines. Compounds 4, 7, and 24 showed less than 50% survival for
more than half of the leukemia cell lines. Interestingly, compounds
4 and 24 might also have preference toward renal and colon
cancers, respectively.
and 100 mM were summarized in Table 1. We have also determined
the cell viability of the drug-treated H1299 cells in parallel using
MTTassay. Significantly, quite a numbers of our compounds showed
selective inhibitory effects on hTERT expression. Among these
compounds, we were most interested in compounds 16, 39, and 40
because they affected the SEAP expression without significantly
affecting the proliferation of the treated H1299 cells (Fig. 2). Thus,
these compounds might selectively repress hTERT expression. The
cytotoxic effects of these three compounds toward normal human
diploid fibroblast IMR90 were also determined. All three
4. Telomerase inhibition and hTERT repression activities
The inhibitory effects of our synthesized imidazole-fused
compounds on telomerase activities were also analyzed using the
cell free extracts prepared from H1299 cells. We first tested the
inhibitory activity of compounds 1e40 and the starting material
compounds showed IC50 at w100 mM toward IMR90, suggesting that
they did not affect the general growth of normal cells (data not
shown). Compound 40 appeared to show the best selectivity toward
repressing hTERTexpression. Interestingly, since a higher fraction of
these compounds with long side chain (compounds 36 and 38e40)
showed selective hTERT repressing activities, the results suggested
that the side chain length might play an important role in deter-
mining the hTERT repressing activity.
1,2-diaminoanthraquinone at 100
mM concentration (data not
shown). Among them, only compounds 20, 24, and 25 showed
significant telomerase inhibition activities. We next analyzed the
concentration effects of compounds 20, 24 and 25 against telo-
merase (Fig. 1). We found compound 20 showed potent telomerase
inhibition activity with IC50 at w5 mM. Compounds 24 and 25 were

32
C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
Table 1
Effects of anthra[1,2-d]imidazole-6,11-dione derivatives on cytotoxicity by MTT assay and repressing hTERT expression activity by SEAP assay.
Compounds
eR
Cell type/H1299
MTT assay (viability % ꢀ SD)a
SEAP activity (% ꢀ SD)a
1 (m
M)
10 (m
M)
100 (
mM)
1 (
m
M)
10 (m
M)
100 (mM)
1,2-diaminoanthraquinone
105 ꢀ 9
82 ꢀ 6
99 ꢀ 6
99 ꢀ 4
101 ꢀ 7
93 ꢀ 7
96 ꢀ 5
95 ꢀ 4
0 ꢀ 2
39 ꢀ 9
0 ꢀ 1
0 ꢀ 2
0 ꢀ 0
0 ꢀ 0
0 ꢀ 1
0 ꢀ 1
99 ꢀ 12
69 ꢀ 2
84 ꢀ 9
97 ꢀ 12
91 ꢀ 4
96 ꢀ 7
77 ꢀ 3
77 ꢀ 12
0 ꢀ 1
18 ꢀ 4
3 ꢀ 1
0 ꢀ 1
0 ꢀ 1
0 ꢀ 0
0 ꢀ 1
0 ꢀ 2
1
2
3
4
5
6
eCl
eN(CH3)2
eN(CH3)(CH2CH3)
eN(CH2CH3)(CH2CH3)
eN(CH3)(CH2CH2CH3)
eN(CH3)(CH(CH3)2)
64 ꢀ 3
65 ꢀ 3
55 ꢀ 4
80 ꢀ 4
23 ꢀ 3
45 ꢀ 1
39 ꢀ 0
32 ꢀ 4
34 ꢀ 4
1 ꢀ 1
7
104 ꢀ 1
99 ꢀ 7
0 ꢀ 1
96 ꢀ 6
51 ꢀ 2
0 ꢀ 1
8
9
104 ꢀ 4
97 ꢀ 5
90 ꢀ 2
98 ꢀ 2
28 ꢀ 1
9 ꢀ 5
90 ꢀ 3
69 ꢀ 2
78 ꢀ 2
7 ꢀ 1
0 ꢀ 2
105 ꢀ 3
10
101 ꢀ 6
13 ꢀ 2
0 ꢀ 2
86 ꢀ 4
0 ꢀ 1
0 ꢀ 2
11
12
98 ꢀ 4
99 ꢀ 5
84 ꢀ 6
96 ꢀ 6
42 ꢀ 7
6 ꢀ 2
102 ꢀ 6
87 ꢀ 3
77 ꢀ 6
81 ꢀ 7
12 ꢀ 2
0 ꢀ 0
13
14
114 ꢀ 3
74 ꢀ 4
110 ꢀ 1
11 ꢀ 2
32 ꢀ 2
0 ꢀ 1
87 ꢀ 2
52 ꢀ 1
89 ꢀ 2
8 ꢀ 7
4 ꢀ 1
2 ꢀ 1
15
16
109 ꢀ 2
111 ꢀ 9
103 ꢀ 4
79 ꢀ 6
14 ꢀ 3
20 ꢀ 3
75 ꢀ 14
76 ꢀ 27
75 ꢀ 17
44 ꢀ 16
1 ꢀ 1
7 ꢀ 3
17
18
114 ꢀ 3
104 ꢀ 7
110 ꢀ 1
85 ꢀ 1
32 ꢀ 3
0 ꢀ 1
102 ꢀ 5
107 ꢀ 5
97 ꢀ 5
66 ꢀ 4
26 ꢀ 5
4 ꢀ 0
19
103 ꢀ 2
79 ꢀ 6
15 ꢀ 3
97 ꢀ 2
96 ꢀ 1
14 ꢀ 2
20
21
22
23
97 ꢀ 5
90 ꢀ 3
92 ꢀ 4
89 ꢀ 5
0 ꢀ 0
71 ꢀ 4
77 ꢀ 4
76 ꢀ 7
0 ꢀ 1
13 ꢀ 6
1 ꢀ 1
96 ꢀ 5
102 ꢀ 5
115 ꢀ 2
106 ꢀ 3
8 ꢀ 0
97 ꢀ 5
77 ꢀ 2
86 ꢀ 1
3 ꢀ 0
26 ꢀ 5
5 ꢀ 1
5 ꢀ 2
12 ꢀ 1
24
25
87 ꢀ 6
86 ꢀ 5
35 ꢀ 4
35 ꢀ 4
0 ꢀ 1
0 ꢀ 1
106 ꢀ 3
92 ꢀ 3
23 ꢀ 1
14 ꢀ 1
6 ꢀ 1
0 ꢀ 1

C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
33
Table 1 (continued )
Compounds
eR
Cell type/H1299
MTT assay (viability % ꢀ SD)a
SEAP activity (% ꢀ SD)a
1 (m
M)
10 (
mM)
100 (
m
M)
1 (m
M)
10 (m
M)
100 (mM)
26
27
28
86 ꢀ 3
90 ꢀ 4
91 ꢀ 4
45 ꢀ 4
54 ꢀ 4
69 ꢀ 4
0 ꢀ 1
18 ꢀ 4
20 ꢀ 2
87 ꢀ 2
92 ꢀ 1
91 ꢀ 3
17 ꢀ 4
77 ꢀ 6
57 ꢀ 1
0 ꢀ 1
6 ꢀ 4
1 ꢀ 1
29
30
85 ꢀ 3
88 ꢀ 2
74 ꢀ 2
77 ꢀ 2
28 ꢀ 2
15 ꢀ 2
111 ꢀ 4
103 ꢀ 3
92 ꢀ 3
78 ꢀ 4
19 ꢀ 2
0 ꢀ 1
31
32
33
78 ꢀ 5
81 ꢀ 10
93 ꢀ 2
62 ꢀ 6
58 ꢀ 7
51 ꢀ 8
22 ꢀ 1
24 ꢀ 3
13 ꢀ 3
106 ꢀ 5
105 ꢀ 6
98 ꢀ 7
57 ꢀ 8
30 ꢀ 7
11 ꢀ 1
6 ꢀ 1
3 ꢀ 0
0 ꢀ 0
34
35
e
94 ꢀ 3
91 ꢀ 6
34 ꢀ 5
86 ꢀ 3
6 ꢀ 2
87 ꢀ 5
95 ꢀ 9
13 ꢀ 4
83 ꢀ 5
0 ꢀ 1
8 ꢀ 4
eCH2Cl
40 ꢀ 1
36
87 ꢀ 7
32 ꢀ 6
0 ꢀ 0
74 ꢀ 3
7 ꢀ 5
0 ꢀ 1
37
94 ꢀ 1
96 ꢀ 14
65 ꢀ 9
103 ꢀ 5
93 ꢀ 5
37 ꢀ 2
38
39
98 ꢀ 8
91 ꢀ 6
96 ꢀ 8
93 ꢀ 8
84 ꢀ 8
54 ꢀ 9
57 ꢀ 11
0 ꢀ 2
88 ꢀ 9
83 ꢀ 4
73 ꢀ 8
63 ꢀ 3
35 ꢀ 3
5 ꢀ 5
1 ꢀ 3
0 ꢀ 1
0 ꢀ 0
40
6 ꢀ 3
a
SD: standard derivation, all experiments were independently performed at least three times.
5. Discussion and conclusion
Evaluation of the antiproliferative activity against PC-3 human
prostate cancer cells showed that the addition of small-sized
functional groups to the imidazole ring enhanced the cell growth
inhibition activities of these compounds. The NCI’s 60 human
cancer cell lines screen for compounds 4, 7, 16, 24, 27 and 28
revealed that most of the synthesized compounds moderately
inhibited the growth of all cancer cell lines examined. Compound
24 appears to be the most active member of these compounds,
suggesting that the methylpiperazine moiety of compound 24
might have a role its cytotoxic effects. While the mechanism of how
these compounds rendered their cytotoxic activities is not fully
In the course of our continuing search for new anticancer agents
from anthraquinone moiety, we described an efficient synthesis
and biological evaluation of a new series of rigid analogs of anthra
[1,2-d]imidazole-6,11-dione. In this investigation, we focused our
attention on the role of our systemic synthesized anthra[1,2-d]
imidazole-6,11-dione as core pharmacophore bearing the imid-
azole moiety linked to the side chain. Forty new compounds have
been designed and prepared, among which ten displayed cyto-
toxicity (IC50) below 10
mM range toward PC-3 cells (Table 2).

34
C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
Table 3 (continued)
Table 2
Effects of compounds 1e40 on cytotoxicity of against the growth of PC-3 cells by SRB
assay.
Panel/cell lines
Compounds/growth percenta (at 10
m
M)
27
80.44
105.37 92.70
112.36 104.29
103.62 81.00
4
7
16
24
28
Cell type/PC-3 (Inhibition
Compounds
m
M)a
IC50
SF-268
SF-295
SF-539
SNB-19
SNB-75
74.04
89.56
85.26
75.06
82.03
70.77
85.87
105.86
102.32 82.47
87.77
81.62
81.83
58.46
e
51.16
81.61
63.88
65.60
69.87
65.41
79.15
Compounds
IC50
1
2
3
4
5
6
7
8
5.1
8.8
7.5
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
e
6.7
12.1
6.5
76.33
86.42
72.67
70.93
84.83
82.14
86.28
10.3
17.1
6.7
14.2
21
18.7
6.4
>30
>30
>30
7.6
22.5
10.3
>30
10.5
21.6
13.8
12.4
6.5
U251
12.8
>30
>30
27.9
16.7
29
15.3
>30
>30
5.2
17.3
>30
>30
>30
16.0
e
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
SKeOV-3
Renal cancer
786e0
A489
ACHN
SN12C
TK-10
UO-31
CAKI-1
RXF 393
Prostate cancer
PC-3
61.98
98.45
97.86
88.53
89.67
62.99
43.47
95.76
82.25
107.00 119.68 89.63
106.97 100.82 97.20
93.53
119.09 124.77
106.33 103.99
101.37 94.85
101.39 98.34
127.46 114.23
74.84
93.36
e
9
102.02 86.53
113.29 107.95
107.08 96.38
65.73
74.61
e
10
11
12
13
14
15
16
17
18
19
20
21
102.69 95.03
92.41
68.20
68.47
e
97.01
75.48
89.67
e
77.46
81.12
e
83.56
85.13
88.91
13.61
63.32
65.69
96.50
e
54.26
83.57
80.99
54.22
66.26
65.27
12.05
52.79
50.98
69.25
76.56
67.72
91.25
88.67
64.57
109.14 101.70 97.72
107.63 110.77
80.59
86.11
106.7
e
e
80.89
80.36
98.95
88.07
81.16
93.16
e
e
65.32
86.81
73.52
92.54
62.93
74.33
a
IC50 is the concentration of drug (mM) required to inhibit cell growth by 50% of
the mean (N ¼ 3).
64.88
49.50
48.41
69.04
98.12
27.21
e
89.74
69.60
80.20
91.19
62.88
61.84
47.11
96.96
59.13
69.36
33.08
48.50
88.48
75.09
86.32
89.91
118.37 120.65
58.33
e
91.03
68.23
80.61
79.85
clear to us, the information is valuable for future development of
these compounds into anti-cancer therapies.
130.74 115.68 73.05
49.49
e
Of the series of compounds analyzed, we found compounds 20,
24 and 25 exhibited telomerase inhibitory activity. In principle,
telomerase inhibition should not lead to acute cell cytotoxicity. A
long lag between telomerase inhibition and detrimental cellular
effects is anticipated. For example, inhibition of telomerase activity
using anti-sense peptide nucleic acid or 20-O-MeRNA oligomers led
to progressive telomere shortening and only caused cellular
response after weeks of treatments [33]. Thus, the cytotoxic effects
of H1299 cells by compounds 20, 24 and 25 should not be simply
due to their telomerase inhibition activities. The mechanism of how
these compounds cause cytotoxic effects is not clear to us. However,
since the anthracycline compounds were also known to inhibit
42.93
e
22.93
e
60.42
68.83
89.67
e
e
e
e
e
73.54
e
83.70
99.83
74.90
81.94
60.08
e
90.69
77.91
81.46
89.10
DU-145
Breast cancer
MCF7
MDA-MB-231/ 75.52
AHS 578-T
BT-549
T-47D
MDA-MB-468
Mean
Delta
51.13
69.06
88.98
108.96 50.98
42.74
77.93
36.71
61.66
51.48
66.08
90.47
80.56
81.99
72.65
111.04
70.37
105.50 114.38 109.47 100.53
110.22 102.42
83.55
54.46
69.66
79.09
68.45
77.09
89.07
49.16
69.11
58.32
75.35
52.58
96.91
97.08
71.59
59.85
73.29
121.39
66.35
72.75
90.15
47.04
56.78
53.92
87.65
33.73
Table 3
Range
114.93 90.83
135.50 70.85
Cytotoxicity of selected compounds in NCI’s drug screen program.
a
Data obtained from NCI in vitro 60-cell line Program at 10
Not test: (ꢁ).
m
M.
b
Panel/cell lines
Compounds/growth percenta (at 10
m
M)
27
4
7
16
24
28
Leukemia
CCRF-CEM
HL-60(TB)
MOLT-4
SR
topoisomerase activity and might inhibit telomerase activities
through the stabilization of G-quadruplex structure formed by
human G-rich DNA sequences, it is likely that these compounds
cause cytotoxic effects through these mechanisms.
47.88
68.18
ꢁ9.43
42.38
e
39.91
120.08 56.89
47.54
70.10
e
102.47 34.45
65.29
74.42
107.54 97.31
eb
22.77
57.64
e
ꢁ13.44 55.21
61.76
e
21.48
43.11
e
Compounds 16, 39, and 40 were identified to repress hTERT
expressions in H1299 cells. We consider the hTERT repressing effects
of these compounds are specific as the same amounts of these
compounds did not show equivalent level of cytotoxic effects. These
compounds might repress hTERT expression directly through
stabilizing the G-quadruplex structure located within the hTERT
promoter. It is also likely that they might stabilize the G-quadruplex
structure located within the c-myc promoter to indirectly repress
hTERTexpression. Thedetailed mechanism of howthese compounds
affect hTERT expression remains to be characterized. SARs analysis
also revealed that the piperazine linker with a one- or two-carbon
spacer between imidazole and amine might be an important func-
tional moiety for hTERT repression or telomerase inhibition.
The cellular activities of the anthra[1,2-d]imidazole-6,11-dione
derivatives appear to be greatly affected by its imidazole ring and
various substituents on the core structure. We did not find apparent
K562
e
83.48
Non-small cell lung cancer
EKVX
HOP-62
HOP-92
73.22
85.27
56.11
77.20
75.96
90.06
55.50
73.14
e
82.24
105.37 92.69
101.41 80.97
81.36
90.81
102.29 92.47
90.27
86.39
106.30
77.14
65.93
60.84
9.17
88.70
98.40
178.61 93.87
82.98
92.11
91.96
94.45
73.61
63.00
103.18
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
A549/ATCC
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
78.85
81.32
64.48
72.97
82.41
57.35
64.61
e
91.88
81.65
93.77
79.98
94.06
64.61
84.14
e
111.76 91.07
81.00
80.61
48.22
65.75
56.02
76.89
60.46
117.88 74.65
53.66
61.90
27.73
44.98
22.64
55.51
44.22
128.37 110.79
e
95.82
83.56
80.78
84.18
89.03
82.81
62.12
54.20
61.38
77.40
92.28
84.30
86.02
75.79
95.03
85.97
78.28
74.39
87.57
85.80
90.92
HT29
KM12
SW-620
CNS cancer
100.52 71.72

C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
35
Fig. 1. Structures and the effects of compounds 20, 24, and 25 on telomerase activities. (A) In standard TRAP assays (top panels), indicated amounts of compounds were incubated
with telomerase-active cell extracts for 5 min at room temperature before the telomerase-extension and PCR-amplification reactions. Telomerase-active cell extracts were prepared
from H1299 cells (P) and the RNase A-treated extracts were used as negative controls (N). In TRAP-m assays (bottom panels), indicated amounts of the compounds were added right
after the telomerase extension step and before the PCR-amplification step. (B) Structure of compounds 20, 24, and 25.
correlation among telomerase inhibition, hTERT repression, and
anti-proliferation of these compounds. Nevertheless, our analysis
indicates that the imidazole-fused chromophore is a novel lead for
anti-cancer therapies. Our analysis also provides further insight
into designing the lead compounds for future drug development.
300 (300 MHz) or Agilent 400 MR DD2 (400 MHz). Mass spectra
were obtained on Finnigan MAT 95 XL HRMS and MAT 95 MS
HRMS. These compounds were synthesized, starting from 1,2-
diaminoanthraquinone and this starting material was purchased
from Aldrich Chemical (Milwaukee, WI, USA). Reagents and
solvents were purchased from Merck and Aldrich used without
further purification.
6. Materials and methods
6.1. Chemistry
6.2. General synthetic methods
Melting points were determined on a Büchi 545 melting point
apparatus and are uncorrected. All reactions were monitored by
TLC, which were performed on Silica Gel F254 plates (Merck). 1H
NMR and 13C NMR spectra were recorded with a Varian GEMINI-
6.2.1. General procedure (I): preparation of compound 1
To a solution of 1,2-diaminoanthraquinone (1.19 g, 5 mmol) in
DMF 20 mL was added chloroacetyl chloride (0.5 mL, 6 mmol)
dropwise with stirring. The reaction mixture was stirred at 80 ꢂC for
Fig. 2. Structures and the inhibition of PhTERT-SEAP by compounds 16, 39, and 40. About 2 ꢃ 103 cells of H1299 harboring PhTERT-SEAP were seeded in 96-well plates and incubated at
37 ꢂC for 24 h. Cells were then washed with PBS, re-cultured in fresh media, and incubated with varying amounts of compounds 16, 39, and 40 for another 48 h. The culture media
were collected and subjected to SEAP activity analysis. The level of cell growth was also determined using MTT assay. The values are obtained from three experiments using the
values without drug treatment as 100%.

36
C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
10 h, and then poured into ice water (200 mL). The precipitate was
collected on a filter, washed with ethanol.
6.2.9. 2-((Ethylmethylamino)methyl)-1H-anthra[1,2-d]imidazole-
6,11-dione (3)
The pure compound was obtained as yellow powder (yield 51%).
6.2.2. General procedure (II): preparation of compounds 2e15
To a solution of compound 1 (1.18 g, 4 mmol) in THF 30 mL was
added TEA (1.1 mL, 8 mmol) and series amine (8 mmol) dropwise
with stirring. The reaction mixture was refluxed for 6 h. The
mixture was concentrated and extracted with ethyl acetate, dried
over MgSO4, and the solvent was removed in vacuo. The formed
precipitate was filtered off and dried in vacuo. Recrystallization
from hexane and ethyl acetate, washed with acetone.
Mp 160e161 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 1.16 (t, J ¼ 7.2 Hz,
3H), 2.35 (s, 3H), 2.60 (q, J ¼ 7.2 Hz, 2H), 3.89 (s, 2H), 7.77e7.80 (m,
2H), 8.01 (d, J ¼ 8.4 Hz, 1H), 8.18 (d, J ¼ 8.4 Hz, 1H), 8.19e8.32 (m,
2H). 13C NMR (75 MHz, CDCl3):
d ppm 12.36, 42.43, 52.00, 55.64,
118.26, 121.49, 125.50, 126.55, 127.62, 128.64, 132.50, 133.55, 133.77,
134.19, 134.32, 149.32, 159.72, 182.98, 185.07. HRMS (ESI) m/z calcd
for [M]þ: 319.1321, found: 320.1423 [M þ H]þ, 342.1245 [M þ Na]þ.
6.2.10. 2-((Diethylamino)methyl)-1H-anthra[1,2-d]imidazole-6,11-
dione (4)
6.2.3. General procedure (III): preparation of compounds 16e33
To a solution of compound 1 (1.18 g, 4 mmol) in THF 30 mL was
added DIPEA (1.4 mL, 8 mmol) and series amine (8 mmol) dropwise
with stirring. The reaction mixture was refluxed for 8 h. The
mixture was concentrated and extracted with ethyl acetate, dried
over MgSO4, and the solvent was removed in vacuo. The formed
precipitate was filtered off and dried in vacuo. Recrystallization
from hexane and ethyl acetate, washed with acetone.
The pure compound was obtained as yellow powder (yield 45%).
Mp 159e160 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 1.12 (t, J ¼ 7.2 Hz,
6H), 2.68 (q, J ¼ 7.2 Hz, 4H), 3.97 (s, 2H), 7.78e7.81 (m, 2H), 8.02 (d,
J ¼ 8.4 Hz, 1H), 8.20 (d, J ¼ 8.4 Hz, 1H), 8.25e8.34 (m, 2H). 13C NMR
(75 MHz, CDCl3):
d ppm. 12.10, 48.40, 52.18, 118.26, 121.51, 125.40,
126.56, 127.64, 128.56, 132.37, 133.61, 133.79, 134.25, 134.33, 149.59,
160.80, 183.05, 185.14. HRMS (ESI) m/z calcd for [M]þ: 333.1477,
found: 334.1580 [M þ H]þ, 356.1407 [M þ Na]þ.
6.2.4. General procedure (IV): preparation of compound 34
To a solution of 1,2-diaminoanthraquinone (0.95 g, 4 mmol) in
DMF 30 mL was added pyridine (0.5 mL) and 3-chloropropionyl
chloride (1.2 mL, 12 mmol) dropwise with stirring in ice-bath.
The reaction mixture was stirred at room temperature under
nitrogen for 24 h, and then poured into ice water (200 mL). The
resulting precipitate was collected and purified by crystallization
from hot ethanol.
6.2.11. 2-((Methylpropylamino)methyl)-1H-anthra[1,2-d]
imidazole-6,11-dione (5)
The pure compound was obtained as yellow powder (yield 43%).
Mp 141e142 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 0.97 (t, J ¼ 7.4 Hz,
3H), 1.59e1.61 (m, 2H), 2.42 (s, 3H), 2.55 (t, J ¼ 7.5 Hz, 2H), 3.98 (s,
2H), 7.76e7.79 (m, 2H), 7.99 (d, J ¼ 8.4 Hz, 1H), 8.16 (d, J ¼ 8.4 Hz,
1H), 8.24e8.31 (m, 2H). 13C NMR (75 MHz, CDCl3):
d ppm 11.54,
20.22, 42.64, 55.90, 59.87, 118.34, 121.49, 125.44, 126.60, 127.56,
128.67, 132.45,133.46, 133.80,134.04,134.30,149.06,158.76,182.98,
184.84. HRMS (ESI) m/z calcd for [M]þ: 333.1477, found: 334.1579
[M þ H]þ, 356.1405 [M þ Na]þ.
6.2.5. General procedure (V): preparation of compound 35
Compound 34 (2.10 g, 5 mmol) was added to a 50% H2SO4
solution (10 mL) at 0 ꢂC, and the reaction mixture was then heated
at 110 ꢂC for 2 h in mini-reactor. After cooling, the reaction mixture
was extracted with dichloromethane, dried over MgSO4, and the
solvent was removed in vacuo. The formed precipitate was filtered
off and dried in vacuo, washed with acetone.
6.2.12. 2-((Ethylisopropylamino)methyl)-1H-anthra[1,2-d]
imidazole-6,11-dione (6)
The pure compound was obtained as yellow powder (yield 47%).
Mp 165e166 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 1.14 (d, J ¼ 6.6 Hz,
6.2.6. General procedure (VI): preparation of compounds 36e40
To a solution of compound 35 (1.24 g, 4 mmol) in THF 30 mL was
added DIPEA (1.4 mL, 8 mmol) and series amine (8 mmol) dropwise
with stirring. The reaction mixture was refluxed for 6 h under
nitrogen. The mixture was concentrated and extracted with ethyl
acetate, dried over MgSO4, and the solvent was removed in vacuo.
The formed precipitate was filtered off and dried in vacuo. Recrys-
tallization from hexane and ethyl acetate, washed with acetone.
6H), 2.32 (s, 3H), 2.96e3.04 (m, 1H), 3.93 (s, 2H), 7.75e7.83 (m, 2H),
8.01 (d, J ¼ 8.4 Hz, 1H), 8.19 (d, J ¼ 8.1 Hz,1H), 8.17e8.33 (m, 2H). 13C
NMR(75 MHz, CDCl3):d ppm18.04, 37.91, 51.99, 54.15,118.26,121.49,
125.41, 126.54, 127.62, 128.57, 132.45, 133.58, 133.79, 134.21, 134.32,
149.52, 160.61, 183.03, 185.12. HRMS (ESI) m/z calcd for [M]þ:
333.1477, found: 334.1578 [M þ H]þ, 356.1402 [M þ Na]þ.
6.2.13. 2-((N-Methyl-2-(pyridin-2-yl)ethanamino)methyl)-1H-
anthra[1,2-d]imidazole-6,11-dione (7)
6.2.7. 2-(Chloromethyl)-1H-anthra[1,2-d]imidazole-6,11-dione (1)
The pure compound was obtained as yellow powder (yield 48%).
The pure compound was obtained as yellow powder (yield 65%).
Mp 150e151 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.44 (s, 3H),
Mp 272e273 ꢂC [13]. 1H NMR (300 MHz, CDCl3):
d
ppm 4.92 (s, 2H),
7.80e7.83 (m, 2H), 8.08 (d, J ¼ 8.4 Hz, 1H), 8.24 (d, J ¼ 8.4 Hz, 1H),
8.26e8.35 (m, 2H), 11.21 (br, 1H). 13C NMR (75 MHz, CDCl3):
ppm
3.01e3.11 (m, 4H), 4.00 (s, 2H), 7.13e7.17 (m, 1H), 7.21 (d, J ¼ 7.5 Hz,
1H), 7.61e7.67 (m, 1H), 7.76e7.80 (m, 2H), 7.99 (d, J ¼ 8.4 Hz, 1H),
8.16 (d, J ¼ 8.7 Hz, 1H), 8.25e8.32 (m, 2H), 8.63e8.65 (m, 1H). 13C
d
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, 183.83. HRMS (EI) m/
z: calcd for [M]þ: 296.0353, found: 296.0344.
NMR (75 MHz, CDCl3): d ppm 36.17, 42.99, 55.51, 57.49, 118.34,
121.41, 121.44, 123.28, 125.37, 126.54, 127.54, 128.59, 132.56, 133.50,
133.77, 134.04, 134.26, 136.61, 149.08, 149.67, 159.35, 160.11, 183.06,
184.73. HRMS (EI) m/z calcd for [M]þ: 396.1586, found: 396.1584.
6.2.8. 2-((Dimethylamino)methyl)-1H-anthra[1,2-d]imidazole-
6,11-dione (2)
6.2.14. 2-(((1,3-Dioxolan-2-yl)-N-methylmethanamino)methyl)-
1H-anthra[1,2-d] imidazole-6,11-dione (8)
The pure compound was obtained as yellow powder (yield 40%).
Mp 171e172 ꢂC.1H NMR (300 MHz, CDCl3):
d
ppm 2.53 (s, 6H), 4.02 (s,
2H), 7.76e7.80 (m, 2H), 7.95 (d, J ¼ 8.4 Hz,1H), 8.13 (d, J ¼ 8.4 Hz,1H),
8.18e8.32 (m, 2H). 13C NMR (75 MHz, CDCl3):
ppm 44.94, 56.69,
The pure compound was obtained as yellow powder (yield 59%).
Mp 161e162 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.56 (s, 3H), 2.79
d
(d, J ¼ 4.2 Hz), 3.98e4.03 (m, 2H), 4.09 (s, 2H), 4.13e4.17 (m, 2H),
5.15 (t, J ¼ 4.2 Hz, 1H), 7.77e7.80 (m, 2H), 8.02 (d, J ¼ 8.4 Hz, 1H),
8.20 (d, J ¼ 8.4 Hz, 1H), 8.26e8.34 (m, 2H). 13C NMR (75 MHz,
117.86, 120.92, 124.98, 126.01, 126.98, 128.25, 131.93, 132.80, 133.23,
133.41,133.74,148.21,156.80,182.34,184.08. HRMS (ESI) m/z calcd for
[M]þ: 305.1164, found: 306.1264 [M þ H]þ, 328.1803[M þ Na]þ.
CDCl3):
d ppm 44.50, 56.03, 59.41, 65.09, 102.96, 118.39, 121.36,

C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
37
125.24, 126.50, 127.46, 128.50, 132.33, 133.48, 133.67, 133.98, 134.15,
6.2.20. 2-((4-(Trifluoromethyl)benzylamino)methyl)-1H-anthra
[1,2-d]imidazole-6,11-dione (14)
The pure compound was obtained as yellow powder (yield 40%).
Mp 158e159 ꢂC. 1H NMR (300 MHz, CDCl3):
ppm 4.00 (s, 2H), 4.22
149.20, 158.92, 183.03, 184.57. HRMS (EI) m/z calcd for [M]þ:
377.1376, found: 377.1395.
d
6.2.15. 2-((3-Methoxy-N-methybenzylamino)methyl)-1H-anthra
[1,2-d]imidazole-6,11-dione (9)
(s, 2H), 7.52 (d, J ¼ 7.8 Hz, 2H), 7.60 (d, J ¼ 8.1 Hz, 2H), 7.78e7.84 (m,
2H), 8.03 (d, J ¼ 8.4 Hz,1H), 8.21 (d, J ¼ 8.1 Hz,1H), 8.25e8.37 (m, 2H).
The pure compound was obtained as yellow powder (yield 39%).
13C NMR (75 MHz, CDCl3):
d ppm 37.84, 46.96, 53.28, 118.61, 121.64,
Mp 150e151 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 2.41 (s, 3H), 3.68
121.98, 125.50, 125.63, 126.27, 126.69, 127.72, 128.60, 129.42, 132.76,
133.29, 133.85, 133.98, 134.43, 134.59, 148.41, 154.59, 182.79, 184.99.
HRMS (ESI) m/z calcd for [M]þ: 435.1195, found: 436.1318 [M þ H]þ,
458.1143 [M þ Na]þ.
(s, 2H), 3.86 (s, 3H), 3.95 (s, 2H), 6.80e6.83 (m, 1H), 6.99e7.02 (m,
2H), 7.27 (t, J ¼ 8.4 Hz, 1H), 7.78e7.82 (m, 2H), 8.02 (d, J ¼ 8.4 Hz,
1H), 8.19 (d, J ¼ 8.4 Hz, 1H), 8.26e8.34 (m, 2H). 13C NMR (75 MHz,
CDCl3):
d ppm 43.22, 55.19, 55.26, 62.40, 113.62, 114.39, 118.30,
121.48, 121.53, 125.55, 126.59, 127.66, 128.75, 129.67, 132.61, 133.40,
133.58, 133.80, 134.22, 134.35, 149.17, 159.03, 160.23, 183.00, 185.04.
HRMS (ESI) m/z calcd for [M]þ: 411.1583, found: 412.1701 [M þ H]þ,
434.1519 [M þ Na]þ.
6.2.21. 2-((Piperonylamino)methyl)-1H-anthra[1,2-d]imidazole-
6,11-dione (15)
The pure compound was obtained as yellow powder (yield 50%).
Mp 166e167 ꢂC.1H NMR (300 MHz, CDCl3):
d ppm 3.83 (s, 2H), 4.18 (s,
2H), 5.92 (s, 2H), 6.74e7.94 (m, 2H), 6.89 (s, 1H), 7.78e7.80 (m, 2H),
6.2.16. 2-((Di-(2-picolyl)amino)methyl)-1H-anthra[1,2-d]
imidazole-6,11-dione (10)
8.00 (d, J ¼ 8.1 Hz, 1H), 8.18 (d, J ¼ 8.4 Hz, 1H), 8.24e8.33 (m, 2H). 13C
NMR (75 MHz, CDCl3):
d ppm 46.74, 53.71, 101.03, 108.31, 108.81,
The pure compound was obtained as yellow powder (yield 45%).
118.26, 121.55, 121.61, 125.44, 126.60, 127.62, 128.62, 132.42, 133.28,
133.52, 133.79, 134.17, 134.35, 147.10, 148.13, 149.23, 159.86, 182.98,
184.99. HRMS (EI) m/z calcd for [M]þ: 435.1219, found: 435.1218.
Mp 175e176 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 4.04 (s, 4H), 4.20
(s, 2H), 7.25e7.29 (m, 2H), 7.46 (d, J ¼ 7.5 Hz, 2H), 7.67e7.73 (m, 2H),
7.78e7.81 (m, 2H), 8.03 (d, J ¼ 8.4 Hz, 1H), 8.21 (d, J ¼ 8.4 Hz, 1H),
8.31e8.39 (m, 2H), 8.85e8.87 (m, 2H). 13C NMR (75 MHz, CDCl3):
6.2.22. 2-((Piperidin-1-yl)methyl)-1H-anthra[1,2-d]imidazole-6,11-
dione (16)
d
ppm 51.87, 59.73, 118.92, 121.32, 122.71, 123.86, 125.02, 126.68,
127.43, 128.64, 132.61, 133.68, 134.02, 134.06, 137.25, 149.03, 149.26,
149.58, 158.07, 159.30, 183.315, 184.44. HRMS (EI) m/z calcd for
[M]þ: 459.1695, found: 459.1700.
The pure compound was obtained as yellow powder (yield 58%).
Mp 204e205 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 1.50e1.53 (m,
2H), 1.65e1.72 (m, 4H), 2.54 (t, J ¼ 5.1 Hz, 4H), 3.86 (s, 2H), 7.79e
7.82 (m, 2H), 8.04 (d, J ¼ 8.4 Hz, 1H), 8.21 (d, J ¼ 8.4 Hz, 1H),
6.2.17. 2-(((Anthracen-10-yl)-N-methylmethanamino)methyl)-1H-
anthra[1,2-d] imidazole-6,11-dione (11)
8.27e8.36 (m, 2H). 13C NMR (75 MHz, CDCl3):
d ppm 23.66, 25.62,
54.98, 56.92, 118.44, 121.51, 125.51, 126.65, 127.59, 128.79, 132.61,
133.55, 133.82, 134.12, 134.32, 149.07, 158.11, 183.03, 184.92. HRMS
(EI) m/z calcd for [M]þ: 345.1477, found: 345.1468.
The pure compound was obtained as yellow powder (yield 66%).
Mp 189e190 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.55 (s, 3H), 3.97
(s, 2H), 4.69 (s, 2H), 7.42e7.47 (m, 2H), 7.60e7.66 (m, 2H), 7.45 (d,
J ¼ 7.8 Hz, 2H), 7.73e7.76 (m, 2H), 7.85 (d, J ¼ 8.4 Hz, 1H), 7.92 (d,
J ¼ 8.4 Hz, 2H), 8.07 (d, J ¼ 8.1 Hz, 1H), 8.19e8.27 (m, 2H), 8.32 (s,
6.2.23. 2-((4-Methylpiperidin-1-yl)methyl)-1H-anthra[1,2-d]
imidazole-6,11-dione (17)
1H), 8.51 (d, J ¼ 9.3 Hz, 2H). 13C NMR (75 MHz, CDCl3):
d
ppm 43.60,
The pure compound was obtained as yellow powder (yield 63%).
53.72, 54.42, 118.00, 121.27, 124.29, 125.09, 126.50, 126.55, 127.46,
128.24, 128.39, 129.25, 131.37, 131.46, 132.22, 133.38, 133.68, 133.93,
134.15, 148.78, 158.99, 182.59, 184.37. HRMS (EI) m/z calcd for [M]þ:
481.1790, found: 481.1790.
Mp 208e209 ꢂC.1H NMR (300 MHz, CDCl3):
d
ppm 0.97 (d, J ¼ 5.7 Hz,
3H),1.36e1.43 (m, 3H),1.68 (d, J ¼ 9.9 Hz, 2H), 2.23 (t, J ¼ 11.7 Hz, 2H),
2.91 (d, J ¼ 11.7 Hz, 2H), 3.89 (s, 2H), 7.61e7.83 (m, 2H), 8.03 (d,
J ¼ 8.4 Hz, 1H), 8.20 (d, J ¼ 8.4 Hz, 1H), 8.25e8.34 (m, 2H). 13C NMR
(75 MHz, CDCl3):dppm 21.57, 30.33, 34.22, 54.54, 56.81,118.35,121.51,
6.2.18. 2-((2-(Trifluoromethyl)benzylamino)methyl)-1H-anthra
[1,2-d]imidazole-6,11-dione (12)
125.53, 126.60, 127.64, 128.73, 132.61, 133.60, 133.80, 134.22, 134.33,
149.26, 159.03, 183.02, 185.09. HRMS (ESI) m/z calcd for [M]þ:
359.1634, found: 360.1742 [M þ H]þ, 382.1562 [M þ Na]þ.
The pure compound was obtained as yellow powder (yield 33%).
Mp 143e144 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 4.09 (s, 2H), 4.21
6.2.24. 2-((Azepan-1-yl)methyl)-1H-anthra[1,2-d]imidazole-6,11-
dione (18)
(s, 2H), 7.39 (t, J ¼ 7.5 Hz, 1H), 7.57 (t, J ¼ 7.5 Hz, 1H), 7.65e7.68 (m,
2H), 7.71e7.81 (m, 2H), 7.98 (d, J ¼ 8.1 Hz, 1H), 8.13 (d, J ¼ 8.1 Hz,
1H), 8.16e8.31 (m, 2H). 13C NMR (75 MHz, CDCl3):
d ppm 47.01,
The pure compound was obtained as yellow powder (yield 53%).
Mp 183e184 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 1.72e1.81 (m,
50.11, 118.28, 121.57, 122.84, 125.48, 126.25, 126.33, 126.40, 126.48,
126.59, 127.59, 128.57, 128.65, 128.97, 130.86, 132.19, 132.39, 133.48,
133.77, 134.13, 134.33, 137.83, 149.19, 159.53, 182.93, 184.96. HRMS
(EI) m/z calcd for [M]þ: 435.1195, found: 435.1189.
8H), 2.92 (t, J ¼ 5.3 Hz, 4H), 4.19 (s, 2H), 7.77e7.80 (m, 2H), 8.00 (d,
J ¼ 8.4 Hz, 1H), 8.18 (d, J ¼ 8.4 Hz, 1H), 8.27e8.33 (m, 2H). 13C NMR
(75 MHz, CDCl3):
d ppm 26.81, 28.15, 56.33, 56.50, 118.16, 121.42,
125.37, 126.55, 127.54, 128.49, 132.37, 133.42, 133.76, 134.02, 134.28,
149.19, 159.78, 182.96, 184.96. HRMS (ESI) m/z calcd for [M]þ:
359.1634, found: 360.1746 [M þ H]þ, 382.1559 [M þ Na]þ.
6.2.19. 2-((3-(Trifluoromethyl)benzylamino)methyl)-1H-anthra
[1,2-d]imidazole-6,11-dione (13)
The pure compound was obtained as yellow powder (yield 38%).
6.2.25. 2-((1,4-Dioxa-8-azaspiro[4.5]decane-8-yl)methyl)-1H-
anthra[1,2-d]imidazole-6,11-dione (19)
Mp 155e156 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 4.00 (s, 2H), 4.23
(s, 2H), 7.45e7.54 (m, 2H), 7.60 (d, J ¼ 6.9 Hz, 1H), 7.66 (s, 1H), 7.82e
The pure compound was obtained as yellow powder (yield 60%).
7.85 (m, 2H), 8.04 (d, J ¼ 8.4 Hz, 1H), 8.22 (d, J ¼ 8.4 Hz, 1H), 8.26e
Mp 219e220 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 1.87 (t,
8.36 (m, 2H). 13C NMR (75 MHz, CDCl3):
d ppm 46.98, 53.35, 118.27,
J ¼ 5.6 Hz, 4H), 2.78 (t, J ¼ 5.4 Hz, 4H), 3.97 (s, 4H), 4.01 (s, 2H),
121.60, 122.38, 124.32, 124.37, 124.41, 124.46, 124.99, 125.04, 125.10,
125.14,125.50, 125.98, 126.59, 127.61, 128.69, 131.71, 132.40, 133.43,
133.82, 134.10, 134.38, 140.28, 149.03, 159.17, 182.92, 184.97. HRMS
(EI) m/z calcd for [M]þ: 435.1195, found: 435.1190.
7.76e7.79 (m, 2H), 7.99 (d, J ¼ 8.4 Hz, 1H), 8.16 (d, J ¼ 8.4 Hz, 1H),
8.22e8.31 (m, 2H). 13C NMR (75 MHz, CDCl3):
d ppm 34.47, 51.92,
55.77, 64.37, 106.36, 118.48, 121.54, 125.57, 126.66, 127.57, 128.87,

38
C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
132.60, 133.45, 133.85, 134.03, 134.33, 148.88, 182.97, 184.81. HRMS
2H), 7.75e7.77 (m, 2H), 7.99 (d, J ¼ 8.4 Hz, 1H), 8.14 (d, J ¼ 8.4 Hz,
1H), 8.19e8.29 (m, 2H). 13C NMR (75 MHz, CDCl3):
ppm 11.75,
(EI) m/z calcd for [M]þ: 403.1532, found: 403.1530.
d
52.13, 52.55, 53.49, 56.35, 118.17, 121.48, 125.57, 126.49, 127.57,
128.62, 132.48, 133.36, 133.79, 134.00, 134.35, 148.97, 158.31, 182.88,
185.01. HRMS (EI) m/z calcd for [M]þ: 374.1743, found: 374.1736.
6.2.26. 2-((4-(3-(Piperidin-4-yl)propyl)piperidin-1-yl)methyl)-1H-
anthra[1,2-d] imidazole-6,11-dione (20)
The pure compound was obtained as yellow powder (yield 40%).
Mp 172e173 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 1.04e1.28 (m,
6.2.32. 2-((4-Allylpiperazin-1-yl)methyl)-1H-anthra[1,2-d]
imidazole-6,11-dione (26)
12H), 1.64e1.68 (m, 4H), 2.14e2.21 (m, 2H), 2.54e2.62 (m, 2H), 2.90
(d, J ¼ 11.4 Hz, 2H), 3.07 (d, J ¼ 12.3 Hz, 2H), 3.86 (s, 2H), 4.22 (br,
1H), 7.74e7.81 (m, 2H), 8.00 (d, J ¼ 8.4 Hz, 1H), 8.17 (d, J ¼ 8.4 Hz,
The pure compound was obtained as yellow powder (yield
54%). Mp 175e176 ꢂC. 1H NMR (300 MHz, CDCl3)
d ppm 2.81 (s,
1H), 8.20e8.32 (m, 2H). 13C NMR (75 MHz, CDCl3):
d ppm 23.49,
8H), 3.22 (d, J ¼ 6.6 Hz, 2H), 3.97 (s, 2H), 5.27e5.33 (m, 2H), 5.97
(m, 1H), 7.77e7.84 (m, 2H), 8.04 (d, J ¼ 8.4 Hz, 1H), 8.21 (d,
J ¼ 8.7 Hz, 1H), 8.23e8.35 (m, 2H). 13C NMR (75 MHz, CDCl3):
32.34, 33.26, 35.29, 36.02, 36.56, 37.22, 46.53, 54.60, 56.86, 118.20,
121.44, 125.46, 126.52, 127.59, 128.57, 132.51, 133.44, 133.80, 134.05,
134.33, 149.10, 159.17, 182.99, 185.05. HRMS (EI) m/z calcd for [M]þ:
470.2682, found: 470.2689.
d
ppm 29.60, 52.37, 52.62, 56.10, 61.02, 118.34, 120.25, 121.64,
125.76, 126.57, 127.69, 128.86, 132.58, 133.46, 133.86, 134.13,
134.46, 149.03, 157.66, 182.93, 185.16. HRMS (EI) m/z calcd for
[M]þ: 386.1743, found: 386.1734.
6.2.27. 2-(Morpholinomethyl)-1H-anthra[1,2-d]imidazole-6,11-
dione (21)
The pure compound was obtained as yellow powder (yield 55%).
Mp 241e242 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 2.64 (t,
6.2.33. 2-((4-Phenylpiperazin-1-yl)methyl)-1H-anthra[1,2-d]
imidazole-6,11-di-one (27)
J ¼ 4.5 Hz, 4H), 3.81 (t, J ¼ 4.7 Hz, 4H), 3.93 (s, 2H), 7.76e7.80 (m,
2H), 8.00 (d, J ¼ 8.4 Hz, 1H), 8.16 (d, J ¼ 8.4 Hz, 1H), 8.21e8.31 (m,
The pure compound was obtained as yellow powder (yield 68%).
2H). 13C NMR (75 MHz, CDCl3):
d
ppm 53.90, 56.74, 66.72, 118.26,
Mp 212e213 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.82 (t,
121.57, 125.65, 126.54, 127.61, 128.77, 132.52, 133.35, 133.85, 134.00,
134.42, 148.84, 157.44, 182.87, 185.02. HRMS (ESI) m/z calcd for
[M]þ: 347.1270, found: 348.1379 [M þ H]þ, 370.1198 [M þ Na]þ.
J ¼ 4.8 Hz, 4H), 3.31 (t, J ¼ 4.8 Hz, 4H), 4.02 (s, 2H), 6.88e6.95 (m,
3H), 7.24e7.30 (m, 2H), 7.76e7.79 (m, 2H), 8.07 (d, J ¼ 8.7 Hz, 1H),
8.17 (d, J ¼ 8.1 Hz,1H), 8.20e8.31 (m, 2H). 13C NMR (75 MHz, CDCl3):
d
ppm 49.06, 53.52, 56.30, 116.35, 118.28, 120.15, 121.54, 125.60,
6.2.28. 2-(Thiomorpholinomethyl)-1H-anthra[1,2-d]imidazole-
6,11-dione (22)
126.54, 127.57, 128.74, 129.22, 132.52, 133.33, 133.84, 133.96, 134.38,
148.85, 151.12, 157.58, 182.88, 184.93. HRMS (EI) m/z calcd for [M]þ:
422.1743, found: 422.1744.
The pure compound was obtained as yellow powder (yield 59%).
Mp 216e217 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 2.82 (t, J ¼ 4.7 Hz,
4H), 2.96 (t, J ¼ 4.8 Hz, 4H), 4.02 (s, 2H), 7.78e7.81 (m, 2H), 8.01 (d,
6.2.34. 2-((4-(Pyridin-2-yl)piperazin-1-yl)methyl)-1H-anthra[1,2-
d]imidazole-6,11-dione (28)
J ¼ 8.4 Hz, 1H), 8.18 (d, J ¼ 8.7 Hz, 1H), 8.23e8.33 (m, 2H). 13C NMR
(75 MHz, CDCl3):
d ppm 27.56, 55.32, 56.93, 118.45, 121.64, 125.67,
The pure compound was obtained as yellow powder (yield 63%).
Mp 200e201 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.80 (t,
126.65, 127.64,128.95, 132.59,133.44,133.89, 134.06,134.42,148.87,
156.97, 182.91, 184.96. HRMS (ESI) m/z calcd for [M]þ: 363.1041,
found: 364.1141 [M þ H]þ, 386.0907 [M þ Na]þ.
J ¼ 4.8 Hz, 4H), 3.69 (t, J ¼ 5.0 Hz, 4H), 4.04 (s, 2H), 6.64e6.68 (m,
2H), 7.48e7.51 (m, 1H), 7.77e7.80 (m, 2H), 8.03 (d, J ¼ 8.4 Hz, 1H),
8.33e8.17 (m, 3H). 13C NMR (75 MHz, CDCl3):
d ppm 45.17, 53.36,
6.2.29. 2-((Thiazolidin-3-yl)methyl)-1H-anthra[1,2-d]imidazole-
6,11-dione (23)
56.42, 107.23, 113.64, 118.34, 121.57, 125.64, 126.57, 127.60, 128.82,
132.58, 133.42, 133.83, 134.06, 134.38, 137.67, 148.00, 148.96, 157.66,
159.37, 182.90, 184.99. HRMS (EI) m/z calcd for [M]þ: 423.1695,
found: 423.1694.
The pure compound was obtained as yellow powder (yield 48%).
Mp 195e196 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 3.10 (t, J ¼ 6.5 Hz,
2H), 3.31 (t, J ¼ 6.5 Hz, 2H), 4.13 (s, 2H), 4.24 (s, 2H), 7.79e7.83 (m,
2H), 8.01 (d, J ¼ 8.4 Hz, 1H), 8.19 (d, J ¼ 8.4 Hz, 1H), 8.23e8.35 (m,
6.2.35. 2-((4-(Pyrimidin-2-yl)piperazin-1-yl)methyl)-1H-anthra
[1,2-d]imidazole-6,11-dione (29)
2H). 13C NMR (75 MHz, CDCl3):
d ppm 29.47, 52.11, 58.09, 60.86,
118.41, 121.69, 125.79, 126.59, 127.69, 128.92, 132.60, 133.49, 133.86,
134.16, 134.45, 149.07, 158.11, 182.92, 185.12. HRMS (ESI) m/z calcd
for [M]þ: 349.0885, found: 350.0999 [M þ H]þ, 372.0816 [M þ Na]þ.
The pure compound was obtained as yellow powder (yield 59%).
Mp 223e224 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.70 (t,
J ¼ 5.0 Hz, 4H), 3.95 (t, J ¼ 5.0 Hz, 4H), 3.99 (s, 2H), 6.51 (t, J ¼ 4.8 Hz,
1H), 7.79e7.82 (m, 2H), 8.06 (d, J ¼ 8.4 Hz, 1H), 8.22 (d, J ¼ 8.7 Hz,
6.2.30. 2-((4-Methylpiperazin-1-yl)methyl)-1H-anthra[1,2-d]
imidazole-6,11-di-one (24)
1H), 8.26e8.35 (m, 4H). 13C NMR (75 MHz, CDCl3):
d ppm. 43.58,
53.48, 56.56, 110.18, 118.34, 121.60, 125.69, 126.59, 127.65, 128.82,
132.61, 133.44, 133.87, 134.07, 134.43, 148.97, 157.85, 161.82, 182.96,
185.10. HRMS (EI) m/z calcd for [M]þ: 424.1648, found: 424.1652.
The pure compound was obtained as yellow powder (yield 70%).
Mp 231e232 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.37 (s, 3H),
2.61e2.69 (m, 8H), 3.92 (s, 2H), 7.76e7.79 (m, 2H), 8.00 (d,
J ¼ 8.4 Hz, 1H), 8.16 (d, J ¼ 8.4 Hz, 1H), 8.22e8.32 (m, 2H). 13C NMR
6.2.36. 2-((4-(2-Fluorophenyl)piperazin-1-yl)methyl)-1H-anthra
[1,2-d]imidazole-6,11-dione (30)
(75 MHz, CDCl3):
d ppm 45.64, 53.27, 54.85, 56.28, 118.26, 121.54,
125.66, 126.53, 127.63, 128.75, 132.55, 133.47, 133.80, 134.16, 134.38,
149.07, 158.18, 182.89, 185.10. HRMS (EI) m/z calcd for [M]þ:
360.1586, found: 360.1585.
The pure compound was obtained as yellow powder (yield 67%).
Mp 163e164 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.89 (t,
J ¼ 4.4 Hz, 4H), 3.25 (t, J ¼ 4.7 Hz, 4H), 4.08 (s, 2H), 6.94e7.07 (m,
4H), 7.77e7.80 (m, 2H), 8.03 (d, J ¼ 8.4 Hz, 1H), 8.18 (d, J ¼ 8.4 Hz,
6.2.31. 2-((4-Ethylpiperazin-1-yl)methyl)-1H-anthra[1,2-d]
imidazole-6,11-dione (25)
1H), 8.23e8.33 (m, 4H). 13C NMR (75 MHz, CDCl3):
d ppm 50.17,
53.61, 56.23, 116.13, 116.40, 118.39, 119.21, 119.25, 121.59, 122.82,
122.92, 124.55, 124.60, 125.67, 126.59, 127.61, 128.85, 132.58, 133.41,
133.84, 134.03, 134.39, 139.74, 139.86, 148.90, 154.29, 157.56, 182.91,
184.96. HRMS (EI) m/z calcd for [M]þ: 440.1649, found: 440.1643.
The pure compound was obtained as yellow powder (yield 64%).
Mp 204e205 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 1.09 (t,
J ¼ 7.2 Hz, 3H), 2.46 (q, J ¼ 7.2 Hz, 2H), 2.58e2.67 (m, 8H), 3.91 (s,

C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
39
6.2.37. 2-((4-(2-Cyanophenyl)piperazin-1-yl)methyl)-1H-anthra
[1,2-d]imidazole-6,11-dione (31)
6.2.43. 2-(2-(4-Phenylpiperazin-1-yl)ethyl)-1H-anthra[1,2-d]
imidazole-6,11-dione (37)
The pure compound was obtained as yellow powder (yield 67%).
Mp 211e212 ꢂC. 1H NMR (300 MHz, CDCl3):
ppm 2.95 (s, 4H), 3.09
The pure compound was obtained as yellow powder (yield 45%).
Mp 230e231 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 2.94 (t,
d
J ¼ 4.2 Hz, 4H), 3.37 (t, J ¼ 4.7 Hz, 4H), 4.10 (s, 2H), 7.47e7.59 (m,
(s, 2H), 3.33 (t, J ¼ 5.6 Hz, 2H), 3.56 (t, J ¼ 4.8 Hz, 4H), 6.93 (t,
J ¼ 7.4 Hz, 1H), 7.05 (d, J ¼ 7.8 Hz, 2H), 7.31e7.37 (m, 2H), 7.73e7.77
(m, 2H), 7.93 (d, J ¼ 8.4 Hz,1H), 8.14 (d, J ¼ 8.4 Hz,1H), 8.18e8.31 (m,
2H), 7.77e7.80 (m, 2H), 8.02 (d, J ¼ 8.4 Hz, 1H), 8.18 (d, J ¼ 8.4 Hz,
1H), 8.22e8.32 (m, 4H). 13C NMR (75 MHz, CDCl3):
d ppm 51.15,
53.53, 56.09, 106.59, 118.22, 118.48, 119.03, 121.67, 122.37, 125.81,
126.63, 127.67, 129.00, 132.65, 133.48, 133.87, 133.91, 134.12, 134.43,
134.47, 134.68, 148.94, 155.35, 182.93, 185.03. HRMS (EI) m/z calcd
for [M]þ: 447.1695, found: 447.1712.
2H). 13C NMR (75 MHz, CDCl3):
d ppm. 25.34, 49.22, 52.93, 55.07,
116.47, 117.39, 118.25, 120.02, 121.34, 124.89, 126.75, 127.43, 128.30,
129.30, 129.49, 132.39, 133.57, 133.65, 134.11, 148.77, 151.36, 183.19,
184.50. HRMS (EI) m/z calcd for [M]þ: 436.1899, found: 436.1896.
6.2.38. 2-((4-(3-Methoxyphenyl)piperazin-1-yl)methyl)-1H-anthra
[1,2-d]imidazole-6,11-dione (32)
6.2.44. 2-(2-(4-(Pyridin-2-yl)piperazin-1-yl)ethyl)-1H-anthra[1,2-
d]imidazole-6,11-dione (38)
The pure compound was obtained as yellow powder (yield 58%).
The pure compound was obtained as yellow powder (yield 65%).
Mp 150e151 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 2.89 (s, 4H), 3.35
Mp 234e235 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.87 (s, 4H), 3.02
(t, J ¼ 4.8 Hz, 4H), 3.79 (s, 3H), 4.13 (s, 2H), 6.43e6.56 (m, 3H), 7.18
(t, J ¼ 5.3 Hz, 2H), 3.31 (t, J ¼ 5.9 Hz, 2H), 3.93 (s, 4H), 6.69 (t, J ¼ 6 Hz,
1H), 6.76(d, J¼ 8.4 Hz,1H), 7.53e7.58 (m,1H), 7.73e7.77 (m, 2H), 7.99
(d, J ¼ 8.4 Hz,1H), 8.17 (d, J ¼ 8.4 Hz,1H), 8.22e8.32 (m, 3H).13C NMR
(t, J ¼ 8.1 Hz, 1H), 7.77e7.80 (m, 2H), 8.02 (d, J ¼ 8.1 Hz, 1H), 8.19 (d,
J ¼ 8.4 Hz, 1H), 8.23e8.33 (m, 4H). 13C NMR (75 MHz, CDCl3):
d ppm
48.80, 53.36, 55.18, 56.09, 103.06, 105.21, 109.23, 118.51, 121.63,
125.68, 126.67, 127.61, 128.97, 129.99, 132.63, 133.42, 133.88, 134.03,
134.39,148.80, 160.88, 182.94, 184.86. HRMS (EI) m/z calcd for [M]þ:
452.1848, found: 452.1846.
(75 MHz, CDCl3): d ppm. 25.21, 45.05, 52.68, 55.10, 107.28, 113.63,
118.26, 121.37, 124.92, 126.76, 127.46, 128.32, 132.39, 133.54, 133.68,
134.07, 134.14, 137.80, 148.08, 148.72, 159.32, 160.07, 183.20, 184.51.
HRMS (EI) m/z calcd for [M]þ: 437.1852, found: 437.1856.
6.2.39. 2-((4-Benzylpiperazin-1-yl)methyl)-1H-anthra[1,2-d]
imidazole-6,11-dione (33)
6.2.45. 2-(2-(4-(Pyrimidin-2-yl)piperazin-1-yl)ethyl)-1H-anthra
[1,2-d]imidazole-6,11-dione (39)
The pure compound was obtained as yellow powder (yield 62%).
The pure compound was obtained as yellow powder (yield 61%).
Mp 194e195 ꢂC. 1H NMR (300 MHz, CDCl3):
d
ppm 2.64e2.69 (m,
Mp 230e231 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.81 (s, 4H), 3.01
8H), 3.60 (s, 2H), 3.92 (s, 2H), 7.27e7.36 (m, 5H), 7.76e7.80 (m, 2H),
(t, J ¼ 5.6 Hz, 2H), 3.30 (t, J ¼ 5.6 Hz, 2H), 4.20 (s, 4H), 6.54 (t,
J ¼ 4.7 Hz, 1H), 7.73e7.76 (m, 2H), 7.97 (d, J ¼ 8.4 Hz, 1H), 8.16 (d,
J ¼ 8.4 Hz, 1H), 8.22e8.31 (m, 2H), 8.36 (d, J ¼ 4.8 Hz, 2H). 13C NMR
8.00 (d, J ¼ 8.1 Hz,1H), 8.16 (d, J ¼ 8.4 Hz,1H), 8.22e8.31 (m, 4H). 13C
NMR (75 MHz, CDCl3):
d ppm. 52.84, 53.50, 56.32, 62.81, 118.17,
121.48, 125.55, 126.49, 127.27, 127.57, 128.34, 128.61, 129.26, 132.46,
133.36, 133.78, 134.00, 134.35, 137.78, 148.98, 158.34, 182.87, 185.00.
HRMS (EI) m/z calcd for [M]þ: 436.1899, found: 436.1902.
(75 MHz, CDCl3): d ppm. 25.20, 43.57, 52.77, 55.10, 110.15, 118.22,
121.34, 124.89, 126.72, 127.45, 128.27, 132.35, 133.55, 133.66, 134.06,
134.13, 148.71, 157.93, 160.27, 161.81, 183.19, 184.51. HRMS (EI) m/z
calcd for [M]þ: 438.1804, found: 438.1806.
6.2.40. 1,2-Bis-(3-chloropropionamido)anthraquinone (34)
The pure compound was obtained as yellow powder (yield 51%).
6.2.46. 2-(2-(4-Benzylpiperazin-1-yl)ethyl)-1H-anthra[1,2-d]
imidazole-6,11-dione (40)
Mp 179e180 ꢂC [22]. 1H NMR (300 MHz, DMSO-d6):
d ppm 2.94e
3.03 (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),
The pure compound was obtained as yellow powder (yield 51%).
Mp 177e178 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.70 (s, 4H), 2.83
10.02 (s, 1H). 13C NMR (75 MHz, DMSO-d6):
d
ppm. 40.20, 40.26,
(s, 4H), 2.90 (t, J ¼ 6.0 Hz, 2H), 3.19 (t, J ¼ 6.0 Hz, 2H), 3.71 (s, 2H),
7.28e7.44 (m, 5H), 7.77e7.80 (m, 2H), 7.96 (d, J ¼ 8.4 Hz, 1H), 8.15
(d, J ¼ 8.4 Hz, 1H), 8.28e8.32 (m, 2H). 13C NMR (75 MHz, CDCl3):
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 for [M]þ: 418.0487, found: 418.0494.
d
ppm. 25.22, 52.21, 52.61, 54.71, 62.68, 118.19, 121.32, 124.91,
126.59,127.50,127.68,128.26,128.55,129.09,129.66,132.36,133.58,
133.63, 134.11, 134.16, 148.75, 160.29, 183.16, 184.48. HRMS (EI) m/z
calcd for [M]þ: 450.2056, found: 450.2050.
6.2.41. 2-(2-Chloroethyl)-1H-anthra[1,2-d]imidazole-6,11-dione
(35)
The pure compound was obtained as yellow powder (yield 68%).
Mp 261e262 ꢂC.1H NMR (400 MHz, CDCl3):
d
ppm 3.53 (t, J ¼ 6.4 Hz,
6.3. Cell cultures and assessment of hTERT
2H), 4.07 (t, J ¼ 6.4 Hz, 2H), 7.79e7.85 (m, 2H), 8.09 (d, J ¼ 8.4 Hz,1H),
8.25 (d, J ¼ 8.1 Hz,1H), 8.26e8.36 (m, 2H).13C NMR(100 MHz, DMSO-
Non-small lung cancercell H1299 was grown in RPMI1640 media
supplemented with 10% fetal bovine serum, 100 units/mL penicillin
and 100 mg/mL streptomycin in a humidified atmosphere with 5%
CO2 at 37 ꢂC. Culture media were changed every 3 days. To establish
stable cell lines 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 SEAP gene
and transfected 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. 31.79, 42.13, 118.24, 120.52, 120.99, 124.32, 125.44,
126.22, 126.78, 127.70, 132.94, 133.10, 134.25, 134.48, 158.75, 182.41,
183.27. HRMS (EI) m/z calcd for [M]þ: 310.0509, found: 310.0511.
6.2.42. 2-(2-(4-Methylpiperazin-1-yl)ethyl)-1H-anthra[1,2-d]
imidazole-6,11-dione (36)
The pure compound was obtained as yellow powder (yield 71%).
Mp 190e191 ꢂC. 1H NMR (300 MHz, CDCl3):
d ppm 2.54 (s, 3H), 2.70
(s, 4H), 2.89 (s, 4H), 2.93 (t, J ¼ 6.0 Hz, 2H), 3.20 (t, J ¼ 5.7 Hz, 2H),
7.75e7.78 (m, 2H), 7.96 (d, J ¼ 8.4 Hz, 1H), 8.15 (d, J ¼ 8.1 Hz, 1H),
8.16e8.31 (m, 2H). 13C NMR (75 MHz, CDCl3):
d
ppm. 25.26, 45.79,
6.4. Telomere repeat amplification protocol (TRAP assay)
52.43, 54.74, 54.90, 118.17, 121.31, 124.91, 126.57, 127.49, 128.23,
132.35, 133.62, 134.13, 148.78, 160.55, 183.19, 184.48. HRMS (EI) m/z
calcd for [M]þ: 374.1743, found: 374.1740.
Telomerase activity was detected by modifying version of the
TRAP protocol [27,28]. Telomerase products were resolved by 10%

40
C.-L. Chen et al. / European Journal of Medicinal Chemistry 60 (2013) 29e41
polyacrylamide gel electrophoresis and visualized by staining with
SYBER Green. As a source of telomerase, the total cell lysates
derived from lung cancer cell line (H1299) was used. Protein
concentration of the lysates was assayed using Bio-Rad protein
assay kit using BSA standards.
Acknowledgments
The present study was supported by National Science Council
Grants NSC 100-2113-M-016, 101-2113-M-016, 100-2311-B-010-
001, 98-2113-M-016-001, and National Health Research Institutes
grant NHRI EX100-10050SI. We are grateful to thank NIH-NCI for
their supports and to credit DTP of the NCI for performing the
screening of our compounds.
6.5. MTT assay for cell viability
The tetrazolium reagent (MTT: 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide, USB) was designed to yield
a colored formazan upon metabolic reduction by viable cells
[23,24]. Approximately 2 ꢃ 103 cells were plated onto each well of
a 96-well plate and incubated in 5% CO2 at 37 ꢂC 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 desig-
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.11.032.
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