2-Arylbenzofuran-based molecules as multipotent Alzheimer's disease modifying agents

Article abstract of DOI:10.1016/j.ejmech.2012.10.045

The complex etiology of Alzheimer's disease prompts scientists to develop multi-target strategies to combat causes and symptoms. In line with this modern paradigm and as a follow-up to our previous studies, we designed and synthesized a focused collection

Full text of DOI:10.1016/j.ejmech.2012.10.045

European Journal of Medicinal Chemistry 58 (2012) 519e532
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2-Arylbenzofuran-based molecules as multipotent Alzheimer’s disease modifying
agents
,
Stefano Rizzo a 1, Andrea Tarozzi b, Manuela Bartolini a, Gregory Da Costa c, Alessandra Bisi a, Silvia Gobbi a,
Federica Belluti a, Alessia Ligresti d, Marco Allarà d, Jean-Pierre Monti c, Vincenza Andrisano a,
,
Vincenzo Di Marzo d, Patrizia Hrelia b, Angela Rampa a
*
a Department of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b Department of Pharmacology, Alma Mater Studiorum, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy
c Laboratoire de Physique et Biophysique, GESVAB EA3675 ISVV, University of Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux cedex, France
d Endocannabinoid Research Group, Institute of Biomolecular Chemistry, National Research Council, Via Campi Flegrei 34, Comprensorio Olivetti, 80078 Pozzuoli, NA, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The complex etiology of Alzheimer’s disease prompts scientists to develop multi-target strategies to
combat causes and symptoms. In line with this modern paradigm and as a follow-up to our previous
studies, we designed and synthesized a focused collection of new 2-arylbenzofurans and evaluated their
biological properties towards specific targets involved in AD, namely human AChE and human BuChE,
Received 20 August 2012
Received in revised form
11 October 2012
Accepted 26 October 2012
Available online 2 November 2012
and A
b
fibril formation. Selected compounds were also tested for their ability to inhibit A
b neurotoxicity
in terms of neuronal viability loss, and to prevent A
b
peptide-binding to cell membrane and intracellular
reactive oxygen species (ROS) formation. The different modifications introduced in the structure of our
lead compound led to an increase in activity towards one or more of the selected targets: the anticho-
linesterase activity of some compounds was found to be significantly higher than previously obtained
Keywords:
Alzheimer’s disease
2-Arylbenzofurans
Drug design
related molecules, and the compounds also proved to possess Ab anti-aggregating properties and neu-
roprotective effects. The most interesting multi-target compounds were 18, and 1. Interestingly, 1 also
showed good selectivity and moderate affinity for CB1 receptor, opening new perspectives in the field of
research on AD, since cannabinoid ligands have been widely reported to have neuroprotective properties.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Ab peptide
Neuroprotection
CB receptors
1. Introduction
decades if no efficacious treatments is discovered that can stop the
progression of the disease.
In the vast plethora of neurodegenerative disorders, Alzheimer’s
disease (AD) stands out as the most common form of dementia,
afflicting more than 24 million of individuals worldwide. AD is
characterized by an insidious onset and a chronic progression [1].
Symptoms worsen with the advancement of the disease, pro-
gressing from mild forgetfulness to widespread severe brain
impairment. Although the highest peak of incidence occurs among
elderly people (aged over 65, sporadic AD), cases of early onset have
been also reported (5% of all cases) and are frequently associated
with genetic predisposition (familial AD) [1]. Reflecting the world
population ageing, the scenario is expected to worsen in the next
In AD brains, the cholinergic system is the most dramatically
affected, showing remarkable depletion of acetylcholine (ACh) and
other markers of the cholinergic activity (cholinergic hypothesis)
[2]. Based on this observation, four drugs (tacrine, donepezil,
rivastigmine, galantamine) have been approved by the FDA and are
currently marketed for the symptomatic treatment of AD. Their
mechanism of action is the inhibition of the active site of acetyl-
cholinesterase (AChE), the enzyme responsible for the degradation
of ACh, resulting in an increased level of the neurotransmitter in the
synaptic cleft. Such a therapeutic strategy, though, exerts only
palliative effects and does not stop the progression of the disease,
indicating that the cholinergic impairment could be a downstream
event of a more complex and multifactorial sequence, still not fully
understood.
Two main neuropathological hallmarks are characteristically
present in AD brains, namely intraneuronal neurofibrillary tangles,
composed of hyper-phosphorylated forms of the microtubule-
associated protein tau and extracellular senile plaques, composed
* Corresponding author. Tel.: þ39 051 2099710; fax: þ39 051 2099734.
E-mail address: angela.rampa@unibo.it (A. Rampa).
Present address: Max-Planck-Institut für Molekulare Physiologie, Abteilung
1
Chemische Biologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.045

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S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
of beta amyloid protein (A
to tau remain undefined, a growing body of genetic and biochem-
ical evidence suggests that abnormal accumulation of A could
b
) [3]. Although the steps connecting A
b
well as, under certain conditions, of inhibitors of endocannabinoid
inactivation, for the treatment of A -induced toxicity and AD [7,8].
b
b
In the awareness of AD’s multifaceted nature, one of the most
promising therapeutic strategies to tackle this disorder is now
based on the multi-target-directed ligand (MTDL) approach [9,10].
In line with this paradigm, in a previous communication, we re-
ported the design, synthesis and biological evaluation of a small
series of 2-arylbenzofurans of hybrid molecules [11]. From this
collection, compound 1 (Fig. 1) emerged as a promising multi-
trigger tau pathology, therefore placing amyloid aggregates
upstream of neurofibrillary tangles and giving the former a central
role in initiating the pathological cascade (amyloid hypothesis) [4].
Of considerable interest is also the role played by butyr-
ylcholinesterase (BuChE), the second member of the cholinester-
ases family. In fact BuChE seems to be involved in the hydrolysis of
ACh during the late stages of the disease (in compensation for the
reduced levels of AChE) contributing to the breakdown of the
neurotransmitter. Moreover, BuChE has been found to be respon-
sible for up-regulating the expression of the amyloid precursor
protein (APP) in cell membranes. Thereby, inhibition of BuChE
could be regarded as an additional approach for the treatment of
moderate forms of AD as it would result in the increase of ACh
target-directed lead, displaying anticholinesterase activity and A
b
anti-aggregation properties, along with an additional neuro-
protective effect in living human neuronal SHeSY5Y cells [11]. The
benzofuran heterocyclic framework is now considered as a privi-
leged structure, which is a common moiety found in many bio-
logically active natural and therapeutic products and thus
represents a very important pharmacophore. In particular, this
scaffold is present in LY320135 (Fig. 1), a high affinity antagonist/
inverse agonist at CB1 receptor [12]. Therefore, as a follow-up to our
previous study, here we present the design, synthesis and biolog-
ical evaluation of an extended collection of 2-arylbenzofuran
derivatives, focused on the structure of 1. Aimed at optimizing/
improving the biological activities, and further exploring the
chemical space of the selected targets, some modifications of the
lead 1 were performed as reported in Fig. 1. The spacer between the
2-arylbenzofuran and the N-methyl-N-benzylamine moieties was
varied to determine its optimal length, and the substituent in 3
position of the benzofuran scaffold was changed. Moreover, the
N-methyl-N-benzylamine heptyloxy side chain was moved from
the para to the meta position. In summary, a small library of 23 new
derivatives was synthesized, whose structures are collected in
Table 1. All compounds were tested for their biological activities
towards selected targets involved in AD, namely human AChE and
synaptic levels and the decrease of neurotoxic Ab fibrils [5,6].
Recently, the endocannabinoid system has attracted much
interest as a novel therapeutic target involved in AD and other
neurodegenerative diseases, due to potentially neuroprotective,
anti-inflammatory and neurotrophic effect of certain cannabinoids.
In particular, compounds that activate cannabinoid receptors of
type 2 (CB2), either directly or by inhibiting endocannabinoid
cellular reuptake or endocannabinoid inactivation by hydrolytic
enzymes, have been suggested to produce beneficial effects in
terms of both anti-inflammatory and neuroprotective effects and,
subsequently, of anti-amnesic actions [7,8]. On the other hand,
activation of cannabinoid receptors of type 1 (CB1) was suggested
to either contribute to the amnesic actions of A
these actions, depending on the timing of the administration, and
to inhibit A toxicity in vitro. These observations prompt the further
b or to counteract
b
testing of both CB1 agonists and antagonists and CB2 agonists, as
NC
O
OMe
Me
R
O
Me
MeO
O(CH2)7N
LY320135
CB1 antagonist
O
AChEI general formula
O(CH2)3NEt2
O
SKF-64346
Aβ antiaggregant
hybridization
O
Me
O(CH2)7N
O
1
diversification studies
R1
p-
Me
O(CH2)nN
O
R2
m-
3-25
Fig. 1. Design of the studied compounds.

S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
521
Table 1
Inhibitory activities on human AChE and BuChE expressed as IC50 values of the studied compounds.
Comp.
R1
R2
H
N
7
Chain
hAChE IC50
m
M BuChE
hBChE IC50 mM
1a
para
40.7 ꢀ 3.5
38.1 ꢀ 2.2
2a
3
4
5
6
H
H
H
H
H
H
H
H
H
H
H
H
7
3
5
8
9
7
para
para
para
para
para
meta
32.6 ꢀ 11.9
n.a.b
0.28 ꢀ 0.02
136 ꢀ 5
215 ꢀ 1
6.60 ꢀ 0.23
7.47 ꢀ 0.34
55.6 ꢀ 7.4
3.00 ꢀ 0.17
n.a.b
n.a.b
7
n.a.b
O
8
9
H
H
7
7
para
para
101 ꢀ 21
60.1 ꢀ 6.3
89.1 ꢀ 9.9
n.a.b
10
11
H
H
7
7
para
para
n.a.b
88.9 ꢀ 14.3
81.2 ꢀ 0.9
67.7 ꢀ 1.3
12
13
14
15
H
H
H
H
7
7
7
7
para
para
para
para
53 ꢀ 10
3.18 ꢀ 0.46
9.42 ꢀ 1.32
1.81 ꢀ 0.08
23.0 ꢀ 2.6
100 ꢀ 18
37.1 ꢀ 6.0
78 ꢀ 21
O
O
O
O
NEt2
NEt2
16
H
7
para
39.0 ꢀ 0.5
21.0 ꢀ 2.8
O
O
17
18
19
OCONHCH3
7
7
7
para
meta
meta
0.34 ꢀ 0.03
0.24 ꢀ 0.02
102 ꢀ 18
0.88 ꢀ 0.10
2.88 ꢀ 0.26
0.40 ꢀ 0.07
H
H
20
H
7
meta
n.a.b
43.9 ꢀ 5.31
(continued on next page)

522
S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
Table 1 (continued )
Comp.
R1
R2
H
N
7
Chain
hAChE IC50
n.a.b
m
M BuChE
hBChE IC50 mM
21
meta
1.97 ꢀ 0.28
22
23
H
H
7
7
meta
meta
n.a.b
n.a.b
1.37 ꢀ 0.57
3.01 ꢀ 0.47
24
25
H
H
7
7
meta
meta
n.a.b
n.a.b
2.51 ꢀ 0.42
0.048 ꢀ 0.008
Rivastigminec
Tacrinec
3.01 ꢀ 0.21
0.42 ꢀ 0.02
2.01 ꢀ 0.15
0.30 ꢀ 0.01
0.046 ꢀ 0.003
20.7 ꢀ 1.5
Galantaminec
Human recombinant AChE and BuChE 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/three independent measurements, each performed in triplicate.
a
From ref. [11].
b
n.a. ¼ not active. Compounds defined “not active” did not show any inhibitory activity when tested at a concentration equal to their maximum solubility in the assay
conditions.
c
From ref. [37].
human BuChE, and A
some selected compounds were also tested for their ability to
inhibit A neurotoxicity in terms of neuronal viability loss, and to
prevent A peptide-binding to cell membrane and intracellular ROS
b
fibril formation. On the basis of the results,
with 3-methylaminomethylphenol afforded 45 subsequently
transformed into the N-methylcarbamate derivative 17, by treat-
ment with methyl isocyanate.
b
b
formation in human neuronal SHeSY5Y cells.
3. Biological evaluation
Moreover, taking advantage of the presence of the benzofuran
scaffold, we decided to evaluate the feasibility of extending the
biological profile of the new compounds, merging in a single
molecule potential activity at CB receptors as well. To this aim we
tested all compounds for their affinity for CB1 and CB2 receptors.
The inhibitory activities against both cholinesterases of the
newly synthesized compounds were studied using the method
described by Ellman [14] to determine the rate of acetylthiocholine
or butyrylthiocholine hydrolysis in the presence of the inhibitor.
The inhibition of A
assay that uses UVevis measurements [15]. In this assay the Ab25e
peptide, which preserves the properties of neurotoxicity and
b fibril formation was studied with an in vitro
2. Chemistry
35
The synthesis of the studied compounds was accomplished as
shown in Scheme 1. Previously described benzofuran-based
phenols [11,13] were O-alkylated by treatment with selected
dihaloalkanes to afford the corresponding halo-alkyloxy-
derivatives 26e31. Condensation with N-benzyl-N-methylamine
in refluxing toluene afforded derivatives 3e7. Next, a selection of
acylchlorides was used for the tin(IV) chloride-promoted Friedele
Crafts acylation of the benzofuran scaffold, which occurred as ex-
pected only at position 3 of the heterocycle. Unfortunately, the
resulting acyl derivatives 12e16 could be obtained only in poor
yields (8e21%), most likely because the high affinity of the Lewis
acid for the amino group in the side chain of 3e7 prevented its
regeneration. To overcome this problem we performed the critical
FriedeleCrafts acylation step prior to the condensation with
N-benzyl-N-methylamine, although to the cost of a premature
divergent step, requiring more purification efforts. Therefore,
intermediates 28 and 31 were first converted into the 3-acyl-
derivatives 32e44, then treated with N-benzyl-N-methylamine to
afford the desired products 8e11, and 18e25. Condensation of 36
aggregation, was used [16,17]. For the compounds exhibiting an
inhibitory activity at least equal to that of curcumin (as reference
compound endowed with antiamyloidogenic properties [18]), IC50
values were calculated. The neuroprotective effects of the most
interesting A
b antiaggregating compounds against the Ab25e35
peptide induced toxicity in human neuronal SHeSY5Y cells were
also determined using a colorimetric MTT assay [19]. In parallel, the
ability of the same compounds to inhibit Ab25e35 peptide-binding
to cell membrane and Ab25e35 peptide-induced ROS formation
was investigated using Congo Red (CR) and DHE assay, respectively
[20].
Finally, the effect of the new compounds on [3H]-CP55940
binding to human recombinant cannabinoid CB1 and CB2 recep-
tors was analysed. The activities, expressed as IC50, were deter-
mined by non-linear regression of the inhibition of radioligand
binding exerted by increasing concentrations of test compounds.
When the IC50 value was lower than 10
mM, the Ki value was
calculated by applying the ChengePrusoff equation to the IC50
value.

S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
523
OH
O
R1
i
O
O(CH2)7X
O(CH2)nX
iii
O
O
26: p; n=3; X=Cl
27: p; n=5; X=Cl
28: p; n=7; X=Br
29: p; n=8; X=Br
30: p; n=9; X=Br
31: m; n=7; X=Br
32-44
ii
ii
R1
Me
O(CH2)nN
O
Me
O(CH2)7N
O
R2
O
3: p; n=3
4: p; n=5
2: p; n=7
5: p; n=8
6: p; n=9
7: m; n=7
8-11; 18-25 (see Table 1)
iii
45 R1=CH3; R2 = OH
17 R1=CH3; R2 = OCONHCH3
iv
R1
p-
m-
O
Me
O(CH2)7N
O
12-16 (see Table 1)
Reagents and conditions: i) Br(CH2)nX, K2CO3, acetone, reflux, 24 h; ii) N-methyl-N-benzylamine
or N-(3-hydroxybenzyl)methylamine, toluene, reflux; iii) selected acylchloride, SnCl4, DCM, r.t.;
iv) CH3NCO, NaH, rt, 24 h.
Scheme 1. Synthesis of the studied compounds.
4. Results and discussion
Moving the heptyloxy chain of 2 from the para to the meta
position of the phenyl ring (compound 7) lowered the activity
toward hBuChE and abolished hAChE inhibition. Nevertheless,
compound 7 was acylated with the groups that proved to be
favourable for activity in the para series, to obtain compounds 18e
25. Interestingly, compound 18, bearing the same unsubstituted
benzoyl group of 1, was the most active of the series on hAChE
inhibition, showing a sub-micromolar IC50 value, thus being 180
fold more potent than 1. The insertion of substituents on the
benzoyl moiety (compounds 19e25) led to almost inactive deriv-
atives on this target. Conversely, compounds 19 (with a 2-
methylbenzoyl group) and 25 (with a 2,4 dimethoxybenzoyl
substituent) emerged for their remarkable activity on hBuChE,
being 25 the most active (IC50 ¼ 48 nM) of the series and highly
selective. Finally, compound 17 bearing a methylcarbamic group
(acting as a pseudo-irreversible inhibitor, as already reported in
previous papers for carbamate analogues [21,22]), showed an
improvement of potency of one order of magnitude on hAChE and
retained a very good activity toward hBuChE, when compared to
the marketed drug rivastigmine.
The inhibitory activities against both recombinant human AChE
(hAChE) and BuChE from human serum (hBuChE) of new deriva-
tives, together with those of the lead compound 1, and the refer-
ence compounds rivastigmine, tacrine and galantamine, are
reported in Table 1, and are expressed as IC50 values.
To assess the importance of the heptyloxy chain spacer of the
lead 1, its length was varied, to obtain compounds 3e6, where
n ¼ 3, 5, 8, 9. All compounds proved to be almost inactive on hAChE,
and remarkably less active toward hBuChE than the reference
compound 2 (not acylated precursor of 1, bearing a heptyloxy
spacer [11]). Then, once the best chain length was established,
different acyl groups were inserted in position 3 of the benzofuran
scaffold of compound 2, to explore the chemical space on the
different targets. Compounds 8e11, bearing bulky and lipophilic
groups, showed lower activity than the lead compound 1 on both
hAChE and hBuChE. Compounds 12e16, carrying an additional
amino moiety, did not show an improvement of AChE inhibition,
still keeping a fairly good BuChE inhibition.

524
S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
The benefit of having cholinesterase inhibitors able of modu-
80
40
lating the brain activity of a single enzyme in a highly selective
manner or both cholinesterases has been debated since non-
cholinergic activities of AChE and the cholinergic role of BuChE over
AD progression were discovered. Indeed, AChE is principally asso-
ciated with neurons and axons, while BuChE is primarily expressed
and secreted by glial cells within the brain [23]. In the healthy
human brain, AChE and BuChE are found in the ratio of 4:1.
However, in the brains of AD patients AChE activity can decline by
up to 45% during disease progression, reflecting the disappearance
of neurons and axons to which it is associated, while BuChE activity
can be elevated by up to 2-fold [5], thereby considerably altering
this ratio. Furthermore, the dual AChE/BuChE inhibitor rivastigmine
demonstrated beneficial effects on memory acquisition and
consolidation. This may involve direct classical cholinergic
augmentation, together with a cholinergically-mediated protective
action, potentially via the reduction of inflammation [24]. There-
fore, a comparable profile could also be envisaged for the dual
AChE/BuChE inhibitor 17 and for the BuChE selective derivatives 19
and 25.
0
-40
-80
-120
Compounds
Fig. 3. Ab25e35 fibril inhibition by meta substituted compounds compared to that of
curcumin (Cur). Means and SD of three independent experiments are shown.
compounds 18 and 19 at 10 and 30 mM significantly reduced the
neuronal viability loss evoked by Ab25e35 peptide, in a dose-
dependent manner. Since the same trend was observed with
compound 1, these results proved that moving the heptyloxy chain
from the para to the meta position of the phenyl ring did not influ-
ence the observed neuroprotective effects. Therefore, the benzoyl
group of compound 1 (maintained also in 18, and with a little change
in 19) is crucial for the interaction with hydrophobic residues of
Regarding Ab fibril inhibition, the chain length turned out to
play an important role, since the propyloxy derivative 3 showed
poor inhibition, while moving through the homologous series the
activity rose. Pentyloxy (4) and octyloxy (5) derivatives showed the
same activity as curcumin, while the nonyloxy derivative 6 was
A
b25e35 peptide, such as Ile31, Ile32 and Met35, that are critical for
more active (IC50 ¼ 5.5
mM). Regarding compounds 8e11, with
both neurotoxicity and aggregation processes [17,28]. The lack of
neuroprotective effect shown in this cellular assay by 7, that proved
to be the most potent antiaggregating compound in the vitro assay,
could probably be ascribed to its lower log P (6.80) with respect to 1
(log P ¼ 8.00), 18 (log P ¼ 8.00) and 19 (log P ¼ 8.49), as already
pointed out in a previous paper [11].
In this context, recent studies have suggested that unaggregated
b25e35 and Ab31e35 peptides could initiate a cascade of events
leading to neurotoxicity solely after their internalization within the
neuronal cells [29]. These findings prompted us to evaluate the
ability of compounds 1, 18 and 19 to prevent the binding between
the Ab25e35 peptide and the plasma membrane surface. The binding
bulky and aromatic groups in 3 position of the benzofuran scaffold,
only compound 8 retained the activity of the lead compound 1,
while 11 surprisingly showed a proaggregatory activity (Fig. 2).
Compounds 12e16, carrying an additional amino moiety,
proved to weakly inhibit amyloid aggregation. The most potent
inhibitor of the series was 7, obtained by moving the heptyloxy
chain to the meta position (Fig. 3), 2.5 times more active when
A
compared to curcumin (IC50 ¼ 3.9 and 10
mM, respectively). In the
meta series, 18 and 19 retained the activity of the lead compound 1.
For compounds exhibiting an inhibitory activity at least equal to
that of curcumin, IC50 values were calculated as reported in Table 2.
Aggregation of monomeric A
weight oligomers produces the primary neurotoxic species in AD
[25]. Indeed, oligomer species of aggregated A exert toxic effects
b species into higher molecular
of
a maximum in 30 min and was significantly reduced by co-
treatment with compounds 1, 18 and 19 (30 M), as determined
Ab25e35 peptide (10 mM) with SHeSY5Y cells reached
b
m
on synaptic and cellular functions [26], finally leading to neuro-
degeneration and cognitive, as well as neuropsychiatric, symptoms.
Thus, compounds that are able to slow down or block the amyloid
polymerization process could be considered potential drugs for
inhibition of AD progression [27].
by the CR assay (Fig. 5). In parallel, the oxidative stress in SHeSY5Y
cells, in terms of intracellular ROS formation evoked by Ab25e35
peptide (10 mM) was also investigated. Recent studies show that
ROS formation is directly related to membrane perturbation by
The neuroprotective effects of the most interesting A
aggregating compounds (7, 8, 18, 19) were determined against the
b25e35 peptide induced toxicity in human neuronal SHeSY5Y cells.
b anti-
Table 2
A
Ab
fibril formation inhibitory activity and effects of selected compounds on Ab25e35
Compounds 18 and 19, but not 7 and 8, showed a similar neuro-
protective effect against Ab25e35 peptide induced neurotoxicity
(Table 2). As reported in Fig. 4, treatment of SHeSY5Y cells with both
peptide induced neurotoxicity in SHeSY5Y cells.
Compound
A
b25e35 fibril
A
b25e35 IC50
m
Mb
SHeSY5Y cells PIc
inhibition (%)a
1
7
8
18
19
47
81
48
35
48
12.5
3.9
e
58.16 ꢀ 3.45
n.d.
n.d.
80
60
40
20
0
e
48.35 ꢀ 4.68
e
46.20 ꢀ 5.40
n.d. ¼ PI not determined because protective effects were not observed at highest
tested concentration.
a
see Figs. 2 and 3.
-20
-40
-60
b
IC50 values defined as the concentrations of inhibitor to inhibit the formation of
Ab25e35 fibrils to 50% of the control value (reference compound: curcumin
IC50 ¼ 10.0
m
M).
c
-80
Percentage Inhibition (PI) observed at highest tested concentration. The
neuronal viability in SHeSY5Y cells was determined by MTT assay (as described in
Compounds
Experimental section), after 3 h of incubation with Ab25e35 peptide (10
m
M) in the
Fig. 2. Ab25e35 fibril inhibition by para substituted compounds compared to that of
curcumin (Cur). Means and SD of three independent experiments are shown.
presence or absence of various compounds (30
at least two independent experiments.
m
M). The values are the mean ꢀ SD of

S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
525
100
80
60
40
20
0
100
80
60
40
20
0
0
1
3
10
30
0
1
3
10
30
Compound 7 ( μM)
Compound 8 ( μM)
100
80
60
40
20
0
100
80
60
40
20
0
**
**
**
*
*
0
1
3
10
30
0
1
3
10
30
Compound 18 (μM)
Compound 19 (μM)
Fig. 4. Effects of selected compounds on Ab25e35 peptide-induced neurotoxicity in SHeSY5Y cells. The neuronal viability in SHeSY5Y cells was determined by MTT assay (as
described in the Experimental section), after 3 h of incubation with Ab25e35 peptide (10 M) in the presence or absence of various concentrations of compounds (1e30 M). The
values are reported as mean ꢀ SD of three independent experiments (*p < 0.05, **p < 0.01, vs. untreated cells, ANOVA with Dunnett’s test).
m
m
amyloid peptide [30,31]. Remarkably, compounds 1, 18 and 19
(30 M) significantly inhibited the Ab25e35 peptide-induced ROS
formation (Fig. 6). Taken together, these results showed the ability
peptides, suggesting their peculiar ability to prevent the interaction
between Ab25e35 peptides and the cell membrane of SHeSY5Y cells,
responsible fort the ROS formation as well as neuronal viability loss.
Finally, with respect to the feasibility of merging in a single
molecule different biological profiles, the new compounds were
tested for their ability of targeting cannabinoid receptors (Table 3).
As expected from their similarity to the heterocyclic pharmaco-
phore present in LY320135, compound 1, and to a lesser extent its
precursor compound 2, exhibited a good selectivity and a moderate
affinity for human recombinant CB1 receptors. When replacing the
phenyl ring in position 3 of 1 with bulkier acyl groups (compounds
8e11), only compound 10, bearing the biphenyl moiety, showed an
appreciable, although unselective, affinity for CB2. Furthermore,
the introduction of an amino moiety on the benzoyl group
(compounds 12e16), could improve the activity, depending on the
type and the position of the substituent: the position (4 vs. 3) of the
amine group appeared to modulate affinity and strongly increase
selectivity for CB2 vs. CB1 (see 14 vs. 15 or 13). On the contrary, the
compounds of the meta series (18e25) were inactive (data not
shown). These results prove the moving the heptyloxy chain from
the para to the meta position is detrimental for activity.
m
of compound 1, 18 and 19 to counteract the neurotoxicity of Ab25e35
Aβ
25-35
Aβ
Aβ
Aβ
+ compound 1
+ compound 19
+ compound 18
25-35
25-35
25-35
140
130
120
110
**
*
100
80
Taking into account our aim of identifying new multi-target
directed ligands, 18 emerged as a promising compound, since it
proved to be remarkably more active than 1 on both AChE and
40
0
BuChE, maintained good Ab antiaggregating properties and showed
0
10
20
30
the best neuroprotective profile, being capable of restoring cell
viability by 48%. Moreover, it also proved to be endowed with
interesting effects in additional biological studies, since it was able
to prevent the binding of the Ab25e35 peptide to the plasma
membrane surface and to inhibit Ab25e35 peptide-induced ROS
formation. Although it is generally expected for a multi-target
ligand that the activities on the different targets would be appro-
priately balanced, the levels of these biological counterparts should
Time (min)
Fig. 5. Effects of selected compounds on the binding of Ab25e35 peptide with SHeSY5Y
cells. SHeSY5Y cells were treated with Ab25e35 peptide (10 M) for different time in
the presence or absence of compounds 1, 18 and 19 (30 M). At the end of incubation,
m
m
the Ab25e35 peptide binding to plasma membrane was determined by CR assay and the
results are expressed as percentage of control cells (*p < 0.05, **p < 0.01, vs. untreated
cells with Ab25e35 peptide at Student’s t-test).

526
S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
Fig. 6. Effects of selected compounds on Ab25e35 peptide-induced ROS formation in SHeSY5Y cells. (a) SHeSY5Y cells were treated with Ab25e35 peptide (10
presence of compounds 1, 18 and 19 (30 M) for 3 h. At the end of incubation, ROS formation was determined using a fluorescence probe, DHE, as described in the Experimental
section. Four randomly selected areas with 50e100 cells in each were analysed under a fluorescence microscope and the values obtained are expressed as densitometry/cell. Values
are shown as mean ꢀ SD of three independent experiments (**p < 0.01, vs. untreated cells at Student’s t-test). (b) Representative images of ROS formation. Scale bars: 100 m.
mM) in the absence or
m
m
also be taken into account in order to appropriately evaluate its
efficacy. Considering the levels of amyloid-peptide in the cerebro-
spinal fluid of patients afflicted with mild cognitive impairment or
sporadic AD are in the sub-nanomolar range [32] new inhibitors
might potentially exert their neuroprotective effects when a sub-
nanomolar concentration is reached. Moreover, on the basis of
the cholinesterase level in AD patients [33], it also seems plausible
that compounds with anti-cholinesterase activity in the sub-
micromolar range might simultaneously inhibit amyloid aggrega-
tion if a concentration close to their IC50 values is locally reached.
Noteworthy, the extension of the biological studies by exploi-
tation of these new assays allowed us to broaden the profile of the
lead compound 1, which was also able to counteract the
neurotoxicity of Ab25e35 peptide by preventing the interaction
between this peptide and the cell membrane of SHeSY5Y cells,
responsible for ROS formation and neuronal viability loss. In addi-
tion, 1 showed good selectivity and moderate affinity for CB1
receptors. The higher cytoprotection activity of 1, with respect to 18
and 19, could thus be ascribed to its affinity for CB1 receptors,
known to be expressed in SHeSY5Y cells [34,35]. In this regard,
a recent study suggests that CB1 receptor activation exerts a strong
neuroprotection action against Ab-induced neurotoxicity [36].
5. Conclusions
In continuing our studies on the design of new multi-target
compounds for the treatment of AD, we performed several modi-
fications of the promising hit 1, previously identified among a new
series of hybrid molecules based on the frameworks of our AChE/
BuChE inhibitors and of SKF-64346 [11].
Table 3
Effect of studied compounds on [3H]-CP55940 binding to the human recombinant
cannabinoid receptors. Percent of binding displacement at the maximum concen-
tration tested is also reported.
With the aim of further optimizing this scaffold and broadening
the biological profile of the molecules by investigating their
potential action on additional targets involved in AD, a small library
of compounds was prepared. The different modifications intro-
duced in the structure of the lead compound led to an increase in
activity towards one or more of the selected targets, compounds 19
and 25 being potent BuChE inhibitors, carbamate 17 showing
higher activity than the marketed drug rivastigmine on both AChE
Comp EC50 on Ki on
Max tested (%
M displacement)
EC50 on Ki on
Max tested (%
M displacemet)
CB1
m
M
CB1
m
CB2
m
M
CB2
m
1
2
8
9
10
11
12
13
14
15
16
0.79
6.95
>10
>10
7.74
>10
>10
>10
6.38
>10
1.37
0.32
2.80
>10
>10
3.12
>10
>10
>10
2.57
>10
0.55
10
10
10
10
25
10
10
10
10
10
10
m
m
m
m
m
m
m
m
m
m
m
M (8.72%)
>10
>10
>10
>10
>10
1.79
>10
>10
1.12
0.69
1.18
0.58
10
10
10
10
25
10
10
10
10
10
10
m
m
m
m
m
m
m
m
m
m
m
M (47.32%)
M (21.78%)
M (8.90%)
M (6.87%)
M (86.00%)
M (9.87%)
M (47.27%)
M (65.17%)
M (77.91%)
M (92.00%)
M (70.58%)
M (57.83%) >10
M (13.81%) >10
M (35.06%) >10
M (80.00%) 7.06
M (30.34%) >10
M (29.75%) >10
M (47.70%) 4.40
M (76.50%) 2.71
M (35.18%) 4.65
M (83.12%) 2.27
and BuChE and 7 inhibiting A
but failed to protect neuronal cells from A
b
fibril formation in vitro up to 81%,
toxicity.
b
The most interesting multi-target compounds proved to be 18,
and 1, being able to address all the selected targets. Noteworthy, 1
also showed good selectivity and moderate affinity for CB1 recep-
tors, opening new perspectives in the field of research on AD.
Data are reported as means of at least n ¼ 3 experiments. SEMs are not shown, but
they were always less than 10% of the mean.

S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
527
In conclusion, these studies indicate that it is possible to obtain
compounds capable of modulating both classic and novel,
emerging targets involved in AD. These new compounds could
serve as a platform for further studies aimed at fine-tuning their
multifaceted activities for the identification of new molecular
entities endowed with broader biological profile in awareness of
the emerging multi-target paradigm for drug discovery.
6.1.5. 2-[4-(9-Bromononyloxy)phenyl]benzofuran (30)
Using the previous procedure and starting from 4-benzofuran-
2-yl-phenol [11] (0.57 g, 2.7 mmol) and 1,9-dibromononane
(1.1 mL, 5.4 mmol), 30 (0.82 g, 73%) was obtained as a white
solid. mp 108 ꢁC. 1H NMR
d: 1.34e1.56 (m, 10H), 1.76e1.95 (m, 4H),
3.41 (t, J ¼ 6.0 Hz, 2H), 4.00 (t, J ¼ 6.6 Hz, 2H), 6.88 (s, 1H), 6.96 (d,
J ¼ 8.3 Hz, 2H), 7.20e7.25 (m, 2H), 7.48e7.56 (m, 2H), 7.75 (dd,
J1 ¼ 2.1 Hz; J2 ¼ 6.9 Hz, 2H).
6. Experimental section
6.1.6. 2-[3-(7-Bromoheptyloxy)phenyl]benzofuran (31)
6.1. Chemistry. General methods
Using the previous procedure and starting from 3-benzofuran-
2-yl-phenol [13] (2.65 g, 13 mmol) and 1,7-dibromoheptane
(4.3 mL, 25 mmol), 31 (4 g, 81%) was obtained as a white solid.
Melting points were measured in glass capillary tubes on
a Büchi SMP-20 apparatus and are uncorrected. Direct infusion ES-
MS spectra were recorded on a Waters Micromass ZQ 4000 appa-
ratus. 1H NMR spectra were recorded in CDCl3 solution on a Varian
Gemini 300/400 MHz spectrometer. Chemical shifts are reported in
parts per million (ppm) relative to tetramethylsilane (TMS), and
spin multiplicities are given as s (singlet), d (doublet), t (triplet), m
(multiplet) or br (broad). Chromatographic separations were per-
formed on silica gel columns (Kieselgel 40, 0.040e0.063 mm;
Merck) by flash chromatography. Analyses indicated by the
symbols of the elements were within ꢀ0.4% of the theoretical
values. Compounds were named following IUPAC rules as applied
by Beilstein-Institut AutoNom (version 2.1), a PC integrated soft-
ware package for systematic names in organic chemistry.
mp 145 ꢁC. 1H NMR
d: 1.23e1.58 (m, 6H), 1.69e1.98 (m, 4H), 3.37e
3.42 (m, 2H), 4.01 (t, J ¼ 6.4 Hz, 2H), 6.89 (s, 1H), 6.93 (d, J ¼ 8.4 Hz,
2H), 7.24e7.31 (m, 2H), 7.50e7.56 (m, 2H), 7.79 (d, J ¼ 8.4, 2H).
6.1.7. [3-(4-Benzofuran-2-yl-phenoxy)propyl]benzylmethylamine
(3)
A stirred solution of 26 (0.9 g, 3.1 mmol) and N-benzyl-N-
methylamine (0.80 mL, 6.2 mmol) in toluene (100 mL) was refluxed
in the presence of a catalytic amount of NaI for 20 h. The mixture
was washed with water (3 ꢂ 25 mL) and the organic layer was dried
over Na2SO4. The solvent was removed under reduced pressure and
the residue was purified by flash chromatography on silica gel
(toluene/acetone 96:4), affording 3 as a yellowish solid (1.1 g, 95%).
mp 84 ꢁC. 1H NMR
d: 1.96e2.04 (m, 2H), 2.23 (s, 3H), 2.52e2.60 (m,
6.1.1. 2-[4-(3-Chloropropoxy)phenyl]benzofuran (26)
2H), 3.51 (s, 2H), 4.09 (t, J ¼ 6.6 Hz, 2H), 6.86 (s, 1H), 6.96 (d,
J ¼ 8.0 Hz, 2H), 7.15e7.30 (m, 7H), 7.45e7.55 (m, 2H), 7.75e7.80 (m,
2H). ES-MS m/z: 372 (M þ 1). Anal. C25H25NO2 (C, H, N).
A
stirred mixture of 4-benzofuran-2-yl-phenol [11] (1 g,
4.7 mmol), 1-bromo-3-chloropropane (0.94 mL, 9.5 mmol) and
K2CO3 (1.2 g) was refluxed in acetone (100 mL) for 20 h. The
suspension was filtered while hot and the solvent was removed
under reduced pressure. After adding petroleum ether, the residue
was kept in the freezer overnight and the white solid that formed
was filtered off, affording 26 (0.93 g, 69%). mp 137e138 ꢁC. 1H NMR
6.1.8. [5-(4-Benzofuran-2-yl-phenoxy)pentyl]benzylmethylamine (4)
Using the previous procedure and starting from 27 (0.34 g,
1.6 mmol), 4 was obtained as a yellowish solid (0.26 g, 40%). mp
57 ꢁC. 1H NMR
d: 1.47e1.59 (m, 4H), 1.75e1.81 (m, 2H), 2.18 (s, 3H),
d
: 2.23e2.31 (m, 2H), 3.77e3.80 (m, 2H), 4.20 (t, J ¼ 6.0 Hz, 2H), 6.87
2.34e2.41 (m, 2H), 3.47 (s, 2H), 3.92e3.99 (t, J ¼ 6.2 Hz, 2H), 6.84 (s,
1H), 6.90e6.95 (d, J ¼ 8.8 Hz, 2H), 7.18e7.31 (m, 7H), 7.46e7.51 (m,
2H), 7.73e7.78 (d, J ¼ 8.8 Hz, 2H). ES-MS m/z: 400 (M þ 1). Anal.
(s, 1H), 6.96 (d, J ¼ 8.3 Hz, 2H), 7.18e7.32 (m, 2H), 7.47e7.58 (m, 2H),
7.78 (d, J ¼ 8.4, 2H).
C27H29NO2 (C, H, N).
6.1.2. 2-[4-(5-Chloropentyloxy)phenyl]benzofuran (27)
Using the previous procedure and starting from 4-benzofuran-
2-yl-phenol [11] (0.34 g, 1.6 mmol) and 1-bromo-5-chloropentane
(0.42 mL, 3.2 mmol), 27 (0.35 g, 69%) was obtained as a white
6.1.9. [8-(4-Benzofuran-2-yl-phenoxy)octyl]benzylmethylamine (5)
Using the previous procedure and starting from 29 (0.51 g,
1.3 mmol), 5 was obtained as a white solid (0.2 g, 36%). mp 69e
solid. mp 115 ꢁC. 1H NMR
d
: 1.60e1.71 (m, 2H), 1.83e1.90 (m, 4H),
70 ꢁC. 1H NMR
d: 1.32e1.58 (m, 10H), 1.75e1.85 (m, 2H), 2.18 (s,
3.58 (t, J ¼ 6.3 Hz, 2H), 4.03 (t, J ¼ 6.0 Hz, 2H), 6.88 (s, 1H), 6.96 (d,
J ¼ 8.4 Hz, 2H), 7.21e7.26 (m, 2H), 7.48e7.56 (m, 2H), 7.76 (d, J ¼ 8.4,
2H).
3H), 2.32e2.39 (m, 2H), 3.47 (s, 2H), 4.00 (t, J ¼ 6.2 Hz, 2H), 6.84 (s,
1H), 6.96 (d, J ¼ 8.8 Hz, 2H), 7.19e7.32 (m, 7H), 7.47e7.54 (m, 2H),
7.78 (d, J ¼ 8.8 Hz, 2H). ES-MS m/z: 442 (M þ 1). Anal. C30H35NO2 (C,
H, N).
6.1.3. 2-[4-(7-Bromoheptyloxy)phenyl]benzofuran (28)
Using the previous procedure and starting from 4-benzofuran-
2-yl-phenol [11] (0.35 g, 1.6 mmol) and 1,7-dibromoheptane
(0.57 mL, 3.3 mmol), 28 (0.34 g, 58%) was obtained as a white
6.1.10. [9-(4-Benzofuran-2-yl-phenoxy)nonyl]benzylmethylamine (6)
Using the previous procedure and starting from 30 (0.82 g,
2.0 mmol), 6 was obtained as a white solid (0.53 g, 58%). mp 79 ꢁC.
solid. mp 92 ꢁC. 1H NMR
d
: 1.25e1.60 (m, 6H), 1.71e1.98 (m, 4H),
1H NMR
d: 1.34e1.53 (m,12H),1.75e1.86 (m, 2H), 2.22 (s, 3H), 2.37e
3.42 (t, J ¼ 5.0 Hz, 2H), 4.01 (t, J ¼ 6.0 Hz, 2H), 6.87 (s, 1H), 6.91 (d,
J ¼ 8.4 Hz, 2H), 7.23e7.29 (m, 2H), 7.49e7.58 (m, 2H), 7.76 (d,
J ¼ 8.4 Hz, 2H).
2.42 (m, 2H), 3.51 (s, 2H), 4.04 (t, J ¼ 6.6 Hz, 2H), 6.91 (s,1H), 6.97 (d,
J ¼ 8.8 Hz, 2H), 7.24e7.36 (m, 7H), 7.52e7.58 (m, 2H), 7.82 (dd,
J1 ¼ 2.0 Hz; J2 ¼ 8.0 Hz, 2H). ES-MS m/z: 456 (M þ 1). Anal.
C31H37NO2 (C, H, N).
6.1.4. 2-[4-(8-Bromooctyloxy)phenyl]benzofuran (29)
Using the previous procedure and starting from 4-benzofuran-
2-yl-phenol [11] (0.45 g, 2.1 mmol) and 1,8-dibromooctane
(0.79 mL, 4.3 mmol), 29 (0.51 g, 61%) was obtained as a white
6.1.11. [7-(3-Benzofuran-2-yl-phenoxy)heptyl]benzylmethylamine (7)
Using the previous procedure and starting from 31 (4 g,
10 mmol), 7 was obtained as a yellowish oil (0.84 g, 20%). 1H NMR
d:
solid. mp 112 ꢁC. 1H NMR
d: 1.37e1.58 (m, 8H), 1.76e1.92 (m, 4H),
1.20e1.59 (m, 8H), 1.69e1.87 (m, 2H), 2.17 (s, 3H), 2.32e2.39 (m,
2H), 3.42 (s, 2H), 3.97 (t, J ¼ 6.2 Hz, 2H), 6.84 (s, 1H), 6.93 (d,
J ¼ 8.8 Hz, 2H), 7.18e7.31 (m, 7H), 7.46e7.51 (m, 2H), 7.75 (d,
J ¼ 8.8 Hz, 2H). ES-MS m/z: 428 (M þ 1). Anal. C29H33NO2 (C, H, N).
3.42 (t, J ¼ 6.0 Hz, 2H), 4.00 (t, J ¼ 6.6 Hz, 2H), 6.88 (s, 1H), 6.96 (d,
J ¼ 8.0 Hz, 2H), 7.20e7.25 (m, 2H), 7.48e7.56 (m, 2H), 7.75 (dd,
J1 ¼ 2.1 Hz; J2 ¼ 8.4 Hz, 2H).

528
S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
6.1.12. (2-{4-[7-(Benzylmethylamino)heptyloxy]phenyl}
(m, 8H), 1.60e1.85 (m, 2H), 2.19 (s, 3H), 2.30e2.37 (t, 2H), 3.45 (s,
2H), 3.77 (t, J ¼ 6.2 Hz, 2H), 3.92 (t, J ¼ 6.4 Hz, 2H), 4.20 (t, J ¼ 6.4 Hz,
2H), 6.74e6.83 (m, 4H), 7.00e7.96 (m, 13H). A stirred solution of
this compound (0.46 g, 0.75 mmol) and diethylamine (0.32 mL,
3.0 mmol) was refluxed in toluene (100 mL) for 20 h in the presence
of a catalytic amount of NaI. The mixture was washed with water
(3 ꢂ 25 mL), then with brine (3 ꢂ 25 mL). The organic layer was
collected and dried over Na2SO4. The solvent was removed and the
residue was purified by flash chromatography (toluene/acetone
benzofuran-3-yl)-[3-(diethylaminomethyl)-phenyl]methanone (12)
To a cooled solution (0 ꢁC) of 2 [11] (0.43 g, 1.0 mmol) and
3-chloromethylbenzoyl chloride (0.24 g, 1.25 mmol) in dry CH2Cl2
(50 mL), SnCl4 (0.32 g, 1.25 mmol) was added dropwise with stir-
ring. The mixture was allowed to reach room temperature then
stirred overnight. The reaction was quenched with ice/water and
stirred for 30 min. The organic layer was washed with water
(3 ꢂ 10 mL) and with brine (3 ꢂ 10 mL), then dried over Na2SO4 and
the solvent was removed under reduced pressure. The crude was
purified by flash chromatography (toluene/acetone 60:40), afford-
ing (2-{4-[7-(benzylmethylamino)heptyloxy]phenyl}benzofuran-
3-yl)-[3-(2-chloroethoxy)phenyl]methanone as a dark yellow oil
20:80), affording 14 as a yellow oil (0.04 g, 9%). 1H NMR
d: 1.10 (t,
J ¼ 6.4 Hz, 6H), 1.18e1.62 (m, 8H), 1.63e1.85 (m, 2H), 2.19 (s, 3H),
2.32e2.43 (m, 2H), 2.58e2.71 (m, 4H), 2.89 (t, J ¼ 6.4 Hz, 2H), 3.53
(s, 2H), 3.91 (t, J ¼ 6.2 Hz, 2H), 4.08 (t, J ¼ 6.4 Hz, 2H), 6.74e6.83 (m,
4H), 7.00e7.96 (m, 13H). ES-MS m/z: 647 (M þ 1). Anal. C42H50N2O4
(C, H, N).
(0.26 g, 43%). 1H NMR
d: 1.02e1.60 (m, 8H), 1.62e1.88 (m, 2H), 2.20
(s, 3H), 2.33e2.44 (m, 2H), 3.46 (s, 2H), 3.91 (t, J ¼ 6.4 Hz, 2H), 4.58
(s, 2H), 6.80 (d, J ¼ 8.4 Hz, 2H), 7.05e7.38 (m, 11H), 7.50e7.63 (m,
4H), 7.79e7.92 (m, 2H). A stirred solution of this compound (0.26 g,
0.45 mmol) and diethylamine (0.1 mL, 0.90 mmol) was refluxed in
toluene (100 mL) for 20 h in the presence of NaI. The mixture was
washed with water (3 ꢂ 25 mL), then with brine (3 ꢂ 25 mL). The
organic layer was collected and dried over Na2SO4. The solvent was
removed under reduced pressure and the residue was purified by
flash chromatography (toluene/acetone 60:40), affording 12 as
6.1.15. (2-{4-[7-(Benzylmethylamino)heptyloxy]phenyl}
benzofuran-3-yl)-[4-(2-chloroethoxy)phenyl]-methanone (15)
Using the previous procedure and starting from 2 [11] (0.62 g,
1.45 mmol) and 4-(2-chloroethoxy)benzoyl chloride (0.38 g,
1.7 mmol), (2-{4-[7-(benzylmethylamino)heptyloxy] phenyl}
benzofuran-3-yl)-[4-(2-chloroethoxy)phenyl]methanone was ob-
tained as a dark yellow oil (0.53 g, 60%). 1H NMR
d: 1.17e1.59 (m,
a yellow oil (0.05 g,18%). 1H NMR
d
: 1.01 (t, J ¼ 6.4 Hz, 6H),1.22e1.58
8H), 1.56e1.79 (m, 2H), 2.15 (s, 3H), 2.32e2.39 (m, 2H), 3.47 (s, 2H),
3.81 (t, J ¼ 6.2 Hz, 2H), 3.89 (t, J ¼ 6.4 Hz, 2H), 4.21 (t, J ¼ 6.4 Hz, 2H),
6.72e6.87 (m, 4H), 7.05e8.02 (m, 13H). Using the previous proce-
dure and starting from (2-{4-[7-(benzylmethylamino)heptyloxy]
phenyl}benzofuran-3-yl)-[3-(2-chloro ethoxy) phenyl]methanone
(0.46 g, 0.75 mmol), 15 was obtained as a yellow oil (0.03 g, 8%). 1H
(m, 8H), 1.62e1.82 (m, 2H), 2.20 (s, 3H), 2.35e2.46 (m, 6H), 3.45 (s,
2H), 3.58 (s, 2H), 3.89 (t, J ¼ 6.6 Hz, 2H), 6.81 (d, J ¼ 8.4 Hz, 2H),
7.10e7.38 (m, 10H), 7.49e7.61 (m, 4H), 7.81e7.93 (m, 2H). ES-MS m/
z: 617 (M þ 1). Anal. C41H48N2O3 (C, H, N).
6.1.13. (2-{4-[7-(Benzylmethylamino)heptyloxy]phenyl}
NMR d: 1.02e1.12 (m, 6H),1.17e1.61 (m, 8H),1.62e1.86 (m, 2H), 2.21
benzofuran-3-yl)-[4-(diethylaminomethyl)-phenyl]methanone (13)
Using the same procedure and starting from 2 [11] (0.43 g,
1.0 mmol) and 4-chloromethylbenzoylchloride (0.24 g, 1.25 mmol),
(2-{4-[7-(benzylmethylamino)heptyloxy]phenyl}benzofuran-3-
yl)-[4-(2-chloroethoxy)phenyl]methanone was obtained after flash
chromatography (toluene/acetone 60:40), as a dark yellow oil
(s, 3H), 2.35e2.46 (m, 2H), 2.60e2.73 (m, 4H), 2.80e2.92 (m, 2H),
3.55 (s, 2H), 3.93 (t, J ¼ 6.2 Hz, 2H), 4.05 (t, J ¼ 6.4 Hz, 2H), 6.75e
6.86 (m, 4H), 7.00e7.94 (m, 13H). ES-MS m/z: 647 (M þ 1). Anal.
C42H50N2O4 (C, H, N).
6.1.16. (2-{4-[7-(Benzylmethylamino)heptyloxy]phenyl}
(0.27 g, 45%). 1H NMR
d
: 1.00e1.58 (m, 8H), 1.64e1.90 (m, 2H), 2.19
benzofuran-3-yl)-[4-(2-morpholinoethoxy)-phenyl]methanone (16)
(2-{4-[7-(Benzylmethylamino)heptyloxy]phenyl}benzofuran-3-
yl)-[4-(2-chloroethoxy)phenyl]methanone (0.4 g, 0.57 mmol) and
morpholine (0.1 g, 1.14 mmol), were refluxed in toluene for 20 h in
the presence of NaI. The mixture was washed with water
(3 ꢂ 25 mL), then with brine (3 ꢂ 25 mL). The organic layer was
collected and dried over Na2SO4. The solvent was removed and the
residue was purified by flash chromatography (toluene/acetone
(s, 3H), 2.35e2.46 (m, 2H), 3.45 (s, 2H), 3.86 (t, J ¼ 6.8 Hz, 2H), 4.59
(s, 2H), 6.79 (d, J ¼ 8.4 Hz, 2H), 7.05e7.38 (m, 11H), 7.49e7.61 (m,
4H), 7.81e7.93 (m, 2H). A stirred solution of this compound (0.26 g,
0.45 mmol) and diethylamine (0.1 mL, 0.90 mmol) was refluxed in
toluene (100 mL) for 20 h in the presence of a catalytic amount of
NaI. The mixture was washed with water (3 ꢂ 25 mL), then with
brine (3 ꢂ 25 mL). The organic layer was collected and dried over
Na2SO4. The solvent was removed and the residue was purified by
flash chromatography (toluene/acetone 60:40), affording 13 as
20:80), affording 16 as a yellow oil (0.08 g, 21%). 1H NMR
d. 1.21e
1.58 (m, 8H), 1.62e1.83 (m, 2H), 2.20 (s, 3H), 2.30e2.42 (m, 2H),
2.56e2.62 (m, 4H), 2.73e2.83 (m, 2H), 3.43 (s, 2H), 3.65e3.80 (m,
4H), 3.93 (t, J ¼ 6.3 Hz, 2H), 4.13 (t, J ¼ 6.2 Hz, 2H), 6.81 (d, J ¼ 8.4 Hz,
2H), 7.12e7.41 (m, 10H), 7.50e7.62 (m, 4H), 7.83e7.95 (m, 2H). ES-
MS m/z: 661 (M þ 1). Anal. C42H48N2O5 (C, H, N).
a yellow oil (0.05 g,18%). 1H NMR
d
: 1.01 (t, J ¼ 6.4 Hz, 6H),1.22e1.58
(m, 8H), 1.62e1.82 (m, 2H), 2.20 (s, 3H), 2.35e2.46 (m, 6H), 3.45 (s,
2H), 3.58 (s, 2H), 3.89 (t, J ¼ 6.4 Hz, 2H), 6.81 (d, J ¼ 8.4 Hz, 2H),
7.10e7.38 (m, 10H), 7.49e7.61 (m, 4H), 7.81e7.93 (m, 2H). ES-MS
m/z: 617 (M þ 1). Anal. C41H48N2O3 (C, H, N).
6.1.17. General procedure for the synthesis of compounds 32e36
SnCl4 (1.2 eq.) was added dropwise to a mixture of 28 (1 eq.) and
the selected acylchloride (1.2 eq.) in dry dichloromethane and the
resulting solution was stirred at room temperature overnight. The
reaction was quenched with ice/water and stirred for 1 h. The
organic layer was separated and the aqueous one was extracted
with dichloromethane. The combined organic extracts were dried
(Na2SO4), filtered and concentrated under reduced pressure to
afford the desired 3-acylated benzofurans (32e36), generally as
honey coloured oils purified by flash chromatography.
6.1.14. (2-{4-[7-(Benzylmethylamino)heptyloxy]phenyl}benzofuran-
3-yl)-[3-(2-diethylaminoethoxy)phenyl]methanone (14)
To a cooled solution (0 ꢁC) of 2 [11] (1.2 g, 2.8 mmol) and 3-(2-
chloroethoxy)benzoyl chloride (0.73 g, 3.4 mmol) in dry CH2Cl2
(50 mL), SnCl4 (0.87 g, 3.4 mmol) was added dropwise with stirring.
The mixture was allowed to reach room temperature then stirred
overnight. The reaction was quenched with ice/water and stirred
for 30 min. The organic layer was washed with water (3 ꢂ 10 mL)
and brine (3 ꢂ 10 mL), then dried over Na2SO4 and the solvent was
removed. The crude was purified by flash chromatography
(toluene/acetone 20:80), affording (2-{4-[7-(benzylmethylamino)
heptyloxy]phenyl}benzofuran-3-yl)-[4-(2-chloroethoxy)phenyl]
6.1.17.1. {2-[4-(7-Bromoheptyloxy)phenyl]benzofuran-3-yl}naph-
thalen-1-yl-methanone (32). (0.06 g, 16%). (petroleum ether/ethyl
methanone as a dark yellow oil (0.92 g, 54%). 1H NMR
d: 1.17e1.59
acetate 98:02). 1H NMR
d: 1.25e1.58 (m, 6H), 1.62e1.95 (m, 4H),

S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
529
3.41 (t, J ¼ 6.6 Hz, 2H), 3.86 (t, J ¼ 6.6 Hz, 2H), 6.60e6.64 (m, 2H),
7.20e7.38 (m, 4H), 7.50e7.65 (m, 7H), 7.84e7.90 (m, 2H), 8.55e8.60
(m, 1H).
J ¼ 8.4 Hz, 1H), 6.84e6.92 (m, 2H), 7.12e7.38 (m, 6H), 7.45e7.56 (m,
2H), 7.73e7.78 (m, 1H).
6.1.18.5. {2-[3-(7-Bromoheptyloxy)phenyl]benzofuran-3-yl}-3-
6.1.17.2. {2-[4-(7-Bromoheptyloxy)phenyl]benzofuran-3-yl}naph-
methoxyphenylmethanone (41). (0.07 g, 21%). (petroleum ether/ethyl
thalen-2-yl-methanone (33). (0.16 g, 47%). (petroleum ether/ethyl
acetate 98:2). 1H NMR
d: 1.40e1.58 (m, 6H), 1.60e1.95 (m, 4H), 3.42
acetate 98:02). 1H NMR
d: 1.29e1.56 (m, 6H), 1.64e1.92 (m, 4H), 3.40
(t, J ¼ 6.6 Hz, 2H), 3.73 (s, 3H), 3.81 (t, J ¼ 6.2 Hz, 2H), 6.82e6.88 (m,
(t, J ¼ 6.6 Hz, 2H), 3.86 (t, J ¼ 6.6 Hz, 2H), 6.73e6.78 (m, 2H), 7.21e
7.38 (m, 3H), 7.42e7.63 (m, 4H), 7.64e7.90 (m, 4H), 7.95e8.01 (m,
1H), 8.34 (s, 1H).
1H), 7.01e7.06 (m, 1H), 7.15e7.41 (m, 8H), 7.56e7.62 (m, 2H).
6.1.18.6. 2-[3-(7-Bromoheptyloxy)phenyl]benzofuran-3-yl}-2,3-
dimethoxyphenylmethanone (42). (0.17 g, 47%). (petroleum ether/
6.1.17.3. Biphenyl-4-yl-{2-[4-(7-bromoheptyloxy)phenyl]benzofuran-
ethyl acetate 90:10). 1H NMR
d: 1.42e1.51 (m, 6H), 1.72e1.92 (m,
3-yl}methanone (34). (0.22 g, 58%). (petroleum ether/ethyl acetate
4H), 3.40 (t, J ¼ 6.6 Hz, 2H), 3.74 (s, 3H), 3.82 (s, 3H), 3.91 (t,
J ¼ 6.6 Hz, 2H), 6.81e6.93 (m, 4H), 7.13e7.40 (m, 5H), 7.51e7.65 (m,
2H).
98:02). 1H NMR
d: 1.27e1.58 (m, 6H), 1.62e1.95 (m, 4H), 3.39 (t,
J ¼ 6.6, 2H), 3.90 (t, J ¼ 6.6 Hz, 2H), 6.78e6.82 (dd, J1 ¼ 2.2 Hz,
J2 ¼ 8.4 Hz, 2H), 7.24e7.50 (m, 5H), 7.51e7.71 (m, 8H), 7.90e7.93 (m,
2H).
6.1.18.7. {2-[3-(7-Bromoheptyloxy)phenyl]benzofuran-3-yl}-3,4-
dimethoxyphenylmethanone (43). (0.13 g, 36%). (petroleum ether/
6.1.17.4. Anthracen-9-yl-{2-[4-(7-bromoheptyloxy)phenyl]benzo-
ethyl acetate 90:10). 1H NMR
d: 1.30e1.56 (m, 6H), 1.60e1.98 (m,
furan-3-yl}methanone (35). (0.08 g, 18%). (petroleum ether/ethyl
4H), 3.40 (t, J ¼ 6.6 Hz, 2H), 3.80e3.90 (m, 8H), 6.72e6.88 (m, 2H),
acetate 98:02). 1H NMR
d: 1.31e1.61 (m, 6H), 1.62e1.95 (m, 4H), 3.43
7.16e7.59 (m, 9H).
(t, J ¼ 6.6, 2H), 3.80 (t, J ¼ 6.2 Hz, 2H), 6.29 (br, 2H), 6.72e7.82 (m,
10H), 7.84e8.12 (m, 4H), 8.34 (s, 1H).
6.1.18.8. {2-[3-(7-Bromoheptyloxy)phenyl]benzofuran-3-yl}-3,5-
dimethoxyphenylmethanone (44). (0.07 g, 16%). (petroleum ether/
6.1.17.5. {2-[4-(7-bromoheptyloxy)phenyl]benzofuran-3-yl}-p-tol-
ethyl acetate 90:10). 1H NMR
d: 1.41e1.58 (m, 6H), 1.62e1.95 (m,
ylmethanone (36). (0.26 g, 62%). (petroleum ether/ethyl acetate
4H), 3.41 (t, J ¼ 6.6 Hz, 2H), 3.68 (s, 6H), 3.81 (t, J ¼ 6.2 Hz, 2H),
6.56e6.58 (m, 1H), 6.83e6.90 (m, 1H), 6.98 (s, 1H), 6.99 (s, 1H),
7.13e7.41 (m, 5H), 7.56e7.66 (m, 2H).
98:2). 1H NMR
d: 1.41e1.59 (m, 6H), 1.70e1.95 (m, 4H), 2.28 (s, 3H),
3.42 (t, J ¼ 6.6 Hz, 2H), 3.81 (t, J ¼ 6.6 Hz, 2H), 6.81e6.87 (m, 1H),
7.14e7.40 (m, 7H), 7.56e7.67 (m, 4H).
6.1.19. Generalprocedureforthesynthesis ofcompounds8e11,18e25
A stirred solution of opportune compounds 32e35, 37e44 (1
eq.) and N-benzyl-N-methylamine (2 eq.) in toluene was refluxed
for 48 h. The mixture was washed with water. The organic layer was
dried over Na2SO4 and the solvent was removed. The residue was
purified by flash chromatography (toluene/acetone 98:2) to afford
8e11, 18e25 generally as yellowish oil.
6.1.18. General procedure for the synthesis of compounds 37e44
SnCl4 (1.2 eq.) was added dropwise to a mixture of 31 (1 eq.) and
the selected acylchloride (1.2 eq.) in dry dichloromethane and the
resulting solution was stirred at room temperature overnight. The
reaction was quenched with ice/water and stirred for 1 h. The
organic layer was separated and the aqueous one was extracted
with dichloromethane. The combined organic extracts were dried
(Na2SO4), filtered and concentrated under reduced pressure to
afford the desired 3-acylated benzofurans (37e44), generally as
honey coloured oils purified by flash chromatography.
6.1.19.1. (2-{4-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
furan-3-yl)naphthalen-1yl-methanone (8). (0.04 g, 62%). 1H NMR
d:
1.21e1.62 (m, 8H), 1.63e1.79 (m, 2H), 2.18 (s, 3H), 2.35 (t,
J ¼ 6.2 Hz, 2H), 3.47 (s, 2H), 3.84 (t, J ¼ 6.6 Hz, 2H), 6.59e6.64 (m,
2H), 7.16e7.35 (m, 8H), 7.52e7.63 (m, 7H), 7.85 (d, J ¼ 8.2 Hz, 2H),
8.57 (d, J ¼ 8.2 Hz, 1H). ES-MS m/z: 582 (M þ 1). Anal. C40H39NO3
(C, H, N).
6.1.18.1. {2-[3-(7-Bromoheptyloxy)phenyl]benzofuran-3-yl}phenyl-
methanone (37). (0.09 g, 28%). (petroleum ether/ethyl acetate
98:2). 1H NMR
d: 1.25e1.58 (m, 6H), 1.62e1.95 (m, 4H), 3.38 (t,
J ¼ 6.6 Hz, 2H), 3.83 (t, J ¼ 6.2 Hz, 2H), 6.85 (dt, J1 ¼ 1.8 Hz,
J2 ¼ 8.4 Hz, 1H), 7.11e7.40 (m, 7H), 7.47e7.58 (m, 2H), 7.78 (d,
J ¼ 8.0 Hz, 2H).
6.1.19.2. (2-{4-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
furan-3-yl)naphthalen-2yl-methanone (9). (0.17 g, 99%). 1H NMR
d:
1.25e1.60 (m, 8H), 1.64e1.80 (m, 2H), 2.18 (s, 3H), 2.35 (t, J ¼ 6.4 Hz,
2H), 3.48 (s, 2H), 3.84 (t, J ¼ 6.6 Hz, 2H), 6.75 (d, J ¼ 8.8 Hz, 2H),
7.18e7.38 (m, 8H), 7.41e7.85 (m, 8H), 7.96e8.02 (m, 1H), 8.33 (s,
1H). ES-MS m/z: 582 (M þ 1). Anal. C40H39NO3 (C, H, N).
6.1.18.2. {2-[3-(7-Bromoheptyloxy)phenyl]benzofuran-3-yl}-m-tol-
ylmethanone (38). (0.06 g, 18%). (petroleum ether/ethyl acetate
98:2). 1H NMR
d: 1.41e1.59 (m, 6H), 1.70e1.95 (m, 4H), 2.28 (s, 3H),
3.42 (t, J ¼ 6.6 Hz, 2H), 3.81 (t, J ¼ 6.6 Hz, 2H), 6.81e6.87 (m, 1H),
7.14e7.40 (m, 7H), 7.56e7.67 (m, 4H).
6.1.19.3. (2-{4-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
furan-3-yl)biphenyl-4-yl-methanone (10). (0.18 g, 73%). 1H NMR
d:
6.1.18.3. {2-[3-(7-Bromoheptyloxy)phenyl]benzofuran-3-yl}-p-tol-
1.21e1.62 (m, 8H), 1.64e1.80 (m, 2H), 2.20 (s, 3H), 2.40 (t, J ¼ 6.4 Hz,
2H), 3.50 (s, 2H), 3.90 (t, J ¼ 6.6 Hz, 2H), 6.80 (d, J ¼ 8.8 Hz, 2H),
7.19e7.45 (m, 11H), 7.54e7.68 (m, 7H), 7.91 (d, J ¼ 8.2 Hz, 2H). ES-
MS m/z: 608 (M þ 1). Anal. C42H41NO3 (C, H, N).
ylmethanone (39). (0.09 g, 27%). (petroleum ether/ethyl acetate
98:2). 1H NMR
d: 1.25e1.58 (m, 6H), 1.62e1.95 (m, 4H), 2.36 (s, 3H),
3.40 (t, J ¼ 6.6 Hz, 2H), 3.82 (t, J ¼ 6.2 Hz, 2H), 6.84 (dt, J1 ¼ 1.8 Hz,
J2 ¼ 8.6 Hz, 1H), 7.12e7.38 (m, 7H), 7.48e7.59 (m, 2H), 7.77 (d,
J ¼ 8.0 Hz, 2H).
6.1.19.4. Anthracen-9-yl-(2-{4-[7-(benzylmethylamino)heptyloxy]
phenyl}benzofuran-3-yl)methanone (11). (0.06 g, 70%). 1H NMR
d:
6.1.18.4. {2-[3-(7-Bromoheptyloxy)phenyl]benzofuran-3-yl}-2-
1.25e1.62 (m, 8H), 1.64e1.80 (m, 2H), 2.22 (s, 3H), 2.40 (t, J ¼ 6.4 Hz,
2H), 3.52 (s, 2H), 3.79 (t, J ¼ 6.6 Hz, 2H), 6.18e6.40 (m, 2H), 7.25e
7.52 (m, 15H), 7.89e7.93 (m, 4H), 8.34 (s, 1H). ES-MS m/z: 632
(M þ 1). Anal. C44H41NO3 (C, H, N).
methoxyphenylmethanone (40). (0.2 g, 59%). (petroleum ether/ethyl
acetate 94:6). 1H NMR
d: 1.42e1.58 (m, 6H), 1.62e1.98 (m, 4H), 3.41
(t, J ¼ 6.6 Hz, 2H), 3.55 (s, 3H), 3.84 (t, J ¼ 6.2 Hz, 2H), 6.69 (d,

530
S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
6.1.19.5. (2-{3-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
furan-3-yl)phenylmethanone (18). (0.09 g, 65%). 1H NMR
: 1.21e
organic layer was dried over Na2SO4 and the solvent was removed.
d
The residue was purified by flash chromatography (toluene/acetone
1.62 (m, 8H), 1.63e1.79 (m, 2H), 2.18 (s, 3H), 2.35 (t, J ¼ 6.4 Hz,
2H), 3.47 (s, 2H), 3.84 (t, J ¼ 6.6 Hz, 2H), 6.89e6.94 (m, 1H), 7.16e
7.62 (m, 15H), 7.80e7.93 (m, 2H). ES-MS m/z: 532 (M þ 1). Anal.
90:10), to afford 45 as a yellowish oil (0.16 g, 21%). 1H NMR
d: 1.20e
1.60 (m, 8H), 1.62e1.83 (m, 2H), 2.19 (s, 3H), 2.23e2.41 (m, 5H), 3.42
(s, 2H), 3.83e3.97 (m, 2H), 6.63e6.87 (m, 4H), 7.11e7.38 (m, 6H),
7.39e7.58 (m, 2H), 7.62e7.81 (m, 4H).
C
36H37NO3 (C, H, N).
6.1.19.6. (2-{3-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
furan-3-yl)-m-tolylmethanone (19). (0.05 g, 77%). 1H NMR
: 1.22e
1.62 (m, 8H), 1.64e1.80 (m, 2H), 2.20 (s, 3H), 2.28 (s, 3H), 2.37 (t,
J ¼ 6.4 Hz, 2H), 3.50 (s, 2H), 3.80 (t, J ¼ 6.6 Hz, 2H), 6.84 (d,
J ¼ 8.2 Hz, 1H), 7.16e7.42 (m, 12H), 7.56e7.67 (m, 4H). ES-MS m/z:
546 (M þ 1). Anal. C37H39NO3 (C, H, N).
6.1.21. Methylcarbamic acid 3-{[methyl-(7-{4-[3-(4-methylbenzoyl)
benzofuran-2-yl]phenoxy}heptyl)-amino]methyl}phenyl ester (17)
To a solution of 45 (0.1 g, 0.18 mmol) in CH2Cl2, NaH (0.004 g,
0.18 mmol) and methyl isocyanate (0.010 g, 0.18 mmol) were added.
The mixture was stirred for 24 h, then quenched with ice/water and
extracted with CH2Cl2. The organic layer was dried and evaporated.
The crude was purified by flash chromatography (toluene/acetone
d
6.1.19.7. (2-{3-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
60:40), affording 17 as a clear oil (0.06 g, 54%). 1H NMR
d: 1.21e1.59
furan-3-yl)-p-tolylmethanone (20). (0.08 g, 82%). 1H NMR
d: 1.25e
(m, 8H), 1.63e1.82 (m, 2H), 2.18 (s, 3H), 2.23e2.41 (m, 5H), 2.82 (s,
3H), 3.45 (s, 2H), 3.83e3.97 (m, 2H), 4.91e5.02 (br, 1H), 6.62e6.85
(m, 4H), 7.09e7.41 (m, 6H), 7.47e7.59 (m, 2H), 7.61e7.84 (m, 4H).
ES-MS m/z: 619 (M þ 1). Anal. C39H42N2O5 (C, H, N).
1.62 (m, 8H), 1.64e1.80 (m, 2H), 2.19 (s, 3H), 2.35e2.40 (m, 5H),
3.48 (s, 2H), 3.80 (t, J ¼ 6.6 Hz, 2H), 6.82e6.86 (m, 1H), 7.11e7.42 (m,
12H), 7.54 (t, J ¼ 8.8 Hz, 2H), 7.77 (d, J ¼ 8.8 Hz, 2H). ES-MS m/z: 546
(M þ 1). Anal. C37H39NO3 (C, H, N).
6.2. Biological evaluation methods
6.1.19.8. (2-{3-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
furan-3yl)-2-methoxyphenylmethanone (21). (0.15 g, 70%). 1H NMR
6.2.1. Inhibition of AChE and BuChE
d
: 1.28e1.62 (m, 8H), 1.65e1.82 (m, 2H), 2.19 (s, 3H), 2.37 (t,
The capacity of 3e25 to inhibit hAChE activity was assessed
using the Ellman’s method [14]. Compounds 1 and 2, galantamine,
tacrine and rivastigmine were used as reference compounds. Initial
rate assays were performed at 37 ꢁC with a Jasco V-530 double
beam spectrophotometer. Stock solutions of the tested compound
(1 mM) were prepared in methanol and diluted in methanol. The
assay solution consisted of a 0.1 M phosphate buffer, pH 8.0, with
J ¼ 6.6 Hz, 2H), 3.48 (s, 2H), 3.53 (s, 3H), 3.84 (t, J ¼ 6.6 Hz, 2H), 6.68
(d, J ¼ 8.0 Hz, 1H), 6.80e6.91 (m, 2H), 7.10e7.35 (m, 11H), 7.44e7.55
(m, 2H), 7.75e7.80 (m, 1H). ES-MS m/z: 562 (M þ 1). Anal.
C
37H39NO4 (C, H, N).
6.1.19.9. (2-{3-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
furan-3yl)-3-methoxyphenylmethanone (22). (0.05 g, 66%). 1H NMR
the addition of 340
unit/mL human recombinant AChE or human serum BuChE (Sigma
Chemical, St. Louis, MO), and 550 M substrate (acetylthiocholine
m
M 5,50-dithiobis(2-nitrobenzoic acid), 0.02
(CDCl3) d: 1.25e1.61 (m, 8H), 1.65e1.82 (m, 2H), 2.20 (s, 3H), 2.38 (t,
J ¼ 6.4 Hz, 2H), 3.49 (s, 2H), 3.73 (s, 3H), 3.81 (t, J ¼ 6.2 Hz, 2H),
6.83e6.87 (m, 1H), 6.98e7.05 (m, 1H), 7.16e7.20 (m, 2H), 7.22e7.32
(m, 8H), 7.37e7.41 (m, 3H), 7.56e7.63 (m, 2H). ES-MS m/z: 562
(M þ 1). Anal. C37H39NO4 (C, H, N).
m
iodide or butyrylthiocholine iodide, respectively). Assay solutions
with and without inhibitor were preincubated at 37 ꢁC for 20 min
followed by the addition of substrate. Blank solutions containing all
components except AChE or BuChE were prepared in parallel to
account for the non-enzymatic hydrolysis of the substrate. Five
increasing concentrations of the inhibitor were used, able to give an
inhibition of the enzymatic activity in the range of 20e80%. The
results were plotted by placing the percentage of inhibition in
function of the decimal log of the final inhibitor concentration.
Linear regression and IC50 values were calculated using Microcal
Origin 3.5 software (Microcal Software, Inc).
6.1.19.10. (2-{3-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
furan-3yl)-2,3-dimethoxyphenyl methanone (23). (0.14 g, 77%). 1H
NMR d: 1.27e1.61 (m, 8H), 1.65e1.83 (m, 2H), 2.18 (s, 3H), 2.36 (t,
J ¼ 6.6 Hz, 2H), 3.48 (s, 2H), 3.73 (s, 3H), 3.79 (s, 3H), 3.90 (t,
J ¼ 6.6 Hz, 2H), 6.80e6.95 (m, 4H), 7.12e7.34 (m,10H), 7.51e7.55 (m,
1H), 7.63e7.68 (m, 1H). ES-MS m/z: 592 (M þ 1). Anal. C38H41NO5
(C, H, N).
6.1.19.11. (2-{3-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
6.2.2. Inhibition of
6.2.2.1. Preparation of solutions. Stock solution of
b-amyloid aggregation
furan-3yl)-3,4-dimethoxyphenyl methanone (24). (0.09 g, 65%). 1H
1 mM was
NMR
d
: 1.25e1.62 (m, 8H), 1.69e1.80 (m, 2H), 2.20 (s, 3H), 2.37 (t,
prepared by solubilizing the lyophilized Ab25e35 peptide by brief
vortexing in sterile water at 4 ꢁC, then by sonication for 1 min. The
peptide stock solution was aliquoted and stored at ꢃ20 ꢁC. All steps
were carried out at 4 ꢁC to prevent Ab25e35 polymerization. The
new compounds were solubilized in MeOH solution to a concen-
tration of 1 M. The stock solution was diluted to obtain aliquots
J ¼ 6.6 Hz, 2H), 3.49 (s, 2H), 3.80e3.89 (m, 8H), 6.74 (d, J ¼ 8.6 Hz,
1H), 6.83e6.88 (m, 1H), 7.23e7.39 (m, 10H), 7.43e7.60 (m, 4H). ES-
MS m/z: 592 (M þ 1). Anal. C38H41NO5 (C, H, N).
6.1.19.12. (2-{3-[7-(Benzylmethylamino)heptyloxy]phenyl}benzo-
furan-3yl)-3,5-dimethoxyphenyl methanone (25). (0.05 g, 67%). 1H
with concentrations between 0.1 and 50
m
M, then stored at ꢃ20 ꢁC.
NMR d: 1.25e1.61 (m, 8H), 1.64e1.81 (m, 2H), 2.20 (s, 3H), 2.38 (t,
J ¼ 6.6 Hz, 2H), 3.50 (s, 2H), 3.67 (s, 6H), 3.80 (t, J ¼ 6.2 Hz, 2H), 6.57
(s, 1H), 6.84e6.88 (m, 1H), 6.98 (d, J ¼ 8.2 Hz, 2H), 7.14e7.41 (m,
10H), 7.56e7.67 (m, 2H). ES-MS m/z: 592 (M þ 1). Anal. C38H41NO5
(C, H, N).
6.2.3. Measurementof inhibitoryactivityby UVevisiblespectroscopy
To study the kinetic of Ab25e35 polymerization alone, experi-
ments were carried out by using a reaction mixture containing
80
10
m
L
phosphate buffer (10 mM final concentration) and
m
L Ab25e35 (100 M final concentration), pH 7.2. When Ab25e35
m
6.1.20. [2-(4-{7-[(3-Hydroxybenzyl)methylamino]heptyloxy}
phenyl)benzofuran-3-yl]-p-tolylmethanone (45)
was added to the buffer solution, we performed sonication for
1 min to avoid any peptide aggregation. 10 L MeOH were added to
m
A
stirred solution of 36 (0.7 g, 1.38 mmol) and 3-
the solution to have the same conditions for the experiments with
the new compounds. To study the inhibitory activity of the new
compounds, experiments were carried out by using a reaction
methylaminomethylphenol (0.38 g, 2.77 mmol) in toluene (120 mL)
was refluxed for 20 h. The mixture was washed with water, the

S. Rizzo et al. / European Journal of Medicinal Chemistry 58 (2012) 519e532
531
mixture containing 80
tration), 10 L MeOH containing 10
the new compounds or 10 L MeOH containing 0.1, 1, 10, 20 and
50 M final concentration for IC50 determination and 10 L Ab25e35
(100 M final concentration), pH 7.2. All steps were carried out at
ꢁC to prevent Ab25e35 polymerization. UVevisible spectroscopy
m
L phosphate buffer (10 mM final concen-
6.2.4.5. Determination of Ab25e35 peptide binding to the cell surface.
SHeSY5Y cells were seeded in 96-well plates at 5 ꢂ 103 cells/well
for 24 h. At the end of incubation, the medium was changed with
m
mM final concentration of one of
m
m
m
a fresh one with Ab25e35 peptide (10
(30 M) for 10, 20 and 30 min and then washed twice with PBS.
The residual Ab25e35 peptide cell complex was stained with CR in
mM) and various compounds
m
m
4
was performed on a Cary 300 bio UVevisible spectrophotometer.
Polymerization kinetics were monitored in the range of 190e
380 nm between 0 and 6 h. For each inhibition experiment, one
sample containing Ab25e35 alone, another containing the new
compound alone and a third with curcumin as inhibitor reference
were used in parallel as controls in the same experimental condi-
tions. Moreover, to rule out any influence due to the new
compounds absorbance, their UVevisible spectra were subtracted
from the Ab25e35 absorption spectra. At least three independent
measurements were made for all cases. All results are presented
with means and standard deviation. IC50 was calculated by using
a least-square fitting technique to match the experimental data
with a sigmoidal curve. IC50 was the concentration of the new
PBS for 20 min and measured with a spectrophotometer
(TECANÒ, GENios) at 540 nm (bound CR). CR values were
reported as percent increases in treated cells vs. untreated cells
(taken as 100%).
6.2.4.6. Determination of Ab25e35 peptide-induced intracellular ROS
formation. ROS formation was determined using the fluorescent
probe DHE (lexcitation ¼ 380 nm, lemission ¼ 445 nm). SHeSY5Y cells
were cultured in BD FalconÔ 8-well Culture slides (surface area
0.7 cm2/well) at 1 ꢂ104 cells/well for 24 h. At the end of incubation,
SHeSY5Y cells were washed and incubated with DHE (10 mM) for
15 min in the dark. After removal of the probe, cells were washed
with PBS and incubated with DMEM serum free for 1 h at 37 ꢁC.
Intracellular ROS formation was measured under a fluorescence
microscope (Zeiss Axio Imager M1). Fluorescence images were
captured with an AxioVision image recording system computer.
Four randomly selected areas with 50e100 cells in each were
analysed and the values obtained are expressed as fold increases of
ROS vs. untreated cells.
compound inhibiting the formation of A
control value.
b fibrils to 50% of the
6.2.4. The neuroprotective effects against Ab25e35 peptide
6.2.4.1. Chemicals. Ab25e35 peptide, 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT), dihydroethidium
(DHE) and Congo red (CR) were purchased from Sigma Chemical Co.
All other reagents were of the highest grade of purity commercially
available.
6.2.5. Binding studies to CB receptors
Membranes from HEK-293 cells stably transfected with the
human recombinant CB1 receptor (Bmax ¼ 2.5 pmol/mg protein) or
the human recombinant CB2 receptor (Bmax ¼ 4.7 pmol/mg protein)
were incubated with [3H]-CP-55, 940 (0.14 nM/Kd ¼ 0.18 nM and
0.084 nM/Kd ¼ 0.31 nM respectively for CB1 and CB2 receptor) as
6.2.4.2. Ab25e35 peptide preparation for neurotoxicity assay.
A
b25e35 peptide was dissolved in hexafluoroisopropanol (HFIP) to
1 mg/mL, sonicated and incubated at room temperature for 24 h
to produce unaggregated A peptide. The HFIP was dried under
b
the high affinity ligand and displaced with 10 mM WIN 55212-2 as
vacuum in a Speed Vac and the resulting peptide film was
dissolved in DMSO to 1 mM. The unaggregated Ab25e35 stock
solution was then aliquoted and stored at ꢃ20 ꢁC until use. For
various biological assay, the Ab25e35 stock solution was diluted
directly into cell culture media.
the heterologous competitor to measure non-specific binding (Ki
values 9.2 nM and 2.1 nM respectively for CB1 and CB2 receptor).
All compounds were tested following the procedure described by
the manufacturer (Perkin Elmer, Italia). Displacement curves were
generated by incubating membranes with [3H]-CP-55, 940 and
increasing concentrations of test compounds for 90 min at 30 ꢁC. Ki
values were calculated by applying the ChengePrusoff equation to
the IC50 values (obtained by GraphPad) for the displacement of the
bound radioligand by increasing concentrations of the test
compounds. Data are reported as means of at least n ¼ 3
experiments.
6.2.4.3. Cell culture. Human neuronal SHeSY5Y cells were
routinely grown at 37 ꢁC in a humidified incubator with 5% CO2 in
Dulbecco’s modified Eagle’s medium supplemented with 10% foetal
bovine serum, 2 mM glutamine, 50 U/mL penicillin and 50
streptomycin.
mg/mL
6.2.4.4. Determination of
A
b25e35 peptide-induced neurotoxicity.
6.2.6. Statistical analysis
to evaluate the protective effects of compounds against Ab25e35
peptide induced neurotoxicity, the SHeSY5Y cells were seeded in
96-well plates at 3 ꢂ 104 cells/well, incubated for 24 h and subse-
Data are reported as mean ꢀ SD of at least 3 independent
experiments. Statistical analysis was performed using one-way
ANOVA with Dunnett post hoc test and Student’s t-test, as appro-
priate. Pearson’s test was also used to assess correlation. Differ-
ences were considered significant at p < 0.05. Analyses were
performed using PRISM 3 software on a Windows platform.
quently treated with 10 mM of unaggregated Ab25e35 peptide for 3 h
at 37 ꢁC in 5% CO2, in presence or absence of various concentrations
of compounds (1e30 M). The neuronal viability in terms of
m
mitochondrial metabolic function was evaluated by the reduction
of MTT to formazan as previously described [18]. The cellular
reduction of MTT represents an indicator of the initial events
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