Water soluble extended naphthalene diimides as pH fluorescent sensors and G-quadruplex ligands

Article abstract of DOI:10.1039/c2ob07006e

Extended naphthalene diimides (NDIs) fused to 1,4-dihydropyrazine-2,3- dione, containing two solubilizing moieties, have been synthesized. Fluorescence spectra of the new NDIs were remarkably affected by pH, as the second deprotonation of the dihydropyrazinedione moiety (pK_a 6.9) switched off the emission. Binding to a G-quadruplex folded oligonucleotide and stoichiometry were evaluated by FRET melting assay and CD analysis. G-quadruplex binding was strongly enhanced shifting from pH 7.4 to pH 6.0 as a consequence of the dihydropyrazinedione moiety protonation. Cytotoxicity studies using two human telomerase-positive cell lines (HT29 and A549) revealed that the best G-quadruplex ligand was very active against the colon cell line, with an EC ₅₀ of 300 nM.

Full text of DOI:10.1039/c2ob07006e

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PAPER
Water soluble extended naphthalene diimides as pH fluorescent sensors and
G-quadruplex ligands†
Filippo Doria,a Matteo Nadai,b Giovanna Sattin,b Luca Pasotti,a Sara N. Richter*b and Mauro Freccero*a
Received 29th November 2011, Accepted 8th March 2012
DOI: 10.1039/c2ob07006e
Extended naphthalene diimides (NDIs) fused to 1,4-dihydropyrazine-2,3-dione, containing two
solubilizing moieties, have been synthesized. Fluorescence spectra of the new NDIs were remarkably
affected by pH, as the second deprotonation of the dihydropyrazinedione moiety (pKa 6.9) switched off
the emission. Binding to a G-quadruplex folded oligonucleotide and stoichiometry were evaluated by
FRET melting assay and CD analysis. G-quadruplex binding was strongly enhanced shifting from pH 7.4
to pH 6.0 as a consequence of the dihydropyrazinedione moiety protonation. Cytotoxicity studies using
two human telomerase-positive cell lines (HT29 and A549) revealed that the best G-quadruplex ligand
was very active against the colon cell line, with an EC50 of 300 nM.
the need to develop probes capable of direct detection of G-4
Introduction
structures in vivo.28–30
Fluorescent molecules have been intensively developed as non-
invasive probes for biological applications.1–4 Of particular inter-
est are those that can reversibly switch between an emitting and
a non-emitting state.5–9 Among them, the pH-responding fluor-
escent sensors are still holding a prominent position in the
current literature, as biochemical processes frequently involve
acid–base equilibria with concomitant changes in the pH of the
environment.10–12 Limitations of the available pH probes include
low sensitivity, excitation profiles in the ultraviolet region, and a
switch of the response at a quite different pH from neutrality.
Among the reported sensitive fluorescent probes, core-substi-
tuted NDIs (1,4,5,8-naphthalenetetracarboxylic acid diimides)13
have been thoroughly investigated by Würthner’s and Matile’s
groups as fluorescent dyes, thanks to their remarkably tunable
emission properties.14–18 In particular, they showed that functio-
nalization of the NDI core can induce tuning of the optical and
redox properties of this class of dyes extending the range of their
application.19,20 In fact, tri- and tetra-substituted NDIs have
recently been exploited as G-quadruplexes (G-4) ligands and cel-
lular fluorescent labelling agents.21–25 G-4 are DNA secondary
structures with a crucial biological role being involved in telo-
merase maintenance and transcriptional regulation of critical
oncogenes.26,27 The growing interest in biology had propelled
Our interest in substituted NDIs led us to develop a new class
of NDIs as G-4 ligands exhibiting absorption and emission prop-
erties strongly affected by the surrounding medium (solvent and
pH). The first type of these modified T-shaped NDIs (1 and 2,
Fig. 1) showed strong green fluorescence which could be
switched on/off by a pH trigger, but they were not suited for bio-
logical application due to negligible solubility in water.13
In the current study, we conferred water solubility to the T-
shaped NDI core, in order to explore its dual potential as fluor-
escent probe under physiological conditions and G-4 ligands.
Three water-soluble NDIs (4a–4c) have been synthesized accord-
ing to an optimized synthetic protocol. They bear the hetero-
cyclic 2,7-dialkyltetraazabenzo[e]pyrene-1,3,6,8,10,11-hexaone
core, tethered to two cationic (4a,b) or anionic (4c) chains at the
imide moieties under physiological conditions (Scheme 1).
Results and discussion
The five-steps procedure for the preparation of the T-shaped
heterocyclic
2,7-dialkyltetraazabenzo[e]pyrene-1,3,6,8,10,11-
hexaones 1 and 2 (Fig. 1)13 has been modified for the synthesis
of the water soluble NDIs 4a–c (Scheme 1). The key-step of
these syntheses is a Jones reaction resulting in the oxidation of
both the methylene-groups of the amines 3a–c, without further
oxidation of the solubilizing moieties (Scheme 2).
aDipartimento di Chimica, Università di Pavia, V.le Taramelli 10, 27100
Pavia, Italy. E-mail: filippo.doria@unipv.it, luca.pasotti@unipv.it,
mauro.freccero@unipv.it
Synthesis
bDipartimento di Medicina Molecolare, Università di Padova, via
Gabelli 63, 35121 Padua, Italy. E-mail: matteo.nadai@unipd.it,
giovanna.sattin@unipd.it, sara.richter@unipd.it
The commercially available anhydride was brominated with
dibromocyanuric acid and the resulting product mixture contain-
ing the 2,6-dibromo-1,4,5,8-naphthalenetetracarboxylic acid
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob07006e
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Fig. 1 Structures of T-shaped NDIs (1 and 2) fused to a 1,4-dihydropyrazine-2,3-dione exhibiting alkyl moieties. Absorption spectra of the neutral
and anionic forms of 1 in DMSO.
good green fluorescent compounds in water at pH 1 [λem
=
514 nm; fluorescence quantum yield, Φfl(3a) = 0.10] with an
absorption spectra characterized by an electronic transition cen-
tered at 491 nm. On the contrary, they were very weakly fluor-
escent in chloroform [λem = 497 nm; Φfl(3a) = 0.04]. Compared
to 2,6-dialkylamino NDIs (λabs = 620 nm; λem = 650 nm),14 the
absorption maximum was blue-shifted by more than 130 nm.
The UV-Vis absorption of 3a (λabs = 491 nm) was slightly more
red shifted than that of 2,6-dialkoxy NDIs (λabs = 470 nm).14
This evidence suggests that cyclic amine substituents at 2,3 pos-
itions on the NDI core are much less efficient electron donors
than amine moieties at 2,6 positions, and that 3a cannot benefit
of the cross-conjugation effect on the absorption spectra due to
two donors symmetrically located on the NDI core.17 After oxi-
dation, both UV-Vis spectra of the resulting 4a and 4c, were just
30 nm blue-shifted (λabs = 455 and 453 nm, respectively, in
acidic water) in comparison to 3a and 3c, in spite of the reduced
electron donor properties of the amide N-substituent. The
UV-Vis spectra of both NDI 4a and 4c were investigated in
several organic solvents: chloroform, methanol, acetonitrile
(ACN), DMSO, acidic water (Fig. 2), and as a function of pH in
water (Fig. 3).
Scheme 1 Generation of the T-shaped heterocyclic 2,7-dialkyltetraaza-
benzo[e]pyrene-1,3,6,8,10,11-hexaones 4a–c by oxidation.
dianhydride (5) was directly used for preparation of the 2,6-
dibromo-substituted NDIs (6a–6c), synthesized by a classical
imidation procedure under acid condition.31,32 The following
nucleophilic aromatic substitution (SNAr) and subsequent
Chichibabin-like ring-closure yielding 7a–c, were carried out in
pure ethylenediamine, at room temperature. Under these mild
conditions only a mono-functionalization of the 2,6-dibromo-
NDIs was efficiently achieved. Further quantitative mild reduc-
tive dehalogenation of 7a–c was run on the crude reaction
mixture, using Na2S2O4 dissolved in aqueous DMSO, yielding
the diimide analogues 3a–c in quantitative yields. The final step
of the synthesis was carried out using the Jones oxidation proto-
col, giving 4a–c in good yield. 8a was synthesized starting from
7a according to an identical oxidization protocol.
The absorbance in organic solvents and in water under very
acidic conditions (pH ≤ 1) was only marginally affected by the
nature of the solvent (453 < λmax < 461 nm; Fig. 2).
However, absorbance spectra of 4a in water at higher pH (pH
> 1.5) were remarkably different (Fig. 3a and b). In more detail,
there was a progressive reduction of the absorbance at the λmax
(455 nm) passing from pH 1.5 to pH 4.85 (Fig. 3a), which
resemble the behavior of 1 during a titration in DMSO (Fig. 1).13
In addition, the appearance of a new and red shifted broad band
extending up to 580 nm became evident at pH > 6.9 (Fig. 3b).
Both the NDIs 4a and 4c exhibited a weak fluorescence emis-
sion centered at λem = 468 nm and 454 nm, respectively, in neat
ACN. On the contrary, 4a and 4c showed an enhanced light-
green fluorescence emission in water solution at pH 1 (Fig. 4),
Spectroscopic properties in organic and aqueous solvents
In order to evaluate the possibility of exploiting the new NDIs as
bio-analytical probes, and more specifically as probes for G-4
sensing, we measured the absorbance and emission spectra of
the NDIs 4a and 4c and their precursors 3a and 3c, in organic
solvents and water. The disubstituted NDIs 3a and 3c were fairly
which was five time more intense [Φfl(4a) = 0.54, pH 1, λem
=
458 nm] than the related amine 3a [Φfl(3a) = 0.10, pH 1; λem
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Scheme 2 Reagents and conditions: (a) amines a–c, glacial acetic acid, 130 °C, 30 min; (b) ethylene diamine, 25 °C, 16 h, under argon; (c) sodium
dithionite, DMSO–H2O = 20 : 80, 40 °C, 2 h, under argon, (d) K2Cr2O7, H2SO4, H2O–acetone, 100 °C, 2 h.
Fig. 2 UV-Vis absorption spectra of 4a and 4c in organic solvents and water at pH 1.
479 nm]. Br-NDI 8a was one of the least fluorescent NDIs
[Φfl(8a) = 0.09, pH 1; λem = 477 nm].
potentiometric and fluorescence titrations (Fig. 7B). Fluor-
escence intensity change as a function of pH was analyzed by
the Henderson Hasselbalch equation: log[(FImax − FI)/(FI −
FImin) = pH − pKa, where FI is the observed fluorescence inten-
sity at a fixed wavelength, FImax and FImin are the corresponding
maximum and minimum respectively. This fluorescence titration
yielded a pKa of 6.88 (Fig. 7B). The UV-Vis titration yielded a
very similar pKa = 6.9 revealing a further additional pKa = 11.6
(Fig. 7B). Potentiometric titration yielded pKa1 = 2.8, pKa2 = 7.3
and pKa3 = 10.6. The potentiometric pKas were in fairly good
agreement with the pKas evaluated from fluorescence and spec-
trophotometric titrations.
The fluorescence intensity of 4a monitored at 458 nm slightly
decreased at pH higher than 2 (inset B, Fig. 5). The quenching
of the emission was more pronounced at 358 nm (inset A,
Fig. 5). The emission intensity rapidly dropped at pH above neu-
trality and it was completely switched off at pH 8.5. The fluor-
escence intensity raised again at pH higher than 9.0, with a new
emission centered at longer wavelength: 535 nm (Fig. 5 and
inset C). This new emission became much more intense using an
excitation wavelength centered at 458 nm instead of 294 nm
(Fig. 6).
Taking into account that (i) the non-water soluble NDI 1, pre-
viously investigated, exhibited a very low pKa in water–DMSO
mixture (pKa ∼ 1),13 and that (ii) the change in the spectrum at
455 nm between pH 1.4 and 5 mimics the behavior of 1 in
DMSO (Fig. 1),13 the pKa1 has been assigned to the dissociation
Absorbance and fluorescence titration
The mode of protonation/deprotonation of 4a (Scheme 3) was
investigated by spectrophotometric (inset of Fig. 3B and 7A),
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Fig. 3 UV-Vis absorption spectra of compound 4a in water as a function of pH (A) 4a (5.1 × 10−5 M) 1.50 < pH < 4.85 and (B) 4a (2.0 × 10−5 M),
5.70 < pH < 10.00. Absorbance monitored at 419 nm as a function of pH is reported in the inset.
Fig. 4 Absorption (dotted-line) and emission spectra (continuous line) of 4a and 4c in water, at pH 1.
+
of the dicationic 4a-H2 (Scheme 3). pKa2 should describe the
acidity of the second proton of the dihydropyrazinedione moiety.
In fact, the deprotonation of 4a-H+ caused the most significant
change in the absorption, as the aromaticity of the resulting 4a-
ZW (or related tautomers) was further extended. Vice versa, the
deprotonation of the ammonium moiety should not cause such
an extensive change. pKa3 should be associated to the deprotona-
tion of the zwitterionic 4a-ZW (or related tautomers), due to the
similar acidity of an alkyl ammonium. The pKa2 value suggested
that the NDI 4a should be mainly neutral and non-fluorescent at
pH higher than 6.9.
The increase in the melting temperature (ΔTm) of the labelled
G-4 oligonucleotide by 1.0 μM NDIs 4a–c was 13.0 0.5 °C,
12.0 1 °C and 3.0 0.5 °C for 4a–c, respectively. Since 4c is
at least di-anionic at pH 7, it was hypothesized that negative
electrostatic repulsion prevented efficient compound binding to
the DNA: in fact, when 4c was incubated with the labelled hTel
DNA in the presence of 50 and 100 mM Mg2+, ΔTm increased
by 5.5 and 7.0 °C, respectively.
To check the preferred G-4 conformation stabilized by the
best performing compound, 4a, we carried out CD analysis of
the hTel oligonucleotide upon binding with the NDI. 4a was
added before G-4 DNA folding (around 80 °C) and samples
were left to equilibrate 24 h prior to CD spectra measurement. In
these conditions hTel oligonucleotide alone was present in its
mixed antiparallel/parallel form as attested by a major positive
peak at 290 nm and a shoulder at 265 nm (Fig. 8a). The addition
of compound 4a, induced a stabilization of the antiparallel G-4
NDIs 4a–c bind G-4 DNAwith a preference for the antiparallel
conformation, and a 3 : 1 drug–DNA stoichiometry
To evaluate the stabilization of NDI derivatives for telomeric
G-4 DNA (hTel), FRET melting experiments were carried out.
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Fig. 5 Fluorescence response of 4a (2 × 10−5 M) to pH variation at 25 °C in water solution from pH 2 to pH 11.7. Excitation wavelength 294 nm.
Inset A, B and C: Fluorescence emissions monitored at 358, 458 and 535 nm, respectively, as function of pH.
Fig. 6 Fluorescence response of 4a (2 × 10−5 M) to pH variation at 25 °C in water solution from pH 2 to pH 11.7. Excitation wavelength 458 nm.
structure as proved by an increase in the positive peak at 290 nm
(Fig. 8a).
binding stoichiometry of 3 molecules of NDI bound to 1 mol-
ecule of DNA (Fig. 8b).
Reaction stoichiometry of compound 4a against hTel DNA in
10 mM Li cacodylate and 50 mM KCl buffer, at 20 °C, was
evaluated by the continuous variation method (Job plot) by
mixing DNA and NDI in different proportions, while maintain-
ing constant the whole amount of DNA + NDI. The peak was
observed at a DNA fraction of 0.25 which corresponded to a
As shown above, pKa2 of the dihydropyrazinedione moiety
was 6.9, just 0.5 units lower than the pH of our working sol-
utions. We therefore argued that decreasing the pH of the
working buffer would augment DNA binding due to reduction
of the anion charge on the aromatic core, increasing the popu-
lation of the monocationic 4a-H+. To check for binding stability
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Scheme 3 Acid–base equilibria involving 4a. Reported pKa values have been measured by both potentiometric and spectrophotometric titrations at
25 °C. pKa3 value is the average of the data from UV-Vis titration (11.6) and potentiometric titration (10.6). For the neutral 4a-ZW and anionic 4a-A
species, only the most charged structures have been drawn.
Fig. 7 UV-Vis (A) and fluorescence (B) titrations of 4a (2 × 10−5 M).
of compound 4a towards hTel at different pH values, FRET
melting assay could not be used due to interference of NDI fluor-
escence (which increased at lower pH) with the labelled DNA
probe. We hence performed CD melting analysis by incubating
hTel with or without 4a at different pH and analyzing molar
ellipticity values at 295 nm at increasing temperatures. Tm values
were calculated according to the vant’Hoff equation. As shown
in Fig. 8c, ΔTm remarkably increased at lower pH, with a
maximum ΔTm value of 21.2 °C and stabilization at pH 6.0. ΔTm
values above 20 °C indicate good G-4 binders.
The ability of compounds 4a to enter mammalian cells was
tested by confocal microscopy taking advantage of its intense
fluorescence emission properties. We observed a significant cell
entry and striking accumulation into the nucleus after 30 min
exposure (Fig. 9).
compound 4a was one of the most cytotoxic compound among
several NDIs developed by our groups21,22 and in line with NDI
cytotoxicity reported by others.24
Fluorescence titration
We performed fluorescent titrations to evaluate the effect of hTel
interaction on the emission properties of the extended NDI 4a.
Fig. 10 shows fluorescence titration of 0.7 μM 4a by adding
increasing molar ratios of hTel (0–8), at pH 6.0. A 2 fold
decrease in fluorescence intensity (FI), monitored at 475 nm,
was recorded when hTel was added in equimolar amount. Upon
saturation with G-quadruplex DNA photoluminescence from 4a
was quenched.
Cytotoxicity was next tested against two telomerase-positive
human carcinoma cell lines, HT29 and A549 (colon and lung,
respectively). Compound 4a was very active against the colon
cell line, with an EC50 of 300 nM, and maintained a good
activity against lung tumor cells (EC50 1.5 μM). 4b was slightly
less active against both cell lines, with EC50 of 3 μM and
6 μM against colon and lung cells, respectively. Remarkably,
Conclusion
In summary, our investigation led to the synthesis of three water
soluble extended NDIs, with the naphthalene diimide core fused
to the heterocyclic 1,4-dihydropyrazine-2,3-dione (4a–4c).
These NDIs behaved as efficient fluorescent pH sensors in water
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Fig. 9 Cell uptake detection using fluorescence confocal microscopy.
Image of 4a localized in the nucleus after 30 min at 1 μM. Emission was
recorded at λ > 470 nm.
Fig. 10 Fluorescence titration of 4a (2 μM) by adding increasing
molar ratios (as shown in legend) of hTel, at pH 6.0.
Fig. 8 CD analysis of compound 4a binding towards hTel. (a) CD
spectra of hTel (4 μM) in Li cacodylate buffer (10 mM, pH 7.4) with
50 mM KCl, with (dotted line) or without (solid line) compound 4a
(16 μM); (b) the Job plot performed with a total amount of hTel + 4a of
10 μM, and varying DNA and drug proportions. The two fitting straight
lines intersect at DNA fraction of 0.25, which corresponds to 3 molecules
of 4a bound to 1 molecule of hTel; (c) CD melting analysis of hTel with
or without compound 4a at increasing temperature in 10 mM Li cacody-
late buffer, 50 mM KCl, at different pH. Tm values were obtained by
plotting molar ellipticities at 295 nm versus temperature and fitting with
the van’t Hoff equation. ΔTm values were obtained as hTel+4a Tm minus
hTel Tm values.
due to the favourable electrostatic interaction of the former.
Among the NDIs tested, 4a was identified as a reasonably good
G-4 ligand, being able to stabilize G-4 folded DNA with a ΔTm
at 1 μM of 21.2 °C, at pH 6.0, with a preference for the antipar-
allel conformation, and a 3 : 1 ligand–DNA stoichiometry.
Unfortunately, the strong emission of the good G-4 binder 4a,
both at physiological pH and pH = 6, is quenched by the DNA.
Work is in progress in order to avoid this fluorescence quench-
ing, by substitutions on the NDI core. Nevertheless, the remark-
able cell entry and accumulation into the nucleus of mammalian
cells of compound 4a was detected taking advantage of its
intense fluorescence. NDI 4a was also very cytotoxic towards
the colon cell line HT29, with an EC50 of 300 nM. This suggests
dual potential applications of these extended-NDIs as fluor-
escence probe and drug candidate.
solution. Importantly, 4a exhibited on/off switch of the fluor-
escence emission between pH 5.0 and pH 8.5, which is appropri-
ate for biological applications. In fact, the monocationic 4a-H+
acts as a fluorescent weak acid (pKa = 6.9), but its conjugate
base (4a-ZW) is a non-fluorescent orange dye. Compound 4a
binding to G-4 folded DNA parallels its emission properties, as
both are strongly affected by pH in the neutrality window. It is
worthy of note that the monocationic and emitting 4a-H+ also
binds the G-4 DNA much better than its conjugate base 4a-ZW,
Materials and methods
The naphthalene bisanhydride were synthesized according to
standard published procedures.32
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To a stirred suspension of dibromodianhydride 5 (600 mg,
0.001 mol) in glacial acetic acid (15 ml) the amine a, b or
glycine (c) (0.003 mol) was added. The reaction mixture was
stirred at 130 °C, and after 30 min was cooled to r.t., quenched
into ice, neutralized with sodium bicarbonate and extracted three
times with CHCl3. The organic layer was dried under vacuum to
give an orange crude, which was solid purified by column
chromatography (CHCl3–MeOH), yielding 6a (63%), 6b (58%),
6c (43%), respectively.
53.05; H, 5.01; Br, 14.70; N, 15.47; O, 11.78. Found: C, 52.90;
H, 5.13; Br, 15.00; N, 15.59%.
N,N’-Di-(N,N-dimethylpropyl)-6-bromo-5,8,9,10-(1,4-diaza-
1,2,3,4-tetrahydroanthracene)tetracarboxylic acid bisimide (7b)
1
Orange solid, yield: 69%. H NMR (300 MHz, CDCl3, 25 °C,
TMS): δ = 10.85 (s, 1H), 10.53 (s, 1H), 8.20 (s, 1H), 4.11 (m,
4H), 3.78 (s, 4H), 2.42 (m, 4H), 2.27 (s, 12H) 1.86 (m, 4H). 13
C
NMR (CDCl3): δ = 166.0; 165.5; 162.3; 161.3; 144.3; 143.4;
135.0; 131.5; 131.29; 125.1; 122.1; 121.5; 119.9; 97.6; 57.1;
45.1; 29.6; 25.7; 25.5. Anal. Calcd for C26H31BrN6O4: C, 54.65;
H, 5.47; Br, 13.98; N, 14.71; O, 11.20. Found: C, 54.34; H,
5.35; Br, 14.17; N, 14.98%.
N,N′-Di-(N,N-dimethylethyl)-2,6-dibromonaphthalene-1,4,5,8-
tetra-carboxylic acid bisimide (6a)22,33
Yellow-orange solid. 1H NMR (200 MHz, CDCl3, 25 °C, TMS).
δ = 9.0 (s, 2H); 4.35 (m, 4H); 2.72 (m, 4H); 2.37 (s, 12H). Anal.
Calcd for C22H22Br2N4O4: C, 46.66; H, 3.92; Br, 28.22; N,
9.89; O, 11.30. Found: C, 46.69; H, 3.89; Br, 28.18; N, 9.92%.
N,N′-Di-(carboxymethyl)-6-bromo-,5,8,9,10-(1,4-diaza-1,2,3,4-
tetrahydroanthracene)tetracarboxylic acid bisimide (7c)
Yellow solid, yield: 65%. 1H NMR (300 MHz, DMSO): δ =
10.89 (s, 1H), 10.60 (s, 1H), 8.54 (s, 1H), 4.72 (s, 4H), 3.72 (s,
4H). 13C NMR (DMSO):δ = 169.3; 169.1; 164.2; 163.7; 161.2;
160.1; 144.7; 143.7; 136.3; 130.0; 129.2; 125.3; 125.0; 120.7;
117.7; 95.3; 53.1; 30.6. Anal. Calcd for C20H13BrN4O8: C,
46.44; H, 2.53; Br, 15.45; N, 10.83; O, 24.75. Found: C, 46.82;
H, 2.43; Br, 15.66; N, 10.71%.
N,N′-Di-(N,N-dimethylpropyl)-2,6-dibromonaphthalene-1,4,5,8-
tetra-carboxylic acid bisimide (6b)
Yellow solid. 1H NMR (300 MHz, CDCl3, 25 °C, TMS): δ = 8.7
(s, 2H), 4.65 (m, 2H), 2.49 (m, 4H), 2.28 (s, 12H), 1.95 (m, 4H).
Anal. Calcd for C24H26Br2N4O4: C, 48.50; H, 4.41; Br, 26.89;
N, 9.43; O, 10.77. Found: C, 48.34; H, 4.58; Br, 27.02; N, 9.31.
This compound was characterized also as hydrochloride after
HPLC purification: N,N′-Di-(N,N-dimethylpropyl)-2,6-dibromo-
naphthalene-1,4,5,8-tetra-carboxylic acid bisimide (6b) 2HCl.
1H NMR (300 MHz, CD3OD): δ = 8.9 (s, 2H), 4.31 (m, 4H),
3.32 (m, 4H), 2.95 (s, 12H), 2.21 (m, 4H). 13C NMR (CD3OD):
δ = 163.2; 163.0; 139.6; 129.7; 128.9; 127.4; 126.1; 57.1; 43.9;
39.2; 24.8.
Mild reductive dehalogenation for the synthesis of 3a–3c.
General procedure
The reactant 7a–c (0.07 mmol) was suspended in a solution of
34 ml of DMSO and 6 ml of water, out-gassed by argon and
stirred at r.t. Sodium dithionite (0.6 mmol) was added and the
new mixture was stirred for 2 h, at 40 °C. After this period the
reaction was worked up admitting oxygen atmosphere and was
induce the precipitation of the product with water. The resulting
suspension was filtered and purified by column chromatography
(CHCl3–MeOH = 6 : 4) as a yellow solid.
N,N′-Di-(carboxymethyl)-2,6-dibromonaphthalene-1,4,5,8-tetra-
carboxylic acid bisimide (6c)
1
Grey solid. H NMR (300 MHz, DMSO): δ = 8.73 (s, 2H), 4.77
(s, 4H), 2.63–2.48 (m, 4H). 13C NMR (DMSO): δ = 168.6;
160.4; 160.2; 137.7; 131.3; 127.8; 127.2; 127.0; 125.2; 123.9;
47.6. Anal. Calcd for C18H8Br2N2O8: C, 40.03; H, 1.49; Br,
29.59; N, 5.19; O, 23.70. Found: C, 41.15; H, 1.54; Br, 29.21;
N, 5.11%.
N,N′-Di-(N,N-dimethylethyl)-5,8,9,10-(1,4-diaza-1,2,3,4-
tetrahydroanthracene)tetra-carboxylic acid bisimide (3a)
Yellow-gold solid, yield: 99%. 1H NMR (300 MHz, CDCl3,
25 °C, TMS): δ = 10.60 (s, 2H), 8.30 (s, 2H), 4.37 (m, 4H), 3.80
(s, 4H), 2.74 (m, 4H), 2.45 (s, 12H). 13C NMR (CDCl3, 25 °C,
TMS): δ = 166.3; 163.4; 143.9; 124.6; 122.5; 122.2; 56.6; 45.4;
38.1; 37.5; 29.6. Anal. Calcd for C24H28N6O4: C, 62.06; H,
6.08; N, 18.09; O, 13.78 Found: C, 61.95; H, 6.12; N, 17.98%.
N,N′-Di-(N,N-dimethylethyl)-6-bromo-5,8,9,10-(1,4-diaza-
1,2,3,4-tetrahydroanthracene)tetracarboxylic acid bisimide (7a)
Compound 6a (200 mg, 0.35 mmol) was placed in a flask con-
taining ethylene diamine (15 ml) and the mixture was stirred at
r.t. for 16 h under argon. The resulting red mixture was poured
into a solution of HCl (1 N, 100 ml), to induce the precipitation
of the product. The collected solid was washed with water and
purified by column chromatography (CHCl3) as an orange solid,
yield: 78%. 1H NMR (300 MHz, CDCl3, 25 °C, TMS): δ =
10.96 (s, 1H), 10.62 (s, 1H), 8.50 (s, 1H), 4.33 (m, 4H), 3.80 (s,
4H), 2.66 (m, 4H), 2.53 (broad s, 12H). 13C NMR (CDCl3,
25 °C, TMS): δ = 166.0; 165.4; 162.3; 161.3; 144.3; 143.3;
134.3; 131.5; 131.3; 126.9; 126.1; 121.4; 119.9; 97.5; 56.8;
56.5; 45.6; 38.2; 37.9; 29.9. Anal. Calcd for C24H27BrN6O4: C,
N,N′-Di-(N,N-dimethylpropyl)-5,8,9,10-(1,4-diaza-1,2,3,4-
tetrahydroanthracene)tetra-carboxylic acid bisimide (3b)
Yellow-gold solid, yield: 99%. 1H NMR (300 MHz, CDCl3,
25 °C, TMS): δ = 10.64 (s, 2H), 8.15 (s, 2H), 4.20 (m, 4H), 3.80
(s, 4H), 2.41 (m, 4H), 2.30 (s, 12H), 1.85 (m, 4H). Anal.
Calcd for C26H32N6O4: C, 63.40; H, 6.55; N, 17.06; O, 12.99.
Found: C, 63.84; H, 6.31; N, 16.95. This compound was
characterized also as hydrochloridric salt after HPLC purifi-
cation: N,N′-di-(N,N-dimethylpropyl)-5,8,9,10-(1,4-diaza-1,2,3,4-
This journal is © The Royal Society of Chemistry 2012
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tetrahydroanthracene)tetra-carboxylic acid bisimide (3b) 2HCl.
Orange solid. H NMR (300 MHz, CD3OD): δ = 7.7 (s, 2H),
4.13 (m, 4H), 3.80 (s, 4H), 3.28 (m, 4H), 2.98 (s, 12H), 2.17 (m,
4H). 13C NMR (CD3OD): δ = 166.9; 164.9; 145.3; 124.6;
123.7; 122.4; 97.6; 57.2; 43.8; 39.2; 38.5; 25.0.
136.9; 130.9; 127.6; 125.9; 102.0; 57.1; 43.8; 38.3; 25.0. Anal.
Calcd for C26H30Cl2N6O6: C, 52.62; H, 5.10; Cl, 11.95; N,
14.16; O, 16.18. Found: C, 52.81; H, 5.21; Cl, 12.04; N, 14.04.
1
2,7-N,N′-Di-(carboxymethyl)-tetraazabenzo[e]pyrene-
1,3,6,8,10,11-hexaone (4c)
N,N′-Di-(carboxymethyl)-5,8,9,10-(1,4-diaza-1,2,3,4-
tetrahydroanthracene)tetra-carboxylic acid bisimide (3c)
Yellow solid, yield: 46%. 1H NMR (300 MHz, DMSO): δ =
11.53 (s, 2H), 10.56 (s, 2H), 8.53 (s, 2H), 4.75 (s, 4H). Anal.
Calcd for C20H10N4O10: C, 51.51; H, 2.16; N, 12.01; O, 34.31.
Found: C, 51.69; H, 2.32; N, 11.83.
Fluorescence spectra were taken on a Perkin-Elmer LS50B
spectrofluorimeter. Potentiometric titrations, pH measurements,
and electrode calibrations were carried out with a Radiometer
TitraLab 90 titration system. Spectrophotometric spectra were
taken on a spectrophotometer Varian CARY 100.
Yellow solid, yield: 99%. 1H NMR (300 MHz, DMSO): δ =
10.58 (s, 2H), 8.13 (s, 2H), 4.72 (s, 4H), 3.74 (s, 4H). 13C NMR
(DMSO): δ = 169.8; 164.7; 162.4; 144.3; 123.5; 122.6; 121.1;
99.1; 95.7; 44.5; 30.7. Anal. Calcd for C20H14N4O8: C, 54.80;
H, 3.22; N, 12.78; O, 29.20. Found: C, 55.01; H, 3.12; N,
12.54%.
Synthesis of 4a–4c, 8a by oxidation. General procedure
Potentiometric titration
The reactant 3a–3c, 7a (0.3 mmol) was solved in 30 ml H2O,
5 ml H2SO4 conc. and 5 ml acetone, then was added the
K2Cr2O7 (200 mg, 0.7 mmol). The brown mixture was reflux for
1 h. After this period the solution become clear, then an
additional amount of K2Cr2O7 was added, and the solution was
heated to reflux. After 2 h the reaction mixture was cooled to r.t.,
ice and Na2CO3 were added, and then it was extracted with
CHCl3 (3 × 50 ml). The combined organic phases were washed
with brine, dried over MgSO4 and the solvent was removed
under vacuum. The crude product was collected and purified by
column chromatography (CHCl3).
A 1 × 10−4 M solution (20 mL) of the chosen molecule was pre-
pared in a aqueous/methanol 8/2 solution of 0.05 M NaNO3.
The solution was placed in the cell of the automatic titrating
system, thermostatted at 25 °C and kept under a N2 atmosphere.
Excess acid (10 μL of 1.00 M standard HNO3) was added, and
the acidic solution was titrated automatically with portions
(10 μL) of 0.020 M standard NaOH. The results were obtained
as E (potential in mV at the glass electrode) versus volume of
added base (in mL) and the protonation constants were calcu-
lated using the nonlinear fitting program Hyperquad.34 Before
titrations, the cell E° values for the hydrogen glass electrode
were determined by means of the Gran method.35
2,7-N,N′-Di-(N,N-dimethylethyl)-4-bromotetraazabenzo[e]
pyrene-1,3,6,8,10,11-hexaone 2HCl (8a)
Coupled pHmetric–spectrophotometric titration
1
Yellow solid, yield: 75%. H NMR (300 MHz, CD3OD): δ =
A 1 × 10−4 M solution (20 mL) of the chosen molecule was pre-
pared in an aqueous/methanol 8/2 solution of 0.05 M NaNO3.
Excess acid (100 μL of 1.00 M standard HNO3) was added and
the acidic solution was titrated with addition of 0.1 M standard
NaOH (100 μL in the ranges of pH 2–4.5, 9.5–12 and 5 μL in
the range of pH 4.5–9.5). While the pH was monitored by using
a double-electrode pH meter, after each addition of base a
portion (3 mL) was withdrawn and placed in a quartz cell to
record the absorption spectrum, after which the sample was
returned to the bulk solution.
8.93 (s, 1H), 4.64 (t, J = 5.6 Hz, 4H), 3.62 (t, J = 5.6 Hz, 4H),
3.31 (s, 12H). 13C NMR (CD3OD): δ = 167.5; 167.0; 163.4;
162.6; 155.0; 138.2; 134.7; 128.0; 127.1; 126.8; 124.6; 120.3;
107.9; 107.4; 57.1; 44.5; 37.3. Anal. Calcd for
C24H25BrCl2N6O6: C, 44.74; H, 3.91; Br, 12.40; Cl, 11.01; N,
13.04; O, 14.90. Found: C, 44.41; H, 4.02; Br, 12.44; Cl, 11.05;
N, 13.12.
2,7-N,N′-Di-(N,N-dimethylethyl)-tetraazabenzo[e]pyrene-
1,3,6,8,10,11-hexaone 2HCl (4a)
1
Yellow solid, yield: 75%. H NMR (300 MHz, CD3OD): δ =
Coupled pHmetric–fluorimetric titration
8.90 (s, 2H), 4.76 (t, J = 5.6 Hz, 4H), 3.75 (t, J = 5.6 Hz, 4H),
3.46 (s, 12H). 13C NMR (CD3OD): δ = 167.9; 164.6; 155.1;
135.1; 131.3; 127.5; 124.9; 108.0; 57.3; 44.6; 37.2. Anal. Calcd
for C24H26Cl2N6O6: C, 50.98; H, 4.63; Cl, 12.54; N, 14.86; O,
16.98. Found: C, 51.15; H, 4.48; Cl, 12.24; N, 15.06.
A 1 × 10−5 M solution (20 mL) of the chosen molecule was pre-
pared in an aqueous/methanol 8/2 solution of 0.05 M NaNO3.
Excess acid (100 μL of 1.00 M standard HNO3) was added and
the acidic solution was titrated with addition of 0.1 M standard
NaOH (100 μL in the ranges of pH 2–4.5, 9.5–12 and 5 μL in
the range of pH 4.5–9.5). While the pH was monitored by using
a double-electrode pH meter, after each addition of base a
portion (3 mL) was withdrawn and placed in a quartz cell to
record the fluorescence emissions spectra at three different wave-
length of excitation. After which the sample was returned to the
bulk solution.
2,7-N,N′-Di-(N,N-dimethylpropyl)-tetraazabenzo[e]pyrene-
1,3,6,8,10,11-hexaone 2HCl (4b)
1
Yellow solid, yield: 58%. H NMR (300 MHz, CD3OD): δ =
8.80 (s, 2H), 4.31 (m, 4H), 3.70 (m, 4H), 3.30 (s, 12H), 2.71 (m,
4H). 13C NMR (CD3OD): δ = 166.9; 164.7; 161.8; 155.15;
3838 | Org. Biomol. Chem., 2012, 10, 3830–3840
This journal is © The Royal Society of Chemistry 2012

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The experimental data of each titration, If vs. pH and Abs vs.
pH, were fitted with a sigmoid profile described by equation
were prepared in Li cacodylate buffer (10 mM, pH 7.4)
with 50 mM KCl. The mole fraction of the DNA was plotted
against the molar ellipticity at 290 nm of the DNA–NDI
complex. In the plot, the mole fraction of the DNA at which the
molar ellipticity of the DNA–NDI complex is maximum, gives
the stoichiometry of the complex. The number of NDI binding
sites is equivalent to (1 − x)/x, where x is the DNA fraction at
the intersection of the two fitting straight lines. All samples were
allowed to equilibrate overnight. A buffer baseline was collected
in the same cuvette and subtracted from the sample spectra.
Final analysis of the data was carried out using Excel and Sigma
Plot software.
A
If ðor AbsÞ ¼ y0 þ
1 þ eðpHꢀx Þ=ꢀB
0
The parameters ‘A’ and ‘B’ are related to the height and
smoothness of the sigmoidal, y0 is the initial If or Abs, and x0
represents the pH of the inflection point which fall at a pH value
coincident with the pKa values.
FRET-melting assay
All oligonucleotides were purchased from Sigma-Aldrich (Italy);
after an initial dilution at 1 mM in purified water, further
dilutions were carried out in the relevant buffer. FRET assay was
performed with F21T (5′-d(FAM -G3[T2AG3]3-Tamra)-3′), with
FAM: 6-carboxyfluorescein and Tamra: 6-carboxy-tetramethylr-
hodamine. Fluorescence melting curves were determined with a
LightCycler II (Roche) real-time PCR machine, using a total
reaction volume of 20 μL, with 0.20 μM of tagged oligonucleo-
tide in a buffer containing 10 mM lithium cacodylate pH 7.4 and
50 mM KCl. After a first equilibration step at 30 °C during
2 minutes, a stepwise increase of 1 °C every minute for 65
cycles to reach 95 °C was performed and measurements were
made after each cycle with excitation at 470 nm and detection at
530 nm. The melting of the oligonucleotides was monitored by
emission of FAM, which was normalized between 0 and 1. Tm
was defined as the temperature for which the normalized emis-
sion is 0.5. ΔTm values are mean of 2–3 experiments standard
deviation.
Cytotoxicity
Telomerase-positive human cells were: lung carcinoma cells
A549 (ATCC number CCL-185) and human colorectal adenocar-
cinoma cells HT-29 (ATCC number HTB-38). Cell lines were
obtained from American Type Culture Collection (Rockville,
MD). Cells were maintained as a monolayer in the logarithmic
growth phase at 37 °C in a 5% CO2 humidified atmosphere,
using Dulbecco’s modified Eagle’s medium supplemented with
10% fetal calf serum and penicillin (100 units mL−1)/streptomy-
cin (100 μg mL−1). The cytotoxic effect on tumor cell growth
was determined by a MTT assay. NDI compounds were dis-
solved and diluted into working concentrations with DMSO.
Cells (1.75 × 104 cells per well) were plated onto 96-microwell
plates to a final volume of 100 μL and allowed an overnight
period for attachment. At day 1, 1 μL of each dilution of tested
compounds was added per well to get a 1% final concentration
of drug solvent per well. Control cells (without any compound
but with 1% drug solvent) were treated in the exact same con-
ditions. Cell survival was evaluated by a MTT assay: 10 μL of
freshly dissolved solution of MTT (5 mg mL−1 in PBS) were
added to each well, and after 4 h of incubation, 100 μL of solu-
bilization solution [10% sodium dodecyl sulfate (SDS) and 0.01
M HCl] were added. After overnight incubation at 37 °C, absor-
bance was read at 540 nm. Data were expressed as mean values
of three individual experiments conducted in triplicate. The per-
centage of cell survival was calculated as follows: cell survival =
(Awell − Ablank)/(Acontrol − Ablank) × 100, where blank
denotes the medium without cells.
CD analysis
CD experiments were performed on a Jasco J 810 spectropolari-
meter equipped with a NESLAB temperature controller and
interfaced to a PC 100. A quartz cuvette with 5 mm path length
was used for spectra recorded from 230 to 350 nm at 2 nm band-
width, 0.1 nm step size, and 4 s time per point. The reported
spectrum of each sample represents the average of 2 scans.
Observed ellipticities were converted to mean residue ellipticity
(θ) = deg × cm2 × dmol−1 (mol. ellip.). The oligomer hTel (5′-d
(AGGG[TTAGGG]3)-3′) was diluted from stock to the final con-
centration (4 μM) in Li cacodylate buffer (10 mM, pH 7.4) with
50 mM KCl and then annealed by heating at 95 °C for 5 min,
gradually cooled to room temperature, and incubated at 4 °C
overnight. Compounds at 16 μM final concentration were added
before hTel annealing. For the CD melting experiments, spectra
were recorded from 20 °C to 95 °C, with temperature increase of
5 °C. Tm values were calculated according to the van’t Hoff
equation, applied for a two state transition from a folded to
unfolded state, assuming that the heat capacity of the folded and
unfolded states are equal.36 The binding stoichiometry was deter-
mined by the continuous variation method (Job plot). Solutions
of hTel and compound 4a were mixed in different proportions
maintaining a total concentration of hTel + 4a of 10 μM.
The mole fractions for DNA/(DNA + NDI) were 0.13, 0.15,
0.21, 0.25, 0.30, 0.34, 0.38, 0.42, 0.50, 0.55. All solutions
For drug uptake experiments, 2.5 × 105 of A549 cells were
seeded in a 6-well plate on a coverslip in 2 ml of medium and
incubated for 24 hours. The next day cells were treated with
compounds and incubated for 30 minutes. The medium was then
removed, cells were washed with PBS and fixed by incubation
with 2% PFA for 20 min at room temperature in the dark. Sub-
sequently, cells were washed 5 times with PBS, the coverslip
mounted on a microscope slide using mounting gel (Vectashield)
and sealed. Slides were visualized using a Leica TCS SP2 confo-
cal microscope with a 63× oil submersion lens, excitation wave-
length 458 nm and emission range 470–600 nm.
Fluorescence analysis
Compound 4a was diluted in 10 mM lithium cacodylate pH 6.0
and 50 mM KCl to a final concentration of 2 μM. hTel DNAwas
This journal is © The Royal Society of Chemistry 2012
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induce folding. The G-4 folded DNA was added at increasing
molar ratios (1, 2, 4 and 8) to the previously prepared solution of
compound 4a. Fluorescence spectra were measured at 25 °C
with a LS55 Fluorescence Spectrometer (PerkinElmer), equipped
with a temperature controller (FA90, FALC). Spectra were
obtained by exciting at 457 nm and read at 465–700 nm with
excitation and emission slides of 5 nm. Data were acquired and
analysed with FLWinLAb and SigmaPlot software.
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This work was supported by MIUR, Grant FIRB-Ideas
RBID082ATK, Grants AIRC 5826 and 8861 (Associazione Itali-
ana per la Ricerca sul Cancro) and by University of Padua and
University of Pavia. We thank Dr L. Mosca (Pavia University)
for the potentiometric titrations.
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