Tetrasubstituted naphthalene diimide ligands with selectivity for telomeric G-quadruplexes and cancer cells

Article abstract of DOI:10.1016/j.bmcl.2010.09.066

A series of tetrasubstituted naphthalene diimide compounds with N-methylpiperazine end groups has been synthesized and evaluated as G-quadruplex ligands. They have high affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA. CD studies show that they induce formation of a parallel G-quadruplex topology. They inhibit the binding of hPOT1 and topoisomerase IIIα to telomeric DNA and inhibit telomerase activity in MCF7 cells. The compounds have potent activity in a panel of cancer cell lines, with typical IC₅₀ values of ～0.1 μM, and up to 100-fold lower toxicity in a normal human fibroblast cell line.

Full text of DOI:10.1016/j.bmcl.2010.09.066

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6459–6463
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tetrasubstituted naphthalene diimide ligands with selectivity for telomeric
G-quadruplexes and cancer cells
Sonja M. Hampel a, Assitan Sidibe b, Mekala Gunaratnam a, Jean-François Riou b, Stephen Neidle a,
⇑
a CRUK Biomolecular Structure Group, The School of Pharmacy, University of London, London WC1N 1AX, UK
b Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, INSERM U655, CNRS UMR 7196, 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 tetrasubstituted naphthalene diimide compounds with N-methylpiperazine end groups has
been synthesized and evaluated as G-quadruplex ligands. They have high affinity and selectivity for telo-
meric G-quadruplex DNA over duplex DNA. CD studies show that they induce formation of a parallel G-
Received 11 August 2010
Revised 10 September 2010
Accepted 13 September 2010
Available online 17 September 2010
quadruplex topology. They inhibit the binding of hPOT1 and topoisomerase III
a to telomeric DNA and
inhibit telomerase activity in MCF7 cells. The compounds have potent activity in a panel of cancer cell
lines, with typical IC50 values of ꢀ0.1
l
M, and up to 100-fold lower toxicity in a normal human fibroblast
Keywords:
cell line.
Quadruplex
Naphthalene diimide
Telomerase
Cancer
Ó 2010 Elsevier Ltd. All rights reserved.
The immortality of cancer cells is at least in part dependent on
the maintenance of telomeres, the nucleoprotein complexes at the
ends of chromosomes.1 Telomeres are shortened in somatic cells,
leading to their limited life span, whereas they are stabilized in
cancer cells. The function of telomerase, which is over-expressed
in >80% of cancer cells, is to both physically protect and maintain
telomere length.2 As telomeric DNA comprises repeated short G-
tracts, it is able to fold into G-quadruplex structures, in which three
planes of hydrogen-bonded G-quartets are held together by
p–p
interactions. Depending on the coordinating ion, the presence of
small-molecule ligands, the DNA sequence and other factors such
as molecular crowding, telomeric DNA G-quadruplexes can have
a diversity of topologies.3
Quadruplex formation can affect a wide range of cellular pro-
cesses. The stabilization of G-quadruplex structures in the single-
stranded 30 telomeric DNA overhang by small-molecule ligands
has been shown to indirectly inhibit telomerase and telomere
maintenance in cancer cells.4 Quadruplex formation along the
overhang can also displace the single-stranded binding protein
hPOT1.5 The function of telomere-associated Holliday junction
Figure 1. Modeled structure of compound 1 (orange) docked on the 30 face of the
parallel human telomeric G-quadruplex 22-mer DNA (side view) using the crystal
structure of a known ND complex as a starting-point.11 Positive and negative charge
regions on the solvent-accessible surface of the DNA quadruplex are marked in blue
and red, respectively.
decatenases such as topoisomerase III
a (Topo IIIa), (essential for
and their stabilization can down-regulate oncogene expression.7
The multiple pathways caused by the stabilization of telomeric
G-quadruplexes can lead to uncapping and end-to-end fusions of
chromosomes, followed by cell cycle arrest, senescence and growth
inhibition, as well as DNA damage response and apoptosis in
cancer cells.5b,c
the maintenance of telomeres in the ALT (alternative lengthening
of telomeres) pathway,6 can be affected by quadruplex-binding
small molecules.21c Promoter regions of oncogenes such as c-myc
and c-kit contain sequences that may fold into G-quadruplexes,
A large number of G-quadruplex binding small molecules have
⇑
Corresponding author. Tel.: +44 207 7535969; fax: +44 207 7535970.
E-mail address: stephen.neidle@pharmacy.ac.uk (S. Neidle).
been reported.8 Tetrasubstituted naphthalene diimides (NDs) are
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.066

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S. M. Hampel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6459–6463
Scheme 1. Synthesis of the side chain amines 7b and 7c. Reagents and conditions:
(i) Na2CO3, toluene, reflux, 16–18 h, 58–85%. (ii) (1) N2H4, EtOH, reflux, 3 h; (2) HCl,
reflux, 30 min, 46–61%. The amine 7a (n = 3) was commercially available.
Figure 2. Competition FRET experiment showing the selectivity of 1–3 for the F21T
G-quadruplex DNA sequence over duplex DNA. Different ratios of excess duplex
DNA were added (concentrations of G-quartets vs nucleotide phosphates of duplex
DNA). The reference compound 9 is a derivative of compound 1 with dimethyl-
amine end groups (see Supplementary data).
Scheme 2. Synthesis of the NDs 1–3. Reagents and conditions: (i) microwave,
150 °C, 20 min–2 h, 11–19%. The starting material 8 was synthesized according to
the established procedure.14
eling (Fig. 1), and demonstrated by X-ray crystallography.11 One
compound from these series, which has high affinity for the G-
quadruplexes found in the promoter region of the c-kit gene, has
been studied for its effects on gastrointestinal stromal tumor cell
lines.12 We report here on a novel subset of NDs with N-methylpi-
perazine end groups, which have enhanced telomeric quadruplex
and cellular selectivity.
Initially, novel ND derivatives with enhanced selectivity for
telomeric G-quadruplexes were conceived by qualitative molecu-
lar modeling using a parallel G-quadruplex from a ND co-crystal
structure.11 Side-chains of 3–5 carbon atoms with bulky methylpi-
perazine end groups gave optimal groove interactions. Overlay of 1
onto the known position of the related ND compound (Fig. 1) indi-
cated that the N-methylpiperazine groups, which are bulky and
can be protonated easily, reach deep into the grooves at the sides
of the G-quadruplex and interact strongly with phosphate
moieties.
Table 1
G-quadruplex stabilization of compounds 1–3 in the FRET melting temperature assay.
Esds in
DTm are 0.1 K.
DNA type
FRET
D
Tm [K], c = 0.5 lM
1
2
3
F21T G4
c-kit1 G4
c-kit2 G4
T-loop
28.3
1.8
15.2
1.3
24.7
4.9
16.7
0.1
23.8
1.5
7.7
0.2
very potent G-quadruplex ligands with high cellular toxicity.9 NDs
have been of broader interest because of their tunable electro- and
photo-chemical properties and their applications in supramolecu-
lar chemistry.10 They have a delocalized electron system, which
is able to effectively stabilize the terminal G-quartets of a G-quad-
ruplex by stacking interactions. Four substituents with amino end
groups can fit into the four grooves at the sides of the G-quadru-
plex and interact electrostatically, as indicated by molecular mod-
The amines 7b and 7c, which were used as side-chains, were
obtained via Gabriel synthesis (Scheme 1).13 For this, N-methylpi-
perazine was added to the bromoalkylphthalimides 5b or 5c in
Figure 3. Circular dichroism (CD) spectra of a human telomeric 23-mer DNA with various ratios of compound 1, at pH 7.4. (A) CD spectrum, 100 mM K+. (B) CD spectrum,
100 mM Na+. Compounds 2 and 3 gave similar results (see Supplementary data).

S. M. Hampel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6459–6463
6461
Figure 4. (A, D, E) Inhibition of POT1 (30 nM) and Topo IIIa (75 nM) binding to the telomeric G-quadruplex DNA sequence WT26 (20 nM) by 1 and 3 in an in vitro EMSA
assay.21 (B) The duplex DNA binding molecule does not inhibit the binding of Topo III
a to WT26. (C) Compounds 1 and 3 do not inhibit the binding of Topo IIIa to duplex DNA.
toluene to give the imides 6b and 6c, respectively. Those were
cleaved with hydrazine in ethanol to give the amines 7b and 7c.
The propylamine analog 7a was commercially available. The
tetrasubstituted NDs 1–3 were synthesized from the dibromo-
dianhydride 8 with the respective aminoalkyl-methylpiperazine
7 as the solvent in the microwave (Scheme 2) as variants of the
established method.14 The tetrasubstituted species was isolated
from the side products, containing a trisubstituted species, by
HPLC with yields of 11–19%. In contrast to previously reported
NDs, compounds 1–3 are highly water-soluble, which simplified
their evaluation.
DNA from calf thymus were added to the classic FRET experiment
with the telomeric sequence F21T (Fig. 2). This showed that a 10:1
duplex:quadruplex ratio does not interfere with the stabilization of
the G-quadruplex by 1–3, and even at a 300-fold excess of duplex
DNA, G-quadruplex stabilization is reduced by ca. 50%. The meth-
ylpiperazine compounds have higher selectivity for G-quadruplex
DNA over duplex DNA than earlier NDs9 such as the reference com-
pound 9, which contains dimethylamine end groups.
The effect of the ligands on the topology of a telomeric G-quad-
ruplex 23-mer DNA was investigated by circular dichroism (CD).17
The ability of compounds 1–3 to stabilize G-quadruplex DNAs
was evaluated in a FRET (Fluorescence Resonance Energy Transfer)
melting temperature assay.15 Melting temperature shifts in Table 1
Table 2
Short term cytotoxicity of compounds 1–3 in a panel of cancer cell lines (WI38 is a
normal fibroblast line), determined by 96 h SRB assays.
were compared at a drug concentration of 0.5
melting curves were already saturated at the standard comparison
concentration of 1 M. All three compounds show very high affin-
lM, as some of the
Cell line
SRB IC50 (nM)
2
l
1
3
ity for the human telomeric G-quadruplex sequence F21T, leading
to increases in melting temperature of 24–28 K. Moderate affinity
for the c-kit2 sequence was observed, whereas there was only
low affinity for the c-kit1 sequence and the duplex DNA T-loop
sequence.
MCF7
A549
167 30
108 17
111 24
148 45
186 17
100 20
69 28
73 11
106 33
117 28
1020 120
5500 1234
196
8
258 42
121 18
193 46
205 30
759 134
MIA-Pa-Ca-2
PANC-1
HPAC
BxPc-3
WI38
1520 133
9042 3197
The selectivity of 1–3 for G-quadruplex DNA was evaluated in a
8430 2904
competition FRET experiment,16 in which different ratios of duplex

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S. M. Hampel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6459–6463
A
B
MCF7+SMH14.6
100
90
80
70
60
50
40
30
20
10
0
VC
50nM W1
50nM W2
Figure 5. Effects of compound 1 on telomerase activity in MCF7 cells. (A) Denaturing gel from the TRAP–LIG assay22 with compound 1 at a concentration of 50 nM. The left-
hand lane shows a positive control with telomerase and no ligand. The middle and right-hand lanes are for 1 and 2 weeks exposure, respectively. (B) The quantitation of the
gel.23
The 23-mer DNA with K+ as the coordinating ion formed a G-quad-
ruplex in solution with a mixed/hybrid type topology,3b indicated
by a negative peak at 235 nm, a shoulder peak at 270 nm and a
positive peak at 290 nm (Fig. 3A, black graph).18 Similar sequences
form a parallel quadruplex in the crystalline state19 and in crowded
solution.3c Addition of 1–3 equiv of compound 1 did not lead to
immediate changes in the spectrum, but after 6 h of incubation,
the equilibrium shifted in the direction of a parallel-type topology,
which was found in DNA annealed in the presence of 1, indicated
by a negative peak at 240 nm and a positive peak at 260 nm
(Fig. 3A, red graph). The Na+ coordinated 23-mer DNA formed a
G-quadruplex with an anti-parallel topology, with a positive peak
at 245 nm, a negative peak at 265 nm, and a positive peak at
290 nm (Fig. 3B, black graph). Upon addition of compound 1, the
ratio of the parallel arrangement increased instantly (Fig. 3B, red
graph). The equilibration towards the parallel topology was more
rapid than for the more stable K+ complex. Compounds 2 and 3
gave very similar results, but with the absence of the shoulder at
290 nm in the CD spectrum of 1 in the presence of sodium,
suggesting a more complete shift of the equilibrium towards the
tested cancer cell lines, including the pancreatic cancer cell lines
MIA-Pa-Ca-2, PANC-1 and HPAC, is high with IC50 values of
0.1–0.2
l
M. The pancreatic cancer cell line BxPc-3 is ꢀ10-fold less
sensitive than the other pancreatic lines. The compounds are sig-
nificantly less toxic to the normal human fibroblast cell line
WI38 (which does not express telomerase) than to the panel of
cancer cell lines examined here.
The effects of compound 1 on telomerase activity in the MCF7
cell line were also examined (Fig. 5). Incubation with a 50 nM con-
centration of 1 (<50% of the IC50 value) resulted in a 50% reduction
in telomerase activity after 1 week, using a modified TRAP (Telo-
merase Repeat Amplification Protocol) assay,22 which was main-
tained after a further week. Since cell populations tend to have
telomere length heterogeneity,24 the high potency of this com-
pound may be attributable to its effect on a sub-population of
susceptible cells with short telomeres.
The compounds reported in this paper show high selectivity
and affinity for telomeric G-quadruplex DNA, together with selec-
tive toxicity in a panel of cancer cell lines. They inhibit the binding
of hPOT1 and Topo IIIa to telomeric G-quadruplex DNA, and stabi-
parallel structure (see Supplementary data).
A
number of
lize G-quadruplexes with a parallel-type topology. The observation
of inhibition of cellular telomerase activity is consistent with the
hPOT1 displacement, in particular in accord with the hypothesis
that these compounds act by displacing hPOT (and telomerase)
from the telomeric DNA overhang and stabilizing quadruplex
structures along its length or during telomere replication,25 which
act as signals of DNA damage.26 The compounds are notably potent
against the pancreatic cancer cell lines used here, and they are cur-
rently being evaluated in a xenograft model for pancreatic cancers,
which are especially challenging to treat.27
G-quadruplex ligands have been reported, which stabilize either
the parallel or the anti-parallel form of G-quadruplex DNA.17,19,20
The potent ligands telomestatin and RHPS4 for instance stabilize
the anti-parallel form.19,20 The crystal structure of a complex of a
telomeric G-quadruplex 23-mer DNA and an ND has a parallel
topology, which is in agreement with the results of the CD study.11
The inhibition of hPOT1 and Topo III
a binding to a telomeric
DNA sequence WT26 by the ligands 1 and 3 was examined
in vitro using EMSA assays.21 Both compounds inhibit the binding
of hPOT1 and Topo IIIa at low lM concentrations, probably due to
G-quadruplex stabilization (Fig. 4A, D, E), by analogy with earlier
compounds examined in this assay.21c In vivo, interference with
these targets may lead to telomere uncapping and end-to-end fu-
sions of chromosomes. This will hinder maintenance of telomeres
both in telomerase-positive and in ALT cells18; these effects have
yet to be examined for 1 and 3. Compounds 1 and 3 do not inhibit
Acknowledgements
This work was supported by grants from CRUK (No. C129/
A4489) and from Dr. Philip Brown to S.N. and by a grant from LNCC
(équipe labellisée 2010) to J.F.R. and A.S.
the binding of Topo III
be examined for 1 and 3. The inhibition of Topo III
a
to duplex DNA (Fig. 4C). effects have yet to
binding to the
Supplementary data
a
G-quadruplex DNA sequence WT26 is specific, as the duplex bind-
ing molecule ethidium bromide does not inhibit the binding of
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.066.
Topo III
the binding of Topo III
a
to WT26 (Fig. 4B), and compounds 1 and 3 do not inhibit
to duplex DNA (Fig. 4C).
a
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dride 8 (190 mg, 446.0 lmol) in 1-(3-aminopropyl)-4-methylpiperazine 7a
(1.0 mL) was heated (microwave at 150 °C) under argon with stirring for 2 h.
The amine was removed i. vac., and compound 1 (37.0 mg, 0.0852 mmol, 19%)
was isolated by preparative HPLC as
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a
dark blue solid. 1H NMR (CDCl3,