Synthesis of mono- and bis-substituted anthraquinones as inhibitors of human telomerase

Article abstract of DOI:10.1211/146080899128734361

A number of anthraquinone derivatives have been synthesized in good yield and tested for their ability to inhibit the enzymes telomerase and Taq polymerase using a modified telomeric repeat amplification protocol. In addition, all the synthesized compounds were screened against a panel of ovarian carcinoma cell lines (A2780, CH1 and SKOV-3) to determine their cytotoxicity. All compounds tested inhibited telomerase at a concentration of 10 μM, but showed negligible inhibition of Taq polymerase. None of the compounds tested displayed significant general cell toxicity in ovarian cancer cell lines. The synthesized compounds are potential selective inhibitors of human telomerase.

Full text of DOI:10.1211/146080899128734361

Pharm. Pharmacol. Commun. 1999, 5: 669±671
Received October 1, 1999
Accepted November 2, 1999
# 1999 Pharm. Pharmacol. Commun.
Synthesis of Mono- and Bis-substituted Anthraquinones as
Inhibitors of Human Telomerase
V. GIBSON, R. J. ANDERSON, T. C. JENKINS* AND D. CAIRNS
Institute of Pharmacy and Chemistry, University of Sunderland, Sunderland and
*Department of Biomedical Sciences, University of Bradford, Bradford, UK
Abstract
A number of anthraquinone derivatives have been synthesized in good yield and tested for
their ability to inhibit the enzymes telomerase and Taq polymerase using a modi®ed
telomeric repeat ampli®cation protocol. In addition, all the synthesized compounds were
screened against a panel of ovarian carcinoma cell lines (A2780, CH1 and SKOV-3) to
determine their cytotoxicity.
All compounds tested inhibited telomerase at a concentration of 10 mM, but showed
negligible inhibition of Taq polymerase. None of the compounds tested displayed
signi®cant general cell toxicity in ovarian cancer cell lines.
The synthesized compounds are potential selective inhibitors of human telomerase.
Telomerase is an RNA-dependent DNA poly-
merase enzyme responsible for the elongation of
repeating telomeric sequences found at the 30 end
of eukaryotic chromosomes (Blackburn 1991). The
length of the telomere sequence is reduced every
time a cell divides and, once a critical length is
reached, the cell becomes unable to continue cell
division and enters a programmed cell death (Kim
et al 1994). Telomerase is not active in healthy
somatic cells but is overexpressed in almost all
human cancers (Kim et al 1994), allowing neo-
plastic cells to continue to divide in a sustained and
uncontrolled manner. Telomerase has thus attracted
considerable attention as a possible target for
anticancer chemotherapy (Raymond et al 1996;
Perry & Kelland 1998; Perry et al 1999).
As part of our work on the use of anthraquinones
as anticancer agents, a number of mono- and bis-
substituted anthraquinones have been synthesized,
fully characterized and tested for their ability to
inhibit telomerase and the related Taq polymerase.
In addition, all synthesized compounds were eval-
uated for general cell toxicity against a panel of
ovarian carcinoma cells.
1-(Hydroxypentylamino)anthraquinone (1A) was
prepared in good yield by re¯uxing 1-chloro-
anthraquinone with an excess of 5-aminopentanol.
The symmetrically substituted compounds, 1,5AA
and 1,8AA were prepared by reacting an excess of
5-aminopentanol with 1,5- and 1,8-dichloroanthra-
quinone. The bis-substituted derivatives, 1,5AC,
1,4AC, and 1,8AC were synthesized from the
appropriate dichloroanthraquinone in two steps by
re¯ux with 5-aminopentanol to yield the mono-
substituted derivative. These compounds were then
isolated and puri®ed before subsequent attachment
of the second sidechain with N,N-diethylamino-
ethylenediamine. 1,4-Bis (5-hydroxypentylamino)
anthraquinone, 1,4AA, was prepared from quinizarin
by reduction to the leuco form and attachment
of 5-aminopentanol (Figure 1). All compounds
were puri®ed by ¯ash column chromatography
and fully characterized by IR, NMR and mass spec-
trometry (Gibson et al 1996).
Materials and Methods
Modi®ed telomeric repeat ampli®cation protocol
(TRAP) assay
This procedure was carried out by the method of
Perry et al (1998) using extracts from exponentially
growing A2780 human ovarian carcinoma cells.
Correspondence: D. Cairns, Institute of Pharmacy and
Chemistry, University of Sunderland, Sunderland, UK.

670
V. GIBSON ET AL
Figure 1. Structure of synthesized anthraquinone derivatives.
Brie¯y, telomerase mediated extension of a primer
was initiated followed by the addition of protein
and incubation of the mixture with the synthesized
anthraquinone. Controls containing no protein,
heat-inactivated protein and protein at a con-
centration of 50% were included in each assay.
Taq DNA polymerase activity was determined by
heating the reaction mixture to 80ꢀC, to inactivate
telomerase, and addition of a reverse CX primer
along with Taq polymerase.
Table 1. Telomerase and Taq polymerase inhibition of
synthesized anthraquinone derivatives.
Compound
Taq inhibition
Telomerase inhibition (%)
10 mM
50 mM
10 mM
50 mM
1A
±
±
±
±
±
±
±
±
±
±
±
±
±
±
6Á4
22Á0
31Á5
IS
18Á1
27Á8
43Á3
36Á1
Sol
1,4AA
1,5AA
1,8AA
1,4AC
1,5AC
1,8AC
29Á9
24Á5
93Á5
57Á3
52Á8
A three-step polymerase chain reaction (PCR)
was performed and the presence or absence of
telomerase extended PCR products determined by
acrylamide gel electrophoresis.
±, Negligible inhibition detected; IS, insuf®cient sample;
Sol, compound not soluble at 50 mM.
Table 2. Cytotoxicity of anthraquinone derivatives in ovarian
carcinoma cell lines.
Growth inhibition assay
Growth inhibition was determined in three human
ovarian carcinoma cell lines (A2780, CH1 and
SKOV-3) using a sulphorhodamine B assay (Kel-
land et al 1993). The anthraquinone derivatives
were tested as water soluble mono- or dihydro-
chloride acid addition salts at a concentration of 4±
> 100 mM. After incubation, cells were stained with
1% sulphorhodamine B in acetic acid and the
absorbance at 540 nm measured. This value was
expressed as a percentage of the untreated control
cells and IC50 values determined for each com-
pound.
Compound
IC50 (mM)
A2780 A2780CisR CH1 CH1CisR SKOV-3
1A
> 100
> 100
> 100
> 100
4Á3
> 100
> 100
> 100
> 100
10Á5
> 100 > 100
> 100 > 100
> 100 > 100
> 100 > 100
> 100
> 100
> 100
> 100
10Á5
1,4AA
1,5AA
1,8AA
1,4AC
1,5AC
1,8AC
6Á8
2Á7
12Á5
13Á0
17Á0
8Á0
23Á5
> 100
> 100
> 100
11Á5
> 100
Table 3. Cell growth as percentage control at 100 mM.
Compound A2780 A2780CisR CH1 CH1CisR SKOV-3
1A
84Á6
92Á8
98Á6
97Á0
0Á5
86Á3
98Á4
100Á1
98Á8
6Á4
94Á0
104Á1
99Á6
71Á5
102Á0
101Á1
116Á0
2Á4
89Á1
98Á8
102Á1
99Á2
6Á7
Results and Discussion
1,4AA
1,5AA
1,8AA
1,4AC
1,5AC
1,8AC
The results of the modi®ed TRAP assay are sum-
marized in Table 1. All compounds tested inhibited
telomerase at concentrations of 10 mM (43Á3% for
1,8AC). None of the synthesized compounds
104Á2
20Á3
10Á9
94Á5
12Á5
96Á9
36Á9
1Á2
48Á8
107Á4
89Á4
12Á9

ANTHRAQUINONES AS INHIBITORS OF HUMAN TELOMERASE
671
inhibited the related enzyme, Taq polymerase, at
concentrations up to 50 mM, suggesting selective
inhibition of telomerase.
Sunderland, and Dr L. R. Kelland, CRC, Sutton, for
helpful discussions.
The general cytotoxicity data for the synthesized
anthraquinones are shown in Tables 2 and 3 . With
the exception of compounds 1,4AC and 1,5AC, all
of the compounds were remarkably non-toxic in all
of the tested ovarian carcinoma cell lines. It appears
that the compounds substituted with the N,N-
diethylaminoethylamino sidechain (1,4AC and
1,5AC) display greater cytotoxicity than the cor-
responding hydroxypentylamino substituted com-
pounds. This may in part be due to the presence of
an ionizable nitrogen in the sidechain of 1,4AC and
1,5AC, which will stabilize interaction with the
polyanionic backbone of DNA leading to non-
speci®c cell toxicity. The 1,8 disubstituted com-
pound, 1,8AC, appears anomalous in that it
demonstrates signi®cant inhibition of human telo-
merase at a concentration of 10 mM with no asso-
ciated cell toxicity even though it possesses the
ionizable N,N-diethylaminoethylamino sidechain.
This may be due to the fact that 1,8-disubstituted
anthraquinones can only intercalate with the long
axis of the chromophore aligned parallel to the
base-pair axis. The other disubstituted anthraqui-
nones are unable to intercalate in this way (Islam et
al 1985). Alternatively, the telomerase inhibition of
these compounds may be mediated through a non-
speci®c inhibition of the enzyme which is unrelated
to intercalation.
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Acknowledgements
We are grateful to Dr S. P. Mackay, Institute
of Pharmacy and Chemistry, University of