Synthesis, molecular modeling and evaluation of novel N′-2-(4- benzylpiperidin-/piperazin-1-yl)acylhydrazone derivatives as dual inhibitors for cholinesterases and Aβ aggregation

Article abstract of DOI:10.1016/j.bmcl.2012.11.064

To develop new drugs for treatment of Alzheimer's disease, a group of N′-2-(4-Benzylpiperidin-/piperazin-1-yl)acylhydrazones was designed, synthesized and tested for their ability to inhibit acetylcholinesterase, butyrylcholinesterase and aggregation of a

Full text of DOI:10.1016/j.bmcl.2012.11.064

Accepted Manuscript
Synthesis, Molecular Modeling and Evaluation of Novel N’-2-(4-Benzylpiper‐
idin-/piperazin-1-yl)acylhydrazone Derivatives as Dual Inhibitors for Cholines‐
terases and Aβ Aggregation
Eda Özturan Özer, Oya Unsal Tan, Keriman Ozadali, Tuba Kü ç ükkılınç, Ayla
Balkan, Gülberk Uçar
PII:
S0960-894X(12)01516-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2012.11.064
BMCL 19828
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
25 September 2012
15 November 2012
18 November 2012
Please cite this article as: Özer, E., Tan, O.U., Ozadali, K., Kü ç ükkılınç, T., Balkan, A., Uçar, G., Synthesis,
Molecular Modeling and Evaluation of Novel N’-2-(4-Benzylpiperidin-/piperazin-1-yl)acylhydrazone Derivatives
as Dual Inhibitors for Cholinesterases and Aβ Aggregation, Bioorganic & Medicinal Chemistry Letters (2012), doi:
http://dx.doi.org/10.1016/j.bmcl.2012.11.064
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis, Molecular Modeling and Evaluation
Leave this area blank for abstract info.
of Novel N'-2-(4-Benzylpiperidin-/piperazin-1-yl)
acylhydrazone Derivatives as Dual Inhibitors for Cholinesterases and Aβ Aggregation
Eda Özturan Özer, Oya Unsal Tan, Keriman Ozadali, Tuba Küçükkılınç, Ayla Balkan, Gülberk Uçar

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis, Molecular Modeling and Evaluation of Novel N'-2-(4-Benzylpiperidin-
/piperazin-1-yl)acylhydrazone Derivatives as Dual Inhibitors for Cholinesterases
and Aβ Aggregation
Eda Özturan Özera, Oya Unsal Tanb, Keriman Ozadalib, Tuba Küçükkılınçc, Ayla Balkanb, Gülberk Uçarc
a Department of Biochemistry, Faculty of Medicine, Başkent University, Ankara, 06530,Turkey.
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, Ankara, 06100, Turkey.
c Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, Ankara, 06100, Turkey.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
To develop new drugs for treatment of Alzheimer's disease, a group of N'-2-(4-Benzylpiperidin-
/piperazin-1-yl)acylhydrazones was designed, synthesized and tested for their ability to inhibit
acetylcholinesterase, butyrylcholinesterase and aggregation of amyloid beta peptides (1-40, 1-42
and 1-40_1-42). The enzyme inhibition assay results indicated that compounds moderately
inhibit both acetylcholinesterase and butyrylcholinesterase. β-Amyloid aggregation results
showed that all compounds exhibited remarkable Aβ fibril aggregation inhibition activity with a
nearly similar potential as the reference compound rifampicin, which makes them promising
anti-Alzheimer drug candidates. Docking experiments were carried out with the aim to
understand the interactions of the most active compounds with the active site of the
cholinesterase enzymes.
Keywords:
Alzheimer’s disease
Cholinesterase
Beta-amyloid fibril
N-Acylhydrazone
Molecular modeling
2009 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD), the most common cause of
dementia, is a neurodegenerative disorder characterized by
progressive deterioration of memory and cognition1. One of the
major therapeutic strategies adopted for primarily symptomatic
AD is based on the cholinergic hypothesis targeting
Aβ formation inhibitor activity is weak (%22)4. Recently, the
accumulation of the β-amyloid peptide (Aβ) in the brain has been
thought to be a key factor in the pathogenesis of the disease5. It
has been shown that AChE interacts with Aβ and promotes
amyloid fibril formation by a hydrophobic environment close to
the peripheral anionic binding site (PAS) of the enzyme6, 7. It is
well known that two distinct binding sites exist in the active
pocket of AChE peripheral binding site (PAS) and catalytic
active site (CAS), located at the entrance and the bottom of the
active-site gorge, respectively. These sites are characterized by
two tryptophan residues, Trp84 at the active site and Trp279 at
cholinesterase
butyrylcholinesterase)2. Inhibition of the hydrolysis of
acetylcholine by blocking its metabolic enzyme
enzymes
(acetylcholinesterase
and
acetylcholinesterase (AChE) increases the ACh concentration
and provides a possible symptomatic treatment option for AD.
On the other hand, butyrylcholinesterase (BuChE) has recently
been considered as a potential target because it also plays an
important role in regulating ACh level3. AChE inhibitors
currently approved as drugs for the treatment of Alzheimer’s
disease are donepezil, rivastigmine, galanthamine and tacrine.
the mouth of the gorge (PAS)8-11
.
The dual-binding inhibitors, which target simultaneously both
the catalytic and the peripheral-binding sites, not only inhibit the
Although, donepezil is most commonly used AChE inhibitor, its
———
 Corresponding author. Tel.: +90-312-305-1872; fax: +90-312-305-1872; e-mail: kozadali@hacettepe.edu.tr

hydrolysis of ACh by AChE, but also prevent self aggregation of
Aβ12, 13
benzylpiperidin-/piperazin-1-yl)acetohydrazide 3 or 4. Finally,
these hydrazides were treated with appropriate aldehydes in the
presence of hydrochloric acid to obtain 5a-6c with yields
between 37-62%.
.
Lately, it was found that the presence of benzyl piperidine
moiety in the AChE inhibitors contribute to inhibitor activity by
interacting with the catalytic site of the AChE14-16. On the other
hand, it is known that potent dual-binding inhibitors of AChE
were established to have aromatic stacking interactions with
aromatic residues lining the wall of the AChE gorge.
The compounds (5a-6c) were tested for their in vitro
inhibitory activities against human recombinant AChE and
equine BuChE according to the Ellman method. The ChE results
were summarized in Table 1. As shown in the Table 1, IC50
values were failed to meet the expectations since all compounds
inhibited human recombinant AChE at higher concentrations
than donepezil (donepezil IC50: 23.1 nM for human AChE19,
IC50: 7.4 µM for equine BuChE20). On the other hand equine
BuChE inhibitory activities of the compounds were more
promising. The inhibitory potential of the compounds in this
series showed that none of them was selective for both enzymes.
In this study we designed N'-2-(4-benzylpiperidin-/piperazin-
1-yl)acylhydrazone derivatives, which were expected to inhibit
both AChE, BuChE, and Aβ aggregation by combining 4-
benzylpiperidine-/piperazine and 3,4-dimethoxybenzyl scaffolds
linked with N-acylhydrazone moiety (Figure 1).
O
O
6c and 6b were selected for further kinetic analysis due to
slightly better activities against AChE and BuChE, respectively.
The Lineweaver-Burk plot of 6b for BuChE indicated the mixed
type inhibition and Dixon plot of 6b for AChE indicated a
uncompetitive inhibition (Figure 2). Ki values were determined
for both enzymes with both inhibitors using inhibition data and
results were summarized in Table 2. 6b was found to act as an
uncompetitive and linear mixed type inhibitor against AChE and
BuChE with Ki values of 17.9 and 4.3 µM respectively (Figure
2). On the other hand, 6c showed uncompetitive and competitive
inhibition against AChE and BuChE with Ki values of 35.8 and
9.49 µM respectively. Uncompetitive and linear mixed type
inhibitory patterns of the compounds indicated their interactions
with the residues at the gorge other than the catalytic active site.
These findings were compatible with their destabilising effects
on amyloid fibrils since amino acids at the peripheral anionic
sites (PAS) of the cholinesterases could promote Aβ fibrillation.
N
O
Donepezil
H
N
O
O
N
N
O
N
5a
Figure 1. Design strategy for the target compounds
To predict the drug-likeness of the designed compounds,
Lipinski's "rule of five" was calculated. All the compounds (5a-
6c) were in agreement with "rule of five" (cLogP ≤ 5, MW ≤ 500,
number of hydrogen bond donors ≤ 5 and number of hydrogen
acceptors ≤ 10) (Table 1). It has been shown that the compounds
penetrating the blood-brain barrier typically have polar surface
area (PSA) values less than 70Å17, 18. For this reason, PSA values
of the target compounds were also calculated. The calculated
theoretical PSA values (Table 1) for the compounds (5a-6c) may
support their ability to penetrate the blood-brain barrier according
to this hypothesis.
Analysis of AChE with 6c showed a concentration dependent
inhibition pattern. At concentrations below IC50 (53 M) it was
shown that the interaction of the inhibitor with the enzyme was
possible only after the formation of enzyme-substrate complex
(uncompetitive inhibition).
It was concluded that the inhibitor interacted with a site other
than the active site due to a new conformation depending on the
formation of enzyme-substrate complex. A noncompetitive
inhibition was observed at high inhibitor concentrations above
IC50. It can be assumed that this concentration dependent
inhibition type might be due to the multi-interaction sites of the
enzyme. An average of inhibitor binding sites calculated as 1.35
by using Hill plot. The obtained n values for 20-40 and 60-100
M inhibitor concentrations were 0.73 and 2.5 respectively.
The target compounds (5a-6c), were synthesized via the route
outlined in Scheme 1. The commercially available secondary
amines were transformed into ethyl 2-(4-benzylpiperidin-
/piperazin-1-yl)acetate 1 or 2 by the reaction with ethyl
bromoacetate in the presence of potassium carbonate. The
reaction of 1 or 2 with excess hydrazine hydrate gave 2-(4-
Table 1. The physicochemical parameters and in vitro AChE / BuChE enzyme inhibition data for the target compounds
IC50 (µM)b ± SEM
H-Bond
Donor/Acceptor
Comp.
X
R
PSAa
LogPa
MW
AChE
BuChE
5a
5b
5c
6a
6b
6c
N
N
N
C
C
C
3,4-diOCH3
4-OCH3
66,4
57,1
57,2
63,2
53,9
53,9
2,24
2,23
2,62
3,11
3,10
3,49
396
366
380
395
365
379
1/6
1/5
1/5
1/5
1/4
1/4
81,1 ± 4,82
71,7 ± 3,69
88,5 ± 3,61
81,4 ± 3,73
62,8 ± 6,07
73,5 ± 4,62
53,1 ± 4,56
88,3 ± 4,41
98,8 ± 4,09
55,1 ± 6,11
48,8 ± 4,99
67,3 ± 5,24
4-OC2H5
3,4-diOCH3
4-OCH3
4-OC2H5
a PSA and LogP calculated using MOE 2011.10 (Molecular Operating Environment, Chemical Computing Group)
bIC50 values of compounds represent the concentration that caused 50% enzyme activity loss

X
O
X
ii
i
N
NH
O
1, 2
X
O
X
O
iii
R
N
N
N
NH2
NH
5a-c, 6a-c
NH
3, 4
Scheme 1. Synthesis of the compounds. Reagents and conditions: (i) BrCH2COOC2H5, K2CO3, reflux (ii) NH2NH2.H2O, reflux (iii) appropriate carbonyl
compounds, rt.
difference in crystal structures of torpedo californica AChE
(1EVE) and human AChE (1B41) is the conformations of
Phe330 and Tyr337 respectively21. Phe330 is open-gate
conformation in 1EVE while the analogous, Tyr337, is closed in
1B41. It is known that the side-chain orientation of Phe330 is
responsible for substrate trafficking down the gorge. The similar
behavior can be expected by Tyr 337. That is why, the
conformation of Tyr337 was changed before docking studies for
human AChE. Previous studies on X-ray crystal structure of the
E2020-TcAChE complex showed that bivalent inhibitor
donepezil span along the active center to peripheral binding site.
All three major functional groups of donepezil, the benzyl
moiety, the piperidine nitrogen, and the dimethoxyindanone
moiety, interact with residues Trp84 at the bottom of the gorge,
Phe330, and Trp279 in the peripheral binding site respectively9.
Figure 3. Inhibitory effects of the compounds on the aggregation of 1-40_1-
Figure 2. A) Dixon plot for the inhibition of AChE by 6b, B) Lineaweaver-
42 amyloid peptides.
Burk plot for the inhibition of BuChE by 6b
All newly synthesized compounds were tested for their ability
to inhibit aggregation of amyloid beta peptides (1-40, 1-42 and 1-
40_1-42) by using a Thioflavin T fluorescence method.
Compared with the reference compound rifampicin, all target
compounds showed significant destabilization effects on the
aggregation of fibriles (Figure 3). The percentage of inhibition
was calculated by taking the non-inhibitor fibril fluorescence
intensity comparing with the reference and the results are given
in Figure 4. Compared with rifampicin for the inhibition of Aβ
aggregation, all compounds showed similar inhibitory activity at
100 µM, with inhibition ratio from 69% to 90%.
Figure 4. Amyloid fibril inhibition of the compounds compared to rifampicin
The docking study was performed by using MOE software to
clarify the binding mode of the active ligands (6c and 6b) in
human AChE and BuChE enzymes respectively. The major
Table 2. Cholinesterase inhibitory effects of 6b and 6c
Inhibitor
Enzyme
Inhibition Type
Ki, M

6b
6b
6c
6c
BuChE
AChE
BuChE
Linear mixed type
Uncompetitive
Competitive
Uncompetitive (20-40 M of I )
Noncompetitive (60-100 M of I )
30,5 ± 0,08
19,5 ± 0,06
31,5 ± 0,05
50,8 ± 0,04
83,8 ± 0,04
12,5 ± 0,18
-
-
-
AChE

Figure 5. Best docking pose of A) 6c in AChE B) 6b in BuChE
5. Hardy, J.; Selkoe, D. J. Science 2002, 297, 353.
6. Ferrari, D.; Yang, L. H.; Miles, E. W.; Dunn, M. F.
Biochemistry-Us 2001, 40, 7421.
7. Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.;
Vicente, M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J.
Neuron 1996, 16, 881.
8. Kreienkamp, H. J.; Weise, C.; Raba, R.; Aaviksaar, A.; Hucho,
F. P Natl Acad Sci USA 1991, 88, 6117.
9. Kryger, G.; Silman, I.; Sussman, J. L. Struct Fold Des 1999, 7,
297.
10. Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.;
Toker, L.; Silman, I. Science 1991, 253, 872.
11. Harel, M.; Schalk, I.; Ehretsabatier, L.; Bouet, F.; Goeldner, M.;
Hirth, C.; Axelsen, P. H.; Silman, I.; Sussman, J. L. P Natl Acad
Sci USA 1993, 90, 9031.
12. Bolognesi, M. L.; Andrisano, V.; Bartolini, M.; Cavalli, A.;
Minarini, A.; Recanatini, M.; Rosini, M.; Tumiatti, V.;
Melchiorre, C. Farmaco 2005, 60, 465.
It was demonstrated that 6c interacts with PAS and CAS of
human AChE. At the entrance of the enzyme, π-π interaction was
formed between the 4-ethoxybenzylidene group of the compound
and the indole ring of PAS residue Trp286 while the
benzylpiperidine moiety of the compound interacted with Trp86,
by means of a π-π stacking (Figure 5A). When we looked into
orientations of 6b which had remarkable inhibitory activity
against BuChE, only the terminal benzyl group of 6b established
π-π interaction with Trp82 due to lack of Trp279 in BuChE
(Figure 5B). This may explain the similar inhibition potential of
our newly synthesized compounds against both AChE and
BuChE.
In summary, six new N'-2-(4-benzylpiperidin-/piperazin-1-
yl)acylhydrazone derivatives (5a-6c) were synthesized and tested
for
their
ability
to
inhibit
acetylcholinesterase,
butyrylcholinesterase and Aβ aggregation. The results showed
that these compounds possessed a moderate and non-selective
inhibitory activity against both enzymes. Analysis of kinetics
data exhibited that, 6b was a mixed type and uncompetitive
inhibitor of BuChE and AChE, respectively whereas 6c was
competitive and uncompetitive inhibitor in the order mentioned.
Also the docking studies indicated that 6c interacted with PAS
and CAS of human AChE. The results of kinetic analysis and
docking studies support the multi-site binding of the compounds
to AChE. On the other hand, β-amyloid aggregation results
showed that all compounds exhibited a good activity against Aβ
aggregation. Although our novel compounds showed weak
cholinesterase inhibitory activity, their remarkable Aβ fibril
aggregation inhibitory activities make us hopeful to study on
these compounds for further optimization.
13. De Ferrari, G. V.; Canales, M. A.; Shin, I.; Weiner, L. M.;
Silman, I.; Inestrosa, N. C. Biochemistry-Us 2001, 40, 10447.
14. Contreras, J. M.; Parrot, I.; Sippl, W.; Rival, Y. M.; Wermuth,
C. G. J Med Chem 2001, 44, 2707.
15. Shao, D.; Zou, C. Y.; Luo, C.; Tang, X. C.; Li, Y. C. Bioorg
Med Chem Lett 2004, 14, 4639.
16. Zhu, Y. P.; Xiao, K.; Ma, L. P.; Xiong, B.; Fu, Y.; Yu, H. P.;
Wang, W.; Wang, X.; Hu, D. Y.; Peng, H. L.; Li, J. Y.; Gong,
Q.; Chai, Q.; Tang, X. C.; Zhang, H. Y.; Li, J.; Shen, J. K.
Bioorg Med Chem 2009, 17, 1600.
17. Muchmore, S. W.; Edmunds, J. J.; Stewart, K. D.; Hajduk, P. J.
J Med Chem 2010, 53, 4830.
18. Kelder, J.; Grootenhuis, P. D. J.; Bayada, D. M.; Delbressine, L.
P. C.; Ploemen, J. P. Pharmaceut Res 1999, 16, 1514.
19. Bolognesi, M. L.; Banzi, R.; Bartolini, M.; Cavalli, A.; Tarozzi,
A.; Andrisano, V.; Minarini, A.; Rosini, M.; Tumiatti, V.;
Bergamini, C.; Fato, R.; Lenaz, G.; Hrelia, P.; Cattaneo, A.;
Recanatini, M.; Melchiorre, C. J Med Chem 2007, 50, 4882.
Supplementary Data
20.
-
, C.; Chioua, M.;
Supplementary data associated with this article can be found
in the online version, at doi:
Pouplana, R.; Luque, F. J.; Unzeta, M.; Marco-Contelles, J.;
Samadi, A. J Med Chem 2011, 54, 8251.
21. Kryger, G.; Harel, M.; Giles, K.; Toker, L.; Velan, B.; Lazar,
A.; Kronman, C.; Barak, D.; Ariel, N.; Shafferman, A.; Silman,
I.; Sussman, J. L. Acta Crystallogr D 2000, 56, 1385.
References
1. Terry, A. V.; Buccafusco, J. J. J Pharmacol Exp Ther 2003,
306, 821.
2. Cummings, J. L. New Engl J Med 2004, 351, 56.
3. Mesulam, M. M.; Guillozet, A.; Shaw, P.; Levey, A.; Duysen,
E. G.; Lockridge, O. Neuroscience 2002, 110, 627.
4. Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. Biochem
Pharmacol 2003, 65, 407.