Synthesis and screening for antiacetylcholinesterase activity of (1-benzyl-4-oxopiperidin-3-ylidene)methylindoles and -pyrroles related to donepezil

Article abstract of DOI:10.1021/jm0109356

The design, synthesis, and rapid evaluation of a new class of acetylcholinesterase (ACHE) inhibitors related to donepezil are reported. A molecular dynamics simulation of the complex between AChE and one representative compound of the series showed a possible inhibitor binding mode in which favorable interactions are formed between the benzylpiperidinone moiety and some active-site residues. The biochemical evaluation of this newly synthesized series was performed using a chemiluminescent method suitable for high-throughput screening.

Full text of DOI:10.1021/jm0109356

J . Med. Chem. 2001, 44, 4011-4014
4011
Brief Articles
Syn th esis a n d Scr een in g for An tia cetylch olin ester a se Activity of
(1-Ben zyl-4-oxop ip er id in -3-ylid en e)m eth ylin d oles a n d -p yr r oles Rela ted to
Don ep ezil
Aldo Andreani,* Andrea Cavalli, Massimiliano Granaiola, Massimo Guardigli, Alberto Leoni,
Alessandra Locatelli, Rita Morigi, Mirella Rambaldi, Maurizio Recanatini, and Aldo Roda
Dipartimento di Scienze Farmaceutiche, Universita` di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received May 21, 2001
The design, synthesis, and rapid evaluation of a new class of acetylcholinesterase (AChE)
inhibitors related to donepezil are reported. A molecular dynamics simulation of the complex
between AChE and one representative compound of the series showed a possible inhibitor
binding mode in which favorable interactions are formed between the benzylpiperidinone moiety
and some active-site residues. The biochemical evaluation of this newly synthesized series was
performed using a chemiluminescent method suitable for high-throughput screening.
Ch a r t 1
In tr od u ction
Alzheimer’s disease (AD) is a neurodegenerative
disease of the central nervous system (CNS) character-
ized especially by premature mental deterioration. A
wide range of evidence shows that acetylcholinesterase
(AChE) inhibitors can interfere with the progression of
AD.1,2 The successful development of these compounds
was based on the well-accepted hypothesis that the
decline in cognitive and mental functions associated
with AD is related to the loss of cortical cholinergic
neurotransmission. Donepezil (E2020, Chart 1)3 inau-
gurates a new class of AChE inhibitors with longer and
more selective action with manageable adverse effects.
The recently published4 crystal structure of donepezil
bound to AChE revealed that donepezil binds along the
active-site gorge where interactions with the enzyme
occur through the benzylpiperidine and indanone
moieties. Donepezil forms no direct contact with the
protein via hydrogen bonds/salt bridge; all hydrogen
bonds are mediated via water molecules.4
Resu lts a n d Discu ssion
The potent AChE inhibitor donepezil was modified by
replacing the indanone moiety with an indole and by
introducing on the piperidin(on)e ring a double bond
connecting the basic ring to the indole fragment (Table
1). This modification was aimed at obtaining less flexible
molecules devoid of stereogenic centers. To verify the
possibility that such compounds can bind into the AChE
gorge, we built a three-dimensional model of the docking
complex between the N-benzoylated derivative 5b and
AChE. The inhibitor was positioned into the AChE
gorge in an orientation similar to that of donepezil, as
revealed by the X-ray crystallographic analysis4 (Figure
1a). The benzyl moiety was stacked against the indole
ring of Trp84, and the charged nitrogen of the piperi-
dinone ring was oriented in a position suitable for a
π-cation interaction with the phenyl ring of Phe330.
However, after 100 ps of molecular dynamics simula-
tions, the inhibitor assumed a different orientation
(Figure 1b) with respect to that of donepezil, even if it
still maintained a position in the gorge compatible with
the possibility of binding. The resulting enzyme-
inhibitor interaction seems to be of an electrostatic
nature and involves only the benzylpiperidinone moiety.
The π-stacking interaction of the benzyl ring with Trp84
is maintained, but the positive charge of the protonated
nitrogen atom seems to be neutralized by the negative
In an attempt to obtain analogues of donepezil that
could maintain its main spatial and physicochemical
characteristics while being more compact and less
flexible, we applied a strategy to synthesize and rapidly
evaluate a series of molecules to be eventually developed
as AChE inhibitors for use in AD. We designed the small
library of benzylpiperidinone derivatives 4a-17 (Schemes
1-3), where the indanone group was replaced by a
bioisosteric indole or pyrrole ring bearing different
substituents. The biochemical evaluation of the newly
synthesized series was performed using a chemilumi-
nescent (CL) method suitable for high-throughput screen-
ing (HTS) of AChE inhibitors.
* To whom correspondence should be addressed. Phone: +39-051-
2099714. Fax: +39-051-2099734. E-mail: aldoandr@alma.unibo.it.
10.1021/jm0109356 CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/02/2001

4012 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Brief Articles
Sch em e 1
Sch em e 2
Sch em e 3
side chains of Asp72 and, eventually, Tyr334. Compared
to donepezil, the important π-stacking interaction of the
indanone ring with Trp279 is lost because neither the
indole nor the benzoyl fragments of 5b are correctly
oriented to be in contact with that residue. On the basis
of this docking model, we concluded that the [(1-benzyl-
4-oxopiperidin-3-ylidene)methyl]-1H-indole scaffold was
suitable for the exploration of a new class of AChE
inhibitors analogues of donepezil. Their syntheses and
Table 2 containing IR and NMR data are in Supporting
Information.
The CL method used for the evaluation of the biologi-
cal activity of the newly synthesized compounds is based
on a series of coupled enzymatic reactions involving
AChE, choline oxidase, and horseradish peroxidase,
with luminol as the chemiluminescent substrate. Such
a CL system was previously used for the measurement
of AChE activity5 and ACh concentration;6 preliminary
results concerning its use for the evaluation of AChE
inhibition were also published.7 The assay is performed
in 384-well microtiter plates, and a low-light imaging
luminograph is used in order to acquire the CL signal

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 4013
Ta ble 1. Compounds 2, 4, 5, 8-12, 14, and 17
compd
R
R1
formula (MW)
methoda or lit.
mp, °C
yield, %
solventb
2a
2b
2c
2d
2e
2f
2g
2h
4a
4b
4c
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
8
9
10
11
12
14
17
H
H
H
H
CH2C6H5
COC6H5
CO(C6H4)F-p
CO(C6H4)Cl-p
COC6H5
COCH3
COCH3
COCH3
19, 20
21, 22
A
23, 24
C16H10FNO2 (267.3)
C23H17NO3 (355.4)
135-138
147-149
82
70
F
F
OCH2C6H5
H
OCH3
OCH2C6H5
H
OCH3
OCH2C6H5
H
H
H
H
H
H
H
H
H
A
25
26
A
C
C
C
D
B
B
B
A
A
A
A
E
A
A
B
B
D
D
C
A
C
C18H15NO3 (293.3)
C21H20N2O (552.5)
C22H22N2O2 (346.4)
C28H26N2O2 (422.5)
C28H26N2O (406.5)
C28H24N2O2 (420.5)
C28H23FN2O2 (438.5)
C28H23ClN2O2 (455.0)
C29H26N2O3 (450.5)
C28H23N3O4 (465.5)
C29H23N3O2 (445.5)
C27H24N2O3S (456.6)
C26H28N2O3 (416.5)
C23H22N2O2 (358.4)
C29H26N2O3 (450.5)
C35H30N2O3 (526.6)
114-117
198-200
175-178
160-163
170-172
160-163
131-133
155-158
205-210 dec
210-213 dec
142-145
158-161
139-142
128-131
144-148
130-133
153-156
189-192
175-176
156-158 dec
122-124
132-134
185-186
70
15
15
15
60
15
20
15
15
30
45
80
15
20
50
15
50
5
F
H
I
H
H
H
I
H
H
I
I
G
I
H
I
H
H
H
H
J
H
I
H
CH2C6H5
COC6H5
CO(C6H4)F-p
CO(C6H4)Cl-p
CO(C6H4)OCH3-p
CO(C6H4)NO2-p
CO(C6H4)CN-p
SO2C6H5
COOC(CH3)3
COCH3
COC6H5
H
OCH3
OCH2C6H5
COC6H5
C
C
24H22N2O2 (370.4)
22H21N3O (343.4)
C17H18N2O (266.3)
10
40
32
10
35-40
C
C
17H18N2O (266.3)
24H22N2O2 (370.4)
C22H21N3O (343.4)
20H22N2O2 (322.4)
C
B, C, D
a
b
See Scheme 1; literature references available in Supporting Information. Recrystallization solvent (F ) acetone/petroleum ether, G
) ethyl acetate) or eluent for column chromatography (H ) petroleum ether/acetone 80/20; I ) petroleum ether/ethyl acetate 80/20; J )
petroleum ether/ethyl ether 90/10).
F igu r e 1. Interactions of (a) donepezil (magenta) and (b) 5b (yellow) with some selected residues of the AChE gorge. Hydrogen
atoms are not shown for clarity.
from the whole plate, making it possible to examine up
to 30 compounds in the same analytical session.
The analytical procedure relies on the analysis of the
kinetics of the CL emission. It was proven that the
initial slope of the luminescence kinetic profile is
directly proportional to the activity of AChE in the
sample. Therefore, the residual AChE activity in the
presence of an AChE inhibitor is simply given by the
ratio of the slope of the luminescence kinetic profile in
the presence of the inhibitor to that in the absence of

4014 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Ta ble 3. Antiacetylcholinesterase Activitya
Brief Articles
microtiter plates and the high detectability and fast
kinetics of the CL signal. Moreover, the possibility of
using a DMSO/water 1:10 (v/v) mixed solvent in the
preparation of AChE inhibitor solutions facilitates the
analysis of compounds with a relatively low solubility
in water. The procedure proved to be reliable in terms
of reproducibility of the data, and the obtained IC50
values were in reasonable agreement with those re-
ported in the literature for the known compounds.
In conclusion, we have explored an integrated ap-
proach for the design, synthesis, and rapid evaluation
of a new class of AChE inhibitors structurally related
to donepezil. The availability of a simple, rapid, and
sensitive CL high-throughput screening method for
AChE inhibitors facilitated the study of the biological
activity of such compounds and the identification of the
most promising molecules. The new derivatives show a
rather low degree of potency against the enzyme, but
the molecular skeleton allows for structural modifica-
tions that are presently under study to explore the
potential of the series toward new cholinergic agents
useful in the treatment of AD.
compd
IC50, µΜ
compd
IC50, µΜ
11 ( 2
4a
4b
4c
5a
5b
5c
5d
5e
5f
8 ( 2
6 ( 2
25 ( 5
>100
70 ( 15
60 ( 10
50 ( 20
70 ( 5
50 ( 5
20 ( 10
insoluble
30 ( 12
5j
5k
5l
80 ( 10
insoluble
30 ( 10
18 ( 4
8
9
10
11
12
14
11 ( 3
6 ( 2
30 ( 5
6 ( 3
5g
5h
5i
17
30 ( 10
0.18 ( 0.05b
0.04 ( 0.01c
tacrine
donepezil
a
b
Mean ( SD of at least three independent measures. IC50
values reported in the literature: 4-0.05 µM. c IC50 values
reported in the literature: 0.014-0.005 µM.
the inhibitor. Evaluation of AChE inhibition in the
presence of different concentrations of inhibitor allows
one to obtain the dose/inhibition curve, and thus the IC50
value of the inhibitor. The method was used to deter-
mine the IC50 values of the well-known AChE inhibitors
tacrine and donepezil. These inhibitors, as well as the
other compounds, were tested as free bases. The result
obtained for tacrine (Table 3) was in good agreement
with those reported in the literature, whereas the IC50
value obtained for donepezil was somewhat higher than
those previously reported, which ranged from 0.005 to
0.014 µM.
Table 3 shows the IC50 values for the newly synthe-
sized compounds measured using the described HTS
method; the data reported represent the mean ( SD of
at least three independent measures; compounds 5h and
5l could not be measured because of their low solubility.
In many cases the coefficients of variation were lower
than 20%, which can be considered a satisfying value
for a first-level screening method.
As regards the AChE inhibition data (Table 3), we
observe that the parent compound 3-[(1-benzyl-4-oxopi-
peridin-3-ylidene)methyl]-1H-indole (4a ) showed activ-
ity in the micromolar range. This is a degree of potency
lower than that of both tacrine and donepezil, and the
loss of interaction with the Trp279 residue might be one
of the reasons for the decreased inhibitory activity.
Another reason for the low potency displayed by the
series with respect to donepezil might be the relative
rigidity of the [(4-oxopiperidin-3-ylidene)methyl]indole
scaffold, which could hinder the penetration into the
AChE gorge to reach the binding site. However, the new
derivatives confirmed the possibility of a binding inter-
action with the target enzyme, maintaining also an
appropriate degree of lipophilicity (calculated log P 8 for
4a is 3.35) in view of the distribution into the CNS.
The CL method allowed us to determine the IC50
values of the newly synthesized compounds in very short
times (5-10 min) and to consume small quantities of
samples and reagents, thanks to the use of 384-well
Ack n ow led gm en t. This work was supported by
University of Bologna (funds for selected research
topics) and MURST (Ministero dell’Universita` e della
Ricerca Scientifica e Tecnologica).
Su p p or tin g In for m a tion Ava ila ble: Procedure for the
synthesis of AChE compounds and Table 2 containing IR and
NMR data; literature references for Table 1. This material is
available free of charge via the Internet at http://
www.pub.acs.org.
Note Ad d ed a fter ASAP
Literature references for Table 1 have been added to
the Supporting Information. The Supporting Informa-
tion paragraph has been corrected to reflect this edition
and additional Supporting Information has been posted
on October 5, 2001.
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(4) Kryger, G.; Silman, I.; Sussman, J . L. Three-Dimensional
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J M0109356