Synthesis and biological evaluation of benzimidazole acridine derivatives as potential DNA-binding and apoptosis-inducing agents

Article abstract of DOI:10.1016/j.bmc.2015.02.036

The discovery of new effective DNA-targeted antitumor agent is needed because of their clinical significance. As acridines can intercalate into DNA and benzimidazoles have the ability to bind in the DNA minor groove, a series of novel benzimidazole acridine derivatives were designed and synthesized to be new DNA-targeted compounds. MTT assay indicated that most of the synthesized compounds displayed good antiproliferative activity, among which compound 8l demonstrated the highest activity against both K562 and HepG-2 cells. Further experiments showed that 8l displayed good DNA-binding capability and inhibited topoisomerase I activity. Moreover, compound 8l could induce apoptosis in K562 cell lines through mitochondrial pathway. These data suggested that compound 8l might be potential as new DNA-binding and apoptosis-inducing antitumor agents.

Full text of DOI:10.1016/j.bmc.2015.02.036

Accepted Manuscript
Synthesis and biological evaluation of benzimidazole acridine derivatives as
potential DNA-binding and apoptosis-inducing agents
Chunmei Gao, Bin Li, Bin Zhang, Qinsheng Sun, Lulu Li, Xi Li, Changjun Chen,
Chunyan Tan, Hongxia Liu, Yuyang Jiang
PII:
S0968-0896(15)00130-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.02.036
BMC 12099
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
27 November 2014
13 February 2015
17 February 2015
Please cite this article as: Gao, C., Li, B., Zhang, B., Sun, Q., Li, L., Li, X., Chen, C., Tan, C., Liu, H., Jiang, Y.,
Synthesis and biological evaluation of benzimidazole acridine derivatives as potential DNA-binding and apoptosis-
inducing agents, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.02.036
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Synthesis and biological evaluation of benzimidazole acridine derivatives as
potential DNA-binding and apoptosis-inducing agents
a,b,c,*
a,b,†
a,b,†
b
b
a,b
Chunmei Gao
, Bin Li , Bin Zhang , Qinsheng Sun , Lulu Li , Xi Li ,
a,b
b,c
b,c
b,c,d,*
Changjun Chen , Chunyan Tan , Hongxia Liu , Yuyang Jiang
a
Tsinghua University, Department of Chemistry, Beijing 100084, PR China
The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key
Laboratory of Chemical Biology, Shenzhen 518055, P. R. China
b
c
National & Local United Engineering Lab for Personalized anti-tumor drugs, the
Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P. R. China
School of Medicine, Tsinghua University, Beijing 100084, PR China
d
*
Corresponding author. Tel.: +86 755 2603 2094; fax: +86 755 2603 2094;
Email address: jiangyy@sz.tsinghua.edu.cn; chunmeigao@sz.tsinghua.edu.cn
Authors with equal contributions.
†
1

Abstract
The discovery of new effective DNA-targeted antitumor agent is needed because of
their clinical significance. As acridines can intercalate into DNA and benzimidazoles
have the ability to bind in the DNA minor groove, a series of novel benzimidazole
acridine derivatives were designed and synthesized to be new DNA-targeted
compounds. MTT assay indicated that most of the synthesized compounds displayed
good antiproliferative activity, among which compound 8l demonstrated the highest
activity against both K562 and HepG-2 cells. Further experiments showed that 8l
displayed good DNA-binding capability and inhibited topoisomerase I activity.
Moreover, compound 8l could induce apoptosis in K562 cell lines through
mitochondrial pathway. These data suggested that compound 8l might be potential as
new DNA-binding and apoptosis-inducing antitumor agents.
Keywords: Acridine, benzimidazole, DNA, topoisomerase I, antitumor
2

1
. Introduction
Cancer, which is mostly caused by a mutation of normal cell, has become the
1
disease mostly urgent to be solved because of its high mortality. Novel effective
anticancer drugs with fewer side effects are urgently needed to be developed. As a large
percentage of chemotherapeutic drugs currently used in cancer therapeutics are
DNA-binding and/or DNA-modifying agents, such as cisplatin, topotecan, adriamycin,
2
et al, DNA has been regarded as one of the most significant targets in cancer
3
treatment. In addition, once drugs bind to DNA, the DNA structure will be changed,
which will influence the activity of DNA-related enzymes, such as DNA
topoisomerases. DNA topoisomerases regulate the winding of DNA and play critical
4
roles in DNA replication, transcription, recombination, et al. As cancer cells express
high level of topoisomerase activity and show remarkable sensitivity to DNA-targeted
drugs, a large number of anticancer drugs targeted DNA and topoisomerases have been
5
designed and synthesized in these years. In spite of the side effects caused by
DNA-targeted compounds and topoisomerase inhibitors, it is still recognized as the
main choice to prolong the patents’ life. Therefore, the search for novel DNA-binding
agents and topoisomerase inhibitors remains a major role in the fight against cancer.
Acrdine is a kind of polycyclic aromatic compound with π-conjugate structure.
Acridine and its derivatives were firstly used as dyes and antibacterial materials.
Nowadays more and more acridine derivatives and their synthetic method have been
developed as chemotherapeutic drugs especially as antitumor agents due to their ability
6
-11
to intercalate into DNA, leading to the inhibition of topoisomerases.
Among the
3

acridine derivatives, the 9-amino acridine scaffold plays an important role and a series
of 9-amino acridine analogues have entered clinical or preclinical phases. Among
them, m-AMSA is one of the most representative acridine drugs in clinical trials and it
1
2
is also the first synthetic DNA-targeted drug in clinical trial. Now many m-AMSA
derivatives have been developed to improve the activity and reduce the side effects,
1
3
such as AHMA and its analogues. In order to further study the structure–activity
relationship of m-AMSA, our group has synthesized a series of 9-anilinoacridines and
9
-benzylaminoacridines, among which most of synthesized acridines can interact with
1
4-16
DNA and display antitumor activity with low micromolar IC values.
5
0
Benzimidazoles are also a class of interesting pharmacophores with various useful
pharmacological properties, such as antiinflammatory, antiviral, antimicrobial and
1
7-21
anticancer activities.
Recent results indicate that the benzimidazole structure can
2
2, 23
bind in the DNA minor groove.
As acridine can intercalate into DNA, we think
that the combination of acridine ring and benzimidazole ring by an appropriate linker
may increase the DNA binding affinity and increase the antitumor activity. In this
work, we designed and synthesized a series of benzimidazole acridine compounds
(8a-8q) as novel DNA binders and topoisomerase inhibitors. The antiproliferative
effect of benzimidazole acridine compounds on K562 and HepG2 cells were evaluated
and the structure-activity relationship (SAR) was discussed. The most active
compound 8l was selected to investigate the DNA binding ability and apoptosis
inducing capability. The results showed that the synthesized benzimidazole acridines
can inhibit topoisomerase I activity and induce apoptosis through mitochondrial
4

pathway.
2
2
. Results and discussion
.1. Chemistry
The synthesis of compounds 8a-8l was shown in Scheme 1(a). The Ullmann
coupling of benzoic acids 1 with the anilines 2a-2l in DMF produced compounds 3a-3l,
which were refluxed in POCl to give 9-chloroacridine derivatives 4a-4l in high yields.
3
The 2-aminomethyl benzimidazole 7 was synthesized from the condensation of
o-phenylenediamine 5 and glycine 6 in concentration hydrochloric acid under reflux
24
conditions. The reaction of 2-aminomethyl benzimidazole 7 and 9-chloroacridine
derivatives 4a-4l in phenol achieved the desired compounds 8a-8l.
As can ben seen in Scheme 1 (b), compounds 8m, 8n, 8p and 8q were obtained by
the reaction of the commercially available materials 9a-9b and 9-chloroacridine
derivatives 4a-4b.
Compound 8o was synthesized by three steps, as shown in Scheme 1 (c). The
cyclization of the compounds 4-nitro-benzaldehyde 9 and o-phenylenediamine 5 gave
2
-(4-nitrophenyl)-benzoimidazole 10, which was then reduced to the amino
compounds 11 by 80% hydrazine hydrate in ethanol. Compound 11 was heated with
9
-chloroacridine to give the desired compound 8o.
.2. In vitro cytotoxicity
2
MTT assay is widely used to determine the antiproliferative activity of drugs,
which was choosed to evaluate the in vitro cytotoxicity of all the benzimidazole
5

acridine derivatives against human leukemia K562 and hepatocellular carcinoma
HepG-2 cells. Colchicin and imatinib were used as the positive controls. The structures
and bioactivity results were shown in Table 1. Most of the synthesized acridine
compounds are more sensitive to K562 cells than HepG-2 cells, which was in
1
4, 25-27
accordance with our previous results.
Therefore, the IC values for K562 cells
50
were selected to study the structure-activity relationships.
Data in Table 1 indicated that both the substitution of the acridine ring and the
linker between benzimidazole ring and acridine ring had great effect on the antitumor
activity. First, the substituent group on C2 position of acridine ring played an
important role on the antiproliferative activity. When R was a butyl group (8l), it
1
displayed the best antitumor activity (IC , 2.68 μM). By changing the butyl group to
5
0
ethyl (8k) or methyl (8c), compounds showed decreasing inhibitory effect. 8k was
about 3-fold less potent than 8l, while 8c was determined to be about 5-fold less potent
than 8l. Compound 8a with no substituents displayed similar activity to 8c with
methyl substituent. The results indicated that the size and length of substituents greatly
influenced the cytotoxicity. In addition, the electron-negativity of the substituents also
significantly affected the antiproliferative activity. Compound 8b with 2-methoxy
substituent showed about 3-fold more potent than 8c with 2-methyl group and 8h with
2
-chloro group. The similar trend can also be seen from 8f and 8g, 8e and 8i.
Moreover, the position of the substituents may change the cytotoxic profile, which can
be seen from the data of compounds 8b, 8e and 8g. In spite of the substitution pattern
of the acridine ring, the linker between the benzimidazole ring and acridine ring is also
6

important in the cytotoxicity, which can be seen from the IC value of compound 8a,
5
0
8
m, 8n and 8o, among which compound 8o with NH-benzene- linker showed best
antiproliferative activity.
As compound 8l showed the best activity against K562 cells (IC = 2.68 μM) and
5
0
HepG-2 cells (IC = 8.11 μM) among the synthesized compounds, it was selected to
5
0
study its antiproliferative activity against other cell lines. Table 2 indicated that
compound 8l exhibited broad-spectrum antitumor activity and it can inhibit the cells
selected with low micromolar IC values, therefore it was selected as a lead compound
5
0
to study DNA binding and apoptosis inducing capability.
2
.3. UV-visible spectral absorbance
UV-visible spectral absorbance has been widely used to study the interaction
between DNA and drugs. To determine the binding interaction between compound 8l
and DNA, the UV-visible absorption spectroscopy was carried out. The results can be
seen in Figure 1a. Compound 8l had strong absorbance peaks at about 415 nm and 440
nm. When DNA was added, the absorption of compound 8l decreased and a slight red
shift was also observed. In addition, with the increase of the concentration of ct DNA,
the intensity of the absorption spectrum of 8l decreased evidently, which suggested that
compound 8l could interact with DNA.
To determine the binding affinity of compound 8l with DNA quantitatively, the
2
5
binding constant was also evaluated (Figure 1b). The equation (1) was used to
calculate the binding constant K . In this equation, [DNA] represents the DNA
b
concentrations in base pair, while ε , ε and ε are the extinction coefficient of the
a
f
b
7

compound absorption band at a given DNA concentration, the complex free in solution
and the complex when fully bound to DNA, respectively. The K is calculated by the
b
5
gradient ratio to the intercept. The binding constant K of compound 8l was 1.88×10
b
-
1
M .
[
DNA] / (ε -ε ) = [DNA] / (ε -ε ) + 1 / K (ε -ε )
(1)
a
f
b
f
b
b
f
2
.4. Fluorescence spectra
To further investigate the binding ability of compound 8l and DNA, the
fluorescence absorption spectroscopy was employed. Compound 8l was dissolved in
Tris-HCl (pH 7.0) buffer solution while the concentration of ct DNA was gradually
increased. The results can be seen in Figure 1c. The fluorescence of compound 8l was
gradually quenched with the concentration of ct DNA increased. The above results
indicated that there existed interaction between 8l and ct DNA.
To confirm the binding affinity of compound 8l with DNA quantitatively, the
2
8
classical Stern-Volmer equation (2) was used to calculate the binding constant
Figure 1d)
F /F = 1 + K [Q]
(
(2)
0
b
In the equation above F is the fluorescence emission intensity in the absence of the
0
quencher, while F is the fluorescence emission intensity in the presence of DNA and [Q]
5
is the concentration of DNA. The quenching constant K of compound 8l was 1.49×10
b
-1
M , which is in accordance with the absorption spectra.
2
.5. DNA topoisomerase I inhibition assay
As most acridine derivatives can interact with DNA and inhibit the activity of
8

topoisomerases, the relative affinity of the benzimidazole acridine compounds on the
relaxation of plasmid pBR322 DNA mediated by topo I was performed. The results
were shown in Figure 2. From the results we can see that most of the compounds
displayed good topo I inhibitory activity at 50 μM. These data suggest that these
compounds may have antiproliferative activity by interacting with DNA and then
inhibiting topo I, being potential leading compounds to be developed into new topo I
inhibitors.
2
.6. Apoptosis induced by compound 8l
The results above showed that compound 8l could interact with DNAand inhibit the
activity of topo I, which may induce apoptosis in cancer cell lines. As we know that
cells die through necrosis and/or apoptosis, in order to distinguish between necrosis
and apoptosis induced by compound 8l, an Annexin-V/PI binding assay using K562
cells was conducted. The results can be seen in Figure 3. The lower right-hand
quadrants displayed the early stage of apoptotic cells. The upper right-hand quadrants
displayed the late stage of apoptotic or necrotic cells. K562 cells were treated with 8l at
the concentrations of 0, 2.5 and 10 μM for 48 h. In Figure 3 we can clearly see that as
the concentration of compound 8l increased, the percentage of early stage apoptotic
cells increased dramatically. At 2.5 μM, 14.9% K562 cells were early apoptotic,
whereas only 1.27% cells were late apoptotic or necrotic. In addition, when the
concentration of 8l was increased to 10 μM, the amount of early apoptotic cells was
up to 57.3%. From the results above we conclude that compound 8l can effectively
induce K562 cells apoptosis.
9

Apoptosis can be triggered by mitochondrial pathway, which results in the
activation of a series of caspases. Caspases, also called crysteine-dependent
aspartate-directed proteases, are a family of cystein proteases which are absolutely
necessary for the process of cell apoptosis. To investigate whether compound 8l
mediated cells through mitochondrial pathway, the activity of cleaved caspase-3, 7
and 9 was tested. As shown in Figure 4, compound 8l at 10 μM for 48 h induced
significant activation of cleaved caspase-3, caspase-7 and caspase-9, suggesting that
mitochondrial pathway was involved in the 8l-induced apoptosis. Since PARP (poly
ADP-ribose polymerase) is the substrate of caspases, we tested the induction of PARP
cleavage by compound 8l. The results in Figure 4 revealed the generation of the
cleaved PARP. In addition Bcl-XL, an important member of Bcl-2 family proteins,
plays an important role in controlling the mitochondrial pathway. As shown in Figure 4,
compound 8l at 10 μM effectively decreased the expression of BcL-XL protein. In
order to verify whether apoptosis is through the mitochondrial pathway the
mitochondrial
membrane
potential
(MMP)
was
analyzed.
5
,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1 ) is a
kind of fluroscent probe that can detect the MMP changes in cells rapidly. JC-1 would
produce red fluorescence when the potential of mitochondrial membrane is high,
while it would produce green fluroscence when the potential of mitochondrial
membrane is low. So we can easily detect the MMP changes by the colour of the
fluorescence. Carbonylcyanide-m-chlorophenylhydrazone (CCCP) was used as the
positive control. As shown in Figure 5, the red fluorescence decreased and the green
10

fluorescen increased with compound 8l added. The results above suggest that
compound 8l induced apoptosis through mitochondrial pathway.
3
. Conclusion
The combination of benzimidazole and acridine resulted in a series of novel potent
DNA-binding anticancer agents. Most compounds showed good cytotoxicity against
K562 and HepG-2 cells. Among all the compounds, compound 8l displayed the highest
cytotoxicity against both K562 and HepG-2 cells. DNA-binding and topo I inhibition
assay demonstrated that 8l interacted with ct DNA and inhibited topoisomerase I
activity, inducing apoptosis in K562 cells. Our studies suggested that compound 8l was
a potent lead compound for further optimization as DNA binding and apoptotic-
inducing agents.
4
4
. Experimental section
.1. General experimental section
NMR spectra were recorded on a Bruker 400 (400 MHz) spectrometer at room
temperature. Chemical shifts are given in ppm (δ) relative to SiMe4 as internal standard.
Coupling constants (J) are in hertz (Hz), and signals are designated as follows: s, singlet;
d, doublet; t, triplet; m, multiplet; br s, broad singlet, etc. The mass spectra were
obtained on a Waters Micromass Q-TOF Premier Mass Spectrometer. Melting points
were determined with a SGW X-4 digital apparatus and are uncorrected. Thin layer
chromatography was carried out using plate silica gel F254.
11

4
.2. Synthesis and characterization
The general preparation of compounds (3a-3l, 4a-4l) can be seen in the supporting
information.
.2.1. General procedure for compounds 8a-8n, 8p-8q
-chloroacridine or its derivatives (1.0 mmol) and phenol (10.0 mmol) were added
4
9
into a 100 ml round-bottom flask and the mixture was stirred for 1 h at 60 ºC under
argon atomsphere. Then benzimidazole derivatives (8a-8n, 8p-8q, 1.2 mmol) were
added. The mixture was stirred under 120 ºC for 2 h. Then the mixture was poured into
a mixture of ethyl acetate (50 mL) and N-methyl morpholine (1 ml) to get the crude
product as yellow precipitation. The crude product was recrystallized from ethyl
acetate.
4
.2.1.1. N-((1H-benzo[d]imidazol-2-yl)methyl)acridin-9-amine (8a). Yield 32%;
1
mp 262-264 ºC; H NMR (400 MHz, d -DMSO) δ 8.66 (m, 2H), 7.98 (m, 4H), 7.54 (m,
6
+
4
3
4
H), 7.19 (m, 2H), 5.54 (s, 2H). HR-MS(ESI): calcd for [M+H] 325.1453; found:
25.1447.
.2.1.2. N-((1H-benzo[d]imidazol-2-yl)methyl)-2-methoxyacridin-9-amine (8b).
1
Yield 25%; mp 265-268 ºC; H NMR (400 MHz, d -DMSO) δ 8.61 (m, 1H), 8.07 (s,
6
1
3
1
4
4
H), 7.93-7.96 (m, 3H), 7.69 (m, 1H), 7.57– 7.49 (m, 3H), 7.20 (m, 2H), 5.54 (s, 2H),
1
3
.88 (s, 3H). C NMR (100 MHz, d -DMSO) δ 156.68, 155.98, 139.71, 135.58,
6
34.72, 127.86, 126.08, 123.84, 122.43, 121.18, 119.40, 114.17, 12.41, 104.16, 56.64,
+
6.42. HR-MS(ESI): calcd for [M+H] 355.1559; found: 355.1551.
.2.1.3. N-((1H-benzo[d]imidazol-2-yl)methyl)-2-methylacridin-9-amine (8c).
12

1
Yield 27%; mp 252-253 ºC; H NMR (d -400 MHz, DMSO) δ 8.65 (m, 1H), 8.51 (s,
6
1
3
4
H), 7.97 – 7.83 (m, 4H), 7.57 (m, 2H), 7.50 (m, 1H), 7.19 (m, 2H), 5.55 (s, 2H), 2.47 (s,
+
H). HR-MS(ESI): calcd for [M+H] 339.1610; found: 339.1611.
.2.1.5. N-((1H-benzo[d]imidazol-2-yl)methyl)-2,3-difluoroacridin-9-amine (8d).
1
Yield 25%; mp 247-249 ºC; H NMR (400 MHz, d -DMSO) δ 8.96 (s, 1H), 8.62 (s, 1H),
6
13
7
.97-7.95 (m, 3H), 7.57 (m, 2H), 7.51 (m, 1H), 7.19-7.21 (m, 2H), 5.55 (s, 2H). C
NMR (100 MHz, d -DMSO) δ 157.47, 153.20, 150.40, 140.51, 138.33, 165.70, 131.23,
6
1
26.24, 124.37, 122.62, 120.96, 119.11, 114.46, 112.69, 106.44, 106.23, 46.42.
+
HR-MS(ESI): calcd for [M+H] 361.1265; found: 361.1267.
4
.2.1.6. N-((1H-benzo[d]imidazol-2-yl)methyl)-3-methoxyacridin-9-amine (8e).
1
Yield 33%; mp 264-268 ºC; H NMR (400 MHz, d -DMSO) δ 8.63 (d, J = 8.7 Hz, 1H),
6
8
7
1
.52 (d, J = 9.5 Hz, 1H), 8.02 – 7.87 (m, 2H), 7.60 (m, 1H), 7.51-7.55 (m, 2H), 7.27 –
1
3
.13 (m, 4H), 5.49 (s, 2H), 3.98 (s, 3H). C NMR (100 MHz, d -DMSO) δ 155.58,
6
53.00, 140.66, 133.82, 132.02, 129.76, 126.78, 123.68, 123.00, 121.69, 120.18,
+
119.58, 117.23, 115.67, 111.80, 104.37, 55.72, 31.11. HR-MS(ESI): calcd for [M+H]
3
55.1559; found: 355.1551.
4
.2.1.7. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-methyl-acridin-9-amine (8f).
1
Yield 32%; mp 252-255 ºC; H NMR (400 MHz, d -DMSO) δ 8.67 (m, 1H), 8.55 (m,
6
1
H), 8.42 (m, 1H), 7.98 (m, 1H), 7.84 (m, 1H), 7.61 – 7.40 (m, 4H), 7.20 (m, 2H), 5.53
1
3
(
s, 2H), 2.76 (s, 3H). C NMR (100 MHz, d -DMSO) δ 157.73, 150.64, 146.70,
6
1
35.24, 133.54, 126.38, 126.33, 126.06, 124.01, 123.88, 123.20, 122.42, 121.21,
+
119.03, 117.81, 117.72, 116.97, 46.42, 21.97. HR-MS(ESI): calcd for [M+H]
13

3
39.1611; found: 339.1610.
4
.2.1.8. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-methoxyacridin-9-amine (8g).
1
Yield 28%; mp 240-241 ºC; H NMR (400 MHz, d -DMSO) δ 8.71 (m, 1H), 8.29 (m,
6
1
3
2
H), 7.95 (m, 1H), 7.62 – 7.41 (m, 5H), 7.19 (m, 2H), 5.53 (s, 2H), 4.14 (s, 3H). C
NMR (100 MHz, d -DMSO) δ 158.2, 150.7, 148.5, 139.6, 135.2, 129.4, 126.1, 124.5,
6
1
4
4
24.1, 123.8, 122.6, 122.4, 121.0, 120.5, 120.4, 119.9, 116.3, 115.7, 113.9, 113.5, 57.2,
+
6.6. HR-MS(ESI): calcd for [M+H] 355.1559; found: 355.1551.
.2.1.9. N-((1H-benzo[d]imidazol-2-yl)methyl)-2-chloroacridin-9-amine (8h).
1
Yield 29%; mp 247-249 ºC; H NMR (400 MHz, d -DMSO) δ 8.89 (m, 1H), 8.64 (m,
6
1
H), 8.02 (m, 4H), 7.57 (m, 2H), 7.50 (m, 1H), 7.21 (m, 2H), 5.56 (s, 2H). HR-MS(ESI):
+
calcd for [M+H] 359.1063; found: 359.1048.
4
.2.1.10. N-((1H-benzo[d]imidazol-2-yl)methyl)-3-methylacridin-9-amine (8i).
1
Yield 28%; mp 232-235 ºC; H NMR (400 MHz, d -DMSO) δ 8.66 (m, 1H), 8.54 (m,
6
1
2
1
4
3
4
2
7
H), 7.98 (m, 2H), 7.72 (m, 1H), 7.55 (m, 3H), 7.37 (m, 1H), 7.20 (dm, 2H), 5.53 (s,
1
3
H), 2.75 (s, 3H). C NMR (100 MHz, d -DMSO) δ 157.71, 150.66, 146.66, 135.23,
6
33.54, 126.38, 126.05, 124.00, 123.87, 123.20, 122.40, 119.03, 117.80, 117.72, 116.97,
+
6.43, 21.98. HR-MS(ESI): calcd for C H ClN O [M+H] 355.1559; found:
2
1
17
2
55.1551.
.2.1.11. N-((1H-indol-2-yl)methyl)-3-fluoroacridin-9-amine (8j). Yield 32%; mp
1
37-240 ºC; H NMR (400 MHz, d -DMSO) δ 8.69-8.79 m, 2H), 7.96-7.99 (m, 2H),
6
1
3
.73-7.75 (m, 1H), 7.57-7.58 (m, 4H), 7.20-7.22 (m, 2H), , 5.58 (s, 2H). C NMR (100
MHz, d -DMSO) δ 166.97, 158.20, 150.32, 135.75, 124.72, 124.27, 122.49, 120.59,
6
14

+
118.93, 113.82, 111.41, 103.58, 103.33, 46.73. HR-MS(ESI): calcd for [M+H]
3
43.1359; found: 343.1367.
4
.2.1.12. N-((1H-benzo[d]imidazol-2-yl)methyl)-2-ethylacridin-9-amine (8k).
1
Yield 45%; mp 252-256 ºC; H NMR (400 MHz, d -DMSO) δ 8.83 (m, 1H), 8.71 (m,
6
1
2
H), 7.97 (m, 2H), 7.74 (m, 1H), 7.58 (m, 2H), 7.53 (m, 1H), 7.44 (m, 1H), 7.21 (m,
H), 5.58 (s, 2H), 2.71(q, J = 7.2 Hz, 2H), 1.21(t, J = 7.2 Hz, 3H). HR-MS(ESI): calcd
+
for [M+H] 353.1753; found: 353.1756.
4
4
7
2
3
4
2
7
.2.1.13. N-((1H-benzo[d]imidazol-2-yl)methyl)-2-butylacridin-9-amine (8l). Yield
1
5%; mp 262-264 ºC; H NMR (400 MHz, d -DMSO) δ 8.63 (m, 1H), 8.38 (s, 1H),
6
.85-7.99 (m, 4H), 7.52-7.60(m, 3H), 7.20 (m, 2H), 5.52 (s, 2H), 2.71(m, 2H), 1.51(m,
+
H), 1.23 (m, 2H), 0.81 (m, 3H). HR-MS(ESI): calcd for [M+H] 381.2079; found:
81.2087.
.2.1.14. N-(1H-benzo[d]imidazol-2-yl)acridin-9-amine (8m). Yield 62%; mp
1
71-274 ºC; H NMR (400 MHz, d -DMSO) δ 7.99-8.05(m, 4H), 7.88-7.92 (m, 2H),
6
+
.38-7.42 (m, 4H), 7.26 – 7.28 (m, 2H). HR-MS(ESI): calcd for [M+H] 311.1297;
found: 311.1307.
4
.2.1.15. 9-(1H-benzo[d]imidazol-2-ylthio)acridine (8n). Yield 72%; mp 285-287 ºC;
1
H NMR (400 MHz, d -DMSO) δ 8.63 (m, 2H), 8.29 (m, 2H), 7.91 – 7.95 (m, 2H),
6
1
3
7
.70-7.74 (m, 2H), 7.30-7.31 (m, 2H), 7.04-7.06 (m, 2H). C NMR (100 MHz,
d6-DMSO) δ149.04, 133.85, 131.06, 130.37, 128.83, 138.13, 126.55, 121.86, 121.41,
+
1
17.76, 114.79, 31.11. HR-MS(ESI): calcd for C21H17ClN2O [M+H] 328.0908;
found: 328.0906.
15

4
2
2
.2.1.16. N-(1H-benzo[d]imidazol-2-yl)-2-methoxyacridin-9-amine (8p). Yield
1
2%; mp 234-236 ºC; H NMR (400 MHz, d -DMSO) δ 8.00 (m, 2H), 7.97 – 7.88 (m,
6
H), 7.69 (m, 1H), 7.52 (m, 1H), 7.41 (m, 1H), 7.37 (m, 2H), 7.26 (m, 2H), 3.74 (s, 3H).
1
3
C NMR (100 MHz, d -DMSO) δ 158.1, 152.2, 148.6, 139.6, 135.6, 132.0, 131.4,
6
1
29.4, 125.3, 124.4, 124.0, 122.4, 120.4, 120.3, 120.0, 116.3, 113.8, 112.7, 112.3,
+
1
11.7, 57.1. HR-MS(ESI): calcd for [M+H] 341.1402; found: 341.1400.
4
.2.1.17. N-(1H-benzo[d]imidazol-4-yl)-4-methoxyacridin-9-amine (8q). Yield
1
2
1%; mp 233-237 ºC; H NMR (400 MHz, d -DMSO) δ 8.83 (d, J = 8.5 Hz, 1H), 8.35
6
(d, J = 8.3 Hz, 1H), 8.29 (d, J = 8.6 Hz, 1H), 8.01 – 7.96 (m, 1H), 7.48-7.59 (m, 3H),
1
3
7
1
1
.31-7.32 (m, 2H), 7.10-7.13 (m, 2H), 4.13 (s, 3H). C NMR (100 MHz, d -DMSO) δ
6
55.7, 152.5, 140.2, 136.2, 134.3, 131.1, 129.8, 127.3, 126.2, 123.9, 123.4, 123.3,
21.3, 119.8, 119.5, 119.2, 116.9, 115.7, 111.8, 111.7, 55.8. HR-MS(ESI): calcd for
+
[
M+H] 341.1402; found: 341.1400.
4
.2.2. Preparation of compounds 8o.
A mixture of o-phenylenediamine (1.0 mmol) and p-nitrobenzaldehyde (1.1 mmol)
was heated in DMF (50 ml) under 70 ºC overnight using ammonium acetate (3 mmol)
as auxiliary. Then the mixture was poured into water after cooling. The red
precipitation was collected and dissolved in ethanol (50 ml) and 80% hydrazine hydrate
(4 ml) was added dropwise. Then the mixture was refluxed under 80 ºC overnight using
Palladium Carbon as catalysts. The catalysts were separated by filtration and the
ethanol was removed by vacuum evaporation. The crude product was purified by
column chromatography using petroleum ether and ethyl acetate (1:2 v/v). Yield 22%;
16

1
mp >300 ºC; H NMR (400 MHz, d -DMSO) δ 8.15-8.17 (m, 2H), 7.98 – 8.00 (m, 2H),
6
1
3
7
.46 (m, 4H), 7.37 (m, 2H), 6.90-6.92 (m, 2H), 6.74-6.80 (m, 4H). C NMR (100 MHz,
d6-DMSO) δ 159.6, 154.8, 152.3, 150.4, 131.8, 131.3, 130.7, 129.9, 129.8, 128.5,
1
27.3, 126.9, 126.4, 123.3, 122.6, 122.1, 121.4, 121.0, 120.1, 119.2, 118.5, 117.9,
+
1
15.8, 115.7. HR-MS(ESI): calcd for [M+H] 387.1610; found: 387.1606.
4
4
.3. Bioassay
.3.1 Cell culture
K562 was cultured in IMDM; HepG-2, A-375, U-118 MG, and A172 were cultured
in DMEM; CNE-2 and Hela were cultured in RPMI 1640; U251 was cultured in
MEM-EBSS. All cells were cultured with 10% fetal bovine serum (FBS), 100 μg/mL
penicillin, and 100 μg/mL streptomycin in humidified air at 37 ºC with 5% CO2.
4
.3.2 Cell growth inhibition assay
The cells were seeded into 96-well plates at 8.0×10 -1.5×10 cells/well, treated
with the synthesized compounds with the final concentrations at 50, 25, 10, 1.0 and
3
4
0
5
.1 μM. After 48 h treatment, the cells were incubated with 10-15 μL MTT (3-(4,
-dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide from Sigma) solution (5
mg/mL) for 4 h at 37 ºC. The formazan precipitates were dissolved in 100 μL DMSO
and the absorbance at 490 nm was measured by a Benchmark microplate reader
(
Molecular Devices Corporation). IC values are the concentration at which cell
50
growth was inhibited by 50%.
4
4
.4. Biophysical evaluation
.4.1. Materials
17

The buffer used in the measurements of the UV-visible Absorption spectra and the
Fluorescence emission spectra is composed of 10 mM Tris and 10 mM NaCl dissolved
in Ultra-pure water(1L) , and a few drop of hydrochloric acid was added to adjust the
pH to 7.0. The purity and concentration of the ct DNA used below were determined by
UV–vis spectrometer.
4
.4.2. UV-visible Absorption spectra
All the emission spectra were measured on a computer-controlled Beckman Coulter
DU 800 spectrophotometer. The pathway of the cell is 1.0 cm. The solution of tested
compound 8l (5 mM in DMSO, 16 μL) and the buffer above were transferred to the
quartz, then a range of ct DNA solution (2.5 mM in Tris-HCl) (4, 8, 16, 24, 32 μL) was
mixed with the solution in the cell. The incubating time before tesing was 3 min.
4
.4.3. Fluorescence emission spectra
The emission spectra were carried out on Fluorolog spectrometer. The solution of
tested compound 8l (5 mM in DMSO, 1.5 μL) and the buffer above (2 mL) were
transferred to the quartz with the final concentration at 3.75μM, then a range of ct DNA
solution (2 mM in Tris-HCl) (0, 1, 2, 3, 4, 5 μL) was mixed with the solution in the cell
with the final concentration at 0, 1, 2, 3, 4, 5 μM. The incubating time before testing
was 3 min.
4
.5. DNA topoisomerase I inhibition assay
The solutions of a mixture of 100 ng of plasmid DNA pBR322 (commercial
available from Takara), 1.0 units of recombinant human DNA topoisomerase I (from
Takara) and with compounds of different amount were incubated at 37 ºC for 30 min in
18

the relaxation buffer (35 mM Tris–HCl (pH 8.0), 5 mM MgCl , 72 mM KCl, 0.01%
2
bovine serum albumin, 2 mM spermidine, 5 mM dithiothreitol). DNA samples were
then electrophoresed on a 1% agarose gel at 100 V for 25 min with a running
buffer( Tris-Acetic acid- EDTA). EB (ethidium bromide) was used as the staining agent
to make the Gels visible ultraviolet light.
4
.6. Western blot analysis
K562 cells were cultured in 6 cm dishes, and then compound 8l was added with the
final concentration at 1 μM, 5 μM and 10 μM independantly, and the resulting
solutions were incubated for 48 h. Then the cells were centrifuged and was dialysised
with lysis buffer at 0 ºC for 0.5 h, followed by centrifugation at 20,000 g for 10 min.
Protein concentrations in the supernatant were determined using bicinchonininc acid
(BCA). Lysate proteins were subjected to 12% sodium dodecylsulfate
(SDS)–polyacrylamide gel electrophoresis (PAGE), and electrophoretically transferred
to PVDF membrane (amc Biobind NT-200). After blotting, the membrane was blocked
in 5% milk for 1 h, and incubated with the specific primary antibody for overnight at 4
ºC. Protein bands were detected using the BIO-RAD GelDoc XR after hybridization
with the antibody.
4
.7. Mitochondrial membrane potential detection
The mitochondrial membrane potential detection is tested using the Mitochondrial
membrane potential detection kit (JC-1).
4
.8. Flow cytometry assay
The Phosphatidylserine externalization was determined with the scheme which is
19

designed as the instructions of the manufacturer by AnnexinV-FITC/PI apoptosis
detection kit.
Acknowledgments
The authors would like to thank the financial supports from the the Ministry of
Science and Technology of China (2012AA020305, 2013AA092902), the Chinese
National Natural Science Foundation (21272134 and 21372141), and Shenzhen Sci &
Tech Bureau (JCYJ20130402164027386 and JCYJ20120831165730905).
Supplementary data
1
Supplementary data (the preparation of compounds 3, 4, H NMR spectrum, High
resolution mass spectrometry) associated with this article can be found, in the online
version.
References and notes
1
. Barot, K. P.; Nikolova, S.; Ivanov, I.; Ghate, M. D. Mini-Rev. Med. Chem. 2013,
3, 1239.
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. Gurova, K. Future Oncol. 2009, 5, 1685.
. Palchaudhuri, R.; Hergenrother, P. J. Curr. Opin. Biotech. 2007, 18, 497.
. Pommier, Y. Chem. Rev. 2009, 109, 2894.
. Kathiravan, M. K.; Khilare, M. M.; Nikoomanesh, K.; Chothe, A. S.; Jain, K. S. J.
Enzym. Inhib. Med. Chem. 2013, 28, 419.
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7
. Belmont, P.; Dorange, I. Expert Opin. Ther. Pat. 2008, 18, 1211.
. Galdino-Pitta, M. R.; Pitta, M. G. R.; Lima, M. C. A.; Galdino, S. L.; Pitta, I. R.
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Mini-Rev. Med. Chem. 2013, 13, 1256.
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9
. Zhang, B.; Li, X.; Li, B.; Gao, C.; Jiang, Y. Expert Opin. Ther. Pat. 2014, 24, 647.
. Cholewinski, G.; Dzierzbicka, K.; Kolodziejczyk, A. M. Pharmacol. Rep. 2011, 63,
3
05.
0. Belmont, P.; Bosson, J.; Godet, T.; Tiano, M. Anti-Cancer Agent. Med. Chem.
007, 7, 139.
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1. Jiang, B.; Wang, X.; Li, M. Y.; Wu, Q.; Ye, Q.; Xu, H. W.; Tu, S. J. Org. Biomol.
Chem. 2012, 10, 8533.
2. Ketron, A. C.; Denny, W. A.; Graves, D. E.; Osheroff, N. Biochem. 2012, 51,
1
730.
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1
3. Redko, B.; Albeck, A.; Gellerman, G. New J. Chem. 2012, 36, 2188.
4. Lang, X.; Li, L.; Chen, Y.; Sun, Q.; Wu, Q.; Liu, F.; Tan, C.; Liu, H.; Gao, C.;
Jiang, Y. Bioorg. Med. Chem. 2013, 21, 4170.
1
1
5. Lang, X. L.; Luan, X. D.; Gao, C. M.; Jiang, Y. Y. Prog. Chem. 2012, 24, 1497.
6. Gao, C. M.; Li, S. F.; Lang, X. L.; Liu, H. X.; Liu, F.; Tan, C. Y.; Jiang, Y. Y.
Tetrahedron 2012, 68, 7920.
1
1
1
7. Narasimhan, B.; Sharma, D.; Kumar, P. Med. Chem. Res. 2012, 21, 269.
8. Gaba, M.; Singh, S.; Mohan, C. Eur. J. Med. Chem. 2014, 76, 494.
9. Shah, K.; Chhabra, S.; Shrivastava, S. K.; Mishra, P. Med. Chem. Res. 2013, 22,
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0. Barot, K. P.; Nikolova, S.; Ivanov, I.; Ghate, M. D. Mini-Rev. Med. Chem. 2013,
3, 1421.
2
1
21

2
2
1. Fei, F.; Zhou, Z. M. Expert Opin. Ther. Pat. 2013, 23, 1157.
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Biomol. Struct. Dyn. 2013, 31, 52.
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3. Miao, Y.; Lee, M. P. H.; Parkinson, G. N.; Batista-Parra, A.; Ismail, M. A.;
Neidle, S.; Boykin, D. W.; Wilson, W. D. Abstr. Pap. Am. Chem. Soc. 2006, 231.
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1
22

Legends
Scheme 1(a). Reagents and conditions: (i) K CO , Cu, DMF, 130 ºC, 4-8 h ; (ii) POCl ,
2
3
3
1
05 ºC, reflux, 2 h ; (iii) 6 M HCl , 100 ºC , reflux , 72 h ; (iv) phenol, 120 ºC, 2 h.
Scheme 1(b). Reagents and conditions: (iv) phenol, 120 ºC, 2 h.
Scheme 1(c). Reagents and conditions: (v) NH Ac, DMF, 70 ºC, 8 h; (vi) N H ·H O,
4
2
4
2
ethanol, 80 ºC, reflux, 8 h; (iv) phenol, 120 ºC, 2 h.
Figure 1. (a) UV-vis absorption spectra of compound 8l (40 μM) in the presence of
increasing amounts of ct DNA; [DNA]/[8l] = 0, 0.125, 0.25, 0.5, 0.75, 1.0. DNA
titration of the compound was performed in 10 mM Tris-HCl buffer containing 10 mM
NaCl at pH 7.0. The arrow indicates the absorbance changes upon increasing DNA
concentrations. (b) The plot of [DNA]/( ε -ε ) as a function of DNA concentrations as
a
f
determined from the absorption data. (c) Spectrofluorimetric titration of compound 8l
3.75 μM) in 10 mM Tris-HCl buffer containing 10 mM NaCl (pH 7.0) by increasing
(
the concentrations of ct DNA ([DNA]=0, 1, 2, 3, 4, 5 μM). The arrow indicates the
fluorescence emission changes upon increasing DNA concentrations. (d) Stern-Volmer
quenching plots of the fluorescence titration.
Figure 2. Effect of the compounds on the relaxation of plasmid DNA by human
23

topoisomerase I. (a) Lane 1, DNA pBR322; Lane 2, topo 1 + DNA pBR322 + DMSO;
Lanes 317, DNA pBR322 relaxation by topo 1 and 8a-8i, 8k-8o at concentrations of 50
μM, respectively. (b) Lane 1, DNA pBR322; Lane 2, topo 1 + DNA pBR322 + DMSO;
Lanes 35, DNApBR322 relaxation by topo 1 and 8j, 8p, 8q at concentrations of 50 μM,
respectively.
Figure 3. Flow cytometric analysis of phosphoatidylserine externalization (Annexin-V
binding) and cell membrane integrity (PI staining). K562 cells were treated with
compound 8l at A)0, B)2.5, C)10 μM, respectively.
Figure 4. K562 cells were treated with different concentrations of compound 8l, and
Western blot analysis was used to evaluate the levels of Cleaved-Caspase-3, Cleaved
Caspase-7, Cleaved Caspase-9, C-PARP, and BCL-XL.
Figure 5. Detection of the mitochondrial membrane potential using JC-1. K562 cells
were treated with compound 8l at 0 μM (A), 2.5 μM (C), 10 μM (D), CCCP at 10 μM
(B) respectively.
24

0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
5
4
3
2
1
a
5
-1
K = 1.88 ×10 M
b
b
0
.5
1.0
1.5
2.0
2.5
3.0
3
50
400
450
500
5
Wavelength/nm
[DNA] ×10 M
7
1
.8
1
8
6
4
2
.0x10
.0x10
.0x10
.0x10
.0x10
5
K = 1.49 × 10 M
-1
d
q
6
6
6
6
1.6
1
1
1
.4
.2
.0
0
.0
0
1
2
Q] × 10
36
4
5
450
500
550
600
[
Figure 1

Figure 2

Figure 3

Figure 4

Figure 5