Benzophenone-based derivatives: A novel series of potent and selective dual inhibitors of acetylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation

Article abstract of DOI:10.1016/j.ejmech.2011.02.019

The leading mechanistic theory of Alzheimer's disease (AD) is the "amyloid hypothesis" which states that the accumulation of the amyloid β protein (Aβ), and its subsequent aggregation into plaques, is responsible for the initiation of a cascade of events

Full text of DOI:10.1016/j.ejmech.2011.02.019

European Journal of Medicinal Chemistry 46 (2011) 1682e1693
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Benzophenone-based derivatives: A novel series of potent and selective dual
inhibitors of acetylcholinesterase and acetylcholinesterase-induced
beta-amyloid aggregation
,
a
b
a
Federica Belluti a , Manuela Bartolini , Giovanni Bottegoni , Alessandra Bisi ,
*
Andrea Cavalli a b, Vincenza Andrisano a, Angela Rampa a
,
a Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b Unit of Drug Discovery and Development, Italian Institute of Technology, Via Morego 30, 16163 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The leading mechanistic theory of Alzheimer’s disease (AD) is the “amyloid hypothesis” which states that
Received 13 October 2010
Received in revised form
14 January 2011
Accepted 12 February 2011
Available online 22 February 2011
the accumulation of the amyloid
responsible for the initiation of a cascade of events resulting in neurodegeneration and dementia. The
anti-amyloid disease-modifying approach, based on the decrease in the production of A , gained thus
a paramount importance. The aim of this study was the design and synthesis of a new series of
acetylcholinesterase inhibitors (AChEIs) endowed with anti-A aggregating capability. These dual
binding inhibitors, being able to interact both with the peripheral anionic site (PAS) of AChE and the
catalytic subsite, proved to be able to inhibit the AChE-induced A aggregation. Thus, starting from the
b protein (Ab), and its subsequent aggregation into plaques, is
b
b
Keywords:
b
Acetylcholinesterase
Alzheimer’s disease
Amyloid
Benzophenone
Drug design
lead compound 1, an AChEI composed by a benzophenone scaffold and a N,N0-methylbenzylamino group,
a substantial modification aimed at targeting the PAS was performed. To this aim, different amino-
terminal side chains were incorporated into this main framework, in order to mimic the dieth-
ylmethylammonium alkyl moiety of the pure PAS ligand propidium. The synthesized compounds proved
to effectively and selectively inhibit AChE. Moreover, compounds 16aec and 18a,b, with a propoxy and
Molecular modeling
a hexyloxy tether respectively, showed a good activity against the AChE-induced A
b aggregation. In
particular, molecular modeling studies confirmed that compounds carrying the diethylaminopropoxy
and the diethylaminohexyloxy side chains (compounds 16a and 19a, respectively) could suitably contact
the PAS pocket of the enzyme.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
rivastigmine, and galantamine were developed to purposely treat
AD, and currently constitute the only FDA-approved therapeutic
Alzheimer’s disease (AD) is a neurodegenerative disorder that
results in the progressive and irreversible cognitive impairment,
memory loss, and decline in language [1]. Several factors, both
genetic and environmental, such as reduced levels of the neuro-
approach. The NMDA (N-methyl-D-aspartate) antagonist mem-
antine, has also been shown to improve cognitive function and
reached the market in 2004 [5]. Nevertheless AChEIs, even if
valuable in improving the patient’s quality of life, represent only
symptomatic and palliative tools by which slowing down the
progression of the disease.
In recent years, treatment strategies aimed to interfere in the
mechanisms that drive the progression of AD, thus blocking or
reducing the gradual but slowly increasing cognitive decline,
transmitter acetylcholine (ACh), amyloid b protein (Ab) aggregation
with consequent extraneuronal neuritic senile plaques (SP) devel-
opment, tau hyperphosphorylation with intraneuronal neurofi-
brillary tangles (NFTs) formation have been considered to play
a crucial role in its pathogenesis [2]. The current standard of care for
mild to moderate AD, based on the so-called cholinergic hypoth-
esis, includes treatment with AChE inhibitors (AChEIs) to improve
cognitive function [3,4]. Several classes of AChEIs such as donepezil,
gained
a
paramount importance. These disease-modifying
approaches have become an important focus of research since they
allow to interrupt the early pathologic events involved in the onset
of the disease, and to hamper the neurotoxic cascade [6,7]. Ab is
thought to play an important role in the disease pathogenesis. In
particular, in amyloid plaques the predominant form is Ab42, that in
vitro exhibits lower solubility and a greater propensity to form
* Corresponding author. Tel.: þ39 051 2099732; fax: þ39 051 2099734.
E-mail address: federica.belluti@unibo.it (F. Belluti).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.019

F. Belluti et al. / European Journal of Medicinal Chemistry 46 (2011) 1682e1693
1683
O
fibrillar aggregates than the variant Ab40 [8,9]. Multiple lines of
evidence, leading to the “amyloid hypothesis”, suggest that the
production and the accumulation of oligomeric aggregates of A
O
O
H3C
H3C
b
may be a central event in the pathogenesis of AD, since they are
thought to be able to initiate the pathogenic cascade, ultimately
leading to neuronal loss and dementia [10]. Current revisions to the
amyloid hypothesis revealed the fundamental role also for soluble
Donepezil
N
H2N
CH3
A
b
aggregates, that proved to be the proximate effectors of synapse
loss and neuronal injury, exerting thus neurotoxic activities [11,12].
Inhibitors of the A aggregation and toxicity may be effective in
blocking the progression of the pathology. Based on this evidence,
several A -targeted therapeutic strategies are being pursued, such
NH2
O
N
+
O
O
b
H3C
H3C
CH3
+
H3C
N
N
b
1
Propidium
CH3
as modulation of A
and inhibition of A
ergic role, binds to A
b
b
production, enhancement of A
aggregation [13]. AChE, with its non cholin-
and as such promotes its assembly and
b degradation
b
O
deposition into insoluble fibrils by means of a proteineprotein
interaction. AChE is integrated into the amyloid aggregates and
forms stable complexes that, besides exerting a fibrillogenic effect,
CH3
N
R1
( )
N
nX
R2
boost the neurotoxicity of Ab fibrils [14,15]. The Ab proaggregating
action seems to be associated with a specific motif, located close to
the entrance of the AChE active-site gorge, termed the peripheral
anionic site (PAS) [16,17]. Blockade of this site by specific inhibitors
has emerged as promising disease-modifying therapeutic strate-
gies for AD. Based on these assumptions, the dual binding AChEIs,
molecules able to interact simultaneously with both the catalytic
and the peripheral binding sites of the enzyme, emerged as valu-
able tool to pursue a disease-modifying approach [18].
7a,b,c: X=NHCO, n=1
8b: X=NHCO, n=4
16a-d: X=O, n=3
17a,b: X=O, n=5
18a,b: X=O, n=6
19a,b: X=O, n=7
O
NH
N
O
NR1R2
N
N
N
N
d
c
a
b
22: X= p-OC6H4, n=2, NR1R2=a
Fig. 1. Donepezil, propidium, lead compound 1, and general structure of the benzo-
At this regard, several classes of dual binding site AChEIs have
been designed and developed and proved to be endowed with
a strong inhibitory activity due to the increased capability to
interact with the active sites of the target [19e21]. In a previous
project, aimed at the identification of new AChEIs, we reported the
synthesis and the biological activity of some benzophenone-based
derivatives bearing a [benzyl(methyl)amino]methyl moiety [22].
This function, targeted at the AChE catalytic binding site, has been
previously introduced into a series of scaffolds and emerged as an
essential structural requirement for obtaining a potent inhibition of
the enzyme [23,24]. The benzophenone moiety, a structural
element often seen in compounds from natural sources, presents
a variety of biological activities such as anti-inflammatory, anti-
malarial and anticancer [25e27] and demonstrated to be a versatile
pharmacophoric nucleus, largely used in medicinal chemistry
programs l [28e31]. Our initial design idea for obtaining AChEIs
was to employ the benzophenone nucleus as the core structure,
phenone derivatives described in this study.
peripheral-site ligand (Fig. 1), which inhibits by 82% (at 100
mM)
the AChE-induced A aggregation [32]. Specifically, on the basis of
b
the binding mode of propidium at the PAS, with particular regard
to the position assumed by its side chain, the features of both the
amino moiety and the linker were designed [33]. The main role of
the tether was to locate the terminal amino function at the
entrance of the gorge in a correct orientation to reach the Trp286
residue. Moreover, it could also allow to explore or to fill the
narrow space of this region of the enzyme. In this view, as first line
of search, the diethylamino and piperidine groups, as amino
functions, together with the alkylamide and alkoxy functions as
linkers were introduced. The design strategy and the general
formula of the synthesized compounds are depicted in Fig. 1.
since this aromatic system established
pe
p
interactions with the
2. Results and discussion
aromatic residues lining the wall of the AChE gorge. Compound 1
(Fig.1) [22], bearing a dimethoxybenzophenone skeleton, proved to
be able to inhibit the enzyme activity at a sub-micromolar level
2.1. Chemistry
(IC50 ¼ 0.46
m
M). Nevertheless, its inability to interfere with the
The synthetic route followed for the preparation of the benzo-
phenone derivatives 7aec, 8b, 16aed, 17e19a,b, and 22 is depicted
in Schemes 1 and 2.
AChE-induced A
b
aggregation, likely related to the fact that it could
not properly contact the aminoacids involved in the proaggregating
action of AChE, represented a focal drawback (even though its
binding mode, emerged from docking studies, showed a weak
interaction with Trp286, an important residue in the PAS region of
human AChE) [16]. In the light of this outcome, in an ongoing
project aimed at developing new dual binding AChEIs, we set out to
further study this new class of compounds, modifying the active-
site AChEI 1.
To this purpose, the approach followed to convert this deriva-
tive into a chemical entity able to modulate the AChE proag-
gregating activity involved the insertion, through spacers, into the
benzophenone scaffold of a number of purposely selected amino
functions as peripheral-site interacting functions. The amino
moiety was inserted in an attempt to mimic the function of the
diethylmethylammonium alkyl group of propidium, a potent
The subset of compounds with the amide side chain (7aec and
8b) was synthesized according to Scheme 1. 4-methyl-40-nitro-
benzophenone and the corresponding bromomethyl derivative (2)
were synthesized using a previously reported procedure and
spectral data are in agreement with the literature characterizations
[34]. Derivative 2 was then subjected to nucleophilic attack by N,N0-
benzylmethylamine to give compound 3. The reduction of the nitro
group, achieved with H2 in the presence of Pd/CaCO3 as catalyst,
allowed to obtain the anilino derivative 4. The subsequent acylation
with the selected
u-chloroalkyl acyl chlorides gave the interme-
diates 5 and 6, which were finally reacted with the selected amines,
to afford the desired final compounds 7aec and 8b.
The synthetic procedure employed to achieve the alkoxy and
phenoxy subset of derivatives (16aed, 17e19a,b) is illustrated in

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F. Belluti et al. / European Journal of Medicinal Chemistry 46 (2011) 1682e1693
O
O
O
a
b
CH3
N
Br
Cl
O2N
O2N
O2N
CH3
3
2
O
O
O
CH3
N
d
c
CH3
N
( )n
N
H
5,6
H2N
4
O
e
R1
N
O
CH3
N
5,7a-c: n=1
6,8b: n=4
( )n
R2
N
H
7a-c, 8b
Scheme 1. Synthesis of compounds 7aec and 8b. Reagents and conditions: (a) NBS, (PhCOO)2O2, CCl4, reflux; (b) N,N0-benzylmethylamine, TEA, toluene, reflux; (c) H2, Pd/CaCO3 10%,
THF, r. t.; (d) -chloroalkyl chloride, CH3CN, reflux; (e) selected amine, 1-propanol, reflux.
u
Scheme 2. Lithiation of the aryl bromide 9, obtained from reaction
of 4-bromomethylbromobenzene and N,N0-benzylmethylamine,
with n-BuLi, followed by addition to 4-methoxybenzonitrile gave,
upon acid hydrolytic workup, compound 10. Cleavage of the
methoxy group was accomplished by treatment with BBr3 to obtain
the key intermediate phenol 11 that, through condensation
amines to provide the desired final compounds. The aryloxy
derivative 20 was obtained by Ullmann reaction of the key inter-
mediate 11 with 2-(4-bromophenyl)ethanol, in the presence of
.
Cs2CO3, and using (CuOTf)2 PhH and EtOAc as catalysts. The
substitution of the alcohol moiety with a chlorine atom, by means
of reaction with thionylchloride, afforded the intermediate 21.
The reaction of 21 with diethylamine gave the desired final
compound 22.
with the selected bromo-
u-halo alkanes in the presence of K2CO3,
gave the intermediates 12e15, that were reacted with the selected
O
Br
Br
a
CH3
N
b
CH3
N
H3C
Br
O
10
9
O
O
X
N
c
CH3
N
CH3
N
f
O
HO
11
20: X = OH
21: X = Cl
g
d
e
O
O
CH3
N
CH3
N
( )
nO
X
O
22
12-15
e
12,16a-d: n=3
13,17a,b: n=5
14,18a,b: n=6
15,19a,b: n=7
O
CH3
N
R1
( )n
R2
N
O
16a-d,17-19a,b
Scheme 2. Synthesis of the alkoxy and aryloxy compounds 16aed, 17e19a,b, and 22. Reagents and conditions: (a) N,N-benzylmethylamine, TEA, toluene, reflux; (b) 4-methoxy-
benzonitrile, n-BuLi, THF, ꢀ78 ꢁC, r.t., conc H2SO4, H2O, dioxane, reflux; (c) BBr3, DCM, 0 ꢁC, r. t.; (d) X-(CH2)n-Br, K2CO3, acetone, reflux; (e) selected amine, TEA, toluene, reflux. (f) 2-
(4-bromophenyl)ethanol, Cs2CO3, (CuOTf)2.PhH, 1-naphtoic acid, AcOEt, toluene, 110 ꢁC; (g) SOCl2, TEA, CH2Cl2, 0 ꢁC, r.t.

F. Belluti et al. / European Journal of Medicinal Chemistry 46 (2011) 1682e1693
1685
2.2. Biology
The synthesized benzophenone derivatives 7aec, 8b, 16aed,
17e19a,b and 22 were tested to evaluate their inhibitory potency on
recombinant human AChE according to the method of Ellman et al.
[35]. For the same set of compounds the inhibition of human
butyrylcholinestarase (BChE) was also evaluated to assess the
selectivity profile. Moreover, only for a small number of compounds
2.2.1. AChE and BChE in vitro inhibition
In this work, the previously reported lead compound 1,
endowed with a valuable AChE inhibitory potency, was trans-
formed into a dual binding site inhibitor by the insertion of
a terminal amino side chain. This transformation imparted to the
new molecules the capability to contact the PAS and thus to hamper
the AChE-induced proaggregating activity. The design rationale was
validated by the observation that compound 10, bearing a methoxy
instead of the amino side chain, was a very weak AChEI, showing
that the terminal amino function plays indeed an important role in
the process of interaction with the enzyme. The new benzophe-
none derivatives 7aec, 8b, 16aed, 17e19a,b and 22 proved to be
potent AChEIs; some of them were equipotent to the reference
compound donepezil. The AChE inhibition fluctuated, in terms of
(16aec and 18a,b) the capability to inhibit the Ab(1e40) aggrega-
tion induced by human AChE was established by means of a thio-
flavin-T based fluorometric method, using both propidium and
donepezil as reference compounds [32]. A
nant form of amyloid in biological fluids, representing almost 90%
of the total amount of A [36,37]. It is therefore conceivable that, in
sporadic form of AD, A (1e40) constitutes the most abundant
b(1e40) is the predomi-
b
b
b
IC50, from 0.020 to 0.800 mM. Remarkably, the BChE inhibition, of
substrate on which AChE might exert its proaggregating action. Due
to the large amount of substrates needed, this assay resulted to be
very expensive; thus, the accurate selection of the compounds to be
evaluated represents a common and general trend in the academic
context [38]. In this study, the criteria of choice were guided by the
high inhibitory potency of the derivatives against AChE and by their
binding mode emerged from docking simulations.
In this paper we describe the synthesis, the biological evaluation
and the molecular modeling studies on a new series of benzo-
phenone derivatives designed to contact both the catalytic and the
peripheral sites of AChE. The biological profile of the compounds,
including AChE and BChE inhibitory potencies, expressed as IC50
two or three orders of magnitude lower than that against AChE,
revealed an extremely selective inhibitory profile. This feature, in
the light of the recent findings regarding the ability of BChE of
delaying the onset and decreasing the rate of Ab fibril formation in
vitro (inverse role with respect to AChE), resulted to be particularly
interesting and could represent an added value, as supplementary
favourable therapeutic effect [39,40]. This suggestion is in contrast
with other lines of evidence that indicate as the inhibition of BChE
might represent an advantage for the treatment of patients with
advanced AD, in view of the fact that some studies revealed for this
enzyme a co-regulatory function of the activity of AChE [41]. At
a first glance, the amide subset of compounds (7aec and 8b)
showed a similar inhibitory profile, exhibiting IC50 values in the
sub-micromolar range and selectivity for AChE with respect to
BChE of three orders of magnitude. For the derivatives 7aec,
bearing an acetamide spacer, by varying the nature of the amino
values, and inhibitory activity against AChE-induced Ab aggrega-
tion, expressed as % values, were investigated. Data are reported in
Table 1, together with those of the reference compounds donepezil
and propidium.
Table 1
Inhibition of AChE and BChE activities and AChE-mediated Ab aggregation by benzophenone derivatives.
O
O
NH
N
CH3
N
O
NR1R2
N
N
R1
N
N
( )
R2
N
nX
d
c
a
b
Compd
X
n
NR1R2
IC50
[
m
M] ꢂ SEM
Ratio IC50 BChE/AChE
Inhibition of A
b
aggregation [%]b ꢂ SEM
hAChEa
hBChEa
7a
7b
7c
8b
NHCO
NHCO
NHCO
NHCO
1
1
1
4
3
3
3
3
5
5
6
6
7
7
a
b
c
b
a
b
c
d
a
b
a
b
a
b
0.21 ꢂ 0.01
0.55 ꢂ 0.06
144 ꢂ 13
145 ꢂ 17
179 ꢂ 20
68 ꢂ 4
585
261
155
312
1372
490
198
581
236
72.7
85.2
46.3
28.4
210
n.t.
n.t.
n.t.
n.t.
34.3 ꢂ 6.0
28.1 ꢂ 0.2
n.t.
0.72 ꢂ 0.04
0.217 ꢂ 0.007
0.025 ꢂ 0.001
0.024 ꢂ 0.002
0.107 ꢂ 0.009
0.068 ꢂ 0.003
0.144 ꢂ 0.009
0.143 ꢂ 0.008
0.257 ꢂ 0.007
0.21 ꢂ 0.02
16a
16b
16c
16d
17a
17b
18a
18b
19a
19b
O
O
O
O
O
O
O
O
O
O
35 ꢂ 1
11 ꢂ 1
21 ꢂ 4
39 ꢂ 2
30.5 ꢂ 4.9
34 ꢂ 2
n.t.
10.4 ꢂ 0.5
21.9 ꢂ 0.7
9.7 ꢂ 0.5
23 ꢂ 2
n.t.
35.7 ꢂ 2.5
40.0 ꢂ 2.7
n.t.
0.81 ꢂ 0.05
0.11 ꢂ 0.02
24.2 ꢂ 0.1
n.t.
22
2
a
0.13 ꢂ 0.02
209 ꢂ 19
146
n.t.
O
10
1
OCH3
e
e
1.82 ꢂ 0.08
0.460 ꢂ 0.04c
0.023 ꢂ 0.005
32 ꢂ 2
>10
>5.5
132
321
0.4
n.t.
61 ꢂ 1c
7.4 ꢂ 0.4
13.2 ꢂ 0.4
4 ꢂ 1
Donepezil
Propidium
22 ꢂ 1d
82 ꢂ 2d
SEM: Standard Error of the Mean; n.t.: not tested.
a
Human recombinant AChE and BChE from human serum were used. IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and
are the mean of two independent measurements, each performed in triplicate.
b
Inhibition of AChE-induced Ab(1-40). The concentration of the tested inhibitor and Ab(1-40) was 100 mM and 230 mM, respectively, whereas the Ab(1-40):AChE ratio was
equal to 100:1.
c
Data obtained from Ref. [22].
Data obtained from Ref. [32].
d

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F. Belluti et al. / European Journal of Medicinal Chemistry 46 (2011) 1682e1693
function, the inhibition properties remained unaffected. Moreover,
the lengthening of the tether of 7b from one to four methylene
units (8b) did not remarkably affect the AChE inhibitory potency.
The three-methylene tether series (16aed), offered preliminary
useful information about the importance of the nature of the
terminal amino group: derivatives 16aeb, bearing the diethyl-
amine and the piperidine functions respectively, with a IC50 values
concentration of 16a gave estimate of competitive inhibition
constant (Ki), which resulted to be 24.3 ꢂ 3.5 nM (n ¼ 4).
2.2.2. AChE-induced Ab aggregation inhibition
Due to the high cost of the in vitro assay used for the assessment
of the inhibitory potency against the AChE-induced amyloid
aggregation, few compounds were selected. Therefore, docking
results and the inhibitory potency against AChE were used as
parameters of selection. In particular, the propoxy derivatives
16a,b,d, being the most active compounds of the series, and the
hexyloxy derivatives 18a,b which, on the basis of docking studies,
seemed to be the most appropriate to locate the terminal amino
function near the PAS region, were selected for evaluation. The
of 0.02 mM, proved to be the most potent of the series. A slight (1.5
fold) reduction of inhibitory potency was observed when
a hindering amino function was introduced (16d). A further drop in
activity (4 fold) was noted for derivative 16c, with the morpholino
group. These data suggest that the diethylamine and the piperidine
functions should be regarded as essential features, able to confer
high potency and selectivity. A decrease of affinity and selectivity
was observed by lengthening the side chain from 3 to 5e7 meth-
ylene units (compounds 17e19a,b, respectively). The role of the
side chain was further analyzed by increasing its steric hindrance:
an aryl moiety was introduced. This modification, that was carried
out in order to further evaluate a potential lipophilic interaction of
the tether with the gorge of the enzyme, did not result to be
beneficial for AChE inhibition: for compound 22 a drop in activity of
one order of magnitude with respect to 16a was observed.
Since the new derivatives were designed with the final aim of
obtaining dual binding site inhibitors, the mechanism of action of
compound 16a was investigated. The graphical analysis of steady-
state inhibition data for 16a on human AChE is shown in Fig. 2.
Reciprocal plots involving 16a inhibition show both increasing
slopes (decreased vmax at increasing inhibitor concentrations) and
increasing intercepts (higher Km) with higher inhibitor concentra-
tion. This pattern indicates mixed inhibition, arising from signifi-
cant inhibitor interaction with both the free enzyme and the
acetylated enzyme. Thus, the pattern in the graphical representa-
tion shows that 16a is able to bind to the peripheral anionic site as
well as the active site of AChE. Replots of the slope versus the
data, reported in Table 1, show that, at a concentration of 100
mM,
the percentage of inhibition of the fibrilization of the A peptide
b
induced by AChE, for the five selected compounds, ranged between
28% and 40%, being from 1.2 to 1.5 folds higher than the reference
compound donepezil. The derivatives obtained by incorporating
the diethylaminopropoxy and the diethylaminohexyloxy side
chains into the benzophenone scaffold (16a and 18a, respectively)
proved to inhibit the Ab aggregation process by approximately 35%.
On the basis of these results, the length of the linker did not seem to
play a pivotal role. A different behaviour was observed for the
analogues bearing the piperidin-1-ylpropoxy and the piperidin-1-
ylhexyloxy fragments (16b and 18b, respectively). In particular, 18b
proved to be 1.5 fold more potent than 16b. The six-methylene-unit
tether of 18b proved to favourably place the piperidine function,
allowing it to contact Trp286 at PAS.
2.3. Computational studies
To investigate the binding mode of the present series of AChEIs,
docking simulations were carried out by means of the ICM software
(v. 3.6) [42]. The model of the receptor was based on the crystal
structure of human AChE (hAChE) in complex with fasciculin
(PDB code 1B41) [43]. The outcomes, referred to the alkoxy series
of derivatives, showed that this flexible linker, ranging from 3 to
7 methylene units, was enough to mimic the diethylmethyl-
ammonium group of propidium. On the basis of the design rationale
pursued to obtain the dual binding site inhibitor, the attention was
focused on the terminal N,N-diethylamino derivatives 16a and 18a
bearing a propoxy and a hexyloxy spacer, respectively, that under-
went molecular modeling studies. In Fig. 3, superimposition
between the docking outcome of 16a (Fig. 3A) and 18a (Fig. 3B) and
the crystallographic structure of propidium in complex with murine
AChE is reported [44]. It can be seen that the considered molecules
could suitably interact with the catalytic pocket and the PAS of the
enzyme. Derivatives 16a and 18a both displayed some major inter-
actions putatively responsible for their inhibiting profile: a) the
terminal benzyl group of the inhibitors establishes hydrophobic
interactions with Trp86; b) the protonated nitrogen atom forms
a hydrogen bond with the hydroxyl group of Tyr337; c) the benzo-
phenone spacer can establish a
of Tyr341, a interaction with the indole ring of Trp286, and an
OHe interaction with the hydroxyl group of Tyr124 [45]. Our
pep interactionwith the phenol ring
pep
p
derivatives were able to mimic propidium in terms of interaction
with AChE, and they were also able to protrude out of the AChE
gorge, a featurewhich is considered important for the AChE-induced
Ab aggregation inhibition [17]. Moreover, the N,N-diethylamino
group of 18a forms a hydrogen bond with the backbone of Leu 289.
3. Conclusions
Fig. 2. Steady-state inhibition of AChE hydrolysis of acetylthiocholine (ATCh) by 16a.
Overlaid reciprocal plots of initial velocity versus substrate concentration are reported.
Lines were derived from a weighted least-squares analysis of the data points.
The identification of the benzophenone nucleus as scaffold for
a new class of AChEIs prompted us to synthesize a rationally-

F. Belluti et al. / European Journal of Medicinal Chemistry 46 (2011) 1682e1693
1687
compared to the lead compound 1, showed a remarkable inhibition
of the AChE-induced A aggregating activity, superior to that of
donepezil. The data clearly suggested positive contribute
b
a
provided by the newly introduced side chains. The presence of
a positive charge on the side chain, such as that occurring in pro-
pidium, appears to be critical for a potent antiaggregating activity.
All the new compounds resulted highly selective for AChE with
respect to BChE.
4. Experimental section
4.1. Chemistry
Starting materials, unless otherwise specified in the
Experimental Section, were used as high grade commercial prod-
ucts. Solvents were of analytical grade. Melting points were deter-
mined in open glass capillaries, using a Büchi apparatus and are
uncorrected. 1H NMR and 13C NMR spectra were recorded on a Var-
ian Gemini spectrometer and chemical shifts are reported as parts
per million (ppm d value) relative to the peak for tetramethylsilane
(TMS) as internal standard. Standard abbreviations indicating spin
multiplicities are given as follow: s (singlet), d (doublet), t (triplet),
br (broad), q (quartet) or m (multiplet). Mass spectra were recorded
on Waters ZQ 4000 apparatus operating in electrospray mode (ES).
Elemental analysis was performed using a PerkineElmer 2400 CHN
Elemental Analyzer. Chromatographic separations were performed
on silica gel columns by flash method (Kieselgel 40,
0.040e0.063 mm, Merck). Reactions were followed by thin layer
chromatography (TLC) on precoated silica gel plates (Merck Silica
Gel 60 F254) and then visualized with a UV lamp. The purity of the
tested compounds was determined by HPLC analysis, performed on
a Jasco LC 1500 PU-1587; the column used was a Phenomenex Luna
C18(2) 5
H2O; the flow-rate was 0.8 mL/min and the injection volume was
L; peaks were detected at 250 nm and results were >98% purity.
mm 4.60 mm ꢃ 150 mm; elution conditions: 30:70 CH3CN/
5
m
Compounds were named following IUPAC rules as applied by Beil-
stein-Institute AutoNom (version 2.1), a PC-integrated software
package for systematic names in organic chemistry.
4.1.1. 4-Bromomethyl-40-nitrobenzophenone (2)
A
mixture of 4-methyl-40-nitrobenzophenone (1.77 g,
7.4 mmol), N-bromosuccinimide (1.45 g, 8.14 mmol), a catalytic
amount of benzoyl peroxyde, in CCl4 (40 mL) was heated at 60 ꢁC
for 6 h. The reaction mixture was hot filtered and the filtrate was
concentrated under reduced pressure to obtain the crude product,
which was purified by crystallization from toluene/petroleum ether
to afford the desided compound 2 (1.98 g, 84%); mp: 115e118 ꢁC; 1H
NMR (300 MHz, CDCl3):
(d, 2H, J ¼ 8.4 Hz), 7.95 (d, 2H, J ¼ 8.8 Hz), 8.37 (d, 2H, J ¼ 8.8 Hz).
d
4.56 (s, 2H), 7.56 (d, 2H, J ¼ 8.4 Hz), 7.81
4.1.2. (4-((Benzyl(methyl)amino)methyl)phenyl)(4-nitrophenyl)
methanone (3)
Fig. 3. A and B. Binding mode of derivatives 16a and 18a at the human AChE gorge.
Compound 2 (1.66 g, 5.2 mmol) was dissolved in toluene
(25 mL), N,N0-benzylmethylamine (0.67 mL, 5.2 mmol) and Et3N
(0.72 mL, 5.2 mmol) were added. The mixture was stirred under
reflux for 12 h while monitoring with TLC. The reaction mixture
was poured into 3 N HCl (25 mL) and the organic phase was taken
away. The aqueous phase was made alkaline with K2CO3 and then
extracted with CH2Cl2 (3 ꢃ 30 mL). The combined organic extracts
were washed with brine (1 ꢃ30 mL), dried over Na2SO4, filtered
and concentrated to dryness. Purification of the residue was ach-
ieved by flash chromatography on silica gel using petroleum ether/
EtOAc (4:1) as eluant to yield pure 3 (1.7 g, 90%) as a brown oil; 1H
designed series of analogues, derived by modifications of the
structure of the lead compound 1 and containing an amino-
terminal side chain. This additional feature was introduced to
enable the molecules to contact the PAS of the enzyme, leading to
dual binding site inhibitors endowed with an increased inhibitory
potency and an expanded biological profile. Compounds 16a,bed,
equipotent to the reference compound donepezil, proved to be the
most active of the series at inhibiting AChE; an improvement of the
potency of one order of magnitude with respect to t 1 was thus
achieved. Interestingly, the derivatives 16a,bed and 18a,b, when
NMR (300 MHz, CDCl3):
d 2.65 (s, 3H), 4.04 (s, 2H), 4.30 (s, 2H),
7.47e7.51 (m, 2H), 7.60e7.64 (m, 2H), 7.90e7.95 (m, 5H), 7.98 (d,

1688
F. Belluti et al. / European Journal of Medicinal Chemistry 46 (2011) 1682e1693
2H, J ¼ 8.6 Hz), 8.35 (d, 2H, J ¼ 8.6 Hz)); 13C NMR (300 MHz, CDCl3):
2H), 3.60e3.80 (m, 4H), 3.75e3.85 (m, 4H), 7.25e7.35 (m, 5H), 7.52
(d, 2H, J ¼ 6.8 Hz), 7.70 (d, 2H, J ¼ 8.6 Hz), 7.75 (d, 2H, J ¼ 8.7 Hz),
7.85 (d, 2H, J ¼ 6.9 Hz), 9.60 (br, 1H). 13C NMR (300 MHz, CDCl3):
d
42.7, 61.88, 123.61, 126.64, 128.11, 128.65, 128.71, 128.11, 129.58,
131.26, 131.54, 131.76, 137.32, 139.75, 144.17, 151.33, 196.65.
d
12.5, 26.2, 41.5, 49.5, 50.6, 61.5, 61.7, 120.1, 127.0, 129.0, 129.2,
4.1.3. (4-Aminophenyl)(4-((benzyl(methyl)amino)methyl)phenyl)
methanone (4)
130.3, 130.6, 128.7, 128.9, 129.8, 133.4, 136.3, 136.8, 140.0, 144.5,
168.2, 187.0. MS (ESIþ): m/z: 458 [M þ H]þ and 480 [M þ Na]þ;
Anal. calcd for C29H33N3O2: C 76.45, H 7.30, N 9.22, found: C 76.23, H
7.48, N 8.96.
Compound 3 (1.7 g) was hydrogenated over Pd/CaCO3 10%
(0.17 g) in anhydrous THF (170 mL). After completion, the catalyst
was removed via filtration on celite pad. The residue, obtained on
evaporation under reduced pressure, gave compound 3 (1.3 g, 83%)
4.1.8. N-(4-{4-[(Benzylmethylamino)methyl]benzoyl}phenyl)-2-
morpholin-4-yl-acetamide (7c)
as an oil; 1H NMR (300 MHz, CDCl3):
d 2.23 (s, 3H), 3.58 (s, 2H), 3.60
(s, 2H), 4.13 (br, 2H), 6.66 (d, 2H, J ¼ 6.6 Hz), 7.25e7.38 (m, 5H), 7.46
Compound 7c was prepared following the procedure described
for 7a, starting from 5 (0.2 g, 0.39 mmol) and morpholine (0.05 mL,
0.6 mmol), and isolated as an oil (0.1 g, 56%); 1H NMR (300 MHz,
(d, 2H, J ¼ 8.4 Hz), 7.68e7.73 (m, 4H) , 13C NMR (300 MHz, CDCl3):
d
42.5, 61.86, 113.41, 121.34, 126.61, 128.01, 128.65, 128.73, 129.55,
132.96, 134.44, 139.25, 144.85, 151.31, 196.62.
CDCl3): d 2.26 (s, 3H), 2.62e2.65 (m, 4H), 3.18 (s, 2H), 3.50e3.70 (m,
4H), 3.65e3.85 (m, 4H), 7.20e7.30 (m, 5H), 7.50 (d, 2H, J ¼ 6.8 Hz),
4.1.4. N-(4-{4-[(Benzylmethylamino)methyl]benzoylphenyl)-2-
chloroacetamide (5)
7.68 (d, 2H, J ¼ 8.4 Hz), 7.76 (d, 2H, J ¼ 8.0 Hz), 7.84 (d, 2H,
J ¼ 8.4 Hz), 9.30 (br, 1H); 13C NMR (300 MHz, CDCl3):
d 51.7, 58.2,
A solution of 4 (0.2 g, 0.6 mmol) and 3-chloroacethyl chloride
(0.07 mL, 0.9 mmol) in acetonitrile (10 mL) was refluxed for 6 h;
the solvent was removed under reduced pressure and the crude
product was purified by flash chromatography on silica gel using
a mixture of CH2Cl2/CH3OH/NH4OH (97:2.5:0.5) as eluant to yield
61.0, 61.6, 71.2, 113.3, 127.0, 127.7, 128.1, 128.5, 128.7, 128.8, 129.7,
130.0, 132.2, 135.9, 138.4, 143.0, 168.9, 198.8. MS (ESIþ): m/z: 480
[M þ Na]þ; Anal. calcd for C28H31N3O3: C 73.50, H 6.83, N 9.18,
found: C 73.80, H 6.64, N 9.01.
pure 5 (0.2 g, 82%) as an oil; 1H NMR (200 MHz, CDCl3):
d
2.40 (s,
4.1.9. 5-Piperidin-1-ylpentanoicacid(4-{4-[(benzylmethylamino)
methyl]benzoyl}phenyl)amide (8b)
Compound 8b was prepared following the procedure described
for 7b, starting from 6 (0.20 g, 0.45 mmol) and piperidine (0.07 mL,
0.68 mmol), and isolated as a an oil (0.15 g, 73%); 1H NMR
3H), 3.56 (s, 2H), 3.57 (s, 2H), 4.32 (s, 2H), 7.25e7.45 (m, 4H), 7.55
(d, 2H, J ¼ 8.4 Hz), 7.71e7.92 (m, 7H), 8.55 (br, 1H); 13C NMR
(300 MHz, CDCl3): d: 42.4, 43.06, 61.83, 120.31, 126.72, 127.71,
128.05, 128.70, 129.53, 131.46, 139.20, 142.11, 144.97, 166.31,
196.65.
(300 MHz, CDCl3):
d 1.45e1.55 (m, 2H), 1.75e1.90 (m, 8H), 2.18 (s,
3H), 2.40e2.60 (m, 2H), 2.75e2.95 (m, 3H), 3.55 (s, 2H), 3.58 (s, 2H),
7.25e7.40 (m, 7H), 7.47 (d, 2H, J ¼ 8.2 Hz), 7.70e7.85 (m, 4H), 9.20
4.1.5. 5-Chloropentanoic acid (4-{4-[(benzylmethylamino)methyl]
benzoyl}phenyl)amide (6)
(br, 1H). 13C NMR (300 MHz, CDCl3):
d 13.0, 26.2, 23.7, 24.9, 33.6,
From compound 4 (0.2 g, 0.6 mmol) and 5-chlorovaleroyl chlo-
ride (0.11 mL, 0.9 mmol), a similar procedure as that described for 5
gave pure 6 (0.22 g, 82%) as an oil; 1H NMR (200 MHz, CDCl3):
38.5, 41.9, 61.5, 61.5, 120.1, 127.0, 129.0, 128.2, 128.6, 130.3, 128.7,
129.8, 129.9, 133.4, 136.7, 136.8, 140.0, 144.5, 172.0, 187.0. MS (ESIþ):
m/z: 458 [M þ H]þ, 520 [M þ Na]þ; Anal. calcd for C32H39N3O2: C
77.23, H 7.90, N 8.44, found: C 77.54, H 8.05, N 8.18.
d
1.75e1.95 (m, 4H), 2.35e2.45 (m, 2H), 3.50 (s, 2H), 3.60 (s, 2H),
3.60e3.65 (m, 2H), 7.30e7.65 (m, 5H), 7.52 (d, 2H, J ¼ 8.2 Hz), 7.64
(d, 2H, J ¼ 8.8 Hz), 7.75 (d, 2H, J ¼ 8.2 Hz), 7.81 (d, 2H, J ¼ 8.4 Hz); 13C
4.1.10. Benzyl-(4-bromobenzyl)methylamine (9)
NMR (300 MHz, CDCl3):
d
22.25, 31.52, 36.84, 42.12, 43.16, 61.87,
Compound 9 was prepared following the procedure described
for 3, starting from 4-bromomethylbenzene (6.22 g, 24.9 mmol)
and isolated as a brown oil (5.8 g, 80%), 1H NMR (200 MHz, CDCl3):
120.01, 126.74, 127.81, 128.05, 128.69, 129.59, 130.46, 131.35, 139.25,
144.14, 144.96, 171.33, 195.95.
d
2.35 (s, 3H), 3.55 (s, 2H), 3.60 (s, 2H), 7.45e7.67 (m, 9H).
4.1.6. N-(4-{4-[(Benzylmethylamino)methyl]benzoyl}phenyl)-2-
diethylaminoacetamide (7a)
4.1.11. {4-[(Benzylmethylamino)methyl]phenyl}(4-methoxyphenyl)
methanone (10)
To a stirred mixture of 5 (0.15 g, 0.39 mmol) in 1-propanol
(10 mL), diethylammine (0.06 mL, 0.6 mmol) was added. The
reaction mixture was heated under reflux for 8 h. Solvent was
removed under reduced pressure, the crude product was purified
by flash chromatography using a mixture of CH2Cl2/CH3OH/NH4OH
(97:2.5:0.5) as eluant to yield pure 7a as an oil (0.05 g, 57%); 1H
n-BuLi (1.6 M hexane solution, 1 mL) wad added dropwise, at
ꢀ78 ꢁC and under N2 atmosphere, to a stirred solution of 9 (1.68 g,
5.5 mmol) in anhydrous THF (55 mL). The mixture was stirred for
1 h at the same temperature, then 4-methoxybenzonitrile (0.96 g,
7.2 mmol) in THF (10 mL) was added and the stirring was continued
for further 1 h under the same conditions. The mixture was then
allowed to warm to room temperature, an acidic solution (15 mL:
conc H2SO4/H2O/dioxane, 10:1:4) was added dropwise and the
resulting solution was heated at reflux for 2 h. On cooling, 2 N
NaOH aq solution was added until pH 10, and the mixture was
extracted with CH2Cl2 (3 ꢃ 50 mL). The combined organic layers
were washed with brine (50 mL), dried over Na2SO4, evaporated
under reduced pressure. The crude product was purified by column
chromatography on silica gel with CH2Cl2/CH3OH 97.5:2.5 as eluant,
NMR (200 MHz, CDCl3):
d
1.01 (t, 6H, J ¼ 7.2 Hz), 2.13 (s, 3H), 2.58 (q,
4H, J ¼ 7.2 Hz), 3.09 (s, 2H), 3.47 (s, 2H), 3.50 (s, 2H), 7.16e7.27 (m,
5H), 7.38 (d, 2H, J ¼ 6.8 Hz), 7.58e7.66 (m, 4H), 7.73 (d, 2H, J ¼ 6.9
H), 9.60 (br, 1H); 13C NMR (300 MHz, CDCl3):
d 13.0, 41.9, 45.5, 49.5,
55.5, 61.0, 61.6, 113.4, 127.2, 127.7, 128.2, 128.6, 128.8, 128.9, 129.5,
130.1, 132.2, 136.9, 138.3, 140.1, 145.6, 170.5, 197.8. MS (ESIþ): m/z:
444 [M þ H]þ and 466 [M þ Na]þ; Anal. calcd for C28H33N3O2: C
75.81, H 7.50, N 9.47, found: C 75.98, H 7.73, N 9.68.
4.1.7. N-(4-{4-[(Benzylmethylamino)methyl]benzoyl}phenyl)-2-
piperidin-1-yl-acetamide (7b)
Compound 7b was prepared following the procedure described
for 7a, starting from 5 (0.2 g, 0.39 mmol) and piperidine (0.06 mL,
0.6 mmol), and isolated as an oil (0.13 g, 75%); 1H NMR (300 MHz,
affording 10 as an oil (1.3 g, 65%); 1H NMR (300 MHz, CDCl3):
d 2.20
(s, 3H), 3.55 (s, 2H), 3.60 (s, 2H), 3.80 (s, 3H), 7.10 (d, 2H, J ¼ 8.8 Hz),
7.20e7.40 (m, 5H), 7.35 (d, 2H, J ¼ 8.0 Hz), 7.60 (d, 2H, J ¼ 8.4 Hz),
7.70 (d, 2H, J ¼ 9.2 Hz); 13C NMR (300 MHz, CDCl3):
d 41.9, 55.5, 61.0,
61.6, 113.6, 127.0, 127.7, 128.1, 128.5, 128.7, 128.8, 129.7, 130.0, 132.2,
136.9, 138.3, 143.1, 162.9, 197.8.
CDCl3): d 1.50e1.70 (m, 2H), 2.26 (s, 3H), 2.60e2.70 (m, 4H), 3.20 (s,

F. Belluti et al. / European Journal of Medicinal Chemistry 46 (2011) 1682e1693
1689
4.1.12. {4-[(Benzylmethylamino)methyl]phenyl}-(4-hydroxyphenyl)
methanone (11)
J ¼ 8.0 Hz), 7.75 (d, 2H, J ¼ 8.4 Hz), 7.85 (d, 2H, J ¼ 9.2 Hz); 13C NMR
(300 MHz, CDCl3):
d 25.46, 28.04, 28.62, 30.93, 41.98, 44.66, 61.86,
To compound 10 (1.0 g, 1.9 mmol) in anhydrous CH2Cl2 (20 mL),
at 0 ꢁC and under N2 atmosphere, BBr3 (1 M solution in CH2Cl2,
6.0 mL, 6.0 mmol) was added dropwise. The solution was stirred for
4 h at room temperature. The mixture was neutralized with
NaHCO3 saturated solution and then extracted with CH2Cl2
(3 ꢃ 50 mL), the combined organic layers were dried (Na2SO4) to
afford a crude residue, which was purified by flash column chro-
matography using CH2Cl2/CH3OH 95:5 as eluant to afford pure 11 as
68.14, 116.16, 124.81, 126.76, 128.04, 128.08, 128.77, 128.81, 129.56,
136.36, 139.28, 144.92, 160.26, 195.61.19, 124.81, 126.71, 128.04,
128.06, 128.64, 128.96, 129.46, 136.22, 139.52, 144.87, 160.54,
195.62.
4.1.17. {4-[(Benzylmethylamino)methyl]phenyl}-
[4-(3-diethylaminopropoxy)phenyl]methanone (16a)
Compound 16a was prepared following the procedure described
for 3, starting from 12 (0.15 g, 0.37 mmol), and diethylamine
(0.05 mL, 0.44 mmol). The crude product was purified by flash
column chromatography using a mixture of CH2Cl2/CH3OH/NH4OH
(97:2.5:0.5) as eluant to afford 16a as a semisolid product (0.10 g,
61%); mp: 128e130 ꢁC (hydrochloride salt); 1H NMR (300 MHz,
an oil (0.5 g, 79%); 1H NMR (300 MHz, CDCl3):
d 2.20 (s, 3H), 3.55 (s,
2H), 3.60 (s, 2H), 7.10 (d, 2H, J ¼ 8.8 Hz), 7.20e7.40 (m, 5H), 7.35 (d,
2H, J ¼ 8.0 Hz), 7.60 (d, 2H, J ¼ 8.4 Hz), 7.70 (d, 2H, J ¼ 9.2 Hz)); 13C
NMR (300 MHz, CDCl3): d 42.21, 61.83,116.71,124.01,126.74,128.08,
128.76, 128.48, 129.54, 136.36, 139.25, 144.92, 164.35, 196.62.
CDCl3):
d
1.15 (t, 6H, J ¼ 7.4 Hz), 1.90e2.05 (m, 2H), 2.20 (s, 3H),
4.1.13. {4-[(Benzylmethylamino)methyl]phenyl}-
[4-(3-chloropropoxy)phenyl]methanone (12)
2.60e2.80 (m, 6H), 3.55 (s, 2H), 3.60 (s, 2H), 4.10 (t, 2H, J ¼ 6.0 Hz),
6.95 (d, 2H, J ¼ 8.8 Hz), 7.20e7.40 (m, 5H), 7.45 (d, 2H, J ¼ 8.4 Hz),
7.70 (d, 2H, J ¼ 8.4 Hz), 7.80 (d, 2H, J ¼ 8.8 Hz); 13C NMR (300 MHz,
Compound 11 (0.5 g, 1.5 mmol) was dissolved in acetone
(30 mL) and anhydrous K2CO3 (0.2 g, 1.5 mmol) and 1-bromo-3-
chloropropane (0.15 mL, 1.5 mmol) were added. After refluxing for
8 h, the mixture was hot filtered, the solvent was removed under
reduced pressure and the crude was purified by flash column
chromatography using CH2Cl2/CH3OH (97.5:2.5) as eluant, to afford
CDCl3): d 23.5, 30.5, 41.9, 45.5, 47.7, 55.9, 61.0, 61.8, 68.9,113.4,127.4,
127.7, 128.2, 128.4, 128.7, 128.8, 129.7, 130.4, 132.4, 136.9, 138.2,
143.6, 162.5, 197.5. MS (ESIþ): m/z: 445 [M þ H]þ; Anal. calcd for
C29H36N2O2: C 78.34, H 8.16, N 6.30, found: C 78.55, H 8.25, N 6.21.
12 as an oil (0.5 g, 82%); 1H NMR (300 MHz, CDCl3):
d
2.00e2.05 (m,
4.1.18. {4-[(Benzylmethylamino)methyl]phenyl}-[4-(3-piperidin-1-
ylpropoxy)phenyl]methanone (16b)
Compound 16b was prepared following the procedure described
for 3, starting from 12 (0.15 g, 0.37 mmol) and piperidine (0.04 mL,
0.44 mmol). The crude product was purified by flash column
2H), 2.20 (s, 3H), 3.40e3.55 (m, 6H), 4.10 (t, 2H, J ¼ 6.0 Hz), 7.10 (d,
2H, J ¼ 8.8 Hz), 7.20e7.40 (m, 5H), 7.45 (d, 2H, J ¼ 8.0 Hz), 7.60 (d,
2H, J ¼ 8.4 Hz), 7.70 (d, 2H, J ¼ 9.2 Hz); 13C NMR (300 MHz, CDCl3):
d
33.73, 41.45, 42.21, 61.84, 68.92, 116.46, 124.84, 126.76, 128.05,
128.66, 128.78, 129.56, 136.36, 139.26, 144.96, 162.25, 196.68.
chromatography using a mixture of CH2Cl2/CH3OH/NH4OH
(97:2.5:0.5) as eluant to afford 16b as a semisolid product (0.13 g,
4.1.14. 4-[(Benzylmethylamino)methyl]phenyl}-
77%); mp: 181e183 ꢁC (hydrochloride salt). 1H NMR (300 MHz,
[4-(5-chloropentyloxy)phenyl]methanone (13)
CDCl3): d 1.40e1.50 (m, 2H), 1.55e1.75 (m, 4H), 1.95e2.10 (m, 2H),
Compound13 waspreparedfollowingtheproceduredescribedfor
12, starting from 11 (0.5 g,1.5 mmol) and 1-bromo-5-chloropenthane
(0.2 mL, 1.5 mmol), and isolated as an oil (0.37 g, 56%); 1H NMR
2.20 (s, 3H), 2.30e2.55 (m, 6H), 3.55 (s, 2H), 3.60 (s, 2H), 4.10 (t, 2H,
J ¼ 6.3 Hz), 6.95 (d, 2H, J ¼ 9.0 Hz), 7.20e7.40 (m, 5H), 7.47 (d, 2H,
J ¼ 8.4 Hz), 7.70 (d, 2H, J ¼ 8.4 Hz), 7.80 (d, 2H, J ¼ 8.7 Hz); 13C NMR
(200 MHz, CDCl3):
d
1.55e1.95 (m, 6H), 2.05 (s, 3H), 3.50e3.60 (m,
(300 MHz, CDCl3): d 23.5, 28.5, 28.8, 30.5, 41.9, 50.9, 51.2, 55.9, 61.3,
6H), 4.05 (t, 2H, J ¼ 6.0 Hz), 6.90 (d, 2H, J ¼ 8.8 Hz), 7.20e7.40 (m, 5H),
61.6, 68.6, 113.4, 126.9, 128.1, 128.5, 128.7, 128.8, 129.7, 130.4, 132.4,
136.9, 138.2, 143.6, 162.5, 197.5. MS (ESIþ): m/z: 457 [M þ H]þ; Anal.
calcd for C30H36N2O2: C 78.91, H 7.95, N 6.13, found: C 79.05, H 8.15,
N 5.99.
7.45 (d, 2H, J ¼ 8.0 Hz), 7.70 (d, 2H, J ¼ 8.4 Hz), 7.85 (d, 2H, J ¼ 9.2 Hz);
13C NMR (300 MHz, CDCl3):
d 23.56, 28.74, 32.23, 41.99, 44.21, 61.84,
68.54, 116.16, 124.81, 126.71, 128.01, 128.66, 128.74, 129.56, 136.46,
139.22, 144.92, 160.26, 195.61.19, 124.81, 126.71, 128.04, 128.06,
128.64, 128.96, 129.46, 136.22, 139.52, 144.27, 160.54, 195.61.
4.1.19. {4-[(Benzylmethylamino)methyl]phenyl}-[4-(3-morpholin-
4-ylpropoxy)phenyl]methanone (16c)
4.1.15. {4-[(Benzylmethylamino)methyl]phenyl}-
[4-(6-chlorohexyloxy)phenyl]methanone (14)
Compound 14 was prepared following the procedure described
for 12, starting from 11 (0.5 g, 1.5 mmol) and 1-bromo-6-chlor-
ohexane (0.2 mL, 1.5 mmol), and isolated as an oil (0.53 g, 79%); 1H
Compound 16c was prepared following the procedure described
for 3, starting from 12 (0.15 g, 0.37 mmol) and morpholine
(0.04 mL, 0.44 mmol). The crude product was purified by flash
column chromatography using a mixture of CH2Cl2/CH3OH/NH4OH
(94.5:5:0.5) as eluant to afford 16c as a semisolid product (0.10 g,
59%); mp: 232e234 ꢁC (hydrochloride salt), 1H NMR (300 MHz,
NMR (300 MHz, CDCl3):
d 1.25e1.60 (m, 4H), 1.80e1.95 (m, 4H),
2.05 (s, 3H), 3.55e3.60 (m, 6H), 4.10 (t, 2H, J ¼ 6.0 Hz), 6.95 (d, 2H,
J ¼ 8.8 Hz), 7.20e7.40 (m, 5H), 7.44 (d, 2H, J ¼ 8.0 Hz), 7.65 (d, 2H,
J ¼ 8.4 Hz), 7.80 (d, 2H, J ¼ 9.2 Hz); 13C NMR (300 MHz, CDCl3):
CDCl3): d 1.95e2.10 (m, 2H), 2.20 (s, 3H), 2.30e2.60 (m, 6H), 3.55 (s,
2H), 3.60 (s, 2H), 3.80e3.90 (m, 6H), 4.10 (t, 2H, J ¼ 6.3 Hz), 6.95 (d,
2H, J ¼ 8.8 Hz), 7.20e7.40 (m, 5H), 7.47 (d, 2H, J ¼ 8.4 Hz), 7.72 (d,
2H, J ¼ 8.4 Hz), 7.81 (d, 2H, J ¼ 8.8 Hz); 13C NMR (300 MHz, CDCl3):
d
25.06, 26.74, 28.32, 31.43, 41.98, 44.26, 61.89, 67.84, 116.16, 124.81,
126.71, 128.01, 128.64, 128.77, 129.56, 136.36, 139.28, 144.92, 160.26,
195.61.
d 25.5, 31.3, 42.2, 44.0, 48.4 56.2, 61.3, 61.9, 67.9, 75.8, 113.1, 127.1,
128.2, 128.5, 128.7, 128.8, 128.9, 129.3, 131.4, 132.4, 136.9, 138.5,
143.7 162.8, 197.9. MS (ESIþ): m/z: 459 [M þ H]þ; Anal. calcd for
C29H34N2O3: C. 75.95, H 7.47, N 6.11, found: C. 76.27, H 7.54, N 6.28.
4.1.16. {4-[(Benzylmethylamino)methyl]phenyl}-
[4-(7-bromoheptyloxy)phenyl]methanone (15)
Compound 15 was prepared following the procedure described
for 12, starting from 11 (0.5 g, 1.5 mmol) and 1,7-dibromoheptane
(0.25 mL, 1.5 mmol), and isolated as an oil (0.43 g, 56%); 1H NMR
4.1.20. 1-{1-[3-(4-{4-[(Benzylmethylamino)methyl]benzoyl}
phenoxy)propyl]piperidin-4-yl}-1,3-dihydrobenzoimidazol-2-
one (16d)
(200 MHz, CDCl3):
d
1.25e1.60 (m, 6H), 1.80e1.95 (m, 4H), 2.08 (s,
Compound 16d was prepared following the procedure described
for 3, starting from 12 (0.15 g, 0.37 mmol) and 1-(4-piperidinyl)-
1,3-dihydro-2H-benzimidazol-2-one (0.1 g, 0.44 mmol). The crude
3H), 3.45 (t, 2H, J ¼ 6.0 Hz), 3.55 (s, 2H), 3.60 (s, 2H), 4.10 (t, 2H,
J ¼ 6.0 Hz), 6.95 (d, 2H, J ¼ 8.8 Hz), 7.20e7.50 (m, 7H), 7.44 (d, 2H,

1690
F. Belluti et al. / European Journal of Medicinal Chemistry 46 (2011) 1682e1693
product was purified by flash column chromatography using
a mixture of CH2Cl2/CH3OH/NH4OH (94.5:5:0.5) as eluant to afford
16d as a semisolid (0.14 g, 77%); mp: 220e222 ꢁC (hydrochloride
4.1.24. {4-[(Benzylmethylamino)methyl]phenyl}-[4-(6-piperidin-1-
ylhexyloxy)phenyl]methanone (18b)
Compound 18b was prepared following the procedure described
for 3, starting from 14 (0.16 g, 0.37 mmol) and piperidine (0.04 mL,
0.44 mmol). The crude product was purified by flash column
salt); 1H NMR (300 MHz, CDCl3):
d 1.60e1.85 (m, 2H), 1.90e2.20 (m,
7H), 2.30e2.50 (m, 2H), 2.60 (t, 2H, J ¼ 6.9 Hz), 3.00e3.15 (m, 2H),
3.50 (s, 2H), 3.53 (s, 2H), 4.08 (t, 2H, J ¼ 6.0 Hz), 4.12e4.20 (m, 1H),
6.90 (d, 2H, J ¼ 9.0 Hz), 6.98e7.03 (m, 3H), 7.15e7.35 (m, 6H), 7.41
(d, 2H, J ¼ 8.4 Hz), 7.66 (d, 2H, J ¼ 8.4 Hz), 7.76 (d, 2H, J ¼ 9.0 Hz),
chromatography using
a mixture of CH2Cl2/CH3OH/NH4OH
(97:2.5:0.5) as eluant to afford 18a as a semisolid product (0.14 g,
76%), mp: 187e188 ꢁC (hydrochloride salt). 1H NMR (300 MHz,
8.95 (br, 1H); 13C NMR (300 MHz, CDCl3):
d
23.5, 28.5, 30.5, 41.9,
CDCl3): d 1.20e1.95 (m, 14H), 2.20 (s, 3H), 2.50e2.90 (m, 6H), 3.55
48.9, 51.6, 51.6, 55.9, 61.3, 61.6, 68.6, 113.4, 122.3, 125.1, 126.5, 128.5,
128.7, 128.8, 129.7, 130.1, 130.4, 132.4, 136.9, 138.2, 143.6, 159.2,
162.5, 198.1. MS (ESIþ): m/z: 589 [M þ H]þ; Anal. calcd for
C37H40N4O3: C 75.48, H 6.85, N 9.52, found: C 75.32, H 6.66, N 9.78.
(s, 2H), 3.60 (s, 2H), 4.05 (t, 2H, J ¼ 6.4 Hz), 6.93 (d, 2H, J ¼ 8.8 Hz),
7.20e7.40 (m, 5H), 7.47 (d, 2H, J ¼ 8.4 Hz), 7.72 (d, 2H, J ¼ 8.4 Hz),
7.80 (d, 2H, J ¼ 8.7 Hz); 13C NMR (300 MHz, CDCl3):
d 23.6, 24.3,
24.8, 26.9, 28.5, 28.8, 30.5, 41.9, 51.4, 52.2, 55.5, 61.6, 61.7, 68.8,113.8
125.4, 128.5, 128.6, 128.9, 129.1, 129.7, 129.9, 131.1, 132.6, 136.9,
138.2, 144.1, 162.9, 197.5. MS (ESIþ): m/z 499 [M þ H]þ; Anal. calcd
for C33H42N2O2: C 79.48, H 8.49, N 5.62, found: C 79.21, H 8.22,
N 5.58.
4.1.21. {4-[(Benzylmethylamino)methyl]phenyl}-[4-(5-
diethylaminopentyloxy)phenyl]methanone (17a)
Compound 17a was prepared following the procedure described
for 3, starting from 13 (0.16 g, 0.37 mmol) and diethylamine
(0.04 mL, 0.44 mmol). The crude product was purified by flash
column chromatography using a mixture of CH2Cl2/CH3OH/NH4OH
(97:2.5:0.5) as eluant to afford 17a as a semisolid product (0.10 g,
57%); mp: 167e168 ꢁC (hydrochloride salt); 1H NMR (300 MHz,
4.1.25. {4-[(Benzylmethylamino)methyl]phenyl}-[4-(7-
diethylaminoheptyloxy)phenyl]methanone (19a)
Compound 19a was prepared following the procedure described
for 3, starting from 15 (0.16 g, 0.37 mmol) and diethylamine
(0.04 mL, 0.44 mmol). The crude product was purified by flash
column chromatography using a mixture of CH2Cl2/CH3OH/NH4OH
(94.5:5:0.5) as eluant to afford 19a as a semisolid product (0.14 g,
76%), mp: 160e162 ꢁC (hydrochloride salt), 1H NMR (300 MHz,
CDCl3):
d
1.05 (t, 6H, J ¼ 7.2 Hz) 1.50e1.95 (m, 6H), 2.15 (s, 3H),
2.40e2.70 (m, 6H), 3.50 (s, 2H), 3.55 (s, 2H), 3.95 (t, 2H, J ¼ 6.6 Hz),
6.87 (d, 2H, J ¼ 8.4 Hz), 7.20e7.40 (m, 5H), 7.40 (d, 2H, J ¼ 8.1 Hz),
7.65 (d, 2H, J ¼ 8.1 Hz), 7.74 (d, 2H, J ¼ 8.4 Hz); 13C NMR (300 MHz,
CDCl3):
d
14.7, 23.9, 31.4, 42.3, 46.1, 48.8, 53.4, 61.5, 62.1, 68.8, 112.4,
CDCl3): d 1.35e1.55 (m, 12H), 1.80e1.95 (m, 4H), 2.30 (s, 3H),
127.3,127.6,128.5,128.6,128.9,129.1,129.5,130.8,132.8,137.4,138.2,
143.7, 162.9, 197.9. MS (ESIþ): m/z: 474 [M þ H]þ; Anal. calcd for
C31H40N2O2: C 78.77, H 8.53, N 5.93, found: C 78.55, H 8.36, N 5.76.
2.95e3.05 (m, 2H), 3.14 (q, 4H, J ¼ 7.2 Hz), 3.70 (s, 2H), 3.75 (s, 2H),
4.04 (t, 2H, J ¼ 6.4 Hz), 6.94 (d, 2H, J ¼ 9.0 Hz), 7.20e7.45 (m, 5H),
7.53 (d, 2H, J ¼ 8.0 Hz), 7.77 (d, 2H, J ¼ 8.0 Hz), 7.80 (d, 2H,
J ¼ 8.8 Hz); 13C NMR (300 MHz, CDCl3):
d 23.5, 25.5, 26.7, 30.7, 42.1,
4.1.22. {4-[(Benzylmethylamino)methyl]phenyl}-[4-(5-piperidin-1-
ylpentyloxy)phenyl]methanone (17b)
Compound 17b was prepared following the procedure described
for 3, starting from 13 (0.16 g, 0.37 mmol) and piperidine (0.04 mL,
0.44 mmol). The crude product was purified by flash column
45.8, 48.1, 53.9, 62.2, 62.9, 70.5,112.1,128.5,129.1,129.6,129.7,130.7,
130.8, 131.2, 132.4, 134.4, 138.9, 139.6, 144.7, 168.8, 196.2. MS (ESIþ):
m/z: 501 [M þ H]þ; Anal. calcd for C33H44N2O2: C 79.16, H 8.86, N
5.59, found: C 79.25, H 8.86, N 5.77.
chromatography using
a
mixture of CH2Cl2/CH3OH/NH4OH
4.1.26. {4-[(Benzylmethylamino)methyl]phenyl}-[4-(7-piperidin-1-
ylheptyloxy)phenyl]methanone (19b)
Compound 19b was prepared following the procedure described
for 3, starting from 15 (0.16 g, 0.37 mmol) and piperidine (0.04 mL,
0.44 mmol). The crude product was purified by flash column
(97:2.5:0.5) as eluant to afford 17b as a semisolid product (0.13 g,
72%); mp: 166e168 ꢁC (hydrochloride salt); 1H NMR (200 MHz,
CDCl3):
d 1.40e1.50 (m, 2H), 1.60e1.75 (m, 6H), 1.20e1.95 (m, 2H),
2.20 (s, 3H), 2.50e2.75 (m, 6H), 3.55 (s, 2H), 3.60 (s, 2H), 4.05 (t, 2H,
J ¼ 6.3 Hz), 6.93 (d, 2H, J ¼ 9.0 Hz), 7.20e7.40 (m, 5H), 7.47 (d, 2H,
J ¼ 8.4 Hz), 7.72 (d, 2H, J ¼ 8.4 Hz), 7.80 (d, 2H, J ¼ 8.7 Hz); 13C NMR
chromatography using
a mixture of CH2Cl2/CH3OH/NH4OH
(94.5:5:0.5) as eluant to afford 19b as a semisolid product (0.14 g,
(300 MHz, CDCl3): d 15.5, 24.5, 28.5, 28.9, 30.5, 41.5, 51.4, 52.4, 54.9,
76%), mp: 170e171 ꢁC (hydrochloride salt); 1H NMR (300 MHz,
61.9, 62.4, 67.7, 113.8, 127.9, 129.2, 129.5, 129.8, 130.7, 130.8, 131.7,
132.5, 133.9, 137.4, 138.8, 144.1, 163.3, 199.1. MS (ESIþ): m/z: 485
[M þ H]þ; Anal. calcd for C32H42N2O2: C 79.30, H 8.32, N 5.78,
found: C 79.24, H 8.49, N 5.65.
CDCl3): d 1.20e1.95 (m, 16H), 2.20 (s, 3H), 2.40e2.70 (m, 6H), 3.55
(s, 2H), 3.60 (s, 2H), 4.05 (t, 2H, J ¼ 6.40 Hz), 6.95 (d, 2H, J ¼ 8.8 Hz),
7.20e7.40 (m, 5H), 7.45 (d, 2H, J ¼ 8.4 Hz), 7.68 (d, 2H, J ¼ 8.4 Hz),
7.82 (d, 2H, J ¼ 8.7 Hz); 13C NMR (300 MHz, CDCl3):
d 23.5, 25.5, 26.7
28.5, 28.8,29.5, 30.5, 41.8, 50.2, 52.6, 55.6, 62.2, 62.7, 65.6, 113.6,
126.7, 128.1, 128.4, 128.5, 128.7, 128.9, 129.1, 131.1, 131.9, 137.9, 138.6,
143.9 165.7, 198.8. MS (ESIþ): m/z: 513 [M þ H]þ; Anal. calcd for
C34H44N2O2: C 79.65, H 8.65, N 5.46, found: C 79.12, H 8.61, N 5.97.
4.1.23. {4-[(Benzylmethylamino)methyl]phenyl}-[4-(6-
diethylaminohexyloxy)phenyl]methanone (18a)
Compound 18a was prepared following the procedure described
for 3, starting from 14 (0.16 g, 0.37 mmol) and diethylamine
(0.04 mL, 0.44 mmol). The crude product was purified by flash
column chromatography using a mixture of CH2Cl2/CH3OH/NH4OH
(97:2.5:0.5) as eluant to afford 18a as a semisolid product (0.10 g,
55%); mp: 107e109 ꢁC (hydrochloride salt). 1H NMR (300 MHz,
4.1.27. {4-[(Benzylmethylamino)methyl]phenyl}-{4-[4-(2-
hydroxyethyl)phenoxy]phenyl}methanone (20)
To a solution of 11 (0.5 g, 1.5 mmol) in toluene (15 mL), 2-(4-
bromophenyl)ethanol (0.15 g, 0.75 mmol), Cs2CO3 (1.0 g,
3.0 mmol), Copper (I) trifluoromethanesulfonate toluene complex
(0.04 g, 0.735 mmol), ethyl acetate (0.04 g, 0.075 mmol), 1-naph-
CDCl3):
d
1.05 (t, 6H, J ¼ 7.2 Hz) 1.50e1.95 (m, 8H), 2.15 (s, 3H),
2.35e2.65 (m, 6H), 3.60 (s, 2H), 3.65 (s, 2H), 4.05 (t, 2H, J ¼ 6.6 Hz),
6.93 (d, 2H, J ¼ 8.8 Hz), 7.20e7.40 (m, 5H), 7.45 (d, 2H, J ¼ 8.4 Hz),
7.75 (d, 2H, J ¼ 8.4 Hz), 7.78 (d, 2H, J ¼ 8.8 Hz); 13C NMR (300 MHz,
ꢀ
toic acid (0.51 g, 3.0 mmol) and 4 A molecular sieves were added.
The reaction mixture was heated at 110 ꢁC under N2 atmosphere for
24 h. On cooling, NaOH 2 N (50 mL) was added and the aqueous
layer was extracted with CH2Cl2 (3 ꢃ 50 mL), the combined organic
layers were washed with brine, dried over anhydrous Na2SO4 and
evaporated to give a crude product that was purified by flash
CDCl3):
d 15.5, 16.6, 26.5, 31.3, 42.4, 45.7, 48.2, 56.7, 64.1, 64.8, 67.7,
115.3,129.4,129.9,128.4,130.7,130.2,130.8,131.6,132.4,135.4,135.9,
139.2, 145.6, 162.5, 198.4. MS (ESIþ): m/z: 487 [M þ H]þ; Anal. calcd
for C32H42N2O2: C 78.97, H 8.70, N 5.76, found: C 78.74, H 8.81, N 5.82.

F. Belluti et al. / European Journal of Medicinal Chemistry 46 (2011) 1682e1693
1691
chromatography on silica gel using a mixture of CH2Cl2/EtOAc (9:1)
with a Jasco V-530 double beam Spectrophotometer. Absorbance
value at 412 nm was recorded for 5 min and enzyme activity was
calculated from the slope of the obtained linear trend. Assays were
carried out with a blank containing all components except AChE or
BChE to account for the non-enzymatic reaction. The reaction rates
were compared and the percent inhibition due to the presence of
tested inhibitors was calculated. Each concentration was analyzed
in duplicate, and IC50 values were determined graphically from log
concentrationeinhibition curves (GraphPad Prism 4.03 software,
GraphPad Software Inc.).
as eluant to afford pure 20 as an oil (0.3 g, 20%). 1H NMR (200 MHz,
CDCl3):
d 2.20 (s, 3H), 3.40 (s, 2H), 3.45 (s, 2H), 3.55 (t, 2H,
J ¼ 6.6 Hz), 3.65 (t, 2H, J ¼ 6.60 Hz), 6.95 (d, 2H, J ¼ 8.8 Hz),
7.00e7.40 (m, 9H), 7.45 (d, 2H, J ¼ 8.4 Hz), 7.75 (d, 2H, J ¼ 8.4 Hz),
7.80 (d, 2H, J ¼ 8.7 Hz); 13C NMR (300 MHz, CDCl3):
d 38.94, 42.18,
61.87, 119.11, 125.34, 125.51, 126.74, 126.98, 128.05, 128.65, 128.78,
129.58, 130.14, 131.66, 133.85, 139.24, 144.97, 157.05, 159.16, 196.62.
4.1.28. {4-[(Benzylmethylamino)methyl]phenyl}-{4-[4-(2-
chloroethyl)phenoxy]phenyl}methanone hydrochloride salt (21)
To a solution of 20 (0.3 g, 0.66 mmol) in CH2Cl2 (30 mL), trie-
thylamine (0.2 mL, 1.6 mmol) and thionylchloride (0.12 mL,
1.6 mmol) were added dropwise at 0 ꢁC. The reaction mixture was
stirred for 18 h while warming at room temperature. The solvent
was removed under reduced pressure and purification was ach-
ieved by flash chromatography on silica gel using a mixture of
CH2Cl2/EtOAc (4:1) as eluant to yield 21 (0.2 g, 65%), mp: 75e77 ꢁC;
4.3. Determination of steady-state inhibition constant
To obtain estimates of the competitive inhibition constant Ki,
reciprocal plots of 1/v versus 1/[S] were constructed at relatively
low concentration of substrate (below 0.5 mM). The plots were
assessed by a weighted least-squares analysis that assumed the
variance of v to be a constant percentage of v for the entire data set.
Slopes of these reciprocal plots were then plotted against 16a
(range 0e1.93 nM) or donepezil concentration (range 0e47.5 nM)
in a similar weighted analysis, and Ki was determined as the ratio of
the replot intercept to the replot slope.
1H NMR (200 MHz, CDCl3):
d
2.60 (d, 3H, J ¼ 4.4 Hz), 3.35 (t, 2H,
J ¼ 6.6 Hz), 3.55 (t, 2H, J ¼ 6.6 Hz), 4.15e4.20 (m, 2H), 4.25e4.30 (m,
2H), 6.90e7.00 (m, 4H), 7.35e7.45 (m, 3H), 7.60e7.70 (m, 2H),
7.80e7.90 (m, 6H); 13C NMR (300 MHz, CDCl3):
d 38.64, 42.17, 61.87,
119.15,125.36,126.75,126.98,128.05,128.25,128.68,128.78,129.55,
130.14, 130.76, 139.24, 144.927, 157.05, 159.16, 196.62.
4.4. Determination of the inhibitory potency on A
aggregation induced by AChE
b (1e40)
4.1.29. {4-[(Benzylmethylaminomethyl]phenyl}-{4-[4-(2-
diethylaminoethyl)phenoxy]phenyl}methanone hydrochloride
salt (22)
Compound 22 was prepared following the procedure described
for 3, starting from 21 (0.20 g, 0.43 mmol) and diethylamine
(0.05 mL, 0.51 mmol). The crude product was purified by flash
column chromatography using a mixture of CH2Cl2/CH3OH (95:5)
as eluant to afford 22 (0.10 g, 46%), mp: 110e112 ꢁC; 1H NMR
Aliquots of 2
ilized from 2 mg/mL HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) and
dissolved in DMSO at a final concentration of 230 M, were incu-
mL of Ab(1e40) (Bachem AG, Switzerland), lyoph-
m
bated for 24 h at room temperature in 0.215 M sodium phosphate
buffer (pH 8.0). For coincubation experiments, aliquots of AChE
(2.30
(100
m
M) and AChE in the presence of the tested compound
m
M) were added. Blanks containing A , AChE, A plus the
b
b
(300 MHz, CDCl3):
d
1.15 (t, 6H, J ¼ 7.4 Hz), 2.20 (s, 3H), 3.45e3.55
tested compound, and AChE plus the tested compound in 0.215 M
sodium phosphate buffer (pH 8.0) were prepared [32]. The final
volume of each vial was 20 mL. To quantify amyloid fibril formation,
the thioflavin-T fluorescence method was used [46,47]. Thioflavin-T
binds to amyloid fibrils, giving rise to an intense specific emission
band at 490 nm in its fluorescent emission spectrum. Therefore,
after incubation, the samples were diluted to a final volume of 2 mL
(m, 8H), 3.75 (t, 2H, J ¼ 6.3 Hz), 4.09 (t, 2H, J ¼ 6.3 Hz), 6.95 (d, 2H,
J ¼ 8.8 Hz), 7.00e7.40 (m, 9H), 7.45 (d, 2H, J ¼ 8.4 Hz), 7.73 (d, 2H,
J ¼ 8.4 Hz), 7.81 (d, 2H, J ¼ 8.7 Hz); 13C NMR (300 MHz, CDCl3):
d
22.5, 34.5, 41.7, 45.6, 47.6, 61.1, 61.5, 61.7, 68.9, 116.4, 116.6, 127.7,
128.2, 128.4, 128.7, 128.9, 129.6, 130.3, 132.3, 132.4, 137.2, 137.9,
144.6, 155.6, 164.5, 187.5. MS (ESIþ): m/z: 528 [M þ Na]þ; 544
[M þ K]þ; Anal. calcd for C34H38N2O2: C 80.60, H 7.56, N 5.53, found:
C 80.80, H 7.43, N 5.47.
with 50 mM glycine-NaOH buffer (pH 8.5) containing 1.5 mM thi-
oflavin-T. A 300 s time scan of fluorescence intensity was carried
out (lexc ¼ 446 nm, lem ¼ 490 nm), and values at the plateau were
averaged after subtraction of the background fluorescence of the
4.2. Determination of the inhibitory potency on AChE and BChE
activities
1.5 mM thioflavin-T solution. The percent inhibition of the AChE-
induced aggregation due to the presence of the test compound was
The capacity of compounds 16aed, 17e19ab to inhibit AChE and
BChE activities was assessed by Ellman’s method [35]. AChE stock
solution was prepared by dissolving human recombinant AChE (E.C.
3.1.1.7) lyophilized powder (Sigma, Italy) in 0.1 M phosphate buffer
(pH ¼ 8.0) containing Triton X-100 (0.1%). Stock solution of BChE
(E.C. 3.1.1.8) from human serum (Lee BioSolutions, USA) was
prepared by dissolving the lyophilized powder in an aqueous
solution of gelatine (0.1%). Stock solutions of tested compounds
(1 mM) were prepared in methanol and diluted in bidistilled water.
Five increasing concentrations of inhibitor were assayed to obtain %
inhibition of the enzymatic activity in the range of 20e80. The
assay solution consisted of a 0.1 M phosphate buffer pH 8.0, with
calculated by the following expression: 100 ꢀ (IFi/IFo ꢀ 100) where
IFi and IFo are the fluorescence intensities obtained for Ab plus
AChE in the presence and in the absence of inhibitor, respectively,
minus the fluorescence intensities due to the respective blanks.
4.5. Computational studies
The docking simulations were carried out by means of ICM 3.6
[42]. The protein structure was prepared starting from the crys-
tallographic complex of human AChE with fasciculin (PDBid: 1B41)
[43]. Fasciculin and other non protein molecules were deleted and
only the enzyme chain was retained. Hydrogen atoms and missing
heavy atoms were added. Zero occupancy side chains, polar
hydrogen atoms, and the positions of asparagine and glutamine
side chain amidic groups were optimized and assigned the lowest
energy conformation. After optimization, histidines were assigned
the tautomerization state which improved the hydrogen bonding
pattern.
the addition of 340
0.02 unit/mL of human recombinant AChE, or BChE from human
serum and 550 M of substrate (acetylthiocholine iodide, ATCh or
mM
5,50-dithio-bis(2-nitrobenzoic acid),
m
butyrylthiocholine iodide, BTCh, respectively). Increasing concen-
tration of tested inhibitor were added to the assay solution and pre-
incubated for 20 min at 37 ꢁC with the enzyme followed by the
addition of substrate. Initial rate assays were performed at 37 ꢁC

1692
F. Belluti et al. / European Journal of Medicinal Chemistry 46 (2011) 1682e1693
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[18] D. Munoz-Torrero, Acetylcholinesterase inhibitors as disease-modifying
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modified to reproduce the rearrangement displayed in the murine
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defining the boundaries of the binding box were assumed to be
known and directly derived from the bound poses of two known
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The docking engine employed was the Biased Probability Monte
Carlo (BPMC) stochastic optimizer as implemented in ICM [50e52].
The ligand binding site at the receptor was represented by pre-
ꢀ
calculated 0.5 A spacing potential grid maps, representing van der
Waals potentials for hydrogens and heavy probes, electrostatics,
hydrophobicity, and hydrogen bonding. The van der Waals inter-
actions were described by a smoother form of the 6e12 Lennard-
Jones potential with the repulsive contribution capped at 4.0 kcal/
mol. The electrostatic contribution was buffered, artificially
increasing the distance between oppositely charged atoms in order
to avoid their collapse when the electrostatic attractive energy
prevailed on the softened van der Waals repulsion. The molecular
conformation was described by means of internal coordinate vari-
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3 force field with a distance-dependent dielectric constant [53].
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number of BPMC steps to be carried out was calculated by an
adaptive algorithm (thoroughness 1.0). The binding energy was
assessed by means of the standard ICM empirical scoring function
[54,55].
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