Synthesis and biological research of novel azaacridine derivatives as potent DNA-binding ligands and topoisomerase II inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.04.030

DNA and DNA-related enzymes are one of the most effective and common used intracellular anticancer targets in clinic and laboratory studies, however, most of DNA-targeting drugs suffered from toxic side effects. Development of new molecules with good antitumor activity and low side effects is important. Based on computer aided design and our previous studies, a series of novel azaacridine derivatives were synthesized as DNA and topoisomerases binding agents, among which compound 9 displayed the best antiproliferative activity with an IC₅₀ value of 0.57?μM against U937 cells, which was slightly better than m-AMSA. In addition, compound 9 displayed low cytotoxicity against human normal liver cells (QSG-7701), the IC₅₀ of which was more than 3 times lower than m-AMSA. Later study indicated that all the compounds displayed topoisomerases II inhibition activity at 50?μM. The representative compound 9 could bind with DNA and induce U937 apoptosis through the exogenous pathway.

Full text of DOI:10.1016/j.bmc.2017.04.030

Accepted Manuscript
Synthesis and biological research of novel azaacridine derivatives as potent
DNA-binding ligands and topoisomerase II inhibitors
Dan Li, Zigao Yuan, Shaopeng Chen, Cunlong Zhang, Lu Song, Chunmei Gao,
Yuzong Chen, Chunyan Tan, Yuyang Jiang
PII:
S0968-0896(17)30053-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.030
BMC 13700
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
11 January 2017
8 March 2017
25 April 2017
Please cite this article as: Li, D., Yuan, Z., Chen, S., Zhang, C., Song, L., Gao, C., Chen, Y., Tan, C., Jiang, Y.,
Synthesis and biological research of novel azaacridine derivatives as potent DNA-binding ligands and topoisomerase
II inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.04.030
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Synthesis and biological research of novel azaacridine derivatives as
potent DNA-binding ligands and topoisomerase II inhibitors
a, b, c
a, b, c
b, c
, Shaopeng Chen , Cunlong Zhang , Lu Song
c
a, b, c
Dan Li
, Zigao Yuan
,
b, c*
b, c
, Yuzong Chen , Chunyan Tan , Yuyang Jiang
b, c
b, c, d*
Chunmei Gao
a
Department of Chemistry, Tsinghua University, Beijing 100084, PR China
b
The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key
Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University,
Shenzhen 518055, PR China
c
National & Local United Engineering Lab for Personalized anti-tumor drugs,
Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China
d
Department of Pharmacology and Pharmaceutical Sciences, School of Medicine,
Tsinghua University, Beijing, 100084, PR China
*
Corresponding authors. Tel./Fax: +86 (755) 26032094
e-mail:chunmeigao@sz.tsinghua.edu.cn, jiangyy@sz.tsinghua.edu.cn

Abstract
DNA and DNA-related enzymes are one of the most effective and common used
intracellular anticancer targets in clinic and laboratory studies, however, most of
DNA-targeting drugs suffered from toxic side effects. Development of new molecules
with good antitumor activity and low side effects is important. Based on computer
aided design and our previous studies, a series of novel azaacridine derivatives were
synthesized as DNA and topoisomerases binding agents, among which compound 9
displayed the best antiproliferative activity with an IC50 value of 0.57 µM against
U937 cells, which was slightly better than m-AMSA. In addition, compound 9
displayed low cytotoxicity against human normal liver cells (QSG-7701), the IC50 of
which was more than 3 times lower than m-AMSA. Later study indicated that all the
compounds displayed topoisomerases II inhibition activity at 50 µM. The
representative compound 9 could bind with DNA and induce U937 apoptosis through
the exogenous pathway.
Keywords ： Azaacridine derivatives, Molecular docking, DNA binding,
Topoisomerase II inhibitor, Antiproliferative activity

1.
Introduction
Cancer has been reported as a leading cause of death in many countries
1
especially in less developed countries. Chemotherapy, radiotherapy, and surgery are
the three traditional methods for cancer, among which chemotherapy maintains the
primary choice in cancer treatment. Many targets have been discovered for cancer
therapy. DNA and its related enzymes (including topoisomerases, tubulin, telomerase,
etc.) are one of the most effective and successful families of drug targets in clinic and
2
laboratory studies. Topoisomerases are universal and necessary enzymes that alter
-5
DNA topology during DNA replication, transcription, chromatin assembly by forming
6
-9
a reversible covalent Topoisomerases-DNA (Top-DNA) complex.
One of the
mechanism of topoisomerases targeted drugs is that molecules can bind with DNA,
and then inhibit the relaxation process catalyzed by DNA topoisomerases. Most of
them have polycyclic conjugated structures with the ability to intercalate into
9
-11
DNA.
With a conjugated planar structure, acridine and acridone derivatives have been
discovered as DNA targeted drugs since World War I. Many acridine analogues have
been developed to target not only DNA but also its correlated enzymes. Now some
acridine compounds, such as m-AMSA, DACA, PZA, BRACO-19 and AS1410, have
11-16
entered the clinical or preclinical stages (Figure 1).
Our research group has also
1
7-22
developed series of acridine agents with good antitumor activity.
Compared with
the huge amount of acridines, little attention has been paid on azaacridines, although
limited azaacridines have been studied in pharmaceutical field such as antibacterial,
11, 23
antimalarial, antiparasitic, as well as antitumor agents.
Like acridines, benzimidazoles were also discovered to have the ability to
interact with some key parts in biological systems such as DNA, motor domain of
spindle kinesin, poly (ADP ribose) polymerase-1 (PARP-1), and microtubules, some
2
4-26
of which have been developed as potent antitumor agents.
Our group had also put
efforts in synthesizing several series of benzimidazoles with good antitumor activity,
and found that the combination of acridine ring and benzimidazole ring by an NHCH
2
2
1, 27-28
linker could increase the DNA binding affinity.
We think that combining

2
azaacridine and benzimidazole ring by the NHCH linker might offer a better
therapeutic effect, because the introduction of another nitrogen in the acridine ring
might increase the interaction between compounds and the targets. Therefore, the
molecular docking scores (Table 1S) were preliminary investigated and illustrated that
the introduction of benzimidazole group to the 9-position of azaacridine led to better
DNA-Top binding ability than the corresponding 9-benzimidazole acridine. Therefore,
as a part of our ongoing work in developing antitumor agents, this paper designed and
synthesized two 9-benzimidazole azaacridines, as well as several 9-substituted phenyl
azaacridines to generate compounds with better antitumor activity. The
antiproliferative activity, the DNA binding ability, inhibition of topoisomerase
activity and induction of apoptosis were also evaluated.
Figure 1. The typical structures of acridine analogues in clinical or preclinical stages
2.
Results and discussion
2.1.
Chemistry
2
9
The synthetic routes of target compounds 5a-5g were carried out in Scheme 1a.
Anthranilic acid 3 was synthesized by using the general procedure of Ullmann
reaction with Cu as a catalyst. Then 3 reacted with POCl at 120 °C to provide the
-chloroacridine derivative 4 in 2 hours. In the presence of phenol, compound 4
3
9
reacted with aniline derivatives to give the corresponding 5a-5g.
As described in Scheme 1(b), 2-aminomethyl benzimidazole 8 was obtained by

commercially available o-phenylenediamine 6 reacting with glycine 7 in
concentration hydrochloric acid under reflux conditions. Compound 8 reacted with
9
-chloroacridine derivatives 4 in phenol giving compound 9 and 12. As shown in
Scheme 1(c), compound 11 was produced by a nucleophilic substitution reaction
30
between compound 9 and 2-dimethylaminoethyl chloride.
COOH
X
OCH3
COOH
Cl
(
i)
X
(
a)
Cl
NH
OCH3
H N
Cl
2
1
2
3
R
Cl
N
NH
(
ii)
X
OCH3
(iii)
X
OCH3
Cl
Cl
N
4
5a-5g
X=N
H
N
NH2
NH2
(
iv)
(
b)
H N
COOH
2
NH2
N
7
8
6
Cl
N
HN
HN
N
X
OCH3
X=N
X=CH 12
9
Cl
(
v)
X
OCH3
Cl
N
N
HN
HN
N
N
N
(
vi)
N
(
c)
Cl
HN
10
N
OCH3
N
OCH3
Cl
N
Cl
N
9
11
Scheme 1. Synthetic routes of the azaacridine derivatives. Reagents and conditions: (a)
i) K CO , Cu, DMF, 130 °C, overnight; (ii) POCl , 120 °C, 2 h; (iii) CHCl , ethanol,
HCl, rt, overnight. (b) (iv) 6 M HCl, 100 °C, reflux, 72 h; (v) phenol, 120 °C, 2 h. (c)
vi) KOH, K CO , acetone, reflux, overnight
(
2
3
3
3
(
2
3

2.2.
Antiproliferative activity in cellular assays
MTT assay, a widely used method to detect the in vitro cytotoxicity of drugs, was
selected to evaluate the antiproliferative activity of the compounds in hand against
human leukaemia K562, U937 and hepatocellular carcinoma HepG2 cells with
m-AMSA, CPT served as the positive drugs. As the result showed in Table 1, all the
compounds displayed moderate antiproliferative activity against K562 cells, among
which compound 9 with benzimidazole and azaacridine scaffold displayed the best
activity. The results confirmed that the introduction of benzimidazole could improve
cell cytotoxicity. After introducing the dimethylaminoethyl group to the
benzimidazole group, the cytotoxicity was slightly decreased. However, the water
solubility was greatly increased. When R was the phenyl group, the substituent group
in the phenyl ring affected the cytotoxicity greatly.
Since in medicinal chemistry study, fluorinated compounds showed remarkable
record, compounds 5a-5c were firstly synthesized to evaluate their antiproliferative
activity. The result demonstrated the ortho-position was more preferable than meta- or
1
7
para-position that was in accordance with our previous results. Compound 5d with a
methoxyl group in para-position and 5f with two methyl groups in meta- and
para-positions displayed similar IC50 values. Unexpectedly, chlorinated compound 5b
presented the best antitumor activity among 5a-5g with an IC50 value of 6.10 µM.
In addition, compound 5g with a pyridine group was also synthesized, which
displayed similar antiproliferative activity comparing with compounds 5b-5d. To
evaluate whether the azaacridine had the influence on the cell toxicity, compound 12
with acridine was synthesized. As shown in Table 1, 12 displayed the lowest
antiproliferative activity, which indicated that azaacridine contributed much to the
bioactivity.
Among all the synthesized compounds, only compounds 9 and 11 displayed good
antiproliferative activity against K562, HepG2 and U937. Especially, 9 and 11
displayed nM IC50 values against U937, which were better or compatible to m-AMSA.
Therefore, they were further tested against A549, Hela and QSG-7701 (Table 2). The
results showed that they were more sensitive to U937. Importantly, they displayed low

31
activity against human normal liver cells (QSG-7701), which indicated that they had
the potential to be developed as antitumor agents.
Table 1. Results of the IC50 values of compounds 5a-5g, 9, 11, 12 against K562,
HepG2 and U937
a
IC50(µM)
Compound
R
K562
HepG2
U937
5
5
a
10.92 ± 0.45
>20
>20
b
15.51 ± 0.34
>20
>20
5
c
16.53 ± 1.34
13.82 ± 0.52
>20
>20
>20
>20
5
d
5
e
6.10 ± 1.22
>20
>20
>20
>20
5
f
14.01 ± 0.23
5
g
13.66 ± 1.02
6.06 ± 0.89
>20
>20
9
6.62 ± 1.89
0.57 ± 0.01
1
1
7.59 ± 0.67
>20
>20
>20
0.76 ± 0.13
ND
1
2
m-AMSA
0.88 ± 0.33
5.21 ± 0.74
0.61 ± 0.01

CPT
ND
4.92 ± 1.18
ND
ND : not detect
a
Each data represents mean ± S.D. from three different experiments performed in
triplicate.
Table 2. Antiproliferative activities of compounds 9, 11 against A549, Hela and
QSG-7701 cell lines
a
IC50 (µM)
Compound
A549
4.80 ± 0.18
13.12 ± 2.24
ND
Hela
QSG-7701
29.36 ± 3.34
21.23 ± 0.47
6.43 ± 0.05
< 1.0
9
12.62 ± 1.39
14.84 ± 1.60
19.19 ± 1.66
3.80 ± 0.84
11
m-AMSA
CPT
< 1.0
ND : not detect
a
Each data represents mean ± S.D. from three different experiments performed in
triplicate.
2.3.
Molecular modeling
To confirm if the nitrogen in the azaacridine ring had an effect on the binding
mode, the comparison between compounds 9 and 12 was taken based on the results of
molecular docking. SYBYL-X 1.3 protocol was used to study the interaction of
compounds 9 and 12 with Top-DNA complex. As seen in Figure 2A and 2B, both
compounds 9 and 12 have one hydrogen bonding interaction with Arg364 which is a
3
2-33
key residue of Top I-DNA complex.
Molecular docking results (Figure 1S)
indicated that the geometry configuration of compound 9 is more similar to the typical
Top I drug CPT than that of 12. Therefore, compound 9 might display stronger Top
I-DNA binding ability than compound 12.
The docking study of compound 9 and 12 with Top II-DNA complex was shown
in Figure 2C and 2D. Three hydrogen bonds were formed between compound 9 and
Top II-DNA complex, however, only one hydrogen bond was found between
compound 9 and Top II-DNA complex. The result indicated that the azaacridine may

contribute much to the binding ability, which was in accordance with the data in Table
S.
1
Figure 2. Molecular docking of compounds 9 and 12 with Top-DNA complex. (A)
Compound 9 with Top I-DNA complex. (B) Compound 12 with Top I-DNA complex.
The DNA phosphate backbone and bases are shown in purple. (C) Compound 9 with
Top II-DNA complex. (D) Compound 12 with Top II-DNA complex. The DNA
phosphate backbone and bases are shown in green.
2.4.
DNA binding analysis
As compound 9 showed the best antiproliferative activity against several cancer
cell lines, as well as low cytotoxicity against normal cells, it is necessary to explore
whether the interaction with DNA contributed to the cell activity as our previous
1
7, 19, 21
reported acridine derivatives.
UV-visible spectral absorbance is a kind of widely and convenient measure to
detect the interaction between drugs and DNA. Figure 3A displayed compound 9 had

the maximal peak at around 410 nm while DNA did not absorb light in this region.
With the concentration of DNA increased, a significant decline in the absorption
spectrum was observed. Beside, a slight bathochromic shift was shown, which
29
indicated that an interaction between compound 9 and DNA occurred.
In order to give a quantitative binding affinity of compound 9 with DNA, the E
1) was used to calculate the binding constant K where [DNA] stands for the DNA
q
(
b
concentration, meanwhile ε
in solution, and ε is the extinction coefficient of the complex when fully bound to
DNA respectively. The K
a
f
, ε represent the extinction coefficient of the complex free
b
b
of compound 9 showed in Figure 3B indicated it had a
better DNA binding capability.
DNA] / (ε -ε ) = [DNA] / (ε
To confirm the binding properties of compound 9, further study of fluorescence
[
a
f
b
-ε
f
) + 1 / K
b
(ε
b f
-ε
)
(1)
emission spectra was employed. In Figure 3C compound 9 exhibited an emission peak
around 450 nm with an excitation wavelength at 300 nm. Along with the
concentration of DNA increased, the fluorescence of 9 was gradually quenched.
Formula (2) was a classical way to calculate the quenching constant K. The
5
quenching constant K of compound 9 was 2.34×10 M which was in accordance
-1
with the absorption spectra.
F
0
/ F = 1 + K
q
[Q]
(2)

Figure 3. (A) UV-visible absorption spectra of 9 (40 µM) in the presence of
increasing amounts of ct DNA ([DNA] = 0, 0.75, 2.25, 3.75, 6.00, 9.75 µM) in
Tris-HCl buffer (pH 7.4). The arrow indicates the absorbance changes upon
increasing DNA concentrations. (B) The plot of absorption data, [DNA], DNA
concentration. (C) Spectrofluorimetric titration of 9 (40 µM) in Tris–HCl buffer (pH
7.4) by increasing the concentrations of ct DNA ([DNA] = 0, 0.2, 0.7, 1, 1.5, 2.5 µM).
The arrow indicates the fluorescence emission changes upon increasing DNA
concentrations. (D) The plots of the fluorescence titration, [Q], DNA concentration.
2.5.
DNA Top I and Top II inhibition assay
In previous study most acridine derivatives could interact with DNA and inhibit
3
4-35
the activity of topoisomerase.
Inhibitory activities were firstly evaluated at 100
µM and all our newly synthesized compounds exhibited apparent Top II inhibitory
activity while only compound 11 showed Top I inhibitory at 100 µM (Figure 4A and
4
B). Further investigation displayed that all the compounds presented Top II
inhibitory activity at 50 µM (Figure 4C). To make a more intuitive and visualized

result of Top II inhibitory, the extent of remained relaxed DNA was quantitated
Figure 5). As showed in Figure 5A, most of the compounds expressed Top II
(
inhibitory activity over 50% except compound 5a, which displayed 28.1% Top II
inhibition activity. Compounds 5b, 5c, 5d, 9, 11 displayed a comparable Top II
inhibitory activity to the positive Top II drug m-AMSA. At 10 µM, the inhibitions of
Top II by compounds 5b, 5c, 5e, 5g, 11 were over 40% and compound 11 showed the
strongest Top II inhibitory activity (75.1%), which was better than m-AMSA (56.9%).
The results suggested all the compounds may have antiproliferative activity through
interacting with DNA and then inhibiting Top II.
Figure 4. Top I and Top II inhibitory activities. (A) Top I: lane B, Top I + pBR322
DNA; lane D, pBR322 DNA; lane C, CPT + Top I + pBR322DNA; the others, tested
compounds + Top I + pBR322 DNA. (B) Top II: lane B, Top II + pBR322 DNA; lane
D, pBR322 DNA; lane A, m-AMSA + Top II + pBR322 DNA; the others, tested
compounds + Top II + pBR322 DNA.

Figure 5. (A) Top II inhibitory activities at 50 µM; (B) Top II inhibitory activities at
10 µM.
2.6.
Apoptosis induced by compound 9
Compound 9 could interact with DNA and inhibit the activity of Top II, which
may induce apoptosis in cancer cell lines. An AnnexinV-FITC/PI binding assay using
U937 cells was conducted. The results can be seen in Figure 6. Q2 is the early stage
of apoptotic cells while Q3 represents the late stage of apoptotic or necrotic cells.
After U937 cells were treated with compound 9 at the concentrations of 0, 1 and 2.5
µM for 48 h, we can clearly see that the percentage of apoptotic cells increased
dramatically with the concentration of compound 9 increased. At 1 µM, 17.5% U937
cells were early apoptotic, and 6.48% cells were late apoptotic or necrotic. At 2.5 µM,
early apoptosis and late apoptosis or necrotic cells were improved to 26.2% and
12.2%. The result indicated that compound 9 can induce U937 cell apoptosis
efficiently.
Figure 6. Flow cytometric analysis of phosphatidylserine externalization (Annexin-V
binding) and cell membrane integrity (PI staining). U937 cells were treated with

compound 9 at (A) 0; (B) 1; (C) 2.5 µM respectively.
Apoptosis can be triggered by two key molecular signaling pathways: intrinsic
pathway (mitochondrial pathway), extrinsic pathway (death-receptor-mediated
3
6
pathway). In order to investigate which pathway that cellular apoptosis was induced
by compound 9, the expressions of regulatory proteins related to apoptotic pathway
such as cleaved caspase-3, cleaved caspase-7 and cleaved caspase-8 in U937 cells
were monitored by immunoblotting. Caspase-8 is an initiator in
death-receptor-mediated cell death pathway and caspase-3, caspase-7 show significant
3
7
roles in cell apoptosis once activated. In Figure 7, after treating with compound 9 at
the concentrations of 0, 1 and 2.5 µM for 48 h, a significant activation of cleaved
caspase-3, 7, 8 was detected. The results above indicated compound 9 induced
apoptosis through an extrinsic pathway.
Figure 7. U937cells were treated with compound 9 at indicated concentrations and
western blot analysis was used to evaluate the expression of cleavage caspase-3/7/8 in
U937 after 48 h. β-actin was used as an equal loading control.
3.
Conclusion
Depending on rational analysis and moleculardocking studies, a new series of
9
-azaacridine derivatives with phenyl or benzimidazole moities were synthesized.

Most of the compounds displayed moderate to good cytotoxicity against K562 cells.
Compounds 9 and 11 with benzimidazole showed good antitumor activity against
other cancer cell lines. In addition, compounds 9 and 11 displayed lower toxicity
against normal cells QST-7701 compared to m-AMSA. All of the compounds strongly
inhibited Top II activity and only compound 11 exhibited Top I inhibitory activity at
100 µM. Most of the compounds displayed moderate to good Top II inhibitory
activity at 10 µM. The representative compound 9 could strongly interact with DNA
and induce U937 cells apoptosis through an extrinsic pathway. Further structural
optimization of compound 9 is on the way.
4.
Experimental section
4.1. Chemistry
The general preparation of compound 4 can be seen in supporting information.
.1.1. General procedure for compound 5a-5g
4
A few drops of concentrated hydrochloric acid as catalysts were added dropwise
to a mixture of substituted 6, 9-dichloro-2-methoyl-acridine (50 mg, 0.20 mmol) and
benzenamine (0.97 mmol) in ethanol/chloroform (V/V= 4/1, 50 ml). The reaction
mixture was stirred overnight at room temperature. Then the liquid was evaporated to
give the crude products, which were purified by washing with ethanol twice.
4.1.1.1. 6-Chloro-2-methyl-9-(2-fluoro) phenylamino-azaacridine (5a) Yield 62%;
1
mp: 281-283 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.38 (d, J = 8.6 Hz, 1H), 8.27 (d, J
=
9.1 Hz, 1H), 8.03 (s, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.53-7.23 (m, 5H), 3.42 (s, 4H).
1
3
C NMR (101 MHz, DMSO-d ) δ 158.75, 136.02, 129.54, 128.74, 128.54, 128.42,
6
127.55, 127.33, 126.38, 125.42, 125.40, 124.48, 124.44, 124.35, 119.95, 116.14,
+
15.63, 115.44, 52.84. HRMS (ESI): [M+H] 354.0809; found 354.0806
1
4.1.1.2. 6-Chloro-2-methyl-9-(3-fluoro) phenylamino-azaacridine (5b) Yield 65%;
1
mp: 276-278 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.42 (d, J=9.2 Hz, 1H), 8.25 (d,
J=9.3 Hz, 1H), 8.18 (d, J=1.9 Hz, 1H), 7.60-7.47 (m, 3H), 7.33 (d, J=10.2 Hz, 1H),
1
3
7
.26 (d, J=7.9 Hz, 1H), 7.19 (t, J=8.4 Hz, 1H), 3.62 (s, 3H). C NMR (101 MHz,
DMSO-d ) δ 163.64, 161.24, 159.41, 147.53, 146.88, 143.77, 141.55, 140.43, 133.94,
6

1
1
4
29.76, 129.35, 127.67, 127.38, 126.22, 124.86, 119.33, 118.30, 115.56, 108.05,
+
07.86, 106.61, 106.41, 53.22. HRMS (ESI): [M+H] 354.0809; found 354.0805
.1.1.3. 6-Chloro-2-methyl-9-(4-fluoro) phenylamino-azaacridine (5c) Yield 59%;
1
mp: 268-270 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.40 (d, J=9.2 Hz, 1H), 8.13 (d,
1
J=1.8 Hz,1H), 8.05 (s, 1H), 7.65-7.48 (m, 4H), 7.39 (t, J=8.7 Hz, 2H), 3.75 (s,3H). C
3
NMR (101MHz, DMSO-d
6
) δ 159.27, 159.07, 156.90, 147.32, 143.19, 142.93, 142.79,
140.25, 139.95, 133.90, 128.33, 127.30, 127.14, 126.30, 124.00, 123.16, 123.09,
+
19.09, 116.73, 115.28, 115.06, 53.22. HRMS (ESI): [M+H] 354.0809; found
1
3
4
6
54.0806.
.1.1.4. 6-Chloro-2-methoyl-9-(4-methoyl) phenylamino-azaacridine (5d) Yield
1
2%; mp: 285-287 °C; H NMR (400 MHz, DMSO-d
6
) δ 11.16 (s, 1H), 8.36 (d, J=9.2
Hz, 1H), 8.05 (d, J=2.1 Hz, 1H), 7.84 (s, 1H), 7.60 (d, J=9.1 Hz, 1H), 7.49-7.35 (m,
1
H), 7.12 (d, J=8.8 Hz, 2H), 3.92 (s, 3H), 3.84 (s, 3H). C NMR (101 MHz,
3
3
DMSO-d
6
) δ 159.57, 158.11, 158.08, 137.91, 133.43, 133.34, 127.45, 126.66, 126.62,
+
1
3
4
6
26.59, 124.07, 121.78, 114.68, 112.78, 55.41, 54.14. HRMS (ESI): [M+H]
66.1009; found 366.1001.
.1.1.5. 6-Chloro-2-methoyl-9-(2-chloro) phenylamino-azaacridine (5e) Yield
1
9%; mp: 289-291 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.40 (d, J=9.2 Hz, 2H), 8.17
(
s, 1H), 7.74-7.59 (m, 3H), 7.59-7.49 (m, 2H), 7.45 (t, J=7.2 Hz, 1H), 3.48 (s, 3H).
1
3
C NMR (101MHz, DMSO-d ) δ 158.63, 136.55, 133.94, 129.36, 128.79, 128.48,
6
128.33, 127.50, 127.16, 126.93, 125.03, 124.04, 124.02, 119.79, 52.86. HRMS (ESI):
+
M+H] 370.0514; found 370.0507.
[
4.1.1.6. 6-Chloro-2-methyl-9-(2,4-dimethyl) phenylamino-azaacridine (5f) Yield
1
5
3%; mp：>300 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.22 (s, 1H), 7.94 (s, 1H), 7.77
(
s,1H), 7.37-7.35 (d, J=7.8 Hz, 1H), 7.28-7.26 (d, J=9.1 Hz, 1H), 7.20 (s, 1H), 7.03 (m,
1
3
2
1
1
6
H), 3.81 (s, 3H), 2.27 (s, 3H), 2.20 (s, 3H). C NMR (101 MHz, DMSO-d ) δ
59.65, 151.90, 139.54, 138.94, 137.75, 136.30, 134.49, 131.52, 131.28, 129.22,
27.59, 126.96, 126.56, 125.27, 124.57, 122.36, 118.31, 54.11, 20.75, 17.58. HRMS
+
ESI): [M+H] 364.1217; found 364.1211
(
4.1.1.7. 6-Chloro-2-methyl-9-(4-pyridine) amino-azaacridine (5g) Yield 67%; mp:

1
45-247 °C; H NMR (400 MHz, DMSO-d
2
6
/D
2
O) δ 8.17-8.10 (m, 4H), 7.98 (s, 1H),
7.50-7.48 (d, J=9.2Hz, 1H), 7.27-7.24 (d, J=9.2, 2H), 6.79-6.78 (m, 2H), 3.54 (s, 3H).
1
3
6 2
C NMR (101 MHz, DMSO-d /D O) δ 171.98, 159.64, 157.24, 148.01, 139.95,
134.41, 130.48, 129.21, 126.36, 125.69, 120.53, 119.45, 118.66, 115.15, 112.24, 53.07.
+
HRMS (ESI): [M+H] 336.0788; found 336.0857.
4.1.2. Preparation of compound 9, 12
A mixture of 6,9-dichloro-2-methoyl-anzacridine (278 mg, 1 mmol) and
benzimidazoles (220 mg, 1 mmol) was heated in phenol under 120 °C for 2 hours
with nitrogen protection. Then the mixture was poured into ethyl acetate to give
yellow precipitates, which were purified by washing with ethyl acetate twice.
4.1.2.1. 6-Chloro-2methyl-9-((1H-benzo[d]imidazol-2-yl)methyl)-azaacridine (9)
1
Yield 73%; mp: 289-291 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.51-8.48 (d, J=14.4
Hz 1H), 8.37 (s, 1H), 8.13-8.11 (d, 1H), 7.92 (s, 1H), 7.47-7.42 (m, 3H), 7.22-7.20 (d,
1
3
J=8.8 Hz, 1H), 7.12-7.10 (m, 2H), 3.81 (s, 3H). C NMR (101 MHz, DMSO-d
6
) δ
1
1
3
4
57.82, 153.67, 147.62, 146.32, 143.49, 140.36, 140.33, 140.28, 133.72, 127.24,
+
26.19, 125.45, 123.24, 118.35, 115.08, 53.37, 44.72. HRMS (ESI): [M+H]
90.1043; found 390.1115.
.1.2.2. 6-Chloro-2methyl-9-((1H-benzo[d]imidazol-2-yl)methyl)-acridine
(12)
1
Yield 22%; mp: 237-238 °C; H NMR (400 MHz, DMSO-d
6
) δ 8.63-8.61 (d, J = 8.1
Hz, 2H), 8.03 (s, 1H) 7.94-7.89 (m, 2H), 7.74-7.73 (d, J = 4.8 Hz, 1H), 7.62-7.53 (m,
1
3
2
1
1
4
6
H), 5.54 (s, 2H) 3.88 (s, 3H) C NMR (101 MHz, DMSO-d ) δ 156.13, 155.86,
39.16, 138.90, 134.79, 134.06, 127.93, 126.86, 123.94, 120.43, 117.32, 112.49,
+
09.67, 103.22, 56.28. HRMS (ESI): [M+H] 389.1169; found 389.1163.
.1.3. Preparation of compound 11
2 3
KOH (30 mg) and K CO (60 mg) as bases were added to an acetone suspension
containing compound 9 (100 mg, 0.31 mmol) and 2-Dimethylaminoethyl chloride
hydrochloride (45 mg, 0.31 mmol), which were refluxed overnight. After cooling to
room temperature, water was added into the mixture under ice-water bath. Purified
yellow solids were filtrated after washing by water once.
4.1.3.1. 6-Chloro-2methyl-9-((1-Dimethylaminoethyl-benzo[d]imidazol-2-yl)met

1
hl)-azaacridine (11) Yield 43% ; mp: 182-184 °C; H NMR (400 MHz, DMSO-d
6
) δ
8
7
2
1
1
4
4
.27 (s, 1H), 8.25 (s, 1H), 8.18-8.16 (m, 1H), 8.05-8.03 (m, 1H), 7.39-7.36 (m, 1H),
.33-7.31 (m, 1H), 7.30-7.26 (m, 1H), 7.19-7.17 (m, 1H), 5.54 (s, 1H), 4.24-4.22 (m,
1
3
H), 4.21 (s, 1H), 2.67-2.64 (m,2H), 2.21 (s, 6H). C NMR (101 MHz, DMSO-d ) δ
6
58.97, 150.95, 148.09, 146.43, 142.28, 141.78, 139.62, 134.95, 134.49, 127.84,
26.94, 124.14, 123.50, 122.38, 121.81, 119.28, 118.11, 114.45, 108.73, 57.96, 53.32,
+
5.26, 44.46, 41.90, 29.10. HRMS (ESI): [M+H] 461.1778; found 461.1853.
.2. Cell proliferation assay
Cell proliferation assays based on MTT were performed with 6 cell lines. Briefly,
3
cells were seeded in 96-well plates at a density of 5-10×10 cells per well with
2
incubation overnight in a 5% CO incubator at 37 °C, the growth medium in each well
was then added with 50 µL of fresh medium containing graded concentrations of
compounds to be tested or equal DMSO and incubated continuously for 48 h. Then 15
µL MTT solution (5 mg/mL) was added to each well, and the cells were incubated for
additional 4 h. The MTT-formazan crystals were dissolved in 100 mL of DMSO, the
absorbance of each well was measured at 490 nm using an automatic ELISA reader
system (TECAN, CHE).
4.3. 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 9 (5 mM in DMSO, 8 µL) and the buffer above were transferred to
the quartz, then a range of ct DNA solution (2.5 mM in Tris–HCl) was mixed with the
solution in the cell with different final concentration ([DNA] = 0, 0.75, 2.25, 3.75,
6.00, 9.75 µM). The incubating time before testing was 5 min.
4.4. Fluorescence emission spectra
The emission spectra were carried out on Fluorolog spectrometer. The solution of
tested compound 9 (5 mM in DMSO, 8 µL) and the buffer above (2 mL) were
transferred to the quartz with the final concentration at 2 µM, then a range of ct DNA
solution (2.0 mM in Tris–HCl) was mixed with the solution in the cell with the final
concentration ([DNA] = 0, 0.2, 0.7, 1, 1.5, 2.5 µM). The incubating time before

testing was 5 min.
4.5. DNA Top I and Top II inhibition assay
The inhibition of compounds to Top I and Top II were determined by assessing
the relaxation of supercoiled pBR322 plasmid DNA. The assay was performed in a
final volume of 20 µL reaction volume containing 500 ng of supercoiled DNA
pBR322 (Takara Biotechnology, Japan) and 1 unit of human Top I (Takara
Biotechnology, Japan) or Top II (USB Corp, USA) with or without our compounds in
the reaction buffer. The reaction mixtures were incubated at 37 °C or 10-15 min and
terminated by adding 4 mL of DNA loading buffer. Electrophoresis was performed on
a 1% agarose gel at 90 V for 25 min in TAE buffer. Then stained gels for 10 min with
ethidium bromide (2.5 mg/mL) and destained in water for 5 min. DNA bands were
visualized with UV light. CPT (Sigma, USA) was used as a positive control as Top I
inhibitor and m-AMSA (Sigma, USA) were used as a Top II inhibitor, respectively.
4.6. Flow cytometry assay
The Phosphatidylserine externalization was determined with the scheme which is
designed as the instructions of the manufacturer by AnnexinV-FITC/PI apoptosis
detection kit.
4.7. Western blot analysis
U937 cells were cultured in 6 cm dishes, and then compound 9 was added with
the final concentration at 1 µM, 2.5 µM independently, and the resulting solutions
were incubated for 48 h. Then the cells were centrifuged and was dialyzed with lysis
buffer at 0 °C for 0.5 h, followed by centrifugation at 20,000 g for 10 min. Protein
concentrations in the supernatant were determined using bicinchoninic 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 °C. Protein bands
were detected using the BIO-RAD Gel Doc XR after hybridization with the antibody.

Acknowledgements
The authors would like to thank the financial supports from Shenzhen Municipal
Development and Reform Commission (Disciplinary Development Program for
Chemical Biology), the financial supports from the Chinese National Natural Science
Foundation (21372141), and Shenzhen Sci & Tech Bureau (CXB201104210013A，
CXZZ20150529165045064, JCYJ20150331151358131 and JCYJ2016030115375326
9
).
Supplementary data
Supplementary data (Molecular modeling of compound 9, 12 and CPT with Top I
1 13
DNA complex, synthesis of compound 4, H NMR and C NMR spectra and high
-
resolution mass spectrometry of compounds 5a-5g, 9, 11 and 12, associated with this
article can be found in the online version.
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Synthesis and biological research of novel azaacridine
derivatives as potent DNA-binding ligands and topoisomerase II
inhibitors
A series of azaacridine derivatives were synthesized and evaluated as good
antiproliferative inhibitors. Compared to m-AMSA, the typical compound 9 showed
comparable IC50 values against cancer cells (HepG-2 and U937 cells) while displayed
lower cytotoxicity against human normal liver cells (QSG-7701).

Highlights
ꢀ
ꢀ
ꢀ
A series of azaacridine derivatives were synthesized.
Most of the compounds displayed good antiproliferative activity.
The typical compound 9 showed low cytotoxicity against human normal liver
cells.
ꢀ
ꢀ
All the compounds displayed good topoisomerase II inhibition activity.
Compound 9 induced apoptosis through the exogenous pathway.