Synthesis, human telomerase inhibition and anti-proliferative studies of a series of 2,7-bis-substituted amido-anthraquinone derivatives

Article abstract of DOI:10.1016/j.bmc.2008.05.072

Telomerase is important in tumor initiation and cellular immortalization. Given the striking correlations between telomerase activity and proliferation capacity in tumor cells, telomerase had been considered as a potentially important molecular target in

Full text of DOI:10.1016/j.bmc.2008.05.072

Bioorganic & Medicinal Chemistry 16 (2008) 6976–6986
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, human telomerase inhibition and anti-proliferative studies of a series
of 2,7-bis-substituted amido-anthraquinone derivatives
b
a
c
c
c
Hsu-Shan Huang a, , Kuo-Feng Huang , Cho-Lu Li , Yi-Yuan Huang , Yi-Hsuan Chiang , Fong-Chun Huang ,
*
Jing-Jer Lin c,
*
a School of Pharmacy, National Defense Medical Center, Neihu, Taipei 114, Taiwan, ROC
b Chi-Mei Medical Center, Tainan 710, Taiwan, ROC
c Institute of Biopharmaceutical Sciences, National Yang-Ming University, Shih-Pai, Taipei 112, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Telomerase is important in tumor initiation and cellular immortalization. Given the striking correlations
between telomerase activity and proliferation capacity in tumor cells, telomerase had been considered as
a potentially important molecular target in cancer therapeutics. A series of 2,7-diamidoanthraquinone
were designed and synthesized. They were evaluated for their effects on telomerase activity, hTERT
expression, cell proliferations, and cytotoxicity. In the series, compounds (6, 10, 13, 16, 18, 19, 20–22,
and 24) showed potent telomerase inhibitory activity, while compounds 19, 21, and 22 activated hTERT
expression in normal human fibroblasts. The results indicated that 2,7-diamidoanthraquinones represent
an important class of compounds for telomerase-related drug developments. Compounds 8, 16, 18, 26,
and 32 were also selected by the NCI for Screening Program and demonstrated high anti-proliferative
activity against 60 human cancer cell lines. Structure–activity relationships (SAR) study revealed that
the test compounds with side chains two carbon spacer between amido and amine are important struc-
tural moiety for telomerase inhibition. Although the exact mechanism of how this amine group contrib-
utes to its activity is still unclear, however, the amine group in the extended arm of the bis-substituted
anthraquinone might contribute to proper binding to the residues within the grove of G-quadruplex
structure. Our results indicated that the 2,7-disubstituted amido-anthraquinones are potent telomerase
inhibitors that have the potential to be further developed into novel anticancer chemotherapeutic agents.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 16 April 2008
Revised 20 May 2008
Accepted 21 May 2008
Available online 2 June 2008
Keywords:
Telomerase inhibition
Cytotoxicity
Anthraquinones
hTERT
G-quadruplex
SEAP assay
Telomerase assay
TRAP assay
1. Introduction
immortalization in large part through the illegitimate activation
of telomerase expression. The reactivation of telomerase activity
Telomerase inhibitors have been touted as a novel cancer spe-
cific therapy, as most tumor cells have high expression of telome-
rase, whereas most normal somatic cells express low or
undetectable levels of telomerase.1 Telomerase is a reverse trans-
criptase that prevents the mortality checkpoints evoked by pro-
grammed telomere shortening during each round of cell
division.2 Expression of human telomerase reverse transcriptase
(hTERT), the catalytic subunit of telomerase, appears to be a key
determinant for telomerase activity. Insufficient hTERT expression
in most mortal somatic cells cannot produce enough telomerase to
maintain telomere length during cycles of chromosome replica-
tion.3,4 The hTERT is highly expressed approximately 90% in stem
cells, germ cell lines, and most human tumors, and its inhibition
represents a strategy for the development of selective anti-cancer
drugs.5–9 It is also well accepted that human cancer cells achieve
in most cancer cells supports the concept that telomerase is a rel-
evant target in oncology, and telomerase inhibitors have been pro-
posed as new potential anti-cancer agents.10 The important roles
of telomerase in tumor initiation and cellular immortalization
have led to the identification of telomerase as a potentially
important molecular target in cancer therapeutics. The develop-
ment of telomerase inhibitors and G-quadruplex stabilizers has
emerged as a highly promising approach.11–14
Dye ligands have been considered as one of the important alter-
natives to natural counterparts for binding most types of proteins,
because they interact with the active sites of many proteins mim-
icking the structure of the substrates, cofactors, or binding agents
for those proteins.15–17 Interactions between the dye ligand and
proteins can be achieved by complex combination of electrostatic,
hydrophobic, hydrogen bonding. Most of these reactive dyes con-
sist of a chromophore (either azo dyes, anthraquinone, or phat-
halocyanine), linked to
a reactive group (often a mono- or
* Corresponding authors. Tel.: +886
(H.-S.H.); tel.: +886 2826 7258; fax: +886
E-mail addresses: huanghs@ndmctsgh.edu.tw (H.-S. Huang), jjlin@ym.edu.tw
(J.-J. Lin).
2
87926454; fax: +886
2 87923169
dichlorotriazine ring).18 Anthraquinone-containing extracts from
different plant sources such as senna, cascara, aloe, frangula, and
rhubarb have been found to have wide variety of pharmacological
2
2
2820 0067 (J.-J.L.).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.072

H.-S. Huang et al. / Bioorg. Med. Chem. 16 (2008) 6976–6986
6977
H
N
O
O
OH
OH
O
X
X
O
O
HN
HN
OH
OH
O
R
OH
OR'
O
O
N
H
O
1-34
NH2
H3C
OH
Doxorubicin (R= OH, R'= Me)
Daunorubicin (R= H, R'= Me)
Mitoxantrone (X= OH)
Ametantrone (X= H)
Chart 1. Structures of bioactive chromophoric anthraquinones.
activities such as antiinflammatory, wound healing, analgesic, anti-
2. Chemistry
pyretic, antimicrobial, and antitumor activities.19 Furthermore,
anthraquinones, porphyrins, ethidium bromide derivatives, fluore-
nones, heteroaromatic polycyclic systems, quinolines, acridines,
and telomestatins have been identified with high affinity and
selectivity for human quadruplex telomeric sequence.14,20,21 Sev-
eral of the anthraquinone derivatives were also been used as che-
motherapeutic agents in anticancer treatments (see Chart 1). The
anthracycline antibiotics (daunorubicin and doxorubicin) have
been introduced in clinical use more than 30 years ago for treat-
ment of wide variety of cancers.22 Mitoxantrone and ametantrone
are another member of 1,4-disubstituted anthraquinones family
developed to treat malignancies but has rather narrow spectrum
of antitumor activities that limits their clinical usage. They are syn-
thetic representatives of a group of compounds termed amino-
anthraquinones.23
It was first reported by Neidle and co-workers that anthraqui-
nones are potent human telomerase inhibitors.24–28 Previous work
by their laboratory had demonstrated that disubstituted anthra-
quinones with progressively increasingly bulky substituents have
close relationship between telomerase inhibition and binding en-
ergy for the G-quadruplex structure form by human telomeric
DNA sequences.29 To date, a number of families of compounds
have been developed and their telomerase activity and cytotoxicity
have been extensively studied.30–33 It was also shown that the pla-
nar moieties of anthraquinones bind onto the end of the G-quadru-
In previous papers in this series we have described a range of
synthetic route to, biological properties of anthraquinones gener-
ally symmetrical substituted in the 1,4-, 1,5-, 1,8-, 2,6-positions
including benzo-annelation, as well as their mono substituted
derivatives.32,33,39–47 The backbone of these compounds resembles
some of the anti-cancer agents mitoxantrone and ametantrone,
and were prepared by two-stage reactions which are similar to
our previous papers.32,44,46 Our strategy for achieving this objec-
tive is symmetric synthesis of 2,7-diamidoanthraquinone deriva-
tives and
a number of alkylacyl chlorides for acylation of
aromatic amine 2 via N-acylation have been synthesized. After
considerable effort we were able to develop a process by which
a 2,7-disubstituted anthraquinones can be synthesized and the
preparation involved two-step synthetic route with appropriate
yields (overall 20–74% in three steps) (Scheme 1): (1) acylation
reaction of 2,7-diaminoanthraquinone 2 with various alkylacyl
chlorides yielded the corresponding 2,7-bis-(x-chloroacylamido)
side chain compounds 3 and 15; (2) followed by nucleophilic dis-
placement of the chloride with primary or secondary amines to
produce symmetrical peptoid dimer analogs 4–14, and 16–24,
respectively; (3) compounds 26–34 were synthesized by direct
one-step acylation reaction using acid chlorides to obtain various
amido-substituted derivatives. The chloroacetamido- and chloro-
propionami-do-substituted ( 3 and 15) were then converted to
the aliphatic amido-anthraquinones (4–14 16–24) upon heating
in mini-reactor at 130 °C. However, under these reaction condi-
tions significant amounts of the reaction products were isolated
along with the desired reaction products. The quantity of the
byproducts isolated was substrate dependant and purification of
the reaction mixtures required tedious recrystallization and chro-
matography. Under these conditions, a more effective acylation
catalyst can achieve the desired amide linker derivatives (26–34)
with appropriate yields.
plex structure through p–p stacking interactions with the guanine
residues and the substituents bind to the grooves of the G-quadru-
plex structures.24,26,30,34–36 Among the quadruplex ligands studies,
disubstituted amido-anthraquinones represent one of the best
small molecules of how the pattern of disubstitution modulates
DNA duplex versus quadruplex selectivity.24,26,37 Thus, anthraqui-
nones might inhibit telomerase-dependent telomere elongation
in vitro by stabilizing of G-quadruplex structures by sequestering
the DNA substrate and making it inaccessible to the telomerase.38
To explore novel and potent telomerase inhibitors, we have re-
cently synthesized a series of anthraquinones and examines their
toxicity to cancer cells as well as their telomerase inhibitory activ-
ity. In past work we have reported the synthesis of a range of struc-
turally diverse anthraquinones.32,33,39–47 In the present study, we
have designed and synthesized a series of 2,7-disubstituted amid-
oanthraquinone derivatives and evaluated their effects to telome-
rase activity as inferred from the TRAP assay and collated some
selected compounds growth-percent against tumor cell lines in vi-
tro (NCI60 assays). The antiproliferative effect and hTERT repress-
ing activity of these newly synthesized compounds were also
determined. Our results indicated that the 2,7-disubstituted ami-
do-anthraquinones are potent telomerase inhibitors that have the
potential to be further developed to be novel anticancer chemo-
therapeutic agents.
3. Telomerase activity
We evaluate the effects of these 2,7-diamidoanthraquinones on
telomerase inhibition using PCR-based telomerase assay, and TRAP
(telomeric repeat amplification protocol) assay. We also designed
and expressed SEAP assay, MTT assay, and TRAP assay to provide
a suitable target for telomerase activity assay. Results are summa-
rized in Table 1 and all test derivatives showed MTT and SEAP inhi-
bition activity in micromolar range. This strategy takes advantage
of the anthraquinone-base structure for analysis. A related con-
struct was used as a telomerase inhibitor, in studies mentioned
above. Given the striking correlations between telomerase activity
and anti-proliferation capacity in tumor cells, we expected that
analysis of anthraquinone-base chromophore telomerase might

6978
H.-S. Huang et al. / Bioorg. Med. Chem. 16 (2008) 6976–6986
O
O
H
N
H
N
H
N
H
N
R1
R1
ii
Cl
Cl
N
H
N
H
O
O
O
O
O
O
3
4-14
O
H
N
H
N
i
Cl
Cl
O
O
O
O
O
O
i
O
O
25
H2N
NH2
O
H
N
H
N
i
O
2
R3
R3
i
O
26-34
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
Cl
Cl
ii
R2
R2
O
O
O
15
16-24
Scheme 1. Reagents and conditions: (i) RCOCl, DMF, pyridine, rt, 24 h; (ii) RNH2, pyridine, mini-reactor, 130 °C, 40 min.
yield further insight into this relationship. We screened a series of
anthraquinone-base derivatives, searching for compounds that ex-
hibit inhibition of telomerase in SEAP assay and less cytotoxicity in
MTT assay. As shown in Table 1, compound 3, which contains sim-
ple side chain of –CH2Cl unit appended to the substituent in the
2,7-positions, showed high selective inhibition activity toward
H1299 cancer cells over hTERT-immortalized normal cells. There-
fore, the torsion angles of various compounds substituted on the
anthraquinone might be related to the inhibitory activity on recep-
tor binding site. As an alternative possibility, the substituent at the
appropriate position might fill a cavity of the receptor site. The re-
sults of a structure–activity relationships (SAR) study indicated
that the torsion angle between the central chromophore ring and
the chloroacetamido substituent, and the bond length of linker at
the anthraquinone moiety, might be important for G-quadruplex
binding activity. We also evaluate the effects of these disubstituted
2,7-amidoanthraquinones on telomerase activity using PCR-based
telomerase assay as well as TRAP assay. Comparative in vitro telo-
merase inhibitory screening of this group of compounds provided
inhibition of G-quadruplex-induced telomerase at the IC50 levels
of 16.0, 13.3, and 9.7 M, respectively. Compound with side chain
pyrrole 18 (IC50 6.7 M) and piperidine 19 (IC50 8.4 M) is the most
potent telomerase inhibitors in this series of compounds. As
shown in Figure 2, the SEAP activities are elevated in cells that
are comparison with MTT and SEAP. Compounds with side chains
–NHCO–(CH2)2NC5H10 19, –NHCO–(CH2)2NHC3H7 21, and –NHCO
–(CH2)2NHC2H5 22 show significant activity of PhTERT-SEAP.
l
l
l
4. Antitumor activity
The 2,7-disubstituted anthraquinone derivatives were studied
for anti-proliferative activity against human cancer cell lines in Na-
tional Cancer Institute Drug Screen Program. Out of the synthe-
sized derivatives, compounds 8, 16, 18, 26, and 32 have been
selected by NCI. Those potential compounds were evaluated in
the full panel of human tumor cell lines derived from nine cancer
cell types (non-small cell lung cancer, colon cancer, breast cancer,
ovarian cancer, leukemia, renal cancer, melanoma, prostate cancer,
CNS cancer). They were evaluated in the 60 cell line panel and anti-
cancer assay was performed in accordance with the protocol of
developmental therapeutics program (one dose mean graph). Re-
sults for each compound were reported as the growth percentage
of the treated cells when compared to that of the untreated control
cells. Our results indicated that only compounds 8, 16, and 18
showed significant cytotoxic activity. The growth percent values
at 10ꢀ5 molar for 60 cancer cell lines are shown in Table 2. It is
especially notable that compound 8 with side chain –NHCO–
CH2N(CH3)2 displayed relatively potent and differential cytotoxic
activity. The colon cancer panel, leukemia, renal, melanoma and
CNS cancer lines were quite sensitive to 8, while the non-small cell
lung cancer lines, breast, ovarian and prostate panels were less
sensitive. It is surprising since compound 16 with side chain
–NHCO–(CH2)2N(C2H5)2 has similar differences in cytotoxicity
which might have a significant effect on their interaction with
DNA. It is especially notable that compound 16 had both cytotox-
icity and telomerase activity whereas for cytocidal effects this dif-
ference was significant. However, it had significant influence on
some intriguing observations of inhibitory profiles. At 1–20
lM
concentrations, among total of 2,7-diamidoanthraquinones
a
tested, we found 10 of them showed telomerase inhibitory effect.
The inhibitory effects of these compounds on telomerase are spe-
cific as they did not affect the internal controls (ICs) for Taq poly-
merase in our assays (Fig. 1). Among a total of compounds tested,
several trends were also evident from the results of testing this
group of compounds, we found that seven compounds (16, 18–
19, 20, 21, 22, and 24) showed telomerase inhibitory effect at the
concentrations of 1, 5, 10, and 20 lM. These seven compounds of
2,7-diamidoanthraquinones with side chains two carbon spacer
between amido and amine. It is interesting to note that most of
these compounds with side chain –CH2CH2N– did affect telome-
rase inhibitory activity. Thus, in addition to anti-cancer function,
our finding indicated that the spacer and the nature of the center
nitrogen atom of the side chain might have potential SAR for telo-
merase inhibitory activity and antineoplastic activity. As shown in
Figure 1, compounds with side chains –NHCO–CH2NHC2H5 10,
–NHCO–(CH2)2NHC3H7 21, and –NHCO–(CH2)2NHC2H5 22 show

Table 1
Effects of symmetrical 2,7-diamidoanthraquinone analogs on activating or repressing hTERT expression and telomerase activity
Compound
R (R1, R2, R3)
Cell type (inhibition
l
M
SD)a
H1299
BJ1
MTT
8
SEAP
MTT
SEAP
100
69 10
65 10
10
95
1
100
62
63 12
36
37 10
20 13
10
85 21
1
100
10
91
1
100
65 23
10
60 35
ND
69 34
131 57
24 24
1
1
2
3
4
5
2,7-Dinitroanthraquinone
2,7-Diaminoanthraquinone
CH2Cl
CH2N(CH2CH3)2
CH2NHCH2CH2CH2CH3
97 15
90 17
58 44
101
91 16
8
87 13
87 10
72
90
86
80
88
50
43
3
2
2
1
1
2
1
3
2
1
91
95
105
116
103
2
2
2
4
1
55 21
80 16
75
40
8
1
83
89
67
41
ND
ND
ND
26
1
25
0
3
5
47
17
3
7
9
1
3
3
135 21
116 34
80 14
8
61 11
26 17
4
15 11
77 18
27 18
N
N
6
7
23 14
52 13
37 15
99
9
30 10
63 15
48 13
69 20
88 10
39
22
1
2
60
78
2
3
99
89
2
1
ND
ND
ND
35 11
50 16
74 19
103
5
121
3
118 16
8
9
10
CH2N(CH3)2
CH2NHCH2CH2CH3
CH2NHCH2CH3
15 12
14 10
55
54 11
24
71
9
92 13
89 15
52 11
21 14
24 10
40 20
44 16
87 12
73 17
60 10
39
42
37
1
1
1
45
44
56
1
0
2
96
96
72
3
3
2
3
ND
17 21
13
44
ND
41
5
4
96
ND
90
1
5
3
4
5
4
34
3
35
5
N
N
11
12
37
6
90 14
23
9
50 11
72 20
76 21
89 17
47
76
3
2
69
1
86
88
1
2
97 30
68 21
61 21
N
88 11
85 13
88 19
78 10
60 12
85 15
ND
ND
ND
NH
13
14
15
16
17
CH2NHCH(CH3)2
CH2NHCH2CH(CH3)2
CH2CH2Cl
CH2CH2N(CH2CH3)2
CH2CH2NHCH2CH2CH2CH3
6
11
11
5
26 15
39 10
44 15
41 20
21 16
26 10
28 18
38 15
73 31
73 20
94 11
76 19
56 14
39
39
39
36
40
1
0
1
1
0
41
37
65
55
70
2
0
1
3
1
89
107
77
103
80
1
3
2
2
3
45 11
55 19
79 14
108
8
77 13
106 15
82 12
40
ND
37
3
44
ND
49
2
83
ND
84
5
29 11
87
9
94
35 17
33
7
1
1
26 11
22 17
5
3
5
14 10
52
4
9
111 25
95 46
88 34
18
19
12 10
54
7
87 15
87 15
17 13
25 10
29 15
73 11
55 23
39
41
0
2
68
54
0
2
82
92
4
6
109 18
67 16
ND
N
N
18
1
9
1
44
7
33
8
234 20
226 51
186 47
20
21
22
CH2CH2N(CH3)2
CH2CH2NHCH2CH2CH3
CH2CH2NHCH2CH3
53 13
50
59
1
104
1
22
13 30
22 14
33 24
5
94
22 13
38
42
30 14
65
73
94
75
82
7
1
2
38
0
61
2
86
ND
211 38
2
13 10
6
3
80 15
86 10
13 15
22 13
52 13
68 29
2
1
2
3
217 40
97 87
133 48
217 133
34
7
23
24
39
6
80 15
95 18
21 10
71 15
52 11
71 21
75 16
75 26
61
88
1
4
80
2
3
95
96
2
2
232 66
138 115
ND
N
N
N
102 13
94
40
8
2
97
8
100
131 29
75 16
118 26
42 45
163 22
13 58
NH
25
26
27
28
CH2CH2CH2Cl
CH3
22
2
84 18
117 15
70 25
93 14
78 17
74 18
85 16
86 13
87 14
95 14
88 19
97 10
90 23
58
91
48
50
3
2
2
6
62
91
57
51
2
2
2
8
72
106
77
3
1
2
2
55 11
35 15
40 15
83 13
38 17
59 13
117
51
5
8
117
64
ND
3
8
123
78 10
ND
(continued on next page)
5
CH2CH3
CH2CH2CH3
83
4
83
6
90
8
84
ND

6980
H.-S. Huang et al. / Bioorg. Med. Chem. 16 (2008) 6976–6986
the spectrum of anti-proliferative activity. Compound 18 had
selective cytotoxicity for colon and melanoma cell lines, and com-
pounds 26 and 32 did not have significant growth inhibitory activ-
ities. The lack of anti-proliferative activity of compounds 26 and 32
demonstrates the important role of the spacer length between the
amino group and chromophore for cytotoxic activity.48 The opti-
mal number of atoms between amido nitrogen atom and terminal
side chain nitrogen is 2, which corresponds to ꢁ4 Å distance. NCI60
cell line panel assays from some selective compounds give only
limited information and observations on mechanisms of drug ac-
tion; for these DNA-affinic tricyclic ring systems more precise
physicochemical measurements are required which DNA polymor-
phic form is the target.
5. Results and discussion
At present, we described a method of synthesizing 2,7 bis-
substituted anthraquinones and compared to their cytotoxicity
and telomerase activity. A structure–activity relationship study
was also conducted on a number of amidoanthraquinone deriva-
tives.32,33,41,45,46,49–53 Appropriately substituted amido-anthraqui-
nones are effective G-quadruplex stabilizers, but without
sufficient information is available as yet on the possible modifica-
tion of G-quadruplex recognition and telomerase inhibition pro-
duced by the position of the amide bond.31 In this investigation,
we continue to focus our attention on the role of our systematic
synthesized 2,7-diamidoanthraquinones bearing the –CO–NH–
group and spacer linked to the planar anthraquinone moiety and
to understand the basis of amido-anthraquinones selectivity. The
chemical and biological activities of anthraquinone compounds
are greatly affected by its various substituents of the planar tricy-
clic ring system which considered as aglycon analogs of anthracy-
cline antibiotics. Since telomerase is an important component of
cancers, we are interested in examining the effects of these com-
pounds on telomerase activity in the cell free extracts prepared
from H1299 cells. The structure–activity analysis of these com-
pounds also revealed that a terminal long aliphatic chain is not a
favorable inhibitor for telomerase (compounds 5, 17). In contrast,
a cyclic or branched terminal chain might favor telomerase
inhibition.
The telomerase activity is regulated mostly at the transcrip-
tional level for its catalytic subunit, hTERT, and partly at the
post-translational level.54 Since the expression of human telome-
rase catalytic component is the key regulator in telomerase
activity, we analyzed the expression of telomerase by monitoring
the expression of hTERT as the criteria. To facilitate the analysis,
we constructed a reporter system that enables the evaluation of
the hTERT promoter activity using a reporter gene, SEAP, in can-
cer cell line H1299 and hTERT-immortalized normal cell line
hTERT-BJ1. The expression of SEAP in H1299 cells harboring
PhTERT-SEAP was used as the criteria to evaluate if anthraquinone
derivatives inhibited the expression of hTERT in cancer cells. The
level of cell viability in these cells was also determined using
MTT assay. As shown in Table 1, most of the compounds did
show various effects on SEAP expression. However, all of them
also affect the proliferation of the treated cells. Thus, although
it is not apparent whether the telomerase repression effects of
these compounds are caused by their cytotoxicity, it is conceiv-
able that inhibition of telomerase expression by these com-
pounds inhibits cell proliferation.
We then used the expression of SEAP in hTERT-BJ1 cells harbor-
ing PhTERT-SEAP as the criteria to evaluate if anthraquinones acti-
vate the expression hTERT in normal cells. The level of cell
viability in these cells was also determined using MTT assay and
the results are shown in Table 1. The cytotoxicity levels in

H.-S. Huang et al. / Bioorg. Med. Chem. 16 (2008) 6976–6986
6981
O
O
H
N
H
N
H
N
H
N
A
N
N
N
N
H
N
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
18
10
O
O
H
N
H
N
H
N
H
N
N
N
H
N
H
O
O
19
13
O
O
H
N
H
N
H
N
H
N
HN
NH HN
NH
O
O
21
22
O
O
H
N
H
N
H
N
H
N
N
N
N
N
N
N
O
O
6
16
N
N
O
O
H
N
H
N
H
N
H
N
O
O
N
H
N
H
24
20
18
19
21
22
24
13
20
10
16
6
B
P
N
Telomere
ladder
IC
6.7
8.4
13.3
9.7
9.1
15.5
15.8
16.0
14.2
13.1
Figure 1. Inhibition of telomerase activity by symmetrical 2,7-diamidoanthraquinone. (A) Structure of 2,7-diamidoanthraquinones that showed telomerase inhibitory
activities. (B) TRAP assay was conducted using cell extracts prepared from H1299 cells and 2 g of extracts were used in each assay. Extended products were separated on a
10% polyacrylamide gel and visualized with SYBER Green staining. The photo pictures of the results are presented. The concentration of test compounds was 1, 5, 10, 20 M,
l
l
respectively. Because the internal control (IC) shares one oligonucleotide with the reaction, the IC products became apparent when telomerase activities were inhibited. P is
positive control and N is negative control.
hTERT-BJ1 cells are moderately higher than that in human tumor
cell lines for most of the tested compounds. Strikingly, compound
3 showed high selective inhibiting activity toward H1299 cancer
cells over hTERT-immortalized normal cells (Table 1). Although
the mechanism of preferential inhibition of compound 3 is still un-
clear to us, the compound might has the potential to be developed
into a drug for anticancer therapies. We also found several 2,7-
diamidoanthraquinone derivatives activated the SEAP levels (com-
pounds 19, 21, 22). Interestingly, these three compounds all carry R
@ CH2CH2N– groups, suggesting that the structural moiety might
be important for its activity in activate hTERT expression. Among
these compounds, we are most interested in compound 22 because
it activated the SEAP levels without greatly affecting the cell viabil-
ity at low concentrations (Fig. 2).
6. Conclusion
In summary, we have designed and synthesized a series of 2,7-
diamidoanthraquinones and evaluated their effects on telomerase
activity, hTERT expression, cell proliferations. Given the striking
correlations between telomerase activity and proliferation capacity
in tumor cells, we expected that analysis of anthraquinone-base
chromophore telomerase might yield further insight into designing
better compounds for anticancer therapies. Of the series of com-

6982
H.-S. Huang et al. / Bioorg. Med. Chem. 16 (2008) 6976–6986
Table 2
Cytotoxicity of symmetrical 2,7-diamidoanthraquinone derivatives in the NCI in vitro
60-cell drug screen program
Growth percenta (at 10ꢀ5 M)
Compound
8
16
18
26
32
Non-small cell lung cancer
EKVX
HOP-62
HOP-92
NCl-H226
NCI-H23
NCl-H322M
NCl-H460
NCl-H522
102.30
59.99
11.65
9.70
49.89
30.34
44.75
9.50
58.72
18.79
7.04
41.10
31.49
45.79
25.52
29.16
95.28
94.31
28.97
56.14
89.14
51.78
32.42
5.01
110.60
101.26
104.51
94.24
97.50
109.29
93.56
91.64
86.18
100.60
95.85
105.77
89.03
89.41
61.82
27.98
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
ꢀ73.57
ꢀ73.97
19.07
16.26
11.73
66.95
23.36
ꢀ49.69
ꢀ87.42
ꢀ32.45
17.42
3.30
ꢀ29.04
19.99
10.51
56.14
19.47
32.55
20.01
119.85
105.35
101.91
93.93
107.23
105.94
103.21
91.92
132.37
87.14
86.35
106.26
92.67
21.82
16.93
104.75
Breast cancer
BT-549
HS 578T
85.36
ꢀ6.11
7.92
57.04
ꢀ23.77
9.34
31.32
71.07
59.52
68.62
76.91
5.26
93.29
133.05
96.90
103.99
109.48
114.26
96.17
92.51
118.90
77.38
97.05
97.35
90.83
79.75
MCF7
13.35
30.47
68.84
80.59
58.98
MDA-MB-231/ATCC
MDA-MB-435
NCI/ADR-RES
T-47D
4.49
68.36
47.84
24.62
Ovarian cancer
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
43.63
45.70
73.15
32.31
70.26
10.39
24.92
33.63
4.30
36.88
49.82
18.81
19.58
27.14
111.06
103.60
102.27
99.85
103.35
97.54
112.01
94.34
93.16
44.02
101.12
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
ꢀ13.21
ꢀ64.97
34.91
29.12
34.97
ꢀ6.02
14.01
8.10
3.74
85.84
6.40
22.97
47.90
19.75
99.77
38.74
98.54
96.96
121.47
3.61
90.67
62.45
91.60
94.29
88.38
1.69
Figure 2. Activation of PhTERT-SEAP by 2,7-diamidoanthraquinone. About
2 ꢂ 103 cells of hTERT-BJ1 harboring PhTERT-SEAP or PCMV-SEAP were seeded in
96-well plates and incubated at 37 °C for 24 h. Cells were then washed with PBS,
recultured in fresh media, and incubated with varying amounts of compounds 19,
21, or 22 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.
Values were presented from six experiments using the values without drug
treatment as 100%.
5.42
ꢀ29.02
-46.45
ꢀ0.39
Renal cancer
786-0
A498
68.60
ꢀ6.39
66.43
59.26
27.99
73.47
ꢀ82.55
24.72
ꢀ38.76
11.09
3.27
8.63
17.16
ꢀ31.53
40.51
ꢀ0.91
29.65
55.55
6.68
97.72
115.60
96.93
105.10
113.35
118.38
96.42
94.92
92.35
86.70
88.43
82.93
136.20
95.55
ACHN
CAK1-1
SN12C
TK-10
pounds analyzed, compounds (6, 10, 13, 16, 18–22 and 24) showed
potent telomerase inhibitory activity, compounds (19, 21 and 22)
activated hTERT expression in normal human fibroblasts. The re-
sults indicated that 2,7-diamidoanthraquinones represent an
important class of compounds for telomerase-related drug devel-
opments. The results of structure–activity relationships (SAR)
study also revealed that an amine with two carbon space is an
important structural moiety for telomerase inhibition. The exact
mechanism of how this amine group contributes to its activity is
unclear. However, the amine group in the extended arm of the
disubstituted anthraquinone might contribute to proper binding
to the residues within the grove of G-quadruplex structure. The
number of C might determine the length of the extended arm for
its proper bindings.
31.22
5.27
UO-31
Melanoma
LOX IMVI
M14
MALME-3M
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
ꢀ52.34
75.04
96.21
ꢀ30.35
32.11
82.59
ꢀ1.84
22.31
3.39
62.79
62.39
ꢀ60.38
2.56
82.75
ꢀ14.46
44.45
30.74
53.88
47.00
99.05
105.11
111.26
30.15
117.60
108.87
26.97
90.27
98.89
91.76
16.85
108.51
75.67
107.63
81.00
ꢀ45.21
14.99
56.94
ꢀ17.88
42.72
101.24
Prostate cancer
DU-145
PC-3
78.68
59.00
10.20
13.59
29.53
21.66
116.79
102.73
98.83
106.89
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
Mean
Delta
Range
The mechanism of how 2,7-diamidoanthraquinones affect
hTERT expression and behind these diversified biological effects
is still unclear. Since several of anthraquinone derivatives were
shown to have G-quadruplex stabilizing activity, it is possible that
compounds 19, 21,22 affect hTERT expression through stabilizing
of G-quadruplex structure in the promoter region. Indeed, stabil-
ization of G-quadruplex structures by small molecule compounds
was shown to repress the expression of c-myc.55 A bioinformatic
survey of hTERT promoter for putative G-quadruplex-forming
sequences has identified one such sequence located at ꢀ2963
64.92
ꢀ83.51
ꢀ3.72
79.57
76.12
16.26
28.70
112.21
185.81
35.18
ꢀ19.91
37.15
75.84
46.62
21.65
16.33
103.75
170.17
26.67
48.62
33.75
66.97
77.47
18.16
30.97
76.18
131.05
113.68
103.25
103.04
94.40
106.91
95.14
99.06
95.45
129.44
95.09
86.60
93.24
98.79
89.23
72.62
91.54
89.85
134.51
a
Data obtained from NCI in vitro 60-cell drug screen program at 10ꢀ5
M
concentration.

H.-S. Huang et al. / Bioorg. Med. Chem. 16 (2008) 6976–6986
6983
position. Even though it remains to be tested for the biological
relevance of this sequences in hTERT promoter, several of these
G-quadruplex-forming sequences are indeed capable of forming
G-quadruplex structures in other genes. For example, the G-rich
region within the promoter sequences of c-myc, VEGF, c-kit, BCL2,
and KRAS proto-oncogenes was capable of forming G-quadruplex
structures.55–59 It will be interesting to test the role of this putative
G-quadruplex-forming sequence in hTERT expression. Anthraqui-
none-base derivatives are known for their potential antitumor
and telomerase activities. Since telomerase reverse transcriptase
activity depends on the 30 single-stranded telomeric DNA end act-
ing as a primer,31 disubstituted amidoanthraquinone molecules
represent an example that might bind and stabilize the folded
quadruplex form of the primer and DNA duplex. The aim of this
study was to elucidate the structure–activity relationships of sim-
ple 2,7-symmetrical bis-substituted analogs of amido-anthraqui-
none to further delineate the nature of the requirements of the
pharmacophore. A striking feature of the results given in Tables 1
and 2 is the marked repressing hTERT expression [PhTERT-SEAP
(H1299)], [PhTERT-SEAP (BJ-1)] and cytotoxicity. The inhibitory ef-
fect and cytotoxicity do not seem to correlate with the modifying
groups. As a whole, these data support the conclusion that all the
tested molecules, including those reported here, share a common
target in telomerase and cytotoxicity. These results confirm amide
bond direction in amido-anthraquinones that would favor G-quad-
ruplex recognition, DNA-interactive, and targeting telomerase in
preventive and/or curative therapy.
ppm 42.99, 115.61, 123.50, 127.87, 127.97, 133.63, 143.21,
165.08, 179.79, 181.67.
7.2.2. 2,7-Bis[2-(diethylamino)acetamido]anthraquinone (4)
Product 4 was obtained as yellow powder (yield 61%): mp
241 °C (EtOH). IR (KBr) (cmꢀ1
) 1679, 1702, 3250. MS (EI,
70 eV) = 464 (M+). 1H NMR (300 MHz, DMSO): d ppm 1.01 (t,
J = 7.0 Hz, 12H), 2.61 (q, J = 6.9 Hz, 8H), 3.22 (s, 2H), 8.09
(d,J = 8.4 Hz, 2H), 8.13 (dd, J = 9.6, 1.2 Hz, 2H), 8.50 (s, 2H), 10.27
(s, 2H). 13C NMR (300 MHz, DMSO): d ppm 11.30, 47.17, 56.80,
115.73, 123.56, 127.57, 127.62, 133.58, 143.15, 170.41, 179.75,
181.80.
7.2.3. 2,7-Bis[2-(butylamino)acetamido]anthraquinone (5)
Product 5 was obtained as yellow powder (yield 40%): mp
125 °C (EtOH). IR (KBr) (cmꢀ1) 1671, 3331. MS (EI, 70 eV) = 464
(M+). 1H NMR (300 MHz, CDCl3): d ppm 0.94 (t, J = 7.2 Hz, 6H),
1.41 (m, 4H), 1.52 (m, 4H), 2.69 (t, J = 6.8 Hz, 4H), 3.41 (s, 4H),
8.09 (d, J = 1.5Hz, 2H), 8.25 (d, J = 8.4Hz, 2H), 8.31 (dd, J = 8.4,
2.1 Hz, 2H), 9.85 (s, 2H). 13C NMR (300 MHz, CDCl3): d ppm
13.82, 20.23, 32.15, 50.04, 53.03, 116.29, 123.88, 128.96, 129.14,
34.53, 142.79, 170.73, 180.72, 182.45.
7.2.4. 2,7-Bis[2-(pyrrolidino)acetamido]anthraquinone (6)
Product 6 was obtained as pale yellow powder (yield 33%): mp
201 °C (EtOH). IR (KBr) (cmꢀ1
) 1674, 1699, 3358. MS (EI,
70 eV) = 460.2 (M+). 1H NMR (300 MHz, CDCl3): d ppm 1.88 (m,
8H), 2.71 (t, 8H), 3.32 (s, 4H), 8.09 (d, J = 2.1 Hz, 2H), 8.28 (d,
J = 8.4Hz, 2H), 8.37 (dd, J = 8.7, 2.4 Hz, 2H), 9.58 (s, 2H). 13C NMR
(300 MHz, CDCl3): d ppm 24.11, 54.65, 59.79, 116.40, 124.07,
129.00, 129.17, 134.50, 142.88, 169.68, 180.71, 182.60.
7. Experimental
7.1. General procedures
7.2.5. 2,7-Bis[2-(piperidino)acetamido]anthraquinone (7)
Melting points were determined with a Büchi 545 melting point
apparatus and are uncorrected. All reactions were monitored by
TLC, which were performed on precoated sheets on silica gel 60
F254, and flash column chromatography was done in silica gel (E.
Merck, 70–230 mesh) with CH2Cl2 as eluant, unless otherwise sta-
ted. 1H NMR spectra were recorded with a Varian GEMINI-300
(300 MHz). d values are in ppm relative to a tetramethylsilane
internal standard. Mass spectra (EI, 70 eV, unless otherwise stated)
were obtained on a Finnigan MAT TSQ-46 and Finnigan MAT TSQ-
700. All other reagents and solvents were purchased from either
Aldrich or Merck and used without further purification.
Product 7 was obtained as yellow powder (yield 51%): mp
243 °C (EtOH). IR (KBr) (cmꢀ1
) 1678, 1706, 3242. MS (EI,
70 eV) = 488 (M+). 1H NMR (300 MHz, CDCl3): d ppm 1.49 (m,
4H), 1.65 (m, 8H), 2.54 (t, J = 4.8 Hz, 8H), 3.09 (s, 4H), 8.07 (d,
J = 2.1 Hz, 2H), 8.23 (d, J = 8.4 Hz, 2H), 8.28 (dd, J = 8.7, 1.8 Hz,
2H), 9.68 (s, 2H). 13C NMR (300 MHz, CDCl3): d ppm 23.52, 26.20,
54.89, 62.77, 116.40, 124.11, 129.12, 129.25, 134.62, 142.87,
169.58, 180.83, 182.67.
7.2.6. 2,7-Bis[2-(dimethylamino)acetamido]anthraquinone (8)
Product 8 was obtained as yellow powder (yield 26%): mp
218 °C (EtOH). IR (KBr) (cmꢀ1) 1630, 1672, 1701, 3330. MS (EI,
70 eV) = 408 (M+). 1H NMR (300 MHz, CDCl3): d ppm 2.38 (s,
12H), 3.09 (s, 4H), 8.04 (d, J = 2.1 Hz, 2H), 8.19 (d, J = 8.4 Hz, 2H),
8.28 (dd, J = 8.4, 2.1 Hz, 2H), 9.55 (s, 2H). 13C NMR (300 MHz,
CDCl3): d ppm 45.98, 63.56, 116.39, 123.99, 129.05, 129.20,
134.54, 142.84, 169.32, 180.74, 182.50.
7.2. General synthetic methods
2,7-Diaminoanthraquinone (2) were prepared according to the
literature.26 All anthraquinone-base derivatives were synthesized
by using a simply acylation or two-stage reaction in mini-reactor.
These compounds were obtained in good yield and their purity
was determined using mass spectrometry, 1H NMR, and 13C
NMR.
7.2.7. 2,7-Bis[2-(propylamino)acetamido]anthraquinone (9)
Product 9 was obtained as dark yellow powder (yield 30%): mp
143 °C (EtOH). IR (KBr) (cmꢀ1) 1670, 3333. MS (EI, 70 eV) = 436.2
(M+). 1H NMR (300 MHz, CDCl3): d ppm 0.98 (t, J = 7.5Hz, 6H),
1.56 (m, 4H), 2.66 (t, J = 6.9 Hz, 4H), 3.41 (s, 4H), 8.10 (d,
J = 1.8 Hz, 2H), 8.25 (d, J = 8.1 Hz, 2H), 8.30 (dd, J = 8.4, 1.8 Hz,
2H), 9.84 (s, 2H). 13C NMR (300 MHz, DMSO): d ppm 11.17,
22.04, 50.51, 52.35, 115.45, 123.36, 127.62, 127.83, 133.71,
143.50, 171.08, 179.90, 181.98.
7.2.1. 2,7-Bis(chloroacetamido)anthraquinone (3)
2,7-Diaminoanthraquinone (0.238 g, 1 mmol) was dissolved in
DMF (20 mL) in ice-bath and pyridine (0.5 mL) and chloroacetyl
chloride (0.5 mL, 6 mmol) were added under nitrogen. The mixture
was stirred for 24 h. Ice was added to precipitate out the crude
product. The resulting precipitate was collected by filtration,
washed with diethyl ether, and purified by crystallization from
ethanol. Product 3 was obtained as slightly yellow powder (yield
59%): mp 286 °C (EtOH). IR (KBr) (cmꢀ1) 1672, 1718, 3315. MS
(EI, 70 eV) = 390.0 (M+). 1H NMR (300 MHz, DMSO): d ppm 4.32
(s, 4H), 7.97 (dd, J = 8.4, 2.1 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 8.33
(d, J = 2.1 Hz, 2H), 10.86 (s, 2H). 13C NMR (300 MHz, DMSO): d
7.2.8. 2,7-Bis[2-(ethylamino)acetamido]anthraquinone (10)
Product 10 was obtained as dark brown powder (yield 55%): mp
145 °C (EtOH). IR (KBr) (cmꢀ1) 1670, 3330. MS (EI, 70 eV) = 408
(M+). 1H NMR (300 MHz, CDCl3): d ppm 1.18 (t, J = 7.2 Hz, 6H),
2.75 (q, J = 7.2 Hz, 4H), 3.42 (s, 4H), 8.12 (d, J = 1.8 Hz, 2H), 8.28

6984
H.-S. Huang et al. / Bioorg. Med. Chem. 16 (2008) 6976–6986
(d, J = 8.4 Hz, 2H), 8.35 (dd, J = 8.7, 2.1 Hz, 2H), 9.85 (s, 2H). 13C
NMR (300 MHz, DMSO): d ppm 14.39, 42.78, 52.18, 115.50,
123.39, 127.62, 127.77, 133.69, 143.48, 171.07, 179.89, 181.96.
(300 MHz, CDCl3): d ppm 2.41 (s, 18H), 2.53 (t, J = 5.4 Hz, 4H),
2.67 (t, J = 5.4 Hz, 4H), 8.01 (d, J = 1.5 Hz, 2H), 8.23 (s, J = 3.0 Hz,
4H), 11.68 (s, 2H). 13C NMR (300 MHz, CDCl3): d ppm 13.30,
19.38, 30.82, 34.37, 45.04, 46.43, 115.33, 123.23, 127.50, 127.83,
133.73, 143.38, 170.04, 179.36, 182.09.
7.2.9. 2,7-Bis[2-(N-methylpiperazino)acetamido]anthraqui-
none (11)
Product 11 was obtained as yellow powder (yield 46%): mp
173 °C (EtOH). IR (KBr) (cmꢀ1) 1672, 1691, 1710, 3241. MS (EI,
70 eV) = 518 (M+). 1H NMR (300 MHz, CDCl3): d ppm 2.35 (s, 6H),
2.55 (s, 8H), 2.68 (s, 8H), 3.19 (s, 4H), 8.12 (d, J = 1.5 Hz, 2H), 8.28
(d, J = 8.7 Hz, 2H), 8.32 (dd, J = 8.7, 1.8 Hz, 2H), 9.55 (s, 2H). 13C
NMR (300 MHz, DMSO): d ppm 45.27, 52.17, 54.02, 61.35, 115.83,
123.67, 127.77, 127.82, 133.73, 143.51, 169.05, 180.01, 182.06.
7.2.16. 2,7-Bis[3-(pyrrolidino)propionamido]anthraquinone
(18)
Product 18 was obtained as brown powder (yield 49%): mp
233 °C (EtOH) (lit.26 mp 232 °C). IR (KBr) (cmꢀ1) 1645, 1672,
1698, 3331. MS (EI, 70 eV) = 488 (M+). 1H NMR (300 MHz, CDCl3):
d ppm 1.95 (s, 8H), 2.57 (t, J = 6.0 Hz, 4H), 2.71 (s, 8H), 2.87 (t,
J = 5.7 Hz, 4H), 7.98 (d, J = 2.1 Hz, 2H), 8.17 (dd, J = 8.4, 2.1 Hz,
2H), 8.24 (d, J = 8.7 Hz, 2H), 11.98 (s, 2H). 13C NMR (300 MHz,
CDCl3): d ppm 22.36, 35.73, 50.79, 57.83, 115.31, 123.15, 127.51,
127.87, 133.74, 143.37, 170.73, 179.93, 182.00.
7.2.10. 2,7-Bis[2-(piperazino)acetamido]anthraquinone (12)
Product 12 was obtained as dark yellow powder (yield 19%): mp
173 °C (EtOH). IR (KBr) (cmꢀ1) 1643, 1668, 1699, 3332. MS (EI,
70 eV) = 490 (M+). 1H NMR (300 MHz, CDCl3): d ppm 2.60 (s, 8H),
3.00 (t, J = 4.8 Hz, 8H), 3.16 (s, 4H), 8.11 (d, J = 1.8 Hz, 2H), 8.28
(d, J = 8.7 Hz, 2H), 8.33 (dd, J = 8.7, 2.1 Hz, 2H), 9.58 (s, 2H). 13C
NMR (300 MHz, DMSO): d ppm 44.81, 53.51, 62.07, 115.85,
123.71, 127.73, 127.77, 133.71, 143.43, 169.09, 179.98, 182.02.
7.2.17. 2,7-Bis[3-(piperidino)propionamido]anthraquinone (19)
Product 19 was obtained as yellowish brown powder (yield
34%): mp 266 °C (EtOH) (lit.26 mp 240 °C). IR (KBr) (cmꢀ1
)
1670, 1694, 3315. MS (EI, 70 eV) = 516 (M+). 1H NMR
(300 MHz, CDCl3): d ppm 1.75 (t, J = 5.1 Hz, 12H), 2.53–2.65
(m, 12H), 2.69 (t, J = 4.8 Hz, 4H), 8.10 (d, J = 1.5 Hz, 2H), 8.19
(dd, J = 8.4, 2.4 Hz, 2H), 8.25 (d, J = 8.7 Hz, 2H), 12.05 (s, 2H).
13C NMR (300 MHz, CDCl3): d ppm 24.09, 26.21, 32.44, 53.57,
54.03, 116.45, 124.34, 128.93, 129.14, 134.78, 144.23, 171.55,
181.28, 182.91.
7.2.11. 2,7-Bis[2-(isopropylamino)acetamido]anthraquinone
(13)
Product 13 was obtained as brown powder (yield 44%): mp
158 °C (EtOH). IR (KBr) (cmꢀ1) 1672, 3323. MS (EI, 70 eV) = 436
(M+). 1H NMR (300 MHz, DMSO): d ppm 1.01 (d, J = 6.0 Hz, 12H),
2.76 (m, 2H), 3.35 (s, 4H), 8.07 (dd, J = 8.4, 2.1 Hz, 2H), 8.14 (d,
J = 8.7 Hz, 2H), 8.49 (d, J = 1.8 Hz, 2H). 13C NMR (300 MHz, DMSO):
d ppm 21.99, 47.65, 50.06, 115.45, 123.36, 127.64, 127.75, 133.70,
143.37, 171.20, 179.86, 181.93.
7.2.18. 2,7-Bis[3-(dimethylamino)propionamido]anthra-
quinone (20)
Product 20 was obtained as yellowish powder (yield 20%): mp
196 °C (EtOH) (lit.26 mp 202–203 °C). IR (KBr) (cmꢀ1) 1651, 1673,
1698, 3325. MS (EI, 70 eV) = 436 (M+). 1H NMR (300 MHz, CDCl3):
d ppm 2.41 (s, 12H), 2.53 (t, J = 5.4 Hz, 4H), 2.67 (t, J = 5.4 Hz,
4H), 8.00 (s, 2H), 8.23 (s, 4H), 11.65 (s, 2H). 13C NMR (300 MHz,
7.2.12. 2,7-Bis[2-(isobutylamino)acetamido]anthraquinone
(14)
Product 14 was obtained as brown powder (yield 73%): mp
146 °C (EtOH). IR (KBr) (cmꢀ1) 1670, 3335. MS (EI, 70 eV) = 465
(M+). 1H NMR (300 MHz, CDCl3): d ppm 0.99 (d, J = 6.6Hz, 12H),
1.79 (m, 2H), 2.50 (d, J = 6.3 Hz, 4H), 3.41 (s, 4H), 8.13 (s, 2H),
8.28 (s, 4H), 9.86 (s, 2H). 13C NMR (300 MHz, DMSO): d ppm
20.06, 27.49, 52.60, 56.74, 115.38, 123.29, 127.61, 127.85, 133.71,
143.47, 171.13, 179.86, 181.95.
DMSO): d ppm 33.40, 44.38, 54.89, 116.87, 124.70, 129.08,
134.71, 144.04, 171.49, 179.99, 182.08.
7.2.19. 2,7-Bis[3-(propylamino)propionamido]anthraquinone
(21)
Product 21 was obtained as yellow powder (yield 25%): mp
170 °C (EtOH). IR (KBr) (cmꢀ1) 1651, 1671, 1698, 3329. MS (EI,
70 eV) = 462 (M+). 1H NMR (300 MHz, CDCl3): d ppm 2.41 (s,
14H), 2.54 (t, J = 6.0 Hz, 4H), 2.67 (t, J = 6.0 Hz, 4H), 8.01 (s, 2H),
8.23 (d, J = 1.8 Hz, 4H), 11.68 (s, 2H). 13C NMR (300 MHz, DMSO):
d ppm 29.17, 29.57, 33.39, 44.37, 54.85, 116.87, 124.66, 129.04,
134.69, 144.04, 171.49, 181.22, 183.15.
7.2.13. 2,7-Bis(3-chloropropionamido)anthraquinone (15)
Product 15 was obtained as yellow brown powder (yield 58%):
mp 281 °C (EtOH). IR (KBr) (cmꢀ1) 1672, 1702, 3333. MS (EI,
70 eV) = 418.0 (M+). 1H NMR (300 MHz, DMSO): d ppm 2.91 (t,
J = 5.9 Hz, 4H), 3.90 (t, J = 5.9 Hz, 4H), 8.01 (d, J = 8.7 Hz, 2H), 8.09
(d, J = 8.7 Hz, 2H), 8.40 (s, 2H), 10.72 (s, 2H). 13C NMR (300 MHz,
DMSO): d ppm 38.86, 39.82, 115.31, 123.14, 127.54, 127.67,
133.53, 143.44, 168.33, 179.63, 181.66.
7.2.20. 2,7-Bis[3-(ethylamino)propionamido]anthraquinone
(22)
Product 22 was obtained as brown powder (yield 23%): mp
181 °C (EtOH). IR (KBr) (cmꢀ1) 1670, 3332. 1H NMR (300 MHz,
CDCl3): d ppm 2.41 (s, 14H), 2.53 (t, J = 5.3 Hz, 4H), 2.67 (t,
J = 5.3 Hz, 4H), 8.01 (s, 2H), 8.23 (s, 4H), 11.68 (s, 2H). 13C NMR
(300 MHz, DMSO): d ppm 29.67, 33.38, 44.38, 54.88, 116.85,
124.67, 129.05, 134.68, 144.02, 171.47, 180.03, 182.07.
7.2.14. 2,7-Bis[3-(diethylamino)propionamido]anthraquinone (16)
Product 16 was obtained as yellow powder (yield 55%): mp
193 °C (EtOH) (lit.26 mp 215 °C). IR (KBr) (cmꢀ1) 1672, 1697,
3323. MS (EI, 70 eV) = 492.3 (M+). 1H NMR (300 MHz, CDCl3): d
ppm 1.15 (t, J = 7.2 Hz, 12H), 2.54 (t, J = 5.7 Hz, 4H), 2.70 (q,
J = 7.2 Hz, 8H), 2.79 (t, J = 5.6 Hz, 4H), 8.02 (s, 2H), 8.23 (s, 4H),
12.01 (s, 2H). 13C NMR (300 MHz, CDCl3): d ppm 11.44, 33.27,
46.12, 48.79, 116.35, 124.37, 128.99, 129.11, 134.78, 144.04,
171.32, 181.06, 182.85.
7.2.21. 2,7-Bis[3-(N-methylpiperazino)propionamido]anthra-
quinone (23)
Product 23 was obtained as yellow powder (yield 61%): mp
)
284 °C (EtOH). IR (KBr) (cmꢀ1 1669, 1701, 3273. 1H NMR
(300 MHz, DMSO): d ppm 2.40 (s, 6H), 2.57–2.76 (m, 24H), 8.15
(d, J = 9.6 Hz, 4H), 8.26 (d, J = 8.1 Hz, 2H), 11.69 (s, 2H). 13C NMR
(300 MHz, DMSO): d ppm 23.51, 32.46, 45.35, 52.26, 55.32,
116.53, 124.38, 129.13, 129.21, 134.55, 144.10, 171.12, 181.29,
182.51.
7.2.15. 2,7-Bis[3-(butylamino)propionamido]anthraquinone
(17)
Product 17 was obtained as yellow powder (yield 22%): mp
)
168 °C (EtOH). IR (KBr) (cmꢀ1 1648, 1671, 3333. 1H NMR

H.-S. Huang et al. / Bioorg. Med. Chem. 16 (2008) 6976–6986
6985
7.2.22. 2,7-Bis[3-(piperazino)propionamido]anthraquinone
(24)
7.2.29. 2,7-Bis(phenylpropionamido)anthraquinone (31)
Product 31 was obtained as yellow powder (yield 45%): mp
268 °C (EtOH). IR (KBr) (cmꢀ1) 1672, 3336. MS (EI, 70 eV) = 502.1
(M+). 1H NMR (300 MHz, DMSO): d ppm 2.71 (t, J = 7.8 Hz, 4H),
2.94 (t, J = 7.8 Hz, 4H), 7.23 (m, 10H), 8.02 (dd, J = 8.4, 2.1 Hz,
2H), 8.12 (d, J = 8.4 Hz, 2H), 8.43 (d, J = 2.1 Hz, 2H), 10.53 (s, 2H).
13C NMR (300 MHz, DMSO): d ppm 30.10, 41.17, 115.47, 123.41,
125.73, 127.68, 127.96, 128.07, 133.94, 140.65, 144.09, 171.22,
180.15, 182.30.
Product 24 was obtained as yellow powder (yield 41%): mp
)
218 °C (EtOH). IR (KBr) (cmꢀ1 1671, 1694, 3338. 1H NMR
(300 MHz, DMSO): d ppm 2.33 (s, 8H), 2.54 (d, J = 5.7 Hz, 6H),
2.60 (d, J = 5.7 Hz, 4H), 2.67 (t, J = 4.5 Hz, 8H), 8.02 (dd, J = 8.4,
2.1 Hz, 2H), 8.13 (d, J = 8.4 Hz, 2H), 8.42 (d, J = 2.1 Hz, 2H), 10.75
(s, 2H). 13C NMR (300 MHz, DMSO): d ppm 33.60, 45.13, 53.37,
53.77, 115.36, 123.32, 127.61, 128.07, 133.91, 144.05, 171.03,
180.08, 182.23.
7.2.30. 2,7-Bis(cyclopropanecarbonamido)anthraquinone (32)
Product 32 was obtained as yellowish brown powder (yield
68%): mp 352 °C (EtOH). IR (KBr) (cmꢀ1) 1663, 3292. MS (EI,
70 eV) = 374.9 (M+). 1H NMR (300 MHz, DMSO): d ppm 0.86 (d,
J = 6.3 Hz, 8H), 1.83 (m, 2H), 8.02 (dd, J = 8.7, 2.1 Hz, 2H), 8.10 (d,
J = 8.7 Hz, 2H), 8.42 (d, J = 2.1 Hz, 2H), 10.80 (s, 2H). 13C NMR
(300 MHz, DMSO): d ppm 7.45, 14.37, 115.38, 123.25, 127.53,
128.05, 133.92, 144.11, 172.45, 180.09, 182.27.
7.2.23. 2,7-Bis(4-chlorobutyramido)anthraquinone (25)
Product 25 was obtained as brown powder (yield 73%): mp
234 °C (EtOH). IR (KBr) (cmꢀ1) 1645, 1664, 1698, 3345. MS (EI,
70 eV) = 446.0 (M+). 1H NMR (300 MHz, DMSO): d ppm 2.05 (m,
4H), 2.54 (t, J = 7.2 Hz, 4H), 3.71 (t, J = 6.5 Hz, 4H), 7.97 (dd,
J = 8.7, 2.1 Hz, 2H), 8.05 (d, J = 8.7 Hz, 2H), 8.36 (d, J = 1.8 Hz, 2H),
10.54 (s, 2H). 13C NMR (300 MHz, DMSO): d ppm 21.18, 26.86,
67.72, 110.74, 119.04, 122.88, 128.34, 134.24, 150.95, 177.22,
178.72, 183.24.
7.2.31. 2,7-Bis(cyclopentanecarbonamido)anthraquinone (33)
Product 33 was obtained as yellowish brown powder (yield
51%): mp 281 °C (EtOH). IR (KBr) (cmꢀ1) 1647, 1674, 1702, 3342.
MS (EI, 70 eV) = 430.9 (M+). 1H NMR (300 MHz, DMSO): d ppm
1.54–1.89 (m, 16H), 2.83 (m, 2H), 8.04 (dd, J = 8.4, 2.1 Hz, 2H),
8.22 (d, J = 8.4 Hz, 2H), 8.46 (d, J = 2.1 Hz, 2H), 10.47 (s, 2H). 13C
NMR (300 MHz, DMSO): d ppm 25.14, 29.45, 44.95, 115.42,
123.20, 127.39, 127.80, 133.70, 144.22, 174.95, 179.92, 182.12.
7.2.24. 2,7-Bis(acetamido)anthraquinone (26)
Product 26 was obtained as yellowish brown powder (yield
42%): mp 340 °C (EtOH). IR (KBr) (cmꢀ1) 1645, 1670, 1691, 3333.
MS (EI, 70 eV) = 322.1 (M+). 1H NMR (300 MHz, DMSO): d ppm
2.21 (s, 6H), 8.00 (dd, J = 8.7, 2.1 Hz, 2H), 8.09 (d, J = 8.7 Hz, 2H),
8.38 (d, J = 2.1 Hz, 2H), 10.54 (s, 2H). 13C NMR (300 MHz, DMSO):
d ppm 23.59, 115.02, 122.81, 127.21, 127.51, 133.42, 143.76,
168.56, 179.52, 181.67.
7.2.32. 2,7-Bis(cyclohexanecarbonamido)anthraquinone (34)
Product 34 was obtained as yellow powder (yield 42%): mp
269 °C (EtOH). IR (KBr) (cmꢀ1) 1670, 3323. MS (EI, 70 eV) = 458.9
(M+). 1H NMR (300 MHz, DMSO): d ppm 1.16–1.46 (m, 12H),
1.63–1.84 (m, 8H), 2.37 (m, 2H), 8.02 (dd, J = 8.4, 1.8 Hz, 2H),
8.09 (d, J = 8.4 Hz, 2H), 8.44 (d, J = 1.8 Hz, 2H), 10.43 (s, 2H). 13C
7.2.25. 2,7-Bis(propionamido)anthraquinone (27)
Product 27 was obtained as yellowish brown powder (yield
48%): mp 291 °C (EtOH). IR (KBr) (cmꢀ1) 1672, 1706, 3368. MS
(EI, 70 eV) = 350.1 (M+). 1H NMR (300 MHz, DMSO): d ppm 1.10
(t, J = 7.5 Hz, 6H), 2.39 (q, J = 7.5 Hz, 4H), 8.02 (dd, J = 8.7, 2.1 Hz,
2H), 8.10 (d, J = 8.7 Hz, 2H), 8.41 (d, J = 1.8 Hz, 2H), 10.46 (s, 2H).
13C NMR (300 MHz, DMSO): d ppm 8.72, 29.12, 115.12, 122.93,
127.20, 127.55, 133.50, 143.87, 172.26, 179.60, 181.77.
NMR (300 MHz, DMSO):
d ppm 24.53, 24.76, 28.37, 44.42,
115.47, 123.29, 127.43, 127.76, 133.73, 144.26, 174.82, 179.97.
7.2.33. Cell culture and assessment of hTERT
Non-small lung cancer cells H129960 were grown in RPMI 1640
media supplemented with 10% fetal bovine serum, 100 U/mL pen-
icillin, and 100 mg/mL streptomycin in a humidified atmosphere
with 5% CO2 at 37 °C. The hTERT immortalized hTERT-BJ1 (BD Bio-
sciences Clontech)61 was grown in DMEM supplemented with 10%
fetal calf serum, 100 U/mL penicillin and 100 mg/mL streptomycin,
7.2.26. 2,7-Bis(butyramido)anthraquinone (28)
Product 28 was obtained as yellowish brown powder (yield
63%): mp 275 °C (EtOH). IR (KBr) (cmꢀ1) 1664, 1674, 1706, 3339.
MS (EI, 70 eV) = 378.2 (M+). 1H NMR (300 MHz, DMSO): d ppm
0.93 (t, J = 7.5 Hz, 6H), 1.63 (m, 4H), 2.36 (q, J = 7.2 Hz, 4H), 8.03
(dd, J = 8.4, 2.1 Hz, 2H), 8.10 (d, J = 8.4 Hz, 2H), 8.43 (d, J = 2.1 Hz,
2H), 10.48 (s, 2H). 13C NMR (300 MHz, DMSO): d ppm 12.97,
17.73, 37.86, 115.11, 122.87, 127.19, 127.48, 133.44, 143.77,
171.36, 179.50, 181.69.
1 mM sodium pyruvate, and 4 mM L-arginine in a humidified
atmosphere with 5% CO2 at 37 °C. 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 ꢁ3.3 kbp DNA
fragment ranging from ꢀ3338 to +1 bp of the hTERT gene was sub-
cloned upstream to a secreted alkaline phosphatase gene (SEAP)
and transfected into H1299 or hTERT-BJ1 by electroporation. The
stable clones were selected using G418. The stable clones derived
from H1299 or hTERT-BJ1 were cultured using conditions that
are similar to their parental cells.
7.2.27. 2,7-Bis(benzoamido)anthraquinone (29)
Product 29 was obtained as red brown powder (yield 30%): mp
224 °C (EtOH). IR (KBr) (cmꢀ1
) 1678, 1703, 3320. MS (EI,
70 eV) = 446.1 (M+). 1H NMR (300 MHz, DMSO): d ppm 7.57 (m,
6H), 8.00 (d, J = 8.1 Hz, 4H), 8.15 (d, J = 8.4 Hz, 2H), 8.30 (dd,
J = 8.4, 1.8 Hz, 2H), 8.65 (d, J = 2.1 Hz, 2H), 10.80 (s, 2H). 13C NMR
(300 MHz, DMSO): d ppm 116.69, 124.39, 127.44, 127.68, 127.81,
128.03, 131.66, 133.73, 144.15, 165.74, 180.12, 182.14.
7.2.34. Cytotoxicity assay
The tetrazolium reagent (MTT; 3-(4,5-di-methylthiazol)-2,5-
diphenyltetrazolium bromide, USB) was designed to yield a colored
formazan upon metabolic reduction by viable cells.62,63 Approxi-
mately 2 ꢂ 103 cells were plated onto each well of a 96-well plate
and incubated in 5% CO2 at 37 °C for 24 h. To assess the in vitro
cytotoxicity, each compound was dissolved in DMSO and prepared
immediately before the experiments and was diluted into the com-
plete medium before addition to cell cultures. Test compounds
were then added to the culture medium for designated various
7.2.28. 2,7-Bis(phenylacetamido)anthraquinone (30)
Product 30 was obtained as yellow powder (yield 74%): mp
224 °C (EtOH). IR (KBr) (cmꢀ1
) 1644, 1668, 3322. MS (EI,
70 eV) = 474.2 (M+). 1H NMR (300 MHz, DMSO): d ppm 3.72 (s,
4H), 7.30 (m, 10H), 8.05 (dd, J = 8.4, 2.1 Hz, 2H), 8.13 (d,
J = 8.4 Hz, 2H), 8.44 (d, J = 2.1 Hz, 2H), 10.79 (s, 2H). 13C NMR
(300 MHz, DMSO): d ppm 42.83, 115.54, 123.42, 126.26, 127.73,
128.74, 133.80, 134.95, 143.97, 169.72, 180.01, 182.08.
concentrations. After 48 h, an amount of 25 lL of MTT was added

6986
H.-S. Huang et al. / Bioorg. Med. Chem. 16 (2008) 6976–6986
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100 L 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.
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7.2.35. Telomere repeat amplification protocol (TRAP) assays
Telomerase activity was detected by a modified version of the
TRAP protocol.8,64,65 Telomerase products were resolved by 10%
polyacrylamide gel electrophoresis and visualized by staining with
SYBER Green. As a source of telomerase, the total cell lysates de-
rived from lung cancer cell line H1299 cells were used. Protein con-
centration of the lysates was assayed using Bio-Rad protein assay
kit using BSA standards.
7.2.36. SEAP assay66
Secreted alkaine phosphatase was used as the reporter system
to monitor the transcriptional activity of hTERT. Here, about 104
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 buffer (2 M
diethanolamine, 1 mM MgCl2, and 20 mM L-homoarginine) 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 is determined.
Acknowledgments
The present study was supported by National Science Council
Grants NSC 95-2113-M-016-002-MY2, NSC 96-2923-M-016-001-
MY3, NSC 96-3112-B-010-002, and NSC 96-2311-B-010-005, and
also by National Health Research Institute grant NHRI-EX96-
9625SI. The first author cordially dedicated to Prof. Dr. Kang-Chien
Liu on the occasion of his 80th birthday and Prof. Dr. Wolfgang
Wiegrebe (University of Regensburg/Germany) on the occasion of
his 75th birthday.
47. Huang, H. S.; Hwang, J. M.; Jen, Y. M.; Lin, J. J.; Lee, K. Y.; Shi, C. H.; Hsu, H. C.
Chem. Pharm. Bull. (Tokyo) 2001, 49, 969.
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