Activation of human telomerase reverse transcriptase expression by some new symmetrical bis-substituted derivatives of the anthraquinone

Article abstract of DOI:10.1021/jm020492l

As a part of our program aimed at exploring the biological activity of symmetrical substitution of side chains into the anthracene-9,10-dione chromophore, we have synthesized a series of 1,5-bisthioanthraquinones 2 and 1,5-bisacyloxyanthraquinones 3 that are related to the antitumor agent mitoxantrone. Since the telomerase enzyme is a novel target for potential anticancer therapy and stem cell expansion, we explore the biological effects of these compounds by evaluating their effects on telomerase activity and telomerase expression. Telomerase is required for telomere maintenance and is active in most human cancers and in germinal cells but not in most of the normal human somatic tissues. We found that most of the 1,5-disubstituted anthraquinones did not exhibit inhibitory activity at the concentration ranging from 20 to 30 μM. To facilitate the analysis of the expression of telomerase, we used cancer and normal cell lines that carry secreted alkaline phosphatase (SEAP) gene under the control of human telomerase reverse transcriptase (hTERT). The effects of these compounds on the expression of telomerease were analyzed using the cell-based reporter systems. While most of these compounds did not appear to selectively repress the expression of hTERT in cancer cells, compounds 3a, 3d, and 3i activated hTERT expression in normal cells. The effects of these three compounds on hTERT expression appear to be specific because they did not increase the expression of a CMV promoter-driven SEAP. Thus, in addition to anticancer functions, our finding raises the possibility that these compounds might also have a role in cell immortilization. The application of these anthraquinone derivatives in stem cell research and tissue engineering is also discussed.

Full text of DOI:10.1021/jm020492l

3300
J . Med. Chem. 2003, 46, 3300-3307
Activa tion of Hu m a n Telom er a se Rever se Tr a n scr ip ta se Exp r ession by Som e
New Sym m etr ica l Bis-Su bstitu ted Der iva tives of th e An th r a qu in on e
Hsu-Shan Huang,*,‡ J eng-Fong Chiou,‡ Yaou Fong,‡ Ching-Cheng Hou,‡ Yu-Cheng Lu,† J en-Yi Wang,†
J ing-Wen Shih,† Yen-Ru Pan,† and J ing-J er Lin*,†
School of Pharmacy, National Defense Medical Center, 161 Section 6, Minchuan E. Road, Neihu, 114, Taipei, Taiwan, R.O.C.,
and Institute of Biopharmaceutical Science, Institute of Pharmacology, National Yang-Ming University,
ShihPai, 112, Taipei, Taiwan, R.O.C.
Received October 31, 2002
As a part of our program aimed at exploring the biological activity of symmetrical substitution
of side chains into the anthracene-9,10-dione chromophore, we have synthesized a series of
1,5-bisthioanthraquinones 2 and 1,5-bisacyloxyanthraquinones 3 that are related to the
antitumor agent mitoxantrone. Since the telomerase enzyme is a novel target for potential
anticancer therapy and stem cell expansion, we explore the biological effects of these compounds
by evaluating their effects on telomerase activity and telomerase expression. Telomerase is
required for telomere maintenance and is active in most human cancers and in germinal cells
but not in most of the normal human somatic tissues. We found that most of the 1,5-
disubstituted anthraquinones did not exhibit inhibitory activity at the concentration ranging
from 20 to 30 µM. To facilitate the analysis of the expression of telomerase, we used cancer
and normal cell lines that carry secreted alkaline phosphatase (SEAP) gene under the control
of human telomerase reverse transcriptase (hTERT). The effects of these compounds on the
expression of telomerease were analyzed using the cell-based reporter systems. While most of
these compounds did not appear to selectively repress the expression of hTERT in cancer cells,
compounds 3a , 3d , and 3i activated hTERT expression in normal cells. The effects of these
three compounds on hTERT expression appear to be specific because they did not increase the
expression of a CMV promoter-driven SEAP. Thus, in addition to anticancer functions, our
finding raises the possibility that these compounds might also have a role in cell immortilization.
The application of these anthraquinone derivatives in stem cell research and tissue engineering
is also discussed.
In tr od u ction
tissues has generated considerable interest in using
telomerase inhibitors as cancer therapeutic agents.
Recently, telomerase activation was considered as a
means for the expansion of somatic stem cells. Indeed,
several studies have already investigated adrenocortical
cells,6 endothelial cells,7 and bone marrow stromal stem
cells in vitro by introducting hTERT into these cells and
restoring the cellular function.8,9 The expression of
TERT, the reverse transcriptase component of telom-
erase, is necessary to convert normal human cells into
cancer cells.10 Thus, inhibition or activation of the
reverse transcriptase telomerase can profoundly affect
the proliferative capacity of normal cells and cancers.11
Telomerase is a ribonucleoprotein that contains both
RNA and protein component for the maintenance of
telomere length. Telomerase activation in some cell
types appears to offset proliferation-dependent telomere
shortening, which delays the inherent mitotic clock.1
This enzyme comprises a template-containing RNA
subunit, hTR (human telomerase RNA component), and
catalytic protein subunit hTERT (human telomerase
reverse transcriptase) in human. In most normal human
cells, telomerase activity is low or not detectable because
telomeric DNA is progressively shortened with each cell
division. It is hypothesized that progressive shortening
of telomeres eventually causes cellular senescence. This
is supported by the immortalization of normal human
cells with the introduction of hTERT gene into these
cells.2-5 Moreover, telomerase activity was detected in
germinal cells, immortalized cell lines, and in 85-90%
of human cancer specimens, further supporting the role
of telomerase in cell immortalization and in cancer
progression. The presence of telomerase activity in
tumors combined with its absence in most normal
The hTERT expression appears to be the key regula-
tor for telomerase activity. The expression of hTERT is
regulated by several factors, and recent interest in the
regulation of telomerase, the enzyme that maintains
chromosomal termini, has led to the discovery and
characterization of the catalytic subunit of hTERT.12 For
example, expression of c-Myc was shown to induce the
expression of hTERT, whereas the expression of Sp1
(stimulating protein 1) suppresses it. However, it re-
mains unclear how hTERT is inactivated during devel-
opment and how it is reactivated during tumorigenesis.
Research into the regulation of telomerase may lead to
the development of methods for accurate diagnosis of
cancer and to the development of novel antitelomerase
cancer therapeutics.13-16 Consequently, telomerase be-
* To whom correspondence should be addressed. For H.-S.H.: phone,
+886-2-87926454; fax, +886-2-87923169; e-mail, huanghs@
ndmctsgh.edu.tw. For J .-J .L.: phone, +886-2-28267258; fax, +886-
2-28220084; e-mail: jjlin@ym.edu.tw.
‡ National Defense Medical Center.
† National Yang-Ming University.
10.1021/jm020492l CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/13/2003

hTERT Expression
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3301
Ch a r t 1. Structures of Mitoxantrone and
Sch em e 2a
Anthraquinone Derivatives
a
Reagents: (b) method A, pyridine; method B, NaH, THF.
Sch em e 1a
stitutions. Anthraquinone may be considered to consist
of two benzoyl chromogens in which little or no interac-
tion would be expected between substituents located in
different benzene rings. In Scheme 1, the synthesis of
1,5-bisthioanthraquinones was carried out by a nucleo-
philic substitution at the 1 and 5 positions with the
appropriate thiols in the presence of sodium methoxide
and THF at room temperature or after refluxing for 1-2
h. In these reactions, sodium methoxide ionized the
thiols that could then serve as nucleophiles for nucleo-
philic substitutions. The synthesis of 1,5-bisacyloxyan-
thraquinones shown in Scheme 2 was based on simple
acylation involving 1,5-dihydroxyanthraquinone (an-
thrarufin) with an excess of the appropriate acyl chlo-
rides in the presence of pyridine and dichloromethane
at room temperature for 1-2 h or in the presence of
NaH and THF at room temperature or after refluxing
for 1-2 h. All final products were characterized by
NMR, IR, UV, and mass spectrometry.
a
Reagents: (a) sodium methoxide/methanol, tetrahydrofuran,
reflux.
came an obligate focus for anticancer research because
it promised a unique target for chemotherapy without
the associated side effects of conventional chemothera-
peutics.17
Anthraquinone-based compounds currently occupy a
prominent position in cancer chemotherapy, and the
anthraquinone mitoxantrone (1, Chart 1) is an impor-
tant compound used clinically as an anticancer agent.18
It has been used in treating human leukemia, lympho-
mas, and advanced breast and ovarian cancers in
combination with other treatments.19 Although the
reaction mechanism of the antitumor activity of an-
thraquinone is probably multimodal in nature, studies
have indicated that its intercalative interaction with
DNA may play a major role.20-22 Human telomerase has
been proposed as a highly selective target for antitumor
drug design.23,24 This hypothesis is supported by experi-
ments with antisense constructs against telomerase
RNA in HeLa cells, which show that telomere length is
progressively shortened, together with the death of
these otherwise immortal cells.25 From the studies on
the structure-activity relationships of anthraquinones,
we have recently reported the synthesis of a number of
1,5-bisthioanthraquinones and 1,5-bisacyloxyanthra-
quinones (2, 3; Chart 1) that exhibited potent cytotox-
icity against rat glioma C6 cells, human hepatoma G2
cells, and lipid peroxidation compared to mitoxantro-
ne.26,27 Here, we present the effects of this new class of
compounds on a biologically important enzyme telom-
erase. We also examine the effects of these compounds
on hTERT expression using cell-based assay systems.
We found that several of these bis-substituted an-
thraquinone analogues activate hTERT expression. Our
results suggest that the compounds with anthraquinone
moieties could have a role in cell immortalization that
was not described before. Side chains have been exam-
ined to establish structure-activity relationships as a
basis for subsequent rational drug design.
Biologica l Resu lts a n d Discu ssion
The chemical and biological activities of anthraquino-
ne compounds are greatly affected by various substit-
uents of the planar ring system.28-35 We have previously
examined the cytotoxic activity in several tumor cell
lines and their inhibitory effects on lipid peroxidation.
Since telomerase is an important component of cancers,
we are interested in examining the effects of these
compounds on telomerase activity in the cell-free ex-
tracts prepared from H1299 cells. We applied a PCR-
based telomerase assay, TRAP (telomeric repeat am-
plification protocol) assay, as a system for assaying the
telomerease activity. The inhibitory effect of anthraquino-
nes at 20-30 µM concentrations was tested. We rea-
soned that an effective telomerase inhibitor should give
good inhibitory effect at this concentration. Among a
total of more than 30 pure compounds tested, none of
them exhibited significant telomerase inhibitory activity
(data not shown). According to our previous study on
9-acyloxy-1,5-dichloroanthracene derivatives, we did not
observe any specific telomerase inhibitory activity.36 It
is interesting to note that even though the compounds
we synthesized do not inhibit telomerase, a report on
amino-disubstituted anthracenc derivatives indicates
that some of these compounds inhibit telomerase.37
Because telomerase expression is a hallmark of
cancer, the effect of anthraquinones on telomerase
expression was determined. The telomerase activity is
regulated mostly at the transcriptional level for its
catalytic subunit, hTERT, and partly at the posttrans-
lational level.38 Since the expression of human telom-
erase catalytic component is the key regulator in
Ch em istr y
The synthesis of the target compounds is illustrated
in Schemes 1 and 2. Various side chains are attached
to anthraquinone using electrophilic additions or sub-

3302 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Huang et al.
F igu r e 1. Activation of hTERT promoter-driven SEAP ex-
pression by c-Myc. About 1 × 107 hTERT-BJ 1 cells were
transfected with 13.5 µg each of plasmid pSEAP or pPhTERT-
SEAP and of plasmid pMT2T or pMT2T-cMyc by electropora-
tion. After 24 h, viable cells were harvested and reinoculated
at a density of 3 × 105/mL, and the SEAP activity was
determined after 24 h at 37 °C. The transfection efficiency of
each experiment was determined by cotransfection with 1.5
µg of plasmid pCMVâ. The values were determined from three
experiments. P < 0.05 is presented by an asterisk.
F igu r e 2. Stable cell lines harboring PhTERT-SEAP did not
affect the growth rate of their parental cell lines. H1299,
hTERT-BJ 1, and stable cell lines harboring PhTERT-SEAP were
grown at 37 °C in the presence of 5% CO2. The cell growth
was monitored for a period of 96 h using MTT assay. The
values are determined from four experiments.
telomerase activity, we analyzed the expression of
telomerase by monitoring the expression of hTERT as
the criteria. To facilitate the analysis, we ligated
downstream to the hTERT promoter a reporter gene,
secreted alkaline phosphatase (SEAP), to generate the
hTERT promoter-driven reporter construct, PhTERT
-
SEAP. In this construct, the 3.4 kbp hTERT promoter
region ranging from position -3338 to +1 was used.
This DNA fragment contains the cis regulatory elements
of hTERT transcription. To test if our reporter is
functional, the SEAP expression was monitored in the
presence of c-Myc because it was shown to be a positive
regulator of hTERT. As shown in Figure 1, the SEAP
activity is elevated in cells that are cotransfected with
F igu r e 3. Expression of PhTERT-SEAP in H1299 cells and lack
of PhTERT-SEAP expression in hTERT-BJ1 cells. H1299, hTERT-
BJ 1, and stable cell lines harboring PhTERT-SEAP from these
two cells were analyzed for SEAP activity. The values are
determined from three experiments.
P
hTERT-SEAP and plasmid that expresses c-Myc. This
result indicated that our reporter construct is capable
of activating the expression of SEAP activity.
Plasmid construct pPhTERT-SEAP or a CMV (cytome-
galovirus) promoter-driven SEAP construct, pPCMV
-
in Tables 1 and 2, most of the compounds did not show
significant effects on SEAP expression. Only compounds
2b, 2c, 2f, 2l, 3a , and 3n affected the SEAP expression.
These six compounds 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 conceivable that
inhibition of telomerase expression by these compounds
inhibit cell proliferation.
We then used the expression of SEAP in hTERT-BJ 1
cells harboring PhTERT-SEAP as the criterion to evaluate
if anthraquinones activate 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 Tables 1 and 2. Interestingly, cytotoxicity
levels in human tumor cell lines were at comparable
levels for several compounds; nearly half of the an-
thraquinone derivatives activated the SEAP levels.
Among these compounds, we are most interested in
compounds 3a , 3d , and 3i because these compounds
activated the SEAP levels without affecting the cell
viability (Figure 4). To test if these three compounds
affect the expression of the other promoter, we analyzed
the effects of these three compounds on the activity of
the CMV promoter. Here, we introduce a CMV promoter-
driven SEAP, PCMV-SEAP, into hTERT-BJ 1 to generate
stable cell lines and used these cell lines for analysis.
SEAP were introduced into cancer cell line H1299 and
hTERT-immortalized normal cell line hTERT-BJ1. Stable
cell lines harboring PhTERT-SEAP or PCMV-SEAP were
then isolated. We selected one cell line from each of
these stable lines for further characterization. We first
examined the global phenotype of these cells. Integra-
tion of pPhTERT-SEAP or pPCMV-SEAP did not appear to
affect the morphology and the growth rate of their
parental cell H1299 or hTERT-BJ 1 (Figure 2 and data
not shown). Since telomerase is active in cancer cells
and inactive in normal cells, we determine the expres-
sion of SEAP in these stable cell lines. As shown in
Figure 3, H1299 cells harboring PhTERT-SEAP expressed
a high level of SEAP activity, whereas hTERT-BJ 1 cells
harboring PhTERT-SEAP only expressed low-to-zero level
of SEAP activity. These results indicate that the 3.4 kbp
DNA fragment used in our study is sufficient to confer
the properties of hTERT promoter. Moreover, our
results demonstrate that cells harboring PhTERT-SEAP
could be used as a tool to monitor the expression of
hTERT.
The expression of SEAP in H1299 cells harboring
P
hTERT-SEAP was used as the criterion 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

hTERT Expression
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3303
Ta ble 1. Effects of Symmetrical 1,5-Bisthio-Substituted Anthraquinones (2a -o) on Respressing and Activating hTERT Expression
P
hTERT-SEAP (H1299)b
P
hTERT-SEAP (hTERT-BJ 1)c
relative
viability (%)
relative SEAP
activity (%)
relative
viability (%)
relative SEAP
activity (%)
compd
R
concna (µM)
2a
CH2CH3
3.0
30
300
2.8
28
280
2.8
28
280
2.4
24
240
2.1
21
210
2.2
22
220
2.2
22
220
2.2
22
220
2.2
22
220
2.2
22
220
2.2
22
220
2.3
23
230
2.3
23
230
2.2
22
220
2.2
22
111 ( 2.8
44 ( 7.3
25 ( 2.3
54 ( 4.0
29 ( 7.0
36 ( 7.2
96 ( 5.9
44 ( 2.5
25 ( 2.4
102 ( 6.2
103 ( 4.2
83 ( 18.2
99 ( 6.2
94 ( 3.9
36 ( 4.2
94 ( 3.5
50 ( 3.3
14 ( 2.5
92 ( 3.5
68 ( 1.9
32 ( 4.9
103 ( 4.3
76 ( 4.0
42 ( 2.3
83 ( 5.5
44 ( 0.9
34 ( 3.3
89 ( 6.1
59 ( 5.4
42 ( .3
93 ( 6.2
51 ( 9.3
27 ( 2.9
107 ( 5.1
99 ( 5.8
44 ( 6.3
79 ( 12.2
45 ( 7.1
29 ( 1.5
99 ( 4.0
95 ( 4.9
51 ( 16.7
84 ( 5.9
41 ( 2.4
25 ( 2.4
134 ( 14.4
111 ( 7.7
99 ( 14.3
97 ( 15.7
76 ( 12.4
45 ( 2.7
112 ( 9.2
98 ( 12.3
34 ( 18.1
94 ( 9.3
49 ( 2.9
23 ( 2.9
103 ( 6.3
97 ( 3.0
39 ( 4.9
98 ( 10.7
86 ( 5.5
79 ( 8.2
99 ( 6.2
70 ( 2.2
40 ( 4.7
103 ( 7.9
107 ( 5.5
25 ( 3.3
104 ( 5.1
9 ( 3.4
109 ( 22.4
110 ( 14.3
104 ( 20.6
104 ( 15.7
98 ( 10.9
71 ( 5.0
2b
2c
2d
2e
2f
CH2CH2OH
CH2CH2CH3
CH2CH(OH)CH2OH
(CH2)6OH
73 ( 5.4
132 ( 21.0
110 ( 13.2
122 ( 14.3
144 ( 16.9
136 ( 10.0
142 ( 9.1
140 ( 9.7
128 ( 14.4
75 ( 17.4
136 ( 17.5
141 ( 18.2
115 ( 29.7
118 ( 9.9
41 ( 9.5
29 ( 2.5
17 ( 13.9
105 ( 21.6
90 ( 5.7
81 ( 6.1
110 ( 6.1
100 ( 5.5
68 ( 5.9
2-NH2C6H4
3-NH2C6H4
4-NH2C6H4
CH2C6H5
108 ( 12.2
96 ( 8.4
59 ( 6.6
106 ( 7.6
109 ( 11.7
101 ( 7.3
100 ( 5.7
95 ( 2.6
84 ( 5.3
97 ( 6.0
100 ( 7.2
100 ( 12.9
92 ( 3.3
96 ( 8.5
88 ( 5.6
108 ( 0.5
102 ( 0.5
97 ( 4.8
105 ( 6.6
110 ( 8.0
49 ( 10.0
104 ( 12.6
103 ( 15.8
89 ( 9.3
92 ( 7.1
97 ( 2.9
93 ( 12.1
117 ( 12.5
103 ( 10.5
90 ( 11.6
2g
2h
2i
4 ( 1.5
8 ( 30.6
86 ( 11.9
65 ( 12.6
56 ( 13.8
121 ( 4.7
98 ( 4.2
47 ( 9.1
119 ( 9.4
98 ( 13.4
62 ( 6.4
91 ( 3.9
54 ( 4.4
35 ( 4.1
104 ( 8.9
103 ( 6.4
73 ( 8.3
112 ( 6.4
91 ( 7.3
55 ( 11.3
104 ( 7.6
106 ( 6.3
89 ( 3.5
111 ( 5.4
77 ( 6.9
22 ( 8.5
97 ( 17.9
97 ( 14.5
26 ( 12.6
117 ( 11.3
112 ( 9.3
87 ( 11.1
142 ( 27.7
141 ( 22.6
118 ( 19.2
119 ( 12.2
109 ( 23.4
110 ( 30.6
137 ( 12.3
125 ( 6.9
61 ( 8.7
2j
CH2C6H4(OCH3)(p)
CH2CH2C6H5
C4H3N2
2k
2l
2m
2n
2o
C5H4N
98 ( 12.3
133 ( 6.8
121 ( 13.8
93 ( 7.9
116 ( 15.4
145 ( 20.7
132 ( 19.3
109 ( 5.7
87 ( 29.6
C4H2N2(OH)(m)
C6H4CH3
220
a
Values are in µM and represent an average of three experiments. The variance for the relative viability (%) and relative SEAP activity
(%) values was less than (20%. Activity of PhTERT-SEAP (H1299) and (hTERT-BJ 1) cell growth was significantly different with respect
to that of the control; n ) 3 or more, P < 0.01. Relative percentage of inhibition was not compared with that of the control, P < 0.01, mean
b
( SE, n ) 4. Values are the mean percent activity at the indicated concentration and include standard errors. Non-small-cell lung
cancer cells H1299. c The hTERT-immortalized hTERT-BJ 1 was purchased from BD Biosciences Clontech.
As shown in Table 3 and Figure 4, none of these three
compounds affected the expression of the CMV pro-
moter, suggesting that the effects of these three com-
pounds on hTERT promoter are specific.
in vitro cultured cells, it has been hypothesized that
telomerase therapy may delay age-associated tissue
degeneration or reverse organ failure in chronic high-
turnover diseases.40,41 However, since telomerase also
has an important role in cancer, it is possible that
hTERT transgene expression might be carcinogenic.
Indeed, neoplastic lesions are developed in the mam-
mary gland of transgenic mice with TERT expression,
suggesting that constitutive telomerase expression might
be tumorigenic.42 Moreover, the risk of genetic alteration
upon hTERT integration into chromosome is unknown.
Con clu sion s
Normal human cells undergo a finite number of cell
divisions and ultimately enter a nondividing state called
replication senescence. It has been proposed that te-
lomere shortening is the molecular clock that triggers
senescence.39 Since telomerase extends the life span of

3304 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Huang et al.
Ta ble 2. Effects of Symmetrical 1,5-Bisacyloxyanthraquinones (3a -n ) on Respressing and Activating hTERT Expression
P
hTERT-SEAP (H1299)b
P
hTERT-SEAP (hTERT-BJ 1)c
relative
viability (%)
relative SEAP
activity (%)
relative
viability (%)
relative SEAP
activity (%)
compd
R
concna (µM)
3a
COCH2CH3
2.8
28
280
2.6
26
260
2.3
23
230
2.4
24
240
2.2
22
220
1.9
19
190
1.9
19
190
1.9
19
190
1.8
18
180
2.1
21
210
2.1
21
210
2.1
21
210
2.1
21
210
2.0
20
116 ( 8.0
116 ( 7.4
87 ( 6.6
107 ( 5.6
106 ( 3.2
87 ( 6.5
117 ( 5.1
114 ( 4.7
106 ( 4.3
106 ( 8.9
97 ( 7.1
70 ( 5.2
99 ( 7.3
60 ( 11.9
33 ( 4.7
74 ( 5.3
34 ( 3.4
30 ( 1.4
98 ( 3.9
88 ( 7.2
46 ( 3.5
91 ( 10.0
57 ( 1.8
31 ( 1.0
108 ( 3.8
103 ( 5.9
77 ( 2.8
72 ( 4.9
36 ( 9.7
45 ( 8.0
26 ( 3.7
28 ( 5.0
29 ( 3.3
61 ( 5.8
33 ( 2.8
32 ( 5.7
91 ( 4.2
53 ( 2.3
30 ( 1.6
111 ( 0.8
101 ( 4.3
54 ( 4.1
103 ( 5.8
107 ( 4.9
97 ( 3.1
114 ( 7.9
118 ( 8.4
111 ( 4.4
91 ( 9.9
95 ( 17.4
95 ( 10.5
105 ( 4.9
78 ( 14.8
80 ( 10.1
88 ( 10.0
94 ( 8.0
86 ( 4.7
93 ( 4.4
101 ( 4.9
97 ( 5.3
83 ( 5.7
92 ( 8.1
81 ( 2.5
106 ( 4.5
106 ( 4.9
97 ( 4.9
100 ( 5.2
102 ( 5.4
96 ( 4.4
97 ( 4.4
91 ( 7.4
90 ( 4.2
104 ( 5.4
116 ( 12.8
110 ( 16.4
98 ( 1.1
95 ( 4.3
95 ( 8.9
98 ( 0.3
101 ( 6.8
142 ( 14
94 ( 2.5
98 ( 4.7
89 ( 5.2
99 ( 11.9
109 ( 8.7
93 ( 10.8
109 ( 5.1
106 ( 5.6
85 ( 6.6
119 ( 9.4
98 ( 13.4
62 ( 6.4
94 ( 3.4
93 ( 6.0
100 ( 9.0
103 ( 6.5
87 ( 9.8
51 ( 4.6
107 ( 5.5
92 ( 4.3
40 ( 4.0
101 ( 2.3
80 ( 6.7
44 ( 6.0
111 ( 6.5
89 ( 14.3
48 ( 9.0
107 ( 7.2
102 ( 4.1
96 ( 7.0
118 ( 11.6
82 ( 8.5
39 ( 13.0
98 ( 9.4
54 ( 7.5
47 ( 4.8
102 ( 13.9
98 ( 6.3
56 ( 6.2
106 ( 9.0
97 ( 8.2
64 ( 10.2
111 ( 7.3
106 ( 6.6
76 ( 7.2
131 ( 7.0
152 ( 16.7
161 ( 12.2
107 ( 11.9
119 ( 8.3
134 ( 3.8
147 ( 18.8
149 ( 16.1
124 ( 5.3
147 ( 11.1
165 ( 18.7
141 ( 14.4
137 ( 10.0
122 ( 9.6
84 ( 21.7
120 ( 10.5
116 ( 3.7
97 ( 10.8
154 ( 23.0
152 ( 15.8
91 ( 25.2
136 ( 5.7
123 ( 6.2
107 ( 6.0
155 ( 16.3
150 ( 13.9
160 ( 36.7
129 ( 13.0
120 ( 24.0
94 ( 27.1
141 ( 10.5
124 ( 15.6
86 ( 17.2
130 ( 8.8
126 ( 13.3
99 ( 15.8
129 ( 4.5
125 ( 4.3
100 ( 17.3
126 ( 6.1
124 ( 13.4
110 ( 10.5
3b
3c
3d
3e
3f
COCH2CH2CH3
CO(CH2)4CH3
COC(CH3)3
COC6H5
COC6H4Cl(o)
COC6H4Cl(m)
COC6H4Cl(p)
COC6H4Cl2(o,p)
COC6H4CH3(o)
COC6H4CH3(m)
COC6H4CH3(p)
COCH2C6H5
3g
3h
3i
3j
3k
3l
3m
3n
COCH2CH2C6H5
200
a
Values are in µM and represent an average of three experiments. The variance for the relative viability (%) and relative SEAP activity
(%) values was less than (20%. Activity of PhTERT-SEAP (H1299) and (hTERT-BJ 1) cell growth was significantly different from that of
the control; n ) 3 or more, P < 0.01. Relative percentage of inhibition was not compared with that of the control, P < 0.01, mean ( SE,
b
n ) 4. Values are the mean percent activity at the indicated concentration and include standard errors. Non-small-cell lung cancer cells
H1299. c The hTERT immortalized hTERT-BJ 1 was purchased from BD Biosciences Clontech.
We report here the identification of small-molecule
compounds that activate hTERT expression without
going through procedures that cause genetic alteration
in cells. The telomerase activating compounds should
be valuable in the fields of stem cell and tissue engi-
neering research in expending target cells. They may
also be applied in treating age-associated tissue degen-
eration or reverse organ failure in chronic high-turnover
diseases. This unique property should definitely be
noted in future drug design.
macophore. A striking feature of the results given in
Tables 1-3 are the marked repressing hTERT expres-
sion [PhTERT-SEAP (H1299)] and activating hTERT
expression [PhTERT-SEAP (hTERT-BJ 1)]. Overall, com-
pounds 3a , 3d , and 3i of these two series have the best
activating hTERT expression and compares favorably
in relative SEAP activity and relative viability in Table
3 and Figure 4. The optimal substituents of the various
homologues of 1,5-bisacyloxyanthraquinone were pro-
pionyloxy (3a ), pivaloyoxy (3d ), and 2,4-dichlorobenzoyl
(3i), and 1,5-bisthio anthraquinones did not exhibit any
significant activation hTERT expression. Anthraquino-
ne derivatives are known for their antitumor activities.
Here, we found that about half (14/30) of the compounds
The aim of this study was to elucidate the structure-
activity relationships of simple 1,5-symmetrical bis-
substituted analogues of anthraquinone to further
delineate the nature of the requirements of the phar-

hTERT Expression
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3305
inhibitory effect does not seem to correlate with the
modifying groups. Also, these effects are very sensitive
to a slight modification of anthraquinone derivatives.
Thus, it appears that the anthraquinone chromophore
itself causes this activity. Finally, the effects of an-
thraquinones on the cytotoxicity, the telomerase-repres-
sion activity in cancer cells, and the telomerase-
activation activity in normal cells are very diverse. A
simple correlation between the modifications of an-
thraquinones with their biological effects cannot be
achieved. The molecular mechanisms behind these
diversified biological effects are unclear. In light of these
findings, it is suggested that activation of hTERT
expression by some of the anthraquinones is not suf-
ficient for potent proliferative action. Nevertheless, the
potential application of these compounds in tumor
biology, stem cell research, and tissue engineering made
them a group of compounds that are worth an extensive
study.
Exp er im en ta l Section
Melting points were determined with a Bu¨chi 530 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 stated. 1H NMR spectra were re-
corded with a Varian GEMINI-300 (300 MHz). δ values are
in ppm relative to a tetramethylsilane internal standard.
Fourier transform IR spectra (KBr) were recorded on a Perkin-
Elmer 983G spectrometer. Mass spectra (EI, 70 eV, unless
otherwise stated) were obtained on a Finnigan MAT TSQ-46
and Finnigan MAT TSQ-700.
F igu r e 4. Specific activation of PhTERT-SEAP by analogues of
anthraquinone. About 2 × 103 cells of hTERT-BJ 1 harboring
P
hTERT-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 3i, 3a , or 3d 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 six experiments using the values
without drug treatment as 100%.
The following anthraquinones were prepared by previously
described procedures: 2 and 3. All other compounds were
commercial materials.
Ta ble 3. Effects of Anthraquinones on the CMV Promoter
Cell Cu ltu r e a n d Assessm en t of h TERT. Nonsmall lung
cancer cells H1299 (telomerase positive) were grown in RPMI
1640 media supplemented with 10% fetal bovine serum, 100
units/mL penicillin and 100 mg/mL streptomycin in a humidi-
fied atmosphere with 5% CO2 at 37 °C. The hTERT immortal-
ized hTERT-BJ 1 (BD Biosciences Clontech)39 were grown in
DMEM media supplemented with 10% fetal calf serum, 100
units/mL penicillin and 100 mg/mL streptomycin, 1 mM
Activity
P
CMV-SEAP (hTERT-BJ 1)b
relative
viability (%)
relative SEAP
activity (%)
compd
concna (µM)
2f
2.2
22
220
2.0
22
220
2.2
22
220
2.8
28
280
2.4
24
240
1.8
18
108 ( 9
109 ( 4
48 ( 6
116 ( 13
67 ( 18
52 ( 7
107 ( 10
118 ( 9
78 ( 18
114 ( 16
110 ( 12
94 ( 21
115 ( 7
104 ( 8
103 ( 11
105 ( 8
105 ( 7
108 ( 15
103 ( 8
102 ( 10
122 ( 8
103 ( 15
102 ( 8
100 ( 8
112 ( 15
105 ( 16
120 ( 14
101 ( 6
112 ( 6
138 ( 12
102 ( 10
114 ( 18
141 ( 11
99 ( 15
111 ( 11
121 ( 17
sodium pyruvate, and 4 mM L-arginine in
a humidified
2j
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 subcloned upstream to a secreted
alkaline phosphatase gene (SEAP) and transfectectd into
H1299 or hTERT-BJ 1 by electroporation. The stable clones
were selected using G418. The stable clones derived from
H1299 or hTERT-BJ 1 were cultured using conditions that are
similar to their parental cells.
Cytotoxicity Assa y. 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.43,44 Approximately 2 × 103 cells were plated
onto each well of a 96-well plate and incubated in 5% CO2 at
37 °C for 24 h. To assess the in vitro cytotoxicity, each
compound was dissolved in DMSO and prepared immediately
before the experiments and was diluted into the complete
medium before addition to cell cultures. Test compounds were
then added to the culture medium for designated various
concentrations. After 48 h, an amount of 25 µL of MTT was
added to each well, and the samples were incubated at 37°C
for 4 h. A 100 µL 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.
2n
3a
3d
3i
180
a
Values are in µM and represent an average of three experi-
ments. The variance for the relative viability (%) and relative
SEAP activity (%) values was less than (20%. Activity of PCMV
-
SEAP (hTERT-BJ 1) cell growth was significantly different from
that of the control; n ) 3 or more, P < 0.01. Relative percentage
of inhibition was not compared with that of the control, P < 0.01,
mean ( SE, n ) 4. Values are the mean percent activity at the
b
indicated concentration and include standard errors. CMV
(cytomegalovirus); SEAP (secreted alkaline phosphatase).
preferentially inhibit the growth of a cancer cell line
H1299 compared with the skin fibroblast BJ -1. The

3306 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
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Ack n ow led gm en t. The present study was sup-
ported by National Science Council Grants NSC 91-
2311-B-010-004 and NSC 91-2311-B-010-008 and by
Taipei Veteran Hospital Grants 390-13 and 89-B-FA22-
2-4 (Program for Promoting Academic Excellence of
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