Design, synthesis and biological evaluation of novel 7-alkylamino substituted benzo[ a[phenazin derivatives as dual topoisomerase I/II inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.01.024

A novel series of benzo[a]phenazin derivatives bearing alkylamino side chains were designed, synthesized and evaluated for their topoisomerases inhibitory activity as well as cytotoxicity against four human cancer cell lines (HL-60, K-562, HeLa, and A549)

Full text of DOI:10.1016/j.ejmech.2015.01.024

European Journal of Medicinal Chemistry 92 (2015) 540e553
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of novel 7-alkylamino
substituted benzo[a]phenazin derivatives as dual topoisomerase I/II
inhibitors
Bing-Lei Yao 1, Yan-Wen Mai 1, Shuo-Bin Chen, Hua-Ting Xie, Pei-Fen Yao, Tian-Miao Ou,
Jia-Heng Tan, Hong-Gen Wang, Ding Li, Shi-Liang Huang*, Lian-Quan Gu, Zhi-Shu Huang*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of benzo[a]phenazin derivatives bearing alkylamino side chains were designed, synthe-
sized and evaluated for their topoisomerases inhibitory activity as well as cytotoxicity against four hu-
man cancer cell lines (HL-60, K-562, HeLa, and A549). These compounds were found to be dual inhibitors
of topoisomerase (Topo) I and Topo II, and exhibited excellent antiproliferative activity, in particular
against HL-60 cells with submicromolar IC50 values. Further mechanistic studies showed that this class of
compounds acted as Topo I poisons by stabilizing the Topo I-DNA cleavage complexes and Topo II cat-
alytic inhibitors by inhibiting the ATPase activity of hTopo II. Molecular docking studies revealed the
binding modes of these compounds for Topo I and Topo II.
Received 21 December 2014
Received in revised form
10 January 2015
Accepted 11 January 2015
Available online 12 January 2015
Keywords:
Benzo[a]phenazin derivatives
Topoisomerase I
© 2015 Elsevier Masson SAS. All rights reserved.
Topoisomerase II
ATPase catalytic inhibitor
Antitumor
1. Introduction
and act at any one of several steps within the Topo catalytic cycle
[9e11].
Topoisomerases are nuclear enzymes that modulate the topo-
logical state of DNA during various key cellular processes such as
replication, transcription, recombination, repair, and chromatin
assembly [1e4]. Topoisomerases have been recognized as one of
the most promising targets in anticancer drug discovery because of
their highly over-expression in cancer cells [5]. There are two major
classes of topoisomerases, namely, topoisomerase type I (Topo I)
and type II (Topo II). Topo I catalyzes the formation of DNA single-
strand breaks while Topo II engenders DNA double-strand breaks
[6e8]. On the basis of their mechanism of action, Topo inhibitors
are classified into two categories, Topo poisons, which interfere
with the breakingerejoining reaction of the enzyme by stabilizing
the cleavable complex of Topo with DNA, and catalytic inhibitors
that impair Topo activity without stabilizing the cleavable complex
Drugs that specifically target Topo I, principally the campto-
thecin analogs, have made an important impact more recently for
the treatment of colon cancer [12], while those that target Topo II,
for example etoposide and doxorubicin, have been used for treat-
ment of a wide variety of malignancies [13]. Since both Topo I and
Topo II are excellent targets, it would be desirable to jointly inhibit
them. In this regard, a single compound able to inhibit both Topo I
and II appears to combine the properties of the individual specific
inhibitor, thus leading to a more promising anti-tumor activity [3].
A number of drugs such as the acridine DACA [14], the benzophe-
nazine XR11576 [12], the benzopyridoindole intoplicine [15], the
indenoquinolinone TAS-103 [16], the pyrazoloacridine NSC 366140
[17], and the tafluposide (F-11782) [18], have now been identified to
target both Topo I and Topo II simultaneously, and some of these
drugs have been advanced to clinical trial [19].
Benzophenazine derivatives are considered to be potentially
bioactive compounds. NC-190 is an anti-tumor compound having a
benzo[a]phenazine ring and displays strong Topo II inhibition and
weak Topo I inhibition. Further studies have revealed NC-190 could
induce Topo II-mediated DNA cleavage and DNA fragmentation in
* Corresponding authors.
E-mail addresses: lsshsl@mail.sysu.edu.cn (S.-L. Huang), ceshzs@mail.sysu.edu.
cn (Z.-S. Huang).
1
These authors contributed equally.
http://dx.doi.org/10.1016/j.ejmech.2015.01.024
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
541
2. Chemistry
Abbreviations
The designed compounds were synthesized by following exist-
ing procedures with modifications [25], as shown in Scheme 1.
Treatment of 2-fluoro-nitrobenzene (1a,1e-1 ~ 1e-3) or 2-chloro-3-
nitropyridine (1g) with the appropriate diamines in the presence of
tetrahydrofuran afforded the intermediates 2. The nitro group of
intermediates 2 was reduced to amines to provide the corre-
sponding intermediates 3 by using Zn/acetic acid as a reducing
agent. In the presence of potassium tert-butoxide, 2-hydroxy-1,4-
naphthoquinone derivatives 5 (5a, 5b) were readily prepared by
autoxidation of methoxyl tetralones 4 (4a, 4b). 2-Hydroxy-1,4-
naphthoquinone anologs 5 and intermediates 3 were condensed
in acetic acid for 60 min at 60 ꢀC to give benzo[a]phenazin de-
rivatives 6. All structures of compounds 6 were confirmed using 1H
NMR, 13C NMR and HRMS (ESI).
CPT
EB
HPLC
HRMS
MTT
NMR
camptothecin
ethidium bromide
high performance liquid chromatography
high resolution mass spectrometer
methyl thiazolyl tetrazolium
nuclear magnetic resonance
1,4-NQ 1,4-naphthoquinone
SARs
Topo
Topo I
structureeactivity relationship
topoisomerase
topoisomerase type I
Topo II topoisomerase type II
VP-16 etoposide
3. Results and discussion
HL-60 cells [20,21]. Similarly, XR 11576, a lipophilic benzo[a]
phenazine developed by Xenova and Millennium, has been proved
to be a potent dual inhibitor of Topo I and II, inducing cleavage of
complex formed by both Topo I and Topo II as dual poison at low
concentrations [19,22,23].
To discover novel anticancer agent as dual Topo inhibitors, we
have previously reported the modification strategy on ring B and
ring D of the benzo[a]phenazine scaffold and successfully devel-
oped compound 5d-1 which has been proved to be a rare dual Topo
inhibitors by acting as Topo I poisons and Topo II catalytic inhibitors
[24]. In an effort to gain more insignt into the substitution effects on
ring A, ring C and ring D and make change from phenolic ether form
to quinone imide form, we synthesized a series of benzo[a]phe-
nazin derivatives by attaching alkylamino side chains to the ni-
trogen group on ring C with or without different substitutive group
on ring A and ring D (Fig. 1).
In this study we report the design and synthesis of benzo[a]
phenazin derivatives and evaluation of their Topo I and II inhibitory
activity, and cytotoxicity against four human cancer cell lines. To
further investigate the action mechanism of these compounds,
several in vitro experiments including cleavage complex assays and
ATPase activity assays were performed. Our results indicated that
this type of compounds were dual Topo inhibitors by acting as Topo
I poisons and Topo II catalytic inhibitors.
3.1. Topo I and II inhibitory activity
Topo I and II Inhibitory activity was measured by detecting the
conversion of supercoiled pBR322 plasmid DNA to its relaxed form
in the presence of the tested compounds as shown in Figs. 2 and 3.
Camptothecin and etoposide, well known Topo I and II inhibitors,
respectively, were used as positive controls. As shown in Fig. 2A,
most of compounds showed good Topo I inhibitory activity at
50
significant Topo I inhibitory activity in further screening assay at
25 M concentrations (Fig. 2B).
The results of Topo I inhibitory activities indicated that intro-
mM concentration, and compounds 6c-1, 6d-4, and 6e-3 showed
m
duction of the terminal amino moiety into the side chain at N-7
position of benzo[a]phenazine scaffold could increase the inhibi-
tory activity of the compounds compared to the compound 6a-6
without terminal amino moiety. Those compounds with the
dimethylamino terminal (6a-1, 6b-1, 6c-1) of the side chain at N-7
position showed good inhibitory activity. Comparing the activity of
three series compounds 6a-1 ~ 6a-6 (n ¼ 1), 6b-1 ~ 6b-6 (n ¼ 2) and
6c-1 ~ 6c-2 (n ¼ 3), it could be found that the activities of these
tested compounds were not affected by the length of the side
chains. By comparing with 6b-1 at inhibitory concentration of
25
mM (Fig. 2B), we could find that the introduction of eOCH3
Fig. 1. Structure of benzophenazine derivatives.

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B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
Scheme 1. Syntheses of benzo[a]phenazin derivatives. Reagents and conditions: (a) THF, 30 ꢀC, 12 h; (b) AcOH, Zn, 0 ꢀC, 1 h; (c) t-BuOK, t-BuOH, O2, 2 h, 42%~45%; (d) AcOH, 60 ꢀC,
1 h, 41% ~ 48%.
groups at position of C-2 (6d-4) or C-9 (6e-3) increased the inhib-
itory activity of the tested compounds, while other substitution
patterns did not produce favorable effects on inhibitory activity.
The effect of the tested compounds on human DNA Topo II was
shown in Fig. 3. Most of the compounds showed good Topo II
dimethylamino terminal (6a-1, 6b-1, 6c-1) and piperidine terminal
(6a-5, 6b-5) side chain at N-7 position exhibited significant inhib-
itory activity and the length of side chain was not important to
inhibitory activity. Compound 6d-4 with eOCH3 group at position
C-2 showed both good Topo I and Topo II inhibitory activity at
inhibitory activity at 50
2, 6a-3, 6a-6, 6d-2, 6d-3, and 6f-2 (Fig. 3A). Further screening assay
showed that compounds 6c-1, 6d-4, and 6f-3 could inhibit
m
M concentrations except compounds 6a-
25
position C-3 and C-9 displayed good Topo II inhibitory activity at
25 M. Moreover, compounds 6g-1 and 6g-2, which possess N at 8
position of ring D of benzo[a]phenazine scaffold, did not show
significant Topo I and Topo II inhibitory activity at 25 M, indicating
that the inhibitory activities of the test compounds were not
affected by the introduction of N into 8 position of ring D of benzo
[a]phenazine scaffold.
mM. Additionally, compound 6f-3 with eOCH3 group at the
m
completely Topo II at 25 mM concentration (Fig. 3B).
The structureeactivity relationship (SARs) analysis on Topo II
was similar to that on Topo I. The results indicated that the terminal
amino moiety is important for inhibitory activity, as compound 6a-
m
6
had lower activity against Topo II. Compounds with the

B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
543
Fig. 2. The Topo I inhibitory activity of benzo[a]phenazine derivatives. Supercoiled pBR322 plasmid was incubated with Topo I in the presence or absence of indicated compounds at
37 ꢀC for 30 min. (A) Effect of 6a-1 ~ 6a-6, 6b-1 ~ 6b-6, 6c-1 ~ 6c-2, 6d-1 ~ 6d-4, 6e-1 ~ 6e-3, 6f-1 ~ 6f-3, and 6g-1 ~ 6g-2 on DNA relaxation catalyzed by Topo I at the concentration
of 50 mM. (B) Effect of 6a-1, 6a-4, 6a-5, 6b-1, 6b-5, 6c-1, 6d-1, 6d-2, 6d-4, 6e-1, 6e-3, 6f-1, 6g-1, and 6g-2 on DNA relaxation catalyzed by Topo I at the concentration of 25 mM.
3.2. Cytotoxicity
moiety and 10-Cl substituent at D ring decreased their cytotoxicity.
For example, 6e-2 (IC50 ¼ 1.08 M) and 6f-2 (IC50 ¼ 6.43 M) had
decreased activity compare with the corresponding compounds
M).
To some extent, the SARs have shown some correlation between
the cytotoxicity and Topo inhibitory activity. The compounds
m
m
The synthesized compounds were evaluated for their cytotoxic
activities against four cancer cell lines including HL-60 (human
myeloid leukemic tumor cell line), K562 (human myeloid leukemic
tumor cell line), HeLa (human cervix tumor cell line) and A549
(adenocarcinomic human alveolar basal epithelial cell line) using
the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazo-
lium bromide] assay as described by Mosmann with modifications
[26]. Camptothecin (CPT) and etoposide (VP-16) were used as
positive controls. The inhibitory activities (IC50) of the tested
compounds are shown in Table 1.
6b-1 (IC50 ¼ 0.21
m
M) and 6d-1 (IC50 ¼ 0.15
m
without Topo I and Topo II inhibitory activity at 25
tion also exhibited low cytotoxicity (The IC50 values for 6a-3, 6a-6,
6b-2 ~ 4, 6d-3, 6e-2, 6f-2 were all more than 1.0 M for HL-60 cell
mM concentra-
m
line). Although compounds 6c-1 and 6d-4 displayed the highest
Topo I and II inhibitory activity, they were not the most potent
compounds in cytotoxicity against HL-60 cell line. In addition,
compounds 6a-2, 6c-2, and 6d-1 which showed moderate or weak
Topo inhibition, also possessed potent cytotoxicity against HL-60
cell line. Therefore, for most of the compounds, this might be due
to different expression levels of Topo I and II in the tested cell lines
and the diverse mechanisms in inhibiting human Topo I and II [27].
From the IC50 values, it is clear that most compounds were more
sensitive against HL-60 cell line than against other three cell lines.
This result is consistent with that observed for the positive controls.
For HL-60 cell line, twenty compounds displayed potent inhibitory
activities with the IC50 values range from 0.09
mM to 0.96 mM.
Among these compounds, eleven compounds for A549 cell line, five
compounds for HeLa cell line, and only one compound for K562 cell
line showed better inhibitory activities than positive control CPT.
Base on the MTT data of sensitive cell HL-60, the cytotoxicity of
compounds were not affected by length of side chain. Compounds
6a-1 (n ¼ 1), 6b-1 (n ¼ 2), and 6c-1 (n ¼ 3) have similar cytotoxic
activity. In addition, the terminal amino moiety of side chain is
important for their cytotoxicity. For example, 6a-6, without ter-
minal amino moiety hardly possessed any cytotoxicity for four
cancer cell lines. The morphorine substituent in terminal amino
3.3. Topo I-mediated DNA cleavage assay
The results of Topo I mediated DNA relaxation assays suggested
that Topo I is a target for our compounds. With the aim to further
understand the mechanism of action of our compounds, we per-
formed the Topo I cleavage assay to see if our compounds can act as
Topo I poisons by stabilizing the Topo I-DNA cleavage complexes.
Compound 6c-1 was chosen to evaluate the occurrence of the
cleavage complexes by the induction of nicked DNA. Camptothecin,

544
B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
Fig. 3. The Topo II inhibitory activity of benzo[a]phenazine derivatives. Supercoiled pBR322 plasmid was incubated with Topo II in the presence or absence of indicated compounds
at 37 ꢀC for 30 min. (A) Effect of 6a-1 ~ 6a-6, 6b-1 ~ 6b-6, 6c-1 ~ 6c-2, 6d-1 ~ 6d-4, 6e-1 ~ 6e-3, 6f-1 ~ 6f-3, and 6g-1 ~ 6g-2 on DNA relaxation catalyzed by Topo II at concentration
of 50
mM. (B) Effect of 6a-1, 6a-4, 6a-5, 6b-1, 6b-5, 6b-6, 6c-1, 6c-2, 6d-1, 6d-4, 6e-1, 6e-2, 6e-3, 6f-1, 6f-3, 6g-1, and 6g-2 on DNA relaxation catalyzed by Topo II at concentration of
25 mM.
a well-known Topo I poison, was used as positive control. As shown
in Fig. 4A, both 6c-1 and CPT could stabilize the cleavage complex
by inducing the formation of nicked DNA. Analysis of optical den-
sity of the nicked DNA induced by CPT revealed that CPT could
stabilize the cleavage complex in a dose-dependent manner. As
shown in Fig. 4B, the amount of nicked DNA increased significantly
with increasing concentrations of camptothecin. By comparison,
although compound 6c-1 could induce the formation of nicked
DNA but the amount of nicked DNA increased with increasing
experiment indicating that our compounds act as Topo I poisons by
trapping Topo I-DNA cleavage complexes.
3.4. Topo II-mediated DNA cleavage assay
Similar to Topo I, our compounds showed good inhibition
against relaxation activity of Topo II. In order to investigate the
mode of action of our compounds, Topo II-mediated cleavage assays
were performed to differentiate Topo II poisons and catalytic in-
hibitors based on whether or not linear DNA is formed.
Compound 6c-1 was chosen to test for its ability to induce the
formation of linear DNA in Topo II-mediated DNA cleavage assays.
In this assay, VP-16 was chosen as a positive control. As shown in
Fig. 5, VP-16 induced the formation of linear DNA, in contrast,
treatment with compound 6c-1 did not generate any linear DNA at
concentrations of 6c-1 at lower concentration (0.5e10
as obviously as that of CPT. 6c-1 exhibited its maximal cleavage
enhancement at 10 M by increasing the amount of nicked DNA of
2.5 fold and showed a decline of nicked DNA at higher concentra-
tion (25e50 M). The lower concentration of 6c-1 producing a
mM) was not
m
m
weakly increase of nicked DNA and the higher concentration of 6c-
1 leading to a gradually decrease of nicked DNA might be because
the intercalative ability of compound hampered the interaction
between Topo I and DNA [28].
To examine if our compound could induce the formation of Topo
I-DNA cleavage complexes in vivo, we further performed the ICE
(immunodetection of complexes of enzyme-to-DNA) assay. As
shown in Fig. 4C, after the exposure to our compounds for 1 h, both
CPTand the selected compound 6c-1 induced the formation of Topo
I-DNA cleavage complexes in a dose-dependent manner at lower
concentration. These results were consistent with those of in vitro
10 and 50 mM, thus indicating its inability to induce the formation
of cleavage complex. Based on these observations, it is possible to
deduce that compound 6c-1 might not be a Topo II poison but a
catalytic inhibitor.
3.5. ATPase domain of hTopo II related assays
In order to investigate the interaction between our compounds
and the ATPase domain of Topo II, we examined the effect of our
compounds on the ATPase activity to see if our compounds can

B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
545
Table 1
IC50 values (mM) of the benzo[a]phenazin derivatives against cancer cells.
Compd.
n
R
R1
R2
R3
R4
X
IC50 (m
M)a
HL-60
K562
24.56
Hela
9.91
A549
22.31
6a-1
6a-2
6a-3
6a-4
6a-5
6a-6
6b-1
6b-2
6b-3
6b-4
6b-5
6b-6
6c-1
6c-2
6d-1
6d-2
6d-3
6d-4
6e-1
6e-2
6e-3
6f-1
6f-2
6f-3
6g-1
6g-2
VP-16
CPT
1
1
1
1
1
1
2
2
2
2
2
2
3
3
2
2
1
2
2
2
2
2
2
2
2
2
e
e
eN(CH3)2
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eOCH3
eCl
eH
eOCH3
eCl
eH
eH
eH
e
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eOCH3
eH
eH
eOCH3
eH
eH
e
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eOCH3
eOCH3
eOCH3
eH
eH
eH
eH
eOCH3
eOCH3
eOCH3
eH
eH
e
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eH
eOCH3
eH
eH
eH
eH
eH
eH
eH
eH
e
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eC
eN
eN
e
0.31
0.31
6.91
0.38
0.93
32.12
0.21
1.50
1.28
1.22
0.52
0.22
0.38
0.22
0.15
0.23
2.85
0.22
0.96
1.08
0.18
0.65
6.43
0.09
0.62
0.63
0.06
0.07
eN(CH2CH3)2
eN(CH2CH2)2O
eN(CH2CH2)2NCH3
eN(CH2CH2)2CH2
eCH3
25.57
>40
21.56
25.17
25.45
>50
28.95
31.96
>50
8.41
29.24
>50
25.26
37.31
>40
>50
eN(CH3)2
5.93
24.76
25.17
23.96
10.34
25.17
4.73
25.97
6.12
21.96
18.35
7.13
20.16
20.36
5.53
21.96
22.76
8.73
36.79
38.39
1.64
13.51
37.41
45.35
23.57
32.96
35.68
16.28
18.85
24.56
22.94
38.47
32.96
25.95
38.41
30.46
34.46
31.46
26.45
20.44
19.93
8.91
eN(CH2CH3)2
eN(CH2CH2)2O
eN(CH2CH2)2NCH3
eN(CH2CH2)2CH2
eN(CH2CH2)2
eN(CH3)2
eN(CH2CH3)2
eN(CH3)2
eN(CH2CH3)2
eN(CH3)2
eN(CH3)2
eN(CH3)2
eN(CH3)2
eN(CH3)2
eN(CH3)2
eN(CH3)2
eN(CH3)2
eN(CH3)2
44.48
>50
26.74
27.45
30.26
21.44
29.45
28.38
25.95
18.93
29.95
24.94
29.56
5.51
6.52
42.98
18.23
4.52
20.44
0.54
eN(CH2CH2)2CH2
e
e
e
e
e
e
e
5.37
11.62
26.45
a
The IC50 represents compound concentration giving 50% survival of each cell line.
inhibit ATP hydrolysis. As shown in Fig. 6A, all of the selected
compounds with significant Topo II inhibitory activity at 25 mM,
We performed the DNA unwinding assay by using eukaryotic DNA
Topo I to clarify the intercalation property of the compounds. In the
presence of the DNA intercalators, a relaxed plasmid DNA appears
to be supercoiled plasmid DNA by treatment with Topo I. As shown
in Fig. 7A, ethidium bromide (EB), a well-known DNA intercalator,
intercalated into plasmid DNA by converting relaxed DNA to
supercoiled DNA substrate in the presence of Topo I in dose-
dependent manner. In the presence of the selected compound 6c-
1, relaxed DNA was transformed into supercoiled DNA substrate by
treatment with Topo I, which was identical to the action of
ethidium bromide (EB). This finding provided evidence that com-
pound 6c-1 could intercalate into DNA. However, the ability of
compound 6c-1 to intercalate into DNA was far less than that of EB,
and had less Topo I/II inhibitory activity and cytotoxicity towards
cancer cell lines.
The intercalating ability of the compounds was also assessed by
using UVeVis spectrometry characterization of the compounds in
absence or presence of CT-DNA. It is widely accepted that if the
compounds could intercalate into DNA, the UVeVis curve of their
complex would demonstrate hypochromicity and bathochromic
shifts [30]. As shown in Fig. 7B, when increasing the concentration
of DNA, the absorption spectra of compound 6c-1 showed a sig-
nificant hypochromicity but without an obvious bathochromic
shift, indicating that compound 6c-1 had a strong interaction with
showed good inhibitory activities higher than the positive control
1,4-naphthoquinone (1,4-NQ) [29]. Especially, compounds 6f-3
showed the best inhibitory activity of 78% on ATP hydrolysis,
comparing with 56% for 1,4-NQ.
To further determine the mode of action for this class of com-
pounds towards ATPase of hTopo II, an in vitro DNA independent
ATP hydrolysis assay was performed using compound 6f-3.
Different concentrations of ATP (0.25, 0.5, 0.8, 1.0, and 2.0 mM)
were incubated with compound 6f-3 of a fixed concentration at 0,
5, 25, 50, and 100 mM, respectively. As shown in Fig. 6B, compound
6f-3 effectively inhibited ATPase of hTopo II in a concentration-
dependent manner. The double reciprocal LineweavereBurk plot
revealed a decrease in Km while no change in Vmax. This result
indicated that compound 6f-3 acted as a competitive inhibitor of
ATP.
3.6. DNA-binding properties
Since the DNA intercalators can interfere with the DNA relaxa-
tion reaction by altering the apparent topological state of nega-
tively supercoiled DNA substrate, they induce constrained negative
and unstrained positive superhelical twists in plasmids DNA [16].

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B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
Fig. 4. (A) Effects of CPT and 6c-1 on Topo I-DNA cleavage complexes formation at various concentrations. Agarose gel electrophoresis of pBR322 (0.1
mg) DNA containing increasing
concentration of CPT and 6c-1 upon treatment with excess Topo I (10 units). (B) Optical density analysis of the nicked DNA induced by CPT and 6c-1 at various concentrations. (C)
Analysis of cellular Topo I-DNA cleavage complexes induced by CPT and 6c-1 at concentrations of 2, 5 and 10 mM, respectively. 0 mM represents the blank control group (upper) and
1.0% DMSO (v/v) control group (bottom).
DNA. The further analysis of the results revealed the Kapp value of
charge on nitrogen of side chain formed hydrogen bonding inter-
action with Thr147. Moreover, the carbonyl group at C-5 position of
ring B of benzo[a]phenazin scaffold was close to Ser148 and Ser149,
indicating that it may have the potential to form hydrogen bonding
interaction with these two amino acid residues. The docking results
further confirmed the importance of the terminal amino side chain
in inhibitory activity against Topo I and II.
compound 6c-1 and DNA was 4.83 ꢁ 105 Mꢂ1
.
3.7. Molecular modeling
Since the mechanism studies revealed that this class of com-
pounds could stabilize the Topo I-DNA cleavage complexes and
inhibit the ATPase activity of Topo II, molecular docking stimula-
tions were performed to better understand the potential interac-
tion mode of these compounds with the Topo I-DNA cleavage
complexes (PDB code: 1K4T) and the ATPase domain of Topo II (PDB
code: 1ZXM), respectively.
Compound 6c-1 was chosen to simulate the interaction with the
Topo I-DNA cleavage complexes. As shown in Fig. 8A, it was found
that 6c-1 was well-fitted into the site of DNA cleavage and formed
base-stacking interactions with both the ꢂ1 (upstream) and þ1
(downstream) base pairs of DNA. Besides, the nitrogen cation of
side chain formed hydrogen bonding interaction with TGP 11 and
had electrostatic interaction with the carboxyl group of Asp533.
The interaction mode of the selected compound 6f-3 with the
ATPase domain of Topo II was also explored as shown in Fig. 8B.
Compound 6f-3 was docked into the ATP binding site of the ATPase
domain of Topo II. It was found that 6f-3 could fit into the ATP
binding pocket, and form hydrophobic interaction with Ile141,
Val137, Phe142, Pro126, Ile125, and Asn91. In addition, the positive
4. Conclusion
In this paper, a series of novel benzo[a]phenazin derivatives were
designed, synthesized and evaluated for Topo I and II inhibitory
activity and cytotoxicity against several human cancer cell lines.
Some of the compounds exhibited significant Topo I and Topo II
inhibitory activity at 25 mM concentration. Our MTT assays showed
that most of these compounds had better activity for HL-60 cells
than the other three tested cell lines, with IC50 values in the sub-
micromolar range. The cytotoxicity of these compounds had some
relationship with their Topo I and II inhibitory activity. Investigation
of SARs revealed that the substitution of amino groups on terminal
of side chain at N-7 position could significantly enhance the Topo I
and II inhibitory activity and cytotoxicity of the compounds. Among
these compounds, the compounds with the dimethylamino termi-
nal (6a-1, 6b-1, 6c-1) showed good Topo I and Topo II inhibitory
activity while the compounds with other terminal groups exhibited
partial or moderate inhibition of Topo I and Topo II. Our results
indicated that the type of terminal amino moiety had a crucial role in
the inhibitory activity. Introduction of different substitutive group
on the ring A or ring D of the scaffolds did not produce favorable
effects on Topo I and Topo II activity. Only compound 6d-4 with
eOCH3 group at position C-9 exhibited good Topo I and Topo II
inhibitory activity at 25
mM concentration. Further mechanistic
studies showed that this class of compounds could stabilize the Topo
I-DNA cleavage complexes and inhibit the ATPase activity of hTopo II,
indicating that these compounds are dual Topo inhibitors by func-
tioning as Topo I poisons and Topo II catalytic inhibitors. The ICE
assay further demonstrated 6c-1 could stabilize the Topo I-DNA
cleavage complexes in vivo, and kinetic studies revealed that 6f-3
acted as a competitive inhibitor of ATP. Molecular docking studies
Fig. 5. Topo II mediated cleavage assay of plasmid pBR322 DNA. Lane 1: pBR322 DNA
only. Lane 2: pBR322 DNA and Topo II. Lane 3: 100
mM VP-16, Topo II and pBR322 DNA.
Lane 4e5: 6c-1 (10 M, 50 M), Topo II and pBR322 DNA.
m
m

B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
547
Fig. 6. (A) ATPase inhibitory activity of 6c-1, 6d-4, and 6f-3 in the presence of 1 mM ATP. The inhibition of ATPase activity was measured by adding 1 mM of each compound to the
reaction at final concentration of 100 M. The optical density (OD620) values obtained for the compounds were compared against the background group without enzyme, which was
m
set as 100% inhibitory rate, while the enzyme only group was set as 0% inhibitory rate. (B) The double reciprocal LineweavereBurk plot for ATP-hydrolysis activity of ATPase in the
absence of DNA.
Fig. 7. Effect of 6c-1 on DNA unwinding. (A) Agarose gel electrophoresis of relaxed pBR322 DNA containing increasing concentration of EB and 6c-1. (B) Absorption spectra of 6c-1
upon addition of CT-DNA. [6c-1] ¼ 15
mM, [DNA] ¼ 0e24
mM. Arrow indicated the absorption change upon increasing amount of CT-DNA. Insert graph shows the plot of [DNA]/
(εaꢂεf) versus [DNA].
suggested that these novel compounds exertedtheir Topo I and Topo
II inhibitory activity via intercalating into the cleavage site of the
Topo I-DNA cleavage complexes and blocking the ATP-binding site of
Topo II, respectively. Our present results could provide useful in-
formation for further structural modification of benzophenazin for
discovery of potent anticancer drugs.
5. Experimental section
5.1. Chemistry
All commercial chemicals used as starting materials were
analytical grade and utilized without further purification. Melting
Fig. 8. Results of the docking simulation for compounds 6c-1 and 6f-3. (A) Binding mode of 6c-1 with Topo I-DNA complex. (B) Binding mode of 6f-3 with ATPase binding site of
Topo II. The green dashed lines indicate the hydrogen bonding interaction, and the red dashed lines represent the electrostatic interaction. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

548
B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
points were determined by using capillary tubes with a MSRS-
OptiMelt Automated Melting point system and are uncorrected.
1H and 13C NMR spectra were recorded using TMS as the internal
standard in CDCl3, D2O, DMSO-d6 or CD3OD with a Bruker BioSpin
GmbH spectrometer at 400 MHz. High resolution mass spectra
(HRMS) were recorded on Shimadzu LCMS-IT-TOF. The purity of
synthesized compounds was proved to be more than 95% by using
analytical HPLC equipped with Shimadzu LC-20AB system and an
5.1.4.1. 7-(2-(Dimethylamino)ethyl)benzo[a]phenazin-5(7H)-one
(6a-1). Red solid, yield 47.2%. m.p. 168.4e171.3 ꢀC. 1H NMR
(400 MHz, DMSO-d6)
d
8.84 (d, J ¼ 6.7 Hz, 1H), 8.21 (d, J ¼ 5.7 Hz,
1H), 8.01 (d, J ¼ 7.4 Hz, 1H), 7.89e7.69 (m, 4H), 7.49 (t, J ¼ 7.2 Hz,
1H), 6.18 (s, 1H), 4.44 (s, 2H), 2.71 (s, 2H), 2.34 (s, 6H). 13C NMR
(101 MHz, MeOD)
d 183.1, 146.4, 140.3, 136.2, 133.3, 132.9, 132.1,
132.0, 126.0, 125.8, 125.7, 115.1, 98.8, 54.6, 45.8, 45.4. HRMS (ESI):
calcd for C20H19N3O [MþH]þ 318.1601, found 318.1587. HPLC purity:
98.37%.
Ultimate XB-C18 column (4.6 ꢁ 250 mm, 5
mm), which was eluted
with methanolewater (70:30e90:10) containing 0.1% TFA at a flow
rate of 0.2 mL/min.
5.1.4.2. 7-(2-(Diethylamino)ethyl)benzo[a]phenazin-5(7H)-one (6a-
2). Red solid, yield 43.1%. m.p. 169.1e171.2 ꢀC. 1H NMR (400 MHz,
5.1.1. General procedure for the preparation of intermediate 2a-
1 ~ 2a-6, 2b-1 ~ 2b-6, 2c-1 ~ 2c-2, 2e-1 ~ 2e-3, 2g-1 ~ 2g-2
Compound 1 (20 mmol) and proper amine (20 mmol) were
dissolved in tetrahydrofuran, and the reaction mixture was stirred
at 30 ꢀC for 12 h under nitrogen. After completion of the reaction,
the mixture was then extracted with dichloromethane after evap-
orating the tetrahydrofuran, washed with water, and concentrated
to provide crude oil product. The residue was used for next syn-
thetic step without further purification.
CD3OD)
d
9.16 (d, J ¼ 7.8 Hz, 1H), 8.37 (d, J ¼ 8.5 Hz, 2H), 8.25 (d,
J ¼ 8.6 Hz, 1H), 8.06 (td, J ¼ 7.2, 1.6 Hz, 1H), 7.95 (td, J ¼ 7.2, 1.2 Hz,
1H), 7.89 (td, J ¼ 7.2, 1.2 Hz, 1H), 7.84 (t, J ¼ 7.2 Hz, 1H), 5.39e5.27
(m, 2H), 3.67e3.57 (m, 2H), 3.45 (q, J ¼ 7.2 Hz, 4H),1.40 (t, J ¼ 7.3 Hz,
6H). 13C NMR (101 MHz, CD3OD)
d 182.8, 146.2, 140.2, 136.0, 133.3,
133.1, 132.8, 132.0, 131.9, 131.8, 125.9, 125.6, 125.6, 115.2, 98.8, 49.7,
48.4, 46.1, 12.2. HRMS (ESI): calcd for C22H23N3O [MþH]þ 346.1914,
found 346.1903. HPLC purity: 99.19%.
5.1.4.3. 7-(2-Morpholinoethyl)benzo[a]phenazin-5(7H)-one (6a-3).
Red solid, yield 42.3%. m.p. 170.3e172.4 ꢀC. 1H NMR (400 MHz,
5.1.2. General procedure for the preparation of intermediates 3a-
1 ~ 3a-6, 3b-1 ~ 3b-6, 3c-1 ~ 3c-2, 3e-1 ~ 3e-3, 3g-1 ~ 3g-2
Above intermediate 2 was dissolved in 100 mL acetic acid, and
cooled in ice bath. 100 mmol of Zinc powder was added to the
solvent slowly in three portions over 30 min. The reaction mixture
was warmed to room temperature and stirred for another 30 min.
After the reaction was completed, the mixture was filtered and
filtrate was used for next step without further purification.
CD3OD)
d
8.98 (d, J ¼ 7.9 Hz, 1H), 8.25 (d, J ¼ 7.2 Hz, 1H), 8.16 (d,
J ¼ 8.1 Hz, 1H), 7.95 (d, J ¼ 8.6 Hz, 1H), 7.88e7.75 (m, 3H), 7.62 (t,
J ¼ 7.6 Hz, 1H), 6.51 (s, 1H), 4.98 (s, 2H), 3.97 (s, 4H), 3.62e3.57 (m,
2H), 3.53 (s, 4H). 13C NMR (101 MHz, DMSO-d6)
d 180.4,146.0,138.9,
134.2, 132.3, 132.1, 131.4, 131.1, 130.9, 130.4, 124.7, 124.3, 114.5, 98.8,
63.4, 51.2, 49.1. HRMS (ESI): calcd for C22H21N3O2 [MþH]þ 360.1707,
found 360.1714. HPLC purity: 99.23%.
5.1.3. General procedure for the preparation of intermediates 5a
and 5b
5.1.4.4. 7-(2-(4-Methylpiperazin-1-yl)ethyl)benzo[a]phenazin-
5(7H)-one (6a-4). Red solid, yield 45.2%. m.p. 184.6e185.4 ꢀC. 1H
A solution of compound 4 (20 mmol) in dry t-BuOH (50 mL) was
added to a 1 M solution of KO-t-Bu in dry t-BuOH (50 mL) which
had previously been saturated with O2 and the red solution vigor-
ously stirred in an atmosphere of O2 until 2 equiv were absorbed.
After the reaction was completed, the cooled solution was acidified
with concentrated HCl, and yellow precipitate was formed imme-
diately. Then the reaction mixture was filtered and the precipitate
was washed with water, dried over anhydrous sodium sulfate, and
concentrated to give the desired product [31,32].
NMR (400 MHz, CDCl3)
d
8.95 (dd, J ¼ 6.0, 3.3 Hz, 1H), 8.42 (dd,
J ¼ 6.0, 3.2 Hz, 1H), 8.06 (dd, J ¼ 8.0, 1.4 Hz, 1H), 7.83e7.75 (m, 2H),
7.64 (td, J ¼ 7.2, 1.6 Hz, 1H), 7.53 (d, J ¼ 8.1 Hz, 1H), 7.44 (t, J ¼ 7.5 Hz,
1H), 6.30 (s, 1H), 4.40 (t, J ¼ 7.6 Hz, 2H), 2.86 (t, J ¼ 7.6 Hz, 2H), 2.71
(s, 4H), 2.52 (s, 4H), 2.33 (s, 3H). 13C NMR (101 MHz, CD3OD)
d 183.0,
146.4, 140.4, 136.2, 133.4, 133.2, 132.9, 132.2, 132.1, 132.0, 131.9,
126.0, 125.7, 115.4, 98.9, 55.9, 54.0, 53.8, 46.0, 45.6. HRMS (ESI):
calcd for C23H24N4O [MþH]þ 373.2023, found 373.1998. HPLC pu-
rity: 97.57%.
5.1.3.1. 2-Hydroxy-6-methoxynaphthalene-1,4-dione
(5a).
8.06 (d,
Yellow solid, yield 45.2%. 1H NMR (400 MHz, CDCl3)
d
5.1.4.5. 7-(2-(Piperidin-1-yl)ethyl)benzo[a]phenazin-5(7H)-one (6a-
5). Red solid, yield 46.7%. m.p. 183.7e185.0 ꢀC. 1H NMR (400 MHz,
J ¼ 8.4 Hz, 1H), 7.57 (s, 1H), 7.43 (s, 1H), 7.16 (d, J ¼ 6.3 Hz, 1H), 6.30
CD3OD)
d
8.67 (d, J ¼ 7.7 Hz, 1H), 8.00 (d, J ¼ 7.6 Hz, 1H), 7.86 (d,
(s, 1H), 3.97 (s, 3H).
J ¼ 7.6 Hz, 1H), 7.74 (d, J ¼ 8.0 Hz, 1H), 7.67 (t, J ¼ 7.3 Hz, 1H), 7.61 (t,
J ¼ 7.5 Hz, 1H), 7.54 (t, J ¼ 7.1 Hz, 1H), 7.40 (t, J ¼ 7.5 Hz, 1H), 6.14 (s,
1H), 3.41 (s, 2H), 3.21 (m, 6H), 1.92 (s, 4H), 1.68 (s, 2H). 13C NMR
5.1.3.2. 2-Hydroxy-7-methoxynaphthalene-1,4-dione
(5b).
7.88 (d,
Yellow solid, yield 42.7%. 1H NMR (400 MHz, DMSO-d6)
d
(101 MHz, DMSO-d6)
d 180.3, 146.0, 138.8, 134.2, 132.3, 132.0, 131.5,
J ¼ 8.6 Hz, 1H), 7.41 (d, J ¼ 2.6 Hz, 1H), 7.35 (dd, J ¼ 8.6, 2.7 Hz, 1H),
131.4, 131.1, 130.9, 130.4, 124.6, 124.2, 114.5, 98.7, 52.3, 49.5, 49.3,
22.7, 21.5. HRMS (ESI): calcd for C23H23N3O [MþH]þ 358.1914,
found 358.1891. HPLC purity: 99.79%.
6.14 (s, 1H), 3.92 (s, 3H).
5.1.4. General procedure for the preparation of 6a-1 ~ 6a-6, 6b-
1 ~ 6b-6, 6c-1 ~ 6c-2, 6d-1 ~ 6d-4, 6e-1 ~ 6e-3, 6f-1 ~ 6f-4, and 6g-
1 ~ 6g-2
5.1.4.6. 7-Propylbenzo[a]phenazin-5(7H)-one(6a-6). Red
solid,
To a stirred solution of above intermediates 3 in 100 mL acetic
acid, 2-hydroxy-1,4-naphoquinone anologs 5 (20 mmol) was added
at room temperature. Precipitate was formed immediately. Stirring
was continued for 60 min at 60 ꢀC. Then, the mixture was filtered
and the precipitate was washed with water to give crude solid
product. The solid residue was purified by using chromatography
with PE/CH2Cl2/MeOH/NH3$H2O elution to give the desired product
[25].
yield 48.2%. m.p. 158.9e160.3 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d
8.86e8.79 (m, 1H), 8.24e8.17 (m, 1H), 8.01 (dd, J ¼ 8.0, 1.4 Hz, 1H),
7.88e7.79 (m, 3H), 7.73 (t, J ¼ 7.1 Hz, 1H), 7.49 (t, J ¼ 7.1 Hz, 1H), 6.22
(s, 1H), 4.33e4.24 (m, 2H), 1.85e1.75 (m, 2H), 1.11 (t, J ¼ 7.4 Hz, 3H).
13C NMR (101 MHz, DMSO-d6)
d 179.6, 145.7, 138.7, 134.2, 132.4,
132.0, 131.4, 131.2, 130.8, 130.3, 124.6, 124.5, 124.0, 114.7, 98.1, 47.1,
18.6,10.7. HRMS (ESI): calcd for C19H16N2O [MþH]þ 289.1335, found
289.1332. HPLC purity: 99.39%.

B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
549
5.1.4.7. 7-(3-(Dimethylamino)propyl)benzo[a]phenazin-5(7H)-one
5.1.4.13. 7-(4-(Dimethylamino)butyl)benzo[a]phenazin-5(7H)-one
(6b-1). Red solid, yield 48.1%. m.p. 163.7e165.6 ꢀC. 1H NMR
(6c-1). Red solid, yield 46.5%. m.p. 150.3e152.5 ꢀC. 1H NMR
(400 MHz, CDCl3)
d
9.02e8.87 (m, 1H), 8.44e8.34 (m, 1H), 8.04 (d,
(400 MHz, CDCl3) d 8.98e8.93 (m, 1H), 8.45e8.39 (m, 1H), 8.06 (d,
J ¼ 7.9 Hz, 1H), 7.79e7.74 (m, 2H), 7.68e7.58 (m, 2H), 7.41 (t,
J ¼ 7.2 Hz, 1H), 6.29 (s, 1H), 4.34 (t, J ¼ 8.0 Hz, 2H), 2.48 (t, J ¼ 6.4 Hz,
2H), 2.34 (s, 6H), 2.12e1.97 (m, 2H). 13C NMR (101 MHz, CD3OD)
J ¼ 7.8 Hz, 1H), 7.81e7.75 (m, 2H), 7.64 (t, J ¼ 7.2 Hz, 1H), 7.56 (d,
J ¼ 8.6 Hz,1H), 7.43 (t, J ¼ 7.6 Hz,1H), 6.27 (s,1H), 4.31e4.21 (m, 2H),
2.48 (t, J ¼ 7.0 Hz, 2H), 2.35 (s, 6H), 2.03e1.90 (m, 2H), 1.83e1.73 (m,
d
183.1, 146.4, 140.3, 136.3, 133.5, 133.2, 133.0, 132.0, 131.9, 126.0,
2H). 13C NMR (101 MHz, CD3OD)
d 182.9, 146.2, 140.1, 136.1, 133.4,
125.7, 115.3, 98.7, 57.1, 45.8, 45.6, 24.7. HRMS (ESI): calcd for
C
133.2, 132.8, 132.0, 131.8, 131.8, 125.9, 125.6, 125.5, 115.3, 98.6, 59.9,
47.5, 45.5, 25.2, 24.6. HRMS (ESI): calcd for C22H23N3O [MþH]þ
346.1914, found 346.1924. HPLC purity: 99.62%.
19H16N2O [MþH]þ 332.1757, found 332.1740. HPLC purity: 99.34%.
5.1.4.8. 7-(3-(Diethylamino)propyl)benzo[a]phenazin-5(7H)-one
5.1.4.14. 7-(4-(Diethylamino)butyl)benzo[a]phenazin-5(7H)-one (6c-
2). Red solid, yield 45.2%. m.p. 151.7e153.4 ꢀC. 1H NMR (400 MHz,
(6b-2). Red solid, yield 43.9%. m.p. 163.1e164.5 ꢀC. 1H NMR
(400 MHz, CDCl3) d 8.97e8.93 (m, 1H), 8.42e8.39 (m, 1H), 8.05 (dd,
CDCl3)
d
8.97e8.93 (m, 1H), 8.43e8.40 (m, 1H), 8.04 (d, J ¼ 7.4 Hz,
J ¼ 8.0, 1.4 Hz, 1H), 7.79e7.75 (m, 2H), 7.65 (d, J ¼ 7.6 Hz, 1H), 7.60
(td, J ¼ 6.8, 1.6 Hz, 1H), 7.42 (td, J ¼ 8.0, 1.6 Hz, 1H), 6.33 (s, 1H),
4.42e4.34 (m, 2H), 3.81 (t, J ¼ 4.4 Hz, 4H), 2.53 (s, 6H), 2.14e2.02
1H), 7.79e7.75 (m, 2H), 7.65e7.58 (m, 2H), 7.42 (dd, J ¼ 7.2, 2.2 Hz,
1H), 6.27 (s, 1H), 4.29e4.21 (m, 2H), 2.67e2.52 (m, 6H), 2.00e1.86
(m, 2H), 1.81e1.65 (m, 2H), 1.09 (t, J ¼ 7.1 Hz, 6H). 13C NMR
(m, 2H), 1.58 (s, 2H). 13C NMR (101 MHz, DMSO-d6)
d 180.1, 145.9,
(101 MHz, CD3OD)
d 183.0, 146.5, 140.3, 136.3, 133.5, 133.2, 133.0,
138.7, 134.2, 132.3, 131.9, 131.4, 131.3, 131.1, 130.9, 130.4, 124.6, 124.1,
114.4, 98.4, 63.3, 52.8, 51.2, 42.9, 19.9. HRMS (ESI): calcd for
132.1, 132.0, 131.9, 126.0, 125.7, 125.6, 115.5, 98.7, 53.2, 47.7, 47.6,
24.8, 24.4, 11.4. HRMS (ESI): calcd for C24H27N3O [MþH]þ 374.2227,
found 374.2219. HPLC purity: 98.26%.
C
23H25N3O [MþH]þ 360.2070, found 360.2066. HPLC purity:
99.96%.
5.1.4.15. 7-(3-(Dimethylamino)propyl)-3-methoxybenzo[a]phenazin-
5(7H)-one (6d-1). Red solid, yield 45.6%. m.p. 172.6e174.1 ꢀC. 1H
5.1.4.9. 7-(3-Morpholinopropyl)benzo[a]phenazin-5(7H)-one (6b-3).
Red solid, yield 42.1%. m.p. 164.1e165.7 ꢀC. 1H NMR (400 MHz,
NMR (400 MHz, CDCl3)
d
8.88 (d, J ¼ 8.7 Hz, 1H), 8.03 (d, J ¼ 7.9 Hz,
CDCl3)
d
8.97e8.93 (m, 1H), 8.42e8.37 (m, 1H), 8.05 (d, J ¼ 8.0 Hz,
1H), 7.84 (d, J ¼ 2.3 Hz, 1H), 7.67 (d, J ¼ 8.4 Hz, 1H), 7.61 (t, J ¼ 7.6 Hz,
1H), 7.42 (t, J ¼ 7.5 Hz, 1H), 7.31 (dd, J ¼ 8.7, 2.4 Hz, 1H), 6.30 (s, 1H),
4.37 (s, 2H), 4.00 (s, 3H), 2.57e2.51 (m, 2H), 2.38 (s, 6H), 2.14e2.06
1H), 7.79e7.75 (m, 2H), 7.68e7.57 (m, 2H), 7.42 (t, J ¼ 7.3 Hz, 1H),
6.33 (s, 1H), 4.40e4.34 (m, 2H), 3.81 (s, 4H), 2.53 (s, 6H), 2.11e2.05
(m, 2H). 13C NMR (101 MHz, CDCl3)
d 182.0,146.8,139.1,135.0,132.9,
(m, 2H). 13C NMR (101 MHz, DMSO-d6)
d 179.5, 161.9, 145.4, 138.4,
132.1, 131.5, 131.3, 131.2, 131.1, 130.7, 125.3, 125.0, 123.8, 113.3, 99.0,
67.0, 55.3, 53.8, 44.2, 22.6. HRMS (ESI): calcd for C23H23N3O2
[MþH]þ 374.1863, found 374.1864. HPLC purity: 99.77%.
134.5, 134.3, 131.2, 130.9, 130.0, 126.9, 124.6, 123.9, 118.8, 114.4,
106.7, 98.1, 55.6, 45.0, 43.9, 23.1. HRMS (ESI): calcd for C22H23N3O2
[MþH]þ 362.1863, found 362.1867. HPLC purity: 99.75%.
5.1.4.16. 7-(3-(Diethylamino)propyl)-3-methoxybenzo[a]phenazin-
5.1.4.10. 7-(3-(4-Methylpiperazin-1-yl)propyl)benzo[a]phenazin-
5(7H)-one (6d-2). Red solid, yield 42.8%. m.p. 168.2e170.3 ꢀC. 1H
5(7H)-one (6b-4). Red solid, yield 46.4%. m.p. 170.2e172.3 ꢀC. 1H
NMR (400 MHz, CDCl3)
d
8.88 (d, J ¼ 8.8 Hz, 1H), 8.08 (d, J ¼ 8.0 Hz,
NMR (400 MHz, CDCl3)
d 9.00e8.92 (m, 1H), 8.42e8.38 (m, 1H),
1H), 8.03 (d, J ¼ 8.4 Hz, 1H), 7.81 (t, J ¼ 7.6 Hz, 1H), 7.70 (s, 1H), 7.57
8.05 (dd, J ¼ 8.0, 1.2 Hz, 1H), 7.80e7.75 (m, 2H), 7.68e7.58 (m, 2H),
7.42 (td, J ¼ 6.8, 1.6 Hz, 1H), 6.38 (s, 1H), 4.36 (t, J ¼ 7.2 Hz, 2H), 2.61
(br, 8H), 4.53 (t, J ¼ 6.4 Hz, 2H), 2.40 (s, 3H), 2.06 (m, 2H). 13C NMR
(t, J ¼ 7.5 Hz, 1H), 7.32 (d, J ¼ 8.8 Hz, 1H), 6.99 (s, 1H), 4.56 (s, 2H),
3.93 (s, 3H), 3.50 (s, 2H), 2.45 (br, 4H), 1.39 (t, J ¼ 6.9 Hz, 6H). 13
C
NMR (101 MHz, CD3OD)
d 181.7, 163.3, 145.4, 139.1, 136.5, 132.9,
(101 MHz, CD3OD)
d 183.0, 146.5, 140.4, 136.3, 133.6, 133.1, 133.0,
131.8, 130.9, 128.1, 126.1, 125.7, 119.9, 117.5, 115.3, 107.1, 82.3, 56.0,
50.1, 44.8, 30.7, 22.0, 9.3. HRMS (ESI): calcd for C24H27N3O2 [MþH]þ
390.2176, found 390.2163. HPLC purity: 98.73%.
132.2, 132.1, 132.0, 131.9, 126.1, 125.7, 125.6, 115.5, 98.9, 55.9, 55.7,
53.8, 46.0, 45.7, 24.0. HRMS (ESI): calcd for C24H26N4O [MþH]þ
387.2179, found 387.2177. HPLC purity: 99.66%.
5.1.4.17. 7-(2-(Dimethylamino)ethyl)-3-methoxybenzo[a]phenazin-
5.1.4.11. 7-(3-(Piperidin-1-yl)propyl)benzo[a]phenazin-5(7H)-one
5(7H)-one (6d-3). Red solid, yield 42.1%. m.p. 180.1e182.4 ꢀC. 1H
(6b-5). Red solid, yield 43.5%. m.p. 163.5e165.4 ꢀC. 1H NMR
NMR (400 MHz, CD3OD)
d
9.16 (d, J ¼ 9.0 Hz, 1H), 8.42 (d, J ¼ 8.2 Hz,
(400 MHz, CDCl3) d 8.98e8.92 (m, 1H), 8.43e8.39 (m, 1H), 8.04 (dd,
1H), 8.31 (d, J ¼ 8.7 Hz, 1H), 8.11 (t, J ¼ 7.9 Hz, 1H), 7.92 (t, J ¼ 7.6 Hz,
1H), 7.86 (d, J ¼ 2.7 Hz, 1H), 7.59 (dd, J ¼ 9.0, 2.7 Hz, 1H), 7.06 (s, 1H),
J ¼ 8.0, 1.4 Hz, 1H), 7.82e7.71 (m, 3H), 7.62 (td, J ¼ 7.2, 1.2 Hz,1H),
7.42 (td, J ¼ 7.2,1.2 Hz,1H), 6.30 (s,1H), 4.35 (s, 2H), 2.49 (s, 6H), 2.08
5.40e5.33 (m, 2H), 4.04 (s, 3H), 3.80e3.71 (m, 2H), 3.21 (s, 6H). 13
C
(s, 2H), 1.70 (s, 4H), 1.51 (s, 2H). 13C NMR (101 MHz, CD3OD)
d 183.0,
NMR (101 MHz, CD3OD)
d 182.6, 163.6, 146.0, 139.8, 136.4, 135.1,
146.4, 140.3, 136.2, 133.5, 133.1, 132.9, 132.1, 132.0, 131.8, 126.0,
125.7, 125.6, 115.4, 98.8, 56.6, 55.7, 45.9, 26.9, 25.3, 24.0. HRMS
(ESI): calcd for C24H25N3O [MþH]þ 372.2070, found 372.2076. HPLC
purity: 98.38%.
132.6, 131.8, 131.5, 128.1, 126.0, 125.7, 120.0, 115.0, 107.6, 98.7, 56.0,
54.8, 45.9, 45.7. HRMS (ESI): calcd for
348.1707, found 348.1712. HPLC purity: 97.87%.
C
21H21N3O2 [MþH]þ
5.1.4.18. 7-(3-(Dimethylamino)propyl)-2-methoxybenzo[a]phenazin-
5.1.4.12. 7-(3-(Pyrrolidin-1-yl)propyl)benzo[a]phenazin-5(7H)-one
5(7H)-one (6d-4). Red solid, yield 44.6%. m.p. 160.2e161.8 ꢀC. 1H
(6b-6). Red solid, yield 43.8%. m.p. 163.9e165.7 ꢀC. 1H NMR
NMR (400 MHz, CDCl3)
d
8.36 (d, J ¼ 2.8 Hz, 1H), 8.31 (d, J ¼ 8.8 Hz,
(400 MHz, CD3OD)
d
8.41e8.33 (m,1H), 7.88 (dd, J ¼ 5.4, 3.2 Hz,1H),
1H), 8.03 (d, J ¼ 7.6 Hz, 1H), 7.68e7.60 (m, 2H), 7.40 (td, J ¼ 7.6,
2.0 Hz, 1H), 7.29 (dd, J ¼ 8.7, 2.7 Hz, 1H), 6.16 (s, 1H), 4.39e4.26 (m,
7.58 (d, J ¼ 7.7 Hz, 1H), 7.46e7.33 (m, 4H), 7.18 (t, J ¼ 6.9 Hz, 1H),
5.89 (s, 1H), 4.02 (s, 2H), 2.52 (t, J ¼ 7.2 Hz, 2H), 2.47 (s, 2H),
2H), 4.04 (s, 3H), 2.60 (s, 2H), 2.42 (s, 6H), 2.15e2.09 (m, 2H). 13
C
1.86e1.76 (m, 2H), 1.72 (s, 4H). 13C NMR (101 MHz, CD3OD)
d
183.1,
NMR (101 MHz, CD3OD) d 189.4, 163.4, 145.5, 139.8, 137.00, 134.5,
146.5,140.4,136.3,133.5,133.2,133.0,132.1,132.1,131.9,126.0,125.7,
125.6, 115.4, 98.8, 55.1, 53.9, 45.8, 26.0, 24.4. HRMS (ESI): calcd for
C
132.4, 131.7, 127.4, 126.9, 119.9, 115.9, 108.7, 92.8, 81.9, 71.5, 56.3,
55.7, 45.3, 43.9, 30.7. HRMS (ESI): calcd for C22H23N3O2 [MþH]þ
362.1863, found 362.1888. HPLC purity: 99.77%.
23H23N3O [MþH]þ 358.1914, found 358.1914. HPLC purity: 99.89%.

550
B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
5.1.4.19. 7-(3-(Dimethylamino)propyl)-10-methoxybenzo[a]phena-
5.1.4.25. 7-(3-(Dimethylamino)propyl)benzo[f]pyrido[2,3-b]quinox-
zin-5(7H)-one (6e-1). Red solid, yield 46.4%. m.p.164.1e165.7 ꢀC. 1H
alin-5(7H)-one (6g-1). Red solid, yield 43.2%. m.p. 157.1e159.4 ꢀC.
NMR (400 MHz, D2O)
d
7.62 (d, J ¼ 7.1 Hz, 1H), 7.24 (d, J ¼ 8.1 Hz,
1H NMR (400 MHz, CDCl3)
d
8.96 (d, J ¼ 7.0 Hz, 1H), 8.75 (s, 1H),
1H), 7.18 (d, J ¼ 9.5 Hz, 1H), 7.11e7.01 (m, 2H), 6.93 (dd, J ¼ 9.3,
2.7 Hz, 1H), 6.49 (d, J ¼ 2.6 Hz, 1H), 3.98 (s, 2H), 3.61 (s, 3H),
3.29e3.18 (m, 2H), 2.81 (s, 6H), 1.99e1.86 (m, 2H). 13C NMR
8.43e8.36 (m, 2H), 7.88e7.77 (m, 2H), 7.59e7.49 (m, 1H), 7.04 (s,
1H), 4.83 (s, 2H), 3.40 (s, 2H), 2.90 (s, 6H), 2.41 (s, 2H). 13C NMR
(101 MHz, CD3OD)
d 183.8, 152.1, 148.3, 142.9, 141.4, 139.5, 133.3,
(101 MHz, D2O)
d
174.7, 157.3, 142.3, 136.9, 135.9, 131.3, 130.9, 129.0,
133.0, 132.7, 132.5, 131.2, 126.4, 125.9, 122.2, 100.7, 56.2, 43.6, 41.7,
22.6. HRMS (ESI): calcd for C20H20N4O [MþH]þ 333.1710, found
333.1716. HPLC purity: 99.66%.
128.3, 124.6, 124.1, 123.8, 123.5, 115.7, 109.8, 100.0, 55.9, 53.9, 44.8,
42.9, 21.5. HRMS (ESI): calcd for C22H23N3O2 [MþH]þ 362.1863,
found 362.1870. HPLC purity: 99.31%.
5.1.4.26. 7-(3-(Piperidin-1-yl)propyl)benzo[f]pyrido[2,3-b]quinox-
alin-5(7H)-one (6g-2). Red solid, yield 42.1%. m.p. 156.4e158.2 ꢀC.
5.1.4.20. 10-Chloro-7-(3-(dimethylamino)propyl)benzo[a]phenazin-
1H NMR (400 MHz, CDCl3)
d
9.13e8.95 (m, 1H), 8.80 (dd, J ¼ 4.5,
5(7H)-one (6e-2). Red solid, yield 42.6%. m.p. 173.0e174.8 ꢀC. 1H
1.6 Hz, 1H), 8.51e8.39 (m, 2H), 7.95e7.80 (m, 2H), 7.59 (dd, J ¼ 8.0,
4.5 Hz, 1H), 7.28 (s, 1H), 4.86 (s, 2H), 3.64 (d, J ¼ 11.2 Hz, 2H),
3.42e3.31 (m, 2H), 2.73 (t, J ¼ 12.2 Hz, 2H), 2.45 (s, 2H), 2.04e1.79
NMR (400 MHz, CD3OD)
d
9.26 (d, J ¼ 7.9 Hz, 1H), 8.51e8.45 (m,
2H), 8.37 (dd, J ¼ 9.4, 1.5 Hz, 1H), 8.13e7.95 (m, 3H), 7.11 (s, 1H),
5.05e4.96 (m, 2H), 3.62e3.54 (m, 2H), 2.99 (s, 6H), 2.54e2.45 (m,
(m, 6H). 13C NMR (101 MHz, CD3OD)
d 183.5, 152.5, 148.0, 142.5,
2H). 13C NMR (101 MHz, DMSO-d6)
d 179.1, 146.9, 138.5, 135.3, 131.7,
141.2, 139.6, 133.2, 132.9, 132.7, 132.6, 131.0, 126.4, 125.9, 122.4,
100.7, 55.4, 54.5, 42.2, 24.3, 22.8, 22.0. HRMS (ESI): calcd for
131.6, 131.2, 131.1, 130.0, 128.9, 128.3, 124.8, 124.5, 116.8, 98.5, 53.3,
43.6, 42.0, 20.4. HRMS (ESI): calcd for C21H20N3OCl [MþH]þ
366.1368, found 366.1368. HPLC purity: 99.08%.
C
23H24N4O [MþH]þ 373.2023, found 373.2021. HPLC purity: 99.87%.
5.2. Topo I inhibitory activity
5.1.4.21. 7-(3-(Dimethylamino)propyl)-9-methoxybenzo[a]phenazin-
5(7H)-one (6e-3). Red solid, yield 45.3%. m.p. 169.3e171.4 ꢀC. 1H
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
camptothecin as a positive control. The reaction mixture was pre-
pared according to the provided protocol, and incubated at 37 ꢀC for
30 min. The reactions were terminated by the addition of dye so-
lution containing 1% SDS, 0.02% bromophenol blue and 50% glyc-
erol. The mixtures were applied to 1% agarose gel and subjected to
electrophoresis for 1 h, in TAE buffer (40 mM Tris-acetate, 2 mM
EDTA). Gels were stained for 30 min in 60 mL 1ꢁ TAE buffer with
NMR (400 MHz, CD3OD)
d
9.28 (d, J ¼ 8.1 Hz, 1H), 8.47 (d, J ¼ 9.1 Hz,
2H), 8.06 (td, J ¼ 7.2,1.2 Hz,1H), 7.98 (td, J ¼ 7.2,1.2 Hz,1H), 7.74 (dd,
J ¼ 9.2, 2.4 Hz,1H), 7.70 (d, J ¼ 2.4 Hz,1H), 7.48 (s,1H), 5.27e5.16 (m,
2H), 4.27 (s, 3H), 3.67e3.62 (m, 2H), 2.99 (s, 6H), 2.61e2.50 (m, 2H).
13C NMR (101 MHz, DMSO-d6)
d 174.2, 164.0, 140.8, 138.0, 132.9,
132.6, 132.4, 131.3, 131.2, 130.8, 129.3, 124.4, 124.1, 117.1, 97.7, 97.0,
57.0, 53.4, 44.9, 42.1, 20.8. HRMS (ESI): calcd for C22H23N3O2
[MþH]þ 362.1863, found 362.1858. HPLC purity: 98.63%.
5.1.4.22. 7-(3-(Dimethylamino)propyl)-3,10-dimethoxybenzo[a]phe-
2 mL Gel Red. DNA bands were visualized by transillumination with
nazin-5(7H)-one (6f-1). Red solid, yield 44.8%. m.p. 172.3e173.5 ꢀC.
UV light and then photographed by Alpha Innotech digital imaging
system.
1H NMR (400 MHz, CD3OD)
d
8.72 (d, J ¼ 8.9 Hz, 1H), 7.90 (d,
J ¼ 9.3 Hz, 1H), 7.49e7.38 (m, 3H), 7.25 (dd, J ¼ 8.9, 2.7 Hz, 1H),
4.63e4.57 (m, 2H), 3.95 (s, 3H), 3.85 (s, 3H), 3.49e3.42 (m, 2H), 2.93
5.3. Topo II inhibitory activity
(s, 6H), 2.36e2.23 (m, 2H). 13C NMR (101 MHz, DMSO-d6)
d 178.8,
161. 8, 155.8, 145.4, 137.5, 135.4, 134.3, 126.7, 124.9, 124.2, 120.5,
118.5, 115.6, 110. 8, 106.4, 96.9, 55.6, 55.4, 53.4, 43.2, 42.2, 20.6.
We used the Topo II assay kit from TopoGEN to determine the
effects of compounds on DNA relaxation catalyzed by hTopo II.
Relaxation assays were carried out according to the manufacturer's
instructions with minor modifications. The assay was performed in
HRMS (ESI): calcd for
C
23H25N3O3 [MþH]þ 392.1969, found
392.1958. HPLC purity: 99.79%.
a final volume of 20
buffer ¼ 50 mM TriseHCl, pH 8.0, 150 mM NaCl, 10 mM MgCl2,
2 mM ATP, 0.5 mM dithiothreitol, and 30 g/mL BSA) with 0.2 mg
m
L in Topo II reaction buffer (1ꢁ Topo II
5.1.4.23. 10-Chloro-7-(3-(dimethylamino)propyl)-3-methoxybenzo
[a]phenazin-5(7H)- one (6f-2). Red solid, yield 41.2%. m.p.
m
157.9e159.6 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d 8.72e8.66 (m, 1H),
pBR322 DNA. Compounds were included in the reactions at a
constant solvent volume. Reactions were initiated by addition of 1U
7.97 (d, J ¼ 6.3 Hz, 1H), 7.86 (d, J ¼ 9.0 Hz, 1H), 7.72e7.67 (m, 1H),
7.60e7.55 (m, 1H), 7.42e7.34 (m, 1H), 6.35 (s, 1H), 4.34 (s, 2H), 3.93
(s, 3H), 3.36 (s, 2H), 2.81 (s, 6H), 2.18e2.06 (m, 2H). 13C NMR
human Topo IIa
, and incubated for 30 min at 37 ꢀC. Reaction was
terminated with 5ꢁ stop buffer (5
mL per 20 mL reaction volume).
(101 MHz, DMSO-d6)
d 179.9, 162.2, 146.8, 138.0, 135.0, 134.4, 130.5,
Stop buffer contained 5% sarkosyl, 0.0025% bromophenol blue and
25% glycerol. Reaction products were analyzed on a 1% agarose gel
in TAE buffer (40 mM Tris-acetate, 2 mM EDTA). Gels were stained
129.7, 128.6, 127.6, 127.1, 124.3, 119.0, 116.2, 106.8, 98.8, 55.6, 53.5,
43.0, 42.3, 20.5. HRMS (ESI): calcd for C22H22N3O2Cl [MþH]þ
396.1473, found 396.1462. HPLC purity: 99.96%.
for 30 min in 60 mL 1ꢁ TAE buffer with 2
mL Gel Red DNA bands
were visualized through transillumination with UV light and then
photographed by Alpha Innotech digital imaging system.
5.1.4.24. 7-(3-(Dimethylamino)propyl)-3,9-dimethoxybenzo[a]phe-
nazin-5(7H)-one (6f-3). Red solid, yield 44.6%. m.p. 154.4e156.5 ꢀC.
1H NMR (400 MHz, CD3OD)
J ¼ 9.1 Hz, 1H), 7.65 (d, J ¼ 2.6 Hz, 1H), 7.45e7.39 (m, 2H), 7.37 (d,
J ¼ 2.2 Hz, 1H), 4.82e4.76 (m, 2H), 4.15 (s, 3H), 3.98 (s, 3H),
3.60e3.51 (m, 2H), 2.99 (s, 6H), 2.47e2.38 (m, 2H). 13C NMR
d
8.88 (d, J ¼ 8.9 Hz, 1H), 8.13 (d,
5.4. Cell growth inhibition assay
The growth inhibitory effect of benzo[a]phenazin derivatives
toward four different human cancer cell lines, including human
promyelocytic leukemia cell line (HL-60), human chronic myelog-
enous leukemia cell (K562), cervical cancer cell line (HeLa), and
adenocarcinomic human alveolar basal epithelial cell line (A549),
were evaluated by using the MTT [3-(4,5-dimethylthiazol-2-yl)-
(101 MHz, DMSO-d6)
d 188.1, 162.4, 161.1, 141.0, 137.5, 134.6, 132.4,
131.9, 131.7, 130.5, 126.3, 124.5, 119.1, 114.3, 105.9, 97.3, 56.3, 55.4,
53.5, 43.5, 42.3, 20.5. HRMS (ESI): calcd for C23H25N3O3 [MþH]þ
392.1969, found 392.1968. HPLC purity: 99.83%.

B.-L. Yao et al. / European Journal of Medicinal Chemistry 92 (2015) 540e553
551
2,5-diphenyl-2H-tetrazolium-bromide] assay as described by
5.7. Topo II-mediated DNA cleavage assays
Mosmann with minor modifications [26]. The cells were plated at a
density of 1 ꢁ 104 per well in 96-well microplates, and allowed to
incubate overnight. Benzo[a]phenazin derivatives were added to
The assays were carried out as described previously [16]. Briefly,
10 units of human Topo II , 0.1 g negatively supercoiled pBR322
DNA, and 10e50 M benzo[a]phenazin derivatives (or 100 M VP-
16) were added in a total of 20 L Topo II buffer (50 mM TriseHCl,
pH 8.0, 150 mM NaCl, 10 mM MgCl2, 2 mM ATP, 0.5 mM dithio-
threitol, and 30 g/mLBSA). Sample treatment (equilibrate, trap-
a
m
the wells at increasing concentrations (0e50
well was treated with 20 L of 5 mg/mL MTT solution, and the cells
were further incubated at 37 ꢀC for 4 h. At the end of the incubation,
the untransformed MTT was removed, and 150 L DMSO was
mM). After 48 h, each
m
m
m
m
m
m
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, and the IC50 values were derived from the mean
OD values of the triplicate tests versus compound concentration
curves.
ped, and digested) procedures were as described above. Cleavage
was monitored with the conversion of negatively supercoiled DNA
to linear molecules. Gel photography and quantitation of DNA
bands were as described above.
5.8. ATPase domain related assays
The HsATPase plasmid and yeast strain, BCY123, for expressing
human Topo II ATPase domain (HsATPase) were presented by Dr.
Jian Ding from Shanghai Institute of Materia Medica. HsATPase was
overexpressed and purified as described previously [34]. The ATP
hydrolysis catalyzed by HsATPase was measured by using a non-
isotopic malachite green assay. Briefly, a reagent containing mala-
chite green (0.0812%, wt/vol), polyvinyl alcohol (2.32%, wt/vol),
ammonium molybdate (5.72%, wt/vol, in 6 M HCl), and water mixed
in a ratio of 2:1:1:2 was freshly prepared and kept at 4 ꢀC. 100 mM
ATP stock solution was freshly prepared in the assay buffer con-
taining 100 mM TriseHCl pH 7.4, 20 mM KCl, and 6 mM MgCl2. A
5.5. Topo I-mediated DNA cleavage assays
The assays were carried out as described previously with minor
modification [16]. In brief, calf thymus Topo I (10 units), 0.1
negatively supercoiled pBR322 DNA, and 0.5e50 M benzo[a]
phenazin derivatives (or 0.1e50 M camptothecin) were added in a
total of 20 L of Topo I buffer (50 mM TriseHCl, pH 7.5, 50 mM KCl,
mg
m
m
m
10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA, and 30 mg/mL bovine
serum albumin). After incubating for 6 min at 37 ꢀC to reach the
cleavage/religation equilibrium, cleavage intermediates were
trapped by adding 2
NaEDTA, pH 8.0. Proteinase K was added (2
reactions were incubated for 30 min at 45 ꢀC to digest the topo-
isomerase I. Samples were mixed with 2 L of agarose gel loading
mL of 1% SDS, followed by 2
mL of 250 mM
L of 0.8 mg/mL), and
typical 25 mL reaction volume for measuring specific ATPase activity
m
contained different concentrations of compounds. Reactions were
started by the addition of 1 mM ATP, and incubated for 1 h at 37 ꢀC,
m
and terminated by the addition of 80 mL of malachite green reagent,
buffer (30% sucrose, 0.5% bromophenol blue, and 0.5% xylene cya-
nole FF in 10 mM TriseHCl, pH 7.9), heated at 70 ꢀC for 2 min, and
subjected to electrophoresis in a 1% agarose gel in TAE buffer
and the absorbance was recorded at 620 nm on a microtiter plate
reader (PowerWave XS2, Biotek). For kinetic studies, the same
protocol was followed for increasing concentrations of ATP (250,
500, 800, 1,000, and 2000 mM) at each respective fixed concentra-
tion of compound 6f-3 (0, 5, 25, 50, and 100 mM) [9,35,36].
(40 mM Tris-acetate, 2 mM EDTA) containing 0.5 mg/mL ethidium
bromide. Cleavage was monitored with the conversion of nega-
tively supercoiled plasmid to nicked DNA. DNA bands were visu-
alized by using UV light, photographed by using Alpha Innotech
digital imaging system, and quantitated with Image J software
(NIH). The intensity of bands in the negative was proportional to
the amount of DNA present.
5.9. Topoisomerase I DNA unwinding assays
The ability of benzo[a]phenazin derivatives to unwind plasmid
DNA was determined as described by John M. Fortune et al. [16,20]
with modification. Relaxed pBR322 plasmid DNA utilized in un-
winding assays was generated by treating negatively supercoiled
pBR322 with topoisomerase I in topoisomerase I reaction buffer
(50 mM TriseHCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 0.5 mM DTT,
5.6. ICE assays
The assays were carried out as described previously [33]. In
brief, mid-log phase HL-60 cells were incubated with DMSO
0.1 mM EDTA, and 30
addition of other reaction components. Assay mixtures contained
0.1 g relaxed pBR322 plasmid DNA, topoisomerase I (10 units), and
compounds in 20 L of topoisomerase I reaction buffer. Following a
mg/mL bovine serum albumin) prior to the
(vehicle, 1.0%, v/v), CPT (2, 5, and 10
mM), or benzo[a]phenazin
derivatives (2, 5, and 10 M) for 1 h. Cells were lysed with 1 mL of
m
m
DNAzol (Invitrogen Life Technologies), and ethanol (0.5 mL, 100%)
was then added and mixed with the lysate, and the solution was
incubated overnight at ꢂ20 ꢀC. The precipitated DNA was collected
by centrifugation (13,000ꢁ g) at 25 ꢀC for 10 min followed by
washing twice with 75% ethanol and air drying. The DNA pellet was
dissolved in 0.2 mL NaOH (8 mM) and then the pH was adjusted to
7.2 by HEPES (1 M, 25 mL). After centrifugation at 30,000ꢁ g for
10 min, the supernatant was used to quantify the DNA concentra-
m
10 min incubation of DNA and compound at room temperature,
topoisomerase I was added, and reactions were incubated for
30 min at 37 ꢀC. Reactions were stopped by adding an equal volume
of phenol chloroform. Aqueous samples (20
the reactions, and 3 L of stop solution (0.77% SDS, 77 mM NaEDTA,
pH 8.0) followed by 2 L of agarose gel loading buffer (30% sucrose,
mL) were removed from
m
m
in 10 mM TriseHCl, pH 7.9) was added to each. Samples were
subjected to electrophoresis in a 1% agarose gel in TAE buffer
(40 mM Tris-acetate, 2 mM EDTA). DNA bands were stained with an
aqueous solution of ethidium bromide (0.5 mg/mL), visualized with
UV light, and photographed by Alpha Innotech digital imaging
system.
tion. DNA (2 mg) dissolved to a final volume of 30 mL in NaH2PO4
buffer (25 mM, pH 6.5) was then loaded onto nitrocellulose
membranes. Membranes were incubated overnight at 4 ꢀC with
rabbit monoclonal antibody to human Topo I (1:1000; Epitomics,
USA) prepared as described previously [34], and then incubated for
1 h with human anti-rabbit secondary horseradish peroxidase-
conjugated antibody (1:3000; Cell Signaling Technology, USA).
Reactive bands were detected using SuperSignal West Pico
chemiluminescent substrate (Thermo Scientific, USA), and chem-
iluminescence was exposed on Kodak X-Omat films.
5.10. UVevis titration
Absorbance titration experiment was performed as previously
described [16,37]. Binding assays were carried out in DPBS

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(2.67 mM KCl, 1.47 mM KH2PO4, 138 mM NaCl, 8.06 mM Na2HPO4,
pH 7.4). Solution of CT-DNA (SigmaeAldrich) in DPBS gave a ratio of
UVevis absorbance of 1.8e1.9 at 260 and 280 nm, indicating that
the DNA was sufficiently free of protein. The calf thymus DNA
concentration was determined with UV absorbance at 260 nm us-
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compounds (15
presence or absence of increasing concentrations of CT-DNA
(0e24 M). Absorption spectra were recorded in the
mM) were prepared in DPBS with 1% DMSO in the
m
400ꢂ600 nm spectral range after equilibration at room tempera-
ture for 5 min using UVevisible spectrophotometer UV-2450
(Shimadzu Instruments, Inc.).
5.11. Molecular docking
The structures of human Topo I and Topo IIa were generated
base on their X-ray structures (PDB ID: 1K4T [38] and 1ZXM [39])
using SYBYL software package (Tripos, Inc. St. Louis, MO). The res-
idues were corrected for physiological pH. In Topo IIa, Mg ion and
its two binding water molecules were conserved. Molecular dock-
ing was carried out with Surflex-dock. For both proteins, the pro-
tomol that characterizes the binding site of the receptor were
generated using a ligand-based approach. All other parameters
accepted default settings. The docking results were visualized using
the Discovery Studio software package.
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We thank the Natural Science Foundation of China (91213302
and 21172272 for Z.-S. Huang, 21272288 for S.-L. Huang), the
Guangdong Provincial Medicinal Research Foundation (A2011155
for S.-L. Huang), and Guangdong Provincial Key Laboratory of
Construction Foundation (Grant 2011A060901014) for financial
support of this study. We also thank Professor Jian Ding (State Key
Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences) for the plasmid gifts.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.01.024.
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