Design, synthesis and biological evaluation of novel triazole N-acylhydrazone hybrids for alzheimer's disease

Article abstract of DOI:10.3390/molecules25143165

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that involves different pathogenic mechanisms. In this regard, the goal of this study was the design and synthesis of new compounds with multifunctional pharmacological activity by molecular hybridization of structural fragments of curcumin and resveratrol connected by an N-acyl-hydrazone function linked to a 1,4-disubstituted triazole system. Among these hybrid compounds, derivative 3e showed the ability to inhibit acetylcholinesterase activity, the intracellular formation of reactive oxygen species as well as the neurotoxicity elicited by A?42 oligomers in neuronal SH-SY5Y cells. In parallel, compound 3e showed a good profile of safety and ADME parameters. Taken together, these results suggest that 3e could be considered a lead compound for the further development of AD therapeutics.

Full text of DOI:10.3390/molecules25143165

molecules
Article
Design, Synthesis and Biological Evaluation
of Novel Triazole N-acylhydrazone Hybrids
for Alzheimer’s Disease
1
,
1
2
, Newton G. Castro 3
Matheus de Freitas Silva * , Ellen Tardelli Lima , Letizia Pruccoli
,
3
3
4
Marcos Jorge R. Guimarães
, Fernanda M. R. da Silva , Nathalia Fonseca Nadur ,
4
4
5
Luciana Luiz de Azevedo , Arthur Eugen Kümmerle , Isabella Alvim Guedes ,
5
, Vanessa Silva Gontijo 1 , Andrea Tarozzi *
2,
Laurent Emmanuel Dardenne
and Claudio Viegas Jr. *
1
,
1
Laboratory of Research in Medicinal Chemistry (PeQuiM), Federal University of Alfenas,
Jovino Fernandes Sales Avenue, 2600, Alfenas 37130000, MG, Brazil; ellentflima17@gmail.com (E.T.L);
vanessagontijo@yahoo.com.br (V.S.G.)
2
3
Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Corso d’Augusto 237,
7921 Rimini, Italy; letizia.pruccoli2@unibo.it
Laboratory of Molecular Pharmacology, Federal University of Rio de Janeiro, Avenida Carlos Chagas Filho,
73, Rio de Janeiro 21941590, RJ, Brazil; ngcastro@icb.ufrj.br (N.G.C.); mj.jorge93@gmail.com (M.J.R.G.);
4
3
dasilvafmr@gmail.com (F.M.R.d.S.)
4
Laboratory of Molecular Diversity and Medicinal Chemistry (LaDMol-QM), Federal Rural
University of Rio de Janeiro—UFRRJ, BR-465, Km 7 Seropédica-Rio de Janeiro 23890000, RJ, Brazil;
nathaliafn18@gmail.com (N.F.N.); lucianaluizazevedo@gmail.com (L.L.d.A.);
akummerle@yahoo.com.br (A.E.K.)
Grupo de Modelagem Molecular em Sistemas Biológicos (GMMSB),
National Laboratory for Scientific Computing—LNCC, Avenida Getúlio Vargas, 333,
Petrópolis 25651-076, RJ, Brazil; isabella.alvimg@gmail.com (I.A.G.); dardenne@lncc.br (L.E.D.)
Correspondence: defreitassilva.matheus@gmail.com (M.d.F.S.); andrea.tarozzi@unibo.it (A.T.);
cvjviegas@gmail.com (C.V.J.)
5
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Federica Belluti
Received: 5 June 2020; Accepted: 9 July 2020; Published: 10 July 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder that involves
different pathogenic mechanisms. In this regard, the goal of this study was the design and synthesis
of new compounds with multifunctional pharmacological activity by molecular hybridization of
structural fragments of curcumin and resveratrol connected by an N-acyl-hydrazone function linked to
a 1,4-disubstituted triazole system. Among these hybrid compounds, derivative 3e showed the ability
to inhibit acetylcholinesterase activity, the intracellular formation of reactive oxygen species as well
as the neurotoxicity elicited by A
β
42
oligomers in neuronal SH-SY5Y cells. In parallel, compound 3e
showed a good profile of safety and ADME parameters. Taken together, these results suggest that 3e
could be considered a lead compound for the further development of AD therapeutics.
Keywords: molecular hybridization; Alzheimer’s disease; curcumin; resveratrol
1
. Introduction
Alzheimer’s disease (AD) is an age-related neurological disorder with complex pathophysiology
and clinical symptoms that causes memory loss, decline in thinking ability, motor and executive
functions. The hallmark brain changes related to AD are due to loss of inter-neuronal connectivity
and neuronal death, especially in areas such as the frontal cortex and hippocampus [
1]. The pathology
begins with a series of events at the molecular level, which results in the production of a functional
Molecules 2020, 25, 3165; doi:10.3390/molecules25143165
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3165
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mismatch between different neurotransmitter systems, especially in the cholinergic system, causing
the peculiar cognitive and psychiatric impairments of the disease [ ]. At the molecular level,
acetylcholine (ACh) deficits are a response to the loss of cholinergiC-Neurons and the presence of
acetylcholinesterase (AChE) levels in the synaptic clefts that hydrolyze ACh into choline and acetate [ ].
Aggregation and deposition of -amyloid (A ) peptide fragments in the neuronal extracellular
environment are currently accepted as an important cause of AD. Due to a not yet clearly understood
dysfunction, enzymes such as - and -secretases can cleave the amyloid precursor protein (APP),
generating A 40 and A peptides that have a high aggregation capacity, producing insoluble
protofibrils, responsible for the inflammatory process, among other pathological effects. Recent studies
suggest that A oligomers diffuse into the brain parenchyma and alter synaptic function, with selective
neuronal loss in the cortex and hippocampus, regions characteristically affected in AD [ ]. Among
the mechanisms of neurotoxicity mediated by A peptides, the formation of reactive oxygen species
ROS) and oxidative stress (OS) contribute to neuronal death. Several studies indicate that redox metals
and mitochondrial respiratory chain activity can impair the secretase activity with the formation of
creating a vicious circle that reinforces both the OS and neuronal death in AD [ ]. In addition,
2
3,4
β
β
β
γ
β
β
42
β
5–7
β
(
A
β
8,9
the cerebral inflammatory process plays a very important role in the pathology of AD, involving
several inflammatory mediators, such as cytokines, chemokines and complement proteins, influencing
the formation of senile plaques. The presence of senile plaques in the brain triggers the activation of
microglia and then an inflammatory process through the interleukin 1, interleukin 6, tumour necrosis
factor
α and cyclooxygenases-1 and 2. The inflammation at brain level also promotes an increase of
APP expression and formation of new senile plaques [10–12].
AD shows a profile of multifactorial hallmarks involving physiological, biochemical and chemical
changes, mediated by different activation pathways [13]. In this regard, there are several therapeutic
strategies for treating the illness condition such as the use of two or more associated drugs or a single
drug-containing structural attribute that guarantee it to act in desired multiple targets. This latest one
is a strategy based on the concept of polypharmacology, and in the last decade has aroused the interest
of the scientific community as a new paradigm in the search for rationally designed molecules for
chronic diseases like AD [14]. In this context, natural products have been used as structural models
in the drug design of ligands against AD, as evidenced by the high volume of studies published in
the literature [15–19].
Curcumin (
potential action against AD, given the multiple activity profiles of themselves and their derivatives
reported in the literature, acting on A , OS, AChE inhibition and neuroprotection [20 21]. Herein
we report the synthesis and pharmacological evaluation of a family of compounds 3a 3m (Figure 1),
1) and resveratrol (2, Figure 1) are prominent examples of natural products with
β
,
–
rationally designed by molecular hybridization (MH) of the structures of curcumin and resveratrol, to
obtain novel molecules with multifunctional antioxidant, anti-inflammatory, neuroprotective and AChE
inhibitory properties. The hybrid series 3a
–
3m was planned by the combination of the ring patterns
A,
B
and from prototypes and as initial inspiration, adding different substituents in rings
C
1
2
B
and C in
order to evaluate potential structure-activity relationships. As a spacer subunit, an N-acylhydrazone
subunit coupled to a 1,4-disubstituted 1,2,3-triazole ring was used, with the expectancy that this
structural pattern guarantees a relative conformational freedom by the presence of an sp3 carbon
atom between ring
and acceptors sites.
A and the triazole subunit, as well as the insertion of additional H-bond donor

Molecules 2020, 25, 3165
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Figure 1. Planning by molecular hybridization of the molecular family synthesized in this work.
. Results and Discussion
2
2
.1. Chemistry
The synthesis of triazole N-acylhydrazone derivatives 3a
aldehyde ( ) was reduced to the corresponding primary alcohol
in the presence of activated carbon in THF and water [22]. Alcohol
in dichloromethane leading to the formation of the correspondent reactive chloride, which is then
converted to the azide 6 [23]. A click reaction between intermediate azide and ethyl propiolate ( ) lead
to the formation of the correspondent triazole ester , which in turn was converted to the intermediate
hydrazide by reaction with hydrazine monohydrate [24 25]. Finally the hydrazide was coupled
with different functionalized aldehydes 10 to give the desired triazole N-acylhydrazone derivatives
a–m (Figure 2) [12].
–m
comprised five steps: Initially, vanillic
4
5 by reaction with sodium borohydride
5
was reacted with thionyl chloride
6
7
8
9
,
9
3
Figure 2. Synthetic route for the preparation of the target-compounds 3a–m.

Molecules 2020, 25, 3165
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2
.2. Biological Evaluation and Computational Study
2
.2.1. Evaluation of AChE Inhibition
Initially, all compounds were screened at a standard screening concentration of 30
and the resulting percentages of AChE inhibition by the triazole derivatives 3a are shown in
Figure 3. Compound 3e was identified as the most active in the series, inhibiting more than 50% of
the AChE activity at the screening concentration and exhibiting an IC50 value of 26.30 M, being more
potent than curcumin (IC = 132.13 M [26]), but less potent than donepezil (IC50= 0.026 M [26]).
µM
–
m
µ
µ
µ
50
A study was also performed to verify the ability of 3e to inhibit BuChE, but no relevant activity was
detected, being selective against AChE.
Figure 3. Effect of hybrid compounds on AChE activity (screened at 30 µM). The result is expressed as
AChE maximum velocity (Vmax) in the presence of the indicated compound relative to uninhibited
enzyme (control).
For a better understanding of the results towards AChE inhibition by the series of hybrid
compounds, a docking study was performed with the most active compounds 3b and 3e, and for
comparison purposes, with the weaker AChE inhibitor 3d. The docking score results are shown in
Table 1.
Table 1. Ensemble docking results of compounds 3b, 3e, 3d and donepezil. The top-scored result of
each compound is given for the AChE conformation that provided the best docking score according to
the Glide XP scoring function (given in kcal/mol).
Ensemble Docking
Top-Scored
Compounds
1
ZGC
1Q84
2CKM
4EY7
Score
PDB ID
3
3
3
b
d
e
−10.67
−8.79
−9.63
−9.15
−9.39
−10.06
−8.19
−8.47
−8.10
−12.57
−10.69
−8.83
−10.69/−10.67
−9.15
4EY7/1ZGC
1Q84
−10.20
−11.14
−8.78
−10.20
1ZGC
4EY7
Donepezil
−17.94
−17.94
According to the ensemble docking results, the targeted compounds bind to the AChE cavity
with the 4-hydroxy-3-methoxyphenyl ring from the intermediate hydrazide positioned at the bottom
of the catalytic gorge, packed between the aromatic residues Trp84 and Phe330 through π-stacking
interactions and the hydroxyl group donating a hydrogen bond to the His440 main chain (~3.0 Å).
On the other side, the substituted phenyl ring from the hydrazone subunit is more exposed to the solvent
near the peripheral anionic site (PAS) at the entrance of the gorge.
The interaction of compound 3b was predicted with similar docking scores against the AChE
conformations 4EY7 and 1ZGC (
−
10.69 kcal/mol and 10.67 kcal/mol, respectively), but with distinct
−
binding modes. In the binding mode of 3b against the 4EY7 conformation, the 2-methoxyphenol
subunit is not capable of interacting with the Trp84 side chain at the bottom of the binding site, while
the resorcinol moiety is exposed to the solvent (Figure 4A). We will focus the discussion for the docked

Molecules 2020, 25, 3165
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pose of 3b against the 1ZGC conformation, which has the 4-hydroxy-3-methoxyphenyl ring from
the intermediate hydrazide interacting at the bottom of the enzymatic cavity similarly to the donepezil
binding mode (Figure 4B). In the docking pose of compound 3b with 1ZGC (docking score = 10.67
−
kcal/mol, Figure 4B), one hydroxyl group of the resorcinol moiety attached at the meta position makes
two hydrogen bond interactions with AChE, donating one hydrogen bond to the Ser286 side chain
and accepting another one from the Arg289 main chain, whereas the other hydroxyl group is exposed
to the solvent. The compound 3e, which contains only one hydroxyl group at the para position, is
capable of making only one hydrogen bond with the Ser286 side chain (2.97 Å) whereas the methoxy
group is exposed to the solvent. (Figure 4C).
Figure 4. Docking results for compounds 3b complexed with 4EY7 (A) and 1ZGC (B), 3e complexed
with 1ZGC ( ), and 3d complexed with 1Q84 ( ). Hydrogen bonds are represented as yellow dashes.
C
D
For compound 3d, the presence of two methoxy groups attached to the phenyl ring exposed
to the solvent and the absence of hydrogen bonds with PAS (Figure 4D), might be deleterious for
its binding affinity against AChE when compared to 3b and 3e.Comparative analysis with potent
AChE inhibitors described in the literature, such as donepezil, also indicates that the absence of
a positively charged group, important to create a dipole moment compatible to that observed for
the AChE gorge [27,28] and to make cation-π interactions with aromatic residues at the bottom of
the cavity, together with the high desolvation cost of polar groups not performing hydrogen bonds
with the binding site present in these compounds (i.e., NAH and triazole group), could be related with
the weaker binding affinities predicted for this class of compounds.
2
.2.2. Toxicity
Initially, we evaluated the toxic effects of compounds 3a
2.5-80 M) in human neuronal (SH-SY5Y) cells after 24 h of treatment by 3-(4,5-dimethylthiazol
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [29]. As shown in Figure 5, some compounds,
–
m at different concentrations
(
-
µ

Molecules 2020, 25, 3165
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such as 3c (IC50 = 10.30
µM), 3g (IC50 = 10.35
µM), 3h (IC50 = 3.02
µM), 3i (IC50 =10.40
µM) and 3k
(
IC50 = 3.62
effects at 80
AChE inhibition, we selected 3e to also evaluate its cytotoxicity in non-neuronal cells.
µ
M), exerted a strong toxicity, while compounds, 3a
, 3b and 3e, did not show any toxic
µM. Among the compounds not associated to toxicity, and considering its best result on
We therefore selected the compounds 3a, 3b and 3e without toxicity to perform the following
assays in SH-SY5Y cells.
Figure 5. Toxicity of compounds 3a
with different concentrations of the compounds (2.5–80
viability was measured using MTT assay. Data are reported as IC50 values (concentration resulting in
0% inhibition of neuronal viability) of the compounds.
–
3m in neuronal SH-SY5Y cells. Cells were incubated for 24 h
µM). At the end of incubation, the neuronal
5
2
.2.3. In Silico ADME Properties
In parallel to evaluation of hybrid compound neurotoxicity we performed an in silico prediction
study of ADME (Table 2) properties with the QikProp tool from Maestro (Schrödinger Release 2018-4,
Schrödinger, LLC, New York, NY, USA, 2018). In particular, the hybrid compounds 3a 3b and 3e that
,
did not show any toxic effect at the neuronal level also showed acceptable properties predicted of oral
absorption as well as permeation of the blood-brain barrier. In addition, the Lipinski’s rule of five
suggested the potential druggability of 3a, 3b and 3e.
Table 2. ADME parameters predicted by computational studies.
QPlogP
o/w
Compounds
HBA
HBD
PSA
% HOA
QPlogS
QPPCaco QPlogBB
3
a
1.73
1.07
2.48
2.54
1.79
2.45
3.58
2.92
2.84
2.68
2.67
2.47
2.62
7.3
8.0
7.3
8.0
8.0
6.5
7.5
6.5
6.5
6.5
6.5
6.5
6.5
3
4
2
2
3
2
2
2
2
2
2
2
2
124.8
147.3
111.2
120.1
133.0
102.4
104.4
102.4
102.4
100.7
102.4
102.1
102.4
72.28
61.12
85.85
86.24
73.14
85.70
92.35
88.48
87.96
88.97
86.99
85.90
86.73
-3.595
-3.282
-3.911
-4.056
-3.708
-3.817
-5.709
-4.519
-4.287
-3.839
-4.154
-3.837
-4.045
92.7
36.2
303.7
303.7
98.7
303.2
303.2
303.2
303.2
388.8
303.2
305.7
303.2
−2.09
−2.57
−1.56
−1.65
−2.15
−1.48
−1.63
−1.34
−1.34
−1.30
−1.38
−1.46
−1.39
3
b
3
c
3
d
3
e
3
f
3
g
3
h
3
3
i
j
3
k
3
l
3
m
QPlogP o/w: Predicted octanol/water partition coefficient (
HBD: Hydrogen bonding donor (0 to 6). PSA: Van der Waals surface area of polar nitrogen and oxygen atoms (7 to
−
2.0 to 6.5). HBA: Hydrogen bonding acceptors (2 to 20).
2
00). % HOA: Percentage of human absorption by oral route (<25%-low; >80%-high). QPlogS: Aqueous solubility
6.5 to 0.5); QPPCaco: Permeability in Caco-2 cell assay, model for intestinal absorption (<25–low; >500–high);
(−
QPlogBB: Permeability in the blood-brain barrier (−3.0 to 1.2).

Molecules 2020, 25, 3165
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2
.2.4. Antioxidant Activity
As reported in the Introduction, the OS plays a critical role in several neurodegenerative processes
of AD. In this context, we evaluated the direct and indirect antioxidant activity of the compounds in
terms of ability to scavenge the radical species and increase the intracellular antioxidant defenses,
respectively, in SH-SY5Y cells.
Initially, the direct antioxidant activities of all compounds 3a–m (1.56–200 mg/L) were assessed
against the DPPH radical in the absence of neuronal cells. Ascorbic acid and Trolox were used as
reference compounds (Table 3).
Table 3. Antioxidant activity of compounds against the DPPH radical and ROS formation induced by
t-BOOH in neuronal SH-SY5Y cells a.
DPPH
EC50 (mg/L)
Direct Antioxidant Activity
in SH-SY5Y Cells b
Indirect Antioxidant Activity
Compound
c
in SH-SY5Y Cells
In d
In
-
-
3
a
>200
>200
>200
>200
30.44
>200
81.51
131.5
190.9
>200
59.00
138.5
62.65
14.92
27.76
20.58
14.07
-
-
3
b
3
c
3
d
3e
29.79
35.16
3
f
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
g
3
h
3
3
i
j
3k
3
l
3
m
Ascorbic acid
Trolox
a
The antioxidant activity is expressed as percentage inhibition of ROS formation induced by t-BOOH in SH-SY5Y
cells; b the direct antioxidant activity was evaluated after a simultaneously treatment (30 min) with the compound
(
10
µM) and t-BOOH (100
µ
M); c the indirect antioxidant activity was evaluated after a chronic treatment (24 h) with
d
the compound (10 µM) then exposed to t-BOOH (100 µM for 30 min); In: inactive.
The compounds 3a
the compound 3e scavenged the DPPH radical in a similar manner to the Trolox positive control
3e, EC50 = 30.44 M vs Trolox, EC50 = 27.76 M). In parallel, we evaluated the antioxidant activity of
–d and 3f–m did not show relevant direct antioxidant activity. In contrast
(
µ
µ
the compounds not associated with toxicity (Figure 5) against the ROS formation induced by t-BOOH
in SH-SY5Y cells. To appraise the direct and indirect antioxidant activity, the SH-SY5Y cells were
simultaneously treated for 30 min with 3a
treated for 24 h (the time necessary to activate the endogenous antioxidant system) with 3a
10 M) and then exposed to t-BOOH (100 M) for 30 min, respectively. Then, the intracellular ROS
formation was detected by using the fluorescent probe 2 ,7 -dichlorodihydrofluorescein diacetate
H DCF-DA). As shown in Table 2, compound 3e, but not 3a and 3b, exerted direct antioxidant effects
,
3b and 3e (10
µM) and t-BOOH (100
µM) or chronically
,
3b and 3e
(
µ
µ
0
0
(
2
against the ROS formation evoked by t-BOOH in SH-SY5Y cells, confirming the data obtained with
DPPH assay. All tested compounds showed indirect antioxidant effects, in terms of increased neuronal
resistance against the ROS formation, with a maximum activity for 3e (35.16%). Taken together,
these results suggest that compound 3e exhibits both direct and indirect antioxidant activity against
the oxidative stress in neuronal cells.
Among the endogenous antioxidants in the central nervous system, glutathione (GSH) plays
an important role to maintain the redox balance by directly interacting with ROS formed during
the physiological metabolic processes [30
also associated with low levels of endogenous antioxidants, including GSH, in the brain. We therefore
evaluated the ability of compounds 3a 3b and 3e to modulate the intracellular GSH levels in SH-SY5Y
–32]. Recent studies suggest that the OS increase in AD is
,

Molecules 2020, 25, 3165
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cells, after 24 h of treatment using the fluorescent probe monochlorobimane (MCB). The treatment of
SH-SY5Y cells for 24 h with the compounds increased the GSH levels, with a maximum of percentage
increase for 3e (Figure 6). These results confirm the profile of indirect antioxidant activity described in
Table 3.
Figure 6. Effects of compounds 3a
4 h with different concentrations of the compounds (2.5–10
levels were measured using MCB assay. Data are expressed as a percentage of increase.
,
3b and 3e on GSH levels in SH-SY5Y cells. Cells were incubated for
2
µM). At the end of incubation, the GSH
In this regard, 3a, 3b and 3e have structural differences that justify their different antioxidant
activity. Among the hybrid compounds, 3e shows the best aspect of free radical resonance: (i) 3a and 3e
can relocate the radical from the phenolic hydroxyl to the N atom of the N-acylhydrazone group, due
2
to the hydroxyl’s para position in relation to the sp carbon of N-acylhydrazone; (ii) 3a shows less
resonance hybrids than 3e, due to the absence of methoxy next to the phenolic hydroxyl; (iii) 3b does
not have the ability to relocate the free radical outside the aromatic ring or between the hydroxyls on
the same aromatic ring.
2
.2.5. Neuroprotective Activity
Given the best antioxidant profile of compound 3e we also evaluated its ability to protect
the SH-SY5Y cells against the neurotoxicity elicited by A 42
β
peptide oligomers (OAβ42), soluble
aggregates of A peptides involved in the pathogenesis of AD. As shown in Figure 7, the concomitant
β
treatment of SH-SY5Y cells with 3e (5
induced by OAβ42 (10 µM).
µM) for 4 h significantly reduced the loss of neuronal viability
Figure 7. Compound 3e counteracts the neurotoxicity induced by OA
β
42
in SH-SY5Y cells. Cells were
treated for 4 h with 3e (5 µM) and OβA42 (10 µM). At the end of incubation, the neuronal viability was

Molecules 2020, 25, 3165
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measured using MTT assay (§§§ p < 0.001 vs cells untreated; ** p < 0.01 vs cells treated with OA 4
β
2
at one-way ANOVA with Bonferroni post hoc test). On the right representative images of formazan
crystals from MTT in living SH-SY5Y cells.
The results suggest that hybrid compound 3e could halt the interaction between the OA 1
β
–42
and the neuronal membrane, an early step of the neurotoxicity mediated by OAβ .
42
3
. Conclusions
Among these new triazole N-acylhydrazone hybrid series, compound 3e showed a potential
profile of a multifunctional compound with the ability to inhibit AChE activity and counteract the ROS
formation through direct and indirect antioxidant mechanisms. In particular, some structural attributes
of 3e, such as the position of the hydroxyls on aromatic ring, were indispensable for its antioxidant
activity in neuronal cells. This compound also showed a good profile of safety in the same neuronal
model and in silico ADME parameters. Taken together, these results suggest that compound 3e could
be considered a lead compound for the development of further AD therapeutics.
4
. Experimental Section
4
.1. General Information
IR spectra were generated on a Nicolet iSso infrared spectrometer (Thermo Scientific, Madison,
WI, USA) coupled to Gladi ATR device (Pike, Madison, WI, USA) at the Analysis and Characterization of
1
13
Drugs Laboratory (LACFar) at the Federal University of Alfenas (UNIFAL-MG). The H- and C-NMR
spectra were obtained on an AC-300 spectrometer (Bruker, Billerica, MA, USA) operating at 300 MHz for
1
13
H-NMR and 75 MHz for C-NMR at the Nuclear Magnetic Resonance Laboratory at the UNIFAL-MG.
Chromatograms for purity testing of the final compounds were recorded on a Prominence model
LC-10 HPLC (Shimadzu, Tokyo, Japan) equipped with an automatic injector, a 300 nm UV/VIS detector
and a C18 analytical column (Shimadzu CLC-ODS, 250 mm
The analyses were conducted in a two-phase isocratic system with a constant concentration of 90%
methanol (A) and 10% acetonitrile (B). Assays were conducted at 20 C at a flow rate of 0.7 mL/min.
×
4.4 mm, inner diameter
×
5 µm).
◦
All reagents used in synthesis were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used
without further purifications. Thin-layer chromatography experiments were performed on silica gel
sheet 60 F254 (Merck, Darmstadt, Germany) and chromatography column was performed on flash silica
gel (220–440 mesh, 0.035–0.075 mm, Sigma-Aldrich). The visualization of the substances was done in
an UV chamber (λ = 254 or 365 nm). The solvents dichloromethane, ethanol, and dimethylformamide
were distilled and dried according to literature [33]. Melting points were determined on Mars
equipment (Pulse Frequency Modulation, PFM II) with crushed samples packed into capillary tubes
and are reported without correction. All spectra, including those of intermediates, are available in
the Supplementary Materials.
4
.2. Chemistry
4
.2.1. 4-(Hydroxymethyl)-2-methoxyphenol (5)
Initially, a solution of vanillic aldehyde (2 g, 13.15 mmol, 1 eq.) in THF (10 mL) was placed in
a round bottomed flask under vigorous stirring, followed by the addition of activated carbon (158 mg,
eq.) and NaBH (249 mg, 0.5 eq.). The resulting solution was stirred for 1 min and then 2 mL of
1
4
distilled H O were slowly added to the reaction. The reaction mixture was kept under stirring for
2
a further 2 min. Finally, an additional 5 mL of distilled H O was added and the solution was stirred
2
for a further 5 min until total consumption of starting material [22]. For isolation of the desired alcohol,
1
0 mL of distilled H O was added and the aqueous solution was filtered off the activated carbon
2

Molecules 2020, 25, 3165
10 of 18
and exhaustively extracted with CH Cl . The organic phase was concentrated and the pure product
2
2
was obtained as white crystals (1.763 g, 11.44 mmol).
4
.2.2. 4-(Azidomethyl)-2-methoxyphenol (6)
In a round bottomed flask, alcohol (1 g, 6.50 mmol, 1 eq.) was reacted with thionyl chloride
1 mL, 2 eq.) and DMF (4 drops) in CH Cl (15 mL). The reaction mixture was kept under magnetic
5
(
2
2
stirring for approximately two hours until TLC analysis indicated the completion of the reaction.
The solvent and the excess of thionyl chloride were removed under vacuum. Subsequently, a 0.5 M
solution of NaN (26 mL, 13 mmol, 2 eq.) in DMSO was added to the flask, and the resulting solution
3
was kept under magnetic stirring for approximately 16 h [23]. When TLC analysis indicated the end of
the reaction, distilled H O (20 mL) was added, followed by exhaustive extraction with chloroform.
2
The organic phase was concentrated under vacuum and the resulting crude product was purified by
flash chromatography in silica gel, eluted with ethyl acetate: hexane (9:1) to give a brown oil (827 mg,
4
.62 mmol).
4
.2.3. Ethyl 1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carboxylate (8)
In a round bottomed flask, azide
H O/CH Cl (8 mL, 1:1 v:v), followed by the addition of ethyl propiolate (7.205 mg, 1 eq.), CuSO
4
6 (375 mg, 2.09 mmol, 1 eq.) was dissolved in a mixture of
2
2
2
(
133 mg, 0.4 eq.) and sodium ascorbate (83 mg, 0.2 eq.). The reaction mixture was kept under
◦
magnetic stirring at 20 C, for 20 h [24]. The reaction product was isolated by adding distilled H O
2
(
5 mL), followed by extraction with CH Cl (3
×
8 mL). The resulting organic phase was washed with
2
2
an aqueous solution of EDTA 50% v/v and concentrated NH OH (1:1) for removing the residual Cu(II).
4
The combined organic phases were dried with anhydrous Na SO and, after filtration, the solvent
2
4
was removed under reduced pressure. The crude product was purified by column chromatography in
silica gel, eluted with 100% CH Cl , affording the correspondent triazole (365 mg, 1.32 mmol) as
8
2
2
a yellowish solid.
4
.2.4. 1-(4-Hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carbohydrazide (9)
In a round bottomed flask, the triazole (300 mg, 1.08 mmol, 1 eq.) was dissolved in ethanol
8
(
10 mL), followed by the addition of hydrazine monohydrate (0.640 mL, 10 eq.). The reaction mixture
◦
was kept under magnetic stirring at 20 C for 17 h [25]. After TLC analysis indicated the total
consumption of the starting material, the reaction flask was refrigerated for 5 h and the reaction product
was isolated by filtration, washed with cold ethanol, providing the desired hydrazide
9 (200 mg,
0
.756 mmol) as a pale yellow solid.
4
.2.5. General procedure for obtaining the final triazoles 3a–m
In a round bottom flask, the appropriate aldehyde 10 (1.2 eq.) was dissolved in dry ethanol
10 mL), followed by the addition of concentrated HCl (5 eq.). Then, hydrazide (100 mg, 0.3801 mmol,
(
9
1
eq.), previously solubilized in the smallest possible volume of dry ethanol, was added. The resulting
◦
solution was kept under magnetic stirring and 20 C for 24h [12]. After TLC analysis indicated the end
of the reaction, the pure triazole derivative was obtained after filtration and exhaustive washing with
ice-cold ethanol.
(
E)-1-(4-Hydroxy-3-methoxybenzyl)-N’-(4-hydroxybenzylidene)-1H-1,2,3-triazole-4-carbohydrazide (3a):
◦
Yellow solid (yield 20%), m.p. 274 C, purity: 99.1%. IR (ATR):
and 1036 cm . H-NMR (DMSO-d )
s, 1H, N-C
.87–6.75 (m, 4H, Ar-
ν
3331, 3237, 1645, 1600, 1433, 1367
); 9.93 (s, 1H, O ), 9.13 (s, 1H, O ), 8.72
), 7.53 (d, J = 8.61 Hz, 2H, Ar- ), 7.05 (d, J = 1.59 Hz, 1H, Ar- ),
), 5.53 (s, 2H, Ar-CH2-N) and 3.77 (s, 3H, O-CH3). C-NMR (DMSO-d6) 159.3,
−1 1
δ
11.83 (s, 1H, N-N
H
H
H
6
(
6
H
=C), 8.41 (s, 1H, N=C
H
H
H
13
H
δ
1
55.8, 148.4, 147.6, 146.7, 142.0, 128.8, 126.9, 126.0, 125.2, 121.1, 115.6, 115.5, 112.6, 55.5 and 53.2. HR-MS
+
(ESI) m/z: Calcd for [M + H] 368.1359 g/mol, found 368.1351 g/mol.

Molecules 2020, 25, 3165
11 of 18
(
E)-N’-(3,5-Dihydroxybenzylidene)-1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carbohydrazide
◦
(
3b): Brown solid (yield 20%), m.p. 185 C, purity: 98.1%. IR (ATR):
and 1009 cm . H-NMR (DMSO-d ) δ 11.97 (s, 1H, N-NH), 9.50 (s, 2H, OH), 9.20 (s, 1H, OH), 8.77 (s,
ν
3536, 3217, 1671, 1590, 1470, 1323
−1 1
6
1
H, N-C
H
=C), 8.33 (s, 1H, N=C
), 5.54 (s, 2H, Ar-CH2-N), 3.77 (s, 3H, O-CH3). C-NMR (DMSO-d6)
47.6, 146.7, 141.8, 135.9, 127.0, 126.0, 121.1, 115.5, 112.6, 105.1, 104.4, 55.5 and 53.2. HR-MS (ESI) m/z:
H
), 7.05 (s, 1H, Ar-
H
), 6.87–6.73 (m, 2H, Ar-
H
), 6.58 (s, 2H, Ar-
H), 6.28
13
(s, 1H, Ar-
H
δ
158.6, 156.0, 148.5,
1
+
Calcd for [M + H] 384.1308 g/mol, found 384.1292 g/mol.
(
E)-1-(4-Hydroxy-3-methoxybenzyl)-N’-(4-methoxybenzylidene)-1H-1,2,3-triazole-4-carbohydrazide (3c):
◦
Light yellow solid (yield 22%), m.p. 196 C, purity: 95%. IR (ATR):
ν
3340, 3298, 1652, 1600, 1436,
); 8.74 (s, 1H, N-C =C); 8.64 (s, 1H,
); 7.64 (d, J = 8.77 Hz, 2H, Ar- ); 7.10–6.97 (m,
); 5.54 (s, 2H, Ar-CH2-N); 3.81 (s, 3H, O-CH3) and 3.77 (s, 3H, O-CH3). C-NMR (DMSO-d6)
161.6; 160.4; 155.9; 148.0; 146.7; 141.9; 129.9; 128.6; 127.0; 126.0; 121.1; 115.5; 114.2; 112.6; 55.5; 55.2
−1 1
1
O
3
δ
370 and 1022. cm . H-NMR (DMSO-d )
δ
11.91 (s, 1H, N-N
H
H
6
H
), 8.47 (s, 1H, N=C
H
); 7.82 (d, J = 8.81 Hz, 2H, Ar-
H
H
13
H, Ar-
H
+
and 53.2. HR-MS (ESI) m/z: Calcd for [M + H] 382.1515 g/mol, found 382.1494 g/mol.
(
E)-N’-(3,5-Dimethoxybenzylidene)-1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carbohydrazide
◦
(
1
8
1
3d): Light yellow solid (yield 20%), m.p. 220 C, purity: 97.9%. IR (ATR):
310 and 1034 cm . H-NMR (DMSO-d )
,64 and 8,45 (s, 1H, N=C
H, Ar-
13C-NMR (DMSO-d6)
ν
3413, 3243, 1647, 1591, 1453,
), 9.15 (s, 1H, O ), 8.76 (s, 1H, N-C =C),
), 6.86–6.78 (m, 3H, Ar- ), 6.67 (t, J = 2.28 Hz,
), 5.54 (s, 2H, Ar-CH2-N), 3.80 (s, 3H, O-CH3), 3.79 (s, 6H, O-CH3) and 3.77 (s, 3H, O-CH3).
−1 1
δ
12.06 (s, 1H, N-N
H
H
H
6
H
), 7.06 (d, J = 2,25 Hz, 2H, Ar-
H
H
H
δ
161.5, 160.6, 156.1, 148.0, 146.7, 141.7, 136.2, 127.1, 121.1, 115.5, 112.6, 106.0, 104.7,
+
1
02.3, 55.3 and 53.2. HR-MS (ESI) m/z: Calcd for [M + H] 412.1621 g/mol, found 412.1603 g/mol.
(
E)-1-(4-Hydroxy-3-methoxybenzyl)-N’-(4-hydroxy-3-methoxybenzylidene)-1H-1,2,3-triazole-4-carbo
◦
-
1
(
7
hydrazide (3e): Light yellow solid (yield 22%), m.p. 222 C, purity: 99.9%. IR (ATR):
ν
3473, 3346, 3255,
11.90 (s, 1H, N-N ), 9.62
), 7.29 (d, J = 1.55 Hz, 1H, Ar- ),
), 6.75 (d, J = 8.05 Hz, 1H, Ar- ), 5.53 (s, 2H, Ar-CH2-N),
.82 (s, 3H, CH3) and 3.76 (s, 3H, CH3). 13C-NMR (DMSO-d6)
156.1, 149.1, 148.9, 148.1, 147.8, 146.9,
42.1, 127.2, 126.2, 125.8, 122.4, 121.3, 115.7, 115.5, 112.8, 109.0, 55.7, 55.6 and 53.4. HR-MS (ESI) m/z:
−1 1
652, 1601, 1572, 1465, 1278 and 1027 cm . H-NMR (300 MHz, DMSO-d )
δ
H
6
s, 1H, O
H
), 9.22 (s, 1H, O
H
), 8.74 (s, 1H, N-C
H
=C), 8.39 (N=C
H
H
.07–7.01 (m, 2H, Ar- ), 6.85-6.80 (m, 2H, Ar-
H
H
H
3
1
δ
+
Calcd for [M + H] 398.1464 g/mol, found 398.1446 g/mol.
(
E)-N’-Benzylidene-1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carbohydrazide (3f) Light brown
◦
solid (yield 17%), m.p. 215 C, purity: 96.8%. IR (ATR):
ν
3376, 3264, 1662, 1567, 1435, 1373 and 1044
=C), 8.54 (s, 1H, N=C ), 7.70 (d, J = 5.42 Hz, 2H, Ar- ),
), 6.81 (dd, J = 8.15 and 18.24 Hz, 2H, Ar- ), 5.54 (s, 2H,
Ar-CH2-N), and 3.77 (s, 3H, O-CH3). C-NMR (DMSO-d6) 156.1, 148.1, 147.6, 146.7, 141.8, 134.2,
30.0, 128.7, 127.1, 127.0, 126.0, 121.1, 115.5, 112.6, 55.5 and 53.2. HR-MS (ESI) m/z: Calcd for [M+Na]+
74.1229 g/mol, found 374.1217 g/mol.
−
1 1
cm . H-NMR (DMSO-d )
δ
8.78 (s, 1H, N-C
H
H
H
6
7
.48–7.41 (m, 3H, Ar-
H
), 7.06 (s, 1H, Ar-
H
H
13
δ
1
3
(
E)-1-(4-Hydroxy-3-methoxybenzyl)-N’-(4-(piperidin-1-yl)benzylidene)-1H-1,2,3-triazole-4-carbohydrazide
◦
(
1
8
2
(
5
3g): Yellow solid (yield 23%), m.p. 225 C, purity: 97.4%. IR (ATR):
ν
3386, 3281, 2425, 2357, 1663, 1570,
12.05 and 11.68 (s, 1H, N-N ), 8.87 (s, 1H, N-C =C),
), 7.96–7.47 (m, 4H, Ar- ), 7.05 (s, 1H, Ar- ), 6.86-6.76 (m, 3H, Ar- ), 5.55 (s,
H, Ar-CH2-N), 3.76 (s, 3H, O-CH3), 3.52-3.36 (m, 4H, Pip- ) and 1.94-1.53 (m, 6H, Pip-
158.9, 156.0, 147.6, 146.8, 141.8, 139.4, 128.3, 127.5, 127.0, 126.0, 125.8, 121.1, 115.5, 112.7,
−1 1
451, 1276 and 1011 cm . H-NMR (DMSO-d )
δ
H
H
6
.78 and 8.49 (s, 1H, N=C
H
H
H
H
13
H
H). C-NMR
DMSO-d6)
δ
+
5.6, 53.2, 23.7 and 22.0. HR-MS (ESI) m/z: Calcd for [M + H] 435.2145 g/mol, found 435.2119 g/mol.
(
E)-N’-(4-Chlorobenzylidene)-1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carbohydrazide (3h):
◦
White solid (yield 22%), m.p. 268 C, purity: 99.9%. IR (ATR):
461, 1240 and 1029 cm . H-NMR (DMSO-d )
ν
3427, 3263, 3066, 1662, 1607, 1553,
12.16 (s, 1H, N-N ), 9.17 (s, 1H, O ), 8.78 (s, 1H,
), 7.72 (d, J = 8.32 Hz, 2H, Ar- ), 7.52 (d, J = 8.32 Hz, 2H, Ar- ), 7.06
), 6.91–6.69 (m, 2H, Ar- ), 5.54 (s, 2H, Ar-CH2-N), and 3.77 (s, 3H, O-CH3). C-NMR
156.1, 147.6, 146.8, 146.7, 141.7, 134.4, 133.2, 128.9, 128.6, 127.2, 126.0, 121.1, 115.5, 112.6,
−
1 1
1
N-C
(
(
5
δ
H
H
6
H
=C), 8.52 (s, 1H, N=C
s, 1H, Ar-
DMSO-d6)
5.5 and 53.2. HR-MS (ESI) m/z: Calcd for [M + H] 386.1020 g/mol, found 368.1000 g/mol.
H
H
H
13
H
H
δ
+

Molecules 2020, 25, 3165
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(
E)-N’-(3-Chlorobenzylidene)-1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carbohydrazide (3i):
◦
White solid (yield 21%), m.p. 234 C, purity: 99.9%. IR (ATR):
341, 1240 and 1034 cm . H-NMR (DMSO-d )
ν
3317, 3212, 3055, 1682, 1596, 1568, 1462,
12.21 (s, 1H, N-N ), 9.16 (s, 1H, O ), 8.79(s, 1H,
), 7.64 (d, J = 3.67 Hz, 1H, Ar- ), 7.49 (d, J = 4.54 Hz,
), 6.81 (q, J = 8.24 Hz, 2H, Ar- ), 5.55 (s, 2H, Ar-CH2-N), and 3.77 (s, 3H,
−
1 1
1
δ
H
H
6
H
N-C
H, Ar-
O-CH3). 13C-NMR (75 MHz, DMSO-d6)
H
=C), 8.52 (s, 1H, N=C
H
H
), 7.75 (s,1H, Ar-
H
2
H
), 7.06 (s, 1H, Ar-
H
δ
156.2, 147.6, 146.7, 146.4, 141.6, 133.6, 130.7, 129.6, 127.3, 126.1,
+
1
26.0, 125.8, 121.1, 115.5, 112.6, 55.6 and 53.2. HR-MS (ESI) m/z: Calcd for [M + H] 386.1020 g/mol,
found 386.0998 g/mol.
(
E)-N’-(2-Chlorobenzylidene)-1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carbohydrazide (3j
)
◦
White solid (yield 22%), m.p. 229 C, purity: 99.2%. IR (ATR):
368, 1244 and 1047 cm . H-NMR (300 MHz, DMSO-d )
s, 1H, N-C
H, Ar-
13C-NMR (DMSO-d6)
ν
3402, 3249, 1654, 1606, 1517, 1434,
12.37 (s, 1H, N-N ), 9.16 (s, 1H, O ), 9.96
), 8.04-7.97 (m, 1H, Ar- ), 7.55–7.50 (m, 1H, Ar- ), 7.49-7.41 (m,
), 5.55 (s, 2H, Ar-CH2-N), and 3.77 (s, 3H, O-CH3).
-
1 1
1
(
2
δ
H
H
6
H
=C), 8.78 (s, 1H, N=C
), 7.06 (s, 1H, Ar-
H
H
H
H
H
), 6.89-6.72 (m, 2H, Ar-H
δ 156.2, 147.6, 146.7, 144.2, 141.6, 131.4, 129.8, 127.5, 127.2, 126.8, 126.0, 121.1,
+
1
15.5, 112.6, 55.6 and 53.2. HR-MS (ESI) m/z: Calcd for [M + H] 386.1020 g/mol, found 386.1003 g/mol.
(
E)-N’-(4-Fluorobenzylidene)-1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carbohydrazide (3k):
◦
With solid (yield 20%), m.p. 257 C, purity: 99.9%. IR (ATR):
373, 1240 and 1030 cm . H-NMR (DMSO-d )
ν
3423, 3263, 3069, 1663, 1603, 1440,
12.09 (s, 1H, N-N ), 9.17 (s, 1H, O ), 8.77 (s, 1H,
), 7.76 (dd, J = 5.97 and 7.98 Hz, 2H, Ar- ), 7.30 (t, J = 8.66 Hz, 2H, Ar- ),
), 6.89–6.71 (m, 2H, Ar- ), 5.54 (s, 2H, Ar-CH2-N) and 3.77 (s, 3H, O-CH3). C-NMR
163.0 (d, J = 248.08 Hz), 156.1, 147.6, 147.0, 146.7, 141.8, 130.8 (d, J = 1.98 Hz), 129.2 (d,
−
1 1
1
δ
H
H
H
6
N-C
.06 (s, 1H, Ar-
DMSO-d6)
H
=C), 8.53 (s, 1H, N=C
H
H
13
7
(
H
H
δ
J = 8.61 Hz), 127.1, 126.0, 121.1, 116.0, 115.6 (d, J = 14.33 Hz), 112.6, 55.5 and 53.2. HR-MS (ESI) m/z:
+
Calcd for [M+Na] 392.1134 g/mol, found 392.1124 g/mol.
(
E)-N’-(2-Fluorobenzylidene)-1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carbohydrazide (3l):
◦
White solid (yield 19%), m.p. 225 C, purity: 99.9%. IR (ATR):
370, 1239 and 1034 cm . H-NMR (DMSO-d )
ν
3373, 3261, 3065, 1651, 1567, 1450,
12.27 (s, 1H, N-N ), 9.16 (s, 1H, O ), 8.79 (s, 2H,
), 7.55-7.43 (m, 1H, Ar- ), 7.29 (t, J = 9.06 Hz, 2H,
), 5.55 (s, 2H, Ar-CH2-N) and 3.77 (s, 3H, O-CH3).
160.7 (d, J = 249.86 Hz), 156.1, 147.6, 146.7, 141.6, 140.8, 131.9 (d, J = 8.17 Hz),
−
1 1
1
δ
H
H
6
N-C
Ar-
13C-NMR (DMSO-d6)
H
=C and N=C
H
), 7.94 (t, J = 7.46 Hz, 1H, Ar-
H
H
H
), 7.06 (s, 1H, Ar-
H
), 6.86–6.74 (m, 2H, Ar-H
δ
1
5
27.2, 126.3, 126.0, 124.8 (d, J = 2.43 Hz), 121.8 (d, J = 9.97 Hz), 121.1, 115.9 (d, J = 20.82 Hz), 115.5, 112.6,
5.5 and 53.2. HR-MS (ESI) m/z: Calcd for [M + H] 370.1315 g/mol, found 370.1292 g/mol.
+
(
E)-N’-(3-Fluorobenzylidene)-1-(4-hydroxy-3-methoxybenzyl)-1H-1,2,3-triazole-4-carbohydrazide (3m):
◦
With solid (yield 17%), m.p. 251 C, purity: 99.7%. IR (ATR):
463, 1357, 1253 and 1032 cm . H-NMR (DMSO-d )
s, 1H, N-C
ν
3320, 3149, 3066, 2836, 1682, 1598,
12.19 (s, 1H, N-N ), 9.16 (s, 1H, O ), 8.78
), 7.55-7.46 (m, 3H, Ar- ), 7.31–7.24 (m, 1H, Ar- ), 7.06 (s 1H,
), 5.55 (s, 2H, Ar-CH2-N), and 3.77 (s, 3H, O-CH3). C-NMR (DMSO-d6)
−
1 1
1
(
Ar-
δ
H
H
6
H
=C), 8.54 (s, 1H, N=C
H
H
H
13
H
), 6.86-6.75 (m, 2H, Ar-
H
δ
162.3 (d, J = 244.08 Hz), 156.2, 147.6, 146.7, 141.7, 136.8 (d, J = 7.61 Hz), 130.8 (d, J = 7.62 Hz), 127.2,
1
26.0, 123.5, 121.1, 116.8 (d, J = 21.29 Hz), 115.5, 113.0, 112.7, 55.6 and 53.2. HR-MS (ESI) m/z: Calcd for
+
[M+Na] 392.1134 g/mol, found 392.1113 g/mol.
4
.3. Pharmacological Activity
4
.3.1. Evaluation of AChE Inhibition
For in vitro evaluation of inhibition of EeAChE (EC3.1.1.7, type V-S, purified from Electrophorus
electricus) and eqBuChE (EC 3.1.1.8, purified from equine serum, both from Sigma-Aldrich), the kinetic
assay was performed according to the Ellman method [34] modified using 96-well plates as previously
described [35
,5’-dithiobis-(2-nitrobenzoic acid) (10
the evaluated compound solutions (twice as concentrated), together with 55
pH 7.4) reaching a volume of 180 L. After 10 min, 20 L of the appropriate substrate (acetylthiocholine
,
36]. To each well of the microplate were added 20
M) in sodium phosphate buffer (0.1 M, pH 7.4), 100
L phosphate buffer (0.1 M,
µ
L enzyme (0.5 U/mL) and 5
µL
5
µ
µL of
µ
µ
µ

Molecules 2020, 25, 3165
13 of 18
or butyrylthiocholine iodide, final concentration 0.5 M) was added and the absorbance was read on
a SpectraMax 250 spectrophotometer (Molecular Devices, San Jose, CA, USA) at 412 nm for 5 min
at 12-s intervals. Progression curves were acquired using the Softmax PRO 5.0 software (Molecular
Devices) from which the maximum hydrolysis velocity was calculated and the analysis performed with
the Prism 5 software (GraphPad Inc., La Jolla, CA, USA). The compounds evaluated were solubilized
in DMSO (0.05 M) and diluted in phosphate buffer to the concentrations described immediately prior
to the assay. The solvent had no detectable effect at the highest concentration used (0.2% v/v).
4
.3.2. Determination of DPPH Scavenging Ability
The ability of compounds to sequester DPPH free radicals was evaluated according to the method
described by Gontijo and co-workers with some modifications [37]. The compounds were evaluated
at concentrations of 100, 50, 25 and 12.5 M. A 4 mL aliquot of the sample was added to 1 mL of
µ
the DPPH solution (0.5 mM in ethanol). The solution was vortexed and after 30 min the absorbance
was measured at 517 nm. Each solution was analyzed in triplicate and the mean values were plotted to
obtain the EC . Trolox and ascorbic acid were used as standards. The sequestering capacity of radicals
50
was represented as the inhibition percentage according to the Equation (1):
Inhibition percentage = [(CA - SA)/CA)] × 100
(1)
CA: control absorbance; SA: sample absorbance.
4
.3.3. Cell Cultures
Human neuronal SH-SY5Y cells were routinely grown in Dulbecco’s modified Eagle’s Medium
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin and 50
µg/mL
◦
streptomycin at 37 C in a humidified incubator with 5 % CO .
2
4
.3.4. Determination of Neuronal Viability
4
SH-SY5Y cells were seeded in a 96-well plate at 2
×
10 cells/well, incubated for 24 h and then
treated with the compounds for 24 h. Cell viability, in terms of mitochondrial metabolic function, was
evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, as previously
described [38].
4
.3.5. Determination of Intracellular GSH Levels
4
SH-SY5Y cells were seeded in a black 96-well plate at 2
×
10 cells/well, incubated for 24 h and then
treated with the compounds for 24 h. GSH levels were evaluated by using monochlorobimane, as
previously described [39]
4
.3.6. Determination of Intracellular ROS Formation
SH-SY5Y cells were seeded in a 96-well plate at 2
4
×
10 cells/well, incubated for 24 h, and then
treated with compounds for 24 h before or during the treatment of 30 min with t-BOOH. ROS formation
was evaluated by using the fluorescent probe H DCF-DA, as previously described [26].
2
4
.3.7. Determination of Neuroprotective Activity Against Aβ42 Oligomers
peptide (AnaSpec, Fremont, CA, USA) was first dissolved in 1,1,1,3,3,3-hexa
A
β
42
fluoroisopropanol to 1 mg/mL, sonicated, incubated at room temperature for 24 h, and lyophilised
◦
to obtain an unaggregated A 42
β
peptide film that was solubilised with DMSO and stored at
−20 C
until use. The aggregation of A
β
4
peptide into oligomers was performed as previously described [40].
2
4
SH-SY5Y cells were seeded in a 96-well plate at 3
the compounds for 24 h. The cells were then treated for 4 h with 3e [5
×
10 cells/well, incubated for 24 h, and treated with
µ
M] and OA 1
β
[10 µM].
-42

Molecules 2020, 25, 3165
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The neuroprotective activity, in terms of increase in intracellular MTT granules, was measured by MTT
assay, as previously described [12].
4
.4. In Silico Studies
We evaluated the possible binding modes of the compounds in the AChE binding site through
molecular docking studies. Due to significant conformational changes observed on the peripheral
anionic site (PAS) [41], we selected three representative conformations of AChE following an ensemble
docking strategy similar to that adopted on previous studies [42–44] in addition to the structure
of human AChE complexed with donepezil [45] (PDB code 4EY7). The ensemble docking strategy
consists in docking the compounds into each representative conformation of the receptor aiming to
consider the protein flexibility [46
–48]. The structures selected were 1ZGC [49] (Torpedo californica),
2CKM [50] (Torpedo californica), 1Q84 [51] (Mus musculus) and 4EY7 [45] (Homo sapiens). All inhibitors
from the four representative conformations of AChE interact with both CAS and PAS. Conserved
waters were identified through the superposition of the structures and considered explicitly during
the docking experiments (Table 4).
Table 4. Conserved waters considered in the docking experiments.
Water
1ZGC
1Q84
2CKM
4EY7
Wat1
Wat2
Wat3
Wat4
1468
1481
1489
1531
1708
1755
1715
1735
2062
2054
2061
2035
729
737
722
731
The receptor structures were prepared with the Protein Preparation Wizard tool from
the Schrödinger Suite 2018–4 [52] and the protonation states of the amino acid residues were predicted
using PROPKA with pH 7. Finally, an optimization of the hydrogen bond network of the protein-ligand
complexes was performed to adjust the orientation of the hydrogen atoms, followed by an energy
minimization of the hydrogen atoms. The compounds were designed and prepared with LigPrep
from Maestro to set up the isomers, protonation states and tautomers with Epik [53
,
54] at pH 7.0
±
0
.4. We applied torsional constraints to some rotatable bonds to keep the planarity observed for some
compounds during the docking experiments (Figure 8). The rotatable bond from the amide group was
kept fixed to the trans conformation on all compounds.
Figure 8. Compounds with rotatable bonds fixed during the docking experiments (orange arrows).
Dihedrals not highlighted in this picture were defined as free to rotate, except amide bonds.
The ensemble docking experiments were performed with the molecular docking program
Glide [55,56] from Maestro in the XP precision mode – indicated for highly flexible ligands and to
reduce false-positives. All the structures were aligned to the 1ZGC conformation using the super tool
from Pymol. The grid box of the receptors was centered on the native ligand present in the 1Q84 complex
(
X: 98.06, Y: 53.14 and Z: 22.06). We also redocked the co-crystallized ligands into their respective AChE
conformation and to the non-native structures (i.e., cross-docking) to validate the docking protocol

Molecules 2020, 25, 3165
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adopted in this work. We selected the ligand pose according to the lowest XP Score among all the four
AChE conformations.
Ensemble Docking Validation
We docked the four reference ligands into all the representative AChE conformations selected
for the ensemble (i.e., redocking and cross-docking experiments) to validate the ensemble docking
strategy. It is possible to note that this strategy allowed us to select the correct binding pose (RMSD
≤
2.0 Å) of all ligands with the lowest docking score (Table 5). Interestingly, despite donepezil being
the smallest reference compound, the experimental binding mode was only found against its native
AChE conformation (i.e., 4EY7). This can be due to the complete distinct chemical profile from the other
tacrine-based ligands, inducing the enzyme to adopt a different conformation. Thus, the ensemble
docking results reinforce the importance of considering multiple representative structures of AChE to
consider the receptor flexibility, increasing the chance of finding the experimentally observed binding
mode of ligands from different chemical classes.
Table 5. Ensemble docking results for the reference ligands against the four AChE conformations.
The binding poses selected according to the ensemble docking strategy (i.e., the AChE conformation
that provides the lowest XP Score pose) are highlighted in bold, achieving 100% of success rate when
considering RMSD ≤ 2.0 Å.
1
ZGC
1Q84
Score
2CKM
Score
4EY7
Score
Reference Ligand
Score
RMSD
RMSD
RMSD
RMSD
1
1
2
4
ZGC
Q84
CKM
EY7
−
18.873
18.246
17.954
11.136
1.411
1.439
4.255
2.436
−
18.553
1.515
0.833
3.608
3.250
−18.989
1.618
4.773
1.234
4.235
−
17.273
11.546
13.255
12.382
6.852
5.905
0.594
−
−
−
−19.085
−
12.208
−
−
−
−
16.969
10.064
−20.525
−
12.575
−17.941
Success Rate
50%
50%
50%
25%
Supplementary Materials: The following are available online, Figure S1–S55: HR-MS spectra, absorption spectra
in the infrared region and chromatograms.
Author Contributions: Conceptualization, C.V.J, A.T. and M.d.F.S.; methodology, M.d.F.S., E.T.L., L.P., M.J.R.G.,
F.M.R.d.S., N.F.N., L.L.d.A., V.S.G. and I.A.G.; software, L.E.D. and I.A.G.; validation, A.T., A.E.K., V.S.G.
and M.d.F.S.; formal analysis, A.T. and C.V.J; investigation, C.V.J., A.T. and M.d.F.S.; writing—original draft
preparation, M.d.F.S., C.V.J. and V.S.G.; writing—review and editing, M.d.F.S., C.V.J. and A.T.; supervision, C.V.J.,
A.T., L.E.D., N.G.C. and A.E.K.; project administration, C.V.J. and A.T.; funding acquisition, C.V.J., A.T., A.E.K.
and L.E.D. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Brazilian Agencies CNPq (#454088/2014-0, #400271/2014-1, #310082/2016-1),
FAPEMIG (#CEX-APQ-00241-15), FINEP, INCT-INOFAR (#465.249/2014-0) and FAPERJ (#E-26-010.002487-2016).
This research was also funded in part by the Coordenaç
ão de Aperfeiçoamento de Pessoal de Nível Superior -
Brazil (CAPES) - Finance Code 001.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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2020 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 (http://creativecommons.org/licenses/by/4.0/).