Synthesis and human telomerase inhibition of a series of regioisomeric disubstituted amidoanthraquinones

Article abstract of DOI:10.1248/cpb.55.284

Telomerase is the enzymatic activity that maintains the ends of eukaryotic chromosomes. Telomerase activity is detected in most tumor cells whereas it is low or undetectable in most normal somatic cells. Expression of the telomerase catalytic component, the human telomerase reverse transcriptase (hTERT), is believed to be controlled primarily at the level of transcription. Because of this selective expression property of telomerase, it has been touted as a specific target for antitumor chemotherapeutics. However, a concern for the applicability of telomerase inhibitors is that they require a long lag time for telomeres to be shortened to critical length before cancer cells stop proliferating. Here we investigate telomerase inhibitory, cytotoxicity and the hTERT repressing effects on a number of synthesized 2,6-diamidoanthraquinones and 1,5-diamidoanthraquinones as compared to their disubstituted homologues. We found that several of the 1,5-diamidoanthraquinones and 2,6- diamidoanthraquinones inhibited telomerase activity effectively with IC ₅₀ at the sub-micro to micro molar range and caused acute cytotoxicity to cancer cells with EC₅₀ similar or better than that of mitoxantrone. Particularly, 2,6-diamidoanthraquinone with 2-ethylaminoacetamido side chains 33, even though not affecting cell proliferation, showed to be endowed with a strong telomerase effect, probably related to a marked stabilization of the G-quadruplex-binding structure. The results suggested that these compounds caused multiple effects to cancer cells. More significantly, they overcome the long lag period problem of classical telomerase inhibitors that they are also potent cytotoxic agents. These results greatly expand the potential of tricyclic anthraquinone pharmacophore in preventive and/or curative therapy.

Full text of DOI:10.1248/cpb.55.284

284
Chem. Pharm. Bull. 55(2) 284—292 (2007)
Vol. 55, No. 2
Synthesis and Human Telomerase Inhibition of a Series of Regioisomeric
Disubstituted Amidoanthraquinones
Hsu-Shan HUANG, ,a In-Been CHEN,a Kuo-Feng HUANG,b Wei-Chih LU,c Fu-Ying SHIEH,d
*
d
d
,d
*
Yi-Yuan HUANG, Fong-Chun HUANG, and Jing-Jer LIN
a School of Pharmacy, National Defense Medical Center; Taipei 114, Taiwan: b Chi-Mei Medical Center; Tainan 710,
c
d
Taiwan: Cheng-Hsin Medical Center; Taipei 112, Taiwan: and Institute of Biopharmaceutical Science, National Yang-
Ming University; Taipei 112, Taiwan, R.O.C. Received September 28, 2006; accepted November 16, 2006
Telomerase is the enzymatic activity that maintains the ends of eukaryotic chromosomes. Telomerase activ-
ity is detected in most tumor cells whereas it is low or undetectable in most normal somatic cells. Expression of
the telomerase catalytic component, the human telomerase reverse transcriptase (hTERT), is believed to be con-
trolled primarily at the level of transcription. Because of this selective expression property of telomerase, it has
been touted as a specific target for antitumor chemotherapeutics. However, a concern for the applicability of
telomerase inhibitors is that they require a long lag time for telomeres to be shortened to critical length before
cancer cells stop proliferating. Here we investigate telomerase inhibitory, cytotoxicity and the hTERT repressing
effects on a number of synthesized 2,6-diamidoanthraquinones and 1,5-diamidoanthraquinones as compared to
their disubstituted homologues. We found that several of the 1,5-diamidoanthraquinones and 2,6-diamidoan-
thraquinones inhibited telomerase activity effectively with IC50 at the sub-micro to micro molar range and
caused acute cytotoxicity to cancer cells with EC50 similar or better than that of mitoxantrone. Particularly, 2,6-
diamidoanthraquinone with 2-ethylaminoacetamido side chains 33, even though not affecting cell proliferation,
showed to be endowed with a strong telomerase effect, probably related to a marked stabilization of the G-
quadruplex-binding structure. The results suggested that these compounds caused multiple effects to cancer
cells. More significantly, they overcome the long lag period problem of classical telomerase inhibitors that they
are also potent cytotoxic agents. These results greatly expand the potential of tricyclic anthraquinone pharma-
cophore in preventive and/or curative therapy.
Key words telomerase inhibition; cytotoxicity; anthraquinone; human telomerase reverse transcriptase (hTERT); G-quadruplex;
secreted alkaline phosphatase (SEAP) assay; telomerase assay; TRAP assay
Most human somatic cells divide 50—60 times before immortal cells, including cancer cells, it has become the
senescence occurs. These cells invariably enter a state of irre- focus of much attention as a novel and potentially highly-
versibly arrested growth. This process, termed replicative specific target for the development of new anticancer
senescence, is thought to be a tumor-suppressive mechanism chemotherapeutics. In this respect, antisense oligonu-
and an underlying cause of aging.1) The molecular mecha- cleotides or small molecular inhibitors have been identified
nism underlying cellular senescence has been characterized. as inhibitors to telomerase.8) Several of them effectively in-
It is well accepted that telomeres play a major role in the hibit telomerase in vitro and limit the proliferation of cancer
process. Telomeres are the physical ends of linear eukaryotic cells in vivo.9) However, because a lag period is required for
chromosomes. They consist of hundreds to thousands of tan- telomeres to be shortened to critical short length, the clinical
dem repeats of the sequence TTAGGG, that are essential for applicability of telomerase inhibitor was questioned. For ex-
stabilizing chromosomes.2) Because conventional DNA poly- ample, it takes ca. 20 additional cell divisions for telomeres
merase cannot replicate the very end of chromosomes, of HeLa cells to be shortened to critical length.8) It is not re-
telomere length is shortened upon each DNA replication. alistic for telomerase inhibitor to be used in treating cancers.
Telomerase is the enzyme activity that elongates short telom- Thus, it was proposed that telomerase inhibitors should work
eres. Because telomerase activity is low or not detectable in with other chemotherapeutic agents for treating cancers.10)
most normal human somatic cells, telomeric DNA is pro- The use of cytotoxic agents to kill cancer cells and telom-
gressively shortened with each cell division. The shortened erase inhibitor to limit the proliferative potential of residual
telomeres then signal cells to enter senescence through a cancer cells should be a better approach in cancer
DNA damage signaling pathway. Telomere length is consid- chemotherapy. Interestingly, guanine-quadruplex (G-quadru-
ered as a biological clock that is capable of determining the plex) stabilizers such as 2,6-pyridine-dicarboxamide deriva-
proliferative capacity of most human somatic cells.3,4)
tives and 3,6,9-trisubstituted acridine compounds caused ac-
While telomerase is not detected or is low in most of the celerated senescence in cancer cells. Molecules able to stabi-
normal human cells, it is detected in ca. 85—90% of the im- lize the G-quadruplex (G4), a structure adopted by the 3ꢀ-
mortalized or tumor cells. In humans, telomerase activity is overhang of telomeres, are thought to inhibit telomerase by
tightly regulated by expression of the human telomerase re- blocking its access to telomeres.11,12) Thus, the phenotypical
verse transcriptase (hTERT) gene, which appears to be the lag for typical telomerase inhibitors could be by-passed by
key regulator for telomerase activity. Inhibition or activation these types of compounds.
of the hTERT expression could profoundly affect the prolif-
In a continuation of our work on anthraquinones that
erative capacity of normal cells and cancers.5—7) Because widely occur in the plant kingdom and anthraquinone-based
telomerase is required for the sustained proliferation of most compounds currently occupy a prominent position in anti-
∗ To whom correspondence should be addressed. e-mail: huanghs@ndmctsgh.edu.tw; jjlin@ym.edu.tw
© 2007 Pharmaceutical Society of Japan

February 2007
285
Chart 2
We have identified these anthraquinones that inhibit telom-
erase and cytotoxic effects. Significantly, several of the most
potent cytotoxic anthraquinones process potent telomerase
inhibitory activities. These compounds have potential in fu-
ture anticancer drug developments that not only cause cyto-
toxic effects but also inhibit telomerase activity in cancer
cells.
Chart 1
Chemistry
cancer drugs development. Mitoxantrone and ametantrone
The general synthetic approach for the 2,6-disubstituted
are antitumor 1,4-bis[(aminoalkyl)amino]anthraquinones that amidoanthraquinones is shown in Chart 2. Starting material
were discovered by molecular simplification of the anthracy- 2,6-diaminoanthraquinone was first acylated with acyl chlo-
cline pharmacophore.13—15) However, adriamycin (doxoru- rides in N,N-dimethylacetamide with a catalytic quantity of
bicin, Chart 1) has structural characteristics that limit its effi- pyridine and subsequently reacted with appropriate amines in
cacy and safety. New anthracyclines with distinct structure, DMSO to obtain the desired compounds. Here the reactions
pharmacokinetics, pharmacodynamics, and toxicity profiles of electrophilic additions/substitutions and nucleophilic addi-
have been developed to overcome the limitations of doxoru- tions occurred at different position states of anthraquinones.
bicin and to further exploit the activity of anthracyclines.16) These compounds were obtained in good yields and their pu-
1
Although the reaction mechanism of the antitumor activity of rity was determined using mass spectrometry, H- and 13C-
anthraquinone is probably multimodal in nature, a number of NMR.
studies have indicated that its interaction with DNA may play
a major role.17—19) Through stabilizing G-quadruplex com- Biological Activity and Discussion
plexes formed by telomeric DNA sequences, it has been
It was shown that some of disubstituted anthraquinones in-
shown that several 1,4-disubstituted amidoanthraquinones, hibit telomerase, affect cell growth, and modulate hTERT ex-
and some 1,4- and 1,5-disubstituted aminoanthraquinones in- pression.23,25,28,29) By comparing these results with our com-
hibited telomerase activity.20—26) Anthraquinones could also pounds,20,27,30,31) it is clear that the chemical and biological
modulate hTERT expression. Using a reporter system, our activities of anthraquinones are greatly affected by various
previous studies indicated that several of the symmetrically substituents of the planar ring system.17,22,25,29,32—36) Neidle et
disubstituted 1,5-diacyloxyanthraquinones activate hTERT al.23,25,28,29) have also indicated that some of disubstituted an-
expression.27) These results provide a new clue on the effects thraquinones inhibit telomerase which provide us rational de-
of the anthraquinone compounds on modulation of hTERT sign for the further investigation of SARs and comparison
gene expression.
with their series of disubstituted homologues. Previously
The study of molecules structurally related to antitumor we described a method of synthesizing 1,5-diamidoanthra-
anthraquinone is expected to provide useful information in quinones (2—17) and comparing to their cytotoxicity,31)
the biological activity and a rational basis for further ana- these compounds considered as aglycon analogues of anthra-
logue development. The present study explores the effects on cycline antibiotics in which the side chains substitutes for
cytotoxicity, telomerase inhibitory, and hTERT expression small-molecule planar tricyclic anthraquinone structural mo-
using cell-based assay systems with two distinct series of tifs. In this investigation, we continue to focus our attention
amidoanthraquinones substituted at the 1,5-, and 2,6-posi- on the role of our newly synthesized 2,6-diamidoanthra-
tions as compare to their homologues. A range of side chains quinones. In addition, the analysis is further extended to the
has been examined to establish structure–activity relation- potential functions of anthraquinones with substitutions on
ships (SARs) as a basis for subsequent rational drug design. the different positions. The backbone of these compounds

286
Vol. 55, No. 2
also resemble the anticancer agents mitoxantrone and
ametantrone, and were prepared by a two-stage reaction
which similar to our previous paper.30,31) We first evaluate the
effects of these disubstituted amidoanthraquinones on telom-
erase activity using PCR-based telomerase assay, and TRAP
(telomeric repeat amplification protocol) assay. Here, a modi-
fied telomerase assay (TRAP-G4) is used to evaluate the ef-
fects of anthraquinones on G-quadruplex-induced telomerase
activity.37) In the TRAP-G4 assay, a G-quadruplex sequence
was introduced into the telomerase extension primer that is
susceptible to forming an intramolecular G-quadruplex. Be-
cause the formation of G-quadruplex blocks telomerase ex-
tension, this TRAP-G4 assay enables the evaluating the ef-
fects of G-quadruplex stabilizing agents on telomerase activ-
ity. The inhibitory effect of anthraquinones at 15—30 mM
concentrations was tested. Among a total of 1,5-diamidoan-
thraquinones and 2,6-diamidoanthraquinones were tested, we
found that 1,5-diamidoanthraquinones 5, 17, and 2,6-diami-
doanthraquinones 33 and 34 showed telomerase inhibitory
effect at the concentrations of 0.1 and 5.0 mM. The 1,5-diami-
doanthraquinones with side chains CH2NHCH2CH3 5 and
(CH)CH3NH(CH2)3N(CH3)2 17, show inhibition of G-
quadruplex-induced telomerase at the IC50 levels of 5.0 and
2.0 mM, respectively, whereas 2,6-diamidoanthraquinones
with side chains CH2NHCH2CH3 33 and CH2NH(CH2)2CH3
34, show IC50 levels of 0.1 and 2.0 mM, respectively. The in-
hibitory effects of these compounds on telomerase are spe-
cific as they did not affect the internal controls (ICs) for Taq
polymerase in our assays. These compounds are the most po-
tent telomerase inhibitors in this series of compounds. Inter-
estingly, most of 1,5-dithioanthraquinones and 1,5-diacyl-
oxyanthraquinones did not affect telomerase activity, on
Chart 3. Inhibition of Telomerase Activity by Disubstituted Amidoan-
thraquinones
TRAP-G4 assay was conducted using cell extracts prepared from H1299 cells and
2 mg 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 were 0.001, 0.01, 0.1, 1,
10 mM, respectively. Because the internal control (IC) shares one oligonucleotide with
the reaction, the IC products became apparent when telomerase activities were inhib-
ited. P is positive control and N is negative control.
the contrary 1,5-diacyloxyanthraquinones with side chains cial for the cytotoxic effects.
–COCH2CH3, –COC(CH3)3 and –COC6H3Cl2(o,p) activate
The mechanism of mitoxantrone action is cell cycle-inde-
hTERT expression in normal cells.27) Thus, in addition to an- pendent that it kills both proliferating and non-proliferating
ticancer functions, our finding raises the possibility that these cells. This property makes it different from many other cyto-
compounds might have potential application for cell immor- toxic agents.38) By comparing the telomerase inhibitory of
talization.
1,5-diamido-, 2,6-diamido-, 1,5-diamino-, and 1,4-diamido-
The analysis systems used cell lines derived from H1299 disubstituted anthraquinones in Table 2, it appears that on
(non-small cell lung cancer cells) and hTERT-BJ1 (human each position the bulkier or similar to the substituent of mi-
normal skin fibroblast cells immortalized with hTERT gene) toxantrone, the higher the activity.
cells. These cells were generated by introducing H1299 or
hTERT-BJ1 cells with DNA constructs harboring PhTERT
Previously we have identified several 1,5-diacyloxy-, 1,5-
dithio-, and 1,4-diamidoanthraquinones that activated the
-
SEAP (secreted alkaline phosphatase), so that the 3.4 kbp SEAP expression in hTERT-BJ1 cells. To our surprise, none
hTERT promoter fused upstream to a reporter gene, SEAP. of the 1,5-diamido and 2,6-diamidoanthraquinones affect
Thus, the expression of SEAP in H1299 cells harboring hTERT expression in hTERT-BJ1 and H1299 cells. Thus, our
PhTERT-SEAP could be used as the criteria to evaluate if an- results indicate that diamido substitution at 2,6 positions of
thraquinone derivatives inhibited the expression of hTERT in anthraquinones were not capable of affecting hTERT expres-
cancer cells. Similarly, the activation of hTERT expressions sion. Moreover, while substitution at 1,5 positions are impor-
could be monitored by the SEAP expressions using hTERT- tant for activating hTERT expression, amido group might not
BJ1 cells. The levels of cell viabilities in these cells upon be the choice for future design of these types of compounds.
drug treatments were also determined using MTT assay. We Our finding provided a new clue on future design of com-
showed that mitoxantrone gave an EC50 of ca. 20 mM under pounds for potential application in cell immortalization.
our assay conditions. Our results indicated that the 1,5-di-
amidoanthraquinone compounds 5, 8, 12, 15, and 20 showed Conclusions
EC50 ꢁ2 mM, that is more than 10 fold effective than that by
Anthraquinone-base molecules have been reported to bind
mitoxantrone. In contrast, most of the 2,6-diamidoanthra- the G-quadruplex DNA formed by telomeric DNA and in-
quinones did not show significant cytotoxic activity, only hibit telomerase activity.25,26,28,39,40) Telomerase inhibitor
compounds 33 and 34 showed EC50 values similar to or a lit- causes the attrition of telomere length and consequently lead-
tle better than mitoxantrone. Thus, it appears that diamido ing to senescence which require a lag period for cancer cells
substitutions at 2,6-positions of anthraquinones are not cru- to stop proliferating. Such a mechanism and result, if it is to

February 2007
287
Table 1. Effects of Symmetrical Diamidoanthraquinones on Activating or Repressing hTERT Expression and Telomerase Activity
PhTERT-SEAP (hTERT-BJ1)b)
PhTERT-SEAP (H1299)c)
Viability Relative SEAP
Concn.
TRAP assay IC50
Compd.
R
(mM)a)
Viability
(%)
Relative SEAP
activity (%)
(mM)
>25.6
>23.9
>22.4
5.0
(%)
activity (%)
2
3
CH2Cl
2.6
25.6
256
2.4
23.9
239
2.2
22.4
224
2.4
24.5
245
2.3
23
229
2.2
21.5
215
2.0
20
203
1.9
19.2
192
2.2
21.5
215
2.4
23.9
239
2.3
22.9
229
2.2
21.5
215
2.0
20.3
203
1.9
19.2
192
1.8
18.2
182
1.8
18.2
182
3.1
31
310
2.6
26.4
264
2.1
21.1
211
2.2
63ꢂ8
38ꢂ7
8ꢂ8
65ꢂ10
23ꢂ6
8ꢂ2
84ꢂ10
72ꢂ11
16ꢂ12
76ꢂ3
43ꢂ6
9ꢂ5
31ꢂ5
20ꢂ5
11ꢂ7
75ꢂ6
61ꢂ8
16ꢂ3
79ꢂ6
39ꢂ8
26ꢂ7
37ꢂ6
12ꢂ4
6ꢂ6
68ꢂ4
34ꢂ2
14ꢂ2
76ꢂ13
47ꢂ11
12ꢂ2
75ꢂ5
53ꢂ4
10ꢂ2
30ꢂ5
9ꢂ6
61ꢂ5
31ꢂ5
18ꢂ10
89ꢂ12
32ꢂ6
21ꢂ7
72ꢂ6
55ꢂ6
9ꢂ5
33ꢂ4
12ꢂ7
6ꢂ5
97ꢂ5
37ꢂ4
4ꢂ4
72ꢂ5
35ꢂ6
20ꢂ3
36ꢂ5
12ꢂ4
3ꢂ3
92ꢂ3
90ꢂ4
69ꢂ1
80ꢂ7
59ꢂ4
31ꢂ2
88ꢂ4
64ꢂ10
23ꢂ4
35ꢂ9
9ꢂ9
(CH2)2Cl
(CH2)3Cl
83ꢂ11
84ꢂ8
16ꢂ2
80ꢂ3
47ꢂ5
13ꢂ4
28ꢂ5
18ꢂ5
12ꢂ6
68ꢂ7
62ꢂ6
15ꢂ4
77ꢂ6
40ꢂ6
24ꢂ5
32ꢂ9
16ꢂ5
10ꢂ4
83ꢂ6
79ꢂ8
5ꢂ4
70ꢂ6
61ꢂ7
40ꢂ5
103ꢂ5
76ꢂ8
31ꢂ2
43ꢂ5
21ꢂ6
5ꢂ3
4
5
CH2NHCH2CH3
8ꢂ4
6
CH2NH(CH2)2CH3
CH2NH(CH2)3CH3
CH2NH(CH2)4CH3
CH2NH(CH2)5CH3
CH2N(CH2CH3)2
(CH)CH3Cl
91ꢂ8
36ꢂ2
8ꢂ3
70ꢂ3
45ꢂ1
28ꢂ2
29ꢂ1
13ꢂ2
4ꢂ3
95ꢂ2
88ꢂ1
71ꢂ3
75ꢂ4
53ꢂ3
35ꢂ5
81ꢂ4
70ꢂ4
24ꢂ2
21ꢂ5
8ꢂ4
>22.9
>21.5
>20.3
>19.2
>21.5
>23.9
>22.9
>21.5
>20.3
>19.2
>18.2
2.0
7
8
9
86ꢂ5
89ꢂ5
6ꢂ4
10
11
12
13
14
15
16
17
19
20
21
22
75ꢂ5
63ꢂ6
38ꢂ4
96ꢂ6
69ꢂ7
25ꢂ8
39ꢂ5
19ꢂ7
3ꢂ4
74ꢂ2
64ꢂ2
38ꢂ2
68ꢂ3
33ꢂ2
41ꢂ1
59ꢂ7
15ꢂ5
13ꢂ6
95ꢂ2
22ꢂ3
2ꢂ5
(CH)CH3NHCH2CH3
(CH)CH3NH(CH2)2CH3
(CH)CH3NH(CH2)3CH3
(CH)CH3NH(CH2)4CH3
(CH)CH3NH(CH2)5CH3
(CH)CH3NH(CH2)3N(CH3)2
CH3
4ꢂ4
4ꢂ5
80ꢂ9
64ꢂ4
42ꢂ5
64ꢂ8
40ꢂ7
39ꢂ5
55ꢂ9
9ꢂ6
10ꢂ6
98ꢂ3
27ꢂ8
4ꢂ4
100ꢂ11
86ꢂ7
53ꢂ5
73ꢂ8
70ꢂ3
28ꢂ7
49ꢂ4
11ꢂ5
6ꢂ4
71ꢂ10
60ꢂ9
43ꢂ3
59ꢂ9
35ꢂ10
21ꢂ3
98ꢂ10
68ꢂ3
40ꢂ2
40ꢂ9
10ꢂ4
9ꢂ6
97ꢂ5
80ꢂ7
61ꢂ10
71ꢂ8
64ꢂ9
20ꢂ10
54ꢂ6
10ꢂ1
7ꢂ1
72ꢂ8
66ꢂ10
44ꢂ8
46ꢂ8
25ꢂ6
6ꢂ5
94ꢂ7
65ꢂ4
36ꢂ5
39ꢂ11
14ꢂ9
5ꢂ9
83ꢂ3
39ꢂ8
18ꢂ7
66ꢂ12
73ꢂ8
55ꢂ5
28ꢂ3
6ꢂ1
31ꢂ7
12ꢂ7
6ꢂ3
106ꢂ11
70ꢂ10
52ꢂ9
52ꢂ9
29ꢂ4
18ꢂ5
90ꢂ11
61ꢂ8
18ꢂ7
89ꢂ7
92ꢂ3
100ꢂ8
85ꢂ12
53ꢂ13
52ꢂ7
30ꢂ3
16ꢂ4
84ꢂ9
52ꢂ8
18ꢂ4
84ꢂ6
84ꢂ5
>31.0
>26.4
>21.1
>21.8
CH2CH3
3-C6H4CH3
80ꢂ5
52ꢂ3
17ꢂ5
71ꢂ5
68ꢂ7
C6H10 (cyclohexane)
21.8

288
Vol. 55, No. 2
Table 1. Continued
PhTERT-SEAP (hTERT-BJ1)b)
PhTERT-SEAP (H1299)c)
Viability Relative SEAP
Concn.
TRAP assay IC50
Compd.
R
(mM)a)
Viability
(%)
Relative SEAP
activity (%)
(mM)
(%)
activity (%)
22
23
C6H10 (cyclohexane)
(CH2)2C5H9
218
2.1
20.6
206
2.3
23.2
232
2.7
26.7
267
1.9
19
190
1.9
18.6
186
2.6
25.6
256
2.4
23.9
239
2.4
24.5
245
2.3
22.9
229
2.2
21.5
215
2.4
23.9
239
3.1
31
310
2.2
29ꢂ7
79ꢂ6
81ꢂ5
19ꢂ2
92ꢂ11
92ꢂ8
9ꢂ6
97ꢂ5
59ꢂ7
40ꢂ3
34ꢂ10
20ꢂ7
4ꢂ4
76ꢂ7
57ꢂ6
34ꢂ8
58ꢂ6
42ꢂ5
5ꢂ7
34ꢂ4
22ꢂ5
10ꢂ7
63ꢂ5
25ꢂ5
1ꢂ3
26ꢂ4
72ꢂ3
70ꢂ4
16ꢂ3
87ꢂ4
83ꢂ5
3ꢂ3
89ꢂ6
55ꢂ3
39ꢂ6
40ꢂ5
15ꢂ4
3ꢂ5
71ꢂ5
50ꢂ4
41ꢂ6
60ꢂ5
42ꢂ4
6ꢂ5
33ꢂ7
24ꢂ4
13ꢂ3
68ꢂ6
21ꢂ3
4ꢂ7
15ꢂ1
75ꢂ6
73ꢂ6
15ꢂ2
81ꢂ5
65ꢂ4
14ꢂ2
81ꢂ4
46ꢂ3
22ꢂ3
79ꢂ5
46ꢂ3
19ꢂ2
80ꢂ4
49ꢂ3
31ꢂ5
76ꢂ4
17ꢂ6
4ꢂ5
10ꢂ7
52ꢂ11
55ꢂ11
4ꢂ8
84ꢂ9
53ꢂ9
36ꢂ8
68ꢂ8
31ꢂ4
6ꢂ4
66ꢂ4
45ꢂ9
3ꢂ5
75ꢂ4
46ꢂ9
24ꢂ8
79ꢂ5
20ꢂ3
5ꢂ1
81ꢂ3
56ꢂ3
23ꢂ3
62ꢂ7
24ꢂ6
4ꢂ3
74ꢂ9
41ꢂ13
8ꢂ4
73ꢂ2
54ꢂ2
20ꢂ3
98ꢂ7
50ꢂ9
24ꢂ7
96ꢂ6
51ꢂ5
40ꢂ6
53ꢂ7
19ꢂ5
16ꢂ6
98ꢂ5
95ꢂ7
44ꢂ4
81ꢂ3.8
66ꢂ4.0
47ꢂ3.9
>20.6
>23.2
>26.7
>19.0
>18.6
>25.6
>23.9
0.1
24
C5H9
25
C3H5
26
trans-CH(CH2)CHC6H5
CH2SC6H5
CH2Cl
27
31
32
CH2CH2Cl
CH2NHCH2CH3
CH2NH(CH2)2CH3
CH2NH(CH2)3CH3
CHCH3Cl
77ꢂ7
57ꢂ5
24ꢂ4
59ꢂ6
22ꢂ7
7ꢂ8
33
34
61ꢂ5
31ꢂ6
7ꢂ6
64ꢂ7
33ꢂ7
8ꢂ6
67ꢂ7
40ꢂ6
16ꢂ5
70ꢂ9
56ꢂ3
23ꢂ7
103ꢂ8
57ꢂ8
19ꢂ9
89ꢂ5
54ꢂ5
39ꢂ3
57ꢂ10
26ꢂ5
14ꢂ8
91ꢂ4
89ꢂ12
31ꢂ11
100ꢂ5.6
57ꢂ4.3
39ꢂ3.2
2.0
35
63ꢂ10
50ꢂ8
12ꢂ5
98ꢂ5
45ꢂ7
25ꢂ6
89ꢂ8
66ꢂ9
36ꢂ10
54ꢂ9
19ꢂ8
10ꢂ5
86ꢂ5
88ꢂ8
39ꢂ4
75ꢂ2.9
56ꢂ3.1
10ꢂ2.0
69ꢂ8
45ꢂ5
14ꢂ7
100ꢂ5
42ꢂ7
29ꢂ5
93ꢂ12
65ꢂ11
45ꢂ13
51ꢂ7
15ꢂ9
7ꢂ6
89ꢂ6
85ꢂ7
31ꢂ5
30ꢂ5.8
13ꢂ9.2
4ꢂ14.2
>21.5
>23.9
>31.0
>22.4
>19.4
2.0
39
45
CH3
46
47
C6H5
22.4
224
1.9
19.4
194
1.9
C6H4Cl-o
Mitoxantrone
19
193
a) Values are in mM and represent an average of three experiments. b) The hTERT immortalized hTERT-BJ1 was purchased from BD Biosciences Clontech. The results in
this column are shown as meansꢂS.E. of experiments repeated five times. c) Values are mean percent activity at the indicated concentration, and standard errors. The variance
for the relative viability (%) and relative SEAP activity (%) values was less than ꢂ20%. All of SEAP data are shown as the result that drug-self interference has been subtracted.
be therapeutically useful, requires a lag period for cancer hibitors with this special type of property. The unique prop-
cells to stop proliferating in which compounds also have the erty of these anthraquinones make them as the best candi-
ability to effectively discriminate between duplex and dates for future developments of anticancer drugs. Mitox-
quadruplex DNA. Thus, the acute cytotoxicity of anthra- antrone is useful for chemotherapy because it inhibits both
quinones should not be caused solely by their effects on DNA replication and DNA-dependent RNA synthesis by in-
telomerase. Interestingly, our results showed that the spec- tercalating into DNA and causing crosslinking and strand
trum of some anthraquinones for cytotoxicity and telomerase breaks.41,42) It also inhibits topoisomerase II and conse-
inhibitory are quite similar. These anthraquinones described quently interferes with DNA repair.42—44) Previous SAR stud-
here exhibit 1,5-diamidoanthraquinone 5 and 2,6-diamidoan- ies have indicated the crucial role of diaminoalkyl group in
thraquinones 33, 34 are potent cytotoxic compounds and ef- side chains of anthraquinones for their cytotoxic activi-
fective telomerase inhibitors which have IC50 values in the ties,44,45) but the importance of this diaminoalkyl group for
low-micromolar range compared with mitoxantrone. They any of the proposed mechanisms of action is still not clear.
represent the first small-molecule structures of telomerase in- The present cytotoxic studies on 1,5-diamido and 2,6-di-

February 2007
289
Table 2. Structures and Inhibition (IC50) of Some Selected Telomerase-Inhibitory Anthraquinones
PhTERT-SEAP (hTERT-BJ1)
Concn.
PhTERT-SEAP (H1299)
Viability Relative SEAP
TRAP assay
IC50 (mM)
Isomers No.
R
(mM)
Viability
(%)
Relative SEAP
activity (%)
(%)
activity (%)
1,4-diamido20)
1,4-diamido20)
CH2CH2N(CH2CH3)2
2,4,6-Cl3C6H2
2.0
20
83ꢂ9.2
15ꢂ3.9
6ꢂ3.9
98ꢂ8.5
63ꢂ7.0
40ꢂ22.2
28ꢂ5
18ꢂ5
12ꢂ6
55ꢂ5
28ꢂ3
6ꢂ1
63ꢂ5
25ꢂ5
1ꢂ3
61ꢂ5
31ꢂ6
7ꢂ6
92ꢂ4.8
76ꢂ5.9
7ꢂ18.2
84ꢂ19.8
60ꢂ11.2
44ꢂ12.9
72ꢂ7.4
39ꢂ10.5
26ꢂ15.9
85ꢂ6.9
3ꢂ6.9
(ꢃ2)ꢂ7.7
101ꢂ10.5
101ꢂ8.8
91ꢂ11.8
75ꢂ2.9
56ꢂ3.1
10ꢂ2.0
2ꢂ11.1
(ꢃ2)ꢂ11.2
(ꢃ2)ꢂ6.5
31ꢂ23.0
12ꢂ12.5
4ꢂ15
31ꢂ5
20ꢂ5
11ꢂ7
31ꢂ7
12ꢂ7
6ꢂ3
68ꢂ6
21ꢂ3
4ꢂ7
64ꢂ7
33ꢂ7
8ꢂ6
11ꢂ22.4
ꢃ15ꢂ18.2
ꢃ26ꢂ16.9
60ꢂ11.6
40ꢂ17.3
52ꢂ19.1
41ꢂ12.5
0ꢂ22.1
ꢃ3ꢂ10.0
103ꢂ19.8
47ꢂ20.5
60ꢂ15.1
114ꢂ20.5
113ꢂ21.6
127ꢂ19.9
30ꢂ5.8
13ꢂ9.2
4ꢂ14.2
92ꢂ6.1
11ꢂ2.5
1ꢂ2.4
113ꢂ4.8
107ꢂ5.4
20ꢂ3.3
30ꢂ5
9ꢂ6
8ꢂ4
59ꢂ9
35ꢂ10
21ꢂ3
59ꢂ6
22ꢂ7
7ꢂ8
67ꢂ7
40ꢂ6
16ꢂ5
107ꢂ9.1
81ꢂ8.3
29ꢂ2.6
97ꢂ8.7
37ꢂ3.9
11ꢂ4.1
107ꢂ5.0
49ꢂ3.5
40ꢂ10.4
85ꢂ11.5
32ꢂ9.3
16ꢂ2.1
114ꢂ8.1
110ꢂ7.1
16ꢂ3.4
100ꢂ5.6
57ꢂ4.3
39ꢂ3.2
73ꢂ2.7
53ꢂ2.7
39ꢂ0.8
98ꢂ4.5
66ꢂ2.4
41ꢂ3.9
33ꢂ4
12ꢂ7
6ꢂ5
46ꢂ8
25ꢂ6
6ꢂ5
62ꢂ7
24ꢂ6
4ꢂ3
74ꢂ9
41ꢂ13
8ꢂ4
103ꢂ6.0
54ꢂ5.9
40ꢂ5.9
87ꢂ3.8
43ꢂ5.5
40ꢂ6.7
88ꢂ4.9
69ꢂ5.4
33ꢂ2.2
102ꢂ4.9
44ꢂ7.5
38ꢂ6.0
89ꢂ4.9
71ꢂ9.8
27ꢂ2.0
81ꢂ3.8
66ꢂ4.0
47ꢂ3.9
2.0
40
203
1.5
15
153
2.4
24.5
245
1.8
18.2
182
2.4
24.5
245
2.3
22.9
229
2.6
26
262
2.4
24
242
2.8
28
282
2.8
28
283
2.6
26
265
1.9
19
193
1,5-diamido31)
CH2NHCH2CH3
5.0
2.0
0.1
2.0
20
5
1,5-diamido31)
(CH)CH3NH(CH2)3N(CH3)2
CH2NHCH2CH3
17
2,6-diamido
33
2,6-diamido
CH2NH(CH2)2CH3
CH2CH2N(CH3)2
CH2CH2NH(CH2)2OH
CH2CH2CH2OH
34
1,5-diamino20)
1,5-diamino20)
1,5-diamino20)
1,5-diamino20)
1,5-diamino20)
0.2
30
CH2CH2CH2NH2
CH2CH2CH2CH2NH2
Mitoxantrone
0.5
5.0
2.0
amido-anthraquinones indicated that many of them are more been shown to have various effects on G-quadruplex forma-
effective than mitoxantrone at concentration 4—20 fold tion. Perry and Neidle25) have shown that amidoanthra-
lower. Our result is consistent with early SAR analysis that quinones favoring duplex binding are related to the inhibition
diaminoanthraquinones are effective cytotoxic agents. Since of telomerase activity and assumed to be nonselective. In our
our results showed that 1,5-diamidoanthraquinone 5 and 2,6- previous papers, we reported the inhibitory effects of human
diamidoanthraquinones 33, 34 have multiple functions on telomerase by a series of difunctionalized substituted an-
cells. In addition to their telomerase inhibitory effects, they thraquinone derivatives.20,27,30,46—49) From a SAR point of
might affect cancer cells in a way similar to that of mito- view cytotoxicity is evident from the remarkable results pre-
xantrone. It is also noteworthy that more 1,5-diamidoan- sented here and previously reported. In combination with our
thraquinones were identified as effective cytotoxic agents present studies on a range of 2,6-diamidoanthraquinones and
than 2,6-diamidoanthraquinones. Keppler et al.17) have also 1,5-diamidoanthraquinones in inhibiting telomerase activity,
indicated that anthraquinones interacted with duplex DNA in some indications of SARs for telomerase inhibition are
molecular modeling studies and have examined how the po- evident. For example, the most active compound from
sition of the attached base-functionalized substituents affects the TRAP assay, with a IC50 of 0.1 mM, is the 2-(N-
their ability to stabilize DNA duplexes. The apparent bias ethylamino)acetamido side chains of 2,6-diamidoanthra-
might provide a direction for future design of anthraquinone- quinone 33. This is thus among the most potent nonnu-
based anticancer agents.
cleoside telomerase inhibitors reported to date. 1,5-Di-
Substitutions on the planar ring of anthraquinone have aminoanthraquinones 6 and 10 with CH2CH2NH(CH2)2OH

290
Vol. 55, No. 2
perature under N2. The resulting precipitate was collected by filtration,
washed with diethyl ether and purified by crystallization from ethyl acetate
to afford desired compounds.
and CH2CH2CH2NH2 side chains also show good inhibition
of telomerase at IC50 level of 0.2 and 0.5 mM, respectively.
The activities of the 1,5-diamidoanthraquinone 17 with
(CH)CH3NH(CH2)3N(CH3)2 side chains, 2,6-diamidoanthra-
quinone 34 with CH2NH(CH2)2CH3 side chains, and 1,4-di-
amidoanthraquinone with CH2CH2N(CH2CH3)2 side chains
show telomerase activity at levels similar to mitoxantrone
with IC50 value of 2.0 mM. Even with substitutions at different
positions, these derivatives share a common feature with
bulky long substituent at the terminus of the side chain. This
feature may be important for inhibition of telomerase activ-
ity, at least in the 2,6-diamidoanthraquinones, 1,4-diami-
doanthraquinones, and 1,5-diamidoanthraquinones series ex-
amined. Molecular modeling studies suggested that the tri-
cyclic structure of anthraquinone bound to the terminal gua-
nine-quartet.26,50,51) Substitutions of anthraquinones then
bound to the DNA grooves to further stabilize the complex.9)
The bulky long substitutions of anthraquinones might serve
as two arms to stabilize the interactions between these com-
pounds and G-quadruplex structures.
Amination (Compounds 33—38, 40—44): A solution of an appropriate
amines (20 mmol) in DMF was added dropwise under N2 to a suspended so-
lution of compounds 31—32, or 39 (1 mmol) in 40 ml of DMF and triethyl-
amine (0.5 ml). The reaction mixture was heated and refluxed at 130 °C in
miniclave (Büchi®) for 1 h. After cooling and the reaction mixture was
treated with crushed ice and the resulting precipitate was collected by filtra-
tion, washed and purified by crystallization from ethyl acetate to afford de-
sired compounds.
2,6-Bis(2-chloroacetamido)anthraquinone (31) 90% yield. mp 323 °C
1
(Ethanol). H-NMR (CDCl3): d: 4.35 (4H, s, CH2), 8.06 (2H, d, Jꢄ8.4 Hz),
8.18 (2H, d, Jꢄ8.4 Hz), 8.42 (2H, s), 10.94 (2H, s, NH). 13C-NMR (CDCl3):
d: 181.30, 165.67, 144.09, 134.46, 128.71, 128.71, 123.89, 116.25, 43.68,
40.53, 39.96, 39.70, 39.41, 39.14, 38.86. IR (KBr) cmꢃ1: 1695, 3300. MS
m/z: 394 (Mꢅ), 393, 392, 391, 389.9, 314, 238.
2,6-Bis(2-chloropropionamido)anthraquinone (32) 90% yield. mp
1
186 °C (Ethanol). H-NMR (CDCl3): d: 2.92 (2H, t, Jꢄ6.9 Hz, CH2), 3.91
(2H, t, Jꢄ6.3 Hz, CH2), 8.08 (2H, d, Jꢄ8.4 Hz), 8.17 (2H, d, Jꢄ8.4 Hz),
8.45 (2H, s), 10.88 (2H, s, NH). 13C-NMR (CDCl3): d: 181.33, 169.04,
144.46, 134.45, 128.61, 128.26, 123.56, 115.98, 40.53, 40.26, 39.96, 39.70,
39.43, 39.14, 38.87. IR (KBr) cmꢃ1: 1666, 3098. MS m/z: 419 (Mꢅ), 389,
314.
2,6-Bis(2-ethylaminoacetamido)anthraquinone (33) 87% yield. mp
379 °C (Ethanol). 1H-NMR (CDCl3): d: 0.88 (6H, t, Jꢄ6.9 Hz, CH3), 1.42—
1.49 (4H, m, CH2), 2.51 (4H, t, Jꢄ6.3 Hz, CH2), 8.09 (2H, d, Jꢄ9.0 Hz),
8.16 (2H, d, Jꢄ8.4 Hz), 8.49 (2H, s). 13C-NMR (CDCl3): d: 181.37, 171.59,
144.30, 134.46, 128.52, 128.22, 123.67, 116.40, 53.08, 43.65, 40.84, 40.56,
40.28, 40.01, 39.72, 39.45, 39.17, 15.29. IR (KBr) cmꢃ1: 1585, 1700, 2956,
3282. MS m/z: 408 (Mꢅ), 346, 292.
We have previously shown that 1,5-diaminoanthraquinones
have good telomerase inhibition using TRAP assay. However,
the present study shows that 2,6-diamidoanthraquinone 33
significantly inhibit telomerase activity. In common with
other similarly behaving compounds in Tables 1 and 2, it was
evaluated in the TRAP assay in view of its potential nonse-
lective affinity for DNA polymerase. The crystal structure of
Taq polymerase indicates that its active site shares at least
some common features with other DNA polymerases.52,53) If
such compounds are to have applications as antitumor
agents, with telomerase inhibition in tumor cells leading to
the attrition of telomere length and consequent senescence.
By contrast the established anthraquinone-based anticancer
2,6-Bis(2-propylamino)ethanamido)anthraquinone (34) 80% yield.
1
mp 149 °C (Ethanol). H-NMR (CDCl3): d: 0.88 (6H, t, Jꢄ6.9 Hz), 1.42—
1.49 (4H, m), 2.49—2.51 (4H, m), 3.34 (4H, d), 8.07 (2H, d, Jꢄ9.0 Hz),
8.16 (2H, d, Jꢄ8.4 Hz), 8.49 (2H, s). 13C-NMR (CDCl3): d: 181.37, 171.59,
144.30, 134.45, 128.52, 128.22, 123.67, 116.02, 53.01, 51.06, 40.54, 40.26,
39.98, 39.70, 39.43, 39.14, 38.87, 22.63, 11.75. IR (KBr) cmꢃ1: 1589, 1652,
1700, 2956, 3282. MS m/z: 436 (Mꢅ), 337, 238.
2,6-Bis(2-N-n-butylaminoacetamido)anthraquinone (35) 76% yield.
mp 171 °C (Ethanol). 1H-NMR (CDCl3): d: 0.87 (6H, t, Jꢄ7.2 Hz, CH3),
drugs mitoxantrone, even though it shows telomerase activity 1.28—1.36 (4H, m, CH2), 1.38—1.45 (4H, m, CH2), 2.48—2.56 (4H, m,
CH2), 3.33 (4H, d, Jꢄ7.0 Hz, CH2), 8.08 (2H, dd, Jꢄ6.6, 2.1 Hz,), 8.15 (2H,
at levels 2.0 mM. It is also noteworthy that the tricyclic an-
thraquinone structure motif itself might contribute to the bio-
logical activity. The telomerase effects induced by an-
d, Jꢄ8.4 Hz), 8.48 (2H, d, Jꢄ1.8 Hz). 13C-NMR (CDCl3): d: 181.37 (CO),
171.63 (NCO), 151.52, 144.31, 134.44, 128.52, 128.21, 123.66, 116.02,
53.11, 48.80, 40.54, 40.26, 39.98, 39.70, 39.43, 39.14, 38.87, 31.70, 19.92,
13.93. IR (KBr) cmꢃ1: 1595, 1655, 1705, 2965, 3285. MS m/z: 464 (Mꢅ),
351.
thraquinone derivatives do not closely resemble what we pre-
viously reported.20,27) As a whole, these data support the con-
clusion that all the tested molecules, including those reported
here, share a common target in telomerase. These results
confirm once more the view that tricyclic anthraquinone
pharmacophore targeting telomerase, DNA-interactive and
fold into a wide variety of four-stranded quadruplex struc-
tures may be regarded as novel potential anticancer agents.
2,6-Bis(2-N-pentylaminoacetamido)anthraquinone (36) 77% yield.
mp 159 °C (Ethanol). 1H-NMR (CDCl3): d: 0.86 (6H, t, Jꢄ6.9 Hz, CH3),
1.28 (8H, t, Jꢄ6.8 Hz, CH2), 1.43 (4H, t, Jꢄ6.8 Hz, CH2), 2.51 (8H, t,
Jꢄ6.8 Hz), 8.08 (2H, dd, Jꢄ8.7, 1.5 Hz), 8.14 (2H, d, Jꢄ8.4 Hz), 8.46 (2H,
d, Jꢄ1.8 Hz). 13C-NMR (CDCl3): d 181.37, 171.66, 144.34, 134.45, 128.54,
128.21, 123.66, 116.03, 53.14, 49.16, 40.54, 40.26, 39.98, 39.70, 39.43,
39.15, 38.87, 29.22, 29.06, 22.13, 14.02. IR (KBr) cmꢃ1: 1690, 1700, 3285.
MS m/z: 492 (Mꢅ), 365, 266, 238.
2,6-Bis(2-N-hexylaminoacetamido)anthraquinone (37) 77% yield.
mp 135 °C (Ethanol). 1H-NMR (CDCl3): d: 0.84 (t, 6H, Jꢄ6.6 Hz, CH3),
1.25—1.43 (18H, m, CH2), 2.53 (4H, t, Jꢄ7.2 Hz, CH2), 3.34 (2H, s, CH2),
Experimental
Melting points were determined with a Büchi 530 melting point apparatus
and are uncorrected. All reactions were monitored by TLC, which were per- 8.08 (2H, dd, Jꢄ8.7, 1.8 Hz), 8.15 (2H, d, Jꢄ7.8 Hz), 8.47 (2H, d,
formed on precoated sheets on silica gel 60 F254, and flash column chro-
Jꢄ1.8 Hz). 13C-NMR (CDCl3): d: 181.35, 171.66, 144.33, 134.44, 128.50,
matography was done in silica gel (E. Merck, 70—230 mesh) with CH2Cl2 128.19, 123.65, 116.02, 53.14, 49.18, 40.53, 40.26, 39.98, 39.70, 39.43,
as eluant, unless otherwise stated. 1H-NMR spectra were recorded with a 39.14, 38.87, 31.29, 29.51, 26.47, 22.14, 13.96. IR (KBr) cmꢃ1: 1695, 2909,
Varian GEMINI-300 (300 MHz). d values are in ppm relative to a tetra- 3339. MS m/z: 520 (Mꢅ), 405, 379, 238.
methylsilane internal standard. Fourier transform IR spectra (KBr) were
2,6-Bis(2-N-ethylaminopropionamido)anthraquinone (38) 75% yield.
recorded on a Perkin-Elmer 983G spectrometer. Mass spectra (EI, 70 eV, un- mp 349 °C (Ethanol). 1H-NMR (CDCl3): d: 1.00 (6H, t, Jꢄ7.5 Hz, CH3),
less otherwise stated) were obtained on a Finnigan MAT TSQ-46 and Finni- 2.47—2.57 (8H, m), 2.80 (4H, t, Jꢄ6.6 Hz), 8.02 (2H, d, Jꢄ8.4 Hz), 8.12
gan MAT TSQ-700. The following 1,5-diamidoanthraquinones (2—27) were (2H, d, Jꢄ8.7 Hz), 8.38 (2H, s). 13C-NMR (CDCl3): d: 181.37, 171.62,
prepared by previously described procedures.31) All other compounds were 144.81, 134.44, 128.55, 127.98, 123.41, 115.83, 45.06, 43.26, 40.54, 40.26,
commercial materials.
39.98, 39.70, 39.43, 39.15, 38.87, 37.27, 15.21. IR (KBr) cmꢃ1: 1700, 2910,
General Procedure for the Preparation of 2,6-Diamidoanthraquinone 3340. MS m/z: 436 (Mꢅ), 346, 292, 238.
Derivatives Acylation (Compounds 31—32, 39, 45—47): Chloroacyl
chloride (12 mmol) was added dropwise at 0 °C under N2 to a solution of
2,6-diaminoanthraquinone (1 mmol) and pyridine (0.5 ml) in N,N-dimethyl- (2H, q, Jꢄ6.6 Hz, CH), 8.09 (2H, dd, Jꢄ6.6 Hz), 8.20 (2H, d, Jꢄ8.1 Hz),
formamide (20 ml). The reaction mixture was stirred for 24 h at room tem-
2,6-Bis(2-chloropropionamido)anthraquinone (39) 90% yield. mp
290 °C (Ethanol). H-NMR (CDCl3): d: 1.65 (6H, d, Jꢄ6.9 Hz, CH3), 4.72
1
8.46 (2H, s). 13C-NMR (CDCl3): d: 181.30, 168.29, 144.15, 134.46, 128.71,

February 2007
291
127.98, 124.37, 116.43, 54.76, 20.93. IR (KBr) cmꢃ1: 1671, 2910, 3340. MS
m/z: 419 (Mꢅ), 348.
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2,6-Bis(benzamido)anthraquinone (46) 75% yield. mp 321 °C
(Ethanol). 1H-NMR (CDCl3): d: 7.54—7.64 (6H, m, CH), 8.02 (2H, d,
Jꢄ8.1 Hz), 8.23 (4H, d, Jꢄ8.1 Hz), 8.34 (2H, d, Jꢄ8.7 Hz), 8.67 (2H, s, Ar-
H), 10.86 (2H, s, NH). 13C-NMR (CDCl3): d: 181.30, 166.26, 144.95,
134.28, 132.19, 128.56, 128.41, 127.97, 124.61, 117.19. IR (KBr) cmꢃ1
:
1580, 1690, 3320. MS m/z: 446 (Mꢅ), 105.
2,6-Bis(2-chlorobenzamido)anthraquinone (47) 80% yield. mp
1
343 °C (Ethanol). H-NMR (CDCl3): d: 7.49—7.62 (6H, m, CH), 7.67 (2H,
d, Jꢄ7.5 Hz), 8.18 (4H, d, Jꢄ8.1 Hz), 8.23 (2H, d, Jꢄ8.1 Hz), 8.62 (2H, s,
Ar-H), 11.17 (2H, s, NH). IR (KBr) cmꢃ1: 1690, 2363, 3320. MS m/z: 516
(Mꢅ), 141, 139.
Cell Culture and Assessment of hTERT Nonsmall lung cancer cells
H1299 (telomerase positive) were grown in RPMI 1640 media supple-
mented with 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml
streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. The hTERT
immortalized hTERT-BJ1 (BD Biosciences Clontech)54) were grown in
DMEM media supplemented with 10% fetal calf serum, 100 units/ml peni-
cillin and 100 mg/ml streptomycin, 1 mM sodium pyruvate, and 4 mM l-argi-
nine in a humidified atmosphere with 5% CO2 at 37 °C. Culture media were
changed every 3 d. To establish stable cell lines that the expression of
hTERT could be monitored by a reporter system, a ca. 3.3 kbp DNA frag-
ment ranging from ꢃ3338 to ꢅ1 bp of the hTERT gene was subcloned up-
stream 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 cul-
tured using conditions that are similar to their parental cells.
Cytotoxicity Assay The tetrazolium reagent (MTT; 3-(4,5-di-methylthi-
azol)-2,5-diphenyltetrazolium bromide, USB) was designed to yield a col-
ored formazan upon metabolic reduction by viable cells.55,56) Approximately
2ꢆ103 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 various desig-
nated concentrations. After 48 h, an amount of 25 ml of MTT was added to
each well, and the samples were incubated at 37 °C for 4 h. A 100 ml solu-
tion 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 ab-
sorbency at 550 nm was measured using an ELISA reader.
Telomerase Assay The telomeric repeat amplification protocol (TRAP)
is commonly used to evaluate telomerase activity in tissues or cell extracts
and also to determine the inhibitory properties of small molecules against
telomerase. A modified telomeric-repeat-amplification protocol (TRAP-G4)
was utilized for G-quadruplex-induced telomerase activity assay.37) Telom-
erase products were resolved by 10% polyacrylamide gel electrophoresis
and visualized by staining with SYBER Green. As a source of telomerase,
the total cell lysates derived from lung cancer cell line H1299 cells were
used. Protein concentration of the lysates was assayed using Bio-Rad protein
assay kit using BSA standards.
SEAP Assay57) Secreted alkaline 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 was determined.
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Acknowledgements The present study was supported by National Sci-
ence Council Grants NSC 94-2113-M-016-003, 95-2113-M-016-002-MY2,
94-2311-B-010-012 and 94-3112-B-010-020 and also by National Health
Research Institute grant NHRI-EX95-9436SI. The authors are also indebted
to Dr. K. K. Mayer (Universität Regensburg, Germany) for the mass spec-
trometry analytical determinations.
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