Design, synthesis and biological evaluation of novel perimidine o-quinone derivatives as non-intercalative topoisomerase II catalytic inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103131

For the development of novel anticancer agents, we designed and synthesized a total of 37 perimidine o-quinone derivatives containing the o-quinone group at the A or B ring and different substituents (alkyl groups, aryl groups or heterocycles) at the C ri

Full text of DOI:10.1016/j.bioorg.2019.103131

Accepted Manuscript
Design, synthesis and biological evaluation of novel perimidine o-quinone de-
rivatives as non-intercalative topoisomerase II catalytic inhibitors
Du-Chao Zhou, Yu-Ting Lu, Yan-Wen Mai, Chen Zhang, Jie Xia, Pei-Fen Yao,
Hong-Gen Wang, Shi-Liang Huang, Zhi-Shu Huang
PII:
S0045-2068(19)30730-8
https://doi.org/10.1016/j.bioorg.2019.103131
103131
DOI:
Article Number:
Reference:
YBIOO 103131
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 May 2019
18 July 2019
18 July 2019
Please cite this article as: D-C. Zhou, Y-T. Lu, Y-W. Mai, C. Zhang, J. Xia, P-F. Yao, H-G. Wang, S-L. Huang, Z-
S. Huang, Design, synthesis and biological evaluation of novel perimidine o-quinone derivatives as non-intercalative
topoisomerase II catalytic inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103131
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Title Page
Design, synthesis and biological evaluation of novel perimidine o-quinone
derivatives as non-intercalative topoisomerase II catalytic inhibitors
Du-Chao Zhou, Yu-Ting Lu, Yan-Wen Mai, Chen Zhang, Jie Xia, Pei-Fen Yao, Hong-Gen Wang,
Shi-Liang Huang*, Zhi-Shu Huang
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s
Republic of China
*To whom correspondence should be addressed. Tel.: 8620- 39943053, Fax.: 8620-39943056,
e-mail: lsshsl@mail.sysu.edu.cn.
Abstract
For the development of novel anticancer agents, we designed and synthesized a total of 37
perimidine o-quinone derivatives containing the o-quinone group at the A or B ring and different
substituents (alkyl groups, aryl groups or heterocycles) at the C ring of the compounds. The
structure-activity relationships (SARs) were established based on the cytotoxicity data of
compounds from the HL-60, Huh7, Hct116, and Hela cell lines. The cytotoxicity results showed
that most compounds exhibited potent cytotoxicity. In particular, compound b-12 showed the best
anti-proliferative activity (IC50 ≤ 1 M) against four cancer cell lines and strong potency against
the HL-60/MX2 (0.47 M) cell line, which is resistant to Topo II poisons. Further studies showed
that b-12 exhibited potent Topo IIα inhibitory activity (IC50 = 7.54 M) compared with Topo I,
which acted as a class of non-intercalative Topo IIα catalytic inhibitor by inhibiting the ATP
binding site of Topo II. Cell apoptosis and cell cycle assays confirmed that b-12 could induce the
apoptosis of Huh7 cells in a dose-dependent manner.
Keywords: Topoisomerase II; Anti-proliferation; Catalytic inhibitor; Perimidine o-Quinone;
Mansonone; Apoptosis

1. Introduction
Human topoisomerase (Topo) has been regarded as an important target in anticancer drug
discovery for more than 30 years. Topo can transiently interrupt one or both strands of DNA to
solve the topology that arises in double-stranded DNA, which is crucial for the replication,
transcription and other cellular transactions of DNA in cell life [1]. Numerous researchers have
been focusing on the search for promising anticancer agents targeting at Topo II because of its
high expression levels in proliferating cancer cells, which is essential to cancer cell survival.
On the basis of different mechanisms of action, Topo II-targeting agents could classified into
two major classes, namely, “Topo II poisons” and “Topo II catalytic inhibitors”. Poisons, such as
etoposide (VP-16), are believed to stabilize the drug-Topo-DNA cleavable complexes and induce
DNA breakage. Agents acting on any other steps in the catalytic cycle are called catalytic
inhibitors[2]. For example, Topo II catalytic inhibitors interfere with the binding between DNA
and Topo II (aclarubicin [3, 4]), inhibit ATP binding (novobiocin), block DNA breakage
(merbarone) [2], or hinder the ATP hydrolysis (ICR-187 and structurally related
bisdioxopiperazine derivatives) [5]. To date, Topo II poisons, such as VP-16 and doxorubicin,
have been extensively and effectively used in clinical oncology. However, these poisons present
numerous side effects, such as frequent tumor recurrence and resistance, especially cardiotoxicity
and secondary malignancies, which substantially limits their application [6, 7]. Therefore, Topo
II catalytic inhibitors are superior to Topo II poisons for clinical cancer therapy [2].
HO
O
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
-lapachone
Duinnone
β
Mansonone F
Salvicine
Mansonone E
HN
NH
O
N
OH
OH
N
NH
NH
N
NH
O
N
N
N
N
NH
N
1
2
3

Figure 1. The structures of o-quinone compounds and perimidine derivatives.
O
O
A
B
R3
N
NH
C
N
N
R2
R
R1
2-substituted Perimidines
(show potent cytotoxicity)
Series A
Fusion
O
O
O
O
A
C
B
N
N
R2
O
R1
Series B
Mansonone F
(Topo II catalytic inhibitor)
Figure 2. Design of novel perimidine o-quinone agents as potential Topo II catalytic
inhibitors
o-Quinone-containing compounds have attracted considerable research attention [8].
Compounds such as salvicine [9], β-lapachone [10], dunione and mansonone [11], all showed
potent antitumor activity and strong Topo II inhibitory effects. The SARs confirmed the
importance of the o-quinone moiety to the cytotoxicity against cancer cell lines of these
compounds. Furthermore, the structure of perimidines leads to their consideration as potential
antitumor compounds, and a large number of 2-substituted perimidines (1, 2) [12, 13] and some
other perimidine derivatives (3) [14] (Figure 1), which all showed potent cytotoxicity against
different cancer cell lines, have been designed and synthesized. However, the mechanism of
action of these perimidine derivatives has yet to be elucidated.
In an effort to discover novel anticancer agents as non-intercalative Topo II catalytic inhibitors,
37 novel perimidine o-quinone derivatives were designed by the combination of the perimidine
skeleton and o-quinone structure (Figure 2). The synthesized compounds were evaluated for their
antiproliferation against four cancer cell lines and inhibitory activity against Topo enzymes. DNA
unwinding, Topo II cleavage, and ATP-dependent relaxing assays were carried out to investigate
the inhibitory mechanisms of compound b-12.

2. Chemistry
In this study, 37 perimidine o-quinone derivatives (a-1 to a-17, b-1 to b-17, and d-1 to d-3)
were designed and synthesized, and the general structures are shown in Table 1. These compounds
were prepared following the procedures described in Scheme 1. Intermediates I (I-1 to I-17) were
easily prepared in high yields by refluxing a mixture of sodium pyrosulfite, an aldehyde and 1,8-
diaminonaphthalene in ethanol [15]. Then, intermediates I and CH3I or CH3CH2I were reacted in
DMF to give the methylated or ethylated products II (II-1 to II-17). Intermediate II was subjected
to NaNO2 in the presence of acetic acid to yield two nitro intermediates IIIa (6-nitro substituent)
and IIIb (7-nitro substituent), which were used without further purification. The nitro group of
the two intermediates was reduced to the corresponding amines IVa and IVb and further oxidized
by Fremy’s salt to yield the two series isomers (series A and series B) of target products, which
could be separated by column chromatography through a gradient elution using dichloromethane:
methanol (100:1~50:1).
O
7
NH2
6
NO2
8
O
5
4
9
1
N
2
N
3
R2
N
N
N
N
R2
R2
R1
R1
R1
IIIa
IVa
Series A
a-1~a-17
7
8
e
d
a
b
c
O
NH2
NO2
6
5
O
NH2 NH2
N
NH
N
N
R2
9
R1
R1
4
1
N
N
N
N
N
N
R2
2
I-1~I-17
II-1~II-17
R2
R2
3
R1
R1
R1
IVb
IIIb
Series B
b-1~b-17
O
R1 (R2= -CH3)
Compd. R1 (R2= -CH3)
O
Compd.
a-11, b-11
a-12, b-12
a-13, b-13
a-14, b-14
a-15, b-15
a-16, b-16
a-17, b-17
d-1, d-2
Cl
a-1, b-1
a-2, b-2
a-3, b-3
a-4, b-4
a-5, b-5
a-6, b-6
a-7, b-7
a-8, b-8
a-9, b-9
3-ClC6H4
4-ClC6H4
2-FC6H4
-CH3
f
O
N
N
O
-CH2CH3
-Cyclopropyl
-CH2C6H5
-C6H5
R2
O
R1
d-2
3-FC6H4
4-FC6H4
g
N
N
O
2,4-di-FC6H3
2,3,4-tri-FC6H2
2-ClC6H4
-CH2CH3
4-CH3C6H4
4-OCH3C6H4
2-Thienyl
2-ClC6H4
R2
R1
Br
N
N
d-3
R2
R1
R1= 2-ClC6H4 R2= -CH2CH3
a-10, b-10
d-1,d-3
Scheme 1. Reagents and conditions: (a) RCHO, Na2S2O5, EtOH, reflux; (b) CH3I or CH3CH2I,
NaOH, DMF, 80 oC; (c) NaNO2, AcOH, rt; (d) H2, Pd/C, Ethyl acetate, 50 oC; (e) Acetone, 0.06
M KH2PO4, Fremy's salt, 10.4%-44.4% (three-steps); (f) NCS, BPO, CHCl3, 80 oC, 63%; (g) NBS,
BPO, CHCl3, 80 oC, 71%-77.5%.
Furthermore, substitutions at the C-9 position of several series B derivatives were obtained
by different methods. Chloro-substituted and bromo-substituted perimidine o-quinone derivatives

were obtained by treatment with N-chlorosuccinimide and N-bromosuccinimide, respectively.
All new compounds were confirmed by chromatographic (HPLC) and spectroscopic (NMR
and HRMS) methods. As an example, the additional HMQC and HMBC spectra of the
compounds a-1 and b-1 were provided to prove the position of o-quinone group (SI file).
3. Results and Discussion
3.1 In Vitro Cytotoxicity
The synthesized compounds were evaluated for their cytotoxicity against four tested cancer
cell lines including HL-60, Huh7, Hct116 and Hela, by using the MTT assay. VP-16 was used as
a positive control. The inhibitory activities (IC50) of the tested compounds are shown in Table 1.
As is evident from the IC50 values, most compounds showed stronger sensitivity against the
suspended cell line ( HL-60 ) than the attached cell lines (Huh7, Hct116, and Hela) and stronger
cytotoxicity than the positive control (VP-16). Among them, compounds b-12 and b-17 showed
the best anti-proliferation against the four cancer cell lines with IC50 values lower than 1 μM. To
gain insight into involvement of Topo II inhibition in the cytotoxicity of b-12 and b-17, we
assessed the anti-proliferative activity by using the HL-60/MX2 cell line, which is resistant to
mitoxantrone and shows altered catalytic activity and reduced levels of Topo II. HL-60/MX2 cells
generally remain sensitive to Topo II catalytic inhibitors and show resistance to Topo II poisons.
The MTT assay demonstrated that b-12 and b-17 retained activity in the HL-60/MX2 cell line.
The IC50 values of b-12 and b-17 were less than 1 µM, and their resistance indices (HL-
60/MX2/HL-60) were 1.5 and 2.6, respectively. However, the IC50 value of VP-16 was 0.52 µM
for HL-60 and 10.3 µM for HL-60/MX2, so the resistance index (HL-60/MX2/HL-60) was 19.8.
These results implied that our compounds were Topo II catalytic inhibitors.
3.2 SARs based on the cytotoxicity data
(1) When R1 was substituted by an alkyl, the compounds possessing o-quinone at ring A
(series A) showed better cytotoxicity than those substituted at ring B (series B). On the other hand,
the compounds (a-9, b-9, a-10, and b-10) with the R2 substituted by methyl or ethyl groups
exhibited similar cytotoxicity.

Table 1. IC50 values () of perimidine o-quinone derivatives against cancer cells and inhibition
rate (I.R.%) of Topo II
IC50 (µM)a
Hct116
Topo II
I.R(%)b
(5μΜ)
20
Compd
R1
R2
R3
HL-60
Huh7
Hela
HL-60/MX2
a-1
b-1
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH2CH3
-CH2CH3
CH3
-
0.73±0.11
1.32±0.31
0.80±0.18
2.91±0.12
0.67±0.11
0.83±0.11
0.84±0.15
0.78±0.09
3.72±0.29
0.64±0.09
3.73±0.49
1.71±0.16
1.23±0.24
0.43±0.06
1.45±0.32
0.77±0.15
0.54±0.13
0.49±0.09
0.86±0.07
0.54±0.11
0.87±0.17
0.63±0.10
1.30±0.19
0.31±0.08
1.12±0.26
0.36±0.07
1.18±0.28
0.41±0.12
2.58±0.43
1.01±0.19
0.69±0.16
0.47±0.06
0.72±0.16
0.36±0.10
0.80±0.20
9.14±3.67
1.93±0.16
3.87±0.56
3.87±0.27
5.45±1.37
1.70±0.17
0.81±0.13
1.13±0.15
1.20±0.29
1.81±0.20
2.78±0.63
10.8±2.65
2.30±0.18
5.89±1.13
2.71±0.32
2.54±0.24
3.01±0.73
1.85±0.38
1.03±0.13
2.46±0.47
2.47±0.26
1.13±0.11
0.45±0.05
7.22±1.80
1.69±0.15
3.14±0.85
1.44±0.14
1.65±0.27
1.28±0.32
2.51±0.41
1.99±0.60
3.48±0.44
0.51±0.15
1.88±0.21
4.87±0.38
6.59±1.11
12.9±1.92
3.74±0.33
18.6±4.00
2.48±0.87
5.41±0.76
1.86±0.30
3.17±1.00
3.14±1.15
4.40±0.42
3.35±0.63
2.39±0.20
3.29±0.84
1.83±0.30
6.24±1.30
2.96±0.77
4.34±1.27
1.70±0.60
1.21±0.08
1.45±0.82
1.22±0.27
1.59±0.43
1.56±0.37
0.63±0.20
2.18±0.15
0.97±0.20
4.34±1.80
2.39±0.69
2.35±0.03
1.46±0.58
1.20±0.56
1.78±0.24
0.96±0.10
1.38±0.37
2.14±0.48
0.43±0.11
1.04±0.40
2.90±0.79
3.91±0.70
13.5±1.22
2.27±0.33
14.7±3.09
1.34±0.40
7.67±1.00
4.32±0.72
6.15±1.10
2.01±0.48
1.63±0.84
1.88±0.89
1.45±0.47
5.01±1.98
3.06±0.70
8.48±1.53
3.28±0.62
6.59±1.47
14.13±3.96
2.99±0.66
2.58±0.22
3.00±0.68
1.41±0.50
2.86±0.63
1.52±0.55
2.14±0.21
1.08±0.35
16.6±4.16
2.39±0.35
2.81±0.64
2.09±0.88
2.20±0.78
1.24±0.24
3.50±1.15
3.17±1.27
4.77±1.50
0.69±0.17
2.22±0.68
7.09±1.47
5.97±1.36
18.7±5.17
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
23
a-2
-CH2CH3
-CH2CH3
-
< 20
< 20
30
b-2
-
a-3
-
b-3
-
< 20
54
a-4
-CH2-C6H5
-CH2-C6H5
-C6H5
-
b-4
-
55
a-5
-
57
b-5
-C6H5
-
57
a-6
4-CH3C6H4-
4-CH3C6H4-
4-OCH3C6H4-
-
< 20
45
b-6
-
a-7
-
< 20
< 20
< 20
< 20
42
b-7
4-OCH3C6H4-
-
S
a-8
-
S
b-8
-
a-9
2-ClC6H4-
2-ClC6H4-
2-ClC6H4-
2-ClC6H4-
3-ClC6H4-
3-ClC6H4-
4-ClC6H4-
4-ClC6H4-
2-FC6H4-
2-FC6H4-
-
b-9
-
< 20
< 20
53
a-10
b-10
a-11
b-11
a-12
b-12
a-13
b-13
a-14
b-14
a-15
b-15
a-16
b-16
a-17
b-17
d-1
-
-
-
38
CH3
-
33
CH3
-
50
CH3
0.47±0.08
60
CH3
-
52
CH3
-
36
3-FC6H54
-
CH3
-
39
3-FC6H4-
4-FC6H4-
CH3
-
51
CH3
-
< 20
31
4-FC6H4-
CH3
-
2,4-di-FC6H3-
2,4-di-FC6H3-
2,3,4-tri-FC6H2-
2,3,4-tri-FC6H2-
2-ClC6H4-
2-ClC6H4-
-CH2CH3
CH3
-
32
CH3
-
32
CH3
-
43
CH3
0.93±0.15
69
CH3
-Br 0.71±0.11
-Cl 3.27±0.46
-Br 0.65±0.06
-
54
d-2
CH3
-
37
d-3
CH3
-
61
VP-16
-
-
-
0.52±0.13
10.3±2.54
85

a The IC50 represents the compound concentration yielding 50% survival of each cell line after 48h.
b The inhibition rate (I.R. %) is calculated base on optical density analysis of agarose gel electrophoretogram of the Topo II mediated
DNA relaxation assay in the presence of different compounds (5 μM) (Figure S1 of SI file).
(2) In general, most compounds displayed poor cytotoxicity activity when R1 wasreplaced by
an aryl moiety substituted with electron-donating group (4-CH3OC6H4 and 4-CH3C6H4) and an
electronically rich heterocyclic group (thiophene). By contrast, when R1 was replaced by an aryl
moiety substituted with electron-withdrawing group (chlorophenyl, and fluorophenyl), most of
the compounds showed better cytotoxicity. Similarly, the SARs of the compounds in the Topo II
inhibitory activity (except b-6 and a-15) are similar to the rules obtained on the cell, and the
compounds b-12 and b-17 are excellent in both enzyme inhibition and cytotoxicity.
(3) The cytotoxicity of series B compounds in the HL-60 cell line was better than that of the
series A compounds. However, this trend was not observed for the other three cell lines.
Compounds b-12 (R1 p-chlorophenyl) and b-17 (R1 = trifluorophenyl) showed the best anti-
=
proliferative activity against the four cancer cell lines. However, unlike the SARs at the cellular
level, there is no obvious rule for the inhibitory activity of the A series and B series compounds
on the enzyme, which may be related to the complexity of the action of the compounds in the cell
and specificity and selectivity of the compounds against Topo II.
(4) Furthermore, to investigate the effect of different substitutions at the C-9 position on the
inhibitory activity of cancer cells, several chlorine or bromine replacement compounds were
synthesized. Compounds (d-1, d-2 and d-3) substituted at the C-9 position exhibited similar or
weaker activity compared with the corresponding unsubstituted compounds (a-9 and a-2).
3.3 Compound b-12 As a Topo II Inhibitor
The conversion of supercoiled plasmid DNA to relaxed DNA by Topo I and IIα in the
presence of the prepared perimidine o-quinone derivatives (b-12 and b-17) was evaluated as
shown in Figure 3. Camptothecin (CPT) and VP-16, respectively known as Topo I and Topo IIα
inhibitors, were used as positive controls. The results (Figure 3A) showed that b-12 and b-17
exhibited stronger Topo IIα inhibitory activity compared with the positive control. The IC50 values
of the Topo IIα inhibition of b-12 and b-17 were 7.54 and 4.68 μM, respectively. However, in the
Topo I relaxation and cleavage assay (Figures 3B and 3C), compound b-12 did not show Topo I
inhibitory activity even at a high concentration, indicating that our compounds are a class of

selective Topo II inhibitors. Because of the poor solubility of b-17, which will affect the follow-
up experiment, b-12 was selected as a candidate compound for further study.
Figure 3. (A) Topo IIα inhibitory activity of compounds b-12 and b-17 at different concentrations
and VP-16 as a positive control. Supercoiled pBR322 plasmid was incubated with Topo IIα in the
presence or absence of the indicated compounds at 37 °C for 30 min. (B) Topo I inhibitory activity
of compound b-12 at the concentrations of 200, 100, 50, 25, and 12.5 μM. Supercoiled pBR322
plasmid was incubated with Topo I in the presence or absence of the indicated compounds at
37 °C for 30 min. (C) Effect of b-12 on the Topo I–DNA cleavage complex formation. Lanes 1
to 2: control group of supercoiled pBR322 without or with excess Topo I; Lanes 3 to 6: effect of
CPT (10 and 20 μM) and b-12 (10 and 20 μM) on excess Topo I with supercoiled pBR322. The
positions of supercoiled DNA (Sc), relaxed DNA (Rx), and nicked DNA (N) are indicated.
3.4 Compound b-12 As a non-Intercalative Topo II Catalytic Inhibitor
3.4.1 Compound b-12 As a Topo II Catalytic Inhibitor
Topo II poisons exhibit activity by stabilizing Topo II-DNA covalent complexes in vitro and
in vivo and causing the formation of linear DNA [16, 17]. As shown in Figure 4A, VP-16 induced
linear DNA formation at 100 µM. By contrast, b-12 did not generate linear DNA at 20 µM. With
VP-16 and b-12 co-treatment, b-12 did not markedly reduce the amount of linear DNA caused
by VP-16, suggesting that b-12 may act on the step after formation of Topo II-DNA complexes.
γ-H2AX is considered as a marker of DNA double-strand breaks (DSBs) [18], so the ability of b-
12 to induce DNA damage in the Huh7 cell line was also examined to confirm this mode of

inhibition by immunofluorescence assay. The results (Figure 4B) showed that VP-16 (20 µM)
induced DNA DSBs, whereas b-12 (2 µM) did not induce γ-H2AX formation. These findings are
consistent with the results obtained from the cleavage assay. The two experiments indicated that
b-12 may not be a Topo II poison but a catalytic inhibitor.
Figure 4. (A) Effect of b-12 on Topo II–DNA cleavage complex formation. Lanes 1 to 2: control
group of supercoiled pBR322 without or with excess Topo II; Lanes 3 to 5: effect of VP-16 (100
μM), b-12 (20 μM) and VP-16 (100 μM) and b-12 (20 μM) together on excess Topo II with
supercoiled pBR322. The positions of supercoiled DNA (Sc), relaxed DNA (Rx), linear DNA (L)
and nicked DNA (N) are indicated. (B) Immunofluorescence assay. Images of blank (nontreated),
DMSO, VP-16 (Topo II poison), and compound b-12 in treated Huh7 cells. (C) The unwinding
assay of b-12, where the positions of supercoil (Sc) and relax (Rx) DNA are indicated. (D)
Absorption spectra of b-12 upon addition of CT-DNA . [b-12] = 40 , [DNA] = 0 - 80 . The
arrow indicates the absorption change upon increasing the amount of CT-DNA . (E) Insert graph
shows the plot of [DNA]/( εa- εf) versus [DNA].
3.4.2 The Binding Properties between Compound b-12 and DNA

The unwinding effect is caused by certain compounds intercalating into the DNA, and the
effect may yield false positive outcomes in the Topo-mediated DNA relaxation assay. Therefore,
the DNA unwinding assay was performed using eukaryotic DNA (CT-DNA) with Topo I to
identify whether compounds intercalate into the DNA. As shown in Figure 4C, ethidium bromide
(EB), a classic DNA intercalator, intercalated into the plasmid DNA by converting relaxed DNA
to supercoiled DNA in a dose-dependent manner (0.5-2.5 µM) and was selected as a positive
control. By contrast, compound b-12 did not change the amount of supercoiled DNA even at a
high concentration (20 µM) compared with the positive control. This finding provides evidence
that b-12 did not intercalate into DNA and is therefore not a DNA intercalator.
In addition, a UV-titration assay was conducted to measure the binding ability of b-12 to CT-
DNA. If the compound intercalated into DNA, the UV curve of the compound would exhibit
hypochromicity and bathochromic shift. In Figure 4D, significant hypochromicity was not
observed with increasing DNA concentration, and the analysis revealed that the binding constant
(Kapp) of b-12 with DNA was 1.3×104 M-1. Taken together, these findings indicate that b-12 is a
nonintercalative Topo II catalytic inhibitor.
3.5 Compound b-12 May Target the ATPase of Topo II
To study the interaction between compounds and the ATPase domain of Topo II, we first
conducted an ATP competitive assay. VP-16 is a Topo II poison that does not bind to the ATPase
domain, so the inhibitory activity would not change with increasing ATP concentrations in Topo
II-mediated DNA relaxation (Figure 5A). The results of Figure 5B showed that the inhibitory
activity of b-12 decreased with increasing ATP concentrations from 0.5 mM to 4.5 mM, and the
corresponding inhibitory activity IC50 value for Topo II increased from approximately 2.5 μM to
exceed 20 μM. These results revealed that b-12 may act as an ATP competitive inhibitor by
blocking the ATP-binding site of the enzyme.
Meanwhile, a docking study was carried out for compound b-12 in the ATP-binding domain
of human topoisomerase IIα [19] (PDB code : 1ZXM) by using Glide of Schrodinger. As seen
from the images (Figure 5C, D), the residues that are in close contact with b-12 are Asn120,
Thr215, Asn91, Asn95, Lys123, Gly124, Ile125, Ile141, Phe142, Ser149, Asn150, Ala167,
Lys168. Hydrogen bonding interaction occurs between the oxygen atoms of C6 and Asn120, C5

and Thr215, whereas the C-2 phenyl of compound b-12 could form an interaction with Mg2+. In
addition, compound b-12 and ATP could overlap well, and this finding could further explain that
our compounds may target the ATPase domain of Topo IIα.
Figure 5. (A) The effects of VP-16 on DNA relaxation catalyzed by Topo II at 37 °C for 30 min
with 0.5 mM, 1.5 mM and 4.5 mM ATP. (B) The effects of b-12 on DNA relaxation catalyzed by
Topo II at 37 °C for 30 min with 0.5 mM, 1.5 mM and 4.5 mM ATP. (C) Interaction of compound
b-12 and the ATPase domain of topoisomerase IIα was displayed in the 2D diagram. (D) Overlay
of compound b-12 and ATP. The compound and the protein binding site are represented as sticks.
Compound b-12 and ATP are shown in green and pink, respectively.
3.6 Compound b-12 Induces Cell Apoptosis
Apoptosis is the process of programmed cell death, which involves a series of biochemical
events. We carried out cell apoptosis and cycle arrest assays to study the activity of b-12 on the
apoptosis of Huh7 cells. From the results (Figure 6A) of the cell apoptosis assay, the percentage
of apoptotic cells substantially increased in a dose-dependent manner, when cells were incubated
with b-12 (0.375, 0.5, 0.75, and 1 µM). DNA fragmentation could be caused by cell apoptosis,
which is expressed as an apoptotic peak of Sub-G1 in the cell cycle assay. In Figure 6B, the
percentage of Sub-G1 phase evidently increased at a b-12 concentration of 0.75 µM, whereas VP-

16 could block the cell cycle in the G2/M phase. Topo II poisons can increase the level of DNA-
enzyme-drug cleavage complexes. This DNA break can trigger recombination/repair pathways in
G2 phase, thus showing G2/M phase arrest in cell cycle assay. However, Topo II catalytic
inhibitor can stabilize DNA in the state of enzyme locking and block the cleavage/replication
cycle of DNA, are not able to untangle daughter chromosomes and ultimately die of mitotic failure
[20]. So compound b-12 is no obvious effect on specificity of cell cycle. Taken together, all the
results suggested that our compounds suppressed Huh7 cell proliferation by inducing apoptosis.
Figure 6. (A) Apoptotic cells were detected with Fluorescein isothiocyanate (FITC) Annexin V/
propidium iodide (PI) double staining after incubation of Huh7 cell with compound b-12 for 24
h. (B) Cell arrest of Huh7 cell treated with b-12 for 24 h.
4. Conclusion
A series of novel perimidine o-quinone derivatives was designed, synthesized and evaluated
for cytotoxicity against four human cancer cell lines. MTT assays showed that most prepared
compounds exhibited potent cytotoxicity, and the SARs showed that the substitution of an
electron-withdrawing group (chlorophenyl or fluorophenyl) on the C-2 position and the location
of o-quinone in ring A or ring B were critical for the anti-proliferative activity of cancer cells.
Among these compounds, b-12 showed the best cytotoxicity against several cancer cell lines and
strong potency against the HL-60/MX2 (0.47 μM) cell line, which is resistant to Topo II poisons.
Moreover, b-12 showed good Topo IIα inhibitory activity (IC50 = 7.54 μM). Further mechanistic

studies indicated that b-12 was a non-intercalative Topo IIα catalytic inhibitor. The ATP
competitive assay and docking study revealed that b-12 may target the ATP binding site of Topo
II. The cell apoptosis assay and cell cycle assay confirmed that b-12 showed strong anti-
proliferation against Huh7 cells by inducing apoptosis in a dose- dependent manner.
The present results indicate that perimidine o-quinone derivatives are potential novel
anticancer agents. Given that Topo II is an important target for cancer chemotherapy, these
findings may provide advanced opportunities for the design and development of new
chemotherapy agents.
5. Experimental Section
5.1. General
Compounds used as starting materials and reagents were from aladdin, innochem or other
chemical companies and used without further purification. 1HNMR, 13CNMR, HMBC or HMQC
spectra were recorded using TMS as the internal standard in CDCl3 or DMSO-d6 with a Bruker
BioSpin GmbH spectrometer at 400 MHz. High resolution mass spectra (HRMS) were recorded
on Shimadzu LCMS-IT-TOF. HPLC analysis showed that the purity of all the compounds was
proved to be more than 95% by using analytical HPLC.
5.2. Procedure for the Preparation of Intermediate I
Sodium pyrosulfite (10 mmol) was dissolved in water (20 ml), 1,8-diaminonaphthalene (20
mmol) was dissolved in ethanol (100 ml) and the aldehyde (10 mmol) were successively added.
The mixture was heated under reflux for 4 h. After completion of the reaction, the reaction mixture
was warmed to room temperature and poured into the ice water. Solid would appear and filter,
then the filtrate was used without further purification.
5.3. Procedure for the Preparation of Intermediate II
Intermediate I (10 mmol) and NaOH (20 mmol) were dissolved in dry DMF, CH3I or
CH3CH2I (15 mmol) was successively added. The reaction mixture was stirred at 80 °C for 4 h in
the pressure tube. After completion of the reaction, the mixture was extracted with ethyl acetate,
washed with saturated salt water for three times and concentrated to provide black yellow oil

product, which were used for further purification with column chromatography to give the product.
The 1H NMR spectra of II-1 to II-17 were shown in SI.
5.4. Procedure for the Preparation of targeted Compounds (a-1～a-17, b-1～b-17)
Firstly, above intermediate II (10 mmol) and NaNO2 (10 mmol) were dissolved in 15 mL
acetic acid, which was stirred in the room temperature for 30 min. The reaction solution changed
quickly from yellow to red and detected by TLC. Then the reaction mixture was concentrated
directly by using rotary evaporator to give the red intermediate III withuot further purification.
Then, a solution of intermediate III (20 mmol) in ethyl acetate (30 mL) was added to Pd/C (2
mmol), which was stirred at 50 °C in an atmosphere of H2 until the reaction solution changed from
red to yellow. After the reaction was completed, ethyl acetate was evaporated, then the product
(intermediate IV) was used for next step without further purification. Finally, the intermediate IV
(1 mmol) was dissolved in acetone (60 mL) and a solution of Fremy’s salt (2.5 mmol) in 0.06-M
KH2PO4 solution (40 mL) was added with vigorous stirring. After stirring for 30 min, the mixture
was extracted by using chloroform with three times. The combined extracts were washed with
saturated salt water (three times), dried over Na2SO4, filtered and evaporated to dryness. The solid
residue was purified to give a pair of isomers (purple and red solid).
1
1,2-Dimethyl-1H-perimidine-7,8-dione (a-1): Red solid, Yield 24%. H NMR (400 MHz,
DMSO-d6) δ 7.97 (dd, J = 5.4, 3.2 Hz, 1H), 7.80 – 7.74 (m, 2H), 5.84 (s, 1H), 3.52 (s, 3H), 2.60
(s, 3H).13C NMR (101 MHz, DMSO-d6) δ 180.7, 177.2, 155.8, 151.7, 142.3, 134.3, 133.6, 129.7,
129.6, 117.9, 99.8, 36.6, 24.1. HRMS (ESI): calcd for C13H10N2O2 [M+H]+ 227.0815, found
227.0809. HPLC purity: 99.6%.
1,2-Dimethyl-1H-perimidine-5,6-dione (b-1): Purple solid, Yield 21%. 1H NMR (400 MHz,
DMSO-d6) δ 7.99 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 7.3 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 5.74 (s,
1H)，3.74 (s, 3H), 2.58 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 182.0, 177.8, 160.4, 158.1,
137.7, 133.7, 130.0, 128.3, 123.6, 116.5, 108.9, 35.8, 24.4. HRMS (ESI): calcd for C13H10N2O2
[M+H]+ 227.0815, found 227.0811. HPLC purity: 99.1%.
2-Ethyl-1-methyl-1H-perimidine-7,8-dione (a-2): Red solid, Yield 27%. 1H NMR (400 MHz,

DMSO-d6) δ 7.97 (dd, J = 6.3, 2.3 Hz, 1H), 7.84 – 7.73 (m, 2H), 5.84 (s, 1H), 3.51 (s, 3H), 2.92
(q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 180.7, 177.2,
158.6, 151.9, 142.1, 134.5, 133.6, 129.6, 129.6, 117.8, 99.8, 35.7, 28.6, 11.2. HRMS (ESI): calcd
for C14H12N2O2 [M+Na]+ 241.0947, found 241.0944. HPLC purity: 99.5%.
1
2-Ethyl-1-methyl-1H-perimidine-5,6-dione (b-2): Purple solid, Yield 23.5%. H NMR (400
MHz, DMSO) δ 7.98 (dd, J = 19.9, 8.0 Hz, 2H), 7.85 – 7.80 (m, 1H), 5.77 (s, 1H), 3.75 (s, 3H),
2.89 (q, J = 7.4 Hz, 2H), 1.25 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 182.0, 177.8,
163.3, 158.1, 137.9, 133.7, 130.0, 128.4, 123.7, 116.4, 109.2, 35.1, 29.0, 11.3. HRMS (ESI): calcd
for C14H12N2O2 [M+H]+ 241.0966, found 241.0972. HPLC purity: 98.2%.
1
2-Cyclopropyl-1-methyl-1H-perimidine-7,8-dione (a-3): Red solid, Yield 10.4%. H NM R
(400 MHz, DMSO) δ 7.95 (dd, J = 6.9, 1.5 Hz, 1H), 7.80 – 7.69 (m, 2H), 5.87 (s, 1H), 3.72 (s,
3H), 2.32 (m, 1H), 1.18 – 1.02 (m, 4H).13C NMR (101 MHz, CDCl3) δ 180.5, 177.7, 156.9, 152.2,
142.1, 134.4, 133.0, 130.2, 129.4, 117.7, 99.8, 35.4, 15.1, 8.6 (2C). HRMS (ESI): calcd for
C15H12N2O2 [M+H]+ 253.0972, found 253.0970. HPLC purity: 100%.
2-Cyclopropyl-1-methyl-1H-perimidine-5,6-dione (b-3): Purple solid, Yield 16.4%. 1H NMR
(400 MHz, DMSO) δ 8.00 (d, J = 8.5 Hz, 1H), 7.93 (d, J = 7.3 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H),
5.68 (s, 1H), 3.92 (s, 3H), 2.34 (d, J = 4.7 Hz, 1H), 1.47 – 1.01 (m, 4H). 13C NMR (101 MHz,
DMSO) δ 182.1, 177.5, 163.1, 158.1, 138.0, 133.7, 129.9, 128.1, 123.6, 116.4, 108.8, 35.3, 14.7,
10.2 (2C). HRMS (ESI): calcd for C15H12N2O2 [M+H]+ 253.0972, found 253.0975. HPLC purity:
99.6%.
1
2-Benzyl-1-methyl-1H-perimidine-7,8-dione (a-4): Red solid, Yield 28%. m.p. 173.3- H
NMR (400 MHz, DMSO) δ 8.03 (dd, J = 7.0, 1.5 Hz, 1H), 7.86 (dd, J = 8.1, 1.5 Hz, 1H), 7.84 –
7.79 (m, 1H), 7.41 – 7.22 (m, 5H), 5.84 (s, 1H), 4.35 (s, 2H), 3.44 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 180.2, 177.7, 154.9, 152.1, 141.8, 134.7, 134.3, 133.2, 130.8, 129.5, 129.4 (2C), 128.1

(2C), 127.8, 118.2, 100.0, 43.0, 36.1. HRMS (ESI): calcd for C19H14N2O2 [M+H]+ 303.1128,
found 303.1128. HPLC purity: 97.9%.
1
2-Benzyl-1-methyl-1H-perimidine-5,6-dione (b-4): Purple solid, Yield 26%. H NMR (400
MHz, DMSO) δ 7.94 (d, J = 7.4 Hz, 2H), 7.88 – 7.78 (m, 1H), 7.32 (d, J = 26.5 Hz, 5H), 5.81 (s,
1H), 4.30 (s, 2H), 3.68 (s, 3H). 13C NMR (101MHz, DMSO) δ 181.7, 178.0, 160.9, 157.7, 137.9,
135.8, 133.7, 130.1, 129.3 (2C), 129.0 (2C), 128.6, 127.5, 123.8, 116.7, 109.6, 42.1, 35.9. HRMS
(ESI): calcd for C19H14N2O2 [M+H]+ 303.1128, found 303.1120. HPLC purity: 95.9%.
1
1-Methyl-2-phenyl-1H-perimidine-7,8-dione (a-5):Red solid, Yield 41.4%. H NMR (400
MHz, CDCl3) δ 8.24 (dd, J = 7.4, 1.0 Hz, 1H), 7.89 (dd, J = 8.1, 0.9 Hz, 1H), 7.74 (t, J = 7.8 Hz,
1H), 7.66 – 7.56 (m, 5H), 5.96 (s, 1H), 3.50 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 179.1, 176.8,
154.9, 151.1, 140.9, 133.9, 133.4, 132.3, 129.9, 129.8, 128.4, 128.2 (2C), 127.2 (2C), 117.0, 99.5,
38.0. HRMS (ESI): calcd for C18H12N2O2 [M+H]+ 289.0972, found 289.0974. HPLC purity:
99.1%.
1-Methyl-2-phenyl-1H-perimidine-7,8-dione (b-5):Purple solid, Yield 44.4%. 1H NMR (400
MHz, DMSO) δ 8.02 (d, J = 7.9 Hz, 2H), 7.96 – 7.84 (m, 1H), 7.71 (d, J = 5.9 Hz, 2H), 7.59 (d,
J = 6.9 Hz, 3H), 5.84 (s, 1H), 3.63 (s, 3H). 13C NMR (101 MHz, DMSO) δ 181.6, 178.0, 160.1,
157.6, 138.1, 135.2, 133.9, 131.1, 129.9, 129.3 (2C), 129.0 (2C), 128.9, 124.5, 116.8, 109.9, 38.7.
HRMS (ESI): calcd for C18H12N2O2 [M+H]+ 289.0972, found 289.0966. HPLC purity: 98.0%.
1
1-Methyl-2-(p-tolyl)-1H-perimidine-7,8-dione (a-6): Red solid, Yield 30%. H NMR (400
MHz, DMSO) δ 8.04 (dd, J = 7.1, 1.3 Hz, 1H), 7.85 (dd, J = 8.1, 1.3 Hz, 1H), 7.83 – 7.78 (m,
1H), 7.57 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 5.86 (s, 1H), 3.39 (s, 3H), 2.39 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 180.2, 177.8, 156.2, 152.2, 142.0, 141.3, 135.0, 133.3, 131.5,
130.8, 129.8 (2C), 129.4, 128.2 (2C), 118.0, 100.5, 39.1, 21.5. HRMS (ESI): calcd for C19H14N2O2
[M+H]+ 303.1128 found 303.1122. HPLC purity: 99.3%.

1-Methyl-2-(p-tolyl)-1H-perimidine-5,6-dione (b-6): Purple solid, Yield 29%. 1H NMR (400
MHz, DMSO) δ 8.01 (d, J = 7.9 Hz, 2H), 7.9 – 7.86 (t, J = 8 Hz 1H), 7.61 (d, J = 8.1 Hz, 2H),
7.39 (d, J = 8.0 Hz, 2H), 5.82 (s, 1H), 3.65 (s, 3H), 2.42 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
181.0, 178.3, 160.2, 157.3, 141.6, 137.5, 133.4, 131.4, 129.7 (3C), 128.9, 128.8 (2C), 122.4, 117.1,
111.4, 38.2, 21.6. HRMS (ESI): calcd for C19H14N2O2 [M+H]+ 303.1128, found 303.1121. HPLC
purity: 99.9%.
1
2-(4-Methoxyphenyl)-1-methyl-1H-perimidine-7,8-dione (a-7): Red solid, Yield 32.9%. H
NMR (400 MHz, DMSO) δ 8.05 (d, J = 7.0 Hz, 1H), 7.87 (d, J = 7.1 Hz, 1H), 7.85 – 7.80 (m,
1H), 7.67 (d, J = 8.6 Hz, 2H), 7.12 (d, J = 8.6 Hz, 2H), 5.88 (s, 1H), 3.86 (s, 3H), 3.45 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 180.2, 177.8, 161.5, 155.9, 152.4, 142.1, 134.8, 133.2, 130.6,
130.1 (2C), 129.4, 126.6, 117.8, 114.5 (2C), 100.5, 55.6, 39.3. HRMS (ESI): calcd for C19H14N2O3
[M+H]+ 319.1077, found 319.1073. HPLC purity: 99.6%.
2-(4-Methoxyphenyl)-1-methyl-1H-perimidine-5,6-dione (b-7): Purple solid, Yield 25.7%.
1H NMR (400 MHz, DMSO) δ 8.02 (d, J = 7.9 Hz, 2H), 7.89 (dd, J = 8.6, 7.3 Hz, 1H), 7.76 –
7.66 (m, 2H), 7.15 – 7.10 (m, 2H), 5.84 (s, 1H), 3.86 (s, 3H), 3.69 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 181.9, 177.9, 161.6, 160.1, 157.8, 138.5, 133.8, 131.6 (2C), 129.9, 128.7, 127.1, 124.5,
116.8, 114.4 (2C), 109.7, 56.0, 39.1. HRMS (ESI): calcd for C19H14N2O3 [M+H]+ 319.1077, found
319.1075. HPLC purity: 99.6%.
1-Methyl-2-(thiophen-2-yl)-1H-perimidine-7,8-dione (a-8): Red solid, Yield 14.4%. 1H NMR
(400 MHz, CDCl3) δ 8.19 (d, J = 7.3 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H),
7.63 (d, J = 4.8 Hz, 1H), 7.53 (d, J = 2.7 Hz, 1H), 7.24 – 7.18 (m, 1H), 5.97 (s, 1H), 3.73 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 180.1, 177.9, 152.5, 150.1, 141.9, 135.7, 134.9, 133.3, 131.0,
130.5, 130.3, 129.4, 127.7, 117.8, 100.9, 39.4. HRMS (ESI): calcd for C16H10N2O2S [M+H]+

295.0536, found 295.0534. HPLC purity: 99.1%.
1
1-Methyl-2-(thiophen-2-yl)-1H-perimidine-5,6-dione (b-8): Purple solid, Yield 12.4%. H
NMR (400 MHz, DMSO) δ 8.02 (dd, J = 8, 4 Hz, 3H), 7.86 (t, J = 7.2 Hz, 2H), 7.31 (s, 1H), 5.82
(s, 1H), 3.94 (s, 3H). 13C NMR (101 MHz, DMSO) δ 181.7, 177.8, 157.3, 154.1, 138.6, 137.0,
133.9, 133.7, 133.4, 129.9, 128.7, 128.7, 124.6, 116.8, 109.5, 38.8. HRMS (ESI): calcd for
C16H10N2O2S [M+H]+ 295.0536, found 295.0531. HPLC purity: 99.7%.
1
2-(2-Chlorophenyl)-1-methyl-1H-perimidine-7,8-dione (a-9): Red solid, Yield 32%. H
NMR (400 MHz, CDCl3) δ 8.27 (dd, J = 7.4, 1.2 Hz, 1H), 7.88 (dd, J = 8.1, 1.2 Hz, 1H), 7.80 –
7.73 (t, J =7.6 Hz, 1H), 7.62 – 7.43 (m, 4H), 5.95 (s, 1H), 3.39 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 180.0, 177.8, 153.5, 151.3, 141.8, 135.0, 133.8, 133.3, 132.4, 131.9, 131.2, 130.1, 130.0,
129.6, 128.1, 118.4, 100.4, 36.9. HRMS (ESI): calcd for C18H11ClN2O2 [M+H]+ 323.0582, found
323.0575. HPLC purity: 97.8%.
1
2-(2-Chlorophenyl)-1-methyl-1H-perimidine-5,6-dione (b-9): Purple solid, Yield 34%. H
NMR (400 MHz, CDCl3) δ 8.12 (d, J = 7.4 Hz, 1H), 7.75 – 7.70 (t, J = 8.4 Hz, 1H), 7.63 (dd, J =
5.9, 3.0 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.55 – 7.47 (m, 3H), 6.16 (s, 1H), 3.55 (s, 3H). 13C
NMR (101 MHz, DMSO) δ 181.2, 178.3, 157.4, 157.1, 137.3, 134.3, 134.1, 132.4, 131.2, 130.6,
130.2, 130.1, 129.3, 128.5, 124.2, 117.0, 110.4, 36.7. HRMS (ESI): calcd for C18H11ClN2O2
[M+H]+ 323.0582, found 323.0575. HPLC purity: 97.6%.
2-(2-Chlorophenyl)-1-ethyl-1H-perimidine-7,8-dione (a-10): Red solid, Yield 29%. 1H NMR
(400 MHz, CDCl3) δ 8.24 (d, J = 7.4 Hz, 1H), 7.85 (d, J = 8.1 Hz, 1H), 7.74 (t, J = 7.8 Hz, 1H),
7.62 – 7.42 (m, 4H), 5.98 (s, 1H), 3.92 (m, 1H), 3.78 (m, 1H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 180.1, 177.9, 153.2, 150.2, 141.8, 135.0, 133.8, 133.4, 132.3, 131.8, 131.4,
130.2, 129.9, 129.5, 127.7, 118.6, 100.5, 44.3, 12.1. HRMS (ESI): calcd for C19H13ClN2O2
[M+H]+ 337.0738, found 337.0735. HPLC purity: 99.9%.

1
2-(2-Chlorophenyl)-1-ethyl-1H-perimidine-5,6-dione (b-10): Purple solid, Yield 22%. H
NMR (400 MHz, DMSO) δ 8.09 (d, J = 8.6 Hz, 1H), 8.05 (d, J = 7.4 Hz, 1H), 7.89 (t, J = 7.6 Hz,
1H), 7.77 (dd, J = 7.4, 1.6 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.67 – 7.52 (m, 2H), 5.85 (s, 1H),
4.21 (m, 1H), 3.82 (m, 1H), 1.19 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 181.2, 178.3,
157.0 (2C), 136.0, 134.2, 134.2, 132.3, 131.0, 130.6, 130.2, 130.2, 129.4, 128.4, 124.1, 117.3,
110.4, 44.0, 14.2. HRMS (ESI): calcd for C19H13ClN2O2 [M+H]+ 337.0738, found 337.0731.
HPLC purity: 97.5%.
1
2-(3-Chlorophenyl)-1-methyl-1H-perimidine-7,8-dione (a-11): Red solid, Yield 36%. H
NMR (400 MHz, CDCl3) δ 8.26 (dd, J = 7.4, 1.2 Hz, 1H), 7.87 (dd, J = 8.1, 1.2 Hz, 1H), 7.79 –
7.73 (t, J = 8 Hz 1H), 7.62 (t, J = 1.6 Hz, 1H), 7.59 – 7.55 (m, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.47
(m, 1H), 5.95 (s, 1H), 3.49 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 179.9, 177.8, 154.5, 151.8,
141.7, 136.0, 135.5, 134.9, 133.4, 131.1, 131.0, 130.5, 129.5, 128.5, 126.4, 118.2, 100.7, 38.9.
HRMS (ESI): calcd for C18H11ClN2O2 [M+H]+ 323.0582, found 323.0577. HPLC purity: 99.8%.
1
2-(3-Chlorophenyl)-1-methyl-1H-perimidine-5,6-dione (b-11): Purple solid, Yield 31%. H
NMR (400 MHz, DMSO) δ 8.08 – 7.96 (m, 2H), 7.87 (m, 1H), 7.77 (s, 1H), 7.65 (d, J = 7.3 Hz,
2H), 7.58 (dd, J = 8.8, 6.6 Hz, 1H), 5.83 (s, 1H), 3.59 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
181.0, 179.0, 157.3, 156.7, 137.0, 134.0, 133.3, 132.2, 132.0, 130.4, 130.2, 130.1, 129.6, 128.2,
121.9, 117.9, 112.4, 36.1. HRMS (ESI): calcd for C18H11ClN2O2 [M+H]+ 323.0582, found
323.0575. HPLC purity: 100%.
1
2-(4-Chlorophenyl)-1-methyl-1H-perimidine-7,8-dione (a-12): Red solid, Yield 14%. H
NMR (400 MHz, DMSO) δ 8.06 (dd, J = 7.2, 1.3 Hz, 1H), 7.88 (dd, J = 8.1, 1.3 Hz, 1H), 7.86 –
7.81 (m, 1H), 7.77 – 7.73 (m, 2H), 7.68 – 7.63 (m, 2H), 5.89 (s, 1H), 3.39 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 180.0, 177.9, 154.9, 152.0, 141.8, 137.2, 135.0, 133.4, 132.8, 131.1, 129.8 (2C),

129.6 (2C), 129.4, 118.1, 100.7, 39.0. HRMS (ESI): calcd for C18H11ClN2O2 [M+H]+ 323.0582,
found 323.0576. HPLC purity: 100%.
1
2-(4-Chlorophenyl)-1-methyl-1H-perimidine-5,6-dione (b-12): Purple solid, Yield 28%. H
NMR (400 MHz, DMSO) δ 8.02 (d, J = 7.9 Hz, 2H), 7.94 – 7.82 (m, 1H), 7.75 (d, J = 8.6 Hz,
2H), 7.66 (d, J = 8.6 Hz, 2H), 5.84 (s, 1H), 3.62 (s, 3H). 13C NMR (101 MHz, DMSO) δ 181.6,
178.1, 159.2, 157.5, 138.1, 135.9, 134.0, 133.9, 131.4 (2C), 129.9, 129.1 (2C), 129.0, 124.5, 116.9,
110.0, 38.7. HRMS (ESI): calcd for C18H11ClN2O2 [M+H]+ 323.0588, found 323.0582. HPLC
purity: 95.2%.
1
2-(2-Fluorophenyl)-1-methyl-1H-perimidine-7,8-dione (a-13): Red solid, Yield 18%. H
NMR (400 MHz, DMSO) δ 8.10 (dd, J = 7.1, 1.5 Hz, 1H), 7.91 (dd, J = 8.1, 1.5 Hz, 1H), 7.89 –
7.84 (t, J = 6.8 Hz 1H), 7.70 (m, 2H), 7.51 – 7.42 (m, 2H), 5.92 (s, 1H), 3.38 (d, J = 1.3 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 180.0, 177.8, 159.3 (d, J = 249.8 Hz), 151.7, 151.5, 141.9, 135.0,
133.1 (d, J = 8.2 Hz),, 133.0, 131.2, 130.7 (d, J = 1.8 Hz), 129.6, 125.5 (d, J = 3.3 Hz), 122.6 (d,
J = 15.1 Hz), 118.3, 116.2 (d, J = 20.6 Hz), 100.5, 37.5 (d, J = 2.7 Hz). HRMS (ESI): calcd for
C18H11FN2O2 [M+H]+ 307.0877, found 307.0869. HPLC purity: 95.6%.
1
2-(2-Fluorophenyl)-1-methyl-1H-perimidine-5,6-dione (b-13): Purple solid, Yield 22%. H
NMR (400 MHz, DMSO) δ 8.04 – 7.97 (m, 2H), 7.86 (dd, J = 8.6, 7.4 Hz, 1H), 7.73 – 7.59 (m,
2H), 7.50 – 7.30 (m, 2H), 5.82 (s, 1H), 3.56 (d, J = 1.4 Hz, 3H). 13C NMR (101 MHz, DMSO) δ
181.3, 178.3, 158.9 (d, J = 247.8 Hz), 157.2, 155.6, 137.6, 134.0, 133.5 (d, J = 8.3 Hz), 131.3 (d,
J = 1.8 Hz), 130.1, 129.3, 125.7 (d, J = 3.2 Hz), 124.3, 123.1 (d, J = 15.0 Hz), 117.0, 116.5 (d, J
= 20.6 Hz), 110.3, 37.5 (d, J = 1.7 Hz). HRMS (ESI): calcd for C18H11FN2O2 [M+H]+ 307.0877,
found 307.0874. HPLC purity: 99.7%.
1
2-(3-Fluorophenyl)-1-methyl-1H-perimidine-7,8-dione (a-14): Red solid, Yield 19%. H

NMR (400 MHz, DMSO) δ 8.12 – 8.04 (m, 1H), 7.94 – 7.83 (m, 3H), 7.76 – 7.65 (m, 2H), 7.52
– 7.37 (m, 2H), 5.92 (s, 1H), 3.38 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 180.0, 177.9, 159.3 (d,
J = 250.0 Hz), 151.7, 151.5, 141.9, 133.1 (d, J = 8.3 Hz), 133.1, 133.0, 131.2, 130.6 (d, J = 1.8
Hz), 129.6, 125.5 (d, J = 3.2 Hz), 124.0 (d, J = 3.4 Hz), 118.4, 116.2 (d, J = 20.5 Hz), 100.5, 37.5
(d, J = 2.8 Hz). HRMS (ESI): calcd for C18H11FN2O2 [M+H]+ 307.0877, found 307.0871. HPLC
purity: 99.7%.
2-(3-Fluorophenyl)-1-methyl-1H-perimidine-5,6-dione (b-14): Purple solid, Yield 26.1%. 1H
NMR (400 MHz, DMSO) δ 8.03 (dd, J = 8, 3.6 Hz, 2H), 7.89 (dd, J = 16.1, 7.5 Hz, 1H), 7.79 –
7.74 (m, 2H), 7.41 (t, J = 8.4 Hz, 2H), 5.88 (s, 1H), 3.39 (s, 3H). HRMS (ESI): calcd for
C18H11FN2O2 [M+H]+ 307.0877, found 307.0872. HPLC purity: 98.8%.
1
2-(4-Fluorophenyl)-1-methyl-1H-perimidine-7,8-dione (a-15): Red solid, Yield 26.1%. H
NMR (400 MHz, DMSO) δ 8.07 (d, J = 6.8 Hz, 1H), 7.89 (d, J = 7.5 Hz, 1H), 7.87 – 7.81 (d, J =
7.6 Hz 1H), 7.81 – 7.76 (t, J = 7.6 Hz 1H), 7.43 (t, J = 8.4 Hz, 2H), 5.90 (s, 1H), 3.41 (s, 2H). 13C
NMR (101 MHz, CDCl3) δ 180.0, 177.8, 164.0 (d, J = 252.6 Hz), 155.1, 152.0, 141.8, 134.9 (2C),
133.3 (2C), 130.8 (d, J = 8.1 Hz), 130.6, 130.5 (d, J = 3.6 Hz), 129.4, 118.0, 116.5 (d, J = 22.1
Hz), 100.7, 39.0. HRMS (ESI): calcd for C18H11FN2O2 [M+H]+ 307.0877, found 307.0872. HPLC
purity: 98.8%.
1
2-(4-Fluorophenyl)-1-methyl-1H-perimidine-5,6-dione (b-15): Purple solid, Yield 21%. H
NMR (400 MHz, DMSO) δ 8.03 (d, J = 7.9 Hz, 2H), 7.89 (t, J = 7.6 Hz, 1H), 7.82 – 7.77 (m,
2H), 7.43 (t, J = 8.9 Hz, 2H), 5.85 (s, 1H), 3.63 (s, 3H). 13C NMR (101 MHz, DMSO) δ 181.7,
178.1, 163.7 (d, J = 248.2 Hz), 159.3, 157.5, 138.2 (2C), 133.9, 132.1 (d, J = 8.8 Hz), 131.7 (d, J
= 3.0 Hz), 130.0 (2C), 129.0, 124.5, 116.9, 116.1 (d, J = 22.1 Hz), 109.9, 38.8. HRMS (ESI):
calcd for C18H11FN2O2 [M+H]+ 307.0877, found 307.0871. HPLC purity: 96.7%.
2-(2,4-Difluorophenyl)-1-methyl-1H-perimidine-7,8-dione (a-16): Red solid, Yield 19.3%.

1H NMR (400 MHz, CDCl3) δ 8.30 – 8.20 (d, J = 8.4, Hz 1H), 7.86 (d, J = 8.1 Hz, 1H), 7.75 (t, J
= 7.8 Hz, 1H), 7.65 (m, 1H), 7.14 (m, 1H), 7.08 – 6.95 (m, 1H), 5.95 (s, 1H), 3.47 (d, J = 2.1 Hz,
3H). 13C NMR (101 MHz, CDCl3) δ 179.9, 177.8, 164.7 (dd, J = 255.4, 11.9 Hz), 159.8 (dd, J =
252.5, 12.4 Hz), 151.5, 150.9, 141.8, 135.0, 133.4, 132.1 (dd, J = 10.1, 4.0 Hz), 131.3, 129.5,
119.0 (dd, J = 15.6, 4.0 Hz), 118.3, 113.1 (dd, J = 21.8, 3.4 Hz), 104.8 (t, J = 25.3 Hz), 100.7,
37.6 (d, J = 3.1 Hz). HRMS (ESI): calcd for C18H10F2N2O2 [M+H]+ 325.0783, found 325.0781.
HPLC purity: 100%.
2-(2,4-Difluorophenyl)-1-methyl-1H-perimidine-5,6-dione (b-16): Purple solid, Yield 17.4%.
1H NMR (400 MHz, CDCl3) δ 8.06 (d, J = 7.4 Hz, 1H), 7.78 (m, 1H), 7.75 – 7.69 (t, J = 8.4 Hz
1H), 7.58 (d, J = 8.4 Hz, 1H), 7.14 (m, 1H), 7.04 – 6.94 (m, 1H), 6.07 (s, 1H), 3.65 (d, J = 2.3
Hz, 3H). 13C NMR (101 MHz, DMSO) δ 180.2, 177.2, 163.1 (dd, J = 250.6, 12.3 Hz), 158.4 (dd,
J = 250.4, 12.9 Hz), 156.0, 153.8, 136.5, 132.9, 131.9 (dd, J = 10.4, 3.7 Hz), 129.0, 128.3, 123.3,
118.7 (dd, J = 15.3, 3.9 Hz), 115.9, 112.0 (dd, J = 22, 3.3 Hz), 109.2, 104.2 (t, J = 25.9 Hz), 36.5
(d, J = 1.9 Hz). HRMS (ESI): calcd for C18H10F2N2O2 [M+H]+ 325.0783, found 325.0775. HPLC
purity: 99.9%.
1-Methyl-2-(2,3,4-trifluorophenyl)-1H-perimidine-7,8-dione (a-17): Red solid, Yield 24%.
1H NMR (400 MHz, DMSO) δ 8.10 (d, J = 6.4 Hz, 1H), 8.01 – 7.79 (m, 2H), 7.61 (m, 2H), 5.94
(s, 1H), 3.40 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 179.7, 177.8, 154.4 – 154.1 (m), 152.1 –
151.4 (m), 151.3, 149.8 (d, J = 2.7 Hz), 148.0 – 147.4 (m), 141.6 (d, J = 3.7 Hz), 135.0, 133.4,
131.4, 129.5, 124.7, 120.9 – 119.6 (m), 118.4, 114.0 (dd, J = 17.7, 2.1 Hz), 100.8, 37.5. HRMS
(ESI): calcd for C18H19F3N2O2 [M+H]+ 343.0689, found 343.0682. HPLC purity: 100%.
1-Methyl-2-(2,3,4-trifluorophenyl)-1H-perimidine-5,6-dione (b-17): Purple solid, Yield
17.4%. 1H NMR (400 MHz, DMSO) δ 8.09 – 8.00 (m, 2H), 7.90 (dd, J = 8.6, 7.4 Hz, 1H), 7.71
– 7.46 (m, 2H), 5.85 (s, 1H), 3.62 (s, 3H). 13C NMR (101 MHz, TFA) δ 174.4 (2C), 160.5, 159.3,
153.7, 151.2 (dddd, J = 263.0, 10.2, 3.8, 2 Hz), 145.3 (dddd, J = 258.2, 12.0, 4.1, 2 Hz), 134.6

(2C), 134.5, 130.0 (2C), 123.2, 122.0 – 120.8 (m), 112.8, 110.4 (dd, J = 19.0, 3.1 Hz), 106.0, 35.8
(d, J = 4.9 Hz). HRMS (ESI): calcd for C18H19F3N2O2 [M+H]+ 343.0689, found 343.0684. HPLC
purity: 100%.
5.5. Procedure for the Preparation of 9-chloro-2-(2-chlorophenyl)-1-methyl-1H-perimidine-
7,8-dione (d-2)
N-Chlorosuccinimide (0.6 mmol, 1.2 equiv.) and benzoyl peroxide (12 mg) were added to a
solution of o-quinone products (0.5 mmol) in chloroform. The reaction mixture was stirred at 80
oC. After the reaction was completed, the chloroform was evaporated. The residue was used for
futher purification to obtain the red product. Red solid, Yield 63%. 1H NMR (400 MHz, CDCl3)
δ 8.23 (d, J = 7.2 Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.73 (t, J = 7.6 Hz, 1H), 7.63 (t, J = 8.6 Hz,
1H), 7.61 – 7.45 (m, 3H), 3.73 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 178.0, 173.7, 155.4, 150.0,
141.5, 135.3, 133.5, 132.9, 132.6, 132.3, 131.5, 130.7, 130.6, 128.0, 127.7, 119.6, 108.4, 43.7.
HRMS (ESI): calcd for C18H10Cl2N2O2 [M+H]+ 357.0192, found 357.0191. HPLC purity: 97.9%.
5.6. General Procedure for the Preparation of d-1 and d-3
N-Bromosuccinimide (0.6 mmol, 1.2 equiv.) and benzoyl peroxide (12 mg) were added to a
solution of o-quinone products (0.5 mmol) in chloroform. The reaction mixture was stirred at 80
oC. After the reaction was completed, the chloroform was evaporated. The residue was used for
futher purification to obtain the red product.
9-Bromo-2-(2-chlorophenyl)-1-methyl-1H-perimidine-7,8-dione (d-1): Red solid, Yield 71%.
1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 7.4 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.73 (t, J = 7.7
Hz, 1H), 7.66 (d, J = 6.9 Hz, 1H), 7.62 – 7.46 (m, 3H), 3.74 (d, J = 5.9 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 177.5, 173.9, 155.4, 152.5, 141.3, 135.2, 133.5, 132.9, 132.6, 132.3, 131.3, 130.9,
130.6, 128.0, 127.7, 120.5, 98.2, 44.5. HRMS (ESI): calcd for C18H10ClBrN2O2 [M+H]+ 400.9687,
found 400.9683. HPLC purity: 97.8%.
9-Bromo-2-ethyl-1-methyl-1H-perimidine-7,8-dione (d-3): Red solid, Yield 77.5%. 1H NMR

(400 MHz, CDCl3) δ 8.12 (d, J = 7.4 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H),
3.91 (s, 3H), 2.89 (q, J = 7.3 Hz, 2H), 1.44 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
177.8, 173.9, 159.7, 153.2, 141.2, 134.6, 132.7, 130.5, 127.5, 120.0, 97.0, 42.7, 28.5, 11.1. HRMS
(ESI): calcd for C14H11BrN2O2 [M+H]+ 319.0077, found 319.0073. HPLC purity: 98.7%.
5.7. Topo I Mediated DNA Relaxation Assay
The effects of compounds on DNA relaxation catalyzed by DNA Topo I (TaKaRa, Kyoto,
Japan) were determined by measuring the relaxation of supercoiled DNA pBR322 (TaKaRa,
Kyoto, Japan) using CPT as the positive control. The reaction solution was prepared according to
o
the provided protocol and incubated at 37 C for 30 min. The reactions were stoped by the
6×loading buffer (provided by TaKaRa) of 4 uL. The products were applied to 1% agarose gel
and subjected to electrophoresis for 1.5 h at 100V in 1×TAE buffer (40 mM Tris-acetate, 2 mM
EDTA). Gels were stained for 30 min in 30 mL 1×TAE buffer with 1 uL Gel Red. DNA bands
were visualized by transillumination with UV light and then photographed by Alpha Innotech
digital imaging system.
5.8. Topo IIα Mediated DNA Relaxation Assay
We used the Topo IIα assay kit from TopoGEN to determine the effects of compounds on
DNA relaxation catalyzed by Topo II. Relaxation assays were carried out according to the
manufacturer's instructions with minor modifications. The assay was performed in a final volume
of 20 uL in Topo II reaction buffer (5×Topo II buffer = 50 mM Trise HCl, pH 8.0, 150 mM NaCl,
10 mM MgCl2 2 mM ATP, 0.5 mM dithiothreitol, and 30 g/mL BSA) with 0.2 g pBR322 DNA
.Then reactions were initiated by 1U human Topo IIα and incubated for 30 min at 37oC. Finally
reaction was stoped with the 6×loading buffer of 4 uL. Reaction products were analyzed on a 1%
agarose gel in 1×TAE buffer. Gels were stained for 30 min in 30 mL 1×TAE buffer with 1 uL
Gel Red. DNA bands were visualized through transillumination with UV light and then
photographed by Alpha Innotech digital imaging system.
5.9. Topo I Mediated DNA Cleavage Assay
In brief, Topo I (10 U), supercoiled pBR322 DNA (0.1 g) and compounds (different

concentrations) were added in Topo I buffer (50 mM Trise-HCl, pH 7.5, 50 mM KCl, 10 mM
MgCl2, 0.5 mM DTT, 0.1 mM EDTA and 30 g/mL bovine serum albumin, provided by TaKaRa)
of 20 Lvolume. After incubating for 6 min at 37 oC and then respectively adding 2 L of 10%
SDS, 2 L of 250 mM NaEDTA, pH 8.0, 2 L of 0.8 mg/mL Proteinase K. Following reactions
were incubated for another 30 min at 45 oC. Samples were mixed with 4 L of 6×loading buffer,
heated at 70 oC for 2 min and subjected to electrophoresis in a 1% agarose gel in 1× TAE buffer
(30 mL) with 1 uL Gel Red . Finally DNA bands were visualized by using UV light, photographed
by using Alpha Innotech digital imaging system.
5.10. Topo II Mediated DNA Cleavage Assay
In brief, Topo IIα (10 U), supercoiled pBR322 DNA (0.2 g) and compounds (different
concentrations) were added in Topo II buffer (5×Topo II buffer = 50 mM Trise HCl, pH 8.0, 150
mM NaCl, 10 mM MgCl2 2 mM ATP, 0.5 mM dithiothreitol, and 30 g/mL BSA) of 20 L
volume. The following procedure was same with that Topo I mediated DNA cleavage Assay.
Finally DNA bands were visualized by using UV light, photographed by using Alpha Innotech
digital imaging system.
5.11. DNA Unwinding Assays
The ability of perimidine derivatives to unwind plasmid DNA was determined as described
by Fortune et al with modifications. The relaxed pBR322 plasmid DNA utilized in unwinding
assays was generated by treating negatively supercoiled pBR322 with Topo I in Topo I reaction
buffer at 37oC for 30 min. Reactions included 0.1 g relaxed pBR322 plasmid DNA and different
concentrations compounds in 20 μL of Topo I reaction buffer were incubated for 10 min in room
temperature. Then Topo I was added and reactions were incubated for another 30 min at 37 oC.
Finally, reactions were stoped by 4 L of 6×loading buffer, subjected to electrophoresis in a 1%
agarose gel in 1× TAE buffer (30 mL) with 1 uL Gel Red And DNA bands were visualized by
using UV light, photographed by using Alpha Innotech digital imaging system.
5.12. UV− Vis Titration Assay
Absorbance titration experiments were performed as previously described [21]. A solution

of CT-DNA (Sigma-Aldrich) in DPBS gave a ratio of UV-Vis absorbance of 1.8-1.9 at 260 and
280 nm, indicating that the DNA was sufficiently free of protein. The CT-DNA concentration
was determined by UV absorbance at 260 nm using a molar absorptivity constant of 6600 M
−1cm−1
.
Compound b-12 (5 μM) was prepared in DPBS in the presence or absence of increasing
concentrations of CT-DNA (0-80  Absorption spectra were recorded in the 450-650 nm
spectral range after equilibration at room temperature for 5 min using UV evisible
spectrophotometer UV-2450 (Shimadzu Instruments, Inc.).
5.13. MTT Assay
The growth inhibitory effects of perimidine derivatives against four cancer cell lines were
evaluated by using the MTT assay as described by Mosmann with modifications [22]. The cells
were plated at a density of 5000 per well in 96-well microplates and allowed to incubate for 24 h
at 37 oC. Compounds were added to the wells at different concentrations and incubated for 48 h
o
at 37 C. To adherent cells, each well was treated with 20 μL of 2.5 mg/mL MTT solution and
incubated for 4 h, and finally 100 mL of DMSO was added. The microplates were well shaken to
dissolve the formazan dye, and the absorbance at 570 nm was measured using a microplate reader
(Bio-Tek). All compound doses were parallel tested in triplicate.
5.14. Cell Apoptosis Assay
Huh7 cells (5×105 cells per mL) were incubated in the presence of compound for 24 h at 37
oC, thenharvested and washed twice with cold 1×PBS, resuspended in 1×binding buffer, and then
stained with 5 μL of Fluorescein isothiocyanate (FITC) Annexin V and 5 μL of propidium iodide
(KeyGEN BioTECH, China) for 15 min in the dark. The stained cells were analyzed by flow
cytometry (BD, FACSCalibur, USA) within 1 h.
5.15. Cell Cycle Assay
Huh7 cells (5×105 cells per mL) were incubated in the presence of compound for 24 h at 37
oC, thenharvested and washed twice with cold 1×PBS, resuspended in 1×binding buffer, and then
stained with 500 μL of propidium iodide (KeyGEN BioTECH, China) for 15 min in the dark. The

stained cells were analyzed by flow cytometry (BD, FACSCalibur, USA) within 1 h.
5.16. Immunofluorescence Assay
Huh7 Cells grown on glass coverslips were fixed in 4% paraformaldehyde/PBS for 15 min,
then permeabilized with 0.5% triton-X100/PBS at 37 °C for 30 min, and finally blocked with 5%
goat serum/PBS at 37 °C for 2h. Immunofluorescence was performed using standard methods,
and the slides were incubated alternately with Phospho-γH2AX(Ser139) (Cell Signaling
Technology) at 37 °C for 2 h. the glass coverslips were washed six times with blocking buffer
and were then incubated with ant-rabbit Alexa 488-conjugated antibody (A21206, Life
Technology), and 2 μg/mL of 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen) at 37 °C for 3h.
The glass coverslips were again washed six times with blocking buffer. Digital images were
recorded using an LSM710 microscope (Zeiss, GER) and analyzed with ZEN software.
Acknowledgements
We thank the Guangdong Provincial Key Laboratory of Construction Foundation
[2017B030314030] for financial support of this study. We also thank Professor Jian Ding and
Hong Liu(State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences) for the HL-60/MX2 cell line gifts.
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Highlights
►Novel perimidine o-quinone derivatives were synthesized as Topo II inhibitors
►Compound b-12 showed potent cytotoxicity (IC50<1 ) against four cancer cell lines
►Compound b-12 showed strong Topo IIα inhibitory activity (IC50 = 5 )
►Compound b-12 is a calss of novel non-intercalative Topo IIα catalytic inhibitor