Design, synthesis, and biological evaluation of substituted naphthalene imides and diimides as anticancer agent∞

Article abstract of DOI:10.1021/jm901131m

Naphthalimmide (NI) and 1,4,5,8-naphthalentetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity. NDI derivatives 1-9 were more cytotoxic than the corresponding NI derivatives 10-18. The molecular mec

Full text of DOI:10.1021/jm901131m

J. Med. Chem. 2009, 52, 7873–7877 7873
DOI: 10.1021/jm901131m
Design, Synthesis, and Biological Evaluation of Substituted Naphthalene Imides and
Diimides as Anticancer Agent¥
Vincenzo Tumiatti,*,†,§ Andrea Milelli,† Anna Minarini,† Marialuisa Micco,† Anna Gasperi Campani,#, Laura Roncuzzi,#,^
Daniela Baiocchi,# Jessica Marinello,‡ Giovanni Capranico,‡ Maddalena Zini,‡ Claudio Stefanelli,‡ and Carlo Melchiorre†
†Department of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, §Polo
Scientifico-Didattico di Rimini, University of Bologna, 40100 Bologna, Italy, ‡Department of Biochemistry “G. Moruzzi”, University of Bologna,
Via Irnerio 48, 40126 Bologna, Italy, #Department of Experimental Pathology, University of Bologna, Via S. Giacomo 14, 40126 Bologna, Italy,
Interdepartmental Center for the Research on Cancer, Policlinico S. Orsola, University of Bologna, Via Massarenti 9, Bologna, Italy, and
^Laboratory of Pathophysiology of Orthopaedic Implants, Istituto Ortopedico Rizzoli, Via di Barbiano 1/10, Bologna, Italy
Received May 21, 2009
Naphthalimmide (NI) and 1,4,5,8-naphthalentetracarboxylic diimide (NDI) derivatives were synthe-
sized and evaluated for their antiproliferative activity. NDI derivatives 1-9 were more cytotoxic than
the corresponding NI derivatives 10-18. The molecular mechanisms of 1 and 2 were investigated in
comparison to mitonafide. They interacted with DNA, were not topoisomerase IIR poisons, triggered
caspase activation, caused p53 protein accumulation, and down-regulated AKT survival. Furthermore,
1 and 2 caused a decrease of ERK1/2 and, unlike mitonafide, inhibited ERKs phosphorylation.
Introduction
The search for novel chemotherapeutic agents and ap-
proaches to cancer treatment is an active research field
stimulated by the discovery of new biological targets and by
the possibility of obtaining new drugs without serious and
undesirable side effects.1 Small molecules able to reversibly
interact with the DNA structure represent a prolific research
area for new potential anticancer agents.2 Such molecules,
Figure 1. Chemical structures of N-DMPrNI and N-BDMPrNDI
known as intercalators, are typically characterized by a planar
derivatives and reference compound mitonafide.
heterocyclic moiety of approximately the size and shape of a
minor grooves.6 In the literature, there are several examples of
naphthalimmide (NIa) and 1,4,5,8-naphtalentetracaboxylic
diimide (NDI) derivatives as intercalating and anticancer
agents.2,7-10 Two important prototypes of NI series are
mitonafide and amonafide (Figure 1) which, although char-
acterized by a valuable anticancer profile, did not pass the
phase II because of their toxicity.11,12 Several attempts were
performed to overcome this limitation by synthesizing diff-
erent analogues.13-15 Two examples of NDI series are
N-DMPrNI and N-BDMPrNDI (Figure 1) which showed
the ability to intercalate into steps containing at least one G:C
base pair.9
These considerations prompted us to design1-18, using NI
and NDI scaffolds as molecular building blocks. 1-18 con-
tain one or two basic polymethylene chains in their structures
whose lengths may have relevance for the anticancer activity.
The protonation of one or two basic nitrogen atoms at
physiological pH strongly influences the DNA binding, as
reported above.6 To enhance the basicity of the terminal
amine functions of lateral chains, a 2-methoxybenzyl substi-
tuent was chosen. This is because, as previously demon-
strated,16 this function improved the basicity when inserted
on terminal nitrogen atoms of related polymethylene amines.
It could also promote additional DNA hydrophobic interac-
tions. Finally, the cytotoxic activity of the most interesting
DNA base pair.3 They insert perpendicularly into the DNA
without forming any covalent bonds, and the formed complex
is stabilized by hydrophobic, van der Waals, hydrogen bond-
ing, and charge transfer forces. It has been demonstrated that
these interactions may induce unwinding, lengthening, and
stiffening of the double helix, introducing structural changes
that can affect the cells’ replication processes, leading to
cellular death and genotoxic effects.4 Several DNA binding
modes were reported and include electrostatic interactions,
groove binding, and threading intercalation.
It is well-known that, at physiological pH, protonated
polyamines interact strongly with the phosphate residues of
DNA.5 Therefore, the inclusion of these basic functionalities
on an intercalating moiety may improve the interaction with
DNA structure by inserting their aromatic system between the
base pairs, while the cationic heads bind with the major and
¥ Dedicated to Professor Fulvio Gualtieri on the occasion of his
retirement.
*To whom correspondence should be addressed. Address: Depart-
ment of Pharmaceutical Sciences, University of Bologna, Via Belmeloro
6, 40126 Bologna, Italy. Phone: þ39-051-2099709. Fax: þ39-051-
2099734. E-mail: vincenzo.tumiatti@unibo.it.
a Abbreviations: NI, naphthalimmide; NDI, 1,4,5,8-naphthalenete-
tracarboxylic diimide, MAPK, mitogen-activated protein kinase; ERK,
extracellular signal regulated kinase.
r
2009 American Chemical Society
Published on Web 09/16/2009
pubs.acs.org/jmc

7874 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Tumiatti et al.
Scheme 1a
Table 1. Cytotoxic Activity of 1-18 against SKBR-3 and CEM Cells
after 72 h and against HL60 after 24 h of Compounds Exposure
IC50 (μM)b
a (i) EtOH-toluene 1:1, reflux, 24 h, quantitative yield; (ii) (a)
2-MeOC6H4CHO, toluene, reflux, 3 h; (b) NaBH4, MeOH, room temp,
4 h, 21-70%.
compda
n
SKBR-3
CEM
HL60d
mitonafide
1
1.6
0.9
2
0.7
0.2
0.7
1.1
1.2
1.7
1.0
0.4
0.2
2
nac
1.7
nac
nac
nac
1.4
0.5
2
1.3
0.4
0.8
1.1
2.2
2.5
1.6
1.4
0.7
nac
nac
3.2
nac
nac
nac
1.2
1.3
8
2
3
1.2
Scheme 2a
3
4
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
4
5
5
6
6
7
7
8
8
9
9
10
2
a (i) EtOH, reflux, 24 h, 20-85%; (ii) (a) 2-MeOC6H4CHO, toluene,
reflux, 3 h; (b) NaBH4, EtOH, room temp, 3 h, 24-36%.
10
11
12
13
14
15
16
17
18
3
4
compounds, 1 and 2, was investigated at the molecular level in
comparison to mitonafide.10
5
6
7
Results and Discussion
8
9
10
Chemistry. Compounds 1-9 were synthesized following
the procedure reported in Scheme 1. Naphthalenetetracar-
boxylic dianhydride was condensed with the correspond-
ing polymethylene diamine (n=4-10) to give 21-27. These
compounds and conjugates 19 and 2017 were treated with
2-methoxybenzaldehyde followed by reduction with sodium
borohydride of formed Schiff bases to afford the correspond-
ing diamine diimides 1-9. The same procedure was adopted
for the synthesis of 10-18, using benzo[de]isochromene-1,
3-dione as starting anhydride, through intermediate amines
28-36 (Scheme 2).
Growth-Inhibiting Activity. Derivatives 1-18 were evalu-
ated by in vitro assays for their antiproliferative activity in
human breast cancer (SKBR-3) and leukemia (CEM) cell
lines. Growth inhibition induced by tested compounds was
assessed by the 3-(4,5-dimethylthiazolyil-2)-2,5-diphenylte-
trazolium bromide (MTT) assay. Cells were treated with the
test compounds at concentrations ranging from 0.1 to 10 μM
for 72 h.
The results shown in Table 1 reveal that bis-substituted
derivatives are more potent than the corresponding mono-
substituted derivatives, with the exception of 7 and 8 which
showed comparable activity values with those of 16 and 17.
Interestingly, the chain length of the side chains of bis-
substituted derivatives 1-9 significantly affected cytotoxic
activity in both cell lines. The most potent were 2 and 9 with
chain lengths of 3 and 10 methylene units, respectively,
separating the nitrogen atoms of the side chains. This finding
suggests that the terminal positive charged nitrogen atoms of
2 and 9 may interact with different anionic counterparts of
DNA. The same pattern was not observed in the monosub-
stituted series, in which the only notable derivative was 17
with an IC50 on SKBR-3 of 0.5 μM, clearly indicating that a
second lateral chain contributes to enhancing cytotoxic
activity.
a 1-9, di-p-toluensulfonate salts; 10-18, p-toluensulfonate salts.
b IC50 values represent the concentration causing 50% growth inhibi-
tion. They were determined by linear regression method. Each sample is
the mean of six experiments. c Not active at 10 μM. d The IC50 values are
reported as the mean of three determinations. Cell death was determined
by dye exclusion. e Not determined.
mechanism. Their choice was dictated by the observation
that 2 was the most potent of both series of compounds on
SKBR-3 and CEM cells, and 1 shares similar chemical
features (side chain length) with mitonafide. Thus, the
cytotoxic activity of 1 and 2 was further assessed against
HL60 leukemia cells in comparison to mitonafide. 1 and 2
confirmed their antiproliferative activity toward this cell
line, with a potency that was comparable to that of mitona-
fide, as revealed by their respective IC50 (μM) values of 0.9,
1.2, and 1.6 (Table 1).
DNA-Binding Properties. Mitonafide and NDI derivatives
are well-known intercalators. To assess the ability of 1 and 2
to interact with DNA, DNA-binding studies were carried
out in comparison to mitonafide, using a fluorometric inter-
calator displacement method.18 DNA-binding activity was
expressed as the drug concentration reducing by 50% the
fluorescence of DNA-bound ethidium bromide. The EC50 is
approximately inversely proportional to the binding con-
stant, and an apparent binding constant18 (Kapp), which
estimates the affinity of the drug for calf thymus DNA, can
be calculated.18,19 1 and 2 turned out to potently bind DNA
and displace ethidium bromide with EC50 values that were
2 orders of magnitude higher than that of the reference
compound mitonafide (Table 2).
Topoisomerase Inhibition. Compounds 1, 2, 10, and 17
were also evaluated by using the DNA SV40 portion as
substrate for the recombinant human DNA topoisomerase
IIR assays to verify whether they act as topoisomerase
IIR poisons.20 It turned out that they were ineffective in
After these first screening assays, 1 and 2 were selected for
further investigation to determine their cytotoxic molecular

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7875
Table 2. Binding to DNA by Ethidium Displacement Test of 1, 2, and
Mitonafide
compda
EC50 (nM)a
Kapp (M-1 b
)
mitonafide
11500 ( 320
93 ( 4
122 ( 6
2.20 ꢀ 106
2.72 ꢀ 108
2.07 ꢀ 108
1
2
a EC50 values are defined as the drug concentrations that reduce the
fluorescence of the DNA-bound ethidium by 50% and are reported as
the mean of three determinations ( SEM. b Apparent binding constant
(Kapp) values have been calculated taking the ethidium binding constant
as 107 M-1
.
Figure 3. Effect of 1, 2, and mitonafide (Ref) on signal transduction
pathway correlated to cell survival in SH-SY5Y neuroblastoma
cells. (A) The cells were incubated for the indicated time in the
presence of 1 and 2 or mitonafide at concentrations ranging from
1 to 5 μM. Then the number of alive viable cells was measured by dye
exclusion. (B) The content of p53 protein was measured in extracts
obtained from cells incubated 20 h in the presence of the indicated
concentration of 1. An amount of 50 μg of protein was separated
and analyzed by Western blotting for each point. (C) The cells were
incubated for 20 h in the presence of 5 μM of the indicated
compound. Then cells were collected. The content and phosphory-
lation status of the indicated proteins in cell extracts were deter-
mined by Western blotting (50 μg of protein/lane).
protein extracted from cells treated with the aforementioned
compounds confirmed the activation of caspase-3
(Figure 2B). In treated cells, we observed a reduction of the
inactive precursor procaspase 3 (32 kDa) associated with the
appearance of the proteolytic fragment, constitutive of the
active enzyme (Figure 2B). It is worth noting that 1 and 2
triggered caspase activation rapidly. After a few hours of
treatment, a large part of the procaspase 3 was processed into
the active form, leading to apoptosis. The early activation of
caspase activity increased in a dose-dependent manner asso-
ciated with the increased number of cells committed to death
(Figure 2C). We further investigated the ability of the most
potent derivative 1 to trigger caspase activation on different
cell lines, such as SH-SY5Y neuroblastoma cells, H9c2 heart
cells, and Jurkat T-cells. Figure 2D shows that 1 elicited
caspase activation in all tested cells. Activation of apoptosis
can be initiated by extracellular apoptogenic compounds or
intracellular insults and often requires the activation of
specific signaling cascades.23 The involvement of certain
putative biochemical pathways in the induction of apoptosis
was examined in SH-SY5Y neuroblastoma cells (Figure 3).
These cells responded to 1 and 2 similarly to the other cell
types previously evaluated, and 5 μM 1 or 2 caused a massive
cell death after 72 h (Figure 3A). Mitonafide toxicity was
very similar to that of 1 and 2. Their IC50 values could be
estimated in the range 1.4-1.9 μM. First, we examined
whether the toxic effect of 1 and 2 was accompanied by
Figure 2. Derivatives 1 and 2 and mitonafide trigger caspase
activation. (A) The activity of caspase proteases acting on the
peptide sequence DEVD (DEVDase activity) was measured in
HL60 cells treated for 4 or 24 h with 5 μM of the indicated
compound. (B) HL60 cells were incubated for 4 h in the presence
of the indicated compound (5 μM). Then the cellular proteins were
extracted and analyzed by Western blotting. (C) HL60 cells were
treated for 4 h with the indicated dose of the test compound.
Afterward, cells were collected for caspase activity determination.
(D) Caspase activation was measured in SH-SY5Y neuroblastoma,
H9c2 heart cells, and Jurkat T-cells following treatment for 24 h
with 5 μM 1. Data are mean values ( SEM of triplicate measure-
ments.
stimulating DNA cleavage by recombinant human topo-
isomerase IIR (Figure 1SI of Supporting Information).
Cytotoxic Effects. NI derivatives can exert their antipro-
liferative effect by multiple independent mechanisms.14,15,21
First, we determined whether 1 and 2 caused the activation of
apoptosis. To this end, HL60 cells were treated with 1, 2, and
mitonafide, followed by the assessment of the activity of
caspase proteases acting on the substrate sequence Asp-Glu-
Val-Asp (DEVD), i.e., mainly effector caspases 3 and 7,
whose activation represents a marker of apoptotic cell
death.22 It was found that 1 and 2, like mitonafide, triggered
caspase activation (Figure 2A). Western blotting analysis of

7876 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Tumiatti et al.
changes in the expression of the p53 protein, whose up-
regulation is generally associated with DNA damage.24,25 In
cells treated for 20 h with the most potent derivative 1, p53
accumulation was evident (Figure 3B) even with the lowest
dose studied (1 μM), which caused a very limited toxicity at
24 h and about 60% of cell survival after 72 h. A large
accumulation of the p53 protein was also evident following
treatment with 2 or mitonafide (5 μM) for 20 h (Figure 3C).
Next, the effect of 1 and 2 on signal transduction pathway
involved in cell death and survival was investigated. In these
studies, the cells were treated with 5 μM of the different
agents for 20 h. At this time point, the amount of viable cells
was quite high, ranging from 70% to 80%, but virtually all
cells were committed to death within 72 h. The kinase Akt, an
oncoprotein correlated to cell survival and proliferation, up-
regulated in several cancers, and responsible for resistance to
apoptotic cell death26 which very recently has been asso-
ciated with the toxic effect of an amonafide analogue, was
examined.21 Interestingly, 1, 2, and mitonafide caused an
intense down-regulation of the kinase Akt and abolished its
phosphorylation (Figure 3C). ERK1/2 mitogen-activated
protein kinases are generally associated with cell growth,
even if in some circumstances they may contribute to activa-
tion of apoptosis.27 In the case of ERK1/2, the effect of 1 and
2 was completely different with respect to mitonafide
(Figure 3C). In fact, mitonafide caused a slight decrease of
p42 and p44 ERK proteins while increasing their phosphory-
lation. In contrast, 1 and 2 caused a large decrease of the p42
ERK2 protein and completely inhibited the phosphorylation
of p42 and p44 ERKs, which are known to influence the
survival of cancer cells.28
or 10-18, respectively) in toluene was refluxed in a Dean-Stark
apparatus for 3 h. Following solvent removal, the residue was
taken up in EtOH, NaBH4 (in a 1:5 or 1:2.5 molar ratio for 1-9
or 10-18, respectively) was added, and the stirring was con-
tinued at room temperature for 4 h. The mixture was then made
acidic with 6 N HCl and the solvent removed. Then the residue
was dissolved in water and the resulting solution was washed
with ether, made basic with K2CO3, and extracted with CH2Cl2
(3 ꢀ 30 mL). Removal of the dried solvent gave the desired
products 1-18 that were converted into the corresponding
p-toluenesulfonates salts (see Supporting Information for the
characterization of compounds).
Acknowledgment. The authors thank Angela De Simone
and Dorina Campana for technical assistance. This research was
supported by a grant from MIUR, Rome (PRIN), University
of Bologna (RFO), and Polo Scientifico-Didattico di Rimini.
Supporting Information Available: Experimental details for
chemistry, biology, characterization, and elemental analyses
of target compounds; experimental details and characterization
of intermediate compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Experimental Section
Melting points were taken in glass capillary tubes on a Buchi
SMP-20 apparatus and are uncorrected. ESI-MS spectra were
1
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