Synthesis and biological evaluation of novel aromatic imide-polyamine conjugates

Article abstract of DOI:10.3390/molecules21121637

Three types of conjugates in which aromatic imide scaffolds were coupled to diverse amine/polyamine motifs were synthesized, and their antitumor activities were evaluated in vitro and in vivo. Results showed that the conjugate 11e of 1,8-naphthilimide with spermine had pronounced effects on inhibiting tumor cell proliferation and inducing tumor cell apoptosis via ROS-mediated mitochondrial pathway. The in vivo assays on three H22 tumor transplant models revealed that compound 11e exerted potent ability in preventing lung cancer metastasis and extending lifespan. Furthermore, the efficacy of 11e in inhibiting tumor growth and improving body weight index were better than that of positive control, amonafide. Our study demonstrates that compound 11e is a valuable lead compound for further investigation.

Full text of DOI:10.3390/molecules21121637

molecules
Article
Synthesis and Biological Evaluation of Novel
Aromatic Imide-Polyamine Conjugates
Ming Li 1,2, Yuxia Wang 3,*, Jianying Zhang 2, Songqiang Xie 2, Chaojie Wang 4 and
Yingliang Wu 1,
*
1
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China;
songxu666@163.com
Pharmaceutical College, Henan University, Kaifeng 475001, China; 18237878517@163.com (J.Z.);
xiesq@henu.edu.cn (S.X.)
College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475001, China
Key Laboratory of Natural Medicine and Immuno-Engineering, Kaifeng 475001, China; wcjsxq@henu.edu.cn
Correspondence: wangyuxia@henu.edu.cn (Y.x.W.); yingliang_1016@163.com (Y.l.W.);
Tel.: +86-378-2388-1589 (Y.x.W.); +86-24-2398-6278 (Y.l.W.)
2
3
4
*
Academic Editors: Derek J. McPhee and Bimal K. Banik
Received: 1 October 2016; Accepted: 23 November 2016; Published: 30 November 2016
Abstract: Three types of conjugates in which aromatic imide scaffolds were coupled to diverse
amine/polyamine motifs were synthesized, and their antitumor activities were evaluated in vitro and
in vivo. Results showed that the conjugate 11e of 1,8-naphthilimide with spermine had pronounced
effects on inhibiting tumor cell proliferation and inducing tumor cell apoptosis via ROS-mediated
mitochondrial pathway. The in vivo assays on three H22 tumor transplant models revealed that
compound 11e exerted potent ability in preventing lung cancer metastasis and extending lifespan.
Furthermore, the efficacy of 11e in inhibiting tumor growth and improving body weight index
were better than that of positive control, amonafide. Our study demonstrates that compound 11e is
a valuable lead compound for further investigation.
Keywords: naphthalimide; polyamine; antitumor; synthesis; amonafide
1. Introduction
The development of effective anticancer agent continues to be a formidable challenge in medicine.
Aromatic imides provide an excellent template for new drug development and have drawn special
attention of medicinal chemists. Some phthalimides such as thalidomide and lenalidomide (Figure 1)
are now in clinical use [1,2]. Thalidomide, initially introduced for treatment of multiple myeloma (MM)
because of its anti-angiogenic properties, has shown remarkable activity alone and in combination
with other drugs in patients across all stages of the disease. Lenalidomide has been shown to benefit
patients with multiple myeloma, myelodysplastic syndromes, and lymphoma [3–5].
Naphthalimides are also a class of intriguing pharmacophores [6,7]. Although many naphthalimides
and their synthetic analogs have reached clinical trials, most of them were abandoned because of
various adverse effects. For example amonafide (Figure 1) had shown potent anticancer activity.
However, due to unexpected central neurotoxicity and limited efficiency, further application in the
clinical trial was abandoned [8]. In order to improve the tumor selectivity, efficiency, and safety, a broad
variety of novel naphthalimide derivatives by modified amonafide at different positions have been
reported [9,10].
Molecules 2016, 21, 1637; doi:10.3390/molecules21121637
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1637
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H2N
XHCl
NH2
O
N
O
O
O
O
N
O
O
N
O
O
XHCl
H
NH
NH
N
N
R2
N
O
N
O
N
H
R2
O
O
O
Thalidomide
Lenalidomide
5d-f
Amonafide
11a-b, e-f
O
O
N
O
XHCl
R2
( )
N
N
k
H
H
N
4HCl
NH2
N
N
H
( )2
N
O
H
H
O
17b-d, k=1 or 2
11e
Figure 1. The structures of 5d
–
f
,
11a
–b
,
e–
f,
17b–d, thalidomide, lenalidomide and amonafide (X = 2,
3, 4; R2 = deprotected polyamine chain).
Currently, extensive researches have emphasized the fact that polyamine conjugates acting as
promising antitumor candidates are becoming increasingly important in the polyamine field [11–15]
.
F14512, an epipodophyllotoxin-spermine conjugate, is perhaps the most promising compound which
is presently being evaluated in phase I trials [16
incorporating an aromatic imide skeleton or connecting to a polyamine linker such as spermidine
or homospermidine, exhibit marked cell selectivity and lung cancer metastasis inhibition [ 22].
–19]. Our work demonstrated that derivatives
2
–
These findings stimulated us to search for more promising imide scaffolds suitable for polyamine
modification. We report herein three kinds of aromatic imide (Figure 1), which are coupled with
diverse polyamine motifs (Figure 2). In addition to the routine in vitro evaluation, in vivo experiments
are designed to provide more data to justify their future drug development.
R1=
( )
( )
( )
( )
( )
N
NHBoc
NHBoc
N
n
N
NHBoc
m
n
m
m
Boc
Boc
Boc
6a
6b m=1, n=1
6c m=2, n=1
6d m=2, n=2
6e m=1, n=2
6f m=2, n=2
Figure 2. Building blocks of the polyamine skeleton.
2. Results and Discussion
2.1. Chemistry
The acetyl-substituted naphthalimide-polyamine compounds 5d
Scheme 1. Intermediates 2 and 3 were prepared by a modified previously reported procedure [23,24].
The respective N-Boc protected intermediates 4d were obtained by the condensation of R1NH2
, the Boc protected polyamines were prepared by a modified previously reported procedure) [25
–f were synthesized according to
–
f
(6
]
(as shown in Figure 2) with 4-acetyl-1,8-naphthalic anhydride (
3). After purification by flash column
chromatography, these intermediates were mixed with 4 M HCl in EtOH at room temperature to obtain
the target compounds 5d–f as their hydrochloride salts with a yield between 70% and 80% [22].
Compounds 11a
Compound obtained by reacting 1,8-naphthalic anhydride with 1,3-propanediamine reacted with
chloroacetyl chloride to give intermediate . Compounds 10a were easily formed by the reaction
of and R1NH2 in acetonitrile. After purification by flash column chromatography, these intermediates
–b, e–f were synthesized following a four-step procedure as shown in Scheme 1.
8
9
–b, e–f
9
were mixed with 4 M HCl at room temperature to obtain the target compounds 11a–b, e–f as their
corresponding hydrochloride salts with a yield between 70% and 80%.

Molecules 2016, 21, 1637
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O
O
O
O
N
O
O
O
N
O
K2Cr2O7
R1NH2
EtOH
4M HCl
EtOH
Cl
R1
R2 XHCl
CH3COOH
O
O
O
O
2
1
5d~f
4d~f
3
Cl
O
O
O
N
O
O
N
O
O
O
Cl
H
R1NH2
N
Cl
H2N
O
NH2
O
N
O
NH2
NH
NH
R1
CH3CN
CH3CN
EtOH
O
8
7
9
10a-b, e-f
O
O
H
XHCl
R2
N
4M HCl
EtOH
N
O
NH
11a-b, e-f
Scheme 1. Synthetic protocol of target compounds 5d
–f
,
11a–b, e–f (X = 2, 3, 4; R1 = N-Boc protected
polyamine chains; R2 = deprotected polyamine chains).
The synthesis of target compounds 17b
were prepared by a modified previously reported procedure [26
–
d
are described as in Scheme 2. Intermediates 12 and 13
27]. Compound 13 was N-alkylated
,
by commercial dibromoalkane to yield intermediate 14 in good yield. Subsequently, compound 14
was condensed with corresponding amines R1NH2, after the completion of the reaction, the solvent
was removed by rotary evaporation yielding a residue. This residue, without further purification, was
N-protected to form 15b
–d
using excess di-tert-butyl dicarbonate (Boc2O). Suzuki coupling of 15b
–d
with phenylboronic acid catalyzed by PdCl2 gave intermediates 16b
–d [
28]. From these compounds,
Boc groups in the polyamine skeleton were subsequently removed with 4 M HCl in EtOH at room
temperature to provide target compounds 17b–d as hydrochloride salts [29].
O
O
O
O
( )
NH2CONH2
Br
N
Br
Br2
k
(CH3CO)2O
reflux
KOH
C2H5OH
Dibromoalkane
acetone
O
NK
O
Br
O
NaOH
Br
O
12
13
O
O
O
14 k=1 or 2
O
O
PhB(OH)2
R1
R2 3HCl
R1NH2 (Boc)2O
4 M HCl
Ph
( )
( )
( )
k
N
N
R1
N
N
N
N
k
k
EtOH
H
MeOH
Ph
Br
Boc
Boc
O
O
O
15b k=1
15c k=1
15d k=2
16b k=1
16c k=1
16d k=2
17b k=1
17c k=1
17d k=2
Scheme 2. Synthetic protocol of target compounds 17b–d (R1 = N-Boc protected polyamine chains;
R2 = deprotected polyamine chains).
2.2. Biological Evaluation
2.2.1. Cytotoxicity in Human Tumor Cell Lines
The inhibitory activity of target compounds toward five tumor cells (HCT-116: human colorectal
cancer cell line; K562: human leukemia cell line; HepG2: human hepatoma cell line; MDA-MB-231:
human breast cancer cell line; QSG-7701: normal hepatocyte cell line) were measured by the
traditional MTT ({3-(4,5-dimethylthiazol-2-yl)}-2,5-diphenyl-tetrazolium bromide) test. Amonafide
was tested as a reference compound. Structures and biological results of target compounds are
shown in Table 1. The results showed that the naphthalimide-polyamine conjugates 5d
antitumor activities as potent as previously reported naphthalimide-polyamine conjugates [
Compounds 11a exhibited mixed results, compound 11e, which displayed improved potency
–
f
exhibited
2,
21].
–b, e–f
in comparison to other analogues in the series, was of comparable potency to amonafide. Surprisingly,
the 5-phenylphthalimide-polyamines 17b–d were not active.

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Table 1. In vitro activity of new compounds against five kinds of tumor cell lines.
O
O
O
N
O
O
H
XHCl
R2
( )
N
N
N
k
N
R2
XHCl
N
H
R2
XHCl
H
O
O
O
11a-b, e-f
5d-f
17b-d, k=1 or 2
IC50 (µM) a
Compd.
R2
X
k
HCT-116 HepG2
K562
MDA-MB-231 QSG-7701
Amo.
5d
5e
15.43
22.92
16.91
19.56
39.85
>50
5.12
>50
>50
6.57
23.85
22.07
30.72
53.46
>50
6.33
>50
>50
6.31
31.05
12.25
18.81
33.19
28.01
2.86
26.18
28.17
>50
7.98
nd b
nd b
nd b
32.38
16.75
11.98
57.08
nd b
nd b
nd b
55.45
23.21
27.65
21.19
39.18
42.89
53.85
50.64
>50
-(CH2)4NH(CH2)4NH2
2
3
3
2
3
4
4
3
3
3
-(CH2)3NH(CH2)4NH(CH2)3NH2
-(CH2)4NH(CH2)4NH(CH2)4NH2
-(CH2)3NH2
5f
11a
11b
11e
11f
17b
17c
17d
-(CH2)3NH(CH2)3NH2
-(CH2)3NH(CH2)4NH(CH2)3NH2
-(CH2)4NH(CH2)4NH(CH2)4NH2
-(CH2)3NH(CH2)3NH2
1
1
2
-(CH2)4NH(CH2)3NH2
-(CH2)4NH(CH2)4NH2
>50
>50
>50
>50
>50
>50
>50
a
IC50 values represent the concentration causing 50% growth inhibition. They were determined by the linear
regression method. Each sample is the mean of three independent experiments. b Not determined. Amo = Amonafide.
2.2.2. Apoptosis
Previous studies revealed that most naphthalimide-polyamine conjugates may trigger tumor
cell death via an apoptotic process [30 31]. To appraise if novel naphthalimide-polyamine conjugates
,
induce cell apoptosis, morphological study was applied after HepG2 cells were stained with AO/EB.
The percentage of apoptotic cells was calculated after observing a total of 300 cells and as shown
in Figure 3A,C, obvious apoptosis induced by 11e was observed in a dose-dependent manner [32].
To furnish more credible information about the apoptosis of HepG2 cells induced by compound
11e, we used the high content screening (HCS) technique to detect related apoptosis indicators.
Mitochondria are vital cell organelles in the process of transmitting apoptosis signals, whereas the
mitochondrial membrane potential (MMP) is a critical parameter related to mitochondrial function
in cells. As a MMP specific cationic fluorescent dye, Rh123 is used to measure the change of MMP,
which accumulates in healthy mitochondria with intact membrane potential and is absent in the
depolarized mitochondria [22]. Lysosomes, as cellular targets in cancer therapy, participate in various
cell death profiles such as apoptosis and senescence [9,33,34]. Thus, the lysosomal mass/pH (Lys/pH)
variation was detected by a basic fluorescent probe Lyso-Tracker Red which selectively accumulates in
acidic lysosomes during cytotoxicity occurs. After treatment with compound 11e for 48 h, the green
fluorescence of Rh123 in cytoplasm was significantly decreased in a dose-dependent manner, indicating
that the destroyed mitochondria lost their mitochondrial transmembrane potential (Figure 3A,D).
Meanwhile, the remarkable accruement of red fluorescence in cells treated with compound 11e implied
the dose-related occurrence of lysosomes impairment (Figure 3B,D).
It is known that polyamine conjugates may produce reactive oxygen species (ROS) [22].
The elevated ROS could bring corresponding influences on MMP loss and LYS/pH destabilization,
which generally activates the cell death. Therefore, the ROS generation was detected by the DCFH-DA
(20,70-dichlorodihydrofluorescein diacetate) experiment. Indeed, the apparently increased ROS was
observed in a dose-dependent pattern as shown in Figure 3B,D.

Molecules 2016, 21, 1637
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Figure 3. The apoptosis and MMP (mitochondrial membrane potential), ROS (reactive oxygen
species) and lysosomal mass/pH (Lys/PH) were investigated in HepG2 cells in vitro. ( ) The cellular
morphous, MMP were detected with AO/EB, Rh123/Hoechst33342 staining using HCS in HepG2 cells;
) ROS, lysosomal mass/pH were detected by HCS using DCFH-DA and Lyso-Tracker Red staining
respectively; ( 11e induced apoptotic cells at different concentrations (3, 6, 12 M) assessed by HCS
using AO/EB staining after 48 h treatment in HepG2 cells (n = 3, x SD, ** p < 0.01, *** p < 0.001);
) Impacts of 11e at different concentrations (3, 6, 12 M) on MMP, ROS, lysosomal mass/pH, detected
by HCS using Rh123, DCFH-DA, and Lyso-Tracker Red staining respectively after 48 h treatment in
A
(
B
C
)
µ
±
(D
µ
HepG2 cells. Each sample is the mean of three independent experiments. (n = 3, x
*** p < 0.001).
±
SD, ** p < 0.01,
2.2.3. Antitumor Activity In Vivo
Several of our in vitro observations reported here demonstrate that 11e has the ability to interfere
with the proliferation of tumor cells. However, few in vivo studies provided limited messages for
rational design of the conjugates in comparison to the plentiful in vitro data. Indeed, there are
significant discrepancies between in vitro and in vivo experiments in many cases. In vivo trials,
based on preliminary in vitro screens, provide more valuable messages for further drug development.
To further evaluate the antitumor activity of 11e in vivo, we chose three H22 (mice hepatoma cell line)
tumor transplant models: solid tumor (tumor growth inhibition evaluation), ascites tumor (live time
evaluation), and pulmonary metastasis tumor (tumor metastasis evaluation). As shown in Figure 4A,B
and Table 2 after treatment of mice bearing hepatoma xenografts (H22 mouse hepatocellular carcinoma
model) with 1 mg/kg of 11e once every day, obvious tumor weight suppression by 70.92% was
observed compared to the negative control group that treated with normal saline. Mice bearing
hepatoma xenografts were also treated with 5 mg/kg of amonafide and the tumor weight suppression
by 43.79% was observed compared to the negative control group. Thus, tumor growth inhibitory rates
in mice treated with 11e and amonafide were dramatically increased, and the in vitro antitumor effect
of 11e was also obviously stronger than that of amonafide.
Table 2. In vivo H22 tumor growth inhibitions by 11e.
Groups
Dose (mg/kg)
Tumor Weight (g)
Inhibition Rate (%)
No. Dead
Control
Amonafide
11e
2.17 ± 0.22
1.22 ± 0.13
0.63 ± 0.21
0/9
0/9
0/9
5
1
43.79
70.92

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Metastasis is the process by which tumors spread from the primary organ in which they arise
to other sites in the body. In most cases, tumors that stay confined to one organ will not be fatal,
tumors that have spread to other organs have a greater potential for causing harm. To test whether
11e was effective against an established lung metastasis, we injected H22 cells intravenously into
24 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. Mice were treated
with 11e, amonafide and normal saline for 7 consecutive days, respectively. Mice treated with 11e
displayed few metastases (62.42%), whereas all mice treated with normal saline had an extensive tumor
burden in the lungs. In contrast, amonafide moderately decreased lung metastasis nodus numbers
(40.7%) (Figure 4C,D; Table 3). These results supported that 11e inhibited the tumor metastasis lighter
than amonafide.
Impacts of 11e on survival time in H22-bearing mice were evaluated by measuring the extension
of the lifespan. The median survival time (MST) in control mice was (12.75
11e or amonafide i.v. administrated, the life span was increased by 2.41-fold ((30.75
1.53-fold (19.5 ± 3.6 days) compared with that of the control group, respectively (Figure 4E).
±
2.5) days. After the
±
4.4) days) or
Figure 4. Antitumor activity of 11e was evaluated in vivo. (
from each treatment group excised on day 10; ( ) Tumor weight in subcutaneous model, compared
with control: (*** p < 0.001, x SD), n = 9; ( ) Lung metastasis photograph; ( ) Lung metastasis
nodus numbers for pulmonary metastasis in mice treatment with 11e, amonafide, and normal saline.
*** p < 0.001, x SD), n = 8; ( ) The changes of body weight of mice treated with 11e, amonafide, and
A) Photographs of tumor obtained
B
±
C
D
(
±
E
normal saline; (
E
) Kaplan-Meier curves showing survival of mice treated with 11e, amonafide, and
normal saline.
Table 3. In vivo lung metastasis inhibitions by 11e.
Groups
Dose (mg/kg)
Metastatic Focus (No.)
Inhibition Rate (%)
No. Dead
Control
Amonafide
110.04 ± 15.38
65.25 ± 13.82
40.25 ± 8.15
0/8
0/8
0/8
5
1
40.70
62.42
11e
2.2.4. Systemic Toxicity
It is reported that amonafide can result in severe adverse effects and which greatly limited
its clinical application [35]. In both cell and animal models, some polyamine conjugates such as

Molecules 2016, 21, 1637
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F14512 has been confirmed to possess favorable cell or tissue selectivity [36], 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 tested the systemic toxicity of 11e (1 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 heart, liver, lung, kidney, spleen and thymus, were taken for the
inspection of systemic toxicity. There was no weight loss in the 11e-treated mice, on the contrary,
the weight increased slightly. Whereas treatment with amonafide was associated with some degree
of weight loss, indicating possibly dose-related toxicity (Figure 5A). Heart, kidney and lung had
no obvious index value changes in the examination of both compounds (Figure 5C). The liver and
spleen index numbers had no significant variation after 11e treatment compared to controls, but
amonafide treatment obviously decreased these index values, implying that 11e had no influence on
liver and immune function, at the same time suggesting that amonafide injured liver function and
caused damage to immune function in some extent (Figure 5B,C). In short, 11e performed better than
amonafide in the experiments of the toxicological profile at the doses that exert antitumor activity
in vivo.
Figure 5. Systemic toxicity of 11e. (
and normal saline; ( ) The organ indexes of immune organ (spleen) were evaluated after treatment
with 11e, amonafide, and normal saline (n = 3, x SD *** p < 0.001); ( ) The organ indexes (heart, liver,
A) The changes of body weight of mice treated with 11e, amonafide,
B
±
C
kidney and lung) were evaluated after treatment with 11e, amonafide, and normal saline (n = 3, x
** p < 0.01).
±
s
2.2.5. ADMET Prediction
The in silico prediction of physicochemical, ADME and toxicity properties of compounds 11e
was calculated using the ACD/Labs Percepta Platform 23 (License#58830) and results were compared
to those obtained for amonafide. As demonstrated in Table 4, the druglikeness of compound 11e
was similar to amonafide except rotatable bonds [37,38]. Regardless of the poor human intestinal
absorption (HIA), compound 11e showed excellent solubility and general permeable based on
predicted permeability across Caco-2 monolayers (Pe). The drug safety profile of these compounds
was also projected using Program ACD/Percepta based on probabilistic predictors. The inhibition
of hERG (human Ether-a-go-go-Related Gene) and the mutagenic profile were calculated and the
results converted in the so called classification scores (Table 5). As depicted in Table 4, compound 11e
was predicted as undefined (score > 0.33 and <0.67) in HLM (human liver microsomes), hERG and
mutagenic, which were similar to amonafide.

Molecules 2016, 21, 1637
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Table 4. Physico-chemistry properties and ADMET profile of compound 11e and amonafide calculated
using the Program ACD/Percepta.
Compounds
Predicted Properties
11e
Amonafide
MW (g/mol)
H-Donors
H-Acceptor
Rot. Bonds
Rings
496.65
6
9
18
3
283.33
2
5
3
3
Lipinski
Log P
1
0.57
0
1.35
Solubility
Caco-2
HIA
HLM
hERG
AMES
CNS
1000 mg/mL
21.1 mg/mL
−6
−6
Pe = 0.1 × 10 cm/s
Pe = 13 × 10 cm/s
1%
0.49
97%
0.46
0.39
0.57
−3.2
0.49
0.41
−4.81
ADMET = Absorption, Distribution Metabolism, Excretion, Toxicity; MW = Molecular weight; HIA = Human
intestinal absorption; HLM = Human liver microsomes; hERG = Human ether-a-go-go-related gene;
AMES = i.e., probability of positive Ames test; CNS = Central nerous system.
Table 5. The meaning of classification score range values for ADMET properties using Program
ACD/Percepta.
Predicted ADMET Properties
Classification Scores
HLM
hERG
AMES
≤0.33
>0.33 and ≤0.67
>0.67
Stable
Undefined
Undefined
Non-inhibitor
Undefined
Inhibitor
Non-mutagenic
Undefined
Mutagenic
3. Materials and Methods
3.1. General Information
All solvents and reagents were purchased from the suppliers and used without further
purification. All NMR spectra were recorded on an AV-400 model spectrometer (Bruker BioSpin,
1
Zürich, Switzerland) in D2O or CDCl3, Chemical shifts for H-NMR spectra are reported in parts
per million to residual solvent protons. ESI-MS spectra were recorded on an ESQUIRE-LC mass
spectrometer (Agilent, Palo Alto, CA, USA). 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 (Elementar ,Langenselbold, Germany), and results were within 0.4% of
the theoretical values.
3.2. General Procedure for the Synthesis of 5d–f
A mixture of 4-acetyl-1,8-naphthalic anhydride
3 (2 mmol) and polyamine 6 (2 mmol) (as shown
in Figure 2) in EtOH (30 mL) 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 purified by chromatography on silica
gel using petroleum ether/ethyl acetate mixture as eluent to obtain the Boc protected intermediates
4d
–
f
. The respective N-Boc protected intermediates 4d
–
f
(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. The solution typically gave a white solid as precipitate.
The solid was filtered, washed several times with absolute ethanol, and target compounds 5d–f were
obtained [19].

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2-[(2-Dimethylamino)ethyl-[4-(4-aminobutylamino)butyl]-6-acetyl-1H-benz[de]isoquinoline-1,3(2H)-dione
1
dihydrochloride (5d). Yield: 83%, white solid. H-NMR (D2O)
δ: 8.67 (d, 1H, J = 8.0 Hz, Ar-H), 8.37 (d,
2H, J = 7.2 Hz, Ar-H), 8.17(d, 1H, J = 7.6 Hz, Ar-H), 7.80 (t, 1H, J = 8.0 Hz, Ar-H), 4.11(t, J = 6.0 Hz, 2H,
CH2), 3.18~3.24 (m, 4H, 2 CH2), 3.11~3.15 (m, 2H, 1 CH2), 2.91 (s, 3H, 1 CH3), 1.85~1.86 (m,
8H, 4 : 206.26, 166.21, 165.60, 140.89, 134.59, 133.39, 132.11, 131.39, 130.69,
CH2); 13C-NMR (D2O)
128.68, 128.48, 125.27, 122.21, 49.63, 49.44, 42.27, 41.35, 31.76, 26.64, 26.49, 25.73, 25.35; ESI-MS m/z:
382.2 [M + H 0.3H2O: C 57.47%, H 6.49%, N 9.14%; found:
2HCl]+. Anal. calcd. for C22H29N3O3Cl2
C 57.32%, H 6.61%, N 9.01%.
1
×
×
×
×
×
δ
−
·
2-{3-[4-(3-Aminopropylamino)butylamino]butyl}-6-acetyl-1H-benz[de]isoquinoline-1,3(2H)-dione trihydrochloride
1
(
5e). Yield: 84%, white solid. H-NMR (D2O)
δ
: 8.59 (d, 1H, J = 8.4 Hz, Ar-H), 8.28~8.31 (m, 2H, Ar-H),
CH2), 3.15~3.25
CH2 ), 1.85~1.87 (m, 4H, 2 CH2);
: 203.86, 163.91, 163.29, 138.65, 132.25, 131.04, 129.75, 128.80, 128.22, 126.44, 126.14,
122.74, 119.75, 47.04, 45.28, 44.56, 37.49, 36.55, 29.33, 27.63, 26.10, 24.14, 23.75, 22.82; ESI-MS m/z: 425.3
[M + H 1.5H2O: C 51.39%, H 6.83%, N 9.99%; found: C
3HCl]+. Anal. calcd. for C24H35N4O3Cl3
51.63%, H 6.94%, N 9.72%.
8.10 (d, 1H, J = 8.1 Hz, Ar-H), 7.74 (t, J = 10 Hz, 1H, Ar-H), 4.15 (t, J = 8.0 Hz, 2H, 1
(m, 10H, 5 CH2), 2.86 (s, 3H, 1 CH3), 2.12~2.20 (m, 4H, 2
13C-NMR (D2O)
×
×
×
×
×
δ
−
·
2-{4-[4-(4-Aminobutylamino)butylamino]butyl}-6-acetyl-1H-benz[de]isoquinoline-1,3(2H)-dione trihydrochloride
5f). Yield: 82%, white solid, 1H-NMR (D2O)
: 8.60 (d, 1H, J = 8.8 Hz, Ar-H), 8.27 (d, J = 8.0 Hz, 2H,
Ar-H), 8.11 (d, 1H, J = 7.6 Hz, Ar-H), 7.73 (t, J = 8.0 Hz, 1H, Ar-H), 4.04 (t, J = 6.0 Hz, 2H, 1 CH2),
3.14~3.21 (m, 8H, 4 CH2), 3.10 (t, J = 8.0 Hz, 2H, 1 CH2), 2.87 (s, 3H, 1 CH3), 1.81~1.84 (m, 12H,
CH2); 13C-NMR (D2O)
: 203.62, 163.58, 162.96, 136.29, 132.01, 130.68, 129.54, 128.85, 128.13, 126.27,
125.64, 122.65, 119.58, 47.11, 46.90, 39.73, 38.80, 29.22, 24.09, 23.92, 23.21, 22.88, 22.76, 22.64; ESI-MS
m/z: 453.2 [M + H 1.2H2O: C 53.51%, H 7.15%, N 9.60%;
3HCl]+. Anal. calcd. for C26H39N4O3Cl3
(
δ
×
×
×
×
6
×
δ
−
·
found: C 53.21%, H 6.94%, N 9.43%.
3.3. General Procedure for the Synthesis of Compounds 11
3.3.1. Synthesis of (3-Aminopropyl)-1H-benz[de]isoquinoline-1,3(2H)-dione (8)
A mixture of 1,8-naphthalic anhydride (10 mmol) and 1,3-propanediamine (10 mmol) in EtOH was
heated at refluxing temperature, and the reaction process was monitored by TLC. After completion, the
solvent was removed by evaporation. The resulting solid (
8) was used in the next step without isolation.
1
(3-Aminopropyl)-1H-benz[de]isoquinoline-1,3(2H)-dione (
8
). Yield: 85%, white solid. H-NMR (DMSO)
δ:
8.44~8.50 (m, 4H, Ar-H), 7.85~7.89 (m, 2H, Ar-H), 4.10 (t, J = 8.0 Hz, 2H, 1
×
CH2), 2.58 (t, J = 6.0 Hz,
2H, 1 × CH2), 1.68~1.75 (m, 2H, 1 × CH2); ESI-MI m/z: 255.2 [M + 1]+.
3.3.2. General Procedure for the Synthesis of 11a–b, e–f
To a solution of intermediate (
8) (5 mmol) and anhydrous K2CO3 (7.5 mmol) in acetonitrile (50 mL),
chloroacetyl chloride (6 mmol) in acetonitrile (5 mL) was added at room temperature. The mixture
was stirred at room temperature overnight, then compound
washed with water (three times with each 10 mL).
9 (1.52 g) was collected by filtration and
A mixture of
(5 mL) and acetonitrile (15 mL) was stirred at room temperature for 0.5 h, then 1 equivalent of Boc
protected polyamine was added and the mixture was stirred overnight. After completion, the reaction
9 (1 mmol), anhydrous K2CO3 (1.5 mmol), a catalytic amount of KI, in DMF
6
mixture was concentrated under vacuum to give an oily residue. The residue was then dissolved
in CH2Cl2 (25 mL) and washed with aqueous Na2CO3 (25 mL). The organic layer was separated,
dried with anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified
by chromatography on silica gel using CH2Cl2/CH3OH (100:3 to 100:5) mixture as eluent to yield
compounds 10a
–b, e–f. The respective N-Boc protected intermediates 10 (1.0 mmol) were dissolved in
◦
◦
EtOH (10 mL) and stirred at 0 C for 10 min. Then 4 M HCl was added dropwise at 0 C. The reaction

Molecules 2016, 21, 1637
10 of 16
mixture was stirred at room temperature overnight. The solution typically gave a white solid as
precipitate. The solid was filtered, washed several times with absolute ethanol, and dried under
vacuum to give the pure target compounds 11a–b, e–f.
2-(3-Aminopropylamino)-N-(3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)acetamide dihydrochloride
(
11a). Yield: 85%, white solid. 1H-NMR (D2O)
δ
: 7.75 (t, J = 8.0 Hz, 4H, Ar-H), 7.28~7.31 (m, 2H,
CH2), 3.69 (t, J = 6.0 Hz, 2H, 1 CH2), 3.24 (t, J = 6.4 Hz, 2H, 1 CH2), 3.17
CH2), 3.07 (t, J = 8.0 Hz, 2H, 1 CH2), 2.05~2.13 (m, 2H, 1 CH2), 1.70~1.75
: 165.68, 163.82, 134.54, 130.66, 129.61, 126.46, 125.19, 119.01,
2HCl]+. Anal. calcd. for
Ar-H), 3.91 (s, 2H, 1
×
×
×
(t, J = 8.0 Hz, 2H, 1
×
×
×
(m, 2H, 1 × CH2); 13C-NMR (D2O)
δ
48.09, 44.60, 37.94, 37.21, 36.54, 26.57, 23.68; ESI-MI m/z: 369.3 [M + 1
−
C20H26Cl2N4O3·0.4H2O: C 53.55%, H 6.02%, N 12.49%; found: C 53.65%, H 5.93%, N 12.26%.
2-(3-(3-Aminopropylamino)propylamino)-N-(3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)acetamide
1
trihydrochloride (11b). Yield: 85%, white solid. H-NMR (D2O)
δ
: 7.82 (t, J = 7.6 Hz, 4H, Ar-H), 7.36
CH2), 3.27 (t, J = 6.4 Hz, 2H,
CH2), 2.13~2.17 (m, 2H, 1 CH2),
CH2); 13C-NMR (D2O)
: 165.62, 163.98, 134.63,
130.76, 129.71, 126.52, 125.32, 119.10, 48.10, 44.71, 44.57, 44.47, 37.96, 37.21, 36.50, 26.57, 23.73, 22.61;
ESI-MI m/z: 426.3 [M + 1 0.35: C 51.04%, H 6.46%,
3HCl]+. Anal. calcd. for C23H34Cl3N5O3
N 12.94%; found: C 51.04%, H 6.78%, N 12.97%.
(t, J = 7.2 Hz, 2H, Ar-H), 3.93 (s, 2H, 1
CH2), 3.15~3.22 (m, 6H, 3 CH2), 3.08 (t, J = 7.6 Hz, 2H, 1
2.06~2.10 (m, 2H, 1 CH2), 1.73~1.76 (m, 2H, 1
×
CH2), 3.74 (t, J = 6.0 Hz, 2H, 1
×
1
×
×
×
×
×
×
δ
−
·
2-(3-(4-(3-Aminopropylamino)butylamino)propylamino)-N-(3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)
acetamide tetrahydrochloride (11e). Yield: 85%, white solid. 1H-NMR (D2O)
: 7.97 (d, J = 6.8 Hz, 4H, Ar-H),
7.48 (t, J = 7.2 Hz, 2H, Ar-H), 3.93 (s, 2H, 1 CH2), 3.83 (t, J = 5.6 Hz, 2H, 1 CH2), 3.28 (t, J = 6.4 Hz
2H, 1 CH2), 3.05~3.21 (m, 12H, 6 CH2), 2.10~2.18 (m, 2H, 1
CH2), 2.04~2.07 (m, 2H, 1 × CH2),
1.76~1.81 (m, 6H, 3 : 165.63, 164.45, 134.63, 130.99, 130.01, 126.69, 125.78,
CH2); 13C-NMR (D2O)
119.55, 48.10, 47.03, 46.98, 44.52, 44.43, 38.00, 37.26, 36.52, 26.63, 23.72, 22.75, 22.63; ESI-MI m/z: 497.4
[M + 1 0.25H2O: C 50.12%, H 6.93%, N 12.99%; found:
4HCl]+. Anal. calcd. for C27H44Cl4N6O3
C 50.37%, H 6.65%, N 12.70%.
δ
×
×
,
×
×
×
×
δ
−
·
2-(4-(4-(4-Aminobutylamino)butylamino)butylamino)-N-(3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)
1
acetamide tetrahydrochloride (11f). Yield: 85%, white solid. H-NMR (D2O)
δ
: 7.71~7.76 (m, J = 8.0 Hz,
CH2), 3.19 (t,
CH2), 1.65~1.71 (m,
: 165.80, 164.53, 134.87, 131.06, 130.19, 126.75, 125.96, 119.74, 47.99,
46.91, 46.81, 38.81, 38.06, 38.02, 37.30, 37.28, 26.69, 26.65, 26.64, 23.92, 22.80, 22.76; ESI-MI m/z: 525.3
[M + 1 1.05H2O: C 50.52%, H 7.32%, N 12.19%; found:
4HCl]+. Anal. calcd. for C29H48Cl4N6O3
4H, Ar-H), 7.28 (t, J = 8.0 Hz, 2H, Ar-H), 3.82 (s, 2H, 1
J = 6.4 Hz, 2H, 1 CH2), 2.99~3.06 (m, 10H, 5 CH2), 2.92 (t, J = 7.6 Hz, 2H, 1
14H, 7
CH2); 13C-NMR (D2O)
×
CH2), 3.64 (t, J = 7.2 Hz, 2H, 1
×
×
×
×
×
δ
−
·
C 50.60%, H 7.12%, N 12.42%.
3.4. General Procedure for the Synthesis of 17b–d
3.4.1. Synthesis of Compounds 14a–b
Intermediates 12, 13 were prepared by a modified previously reported procedure [26,27].
Compound 13 (1.13 g, 5 mmol) added portion wise to 1,3-dibromopropane or 1,4-dibromobutane
(10 mmol) in acetone (30 mL). The solution was refluxed for 12 h; after cooling and filtration, the
solvent was removed under vacuo to give a solid. The latter was recrystallized from ethanol to afford
compound 14a–b.
1
5-Bromo-2-(3-bromopropyl)isoindoline-1,3-dione (14a), Yield: 70%, H-NMR (CDCl3)
δ: 7.97 (d, 1H,
J = 1.8 Hz, Ar-H), 7.85~7.87 (m, 1H, Ar-H), 7.72 (d, 1H, J = 8.0 Hz, Ar-H), 3.71 (t, 2H, J = 6.0 Hz,
1 × CH2), 3.44 (t, 2H, J = 6.4 Hz, 1 × CH2), 1.82~1.91 (m, 4H, 1 × CH2). ESI-MS m/z: 345.9 [M + 1]+.

Molecules 2016, 21, 1637
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1
5-Bromo-2-(4-bromobutyl)isoindoline-1,3-dione (14b), Yield: 68%, H-NMR (CDCl3)
δ: 7.98 (d, 1H,
J = 1.2 Hz, Ar-H), 7.84~7.87 (m, 1H, Ar-H), 7.72 (d, 1H, J = 7.6 Hz, Ar-H), 3.71 (t, 2H, J = 6.8 Hz,
1 × CH2), 3.44 (t, 2H, J = 6.4 Hz, 1 × CH2), 1.81~1.91 (m, 4H, 2 × CH2). ESI-MS m/z: 359.9 [M + 1]+.
3.4.2. Synthesis of Compounds 15b–d
Intermediates 14 (1 mmol), Boc protected polyamine (1.2 mmol), anhydrous K2CO3 (1.5 mmol)
were added to the◦flask in the presence of KI (0.01 mmol) and CH3CN (30 mL). The resulting mixture
was heated at 45 C for 12 h, then the solvent was evaporated in vacuo, water was added to the
residue, and extracted with CH2Cl2. The organic layer was dried over Na2SO4 and evaporated in
vacuo. The residue was dissolved in CH3OH and a solution of di-tert-butyl dicarbonate (1.5 mmol)
in methanol (5 mL) was added dropwise at 0 ◦C then the mixture was stirred for an additional 1 h at
◦
0 C. The temperature was allowed to gradually rise to room temperature, and the reaction was stirred
overnight. The mixture was evaporated in vacuo. The residue was dissolved in CH2Cl2 and washed
with water several times. The organic layer was separated, dried with anhydrous Na2SO4, filtered,
and concentrated. The residue could be purified by flash column chromatography on silica gel.
5-Bromo-2-{3-[3-(3-butoxycarbonylaminopropylbutoxycarbonylamino)-propylbutoxy-carbonyl-amino]propyl}
isoindoline-1,3-dione (15b). Yield: 63%; 1H-NMR (CDCl3)
δ: 7.88 (s, 1H, Ar-H), 7.78 (d, 1H, J = 8.0 Hz,
Ar-H), 7.63 (d, 1H, J = 8.0 Hz), 3.62 (t, 2H, J = 6.4 Hz, 1
×
CH2), 3.03~3.17 (m, 10H, 5 CH2), 1.82 (m,
×
2H, 1 × CH2), 1.35~1.44 (m, 31H, 2 × CH2 + 9 × CH3); ESI-MS m/z: 697.3 [M + 1]+.
5-Bromo-2-{3-[4-(3-butoxycarbonylaminopropylbutoxycarbonylamino)-butylbutoxy-carbonyl-amino]propyl}
isoindoline-1,3-dione (15c). Yield: 56%; 1H-NMR (CDCl3)
: 7.87 (s, 1H, Ar-H), 7.78 (t, 1H, J = 6.40 Hz,
CH2), 3.04~3.18 (m, 10H, 5 CH2), 1.83 (m,
δ
Ar-H), 7.64 (d, 1H, J = 8.0 Hz), 3.65 (t, 2H, J = 6.0 Hz, 1
×
×
4H, 2 × CH2), 1.32~1.40 (m, 31H, 2 × CH2 + 9 × CH3); ESI-MS m/z: 711.3 [M + 1]+.
5-Bromo-2-{4-[4-(4-butoxycarbonylaminobutylbutoxycarbonylamino)-butylbutoxy-carbonyl-amino]butyl}
isoindoline-1,3-dione (15d). Yield: 60%; 1H-NMR (CDCl3)
: 7.88 (s, 1H, Ar-H), 7.77 (t, 1H, J = 6.80 Hz
CH2), 3.02~3.15 (m, 10H, 5 CH2), 1.31~1.401
δ
,
Ar-H), 7.63 (d, 1H, J = 6.4 Hz), 3.64 (t, 2H, J = 6.6 Hz, 1
×
×
(m, 39H, 6 × CH2 + 9 × CH3); ESI-MS m/z: 739.3 [M + 1]+.
3.4.3. Synthesis of Compounds 17b–d
A 50 mL round bottomed flask equipped with a magnetic stirring bar was charged with pure
15 (2 mmol), toluene (20 mL), phenylboronic acid (2 mmol), K2CO3 (3 mmol), PdCl2 (0.03 mmol)
and tetrabutylammonium bromide (0.03 mmol). 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 to obtain the intermediates 16b
–d.
The respective N-Boc protected intermediates 16b (1.2 mmol) were dissolved in EtOH (20 mL) and
–
d
stirred at 0 ◦C for 10 min. Then, 4 M HCl was added dropwise at 0 ◦C The reaction was stirred at
room temperature overnight. 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 17b–d.
5-Phenyl-2-{3-[3-(3-aminopropylamino)-propylamino]propyl}isoindoline-1,3-dione trihydrochloride (17b).
1
Yield: 78%, H-NMR (D2O)
δ
: 7.38 (d, 1H, J = 8.0 Hz, Ar-H), 7.25~7.33 (m, 2H, Ar-H), 7.21 (t, 3H,
CH2), 3.11~3.22 (m,
CH2); 13C-NMR (D2O)
: 169.33,
168.79, 146.39, 137.55, 132.77, 129.81, 129.17, 128.90, 126.48, 123.86, 120.98, 45.30, 44.69, 44.54, 36.51,
34.81, 24.94, 24.80, 23.72, 22.63; ESI-MS m/z: 395.3 [M + 1 0.1H2O: C 54.63%,
3HCl]+. C23H33Cl3N4O2
H 6.62%, N 11.08%; found: C 54.59%, H 6.91%, N 10.99%.
J = 7.8 Hz, Ar-H), 7.07~7.09 (d, 2H, J = 7.6 Hz, Ar-H), 3.68 (t, 2H, J = 6.0 Hz, 1
10H, 5 CH2), 2.09~2.16 (m, 4H, 2 CH2), 1.90~2.01 (m, 2H, 1
×
×
×
×
δ
−
·
5-Phenyl-2-{3-[4-(3-aminopropylamino)-amino]propyl}isoindoline-1,3-dione trihydrochloride (17c). Yield: 76%;
1H-NMR (D2O)
: 7.94~7.96 (m, 1H, Ar-H), 7.90 (d, 1H, J = 7.6Hz, Ar-H), 7.80 (s, 1H, Ar-H), 7.73~7.75
δ

Molecules 2016, 21, 1637
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(m, 1H, Ar-H), 7.68~7.70 (m, 1H, Ar-H), 7.53~7.56 (m, 1H, J = 6.4 Hz, Ar-H), 7.46~7.48 (m, 2H, Ar-H),
3.67~3.74 (m, 2H, 1 CH2), 3.09~3.19 (m, 10H, 5 CH2), 2.02~2.14 (m, 4H, 2 CH2), 1.79~1.80 (m, 4H,
CH2); 13C-NMR (D2O)
: 169.27, 169.01, 145.93, 137.43, 132.52, 132.16, 131.42, 129.04, 126.53, 123.66,
×
×
×
2
×
δ
120.68, 47.04, 45.02, 44.56, 36.56, 34.83, 24.97, 24.79, 23.75, 22.83, 22.77; ESI-MS m/z: 409.3 [M + 1 − 3HCl]+
.
C24H35Cl3N4O2·0.1H2O: C 55.46%, H 6.83%, N 10.78%; found: C 55.77%, H 6.98%, N 10.75%.
5-Phenyl-2-{4-[4-(4-aminobutylamino)-butylamino]butyl}isoindoline-1,3-dione trihydrochloride (17d). Yield:
77%, 1H-NMR (D2O)
: 7.30~7.31 (m, 1H, Ar-H), 7.18~7.23 (m, 2H, Ar-H), 7.10~7.15 (m, 3H, Ar-H),
8.96~7.01 (m, 2H, Ar-H), 3.29 (t, 2H, J = 6.0 Hz, 1 CH2), 2.96~3.06 (m, 10H, 5 CH2), 1.73~1.74 (m,
8H, 4 CH2), 1.60~1.63 (m, 2H, 1 CH2), 1.45~1.47 (m, 2H, 1 : 169.69,
CH2); 13C-NMR (D2O)
169.51, 146.14, 137.29, 132.29, 131.51, 129.10, 129.05, 126.69, 123.61, 120.83, 46.96, 46.89, 46.85, 46.79,
38.75, 36.97, 27.63, 24.87, 23.89, 22.98, 22.79, 22.73; ESI-MS m/z: 437.3 [M + 1
3HCl]+. Anal. calcd. for
δ
×
×
×
×
×
δ
−
C26H39Cl3N4O2: C 57.20%, H 7.20%, N 10.26%; found: C 57.35%, H 7.28%, N 10.15%.
3.5. Cell Culture
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 (Biological Industries Cromwell, CT,
USA). Cells were cultured at 37 ◦C under a 5% CO2 atmosphere.
3.6. Cytotoxicity against Cancer Cell Lines
The antiproliferative ability of compounds was evaluated in HCT-116 cells, QSG-7701,
MDA-MB-231 cells, K562 and HepG2 cells by the conversion of MTT to a purple formazan precipitate
as previously described [39]. Briefly, exponentially growing HCT-116 cells, QSG-7701, MDA-MB-231
cells and HepG2 cells were seeded in 96-well plates (5 × 103 cells to each well) and allowed to attach
overnight. After 24 h, the cells were treated with various concentrations (1, 5, 10, 30, and 50 µM) of
samples for 48 h, and then MTT (100 µL, 1 mg/mL) was added. After incubation for 4 h at 37 ◦C, the
MTT solution was removed and the crystals of viable cells were dissolved with DMSO (150 L) in each
well; 5000/well exponentially growing K562 were seeded in 96-well plates and treated with indicated
µ
concentrations (1, 5, 10, 30, and 50 µ◦M) of samples for 48 h, and then MTT (10 mg/mL, 10
µL) was
added. After incubation for 4 h at 37 C the crystals of viable cells were dissolved overnight with SDS
(sodium dodecylsulfonate, 10%, 100 µL) in each well. The absorbance spectra were measured on an
ELISA Processor II Microplate Reader (Thermo Scientific, Waltham, MA, USA) at a wavelength of
570 nm. The percentage of cytotoxicity was defined with treated and untreated cell lines. The 50%
antitumor activity dose (IC50) was defined as the concentration of samples that reduced the absorbance
of the treated cells by 50%.
3.7. Cellular Apoptotic Evaluation
HepG2 cells were seeded in 96 well plates (6
a confluent monolayer and then treated with various concentrations of tested compounds for 48 h.
Cells were incubated with Acridine Orange (50 M)/ethidium bromide (50 M) for 30 min, then
×
103 cells/well), cultured for 24 h to obtain
µ
µ
washed with PBS to remove unbound dyes. Images were obtained on the High Content Screening
(HCS, ArrayScan, Thermo Scientific) reader using the Target Activation BioApplication software
(Thermo Scientific).
HepG2 cells were seeded in 96 well plates (6
a confluent monolayer, and then treated with various concentrations of tested compounds for 48 h.
×
103 cells/well), cultured for 24 h to obtain
Cells were incubated with Rh123 (0.25 nM), or DCFH-DA (10 µM), or Lyso-Tracker Red (50 nM ) in
the dark at 37 ◦C for 30 min. for the measurement of mitochondrial membrane potential (MMP), or
reactive oxygen species (ROS) or lysosomal mass/pH staining, respectively. After being washed with
PBS to remove excess dyes, cells were further stained with the nuclear dye Hoechst 33,342 (10 µM) in

Molecules 2016, 21, 1637
13 of 16
the dark at 37 ◦C for 20 min. Images were acquired on the ArrayScan HCS reader using the Target
Activation BioApplication software.
3.8. 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 (HUSOM2016-162). For solid tumor development, 30 male
Kunming mice (Laboratory Animal Center of Henan, Zhengzhou, China) 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, 11e group, and amonafide group) and
then were administered by caudal vein injection 11e (1 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 cervical
vertebra dislocation, and solid tumors were removed and weighed. The inhibitory rate was calculated
as follows: inhibitory rate (%) = [(A
group and B was that of the drug treated or positive group.
−
B)/A]
×
100, where A was the mean tumor weight of the control
3.9. H22 Cells Lung Metastasis Models
H22 cells (5
×
106 cells per mouse) for Kunming mice were injected intravenous (i.v.) 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 11e (1 mg/kg), amonafide (5 mg/kg, positive, control), and normal saline
(negative control) (n = 8) for 7 consecutive days. On day 18, mice were killed by cervical vertebra
dislocation, and lungs were removed and weighed, and then fixed in Bouin’s fluid. After the lungs
were fixed, lung metastases nodus was numbered.
3.10. Survival Time in Mice Bearing H22 Cells
For calculation of the survival time, 30 male Kunming mice were inoculated via intraperitoneal
(i.p.) injection with 1
×
106 H22 cells/mouse on day 0 and were randomly divided into three groups.
The treatment with 11e (1 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 the drug was evaluated by measuring the increase
of the lifespan. The extended lifespan rate was calculated as follows: extend rate (%) = (MST treated
group/MST control group) × 100.
3.11. Systemic Toxicity Evaluation
Mice (8 per group) received 11e (1 mg/kg) and amonfide (5 mg/kg, i.v.) for 7 consecutive days
and then were sacrificed. Heart, liver, kidney, lung and spleen were removed and weighed. The organ index
was investigated for systemic toxicity evaluation: organ index (%) = (organ weight/body weight) × 100.
4. Conclusions
Three series of aromatic imide modified polyamines were synthesized, and their induced growth
inhibition of multiple cancer cell lines in vitro was determined using the MTT assay. We found
that both the imide scaffold and the polyamine motif affect the biological activities of the designed
compounds. One naphthalene imide-polyamine conjugate potently inhibited the growth of multiple
cancer cell lines as the reference drug amonafide. The impacts of 11e on cell morphology, mitochondrial
transmembrane potential, and lysosomes indicated that 11e may impair tumor cells via apoptosis.
The present study identified compound 11e induces apoptosis via a ROS mediated mitochondrial
pathway in the preliminary study of cell death profile. Further in vivo experiments on H22 tumor
transplant models showed that 11e prevented the lung cancer metastasis and extended lifespan
compared to amonafide. Encouragingly, 11e inhibited tumor growth and improved body weight

Molecules 2016, 21, 1637
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more efficiently than amonafide, which strongly corroborated that compound 11e is worthy of further
investigation as a new lead compound for cancer. Collectively, the present report may provide new
inspiration for tackling critical issues ranging from naphthalimide/polyamine-based drug design to
antitumor drug screening.
Acknowledgments: This work was supported by Projects of Science and Technology of Henan (152300410058);
NSFC-Henan talented man train union fund (No. U1204829); Program for Science and Technology Innovation
Talents in Universities of Henan Province (No. 14HASTIT033).
Author Contributions: Ming Li, Yuxia Wang, Songqiang Xie, Chaojie Wang and Yingliang Wu conceived
and designed the experiments; Ming Li, Yuxia Wang, Jianying Zhang carried out experiments; Yuxia Wang,
Ming Li, Songqiang Xie, Chaojie Wang and Yingliang Wu analyzed experimental results; Yuxia Wang, Ming Li,
Songqiang Xie and Chaojie Wang wrote the manuscript; all authors who are Yuxia Wang, Ming Li, Jianying Zhang,
Songqiang Xie, Chaojie Wang and Yingliang Wu discussed, edited and approved the final version.
Conflicts of Interest: The authors declare no conflict of interest.
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