3-Nitroacridine derivatives arrest cell cycle at G0/G1 phase and induce apoptosis in human breast cancer cells may act as DNA-target anticancer agents

Article abstract of DOI:10.1016/j.lfs.2018.05.010

DNA is considered to be one of the most promising targets for anticancer agents. Acridine analogues have anticancer activity based on DNA binding and topoisomerases inhibition. However, due to the side effects, resistance and low bioavailability, a few have entered into clinical usage and the mechanisms of action are not fully understood. Novel acridine derivatives are needed for effective cancer therapy. A series of novel 3-nitroacridine-based derivatives were synthesized, their DNA binding and anticancer activities were evaluated. The chemical modifications at position 9 of the 3-nitroacridine were crucial for DNA affinity, thus optimizing anticancer activity. UV–Vis and circular dichroism (CD) spectroscopy indicated interaction of compounds with DNA, and the binding modes were intercalation and groove binding. MTT assay and clonogenic assay showed that compounds 1, 2 and 3 had obvious cell growth inhibition effect. They induced cell apoptosis in human breast cancer cells in a dose-dependent manner, and exhibited anticancer effect via DNA damage as well as cell cycle arrest at G0/G1 phage. Using confocal fluorescent microscope, the apoptotic features were observed. The results suggested that compounds 1–3 with high DNA binding affinity and good inhibitory effect of cancer cell proliferation can be developed as prime candidates for further chemical optimization.

Full text of DOI:10.1016/j.lfs.2018.05.010

Accepted Manuscript
3
-Nitroacridine derivatives arrest cell cycle at G0/G1 phase and
induce apoptosis in human breast cancer cells may act as DNA-
target anticancer agents
Qian Zhou, Chaoqun You, Cong Zheng, Yawen Gu, Hongchao
Gu, Rui Zhang, Hongshuai Wu, Baiwang Sun
PII:
S0024-3205(18)30250-9
doi:10.1016/j.lfs.2018.05.010
LFS 15704
DOI:
Reference:
To appear in:
Life Sciences
Received date:
Revised date:
Accepted date:
27 March 2018
3 May 2018
3 May 2018
Please cite this article as: Qian Zhou, Chaoqun You, Cong Zheng, Yawen Gu, Hongchao
Gu, Rui Zhang, Hongshuai Wu, Baiwang Sun , 3-Nitroacridine derivatives arrest cell cycle
at G0/G1 phase and induce apoptosis in human breast cancer cells may act as DNA-target
anticancer agents. The address for the corresponding author was captured as affiliation for
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ACCEPTED MANUSCRIPT
3
-nitroacridine derivatives arrest cell cycle at G0/G1 phase and
induce apoptosis in human breast cancer cells may act as
DNA-target anticancer agents
a
a
b
b
b
b
Qian Zhou , Chaoqun You , Cong Zheng , Yawen Gu , Hongchao Gu , Rui Zhang ,
a
a
Hongshuai Wu , Baiwang Sun*
a
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210089, China.
Department of Chemical and Pharmaceutical Engineering, Southeast University Chenxian
b
College, Nanjing 210000, China
Corresponding author: E-mail: chmsunbw@seu.edu.cn; Fax: +86-25-52090614;
Tel: +86-25-52090614
Abstract
DNA is considered to be one of the most promising targets for anticancer agents.
Acridine analogues have anticancer activity based on DNA binding and
topoisomerases inhibition. However, due to the side effects, resistance and low
bioavailability, a few have entered into clinical usage and the mechanisms of action
are not fully understood. Novel acridine derivatives are needed for effective cancer
therapy. A series of novel 3-nitroacridine-based derivatives were synthesized, their
DNA binding and anticancer activities were evaluated. The chemical modifications at
position 9 of the 3-nitroacridine were crucial for DNA affinity, thus optimizing
anticancer activity. UV-Vis and circular dichroism (CD) spectroscopy indicated
interaction of compounds with DNA, and the binding modes were intercalation and
groove binding. MTT assay and clonogenic assay showed that compounds 1, 2 and 3
had obvious cell growth inhibition effect. They induced cell apoptosis in human breast
cancer cells in a dose-dependent manner, and exhibited anticancer effect via DNA
damage as well as cell cycle arrest at G0/G1 phage. Using confocal fluorescent
microscope, the apoptotic features were observed. The results suggested that
compounds 1-3 with high DNA binding affintiy and good inhibitory effect of cancer
cell proliferation can be developed as prime candidates for further chemical
optimization.
Keywords: acridine derivatives; synthesis; DNA binding; anticancer activity;
structure-activity relationship (SAR); apoptosis.
1
. Introduction
Cancer is a deadly disease in the world[1], great efforts have been made to
understand the cancer biology and develop the potential anticancer drugs[2-6].
Among the current strategies for cancer treatment, chemotherapy remains the most
mature and effective approach[7].

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Numerous compounds display their anticancer activity through DNA binding and
their effectiveness depends on the mode and strength of the binding[8]. The
interaction between anticancer drugs and DNA can cause DNA damage in cancer cells
and block cell division, thereby leading to cell death[9]. As cancer cells exhibit high
levels of topoisomerase activity and demonstrate remarkable sensitivity to
DNA-targeted drugs, enormous anticancer drugs have been designed and synthesized
which specifically target the interaction between DNA and topoisomerases[10,11]. In
cancer therapy, DNA acts as target for two classes of chemotherapeutical: alkylating
agents and molecules with non-covalent binding properties. For the latter, DNA
recognition is a key stage of anti-tumor action, and for clinic effectiveness of drugs,
such as anthracyclines, anthraquinones, and acridines, intercalation is a kind of
interaction in the recognition of these biomolecules. Therefore, the finding of novel
DNA intercalating agents has been considered as a practical approach and a large
number of molecules have been evaluated intercalation properties[12,13].
Acridine derivatives have been used for commercial purposes for over a
century[14]. The acridines are formed by two benzene rings fused to a pyridine ring in
the centre[15]. Acridine derivatives are promising compounds for cancer therapy with
biological activity based on DNA binding and topoisomerase enzymes inhibition. So
far, a few molecules have entered clinical trials and have been approved for
chemotherapy[16]. Amsacrine (m-AMSA) is an anticancer agent that displays activity
against leukaemia and lymphomas in the clinic due its DNA-intercalation and
inhibition of Topo II[17,18]. However, the side effects, the development of resistance
and poor bioavailability, have limited therapeutic effect. These factors encouraged
chemists to structurally modify acridine and produce different derivatives that have
exhibited obvious antitumor activity[19,20].
The mutagenic activity of 9-aminoacridine has been characterized in the Ames
test[21], and the introduction of a nitro group increases the frameshift
mutagenicity[22]. In this study, we fouced on the development of novel
3
-nitroacridine derivatives that possess bioactive 9-substituents, which can affect or
interfere with cell processes or metabolities. We examined the binding constants of
the compounds with DNA using UV-Vis measurements, and used Circular dichroism
(CD) spectroscopy to confirm the DNA binding mode. The biological activities of the
compounds which include cell apoptosis, cell cycle distribution and morphological
changes were investigated in MCF-7 and MDA-MB-231 cells.
2
. Material and methods
2
.1 Synthesis
All reagents used in the synthesis were obtained from common vendors and used
1
without further purification. H NMR spectra was measured on a Bruker Avance-300
MHz instruments. Mass spectra (MS) were obtained from Agilent 1260. Elemental
analysis was measured on a Perkin Elmer CHN 2400 elemental analyzer.
Synthesis of the 3-nitroacridine derivatives were carried out as shown in Scheme
1
as previously described. Compound 9 was obtained from the Ullmann reaction of

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2
-chloro-4-nitrobenzonic acid with aniline, then Friedel-Crafts acyltion reaction was
conducted inphosphorus oxychloride at 105 ℃ for 4 h to yeild the acridine
intermediate 10[23].
Scheme 1. Reagents and conditions: (i) CuOAc, K CO , DMF, 130 ℃, 4 h; (ii) POCl , 105 ℃, 4 h;
2
3
3
(iii) various amines, NMP, HCl, reflux.
Table 1. Structures of compounds 1-8.
Compound
R
Compound
R
1
5
2
3
4
6
7
8
General Procedure[24]: 3-nitro-9-(4-carbethoxyl)phenylaminoacridine (1). 1 was
prepared by dissolving ethyl-p-aminobenzoate (0.358 g, 2.16 mmol) and compound
1
0 (0.564 g, 2..16 mmol) in N-methyl-2-pyrrolidinone (20 mL) with the addition of 2
drops of concentrated HCl. The reaction mixture was stirred for 4 h at room
temperature; EtOAc (100mL) was then added to precipitate out the crude product. The
crude product was then mixed with 10 mL of N-methyl-2-pyrrolidinone again for 1 h,
followed by addition of EtOAc (150 mL) for complete precipitation. The resulting
1
precipitate was collected. Yeild: 69.1%. H NMR (400 MHz, DMSO) δ 11.68 (br, 1H),

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8
1
.62-8.05 (m, 2H), 8.02-7.30 (m, 6H), 7.30-6.51 (m, 4H), 4.27 (q, J = 7.1 Hz, 2H),
+
.29 (t, J = 7.1 Hz, 3H). LCMS:[M+H] 388. Elemental analysis calcd for C H N O
2
2
17
3
4
(%): C 68.21, H 4.42, N 10.85; found: C 68.41, H 4.54, N 10.76.
9
-(2-carbethoxyl)ethylamino-3-nitroacridine (2). 2 was prepared from glycine ethyl
1
ester and compound 10 according to the procedure for 1. Yeild: 67.8%. H NMR (400
MHz, CDCl ) δ 8.96 (d, J = 1.7 Hz, 1H), 8.32 (d, J = 9.5 Hz, 1H), 8.19 (d, J = 8.7 Hz,
3
1
7
H), 8.13 (d, J = 8.7 Hz, 1H), 8.07 (dd, J = 9.5 Hz, 2.2 Hz, 1H), 7.82 – 7.75 (m, 1H),
.56-7.49 (m, 1H), 4.59 (s, 2H), 4.33 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H).
+
LCMS:[M+H] 326. Elemental analysis calcd for C H N O (%): C 62.76, H 4.65,
17
15
3
4
N 12.92; found: C 63.01, H 4.54, N 12.76.
3
3
7
8
-nitro-9-(3,4-dichloro)phenylaminoacridine (3).
3
was prepared from
,4-dichloroaniline and compound 10 according to the general procedure for 1. Yeild:
1
1.2%. H NMR (400 MHz, DMSO) δ 8.88 (s, 1H), 8.50 (d, J = 8.9 Hz, 1H),
.28-8.17 (m, 1H), 8.14-7.93 (m, 3H), 7.78-7.61 (m, 1H), 7.53-7.42 (m, 1H), 7.33 (d,
+
J = 5.6 Hz, 1H), 7.26 (d, J = 8.6 Hz, 1H), 6.87 (s, 1H). LCMS:[M+H] 383.7.
Elemental analysis calcd for C H Cl N O (%): C 59.39, H 2.89, N 10.94; found: C
1
9
11
2
3
2
6
3
0.11, H 2.84, N 11.76.
-nitro-9-(3-chloro)phenylaminoacridine (4). 4 was prepared from 3-chloroaniline
1
and compound 10 according to the general procedure for 1. Yeild: 72.8%. H NMR
(
400 MHz, DMSO) δ 8.66 (s, 1H), 8.42 (s, 1H), 7.98 (d, J = 5.2 Hz, 4H), 7.48-7.21
+
(m, 5H). LCMS:[M+H] 350.0. Elemental analysis calcd for C H ClN O (%): C
1
9
12
3
2
6
3
2
5.24, H 3.46, N 12.01; found: C 65.11, H 3.34, N 11.86.
-nitro-9-(4-bromo-2-methyl)phenylaminoacridine (5). 5 was prepared from
-methyl-4-bromoaniline and compound 10 according to the general procedure for 1.
1
Yeild: 73.9%. H NMR (400 MHz, Methanol) δ 8.71 (s, 1H), 8.36 (d, J = 9.8 Hz, 1H),
8
7
.15 (d, J = 9.4 Hz, 1H), 8.12 – 8.01 (m, 2H), 7.92 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H),
.57 (d, J = 8.6 Hz, 1H), 7.54 – 7.46 (m, 1H), 7.29 (d, J = 8.3 Hz, 1H), 2.32 (s, 3H).
+
LCMS:[M+H] 409.8. Elemental analysis calcd for C H BrN O (%): C 58.84, H
2
0
14
3
2
3
.46, N 10.29; found: C 59.12, H 3.44, N 10.66.
3
-nitro-9-(3,5-dimethyl)phenylaminoacridine (6).
6
was prepared from
N,N-dimethylaniline and compound 10 according to the general procedure for 1. Yeild:
1
7
1
2
7
3
2
6
7
1.8%. H NMR (400 MHz, DMSO) δ 8.73 (s, 1H), 8.43 (d, J = 8.7 Hz,1H), 8.13 (s,
H), 8.07 (d, J = 8.7 Hz, 2H), 7.96 (s, 2H), 7.43 (s, 1H), 7.06 (s, 1H), 7.00 (s, 2H)
+
.29 (s, 6H). LCMS:[M+H] 344.25. Elemental analysis calcd for C H N O (%): C
3.45, H 4.99, N 12.24; found: C 73.21, H 5.03, N 12.06.
-nitro-9-(2,4-dimethyl)phenylaminoacridine (7).
2
1
17
3
2
7
was prepared from
,4-dimethylaniline and compound 10 according to the general procedure for 1. Yeild:
1
9.1%. H NMR (400 MHz, DMSO) δ 8.86 (s, 1H), 8.42 (s, 1H), 8.06-7.80 (m, 4H),
.41 (s, 1H), 7.33 (s, 1H), 7.19 (t, J = 7.2 Hz, 3H), 2.39 (s, 3H), 2.23 (s, 3H).
ESI-MS:[M+H] 344.1. Elemental analysis calcd for C H N O (%): C 73.45, H
+
2
1
17
3
2
4
3
4
6
.99, N 12.24; found: C 73.11, H 4.84, N 12.36.
-nitro-9-(4-isopropyl)phenylaminoacridine (8).
8
was prepared from
-isopropylaniline and compound 10 according to the general procedure for 1. Yeild:
1
3.8%. H NMR (400 MHz, DMSO) δ 8.70 (s, 1H), 8.38 (s, 1H), 8.04 (s, 2H), 7.94 (s,

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2
6
H), 7.39 (d, J = 7.6 Hz, 3H), 7.28 (s, 2H), 3.01-2.95 (m, 1H), 1.26 (d, J = 2.8 Hz,
H), 0.86 (t, J = 6.4 Hz, 1H). LCMS:[M+H] 358.1. Elemental analysis calcd for
+
C H N O (%): C 73.93, H 5.36, N 11.76; found: C 74.11, H 5.44, N 11.76.
22
19
3
2
2
.2 UV-vis absorption measurements
The concentration of calf thymus DNA (CT-DNA) in Tris-HCl buffer (5 mM
Tris/50 mM NaCl, pH 7.2) was measured as described by Wolfe et al.[25] as
micromolar equivalents of the base pairs. The compounds were diluted to a final
-5
concentration of 1×10 M in Tris-HCl buffer, followed adding CT-DNA. The final
-4
concentrations of CT-DNA were ranged from 0 to 0.9×10 M. Each absorption
spectrum was scanned at a wavelength range of 200-600 nm, and all measurements
were performed at room temperture.
2
.3 Circular dichroism
Circular dichroism (CD) spectra were obtain at room temperature from 220 to
−
5
3
20nm. The samples of the same concentration of CT-DNA (4×10 M) were
−
5
−5
incubated with different concentrations of compounds (0, 1×10 M and 2×10 M) at
7 ℃ for 12h in the dark before the measurements. All measurements were performed
3
in Tris-HCl buffer (5 mM Tris/50 mM NaCl, pH 7.2) in the range of 220-320 nm, the
buffer background was subtracted.
2
.4 Cell lines and cell culture conditions
Human breast cancer cell lines (MCF-7, MDA-MB-231) and human gastric
cancer cell lines (MGC-803, SGC-7901) were purchased from the Cell Bank of the
Chinese Academy of Sciences (Shanghai, China). MCF-7 and MDA-MB-231 cells
were grown in Dulbecco’s Modified Eagle medium (DMEM). MGC-803 and
SGC-7901 cells were cultured in RPMI-1640 medium. Both media were
supplemented with 10% fetal bovine serum (Gibco BRL Co. Ltd, USA) and 1%
penicillin/streptomycin. All cell lines were incubated in a 5% CO humidified
2
incubator at 37 ℃.
2
.5 In vitro cytotoxicity
Compounds 1-8 were screened in vitro for anticancer activity against four cancer
cell lines by the MTT-based cytotoxicity assay. Briefly, cells were seeded in 96-well
3
plates at a density of about 5×10 cells per well. After 24 h, the growth medium in
each well was exchanged with 0.1 mL of fresh medium containing graded
concentrations of compounds to be tested, the complete medium without cells was put
in the wells for blank groups and the culture medium containing DMSO was put in
the wells for control groups. After incubated for 48 h, 10 µL MTT (Sigma-Aldrich)
was added to each well, and the cells were incubated for additional 4 h. The
MTT-formazan crystals were dissolved in 100 µL of DMSO, the absorbance of each
well was recorded at 490 nm using an automatic ELISA reader system. The results are
expressed as the percentage of absorbance of treated wells versus that of vehicle
control. IC , the compound concentration giving 50% growth inhibition was
5
0

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calculated via sigmoid curve fitting using a GraphPad Prism 5.0.
2
.6 Clonogenic assay[26]
MCF-7 and MDA-MB-231 cells were treated with compounds 1-3 at a
concentration of 10 μM for 48 h, with DMSO as control. Then the cells digested with
trypsin and counted, cells were diluted and spread on a 8 cm culture dish. After
incubation for around 2 weeks, cells were washed with PBS twice, fixed with
paraformaldehyde for 15 min, then stained with crystal violet for 15 min at room
temperature. The colony was defined to consist of at least 50 cells and counted.
2
.7 Apoptosis induction
To further investigate the antitumor and growth-inhibitory effects, Annexin
V-FITC/PI assay was performed to evaluate the ability of 1-3 to induce apoptosis in
MCF-7 and MDA-MB-231 cells. Cells were seeded in 6-well plates, treated with the
compounds at a concentration of 5 μM and 10 μM, then incubated for 24 h. Cells
treated with DMSO only were used as control. Cells were washed with cold PBS and
resuspended in 100 μL binding buffer and incubated with 5 μL Annexin-FITC and 10
μL PI in the dark for 15 min. Samples were tested using flow cytometry[27] on a
FACS Calibur (BD Biosciences, USA).
2
.8 Morphological studies
The cells were seeded onto cover slides for 12 h and then treated with the vehicle
control or various concentrations of 1-3 (20, and 40 μM) for 24 h. After the cells were
washed with cold PBS, the cells were fixed in 4% paraformaldehyde for 10 min. The
cells
,6-diamidino-2-phenylindole (DAPI) for 30 min in the dark at room temperature.
Cover slips containing the cells were then washed with PBS-TX (10 mL PBS+10 μL
0% Tritonx-100) three times, and images were taken using a confocal fluorescent
microscope.
were
subsequently
washed
with
PBS
and
stained
with
4
1
2
.9 Cell cycle analyses
Effects of compounds on cell cycle and apoptosis induction were analyzed using
PI staining. Cells under experiments were washed twice with PBS. Cells were then
incubated with 100 μL PI (50 μg/mL), 500 μL 0.1% sodium citrate, and 1500 μL 0.1%
Triton X-100. Apoptosis induction percentage and DNA content in cell cycle were
detected by a FACS Calibur (BD Biosciences, USA).
2
.10 Statistical analysis
The data are expressed as mean ± SD statistical comparisons were performed by
one way analysis of variance (ANOVA), followed by Duncan’s multiple range test
DMRT) using SPSS Statistics 17.0. The results were considered statistically
significant values p < 0.05.
(
3
. Results

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3
.1 Synthesis of new compounds
A series of novel 3-nitroacridine derivatives were designed and synthesized, as
summarized in Table 1. In this series, our work focused on modifying the substituted
group on position 9 of 3-nitroacridine. We attempted to replace the hydrogen by a
variety of amines, such as aromatic amine, heterocyclic amine and alicyclic amine to
screen effective antitumor compounds.
The synthesis of compounds 1-8 was shown in Scheme 1. The Ulmann coupling
of 2-chloro-4-nitrobenzonic acid with aniline in DMF produced compound 9, which
was refluxed in POCl to give 10 in a desirable yield (79.1%). The reaction of
3
intermediate 10 and different amines achieved the desired compounds 1-8.
3
.2 New compounds can interact with DNA
The interaction of the compounds 1-8 with CT-DNA was studied by UV-vis
absorption spectra. The absorption spectras of the compounds in the absence and
presence of CT-DNA at a constant compound concentration are given in Figure S1.
The shapes of the compound absorption bands were quite similar. The changes
observed in the compounds in the presence of increasing concentration of CT-DNA
were used to determine the interaction of compunds with double stranded DNA.
Hyperchromicity with no significant shift of the absorption peaks were observed upon
addition of CT-DNA, which suggested that the compounds could interact with DNA.
To determine the binding affinity of compounds 1-8 with DNA quantitatively, the
intrinsic binding constants (K ) were evaluated by the McGhee-Von Hippel (MVH)
b
model[28]. The binding constants of compounds 1-8 bind to CT-DNA ranged from
5
-1
2
.62-6.61×10 M , showed in Table 2. The range of binding constants characteristic
4
6
-1
for intercalators are from10 to10 M [29], and the binding constants for ligand
agents in DNA grooves range from 10 to 10 M [30,31].
5
9
-1
Table 2. UV-vis absorption data of the compounds in the absence and presence of
CT-DNA. K values are mean ± SD from three independent experiments.
b
-
1
Compound
Intrinsic binding constants (K ± SD, M )
b
5
*
1
2
3
4
5
6
7
8
5.968 ± 0.231×10
5
*
6.61 ± 0.153×10
5
5
*
*
5.439 ± 0.197×10
5.243 ± 0.319×10
5
5.014 ± 0.239×10
3.862 ± 0.323×10
5
5*
4.268 ± 0.092×10
5
2.62 ± 0.108×10
*
p < 0.05 compared to control.
In order to confirm the interaction of these new compounds with CT-DNA, the
binding properties were also investigated by CD spectroscopy. CD spectra of DNA
are usually altered due to conformational changes upon binding of ligands such as

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small organic or drug molecules[32,33]. CD spectroscopy is useful to diagnose
changes in DNA morphology during compound-DNA interaction, because the
positive band due to base stacking (273 nm) and the negative band due to
right-handed helicity (246 nm) are quite sensitive to the interaction mode of DNA
with small molecules[34,35]. The changes in CD signals of DNA observed on
interaction with drugs may often be assigned to the corresponding changes in DNA
structure[36]. Thus, simple groove binding and electrostatic interaction of small
molecules show less or no perturbation on the base-stacking and helicity bands, while
intercalation enhances the intensities of both the bands stabilizing the right-handed B
conformation of CT-DNA as observed for the classical intercalator methylene
blue[15]. The CD spectras of CT-DNA in the absence and presence of the compounds
1
-8 are shown in Figure 1. Significant increase in intensity of positive band and
decrease inintensity of negative band with slight red shift were observed for all the
compounds, which indicating that the intercalation is not the only binding mode.
Replacing the hydrogen by nitro at position 3 added distortion to the planer
acridine, and attaching amines at position 9, which help to attain a better fit between
the protein and DNA grooves. The results showed that arcidine moiety can partially
intercalate into DNA and the flexible 9-amines bind with DNA in the mode of groove
binding.
−
5
Figure 1. CD spectra of the calf thymus DNA (4×10 M) in the absence and presence of the
−
5
−5
complexes (1×10 M, 2×10 M) in Tris-HCl buffer (5 mM Tris/50 mM NaCl, pH 7.2). Blue lines
are for DNA free solutions. A-H: Compounds 1-8.
3
.3 The cytotoxic effect of new compounds
To determine the effects of compounds on cell proliferation and gain a primary
structure-activity relationship, the cytotoxicity of compounds 1-8 was investigated
using standard MTT assays with four cancer cells: human breast cancer cells lines
(MCF-7, MDA-MB-231) and human gastric cancer cells lines (MGC-803,
SGC-7901). Most compounds inhibited the viability of the four cancer cells in a
dose-dependent manner with IC values less than 50 µM, as shown in Table 3. Each
5
0
sample was analyzed in triplicate. Among them, compounds 1, 2 and 3 showed
pronounced cytotoxicity, especially in human breast cancer cell lines. Compound 2
showed the best growth inhibitory activity against MCF-7 cells with IC values at
50
6
.79 µM and MDA-MB-231 cells with IC values at 6.46 µM. Compound 1 also
50

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displayed good cytotoxicity against MCF-7 (8.83 µM) and MDA-MB-231 (8.02 µM)
cells. Compound 3 showed similar cytotoxicity with compound 2 against MCF-7
(10.23 µM) and MDA-MB-231 (9.85 µM) cells.
Table 3. Summary of cytotoxicity data for 3-nitroacridine analogues in four cancer cell lines. IC50
values are mean ± SD from at least three independent experiments.
Cytotoxicity in different cell lines (IC ± SD, μM)
5
0
Compound
MCF-7
8.83 ± 0.88
6.79 ± 2.01
MDA-MB-231
SGC7901
MGC803
13.99 ± 1.25
10.94 ± 2.26
*
*
*
*
*
*
1
2
3
4
5
6
7
8
8.02 ± 0.78
14.47 ± 3.04
*
6.46 ± 2.19
17.25 ± 3.84
*
*
*
10.23 ± 1.48
26.45 ± 3.2
9.85 ± 2.12
9.35 ± 3.26
15.04 ± 5.12
39.6 ± 4.65
30.04 ± 5.43
22.58 ± 6.79
60.86 ± 5.32
25.88 ± 4.32
46.33 ± 5.89
20.57 ± 0.95
24.93 ± 2.13
17.162 ± 0.65
28.32 ± 4.64
30.36 ± 4.75
*
*
15.04 ± 1.63
24.25 ± 3.12
18.33 ± 2.43
10.4 ± 1.66
*
18.32 ± 3.54
25.3 ± 4.67
11.9 ± 3.42
35.75 ± 3.72
*
p < 0.05 compared to control.
The results indicated that the substitution of the acridine ring had great effect on
the DNA binding abilities of the compounds, which also affected the antiproliferative
activity. Compounds 1, 2 and 3 with superior cytotoxicity were selected for further
study.
One of the hallmarks of cancer cells is their ability of separately clonal growth.
The anticancer activities of compounds 1-3 were further analyzed by clonogenic assay
with MCF-7 and MDA-MB-231 cells, as shown in Figure 2. Compounds 1-3 induced
significant inhibition of colony formation in MCF-7 and MDA-MB-231 cells
compared with control.
Figure 2. Clonogenic assay on MCF-7 (A) and MDA-MB-231 (B) cells after treatment with
compounds 1-3. The number of colonies counted after treatment for 12 days. Data represent the
*
**
mean value and SD from three independent experiments. p < 0.05, p < 0.01, versus control.
3
.4 The induction of apoptosis and cell cycle arrest by new compounds
The results above indicated that the compounds could interact with DNA, which
may induce apoptosis in cancer cell lines. Then we evaluated the induced apoptosis

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effects of compounds 1, 2 and 3 in MCF-7 and MDA-MB-231 cells using Annexin
V-FITC/PI assay. UL represents the necrotic or damaged dead cells, UR represents
apoptosis cells in later stage, LR represents apoptosis cells in early stage, and LL
represents live cells.
The apoptotic cells were detected using flow cytometry, as shown in Figure 3.
Cells were treated with compounds 1, 2 and 3 at the concentrations of 5 µM and 10
µM. Compounds 1, 2 and 3 induced cell apoptosis in a dose-dependent manner. They
could induce potent cell apoptosis in MCF-7 cells and MDA-MB-231 cells, the
apoptosis level was superior in treated MDA-MB-231 cells as compared to that in
MCF-7 cells. Compound 2 induced the most potent cell apoptosis, with the apoptosis
rate of 62.2% (5 µM) and 99.4% (10 µM) in MDA-MB-231 cells. The apoptosis level
was slight lower in treated MCF-7 cells, with the apoptosis rate of 72.9% (5 µM) and
8
5.1% (10 µM). This suggests that the selected compounds are suitable for the
resistance of human breast cancer cells.

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Figure 3. Apoptosis induced by compounds in MCF-7 and MDA-MB-231 cells. Cells were
treated with 1, 2 and 3 (5 µM and 10 µM) for 24 h. (A) Flow cytometric analysis of

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phosphatidylserine externalization (Annexin-V binding) and cell membrane intergrity (PI staining).
(
B) The percentage of apoptotic cells is calculated as the percentage of apoptotic cells in early and
*
later stage. Data represent the mean value and SD from three independent experiments. p < 0.05,
*
*
p < 0.01, versus control.
Recent studies show that acridine derivatives exert antitumor activities by
interfering with cell cycle[37]. DNA damage induced by anticancer agents may
activate DNA damage check points, which arrest cell cycle progression, trigger DNA
damage repair, or lead to cell death[38].
To further investigate the cell cycle arrest induced by compounds 1, 2 and 3, the
cell cycle distributions of MCF-7 and MDA-MB-231 cells treated with compounds 1,
2
and 3 were detected using flow cytometry under PI staining. As shown in Figure 4,
compounds 1, 2 and 3 interfered with the cell cycle in G0/G1 phage. Compound 1
moderately interfered with the cell cycle in G0/G1 phage, the cell percentages in the
G0/G1 phage increased from 49.0% to 69.1% in MCF-7 cells, and 37.6% to 58.8% in
MDA-MB-231 cells. Compound 2 induced the cell cycle in G0/G1 phage
remarkablely, the cell percentages in the G0/G1 phage increased from 49.0% to 71.7%
in MCF-7 cells, and 37.6% to 64.3% in MDA-MB-231 cells. Compound 3 also
displayed obvious effect on the cell cycle.
Figure 4. Effect of compounds 1, 2 and 3 on cell cycle phage arrest in MCF-7 and MDA-MB-231
cells. Cells were treated with 10 µM of each compound for 24 h, then the cells were fixed and
stained with PI to analyze the DNA content using flow cytometry.
3
.5 Observation of morphological changes
Apoptosis is a functional configuration of cell death characterized by
morphological structure and extensive DNA damage[39]. As shown in Figure 5, a
change in cell morphology and a decrease in the cell number was observed in MCF-7
and MDA-MB-231 cells, which treated with compounds 1, 2 and 3 (20 µM and 40
µM). Figure 5 showed dose-dependent effects of these compounds on inhibition of
human breast cancer cell growth. Using DAPI staining assay, we examined cells for
morphological features of apoptosis, such as cell shrinkage, chromatin condensation

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and apoptotic bodies (Figure 6). The abilities of 1, 2 and 3 to induce apoptosis were
enhanced with the increase of the concentration. Compound 2 showed the most
obvious changes in both cell morphology and cell number. Collectively, these results
suggested that 1, 2 and 3 exhibited potential antitumor activity.
Figure 5. Morphological changes of MCF-7 and MDA-MB-231 cells treated with 1, 2 and 3 (20
µM and 40 µM) for 24 h.

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Figure 6. Representative analysis of changes in nuclear morphology of MCF-7 and
MDA-MB-231 cells treated with 1, 2 and 3 (20 µM and 40 µM). Cells were stained with DAPI
and visualized under a confocal fluorescent microscope.
4
. Discussion
In the field of DNA binding anticancer agents, a great amount of acridine and
anthracycline derivatives become chemotherapeutic drugs on the market[8]. However,
the clinical usage of these compounds has been limited for side effects and multi-drug
resistance. Thus, new compounds capable of DNA binding are in urgent need of
development.
We have synthesised eight 3-nitroacridine derivatives. According to the different
substituents at position 9, the synthesized compounds could be divided into two
classes: compounds with aromatic amino substituents (1, 3, 4, 5, 6, 7 and 8) and
compound with nonaromatic amino substituent (2). We studied the effects of

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variations of the 9-substituents in 3-nitroacridine derivatives on DNA damage and
anticancer activity. The results showed that the compounds can be effective DNA
5
-1
binding agents, with calculated binding constants of K = 2.62-6.61×10 M . The
b
introduction of nitro group affects the planarity of acridine, which alters the binding
ability of acridine. The flexibilities of glycine ethyl ester enhanced the DNA binding
affinity of compound 2. The 9-anilino ring was twisted away from the acridine ring,
which are suitable for the minor groove of the DNA[8]. The binding constants of
compounds 5-8 decreased for the substituent alkyl increased in benzene ring of the
aromatic amines, which suggested that the presence of hydrophobic substituents will
reduce the DNA binding activity of this class of compounds. Chemical modifications
changed the mode of DNA binding of acridine ring, which were intercalation and
groove binding.
These compounds were tested antiproliferative activity on human breast cancer
cell lines (MCF-7, MDA-MB-231) and human gastric cancer cell lines (MGC-803,
SGC-7901) by MTT assay. The nature of the 9-substituents of the 3-nitroacridine
derivatives had an important effect on inhibition activity, which were well correlated
with the DNA binding assay. The compounds were more active against human breast
cancer cell lines. The compounds 1, 2 and 3 with higher DNA binding constants
exhibited good inhibitory effect of tumor cell proliferation. The most active
compounds 1, 2 and 3 were selected for further assayed. Clonogenic assay showed
that compounds 1-3 inhibited the colony formation of MCF-7 and MDA-MB-231
cells, which was in line with MTT studies.
The induction of cancer cell apoptosis is an important goal of anticancer
therapy[40]. To determine the potential action mechanisms, flow cytometric analysis
was used to estimate the percentage of apoptotic cells after treatment with the selected
compounds. Compound 2 exhibited the highest ability to induce apoptosis in MCF-7
cells and MDA-MB-231 cells at the concentration of 10 µM, with the apoptosis rate
of 85.1% and 99.4%, respectively. Induction of cell apoptosis may be mediated by the
cell cycle arrest, thus we evaluated the effect of the selected compounds on cell cycle
progression by flow cytometry analysis. The compounds 1, 2 and 3 had great
influence on the cell cycle. The results demonstrated that the compounds exhibited
anticancer activity via induction of cell cycle arrest at G0/G1 phage and apoptosis in
human breast cancer cells.
Cell morphology changes and decline in the cell number were observed in
MCF-7 and MDA-MB-231 cells using a confocal fluorescent microscope. The
apoptotic features were found in DAPI staining assay. The early apoptotic cells
showed nuclear concentration, dyed darker, or nuclear chromatin crescent form on the
side of the nuclear membrane, and late apoptotic cells showed fragmentation of nuclei
into small round bodies with different sizes, and were surrounded by cell membranes,
namely apoptotic bodies. The effect of compounds on cell growth inhibition and
morphology change was dose-dependent.
5
. Conclusion
The present study suggested that the new compounds may directly interact with

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DNA and interfere with regulatory machanism to initiate DNA damage.
Structure-activity relationship indicated that substituents on position 9 of
3
-nitroacridine derivatives played an important roles in the DNA binding abilities and
antiproliferative activities. Compounds 1, 2 and 3 with super DNA binding affinity
and good inhibitory effect of tumor cell proliferation can be developed as prime
candidates for further chemical optimization. More detailed researches, especially the
action mechanisms, are ongoing.
Conflict of interest
The authors declare that there are no conflicts of interest related to this work.
Acknowledgements
We gratefully acknowledge A Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD).
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