Design, synthesis and biological evaluation of 4-amidobenzimidazole acridine derivatives as dual PARP and Topo inhibitors for cancer therapy

Article abstract of DOI:10.1016/j.ejmech.2017.07.050

PARP-1 could repair the DNA damages induced by Topo inhibitors, therefore inhibiting Topo and PARP-1 simultaneously might be able to overcome resistance and improve outcomes. In this study a series of 4-amidobenzimidazole acridines were designed and synthesized as dual Topo and PARP-1 inhibitors. Compound 11l displayed good inhibitory activities against Topo and PARP-1, as well as significantly inhibited cancer cells proliferation. Further mechanistic evaluations indicated that 11l treatment in MCF-7 cells induced accumulated DNA double-strand breaks, prompted remarkable apoptosis, and caused prominent G0/G1 cell cycle arrest. Moreover, 11l greatly suppressed tumor growth in mice, and displayed favorable metabolic properties in liver microsomes. Our study suggested that single agents inhibiting Topo and PARP concurrently might be an alternative for cancer therapy and 11l represented a potential lead compound for development of antitumor agents.

Full text of DOI:10.1016/j.ejmech.2017.07.050

Accepted Manuscript
Design, synthesis and biological evaluation of 4-amidobenzimidazole acridine
derivatives as dual PARP and Topo inhibitors for cancer therapy
Zigao Yuan, Shaopeng Chen, Changjun Chen, Jiwei Chen, Chengken Chen, Qiuzi
Dai, Chunmei Gao, Yuyang Jiang
PII:
S0223-5234(17)30572-X
10.1016/j.ejmech.2017.07.050
EJMECH 9613
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 May 2017
Revised Date: 20 July 2017
Accepted Date: 22 July 2017
Please cite this article as: Z. Yuan, S. Chen, C. Chen, J. Chen, C. Chen, Q. Dai, C. Gao, Y. Jiang,
Design, synthesis and biological evaluation of 4-amidobenzimidazole acridine derivatives as dual PARP
and Topo inhibitors for cancer therapy, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.07.050.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of 4-amidobenzimidazole acridine
derivatives as dual PARP and Topo inhibitors for cancer therapy
Zigao Yuana,b,†, Shaopeng Chenb, †, Changjun Chenb, Jiwei Chena,b, Chengken Chena,b,
Qiuzi Daia,b, Chunmei Gaob,d*, Yuyang Jiangb,c,d*
a Department of Chemistry, Tsinghua University, Beijing, 100084, P. R. China
b
The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key
Laboratory of Chemical Biology, the Graduate School at Shenzhen, Tsinghua
University, Shenzhen, 518055, P. R. China
c
Department of Pharmacology and Pharmaceutical Sciences, School of Medicine,
Tsinghua University, Beijing, 100084, P. R. China
d
National & Local United Engineering Lab for Personalized anti-tumor drugs, the
Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055, P. R. China
* Corresponding author. Email address: chunmeigao@sz.tsinghua.edu.cn (C. Gao);
jiangyy@sz.tsinghua.edu.cn (Y. Jiang).
† Authors with equal contributions.

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Abstract
PARP-1 could repair the DNA damages induced by Topo inhibitors, therefore
inhibiting Topo and PARP-1 simultaneously might be able to overcome resistance and
improve outcomes. In this study a series of 4-amidobenzimidazole acridines were
designed and synthesized as dual Topo and PARP-1 inhibitors. Compound 11l
displayed good inhibitory activities against Topo and PARP-1, as well as significantly
inhibited cancer cells proliferation. Further mechanistic evaluations indicated that 11l
treatment in MCF-7 cells induced accumulated DNA double-strand breaks, prompted
remarkable apoptosis, and caused prominent G0/G1 cell cycle arrest. Moreover, 11l
greatly suppressed tumor growth in mice, and displayed favorable metabolic
properties in liver microsomes. Our study suggested that single agents inhibiting Topo
and PARP concurrently might be an alternative for cancer therapy and 11l represented
a potential lead compound for development of antitumor agents.
Key words: Topo; PARP; Multitarget; Acridines; Antitumor bioactivity

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1. Introduction
DNA topoisomerases are ubiquitous nuclear enzymes that regulate DNA
supercoiling and entanglements during important cellular processes such as
transcription, replication, recombination and repair [1]. Two types of DNA
topoisomerases have been identified and classified according to functional mechanism,
during which topoisomerase I (Topo I) and II (Topo II) cleave one or both DNA
strands, leading to transient DNA single-strand breaks (SSBs) or double-strand breaks
(DSBs), respectively. Topoisomerases are widely recognized targets during the
development of anticancer drugs [2, 3]. Topo I/II poisons induce DNA damage by
enhancing the formation of Topo I/II-DNA cleavage complexes or suppressing DNA
religation [4]. Topo I-DNA cleavage complexes trapped by Topo I poisons crash with
DNA replication forks, converting DNA SSBs into potentially lethal and irreversible
DSBs, and ultimately promoting tumor cells death [5, 6].
In another aspect, PARP-1 is a sensitive DNA damage sensor and plays critical
roles during DNA damage repair. It is also a practical therapeutic target for stroke,
cardiac ischemia, diabetes, inflammation, and cancer [7-10]. PARP-1 inhibitors
suppress repair of DNA lesions caused by radiation and/or other chemotherapeutics,
leading to DSBs accumulation and resulting in enhanced treatment response,
especially in tumor cells harboring BRCAness [11-13]. The overexpression of
PARP-1 and other various DNA damage repair proteins may contribute to the repair
of DNA lesions induced by Topo I/II inhibitors, and then help tumor cells develop

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resistance to Topo I/II drugs [1, 14, 15]. The mechanisms that PARP-1 decreases
cytotoxicity of Topo
I
inhibitors include that PARP-1 catalyzes
poly(ADP-ribosylation) of Topo I in Topo I-DNA cleavage complex, leading to
destabilize of the complex, as well as increasing the probability of resolution of the
associated DNA break to DNA repair proteins [16, 17]. Moreover, PARP activity
promotes replication fork reversal and/or restart, thereby limiting the formation of
DSBs upon Topo I poisons treatment and helping cells to recover from Topo
inhibition [6].
The aforementioned evidences provide mechanistic rationales for the
combination of PARP-1 and Topo I/II inhibitors in cancer therapy, which may help to
overcome resistance and improve outcomes. Indeed, combination therapies against
tumor cells indicated that PARP inhibitors displayed synergistic effects with Topo I
poisons (e.g., topotecan, camptothecin) [18-20] and Topo II inhibitors (e.g., C-1305)
[21, 22]. Considering the promising results of combination therapy and the
relationship between PARP and Topo, we speculated that compounds inhibiting PARP
and Topo concurrently might be effective antitumor agents. Despite numerous
inhibitors against attractive targets Topo or PARP have been developed, none dual
PARP and Topo inhibitors have been reported as far as we know. As part of our
ongoing effects in the development of multi-target antitumor agents, we have
discovered some multifunctional inhibitors as antitumor agents [23-26]. In this study,
we intended to develop dual PARP and Topo inhibitors based on rational drug design
strategy.

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2. Rational design of dual PARP and Topo inhibitors
Acridine and its derivatives possess tricyclic planar constructions which could
intercalate into DNA and interact with Topo I/II [27, 28]. Some acridines display
potent anticancer potency, among them Topo I inhibitor amsacrine (m-AMSA) has
been used for the treatment of acute lymphoblastic leukemia. In the last few years, we
have developed some acridine derivatives for cancer therapy [29-33] and reported that
benzimidazole derivative 8o is a good Topo I inhibitor with potential antitumor
activity [29]. On the other hand, benzimidazole carboxamide has been recognized as
an effective scaffold to develop PARP inhibitors since the report of NU-1085 (Figure
1) [11]. The carboxamide of the scaffold formed critical hydrogen bonds with amino
acid residues Gly863 and Ser904 of human PARP-1 [8, 9, 34]. Many benzimidazole
carboxamide derivatives have been reported as potent PARP inhibitors [35], among
them analogue veliparib is under late clinical trials and niraparib has been approved
by the FDA in March, 2017. We noticed that if introducing a carboxamide group to
the benzimidazole part of 8o, the obtained novel chemical entity (compound 11a,
Scheme 1) could be a potential PARP and Topo inhibitor. Furthermore, a series of
analogues of 11a could be obtained by changing the substituent patterns of the
acridine group to improve activities (Scheme 1).

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3. Results and discussion
3.1. Chemistry
The synthesis of target molecules 11a-11n is shown in Scheme 2. Firstly,
2-amino-3-nitrobenzoic acid 1 was converted to 2,3-diaminobenzamide 3 via
amidation reaction and reduction reaction in sequence, which then reacted with 4a-4c
to give benzimidazole carboxamides 5a-5c. Subsequently reduction reactions were
carried out to produce intermediates 6a-6c [36]. On the other side, compounds 9a-9f
were obtained from the Ullmann reactions of 2-chlorobenzoic acid 7 with
phenylamine derivatives 8a-8f, and then Friedel-Crafts acylation reactions were
conducted in phosphorus oxychloride at 105 °C for 4 h to yield corresponding
acridine intermediates 10a-10f [29]. At last, the desired compounds 11a-11n were
produced by the nucleophilic substitution reactions between corresponding
phenylamine derivatives 6a-6c and 9-chloroacridines 10a-10f. Structures were
1
characterized with H NMR, 13C NMR, melting point and high resolution mass
spectrum.
3.2. PARP-1 inhibition assay
The ability of target compounds 11a-11n and intermediates 6a-6c to inhibit
PARP-1 enzymatic activity was tested using an assay that measures incorporation of
biotinylated poly-ADP-ribose onto histone proteins. Olaprib, which was the first
PARP inhibitor to be approved by the FDA in 2014, was used as a positive reference

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compound. As we expected, compound 11a displayed potent PARP-1 inhibitory
activity with IC50 less than 100 nM (Table 1). Derivatives of 11a (i.e., 11b-11f) which
introduced substituents to acridine group exhibited slightly decreased activity with
IC50 values less than 300 nM (the activity order is 11a＞11e＞11d＞11f＞11c＞11b).
Among them 11a showed more than 2-fold more potent activity than compounds 11b
and 11c, which were methoxy substituted at position 2 and 4 in the acridine group,
respectively. However, for compounds 11g-11l, substituents to acridine group may be
helpful to improve activity, as compound 11g displayed weaker activity than the other
five compounds with different substituents (the activity order is 11j＞11l＞11k＞11i
＞11h＞11g). Besides, compounds 11m and 11n showed decreased activities against
PARP-1 compared to 11a-11l. Furthermore, for compounds sharing the same
9-aminoacridines, the p-linked compounds is more potent PARP-1 inhibitors than
m-linked and o-linked compounds (e.g., 11a ＞ 11g ＞ 11m; 11b ＞ 11h ＞ 11n),
indicating that the linking position of 9-aminoacridines attached to the benzene cycle
is critical for PARP-1 inhibitory activity. It also should be noted that intermediates 6a
and 6b had better PARP-1 inhibitory activities than their acridine derivatives 11a-11f
and 11g-11l, respectively. Nevertheless, compound 6c showed pretty weak PARP-1
inhibitory potency, which might due to the o-amino group which could form
intramolecular hydrogen bond with benzimidazole group and thus destroy the
intramolecular hydrogen bond of benzimidazole carboxamide [37]. Acridine
derivatives of 6c (i.e., 11m and 11n) might remain the intramolecular hydrogen bond
of benzimidazole carboxamide and suppress the hydrogen bond of o-amino group via

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steric hindrance of the acridine groups, thereby regaining limited PARP-1 inhibitory
activities.
3.3. In vitro cytotoxicity assay
MCF-7 is a type of breast adenocarcinoma cell line. To obtain potential
antitumor agents for breast cancer therapy, antiproliferative activities of our
synthesized compounds against MCF-7 were assessed utilizing an MTT assay, with
olaparib and m-AMSA used as reference compounds. The result (Table 1) showed that
compound 11a (IC50 = 12.01 µM) displayed improved antiproliferative activity
against MCF-7 than that of parent compound 8o (IC50 = 26.12 µM). Compound 11b
(IC50 = 38.16 µM), which was 2-methoxy substituted, exhibited decreased activity
compared with 11a, while the 4-methoxy substituted analogue 11c (IC50 = 13.22 µM)
showed similar activity to that of 11a. The methyl substituted derivatives 11e and 11f
exhibited 3-fold less potent activity than that of 11a, with IC50 values of 39.27 and
39.14 µM, respectively. Moreover, after changing the linking position of
9-aminoacridines attached to the benzene cycle from p-position to m-position,
compounds 11g (IC50 = 36.09 µM), 11h (IC50 = 45.31 µM) and 11i (IC50 = 25.81 µM)
showed decreased activities compared with their analogues 11a, 11b, and 11c,
respectively, while compounds 11k (IC50 = 29.64 µM) and 11l (IC50 = 2.14 µM) had
improved activity compared with their respective analogues 11e and 11f. It should be
noted that compound 11l (IC50 = 2.14 µM) displayed the most potent antiproliferative
potency in its class, which was 4- and 17-fold more potent than reference compounds

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m-AMSA and olaparib, respectively. For the skeleton of compound 11g (IC50 = 36.09
µM), the methoxy substitution at 2-position (11h, IC50 = 45.31 µM) and 4-position
(11i, IC50 = 25.81 µM) could influence the activity in the opposite direction.
Furthermore, 2-butyl substituted might not be tolerable, as compound 11d and 11j
displayed pretty weak antiproliferative activity (IC50 > 50 µM). Besides, when the
linking position of 9-aminoacridines attached to the benzene cycle was changed to
o-position from p-/m-position, compound 11m (IC50 > 50 µM) displayed weaker
activity than its analogues 11a and 11g, while the 2-methoxy substituted derivative
11n (IC50 = 21.81 µM) exhibited improved activity than 11b and 11h. Moreover,
despite possessing potent PARP-1 inhibitory activities, compounds 11d, 11j, 11m and
intermediates 6a-6b displayed weak activities in MCF-7 cells (IC50 > 50 µM), that
could be related to their physicochemical properties (e.g., solubility, permeability,
lipophilicity, and stability) [38].
Furthermore, it’s reported that breast cancer cell line HCC1937 which harbors
BRCA1 mutation is sensitive to PARP inhibitors [39]. Besides, human colon cancer
cell line HCT116 possesses pretty high levels of Topo I expression and may display
positive response to Topo I inhibitors [40]. To further assess the cytotoxicity of
compounds 11l against tumor cells, MTT assays were conducted in cancer cells
HCC1937 and HCT116. As shown in Table 2, HCC1937 is more sensitive to olaparib
(IC50 = 4.97µM) than that to m-AMSA (IC50 = 7.85 µM), and compound 11l (IC50 =
0.83 µM) showed 6- and 9.5-fold more potent activity than olaparib and m-AMSA,
respectively, although 11l exhibited weaker PARP-1 inhibitory potency than that of

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olaparib. In HCT116 cells m-AMSA (IC50 = 3.89 µM) displayed 3.9-fold more potent
activity than olaparib (IC50 = 15.13 µM), but was 2.7-fold less potent than 11l (IC50 =
1.43 µM). The therapeutic advantages of compound 11l over reference compounds
olaparib and m-AMSA might attribute to collective inhibition of PARP and Topo
activity.
3.4. Inhibitory activities against topo I and II
As compound 11l displayed significant antiproliferative activity against human
cancer cells, it was selected for further evaluations. The topoisomerase inhibitory
activity is directly related to the conversion of supercoiled DNA to its relaxed form
[41]. As shown in Fig. 2A, 11l exhibited comparable Topo I inhibitory potency with
that of reference compound camptothecin (CPT, Topo I inhibitor) at 100 µM, and
displayed good Topo I inhibitory activity at lower concentrations, even at 2 µM.
Moreover, 11l and our other synthesized compounds were also subjected to Topo II
inhibitory assay, using Topo II inhibitor etoposide as a reference compound. Fig. 2B
indicated that 11l and the other tested compounds displayed Topo II inhibitory
activities at 100 µM (except 11g). This results suggested that 11l possesses both Topo
I and Topo II inhibitory activity, alongside with good PARP1 inhibitory potency (IC50
= 0.45 µM).
3.5. Compound 11l prompted DNA DSBs and decreased Topo I/II expression
It`s well known that Topo II inhibitors could cause DNA DSBs [2]. Besides,
unrepaired DNA SSBs induced by Topo I inhibitors result in replication fork collapse

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and the efficient formation of DSBs [42]. Moreover, PARP-1 inhibitors increased
DNA SSBs accumulation, which then converted to more lethal DSBs [43]. As 11l
inhibits Topo I/II and PARP-1 simultaneously, we next conducted western blot assay
to evaluate whether 11l could induce DNA DSBs in MCF-7 cells. γH2AX is a widely
recognized molecular marker for DSBs [44], which was also reported as a
pharmacodynamic marker for Topo I inhibitor activity [45]. Except for γH2AX,
53BP1 also forms nuclear foci in response to DSBs [6, 46]. Furthermore, loss of
53BP1 might result in partial restoration of homologous recombination, thereby
repairing DSBs and acquiring resistance to drugs which cause DSBs [7]. The results
indicated that treatment with 11l induced marked DSBs in MCF-7 cells, given that the
expressions of γH2AX and 53BP1 were significantly increased in a dose-dependent
manner (Fig. 3).
It has been reported that some topoisomerase inhibitors (e.g., camptothecin,
etoposide) could induce topoisomerase degradation [47, 48] . Liu et al. [5] reported
that Topo I cleavage complexes could be degraded by proteasome to generate DSBs.
Topo II poisons could induce covalently formed Topo II-DNA complex, therefore free
Topo II band was not detected by the Topo II antibody in band depletion assays [48].
Herein we discovered that treatment with 11l caused remarkable decrease of both
Topo I and Topo II in dose-dependent manners in MCF-7 cells (Fig. 3), which might
be lethal to tumor cells, providing the critical roles of topoisomerases in various
cellular processes.

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3.6. Apoptosis induction
To further investigate the antitumor and growth-inhibitory effects,
PI-FITC/annexin assay was performed to evaluate the ability of 11l to induce
apoptosis in MCF-7 cells. As shown in Fig. 4, Q1 represents the necrotic or damaged
dead cells, Q2 represents apoptosis cells in later stage, Q3 represents apoptosis cells
in early stage, and Q4 represents live cells. Compared with DMSO controls, MCF-7
cells treated with 11l at different concentrations of 1, 5 and 10 µM resulted in 56.6%,
60.3% and 91.5% cells apoptosis, respectively, suggesting that compound 11l induced
apoptosis in a dose-dependent manner, and the 11l-mediated cell proliferation
inhibition could be due to the increased apoptosis.
We then evaluated the expression of apoptosis-related proteins in MCF-7 cells
which were treated with 11l at various concentrations to dissect the mechanism of
11l-induced apoptosis. Caspase-8 and -9 are initiator caspases which are closely
related to proapoptotic signals and involved in the extrinsic pathway (death
receptor-mediated pathway) and intrinsic pathway (mitochondria-mediated pathway),
respectively [49]. Besides, caspase-3 is an executioner caspase that modifies proteins
ultimately responsible for apoptosis. In addition, PARP is the main substrate of
activated caspase-3 and cleaved PARP is an apoptosis-specific biomarker. Consistent
with the above PI-FITC/annexin assay, the results (Fig. 5) showed that the expressions
of cleaved caspase-3 and cleaved PARP were substantially increased in a
dose-dependent manner in MCF-7 cells treated with 11l, meanwhile the full length
PARP was cleaved and downregulated. Furthermore, both cleaved caspase-8 and

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cleaved caspse-9 were upregulated in MCF-7 after exposing to 11l with increasing
concentrations, indicating that 11l could induce apoptosis through modulating both
extrinsic and intrinsic pathways.
3.7. Compound 11l induced G0/G1 cell cycle arrest
DNA damage caused by antitumor agents may activate DNA damage
checkpoints, which arrest cell cycle progression, initiate DNA damage repair, or lead
to cell death [50]. Given that compound 11l significantly induced DNA DSBs
accumulation, potently suppressed proliferation and induced marked apoptosis of
cancer cells MCF-7, we further measured the effect of 11l on the distribution of cell
cycle using flow cytometry assy. The cell cycle profile of MCF-7 after exposure to
various concentrations of 11l for 24 hours is shown in Fig. 6, which demonstrated that
11l induced G0/G1 arrest in a dose-dependent manner. In fact, the G0/G1-phase
fraction was substantially increased from 17.67% in the untreated cells to 19.96%,
43.62% and 58.27% in the cells treated with 11l at 1, 5, and 10 µM, respectively.
The G0/G1 arrest induced by 11l was further confirmed by evaluation of the
changes in G0/G1 checkpoint-related proteins utilizing western blot analysis. It has
been reported that high-level expression of cyclin A can trigger the G1/S transition in
G1 cells [51], and the levels of cyclin D-CDK4(6) or cyclin E-CDK2 pathways are
decreased when G1 arrest occurs, while the expressions of CDK inhibitors (e,g,, P18,
P21, P27) are increased, thereby silencing the CDKs that are essential for entry into S
phase [48, 52]. As shown in Fig. 7, remarkable increased expressions of P18, P21 and

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P27 were observed in a dose-dependent manner in MCF-7 cells treated with 11l, as
well as the decreased levels of CDKs 2, 4, and 6 and cyclins A, D, and E. These
results indicated that treatment with 11l generated stable G0/G1 arrest in MCF-7 cells.
3.8. In vivo antitumor activity of 11l against MCF-7
Considering that 11l displayed potent antitumor potency in vitro, we further
evaluated the in vivo therapeutic efficacy of 11l on subcutaneously xenografts tumor
models of human breast cancer cell MCF-7 in mice. The drug groups were treated
with 11l at doses of 20 and 40 mg/kg/d, respectively, compared to the control groups
treated with 2% DMSO solution. The results (Fig. 8) revealed that 11l treatment
significantly reduced tumor volume and mass in drug groups (Fig. 8B/C/E). Besides,
little toxicity of 11l was observed, as demonstrated by the absence of weight loss (Fig.
8D) and by the normal growth and behavior of all mice tested. All these data indicated
that 11l exhibited efficacious antitumor activity in vivo.
3.9. Preliminary studies on metabolic profiles of 11l
The potent antitumor potency of 11l in vitro and in vivo warranted it as a
potential antitumor agent. The metabolic stability of 11l in live microsomes to
preliminary estimate its pharmacokinetic properties were evaluated. As shown in
Table 3, 11l exhibited modest stability in liver microsomes from three different
species (human, mouse, and rat). Specially, the half life (T1/2) of 11l in human liver
microsomes is about half an hour, which is a little shorter than that in rat live
microsomes. Moreover, 11l is more stable in mouse liver microsomes, with smaller

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clearance rate and longer half life than that in human or rat liver microsomes. Further
investigation about CYP450 isoenzymes inhibition in human live microsomes
suggested that 11l could mainly be metabolized by CYP2C9, as well as could also
interact with isoenzyme CYP3A4 (Table 4). The property that 11l could be
metabolized by different CYP isoforms may be helpful to minimize the potential for
drug-drug interactions in the clinic [53].
3.10. Binding models of compound 11l with PARP1 and Topo I/II
To explore the binding models of compound 11l with the respective enzymes, 11l
was docked into the active sites of PARP1 (PDB code: 4UND), Topo I (PDB code:
1K4T) and Topo II (PDB code: 4G0U) using the SYBYL-X 1.3 protocol. As shown in
Figure 9A, the 4-amidobenzimidazole group of 11l occupied the nicotinamide-ribose
binding domain of PARP1, forming two critical hydrogen bonds with Gly863 and
essential pi-pi interactions with Tyr907. Besides, the nitrogen atom of the acridine
group formed one more hydrogen bond with Tyr889. Figure 9B suggested that 11l
could form cleavage-complex with Topo I and DNA. The 4-amidobenzimidazole
group of 11l was parallel to DA113 and formed three hydrogen bonds with DA113
and DT10. 11l could also form cleavage-complex with Topo II and DNA, in which the
acridine group of 11l was parallel to DG13 and formed pi-pi interactions (Fig. 9C).
The above docking results may further confirm the inhibitory activity of 11l against
PARP1 and Topo I/II.

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4. Conclusion
PARP-1 is closely related to the repair of DNA damages caused by Topo
inhibitors. The combinations of PARP-1 inhibitor and Topo inhibitor display enhanced
antitumor potency and might help to overcome resistance. In the present study, a
series of 4-amidobenzimidazole acridine derivatives were designed and synthesized.
Among them, compound 11l showed potent inhibitory potency against both PARP and
Topo. Besides, 11l exhibited the most potent antiproliferative activity against MCF-7
in its class. Further evaluations indicated that 11l induced remarkable DNA DSBs
accumulation and caused Topo I/II degradation. Moreover, 11l treatment in MCF-7
prompted PARP cleavage and the activation of caspase-3, -8, and -9, leading to
marked apoptosis through modulating both extrinsic and intrinsic pathways. Cell
cycle analysis and western blot assay indicated that 11l increased the expression of
P18, P21 and P27, as well as downregulated the levels of CDKs 2, 4, and 6 and
cyclins A, D, and E, thereby causing stable G0/G1 phase arrest. The in vivo study
further demonstrated that 11l significantly suppressed the growth of MCF-7 xenograft
tumors, without adverse toxicity as no distinct weight loss of mice was observed.
Furthermore, preliminary pharmacokinetic studies indicated that 11l exhibited proper
metabolic stability in human/mouse/rabbit liver microsomes and could be metabolized
by CYP2C9 and CYP3A4. Taken together, all these thorough evaluations warranted
11l as a promising lead compound worth further optimization and development for
cancer therapy.

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5. Experimental section
5.1. Chemistry
Reagents and solvents were purchased from commercial sources and used
without further purification. All reactions were run under a nitrogen atmosphere.
Nuclear magnetic resonance spectra were obtained using a Bruker 400 (400 MHz)
spectrometer. Chemical shifts were given in ppm (δ) relative to SiMe4 as internal
standard. Coupling constants (J) were in hertz (Hz), and signals were designated as
follows: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad singlet, etc. The mass
spectra were obtained on a Waters Micromass Q-TOF Premier Mass Spectrometer.
Melting points were determined with a Hanon MP430 Melting Point Apparatus. Thin
layer chromatography was carried out using plate silica gel F254. All chemical yields
were unoptimized and generally represented the result of a single experiment.
Intermediates 6a-6c were synthesized following a literature procedure [36], and
9-chloroacridines 10a-10f were prepared as we reported before [29].
5.1.1. General procedure for compounds 11a-11n
Aniline derivatives 6a-6c (1 eq) and respective 9-chloroacridine derivatives
10a-10f (1.1 eq) were added to a round flask, and then excess phenol was added as
solvent. The reaction system was warmed up to 60 °C until phenol was melted and
stirred for 0.5 hour under argon protection. Then the mixture was warmed up to
120 °C and continued to reaction for more 2 hours. After the reaction was completed,

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the mixture was added to ethyl ether drop by drop. The precipitate was filtered and
washed twice with new ethyl ether to give pure products 11a-11n, which were then
1
characterized by H NMR, 13C NMR, melting point and high resolution mass
spectrum (HRMS, ESI).
5.1.1.1. 2-(4-(acridin-9-ylamino)phenyl)-1H-benzo[d]imidazole-4-carboxamide (11a)
1
Yield 64%; mp 240-242 °C; H NMR (400 MHz, DMSO-d6) δ 7.37 (t, J = 8.2
Hz, 1H), 7.52 (t, J = 8.2 Hz, 2H), 7.62 (d, J=8.4 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H),
7.89 (d, J = 8.4 Hz, 1H), 8.04 (t, J = 8.4 Hz, 2H), 8.34-8.39 (m, 3H), 11.69 (s, 1H),
13
13.75 (s, 1H). C NMR (101 MHz, DMSO-d6) δ 166.7, 155.3, 151.7, 140.7, 136.0,
128.9, 127.4, 124.7, 124.5, 123.5, 122.9, 122.7, 119.9, 115.1. HRMS (ESI) m/z
calculated for [M+H]+ 430.1668, found 430.1663.
5.1.1.2.
2-(4-((2-methoxyacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(11b)
Yield 55%; mp 214-216 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.76 (s, 3H), 7.36
(t, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.75-7.80 (m,
3H), 7.88 (d, J = 8.0 Hz, 1H), 7.99 (t, J = 8.0 Hz, 1H), 8.16-8.18 (m, 3H), 8.36 (d, J =
8.4 Hz, 2H), 9.30 (s, 1H), 11.42 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 166.7,
156.4, 153.0, 151.7, 144.2, 139.8, 136.6, 135.0, 129.1, 128.8, 126.8, 126.0, 124.9,
124.1, 123.5, 122.9, 122.7, 122.0, 120.3, 116.9, 115.2, 103.7, 56.3. HRMS (ESI) m/z

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calculated for [M+H]+ 460.1773, found 460.1778.
5.1.1.3.
2-(4-((4-methoxyacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(11c)
Yield 58%; mp 220-222 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.77 (s, 3H), 7.38
(m, 2H), 7.45-7.63(m, 3H), 7.77-7.85 (m, 4H), 7.88-7.96 (m, 2H), 8.02-8.39 (m, 2H),
8.48 (d, J = 8.0 Hz, 1H), 9.25 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 166.7, 155.5,
151.6, 149.0, 140.5, 135.8, 132.6, 128.9, 126.2, 125.0, 124.6, 123.6, 123.0, 120.7,
117.5, 115.9, 115.4, 113.7, 57.4. HRMS (ESI) m/z calculated for [M+H]+ 460.1773,
found 460.1779.
5.1.1.4.
2-(4-((2-butylacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide (11d)
1
Yield 54%; mp 260-261 °C; H NMR (400 MHz, DMSO-d6) δ 0.72-0.77 (m,
2H), 1.20-1.23 (m, 3H), 1.44-1.49 (m, 2H), 2.61-2.67 (m, 2H) 7.33-7.41 (m, 1H),
7.53-7.60 (m, 2H), 7.76-8.09 (m, 7H), 8.37-8.39 (m, 2H), 9.33 (s, 1H), 11.51 (s, 1H),
13.63 (s, 1H), 14.84 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 166.6, 154.9, 151.3,
142.5, 140.2, 139.3, 138.8, 137.4, 135.6, 131.1, 131.0, 126.5, 126.0, 125.6, 124.6,
124.3, 123.5, 123.0, 123.0, 119.8, 119.7, 114.6, 114.4, 35.0, 32.6, 21.9, 14.0. HRMS
(ESI) m/z calculated for [M+H]+ 486.2294, found 486.2287.

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5.1.1.5.
2-(4-((2-methylacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(11e)
1
Yield 48%; mp 197-199 °C; H NMR (400 MHz, DMSO-d6) δ 2.85 (s, 3H),
7.41-7.45 (m, 2H), 7.53 (t, J = 8.8 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.95 (d, J = 8.0
Hz, 2H), 7.78-7.92 (m, 2H), 8.05 (t, J = 7.6 Hz, 1H), 8.27 (d, J = 8.8 Hz, 1H),
8.33-8.38 (m, 3H), 8.59 (d, J=8.8 Hz, 1H), 9.19 (s, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 166.8, 151.5, 141.1, 139.8, 136.6, 135.8, 129.1, 128.5, 126.1, 124.9,
124.5, 124.4, 124.3, 123.6, 123.2, 122.6, 120.8, 116.1, 115.5, 115.2, 19.0. HRMS (ESI)
m/z calculated for [M+H]+ 444.1824, found 444.1827.
5.1.1.6.
2-(4-((4-methylacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(11f)
Yield 46%; mp 213-214 °C; 1H NMR (400 MHz, DMSO-d6) δ 2.44 (s, 3H), 7.38
(t, J = 8.0 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.77-7.80 (m,
2H), 7.88-7.90 (m, 1H), 7.93-7.97 (m, 2H), 8.01-8.03 (m, 2H), 8.05-8.20 (m, 3H),
8.29 (s, 1H), 8.37 (d, J=8.4 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 166.1, 150.8,
140.4, 139.1, 135.9, 135.1, 128.4, 127.8, 125.4, 124.2, 123.8, 123.7, 123.6, 122.9,
122.5, 121.9, 120.1, 115.4, 114.8, 114.5, 18.3. HRMS (ESI) m/z calculated for
[M+H]+ 444.1824, found 444.1825.

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5.1.1.7.
2-(3-(acridin-9-ylamino)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(11g)
1
Yield 60%; mp 319-320 °C; H NMR (400 MHz, DMSO-d6) δ 7.37 (t, J = 8.4
Hz, 1H), 7.48-7.56 (m, 3H), 7.68-7.89 (m, 4H), 8.03 (t, J = 8.4 Hz, 2H), 8.14 (d, J =
8.4 Hz, 2H), 8.29-8.35 (m, 3H), 8.42 (s, 1H), 9.26 (s, 1H), 11.69 (s, 1H), 13.71 (s, 1H).
13C NMR (101 MHz, DMSO-d6) δ 166.6, 155.6, 151.4, 142.6, 140.7, 135.9, 131.3,
131.1, 129.8, 126.4, 126.3, 125.8, 124.6, 123.6, 123.1, 119.9, 115.7, 114.6. HRMS
(ESI) m/z calculated for [M+H]+ 430.1668, found 430.1660.
5.1.1.8.
2-(3-((2-methoxyacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(11h)
Yield 56%; mp 264-266 °C; 1H NMR (400 MHz, DMSO-d6) δ 3.76 (s, 3H), 7.37
(t, J = 8.0 Hz, 1H), 7.44-7.52 (m, 2H), 7.67-7.89 (m, 6H), 7.99 (t, J = 8.4 Hz, 1H),
8.12-8.19 (m, 3H), 8.25 (d, J = 8.0 Hz, 1H), 8.39 (s, 1H), 9.24 (s, 1H), 11.38 (s, 1H),
13
13.65 (s, 1H). C NMR (101 MHz, DMSO-d6) δ 166.7, 151.4, 151.3, 142.5, 141.1,
138.9, 135.7, 134.8, 133.9, 131.0, 130.8, 126.2, 124.8, 124.3, 123.7, 123.4, 122.7,
120.8, 116.3, 114.4. HRMS (ESI) m/z calculated for [M+H]+ 460.1773, found
460.1775.
5.1.1.9.
2-(3-((4-methoxyacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide

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(11i)
1
Yield 50%; mp 255-256 °C; H NMR (400 MHz, DMSO-d6) δ 4.19 (s, 3H),
7.34-7.43 (m, 2H), 7.48-7.50 (m, 2H), 7.67 (t, J = 8.0 Hz, 1H), 7.74-7.76 (m, 2H),
7.80 (s, 2H), 7.99-8.03 (m, 1H), 8.25-8.32 (m, 3H), 8.39 (s, 1H), 8.55 (d, J = 8.0 Hz,
1H), 9.24 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 166.7, 154.8, 153.2, 151.6,
149.0, 140.6, 134.9, 132.6, 131.2, 130.9, 130.1, 129.9, 126.3, 125.4, 124.9, 124.2,
123.8, 123.5, 123.0, 122.0, 120.5, 117.7, 116.0, 115.6, 115.2, 113.1, 57.2. HRMS (ESI)
m/z calculated for [M+H]+ 460.1773, found 460.1777.
5.1.1.10.
2-(3-((2-butylacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide (11j)
1
Yield 60%; mp 228-230 °C; H NMR (400 MHz, DMSO-d6) δ 0.69-0.73 (m,
3H), 1.09-1.18 (m, 2H), 1.39-1.47 (m, 2H), 2.59-2.62 (m, 2H), 7.36 (t, J = 8.0 Hz,
1H), 7.52-7.69 (m, 2H), 7.71-7.92 (m, 5H), 8.03-8.16 (m, 4H), 8.31 (d, J = 8.0 Hz,
1H), 8.39 (s, 1H), 8.41 (s, 1H), 9.22 (s, 1H), 11.57 (s, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 166.7, 154.9, 151.3, 142.5, 140.2, 139.3, 138.9, 137.5, 135.7, 131.2,
131.1, 126.5, 126.1, 124.6, 124.3, 123.6, 123.1, 123.0, 119.9, 119.8, 114.6, 114.4, 35.0,
32.7, 21.9, 14.0. HRMS (ESI) m/z calculated for [M+H]+ 486.2294, found 486.2290.
5.1.1.11.
2-(3-((2-methylacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(11k)

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1
Yield 51%; mp 209-211 °C; H NMR (400 MHz, DMSO-d6) δ 2.84 (s, 3H),
7.34-7.43 (m, 2H), 7.49-7.51 (m, 2H), 7.67 (t, J = 8.0 Hz, 1H), 7.74-7.89 (m, 2H),
8.02 (t, J = 8.0 Hz, 1H), 8.26-8.32 (m, 3H), 8.39 (s, 1H), 8.55 (d, J = 8.0 Hz, 1H),
9.24 (s, 1H), 11.72 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 166.7, 154.8, 151.4,
142.5, 140.5, 138.9, 138.1, 135.6, 134.6, 131.0, 126.4, 125.7, 124.6, 124.4, 123.6,
123.1, 123.0, 119.9, 119.7, 115.0, 114.3, 21.6. HRMS (ESI) m/z calculated for
[M+H]+ 444.1824, found 444.1822.
5.1.1.12.
2-(3-((4-methylacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(11l)
1
Yield 58%; mp 228-230 °C; H NMR (400 MHz, DMSO-d6) δ 2.84 (s, 3H),
7.34-7.43 (m, 2H), 7.49-7.51 (m, 2H), 7.67 (t, J = 8.0 Hz, 1H ), 7.74-7.89 (m, 2H),
8.02 (t, J = 8.0 Hz, 1H), 8.26-8.32 (m, 3H), 8.39 (s, 1H), 8.55 (d, J = 8.0 Hz, 1H),
13
9.24 (s, 1H), 11.72 (s, 1H). C NMR (101 MHz, DMSO-d6) δ 166.7, 154.8, 151.4,
142.5, 140.5, 138.9, 138.1, 135.6, 134.6, 131.0, 126.4, 125.7, 124.6, 124.4, 123.6,
123.1, 123.0, 119.9, 119.7, 115.0, 114.3, 19.0. HRMS (ESI) m/z calculated for
[M+H]+ 444.1824, found 444.1823.
5.1.1.13.
2-(2-(acridin-9-ylamino)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(11m)
1
Yield 61%; mp 289-291 °C; H NMR (400 MHz, DMSO-d6) δ 7.28-7.32 (m,

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2H), 7.40-7.50 (m, 1H), 7.55-7.70 (m, 5H), 7.75-7.77 (m, 2H), 7.90-7.94 (m, 2H),
8.19-8.21 (m, 2H), 8.49 (s, 1H), 14.32 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ
165.8, 155.6, 149.6, 139.7, 135.4, 134.9, 131.7, 130.7, 128.0, 126.7, 125.4, 124.8,
124.5, 123.7, 122.9, 122.6, 122.0, 118.9, 118.7, 115.6, 113.5. HRMS (ESI) m/z
calculated for [M+H]+ 430.1668, found 430.1658.
5.1.1.14.
2-(2-((2-methoxyacridin-9-yl)amino)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(11n)
1
Yield 57%; mp 247-249 °C; H NMR (400 MHz, DMSO-d6) δ 7.28-7.32 (m,
2H), 7.44-7.47 (m, 1H), 7.55-7.61 (m, 2H), 7.61-7.70 (m, 4H), 7.76 (d, J = 8.0 Hz,
13
1H), 7.90-7.94 (m, 3H), 8.19-8.22 (m, 2H), 8.47 (s, 1H). C NMR (101 MHz,
DMSO-d6) δ 165.8, 155.4, 153.8, 149.6, 139.7, 139.3, 138.7, 135.6, 135.0, 134.3,
131.7, 130.8, 128.1, 127.7, 127.0, 124.9, 124.5, 124.1, 122.9, 122.6, 122.0, 120.9,
119.1, 115.6, 114.6, 113.5, 103.2, 55.5. HRMS (ESI) m/z calculated for [M+H]+
460.1773, found 460.1779.
5.2. Bioassay
5.2.1. PARP inhibition assay
PARP enzyme inhibition was measured using an HT Universal Colorimetric
96-well PARP assay kit (catalog no. 4677-096-K; Trevigen, Inc., Gaithersbug, MD),

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according to the manufacturer’s protocol and referring to reported instructions [54].
5.2.2. Cell culture and proliferation assay
The cancer cell lines MCF-7, HCC1937 and HCT116 were obtained from the
Cell Bank of Chinese Academy of Science (Shanghai, China) and cultured according
to the supplier’s protocol. For MTT assays, the cells were seeded into 96-well plates
at a density of 6-8×103 cells per well and cultured for 12 hours, and then treated with
the synthesized compounds at various concentrations. After 72 hours treatment, 15 µL
MTT (3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide from Sigma)
solution (5 mg/mL in PBS) was added to each well and incubated for 4 hours at 37 °C,
5% CO2. The formazan crystals were dissolved in 100 ꢀl DMSO per well. The
absorbance at 490 nm was measured using Multimode Detector (Beckman Coulter,
Fullerton, CA, USA). Three independent experiments were performed.
5.2.3. Topo I/II inhibition assay
The inhibitory activities of compounds against Topo I and Topo II were
determined by assessing the relaxation of supercoiled pBR322 plasmid DNA as we
described before [33]. The assay was performed in a final volume of 20 µL reaction
volume containing 500 ng of supercoiled DNA pBR322 (Takara Biotechnology, Japan)
and 1 unit of human Topo I (Takara Biotechnology, Japan) or Topo II (USB Corp.,
USA) with or without our compounds in the reaction buffer. The reaction mixtures
were incubated at 37 °C for 10-15 min and terminated by adding 4 µL of DNA

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loading buffer. Electrophoresis was performed on a 0.8% agarose gel at 80 V for 30
min in TAE buffer. Then gels were stained for 10 min with ethidium bromide (2.5
µg/mL) and destained in water for 5 min. DNA bands were visualized with UV light.
Camptothecin (Sigma, USA) and etoposide (Sigma, USA) were used as positive
controls during Topo I and Topo II inhibition assay, respectively.
5.2.4. Apoptosis assay
Phosphatidylserine externalization was measured using Annexin V-FITC/PI
apoptosis detection kit (Beyotime Company, China) according to the manufacturer`s
instructions. MCF-7 cells were treated with 11l at defined concentrations for 48 h.
5.2.5. Cell cycle analysis
MCF-7 cells were seeded in six-well plate at a density of 2×105 cells per well
and incubated for 12 hours in serum-free medium, following treated with graded
concentrations of 11l for 24 hours. Cells were harvested by trypsinization, and fixed
in ice cold absolute methanol. Then the cells were stained with 4 mg/mL PI and 0.1
mg/mL RNaseA in PBS. After incubated in the dark at room temperature for 30 min,
samples were subjected to flow cytometry analysis.
5.2.6. Western blot assay
The primary antibodies anti-53BP1 (ab175933), anti-Topo I (ab109374), and
anti-Topo II (ab109524) were purchased from Abcam. Anti-γH2AX (9718),

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anti-PARP (9542), anti-cleaved PARP (5625), anti-cleaved caspase-3 (9664),
anti-cleaved caspase-8 (9496), anti-cleaved caspase-9 (20750), anti-P18 (2896),
anti-P21 (2947), anti-P27 (3686), anti-CDK2 (2546), anti-CDK4 (12790), anti-CDK6
(3136), anti-cyclin A2 (4656), anti-cyclin D3 (2936), and anti-cyclin E2 (4132) were
purchased from Cell Signaling Technology (CST).
MCF-7 cells were cultured in 6 cm dishes, followed by treatment with 11l at 0, 1,
5, and 10 ꢀM for 24 hours. Then the cells were collected and treated with lysis buffer
o
at 0 C for 0.5 h, followed by centrifugation at 20,000 g for 10 min. Protein
concentrations in the supernatant were determined using bicinchonininc acid (BCA).
Lysate proteins were subjected to 12% sodium dodecylsulfate (SDS) polyacrylamide
gel electrophoresis (PAGE), and electrophoretically transferred to PVDF membrane
(amcBiobind NT-200). After blotting, the membrane was blocked in 5% milk for 1 h,
and incubated with the specific primary antibody at 4 oC for overnight. Followed by a
HRP-conjugated anti-mouse or anti-rabbit second antibodies, proteins were visualized
with the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce).
5.2.7. In vivo antitumor activity
Human breast cancer cell line MCF-7 were injected subcutaneously in the flanks
of mice, and the mice were randomized into three different groups (16/group) with
one group used as blank control (treated with 2% DMSO), and the others treated by
tested drug 11l at 20 mg/Kg and 40 mg/Kg, respectively. These groups were dosed via
i.p. injection once every day. Tumor volume and body weight were measured every 3

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days. On the 30th day after the initial treatment, mice were euthanized and the tumor
tissues were harvested and weighed. Tumor growth inhibition (TGI) was calculated in
this experiment and the antitumor effect of the tested drug was evaluated.
5.2.8. Metabolic stability and CYP450 inhibition assay
The metabolic stability of 11l in liver microsomes and inhibitory activities
against CYP450 isoenzymes were evaluated by Suzhou Truway Biotech Co., Ltd. in
Suzhou, China. For metabolic stability in human/mouse/rat liver microsomes
o
(Xenotech), incubations were performed in 96-well plates at 37 C. Compound
working solution contained the tested compound (1.11 ꢀM) and liver microsomes (0.7
mg protein/mL). After warmed in water bath at 37 °C for 5 min, 10 ꢀL NADPH
co-factor solution per well was added to the test plates and incubated at 37 °C for
defined time, then reaction was stopped by adding 300 µL cold (4 °C) stop solution to
the plates. Samples were centrifuged at 4000 rpm for 20 minutes. While centrifuging,
6×new 96-well plate with 300 ꢀL/well 0.1% Formic Acid HPLC water was loaded,
then 100 ꢀL/well supernatant was transferred and mixed with water for LC/MS/MS,
finally transferred to Bioanalytical Services for LC-MS/MS analysis.
For CYP450 inhibition assay, inhibitor solution was prepared with starting
concentration of 10 mM in DMSO and diluted by acetonitrile. CYP450 isoenzyme
solution and inhibitor solution were added to 96-well plates and mixed, followed by
pre-incubated in water bath 37 °C for 10 min. Then the reaction was initated with the
addition of 50 ꢀL of NADPH/Substrate working solution to the samples for defined