A novel inhibitor of human telomerase derived from 10h-indolo[3,2-b]quinoline

Article abstract of DOI:10.1016/S0960-894X(00)00378-4

The bis-dimethylaminoethyl derivative of quindoline (10H-indolo[3,2-b]quinoline), an alkaloid from the West African shrub Cryptolepis sanguinolenta, has been synthesised. This has been shown to have modest cytotoxicity, as well as inhibitory activity agai

Full text of DOI:10.1016/S0960-894X(00)00378-4

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2063±2066
A Novel Inhibitor of Human Telomerase Derived from
10H-Indolo[3,2-b]quinoline
Vittorio Caprio,a Berengere Guyen,b Yaw Opoku-Boahen,a John Mann,a,b
Sharon M. Gowan,c Lloyd M. Kelland,c Martin A. Readd and Stephen Neidled,*
aChemistry Department, Reading University, Whiteknights, Reading RG6 6AD, UK
bSchool of Chemistry, Queen's University Belfast, Belfast BT9 5AG, UK
cCRC Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK
dCRC Biomolecular Structure Unit, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK
Received 19 April 2000; revised 23 June 2000; accepted 27 June 2000
AbstractÐThe bis-dimethylaminoethyl derivative of quindoline (10H-indolo[3,2-b]quinoline), an alkaloid from the West African
shrub Cryptolepis sanguinolenta, has been synthesised. This has been shown to have modest cytotoxicity, as well as inhibitory
activity against the telomerase enzyme. It is hypothesised that the latter activity is due to stabilisation of an intermediate guanine-
quadruplex complex, in accordance with computer modelling. # 2000 Elsevier Science Ltd. All rights reserved.
The specialised DNA sequences at the ends of chromo-
somes comprise tandem repeats of simple oligonucleotide
sequences. The telomeric repeat in vertebrates, including
humans, is 50-TTAGGG.1,2 Telomeres have a number
of functions including protection of the ends of
chromosomes from degradation and recombination,
and an involvement in control of senescence, replication
and the cell cycle clock.3 In normal cells, successive
rounds of cell division are accompanied by telomere
shortening, by around 50±200 nucleotides per division,
due to the inability of DNA polymerase to fully repli-
cate the ends. In cancer cells, although the telomeres are
typically shorter than those of normal (or of germ-line)
cells, their length is maintained by addition of telomere
repeat sequences, a process catalysed by the enzyme
telomerase.4 This enzyme is also directly implicated in
the immortalisation of cancer cells, and it is signi®cant
that around 85±90% of human tumours possess telo-
merase activity while somatic cells invariably lack the
enzyme5 (although stem cells, which tend to have long
telomeres, do have signi®cant telomerase activity).
in tumour cells over a number of generations, leading to
selective inhibition of tumour cell growth. This has now
been demonstrated using (i) PNA antisense molecules
targeted against the RNA template of telomerase,7 and
(ii) with telomerase dominant negative mutants.8
We have developed an approach to the rational design
of telomerase inhibitors which has involved the folding
of the single-stranded telomere primer into four-stranded
quadruplex structures;9 these cannot be substrates for
the enzyme. Folding has been achieved with a range of
small molecules, sharing the structural features of a
planar electron-de®cient chromophore and basic side
chain(s), including derivatives of amido-substituted tri-
cyclic anthraquinones, ¯uorenones and acridines.10
Molecular modelling11 and other biophysical studies12
have shown that these molecules bind to G-quad-
ruplexes, and that strength of binding correlates with
extent of telomerase inhibition.
The original discovery that forms the basis for the work
described here, arises from studies on the West African
shrub Cryptolepis sanguinolenta. This has a long asso-
ciation with Ghanaian folk medicine, and is prized for
its use in the treatment of fevers, including malaria, and
for the control of infections of the urinary and upper
respiratory tracts.13 During the past few years, the
major alkaloidal constituent, cryptolepine 1 (Fig. 1) has
been the object of numerous biological studies, and the
medicinal value of the plant has been fully substantiated.
The possibility of designing drugs with inhibitory activity
against telomerase is emerging as an attractive strategy for
cancer chemotherapy.6 It is proposed that telomerase
inhibition would result in progressive telomere shortening
*Corresponding author. Tel.: +44-020-7970-6043; fax: +44-020-7352-
8039; e-mail: s.neidle@icr.ac.uk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00378-4

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V. Caprio et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2063±2066
Table 1. Cytotoxicity, telomerase inhibition and molecular modelling
data for three tetracyclic compounds compared to the analogous 2,6-
dimethylamino anthraquinone.10 Etotal is the sum of van der Waals
and electrostatic interaction energy between ligand and quadruplex, in
1
kcal mol
A2780 A2780R CH1 CH1R SKOV-3 telIC50 Etotal
Quindoline 2 21.5
24.5 15.5
30
12.5
66
6.3
3.6
2.9
n.d. n.d.
Comp. 14
Comp. 1521
2,6-AQ
12.5
0.15
2.6
10.5
n.d.
n.d.
8.4
0.18 n.d.
1.8 n.d.
16
7.0
4.1
145
168
181
analogues. Our route to analogues of quindoline bearing
one or more such side-chains is shown in the scheme.
The synthesis of 2-bromo-10H-indolo[3,2-b]quinoline-
11-carboxylic acid 9 was accomplished by the same
general route used for the synthesis of quindoline-10-
carboxylic acid, that is by treatment of 5-bromoisatin 10
and O,N-diacetylindoxyl 4 with aqueous KOH solution.
As before, we had great diculty separating the side-
products (mainly indigo) from the product, and the
yield of 9 was rarely above 50% on the 60 mmol scale.
Decarboxylation to provide 2-bromo-10H-indolo[3,2-
b]quinoline 11 could be e€ected (as before) by heating in
liquid paran at 250±300 ꢀC with yields in excess of
80% on the 10 gram scale. Reaction of this bromo-
quindoline with n-butyl lithium in THF, followed by
addition of excess ethylene oxide produced the antici-
pated bis-hydroxyethyl derivative 12 (61%), which was
converted into the bis-tosylate 13 (tosylCl/pyridine;
41%), and thence into the bis-dimethylaminoethyl deri-
vative 14 (Me2NH/DCM; 64%). Preliminary biological
evaluation shows that this compound has about twice
the cell growth inhibitory activity of quindoline.
Figure 1.
Cryptolepine is reported to have useful biological activity
as an antimicrobial agent,14 an anti-im¯ammatory
agent,15 as a vasodilator,16 and most recently, as an
antihyperglycaemic agent.17 Its value as an antimalarial
agent appears so promising18 that several African
countries have proceeded with large-scale therapeutic
programmes. However, the potential of cryptolepine or
others constituents of the plant as anti-tumour agents,
has not, to our knowledge, been investigated. Our syn-
thetic, molecular modelling and biological studies with a
derivative of the parent indoloquinoline compound,
quindoline (2) are reported here.
Compound 14 was also evaluated in a modi®ed TRAP
assay11,12 for its ability to inhibit the extension of telo-
meres by human telomerase, using partially-puri®ed
enzyme extracted from human ovarian carcinoma
A2780 cells. Experiments at a range of concentrations
gave a value of 16 mM for telIC50, the concentration
required to produce 50% inhibition of telomerase. We
have previously studied another tetracyclic system21
based on benzo[b]naphtho[2,3-d]furan (15), with a single
-O-(CH2)2-NR2 substituent (where R=Et or Me). The
telIC50 value for the Et compound (as methiodide deri-
vative), is given in Table 1.
Quindoline has been synthesised by a number of groups,
and we initially used the method of Petrow et al.19 which
involves the reaction between isatin 3 and O,N-diacetyl-
indoxyl 4, in the presence of aqueous KOH, to produce
quindoline-10-carboxylic acid 5. This reaction is not
without problems, because any unreacted indoxyl must
be oxidised (in a stream of air) to produce indigo, which
must then be removed before isolation of the acid. The
isolation procedures are thus plagued by the presence of
highly coloured material. The decarboxylation of 5 was
then achieved by heating the acid in liquid paran at
around 280 ꢀC, and in this way quindoline could be
produced on the multigram scale in a maximum overall
yield of around 50%.
Molecular modelling was used to examine possible
interactions of compound 14 with a guanine quad-
ruplex. Coordinates from the solution NMR structure
of the human telomeric repeat d[AG3(T2AG3)3] G-
quadruplex22 were used to give an initial low energy
starting model. This was optimised by molecular
mechanics energy minimization followed by molecular
dynamics and a ®nal set of molecular mechanics mini-
misations.11 An intercalation site was introduced
The cytotoxicity of quindoline was evaluated20 using
®ve human ovarian cancer cell lines: three parent lines
SKOV-3, A2780 and CH1, and two drug resistant ones.
IC50 values are given in Table 1, which show that quin-
doline has only moderate activity, although it has com-
parable activity against the resistant cell lines. These
results have prompted the synthesis of a range of

V. Caprio et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2063±2066
2065
between the diagonal T2A loop and the G-quartet seg-
ment of the structure (the 50-AG step) by breaking the
phosphate backbones and separating the structure
whilst monitoring the distance between the segments.
The sugar-phosphate chains were reconnected and
molecular mechanics energy minimization was used to
relieve any resulting steric distortion while retaining the
G-quartet and loop motifs with positional restraints.
quadruplex stabilisation. Structure±activity studies on
tricyclic inhibitors has previously demonstrated the
importance of two protonated side-chains; the relatively
poor activity of 14 compared to what might have been
expected for a tetracyclic chromophore (having superior
stacking interactions with the guanine quartet) is due to
its side-chains being of insucient length to avoid some
steric clashes with the bases (Fig. 2). It is notable that
both quindoline and 14 have relatively low acute cyto-
toxicities compared to the other telomerase inhibitors in
the Table, and thus greater selectivity. We have recently
developed a more convergent route that is not only
more ecient but should also allow access to a wide
range of second-generation analogues with varying side-
chains and thus potentially superior selectivity against
telomerase.
A model for compound 14 was created, minimised and
docked into the intercalation site using the DOCKING
module within the INSIGHTII package. This enables
molecular orientation to be explored whilst monitoring
electrostatic and van der Waals interactions between
ligand and quadruplex. Both ligand geometry and that
of the intercalation site were allowed to vary during the
search. Possible starting orientations were chosen by
means of a Monte Carlo algorithm. Individual bases were
constrained at this initial docking stage, although the
backbone around the intercalation site was unconstrained.
The best chromophore positions were subjected to 100
steps of unconstrained molecular mechanics minimisation,
to give the ®nal structure shown in Figure 2, with its
total interaction energy given in Table 1.
Acknowledgements
The authors are grateful to the Cancer Research Cam-
paign for support. We thank the Government of Ghana
(Y. O.-B.) and the Institute of Cancer Research (M. A. R.)
for research studentships.
The quindoline derivative 14 has signi®cant activity
against human telomerase, at a level comparable to
several nucleoside inhibitors of the reverse transcriptase
activity of telomerase.6 It is approximately 10-fold less
active than the best quadruplex-mediated inhibitors
based on tricyclic chromophores10 (which all have two
aminoalkyl substituents), and is 2-fold less active than
the benzo[b]naphtho[2,3-d]furan derivative 15. The
computed energies of interaction with the guanine-
quadruplex are in accord with this ranking order. We
take this as suggestive evidence that the mechanism of
telomerase inhibition by the quindoline 14 does involve
References
1. Blackburn, E. H. Nature 1991, 350, 569.
2. Blackburn, E. H., Greider, C. W., Eds., Telomeres; Cold
Spring Harbor Press: New York, 1995.
3. Counter, C. M.; Avilon, A. A.; LeFeuvre, C. E.; Stewart,
N. G.; Greider, C. W.; Harley, C. G.; Bacchetti, S. EMBO J.
1992, 11, 1921.
4. Bryan, T. M.; Cech, T. R. Curr. Opinion Cell Biol. 1999, 11,
318. O'Reilly, M.; Teichmann, S.; Rhodes, D. Curr. Opinion
Struct. Biol. 1999, 9, 56.
5. Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C.
B.; West, M. D.; Ho, P. L. C.; Coviello, G. M.; Wright, W. E.;
Weinrich, R. L.; Shay, J. W. Science 1994, 266, 2011. Counter,
C. M.; Hirte, H. W.; Bacchetti, S.; Harley, C. B. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 2900. Tang, R.; Cheng, A.-J.;
Wang, J.-Y.; Wang, T.-C. V. Cancer Res. 1998, 58, 4052.
Hoos, A.; Hepp, H. H.; Kaul, S.; Ahlert, T.; Bastert, G.;
Wallwiener, D. Int. J. Cancer 1998, 79, 8. Sano, T.; Asai, A.;
Fujimaki, T.; Kirino, T. Brit. J. Cancer 1998, 77, 1633.
6. Raymond, E.; Sun, D.; Chen, S. F.; Windle, B.; von Ho€,
D. D. Curr. Opin. Biotech. 1996, 7, 583. Pitts, A. E.; Corey, D.
R. Drug Discovery Today 1999, 4, 155. Neidle, S.; Kelland, L.
R. Anti-Cancer Drug Design 1999, 14, 341. Newbold, R. F.
Anti-Cancer Drug Design 1999, 14, 349.
7. Shammas, M. A.; Simmons, C. G.; Corey, D. R.; Reis, R.
J. S. Oncogene 1999, 18, 6191. Herbert, B. S.; Pitts, A. E.;
Baker, S. I.; Hamilton, S. E.; Wright, W. E.; Shay, J. W.;
Corey, D. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14276.
8. Zhang, X.; Mar, V.; Zhou, W.; Harrington, L.; Robinson,
M. O. Genes Development 1999, 13, 2388. Hahn, W. C.; Stew-
art, S. A.; Brooks, M. W.; York, S. G.; Eaton, E.; Kurachi,
A.; Beijersbergen, R. L.; Knoll, J. H. M.; Meyerson, M.;
Weinberg, R. A. Nature Medicine 1999, 10, 1164.
9. Mergny, J.-L.; Helene, C. Nature Medicine 1998, 4, 1366.
Mergny, J.-L.; Mailliet, P.; Riou, J.-F.; Laoui, A.; Helene, C.
Anti-Cancer Drug Design 1999, 14, 327.
10. Sun, D.; Thompson, B.; Cathers, B. E.; Salazar, M.; Ker-
win, S. M.; Trent, J. O.; Jenkins, T. C.; Neidle, S.; Hurley, L.
H. J. Med. Chem. 1997, 40, 2113. Perry, P. J.; Reszka, A. P.;
Figure 2. Plot of compound 14 bound in the global minimum-energy
position in the human G-quadruplex structure, viewed onto the plane
of the chromophore.

2066
V. Caprio et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2063±2066
Wood, A. A.; Read, M. A.; Gowan, S. M.; Dosanjh, H. S.;
Trent, J. O.; Jenkins, T. C.; Kelland, L. R.; Neidle, S. J. Med.
Chem. 1998, 24, 4873. Perry, P. J.; Gowan, S. M.; Reszka, A.
P.; Polucci, P.; Jenkins, T. C.; Kelland, L. R.; Neidle, S. J.
Med. Chem. 1998, 41, 3253. Perry, P. J.; Read, M.; Gowan, S.
M.; Reszka, A. P.; Wood, A. A.; Kelland, L. R.; Neidle, S. J.
Med. Chem. 1999, 42, 2679. Harrison, R. J.; Gowan, S. M.;
Kelland, L. R.; Neidle, S. Bioorg. Med. Chem. Lett. 1999, 9,
2463.
11. Read, M. A.; Wood, A. A.; Harrison, J. R.; Gowan, S.
M.; Kelland, L. R.; Dosanjh, H. S.; Neidle, S. J. Med. Chem.
1999, 42, 4538.
12. Federo€, O. V.; Salazar, M.; Han, H.; Chemeris, V. V.;
Kerwin, S. M.; Hurley, L. H. Biochemistry 1998, 37, 12367.
Han, H.; Cli€, C. L.; Hurley, L. H. Biochemistry 1999, 38,
6891.
14. Cimanga, K.; Pieters, L.; Claeys, M.; Vanden Berghe, D.;
Vlietinck, A. J. Planta Med. 1991, 57(suppl. 2), A98. Boakye-
Yiadom, K.; Hemen-Ackah, S. M. J. Pharm. Sci. 1979, 68,
1510. Paulo, A.; Duarte, A.; Gomes, E. T. Ethnopharmacol.
1994, 44, 127.
15. Bamgbose, S. O. A.; Noamesi, B. K. Planta Med. 1981,
41, 392.
16. Oyekan, A. O. J. Cardiovasc. Pharmacol. 1994, 23, 602.
17. Bierer, D. E. J. Med. Chem. 1998, 41, 894.
18. Kirby, G. C.; Paine, A.; Warhurst, D. C.; Noamese, B. K.;
Phillipson, J. D. Phytother. Res. 1995, 9, 359.
19. Holt S. J.; Petrow, V. Proc. Roy. Soc. B 1946, 148, 607.
Cooper, M. J.; Lovell, J. M.; Joule, J. A. Tetrahedron Lett.
1996, 37, 4283.
20. Kelland, L. R.; Abel, G.; McKeage, M. J.; Jones, M.;
Goddard, P. M.; Valenti, M.; Murrer, B. A.; Harrap, K. R.
Cancer Res. 1993, 53, 2581.
13. Tackie, A. N.; Bove, G.; Sharaf, M. H. M.; Schi€, P. L.;
Crouch, R. C.; Spitzer, T.; Johnson, D. R. L.; Dunn, J.;
Minick, D.; Martin, G. E. J. Nat. Prod. 1993, 56, 553.
21. Perry, P. J.; Gowan, S. M.; Read, M. A.; Kelland, L. R.;
Neidle, S. Anti-Cancer Drug Design 1999, 14, 373.