Synthesis, preliminarily biological evaluation and molecular docking study of new Olaparib analogues as multifunctional PARP-1 and cholinesterase inhibitors

Article abstract of DOI:10.1080/14756366.2018.1530224

A series of new Olaparib derivatives was designed and synthesized, and their inhibitory activities against poly (ADP-ribose) polymerases-1 (PARP-1) enzyme and cancer cell line MDA-MB-436 in vitro were evaluated. The results showed that compound 5l exhibited the most potent inhibitory effects on PARP-1 enzyme (16.10 ± 1.25 nM) and MDA-MB-436 cancer cell (11.62 ± 2.15 μM), which was close to that of Olaparib. As a PARP-1 inhibitor had been reported to be viable to neuroprotection, in order to search for new multitarget-directed ligands (MTDLs) for the treatment of Alzheimer’s disease (AD), the inhibitory activities of the synthesized compounds against the enzymes AChE (from electric eel) and BChE (from equine serum) were also tested. Compound 5l displayed moderate BChE inhibitory activity (9.16 ± 0.91 μM) which was stronger than neostigmine (12.01 ± 0.45 μM) and exhibited selectivity for BChE over AChE to some degree. Molecular docking studies indicated that 5l could bind simultaneously to the catalytic active of PARP-1, but it could not interact well with huBChE. For pursuit of PARP-1 and BChE dual-targeted inhibitors against AD, small and flexible non-polar groups introduced to the compound seemed to be conducive to improving its inhibitory potency on huBChE, while keeping phthalazine-1-one moiety unchanged which was mainly responsible for PARP-1 inhibitory activity. Our research gave a clue to search for new agents based on AChE and PARP-1 dual-inhibited activities to treat Alzheimer’s disease.

Full text of DOI:10.1080/14756366.2018.1530224

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis, preliminarily biological evaluation
and molecular docking study of new Olaparib
analogues as multifunctional PARP-1 and
cholinesterase inhibitors
Cheng-Zhi Gao, Wei Dong, Zhi-Wen Cui, Qiong Yuan, Xia-Min Hu, Qing-Ming
Wu, Xianlin Han, Yao Xu & Zhen-Li Min
To cite this article: Cheng-Zhi Gao, Wei Dong, Zhi-Wen Cui, Qiong Yuan, Xia-Min Hu, Qing-
Ming Wu, Xianlin Han, Yao Xu & Zhen-Li Min (2019) Synthesis, preliminarily biological evaluation
and molecular docking study of new Olaparib analogues as multifunctional PARP-1 and
cholinesterase inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 150-162,
DOI: 10.1080/14756366.2018.1530224
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 150–162
https://doi.org/10.1080/14756366.2018.1530224
RESEARCH PAPER
Synthesis, preliminarily biological evaluation and molecular docking study of new
Olaparib analogues as multifunctional PARP-1 and cholinesterase inhibitors
a
a
a
a
b
a
c
d
ꢀ
ꢀ
Cheng-Zhi Gao , Wei Dong , Zhi-Wen Cui , Qiong Yuan , Xia-Min Hu , Qing-Ming Wu , Xianlin Han , Yao Xu
and Zhen-Li Mina,c
aHubei Province Key Laboratory of Occupational Hazard Identification and Control, Wuhan University of Science and Technology, Wuhan, China;
c
bCollege of Pharmacy, Shanghai University of Medicine & Health Sciences, Shanghai, China; Barshop Institute for Longevity and Aging Studies,
d
University of Texas Health Science Center at San Antonio, San Antonio, TX, USA; College of Life Science and Health, Wuhan University of
Science and Technology, Wuhan, China
ABSTRACT
ARTICLE HISTORY
Received 7 August 2018
Revised 23 September 2018
Accepted 25 September 2018
A series of new Olaparib derivatives was designed and synthesized, and their inhibitory activities against
poly (ADP-ribose) polymerases-1 (PARP-1) enzyme and cancer cell line MDA-MB-436 in vitro were eval-
uated. The results showed that compound 5l exhibited the most potent inhibitory effects on PARP-1
enzyme (16.10 1.25 nM) and MDA-MB-436 cancer cell (11.62 2.15 lM), which was close to that of
Olaparib. As a PARP-1 inhibitor had been reported to be viable to neuroprotection, in order to search for
new multitarget-directed ligands (MTDLs) for the treatment of Alzheimer’s disease (AD), the inhibitory
activities of the synthesized compounds against the enzymes AChE (from electric eel) and BChE (from
equine serum) were also tested. Compound 5l displayed moderate BChE inhibitory activity (9.16 0.91lM)
which was stronger than neostigmine (12.01 0.45lM) and exhibited selectivity for BChE over AChE to
some degree. Molecular docking studies indicated that 5l could bind simultaneously to the catalytic active
of PARP-1, but it could not interact well with huBChE. For pursuit of PARP-1 and BChE dual-targeted inhib-
itors against AD, small and flexible non-polar groups introduced to the compound seemed to be condu-
cive to improving its inhibitory potency on huBChE, while keeping phthalazine-1-one moiety unchanged
which was mainly responsible for PARP-1 inhibitory activity. Our research gave a clue to search for new
agents based on AChE and PARP-1 dual-inhibited activities to treat Alzheimer’s disease.
KEYWORDS
PARP-1 inhibitor; Olaparib;
AChE; BChE;
Alzheimer’s Disease
Introduction
transfer of ADP-ribose units to nuclear target proteins upon oxida-
tive stress and DNA injury. Over-activation of PARP-1 will lead to
cellular depletion of NADþ and ATP, and then, energy deficiency
slows glycolysis and mitochondrial respiration rates, resulting in
controlled nerve cell death. This PARP-related specific neurodege-
nerative mechanism was termed as parthanatos10. Meanwhile,
over-activated PARP-1 will increase its interaction with transcrip-
tion regulators such as nuclear receptors, sirtuins, and other meta-
bolic transcription factors. Further, it will also lead to the
impairment of mitochondrial function that seems to be an early
Poly (ADP-ribose) polymerases-1 (PARP-1) is a pivotal nuclear
enzyme ubiquitously expressed in eukaryotic cells. It can detect
DNA single-strand break and catalyse the addition of ADP-ribose
units to acceptor proteins, thus facilitating DNA repair process
and promoting cell survival1,2. Inhibition of PARP-1 accelerates the
damage of injured DNA, which especially leads to synthetic lethal-
ity in DNA-repairing-deficient cancer cells, for example BRCA1/
2-deficient cells. Therefore, PARP-1 has become an attractive anti-
tumor target3–5. PARP-1 inhibitors can be utilized not only as a
single agent for the treatment of BRCA1- or BRCA2-deficient can-
cers, but in combination with DNA-damaging therapeutics (radi-
ation or chemotherapy) to improve their potencies by blocking
DNA-repairing process. Then, PARP-1 inhibitors have been exten-
and critically important event in the pathogenesis of AD9,11
.
Although the role of PARP-1 in human AD is not fully elucidated
and many mechanisms are under investigations, accumulating evi-
dences suggest that PARP-1 inhibitors are viable to neuroprotec-
tion and PARP-1 has emerged as a potential therapeutic target for
AD12,13. As for AD, it still imposes a great threat to human health.
sively investigated as potential anticancer drugs6,7
.
In addition to being used for cancer treatment, recent Currently, most of the approved drugs for AD treatment are
researches indicate that PARP-1 inhibitors have potentially thera- acetylcholinesterase (AChE) inhibitors, namely donepezil, tacrine,
peutic value in Alzheimer’s disease (AD)8–10. PARP-1 utilizes nico- rivastigmine and galantamine. Nevertheless, these drugs modestly
tinamide adenine dinucleotide (NADþ) as substrate to catalyse the alleviate the symptoms but cannot cure brain damage or stop
CONTACT Zhen-Li Min
and Technology, Wuhan, 430081, China;
minzhenli@wust.edu.cn
Hubei Province Key Laboratory of Occupational Hazard Identification and Control, Wuhan University of Science
Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, TX, 78229,
College of Pharmacy, Shanghai University of Medicine & Health Sciences, Shanghai, China
USA; Xia-Min Hu
These authors contributed equally to this work.
huxm@sumhs.edu.cn
ꢀ
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
151
neuronal degeneration14. Therefore, discovery for effective anti-AD cholinesterases and neuroprotection26–28. Through the structural
drugs remains an enormous challenge to medicinal chemistry optimizations, we hoped to find a more potent PARP-1 inhibitor,
communities. In fact, in addition to AChE, there is another cholin-
esterase, butyrylcholinesterase (BChE), in the synaptic gap of neur-
onal cells to efficiently hydrolyse acetylthiocholine (ACh), and its
inhibitors have been reported to be more beneficial to AD in later
stages of AD than AChE inhibitors15–17. In the light of the multifac-
torially pathological mechanisms of AD, developing multitarget-
directed ligands (MTDLs) may be a more promising approach for
its treatment. Some researches and reviews of MTDLs for AD have
already been reported18,19, but almost none of which is related to
both PARP-1 and cholinesterase inhibitors20. In this regard, the
PARP-1 and cholinesterase dual-targeted inhibitor of AD are wor-
thy to be investigated.
at the same time, in order to explore new MTDLs for the treat-
ment of AD, the inhibitory activities against AChE and BChE
enzymes of these compounds were also evaluated. Here, we
described the synthesis and bioactivities assays of 15 Olaparib
derivatives; meanwhile, molecular dockings were conducted to
investigate their interaction fashion with the correspond-
ing proteins.
Materials and methods
Chemistry
All reagents and solvents were obtained from commercially avail-
able sources and were used without further purification. Reaction
progress was monitored using analytical thin layer chromatog-
raphy (TLC) on precoated silica gel GF254 (Qingdao Haiyang
Chemical Plant, Qing-Dao, China) plates and spots were detected
under UV light (254 nm). Melting points were determined on a
WRS-2B digital melting point apparatus and uncorrected. IR spec-
tra were recorded using a Nicolet 380 Fourier-transform infrared
Olaparib is the first-in-class PARP-1 inhibitor marketed in 2014
as monotherapy for advanced BRCA-deficient ovarian cancer.
However, its anticancer activity was not potent enough7,21, and it
showed dose-limiting toxicity that was more pronounced than
that seen with the chemotherapeutic agents alone when it was
utilized in combination with chemotherapeutic agents for cancer
treatment22. To overcome these shortcomings, several modifica-
tions based on structural skeleton of Olaparib have been reported
(Figure 1)21–24. The reported structure–activity relationship (SAR)
revealed that 4-benzyl phthalazinone was the core scaffold
responsible for moderate PARP-1-inhibiting potency, and a substi-
tuted piperazine at the meta position of benzyl moiety was con-
ducive to enhancing activity and maintaining good oral
bioavailability7,25. In view of the important therapeutic value of
PARP-1 inhibitor for cancer and potentially for AD treatment, it
was interesting and deserved to further explore structural opti-
mization of Olaparib based on the existed SAR. With this aim in
mind, we decided to keep the 4-benzyl phthalazinone group
unchanged and replace the cyclopropane group of Olaparib by
substituted aryl vinyl ones to constitute 3-aromatic a, b-unsatur-
ated carbonyl moiety. On the one hand, the aryl vinyl groups are
hydrophobic, just like cyclopropane group, which is helpful to
improve oral bioavailability7; on the other hand, 3-aromatic a,
1
(FTIR) spectrophotometer (Thermo, USA) from KBr pellets. H NMR
and 13C NMR spectra were recorded on a Bruker AVANCE III-600
NMR spectrometer (Bruker Biospin Co., Switzerland) with tetrame-
thylsilane (TMS) as the internal standard and CDCl3 or DMSO-d6 as
solvent and known chemical shifts of residual proton signals of
deuterated solvents (1H NMR: d: 2.50 for DMSO-d6 and d: 3.33 for
H2O) or carbon signals of deuterated solvents (13C NMR: d: 39.52
for DMSO-d6) as internal standard. MS (ESI) measurement was con-
ducted on an Agilent 1100 LC-MS spectrometer (Agilent, Palo
Alto, USA).
Dimethyl (3-oxo-1, 3-dihydro-isobenzofuran-1-yl)-
phosphonate (7)
Compounds 7, 8 and 9 were prepared according to the literature
b-unsaturated carbonyl moiety is widely included into natural procedure24,29
. A mixture of 2-formylbenzoic acid 6 (2.0 g,
products, especially within chalcone framework that possesses a 15.0 mmol) and dimethyl phosphate (4.0 g, 36.0 mmol) was heated
variety of bioactivities, including anticancer, inhibition of to 100 ꢁC in oil bath and stirred for 8 h. Subsequently, the mixture
O
O
O
NH
N
NH
N
NH
N
O
F
O
O
F
N
N
N
F
N
N
N
F
N
N
N
N
F
CF3
F
O
1 Olaparib
3 Simmiparib
2 AZ0108
O
O
N
O
O-
O
NH
N
NH
N+
N
N
O
F
O
F
NO2
N
N
N
O
Ar
N
N
N+
O-
NO2
5 Target Molecule
4
Figure 1. The structures of Olaparib analogs and target molecules.

152
C.-Z. GAO ET AL.
was cooled to room temperature and then poured into ice-cold The mixture was washed with saturated NaHCO3 solution
(2 ꢂ 15 mL), brine (30 mL), dried over Na2SO4 and evaporated
water (20 mL), which was followed by extraction with dichlorome-
under reduced pressure, and then, the residue was purified by sil-
thane (3 ꢂ 30 mL). The combined organic extracts were dried over
ica gel column chromatography to obtain 12a–o in 40–70% yields.
Finally, 12a–o (12.0 mmol) was dissolved in DCM (30 mL) to which
trifluoroacetic acid (78 mmol) was added, and then, the mixture
was stirred for 8 h at room temperature. Water (30 mL) was added
to the mixture, which was stirred for another 0.5 h and then sepa-
rated. The water layer was extracted with DCM (3 ꢂ 10 mL), the
combined organic layers were dried over Na2SO4 and concen-
trated and purified by silica gel column chromatography to give
13a–o as yellow oils in 50–80% yields1. HNMR data of 12a–o
were shown below:
MgSO4 and were evaporated in vacuum to produce a white solid
which was crystallized in ethanol to yield 2.25 g compound 7.
Yield: 62.3%, mp: 96.1–98.2 ꢁC. 1H-NMR (CDCl3, 600 MHz), d: 7.93
(d, J ¼ 7.68 Hz, 1H, ArH), 7.76–7.70 (m, 2H, ArH), 7.58 (t, J ¼ 7.56 Hz,
1H, ArH), 5.70 (d, J ¼ 10.98 Hz, 1H, CH), 3.91 (d, J ¼ 10.92 Hz, 3H,
OCH3), 3.58 (d, J ¼ 10.62 Hz, 3H, OCH3).
2-Fluoro-5-[(3-oxo-2-benzofuran-1-ylidene) methyl]
benzonitrile (8)
To a mixture of 7 (4.3 g, 17.8 mmol) and 2-fluoro-5-formylbenzoni-
trile (2.6 g, 17.5 mmol) in anhydrous THF (40 mL) was added trie-
thylamine (1.8 mL, 13 mmol) dropwise in 30 min, and the
temperature was maintained below 15 ꢁC. The reaction mixture
was slowly warmed to room temperature. The progress of the
reaction was monitored by TLC. After completion of the reactions,
the reaction mixture was then concentrated in vacuo. The residue
was slurried in water (50 mL) for 30 min, and the solid was col-
lected by filtration, was washed with diethyl ether (2 ꢂ 10 mL) and
was dried to afford 3.6 g of 8 in 78.5% yield. The material was car-
ried forward without further purification.
Tert-butyl 4-((E)-3-phenylacryloyl)piperazine-1-carboxylate (12a)
1
A white solid, yield: 63.3%, mp: 131.1–132.2 ꢁC, H NMR (600 MHz,
CDCl3) d: 7.68 (d, J ¼ 15.4 Hz, 1H, ¼CH), 7.51 (dd, J ¼ 7.8, 1.4 Hz,
2H, ArH), 7.35 (dd, J ¼ 8.3, 6.6 Hz, 2H, ArH), 6.84 (d, J ¼ 15.4 Hz, 1H,
¼CH), 3.66–3.47 (m, 8H, NCH2), 1.47 (s, 9H, C (CH3)3).
Tert-butyl 4-[(E)-3-(3,4-dimethoxyphenyl)acryloyl]piperazine-1-
carboxylate (12b)
1
A white solid, yield: 55.3%, mp: 124.9–125.1 ꢁC, H NMR (600 MHz,
CDCl3) d: 7.87 (d, J ¼ 15.6 Hz, 1H, ¼CH), 7.02 (s, 1H), 6.91 (d,
J ¼ 15.6 Hz, 1H, ¼CH), 6.83 (dt, J ¼ 17.9, 5.9 Hz, 2H, ArH), 3.80 (s,
3H, OCH3), 3.76 (s, 3H, OCH3), 3.65–3.62 (m, 4H, NCH2), 3.50–3.36
(m, 4H, NCH2), 1.45 (s, 9H, C (CH3)3).
2-Fluoro-5-[(4-oxo-3H-phthalazin-1-yl) methyl] benzoic Acid (9)
To a stirred suspension of 8 (2.1 g, 8.0 mmol) in water (20 mL) was
added aqueous NaOH (10 mol/l, 3.5 mL) with subsequent heating
to 90 ꢁC for 1 h. The reaction mixture was cooled to 70 ꢁC into
which hydrazine hydrate (5.4 mL, 94.0 mmol) was added, and the
mixture was stirred for 18 h at 70 ꢁC before TLC indicated the reac-
tion was completed. The mixture was cooled to ambient tempera-
ture and was acidified with HCl (4 mol/L) to pH 4, and then, the
suspension was filtered and was washed with diethyl ether and
dried to get 2.2 g of 9 as a light red powder. Yield: 93.7%, mp:
Tert-butyl 4-[(E)-3-(4-methoxyphenyl)acryloyl]piperazine-1-
carboxylate (12c)
1
A yellow solid, yield: 56.3%, mp: 127.1–128.8 ꢁC, H NMR (600 MHz,
CDCl3) d: 7.90 (d, J ¼ 15.6 Hz, 1H, ¼CH), 7.47 (d, J ¼ 7.6 Hz, 2H,
ArH), 7.33–7.28 (m, 2H, ArH), 6.94 (d, J ¼ 15.6 Hz, 1H, ¼CH), 3.88 (s,
3H, OCH3), 3.68–3.62 (m, 4H, NCH2), 3.53–3.16 (m, 4H, NCH2), 1.46
(s, 9H, C (CH3)3).
1
208.4–210.1 ꢁC。 H-NMR (600 MHz, DMSO-d6), d: 13.05 (s, 1H,
COOH), 12.56 (s, 1H, NH), 8.26–8.20 (m, 1H, ArH), 7.94 (d,
J ¼ 8.0 Hz, 1H, ArH), 7.91–7.84 (m, 1H, ArH), 7.83–7.75 (m, 2H, ArH),
7.58–7.50 (m, 1H, ArH), 7.20 (m, 1H, ArH), 4.32 (s, 2H, CH2).
Tert-butyl 4-[(E)-3-(4-methylphenyl)acryloyl]piperazine-1-
carboxylate (12d)
A light yellow solid, yield: 58.3%, mp: 149.6–151.3 ꢁC, 1H NMR
(600 MHz, DMSO-d6) d: 7.88 (d, J ¼ 15.6 Hz, 1H, ¼CH), 7.82–7.79 (m,
2H, ArH), 7.59–7.56 (m, 2H, ArH), 6.91 (d, J ¼ 15.6 Hz, 1H, ¼CH),
3.77–3.48 (m, 6H, NCH2), 3.21–3.19 (m, 2H, NCH2), 2.31 (s, 3H,
CH3), 1.45 (s, 9H, C (CH3)3);
General procedure for preparation of intermediates 13a–o
Compounds 10a–o, 11a–o, 12a–o and 13a–o were prepared
according to the literature procedures29,30. To a stirred solution of
various substituted benzaldehydes (or 2-furaldehyde) (24.0 mmol)
in pyridine (15 mL), malonic acid (4.9 g, 48.0 mmol) and ammo-
nium acetate (0.2 g, 23 mmol) were added. The mixture was stirred
for 5 h at 85 ꢁC and then cooled to room temperature, slowly
diluted with ice water (100 mL). The solution was acidified with
HCl to pH 2, and the precipitate formed. After filtered, the solid
was washed with water until it had no pyridine smell. It was dried,
which was followed by recrystallization from ethanol to give
10a–o in 50–80% yields. Compounds 10a–o (8.0 mmol) were dis-
solved in DMF (0.25 mL) to which oxalyl chloride was added drop-
wise at 0–5 ꢁC. The mixture was stirred for 1.5 h at room
temperature and then evaporated under reduced pressure. The
residue 11a–o was added dichloromethane (DCM, 15 mL) to dis-
solve and sealed for use in next step. Tert-butoxycarbonylpipera-
zine (1.5 g, 8.0 mmol) and triethylamine (1.1 mL, 8.0 mmol) were
dissolved in DCM (15 mL). After dropwise addition of 11a–n at
Tert-butyl 4-[(E)-3-(2-methoxyphenyl)acryloyl]piperazine-1-
carboxylate (12e)
1
A white solid, yield: 58.1%, mp: 114.7–115.8 ꢁC, H NMR (600 MHz,
CDCl3) d: 7.91 (d, J ¼ 15.6 Hz, 1H, ¼CH), 7.47 (d, J ¼ 7.6 Hz, 1H,
ArH), 7.33–7.28 (m, 1H, ArH), 6.98–6.88 (m, 3H, ArH and ¼ CH), 3.87
(s, 3H, OCH3), 3.68–3.62 (m, 4H, NCH2), 3.53–3.16 (m, 4H, NCH2),
1.46 (s, 9H, C (CH3)3);
Tert-butyl 4-[(E)-3-(2,5-dimethoxyphenyl)acryloyl]piperazine-1-
carboxylate (12f)
A light yellow solid, yield: 60.2%, mp: 147.0–147.6 ꢁC, 1H NMR
0–5 ꢁC, the mixture was stirred for 12 h at room temperature. (600 MHz, CDCl3) d: 7.85 (d, J ¼ 15.6 Hz, 1H, ¼CH), 6.99

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
153
(d, J ¼ 2.9 Hz, 1H, ArH), 6.92 (d, J ¼ 15.6 Hz, 1H, ¼CH), 6.83 (dt, ArH), 7.43 (d, J ¼ 15.4 Hz, 1H, ¼CH), 7.26 (d, J ¼ 15.4 Hz, 1H, ¼CH),
J ¼ 17.9, 5.9 Hz, 2H, ArH), 3.80 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.58–3.31 (m, 4H, NCH2), 3.32 (s, 4H, NCH2), 1.38 (s, 9H, C (CH3)3).
3.65–3.61 (m, 4H, NCH2), 3.50–3.36 (m, 4H, NCH2), 1.45 (s, 9H,
C (CH3)3).
Tert-butyl 4-[(E)-3-(3,4,5-trimethoxyphenyl)acryloyl]piperazine-1-
carboxylate (12n)
Tert-butyl 4-[(E)-3-(4-fluorophenyl)acryloyl]piperazine-1-
carboxylate (12g)
A light yellow solid, yield:52.7%, mp: 137.3–138.2 ꢁC, 1H NMR
(600 MHz, CDCl3) d: 7.58 (d, J ¼ 15.3 Hz, 1H, ¼CH), 6.75–6.70 (m,
1
A white solid, yield: 62.1%, mp: 143.6–144.1 ꢁC, H NMR (600 MHz,
3H, ArH and ¼ CH), 3.90–3.83 (m, 9H, OCH3), 3.69–3.64 (m, 4H,
NCH2), 3.47 (s, 4H, NCH2), 1.46 (s, 9H, C (CH3)3).
CDCl3) d: 7.64 (d, J ¼ 15.4 Hz, 1H, ¼CH), 7.54–7.45 (m, 2H, ArH),
7.09–7.00 (m, 2H, ArH), 6.76 (d, J ¼ 15.4 Hz, 1H, ¼CH), 3.65–3.32(m,
8H, NCH2), 1.46 (s, 9H, C (CH3)3).
Tert-butyl 4-[(E)-3-(furan-2-yl)acryloyl]piperazine-1-
carboxylate (12o)
A brown yellow solid, yield: 42.3%, mp: 103.7–104.7 ꢁC, 1H NMR
Tert-butyl 4-[(E)-3–(4-trifluoromethylphenyl)acryloyl]piperazine-
1-carboxylate (12h)
(600 MHz, CDCl3) d: 7.45 (d, J ¼ 15.1 Hz, 1H, ¼CH), 7.42 (d,
1
A white solid, yield: 56.2%, mp: 173.1–173.6 ꢁC, H NMR (600 MHz,
CDCl3) d: 7.68 (d, J ¼ 15.4 Hz, 1H, ¼CH), 7.64–7.58 (m, 4H, ArH),
6.92 (d, J ¼ 15.5 Hz, 1H, ¼CH), 3.66–3.48 (m, 8H, NCH2), 1.47 (s, 9H,
C (CH3)3).
J ¼ 1.4 Hz, 1H, furan-H), 6.75 (d, J ¼ 15.1 Hz, 1H, ¼CH), 6.53 (d,
J ¼ 3.4 Hz, 1H, furan-H), 6.43 (dd, J ¼ 3.4, 1.8 Hz, 1H, furan-H), 3.63
(t, J ¼ 9.6 Hz, 4H, NCH2), 3.45 (t, J ¼ 7.3 Hz, 4H, NCH2), 1.45 (s, 9H,
C (CH3)3).
General procedure for preparation of target compounds 5a–o
Tert-butyl 4-[(E)-3-(3-fluorophenyl)acryloyl]piperazine-1-
carboxylate (12i)
To a solution of 9 (1.2 g, 4.1 mmol) in DCM (30 mL) were added
N,N-Diisopropylethylamine (DIPEA, 1.1 mL, 6.2 mmol) and hexa-
fluorophosphate benzotriazole tetramethyl uranium (HBTU, 2.4 g,
6.2 mmol), followed by an appropriate solution of 13a–o
(4.1 mmol) in DCM. The reaction mixture was stirred at room tem-
perature for 18 h and was then poured into water (40 mL), which
was followed by extraction with DCM (3 ꢂ 20 mL). The combined
organic extracts were dried over Na2SO4 and evaporated under
reduced pressure to give crude product. The pure product was
isolated by silica gel column chromatography as a white or yellow
powder. Data of these compounds were shown below:
1
A white solid, yield: 55.3%, mp: 121.8–122.2 ꢁC, H NMR (600 MHz,
CDCl3) d: 7.62 (d, J ¼ 15.4 Hz, 1H, ¼CH), 7.32 (d, J ¼ 5.8 Hz, 1H,
ArH), 7.26–7.20 (m, 1H, ArH), 7.03 (t, J ¼ 8.5 Hz, 1H, ArH), 6.83 (d,
J ¼ 15.4 Hz, 1H, ¼CH), 3.65–3.47 (m, 8H, NCH2), 1.46 (s, 9H,
C (CH3)3).
Tert-butyl 4-[(E)-3-(4-chlorophenyl)acryloyl]piperazine-1-
carboxylate (12j)
1
A white solid, yield: 58.2%, mp: 187.1–187.5 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 7.73 (d, J ¼ 8.4 Hz, 2H, ArH), 7.49–7.44 (m, 3H, ArH
and ¼ CH), 7.25 (d, J ¼ 15.4 Hz, 1H), 3.58–3.31 (m, 8H, NCH2), 1.38
(s, 9H, C (CH3)3).
(E)-4-{[3-[4-(3-phenylacryloyl) piperazine-1-carbonyl]-4-
fluorophenyl]methyl}-2H-phthalazin-1-one (5a)
1
A white solid, yield: 18.3%, mp: 270.8–272.4 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.57 (s, 1H), 8.23 (d, J ¼ 7.8 Hz, 1H), 7.95 (d,
J ¼ 8.1 Hz, 1H), 7.87 (t, J ¼ 8.2 Hz, 1H), 7.80 (s, 1H), 7.67–7.70 (m,
2H), 7.48 (d, J ¼ 15.4 Hz, 1H), 7.47–7.35 (m, 4H), 7.29–7.14 (m, 3H),
4.31 (s, 2H), 3.77–3.48 (m, 6H), 3.20 (s, 2H); 13C NMR (150 MHz,
DMSO-d6) d: 165.06, 164.49, 159.82, 157.63, 156.01, 145.27, 142.29,
135.49, 135.28, 133.94, 132.20, 132.00, 130.07, 129.53, 129.41,
129.20, 128.48, 128.34, 126.51, 125.89, 124.06, 123.92, 118.46,
116.46, 47.36, 46.77, 45.61, 45.04, 36.88; IR (KBr, cmꢃ1): 3435, 3011,
2911, 1649, 1606, 1217, 769; ESI-MS: 495.8 [M ꢃ H]ꢃ.
Tert-butyl 4-[(E)-3-(4-bromophenyl)acryloyl]piperazine-1-
carboxylate (12k)
1
A white solid, yield: 65.3%, mp: 207.7–208.4 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 7.65 (d, J ¼ 8.4 Hz, 2H, ArH), 7.57 (d, J ¼ 8.4 Hz, 2H,
ArH), 7.43 (d, J ¼ 15.4 Hz, 1H, ¼CH), 7.26 (d, J ¼ 15.4 Hz, 1H, ¼CH),
3.58–3.39 (m, 4H, NCH2), 3.32 (s, 4H, NCH2), 1.38 (s, 9H, C (CH3)3).
Tert-butyl 4-[(E)-3-(3-nitrophenyl)acryloyl]piperazine-1-
carboxylate (12l)
1
A white solid, yield: 52.4%, mp: 145.1–145.8 ꢁC, H NMR (600 MHz,
(E)-4-{[3-[4–(3-(3,4-dimethoxyphenyl)acryloyl) piperazine-1-
carbonyl]-4-fluorophenyl]methyl}-2H-phthalazin-1-one (5b)
DMSO-d6) d: 7.80 (d, J ¼ 6.6 Hz, 1H, ArH), 7.72 (d, J ¼ 15.4 Hz, 1H,
¼CH), 7.67–7.62 (m, 2H, ArH), 7.59 (t, J ¼ 7.2 Hz, 1H, ArH), 7.45 (d,
J ¼ 15.4 Hz, 1H, ¼CH), 3.60–3.39 (m, 4H, NCH2), 3.35–3.31 (m, 4H,
NCH2), 1.39 (s, 9H, C (CH3)3).
1
A white solid, yield: 20.3%, mp: 197.9–199.3 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.57 (s, 1H), 8.23 (d, J ¼ 7.8 Hz, 1H), 7.95 (d,
J ¼ 8.0 Hz, 1H), 7.87 (t, J ¼ 7.6 Hz, 1H), 7.80 (t, J ¼ 7.3 Hz, 1H),
7.45–7.30 (m, 4H), 7.24–7.19 (m, 3H), 6.94 (d, J ¼ 8.3 Hz, 1H), 4.31
(s, 2H), 3.79–3.76(m, 7H), 3.63–3.48 (m, 5H), 3.20(s, 2H); 13C NMR
(150 MHz, DMSO-d6) d: 165.35, 164.48, 159.83, 157.63, 156.01,
150.79, 149.36, 145.28, 142.72, 135.27, 133.93, 132.25, 131.99,
Tert-butyl 4-[(E)-3-(4-nitrophenyl)acryloyl]piperazine-1-
carboxylate (12m)
A white solid, yield:55.6%, mp: 212.1–213.3 ꢁC, 1H NMR (600 MHz, 129.53, 129.42, 128.34, 128.30, 126.51, 125.88, 123.94, 122.87,
DMSO-d6) d: 7.65 (d, J ¼ 8.4 Hz, 2H, ArH), 7.57 (d, J ¼ 8.4 Hz, 2H, 116.43, 115.69, 111.95, 110.80, 56.13, 55.98, 47.41, 46.87, 45.62,

154
C.-Z. GAO ET AL.
45.07, 36.88; IR (KBr, cmꢃ1): 3443, 3010, 2909, 1638, 1593, 1261, (E)-4-{[3-[4–(3-(4-fluorophenyl) acryloyl) piperazine-1-carbonyl]-
835; ESI-MS: 555.8 [M ꢃ H]ꢃ.
4-fluorophenyl] methyl} -2H -phthalazin-1-one (5g)
1
A white solid, yield: 15.1%, mp: 272.7–272.9 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.56 (s, 1H), 8.23 (d, J ¼ 8.9 Hz, 1H), 7.94 (d,
(E)-4-{[3-[4–(3-(4-methoxyphenyl) acryloyl) piperazine-1-
carbonyl]-4-fluorophenyl] methyl} -2H-phthalazin-1-one (5c)
J ¼ 8.0 Hz, 1H), 7.86 (t, J ¼ 7.6 Hz, 1H), 7.84–7.67 (m, 3H), 7.47 (d,
J ¼ 15.3 Hz, 1H), 7.4–7.31 (m, 2H), 7.27–7.07 (m, 4H), 4.30 (s, 2H),
1
A white solid, yield: 19.7%, mp: 212.5–213.2 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.56 (s, 1H), 8.23 (d, J ¼ 7.8 Hz, 1H), 7.94 (d,
J ¼ 8.0 Hz, 1H), 7.87 (td, J ¼ 7.7, 1.3 Hz, 1H), 7.80–7.69 (m, 3H),
7.44–7.33 (m, 4H), 7.20–7.17 (m, 2H), 7.04 (d, J ¼ 8.3 Hz, 1H), 6.95
(t, J ¼ 7.4 Hz, 1H), 4.31 (s, 2H), 3.82 (s, 3H), 3.62–3.47 (m, 6H), 3.20
3.83–3.45 (m, 6H), 3.24–3.17 (m, 2H); 13C NMR (150 MHz, DMSO-
d6) d: 164.87, 164.49, 159.83, 157.64, 156.01, 145.27, 140.97,
134.81, 133.93, 132.14, 132.00, 130.47, 129.53, 129.43, 128.34,
126.51, 125.88, 124.04, 123.92, 123.28, 119.39, 116.29, 47.37, 46.77,
45.69, 45.12, 36.88; IR (KBr, cmꢃ1): 3435, 3109, 2908, 1654, 1276,
(s, 2H); 13C NMR (150 MHz, DMSO-d6) d: 165.34, 164.49, 159.83, 839; ESI-MS: 513.2 [M ꢃ H]ꢃ.
157.89, 157.63, 156.01, 145.27, 136.91, 135.27, 133.93, 132.24,
132.19, 131.58, 129.53, 128.34, 126.51, 125.88, 123.80, 120.98,
(E)-4-{[3-[4–(3-(4-trifluoromethylphenyl) acryloyl) piperazine-1-
carbonyl]-4-fluorophenyl] methyl} -2H -phthalazin-1-one (5h)
118.20, 116.43, 112.05, 56.03, 47.34, 46.80, 45.58, 45.12, 36.88; IR
(KBr, cmꢃ1): 3455, 3014, 2911, 1660, 1633, 1269, 847; ESI-MS:
526.1 [M ꢃ H]ꢃ.
A light yellow solid, yield: 19.3%, mp: 213.6–214.0 ꢁC, 1H NMR
(600 MHz, DMSO-d6) d: 12.56 (s, 1H), 8.23 (d, J ¼ 7.8 Hz, 1H),
7.96–7.84 (m, 3H), 7.83–7.76 (m, 2H), 7.73 (d, J ¼ 8.3 Hz, 2H), 7.53
(d, J ¼ 15.4 Hz, 1H), 7.47–7.29 (m, 3H), 7.22 (t, J ¼ 9.0 Hz, 1H), 4.31
(s, 2H), 3.82–3.46 (m, 6H), 3.22–3.17 (m, 2H); 13C NMR (150 MHz,
DMSO-d6) d: 164.66, 164.52, 159.83, 157.43, 155.87, 145.27, 140.44,
139.56, 133.93, 131.99, 129.85, 129.53, 129.44, 129.10, 128.34,
126.51, 126.03, 125.88, 121.50, 116.39, 47.34, 46.79, 45.64, 45.18,
36.88; IR (KBr, cmꢃ1): 3453, 3069, 2919, 1658, 1614, 1280, 981; ESI-
MS: 563.2 [M ꢃ H]ꢃ.
(E)-4-{[3-[4-(3-(4-methylphenyl) acryloyl) piperazine-1-carbonyl]-
4-fluorophenyl] methyl} -2H-phthalazin-1-one (5d)
1
A white solid, yield: 15.8%, mp: 245.6–245.9 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.56 (s, 1H), 8.24 (d, J ¼ 7.8 Hz, 1H), 7.94 (d,
J ¼ 8.04 Hz, 1H), 7.90 (t, J ¼ 11.94 Hz, 1H), 7.87 (t, J ¼ 7.38 Hz, 1H),
7.59–7.56 (m, 2H), 7.48–7.36 (m, 3H), 7.23–7.19 (m, 4H), 4.31 (s,
2H), 3.76–3.47 (m, 6H), 3.21–3.18 (m, 2H), 2.30 (s, 3H); 12C NMR
(150 MHz, DMSO-d6) d: 165.18, 164.48, 159.82, 157.63, 156.01,
145.27, 142.33, 139.86, 135.28, 133.93, 132.75, 132.20, 131.99,
129.80, 129.44, 128.47, 128.34, 126.51, 125.88, 124.06, 117.27,
116.43, 47.37, 46.77, 45.59, 45.15, 36.88, 21.41; IR (KBr, cmꢃ1):
3433, 3111, 2906, 1649, 1606, 1277, 847; ESI-MS: 509.8 [M ꢃ H]ꢃ.
(E)-4-{[3-[4–(3-(3-fluorophenyl) acryloyl) piperazine-1-carbonyl]-
4-fluorophenyl] methyl} -2H -phthalazin-1-one (5i)
A white solid, yield: 16.5%, mp: 262.7–263.5 ꢁC, 1H NMR (600MHz,
DMSO-d6) d: 12.56 (s, 1H), 8.23 (d, J ¼ 7.8 Hz, 1H), 7.94 (d,
J ¼ 8.1 Hz, 1H), 7.89–7.84 (m, 1H), 7.82–7.74 (m, 1H), 7.66–7.58 (m,
1H), 7.53–7.31 (m, 5H), 7.23–7.17 (m, 3H), 4.31 (s, 2H), 3.77–3.47
(m, 6H), 3.22–3.17(m 2H); 13C NMR (150 MHz, DMSO-d6) d: 164.79,
164.46, 163.82, 162.16, 159.82, 145.27, 140.91, 138.13, 135.27,
133.94, 132.26, 132.00, 131.17, 129.53, 129.43, 128.34, 126.51,
125.89, 120.12, 116.80, 116.66, 114.58, 114.40, 47.38, 46.81, 45.62,
45.13, 36.88; IR (KBr, cmꢃ1): 3435, 3109, 2907, 1651, 1609, 1251,
974; ESI-MS: 513.2 [M ꢃ H]ꢃ.
(E)-4-{[3-[4-(3-(2-methoxyphenyl) acryloyl) piperazine-1-
carbonyl]-4-fluorophenyl] methyl}-2H-phthalazin-1-one (5e)
A white solid, yield: 15.2%, mp: 243.8–244.0 ꢁC, 1H NMR (600MHz,
DMSO-d6) d: 12.56 (s, 1H), 8.23 (d, J ¼ 7.7 Hz, 1H), 7.94 (d,
J ¼ 8.0 Hz, 1H), 7.89–7.84 (m, 1H), 7.83–7.62 (m, 3H), 7.43–7.33 (m,
3H), 7.23–7.18 (m, 2H), 7.03 (d, J ¼ 8.2 Hz, 1H), 6.95 (t, J ¼ 7.5 Hz,
1H), 4.30 (s, 2H), 3.82 (s, 3H), 3.73–3.47 (m, 6H), 3.23–3.18 (m, 2H);
13C NMR (150 MHz, DMSO-d6) d: 165.35, 164.49, 159.83, 157.89,
157.71, 156.00, 145.28, 136.88, 135.27, 133.93, 132.19, 131.98,
131.58, 129.53, 129.42, 128.34, 126.51, 125.87, 123.94, 123.80,
120.97, 118.20, 116.44, 112.05, 56.03, 47.32, 46.77, 45.59, 45.11,
36.88; IR (KBr, cmꢃ1): 3434, 3012, 2908, 1661, 1634, 1268, 980; ESI-
MS: 525.4 [M ꢃ H]ꢃ.
(E)-4-{[3-[4–(3-(4-chlorophenyl) acryloyl) piperazine-1-carbonyl]-
4-fluorophenyl] methyl} -2H -phthalazin-1-one (5j)
1
A white solid, yield: 17.2%, mp: 239.2–240.4 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.56 (s, 1H), 8.23 (d, J ¼ 7.3 Hz, 1H), 7.94 (d,
J ¼ 8.1 Hz, 1H), 7.89–7.84 (m, 1H), 7.80–7.70(m, 3H), 7.49–7.40 (m,
4H), 7.38–7.26 (m, 2H), 7.21 (t, J ¼ 9.0 Hz, 1H), 4.30 (s, 2H),
3.81–3.43 (m, 6H), 3.22–3.17 (m, 2H); 13C NMR (150 MHz, DMSO-d6)
d: 164.88, 164.50, 159.83, 157.63, 156.01, 145.27, 140.88, 135.26,
134.51, 134.47, 133.92, 132.27, 132.22, 131.98, 130.21, 129.53,
129.21, 128.34, 126.51, 125.87, 124.01, 119.33, 116.29, 47.31, 46.76,
45.63, 45.09, 36.89; IR (KBr, cmꢃ1): 3435, 3108, 2908, 1657, 1492,
1273, 842; ESI-MS: 529.2 [M ꢃ H]ꢃ.
(E)-4-{[3-[4–(3-(2,5-dimethoxyphenyl) acryloyl) piperazine-1-
carbonyl]-4-fluorophenyl] methyl} -2H-phthalazin-1-one (5f)
1
A white solid, yield: 23.9%, mp: 133.9–134.6 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.56 (s, 1H), 8.23 (d, J ¼ 7.9 Hz, 1H), 7.94 (d,
J ¼ 8.1 Hz, 1H), 7.86 (t, J ¼ 7.6 Hz, 1H), 7.82–7.71 (m, 2H), 7.43–7.27
(m, 3H), 7.24–7.10 (m, 2H), 6.97–6.91 (m, 2H), 4.30 (s, 2H), 3.76(s,
3H), 3.73(s, 3H) 3.62–3.47 (m, 6H), 3.23–3.19 (m, 2H); 13C NMR
(150 MHz, DMSO-d6) d: 165.28, 164.46, 159.81, 157.61, 153.63,
152.28, 145.26, 136.70, 135.25, 133.92, 132.23, 131.97, 129.51,
128.32, 126.50, 125.87, 124.41, 123.94, 118.41, 116.97, 116.41,
113.28, 112.97, 56.50, 54.03, 47.38, 46.74, 45.57, 45.09, 36.86; IR
(KBr, cmꢃ1): 3432, 3109, 2909, 1641, 1565, 1224, 844; ESI-MS:
556.6 [M ꢃ H]ꢃ.
(E)-4-{[3-[4–(3-(4-bromophenyl) acryloyl) piperazine-1-carbonyl]-
4-fluorophenyl] methyl} -2H -phthalazin-1-one (5k)
1
A white solid, yield: 18.2%, mp: 225.7–226.0 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.56 (s, 1H), 8.23 (d, J ¼ 7.74 Hz, 1H), 7.94 (d,
J ¼ 8.0 Hz, 1H), 7.88–7.84 (m, 1H), 7.80 (t, J ¼ 7.3 Hz, 1H), 7.67–7.62
(m, 2H), 7.57 (d, J ¼ 8.46 Hz, 2H), 7.46–7.18 (m, 5H), 4.30 (s, 2H),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
155
3.75–3.47 (m, 6H), 3.19–3.13 (m, 2H); 13C NMR (150 MHz, DMSO-d6) 129.44, 129.42, 128.33, 126.51, 125.89, 124.04, 116.44, 116.30,
d: 164.87, 164.50, 159.83, 157.64, 156.01, 145.27, 140.97, 135.27, 114.65, 112.98, 47.02, 46.59, 45.55, 45.18, 36.88; IR (KBr, cmꢃ1):
134.81, 133.93, 132.27, 132.22, 132.14, 131.99, 130.47, 129.53, 3431, 3102, 2908, 2865, 1645, 1275, 844; ESI-MS: 485.5 [M ꢃ H]þ.
129.41, 128.34, 126.51, 125.87, 123.92, 123.28, 119.39, 116.29,
47.41, 46.53, 45.69, 45.12, 36.89; IR (KBr, cmꢃ1): 3461, 3064, 2915,
1657, 1612, 1359, 1286, 1224; ESI-MS: 573.1 [M ꢃ H]ꢃ.
Biological activity
PARP inhibition assay
PARP enzyme inhibition was measured using an HT
F
(E)-4-{[3-[4–(3-(3-nitrophenyl) acryloyl) piperazine-1-carbonyl]-4-
Homogeneous 96-well PARP Inhibition Assay Kit (Trevigen, Cat#
4690–096-K, Gaithersburg, USA), according to the manufacturer’s
protocol. The various synthesized compounds were dissolved in
DMSO and then serially diluted to the required concentrations
with distilled water, keeping the final concentration of DMSO
lower than 1%. Olaparib was used as positive control.
Fluorescence values under the condition of excitation wavelength
(544 nm) and emission wavelength (590 nm) were measured using
a multiwell spectrophotometer (Molecular Devices SpectraMax M5
microplate reader, Careforde, Chicago, USA). Then, the standard
curve was drawn and the inhibition rate of each test compound
was calculated. IC50 value of each compound was calculated using
GraphPad Prism 6 software according to the above results.
fluorophenyl] methyl} -2H -phthalazin-1-one (5l)
1
A white solid, yield: 16.9%, mp: 266.8–268.2 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.56 (s, 1H), 8.58 (d, J ¼ 30.2 Hz, 1H), 8.23 (d,
J ¼ 7.8 Hz, 1H), 8.19–8.08 (m, 2H), 7.94 (d, J ¼ 8.1 Hz, 1H), 7.86 (td,
J ¼ 7.7, 1.3 Hz, 1H), 7.81–7.80 (m, 1H), 7.67 (t, J ¼ 8.0 Hz, 1H), 7.59
(d, J ¼ 15.4 Hz, 1H), 7.50–7.53 (m, 1H), 7.43–7.35 (m, 3H), 7.22 (t,
J ¼ 9.0 Hz, 1H), 4.31 (s, 2H), 3.80–3.49 (m, 6H), 3.21 (s, 2H); 13C
NMR (150 MHz, DMSO-d6) d: 164.61, 159.82, 157.72, 156.47, 148.80,
145.26, 139.92, 137.41, 135.29, 135.00, 133.92, 132.22, 131.98,
130.66, 129.52, 129.40, 128.34, 126.51, 125.87, 124.29, 122.55,
121.61, 47.74, 46.76, 45.67, 45.10, 36.89; IR (KBr, cmꢃ1): 3436, 3109,
2907, 1645, 1596, 1277, 841; ESI-MS: 540.9 [M ꢃ H]ꢃ.
(E)-4-{[3-[4–(3-(4-nitrophenyl) acryloyl) piperazine-1-carbonyl]-4-
fluorophenyl] methyl} -2H -phthalazin-1-one (5m)
MTT assay for cell growth inhibition
The anti-proliferative activities of synthesized compounds were
assessed against breast cancer cell line MDA-MB-436 by the 3-(4,
5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The MDA-MB-436 cell was purchased from Chinese
Academy of Sciences. Briefly, cells were seeded in 96-well plates
at a density of 4 ꢂ 104 cells per mL in Hyclone RPMI medium sup-
plemented with 10% foetal bovine serum and then treated with
different concentrations of synthesized compounds in DMSO.
Olaparib was used as the positive control. After treated with com-
pounds for 72 h, cells were incubated with 20 lL of 5 mg/mL MTT
for 4 h. The culture medium was removed, and 150 lL DMSO was
added to dissolve the formazan crystals. Wells containing only
media were used for background correction. The optical density
was measured spectrophotometrically at 490 nm. EC50 values were
calculated using GraphPad Prism 6 software. Data were expressed
as mean SEM.
1
A white solid, yield: 17.3%, mp: 224.0–224.5 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.59 (s, 1H), 8.24 (t, J ¼ 8.3 Hz, 3H), 8.01–7.95 (m,
3H), 7.89–7.82 (m, 2H), 7.58 (d, J ¼ 15.4 Hz, 1H), 7.45–7.37 (m, 3H),
7.23 (t, J ¼ 8.94 Hz, 1H), 4.32 (s, 2H), 3.81–3.51 (m, 6H), 3.27–3.18
(m, 2H); 13C NMR (150 MHz, DMSO-d6) d: 164.45, 159.83, 157.59,
156.02, 148.01,145.28, 142.10, 139.74, 135.29, 133.94, 132.26,
132.01, 129.52, 129.44, 128.33, 126.52, 125.89, 124.32, 123.99,
123.07, 116.47, 116.33, 47.33, 46.73, 45.68, 45.20, 36.88; IR (KBr,
cmꢃ1): 3436, 3072, 2926, 1766, 1648, 1274, 842; ESI-MS:
539.7 [M ꢃ H]ꢃ.
(E)-4-{[3-[4–(3-(3,4,5-trimethoxyphenyl) acryloyl) piperazine-1-
carbonyl]-4-fluorophenyl] methyl} -2H -phthalazin-1-one (5n)
1
A white solid, yield: 26.8%, mp: 235.3–236.2 ꢁC, H NMR (600 MHz,
DMSO-d6) d: 12.56 (s, 1H), 8.23 (d, J ¼ 7.1 Hz, 1H), 7.94 (d,
J ¼ 8.1 Hz, 1H), 7.90–7.84 (m, 1H), 7.80 (d, J ¼ 7.7 Hz, 1H), 7.47–7.34
(m, 3H), 7.22 (t, J ¼ 9.0 Hz, 2H), 7.03–7.00 (m, 2H), 4.31 (s, 2H), 3.79
(s, 7H), 3.71–3.46 (m, 9H), 3.22–3.16 (m, 2H); 13C NMR (150 MHz,
DMSO-d6) d: 165.13, 164.46, 159.81, 157.62, 155.99, 153.46, 145.26,
142.82, 139.31, 135.27, 133.93, 132.25, 132.19, 131.98, 131.04,
129.51, 128.32, 126.50, 125.87, 124.03, 123.92, 117.37, 116.41,
106.13, 60.52, 56.50, 54.04, 47.32, 46.80, 45.57, 44.90, 36.86; IR
(KBr, cmꢃ1): 3435, 3109, 2908, 1642, 1270, 843; ESI-MS:
586.2 [M ꢃ H]ꢃ.
Anti-cholinesterase activity assays
The acetylcholinesterase (eeAChE, from the electric eel), butyryl-
cholinesterase (eqBChE, from equine serum), 5, 5’-dithiobis-2-nitro-
benzoic acid (Ellman’s reagent, DTNB), S-butyrylthiocholine iodide
(BTCI), acetylthiocholine iodide (ATCI), and Donepezil hydrochlor-
ide and Neoeserine methyl sulfate were purchased from
Sigma-Aldrich.
The capacity of the test compounds 5a–o to inhibit the AChE
and BChE activities were assessed by the Ellman’s method in 96-
well plates with slight modification31. The Donepezil and
Neoeserine were used as reference drugs. The test compounds
were dissolved in a minimum volume of DMSO (1%) and were
diluted using sodium phosphate buffer (0.1 M, pH 7.4). A reaction
mixture containing 20 lL sodium phosphate buffer, 100lL DTMB
(1 mM) and 40 lL AChE (0.2 U/Ml)/BChE (0.3 U/mL) was incubated
with 20 lL of various concentrations of test compounds at 37ꢁC for
15min followed by the addition of the substrate (20lL) acetylthio-
(E)-4-{[3-[4-(3-(furan-2-yl) acryloyl) piperazine-1-carbonyl]-4-
fluorophenyl] methyl} -2H -phthalazin-1-one (5o)
A light yellow solid, yield: 17.8%, mp: 152.5–155.0 ꢁC, 1H NMR
(600 MHz, DMSO-d6) d: 12.56 (s, 1H), 8.23 (d, J ¼ 7.8 Hz, 1H), 7.93
(d, J ¼ 8.0 Hz, 1H), 7.86 (t, J ¼ 7.1 Hz, 1H), 7.80 (td, J ¼ 7.5, 0.78 Hz,
1H), 7.60 (d, J ¼ 1.3 Hz, 1H), 7.43–7.31 (m, 3H), 7.21 (t, J ¼ 9.0 Hz,
1H), 6.92–6.83 (m, 2H), 6.57 (dd, J ¼ 3.2, 1.8 Hz, 1H), 4.30 (s, 2H),
3.67–3.47 (m, 6H), 3.19–3.10 (m, 2H); 13C NMR (150 MHz, DMSO- choline iodide (1 mM) or S-butyrylthiocholine iodide (1 mM) and
d6) d: 164.69, 164.48, 159.83, 157.63, 156.01, 151.57, 145.39, incubation for another 3 min. The activities were determined by
145.28, 135.25, 133.94, 132.25, 132.20, 131.99, 129.57, 129.53, using a Varioskan Flash Multimode Reader (Thermo, Miami, FL, USA)

156
C.-Z. GAO ET AL.
at 412nm. The inhibition per cent was calculated by the following water molecules, non-bonded inhibitors and cofactors.
expression: (1-Ai/Ac) ꢂ 100, where Ai and Ac were the absorbance
Subsequently, both proteins were edited using the ADT program
obtained for AChE/BChE in the presence and absence of inhibitors,
respectively. The concentration of compound producing 50% of
enzyme activity inhibition (IC50) was calculated by nonlinear regres-
sion analysis of the response–concentration (log) curve, using the
Graph Pad Prism program package (Graph Pad Software; San Diego,
CA). Results are expressed as the mean SEM of at least three differ-
ent experiments performed in triplicate.
to add the polar hydrogen atoms and Kollman charges as means
to rigidify protein structures and also saved in the same pdbqt
format as the ligands 5c, 5i, 5l. The torsions for ligands and recep-
tors were set to be rotatable and rigid respectively. The
AutoGridFR software was applied to determine the binding pocket
and the grid coordinate35. After that, docking was performed
using the default settings of Vina and only the minimum pre-
dicted Gibbs binding energy pose was considered for each case.
Analysis and visualization of the docking results were done using
Molecular docking
the PyMOL version 1.78 for 3D diagrams (The PyMOL Molecular
þ
Molecular docking studies were performed using the AutoDock
Vina 1.1.232 along with the AutoDockTools 1.5.6 (ADT) and
AutoGridFR 1.0. The structures of compounds 5c, 5i, 5l were
drawn and then subjected to energy minimization with the
molecular mechanics (MM2) using the ChemBioOffice 13.0
(CambridgeSoft, PerkinElmer Inc., MA, USA). The ligands were fur-
ther processed and saved in the pdbqt format for docking by ADT
program. The X-ray crystallographic structures of PARP-1 (PDB
Code 5DS3)33 and hBChE (PDB Code 5NN0)34 were obtained from
the online PDB database (RCSB) (http://www.rcsb.org). Both recep-
tors were firstly prepared using the Acceryls Discovery Studio
€
Graphics System, Version 1.6. Schrodinger, LLC.) and the LigPlot
version 1.4536 for 2D ones.
Results and discussion
Chemistry
The synthetic route for the desired compounds 5a–o is described
in Scheme 1. 2-Formyl benzoic acid 6 was used as starting mater-
ial for the preparation of the key intermediate 9, which
firstly reacted with dimethyl phosphate to afford the dimethyl
2017 R2 (Accelrys Inc., San Diego, CA, USA) involving removal of (1, 3-dihydro-3-oxoisobenzofuran-1-yl)-1-phosphonate 7, and then,
O
O
O
CHO
NH
N
a
c
b
O
O
CN
F
O
F
COOH
OMe
OMe
P
O
OH
6
7
8
9
O
COCl
COOH
e
CHO
N
R
d
R
R
f
R
N
Boc
12a-n
10a-n
11a-n
O
O
N
O
d-g
O
g
R
N
NH
CHO
NH
13a-n
13o
O
O
NH
N
NH
N
O
F
O
F
h
9
13a-o
+
N
O
N
R
N
N
O
O
5o
5a-5n
5a R=H
5e R=2-OCH3
5i R=3-F
5m R=4-NO2
5b R=3-OCH3, 4-OCH3
5f R=2-OCH3, 5-OCH3
5j R=4-Cl
5c R=4-OCH3
5g R=4-F
5k R=4-Br
5d R=4-CH3
5h R=4-CF3
5l R=3-NO2
5n R=3-OCH3, 4-OCH3,
5-OCH3
Scheme 1. Synthesis route for target compounds. Reactions and conditions: (a) dimethyl phosphite, 100 ꢁC, 8 h, 62.3%; (b) 2-fluoro-5-formylbenzonitrile, Et3N, anhyd-
rous THF, 20 ꢁC, 16 h, 92.6%; (c) (i) H2O, 10mol/l NaOH, 90ꢁC, 1 h, (ii) NH2NH2ꢄH2O, 70 ꢁC, 18h, 4 mol/l HCl, 93.7%; (d) propane diacid, NH4Ac, 85 ꢁC, 5 h, 50.0–80.0%;
(e) oxalyl chloride, DMF, 20 ꢁC, 1.5h; (f) Tert-butoxycarbonylpiperazine, Et3N, DCM, 20 ꢁC, 12 h, 40.0–70.0%; (g) TFA, DCM, 20 ꢁC, 8 h, 50.0–80.0%; (h) HBTU/DIPEA, DCM,
20 ꢁC, 18h, 15.7–26.8%;

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
157
Table 1. PARP-1 inhibitory activity of compounds 5a–5o.
Compd.
Inhibition rate (%)a
IC50 (nM)b
Compd.
Inhibition rate (%)a
IC50 (nM)b
5a
5b
5c
5d
5e
5f
75.21 4.84%
77.23 3.89%
89.23 10.64%
64.21 4.59%
62.56 3.65%
75.62 3.61%
85.63 3.69%
89.52 12.58%
NDc
ND
143.78 5.26
5i
5j
5k
5l
5m
5n
5o
Olaparib
88.23 5.24%
75.45 4.54%
43.21 6.54%
95.72 4.35%
93.15 5.28%
65.96 4.82%
65.23 5.98%
98.20 3.59%
267.22 9.32
ND
ND
ND
ND
ND
16.10 1.25
37.90 1.89
ND
ND
8.20 1.06
5g
5h
396.71 7.01
294.70 8.25
aData were expressed as mean SD, measured at the conc. of 0.5 lM, n¼ 3.
bData were expressed as mean SD, n¼ 3(p<.05);
cND: not determined.
effects, among which 5l exhibited the most potent inhibitory
activity. On the cellular level, incorporation groups containing
fluorine (5g, 5h, 5i) helped to increase potency of target com-
pounds perhaps improving bioavailability.
Table 2. MDA-MB-436 cell inhibitory activities of compounds 5a–5o.
Compd.
EC50 (lM)a,b
Compd.
EC50 (lM)a,b
5a
5b
5c
5d
5e
5f
38.21 2.15
41.23 1.95
14.20 3.16
35.15 3.42
44.23 1.95
30.18 3.25
13.02 2.14
14.83 1.42
5i
5j
5k
5l
5m
5n
5o
Olaparib
14.17 5.21
45.19 1.68
112.01 5.24
11.62 2.15
13.95 1.28
31.25 4.12
32.23 3.58
8.63 1.25
In order to explore the potential of being MTDLs for AD, the
in vitro inhibitory effects of all target compounds on Electrophorus
electricus AChE (EeAChE) and equine serum butyrylcholinesterase
(eqBChE) were investigated according to the method31. Two drugs,
Donepezil and Neostigmine, were used as the standards. The
results are summarized in Table 3. The IC50 values suggested that
most of the synthesized compounds exhibited little-to-moderate
inhibitory activities against cholinesterases. Furthermore, a clear
trend appeared that with exception of compound 5c all the other
compounds showed better inhibition of BChE than AChE. Although
the inhibitory potency against AChE of compound 5m was weaker
than Donepezil and Neostigmine, its inhibitory potency against
BChE (5.93 lM) is more potent than the two drugs (7.64 and
12.01 lM, respectively). Comparison of 3, 4-dimethanoxy derivative
5b with 3, 4, 5-trimethanoxy one 5n, 2-methanoxy compound 5e
with 2, 5-dimethanoxy one 5f, demonstrated the relatively compact
group decreased its inhibitory activities against cholinesterase.
Halogen-substituted compounds 5g–k having F, CF3, Cl, Br on the
benzene ring appear less active than the unsubstituted compound
5a. Both compounds 5l and 5m with a strong electron-withdrawing
nitro group showed the most potent activities among these
derivatives.
5g
5h
aData are expressed as mean SD, n¼ 3. Incubated for 72 h (p<.05).
bThe EC50 value was the concentration required to reduce cell proliferation by
50% in single-agent cytotoxicity assay.
the Wittig reaction of 7 with 2-fluoro-5-formylbenzonitrile was car-
ried out to give compound 8. The benzoic acid derivative 9 was
finally obtained after hydrolysis of 8. Condensations of propane-
dioic acid with various substituted benzaldehydes or furan-2-car-
baldehyde followed by decarboxylation reactions furnished
compounds 10a–o. Treatment of 10a–o with oxalyl chloride fol-
lowed by 1-boc-piperazine gave amides 12a–o, which were subse-
quently deprotected to provide compounds 13a–o. Finally,
intermediates 9 reacted with 13a–o to afford the target com-
pounds 5a–o.
The structures of newly synthesized target compounds 5a–o
were confirmed by various spectral techniques. The IR spectra of
5a–o exhibited absorption bands at around 3400 cmꢃ1 which was
1
assigned to NH group stretching vibration. In the H NMR spectra
The two important cholinesterases, both AChE and BChE, can
hydrolyse acetylthiocholine (ACh) in the synaptic gap of neur-
onal cells, which results in the loss of neurotransmitter and the
decline of cognition during AD. The role of BChE for ACh
hydrolysis is only of minor impact in healthy brain, but in
advanced AD the level of AChE drops dramatically down to 90%
and the levels of BChE in certain parts of the brain increase sig-
nificantly to compensate the loss of neuronal AChE37. Besides
of 5a–o the N–H appeared at about 12.56 ppm. In the 1H NMR
spectra of 12a–o, the vinylic protons appeared as doublet at
7.40–7.90. The large coupling constant (J ¼ 15.4 Hz) of the vinylic
protons confirmed the trans configuration.
Biology
The in vitro PARP-1 enzyme inhibitory activities of all the target that, it has been shown that BChE (over-)expression is associated
compounds were firstly screened at the fixed concentration of with senile plaques and the transformation of non-fibrillar to
0.5 lM and then, the ones with inhibitory rates ꢅ80% were
fibrillary Ab plaques. Hence, inhibition of BChE may be more
selected to further determine their IC50 value. The results are sum-
therapeutically valuable than inhibition of AChE in later stages of
marized in Table 1. It showed that all compounds possessed AD and researches on selective BChE inhibitors are paid wide
PARP-1 inhibitory activities, but none of them were more potent attention to in recent years38,39. In this study, the synthesized
than Olaparib. 5l and 5m exhibited more potent inhibitory effects
on PARP-1 than the other compounds, which might attribute to
the nitro group on the phenyl ring. Introduction of steric groups
compounds to some degree showed inhibitory selectivity over
BChE, although their potencies were not strong enough as nano-
molar level, which at least gave a clue to search for new skel-
into the phenyl ring of aromatic propylene moiety, e.g. the dime- eton frame responsible for multifunctional PARP-1 and
thoxy in 5b, 5f or the trimethoxy group in 5n, decreased the
inhibitory potency; nevertheless, the less steric group in 5o with a
cholinesterase inhibition.
In biological tests, compound 5l showed more potent inhibi-
furyl group also resulted in decreased activity. The cellar inhibitory tory activity than 5m against PARP-1, while 5m exhibited a little
potencies of all target compounds were tested against breast can- more potent than 5l against AChE and BChE; meanwhile, these
cer cell line MDA-MB-436 by MTT assay and the results are shown synthesized compounds show inhibitory selectivity over BChE, and
in Table 2. It indicated that 5c, 5g, 5h, 5i, 5l and 5m had similar taking all together, 5l was chosen to carry on molecular docking

158
C.-Z. GAO ET AL.
Table 3. AChE and BChE inhibitory activities of compounds 5a–5o.
IC50 (lM)a
IC50 (lM)a
Compd.
AChEb
BChEb
Compd.
AChE
BChE
Donepezil
Olaparib
5a
5b
5c
5d
5e
5f
5g
0.03 0.004
50.95 0.46
52.91 3.02
45.19 1.38
55.82 0.60
71.99 3.84
56.94 4.30
76.86 4.13
173.01 29.98
7.64 0.21
34.36 0.64
22.40 1.19
17.75 0.72
178.84 5.89
27.81 0.87
26.40 1.93
35.72 2.98
104.63 12.50
Neostigmine
5h
5i
5j
5k
5l
5m
5n
5o
0.04 0.01
136.43 12.51
110.35 9.18
86.54 5.12
80.69 4.87
24.55 1.10
12.24 0.49
99.09 2.21
113.71 14.95
12.01 0.45
56.76 04.36
33.39 4.14
37.08 2.37
45.75 3.76
9.16 0.91
5.93 0.19
65.22 1.08
77.57 9.06
aData are expressed as mean SD, n¼ 3 (p < .05).
bAChE from electric eel and BChE from equine serum were used.
with PARP-1 and BChE proteins to obtain an insight into the A-NH group, the –NH group as H-bond donor with Gly863
molecular level of the ligand binding. Additionally, in order to fur- A – C¼O group. However, within the other moieties of 5c and
ther investigate the interaction fashion of these new compounds 5i just one position formed hydrogen bond with PARP-1 com-
with PARP-1, molecular docking of 5c and 5i with PARP-
1was performed.
pared to two positions within those of 5l and Olaparib. The dif-
ferences were maybe the reason why 5c and 5l exhibited
weaker potency than 5l. On the basis of the docked conform-
ation, the protein contact potential was generated for the com-
pound 5l (Figure 3(b)) depicting the whole surface of the
protein. Red and blue colours showed negative and positive
charges, respectively. In this depiction, it was shown that com-
pound 5l was bound inside a cavity formed at the interface of
the enzyme.
Molecular modelling studies
Docking simulations were performed for 5c, 5i and 5l into the
PARP-1 (PDB ID: 5DS3) and 5l into the hBChE (PDB ID: 5NNO)
active sites using the AutoDock Vina 1.1.2. The molecular docking
protocols were validated through re-docking of the co-crystallized
ligands in the vicinity of the PARP-1 and hBChE active site. It
reproduced all the key interactions accomplished by the co-crys-
tallized ligand with the key amino acids in the active site as
shown in the crystal, indicating the suitability of the used setup
for the docking study.
Regarding PARP-1, molecular docking of 5l in PARP-1
revealed an optimal binding mode characterized by affinity of
the minimum Gibbs binding energy with –11.9 kcal/mol. As illus-
trated in the 2D diagrams plotted by LigPlotþ (Figure 2), dock-
ing simulation of 5l showed that it fitted into the enzyme
active site almost at the same position of co-crystallized ligand
Olaparib. Three hydrogen bonds in 5l-PARP-1 complex were
formed in the phthalazinone ring which was well recognized as
The crystal structure of the recombinant human BChE (PDB
ID: 5NNO) complexed with ligand 92H has been reported
recently34. The compound 92H, N-((1–(2, 3-dihydro-1H-inden-2-
yl)
piperidin-3-yl)methyl)-N-(2-(dimethyl-amino)ethyl)-2-naphtha-
mide, shows high selectivity and an extremely potent inhibitory
effect on huBChE with picomolar affinity (IC50¼1.03 0.04 nM);
therefore, 5NN0 was used for docking to analyse the key
binding interactions of the synthesized compound 5l for
huBChE inhibition.
The molecular docking of 5l into huBChE revealed an optimal
binding mode characterized by the affinity of the minimum
Gibbs binding energy with –13.5 kcal/mol. As illustrated in the
2D diagrams plotted by LigPlotþ (Figure 4), docking simulation
of 5l showed that it interacted with the enzyme active site at
local positions same as the reference 92H did. The naphthalene
ring of 92H and the phthalazin-1-one moiety of 5l all formed
hydrophobic interactions with residues Leu286, Gly116, Gly117
and Trp231. The –(CH2)2NMe2 side chain of 92H formed hydro-
phobic interactions with residues Trp82 and Glu197 as well as
the fluorophenyl group of 5l did. In particular, the Trp231 and
Trp82 were two important residues located, respectively, in the
acyl-binding pocket and choline-binding pocket of huBChE with
which strong interactions could result in an excellently inhibitory
effect on huBChE. In spite of this, there were some differences
between 5l and 92H about the interactions with residues of
huBChE, which perhaps led to a comparatively weaker activity of
5l. Two hydrogen bonds, the carbonyl group as H-bond acceptor
with the His438 A-NH group (2.84 Å) and the nitro group as H-
bond acceptor with the Tyr128 –OH group (3.22 Å), were estab-
lished between 5l and huBChE, which may be weaken the
hydrophobic interaction of 5l with the key residues Trp82 and
Trp231. On the other hand, the 2, 3-dihydro-1H-inden moiety of
92H formed hydrophobic interactions with residues Ile69 and
a
scaffold mainly responsible for PARP-1 inhibitory activity
(Figures 2(b) and 3(a)): the carbonyl group as H-bond acceptor
with the Gly863 A-NH group (3.04 Å) and Ser904 A-OH group
(2.92 Å), the –NH group as H-bond donor with Gly863 A-C¼O
group (2.88 Å). The fourth identical hydrogen bond was estab-
lished between the C¼O group from acryloyl moiety and
Arg878 A-NH group (3.04 Å). The newly introduced nitro group
formed a hydrogen bond with Gly871 A-NH group (Figures 2(b)
and 3(a)). However, another C¼O group connected to the
piperazine moiety did not form a hydrogen bond with the
Tyr896 A-NH group as compared with Olaparib, which perhaps
was one of the reasons why 5l exhibited a less potent effect
on PARP-1 inhibition than Olaparib as shown in vitro experi-
ments. Besides the interaction of hydrogen bond, compound 5l
formed hydrophobic interactions with residues Tyr907, Lys903,
Phe897, Tyr896, His862, Ile895, Gly894, Leu877 and Asn868. The
minimum Gibbs binding energies of molecular docking of 5c
and 5i into PARP-1 were –11.9 and –11.8 kcal/mol respectively.
As shown in Figure 2, the phthalazinone rings of both com-
pounds 5c (Figure 2(c)) and 5d (Figure 2(d)) formed corre-
sponding two and three hydrogen bonds with PARP-1which Asp70 while 5l did not. As shown in the vacuum electrostatics
were similar with those of 5l and Olaparib: the carbonyl group depiction of huBChE bound to 5l (Figure 5(a)) and 92H (Figure
as H-bond acceptor with the Ser904 A-OH group or Gly863 5(b)), although both 5l and 92H were bound inside a cavity

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
159
Figure 2. Ligplot images showing the interactions of the Olaparib (a), 5l (b), 5c (c) and 5i (d) with the enzyme PARP-1. The commonly interacting amino acid residues
in both interactions were encircled in red circles.

160
C.-Z. GAO ET AL.
Figure 3. (a) 3D representation of different interactions of compound 5l with residues in the binding sites of PARP-1. The compound was rendered in green stick
model and the residues were rendered in purple sticks. Hydrogen bonds were indicated with yellow dashed lines. (b) A vacuum electrostatics depiction of PARP-1
bound to derivative 5l, showing protein contact potential. Surface coloring was according to the electrostatic potential: red, white, and blue correspond to negative,
neutral, and positive potential, respectively. The vacuum electrostatics/protein contact potential was generated by PyMOL. The 5l was depicted by sticks.
Figure 4. Ligplot image showing the interactions of the 92H (a) and 5l (b) with the enzyme huBChE. Arcs with red lines represented amino acid hydrophobic contacts;
green dashed lines represents hydrogen bonds. The commonly interacting amino acid residues in both interactions were encircled in red circles.
formed at the interface of the enzyme, it was obvious for 5l to
Generally, the results of respective docking of 5c, 5i and 5l in
be closely packed. Therefore, it was inferred that the compound complexly PARP-1 and 5l with huBChE indicated that 5l could
with small and flexible groups except for the phthalazin-1-one interact well with the active sites of PARP-1 protein, but it did not
moiety contributed to enhance its inhibitory activity against do well with that of huBChE, which provided a basis and valuable
huBChE in the pursuit of the PARP-1 and AChE dual-tar- information for further studies aimed at identifying inhibitors of
geted inhibitor.
both PARP-1 and huBChE.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
161
Figure 5. A vacuum electrostatics depiction of huBChE bound to derivative 5l (a) and reference 92H (b), showing protein contact potential. Surface colouring was
according to the electrostatic potential: red, white, and blue correspond to negative, neutral, and positive potential, respectively. The vacuum electrostatics/protein con-
tact potential was generated by PyMOL. The 5l and 92H are depicted by sticks.
of tumors with defective homologous recombination. J Med
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(ADP-ribose)polymerase-1 (PARP-1) inhibitors: opportunities
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Conclusion
Considering that inhibition of PARP-1 has therapeutical values not
only for cancer but also for AD, a series of 15 novel Olaparib ana-
logues was synthesized, and their inhibitory activities against the
enzymes PARP-1, AChE (from electric eel), BChE (from equine
serum) and the cancer cell line MDA-MB-436 were tested. Among
these synthesized compounds, 5l exhibited the most potent
inhibitory effects on PARP-1 (16.10 1.25 nM) enzyme and MDA-
MB-436 (11.62 2.15 lM) cancer cell, which were just a little
weaker than Olaparib. Meanwhile, 5l displayed moderate BChE
inhibitory activity (9.16 0.91 lM) which was stronger than
Neostigmine (12.01 0.45 lM) and selectivity for BChE over AChE
to some degree. Molecular docking researches implied that the
formation of hydrogen bond was necessary for PARP-1 inhibitory
potency, but it was not important for BChE inhibitory activity.
While keeping phthalazin-1-one unchanged, small and flexible
non-polar groups seemed to be conducive to improving inhibitory
potency on BChE. Our research gave a clue to search for new
agents based on AChE and PARP-1 dual-inhibited activities to treat
Alzheimer’s disease.
ꢀ
9. Salech FPDP, SanMartın CD, Rogers NK, et al. PARP-1 and
p53 regulate the increased susceptibility to oxidative death
of lymphocytes from MCI and AD patients. Front Aging
Neurosci 2017;9:310.
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senger of death. Front Biosci (Landmark Ed) 2009;14:
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generation: radiosynthesis and radioligand binding in ARC-
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reduce oxidative stress mediated neurodegeneration?
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13. Ruan Q, Ruan J, Zhang W, et al. Targeting
NAD þ degradation: the therapeutic potential of flavonoids
for Alzheimer’s diseaseand cognitive frailty. Pharmacol Res
2018;128:345–58.
Disclosure statement
No potential conflict of interest was reported by the authors.
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