Exploiting the chalcone scaffold to develop multifunctional agents for Alzheimer’s disease

Article abstract of DOI:10.3390/molecules23081902

Alzheimer’s disease still represents an untreated multifaceted pathology, and drugs able to stop or reverse its progression are urgently needed. In this paper, a series of naturally inspired chalcone-based derivatives were designed as structural simplification of our previously reported benzofuran lead compound, aiming at targeting both acetyl (AChE)- and butyryl (BuChE) cholinesterases that, despite having been studied for years, still deserve considerable attention. In addition, the new compounds could also modulate different pathways involved in disease progression, due to the peculiar trans-α,β-unsaturated ketone in the chalcone framework. All molecules presented in this study were evaluated for cholinesterase inhibition on the human enzymes and for antioxidant and neuroprotective activities on a SH-SY5Y cell line. The results proved that almost all the new compounds were low micromolar inhibitors, showing different selectivity depending on the appended substituent; some of them were also effective antioxidant and neuroprotective agents. In particular, compound 4, endowed with dual AChE/BuChE inhibitory activity, was able to decrease ROS formation and increase GSH levels, resulting in enhanced antioxidant endogenous defense. Moreover, this compound also proved to counteract the neurotoxicity elicited by Aβ1–42 oligomers, showing a promising neuroprotective potential.

Full text of DOI:10.3390/molecules23081902

molecules
Article
Exploiting the Chalcone Scaffold to Develop
Multifunctional Agents for Alzheimer’s Disease
Angela Rampa 1,
*
ID , Manuela Bartolini 1 ID , Letizia Pruccoli 2 ID , Marina Naldi 1
,
ID
Isabel Iriepa 3, Ignacio Moraleda 3, Federica Belluti 1 ID , Silvia Gobbi 1 ID , Andrea Tarozzi 2
and Alessandra Bisi 1,
*
ID
1
Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna,
Via Belmeloro 6, 40126 Bologna, Italy; manuela.bartolini3@unibo.it (M.B.); marina.naldi@unibo.it (M.N.);
federica.belluti@unibo.it (F.B.); silvia.gobbi@unibo.it (S.G.)
Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Corso d’Augusto 237,
47921 Rimini, Italy; letizia.pruccoli2@unibo.it (L.P.); andrea.tarozzi@unibo.it (A.T.)
Department of Organic Chemistry and Inorganic Chemistry, School of Sciences, University of Alcalá,
E-28871 Alcalá de Henares, Madrid, Spain; isabel.iriepa@uah.es (I.I.); ignacio.moraleda@uah.es (I.M.)
Correspondences: angela.rampa@unibo.it (A.R.); alessandra.bisi@unibo.it (A.B.)
2
3
*
Academic Editor: Diego Muñoz-Torrero
Received: 29 May 2018; Accepted: 27 July 2018; Published: 30 July 2018
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Alzheimer’s disease still represents an untreated multifaceted pathology, and drugs
able to stop or reverse its progression are urgently needed. In this paper, a series of naturally
inspired chalcone-based derivatives was designed as structural simplification of our previously
reported benzofuran lead compound, aiming at targeting both acetyl (AChE)- and butyryl
(BuChE) cholinesterases that, despite having been studied for years, still deserve considerable
attention. In addition, the new compounds could also modulate different pathways involved in
disease progression, due to the peculiar trans-α,β-unsaturated ketone in the chalcone framework.
All molecules presented in this study were evaluated for cholinesterase inhibition on the human
enzymes and for antioxidant and neuroprotective activities on a SH-SY5Y cell line. The results
proved that almost all the new compounds were low micromolar inhibitors, showing different
selectivity depending on the appended substituent; some of them were also effective antioxidant
and neuroprotective agents. In particular, compound 4, endowed with dual AChE/BuChE
inhibitory activity, was able to decrease ROS formation and increase GSH levels, resulting in
enhanced antioxidant endogenous defense. Moreover, this compound also proved to counteract the
neurotoxicity elicited by Aβ1–42 oligomers, showing a promising neuroprotective potential.
Keywords: Alzheimer’s disease; chalcones; cholinesterase inhibitors; antioxidants; neuroprotection
1. Introduction
Alzheimer’s disease (AD) is the most common cause of dementia and is becoming a global health
concern. Indeed, the continuous rise in the aging population and an exponential increase in AD cases
over the next few decades in Europe are expected to result in immense pressure on the social and
health-care systems of the region [1,2]. Therefore, there is an urgent need for developing curative
or disease-modifying therapies to offset the upcoming AD epidemic. However, despite millions
of dollars of investment in drug discovery and clinical trials, no single molecule has yet been
approved for its treatment since the advent of cholinesterase inhibitors (ChEIs) and memantine,
an N-methyl-d-aspartate (NMDA) receptor antagonist [3].
In AD, atrophy of cells’ nuclei in the key areas of the brain, such as the basal forebrain, leads to
a noticeable decrease in cholinergic function; cholinergic transmission in the brain is an essential
Molecules 2018, 23, 1902; doi:10.3390/molecules23081902
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1902
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component of cognitive function (cholinergic hypothesis). For this reason, drug therapies have been
developed focusing on ChEIs, which increase cholinergic transmission through the inhibition of the
enzymes responsible for breaking down the neurotransmitter acetylcholine. ChEIs are currently
used for the symptomatic treatment of AD, since they may offer some stabilization of the symptoms,
which ultimately could provide patients with better quality of life in their remaining years, even if
they do not alter the underlying disease trajectory [4].
Furthermore, AD is characterized by misfolded protein aggregates [
5], chronic inflammation [6]
and oxidative stress, elicited by increased generation of reactive oxygen species (ROS) [
7].
Oxidative stress merely reflects a disequilibrium between the amount of oxidants produced and
the quantities of antioxidant enzymes—such as superoxide dismutases, catalases and glutathione
peroxidase—required to restore the appropriate balance. This imbalance has its origins in genes
and the ways in which gene expression is regulated: a central role is played by the transcription
factor called nuclear factor (erythroid-derived 2)-like 2 or Nrf2, the “master regulator” of the
antioxidant response that modulates the expression of hundreds of genes, including antioxidant
enzymes [8]. Young healthy individuals possess all the genetic resources required to maintain
a correct oxidative balance, whereas Nrf2 expression appears to decline with aging, leading to a
dysregulation of oxidative stress responses. Accordingly, the use of Nrf2 activators has been attracting
increasing attention as a valuable therapeutic strategy for neurodegenerative diseases. In particular,
cellular Nrf2 levels are tightly regulated by Keap1, a protein acting as an oxidative stress sensor through
modifications of various cysteine residues located in different protein regions. These modifications
lead to conformational changes that disrupt the Keap1/Nrf2 interaction, inhibiting the degradation of
Nrf2 and enabling its binding to antioxidant response elements (ARE) and the following activation of
antioxidant enzymes transcription [8].
Our research group has been involved for many years in the development of drug candidates
for AD treatment. In particular, in previous papers we reported a promising benzofuran-based lead
compound endowed with multiple biologic properties (Figure 1) [9,10]. Here we report the design and
evaluation of chalcone-based compounds and the corresponding hydrogenated derivatives as flexible
open ring analogs of this lead. Chalcones (trans-1,3-diaryl-2-propen-1-ones), found as central core
in a variety of important bioactive compounds [11], belong to the flavonoid family, the largest class
of secondary metabolites in plants, where they serve as a defense mechanism to counteract ROS in
order to survive and prevent molecular damage. These natural products can be defined as “privileged
structures” and they can be properly exploited to develop effective strategies in different fields of drug
discovery [12]. Indeed, chalcones possess a peculiar trans-α,β-unsaturated ketone, a functional group
perceived as a potential Michael acceptor, that allows the molecule to interact with cysteine residues of
different biological targets to obtain the Michael adduct [12–14].
The simpler and more flexible framework of the new chalcone-based derivatives allowed
the obtaining of less constrained molecules, which could better interact with the narrow gorge
of the ChE enzymes. In this new series, the A ring was decorated with a methoxy group or an
additional tertiary amino function, charged at physiological pH and linked through spacers of different
lengths, to evaluate its possible binding to the peripheral anionic site (PAS) located at the top of the
acetylcholinesterase (AChE) gorge. To better address this point, the amino side chain was introduced
in meta or para positions on the benzene ring, as in compounds
1–15. Finally, to assess the role
of the chalcone -double bond and to evaluate the impact of a further increase in flexibility, the
α,β
saturated analogs 16–18 were also designed and synthesized. The new compounds were reported
in Tables 1 and 2.

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Figure 1. Design strategy for the studied compounds 1–18.
2. Results and Discussion
2.1. Chemistry
According to Scheme 1, compound
1
was synthesized starting from 4-hydroxybenzonitrile,
which was alkylated, in the presence of K2CO3, with 1,7-dibromoheptane obtaining 19, that was then
subjected to nucleophilic substitution reaction by N-methylbenzylamine to afford 20. Afterwards,
the cyano group was reduced using the Ni/Raney alloy in formic acid [15] to give the aldehyde
21. Benzylic bromination with N-bromosuccinimide (NBS) of 4-methylacetophenone afforded 22
,
which was subsequently reacted with diethylamine to give 23 Chalcone was obtained via
.
1
Claisen-Schmidt condensation reaction of 23 with the previously prepared aldehyde 21.
Scheme 1. Reagents and conditions: (i) Br(CH2)7Br, K2CO3, acetone, reflux; (ii) N-methylbenzylamine,
Et3N, toluene, reflux; (iii) Ni/Raney, HCOOH, reflux; (iv) NBS, (PhCOO)2, CCl4, reflux; (v) Et2NH,
toluene, reflux; (vi) KOH, EtOH, r.t.

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Table 1. Inhibitory activities against hAChE and hBuChE, as IC50 values, of compounds
reference compounds tacrine (THA) and galantamine (GAL).
1–15 and
IC50 (µM) ± SEM
SI a
Comp.
X
Chain Position
R
hAChE
hBuChE
8.83 ± 0.58
2.85 ± 0.70
19.9 ± 5.5
1
2
3
-
para
para
para
2.81 ± 0.29
98.5 ± 8.8
0.70 ± 0.09
3.14
0.03
28.4
O
O
CH3
4
5
O
O
para
para
0.92 ± 0.07
1.06 ± 0.08
2.38 ± 0.05
3.70 ± 0.08
2.6
3.5
6
7
8
O
O
O
para
para
para
511 ± 37
1.25 ± 0.06
16.5 ± 2.6
11.0 ± 0.5
19.7 ± 0.6
3.87 ± 0.30
0.021
15.8
0.23
9
O
para
13.5 ± 2.3
3.93 ± 0.20
0.29
b
b
10
11
O
O
para
para
-
-
n.a.
n.a.
b
b
n.a.
n.a.
12
13
O
O
meta
meta
CH3
105 ± 17
8.58 ± 0.35
0.92 ± 0.04
0.08
0.62
1.47 ± 0.11
14
15
O
O
meta
meta
5.78 ± 0.67
1.82 ± 0.05
0.31
2.19
1.08 ± 0.17
2.37 ± 0.05
THA
GAL
-
-
-
-
-
-
0.32 ± 0.01
2.10 ± 0.15
0.04 ± 0.00 c
20.7 ± 1.5
0.12
9.85
a SI: selectivity index for AChE, calculated as IC50 hBuChE/hAChE ratio; b n.a. stands for “not active”, i.e., inhibition
lower than 15% at the highest tested concentration; c IC50(BuChE) = 45.8 ± 0.3 nM.

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Table 2. Inhibitory activities against hAChE and hBuChE, as IC50 values, of compounds 16–18.
IC50 (µM) ± SEM
SI a
Comp.
n
hAChE
hBuChE
16
17
18
0
1
2
6.21 ± 0.32
1.66 ± 0.09
2.46 ± 0.11
20.2 ± 1.7
5.04 ± 0.30
12.4 ± 2.4
3.25
3.04
5.04
a
SI: selectivity index for AChE, calculated as IC50 hBuChE/hAChE ratio.
The synthesis of compounds
4-hydroxyacetophenone was alkylated, in the presence of K2CO3, with the selected bromochloroalkane
to afford the chloroalkyloxy derivatives 24 29, which were then subjected to nucleophilic substitution
by diethylamine in refluxing toluene to give compounds 30 35. The chalcones and 13 15 were
obtained via Claisen-Schmidt condensation of 30 35 with the previously described aldehyde 21 (see
Scheme 1). With the same procedure (Scheme 2, right side), compounds and 12 were obtained
were hydrogenated
2–18 were accomplished as shown in Scheme 2. The 3- or
–
–
3–
5
–
–
2
starting from the selected methoxyacetophenone and 21. Finally, compounds
with Pd/CaCO3 to obtain the final derivatives 16–18.
3–5
The derivatives bearing amines other than diethylamine were prepared according to Scheme 3:
24 was first subjected to Claisen-Schmidt condensation reaction with 21 to give chalcone 36 that, via a
parallel synthesis procedure, was then subjected to nucleophilic substitution reaction by the selected
amine (6–11).
2.2. Inhibition of Cholinesterase Activity
The inhibitory potencies of compounds 1–18 toward human recombinant AChE (hAChE) and
BuChE from human serum (hBuChE) were determined using Ellman’s assay [16] and the IC50 values
were reported in Tables 1 and 2.
Regarding hAChE, the introduction of a methoxy group on the chalcone A ring, independently
of its para or meta position, as in compounds
potency with respect to the benzofuran-based lead compound (about 100
The substitution of the methoxy with a methylendiethylamino group, in compound
35-fold increase in inhibitory activity (2.81 M), leading us to speculate on a positive contribution of
2
and 12, respectively, led to a consistent drop in
M vs 40.7 M [ ], Table 1).
, induced a
µ
µ
9
1
µ
the diethylamino group. An additional structural modification, namely the lengthening of the side
chain from 2 to 4 methylene units, combined with the introduction of an oxygen atom, allowed an
increase in activity of one order of magnitude, as in the para-alkoxymethylendiethylamino derivatives
3
–
5, showing potency in the sub-micromolar range. In this context, the meta-substitution seemed
to negatively affect activity, compounds 13 15 being less effective as AChE inhibitors than the
–
corresponding para-substituted compounds. In this small meta- subset, compound 15, with 4-methylene
units, was the most active.
One of the aims of this paper was the design of structurally simpler and more flexible derivatives
with respect to our previously reported benzofuran-based lead compound, in order to exploit this
increased adaptability in a better interaction with AChE. The results indicate that the introduction of a
para-diethylaminoethoxy group (compound 3) led to a 10-fold increase in potency. Moreover, a positive
contribution of the oxygen atom in the side chain can be recognized, maybe due to its H-bond acceptor
feature, since compound 1, bearing a simple diethylaminomethyl group, proved to be less active.

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Scheme 2. Reagents and conditions: (i) Cl(CH2)nBr, K2CO3, acetone, reflux; (ii) selected amine, toluene,
reflux; (iii) 21, KOH, EtOH, r.t.; (iv) H2, Pd/CaCO3, THF, r.t.
Taking this into account, for a deeper evaluation of the contribution of the terminal amino group
in the interaction with PAS, the ethoxy spacer was maintained and diethylamine was replaced with
different bulkier amines (Table 1).
Surprisingly, among the selected amines, only piperidine (
micromolar inhibitory potency, whereas all the other amino functions led to significantly less active
compounds ( 11). In particular, the morpholino derivative ( ) was about 500-fold worse than
In order to explain this unexpected result, docking studies were performed (see below).
Finally, the unsaturation of derivatives was selectively hydrogenated, aiming at evaluating
its role in the orientation of the molecules. This modification, leading to the more flexible compounds
16 18 (Table 2), allowed maintaining a micromolar inhibitory potency, even if lower with respect
to 3–5.
Regarding hBuChE, the introduction of a methoxy group on the A ring, both in para and in meta
positions, as in compounds and 12, led to a consistent increase in potency with respect to AChE
(3.42 M vs. 98.5 M for and 8.58 M vs. 105 M for 12, Table 1). A similar trend was observed for
the benzofuran-based lead compound (3.42 and 8.58 µM, respectively, and 38.1 µM [9]).
7) enabled the compound to retain a
6
,
8
–
6
7.
α
,
β
3–5
–
2
µ
µ
2
µ
µ

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Scheme 3. Reagents and conditions: (i) 21, KOH, EtOH, r.t.; (ii) selected amine, toluene, reflux.
Differently from what was noticed for hAChE inhibition, the substitution of the methoxy with a
diethylamino group, as in compounds and , allowed maintaining of the activity in the micromolar
1
,
4
5
range, although with different selectivity depending on the position of the chain. Indeed, the subset
bearing the diethylamino group in para position showed a greater affinity for hAChE, while the
meta substituted one proved to be more potent on hBuChE. In this small subset of meta-derivatives,
compound 13, with 2-methylene units, was the most active and the most potent of the whole series on
this enzyme.
As expected, the larger size of the active site gorge in BuChE, due to the presence of the smaller
valine and leucine instead of the aromatic phenylanine residues [17], enabled a better fit of molecules
with bulky substituents, with the exception of compounds 10 and 11, which proved to be inactive on
both ChEs.
To complete the picture, the hydrogenated compounds 16–18 (Table 2) were three/four-fold less
potent on BuChE with respect to AChE.
In a healthy brain, AChE is the major player in the breakdown of the neurotransmitter
acetylcholine (ACh). Conversely, in an AD affected brain, AChE levels progressively decrease,
while BuChE increases and becomes prevailing [18]. As a result, the regulation of ACh central
levels in AD patients likely involves both enzymes, which thus represent validated therapeutic targets
to counteract the cholinergic deficit [19
associated with A plaques, due to its accumulation in these structures, and inhibition of this enzyme
could be responsible for a reduced A deposition in subcortical regions of the brain [21]. In this overall
emerges as the most promising, being an effective low micromolar inhibitor
,20]. Moreover, recent studies indicate that BuChE can be
β
β
framework, compound
of both enzymes.
4
2.3. Docking Studies
To explain the binding mode and the affinities of newly synthesized compounds to target enzymes
AChE and BuChE, docking studies were performed and interactions of compounds and with AChE
4
6

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and BuChE were evaluated. Three-dimensional (3D) and two-dimensional (2D) representations of
the most energetically profitable poses of these compounds docked in the active site of hAChE are
presented in Figure 2.
a
b
c
Figure 2. Docked pose of inhibitors
4
and
6
at the active site of hAChE. (
a) Compounds 4 and 6 are
rendered as ball and sticks ( , carbon atoms in pink;
4
6, carbon atoms in blue) and the side chain
conformations of the mobile residues are illustrated in pink and blue, respectively. The catalytic
triad (CT) is colored in green, the oxyanion hole (OH) in magenta, the anionic sub-site (AS) in
orange, except Trp86, the acyl binding pocket (ABP) in yellow and PAS in light pink and blue;
(b
) 2D-representation of interactions established by
4 with hAChE; (c) 2D-representation of interactions
established by 6 with hAChE.
The most energetically favorable binding mode of compound
4 revealed that the
N-benzylmethylamine moiety binds to the CAS while the diethylamino group is located in the pocket
forming PAS and the linker occupied the center of both cavities (Figure 2a). The AChE-4 complex is
stabilized by π-cation and π-π stacking interactions (Figure 2b).

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Analysis of the optimal binding mode for compound
6
places the ligand within the active site of
AChE. The simulations showed a clear preference to accommodate the N-benzylmethylamine moiety
within the CAS while the morpholine moiety is turned away from the PAS. The key factors to stabilize
the enzyme-ligand complex were found to be
bonding interactions (Figure 2c).
π-cation, π–π T-shaped, π–π stacking and hydrogen
Figure 3 illustrates the docked pose of compound
compound , pointing out to the fact that these two molecules do not share a similar binding mode.
Both compounds and could interact with the catalytic active site with Trp86 and Glu202 residues;
however, compound also interacted in the peripheral anionic site. The higher binding of compound
within AChE binding pocket can be attributed to the binding orientation and geometry permitted by
4 superimposed with the docked pose of
6
4
6
4
4
this ligand, spanning both cavities of AChE (CAS and PAS).
Figure 3. 3D surface representation of compounds 4 (pink), 6 (blue) and hAChE (white).
Compounds
4 and 6 were modeled into the structure of hBuChE (PDB: 4BDS, Figure 4).
Both ligands fit into the active site of hBuChE mainly through
π–
π
and hydrogen bonds interactions,
which are found to be essential for binding. Thus, compound
4
interacts inside the hBuChE binding
cavity in the same region occupied by compound 6. These compounds find interactions in the middle
of the active-site gorge and the N-benzylmethylamine moiety is pointed toward the catalytic triad
residues, His438, Ser198 and Glu325.
Figure 4. Proposed binding mode for compounds 4 and 6 inside the gorge cavity of hBuChE. Compound
4
is colored olive green. Compound 6 is colored pink. Different subsites of the active site were colored:
catalytically anionic site (CAS) in green, oxyanion hole (OH) in red, choline binding site in violet (CBS),
acyl binding pocket (ABP) in yellow, and peripheral site (PAS) in blue.

Molecules 2018, 23, 1902
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As shown in Figure 5, it was found that the N-benzylmethylamine moiety of both ligands
occupied the catalytic site of hBuChE through stacking interactions with Trp231 and Phe329.
Furthermore, the binding in compound is also supported by the formation of a hydrogen bond
π–π
6
between a morpholine oxygen atom of the ligand and Asn289. In addition, the other ammonium groups
are located at the beginning of the gorge. These ligands bind to hAChE with extended conformations,
whereas they bind to hBuChE with folded conformations
Figure 5. 2D Schematic representation of different interactions of compounds
hBuChE (right).
4 (left) and 6 with
2.4. Antioxidant Activity
To deeply investigate the biological profile of the new derivatives, a small subset of compounds
was evaluated to establish their antioxidant potential, which could be associated to the chalcone
scaffold. To this aim, derivatives
2
,
4
and 17 were selected, due to their different structural features:
is a dual AChE/BuChE inhibitor bearing an amino terminal
2
possesses a simple methoxy group,
4
group and 17 is devoid of the chalcone peculiar double bond.
To establish the range of concentrations not associated with neurotoxicity, SH-SY5Y cells viability
was evaluated after a 24 h treatment with compounds
2,
4
and 17 at different concentrations (2.5–80
µM)
using the MTT assay. The obtained results highlighted that concentrations up to 2.5
µM did not
affect neuronal viability (data not shown). Therefore, to perform the subsequent antioxidant and
neuroprotective assays, a concentration of 1.25 µM was selected for all the studied compounds.
To determine the antioxidant activity, the intracellular ROS formation induced by t-BuOOH was
evaluated after a 24 h treatment with compounds 2, 4 and 17 (1.25 µM) using the fluorescent probe
H2DCF-DA. Data showed that the studied compounds prevented ROS formation in the order of
strength 4 > 17 > 2, with inhibition percentages of 27%, 23% and 16%, respectively (Figure 6).
In parallel, glutathione (GSH) levels in SH-SY5Y cells were evaluated after a 24 h treatment
with compounds
evaluation was based on experimental evidence indicating that oxidative stress and aging reduced GSH
levels. Data showed that compounds and 17 significantly increased GSH levels, while compound
decreased GSH basal levels (Figure 7).
2, 4 and 17 (1.25 µM) using the fluorescent probe monochlorobimane (MCB). This
4
2

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Figure 6. Antioxidant activity of compounds 2, 4 and 17 against the ROS formation induced by
t-BuOOH in SH-SY5Y cells. Cells were incubated with compounds and 17 (1.25 M) for 24 h
and then treated with t-BuOOH (100 M) for 30 min. At the end of incubation, intracellular ROS
2,
4
µ
µ
formation was detected using the fluorescent probe H2DCF-DA, as described in the Materials and
Methods section. Data are expressed as antioxidant activity in terms of inhibition percentage in ROS
formation induced by t-BuOOH and are reported as mean ± SEM of three independent experiments.
Figure 7. Effects of compounds
compounds and 17 (1.25 M) for 24 h. At the end of incubation, GSH levels were detected using
the fluorescent probe MCB, as described in the Materials and Methods section. Data are expressed as
concentration of GSH ( M) and reported as mean SEM of three independent experiments (* p < 0.05,
2, 4 and 17 on GSH levels in SH-SY5Y cells. Cells were incubated with
2,
4
µ
µ
±
** p < 0.01 and *** p < 0.001 vs. control at one way ANOVA with Dunnett post hoc test).
2.5. Neuroprotective Activity
It is well documented that soluble Aβ oligomers are neurotoxic species, able to trigger cognitive
deficits also in the absence of plaques. Thus, they can be considered critical factors in the pathogenesis
of AD by causing synaptic dysfunction and neuronal death [22].
The neuroprotective activity toward Aβ1–42 oligomers (OAβ1–42) (10
SH-SY5Y cells was evaluated after 4 h treatment with compounds 17 (1.25
formazan exocytosis assay. As shown in Figure 8, compound partially counteracted the neurotoxic
effects induced by OAβ1–42 increasing SH-SY5Y cells viability, while compound reinforced the
neurotoxic effects induced by OAβ1–42. No neuroprotective effect was observed for compound 17.
µM) induced toxicity in
2,
4,
µM) using the MTT
4
2

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Figure 8. Effects of compounds
cells. Cells were incubated with compounds
2
,
4
and 17 on the neurotoxicity induced by OAβ1–42 in SH-SY5Y
17 (1.25 M) and OAβ1–42 (10 M) for 4 h. At the
2,
4,
µ
µ
end of incubation, the neuroprotective activity of the compounds was detected by the solubilization
of intracellular MTT granules in Tween-20, as described in the Materials and Methods section. Data
are expressed as percentage of neuronal viability and reported as mean
±
SEM of three independent
experiments (§§§ p < 0.001 vs. control, * p < 0.05 and ** p < 0.01 vs. cells treated with OAβ1–42 at one
way ANOVA with Bonferroni post hoc test).
Taken together, the data for antioxidant and neuroprotective activities point at compound
most promising one, being endowed with a better profile than compounds and 17, suggesting that
both the amino terminal group and the chalcone peculiar double bond were crucial structural features
for inducing these effects. Undoubtedly, the -unsaturated carbonyl moiety in compound , acting as
4 as the
2
α
,
β
4
a Michael acceptor, could interfere with Keap1-Nrf2 binding, causing the subsequent activation of
Nrf2 signaling pathway [23]. Indeed, several studies postulate for electrophylic compounds a possible
Cys-based modification of Keap1 allowing its dissociation from Nrf2 and leading to the transcription
of cytoprotective genes [24
the substituents introduced, can be considered an important feature to minimize the risk of off-target
effects. On the other hand, compound 17, only devoid of the double bond with respect to
could still increase the GSH levels, probably acting with a different mechanism.
,25]. In this respect, a fine-tuning of the chalcone electrophilicity, due to
α
,β
4,
In summary, the increased flexibility of these newly synthesized chalcone-based derivatives led
to an improved cholinesterase inhibitory activity with respect to the benzofuran lead compound,
possibly due to a better fit into the catalytic ChEs gorges. On the other hand, the presence of the
distinctive
α
,β-unsaturated carbonyl moiety also allowed introducing an appreciable antioxidant
and neuroprotective potential. In this series, compound
4, endowed with a dual AChE/BuChE low
micromolar inhibitory activity and a relevant antioxidant and neuroprotective profile, emerged as an
effective multipotent molecule, suitable to be further developed in view of the multifaceted character
of AD.
3. Materials and Methods
3.1. Chemistry
General Methods. Melting points were measured in glass capillary tubes on a Büchi
SMP-20 apparatus and are uncorrected. 1H-NMR and 13C-NMR spectra were recorded in CDCl3,
unless otherwise indicated, on a Varian Gemini spectrometer 400 MHz and 101 MHz, respectively.
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). Direct infusion

Molecules 2018, 23, 1902
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ES-MS spectra were recorded on a Waters Micromass ZQ 4000 apparatus (Waters Alliance, San Diego,
CA, USA). Chromatographic separations were performed by flash chromatography on silica gel
columns (Kieselgel 40, 0.0400–0.063 mm; Merck, Darmstadt, Germany). Organic solutions were dried
over anhydrous sodium sulfate. All chemicals were purchased from Aldrich Chemistry, Milan (Italy),
or from Alfa Aesar, Milan (Italy), and were of the highest purity. Compounds were named relying on
the naming algorithm developed by CambridgeSoft Corporation (Waltham, MA, USA) and used in
ChemDraw Professional 15.0 (PerkinElmer Inc., Waltham, MA, USA).
General method for the synthesis of final compounds 1–5, 12–15 and of intermediate 36: To a
mixture of the selected acetophenone (0.001 mol) and 21 (0.001 mol) in EtOH (10 mL), a solution of
KOH (1 g, 0.018 mol, in 5 mL of H2O) was added, and the reaction mixture was stirred at rt overnight.
The mixture was poured into ice and the resulting yellow solid was filtered off. The residue was
purified by flash chromatography on silica gel (toluene/acetone 3:2, then MeOH).
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-((diethylamino)methyl)phenyl)prop-2-en-1-one (
Using the previous procedure and starting from 21 and 23 (yield 55%) was obtained as an oil.
1H-NMR
1.03 (t, J = 7.2 Hz, 6H, 2CH3), 1.25–1.61 (m, 8H, 4CH2), 1.64–1.85 (m, 2H, CH2), 2.19 (s, 3H,
NCH3), 2.35 (t, J = 7.2 Hz, 2H, CH2N), 2.42–2.60 (m, 4H, 2CH2), 3.46 (s, 2H, CH2), 3.62 (s, 2H, NCH2),
3.99 (t, J = 6.8 Hz, 2H, OCH2), 6.86–7.99 (m, 15H, Ar). 13C-NMR
11.88, 26.08, 27.31, 27.42, 29.22, 29.37,
1).
,
1
δ
δ
42.25, 47.05, 57.45, 57.50, 62.32, 68.25, 115.02, 119.81, 127.06, 127.60, 128.31, 128.53, 128.62, 129.00, 129.25,
130.31, 137.30, 139.03, 144.59, 145.49, 161.36, 190.35. MS (ES) m/z: 527 [M + H]+. C35H46N2O2.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (
2
). Using the
previous procedure and starting from 4-methoxyacetophenone and 21
,
2
(yield 75%) was obtained as
1
an oil. H-NMR
δ 1.22–1.59 (m, 8H), 1.64–1.82 (m, 2H, CH2), 2.20 (s, 3H, NCH3), 2.36 (t, J = 7.2 Hz,
2H, CH2N), 3.48 (s, 2H, CH2), 3.88 (s, 3H, OCH3), 4.00 (t, J = 6.8 Hz, 2H, OCH2), 6.88 (m, 3H, Ar),
7.20–7.84 (m, 10H, Ar), 8.02 (d, J = 8.8 Hz, 2H, Ar). 13C-NMR
δ
26.18, 27.35, 27.41, 29.28, 29.43, 42.30,
55.72, 57.46, 62.38, 68.22, 112.94, 114.65, 115.36, 119.18, 119.77, 120.96, 121.11, 127.16, 127.56, 128.34,
128.49, 129.37, 129.64, 129.72, 130.39, 138.96, 140.11, 144.96, 160.13, 161.44, 190.76. MS (ES) m/z: 472
[M + H]+. C31H37NO3.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(2-(diethylamino)ethoxy)phenyl)-prop-2-en-1-one (
3
).
Using the previous procedure and starting from 30 and 21
,
3
(yield 70%) was obtained as a solid,
1
mp 40–41 ◦C (ligroin). H-NMR
δ 1.09 (t, J = 7.2 Hz, 6H, 2CH3), 1.45–1.55 (m, 4H, 2CH2), 1.61–1.82
(m, 2H, CH2), 2.19 (s, 3H, NCH3), 2.37 (t, J = 7.2 Hz, 2H, NCH2), 2.63–2.69 (m, 4H, 2CH2),
2.91 (t, J = 6.4 Hz, 2H, NCH2), 3.48 (s, 2H, CH2), 4.00 (t, J = 6.8 Hz, 2H, OCH2), 4.13 (t, J = 5.6 Hz, 2H,
OCH2), 6.92 (d, J = 8.8 Hz, 2H, Ar), 6.98 (d, J = 9.2 Hz, 2H, Ar), 7.24–7.45 (m, 4H, Ar), 6.92 (d, J = 8.8 Hz,
2H, Ar), 7.59 (d, J = 8.8 Hz, 2H, Ar), 7.78 (d, J = 15.6 Hz, 1H), 8.02 (d, J = 8.8 Hz, 2H, Ar). 13C-NMR
δ
12.37, 26.07, 27.37, 29.20, 29.37, 42.31, 46.51, 46.69, 52.31, 57.51, 62.39, 68.05, 68.24, 113.69, 114.65, 114.84,
115.01, 119.49, 119.75, 120.92, 126.99, 127.49, 127.77, 128.27, 129.18, 129.59, 130.35, 139.19, 140.01, 144.90,
159.40, 161.42, 190.43. MS (ES) m/z: 557 [M + H]+. C36H48N2O3.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(3-(diethylamino)propoxy)phenyl)-prop-2-en-1-one (
Using the previous procedure and starting from 31 and 21 (yield 20%) was obtained as an oil.
1H-NMR
1.01 (t, J = 7.2 Hz, 6H, 2CH3), 1.45–1.58 (m, 4H, 2CH2), 1.61–1.81 (m, 2H, CH2), 1.83–2.01
4).
,
4
δ
(m, 2H, CH2), 2.18 (s, 3H, NCH3), 2.36 (t, J = 7.2 Hz, 2H, NCH2), 2.41–2.65 (m, 6H, 2CH2), 3.45 (s, 2H,
CH2), 3.99 (t, J = 6.8 Hz, 2H, OCH2), 4.10 (t, J = 5.6 Hz, 2H, OCH2), 6.92–7.01 (m, 4H, Ar), 7.19–7.42 (m,
6H, Ar), 7.59 (d, J = 8.8 Hz, 2H, Ar), 7.78 (d, J = 15.6 Hz, 1H), 8.02 (d, J = 8.8 Hz, 2H, Ar). 13C-NMR
δ
11.49, 26.38, 27.51, 28.47, 29.28, 29.45, 42.41, 46.56, 46.82, 52.33, 57.60, 62.96, 68.22, 68.63, 113.74, 114.55,
114.99, 115.36, 119.14, 119.85, 120.92, 126.98, 127.52, 127.78, 128.29, 129.18, 129.61, 130.45, 139.29, 140.22,
144.96, 159.40, 161.02, 190.55. MS (ES) m/z: 571 [M + H]+. HRMS Esi + [M + 1]: calcd for C37H51N2O3,
571.3900. Found: 571.3902.

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3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(4-(diethylamino)butoxy)phenyl)prop-2-en-1-one (
Using the previous procedure and starting from 32 and 21 (yield 25%) was obtained as an
oil. 1H-NMR
1.02 (t, J = 7.2 Hz, 6H, 2CH3), 1.351–1.82 (m, 14H, 7CH2), 2.18 (s, 3H, NCH3),
5).
,
5
δ
2.37 (t, J = 7.2 Hz, 2H, NCH2), 2.42–2.64 (m, 6H, 2CH2), 3.45 (s, 2H, CH2), 3.99 (t, J = 6.8 Hz, 2H,
OCH2), 4.10 (t, J = 5.6 Hz, 2H, OCH2), 6.84–7.03 (m, 4H, Ar), 7.21–7.42 (m, 6H, Ar), 7.60 (d, J = 8.8 Hz,
2H, Ar), 7.78 (d, J = 15.6 Hz, 1H), 8.01 (d, J = 8.8 Hz, 2H, Ar). 13C-NMR
δ 11.34, 23.27, 26.05, 27.37,
27.48, 29.20, 29.45, 42.37, 46.58, 46.71, 52.36, 57.54, 62.42, 68.25, 68.44, 113.66, 114.65, 114.92, 115.03,
119.48, 119.76, 120.92, 126.99, 127.47, 127.79, 128.27, 129.19, 129.61, 130.37, 139.03, 140.34, 144.92, 159.43,
161.42, 190.55. MS (ES) m/z: 585 [M + H]+. C38H52N2O3.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(3-methoxyphenyl)prop-2-en-1-one (12). Using the
previous procedure and starting from 3-methoxyacetophenone and 21, 1
2
(yield 68%) was obtained as
1
an oil. H-NMR
δ 1.24–1.58 (m, 8H), 1.69–1.82 (m, 2H, CH2), 2.19 (s, 3H, NCH3), 2.37 (t, J = 7.2 Hz,
2H, NCH2), 3.44 (s, 2H, CH2), 3.88 (s, 3H, OCH2), 4.00 (t, J = 6.8 Hz, 2H, OCH2), 6.88–7.80 (m, 15H,
Ar). 13C-NMR
δ
26.11, 27.30, 27.45, 29.25, 29.40, 42.23, 55.63, 57.44, 62.30, 68.28, 112.94, 114.68, 115.06,
119.19, 119.79, 120.96, 121.09, 127.11, 127.53, 128.34, 128.48, 129.30, 129.64, 129.70, 130.39, 138.96, 140.11,
144.98, 160.01, 161.47, 190.46. MS (ES) m/z: 472 [M + H]+. C31H37NO3.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(3-(2-(diethylamino)ethoxy)phenyl)-prop-2-en-1-one (13).
Using the previous procedure and starting from 33 and 21, 1
1H-NMR
1.10 (t, J = 7.2 Hz, 6H, 2CH3), 1.20–1.90 (m, 10H, 5CH2), 2.20 (s, 3H, NCH3), 2.38 (t, J = 7.2 Hz
2H, NCH2), 2.60–2.73 (m, 4H, 2CH2), 2.90 (t, J = 6.4 Hz, 2H, NCH2), 3.50 (s, 2H, CH2), 4.01 (t, J = 6.8 Hz
3 (yield 20%) was obtained as an oil.
δ
,
,
2H, OCH2), 4.10 (t, J = 5.4 Hz, 2H, OCH2), 6.98 (d, J = 8.8 Hz, 2H, Ar), 7.15–7.82 (m, 13H, Ar). 13C-NMR
δ
12.37, 26.07, 27.37, 29.20, 29.37, 42.31, 46.51, 46.69, 52.31, 57.51, 62.39, 68.05, 68.24, 113.69, 114.65,
114.84, 115.01, 119.49, 119.75, 120.92, 126.99, 127.49, 127.77, 128.27, 129.18, 129.59, 130.35, 139.19, 140.01,
144.90, 159.40, 161.42, 190.43. MS (ES) m/z: 557 [M + H]+. HRMS Esi + [M + 1]: calcd for C36H49N2O3,
557.3743. Found: 557.3741.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(3-(3-(diethylamino)propoxy)phenyl)-prop-2-en-1-one
(
14). Using the previous procedure and starting from 34 and 21, 1
1H-NMR
1.01 (t, J = 7.2 Hz, 6H, 2CH3), 1.20–1.81 (m, 10H, 5CH2), 1.82–2.01 (m, 2H, CH2), 2.18 (s, 3H,
NCH3), 2.36 (t, J = 7.2 Hz, 2H, NCH2), 2.41–2.66 (m, 6H, 3CH2), 3.45 (s, 2H, CH2), 3.95 (t, J = 6.8 Hz,
2H, OCH2), 4.05 (t, J = 5.8 Hz, 2H, OCH2), 6.79–7.80 (m, 15H, Ar). 13C-NMR
11.47, 26.35, 27.37, 28.01,
4 (yield 45%) was obtained as an oil.
δ
δ
29.24, 29.37, 42.38, 46.51, 46.76, 52.29, 57.52, 62.93, 68.15, 68.24, 113.55, 114.47, 114.91, 115.19, 119.04,
119.75, 120.92, 126.99, 127.49, 127.77, 128.27, 129.18, 129.59, 130.38, 139.19, 140.14, 144.90, 159.40, 160.98,
190.36. MS (ES) m/z: 571 [M + H]+. C37H50N2O3.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(4-(diethylamino)butoxy)phenyl)prop-2-en-1-one (15).
Using the previous procedure and starting from 35 and 21, 1
1H-NMR
1.02 (t, J = 7.2 Hz, 6H, 2CH3), 1.22–1.60 (m, 10H, 5CH2), 1.61–1.90 (m, 4H, 2CH2), 2.19 (s, 3H,
NCH3), 2.38 (t, J = 7.2 Hz, 2H, NCH2), 2.42–2.59 (m, 6H, 3CH2), 3.42 (s, 2H, CH2), 3.99–4.13 (m, 4H,
2OCH2), 6.85 (d, J = 8.8 Hz, 2H, Ar), 7.10–7.82 (m, 13H, Ar). 13C-NMR
11.35, 23.21, 26.07, 27.37, 27.41,
5 (yield 55%) was obtained as an oil.
δ
δ
29.20, 29.37, 42.31, 46.51, 46.69, 52.31, 57.51, 62.39, 68.05, 68.24, 113.69, 114.65, 114.84, 115.01, 119.49,
119.75, 120.92, 126.99, 127.49, 127.77, 128.27, 129.18, 129.59, 130.35, 139.19, 140.01, 144.90, 159.40, 161.42,
190.43. MS (ES) m/z: 585 [M + H]+. C38H52N2O3.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(2-chloroethoxy)phenyl)prop-2-en-1-one (36). Using
the previous procedure and starting from 24 and 21
1.22–1.80 (m, 10H), 2.18 (s, 3H), 2.35 (t, J = 7.2 Hz, 2H), 3.45 (s, 2H), 3.80 (t, 2H), 3.99 (t, 2H), 4.30 (t, 2H)
6.85–7.80 (m, 13H, Ar), 8.02 (d, 2H).
,
36 (yield 77%) was obtained as yellow oil. 1H-NMR
δ
,
General method for the synthesis of final compounds
and the selected amine (1 mmol) in toluene (50 mL) was refluxed for 24 h. The mixture was washed
with water (3 25 mL) and the organic layer was dried. The solvent was removed under reduced
6–11: A stirred solution of 36 (0.5 mmol)
×

Molecules 2018, 23, 1902
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pressure and the residue was purified by flash chromatography on silica gel (toluene/acetone 4:1, then
only MeOH).
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(2-morpholinoethoxy)phenyl)prop-2-en-1-one
Using the previous procedure and starting from morpholine, (yield 15%) was obtained as an oil.
1H-NMR
1.25–1.90 (m, 10H, 5CH2), 2.18 (s, 3H, NCH3), 2.35 (t, J = 7.2 Hz, 2H, NCH2), 2.43–2.61
(m, 4H, 2NCH2), 2.83 (t, 2H, NCH2), 3.42 (s, 2H, CH2), 3.70–3.80 (m, 4H, 2OCH2), 3.99 (t, J = 6.8 Hz,
2H, OCH2), 4.15 (t, J = 6.8 Hz, 2H, OCH2), 6.80–8.03 (m, 15H, Ar). 13C-NMR
26.42, 27.33, 27.45, 29.25,
(6).
6
δ
δ
29.40, 42.32, 56.21, 57.54, 57.56, 57.86, 62.38, 66.71, 66.73, 67.98, 68.26, 114.47, 115.21, 114.84, 119.49,
119.55, 127.16, 127.74, 127.77, 128.37, 129.28, 130.24, 130.82, 138.69, 140.34, 144.23, 159.62, 162.65, 189.97.
MS (ES) m/z: 571 [M + H]+. HRMS Esi + [M + 1]: calcd for C36H47N2O4, 571.3536. Found: 571.3535.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(2-(piperidin-1-yl)ethoxy)phenyl)prop-2-en-1-one (
7
).
Using the◦previous procedure and starting from piperidine,
7 (yield 95%) was obtained as a solid,
1.20–1.90 (m, 16H, 8CH2), 2.18 (s, 3H, NCH3), 2.33 (t, J = 7.2 Hz, 2H,
1
mp 58–59 C (ligroin). H-NMR
δ
NCH2), 2.41–2.59 (m, 4H, 2NCH2), 2.79 (t, J = 6.8 Hz, 2H, NCH2), 3.43 (s, 2H, CH2), 3.99 (t, J = 6.8 Hz
2H, OCH2), 4.18 (t, J = 6.8 Hz, 2H, OCH2), 6.82–7.90 (m, 13H, Ar), 8.01 (d, J = 8.8 Hz, 2H, Ar). 13C-NMR
24.26, 26.02, 26.12, 27.01, 27.39, 27.45, 29.26, 29.41, 42.34, 55.21, 57.54, 57.86, 62.41, 66.35, 68.26, 114.49,
,
δ
115.01, 114.84, 119.49, 119.54, 127.06, 127.74, 127.77, 128.33, 129.24, 130.24, 130.80, 138.69, 140.11, 144.03,
159.61, 162.65, 188.91. MS (ES) m/z: 569 [M + H]+. HRMS Esi + [M + 1]: calcd for C37H49N2O3,
569.3743. Found: 569.3744.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(2-(3,4-dihydroisoquinolin-2(1H)-yl)ethoxy)phenyl)
prop-2-en-1-one (
8
8
). Using the previous procedure and starting from 1,2,3,4-tetrahydroisoquinoline,
(yield 30%) was obtained as an oil. 1H-NMR
δ
1.22–1.80 (m, 10H, 5CH2), 2.18 (s, 3H, NCH3),
2.31 (t, J = 7.2 Hz, 2H, NCH2), 2.80–3.02 (m, 6H, 3NCH2), 3.45 (s, 2H, CH2), 3.79 (s, 2H, CH2),
3.99 (t, J = 6.8 Hz, 2H, OCH2), 4.26 (t, J = 6.6 Hz, 2H, OCH2), 6.86–7.85 (m, 17H, Ar), 8.03 (d, J = 8.8 Hz,
2H, Ar). 13C-NMR
δ 25.91, 26.74, 27.22, 29.06, 29.17, 41.74, 51.39, 56.38, 57.01, 61.83, 67.04, 68.07, 114.18,
114.35, 119.29, 125.68, 126.25, 126.57, 126.95, 127.04, 127.27, 128.29, 129.37, 130.11, 130.68, 130.73, 131.81,
132.17, 138.27, 145.19, 158.42, 162.20, 192.42. MS (ES) m/z: 617 [M + H]+. C41H48N2O3.
1-(1-(2-(4-(3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)acryloyl)phenoxy)ethyl)-piperidin-4-yl)-1,3-
dihydro-2H-benzo[d]imidazol-2-one (
-1,3-dihydro-2H-benzo[d]imidazol-2-one,
9
). Using the previous procedure and starting from 1-(piperidin-4-yl)
1
9
(yield 70%) was obtained as an oil. H-NMR
δ
1.20–1.60 (m,
8H, 4CH2), 1.60–1.95 (m, 2H, CH2), 2.18 (s, 3H, NCH3), 2.24–2.60 (m, 4H, 2NCH2), 2.90 (t, J = 6.8 Hz
2H, NCH2), 3.10–3.20 (m, 2H, NCH2), 3.43 (s, 2H, CH2), 3.99 (t, J = 6.8 Hz, 2H, OCH2), 4.20 (t, J = 6.8 Hz
,
,
2H, OCH2), 4.25–4.38 (m, 1H), 6.82–7.82 (m, 17H, Ar), 8.02 (d, J = 8.8 Hz, 2H, Ar), 9.10 (br, 1H, NH).
13C-NMR
26.02, 26.18, 27.11, 27.38, 27.46, 29.26, 29.41, 42.34, 55.21, 57.54, 57.86, 59.01, 62.41, 66.34,
δ
68.26, 111.76, 114.49, 115.13, 114.84, 119.49, 119.54, 124.51, 124.56, 126.81, 127.06, 127.74, 127.77, 128.33,
128.89, 129.24, 130.24, 130.80, 138.69, 140.11, 144.03, 152.62, 159.61, 162.65, 188.91. MS (ES) m/z: 701
[M + H]+. C44H52N4O4.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(2-(4-phenylpiperidin-1-yl)ethoxy)-phenyl)prop-2-en-
1-one (10). Using the previous procedure and starting from phenylpiperidine, 10 (yield 65%) was
1
obtained as a solid, mp 81–82 ◦C (ligroin). H-NMR
δ 1.20–1.61 (m, 10H, 5CH2), 1.62–1.97 (m, 4H,
2CH2), 2.18 (s, 3H, NCH3), 2.20–2.61 (m, 5H), 2.90 (t, J = 6.8 Hz, 2H, NCH2), 3.05–3.20 (m, 2H), 3.46 (s,
2H, CH2), 3.99 (t, J = 6.8 Hz, 2H, OCH2), 4.20 (t, J = 6.8 Hz, 2H, OCH2), 6.86–7.82 (m, 18H, Ar). 8.02 (d,
J = 8.8 Hz, 2H, Ar). 13C-NMR
δ 26.18, 27.11, 27.46, 29.26, 29.41, 30.21, 30.26, 42.07, 42.44, 55.21, 57.54,
57.86, 62.41, 66.34, 68.26, 111.76, 114.49, 115.13, 114.84, 119.49, 119.54, 124.51, 124.56, 126.81, 127.06,
127.74, 127.77, 128.36, 128.89, 129.24, 130.25, 130.80, 138.69, 140.11, 144.03, 159.66, 162.65, 189.75. MS
(ES) m/z: 645 [M + H]+. C43H52N2O3.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(2-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)ethoxy)
phenyl)prop-2-en-1-one (11). Using the previous procedure and starting from 4-(4-chlorophenyl)

Molecules 2018, 23, 1902
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1
piperidin-4-ol, 11 (yield 15%) was obtained as an oil. H-NMR
δ
1.20–1.61 (m, 10H, 5CH2), 1.62–1.90
(m, 4H, 2CH2), 2.05–2.15 (m, 2H), 2.18 (s, 3H, NCH3), 2.30 (t, J = 6.8 Hz, 2H, NCH2), 2.54–2.61 (m, 2H),
2.91 (t, 2H, NCH2), 3.41 (s, 2H, CH2), 3.99 (t, 2H, OCH2), 4.20 (t, 2H, OCH2), 6.86–7.79 (m, 17H, Ar),
8.02 (d, J = 8.8 Hz, 2H, Ar). 13C-NMR
δ 26.78, 27.12, 27.48, 29.26, 29.44, 30.24, 30.30, 42.44, 55.28, 57.53,
57.87, 62.41, 66.37, 68.26, 69.88, 111.66, 114.51, 115.14, 114.84, 119.49, 119.54, 124.51, 124.56, 127.06,
127.74, 127.77, 128.36, 128.89, 129.24, 130.25, 130.80, 131.63, 138.69, 140.11, 144.03, 159.66, 162.65, 189.75.
MS (ES) m/z: 696 [M + H]+. C43H51ClN2O4.
General method for the synthesis of final compounds 16–18: A solution of 2 or 3 or 4 (0.1 mmol) in
THF (50 mL) was hydrogenated at room temperature and pressure over Pd/CaCO3. The solution was
filtered from catalyst and evaporated to dryness. The residue was purified by flash chromatography
on silica gel (only MeOH).
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(2-(diethylamino)ethoxy)phenyl)-propan-1-one (16).
1
Using the previous procedure and starting from
2,
16 (yield 95%) was obtained as an oil. H-NMR
δ
1.05 (t, J = 7.2 Hz, 6H, 2CH3), 1.20–1.61 (m, 8H, 4CH2), 1.63–1.82 (m, 2H, CH2), 2.19 (s, 3H, NCH3),
2.38 (t, J = 7.2 Hz, 2H, NCH2), 2.58–2.69 (m, 4H, 2CH2), 2.85–3.05 (m, 4H, 2CH2), 3.18 (t, J = 8.6 Hz, 2H,
NCH2), 3.46 (s, 2H, CH2), 3.97 (t, J = 8.6 Hz, 2H, OCH2), 4.15 (t, J = 8.6 Hz, 2H, OCH2), 6.63 (s, 1H),
6.80–7.35 (m, 10H, Ar), 7.98 (d, J = 8.8 Hz, 2H, Ar). 13C-NMR
δ 12.37, 26.07, 27.37, 29.20, 29.37, 35.08,
40.65, 42.31, 46.51, 46.69, 52.31, 57.51, 62.39, 68.05, 68.24, 113.69, 114.65, 114.84, 115.01, 119.49, 119.75,
126.99, 127.49, 127.77, 128.27, 129.18, 129.59, 130.35, 139.19, 140.01, 159.40, 161.42, 197.83. MS (ES) m/z:
560 [M + H]+. C36H50N2O3.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(3-(diethylamino)propoxy)phenyl)-propan-1-one (17).
1
Using the previous procedure and starting from
3,
17 (yield 95%) was obtained as an oil. H-NMR
δ
1.01
(t, J = 7.2 Hz, 6H, 2CH3), 1.20–1.61 (m, 8H, 4CH2), 1.63–1.81 (m, 2H, CH2), 1.85–2.02 (m, 2H, CH2), 2.18
(s, 3H, NCH3), 2.35 (t, J = 7.2 Hz, 2H, NCH2), 2.41–2.64 (m, 6H), 2.99 (t, J = 8.6 Hz, 2H), 3.19 (t, J = 8.6 Hz
,
2H), 3.45 (s, 2H, CH2), 3.90–4.08 (m, 4H, 2OCH2), 6.78–7.83 (m, 10H, Ar), 7.95 (d, J = 12.6 Hz, 2H),
8.02 (d, J = 8.8 Hz, 1H, Ar). 13C-NMR
δ 11.37, 26.05, 27.37, 27.84, 29.24, 29.37, 35.15, 40.77, 42.38,
46.51, 46.76, 52.29, 57.52, 62.41, 68.15, 68.26, 113.55, 114.47, 114.91, 115.19, 119.04, 119.75, 126.99, 127.49,
127.77, 128.27, 129.18, 129.59, 130.41, 139.24, 140.14, 159.40, 160.98, 197.86. MS (ES) m/z: 573 [M + H]+.
C37H52N2O3.
3-(4-((7-(Benzyl(methyl)amino)heptyl)oxy)phenyl)-1-(4-(4-(diethylamino)butoxy)phenyl)-propan-1-one (18).
1
Using the previous procedure and starting from
4,
18 (yield 90%) was obtained as an oil. H-NMR
δ
1.05 (t, J = 7.2 Hz, 6H, 2CH3), 1.20–1.85 (m, 14H, 7CH2), 2.19 (s, 3H, NCH3), 2.38 (t, 2H, NCH2),
2.58–2.69 (m, 6H), 2.95–3.05 (m, 2H), 3.18–3.24 (m, 2H), 3.46 (s, 2H, CH2), 4.00 (t, J = 8.6 Hz, 2H, OCH2),
4.11 (t, J = 8.6 Hz, 2H, OCH2), 6.80–7.21 (m, 11H, Ar), 7.98 (d, J = 8.8 Hz, 2H, Ar). 13C-NMR
11.69,
δ
23.21, 26.17, 27.32, 29.46, 29.65, 35.04, 40.54, 42.39, 46.89, 46.69, 52.57, 57.63, 62.47, 68.12, 68.24, 113.72,
114.31, 114.67, 115.22, 119.39, 119.65, 126.98, 127.51, 127.84, 128.30, 129.20, 129.42, 130.43, 139.29, 140.11,
156.20, 160.42, 198.22. MS (ES) m/z: 587 [M + H]+. C38H54N2O3.
4-(7-Bromoheptyloxy)benzonitrile (19). A stirred mixture of 4-hydroxybenzonitrile (2 g, 0.017 mol),
1,7-dibromoheptane (4.3 mL, 0.025 mol) and K2CO3 (4 g) was refluxed in acetone (150 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 19 (1.41 g, 70%). mp 47–49 C (ligroin). 1H-NMR
δ 1.46–1.98 (m, 10H),
3.41 (t, J = 6.8 Hz, 2H), 3.99 (t, J = 6.8 Hz, 2H), 6.5 (d, J = 8.8 Hz, 2H, Ar), 7.61 (d, 2H, J = 6.8 Hz, Ar).
4-((7-(Benzyl(methyl)amino)heptyl)oxy)benzonitrile (20). A stirred solution of 19 (1.14 g, 3.85 mmol),
N-benzyl-N-methylamine (1.5 mL, 3.8 mmol) and TEA (0.4 mL, 3.8 mmol) in toluene (100 mL) was
refluxed for 20 h. The mixture was washed with water (3
dried. The solvent was removed under reduced pressure and the residue was purified by flash
×
25 mL) and the organic layer was
chromatography on silica gel (petroleum ether/ethyl acetate 9:1), affording 20 as an oil (0.9 g, 70%).

Molecules 2018, 23, 1902
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1H-NMR
δ 1.30–1.58 (m, 8H), 1.71–1.82 (m, 2H), 2.18 (s, 3H), 2.37 (t, J = 7.2 Hz, 2H), 3.45 (s, 2H),
3.97 (t, J = 6.8 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H, Ar), 7.19–7.30 (m, 5H, Ar), 7.55 (d, J = 8.8 Hz, 2H, Ar).
4-((7-(Benzyl(methyl)amino)heptyl)oxy)benzaldehyde (21). A mixture of 20 (2.18 g, 6.48 mmol) and
Ni/Raney alloy (3.06 g, 13 mmol) in 75% HCOOH (44 mL) was refluxed for 7 h, and then hot filtered.
The residue was diluted with 100–150 mL of water, basified by K2CO3 and extracted with DCM
(
3 × 25 mL). The combined organic extracts were dried, and concentrated under reduced pressure to
afford 21 (1.9 g, 90%) as yellow oil (purified by flash chromatography with toluene/acetone 9:1 as
eluent). H-NMR
1
δ
1.22–1.59 (m, 8H), 1.65–1.83 (m, 2H), 2.18 (s, 3H), 2.38 (t, J = 7.2 Hz, 2H), 3.43 (s, 2H)
,
4.01 (t, J = 6.8 Hz, 2H), 6.94–6.99 (m, 2H, Ar), 7.18–7.35 (m, 5H, Ar), 7.79 (d, J = 8.8 Hz, 2H, Ar),
9.85 (broad, 1H).
1-(4-(Bromomethyl)phenyl)ethan-1-one (22). A mixture of 4-methylacetophenone (2 g, 0.015 mol),
N-bromosuccinimide (NBS, 2.66 g, 0.015 mol) and a catalytic amount of benzoyl peroxide in CCl4 was
refluxed for 4 h. The mixture was hot filtered and evaporated to dryness to afford 22 (2.9 g, 95%) as
1
brown oil. H-NMR
δ 2.51 (s, 3H), 4.52 (s, 2H), 7.40 (d, J = 8.8 Hz, 2H, Ar), 7.91 (d, J = 8.8 Hz, 2H, Ar).
1-(4-((Diethylamino)methyl)phenyl)ethan-1-one (23). A stirred solution of 22 (1.5 g, 0.007 mol) and
diethylamine (2.2 mL, 0.021 mol) in toluene (30 mL) was refluxed for 24 h. The mixture was washed
with water (3
pressure and the residue was purified by flash chromatography on silica gel (petroleum ether/ethyl
acetate 9:1), affording 23 as an oil used for the next step without further purification. 1H-NMR
×
25 mL) and the organic layer was dried. The solvent was removed under reduced
δ
1.01 (t, J = 6.8 Hz, 6H), 2.43–2.59 (m, 7H), 3.60 (s, 2H), 7.40 (d, J = 8.8 Hz, 2H, Ar), 7.90 (d, J = 8.8 Hz,
2H, Ar).
General method for the synthesis of compounds 24
–
29
:
A stirred mixture of
4-hydroxyacetophenone (0.022 mol), selected bromochloroalkane (0.044 mol) and K2CO3 (6 g)
was refluxed in acetone (150 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 and purified by flash chromatography on
silica gel (petroleum ether/ethyl acetate 9:1).
1-(4-(2-Chloroethoxy)phenyl)ethan-1-one (24). Using the previous procedure and starting from
◦
1
1-bromo-2-chloroethane, 24 (yield 50%) was obtained as a white solid, mp 59–60 C (ligroin). H-NMR
2.58 (s, 3H), 3.85 (t, J = 6.8 Hz, 2H), 4.25 (t, 2H), 6.95 (d, J = 8.8 Hz, 2H, Ar), 7.95 (d, J = 8.8 Hz, 2H, Ar).
δ
1-(4-(3-Chloropropoxy)phenyl)ethan-1-one (25). Using the previous procedure and starting from
◦
1-chloro-3-bromopropane, 25 (yield 78%) was obtained as a white solid, mp 25–26 C (ligroin). 1H-NMR
δ
2.11–2.35 (m, 2H), 2.57 (s, 3H), 3.72 (t, J = 6.8 Hz, 2H), 4.18 (t, J = 6.8 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H,
Ar), 7.95 (d, J = 8.8 Hz, 2H, Ar).
1-(4-(4-Chlorobutoxy)phenyl)ethan-1-one (26). Using the previous procedure and starting from
◦
1
1-chloro-4-bromobutane, 26 (yield 80%) was obtained as a white solid, mp 39–40 C (ligroin). H-NMR
1.98–2.05 (m, 4H), 2.58 (s, 3H), 3.62 (t, J = 6.8 Hz, 2H), 4.17 (t, J = 6.8 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H,
δ
Ar), 7.91 (d, J = 8.8 Hz, 2H, Ar).
1-(3-(2-Chloroethoxy)phenyl)ethan-1-one (27). Using the previous procedure and starting from
1
3-hydroxyacetophenone and 1-bromo-2-chloroethane, 27 (yield 45%) was obtained as an oil. H-NMR
δ 2.59 (s, 3H), 3.81 (t, J = 6.8 Hz, 2H), 4.27 (t, J = 6.8 Hz, 2H), 7.05–7.60 (m, 4H, Ar).
1-(3-(3-Chloropropoxy)phenyl)ethan-1-one (28). Using the previous procedure and starting from
3-hydroxyacetophenone and 1-chloro-3-bromopropane, 28 (yield 70%) was obtained as yellow oil.
1H-NMR
(m, 4H, Ar).
δ 2.12–2.36 (m, 2H), 2.57 (s, 3H), 3.71 (t, J = 6.8 Hz, 2H), 4.17 (t, J = 6.8 Hz, 2H), 7.05–7.58

Molecules 2018, 23, 1902
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1-(3-(4-Chlorobutoxy)phenyl)ethan-1-one (29). Using the previous procedure and starting from
3-hydroxyacetophenone and 1-chloro-4-bromobutane, 29 (yield 50%) was obtained as a white solid,
1
mp 25–26 ◦C (ligroin). H-NMR
δ 1.98–2.05 (m, 4H), 2.58 (s, 3H), 3.60 (t, J = 6.8 Hz, 2H), 4.06 (t, J = 6.8
Hz, 2H), 7.06–7.56 (m, 4H, Ar).
General method for the synthesis of compounds 30
chloroderivative (6.5 mmol) and diethylamine (13 mmol) in toluene (100 mL) was refluxed for 24 h.
The mixture was washed with water (3 25 mL) and the organic layer was dried. The solvent was
–
35
.
A stirred solution of selected
×
removed under reduced pressure and the residue was purified by flash chromatography on silica gel
(toluene/acetone 4:1).
1-(4-(2-(Diethylamino)ethoxy)phenyl)ethan-1-one (30). Using the previous procedure and starting from
30 (yield 40%) was obtained as an oil. 1H-NMR
24, δ 1.03 (t, J = 7.2 Hz, 6H), 2.48–2.70 (m, 7H),
2.89 (t, J = 6.8 Hz, 2H), 4.05 (t, J = 6.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H, Ar), 7.90 (d, J = 8.8 Hz, 2H, Ar).
1-(4-(3-(Diethylamino)propoxy)phenyl)ethan-1-one (31). Using the previous procedure and starting from
1
25
,
31 (yield 70%) was obtained as an oil. H-NMR
δ
1.03 (t, J = 7.2 Hz, 6H), 1.85–2.02 (m, 2H), 2.45–2.64
(m, 9H), 4.09 (t, J = 6.8 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H, Ar), 7.90 (d, J = 8.8 Hz, 2H, Ar).
1-(4-(4-(Diethylamino)butoxy)phenyl)ethan-1-one (32). Using the previous procedure and starting from 26
,
1
32 (yield 90%) was obtained as an oil. H-NMR
δ
1.03 (t, J = 7.2 Hz, 6H), 1.61–1.89 (m, 4H), 2.43–2.61
(m, 9H), 4.02 (t, J = 6.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H, Ar), 7.90 (d, J = 8.8 Hz, 2H, Ar).
1-(3-(2-(Diethylamino)ethoxy)phenyl)ethan-1-one (33). Using the previous procedure and starting from
1
27
,
33 (yield 40%) was obtained as yellow oil. H-NMR
δ
1.06 (t, J = 7.2 Hz, 6H), 2.59–2.72 (m, 7H),
2.95 (t, J = 6.8 Hz, 2H), 4.08 (t, J = 6.8 Hz, 2H), 7.08–7.58 (m, 4H, Ar).
1-(3-(3-(Diethylamino)propoxy)phenyl)ethan-1-one (34). Using the previous procedure and starting from
1
28
,
34 (yield 90%) was obtained as an oil. H-NMR
δ
1.02 (t, J = 7.2 Hz, 6H), 1.88–1.99 (m, 2H), 2.58–2.64
(m, 9H), 4.01 (t, J = 6.8 Hz, 2H), 7.06–7.55 (m, 4H, Ar).
1-(3-(4-(Diethylamino)butoxy)phenyl)ethan-1-one (35). Using the previous procedure and starting from 29
,
1
35 (yield 50%) was obtained as an oil. H-NMR
δ
1.01 (t, J = 7.2 Hz, 6H), 1.52–1.90 (m, 4H), 2.40–2.60
(m, 9H), 4.01 (t, J = 6.8 Hz, 2H), 7.07–7.57 (m, 4H, Ar).
3.2. Inhibition of Human Cholinesterases
The capacity of 1–18 to inhibit human cholinesterase activity was assessed using the Ellman’s
assay [16]. The assays were performed on a Jasco V-530 double beam spectrophotometer connected
to a HAAKE DC30 thermostating system (Thermo Haake, Karlsruhe, Germany). Stock solutions of
the tested compound (2 mM) were prepared in methanol and diluted in methanol. The assay solution
0
consisted of a 0.1 M phosphate buffer, pH 8.0, with the addition of 340
µ
M 5,5 -dithiobis(2-nitrobenzoic
acid), 0.02 unit/mL human recombinant AChE or BuChE from human serum (Sigma-Aldrich, Milan,
Italy), and 550 M substrate, i.e., acetylthiocholine iodide or butyrylthiocholine iodide, respectively
µ
(Sigma-Aldrich). Inhibitors were added to the assay solution at increasing concentrations and
◦
preincubated at 37 C with the enzyme for 20 min before the addition of substrate. The rate of
absorbance increase at 412 nm was followed for 3 min. In parallel, blanks containing all components
except the enzyme were prepared to account for the non-enzymatic hydrolysis of the substrate.
The reaction rates were compared and the percent inhibition due to the presence of the tested compound
was calculated. Each concentration was analyzed at least in duplicate. Inhibition plots for each
compound were obtained by plotting the % inhibition versus the logarithm of inhibitor concentration
in the assay solution. The linear regression parameters were determined for each curve and the IC50
was extrapolated. For each tested compound two independent assessments of the IC50 value were
carried out.

Molecules 2018, 23, 1902
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3.3. Cell Culture
Human neuronal (SH-SY5Y) cells were routinely grown in Dulbecco’s modified Eagle’s Medium
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin and 50
streptomycin at 37 ◦C in a humidified incubator with 5% CO2.
µg/mL
3.4. Determination of Neurotoxicity
To establish the range of concentrations not associated with neurotoxicity, SH-SY5Y cells were
seeded in a 96-well plate at 2
104 cells/well, incubated for 24 h and subsequently treated with
different concentrations of compounds and 17 (2.5–80
M) for 24 h at 37 ◦C in 5% CO2. Neuronal
×
2,
4
µ
cells viability, in terms of mitochondrial metabolic function, was evaluated by the reduction of
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to its insoluble formazan,
as previously described [26]. Briefly, the treatment medium was replaced with MTT in Hank’s
Balanced Salt Solution (HBSS) (0.5 mg/mL) for 2 h at 37 ◦C in 5% CO2. After washing with HBSS,
formazan crystals were dissolved in isopropanol. The amount of formazan was measured (570 nm,
reference filter 690 nm) using a multilabel plate reader (VICTOR
™ X3, PerkinElmer, Waltham, MA,
USA). The quantity of formazan was directly proportional to the number of viable cells.
3.5. Determination of Antioxidant Activity
The intracellular antioxidant activity of the studied compounds was evaluated in SH-SY5Y cells
as previously described [27]. Briefly, SH-SY5Y cells were seeded in a 96-well plate at 2
and incubated for 24 h at 37 ◦C in 5% CO2. Subsequently, SH-SY5Y cells were incubated for 24 h with
compounds and 17 (1.25 M). At the end of incubation, the treatment medium was removed and
100 g/mL),
L of a fluorescent probe, 20-70 dichlorodihydrofluorescein diacetate (H2DCF-DA) (10
was added to each well. After 30 min of incubation at room temperature, H2DCF-DA solution was
replaced with a solution of tert-butyl hydroperoxide (t-BuOOH) (100 M) for 30 min. The reactive
oxygen species (ROS) formation was measured (excitation at 485 nm and emission at 535 nm) using a
multilabel plate reader (VICTOR X3, PerkinElmer). The antioxidant activity in terms of inhibition
percentage in ROS formation induced by t-BuOOH, is calculated using the following formula:
×
104 cells/well
2
,
4
µ
µ
µ
µ
™
ꢀ
ꢁ
Ptc × 100
% of inhibition = 100 −
(1)
Pt
where Ptc = % of increase in ROS formation induced by t-BuOOH in the presence of the studied
compounds; Pt = % of increase in ROS formation induced by t-BuOOH.
3.6. Determination of Glutathione Levels
The GSH levels were evaluated in SH-SY5Y cells as previously described [28]. SH-SY5Y cells
◦
were seeded in a black 96-well plate at 2
CO2. Subsequently, SH-SY5Y cells were incubated for 24 h with compounds
×
104 cells/well and incubated for 24 h at 37 C in 5%
2
,
4
and 17 [1.25
µM].
At the end of incubation, the treatment medium was removed, and 100
µL of a fluorescent probe,
monochlorobimane (MCB), was added to each well. After 30 min of incubation at 37 ◦C in 5% CO2,
the GSH levels were measured (excitation at 355 nm and emission at 460 nm) using a multilabel plate
reader (VICTOR
™ X3, PerkinElmer). Data are expressed as concentration of GSH (µM) obtained by a
GSH standard curve.
3.7. Aβ1–42 Oligomers Preparation for the Determination of Neuroprotective Activity
A
β1–42 peptide was first dissolved in 1,1,1,3,3,3,-hexafluoroisopropanol to 1 mg/mL, sonicated,
incubated at room temperature for 24 h and lyophilized. The resulting unaggregated Aβ1–42 peptide
film was dissolved with DMSO and stored at
20 ◦C until use. The Aβ1–42 peptide aggregation to
oligomeric form was prepared as previously described [29].
−

Molecules 2018, 23, 1902
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3.8. Determination of Neuroprotective Activity toward Aβ1–42 Oligomers
To evaluate the neuroprotective activity of the studied compounds, SH-SY5Y cells were seeded in
a 96-well plate at 3
104 cells/well, incubated for 24 h and subsequently treated with Aβ1–42 oligomers
(OAβ1–42) [10 M] in the presence of compounds and 17 [1.25 M] for 4 h. The neuroprotective
×
µ
2,
4
µ
activity, in terms of increase in intracellular MTT granules, was measured by MTT formazan exocytosis
assay, as previously described [30]. Briefly, the treatment medium was replaced with MTT in HBSS
◦
(
0.5 mg/mL) for 1 h at 37 C in 5% CO2. After the incubation, intracellular MTT granules were
completely solubilized in Tween-20 (10% v/v). The absorbance of Tween-20 soluble MTT was measured
at 570 nm (reference filter 690 nm) using a multilabel plate reader (VICTOR
Data are expressed as percentage of neuronal viability versus control.
™ X3, PerkinElmer).
3.9. Docking Studies
Molecular dockings were performed by using the Autodock Vina [31] software 1, The Scripps
Research Institute., La Jolla, CA, USA). Diprotonated compounds and were prepared with Discovery
4
6
Studio, version 2.1, software package (BIOVIA, San Diego, CA, USA), using standard bond lengths and
bond angles. With the CHARMm force field [32] and partial atomic charges, the molecular geometries
of the compounds were energy-minimized using the adopted-based Newton-Rapson algorithm until
the rms gradient was below 0.01 kcal (mol Å)−1
.
The crystal structure of hAChE complexed with fasciculin-II (PDB: 1B41) was obtained from the
Protein Data Bank (PDB). The protein was prepared by removing all water molecules, heteroatoms,
any co-crystallized solvent and the ligand. Protein model tool in Discovery Studio, version 2.1,
software package was used to assign proper bonds, bond orders, hybridization and charges. CHARMm
force field was applied using the receptor-ligand interactions tool in Discovery Studio, version 2.1,
software package. AutoDockTools (ADT; version 1.5.4, The Scripps Research Institute, La Jolla, CA,
USA) was used to add hydrogens and partial charges for proteins and ligands using Gasteiger charges.
The structures of the ligands were then loaded in ADT Tools and flexible torsions were assigned with
AutoTors module, and the acyclic dihedral angles were allowed to rotate freely. Some selected side
chains into the hAChE macromolecule were also allowed to change their conformations. Using the
AutoTors module, the macromolecule side chains chosen to be flexible are: Trp286, Tyr124, Tyr337,
Tyr72, Asp74, Thr75, Trp86 and Tyr341. The docking box was displayed using ADT and it is big
enough to include the whole protein target (“blind docking”). A grid box of 60
×
60
×
72 with grid
134.181).
Ǻ
point spacing of 1 , was positioned at the middle of the protein (x = 116.546; y = 110.33; z =
−
The three-dimensional structure of hBuChE has been used (PDB ID: 4BDS). Docking calculations
were performed following the same protocol described before for hAChE, not including flexibility at
Ǻ
the receptor. All dockings were performed as blind dockings where a box of 66
×
66
×
70 with grid
Ǻ
points separated 1 , was positioned at the middle of the protein (x = 136.0; y = 123.59; z = 38.56).
Default parameters were used except num_modes, which was set to 40. The docked conformations
of each ligand were ranked based on the binding energy and the top ranked conformations were
visually analyzed. The lowest docking-energy conformation was considered as the most stable
orientation. Finally, the docking results generated were directly loaded into the Discovery Studio and
interactions between docked ligands and macromolecule were analyzed.
Author Contributions: A.R. and A.B. conceived and designed the experiments; F.B. and S.G. contributed
reagents/materials/analysis tools; M.N. supervised the anticholinesterase activity assay; L.P. performed the
antioxidant and neuroprotective assays; M.B. and A.T. analyzed the data; I.I. and I.M. performed the docking
studies, A.R. and A.B. wrote the paper; A.R., A.B., S.G., F.B., M.B. and A.T. reviewed and edited the paper.
Funding: This work was supported by the Italian Ministry for Education, Universities and Research (MIUR) and
by the University of Bologna (RFO).
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2018, 23, 1902
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References
1.
Prince, M.; Wimo, A.; Guerchet, M.; Ali, G.-C.; Wu, Y.-T.; Prina, M. World Alzheimer Disease Report; Alzheimer’s
Disease International: London, UK, 2015; pp. 25–46.
2.
Winblad, B.; Amouyel, P.; Andrieu, S.; Ballard, C.; Brayne, C.; Brodaty, H.; Cedazo-Minguez, A.; Dubois, B.;
Edvardsson, D.; Feldman, H.; et al. Defeating Alzheimer’s disease and other dementias: A priority for
European science and society. Lancet Neurol. 2016, 15, 455–532. [CrossRef]
3.
4.
5.
Bachurin, S.O.; Bovina, E.V.; Ustyugov, A. Drugs in Clinical Trials for Alzheimer’s Disease: The Major Trends.
Med. Res. Rev. 2017, 37, 1186–1225. [CrossRef] [PubMed]
Contestabile, A. The history of the cholinergic hypothesis. Behav. Brain Res. 2011, 221, 334–340. [CrossRef]
[PubMed]
Ashraf, G.M.; Greig, N.H.; Khan, T.A.; Hassan, I.; Tabrez, S.; Shakil, S.; Sheikh, I.A.; Zaidi, S.K.; Akram, M.;
Jabir, N.R.; et al. Protein Misfolding and Aggregation in Alzheimer’s Disease and Type 2 Diabetes Mellitus.
CNS Neurol. Disord. Drug Targets 2014, 13, 1280–1293. [CrossRef] [PubMed]
6.
Akiyama, H.; Barger, S.; Barnum, S.; Bradt, B.; Bauer, J.; Cole, G.M.; Cooper, N.R.; Eikelenboom, P.;
Emmerling, M.; Fiebich, B.L.; et al. Inflammation and Alzheimer’s disease. Neurobiol. Aging 2000, 21,
383–421. [CrossRef]
7.
8.
9.
Rosini, M.; Simoni, E.; Milelli, A.; Minarini, A.; Melchiorre, C. Oxidative Stress in Alzheimer’s Disease:
Are We Connecting the Dots? J. Med. Chem. 2014, 57, 2821–2831. [CrossRef] [PubMed]
Hybertson, B.M.; Gao, B.; Bose, S.K.; McCord, J.M. Oxidative stress in health and disease: The therapeutic
potential of Nrf2 activation. Mol. Aspects Med. 2011, 32, 234–246. [CrossRef] [PubMed]
Rizzo, S.; Rivière, C.; Piazzi, L.; Bisi, A.; Gobbi, S.; Bartolini, M.; Andrisano, V.; Morroni, F.; Tarozzi, A.;
Monti, J.P.; et al. Benzofuran-based hybrid compounds for the inhibition of cholinesterase activity,
β amyloid
aggregation, and Aβ neurotoxicity. J. Med. Chem. 2008, 51, 2883–2886. [CrossRef] [PubMed]
10. Rizzo, S.; Tarozzi, A.; Bartolini, M.; Da Costa, G.; Bisi, A.; Gobbi, S.; Belluti, F.; Ligresti, A.; Allarà, M.;
Monti, J.-P.; et al. 2-Arylbenzofuran-based molecules as multipotent Alzheimer’s disease modifying agents.
Eur. J. Med. Chem. 2012, 58, 519–532. [CrossRef] [PubMed]
11. Singh, P.; Anand, A.; Kumar, V. Recent developments in biological activities of chalcones: A mini review.
Eur. J. Med. Chem. 2014, 85, 758–777. [CrossRef] [PubMed]
12. Zhuang, C.; Zhang, W.; Sheng, C.; Zhang, W.; Xing, C.; Miao, Z. Chalcone: A privileged structure in medicinal
chemistry. Chem. Rev. 2017, 117, 7762–7810. [CrossRef] [PubMed]
13. Mendieta, L.; Picò, A.; Tarragò, T.; Teixidò, M.; Castillo, M.; Rafecas, L.; Moyano, A.; Giralt, E. Novel Peptidyl
Aryl Vinyl Sulfones as Highly Potent and Selective Inhibitors of Cathepsins L and B. ChemMedChem 2010, 5,
1556–1567. [CrossRef] [PubMed]
14. Mertens, M.D.; Schmitz, J.; Horn, M.; Furtmann, N.; Bajorath, J.; Mares, M.; Gutschow, M. A Coumarin-
Labeled Vinyl Sulfone as Tripeptidomimetic Activity-Based Probe for Cysteine Cathepsins. ChemBioChem
2014, 15, 955–959. [CrossRef] [PubMed]
15. Staskun, B.; van Es, T. The Reduction of Nitriles to Aldehydes: Applications of Raney Nickel/Sodium
Hypophosphite Monohydrate, of Raney Nickel/Formic Acid, or of Raney(Ni/Al)Alloy/Formic Acid,
Respectively. S. Afr. J. Chem. 2008, 61, 144–156. [CrossRef]
16. Ellman, G.L.; Courtney, K.D.; Andres, V.; Featherstone, R.M. A new rapid colorimetric determination of
acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [CrossRef]
17. Nicolet, Y.; Lockridge, O.; Masson, P.; Fontecilla-Camps, J.C.; Nachon, F. Crystal structure of human
butyrylcholinesterase and of its complexes with substrate and products. J. Biol. Chem. 2003, 278, 41141–41147.
[CrossRef] [PubMed]
18. Greig, N.H.; Lahiri, D.K.; Sambamurti, K. Butyrylcholinesterase: An Important New Target in Alzheimer’s
Disease Therapy. Int. Psychogeriatr. 2002, 14 (Suppl. 1), 77–91. [CrossRef] [PubMed]
19. Weinstock, M. Selectivity of cholinesterase inhibition: Clinical implications for the treatment of Alzheimer’s
disease. CNS Drugs 1999, 12, 307–323. [CrossRef]
20. Greig, N.H.; Utsuki, T.; Ingram, D.K.; Wang, Y.; Pepeu, G.; Scali, C.; Yu, Q.-S.; Mamczarz, J.; Holloway, H.W.;
Giordano, T.; et al. Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning
and lowers Alzheimer
[CrossRef] [PubMed]
β-amyloid peptide in rodent. Proc. Natl. Acad. Sci. USA 2005, 102, 17213–17218.

Molecules 2018, 23, 1902
22 of 22
21. Darvesh, S.; Reid, G.A. Reduced fibrillar
β-amyloid in subcortical structures in a butyrylcholinesterase-
knockout Alzheimer disease mouse model. Chem. Biol. Interact. 2016, 259 Pt B, 307–312. [CrossRef] [PubMed]
22. Sengupta, U.; Nilson, A.N.; Kayed, R. The Role of Amyloid- Oligomers in Toxicity, Propagation, and
Immunotherapy. EbioMedicine 2016, 6, 42–49. [CrossRef] [PubMed]
β
23. Wu, R.; Hayashi, T.; Cottam, H.; Jin, G.; Yao, S.; Wu, C.; Rosenbach, M.; Corr, M.; Schwab, R.; Carson, D.
Nrf2 responses and the therapeutic selectivity of electrophilic compounds in chronic lymphocytic leukemia.
Proc. Natl. Acad. Sci. USA 2010, 107, 7479–7484. [CrossRef] [PubMed]
24. Satoh, T.; McKercher, S.R.; Lipton, S.A. Nrf2/ARE-mediated antioxidant actions of pro-electrophilic drugs.
Free Radic. Biol. Med. 2013, 65, 645–657. [CrossRef] [PubMed]
25. Hur, W.; Gray, N.S. Small molecule modulators of antioxidant response pathway. Curr. Opin. Chem. Biol.
2011, 15, 162–173. [CrossRef] [PubMed]
26. Tarozzi, A.; Morroni, F.; Merlicco, A.; Hrelia, S.; Angeloni, C.; Cantelli-Forti, G.; Hrelia, P. Sulforafane as an
inducer of glutathione prevents oxidative stress-induced cell death in a dopaminergic-like neuroblastoma
cell line. J. Neurochem. 2009, 111, 1161–1171. [CrossRef] [PubMed]
27. Tarozzi, A.; Morroni, F.; Bolondi, C.; Sita, G.; Hrelia, P.; Djemil, A.; Cantelli-Forti, G. Neuroprotective effects
of erucin against 6-hydroxydopamine-induced oxidative damage in a dopaminergic-like neuroblastoma cell
line. Int. J. Mol. Sci. 2012, 13, 10899–10910. [CrossRef] [PubMed]
28. Morroni, F.; Sita, G.; Djemil, A.; D’Amico, M.; Pruccoli, L.; Cantelli-Forti, G.; Hrelia, P.; Tarozzi, A. Comparison
of Adaptive Neuroprotective Mechanisms of Sulforaphane and its Interconversion Product Erucin in in Vitro
and in Vivo Models of Parkinson’s Disease. J. Agric. Food Chem. 2018, 66, 856–865. [CrossRef] [PubMed]
29. Tarozzi, A.; Bartolini, M.; Piazzi, L.; Valgimigli, L.; Amorati, R.; Bolondi, C.; Djemil, A.; Mancini, F.;
Andrisano, V.; Rampa, A. From the dual function lead AP2238 to AP2469, a multi-target-directed ligand for
the treatment of Alzheimer’s disease. Pharmacol. Res. Perspect. 2014, 2, e00023. [CrossRef] [PubMed]
30. Rampa, A.; Montanari, S.; Pruccoli, L.; Bartolini, M.; Falchi, F.; Feoli, A.; Cavalli, A.; Belluti, F.; Gobbi, S.;
Tarozzi, A.; et al. Chalcone-based carbamates for Alzheimer’s disease treatment. Future Med. Chem. 2017, 9,
749–764. [CrossRef] [PubMed]
31. Trott, O.; Olson, A.J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring
function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31, 455–461. [CrossRef]
[PubMed]
32. Brooks, B.R.; Bruccoleri, R.E.; Olafson, B.D.; States, D.J.; Swaminathan, S.; Karplus, M. CHARMM: A Program
for Macromolecular Energy, Minimization, and Dynamics Calculations. J. Comput. Chem. 1983, 4, 187–217.
[CrossRef]
Sample Availability: Samples of the compounds are not available from the authors.
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