Synthesis and study of antiproliferative, antitopoisomerase II, DNA-intercalating and DNA-damaging activities of arylnaphthalimides

Article abstract of DOI:10.1016/j.bmc.2013.08.039

A series of arylnaphthalimides were designed and synthesized to overcome the dose-limiting cytotoxicity of N-acetylated metabolites arising from amonafide, the prototypical antitumour naphthalimide whose biomedical properties have been related to its ability to intercalate the DNA and poison the enzyme Topoisomerase II. Thus, these arylnaphthalimides were first evaluated for their antiproliferative activity against two tumour cell lines and for their antitopoisomerase II in vitro activities, together with their ability to intercalate the DNA in vitro and also through docking modelization. Then, the well-known DNA damage response in Saccharomyces cerevisiae was employed to critically evaluate whether these novel compounds can damage the DNA in vivo. By performing all these assays we conclude that the 5-arylsubstituted naphthalimides not only keep but also improve amonafide's biological activities.

Full text of DOI:10.1016/j.bmc.2013.08.039

Bioorganic & Medicinal Chemistry 21 (2013) 6484–6495
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and study of antiproliferative, antitopoisomerase II,
DNA-intercalating and DNA-damaging activities of
arylnaphthalimides
Patricia Quintana-Espinoza a,b, Jonay García-Luis b,c, ,à, Ángel Amesty a,b, Patricia Martín-Rodríguez b,d,e
,
Isabel Lorenzo-Castrillejo c, Angel G. Ravelo a,b, Leandro Fernández-Pérez b,d, Félix Machín b,c,
,
⇑
Ana Estévez-Braun a,b,
⇑
a Instituto Universitario de Bio-Orgánica ‘Antonio González’, Centro de Investigaciones Biomédicas de Canarias (CIBICAN), Departamento de Química Orgánica, Universidad de
La Laguna, Avda. Astrofísico Fco. Sánchez 2, 38206 La Laguna, Tenerife, Spain
b Instituto Canario de Investigación del Cáncer (ICIC), Spain
c Unidad de Investigación, Hospital Universitario Nuestra Señora de Candelaria, Carretera del Rosario, 145, 38010 Santa Cruz de Tenerife, Spain
d Departamento de Ciencias Clínicas-Unidad de Farmacología, Facultad de Ciencias de la Salud, Universidad de Las Palmas de G.C., Unidad Asociada de Biomedicina ULPGC-IIBM
‘Alberto Sols’-CSIC, Las Palmas de G.C., Spain
e Centro Atlántico del Medicamento, Spainà
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2013
Revised 20 August 2013
Accepted 21 August 2013
Available online 29 August 2013
A series of arylnaphthalimides were designed and synthesized to overcome the dose-limiting cytotoxicity
of N-acetylated metabolites arising from amonafide, the prototypical antitumour naphthalimide whose
biomedical properties have been related to its ability to intercalate the DNA and poison the enzyme Topo-
isomerase II. Thus, these arylnaphthalimides were first evaluated for their antiproliferative activity
against two tumour cell lines and for their antitopoisomerase II in vitro activities, together with their
ability to intercalate the DNA in vitro and also through docking modelization. Then, the well-known
DNA damage response in Saccharomyces cerevisiae was employed to critically evaluate whether these
novel compounds can damage the DNA in vivo. By performing all these assays we conclude that the 5-
arylsubstituted naphthalimides not only keep but also improve amonafide’s biological activities.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Arylnaphthalimides
Antiproliferative activity
Topoisomerase II
DNA damage
Saccharomyces cerevisiae
1. Introduction
a nuclear enzyme that regulates the topological structure of
chromatin. It is mainly involved in chromosome condensation
Naphthalimides, a class of compounds that bind to DNA by
intercalation have shown high anticancer activity against a variety
of murine and human tumor cells.1–4 Two representative com-
pounds, mitonafide and amonafide (Fig. 1), have reached clinical
trials as potential anticancer agents.5,6 However, amonafide,
although effective in its phase 2 clinical trials when administered
either alone or in combination, suffered from the dose-limiting
bone marrow toxicity caused by the toxic N-acetyl-amonafide gen-
and sister chromatid decatenation in mitosis. Topo II poisons are
well validated chemotherapy agents.10 Epipodophyllotoxins
(etoposide), aminoacridines (amsacrine) and anthracyclines
(doxorubicin, daunorubicin, etc.) are all potent Topo II poisons.
Amonafide is distinct from other Topo II poisons in being able to
evade Glycoprotein 1 (PgP) and related transporters responsible
for multi-drug resistance.11
With the aim of accessing to less cytotoxic naphthalimides
against bone-marrow while keeping their antitumoral activities,
we synthesized several non-amino substituted naphthalimides.
In this paper, we describe the preparation and biological evaluation
of a series of aromatic substituted naphthalimides. We include the
antiproliferative activity against two cancer cell lines of the syn-
thesized naphthalimides together with the antitopoisomerase I
and II in vitro activities. Besides, we carried out docking studies
in order to further confirm the ability of the arylnaphthalimides
to intercalate the DNA and thus complement the results obtained
in vitro during the topoisomerase (I and II)-mediated relaxation.
erated through N-acetylation by the N-acetyltransferase
2
(NAT2).7,8 Amonafide is a DNA intercalating agent that induces
apoptotic signalling by promoting topoisomerase II (Topo II)-
mediated DNA cleavage (i.e., it ‘poisons’ the enzyme).9 Topo II is
⇑
Corresponding authors. Tel.: +34 922318576; fax: +34 922318571.
E-mail addresses: fmacconw@gmail.com (F. Machín), aestebra@ull.es (A. Esté-
vez-Braun).
http://www.icic.es.
http://www.ceamedsa.com.
à
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.08.039

P. Quintana-Espinoza et al. / Bioorg. Med. Chem. 21 (2013) 6484–6495
6485
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
O
O
O
O
O
N
O
N
O
O
N
O
(b)
(a)
mitonafide R=NO2
amonafide R=NH2
NO2
R
NO2
NH2
12
14
13
Figure 1. Structures of mitonafide and amonafide.
(c)
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
O
O
O
O
N
O
N
O
N
O
N
O
O
N
O
(d)
Ar
Ar
Ar
I
Ar
15
16
Scheme 2. Preparation of 5-arylnaphthalimides. Reagents and conditions: (a)
NH2(CH2)2N(CH3)2, EtOH, (67%); (b) HCO2H, TEA, Pd/C (100%); (c) NaNO2, H2SO4,
KI, 0 °C (44%); (d) ArB(OH)2, Pd(PPh3)4, Na2CO3, EtOH.
17 Ar1 (60%)
18 Ar2 (35%)
19 Ar3 (53%)
20 Ar1(31%)
21 Ar2(65%)
22 Ar3 (50%)
23 Ar1(70%)
24 Ar2(65%)
25 Ar3 (73%)
D
Ar1=
Ar2=
Ar3=
tain the 4- and 6-aromatic substituted naphthalimides we followed
the synthetic approach shown in Scheme 1.
1'
OMe
3'
Thus, compounds 10 and 11 were synthesized in six steps. The
nitration of acenaphthene (1)15 and subsequently hydrogenation16
led to a mixture of two isomers: 5- and 3-aminoacenaphthene (2
and 3). In the following Sandmeyer reaction, the iodo derivatives
(4) and (5) were formed and used for Suzuki coupling with com-
mercial aryl boronic acids.17,18 The mixture of aryl derivatives (6)
and (7) was then oxidized with Na2Cr2O7 in AcOH to yield the cor-
responding anhydrides (8) and (9), which were separated by col-
umn chromatography. Finally, each anhydride was treated with
N,N-dimethyl-ethane-1,2-diamine under reflux to yield the desired
aryl naphthalimides (10) and (11).
OMe
OMe
Figure 2. Synthesized arylnaphthalimides.
Finally, we have also evaluated the ability of these compounds to
damage the DNA in vivo by studying the well-known DNA damage
response in Saccharomyces cerevisiae.
2. Results and discussion
To obtain the 5-aromatic substituted naphthalimides, we em-
ployed the commercial 3-nitro-1,8-naphthalic anhydride (12) as
starting material and we followed the synthetic approach shown
in Scheme 2.19–21
Figure 2 shows the structures of the synthesized arylnaphthal-
imides (17–25) and the obtained yields from the corresponding
precursors 8, 9 or 15. These arylnaphthalimides were subjected
to antiproliferative evaluation against the human breast cancer cell
With the aim to explore if an extension of the conjugation of the
naphthalimide core through an aromatic substituent could led to
more active and less toxic derivatives, we decided to prepare 4-, 5-
, and 6-aromatic substituted naphthalimides. There are only a few
examples of 5-and 6- non fused aromatic arylnaphthalimides12,13
and only one example of 4-arylnaphthalimide derivative.14 To ob-
NH2
(c)
I
(a)
+
+
(b)
2
3
1
4
5
NH2
I
CH2CH2N(CH3)2
(d)
O
O
N
O
O
O
(g)
Ar
8
(e)
(f)
10
Ar
Ar
+
+
CH2CH2N(CH3)2
6
7
Ar
O
O
O
O
N
O
Ar
Ar
(g)
11
9
Scheme 1. Preparation of 4- and 6-arylnaphthalimides. Reagents and conditions: (a) HNO3, 15 °C, Ac2O, 3 h (98%); (b) H2, Pd/C (10%), in THF (98%); (c) NaNO2, H2SO4, KI, 0 °C
(71%); (d) ArB(OH)2, Pd(PPh3)4, C7H8//EtOH; (e) Na2Cr2O7/AcOH; (f) separation by column chromatography; (g) NH2(CH2)2N(CH3)2, DCM/C7H8, , DMAP.
D

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P. Quintana-Espinoza et al. / Bioorg. Med. Chem. 21 (2013) 6484–6495
Table 1
Antiproliferative activitya of arylnaphthalimides (17–25) against the Human Breast Cancer MCF-7 and SK-Br-3 cell lines
Compound
MCF-7
SKBr-3
Compound
MCF-7
SKBr-3
Compound
MCF-7
SKBr-3
17
18
19
24.0 4.1
>30
>30
24.0 4.1
>30
>30
20
21
22
25.7 2.9
0.5 0.1
7.6 0.8
11.9 3.1
0.3 0.1
1.5 0.3
23
24
25
>30
>30
>30
>30
>30
27.6 3.1
a
Expressed as IC50 values given in lm and determined as means SD (n = 3).
Figure 3. Inhibition of Topo I- and Topo II-mediated supercoiled DNA relaxation by arylnaphthalimides (17, 19–25). (A) Agarose gel electrophoresis (without ethidium
bromide) to separate substrates (SC form) and products (relaxed topoisomers) of the hTopo II reaction. Covalently closed negative supercoiled pRYG plasmid (SC form) was
treated with hTopo II in the presence of either just 1% (v/v) DMSO, or 100 M of a set of arylnaphthalimides in 1% DMSO. Concentration below 100 M were also used and
the inhibition profile is indicated underneath (ꢀꢀ, no inhibition at 100 M; +, inhibition at 100 M; ++, inhibition at 60 M; +++, inhibition at 30 M). (B) Agarose gel
electrophoresis (upper gel run without ethidium bromide and lower gel run with it) to separate substrates (SC form) and products (relaxed topoisomers) of the wTopoI
reaction. For this assay, covalently closed negative supercoiled pBSKS plasmid (SC form) was treated with wTopoI in the presence of either just 1% (v/v) DMSO, or 100 M of
a
a
l
l
l
l
l
l
l
arylnaphthalimides in 1% DMSO. (C) Inhibition of endonucleolytic cleavage of DNA by restriction enzymes. The assay was carried out like above using the restriction enzyme
XhoI and the plasmid pBSKS as substrate. The linear form of the plasmid is the final product, being the nicked form a reaction intermediate. Actinomycin D (Acti-D) was used
as a control of a DNA intercalating agent capable of inhibiting the endonucleolytic reaction. Note how the presence of the 5-arylnaphthalimide 21 greatly inhibit all hTopo IIa,
wTopo I and XhoI reactions (as compared to controls without enzyme). Nck (nicked plasmids), Rel (fully relaxed plasmids), Lin (linearized plasmids), SC (supercoiled
plasmids).
lines MCF-7 and SKBr-3 (Table 1) and antitopoisomerase I and II
in vitro activities (Fig. 3).
the relevant molecular target for the antiproliferative actions in tu-
mour cells.9,22 Thus, we observed that compound 21 was the best
at inhibiting Topo II (Fig. 3A). Interestingly, the supercoiled form
of the DNA substrate appeared shifted after being incubated with
compound 21 and Topo II. This might indicate that this compound
is also a potent DNA intercalator. To further explore this possibility
we included an anti-Topo I assay (Fig. 3B) and also evaluated the
cutting efficiency of a restriction endonuclease in the presence of
the compounds (Fig. 3C). Anti-Topo I assays can measure both ac-
From the preliminary antiproliferative assay, we concluded that
only the 5-arylsubstituted derivatives show good activities against
MCF-7 and SKBr-3 cell lines. Within the 5-arylsubstituted naph-
thalimides series, the best results were obtained with the com-
pound having a methoxy group at C-30 (compare 21 vs 20 and 22).
We next tested whether the synthesized arylnaphthalimides
were good at inhibiting Topoisomerase II since this enzyme seems

P. Quintana-Espinoza et al. / Bioorg. Med. Chem. 21 (2013) 6484–6495
6487
O
O
O
O
CH2CH2N(CH3)2
O
N
O
27
(a)
CH2CH2N(CH3)2
(c)
O
O
(76%)
30
O
N
O
(b)
(c)
CH2CH2N(CH3)2
(c)
O
N
O
I
I
(100%)
28
15
CH2CH2N(CH3)2
NO2
(c)
O
N
O
(e)
32 (57%)
CH2CH2N(CH3)2
OMe
CH2CH2N(CH3)2
21
O
N
O
(d)
CH2CH2N(CH3)2
(65%)
O
N
O
CF3
O
N
O
NH2
31
(70%)
(100%)
33
OH
(78%)
29
Scheme 3. Preparation of arylnaphthalimides 15, 21, 29–33. Reagents and conditions: (a) H2SO4, Ag2SO4, I2, 65 °C; (b) NH2CH2CH2N(CH3)2, EtOH,
Na2CO3, EtOH; (d) BBr3, DCM, ꢀ78 °C; (e) H2/Pd/C.
D
; (c) ArB(OH)2, Pd(PPh3)4,
tual inhibition of Topo I and DNA intercalation since the latter non-
specifically affects the relaxation activity.23 Compounds 21, 24 and
25 inhibited DNA relaxation by Topo I (Fig. 3B, upper panel). How-
ever, none seemed to poison the enzyme since the nicked form of
DNA (Nckd) was not enriched in this assay when we forced the re-
laxed form (Rel) to become supercoiled again under the presence
of another intercalating agent (i.e., ethidium bromide) (Fig. 3B,
lower panel). Intercalating agents are known to inhibit DNA cleav-
age by restriction enzymes.24 We also used this approach and
found that compound 21 prevented full digestion of a supercoiled
plasmid which carries a single recognition site for the restriction
enzyme XhoI (Fig. 3C).
With these results in our hands, we decided to prepare other 5-
aryl derivatives and evaluate the influence in the activity of other
groups such as, –OH, –NH2, –NO2, CF3, etc. Since the synthetic ap-
proach followed in Scheme 2 led to 5-arylsubstituted naphthali-
mides after four steps in moderated global yield, we planned an
alternative synthetic route. Our objective was to obtain 3-iodo-
1,8-naphthalic anhydride from direct iodination of the correspond-
ing anhydride and to avoid the Sandmeyer reaction following a
modified methodology described by Xie et al.13 After using several
iodination conditions, we obtained quantitatively the desired io-
dide derivative (28) by using of iodine in the presence of Ag2SO4/
H2SO4.25 This compound was converted into the imide (15) by
treating with N,N-dimethyl-ethane-1,2-diamine. The desired 5-
aryl derivatives were obtained under Suzuki coupling conditions
with the corresponding aryl boronic acid in good yields (Scheme 3).
Thus, this new approach permits the access to 5-arylnaphthali-
mides in three steps and in higher global yield than the synthetic
route shown in Scheme 2. The hydroxyl derivative 29 was obtained
by demethylation of 21 using BBr3/DCM and the hydrogenation of
32 yielded the derivative 33.
directly test if our compounds globally keep (or even improve)
amonafide’s biological activities.
From these results, several structure–activity relationships can
be outlined. The presence of hydrogen bond donor groups in the
aromatic ring at C-5 (compounds 29 and 33) led to the lower anti-
proliferative activities. Compounds 31 and 32 having electron
withdrawing groups (NO2 and CF3) showed the higher values for
the HEL cell lines while compounds 21 with a OMe group or com-
pound 30 resulted to be the most active compounds against the
SK-Br-3 cell line. Importantly, most of the 5-aryl derivatives exhib-
ited higher antiproliferative activities than amonafide.
Again, we carried out anti-Topo II assays for this new set of
compounds. We found that compounds 30–32 strongly inhibited
Topo II (Fig. 4A). Compound 32 was the best one and inhibition
was seen even at concentrations as low as 30 lM, a concentration
where both amonafide and 21 cannot completely inhibit the
relaxation activity (Fig. 4A). Once again, the supercoiled form of
the DNA substrate appeared shifted after being incubated with
compounds 30–32 and Topo II. Compound 32 (Fig. 4A) was the
one where that shift was maximum. This result points out that
at least some of these derivatives are also potent DNA intercala-
tors. At this point, we also performed an assay to check whether
these compounds stabilized the cleavage complex during the
Topo II reaction (i.e., poison the enzyme) since it is believed that
amonafide hinders the religation step of topoisomerase II action
and thus stabilizes the cleavable complex.9 We found that all
tested 5-arylnaphthalimides increased the visibility of this reac-
tion intermediate (linearized form) compared to a control with
the vehicle (Fig. 4B). This intermediate was equally present for
amonafide and all the other naphthalimides although slightly less
abundant than when treated with the well-known Topo II poison
etoposide. Overall, we hypothesize that our arylnaphthalimides,
at least those that show the best antiprofilerative activities, act
by intercalating the DNA and then poisoning Topo II in a similar
manner as amonafide does.9
This new set of derivatives was again evaluated for antiprolifer-
ative (Table 2) and antitopoisomerase I and II activities (Fig. 4).
Noteworthy, we also included at this point amonafide in order to

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P. Quintana-Espinoza et al. / Bioorg. Med. Chem. 21 (2013) 6484–6495
Figure 4. Topo II-mediated supercoiled DNA relaxation and cleavage under the presence of 5-arylnaphthalimides related to 21. (A) Agarose electrophoresis run under the
absence of ethidium bromide to separate substrates (SC form) and products (relaxed topoisomers) of the hTopo II reaction. Covalently closed negative supercoiled pRYG
plasmid (SC form) was treated with hTopo II in the presence of 100 M of 5-arylnaphthalimides in 1% (v/v) DMSO. Amonafide (AM) was also included for cross-comparisons.
Concentration below 100 M; +, inhibition at 100 M; ++, inhibition at 60 M;
M were also used and the inhibition profile is indicated underneath (ꢀꢀ, no inhibition at 100
+++, inhibition at 30 M). (B) Agarose electrophoresis run under the presence of ethidium bromide to separate substrates (SC form) and intermediate products (linearized
plasmid) of the hTopo II cleavage assay. Covalently closed negative supercoiled pRYG plasmid (SC form) was treated with high concentrations of hTopo II in the presence of
100 M of the same set of 5-aryl-naphthalimides in 1% (v/v) DMSO. Etoposide (Etop) was used as a positive control for this assay. Note how most of these arylnaphthalimide
inhibit hTopo II . They further poison hTopo II (as compared to controls without enzyme). The shift of the SC form in the Topo II inhibition assay for compounds 30–32
a
a
l
l
l
l
l
l
a
a
l
a
a
further indicate that they intercalate into the DNA even better that amonafide and 21. Nck (nicked plasmids), Rel (fully relaxed plasmids), Lin (linearized plasmids), SC
(supercoiled plasmids).
Table 2
Antiproliferative activitya of amonafide and 5-aryl-naphthalimides (21, 29–33)
against HEL (Human Erythroleukemia) and SK-Br-3 (Human Breast Cancer) cell lines
Compound
HEL
2.7 0.3
7.0 0.9
6.0 0.9
1.6 0.2
0.6 4.1
13.5 2.2
3.4 0.5
SKBr-3
21
29
30
31
32
33
0.3 0.1
4.4 0.8
0.8 0.1
1.2 0.3
1.2 0.4
14.2 2.4
7.4 0.9
Amonafide
a
Expressed as IC50 values given in lm and determined as means of SD (n = 3).
With the aim to explore the structural determinants responsible
for the better intercalating properties of some of these 5-arylnaph-
thalimides, we performed docking calculations by using the Glide
software.26,27 The self-complementary oligodeoxy nucleotide
d(ATGCAT)2 was used as a model.28–30 The best docking scores
were found in the range from ꢀ6.13 to ꢀ8.21 kcal molꢀ1 (see
Table S1 in Supplementary data). The analysis of docking results
reveals that all ligands share a similar binding mode. The common
structural portion, constituted by a polycyclic system, is funda-
mental for the lineup of the complex with the biological target,
through a base pairs intercalation. In this sense, for example, the
planar portions of the structure of the most active derivative 32
establishes
p–p interactions with DNA aromatic rings, through
intercalated naphthalimides and these interactions are extended
thanks to the presence of the new aromatic portion where the nitro
group is present (Fig. 5). The aminoalkyl chain establishes Van der
Waals contacts and the amino groups interact with N7 of G3
through hydrogen bond with nucleic acids.
Drugs that poison Topo II are known to cause DNA damage
in vivo, especially in the form of highly cytotoxic DNA double
strand breaks (DSB).10 It is this type of damage which makes cancer
Figure 5. Compound 32, into the DNA binding site. The figure shows the interaction
of the amino groups of 32 with N7 of G3 through hydrogen bond. The DNA is
represented by tube (coloured by atom type: C, grey; polar H, white; N, dark blue;
O, red). Compound 32 is depicted in green.

P. Quintana-Espinoza et al. / Bioorg. Med. Chem. 21 (2013) 6484–6495
6489
1.2
0.8
0.4
(AM)
(17)
(19)
0.0
1.2
(22)
0.8
0.4
(20)
(23)
(29)
(21)
(24)
0.0
1.2
0.8
0.4
(25)
0.0
1.2
(31)
(30)
0.8
0.4
0.0
1.2
0
0.5
1
1.5 2 2.5
(32)
Log10 of conc. (µM)
0.8
0.4
0.0
(33)
0
0.5 1 1.5 2 2.5
0
0.5
1 1.5 2 2.5
Log10 of conc. (µM)
Log10 of conc. (µM)
Figure 6. Sensitivity to the set of arylnaphthalimides (17, 19–25, 29–33) in wild type and homologous recombination deficient yeast cells. Strains proficient (RAD52) and
deficient (rad52 ) for the homologous recombination pathway were grown for 24 h under increasing concentrations of the tested naphthalimides. Relative growth to a
control with DMSO 1% (v/v) was plotted against the drug concentrations. Note how: (i) compounds 21, 30–32 are stronger than amonafide (AM) against yeast; and (ii) the
rad52 mutant was always hypersensitive. In the graph, open squares depict growth of the RAD52 strain and filled triangles depict the rad52 strain.
D
D
D
cells hypersensitive to these agents. In order to explore whether
our compounds were making DSBs in vivo, we decided to carry
out assays with the model organism Saccharomyces cerevisiae. This
yeast has been extensively used for studies on DSB repair since it
strongly depends on simple pathways that can be knocked out
without compromising cell viability under non-toxic conditions.
The central DBS repair pathway in yeast is homologous recombina-
tion (HR). This repair mechanism fully depends on the RAD52
gene.31 Thus, any yeast-permeable drug that causes DSBs would
make yeast cells deficient in Rad52 hypersensitive to that drug.
Furthermore, if that protein is labelled (e.g., Rad52-YFP) and we
cytologically followed it we would see how it concentrates onto
nuclear factories while DSBs occur.32 Finally, DSBs also trigger
checkpoints that arrest the cell cycle to allow time for repair. This
arrest mainly takes place in G2 unless the number of DSBs is very
high.33 We made used of these three expected phenotypes to
determine if the arylnaphthalimides (17, 19–25, 29–33) generate
DSBs. First, we performed dose–response growth curves for each
naphthalimide (including amonafide), and compared a wild-type
RAD52 yeast strain to the isogenic single rad52D mutant strain
(Fig. 6).
We observed that amonafide did not inhibit the growth of the
RAD52 strain in the conditions we employed (1–100 M concentra-
l
tion range with an initial yeast inoculum of ꢁ105 cells/mL). Never-
theless, many of our arylnaphthalimides did inhibit the growth
(compounds 21, 30–32). Importantly, those that did were the ones
that gave the best antiproliferative profiles against tumor cell lines
(Tables 1 and 2), strongly inhibited Topo II in vitro (Figs. 3 and 4),
and likely are the best DNA intercalating agents (Figs. 3–5).
Remarkably, the rad52D mutant was hypersensitive to most tested
naphthalimides, including those moderately cytotoxic against tu-
mor cell lines but not wild type yeast (e.g., compound 22, 29, 33
and amonafide itself). Arylnaphthalimides which were non-cyto-
toxic against tumor cell lines did not cause growth inhibition in
the rad52D mutant either (e.g., 19 and 23).

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100
80
60
40
20
0
2
1.5
1
0.5
0
PH DM AM (17) (19) (20) (21) (22) (23) (24) (25) (29) (30) (31) (32) (33)
Compound
Figure 7. Cell cycle G2 arrests and homologous recombination nuclear factories in yeast cells treated with the set of naphthalimides (17, 19–25, 29–33). A yeast strain which
expresses Rad52 fused to YFP was treated with 100 M of the novel set of arylnaphthalimides or amonafide (AM) in DMSO 1% (v/v). The vehicle alone (DM) or phleomycin
l
(PH) were used as the negative and positive control, respectively. Note how compounds 21, 30–32 increased both the percentage of cells arrested in G2 and the average
number of Rad52 foci per cell.
Besides, we used a RAD52-YFP strain to follow up the cell cycle
arrest and formation of nuclear Rad52 repair factories after a short
treatment (3 h) with the corresponding naphthalimides (Fig. 7).
We observed that naphthalimides to which RAD52 yeast cells were
hypersensitive (i.e., 21, 30–32) partly triggered a G2 checkpoint as
well (Fig. 7, upper panel). These same compounds led to the forma-
tion of more Rad52 foci than amonafide or other less-cytotoxic
derivatives (Fig. 7, lower panel).
appropriate. Reactions were monitored by TLC (on silica gel POLY-
GRAM SIL G/UV254 foils). Purification by column flash-chromatog-
raphy used Merk Kiesel 60-H (0.063–0.2 mm) as adsorbent and
different mixtures of hexanes–EtOAc as eluent. Pre-coated TLC
plates SIL G-100 UV254 (Machery-Nagel) were used for prepara-
tive-TLC purification. 1H NMR spectra were recorded in CDCl3 or
C6D6 at 300 or 400 MHz, using a Bruker AMX300 or Bruker
AMX400 instruments. For 1H spectra, chemical shifts are given in
parts per million (ppm) and are referenced to the residual solvent
peak. Coupling constants (J) are given in Hertz (Hz) to the nearest
0.5 Hz. 13C NMR spectra were recorded at 75 and 100 MHz using a
Bruker AMX300 or Bruker AMX400 instruments. Carbon spectra
assignments are supported by DEPT-135 spectra, 13C–1H (HMQC)
and 13C–1H (HMBC) correlations where necessary. Chemical shifts
are quoted in ppm and are referenced to the appropriate residual
solvent peak. MS and HRMS were recorded on a VG Micromass
ZAB-2F. IR spectra were taken on a Bruker IFS28/55 spectropho-
tometer. The preparation of 3-nitro-acenaphthene, 5-nitro-
acenaphthene, 5-amino-acenaphthene (2), 3-amino-acenaphthene
3. Conclusions
In short, we have synthesized a set of 4-, 5- and 6-aryl naphthal-
imides. The antiproliferative assay against the Human Breast Cancer
MCF-7 and SK-Br-3 cell lines showed that only the 5-arylsubstituted
derivatives have good activities. We prepared new 5-aryl derivatives
and evaluated the influence of other groups such as, –OH, –NH2, –
NO2, CF3, etc., in the antiproliferative, and antitopoisomerase I and
II activities. We found that all synthesized 5-arylnaphthalimides
but those carrying H-bond donor groups, strongly inhibited Topo II
and that the DNA substrate appeared electrophoretically shifted
after being incubated with these compounds and Topo II, which
points out that these derivatives are also potent DNA intercalators.
These compounds also stabilized the cleavage complex during the
Topo II reaction. Docking studies using a self-complementary oligo-
deoxynucleotide d(ATGCAT)2 as a model corroborated that 5-aryl-
naphthalimides can act as DNA intercalating agents. In order to
explore whether our compounds were making DSBs in vivo, assays
with the model organism Saccharomyces cerevisiae were carried
(3),
5-iodo-acenaphthene
(4),
3-iodo-acenaphthene
(5),
5-aryl-acenaphthenes (6a–6c), 3-aryl-acenaphthenes (7a–7c),
5-aryl-1,8-naphthalic anhydrides (8a–8c), 3-aryl-1,8-naphthalic
anhydride (9a–9c), 2-(2-dimethylamino-ethyl)-5-nitro-benzo[de]
isoquinoline-1,3-dione (13), 2-(2-dimethylamino-ethyl)-5-amino-
benzo[de]isoquinoline-1,3-dione (14), 2-(2-dimethylamino-ethyl)-
5-iodo-benzo[de]isoquinoline-1,3-dione (15) was carried out fol-
lowing the procedure described in Refs. 15–21 and the experimen-
tal procedures and spectroscopic characterization are included in
Supplementary data.
out. Remarkably, the rad52D mutant was hypersensitive to most
tested naphthalimides, including those moderately cytotoxic
against tumor cell lines (e.g., compound 22, 29, 33 and amonafide it-
self). However, some 5-arylnaphthalimides (i.e., 21, 30–32) were the
most cytotoxic derivatives in yeast as well as in tumor cell lines.
They were also able to arrest yeast cells in G2 and promote the for-
mation of Rad52 foci, which fit well with DSBs being generated by
these compounds in vivo.
4.1.1. Preparation of 2-(2-dimethylamino-ethyl)-4-(2-methoxy-
phenyl)-benzo[de]isoquinoline-1,3-dione (17)
15
lL (0.13 mmol) of N,N-dimethyl-ethane-1,2-diamine and
catalytic amount of DMAP were added to 15 mg (0.05 mmol) of
3-(2-methoxyphenyl)-1,8-naphthalic anhydride in 5 mL of DCM/
C7H8 (1:1). The reaction mixture was refluxed under argon for
2 h. Then, the solvent was removed under reduced pressure and
the resulting residue was dissolved in 10 mL of EtOAc and treated
with a 5% solution of HCl and with brine. The aqueous phase was
separated and the organic layer was dried over anhydrous MgSO4,
filtered and concentrated to obtain 20 mg of a residue, which was
purified by preparative-TLC using DCM:MeOH (9:1) to yield
4. Experimental
4.1. Chemistry
All solvents and reagents were purified by standard techniques
reported in Ref. 34 or used as supplied from commercial sources as

P. Quintana-Espinoza et al. / Bioorg. Med. Chem. 21 (2013) 6484–6495
6491
11.4 mg (60%) of compound 17 as an amorphous yellow solid. 1H
NMR (CDCl3) d: 2.41 (6H, s, H-300, H-400), 2.73 (2H, t, J = 7.0 Hz, H-
200), 3.71 (3H, s, OCH3), 4.39 (2H, t, J = 7.1 Hz, H-100), 7.09 (1H, d,
J = 8.2, H-30), 7.14 (1H, t, J = 8.2 Hz, H-50), 7.30 (1H, m, H-60), 7.51
(1H, t, J = 8.2 Hz, H-40), 7.65 (1H, d, J = 8.4 Hz, H-6), 7.67 (1H, d,
J = 7.6 Hz, H-3), 7.97 (1H, d, J = 8.4 Hz, H-5), 8.61 (1H, d,
J = 7.6 Hz, H-4), 8.66 (1H, d, J = 8.4 Hz, H-7). 13C NMR (CDCl3) d:
37.8 (t, C-100), 45.4 (q, C-300, C-400), 55.4 (q, OCH3), 56.7 (t, –C-200),
111.1 (d, C-30), 120.8 (d, C-50), 121.7 (s, C-1a), 122.6 (s, C-7a),
126.4 (d, C-6), 127.5 (s, C-10), 128.4 (d, C-60), 130.1 (d, C-40),
130.2 (s, C-4b), 130.6 (s, C-4a), 130.8 (d, C-3), 131.0 (d, C-7),
131.4 (d, C-5), 133.1 (d, C-4), 144.1 (s, C-2), 156.7 (s, C-20), 164.2
(s, C-1), 164.5 (s, C-8). EIMS m/z (%): 374 (9), 316 (24), 303 (91),
245 (10) 189 (17). HREIMS: 374.1647 (calcd for C23H22N2O3 [M]+
(0.56 mmol) of a 2 M Na2CO3 solution were treated with 64 mg
(0.42 mmol) of 2-methoxyphenyl boronic in 3 mL of EtOH. The
reaction mixture was refluxed under argon for 16 h, next 0.1 mL
of H2O2 (30%) was added and the reaction mixture was stirred
for one additional hour. The aqueous phase was extracted several
times with AcOEt, the combined organic layers were dried over
anhydrous magnesium sulfate, filtered and concentrated. The
resulting residue was purified by flash chromatography using
DCM/MeOH 9:1 to yield 33 mg of compound 20 as a yellow amor-
phous solid (31%). 1H NMR (CDCl3) d: 2.38 (6H, s, H-300, H-400), 2.69
(2H, t, J = 7.2 Hz, H-200), 3.84 (3H, s, OCH3), 4.35 (2H, t, J = 7.2 Hz, H-
100), 7.03 (2H, m, H-50, H-30), 7.41 (2H, m, H-40, H-60), 7.71 (1H, t,
J = 7.2 Hz, H-6), 8.20 (1H, d, J = 7.2 Hz, H-5), 8.29 (1H, s, H-4),
8.54 (1H, d, J = 7.2 Hz, H-7), 8.79 (1H, s, H-2). 13C NMR (CDCl3) d:
38.1 (t, C-100), 45.7 (q, C-300, C-400), 55.7 (q, OCH3), 57.0 (t, C-200),
111.4 (d, C-30), 121.2 (d, C-50), 122.1 (s, C-1a), 122.5 (s, C-7a),
127.0 (d, C-60) 127.2 (s, C-4b), 128.7 (s, C-10), 129.8 (d, C-40),
130.9 (d, C-6), 131.0 (d, C-5), 131.8 (s, C-4a), 133.6 (d, C-7), 134.0
(d, C-2), 134.1 (d, C-4), 137.8 (s, C-3), 156.6 (s, C-20), 164.4 (s, C-
8, C-1). EIMS m/z (%): 374 (17), 316 (13), 303 (8), 189 (15), 71
(96), 58 (100). HREIMS 374.1638 (calcd for C23H22N2O3 [M]+
374.1630). IR m .
max: 826, 1236, 1671, 1699, 2783, 2866, 2989 cmꢀ1
4.1.2. Preparation of 2-(2-dimethylamino-ethyl)-4-(3-
methoxyphenyl)-benzo[de]isoquinoline-1,3-dione (18)
15 lL (0.13 mmol) of N,N-dimethyl-ethane-1,2-diamine and
2 mg of DMAP were added to 10 mg (0.05 mmol) of 3-(3-methoxy-
phenyl)-1,8-naphthalic anhydride in 5 mL of DCM/C7H8 (1:1). The
reaction mixture was refluxed under argon for 6 h. After carrying
out the same procedure described above, the resulting residue was
purified by preparative-TLC using DCM:MeOH (9:1) to yield 4 mg
(35%) of compound 18 as an amorphous yellow solid. 1H NMR
(CDCl3) d: 2.41 (6H, s, H-300, H-400), 2.73 (2H, t, J = 7.0 Hz, H-200),
3.84 (3H, s, OCH3), 4.28 (2H, t, J = 6.5 Hz, H-100), 6.95 (3H, m, H-40,
H-60, H-20), 7.38 (1H, t, J = 8.0 Hz, H-50), 7.55 (1H, d, J = 8.4 Hz, H-4),
7.78 (1H, t, J = 7.6 Hz, H-6), 8.15 (1H, d, J = 8.4 Hz, H-3), 8.21 (1H, d,
J = 8.0 Hz, H-5), 8.66 (1H, d, J = 6.4 Hz, H-7). 13C NMR (CDCl3) d:
32.1 (t, C-100), 45.2 (q, C-300, C-400), 55.4 (q, OCH3), 55.6 (t, –C-200),
113.0 (d, C-40), 113.8 (d, C-20), 119.3 (s, C-7a), 120.5 (d, C-60), 123.1
(s, C-1a), 126.9 (d, C-50), 129.0 (s, C-4b), 129.3 (d, C-3), 131.3 (s, C-
4a), 131.6 (d, C-6), 131.9 (d, C-4), 132.9 (d, C-5), 134.2 (d, C-7),
143.8 (s, C-10), 147.9 (s, C-2), 159.5 (s, C-30), 163.4 (s, C-1), 164.3 (s,
C-8). ESMS m/z (%): 352 (43), 397 (100), 413 (88). HRESMS:
374.1630). IR m .
max: 757, 1029, 1664, 1703, 2774, 2837, 2944 cmꢀ1
4.1.5. Preparation of 2-(2-dimethylamino-ethyl)-5-(3-methoxy-
phenyl)-benzo[de]isoquinoline-1,3-dione (21)
110 mg (0.28 mmol) of compound 15 in 3 mL of dry toluene,
tetrakis(triphenylphosphine)palladium (7 mol %) and 0.3 mL
(0.56 mmol) of a 2 M Na2CO3 solution were treated with 64 mg
(0.42 mmol) of 3-methoxyphenyl boronic acid in 3 mL of EtOH.
The reaction mixture was refluxed under argon for 16 h. Then
0.1 mL of 30% H2O2 solution was added and the reaction mixture
was stirred for one additional hour. After carrying out the same
procedure described for compound 20, the resulting residue was
purified by flash chromatography using DCM/MeOH 9:1 to yield
67 mg of compound 21 as a yellow amorphous solid (65%). 1H
NMR(CDCl3) d: 2.38 (6H, s, H-300, H-400), 2.69 (2H, t, J = 7.2 Hz, H-
200), 3.91 (3H, s, OCH3), 4.35 (2H, t, J = 7.2 Hz, H-100), 6.98 (1H, d,
J = 7.6 Hz, H-40), 7.25 (1H, s, H20), 7.32 (1H, d, J = 7.6 Hz, H-60),
7.42 (1H, d, J = 7.6 Hz, H-50), 7.73 (1H, t, J = 7.3 Hz, H-6), 8.22 (1H,
d, J = 7.4 Hz, H-5), 8.33 (1H, s, H-4), 8.54 (1H, d, J = 7.3 Hz, H-7),
397.1523 (calcd for C23H22N2O3Na [M+Na]+ 397.1528); IR mmax
816, 1246, 1671, 1698, 2763, 2865, 2990 cmꢀ1
:
.
4.1.3. Preparation of 2-(2-dimethylamino-ethyl)-4-(4-
methoxyphenyl)-benzo[de]isoquinoline-1,3-dione (19)
13
8.81 (1H, s, H-2). C NMR (CDCl3) d: 38.0 (t, C-100), 45.6 (q, C-300,
10
l
L (0.09 mmol) of N,N-dimethyl-ethane-1,2-diamine and
C-400), 55.4 (q, OCH3), 56.9 (t, C-200), 112.9 (d, C-20), 113.7 (d, C-40),
119.7 (d, C-60), 122.4 (s, C-1a), 122.9 (s, C-7a), 127.2 (d, C-6),
130.1 (d, C-50), 130.6 (d, C-2), 130.9 (d, C-7), 131.2 (d, C-4), 131.9
(s, C-10, C-4b), 134.0 (d, C-5), 139.7 (s, C-3), 140.5 (s, C-4a), 160.2
(s, C-30), 164.1 (s, C-8, C-1). EIMS m/z (%): 374 (16), 316 (12), 303
(8), 189 (15) 71 (96), 58 (100). HREIMS: 374.1638 (calcd for
catalytic amount of DMAP were added to 10 mg (0.03 mmol) of
3-(4-methoxyphenyl)-1,8-naphthalic anhydride in 5 mL of DCM/
C7H8 (1:1), and the reaction mixture was refluxed under argon
for 6 h. After carrying out the same procedure described for com-
pound 17, the resulting residue was purified by preparative-TLC
to yield 6.0 mg (53%) of compound 19 as an amorphous yellow so-
C
23H22N2O3 [M]+ 374.1630). IR
2849, 2923, 3067 cmꢀ1
mmax: 783, 1015, 1734, 1775,
1
lid. H NMR (CDCl3) d: 2.39 (6H, s, H-300, H-400), 2.70 (2H, t,
.
J = 6.8 Hz, H-100), 3.91 (3H, s, OCH3), 4.29 (2H, t, J = 6.8 Hz, H-200),
7.03 (2H, d, J = 8.5 Hz, H-30, H-50), 7.37 (2H, d, J = 8.5 Hz, H-20, H-
60), 7.58 (1H, d, J = 8.4 Hz, H-3), 7.77 (1H, t, J = 8.0 Hz , H-6), 8.16
(1H, d, J = 8.4 Hz, H-4), 8.22 (1H, d, J = 8.0 Hz, H-5), 8.68 (1H, d,
4.1.6. Preparation of 2-(2-dimethylamino-ethyl)-5-(4-methoxy-
phenyl)-benzo[de]isoquinoline-1,3-dione (22)
110 mg (0.28 mmol) of compound 15 in 3 mL of dry toluene,
tetrakis(triphenylphosphine)palladium (7 mol %) and 0.56 mmol
of a 2 M sodium carbonate solution were treated with 64 mg
(0.42 mmol) of 3-methoxyphenyl boronic acid in 3 mL of EtOH.
The reaction mixture was refluxed for 16 h under argon. Then
0.1 mL of 30% H2O2 was added and the reaction mixture was stir-
red for one additional hour. After carrying out the same procedure
described for compound 20, the resulting residue was purified by
flash chromatography using DCM/MeOH 9:1 to yield 50 mg of
compound 22 as a yellow amorphous solid (50%). 1H NMR (CDCl3)
d: 2.37 (6H, s, H-300, H-400), 2.66 (2H, t, J = 7.4 Hz, H-200), 3.88 (3H, s,
OCH3), 4.32 (2H, t, J = 7.4 Hz, H-100), 7.02 (2H, d, J = 8.4 Hz, H-50, H-
30), 7.65 (2H, d, J = 8.4 Hz, H-40, H-60), 7.68 (1H, m, H-6), 8.17 (1H, d,
J = 8.1 Hz, H-5), 8.24 (1H, s, H-4), 8.45 (1H, d, J = 7.3 Hz, H-7), 8.74
13
J = 8.0 Hz, H-7). C NMR (CDCl3) d: 37.5 (t, C-100), 45.2 (c, C-300, C-
400), 55.2 (c, OCH3), 56.5 (t, C-200), 113.6 (d, C-30, C-50), 119.0 (s, C-
1a), 122.8 (s, C-7a), 126.5 (d, C-3), 129.1 (s, C-4b), 129.5 (d, C-60,
C-20), 130.9 (s, C-4a), 131.6 (d, C-6), 132.0 (d, C-5), 132.7 (d, C-4),
133.9 (d, C-7), 134.3 (s, C-10), 147.9 (s, C-2), 159.1 (s, C-40), 163.6
(s, C-1), 164.1 (s, C-8); EIMS m/z (%): 374 (5), 316 (21), 303 (60),
243 (8) 189 (17); HREIMS: 374.2136 (calcd for C23H22N2O3 [M]+
374.1630); IR m .
max: 819, 1244, 1659, 1698, 2773, 2851, 2924 cmꢀ1
4.1.4. Preparation of 2-(2-dimethylamino-ethyl)-5-(2-methoxy-
phenyl)-benzo[de]isoquinoline-1,3-dione (20)
110 mg (0.28 mmol) of compound 15 in 3 mL of dry toluene,
tetrakis(triphenylphosphine)palladium (7 mol %) and 0.3 mL

6492
P. Quintana-Espinoza et al. / Bioorg. Med. Chem. 21 (2013) 6484–6495
13
(1H, s, H-2). C NMR (CDCl3) d: 38.1 (t, C-100), 45.7 (q, C-300, C-400),
55.3 (q, OCH3), 56.9 (t, C-200), 114.5 (d, C-30 C-50), 122.4 (s, C-1a),
122.8 (s, C-7a), 126.8 (s, C-4b) 127.1 (d, C-6), 128.3 (d, C-60 C-20),
130.1 (d, C-2), 130.2 (d, C-7), 130.5 (d, C-4), 131.3 (s, C-10), 133.7
(d, C-5), 139.0 (s, C-4b), 139.4 (s, C-3), 141.8 (s, C-4a), 159.9 (s,
C-40), 164.1 (s, C-8 or C-1), 164.2 (s, C-8 or C-1). EIMS m/z (%):
374 (5), 316 (26), 303 (100), 189 (14) 69 (17), 58 (5). HREIMS:
(6H, s, H-300, H-400), 2.71 (2H, t, J = 6.8 Hz, H-200), 3.93 (3H, s,
OCH3), 4.38 (2H, t, J = 6.9 Hz, H-100), 7.10 (2H, d, J = 8.5 Hz, H-30,
H-50), 7.45 (2H, d, J = 8.5 Hz, H-20, H-60), 7.69 (1H, dd, J = 7.4 Hz,
J = 8.5 Hz, H-6), 7.71 (1H, d, J = 7.5 Hz , H-3), 8.33 (1H, dd,
J = 1.6 Hz, J = 8.5 Hz, H-5), 8.65 (2H, d, J = 7.4 Hz, H-7, H-2). 13C
NMR (CDCl3) d: 38.0 (t, C-100), 45.6 (q, C-300, C-400), 55.4 (q, OCH3),
56.8 (t, C-200), 114.1 (d, C-30, C-50), 120.9 (s, C-1a), 122.8 (s, C-7a),
126.6 (d, C-3), 127.7 (d, C-6), 128.8 (s, C-4b), 130.1 (s, C-4a),
130.9 (d, C-7), 131.0 (d, C-2), 131.1 (d, C-20, C-60), 132.7 (d, C-5),
146.7 (s, C-4), 158.4 (s, C-10), 159.9 (s, C-40), 164.2 (s, C-8 or C-1),
164.4 (s, C-1 or C-8). EIMS m/z (%): 374 (5), 316 (7), 303 (15),
189 (11), 58 (100). HREIMS: 374.1614 (calcd for C23H22N2O3
374.1638 (calcd for C23H22N2O3 [M]+ 374.1630). IR
1034, 1662, 1698, 2850, 2923, 3065 cmꢀ1
mmax: 787,
.
4.1.7. Preparation of 2-(2-dimethylamino-ethyl)-6-(2-
methoxyphenyl)-benzo[de]isoquinoline-1,3-dione (23)
50 mg (0.16 mmol) of 5-(2-methoxyphenyl)-1,8-naphthalic
anhydride in 5 mL of DCM/C7H8 (1:1), were treated with 26
[M]+374.1630). IR
2962 cmꢀ1
mmax: 673, 788, 1023, 1736, 1777, 2853, 2922,
lL
.
(0.24 mmol) of N,N-dimethyl-ethane-1,2-diamine and 2.0 mg of
DMAP. After carrying out the same procedure described for com-
pound 17, the resulting residue was purified by flash chromatogra-
phy using DCM:MeOH (9:1) to yield 42 mg (70%) of compound 23
as an amorphous yellow solid. 1H NMR (CDCl3) d: 2.41 (6H, s, H-300,
H-400), 2.73 (2H, t, J = 6.8 Hz, H-200), 3.71 (3H, s, OCH3), 4.39 (2H, t,
J = 6.8 Hz, H-100), 7.09 (1H, m, H-30), 7.10 (1H, t, J = 8.3 Hz, H-50),
7.29 (1H, m, H-60), 7.51 (1H, td, J = 1.6 Hz, J = 8.3, H-40), 7.65 (1H,
d, J = 8.3 Hz, H-6), 7.67 (1H, d, J = 7.6 Hz, H-2), 7.69 (1H, d,
J = 7.6 Hz, H-3). 7.97 (1H, d, J = 7.8 Hz, H-5). 8.61 (1H, d,
4.1.10. Preparation of 3-iodo-1,8-naphthalic anhydride (28)
3.0 g (15 mmol) of 1,8-naphthalic anhydride in 60 mL of H2SO4
were treated with 3.4 g of Ag2SO4 (11 mmol) and 6.9 g of I2
(27.5 mmol) pulverized in a mortar. The reaction mixture was stir-
red at 65 °C for 24 h, then it was poured onto ice, and the resulting
yellow precipitate was filtered through a büchner funnel. The pre-
cipitate was washed with cold water, saturated solution of sodium
carbonate, and saturated solution of sodium bisulphite. The ob-
tained solid was dried in an oven at 80 °C to yield quantitatively
compound (28) (5.9 g). 1H NMR (DMSO-d6) d: 7.91 (1H, t,
J = 7.9 Hz H-6), 8.45 (1H, d, J = 7.9 Hz, H-5), 8.51 (1H, d, J = 7.8 Hz,
H-7), 8.60 (1H, d, J = 1.4 Hz, H-2), 8.99 (1H, d, J = 1.4 Hz, H-4). 13C
NMR (DMSO-d6) d: 93.3 (s, C-3), 119.7 (s, C-1a), 121.1 (s, C-7a),
128.8 (d, C-6) y (c, C-4b), 133.1 (d, C-7), 1333.3 (c, C-4a), 134.5
(d, C-5), 139.6 (d, C-2), 143.4 (d, C-4), 160.0 (c, C-1), 160.6 (c, C-
8). EIMS m/z (%): 325.9 (2), 324.9 (13), 323.9 (100), 279.9 (46),
153 (41). HREIMS: 323.9282 (calcd for C12H5IO3 [M]+ 323.9282.
13
J = 7.8 Hz, H-7). C NMR (CDCl3) d: 37.8 (t, C-100), 45.4 (q, C-300, C-
400), 55.4 (q, OCH3), 56.7 (t, C-200), 111.1 (d, C-30), 120.8 (d, C-50),
121.7 (s, C-1a), 122.6 (s, C-7a), 126.4 (d, C-6),127.5 (s, C-4a),
128.4 (d, C-60), 130.1 (d, C-40), 130.6 (s, C-4b), 130.8 (d, C-3),
131.0 (d, C-7), 131.4 (d, C-2), 133.1 (d, C-5) 144.1 (s, C-4), 156.7
(s, C-20), 164.2 (s, C-8 or C-1), 164.5 (s, C-1 or C-8). EIMS m/z (%):
374 (5), 316 (7), 303 (15), 189 (10.7), 58 (100); HREIMS:
374.1614 (calcd for C23H22N2O3 [M]+ 374.1630); IR
768, 1658, 1698, 2852, 2926 cmꢀ1
mmax: 758,
.
IR m .
max: 782, 1012, 1290, 1771, 3056 cmꢀ1
4.1.8. Preparation of 2-(2-dimethylamino-ethyl)-6-(3-
4.1.11. Preparation of 2-(2-dimethylamino-ethyl)-5-iodo
methoxyphenyl)-benzo[de]isoquinoline-1,3-dione (24)
benzo[de]isoquinoline-1,3-dione (15) from 28
45 mg (0.15 mmol) of 6-(3-methoxyphenyl)-1,8-naphthalic
anhydride in 5 mL of DCM/C7H8 (1:1), were treated with 25
To a solution of 3-iodo-1,8-naphthalic anhydride (1.0 g,
lL
3.08 mmol) of 28 in 10 mL of absolute EtOH were added 0.99 mL
of N,N-dimethylethylendiamine and catalytic amounts of DMAP.
The reaction mixture was refluxed under argon for 45 min, then
it was filtered hot, the filtrate was cooled and the resulting orange
precipitate was filtered through a buchner funnel, and washed
with cold EtOH (100 mL) and two portions of hexane (20 mL).
850 mg of compound 15 were obtained without furthermore puri-
fication (70%).
(0.22 mmol) of N,N-dimethyl-ethane-1,2-diamine and 2.0 mg of
DMAP. After carrying out the same procedure described for com-
pound 17, the resulting residue was purified by flash chromatogra-
phy using DCM:MeOH (9:1) to yield 36 mg (65%) of compound 24
as an amorphous yellow solid. 1H NMR (CDCl3) d: 2.38 (6H, s, H-300,
H-400), 2.69 (2H, t, J = 7.0 Hz, H-200), 3.89 (3H, s, OCH3), 4.37 (2H, t,
J = 6.8 Hz, H-100), 7.06 (3H, m, H-40, H-20, H-60), 7.47 (1H, t,
J = 7.3 Hz, H-50), 7.71 (2H, m, H-6, H-3), 8.30 (1H, d, J = 8.3 Hz, H-
5), 8.63 (1H, d, J = 6.5 Hz, H-7), 8.64 (1H, d, J = 7.4 Hz, H-2); 13C
NMR (CDCl3) d: 38.1 (t, C-100), 45.7 (c, C-300, C-400), 55.3 (q, OCH3),
56.9 (t, C-200), 113.7 (d, C-20), 115.6 (d, C-40), 121.7 (s, C-1a),
122.3 (d, C-60), 122.8 (s, C-7a) 126.8 (d, C-3), 127.6 (d, C-6), 128.6
(s, C-4b), 129.6 (d, C-50), 130.0 (s, C-4a), 130.8 (d, C-7), 131.2 (d,
C-2), 132.6 (d, C-5), 140.1 (s, C-10), 146.7 (s, C-4), 159.6 (s, C-30),
164.1 (s, C-8 or C-1), 164.2 (s, C-1 or C-8). EIMS m/z (%): 374 (4),
316 (7), 3.42 (15), 303 (100), 217 (17) 189 (28), 58 (100). HREIMS
4.1.12. Preparation of 2-(2-dimethylamino-ethyl)-5-(3-
hydroxy-phenyl)-benzo[de]isoquinoline-1,3-dione (29)
35 mg (0.09 mmol) of compound 21 in dry DCM at ꢀ78 °C were
treated with 0.55 mL of a 1 M BBr3 solution in DCM. The reaction
mixture was stirred at 0 °C under argon until disappearance of
the starting compound, then was treated with NaHCO3 and stirred
for 30 min. The aqueous phase was extracted several times with
DCM, the organic layers were dried over anhydrous magnesium
sulfate, filtered and concentrated. The resulting residue was puri-
fied by flash chromatography using DCM/MeOH (85:15) to yield
23 mg (60%) of compound 29 as a yellow amorphous solid. 1H
NMR (CDCl3) d: 2.51 (6H, s, H-300, H-400), 2.98 (2H, t, J = 7.2 Hz, H-
200), 4.47 (2H, t, J = 7.2 Hz, H-100), 6.34 (1H, s, H-20), 6.40 (1H, d,
J = 6.6 Hz, H-60), 6.85 (1H, d, J = 7.4 Hz, H-40), 6.92 (1H, t,
J = 7.4 Hz, H-50), 7.72 (1H, t, J = 7.8 Hz, H-6), 8.06 (1H, s, H-4),
8.17 (1H, d, J = 7.8 Hz, H-5), 8.48 (1H, s, H-2), 8.52 (1H, d
374.1623 (calcd for C23H22N2O3 [M]+ 374.1630); IR
1645, 1698, 2882, 2986 cmꢀ1
mmax: 745, 766,
.
4.1.9. Preparation of 2-(2-dimethylamino-ethyl)-6-(4-
methoxyphenyl)-benzo[de]-isoquinoline-1,3-dione (25)
70 mg (0.22 mmol) of 5-(4-methoxyphenyl)-1,8-naphthalic
anhydride in 5 mL of DCM/C7H8 (1:1), were treated with 26 lL
(0.09 mmol) of N,N-dimethyl-ethane-1,2-diamine and catalytic
amount of DMAP. After carrying out the same procedure described
for compound 17, the resulting residue was purified by flash chro-
matography using DCM:MeOH (9:1) to yield 63 mg (73%) of com-
pound 25 as an amorphous yellow solid. 1H NMR (CDCl3) d: 2.39
13
J = 6.5 Hz, H-7). C NMR (CDCl3) d: 37.5 (t, C-100), 45.4 (q, C-300, C-
400), 57.3 (t, C-200), 113.7 (d, C-20), 115.2 (d, C-60), 117.6 (d, C-40),
122.6 (s, C-7a), 122.8 (s, C-1a), 127.1 (d, C-6), 129.6 (d, C-50, C-2),
130.4 (d, C-4), 130.7 (d, C-7), 131.9 (s, C-4a), 134.1 (d+s, C-5, C-

P. Quintana-Espinoza et al. / Bioorg. Med. Chem. 21 (2013) 6484–6495
6493
4b), 139.0 (s, C-3), 139.4 (s, C-10), 157.5 (s, C-30), 164.8 (s, C-8, C-1).
EIMS m/z (%): 361 (25), 360 (89), 189 (15), 71 (54), 58 (100). HRE-
IMS: 361.1538 (calcd for C23H22N2O3 [M+1]+ 361.1530). IR mmax
783, 1015, 1734, 1775, 2849, 2923, 3067 cmꢀ1
4.1.16. Preparation of 2-(2-dimethylamino-ethyl)-5-(3-amino-
phenyl)-benzo[de]isoquinoline-1,3-dione (33)
:
40 mg (0.10 mmol) of compound 32 in 5 mL of dry THF were
hydrogenated in the presence of catalytic amounts of 10% Pd/C
for 6 h. Then the reaction mixture was filtered through Celite and
the solvent eliminated to yield quantitatively compound 33. 1H
NMR (CDCl3) d: 2.37 (6H, s, H-300, H-400), 2.68 (2H, t, J = 6.9 Hz, H-
200), 3.95 (2H, s, NH2), 4.35 (2H, t, J = 7.2 Hz, H-100), 6.75 (1H, d,
J = 7.6 Hz, H-40), 7.06 (1H, s, H-20), 7.14 (1H, d, J = 7.6 Hz, H-60), ,
7.29 (1H, t, J = 7.7 Hz, H-50), 7.74 (1H, t, J = 8.4 Hz, H-6), 8.21 (1H,
d, J = 8.2 Hz, H-5), 8.33 (1H, s, H-4), 8.55 (1H, d, J = 7.3 Hz, H-7),
.
4.1.13. 2-(2-Dimethylamino-ethyl)-5-(phenyl)-
benzo[de]isoquinoline-1,3-dione (30)
50.0 mg (0.125 mmol) of compound 15 in 2 mL of dry toluene,
tetrakis(triphenylphosphine) palladium (10 mol %) and 0.14 mL of
a 2 M solution of Na2CO3 were treated with 62.6 mg (0.42 mmol)
of phenyl boronic acid dissolved in 1 mL of absolute ethanol. The
reaction mixture was refluxed under argon for 6 h. Then 0.05 mL
of 30% H2O2 was added, and the reaction mixture was stirred for
one additional hour. The aqueous phase was extracted several
times with AcOEt, the combined organic layers were dried over
anhydrous magnesium sulfate, filtered and concentrated. The res-
idue was purified by flash chromatography using DCM/MeOH 9:1
to yield 33.0 mg of compound 30 as an orange amorphous solid
(76%). Compound 30 showed identical spectroscopic data to those
previously reported.12,13
13
8.82 (1H, s, H-2). C NMR (CDCl3) d: 38.0 (t, C-100), 45.3 (q, C-300,
C-400), 57.1 (t, C-200), 114.4 (d, C-20), 115.1 (d, C-40), 119.8 (d, C-60),
122.7 (s, C-1a), 123.1 (s, C-7a), 127.3 (d, C-6), 130.2 (d, C-50),
130.9 (d, C-2), 131.0 (d, C-7), 131.2 (d, C-4), 132.2 (s, C-10, C-4b),
134.1 (d, C-5), 140.3 (s, C-3), 140.4 (s, C-4a), 147.3 (s, C-30), 164.4
(s, C-8, C-1); EIMS m/z (%):359 (55), 315 (17) 301 (68), 217 (33),
288 (28), 189 (33), 71 (44), 58 (100); HREIMS: 359.1638 (calcd
for C22H21N3O2 [M]+ 359.1630); IR
2849, 2923, 3067 cmꢀ1
mmax: 783, 1015, 1734, 1775,
.
4.2. Molecular modeling studies
4.1.14. Preparation of 2-(2-dimethylamino-ethyl)-5-(3-
trifluormethyl-phenyl)-benzo[de]isoquinoline-1,3-dione (31)
100 mg (0.25 mmol) of compound 15 in 4 mL of dry toluene
were treated with calatytic amounts of tetrakis(triphenylphos-
phine) palladium (10 mol %), 0.28 mL of a 2 M solution of sodium
carbonate and 64 mg (0.42 mmol) of 3-trifluormethyl-phenyl
boronic acid in 3 mL of absolute ethanol. The reaction mixture
was refluxed under argon for 14 h. Then 0.1 mL of 30% H2O2 was
added, and the reaction mixture was stirred for one additional
hour. The aqueous phase was extracted several times with AcOEt,
the combined organic layers were dried over anhydrous magne-
sium sulfate, filtered and concentrated. The residue was purified
by flash chromatography using DCM/MeOH 9:1 to yield 70 mg of
compound 31 as an orange amorphous solid (70%). 1H NMR (CDCl3)
d: 2.35 (6H, s, H-300, H-400), 2.68 (2H, t, J = 6.9 Hz, H-200), 4.33 (2H, t,
J = 6.9 Hz, H-100), 7.65 (2H, m, H-40, H-60), 7.74 (1H, t, J = 7.5 Hz H 6),
7.89 (1H, d, J = 7.6 Hz, H-50), 7.93 (1H, s, H- 20), 8.20 (1H, d,
J = 8.2 Hz, H-5), 8.30 (1H, d, J = 1.6 Hz, H-4), 8.51 (1H, d,
J = 7.2 Hz, H-7), 8.73 (1H, d, H-2, J = 1.6 Hz).13C NMR (CDCl3) d:
38.1 (t, C-100), 45.6 (q, C-300, C-400), 56.9 (t, C-200), 122.6 (s, C-7a),
123.4 (s, C-1a), 123.1 (d, C-20), 124.1 (d, C-60),127.3 (s, C-4a)
127.5 (d, C-6), 129.7 (d, C-40), 130.1 (d, C-2), 130.6 (d, C-50),
131.3 (d, C-4, C-7), 132.0 (s, C-10, C-4b), 134.0 (d, C-5), 138.3 (s,
C-3), 163.9 (s, C-30), 164.0 (s, C-8, C-1). EIMS m/z (%): 412 (19),
340 (51), 270 (70), 202 (98), 71 (15), 58 (100); HREIMS:
Docking studies were performed using the Glide 5.7. The PDB
structure 1CX3 (d(ATGCAT)2 duplex (D6)) as a template and the
starting coordinates were taken from the Protein Data Bank
(www.rcsb.org). The receptor was optimized in Maestro 9.2 by
using OPLS2005 force field before docking study. A receptor grid
was generated using a 1.00 van der Waals (vdW) radius scaling fac-
tor and 0.25 partial charge cutoff. The radius of 30 Å from the cen-
tral atom of DNA was defined as the binding site that covers the
entire DNA. Ligands were prepared using Lig-Prep 2.5 as imple-
mented in Maestro 9.2 and were docked using the extra precision
mode (XP) without using any constraints and a 0.80 van der Waals
(vdW) radius scaling factor and 0.15 partial charge cutoff and using
Glidescore for ligand ranking. A modified version of ChemScore,35
GlideScore implemented in Glide, was used to estimate binding
affinity and rank ligands. One poses per ligand were generated
and post-docking minimization was carried out.
4.3. Biological assays
The solvent for most stocks of the chemical agents employed
was Dimethyl Sulphoxide (DMSO), especial Molecular Biology
grade (DNase and RNase-free), from Sigma–Aldrich. Etoposide
and Actinomycin D were purchased from Sigma–Aldrich and
stored as a 10 mM stock in DMSO at ꢀ20 °C. Amonafide and related
arylnaphthalimides were also stored in DMSO as a 10 mM stock at
ꢀ20 °C until their use. Phleomycin was also purchased from Sig-
ma–Aldrich but stored as a 1.5 mg/mL stock in H2O at ꢀ20 °C.
412.1389 (calcd for C23H19F3N2O2 [M]+ 412.1399); IR
1075, 1744, 1775, 2850, 2925, 3097 cmꢀ1
mmax: 770,
.
4.1.15. Preparation of 2-(2-dimethylamino-ethyl)-5-(3-nitro-
phenyl)-benzo[de]isoquinoline-1,3-dione (32)
4.3.1. MTT cell viability assay
100 mg (0.25 mmol) of compound 15 in 4 mL of dry toluene
were treated with catalytic amounts of tetrakis(triphenylphos-
phine)palladium (10 mol %), 0.28 mL of a 2 M solution of sodium
carbonate and 62.6 mg (0.38 mmol) of 3-nitro-phenyl boronic acid
dissolved in 2 mL of absolute ethanol. The reaction mixture was re-
fluxed under argon for 12 h, then 0.1 mL of 30% H2O2 was added,
and the reaction mixture was stirred for one additional hour. The
aqueous phase was extracted several times with AcOEt, the com-
bined organic layers were dried over anhydrous magnesium sul-
fate, filtered and concentrated. The residue was purified by flash
chromatography using DCM/MeOH 9:1 to yield 55 mg of com-
pound 32 as an orange amorphous solid (57%). Compound 32
showed identical spectroscopic data to those previously
reported.13
The human cancer cell lines HL60 (promyelocytic leukemia),
MCF-7 (breast adenocarcinoma) and SK-Br3 (breast adenocarci-
noma) were purchased from ATCC and cultured in RPMI or DMEN
containing 10% FBS, respectively. The MTT assay, MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], was
used to test cytotoxicity of naphthalimides and cell viability.36
Briefly, cells were plated in 96-well plates at 10,000 cells/well.
Twenty-four hours after plating, vehicle (0.1% DMSO, final concen-
tration) or compound was added to cells at indicated concentra-
tions. Forty eight hours following compound addition, MTT
(Sigma–Aldrich, St. Louis, MO) was added to each well (0.5 mg/
mL, final concentration) and plates were incubated for an addi-
tional 3 h at 37 °C. Medium was then aspirated and the formazan
product was solubilized in SDS–HCl (20% SDS; HCl 0.02 M). The

6494
P. Quintana-Espinoza et al. / Bioorg. Med. Chem. 21 (2013) 6484–6495
absorbance of each well was measured at 570 nm using a micro-
plate reader. Non linear regression analysis was performed to cal-
culate IC50 according to the GraphPad Prism 5 program (GraphPad
Software, San Diego, CA). The data are expressed by mean SD
(n = 3).
to just DMSO 1% (v/v) was then calculated and plotted against
the logarithm of drug concentrations.
4.3.7. Assay for G2 arrest and DNA repair nuclear Rad52 foci
Strain W3749-14C (RAD52-YFP) form S/G2/M-specific nuclear
YFP foci under different types of DNA damage; especially those
that generate DSBs.32 For this assay, yeast cells were treated with
4.3.2. TopoII-mediated DNA relaxation assay
The hTopoII
UK). The reaction conditions were as described before37 with min-
or modifications. Briefly, the 20 L reaction mixture contained
a
enzyme was purchased from Inspiralis (Norwich,
100
at 25 °C. Chemical treatments with DMSO 1% (v/v) and Phleomycin
25 g/mL were also included as controls. After 3 h, samples were
lM of the tested compounds while asynchronously growing
l
l
250 ng of pRYG plasmid DNA purchased from TopoGEN (Columbus,
taken and micrographed. All cells in the field were then classified
according to the cell cycle stage they were in. Mononucleated bud-
ded cells where the bud size was more than two-thirds that of the
mother were considered to be arrested in G2. Rad52 foci were de-
tected and quantified in the YFP channel by using the CellProfiler
software.41 Briefly, whole YFP images were normalized following
the procedure: most intense foci in the first photo taken for Phleo-
mycin was set to 1, least intense pixel of the background was set to
0. A lower threshold of 0.1 was set for foci recognition. Then, total
number of detected foci were divided by total number of cells to
obtain the average foci number per cell.
OH) and 1 mM ATP in the assay buffer [5 mM TrisꢂHCl (pH 7.5),
12.5 mM NaCl, 1 mM MgCl2, 0.5 mM DTT and 10 lg/mL albumin].
The reaction components were added as follows: assay buffer,
DNA, chemical compound from DMSO stock or just DMSO, and fi-
nally 1–2 units of hTopoIIa. The reaction mixture was incubated at
37 °C for 30 min, and quenched with 1% (w/v) SDS and 25 mM Na2-
EDTA. The mixture was treated with 0.25 mg/mL proteinase K
(Roche) at 55 °C for 30 min to digest the protein. pRYG DNA topoi-
somers were resolved in 1% (w/v) agarose gel electrophoresis
(0.5 V/cm) in 1ꢃ TBE buffer (89 mM Tris–borate and 2 mM Na2-
EDTA, pH 8) without ethidium bromide. The photograph was taken
after staining the gel with ethidium bromide.
Acknowledgments
4.3.3. Stabilization of the TopoII cleavage-complex
The same procedure was used as above but this time 10 units of
hTopoIIa were added 1–3 min before the compound addition. Elec-
trophoresis was run (0.5 V/cm) in TAE buffer with 0.5
dium bromide.
We gratefully acknowledge the financial support from Spanish
Ministry of Science and Innovation (SAF 2009-13296-C02-01 and
SAF 2012-37344-C03-01 to A.E.B. and SAF 2009-13296-C02-02
and SAF 2012-37344-C03-02 to L.F.P.), Instituto de Salud Carlos
III (PS09/00106 and PI12/00280 to F.M.), and the EU Research Po-
tential (FP7-REGPOT-2012-CT2012-31637-IMBRAIN). All projects
to principal investigators are also co-founded by the European Re-
gional Development Fund (ERDF). P.Q.E. thanks Gobierno de Chile
for a predoctoral fellowship. J.G-L. thanks Spanish Ministry of Edu-
cation for a predoctoral fellowship (AP2009-2511). We also thank
Professor M. Fernández-Braña for his interest in our work and
helpful discussions and advices.
lg/mL ethi-
4.3.4. TopoI-mediated DNA relaxation assay
The wTopoI enzyme (Promega) was used. The reaction condi-
tions were as follows: assay buffer [50 mM Tris–HCl (pH 7.5 at
25 °C), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT and 20% glycerol],
DNA (250 ng of pBSKS plasmid), chemical compound from DMSO
stock or just DMSO, and 5 units of wTopoI enzyme were added
in that order. The reaction mixture was incubated at 37 °C for
30 min, and quenched with 1% (w/v) SDS and 25 mM Na2EDTA.
The mixture was treated with 0.25 mg/mL proteinase K (Roche)
at 55 °C for 30 min to digest the protein. pBSKS DNA topoisomers
were resolved in 1% (w/v) agarose gel electrophoresis (0.5 V/cm)
in 1ꢃ TBE buffer (89 mM Tris–borate and 2 mM Na2EDTA, pH 8)
without ethidium bromide. Since wTopoI tended to give a nearly
fully relaxed form of the plasmid in our conditions, we also run a
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.08.039.
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