Novel donepezil?arylsulfonamide hybrids as multitarget-directed ligands for potential treatment of Alzheimer?s disease

Article abstract of DOI:10.3390/molecules26061658

Alzheimer’s disease (AD) is one of the most devastating neurodegenerative disorders, characterized by multiple pathological features. Therefore, multi-target drug discovery has been one of the most active fields searching for new effective anti-AD therapies. Herein, a series of hybrid compounds are reported which were designed and developed by combining an aryl-sulfonamide function with a benzyl-piperidine moiety, the pharmacophore of donepezil (a current anti-AD acetylcholinesterase AChE inhibitor drug) or its benzyl-piperazine analogue. The in vitro results indicate that some of these hybrids achieve optimized activity towards two main AD targets, by displaying excellent AChE inhibitory potencies, as well as the capability to prevent amyloid-β (Aβ) aggregation. Some of these hybrids also prevented Aβ-induced cell toxicity. Significantly, drug-like properties were predicted, including for blood-brain permeability. Compound 9 emerged as a promising multi-target lead compound (AChE inhibition (IC₅₀ 1.6 μM); Aβ aggregation inhibition 60.7%). Overall, this family of hybrids is worthy of further exploration, due to the wide biological activity of sulfonamides.

Full text of DOI:10.3390/molecules26061658

molecules
Article
Novel Donepezil–Arylsulfonamide Hybrids as
Multitarget-Directed Ligands for Potential
Treatment of Alzheimer’s Disease
Fausto Queda 1,†, Sonia Calò 1,2,†, Karolina Gwizdala 1, João D. Magalhães 3 , Sandra M. Cardoso 3,4
,
Sílvia Chaves 1 , Luca Piemontese 2,5 and M. Amélia Santos 1,
*
1
Centro de Química Estrutural and Departamento de Engenharia Química, Instituto Superior Técnico,
Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; fausto.queda@gmail.com (F.Q.);
s.calo19@studenti.uniba.it (S.C.); karo.gwizdala@gmail.com (K.G.); silvia.chaves@tecnico.ulisboa.pt (S.C.)
Dipartimento di Farmacia–Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, Via E. Orabona 4,
I-70125 Bari, Italy; luca.piemontese@uniba.it
CNC–Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal;
joaoduartemagalhaes7@gmail.com (J.D.M.); cardoso.sandra.m@gmail.com (S.M.C.)
Institute of Molecular and Cell Biology, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal
National Research Council (CNR)–ISPA, via Amendola 122/O, 70126 Bari, Italy
Correspondence: masantos@tecnico.ulisboa.pt; Tel.: +351-218419273
2
3
4
5
*
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Alzheimer’s disease (AD) is one of the most devastating neurodegenerative disorders,
characterized by multiple pathological features. Therefore, multi-target drug discovery has been
one of the most active fields searching for new effective anti-AD therapies. Herein, a series of hybrid
compounds are reported which were designed and developed by combining an aryl-sulfonamide
function with a benzyl-piperidine moiety, the pharmacophore of donepezil (a current anti-AD acetyl-
cholinesterase AChE inhibitor drug) or its benzyl-piperazine analogue. The in vitro results indicate that
some of these hybrids achieve optimized activity towards two main AD targets, by displaying excellent
Citation: Queda, F.; Calò, S.;
Gwizdala, K.; Magalhães, J.D.;
Cardoso, S.M.; Chaves, S.;
Piemontese, L.; Santos, M.A. Novel
Donepezil–Arylsulfonamide Hybrids
as Multitarget-Directed Ligands for
Potential Treatment of Alzheimer’s
Disease. Molecules 2021, 26, 1658.
https://doi.org/10.3390/
AChE inhibitory potencies, as well as the capability to prevent amyloid-
these hybrids also prevented A -induced cell toxicity. Significantly, drug-like properties were predicted,
including for blood-brain permeability. Compound emerged as a promising multi-target lead com-
pound (AChE inhibition (IC50 1.6 M); A aggregation inhibition 60.7%). Overall, this family of hybrids
is worthy of further exploration, due to the wide biological activity of sulfonamides.
β (Aβ) aggregation. Some of
β
9
molecules26061658
µ
β
Academic Editors: Tomasz Plech and
Marta Andres-Mach
Keywords: arylsulfonamide; donepezil; anti-neurodegeneratives; Alzheimer´s disease; AChE in-
hibitors; Aβ aggregation
Received: 10 February 2021
Accepted: 11 March 2021
Published: 16 March 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Alzheimer’s disease (AD) is one of the most common devastating age-dependent neu-
rodegenerative disorders of the central nervous system (CNS), characterized by a progres-
sive neuronal degeneration associated to the loss of cognitive function. Multiple etiolog-
ical hypotheses have been proposed for the onset and progression of AD, including ge-
netics, oxidative stress, inflammatory–immunologic dysregulation and neurotransmitter
dysfunctions. Notwithstanding the enormous worldwide research efforts aimed to eluci-
date AD pathology and to find an effective therapy, its multifaceted nature and the still
not fully understood complexity of this disease are certainly the main reasons for the
high rate of failure in drug development programs and the so far consequent absence
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
of cure [1]. There are a few drugs approved by the US Food and Drug Administration
(FDA) for the treatment of AD, but they can only provide some amelioration of memory
deficits. Among them, four are acetylcholinesterase (AChE) inhibitors (tacrine, donepezil,
rivastigmine and galantamine), to compensate for the loss of acetylcholine and one (memantine)
Molecules 2021, 26, 1658. https://doi.org/10.3390/molecules26061658
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1658
2 of 15
is a N-methyl-D-aspartate receptor (NMDAR) antagonist, to regulate the excitatory glu-
tamatergic function [2,3]. Excluding tacrine, which was withdrawn from the market due
to hepatotoxicity, the other mentioned drugs are under current use, as well as the quite
recently (in 2014) FDA approved donepezil-memantine combination drug (Namzaric),
used in fixed doses. However, these drugs are not able to stop or reverse the progression of
neurodegeneration, but only provide a short-term symptomatic relief in prodromal or early
stages of AD [1–3]. Therefore, the identification of valid disease-modifying treatments for
AD represents nowadays a very urgent and still unmet medical global problem [4,5].
The existence of diverse multiple factors, that can contribute to the onset and pro-
gression of AD, has been considered one of the main obstacles for drug development.
However, several pathological hallmarks have been widely recognized, including the
protein misfolding associated to amyloid cascade, with formation of amyloid aggregates
(amyloid plaques) and tau hyperphosphorylation; the collapse of the neurotransmitters
(cholinergic dysfunction), oxidative stress and metal dyshomeosthasis [
6]. Thus, over the
years, the development of a multi-target therapeutics [ ] has become an important ap-
7
proach widely used in the research of new drugs, envisaging the simultaneous interac-
tion with several pathological targets. Among the different drug development strategies,
many hybrid molecules have been explored by combining in the same molecular entity
approved anti-AD drugs, or their determinant pharmacophores, with other biologically
active molecular moieties to enable the hitting of other important AD targets, such as
amyloid beta (Aβ) aggregates, radical oxygen species (ROS), redox-active metal ions and
enzymes (e.g., monoamine oxidase, beta-secretase 1) [8–13].
Following the conceptualization of the multi-target strategy for anti-AD drugs and the
associated interest of this research group on the repositioning of approved drugs [14–16],
the present work is focused on the development of new arylsulfonamide-donepezil like
molecules with potential ability to interact with different targets of AD. The option for
aromatic sulfonamides, as second pharmacophore, is due to their wide clinical applica-
tions, namely as antibacterial, antifungal, anti-cancer and anti-depressant [17]. Further-
more, a number of sulfonamide-containing hybrids, enclosing tacrine/quinoline moieties,
have been recently developed as promising anti-AD drugs, exhibiting good capacity for
the inhibition of cholinesterases and self-induced A
activity [18–20].
β aggregation, as well as antioxidant
In particular, we present herein the design, synthesis and biological evaluation of a
small library of donepezil-sulfonamides, which are based on the attachment of a DNP active
pharmacophore moiety (benzylpiperidine/benzylpiperazine) to a set of para-substituted
aromatic sulfonamides (see Scheme 1). In addition to the molecular design and synthesis,
these novel compounds are biologically evaluated in vitro for inhibition of AChE and A
β
aggregation and then bio-assayed in cell environment for the neuroprotection properties.
The new compounds are also evaluated for their pharmacokinetic properties and drug like-
ness. Discussion on some structural-activity relationships is also provided, namely effects
of para-substituents modification and linker size on the biological activity of the hybrids.
Scheme 1. Donepezil (DNP) and the studied DNP-like hybrids.

Molecules 2021, 26, 1658
3 of 15
2. Results and Discussion
2.1. Molecular Design and Docking
Since donepezil (DNP) is a molecule of high interest in the medical treatment of
AD [ ], its active pharmacophore was selected for the design of several new DNP-like
2,3
hybrid molecules enclosing an aryl sulfonamide moiety. In particular, the rationale for
these new hybrids was based on using, as the first pharmacophores, two donepezil-mimetic
derivatives, namely the benzyl-piperidine moiety ( and
8
9) (the active moiety of Donepezil,
as AChEi) and its isostere benzyl-piperazine (5–7), to interact with the catalytic anionic
site (CAS) of AChE. As secondary pharmacophores, the aryl sulfonamides were supposed
to increase the AChE binding affinity, by mimicking the role of the indanone moiety of
DNP on its extra-interaction with the peripheral active site (PAS) of AChE. In addition,
enabling that potential dual mode of interaction with AChE, the aryl sulfonamides could
also inhibit Aβ aggregation. Furthermore, sulfonamides are well-known moieties present
in many approved drugs due to their wide biological activity [21].
Modelling docking studies of this set of new compounds into AChE were performed
in order to get some insight on their binding interactions with the enzyme active site and
to understand potential structure-activity relationships. Firstly, from an X-ray structure of
a DNP-AChE complex, the co-crystallized ligand (DNP) was docked back into the AChE
binding site of the enzyme and showed good superimposition with the native ligand.
Being satisfied with the good overlay of the simulated and original co-crystallized DNP,
the same docking protocol was used to dock the target compounds (5–9). The best docked
poses of all the synthesized compounds were selected and superimposed with that of DNP,
as shown in Figure 1A–E. Analysis of the docking graphics shows that all the compounds
have the pharmacophore benzyl-piperazinium (
and ) anchored to the CAS of AChE (bottom of the gorge), through binding interac-
tions via aromatic stacking with the phenyl ring from Trp84 (A). Among this set of
compounds, those bearing a benzyl-piperidine group ( (B) and (E)), showed the best
5–7) and benzyl-piperidinium moieties
(
8
9
π-π
8
9
superimposition with the same moiety of DNP, while some other compounds (6 (A), 7 (C))
present a little bending of the benzyl group relatively to that of DNP. At the middle of
the gorge, the charged nitrogen atom can establish a cation–
the phenyl group of Phe330. Compound (E) shows a further superimposition between
the sulfonyl group of the biphenyl-sulfonamide moiety and the carbonyl group of the
DNP indanone. Both these groups, in DNP and (E), can similarly interact with Phe290,
but for some compounds, as (B), this is not favored because they are pointed away from
each other. These differences are reflected in the orientation of the aryl-sulfonyl groups,
which in some compounds, as (D) appears distorted from that of the DNP indanone,
possibly allowing positive interactions with other residues. For the biphenyl derivatives
(C) and (E)), the corresponding biaryl-sulfonyl groups seem to adopt a spatial orienta-
π binding interaction with
9
9
8
5
,
(
7
9
tion similar to that of the indanone moiety of DNP and so a similar
with the peripheral anionic residue, namely Trp279, is kept.
π–π stacking interaction

Molecules 2021, 26, 1658
4 of 15
Figure 1. Superimposition of the different DNP-sulfonamide compounds with the original ligand (DNP-green), as a
result of their docking inside the AChE active site (PDB code: 1EVE). ( ) compound ; ( ) compound ; ( ) compound 7;
A
6
B
8
C
(D) compound 5; (E) compound 9; (F) all the compounds.
Pharmacokinetic Properties
Pharmacokinetic properties were calculated with the software QikProp v.2.5 (Schrödinger,
LLC, New York, NY, USA) [22] and they are depicted in Table 1.
Table 1. Summary of pharmacokinetic properties predicted by the software QikProp v.2.5.
Compound
MW(Da)
373.512
389.512
435.583
374.497
420.568
379.50
clog P
2.259
1.972
3.575
3.080
4.686
4.269
log BB
0.123
Caco Permeability (nm/s)
CNS
5
114
116
118
363
457
893
+
+
+
+
+
+
6
0.094
7
8
0.021
−0.354
−0.319
0.132
9
DNP
All the developed compounds are free from violations of Lipinski’s rule, thus,
predicting their drug-likeness as oral active anti-AD agents and so they can be poten-

Molecules 2021, 26, 1658
5 of 15
tially considered for oral formulation [23]. The molecular weight (under 500) and the
lipophilic character (clog P = 1.9–4.7) are two important parameters that allow anticipa-
tion of a good absorption and bioavailability [24]. Log BB was calculated to evaluate the
capacity of the compounds to cross the blood-brain barrier (BBB) and the values obtained
are also within the range calculated for drugs ( 3 to 1) [22]. Caco permeability indicates
−
the absorption from the intestine to the bloodstream. Considering that values lower than
25 nm/s and higher than 500 nm/s represent, respectively, a poor and a great absorption,
it can be inferred that compounds
8 (363 nm/s) and 9 (457 nm/s) present a good Caco
permeability (gut absorption), though lower than the drug DNP (893 nm/s), while 5, 6 and
7 present a more moderate one.
Overall, these predictions indicate that this set of DNP derivatives presents good
drug-like pharmacokinetic properties, in particular compounds and with good BBB
8
9
permeability (log BB) and activity in CNS (+) as the parent drug, suggesting they can be
good therapeutic candidates, even for oral administration.
2.2. Synthesis
Five hybrid compounds were obtained by conjugation of two DNP mimetic moieties,
namely the N-benzylated piperidine and piperazine derivatives, with para-substituted
arylsulfonamides, following the synthetic procedure depicted in Scheme 2.
◦
Scheme 2. Synthetic plan for compounds
KOH, EtOH, rt, 24 h to obtain
24 h; (d) TEA, dioxane, rt, 12 h.
5
–
9
. Reagents and conditions: (
a
) Phthalic anhydride 160–180 C, 4 h; (
b
) BnBr,
◦
1
or BnBr, TEA, K2CO3, ACN, 50 C, 3 h to obtain
2; (c
) MeNH2 (40% aqueous solution) rt,
For the preparation of the target hybrids, two starting compounds were selected,
namely piperidine and piperazine derivatives bearing alkylamine groups (1a and 2a),
for the subsequent attachment to the arylsulfonyl moieties. So, the first objective was to
introduce a permanent benzyl group at the secondary amine of the piperidine and piper-
azine rings. However, the success of this alkylation required a previous selective temporary
protection of the primary amines of the alkyl amine side groups. Therefore, a phthalimide
group was chosen as protecting group because of its “orthogonality” relative to the benzyl
group. Thus, the preparation of compounds
1 and 2 relied on the phthalamidation of
the bearing primary amines of the corresponding precursors (1a and 2a) by its reaction
with phthalic anhydride at 160–180 ◦C for 4 h, under neat conditions (Scheme 2, step a),
according to previously reported [14,25].
For the subsequent benzylation of the secondary cyclic amines, benzyl bromide (BnBr)
was used with different solvent and base conditions (Scheme 2, step b), mainly due to the
poor solubility of the piperidine derivative in EtOH. Nevertheless, the reactions went on
smoothly, with 50% yield for 1 and 29% yield for 2, after two steps. The subsequent step

Molecules 2021, 26, 1658
6 of 15
towards the preparation of compounds
3
and
4
consisted in the removal of the phthalimide
group, to leave the primary amine free for the next reaction (coupling of the two main
moieties). To perform this, a well reported procedure was carried out, using MeNH2 in
40% aqueous solution, to afford
3 and 4 in 97% and 69% yield, respectively (Scheme
2, step c) [26]. The last reaction (Scheme 2, step d) involved the coupling of the free
amine intermediates (3, 4) with the different commercially available aryl-sulfonyl chlorides,
via sulfonamide formation. This reaction was carried out in dioxane and the final pure
compounds (5–9) were obtained in moderate yields (30–49%).
2.3. Biological Assays in Solution
2.3.1. Acetylcholinesterase Inhibition
In Table 2 the biological activity results for the synthetized compounds (5–9) are
summarized and compared with two well-known standard compounds, namely in terms
of AChE inhibition and inhibition of self-induced Aβ aggregation.
Table 2. Biological activity of the compounds towards the inhibition of AChE and Aβ1–42 aggregation.
AChE Inhib.a
IC50 (µM) ± SD
Aβ1–42 Aggreg.
X
R
n
Inhib.b,c (%)
5
N
N
Me
OMe
Ph
2
2
≥100
89 ± 9
38.7 ± 0.2
6.2 ± 0.9
1.6 ± 0.2
0.15 ± 0.02
0.026
41.8
37.2
34.6
64.2
60.7
54.5
6
7
8
N
2
CH
CH
−
OMe
Ph
1
9
1
Tacrine
DNP d
−
−
−
−
−
<5
b
a
The values are mean of two independent experiments
self-induced Aβ1–42 aggregation (%) by the thioflavin-T fluorescence method in the presence of the inhibitor (40
control; c The values are the mean of three independent measurements in duplicate (SEM < 10%); d references [3,27].
±
SD; AChE from Electric eel; Tacrine was used as a control; Inhibition of
µ
M); Tacrine was used as a
Compounds
5 and 6 do not show significant inhibition of AChE, which can be related
with the lower affinity of the piperazine ring, as compared with the piperidinic one,
for the AChE binding site and also to the higher linker size (n = 2) of these compounds
when compared with that of derivatives
hybrids with higher length linker presented some bending, relatively to DNP, in the CAS
of AChE. The fact that the inhibitory activity of , also containing a piperazine moiety
and n = 2, is higher than that of compounds and should be attributed to its biphenyl
group. Moreover, compounds and presented higher AChE inhibition (IC50 1.6–6.2
M) than the compounds with piperazine moiety (38.7 < IC50 > 100 M) and again the
) is the most potent, which is in accordance with the docking
8 and 9 (n = 1). As already shown in Figure 1,
7
5
6
8
9
µ
µ
biphenyl compound (
9
studies (see Section 2.1). The better inhibitory activity of the biphenyl derivatives can
be explained in two ways: the phenyl substituent of these inhibitors can promote extra
binding interactions inside the active site of the enzyme, namely with the aromatic residues
of PAS, as suggested by the inhibitor-enzyme docking simulations; and, the long size of
this group can force some structural rearrangements of the inhibitors and promote some
additional hydrophobic interaction between the inhibitor and the active site, thus further
strengthening the inhibition.
Although the hybrids studied herein are poorer AChE inhibitors than DNP
(IC50 = 0.026
µM [3]), the proposed series of compounds is still a valid research topic
focused on a multi-target approach for anti-AD drug.

Molecules 2021, 26, 1658
7 of 15
2.3.2. Inhibition of Aβ1–42 Aggregation
Both drugs, tacrine and DNP, have moderate/weak capacity for the inhibition of
A
β1–42 aggregation, in opposition to their good activity as AChE inhibitors, as already
mentioned in Section 2.3.1. Although all compounds (
sults in this assay, compounds and 9, enclosing the benzylpiperidine moiety, are better
inhibitors of A aggregation than tacrine, which is by far superior to DNP and are the
strongest inhibitors among the hybrids under study. Therefore, the hybrids enclosing
the benzylpiperidine moiety and a shorter linker size (n = 1) show better A aggregation
5–9) show moderate to good re-
8
β
β
inhibition when compared to those containing a benzylpiperazine unit and n = 2. Moreover,
for each type of derivative (benzylpiperidine/benzylpiperazine), it is possible to ob-
serve the same sequence of inhibitory capacity depending on the sulfonamide substitute
(methoxy- > biphenyl-). Furthermore, among the benzylpiperazine derivatives, compound
5 with the methyl-arylsulfonamide group seems to be the best one.
The observed inhibitory ability dependence on some ligand structural features may be
mainly explained by the different capacity for intercalation of the compounds into the fibrils
that seems to be more difficult for the benzylpiperazine derivatives herein developed.
2.4. Cell Viability and Neuroprotection
The approved therapy for AD is still scarce and ineffective. Thus, the need for new
drugs that will help treat AD patients is essential. As such, we performed a dose-response
screening to determine the highest non-toxic concentration of several hybrid compounds
(Figure 2). Deposit of amyloid plaques is the most typical histopathological feature of
AD. These extracellular agglomerates are typically constituted by fibrils of oligomerized
A
β1–42
in vitro model, for this study. As observed in Figure 3, Aβ1–42 exposure for 24 h was enough
to decrease cell viability. Giving that A deposition is deleterious to diseased patients,
our results demonstrate that compound was able to relapse the neurotoxicity induced by
peptides in our cells (Figure 3) while compounds and showed more modest results
in reverting neurotoxicity provoked by A . Therefore, this kind of compounds deserves
[28]. Hence, we sought to challenge SH-SY5Y cells with Aβ1–42 peptides, as an
β
7
A
β
8
9
β
further studies, since they can be seen as suitable candidates to impede A
and further toxic effects.
β oligomerization
Figure 2. Dose-response screening of compounds 6–9 on SH-SY5Y neuronal cell line. SH-SY5Y cells were treated with
varying concentrations of compounds from 2.5 M to 50 M for 24 h and cellular viability was measured by using the MTT
reduction assay. Results are expressed relatively to SH-SY5Y untreated cells, with the mean SEM derived from at least
µ
µ
±
three different experiments. ** p < 0.01, *** p < 0.001, **** p < 0.0001 significantly different when compared with control,
untreated SH-SY5Y cells.

Molecules 2021, 26, 1658
8 of 15
Figure 3. Neuroprotective effect of substances on Aβ42-induced toxicity on SH-SY5Y cells. SH-SY5Y cells were treated with
β42 peptide (2.5 M) for 24 h in the absence or in the presence of the compounds. When compounds were present, a pre-
incubation of 1 h preceded the Aβ42 treatment. Cellular viability was measured by using the MTT reduction assay. Results
are expressed relatively to SH-SY5Y untreated cells, with the mean SEM derived from at least 3 different experiments.
A
µ
±
**** p < 0.0001, significantly different when compared with SH-SY5Y untreated cells (CT); # p < 0.05, significantly different
when compared with Aβ42 treated SH-SY5Y cells. Compounds: 6 (40 µM); 7 (15) µM; 8 (30 µM); 9 (2.5 µM).
A
β
plaques formation is an intricate process, yet to be fully discovered. However, gathered
data suggests that the production of reactive oxygen species (ROS) has a prominent role in this
event [29]. For instance, ROS production is considered to be an earlier step than A deposition
β
itself. ROS can disrupt cellular signaling by oxidizing lipids, proteins and DNA. Remarkably,
this is a feature usually found in AD patients’ brains [30]. The combination of Fe/Asc was used
to induce oxidative stress in our cellular model. As observed in Figure 4, Fe/Asc was able to
decrease SH-SY5Y viability. In our assay, neither of the tested substances was able to protect
cells against a Fe/Asc insult (Figure 4). These results might suggest that compounds
provide neuroprotection independently of the oxidative status of cells.
7–9 can
Figure 4. Neuroprotective effect of compounds on Ferrous Sulfate (Fe)/L-Ascorbic Acid (AscH( ))-induced toxicity on
−
SH-SY5Y cells. Overnight cultures were treated with Fe/Asc (2.5 M/5 mM, respectively) for 24 h in the absence or in
the presence of the compounds. When compounds were present, a pre-incubation of 1 h preceded the Aβ42 treatment.
Cellular viability was determined by employing the MTT reduction assay. Results are expressed relatively to SH-SY5Y
untreated cells, with the mean
compared with SH-SY5Y untreated cells (CT). Compounds: 6 (40 µM); 7 (15 µM); 8 (30 µM); 9 (2.5 µM).
±
SEM derived from at least 3 different experiments. ** p < 0.01, significantly different when

Molecules 2021, 26, 1658
9 of 15
3. Materials and Methods
3.1. Chemicals and Apparatus
For the synthesis of the compounds, all the reagents (from Sigma-Aldrich, St. Louis,
MO, USA; Alfa-Aesar, Kandel, Germany; Acros, Thermo Fisher Scientific, Geel, Belgium)
were of analytical grade and used as supplied. Whenever necessary, the solvents were
dried according to standard methods [31]. The chemical reactions were followed by thin
layer chromatography (TLC) using alumina plates coated with silica gel 60 F254 (Macherey-
Nagel, Düren, Germany). Column chromatography purifications were performed on
silica gel (Merck 230–400 mesh, Geduran Si 60, Darmstadt, Germany). The purity of the
compounds was assessed by determination of the melting points (M.P.) and by using
NMR and mass spectral techniques. The melting points were measured with a Leica Galen
1
III (Microsystems, Wetzlar, Germany) hot stage apparatus and are uncorrected. H- and
13C-NMR spectra were recorded either on Bruker AVANCE III-300 (300 MHz and 75.5 MHz)
or Bruker AVANCE III-400 (400 MHz and 100 MHz) NMR spectrometers (Billerica, MA,
◦
USA), at 25 C. Chemical shifts are reported as
δ values (ppm) from internal reference
TMS (tetramethylsilane, Sigma-Aldrich, St. Louis, MO, USA) and coupling constants (J)
in Hertz. The following abbreviations have been used: s = singlet, d = doublet, t = triplet,
m = multiplet. The mass spectra were performed on a 500 MS LC Ion Trap mass spectrom-
eter (Varian Inc., Palo Alto, CA, USA) equipped with an ESI ion source, operated in the
positive ion mode.
3.2. Molecular Modeling: Docking and Pharmacokinetics Studies
Docking simulations were done using the Gold v. 5.1 software (CCDC, Cambridge,
UK) [32]. The AChE active site model structure was retrieved from the protein database
(RCSB) (PDB, entry 1 EVE) [33], as a complex of AChE with Donepezil (DNP), a clinical
drug in use, herein named as the original ligand. Despite of some recognized differences
between the inhibitor-enzyme complexes, described for the human acetylcholine esterase
(hAChE) and the electric ray homologue (Torpedo californica, TcAChE), both enzymes have
shown to be conservative at the main amino acid residues that line the active site gorge [34
]
and so the docking study was performed using the TcAChE.
For the docking simulations, the TcAChE-DNP complex was retrieved from RCSB and
then treated with MAESTRO v. 9.3 (Schrödinger Inc., Portland, OR, USA) [35], to obtain
the receptor-enzyme model, by removing the original ligand, solvent and co-crystallization
molecules and subsequent addition of hydrogen atoms. The program was also used to
create the ligand configurations that were optimized (1000 cycles of random conformational
search and 2500 optimization steps) using Ghemical v 2.0 (Free Software Foundation
Inc., Atlanta, GA, USA) [36]. The ligands were then docked with the AChE active site,
using GOLD program with default parameters and the Astex Statistical Potential (ASP)
scoring function. The docking site was well-defined by selecting a box within 10 Å from the
original ligand at the above stated PDB structure. The top structures generated from these
computations were compared with the original ligand and their interactions evaluated
with the active site residues. The outcomes were pictured using Chimera 1.6 (Free software,
University of California, San Francisco, CA, USA) [37].
Pharmacokinetic molecular properties were assessed in silico. Factors like the ability
to be absorbed through the intestinal tract (Caco-2 cell permeability), blood–brain barrier
partition coefficient (log BB), the octanol–water partition coefficient (clog P) and CNS activ-
ity were estimated. The chemical structures were previously optimized using Maestro v. 9.3
(Schrödinger Inc., Portland, OR, USA) followed by submission in the calculation of these
relevant pharmacokinetic properties and descriptors using QikProp v. 2.5 (Schrödinger,
LLC, New York, NY, USA) [22].

Molecules 2021, 26, 1658
10 of 15
3.3. Synthesis
3.3.1. Synthesis of the Compounds 1 and 2
Equivalent amounts of phthalic anhydride (1 eq) and of 1-(2-aminoethyl)-piperazine
(
1a) or (piperidin-4-yl)methanamine (2a) were heated at 160–180 ◦C for 4 h. The resulting
dark brown solid was dissolved in ethanol 96% and subsequently were added equivalent
amounts of KOH and benzyl bromide. The resulting mixture was stirred at room tempera-
ture (RT) for 24 h. Then, the ethanol was removed in vacuo and the residue was partitioned
between diethyl ether and H2O. The aqueous layer was further extracted with diethyl ether
(2 times) and the collected organic phases were washed with brine, dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure. The crude was purified by
chromatography column (eluent: DCM/MeOH 92:8 or ACN/H2O) and the pure products
were obtained as pale-yellow solids (1 or 2).
2-(2-(4-Benzylpiperazin-1-yl)ethyl)isoindoline-1,3-dione (
CDCl3), (ppm): 2.39–2.52 (m, 8H, piperazine), 2.60 (t, 2H, J = 6.7 Hz, phthalimide-
1
). Yield 50%; 1H NMR (400 MHz,
δ
CH2CH2N), 3.44 (s, 2H, NCH2Ph) 3.78 (t, 2H, J = 6.7 Hz, phthalimide–CH2CH2N), 7.19–7.27
(m, 5H, aromatics, Ph–CH2), 7.67–7.69 and 7.80–7.82 (m, 4H, phthalimide). m/z (ESI-MS):
350 (M + H)+.
2-((1-Benzylpiperidin-4-yl)methyl)isoindoline-1,3-dione (
DMSO-d6), (ppm): 1.07–1.15, 1.58–1.76 and 3.44–3.50 (m, 9H, piperidine), 2.73 (d, 2H,
2
). Yield: 29%; 1H NMR (300 MHz,
δ
NCH2CH) 3.41 (s, 2H, NCH2Ph), 7.15–7.40 (m, 5H, aromatics, Ph–CH2), 7.79–7.91 (m, 4H,
phthalimide). m/z (ESI-MS): 335 (M + H)+.
3.3.2. Synthesis of the Compounds 3 and 4
2-(2-(4-Benzylpiperazin-1-yl)ethyl)isoindoline-1,3-dione (
1) or 2-((1-benzylpiperidin-
4-yl)methyl)isoindoline-1,3-dione ( ) (0.897 mmol) was dissolved in an aqueous solution
2
of MeNH2 40% (w/w, 5 mL) and stirred at room temperature for 60 h. Then, an aqueous
solution of NaOH 20% (w/w, 5 mL) was added and the resulting mixture was stirred
for 2 h. Afterwards NaCl was added (0.897 mmol) and the solution was extracted with
dichloromethane and the organic layer washed with water, dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure affording pale-yellow oils.
2-(4-Benzylpiperazin-1-yl)ethanamine (
(ppm): 2.35–2.43 (m, 10H, piperazine, NH2–CH2CH2N), 2.73 (t, 2H, J = 6.1 Hz, NH2–
3
). Yield: 97%. 1H NMR (400 MHz, CDCl3),
δ
CH2CH2N), 3.46 (s, 2H, NCH2Ph), 7.18–7.27 (m, 5H, aromatics, Ph–CH2). M/z (ESI-MS):
220 (M + H)+.
1
(1-Benzylpiperidin-4-yl)methanamine (
4
). Yield: 69%. H NMR (300 MHz, DMSO-d6),
δ(ppm): 1.00–1.23, 1.61–1.65, 1.82–1.89 and 2.37–2.39 (m, 9H, piperidine), 2,77 (d, 2H,
J = 11.5 Hz, NCH2CH) 3.41 (s, 2H, NCH2Ph), 7.20–7.33 (m, 5H, aromatics, Ph–CH2). M/z
(ESI-MS): 205 (M + H)+.
3.3.3. Synthesis of the Final Conjugates (5–9)
To a solution of 2-(4-benzyl-1-piperazinyl)ethanamine (1) or (1-benzylpiperidin-4-
yl)methanamine ( ) (0.509 mmol) in dioxane (12,7 mL), was added the suitable sulfonyl
2
chloride derivative (in a stoichiometric ratio 1:1.2) and stirred overnight at RT. The mixture
was concentrated under reduced pressure, added to water and extracted with ethyl acetate
for 3 times. The organic layers were washed with a solution of HCl 1M and NaOH 1M and
then dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude
was purified by chromatography column (eluent: DCM/MeOH, 95:5).
N-(2-(4-benzylpiperazin-1-yl)ethyl)-4-methylbenzenesulfonamide (
oil. 1H NMR (400 MHz, MeOD-d4)
(ppm): 7.73 (d, J = 8.2 Hz, 2H: H21, H25), 7.38 (d,
J = 8.0 Hz, 2H: H22, H24), 7.33–7.27 (m, 5H, H1-H6), 3.59 (s, 2H: H7), 2.97 (t, J = 6.7 Hz, 2H:
H15), 2.70–2.44 (m, 10H, H9–H14), 2.42 (s, 3H, H26). 13C NMR (100 MHz, MeOD-d4)
(ppm):
5). Yield: 30%, yellow
δ
δ
144.76, 138.71, 130.90, 130.76, 129.44, 128.77, 128.10, 63.57, 58.04, 53.37, 53.33, 40.91, 21.44. m/z
(ESI-MS): 374.19 (M + H)+.

Molecules 2021, 26, 1658
11 of 15
N-(2-(4-benzylpiperazin-1-yl)ethyl)-4-methoxybenzenesulfonamide (
6
). Yield: 49%, yellow oil.
1H NMR (300 MHz, MeOD-d4)
δ
(ppm): 7.78 (d, J = 8.9 Hz, 2H: H21, H25), 7.33–7.23 (m, 5H:
H1-H6), 7.07 (d, J = 8.9 Hz, 2H: H22, H24), 3.87 (s, 3H: H27), 3.54 (s, 2H: H7), 2.96 (t, J = 6.8 Hz,
2H: H15), 2.56–2.41 (m, 10H: H9–H14). 13C NMR (75.5 MHz, MeOD-d4)
(ppm): 165.45, 137.83,
δ
133.13, 130.81, 130.20, 129.37, 128.60, 115.33, 63.73, 58.09, 56.21, 53.56, 53.47, 40.94. m/z (ESI-MS):
390.18 (M + H)+.
N-(2-(4-benzylpiperazin-1yl)ethyl)-[1,1’-biphenyl]-4-sulfonamide (
7
). Yield: 38%, white solid,
(ppm): 7.98–7.44 (m, 9H: H21-H31),
7.39–7.23 (m, 5H: H1-H6), 3.52 (s, 2H: H7), 3.05 (t, J = 6.3 Hz, 2H: H15), 2.68–2.22 (m, 10H:
H9–H14). 13C NMR (75.5 MHz, MeOD-d4)
(ppm): 146.67, 140.60, 140.40, 137.90, 130.78,
◦
1
M.P. 101-103 C. H NMR (400 MHz, MeOD-d4)
δ
δ
130.16, 129.53, 129.33, 128.67, 128.53, 128.28, 63.78, 58.16, 53.59, 53.51, 41.05. m/z (ESI-MS):
436.36 (M + H)+.
N-((1-benzylpiperidin-4-yl)methyl)-4-methoxybenzenesulfonamide (
M.P. 83-86 ◦C. 1H NMR (400 MHz, MeOD-d4)
(ppm): 7.78 (d, J = 7.9 Hz, 2H: H20, H24),
7.37–7.26 (m, 5H: H1–H6), 7.08 (d, J = 8.0 Hz, 2H: H21, H23), 3.89 (s, 3H: H26), 3.60 (s, 2H:
8). Yield: 39%, white solid.
δ
0
0
H7), 2.95 (d, J = 11.5 Hz, 2H: H9 , H13 ), 2.71 (d, J = 6.7 Hz, 2H: H14), 2.08 (dd, J = 19.3, 7.8 Hz,
0
0
0
0
2H: H10 , H12 ), 1.72 (d, J = 12.7 Hz, 2H: H9 ’ H13 ’), 1.49–1.40 (m, 1H: H11), 1.27–1.18 (m, 2H:
H100’, H120’). 13C NMR (75.5 MHz, MeOD-d4)
δ(ppm): 133.41, 131.08, 130.07, 129.42, 128.81,
115.26, 63.86, 56.18, 54.02, 36.92, 30.05. m/z (ESI-MS): 375.17 (M + H)+.
N-((1-benzylpiperidin-4-yl)methyl)-[1,1’-biphenyl]-4-sulfonamide (9). Yield 48%, white solid.
◦
1
M.P. 139–140 C. H NMR (400 MHz, MeOD)
δ 7.91–7.39 (m, 9H: H20-H30), 7.30–7.20
(m, 5H: H1–H6), 3.51 (s, 2H: H7), 2.88 (d, J = 11.0 Hz, 2H: H9’,H13’), 2.76 (d, J = 6.5 Hz,
2H: H14), 2.04–1.96 (m, 2H: H10’,H12’), 1.70 (d, J = 12.9 Hz, 2H: H9”,H13”), 1.50–1.38 (m, 1H:
H11), 1.29–1.16 (m, 2H: H10”,H12”). 13C NMR (75.5 MHz, MeOD-d4)
δ (ppm): 146.59, 140.66,
140.64, 137.81, 130.95, 130.14, 129.49, 129.31, 128.63, 128.57, 128.28, 64.09, 54.11, 49.45, 37.16,
30.26. m/z (ESI-MS): 421.35 (M + H)+.
3.4. Biological Assays in Solution
3.4.1. Acetylcholinesterase Inhibition
The acetylcholinesterase inhibitory activity was assessed by an adaptation of the Ell-
man’s method [38,39]. The assay solution contained 4-(2-hydroxyethyl)-1-piperazineethane
sulfonic acid (HEPES) buffer (50 mM, pH 8.0), 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB,
3 mM), the stock solution of each compound in methanol (1 mg/mL), AChE (type VI-S,
from electric eel) and the required quantity of methanol to reach the desired volume of
sample mixture in a 1 mL cuvette. Samples were incubated for 15 min and then 75 µL of
acetylthiocholine iodide (AThChI) solution (16 mM) was added. The reaction was moni-
tored for 5 min at 405 nm. Assays were run with a blank containing all the reagents but
AChE. Reaction rates were calculated as well as the enzyme activity. A positive control
reaction was performed using methanol as a sample solution. The fraction of enzyme
inhibition due to the existence of rising concentration of the test compound was calcu-
lated using Equation (1), where vi = starting reaction rate in the presence of inhibitor;
vo = starting reaction rate of the control reaction.
vi
%I = 100 − 100 ×
(1)
(vi − vo)
The inhibition curves were achieved by plotting the percentage of enzymatic inhibition
against inhibitor concentration and a calibration curve was got from which the linear
regression parameters were obtained.
3.4.2. Inhibition of Aβ1–42 Aggregation
Inhibitory capacity of the compounds for the Aβ1–42 aggregation was assessed based
on the fluorescence properties of thioflavine T (ThT), measured with a Varian Cary Eclipse
spectrofluorometer.

Molecules 2021, 26, 1658
12 of 15
A previously prepared solution of Aβ1–42 was used. Firstly. the lyophilized peptide
(1 mg) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol, (HFIP) (1.5 mL) by brief son-
ication and kept overnight at 25 ◦C. The following day, in an ice bath, the solution was
◦
partitioned into 6 Eppendorf tubes (250
µ
L) and left to evaporate overnight at 25 C. Then,
the resulting films were stored at −20 ◦C for months [14].
A film of Aβ1–42 was re-dissolved in a fresh mixture solution of CH3CN/Na2CO3
(300 µM)/NaOH (250 mM) (48.3/48.3/3.4 µL v/v/v) by brief sonication. To the resulting
alkaline Aβ1–42 solution was added phosphate buffer (pH 8.0). The compounds tested were
diluted in MeOH (1 mg/mL) and then further diluted in phosphate buffer. Two different
types of experiments were prepared in a final volume of 60
ligand assay consists in 30 L of phosphate buffer and 30 L of Aβ1–42 solution, to evaluate
the aggregation without any inhibition, while the solution for the ligand assay consists
of 20 L of phosphate buffer, 30 L of Aβ1–42 solution and 10 L of the ligand solution,
µL. The solution for the control
µ
µ
µ
µ
µ
to evaluate the effect of the ligand in the aggregation. For each experiment a blank
sample, without Aβ1–42, is used to monitor the effect of the compounds in the fluorescence.
These samples were left incubating in a water bath (CERTOMAT WR), for 24 h, at 37 ◦C
with gentle shaking [14].
A glycine-NaOH buffer (pH 8.5) containing ThT (5 µM) was added to each sample.
Samples were placed in a microplate and the fluorescence signal was monitored at 446 nm
(λexc) and 490 nm (λem) [40].
The percentage of inhibition was obtained, following the equation (2).
ꢀ
ꢁ
Fi
F0
%I = 100 −
× 100
(2)
where Fi and F0 are the fluorescence intensities in the presence and absence of the com-
pound, respectively, minus the fluorescence intensities of the respective blanks. Statistical
analysis was performed in Microsoft Office Excel®.
3.5. Cell Viability and Neuroprotection
SH-SY5Y human neuroblastoma cells (ATCC-CRL-2266) (American Type Culture
Collection, Manassas, Virgínia, USA) were plated in DMEM/F12 (Dulbecco’s Modified
Eagle Medium/Ham’s F-12 Nutrient Mixture; Thermo Fisher Scientific) with 10% heat
inactivated fetal bovine serum, 50 U/mL penicillin and 50 U/mL streptomycin under
a humidified atmosphere with 95% relative humidity at 37 ◦C at a 0.16
density. Each of the tested substances (6, 7, 8, 9
×
106 cells/mL
) were dissolved in DMSO to prepare a
stock solution at a 25 mM concentration and stored at
20 ◦C until further use. SH-SY5Y
−
cells were treated for 25 h with varying concentrations of compounds, in order to select
the highest non-toxic concentration. The selected concentrations for each compound were:
40
µM final concentration for
6; 15
µM final concentration for
7; 30 µM final concentration
for
8
and 2.5 M final concentration for 9
µ
. The final concentration of DMSO did not exceed
0.05% (v/v) and no alterations on cells were observed. For the neuroprotection assays,
the incubation with Aβ1–42 or L-ascorbic acid/ferrous sulphate treatment was preceded
with 1 h incubation of the compounds when needed. Pre-incubation was followed by 24 h
treatment of Aβ1–42 or Fe/Asc, accounting a total of 25 h. Aβ1–42 was prepared as 276.9
µM stock in sterile water and administrated in the medium at a 2.5 µM final concentration.
Ferrous sulphate was freshly prepared as 0.36 M stock in water and added to the medium at
2.5 mM final concentration. L-ascorbic acid was freshly prepared as 80 mM stock in water
and added to the medium at 5 mM final concentration. Aβ1–42 was purchased from Bachem
(Torrance, CA, USA) and ferrous sulphate and L-ascorbic acid from Sigma Chemical Co.
(St. Louis, MO, USA).
Cellular viability was determined by employing the MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) reduction assay, according to the Mosmann method [41].
This colorimetric test consists in the ability of the succinate dehydrogenase enzyme from
viable cells to metabolize the MTT into a formazan. In the end of treatments, cells medium

Molecules 2021, 26, 1658
13 of 15
was aspirated and cells were washed with 1
dead cells. MTT solution (200
×
PBS (phosphate-buffered saline) to remove
L, 0.5 mg/mL) was added to each well and incubated
µ
for 2 h protected from light. Formazan precipitates were then dissolved in DMSO and
absorbance measured at 570 nm. The ability to reduce MTT was expressed as a percentage
of untreated, control cells.
4. Conclusions
AD is recognized as a multifactorial complex disorder but current approved thera-
peutics, such as the acetylcholinesterase (AChE) inhibitor donepezil (DNP), only pro-
vides symptomatic relief. Following the promising recently adopted strategy of sin-
gle molecule-multiple target drugs associated with the advantage of repositioning al-
ready approved drugs, a new set of DNP-sulfonamide hybrids was herein developed.
These compounds include the DNP pharmacophore nucleus (N-benzylpiperidine or its
analogue N-benzylpiperazine) connected to substituted arylsulfonamide derivatives aimed
to improve the binding with AChE and its inhibition, as well as to inhibit the forma-
tion of amyloid-beta (Aβ) aggregates. The designed series of hybrids was synthesized,
fully characterized and bioassayed. The results of in vitro assays indicated a good inhibitory
potential against AChE and Aβ1–42 aggregation, namely for the benzylpiperidine-aryl sul-
fonamide derivatives (8 and 9), but in particular 9 (AChE (IC50 = 1.6 µM) and 60.7% Aβ1–42
aggregation), substantiating the computational simulation studies. Some compounds evi-
denced neuroprotection of SHSY5Y neuroblastoma cells, namely reverting neurotoxicity
induced by A
β
peptides. Importantly, the compounds also show good drug-like prop-
as the
erties with potential oral formulation. Overall, the results indicate compound
9
most promising DNP-sulfonamide hybrid of this series, which can be considered as a lead
compound for further developments on anti-AD drugs.
Author Contributions: Conceptualization, M.A.S. and L.P.; methodology, M.A.S., S.C. (Sílvia Chaves),
L.P. and S.M.C.; Investigation, F.Q., S.C. (Sonia Calò), K.G. and J.D.M.; Data Curation, S.C. (Sílvia
Chaves) and S.M.C.; Original Draft Preparation, F.Q., S.C. (Sonia Calò) and S.M.C.; Review & Editing,
M.A.S., S.C. (Sílvia Chaves) and L.P.; Supervision, M.A.S., S.C. (Sílvia Chaves), S.M.C. and L.P.
All authors have read and agreed to the published version of the manuscript.
Funding: Fundação para a Ciência e Tecnologia (FCT, Projects UID/QUI/00100/2013, UID/QUI/00100/
2019 and POCI-01-0145-FEDER-030712, Global Thesis Prize and Europe Erasmus. European Regional
Development Fund (ERDF), through the Centro 2020 Regional Operational Programme under project
CENTRO-01-0145-FEDER-000012 (HealthyAging2020) and through the COMPETE 2020-Operational
Programme for Competitiveness and Internationalization.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study is available in the article.
Acknowledgments: The authors acknowledge the Portuguese Fundação para a Ciência e Tecnologia
(FCT) for the projects UID/QUI/00100/2013, UID/QUI/00100/2019, POCI-01-0145-FEDER-030712
and also the Portuguese NMR (IST-UL Center) and Mass Spectrometry Networks (Node IST-CTN)
for providing access to their facilities. The authors S.C. and K.G. are thankful, respectively, to the
Global Thesis Prize and the Europe Erasmus program for providing fellowships.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors
References
1.
Cummings, J.L.; Lee, G.; Mortsdorf, T.; Ritter, A.; Zhong, K. Alzheimer’s disease drug development pipeline. Alzheimer’s Dement.
2019, 5, 272–293. [CrossRef]
2.
Zemek, F.; Drtinova, F.L.; Nepovimova, E.; Sepsova, V.; Korabecny, J.; Klimes, J.; Kuca, K. Outcomes of Alzheimer’s disease
therapy with acetylcholinesterase inhibitors and memantine. Expert Opin. Drug Saf. 2014, 13, 759–774.

Molecules 2021, 26, 1658
14 of 15
3.
Tan, C.C.; Yu, J.T.; Wang, H.F.; Tan, M.S.; Meng, X.F.; Wang, C.; Jiang, T.; Zhu, X.C.; Tan, L. Efficacy and safety of donepezil,
galantamine, rivastigmine, and memantine for the treatment of Alzheimer’s disease: A systematic review and meta-analysis.
J. Alzheimer’s Dis. 2014, 41, 615–631. [CrossRef] [PubMed]
4.
5.
Cummings, J.; Fox, N. Defining disease modifying therapy for Alzheimer’s disease. Prev. Alzheimers Dis. 2017, 4, 109–115.
Yiannopoulou, K.G.; Papageorgiou, S.G. Current and future treatments in Alzheimer disease: An update. J. Centr. Neurv. Syst.
Dis. 2020, 12, 1–12. [CrossRef] [PubMed]
6.
7.
8.
Gan, L.; Cookson, M.R.; Leonard Petrucelli, L.; La Spada, A.R. Converging pathways in neurodegeneration, from genetics to
mechanisms. Nat. Neurosci. 2018, 21, 1300–1309. [CrossRef] [PubMed]
Benek, O.; Korabecny, J.; Soukup, O. A perspective on multi-target drugs for Alzheimer’s disease. Trends Pharm. Sci. 2020, 41,
434–445. [CrossRef] [PubMed]
Savelieff, M.G.; Nam, G.; Kang, J.; Lee, H.J.; Lee, M.; Lim, M.H. Development of multifunctional molecules as potential therapeutic
candidates for Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis in the last decade. Chem. Rev. 2019, 119,
1221–1322. [CrossRef]
9.
Wang, N.; Qiu, P.; Cui, W.; Yan, X.; Zhang, B.; He, S. Recent advances in multi-target anti-Alzheimer disease compounds (2013 up
to the present). Curr. Med. Chem. 2019, 26, 5684–5710. [CrossRef]
10. Santos, M.A.; Chand, K.; Chaves, S. Recent progress in repositioning Alzheimer’s disease drugs based on a multitarget strategy.
Fut. Med. Chem. 2016, 8, 2113–2142. [CrossRef]
11. Ismaili, L.; Refouvelet, B.; Benchekroun, M.; Brogi, S.; Brindisi, M.; Gemma, S.; Campiani, G.; Filipic, S.; Agbaba, D.; Esteban, G.;
et al. Multitarget compounds bearing tacrine- and donepezil-like structural and functional motifs for the potential treatment of
Alzheimer’s disease. Prog. Neurobiol. 2017, 151, 4–34. [CrossRef]
12. Piemontese, L. New approaches for prevention and treatment of Alzheimer’s disease: A fascinating challenge. Neural Regen. Res.
2017, 12, 405–406. [CrossRef] [PubMed]
13. Chaves, S.; Várnagy, K.; Santos, M.A. Recent multi-target approaches on the development of anti-Alzheimer’s agents with metal
chelation activity. Curr. Med. Chem 2021, in press. [CrossRef] [PubMed]
14. Piemontese, L.; Tomas, D.; Hiremathad, A.; Capriati, V.; Candeias, E.; Cardoso, S.M.; Chaves, S.; Santos, M.A. Donepezil
structure-based hybrids as potential multifunctional anti-Alzheimer’s drug candidates. J. Enz. Inhib. Med. Chem. 2018, 33,
1212–1224. [CrossRef]
15. Chand, K.; Rajeshwari; Candeias, E.; Cardoso, S.M.; Chaves, S.; Santos, M.A. Tacrine–deferiprone hybrids as multi-targetdirected
metal chelators against Alzheimer’s disease: A two-in-one drug. Metallomics 2018, 10, 1460–1475. [CrossRef]
16. Chaves, S.; Resta, S.; Rinaldo, F.; Costa, M.; Josselin, R.; Gwizdala, K.; Piemontese, L.; Capriati, V.; Pereira-Santos, A.R.; Cardoso,
S.M.; et al. Design, synthesis and in vitro evaluation of hydroxybenzimidazole-donepezil analogues as multitarget-directed
ligands for the treatment of Alzheimer’s disease. Molecules 2020, 25, 985. [CrossRef]
17. Apaydin, S.; Torok, M. Sulfonamide derivatives as multi-target agents for complex diseases. Bioorg. Med. Chem. Lett. 2019, 29,
2042–2050. [CrossRef] [PubMed]
18. Bag, S.; Tulsan, R.; Sood, A.; Cho, H.; Redjeb, H.; Zhou, W.; LeVine, H., III; Torok, B.; Torok, M. Sulfonamides as multifunctional
agents for Alzheimer’s disease. Bioorg. Med. Chem. Lett. 2015, 25, 626–630. [CrossRef] [PubMed]
19. Ulus, R.; Kurt, B.Z.; Gazioglu, I.; Kaya, M. Microwave assisted synthesis of novel hybrid tacrine-sulfonamide derivatives and
investigation of their antioxidant and anticholinesterase activities. Bioorg. Chem. 2017, 70, 245–255. [CrossRef] [PubMed]
20. Makhaeva, G.F.; Kovaleva, N.V.; Boltneva, N.P.; Lushchekina, S.V.; Astakhova, T.Y.; Rudakova, E.V.; Proshin, A.N.; Serkov, I.V.;
Radchenko, E.V.; Palyulin, V.A.; et al. New hybrids of 4-amino-2,3-polymethylene-quinoline and p-tolylsulfonamide as dual
inhibitors of acetyl- and butyryl-cholinesterase and potential multifunctional agents for Alzheimer’s disease treatment. Molecules
2020, 25, 3915. [CrossRef] [PubMed]
21. Zhao, C.; Rakesh, K.P.; Ravidar, L.; Fang, W.Y.; Qin, H.L. Pharmaceutical and medicinal significance of sulfur (SVI)-containing
motifs for drug discovery: A critical review. Eur. J. Med. Chem. 2019, 162, 679–734. [CrossRef] [PubMed]
22. QikProp, Version 2.5; Schrödinger, LLC: New York, NY, USA, 2005.
23. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug. Deliv. Rev. 1997, 23, 3–25. [CrossRef]
24. Veber, D.F.; Johnson, S.R.; Cheng, H.-Y.; Smith, B.R.; Ward, K.W.; Kopple, K.D. Molecular properties that influence the oral
bioavailability of drug candidates. J. Med. Chem. 2002, 45, 2615–2623. [CrossRef]
25. Zagidullin, R.N. Reactions of N-(B-aminoethyl)piperazine and its derivatives. Chem. Heterocycl. Compd. 1991, 27, 309–312.
[CrossRef]
26. Piemontese, L.; Sergio, R.; Rinaldo, F.; Brunetti, L.; Perna, F.M.; Santos, M.A.; Capriati, V. Deep eutectic solvents as effective
reaction media for the synthesis of 2-hydroxyphenylbenzimidazole-based scaffolds en route to donepezil-like compounds.
Molecules 2020, 25, 574. [CrossRef] [PubMed]
27. Rizzo, S.; Bartolini, M.; Ceccarini, L.; Piazzi, L.; Gobbi, S.; Cavalli, A.; Recanatini, M.; Andrisano, V.; Rampa, A. Targeting
Alzheimer’s disease: Novel indanone hybrids bearing a pharmacophoric fragment of AP2238. Bioorg. Med. Chem. 2010, 18,
1749–1760. [CrossRef] [PubMed]
28. Murphy, M.P.; LeVine, H., 3rd. Alzheimer’s disease and the amyloid-beta peptide. J. Alzheimers Dis. 2010, 19, 311–323. [CrossRef]

Molecules 2021, 26, 1658
15 of 15
29. Zhao, Y.; Zhao, B. Oxidative stress and the pathogenesis of Alzheimer’s disease. Oxid. Med. Cell. Longev. 2013, 2013, 316523.
[CrossRef]
30. Peña-Bautista, C.; Tirle, T.; López-Nogueroles, M.; Vento, M.; Baquero, M.; Cháfer-Pericás, C. Oxidative damage of DNA as early
marker of Alzheimer’s disease. Int. J. Mol. Sci. 2019, 20, 6136. [CrossRef] [PubMed]
31. Armarego, W.L.F.; Perrin, D.D. Purification of Laboratory Chemicals, 4th ed.; Butterworth-Heinemann: Oxford, UK, 1999.
32. Jones, G.; Willett, P.; Glen, R.C.; Leach, A.R.; Taylor, R. Development and validation of a genetic algorithm for flexible docking. J.
Mol. Biol. 1997, 267, 727–748. [CrossRef]
33. Kryger, G.; Silman, I.; Sussman, J.L. Structure of acetylcholinesterase complexed with E2020 (Aricept (R)): Implications for the
design of new anti-Alzheimer drugs. Structure 1999, 7, 297–307. [CrossRef]
34. Cheung, J.; Rudolph, M.J.; Burshteyn, F.; Cassidy, M.S.; Gary, E.N.; Love, J.; Franklin, M.C.; Height, J.J. Structures of human
acetylcholinesterase in complex with pharmacologically important ligands. J. Med. Chem. 2012, 55, 10282–10286. [CrossRef]
35. Maestro, Version 9.3; Schrödinger Inc.: Portland, OR, USA, 2012.
36. Hassinen, T.; Perakyla, M. New energy terms for reduced protein models implemented in an off-lattice force field. J. Comp. Chem.
2001, 22, 1229–1242. [CrossRef]
37. Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF chimera—A visualization
system for exploratory research and analysis. J. Comp. Chem. 2004, 25, 1605–1612. [CrossRef]
38. Quintanova, C.; Keri, R.S.; Marques, S.M.; G-Fernandes, M.; Cardoso, S.M.; Serralheiro, M.L.; Santos, M.A. Design, synthesis
and bioevaluation of tacrine hybrids with cinnamate and cinnamylidene acetate derivatives as potential anti-Alzheimer drugs.
MedChemComm 2015, 6, 1969–1977. [CrossRef]
39. Sebestik, J.; Marques, S.M.; Fale, P.L.; Santos, S.; Arduino, D.M.; Cardoso, S.M.; Oliveira, C.R.; Serralheiro, M.L.M.; Santos, M.A.
Bifunctional phenolic-choline conjugates as anti-oxidants and acetylcholinesterase inhibitors. J. Enz. Inhib. Med. Chem. 2011, 26,
485–497. [CrossRef] [PubMed]
40. Hiremathad, A.; Chand, K.; Esteves, A.R.; Cardoso, S.M.; Ramsay, R.R.; Chaves, S.; Keri, R.S.; Santos, M.A. Tacrine-
allyl/propargylcysteine-benzothiazole trihybrids as potential anti-Alzheimer’s drug candidates. RSC Adv. 2016, 6, 53519–53532.
[CrossRef]
41. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.
J. Immunol. Methods 1983, 65, 55–63. [CrossRef]