Nonhematotoxic naphthalene diimide modified by polyamine: Synthesis and biological evaluation

Article abstract of DOI:10.1021/jm300168w

With the aim of up-regulating antitumor efficacy and down-regulating adverse effects, three types of aromatic imide and diimides were designed to couple with different polyamines. The in vitro assays revealed that two naphthalene diimide-polyamine conjuga

Full text of DOI:10.1021/jm300168w

Article
pubs.acs.org/jmc
Nonhematotoxic Naphthalene Diimide Modified by Polyamine:
Synthesis and Biological Evaluation
Yuxia Wang,† Xingbo Zhang,‡ Jin Zhao,§ Songqiang Xie,*,† and Chaojie Wang*,§
‡
§
†Institute of Chemical Biology, Department of Chemistry, and Key Lab of Natural Medicine and Immune Engineering,
Henan University, Henan, China
ABSTRACT: With the aim of up-regulating antitumor efficacy and
down-regulating adverse effects, three types of aromatic imide and
diimides were designed to couple with different polyamines. The in
vitro assays revealed that two naphthalene diimide−polyamine
conjugates could inhibit the growth of multiple cancer cell lines
more potently than amonafide. 9f, the most potent compound, was
verified to efficiently induce apoptosis via a ROS mediated
mitochondrial pathway in a preliminary mechanistic study. The
comprehensive in vivo trials on three H22 tumor transplant models
demonstrated that 9f improved the indexes in terms of inhibitive
effect and lifespan extension and reduced the hematotoxicity which is
one of main drawbacks of amonafide. More importantly, the
obviously elevated ability in preventing lung cancer metastasis was
observed, which increased the value of 9f as a promising lead
compound. This work supported that the versatile function of polyamines may endow some intriguing biological features to the
parent drugs.
the aromatic ring to prevent hematotoxic results.8 Qian et al.
suggested a strategy to furnish multitargeted naphthalimides by
1. INTRODUCTION
Cancer is well-known as one of the most fatal diseases featured
varying the side chains and substituents with polyamine
by eternal cell proliferation and the spreading of cancerous cells
moieties and lipophilic alkyl chains.9 Naphthalene diimides
to other tissues by invasion or metastasis. Undoubtedly, cancer
such as N-BDMPrNDI (Figure1) are attractive DNA
is an unsolved problem to date, and the rapid growth in cancer
intercalators similar to naphthalimides.10 A recent report of
cases represents a real crisis for public health in the world. In
Tumiatti et al. focused on the naphthalene diimides, which are
spite of side effects caused by chemotherapy treatment such as
found to be more active than their corresponding mono-
the occurrence of myelosuppression and the development of
drug resistance, it is still recognized as the main choice to
prolong the patient’s life. Therefore, the discovery of novel,
imides.11
Diverse polyamine derivatives have been extensively studied
for their intriguing bioactivities.12−15 In recent years, polyamine
selective, efficient, and safe drugs for cancer chemotherapy
conjugates have attracted increasing attention because the
remains an urgency and high priority for medicinal research.
antitumor agents modified by polyamines may possess
In therapeutics, aromatic imides have been recognized as a
improved bioactivity and cell selectivity.16−18 After efforts of
promising group of antitumor agents. Some phathalimides such
as thalidomide and lenalidomide are now in clinical use.1,2
Naphthalimides are also a class of intriguing pharmacophores
because of their broad bioactivities.3,4 Many naphthalimides
reached clinical trials, but most of them were abandoned
because of various adverse effects such as dose-limiting bone
marrow toxicity. As a representative, amonafide (Figure 1) is
currently undergoing phase III clinical trials in combination
with cytarabine in patients with acute myeloid leukemia.5 In
view of both the evidenced antitumor activity and negative
effects of these compounds, extensive efforts including the
modification of side chain, aromatic ring system, and the
substituents on the ring have been attempted to search for
more selective naphthalimides to improve the potency and
reduce the adverse effects (Figure 1).6,7 In an excellent paper
with comprehensive in vivo messages, Kiss et al. reported the
amonafide derivatives by the modification of amino group on
several decades, F14512, an epipodophyllotoxin−spermine
conjugate, is perhaps the most promising compound which is
presently being evaluated in phase I trials.19,20 There are several
reports about naphthalimide polyamine conjugates as well.21,22
Our work demonstrated that the naphthalimide coupled with
spermidine or homospermidine (Figure 1, MNISpd,
MNIHspd) exhibited antineoplastic potency and, more
importantly, excellent cell selectivity in a normal versus
transformed cells screen.23−25 These findings stimulated us to
search for more promising imide scaffolds suitable to polyamine
modification. We report herein three kinds of aromatic imide/
diimides (with anhydrides as starting materials), which are
coupled with diverse polyamine motifs (Figure 2). In addition
Received: February 7, 2012
Published: March 19, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of amonafide, N-BDMPrNDI, MNISpd, and MNIHspd.
Figure 2. Building blocks of the designed library.
a
Scheme 1
a
(a) 1.0 equiv of R1NH2, EtOH, reflux, 3 h; (b) 1.0 equiv of PhB(OH)2, 1.5 equiv of K2CO3, catalytic amount of TBAB and PdCl2, toluene, reflux;
(d) EtOH, 4 M HCl, room temperature, overnight.
a
Scheme 2
a
(a) 1.0 equiv of R1NH2, EtOH/toluene, reflux; (b) EtOH, 4 M HCl, room temperature, overnight.
to the routine in vitro evaluation, in vivo experiments are
designed to provide more data for future drug development.
at room temperature to provide target compounds 7a−g as
hydrochloride salts.
The diimides compounds 9b−g and 11e−g were synthesized
following a similar two-step procedure reported in Scheme 2. The
naphthalene and biphenyl tetracarboxylic dianhydrides (2, 3) were
condensed with corresponding amines (4) to give 8b−g and 10e−
g, respectively.11,28 After purification by flash column chromatog-
raphy, these intermediates were mixed with 4 M HCl at room
temperature to obtain the target compounds (9b−g, 11e−g) in the
form of hydrochloride salts with a yield between 60% and 80%.
2.2. Growth-Inhibiting Activity. Novel compounds were
evaluated by in vitro MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assays for their antiproliferative
2. RESULTS AND DISCUSSION
2.1. Chemistry. The phenyl substituted naphthalimide−
polyamine compounds (7a−g) were synthesized according to
Scheme 1. Intermediates 5a−g were obtained by the
condensation of R1NH2 (4; the Boc protected polyamines
were prepared by a modified procedure reported previ-
ously23,24) with 4-bromo-1,8-naphthalic anhydride (1). The
Suzuki coupling reaction of 5 with phenylboronic acid catalyzed
by PdCl2 gave intermediates 6,26,27 and their Boc groups in the
polyamine skeleton were subsequently removed with 4 M HCl
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Table 1. In Vitro Activity of New Compounds against Four Kinds of Tumor Cell Lines
a
IC50 (μM)
compd
R2, X
HepG2
MDA-MB-231
K562
HCT-116
amonafide
7a
R2 = (CH2)2N(CH3)2, X = 0
36
42
31.7
>50
14.4
14.4
14.2
14.2
1.01
21.2
41.0
21.2
32
nd
nd
nd
nd
nd
b
b
b
b
b
R2 = (CH2)2N(CH3)2, X = l
37.0
17.1
15.6
15.8
32.1
2.83
33.3
43.8
48.5
1.22
>50
>50
>50
>50
39.2
19.2
14.6
14.5
19.1
2.95
20.2
27.6
18.0
0.45
>50
>50
>50
>50
7d
R2 = (CH2)4NH(CH2)4NH2, X = 2
7e
R2 = (CH2)3NH(CH2)3NH(CH2)3NH2, X = 3
R2 = (CH2)3NH(CH2)4NH(CH2)3NH2, X = 3
R2 = (CH2)4NH(CH2)4NH(CH2)4NH2, X = 3
R2 = (CH2)3NH(CH2)3NH2, X = 4
7f
7g
9b
5.02
b
9c
R2 = (CH2)4NH(CH2)3NH2, X = 4
nd
b
9d
R2 = (CH2)4NH(CH2)4NH2, X = 4
nd
b
9e
R2 = (CH2)3NH(CH2)3NH(CH2)3NH2, X = 6
R2 = (CH2)3NH(CH2)4NH(CH2)3NH2, X = 6
R2 = (CH2)4NH(CH2)4NH(CH2)4NH2, X = 6
R2 = (CH2)3NH(CH2)3NH(CH2)3NH2, X = 6
R2 = (CH2)3NH(CH2)4NH(CH2)3NH2, X = 6
R2 = (CH2)4NH(CH2)4NH(CH2)4NH2, X = 6
nd
b
9f
nd
0.13
b
9g
28
nd
b
11e
11f
11g
nd
>50
b
b
nd
nd
b
nd
>50
a
IC50 values represent the concentration causing 50% growth inhibition. They were determined by the linear regression method. Each sample is the
b
mean of three independent experiments. Not determined.
activity against HCT-116 (human colorectal cancer cell line),
K562 (human leukemia cell line), MDA-MB-231 (human
breast cancer cell line), and HepG2 (human hepatoma cell
line). Amonafide was tested as a reference compound. The
structures and biological results of target compounds were
shown in Table 1. The polyamine modified monoimides (7c−
g) exhibited better cytotoxic activity than amonafide, while 7a,
with a simple replacement of 5-amino by 6-phenyl group in
amonafide, did not result in biological improvement. On the
other hand, all 6-phenyl naphthalimide−polyamine conjugates
are less potent in vitro than previously reported naphthali-
mide−polyamine conjugates.23,24 Two series of diimide−
polyamine conjugates displayed contrary inhibitive behaviors
against tested tumor cells. Surprisingly, the biphenyl
diimides (11e−g) were nontoxic, whereas naphthalene
diimide derivatives exhibited mixed results. Compounds 9b
and 9f displayed much improved potency in comparison to
the other analogues in this series, which were just as potent
as amonafide. 9b and 9f, the most active compounds in the
three types of synthesized imide/diimides, were as potent as
previously reported naphthalimide−polyamine conju-
gates.23,24
To furnish more comprehensive and credible information
about the cytotoxicity produced by the tested compounds, we
applied a high content screening (HCS) technique to assess
related cytotoxic indicators in the HepG2 cells. HCS can
simultaneously monitor these multiple indicators and detect the
earliest onset of toxic effects of tested compounds to increase
the predictive power of the in vitro assay. Indeed, cytotoxicity is
a complicated gradual process in which changes in nuclear
morphology, mitochondrial membrane potential, lysosomal
mass/pH, and cell membrane permeability could be observed.29
In the experiments, changes in nuclear morphology are
monitored by staining with the nuclear dye Hoechst 33342.
Cell membrane permeability changes are measured using a
permeability dye that stains nuclei in permeability compro-
mised cells, giving a green fluorescence. Mitochondrial
transmembrane potential changes are measured using a
mitochondrial membrane potential dye that accumulates in
healthy mitochondria with intact membrane potential and is
absent from depolarized mitochondria. Changes in lysosomal
mass/pH are measured using a dye that is a weak base and
accumulates in lysosomes during cytotoxicity occurs.
Results in Figure 3A corroborated that some typical imide−
polyamine derivatives displayed cytotoxicity in HepG2 cell line
in vitro. In Figure 3A, after treatment with the most potent
compound 9f, cell nuclear shrinkage, membrane permeability
augmentation, mitochondrial membrane potential degradation,
and lysosomal mass/pH accruement were detected in a dose
dependent manner. 7a and amonafide with equivalent IC50
values performed similarly in these indicators. No significant
changes of these indicators were found in cells treated by less
toxic compounds such as 11g.
In Figure 3B, the cytotoxicity of 9f was more powerful than
that of amonafide. For example, after treatment with the same
dose of 9f and amonafide (10 μM), the green nucleus and red
fluorescence of 9f were obviously increased, suggesting that the
integrity of cell membrane was broken down and cytolysosome
was activated more potently. Furthermore, the green
fluorescence of Rh123 in cytoplasm was significantly decreased,
indicating that the mitochondria were destroyed and
mitochondrial transmembrane potential was lost.
The measured indicators provide useful information about
the mitochondrial membrane potential and lysosomal mass
change which are valuable preliminary clues for further
mechanistic study. For example, the pronounced variation of
lysosomal mass/pH triggered by some imide−polyamine
conjugates, observed in Qian’s report as well, suggested that
lysosomes may be the target of these compounds. Interestingly,
amonafide affects LMP in HepG2 cells but does not in HeLa
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Figure 3. (A) The in vitro cytotoxicity of some new compounds was evaluated in HepG2 cell line after treatment for 48 h. All data are expressed as
the mean SD from three separate determinations. (B) Representative images showing simultaneous monitoring of changes in cytotoxic indicators.
Changes in mitochondrial transmembrane potential (green cytoplasm), cell membrane permeability (green nucleus), nuclear morphology (blue),
and lysosomal mass/pH (red) were measured in HepG2 cells after treatment with 9f or amonafide for 48 h. The cells were imaged using an
ArrayScan HCS reader. Scale bar = 10 μm.
cells.9 On the other side, the changes of mitochondrial
membrane potential after drug treatment implied the
occurrence of cell apoptosis.
Double staining of HepG2 cells with FITC-labeled annexin V
and propidium iodide (PI) was used to illustrate the drug-
induced apoptosis. In apoptotic cells, the membrane
phospholipid phosphatidylserine (PS) is translocated from
the inner to the outer leaflet of the plasma membrane, thereby
exposing PS to the external cellular environment. Annexin V, a
Ca2+ dependent phospholipid-binding protein that has a high
affinity for PS, can bind to cells with exposed PS. Annexin V
may be conjugated to fluorochromes such as FITC. This format
retains its high affinity for PS and thus serves as a sensitive
probe for the analysis of cells that are undergoing apoptosis.
Since externalization of PS appears in the earlier stages of
2.3. Apoptosis. Apoptosis or programmed cell death, a
favorable ultimate fate of tumor cells, is a complicated cellular
process and a major field of interest in antitumor drug
development.30 We and other groups found that naphthalene
monoimides and diimides could trigger cell apoptosis.11,23,24
The above-mentioned MMP changes induced by these newly
synthesized naphthalimides suggested that they might produce
cytotoxicity through apoptosis. Because of better biological
properties, 9f was chosen for further investigation.
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Figure 4. After treatment with 9f (10 μM) or amonafide (10 μM), annexin V−FITC/PI double staining and caspase activation for apoptosis
evaluation and DCFH-DA staining for ROS assay were detected using HCS in HepG2 cells. Images were acquired on the ArrayScan HCS reader
using Cellomics’ Target Activation BioApplication: scale bar = 10 μm; compared with control, (∗∗) p < 0.01.
apoptosis, annexin V−FITC staining can identify apoptosis at
an earlier stage. Viable cells with intact membranes exclude PI,
whereas the membranes of dead and damaged cells are
permeable to PI and the PI bind to cellular DNA. This
assessment can distinguish cell apoptosis from necrosis.
Caspase 3 is one of the key proteases in early apoptosis, and
it is known that naphthalimides could lead to caspase-
dependent apoptosis.31 Therefore, the activation of caspase 3
was measured by indirect immunofluorescence, using primary
antibody against the cleaved portions of caspase 3.
We previously reported that significant reactive oxygen
species (ROS) accumulation triggered by MNISpd is closely
related to polyamine oxidase (PAO) activity.25 Destabilization
of the lysosome has recently been linked to cytotoxicity involv-
ing ROS generation and associated disruption of mitochondrial
membrane potential.32 On the basis of the above-mentioned
results, ROS generation was measured by the DCFH-DA (2′,7′-
dichlorodihydrofluorescein diacetate) method as well.
As shown in Figure 4, after incubation for 48 h, both 9f and
amonafide did induce HepG2 cell apoptosis, supported by the
significant variation of annexin V, accompanied by a
pronounced generation of ROS and an increase in caspase 3
activation as evidenced by the staining of active (cleaved)
caspase 3. At the same concentration, 9f could lead to more
obvious variations in three detected indexes than amonafide.
These initial data suggested that this apoptosis is triggered via a
ROS-mediated mitochondrial pathway.25,33
Tumiatti et al. also found that some naphthalene diimides
and the reference drug mitonafide (similar to amonafide in
which the 5-NH2 is replaced by 5-NO2) activate caspase 3 and
trigger human HL60 leukemia cells apoptosis.11 Interestingly,
the detailed reports by Kiss et al. revealed that UNBS3157 and
UNBS5162, the 5-urea substituted mononaphthalimide de-
rivatives, exert antitumor effects via nonapoptotic manner,
which is evidently different from that of amonafide.8,34 For
example, 10 μM UNBS5162 (a hydrolyzed product of
UNBS3157) does not trigger apoptosis but induces autophagy
and senescence in tested human DU-145 prostate cancer
cells.34 Thus, even for drugs of the same class, their effect on
cell proliferation and cell death may be diverse and is perhaps
associated with minor structural variation, cell type, and drug
concentration, etc.
2.4. Antitumor Activity in Vivo. As an emerging field of
polyamine, drug−polyamine conjugates that reinforce the
efficacy of vectored drug in addition to the inhibition of
polyamine cycle have attracted more attention.16−18 However,
few in vivo studies provided limited messages for rational
design of the conjugates in comparison to the plentiful in vitro
data. Indeed, in vivo and in vitro experiments may give
contradictory information in many cases. The excellent in vitro
impacts are often not translated into expected potency in vivo.
Therefore, the criteria of lead compounds should include the in
vivo trials which may be more important than in vitro ones. To
further evaluate the antitumor activity of 9f in vivo, we chose
three H22 (mice hepotoma cell line) tumor transplant models:
solid tumor (tumor growth inhibition evaluation), ascites tumor
(live time evaluation), and pulmonary metastasis tumor (tumor
metastasis evaluation). We compared the antitumor effect of 9f
with amonafide in the H22 subcutaneous model. The 5 × 106
H22 cells were inoculated into 30 female Kunming mice to
establish an H22 cancer model, and the treatments were
randomized into three groups (n = 10 mice per group): 9f
group, positive control group, negative control group. The first
group was injected intravenously everyday with 9f (0.2 mg/kg)
from the day of inoculation, and the second treatment group
was injected everyday with amonafide (5 mg/kg) as the positive
control group. The third group was injected everyday with
normal saline as negative control group. The tumor growth
inhibition rates of each group were calculated, and the ratios are
51.8% ((0.68 0.29) g) and 44.7% ((0.78 0.27) g) for 9f
and amonafide compared to the control mice ((1.41 0.18) g),
respectively (Figure 5A and Figure 5B). Thus, tumor growth
inhibitory rates in mice treated with 9f and amonafide were
dramatically decreased, and the in vitro antitumor effect of 9f
was slightly stronger than that of amonafide. Pathologic
examination (HE staining) demonstrated that the survival
cells were more obviously dropped after treatment with 9f than
amonafide (Figure 5C).
The impacts of 9f on survival time in H22-bearing mice were
evaluated by measuring the extension of the lifespan. The
median survival time (MST) in the control mice was (12.7
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Figure 5. Antitumor activity of 9f was evaluated in vivo. (A) Mean tumor weight in subcutaneous model, compared with control: (∗∗) p < 0.01, n =
10. (B) Photographs of tumor were obtained from each treatment group excised on day 10. (C) Representative photograph of histological section
was obtained from each treatment group excised on day 10 (HE stain, ×20). (D) Kaplan−Meier curves showing survival of mice treated with 9f
(red), amonafide (black), and normal saline (blue). (E) Antitumor effect of 9f in the H22 hepatoma pulmonary metastasis model. Photograph (up)
and lung metastasis nodus numbers (down) are shown for pulmonary metastasis in mice treatment with 9f, amonafide, and normal saline. (F)
Representative lung metastasis photograph of histological section was obtained from each treatment group (HE stain, ×20): arrow, lung metastasis.
2.5) days. After the 9f or amonafide was iv administrated, the
lifespan was increased by 2.3-fold ((29.8 4.4) days) or 1.5-
2.5. Systemic Toxicity. It is reported that amonafide can
result in severe irreversible hematotoxicity, which greatly limits
its clinical application.35 In both cell and animal models, some
polyamine conjugates such as F14512 have been confirmed to
possess favorable cell or tissue selectivity,19 which is expected to
reduce the adverse effects of parent drugs. However, there are
few reports about the systemic toxicology of naphthalimide−
polyamine conjugates to support the above hypothesis.
Consequently, we appraised the systemic toxicity of 9f (0.2
mg/kg) with amonafide (5 mg/kg) in tumor-bearing mice for 7
consecutive days (intravenous injection). At the same time, the
body weight was monitored to detect the dose-limited toxicity.
On day 8, these mice were killed by cervical vertebra, and the
organs and tissues, including samples of blood, brain, heart,
liver, lung, kidney, spleen, thymus, and bone marrow, were
taken for inspection of systemic toxicity. We did not observe
any weight loss in the 9f-treated mice, whereas treatment with
amonafide was associated with some weight loss, indicating
possible dose-limited toxicity (Figure 6A). 9f also did not lead
to significant drops in platelet, red blood cell, and white blood
cell numbers compared to controls, while amonafide did.
Furthermore, 9f did not obviously influence bone marrow cell
numbers, which is one of the main adverse effects of amonafide,
suggesting that 9f had no hematotoxicity in this test (Figure
6B). The spleen and thymus index number had no significant
variation after 9f treatment compared to controls, whereas
amonafide obviously decreased these index numbers, implying
that 9f had no influence on immune function and amonafide
inhibited immune function to some extent (Figure 6C). Heart,
liver, kidney, brain, lung, and spleen had no pathological
changes in the pathology examination of both compounds
(Figure 6D and Figure 6E). In short, 9f performed better than
fold ((18.9
3.6) days) compared with that of the control
group, respectively (Figure 5D)
The tumor metastasis is fatal to many patients. On the basis
of the versatile characters of polyamines, we hypothesized that
polyamines may endow extra functions to parent drugs, and
thus, the polyamine conjugates may have broad antitumor
spectra. To test whether 9f was effective against an established
lung metastasis, we injected H22 cells intravenously into 30
Kunming mice (5 × 106 cells each mouse). To ensure that all
mice bore actively growing lung tumors before the drug
treatment, pulmonary cancer was allowed to develop for 10
days. After establishment of tumors, the mice (10 in each
treatment group) were injected intravenously on day 11 with 9f
(0.2 mg/kg), amonafide (5 mg/kg, positive control), and
normal saline (negative control) for 7 consecutive days. On day
18, mice treated with 9f displayed few or no visible metastases,
whereas all mice treated with normal saline had an extensive
tumor burden in the lungs. In contrast, amonafide moderately
decreased lung metastasis nodus numbers (Figure 5E).
Furthermore, pathologic examination (HE staining) also
confirmed that the metastasis focuses were obviously decreased
after treatment with 9f than amonafide (Figure 5F).
The inhibitory effect on metastasis could also be reflected in
a statistically marked reduction in tumor weight in the lung on
day 18 compared with control. The mean values were 0.17
0.08 g for mice treated with 0.2 mg/kg 9f, 0.29 0.11 g for
mice treated with 5 mg/kg amonafide, and 0.34 0.15 g for
mice treated with normal saline. These results supported that 9f
inhibited the tumor metastasis more potently than amonafide.
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Figure 6. Systemic toxicity of 9f. (A) The mean weight change was shown after treatment with 9f, amonafide, and normal saline: compared with
control, (∗∗) p < 0.01, n = 10. (B) The hematotoxicity was evaluated after treatment with 9f, amonafide, and normal saline. (C) The immune organs
(spleen and thymus) were evaluated after treatment with 9f, amonafide, and normal saline. (D) The organ index (heart, liver, kidney, brain, and
lung) was evaluated after treatment with 9f, amonafide, and normal saline. (E) Representative photograph of histological section was obtained from
each treatment group (HE stain, 20×).
amonafide in the experiments of toxicological profile at the
doses that exert antitumor activity in vivo.
extensive study of apoptotic mechanism, the signaling pathway
of inhibiting metastasis by naphthalimides is rarely reported,
which is underway in our laboratory.
It is interesting to compare the results of 9f with UNBS3157
and UNBS5162. To alleviate the toxicity of amonafide, Kiss et
al. transformed the aryl 5-amino group to 5-urea motif to
prevent its metabolization to N-acetylamonafide by the enzyme
N-acetyltransferase 2. Indeed, the synthesized UNBS3157 and
its hydrolyzed product UNBS5162 are nonhematotoxic.8,34 As
for 9f with primary amino groups in polyamine skeletons but
without amino group in the aryl system, the observed non-
hematotoxicity at the therapeutic dose implied that the terminal
amino groups in the polyamine tails did not display negative
functions in the present toxicological assays.
4. EXPERIMENTAL PROTOCOLS
4.1. Synthesis of Aromatic Imide and Diimide−Polyamine
Conjugates. All solvents and reagents were purchased from the
1
suppliers and used without further purification. All H NMR spectra
were recorded on a Bruker AV-400 model spectrometer in D2O or
CDCl3. Chemical shifts for 1H NMR spectra are reported in parts per
million to residual solvent protons. ESI-MS spectra were recorded on
an ESQUIRE-LC mass spectrometer. The target compounds with the
purity being higher than 95% were analyzed using combustion analysis.
Elemental analyses were performed on a Gmbe VarioEL elemental
instrument, and results were within 0.4% of the theoretical values.
4.1.1. General Procedure for the Synthesis of 6a,d−g. A mixture
of 4-bromo-1,8-naphthalic anhydride and polyamine (in a 1:1 molar
ratio) in EtOH was heated at refluxing temperature, and the reaction
process was monitored by TLC. After completion, the reaction
mixture was cooled to room temperature and concentrated under
vacuum to give an oily residue, which was further purified to obtain the
BOC protected intermediates 5a,d−g.
A 50 mL round bottomed flask equipped with a magnetic stirring
bar was charged with pure 5 (2 mmol), phenylboronic acid (2 mmol),
K2CO3 (3 mmol), a catalytic amount of PdCl2, and tetrabutylammo-
nium bromide. The mixture was vigorously stirred for 6 h at the
boiling temperature. After reaction completion, the reaction mixture
was filtered and evaporated to dryness, and the residue was subjected
to column chromatography using ethyl acetate/petroleum ether
mixture as the eluent to yield the compounds 6a,d−g.
3. CONCLUSION
Three series of aromatic imide/diimides modified polyamines
were synthesized, and their antitumor activities were evaluated
in vitro and in vivo. Both the imide/diimides scaffold and the
polyamine motif affect the biological activity of designed
compounds. Two naphthalene diimide−polyamine conjugates
more potently inhibited the growth of multiple cancer cell lines
than the reference drug amonafide. 9f, which was tested more
comprehensively as a representative candidate, was verified to
efficiently induce apoptosis via a ROS mediated mitochondrial
pathway in the preliminary study of cell death profile. The in
vivo trials of H22 tumor transplant models revealed that 9f
expressed improved indexes of inhibitive effect and lifespan
extension compared to amonafide. The dropped hematotox-
icity, which is one of main drawbacks of amonafide, testified
that 9f is a valuable lead compound for further investigation.
More importantly, the obviously improved ability of 9f in
preventing lung cancer metastasis was observed, which
increased its value as a promising lead compound. Present
experiments again indicated that the polyamine motifs may
enhance the antitumor efficacy and alleviate the adverse effects
of vectored drugs in certain cancerous models. In addition, the
versatile function of polyamines may endow some unexpected
biological behaviors to the parent drugs. Compared to the
2-[(2-Dimethylamino)ethyl]-6-phenyl-1H-benz[de]isoquinoline-
1,3(2H)-dione (6a). Yield 84%, white solid. 1H NMR (400 MHz,
CDCl3) δ: 8.62 (t, J = 6.4 Hz, 2H, Ar-H); 8.25 (d, J = 8.4 Hz, 1H, Ar-
H); 7.66−7.70 (m, 2H, Ar-H); 7.48−7.56 (m, 5H, Ar-H); 4.35 (t, J =
7.2 Hz, 2H, 1 × N-CH2), 2.67 (t, J = 7.2 Hz, 2H, 1 × N-CH2); 2.36 (s,
6H, 2 × N-CH3). ESI-MI m/z: 345.1 [M + 1]+.
2-[4-(4-Butoxycarbonylaminobutylbutoxycarbonylamino)butyl]-6-phe-
nyl-1H-benz[de]isoquinoline-1,3(2H)-dione (6d). Yield 85%, pale yellow
1
oil. H NMR (400 MHz, CDCl3) δ: 8.55 (t, J = 6.8 Hz, 2H, Ar-H);
8.19 (d, J = 8.4 Hz, 1H, Ar-H); 7.62 (t, J = 6.2 Hz, 2H, Ar-H); 7.43−
7.51 (m, 5H, Ar-H); 4.16 (t, J = 7.0 Hz, 2H, 1 × N-CH2); 3.08−3.19
(m, 6H, 3 × N-CH2); 1.67−1.74 (m, 2H, 1 × CH2); 1.60−1.62 (m,
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2H, 1 × CH2); 1.47−1.54 (m, 2H, 1 × CH2); 1.39−1.44 (m, 20H, 1 ×
CH2 + 6 × CH3). ESI-MI m/z: 638.3 [M + Na]+.
7.74 (d, J = 7.6 Hz, 1H, Ar-H); 7.58 (d, J = 8.4 Hz, 1H, Ph-H); 7.34−
7.43 (m, 3H, Ar-H); 7.11 (t, J = 7.0 Hz, 1H, Ar-H); 6.94 (d, J = 7.2
Hz, 2H, Ar-H); 6.87 (d, J = 7.6 Hz, 1H, Ar-H); 3.85 (t, J = 6.0 Hz, 2H,
1 × N-CH2); 3.14−3.26 (m, 10H, 5 × N-CH2); 2.13−2.21 (m, 2H,
CH2); 1.95−2.02 (m, 2H, 1 × CH2); 1.87−1.89 (m, 4H, 2 × CH2).
ESI-MI m/z: 459.3 [M + H − 3HCl]+. Anal. Calcd for
C28H37O2N4Cl3·0.5H2O: C 58.29%, H 6.64%, N 9.71%. Found: C
58.09%, H 6.47%, N 9.81%.
2-{3-[3-(3-Butoxycarbonylaminopropylbutoxycarbonylamino)-
propylbutoxycarbonylamino]propyl}-6-phenyl-1H-benz[de]isoquinoline-
1
1,3(2H)-dione (6e). Yield 86%, pale yellow oil. H NMR (400 MHz,
CDCl3) δ: 8.53−8.55 (m, 2H, Ar-H); 8.19 (d, 1H, J = 7.2 Hz, Ar-H);
7.61 (s, 2H, Ar-H); 7.42−7.50 (m, 5H, Ar-H); 4.14 (t, J = 7.8 Hz, 2H,
1 × N-CH2); 3.04−3.32 (m, 10H, 5 × N-CH2); 1.91−1.97 (m, 2H, 1
× CH2); 1.59−1.69 (m, 2H, 1 × CH2); 1.36−1.46 (m, 29H, 1 × CH2
+ 9 × CH3). ESI-MI m/z: 767.4 [M + Na]+.
2-{4-[4-(4-Aminobutylamino)butylamino]butyl}-6-phenyl-1H-benz-
[de]isoquinoline-1,3(2H)-dione Trihydrochloride (7g). Yield 82%, white
1
2-{3-[4-(3-Butoxycarbonylaminopropylbutoxycarbonylamino)-
butylbutoxycarbonylamino]propyl}-6-phenyl-1H-benz[de]isoquinoline-
solid. H NMR (400 MHz, D2O) δ: 7.58 (d, J = 6.4 Hz, 1H, Ar-H);
7.21−7.36 (m, 5H, Ar-H); 6.82−6.84 (m, 1H, Ar-H); 6.74 (d, J = 5.6
Hz, 2H, Ar-H); 6.51 (d, J = 6.0 Hz, 1H, Ar-H); 3.48 (t, J = 2.8 Hz, 2H,
1 × N-CH2); 2.95−3.08 (m, 10H, 5 × N-CH2); 1.73−1.76 (m, 8H, 4
× CH2); 1.62−1.63 (m, 2H, 1 × CH2); 1.23−1.29 (m, 2H, 1 × CH2).
ESI-MI m/z: 487.4 [M + H − 3HCl]+. Anal. Calcd. for
C30H41Cl3N4O2·0.5H2O: C 59.55%, H 7.00%, N 9.26%. Found: C
59.77%, H 7.32%, N 9.07%.
1
1,3(2H)-dione (6f). Yield 83%, pale yellow oil. H NMR (400 MHz,
CDCl3) δ: 8.63 (t, J = 6.6 Hz, 2H, Ar-H); 8.27 (d, 1H, J = 8.4 Hz, Ar-
H); 7.69 (t, J = 7.2 Hz, 2H, Ar-H); 7.49−7.57 (m, 5H, Ar-H); 4.20−
4.23 (t, J = 6.4 Hz, 2H, 1 × N-CH2); 3.09−3.39 (m, 10H, 5 × N-
CH2); 1.95−2.03 (m, 2H, 1 × CH2); 1.63−1.64 (m, 2H, 1 × CH2);
1.42−1.50 (m, 31H, 2 × CH2 + 9 × CH3). ESI-MI m/z: 781.4 [M +
Na]+.
4.1.3. General Procedure for the Synthesis of 9b−g and 11e−g.
A mixture of the respective dianhydride and polyamine (in a 1:1 molar
ratio) in the solvents EtOH and toluene (v:v = 1:1) was heated at
refluxing temperature and monitored by TLC. After completion, the
reaction mixture was cooled to room temperature and concentrated
under vacuum to give an oily residue, which was further purified to
obtain the BOC protected intermediates 8b−g (10e−g).
2-{4-[4-(4-Butoxycarbonylaminobutylbutoxycarbonylamino)-
butylbutoxycarbonylamino]butyl}-6-phenyl-1H-benz[de]isoquinoline-
1
1,3(2H)-dione (6g). Yield 86%, pale yellow oil. H NMR (400 MHz,
CDCl3) δ: 8.56−8.66 (m, 3H, Ar-H); 8.40 (d, 1H, J = 7.6 Hz, Ar-H);
8.04 (t, J = 8.0 Hz, 1H, Ar-H); 7.83−7.87 (m, J = 8.0 Hz, 1H, Ar-H);
7.68−7.73 (m, 1H, Ar-H), 7.49−7.57 (m, 3H, Ar-H); 4.18 (t, J = 7.2
Hz, 2H, 1 × N-CH2); 3.13−3.15 (m, 10H, 5 × N-CH2); 1.62−1.72
(m, 6H, 3 × CH2); 1.41−1.46 (m, 33H, 3 × CH2 + 9 × CH3). ESI-MI
m/z: 809.5 [M + Na]+.
Target compounds 9b−g (11e−g) were obtained in the same way
as described in the synthesis of 7a−g.
2,7-Bis[3-(3-aminopropylamino)propyl]benzo[lmn][3,8]-
phenanthroline-1,3,6,8(2H,7H)-tetraone Tetrahydrochloride (9b). Yield
4.1.2. General Procedure for the Synthesis of 7a,d−g. The
respective N-Boc protected intermediates 7a−g (1.2 mmol) were
dissolved in EtOH (20 mL) and stirred at 0 °C for 10 min. Then 4 M
HCl was added dropwise at 0 °C. The reaction mixture was stirred at
room temperature overnight. (After 0.6 mL of 4 M HCl was added,
the mixture was stirred for 30 min for 7a.) The solution typically gave
a white solid as a precipitate. The solid was filtered, washed several
times with absolute ethanol, and dried under vacuum to give the pure
target compounds 7a,d−g.
1
83%, white solid. H NMR (400 MHz, D2O) δ: 8.45 (S, 4H, Ar-H);
4.22 (t, J = 6.8 Hz, 4H, 2 × N-CH2); 3.20−3.27 (m, 8H, 4 × N-CH2);
3.14 (t, J = 7.8 Hz, 4H, 2 × N-CH2); 2.11−2.21 (m, 8H, 4 × CH2).
ESI-MI m/z: 495.3 [M + H − 4HCl]+. Anal. Calcd for
C26H38Cl4N6O4·2H2O: C 46.16%, H 6.26%, N 12.42%. Found: C
46.33%, H 6.56%, N 12.65%.
2,7-Bis[4-(3-aminopropylamino)butyl]benzo[lmn][3,8]phenanthroline-
1,3,6,8(2H,7H)-tetraone Tetrahydrochloride (9c). Yield 80%, white
1
2-[(2-Dimethylamino)ethyl]-6-phenyl-1H-benz[de]isoquinoline-
solid. H NMR (400 MHz, D2O) δ: 8.28 (S, 4H, Ar-H); 4.07 (t, J
1
1,3(2H)-dione Hydrochloride (7a). Yield 80%, white solid. H NMR
= 7.0 Hz, 4H, 2 × N-CH2); 3.22 (t, J = 7.8 Hz, 8H, 4 × N-CH2); 3.15
(t, J = 7.0 Hz, 4H, 2 × N-CH2); 2.10−2.18 (m, 4H, 2 × CH2); 1.82−
1.89 (m, 8H, 4 × CH2). ESI-MI m/z: 523.3 [M + H − 4HCl]+. Anal.
Calcd for C28H42Cl4N6O4·1.5H2O: C 48.35%, H 6.52%, N 12.08%.
Found: C 48.09%, H 6.70%, N 11.86%.
(400 MHz, D2O) δ: 8.59 (t, J = 6.2 Hz, 2H, Ar-H); 8.22 (d, J = 8.4 Hz,
1H, Ar-H); 7.66 (t, J = 6.8 Hz, 2H, Ar-H); 7.45−7.53 (m, 5H, Ar-H);
4.33 (t, J = 7.0 Hz, 2H, 1 × N-CH2), 2.66 (t, J = 6.8 Hz, 2H, 1 × N-
CH2); 2.35 (s, 6H, 2 × N-CH3). ESI-MI m/z: 345.1 [M + 1 − HCl]+.
Anal. Calcd for C22H21N2O2Cl·0.25H2O: C 68.57%, H 5.62%, N
7.27%. Found: C 68.65%, H 5.92%, N 7.28%.
2,7-Bis[4-(4-aminobutylamino)butyl]benzo[lmn][3,8]phenanthroline-
1,3,6,8(2H,7H)-tetraone Tetrahydrochloride (9d). Yield 83%, white
1
2-[4-(4-Aminobutylamino)butyl]-6-phenyl-1H-benz[de]isoquinoline-
solid. H NMR (400 MHz, D2O) δ: 8.32 (S, 4H, Ar-H); 4.09 (t, J =
1
1,3(2H)-dione Dihydrochloride (7d). Yield 83%, white solid. H NMR
6.6 Hz, 4H, 2 × N-CH2); 3.15−3.23 (m, 8H, 4 × N-CH2); 3.08 (t, J =
6.8 Hz, 4H, 2 × N-CH2); 1.78−1.84 (m, 16H, 8 × CH2). ESI-MI m/z:
551.4 [M + H − 4HCl]+. Anal. Calcd for C30H46Cl4N6O4·0.2H2O: C
51.46%, H 6.68%, N 12.00%. Found: C 51.33%, H 6.67%, N 11.82%.
2,7-Bis{3-[3-(3-aminopropylamino)propylamino]propyl}benzo[lmn]-
[3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone Hexahydrochloride (9e).
(400 MHz, D2O) δ: 7.74 (d, J = 7.2 Hz, 1H, Ar-H); 7.53−7.58 (m,
2H, Ar-H); 7.37−7.44 (m, 3H, Ar-H); 7.04 (t, J = 7.0 Hz, 1H, Ar-H);
6.94 (d, J = 6.8 Hz, 2H, Ar-H); 6.79 (d, J = 7.6 Hz, 1H,Ar-H); 3.61 (t,
J = 6.6 Hz, 2H, 1 × N-CH2); 3.06−3.13 (m, J = 7.8 Hz, 4H, 2 × N-
CH2); 3.02 (t, J = 8.0HZ, 2H, 1 × N-CH2); 1.78−1.83 (m, 4H, 2 ×
CH2); 1.65−1.73 (m, 2H,1 × CH2); 1.37−1.42 (m, 2H, 1 × CH2).
ESI-MI m/z: 416.3 [M + H − 2HCl]+. Anal. Calcd for
C26H31Cl2N3O2·1.8H2O: C 59.95%, H 6.70%, N 8.07%. Found: C
59.63%, H 6.33%, N 8.09%.
2-{3-[3-(3-Aminopropylamino)propylamino]propyl}-6-phenyl-1H-
benz[de]isoquinoline-1,3(2H)-dione Trihydrochloride (7e). Yield 82%,
white solid. 1H NMR (400 MHz, D2O) δ: 7.79 (d, J = 6.8 Hz, 1H, Ar-
H); 7.64 (d, J = 6.8 Hz, 1H, Ar-H); 7.53 (d, 1H, J = 8.4 Hz, Ar-H);
7.30−7.37 (m, 3H, Ar-H); 7.05 (t, J = 6.8 Hz, 1H, Ar-H); 6.90 (d, J =
6.4 Hz, 2H, Ar-H); 6.81 (d, J = 7.2 Hz, 1H, Ar-H); 3.74 (t, 2H, 1 × N-
CH2); 3.06−3.16 (m, 10H, 5 × N-CH2); 2.01−2.14 (m, 2H, 1 ×
CH2); 1.87−1.89 (m, 2H, 1 × CH2); 1.71−1.76 (m, 2H, 1 × CH2).
ESI-MI m/z: 445.3 [M + H − 3HCl]+. Anal. Calcd for
C27H35Cl3N4O2·1.5H2O: C 55.82%, H 6.59%, N 9.64%. Found: C
55.62%, H 6.67%, N 9.68%.
1
Yield 83%, white solid. H NMR (400 MHz, D2O) δ: 8.31 (s, 4H,
Ar-H); 4.14 (t, J = 6.0 Hz, 4H, 2 × N-CH2); 3.15−3.22 (m, 16H, 8 ×
N-CH2); 3.09 (t, J = 7.8 Hz, 4H, 2 × N-CH2); 2.04−2.19 (m, 12H, 6
× CH2). ESI-MI m/z: 609.5 [M + H − 6HCl]+. Anal. Calcd for
C32H54Cl6N8O4·1.5H2O·C2H5OH: C 45.34%, H 7.05%, N 12.44%.
Found: C 45.45%, H 7.22%, N 12.15%.
2,7-Bis{3-[4-(3-aminopropylamino)butylamino]propyl}benzo[lmn]-
[3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone Hexahydrochloride (9f).
1
Yield 82%, white solid. H NMR (400 MHz, D2O) δ: 8.57 (s, 4H,
Ar-H); 4.28 (t, J = 6.4 Hz, 4H, 2 × N-CH2); 3.15−3.29 (m, 20H, 10 ×
N-CH2); 2.12−2.24 (m, 8H, 4 × CH2); 1.87 (br s, 8H, 4 × CH2). ESI-
MI m/z: 637.5 [M
C34H58Cl6N8O4·0.5H2O: C 47.23%, H 6.88%, N 12.96%. Found: C
47.08%, H 6.78%, N 12.74%.
+ H −
6HCl]+. Anal. Calcd for
2,7-Bis{4-[4-(4-aminobutylamino)butylamino]butyl}benzo[lmn][3,8]-
phenanthroline-1,3,6,8(2H,7H)-tetraone Hexahydrochloride (9g). Yield
2-{3-[4-(3-Aminopropylamino)butylamino]propyl}-6-phenyl-1H-benz-
[de]isoquinoline-1,3(2H)-dione Trihydrochloride (7f). Yield 84%, white
1
82%, white solid. H NMR (400 MHz, D2O) δ: 8.24 (s, 4H, Ar-H);
1
solid. H NMR (400 MHz, D2O) δ: 7.88 (d, J = 7.2 Hz, 1H, Ar-H);
4.04 (t, J = 6.2 Hz, 4H, 2 × N-CH2); 3.14−3.24 (m, 16H, 8 × N-
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CH2); 3.09 (t, J = 6.8 Hz, 4H, 2 × N-CH2); 1.80−1.87 (m, 24H, 12 ×
CH2). ESI-MI m/z: 693.5 [M + H − 6HCl]+. Anal. Calcd for
C38H66Cl6N8 O4·1H2O: C 49.09%, H 7.37%, N 12.05%. Found: C
49.18%, H 7.38%, N 11.93%.
Following this, the wells were rinsed twice with PBS to remove excess
dye and 200 μL of PBS was added to each well. Fluorescence was
measured with excitation at 485 nm and emission at 530 nm using a
Tecan Infinite M 200 microplate reader (San Jose, CA) with
measurements done in quadruplicate. The fluorescence intensities of
treated cells were normalized to untreated controls and plotted as
relative fluorescence units (RFUs).
4.2.5. Subcutaneous Xenograft of H22 Cells in Kunming Mice. All
animal care and experimentation conformed to the Guide for the Care
and Use of Laboratory Animals published by Henan University. For
solid tumor development, 30 male Kunming mice (Laboratory Animal
Center of Henan, Zhengzhou) aged 5 weeks (weighing 18−22 g) were
injected subcutaneously with 2 × 106 H22 cells. On the eighth day
after inoculation, the mice were randomly divided into three groups
(control, 9f group, and amonafide group) and then were administered
by caudal vein injection 9f (0.2 mg/kg) or amonafide (5 mg/kg, as
positive control) or physiologic saline for 7 consecutive days. On day
16, the mice were killed by ether anesthesia, and solid tumors were
removed and weighed. The inhibitory rate was calculated as follows:
inhibitory rate (%) = [(A − B)/A] × 100, where A was the mean
tumor weight of the control group and B was that of the drug treated
or positive group. After being weighed, the tumors were fixed in
formalin and stained with hematoxylin and eosin.
2,2′-Bis{3-[3-(3-aminopropylamino)propylamino]propyl}-5,5′-biisoin-
doline-1,1′,3,3′-tetraone Hexahydrochloride (11e). Yield 83%, white solid.
1H NMR (400 MHz, D2O) δ: 7.92 (dd, J1 = J2 = 1.2 Hz, 2H, Ar-H);
7.80 (s, 2H, Ar-H); 7.75 (d, J = 7.6 Hz, 2H, Ar-H); 3.78 (t, J = 6.8 Hz,
4H, 2 × N-CH2); 3.19−3.24 (m, 16H, 8 × N-CH2); 3.13 (t, J =
7.8HZ, 4H, 2 × N-CH2); 2.08−2.20 (m, 12H, 6 × CH2). ESI-MI m/z:
635.4 [M + H − 6HCl]+. Anal. Calcd for C34H56Cl6N8O4·1H2O: C
46.85%, H 6.71%, N 12.86%. Found: C 46.81%, H 6.69%, N 12.70%.
2,2′-Bis{3-[4-(3-aminopropylamino)butylamino]propyl}-5,5′-biisoindo-
line-1,1′,3,3′-tetraone Hexahydrochloride (11f). Yield 80%, white solid.
1H NMR (400 MHz, D2O) δ: 8.09 (d, J = 8.0 Hz, 2H, Ar-H); 8.04 (s,
2H, Ar-H); 7.93 (d, J = 8.0 Hz, 2H, Ar-H); 3.86 (t, J = 6.6 Hz, 4H, 2 ×
N-CH2); 2.14−3.27 (m, 20H, 10 × N-CH2); 2.11−2.19 (m, 8H, 4 ×
CH2); 1.85−1.86 (m, 8H, 4 × CH2). ESI-MI m/z: 663.5 [M + H −
6HCl]+. Anal. Calcd for C36H60Cl6N8O4·0.85H2O: C 48.21%, H
6.93%, N 12.49%. Found: C 48.51%, H 6.72%, N 12.17%.
2,2′-Bis{4-[4-(4-aminobutylamino)butylamino]butyl}-5,5′-biisoindo-
line-1,1′,3,3′-tetraone Hexahydrochloride (11g). Yield 80%, white solid.
1H NMR (400 MHz, D2O) δ: 8.03 (d, J = 2.4 Hz, 2H, Ar-H); 7.95 (d,
4.2.6. Survival Time in Mice Bearing H22 Cells. For calculation of
the survival time, 30 male Kunming mice were inoculated via
intraperitoneal (ip) injection with 1 × 106 H22 cells/mouse on day 0
and were randomly divided into three groups. The treatment with 9f
(0.2 mg/kg), amonafide (5 mg/kg), or physiologic saline by caudal
vein injection was started 24 h after inoculation for 7 consecutive days.
The median survival time (MST) for each group was observed, and
the antitumor activity of drug was evaluated by measuring the increase
of the lifespan. The extended lifespan rate was calculated as follows:
extend rate (%) = (MSTtreated group/MSTcontrol group) × 100.
J = 4.2 Hz, 2H, Ar-H); 7.86 (d, J = 4.8 Hz, 2H, Ar-H); 3.73 (t, J = 6.4
Hz, 4H, 2 × N-CH2); 3.13 (m, 16H, 8 × N-CH2); 3.06 (m, 4H, 2 ×
N-CH2); 1.79−1.81 (m, 24H, 12 × CH2). ESI-MI m/z: 719.6 [M + H −
6HCl]+. Anal. Calcd for C40H68Cl6N8O4·0.5H2O: C 50.75%, H 7.35%, N
11.84%. Found: C 50.62%, H 7.12%, N 11.64%.
4.2. Biological Materials and Methods. 4.2.1. Cell Culture
and MTT Assay. HCT-116 cell line, MDA-MB-231 cell line, K562
cell line, and HepG2 cell line were purchased from Shanghai
Institute for Biological Science, Chinese Academy of Science
(Shanghai, China) and were supplemented with 1 mM
glutamine and 10% or 20% (v/v) FCS. Cells were cultured at
37 °C under a 5% CO2 atmosphere. The antiproliferative ability
of compounds was evaluated in HCT-116 cells, MDA-MB-231
cells, K562 cells, and HepG2 cells by the conversion of MTT to
a purple formazan precipitate as previously described. Cells
were seeded into 96-well plates at 5 × 103 cells/well. After 12 h,
various concentrations (1, 5, 10, 30, and 50 μM) of compounds
were subsequently added and incubated for 48 h. (Note: the
cytotoxicity of 9f was confirmed using 0.1, 1, 10, 30, and 50 μM
when the IC50 values are less than 1 μM.) The inhibition rate
was calculated from plotted results using untreated cells as
100%.
4.2.2. Cytotoxic Assay. The cytotoxicity of compounds was
evaluated in HepG2 cells using the multiparameter cytotoxicity 2 kit
by High Content Screening (HCS, Cellomics ArrayScan Vti, U.S.).
Briefly, cells were seeded into 96-well plates at 5 × 103 cells/well. After
12 h, various concentrations (1, 5, 10, 15, and 30 μM) of compounds
were subsequently added and incubated for 48 h. Cells were stained
according to the manufacturer’s protocol. Images were acquired on the
ArrayScan HCS reader using Multiparameter Cytotoxicity BioAppli-
cation software.
4.2.3. Cellular Apoptotic Evaluation. Cell apoptosis was evaluated
by annexin V−FITC apoptosis detection kit and caspase 9 and caspase
3 activation kits using HCS. Briefly, the HepG2 cells were seeded in
24-well plates and exposed to compound 9f for 48 h, then harvested
and stained according to the manufacturer’s protocol. Images were
acquired on the ArrayScan HCS reader using Target Activation
BioApplication software.
4.2.4. ROS Determination. Cellular generation of ROS was
determined using oxidation of 2′,7′-dichlorodihydrofluorescein diac-
etate (DCFH-DA) as previously described. Specifically, culture
medium from the wells was removed and replaced with 100 μL of
new culture medium containing particles at 100 μg/mL. After 2 h of
incubation with nanomaterials, a time sufficient to stimulate
intracellular ROS production, the cells were rinsed three times with
PBS to remove unbound nanomaterials. The cells were then incubated
with 100 μL of H2DCFDA (5 μM in PBS) at 37 °C for 30 min.
4.2.7. H22 Cells Lung Metastasis Models. H22 cells (5 × 106 cells
per mouse) for Kunming mice were injected iv through the tail vein
for tumor passive metastasis. To ensure all mice bore actively growing
lung tumors before the drug treatment, pulmonary metastasis was
allowed to develop for 10 days. On day 11, mice were injected
intravenously with 9f (0.2 mg/kg), amonafide (5 mg/kg, positive
control), and normal saline (negative control) (n = 10) for 7
consecutive days. On day 18, mice were killed by ether anesthesia and
lungs were removed and weighed and then fixed in Bouin’s fluid. After
the lungs were fixed, lung metastases nodus was numbered.
4.2.8. Systemic Toxicity Evaluation. Mice (10 per group) received
9f (0.2 mg/kg) and amonfide (5 mg/kg, iv) for 7 consecutive days and
then were sacrificed. Blood and bone marrow were collected, and
hematological profiling was undertaken by determining platelet, red
blood cell (RBC), white blood cell (WBC), and bone marrow cell
counts using a Coulter counter T-890 (Coulter Electronics, U.S.).
Heart, liver, kidney, brain, lung, spleen, and thymus were removed and
weighed. The organ index and histopathology were investigated for
systemic toxicity evaluation: organ index (%) = (organ weight/body
weight) × 100.
4.2.9. Histopathologic Evaluation. All tumor and organs from each
group were chosen for histopathologic evaluation at tested times.
Lungs and tumor were fixed in 10% formaldehyde for 24 h and
processed in paraffin. Sections were stained with H&E for examination
for histopathologic change.
4.2.10. Data Analysis. All data are presented as the mean SD and
analyzed using Student’s t test or analysis of variance (ANOVA)
followed by q test: compared with control, (∗) p < 0.05, (∗∗) p < 0.01
as significant.
AUTHOR INFORMATION
■
Corresponding Author
*For S.X.: phone, 86-13938637212; e-mail, kfxsq@yahoo.com.
cn. For C.W.: phone, 86-13619810550; fax, 86-378-2864665;
e-mail, wcjsxq@henu.edu.cn.
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and induce lysosomal membrane permeabilization and apoptosis.
J. Med. Chem. 2010, 53 (6), 2589−2600.
Notes
The authors declare no competing financial interest.
(10) Liu, Z. R.; Hecker, K. H.; Rill, R. L. Selective DNA binding of
(N-alkylamine)-substituted naphthalene imides and diimides to G+C-
rich DNA. J. Biomol. Struct. Dyn. 1996, 14 (3), 331−339.
(11) Tumiatti, V.; Milelli, A.; Minarini, A.; Micco, M.; Gasperi
Campani, A.; Roncuzzi, L.; Baiocchi, D.; Marinello, J.; Capranico, G.;
Zini, M.; Stefanelli, C.; Melchiorre, C. Design, synthesis, and biological
evaluation of substituted naphthalene imides and diimides as
anticancer agent. J. Med. Chem. 2009, 52 (23), 7873−7877.
(12) Casero, R. A. Jr; Woster, P. M. Recent advances in the
development of polyamine analogues as antitumor agents. J. Med.
Chem. 2009, 52 (15), 4551−4573.
ACKNOWLEDGMENTS
This work was supported by National Science Foundation of
China (Grant 90913001) .
■
ABBREVIATIONS USED
■
N-BDMPrNDI, 2,7-bis[(2-dimethylamino)ethyl]benzo[lmn]-
[3,8] phenanthroline-1,3,6,8(2H,7H)-tetraone; MNISpd, 2-{3-
[4-(3-aminopropylamino)butylamino]propyl}-1H-benz[de]-
isoquinoline-1,3(2H)-dione trihydrochloride; MNIHspd, 2-{4-
[4-(4-aminobutylamino)butylamino]butyl}-1H-benz[de]-
isoquinoline-1,3(2H)-dione trihydrochloride; Boc, tert-butyloxy-
carbonyl; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide; HCS, high content screening; MMP,
mitochondrial membrane potential; LMP, lysosomal membrane
permeability; PI, propidium iodide; PS, phosphatidylserine;
FITC, fluorescein isothiocyanate; ROS, reactive oxygen species;
PAO, polyamine oxidase; DCFH-DA, 2′,7′-dichlorodihydro-
fluorescein diacetate; MST, median survival time; PBS,
phosphate buffered saline; CM-H2DCFDA, 5(and 6)-chlor-
omethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester;
RFU, relative fluorescence unit; RBC, red blood cell; WBC,
white blood cell; TBAB, tetrabutyl ammonium bromide; TLC,
thin-layer chromatography; FCS, fetal calf serum
(13) Casero, R. A. Jr.; Woster, P. M. Terminally alkylated polyamine
analogues as chemotherapeutic agents. J. Med. Chem. 2001, 44 (1), 1−
26.
(14) Carta, F.; Temperini, C.; Innocenti, A.; Scozzafava, A.; Kaila, K.;
Supuran, C. T. Polyamines inhibit carbonic anhydrases by anchoring
to the zinc-coordinated water molecule. J. Med. Chem. 2010, 53 (15),
5511−5522.
(15) Weisell, J.; Hyvonen, M. T.; Hakkinen, M. R.; Grigorenko,
̈
̈
N. A.; Pietila, M.; Lampinen, A.; Kochetkov, S. N.; Alhonen, L.;
̈
Vepsalainen, J.; Keinanen, T. A.; Khomutov, A. R. Synthesis and
biological characterization of novel charge-deficient spermine ana-
logues. J. Med. Chem. 2010, 53 (15), 5738−5748.
̈
̈
̈
(16) Tomasi, S.; Renault, J.; Martin, B.; Duhieu, S.; Cerec, V.; Le
Roch, M.; Uriac, P.; Delcros, J. G. Targeting the polyamine transport
system with benzazepine- and azepine-polyamine conjugates. J. Med.
Chem. 2010, 53 (21), 7647−7663.
(17) Palmer, A. J.; Wallace, H. M. The polyamine transport system as
a target for anticancer drug development. Amino Acids 2010, 38 (2),
415−422.
(18) Xie, S.; Wang, J.; Zhang, Y.; Wang, C. Antitumor conjugates
with polyamine vectors and their molecular mechanisms. Expert Opin.
Drug Delivery 2010, 7 (9), 1049−1061.
REFERENCES
■
(1) Giannopoulos, K.; Dmoszynska, A.; Kowal, M.; Wasik-
Szczepanek, E.; Bojarska-Junak, A.; Rolinski, J.; Dohner, H.;
Stilgenbauer, S.; Bullinger, L. Thalidomide exerts distinct molecular
antileukemic effects and combined thalidomide/fludarabine therapy is
clinically effective in high-risk chronic lymphocytic leukemia. Leukemia
2009, 23 (10), 1771−1778.
̈
(19) Kruczynski, A.; Vandenberghe, I.; Pillon, A.; Pesenl, S.; Goetsch,
L.; Barret, J. M.; Guminski, Y.; Le Pape, A.; Imbert, T.; Bailly, C.;
Guilbaud, N. Preclinical activity of F14512, designed to target tumors
expressing anactive polyamine transport system. Invest. New Drugs
2011, 29 (1), 9−21.
(2) Warren, K. E.; Goldman, S; Pollack, I. F.; Fangusaro, J.;
Schaiquevich, P.; Stewart, C. F.; Wallace, D.; Blaney, S. M.; Packer, R.;
Macdonald, T.; Jakacki, R.; Boyett, J. M.; Kun, L. E. Phase I trial of
lenalidomide in pediatric patients with recurrent, refractory, or
progressive primary CNS tumors: Pediatric Brain Tumor Consortium
study PBTC-018. J. Clin. Oncol. 2011, 29 (3), 324−329.
́
(20) Barret, J. M.; Kruczynski, A.; Vispe, S.; Annereau, J. P.; Brel, V.;
Guminski, Y.; Delcros, J. G.; Lansiaux, A.; Guilbaud, N.; Imbert, T.;
Bailly, C. F14512, a potent antitumor agent targeting topoisomerase II
vectored into cancer cells via the polyamine transport system. Cancer
Res. 2008, 68 (23), 9845−9853.
(3) Lv, M.; Xu, H. Overview of naphthalimide analogs as anticancer
agents. Curr. Med. Chem. 2009, 16 (36), 4797−4813.
(21) Oliveira, J.; Ralton, L.; Tavares, J.; Codeiro-da-Silva, A.;
Bestwick, C. S.; McPherson, A.; Thoo Lin, P. K. The synthesis and
the in vitro cytotoxicity studies of bisnaphthalimidopropyl polyamine
derivatives against colon cancer cells and parasite Leishmania infantum.
Bioorg. Med. Chem. 2007, 15 (1), 541−545.
(4) Ingrassia, L.; Lefranc, F.; Kiss, R.; Mijatovic, T. Naphthalimides
and azonafides as promising anti-cancer agents. Curr. Med. Chem.
2009, 16 (10), 1192−1213.
(5) See http://clinicaltrials.gov. Identifiers: NCT01066494,
NCT00715637.
(22) Antonini, I.; Volpini, R.; Dal Ben, D.; Lambertucci, C.; Cristalli,
G. Design, synthesis, and biological evaluation of new mitonafide
derivatives as potential antitumor drugs. Bioorg. Med. Chem. 2008, 16
(18), 8440−8446.
(23) Tian, Z. Y.; Xie, S. Q.; Du, Y. W.; Ma, Y. F.; Zhao, J; Gao, W. Y.;
Wang, C. J. Synthesis, cytotoxicity and apoptosis of naphthalimide
polyamine conjugates as antitumor agents. Eur. J. Med. Chem. 2009, 44
(1), 393−399.
(24) Tian, Z. Y.; Xie, S. Q.; Mei, Z. H.; Zhao, J.; Gao, W. Y.; Wang,
C. J. Conjugation of substituted naphthalimides to polyamines as
cytotoxic agents targeting the Akt/mTOR signal pathway. Org. Biomol.
Chem. 2009, 7 (22), 4651−4660.
(25) Yang, L.; Zhao, J.; Zhu, Y.; Tian, Z.; Wang, C. Reactive oxygen
species (ROS) accumulation induced by mononaphthalimide-
spermidine leads to intrinsic and AIF-mediated apoptosis in HeLa
cells. Oncol. Rep. 2011, 25 (4), 1099−1107.
(6) Peduto, A.; Pagano, B.; Petronzi, C.; Massa, A.; Esposito, V.;
Virgilio, A.; Paduano, F.; Trapasso, F.; Fiorito, F.; Florio, S.; Giancola,
C.; Galeone, A.; Filosa, R. Design, synthesis, biophysical and biological
studies of trisubstituted naphthalimides as G quadruplex ligands.
Bioorg. Med. Chem. 2011, 19 (21), 6419−6429.
(7) Liu, Y.; Norton, J. T.; Witschi, M. A.; Xu, Q.; Lou, G.; Wang, C.;
Appella, D. H.; Chen, Z.; Huang, S. Methoxyethylamino-numonafide
is an efficacious and minimally toxic amonafide derivative in murine
models of human cancer. Neoplasia. 2011, 13 (5), 453−460.
(8) Van Quaquebeke, E.; Mahieu, T.; Dumont, P.; Dewelle, J.;
Ribaucur, F.; Simon, G.; Sauvage, S.; Gaussin, J. F.; Tuti, J.; EI Yazidi,
M.; Van Vynckt, F.; Mijatovic, T.; Lefranc, F.; Darro, F.; Kiss, R. 2,2,2-
Trichloro-N-({2-[2-(dimethylamino)ethyl]-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinolin-5-yl}carbamoyl)acetamide (UNBS3157), a
novel nonhematotoxic naphthalimide derivative with potent antitumor
activity. J. Med. Chem. 2007, 50 (17), 4122−4134.
(26) Sakurai, H.; Tsukuda, T.; Hirao, T. Pd/C as a reusable catalyst
for the coupling reaction of halophenols and arylboronic acids in
aqueous media. J. Org. Chem. 2002, 67 (8), 2721−2722.
(9) Chen, Z.; Liang, X.; Zhang, H.; Xie, H.; Liu, J.; Xu, Y.; Zhu, W.;
Wang, Y.; Wang, X.; Tan, S.; Kuang, D.; Qian, X. A new class of
naphthalimide-based antitumor agents that inhibit topoisomerase II
3511
dx.doi.org/10.1021/jm300168w | J. Med. Chem. 2012, 55, 3502−3512

Journal of Medicinal Chemistry
Article
(27) Xie, L.; Cui, J.; Qian, X.; Xu, Y.; Liu, J.; Xu, R. 5-Non-amino
aromatic substituted naphthalimides as potential antitumor agents:
synthesis via Suzuki reaction, antiproliferative activity, and DNA-
binding behavior. Bioorg. Med. Chem. 2011, 19 (2), 961−967.
(28) Tumiatti, V.; Milelli, A.; Minarini, A.; Rosini, M.; Bolognesi, M. L.;
Micco, M.; Andrisano, V.; Bartolini, M.; Mancini, F.; Recanatini, M.;
Cavalli, A.; Melchiorre, C. Structure−activity relationships of acetylcho-
linesterase noncovalent inhibitors based on a polyamine backbone. 4.
Further investigation on the inner spacer. J. Med. Chem. 2008, 51 (22),
7308−7312.
(29) O’Brien, P. J.; Irwin, W.; Diaz, D.; Howard-Cofield, E.; Krejsa,
C. M.; Slaughter, M. R.; Gao, B.; Kaludercic, N.; Angeline, A.;
Bernardi, P.; Brain, P.; Hougham, C. High concordance of drug-
induced human hepatotoxicity with invitro cytotoxicity measured in a
novel cell-based modelusing high content screening. Arch. Toxicol.
2006, 80 (9), 580−604.
(30) Elmore, S. Apoptosis: a review of programmed cell death.
Toxicol. Pathol. 2007, 35 (4), 495−516.
(31) Wang, J.; Wu, A. B.; Xu, Y. F.; Liu, J. W.; Qian, X. H. M2-A
induces apoptosis and G2-M arrest via inhibiting PI3 K/Akt pathway
in HL60 cells. Cancer Lett. 2009, 283 (2), 193−202.
(32) Sohaebuddin, S. K.; Thevenot, P. T.; Baker, D.; Eaton, J. W.;
Tang, L. Nanomaterial cytotoxicity is composition, size,and cell type
dependent. Part. Fibre Toxicol. 2010, 7 (22), 2−17.
(33) Lin, B.; Chen, Z.; Xu, Y.; Zhang, H.; Liu, J.; Qian, X. 7-b, a novel
amonafide analogue, cause growth inhibition and apoptosis in Raji
cells via a ROS-mediated mitochondrial pathway. Leuk. Res. 2011, 35
(5), 646−656.
̀ ̀
(34) Mijatovic, T.; Mahieu, T.; Bruyere, C.; De Neve, N.; Dewelle, J.;
Simon, G.; Dehoux, M. J.; van der Aar, E.; Haibe-Kains, B.; Bontempi,
G.; Decaestecker, C.; Van Quaquebeke, E.; Darro, F.; Kiss, R.
UNBS5162, a novel naphthalimide that decreases CXCL chemokine
expression in experimental prostate cancers. Neoplasia 2008, 10 (6),
573−586.
(35) Zhu, H.; Miao, Z. H.; Huang, M.; Feng, J. M.; Zhang, Z. X.; Lu,
J. J.; Cai, Y. J.; Tong, L. J.; Xu, Y. F.; Qian, X. H.; Ding, J.
Naphthalimides induce G2 arrest through the ATM-activated Chk2-
executed pathway in HCT116 cells. Neoplasia 2009, 11 (11), 1226−
1234.
3512
dx.doi.org/10.1021/jm300168w | J. Med. Chem. 2012, 55, 3502−3512