Design, synthesis and biological evaluation of novel phthalazinone acridine derivatives as dual PARP and Topo inhibitors for potential anticancer agents

Article abstract of DOI:10.1016/j.cclet.2019.06.019

In this study, we designed and synthesized a series of phthalazinone acridine derivatives as dual PARP and Topo inhibitors. MTT assays indicated that most of the compounds significantly inhibited multiple cancer cells proliferation. In addition, all the compounds displayed Topo II inhibition activity at 10 mol/L, and also possessed good PARP-1 inhibitory activities. Subsequent mechanistic studies showed that compound 9a induced remarkable apoptosis and caused prominent S cell cycle arrest in HCT116 cells. Our study suggested that 9a inhibiting Topo and PARP concurrently can be a potential lead compound for cancer therapy.

Full text of DOI:10.1016/j.cclet.2019.06.019

Accepted Manuscript
Title: Design, synthesis and biological evaluation of novel
phthalazinone acridine derivatives as dual PARP and Topo
inhibitors for potential anticancer agents
Authors: Qiuzi Dai, Jiwei Chen, Chunmei Gao, Qinsheng Sun,
Zigao Yuan, Yuyang Jiang
PII:
DOI:
Reference:
S1001-8417(19)30347-X
https://doi.org/10.1016/j.cclet.2019.06.019
CCLET 5050
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
24 April 2019
6 June 2019
10 June 2019
Please cite this article as: Dai Q, Chen J, Gao C, Sun Q, Yuan Z, Jiang Y, Design,
synthesis and biological evaluation of novel phthalazinone acridine derivatives as dual
PARP and Topo inhibitors for potential anticancer agents, Chinese Chemical Letters
(2019), https://doi.org/10.1016/j.cclet.2019.06.019
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Communication
Design, synthesis and biological evaluation of novel phthalazinone acridine
derivatives as dual PARP and Topo inhibitors for potential anticancer agents
Qiuzi Daia,b,1, Jiwei Chen , Chunmei Gao , Qinsheng Sun , Zigao Yuan , Yuyang Jiang
b,1
b,c
b
b,*
b,d,
a
Department of Chemistry, Tsinghua University, Beijing 100084, China
The State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Biology, the Graduate School at Shenzhen, Tsinghua University, Shenzhen
b
5
18055, China
c
College of Chemistry and Chemical Engineering, Shenzhen University, Shenzhen 518060, China
d
Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China
*
Corresponding authors.
E-mail addresses: yzg12@tsinghua.org.cn (Z. Yuan), jiangyy@sz.tsinghua.edu.cn (Y. Jiang).
1
The two authors contributed equally to this work.
Graphical Abstract
Herein we discovered a new series of phthalazinone acridine derivatives as PARP and Topo dual inhibitors, with compound 9a
exhibited broad antiproliferative activities and induced remarkable apoptosis and S cell cycle arrest in HCT116 cells.
ARTICLE INFO
Article history: Received Received in revised form Accepted Available online
ABSTRACT
In this study, we designed and synthesized a series of phthalazinone acridine derivatives as dual PARP and Topo inhibitors. MTT assays indicated
that most of the compounds significantly inhibited multiple cancer cells proliferation. In addition, all the compounds displayed Topo II inhibition
activity at 10 μmol/L, and also possessed good PARP-1 inhibitory activities. Subsequent mechanistic studies showed that compound 9a induced
remarkable apoptosis and caused prominent S cell cycle arrest in HCT116 cells. Our study suggested that 9a inhibiting Topo and PARP concurrently
can be a potential lead compound for cancer therapy.
Keywords:
Topo PARP Multitarget Acridines Antitumor bioactivity
The structural integrity and stability of DNA are important for cell survival and normal physiological functions [1-3]. However,
some endogenous or exogenous factors could directly or indirectly induce DNA damages or even mutation [4-6], which, if unrepaired,
would lead to the development of various diseases, including cancer. Targeting pathways of DNA damage and repair are feasible
strategy for treatment of cancer [7-9].
Topoisomerases (Topos) are necessary for the genome DNA correctly squeezed in a small cell nuclear. According to their
structure and catalytic mechanism, Topos are classified into two major classes, during which Topo I cleaves one single-stranded DNA
during each catalytic cycle, while Topo II cleaves double stranded DNA to resolve the DNA damage problems [10,11]. Overexpression
of Topos may cause the instability of genome DNA and have been observed in many tumor cells. Targeting Topos have been
demonstrated to be an effective way for cancer therapy [12-14]. Many acridine analogues, such as m-AMSA (Fig. 1), have entered
clinical or preclinical trials as potent Topo inhibitors. Our group has also devoted much efforts in developing acridine-based Topo
inhibitors for cancer therapy [15-19].
At the same time, poly(ADP-ribose) polymerases (PARPs) are well-known sensors of DNA damage and could repair the single-
strand breaks (SSBs) in DNA via the base-excision repair (BER) pathway [20-22]. PARP-1 is a well-established target for developing

anti-tumor drugs. PARP inhibitors could block PARP-mediated DNA damage repair and make tumor cells more sensitivity to
cytotoxic agents. During the last decades, many PARP inhibitors (PARPi) have been reported [23-25]. Olaparib (Fig. 1) is the first
PARP inhibitor approved by the FDA in 2014 for the treatment of advanced ovarian cancer. Different combinations of PARP inhibitors
and genotoxic drugs are proceeded in clinical trials. These combinations including alkylating agents (temozolomide), cross-linking
agents (cisplatin), Topo I inhibitors (topotecan and irinotecan) and Topo II inhibitors (etoposide) [26].
Fig. 1. Acridines and phthalazinone derivatives.
Scheme 1. Design strategy of dual Topo-PARP inhibitors.
o
o
Scheme 2. Synthesis of compounds 9a−9f. Reagent and conditions: (a) (i) Cu, K
2
CO
3
, DMF, 130 C, 4 h; (ii) POCl
3
, 105 C, 2 h; (b) (iii) HATU, trithylamine,
o
DMF, 2 h, room temperature; (iv) HCl, H O, EtOH, 3 h, room temperature; NH ·H
2
3
2
O, CH
Cl
2 2
; (v) Phenol, Ar, 120 C, 1 h.

In this study, we intended to develop dual PARP and Topo inhibitors based on rational drug design strategy and our own studies
reported previously [15,27-31]. We have developed 4-amidobenzimidazole acridine derivatives as first-in-class dual PARP and Topo
inhibitors for cancer therapy [17]. As reported, compound 11l (Fig. 1) showed good in vitro activities targeting Topo and PARP-1 and
could efficiently suppress tumor growth in mice. Considering the relationship between PARP and Topo and the significant results, we
hold the opinion that simultaneously targeting Topo and PARP-1 is a promising strategy for combating cancer. Phthalazinones has
been recognized as an effective scaffold to develop PARP inhibitors especially since the approval of olaparib [32-35]. The
phthalazinone structure of olaparib is a vital functional group to interact with PARP and the piperazine moiety is mainly used to
improve the activity or adjust the physical and chemical properties. Therefore, the piperazine moiety can be modified or optimized
without loss of PARP inhibitory activity based on the retention of the phthalazinone functional group (Scheme 1). Herein we
developed a series of phthalazinone acridine derivatives by changing the substituent pattern of the acridine group and further evaluated
their anti-tumor activities.
The synthesis of target molecules 9a−9f is shown in Scheme 2. Firstly, compounds 3a−3f were obtained by using 2-chlorobenzoic
acid 2a−2b reacted with phenylamine derivatives 1a−1f via the Ullmann reactions. Then Friedel-Crafts acylation reactions were
conducted in phosphorus oxychloride at 105 °C for 4 h to yield the corresponding acridine intermediates 4a−4f [36]. Secondly,
Compound 8 were synthesized according to the reported procedures [37]. At last, the desired compounds 9a−9f were produced by the
nucleophilic substitution reactions between the phthalazinone derivative 8 and corresponding 9-chloroacridines 4a−4f. Target
1
13
compounds were characterized with H NMR, C NMR, melting point and high resolution mass spectrum (Supporting information).
According to our previous studies, the size, length and the electron-negativity of the substituents on the acridine ring and the
linker between two kinds of inhibitors had great effect on the antitumor activity [28]. Besides, methyl and methoxyl substitution at 2-
position and 4-position of acridine could be helpful to its bioactivity as dual inhibitors of PARP and Topo [17]. In this report, PARP
inhibitor of phthalazinone conjugated with Topo inhibitor acridine was used as a new scaffold compound for the development of PARP
and Topo dual inhibitors. Compounds that were substituted by methyl, methoxyl or chloride substituted on the acridine ring were
investigated. Using m-AMSA and olaparib as positive control, the cellular activities of the compounds were evaluated by the MTT
assays. Different kinds of cancer cells including MCF-7 (human breast adenocarcinaoma cell line), K562 (human chronic myelogenous
leukemia cells), HepG2 (human hepatoma cells), HeLa (human cervical cancer cells), HCT116 (human colon cancer cell line) and
HCC1937 (human breast cancer cell line) were tested. Among the compounds we synthesized (Table 1), 9a and 9c displayed
outstanding antiproliferative activities in all the tumor cells mentioned above. In particular, 9a and 9c showed similar activities to
olaparib in MCF-7 and HCC1937 cells and greatly improved activities in the others. Further, compared with 9c, 9a with non-
substitution of acridine showed better activities. While, compound 9f with the N substituted at 1-position of acridine exhibited
significantly reduced activity. In terms of cellular activity, 9a is the most potent one among the compounds.
We then tested the activities of the synthesized compounds targeting PARP1 and Topo I/II enzymes using olaparib, camptothecin
(CPT) and m-AMSA as positive controls. As shown in Fig. 2A and Table 1, compounds 9a and 9c showed slightly weak PARP-1
inhibitory activities compared to olaparib. Compounds 9d and 9e showed similar PARP-1 inhibitory activities compared to olaparib.
Compounds 9b and 9f showed poor PARP-1 inhibitory activities compared to olaparib. Compound 9d showed the most potent PARP-1
inhibitory activity among the synthesized compounds, while its antiproliferative activities against cancer cells were weak (Table 1),
which may attribute to the compound’s physicochemical properties (e.g., solubility, permeability, lipophilicity, and stability) or the
cellular environments (e.g., cell membrane, pH, and metabolism) which could attenuate the amounts of compounds reaching the
therapeutic target. The weak activity of 9f against tumor cells and PARP1 might result from unfavorable solubility (Fig. S1 and Table
S1 in Supporting information). Moreover, all the compounds displayed comparable Topo II inhibitory activities to m-AMSA at 10
μmol/L (Fig. 2B). These data suggest that all the compounds could potently inhibit Topo II, warranting them as dual PARP and Topo
inhibitors.
Fig. 2. PARP-1, Topo I and Topo II inhibitory activities. (A) PARP-1 inhibition activities of compounds 9a−9f and olaparib at different concentrations. The IC50
values were determined by fitting the experiment data to log(inhibitor) vs. response-variable slope (three parameters) model in Prism. (B) Topo I: lane DNA:
pBR322 DNA; lane DMSO: Topo I + pBR322 DNA; lane CPT: camptothecin + Topo I + pBR322 DNA; the others: compounds (100 μmol/L) + Topo I +
pBR322 DNA. Topo II: lane DNA: pBR322 DNA; lane DMSO: Topo II + pBR322 DNA; lane m-AMSM: m-AMSM+ Topo II + pBR322 DNA; the others:
tested compounds at defined concentrations + Topo II + pBR322 DNA.

DNA damage and its repair have been reported to have effect on the apoptosis pathway [38]. We choose 9a for further study as it
showed excellent cellular activities and desirable enzyme activities. To evaluate whether compound 9a could induce apoptosis, an
annexin-V/PI binding assay was conducted in HCT116 cells as shown in Fig. 3. Compared with DMSO controls, HCT116 cells treated
with 9a at different concentrations of 10, 25, and 50 μmol/L resulted in 0.5%, 22.9%, and 81.5% cells apoptosis, respectively,
suggesting that compound 9a could induce apoptosis in a dose dependent manner.
Fig. 3. Compound 9a induced apoptosis. (A) HCT116 cells were incubated with different concentrations of compounds 9a for 24 h followed by annexin V and
PI staining and analyzed by flow cytometry. (B) Apoptosis analyzed by cell population distributions.
Fig. 4. Compound 9a induced S cell cycle arrest.
Fig. 5. Molecular models of Compound 9a docked into (A) PARP-1, (B) Topo I & DNA complex and (C) Topo II & DNA complex.
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 [38]. To evaluate whether compound 9a could induce tumor cell cycle arrest, we then
performed a flow cytometry assay to assess the effect of compound 9a on the distribution of cell cycle. The cell cycle profiles of
HCT116 after treating with compound 9a in different concentrations for 48ꢀh were illustrated in Fig. 4. The S-phase fraction was
gradually increased from 8.90% in the untreated cells to 9.74%, 13.66%, and 35.78% in cells treated with compound 9a at 5, 10, and
2
5ꢀμmol/L, respectively, indicating that compound 9a caused S cell cycle arrest in a dose-dependent manner.
To further understand the mechanism of compound 9a interacting with the respective enzymes, 9a 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 Fig. 5A, the phthalazinone group of 9a occupied the nicotinamideribose binding domain of PARP1, forming a hydrogen bond with
Gly863. Moreover, the nitrogen atom of the acridine group formed one more hydrogen bond with Arg364 of Topo I and the π-π
stacking interaction between acridine with DA113 was observed (Fig. 5B). The docking results suggested that 9a could form cleavage-
complex with Topo I and DNA. Besides, the phthalazinone group of 9a was parallel to DA12 and formed two hydrogen bonds with
DA12 and DG13, leading to the formation of cleavage-complex with Topo II and double strands DNA (Fig. 5C). The above docking
results may further confirm that 9a could act as dual PARP and Topo I/II inhibitors.
In conclusion, Topos and PARPs are important for genome stability. Inhibitors targeting both of the targets may have synergistic
antitumor effects. In this study, we designed and synthesized a new series of phthalazinone acridine derivatives targeting both PARP
and Topo. Some of the compounds displayed potent antiproliferative activities against kinds of cancer cells and showed potential
inhibitory potency against both PARP-1 and Topo I/II in vitro. Compound 9a exhibited the most potent antiproliferative activity in its

class. Further evaluation indicated that 9a induced remarkable apoptosis, and caused prominent S cell cycle arrest. Taken together,
compound 9a is a promising dual-targeted inhibitor and is worth for further optimization for cancer therapy.
Acknowledgments
The authors would like to thank the financial supports from the Shenzhen Development and Reform Committee (Nos. 20151961
and 2019156) and Department of Science and Technology of Guangdong Province (No. 2017B030314083).
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Table 1
In vitro antiproliferative potency and PARP-1 inhibitory activity of compounds 9a−9f.
a
Compd.
R
1
R
2
X
MTT assays (IC50 , μmol/L)
PARP-1
K562
MCF-7
HepG2
HeLa
HCT116
HCC1937
inhibitory
activity
a
(
IC50
,
μmol/L)
9
9
9
9
9
9
a
b
c
d
e
f
H
H
H
H
H
CH
CH
CH
CH
CH
N
3.94 ± 0.38
40.99 ± 2.51
12.89 ± 0.95
>50
22.69 ± 0.05
>50
13.80 ± 0.58
41.65 ± 2.0
15.09 ± 1.0
>50
11.74 ± 0.40
24.57 ± 0.57
12.94 ± 0.47
34.56 ± 1.86
25.53 ± 0.33
>50
13.11 ± 1.02
>50
24.23 ± 0.95
>50
43.59 ± 2.06
>50
5.04 ± 0.66
>50
12.88 ± 0.54
46.10 ± 2.51
36.93 ± 0.79
>50
25.95 ± 0.49
>50
7.96 ± 0.61
48.86 ± 1.97
9.42 ± 2.38
>50
8.65 ± 2.48
>50
11.85 ± 0.65
87.89 ± 0.20
26.69 ± 0.10
2.63 ± 0.03
3.12 ± 0.08
130.7 ± 9.80
4-CH3
4-C2H5
2-CH3
2-OCH3 Cl
2-OCH3 Cl
>50
21.51 ± 1.5
12.97 ± 0.35
32.06 ± 0.62
m-AMSA
Olaparib
1.65 ± 0.10
>50
6.56 ± 0.25
>50
12.73 ± 0.08
>50
15.23 ± 1.10
9.39 ± 0.51
0.98 ± 0.009
aData are expressed as the mean ± SD of at least duplicate determinations.