Design, synthesis and evaluation of telomerase inhibitory, hTERT repressing, and anti-proliferation activities of symmetrical 1,8-disubstituted amidoanthraquinones

Article abstract of DOI:10.1016/j.ejmech.2012.01.044

A series of symmetrical disubstituded 1,8-diamidoanthraquinones were synthesized and evaluated for anti-proliferation, telomerase inhibitory by TRAP assay, and hTERT expression by SEAP assay. All of the compounds tested, except for compounds 3a and 3s were selected by the NCI screening system. In addition, compounds 4e and 4k exhibited inhibitory effects on telomerase activity. Taken together, our findings indicated that the analysis of cytotoxicity and telomerase inhibition might provide information applicable for further developing potential telomerase targeting strategy.

Full text of DOI:10.1016/j.ejmech.2012.01.044

European Journal of Medicinal Chemistry 50 (2012) 102e112
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and evaluation of telomerase inhibitory, hTERT repressing,
and anti-proliferation activities of symmetrical 1,8-disubstituted
amidoanthraquinones
,
Chia-Chung Lee a, Kuo-Feng Huang b, Deh-Ming Chang a c, Jung-Jung Hsu d, Fong-Chun Huang e,
,
Kai-Ning Shih e, Chun-Liang Chen a, Tsung-Chih Chen a, Ruei-Huei Chen f, Jing-Jer Lin e
,
**
,d,
Hsu-Shan Huang a
*
a Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan
b Chi-Mei Medical Center, Tainan 710, Taiwan
c Rheumatology/Immunology/Allergy, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan
d School of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan
e Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan
f Department of Basic Medical Science, HungKuang University, Taichung 433, 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 symmetrical disubstituded 1,8-diamidoanthraquinones were synthesized and evaluated for
anti-proliferation, telomerase inhibitory by TRAP assay, and hTERT expression by SEAP assay. All of the
compounds tested, except for compounds 3a and 3s were selected by the NCI screening system. In
addition, compounds 4e and 4k exhibited inhibitory effects on telomerase activity. Taken together, our
findings indicated that the analysis of cytotoxicity and telomerase inhibition might provide information
applicable for further developing potential telomerase targeting strategy.
Received 20 December 2011
Received in revised form
19 January 2012
Accepted 22 January 2012
Available online 1 February 2012
Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.
Keywords:
Telomerase
TRAP assay
Amidoanthraquinones
hTERT expression
SEAP assay
NCI 60-cell panel assay
1. Introduction
G-quadruplex [2]. The requirement for telomerase for proliferation
by most cancer cells is a very important target in anticancer drug
The anthracycline antibiotics daunorubicin, doxorubicin,
mitoxantrone and ametantrone have been used widely as anti-
cancer drugs for many decades, but their cardiotoxicity limits their
clinical applicability (Scheme 1) [1]. Side effects might be attrib-
utable to the molecular targeted agents, which represent “on-
target” modulation in normal tissues. The precise mechanism of
how these compounds, all of which contain an anthraquinone-
based pharmacophore, have remained elusive, although they
might rendered their effects through forming complexes with the
DNA base pairs by forming a complex with topoisomerase II or
design strategies [3]. Telomerase activity is over-expressed in over
85% of all known human tumors, whereas it is low or not present in
most somatic cells [4]. Telomerase is composed of an RNA subunit,
telomerase-associated protein 1, and the catalyst telomerase
reverse transcriptase (TERT), which is a catalytic subunit of telo-
merase in regulating the activity and key determinant for telome-
rase activity [5]. The close correlation between telomerase activity
and human telomerase reverse transcriptase (hTERT) expression
has caused hTERT to be considered as a selective molecular target
for human cancer therapy [6]. Insufficient hTERT expression in most
mortal somatic cells cannot produce enough telomerase to main-
tain telomere length during cycles of chromosome replication [7,8].
Thus, a potential therapeutic window exists in which cancer cells
can be efficiently targeted by telomerase inhibitors, while normal
telomerase-expressing cells, such as stem and germ-line cells,
remain unaffected as a result of their longer telomeres and slower
* Corresponding author. School of Pharmacy, National Defense Medical Center,
Taipei 114, Taiwan. Tel.: þ886 2 87923100x18179; fax: þ886 2 87923169.
** Corresponding author. Tel.: þ886 2 28267000x5603; fax: þ886 2 28267258.
E-mail addresses: jjlin@ym.edu.tw (J.-J. Lin), huanghs@ndmctsgh.edu.tw
(H.-S. Huang).
0223-5234/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.044

C.-C. Lee et al. / European Journal of Medicinal Chemistry 50 (2012) 102e112
103
Scheme 1. Structures of bioactive chromophoric anthraquinones.
rates of cell division [9e11]. Therefore, telomerase is an important
target for the design of therapeutic strategy for selectively targeting
that might have minimal side effects.
in Scheme 2e4. The starting material of 1,8-diaminoanthraquinone 2
was prepared by treatment of 1,8-dinitroanthraquinone 1 with Na2S
and refluxing for 6 h [20,21]. The acylated compounds 3 and 4 with
primary or secondary amines afforded the symmetrical compounds
3aet, and 4aek, respectively. The nucleophilic substitution between
In our studies, a series of anthraquinone derivatives have
designed and synthesized to develop novel and potent telomerase
inhibitors which were investigated via modified telomerase repeat
amplification protocol (TRAP) assay and other biological evalua-
tions [2,12e17]. To obtain more selective and potent inhibitors that
can be employed as tools for studying telomerase, structure-based
methods have been used to design a series of disubstituted
aminoacyl-linked anthraquinone compounds at positions 1 and 8
with selectivity for human telomeric quadruplexes. These deriva-
tives were evaluated for their inhibitory effects on telomerase
activity in order to identify new molecular or pharmacophoric
targets for anticancer drugs. The anti-proliferative effect and hTERT
repressing activity of these compounds were also determined. This
study also enables us to address the issue of how, and to what
extent, the position of side chain substituents affects telomerase
activity. Moreover, compounds (3a and 3s) were selected by the NCI
for one dose screening program. Both compounds also have
selective inhibitory effects on the proliferation of H1299 cancer cell
lines, and have been shown to inhibit the activity of the telomerase
enzyme, which is selectively expressed in tumor cells, where it
plays a role in maintaining telomere integrity and cellular immor-
talization. The interplay between the cytotoxicity, hTERT repressing
activities and the drug explains the structureeactivity relationships
of anthraquinone derivatives and the molecular basis of drug-
induced activities.
2. Chemistry
There has been continuing interest in the synthesis of
anthraquinone-based derivatives and their systematic dissection
largely on account of their biological activities [14,18,19]. Herein, we
reported on the discovery of an efficiently method for synthesis of
tricyclic anthraquinone structural motif and the functionalized linker
of aliphatic amide or peptidyl framework as part of an effort to
a rational design for potential anticancer agents. A series of
symmetrically 1,8-difunctionalized anthraquinone-containing small
moleculeshave beenprepared, and thesyntheticroutewaspresented
Scheme 2. Overall synthetic routes of 1,8-diamidoanthraquinone analogs. Reagents
and conditions: (i) Na2S, NaOH/EtOH, reflux, 6 h; (ii) chloroacetyl chloride, pyridine, rt,
24 h; (iii) R1NH2, DIPEA or TEA, mini-reactor, 130e150 ꢀC, 30e90 min; (iv) 3-
chloropropionyl chloride, pyridine, rt, 24 h; (v) R2NH2, DIPEA or TEA, mini-reactor,
130e150 ꢀC, 30e90 min.

104
C.-C. Lee et al. / European Journal of Medicinal Chemistry 50 (2012) 102e112
Scheme 3. Synthesis of symmetrical amindoanthraquinone derivatives 3ae3t. Reagents and conditions: (i) methyl ester of a-amino acids (L- or D-form), DMF, DIPEA or TEA, mini-
reactor, 130e150 ꢀC, 30e90 min; (ii) proline methyl ester hydrochloride, DMF, DIPEA, mini-reactor, 130e150 ꢀC, 90 min, 60%; (iii) amine, DMF, DIPEA, mini-reactor, 130e150 ꢀC,
30e90 min.
Scheme 4. Synthesis of symmetrical amindoanthraquinone derivatives 4ae4k. Reagents and conditions: (i) amine, DMF, DIPEA, mini-reactor, 130e150 ꢀC, 30e90 min.

C.-C. Lee et al. / European Journal of Medicinal Chemistry 50 (2012) 102e112
105
the compounds 3, 4 and the appropriated amines under catalysis
conditions in the presence of DIPEA or TEA as well as in dry DMF
resulted in the formation of desired symmetrical compounds.
Besides, it was not only compounds 3 and 4, but also all the final
products of these reactions that were monitored by TLC; the quan-
tities of the byproducts were separated from their physical chemistry
properties and purification by tedious recrystallization and chroma-
tography. All of the structural compounds were determined by FTeIR,
1H NMR, 13C NMR and high resolution mass (HRMS) spectra and the
results are presented in the experimental part.
were added after the telomerase extension step during the TRAP
assays (Fig. 1, TRAP-M). A similar dose-dependent pattern was
observed for both compounds in both TRAP and TRAP-M assays,
suggesting that the inhibitory effect observed for both
compounds might be due to the inhibitory Taq polymerase
activity. Thus, although two of the 1,8-disubstituted amidoan-
thraquinones showed potent inhibitory activities in our TRAP
assays, these inhibitory activities might not due to telomerase
inhibition.
To monitor the expression of hTERT, we established a stable
H1299 cell line that carrying a secreted alkaline phosphatase
(SEAP) reporter gene under the control of hTERT promoter, PhTERT
-
3. Telomerase inhibitory and hTERT repression activities
SEAP. The expression of SEAP in cells harboring PhTERT-SEAP was
used as the criteria to evaluate if anthraquinone-linked derivatives
inhibited the expression of hTERT in cancer cells [4]. The level of
cell viability in these cells was also determined using MTT assay.
As shown in Table 1, parallel comparison of the drug-treated cells
for the cell viability and hTERT expression revealed that quite
a numbers of 1,8-disubstituted amidoanthraquinones showed
selective inhibitory effects on hTERT expression. Among these
compounds, we are most interested in compounds 3a, 3c, 3j, 3p,
3q, and 3r because they affected the SEAP expression without
significantly affecting the proliferation of the treated cells (Fig. 2).
Compounds 3c and 3j appeared to show the best selectivity
toward repressing hTERT expression. Interestingly, compounds
4ae4k did not show potent cytotoxic effects toward H1299 cells
and did not show preferential SEAP inhibitory activity. The results
suggested that only one carbon spacer between the amido and
amine groups of the 1,8-disubstituted amidoanthraquinones
greatly affected the hTERT repressing activity of these compounds.
Of note, none of the compound 3r showed strong cytotoxicity
toward H1299 cells.
We have previously identified several anthraquinone-linked
derivatives that show potent telomerase inhibitory activities.
Given the striking correlations between proliferation capacity
and telomerase activity in tumor cells, we expected that analysis
of anthraquinone-based derivatives might yield further insight
into SARs. Here we first screened this series of symmetrical
anthraquinone-based derivatives for their telomerase inhibition
activities using
protocol (TRAP) assay. The screen started by using the concen-
tration of 10 M for each compound. Among them, compounds
a PCR-based telomeric repeat amplification
m
4e and 4k gave profound telomerase inhibitory activities and
their telomerase IC50 values were subsequently determined
(Fig. 1 and data not shown). Compounds 4e and 4k have IC50
values toward telomerase at 3 and 6 mM, respectively. Since TRAP
assay involves both telomerase extension and PCR-amplification
steps, the effect of these compounds on Taq polymerase were
investigated. It is still a fast and sensitive PCR-based assay for
detection and measurement of telomerase activity associated
with telomere or telomerase dysfunction. Compounds 4e and 4k
Fig. 1. The effects of compounds 4e and 4k on telomerase activities by TRAP assay. (A) Structure of 4e and 4k; (B) In TRAP assays (left 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 (right panels), indicated amounts of the compounds were
added right after the telomerase extension step and before the PCR-amplification step.

106
C.-C. Lee et al. / European Journal of Medicinal Chemistry 50 (2012) 102e112
Table 1
4. In vitro anti-proliferative activities
Effects of symmetrical 1,8-diamidoanthraquinone analogs on activating or repres-
sing hTERT expression and telomerase activity.
All of the synthesized compounds were submitted to the National
Cancer Institute Drug Screen Program for anti-proliferative activity
screens. Among them, compounds 3a (NSC747340) and 3s
(NSC747341) were selected and tested against 60 full panels of
human tumor cell lines derived from nine cancer cell types. Results
from the screen were presented as the mean of growth percentage
Cell type
Compd.
H1299 (inhibition ꢁ SD)a
MTT
SEAP
1 (
mM)
10 (m
M) 100 (
m
M) 1 (
m
M)
10 (mM) 100 (mM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
84 ꢁ 9
99 ꢁ 4
99 ꢁ 9
86 ꢁ 7 68 ꢁ 6
78 ꢁ 8 60 ꢁ 5
90 ꢁ 8 86 ꢁ 5
111 ꢁ 8
105 ꢁ 5
100 ꢁ 6
91 ꢁ 6
93 ꢁ 10 12 ꢁ 3
94 ꢁ 11 24 ꢁ 5
values of 10 mM compound-treated cells using the untreated control
49 ꢁ 5
0 ꢁ 1
88 ꢁ 10 80 ꢁ 3 58 ꢁ 3
112 ꢁ 6 102 ꢁ 6 96 ꢁ 6
98 ꢁ 4 104 ꢁ 5 96 ꢁ 6
56 ꢁ 10 50 ꢁ 12 10 ꢁ 7
95 ꢁ 13 45 ꢁ 5
cells as 100% (Table 2). Both compounds did not show significant
growth inhibition activities against these 60 cells. Compound 3a
showed moderate growth inhibition against leukemia RPMI-8226
101 ꢁ 7 101 ꢁ 13 88 ꢁ 5
99 ꢁ 11 93 ꢁ 12 67 ꢁ 6
83 ꢁ 10 74 ꢁ 9 66 ꢁ 2
97 ꢁ 11 74 ꢁ 8 42 ꢁ 9
cells with 31.45% cell growth inhibition at 10 mM.
77 ꢁ 9
59 ꢁ 3 18 ꢁ 5
127 ꢁ 6 128 ꢁ 1 91 ꢁ 5
88 ꢁ 1 104 ꢁ 7 93 ꢁ 7
98 ꢁ 2
87 ꢁ 3
94 ꢁ 7 98 ꢁ 1
59 ꢁ 3 8 ꢁ 4
3j
3k
3l
71 ꢁ 6
88 ꢁ 9 46 ꢁ 8
106 ꢁ 11 95 ꢁ 14 73 ꢁ 4
5. Discussion and conclusion
53 ꢁ 13 44 ꢁ 14 28 ꢁ 5
110 ꢁ 6
93 ꢁ 4
86 ꢁ 7 77 ꢁ 10
96 ꢁ 5 84 ꢁ 5
3m
3n
3o
3p
3q
3r
3s
3t
4a
4b
4c
4d
4e
4f
72 ꢁ 9
89 ꢁ 3
106 ꢁ 4
97 ꢁ 6
61 ꢁ 7 57 ꢁ 7
86 ꢁ 10 14 ꢁ 8
87 ꢁ 7 17 ꢁ 2
93 ꢁ 6 93 ꢁ 7
103 ꢁ 7 101 ꢁ 9 35 ꢁ 6
Tumor growth addiction to the specific pathway that is effec-
tively targeted may be the link between a cell-based cytotoxicity
and telomerase activity. Here we describe a method to synthesize
1,8-disubstituted amidoanthraquinones and evaluate their biolog-
ical activities including cytotoxicity, telomerase inhibitory, and
hTERT repressing activities. We found compounds 4e and 4k
showed potent telomerase inhibitory activity. The results suggest
that symmetrical 1,8-diamidoanthraquinones might represent an
important class of compounds for telomerase-related drug devel-
opments. It is also important to note that a big fraction of these
compounds showed potent hTERT repressing activities, indicating
that they might be more suitable for developing as hTERT repres-
sors. In particular, compounds 3c and 3j showed the most potent
hTERT repressing activities. Since compounds 3ae3t and 4ae4k
differ by one carbon, the distance on their extended arms might
separate the function of 1,8-diamidoanthraquinones from telome-
rase inhibitors to hTERT repressor. It is also important to note that
although anthraquinone-based compounds are generally of high
toxicity toward cancer cells, we found our newly synthesize 1,8-
diamidoanthraquinones do not show potent cytotoxic effects
toward H1299 cancer cells. Consistently, two compounds, 3a
(NSC747340) and 3s (NSC747341), selected by NCI’s drug screen
103 ꢁ 9
92 ꢁ 9
2 ꢁ 1
75 ꢁ 17 84 ꢁ 5 33 ꢁ 7
100 ꢁ 9 101 ꢁ 4 73 ꢁ 4
108 ꢁ 7 108 ꢁ 4 97 ꢁ 3
102 ꢁ 7
85 ꢁ 7
86 ꢁ 5
95 ꢁ 5 20 ꢁ 1
87 ꢁ 6 45 ꢁ 4
84 ꢁ 6 42 ꢁ 11
86 ꢁ 6
90 ꢁ 7
97 ꢁ 4
93 ꢁ 8
94 ꢁ 3
78 ꢁ 5 50 ꢁ 16
76 ꢁ 6 66 ꢁ 8
105 ꢁ 3 101 ꢁ 9 94 ꢁ 8
30 ꢁ 5
4 ꢁ 2
99 ꢁ 11 35 ꢁ 6
87 ꢁ 7 20 ꢁ 5
0 ꢁ 3
0 ꢁ 3
24 ꢁ 2 11 ꢁ 4
99 ꢁ 5 22 ꢁ 2
100 ꢁ 6 100 ꢁ 14 4 ꢁ 1
117 ꢁ 5 107 ꢁ 8 33 ꢁ 3
101 ꢁ 6
106 ꢁ 9
97 ꢁ 8 22 ꢁ 3
101 ꢁ 8
96 ꢁ 8
73 ꢁ 6
93 ꢁ 6
82 ꢁ 8
29 ꢁ 4 14 ꢁ 5
23 ꢁ 2 12 ꢁ 2
16 ꢁ 4
0 ꢁ 3
0 ꢁ 0
110 ꢁ 10 19 ꢁ 2
4g
4h
4i
4j
4k
4 ꢁ 2
0 ꢁ 6
6 ꢁ 3
96 ꢁ 8
98 ꢁ 8 100 ꢁ 7
100 ꢁ 6 84 ꢁ 12 33 ꢁ 14
29 ꢁ 11 0 ꢁ 2
87 ꢁ 7
3 ꢁ 1
78 ꢁ 5 11 ꢁ 7
122 ꢁ 9 105 ꢁ 2 21 ꢁ 6
95 ꢁ 11 95 ꢁ 10 20 ꢁ 4
98 ꢁ 5
74 ꢁ 5
0 ꢁ 1
0 ꢁ 2
7 ꢁ 6
0 ꢁ 1
0 ꢁ 1
121 ꢁ 9
20 ꢁ 1
33 ꢁ 2
97 ꢁ 12 4 ꢁ 3
mitoxantrone 23 ꢁ 8
9 ꢁ 4
7 ꢁ 1
5 ꢁ 4
doxorubicin
17 ꢁ 5
13 ꢁ 3
a
SD, standard derivation; all experiments were independently performed at least
three times.
Fig. 2. Inhibition of PhTERT-SEAP by 1,8-diamidoanthraquinones (compounds 3a, 3c, 3j, 3p, 3q, and 3r). About 2 ꢂ 103 cells of H1299 harboring PhTERT-SEAP were seeded in 96-well
plates and incubated at 37 ꢀC for 24 h. Cells were then washed with PBS, re-cultured in fresh media, and incubated with varying amounts of 3a, 3c, 3j, 3p, 3q, and 3r 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%.

C.-C. Lee et al. / European Journal of Medicinal Chemistry 50 (2012) 102e112
107
Table 2
In vitro anticancer activity of compounds 3a (NSC747340) and 3s (NSC747341) in the NCI's 60 human cancer cell lines.
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H3CO
NH
O
HN
OCH3 H3CO
NH
O
HN
OCH3
O
O
3a (NSC747340)
3s (NSC747341)
.
Panel/cell line
Growth percenta (at 10ꢃ5 M)
3a (NSC747340)
3s (NSC747341)
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
64.36
107.25
97.12
83.83
37.41
84.63
94.67
106.43
91.21
68.55
88.55
119.92
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
94.31
104.06
107.76
75.76
84.62
95.17
94.52
95.11
96.85
91.67
103.99
88.35
93.50
85.49
97.13
93.76
97.21
87.99
NCl-H226
NCI-H23
NCl-H322M
NCl-H460
NCl-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
101.76
105.65
96.36
114.18
103.23
84.22
HCT-15
94.29
93.42
HT29
KM12
SW-620
96.31
106.53
108.59
101.26
119.47
104.42
Breast cancer
BT-549
HS 578T
99.87
103.66
89.54
90.16
102.22
83.84
92.90
83.78
97.39
111.26
88.66
91.17
102.97
86.63
MCF7
MDA-MB-231/ATCC
MDA-MB-435
MDA-MB-468
NCI/ADR-RES
T-47D
100.90
84.80
Ovarian cancer
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
89.86
87.67
105.95
96.34
161.23
91.35
101.17
91.24
104.18
103.00
Renal cancer
786e0
A498
ACHN
CAK1-1
SN12C
TK-10
UO-31
102.54
85.15
90.73
83.79
90.39
84.90
88.90
86.42
93.34
91.90
88.35
92.11
105.35
87.27
Melanoma
LOX IMVI
M14
88.22
113.02
102.47
107.45
126.58
94.32
89.33
98.62
111.69
83.97
117.25
100.86
105.13
93.51
MALME-3M
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Prostate cancer
DU-145
92.43
90.28
107.44
136.86
(continued on next page)

108
C.-C. Lee et al. / European Journal of Medicinal Chemistry 50 (2012) 102e112
Table 2 (continued )
Panel/cell line
Growth percenta (at 10ꢃ5 M)
3a (NSC747340)
3s (NSC747341)
PC-3
76.59
89.07
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
100.06
97.18
91.88
95.27
89.92
93.72
137.50
88.91
91.71
94.11
81.66
100.10
Mean
Delta
Range
94.95
57.54
89.17
97.96
29.41
92.68
a
Data obtained from NCI in vitro 60-cell drug screen program at 10ꢃ5 M concentration.
program, also showed no significant cytotoxic effects toward a 60-
cell panel of cancer cells.
and their purity was confirmed with FTeIR, 1H NMR, 13C NMR and
high resolution mass (HRMS) spectrometry.
Correlated with superior efficacy in retrospective analyses of
large studies in telomerase and cytotoxicity as an adverse event of
anticancer agents, may also be a marker of effective target inhibi-
tion. These assays have been used for initial screening of telomerase
inhibitors as anticancer drug agents. The aim of this study was to
elucidate the structureeactivity relationships (SARs) of symmet-
rical 1,8-disubstituted amidoanthraquinones to further delineate
the pharmacological requirements of the pharmacophore. The
results of SARs study revealed that symmetrical linker with one- or
two-carbon spacer between amido and amine was an important
functional moiety for hTERT repression or telomerase inhibition.
The exact mechanism of how this spacer affects hTERT repression
and telomerase activities is unclear. Since both telomeric DNA and
promoter of hTERT are capable of forming G-quadruplex structure,
it will be interesting to test whether the G-quadruplex structures
6.2.1. 1,8-Bis(chloroacetamido)anthraquinone (3)
A mixture of 2 (0.476 g, 2 mmol), pyridine (0.5 ml), and anhy-
drous DMF (20 ml) was stirred in ice bath and 3-chloropropioyl
chloride (0.6 ml, 6 mmol) was added under nitrogen for 24 h. Ice
was added to precipitate out the crude product. The resulting
precipitate was collected and purified by crystallization from
ethanol and column chromatography (CH2Cl2) to afford desired
compound 3 as yellow powder (80%). Mp: 280e281 ꢀC (EtOH). 1H
NMR (CDCl3, 300 MHz)
d
(ppm): 4.30 (s, 8H), 7.83 (t, J ¼ 7.8 Hz, 2H),
8.13 (d, J ¼ 7.2 Hz, 2H), 9.17 (d, J ¼ 8.1 Hz, 2H), 13.02 (s, 2H). HRMS
(EI) m/z calcd for [M]þ: 390.0174, found: 390.0174.
6.2.2. 1,8-Bis[2-(glycin methyl ester)acetamido]anthraquinone (3a)
1,8-Bis(chloroacetamido)anthraquinone (0.3 g, 0.75 mmol) was
dissolved in anhydrous DMF (10 ml) and glycine methyl ester
hydrochloride (0.75 g, 6 mmol), DIPEA (12 mmol) were added
dropwise under nitrogen. The reaction mixture was heated and
refluxed at 130 ꢀC in miniclave for 1 h. Upon completion, the mixture
was cooled to room temperature. The solution put into the ice water,
the precipitated and was collected by filtration, washed with ice-cold
water and ethanol and dried in vacuo. The crude compound was
purified by column chromatography (CH2Cl2) and recrystallization
from ethanol provided 3a as dark yellow powder (42%). Mp:
173e174 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3187 (NH), 2938 (NH), 1732
formed by these sequences affect the binding
a series of
compounds 3ae3t or 4ae4k. The biological basis for this correla-
tion can be pharmacological, associated with cytotoxicity and
telomerase activity, and can be contradictory, because hTERT
repression and telomerase activities play an important role in
anticancer drug development, thus, providing novel possible
insight into the mechanism of resistance to telomerase inhibitors in
cancer treatment. However, the most widely used telomeric repeat
amplification protocol (TRAP) assay has been examined for telo-
merase detection.
(CO),1693 (CO). 1H NMR (CDCl3, 300 MHz)
(s, 6H), 7.75 (t, J ¼ 8.1 Hz, 2H), 8.02 (d, J ¼ 7.8 Hz, 2H), 9.18 (d, J ¼ 8.7 Hz,
2H), 12.86 (s, 2H). 13C NMR (CDCl3, 75 MHz)
(ppm): 50.07, 51.23,
d (ppm): 3.60 (s, 8H), 3.75
6. Materials and methods
d
53.13, 118.60, 121.91, 126.18, 132.87, 134.92, 140.63, 171.07, 172.02,
6.1. Chemistry
181.93,189.77. HRMS (EI) m/z calcd. [M]þ: 496.1594, found: 496.1596.
The starting material of 1,8-dinitroanthraquinone was
purchased from Aldrich chemicals. All chemicals and solvents were
purchased from Aldrich and Merck used without further purifica-
tion. The progress of reactions was monitored by TLC (silica gel 60
F254). Melting points were determined on a Büchi 545 melting point
apparatus. Infra-Red spectrum was acquired on a PerkineElmer
FTIR-1615 spectrometer. Both 1H NMR and 13C NMR spectra of
synthesized compounds were performed with a Varian GEMINI-
6.2.3. 1,8-Bis[2-(methioine methyl ester)acetamido]anthraquinone (3b)
The pure compound was obtained as pale yellow powder (40%).
Mp: 126e127 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3194 (NH), 2938 (NH),
1738 (CO), 1694 (CO), 1628 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm):
1.98 (s, 6H), 2.01e2.11 (m, 4H), 2.65e2.71 (m, 4H), 3.48 (t, J ¼ 6.3 Hz,
2H), 3.34 (d, J ¼ 17.1 Hz, 2H), 3.74 (s, 6H), 3.76 (d, J ¼ 17.1 Hz, 2H),
7.67 (t, J ¼ 8.1 Hz, 2H), 7.94 (d, J ¼ 7.2 Hz, 2H), 9.46 (d, J ¼ 8.4 Hz, 2H),
12.61 (s, 2H). 13C NMR (CDCl3, 75 MHz)
d (ppm): 14.58, 29.72, 32.06,
300 MHz. Chemical shift (d) values were in ppm relative to TMS as
51.46, 52.01, 59.76, 118.39, 121.97, 126.20, 132.85, 135.02, 140.80,
171.16, 174.20, 181.90, 189.85. HRMS (EI) m/z calcd for [M]þ:
644.1975, found: 644.1976.
an internal standard. High resolution mass spectra were obtained
on a Finnigan MAT 95 XL and a JEOL JMS-700.
6.2. General synthetic methods
6.2.4. ( )-1,8-Bis[2-(valine methyl ester)acetamido]anthraquinone (3c)
L
The pure compound was obtained as yellow powder (47%). Mp:
1,8-Diaminoanthraquinone (2) were prepared according to the
literature [20,21]. All anthraquinone-based derivatives were
synthesized by using a simply acylation or two-stage reaction in
miniclave (Büchi). These compounds were obtained in good yield
150e151 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3225 (NH), 2961 (NH), 1736
(CO), 1693 (CO), 1630 (CO). 1H NMR (CDCl3, 300 MHz)
d
(ppm): 0.98
(d, J ¼ 6.6 Hz, 6H), 1.02 (d, J ¼ 6.6 Hz, 6H), 2.01e2.08 (m, 2H), 2.27 (s,

C.-C. Lee et al. / European Journal of Medicinal Chemistry 50 (2012) 102e112
109
2H), 3.09 (d, J ¼ 5.4 Hz, 2H), 3.28 (d, J ¼ 16.8 Hz, 2H), 3.63 (d,
J ¼ 16.8 Hz, 2H), 3.71 (s, 6H), 7.63 (t, J ¼ 7.8 Hz, 2H), 7.90 (d,
J ¼ 7.2 Hz, 2H), 9.07 (d, J ¼ 8.4 Hz, 2H),12.44 (s, 2H). 13C NMR (CDCl3,
128.15, 128.47, 132.79, 135.10, 135.43, 141.69, 169.04, 172.20, 181.66,
191.16. HRMS (EI) m/z calcd for [M]þ: 648.2220, found: 648.2226.
75 MHz)
d
(ppm): 18.05, 18.46, 30.94,51.00, 52.52, 66.90, 118.45,
6.2.10. 1,8-Bis[2-(tert-butyl-serine methyl ester)acetamido]
anthraquinone (3i)
121.92, 126.29, 132.87, 134.93, 140.77, 170.95, 174.11, 181.83, 189.69.
HRMS (EI) m/z calcd for [M]þ: 580.2533, found: 580.2532.
The pure compound was obtained as yellow powder (44%). Mp:
139e140 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3206 (NH), 2967 (NH),1691
6.2.5. (D)-1,8-Bis[2-(valine methyl ester)acetamido]anthraquinone
(CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 1.11 (s, 18H), 3.46e3.55
(3d)
(m, 8H), 3.70 (s, 6H), 3.73 (s, 4H), 4.21e4.30 (m, 2H), 7.69 (t,
J ¼ 8.1 Hz, 2H), 7.96 (d, J ¼ 6.3 Hz, 2H), 9.15 (d, J ¼ 8.4 Hz, 2H), 12.85
The pure compound was obtained as yellow powder (35%). Mp:
150e151 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3215 (NH), 2960 (NH), 1732
(s, 2H). 13C NMR (CDCl3, 75 MHz)
d (ppm): 26.69, 42.88, 51.22, 61.17,
(CO), 1694 (CO), 1632 (CO). 1H NMR (CDCl3, 300 MHz)
d
(ppm): 1.01
62.09, 72.81, 118.64, 121.75, 126.14, 132.92, 134.86, 140.79, 165.57,
172.30, 182.08, 189.89. FAB-MS m/z(%): 670 ([Mþ2]þ,100), 671
([Mþ1]þ, 44.33).
(d, J ¼ 6.9 Hz, 6H),1.05 (d, J ¼ 6.6 Hz, 6H), 2.04e2.08 (m, 2H), 3.11 (d,
J ¼ 5.7 Hz, 2H), 3.30 (d, J ¼ 16.8 Hz, 2H), 3.68 (d, J ¼ 17.1 Hz, 2H), 3.75
(s, 6H), 7.73 (t, J ¼ 8.4 Hz, 2H), 8.02 (dd, J ¼ 7.5 Hz, 0.9 Hz, 2H), 9.16
(dd, J ¼ 8.7 Hz, 0.9 Hz, 2H), 12.59 (s, 2H). 13C NMR (CDCl3, 75 MHz)
6.2.11. 1,8-Bis[2-(proline methyl ester)acetamido]anthraquinone (3j)
The pure compound was obtained as yellow powder (60%). Mp:
170e171 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3202 (NH), 2952 (NH), 1732
d
(ppm): 18.04, 18.48, 30.97, 51.02, 52.57, 66.94, 118.61, 122.05,
126.39, 133.00, 135.01, 140.81, 171.01, 174.15, 182.02, 189.81. HRMS
(EI) m/z calcd for [M]þ: 580.2533, found: 580.2527.
(CO), 1694 (CO), 1627 (CO). 1H NMR (300 MHz, CDCl3)
d: 1.92e2.23
(m, 8H), 2.78 (dd, 2H, J ¼ 7.5 Hz,15.3 Hz), 3.23e3.67 (m, 4H), 3.53 (d,
J ¼ 16.8 Hz, 2H), 3.67 (s, 6H), 3.75 (d, J ¼ 16.8 Hz, 2H), 7.62 (t,
J ¼ 8.1 Hz, 2H), 7.86 (d, J ¼ 7.5 Hz, 2H), 9.06 (d, J ¼ 8.7 Hz, 2H), 12.33
6.2.6. 1,8-Bis[2-(leucine methyl ester)acetamido]anthraquinone (3e)
The pure compound was obtained as yellow powder (40%). Mp:
238e239 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3206 (NH), 2955 (NH),
(s, 2H). 13C NMR (CDCl3, 75 MHz)
d (ppm): 23.55, 28.95, 51.03, 53.18,
1737 (CO), 1693 (CO), 1630 (CO). 1H NMR (CDCl3, 300 MHz)
d
(ppm):
57.28, 63.96, 118.22, 121.70, 126.07, 132.77, 134.93, 141.01, 170.34,
173.44, 181.78, 189.97. HRMS (EI) m/z calcd for [M]þ: 576.2220,
found: 576.2224.
0.83 (d, J ¼ 6.6 Hz, 6H), 0.88 (d, J ¼ 6.6 Hz, 6H), 1.54e1.73 (m,
4H),1.88e1.93 (m, 2H), 3.33e3.38 (m, 6H), 3.35 (d, J ¼ 17.1 Hz, 2H),
3.68 (d, J ¼ 17.1 Hz, 2H), 3.75 (s, 6H), 7.80 (t, J ¼ 8.1 Hz, 2H), 8.08 (dd,
J ¼ 7.5 Hz, 1.2 Hz, 2H), 9.26 (dd, J ¼ 8.4 Hz, 1.2 Hz, 2H), 12.82 (s, 2H).
6.2.12. 1,8-Bis[2-(sacrocine methyl ester)acetamido]anthraquinone
(3k)
13C NMR (CDCl3, 75 MHz)
d (ppm): 22.06, 24.15, 42.09, 51.17, 51.90,
59.36, 118.35, 121.91, 126.15, 132.89, 134.99, 140.74, 171.04, 174.97,
181.83, 189.65. HRMS (EI) m/z calcd for [M]þ: 608.2846, found:
608.2832.
The pure compound was obtained as yellow powder (65%). Mp:
129e130 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3206 (NH), 2947 (NH),1739
(CO), 1691 (CO), 1630 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 2.62
(s, 6H), 3.56 (s, 4H), 3.58 (s, 4H), 3.69 (s, 6H), 7.66 (t, J ¼ 7.8 Hz, 2H),
6.2.7. ( )-1,8-Bis[2-(alaline methyl ester)acetamido]anthraquinone (3f)
L
7.92 (d, J ¼ 6.9 Hz, 2H), 9.08 (d, J ¼ 8.1 Hz, 2H),12.52 (s, 2H). 13C NMR
The pure compound was obtained as yellow powder (41%). Mp:
119e120 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3194 (NH), 2938 (NH),
1738 (CO), 1694 (CO), 1628 (CO). 1H NMR (CDCl3, 300 MHz)
(CDCl3, 75 MHz) d (ppm): 42.18, 50.78, 57.15, 60.89, 118.62, 121.92,
126.36, 132.87, 134.84, 140.73, 170.23, 170.31, 181.90, 189.73. HRMS
(EI) m/z calcd for [M]þ: 524.1907, found: 524.1906.
d
(ppm): 1.46 (d, J ¼ 6.9 Hz, 6H), 3.38 (d, J ¼ 17.4 Hz, 2H), 3.46 (q,
J ¼ 6.9 Hz, 2H), 3.70 (d, J ¼ 17.4 Hz, 2H), 3.75 (s, 6H), 7.77 (t,
J ¼ 7.8 Hz, 2H), 8.06 (dd, J ¼ 7.5 Hz, 1.2 Hz, 2H), 9.23 (dd, J ¼ 8.4 Hz,
6.2.13. 1,8-Bis[2-(isobutylamino)acetamido]anthraquinone (3l)
The pure compound was obtained as yellow crystal (40%). Mp:
139e140 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3196 (NH), 2947 (NH), 1693
1.2 Hz, 2H), 12.84 (s, 2H). 13C NMR (CDCl3, 75 MHz)
d (ppm): 18.29,
51.36, 51.52, 55.94, 118.18, 121.79, 126.01, 132.67, 134.93, 140.69,
170.18, 174.86, 181.70, 189.60. HRMS (EI) m/z calcd for [M]þ:
524.1907, found: 524.1904.
(CO), 1630 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 0.95 (d,
J ¼ 6.3 Hz, 12H), 1.85e1.89 (m, 2H), 2.55 (d, J ¼ 6.9 Hz, 4H), 3.55 (s,
4H), 7.77 (t, J ¼ 8.1 Hz, 2H), 8.06 (d, J ¼ 7.5 Hz, 2H), 9.23 (d,
J ¼ 8.4 Hz, 2H), 12.75 (s, 2H). 13C NMR (CDCl3, 75 MHz)
d (ppm):
6.2.8. (D)-1,8-Bis[2-(alaline methyl ester)acetamido]anthraquinone (3g)
19.92, 27.79, 53.72, 57.64, 118.60, 121.93, 126.32, 132.98, 134.69,
140.91, 171.83, 182.12, 189.93. HRMS (EI) m/z calcd for [M]þ:
464.2424, found: 464.2424.
The pure compound was obtained as yellow powder (45%). Mp:
118e119 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3194 (NH), 2952 (NH), 1738
(CO), 1694 (CO), 1628 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 1.45
(d, J ¼ 7.2 Hz, 6H), 3.38 (d, J ¼ 17.4 Hz, 2H), 3.46 (q, J ¼ 6.9 Hz, 2H),
3.70 (d, J ¼ 17.4 Hz, 2H), 3.75 (s, 6H), 7.77 (t, J ¼ 7.8 Hz, 2H), 8.06 (dd,
J ¼ 7.5 Hz, 1.2 Hz, 2H), 9.24 (dd, J ¼ 8.4 Hz, 1.2 Hz, 2H), 12.85 (s,
6.2.14. 1,8-Bis[2-(dimethylamino)acetamido]anthraquinone (3m)
The pure compound was obtained as yellow powder (64%). Mp:
194e195 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3206 (NH), 2779 (NH),1693
2H).13C NMR (CDCl3, 75 MHz)
d
(ppm): 18.23, 51.28, 51.45, 55.89,
(CO), 1633 (CO). 1H NMR (CDCl3, 300 MHz)
d(ppm): 2.49 (s, 8H),
118.03, 121.64, 125.90, 132.54, 134.83, 140.63, 170.08, 174.77, 181.51,
3.25 (s, 4H), 7.77 (t, J ¼ 7.8 Hz, 2H), 8.06 (dd, J ¼ 7.5 Hz, 1.2 Hz, 2H),
189.43. HRMS (EI) m/z calcd for [M]þ: 524.1907, found: 524.1908.
9.24 (dd, J ¼ 8.7 Hz, 1.2 Hz, 2H), 12.69 (s, 2H). 13C NMR (CDCl3,
75 MHz)
d (ppm): 45.46, 64.23, 118.64, 121.79, 126.36, 132.80,
6.2.9. (S)-1,8-Bis[2-(phenylglycin methyl ester)acetamido]
anthraquinone (3h)
134.62, 140.64, 170.50, 181.88, 189.35. HRMS (EI) m/z calcd for [M]þ:
408.1798, found: 408.1798.
The pure compound was obtained as yellow powder (33%). Mp:
256e260 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3249 (NH), 2938 (NH),1741
6.2.15. 1,8-Bis[2-(isopropylamino)acetamido]anthraquinone (3n)
The pure compound was obtained as yellow powder (65%). Mp:
196e197 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3183 (NH), 2962 (NH), 1698
(CO) 1702 (CO), 1629 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 3.42
(d, J ¼ 17.4 Hz, 2H), 3.51 (d, J ¼ 17.7 Hz, 2H), 3.69 (s, 6H), 4.52 (s, 2H),
7.33e7.36 (m, 10H) 7.69 (t, J ¼ 8.1 Hz, 2H), 7.94 (d, J ¼ 7.2 Hz, 2H),
9.07 (d, J ¼ 8.1 Hz, 2H), 11.94 (s, 2H). 13C NMR (CDCl3, 75 MHz)
(CO), 1628 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 1.17 (d,
J ¼ 5.4 Hz, 12H), 2.89e2.93 (m, 2H), 3.55 (s, 4H), 7.76 (t, J ¼ 8.4 Hz,
d
(ppm): 24.89, 51.70, 51.45, 64.39, 117.62, 121.84, 125.98, 127.14,
2H), 8.04 (d, J ¼ 7.5 Hz, 2H), 9.23 (d, J ¼ 8.7 Hz, 2H), 12.76 (s, 2H). 13C

110
C.-C. Lee et al. / European Journal of Medicinal Chemistry 50 (2012) 102e112
NMR (CDCl3, 75 MHz)
d
(ppm): 22.38, 48.67, 51.30, 118.57, 121.95,
d
(ppm): 11.11, 19.53, 56.73, 59.37, 118.63, 121.73, 126.33, 132.97,
126.27, 133.01, 135.04, 140.95, 172.27, 182.22, 189.96. HRMS (EI) m/z
134.70, 140.94, 171.68, 181.95, 189.58. HRMS (EI) m/z calcd for [M]þ:
calcd for [M]þ: 436.2111, found: 436.2101.
520.3050, found: 520.3048.
6.2.16. 1,8-Bis[2-(ethanamino)acetamido]anthraquinone (3o)
The pure compound was obtained as yellow powder (52%). Mp:
182e183 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3175(NH), 2957 (NH), 1694
6.2.22. 1,8-Bis(3-chloropropionamido)anthraquinone (4)
A mixture of 2 (0.476 g, 2 mmol), pyridine (0.5 ml), and anhy-
drous DMF (20 ml) was stirred in ice bath and chloroacetyl chloride
(0.5 ml, 6 mmol) was added under nitrogen for 24 h. Ice was added
to precipitate out the crude product. The resulting precipitate was
collected and purified by crystallization from ethanol and column
chromatography (CH2Cl2) to afford desired compound 4 as orange
powder (58%). Mp: 248e249 ꢀC (EtOH). 1H NMR (300 MHz, CDCl3)
(CO), 1628 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 1.24 (t,
J ¼ 6.9 Hz, 6H), 2.79 (q, J ¼ 6.9 Hz, 4H), 3.55 (s, 4H), 7.75 (t, J ¼ 8.1 Hz,
2H), 8.04 (d, J ¼ 7.5 Hz, 2H), 9.23 (d, J ¼ 8.4 Hz, 2H), 12.82 (s, 2H). 13C
NMR (CDCl3, 75 MHz)
d (ppm): 14.70, 43.71, 53.24, 118.52, 121.63,
126.01, 132.70, 134.75, 140.77, 171.79, 181.73, 189.48. HRMS (EI) m/z
calcd for [M]þ: 408.1798, found: 408.1801.
d
(ppm): 3.06 (t, J ¼ 6.6 Hz, 4H), 3.97 (t, J ¼ 6.6 Hz, 4H), 7.80 (t,
J ¼ 8.1 Hz, 2H), 8.08 (dd, J ¼ 7.8 Hz, 1.2 Hz, 2H), 9.17 (dd, J ¼ 8.7 Hz,
1.2 Hz, 2H), 12.17 (s, 2H). HRMS (EI) m/z calcd for [M]þ: 418.0487,
found: 418.0487.
6.2.17. 1,8-Bis[2-(3-(trifluoromethyl)benzylamino)acetamido]
anthraquinone (3p)
The pure compound was obtained as yellow powder (66%). Mp:
190e191 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3202 (NH), 2836 (NH),1693
6.2.23. 1,8-Bis[3-(diethylamino)propionamido]anthraquinone (4a)
The pure compound was obtained as dark yellow powder (72%).
Mp: 166e167 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3240 (NH), 2969 (NH),
(CO), 1632 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 3.40 (s, 4H),
3.77 (s, 4H), 7.74e7.54 (m, 6H), 7.65 (s, 2H) 7.75 (t, J ¼ 8.1 Hz, 2H),
8.08 (d, J ¼ 7.2 Hz, 2H), 9.23 (d, J ¼ 8.4 Hz, 2H), 12.91 (s, 2H). 13C
1699 (CO), 1625 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 1.05 (t,
NMR (CDCl3, 75 MHz)
d
(ppm): 52.80, 52.87, 114.95, 118.67, 122.17,
J ¼ 7.2 Hz, 12H), 2.60 (q, J ¼ 7.2 Hz, 8H), 2.63 (t, J ¼ 7.2 Hz, 4H), 2.92
(t, J ¼ 7.2 Hz, 4H), 7.66 (t, J ¼ 7.8 Hz, 2H), 7.92 (dd, J ¼ 7.2 Hz, 1.2 Hz,
2H), 9.04 (dd, J ¼ 8.4 Hz, 1.2 Hz, 2H), 12.01 (s, 2H). 13C NMR (CDCl3,
123.87, 124.52, 126.29, 128.50, 131.17, 133.04, 135.21, 139.85, 140.81,
171.05, 182.14, 190.28. HRMS (EI) m/z calcd for [M]þ: 668.1858,
found: 668.1859.
75 MHz)
d (ppm): 11.12, 36.46, 46.40, 48.08, 117.99, 121.77, 126.35,
132.83, 135.18,141.49,171.40,181.79,190.71. HRMS (EI) m/z calcd for
6.2.18. 1,8-Bis[2-(ethylmethylamino)acetamido]anthraquinone (3q)
The pure compound was obtained as yellow powder (79%). Mp:
186e187 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3215 (NH), 2967 (NH), 1665
[M]þ: 492.2737, found: 492.2747.
6.2.24. 1,8-Bis[2-(dimethylamino)propionamido]anthraquinone (4b)
The pure compound was obtained as orange powder (74%). Mp:
116e117 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3251 (NH), 2949 (NH), 1697
(CO). 1H NMR (300 MHz, CDCl3)
d
(ppm): 1.18 (t, J ¼ 7.2 Hz, 6H), 3.48
(s, 6H), 2.64 (q, J ¼ 7.2 Hz, 4H), 3.29 (s, 4H), 7.76 (t, J ¼ 8.4 Hz, 2H),
8.06 (d, J ¼ 7.5 Hz, 2H), 9.22 (d, J ¼ 8.7 Hz, 2H), 12.62 (s, 2H). 13C
(CO), 1626 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 2.36 (s, 12H),
NMR (CDCl3, 75 MHz)
d
(ppm): 11.66, 42.25, 51.15, 61.73, 118.84,
2.71(t, J ¼ 6.3 Hz, 4H), 2.80 (t, J ¼ 6.3 Hz, 4H), 7.76 (t, J ¼ 8.1 Hz, 2H),
121.93, 126.52, 132.94, 134.73, 140.74, 171.20, 182.19, 189.49. HRMS
8.05 (dd, J ¼ 7.5 Hz,1.2 Hz, 2H), 9.14 (dd, J ¼ 8.4 Hz,1.2 Hz, 2H),12.22
(EI) m/z calcd for [M]þ: 436.2111, found: 436.2108.
(s, 2H). 13C NMR (CDCl3, 75 MHz)
d (ppm): 36.29, 44.66, 54.34,
117.89, 121.66, 126.33, 132.61, 135.01, 141.29, 170.84, 181.52, 190.34.
6.2.19. 1,8-Bis[2-(methylethylamino)acetamido]anthraquinone (3r)
The pure compound was obtained as yellow powder (67%). Mp:
159e160 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3215 (NH), 2958 (NH), 1692
HRMS (EI) m/z calcd for [M]þ: 436.2111, found: 436.2111.
6.2.25. 1,8-Bis[2-(dipropylamino)propionamido]anthraquinone (4c)
The pure compound was obtained as orange powder (79%). Mp:
129e130 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3234 (NH), 2959 (NH),1698
(CO), 1631 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 0.92 (t,
J ¼ 7.2 Hz, 6H), 1.61 (q, J ¼ 7.2 Hz, 4H), 2.48 (s, 6H), 2.51 (t, J ¼ 7.5 Hz,
4H), 3.28 (s, 4H), 7.73 (t, J ¼ 8.4 Hz, 2H), 8.02 (d, J ¼ 7.5 Hz, 2H), 9.18
(CO), 1626 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 0.87 (t,
(d, J ¼ 8.7 Hz, 2H), 12.55 (s, 2H). 13C NMR (CDCl3, 75 MHz)
d
(ppm):
J ¼ 7.5 Hz, 12H), 1.46e1.53(m, 8H), 2.46 (t, J ¼ 5.4 Hz, 8H), 2.67 (t,
J ¼ 6.9 Hz, 4H), 2.96 (t, J ¼ 6.9 Hz, 4H), 7.76 (t, J ¼ 8.1 Hz, 2H), 8.04 (d,
J ¼ 7.5 Hz, 2H), 9.15 (d, J ¼ 8.7 Hz, 2H), 12.11 (s, 2H).13C NMR (CDCl3,
11.04, 19.76, 42.80, 59.44, 62.33, 118.90, 121.92, 126.53, 133.04,
134.73, 140.79, 171.07, 182.20, 189.52. HRMS (EI) m/z calcd for [M]þ:
464.2424, found: 464.1654.
75 MHz)
d (ppm): 11.21, 19.66, 36.72, 49.46, 55.61, 117.79, 121.68,
126.19, 132.77, 135.18, 141.58, 171.43, 181.69, 190.78. HRMS (EI) m/z
6.2.20. 1,8-Bis[2-(diethylamino)acetamido]anthraquinone (3s)
calcd for [M]þ: 548.3363, found: 548.3374.
The pure compound was obtained as yellow powder (70%). Mp:
228e229 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3187 (NH), 2968 (NH),
6.2.26. 1,8-Bis[2-(methylamino)propionamido]anthraquinone (4d)
The pure compound was obtained as orange powder (32%). Mp:
233e234 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3249 (NH), 2967 (NH),
1
1693 (CO), 1628 (CO). H NMR (CDCl3, 300 MHz)
d (ppm): 1.13 (t,
J ¼ 6.9 Hz, 12H), 2.75 (q, J ¼ 7.2 Hz, 8H), 3.33 (s, 4H), 7.71 (t,
J ¼ 8.1 Hz, 2H), 8.00 (dd, J ¼ 7.5 Hz, 1.2 Hz, 2H), 9.19 (dd, J ¼ 8.4 Hz,
1690 (CO), 1624 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 2.53 (s,
1.2 Hz, 2H), 12.75 (s, 2H). 13C NMR (CDCl3, 75 MHz)
d
(ppm): 11.28,
6H), 2.78 (t, J ¼ 6.3 Hz, 4H), 3.05 (t, J ¼ 6.3 Hz, 4H), 7.77 (t, J ¼ 8.1 Hz,
47.97, 58.27, 118.88, 121.89, 126.55, 133.03, 134.67, 140.82, 172.01,
182.19, 189.47. HRMS (EI) m/z calcd for [M]þ: 464.2424, found:
464.2424.
2H), 8.05 (dd, J ¼ 7.5 Hz, 1.2 Hz, 2H), 9.14 (dd, J ¼ 8.4 Hz, 1.2 Hz, 2H),
12.17 (s, 2H). 13C NMR (CDCl3, 75 MHz)
d (ppm): 35.65, 38.03, 46.82,
118.06, 121.99, 126.33, 132.90, 135.35, 141.43, 171.19, 181.83, 190.97.
HRMS (EI) m/z calcd for [M]þ: 408.1798, found: 408.1783.
6.2.21. 1,8-Bis[2-(dipropylamino)acetamido]anthraquinone (3t)
The pure compound was obtained as yellow powder (82%). Mp:
129e130 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3167(NH), 2957 (NH), 1694
6.2.27. 1,8-Bis[2-(isopropylamino)propionamido]anthraquinone (4e)
The pure compound was obtained as orange powder (58%). Mp:
105e106 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3249 (NH), 2967 (NH),1689
(CO), 1628 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 0.91 (t,
J ¼ 7.5 Hz, 12H), 1.55e1.62 (m, 8H), 2.62e2.67 (m, 8H), 3.38 (s, 4H),
7.76 (t, J ¼ 8.1 Hz, 2H), 8.07 (dd, J ¼ 7.8 Hz, 1.5 Hz, 2H), 9.23 (dd,
J ¼ 8.7 Hz, 1.5 Hz, 2H), 12.53 (s, 2H). 13C NMR (CDCl3, 75 MHz)
(CO), 1624 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 1.11 (d,
J ¼ 6.3 Hz, 12H), 2.78 (t, J ¼ 6.3 Hz, 4H), 2.88e2.92 (m, 2H), 3.06 (t,
J ¼ 6.3 Hz, 4H), 7.77 (t, J ¼ 7.8 Hz, 2H), 8.05 (dd, J ¼ 7.8 Hz, 1.5 Hz,

C.-C. Lee et al. / European Journal of Medicinal Chemistry 50 (2012) 102e112
111
2H), 9.14 (dd, J ¼ 8.7 Hz, 1.5 Hz, 2H), 12.14 (s, 2H). 13C NMR (CDCl3,
6.2.33. 1,8-Bis[2-(dimethylpropanediamino)propionamido]
anthraquinone (4k)
75 MHz)
d (ppm): 22.20, 38.61, 42.12, 48.04, 117.59, 121.65, 126.04,
132.59, 135.15, 141.36, 171.11, 181.37, 190.55. HRMS (EI) m/z calcd for
The pure compound was obtained as orange powder (44%). Mp:
[M þ H]þ: 464.2424, found: 464.2425.
107e108 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3254 (NH), 2928 (NH),1688
(CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 1.67e1.74 (m, 4H), 2.19 (s,
6.2.28. 1,8-Bis[2-(N-ethylmethylamino)propionamido]
anthraquinone (4f)
12H), 2.32 (t, J ¼ 7.2 Hz, 4H), 2.73 (t, J ¼ 6.9 Hz, 4H), 2.79 (t,
J ¼ 6.3 Hz, 4H), 3.06 (t, J ¼ 6.3 Hz, 4H), 7.76 (t, J ¼ 7.8 Hz, 2H), 8.04
(dd, J ¼ 7.5 Hz, 1.2 Hz, 2H), 9.14 (d, J ¼ 8.4 Hz, 2H), 12.12 (s, 2H). 13C
The pure compound was obtained as orange powder (79%). Mp:
129e130 ꢀC (EtOH). IR (KBr, nmaxcmꢃ1): 3233 (NH), 2969 (NH), 1700
NMR (CDCl3, 75 MHz)
d (ppm): 27.33, 38.41, 44.79, 47.70, 57.33,
(CO), 1624 (CO). 1H NMR (CDCl3, 300 MHz)
d
(ppm): 1.09 (t,
117.93, 121.84, 126.24, 132.85, 135.26, 141.48, 171.19, 181.73, 190.88.
J ¼ 7.2 Hz, 6H), 2.33 (s, 4H), 2.54 (q, J ¼ 7.2 Hz, 4H), 2.70 (t, J ¼ 6.9 Hz,
4H), 2.86 (t, J ¼ 6.6 Hz, 4H), 7.72 (t, J ¼ 8.4 Hz, 2H), 8.00 (dd,
J ¼ 7.5 Hz, 1.2 Hz, 2H), 9.09 (dd, J ¼ 8.7 Hz, 1.2 Hz, 2H), 12.14 (s, 2H).
FAB-MS m/z (%): 552 ([Mþ2]þ,100).
6.3. Cell culture and assessment of hTERT
13C NMR (CDCl3, 75 MHz)
d (ppm): 11.41, 36.20, 40.80, 50.74,51.93,
117.89, 121.70, 126.33, 132.67, 135.08, 141.36, 171.07, 181.60, 190.45.
Non-small lung cancer cells H1299 were grown in RPMI 1640
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 ꢀC [22]. Culture media were changed every 3
days. To establish stable cell lines so that the expression of hTERT
could be monitored by a reporter system, a w3.3 kbp DNA fragment
ranging from ꢃ3338 to þ1 bp of the hTERT gene was subcloned
upstream to a secreted alkaline phosphatase gene (SEAP) and
transfectectd into H1299 by electroporation. The stable clones were
selected using G418. The stable clones derived from H1299 was
cultured using conditions that are similar to their parental cells.
HRMS (EI) m/z calcd for [M]þ: 464.2424, found: 464.2419.
6.2.29. 1,8-Bis[3-(isobutylamino)propionamido]anthraquinone (4g)
The pure compound was obtained as orange powder (54%). Mp:
224e225 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3249 (NH), 2954 (NH),
1690 (CO), 1627 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 0.93 (d
J ¼ 6.6 Hz, 12H), 1.74e1.81 (m, 2H), 2.51(d, J ¼ 6.6 Hz, 4H), 2.78 (t,
J ¼ 6.3 Hz, 4H), 3.06 (t, J ¼ 6.3 Hz, 4H), 7.77 (t, J ¼ 8.1 Hz, 2H), 8.05
(dd, J ¼ 7.8 Hz, 1.2 Hz, 2H), 9.15 (dd, J ¼ 8.4 Hz, 1.2 Hz, 2H), 12.13 (s,
2H). 13C NMR (CDCl3, 75 MHz)
d (ppm): 19.72, 27.28, 38.00,
44.54,57.12, 117.20, 121.36, 125.74, 132.19, 134.91, 141.11, 170.92,
180.98, 190.10. HRMS (EI) m/z calcd for [M]þ: 492.2737, found:
492.2728.
6.4. Secreted alkaline phosphatase (SEAP) assay
Secreted alkaine phosphatase was used as the reporting system
to monitor the transcriptional activity of hTERT [23]. Here, about
2 ꢂ 103 cells each were grown in 96-well plates and incubated at
37 ꢀC for 24 h and changed with fresh media. Varying amounts of
drugs were added and cells were incubated for another 24 h.
Culture media were collected and heated at 65 ꢀC for 10 min to
inactivate heat-labile phosphatases. An equal amount of SEAP
6.2.30. 1,8-Bis[3-(3,5-difluorobenzylamino)propionamido]
anthraquinone (4h)
The pure compound was obtained as orange powder (40%). Mp:
190e191 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3246 (NH), 2909 (NH),1702
(CO), 1626 (CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 2.76 (t,
J ¼ 6.3 Hz, 4H), 3.05 (t, J ¼ 6.3 Hz, 4H), 3.86 (s, 4H), 6.62 (tt,
J ¼ 9.0 Hz, 2.1 Hz, 2H), 6.89 (dd, J ¼ 8.1 Hz, 1.8 Hz, 4H), 7.78 (t,
J ¼ 8.1 Hz, 2H), 8.06 (dd, J ¼ 7.8 Hz, 1.2 Hz, 2H), 9.15 (dd, J ¼ 8.4 Hz,
buffer (2 M diethanolamine, 1 mM MgCl2, and 20 mM L-homo-
arginine) was added to the media and p-nitrophenyl phosphate was
added to a final concentration of 12 mM. Absorptions at 405 nm
were taken, and the rate of absorption increase was determined.
1.2 Hz, 2H), 12.09 (s, 2H). 13C NMR (CDCl3, 75 MHz)
d (ppm): 38.40,
44.15, 52.41, 101.68, 109.76, 110.08, 117.76, 121.88, 126.10, 132.83,
135.36, 141.46, 144.18, 161.16, 164.29, 170.91, 181.53, 191.00. HRMS
(EI) m/z calcd for [M]þ: 632.2047, found: 632.2044.
6.5. MTT assay for cell viability
6.2.31. 1,8-Bis[2-(N-Boc-ethanediamino)propionamido]
anthraquinone (4i)
The tetrazolium reagent (MTT; 3-(4,5-cimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide, USB) was designed to yield a colored
formazan upon metabolic reduction by viable cells [24,25]. Approx-
imately 2 ꢂ103 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 invitro cytotoxicity,
each compound was dissolved in DMSO and prepared immediately
before the experiments and was diluted into the complete medium
before addition to cell cultures. Test compounds were then added to
the culture medium for designated various concentrations. After 48 h,
The pure compound was obtained as orange powder (30%). Mp:
101e102 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3359 (NH), 2967 (NH), 1692
(CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 1.39 (s, 18H), 2.76 (t,
J ¼ 6.3 Hz, 4H), 2.83 (t, J ¼ 5.7 Hz, 4H), 3.08 (t, J ¼ 6.3 Hz, 4H), 3.28
(q, J ¼ 5.7 Hz, 4H), 5.18 (s, 2H), 7.77 (t, J ¼ 8.4 Hz, 2H), 8.05 (dd,
J ¼ 7.5 Hz, 1.2 Hz, 2H), 9.14 (dd, J ¼ 8.7 Hz, 1.2 Hz, 2H), 12.13 (s, 2H).
13C NMR (CDCl3, 75 MHz)
d (ppm): 27.93, 40.11, 49.33, 53.32,78.75,
118.39, 121.77, 126.07, 132.77, 134.90, 140.85, 155.97, 171.92, 181.90,
an amount of 25
m
L of MTT was added to each well, and the samples
189.71. FAB-MS m/z (%): 666 ([M]þ, 2.17), 668 ([Mþ2]þ, 100).
were incubated at 37 ꢀC for 4 h. A 100
mL solution of lysis buffer
containing 20% SDS and 50% N,N-dimethylformamide was added to
each well and incubated at 37 ꢀC for another 16 h. The absorbency at
550 nm was measured using an ELISA reader.
6.2.32. 1,8-Bis[2-(dimethylethanediamino)propionamido]
anthraquinone (4j)
The pure compound was obtained as orange powder (18%). Mp:
100e102 ꢀC (EtOH). IR (KBr, nmax cmꢃ1): 3248 (NH), 2938 (NH),1690
6.6. Telomere repeat amplification protocol (TRAP) assays
(CO). 1H NMR (CDCl3, 300 MHz)
d (ppm): 2.22 (s, 8H), 2.44(t,
J ¼ 6.0 Hz, 4H), 2.77 (t, J ¼ 6.3 Hz, 4H), 2.80 (t, J ¼ 6.0 Hz, 4H), 3.09 (t,
J ¼ 6.3 Hz, 4H), 7.76 (t, J ¼ 8.1 Hz, 2H), 8.04 (dd, J ¼ 7.8 Hz, 1.5 Hz,
2H), 9.14 (dd, J ¼ 8.7 Hz, 1.5 Hz, 2H), 12.13 (s, 2H). 13C NMR (CDCl3,
Telomerase activity was detected by a modified version of the
TRAP protocol [26e28]. Telomerase products were resolved by 10%
polyacrylamide gel electrophoresis and visualized by staining with
SYBER Green. To test the possible inhibitory effects of PCR reactions
caused by G-quarduplex stabilizers, compounds were also added
after the telomerase extension step of the TRAP assays (TRAP-M). As
75 MHz)
d (ppm): 38.60, 44.87, 45.29, 46.83, 58.57, 118.03, 121.82,
126.30, 132.91, 135.23, 141.53, 171.12, 181.77, 190.92. HRMS (EI) m/z
calcd for [M]þ: 522.2955, found: 522.2964.

112
C.-C. Lee et al. / European Journal of Medicinal Chemistry 50 (2012) 102e112
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Acknowledgments
The present study was supported by National Science Council
grants NSC 100-2113-M-016-001, 99-2113-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 NDMC and
NIHeNCI for their supports and efforts.
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