Design, synthesis, and in vitro evaluation of hydroxybenzimidazole-donepezil analogues as multitarget-directed ligands for the treatment of Alzheimer's disease

Article abstract of DOI:10.3390/molecules25040985

A series of multi-target-directed ligands (MTDLs), obtained by attachment of a hydroxyphenylbenzimidazole (BIM) unit to donepezil (DNP) active mimetic moiety (benzylpiperidine /-piperazine) was designed, synthesized, and evaluated as potential anti-Alzheimer's disease (AD) drugs in terms of biological activity (inhibition of acetylcholinesterase (AChE) and β-amyloid (Aβ) aggregation), metal chelation, and neuroprotection capacity. Among the DNP-BIM hybrids studied herein, the structural isomerization did not significantly improve the biological properties, while some substitutions, namely fluorine atom in each moiety or the methoxy group in the benzyl ring, evidenced higher cholinergic AChE activity. All the compounds are able to chelate Cu and Zn metal ions through their bidentate BIM moieties, but compound 5, containing a three-dentate chelating unit, is the strongest Cu(II) chelator. Concerning the viability on neuroblastoma cells, compounds 9 and 10 displayed the highest reduction of Aβ-induced cell toxicity. In silico calculations of some pharmacokinetic descriptors indicate that all the compounds but the nitro derivatives have good potential oral-bioavailability. Overall, it can be concluded that most of the studied DNP-BIM conjugates showed quite good anti-AD properties, therefore deserving to be considered in further studies with the aim of understanding and treating AD.

Full text of DOI:10.3390/molecules25040985

molecules
Article
Design, Synthesis, and In Vitro Evaluation of
Hydroxybenzimidazole-Donepezil Analogues as
Multitarget-Directed Ligands for the Treatment of
Alzheimer’s Disease
Sílvia Chaves 1 , Simonetta Resta 1,2, Federica Rinaldo 1,2, Marina Costa 1 , Romane Josselin 1,
Karolina Gwizdala 1, Luca Piemontese 2 , Vito Capriati 2,3 , A. Raquel Pereira-Santos 4,5
,
Sandra M. Cardoso 4,5 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; silvia.chaves@tecnico.ulisboa.pt (S.C.);
s.resta7@studenti.uniba.it (S.R.); f.rinaldo@studenti.uniba.it (F.R.); marinamcosta91@gmail.com (M.C.);
josselinromane@gmail.com (R.J.); karo.gwizdala@gmail.com (K.G.)
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 (L.P.); vito.capriati@uniba.it (V.C.)
Consorzio C.I.N.M.P.I.S., Via E. Orabona 4, I-70125 Bari, Italy
CNC—Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal;
araqpsantos@gmail.com (A.R.P.-S.); cardoso.sandra.m@gmail.com (S.M.C.)
Institute of Molecular and Cell Biology, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra,
Portugal
2
3
4
5
*
Correspondence: masantos@tecnico.ulisboa.pt; Tel.: +351-218419273
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Diego Muñoz-Torrero
Received: 9 January 2020; Accepted: 18 February 2020; Published: 22 February 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A series of multi-target-directed ligands (MTDLs), obtained by attachment of a
hydroxyphenylbenzimidazole (BIM) unit to donepezil (DNP) active mimetic moiety (benzyl-
piperidine/-piperazine) was designed, synthesized, and evaluated as potential anti-Alzheimer’s
disease (AD) drugs in terms of biological activity (inhibition of acetylcholinesterase (AChE) and
β–amyloid (Aβ) aggregation), metal chelation, and neuroprotection capacity. Among the DNP-BIM
hybrids studied herein, the structural isomerization did not significantly improve the biological
properties, while some substitutions, namely fluorine atom in each moiety or the methoxy group in
the benzyl ring, evidenced higher cholinergic AChE activity. All the compounds are able to chelate Cu
and Zn metal ions through their bidentate BIM moieties, but compound
chelating unit, is the strongest Cu(II) chelator. Concerning the viability on neuroblastoma cells,
compounds and 10 displayed the highest reduction of A -induced cell toxicity. In silico calculations
5, containing a three-dentate
9
β
of some pharmacokinetic descriptors indicate that all the compounds but the nitro derivatives have
good potential oral-bioavailability. Overall, it can be concluded that most of the studied DNP-BIM
conjugates showed quite good anti-AD properties, therefore deserving to be considered in further
studies with the aim of understanding and treating AD.
Keywords: hydroxyphenyl-benzimidazole; donepezil; anti-neurodegeneratives; Alzheimer´s disease;
multifunctional drugs; metal chelation
1. Introduction
Alzheimer’s disease (AD) is a severe age-dependent neurodegenerative disorder, with no cure so
far, characterized by a drastic progressive decline in memory and cognitive abilities. With the increase
of life expectancy of humans, the number of AD patients is growing dramatically and in 2050 it will be
Molecules 2020, 25, 985; doi:10.3390/molecules25040985
www.mdpi.com/journal/molecules

Molecules 2020, 25, 985
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around 150 million worldwide [
1]. In spite of all the research performed to understand and treat AD,
there has been a high rate of failure in AD drug development programs [
therapies consist of acetylcholinesterase (AChE) inhibitors, such as donepezil (DNP, Aricept®), and an
N-methyl-d-aspartate receptor (NMDAR) antagonist (memantine) [ ]. However, they can only lead to
2,3]. The currently available
4
some temporary enhancement of neurotransmission levels (e.g., cholinergic and glutamatergic), and so
a valid therapy for AD remains an urgent unmet medical need. From the research efforts to understand
this disease, several factors have been considered to play important roles in the pathophysiology of
this illness, such as deficits of acetylcholine,
β-amyloid deposits, τ-protein phosphorylation, oxidative
stress, and metal dyshomeostasis [ ]. The disease complexity has been recognized as the main cause
5
for clinical failures of drugs based on the individual pathogenic targets. Therefore, over the last decade,
the need to design chemical tools capable of interacting with multiple pathological elements of AD
has prompted the scientific community and led to extensive research on multi-target-directed-ligands
(MTDLs) [6–8]. Under this strategy, many multifunctional molecules have been discovered with
the aim to combine the control of the symptoms with real disease modifying actions. Thus, various
approved acetylcholinesterase drugs have been repositioned for further derivatizations, by enclosing
or fusing other molecular entities to interact with some other AD targets, namely those related with
the toxic amyloid plaque, such as the inhibition of
-Secretase, BACE-1) and its aggregation, as well as the control of effects of oxidative stress and
metal dyshomeostasis, due to their recognized role in deleterious protein/peptide modification and
aggregation [ 12]. In particular, several DNP derivatives have been recently developed to associate the
cholinesterase inhibition with anti-oxidant capacity or with anti- amyloid cascade properties [13 14].
β-amyloid (Aβ) fibril formation (e.g., inhibition
β
9
–
β
,
Also, the implication of metal ions, such as copper, zinc, and iron, has long been recognized in AD,
associated to the enhancement of deleterious reactive oxygen species (ROS) in the brain (Fe, Cu) and to
intertwined roles (Cu, Zn) in β-amyloid aggregation [15–17]. Therefore, intensive research has recently
been dedicated towards the development of MTDLs enclosing metal chelating functions, as potential
anti-AD drug candidates (for reviews see refs [5,10,18,19]).
This subject has also been one of the main recent aims of our research group, with
MTDLs enclosing a molecular fragment based on AChEi drug (e.g., Tacrine, DNP), which
has been derivatized with a variety of functional moieties, several of them including metal
chelating groups (such as cysteine, hydroxypyridinone, hydroxycinamate, hydroxybenzofurane,
and hydroxyphenylbenzimidazole (BIM)) [19–22]. The interesting properties revealed by the
recently reported novel tacrine-hydroxyphenylbenzimidazole (TAC-BIM) hybrids [22] led to the
development of other MTDL analogues, by attachment of a BIM group to DNP active mimetic
ligands (benzyl-piperidine/benzyl-piperazine), to assure AChEi, inhibition of Aβ aggregation and
modulation of metal dyshomeostasis [23]. As a continuation of our efforts to search for new DNP-BIM
hybrids, herein we present a series of analogues (see Figure 1), which aims to explore the effect of
positional isomerization and introduction of a substituent on each of the above referred main multiple
anti-AD properties as well as on drug-likeness prediction. Discussion is also provided by establishing
comparison with previously reported DNP-BIM hybrids.

Molecules 2020, 25, 985
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O
3
R2
Y
OH
OH
N
N
N
N
N
H
R1
H
N
H
N
X
N
H
n
O
O
X
Y
C
C
C
C
C
C
n
1
2
1
2
2
2
R1
H
H
3-F
3-NO2
3-F
R2
H
H
H
H
H
H
OH
N
N
N
1
2
6
7
8
9
C
N
C
N
N
N
H
N
H
4
N
H
N
O
3-OMe
OH
N
10
11
12
N
N
N
C
C
N
2
2
2
H
H
H
2-F
2-NO2
H
N
H
5
Figure 1. Structures of the studied donepezil-hydroxyphenylbenzimidazole (DNP-BIM) hybrids.
2. Results and Discussion
2.1. Molecular Design and Docking
Taking into account the recognized medical interest on the donepezil (DNP) drug to ameliorate AD
symptoms, it was our inspiration to select N-benzylpiperidine and its isosteres benzylpiperazine and
benzylpyridine as the main pharmacophores in the design and development of new hybrid compounds
with multitarget capacity. The second pharmacophore herein explored is hydroxyphenylbenzimidazole
(BIM), aimed to mimic the indanone scaffold of DNP, and also to provide metal-chelating and
anti-Aβ aggregation capacity, similarly to other hybrid analogues recently reported [22,23]. Thus, the
N-benzylpiperidine and its isostere moieties should account to the most important binding interaction
of DNP within the catalytic anionic site (CAS) of AChE [10], while the BIM moiety should mimic the
indanone moiety in DNP and reinforce the interaction with AChE through its extra-binding to the
peripheral active site (PAS), thus enabling a dual binding mode.
Docking studies were performed with this set of new hybrids in order to gain some insight into
the differences in their binding interactions with the active site of AChE. Specifically, it was intended to
understand the effect of positional isomerization and of specific substitutions in the benzylpiperidine,
benzylpiperazine, and hydroxyphenylbenzimidazole (BIM) moieties on the ligand-enzyme binding,
namely in the dual binding mode. Similarly, as previously reported for compounds
1 and 2, the
docking simulations of this set of hybrids were performed with AChE (PDB code: 1EVE) [24], using
the X-ray crystal structure of the TcAChE complex with DNP. Firstly, the co-crystallized ligand (DNP)
was docked back into the binding site of the enzyme and superimposed with the native ligand (Figure
S1). Being satisfied with the good overlay, the same docking protocol was used for further docking
studies with the target compounds. The best docked poses of each synthesized compound were
selected and superimposed with that of DNP, as shown in Figure 2. Similar to the already published
simple analogues (
all the compounds have the first pharmacophore moieties, namely the benzyl-piperidinium (
-piperazinium ( 11), or -pyridinium (12), anchored to the CAS of AChE (bottom of the gorge), through
binding interactions via aromatic stacking with the phenyl ring from Trp 84A; at the middle of
the gorge, the charged nitrogen atom can establish a cation– binding interaction with the phenyl
1
,
2), they showed a pattern of orientation resembling that of DNP. In particular,
3–6),
7–
π-π
π
group of Phe330. Noteworthy is the observation that the benzyl-piperidinium moieties of positional
isomers (3–5) present some bending relative to that of DNP, though no significant effect appeared on the

Molecules 2020, 25, 985
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complexes with the benzyl-piperazinium substituted (10
,
11) or the benzyl-pyridinium (12) moieties.
Regarding the BIM moieties, similar to the DNP indanone ring, these moieties are at the entrance of the
gorge interacting via stacking with the peripheral anionic residue Trp279. However, these docking
π-π
simulations indicate for the BIM moieties less favorable interactions with PAS than DNP, which can
be mainly attributed to the fact that the tricyclic BIM moieties are longer than the bicyclic indanone
moiety of DNP. Furthermore, some additional distortion seems to occur for positional isomers (3–5
and substitution with big size substituents (-NO2, ; -OMe, ). In fact, the AChE inhibition assays
herein performed (see Section 2.4.1) confirm lower inhibitory activity for , and 11, probably due
to the existence of distorted configurations of these compounds inside the active site of the enzyme.
)
7
9
4, 5, 7
Interestingly, the substitution by the small size fluorine atom (
8,
10) does not seem to contribute to any
further distortion, as compared with the simple analogues (
1,
2), though from the docking studies we
cannot observe any engaged positive binding interactions.
1
2
3
4
5
6
Figure 2. Cont.

Molecules 2020, 25, 985
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7
8
10
9
11
12
Figure 2. Superimposition of the different DNP-BIM hybrids and the original ligand (donepezil-green),
as a result of their docking inside the acetylcholinesterase (AChE) active site (PDB code 1EVE) [24].
2.2. Synthesis
Twelve hybrid compounds resulted from the conjugation of a hydroxyphenylbenzimidazole (BIM)
moiety with benzylpiperidine (PP, 7−11), and also benzylpyridine
3−6), benzylpiperazine (PZ,
12). The hybrids were obtained by following the convergent synthetic approach depicted in Scheme 1.
1
,
2,
(
The preparation of each final intermediate moiety,
10 12b) and -series of carboxyl BIM derivatives (1c
reported [22 23]. More specifically, the synthesis of the benzyl-cyclic amino derivatives (
b
-series of benzyl amine derivatives (
1
–
4b and
–
c
,
, 5–9c), involved mostly procedures previously
b
-series)
involved a first one-pot-two-step reaction, with preliminary protection of the primary alkylamine
group to a phthalimide group by its reaction with phthalic anhydride (FA) in absence of solvent and
subsequent benzylation of the secondary cyclic amine with benzyl bromide in dry acetonitrile under
basic conditions; the final deprotection of the primary amine was performed with hydrazine hydrate in
refluxing ethanol. The preparation of the c-series of intermediates, hydroxyphenylbenzimidazole acid
derivatives, involved the cyclization of diaminobenzoic acid derivatives with diverse salicylaldehydes,
in the presence of sodium metabissulfite and N,N-dimethylacetamide (DMA) (method A) [22]. However,
for some compounds (6c
, 7c, 9c), an eutectic solvent mixture, choline chloride/glycerol (ChCl/Gly), was
used (method B) instead of DMA, with improved effectiveness [25]. The last reaction step involved the

Molecules 2020, 25, 985
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coupling of amine intermediate moieties (
b
-series) with carboxylic intermediate moieties (
c
-series), via
amide formation, and it was carried out in dry DMF, using NHS and DCC as activators.
R2
R2
Y
Y
O
O
O
H
N
N
X
N
X
iii)
i)
k
k
l
j
l
j
ii)
R2
X
Y
O
n
n
n
BrH2C
N
H2N
H2N
n
X
l/j/k
Y
R2
O
1b 1
C
N
C
1/1/1 C
1/1/1 C
0/1/2 C
H
H
H
2b 2
3b 1
4b 1
C
0/0/3 C
H
10b 2
N
N
N
1/1/1 C
1/1/1 C
1/1/1 N
H
H
-
1a-4a
10a-12a
11b 2
12b 2
p
p
H
N
CO2H
iv)
CO2H
H2N
CHO
R1
q
q
R1
N
H2N
OH
R1
OH
p/q R1
1c 1/1
5c 3/0 H
5-F
5-NO2
4-OCH3
H
6c 1/1 5-F
7c 1/1 5-NO2
9c 1/1 4-OCH3
O
H
N
X
N
H
N
v)
N
R1
OH
R2
Y
n
X
l/j/k
Y
p/q R1
R2
1
2
3
4
5
6
7
8
9
1
2
1
1
1
1
2
2
2
C
N
C
C
C
C
N
N
N
N
N
N
1/1/1 C i/j
1/1/1 C 1/1
0/1/2 C 1/1
0/0/3 C 1/1
1/1/1 C 3/0
1/1/1 C 1/1 5-F
1/1/1 C 1/1 5-NO2
1/1/1 C 1/1 5-F
H
H
H
H
H
H
H
H
H
H
H
H
H
1/1/1 C 1/1 4-OMe H
10 2
11 2
12 2
1/1/1 C 1/1
1/1/1 C 1/1
1/1/1 N 1/1
H
H
H
F
NO2
-
Scheme 1. Reagents and conditions: (i) Phthalic anhydride, net, 180 ◦C, 4 h; (ii), TEA, K2CO3, ACN,
50 ◦C, 3 h; (iii) N2H4.xH2O; (iv) (A) Na2S2O5, DMA, 100 ◦C, 12 h; (B) Na2S2O5, ChCl/Gly, 50 ◦C, 30 min;
(v) DCC, N-hydroxysuccinimide, dry DMF, RT, 40 h.

Molecules 2020, 25, 985
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2.3. Metal Chelation
In order to evaluate the chelating capacity of this type of compound towards Cu(II) and Zn(II),
some of them ( and 12) were selected and their acid-base behavior was firstly studied. A working
7
medium of 50% (w/w) DMSO/water was chosen due to solubility reasons and also to enable the
comparison with results for other hybrids, previously reported in the same medium. Although a
50% DMSO/water (w/w) medium has been implemented to perform these solution equilibrium assays,
in terms of cellular studies the concentration of ligand employed is significantly lower (<7 µM) and
therefore the final concentration of DMSO used in culture media would be inferior (<1%) and with no
associated modifications in biological tissues.
The protonation constants of the compounds were determined by potentiometry (see Figure 3
and Table 1) by using Hyperquad [26].
12
10
8
12
10
8
pH
pH
6
6
L
L
4
4
Cu/L 1:2
Zn/L 1:2
Cu/L 1:2
Zn/L 1:2
2
2
-2
-1
0
1
2
3
-2
-1
0
1
2
3
a
a
(A)
(B)
Figure 3. Potentiometric titration curves of compounds
7
(
A
) and 12
(
B
) (CL = 6.7
×
10−4 M, a represents
moles of added base per mole of ligand). Experimental conditions: 50% w/w DMSO/water, I = 0.1 M KCl,
T = 25.0 ± 0.1 ◦C.
Compound
7 was isolated in its neutral mono-protonated form (HL, a = 0 in Figure 3), although
it has three dissociable protons (H3L2+), while compound 12, with four dissociable protons (H4L3+),
was isolated in its mono-positive charged di-protonated form (H2L+, a = 0 in Figure 3). Table 1 shows
three values of protonation constants (log Ki = 6.99, 5.51, and 2.95) calculated for compound
7
and four
values for compound 12 (log Ki = 9.09, 6.44, 3.64, and 3.17).
1H NMR titration studies previously reported revealed that, for compounds
1 and 2, the sequence
of protonation corresponds to the following order: firstly, the phenolic oxygen of the BIM moiety,
afterwards the N-piperazine nitrogen (opposite to benzyl group)/N-piperidine nitrogen atom, and
lastly the imidazole nitrogen N(3) of the BIM moiety. Concerning compound
sequence of protonation, both log K1 (6.99) and log K2 (5.51) are much lower than the corresponding
ones for compound (see Table 1). The decrease of log K value corresponding to the phenolic oxygen is
7, with analogous
2
attributed to the resonance electron withdrawing nature of the nitro group present at the para position
(e.g., phenol 9.98 [27] and 4-nitrophenol 7.15 [28]), that stabilizes the conjugated base. Regarding
compound 12, the first two and the fourth protonation constants correspond to those of the analogous
compound 2, while the third one (log K3 = 3.64) is attributed to the pyridine nitrogen; the decrease of
the log K value, when compared with that of pyridine (5.24, in water [29]), may be due to the electron
withdrawing nature of the neighbor nitrogen atom from the piperazine moiety, that stabilizes the
conjugated base by resonance effect. Finally, for all the hybrids contained in Table 1, the decreasing
of the protonation constants corresponding to the N(3) atom (log K3 = 2.86 3.37) and O-phenol
−
(log K1 = 6.99−9.09), when compared with equivalent standard groups (e.g., imidazole 6.95 [30] and
phenol 9.98 [27]), reflects the existence of intramolecular hydrogen bond interactions involving these
atoms in the BIM moiety.

Molecules 2020, 25, 985
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Table 1. Stepwise protonation constants (log Ki) of compounds 7, 12 and other analogues for comparison
purposes, as well as global formation constants a for their Cu(II) and Zn(II)) complexes (T = 25.0
±
0.1 ◦ C, I = 0.1 M KCl, 50% w/w DMSO/water) and pM b values.
Compound
MmHhLl
log Ki
log βCu
log βZn
mHhLl
mHhLl
(mhl)
(011)
(021)
(031)
(111)
(101)
(122)
(102)
(1–12)
pM
6.99(2)
5.51(5)
2.95(6)
14.23(5)
10.40(8)
-
4.98(8)
21.92(7)
10.22(8)
-
26.90(7)
15.27(8)
7.30(9)
10.9
7
6.5
(011)
(021)
(031)
(041)
(111)
(101)
(102)
(1–12)
pM
9.09(3)
6.44(4)
3.64(5)
3.17(6)
16.26(3)
11.18(5)
17.14(7)
7.88(7)
10.4
12.58(4)
6.26(5)
10.54(5)
2.07(7)
6.3
12
(011)
(021)
(031)
pM
8.671(1) c
7.39(1) c
3.21(1) c
10.7/
10.7
14.3
6.3
6.3
6.4
1 c
(011)
(021)
(031)
pM
9.04(3)
6.76(6)
3.37(7)
2 c
(011)
(021)
(031)
pM
8.98(2)
7.78(4)
2.86(5)
5d
a
βM
= [MmHhLl]/([M]m[H]h[L]l); b pM = log[M] at pH 7.4 (CL/CM = 10, CM = 10−6 M), ref [32]; c ref. [33]; d
−
H
L
l
m
h
ref [31].
Regarding compound
sequence of protonation were found to be similar to those of compound 1.
The metal chelating ability of hybrids and 12, due to the BIM bidentate unity with (N,O)
5 (see Table 1), previously studied [31], its three protonation constants and
7
coordination mode, was studied by potentiometry. Figure 3A,B show a change in the deprotonation
profile of the ligand titration curves due to the presence of the metal ions. In fact, the curves for the
Cu(II)/L systems lie well below that of the ligand, while for the Zn(II)/L systems stay under for a >
−
1 (compound 7) or a > 0 (compound 12), thus supporting the formation of 1:1 and 1:2 M/L ratio
complexes with the deprotonated and mono-protonated forms of the ligand and with relative metal
dependent stability order (Cu > Zn). Table 1 contains the equilibrium models obtained from the fitting
analysis of the potentiometric curves by program Hyperquad [26]. In these equilibrium models, which
the coordination core of the compounds integrates the bidentate (N,O) BIM moiety, MHL and MH2L2

Molecules 2020, 25, 985
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correspond to species with the ligand containing one protonated nitrogen atom at the piperazine
moiety, ML and ML2 correspond to complexes involving the completely deprotonated form of the
ligand while MH-1L2 are mixed ligand-hydroxo metal complexes.
Figure 4 shows the species distribution curves for the 1:2 M(II)/
L (M = Cu, Zn) systems,
involving compounds
7
and 12, by using the potentiometric determined model at the used
experimental conditions.
100
100
ZnL2
Zn
CuH2L2
CuHL
Cu
80
60
40
20
0
80
60
40
20
0
CuL2
ZnH2L2
CuH-1L2
ZnH-2
ZnH-3
ZnL
ZnHL
2
4
6
8
3
5
7
pH
pH
a)
b)
100
80
60
40
20
0
100
80
60
40
20
0
CuHL
Zn
CuL
CuL2
CuH-1L2
8
Cu
ZnL
ZnL2
ZnHL
ZnH-2
ZnH-1L2
2
4
6
4.0
5.0
6.0
pH
7.0
8.0
pH
c)
d)
Figure 4. Species distribution curves for the 1:2 M(II)/L systems with compound
7
and
a) M = Cu,
b
) M
= Zn, as well as with compound 12, and c) M = Cu, d) M = Zn (CL = 6.7 × 10−4 M).
The species distribution curves show that, for the concentrations used in the experimental
conditions, the metal complexation with compounds and 12 starts at pH around 2 for Cu(II) or above
7
3.5–4 for Zn(II), with the formation of MHL species. Moreover, both 1:1 (MHL, ML) and 1:2 (MH2L2,
ML2, MH-1L2) species are formed and the mixed hydroxo-ligand complex (MH-1L2) starts to appear
above pH 6–7. For the Zn(II)/L systems, some competition with metal hydroxide species occurs above
pH 6.5–7. Previous results from UV-VIS spectral data collected for the analogous compounds
1 and 2
pointed towards a chelating core composed by the N(3) imidazole and O-phenol atoms of the ligands,
excluding the hypothesis of metal coordination through the distant nitrogen atoms of piperidine or
piperazine [33]. Therefore, it is expectable that the same coordination core is kept for hybrids
7 and 12.
Comparison of the metal chelating capacity of compounds and 12 with that of the already
7
studied analogous DNP-BIM hybrids (see Table 1) reveals that these kind of ligands are good binders
of Cu(II) and only moderate for Zn(II). In fact, the calculated pM values (at pH 7.4, CL/CM = 10,
C
M = 10−6 M) for the set of DNP-BIM hybrids studied are higher for Cu(II) (pCu = 10.4
−
14.3) than
, it is possible to
see some influence of the p-nitro group on the increase of stability of the metal complexes, although
compound has lower total basicity than . Noteworthy is also the higher chelating capacity towards
Cu(II) of compound vs. (pCu ( ) = 14.3, pCu ( ) = 10.7), attributable to the possibility of compound
being able to establish a tridentate (N,O,O) coordination to the metal ion, by including the adjacent
carbonyl oxygen atom [30], with correspondingly higher stability of its copper complex.
for zinc (pZn = 6.3 6.5). Moreover, by comparing the pM values relative to 7 and 2
−
7
2
5
1
5
1
5

Molecules 2020, 25, 985
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2.4. Biological Studies in Solution
2.4.1. Acetylcholinesterase (AChE) Inhibition
The inhibition of AChE by the DNP-BIM hybrids herein studied was assayed by using a variation
of Ellman s test [23 34] and the IC50 values obtained are included in Table 2. All the inhibitors, but
the benzyl-pyridinium derivative (12), present IC50 values in low micromolar range. Among the
piperidine (PP) series (PP-BIM), the best results were obtained with compounds , and , while
for the piperazine (PZ) series (PZ-BIM), the most potent were compounds , and 10. Although the
present hybrids are weaker AChE inhibitors than DNP (IC50 = 0.026 M), this does not weaken the
´
,
1,
3
6
8, 9
µ
proposed drug design since the advantages of using a multi-target drug in multifactorial diseases can
surpass the drawback of losing some specific property of a single-target drug.
Table 2. Summary of results obtained for inhibition of AChE and β–amyloid (Aβ) aggregation of the
DNP-BIM hybrids.
Aβ
Aggregation Inhibitionb,c
(%)
1–42
TcAChE Inhibition a
Compound
IC50 (µM)
Self-Induced
Cu-Induced
1
2
3
4
5
6
7
8
9
48.8 e
25.3 d
71.4
47.6
38.6 e
51.2
57.6
50.6
43.7
39.1
22.7
30.1
-
69.0 e
37.7 d
81.8
55.9
58.9 e
61.0
71.4
55.2
66.0
75.8
43.5
44.4
-
4.2 ± 0.8 d
6.9 ± 0.6 d
5.0 ± 0.5
14 ± 1
9 ± 1 e
1.67 ± 0.05
21 ± 3
4.3 ± 0.3
1.87 ± 0.8
4.32 ± 0.03
20 ± 1
10
11
12
DNPf
>102
0.026
a
b
The values are means of three independent experiments
β42 aggregation (%) with or without copper (40 M). The thioflavin-T fluorescence method was used, and the
measurements were carried out in the presence of an inhibitor (40
±
SD; AChE from Electric eel. Inhibition of self-mediated
A
µ
c
µM and 20
µ
M for compound
4
). The values
d
are the means of three independent measurements in duplicate (SEM < 10%). Reference [31] (A
β
aggregation
inhibitory assays for Cinhibitor = 80 µM); e reference [31]; f reference [35].
In order to understand the effect of different structural changes on the inhibitory capacity of
AChE such as the existence of positional isomers (compounds , and ) or the inclusion of R1 or R2
1
,
3
4
substituents, a brief structure-activity analysis can be performed based on graphical representation of
the enzymatic activity (IC50) (Figure 5).
Analysis of the data illustrated in Figure 5A (or Table 2), shows that the inhibitory capacity of the
positional isomers on the PP moiety is according to the following sequential order: para (
ortho ( ). Concerning the attachment point in the BIM moiety, the para position ( ) is also preferred
to the ortho one ( ). Therefore, apart from compounds , and , this series of DNP-BIM hybrids is
1) > meta (3) >
4
1
5
3,
4
5
mainly composed by positional para isomers, both in the PP and in the BIM moieties, aimed to be better
accommodated in the enzyme structure and so with higher inhibitory activity against AChE. Figure 5A
also shows that the inclusion of a fluorine in the BIM moiety leads to an activity improvement e.g.,
1 versus 6).

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25
20
15
10
5
0
4
4
5
5
6
6
11
33
para-PP
meta-PP
ortho-PP
R1=H
R1=H
R1=F
R1=F
(A)
(B)
Figure 5. Graphical summary of the effect of different structural parameters on the inhibition of AChE
(AChEi, IC50) for: (A) PP-BIM hybrids; (B) PZ-BIM hybrids.
On the other hand, Figure 5B shows the effect of substituent groups, as R1 in the BIM moiety or
R2 in the benzyl of the PZ unity. In both types of substitutions, it is evident that the fluorine (and also
R1 = -OMe) leads to enhancement of the inhibitory capacity, while the nitro group decreases its value.
Overall, the best AChEi activity was achieved for para-/para- hybrids 6 and 9, with fluorine and
methoxy R1 substituents in the BIM moiety, respectively, which demonstrates the determinant role of
these substituents in the establishment of interactions within the active site of AChE.
2.4.2. Inhibition of Aβ1–42 Aggregation
The anti-amyloidogenic capacity of the compounds was also evaluated in vitro, in the absence
and presence of Cu(II), based on the measurement of the fluorescence emission of Thioflavin T (ThT),
a stain known by its Aβ fibril binding capacity [36,37]. This binding interaction can be analyzed by
fluorimetry, since the presence of ThT-fibrils increases the absorbance and the emission of the ThT dye,
and it also induces red shifts on the absorbance (from 385 to 446 nm) and emission peaks (from 445 to
485 nm) [22]. All the measurements were performed after incubation (24 h, 37 ◦C) of the self-mediated
and Cu2+-induced Aβ aggregates in the presence/absence of the compounds under evaluation.
In fact, it is well known that A
β
binds Cu(II) and, although this interaction has been associated to
the induction of A aggregation [15
β
,16], it has also been admitted that it can lead to the precipitation
of amorphous deposits of the peptide and not to ThT-positive
with different studies being performed on the analysis of the effect of Cu(II) on the propensity for A
fibril formation as well as on the effect of metal chelators on this process [38 39]. Several reported
fluorescence studies based on the ThT dye have been performed in different experimental conditions
(solvent used for A , pH, and incubation time), which turn difficult comparison of results. Under
our experimental method it was observed a tendency for decreasing the fluorescence intensity for A
in the presence of copper, in comparison with its absence, which may be due to some precipitation
of amorphous deposits of the peptide rather than formation of sheets [38 39]. In former studies,
with TAC-BIM derivatives ( ) [22 23 33], a fluorescence-independent method like transmission
β sheet rich amyloid fibril formation
β
,
β
β
β
,
1,
2
, ,
electron microscopy (TEM) was used, due to possible quenching interferences in the emission of the
paramagnetic copper ion, and it was observed that scarcer aggregates appear in the presence of Cu(II)
when comparing Aβ with Aβ + Cu(II). Furthermore, it has been proposed that metal chelators might
either dissolve the deposits in the brain tissue or prevent Aβ aggregation.
Herein, due to solubility problems of some compounds in the phosphate buffer (0.215 M, pH 8.0)
of the working medium, the assays were carried out in the presence of 40 µM concentration of

Molecules 2020, 25, 985
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inhibitor and 20
µM in the case of compound
4, instead of 80
µM as previously reported [33]. The
obtained data, expressed as percentage of aggregation inhibition, is included in Table 2 and graphically
presented in Figure 6. The results show that all the compounds are able to inhibit A aggregation
β
with good-to-moderate activity. The activity dependence on ligand structural features may be mainly
explained by the different ability for intercalation into the fibrils, as already assumed for other
inhibitors [22
and Cu-induced A
. The data obtained for compounds
in the BIM have some small positive effect on the inhibition of A
compounds 10 11, and 12 showed decreased inhibitory activity, which may result from a somehow
,
40]. Among the hybrids under study,
aggregation, although very good inhibitory activity is also presented by
and indicate that the presence of R1 = F, -NO2 substituents
aggregation. On the other hand,
3
exhibited the highest inhibitory activity for self-
β
1, 6, and
7
6
7
β
5,
,
more difficult intercalation into the fibrils, when there is an ortho-attached BIM or when the PZ benzyl
unity is substituted.
90
80
70
60
50
40
30
20
10
0
10
5
9
4
7
12
7
2
8
3
11
6
1
2
Cu-induced aggregation
self-Aβ aggregation
(A)
(B)
Figure 6. Graphical summary of the effect of structural parameters on the inhibition of A
β
aggregation
(self- and Cu-induced, in %) for: (A) PP-BIM hybrids; (B) PZ-BIM hybrids.
Concerning the inhibition of Cu-induced Aβ aggregation, all the compounds induce an increase
when comparing with their corresponding capacity for self-inhibition, in accordance with their
good chelating capacity towards copper. Nevertheless, these chelators have conditional dissociation
constants (K
), 80 nanomolar (
picomolar corresponding to chelators eventually able to retrieve copper from A
picomolar–100 nanomolar for Cu(A ) complexes) [41]. Therefore, it seems that there is no competition
for copper between these BIM hybrids and A peptide, and so the inhibition process must be mainly
due to ligand intercalation between the -sheets of A fibrils. TEM assays previously performed by
us on analogous BIM hybrids [22 23 33] also confirmed the role of these kinds of compounds in the
´
d) for the copper complexes at pH 7.4 of 34 nanomolar (
1
), 24 nanomolar (
2
), 254 picomolar
= 1 10
peptide (K´ = 10
d
(5
7), and 365 picomolar (12). These values are outside the proposed range K
´
d
−
β
β
β
β
β
,
,
reduction of Aβ aggregation, both in the absence and presence of copper.
2.5. Cell Viability and Neuroprotection
The neuroprotective effect of DNP-BIM hybrids designed for AD was evaluated using SH-SY5Y
cell line treated with Aβ1–42 or ferrous sulfate and L-Ascorbic acid (Fe/Asc). A dose-response curve
was performed to select a non-toxic concentration of each compound (Figure 7). For all the compounds,
a concentration of 2.5 µM showed to be the highest non-toxic value.
Extracellular formation of senile plaques of aggregated A
β along with the production of reactive
oxygen species (ROS) are processes involved in the neurodegeneration that characterize AD [42].

Molecules 2020, 25, 985
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Herein, we observed that A
β
induces a decrease in cell viability close to 50% and, remarkably,
compounds
9 and 10 prevented Aβ-induced cell toxicity (Figure 8). Additionally, ROS production
is also one of the hallmarks associated with AD pathogenesis [42]. To study the neuroprotective
effect of DNP-BIM hybrids against oxidative stress, we incubated our cell model with Fe/Asc, which
showed a decrease of 25% in the cell viability when compared with untreated cells (Figure 9). However,
none of the compounds presented a statistically significant neuroprotective effect by preventing ROS
production, even the compounds 9 and 10 that showed to be able to inhibit Aβ aggregation.
1.5
1.0
**
**
*
*
*
***
0.5
0.0
12
7
8
9
10
11
Figure 7. Dose-response screening to select non-toxic concentrations of compounds
7–
12. Cells
were treated with different concentrations of the compounds for 24 h and MTT reduction assay was
performed to evaluate cell viability. Results are expressed relatively to SH-SY5Y untreated cells, with
the mean
±
standard error of the mean (SEM) derived from four different experiments. * p < 0.05;
** p < 0.01; *** p < 0.001, significantly different when compared with SH-SY5Y untreated cells.
1.5
##
####
1.0
0.5
0.0
*
***
*
****
****
****
Aβ1-42
Figure 8. Neuroprotective effect of compounds
Cells were treated with Aβ42 peptide (1 M) for 24 h in the presence or absence of the compounds (1 h
pre-incubation plus 24 h co-incubation). Evaluation of cell viability was performed using the MTT
reduction assay. Results are expressed relatively to SH-SY5Y untreated cells, with the mean SEM
7–12 from Aβ42-induced toxicity on SH-SY5Y cells.
µ
±
derived from four different experiments. * p < 0.05; *** p < 0.001; **** p < 0.0001, significantly different
when compared with SH-SY5Y untreated cells; ## p < 0.01; #### p < 0.0001, significantly different when
compared with Aβ42 treated SH-SY5Y cells. The concentration used for all the compounds was 2.5 µM.

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Figure 9. Neuroprotective effect of compounds 7–12 from L-Ascorbic Acid (AscH (-))/Ferrous sulphate
(Fe) toxicity on SH-SY5Y cells. Cells were treated with Fe/Asc (2.5 mM and 5 mM, respectively) for
24 h in the absence or in the presence of the compounds (1 h pre-incubation plus 24 h co-incubation).
Evaluation of cell viability was performed using the MTT reduction assay. Results are expressed
relative to SH-SY5Y untreated cells, with the mean
The concentration used for all the compounds was 2.5 µM.
±
SEM derived from four different experiments.
2.6. Prediction of Pharmacokinetic Properties
To get some insight on the drug-likeness of the studied hybrid compounds, in silico calculations
of some pharmacokinetic descriptors were performed by the QikProp program [43]. Analysis of the
selection of calculated parameters depicted in Table 3 shows that almost all the compounds present no
violations of Lipinski’s rule of five [44], which can be indicative of their potential oral-bioavailability.
However, compounds
higher than 500 Da (516.55 and 500.55), resulting in one violation of that rule. The range of values
(2.586 4.513) calculated for log P (clog P, octanol-water partition coefficient) are within the normal
range of values calculated for drugs ( 2 to 6.5) [43], although hydrophilic character is generally higher
7 and 11, with a nitro substituent, present a molecular weight (MW) slightly
−
−
for the piperazinic derivatives (X = N), as compared with the piperidinic analogues (X = C). To assess
the capacity of the compound to cross the blood-brain barrier (BBB), log BB was calculated. The range
of values obtained for log BB are also within the range calculated for drugs (
−
3 to 1.2) [43], although it is
generally accepted that compounds with log BB < 1 are poorly distributed in the brain and so with low
−
CNS activity. Regarding the Caco-2 permeability, the values obtained for the piperidinic derivatives
are higher than for the piperazinic analogues (<100), thus indicating for this last series of compounds a
more moderate absorption from the intestinal tract to the blood. Overall, the remarkably low values
obtained for Caco-2 permeability of the nitro-derivatives (<10), together with the lowest values for
clog P and log BB, suggest that these hybrids should be excluded from further drug development.
Overall, excepting the nitro-derivatives, generally, this set of hybrids presents well balanced
lipophilic/hydrophilic character and moderate blood-brain barrier permeability (log BB), thus making
them eligible as potential drug candidates.

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Table 3. Pharmacokinetic properties of the compounds as predicted in silico by software QikProp
v.2.5 [43].
MW
(Da)
Caco-2 Permeability
Violations of
Lipinski’s Rule
Comp.
clog P
log BB
CNS
(nm/s)
1
2
3
4
5
6
7
8
9
440.54
455.55
440.54
440.54
440.54
458.53
516.55
473.54
485.58
473.54
500.55
456.54
379.50
4.386
2.587
4.122
4.202
4.352
4.513
2.421
3.490
2.840
3.455-
2.604
2.686
4.269
−0.730
−0.097
−0.825
−0.981
−0.638
−0.663
−1.148
−0.147
−0.029
−0.385
−1.641
−0.597
0.132
194
48
-
+/−
-
0
0
0
0
0
0
1
0
0
0
1
0
0
155
121
225
177
7
61
79
45
6
-
+/−
+/-
-
+/−
-
10
11
12
DNP
+/−
-
34
893
+/−
+
3. Materials and Methods
3.1. Chemicals
For the synthesis, 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 [45]. 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 60Darmstadt, 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 III (Microsystems,
1
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
spectrometer (Varian Inc., Palo Alto, CA, USA) equipped with an ESI ion source, operated in the
positive ion mode.
3.2. Synthesis of the Compounds
3.2.1. Synthesis of the Benzyl-Piperidine as Well as Benzyl- and Pyridinyl-Piperazine Alkylamino
Derivatives (1–4b,10–12b)
3.2.1.1. Synthesis of the N-benzyl Compounds with Isoindoline-1,3-Dione Protection (1–4a, 10–12a)
General Procedure
Phthalic acid anhydride (1 eq) and the aminoalkyl-piperidines or -piperazine derivatives
(i-(aminomethyl)piperidine (i = 2,3,4) or 1-(2-aminoethyl)piperazine (1 eq) were heated at 160 ◦C for 4 h.
The resulting dark brown solid (1 eq) was mixed with K2CO3 (6.6 eq), triethylamine (1.4 eq), and benzyl
bromide derivatives or 2-(bromomethylpyridine (2.3 eq), and refluxed in acetonitrile for 24 h. Then, the
mixture was cooled at rt, filtered and to the organic phase was added water, and then extracted with
ethyl acetate (three times). The organic layers were collected, dried over anhydrous sodium sulphate,

Molecules 2020, 25, 985
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and concentrated under reduced pressure. The crude material was purified through chromatography
column (with eluent: DCM/MeOH/TEA (98:2:0.1) or ACN/H2O (7:1)) or recrystallization.
2-((1-Benzylpiperidine-4-yl)methyl)isoindoline-1,3-dione (1a). Starting from 4-(aminomethyl)piperidine
and afterwards benzyl bromide the a pure title compound was obtained as a yellow solid (
M.P. = 129 (ppm): 1.24 1.38, 1.52 1.91, 3.44 3.50 (m, 9H,
132 ◦C). 1H RMN (300 MHz, DMSO-d6),
7.40 (m, 5H, Ph), 7.79–7.91
η = 40%),
−
δ
−
−
−
piperidine), 2.73 (d, 2H, J = 12.0 Hz, NCH2CH), 3.41 (s, 2H, NCH2Ph), 7.15
−
(m, 4H, PhTh). M/z (ESI-MS): 335 (M + 1)+.
2-(2-(4-Benzylpiperazin-1-yl)ethyl)isoindoline-1,3-dione (2a). Starting from 1-(2-aminoethyl)piperazine,
the crude product was washed with diethylether affording the pure title compound as a yellow solid
1
(η
= 35%). H NMR (300 MHz, MeOD–d4),
δ
(ppm): 2.384 2.640 (m, 8H, piperazine), 2.675 (t, 2H,
−
J = 6.0 Hz, PhthCH2CH2N), 3.52 (s, 2H, NCH2Ph) 3.801 (t, 2H, J = 6.0 Hz, Phth-CH2CH2N), 7.25
−
7.36
(m, 5H, Ph-CH2), 7.80−7.90 (m, 4H, Phth). M/z (ESI-MS): 350 (M+H)+.
2-((1-Benzylpiperidine-3-yl)methyl)isoindoline-1,3-dione (3a). Starting from 3-(aminomethyl)piperidine
and afterwards benzyl bromide, the crude solid compound was purified by chromatography column
1
(with eluent ACN/H2O, 7/1) affording the pure title compound as yellow solid (
η
= 40%). H RMN
(300 MHz, MeOD-d4),
δ
(ppm): 1.41–1.46, 1.71–1.99, 2.20–2.27 (m, 9H, piperidine), 2.88 (d, 2H,
J = 12.0 Hz, NCH2CH), 3.49 (s, 2H, NCH2Ph), 7.40–7.57 (m, 9H, PhCH2, Phth). M/z (ESI-MS): 335
(M + 1)+.
2-((1-Benzylpiperidine-2-yl)methyl)isoindoline-1,3-dione (4a
afterwards benzyl bromide, the crude solid compound was purified by recrystallization ACN/MeOH,
affording a pure compound as a yellow solid (
= 43%, M.P. = 130 ◦C). 1H RMN (300 MHz, MeOD-d4),
(ppm): 1.37 1.80 (M, 6H, piperidine), 2.02 (t, 2H, J = 12.0 Hz, piperidine, 2.91 (m, 1H, piperidine),
). Starting from 2-(aminomethyl)piperidine and
η
δ
−
3.51 (s, 2H, CH2Ph), 3.58 (d, 2H, J = 12.0 Hz, NCH2CH), 7.34
−
7.29 (m, 5H, Ph), 7.89 7.79 (m, 4H, Phth);
−
M/z (ESI-MS): 335 (M + 1)+.
2-(2-(4-(2-Fluorobenzyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (10a
and afterwards 2-fluorobenzyl-bromide, the crude solid compound was purified by washing with
methanol, affording a pure compound as a beige solid ( = 25%, M.P. = 93
95 ◦C). H1 NMR (300 MHz,
7.11,
). Starting from 1-(2-aminoethyl)piperazine
η
−
DMSO-d6),
δ
(ppm): 2.21
−
2.50 (m, 10H, piperazine, CH2N), 3.68 (t, 2H, J = 6 Hz, CH2NPhth), 7.18
−
7.40−7.27 (2 × m, 4H, Ph), 7.82–7.88 (m, 4H, Phth). M/z (ESI-MS): 368.36 (M + 1)+.
2-(2-(4-(2-Nitrobenzyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (11a
and afterwards 2-nitrobenzyl-bromide, the crude has been purified by washing with methanol affording
a pure compound as a light yellow solid ( = 28%, M.P. = 100
103 ◦C). H1 NMR (300 MHz, MeOD-d4),
(ppm): 2.26 2.58 (m, 8H, H8–11), 2.63 (t, 2H, J = 8 Hz, CH2N), 3.74 (s, 2H, NCH2Ph), 3.80 (t, 2H, J = 8
). Starting from 1-(2-aminoethyl)piperazine
η
−
δ
−
Hz, CH2NPhth), 7.48
−
7.42, 7.56–7.61 (2
×
m, 4H,Ph), 7.78 7.86 (m, 4H, Phth). M/z (ESI-MS): 395.38
−
(M + 1)+.
2-(2-(4-(Pyridin-2-ylmethyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (12a
piperazine and afterwards 2-(bromomethyl)pyridine-HBr, the crude has been purified by washing
with ether, affording a pure compound as a beige solid ( = 20%, M.P. = 93
95 ◦C). H1 NMR (400 MHz,
MeOD–d4) (ppm): 2. 50–2.62 (m, 8H, piperazine), 2.66 (t, 2H, J = 8 Hz, CH2N), 3.63 (s, 2H, CH2Ph).
3.82 (t, 2H, J = 8 Hz, CH2NPhth), 7.28 7.33 (m, 1H, H2-py), 7.51 (d, 1H, J = 4 Hz, H4-Py), 7.86 7.78 (m,
). Starting from 1-(2-aminoethyl)
η
−
δ
−
−
1+4 H, H3-Py + Phth), 8.66 (d, 1H, J = 4 Hz, H1-Py). M/z (ESI-MS): 351.36 (M + 1)+.
3.2.1.3. Deprotection of Primary Amine from the Isoindoline-1,3-Diones (1b−4b,10b−12b)
General Procedure
The benzylpiperidine- or benzylpiperazine- or methylpyridinepiperazine-isoindoline-1,3-dione
derivatives (1a−4a 10a−12a) (1 eq) and hydrazine monohydrate (6 eq) were dissolved in absolute
;

Molecules 2020, 25, 985
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ethanol and warmed at 60 ◦C for 3 h. The reaction mixture was then filtered and the liquid filtrate
was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed with
brine (three times), dried over anhydrous sodium sulphate, and concentrated under reduced pressure
obtaining a yellow oil (η = 35–79%).
(1-Benzylpiperidin-4-yl)methanamine (1b) Starting from 1a, the pure compound was obtained as a yellow
1
oil (
η
= 40%). H RMN (300 MHz, DMSO-d6),
δ
(ppm): 1.25
−
1.39, 1.58
−
1.69, 1.79
−
1.91 e 2.36 2.41 (m,
−
9H, piperidine), 2,77 (d, 2H, J = 12.0 Hz, NCH2CH), 3.42 (s, 2H, NCH2Ph), 7.19–7.36 (m, 5H, Ph-CH2).
M/z (ESI-MS): 205.00 (M + 1)+.
2-(4-Benzylpiperazin-1-yl)ethanamine (2b
)
.
Starting from 1a, the pure compound was obtained as a
(ppm): 2.45 (t, 2H, J = 6 Hz, NH2CH2CH2N),
1
yellow oil (
η
= 36%). H NMR (300 MHz, MeOD-d4),
δ
2.60–2.52 (m, 8H, piperazine), 2.75 (t, 2H, J = 6 Hz, NH2CH2CH2N), 3.53 (s, 2H, NCH2Ph), 7.21–7.37
(m, 5H, Ph). M/z (ESI-MS): 220 (M + 1)+.
(1-Benzylpiperidin-3-yl)methanamine (3b
)
.
Starting from 3a, the title pure product was obtained (η = 37%).
1.96, 2.32–2.39 (m, 7H, piperidine), 2.54 (t, 2H, J = 12 Hz,
1H RMN (300 MHz, MeOD-d4),
δ
(ppm): 1.69
−
piperidine), 2.97 (d, 2H, J = 9 Hz, NCH2CH), 3.56 (s, 2H, NCH2Ph), 7.81
−
7.90 (m, 5H, Ph). M/z (ESI-MS):
205 (M + 1)+.
(1-Benzylpiperidin-2-yl)methanamine (4b
)
.
Starting from 4a, the pure title product was obtained (η = 35%).
1.86 (2 m, 6H, piperidina), 2.40 (t, 2H, J = 12.0 Hz
1H RMN (300 MHz, MeOD-d4),
δ
(ppm): 1.24
−
×
piperidina), 2.78 (m, 1H, J = 12.0 Hz, NCH2CH), 2.91 (m, 1H, piperidina), 3.51 (s, 2H, NCH2Ph),
7.34−7.19 (m, 5H, Ph). M/z (ESI-MS): 205 (M + 1)+.
1
2-(4-(2-Fluorobenzyl)piperazin-1-yl)ethanamine (10b
MeOD-d4),
).
Starting from 10a
,
η
= 73%. H NMR (400 MHz,
δ
(ppm): 2.46 (t, 2H, J = 8 Hz, NCH2CH2NH2), 2.69–2.48 (m, 8H, piperazine), 2.74–2.78 (t,
2H, J = 8 Hz, NCH2CH2NH2), 3.62 (s, 2H, NCH2Ph), 7. 06
−
7.10, 7.13
−
7.17, 7.28
−
7.33, 7.38
−
7.42 (4
×
m,
4H, Ph). M/z (ESI-MS): 238.22 (M + 1)+.
2-(4-(2-Nitrobenzyl)piperazin-1-yl)ethanamine (11b
MeOD-d4), (ppm): 2.41 2.55 (m, 10H, Pyridine+ NCH2CH2NH2), 2.74 (t, 2H, J = 6 Hz, NCH2CH2NH2),
7.50, 7.58 7.60 (m, 3H, Ph), 7.81 (d, 1H, J = 9 Hz, Ph). M/z (ESI-MS): 265
). Starting from 11, η
= 79%. 1H NMR (300 MHz,
δ
−
3.79 (s, 2H, CH2Ph), 7.44
−
−
(M + 1)+.
2-(4-(Pyridin-2-ylmethyl)piperazin-1-yl)ethanamine (12b
)
.
Starting from 12a
2.55 (m, 8H, piperazine), 2.74 (t, 2H, J =
7.34 (m, 1H, Py-H-2), 7.54 (d, 1H, J = 6 Hz, Py-H-4),
, η
= 36.3%. 1H NMR (300 MHz,
MeOD-d4), (ppm): 2.45 (t, 2H, J = 6 Hz, NCH2CH2NH2) 2.64
δ
−
6 Hz, NCH2CH2NH2), 3.67 (s, 2H, CH2Py), 7.30
−
7.80−7.85 (m, 1H, H3Py), 8.48 (d, 1H, J = 6 Hz, Py-H-1). M/z (ESI-MS): 221 (M + 1)+.
3.2.2. Synthesis of 2-(X-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic Acids (X = 5-F, 5-NO2,
4-OMe)
General Procedure (Method B)
3,4-Diaminobenzoic acid (1 eq), a suitable salicylaldehyde (1 eq) and Na2S2O5 (1.4 eq) were
◦
dissolved in choline chloride/glycerol (1:2), mol/mol) and warmed at 50 C for 30 min. Then the
reaction mixture was cooled at rt, added to water and filtrated. The solid obtained was washed with
dichloromethane, affording beige-yellow solids with yield 67%−97%.
2-(5-Fluoro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (6c
salicylaldehyde, the title compound was obtained as a beige solid (
NMR (300 MHz, DMSO-d6), (ppm): 7.08 (s, 1H, BIM-H-7) 7.26 (d, 1H, J = 6.0 Hz, BIM-H-4),7.74 (d,
). Starting from 5-fluoro-
◦
1
η
= 97%), M.P. = 250 C. H
δ
1H, J = 6.0 Hz, BIM-H-5), 7.91 (d, 1H, J = 9.0 Hz, BIM-H-2), 8.00 (d, 1H, J = 9.0 Hz, BIM-H-3),8.25 (s, 1H,
BIM-H-1), 12.78 (s, 1H, COOH). M/z (ESI-MS): 273 (M + 1)+.

Molecules 2020, 25, 985
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2-(5-Nitro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid
(
7c
)
.
Starting from 5-nitro-
◦
1
salicylaldehyde, the title compound was obtained as a beige solid (
NMR (300 MHz, DMSO-d6), (ppm): 7.06 7.11 (m, 1H, BIM-H-2), 7.25–7.32 (m, 1H, BIM-H-4),
7.96 (m, 2H, BIM-H-1, BIM-H-5), 8.24 (s, 1H, BIM-H-7),
η = 78%), M.P. > 300 C. H
δ
−
7.73
−
7.76 (d, 1H, J = 9.0 Hz, BIM-H-2), 7.89
−
12.78 (s, 1H, COOH). M/z (ESI-MS): 300 (M + 1)+.
2-(4-Metoxy-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic acid (9c). Starting from 4-metoxy-
◦
1
salicylaldehyde, the title compound was obtained as a beige solid (
NMR (300 MHz, DMSO-d6), (ppm): 3.82 (sg, 3H, OCH3), 6.62 (s, 1H, BIM-H-4) 6.66 (d, 1H, J = 9.0 Hz,
η = 67%), M.P. > 300 C. H
δ
BIM-H-6), 7.68 (d, 1H, J =9.0 Hz, BIM-H-7), 7.88 (d, 1H, J = 9.0 Hz, BIM-H-2), 7.98 (d, 1H, J = 9.0 Hz,
BIM-H-3), 8.18 (s, 1H, BIM-H-1), 12.96 (s, 1H, COOH). M/z (ESI-MS): 285 (M + 1)+.
3.2.3. Synthesis of the Final Conjugates (1–12)
General Procedure
To a solution of the carboxylic acid derivatives (1c
, 5c, 6c, 7c, 9c) (1 eq) and the amine derivatives
(1–
4b 10 12b) (1 eq) in anhydrous DMF, was added N-hydroxysuccinimide (1 eq) and DCC (1 eq) and
,
–
the reaction mixture was left stirring for 24 h at rt. Then, the precipitate obtained (urea) was filtered off
and the liquid phase was diluted with ethyl acetate and washed with brine (three times). The combined
organic phase was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified
by recrystallisation in EtOAc or Et2O and ACN/MeOH, or column chromatography, affording the pure
final conjugate as a solid.
N-((1-Benzylpiperidin-3-yl)methyl)-2-(2-hydroxyphenyl)-1H-benzo[d]imidazole-6-carboxamide (
from 3b and 1c, the title compound was obtained as a beige solid ( = 22%), M.P. = 220
1H RMN (300 MHz, MeOD-d4),
(ppm): 1.49 1.94 (m, 5H, piperidine), 2.64
(piperidine)), 3.18 (m, 2H, NHCH2, 3.53 (s, 2H, NCH2Ph): 13C RMN (75.5 MHz, MeOD-d4),
3
)
.
Starti◦ng
η
−
222 C.
δ
−
−
2.89 (m, 4H, NCH2
(ppm):
δ
25.41, 27.80, 29.32, 43.06, 56.94, 59.71, 64.82, 114.2, 116.4, 117.9, 118.4, 121.9, 122.4, 127.6, 128.2, 128.9,
129.8, 130.3, 131.8, 139.0, 145.3,152.7, 154.1, 166.9. M/z (ESI-MS): 441.36 (M + 1)+.
N-((1-Benzylpiperidin-2-yl)methyl)-2-(2-hydroxyphenyl)-1H-benzo[d]imidazole-6-carboxamide (
from 4b and 1c, the title compound was obtained as a beige solid (
1H NMR (300 MHz, DMSO-d6),
(ppm): 1.24
J = 12 Hz, CHCH2N), 3.64 (s, 2H, CH2Ph), 4.05 (sb, 1H, NH), 7.05
(m, 6H, H1 a H5 e H25), 7.65 (d, 1H, J = 6 Hz, H27), 7.80 (d, 1H, J = 6 Hz, H18), 8.09 (d, 1H, J = 9 Hz,
H19), 8.26 (s, 1H, H16), 8.41 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-d6),
(ppm): 22.75, 24.38, 28.96,
4
). Starti◦ng
η
= 25%), M. P. = 226 227 C.
1.71 (m, 6H, Pip), 2.63–2.73 (m, 3H, Pip), 3.42 (d, 2H,
7.08 (m, 2H, H24, H26), 7.23 7.45
−
δ
−
−
−
δ
33.96, 50.01 50.58, 60.08, 111.56, 112.94, 117.36, 117.73, 119.74, 122.14, 123.43, 127.00, 128.57, 128.97,
132.60, 135.69, 140.15, 141.03, 153.79, 158.47, 166.91. M/z (ESI-MS): 441.24 (M + 1) +.
N-((1-Benzylpiperidin-4-yl)methyl)-2-(2-hydroxy-5-fluorophenyl)-1H-benzo[d]imidazole-5-carboxamide (
Starting from 1b and 6c, the title compound was obtained as a white solid ( = 24%) M.P. = 241
243 ◦C.
1H NMR (300 MHz, MeOD-d4),
(ppm): 1.01 1.24 (m, 4H, piperidine), 163 1.91 (m, 5H, piperidine),
2.81 (d, 2H, J = 12.0 Hz, NCH2CH), 3.49 (s, 2H, NCH2Ph), 5.59 (b, 1H, NH), 7.12 7.03 (m, 2H, BIM-H-5,6)
7.19
6).
η
−
δ
−
−
−
−
7.39 (m, 5H, Ph-CH2), 7.69 (s, 1H, BIM-H-7), 7.81 (d, 1H, J = 8.0 Hz, BIM-H-4), 7.93 (d, 1H,
J = 8.0 Hz, BIM-H-3), 8.51 (s, 1H, BIM-H-2). 13C NMR (100 MHz, MeOD-d4), δ (ppm): 27.3, 37.2, 43.2,
53.1, 63.3, 115.2, 116.5, 117.6, 119.9, 122.3, 126.6, 128.2, 128.9, 132.3, 138.4, 146.2, 149.3, 151.3, 152.7, 166.8.
M/z (ESI-MS): 459.23 (M + 1)+.
N-(2-(4-Benzylpiperazin-1-yl)ethyl)-2-(2-hydroxy-5-nitrophenyl)-1H-benzo[d]imidazole-5-carboxamide (
Starting from 2b and 7c, with final purification through column chromatography (eluent: DCM/MeOH
98:2) afforded a yellow solid ( (ppm):
= 28%), M.P. = 225–227 ◦C. 1H NMR (300 MHz, MeOD-d4),
2.77–2.94 (m, 10H, 8 piperazine + 2 NH2CH2CH2N), 3.66–3.75 (m, 4H, NCH2Ph and NH2CH2CH2N),
7.07–7.38, 7.77–7.83, and 8.18–9.05 (3 (ppm): 30.3,
m, 11H, Ar-H). 13C NMR (100 MHz, MeOD-d4),
7).
η
δ
×
δ

Molecules 2020, 25, 985
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36.2, 45.7, 53.6, 62.8, 113.3, 117.8, 119.5, 127.0, 127.3, 128.6, 129.3, 129.8, 132.5, 139.1, 143.9, 157.1, 160.8,
167.9. M/z (ESI-MS): 501.24 (M + 1)+.
N-(2-(4-Benzylpiperazin-1-yl)ethyl)-2-(2-hydroxy-5-fluorophenyl)-1H-benzo[d]imidazole-5-carboxamide (
Starting from 2b and 6c, with final recrystallization from ACN/MeOH, afforded the title compound as
a white-beige solid ( = 27%) M.P. = 246 (ppm): 2.77–2.94
248 ◦C. 1H NMR (300 MHz, MeOD-d4),
(m, 10H, piperazine + 2 NH2CH2CH2N), 3.66–3.75 (m, 4H, 2 NCH2Ph + 2 NH2CH2CH2N), 7.03–7.20,
7.34–7.37, and 7.69–8.16 (m, 11H, Ar-H). 13C NMR (100 MHz, MeOD-d4),
(ppm): 30.3, 36.2, 45.7,
8).
η
−
δ
δ
53.6, 62.8, 113.3, 117.8, 119.5, 127.0, 127.3, 128.6, 129.3, 129.8, 132.5, 139.1, 154.6, 157.1, 160.8, 167.9. M/z
(ESI-MS): 474.22 (M + 1)+.
N-(2-(4-Benzylpiperazin-1-yl)ethyl)-2-(4-metoxy-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxamide (
Starting from 2b and 9c, with final recrystallization from ACN/MeOH afforded the pure title compound
as white solid ( = 32%), M.P. = 249 (ppm): 2.77–2.94 (m, 10H,
251 ◦C. 1H NMR (300 MHz, MeOD-d4),
piperazine + NH2CH2CH2N), 3.66–3.75 (m, 4H, 2 NCH2Ph + 2 NH2CH2CH2N), 3.87 (s, 3H, OCH3),
6.70–6.74, 7.29–7.36, and 7.61–8.10 (3 (ppm): 30.3,
m, 11H, Ar-H). 13C NMR (100 MHz, MeOD-d4),
9).
η
−
δ
×
δ
36.2, 45.7, 53.6, 62.8, 113.3, 117.8, 119.5, 127.0, 127.3, 128.6, 129.3, 129.8, 132.5, 139.1, 157.1, 160.8, 162.1,
167.9. M/z (ESI-MS): 486.25 (M + 1)+.
N-(2-(4-(2-Fluorobenzyl)piperazin-1-yl)ethyl)-2-(2-hydroxyphenyl)-1H-benzo[d]imidazole-6-carboxamide (10).
Starting from 10b and 1c, the pure◦product was purified by column chromatography (eluent: ACN/H2O,
1
9:1) (
η
= 47%), M.P. = 219
−
222 C. H NMR (400 MHz, MeOD–d4),
δ
(ppm): 2.63
−
2.90 (m, 10H,
piperazine + NCH2CH2NH), 3.62 (t, 2H, J = 6 Hz, NCH2CH2NH), 3.72 (s, 2H, NCH2Ph), 6.98 -7.20 (m,
4H, H4-5, H24, H26), 7.30 7.44 (m, 3H, H1-2, H14), 7.67 (d, 1H, J = 9 Hz, H27), 7.78 (d, 1H, J = 6 Hz,
H16), 7.97 (d, 1H, J = 9 Hz, H17), 8.14 (s, 1H, H20). 13C NMR (100 MHz MeOD–d4),
ppm: 36.26,
−
δ
51.65, 52.30, 54.32, 56.78, 112.62, 114.87, 115.05, 117.34, 119.41, 123.88, 126.32, 131.88, 132.12, 158.18. M/z
(ESI-MS): 474.31 (M + 1)+.
2-(2-Hydroxyphenyl)-N-(2-(4-(2-nitrobenzyl)piperazin-1-yl)ethyl)-1H-benzo[d]imidazole-6-carboxamide (11).
Starting from 11b and 1c the pure title product was purified by column chromatography (eluent:
AcOEt/MeOH, 9:1), (
(m, 10H, piperazine + NCH2CH2NH) 3.63
8.39 (s, 1H, H20), 7.01 7.06 (m, 2H, H24 and H26), 7.39
7.60 7.70 (m, 3H, H3, H4 and H5), 7.78 (d, 2H, J = 8 Hz, H2), 7.85 (d, 2H, J = 8 Hz, H16), 8.08 (d, 2H,
J = 8 Hz, H17), 8.14 (s, 1H, C-CH-C). 13C NMR (100 MHz, DMSO–d6),
ppm: 34.36, 35.31, 42.99, 46.81,
η
= 40%), M.P. = 242
−
244 ◦C. 1H NMR (400 MHz, DMSO–d6),
δ
(ppm): 2.36
3.74 (m, 4H, NCH2Ph + NCH2CH2NH), 5.51 (s, 1H, OH),
7.42 (m, 1H, H25), 7.50 7.53 (m, 1H, H27),
−
2.53
−
−
−
−
−
δ
62.49, 66.87, 68.25, 122.48, 123.62, 127.31, 129.13, 134.07, 136.31, 138.42, 140.98, 142.11, 142.38, 142.85,
157.52, 157.80, 159.61, 166.38, 167.80, 176.01. M/z (ESI-MS): 501.29 (M + 1)+.
2-(2-Hydroxyphenyl)-N-(2-(4-(pyridin-2-ylmethyl)piperazin-1-yl)ethyl)-1H-benzo[d]imidazole-6-carboxamide
(
12). Starting from 12b and 1c the title product was purified by recrystallization from ACN/MeOH,
(η = 41%), M.P. = 220 (ppm): 2.50 2.62 (m, 8H, piperazine),
222 ◦C. 1H NMR (300 MHz, MeOD–d4),
2.66 (t, 2H, J = 6Hz, H11), 3.58 (t, 2H, J = 6 Hz, H12), 3.68 (s, 2H, NCH2Ph), 6.97 7.05 (m, 2H, H23 and
H25), 7.3 7.4 (m, 2H, H24 and H2), 7.54 (d, 1H, J = 9 Hz, H4), 7.66 (d, 1H, J = 9 Hz, H26), 7.76 7.85 (m,
2H, H3 and H17), 7.96 (d, 1H, J = 9 Hz, H18), 8.13 (s, 1H, H1), 8.47 (d, 1H, J = 6 Hz, H1). 13C NMR
(100 MHz MeOD–d4), ppm: 36.49, 52.41, 52.73, 56.96, 63.20, 112.68, 116.80, 119.12, 121.93, 122.70,
−
δ
−
−
−
−
δ
123.71, 126.29, 128.83, 131.85, 137.11, 148.25, 149.64, 153.83, 155.50, 157.68, 158.44, 161.24, 169.17. M/z
(ESI-MS): 457.31 (M + 1)+.
3.3. Molecular Modeling: Docking and Pharmacokinetics Studies
Docking simulations were carried out using the Gold v. 5.1 package of software [46]. The model
structure for the active site of AChE was retrieved from the Brookhaven protein database (RCSB)
(PDB, entry 1 EVE) [24], in particular, from the X-ray crystal structure of Torpedo Californica AChE
(TcAChE) complexed with the cholinesterase inhibitor drug in clinical use (donepezil, DNP), hereinafter

Molecules 2020, 25, 985
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named as the original ligand. In spite of some known differences between the inhibitor-enzyme
complexes, reported for the human acetylcholine esterase, hAChE, and the electric ray homologue
(Torpedo californica, TcAChE) both enzymes are quite conservative in terms of the main amino acid
residues that line up the active site gorge [47], and so this docking study was performed with the
TcAChE molecular model.
For the simulations, the original complex of TcAChE-DNP, retrieved from RCSB, was treated with
MAESTRO v. 9.3 [48] to get the model structure of the receptor enzyme, by removing the original
ligand (DNP), solvent and co-crystallization molecules, and subsequently adding hydrogen atoms.
This program was also used to build the ligand structures which were then optimized (1000 cycles of
random conformational search and 2500 optimization steps) with the program Ghemical v 2.0 [49,50].
The ligands were subsequently docked into the AChE active site, using GOLD program with the
corresponding default parameters (except “allow early termination” option) and the Astex Statistical
Potential (ASP) scoring function. The docking site was defined by establishing a box within 10 Å from
the original ligand in the above mentioned PDB structure. The first-ranked structures obtained from
these calculations were then compared with the original ligand and analyzed in their interactions with
the active site residues. The results were visualized using Chimera 1.6 [51].
Moreover, pharmacokinetic molecular descriptors were in silico evaluated. Parameters such as
the octanol-water partition coefficient (clog P), blood–brain barrier partition coefficient (log BB), the
ability to be absorbed through the intestinal tract (Caco-2 cell permeability), and CNS activity were
calculated. The chemical structures were previously minimized in Maestro v. 9.3 [48] and then they
were submitted to the calculation of these relevant pharmacokinetic properties and descriptors using
QikProp v. 2.5 [43].
3.4. Bio-Analytical Procedures
3.4.1. Inhibition of Acetylcholinesterase
An adaptation of the Ellman
acetylcholinesterase inhibitory activity [52
1-piperazineethanesulfonic acid (HEPES) buffer (50 mM, pH 8.0), 476
acid) (DTNB, 3 mM), a variable volume (10–50 L) of the stock solution of each compound in methanol
(1 mg/mL), 25 L of AChE (type VI-S, from electric eel) stock solution, and the necessary amount of
methanol to obtain 0.925 mL of sample mixture in a 1 mL cuvette. The samples were incubated for
15 min and then 75 L of acetylthiocholine iodide (AChI) solution (16 mM) was added. The reaction
´
s method, previously described, was used to measure the
53]. The assaysolutioncontained 374 L of 4-(2-hydroxyethyl)-
L of 5,5’-dithiobis-(2-nitrobenzoic
,
µ
µ
µ
µ
µ
was monitored for 5 min at 405 nm. Assays were run with a blank containing all the components
except AChE, which was replaced by HEPES buffer. The rates of the reaction were calculated as well
as the enzyme activity. A control reaction was carried out using methanol as a sample solution and it
was considered as 100% activity. The percentage inhibition of the enzyme activity due to the presence
of increasing test compound concentration was calculated by the following Equation (1),
I% = 100 − 100 × vi/(vi − vo)
(1)
where vi = starting reaction rate in the presence of inhibitor; vo = starting reaction rate of the
control reaction.
The inhibition curves were obtained by plotting the percentage of enzymatic inhibition versus
inhibitor concentration and a calibration curve was got from which the linear regression parameters
were obtained.
3.4.2. Inhibition of Self- and Cu-Mediated Aβ1–42 Aggregation
The A
CH3CN/Na2CO3 (300
β
peptide was treated with 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) and dissolved in a
M)/NaOH (250 M) (48.3:48.3:4.3, v/v/v) mixture in order to have a stable stock
µ
µ

Molecules 2020, 25, 985
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solution, avoiding self-aggregation of Aβ1–42. Then, the prepared 500
µM in phosphate buffer (0.215 M, pH 8.0).
µM solution was diluted at 40
The solutions of the tested compounds were prepared in MeOH (1 mg/mL), being further diluted
in phosphate buffer to a concentration of 480 M. For copper-induced aggregation studies, a solution
of CuCl2 240 M was prepared from a stock standard solution (0.015 M). To perform the assays for the
β1–42 aggregation inhibition, a reported method, based on the fluorescence emission of thioflavin
T (ThT), was followed [36 37 54]. Aβ1–42 (40
M) was incubated at 37 ◦C, for 24 h with or without
Cu(II) (40 M) in a phosphate buffer in presence or absence of the single ligand (40 M and 20
for compound ). Then, the samples were added to a 96-well plate with 180 M of 5 M ThT in
µ
µ
A
,
,
µ
µ
µ
µM
4
µ
µ
50 mM glycine-NaOH (pH 8.5) buffer. Blank samples were prepared without the peptide for each
concentration. The ThT fluorescence was measured at 446 nm (excitation) and 485 nm (emission). The
inhibition percentage of aggregation was calculated by Equation (2), in which IFi and IF0 corresponded
to the fluorescence intensities, in the presence and in the absence of the tested compound, subtracted
of the fluorescence intensities due to the respective blanks.
I% = 100 − (IFi/IF0 × 100)
(2)
The reported values were obtained as the mean
±
SEM of duplicate of three different experiments.
3.4.3. Metal Chelation
Solutions: the aqueous copper (CuCl2, 0.015 M) and zinc (ZnCl2, 0.0156 M) stock solutions
for potentiometric equilibrium studies were prepared from 1000 ppm standards (Titrisol) and their
metal content was evaluated by atomic absorption. The titrant solution (0.1 M KOH) was obtained
from a carbonate-free commercial concentrate (Titrisol) and standardized by potentiometric titration
with potassium hydrogen phthalate. The titrant solution was discarded whenever the percentage of
carbonate (Gran’s method) [55] was about 0.5% of the total amount of base.
Measurements: compounds 7 and 12 were titrated in 50% (w/w) DMSO/H2O medium, at
T = 25.0 ± 0.1 ◦C and ionic strength (I) 0.1 M KCl, using 0.1 M KOH as titrant. Both glass and
Ag/AgCl reference electrodes were previously conditioned in DMSO/H2O mixtures with increasing
amounts of DMSO and the response of the glass electrode was controlled by strong acid–strong base
(HCl/KOH) calibrations with the determination of the Nernst parameters by Gran’s method [55].
The measurements were performed in a final volume of 30.00 mL, with a ligand concentration (CL)
of 6.7
×
10−4 M, under different CM/CL ratios: 0:1 (L), 1:1, and 1:2 (M = Cu, Zn). All titrations
were performed in duplicate and under the stated experimental conditions the pKw value (13.6)
was determined and subsequently used in the computations. The stepwise protonation constant
of the ligand, Ki = [HiL]/([Hi–1L][H]), and the overall metal–complex stability constants, βM
=
mHhLl
[MmHhLl]/([M]m[H]h[L]l), were calculated by fitting the potentiometric titration data with Hyperquad
2008 [26]. The metal hydrolysis constants were determined under the defined experimental conditions
(I = 0.1 M KCl, 50% w/w DMSO/H2O, T = 25.0 ± 0.1 ◦C) and the following values of stability constants
were included in the fitting of experimental data towards the equilibrium models related to the Cu2+/L
and Zn2+/L systems: logβCu H
curves were plotted with the Hyss program [26].
=
−
9.99; logβZnH
=
−
14.79, logβZnH
= −23.48. Species distribution
2
−2
−2
−3
3.4.4. Cell Viability and Neuroprotection
SH-SY5Y human neuroblastoma cell line (ATCC-CRL-2266) was cultured in Dulbecco’s Modified
Eagle Medium (DMEM) (Gibco-Invitrogen, Life Technologies Ltd., Liverpool, UK) supplemented with
10% of heat inactivated fetal bovine serum, 50 U/mL penicillin, and 50 µ
g/mL streptomycin, at 37 ◦C
in 5% CO2. For the cell viability experiments, cells were plated with a density of 0.12 × 106 cells/mL.
All the tested compounds (from 7 to 12) were dissolved in DMSO and aliquots were stored at −20 ◦C
with a stock concentration of 25 mM. To choose the highest non-toxic concentration of the compounds, a

Molecules 2020, 25, 985
22 of 25
concentration screening was performed (from 2.5
µM to 10
µM). For the experiments, all the compounds
were added to the medium at a final concentration of 2.5 µM. The concentration of DMSO in culture
media did not exceed 0.05% (v/v) and no alterations were detected in the SH-SY5Y cell line. Cells
were pre-incubated for 1 h with the compounds and then incubated with Aβ1–42 or with Fe/Asc for
an additional 24 h. Aβ1–42 (Bachem, Torrance, CA, USA) stock with a concentration of 276.9
µM was
prepared in sterile water and a final concentration of 1 M was added to the culture medium. Both
µ
stock solutions of ferrous sulfate (Fe) and L-Ascorbic acid (Asc) (Sigma Chemical Co, St. Louis, MO,
USA) were prepared in sterile water and 2.5 mM of ferrous sulfate and 5 mM of L-Ascorbic acid were
added together to the culture medium. For all the experiments, control conditions were performed in
which the compounds were tested and Aβ1–42 or Fe/Asc were not added.
Cell viability was determined using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay [56]. In viable cells, cellular dehydrogenases metabolize
the MTT into a formazan that absorbs light at 570 nm. After the incubations, the culture medium was
aspirated and 0.3 mL of MTT (0.5 mg/mL) was added to each well. After incubation at 37 ◦C for 3 h,
formazan precipitates were solubilized with 0.3 mL of acidic/isopropanol (0.04 M HCl/Isopropanol).
The absorbance of the plate was measured at 570 nm. Cell reduction ability was normalized to
untreated control cells.
All data were expressed as mean
in duplicates. Statistical analyses were performed using one-way ANOVA followed by Bonferroni
±
SEM of at least four independent experiments performed
multiple comparisons procedure post hoc test. P value < 0.05 was considered statistically significant.
4. Conclusions
Due to the complexity of AD pathogenesis and concomitant absence of cure, we have been
engaged in the search for molecular entities capable of hitting multiple targets of this disease and thus
being possibly explored as chemical tools against AD. The present strategy has been focused on the
repositioning of approved drugs based on AChE inhibition, namely donepezil (DNP). Therefore, the
MTDLs developed herein integrate mimetics of the active molecular fragment of DNP merged with other
moieties, in order to combine the symptomatic amelioration by AChEi with other disease-modifying
roles involved in amyloid plaque formation, namely inhibition of Aβ aggregation, as well as modulation
of metal dyshomeostasis and oxidative stress. In particular, challenged by previous results with simple
(DNP-BIM) conjugates (DNP mimetics coupled with a 2-hydroxyphenylbenzimidazole (BIM)), the
present work exploited the effect on the activity of some structural modifications, namely by positional
isomerization and substituent introduction. Regarding the positional isomers (
3
,
4
,
5
) of compound
, due to some
aggregation. All the
, due
1
, there is a general slight decrease of the AChEi activity, namely for compound 4
apparent structural distortion, although they keep identical capacity to inhibit A
β
compounds are able to chelate metal ions through their BIM moieties and, remarkably, isomer
5
to the closeness of the carbonyl to the bidentate BIM unity provides a three-dentate chelating moiety,
having higher capacity for copper chelation. This may be responsible for the more than 50% increase
on Cu-induced A
of the substituents on the cholinergic activity, the best results were obtained for the conjugates with the
fluorine atom in each moiety ( and 10) and also with the metoxyl group in the benzyl ring ( ), while
lowest activity was revealed by the compounds with the nitro substituents ( 11), but the worst results
were obtained for the pyridinium derivative (12). Regarding the effect of a selection of DNP-BIM
hybrids in neuroblastoma cells, compounds and 10 showed a remarkable reduction of A -induced
β aggregation relative to the self-induced one observed for 5. Concerning the effect
6,
8
9
7,
9
β
cell toxicity, although none of the tested compounds presented a statistically significant neuroprotective
effect by preventing ROS production. Overall, among these DNP-BIM hybrids, structural isomerization
did not bring general improvement of properties; regarding the substituent modification, excluding
the nitro-substituted and pyridinium derivatives, a set of substituted conjugates, namely methoxy and
fluorine substituent groups, showed very good properties and so they deserve to be further explored
in the development of new hybrids aimed at the discovery of novel anti-AD drugs.

Molecules 2020, 25, 985
23 of 25
Supplementary Materials: The following are available online, Figure S1: Redocking of original ligand (DNP)
from PDB code 1EVE [1], inside the TcAChE active site, under the same simulation conditions used for the docking
of the ligands in the present study.
Author Contributions: Conceptualization, M.A.S., S.C., L.P.; formal analysis, S.C., S.R., F.R., M.C., R.J., K.G.,
A.R.P.-S.; investigation, S.R., F.R., M.C., R.J., K.G., A.R.P.-S.; data curation, S.C., M.A.S., S.M.C.; writing—original
draft preparation, M.A.S., S.C., S.R., F.R., M.C., S.M.C.; writing—review and editing, M.A.S., S.C.; supervision,
M.A.S., S.C., L.P., S.M.C.; project administration, M.A.S., S.C., V.C.; funding acquisition, M.A.S., S.C., V.C. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Portuguese Fundação para a Ciência e Tecnologia (FCT), projects
UID/QUI/00100/2013 and UID/QUI/00100/2019, and F.R., S.R., K.G. and R.J. were funded by Europe Erasmus+
program. It was also funded by the 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 Internationalisation and
Portuguese national funds via FCT, under project POCI-01-0145-FEDER-030712 and UID/NEU/04539/2019.
Acknowledgments: The authors acknowledge FCT for the research funding and also the Portuguese NMR
(IST-UL Center) and Mass Spectrometry Networks (Node IST-CTN) for providing access to their facilities.
Conflicts of Interest: The authors have no conflicting interests. The founding sponsors had no role in the design of
the study, in the interpretation of data, in the writing of the manuscript, and in the decision to publish the results.
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