Design, synthesis and bioactivity of novel phthalimide derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.07.052

A series of novel phthalimide derivatives related to benzylpiperazine were synthesized and evaluated as cholinesterase inhibitors. The results showed that all compounds were able to inhibit acetylcholinesterase (AChE), with two of them dramatically inhibiting butyrylcholinesterase (BuChE). Most compounds exhibited potent anti-AChE activity in the range of nM concentrations. In particular, compounds 7aIII and 10a showed the most potent activity with the IC₅₀ values of 18.44 nM and 13.58 nM, respectively. To understand the excellent activity of these compounds, the structure-activity relationship was further examined. The protein-ligand docking study demonstrated that the target compounds have special binding modes and these results are in agreement with the kinetic study.

Full text of DOI:10.1016/j.bmcl.2015.07.052

Accepted Manuscript
Design, synthesis and bioactivity of novel phthalimide derivatives as acetyl-
cholinesterase inhibitors
Weijie Si, Tao Zhang, Lanxiang Zhang, Xiangdong Mei, Mengya Dong, Kaixin
Zhang, Jun Ning
PII:
S0960-894X(15)00756-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.07.052
BMCL 22941
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 April 2015
9 July 2015
18 July 2015
Please cite this article as: Si, W., Zhang, T., Zhang, L., Mei, X., Dong, M., Zhang, K., Ning, J., Design, synthesis
and bioactivity of novel phthalimide derivatives as acetylcholinesterase inhibitors, Bioorganic & Medicinal
Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.07.052
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Bioorganic & Medicinal Chemistry Letters
Design, synthesis and bioactivity of novel phthalimide derivatives as
acetylcholinesterase inhibitors

Weijie Si, Tao Zhang, Lanxiang Zhang, Xiangdong Mei , Mengya Dong, Kaixin Zhang, Jun Ning
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P
.
R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of novel phthalimide derivatives related to benzylpiperazine were synthesized and
evaluated as cholinesterase inhibitors. The results showed that all compounds were able to
inhibit acetylcholinesterase (AChE), with two of them dramatically inhibiting
butyrylcholinesterase (BuChE). Most compounds exhibited potent anti-AChE activity in the
range of nM concentrations. In particcular, compound 7aIII and 10a showed the most potent
activity with the IC50 values of 18.44 nM and 13.58 nM respectively. To understand the
excellent activity of these compounds, the structure-activity relationship was further examined.
The protein-ligand docking study demonstrated that the target compounds have special binding
modes and these results are in agreement with the kinetic study.
Keywords:
substructure combination
phthalimide derivatives
acetylcholinesterase inhibitor
structure-activity relationship
molecular docking
Elsevier Ltd. All rights reserved.

Corresponding author. Tel.: +86-010-6289-9142; e-mail: xdmei@ippcaas.cn

Alzheimer’s disease (AD) is a neurodegenerative disease with
Figure 1. Our previous reported AchE inhibitors.
main symptoms of memory loss, cognition defect and behavioral
1
impairment . There are 18 million people around the world with
AD each year and this population is expected to increase to more
than 34 million in ten years. This disease severely influences the
2
patients’ family life and social activities . AD is mainly
characterized by the pre-synaptic decrease of acetylcholine
(
ACh) due to damage of cholinergic neurons in some especial
3
parts of the brain . The Cholinergic Hypothesis was put up in
1
982, which considered that one of the rational and effective
approaches to retard the AD’s symptoms is increasing the ACh
Figure 2. Structures of reported AChE inhibitors.
through inhibiting acetylcholinesterase (AChE) in pre-synaptic
4
areas . Moreover, some evidences suggest that inhibition of brain
BuChE may represent an important therapeutic target for AD. It
has also been recently reported that dual inhibition of AChE and
BuChE might alleviate AD symptoms owing to the key role of
5
BuChE in hydrolysis of ACh . As reported in some earlier
studies , it is essential to ensure that the drug inhibits both AChE
Figure 3. Design strategy of phthalimide derivatives.
6
and BuChE.
The three-dimensional structure of AChE, as determined by
X-ray crystallography, is composed of two active centers which
are connected by aromatic gorge. It has also been revealed that its
The AChE inhibitory activity was determined by using
electric eel acetylcholinesterase (AChE, E.C. 3.1.1.7) and the
BuChE inhibitory activity was tested by using equine serum
butylcholinesterase (BuChE, E.C. 3.1.1.8). The inhibitory
activities of cholinesterase were measured by the method
active site can be reached only through a deep and narrow
7
‘
catalytic gorge’ . In addition, it was proved that multiple binding
site AChE inhibitors were more potent inhibitors compared to the
2
1
22
compounds that interact with only one site of the enzyme in
described by Ellman et al. ，and Ali Asadipour .
8
organism . According to the structure characteristics of
The IC50 values for AChE inhibitions are summarized in
Table 1. Generally, all synthetic compounds showed excellent
AchE inhibition, nevertheless two of them (7aVII and 7aVIII)
could significantly inhibit BuChE. Due to the poor BuChE
inhibition activity, most of the BuChE inhibition activities of the
compounds were not deep measured. Compound 7aIII and 10a,
the most potent for AChE inhibition, presented an IC50 value of
18.44 nM and 13.58 nM respectively, while 7aVIII, the most
potent for BuChE, showed an IC50 value of 33.08 nM. Through
analysis and comparison, we found that the inhibitory potency is
associated with the length of linker chain: the moderate length of
the linker may give rise to a better activity, but there is no
obvious regularity. As we can see from the kinetic experiment
results, the inhibitory activity of AChE proved to be more
susceptible to the length of linker. The chain length governs the
activity of compound and any departure from the optimum length
value would lead to the decreasing acitivity. The change of
substitution site of benzylpiperazine may also influence the
acetylcholinesterase, several new compounds (which have novel
structure and excellent activity) were syhthesized during our
previous studies (Fig. 1).
9
Phthalimide derivatives are important compounds because of
1
0
their various bioactivities, such as anti-cancer , anti-
1
1
12
13
inflammatory , anticonvulsant , α-glucosidase inhibitory and
1
4
AChE inhibitory . Phthalimide had been proved to combine to
1
5
the peripheral anionic sites of AchE . And, several novel AchE
inhibitors (Compound A and B in Fig 2.) were designed based on
this pharmacophore. Meanwhile, a literature survey revealed that
piperazine derivatives are important pharmacophores across a
1
6
number of different therapeutic areas and they act as
17
18
19
antifungal , antipsychotic , antioxidant , antimalarial and anti-
2
0
HIV protease . Piperazine derivatives were also reported as
AChE inhibitors by combining to catalytic anionic sites. For
instance, compound C and D in Fig. 2 were designed as AChE
inhibitors, and showed excellent activity.
3
inhibitory activity, however, neither Cl nor OCH enhanced the
activity of compounds.
In view of the above studies, based on structural
characteristics of acetylcholinesterase, we designed a series of
novel AchE inhibitors, in which two pharmacophores
(phthalimide and benzylpiperazine) were combined by alkyl
linkers (Fig. 3). We expect that two pharmacophores could bind
two active centers at the same time, and the optimized alkyl chain
could increase the binding affinity. The preparation of the target
compounds was shown in scheme 1.
Scheme1. Synthesis of title compounds.
2 3
Reagents and conditions: (a) THF, reflux; (b) K CO , Aceton, reflux; (c)
°
2 3
K CO , DMF, 60-70 C.

Table 1. AchE inhibitory activity of title compounds in vitro
a
a
Comp.
aI
aII
R
H
n
2
3
4
5
6
7
8
9
10
2
3
4
5
6
7
8
AChE /nM
Comp.
7bVIII
7bIX
7cI
R
Cl
n
9
AChE /nM
7
91.12
644.42
1264.44
465.96
508.29
403.35
547.58
686.52
448.36
698.93
657.23
712.28
13.58
7
H
123.04
18.44
Cl
10
2
7
aIII
aIV
H
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
H
3
3
3
3
3
3
3
3
3
7
H
35.38
7cII
3
7
aV
H
100.21
53.84
7cIII
7cIV
4
7
aVI
H
5
7
aVII
aVIII
aIX
H
204.02
40.66
7cV
6
7
H
7cVI
7
7
H
350.40
1101.23
1432.82
1419.47
720.18
389.18
655.20
440.27
7cVII
7cVIII
7cIX
8
7
bI
Cl
Cl
Cl
Cl
Cl
Cl
Cl
9
7
bII
10
--
--
--
--
--
7
bIII
bIV
10a
7
10b
Cl
383.86
488.67
20
7
bV
10c
OCH
--
3
7
bVI
Donepezil
Ensaculin
7
bVII
--
36
a
5
0% inhibitory concentration (means of three experiments) of AChE from electric eel.
Subsequently, docking studies were carried out using the
2
3
AUTODOCK 4.0 package as displayed in Fig.3 aided by a
2
4
visualization PyMOL program . All compounds exhibited
multiple amino acid residues binding sites with AchE. In the
7aIII/TcAChE (PDB code: 1EVE, from http://www.rcsb.org)
complex, the results showed that compound 7aIII interacted
principally along the gorge of AChE through its major functional
groups. Compound 7aIII, enters the active site of protein through
hydrophobic interaction and interactes with W84, N85, D72,
S122, F288, W279, L282, R289 and other amino acid residues.
Carbonyl oxygen atom of the five-membered ring interacts with a
hydrogen atom of F288 residues forming hydrogen bonds; near
the bottom of the gorge, a hydrogen atom interact with the
carbonyl oxygen atom of D72 residue to form hydrogen bonds.
The formation of hydrogen bonds is conducive to enhance the
ability between the ligands and proteins. Meanwhile, the kinetic
analysis suggests that our compounds show a special mode of
inhibition, which is in agreement with the docking studies.
Figure 3. Docking models of compound–enzyme complex.
In conclusion, a series of novel phthalimide derivatives were
designed and synthesized as AChE inhibitors. The target
compounds might show double binding sites with the active sites,
CAS site and the PAS of the enzymes. However, their inhibitory
activities were jagged. In fact, to design a good cholinesterase
inhibitor with mixed binding modes, structural modification of
molecule should be well ordered to adopt the conformation.
Structure–activity relationships of investigated compounds
showed that the length of linker chain was the most important
factor in controlling the inhibitory activity. It might
simultaneously influence the interaction of phthalimide moiety
and piperazine group with their binding sites. In light of the
results from the docking studies, the linker length of designed
ligands might be appropriate for their preferable dual binding

interactions with AChE but not for BuChE. Lastly, the change of
piperazine group should also be considered. Further study of
phthalimide derivatives linked to different piperazine group to
increase the inhibition of both enzymes and selective BuChE
inhibitors are in progress. Also, the stability, toxicity, safety of
title compounds need further study.
12.
Kamiński, K.; Obniska, J.; Wiklik, B.; Atamanyuk, D. Eur. J. Med.
Chem. 2011, 46, 4634.
1
3.
BIAN, X.-l.; WANG, Q.; LUO, Z.-j.; FAN, X.-n. Chinese. J. Med.
Chem. 2012, 5, 002.
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Hoerr, R.; Noeldner, M. CNS. Drug. Rev. 2002, 8, 143.
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1
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5. Alonso, D.; Dorronsoro, I.; Rubio, L.; Munoz, P.; García-Palomero, E.;
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Acknowledgments
We thank the National Basic Research Program of China (No.
2014CB932200 and 2012CB114104), the NSFC (No. 31321004
6. Berkheij, M.; van der Sluis, L.; Sewing, C.; den Boer, D. J.; Terpstra, J.
W.; Hiemstra, H.; Bakker, W. I. I.; van den Hoogenband, A.; van
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Supplementary data
7.
Upadhayaya, R. S.; Sinha, N.; Jain, S.; Kishore, N.; Chandra, R.;
Arora, S. K. Bioorg. Med. Chem. 2004, 12, 2225.
Supplementary data (Synthetic procedure, structural analysis
data, in vitro AChE and BuChE assay method, kinetic analysis
and molecular docking study) associated with this article can be
found, in the online version, at-----
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Design, synthesis and bioactivity of
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novel phthalimide derivatives as
acetylcholinesterase inhibitors
Weijie Si, Tao Zhang, Lanxiang Zhang, Xiangdong Mei, Mengya Dong, Kaixin Zhang, Jun Ning