Design, synthesis and biological research of novel N-phenylbenzamide-4-methylamine acridine derivatives as potential topoisomerase I/II and apoptosis-inducing agents

Article abstract of DOI:10.1016/j.bmcl.2019.126714

A series of novel N-phenylbenzamide-4-methylamine acridine derivatives were designed and synthesized based initially on the structure of amsacrine (m-AMSA). Molecular docking suggested that the representative compound 9a had affinity for binding DNA topoisomerase (Topo) II, which was comparable with that of m-AMSA, and furthermore that 9a could have preferential interactions with Topo I. After synthesis of 9a and analogues 9b-9f, these were all tested in vitro and the synthesized compounds displayed potent antiproliferative activity against three different cancer cell lines (K562, CCRF-CEM and U937). Among them, compounds 9b, 9c and 9d exhibiting the highest activity with IC₅₀ value ranging from 0.82 to 0.91 μM against CCRF-CEM cells. In addition, 9b and 9d also showed high antiproliferative activity against U937 cells, with IC₅₀ values of 0.33 and 0.23 μM, respectively. The pharmacological mechanistic studies of these compounds were evaluated by Topo I/II inhibition, western blot assay and cell apoptosis detection. In summary, 9b effectively inhibited the activity of Topo I/II and induced DNA damage in CCRF-CEM cells and, moreover, significantly induced cell apoptosis in a concentration-dependent manner. These observations provide new information and guidance for the structural optimization of more novel acridine derivatives.

Full text of DOI:10.1016/j.bmcl.2019.126714

Journal Pre-proofs
Design, synthesis and biological research of novel N-phenylbenzamide-4-meth-
ylamine acridine derivatives as potential topoisomerase I/II and apoptosis-
inducing agents
Bin Zhang, Zhende Dou, Zheng Xiong, Ning Wang, Shan He, Xiaojun Yan,
Haixiao Jin
PII:
S0960-894X(19)30672-9
DOI:
https://doi.org/10.1016/j.bmcl.2019.126714
BMCL 126714
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19 September 2019
Please cite this article as: Zhang, B., Dou, Z., Xiong, Z., Wang, N., He, S., Yan, X., Jin, H., Design, synthesis and
biological research of novel N-phenylbenzamide-4-methylamine acridine derivatives as potential topoisomerase I/
II and apoptosis- inducing agents, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/
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Design, synthesis and biological research of novel
N-phenylbenzamide-4-methylamine acridine derivatives as potential
topoisomerase I/II and apoptosis- inducing agents
Bin Zhang a, Zhende Dou a, Zheng Xiong a, Ning Wang b,*, Shan He a , Xiaojun Yan a,
Haixiao Jin a,**
a Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center,
Department of Marine Pharmacy, College of Food and Pharmaceutical Sciences,
Ningbo University, Ningbo, Zhejiang, 315800, People’s Republic of China
b
Institute of Drug Discovery Technology, Ningbo University, Ningbo, Zhejiang
315211, People’s Republic of China
*
Corresponding author at: Institute of Drug Discovery Technology, Ningbo
University, Ningbo, Zhejiang 315211, People’s Republic of China;
**
Corresponding author at: Li Dak Sum Yip Yio Chin Kenneth Li Marine
Biopharmaceutical Research Center, Department of Marine Pharmacy, College of
Food and Pharmaceutical Sciences, Ningbo University, Ningbo, Zhejiang, 315800,
People’s Republic of China.
E-mail addresses: wangning2@nbu.edu.cn (N. Wang); jinhaixiao@nbu.edu.cn (H.
Jin)
1

Abstract
A series of novel N-phenylbenzamide-4-methylamine acridine derivatives were
designed and synthesized based initially on the structure of amsacrine (m-AMSA).
Molecular docking suggested that the representative compound 9a had affinity for
binding DNA topoisomerase (Topo) II, which was comparable with that of m-AMSA,
and furthermore that 9a could have preferential interactions with Topo I. After
synthesis of 9a and analogues 9b-9f, these were all tested in vitro and the synthesized
compounds displayed potent antiproliferative activity against three different cancer
cell lines (K562, CCRF-CEM and U937). Among them, compounds 9b, 9c and 9d
exhibiting the highest activity with IC50 value ranging from 0.82 to 0.91 μM against
CCRF-CEM cells. In addition, 9b and 9d also showed high antiproliferative activity
against U937 cells, with IC50 values of 0.33 and 0.23 μM, respectively. The
pharmacological mechanistic studies of these compounds were evaluated by Topo I/II
inhibition, western blot assay and cell apoptosis detection. In summary, 9b effectively
inhibited the activity of Topo I/II and induced DNA damage in CCRF-CEM cells and,
moreover, significantly induced cell apoptosis in a concentration-dependent manner.
These observations provide new information and guidance for the structural
optimization of more novel acridine derivatives.
Keywords: Acridine derivatives; Anticancer; Topoisomerase I/II; Molecular docking;
DNA damage
2

Cancer is expected to be the second leading cause of death before age 70 in most
countries of the world in the 21st century, moreover, its incidence and mortality are
increasing rapidly worldwide [1]. Therefore, efforts have been continuously made for
the development of effective chemotherapy drugs that can have profound impacts on
society through human health benefits and economic rewards. Acridine derivatives
have for decades been widely explored in the field of anticancer therapeutics because
of their excellent anticancer activity, especially against leukemia [2, 3]. For instance,
amsacrine (m-AMSA) has been used clinically in a number of countries for the
treatment of leukemia. Several of the coauthors have also previously developed
different series of novel acridine compounds, including pyridyl acridones, benzyl
acridones and 9-aminoacridine derivatives with good antitumor activity and low
toxicity [4-6].
DNA topoisomerase (Topo) I/II directly regulates the topological structure of DNA
and induces DNA damage by enhancing the formation of Topo I/II-DNA cleavage
complexes or suppressing DNA religation [7]. Because of the rapid growth of tumor
cells, the content and activity of Topo I and II in tumor cells are generally upregulated
compared with normal cells [8]. Therefore, compared with other DNA damage agents,
such as alkylating agents, topoisomerase inhibitors may have preferable anti-tumor
specificity. Nowadays, a great many novel Topo I/II inhibitors with different
structural scaffolds have been reported [9-11]. m-AMSA (Fig. 1) is a Topo II inhibitor
with a 9-aminoacridine scaffold that has been used successfully in the treatment of
acute myeloid leukemia (AML), acute lymphoblastic leukemia and acute
promyelocytic leukemia [12, 13]. Specific interactions of m-AMSA with Topo II via
the 9-anilino moiety of the drug seem to be crucial for its inhibitory activity [14]. We
hypothesized that if the aniline group is not directly linked to the acridine ring, for
instance, there is an intermediate chain (linker) between these two groups, the binding
mode between the ligand and the protein could be affected. Based on the structure of
m-AMSA, we designed
a
new representative compound 9a with
N-phenylbenzamide-4-methylamine acridine scaffold (Fig. 1).
3

O
N
O
H
H
H
Structural
Modification
O
O
N
N
S
N
Linker
N
H
N
Representative compound 9a
m-AMSA
Fig. 1. the structures of m-AMSA and the representative compound 9a
Molecular docking study of the m-AMSA and 9a with Topo II-DNA complex
mode (PDB ID: 4G0U) [15] showed that amide NH (CONH-) of 9a has a hydrogen
bond with Ala521, and aminomethyl NH has a hydrogen bond with Arg503 (Fig. 2A).
In addition, it is important that the phenyl in 9a (marked yellow stick) inserted into
the hydrophobic pocket formed by side chain of Ile454 and Ala503 (Fig. 2B).
However, the aniline side chain of m-AMSA (marked purple stick) could not provide
such hydrophobic effect. Therefore, the strategy of introducing the extension linker
between the aniline group and acridine ring is reasonable and feasible.
Since most acridine derivatives generally also have good inhibitory activity against
Topo I, we also aimed to verify whether representative compound 9a has inhibitory
effect on this target. The molecular docking study (Fig. 2C) of 9a with Topo I-DNA
complex (PDB ID: 1SC7) [16] showed that 9a had a hydrogen bond with Arg364, and
the acridine ring is inserted into the cavity formed by two pairs of DNA bases.
Interestingly, benzene ring was extended into hydrophobic subpocket formed by
Pro431, Leu721, Lys751, Phe752 and Glu748 to form π-π and C-H-π interactions.
Therefore, the above docking results indicated that this series of acridine compounds
may have a Topo I/II dual inhibitory effect, which was further verified by subsequent
biological experiments.
4

Fig. 2. Docking mode of representative compound 9a (stick) and Topo I/II using gold
5.2.2. The carbon atom of 9a, residues and base pairs are presented in yellow, grey
and orange, respectively, the carbon atom in m-AMSA is colored in purple. All
oxygen and nitrogen atoms are colored in red and blue respectively. Hydrogen bonds
are represented by red dotted lines. (A) Topo II-9a binding mode; (B) Topo II-9a
(yellow) and Topo II-m-AMSA (purple) binding mode; (C) Topo I-9a binding mode.
To investigate the relationship between the structure and activity of the target
compounds, six novel N-phenylbenzamide-4-methylamine acridine derivatives
(compounds 9a-f) were generated preferentially by introducing different substituents
on the acridine (A and C ring) and benzene (E ring) moieties. These target compounds
were synthesized from the starting materials aniline and 2-chlorobenzoic acid, or its
derivatives, in a sequence of chemical reactions as shown in Scheme 1. First,
compounds 3a-e were obtained in high yields from the Ullmann reaction of
2-chlorobenzoic acid 1a or 2,4-dichlorobenzoic acid 1b with aniline 2a or its
derivatives 2b-d in DMF using Cu as the catalyst. Subsequent Friedel-Crafts acylation
was carried out in POCl3 for 3-5 h to give 9-chloroacridine derivatives 4a-e [17]. The
intermediate 6 was obtained in high yield by protection of the amino group of
4-(aminomethyl)benzoic acid 5, which was carried out by reflux in methanol with
BOC anhydride and triethylamine [18]. Subsequently, 6 was reacted with aniline 2a
or 2b using N,N′-carbonyldiimidazole (CDI) as the condensation agent at room
temperature to afford the intermediate 7a-b [4]. 4-(aminomethyl)-N-phenylbenzamide
8a-b was obtained by the hydrolysis of 7a-b in 3M hydrochloric acid and methanol at
5

room temperature [19]. The desired compounds 9a-f were produced by the
nucleophilic substitution between the corresponding 9-chloroacridines 4a-e and
intermediate 8a-b in phenol at 90-100℃ under an argon atmosphere [11]. Structures
1
of compounds were confirmed by H NMR, 13C NMR and high resolution mass
spectrometry (HR-MS) data.
Cl
1
8
5'
2'
5
9
COOH
6'
4'
R1
3'
COOH
Cl
4
(ii)
7
R1
6
2
3
(i)
6
R2
R2
R2
R1
3
1'
1
N
N
H2N
4
2
5
H
4a R1 = -H, R2 = -H
2a R2 = -H
1a R1 = -H
1b R1 = 4-Cl
3a R1 = -H, R2 = -H
4b R1 = -H, R2 = 4-OCH3
4c R1 = -H, R2 = 3-OCH3
4d R1 = -H, R2 = 2-OCH3
4e R1 = 6-Cl, R2 = 2-OCH3
2b R2 = 2-OCH3
2c R2 = 3-OCH3
2d R2 = 4-OCH3
3b R1 = -H, R2 = 2-OCH3
3c R1 = -H, R2 = 3-OCH3
3d R1 = -H, R2 = 4-OCH3
3e R1 = 4'-Cl, R2 = 4-OCH3
O
O
R3
2a R3 = -H
NH
O
NH2
H
NH
O
N
NH2
(iii)
R3
2b R3 = 2-OCH3
7a R3 = -H
HOOC
4'
O
HOOC
(iv)
7b R3 = 2-OCH3
6
5
(v)
O
R3
3'
R1
7
N
H
8
6
5
Cl
N
NH2
2'
H
H
9
N
N
(vi)
R2
R1
R3
N
1
2
O
4a R1 = -H, R2 = -H
9a R1 = -H, R2 = -H, R3 = -H
8a R3 = -H
8b R3 = 2-OCH3
4
3 R2
4b R1 = -H, R2 = 4-OCH3
4c R1 = -H, R2 = 3-OCH3
4d R1 = -H, R2 = 2-OCH3
4e R1 = 6-Cl, R2 = 2-OCH3
9b R1 = -H, R2 = 2-OCH3, R3 = -H
9c R1 = -H, R2 = 3-OCH3, R3 = -H
9d R1 = -H, R2 = 4-OCH3, R3 = -H
9e R1 = 6-Cl, R2 = 2-OCH3, R3 = -H
9f R1 = -H, R2 = -H, R3 = 2'-OCH3
Scheme 1 Reagents and conditions: (i) K2CO3, Cu, DMF, 130 ºC, overnight; (ii)
POCl3, reflux, 3-5 h; (iii) BOC anhydride, triethylamine, methanol, reflux; (iv) CDI,
DMF, overnight; (v) 3M HCl, methanol, room temperature; (vi) phenol, under the
protection of N2, 90-100 ºC.
Most acridine derivatives, such as m-AMSA, always have been shown potenial
anti-leukemia
activity
[2].
Therefore,
in
this
study,
six
N-phenylbenzamide-4-methylamine acridine derivatives 9a-f and two intermediates
(4e and 8a) were tested against human leukemia K562 and CCRF-CEM cells by the
MTT reduction assay. Furthermore, m-AMSA was used as the positive control. In
order to study the inhibitory effect of these compounds against lymphoma cells, the
6

human histiocytic lymphoma cell line U937 was also selected for MTT assay
screening. The structures and bioactivity testing results are summarized in Table 1.
Most of the test compounds displayed potent activity against all the tested cancer cell
lines, except for the intermediates 4e and 8a (IC50 >10 μM). Among them, compounds
9b-d exhibited the highest activity with IC50 value ranging from 0.82 to 0.90 μM
against CCRF-CEM cells. However, the inhibitory activity of these compounds
against K562 cells was relatively poor, only 9f with methoxy substitution (R3 group)
at position 2 of the E ring had an IC50 value of 0.91 μM, similar to the positive control
m-AMSA (IC50 = 0.71 μM). This suggested that methoxy substitution on the E ring
can enhance the inhibitory activity against K562 cells. When the R1 group on the A
ring was a chlorine atom (9e), the antiproliferative activity against K562 and U937
cells decreased significantly, for example, the IC50 value of 9e against U937 cells
decreased about 6 fold compared with 9a. When the R2 substituent on the C ring was
a methoxy group, the activity of compounds 9b-d against CCRF-CEM cells were
increased about two times compared with those without substitution (9a and 9f). For
instance, 9b had significant cancer cell cytotoxic activity with IC50 of 0.82 μM, while
the IC50 of 9a was 1.56 μM. In addition, 9b and 9d also showed high antiproliferative
activity against U937 cells, with IC50 values of 0.33 and 0.23 μM, respectively. This
indicated that the antiproliferative activity of these compounds against U937 were
significantly increased when the methoxy group was at position 2 and 4 of the C ring.
In summary, the synthesized series of compounds had good antiproliferative activity
against these three kinds of cancer cells. In view of the good inhibitory activity of 9b
against various tumor cells in vitro, it was selected for subsequent biological
evaluation.
As compounds 9a-f displayed good antiproliferative activity against K562,
CCRF-CEM and U937 cells, the in silico predictions of drugability and
pharmacokinetics of 9a-f using Molinspiration property engine (Table 2) and BIOVIA
Discovery Studio 2019 (Table 3) were performed. Lipinski's rule is often used to
predict the drugability properties of small molecule drugs [20]. It can be seen from
7

Table 2 that the Lipinski's violations of compounds 9a-f were 1, which are accorded
with the limit of Lipinski's rule (≤1). The specific parameters are described as follows.
For instance, the number of hydrogen bond donors and acceptors of 9b are 2 and 5,
respectively; the calculated log P (clopP) value is 5.85 (slightly higher), the molecular
weight is 433.51; the number of rotatable bonds is 6, the surface area is 63.25 Å2, all
of which except the slightly larger clopP were within the range specified by the
suggested rule. Therefore, the predicted results suggested that this series of
compounds could have appropriate physicochemical and drugability properties.
Table 1 Antiproliferative activity of N-phenylbenzamide-4-methylamine acridines
9a-f and two intermediates (4e and 8a) against K562, CCRF-CEM and U937 cells.
4'
R3
O
E
R1
7
3'
N
8
6
5
H
2'
D
A
H
9
N
B
N
1
2
3 R2
C
9a-9f
4
IC50 (μM)
Compound
R1
R2
R3
CCRF-CE
M
K562
U937
4e
>10
>10
>10
8a
>10
>10
>10
9a
-H
-H
-H
-H
-H
-H
-H
-H
1.56±0.68
0.82±0.01
0.90±0.04
0.89±0.13
1.30±0.06
1.55±0.41
1.24±0.60
1.32±0.21
1.43±0.42
2.37±0.56
0.91±0.12
0.71±0.09
1.32±0.01
0.33±0.29
1.42±0.05
0.23±0.03
1.80±0.84
1.49±1.77
0.08±0.01
9b
2-OCH3
3-OCH3
4-OCH3
2-OCH3
-H
9c
-H
9d
9e
-H
6-Cl
-H
9f
2'-OCH3 1.28±0.05
0.03±0.01
m-AMSA
8

Table 2 In silico prediction of physicochemical and drugability properties of 9a-f
using Molinspiration property engine v2018.10
Lipinski HBAa HBDb clogPc
MWd NROTBe
TPSAf
(≤140Å2)
54.02
Compound
(≤1)
1
(≤10)
(≤5)
2
(≤5)
5.84
5.85
5.88
5.88
6.53
5.85
(≤500)
403.49
433.51
433.51
433.51
467.96
433.51
(≤10)
9a
9b
9c
9d
9e
9f
4
5
5
5
5
5
5
6
6
6
6
6
1
2
63.25
1
2
63.25
1
2
63.25
1
2
63.25
1
2
63.25
a
b
c
Number of hydrogen-bond acceptors.
Number of hydrogen-bond donors.
Octanol/water partition coefficient. d Molecular weight. e Number of rotatable bonds. f
Topological polar surface area.
The evaluation of predicted pharmacokinetic properties of 9a-f were carried out
using BIOVIA Discovery Studio 2019 [21]. It can be seen from Table 3 that the
predicted solubility level of compounds 9a-f is 1, indicating that the water-solubility
of these compounds should be poor. However, the solubility of these compounds can
be improved by converting them into counter-ionic salt forms, such as hydrochlorides.
The BBB level represents blood-brain barrier permeability after oral administration,
and a predicted value of 1 for 9a-f indicated that these compounds might have high
BBB permeability. Absorption Level predicts intestinal absorption after oral
absorption, and the calculated values of 0 and 1 indicated that compounds 9a-f may
have good intestinal absorption characteristics. CYP2D6 Prediction refers to the
prediction of enzyme inhibition of compounds 9a-f to cytochrome P450 2D6, and
“TRUE/FALSE” was used to determine whether these compounds were inhibitors of
9

CYP2D6. The generated data suggested that compounds 9a-f were each unlikely to
inhibit the CYP2D6 enzyme. In the analysis of a new drug, it is very important to
know the basic pharmacokinetic parameters such as human plasma protein binding
(PPB), and the calculated result of “TRUE” indicated that these compounds likely
have high plasma protein binding rates.
Table 3 In silico prediction of pharmacokinetic properties of 9a-f using BIOVIA
Discovery Studio 2019
Solubility
BBB
Absorption
CYP2D6
Prediction
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
PPB
Prediction
TRUE
TRUE
TRUE
TRUE
TRUE
TRUE
Compound
Level
Level
Level
9a
9b
9c
9d
9e
9f
1
1
1
1
1
1
1
1
1
1
4
1
0
0
0
0
1
0
Molecular docking study indicated that the synthesized acridine derivatives 9a-f
had a good binding effect on Topo I/II. Therefore, it was evaluated whether these
compounds could inhibit the activity of topoisomerases in vitro. Compounds 9a and
9b were selected for an enzymatic Topo I/II activity inhibition test, and camptothecin
(a clinical Topo I inhibitor) and etoposide (a clinical Topo II inhibitor) were selected
as positive controls for this experiment. It can be seen from Table 4 that compounds
9a and 9b have partial inhibitory effects on Topo I/II at 100 μM. For example, the
inhibition rate of compound 9a on Topo I and Topo II reached 44.94% and 23.96%
respectively. However, the inhibitory effects of these two compounds were not as
strong as camptothecin and etoposide, respectively. Future studies should aim to
optimize the structure-activity of these compounds to improve the target inhibition
activity.
10

Table 4 The inhibitory activity of compounds 9a and 9b on Topo I/II
100 μM （Inhibition rate %）
Compound
Topo I
7.20
44.94
74.32
-
Topo II
9b
17.76
23.96
-
9a
Camptothecin
Etoposide
“-” not tested.
40.88
When DNA damage occurs, histone H2AX is phosphorylated to form γ-H2AX,
therefore γ-H2AX is considered as an important biomarker of DNA damage [22]. As
a DNA damage agent, a Topo I/II inhibitor can induce DNA strand breakage and cell
apoptosis. It was next determined whether compound 9b could induce the formation
of γ-H2AX by western blot analysis. For this purpose, CCRF-CEM cells were treated
with 9b with different concentration gradients. Fig. 3 shows that the content of
γ-H2AX in CCRF-CEM cells was significantly increased with the increase of the
concentration of 9b from 0.5 μM to 2 μM.
11

Fig. 3. 9b induced the formation of γ-H2AX in vitro (CCRF-CEM cells-48h)
An Annexin V-FITC/PI kit was used to detect whether compound 9b induced
apoptosis in CCRF-CEM cells. Annexin V-FITC can recognize early apoptotic cells
and show green fluorescence. Propyridine iodide (PI) is used to identify late apoptotic
and necrotic cells, showing red fluorescence. As can be seen from Fig. 4, 9b can
induce early apoptosis at 0.5 μM. When the concentration of 9b was increased to 2
μM, the number of early apoptotic cells increased significantly. Additionally, the
number of late apoptotic cells and necrotic cells also increased in a
concentration-dependent manner. However, the trend was not obvious compared with
early apoptotic cells. Similarly, the results obtained by using Hoechst 33342/PI kit
confirmed that 9b could significantly induce apoptosis (Fig. 1s). Hoechst 33342 can
recognize apoptotic cells (blue fluorescence), and PI is used to identify necrotic cells
(red fluorescence). In addition, it was observed that 9a could also significantly induce
apoptosis of CCRF-CEM cells using AnnexinV-FITC/PI apoptosis detection kit (Fig.
2s). Therefore, it is indicated that the new N-phenylbenzamide-4-methylamine
acridine derivatives synthesized in this study were apoptosis-inducing agents.
12

Fig. 4. 9b induced apoptosis in CCRF-CEM cells (48h). Annexin V-FITC can
recognize early apoptotic cells and show green fluorescence. PI is used to identify late
apoptotic and necrotic cells, showing red fluorescence.
In this study, a series of N-phenylbenzamide-4-methylamine acridine derivatives
(9a-9f) were preferentially designed and synthesized as Topo I/II inhibitors based on
the structure of m-AMSA. MTT screening showed that these compounds exhibited
good antiproliferative activity against three different cancer cell lines (K562,
CCRF-CEM and U937) in vitro. The IC50 values of compound 9b against
CCRF-CEM cells and U937 cells was 0.82 and 0.33 μM, respectively. In silico and in
vitro pharmacological mechanistic studies indicated 9b can inhibit the activity of
topoisomerase I/II and induce DNA damage in CCRF-CEM cells. Moreover, 9b
induces cell apoptosis in a concentration-dependently. Accordingly, the results of this
study provide new perspectives for the further structural optimization of this class of
molecules for use in cancer chemotherapy research.
13

Acknowledgments
The authors thank the financial supports from the National Key Research and
Development Program of China (2018YFC0310900), the National Natural Science
Foundation of China (81850410553, 41776168) , the Natural Science Foundation of
Ningbo City (2018A610410), Foundation of Ningbo University for Grant
(XYL18004), the National 111 Project of China (D16013), the Li Dak Sum Yip Yio
Chin Kenneth Li Marine Biopharmaceutical Development Fund, and the K.C. Wong
Magna Fund in Ningbo University. We appreciate Prof. C. Benjamin Naman of
Ningbo University for careful editing of and thoughtful comments about the
manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version.
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Captions
Fig. 1. the structures of m-AMSA (A) and the representative compound 9a (B).
16

Fig. 2. Docking mode of representative compound 9a (stick) and Topo I/II using gold
5.2.2. The carbon atom of 9a, residues and base pairs are presented in yellow, grey
and orange, respectively, the carbon atom in m-AMSA is colored in purple. All
oxygen and nitrogen atoms are colored in red and blue respectively. Hydrogen bonds
are represented by red dotted lines. (A) Topo II-9a binding mode; (B) Topo II-9a
(yellow) and Topo II-m-AMSA (purple) binding mode; (C) Topo I-9a binding mode.
Fig. 3. 9b induced the formation of γ-H2AX in vitro (CCRF-CEM cells-48h).
Fig. 4. 9b induced apoptosis in CCRF-CEM cells (48h). Annexin V-FITC can
recognize early apoptotic cells and show green fluorescence. PI is used to identify late
apoptotic and necrotic cells, showing red fluorescence.
Scheme 1 Reagents and conditions: (i) K2CO3, Cu, DMF, 130 ºC, overnight; (ii)
POCl3, reflux, 3-5 h; (iii) BOC anhydride, triethylamine, methanol, reflux; (iv) CDI,
DMF, overnight; (v) 3M HCl, methanol, room temperature; (vi) phenol, under the
protection of N2, 90-100 ºC.
17

R3
O
N
O
R1
H
Structural
Modification
H
H
O
O
N
N
S
N
Linker
H
N
N
R2
Target Compounds
m-AMSA
18