Antioxidant and acetylcholinesterase inhibition activity of aliphatic and aromatic edaravone derivatives

Article abstract of DOI:10.1007/s00044-020-02667-5

As Alzheimer disease (AD) is a multifactorial condition, it should be tackled with drugs targeting multiple key pathways. A series of aliphatic (2–8) and aromatic (9–15) edaravone derivatives were synthesized, characterized, and evaluated as antioxidant a

Full text of DOI:10.1007/s00044-020-02667-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02667-5
ORIGINAL RESEARCH
Antioxidant and acetylcholinesterase inhibition activity of aliphatic
and aromatic edaravone derivatives
Victor Wagner Barajas-Carrillo1 Arturo Estolano-Cobián1 Laura Díaz-Rubio1 Rocío Rosario Ayllón-Gutiérrez1
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Ricardo Salazar-Aranda2 Raúl Díaz-Molina3 Víctor García-González3 Horacio Almanza-Reyes4
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Ignacio A. Rivero5 Joaquín G. Marrero6 Iván Córdova-Guerrero
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Received: 16 September 2020 / Accepted: 5 November 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
As Alzheimer disease (AD) is a multifactorial condition, it should be tackled with drugs targeting multiple key pathways. A
series of aliphatic (2–8) and aromatic (9–15) edaravone derivatives were synthesized, characterized, and evaluated as
antioxidant agents using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2-2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonate)
(ABTS·+) assays, as well as acetylcholinesterase (AChE) inhibitors. In both antioxidant assays, even though the starting
compound edaravone was more active, the best derivative was 5 with 50% effective concentration (EC50) of 0.0301 and
0.8106 mM respectively, followed by 3 (EC50 of 0.1920 mM and 3.5311 mM). In the AChE inhibition assay, the derivatives
were not as active as the positive control galantamine, but a general better activity was shown from the aromatic compounds.
The best results were for 10, with 41.9% of inhibition (concentration of 150 μg/mL), and 9 with 31.6%. Docking analysis of
compound 10 showed hydrogen bonds with residues Ser200 and His440 in the AChE catalytic gorge. All synthesized
derivatives 2–15 presented drug-like properties and are capable of crossing the blood–brain barrier and not be pumped out of
it. These results indicate edaravone derivatives can function as scaffolds for AD drugs, though further derivatizations should
be conducted to improve their antioxidant and AChE inhibition profiles.
Graphical Abstract
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02667-5) contains supplementary
material, which is available to authorized users.
* Iván Córdova-Guerrero
4
Facultad de Medicina y Psicología, Universidad Autónoma de
Baja California, 22390 Tijuana, B.C., Mexico
icordova@uabc.edu.mx
5
6
1
2
Centro de Graduados e Investigación en Química, Tecnológico
Nacional de México/Instituto Tecnológico de Tijuana, 22510
Tijuana, B.C., Mexico
Facultad de Ciencias Químicas e Ingeniería, Universidad
Autónoma de Baja California, 22390 Tijuana, B.C., Mexico
Departamento de Química Analítica, Facultad de Medicina,
Universidad Autónoma de Nuevo León, San Nicolás de los Garza,
64460 Monterrey, Mexico
Instituto Politécnico Nacional, UPIIG, Silao de la Victoria,
Guanajuato, Mexico
3
Departamento de Bioquímica, Facultad de Medicina Mexicali,
Universidad Autónoma de Baja California, 21000
Mexicali, Mexico

Medicinal Chemistry Research
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Keywords Edaravone Antioxidant Acetylcholinesterase inhibition Docking ADME calculation
Introduction
antitumor, and antioxidant activities [18, 19]. Edaravone is
a pyrazolone, structure of which can be seen in Fig. 1, along
with a series of related structures such as phenidone, a dual
cyclooxygenase COX-2 and lipoxygenase 5-LOX inhibitor
with chemopreventive profile [20], and dipyrone, a widely
used analgesic, from which recently it has been reported
that one of its metabolites can act as a cannabinoid receptor
1 agonist [21]; these are some examples that show the
importance of the pyrazolone-like moieties in biological
studies.
Edaravone is a free radical scavenger and nootropic, and
its derivatives have been reported to exhibit diverse med-
icinal activities, including antitumoral, antiviral, and pro-
tective effect against sleep deprivation-related memory
impairment [22–26].
Edaravone, which can also be found by the names
methylphenylpyrazolone or norphenazone, to name a cou-
ple, has demonstrated different bioactivities, such as
reduction of inflammation and oxidative stress response in
acute cerebral infarction patients, in the treatment of injury
in acute pancreatitis, in the treatment in craniocerebral
injury, prevention of ferroptosis in amyotrophic lateral
sclerosis (ALS) patients, and in asthma treatment through
anti-inflammatory and antioxidative effects [27–31]. Fur-
thermore, its use with the commercial name Radicava for
the treatment of ALS by the Food and Drug Administration
in USA has been approved [30]. Several of these above-
mentioned edaravone properties point out to its antioxidant
The neurodegenerative disorder Alzheimer’s disease (AD)
is the main cause of dementia in elderly patients [1], AD
being characterized by global cognitive impairment,
including progressive memory loss and declined reasoning
[2, 3]. Several pathogenic mechanisms have been suggested
in the development of AD. Currently, therapeutic inter-
ventions target these proposed mechanisms, namely, the
antioxidant therapy against oxidative stress caused by free
radicals and the inhibition of acetylcholinesterase (AChE)
to compensate the loss of cholinergic function [4].
The aging process diminishes the free radical-scavenging
ability by endogenous mechanisms [5]. Free radicals are
nocent agents able to cause irreversible oxidative damage to
cellular constituents [6]; under normal physiological con-
ditions, the organism exhibits a variety of antioxidant
mechanisms to counterbalance the potential damage of
oxidants [7]. When the balance between free radicals and
antioxidants is disrupted, it leads to a state known as oxi-
dative stress [8], which translates into several pathologies,
including cancer, hypertension, diabetes, and neurological
disorders, including AD [9].
The brain contains an abundance of highly oxidizable
polyunsaturated fatty acids and a high demand for oxygen
[10], hence its vulnerability to damage caused by oxidant
agents. In patients with AD, the brain displays higher levels
of lipid peroxidation products and increased carbonyls
derived from protein oxidation in neuronal cytoplasm; these
abnormalities provide evidence of the connection between
oxidative stress with this neurodegenerative disorder
[11, 12].
AD progression is also related to another pivotal
mechanism: cholinergic dysfunction [13], which states that
deficiency of crucial neurotransmitter acetylcholine (ACh)
in the brain of people with AD is due to insufficient pro-
duction of ACh or the augmented activity of enzyme AChE
[14], leading to the loss of cognitive capacity and general
motor skills [15]. Common treatment for AD relies on drugs
that increase concentration of ACh neurotransmitter by
inhibiting AChE activity, with donepezil, galantamine, and
tacrine, being the most used drugs [16]. Although well
tolerated, AChE inhibitors (AChEIs) cause characteristic
side effects, including nausea, dizziness, and sleep dis-
turbances [17].
Pyrazolone and its derivatives are a pharmacological
important group of compounds; these five-membered het-
erocyclic lactam rings display a wide range of biological
activities, such as antipyretic, analgesic, anti-inflammatory,
Fig. 1 Edaravone and related compounds structures

Medicinal Chemistry Research
capabilities; therefore, it should be an interesting scaffold to
start our search for antioxidant and AChEIs.
δ = 6.26–6.07, in accordance with the reference for the
edaravone ester derivatives [34]. In 13C-NMR, along with
the new signals for the appropriate aliphatic or aromatic
carbons, the carbon position 14 in the appropriate range δ =
172.4–161.9 for ester carbonyls of compounds 2–12, 14,
and 15, with a δ = 149.7 for compound 13 as the incorpo-
rated carbonyl is a carbamate one, can be highlighted.
From the synthesized compounds, 2 [35], 6, 7 [36], 9
[37], 10 [38], 11 [37], and 14 [39], there are previous
reports of their synthesis. However, to the best of our
knowledge products 3, 4, 5, 8, 12, 13, and 15 are reported
for the first time in this work.
The aim of this study is to design and synthesize edar-
avone derivatives, incorporating in the pyrazolone ring
different acyl fragments with aliphatic and aromatic sub-
stitutions, and evaluate in vitro their antioxidant capacity
and AChE inhibition activity. In addition, in silico deter-
minations were conducted, as docking for the exploration of
AChE inhibition results, and the calculation of the drug-like
properties of the synthesized compounds.
Results and discussion
Antioxidant evaluation
Synthesis
Previous antioxidant evaluations of edaravone derivatives
and analogs, both in in vitro assays and computational
analysis [40, 41], point out the single electron transfer and
hydrogen atom transfer as the proposed mechanisms for the
antioxidant properties of these compounds [42]. For the
electron transfer mechanism, this depends on the electron
density of the pyrazolone ring of edaravone [43]. In the case
of the hydrogen atom transfer mechanism, such as the 1,1-
diphenyl-2-picrylhydrazyl (DPPH) assay, edaravone sca-
venges DPPH by donation of the H atom at position 4 [40].
By employing acyl chlorides as reacting agents in the
incorporation of fragments in the pyrazolone ring, the
synthesized products have an ester or carbamate (compound
13) carbonyl attached to the before-mentioned ring. These
groups with alkyl and aryl variations incorporated in these
fragments were proposed to further explore the modulation
of the antioxidant activity of edaravone derivatives, which
was measured with the DPPH and 2-2’-azino-bis-(3-ethyl-
benzothiazoline-6-sulfonate) (ABTS·+) assays.
As stated in the methodology section, the synthesized
compounds were prepared and evaluated in serial dilutions,
their EC50 in mg/mL can be seen in Table 1, and in order to
compare the products accounting their different molecular
weights, their EC50 is also reported as mM. In the DPPH
assay, the positive control quercetin showed the best anti-
oxidant activity with an EC50 of 0.0028 mM, being fol-
lowed by 1 with 0.0180 mM, and the derivatives showed
lower antioxidant activity compared to edaravone. How-
ever, through the DPPH assay compounds 1–15 presented
better antioxidant properties, compared with the results
obtained from the ABTS·+ evaluation.
The synthetic procedure employed was based on previous
works of 4-substituted edaravone derivatives [32, 33] with
some modifications. Reaction between the starting material
edaravone 1 with different alkyl and aryl chlorides was
performed in order to get the 4-substituted compounds. The
main product of each reaction, as seen from the yields
obtained (70–99%), was purified and characterized. How-
ever, no incorporation was detected in position 4 of edar-
avone. Instead, the spectroscopic data shows the
incorporation of the new fragments as ester groups. Ester
derivatives from edaravone after the synthesis by Jensen
[32] have been reported before as side products from the
synthetic procedure, although they can also be synthesized
as the main one [34]. This could be due to the initial basic
conditions, which are intended to remove the acidic
hydrogen in position 4, so that this position can react with
the alkyl and aryl chlorides. After the hydrogen removal,
the pyrazolone ring could rearrange to an enolate form,
where the oxygen would be reacting with the alkyl chlor-
ides, generating the products 2–15, as seen in Scheme 1.
The substitutions in the incorporated carbonyl show ali-
phatic chains with varying lengths (2–8), as well as aro-
matic rings adjacent or separated (9–15) from the
incorporated carbonyl. The proposed structures were ana-
lyzed and confirmed through infrared (IR) spectroscopy and
mass spectroscopy (MS) and with 1H- and 13C-nuclear
magnetic resonance (NMR).
In IR spectroscopy, the synthesized compounds showed
bands for their aromatic C-H bonds between 3023 and
3069 cm−1, as even the aliphatic derivatives have an aro-
matic ring in their core. For the incorporated fragments, the
carbonyl band appears between 1709 and 1784 cm−1 for the
C=O bond. The expected shifts and multiplicities were
Following 1, the better result for a derivative was for
compound 5, with an EC50 of 0.0301 mM. Product 12 and 3
were the next most active derivatives, with EC50 of 0.1603
and 0.1920 mM, respectively. Both 5 and 3 have aliphatic
non-branched R fragments with four and two carbon atoms
(Scheme 1), while 12 is from the aromatic derivatives and
1
observed for the H- and 13C-NMR signals. For the com-
mon core structure, the aromatic hydrogens were present at
δ = 7.62–7.16, with the methyl in position 7 at δ =
2.40–2.28 and the singlet for one hydrogen in position 4 at

Medicinal Chemistry Research
Scheme 1 Synthesis of
edaravone derivatives 2–15
Compound
2
R
Compound
9
R
3
4
10
11
5
12
6
7
13
14
8
15

Medicinal Chemistry Research
Table 1 Percentage of
antioxidant activity of
compounds 1–15
Compound
DPPH
ABTS
EC50 (mg/mL)
EC50 (mM)
EC50 (mg/mL)
EC50 (mM)
1
0.0031 0.001
0.3704 0.243
0.0442 0.073
1.1367 0.156
0.0078 0.017
0.3966 0.309
1.0867 0.101
7.4140 0.953
1.3101 0.272
0.5135 0.063
0.4504 0.108
0.0549 0.053
ND
0.0180
1.7127
0.1920
4.6531
0.0301
1.3849
3.6175
24.6804
4.7074
1.7563
1.5408
0.1603
—
0.0357 0.042
1.0660 0.658
0.8131 0.499
4.6446 0.807
0.2094 0.866
1.5406 0.082
28.765 0.34
31.245 2.08
3.9011 0.072
9.5909 0.831
6.0331 0.594
1.2631 0.133
ND
0.2049
4.9297
3.5311
19.0126
0.8106
5.3796
95.7557
104.0113
14.0171
32.8073
20.6373
3.6890
—
2
3
4
5
6
7
8
9
10
11
12
13
14
0.4220 0.030
0.5522 0.824
0.0008 0.037
1.3866
1.6512
0.0028
6.7344 0.820
12.247 0.20
0.075 0.002
22.1272
36.6216
0.2482
15
aQuercetin
Values are mean SD, n = 3
ND not determined
aServed as reference compound
the only one with two aromatic rings (as 13 could not be
evaluated due to poor solubility in the antioxidant assays).
In the ABTS·+ antioxidant evaluation, although this
radical can be neutralized through different mechanisms,
including also hydrogen transfers [44], in this assay the
results were less active compared with the DPPH assay. The
positive control presented an EC50 of 0.2482 mM, and 1
showed 0.2049 mM. As in the DPPH analysis, the deriva-
tives were not as active as edaravone; nevertheless, in both
techniques the better antioxidant derivative was 5, as in the
ABTS·+ assay it showed an EC50 of 0.8106 mM. It also
concurs that the following more active products are 3
(3.5311 mM) and 12 (3.6890 mM). In each of the anti-
oxidant assays, the less active product was 8, with EC50 of
24.6804 and 104.0113 mM in DPPH and the ABTS·+
assays, respectively. As 8 presented such a low activity, and
4 was not as active as 5 and 3, it could be possible that in
the aliphatic derivatives the antioxidant activity is enhanced
by short and non-branched chains adjacent to the incorpo-
rated carbonyl.
rats injected with Aβ [47]; however, in a study with
cisplatin-treated rats, edaravone inhibited the AChE levels
that were increased by concomitant cisplatin treatment, but
edaravone alone did not alter AChE levels [48]. As AD has
been cataloged as a multifactorial disease and currently
there is no effective drug for it, the suggested therapeutic
approach is to target multiple key pathways [45]. Therefore,
we searched for the synthesized edaravone derivatives both
to display antioxidant properties and to act as AChEIs.
The results from the AChE inhibition assay can be seen
in Table 2, where the positive control galantamine presented
the highest inhibition percentage (91.9%). Edaravone per-
centage could not be determined due to the sample color
interference in the analysis. As for the synthesized deriva-
tives, the most active were 10 and 9, with 41.9 and 31.6% of
inhibition, respectively. These two products share as char-
acteristic an aromatic ring adjacent to the ester carbonyl in
the incorporated fragments. The other compound that shares
this structure is 11, being a position isomer of 9; therefore,
the ortho position of the methyl substitution in the aromatic
ring of 11 could be responsible for the decrease in the
activity, as this product only presented 16.3%. This result is
in agreement with the literature, where in some families of
compounds the ortho substitutions tend to be less active
than the para substitutions [49–51].
AChE inhibitory activity
Edaravone has been related to AD treatment through dif-
ferent mechanisms, mainly by reduction of amyloid-β (Aβ)
peptide deposition, disaggregation of Aβ fibrils, oxidative
stress diminution, attenuating tau hyperphosphorylation,
and neuroinflammation, among other mechanisms [45, 46].
It has been reported that edaravone could inhibit AChE in
From the aliphatic derivatives, compounds 6, 4, 8, and 7
were the less active products (4.8, 3.0, 2.0, and 1.7%,
respectively), so in a similar way to the aliphatic antioxidant
results, a short and linear chain tend to be better for the

Medicinal Chemistry Research
Table 2 Acetylcholinesterase inhibition of compounds 1–15
Table 3 Docking scores of 10 and reference compounds with AChE
(PDB ID: 1EVE)
Compound
% inhibition (150 μg/mL)
Compound
Binding energy
(kcal/mol)
Principal residue interactions
1
ND
2
20.7 4.9
25.8 7.4
3.0 1.0
28.9 8.6
4.8 1.0
1.7 0.8
2.0 1.2
31.6 8.1
41.9 7.3
16.3 3.3
16.9 1.8
23.9 2.8
19.5 4.7
2.8 1.8
91.9 0.82
10
−9.70
−11.2
−9.26
Phe331, Ser200, His440
Trp84, Trp279
3
aDonepezil
aGalantamine
4
Trp84, Ser200,
His440, Glu199
5
6
aServed as reference compound
7
8
9
10
11
12
13
14
15
aGalantamine
Values are mean SD, n = 3
ND not determined
aServed as reference compound
AChE inhibition activity. In the case of the aromatic deri-
vatives 9–15, it can be highlighted that, in general, the
aromatic compounds were better inhibitors compared to the
aliphatic ones. The exception to this observation is product
15, which was almost inactive with 2.8% of inhibition. This
could be explained as for the derivative that shows with its
aromatic ring a larger chain separation to the incorporated
ester carbonyl, and as discussed with the aliphatic deriva-
tives, the long aliphatic chain appears to diminish the AChE
inhibition activity.
Fig. 2 Docking pose and interactions of compound 10 (purple) with
the catalytic gorge of TcAChE. Hydrogen bonds are represented by
black lines
−9.26 kcal/mol was slightly higher than derivative 10,
which showed −9.70 kcal/mol. The in vitro result could be
explained nevertheless as the docked pose is very similar to
the crystallographic one obtained by [53], where galanta-
mine shows hydrogen bonds with Ser200 and His440, two
of the catalytic enzyme residues, and with Glu199. Com-
pound 10 shows the first two interactions but lack the bond
with Glu199. In the case of donepezil, as the co-crystallized
ligand for the TcAChE model, it scored accordingly the best
docking result with −11.2 kcal/mol.
The synthesized compound 10 was the most active
edaravone derivative in vitro with 41.9% inhibition. In the
docking analysis, it showed a score of −9.70 kcal/mol, and
its resulting docked pose in the TcAChE catalytic gorge
present the residues that could interact with them by
hydrogen bonds and hydrophobic and π–π interactions
(Fig. 2). The oxygen in the carbonyl behaves as the
hydrogen bond acceptor for these before-mentioned inter-
actions. Additionally, compound 10 shows its p-methyl
benzyl ring in the vicinity of hydrophobic residues Phe330
and Phe331, where a π–π interaction is almost established
Molecular docking
In order to further explore the results obtained in vitro in the
AChE inhibition assay, the most active synthesized deri-
vative 10 was evaluated in silico through its docking against
AChE. The enzyme molecular structure was selected from
the Protein Data Bank (PDB ID: 1EVE), corresponding to
the AChE from Tetronarce californica (TcAChE), which
shows a high similarity to the human one and therefore can
be employed for the docking analysis [52]. As galantamine
was employed as positive control in the in vitro assay, it
was also docked against the TcAChE model, as well the
enzyme co-crystallized ligand donepezil.
The docking results can be seen in Table 3, where a more
negative docking score represents a better ligand–receptor
affinity. Galantamine, as a positive control in the in vitro
assay, presented the best AChE inhibition percentage
(91.9%), although in the docking analysis its score of

Medicinal Chemistry Research
with Phe331; however, it is very clear that an intramolecular
π–π interaction found as the benzyl ring in the core of 10 is
completely overlapped with the incorporated p-methyl
benzyl ring.
ADME (Absorbed, Distributed, Metabolized, or
Eliminated) calculations
In Table 4 can be seen the predicted pharmacokinetics and
drug-like properties of the compounds 1–15, as predicted
from the SwissADME server. Having Lipinski’s rule [54] as
a good guideline for the drug-like properties of new com-
pounds (considering descriptors such as H bonds donors
and acceptors, molecular weight, and lipophilicity), only
products 12, 13, and 15 show one Lipinski’s rule violation,
having an MLogP >4.15. The rest of the synthesized
compounds comply fully to these guidelines, presenting
drug-like properties. Furthermore, all compounds show a
high predicted gastrointestinal absorption.
Regarding the aim of the synthesized molecules as
AChEI (and potentially leading to AD treatment), it is
important for them to be capable to cross the blood–brain
barrier (BBB), which is usually related to small and lipo-
philic molecules, of <400–500 Da [55]. As shown in Table
4, the molecular weight of compounds 1–15 fulfill this
requirement, and all their Log P (1.64–4.46) show a more
lipophilic character over a hydrophilic one. The Swis-
sADME server predicts that all our compounds are BBB
permeant, which could help in future in vivo studies in
Alzheimer’s treatment due to this activity. Moreover, the
results show that all the products are not substrate for the
permeability glycoprotein (P-gp). As this protein works by
pumping out xenobiotics of the central nervous system
(CNS), and therefore could interfere with AD treatment
[55], the predicted properties of the synthesized compounds
suggest that they can work as scaffolds for further devel-
oping new and better AD drugs.
Conclusion
In the search for new AD treatments, this work proposes the
derivatization of edaravone, in order to obtain products that
can possess different pharmacological mechanisms to
improve their profile as potential AD drugs. Reacting
edaravone with different aliphatic and aromatic acyl halides,
a family of compounds were synthesized, characterized, and
evaluated as antioxidants and AChEIs.
Compounds 1–15 showed through ADME calculations
to have desirable drug-like properties, highlighting their
capacity to cross the BBB and not to be pumped out by the
P-gp, which could facilitate them to exert their activities in
the CNS. This points out that edaravone derivatives with

Medicinal Chemistry Research
aliphatic and aromatic esters in the pyrazol ring can be a
good scaffold in the search of new AD drugs.
Synthesis of edaravone derivatives
Preliminarily, the antioxidant results indicate that in the
aliphatic derivatives the antioxidant activity is improved by
short and non-branched chains, although further explora-
tions are suggested to improve the current antioxidant
profiles. The AChE inhibition of the synthesized derivatives
again points out to short linear fragment derivatives as more
active compounds compared to the ones with longer ali-
phatic chains. However, the enzyme inhibition was
improved with the aromatic derivatives, especially when the
aromatic ring was adjacent to the incorporated ester car-
bonyl (compounds 10 and 9). Docking analysis of 10 sug-
gest that this could be due to hydrogen bonds, with
hydrophobic and π–π interactions in the enzyme catalytic
gorge with the incorporated aromatic ring. As the products
synthesized in this work were mainly hydrocarbon ones,
further derivatizations with heteroatom groups are proposed
to be incorporated, especially in the aromatic derivatives, to
further improve their antioxidant and AChE inhibition
activities.
In a round bottom flask, 5 Eq of KOH were dissolved in an
acetonitrile and water mixture under magnetic stirring. After
complete dissolution, 1 Eq of edaravone (1) was added, and
the mixture was left under strong stirring for 30 min. Sub-
sequently, 2 Eq of the corresponding acyl halide dissolved
in dichloromethane (DCM) was transferred to the reaction
and the stirring continued for 5 min (Scheme 1). After-
wards, the reaction mixture was washed with a saturated
sodium bicarbonate solution, and the product was extracted
with DCM. The reactions were monitored by TLC and the
products were purified by column chromatography. The
structures of the products were confirmed by MS and NMR
spectra as discussed below and in Supplementary Figs.
S1–S28.
3-methyl-1-phenyl-1H-pyrazol-5-yl acetate (2)
Brownish-orange viscous oil (yield 97%); C12H12N2O2; IR
(attenuated total reflection (ATR) diamond, cm−1) 3063 (C-
H aromatic), 2961 (C-H aliphatic), 1706 (C=O), 1498 (C-
C) cm−1; 1H NMR (CDCl3, 400 MHz,): δ = 7.55–7.51 (2H,
m, H-13, H-9), 7.46–7.41 (2H, m, H-12, H-10), 7.34–7.29
(1H, m, H-11) 6.09 (1H, s, H-4), 2.32 (3H, s, H-7), 2.25
(3H, s, H-16); 13C NMR (CDCl3, 100 MHz): δ = 166.1 (C,
C-14), 148.9 (C, C-3), 144.3 (C, C-5), 138.1 (C, C-8), 129.1
(CH, C-12, C-10), 127.1 (CH, C-11), 123.0 (CH, C-13, C-
9), 95.9 (CH, C-4), 20.8 (CH3, C-16), 14.5 (CH3, C-7);
electron ionization MS (EIMS) m/z (rel. int.) 216 [M]+ (1),
174(23), 129(5), 91(2), 77(41), 68(5), 51(28), 43(100).
Experimental/material and methods
Chemistry
All commercial reagents and solvents were purchased and
used without previous purification. The synthesized pro-
ducts were purified through column chromatography with
silica gel 60 Å, 230–400 mesh (Sigma-Aldrich). Purity of
the products was first monitored by thin-layer chromato-
graphy (TLC) carried out in aluminum backed silica plates
(Merck) revealed by ultraviolet (UV) light at 254 nm. For
the chemical characterization of the synthesized products,
melting points were taken on a Mel-Temp melting point
apparatus (Thermo Scientific). Gas chromatography–MS
was performed on a TRACE 1310 chromatograph and
single quadrupole ISQ LT mass spectrometer (both from
Thermo Scientific). Fourier-transform IR spectroscopy was
performed on a Thermo Scientific Nicolet iS 5 spectrometer.
NMR spectra were obtained on a 400 MHz Avance DPX
spectrometer (Bruker). The chemical shifts in NMR spectra
(δ) are presented with tetramethylsilane as the internal
standard (δ: 0.00). Chemical shifts are given in ppm, cou-
pling constants J are expressed in Hertz (Hz) (multiplicity:
s = singlet, d = doublet, dd = doublet of doublet, ddd =
doublet of doublet of doublet, t = triplet, tt = triplet of tri-
plet, q = quadruplet, quint = quintuplet, sext = sextuplet,
sept = septuplet, m = multiplet). UV–visible spectra for the
antioxidant assays were obtained on a Genesys 20 (Thermo
Scientific) spectrophotometer, while a Microplate reader
Multiskan FC was employed for the AChE assay.
3-methyl-1-phenyl-1H-pyrazol-5-yl propionate (3)
Dark orange viscous oil (yield 99%); C13H14N2O2; IR (ATR
diamond, cm−1) 3069 (C-H aromatic), 2928 (C-H aliphatic),
;
1776 (C=O), 1506 (C-C) cm−1 1H NMR (CDCl3,
400 MHz,): δ = 7.54–7.52 (2H, dd, J = 8.8, 1.5 Hz, H-13,
H-9), 7.45–7.41 (2H, m, H-12, H-10), 7.33–7.29 (1H, m, H-
11), 6.09 (1H, s, H-4), 2.52 (2H, q, J = 7.6 Hz, H-16), 2.32
(3H, s, H-7), 1.19 (3H, t, J = 7.5, H-17); 13C NMR (CDCl3,
100 MHz): δ = 169.7 (C, C-14), 148.9 (C, C-3), 144.4 (C,
C-5), 138.0 (C, C-8), 129.0 (CH, C-12, C-10), 127.1 (CH,
C-11), 123.1 (CH, C-13, C-9), 95.8 (CH, C-4), 27.5 (CH2,
C-16), 14.5 (CH3, C-7), 8.8 (CH3, C-17); EIMS m/z (rel.
int.) 230 [M]+ (16), 175(22), 174(100), 129(18), 105(11),
77(75), 57(99), 51(14), 39(8), 29(35).
3-methyl-1-phenyl-1H-pyrazol-5-yl isobutyrate (4)
Dark yellow viscous oil (yield 90%); C14H16N2O2; IR (ATR
diamond, cm−1) 3066 (C-H aromatic), 2980 (C-H aliphatic),
;
1773 (C=O), 1506 (C-C) cm−1 1H NMR (CDCl3,

Medicinal Chemistry Research
400 MHz,): δ = 7.54–7.50 (2H, m, H-9, H-13), 7.45–7.40
(2H, m, H-10, H-12), 7.33–7.31 (1H, m, H-11), 6.08 (1H, s,
H-4), 2.73 (1H, sept, J = 7.0 Hz, H-16), 2.32 (3H, s, H-7),
1.22 (6H, d, J = 7.0 Hz, H-17, H-18); 13C NMR (CDCl3,
100 MHz): δ = 172.4 (C, C-14), 149.1 (C, C-3), 144.8 (C,
C-5), 138.2 (C, C-8), 129.1 (CH, C-10, C-12), 127.3 (CH,
C-11), 123.5 (CH, C-9, C-13), 95.8 (CH, C-4), 34.2 (CH,
C-16), 18.8 (CH3, C-17, C-18), 14.6 (CH3, C-7); EIMS m/z
(rel. int.) 244 [M]+ (1), 174(5), 129(2), 117(1), 105(3), 91
(2), 77(38), 71(73), 51(11), 43(100), 41(9).
H-4), 2.51 (2H, t, J = 7.4 Hz, H-16), 2.34 (3H, s, H-7), 1.67
(2H, quint, J = 7.4 Hz, H-17), 1.33–1.27 (8H, m, H-18, H-
19, H-20, H-21), 0.90 (3H, t, J = 6.7 Hz, H-22); 13C NMR
(CDCl3, 100 MHz): δ = 169.0 (C, C-14), 148.9 (C, C-3),
144.5 (C, C-5), 138.1 (C, C-8), 129.0 (CH, C-10, C-12),
127.1 (CH, C-11), 123.2 (CH, C-9, C-13), 95.8 (CH, C-4),
34.0 (CH2, C-16), 31.5 (CH2, C-19), 28.8 (CH2, C-18), 28.8
(CH2, C-20), 24.5 (CH2, C-17), 22.5 (CH2, C-21), 14.5
(CH3, C-7), 14.0 (CH3, C-22); EIMS m/z (rel. int.) 300 [M]+
(2), 174(100), 157(2), 145(3), 127(84), 109(17), 105(9), 91
(4), 77(62), 67(16), 56(91), 43(36).
3-methyl-1-phenyl-1H-pyrazol-5-yl pentanoate (5)
3-methyl-1-phenyl-1H-pyrazol-5-yl 2-propylpentanoate (8)
Brown solid (yield 92%); C15H18N2O2; mp 108–110 °C; IR
(ATR diamond, cm−1) 3047 (C-H aromatic), 2914 (C-H
aliphatic), 1709 (C=O) cm−1; 1H NMR (CDCl3, 400 MHz,):
δ = 7.55–7.48 (2H, m, H-13, H-9), 7.46–7.37 (2H, m, H-12,
H-10), 7.34–7.25 (1H, m, H-11), 6.08 (1H, s, H-4), 2.49
(2H, t, J = 7.4 Hz, H-16), 2.32 (3H, s, H-7), 1.70–1.55 (2H,
m, H-17), 1.41–1.22 (2H, m, H-18), 0.89 (3H, t, J = 7.1 Hz,
H-19); 13C NMR (CDCl3, 100 MHz): δ = 164.5 (C, C-14),
144.4 (C, C-3), 139.9 (C, C-5), 133.4 (C, C-8), 124.6 (CH,
C-10, C-12), 122.6 (CH, C-11), 118.8 (CH, C-9, C-13), 91.4
(CH, C-4), 29.2 (CH2, C-16), 22.0 (CH2, C-17), 17.5 (CH2,
C-18), 10.0 (CH3, C-7), 9.2 (CH3, C-19); EIMS m/z (rel.
int.) 258 [M]+ (1), 175(1), 174(10), 173(1), 129(1), 105(2),
85(42), 77(39), 57(100), 51(13), 41(36).
Yellow liquid (yield 92%); C18H24N2O2; IR (ATR diamond,
cm−1) 3050 (C-H aromatic), 2926 (C-H aliphatic), 1768
1
(C=O), 1506 (C-C) cm−1; H NMR (CDCl3, 400 MHz,):
δ = 7.55–7.53 (2H, m, H-9, H-13), 7.46–7.42 (2H, m, H-10,
H-12), 7.35–7.31 (1H, m, H-11), 6.08 (1H, s, H-4), 2.56
(1H, tt, J = 8.8, 5.4 Hz, H-16), 2.34 (3H, s, H-7), 1.70–1.60
(2H, m, H-20), 1.53–1.46 (2H, m, H-17), 1.27 (4H, sext,
J = 7.4 Hz, H-18, H-21), 0.88 (6H, t, J = 7.3 Hz, H-19, H-
22); 13C NMR (CDCl3, 100 MHz): δ = 171.7 (C, C-14),
148.9 (C, C-3), 144.6 (C, C-5), 138.0 (C, C-8), 128.9 (CH,
C-10, C-12), 127.2 (CH, C-11), 123.7 (CH, C-9, C-13),
95.6 (CH, C-4), 45.3 (CH, C-16), 34.2 (CH2, C-17, C-20),
20.5 (CH2, C-18, C-21), 14.5 (CH3, C-7), 13.9 (CH3, C-19,
C-22); EIMS m/z (rel. int.) 300 [M]+ (1), 174(1), 127(5), 99
(10), 77(13), 57(100), 43(8).
3-methyl-1-phenyl-1H-pyrazol-5-yl heptanoate (6)
Yellow viscous oil (yield 93%); C17H22N2O2; IR (ATR dia-
mond, cm−1) 3069 (C-H aromatic), 2928 (C-H aliphatic),
1782 (C=O), 1504 (C-C) cm−1; 1H NMR (CDCl3,
400 MHz,): δ = 7.54–7.51 (2H, m, H-9, H-13), 7.45–7.40
(2H, m, H-10, H-12), 7.33–7.29 (1H, m, H-11), 6.08 (1H, s,
H-4), 2.49 (2H, t, J = 7.4 Hz, H-16), 2.32 (3H, s, H-7), 1.65
(2H, quint, J = 7.4 Hz, H-17), 1.33-1.23 (6H, m, H-18, H-19,
H-20), 0.87 (3H, t, J = 6.8 Hz, H-21); 13C NMR (CDCl3,
100 MHz): δ = 169.2 (C, C-14), 149.1 (C, C-3), 144.6 (C, C-
5), 138.3 (C, C-8), 129.1 (CH, C-10, C-12), 127.3 (CH, C-11),
123.4 (CH, C-9, C-13), 95.9 (CH, C-4), 34.2 (CH2, C-16),
31.5 (CH2, C-17), 28.7 (CH2, C-18), 24.7 (CH2, C-19), 22.5
(CH2, C-20), 14.6 (CH3, C-7), 14.1 (CH3, C-21); EIMS m/z
(rel. int.) 286 [M]+ (3), 174(100), 157(2), 145(3), 129(7), 113
(94), 105(8), 95(11), 85(86), 77(61), 57(48), 42(77), 39(8).
3-methyl-1-phenyl-1H-pyrazol-5-yl benzoate (9)
Cream color powder (yield 99%); C17H14N2O2; mp 118 °C; IR
(ATR diamond, cm−1) 3069 (C-H aromatic), 1755 (C=O),
1504 (C-C) cm−1; 1H NMR (CDCl3, 400 MHz,): δ =
8.01–7.96 (2H, m, H-17, H-21), 7.59–7.48 (3H, m, H-9, H-13,
H-19), 7.43–7.29 (4H, m, H-10, H-12, H-18, H-20), 7.25–7.17
(1H, m, H-11), 6.19 (1H, s, H-4), 2.28 (3H, s, H-7); 13C NMR
(CDCl3, 100 MHz): δ = 161.9 (C, C-14), 149.1 (C, C-3),
144.5 (C, C-5), 138.1 (C, C-8), 134.4 (C, C-16), 130.5 (CH,
C-19), 129.1 (CH, C-10, C-12), 128.9 (CH, C-18, C-20),
127.9 (CH, C-11) 127.2 (CH, C-17, C-21), 123.2 (CH, C-9, C-
13), 95.9 (CH, C-4), 14.7 (CH3, C-7); EIMS m/z (rel. int.) 278
[M]+ (1), 106(8), 105(100), 77(62), 51(15).
3-methyl-1-phenyl-1H-pyrazol-5-yl 4-methylbenzoate (10)
3-methyl-1-phenyl-1H-pyrazol-5-yl octanoate (7)
Cream color crystals (yield 99%); C18H16N2O2; mp
Orange-yellow liquid (yield 98%); C18H24N2O2; IR (ATR
diamond, cm−1) 3066 (C-H aromatic), 2926 (C-H aliphatic),
105–106 °C; IR (ATR diamond, cm−1) 3058 (C-H aro-
1
matic), 1752 (C=O), 1504 (C-C) cm−1; H NMR (CDCl3,
1784 (C=O), 1504 (C-C) cm−1
;
1H NMR (CDCl3,
400 MHz,): δ = 7.96–7.90 (2H, m, H-17, H-21), 7.61–7.54
(2H, m, H-9, H-13), 7.44–7.22 (5H, m, H-10, H-11, H-12,
H-18, H-20), 6.24 (1H, s, H-4), 2.40 (3H, s, H-22), 2.34 (3H,
400 MHz,): δ = 7.56–7.53 (2H, m, H-9, H-13), 7.47–7.42
(2H, m, H-10, H-12), 7.35–7.31 (1H, m, H-11), 6.10 (1H, s,

Medicinal Chemistry Research
s, H-7); 13C NMR (CDCl3, 100 MHz): δ = 162.0 (C, C-14),
149.2 (C, C-3), 145.5 (C, C-5), 144.7 (C, C-19), 138.1 (C,
C-8), 130.5 (CH, C-10, C-12), 129.6 (CH, C-18, C-20),
129.2 (CH, C-17, C-21), 127.3 (CH, C-11), 125.2 (C, C-16),
123.3 (CH, C-9, C-13), 96.0 (CH, C-4), 21.9 (CH3, C-22),
14.6 (CH3, C-7); EIMS m/z (rel. int.) 292 [M]+ (1), 119
(100), 91(38), 77(20), 65(15), 51(7).
1747 (C=O), 1504 (C-C), 1303 (C-N) cm−1 1H NMR
;
(CDCl3, 400 MHz,): δ = 7.40–7.32 (5H, m, H-9, H-10, H-
11, H-12, H-13), 7.29–7.20 (10H. m, H-19, H-20, H-21, H-
22, H-23, H-24, H-25, H-26, H-27, H-28), 6.23 (1H, s, H-
4), 2.34 (3H, s, H-7); 13C NMR (CDCl3, 100 MHz): δ =
149.7 (C, C-14), 148.8 (C, C-3), 144.9 (C, C-5), 141.4 (C,
C-17, C-18), 137.9 (C, C-8), 129.3 (CH, C-10, C-12), 128.8
(CH, C-20, C-22, C-25, C-27), 127.1 (CH, C-11, C21, C-
26), 126.6 (CH, C-9, C-13), 122.8 (CH, C-19, C-23, C-24,
C-28), 95.1 (CH, C-4), 14.5 (CH3, C-7); EIMS m/z (rel. int.)
369 [M]+ (1), 196(100), 168(53), 167(27), 139(1), 115(2),
93(8), 77(53), 65(4), 51(12), 39(3).
3-methyl-1-phenyl-1H-pyrazol-5-yl 2-methylbenzoate (11)
Cream color powder (yield 95%); C18H16N2O2; mp 104 °C;
IR (ATR diamond, cm−1) 3066 (C-H aromatic), 2923 (C-H
aliphatic), 1744 (C=O), 1506 (C-C) cm−1 1H NMR
;
(CDCl3, 400 MHz,): δ = 8.02 (1H, dd, J = 7.8, 1.4 Hz, H-
17), 7.62 (2H, dd, J = 8.7, 1.4 Hz, H-9, H-13), 7.49 (1H,
ddd, J = 7.5, 7.5, 1.5 Hz, H-19), 7.46–7.41 (2H, m, H-10,
H-12), 7.35–7.27 (3H, m, H-11, H-18, H-20), 6.26 (1H, s,
H-4), 2.61 (3H, s, H-22), 2.40 (3H, s, H-7); 13C NMR
(CDCl3, 100 MHz): δ = 162.2 (C, C-14), 149.0 (C, C-3),
144.7 (C, C-5), 142.2 (C, C-21), 138.2 (C, C-8), 133.4 (CH,
C-19), 132.1 (CH, C-20), 131.1 (CH, C-17), 129.0 (CH, C-
10, C-12), 127.2 (CH, C-11), 126.8 (C, C-16), 126.1 (CH,
C-18), 123.4 (CH, C-9, C-13), 95.9 (CH, C-4), 21.8 (CH3,
C-22). 14.5 (CH3, C-7); EIMS m/z (rel. int.) 292 [M]+ (1),
173(2), 120(18), 118(100), 106(2), 91(75), 77(31), 65(27),
51(9), 39(8).
3-methyl-1-phenyl-1H-pyrazol-5-yl cinnamate (14)
Cream color powder (yield 93%); C19H16N2O2; mp
107–108 °C; IR (ATR diamond, cm−1) 2920 (C-H ali-
phatic), 1747 (C=O), 1504 (C-C) cm−1
;
1H NMR
(CDCl3, 400 MHz,): δ = 7.83 (1H, d, J = 16 Hz, H-17),
7.60–7.57 (2H, m, H-9, H-13), 7.56–7.53 (2H, m, H-19,
H-23), 7.47–7.39 (5H, m, H-10, H-12, H-20, H-21, H-
22), 7.34–7.29 (1H, m, H-11), 6.51 (1H, d, J = 15.9 Hz,
H-16), 6.20 (1H, s, H-4), 2.35 (3H, s, H-7); 13C NMR
(CDCl3, 100 MHz): δ = 162.1 (C, C-14), 149.0 (C, C-3),
148.5 (CH, C-17), 144.5 (C, C-5), 138.2 (C, C-8), 133.7
(C, C-18), 131.3 (CH, C-21), 129.1 (CH, C-10, C-12),
129.1 (CH, C-20, C-22), 128.5 (CH, C-19, C-23), 127.1
(CH, C-11), 123.1 (CH, C-9, C-13), 115.4 (CH, C-16),
95.8 (CH, C-4), 14.6 (CH3, C-7); EIMS m/z (rel. int.) 304
[M]+ (1), 173(3), 131(61), 104(4), 103(45), 77(100), 51
(37).
3-methyl-1-phenyl-1H-pyrazol-5-yl 2-(naphthalen-1-yl)
acetate (12)
Yellow viscous oil (yield 70%); C22H18N2O2; IR (ATR
diamond, cm−1) 3031 (C-H aromatic), 2920 (C-H aliphatic),
1714 (C=O) cm−1
;
1H NMR (CDCl3, 400 MHz,): δ =
3-methyl-1-phenyl-1H-pyrazol-5-yl 5-phenylpentanoate
7.93–7.89 (2H, m, H-20, H-26), 7.87 (1H, d, J = 8.0 Hz, H-
23), 7.55–7.48 (2H, m, H-10, H-12), 7.46–7.40 (2H, m, H-
24, H-25), 7.26–7.24 (2H, m, H-9, H-13), 7.22–7.18 (3H,
m, H-11, H-21, H-22), 6.16 (1H, s, H-4), 4.26 (2H, s, H-
16), 2.33 (3H, s, H-7); 13C NMR (CDCl3, 100 MHz): δ =
166.5 (C, C-14), 148.8 (C, C-3), 144.3 (C, C-5), 137.8 (C,
C-8), 133.9 (C, C-19), 132.0 (C, C-18), 128.9 (C, C-17),
128.8 (CH, C-10, C-12), 128.7 (CH, C-20), 128.6 (CH, C-
26), 128.5 (CH, C-24), 126.8 (CH, C-22), 126.8 (CH, C-
11), 126.1 (CH, C-21), 125.5 (CH, C-25), 123.3 (CH, C-
23), 122.8 (CH, C-9, C-13), 95.7 (CH, C-4), 39.0 (CH2, C-
16), 14.5 (CH3, C-7); EIMS m/z (rel. int.) 342 [M]+ (2), 174
(3), 168(28), 141(100), 115(23), 105(2), 91(2), 77(46), 63
(5), 51(13), 40(7).
(15)
Light yellow viscous oil (yield 90%); C21H22N2O2; IR (ATR
diamond, cm−1) 3023 (C-H aromatic), 2928 (C-H aliphatic),
1779 (C=O), 1504 (C-C) cm−1
;
1H NMR (CDCl3,
400 MHz,): δ = 7.52–7.49 (2H, m, H-9, H-13), 7.42–7.37
(2H, m, H-10, H-12), 7.31–7.26 (3H, m, H-22, H-23, H-24),
7.21–7.16 (1H, m, H-11), 7.13 (2H, dd, J = 8.2, 1.6 Hz, H-
21, H-25), 6.07 (1H, s, H-4), 2.60 (2H, t, J = 7.5 Hz, H-16),
2.52 (2H, t, J = 7.0 Hz, H-19), 2.32 (3H, s, H-7), 1.74–1.59
(4H, m, H-17, H-18); 13C NMR (CDCl3, 100 MHz): δ =
169.0 (C, C-14), 149.1 (C, C-3), 144.6 (C, C-5), 141.8 (C,
C-8), 129.2 (CH, C-10, C-12), 128.5 (CH, C-22, C-24),
128.5 (CH, C-21, C-25), 127.3 (CH, C-23), 126.1 (CH, C-
11), 123.4 (CH, C-9, C-13), 95.9 (CH, C-4), 35.6 (CH2, C-
16), 34.1 (CH2, C-19), 30.7 (CH2, C-17), 24.3 (CH2, C-18),
14.6 (CH3, C-7); EIMS m/z (rel. int.) 334 [M]+ (1), 175(9),
174(56), 143(3), 133(3), 117(76), 104(5), 91(100), 77(40),
65(8), 51(5), 39(4).
3-methyl-1-phenyl-1H-pyrazol-5-yl diphenylcarbamate (13)
Cream color crystals (yield 98%); C23H19N3O2; mp
95–96 °C; IR (ATR diamond, cm−1) 3061 (C-H aromatic),

Medicinal Chemistry Research
DPPH assay antioxidant evaluation
AChE inhibitory activity
The radical-scavenging activity was analyzed based on the
methodology by Salazar-Aranda [56] with slight modifica-
tions. The products were dissolved with methanol and dif-
ferent concentrations were obtained by serial dilutions. An
aliquot of 0.5 mL from each dilution was mixed with
0.5 mL of a DPPH methanolic solution (prepared at a
concentration 7.5 mg/250 mL). The mixtures were incu-
bated for 30 min in the dark at room temperature. After this
time, the spectrophotometric measurement of the mixtures
was performed at 517 nm, employing methanol as a blank.
The radical-scavenging activity was calculated as the per-
centage of DPPH discoloration with the formula:
AChE inhibitory activity was determined employing a
previously reported methodology [15] with slight mod-
ifications. In a 96-well plate, 75 μL of a 50 mM Trizma-HCl
buffer (pH = 8) was added with 75 μL of a solution of the
synthesized compound to analyze, obtaining at the end a
final concentration of 150 μg/mL (0.15% for dimethyl
sulfoxide). Then, to each well, 25 μL of a buffer solution of
15 mM acetylthiocoline chloride was added, along with
125 μL of a 3 mM buffer solution of Ellman’s reagent
(DTNB), with final concentrations of 1.25 mM for both.
Employing a microplate reader every 45 s, the absorbance
was measured at a wavelength of 405 nm, three consecutive
times. Afterwards, to each well 25 μL of a 2 U/mL AChE in
buffer solution was added, enriched with bovine serum
albumin (0.1 mg/mL), with a final concentration of the
enzyme at 0.15 U/mL. Five consecutive lectures were taken
every 45 s. Six wells from each plate had no tested com-
pound in them in order to serve as the control for the
enzyme 100% activity. To correct the substrate’s sponta-
neous hydrolysis, the absorbance from before the enzyme
addition was subtracted from the absorbance obtained after.
The percentage of inhibition was calculated using the for-
mula:
ꢀ
ꢁ
B
A
DPPH ð%Þ ¼ 1 À
 100;
where the value of a DPPH solution as control is
represented by A and the sample by B. All determinations
were performed in triplicate. The DPPH discoloration was
plotted against the concentration of each dilution, and by
interpolation employing a linear regression analysis, the
concentration needed to decrease the absorbance of DPPH
by 50% (EC50) was obtained. As a reference compound,
quercetin was employed.
ꢀ
ꢁ
Asample
Acontrol
ABTS assay antioxidant evaluation
% of inhibition ¼ 1 À
 100;
The ABTS radical cation (ABTS·+) scavenging assay was
analyzed based on reported methodologies [57, 58] with
slight modifications. An aqueous solution of ABTS was
prepared at a 7 mM concentration. The radical cation
ABTS·+ was produced by the reaction between the ABTS
solution with a 2.45 mM potassium persulfate solution; the
mixture was kept at room temperature in the dark for
16–18 h before use. Then 150 μL of the ABTS·+ solution
was diluted with methanol to obtain an absorbance 0.7
0.02 as an initial absorbance, at a wavelength of 754 nm. A
volume of 980 μL from this solution was mixed with 20 μL
of the different concentrations prepared of the samples. The
mixture was stirred and incubated for 7 min at room tem-
perature, before reading the final absorbance. The percen-
tage of inhibition was calculated using the formula:
where the absorbance difference between time 0 and
225 s in the presence of the compound to test is represented
by ASample and the corresponding absorbance difference of
the 100% AChE activity control is represented by AControl
All experiments were performed in triplicate. As a positive
control, galantamine was employed [60].
.
Molecular docking
The methodology was based on a previous work [61].
Molecular models were generated for the analyzed pro-
ducts through their SMILES strings with the use of UCSF
Chimera [62]. These models were energy minimized
employing Chimera default conditions with Antechamber
parameters [63]. With AutoDock Tools 1.5.6 [64] were
defined the rotatable bonds and the atomic charges for
each model. The crystallographic structure of the AChE
receptor (PDB ID: 1EVE) was retrieved from Protein
Data Bank (http://www.rcsb.org/), which was prepared
with AutoDock Tools removing the co-crystalized ligand
(donepezil) along with the water molecules, hydrogen
addition, and Gasteiger charge calculation. For the
docking analysis, AutoDock Vina was employed [65],
with a grid of 28 × 28 × 28 Å, with center coordinates at
x = 3.11011, y = 66.7013, and z = 63.9449. For each
ꢀ
ꢁ
A1 À A2
% of inhibition ¼
 100;
A1
where the initial absorbance of the ABTS·+ solution is A1
and the final absorbance in the presence of the sample is A2.
All determinations were performed in triplicate. For each
compound, the concentration of each dilution was plotted
against the percentage of inhibition, and the EC50 value was
obtained by interpolation from a linear regression analysis.
As a reference compound, quercetin was employed [59].

Medicinal Chemistry Research
ligand, ten poses were generated with an exhaustiveness
of eight, making the docking runs by triplicate. The
visualization and analysis of the docked poses was made
with UCSF Chimera.
11. Chauhan V, Chauhan A. Oxidative stress in Alzheimer’s disease.
Pathophysiology 2006;13:195–208. https://doi.org/10.1016/j.pa
thophys.2006.05.004.
12. Guan Z-Z. Cross-talk between oxidative stress and modifications
of cholinergic and glutaminergic receptors in the pathogenesis of
Alzheimer’s disease. Acta Pharmacol Sin. 2008;29:773–80.
https://doi.org/10.1111/j.1745-7254.2008.00819.x.
13. Liu Z, Qin G, Mana L, Dong Y, Huang S, Wang Y, et al. GAPT
regulates cholinergic dysfunction and oxidative stress in the brains
of learning and memory impairment mice induced by scopola-
mine. Brain Behav. 2020;10:e01602 https://doi.org/10.1002/brb3.
1602.
14. Singh M, Kaur M, Kukreja H, Chugh R, Silakari O, Singh D.
Acetylcholinesterase inhibitors as Alzheimer therapy: from nerve
toxins to neuroprotection. Eur J Med Chem. 2013;70:165–88.
https://doi.org/10.1016/j.ejmech.2013.09.050.
ADME calculations
The drug-likeness prediction of the synthesized compounds
was made by obtaining their pharmacokinetic and physi-
cochemical properties using the SwissADME web server
[66].
Acknowledgements We gratefully acknowledge the Facultad de
Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja
California for the financing given for the realization of this project.
Additionally, to the Consejo Nacional de Ciencia y Tecnología
(CONACYT) for ITT NMR facilities (Grant INFR-2011-3-173395).
15. Adewusi EA, Moodley N, Steenkamp V. Antioxidant and acet-
ylcholinesterase inhibitory activity of selected southern African
medicinal plants. S Afr J Bot. 2011;77:638–44. https://doi.org/10.
1016/j.sajb.2010.12.009.
16. Holzgrabe U, Kapková P, Alptüzün V, Scheiber J, Kugelmann E.
Targeting acetylcholinesterase to treat neurodegeneration. Expert Opin
Ther Targets. 2007;11:161–79. https://doi.org/10.1517/14728222.11.
2.161.
17. Zambrano P, Suwalsky M, Jemiola-Rzeminska M, Strzalka K,
Sepúlveda B, Gallardo MJ, et al. The acetylcholinesterase (AChE)
inhibitor and anti-Alzheimer drug donepezil interacts with human
erythrocytes. Biochim Biophys Acta Biomembr. 2019;1861:1078–85.
https://doi.org/10.1016/j.bbamem.2019.03.014.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
18. Pal S, Mareddy J, Devi NS. High speed synthesis of pyr-
azolones using microwave-assisted neat reaction technology. J
Braz Chem Soc. 2008;19:1207–14. https://doi.org/10.1590/
S0103-50532008000600023.
19. Schiel MA, Chopa AB, Silbestri GF, Alvarez MB, Lista AG,
Domini CE. Use of ultrasound in the synthesis of heterocycles of
medicinal interest. In: Brahmachari G, editor. Green synthetic
approaches for biologically relevant heterocycles. Boston: Else-
vier; 2015. p. 571–601.
20. Gautam S, Rani S, Aldossary SA, Saeedan AS, Ansari MN,
Kaithwas G. Effects of phenidone (DuCLOX-2/5 inhibitor)
against N-methyl-N-nitrosurea induced mammary gland carci-
noma in albino rats. Toxicol Appl Pharm. 2018;351:57–63.
https://doi.org/10.1016/j.taap.2018.04.019.
21. Russo S, Filgueira, de Azevedo W. Computational analysis of
dipyrone metabolite 4-aminoantipyrine as a cannabinoid receptor
1 agonist. Curr Med Chem. 2020;27:4741–9. https://doi.org/10.
2174/0929867326666190906155339.
References
1. Mangialasche F, Solomon A, Winblad B, Mecocci P, Kivipelto
M. Alzheimer’s disease: clinical trials and drug development.
Lancet Neurol. 2010;9:702–16. https://doi.org/10.1016/S1474-
4422(10)70119-8.
2. Lin HQ, Ho MT, Lau LS, Wong KK, Shaw PC, Wan DCC. Anti-
acetylcholinesterase activities of traditional Chinese medicine for
treating Alzheimer’s disease. Chem Biol Interact. 2008;175:352–4.
https://doi.org/10.1016/j.cbi.2008.05.030.
3. Calissano P, Matrone C, Amadoro G. Apoptosis and in vitro
Alzheimer’s disease neuronal models. Commun Integr Biol.
2009;2:163–9. https://doi.org/10.4161/cib.7704.
4. Shah RS, Lee H-G, Xiongwei Z, Perry G, Smith MA, Castellani
RJ. Current approaches in the treatment of Alzheimer’s disease.
Biomed Pharmacother. 2008;62:199–207. https://doi.org/10.1016/
j.biopha.2008.02.005.
22. Higashi Y, Jitsuiki D, Chayama K, Yoshizumi M. Edaravone (3-
methyl-1-phenyl-2-pyrazolin-5-one), a novel free radical sca-
venger, for treatment of cardiovascular diseases. Recent Pat Car-
diovasc Drug Disco. 2006;1:85–93. https://doi.org/10.2174/
157489006775244191.
5. Butterfield DA, Castegna A, Pocernich CB, Drake J, Scapagnini
G, Calabrese V. Nutritional approaches to combat oxidative stress
in Alzheimer’s disease. J Nutr Biochem. 2002;13:444–61. https://
doi.org/10.1016/s0955-2863(02)00205-x.
6. Christen Y. Oxidative stress and Alzheimer disease. Am J Clin
Nutr. 2000;71:621S–9S. https://doi.org/10.1093/ajcn/71.2.621s.
7. Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O.
Oxidative stress and antioxidant defense. World Allergy Organ J
2012;5:9–19. https://doi.org/10.1097/WOX.0b013e3182439613.
8. Smith MA, Rottkamp CA, Nunomura A, Raina AK, Perry G.
Oxidative stress in Alzheimer disease. Biochim Biophys Acta.
2000;1502:139–44. https://doi.org/10.1016/s0925-4439(00)00040-5.
9. Roberts CK, Sindhu KK. Oxidative stress and metabolic syn-
drome. Life Sci. 2009;84:705–12. https://doi.org/10.1016/j.lfs.
2009.02.026.
23. Watanabe T, Tahara M, Todo S. The novel antioxidant edaravone:
from bench to bedside. Cardiovasc Ther. 2008;26:101–14. https://
doi.org/10.1111/j.1527-3466.2008.00041.x.
24. Marković V, Erić S, Juranić ZD, Stanojković T, Joksović L,
Ranković B, et al. Synthesis, antitumor activity and QSAR studies
of some 4-aminomethylidene derivatives of edaravone. Bioorg
Chem. 2011;39:18–27. https://doi.org/10.1016/j.bioorg.2010.10.
003.
25. Fan X, Zhang X, Zhou L, Keith KA, Kern ER, Torrence PF. A
pyrimidine-pyrazolone nucleoside chimera with potent in vitro
anti-orthopoxvirus activity. Bioorg Med Chem Lett.
2006;16:3224–8. https://doi.org/10.1016/j.bmcl.2006.03.043.
26. Alzoubi KH, Al Mosabih HS, Mahasneh AF. The protective effect
of edaravone on memory impairment induced by chronic sleep
10. Huang W-J, Zhang X, Chen W-W. Role of oxidative stress in
Alzheimer’s disease. Biomed Rep. 2016;4:519–22. https://doi.org/
10.3892/br.2016.630.

Medicinal Chemistry Research
deprivation. Psychiatry Res. 2019;281:112577 https://doi.org/10.
1016/j.psychres.2019.112577.
intramolecular base. Bioorg Med Chem Lett. 2006;16:5939–42.
https://doi.org/10.1016/j.bmcl.2006.09.005.
27. Li K, Chen C, Huang Q, Zhang Y, Yao S. Efficacy of edaravone
combined with rt-PA in treatment of acute cerebral infarction and
the relation with inflammatory response and oxidative stress. Lat
Am J Pharm. 2020;39:511–7.
44. Ilyasov IR, Beloborodov VL, Selivanova IA, Terekhov RP.
ABTS/PP decolorization assay of antioxidant capacity reaction
pathways. Int J Mol Sci. 2020;21:1131 https://doi.org/10.3390/
ijms21031131.
28. Wang B, Lin W. Edaravone protects against pancreatic and
intestinal injury after acute pancreatitis via nuclear factor-κB
signaling in mice. Biol Pharm Bull. 2020;43:509–15. https://doi.
org/10.1248/bpb.b19-00944.
45. Jiao SS, Yao XQ, Liu YH, Wang QH, Zeng F, Lu JJ, et al.
Edaravone alleviates Alzheimer’s disease-type pathologies and
cognitive deficits. Proc Natl Acad Sci USA. 2015;112:5225–30.
https://doi.org/10.1073/pnas.1422998112.
29. Gao Y-K, Gui C-J, Xin W-Q, Hu D, Yang X-Y. Assessment of
mild hypothermia combined with edaravone for the treatment of
severe craniocerebral injury. Trop J Pharm Res. 2019;18:2557–62.
https://doi.org/10.4314/tjpr.v18i12.14.
30. Spasić S, Nikolić-Kokić A, Miletić S, Oreščanin-Dušić Z, Spasić
MB, Blagojević D, et al. Edaravone may prevent ferroptosis in
ALS. Curr Drug Targets. 2020;21:776–80. https://doi.org/10.
2174/1389450121666200220123305.
31. Pan Y, Li W, Feng Y, Xu J, Cao H. Edaravone attenuates
experimental asthma in mice through induction of HO-1 and the
Keap1/Nrf2 pathway. Exp Ther Med. 2020;19:1407–16. https://
doi.org/10.3892/etm.2019.8351.
46. Zondagh LS, Malan SF, Joubert J. Design, synthesis and biolo-
gical evaluation of edaravone derivatives bearing the N-benzyl
pyridinium moiety as multifunctional anti-Alzheimer’s agents. J
Enzym Inhib Med Chem. 2020;35:1596–605. https://doi.org/10.
1080/14756366.2020.1801673.
47. Yang R, Wang Q, Li F, Li J, Liu X. Edaravone injection ame-
liorates cognitive deficits in rat model of Alzheimer’s disease.
Neurol Sci. 2015;36:2067–72. https://doi.org/10.1007/s10072-
015-2314-y.
48. Jangra A, Kwatra M, Singh T, Pant R, Kushwah P, Ahmed S,
et al. Edaravone alleviates cisplatin-induced neurobehavioral
deficits via modulation of oxidative stress and inflammatory
32. Jensen BS. The synthesis of 1-phenyl-3-methyl-4-acyl-pyr-
azolones-5. Acta Chem Scand. 1959;13:1668–70. https://doi.org/
10.3891/acta.chem.scand.13-1668.
33. Zamorano S, Camus J. Síntesis y caracterización RMN 1D y 2D
de derivados sulfurados de la 1-fenil-3-metil-2-pirazolín-5-ona y
su potencial capacidad ligante. Rev Soc Quím Perú.
2015;81:24–32.
mediators in the rat hippocampus. Eur
J
Pharmacol.
2016;791:51–61. https://doi.org/10.1016/j.ejphar.2016.08.003.
49. Chiou S-Y, Huang C-F, Hwang M-T, Lin G. Comparison of
active sites of butyrylcholinesterase and acetylcholinesterase
based on inhibition by geometric isomers of benzene-di-N-sub-
stituted carbamates. J Biochem Mol Toxicol. 2009;23:303–8.
https://doi.org/10.1002/jbt.20286.
34. Holzer W, Guo C, Schalle K. 3-Methyl-1-phenyl-1H-pyrazol-5-yl
2-bromo-3-furan-carboxylate. Molbank 2009;2009:M603 https://
doi.org/10.3390/M603.
35. Xie C, Lu Z, Zhou W, Han J, Pan Y. A facile organocatalyzed
Michael addition of pyrazolines to α,β-unsaturated carbonyl
compounds. Tetrahedron Lett. 2012;53:6650–3. https://doi.org/10.
1016/j.tetlet.2012.09.096.
36. Levin A. Orally bioavailable prodrugs of edaravone with altered
pharmacokinetic properties and methods of use thereof. Patent no.
WO 2019/213335 A1. 2019.
37. Maruoka H, Yamagata K, Okabe F, Tomioka Y. Synthesis of 1-
acyl-1,2-dihydro-3H-pyrazol-3-ones via Lewis acid-mediated
50. Srivastava P, Tripathi PN, Sharma P, Rai SN, Singh SP, Srivas-
tava RK, et al. Design and development of some phenyl ben-
zoxazole derivatives as a potent acetylcholinesterase inhibitor with
antioxidant property to enhance learning and memory. Eur J Med
Chem. 2019;163:116–35. https://doi.org/10.1016/j.ejmech.2018.
11.049.
51. Sarikaya G, Çoban G, Parlar S, Tarikogullari AH, Armagan G,
Erdoğan MA, et al. Multifunctional cholinesterase inhibitors for
Alzheimer’s disease: synthesis, biological evaluations, and dock-
ing studies of o/p-propoxyphenylsubstituted-1H-benzimidazole
derivatives. Arch Pharm Chem Life Sci. 2018;351:e1800076
https://doi.org/10.1002/ardp.201800076.
rearrangement of 3-acyloxypyrazoles.
J
Heterocycl Chem.
52. Kryger G, Silman I, Sussman JL. Structure of acetylcholinesterase
complexed with E2020 (Aricept®: Implications for the design of
new anti-Alzheimer drugs. Structure. 1999;7:297–307. https://doi.
org/10.1016/s0969-2126(99)80040-9.
53. Bartolucci C, Perola E, Pilger C, Fels G, Lamba D. Three-
dimensional structure of a complex of galanthamine (Nivalin®)
with acetylcholinesterase from Torpedo californica: Implication
for the design of new anti-Alzheimer drugs. Proteins
2001;42:182–91. https://doi.org/10.1002/1097-0134(20010201)
42:2<182::aid-prot50>3.0.co;2-1.
54. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental
and computational approaches to estimate solubility and perme-
ability in drug discovery and development settings. Adv Drug
Deliv Rev. 2001;46:3–26. https://doi.org/10.1016/s0169-409x(00)
00129-0.
55. Jakki SL, Senthil V, Yasam VR, Chandrasekar MJN, Vijayar-
aghavan C. The blood brain barrier and its role in Alzheimer’s
therapy: an over-view. Curr Drug Targets. 2018;19:155–69.
https://doi.org/10.2174/1389450118666170612100750.
56. Salazar-Aranda R, Granados-Guzmán G, Pérez-Meseguer J, Gon-
zález GM, Waksman, de Torres N. Activity of polyphenolic com-
pounds against Candida glabrata. Molecules 2015;20:17903–12.
https://doi.org/10.3390/molecules201017903.
2006;43:859–65. https://doi.org/10.1002/jhet.5570430408.
38. Bai Y, Lu J, Gan H, Wang Z. Rapid microwave-enhanced
synthesis of 1-phenyl-3-methyl-5-acyloxypyrazoles under solvent-
free conditions. Synth Commun. 2002;32:2549–53. https://doi.
org/10.1081/SCC-120005938.
39. Terebenina A, Todorova O, Simova S, Borisov G. Reaction of 3-
methyl-1-phenyl-5-pyrazolone with some unsaturated aliphatic
carboxylic acid chlorides. Izv Khim. 1989;22:236–45.
40. Borges RS, Queiroz AN, Mendes APS, Araújo SC, França LCS,
Franco ECS, et al. Density functional theory (DTF) study of
edaravone derivatives as antioxidants. Int
J
Mol Sci.
2012;13:7594–606. https://doi.org/10.3390/ijms13067594.
41. Polkam N, Ramaswamy VR, Rayam P, Allaka TR, Anantaraju
HS, Dharmarajan S, et al. Synthesis, molecular properties pre-
diction and anticancer, antioxidant evaluation of new edaravone
derivatives. Bioorg Med Chem Lett. 2016;26:2562–8. https://doi.
org/10.1016/j.bmcl.2016.03.024.
42. Minelli C, Laudadio E, Galeazzi R, Rusciano D, Armeni T, Stipa
P, et al. Synthesis, characterization and antioxidant properties of a
new lipophilic derivative of edaravone. Antioxidants 2019;8:258
https://doi.org/10.3390/antiox8080258.
43. Nakagawa H, Ohyama R, Kimata A, Suzuki T, Miyata N.
Hydroxyl radical scavenging by edaravone derivatives: efficient
scavenging by 3-methyl-1-(pyridin-2-yl)-5-pyrazolone with an
57. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-
Evans C. Antioxidant activity applying an improved ABTS radical

Medicinal Chemistry Research
cation decolorization assay. Free Radic Biol Med.
1999;26:1231–7. https://doi.org/10.1016/s0891-5849(98)00315-3.
58. Kuskoski EM, Asuero AG, Troncoso AM, Mancini-Filho J, Fett R.
Aplicación de diversos métodos químicos para determinar actividad
antioxidante en pulpa de frutos. Food Sci Technol. 2005;25:726–32.
https://doi.org/10.1590/S0101-20612005000400016.
59. Noriega-Iribe E, Díaz-Rubio L, Estolano-Cobián A, Barajas-
Carrillo VW, Padrón JM, Salazar-Aranda R, et al. In vitro and in
silico screening of 2,4,5-trisubstituted imidazole derivatives as
potential xanthine oxidase and acetylcholinesterase inhibitors,
antioxidant, and antiproliferative agents. Appl Sci. 2020;10:2889
https://doi.org/10.3390/app10082889.
60. Díaz-Rubio L, Hernández-Martínez R, Estolano-Cobián A,
Chávez-Velasco D, Salazar-Aranda R, Waksman de Torres N,
et al. Synthesis, biological evaluation and docking studies of
chalcone and flavone analogs as antioxidants and acet-
ylcholinesterase inhibitors. Appl Sci. 2019;9:410 https://doi.org/
10.3390/app9030410.
61. Galindo-Hernández O, Córdova-Guerrero I, Díaz-Rubio LJ,
Pulido-Capiz A, Díaz-Villanueva JF, Castañeda-Sánchez CY,
et al. Protein translation associated to PERK arm is a new target
for regulation of metainflammation: a connection with hepatocyte
cholesterol. J Cell Biochem. 2019;120:4158–71. https://doi.org/
10.1002/jcb.27701.
62. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt
DM, Meng EC, et al. UCSF Chimera–a visualization system for
exploratory research and analysis.
J
Comput Chem.
2004;25:1605–12. https://doi.org/10.1002/jcc.20084.
63. Wang J, Wang W, Kollman PA, Case DA. Automatic atom type
and bond type perception in molecular mechanical calculations. J
Mol Graph Model. 2006;25:247–60. https://doi.org/10.1016/j.
jmgm.2005.12.005.
64. Sanner MF. Python: A programming language for software inte-
gration and development. J Mol Graph Model. 1999;17:57–61.
https://doi.org/10.1016/S1093-3263(99)99999-0
65. Trott A, Olson AJ. AutoDock Vina: Improving the speed and
accuracy of docking with a new scoring function, efficient opti-
mization, and multithreading. J Comput Chem. 2010;31:455–61.
https://doi.org/10.1002/jcc.21334.
66. Daina A, Michielin O, Zoete V. SwissADME: a free web tool to
evaluate pharmacokinetics, drug-likeness and medicinal chemistry
friendliness of small molecules. Sci Rep. 2017;7:42707 https://
doi.org/10.1038/srep42717.
67. Ali J, Camilleri P, Brown MB, Hutt AJ, Kirton SB. Revisiting the
general solubility equation: in silico prediction of aqueous solu-
bility incorporating the effect of topographical polar surface area.
J
Chem Inf Model. 2012;52:420–8. https://doi.org/10.1021/
ci200387c.