Design, synthesis and evaluation of 4,5-di-substituted acridone ligands with high G-quadruplex affinity and selectivity, together with low toxicity to normal cells

Article abstract of DOI:10.1016/j.bmcl.2009.07.033

A series of 4,5-di-substituted acridones have been designed and synthesized. Several compounds show high affinity for telomeric G-quadruplex DNA in classical and competition FRET assays, together with low duplex DNA affinity, although they do not show activity in a telomerase assay or evidence of telomere shortening. They have low toxicity against a panel of cancer cell lines and a normal human fibroblast line, and produce potent senescence-based long-term growth arrest in the MCF7 and A549 cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2009.07.033

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5109–5113
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of 4,5-di-substituted acridone ligands
with high G-quadruplex affinity and selectivity, together with low toxicity
to normal cells
Francisco Cuenca a, Michael J. B. Moore a, Karin Johnson a, Bérangère Guyen a, Anne De Cian b,
Stephen Neidle a,
*
a CRUK Biomolecular Structure Group, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK
b Laboratoire de Régulation et Dynamique des Génomes, USM503 INSERM U565, CNRS UMR5153, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4,5-di-substituted acridones have been designed and synthesized. Several compounds show
high affinity for telomeric G-quadruplex DNA in classical and competition FRET assays, together with
low duplex DNA affinity, although they do not show activity in a telomerase assay or evidence of telo-
mere shortening. They have low toxicity against a panel of cancer cell lines and a normal human fibro-
blast line, and produce potent senescence-based long-term growth arrest in the MCF7 and A549
cancer cell lines.
Received 12 June 2009
Revised 23 June 2009
Accepted 2 July 2009
Available online 10 July 2009
Keywords:
Quadruplex ligands
Acridones
Ó 2009 Elsevier Ltd. All rights reserved.
DNA binding
Telomeres are highly specialized DNA–protein structures at the
end of eukaryotic chromosomes.1 Telomeric DNA erosion in each
replication cycle due to the end-replication problem2 occurs in
normal somatic cells, so that eventually telomeric DNA reaches a
critically short length, the Hayflick limit, and cells enter the repli-
cative senescence state.3,4 The senescent phenotype must be over-
come for cells to reach the immortalized state that characterizes
cancer,5 in most cases by activation of the reverse transcriptase en-
zyme telomerase,6 which is expressed in 85–90% of cancer cells yet
is not significantly expressed in somatic tissue.7 Telomerase main-
tains telomeric DNA length and its inhibition induces cell senes-
cence and is thus a target for therapeutic intervention.8,9
One approach to selectively inhibit telomere maintenance in
cancer cells is to target the telomerase substrate, the short se-
quence of single-stranded DNA at the extreme 30 end of telomeric
DNA. A wide range of small-molecule ligands have been found to
stabilize the G-quadruplex (G4) DNA conformation that the sin-
gle-stranded 30 end of telomeric DNA can adopt. G4 formation
can displace bound hPOT1 protein at the single-stranded end,
which is involved in the regulation of telomerase activity,10 and
may dissociate telomerase from its telomere capping function.11
The majority of G4-binding ligands12 have a planar aromatic
chromophore that complements the G-quartet surface of a human
telomeric DNA quadruplex structure.13 Crystal structures are now
available for human telomeric G4 DNAs and their ligand complexes
with a tri-substituted acridine14 and a tetra-substituted naphtha-
lenediimide compound,15 and can be used in structure-based drug
design studies.
Several series of 2,6, 2,7 and 3,6-di-substituted acridone com-
pounds have been reported previously as G4 ligands and show pat-
terns of activity that are similar to their di-substituted acridine
counterparts,16 with short-term toxicities in the low
lM range
but with only low levels of discrimination between cancer and nor-
mal cell lines. In this work we explore further the potential of acri-
done-based compounds as G4 ligands and selective telomere
targeting agents through the synthesis and biological evaluation
of a novel series of 4,5-di-substituted acridone derivatives. We
postulated that the incorporation of aromatic side chains to the
acridone core in the 4 and 5 positions, conjugated by the use of
amide bonds, would confer enhanced planarity on the molecule,
hence improving p–p stacking interactions with a G-quartet. Com-
pounds were designed with these considerations in mind (Fig. 1),
and also contain several features that were anticipated to contrib-
ute to the hypothesized improvements in G4 affinity, namely: (i)
the NH groups attached to the acridone would play a role in substi-
tuent planarity by participating in a pair of bifurcated hydrogen
bonds to the carbonyl groups of the 4- and 5-substituted amides,
which themselves would be conjugated with the acridone moiety,
and (ii) alkyl chains bearing a tertiary amine, a common feature of
G4 ligands.17,18
* Corresponding author.
E-mail address: stephen.neidle@pharmacy.ac.uk (S. Neidle).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.033

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F. Cuenca et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5109–5113
had been made when designing the library (Table 1). The benzene
rings in the side chains had been postulated as important for G4
interaction. This is supported by the low increase in melting tem-
perature (
Tm(1 M) = 7°C). Compound 25, which has N-methyl pyrrole
substituting for benzene, shows only limited stabilization
Tm(1 M) = 15 °C) possibly due to a less favourable out-of plane
DTm) for the compound with nonaromatic side chains 26
(D
l
(D
l
conformation as a result of the methyl groups. Compounds 9, 11,
12, 13, 17, 19, 20 and 24 all showed similar high levels of stabiliza-
tion ability for the telomeric G4 sequence with
between 28 and 34 °C. These are comparable to the 32 °C
Tm(1 M)) for the tri-substituted acridine compound BR-19.
All these acridones have side chains terminating in a basic ter-
tiary amine, which is protonated at pH 7. Comparison with the less
basic morpholine analogues 10 and 18 ( Tm(1 M) of 19 and 20 °C,
respectively) and especially with the neutral amide analogue 16
Tm(1 M) = 0 °C) highlights the importance of the positively
charged groups. Compound 14 with a secondary cyclohexyl amine
DTm(1 lM) values
(D
l
D
l
(D
l
(
D
Tm(1
Tm(1
l
l
M) = 24.5 °C) and 15 with an imidazole group
M) = 15 °C) were less effective than those with aliphatic
Figure 1. Qualitative molecular model of compound 9 stacked onto a terminal G-
(D
quartet at the 30 face of the human intramolecular G4 structure.13–15
tertiary amines. Interestingly, the length of the side chains is crit-
ical for their potency. Compounds 9, 11, 12, 13, 17, 19, 20 and 24 all
have the same number of side-chain atoms between the acridone
and the tertiary amines. Comparing these values with the obtained
Compounds 9–27 were chosen as synthetic targets, (Table 1), in
order to examine in particular diversity in side-chain substituents
and side-chain length, as well as substitution pattern in the ben-
zene ring and presence or absence of the carbonyl group. The syn-
thesis of the acridone core and the side chains was conducted
independently and converged in the last and key step, amide bond
formation between the 4,5-dicarboxyl acridone 1 (or the 4-car-
boxyl acridone 2 for the mono-substituted compound 27) and
the side chains (Scheme 1).
for the shorter side-chain analogue 21 (
those with longer side chains 22 ( Tm(1
Tm(1 M) = 22 °C) shows that the optimum length is n = 2 (Table
1), in accord with indications from preliminary molecular model-
ling. The similarity of the Tm(1 M) values between compounds
D
Tm(1
lM) = 25 °C) and
D
l
M) = 15 °C) and 23
(D
l
D
l
9 and 17, 10 and 18, 11 and 19 and 12 and 20 shows that the pres-
ence or absence of the carbonyl group in the side chains does not
affect G4 stabilization. The amide bond is a potential site for
metabolism so this finding has relevance for further development
of these compounds. Mono-substitution of the acridone core (com-
pound 27) results in reduced G4 stabilization (DTm(1 lM) = 11 °C).
Duplex-binding experiments show that the compounds as a
class have high selectivity with those having the largest G4
Compound 1 was synthesized using the reported method.19
Although the overall yield was low (16% for 3 steps), in our hands
this method proved to be more time efficient than an alternative
literature method.20 Compound 2 was synthesized by modification
of the reported method19 using the Jourdan–Ullmann reaction with
2-bromobenzoic acid and anthranilic acid followed by ring closure
with concentrated H2SO4.
D
Tm(1
3 °C. At 10
DNA stabilization with the exception of the meta-substituted com-
pound 24 ( Tm(10 M) = 30 °C). The shape of this compound may
l
M) values having duplex DNA
DTm(1 lM) values of 1.5–
lM ligand concentration they showed low duplex
The general synthesis pathway for the anilinic side chains is
shown in Scheme 2.21
D
l
The synthesis of the side chain leading to compound 16 in-
volved reaction of 4-nitroaniline with succinic anhydride, followed
by amide formation with pyrrolidine and reduction of the nitro
group using ammonium formate (Scheme 3). Synthesis of the side
chain leading to compound 25 involved synthesis of compound 622
followed by amide formation and reduction of the nitro group
using ammonium formate (Scheme 3).
For the coupling reactions an approach using PyBOP was found
to be the most efficient for this particular reaction. Several solvent
systems (DMF/DMA/DCM/MeCN mixtures) were trialed and a 3:1
DMF:MeCN mixture gave the best results. Up to 4 equiv of the cor-
responding amines were used to increase conversion, as initial
reactions using fewer equivalents gave unwanted mono-coupled
byproducts.
be more adaptable to one of the grooves of duplex DNA. Competi-
tion experiments with calf thymus DNA (Fig. 2 and Supplementary
data) show that G4
DTm values are little changed in the presence of
>400 excess of duplex DNA, confirming that this family of 4,5-acri-
dones have unusually high selectivity for G4 DNAs. Their selectiv-
ity for individual G4s, for example, those found in promoter
regions, remains to be established. The reference compound BRAC-
O-19 (BR-19) showed significant duplex DNA affinity in the FRET
assay (DTm(1 lM) = 6 °C), in contrast to the behavior of the 4,5-
acridones (Fig. 2).
The short-term (72 h) toxicity of the 4,5-acridones was evalu-
ated using the SRB assay.25 None of the 4,5-acridones examined
(Table 1) show significant toxicity in the somatic fibroblast cell line
IMR90, apart from the abnormal behavior shown by compound 27
Compounds 9–27 were successfully synthesized with this
methodology.23 Isolation involved precipitation of the product
from the reaction mixture by EtOAc addition, and purified by re-
peated precipitation of the product from a DMF solution with
EtOAc and preparative HPLC when required (when the purity
was lower than 95%). Typically the purity obtained after reprecip-
itation was >90% and the yield was >50%.
G4 DNA binding of compounds 9–27 and a reference compound
(BRACO-19: BR-19) were assessed with a FRET (Fluorescence Res-
onance Electron Transfer) assay using a human telomeric G4 DNA
sequence.24 The results confirm and extend the assumptions that
which is active in all three cell lines and also has low DTm activity.
This suggests the possibility of a therapeutic window for cancer
versus somatic cells. Several compounds are selectively toxic to
the two cancer cell lines examined, A549 (lung) and MCF7 (breast).
The latter was overall the most sensitive cell line with compounds
9, 15, 16, 22, 23, 25 and 27 having IC50 values lower than 25
The A549 line was overall less sensitive although compounds 9,
11, 15, 22, 25 and 27 have IC50 values <25 M.
Overall there is not a high correlation between G4 affinity and
short-term toxicity. Two compounds with the highest Tm values
in the G4 FRET assay, 9 and 11 (34 and 32 °C, respectively) were
lM.
l
D

Table 1
The 4,5-acridones synthesized together with details of their G4 and duplex DNA melting behaviour and cell growth inhibition in two cancer cell lines (MCF7 and A549) and a human fibroblast cell line (IMR90)
O
N
H
HN
HN
O
O
O
NH
A
B
HN
O
C
D
n
R
n
R
Compound
A group at 1st sector
B Presence of carbonyl group
C linker length (n)
D end group (R)
G4
D
Tm (°C)
Duplex
D
Tm (°C)
MCF7 IC50
(l
M)
A549 IC50
(
lM)
IMR90 IC50 (lM)
9
p-Pha
p-Ph
p-Ph
p-Ph
p-Ph
p-Ph
p-Ph
p-Ph
p-Ph
p-Ph
p-Ph
p-Ph
p-Ph
p-Ph
p-Ph
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
2
2
2
2
2
2
2
2
3
3
3
3
1
3
4
2
Pyrrolidine
Morpholine
Dimethylamine
N-methylpiperazine
Piperidine
Cyclohexaneamine
Imidazole
C@O-pyrrolidine
Pyrrolidine
Morpholine
Dimethylamine
N-Methylpiperazine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
Pyrrolidine
n.a.
Pyrrolidine
n.a.
34
19
32
31
31
24.5
15
0
33
20
31
28
25.5
15
22.5
29
15
7
2.5
0
2.5
1.5
1.5
0
0.5
0
2.5
0
2.5
1.5
0.5
0
0.75
3
23
>100
55
99
62
28
17
21
60
>100
53
68
72
15
2
16
>100
15
>100
>100
31
19
27
66
>100
>100
>100
>100
8
43
>100
21
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
7.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27e
BR-19
No
No
No
Yes
Yes
Yes
Yes
n.a.
n.a.
Yes
n.a.
m-Phb
N-MePyc
N-DMPDAd
p-Ph
84
19
40
9
3
n.a.
2
0
1
0.5
6
62
16
2.4
11
32
n.a.
n.a.
2.5
26
The figure above shows the general structure of the final compounds. The table includes thermal stabilization (
D
Tm) data for each compound, with a human telomeric G4 and a duplex DNA, both determined by FRET methods.24 The
M for 96 hr exposures with the MCF7 and A549 cancer cell lines, and the human fibroblast normal cell line IMR90. Esds for Tm are 0.5°, and for IC50 are M.
three columns on the right give short-term (IC50) data in
n.a. = not applicable.
l
D
2 l
a
para-Phenyl.
meta-Phenyl.
4-Amino-1-methyl-2-carboxamide-1H-pyrrole.
N,N-Dimethylpropane-1,3-diamine.
b
c
d
e
Mono-substituted analogue.

5112
F. Cuenca et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5109–5113
O
O
N
no competitor
ds26 3µM (1:156 ratio)
ds26 10µM (1:433 ratio)
N
H
120
100
80
60
40
20
0
H
O
HN
O
O
O
O
NH
O
HN
O
O
a
a
N
H
N
H
HN
N
HN
N
NH
N
HO
O
O
OH
HO
O
1
2
9
27
Scheme 1. Synthetic route for final compounds 9 and 27. Reagents and conditions:
(a) side chain N-(4-aminophenyl)-3-(pyrrolidin-1-yl)propanamide, PyBOP, DMF/
acetonitrile, rt, 24 h.
O2N
O2N
O2N
b
O
a
N
Cl
Cl
NH2
N
n
H
H
Compounds (1µM)
c
c
Figure 2. FRET data25 on BRACO-19 and compounds 9, 11 in competition with
varying ratios of duplex DNA in excess, showing the high G4 selectivity of the
acridones.
X
X
O
R
N
R
N
H
n
H
X=NO2
X=NH2
X=NO2
X=NH2
d
d
16
VC
Scheme 2. General synthetic route for side chains. Please refer to Supplementary
data for specific structures. Reagents and conditions: (a) chloroacyl chloride, ; (b)
Me2S.BH3, THF; (c) amine R, neat/THF; (d) ammonium formate, Pd/C, ethanol,
14
12
10
8
10 µM
25 µM
35 µM
50 µM
D
D
.
O2N
X
O2N
a
O
O
b
OH
N
N
N
NH2
O2N
H
H
O
O
4
5
6
X=NO2
X=NH2
c
3
4
X
2
H
N
OH
d
N
N
N
0
O
O
0
7
14
21
X=NO2
X=NH2
7
8
6
c
Time (days)
Figure 3. Effects of long-term exposure to compound 24 on proliferation in the
MCF7 cell line. 24 was added initially to cells, then three times weekly (on days 2, 4
and 6). At each re-treatment the medium was discarded and the cells washed with
PBS. Fresh media and compound solution were then added. VC represents the
control experiment in the absence of ligand.
Scheme 3. Synthetic route for side chains 5 and 8. Reagents and conditions: (a)
succinic anhydride, toluene, reflux, 1 h; (b) pyrrolidine, HOBt, EDCI, DMF, rt, 16 h;
(c) ammonium formate, Pd/C, ethanol, 120 °C, 4 min; (d) 3-(pyrrolidin-1-yl)propan-
1-amine, HOBt, EDCI, acetonitrile, rt, 16 h.
amongst the most active in the SRB assay with the two cancer cell
lines used here. The morpholine compounds 10 and 18 had more
notype.27 To gain an insight into the mechanism of action of one of
these ligands (compound 24), the number of senescent cells at dif-
ferent stages during the long-term experiments in the MCF7 and
modest
DTm capabilities and showed no cellular toxicity
<100 M, although uptake problems cannot be discounted. Several
l
A549 cell lines was quantified.28 The former after 25
lM of com-
compounds showed high cancer cell toxicity while being poor G4
ligands and others stabilized the G4 strongly but showed high
IC50 values.
Anti-proliferative effects were further examined by long-term
incubation of selected compounds in the cancer cell lines MCF7
pound 24 treatment had 25% of cells undergoing senescence after
three weeks; lower concentrations caused no senescence. Com-
pound 24 also caused senescence in A549 cells in a concentra-
tion-dependent manner (Fig. 4). However it is not clear from this
data alone whether these relatively low levels of senescence are
sufficient to explain the inhibition of cell proliferation observed
in the long-term studies.
and A459 at sub-toxic doses. For compound 24 (IC50 = 84
lM)
and the MCF7 cell line, a concentration of 10 M showed an effect
l
on cell proliferation (Fig. 3); after five weeks cells receiving this
treatment had gone through 17.5 pd while the control cells under-
went 22.5 pd. Experiments with concentrations >25 lM had to be
discontinued after three weeks or less; cell growth was profoundly
inhibited after seven days. Similar behavior was observed for A549
cell (see Supplementary data).
Inhibition of telomerase or telomere uncapping can result in
telomere attrition. Thus potentially, G4 ligands can cause telomere
shortening by either of these mechanisms and such results have
been reported for several ligands.11 Telomere length measure-
ments were undertaken on DNA extracted from cells following
long-term exposure to compounds 11 and 24 but no telomere
shortening was detected in cells treated for up to six weeks (results
not shown). Measurements of telomerase inhibitory activity using
The targeting of telomeres with G4 ligands can result in cells
with dysfunctional telomeres26 and the onset of the senescent phe-

F. Cuenca et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5109–5113
5113
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Temime-Smaali, N.; Trentesaux, C.; Mergny, J.-L.; Gueritte, F.; Riou, J.-F. Mol.
Pharmacol. 2007, 72, 631.
11. Gunaratnam, M.; Greciano, O.; Martins, C.; Reszka, A. P.; Schultes, C. M.;
Morjani, H.; Riou, J. F.; Neidle, S. Biochem. Pharmacol. 2007, 74, 679.
12. De Cian, A.; Lacroix, L.; Douarre, C.; Temime-Smaali, N.; Trentesaux, C.; Riou, J.;
Mergny, J. Biochimie 2008, 90, 131.
13. Parkinson, G. N.; Lee, M. P.; Neidle, S. Nature 2002, 417, 876.
14. Campbell, N. H.; Parkinson, G. N.; Reszka, A. P.; Neidle, S. J. Am. Chem. Soc. 2008,
130, 6722.
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16. Harrison, R. J.; Reszka, A. P.; Haider, S. M.; Romagnoli, B.; Morrell, J.; Read, M.
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Figure 4. Micrograph of A549 cells after treatment with compound 24 and after
staining for senescence. Red arrows indicate cells with the characteristic blue colour
associated to senescent cells.
19. Rewcastle, G. W.; Denny, W. A. Synthesis 1985, 2, 217.
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both the TRAP-LIG29 and direct telomerase assays30 did not show
any activity for any of the 4,5-acridones.31
21. Acylation of either 3- or 4-nitro aniline used the correspondent chloroacyl
chloride as a solvent under mild heating for 8–16 h. For the substitution
reaction the chloro compounds were treated with the different amines as
solvent, except for dimethylamine when a 2 M solution in THF was used, with
mild heating for 24 h. The nitro compounds were reduced with ammonium
formate and Pd/C in ethanol in a microwave with very short reaction times (2–
3 min) at 120ꢀC to give the targeted anilines. When necessary, an extra step
was performed for carbonyl group reduction with a Me2SꢀBH3 complex in THF,
prior to the reaction with the amines.
Inhibition of cell proliferation in long-term culture experiments,
together with evidence of cellular senescence, is suggestive of a
telomere-targeted mechanism of cancer cell toxicity, which is sup-
ported by the high G4 affinity of some (but not all) of the 4,5-acri-
done compounds. The lack of telomerase inhibition and of
telomere shortening, demonstrates that the catalytic function of
telomerase is not involved, although telomerase uncapping from
the 30 end of telomeres may be a contributor to the observed can-
cer cell selectivity. We cannot also discount other mechanisms of
action, including the involvement of promoter G4s since these
compounds are not specific for telomeric G4s.31 The properties of
these 4,5-acridones are in striking contrast with a recently- re-
ported series of 4,5-di-substituted acridines (with amidoalkylami-
no side chains).32 These show low G4 stabilization ability, with
22. Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6141.
23. Synthesis and analytical data for compound 9. Information on other compounds
is provided in the Supplementary data.
N4,N5-Bis(4-(3-(pyrrolidin-1-yl)propanamido) phenyl)-9,10-dihydro-9-oxoac-
ridine-4,5-dicarboxamide (9): A solution of 1 (20 mg, 0.071 mmol), PyBOP
(3 equiv, 0.213 mmol, 111 mg) and N-(4-aminophenyl)-3-(pyrrolidin-1-
yl)propanamide (4 equiv, 1.818 mmol, 69 mg) in a mixture of anhydrous DMF
(3 ml) and acetonitrile (1 ml) under N2 atmosphere was stirred at rt for 24 h.
EtOAc (30 ml) was added and the yellow solid formed was filtered, washed with
EtOAc (3 ꢁ 5 ml) and ether (3 ꢁ 5 ml). Yield: 42.8 mg, 84.5%. Analytical data:
decomp. at 250 °C; IR
l
(cmꢂ1) 3283, 1644, 1610, 1510, 1433, 1406, 1311, 1238,
1022, 832; 1H NMR (DMSO-d6) d: 1.89 (m, 8H), 2.79 (t, 4H, J = 6.9 Hz), 3.10 (m,
8H), 3.33 (m, 4H), 7.46 (t, 2H, J = 7.7 Hz), 7.61 (d, 4H, J = 9.1 Hz), 7.67 (d, 4H,
J = 9.1 Hz), 8.34 (dd, 2H, J = 7.5, 1.4 Hz), 8.50 (dd, 2H, J = 8.1, 1.3 Hz), 10.29 (s, 2H),
10.62 (s, 2H), 13.26 (s, 1H); 13C NMR (DMSO-d6) d: 23.02 (4 ꢁ CH2), 35.52
(2 ꢁ CH2), 51.28 (2 ꢁ CH2), 53.28 (2 ꢁ CH2), 119.19 (4 ꢁ CH), 120.58 (2 ꢁ C),
120.63 (2 ꢁ C), 121.06 (2 ꢁ C), 121.29 (4 ꢁ CH), 129.78 (2 ꢁ CH), 133.66
(2 ꢁ CH), 134.05 (2 ꢁ CH), 135.47 (2 ꢁ C), 139.25 (2 ꢁ C), 165.56 (2 ꢁ C@O),
169.60 (2 ꢁ C@O), 176.19 (C@O); HRMS (ESI+) calcd C41H44N7O5 [M+H]+
714.3398. Found: 714.3395.
D
Tm(1 lM) values in the range 0–3 °C, and with some compounds
having potent in vitro telomerase inhibitory activity.
Acknowledgements
We thank CRUK and EU FP6 (Molecular Cancer Medicine LSHC-
CT-2004-502943) for support, Jean-Louis Mergny for useful discus-
sions and Joachim Lingner for a kind gift of supertelomerase
extracts.
24. The FRET DNA melting assay was performed as described previously (Schultes,
C. M.; Guyen, B.; Cuesta, J.; Neidle, S. Bioorg. Med. Chem. Lett. 2004, 14, 4347).
The full protocol is given in the Supplementary data. The tagged DNA
sequences used were: 50-FAM-d(GGG[TTAGGG]3)-TAMRA-30 for the G4 and
50-FAM-dTATAGCTATA-HEG-TATAGCTATA-TAMRA-30 (HEG linker: [(–CH2–
CH2–O–)6]) for the duplex experiment.
Supplementary data
25. See Supplementary data for more information.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.033.
26. Leonetti, C.; Amodei, S.; D’Angelo, C.; Rizzo, A.; Benassi, B.; Antonelli, A.; Elli, R.;
Stevens, M.; D’Incalci, M.; Zupi, G.; Biroccio, A. Mol. Pharmacol. 2004, 8, 1063.
27. D’adda di Fagagna, F. D.; Reaper, P. M.; Clay-Farrace, L.; Fiegler, H.; Carr, P.; von
Zglinicki, T.; Saretzki, G.; Carter, N. P.; Jackson, S. P. Nature 2003, 426, 194.
28. 1 ꢁ 105 cells per well were cultivated in 2 ml of medium plus compound in 96-
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