Synthesis, in vitro assay, and molecular modeling of new piperidine derivatives having dual inhibitory potency against acetylcholinesterase and Aβ1-42 aggregation for Alzheimer's disease therapeutics

Article abstract of DOI:10.1016/j.bmc.2007.07.003

With the goal of developing Alzheimer's disease therapeutics, we have designed and synthesized new piperidine derivatives having dual action of acetylcholinesterase (AChE) and beta-amyloid peptide (Aβ) aggregation inhibition. For binding with the catalytic site of AChE, an ester with aromatic group was designed, and for the peripheral site, another aromatic group was considered. And for intercalating amyloid-beta oligomerization, long and linear conformation with a lipophilic group was considered. The synthetic methods employed for the structure with dual action depended on alcohols with an aromatic ring and the substituted benzoic acids, which are esterificated in the last step of the synthetic pathway. We screened these new derivatives through inhibition tests of acetylcholinesterase, butyrylcholinesterase (BChE), and Aβ_1-42 peptide aggregation, AChE-induced Aβ_1-42 aggregation. Our results displayed that compound 12 showed the best inhibitory potency and selectivity of AChE, and 29 showed the highest selectivity of BChE inhibition. Compounds 15 and 12 had inhibitory activities against Aβ_1-42 aggregation and AChE-induced Aβ aggregation. In the docking model, we confirmed that 4-chlorobenzene of 12 plays the parallel π-π stacking against the indole ring of Trp84 in the bottom gorge of AChE. Because the benzyhydryl moiety of 12 covered the peripheral site of AChE in a funnel-like shape, 12 showed good inhibitory potency against AChE and could inhibit AChE-induced Aβ_1-42 peptide aggregation.

Full text of DOI:10.1016/j.bmc.2007.07.003

Bioorganic & Medicinal Chemistry 15 (2007) 6596–6607
Synthesis, in vitro assay, and molecular modeling of new
piperidine derivatives having dual inhibitory potency
against acetylcholinesterase and Ab1–42 aggregation for
Alzheimer’s disease therapeutics
Young Ee Kwon,a,*, Jung Youl Park,b, Kyung Tai No,c,d Jae Hong Shin,d
Sung Kwang Lee,d Jae Soon Eun,a Jae Heon Yang,a Tae Yong Shin,a Dae Keun Kim,a
Byung Sook Chae,a Jae-Yoon Leema and Kuk Hwan Kime
aCollege of Pharmacy, Woosuk University, Jeonbuk, Republic of Korea
bC&C Research Laboratories, Kyung Gi, Republic of Korea
cDepartment of Biotechnology, Yonsei University, Seoul, Republic of Korea
dBioinformatics & Molecular Design Research Center, Seoul, Republic of Korea
eK&C Medicine Research Center, Bucheon, Republic of Korea
Received 10 May 2007; revised 30 June 2007; accepted 6 July 2007
Available online 25 July 2007
Abstract—With the goal of developing Alzheimer’s disease therapeutics, we have designed and synthesized new piperidine deriva-
tives having dual action of acetylcholinesterase (AChE) and beta-amyloid peptide (Ab) aggregation inhibition. For binding with the
catalytic site of AChE, an ester with aromatic group was designed, and for the peripheral site, another aromatic group was consid-
ered. And for intercalating amyloid-beta oligomerization, long and linear conformation with a lipophilic group was considered. The
synthetic methods employed for the structure with dual action depended on alcohols with an aromatic ring and the substituted ben-
zoic acids, which are esterificated in the last step of the synthetic pathway. We screened these new derivatives through inhibition tests
of acetylcholinesterase, butyrylcholinesterase (BChE), and Ab1–42 peptide aggregation, AChE-induced Ab1–42 aggregation. Our
results displayed that compound 12 showed the best inhibitory potency and selectivity of AChE, and 29 showed the highest selec-
tivity of BChE inhibition. Compounds 15 and 12 had inhibitory activities against Ab1–42 aggregation and AChE-induced Ab aggre-
gation. In the docking model, we confirmed that 4-chlorobenzene of 12 plays the parallel p–p stacking against the indole ring of
Trp84 in the bottom gorge of AChE. Because the benzyhydryl moiety of 12 covered the peripheral site of AChE in a funnel-like
shape, 12 showed good inhibitory potency against AChE and could inhibit AChE-induced Ab1–42 peptide aggregation.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
can interfere with the progression of AD.1–3 The more
basic pathological abnormalities in AD are amyloid pla-
ques, neurofibrillary tangles, and neuronal death.4,5 In
the past two decades, many efforts have been made to
understand the molecular pathogenesis of AD, and to
carry out its early diagnosis and therapeutic control.
Most relevant pathogenic events in AD can be classified
into the following four categories, (1) genetic alteration,
(2) beta-amyloid (Ab) deposition in senile plaques, (3)
neuroimmune dysfunction, and (4) cerebrovascular dys-
function.4–6 All of these pathogenic events are potential
targets for AD therapy, but the cholinergic neurotrans-
mission and beta-amyloid peptide are regarded as main
targets for more effective treatment strategies. The cho-
linergic hypothesis is still the most successful approach
Alzheimer’s disease (AD), the most common dementia
in elderly people, is a complex neurodegenerative disor-
der of central nervous system. It is associated with a
selective loss of cholinergic neurons and reduced levels
of acetylcholine neurotransmitter. A wide range of evi-
dence shows that acetylcholinesterase (AChE) inhibitors
Keywords: Piperidine derivatives; Alzheimer’s disease; Acetylcholines-
terase; Butyrylcholinesterase; Ab1–42 aggregation; Dual action.
Corresponding author. Tel.: +82 63 290 1566/+82 17 257 1006; fax:
+82 63 290 1812; e-mail: yekwon@woosuk.ac.kr
These authors contributed equally to this work.
*
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.003

Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
6597
for the symptomatic treatment of AD. This hypothesis
postulates that at least some of the cognitive decline
experienced by AD patients results from a deficiency
in acetylcholine and thus in cholinergic neurotransmis-
sion. Therefore, inhibition of AChE appears to be a nat-
ural therapeutic strategy to palliate the cognitive deficit
in AD. Thus, the AChE inhibitors such as tacrine,7
donepezil,8 rivastigmine9, and galantamine10 have been
launched on the market for the symptomatic treatment
of AD (Fig. 1).
of Ab are presently pursued for the inhibition of amy-
loid production by inhibiting the enzyme cleaving
beta-amyloid protein precursor, immunizing against
AD, and inhibition of amyloid polymerization.12 An
attractive therapeutic strategy is to inhibit peptide
aggregation itself, because this appears to be the first
step in the pathogenic process of amyloidosis, which
is not associated with natural biological function.13
Figure 2 shows examples of some small molecule Ab
inhibitors. Current NMR and molecular modeling
studies are being conducted to unravel the structure
and dynamics of Ab peptide and to understand the
molecular basis of the amyloid fibril formation.14–17
But the lack of structural similarity among the Ab
inhibitors is striking, suggesting that they bind to dif-
ferent sites within amyloid in contrast to most drugs.
This interferes in making conclusions from rational
structure–activity relationships. Recent studies have
identified that AChE enhances the aggregation of Ab
peptide fragments18 and accelerates the assembly of
Ab1–42 peptide into the amyloid fibrils that form the
senile plaques characteristic of AD. These results, to-
gether with binding assays, have suggested that AChE
may contribute to the generation of amyloid deposits
and/or physically affects fibril assembly. Moreover, it
has also been shown that the neurotoxicity of Ab1–42
peptide aggregates depends on the amount of AChE
bound to the complexes, suggesting that AChE may
play a key role in the neurodegeneration observed in
an AD patient’s brain.19,20
Beta-amyloid peptide is a main component of the se-
nile plaques and fibrillary tangles that constitute one
of the neurohistopathological features of AD. An
overproduction of Ab peptide and its subsequent
deposition as insoluble amyloid plaques may represent
the key pathological pathway.11 Accordingly, Ab pep-
tide has become a primary target in the development
of effective therapies.6,11 Amyloid targeted therapeutic
approaches aimed at blocking the neurotoxic activity
NH2
O
N
N
O
N
rivastigmine
tacrine
OH
N
O
OCH3
OCH3
We designed and synthesized new compounds with dual
action of effective anti-acetylcholinesterase and Ab1–42
peptide aggregation inhibition. Specifically, we focused
our efforts on piperidine derivatives binding with a cat-
alytic and a peripheral site of AChE, and having inhib-
itory potency against Ab aggregation. We report here
the synthesis, pharmacological evaluation, and molecu-
lar modeling of new piperidine compounds.
O
O
N
donepezil
galantamine
Figure 1. Structures of the acetylcholinesterase inhibitors as FDA
approved Alzheimer’s disease therapeutics.
O
O
HN
N
O
N
S
Cl
N
H
O
NO2
O
9-Acridone derivative
Benzofuran derivative, SKF-74652
NH2
NH2
HN
N N
N N
O
H3CO
O
S
O
O S O
O- Na+
N
H
O- Na+
melatonine
Congo-red
Figure 2. Structures of the effective beta-amyloid fibril aggregation inhibitors in vitro.

6598
Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
2. Results and discussion
reduction using LiAlH4 of the esters 7, 8. The esters 7,
8 were obtained by substitution reaction of the ether
containing piperidine and aromatic ring 5, 6. Deprotec-
tion of the tert-butyl carbonate group in piperidine com-
pounds 3, 4 gave the compounds 5, 6. The ethers 3, 4
were obtained by substitution reaction of benzylbromide
or benzhydrylbromide with N-protected 4-hydroxypi-
peridine 2. The key intermediate compounds 9 and 10
were prepared in five steps as described in Scheme 1.
All the piperidine ester derivatives 11–18 and 21–28 were
synthesized with intermediate compounds 9, 10 and the
substituted benzoic acids that are commercially avail-
able. Because the reaction condition is mild and simple,
there may be no problem in large scale syntheses
(Scheme 2). The mixture of the corresponding substi-
tuted benzoic acid (4-chlorobenzoic acid, 4-nitrobenzoic
acid, 3-fluorobenzoic acid, etc.) and compound 9 or 10
with 4-N,N-dimethylaminopyridine and N-[3-(dimeth-
2.1. Chemistry
Several possible chemical variations were considered on
the basis of piperidine derivatives (Fig. 3). For binding
with catalytic site of AChE, the ester with an aromatic
group was designed, and for binding with peripheral
site, another aromatic group was considered. As the
pocket of AChE is deep, a few carbon chains as a linker
were needed at the middle site between the gorge and the
entrance of AChE. Several groups studied the peptidic
Ab inhibitors by modifying core regions of Ab residue,
and the numerous anti-amyloidogenic compounds have
been developed.13 Unfortunately, these peptidic amyloid
aggregation inhibitors were not stable at oral adminis-
tration in vivo. However, they have a hydrophobic
group in common for binding with the core regions of
Ab residue.12,13 Therefore, we considered long and lin-
ear conformation with some lipophilic groups for inter-
calating against Ab peptide oligomerization. The
synthetic methods for the dual action depended on the
alcohols with an aromatic ring and the substituted ben-
zoic acids, which are esterificated in the last step of the
synthetic pathway.21 Preparation of the alcohols, the
key intermediate compounds 9, 10 containing a piperi-
dine and aromatic group, was carried out by hydride
ylamino)propyl]-N0-ethylcarbodiimide
hydrochloride
was allowed to react at room temperature. The resulting
products 11–18 and 21–28 were obtained. Separately,
compounds 9 or 10 were reacted with halide substituted
phenyl isocyanate at para position in acetonitrile as sol-
vent and then the resulting products were obtained as
compounds 19–20 and 29–30 (Scheme 3). The chemical
structures of all new compounds synthesized herein were
fully characterized by mass analysis and proton, carbon
O
Ar
O
N
O
Ar'
Linker & Middle site Catalytic site
binding binding
Peripheral site
binding
AChE binding
Linear conformation with Aromatic group and
electron donor and/or acceptors
A-beta intercalating
Figure 3. Design of the molecules with dual inhibitory action for AChE and amyloid-beta aggregation.
O
O
a
b
c
NH
N
O
N
O
HO
HO
RO
2
1
R = (C6H5)2CH : 3
C6H5CH2 : 4
OEt
d
e
f
NH
N
O
RO
RO
R = (C6H5)2CH : 7
C6H5CH2 : 8
R = (C6H5)2CH : 5
C6H5CH2 : 6
OH
g
N
RO
R = (C6H5)2CH : 9
C6H5CH2 : 10
Scheme 1. Reagents and conditions: (a) Et3N, DIBOC, CHCl3, 1 h; (b) (C6H5)2CHBr, KI (cat), Na2CO3 1,4-dioxane, reflux, 10 h; (c) C6H5CH2Br,
Na2CO3, THF, 4 h; (d) KOH, IPA, reflux, 14 h; (e) NaOH, EtOH, reflux, 6 h; (f) NaHCO3, KI (cat), Cl(CH2)3CO2C2H5, DMF, reflux, 4 h; (g) LAH,
ether, 2 h.

Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
6599
AChE. The role of a chlorine atom at para position
may be an anchor in the hydrophobic pocket of
AChE.23 The hydrophobic interaction between Trp279
and phenyl ring of rigid benzhydryl is more stable than
flexible benzoate phenyl ring. Compound 12 could
locate upside-down to the showed binding mode. We
performed energy minimizing (no data), however, the
result was more in preference of the suggested modeling
(Figs. 4 and 5). When the compound 12 is docked with
enzyme, the benzoate group could enter easier than the
bulky benzhydryl group. Current studies reported that
AChE is greatly reduced in specific brain regions, while
BChE increases in AD patients with severe pathology.
Recent evidence suggests that both AChE and BChE
may have roles in the etiology and progression of AD
beyond the regulation of synaptic acetylcholine levels.24
Therefore, the synthesized compounds were screened
against BChE. Compound 29 having carbamate group
(X = NH) presented the best BChE inhibitory potency
(IC50 0.11 lM). To further characterize the pharmaco-
logical profile of the synthesized compounds, their selec-
tivity to inhibit AChE versus BChE was determined.
The selectivity for AChE of compound 12 is similar to
donepezil and showed the best AChE inhibition and
selectivity. Considering the selectivity for BChE, com-
pound 29 showed the best BChE inhibition and is above
39-fold more potent than tacrine. The structural differ-
ences between compounds 12and 19 are that R is benz-
hydryl or benzyl and other functional group is ester or
carbamate. The peripheral anionic site (PAS) of AChE,
the subsite outside of the active site gorge, mediates sub-
strate inhibition of AChE.25 Three aromatic amino acids
located at the entrance to the active site gorge are the
key residues of this subsite. The PAS of BChE, which
is different from that of AChE, has weaker affinity than
AChE for the typical PAS ligands and mediates sub-
strate activation. The three aromatic residues of the
AChE PAS are missing in the PAS of BChE, which is
formed by two amino acid residues (Asp70 and
Tyr332) in the human enzyme.26 Recent evidence sug-
gested that rivastigmine with carbamate group has a
high BChE preference (selectivity 122 BChE, ref 26). Ex-
cept some compounds, many carbamates of benzofura-
ne, benzodioxepine, and physostigmine showed BChE
preference.27 Because compounds 12–20 have one more
aromatic moiety than compounds 21–30, their benzene
rings seem to play a key role of interaction with the three
aromatic residues of the AChE peripheral site and have
an AChE preference. Compounds 19, 20, and 29, 30
having carbamate group showed BChE inhibitory activ-
ity. Among them, compounds 29 and 30 showed high
BChE preference (selectivity >400 BChE).
O
OH
N
+
OH
R1
RO
R1
4-DMAP, EDCI
CHCl3, rt, 2h
O
N
O
RO
R = (C6H5)2CH
11 R1 = H
12 R1 = 4-Cl
13 R1 = 4-F
14 R1 = 4-NO2
R = C6H5CH2
21 R1 = H
22 R1 = 4-Cl
23 R1 = 4-F
24 R1 = 4-NO2
15 R1 = 4-tert-butyl 25 R1 = 4-tert-butyl
16 R1 = 4-isopropyl 26 R1 = 4-isopropyl
17 R1 = 3-Cl
18 R1 = 3-F
27 R1 = 3-Cl
28 R1 = 3-F
Scheme 2.
OH
NCO
N
+
RO
R2
H
N
O
CH3CN
N
O
reflux, 4h
RO
R2
R = (C6H5)2CH2
19 R2 = 4-Cl
20 R2 = 4-F
R = C6H5CH2
29 R2 = 4-Cl
30 R2 = 4-F
Scheme 3.
NMR spectroscopic data reported in the experimental
section.
2.2. AChE, BChE inhibition and selectivity for AChE or
BChE
To determine the therapeutic potency of the new piper-
idine derivatives (compounds 11–30) for the AD thera-
peutics, their anticholinesterase activities were assayed
according to Ellmann’s method22 against freshly pre-
pared AChE from Electrophorus electricus and human
BChE from plasma using tacrine and donepezil as refer-
ence compounds. Table 1 summarizes the data compar-
ing AChE and BChE inhibition as well as the selectivity
for AChE or BChE inhibitory activities from IC50 val-
ues for the new piperidine derivatives. From the IC50
values of compounds 11–30, it appears that variations
of aromatic phenyl group influenced AChE or BChE
activities. In particular, compounds 11–20 with benzhy-
dryl group (R) have shown AChE inhibitory activities,
while compounds 21–30 with benzyl group showed
BChE inhibitory activities. Compounds 12 and 13 pos-
sessing halogen group (R1 = 4-Cl, 4-F) at para position
showed best activities against AChE inhibition assay
and their IC50 values are 0.32 and 0.84 lM, respectively.
The meta substituted compounds 17 and 18 (R1 = 3-Cl,
3-F) showed less activity of about 1/10-fold than para
substituted compounds. Insertion of a chlorine atom
at para position seems to enhance binding affinity with
2.3. Molecular modeling of AChE and compound 12 with
donepezil
In order to gain further insight into the mechanism of
inhibition, we performed the docking study to generate
the binding model for compound 12 on the basis of
the existing X-ray crystal structure of AChE (PDB code:
1EVE).28 The proposed binding model of 12 with the
key residues in the gorge site is shown in Figure 4.
The binding model suggests the 4-chlorobenzene of 12

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Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
Table 1. Inhibition of AChE and BChE activities and Selectivity Ratios of the synthesized compounds
O
X
N
O
RO
R1
Compound
R
R1
X
AChE inhibitiona (lM, IC50
)
BChE inhibitiona (lM, IC50
)
Selectivityb
11
12
Benzhydryl
Benzhydryl
Benzhydryl
Benzhydryl
Benzhydryl
Benzhydryl
Benzhydryl
Benzhydryl
Benzhydryl
Benzhydryl
Benzyl
H
4-Cl
1.31 0.21
0.32 0.12
0.84 0.22
7.25 1.31
2.41 0.2
3.89 0.31
3.39 0.39
7.72 2.12
2.45 0.32
2.45 0.46
>100
31.55 3.53
38.4 3.72
22.19 1.85
26.83 1.92
78.92 5.44
23.21 2.73
57.87 6.23
35.88 3.1
13.65 1.2
15.67 1.21
1.89 0.56
3.52 0.89
0.56 0.21
5.23 0.78
15.23 2.01
10.79 1.86
1.35 0.35
1.58 0.54
0.25 0.05
0.78 0.25
0.03 0.01
10.05 1.72
24 AChE
120 AChE
26 AChE
5 AChE
13
14
4-F
4-NO2
4-tert-Butyl
4-Isopropyl
3-Cl
15
16
33 AChE
6 AChE
17
18
17 AChE
5 AChE
6 AChE
3-F
4-Cl
19
20
NH
NH
4-F
6 AChE
21
22
H
4-Cl
>53 BChE
>28 BChE
>179 BChE
>19 BChE
1.2 AChE
1.4 BChE
>74 BChE
>63 BChE
>400 BChE
>128 BChE
5 BChE
Benzyl
Benzyl
>100
>100
>100
23
24
4-F
Benzyl
Benzyl
4-NO2
4-tert-Butyl
4-Isopropyl
3-Cl
25
26
12.22 1.55
14.78 1.62
>100
Benzyl
Benzyl
27
28
Benzyl
Benzyl
Benzyl
3-F
4-Cl
>100
>100
29
30
NH
NH
4-F
>100
0.15 0.11
0.08 0.01
Tacrine
Donepezil
126 AChE
The activities of AChE and BChE inhibition were screened by Ellman’s method. IC50 is defined as the concentration of compounds that reduces by
50% of with respect to that without inhibitors. The dose–response curves have been fit by Probit analysis in StatsDirect statistical software (ver 2.5.5).
a Values are expressed as mean standard error of the mean of at least three experiments and the data showed proportional response with 95%
confidence intervals.
b Because a smaller IC50 represents a higher activity, the selectivity is defined as: selectivity for AChE = IC50(BChE)/IC50 (AChE), and selectivity for
BChE = IC50 (AChE)/IC50 (BChE).
˚
is bound to near the bottom of the gorge, and it shows
parallel p–p stacking against the six-membered ring of
Trp84 indole. The average distances between rings were
3.8 A. In the middle of the gorge, the constricted region,
the aliphatic alkyl chains of 12 were located as a linker
and surrounded with phenyl rings of Tyr121, Phe330,
Figure 4. Proposed binding model of compound 12 with AChE. 12 is
represented as a ball-and-stick model (purple) and other amino acid
residues are displayed as stick model. (Color of the chloride atom is
green, nitrogen is blue, hydrogen is white, and oxygen is red.)
Figure 5. The superposition of compound 12 (purple) and donepezil
(yellow) in the active site of AChE pocket.

Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
6601
Table 2. Inhibition of Ab1–42 aggregation without and with AChE by
the piperidine derivatives and reference compounds
and Tyr334. The nitrogen of piperidine ring could have
a hydrogen bond with Tyr121 OH with the distance
˚
3.1 A. The piperidine ring may have van der Waals con-
tact with Trp279 and Tyr334 (distances between the
Compound
Ab1–42 aggregation
inhibition by ThT
assaya (lM, IC50
Inhibition with AChE
0.02 Ub
˚
closest carbon atoms of 3.9 and 3.8 A). At the mouth
)
100 lMc (%)
1 lMd (%)
of the gorge, the benzene ring of benzhydryl group
showed the hydrophobic interaction with the residues
Trp279 and Leu282 (distances between the closest car-
11
12
40.33 3.53
28.21 3.12
36.33 4.1
53.14 5.26
20.02 1.84
76.86 6.4
91.96 10.2
33.72 3.23
37.32 2.8
113.64 8.9
105.32 9.53
>150
nde
55.5
41.8
nde
72.4
nde
nde
52.0
34.6
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
99.9
0
nde
42.4
28.4
nde
61.2
nde
nde
41.4
15.5
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
nde
99.5
0
13
14
˚
bon atoms of 3.5 and 4.1 A). In case of compound 22
possessing one benzene ring at the peripheral binding
site, the hydrophobic interaction with Trp279 and
Leu282 may be weak and have low potency, because
of flexibility of the benzyl group. We compared donepe-
zil (E2020) and 12 from the aspect of binding model and
they were shown to be similar. Especially, the location of
phenyl group to parallel p–p stacking against Trp84 was
similar to 12 and donepezil. The superposition of 12 and
donepezil is shown in Figure 5. In the chemical struc-
ture, both of 12 and donepezil have piperidine-nitrogen
atom, but different position of piperidine-nitrogen
showed the variation of potency. It seems likely that
the para-chloro group of compound 12plays an impor-
tant role in the activity. The distance between para-posi-
tion chlorine and hydrogen in Gly117 or hydrogen in
15
16
17
18
19
20
21
22
23
24
>150
>150
25
26
78.5 5.7
85.92 6.9
69.4 6.75
74.94 7.4
>150
27
28
29
30
>150
Congo-red
Tacrine
Donepezil
1.65 0.2
>150
86.5 7.87
˚
Tyr130 is 2.5 and 3.1 A, respectively. Therefore, the
para-chlorine and hydrogen can take place hydrogen
bonding. The evidences of halogen and hydrogen bond-
ing were found in a lot of research results.29 The meta-
chloro substituent has no amino acids for hydrogen
bonding nearby. Because the pocket of AChE is in the
shape of a deep pitfall, 12 could locate from entrance
to bottom gorge. Compound 12 was about 37-fold less
potent than donepezil, because the piperidine-nitrogen
of 12 (unlike donepezil which showed quaternary-p
0
0
a Each value of Ab1–42 aggregation inhibition was from Thioflavin T
(ThT) assay using fluorospectrometer. The dose–response curves
have been fit by Probit analysis in StatsDirect statistical software (ver
2.5.5). The values are expressed as means standard error of the
mean at least three experiments and the data showed proportional
response with 95% confidence intervals.
b Co-aggregation inhibition of Ab1–42 and AChE 0.02 U was detected
by ThT assay.
˚
c The data (%) showed that the test compounds inhibited the co-
aggregation at 100 lM.
interaction with Phe330) was the about 4.6 A distance
from the piperidine-nitrogen of donepezil towards the
mouth of the gorge. The backbone length of 12 is longer
than donepezil. Compound 12 and donepezil have the
d The data (%) showed that the test compounds inhibited the co-
aggregation at 1 lM.
˚
e nd, not determined.
length of 18.9 and 15.6 A, respectively. It showed that
12 had enough length to cover the peripheral site as
˚
the active site gorge is about 20 A deep from the mouth
of gorge.29 The benzhydryl moiety of 12 also had en-
ough size and shape to cover the peripheral site, which
was the funnel-like entrance to the gorge. The peripheral
site at the mouth of the gorge has been recently postu-
lated to regulate the Ab assembly.30 The result of the
Ab aggregation inhibition assay showed that 12 had
good inhibitory potency. This might be due to the size
differences between the benzhydryl of 12 and the
dimethoxyindanone of donepezil. Thus, we suggest com-
pound 12 could be a ‘dual action’ inhibitor.
compound. In the screening results of ThT assay for
Ab1–42 aggregation inhibition, the most effective com-
pound was 15, followed by 12, 18, 13, and 19, and their
IC50 values (lM) were 20.02 1.84, 28.21 3.12,
33.72 3.23, 36.33 4.1, and 37.32 2.8, respectively.
To further explore the dual action of these compounds,
AChE-induced Ab1–42 aggregation inhibitory activity
was examined employing the same ThT-based fluoro-
metric assay.30,32 In the co-aggregation study, com-
pounds 15and 12 were more effective than tacrine or
donepezil. Considering the results, two aromatic rings,
benzhydryl group of the synthesized compounds, could
play a major role against Ab1–42 peptide aggregation
inhibition. A number of inhibitors of Ab fibril formation
have been described that prevent the formation of a bio-
logically active species of peptide. Short peptide frag-
ments, based primarily on the central hydrophobic
region of Ab, have been proposed as being capable of
preventing b-sheet formation.33,34 SKF-74652, a repre-
sentative benzofuran analogue, and 9-acridone deriva-
tives are known as potent Ab aggregation inhibitors
(Fig. 2).35 Because of their hydrophobic regions and
2.4. Effects on the Ab1–42 peptide aggregation without and
with AChE
To determine the amyloid-beta(1–42) aggregation inhi-
bition of the new piperidine derivatives (compounds
11–30), thioflavinT (ThT) assay was performed compar-
ing with Congo-red, tacrine and donepezil as reference
compound.30,31 Table 2 summarizes the data for effects
on the Ab1–42 peptide aggregation from IC50 values
and effects on the co-aggregation of Ab1–42 with AChE
from percent (%) inhibition at 100 and 1 lM of each

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Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
hydrogen bond acceptors, they are capable of multiple
binding for inhibition on the Ab peptide. Other studies
suggested the role of AChE not only in the hydrolysis
of neurotransmitter ACh, but also in accelerating the
aggregation of Ab into amyloid fibrils (AChE-induced
Ab-fibrillogenesis).32,36 In the recent study, the benzyle-
thers of pyridinium-type compounds showed high activ-
ity against AChE and Ab aggregation dual inhibition.37
Although the benzofuran, the benzylether and the piper-
idine derivatives are not of the same structure, they have
common hydrophobic regions, hydrogen bond accep-
tors, and the proper molecular length. These structural
characters could give effect for multiple binding on the
Ab peptide and a further study using pharmacophore
mapping and virtual screening is needed.
gen bond acceptor may be having neuroprotective effect.
Because compound 13 has a strong electron-withdraw-
ing fluoro group, it is likely to act as a cytotoxic factor.
The exact reason for the neuroprotective effect is un-
known; therefore its mechanism needs further study.
3. Conclusion
We have synthesized new piperidine derivatives having
dual inhibitory potency of AChE and Ab1–42 peptide
aggregation. Compound 12 displayed the most inhibi-
tory potency against AChE (IC50 = 0.32 lM) and selec-
tivity (AChE relative to BChE) of 120 times. This
contains a benzhydryl group as a peripheral site interac-
tion unit and a phenyl ring as a catalytic site binding
unit, and the two units are connected with a piperidine
and alkyl chains as a linker. In the docking model,
4-chlorobenzene of 12 showed parallel p–p stacking
against the indole ring of Trp84 in the bottom gorge
of AChE. 29 and 30 showed high selectivity for BChE
and had structure of carbamate with benzylpiperidinyl
ether. Unlike AChE, the physiological function of BChE
is still unclear. Therefore, the selective BChE inhibitor
may be an interesting compound for the new AD thera-
peutic area. Compounds 12 and 15 showed good inhibi-
tion effects on Ab1–42 peptide oligomerization and the
AChE-induced aggregation. In the molecular modeling,
compound 12 had enough length to cover the mouth
gorge of AChE and the benzyhydryl moiety of 12 cov-
ered the entrance to the gorge of the peripheral site in
a funnel-like shape. Therefore, 12 and 15 could inhibit
the AChE-induced Ab1–42 peptide aggregation. The ob-
tained results are valuable for further study focusing on
investigating the dual inhibitor for Alzheimer’s disease
therapeutics.
2.5. Neuroprotective effects on the IMR-32 cell line
The cytotoxic effect of the new piperidine derivatives
was screened in human neuroblastoma cell line, IMR-
32.32,38 Table 3 represents cell viability (%) by treatment
of the synthesized compounds comparing with control
cells. Control cells were treated with Ab1–42 peptide
only. When the viability of control cells became about
50% compared with standard cells (untreated peptide
or inhibitors), the viability of compound treated cells
was determined by MTT assay. The most neuroprotec-
tive compound was 12, followed by 15, donepezil, and
11. For tacrine and 13, cytotoxicity was found at
0.1 lmol/mL against IMR-32 cells. It is well known that
tacrine has hepatotoxicity and cytotoxicity against hepa-
tocellular carcinoma, HepG2, however, donepezil has
lesser toxicity in vivo and in vitro.39,40 In this study, ta-
crine has little neuroprotective effect (42.0%) against
Ab1–42 peptide treated human neuroblastoma cells at
similar level with control cells (49.1%) in vitro. On the
other hand, compounds 12 (90.5%), 15 (85.2%), and
donepezil (82.6%) showed good neuroprotective effect.
The cytotoxic mechanism of tacrine is not clear, but it
is reported that tacrine and some polycyclic aromatic
compounds may be inactivating CYP1A enzymes and
inhibiting drug metabolism pathway and the increased
toxicity is NADPH dependent.41 The piperidine deriva-
tives aren’t seem to be related with CYP1A enzymes by
the reason that the compounds are not polycyclic com-
pounds. Their hydrophobic aromatic group and hydro-
4. Experimental
4.1. Chemistry
Reaction progress was monitored using analytical thin-
layer chromatography (TLC) on precoated Merck silica
gel Kiesegel 60 F254 plates and the spots were detected
under UV light (254 nm). The flash chromatography
was conducted using silica gel 230–400 mesh. IR spectra
were measured on a Jasco FT/IR-430 spectrophotome-
ter. 1H and 13C NMR spectra were recorded at
300 MHz on a Bruker ARX 300 spectrometer. The
chemical shifts are reported downfield in ppm (d) rela-
tive to internal TMS, and coupling constants are re-
ported in Hertz (Hz). Mass spectra analysis was
performed on a Quattro micro MS Micromass UK mass
spectrometer and recorded on an electrospray ionization
mass spectrometer as the value m/z. All chemicals and
solvents were purchased from Sigma–Aldrich.
Table 3. Neuroprotective effects on the neuron cell line, IMR-32
in vitro
Compound
Cell viability (0.1 lmol/ml) (%)
Control
11
49.1a
80.5b
90.5
36.3
85.2
56.5
42.0
82.6
12
13
15
19
Tacrine
Donepezil
4.2. tert-Butyl 4-hydroxypiperidine-1-carboxylate (2)
a Control group was treated with Ab1–42 peptide only.
b Test group was treated with Ab1–42 plus the synthesized compound or
reference drugs on 0.1 lmol/mL concentration. The neuroprotective
effect against neuron cells was determined by MTT assay.
4-Hydroxypiperidine 1 (10.0 g, 98.86 mmol) was dis-
solved in chloroform (150 mL) and cooled at 0 ꢁC. Tri-
ethylamine (16.5 mL, 118.63 mmol) was added above

Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
6603
the solution slowly and stirred for 1 h at room tempera-
ture with di-tert-butyl dicarbonate (DIBOC, 25.8 g,
118.63 mmol). The reaction was completed with water
and the resultant solution was acidified by concentrated
HCl in ice bath and extracted with chloroform. The
chloroform layer was dried with anhydrous Na2SO4
and evaporated under reduced pressure. The flash chro-
matography was performed and obtained brown-yel-
lowish solid product 2 (yield 83%). Mp: 56–59 ꢁC. IR
(m, 2H), 3.31–3.42 (m, 2H), 3.45 (m, 1H), 5.24 (s, 1H),
7.28–7.36 (m, 5H).
4.5. General procedure for the preparation of derivatives 7
and 8
To 12–28 mL of N,N-dimethylformamide (DMF) were
added compound 5 or 6 (1.0 g, 3.74 mmol), NaHCO3
(1.42 g), potassium iodide (catalyst), and ethyl-4-chlo-
robutyrate (5.61 or 7.84 mmol). The mixture was re-
fluxed for 4 h and cooled down to room temperature.
The resulting solution was extracted with chloroform
and washed with water. The organic layer was dried
over anhydrous Na2SO4 and evaporated under reduced
pressure. The flash chromatography was performed and
obtained liquid product.
(KBr): 3485, 2934, 1671, 1434, 1170 cmꢀ1 1H NMR
.
(CDCl3): d 1.52 (s, 9H), 1.81–1.91 (m, 4H), 2.95–3.08
(m, 2H), 3.83–3.89 (m, 3H). 13C NMR (CDCl3): d
28.3, 34.1, 41.2, 67.6, 79.5, 154.7.
4.3. General procedure for the preparation of derivatives 3
and 4
To 90 mL of 1,4-dioxane were added compound 2 (9.7 g,
26.4 mmol), Na2CO3 (3.6 g, 34.38 mmol), KI (catalyst)
and diphenylbromomethane (8.5 g, 34.28 mmol), or
benzylbromide (1.3 equiv, 34.28 mmol). The mixture
was refluxed for 10 h and then the resultant was evapo-
rated. The concentrated solution was poured into water
and extracted with methylene chloride. The methylene
chloride layer was dried with anhydrous Na2SO4 and
evaporated under reduced pressure. The flash chroma-
tography was performed and obtained liquid products.
4.5.1. Ethyl-4-[4-(benzhydryloxy)piperidino]butanoate (7).
Yield 66%, yellowish liquid, H NMR (CDCl3): d 1.26
1
(t, J = 7.2 Hz, 3H), 1.77–1.93 (m, 6H), 2.05–2.12 (m,
2H), 2.28–2.33 (m, 4H), 2.69–2.77 (m, 2H), 3.38–3.46
(m, 1H), 4.14 (q, J = 7.2 Hz, 2H), 5.53 (s, 1H), 7.23–
7.37 (m, 10H). 13C NMR (CDCl3): d 14.2, 22.1, 22.4,
27.6, 27.7, 31.0, 31.3, 32.3, 51.0, 57.6, 60.2, 60.4, 62.0,
72.5, 68.4, 79.9, 127.0, 127.2, 128.2, 142.8, 173.5,
177.6; ESI-MS: 382.2 (M+1).
4.5.2. Ethyl 4-[4-(benzyloxy)piperidino]butanoate (8).
1
4.3.1. tert-Butyl 4-(benzhydryloxy)piperidine-1-carboxyl-
ate (3). Yield 72%, brownish liquid, IR (NaCl): 2941,
Yield 82%, yellowish liquid, H NMR (CDCl3): d 1.22
(t, J = 7.2 Hz, 3H), 1.67–1.91 (m, 6H), 2.09–2.19 (m,
2H), 2.27–2.36 (m, 4H), 2.72–2.78 (m, 2H), 3.38–3.42
(m, 1H), 4.10 (q, J = 7.2 Hz, 2H), 4.51 (s, 2H), 7.23–
7.34 (m, 5H). 13C NMR (CDCl3): d 14.1, 22.4, 31.2,
32.3, 51.1, 57.5, 60.1, 69.5, 127.3, 128.2, 138.9, 173.5;
ESI-MS: 306.2 (M+1).
1
1732, 1492, 1452, 1180, 1071 cmꢀ1. H NMR (CDCl3):
d 1.53 (s, 9H), 1.57–1.86 (m, 2H), 3.04–3.17 (m, 2H),
3.53–3.61 (m, 1H), 3.67–3.79 (m, 2H), 5.51 (s, 1H),
7.18–7.36 (m, 10H). 13C NMR (CDCl3): d 28.3, 31.0,
71.8, 80.1, 126.8, 126.9, 127.2, 128.2, 142.5, 184.0.
4.3.2. tert-Butyl 4-(benzyloxy)piperidine-1-carboxylate
(4). Yield 82%, yellowish liquid, IR (NaCl): 3125,
4.6. General procedure for the preparation of derivatives 9
and 10
1750, 1510, 1180, 1060 cmꢀ1 1H NMR (CDCl3): d
.
1.53 (s, 9H), 1.61–1.82 (m, 2H), 3.04–3.17 (m, 2H),
3.58 (m, 1H), 3.67–3.79 (m, 2H), 5.51 (s, 1H), 7.21–
7.38 (m, 5H).
The compound 7 or 8 (1.0 g, 2.62 or 3.27 mmol) was dis-
solved in 10 mL of ether and cooled in an ice bath.
LiAlH4 (0.2 or 0.25 g, 5.24 or 6.54 mmol) was added
to the solution slowly. The mixture was stirred at room
temperature for 2 h and added water to end the reaction.
The resulting solution was then extracted with ether and
washed with water. The organic layer was dried over
magnesium sulfate and evaporated under reduced pres-
sure. The flash chromatography was performed and ob-
tained liquid product.
4.4. General procedure for the preparation of derivatives 5
and 6
To the dried isopropyl alcohol was added compound 3
or 4 (1 equiv) and KOH (10 equiv). The mixture was re-
fluxed for 14 h, cooled down to room temperature, and
distilled water was added for ending the reaction. The
resultant was extracted with chloroform and evaporated
under reduced pressure. The flash chromatography was
performed and obtained liquid product.
4.6.1. 4-[4-(Benzhydryloxy)piperidino]-1-butanol (9).
Yield 43%, yellowish liquid, 1H NMR (CDCl3): d
1.68–1.77 (m, 4H), 1.78–1.94 (m, 4H), 2.27–2.38 (m,
4H), 2.76–2.79 (m, 2H), 3.45–3.58 (m, 3H), 5.51 (s,
1H), 7.23–7.36 (m, 10H). 13C NMR (CDCl3): d 25.7,
30.5, 32.6, 58.4, 62.6, 80.1, 127.0, 127.2, 128.2, 142.7;
ESI-MS: 340.2 (M+1).
4.4.1. 4-Benzhydryloxypiperidine (5). Yield 70%, yellow-
ish liquid, 1H NMR (CDCl3): d 1.49–1.62 (m, 2H), 1.88–
1.96 (m, 2H), 2.48–2.62 (m, 2H), 3.04–3.12 (m, 2H),
3.45–3.49 (m, 2H), 5.54 (s, 1H), 7.21–7.40 (m, 10H).
13C NMR (CDCl3): d 33.06, 44.3, 73.0, 79.8, 127.0,
127.2, 128.2, 142.7; ESI-MS: 268.1 (M+1).
4.6.2. 4-[4-(Benzyloxy)piperidino]-1-butanol (10). Yield
1
83%, yellowish liquid, H NMR (CDCl3): d 1.62–1.76
(m, 6H), 1.84–1.91 (m, 2H), 2.21–2.31 (m, 4H), 2.70–
2.76 (m, 2H), 3.42–3.56 (m, 3H), 4.48 (s, 2H), 7.19–
7.30 (m, 5H). 13C NMR (CDCl3): d 25.8, 30.4, 32.7,
4.4.2. 4-Benzyloxypiperidine (6). Yield 80%, yellowish li-
quid, 1H NMR (CDCl3): d 1.65–2.05 (m, 4H), 3.04–3.12

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Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
50.4, 50.5, 58.4, 62.6, 69.9, 127.3, 128.3, 138.8; ESI-MS:
266.1 (M+1).
(CDCl3): d 1.41 (s, 9H), 1.72–1.86 (m, 8H), 2.05–2.15
(m, 2H), 2.34 (t, J = 7.4 Hz, 2H), 2.71–2.77 (m, 2H),
3.39–3.47 (m, 1H), 4.3 (t, J = 6.4 Hz, 2H), 5.51 (s, 1H),
7.21–7.37 (m, 10H), 7.42 (d, J = 8.3 Hz, 2H), 7.96 (d,
J = 8.3 Hz, 2H). 13C NMR (CDCl3): d 23.7, 26.8, 31.0,
31.3, 35.0, 51.1, 58.1, 64.6, 79.9, 125.2, 127.0, 127.2,
127.6, 128.2, 129.3, 142.8, 165.7; ESI-MS: 500.3 (M+1).
4.7. General procedure for the preparation of derivatives
11–18 and 21–28
To 15 mL of chloroform was added compound 9 or 10
(0.619 or 1.135 mmol), each of benzoic acid derivatives
(1.5 equiv), 4-dimethylaminopyridine (3 equiv), and 1-
4.7.6. 4-[4-(Benzhydryloxy)piperidino]butyl 4-isopropyl-
benzoate (16). Yield 66%, yellowish liquid, 1H NMR
(CDCl3): d 1.24 (d, J = 7.2 Hz, 6H), 1.58–1.86 (m,
8H), 2.04–2.18 (m, 2H), 2.34 (t, J = 7.6 Hz, 2H), 2.72–
2.78 (m, 2H), 2.90–2.97 (m, 1H), 3.46–3.40 (m, 1H),
4.28 (t, J = 6.4 Hz, 2H), 5.50 (s, 1H), 7.19–7.38 (m,
12H), 7.93 (d, J = 8.4 Hz, 2H). 13C NMR (CDCl3): d
23.6, 23.7, 26.7, 31.3, 34.1, 40.8, 51.0, 58.0, 64.5, 72.4,
89.9, 126.3, 126.9, 127.1, 127.9, 128.1, 129.5, 142.7,
154.1, 166.5; ESI-MS: 486.2 (M+1).
[3-(dimethylamino)propyl]-3-ethylcarboimide
hydro-
chloric acid (1.1 equiv). The mixture was allowed to
react at room temperature for 2 h and added water to
end the reaction. The resulting solution was acidified
with 1 N HCl and extracted using chloroform. An
organic layer was washed with 1 N NaOH, dried over
magnesium sulfate, and evaporated under reduced
pressure. The flash chromatography was performed
and obtained each product.
4.7.1. 4-[4-(Benzhydryloxy)piperidino]butyl benzoate (11).
Yield 63%, yellowish liquid, H NMR (CDCl3): d 1.67–
4.7.7. 4-[4-(Benzhydryloxy)piperidino]butyl 3-chloroben-
zoate (17). Yield 33%, yellowish liquid, 1H NMR
(CDCl3): d 1.57–1.92 (m, 8H), 2.10–2.18 (m, 2H), 2.34
(t, J = 7.4 Hz, 2H), 2.73–2.79 (m, 2H), 3.41–3.48 (m,
1H), 4.31 (t, J = 6.4 Hz, 2H), 5.50 (s, 1H), 7.20–7.39
(m, 11H), 7.50 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 7.6 Hz,
1H), 7.98 (s, 1H). 13C NMR (CDCl3): d 23.7, 26.7,
31.3, 51.1, 58.0, 65.2, 72.3, 79.9, 116.2, 116.5, 119.7,
119.9, 125.2, 127.0, 127.2, 127.6, 128.2, 129.5, 129.6,
132.1, 132.8, 134.4, 142.8, 165.3; ESI-MS: 478.2 (M+1).
1
1.91 (m, 8H), 2.12–2.20 (m, 2H), 2.40 (t, J = 7.4 Hz,
2H), 2.77–2.80 (m, 2H), 3.44–3.50 (m, 1H), 4.35 (t,
J = 6.4 Hz, 2H), 5.54 (s, 1H), 7.25–7.37 (m, 10H),
7.42–7.47 (m, 2H), 7.54–7.57 (m, 1H), 8.05 (d,
J = 7.2 Hz, 2H). 13C NMR (CDCl3): d 23.7, 26.8, 31.2,
51.0, 58.1, 64.8, 79.9, 127.0, 127.2, 128.2, 129.5, 130.3,
132.8, 142.7; ESI-MS: 444.2 (M+1).
4.7.2. 4-[4-(Benzhydryloxy)piperidino]butyl-4-chlorobenzo-
ate (12). Yield 71%, yellowish liquid, 1H NMR (CDCl3):
d 1.62–1.90 (m, 8H), 2.14–2.22 (m, 2H), 2.39 (t,
J = 7.4 Hz, 2H), 2.74 (m, 2H), 3.45–3.51 (m, 1H), 4.3
(t, J = 6.3 Hz, 2H), 5.53 (s, 1H), 7.27–7.37 (m, 10H),
7.41 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 8.5 Hz, 2H). 13C
NMR (CDCl3): d 23.7, 26.7, 31.3, 51.1, 58.1, 65.1,
80.0, 127.2, 127.6, 128.2, 128.6, 128.8, 130.9, 139.2,
142.8, 165.7; ESI-MS: 478.2 (M+1).
4.7.8. 4-[4-(Benzhydryloxy)piperidino]butyl 3-fluoroben-
zoate (18). Yield 14%, yellowish liquid, 1H NMR
(CDCl3): d 1.66–1.92 (m, 8H), 2.17–2.24 (m, 2H), 2.40
(t, J = 7.2 Hz, 2H), 2.76–2.82 (m, 2H), 3.44–3.49 (m,
1H), 4.35 (t, J = 6.4 Hz, 2H), 5.53 (s, 1H), 7.23–7.43
(m, 12H), 7.72(d, J = 7.7 Hz, 1H), 7.84 (d, J = 7.7 Hz,
1H).13C NMR (CDCl3): d 23.7, 26.7, 31.3, 51.1, 58.0,
65.2, 80.0, 116.2, 116.5, 119.7, 120.0, 125.2, 127.0,
127.2, 128.2, 129.8, 129.9, 132.5, 132.6, 142.8, 160.8,
165.4; ESI-MS: 462.2 (M+1).
4.7.3. 4-[4-(Benzhydryloxy)piperidino]butyl 4-fluorobenzo-
ate (13). Yield 74%, yellowish liquid, 1H NMR (CDCl3):
d 1.61–1.89 (m, 8H), 2.11–2.14 (m, 2H), 2.34 (t,
J = 7.4 Hz, 2H), 2.69–2.77 (m, 2H), 3.38–3.45 (m, 1H),
4.29 (t, J = 6.4 Hz, 2H), 5.50 (s, 1H), 7.07 (dd, J = 8.9,
8.3 Hz, 2H), 7.21–7.39 (m, 10H), 8.02 (dd, J = 8.9,
5.5 Hz, 2H). 13C NMR (CDCl3): d 23.7, 26.7, 31.3,
51.1, 58.0, 64.9, 79.9, 115.2, 115.5, 126.5, 127.0, 127.2,
128.2, 131.9, 132.0, 142.8, 163.9, 165.5; 167.2; ESI-MS:
462.2 (M+1).
4.7.9. 4-[4-(Benzyloxy)piperidino]butyl benzoate (21).
Yield 69%, yellowish liquid, 1H NMR (CDCl3): d
1.58–1.79 (m, 6H), 1.88–1.96 (m, 2H), 2.08–2.16 (m,
2H), 2.35 (t, J = 7.4 Hz, 2H), 2.74–2.78 (m, 2H), 3.36–
3.44 (m, 1H), 4.30 (t, J = 6.4 Hz, 2H), 4.51 (s, 2H),
7.21–7.33 (m, 5H), 7.38–7.45 (m, 2H), 7.50–7.54 (m,
1H), 8.01 (d, J = 9.6 Hz, 2H).13C NMR (CDCl3): d
23.8, 26.8, 31.2, 51.1, 58.1, 64.8, 69.6, 127.4, 128.3,
129.5, 130.3, 132.8, 138.9, 166.6; ESI-MS: 368.2 (M+1).
4.7.4. 4-[4-(Benzhydryloxy)piperidino]butyl-4-nitrobenzo-
ate (14). Yield 76%, yellowish liquid, 1H NMR (CDCl3):
d 1.65–1.89 (m, 8H), 2.06–2.16 (m, 2H), 2.39 (t,
J = 7.4 Hz, 2H), 2.74–2.77 (m, 2H), 3.45–3.48 (m, 1H),
4.40 (t, J = 6.4 Hz, 2H), 5.53 (s, 1H), 7.23–7.37 (m,
12H), 8.21 (d, J = 8.8 Hz, 2H), 8.29 (d, J = 8.7 Hz,
2H). 13C NMR (CDCl3): d 23.6, 26.6, 31.2, 51.0, 57.9,
65.7, 72.3, 80.0, 126.5, 122.7, 123.4, 126.7, 127.0,
127.2, 127.8, 128.2, 128.5, 129.9, 130.6, 135.7, 142.7,
150.4, 164.6; ESI-MS: 489.2 (M+1).
4.7.10. 4-[4-(Benzyloxy)piperidino]butyl-4-chlorobenzoate
(22). Yield 61%, yellowish liquid, H NMR (CDCl3): d
1
1.68–1.94 (m, 8H), 2.15–2.22 (m, 2H), 2.40 (t,
J = 7.4 Hz, 2H), 2.78–2.84 (m, 2H), 3.42–3.46 (m, 1H),
4.34 (t, J = 6.4 Hz, 2H), 4.56 (s, 2H), 7.28–7.37 (m,
5H), 7.42 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H).
13C NMR (CDCl3): d 22.2, 23.7, 25.6, 26.8, 31.2, 51.1,
58.1, 65.1, 69.6, 127.3, 127.4, 128.3, 128.6, 128.8,
130.9, 133.6, 138.9, 139.2; ESI-MS: 402.1 (M+1).
4.7.5. 4-[4-(Benzhydryloxy)piperidino]butyl-4-(tert-butyl)ben-
zoate (15). Yield 96%, yellowish liquid, 1H NMR
4.7.11. 4-[4-(Benzyloxy)piperidino]butyl-4-fluorobenzoate
(23). Yield 63%, yellowish liquid, H NMR (CDCl3): d
1

Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
6605
1.61–1.78 (m, 6H), 1.84–1.90 (m, 2H), 2.10–2.16 (m,
2H), 2.34 (t, J = 7.6 Hz, 2H), 2.71–2.78 (m, 2H), 3.38–
3.44 (m, 1H), 4.29 (t, J = 6.8 Hz, 2H), 4.51 (s, 2H),
7.07 (dd, J = 8.7, 8.4 Hz, 2H), 7.21–7.34 (m, 5H), 8.02
(dd, J = 8.9, 8.7 Hz, 2H).13C NMR (CDCl3): d 23.7,
26.8, 31.2, 51.1, 58.1, 65.0, 69.6, 74.3, 115.2, 115.5,
126.5, 126.6, 127.4, 128.3, 131.9, 138.9, 164.0, 165.6,
167.3, 184.9; ESI-MS: 386.2 (M+1).
4.8. General procedure for the preparation of derivatives
19–20 and 29–30
To 25 mL of acetonitrile was added compound 9 or 10
(1.945 or 2.658 mmol), 4-chloro- or fluoro-phenyl isocy-
anate (1.2 equiv). The mixture was refluxed for 4 h,
cooled, and evaporated under reduced pressure. The
resulting solution was then extracted by using water
and chloroform. An organic layer was dried with mag-
nesium sulfate, and evaporated under reduced pressure.
The flash chromatography was performed and obtained
product.
4.7.12. 4-[4-(Benzyloxy)piperidino]butyl-4-nitrobenzoate
1
(24). Yield 64%, yellowish liquid, H NMR (CDCl3): d
1.64–1.91 (m, 8H), 2.12–2.18 (m, 2H), 2.40 (t,
J = 7.4 Hz, 2H), 2.76–2.82 (m, 2H), 3.42–3.48 (m, 1H),
4.40 (t, J = 6.4 Hz, 2H), 4.55 (s, 2H), 7.27–7.36 (m,
5H), 8.21 (d, J = 8.6 Hz, 2H), 8.29 (d, J = 8.6 Hz, 2H).
13C NMR (CDCl3): d 23.7, 26.7, 31.2, 51.1, 58.0, 65.8,
69.6, 123.4, 127.4, 128.3, 130.6, 135.7, 138.8, 150.4,
164.6, 184.8; ESI-MS: 413.2 (M+1).
4.8.1. 4-[4-(Benzhydryloxy)piperidino]butyl-N-(4-chloro-
phenyl)carbamate (19). Yield 24%, yellowish liquid, H
1
NMR (CDCl3): d 1.68–1.94 (m, 6H), 2.04–2.12 (m,
2H), 2.22–2.28 (m, 2H), 2.43 (t, J = 7.2 Hz, 2H), 2.76–
2.82 (m, 2H), 3.47–3.51 (m, 1H), 4.18 (t, J = 5.4 Hz,
2H), 5.52 (s, 1H), 6.85 (s, 1H), 7.24–7.43 (m, 14H).
13C NMR (CDCl3): d 20.9, 26.4, 26.5, 30.6, 57.5, 57.8,
60.7, 64.0, 64.6, 80.2, 126.9, 127.0, 127.3, 128.3, 128.9,
136.6, 142.5, 142.4, 142.6, 152.0, 153.4, 171.1; ESI-MS:
493.2 (M+1).
4.7.13.
4-[4-(Benzyloxy)piperidino]butyl-4-(tert-butyl)-
benzoate (25). Yield 76%, yellowish liquid, 1H NMR
(CDCl3): d 1.32 (s, 9H), 1.64–1.78 (m, 6H), 1.86–1.92
(m, 2H), 2.08–2.14 (m, 2H), 2.35 (t, J = 7.4 Hz, 2H),
2.72–2.77 (m, 2H), 3.37–3.41 (m, 1H), 4.29 (t,
J = 6.4 Hz, 2H), 4.51 (s, 2H), 7.23–7.36 (m, 5H), 7.42
(d, J = 8.0 Hz, 2H), 7.94 (d, J = 8.0 Hz, 2H). 13C
NMR (CDCl3): d 23.7, 26.8, 31.1, 31.2, 35.0, 51.2,
57.4, 58.1, 64.6, 69.6, 125.2, 127.3, 127.4, 127.6, 128.3,
129.3, 138.9, 156.4, 166.6; ESI-MS: 424.2 (M+1).
4.8.2. 4-[4-(Benzhydryloxy)piperidino]butyl-N-(4-fluoro-
phenyl)carbamate (20). Yield 24%, yellowish liquid, H
1
NMR (CDCl3): d 1.66–1.92 (m, 6H), 1.81–1.97 (m,
2H), 2.09–2.16 (m, 2H), 2.31 (t, J = 6.1 Hz, 2H), 2.74–
2.82 (m, 2H), 3.39–3.42 (m, 1H), 4.12 (t, J = 5.6 Hz,
2H), 5.49 (s, 1H), 6.67 (s, 1H), 6.98 (t, J = 8.8 Hz,
2H), 7.14–7.25 (m, 12H). 13C NMR (CDCl3): d 22.6,
26.5, 29.6, 30.2, 50.3, 57.6, 64.1, 64.5, 80.2, 115.3,
115.6, 120.2, 126.9, 127.3, 128.2, 128.3, 133.9, 138.6,
142.5, 142.6, 153.7, 171.1; ESI-MS: 477.2 (M+1).
4.7.14. 4-[4-(Benzyloxy)piperidino]butyl-4-isopropylben-
zoate (26). Yield 64%, yellowish liquid, 1H NMR
(CDCl3): d 1.24 (d, J = 7.3 Hz, 6H), 1.63–1.77 (m,
6H), 1.80–1.94 (m, 2H), 2.10–2.14 (m, 2H), 2.39 (t,
J = 7.4 Hz, 2H), 2.76–2.81 (m, 2H), 2.90–2.97 (m, 1H),
3.38–3.44 (m, 1H), 4.33 (t, J = 6.4 Hz, 2H), 4.56 (s,
2H), 7.29–7.37 (m, 7H), 7.94 (d, J = 7.9 Hz, 2H). 13C
NMR (CDCl3): d 23.6, 23.7, 26.8, 31.2, 34.2, 51.2,
58.1, 64.6, 69.6, 126.4, 127.3, 127.4, 128.0, 128.3,
129.6, 138.9, 154.2, 166.6; ESI-MS: 410.2 (M+1).
4.8.3. 4-[4-(Benzyloxy)piperidino]butyl-N-(4-chlorophe-
nyl)carbamate (29). Yield 61%, yellowish liquid, 1H
NMR (CDCl3): d 1.53–1.69 (m, 6H), 1.89–1.99 (m,
2H), 2.08–2.16 (m, 2H), 2.32 (t, J = 7.2 Hz, 2H), 2.68–
2.74 (m, 2H), 3.38–3.43 (m, 1H), 4.14 (t, J = 6.1 Hz,
2H), 4.51 (s, 2H), 6.97 (s, 1H), 7.21–7.32 (m, 9H).13C
NMR (CDCl3): d 23.2, 26.8, 30.8, 50.8, 57.7, 64.9,
69.6, 73.8, 119.8, 127.4, 128.0, 128.1, 128.3, 128.9,
136.5, 138.7, 151.2, 153.4, 174.6, 184.9; ESI-MS: 417.1
(M+1).
4.7.15. 4-[4-(Benzyloxy)piperidino]butyl-3-chlorobenzoate
1
(27). Yield 47%, yellowish liquid, H NMR (CDCl3): d
1.57–1.77 (m, 6H), 1.89–1.96 (m, 2H), 2.08–2.14 (m,
2H), 2.31 (t, J = 7.4 Hz, 2H), 2.71–2.78 (m, 2H), 3.36–
3.42 (m, 1H), 4.30 (t, J = 6.4 Hz, 2H), 4.51 (s, 2H),
7.21–7.38 (m, 6H), 7.48 (d, J = 8.0 Hz, 1H), 7.86 (d,
J = 7.6 Hz, 1H), 7.94 (s, 1H). 13C NMR (CDCl3): d
23.7, 26.7, 31.2, 51.1, 58.0, 65.3, 67.3, 67.4, 69.6, 74.3,
127.3, 127.4, 127.6, 128.3, 129.6, 132.1, 132.8, 134.4,
138.9, 165.3; ESI-MS: 402.1 (M+1).
4.8.4. 4-[4-(Benzyloxy)piperidino]butyl-N-(4-fluorophe-
nyl)carbamate (30). Yield 48%, yellowish liquid, 1H
NMR (CDCl3): d 1.59–1.71 (m, 6H), 1.88–1.94 (m,
2H), 2.12–2.18 (m, 2H), 2.36 (t, J = 7.2 Hz, 2H), 2.78–
2.88 (m, 2H), 3.42–3.48 (m, 1H), 4.18 (t, J = 6.1 Hz,
2H), 4.56 (s, 2H), 7.02 (t, J = 8.6 Hz, 2H); 7.28–7.36
(m, 8H). 13C NMR (CDCl3): d 23.1, 26.8, 30.8, 50.8,
53.3, 57.7, 64.7, 69.5, 73.8, 73.9, 115.2, 115.5, 120.3,
127.3, 128.1, 133.9, 138.6, 153.8, 157.1, 160.3, 184.5;
ESI-MS: 401.2 (M+1).
4.7.16. 4-[4-(Benzyloxy)piperidino]butyl-3-fluorobenzoate
1
(28). Yield 48%, yellowish liquid, H NMR (CDCl3): d
1.64–1.79 (m, 6H), 1.89–1.94 (m, 2H), 2.08–2.17 (m,
2H), 2.35 (t, J = 7.4 Hz, 2H), 2.73–2.84 (m, 2H),
3.39–3.44 (m, 1H), 4.31 (t, J = 6.4 Hz, 2H), 4.51 (s,
2H), 7.24–7.66 (m, 7H), 7.80 (ddd, J = 8.0, 2.5,
1.5 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H). 13C NMR
(CDCl3): d 23.7, 26.7, 31.2, 51.1, 58.0, 65.2, 69.6,
116.2, 116.5, 119.7, 120.0, 125.2, 127.3, 127.4, 128.3,
129.8, 129.9, 132.4, 132.5, 138.9, 160.8, 164.1, 165.4;
ESI-MS: 386.2 (M+1).
4.9. AChE (or BChE) inhibition assay in vitro
Inhibitory activity against AChE (or BChE) was evalu-
ated at 37 ꢁC by the colorimetric method reported by
Ellman et al.22 The final concentration containing test
compound of the assay solution consisted of 0.1 M

6606
Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
sodium phosphate buffer (pH 8.0), 0.3 mM 5,50-dithio-
bis-2-nitrobenzoic acid (DTNB, Ellman’s reagent),
0.02 U of AChE from Electrophorus electricus (or
0.01 U BChE from human serum, Sigma Chemical
Co.), and 0.5 mM acetylthiocholine iodide (or 0.5 mM
butyrylthiocholine) as substrate of the enzymatic reac-
tion, respectively. The assay solutions except substrate
were preincubated with the enzyme for 10 min at
37 ꢁC. After preincubation, the substrate was added.
The absorbance changes at 405 nm were recorded for
5 min with a microplate reader GENios F129004 (Tecan
Ltd, Austria). The AChE inhibition (or BChE inhibi-
tion, IC50) was determined for each compound. Each as-
say was run in triplicate and each reaction was repeated
at least three independent times. The dose–response
curves have been fit by Probit analysis in StatsDirect sta-
tistical software (ver 2.5.5) and the data showed propor-
tional response with 95% confidence intervals.
formation, thioflavin T (ThT) fluorescence method was
used.30,31 Analyses were performed with an Infinite
F200 (Tecan Ltd, Austria) and fluorescence was mea-
sured at 450 nm (k excitation, slit width 5 nm) and
490 nm (k emission, slit width 10 nm). To determine
amyloid fibril formation, after incubation, to the solu-
tion (20 lL) containing Ab or Ab plus inhibitors with
0.01 M sodium phosphate buffer (pH 8.0) was added
5 lM ThT 200 lL. Each assay was run in triplicate
and each reaction was repeated at least three indepen-
dent times. The fluorescence intensities were recorded,
and the percent aggregation was calculated by the fol-
lowing equation: 100 ꢀ {(IFi ꢀ IFb)/(IFo ꢀ IFb) · 100}
where IFi, IFo and IFb are the fluorescence intensities
obtained for Ab1–42 aggregation in the presence of inhib-
itors, in the absence of inhibitors and the blanks, respec-
tively. The IC50 is defined as the concentration of
compounds that reduces by 50% of with respect to that
without inhibitors. The dose–response curves have been
fit by Probit analysis in StatsDirect statistical software
(ver 2.5.5) and the data showed proportional response
with 95% confidence intervals.
4.10. Molecular modeling
Molecular modeling studies were performed on Silicon
Graphics Octane2 R12000 workstation by using Cerius2
(version 4.10) and InsightII (version 2000.2) which were
molecular modeling software packages. To determine
the binding model, we used Cerius2. On the other hand,
to get an insight into the binding mode of known ligands
from the X-ray crystal structures, we used InsightII and
performed structure alignments for reported AChE
complex structures and their PDB codes were 1EVE,
1GPK, 1GPN, 2ACE, and 1ODC. In order to obtain
a binding model of compound 12 in the active site of
AChE, we performed the following steps. First, the 3D
structure of 12 was minimized by using CFF1.02 which
was available in Cerius2, and atomic charges were as-
signed by CFF1.02. We generated conformationally di-
verse 3D structures of 12 and chose one of the
structures which had a conformationally similar struc-
ture to donepezil which bound at the active site of AChE
structure (1EVE). Second, the protein structure was
modeled by adding hydrogen atom followed by ‘the
templates for protein residues’ in Cerius2, and all water
molecules were removed. Third, to designate an initial
position of 12 in the active site, we used donepezil as a
template structure from 1EVE. We manually set the ini-
tial position of 4-chlorobenzene of 12 to be overlapped
with the benzyl ring of donepezil. Lastly, energy minimi-
zation was performed to obtain a binding mode of 12 to
the active site in AChE. For parameters, we set ‘Smart
Minimizer,’ as an energy minimization method which
was available in Cerius2, and set the convergence criteria
to 5000 cycles with RMSD 0.1 cal/mol. We also set the
ligand to be flexible to move and the protein to be rigid.
4.12. Inhibition of AChE-induced Ab1–42 peptide aggre-
gation assay
For co-incubation experiments,30,32 of Ab1–42 peptide
(American Peptide Company, USA) and AChE from
Electrophorus electricus, the mixtures of Ab1–42 peptide
and AChE in presence or absence of the test inhibitors
were incubated for 6 h at 37 ꢁC. The final concentrations
of Ab (dissolved in DMSO and diluted 0.215 M sodium
phosphate buffer, pH 8.0) and AChE (dissolved in 0.1 M
sodium phosphate buffer, pH 8.0) are 20 lM and
0.02 U, respectively. To analyze co-aggregation inhibi-
tion, the ThT fluorescence method was used30,31 and
the fluorescence was measured at 450 nm (k excitation,
slit width 5 nm) and 490 nm (k emission, slit width
10 nm). After co-incubation, to the mixture solutions
of 20 lL was added 5 lM ThT 200 lL. Each assay
was run in triplicate and each reaction was repeated at
least three independent times.
4.13. Cell viability
IMR-32, human neuroblastoma cells were obtained from
the American Type Culture Collection (Rockville, MD).
The cells were cultured in Minimum Essential Medium
with Earle’s salt with L-glutamine (EMEM), 10% fetal
bovine serum, and 1% penicillin/streptomycin in a highly
humidified atmosphere having 5% CO2at 37 ꢁC. The
compounds’ ability to protect IMR-32 cells from Ab1–42
insult was investigated according to the published proce-
dure.32,33 To determine the cell viability, exponentially
growing cells were plated at about 5000 cells per well
100 lL of fresh medium in 96-well tissue culture plates
for 2 h. Cells were incubated with Ab1–42 (20 lM from
a stock solution in DMSO) or Ab plus 0.1 lM of each
inhibitor for 48 h. The final DMSO concentration was
less than 1%. After the incubation of cells in MTT solu-
tion (20 lL per well from 1 mg/mL stock solution) for 4 h
at 37 ꢁC. Cell lysis buffer (100 lL per well, 20% SDS/50%
N,N-dimethylformamide, pH 4.7) was added, mixed, and
4.11. Inhibition of Ab1–42 peptide aggregation (Thioflavin
T-based fluorometric assay)
Ab1–42 peptide (American Peptide Company, USA) was
dissolved in DMSO to obtain a 2.3 mM solution. Ali-
quots of Ab1–42 in DMSO were then incubated for
16 h at 37 ꢁC in 0.215 M sodium phosphate buffer (pH
8.0) at a final Ab concentration of 20 lM in the presence
or absence of test inhibitors. To quantify amyloid fibril

Y. E. Kwon et al. / Bioorg. Med. Chem. 15 (2007) 6596–6607
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