Novel alkyl- and arylcarbamate derivatives with N-benzylpiperidine and N-benzylpiperazine moieties as cholinesterases inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.09.010

The study presents synthesis and biological activity of novel alkyl- and arylcarbamate derivatives with N-benzylpiperidine and N-benzylpiperazine moieties designed as cholinesterases inhibitors. These fragments turned out to determine compounds' selectivi

Full text of DOI:10.1016/j.ejmech.2010.09.010

European Journal of Medicinal Chemistry 45 (2010) 5602e5611
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel alkyl- and arylcarbamate derivatives with N-benzylpiperidine and
N-benzylpiperazine moieties as cholinesterases inhibitors
*
Anna Wie˛ckowska, Marek Bajda, Natalia Guzior, Barbara Malawska
Department of Physicochemical Drug Analysis, Chair of Pharmaceutical Chemistry, Jagiellonian University Medical College, Medyczna 9 St. 30-688 Kraków, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The study presents synthesis and biological activity of novel alkyl- and arylcarbamate derivatives with
N-benzylpiperidine and N-benzylpiperazine moieties designed as cholinesterases inhibitors. These
fragments turned out to determine compounds’ selectivity between AChE and BuChE. Derivatives of
N-benzylpiperazine (16e25) were selective BuChE inhibitors with 3-(2-(4-benzylpiperazin-1-yl)-2-
oxoethyl)-phenyl butylcarbamate (22) being the most potent compound (pIC50 ¼ 5.00) while a series of
carbamate derivatives of N-benzylpiperidine (5e14) displayed non-selective BuChE/AChE inhibitory
activity. Molecular modelling studies point out significant differences between orientations of these two
groups of compounds in the active site of AChE, which can be an explanation of their different biological
activity.
Received 10 July 2009
Received in revised form
12 August 2010
Accepted 7 September 2010
Available online 17 September 2010
Keywords:
Acetylcholinesterase inhibitors
Butyrylcholinesterase inhibitors
Carbamate derivatives
Alzheimer’s disease
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
phosphorylation [12,13], and inflammatory processes [14], which
are all important for the AD pathogenesis. Influence on the amyloid
Acetylcholinesterase inhibitors are still the most frequently used
drugs in treatment of Alzheimer’s disease (AD) [1,2]. Their intro-
duction into the AD treatment arose from the cholinergic hypothesis
of AD [3,4] blaming deficit in central cholinergic transmission for
cognitive and non-cognitive symptoms observed in patients. As
acetylcholinesterase (AChE) was thought to be the only enzyme
responsible for the hydrolysis of acetylcholine in the central nervous
system (CNS), it was the main target in search for anti-AD drugs. As
a consequence, four AChE inhibitors belonging to different chemical
groups have been approved for the symptomatic treatment of mild
to moderate stages of AD: tacrine, donepezil, rivastigmine and gal-
antamine. Recently, significant evidence pointing out the role of
butyrylcholinesterase (BuChE) in the cholinergic system function
has appeared [5]. In physiological conditions cholinesterase activity
in the brain is mostly related to AChE. However, over the course of
AD, AChE activity progressively decreases in certain brain regions,
whereas BuChE activity increases and BuChE may then act as
a compensatory mechanism for acetylcholine hydrolysis [6]. It has
also been proved, that aside from their typical enzymatic function,
cholinesterases display several non-classical activities like: regula-
tion of cerebral blood flow and metabolism [7,8], modulatory effects
on the amyloid cascade [9e11], glial proliferation, tau protein
cascade is of special interest as it has been observed that in
Alzheimer’s disease AChE and BuChE are mostly located within
neuritic plaques [15] and both seem to be associated with the
formation of cytotoxic
ments it has been shown that AChE not only initiates transformation
of relatively inert A into pathogenic plaques [9,11], but also it
b-amyloid (Ab) fibrills. In numerous experi-
b
increases neurotoxicity of such aggregates [16e18].
Due to cholinergic hypothesis and numerous reports regarding
multiple functions of cholinesterases in pathogenesis and devel-
opment of AD, AChE and BuChE are very attractive targets for the
development of anti-AD drugs. Recently developed cholinesterases
inhibitors for the treatment of AD represent structural modifica-
tions of existing drugs and other compounds [19e25]. Taking into
consideration that the role of BuChE in AD is not clearly understood
[6,26], and relatively few selective inhibitors of this enzyme have
been investigated [27e29], there is a constant need for new BuChE
inhibitors, which can serve both as potential anti-AD drugs and
pharmacological tools.
The presented study describes a synthesis and preliminary
in vitro activity screening of 4 new series of potential cholinester-
ases inhibitors. The structures were designed as a combination of
cholinesterases inhibitors pharmacophores, carbamates and ary-
lalkylamines linked by a phenylacetamide fragment. Carbamate
moieties, present in the structure of pseudoirreversible AChE
inhibitors, rivastigmine and physostigmine, play a crucial role in
* Corresponding author. Tel.: þ48 12 62 05 450; fax: þ48 12 6570 262.
E-mail address: mfmalaws@cyf-kr.edu.pl (B. Malawska).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.010

A. Wie˛ckowska et al. / European Journal of Medicinal Chemistry 45 (2010) 5602e5611
5603
their activity due to carbamylation of serine in the catalytic triad of
the enzyme. N-benzylpiperidine, N-benzylpiperazine and amide
fragments were introduced into structures of the new compounds
to provide hydrophobic interactions and hydrogen bonds formation
within cholinesterases active sites (Fig. 1).
as well as for meta- or para- substituted analogues. The results
suggest that the larger and more lipophilic butyl substituent seems
to be beneficial for the activity of the compounds (11, 22) while other
structural changes do not have such a strong influence. Interestingly,
among the tested compounds only the N-benzylpiperidine series
(5e14) displayed activity against AChE. Inhibitory potencies of
compounds in this group were ranging from 19.21 to 50.45% at
2. Results and discussion
100 mM concentration, which makes them weak AChE inhibitors
with the most potent compounds bearing a small dimethyl
substituent in the carbamate group, 9 (pIC50 ¼ 4.00) and 14
(pIC50 ¼ 3.98). The same tendency was noticed for intermediates
1e4, lacking carbamoyl moieties, although their activities were
lower (Table 1). These results point out the importance of the N-
benzylpiperazine and N-benzylpiperidine fragments for activityand
selectivity of these two groups of compounds, whereas carbamoyl
groups, though able to modify activity, seem not to be crucial.
2.1. Chemistry
New carbamate derivatives (5e25) were synthesized via the
route outlined in Scheme 1. In the first step, activation of the
carboxylic groups of hydroxyphenylacetic acids using N-(3-dime-
thylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC) or
1,10-carbonyldiimidazole (CDI) followed by the coupling with the
1-benzylpiperazine and 4-amino-1-benzylpiperidine lead to
amides 1e4 in moderate yields (30e55%). In the next step the
obtained phenols were transformed into the target compounds
(5e8, 10e13, 15e19, 21e24) in reactions with alkyl- and aryl
isocyanates. Carbamoylation of compounds 1e4 with dime-
thylcarbamoyl chloride gave compounds 9, 14, 20 and 25. Further
purification by recrystallization or column chromatography affor-
ded the desired carbamates in 44e99% yields.
2.3. Molecular modelling
In order to explain possible interactions between the newly
synthesized compounds and the active site of acetylcholinesterase
and butyrylcholinesterase, molecular modelling studies were per-
formed. Using Glide 4.5 (Schrödinger LLC, 1999e2007), compounds
(5e25) were docked to the active site of AChE derived from complex
of the enzyme with donepezil (PDB code: 1EVE) and to BuChE
derived from complex of the enzyme with butyrate (PDB code:
2J4C). Water molecules were deleted beyond the radius of 5 Å from
reference ligand (donepezil or butyrate, respectively) by Protein
Preparation Wizard and then AChE or BuChE was used to prepare
the Receptor Grid. All ligands were optimized with OPLS-2005
method by LigPrep. Docking was carried out on all derivatives
resulting in 10 poses of each ligand, sorted by increasing scoring
function.
Molecular modelling and docking studies showed significant
differences between orientations of compounds of N-benzylpiper-
idine (5e15) and N-benzylpiperazine (16e25) series in the active site
of AChE. Types of interactions for selected representatives of each
series (9, 20) are presented in Fig. 2 (visualized by PyMOL program).
Compound 9 orientates along the active site gorge, extending from
the active site at the bottom near Trp84 (N-benzylpiperidine frag-
ment), to the peripheral binding site at the top near Trp279 (carba-
moyl fragment). Three major functional moieties of this molecule, i.e.
the benzyl group, the piperidine nitrogen atom and the phenyl ring
2.2. Biological activity
Inhibitory potencies of the newly synthesized compounds against
AChE from electric eel and BuChE from horse serum were evaluated
by the spectroscopic method of Ellman et al. [30]. This test is based on
the reaction of 5,50-dithio-bis-(2-nitrobenzoic) acid, known as DTNB
or Ellman’s reagent, with the sulfhydryl group of acetylthiocholine or
butyrylthiocholine which results in formation of a yellow-coloured
product, i.e. 2-nitro-5-thiobenzoic acid. Changes of absorbance
recorded at 412 nm determine the activity of tested compounds.
The assays were performed at a 100 mM concentration of the
potential inhibitors. The activity measured is given in percentage of
enzyme inhibition and as pIC50 values. pIC50 values were only
determined for compounds with more than 60% inhibitory activity.
As shown in Table 1 almost all carbamate derivatives displayed
moderate or good BuChE inhibitory activities ranging from
pIC50 ¼ 4.26 to 5.24 (62.49e91.97%). Only compounds 10 and 20
showed activities lower than 50%. Similar activities against BuChE
appear for both N-benzylpiperidine and N-benzylpiperazine series
Physostigmine
Rivastigmine
H3C
H
N
CH3
N
CH3
O
H3C
O
H3C
CH3
N
N
CH3
O
N
H
CH3
O
CH3
Novel carbamates
H/R
N
H/R
N
O
O
O
R/Ar
.
R/Ar
O
N
.
O
O
N
N
H
N
Cmp. 5-15
Cmp. 16-25
Fig. 1. Structures of cholinesterases inhibitors, physostigmine and rivastigmine, and new carbamate derivatives 5e25.

5604
A. Wie˛ckowska et al. / European Journal of Medicinal Chemistry 45 (2010) 5602e5611
O
HO
.
OH
a
b
O
HO
.
N
O
HO
.
N
N
H
N
1, 3 2, 4
Cmp.
1, 2
3, 4
4-OH 3-OH
c or d
c or d
R
O
R
O
O
N
O
O
O
N
N
H
N
5 - 10
16 - 21
Cmp.
Cmp.
5
6
7
8
9
10
16
17
18
19
20
21
Me
N
Me
N
H
N
H
H
N
H
N
H
N
H
N
H
H
H
N
H
H
N
H
N
N
N
N
N
Pr
i-Pr
Pr
i-Pr
.
Bu
.
Et
.
. Me
N
. o-tolyl
.
Bu
O
.
Et
.
. Me
. o-tolyl
.
R
.
.
R
.
O
O
O
N
O
R
N
R
O
H
N
22 - 25
11 - 15
Cmp.
Cmp.
11
12
13
14
15
22
23
24
25
Me
N
Me
N
H
N
H
N
H
N
H
N
H
N
H
H
H
N
H
N
N
N
i-Pr
Bu
.
.
Et
.
. Me
.
i-Pr
R
Bu
.
.
Et
.
. Me
. o-tolyl
.
R
Scheme 1. Synthesis of compounds 1e25. Reagents and conditions: (a) 1-benzylpiperidin-4-ylamine, CDI/THF or EDC/CH2Cl2; (b) 1-benzylpiperazine, CDI/THF or EDC/CH2Cl2;
(c) alkyl- or arylisocyanate, Et3N; CH2Cl2, (d) dimethylcarbamoyl chloride, K2CO3, Me2CO.
make principal interactionswith the active site gorge of TcAChE. Close
to the bottom of the gorge the benzyl moiety creates hydrophobic
between the carbonyl group of piperazine amide and the Tyr121
residue. The Trp279 residue creates -cation interaction with the
p
interactions and
p
-
p
stacking with Phe330 and Trp84 residues
protonatedpiperazinenitrogen atom. No interactionswiththebenzyl
moiety are observed. These results show significant differences in
types and energy of interactions between the compounds and the
amino acids of the active site, which can explain differences in AChE
inhibitory activity between two series of compounds.
(aromatic rings of amino acids are not situated directly over the
phenyl ring but they are slightly shifted). The protonated piperidine
nitrogen atom creates cation-p interactions with Tyr334 and an ionic
bond with Asp72. These interactions resemble arrangement of
donepezil in the catalytic center of AChE, however this inhibitor is not
placed so deep in the gorge of the enzyme, therefore its interactions
are weaker, which also explains lower activity of the compounds. The
phenyl ring, to which the carbamoyl group is attached, stacks against
the indole moiety of Trp279 in the peripheral binding site, in a classic
We did not notice such divergency when docking to BuChE. Both
series with N-benzylpiperidine (5e15) and N-benzylpiperazine
(16e25) moieties display similar interactions within the active site
of the enzyme, which are presented on example of the most active
compound (11) (Fig. 3). The carbamoyl moiety of the ligand is
orientated towards catalytic triad and its arrangement enables its
transfer onto Ser198 residue. The n-butyl substituent creates
hydrophobic interactions with Trp82 and additionally with Ile442,
Met437 and Tyr440. Oxygen atoms of amide and carbamate
moieties can create hydrogen bonds with water molecules located
in the active center. The phenyl ring of the phenylacetamide (11)
can interact with Thr120 while the benzyl moiety interacts with
Pro285 via hydrophobic interactions.
parallel p-p interaction. The carbamoyl moiety remains outside the
gorge and may probably not create significant interactions with
Tyr70. Reverse arrangement, i.e. the carbamate group towards cata-
lytic center is not preferred and characterized by higher energy
values. Compound 20 represents N-benzylpiperazine derivatives,
which were not active against AChE, and its interactions are different
from those of N-benzylpiperidine series. The carbamoyl moiety of
compound 20 is orientated towards the catalytic center. In this case
such orientation is more favorable as regards energy. However, the
distance between the carbamoyl group and the catalytic triad is too
large to enable transfer to Ser200. The phenyl ring may create
hydrophobic interactions with Phe330, Phe331 and Tyr334 residues
but they are less significant due to non-parallel orientation and long
distances between rings. The essential interaction is a hydrogen bond
2.4. Kinetic studies of AChE and BuChE inhibition
The mechanism of AChE and BuChE inhibition was investigated
in enzyme kinetic studies [23,31,32] using Ellman’s test and
compound 14, the most potent, non-selective inhibitor of both

A. Wie˛ckowska et al. / European Journal of Medicinal Chemistry 45 (2010) 5602e5611
5605
Table 1
Cholinesterases inhibitory activity of tested compounds 1e25 and reference compounds, tacrine and rivastigmine.
O
N
O
R
O
N
R
O
N
H
N
H
2, 11 - 15
1, 5 - 10
O
O
N
O
N
R
R
O
N
N
4, 22 - 25
3, 16 - 21
Cmp.a
R
%Ib AChE
ꢀ SEM
%Ib BuChE
ꢀ SEM
BuChE pIC50
ꢀ SEM
1
2
3
4
15.16 ꢀ 0.12
38.14 ꢀ 0.39
45.63 ꢀ 1.91
49.82 ꢀ 1.27
45.33 ꢀ 0.91
31.48 ꢀ 2.13
e
H
H
H
H
e
e
e
na
na
O
H3C
H3C
N
H
O
5
6
7
8
43.72 ꢀ 1.31
33.81 ꢀ 0.26
19.21 ꢀ 0.98
23.43 ꢀ 0.10
63.37 ꢀ 0.16
95.07 ꢀ 0.13
63.15 ꢀ 2.67
90.37 ꢀ 1.39
4.93 ꢀ 0.36
5.03 ꢀ 0.05
4.74 ꢀ 0.03
4.97 ꢀ 0.16
N
H
CH3
O
H3C
H3C
N
H
O
N
H
O
H3C
N
9
46.18 ꢀ 0.73
24.69 ꢀ 0.33
89.76 ꢀ 0.27
46.93 ꢀ 0.76
5.05 ꢀ 0.02
CH3
O
N
H
10
e
CH3
O
H3C
N
H
O
11
12
13
39.86 ꢀ 5.16
34.86 ꢀ 1.04
28.53 ꢀ 0.14
91.97 ꢀ 0.33
72.22 ꢀ 0.40
68.59 ꢀ 0.41
5.24 ꢀ 0.02
4.53 ꢀ 0.04
4.43 ꢀ 0.04
CH3
H3C
N
H
O
H3C
N
H
O
H3C
N
14
15
50.45 ꢀ 0.49
82.13 ꢀ 0.57
4.66 ꢀ 0.02
CH3
O
N
H
nd
nd
nd
CH3
(continued on next page)

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A. Wie˛ckowska et al. / European Journal of Medicinal Chemistry 45 (2010) 5602e5611
Table 1 (continued )
Cmp.a
R
%Ib AChE
ꢀ SEM
%Ib BuChE
ꢀ SEM
BuChE pIC50
ꢀ SEM
O
H3C
N
H
16
na
83.09 ꢀ 0.31
4.78 ꢀ 0.02
O
H3C
N
17
18
19
na
na
na
88.26 ꢀ 0.38
72.18 ꢀ 1.59
83.37 ꢀ 0.67
4.49 ꢀ 0.06
4.28 ꢀ 0.03
4.36 ꢀ 0.05
H
CH3
O
H3C
N
H
O
O
H3C
N
H
O
H3C
N
20
21
na
nd
32.47 ꢀ 0.61
e
CH3
N
H
nd
nd
CH3
O
H3C
N
H
O
22
23
24
na
na
na
92.70 ꢀ 0.10
77.73 ꢀ 0.16
62.49 ꢀ 0.36
5.00 ꢀ 0.01
4.45 ꢀ 0.02
4.26 ꢀ 0.03
CH3
H3C
N
H
O
H3C
N
H
O
H3C
N
25
na
78.95 ꢀ 0.58
4.63 ꢀ 0.03
CH3
AChE pIC50
7.76 ꢀ 0.02
5.38e6.03c
BuChE pIC50
8.31 ꢀ 0.01
6.08e7.43c
Tacrine
Rivastigmine
enzymes. Enzymes kinetics were analyzed by recording sub-
strateevelocity curves in absence and presence of compound 14 or
a referenceetacrineereversible non-competitive inhibitor. Graph-
ical analysis of LineweavereBurk plots of AChE activity (Fig. 4)
showed increasing slopes (decreased Vmax) with increasing inhibitor
concentration. This pattern indicates non-competitive inhibition.
LineweavereBurk plots of BuChE activity (Fig. 5) showed increasing
slopes (lower Vmax) and increasing intercepts (higher Km), indicating
mixed-type inhibition.
Fig. 2. Hypothetical binding mode of compound 9 (left) and 20 (right) in the AChE
active site gorge.
Fig. 3. Hypothetical binding mode of compound 11 in the BuChE active site gorge.

A. Wie˛ckowska et al. / European Journal of Medicinal Chemistry 45 (2010) 5602e5611
5607
60
40
20
enzyme. Overall, these results indicate that the novel carbamate
derivatives of 1-(4-benzylpiperazin-1-yl)-2-(4- or 3-hydrox-
yphenyl)-ethanone are interesting structures for further research
for selective and more potent BuChE inhibitors.
3.1. Experimental protocols
0
0.1 mM
1 mM
3.1.1. Chemistry
Reagents and solvents were purchased from common
commercial suppliers and were used without further purification.
Melting points were determined in open capillaries on an Elec-
trothermal 9300 apparatus and are uncorrected. Merck silica gel 60
(70e230 mesh ASTM) was used for column chromatography.
Analytical thin layer chromatography was performed on Merck TLC
plates, silica gel 60 F254. TLC visualization was achieved with a UV
lamp or with ninhydrin solution. Nuclear magnetic resonance
spectra (1H NMR) were recorded with Varian Mercury VX 300 MHz
-10
10
20
30
-1
1/[S] (mM)
Fig. 4. LineweavereBurk plots resulting from substrateevelocity curves of AChE
activity with different substrate concentrations (40e500 mM) in the absence and
presence of compound 14 in concentrations of 0.1 mM and 1 mM.
spectrometer using CDCl3 or DMSO-d6. The chemical shifts (d) are
reported in parts per million (ppm) relative to the internal standard
(TMS). Elemental analyses were performed on Vario EL III
Elemental analyser. Mass spectra were recorded on MDX SCILEX
API 2000 (Concord, ON, Canada) by using ESI method or on Fin-
nigan MAT 95S (Bremen, Germany) by using EI method.
80
60
4. General procedure for the synthesis of compounds 1e4
0
40
0.01 mM
Procedure A1. To a stirred solution of 3-hydroxyphenylacetic acid
or 4-hydroxyphenylacetic acid (1 equiv.) and either 1-benzylpiper-
azine or 4-amino-1-benzylpiperidine (1 equiv.) in dichloromethane,
0.1 mM
20
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydrochloride
(EDCI) (1.1 equiv.) was added at 0 ꢁC. The reaction mixture was
warmed to room temperature and stirred for 24 h. The solvent was
evaporated and the residue was purified by column chromatography
(CH2Cl2/MeOH, 9.5:0.5) to afford the pure product.
-10
10
20
30
-1
1/[S] (mM)
Procedure A2. To the solution of 3-hydroxyphenylacetic acid or
4-hydroxyphenylacetic acid (1 equiv.) in anhydrous tetrahydro-
furan (THF), 1,10-carbonyldiimidazole (CDI) (1 equiv.) was added
and the mixture was stirred at room temperature. After 3 h, 1-
benzylpiperazine or 4-amino-1-benzylpiperidine (1 equiv.) was
added and the resulting solution was stirred for 24 h. After evap-
oration under reduced pressure the crude product was purified by
column chromatography (CH2Cl2/MeOH, 9.5:0.5) to give the pure
product.
Fig. 5. LineweavereBurk plots resulting from substrateevelocity curves of BuChE
activity with different substrate concentrations (40e500 mM) in the absence and
presence of compound 14 in concentrations of 0.01 mM and 0.1 mM.
3. Conclusions
As AD progresses, acetylcholine regulation becomes increas-
ingly dependent on BuChE, thus the enzyme is considered as one of
the important factors implicated in the pathomechanism of AD.
New selective inhibitors of the enzyme could serve as biological
tools in research regarding physiological function of the enzyme
and its role in AD and its treatment. This study has resulted in series
of new cholinesterases inhibitors, both selective BuChE inhibitors
(16e20, 22e25) and non-selective AChE/BuChE inhibitors (5e14).
Structures of compounds presented in this paper contain well-
known cholinesterases inhibitors pharmacophoresealkyl- and
arylcarbamates that improved the activity but were not crucial for
it. N-Benzylpiperidine, N-benzylpiperazine and amide moieties
turned out to be important for selectivity of the compounds.
Molecular modelling studies suggest that a reason for selectivity of
N-benzylpiperazine compounds may be the long distance between
carbomoyl group and the catalytic site of AChE, which makes an
interaction between them impossible. In the other series, the
N-benzylpiperidine fragment is oriented towards the catalytic
triad, creating interactions with amino acids of the active site.
Preliminary kinetic studies performed for the most potent cholin-
esterases inhibitor obtained (14) indicated non-competitive type
inhibition against AChE and mixed-type inhibition against (BuChE),
which is in agreement with molecular modelling studies that
showed that compound 14 can bind to the peripheral site of the
4.1. N-(1-Benzylpiperidin-4-yl)-2-(4-hydroxyphenyl)-
acetamide (1)
Procedure: A2; Reagents: 4-hydroxyphenylacetic acid (1.52 g,
10 mmol), THF (30 mL), CDI (1.62 g, 10 mmol), 4-amino-1-benzyl-
piperidine (1.90 g, 10 mmol); pale yellow solid; yield: 40%; mp:
215e217 ꢁC; 1H NMR (300 MHz, CDCl3)
d (ppm): 1.24e1.37 (m, 2H),
1.81e1.84 (m, 2H), 2.07e2.15 (m, 2H), 2.83 (d, J¼ 11.82 Hz, 2H), 3.46
(s, 2H), 3.49 (s, 2H), 3.73e3.86 (m, 1H), 5.29 (d, J¼ 8.28 Hz, 1H),
6.63e6.68 (m, 2H), 6.97e7.01 (m, 2H), 7.23e7.28 (m, 5H), OH signal
not detected; ESI-MS (m/z) 325.4 [M þ H]þ. The HCl salt was
prepared in ethanol; mp: 266e268 ꢁC; Anal. calcd. for
C20H25N2O2Cl$H2O: C, 63.40; H, 7.18; N, 7.39. Found: C, 62.92; H,
7.36; N, 7.60.
4.2. N-(1-Benzylpiperidin-4-yl)-2-(3-hydroxyphenyl)-
acetamide (2)
Procedure: A1; Reagents: 3-hydroxyphenylacetic acid (0.76 g,
5 mmol), CH2Cl2 (50 mL), EDC (1.05 g, 5.5 mmol), 4-amino-1-ben-
zylpiperidine (0.95 g, 5 mmol); white solid; yield: 55.5%; mp:

5608
A. Wie˛ckowska et al. / European Journal of Medicinal Chemistry 45 (2010) 5602e5611
137e140 ꢁC; 1H NMR (300 MHz, CDCl3)
d
(ppm): 1.33e1.46 (m, 2H),
5.2. 4-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl
propylcarbamate (6)
1.79e1.84 (m, 2H), 2.11e2.20 (m, 2H), 2.88 (d, J¼ 11.83 Hz, 2H), 3.44
(s, 2H), 3.54 (s, 2H), 3.73e3.86 (m, 1H), 5.50 (d, J ¼ 8.12 Hz, 1H),
6.59e6.70 (m, 3H), 7.13 (t, J¼ 7.78 Hz, 1H), 7.27e7.30 (m, 5H), OH
signal not detected; ESI-MS (m/z) 325.7 [M þ H]þ. The HCl salt was
prepared in ethanol; mp: decomposition > 234 ꢁC; Anal. calcd. for
C20H25N2O2Cl$H2O: C, 63.40; H, 7.18; N, 7.39. Found: C, 63.22; H,
7.68; N, 6.91.
Procedure: K1; Reagents: comp. 1 (0.320 g, 1 mmol), propyl
isocyanate (0.085 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); white
solid; yield: 81%; mp: 184e186 ꢁC; 1H NMR (300 MHz, CDCl3)
d
(ppm): 0.97 (t, J ¼ 7.40 Hz, 3H), 1.23e1.41 (m, 2H), 1.54e1.66 (m,
2H), 1.80e1.85 (m, 2H), 2.03e2.11 (m, 2H), 2.71 (d, J ¼ 11.66 Hz, 2H),
3.23 (dd, J ¼ 6.63, 13.53 Hz, 2H), 3.45 (s, 2H), 3.51 (s, 2H), 3.74e3.83
(m, 1H), 5.05e5.08 (m, 1H), 5.27 (d, J ¼ 7.64 Hz, 1H), 7.08e7.11 (m,
2H), 7.19e7.22 (m, 2H), 7.25e7.32 (m, 5H); ESI-MS (m/z) 410.5
[M þ H]þ. The HCl salt was prepared in ethanol; mp: 205e207 ꢁC;
Anal. calcd. for C24H32N3O3Cl: C, 64.63; H, 7.23; N, 9.42. Found: C,
64.51; H, 7.75; N, 9.46.
4.3. 1-(4-Benzylpiperazin-1-yl)-2-(4-hydroxyphenyl)-ethanone (3)
Procedure: A2; Reagents: 4-hydroxyphenylacetic acid (1.52 g,
10 mmol), THF (30 mL), CDI (1.62 g, 10 mmol), 1-benzylpiperazine
(1.76 g, 10 mmol); white solid; yield: 30%; mp: 151e153 ꢁC; 1H NMR
(300 MHz, CDCl3)
d
(ppm): 2.30 (t, J ¼ 4.95 Hz, 2H), 2.43 (t,
5.3. 4-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl
isopropylcarbamate (7)
J ¼ 5.07 Hz, 2H), 3.45 (t, J ¼ 5.00 Hz, 2H), 3.48 (s, 2H), 3.63 (s, 2H), 3.66
(t, J ¼ 5.06 Hz, 2H), 6.65e6.70 (m, 2H), 6.99 (d, J ¼ 8.50 Hz, 2H),
7.22e7.34 (m, 5H), OH signal not detected; EI-MS (m/z) 310,1 [M]þ.
The HCl salt was prepared in ethanol; mp: 245e247 ꢁC; Anal. calcd.
for C19H23N2O2Cl$H2O: C, 62.55; H, 6.91; N, 7.68. Found: C, 62.30; H,
7.05; N, 7.80.
Procedure: K1; Reagents: comp. 1 (0.320 g, 1 mmol), isopropyl
isocyanate (0.085 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL);
white solid; yield: 83%; mp: 197e199 ꢁC; 1H NMR (300 MHz,
CDCl3)
d
(ppm): 1.24 (d, J ¼ 6.55 Hz, 6H), 1.28e1.39 (m, 2H),
1.80e1.85 (m, 2H), 2.03e2.13 (m, 2H), 2.71 (d, J ¼ 11.73 Hz, 2H),
3.45 (s, 2H), 3.51 (s, 2H), 3.73e3.83 (m, 1H), 3.84e3.95 (m, 1H),
4.88 (d, J ¼ 6.78 Hz, 1H), 5.24 (d, J ¼ 7.70 Hz, 1H), 7.09e7.11 (m,
2H), 7.20e7.23 (m, 2H), 7.27e7.32 (m, 5H); ESI-MS (m/z) 410.5
[M þ H]þ. The HCl salt was prepared in ethanol; mp: 216e219 ꢁC;
Anal. calcd. for C24H32N3O3Cl$H2O: C, 62.13; H, 7.39; N, 9.06.
Found: C, 62.78; H, 7.50; N, 9.09.
4.4. 1-(4-Benzylpiperazin-1-yl)-2-(3-hydroxyphenyl)-ethanone (4)
Procedure: A1; Reagents: 3-hydroxyphenylacetic acid (0.76 g,
5 mmol), CH2Cl2 (50 mL), EDC (1.05 g, 5.5 mmol), 1-benzylpiper-
azine (0.88 g, 5 mmol); white solid; yield: 45%; mp: 122e125 ꢁC; 1H
NMR (300 MHz, CDCl3)
d
(ppm): 2.25 (t, J ¼ 5.00 Hz, 2H), 2.41 (t,
J ¼ 5.10 Hz, 2H), 3.42e3.46 (m, 4H), 3.64e3.67 (m, 4H), 6.63e6.66
(m, 1H), 6.69e6.73 (m, 1H), 6.85e6.87 (m, 1H), 7.09e7.14 (m, 1H),
7.21e7.3þ3 (m, 5H), OH signal not detected; ESI-MS (m/z) 311.7
[M þ H] . The HCl salt was prepared in ethanol; mp: 194e198 ꢁC;
Anal. calcd. for C19H23N2O2Cl: C, 65.79; H, 6.68; N, 8.08. Found: C,
65.79; H, 7.05; N, 8.11.
5.4. 4-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl
ethylcarbamate (8)
Procedure: K1; Reagents: comp. 1 (0.320 g, 1 mmol), ethyl isocy-
anate (0.071 g,1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); white solid;
yield: 99%; mp: 174e176 ꢁC; 1H NMR (300 MHz, CDCl3)
d (ppm): 1.21
5. General procedure for the synthesis of compounds 5e25
(t, J ¼ 7.25 Hz, 3H), 1.30e1.42 (m, 2H), 1.80e1.85 (m, 2H), 2.05e2.14
(m, 2H), 2.74 (d, J ¼ 11.60 Hz, 2H), 3.27e3.36 (m, 2H), 3.47 (s, 2H),
3.51 (s, 2H), 3.72e3.84 (m, 1H), 5.03e5.07 (m, 1H), 5.32 (d,
J ¼ 7.23 Hz, 1H, amide-NH), 7.08e7.11 (m, 2H), 7.20e7.23 (m, 2H),
7.28e7.32 (m, 5H); ESI-MS (m/z) 396.5 [M þ H]þ. The HCl salt was
prepared in ethanol; mp: 181e185 ꢁC; Anal. calcd. for C23H30N3O3Cl:
C, 63.95; H, 7.00; N, 9.73. Found: C, 63.55; H, 7.25; N, 9.70.
Procedure K1. A solution of compound 1, 2, 3 or 4 (1 equiv.),
isocyanate (1 equiv.), and triethylamine (Et3N) in CH2Cl2 was
allowed to stir at room temperature overnight. The reaction
mixture was concentrated in vacuo and the resulting residue was
washed with diethyl ether and if necessary purified via column
chromatography or recrystallization.
Procedure K2. A mixture of compound 1, 2, 3 or 4 (1 equiv.),
dimethylcarbamoyl chloride (1 equiv.) and K2CO3 (3 equiv.) in
acetone was stirred at room temperature for 24 h. The mixture was
filtered and the organic solvent was evaporated in vacuo. The
resulting residue was purified by recrystallization or column
chromatography.
5.5. 4-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl
dimethylcarbamate (9)
Procedure: K2; Reagents: comp. 1 (0.320 g, 1 mmol), dime-
thylcarbamoyl chloride (0.107 g, 1 mmol), K2CO3 (0.410 g, 3 mmol),
acetone (10 mL); Purification: recrystallization from EtOAc; white
solid; yield: 89%; mp: 126e128 ꢁC; 1H NMR (300 MHz, CDCl3)
5.1. 4-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl
butylcarbamate (5)
d (ppm): 1.27e1.40 (m, 2H), 1.79e1.83 (m, 2H), 2.03e2.11 (m, 2H),
2.73 (d, J ¼ 11.77 Hz, 2H), 3.01 (s, 3H), 3.10 (s, 3H), 3.45 (s, 2H), 3.51
(s, 2H), 3.71e3.84 (m, 1H), 5.32 (d, J ¼ 7.92 Hz, 1H), 7.06e7.09 (m,
2H), 7.19e7.23 (m, 2H), 7.24e7.33 (m, 5H); ESI-MS (m/z) 396.5
[M þ H]þ. The HCl salt was prepared in ethanol; mp: 218e222 ꢁC;
Anal. calcd. for C23H30N3O3Cl$3H2O: C, 56.84; H, 7.47; N, 8.65.
Found: C, 57.17; H, 7.16; N, 8.43.
Procedure: K1; Reagents: comp. 1 (0.32 g, 1 mmol), butyl isocy-
anate (0.10 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); white solid;
yield: 83%; mp: 188e190 ꢁC; 1H NMR (300 MHz, CDCl3)
d (ppm):
0.95 (t, J ¼ 7.23 Hz, 3H), 1.31e1.45 (m, 4H), 1.51e1.60 (m, 2H),
1.80e1.85 (m, 2H), 2.03e2.12 (m, 2H), 2.72 (d, J ¼ 11.71 Hz, 2H), 3.27
(dd, J ¼ 6.89, 13.08 Hz, 2H), 3.45 (s, 2H), 3.51 (s, 2H), 3.71e3.84 (m,
1H), 5.03e5.07 (m, 1H), 5.28 (d, J ¼ 7.89 Hz, 1H), 7.08e7.11 (m, 2H),
7.19e7.22 (m, 2H), 7.24e7.32 (m, 5H); ESI-MS (m/z) 424.6 [M þ H]þ.
The HCl salt was prepared in ethanol; mp: 185e188 ꢁC; Anal. calcd.
for C25H34N3O3Cl$H2O: C, 62.82; H, 7.59; N, 8.79. Found: C, 63.49; H,
7.63; N, 9.23.
5.6. 4-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl o-
tolylcarbamate (10)
Procedure: K1; Reagents: comp. 1 (0.32 g, 1 mmol), o-tolyl
isocyanate (0.13 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); Puri-
fication: column chromatography (CHCl3/MeOH, 9:1); white solid;

A. Wie˛ckowska et al. / European Journal of Medicinal Chemistry 45 (2010) 5602e5611
5609
yield: 50%; mp: 178e181 ꢁC; 1H NMR (300 MHz, CDCl3)
d
(ppm):
(m, 3H), 7.22e7.32 (m, 6H); ESI-MS (m/z) 396.7 [M þ H]þ. The HCl
salt was prepared in ethanol; mp: 190e193 ꢁC; Anal. calcd. for
C23H30N3O3Cl$2H2O: C, 59.03; H, 7.32; N, 8.98. Found: C, 59.71; H,
7.49; N, 8.93.
1.30e1.43 (m, 2H), 1.81e1.87 (m, 2H), 2.05e2.13 (m, 2H), 2.34 (s,
3H), 2.74 (d, J ¼ 11.77 Hz, 2H), 3.47 (s, 2H), 3.53 (s, 2H), 3.70e3.86
(m, 1H), 5.29 (d, J ¼ 7.92 Hz, 1H), 6.72e6.81 (m, 1H), 7.05e7.10 (m,
1H), 7.17e7.32 (m, 11H), 7.78e7.89 (m, 1H); ESI-MS (m/z) 458.4
[M þ H]þ. The HCl salt was prepared in ethanol; mp: 205e208 ꢁC;
Anal. calcd. for C28H32N3O3Cl$H2O: C, 65.68; H, 6.69; N, 8.21. Found:
C, 65.54; H, 7.15; N, 7.87.
5.11. 3-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl o-
tolylcarbamate (15)
Procedure: K1; Reagents: comp. 2 (0.32 g, 1 mmol), o-tolyl
isocyanate (0.13 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); Puri-
fication: column chromatography (CHCl3/MeOH, 9:1); white solid;
5.7. 3-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl
butylcarbamate (11)
yield: 44%; mp: 151e154 ꢁC; 1H NMR (300 MHz, CDCl3)
d (ppm):
Procedure: K1; Reagents: comp. 2 (0.32 g, 1 mmol), butyl
isocyanate (0.10 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL);
Purification: recrystallization from EtOAc; white solid; yield: 90%;
1.29e1.43 (m, 2H), 1.81e1.85 (m, 2H), 2.04e2.12 (m, 2H), 2.33 (s,
3H), 2.73 (d, J ¼ 11.74 Hz, 2H), 3.46 (s, 2H), 3.54 (s, 2H), 3.72e3.86
(m, 1H), 5.43 (d, J ¼ 7.52 Hz, 1H), 6.78e6.87 (m, 1H), 7.05e7.38 (m,
12H), 7.77e7.89 (m, 1H); ESI-MS (m/z) 459 [M þ H]þ. The HCl salt
was prepared in ethanol; mp: 172e175 ꢁC; Anal. calcd. for
C28H32N3O3Cl$5H2O: C, 57.58; H, 7.25; N, 7.19. Found: C, 57.17; H,
6.72; N, 7.12.
mp: 127e128 ꢁC; 1H NMR (300 MHz, CDCl3)
d (ppm): 0.95 (t,
J ¼ 7.25 Hz, 3H), 1.31e1.45 (m, 4H), 1.50e1.60 (m, 2H), 1.81e1.85
(m, 2H), 2.06e2.13 (m, 2H), 2.74 (d, J ¼ 11.61 Hz, 2H), 3.26 (dd,
J ¼ 6.95, 13.06 Hz, 2H), 3.47 (s, 2H), 3.52 (s, 2H), 3.71e3.83 (m, 1H),
5.08 (t, J ¼ 5.12 Hz, 1H), 5.40 (d, J ¼ 7.41 Hz, 1H), 7.02e7.06
(m, 3H), 7.20e7.33 (m, 6H); ESI-MS (m/z) 425.1 [M þ H]þ. The HCl
salt was prepared in ethanol; mp: 205e206 ꢁC; Anal. calcd. for
C25H34N3O3Cl$H2O: C, 62.82; H, 7.59; N, 8.79. Found: C, 63.11; H,
7.75; N, 8.72.
5.12. 4-(2-(4-Benzylpiperazin-1-yl)-2-oxoethyl)-phenyl
butylcarbamate (16)
Procedure: K1; Reagents: comp. 3 (0.31 g, 1 mmol), butyl isocy-
5.8. 3-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl
isopropylcarbamate (12)
anate (0.10 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); white solid;
yield: 85%; mp: 111e113 ꢁC; 1H NMR (300 MHz, CDCl3)
d (ppm):
0.94 (t, J ¼ 7.25 Hz, 3H),1.33e1.45(m, 2H),1.50e1.60 (m, 2H), 2.27 (t,
J ¼ 4.92 Hz, 2H), 2.40 (t, J ¼ 5.09 Hz, 2H), 3.26 (dd, J ¼ 13.07, 6.89 Hz,
2H), 3.43 (t, J ¼ 4.99 Hz, 2H), 3.47 (s, 2H), 3.64 (t, J ¼ 5.06 Hz, 2H),
3.69 (s, 2H), 5.02 (t, J ¼ 5.05 Hz, 1H), 7.05e7.08 (m, 2H), 7.19e7.23
(m, 2H), 7.24e7.33 (m, 5H); ESI-MS (m/z) 410.5 [M þ H]þ. The HCl
salt was prepared in ethanol; mp: 215e218 ꢁC; Anal. calcd. for
C24H32N3O3Cl: C, 64.63; H, 7.23; N, 9.42. Found: C, 64.21; H, 7.27;
N, 9.35.
Procedure: K1; Reagents: comp. 2 (0.320 g, 1 mmol), isopropyl
isocyanate (0.085 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL);
Purification: recrystallization from EtOAc; white solid; yield: 80%;
mp: 153e155 ꢁC; 1H NMR (300 MHz, CDCl3)
d (ppm): 1.23 (d,
J ¼ 6.54 Hz, 6H),1.32e1.45 (m, 2H),1.81e1.86 (m, 2H), 2.07e2.14 (m,
2H), 2.75 (d, J ¼ 11.62 Hz, 2H), 3.48 (s, 2H), 3.52 (s, 2H), 3.72e3.93
(m, 2H), 4.91 (d, J ¼ 6.98 Hz, 1H), 5.40 (d, J ¼ 7.41 Hz, 1H), 7.03e7.06
(m, 3H), 7.23e7.33 (m, 6H); ESI-MS (m/z) 410.3 [M þ H]þ. The HCl
salt was prepared in ethanol; mp: 178e182 ꢁC; Anal. calcd. for
C24H32N3O3Cl$H2O: C, 62.13; H, 7.39; N, 9.06. Found: C, 61.61; H,
7.42; N, 8.74.
5.13. 4-(2-(4-Benzylpiperazin-1-yl)-2-oxoethyl)-phenyl
propylcarbamate (17)
5.9. 3-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl
ethylcarbamate (13)
Procedure: K1; Reagents: comp. 3 (0.310 g, 1 mmol), propyl
isocyanate (0.085 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); white
solid; yield: 80%; mp: 130e132 ꢁC; 1H NMR (300 MHz, CDCl3)
Procedure: K1; Reagents: comp. 2 (0.320 g, 1 mmol), ethyl
isocyanate (0.071 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL);
Purification: recrystallization from EtOAc; white solid; yield: 88%;
d
(ppm): 0.97 (t, J ¼ 7.40 Hz, 3H), 1.53e1.65 (m, 2H), 2.26e2.29 (m,
2H), 2.39e2.42 (m, 2H), 3.23 (dd, J ¼ 6.72, 13.44, Hz, 2H), 3.42e3.46
(m, 2H), 3.48 (s, 2H), 3.62e3.66(m, 2H), 3.69 (s, 2H), 5.04 (t,
J ¼ 5.29 Hz,1H), 7.05e7.08 (m, 2H), 7.19e7.22 (m, 2H), 7.24e7.34 (m,
5H); EI-MS (m/z) 395.2 [M]þ. The HCl salt was prepared in ethanol;
mp: 235e238 ꢁC; Anal. calcd. for C23H30N3O3Cl: C, 63.95; H, 7.00; N,
9.73. Found: C, 63.88; H, 7.11; N, 9.80.
mp: 125e128 ꢁC; 1H NMR (300 MHz, CDCl3)
d (ppm): 1.21 (t,
J ¼ 7.24 Hz, 3H), 1.41e1.50 (m, 2H), 1.83e1.87 (m, 2H), 2.12e2.19 (m,
2H), 2.79 (d, J ¼ 11.67 Hz, 2H), 3.25e3.34 (m, 2H), 3.52 (s, 2H), 3.53
(s, 2H), 3.73e3.85 (m, 1H), 5.08e5.14 (m, 1H), 5.52 (d, J ¼ 6.89 Hz,
1H), 7.02e7.07 (m, 3H), 7.22e7.33 (m, 6H); ESI-MS (m/z) 396.1
[M þ H]þ. The HCl salt was prepared in ethanol; mp: 180e183 ꢁC;
Anal. calcd. for C23H30N3O3Cl$2H2O: C, 59.03; H, 7.32; N, 8.98.
Found: C, 59.10; H, 7.32; N, 8.53.
5.14. 4-(2-(4-Benzylpiperazin-1-yl)-2-oxoethyl)-phenyl
isopropylcarbamate (18)
5.10. 3-(2-(1-Benzylpiperidin-4-ylamino)-2-oxoethyl)-phenyl
dimethylcarbamate (14)
Procedure: K1; Reagents: comp. 3 (0.310 g, 1 mmol), isopropyl
isocyanate (0.085 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); white
solid; yield: 70%; mp: 127e129 ꢁC; 1H NMR (300 MHz, CDCl3)
Procedure: K2; Reagents: comp. 2 (0.320 g, 1 mmol), dime-
thylcarbamoyl chloride (0.107 g, 1 mmol), K2CO3 (0.410 g, 3 mmol),
acetone (10 mL); Purification: recrystallization from EtOAc; white
solid; yield: 81%; mp: 85e88 ꢁC; 1H NMR (300 MHz, CDCl3)
d
(ppm): 1.23 (d, J ¼ 6.55 Hz, 6H), 2.27 (t, J ¼ 4.99 Hz, 2H), 2.40 (t,
J ¼ 5.11 Hz, 2H), 3.42 (t, J ¼ 5.03 Hz, 2H), 3.46 (s, 2H), 3.64
(t, J ¼ 5.07 Hz, 2H), 3.69 (s, 2H), 3.80e3.93 (m, 1H), 4.90 (d, J
¼ 7.23 Hz, 1H), 7.05e7.08 (m, 2H), 7.18e7.22 (m, 2H), 7.26e7.33 (m,
5H); ESI-MS (m/z) 396.1 [M þ H]þ. The HCl salt was prepared in
ethanol; mp: 239e243 ꢁC; Anal. calcd. for C23H30N3O3Cl: C, 63.95;
H, 7.00; N, 9.73. Found: C, 63.51; H, 7.03; N, 9.64.
d
(ppm): 1.30e1.43 (m, 2H), 1.78e1.83 (m, 2H), 2.03e2.12 (m, 2H),
2.73 (d, J ¼ 11.83 Hz, 2H), 2.98 (s, 3H), 3.07 (s, 3H), 3.45 (s, 2H), 3.50
(s, 2H), 3.68e3.82 (m, 1H), 5.48 (d, J ¼ 7.82 Hz, 1H), 6.99e7.05

5610
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5.15. 4-(2-(4-Benzylpiperazin-1-yl)-2-oxoethyl)-phenyl
ethylcarbamate (19)
7.20e7.35 (m, 6H); ESI-MS (m/z) 396.6 [M þ H]þ. The HCl salt was
prepared in ethanol; mp: 210e215 ꢁC; Anal. calcd. for C23H30N3O3Cl:
C, 63.95; H, 7.00; N, 9.73. Found: C, 63.36; H, 6.87; N, 9.53.
Procedure: K1; Reagents: comp. 3 (0.310 g, 1 mmol), ethyl
isocyanate (0.071 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); white
solid; yield: 97%; mp: 108e112 ꢁC; 1H NMR (300 MHz, CDCl3)
5.20. 3-(2-(4-Benzylpiperazin-1-yl)-2-oxoethyl)-phenyl
ethylcarbamate (24)
d
(ppm): 1.21 (t, J ¼ 7.24 Hz, 3H), 2.27 (t, J ¼ 4.97 Hz, 2H), 2.40
(t, J ¼ 5.10 Hz, 2H), 3.25e3.34 (m, 2H), 3.43(t, J ¼ 5.02 Hz, 2H), 3.47
(s, 2H), 3.64 (t, J ¼ 5.05 Hz, 2H), 3.69 (s, 2H), 5.07e5.10 (m, 1H),
7.04e7.07 (m, 2H), 7.18e7.21 (m, 2H), 7.24e7.33 (m, 5H); ESI-MS (m/
z) 381.3 [M þ H]þ. The HCl salt was prepared in ethanol; mp:
218e220 ꢁC; Anal. calcd. for C22H28N3O3Cl: C, 63.23; H, 6.75; N,
10.05. Found: C, 62.88; H, 6.75; N, 10.01.
Procedure: K1; Reagents: comp. 4 (0.310 g, 1 mmol), ethyl
isocyanate (0.071 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); oil;
yield: 99%; 1H NMR (300 MHz, CDCl3)
d
(ppm): 1.21 (t, J ¼ 7.25 Hz,
3H), 2.27(t, J ¼ 4.97 Hz, 2H), 2.40 (t, J ¼ 5.11 Hz, 2H), 3.24e3.34 (m,
2H), 3.42(t, J ¼ 5.02 Hz, 2H), 3.46 (s, 2H), 3.64 (t, J ¼ 5.06 Hz, 2H),
3.71 (s, 2H), 5.03e5.06 (m, 1H), 7.00e7.06 (m, 3H), 7.21e7.33
(m, 6H); ESI-MS (m/z) 382.3 [M þ H]þ. The HCl salt was prepared in
ethanol; mp: 188e191 ꢁC; Anal. calcd. for C22H28N3O3Cl$H2O: C,
60.61; H, 6.94; N, 9.64. Found: C, 61.10; H, 6.83; N, 9.42.
5.16. 4-(2-(4-Benzylpiperazin-1-yl)-2-oxoethyl)-phenyl
dimethylcarbamate (20)
Procedure: K2; Reagents: comp. 3 (0.310 g, 1 mmol), dime-
thylcarbamoyl chloride (0.107 g, 1 mmol), K2CO3 (0.410 g, 3 mmol),
acetone (10 mL); Purification: recrystallization from EtOAc; white
5.21. 3-(2-(4-Benzylpiperazin-1-yl)-2-oxoethyl)-phenyl
dimethylcarbamate (25)
solid; yield: 89%; mp: 81e84 ꢁC; 1H NMR (300 MHz, CDCl3)
d
(ppm):
Procedure: K2; Reagents: comp. 4 (0.310 g, 1 mmol), dime-
thylcarbamoyl chloride (0.107 g, 1 mmol), K2CO3 (0.410 g, 3 mmol),
acetone (10 mL); Purification: column chromatography (CH2Cl2/
2.26 (t, J ¼ 4.99 Hz, 2H), 2.39 (t, J ¼ 5.09 Hz, 2H), 2.99 (s, 3H), 3.08 (s,
3H), 3.41 (t, J ¼ 4.91 Hz, 2H), 3.45 (s, 2H), 3.62 (t, J ¼ 5.00 Hz, 2H), 3.68
(s, 2H), 7.01e7.06 (m, 2H), 7.18e7.21 (m, 2H), 7.24e7.32 (m, 5H); ESI-
MS (m/z) 381.3 [M þ H]þ. The HCl salt was prepared in ethanol; mp:
237e240 ꢁC; Anal. calcd. for C22H28N3O3Cl: C, 63.23; H, 6.75; N,
10.05. Found: C, 63.10; H, 6.98; N, 10.17.
MeOH, 9.5:0.5); oil; yield: 84%; 1H NMR (300 MHz, CDCl3)
d (ppm):
2.27 (t, J ¼ 5.03 Hz, 2H), 2.40 (t, J ¼ 5.10 Hz, 2H), 2.99 (s, 3H), 3.07 (s,
3H), 3.42 (t, J ¼ 5.04 Hz, 2H), 3.46 (s, 2H), 3.64 (t, J ¼ 5.05 Hz, 2H),
3.71 (s, 2H), 6.98e7.05 (m, 3H), 7.20e7.32 (m, 6H); ESI-MS (m/z)
382.7 [M þ H]þ. The HCl salt was prepared in ethanol; mp:
218e220 ꢁC; Anal. calcd. for C22H28N3O3Cl: C, 63.23; H, 6.75; N,
10.05. Found: C, 63.52; H, 7.02; N, 10.14.
5.17. 4-(2-(4-Benzylpiperazin-1-yl)-2-oxoethyl)-phenyl
o-tolylcarbamate (21)
Procedure: K1; Reagents: comp. 3 (0.31 g, 1 mmol), o-tolyl
isocyanate (0.13 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); Purifi-
cation: column chromatography (AcOEt/MeOH, 10:0.5); oil; yield:
5.22. AChE/BuChE inhibition activity
AChE and BuChE inhibitory activities were evaluated by spec-
trophotometrical Ellman’s method [30] using AChE from electric
eel and BuChE from horse serum (2.5 units/1 mL). The reaction took
place in a final volume of 3.32 mL of 100 mM phosphate buffer, pH
8.0, containing 0.25 unit of AChE or BuChE, 0.3 mmol 5,50-dithio-bis
(2-nitrobenzoic) acid (DTNB, Ellman’s reagent) and 0.45 mmol
acetylthiocholine or butyrylthiocholine as substrates. The tested
compounds were preincubated with the enzyme for 5 min at 20 ꢁC
before starting the reaction by adding a substrate. Enzyme activity
was determined by measuring the absorbance at 412 nm during
5 min with the Perkin Elmer Lambda 12. As a reference, sample
without inhibitor was used (100% enzyme activity). Each
compound was assayed at 6 concentrations in triplicate. The reac-
tion rates were compared and the percent of inhibition due to the
presence of the test compounds was calculated. Data from
concentration-inhibition experiments were calculated by non-
linear regression analysis using the GraphPad Prism program
(GraphPad Prism Software Inc. 2005), which gave estimates of IC50
(the compound concentration producing 50% of enzyme inhibi-
tion). DNTB, acetylthiocholine, butyrylthiocholine and the enzymes
were purchased from SigmaeAldrich.
47%; 1H NMR (300 MHz, CDCl3)
d
(ppm): 2.29 (t, J ¼ 5.03 Hz, 2H), 2.34
(s, 3H), 2.41 (t, J ¼ 5.13 Hz, 2H), 3.45 (t, J ¼ 5.00 Hz, 2H), 3.48 (s, 2H),
3.65(t, J ¼ 5.15 Hz, 2H), 3.72 (s, 2H), 7.06e7.34 (m, 13H), 7.80e7.87
(m, 1H); ESI-MS (m/z) 444.6 [M þ H]þ. The HCl salt was prepared in
ethanol; mp: 200e206 ꢁC; Anal. calcd. for C27H30N3O3Cl$0.5H2O: C,
66.25; H, 6.33; N, 8.58. Found: C, 66.41; H, 6.84; N, 8.91.
5.18. 3-(2-(4-Benzylpiperazin-1-yl)-2-oxoethyl)-phenyl
butylcarbamate (22)
Procedure: K1; Reagents: comp. 4 (0.31 g, 1 mmol), butyl isocy-
anate (0.10 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); oil; yield:
88%; 1H NMR (300 MHz, CDCl3)
d
(ppm): 0.94 (t, J ¼ 7.28 Hz, 3H),
1.32e1.45 (m, 2H), 1.50e1.60 (m, 2H), 2.27 (t, J ¼ 4.97 Hz, 2H), 2.40
(t, J ¼ 5.08 Hz, 2H), 3.25 (dd, J ¼ 6.96, 13.05 Hz, 2H), 3.39e3.43 (m,
2H), 3.46 (s, 2H), 3.62e3.65 (m, 2H), 3.71 (s, 2H), 5.05 (t, J ¼ 5.39 Hz,
1H), 6.9þ9e7.05 (m, 3H) 7.22e7.33 (m, 6H); ESI-MS (m/z) 410.8
[M þ H] . The HCl salt was prepared in ethanol; mp: 203e206 ꢁC;
Anal. calcd. for C24H32N3O3Cl$H2O: C, 62.13; H, 7.39; N, 9.06. Found:
C, 62.77; H, 7.29; N, 9.23.
5.19. 3-(2-(4-Benzylpiperazin-1-yl)-2-oxoethyl)-phenyl
isopropylcarbamate (23)
5.23. Kinetic studies of AChE and BuChE inhibition
Kinetic studies were performed using Ellman’s method for
compound 14, which was the most potent inhibitor of both AChE
and BuChE. The test was carried out without the inhibitor and in
0.1 mM and 1 mM concentration of the inhibitor for AChE and
0.01 mM and 0.1 mM concentration for BuChE. Substrates of the
reaction, acetylthiocholine and butyrylthiocholine, were used in
following concentrations: 40, 50, 100, 200, 300, 400 and 500 mM.
The obtained data were used to create substrateevelocity curves,
Procedure: K1; Reagents: comp. 4 (0.310 g, 1 mmol), isopropyl
isocyanate (0.085 g, 1 mmol), Et3N (5 drops), CH2Cl2 (10 mL); Puri-
fication: recrystallization from EtOAc; white solid; yield: 81%; mp:
166e168 ꢁC; 1H NMR (300 MHz, CDCl3)
d
(ppm): 1.23 (d, J ¼ 6.55 Hz,
6H), 2.27 (t, J ¼ 4.96 Hz, 2H), 2.40 (t, J ¼ 5.10 Hz, 2H), 3.42 (t,
J ¼ 4.99 Hz, 2H), 3.46(s, 2H), 3.64 (t, J ¼ 5.05 Hz, 2H), 3.71 (s, 2H),
3.80e3.95 (m, 1H), 4.88 (d, J ¼ 6.74 Hz, 1H), 7.00e7.06 (m, 3H),

A. Wie˛ckowska et al. / European Journal of Medicinal Chemistry 45 (2010) 5602e5611
5611
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Acknowledgments
This work was supported by funds from grant of Ministry of
Science and High Education no 3254/P01/2006/31 and the project
of Jagiellonian University Collegium Medicum no K/ZDZ/000723.
We thank Dr. Krzysztof Wie˛ckowski for critical review of this article
and Dr. Malte Behrends for linguistic advice.
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