Novel arylcarbamate-N-acylhydrazones derivatives as promising BuChE inhibitors: Design, synthesis, molecular modeling and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2020.115991

A novel series of arylcarbamate-N-acylhydrazones derivatives have been designed and synthesized as potential anti-cholinesterase agents. In vitro studies revealed that these compounds demonstrated selective for butyrylcholinesterase (BuChE) with potent inhibitory activity. The compounds 10a-d, 12b and 12d were the most potent BuChE inhibitors with IC₅₀ values of 0.07–2.07 μM, highlighting the compound 10c (IC₅₀ = 0.07 μM) which showed inhibitory activity 50 times greater than the reference drug donepezil (IC₅₀ = 3.54 μM). The activity data indicates that the position of the carbamate group in the aromatic ring has a greater influence on the inhibitory activity of the derivatives. The enzyme kinetics studies indicate that the compound 10c has a non-competitive inhibition against BuChE with Ki value of 0.097 mM. Molecular modeling studies corroborated the in vitro inhibitory mode of interaction and show that compound 10c is stabilized into hBuChE by strong hydrogen bond interaction with Tyr128, π-π stacking interaction with Trp82 and CH?O interactions with His438, Gly121 and Glu197. Based on these data, compound 10c was identified as low-cost promising candidate for a drug prototype for AD treatment.

Full text of DOI:10.1016/j.bmc.2020.115991

Bioorg. Med. Chem. 32 (2021) 115991
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel arylcarbamate-N-acylhydrazones derivatives as promising BuChE
inhibitors: Design, synthesis, molecular modeling and biological evaluation
a
a
a
a
a
Diego A.S. Yamazaki , Andrew M.F. Rozada , Paula Bar ´e a , Elaine C. Reis , Ernani A. Basso ,
a
b
a,*
Maria Helena Sarragiotto , Fl ´a vio A.V. Seixas , Gisele F. Gauze
a
Department of Chemistry, State University of Maring a´ , Maring a´ , PR, Brazil
Department of Technology, State University of Maring a´ , Umuarama, PR, Brazil
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
A novel series of arylcarbamate-N-acylhydrazones derivatives have been designed and synthesized as potential
anti-cholinesterase agents. In vitro studies revealed that these compounds demonstrated selective for butyr-
ylcholinesterase (BuChE) with potent inhibitory activity. The compounds 10a-d, 12b and 12d were the most
potent BuChE inhibitors with IC50 values of 0.07–2.07 µM, highlighting the compound 10c (IC50 = 0.07 µM)
which showed inhibitory activity 50 times greater than the reference drug donepezil (IC50 = 3.54 µM). The
activity data indicates that the position of the carbamate group in the aromatic ring has a greater influence on the
inhibitory activity of the derivatives. The enzyme kinetics studies indicate that the compound 10c has a non-
competitive inhibition against BuChE with Ki value of 0.097 mM. Molecular modeling studies corroborated
the in vitro inhibitory mode of interaction and show that compound 10c is stabilized into hBuChE by strong
Butyrylcholinesterase inhibitors
Carbamates
N-acylhydrazones
Molecular modeling
hydrogen bond interaction with Tyr128,
π π stacking interaction with Trp82 and CH⋯O interactions with
-
His438, Gly121 and Glu197. Based on these data, compound 10c was identified as low-cost promising candidate
for a drug prototype for AD treatment.
1
. Introduction
treatment of AD, with the current annual cost of dementia is estimated at
1
0
US $1trillion, and the projection for 2030 a figure set to double.
Cholinesterase inhibitors (ChEI) are a group of drugs that block the
Depending on the AD stage, there is a decline in acetylcholinesterase
(AChE) levels in the brain and a progressive increase of butyr-
normal hydrolysis (cholinesterase-induced) of the neurotransmitter
acetylcholine (ACh) in acetate and choline, and subsequent increasing
the levels and duration of the acetylcholine in the central nervous sys-
ylcholinesterase (BuChE) which becomes responsible for the hydrolysis
of ACh.1
2–14
According to amyloid hypothesis, fibrillar β-amyloid dis-
1
–4
tem.
These inhibitor act in the synaptic cleft, inhibiting the cholin-
order is a neuropathology of AD that may be related to neural
esterase enzymes.3 ChEI are the most promising agents developed until
,4
decrease.
15,16
The increase BuChE levels may be associated with the
today, for the symptomatic treatment of diseases related to cholinergic
formation of these toxic fibrillar β-amyloid characteristic of AD, how-
3
,4
glaucoma5 and myas-
17,18
systems, such as: Alzheimer’s disease (AD),
ever, a mechanism for this process has not been elucidated.
6
thenia gravis . The use of these therapeutic agents has improved the
cognitive functions of patients affected by AD.3,4
AChE knockout mice studies have shown the importance role of
BuChE in the nervous system as coregulator of ACh. BuChE is able to
AD is a multifactorial neurodegenerative disorder characterized by
cognitive impairment, mainly affects elderly.7 Due to the aging of the
world’s populations is a public health problem, with a great human,
compensate for the lack of AChE, allowing the continued regulation of
,8
19
cholinergic neurotransmission.
Moreover, BuChE knockout mice
studies demonstrated the fibrillar β-amyloid plaques deposition in
subcortical regions of the brain was reduced with BuChE deficiency. In
9
social and economic burden. According World Alzheimer Report 2019,
5
0 million people are living with dementia worldwide, and the projec-
this way, the decrease BuChE levels contributed to increase learning
1
0,11
20–23
tion for 2050 is estimated to increase to more than 152 million.
capacity and lowered the vulnerability β-amyloid toxicity.
There-
Thus, enormous material and financial resources are devoted into
fore, innumerous studies have been performed to development of
*
Corresponding author.
E-mail address: gfgbandoch@uem.br (G.F. Gauze).
https://doi.org/10.1016/j.bmc.2020.115991
Received 4 September 2020; Received in revised form 14 December 2020; Accepted 28 December 2020
Available online 2 January 2021
0
968-0896/© 2021 Elsevier Ltd. All rights reserved.

D.A.S. Yamazaki et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115991
selective BuChE inhibitors.24–28 Since the reduction of BuChE levels can
be an important cholinergic therapeutic approach aimed at controlling
diastereomers in N-acylhydrazone derivatives. Compound 12d was
selected to identify the stereochemistry by 1D NOESY analysis by se-
the development of AD pathology.2
9
lective irradiation of H-2 (
(Fig. 2) an interaction between the H-2 (
N–
–
N
NH ). We verified in the spectra
–
–
–
–
– –
The anticholinesterasic property of carbamates has been known for
decades. The first carbamate used clinically as ChEI in the treatment of
AD was physostigmine. However, its use has been discontinued due to its
high doses and side effects.16 Other carbamates such as neostigmine and
pyridostigmine are used to treat myasthenia gravis, and neostigmine is
also used in the glaucoma treatment. In addition, rivastigmine is one of
–
N
NH , δ = 11,94) and H-1
–
–
–
(
CH
, δ = 8,57) that can be observed in E isomer. Furthermore, an
–
–
NH , δ =
– –
evident NOE signal was observed between the H-2 (
N
11.94) and H-3 (δ = 7.61) and H-4 (δ = 7.56) in the aromatic ring
attached to carbonyl group of the N-acylhydrazone subunit. These in-
teractions can be observed in anti conformer, but should not be observed
in the putative syn conformer. Therefore, N-acylhydrazones 10a-12d
were clearly confirmed as anti-E isomers.
the four drugs approved by the Food and Drug Administration (FDA) for
the treatment of AD.3
0,31
In the discovery of new bioactive compounds,
the N-acylhydrazone moiety is considered a privileged structure,
capable of acting as a pharmacophore or auxophore subunit in different
2.2. In vitro inhibition of AChE and BuChE
3
2
pharmaceutical classes. In the last years, compounds containing the N-
acylhydrazone portion have been studied as ChEI.3
3–36
All new synthesized hybrids were evaluated for their AChE and
4
1
Based on these considerations, a series of arylcarbamate-N-acylhy-
drazone derivatives have been designed, synthesized and evaluated in
vitro which were expected to inhibit cholinesterases. These compounds
were designed by combining arylcarbamate and 3,4-dimethoxybenzyl
scaffolds linked with N-acylhydrazone moiety (see Fig 1).
BuChE inhibitory activities according to modified Ellman method,
using the enzymes AChE (eeAChE, EC.3.1.1.7 from electric eel) and
BuChE (eqBChE, EC.3.1.1.8 from equine serum). Initially, the com-
pounds were tested at concentrations of 10 and 100 µM, for both en-
zymes. For compounds that showed an inhibition greater than 50% at
1
00 µM, new tests were performed to determine the IC50 values. Done-
2
. Results and discussion
pezil was used as the reference and results are summarized in Table 1.
We can observe that the inhibition data for donepezil, are in good
agreement with the literature data.3
8,42–44
2
.1. Synthesis
The tested compounds show low inhibition for AChE and not inhibit
50% of the enzymatic activity at 100 µM (the highest concentration
analyzed). On the other hand, all compounds inhibited the BuChE
enzyme at concentration of 100 µM, and the most of them showed an
inhibition percentage of the BuChE activity greater than 50% at 100 µM
(62.25–82.00%). In addition, eight compounds tested, showed inhibi-
tion greater than 50% at concentration of 10 µM (50.12–76.40%),
except to derivatives with the carbamate group in the para-position
(11a-d). These results indicate that the new arylcarbamate-N-acylhy-
drazones exhibit greater affinity for BuChE and were selective for this
enzyme. The IC50 values were determined for compounds that inhibited
more than 40% of BuChE at concentration of 10 µM (Table 1).
The target compounds (10a-12d) were synthesized via the route
outlined in Scheme 1. Carboxylic acids 1a-c were used as starting ma-
terials to prepare carbohydrazide intermediates 3a-d. In the route A,
carboxylic acids 1a-1c were reacted with SOCl
2
at reflux of ethanol for
2 h to provide ester derivatives 2a-c (72 – 89% yield) by one pot
esterification reaction. To obtain methyl 3,4-dimethoxybenzoperoxoate
d, the carboxylic acid 1c was reacted with Me SO in the presence of
at reflux of acetone for 6 h affording ester 2d in 87% yield.
was used as methylating agent,38 due to the possibility of
OH and
OH of the carboxylic group. In the second step, the esters 2a-
3
7
1
2
2
4
K
2
CO
3
Me
2 4
SO
simultaneously carrying out the methylation of the phenolic
the
–
–
3
7
d were subjected to hydrazinolysis reaction with hydrazine mono-
In general, the new arylcarbamate-N-acylhydrazones showed excel-
lent BuChE inhibitory activity with IC50 values ranging from 0.07 to
29.25 µM. The compounds 10a-d, 12b and 12d were the more actives in
the series with IC50 values ranging from 0.07 to 2.07 µM. These com-
pounds show higher inhibitory activity than reference drug donepezil
(IC50 of 3.54 µM). The most potent inhibitor 10c was 50 times more
active than donepezil, showing a promising anticholinesterasic agent.
Analyzing the IC50 values, its possible note a tendency in the inhib-
itory behavior regarding the position of the substituents in the aromatic
ring of the carbamate group (ring A). The derivatives 10a-d with the
ortho-substituted groups in aromatic ring A showed potent BuChE
inhibitory activity, being the most promising compounds in the series
(IC50 0.07 to 0.56 to µM). On the other hand, the derivatives 11a-d with
the para-substituted groups in aromatic ring A were the least active in
the series, with two of them (11b and 11d) showing a low inhibition of
BuChE at 100 µM. The compounds 12a-d that has the tri-substituted ring
A, showed potent to moderate inhibitory activity with IC50 values
ranging from 0.94 to 9.98 to µM. On the other hand, it is not possible to
observe a trend in relation of the substitutions in the aromatic ring from
the carbohydrazide portion (ring B), suggesting that the position of the
carbamate group in the aromatic ring A has a greater influence on the
inhibitory activity of the compounds.
hydrate (80%) at reflux of ethanol for 12 h to obtain the carbohydrazides
3
a-d in 64–87% yield. In the routes B and C, the carbamoylation reac-
tion of compounds 4–6 with N, N-dimethylcarbamoyl chloride in the
presence of K
3
8
2
CO
3
affording the aldehydes 7–9 in 82–89% yield. In a
final step, condensation reactions of the carbohydrazides 3a-d with the
aldehydes 7, 8 and 9 in DMSO and acid catalysis39 at room temperature
were carried out to provide the new hybrids 10a-d, 11a-d and 12a-
d with excellent yields (62–93%).
Compounds containing the N-acylhydrazones subunit can exist as E/
Z diastereoisomers and anti/syn conformers, leading to four possible
1
isomers: anti-E, anti-Z, syn-E and syn-Z (Fig. 2). The H NMR spectra of N-
acylhydrazones 10a-12d showed only a set of signals, indicating that
only one of the possible isomeric forms is present in solution. According
to the literature40 this was attributed to the presence of only (E)-
2
.3. Kinetic studies of BuChE inhibition
Kinetics studies were carried out for the most potent inhibitor
(
compound 10c) of BuChE to investigate the inhibition mechanism and
determine the inhibition constant (Ki). To assess the kinetic parameters,
we measured the initial rate of enzyme activity at different concentra-
2
5
Fig. 1. Design strategy for the target compounds.
tions of substrate butyrylthiocholine (0.025–2.0 mM) in the absence
2

D.A.S. Yamazaki et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115991
Scheme 1. Synthesis of arylcarbamate-N-acylhydrazones derivatives 10a-12d. Reagents and conditions: i. 1a, 1b or 1c, SOCl
2
, EtOH, reflux, 12 h. ii. 1c, K
2 3
CO ,
MeSO
4
, acetone, reflux, 6 h. iii. NH
2
NH
2
2 2 3 2
⋅H O, EtOH, reflux, 12 h. iv. K CO (2 eq), acetone, reflux, 1 h; (Me) NCOCl, reflux. 8 h. v. DMSO, HCl, 3 h.
1
by selective irradiation of H-2 (–
N
–
Fig. 2. 1D NOESY (top) and H NMR spectra (bottom) for representative compound 12d in DMSO‑d
6
–
NH ) in 11.94 ppm.
–
and presence of the inhibitor 10c (0.5 and 1.0 µM). Lineweaver-Burk
10c have very close scores. So, the simulation was also performed by
programs Gold and Molegro to investigate which pose is the most
frequent. These programs were able to find and reproduce the same pose
for ligand 10c in all attempts. Therefore, the most frequent pose found
by these three programs, which is also the best ranked pose found by
Molegro and Gold was used to perform the analysis of enzyme-inhibitor
interactions.
-
1
0
plots (Fig. 3) were built by the reciprocal of the initial rate (V )
-
1
against the reciprocal of substrate concentrations ([S] ) for the different
inhibitor concentrations resulting from the substrate-velocity curves for
BuChE. Lineweaver-Burk shows the lines intersect at the x-axis intercept
(
ꢀ 1/K
m m
), indicating that the K value has practically not changed with
the increase in the inhibitor concentration, differently of Vmax that has
been reduced. Thus, it is possible to infer that the inhibition caused by
compound 10c is related to a non-competitive process with Ki value of
In the hBuChE binding pocket the compound 10c (Fig. 4A and B) is
complexed by a strong hydrogen bond interaction between the
the Tyr128 with OH and OCH groups of the aromatic ring B in a
distance of 2.8 and 2.6 Å, respectively. The aromatic ring B forms a
stacking interaction with Trp82, an essential residue in the hydrolysis
process.48 The aromatic ring A is stabilized by
-alkyl interaction with
–
OH of
0
.097 mM for BuChE. In this process, the inhibitor and the substrate
–
–
3
bind to different sites in the enzyme, forming an enzyme-inhibitor-
substrate complex (EIS).
π-π
π
Ala328. In addition, ligand 10c forms important CH⋯O interactions
2
.4. Molecular docking studies
with His438, Gly121 and Glu197.
The molecular docking simulations were performed to compound
3
1
. Conclusions
4
5
1
0c in the active site of hBuChE (PDB: 4BDS) using the Autodock Vina,
4
6
47
Molegro (v.2013.6.0.1) and Gold (v.2020.2.0) to assess the enzyme-
inhibitor interactions.
A series of new arylcarbamate-N-acylhidrazones derivatives (10a-
2d) was designed and synthesized using low-cost starting materials and
The first- and second-best poses found by Autodock Vina for ligand
3

D.A.S. Yamazaki et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115991
Table 1
ChEs inhibitory activity of compounds arylcarbamate-N-acylhydrazones 10-12d.
Compound
AChE
inhibition
00 µM
BuChE
%
IC50 (µM)
% inhibition
10 µM
IC50 (µM)
1
100 µM
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
1a
1b
1c
1d
2a
2b
2c
2d
18.18 ± 0.51
11.06 ± 0.48
24.95 ± 1.37
21.86 ± 5.61
9.65 ± 0.36
11.74 ± 3.28
20.15 ± 1.88
28.34 ± 2.46
19.92 ± 1.25
23.62 ± 3.68
29.25 ± 1.83
24.70 ± 2.28
–
>100
74.92 ± 0.36
76.40 ± 3.03
73.02 ± 1.17
62.11 ± 0.39
42.12 ± 5.17
10.43 ± 11.38
44.65 ± 2.12
11.44 ± 2.54
50.12 ± 2.60
68.36 ± 0.84
57.26 ± 1.76
74.78 ± 1.93
–
82.00 ± 5.26
80.94 ± 2.32
77.81 ± 0.87
74.03 ± 1.24
75.18 ± 1.71
31.00 ± 7.38
63.92 ± 1.37
37.77 ± 4.28
62.25 ± 4.50
73.38 ± 2.66
49.68 ± 0.95
78.47 ± 2.12
–
0.56 ± 0.004
0.18 ± 0.01
0.07 ± 0.02
0.31 ± 0.07
29.25 ± 5.27
> 100
>100
>100
>100
>100
>100
>100
21.86 ± 0.67
>100
>100
>100
7.65 ± 0.40
0.94 ± 0.27
9.98 ± 0.78
2.07 ± 0.44
3.54 ± 0.54
>100
>100
>100
Donepezil
0.01 ± 0.003
4
. Experimental
4
.1. Chemicals
Starting materials and reagents were purchased from Sigma-Aldrich
and Acros. For column chromatography, silica gel 60, 230–400 mesh
Merck) was used. Nuclear magnetic resonance (NMR) spectra were
(
acquired with Varian Mercury Plus BB 300 MHz and Bruker Avance III
HD 300 and 500 MHz. The spectra were recorded in 20 mg cm-3 solu-
tions of DMSO‑d
6
, with a probe temperature of ca. 300 K and tetrame-
thylsilane (TMS) as reference. High-resolution mass spectrometry
(
(
HRMS) analysis was performed in a high-resolution mass spectrometer
Impact II; Bruker Daltonik GmbH, Leipzig, Germany) equipped with an
electrospray ionization source (ESI).
4
.1.1. General procedure for the synthesis of ethyl benzoates (2a-c)37
To a solution of carboxylic acid 1a-c (10 mmol) in ethanol (20 ml)
thionyl chloride (11 mmol) was added dropwise under stirred and the
reaction mixture was heated under reflux for 12 h. The remaining
ethanol was evaporated and the reaction mixture was washed with
water (25 ml) and extracted twice with ethyl acetate (3 × 20 ml). The
combined organic layers were then washed with saturated sodium bi-
carbonate solution (2 × 15 ml) and brine (2 × 15 ml) and dried over
sodium sulphate. Removal of the dried organic solvent provide the esters
Fig. 3. Lineweaver-Burk plot for the inhibition of BuChE with different
butyrylthiocholine concentrations (0.025–2.0 mM) in the absence and presence
of compound 10c in concentrations of 0.5 and 1.0 µM.
2
a-c with 72–89% yield.
simple methodologies. Enzymatic inhibition studies show that the
compounds 10a-d, 12b and 12d were selective and potent BuChE in-
hibitors, with IC50 values of 0.07–2.07 µM. The greater inhibitory po-
tency towards BuChE is showed by compound 10c (Ki 0.097 mM), with
orto-carbamate-N-acylhydrazone groups in the ring A and and 3-
methoxy-4-hydroxybenzyl scaffold linked with N-acylhydrazone moi-
ety. The position of the carbamate group in the aromatic ring A has a
greater influence on the inhibitory activity of the derivatives. Analysis of
kinetics data indicated that 10c was a non-competitive inhibitor of
BuChE. The binding mode was supported by the molecular modeling
studies indicated compound 10c complexed to hBuChE by hydrogen
4
.1.2. General procedure for synthesis of methyl 3,4-dimethoxybenzoate
3
8
(
2d)
4
-hydroxy-3-methoxybenzoic acid 1c (1.68 g, 10 mmol) were solu-
bilized in 20 ml of acetone and potassium carbonate (1.38 g, 10 mmol)
was added. The mixture was stirred for 10 min at room temperature.
Then of dimethyl sulfate (3.8 ml, 40 mmol) was added dropwise,
keeping the reaction at reflux for 6 h. After this time, the reaction
2 3
mixture was then filtered to remove K CO and acetone was evaporated.
The filtrate was diluted with distilled water (25 ml) and extracted with
ethyl acetate (3 × 20 ml). The organic phase was dried over anhydrous
sodium sulfate, and the solvent was removed in a rotary evaporator. The
compound was obtained pure in 70% yield.
bond interaction with Tyr128,
π
-π stacking interaction with Trp82 and
CH⋯O interactions with His438, Gly121 and Glu197. Our results might
be of relevance to be applied for development of improved and selective
BuChE inhibitors.
4

D.A.S. Yamazaki et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115991
Fig. 4. Binding mode of the compound 10c and BuChE. (A) 3D diagram of 10c into hBuChE (PDB: 4BDS) performed using CCP4mg program. The compound is
rendered in sphere and gray stick models, and the residues are rendered in coral sticks. (B) 2D mode of interaction of the compound 10c into hBuChE (PDB: 4BDS)
analyzed by Discovery Studio Client v20.1.0.
4
.1.3. General procedure for synthesis of carbohydrazides (3a-d)37
A mixture of ester 2a-d (10 mmol) and hydrazine monohydrate 80%
(E)-2-((2-(4-hydroxybenzoyl)hydrazono)methyl)phenyl
dime-
thylcarbamate (10b)
◦
1
(
200 mmol) in ethanol (20 ml) was stirred vigorously for 12 h at reflux.
Yellow powder; yield 87%; m.p. 239.6–241.4 C. H NMR (300 MHz,
DMSO‑d ) δ 11.70 (sl, 1H), 10.14 (sl, 1H), 8.50 (sl, 1H), 7.91 (m, 1H),
After this time, the reaction mixture was kept in the freezer for 1 h. The
solid formed was filtered and washed with cold ethanol. Compounds 3a-
d were obtained pure in 72–89% yield.
6
7.80 (d, J = 8.71 Hz, 2H), 7.43 (ddd, J = 8.14, 7.32, 1.78 Hz 1H), 7.31
(m, 1H), 7.17 (dd, J = 8.15, 1.20 Hz 1H), 6.87 (d, J = 8.72 Hz, 2H) 3.14
(
s, 3H), 2.94 (s, 3H). 13C (75 MHz, DMSO‑d
) δ 163.5, 161.4, 154.5,
6
4
.1.4. General procedure for synthesis of N, N-dimethylcarbamate
150.5, 142.1, 131.2, 130.4, 127.9, 126.7, 126.4, 124.5, 124.2, 115.7,
3
8
, [M + H]+:
benzaldehydes (7–9)
To a mixture of aldehyde 4–6 (1 mmol), and potassium carbonate (2
mmol) in acetone (20 ml) was added dropwise N, N-dimethylcarbamoyl
chloride (2 mmol) and the reaction mixture was heated under reflux for
115.7, 37.2, 37.0. HRMS (ESI+): calculated for C17
17 3 4
H N O
328.1297, found 328.1304.
(E)-2-((2-(4-hydroxy-3-methoxybenzoyl)hydrazono)methyl)phenyl
dimethylcarbamate (10c)
◦
1
1
2 h. The reaction mixture was then filtered to remove K
2
CO
3
and
White powder; yield 88%; m.p. 180.5–181.3 C. H NMR (300 MHz,
DMSO‑d ) δ 11.70 (sl, 1H), 9.75 (sl, 1H), 8.51 (sl, 1H), 7.92 (m, 1H),
7.45 (m, 3H), 7.31 (m, 1H), 7.17 (m, 1H), 6.88 (d, J = 8.11 Hz, 1H), 3.85
(s, 3H), 3.13 (s, 3H), 2.94 (s, 3H). 13C (75 MHz, DMSO‑d
) δ 163.5,
acetone was evaporated. The filtrate was diluted with distilled water
6
(
25 ml) and extracted with dichloromethane (3 × 20 ml). The combined
organic layers were then washed with saturated sodium bicarbonate
solution (2 × 15 ml), dried with anhydrous sodium sulfate, and the
solvent removed in a rotary evaporator. Compounds 7–9 were obtained
pure in 82–89% yields.
6
154.5, 150.9, 150.5, 148.0, 142.2, 131.2, 127.9, 126.8, 126.4, 124.8,
124.2, 122.0, 115.6, 112.5, 56.4, 37.2, 37.0. HRMS (ESI+): calculated
+
for C18
H
19
3
N O
5
, [M + H] : 358.1402, found 358.1408.
(
E)-2-((2-(3,4-dimethoxybenzoyl)hydrazono)methyl)phenyl dime-
thylcarbamate (10d)
White powder; yield 90%; m.p. 167.1–169.0 C. H NMR (300 MHz,
DMSO‑d ) δ 11.77 (sl, 1H), 8.53 (sl, 1H), 7.93 (m, 1H), 7.57 (m, 1H),
4
.1.5. General procedure for synthesis of N-acylhydrazones (10a-d, 11a-
3
9
◦
1
d and 12a-d)
To a carbohydrazide 3a-d (1 mmol) in DMSO (2 ml) were added the
aldehyde 7–9 (1 mmol) and two drops of HCl. The reaction was stirred to
room temperature for 3 h. Then, cold water (20 ml) was added to a
reactional mixture and the pH was corrected to 7 with aqueous solution
6
7.48 (m, 1H), 7.43 (m, 1H), 7.32 (m, 1H), 7.18 (m, 1H), 7.09 (d, J =
8.45 Hz, 1H), 3.84 (s, 6H), 3.13 (s, 3H), 2.94 (s, 3H). 1 C (75 MHz,
3
6
DMSO‑d ) δ 163.4, 154.5, 152.5, 150.6, 149.1, 142.5, 131.3, 127.8,
K
2
CO
3
solution 10%. The precipitated resulting was washed with cold
126.9, 126.4, 126.1, 124.2, 121.7, 111.7, 111.6, 56.3, 37.2, 37,0. HRMS
+
water and filtrated to obtained pure N-acylhydrazones 10a-d, 11a-
d and 12a-d in 62 – 93 yield.
(ESI+): calculated for C19
H
21
N
3
O
5
, [M + H] : 372.1559, found
372.1560.
(
E)-2-((2-(3-hydroxybenzoyl)hydrazono)methyl)phenyl
dime-
(E)-4-((2-(3-hydroxybenzoyl)hydrazono)methyl)phenyl
dime-
thylcarbamate (10a)
White powder; yield 92%; m.p. 123.2–125.4 C. H NMR (300 MHz,
DMSO‑d ) δ 11.83 (sl, 1H), 9.78 (sl, 1H), 8.53 (sl, 1H), 7.93 (m, 1H),
.45 (m, 1H), 7.31 (m, 4H), 7.18 (m, 1H), 6.98 (m, 1H), 3.13 (s, 3H),
.94 (s, 3H). 13C (75 MHz, DMSO‑d
) δ 163.9, 158.1, 154.5, 150.6,
thylcarbamate (11a)
◦
1
◦
1
Yellow powder; yield 88%; m.p. 180.1–182.6 C. H NMR (300 MHz,
DMSO‑d
) δ 11.77 (sl, 1H), 9.76 (sl, 1H), 8.44 (sl, 1H), 7.73 (d, J = 8.21
Hz, 2H), 7.31 (m, 3H), 7.21 (d, J = 8.22 Hz, 2H), 6.96 (m, 1H), 3.05 (s,
6
6
7
2
1
1
3
13
6
6
3H), 2.92 (s, 3H). C (75 MHz, DMSO‑d ) δ 163.8, 158.1, 154.4, 153.3,
43.0, 135.4, 131.4, 130.3, 127.7, 126.8, 126.5, 124.3, 119.4, 118.8,
147.6, 135.5, 132.0, 130.2, 128.7, 123.0, 123.0, 119.4, 118.8, 115.2,
15.2, 37.2, 37.0. HRMS (ESI+): calculated for C17
H
17
N
3
O
4
, [M + H]+:
37.0, 36.8. HRMS (ESI+): calculated for C17
H
17
N
3
O
4
, [M + H]+:
28.129725, found: 328.1302.
328.1297, found 328.1298.
5

D.A.S. Yamazaki et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115991
(
E)-4-((2-(4-hydroxybenzoyl)hydrazono)methyl)phenyl
thylcarbamate (11b)
Yellow powder; yield 91%; m.p. 250.9–252.4 C. H NMR (300 MHz,
DMSO‑d
) δ 11.63 (sl, 1H), 10.12 (sl, 1H), 8.43 (sl, 1H), 7.81 (d, J = 8.71
dime-
4.2. ChEs inhibition assays
◦
1
AChE (from electrophorus electricus, type VI-S, lyophilized powder,
lot 041M7009V), BuChE (from equine serum, lyophilized powder, lot
6
′
Hz, 2H), 7.72 (m, 2H), 7.20 (d, J = 8.38 Hz, 2H), 6.86 (d, J = 8.74 Hz,
SLBB2114V). 5,5 - dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman’s re-
2
1
3
H), 3.05 (s, 3H), 2.92 (s, 3H). 13C (75 MHz, DMSO‑d
6
) δ 163.4, 161.3,
agent), acetylthiocholine iodide, Sbutyrylthiocholine iodide were pur-
chased from Sigma Aldrich. Absorbance measurements were performed
in a Molecular Devices FlexStation 3 Microplate Reader using Softmax
Pro 5.3 software.
54.4, 153.1, 146.7, 132.1, 130.3, 128.5, 124.6, 123.0, 115.7, 37.0,
+
6.8. HRMS (ESI+): calculated for C17
H
17
N
3
O
4
, [M + H] : 328.1297,
found: 328.1306.
E)-4-((2-(4-hydroxy-3-methoxybenzoyl)hydrazono)methyl)phenyl
dimethylcarbamate (11c)
White powder; yield 87%; m.p. 197.7–200.3 C. H NMR (300 MHz,
DMSO‑d
) δ 11.62 (sl, 1H), 9.72 (sl, 1H), 8.44 (sl, 1H), 7.73 (d, J = 8.21
Hz, 2H), 7.49 (m, 1H), 7.45 (m, 1H), 7.21 (d, J = 8.65 Hz, 2H), 6.88 (d, J
(
AChE and BuChE inhibitory activities were evaluated by spectro-
photometrical Ellman’s modified method and donepezil was used as
reference compound. Stock solutions of tested compounds 10a-d, 11a-
d and 12a-d were prepared in methanol and phosphate buffer solution
at pH 8.00, AChE and BuChE were prepared in phosphate buffer solution
◦
1
6
8.19 Hz, 1H), 3.85 (s, 3H), 3.05 (s, 3H), 2.92 (s, 3H). 1 C (75 MHz,
3
at pH 8.00 with concentration of 0.23 U mL . The tests were performed
ꢀ 1
=
DMSO‑d ) δ 163.4, 154.4, 153.1, 150.8, 148.0, 146.8, 132.1, 128.5,
6
in polystyrene 96-well plate, in each well were added 125 µL of DTNB
(0.5 mM), 50 µL of phosphate buffer solution (pH 8), 25 µL of sample
solution at different concentrations and without the inhibitor (control),
25 µL of enzyme solution of AChE or BuChE. The plate was incubated at
1
24.8, 123.0, 122.0, 115.6, 112.3, 56.4, 37.0, 36.8. HRMS (ESI+):
+
calculated for C18
H
19
3
N O
4
, [M + H] : 358.1402, found: 358.1409.
(
E)-4-((2-(3,4-dimethoxybenzoyl)hydrazono)methyl)phenyl dime-
◦
thylcarbamate (11d)
White powder; yield 62%; m.p. 296 C (decompose). 1H NMR (300
MHz, DMSO‑d
) δ 11.73 (sl, 1H), 8.42 (sl, 1H), 7.74 (d, J = 8.26 Hz, 2H),
.55 (dd, J = 8.40, 2.04 Hz,1H), 7.47 (d, J = 2.01 Hz, 1H), 7.18 (d, J =
.41 Hz, 2H), 7.06 (d, J = 8.49 Hz, 1H), 3.81 (s, 6H), 3.02 (s, 3H), 2.89
) δ 163.5, 154.5, 153.2, 152.4, 149.0,
30 C for 15 min under stirring, and then absorbance was measured at a
◦
wavelength of 412 nm. After this time, 25 µL of substrate (acetylth-
iocholine or butyrylthiocholine, 5 mM, prepared in milli-Q water) were
6
◦
7
8
added to each well. The plate was kept at 30 C, under agitation and the
absorbance was measured for 4 min, following the standard parameters
s, 3H). 13C (75 MHz, DMSO‑d
of the incubation. The tests were performed in triplicate.
49
(
6
1
3
3
47.3, 132.0, 128.7, 125.9, 123.0, 121.7, 111.6, 111.4, 56.3, 56.2, 36.9,
The rates of the reactions were calculated using appropriate software
(Origin 6.1). The inhibition percentages were calculated by comparing
of control reaction rate with the samples reaction rate using Eq. (1):
+
6.8. HRMS (ESI+): calculated for
C
19
H
21
N
3
O
5
Na, [M
+
Na] :
94.1373, found: 394.1367
(
E)-4-((2-(3-hydroxybenzoyl)hydrazono)methyl)-2-methoxyphenyl
dimethylcarbamate (12a)
Yellow powder; yield 88%; m.p. 211.4–212.7 C. H NMR (300 MHz,
DMSO‑d ) δ 11.79 (sl, 1H), 9.77 (sl, 1H), 8.43 (sl, 1H), 7.43 (m, 1H),
.31 (m, 3H), 7.25 (m, 1H), 7.16 (m, 1H), 6.98 (m, 1H), 3.84 (s, 3H),
.04 (s, 3H), 2.90 (s, 3H). 13C (75 MHz, DMSO‑d
) δ 163.9, 158.1, 154.2,
%
inhibition = ((control reaction rate
ꢀ sam ple reaction rate)/control reaction rate )*100
The inhibition curve was obtained by plotting an inhibition per-
centage graph versus the logarithm of the inhibitor concentration.
◦
1
(1)
6
7
3
1
1
6
52.4, 147.9, 142.4, 135.5, 133.3, 130.2, 124.3, 121.2, 119.4, 118.8,
4
.3. Kinetic studies of ChEs inhibition
15.2, 110.2, 56.5, 37.0, 36.8. HRMS (ESI+): calculated for
+
C
18
H
19
3
N O
4
, [M + H] : 358.1402, found 358.1409.
Kinetic studies were carried out by Ellman’s modified method for
(
E)-4-((2-(4-hydroxybenzoyl)hydrazono)methyl)-2-methoxyphenyl
dimethylcarbamate (12b)
White powder; yield 93%; m.p. 261.0–262.1 C. H NMR (300 MHz,
DMSO‑d
) δ 11.66 (sl, 1H), 10.13 (sl, 1H), 8.42 (sl, 1H), 7.81 (d, J = 8.67
ꢀ 1
compound 10c, using a 0.23 U mL solution of BuChE from equine. The
test was performed without the inhibitor, in 0.5 and 1.0 µM concen-
tration of the inhibitor 10c for BuChE. Butyrylthiocoline iodine was used
as substrate of reaction in the following final concentrations: 0.025,
0.05, 0.125, 0.50, 0.75, 1.0 and 2.0 µM. The absorbance was measured
in 10 s for 6 min. The obtained data were used to create substrate-
velocity curves which were transformed in Origin 6.1 program to
Linerweaver-Burk plots.25
◦
1
6
Hz, 2H), 7.42 (m, 3H), 7.23 (m, 1H), 7.15 (m, 1H), 6.86 (d, J = 8.68 Hz,
H), 3.83 (s, 3H), 3.04 (s, 3H), 2.90 (s, 3H). 1 C (75 MHz, DMSO‑d
3
2
1
1
6
) δ
63.5, 161.4, 154.2, 152.4, 147.1, 142.2, 133.5, 130.4, 124.5, 124.3,
21.0, 115.7, 115.7, 110.2, 56.5, 37.0, 36.8. HRMS (ESI+): calculated
+
for C18
H
19
N
3
O
4
, [M + H] : 358.1402, found 358.1407.
(
E)-4-((2-(4-hydroxy-3-methoxybenzoyl)hydrazono)methyl)-2-
methoxyphenyl dimethylcarbamate (12c)
Yellow powder; yield 85%; m.p. 132.1 ꢀ 133 C. H NMR (300 MHz,
DMSO‑d ) δ 11.64 (sl, 1H), 9.73 (sl, 1H), 8.48 (sl, 1H), 7.46 (m, 3H),
.24 (m, J = 8.17 Hz, 1H), 7.15 (m, J = 8.08 Hz, 1H), 6.88 (d, J = 8.88
4.4. Molecular modeling
◦
1
6
The crystal structures of BuChE complexed with tacrine (PDB ID:
4BDS) was obtained from the Protein Data Bank. All small molecules
such as water, salts and ligands (except the main ligand tacrine) were
removed. The compound structure was drawn with the Marvin Sketch
7
1
3
Hz, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.04 (s, 3H), 2.90 (s, 3H). C (75
MHz, DMSO‑d
6
) δ 163.4, 154.2, 152.4, 150.8, 147.9, 147.2, 142.2,
Version 14.8.25 ChemAxon.50 The molecular docking simulations were
1
3
33.5, 124.8, 124.3, 122.0, 121.0, 115.6, 112.3, 110.2, 56.5, 56.4, 37.0,
+
45
6.8. HRMS (ESI+): calculated for C19
H
21
N
3
O
6
, [M + H] : 358.1508,
performed using three programs: Autodock Vina,
Molegro
(v.2013.6.0.1)46 and Gold (v.2020.2.0). The redocking was performed
to validate the programs and protocols that would be used in the
simulations.
47
found 358.1512.
(
E)-4-((2-(3,4-dimethoxybenzoyl)hydrazono)methyl)-2-methox-
yphenyl dimethylcarbamate (12d)
Yellow powder; yield 88%; m.p. 174.5–175.3 C. H NMR (300 MHz,
DMSO‑d
) δ 11.86 (sl, 1H), 8.53 (sl, 1H), 7.60 (dd, J = 8.4 Hz, 1,78 Hz,
H), 7.54 (m, 1H), 7.42 (m, 1H), 7.24 (m, 1H), 7.16 (d, J = 8.1 Hz, 1H),
◦
1
The Autodock Vina program was implemented in PyRx 0.9.8
graphical interface51 with a search box of size 15 × 15 × 15 Å and the
center of the grid box placed at coordinates [133.0; 116.0; 41], using
default settings. In the Molegro program, the search algorithm was
MolDock Optimizer, PLANTS Score [grid] for ranking, and a search
sphere with 8.0 radius centered on the coordinates [133.17, 116.22;
0.98]. The best poses were ordered according to the best MolDock Score.
The simulation protocol for Gold program was ASP algorithm with
6
1
7
1
3
.08 (d, J = 8.5, 1H) 3.83 (s, 9H), 3.05 (s, 3H), 2.90 (s, 3H). C (75 MHz,
DMSO‑d
6
) δ 163.1, 153.8, 152.0, 148.5, 147.3, 141.8, 133.0, 125.3,
1
23.9, 121.5, 120.7, 111.2, 110.0, 56.1, 55.9, 36.6, 36.4. HRMS (ESI+):
+
calculated for C20
H
23
3
N O
6
, [M + H] : 402.1659, found: 402.1656
6

D.A.S. Yamazaki et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115991
2
00% efficiency and a search radius of 10 Å. Four repetitions were made
19 Li B, Stribley JA, Ticu A, et al. Abundant tissue butyrylcholinesterase and its possible
function in the acetylcholinesterase knockout mouse. J Neurochem. 2000;75:
with for the ligand in each program.
1
320–1331. https://doi.org/10.1046/j.1471-4159.2000.751320.x.
The protein ligand interactions were analyzed and represented using
2
2
0
1
Reid GA, Darvesh S. Butyrylcholinesterase-knockout reduces brain deposition of
fibrillar β-amyloid in an Alzheimer mouse model. Neuroscience. 2015;298:424–435.
https://doi.org/10.1016/j.neuroscience.2015.04.039.
5
2
CCP4 Molecular Graphics software
v20.1.0.53
and Discovery Studio Client
Maurice T, Strehaiano M, Sim ´e on N, Bertrand C, Chatonnet A. Learning
performances and vulnerability to amyloid toxicity in the butyrylcholinesterase
knockout mouse. Behav Brain Res. 2016;296:351–360. https://doi.org/10.1016/j.
bbr.2015.08.026.
Declaration of Competing Interest
2
2
2
3
Darvesh S, Reid GA. Reduced fibrillar β-amyloid in subcortical structures in a
butyrylcholinesterase-knockout Alzheimer disease mouse model. Chem Biol Interact.
2016;259:307–312. https://doi.org/10.1016/j.cbi.2016.04.022.
DeBay DR, Reid GA, Macdonald IR, et al. Butyrylcholinesterase-knockout reduces
fibrillar β-amyloid and conserves 18FDG retention in 5XFAD mouse model of
Alzheimer’s disease. Brain Res. 2017;1671:102–110. https://doi.org/10.1016/j.
brainres.2017.07.009.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
2
2
4
5
Bagatin MC, C ˆa ndido AA, Pinheiro GMS, et al. Molecular modeling and
anticholinesterasic activity of novel 2-arylaminocyclohexyl N, N
We would like to thank Fundaç a˜ o Arauc ´a ria (002/17) and CNPQ
-
Dimethylcarbamates. J Braz Chem Soc. 2013;24:1798–1807. https://doi.org/
(
404455/2016-6) for the financial support of this research, and CAPES
10.5935/0103-5053.20130225.
Yamazaki DAS, C aˆ ndido AA, Bagatin MC, et al. Cholinesterases inhibition by novel
cis- and trans-3-arylaminocyclohexyl N, N-dimethylcarbamates: Biological
evaluation and molecular modeling. J Braz Chem Soc. 2016;27. https://doi.org/
10.5935/0103-5053.20160041.
for a scholarship (Yamazaki, D. A. S.; Bar ´e a, P.; Reis, E. C. and Rozada, A.
M. F.) and to Complexo de Centrais de Apoio a` Pesquisa of Universidade
Estadual de Maring ´a (COMCAP-UEM) for the facilities.
2
2
2
2
3
3
3
3
6
7
8
9
0
1
2
3
Li Q, Yang H, Chen Y, Sun H. Recent progress in the identification of selective
butyrylcholinesterase inhibitors for Alzheimer’s disease. Eur J Med Chem. 2017;132:
Appendix A. Supplementary material
2
94–309. https://doi.org/10.1016/j.ejmech.2017.03.062.
Xu Y, Zhang Z, Jiang X, et al. Discovery of δ-sultone-fused pyrazoles for treating
Alzheimer’s disease: Design, synthesis, biological evaluation and SAR studies. Eur J
Med Chem. 2019;181, 111598. https://doi.org/10.1016/j.ejmech.2019.111598.
Zhang Z, Min J, Chen M, et al. The structure-based optimization of δ-sultone-fused
pyrazoles as selective BuChE inhibitors. Eur J Med Chem. 2020;201, 112273. https://
doi.org/10.1016/j.ejmech.2020.112273.
Johnson G, Moore SW. Why has butyrylcholinesterase been retained? Structural and
functional diversification in a duplicated gene. Neurochem Int. 2012;61:783–797.
https://doi.org/10.1016/j.neuint.2012.06.016.
Bullock R. The clinical benefits of rivastigmine may reflect its dual inhibitory mode
of action: an hypothesis. Int J Clin Pract. 2002;56:206–214. http://www.ncbi.nlm.
nih.gov/pubmed/12018828.
Pope C, Karanth S, Liu J. Pharmacology and toxicology of cholinesterase inhibitors:
uses and misuses of a common mechanism of action. Environ Toxicol Pharmacol.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2020.115991.
References
1
2
3
4
Ellis JM. Cholinesterase inhibitors in the treatment of dementia. J Am Osteopath
Assoc. 2005;105:145–158. http://www.ncbi.nlm.nih.gov/pubmed/15863734.
Anand P, Singh B. A review on cholinesterase inhibitors for Alzheimer’s disease. Arch
Pharm Res. 2013;36:375–399. https://doi.org/10.1007/s12272-013-0036-3.
Minett TSC, Bertolucci PHF. Terapia colin ´e rgica na doença de Alzheimer. Rev
Neuroci ˆe ncias. 2000;8:11–14.
Colovic MB, Krstic DZ, Lazarevic-Pasti TD, Bondzic AM, Vasic VM.
Acetylcholinesterase inhibitors: pharmacology and toxicology. Curr Neuropharmacol.
2
005;19:433–446. https://doi.org/10.1016/j.etap.2004.12.048.
Duarte C, Barreiro E, Fraga C. Privileged structures: a useful concept for the rational
design of new lead drug candidates. Mini-Rev Med Chem. 2007;7:1108–1119. https://
doi.org/10.2174/138955707782331722.
2
013;11:315–335. https://doi.org/10.2174/1570159X11311030006.
Hoyng PFJ, van Beek LM. Pharmacological therapy for glaucoma. Drugs. 2000;59:
11–434. https://doi.org/10.2165/00003495-200059030-00003.
Richman DP, Agius MA. Treatment of autoimmune myasthenia gravis. Neurology.
5
6
7
4
¨
¨
Ozturan Ozer E, Unsal Tan O, Ozadali K, Küçükkılınç T, Balkan A, Uçar G. Synthesis,
′
molecular modeling and evaluation of novel N -2-(4-benzylpiperidin-/piperazin-1-yl)
2
003;61:1652–1661.
acylhydrazone derivatives as dual inhibitors for cholinesterases and Aβ aggregation.
Bioorg Med Chem Lett. 2013;23:440–443. https://doi.org/10.1016/j.
bmcl.2012.11.064.
Karaman N, Sıcak Y, Ta s¸ kın-Tok T, et al. New piperidine-hydrazone derivatives:
Synthesis, biological evaluations and molecular docking studies as AChE and BChE
inhibitors. Eur J Med Chem. 2016;124:270–283. https://doi.org/10.1016/j.
ejmech.2016.08.037.
da Silva GS, Figueir o´ M, Tormena CF, Coelho F, Almeida WP. Effects of novel
acylhydrazones derived from 4-quinolone on the acetylcholinesterase activity and A
β 42 peptide fibrils formation. J Enzyme Inhib Med Chem. 2016;31:1464–1470.
https://doi.org/10.3109/14756366.2016.1144597.
Dias Viegas FP, de Freitas Silva M, Divino da Rocha M, et al. Design, synthesis and
pharmacological evaluation of N-benzyl-piperidinyl-aryl-acylhydrazone derivatives
as donepezil hybrids: Discovery of novel multi-target anti-alzheimer prototype drug
candidates. Eur J Med Chem. 2018;147:48–65. https://doi.org/10.1016/j.
ejmech.2018.01.066.
Larson EB, Kukull WA, Katzman RL. Cognitive impairment: dementia and
Alzheimer’s disease. Annu Rev Public Health. 1992;13:431–449. https://doi.org/
1
0.1146/annurev.pu.13.050192.002243.
3
3
3
4
5
6
8
9
Kumar A, Singh A, Ekavali. A review on Alzheimer’s disease pathophysiology and its
management: an update. Pharmacol Reports. 2015;67:195–203. https://doi.org/
1
0.1016/j.pharep.2014.09.004.
Wimo A, Guerchet M, Ali GC, et al. The worldwide costs of dementia 2015 and
comparisons with 2010. Alzheimer’s Dement. 2017;13:1–7. https://doi.org/10.1016/
j.jalz.2016.07.150.
1
0
1
2
International AD. World Alzheimer Report 2019 Attitudes to dementia. World
Alzheimer Rep 2019. Published online 2019: 13. doi:10.1007/978-3-030-10814-4_
2
3.
1
1
Kuca K, Soukup O, Maresova P, et al. Current approaches against Alzheimer’s disease
in clinical trials. J Braz Chem Soc. 2016;27:641–649. https://doi.org/10.5935/0103-
5
053.20160048.
Atack JR, Perry EK, Bonham JR, Candy JM, Perry RH. Molecular forms of
acetylcholinesterase and butyrylcholinesterase in the aged human central nervous
system. J Neurochem. 1986;47:263–277. https://doi.org/10.1111/j.1471-4159.1986.
tb02858.x.
3
3
3
7
8
9
Bagatin MC, Rozada AMF, Rodrigues FAV, et al. New 4-methoxy-naphthalene
derivatives as promisor antifungal agents for paracoccidioidomycosis treatment.
Future Microbiol. 2019;14:235–245. https://doi.org/10.2217/fmb-2018-0276.
Liew KF, Chan KL, Lee CY. Blood-brain barrier permeable anticholinesterase aurones:
Synthesis, structure-activity relationship, and drug-like properties. Eur J Med Chem.
1
1
1
3
4
5
Ballard C, Greig N, Guillozet-Bongaarts A, Enz A, Darvesh S. Cholinesterases: roles in
the brain during health and disease. Curr Alzheimer Res. 2005;2:307–318. https://
doi.org/10.2174/1567205054367838.
Lane RM, Potkin SG, Enz A. Targeting acetylcholinesterase and butyrylcholinesterase
in dementia. Int J Neuropsychopharmacol. 2006;9:101–124. https://doi.org/10.1017/
S1461145705005833.
Montine TJ, Phelps CH, Beach TG, et al. National institute on aging-Alzheimer’s
association guidelines for the neuropathologic assessment of Alzheimer’s disease: A
practical approach. Acta Neuropathol. 2012;123:1–11. https://doi.org/10.1007/
s00401-011-0910-3.
2
015;94:195–210. https://doi.org/10.1016/j.ejmech.2015.02.055.
Rozada AMF, Rodrigues FAV, Sampiron EG, et al. Novel 4-methoxynaphthalene-N-
acylhydrazones as potential agents for the treatment of paracoccidioidomycosis and
tuberculosis co-infection. Future Microbiol. 2019;14:587–598. https://doi.org/
1
0.2217/fmb-2018-0357.
4
4
4
4
0
1
2
3
Lima PC, Lima LM, da Silva KCM, et al. Synthesis and analgesic activity of novel N-
acylarylhydrazones and isosters, derived from natural safrole. Eur J Med Chem. 2000;
3
5:187–203. https://doi.org/10.1016/S0223-5234(00)00120-3.
Ellman GL, Courtney KD, Andres V, Featherstone RM. A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88–95.
https://doi.org/10.1016/0006-2952(61)90145-9.
1
1
1
6
7
8
Francis PT, Palmer AM, Snape M, Wilcock GK. The cholinergic hypothesis of
Alzheimer’s disease: a review of progress. J Neurol Neurosurg Psychiatry. 1999;66:
1
37–147. https://doi.org/10.1136/jnnp.66.2.137.
Cavalli A, Bolognesi ML, Minarini A, et al. Multi-target-directed ligands to combat
neurodegenerative diseases. J Med Chem. 2008;51:347–372. https://doi.org/
Mesulam-Marsel, Geula C. Butyrylcholinesterase reactivity differentiates the amyloid
plaques of aging from those of dementia. Ann Neurol. 1994;36:722–727. https://doi.
org/10.1002/ana.410360506.
Guillozet AL, Mesulam M-M, Smiley JF, Mash DC. Butyrylcholinesterase in the life
cycle of amyloid plaques. Ann Neurol. 1997;42:909–918. https://doi.org/10.1002/
ana.410420613.
1
0.1021/jm7009364.
Xie SS, Lan JS, Wang X, et al. Design, synthesis and biological evaluation of novel
donepezil-coumarin hybrids as multi-target agents for the treatment of Alzheimer’s
7

D.A.S. Yamazaki et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115991
disease. Bioorganic Med Chem. 2016;24:1528–1539. https://doi.org/10.1016/j.
48 Bajda M, Wi A, Hebda M, Guzior N. Structure-based search for new inhibitors of
cholinesterases. Int J Mol Sci. 2013;14:5608–5632. https://doi.org/10.3390/
ijms14035608.
49 Barbosa VA, Bar ´e a P, Mazia RS, et al. Synthesis and evaluation of novel hybrids β
-carboline-4-thiazolidinones as potential antitumor and antiviral agents. Eur J Med
Chem. 2016;124:1093–1104. https://doi.org/10.1016/j.ejmech.2016.10.018.
50 Marvin Sketch Version 14.8.25, 2014, ChemAxon (http://www.chemaxon.com).
http://www.chemaxon.com.
bmc.2016.02.023.
4
4
Li F, Wang Z-M, Wu J-J, et al. Synthesis and pharmacological evaluation of
donepezil-based agents as new cholinesterase/monoamine oxidase inhibitors for the
potential application against Alzheimer’s disease. J Enzyme Inhib Med Chem. 2016;31
(
sup3):41–53. https://doi.org/10.1080/14756366.2016.1201814.
4
4
4
5
6
7
Anil KTJW. Autodock vina: improving the speed and accuracy of docking. J Comput
Chem. 2019;31:455–461. https://doi.org/10.1002/jcc.21334.AutoDock.
Thomsen R, Christensen MH. MolDock: a new technique for high-accuracy molecular
docking. J Med Chem. 2006;49:3315–3321. https://doi.org/10.1021/jm051197e.
Jones G, Willett P, Glen RC, Leach AR, Taylor R. Development and validation of a
genetic algorithm for flexible docking 1 1Edited by F. E. Cohen. J Mol Biol. 1997;267:
51 Wolf L. newscripts. Chem Eng News. 2009;87:48. https://doi.org/10.1021/cen-
v087n031.p048.
52 McNicholas S, Potterton E, Wilson KS, Noble MEM. Presenting your structures: the
CCP 4 mg molecular-graphics software. Acta Crystallogr Sect D Biol Crystallogr. 2011;
67:386–394. https://doi.org/10.1107/S0907444911007281.
7
27–748. https://doi.org/10.1006/jmbi.1996.0897.
5
3
BIOVIA, Dassault Syst `e mes, Discovery Studio Client, v.20.1.0.199295, San Diego:
Dassault Syst `e mes, 2020.
8