Fluorinated benzophenone derivatives: Balanced multipotent agents for Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2014.03.042

In an effort to develop multipotent agents against β-secretase (BACE-1) and acetylcholinesterase (AChE), able to counteract intracellular ROS formation as well, the structure of the fluorinated benzophenone 3 served as starting point for the synthesis of a small library of 3-fluoro-4-hydroxy- analogues. Among the series, derivatives 5 and 12, carrying chemically different amino functions, showed a balanced micromolar potency against the selected targets. In particular, compound 12, completely devoid of toxic effects, seems to be a promising lead for obtaining effective anti-AD drug candidates.

Full text of DOI:10.1016/j.ejmech.2014.03.042

European Journal of Medicinal Chemistry 78 (2014) 157e166
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Fluorinated benzophenone derivatives: Balanced multipotent agents
for Alzheimer’s disease
,
b
b
a
Federica Belluti a , Angela De Simone , Andrea Tarozzi , Manuela Bartolini ,
Alice Djemil b, Alessandra Bisi a, Silvia Gobbi a, Serena Montanari a, Andrea Cavalli a
Vincenza Andrisano b, Giovanni Bottegoni c, Angela Rampa a
*
,
c
,
a Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b Department for Life Quality Studies, Alma Mater Studiorum, University of Bologna, Corso d’Augusto 237, 47900 Rimini, Italy
c Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, 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:
In an effort to develop multipotent agents against b-secretase (BACE-1) and acetylcholinesterase (AChE),
Received 29 September 2013
Received in revised form
12 March 2014
Accepted 14 March 2014
Available online 16 March 2014
able to counteract intracellular ROS formation as well, the structure of the fluorinated benzophenone 3
served as starting point for the synthesis of a small library of 3-fluoro-4-hydroxy- analogues. Among the
series, derivatives 5 and 12, carrying chemically different amino functions, showed a balanced micro-
molar potency against the selected targets. In particular, compound 12, completely devoid of toxic ef-
fects, seems to be a promising lead for obtaining effective anti-AD drug candidates.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Acetylcholinesterase
Alzheimer’s disease
Antioxidant activity
BACE-1
Benzophenone
Drug design
Lead identification
1. Introduction
the last few years, this lack of an effective cure has fuelled an
intense search for disease-modifying agents that, by targeting the
Alzheimer’s disease (AD) is a devastating neurodegenerative
disorder that accounts for the majority of cases of dementia. It
afflicts over 30 million people worldwide and these figures are
expected to quadruple by 2050. At present, the only available
treatments treat the symptoms of AD. Affecting neurotransmission
by means of inhibitors of acetylcholinesterase (AChE), a well
known molecular target involved in AD pathology, and N-methyl-
underlying pathophysiology of AD, could control the disease pro-
cess and slow down its clinical course [1]. In this scenario, the
development of multitarget agents, chemical entities able to
simultaneously modulate multiple biological targets significantly
involved in AD neurotoxic pathway, has clearly emerged as a
successful strategy [2].
A key event in AD pathogenesis is the accumulation of amyloid-
D-aspartate (NMDA) receptor antagonists, makes it possible to
b (Ab) peptide in the brain. The Ab42 monomers aggregate into toxic
slow down the cognitive decline associated with AD, yet showing
only modest palliative clinical efficacy without affecting the dis-
ease progression or correcting the neurodegenerative process. In
extra-cellular oligomeric species, which, in turn, form insoluble
fibrillar aggregates, that compose the core of the dense amyloid
plaques [3,4]. These structures insert themselves into neuronal
membranes and induce lipid peroxidation, protein oxidation and
increased production of reactive oxygen species (ROS) and reactive
nitrogen species (RNS), together with loss of function of many
antioxidant defence enzymes, thus contributing to oxidative stress
and neurotoxicity [5,6]. A common concept of the amyloid cascade
hypothesis is that the aggregation of Ab42 peptide into toxic fibrils
is the main initiating event that sets off a cascade of neurobiological
processes, such as neurotoxicity, oxidative damage and
Abbreviations: AChE, acetylcholinesterase; AD, Alzheimer’s disease; ABP, ami-
nobenzylpiperidine; Ab, amyloid-b; APP, amyloid precursor protein; BACE-1, b-
secretase; CNS, central nervous system; HE, hydroxyethylene; FRET, fluorescence
resonance energy transfer; PDB, protein data bank; ROS, reactive oxygen species;
SAR, structureeactivity relationships; t-BuOOH, tert-butylhydroperoxide.
* Corresponding author.
E-mail address: federica.belluti@unibo.it (F. Belluti).
http://dx.doi.org/10.1016/j.ejmech.2014.03.042
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

158
F. Belluti et al. / European Journal of Medicinal Chemistry 78 (2014) 157e166
inflammation, ultimately culminating with extensive brain atrophy,
neuronal dysfunction, and cognitive decline [7]. The central ner-
vous system (CNS) accumulation of unbound transition metals,
such as iron and copper, has also been considered a significant
source of oxidant species [8]. Recent in vivo studies demonstrated
potential of the benzophenone core structure to hit several targets
involved in AD. This scaffold has indeed proved to be a privileged
structure, a versatile pharmacophore nucleus, that could be
exploited through suitable modifications to provide ligands for an
array of biological targets [22,23]. Benzophenone has recently been
employed by the authors as a starting point for obtaining com-
pounds able to modulate the actions of AChE [24,25]. Following a
previous research project aimed at identifying new chemical en-
tities able to inhibit both BACE-1 and AChE enzymes [26], our in-
ternal benzophenone-based collection of AChE inhibitors, bearing a
N,N0-benzylmethylamine function to target the catalytic binding
site of the enzyme, was screened against BACE-1. Given that the
cyclic amines proved to be suitable for specific hydrogen bonding
with the catalytic aspartic acid, this tertiary amine function could
hold promise for interacting with the enzyme, improving the
compound solubility as well. Among the tested compounds, de-
rivatives 1e4, with AChE inhibition values ranging from sub-micro
to low micromolar (Table 1), showed a promising trend of BACE-1
inhibition, that made it possible to gain insight into the benzo-
phenone substitution pattern essential for this enzyme. Compound
1, with a 3,4-dimethoxy benzophenone nucleus, endowed with
the presence of Ab in mitochondrial membranes, where it was
thought to be responsible for both the disruption of the electron
transport chain and the irreversible cell damage [9]. All these fac-
tors are not independent of each other, and it is plausible that,
especially in the early stages of the disease process, Ab could enter
the mitochondria where it would increase the generation of ROS
and induce oxidative stress. Hence, the “oxidative stress hypothe-
sis” states that the increased production of free radicals in AD is a
potential target for therapeutic strategies; as such, therapeutic
modalities involving antioxidants may be an effective approach to
the treatment of this neurodegenerative disease [10].
A
b is generated by the proteolytic processing of a larger
membrane-bound precursor protein, known as the amyloid pre-
cursor protein (APP), upon sequential cleavage by two aspartyl
proteases,
b
-secretase (also known as
b-site APP cleaving enzyme-
1, or BACE-1) and
g
-secretase. It has been demonstrated that a
variety of stress factors, including hypoxia, ischaemic injury and
inflammation, can induce BACE-1 expression in experimental
models of sporadic AD [11e14]. In particular, recent studies support
sub-micromolar AChE inhibitory potency [24], when tested at 5 mM
concentration showed low BACE-1 inhibition (20%). Removal of the
methoxy group in position 3 (compound 2) led to a notable
decrease in potency. Interestingly compound 3, with a fluorine
atom instead of the 3-methoxy substituent, was identified as a
the hypothesis that oxidative stress, secretase function and A
b
production are strictly interrelated events and suggest that inhi-
bition of BACE-1 may have a synergic therapeutic effect with
antioxidant compounds [10,11,15]. In this context, novel multi-
potent agents against BACE-1 and oxidative stress have gained
attention for their potential as effective anti-AD drug candidates.
The first generation of BACE-1 inhibitors focused on compounds
with a peptide or peptidomimetic structure, designed as transition-
state (TS) mimetics, and different substrate models were designed
to closely interact with the BACE-1 catalytic aspartic acids (Asp 32
and Asp 228, catalytic dyad) [16] such as OM99-2, a 8-aminoacid
residue hydroxyethylene (HE)-based analogue that spans the P4
to P40 binding pockets of BACE-1, showing a noteworthy hydrogen
bonding network within the active site, together with a direct
interaction among the OH of the HE function and the catalytic dyad.
Notwithstanding its nanomolar inhibitory potency, this compound
showed suboptimal in vivo pharmacokinetics and low brain pene-
tration [17]. Generally, the majority of the early BACE-1 inhibitors
were characterized by complex, high molecular weight structural
motifs, lacking drug-like properties [18]. Since the first X-ray crystal
structures of BACE-1 were reported, intensive efforts have focused
on the development of potent enzyme inhibitors that possess ideal
properties such as oral bioavailability and a good pharmacokinetic
profile [17]. The identification of small molecule inhibitors of BACE-
1 with CNS permeability represents an important and difficult
weak BACE-1 inhibitor (10.72% of inhibition at 3.38 mM concen-
tration). Notwithstanding its poor activity against BACE-1, com-
pound 3 might be interesting from a pharmacokinetic perspective,
since the presence of a fluorine substituent on an aromatic ring
could impart a variety of properties, including enhanced binding
interactions, metabolic stability, and selective reactivity [27]. For
these reasons, 3 could be regarded as a hit compound to be further
modified to obtain more potent analogues, and given that the
strongly electron withdrawing effect of the fluorine atom markedly
influences the acidity of neighbouring functional groups, we then
synthesized the corresponding des-methyl analogue 5 (Table 2).
Gratifyingly, this compound was able to potently inhibit BACE-1,
with an IC50 value in the low micromolar range, providing a five-
Table 1
hBACE-1 and hAChE inhibition profiles of compounds 1e4.
challenge since, in order to mediate brain Ab lowering, inhibitors
ought to be able to cross the bloodebrain barrier (BBB) [19]. A 3-D
pharmacophore map of BACE-1 has also been proposed, to guide
the design and optimization of inhibitors [20]. Currently, several
crystal structures of the catalytic domain of BACE-1, alone or in
complex with an inhibitor, have been deposited in the Protein Data
Bank (PDB). The structural information that emerged from these
studies was employed for structure-based drug discovery projects,
leading to the identification of several classes of non-peptidic
BACE-1 inhibitors with novel core templates and with improved
pharmacokinetics profile [16,18,21].
Cmpd
R1
R2
hBACE-1 inhibition (%)a,b
hAChE inhibitionb,c
IC50 M)
ꢀ SEM
(
m
1
2
3
4
OCH3
H
F
OCH3
OCH3
OCH3
OH
20 (at 5
n.i.d
m
M)
0.46 ꢀ 0.04
1.82 ꢀ 0.08
1.57 ꢀ 0.08
2.10 ꢀ 0.09
10.72 (at 3.38
mM)
H
n.i.d
a
% inhibition of BACE-1 activity at the reported concentration of the tested
compounds.
b
Values are mean of two independent measurements, each performed in
triplicate.
2. Design
c
See Refs. [24,25]. IC50 values represent the concentration of inhibitor required to
decrease enzyme activity by 50% IC50 values were determined by following Ellman’s
In a drug discovery effort to obtain single multitarget small
molecules as anti-AD drug candidates, we further investigated the
method.
d
n.i.: not inhibiting up to 4 mM.

F. Belluti et al. / European Journal of Medicinal Chemistry 78 (2014) 157e166
159
Table 2
fold enhancement in activity over 3 and being more active than the
standard bis(7)-tacrine, which displayed an IC50 of 7.5 M.
hBACE-1 inhibition profiles of compounds 5e15.
m
The pivotal importance of the fluorine substituent on BACE-1
inhibition was underlined by the finding that its removal (4)
resulted in a loss of activity. 5 was docked into the active site of
BACE-1 (Fig. 2A) to validate its capability to interact with key res-
idues such as the catalytic dyad (see the Computational Studies
paragraph for further details). As expected, the N,N0-benzylme-
thylamine portion turned out to be oriented towards the centre of
the BACE-1 binding pocket by contacting the catalytic Asp 32 by
means of electrostatic and H-bond interactions. Moreover, the 3-
fluoro-4-hydroxybenzophenone framework established essential
Cmpd
R
hBACE-1
Inhibition (%) at 3 m (m
Ma,b IC50 M)b
ꢀ SEM
ꢀ SEM
0
interactions at the S2 subsite, interacting with the side chains of
Tyr71, Tyr198, and Arg128, and to validate the impact of this mo-
lecular fragment on BACE-1 inhibition, the simplified analogue (6),
lacking the N,N0-benzylmethylamine moiety, was tested. Inhibition
data, listed in Table 2, were in agreement with the previous docking
simulation performed on 5, since the compound retained a some-
5
6
7
40.75 ꢀ 1.05
35.38 ꢀ 4.09
n.i.c
3.66 ꢀ 0.29
H
what good activity (35.38% inhibition at 3.0 mM), although lower
relative to the lead 5, and supported the presence of an appropriate
tertiary amine function for obtaining effective BACE-1 inhibitors.
The 3-fluoro-4-hydroxy-benzophenone nucleus, identified as an
essential chemical feature for the binding within the BACE-1
enzyme, due to its good synthetic accessibility and low molecular
weight (MW w 230) turned out to be an appropriate synthon for
performing a parallel chemical synthesis; in particular, the methyl
group could provide the insertion point for the amino group. With
this idea we generated a small library of 3-fluoro-4-hydroxy-
benzophenone-based compounds (7e15) in which the N,N0-ben-
zylmethylamine group (R, Table 2) was replaced with a number of
tertiary amines bearing different functional groups and flexible
chains at the tertiary nitrogen atom, with the aim of performing a
quick SAR study and of effectively determining the essential fea-
tures of the R moiety required for an optimal interaction with the
BACE-1 binding pocket.
8
9
46.69 ꢀ 5.12
32.78 ꢀ 4.06
10
n.i.c
3. Chemistry
11
12
n.i.c
The synthetic route followed for the preparation of the benzo-
phenone derivatives (5e15) is depicted in Scheme 1. Intermediates
16 and 17 were synthesized according to a previously described
procedure [24]. Cleavage of the methoxy group of 17 was accom-
plished by treatment with BBr3 to obtain the key intermediate 18,
that was then subjected to nucleophylic attack by different amines
to give, in parallel, the final compounds.
58.72 ꢀ 3.22
2.32 ꢀ 0.44
n.i.c
4. Biology
13
14
The benzophenone-based analogues presented in this study
were first evaluated to investigate the ability to inhibit BACE-1
enzyme activity, by means of a biochemical assay performed us-
ing the fluorescence resonance energy transfer (FRET) methodol-
ogy. FRET assay is based on the use of synthetic peptides carrying a
fluorophore (donor group) and a quencher (acceptor group)
bearing the Swedish mutated sequence of APP (-Leu w Asp- instead
of Met w Asp) as substrate. The substrate becomes highly fluo-
rescent upon enzymatic cleavage and the increase in fluorescence is
linearly related to the rate of proteolysis. The methoxycoumarin
based peptide M-2420 was used to evaluate enzyme inhibition by
measuring the loss of fluorescence due to the presence of test
compounds [28]. In particular, compounds 6e15 were tested at a
13.50 ꢀ 5.50
15
36.16 ꢀ 3.05
tacrine (THA)
bis-(7)- THA
n.i. (at 5
27.2 ꢀ 0.2 (at 10
m
M)d
m
M)
7.5 ꢀ 0.4d
a
% inhibition of BACE-1 activity (at 3
Values are mean ꢀ SEM of two independent measurements, each performed in
m
M concentration) of the tested compounds.
b
concentration of 3 mM and their BACE-1 inhibition percentages are
reported in Table 2. The IC50 values of the most active compounds
(5 and 12) were determined by using the linear regression pa-
rameters. Subsequently, to determine the multitarget profile of 5
triplicate. SEM ¼ standard error of the mean.
c
n.i.: not inhibiting up to 4
See Ref. [45].
mM.
d

160
F. Belluti et al. / European Journal of Medicinal Chemistry 78 (2014) 157e166
Fig. 1. Benzophenone BACE-1 inhibitors fragment evolution (inhibitory activities are reported in brackets), and structure of the ABP-based derivative employed for the compu-
tational studies.
Fig. 2. The binding mode of 5 and 12 at the binding site of BACE-1. A) The bound conformation of 5 (carbon atoms in yellow). B) The bound conformation of 12 (carbon atoms in
green). Residues of the binding site making relevant interactions are reported and labelled explicitly. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
and 12, we investigated their capability to inhibit the activity of
human recombinant AChE by means of Ellman’s method [29].
Finally, the antioxidant activity of 5 and 12 was evaluated
against ROS formation in human neuronal SH-SY5Y cells following
5. Results and discussion
5.1. BACE-1 inhibition
exposure to t-BuOOH or Ab25e35 peptides, the toxic core of A
using compound 3 as a negative reference compound. In particular,
the t-BuOOH is an organic peroxide that generates a pattern of ROS
similar to that involved in the oxidative stress induced by Ab [30].
In the same cells the neurotoxicity of the compounds was also
determined.
b
,
Having established one of the important features required for
BACE-1 inhibition, and in order to both explore the chemical space
of the target and enhance the binding affinity of the designed 3-
fluoro-4-hydroxybenzophenone framework, different R portions
were introduced on this scaffold. BACE-1 inhibition data are re-
ported in Table 2. The presence of additional hydroxyl and amino
Scheme 1. Reagents and conditions: i) NBS, (PhCOO)2O, CCl4, reflux; ii) BBr3; DCM, r.t.; iii) selected amine, TEA, toluene, reflux.

F. Belluti et al. / European Journal of Medicinal Chemistry 78 (2014) 157e166
161
groups might make it possible to form favourable interactions with
residues of the S1 subsite. With this strategy in mind, we attempted
to modify the electronic and hydrophobic/hydrophilic properties of
the R portion. The effect of the introduction of a hydroxy appendage
on the benzyl function was investigated with derivative 7; this
modification was not tolerated since no inhibition was detected at
piperidinethanol function appears flexible enough to efficiently
bind at the S1 subsite and establish a hydrogen bond with the
backbone of Phe108. The proposed binding modes are in fairly good
agreement with the reported activities for the other members of
the library.
the tested concentration of 3
m
M. In this case the substituent would
5.3. AChE inhibition
alter the geometry of the designed molecule, thus hampering the
interaction of the tertiary nitrogen with Asp 32 of the enzyme. The
contribution of an alcohol function on the R portion was also
evaluated with a subset of compounds (8, 9, and 12). In this regard,
analogue 8, bearing a 2-methylaminoethanol function, proved to be
more active in comparison with the corresponding conformation-
ally constricted piperidin-4-ol derivative 9 (46.69 and 32.78% of
inhibition, respectively). A gain in potency with respect to 8 was
achieved with compound 12, bearing a 2-piperidin-4-ylethanol
function, that proved to inhibit BACE-1 to a good extent (58.72%,
The ability of the newly synthesized compounds to inhibit hu-
man AChE catalytic activity was also investigated. Interestingly,
only the most potent BACE-1 inhibitors of the series (5 and 12)
proved to be weak inhibitors of AChE, with IC50 values in the low
micromolar range (7.00 and 2.52
mM, respectively) less active than
tacrine (IC50 ¼ 0.25
m
M). The remaining molecules showed only a
slight ability to inhibit human AChE (data not shown).
5.4. Antioxidant activity and neurotoxicity in human neuronal SH-
SY5Y cells
IC50 ¼ 2.32
mM), showing potency comparable to that of the lead 5.
On these bases, we assumed that this flexible moiety could play a
crucial role, allowing an appropriate docking of the new compound
into the BACE-1 binding site. In an attempt to support this hy-
pothesis, a computational study performed on 12 showed that this
R portion appears to be able to efficiently interact with the S1
subsite (Fig. 2B; see the Computational Studies paragraph for
further details). To probe the effects of an additional protonable
nitrogen atom on the R portion, compounds 10, 11, and 13, carrying
different substituted piperazine moieties, were synthesized, but
this functional group alteration was detrimental for the activity. A
slight gain in potency, in comparison to the conformationally
restricted methylpiperazine derivative analogue (13), was obtained
with the corresponding ‘open’ analogue 14, carrying a dimethyla-
minoethylmethylamino side chain (13.50% inhibition). Finally,
analogue 15, bearing a cyclohexylmethyl amine group, showed the
same potency as the simplified 3-fluoro-4-hydroxy-40-methyl-
benzophenone 6 confirming that the amino function could lead to a
conformational modification that hampers the interaction with the
enzyme residues.
We evaluated the ability of compounds 3, 5 and 12 to counteract
intracellular ROS formation evoked by t-BuOOH (100
mM) and
A
b25e35 peptides (5 M) in human neuronal SH-SY5Y cells using
m
DHE assays. As shown in Fig. 3, compounds 5 and 12, but not 3,
significantly inhibited the ROS formation elicited by both t-BuOOH
and Ab25e35 peptides in SH-SY5Y cells at concentrations of 3
mM
and 5 M. In particular, the maximum inhibition of ROS formation
m
elicited by Ab25e35 peptides was 30% for both compounds 5 and 12.
In similar experimental conditions, the well-known antioxidant N-
Acetylcysteine (500 mM) decreases ROS formation by 35% (data not
shown). In parallel, the neurotoxicity of the same compounds in
SH-SY5Y cells was then evaluated by the reduction of MTT to for-
mazan. The persistent treatment of SH-SY5Y cells with 5, but not 3
and 12, significantly decreased the neuronal viability at higher
concentrations of 10 and 50 mM (Fig. 4). Taken together, these re-
sults suggest that the des-methylation of 3 contributes to give 5 and
12 antioxidant properties. In particular, the presence of hydroxy
groups bound to the aromatic ring could explain the ability of both
compounds to counteract ROS formation at neuronal level. Inter-
estingly, the affected neuronal viability recorded with 5, bearing a
N,N0-benzylmethylamine moiety, can be overcome with the 2-
(piperidin-4-yl) ethanol function present in 12.
5.2. Computational studies
To understand how our benzophenone-based derivatives inhibit
BACE-1 enzyme, computational studies were performed on 5 and
12. We carried out docking simulations modelling the binding site
of BACE-1 according to the geometry displayed in the crystallo-
graphic complex with an aminobenzylpiperidine-based (ABP)
BACE-1 inhibitor bearing a 3-sulfonamidephenoxy substituent
(Fig. 1). X-ray crystal structure coordinates have been deposited in
the PDB as entry 2ZJM (PDBid code: 2ZJM) [31]. This specific rear-
rangement of the enzyme was selected because this ABP derivative
turned out to most closely match the pharmacophoric features of
the selected compounds, among the 160 co-crystallized inhibitors
available at the time these calculations were carried out (see
Supporting Information for details). The putative bound confor-
6. Conclusions
In this paper, a new series of small molecules based on the 3-
fluoro-4-hydroxybenzophenone core structure was developed to
obtain BACE-1 inhibitors. This main framework was identified upon
a structural modification performed on the weak BACE-1 inhibitor
3, devoid also of antioxidant activity. The resulting compound 5
showed a remarkable change in the biological profile, such as a
noteworthy gain in both BACE-1 inhibition and antioxidant activity.
Computational studies confirmed that the 3-fluoro-4-hydroxy
substitution pattern of the benzophenone scaffold seemed to be
suitable for interactions with the biological counterpart. Moreover,
the presence of a protonable tertiary nitrogen atom, having the
capability to establish H-bonds with a catalytic aspartate, further
reinforced the BACE-1 binding. In this respect, the nature of the
substituent on this atom strongly influenced the capability to
inhibit the enzyme. Among the novel derivatives, compounds 5 and
12, albeit carrying chemically different amine functions, showed
comparable potencies against the selected targets (balanced
inhibitory potencies). In particular, they proved to be able to inhibit
mation of
5
is reported in Fig. 2A. The 3-fluoro-4-
0
hydroxybenzophenone group binds at the S2 subsite interacting
with the side chains of Tyr71, Tyr198, and Arg128 [32]. In particular,
the 4-hydroxy group directly interacts with the side chain of
Arg128. The basic nitrogen of the benzylmethylamine group es-
tablishes a hydrogen bond interaction with the outer oxygen of Asp
32, in the catalytic dyad. Finally, the benzylmethylamine group
points toward the S1 subpocket binding the hydrophobic region
described by the side chains of Tyr71, Phe108, and Trp115. The
docking of 12 with respect to the BACE-1 key residues is depicted in
Fig. 2B, in which the benzophenone scaffold shows the same
interaction pattern as that observed for 5, whilst the
human recombinant BACE-1 enzyme to a good extent (low
mM
range) and the same was observed for the inhibition of human
AChE and for antioxidant activity in human neuronal SH-SY5Y cells.

162
F. Belluti et al. / European Journal of Medicinal Chemistry 78 (2014) 157e166
Fig. 3. Antioxidant activity of compounds 3, 5 and 12 in SH-SY5Y cells. (a) SH-SY5Y cells were co-treated with various concentrations of compounds and t-BuOOH (100
mM) for
30 min. (b) SH-SY5Y cells were co-treated for 3 h with various concentrations of compounds and Ab25e35 peptides (5 M). At the end of incubation, intracellular ROS formation was
m
determined using the probe DHE (as described in the experimental section). The results obtained are expressed as fold increases of ROS formation induced by exposure to t-BuOOH
or Ab25e35 peptides. The values are shown as mean ꢀ SD of three independent experiments (*P < 0.05 vs. untreated cells at ANOVA with the Dunnett post hoc test). (c) Repre-
sentative images of ROS formation induced by Ab25e35 peptides. Scale bars: 100 mm.
It is noteworthy that the overload of ROS induces accumulation of
, establishing a vicious circle that reinforces the oxidative stress
induced by A
may provide a high background cognitive influence and obscure the
benefit of A reduction [37]. In this context, the use of -secretase
inhibitors with added antioxidant properties could extend their
therapeutic window to the intermediate and late stages of AD. It is
b, such as neuroinflammation and oxidative stress,
A
b
with strengthening of oxidative damage at neuronal level [33].
Moreover, since final targets of this study are located in the CNS,
the possibility for the designed compounds to penetrate the BBB
was also estimated by calculation of molecular lipophilicity, a
physico-chemical property well known to influence BBB penetra-
tion. In particular, logP values were predicted by means of Chem-
BioDraw Ultra 12.0 and proved to be ꢁ5 (4.69 and 3.24 for com-
pounds 5 and 12, respectively). With a number of hydrogen bond
donors ꢁ3 (n. H-bond donors ¼ 1 and 2 for compounds 5 and 12,
respectively), number of hydrogen bond acceptors largely below 7
(n. H-bond acceptors ¼ 2 for both compounds), and molecular
weights below 400 g/mol (349.40 and 357.42 g/mol for compounds
5 and 12, respectively), the physico-chemical properties profiles of
5 and 12 are in compliance with Lipinski’s and Wenlock’s guidelines
for good passive CNS penetration [34,35].
b
b
noteworthy that the overload of ROS induces accumulation of Ab,
establishing a vicious circle that reinforces the oxidative stress with
strengthening of oxidative damage at neuronal level [33].
Based on these considerations, compound 12, with a balanced
inhibitory potency against these selected targets, together with a
complete lack of toxic effects, might be considered a potential
therapeutic agent to modify the course of AD.
7. Experimental section
7.1. Chemistry
Given that AChE, BACE-1 and oxidative stress have been
recognized to play a pivotal role for both onset and progression of
AD, the development of non-peptidic small molecules able to
inhibit these enzymes, and to tackle the oxidative stress, is a
challenging area of drug discovery. Recent clinical studies suggest
that the efficacy of b-secretase inhibitors has a better chance to be
observed in the treatment of the early stages of AD [36]. During the
progression of AD, secondary pathological events not directly
Starting materials, unless otherwise specified, were used as high
grade commercial products. Solvents were of analytical grade. Re-
action progress was followed by thin layer chromatography (TLC)
on precoated silica gel plates (Merck Silica Gel 60 F254) and then
visualized with a UV254 lamplight. Chromatographic separations
were performed on silica gel columns by flash method (Kieselgel
40, 0.040e0.063 mm, Merck). Melting points were determined in
open glass capillaries, using a Büchi apparatus and are uncorrected.

F. Belluti et al. / European Journal of Medicinal Chemistry 78 (2014) 157e166
163
3H, H-5, H-50 and H-30), 7.90e7.95 (m, 5H, phenyl), 7.98 (d,
J ¼ 8.6 Hz, 2H, H-20 and H-60), 8.35 (d, J ¼ 8.6 Hz, 1H, H-6). 13C NMR
(CDCl3):
d 55.25, 59.35, 60.32, 61.82, 112.17, 112.42, 117.82, 120.52,
127.48, 128.67, 129.78, 130.43, 130.48, 132.88, 136.31, 143.95, 147.29,
148.75, 150.47, 151.37, 151.48, 152.93, 170.15. ESI-MS (m/z): 350
(M þ Hþ).
7.2.2. 3-Fluoro-4-hydroxyphenyl-4-(3-hydroxybenzyl)
methylaminomethylphenylmethanone 7
Reaction of 18 (0.5 mmol) and 3-methylaminomethylphenol
(0.097 g) gave the crude final product 7 that was purified by flash
chromatography (CH2Cl2/CH3OH/NH4OH 9/1/0.1). Yield 75%,
yellowish solid, mp 135e137 ꢂC (AcOEt/n-hexane). 1H NMR
(DMSO):
d 2.55 (s, 3H, NeCH3), 3.77 (s, 2H, CH2eN), 3.79 (s, 2H,
CH2eN), 6.66 (dd, J ¼ 1.8 and 8.6 Hz, 1H, H-400), 6.87 (d, J ¼ 8.6 Hz,
1H, H-600), 6.90 (s, 1H, H-200), 7.03e7.15 (m, 2H, H-2 and H-500), 7.43
(d, J ¼ 8.6 Hz, 1H, H-5), 7.51e7.58 (m, 3H, H-6, H-50 and H-30), 7.75
(d, J ¼ 8.6 Hz, 2H, H-20 and H-60). 13C NMR (DMSO):
d 58.28, 59.88,
62.33, 63.83, 111.12, 111.92, 115.81, 121.11, 125.32, 128.67, 129.78,
130.39, 130.42, 132.78, 135.99, 144.55, 147.29, 148.75, 151.47, 151.77,
152.54, 154.12, 170.33. ESI-MS (m/z): 366 (M þ Hþ).
7.2.3. 3-Fluoro-4-hydroxyphenyl-4-(2-hydroxyethyl)
methylaminomethylphenylmethanone 8
Reaction of 18 (0.5 mmol) and 2-methylaminoethanol (0.054 g)
gave the crude final product 8 that was purified by flash chroma-
tography (CH2Cl2/CH3OH/NH4OH 9.0/1.0/0.1). Yield 64%; white
solid, mp 65e67 ꢂC (AcOEt/n-hexane). 1H NMR (CDCl3):
d 2.30 (s,
Fig. 4. Neurotoxicity of compounds 3, 5 and 12 in SH-SY5Y cells. The neuronal viability
in SH-SY5Y cells was determined by MTT assay (as described in the experimental
section) after 24 h of incubation with various concentrations of compounds (0.1e
3H, NeCH3), 2.65 (t, J ¼ 5.8 Hz, 2H, CH2eN), 3.66 (t, J ¼ 5.8 Hz, 2H,
CH2OH), 3.68 (s, 2H, CH2eN), 4.99 (br,1H, OH), 7.14 (t, J ¼ 8.4 Hz,1H,
H-2), 7.42 (d, J ¼ 8.4 Hz, 2H, H-30 and H-50), 7.51 (d, J ¼ 8.4 Hz,1H, H-
5), 7.60 (dd, J ¼ 1.8 and 8.4 Hz, 1H, H-6), 7.70 (d, J ¼ 8.4 Hz, 2H, H-20
50 mM). The results are expressed as a percentage of control cells and the values are
reported as mean ꢀ SD of three independent experiments (**P < 0.01 vs. cells treated
with compound 3 at Student’s t-test).
and H-60). 13C NMR (acetone-d6):
d 40.93, 58.35, 58.50, 61.02,
116.60, 116.66, 116.84, 127.03, 127.98, 128.66, 136.05, 142.81, 147.99,
1H NMR and 13C NMR spectra were recorded on a Varian Gemini
spectrometer 400 MHz, and chemical shifts (d) are reported as parts
149.26, 149.52, 152.82, 192.39. ESI-MS (m/z): 304 (M þ Hþ).
per million (ppm) values relative to tetramethylsilane (TMS) as
internal standard; coupling constants (J) are reported in Hertz (Hz).
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). The purity of
compounds was determined by elemental analysis; purity for all
the tested compounds was superior to 95%. Compounds were
named relying on the naming algorithm developed by Cam-
bridgeSoft Corporation and used in Chem-BioDraw Ultra 12.0.
7.2.4. Fluoro-4-hydroxyphenyl-4-(4-hydroxypiperidin-1-ylmethyl)
phenylmethanone 9
Reaction of 18 (0.5 mmol) and piperidin-4-ol (0.078 g) gave the
crude final product 9 that was purified by flash chromatography
(CH2Cl2/CH3OH/NH4OH 9.0/1.0/0.1). Yield 83%; white solid, mp 66e
69 ꢂC (AcOEt/n-hexane). 1H NMR (acetone-d6):
d 1.54e1.65 (m, 2H,
piperidine), 1.83e1.94 (m, 2H, piperidine), 2.08e2.33 (m, 2H,
piperidine), 2.60e2.90 (m, 2H, piperidine), 3.30 (s, 2H, CH2eN),
3.63e3.66 (m, 1H, piperidine), 4.26 (br, 1H, OH), 7.18 (t, J ¼ 8.4 Hz,
1H, H-2), 7.42e7.58 (m, 4H, H-5, H-6, H-30, H-50), 7.82 (d, J ¼ 8.4 Hz,
2H, H-20, H-60). 13C NMR (acetone-d6):
d 32.96, 50.07, 61.01, 65.54,
7.2. General parallel procedure for the synthesis of compounds 5,
7e15
107.31, 117.39, 117.93, 128.25, 128.35, 128.89, 129.65, 133.16, 141.22,
151.23, 151.51, 219.29. ESI-MS (m/z): 330 (M þ Hþ).
In distinct reactors the bromomethyl 18 (0.5 mmol, 0.15 g) was
dissolved in toluene (5 mL), then the selected amine (0.75 mmol)
and Et3N (0.75 mmol) were added to the corresponding reactors.
The mixtures were stirred under reflux for 12e20 h while moni-
toring with TLC. Purification of the crudes was achieved by flash
chromatography on silica gel.
7.2.5. 3-Fluoro-4-hydroxyphenyl-4-(4-phenylpiperazin-1-
ylmethyl)phenylmethanone 10
Reaction of 18 (0.5 mmol) and 1-phenylpiperazine (0.13 g) gave
the crude final product 10 that was purified by flash chromatog-
raphy (CH2Cl2/CH3OH/NH4OH 9.0/1.0/0.1). Yield 73%; white solid,
mp 93e96 ꢂC (AcOEt/n-hexane). 1H NMR (CDCl3):
d 2.68e2.77 (m,
7.2.1. 4-(benzylmethylaminomethylphenyl)-3-fluoro-4-
hydroxyphenylmethanone 5
4H, piperazine), 3.15e3.30 (m, 6H, CH2eN and piperazine), 6.82e
6.98 (m, 3H, phenyl), 7.14 (t, J ¼ 8.4 Hz, 1H, H-2), 7.25e7.32 (m, 2H,
phenyl), 7.49e7.61 (m, 4H, H-5, H-6, H-30, H-50), 7.77 (d, J ¼ 8.4 Hz,
Reaction of 18 (0.5 mmol) and N,N’-benzylmethylamine
(0.096 g) gave the crude final product 5 that was purified by flash
chromatography (CH2Cl2/CH3OH/NH4OH 9.5/0.5/0.1). Yield 65%;
2H, H-20, H-60). 13C NMR (CDCl3):
d 45.22, 51.98, 53.68, 54.55, 62.28,
111.11, 117.45, 118.00, 127.02, 127.36, 128.60, 129.49, 129.50, 130.15,
130.25, 130.89, 131.65, 136.37, 137.02, 141.72, 150.50, 151.51, 194.29.
ESI-MS (m/z): 391 (M þ Hþ).
brown semisolid. 1H NMR (CDCl3):
d
2.65 (s, 3H, NeCH3), 4.04 (s,
2H, CH2eN), 4.30 (s, 2H, CH2eN), 7.00 (m, 1H, H-2), 7.47e7.64 (m,

164
F. Belluti et al. / European Journal of Medicinal Chemistry 78 (2014) 157e166
7.2.6. 4-(4-Benzylpiperazin-1-ylmethyl)phenyl-3-fluoro-4-
hydroxyphenylmethanone 11
2H, H-20, H-60). 13C NMR (CDCl3):
d 22.11, 24.32, 24.36, 25.45, 25.89,
40.93, 43.12, 59.35, 59.65, 61.02, 116.60, 116.68, 116.88, 127.24,
127.47, 128.47, 136.35, 142.54, 147.27, 149.95, 149.99, 152.99, 191.16.
ESI-MS (m/z): 342 (M þ Hþ).
Reaction of 18 (0.5 mmol) and 1-benzylpiperazine (0.13 g) gave
the crude final product 11 that was purified by flash chromatog-
raphy (CH2Cl2/CH3OH/NH4OH 9.5/0.5/0.1). Yield 78%; white solid,
mp 101e103 ꢂC (AcOEt/n-hexane). 1H NMR (CDCl3):
d
2.61e2.73 (m,
7.3. General procedure for the synthesis of compounds 6 and 18
8H, piperazine), 3.58 (s, 2H, CH2eN), 3.61 (s, 2H, CH2eN), 6.94 (t,
J ¼ 8.4 Hz, 1H, H-2), 7.21e2.35 (m, 5H, phenyl), 7.40 (d, J ¼ 8.4 Hz,
2H, H-30, H-50), 7.42e7.56 (m, 2H, H-5, H-6), 7.66 (d, J ¼ 8.4 Hz, 2H,
BBr3 (1 M solution in CH2Cl2, 1.5 equiv) was added dropwise to a
solution of the methoxy derivative (1 equiv) in anhydrous CH2Cl2
(2 mL), at 0 ꢂC and under N2 atmosphere. The solution was stirred
for 2 h at the same temperature, then overnight at room temper-
ature. The mixture was neutralized with NaHCO3 saturated solution
and then extracted with CH2Cl2, the combined organic layers were
dried (Na2SO4) to afford a crude residue, which was purified by
flash column chromatography.
H-20, H-60). 13C NMR (CDCl3):
d 45.28, 52.00, 54.01, 54.15, 58.31,
62.55, 111.47, 117.21, 118.54, 127.72, 127.84, 128.11, 128.81, 129.13,
129.15, 129.25, 130.89, 131.65, 136.37, 137.02, 141.78, 150.12, 151.65,
195.46. ESI-MS (m/z): 405 (M þ Hþ).
7.2.7. 3-Fluoro-4-hydroxyphenyl-4-[4-(2-hydroxyethyl)piperidin-
1-ylmethyl]phenylmethanone 12
Reaction of 18 (0.5 mmol) and 2-piperidin-4-ylethanol (0.125 g)
gave the crude final product 12 that was purified by flash chro-
matography (CH2Cl2/CH3OH/NH4OH 9.5/0.5/0.1). Yield 78%; white
7.3.1. 3-Fluoro-4-hydroxyphenyl-p-tolylmethanone 6
Reaction of 16 [19] gave the crude product 6 that was purified by
flash chromatography (petroleum ether/AcOEt 9/1). Yield 71%;
solid (AcOEt/n-hexane), mp 101e103 ꢂC; 1H NMR (CDCl3):
d
1.22e
white solid, mp 100e101 ꢂC. 1H NMR (CDCl3):
d 2.37 (s, 3H, CH3),
1.83 (m, 5H, piperidine), 2.04e2.13 (m, 2H, CH2-piperidine), 2.90e
3.05 (m, 4H, piperidine), 3.65 (t, J ¼ 6.4 Hz, 2H, CH2OH), 3.69 (s, 2H,
CH2eN), 5.12 (br, 1H, OH), 7.09 (t, J ¼ 8.4 Hz, 1H, H-2), 7.43e7.57 (m,
4H, H-5, H-6, H-30, H-50), 7.76 (d, J ¼ 8.4 Hz, 2H, H-20, H-60). 13C NMR
4.50 (br, 1H, OH), 6.94 (t, J ¼ 8.4 Hz, 1H, H-2), 7.21 (d, J ¼ 8.4 Hz, 2H,
H-30, H-50), 7.46e7.56 (m, 4H, H-20, H-60, H-5, H-6). 13C NMR
(CDCl3):
d 21.50, 117.13, 117.19, 117.65, 118.04, 128.94, 129.87, 135.07,
148.50, 148.78, 149.07, 207.32. ESI-MS (m/z): 231 (M þ Hþ).
(CDCl3):
d 25.11, 32.95, 50.09, 61.09, 65.59, 107.21, 117.39, 117.93,
128.25, 128.36, 128.88, 129.66, 133.12, 141.26, 151.26, 151.58, 219.27.
7.3.2. 4-bromomethylphenyl-3-fluoro-4-hydroxyphenylmethanone
18
ESI-MS (m/z): 405 (M þ Hþ).
Reaction of 17 [19] gave the crude product 18, that was purified
by flash column chromatography (petroleum ether/AcOEt 7/3).
7.2.8. 3-Fluoro-4-hydroxyphenyl-4-(4-methylpiperazin-1-
ylmethyl)phenylmethanone 13
Yield 92%; white solid, mp 145e147 ꢂC. 1H NMR (CDCl3):
d 4.53 (s,
Reaction of 18 (0.5 mmol) and 1-methylpiperazine (0.13 g) gave
the crude final product 13 that was purified by flash chromatog-
raphy (CH2Cl2/CH3OH/NH4OH 9.5/0.5/0.1). Yield 74%; white solid,
2H, CH2Br), 5.90 (br, 1H, OH), 7.08 (t, J ¼ 8.4 Hz, 1H, H-2), 7.50 (d,
J ¼ 8.0 Hz, 2H, H-30, H-50), 7.59 (d, J ¼ 8.0 Hz, 1H, H-5), 7.65e7.68 (m,
1H, H-6), 7.73 (d, J ¼ 8.4 Hz, 2H, H-20, H-60).
mp 88e89 ꢂC (AcOEt/n-hexane). 1H NMR (CDCl3):
d 2.44 (s, 3H,
CH3), 2.56e2.69 (m, 8H, piperazine), 3.60 (s, 2H, CH2), 6.91 (t,
J ¼ 8.4 Hz,1H, H-2), 7.40 (d, J ¼ 8.4 Hz, 2H, H-30, H-50), 7.46e7.56 (m,
2H, H-5, H-6), 7.68 (d, J ¼ 8.4 Hz, 2H, H-20, H-60). 13C NMR (CDCl3):
7.4. BACE-1 inhibition. FRET inhibition assay
FRET inhibition studies were performed using the following
d
29.16, 45.19, 51.92, 53.72, 54.55, 62.25, 111.46, 117.59, 118.03,
procedures: 5
with 175 L of BACE-1 (17.2 nM, final concentration) in 20 mM
sodium acetate pH 4.5 containing CHAPS (0.1% w/v) for 1 h at room
temperature. M-2420 (3 M, final concentration) was then added
mL of test compound (or DMSO) were pre-incubated
128.15, 128.25, 128.89, 129.65, 137.13, 147.72, 151.23, 151.51, 194.29.
m
ESI-MS (m/z): 231 (M þ Hþ).
m
7.2.9. 4-(2-dimethylaminoethylmethylaminomethylphenyl)-3-
fluoro-4-hydroxyphenylmethanone 14
and left to react for 15 min at 37 ꢂC. The fluorescence signal was
read at lem ¼ 405 nm (lexc ¼ 320 nm). DMSO concentration in the
final mixture was maintained below 5% (v/v) to guarantee no sig-
nificant loss of enzyme activity. Fluorescence intensities with and
without inhibitors were registered and compared. The percent in-
hibition due to the presence of test compounds was calculated. The
background signal was measured in control wells containing all the
reagents, except hrBACE-1, and subtracted. The % inhibition due to
the presence of test compound was calculated by the following
expression: 100 ꢃ (IFi/IFo ꢄ 100) where IFi and IFo are the fluo-
rescence intensities obtained for hrBACE-1 in the presence and in
the absence of inhibitor, respectively [28]. The linear regression
parameters were determined and the IC50 interpolated (GraphPad
Prism 4.0, GraphPad Software Inc.).
Reaction of 18 (0.5 mmol) and N1,N1,N2-trimethylethane-1,2-
diamine (0.075 g) gave the crude final product 14 that was puri-
fied by flash chromatography (CH2Cl2/CH3OH/NH4OH 9.5/0.5/0.1).
Yield 74%; white solid mp 73e75 ꢂC (AcOEt/n-hexane). 1H NMR
(CDCl3):
d
2.50 (s, 3H, CH3), 2.89 (s, 6H, CH3), 2.99 (t, J ¼ 6.2 Hz, 2H,
CH2), 3.56 (t, J ¼ 6.2 Hz, 2H, CH2), 3.72 (s, 2H, CH2), 4.09 (br, 1H, OH),
7.30 (t, J ¼ 8.2 Hz, 1H, H-2), 7.46e7.56 (m, 4H, H-5, H-6, H-30, H-50),
7.65 (d, J ¼ 8.4 Hz, 2H, H-20, H-60). 13C NMR (CDCl3):
d 40.93, 58.23,
58.35, 58.50, 67.02, 116.55, 116.58, 116.98, 127.54, 127.75, 128.88,
136.12, 142.41, 147.57, 149.32, 149.36, 152.24, 193.11. ESI-MS (m/z):
331 (M þ Hþ).
7.2.10. 4-Cyclohexylmethylaminomethylphenyl-3-fluoro-4-
hydroxyphenylmethanone 15
7.5. AChE inhibition
Reaction of 18 (0.5 mmol) and N-methylcyclohexylamine
(0.08 g) gave the crude final product 15 that was purified by flash
chromatography (CH2Cl2/CH3OH/NH4OH 9.5/0.5/0.1). Yield 73%;
white solid mp 65e67 ꢂC (AcOEt/n-hexane). 1H NMR (acetone-d6):
The capacity of compounds 5, 7e15 to inhibit AChE activity was
assessed by Ellman’s method [29]. AChE stock solution was pre-
pared by dissolving human recombinant AChE (E.C.3.1.1.7) lyophi-
lized powder (Sigma, Italy) in 0.1 M phosphate buffer (pH ¼ 8.0)
containing Triton X-100 (0.1%). Five increasing concentrations of
inhibitor were assayed to obtain % inhibition of the enzymatic ac-
tivity in the range of 20e80. The assay solution consisted of a 0.1 M
d
1.20e1.41 (m, 6H, cyclohexane C-3, C-4, C-5), 1.75e1.98 (m, 4H,
cyclohexane C-2, C-6), 2.21 (s, 3H, CH3), 2.43e2.51 (m, 1H, cyclo-
hexane C-1), 3.51 (s, 2H, CH2), 4.90 (br, 1H, OH), 7.09 (t, J ¼ 8.4 Hz,
1H, H-2), 7.65e7.68 (m, 4H, H-5, H-6, H-30, H-50), 7.73 (d, J ¼ 8.4 Hz,

F. Belluti et al. / European Journal of Medicinal Chemistry 78 (2014) 157e166
165
phosphate buffer pH 8.0, with the addition of 340
bis(2-nitrobenzoic acid), 0.02 unit/mL of human recombinant AChE
from human serum and 550 M of substrate (acetylthiocholine
iodide, ATCh). Increasing concentrations of tested inhibitor were
added to the assay solution and pre-incubated for 20 min at 37 ꢂ
with the enzyme followed by the addition of substrate. Initial rate
assays were performed at 37 ꢂC 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 con-
taining all components except AChE 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 analysed in duplicate, and IC50
values were determined graphically from log concentrationeinhi-
bition curves (GraphPad Prism 4.03 software, GraphPad Software
Inc.).
m
M 5,50-dithio-
7.6.5. Ab25e35 peptide preparation for intracellular ROS formation
assay
m
A
b25e35 peptides were first dissolved in hexafluoroisopropanol
to 1 mg mLꢃ1, sonicated, incubated at room temperature for 24 h
and lyophilized. The resulting unaggregated Ab25e35 peptide film
was dissolved with dimethylsulfoxide and stored at ꢃ20 ꢂC until
use.
C
7.6.6. Determination of Ab25e35 peptide-induced intracellular ROS
formation
The intracellular ROS formation induced by Ab25e35 peptides at
SH-SY5Y cell level was determined using the probe DHE as previ-
ously described [39]. Briefly, SH-SY5Y cells were cultured in BD
FalconÔ 8-well Culture slides (surface area 0.7 cm2/well) at
1 ꢄ 104 cells/well for 24 h. The cells were then co-treated with
various concentrations of compounds (0.5e5
peptides (5
incubated with DHE (10
m
M) and Ab25e35
M). At the end of treatment, the cells were washed and
M) in PBS for 30 min in the dark. After
m
m
removal of the probe, cells were washed with PBS and incubated
with DMEM serum free for 1 h at 37 ꢂC. 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 50e
100 cells in each were analysed and the values obtained are
expressed as fold increases of ROS formation vs. untreated cells.
7.6. Antioxidant activity and neurotoxicity in human neuronal SH-
SY5Y cells
7.6.1. Chemicals
A
b25e35 peptide, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT), dihydroethidium (DHE) and tert-
butylhydroperoxide (t-BuOOH) were purchased from Sigma
Chemical Co. All other reagents were of the highest grade of purity
commercially available.
7.6.7. Statistical analysis
Data are reported as mean ꢀ SD of at least 3 independent ex-
periments. Statistical analysis was performed using one-way
ANOVA with the Dunnett post hoc test and Student’s t-test, as
appropriate. Differences were considered significant at p < 0.05.
Analyses were performed using GraphPad Prism 4.0 software.
7.6.2. Cell cultures
Human neuronal (SH-SY5Y) cells were routinely grown in Dul-
becco’s modified Eagle’ medium supplemented with 10% fetal
bovine serum, 2 mM glutamine, 50 U mLꢃ1 penicillin, and
50
m
g mLꢃ1 streptomycin at 37 ꢂC in a humidified incubator with 5%
7.7. Molecular docking
CO2.
The docking simulations were carried out by means of ICM 3.7
[40]. The protein structure was prepared starting from the crys-
tallographic complex of BACE-1 2ZJM [31]. Hydrogen atoms were
added. 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 automatically assigned the tautomerization state that
improved the hydrogen bonding pattern. Ligands were built
defining the right bond orders, stereochemistry, hydrogen atoms,
and protonation states. Each ligand was assigned the MMFF force
field atom types and charges [41]. The residues with at least one
7.6.3. Determination of neurotoxicity induced by compounds
To evaluate the neurotoxic effects of compounds, the SH-SY5Y
cells were seeded in 96-well plates at 2 ꢄ 104 cells/well, incu-
bated for 24 h and subsequently treated with various concentra-
tions of compounds (0.1e50 mM). The neuronal viability in terms of
mitochondrial metabolic function was evaluated by the reduction
of MTT to formazan as previously described [38]. Briefly, the
treatment medium was replaced with MTT (5 mg/mL) in phosphate
buffered saline (PBS) for 2 h at 37 ꢂC in 5% CO2. After washing with
PBS, the formazan crystals were dissolved with isopropanol. The
amount of formazan was measured (570 nm, ref. 690 nm) with a
spectrophotometer (TECANÒ, GENios, Salzburg, Austria). The
neuronal viability is expressed as a percentage of control cells.
ꢀ
heavy atom within 5 A from the bound conformation of the co-
crystallized inhibitors were considered to define the boundaries
of the binding box. The docking engine employed was the Biased
Probability Monte Carlo (BPMC) stochastic optimizer as imple-
mented in ICM [42]. The ligand binding site at the receptor was
7.6.4. Determination of t-BuOOH-induced intracellular ROS
formation
ꢀ
represented by pre- calculated 0.5 A spacing potential grid maps,
The intracellular ROS formation induced by t-BuOOH at SH-
SY5Y cell level was determined using DHE (lexc ¼ 380 nm,
lem ¼ 445 nm). Briefly, SH-SY5Y cells were cultured in 96-well
microtiter plates at 3 ꢄ 104 cells/well for 24 h. The medium was
then removed and the cells were washed with PBS and then
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 repul-
sion. The molecular conformation was described by means of in-
ternal coordinate variables. The adopted force field was a modified
version of the ECEPP/3 force field with a distance-dependent
dielectric constant [43]. Given the number of rotatable bonds in
the ligand, the basic number of BPMC steps to be carried out was
incubated with DHE (10
removal of the probe and further washing, the cells were co-treated
with various concentrations of compounds (0.5e5 M) and t-
BuOOH (100 M) for 30 min at room temperature in the dark. At the
mM) in PBS for 30 min in the dark. After
m
m
end of incubation, the red fluorescence of the cells from each well
was measured with a spectrofluorometer (TECANÒ, GENios). The
results are expressed as fold increase of intracellular ROS evoked by
exposure to t-BuOOH.

166
F. Belluti et al. / European Journal of Medicinal Chemistry 78 (2014) 157e166
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.042.
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