Design, synthesis, biophysical and biological studies of trisubstituted naphthalimides as G-quadruplex ligands

Article abstract of DOI:10.1016/j.bmc.2011.08.062

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)3 binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UVmelting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.

Full text of DOI:10.1016/j.bmc.2011.08.062

Bioorganic & Medicinal Chemistry 19 (2011) 6419–6429
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, biophysical and biological studies of trisubstituted
naphthalimides as G-quadruplex ligands
Antonella Peduto a, , Bruno Pagano a, , Carmen Petronzi a, Antonio Massa b, Veronica Esposito c,
Antonella Virgilio c, Francesco Paduano d, Francesco Trapasso d, Filomena Fiorito e, Salvatore Florio f,
a,
Concetta Giancola g, Aldo Galeone c, , Rosanna Filosa
⇑
⇑
a Dipartimento di Scienze Farmaceutiche e Biomediche, Università di Salerno, Via Ponte don Melillo, 84084 Fisciano (SA), Italy
b Dipartimento di Chimica, Università di Salerno, Via Ponte don Melillo, 84084 Fisciano (SA), Italy
c Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli Federico II, Via D. Montesano, 49 80131 Napoli, Italy
d Dipartimento di Medicina Sperimentale e Clinica, Università Magna Grecia di Catanzaro, Viale Europa, 88100 Catanzaro, Italy
e Dipartimento di Patologia e Sanità Animale, Università degli Studi di Napoli Federico II, Via Delpino 1, 80137 Napoli, Italy
f Dipartimento di Strutture, Funzioni e Tecnologie Biologiche, Università degli Studi di Napoli Federico II, Via Delpino 1, 80137 Napoli, Italy
g Dipartimento di Chimica ‘P. Corradini’, Università degli Studi di Napoli Federico II, Via Cintia, 80126 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 April 2011
Revised 28 July 2011
Accepted 28 August 2011
Available online 1 September 2011
A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadru-
plex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)3 binding
was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex
with compounds showing the best affinity has been studied by isothermal titration calorimetry and UV-
melting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a
2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit tel-
omerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human
lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell
proliferation was observed for all cellular lines during short-term treatment.
Keywords:
Naphthalimides
G-Quadruplex
Telomerase
Cancer
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
researches.3 The key function of telomeres is to protect chromo-
somal termini from exonucleases and ligases, thus preventing deg-
G-Quadruplexes are unique DNA secondary structures based on
the building block called G-quartet (also known as G-tetrad). This
substructure is formed by the association of four guanines into a
cyclic arrangement in which the bases are held together by eight
Hoogsteen hydrogen bonds. Several G-quartets can stack each
other to generate a range of structures (monomolecular, bimolecu-
lar and tetramolecular) exhibiting an extensive structural diversity
and polymorphism compared to duplex DNA. Recent researches
suggest that human genome could contain over 300,000 sequences
potentially able to form G-quadruplexes1 and a number of relevant
regions has been reported to adopt these structures including sev-
eral oncogene promoter regions, telomeric ends, ribosomal DNA,
mini-satellites and the immunoglobulin heavy chain switch region.
For these reasons, the G-quadruplex structures adopted by these
sequences can be regarded as potential new pharmacological tar-
gets.2 Among these, telomeric G-quadruplexes represent particu-
larly appealing targets and they have been the topic of several
radation or end-fusion and maintaining stability.4,5 Several
intramolecular G-quadruplex structures formed by the human
telomeric repetitive DNA sequence (TTAGGG)n have been de-
scribed, depending on the conditions and technique employed.6–9
Potassium-stabilized G-quadruplex structures formed by the telo-
mere substrate inhibit in vitro telomere maintenance by the en-
zyme telomerase.10 Since elevated telomerase activity has been
implicated in ꢀ85% of cancers,11 its inhibition represents an attrac-
tive strategy for the development of selective anticancer drugs.12,13
In fact, G-quadruplex-interactive inhibitors of telomerase may of-
fer a less challenging developmental pathway to the clinic than
antisense agents targeted directly against components of the telo-
merase ribonucleoprotein complex.14,15 The formation and stabil-
ization of G-quadruplex structures at the telomeric ends have
been reported to impede the telomerase association and to in-
crease the genomic instability by hampering the recognition of
telomere-associated proteins with their targets.16,17 Although G-
quadruplex structures can be targeted by several types of organic
small ligands through various binding modes, the development
of most of small molecules as G-quadruplex binders has been lar-
gely based on polycyclic planar aromatic compounds with at least
⇑
Corresponding authors.
E-mail addresses: galeone@unina.it (A. Galeone), rfilosa@unisa.it (R. Filosa).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.062

6420
A. Peduto et al. / Bioorg. Med. Chem. 19 (2011) 6419–6429
one substituent, usually two, terminating in a cationic group.18,19
The rationale for the planar moiety has been that this would stack
effectively onto planar G-quartets, which has been confirmed by
several crystallographic and NMR studies of G-quadruplex–ligand
complexes.20–25 Different structures with these characteristics
have been identified with high affinity and selectivity for the hu-
man telomeric quadruplex.26 However, there is still a growing
interest in the design and screening of newer chemical structures
as potential G-quadruplex ligands, with the aim of developing
more potent and selective molecules. Recently, tri-, tetra- and hep-
tacyclic perylene analogues as DNA telomerase inhibitors have
been reported.27 Among these, the monosubstituted naphthali-
mides showed poor telomerase inhibition properties. However,
the potential of naphthalimido derivatives as effective G-quadru-
plex ligands and their possible therapeutic properties have not
exhaustively investigated. In the present paper, we describe the
design and the synthesis of several trisubstituted naphthalimides
and their ability to interact with a telomeric quadruplex, namely
AGGG(TTAGGG)3.9 The evaluation of the capacity of these com-
pounds to inhibit telomerase and their activity against human can-
cer cells are reported, as well.
2.1.2.2. 5,8-Diamino-2-(2-(pyrrolidin-1-il)ethyl)-1H-benzo[de]
isoquinoline-1,3(2H)-dione (5).
Compound 5 was obtained by precipitation from reaction mix-
ture (yield: 93%). 1H NMR (CDCl3) 1.90 (br, 4H); 2.73 (br, 4H); 2.98
(t, 2H, J = 7.45 Hz); 4.15 (s, 2NH2); 4.41 (t, 2H, J = 7.45 Hz); 7.15 (d,
2H, J = 2.19 Hz); 7.86 (d, 2H, J = 2.19 Hz).
2.1.2.3. 5,8-Diamino-2-(2-(piperidin-1-yl)ethyl)-1H-benzo[de]
isoquinoline-1,3(2H)-dione (6).
Solvent was evaporated and the residue was chromatographed
on silica gel eluting with CHCl3/MeOH/NH4OH 9.5/0.15/0.25 to give
6 (63%). 1H NMR (CDCl3) 1.52 (br, 6H); 2.52 (br, 4H); 2.73 (t, 2H,
J = 7.45 Hz); 4.12 (s, 2NH2); 4.39 (t, 2H, J = 7.45 Hz); 7.12 (d, 2H,
J = 2.19 Hz); 7.88 (d, 2H, J = 2.19 Hz).
2.1.2.4. 5,8-Diamino-2-(2-morpholinoethyl)-1H-benzo[de]iso-
quinoline-1,3(2H)-dione (7).
Solvent was evaporated and the residue was chromatographed
on silica gel eluting with CHCl3/MeOH/NH4OH 9.5/0.15/0.25 to give
7 (60%). 1H NMR (DMSO-d6) 2.52 (br, 6H); 3.53 (t, 4H, J = 5.92 Hz);
4.16 (t, 2H, J = 7.45 Hz); 5.85 (s, 2NH2); 6.98 (d, 2H, J = 2.19 Hz);
7.56 (d, 2H, J = 2.19 Hz).
2. Materials and methods
2.1. Chemistry
2.1.2.5. 5,8-Diamino-2-(3-(piperazin-1-yl)ethyl)-1H-benzo[de]
isoquinoline-1,3(2H)-dione (8).
Compound 8 was obtained by precipitation from reaction mix-
ture (yield: 75%). 1H NMR (CD3OD) 2.52 (br, 4H); 2.92 (br, 6H); 4.26
(t, 2H, J = 7.45 Hz); 7.12 (d, 2H, J = 2.19 Hz); 7.76 (d, 2H,
J = 2.19 Hz).
All reagents were analytical grade and purchased from Sig-
ma–Aldrich (Milano, Italy). Flash chromatography was performed
on Carlo Erba Silica Gel 60 (230ꢁ400 mesh; Carlo Erba, Milano,
Italy). TLC was carried out using plates coated with Silica Gel
60F 254 nm purchased from Merck (Darmstadt, Germany). 1H
and 13C NMR spectra were registered on a Brucker AC 300.
Chemical shifts are reported in ppm. The abbreviations used
are as follows: s, singlet; d, doublet; dd double doublet; br,
broad signal. MS spectrometry analysis ESI-MS was carried out
on a Finnigan LCQ Deca ion trap instrument. Elemental analysis
was performed by Desert Analytics (Tucson, AZ). Melting points
were determined in open capillary tubes on an Electrothermal
9100 apparatus and are uncorrected.
2.1.2.6.
5,8-Diamino-2-(3-(dimethylamino)propyl)-1H-benzo
[de]isoquinoline-1,3(2H)-dione (9).
Solvent was evaporated and the residue was chromatographed
on silica gel eluting with CHCl3/MeOH/NH4OH 9.5/0.15/0.25 to give
9 (63%). 1H NMR (CDCl3) 1.82–1.93 (m, 2H); 2.24 (s, 6H); 2.47 (t,
2H, J = 7.23 Hz); 4.00 (2NH2); 4.19 (t, 2H, J = 7.23 Hz); 7.02 (d, 2H,
J = 2.19 Hz); 7.76 (d, 2H, J = 2.19 Hz).
2.1.2.7.
5,8-Diamino-2-(3-(pyrrolidin-1-yl)propyl)-1H-benzo
[de]isoquinoline-1,3(2H)-dione (10).
Solvent was evaporated and the residue was chromatographed
on silica gel eluting with CHCl3/MeOH/ NH4OH 9/1/0.5 to give 10
(65%). 1H NMR (CDCl3) 1.88 (br, 4H); 2.00 (m, 2H); 2.57 (br, 4H);
2.66 (t, 2H, J = 7.45 Hz); 4.15 (s, 2NH2); 4.24 (t, 2H, J = 7.45 Hz);
7.15 (d, 2H, J = 2.19 Hz); 7.78 (d, 2H, J = 2.19 Hz).
2.1.1. 3,6-Diamino-1,8-naphthalic anhydride (3)
To
a
solution of 3,6-dinitro-1,8-naphthalic anhydride28
(7.42 g, 25.7 mmol) in 188 mL of DMF/MeOH (4:1) ammonium
formate (15.07 g, 0.231 mol) and 10% palladium charcoal
(0.151 g, 1.42 mmol) were added. The mixture was stirred for
2 h at room temperature. The solid was filtered off and the fil-
trate was concentrated on a rotary evaporator. To the resulting
concentrate, containing DMF, cold water was added. The precip-
itate was filtered and crystallized from DMF/H2O to give 3,6-dia-
mino-1,8-naphthalic anhydride as a yellow powder (5.21 g, 89%).
1H NMR (DMSO-d6) 5.79 (s, 2NH2) 6.97 (d, 2H, J = 2.19 Hz); 7.53
(d, 2H, J = 2.19 Hz).
2.1.2.8. 5,8-Diamino-2-(3-(piperidin-1-yl)propyl)-1H-benzo[de]
isoquinoline-1,3(2H)-dione (11).
Solvent was evaporated and the residue was chromatographed
on silica gel eluting with CHCl3/MeOH/NH4OH 9.5/0.15/0.25 to give
11 (62%). 1H NMR (CDCl3) 1.52 (br, 6H); 1.98 (m, 2H); 2.47 (br, 4H);
2.51 (t, 2H, J = 7.45 Hz); 4.00 (s, 2NH2); 4.21(t, 2H, J = 7.45 Hz); 7.10
(d, 2H, J = 2.19 Hz); 7.78 (d, 2H, J = 2.19 Hz).
2.1.2. General procedures for 4–13
2.1.2.9.
5,8-Diamino-2-(3-morpholinopropyl)-1H-benzo[de]
To a suspension of compound 3 (0.200 g, 0.87 mmol) in toluene
(5 mL), a solution of desired amine (3 equiv) dissolved in 3 mL of
EtOH was added. The reaction was refluxed for 3 h. The mixture
was allowed to cool to room temperature.
isoquinoline-1,3(2H)-dione (12).
Solvent was evaporated and the residue was chromatographed
on silica gel eluting with CHCl3/MeOH/NH4OH 9.5/0.15/0.25 to give
12 (63%). 1H NMR (CDCl3) 1.88 (m, 2H); 2.48 (br, 4H); 2.50 (t, 2H,
J = 7.45 Hz); 3.53 (t, 4H, J = 5.92 Hz); 3.98 (s, 2NH2); 4.12 (t, 2H,
J = 7.45 Hz); 6.98 (d, 2H, J = 2.19 Hz); 7.62 (d, 2H, J = 2.19 Hz).
2.1.2.1.
5,8-Diamino-2-(2-(dimethylamino)ethyl)-1H-benzo
[de]isoquinoline-1,3(2H)-dione (4).
Compound 4 was obtained by precipitation from reaction mix-
ture (yield: 67%). 1H NMR (DMSO-d6): 2.22 (s, 6H); 2.52 (t, 2H,
J = 7.23 Hz); 4.11 (t, 2H, J = 7.23 Hz); 5.65 (s, 2NH2); 6.98 (d, 2H,
J = 2.19 Hz); 7.56 (d, 2H, J = 2.19 Hz).
2.1.2.10. tert-Butyl-4-(3-(5,8-diamino-1,3-dioxo-1H-benzo[de]
isoquinolin-2(3H)-yl)propyl)piperazine-1-carboxylate (13).
Solvent was evaporated and the residue was chromatographed
on silica gel eluting with CHCl3/MeOH/NH4OH 9.5/0.25/0.15 to give

A. Peduto et al. / Bioorg. Med. Chem. 19 (2011) 6419–6429
6421
13 (80%). 1H NMR (CDCl3) 1.56 (s, 9H); 1.93 (br, 2H); 2.42 (br, 4H);
2.53 (t, 2H, J = 7.2 Hz); 3.33 (br, 4H); 4.02 (s, 2H); 4.21(t, 2H,
J = 7.2 Hz); 7.12 (d, 2H, J = 2.19 Hz); 7.76 (d, 2H, J = 2.19 Hz).
2H, J = 7.45 Hz); 3.88 (t, 4H, J = 6.03 Hz); 4.12 (t, 2H, J = 7.45 Hz);
8.72 (d, 4H, J = 2.19 Hz); 10.78 (s, 2H).
2.1.3.9.
N,N0-(2-(3-Morpholinopropyl)-1,3-dioxo-2,3-dihydro-
2.1.3. General procedures for 14–23
1H-benzo[de]isoquinoline-5,8-diyl)bis(3-chloropropanamide)
(22).
A solution of compounds 4–13 (0.150 g) in 3-chloropropionyl
chloride (20 equiv) was refluxed for 2 h, cooled to 0 °C, filtered,
and washed with ether. Recrystallization from ether gave the pro-
pionamides 14–23.
Yield: 87%. 1H NMR (DMSO-d6) 2.02 (m, 2H); 2.92 (t, 4H,
J = 6.03 Hz); 3.02 (m, 2H); .3.22 (m, 2H); 3.41 (m, 2H); 3.78 (t,
4H, J = 5.45 Hz); 3.97 (t, 4H, J = 6.03 Hz); 4.12 (t, 2H, J = 7.45 Hz);
8.62 (d, 4H, J = 2.19 Hz); 10.78 (s, 2H).
2.1.3.1. N,N0-(2-(2-(Dimethylamino)ethyl)-1,3-dioxo-2,3-dihy-
dro-1H-benzo[de]isoquinoline-5,8-diyl)bis(3-chloropropana-
mide) (14).
2.1.3.10. N,N0-(2-(3-(4-(3-Chloropropanoyl)piperazin-1-yl)pro-
pyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5,8-diyl)-
bis(3-chloropropanamide) (23).
Yield: 93%. 1H NMR (DMSO-d6) 2.22 (s, 6H); 3.15 (t, 4H.
J = 6.03 Hz); 3.52 (t, 2H, J = 7.45 Hz); 4.11 (t, 4H, J = 6.03 Hz); 4.52
(t, 2H, J = 7.45 Hz); 8.63(d, 4H, J = 2.19 Hz); 10.81 (s, 2H).
Yield: 98%. 1H NMR (DMSO-d6) 2.10 (m, 2H); 2.80 (t, 2H,
J = 7.45 Hz); 3.00 (t, 4H, J = 6.03 Hz); 3.80 (t, 2H J = 7.45 Hz);3.78
(br, 2H); 3.88 (t, 4H, J = 6.03 Hz); 4.42 (br, 2H); 8.72 (s, 4H);
10.78 (s, 2H).
2.1.3.2.
N,N0-(1,3-Dioxo-2-(2-(pyrrolidin-1-yl)ethyl)-2,3-dihy-
dro-1H-benzo[de]isoquinoline-5,8-diyl)bis(3-chloropropana-
mide) (15).
2.1.4. General procedures for I–X
Yield: 96%.1H NMR (DMSO-d6) 1.98 (br, 2H); 2.10 (br, 2H); 3.00
(t, 4H, J = 6.03 Hz); 3.11 (br, 2H); 3.53 (t, 2H, J = 7.45 Hz); 3.72 (br,
2H); 4.00 (t, 4H, J = 6.03 Hz); 4.49 (t, 2H, J = 7.45 Hz); 8.61 (s, 4H);
10.81 (s, 2H).
To a stirred suspension of 14–23 (0.150 g) in EtOH (5 mL), NaI
(0.5 equiv) and pyrrolidine (8 equiv) were added. The mixture
was stirred at reflux for 3 h and allowed to cool to room
temperature.
2.1.3.3. N,N0-(1,3-Dioxo-2-(2-(piperidin-1-yl)ethyl)-2,3-dihydro-
1H-benzo[de]isoquinoline-5,8-diyl)bis(3-chloropropanamide)
(16).
2.1.4.1. N,N0-(2-(2-(Dimethylamino)ethyl)-1,3-dioxo-2,3-dihy-
dro-1H-benzo[de]isoquinoline-5,8-diyl)bis(3-(pyrrolidin-1-
yl)propanamide) (I).
Yield: 96%. 1H NMR (DMSO-d6) 1.52 (br, 2H); 2.12 (br, 2H); 2.98
(br, 2H); 3.00 (t, 4H, J = 6.03 Hz); 3.20 (br, 2H); 3.52 (br, 2H); 3.88
(t, 4H, J = 6.03 Hz); 4.12 (br, 2H); 8.72 (s, 4H); 10.78 (s, 2H).
The residue was chromatographed on silica gel using CHCl3/
MeOH/NH4OH 9/1/0.5 as an eluent affording I in 60% yield. Mp
192.8 °C; 1H NMR (CDCl3) 2.00 (br, 8H); 2.40 (s, 6H); 2.67 (br,
6H); 2.81 (br, 8H); 2.97 (t, 4H, J = 6.03 Hz); 4.32 (t, 2H,
J = 7.23 Hz); 6.86 (d, 2H, J = 8.99 Hz); 7.18 (d, 2H, J = 8.99 Hz);
8.12 (d, 2H, J = 2.19 Hz); 8.78 (d, 2H, J = 2.19 Hz); 11.80 (br,
2NH); 13C NMR (CDCl3) 22.1, 24.5, 37.1, 43.8, 49.6, 51.9, 55.4,
119.9, 121.2, 131.8, 135.8, 162.1, 169.9; MS (ESI) m/z: 549.35; Anal.
Calcd for C30H40N6O4: C, 65.67; H, 7.35; N, 15.32; O, 11.66. Found:
C, 65.55; H, 7.41; N, 15.40; O, 11.50.
2.1.3.4. N,N0-(2-(2-Morpholinoethyl)-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinoline-5,8-diyl)bis(3-chloropropanamide) (17).
Yield: 87%. 1H NMR (DMSO-d6) 2.95 (t, 4H, J = 6.03 Hz); 3.02 (m,
2H); 3.22 (m, 2H); 3.41 (m, 2H); 3.97 (t, 4H, J = 6.03 Hz); 4.01 (t,
4H, J = 5.45 Hz); 4.12 (t, 2H, J = 7.45 Hz); 8.62 (d, 4H, J = 2.19 Hz);
10.79 (s, 2H).
2.1.3.5. N,N0-(2-(2-(4-(3-Chloropropanoyl)piperazin-1-yl)ethyl)-
1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5,8-diyl)-
bis(3-chloropropanamide) (18).
2.1.4.2.
N,N0-(1,3-Dioxo-2-(2-(pyrrolidin-1-yl)ethyl)-2,3-dihy-
dro-1H-benzo[de]isoquinoline-5,8-diyl)bis(3-(pyrrolidin-1-
yl)propanamide) (II).
Yield: 97%. 1H NMR (DMSO-d6) 2.80 (t, 2H, J = 7.45 Hz); 3.00 (t,
4H, J = 6.03 Hz); 3.80 (t, 2H, J = 7.45 Hz);3.78 (br, 2H); 3.88 (t, 4H,
J = 6.03 Hz); 4.42 (br, 2H); 8.72 (s, 4H); 10.78 (s, 2H).
The residue was chromatographed on silica gel using CHCl3/
MeOH/NH4OH 9/1/0.5 as an eluent affording II in 80% yield. Mp
192.8 °C; 1H NMR (CDCl3) 2.00 (br, 8H); 2.40 (s, 6H); 2.67 (br,
6H); 2.81 (br, 8H); 2.97 (t, 4H, J = 6.03 Hz); 4.32 (t, 2H,
J = 7.23 Hz); 6.86 (d, 2H, J = 8.99 Hz); 7.18 (d, 2H, J = 8.99 Hz);
8.12 (d, 2H, J = 2.19 Hz); 8.78 (d, 2H, J = 2.19 Hz); 11.80 (br,
2NH); 13C NMR (CDCl3) 24.1, 24.2, 30.5, 39.4, 51.7, 53.8, 54.1,
54.8, 122.2, 123.4, 134.3, 138.7, 164.4, 172.2; MS (ESI) m/z:
549.35; Anal. Calcd for C32H42N6O4: C, 66.88; H, 7.37; N, 14.62;
O, 11.14. Found: C, 66.68; H, 7.44; N, 14.59; O, 11.09.
2.1.3.6. N,N0-(2-(3-(Dimethylamino)propyl)-1,3-dioxo-2,3-dihy-
dro-1H-benzo[de]isoquinoline-5,8-diyl)bis(3-chloropropana-
mide) (19).
Yield: 80%. 1H NMR (DMSO-d6) 2.00 (m, 2H); 2.72 (s, 6H); 2.96
(t, 4H, J = 6.03 Hz); 3.13 (t, 2H, J = 7.45 Hz); 3.98 (t, 4H, J = 6.03 Hz);
4.12 (t, 2H, J = 7.45 Hz); 8.58(d, 4H, J = 2.19 Hz); 10.78 (s, 2H).
2.1.3.7. N,N0-(1,3-Dioxo-2-(3-(pyrrolidin-1-yl)propyl)-2,3-dihy-
dro-1H-benzo[de]isoquinoline-5,8-diyl)bis(3-chloropropana-
mide) (20).
2.1.4.3. N,N0-(1,3-Dioxo-2-(2-(piperidin-1-yl)ethyl)-2,3-dihydro-
1H-benzo[de]isoquinoline-5,8-diyl)bis(3-(pyrrolidin-1-yl)pro-
panamide) (III).
Yield: 94%. 1H NMR (DMSO-d6) 1.82 (m, 2H) 1.98 (m, 2H); 2.10
(m, 2H); 3.00 (t, 4H, J = 6.03 Hz); 3.11 (br, 2H); 3.53 (t, 2H,
J = 7.45 Hz); 3.62 (br, 2H); 3.94 (t, 4H, J = 6.03 Hz); 4.12 (t, 2H,
J = 7.45 Hz); 8.61 (d, 4H, J = 2.19 Hz); 10.83 (s, 2H).
The residue was chromatographed on silica gel using CHCl3/
MeOH/NH4OH 9.5/0.5/0.25 as an eluent affording III in 70% yield.
Mp 181.4 °C; 1H NMR (CDCl3) 1.45 (m, 2H); 1.6 (m, 4H); 2.00 (br,
8H); 2.48 (br, 4H); 2.52 (t, 2H, J = 7.23 Hz); 2.62 (t, 4H,
J = 6.03 Hz); 2.77 (br, 8H); 2.87 (t, 4H, J = 6.03 Hz); 4.18 (t, 2H,
J = 7.23 Hz); 8.12 (d, 2H, J = 2.19 Hz); 8.78 (d, 2H, J = 2.19 Hz);
11.78 (br, 2NH); 13C NMR (CDCl3) 24.3, 24.5, 25.8, 35.6, 40.1,
51.5, 53.5, 55.9, 57.2, 122.3, 123.6, 134.3, 138.6, 164.6, 171.8; MS
(ESI) m/z: 589.36; Anal. Calcd for C33H44N6O4: C, 67.32; H, 7.53;
N, 14.27; O, 10.87. Found: C, 67.20; H, 7.66; N, 14.18; O, 10.91.
2.1.3.8. N,N0-(1,3-Dioxo-2-(3-(piperidin-1-yl)propyl)-2,3-dihy-
dro-1H-benzo[de]isoquinoline-5,8-diyl)bis(3-chloropropana-
mide) (21).
Yield: 98%. 1H NMR (DMSO-d6) 1.52 (m, 2H); 1.95 (m, 2H), 2.12
(m, 2H); 2.94 (m, 2H); 3.02 (t, 4H, J = 6.03 Hz); 3.20 (m, 2H); 3.52 (t,

6422
A. Peduto et al. / Bioorg. Med. Chem. 19 (2011) 6419–6429
2.1.4.4. N,N0-(2-(2-Morpholinoethyl)-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinoline-5,8-diyl)bis(3-(pyrrolidin-1-yl)propana-
mide) (IV).
11.78 (br, 2NH); 13C NMR (CDCl3) 24.5, 24.8, 25.7, 26.4, 35.2,
39.9, 51.8, 53.7, 55.1, 57.2, 122.0, 123.4, 134.2, 138.5, 164.2,
171.6; MS (ESI) m/z: 603.4; Anal. Calcd for C34H46N6O4: C, 67.75;
H, 7.69; N, 13.94; O, 10.62. Found: C, 67.58; H, 7.77; N, 13.89; O,
10.50.
The residue was chromatographed on silica gel using CHCl3/
MeOH/NH4OH 9.5/0.5/0.25 as an eluent affording IV in 77% yield.
Mp 186.0 °C; 1H NMR (CDCl3) 2.00 (br, 8H); 2.48 (br, 4H); 2.51 (t,
2H, J = 7.23 Hz); 2.70 (t, 4H, J = 5.92 Hz,); 2.80 (br, 8H); 3.01 (t,
4H, J = 5.92 Hz); 3.66 (m, 4H); 4.25 (t, 2H, J = 7.23 Hz); 8.18 (d,
2H, J = 2.19 Hz); 8.79 (d, 2H, J = 2.19 Hz); 11.56 (br, 2NH); 13C
NMR (CDCl3) 23.4, 23.5, 32.7, 34.5, 38.5, 41.7, 45.4, 51.1, 52.0,
52.8, 53.1, 53.3, 54.5, 55.7, 121.8, 122.7, 133.6, 138.4, 164.0,
169.9, 171.5; MS (ESI) m/z: 592.66; Anal. Calcd for C33H44N6O5:
C, 65.06; H, 7.17; N, 14.23; O, 13.54. Found: C, 65.19; H, 7.08; N,
14.39; O, 13.70.
2.1.4.9.
N,N0-(2-(3-Morpholinopropyl)-1,3-dioxo-2,3-dihydro-
1H-benzo[de]isoquinoline-5,8-diyl)bis(3-(pyrrolidin-1-yl)pro-
panamide) (IX).
Compound IX was obtained by recrystallization from EtOH
(yield: 82%). Mp 192.0 °C; 1H NMR (CDCl3) 1.98 (m, 2H); 2.00 (br,
8H); 2.48 (br, 4H); 2.51 (t, 2H, J = 7.23 Hz); 2.70 (t, 4H,
J = 5.92 Hz,); 2.80 (br, 8H); 3.01 (t, 4H, J = 5.92 Hz); 3.66 (m, 4H);
4.25 (t, 2H, J = 7.23 Hz); 8.18 (d, 2H, J = 2.19 Hz); 8.78 (d, 2H,
J = 2.19 Hz); 11.55 (br, 2NH); 13C NMR (CDCl3) 23.3, 23.8, 24.9,
32.8, 34.7, 38.8, 41.5, 45.2, 51.3, 51.8, 52.7, 53.0, 53.2, 54.4, 55.9,
121.6, 122.8, 133.7, 138.2. 164.1, 169.8, 171.7; MS (ESI) m/z:
605.4; Anal. Calcd for C33H44N6O5: C, 65.54; H, 7.33; N, 13.90; O,
13.23. Found: C, 65.41; H, 7.18; N, 13.78; O, 13.41.
2.1.4.5. N,N0-(1,3-Dioxo-2-(2-(4-(3-(pyrrolidin-1-yl)propanoyl)
piperazin-1-yl)ethyl)-2,3-dihydro-1H-benzo[de]isoquinoline-
5,8-diyl)bis(3-(pyrrolidin-1-yl)propanamide) (V).
The residue was chromatographed on silica gel using CHCl3/
MeOH/TEA 9.5/1/0.5 as an eluent affording V in 63% yield. Mp
124.0 °C; 1H NMR (CDCl3) 1.81 (br, 4H); 2.00 (br, 8H); 2.50–2.61
(m, 10H); 2.65 (t, 4H, J = 6.03 Hz); 2.70–2.88 (m, 12H); 2.95 (t,
4H, J = 6.03 Hz); 3.47 (br, 2H); 3.62 (br, 2H); 4.32 (t, 2H,
J = 7.23 Hz); 8.17 (d, 2H, J = 2.19 Hz); 8.75 (d, 2H, J = 2.19 Hz);
11.95 (br, 2NH); 13C NMR (CDCl3) 24.6, 24.8, 32.8, 35.1, 38.6,
42.3, 46.5, 52.3, 52.1, 54.2, 55.1, 55.7, 122.0, 122.3, 123.8, 124.2,
135.3, 138.1, 164.3, 170.2, 172.0; MS (ESI) m/z: 715.6; Anal. Calcd
for C40H56N8O5: C, 65.91; H, 7.74; N, 15.37; O, 10.97. Found: C,
66.10; H, 7.58; N, 15.47; O, 10.70.
2.1.4.10. N,N0-(1,3-Dioxo-2-(3-(4-(3-(pyrrolidin-1-yl)propanoyl)
piperazin-1-yl)propyl)-2,3-dihydro-1H-benzo[de]isoquinoline-
5,8-diyl)bis(3-(pyrrolidin-1-yl)propanamide) (X).
The residue was chromatographed on silica gel using CHCl3/
MeOH/NH4OH 9.5/1/0.5 as an eluent affording X in 63% yield. Mp
148.0 °C; 1H NMR (CDCl3) 1.81 (br, 4H); 1.98 (m, 2H); 2.00 (br,
8H); 2.38 (br, 4H); 2.50–2.6 (m, 8H); 2.63 (t, 4H, J = 6.03 Hz);
2.70–2.88 (m, 10H); 2.95 (t, 4H J = 6.03 Hz); 3.47 (br, 2H); 3.62
(br, 2H); 4.32 (t, 2H, J = 7.23 Hz); 8.17 (d, 2H, J = 2.19 Hz); 8.75
(d, 2H, J = 2.19 Hz); 11.95 (br, 2NH); 13C NMR (CDCl3) 24.2, 24.6,
25.2, 32.7, 34.8, 39.2, 51.6, 52.2, 53.1, 53.5, 53.8, 54.5, 56.1,
121.0, 121.8, 122.8, 123.2, 134.0, 138.8, 164.2, 170.3, 172.8; MS
(ESI) m/z: 729.49; Anal. Calcd for C39H54N8O5: C, 65.52; H, 7.61;
N, 15.67; O, 11.19. Found: C, 65.44; H, 7.51; N, 15.78; O, 11.08.
2.1.4.6. N,N0-(2-(3-(Dimethylamino)propyl)-1,3-dioxo-2,3-dihy-
dro-1H-benzo[de]isoquinoline-5,8-diyl)bis(3-(pyrrolidin-1-
yl)propanamide) (VI).
The residue was chromatographed on silica gel using CHCl3/
MeOH/NH4OH 9.5/0.5/0.15 as an eluent affording VI in 80% yield.
Mp 172.0 °C; 1H NMR (CDCl3) 1.98 (m, 2H); 2.02 (br, 8H); 2.43 (s,
6H); 2.61 (br, 6H); 2.82 (br, 8H); 2.97 (t, 4H, J = 6.03 Hz); 4.32 (t,
2H, J = 7.23 Hz); 6.86 (d, 2H, J = 8.99 Hz); 7.18 (d, 2H, J = 8.99 Hz);
8.12 (d, 2H, J = 2.19 Hz); 8.78 (d, 2H, J = 2.19 Hz); 11.78 (br,
2NH); 13C NMR (CDCl3) 22.1, 24.5, 37.1, 43.8, 49.6, 51.9, 55.4,
119.9, 121.2, 131.8, 135.8, 162.1, 169.9; MS (ESI) m/z: 563.33; Anal.
Calcd for C31H42N6O4: C, 66.17; H, 7.52; N, 14.94; O, 11.37. Found:
C, 66.22; H, 7.69; N, 14.77; O, 11.21.
2.2. Preparation of DNA for the biophysical studies
Oligodeoxynucleotides used for biophysical experiments were
synthesized on a Millipore Cyclone Plus DNA synthesizer using so-
lid phase b-cyanoethyl phosphoramidite chemistry at 15 lmol
scale and purified by standard methods. Quadruplex and duplex
samples were prepared by dissolving the lyophilised oligonucleo-
tide in a buffer solution containing 20 mM potassium phosphate,
70 mM KCl, 0.1 mM EDTA, at pH 7.0. The resulting solutions were
heated at 90 °C for 5 min and subsequently allowed to slowly cool
to room temperature. The solutions were then equilibrated for 24 h
at 4 °C. The concentration of oligonucleotides was determined by
UV adsorption measurements at 90 °C using molar extinction coef-
ficient values e(260nm) of 228,500 and 110,700 Mꢂ1 cmꢂ1 for
AGGG(TTAGGG)3 and CGCGAATTCGCG, respectively. The molar
extinction coefficient was calculated by the nearest neighbour
model.29
2.1.4.7. N,N0-(1,3-Dioxo-2-(3-(pyrrolidin-1-yl)propyl)-2,3-dihy-
dro-1H-benzo[de]isoquinoline-5,8-diyl)bis(3-(pyrrolidin-1-
yl)propanamide) (VII).
The residue was chromatographed on silica gel using CHCl3/
MeOH/NH4OH 9.5/0.15/0.25 as an eluent affording VII in 88% yield.
Mp 165.0 °C; 1H NMR (CDCl3) 1.82 (br, 4H); 2.00 (br, 10H); 2.61 (br,
4H); 2.67 (m, 6H); 2.81 (br, 8H); 2.97 (t, 4H, J = 6.03 Hz); 4.32 (t,
2H, J = 7.23 Hz); 8.13 (d, 2H, J = 2.19 Hz); 8.78 (d, 2H, J = 2.19 Hz);
11.78 (br, 2NH); 13C NMR (CDCl3) 23.8, 24.2, 27.9, 35.1, 39.5,
52.2, 53.8, 54.5, 54.6, 122.0, 123.4, 134.2, 138.4, 164.5, 172.0. MS
(ESI) m/z: 589.20; Anal. Calcd for C33H44N6O4: C, 67.32; H, 7.53;
N, 14.27; O, 10.87. Found: C, 67.19; H, 7.44; N, 14.39; O, 10.91.
2.3. Fluorescence titrations
Fluorescence titrations were performed on a Perkin Elmer
LS50B fluorescence spectrometer, using a 1 cm path length quartz
cuvette. The ligands were dissolved in DMSO to produce stock
solutions at 0.001 M (concentration was calculated using their
respective molecular weights) and then diluted into the appropri-
ate amount of aqueous buffer to yield a ligand concentration of
2.1.4.8. N,N0-(1,3-Dioxo-2-(3-(piperidin-1-yl)propyl)-2,3-dihy-
dro-1H-benzo[de]isoquinoline-5,8-diyl)bis(3-(pyrrolidin-1-
yl)propanamide) (VIII).
The residue was chromatographed on silica gel using CHCl3/
MeOH/NH4OH 9.5/0.5/0.25 as an eluent affording VIII in 76% yield.
Mp 178.0 °C; 1H NMR (CDCl3) 1.45 (m, 2H); 1.6 (m, 4H); 1.98 (m,
2H); 2.00 (br, 8H); 2.48 (br, 4H); 2.52 (t, 2H, J = 7.23 Hz); 2.62 (t,
4H, J = 6.03 Hz); 2.77 (br, 8H); 2.87 (t, 4H, J = 6.03 Hz); 4.18 (t,
2H, J = 7.23 Hz); 8.12 (d, 2H, J = 2.19 Hz); 8.78 (d, 2H, J = 2.19 Hz);
10
umes of 5 or 10
100–160 M were added into a ligand solution (500
l
M and a final DMSO concentration of 2%. In each titration, vol-
L of quadruplex solution at a concentration of
L). After each
l
l
l
addition of DNA, the solution was stirred and allowed to equili-
brate for at least 3 min and fluorescence emission spectra were col-

A. Peduto et al. / Bioorg. Med. Chem. 19 (2011) 6419–6429
6423
lected from 400 to 600 nm, at 10 nm excitation and emission slit
widths. Each experiment was performed at least three times, each
spectrum was recorded three times and the average of three scans
time constant of 4 s, a 2 nm bandwidth, and a scan rate of
20 nm minꢂ1 were used to acquire the data.
was taken. The fraction of free ligand (
a) was calculated for each
2.6. UV-melting experiments
sample, following the changes in fluorescence emission maximum
upon quadruplex addition, using the following relationship:
UV-melting experiments were carried out using a Jasco V-530
UV–visible spectrophotometer equipped with a Jasco ETC-505-T
Fmax ꢂ Fb
max
temperature controller. Solutions of quadruplex DNA (25
lM)
a
¼
Ffmax ꢂ Fbmax
and of 1:1 or 2:1 ligand/DNA mixture samples (300 L) were
l
placed in quartz cuvettes of 1 mm path length. UV-melting exper-
iments were performed in a temperature range of 20–85 °C by
monitoring absorbance at 295 nm,31 at a 0.5 °C/min heating rate.
Each experiment was performed at least three times. Thermal
denaturation temperatures (Tm) for the DNA and DNA–ligand com-
plexes were determined using previously reported procedure,32
and represent the mean SD.
where Fmax is the maximum fluorescence intensity observed, Fmf ax
and Fbmax are the respective fluorescence intensities of free ligand
and fully bound ligand, respectively. To obtain the binding con-
stants, the resulting titration curves, obtained by plotting the frac-
tion of free ligand versus quadruplex concentration, were fitted
with the ORIGIN 7.0 program by using the following equation:30
2
3
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢀ
ꢁ ꢀ
ꢁ
ꢀ
ꢁ
2
1
2L0
1
Kb
1
Kb
4
5
a
¼
L0 þ nQ þ
ꢂ
L0 þ nQ þ
ꢂ 4L0nQ
2.7. Enzyme assays. Telomeric repeat amplification protocol
(TRAP assay) on A549 cells
where L0 is the total ligand concentration, Q the added G-quadru-
plex concentration, n the number of binding sites and Kb the bind-
ing constant.
Telomerase activity was evaluated by Telomeric Repeat Ampli-
fication Protocol (TRAP) assay using the TeloTAGG Telomerase PCR
ELISAPLUS (Roche Diagnostics)33 which is an extension of the ori-
ginal method described by Kim et al.34 Cell extract (1–3 ꢃ 105 cell
equivalents), at 72 h post exposure to examined compounds, in dif-
ferent concentrations, were employed in the first step, in which
telomerase adds telomeric repeats (TTAGGG) to the 30-end of the
biotin-labeled synthetic P1-TS primer, according to manufacturer’s
protocol for the TeloTAGGG Telomerase PCR ELISAPLUS kit analysis.
Briefly, reaction mixtures contained telomerase extracts, oligonu-
cleotide primers (P1-TS and P2) for amplification of telomeric re-
peats and internal standard (IS), Taq DNA polymerase (5 units/
2.4. Isothermal titration calorimetry
ITC experiments were performed at 298 K using a high-sensitiv-
ity CSC-5300 Nano-ITC microcalorimeter from Calorimetry Science
Inc. (Lindon, Utah) with a cell volume of 1 ml. Ligand solutions
were prepared by dissolving the powder in the same buffer used
for DNA, and the concentration has been estimated by UV spectros-
copy using the calculated extinction coefficients (Table S1). Mea-
surement from the first injection was discarded from the analysis
of the data, to avoid artifacts due to the diffusion through the injec-
tion port occurring during the equilibration period, locally affect-
ing the quadruplex concentration near the syringe needle tip. In
lL) (Roche), dNTP Mix and TRAP reaction buffer. After 20 min incu-
bation at 25 °C for telomerase extension of the P1-TS primer, the
PCR cycling conditions were 94 °C for 5 min followed by 30 cycles
at 94 °C for 30 s, 50 °C for 30 s and 72 °C for 90 s with final step at
72 °C for 10 min. The PCR products derived from telomerase elon-
gation and internal standard are quantitatively determined using
ELISA assay. The ELISA kit allows the highly specific amplification
of telomerase-mediated elongation products combined with non-
radioactive detection following an ELISA protocol. The levels of tel-
omerase activity were within the linear range of the TRAP assay. A
heat-inactivated cell extract was also tested as a negative control.
Data are presented as mean SEM. The paired Student’s t-test was
used for comparison between control and experimental groups. P
value <0.05 was considered statistically significant.
each titration, volumes of 5–10
tration of 480–640 M were injected into a quadruplex or duplex
solution (30–40 M) every 300 or 400 s up to a total of 25–50
injections, using a computer-controlled 250 L microsyringe, with
lL of ligand solution at a concen-
l
l
l
stirring at 150 rpm. Heat produced by ligands dilution was evalu-
ated by performing ligand dilution experiment, titrating each li-
gand into the buffer alone. The interaction heats were calculated
after correction for the heat of ligand dilution. The corrected heat
values were plotted as a function of the molar ratio, to give the cor-
responding binding isotherms. The resulting isotherms were fitted
with an independent and equivalent-sites model or a multiple-
sites model employing a nonlinear least-squares minimization
algorithm, by using the Bindwork software from Calorimetry Sci-
ence Inc., to give the binding enthalpy (DbH°), binding constant
(Kb) and stoichiometry (n). The Gibbs energy change and the entro-
pic contribution were calculated using the relationships
2.8. Biological evaluation-cell growth inhibition and toxicity
assays
M14 human melanoma and A549 human lung cancer cell lines
were maintained in logarithmic growth phase at 37 °C in a 5% CO2
humidified atmosphere in RPMI-1640 medium containing 10% fe-
DbG° = ꢂRTlnKb
DbS° = DbH° ꢂ DbG°. Each ITC experiment was performed at least
three times.
(R = 8.314 J molꢂ1 Kꢂ1
,
T = 298 K)
and
T
tal calf serum, 2 mM of L-glutamine and 0.1 mg/ml gentamycin.
Compounds, synthesized as previously described, were reconsti-
tuted in sterile DMSO and then diluted with sterile water to the de-
sired concentrations, immediately before each experiment. To
evaluate the toxic profiles of the potential antitelomerasic com-
pounds, a colorimetric assay was used as described by Mosmann.35
The assay is based on the tetrazolium salt 3-(4,5-dimethylthiazol-
yl-2)-2,5-diphenyltetrazoliumbromide (MTT), (Sigma, Milan, Italy)
a pale yellow substrate that is cleaved by active mitochondria to
produce a dark blue formazan product. MTT assays were per-
formed as follows: cells were plated in 96-well plates at 1000–
3000 cells/well and allowed to attach for 24 h. Cells were exposed
2.5. Circular dichroism
CD spectra were recorded with a Jasco J-715 spectropolarimeter
equipped with a Peltier-type temperature control system (model
PTC-348WI) and calibrated with an aqueous solution of 0.06% d-
10-(1)-camphorsulfonic acid at 290 nm. CD spectra of quadruplex
DNA solutions (40 lM) and of solutions containing DNA in the
presence of an excess of ligand were recorded between 220 and
340 nm, using 1 mm path-length cuvettes and were averaged over
three scans. Buffer baseline was subtracted from each spectrum. A
to 0.1–100 lM of drugs at 37 °C for 5 days. In each experiment con-

6424
A. Peduto et al. / Bioorg. Med. Chem. 19 (2011) 6419–6429
trol samples were run with 0.2% DMSO. At the end of this period,
MTT was added to a final concentration of 50 g/ L, and four addi-
tional hours of incubation were performed. After that, medium was
aspirated carefully and 100 L of acidified isopropanol was added
pounds have capability for binding to quadruplex DNA and that
compounds VII and VIII show the best affinity for the quadruplex
(Table 1). Our experimental results suggest that the length of the
chain seems to be important for quadruplex-binding ability of
these drugs, indicating the derivatives with propyl chains as the
ones with more affinity for the quadruplex. Fluorescence titration
data showed that the binding to the quadruplex structure AGGG(T-
TAGGG)3 varied over a fivefold range between the strongest com-
l
l
l
per well. Soluble formazan salts were homogenized by manual
pipetting and absorbance at 570 nm was read. Each experimental
point was run eight times. The results were expresses as the absor-
bance values of treated samples compared with those of controls.
The in vitro activity of the drugs was expressed in terms of concen-
tration able to inhibit cell proliferation by 50% (IC50). IC50 values
were determined graphically from the growth inhibition curves
obtained after 5 days of exposure of the cells to each drug. Data
represent mean values SD of three independent experiments.
The same procedure applied in the case of A549 and M14 cell lines
was conducted for normal fibroblasts NIH3T3 (mouse embryo
fibroblast), except for using bovine calf serum (BCS) instead of FBS.
pound VII (Kb = 30 ꢃ 105 Mꢂ1
)
and weakest compound
I
(Kb = 6 ꢃ 105 Mꢂ1). The results of the interpolation analysis indi-
cate that the data concerning the compounds VII and VIII could
not be fitted with a single binding site, but they are well fitted with
two independent binding sites per quadruplex molecule.
In order to obtain a complete thermodynamic characterization
of the interaction between the best naphthalimido derivatives
(compounds VII and VIII) and human telomeric quadruplex, and
to evaluate the individual binding constants, a calorimetric analy-
sis has been carried out by means of isothermal titration experi-
3. Results and discussion
3.1. Chemistry
ments. Indeed, ITC is
a
high-accuracy method for the
measurement of thermodynamic parameters in biological interac-
tions and it is the only technique capable of quantifying both
enthalpic and entropic components of the interaction, revealing
the overall nature of the forces that drive the molecular recogni-
tion.40 ITC has been used to characterize the energetics of several
drug–quadruplex interactions,41–44 and it has been successfully ap-
plied to extract the individual binding constants when the binding
process comprises two sequential events resulting in the formation
of a complex.45,46
Synthesis of the trifunctionalized derivatives I–X (Scheme 1)
was accomplished using procedures described in Scheme 2. The
key intermediate, 3,6-diamino-1,8-naphthalic anhydride (3), was
obtained from 3-nitro-1,8-naphthalic anhydride (1), which was
subjected to nitration followed by reduction of both nitro groups
to obtain intermediate 3. Intermediate 3 was treated with the
opportune amines to provide naphthalimides 4–13. Amines 3-
(piperidin-1-yl)propan-1-amine and tert-butyl 4-(3-aminopro-
pyl)piperazine-1-carboxylate, were prepared according to the pro-
cedure described in the literature.36,37 The remaining amines were
commercially available. Acylation of diamines 4–13 by 3-chloro-
propionyl chloride and subsequent aminolysis by reflux treatment
with pyrrolidine, in the presence of NaI as catalyst, gave the de-
sired derivatives I–X. Compounds were obtained in good yields
and were adjudged pure by elemental analysis, mass spectrometry,
1H and 13C NMR.
It should be underlined that all tested compounds have side
chains bearing pyrrolidine rings (pKa ꢄ11), that can be considered
totally charged in the experimental conditions we adopted in titra-
tion experiments (pH 7.0) (see below). Similar considerations can
be made for side chains containing piperidine rings (III and VIII)
and dimethylamino moieties (I and VI) for which a comparable
pKa could be envisaged. On the other hand, for compounds charac-
terized by side chains containing a morpholine ring (IV and IX), the
expected pKa (about 8) provide about 90% of charged chains.
To gain a deeper understanding of the quadruplex-binding
behaviour of these analogues, a direct comparison with the binding
behaviour of compound IX was performed. Compound IX was se-
lected for the successive ITC study because it belongs to the series
with propyl side-chain moieties, as compounds VII and VIII that
showed the highest binding constants. Examples of the raw ITC
and integrated heat data for the titration of human telomeric quad-
ruplex with compounds VII, VIII and IX are shown in Figure 2. The
raw ITC data (insets in Fig. 2) indicate exothermic interactions. In
all cases, with each injection of ligand, less and less heat release
was observed until constant values were obtained, reflecting a sat-
urable process. The normalized heat binding curves of compounds
VII and VIII (Fig. 2, panels A and B) show biphasic profiles, with
two separate binding events, well fitted using a multiple-sites
model. In the first event, one molecule of ligand interacts with
the quadruplex structure, followed by the binding of a further li-
gand molecule, allowing the formation of a final complex com-
posed of one molecule of quadruplex and two molecules of
ligand. The analysis suggests that the first binding event
(Kb = 2 ꢃ 107 Mꢂ1 and 5 ꢃ 107 Mꢂ1 for VII and VIII, respectively)
is about two order of magnitude stronger than the second one
(Kb = 5 ꢃ 105 Mꢂ1 and 6 ꢃ 105 Mꢂ1 for VII and VIII, respectively).
Considerably different results were obtained for the interaction
of compound IX. Indeed, the binding isotherm of compound IX
(Fig. 2, panel C) shows a simpler profile compared to the ones of
VII and VIII, with a single sigmoidal curve centered at 1:1 stoichi-
ometry. The binding curve obtained is, in this case, well fitted using
an independent and equivalent-sites model. The analysis of the
binding isotherm of IX suggests that the affinity of this compound
for the quadruplex (Kb = 4 ꢃ 105 Mꢂ1) is comparable with the one
measured for the second binding event of VII and VIII.
3.2. Biophysical analysis
Fluorescence emission spectra of naphthalimido derivatives in
the absence and presence of increasing amounts of quadruplex
were recorded in order to screen such collection of molecules for
their quadruplex affinity. Fluorescence titration experiments were
performed with the quadruplex-forming human telomeric se-
quence AGGG(TTAGGG)3, already used in similar investiga-
tions,30,38 under experimental conditions highly favorable to
quadruplex formation (70 mM KCl),39 arguing against binding to
the single-stranded form of oligonucleotide. Naphthalimido deriv-
atives show fluorescence quenching when binding to quadruplex
DNA. The binding constants were determined from the variation
of fluorescence properties. The binding curves for all the systems
were obtained by plotting the fraction of free ligand versus quad-
ruplex concentration (Fig. 1). The resulting titration curves were
fitted using the independent and equivalent-sites model (see Sec-
tion 2). Fluorescence titrations showed that eight out of ten com-
The values of Kb and DbH° derived by ITC enable us to complete
the thermodynamic characterization by calculating corresponding
values of TDbS° and DbG°. The resulting thermodynamic parame-
ters are showed in Table 2. In the case of VII and VIII, the thermo-
dynamic profiles of the ligand–quadruplex interactions are
qualitatively similar. Inspection of the data reveals that the two
binding events of VII and VIII are driven by both enthalpic and

A. Peduto et al. / Bioorg. Med. Chem. 19 (2011) 6419–6429
6425
R
O
N
O
O
O
N
NH
NH
N
I-X
compound
VI
R
compound
I
R
N
N
N
II
N
VII
III
N
VIII
N
N
IV
V
N
IX
X
O
O
N
N
N
N
N
N
O
O
Scheme 1. Chemical structures of compounds I–X.
R
N
R
N
R
N
O
O
3
O
O
O
O
O
2
O
O
O
O
O
O
O
1
O
c
d
a
b
e
O
O
O
O
R-NH2
NO2
H2N
NH2
H2N
NH2 Cl
N
H
N
Cl
O2
N
N
N
H
N
H
N
NO2
H
4-13
14-23
I-X
X
O
N
*
N
*
N
*
N
*
N
R:
*
N
n
n
n
n
n
4,14 n=2
5,15 n=2
6,16
n=2
7,17
n=2
8,18
9,19 n=3
n=2, X=H
10,20
n=3
11,21 n=3
12,22 n=3
13,23 n=3, X=Boc
Scheme 2. Reagents and conditions: (a) H2SO4/HNO3, from ꢂ10 °C to 55 °C, 1 h; (b) HCOONH4, Pd(C), DMF/MeOH, rt, 2 h; (c) toluene/EtOH,
chloride, , 2 h; (e) pyrrolidine, NaI, EtOH, , 2.5 h.
D, 2 h; (d) 3-chloropropionyl
D
D
entropic contributions, as well as the single binding event of com-
pound IX. However, the enthalpic component for the binding of IX
is more favourable than the other two ligands, probably because
the oxygen atom of the morpholinopropyl side-chain is able to gain
an additional H-bond with DNA. This could lead to the formation of
a more rigid complex with respect to the others, consistent with
the low entropic contribution observed by ITC.
The ITC data confirm that VII and VIII interact with quadruplex
DNA more avidly than IX. Inspection of Tables 1 and 2 shows that
the Kb values determined from the fluorescence and ITC data differ,
despite the use of identical salt and buffer conditions. To explain
those apparent discrepancies, some considerations need to be
done. A binding constant can be accurately determined when the
titrant, for example, a ligand, is added to a fixed and constant con-
centration of DNA (or vice versa), such that this concentration is in
the range of 1/Kb. In fluorescence titrations, the concentration used
(10
l
M) is closer to 1/Kb (about 1
lM) than the one used in ITC
(30–40
lM). Thus, in the case of IX (1:1 stoichiometry), we should
consider the spectroscopic binding constant more accurate than
the calorimetric one. On the other hand, in the case of VII and VIII
(2:1 stoichiometry), thanks to ITC we were able to extract the indi-
vidual binding constants associated to each binding event,
whereas, by fluorescence we observe almost certainly an average
of the two constants.
To investigate the selectivity of compounds VII and VIII, the
binding of these compounds to duplex DNA was preliminary eval-

6426
A. Peduto et al. / Bioorg. Med. Chem. 19 (2011) 6419–6429
Figure 1. Example of fluorescence titration curve for compound VII (10
obtained by plotting the fraction of free ligand as function of quadruplex
concentration ( M). The experimental data (circles) are fitted using an independent
lM),
a
l
and equivalent-sites model (solid line) to obtain the binding constants. All the
titration experiments were performed at 25 °C in buffer solution containing 20 mM
potassium phosphate, 70 mM KCl, 0.1 mM EDTA, at pH 7.0.
Table 1
Binding constants for the interaction of naphtalimido derivatives with quadruplex
AGGG(TTAGGG)3 determined by fluorescence titrations
Compound
Kb ꢃ 105 (Mꢂ1
)
Compound
Kb ꢃ 105 (Mꢂ1
)
I
6
12
9
nd
7
1
1
1
VI
nd
II
III
IV
V
2
VII
VIII
IX
30
24
10
9
3
3
1
X
1
nd = not detected.
uated by UV titration experiments with the genomic STDNA, which
was selected as it presents 42% of GC bases and it is commonly re-
garded as a reference duplex. UV studies have indicated a low
affinity of VII and VIII for duplex structure (data not shown), sug-
gesting that the two compounds have preference for the quadru-
plex structure over the duplex.
Additionally, in order to support these results, we evaluated the
affinity of VII for the duplex model formed by the Dickerson se-
quence CGCGAATTCGCG by ITC (Fig. S1). The binding constant ob-
tained (5 ꢃ 105 Mꢂ1, Table S2) is about two orders of magnitude
lower than for AGGG(TTAGGG)3 quadruplex, thus confirming the
preference for the quadruplex structure.
Furthermore, CD experiments were carried out to evaluate if the
structure of the quadruplex is maintained upon ligands addition.
The investigated quadruplex-forming sequence shows the typical
CD spectrum of the hybrid conformation (Fig. 3), with a maximum
around 290 nm, a shoulder centered around 270 nm and a weak
minimum around 240 nm.9 The addition of VII, VIII or IX causes
the increase of the shoulder at 270 nm in the CD spectra, thus sug-
gesting that the structure of the quadruplex is maintained upon li-
gands addition, although their interaction could promote some
conformational changes in the quadruplex DNA structure.
In order to test the ability of the ligands to stabilize quadruplex
upon binding, thermal denaturation experiments were performed,
as well. Since quadruplex structure melts with a characteristic
hypochromic shift at 295 nm,31 UV-melting experiments were per-
formed by recording the absorbance at that wavelength. The
experiments were carried out at two different ligand/DNA ratio
(1:1 and 2:1). All the UV-melting profiles were consistent with
Figure 2. Raw ITC data (insets) and binding isotherms for titration of AGGG(T-
TAGGG)3 quadruplex with compounds VII (A), VIII (B) and IX (C), obtained at 25 °C
in a buffer solution containing 20 mM potassium phosphate, 70 mM KCl, 0.1 mM
EDTA, at pH 7.0. The circles represent the experimental data obtained by integrating
the raw ITC data and subtracting the heat of ligand dilution into the buffer. The
line represents the best fit obtained by a nonlinear least-squares procedure based
on a multiple-sites model (A and B) and on an independent and equivalent-sites
model (C).
the G-quadruplex folding (Fig. S2). Relative stabilities of the quad-
ruplexes upon ligand binding are reported in Table 3. In all the
cases, the addiction of one equivalent of ligand to quadruplex
DNA solutions leads to Tm values higher than the uncomplexed
quadruplex (DTm = 3 °C). In the cases of VII and VIII, the addition
of two equivalents of ligand leads to a further enhancement of
Tm values (62 and 63 °C, respectively). On the other hand, in the
case of IX the Tm value at 2:1 ligand/DNA ratio is indistinguishable

A. Peduto et al. / Bioorg. Med. Chem. 19 (2011) 6419–6429
6427
Table 2
Thermodynamic parameters for the interaction of naphtalimido derivatives with AGGG(TTAGGG)3 quadruplex determined by ITC at 25 °C and pH 7.0a
Compd
First binding event
Second binding event
DbGꢅ
(kJ molꢂ1
)
DbGꢅ
(kJ molꢂ1
)
298K
b
b
n1
Kb (Mꢂ1
)
DbH° (kJ molꢂ1
)
ꢂTDbS° (kJ molꢂ1
)
n2
Kb (Mꢂ1
)
DbH° (kJ molꢂ1
)
ꢂTDbS° (kJ molꢂ1
)
298K
VII
VIII
IX
1
1
1
2 ꢃ 107
5 ꢃ 107
4 ꢃ 105
ꢂ19
ꢂ18
ꢂ23
ꢂ23
ꢂ26
ꢂ9
ꢂ42
ꢂ44
ꢂ32
1
1
5 ꢃ 105
6 ꢃ 105
ꢂ6
ꢂ6
ꢂ26
ꢂ27
ꢂ32
ꢂ33
a
The experimental error for each thermodynamic parameter is <8%.
n1 and n2 are referred to the number of ligand molecules interacting in each binding event; the final stoichiometry of the complexes is ntot = n1 + n2.
b
3.3. Evaluation of TRAP-inhibitory activity
Compounds VII, VIII and IX, were also tested as telomerase
inhibitors.47–49 As shown in Figure 4, after 72 h of exposure, we ob-
tained a significant decrease of telomerase activity (black bars) for
all the compounds tested in a dose-dependent manner, with an
appreciable selectivity comparing to Taq polymerase inhibition
(white bars) calculated from internal standard level amplification
quantification for compounds VII and VIII. In particular, in the
presence of 5
of 50%. In the presence of 5
reduced of 26%. According to the results shown in Figure 4 we ob-
served that in the range of concentrations 0.1–50 M, compound
lM of compound VII, we detected reducing values
l
M of VIII telomerase activity has been
l
VII decreased the activity of telomerase with an appreciable selec-
tivity comparing to Taq polymerase inhibition. At higher concen-
trations of compound VIII (10–100 lM) the inhibition of Taq
polymerase is also observed with a loss in selectivity. For com-
pound IX no selectivity between telomerase and Taq polimerase
inhibition has been detected. These results are consistent with
the studies about the interaction drug/quadruplex in which com-
pounds VII and VIII showed the best binding constants towards
telomeric quadruplex.
Figure 3. CD spectra of AGGG(TTAGGG)3 quadruplex-forming sequence before
(black line) and after the addition of an excess of VII (red line), VIII (blue line) and
IX (green line). All the spectra were recorded at 25 °C in a buffer solution containing
20 mM potassium phosphate, 70 mM KCl, 0.1 mM EDTA, at pH 7.0.
from the one at 1:1 ratio (Tm = 60 °C). Our thermal denaturation re-
sults confirm that two molecules of VII and VIII bind and stabilize
the human telomeric quadruplex structure, whereas, in the case of
IX, just one molecule interacts with the investigated quadruplex.
In summary, fluorescence titration experiments allowed us to
screen a collection of naphthalimido derivatives for binding affinity
to the quadruplex structure formed by the human telomeric se-
quence. The combination of spectroscopic and calorimetric results
enabled us to shed light on the recognition processes between the
quadruplex and the derivatives showing the higher affinity. ITC
experiments showed that, from a thermodynamic point of view,
the interactions of the best naphthalimido derivatives with the
quadruplex are strongly favoured and that the final stoichiometry
of the complexes is 2:1 (ligand/DNA). Interestingly, ITC experi-
ments reveal that the two molecules of ligand do not simulta-
neously bind the quadruplex structure, but the interaction is
sequential and composed by two distinct binding events. CD and
UV experiments revealed that the structure of the quadruplex is
preserved upon binding and that the drugs are also able to induce
quadruplex stabilization.
3.4. Cell cytotoxicity
Antiproliferative experiments were carried out in order to get
some insights on the in vitro properties of the best binding com-
pounds, namely VII and VIII, and compound IX, as well, against
the lung cancer A549 and the human melanoma M14 cell lines,
when these were exposed to different drug concentrations (from
0.1 to 100 lM) for 5 days. We have selected these two cell lines be-
cause they are the most widely studied cells among non-small cell
lung carcinoma and melanoma panels, respectively. Moreover,
M14 Melanoma cells are still a valuable model for investigating
cancer metastasis.50 A marked dose-dependent inhibition of cell
growth was consistently observed in both cell lines. The detailed
profile of the inhibition of these cell lines by our compounds is re-
ported in Figure 5.
The aim of this experiment was to evaluate cell growth as a
function of drug concentration and to calculate the IC50 values as
detailed in the experimental section. The obtained results are sum-
marized in Table 4, with IC50 values ranging from 1.5 to 34.7
lM in
M14 melanoma cells and from 5.15 to 40.3 M in A549 lung cancer
l
Table 3
cells. Generally, the in vitro experiments revealed a good activity of
tested compounds. In particular, M14 melanoma cells appeared to
be more sensitive to the growth inhibition by tested compounds
than A549 lung cancer cells. The most interesting result was re-
lated to compound VII, which was found to be the best inhibitor
of this series against both M14 and A549 cell lines with IC50 of
1.5 0.7 and 5.15 0.91, respectively. Moreover, selected com-
pounds were tested against NIH3T3 normal fibroblasts in order
to determine their selectivity for cancer cells compared with nor-
mal cells. Compounds VII and VIII showed higher selectivity for
A549 and M14 cells than for NIH3T3 fibroblasts. On the other hand,
compound IX, showing a better affinity towards DNA duplex, is
Melting temperatures for AGGG(TTAGGG)3 quadruplex-
forming sequence (TQ) and 1:1 or 1:2 DNA/ligand
mixture samples calculated from UV-melting curves
recorded at 295 nm
Sample
Tm (°C) ( 0.2)
TQ
57.0
60.0
63.0
60.0
62.0
60.0
60.0
TQ + VII (1:1)
TQ + VII (1:2)
TQ + VIII (1:1)
TQ + VIII (1:2)
TQ + IX (1:1)
TQ + IX (1:2)

6428
A. Peduto et al. / Bioorg. Med. Chem. 19 (2011) 6419–6429
Table 4
400
350
300
250
200
150
100
50
Telomerase activity
A
In vitro results of selected compounds against melanoma (M14), lung (A549) cancer
cell lines and normal fibroblasts (NIH3T3) expressed as IC50 in M.
l
Taq polymerase activity
Cpd
M14 (
l
M + S.D.)
A549 (
l
M + S.D.)
NIH3T3 (
NOT ACTIVE
43.3 4.3
4.6 0.1
lM + S.D.)
VII
VIII
IX
1.5 0.7
8.0 0.8
34.7 0.6
5.2 0.9
9.9 1.8
40.3 1.6
0
Control 0.05
0.1
0.5
1
5
10
50
100
meric binding agents. An initial screening, performed by
fluorescence titration experiments involving the telomeric quadru-
plex AGGG(TTAGGG)3, has allowed us to select two compounds
showing the best affinities (VII and VIII), to be further investigated.
Isothermal titration calorimetry measurements strongly suggest
that VII and VIII are able to bind the telomeric quadruplex with
a drug/DNA ratio 2:1. In both cases the association constants for
the 1:1 complexes are two orders of magnitude higher than those
for the 2:1 complexes. On the other hand, data for derivative IX
point only to the formation of a 1:1 drug/DNA complex character-
ized by an association constant two order of magnitude lower com-
pared to VII and VIII. Interestingly, the interaction stoichiometry of
compounds VII and VIII with the telomeric quadruplex AGGG(T-
TAGGG)3 is similar to that observed for drug BRACO-1951 that
was shown to inhibit tumour growth consistently with telomere
targeting.52 Derivatives VII and VIII have been further evaluated
for their ability to inhibit telomerase by a TRAP assay. Particularly,
compound VII comes out more active and selective than the other
ones. Described results have encouraged us to estimate their cell
toxicity against the lung cancer A549 and the human melanoma
M14 cell lines. Compound VII shows the best activity for both cell
lines and is not toxic for normal cell line. These results are in good
agreement with data collected both from physico-chemical mea-
surements and TRAP assays. Recently, the properties of a series
of tetrasubstituted naphthalene diimide derivatives has been re-
ported,53 that have been shown to be more active than compound
VII against the lung cancer A549 cell lines. However it should be
noted that these compounds are characterized by four substituents
and side chains different from those ones introduced in our com-
pounds. It is our intention take into consideration these results in
designing new derivatives. In conclusion, our results show that tri-
substituted naphthalimido derivatives are a promising class of li-
gands able to bind telomeric G-quadruplex and to remarkably
inhibit telomerase, thus supporting the potential of this class of
quadruplex-interactive compounds for use in anticancer ap-
proaches. On the basis of these findings molecular modelling will
be envisaged towards the development of drugs characterized by
higher affinities to telomeric G-quadruplex structures and en-
dowed by higher antiproliferative properties. As a matter of fact,
the described synthetic approach for their preparation is quite ver-
400
350
300
250
200
150
100
50
Conc. compound VII (µM)
B
0
Control 0.05
0.1
0.5
1
5
10
50
100
Conc. compound VIII (µM)
400
350
300
250
200
150
100
50
C
0
Control 0.05
0.1
0.5
1
5
10
50
100
Conc. compound IX (µM)
Figure 4. Effect of the increasing concentrations of compounds VII (A), VIII (B) and
IX (C) on the human telomerase activity, represented by the absorbance measure-
ments following the ELISA protocol in TRAP assay. TRAP assay was performed on
the extract of A549 cells exposed for 72 h to the examined compounds. All
experiments were performed in triplicates, and error bars show the standard error
of the mean values ( SEM). Black bars, telomerase activity; white bars, Taq
polymerase activity.
toxic for NIH3T3 fibroblasts. So it can be concluded that derivatives
VII and VIII are promising leads for design of highly potent and
highly selective anti-proliferative agent for treatment of melanoma
and lung cancer.
4. Conclusions
G-Quadruplex ligands based on the trisubstituted naphthalimi-
do aromatic nucleus have been synthesized and evaluated as telo-
Figure 5. Dose–response curves obtained from M14 cells (A), A549 (B) and NIH3T3 normal fibroblasts (C) exposed to VII (ꢀ), VIII (N) and IX (j) for 5 days. Points represent
mean values SD of three independent experiments.

A. Peduto et al. / Bioorg. Med. Chem. 19 (2011) 6419–6429
24. Parkinson, G. N.; Cuenca, F.; Neidle, S. J. Mol. Biol. 2008, 381, 1145.
6429
satile to allow the synthesis of new series of derivatives character-
ized by different lateral chains. These compounds can be regarded
as a further class of G-quadruplex ligands providing proof of con-
cept for the inhibition of telomerase in anticancer therapy. How-
ever, our interest is also focused on the interaction of these
compounds with other type of biologically important quadruplex
structures formed by human genetic regions as c-myc, c-kit and
bcl-2.26 At the moment, these investigations are in progress in
our laboratories.
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