Design and synthesis of new piperidone grafted acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.11.065

Alzheimer's disease (AD) is a neurodegenerative disorder affecting 35?million people worldwide. A common strategy to improve the well-being of AD patients consists on the inhibition of acetylcholinesterase with the concomitant increase of the neurotransmitter acetylcholine at cholinergic synapses. Two series of unreported N-benzylpiperidines 5(a–h) and thiazolopyrimidines 9(a–q) molecules were synthesized and evaluated in vitro for their acetylcholinesterase (AChE) inhibitory activities. Among the newly synthesized compounds, 5h, 9h, 9j, and 9p displayed higher AChE enzyme inhibitory activities than the standard drug, galantamine, with IC₅₀values of 0.83, 0.98, and 0.73?μM, respectively. Cytotoxicity studies of 5h, 9h, 9j, 9n and 9p on human neuroblastoma cells SH-SY5Y, showed no toxicity up to 40?μM concentration. Molecular docking simulations of the active compounds 5h and 9p disclosed the crucial role of π-π-stacking in their binding interaction to the active site AChE enzyme. The presented compounds have potential as AChE inhibitors and potential AD drugs.

Full text of DOI:10.1016/j.bmcl.2016.11.065

Accepted Manuscript
Design and Synthesis of New Piperidone Grafted Acetylcholinesterase Inhibi-
tors
Alireza Basiri, Michelle Xiao, Alec McCarthy, Debashis Dutta, Siddappa N.
Byrareddy, Martin Conda-Sheridan
PII:
S0960-894X(16)31221-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.065
BMCL 24460
Reference:
Bioorganic & Medicinal Chemistry Letters
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3 November 2016
21 November 2016
22 November 2016
Please cite this article as: Basiri, A., Xiao, M., McCarthy, A., Dutta, D., Byrareddy, S.N., Conda-Sheridan, M.,
Design and Synthesis of New Piperidone Grafted Acetylcholinesterase Inhibitors, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.065
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Design and Synthesis of New Piperidone Grafted
Acetylcholinesterase Inhibitors
Alireza Basiria, Michelle Xiaob, Alec McCarthyc, Debashis Duttad, Siddappa N. Byrareddyd and
Martin Conda-Sheridana,Ŧ
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical
Center, Omaha, NE 68198, United States
bSchool of Engineering, Stanford University. Stanford, CA 94305, United States
cSchool of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588,
United States
dDepartment of Pharmacology and Experimental Neuroscience, University of Nebraska Medical
Center, Omaha, NE 68198, United States
Alzheimer’s disease (AD) is a neurodegenerative disorder affecting 35 million people worldwide.
A common strategy to improve the well-being of AD patients consists on the inhibition of
acetylcholinesterase with the concomitant increase of the neurotransmitter acetylcholine at
cholinergic synapses. Two series of unreported N-benzylpiperidines 5(a-h) and
thiazolopyrimidines 9(a-q) molecules were synthesized and evaluated in vitro for their
acetylcholinesterase (AChE) inhibitory activities. Among the newly synthesized compounds, 5h,
9h, 9j, and 9p displayed higher AChE enzyme inhibitory activities than the standard drug,
galantamine, with IC50 values of 0.83, 0.98, and 0.73 µM, respectively. Cytotoxicity studiess of
5h, 9h, 9j, 9n and 9p on human neuroblastoma cells SH-SY5Y, showed no toxicity up to 40µM
concentration. Molecular docking simulations of the active compounds 5h and 9p disclosed the
crucial role of π-π-stacking in their binding interaction to the active site AChE enzyme. The
presented compounds have potential as AChE inhibitors and potential AD drugs.
Keywords: N-benzylpiperidines, Thiazolopyrimidinium, AChE activity, Molecular modeling, π-π-
stacking interactions, SH-SY5Y cell, Cytotoxicity.
ŦCorresponding author: martin.condasheridan@unmc.edu

Alzheimer’s disease (AD) is a prevalent, irreversible neurodegenerative disorder that affects
more than 35 million people worldwide. It is estimated that AD will be the cause of more than
700,000 deaths in 2016, making it one of the top ten leading causes of death in the United
States. 1 Despite the tremendous efforts in search for novel and potent therapeutic agents, the
pipeline of available treatments is very low.2, 3 The etiology of AD is not completely understood.
However, the appearance of extracellular β-amyloid plaques and the imbalance of acetylcholine
(ACh) neurotransmitters (due to the severe loss of cholinergic neurons) are two major
hallmarks.4, 5 The cholinergic system is severely affected in AD patients, leading to a reduced
concentration of the neurotransmitter acetylcholine (ACh) at the synaptic clefts.6
Acetylcholinesterase (AChE), the enzyme responsible for degradation and regulation of
acetylcholine in the human body, has been actively investigated as a target for AD treatments.7
Previous reports suggest that increasing acetylcholine levels by inhibiting AChE activity partially
restores the substantial impairment of memory and cognitive dysfunctions.8 In fact, the main AD
treatments are cholinesterase inhibitors such as Donepezil, which works by increasing
acetylcholine concentration at synaptic clefts 9, 10. Therefore, compounds that can inhibit the
action of AChE could rival Donepezil as promising AD treatments.
The active site of AChE is located at the bottom of a 20 Å long, narrow gorge comprising of five
regions: the catalytic triad (composed of Glu327, His440 and Ser200), the oxyanion hole
(Gly121, Gly122 and Ala201), the choline binding site (Trp84 and Phe330), the acyl binding
pocket (Phe288 and Phe290), and the peripheral anionic site (PAS) (Trp279, Phe330 and
Tyr70).11-14 The PAS of AChE catalyzes a series of reactions on the Aβ fibrils that lead to β-
sheets with high aggregating potential.15-17 AChE inhibitors that bind to the PAS of the enzyme
decrease aggregation rates of amyloid peptides and facilitates their clearance when compared
to inhibitors that only bind the catalytic triad of enzyme.17-19 The AChE active site is highly
hydrophobic20 and it is hypothesized molecules with multiple aromatic cores can bind to more
21, 22
23, 24
effectively
due to hydrophobic interactions.
Studies also showed that compounds
having α, β-unsaturated moiety or the thiazolopyrimidine core structure are capable of inhibiting
AChE.23, 25-27 Moreover, the N-benzylpiperidine moiety is a structure found in donepezil, a FDA-
approved drug for treatment of mild to moderate AD.28 The crystal structure of AChE in complex
with donepezil, revealed a π-π stacking as well as hydrogen bonding interaction of N-
benzylpiperidine to the active site of AChE.29 Inspired by these precedents, and the
characteristics of the enzyme’s binding pocket, we used computer modeling to design potential
inhibitors of AChE enzyme. In this study, we wish to report the synthesis, AChE inhibitory
activity, and toxicity towards human neuroblastoma SH-SY5Y cells of these new N-

benzylpiperidines and thiazolopyrimidines derivatives. Finally, the plausible binding interactions
of the most active derivatives were investigated using molecular docking.
Molecular modeling techniques were employed for designing various inhibitors. Energy
minimizations lead us to two potential cores; 5 and 9. The in silico studies showed the critical
role of (i) the benzylamine moiety in 5(a-h) and (ii) sulfur atom in 9(a-q) to form favorable
π-stacking and hydrogen bonding interactions to the active site residues of AChE enzyme,
respectively.
The common α,β-unsaturated ketone precursors (3) were prepared by the Claisen-Schmidt
condensation of 4-piperidone hydrochloride (1) with aromatic aldehydes (2) in the presence of
HCl in acetic acid (Scheme 1).30 The reaction of the 3 with benzyl bromides (4) under basic
conditions provided N-benzylpiperidines 5(a-h). Also, the unsaturated ketone 3 was reacted with
thiourea (6) in presence of sodium ethoxide24 to yield 7. These compounds were reacted with
α-Bromoacetophenones 8(a-d) in refluxing ethanol for 12 h to afford thiazolopyrimidinium salts
9(a-q).
Scheme 1: Reagent and conditions: (a) NaOH, EtOH, rt, 24 h; (b) EtOH, K2CO3, rt, 2 h; (c) Heat, NaOEt,
15 min; (d) EtOH, Reflux, 12 h.

The synthesis of the thiazolopyrimidinium molecules (9) can yield two possible isomers (see SI).
Thus, we prepared 9q and studied its spectroscopic properties to assign the correct
1
stereochemistry (Figure 1). In the H NMR spectrum of 9q,31 the one hydrogen singlet at 6.27
ppm is due to H-5 and assigned to the carbon peak at 63.2 ppm using HMQC. This proton
showed HMBC correlations to C-6 at 42.8 ppm besides showing correlation with C-5a and C-3
at 106.6 and 140.0 ppm, respectively. From HMQC, the doublets at 3.38 and 4.08 ppm (J=16.5
Hz) are assigned to H-6a and H-6b whilst the other doublets at 4.27 and 4.42 ppm (J=14.5 Hz)
can be attributed to H-8a and H-8b. The singlet at 7.43 ppm is due to H-12 and H-2, which
showed HMBC correlation to C=N at 163.8 ppm. The aromatic protons for ring A showed up as
two singlets at 6.01 and 6.40 ppm in proton NMR having HMQC correlation with corresponding
carbons at 102.0 and 112.3 ppm in 13C NMR. On the same basis, the protons of ring B were
1
elucidated as two singlets at 6.52 and 6.55 ppm in H NMR, having correlation to carbons at
1
112.3 and 101.6 ppm through HMQC. The H and 13C chemical shifts as well as selected
ROESY and HMBC correlations of 9q are depicted in Figure 1 and Figure 2a/2b, respectively.
The infrared spectroscopy (IR) for 9q showed three medium peaks at 3469 cm-1, 1654 cm-1 and
755 cm-1 specific for free NH, C=N and C-S moieties, respectively. This data helped us to
conclude the spatial arrangement of the atoms in 9q (and all the other analogues).
Figure 1: Selected 1H and 13C chemical shifts of 9q

Figure 2: Selected ROESY (a) and HMBC (b) correlations of 9q
All the newly synthesized N-benzylpiperidines 5 and thiazolopyrimidinium bromide salts 9 were
evaluated for their inhibitory activity against AChE (Table 1) using Ellman’s method,32 which
uses acetylthiocholine and 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB). In presence of AChE
enzyme, the reaction produces the yellow compound 5-thio-2-nitrobenzoate. AChE inhibitors
decrease the rate of the reaction resulting in a less colored solution, the absorption difference is
correlated to inhibitory activity (see SI). Compound 9p showed the highest inhibitory activity
among both series, with an IC50 value of 0.73 µM, nearly two fold better than the standard drug
(IC50 = 1.31 µM). Other active compounds are: 5h, 9h, 9n, and 9j with IC50 values of 0.83, 0.98,
1.01 and 1.09 µM. Compounds bearing 2-naphtyl moiety (except for 5f and 9d), showed good
IC50 values suggesting the importance of π-π interactions. Based on the above results, it can be
deduced that those compounds having extended aromatic systems on either R1 or R3 positions,
may display more van der Waals interactions on the active site of AChE enzyme. It was also
noticed that compounds 5h and 9p, bearing bromine atoms, showed the highest inhibitory
potencies among their series. This may suggest an electronic effect due to the halide. Studies
are undergoing to decipher the role electronically diverse substituents have on AChE inhibitory
activity.

Table 1: AChE inhibitory activities of 5(a-h) and 9(a-q)
AChE inhibition
(IC50)
AChE
inhibition %
at 5 µg/ml
Entry
Compound
R1
R2
R3
(µg/ml)
(µM)
1
2
3
4
5
6
7
8
9
5a
5b
5c
5d
5e
5f
5g
5h
9a
9b
9c
3.18
1.93
3.22
1.59
-
-
-
3-CH3
2-naphtyl
3-CH3
2-naphtyl
3-CH3
2-naphtyl
3-CH3
2-naphtyl
-
-
-
-
-
-
-
-
-
H
63.1
67.9
59.8
72.6
41.6
42.4
45.7
67.2
48.6
42.1
48.7
1.2±0.08
0.80±0.05
1.31±0.10
0.71±0.07
H
4-CH3
4-Cl
-
-
-
4-Br
0.83
0.47±0.03
-
-
-
-
-
-
10
11
-
-
4-CH3
4-Br
H
12
9d
-
-
2-naphtyl
41.8
-
13
14
15
16
17
18
19
20
21
22
23
24
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
-
-
-
-
-
-
-
-
-
-
-
-
-
H
4-CH3
4-Br
45.7
60.4
64.6
69.6
41.6
74.7
41.6
62.8
66.7
68.3
48.3
65.7
-
1.80
2.27
0.98
-
1.09
-
1.35
1.48
1.01
-
0.86±0.09
1.23±0.08
0.51±0.04
-
0.57±0.03
-
0.74±0.07
0.87±0.09
0.61±0.04
-
4-CH3
4-Cl
2-naphtyl
H
4-CH3
4-Br
2-naphtyl
H
4-CH3
4-Br
4-Br
0.73
2-naphtyl
0.47±0.03
3,5-
OCH3
-
25
9q
-
-
-
4-NO2
-
-
-
-
Galantamine
1.30
0.48±0.01
The cytotoxicity of the most active inhibitors, namely 5h, 9h, 9j, 9n and 9p, was evaluated on
the neuronal cell line SH-SY5Y using the MTT assay, which measures the cell metabolic
activity. The assay was performed after 24 hours incubation of neuronal cells with
concentrations ranging from 0.2 to 40 M. The molecules showed significant cell viability at this
range of concentrations (Figure 3).

Figure 3: Effect of compounds 5h, 9h, 9j, 9n and 9p on apoptotic neuronal cell death in SH-SY5Y. MTT
assay results are plotted for different concentration of compounds treated for 24 hours
The most active compounds from each series (5h and 9p) were docked into the groove of the
AChE enzyme. Compound 5h (Figure 4) mainly displayed interaction to the amino acids
composing the PAS of the
enzyme. As predicted, the main
interactions occur between Tyr 70,
Trp 279 with the 2-naphthyl
moiety of 5 h (distances of 3.69 Å
and 3.36 Å). The B ring of the
molecules also interacts with Phe
330 and Tyr 334 (3.31 Å and 3.67
Å). Compound 9p also sits on the
PAS of the AChE enzyme
(Figure 5). The amide nitrogens
Figure 4: Binding interaction of 5h with major active site residues
of AChE Enzyme
of Phe 288 and Arg display a
H-bond type interaction to the sulfur of the thiazoline ring with contact distance of 2.02 Å and
2.33 Å. Phe 331 displays a strong “T-shaped” stacking interaction to ring A at the left side of the
molecule (3.31 Å). Trp 279 shows a “T-shaped” stacking with ring B of the molecule (3.49 Å).
Finally, there is a potential cation-π interaction between the piperidine ring and Tyr 334 (3.58 Å).

Figure 5: Binding interaction of 9p with major active site residues of AChE Enzyme
In conclusion, two series of piperidone grafted heterocyclic compounds were prepared
employing efficient synthetic methodologies in pure form and were tested for their inhibitory
activity against AChE enzyme. Compound 5h and 9p displayed the highest inhibitory activities
among their series with IC50 values of 0.73 and 0.83 µM, respectively. The most active
compounds did not show cell toxicity at the tested concentrations. Molecular modeling disclosed
binding interaction patterns to the active site of AChE suggesting the positive effect of aromatic
core multiplicity.
Acknowledgements
This work was support by the University of Nebraska Medical Center, this work was also
supported by the NIH, R01AI113883 (SNB). The work was partially funded by the UNMC High
School Alliance program with a grant from The Sherwood Foundation (MX). Research reported
in this publication was supported by the NIGMS-NIH, GM103427 (AM). The content is solely
the responsibility of the authors and does not necessarily represent the official views of the
National Institutes of Health. Sh-SY5Y cells were obtained from Dr. Buch's laboratory, UNMC

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Graphical Abstract
Design, Synthesis and Inflammation Mediated Neurotoxicity Studies of New
Piperidone Grafted Acetylcholinesterase inhibitors
Alireza Basiri, Michelle Xiao, Alec McCarthy, Debashis Dutta, Siddappa Byrareddy and Martin
Conda-Sheridan
N-benzylpiperidines 5 and thiazolopyrimidinium salts 9 derivatives, possessing multiple aromatic
cores were synthesized and evaluated for AChE inhibitory activity. Compounds 5h and 9p
displayed better inhibitory activities than galantamine having IC50 values of 0.73 and 0.83 µM,
respectively.

 A simple and efficient method for the synthesis of heterocycles is reported
 Some compounds showed higher AChE inhibitory activity than galantamine
 The most active inhibitors did not show toxicity towards human neuroblastoma cells