Acylhydrazones as isoniazid derivatives with multi-target profiles for the treatment of Alzheimer's disease: Radical scavenging, myeloperoxidase/acetylcholinesterase inhibition and biometal chelation

Article abstract of DOI:10.1016/j.bmc.2020.115470

Acylhydrazones 1a-o, derived from isoniazid, were synthesized and evaluated for Myeloperoxidase (MPO) and Acetylcholinesterase (AChE) inhibition, as well as their antioxidant and metal chelating activities, with the purpose of investigating potential multi-target profiles for the treatment of Alzheimer's disease. Synthesized compounds were tested using the 2,2-diphenyl-2-picrylhydrazyl (DPPH) method and 1i, 1j and 1 m showed radical scavenging ability. Compounds 1b, 1 h, 1i, 1 m and 1o inhibited MPO activity (10 μM) at 96.1 ± 5.5%, 90 ± 2.1%, 100.3 ± 1.7%, 80.1 ± 9.4% and 82.2 ± 10.6%, respectively, and only compound 1 m was able to inhibit 54.2 ± 1.7% of AChE activity (100 μM). Docking studies of the most potent compound 1 m were carried out, and the computational results provided the theoretical basis of enzyme inhibition. Furthermore, compound 1 m was able to form complexes with Fe²⁺ and Zn²⁺ ions in a 2:1 ligand:metal ratio according to the Job Plot method.

Full text of DOI:10.1016/j.bmc.2020.115470

Bioorganic&MedicinalChemistry28(2020)115470
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Acylhydrazones as isoniazid derivatives with multi-target profiles for the
treatment of Alzheimer’s disease: Radical scavenging, myeloperoxidase/
acetylcholinesterase inhibition and biometal chelation
Daniela Corrêa Santosa, Ruan Roberto Henriquesb, Marcos Antonio de Abreu Lopes Juniora,c
,
André Borges Fariasa,d, Thayssa Lisboa do Couto Nogueiraa, João Victor Fernandes Quimasa,
⁎
⁎
Nelilma Correia Romeiroa,c, Leandro Louback da Silvaa,1, , Andréa Luzia Ferreira de Souzaa,c,1,
a Universidade Federal do Rio de Janeiro, Campus Macaé Professor Aloísio Teixeira, Avenida Aluizio da Silva Gomes, 50, CEP 27930-560, Granja dos Cavaleiros, Macaé,
RJ, Brazil
b Universidade Federal do Rio de Janeiro, Cidade Universitária, Avenida Horácio Macedo, 2030, CEP 21941-598, Rio de Janeiro – RJ, Brazil
c Programa de Pós-Graduação em Produtos Bioativos e Biociências - PPG-PRODBIO, Universidade Federal do Rio de Janeiro, Campus Macaé Professor Aloísio Teixeira,
Avenida Aluizio da Silva Gomes, 50, CEP 27930-560, Granja dos Cavaleiros, Macaé – RJ, Brazil
d Programa de Pós-Graduação em Química – PPGQu, Universidade Federal do Rio de Janeiro, Cidade Universitária, Avenida Athos da Silveira Ramos, 149, Bloco A, CEP
21941-909, Rio de Janeiro – RJ, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Acylhydrazones 1a-o, derived from isoniazid, were synthesized and evaluated for Myeloperoxidase (MPO) and
Acetylcholinesterase (AChE) inhibition, as well as their antioxidant and metal chelating activities, with the
purpose of investigating potential multi-target profiles for the treatment of Alzheimer’s disease. Synthesized
compounds were tested using the 2,2-diphenyl-2-picrylhydrazyl (DPPH) method and 1i, 1j and 1 m showed
Acylhydrazone
Isoniazid
Antioxidant
Acetylcholinesterase
Myeloperoxidase
Molecular docking
Biometal chelation
radical scavenging ability. Compounds 1b, 1 h, 1i, 1 m and 1o inhibited MPO activity (10 μM) at 96.1
5.5%,
90
2.1%, 100.3
1.7%, 80.1
9.4% and 82.2
10.6%, respectively, and only compound 1 m was able
to inhibit 54.2
1.7% of AChE activity (100 μM). Docking studies of the most potent compound 1 m were
carried out, and the computational results provided the theoretical basis of enzyme inhibition. Furthermore,
compound 1 m was able to form complexes with Fe2+ and Zn2+ ions in a 2:1 ligand:metal ratio according to the
Job Plot method.
1. Introduction
the tau protein and consequent formation of neurofibrillary tails, neu-
ronal and synaptic loss, glial activation and inflammation.3 Based on
Alzheimer's disease (AD) is the most frequent neurodegenerative
disorder associated with age which cognitive and neuropsychiatric
manifestations result in progressive disability and eventual in-
capacitation.1 In general, the first clinically observed aspect is the re-
cent memory deficiency, while remote memories are preserved until a
certain stage of the disease. AD is devastating, since it is characterized
by a progressive loss of brain functions such as memory and language
skills, and ultimately by death, which occurs from 8 to 10 years on
average after diagnosis.2 The histopathological features present in the
cerebral parenchyma of patients with AD include amyloid fibrillar de-
posits located in the walls of blood vessels, associated with a variety of
different types of senile plaques, accumulation of abnormal filaments of
these neuropathological markers, two main hypotheses have been
proposed in order to explain the etiology of the disease. Brains of pa-
tients with AD showed degeneration of cholinergic neurons, as well as a
reduction of cholinergic markers in the cerebral cortex, such as choline
acetyltransferase and acetylcholinesterase.4 Despite the several hy-
potheses about the pathogenesis of AD, the cholinergic one maintains
its relevance, considering that, despite the fact that there are no ef-
fective treatments for AD, the only available medications are acet-
ylcholinesterase inhibitors (e.g., tacrine, donepezil, rivastigmine, and
galantamine) and the NMDA receptor antagonist, memantine, although
these drugs only marginally reduce the symptoms of AD, exerting no
effects on the course of the disease.5
⁎ Corresponding authors at: Universidade Federal do Rio de Janeiro, Campus Macaé Professor Aloísio Teixeira, Avenida Aluizio da Silva Gomes, 50, CEP 27930-
560, Granja dos Cavaleiros, Macaé, RJ, Brazil.
E-mail addresses: lelouback@macae.ufrj.br (L.L.d. Silva), andrealuzia@iq.ufrj.br (A.L.F.d. Souza).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2020.115470
Received 14 August 2019; Received in revised form 24 March 2020; Accepted 26 March 2020
Availableonline31March2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.

D.C. Santos, et al.
Bioorganic&MedicinalChemistry28(2020)115470
According to the amyloid cascade hypothesis, neurodegeneration in
as potential Acetylcholinesterase and Myeloperoxidase inhibitors with
antioxidant activity. Molecular docking and biometal chelation studies
were also conducted for the derivative with the best multi-target profile.
Alzheimer's disease begins with the proteolytic cleavage of the amyloid
precursor protein (APP) and results in the production, aggregation and
deposition of β-amyloid (Aβ) substance and formation of senile pla-
ques.6 In recent years, with the wide discussion about the amyloid
cascade hypothesis, evidences have suggested that endogenous metal
ions, particularly those with redox activity, such as copper(II) and iron
(II) ions, in addition to non-redox active ions, such as zinc(II), may
contribute to the evolution of neurodegenerative diseases, favoring the
aggregation of Aβ and increasing its toxicity.7 Several studies have
shown that zinc and copper compete for the same Aβ residues, although
zinc has demonstrated greater relevance in the rapid aggregation of the
peptide than copper, which, in turn, mainly induces conformational
changes in the peptide.8
2. Results and discussion
2.1. Chemistry
The acylhydrazones reported in this work have already been de-
scribed in the literature and their antimicrobial, anticancer and anti-
inflammatory activities have been investigated.21 However, prior to the
current work, these compounds have not been tested for AChE and MPO
inhibition and biometals chelating activity. Generally, condensation
reactions for the formation of acylhydrazones are carried out by heating
under reflux for more than 1 hour21, so to accelerate the formation of
derivatives 1a-o, the syntheses were carried out in a microwave reactor
under high pressure. As depicted in Scheme 1, isoniazid and the re-
spective aldehydes were reacted under microwave irradiation in
ethanol as solvent at 140 °C for 45 min. The acylhydrazone derivatives
1a-o were then obtained in good yields (65–95%). The spectroscopic
data obtained are in accordance with the predicted structures (sup-
plementary material). In the proton nuclear magnetic resonance
spectra, the resonance signals of azomethine protons are registered as
singlets in the range of 8.33 to 8.67 ppm and the NeH signals are
singlets in the range of 11.78 to 12.37 ppm for compounds 1a–o.21 In
the positive electron impact (EI) mass spectra, the molecular ion peaks
[M + H] have shown that the predicted compounds have been formed.
Reaction of H2O2 with reduced transition metal ions via Fenton and
Haber–Weiss reaction leads to the generation of the hydroxyl radical
·OH, which induces oxidative modifications associated with AD neu-
ronal cytoplasm and lesions are consistent with the chronic dysregu-
lation of metal ion binding in the cytoplasm of pyramidal neurons and
lesions of the AD brain. In addition, metal ion accumulation and oxi-
dative stress have been found to correlate with changes in the con-
centration of both soluble and deposited Aβ.9
Based on those reports, a good approach would be to develop
compounds that can disrupt specific, abnormal metal–protein interac-
tions. These compounds, called MPACs (Metal–Protein Attenuating
Compounds), are related to the repartition of the metal ion distribu-
tion.10 Furthermore, some hydrazone derivatives containing an iso-
niazid skeleton resulted in interesting ligands, capable of coordinating
metal ions of biological importance through several N/O donor
atoms.11 Isoniazid is a known anti-tuberculosis drug and hydrazones
derived from this drug are known to be iron chelators.12 Some chelators
such as clioquinol are referred to as MPAC, assuming that they cause
metal release from peptides and resumption of Aβ clearance.13 Un-
fortunately, the use of clioquinol generates adverse side-effects.14 Re-
cently, an isonicotinoyl hydrazone of 8-hydroxyquinoline-2-carbox-
aldehyde has been considered as a MPAC.15a The same research group
confirmed, in an ensuing publication, that the chemical class of acyl-
hydrazones constitute, in fact, a promising group of MPACs for the
treatment of Alzheimer's disease.15b
2.2. Biological activities
Target compounds 1a-o were tested for their antioxidant activity
and inhibitory effects against Acetylcholinesterase (AChE) and
Myeloperoxidase (MPO) enzymes (Table 1).
2.2.1. Antioxidant activity
The in vitro antioxidant activity of the acylhydrazones was evaluated
at 100 µM using the DPPH (1,1-Diphenyl-2-picrylhydrazyl) stable free
radical method.22 DPPH radicals are considered as a representative
method for the preliminary screening of compounds able to scavenge
activated oxygen species, since they are more stable and easier to
handle than oxygen free radicals and have been largely used to measure
the antioxidant activity of different compounds. DPPH• absorbance
decrease in the presence of antioxidants is due to hydrogen transfer
from the antioxidant, thus forming the DPPH-H stable compound.
Methanolic DPPH• solutions used at different concentrations were
stable (checked over a 30 min period). Three of the fifteen compounds
Oxidative stress is one of the earliest events in AD pathogenesis. It
plays an important role in neuronal degeneration, being involved in the
damage of biological molecules such as proteins, DNA, and lipids, thus
accelerating cancer, aging, inflammation, cardiovascular and neurode-
generative diseases.16 Therefore, the successful protection of neuronal
cells from oxidative damage could potentially prevent AD. The pro-
tection against oxidative stress-induced toxicities can be accomplished
by direct antioxidants, which are redox active, short-lived, and are
consumed in the process of their antioxidative actions, and indirect
antioxidants, that may or may not be redox- active through the sti-
mulation of the enzymatic antioxidant system or inhibition of proox-
idant enzymes.17 Sources of ROS include redox active metals, mi-
tochondria, and enzymes like NADPH oxidase and Myeloperoxidase
(MPO).18 The main product of MPO is hypochlorous acid (HOCl), an
extremely oxidizing species, both of which are increased in neurode-
generative diseases, leading to severe tissue damage. Increased levels of
3-chlorotyrosine (biomarker of HOCl) in damaged brain areas has been
shown in the literature. HOCl causes cellular damage and tissue de-
struction that can lead to cell death. Thus, MPO has been considered an
important target for the inhibition of oxidative stress and its con-
sequences in the physio pathological process of neurodegenerative
diseases, such as AD.19,20
showed radical scavenging ability: 1j (92.4
(76.7 1.3%) and 1 m (82.2 1.7%) as shown in Table 1.
Compared to quercetin and isoniazid, which had IC50 values of
15.3 0.6 µM and 22.9 0.7 µM, respectively, compound 1j was
equipotent, with an IC50 value of 18.3 1.5 µM, while 1 k and 1 m
were less potent, with IC50 values of 33.6 2.3 µM and
42.4 1.9 µM, respectively (Table 2). The hydrogen atom of the
1.2%), 1 k
isoniazid moiety in α position from the acylhydrazone function is
highly activated. Therefore, reaction of DPPH radicals may create a
nitrogen radical on the antioxidant under consideration. For those
possessing at least one hydrogen atom bound to nitrogen, there may be
formation of an N• radical (hydrazonyl radical) which is stabilized
through conjugation forming delocalized C• radicals. The different
DPPH• scavenging properties (IC50) of the synthesized hydrazones may
reflect the relative ability for the NeH hydrogen atom abstraction from
the considered compounds.23
Alzheimer's disease is a multifactorial neurodegenerative disorder.
Thus, the development of agents that affect two or more AD-relevant
targets has drawn considerable attention for their potential to enhance
efficacy and/or improve safety in the therapy of AD. In this context, we
investigated some Acylhydrazones derived from isoniazid (1a-o, Fig. 1)
2.2.2. Myeloperoxidase inhibitory activity
Inhibition of MPO chlorinating activity of all acylhydrazones was
examined by the chlorotaurine assay employing MPO from rat bone
2

D.C. Santos, et al.
Bioorganic&MedicinalChemistry28(2020)115470
Fig 1. Structures of Acylhydrazone derivatives 1a-o.
Scheme 1. Obtention of acylhydrazone de-
rivatives 1a-o from isoniazid under micro-
wave irradiation.
O
O
NH2
EtOH/HOAc, 45 min
MW
N
Ar
NH
NH
Ar
+
N
O
N
marrow.24 Isoniazid was used as the reference standard. The % of in-
hibition values at 10 µM are summarized in Table 1. Five of the fifteen
acylhydrazone derivatives decreased MPO chlorinating activity by
more than 80%. 1b, 1i, 1j, 1 m and 1o inhibited MPO activity by
2.2.3. Acetylcholinesterase inhibitory activity
The AChE inhibitory activity of all acylhydrazones was examined by
the Ellmańs method25
, using AChE from Electrophorus electricus.
Rivastigmine was used as the reference standard. The inhibition %
values at 100 µM are summarized in Table 1. Only one of the evaluated
derivatives was able to inhibit more than 50% of AChE activity. Com-
96.1
82.2
5.5%, 90
2.1%, 100.3
1.7%, 80.1
9.4% and
10.6%, respectively. The potency of these compounds was
determined and IC50 values are presented in Table 2. Compared to
pound 1 m reduced AChE activity at 54.2
1.7% compared to riv-
2.6%. In order to
isoniazid, which has an IC50 value of 3.9
0.3 µM, derivatives 1b and
0.3 µM
astigimine which reduced AChE activity at 59.6
1o were more potent MPO inhibitors, with IC50 values of 1.4
investigate the mechanism of action of derivative 1 m, kinetic analysis
was performed. The experiments were carried out using combinations
of five substrate concentrations and two inhibitor concentrations and
the double-reciprocal Lineweaver–Burk plot of the data was obtained
and 1.5
0.5 µM, respectively, while compounds 1i, 1j and 1 m were
as potent as isoniazid, with IC50 values of 4.3
1 µM, 4.8
0.3 µM
and 5.3
0.5 µM, respectively.
Table 1
AChE and MPO inhibitory activities and antioxidant capacity (DPPH assay) of acylhydrazone derivatives 1a-o.
Compound
Ar
DPPH
MPO inhibition
AChE inhibition (% at 100 µmol.L)
scavenger
(% at 10 µmol.L)
(% at 100 µmol.L)
1a
Phenyl
3.3
1.2
1.2
1.3
0.5
0.8
5.1
12.9
5.5a
7.7
16.5
6.9
16.3
8
7.3
10.4
1.1
1b
4-dimethylaminophenyl
4-fluorophenyl
4-methoxyphenyl
4-nitrophenyl
4-hydroxyphenyl
3-nitrophenyl
2-hydroxyphenyl
2-hydroxy-5-bromophenyl
2,5-dihydroxyphenyl
3-methoxy-4-hydroxyphenyl
3,4-dichlorophenyl
3-chromonyl
2-pyridyl
6.3
96.1
4.6
1c
8.1
1d
2.4
6
10.6
8.7
1e
2.5
32
21.6
12.7
10
11.3
3.6
1f
28.7
2.0
1.1
−63.9
−10
45.3
90
24.5a
1 g
1.8
2
4.5
36.2
3
7.9
1 h
5.3
15.5
14.5
4.8
1i
3.9
1
2.1a
6.8
0.3
23.1
14
1j
92.4
76.7
3.9
1.2a
100.3
17.3
−2.4
80.1
0.3
1.7a
15.7
1 k
1.3a
6
12.2
1 L
1.6
4.7
9.4
7.2
8
a
1 m
82.2
−0.6
28.8
1.9
1.7a
1.3
0.8
54.2
24.5
5.5
7
1.7a
9.1
1n
a
1o
3-indolyl
82.2
−1.4
17.1
99
10.6
2.4
DMSO
Rivastigmine
Isoniazid
Quercetin
–
1.2
0.4
1.4
1.2
12.7
11.4
7.1
–
0.2
59.6
5.8
ND
2.6a
a
a
–
91.3
96.3
4.8a
12.5
a
–
96.3
2.4
ND, not determined; aStatistically different from DMSO.
3

D.C. Santos, et al.
Bioorganic&MedicinalChemistry28(2020)115470
Table 2
Phe147 (Fig. 3B). It is worth of note that binding near the distal Heme
pocket in MPO has been considered important for aromatic mole-
cules.26
IC50 values of the most active acylhydrazone derivatives in MPO and DPPH
assays.
The active site of AChE contains the catalytic site (Ser203, His447
and Glu334), the peripheral anionic site – PAS (Tyr74, Tyr124, Trp286
and Tyr341) and the anionic site (Trp86, Tyr133, Tyr337 and Phe338).
In the study by Lu and coworkers,27 the virtual screening of several
substances with anticholinesterase activity has shown that π-stacking
interactions with the indole ring of Trp286 and hydrogen bonds with
residues Ser203, Gly122, Tyr124, Tyr72 and Phe337 are the most fre-
quently encountered.
Compound
DPPH scavenger
MPO Inhibition IC50 (µmol.L)
IC50 (µmol.L)
1b
ND
ND
1.4
4.3
4.8
ND
5.3
1.5
ND
3.9
0.3
1
1i
1j
18.3
33.6
42.4
ND
1.5
2.3
1.9
0.3
1 k
1 m
0.5
0.5
1o
Quercetin
Isoniazid
15.3
22.9
0.6
0.7
Also, previous works have analyzed the structure–activity relation-
ships of a series of MPO inhibitors by molecular docking demonstrating
that two molecules with amino substituents at the terminal part of the
alkyl group attached to the indole ring performed hydrogen bonding
interactions with Glu102 and showed IC50 values of 3 and 6 nM, in
Taurine Chlorination inhibition assays.28 In addition, another study has
also shown that potent inhibition featured ligand interaction with
Glu102.29 Thus, amino acid residue Glu102 seems to play an important
role in MPO activity, since the active molecules of the present work
were oriented towards this residue, while the inactive ones had less
effective interactions (Supplementary Material).
0.3
ND, not determined.
2.0
1.5
1.0
0.5
0.0
1m(60 µM)
1m(45 µM)
DMSO (0,02%)
2.4. Metal-chelating properties of derivative 1 m
-10
-5
0
5
10
15
The complexation ability of derivative 1 m with biometals such as
Ca2+, Cu2+, Fe2+, Mg2+ and Zn2+ ions was performed. The pre-
liminary metal chelation assay was carried out by UV–vis spectro-
photometer (200 to 600 nm) in dimethyl sulfoxide (DMSO) (Fig. 4). The
UV absorption spectra of the compound in DMSO changed with the
titration of Cu2+, Fe2+ and Zn2+ ions.
1/[acetylthiocholine] mM
Fig. 2. Lineweaver–Burk plots representing the reciprocal of initial enzyme
velocity versus the reciprocal of acetylthiocholine concentration in the absence
and presence of different concentrations of 1 m. Each point is the mean of three
different experiments.
Despite the complexation of the derivative 1 m with Cu2+ ions, the
stoichiometry of the complex analyzed by the Job Plot method30 was
performed on the derivative 1 m with Fe2+ and Zn2+ ions. The Fe2+
ions have been investigated as an example of ions with redox activity
and Zn2+ ions as an example of ions with non-redox activity. According
to the literature on the amyloid cascade hypothesis, evidence suggests
that endogenous metal ions, those with redox activity such as copper(II)
and iron(II) ions, as well as non-redox active ions such as zinc(II) ions,
can contribute to the evolution of Alzheimer’s disease, favoring the
aggregation of Aβ7. In order to avoid solvent interference (DMSO) as a
ligand in the complexation of derivative 1 m with metal ions, we have
also performed the Job Plot in methanol. The stoichiometry of the
1 m–Fe2+ and 1 m-Zn2+ complexes was determined using the molar
ratio method by preparing solutions of compound 1 m (20 μM in
MeOH) with increasing amounts of FeCl2 and ZnCl2 (5–80 μM in
MeOH). The UV spectra were used to obtain the absorbance of the
complex of 1 m with FeCl2 and ZnCl2 at different concentrations at
403 nm and 371 nm respectively. As shown in Figs. 5 and 6, initially,
the absorbance increased linearly and then became stable. The two
straight lines intersected at a molar fraction of 2, revealing a 2:1 stoi-
chiometry for the complexes.
(Fig. 2) in which we can see intersects above or at the x-axis which is
typical of non-competitive mixed inhibitors, suggesting that 1 m binds
to an allosteric site, in addition to the catalytic site.
2.3. Molecular docking studies
Molecular docking was validated by redocking calculations and, in
the process, the best poses have been evaluated using different scoring
functions and binding site radius around His95 and Ser203 for MPO and
AChE, respectively. Thus, the best schemes obtained in the redocking
process have been chosen for the docking studies with the acylhy-
drazone derivatives; that is the ChemPLP function with a 10 Å binding
site radius, which exhibited an RMSD value of 0.43 Å for MPO and
0.22 Å for AChE in the redocking step, respectively, reflecting the
ability of the software to find the binding pose of the co-crystallized
ligand.
Figure 3 shows MPO and AChE active site cavities with the hit
compound revealed in this work, derivative 1 m. It is worth of note that
1 m presents important interactions with residues in the AChE gorge
such as π-stacking of the 3-chromonyl nucleus with residues Tyr341,
Phe297 and Trp286 (part of the Peripheral Anionic Site, PAS), in ad-
dition to hydrogen bonding involving the ether oxygen of the 3-chro-
monyl nucleus and the oxygen atom of the carbonyl group of the
acylhydrazone moiety with Ser293 and Phe295 (Fig. 3A).
3. Conclusions
Several acylhydrazone isoniazid derivatives showed important ac-
tivities on the targets evaluated, in particular DPPH scavenging, AChE
and MPO inhibition. However, compound 1 m was the only studied
compound that exhibited significant inhibition against AChE (54%,
100 μM), MPO inhibition related to the chlorination mechanism (IC50
Besides that, the top binding pose of 1 m with MPO shows hydrogen
bond interactions between the –NH moiety of the acylhydrazone
function and the oxygen atom of the side chain of Glu102 and also
between the ether oxygen of the 3-chromonyl nucleus and the side
chain of Arg239. Also, the oxygen atom of the amide carbonyl of the
ligand and the oxygen atom of Gln291 are within van der Waals contact
which, in the dynamics of ligand-enzyme interaction, is most likely
relieved. Finally, another important interaction, of π-stacking type, was
observed between the 3-chromonyl nucleus, the Heme group and
5.3
0.5 μM), antioxidant capacity (IC50 42.4
1.9 μM) and good
biometal (Fe2+ and Zn2+ ions) chelating properties. In addition, its
putative interactions in the active sites of AChE and MPO revealed by
docking studies have shown important complementary features that
shall be further explored with the aim to obtain more potent com-
pounds.
4

D.C. Santos, et al.
Bioorganic&MedicinalChemistry28(2020)115470
Fig. 3. Binding poses of 1 m (purple) with AChE (A) and MPO (B) as suggested by docking studies. Hydrogen bonds are shown in yellow dashed lines, with respective
interacting amino acid in olive green. Distance of π-stacking and π- π interactions are shown in green dashed lines with respective interacting amino acid in pink. The
Van der Waals interactions are shown by cyan amino acid carbon atoms.
Fig 6. Determination of the stoichiometry of the 1 m–Zn2+ complex by the
Fig 4. UV spectrum of compound 1 m (20 μM in DMSO) alone or in the pre-
molar ratio method. The final concentration of the tested compound was 20 μM,
and the final concentration of Zn2+ ions ranged from 5 to 80 µM.
sence of Ca2+, Cu2+, Fe2+, Mg2+ and Zn2+ ions (20 μM in DMSO).
4. Experimental section
4.1. General
The chemical reagents and solvents used in this study were pur-
chased from Sigma-Aldrich. Microwave apparatus used Monowave 300
Anton Paar (vial G10 – vial used for reaction). Melting points of the
compounds were obtained on Fisatom melting-point apparatus. The 1H
and 13C NMR spectra were recorded with tetramethylsilane (TMS) as
the internal standard on a Bruker FT-400(1 0 0) and FT-500(1 2 5) MHz
spectrometers using deuterated dimethyl sulphoxide (DMSO‑d6) as
solvent. Mass spectra were recorded on Agilent 1200 mass spectrometer
at 10 eV. The UV and Job Plot were recorded on a UV–Vis Shimadzu
UV2600 spectrometer.
4.2. General procedure for the synthesis of compounds 1a-o
A
mixture of isoniazid (1.0 mmol), the respective aldehyde
Fig 5. Determination of the stoichiometry of the 1 m–Fe2+ complex by the
molar ratio method. The final concentration of the tested compound was 20 μM,
and the final concentration of Fe2+ ions ranged from 5 to 80 µM.
(1.0 mmol) and one drop of glacial acetic acid in ethanol (6 mL) was
magnetic stirred under microwave irradiation for 45 min (140 °C,
1000 rpm). The reaction mixture was filtered off and the solid was re-
crystallized with ethanol. The synthesis pathway is shown on scheme 1.
5

D.C. Santos, et al.
Bioorganic&MedicinalChemistry28(2020)115470
4.2.1. N’-Benzylidene Isonicotinohydrazide (1a)
4.2.8. N’-(2-Hydroxybenzylidene) Isonicotinohydrazide (1 h)
white solid, yield 84%, 98.9% HPLC purity, MP: 189–190 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 12.02 (s, 1H, NH), 8.48 (s, 1H, CH = N),
8.77 (d, 2H, J = 3.0 Hz, Ar-H), 7.82 (d, 2H, J = 6.0 Hz, Ar-H), 7.73 (d,
2H, J = 3.0 Hz, Ar-H), 7.45–7.16 (m, 3H, Ar-H). 13C NMR (100 MHz,
DMSO‑d6) δ: 161.63, 150.33, 149.03, 140.46, 134.02, 130.38, 128.87,
127.26, 121.53. HRMS (ESI) m/z calcd for C13H11N3O [M + H]:
226.2459, found: 226.0974.
white solid, yield 90% 98.1% HPLC purity, MP: 244–246 °C. 1H
NMR (500 MHz, DMSO‑d6) δ: 11.88 (s, 1H, NH), 10.00 (s,1H, OH), 8.79
(d, 2H, J = 5.5 Hz, Ar-H), 8.38 (s,1H, CH = N), 7.83 (d, 2H,
J = 6.0 Hz, Ar-H), 7.61 (d, 2H, J = 8.5 Hz, Ar-H), 6.87 (d, 2H,
J = 8.5 Hz, Ar-H). 13C NMR (125 MHz, DMSO‑d6) δ: 161.27, 159.70,
150.28, 149.49, 145.01, 140.67, 128.62, 124.98, 123.13, 121.48,
115.76. HRMS (ESI) m/z calcd for C13H11N3O2 [M + H]: 242.2453,
found: 242.0913.
4.2.2. N’-(4-Dimethylaminobenzylidene) Isonicotinohydrazide (1b)
yellow solid, yield 81%, 98.9% HPLC purity, MP: 203–205 °C. 1H
NMR (500 MHz, DMSO‑d6) δ: 11.82 (s, 1H, NH), 8.78 (d, 2H,
J = 5.0 Hz, Ar-H), 8.33 (s, 1H, CH = N), 7.83 (d, 2H, J = 5.0 Hz, Ar-
H), 7.58 (d, 2H, J = 10.0 Hz, Ar-H), 6.78 (d, 2H, J = 10.0 Hz, Ar-H),
2.98 (s, 6H, N-CH3). 13C NMR (125 MHz, DMSO‑d6) δ: 167.57, 161.51,
151.88, 150.32, 149.92, 141.10, 128.63, 121.94, 121.66, 111.98.
HRMS (ESI) m/z calcd for C13H11N3O [M + H]: 269.3137, found:
269.1396.
4.2.9. N’-(2-Hydroxy-5-bromobenzylidene) Isonicotinohydrazide (1i)
yellowish solid, yield 88%, 98.8% HPLC purity, MP: 256–258 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 12.37 (s, 1H, NH), 11.15 (s, 1H, OH),
8.82 (d, 2H, J = 5.3 Hz, Ar-H), 8.67 (s, 1H, CH = N), 7.87 (d, 2H,
J = 6.0 Hz, Ar-H), 7.84 (d, 1H, J = 2.5 Hz, Ar-H), 7.47 (dd, 1H, J = 2.5
and 8.7 Hz, Ar-H), 6.93 (d, 1H, J = 8.7 Hz, Ar-H). 13C NMR (100 MHz,
DMSO‑d6) δ: 161.48, 156.46, 150.37, 146.34, 139.89, 133.89, 130.12,
121.52, 121.29, 118.70, 110.55. HRMS (ESI) m/z calcd for
C13H10BrN3O2 [M + H]: 320.1414, found: 320.0013.
4.2.3. N’-(4-fluorobenzylidene) Isonicotinohydrazide (1c)
white solid, yield 65%, 98.5% HPLC purity, MP: 184–186 °C. 1H
NMR (500 MHz, DMSO‑d6) δ: 12.10 (s, 1H, NH), 8.81 (d, 2H,
J = 6.0 Hz, Ar-H), 8.48 (s, 1H, CH = N), 7.84 (m, 4H, Ar-H), 7.34 (m,
2H, Ar-H). 13C NMR (125 MHz, DMSO‑d6) δ: 164.30, 161.57, 150.26,
147.94, 140.64, 130.64, 129.47, 121.65, 115.90. HRMS (ESI) m/z calcd
for C13H10FN3O [M + H]: 244.2364, found: 244.0880.
4.2.10. N’-(2,5-Dihydroxybenzylidene) isonicotinohydrazide (1j)
yellow solid, yield 80%, 98.3% HPLC purity, MP: 294–295 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 12.20 (s, 1H, NH), 10.23 (s, 1H, OH),
9.03 (s, 1H, OH), 8.81 (d, 2H, J = 1.5 and 4.5 Hz, Ar-H), 8.64 (s, 1H,
CH = N), 7.87 (dd, 2H, J = 1.6 and 4.5 Hz, Ar-H), 7.07 (d, 1H,
J = 1.7 Hz, Ar-H), 6.78 (d, 2H, J = 2.5 Hz, Ar-H). 13C NMR (100 MHz,
DMSO‑d6) δ: 161.32, 150.38, 150.32, 149.98, 148.35, 140.16, 121.54,
119.38, 119.08, 117.19, 113.41. HRMS (ESI) m/z calcd for C13H11N3O3
[M + H]: 258.2447, found: 258.0863.
4.2.4. N’-(4-Methoxybenzylidene) Isonicotinohydrazide (1d)
white solid, yield 75%, 97,5.% HPLC purity, MP: 171–173 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 11.95 (s, 1H, NH), 8.80 (dd, 2H, J = 1.5
and 4.5 Hz, Ar-H), 8.42 (s, 1H, CH = N), 7.84 (dd, 2H, J = 1.5 and
4.4 Hz, Ar-H), 7.72 (d, 2H, J = 8.7 Hz, Ar-H), 7.05 (d, 2H, J = 8.7 Hz,
Ar-H), 3.82 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO‑d6) δ: 161.40,
161.08, 150.30, 148.92, 140.62, 128.92, 126.56, 121.50, 114.39,
55.32. HRMS (ESI) m/z calcd for C14H13N3O2 [M + H]: 256.27192,
found: 256.108053.
4.2.11. N’-(3-Hydroxy-4-methoxybenzylidene) isonicotinohydrazide (1 k)
yellow solid, yield 80%, 98.8% HPLC purity, MP: 226–228 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 11.89 (s, 1H, NH), 9.62 (s, 1H, OH), 8.79
(d, 2H, J = 5.9 Hz, Ar-H), 8.36 (s, 1H, CH = N), 7.83 (dd, 2H, J = 1.5
and 4.5 Hz, Ar-H), 7.34 (d, 1H, J = 1.7 Hz, Ar-H), 7.14 – 7.12 (dd, 1H,
J = 1.7 and 8.0 Hz, Ar-H), 6.88 (d, 1H, J = 8.0 Hz, Ar-H), 3.84 (s, 3H,
OCH3). 13C NMR (100 MHz, DMSO‑d6) δ: 161.32, 150.29, 149.58,
149.31, 148.08, 140.69, 125.39, 122.46, 121.50, 115.48, 109.06,
55.56. HRMS (ESI) m/z calcd for C14H13N3O3 [M + H]: 272.2713,
found: 272.1035.
4.2.5. N’-(4-Nitrobenzylidene) Isonicotinohydrazide (1e).
yellow solid, yield 92%, 98.1% HPLC purity, MP: 265–267 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 12.36 (s, 1H, NH), 8.82 (d, 2H,
J = 6.0 Hz, Ar-H), 8.57 (s, 1H, CH = N), 8.33 (d, 2H, J = 8.7 Hz, Ar-
4.2.12. N’-(3,4-Dichlorobenzylidene) isonicotinohydrazide (1 L)
H), 8.04 (d, 2H, J = 8.7 Hz, Ar-H), 7.85 (d, 2H, J = 6.0 Hz, Ar-H). 13
C
white solid, yield 89%, 99.2% HPLC purity, MP: 248–250 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 12.27 (s, 1H, NH), 8.81 (d, 2H,
J = 4.6 Hz, Ar-H), 8.44 (s, 1H, CH = N), 7.99 (d, 1H, J = 1.2 Hz, Ar-
H), 7.84 (dd, 2H, J = 1.5 and 4.5 Hz, Ar-H), 7.78 (m, 2H, Ar-H). 13C
NMR (100 MHz, DMSO‑d6) δ: 161.81, 150.35, 146.23, 140.19, 134.86,
132.57, 131.76, 131.14, 128.73, 127.00, 122.46, 121.54. HRMS (ESI)
m/z calcd for C13H9Cl2N3O [M + H]: 294.1361, found: 294.0187.
NMR (100 MHz, DMSO‑d6) δ: 161.96, 150.40, 148.05, 146.48, 140.27,
128.21, 124.09, 121.56. HRMS (ESI) m/z calcd for
C13H10N4O3
[M + H]: 271.2440, found: 271.0827.
4.2.6. N’-(4-hydroxybenzylidene) Isonicotinohydrazide (1f)
yellowish solid, yield 91%, 97.1% HPLC purity, MP: 299–301 °C. 1H
NMR (500 MHz, DMSO‑d6) δ: 11.89 (s, 1H, NH), 10.02 (s, 1H, OH),
8.79 (d, 2H, J = 1.4 and 4.5 Hz, Ar-H), 8.38 (s, 1H, CH = N), 7.83 (d,
2H, J = 1.5 and 4.5 Hz, Ar-H), 7.61 (d, 2H, d, J = 8.6 Hz, Ar-H), 6.88
(2H, d, J = 8.6 Hz, Ar-H). 13C NMR (125 MHz, DMSO‑d6) δ: 161.33,
159.73, 150.30, 149.39, 140.71, 129.12, 125.01, 121.51, 115.80.
HRMS (ESI) m/z calcd for C13H11N3O2 [M + H]: 242.2453, found:
242.0924.
4.2.13. N’-(chromone-3lmethylene) hydrazide (1 m)
white solid, yield 82%, 98.6% HPLC purity, MP: 215–217 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 12.16 (s, 1H, NH), 8.87 (s, 1H, Ar-H),
8.80 (dd, 2H, J = 1.4 and 4.6 Hz, Ar-H), 8.66 (s, 1H, CH = N), 8.15
(dd, 1H, J = 1.3 and 7.9 Hz, Ar-H), 7.89 (m, 3H, Ar-H), 7.75 (d, 1H,
J = 8.3 Hz, Ar-H), 7.58 (t, 1H, Ar-H). 13C NMR (100 MHz, DMSO‑d6) δ:
175.01, 161.40, 155.78, 154.85, 150.35, 141.68, 140.20, 134.75,
132.57, 131.76, 126.15, 125.21, 123.32, 121.50, 118.75, 118.09.
HRMS (ESI) m/z calcd for C16H11N3O3 [M + H]: 294.2768, found:
316.0701 (C16H11N3NaO3).
4.2.7. N’-(3-Nitrobenzylidene) Isonicotinohydrazide (1 g)
yellow solid, yield 82%, 99.5% HPLC purity, MP: 248–250 °C. 1H
NMR (400 MHz, DMSO‑d6) δ: 12.34 (s, 1H, NH), 8.82 (d, 2H,
J = 6.0 Hz, Ar-H), 8.58 (s, 1H, CH = N), 8.30 (d, 2H, J = 9.0 Hz, Ar-
H), 8.20 (s, 1H, Ar-H), 7.86 (d, 2H, J = 9.0 Hz, Ar-H), 7.77 (d, 2H,
J = 6.0 Hz, Ar-H). 13C NMR (100 MHz, DMSO‑d6) δ: 161.94, 150.39,
148.24, 146.57, 140.17, 135.89, 133.53, 130.52, 124.55, 121.58,
121.14. HRMS (ESI) m/z calcd for C13H10N4O3 [M + H]: 271.2435,
found: 271.0826.
4.2.14. Nˈ-(pyridine-2-carboxaldehyde)isonicotinoylhydrazone (1n)
white solid, yield 60%, 95% HPLC purity, MP: 160–161 °C. 1H NMR
(400 MHz, DMSO‑d6) δ: 12.27 (s, 1H, NH), 8.82 (d, 2H, J = 1,4 and 4,5
Hz, Ar-H), 8.65 (d, 1H, J = 4,7 Hz, Ar-H), 8.50 (s, 1H, CH = N), 8.03
(d, 1H, J = 7.9 Hz, Ar-H), 7.94 (td, 1H, Ar-H), 7.86 (d, 2H, J = 4.5 Hz,
6

D.C. Santos, et al.
Bioorganic&MedicinalChemistry28(2020)115470
addition of FeCl2 and ZnCl2 (20 μM in MeOH). The UV spectra was
recorded ranging from 403 to 371 nm after incubating for 2 h at room
temperature and treated by numerical subtraction of metal salts and
tested compounds at corresponding concentrations, plotted versus the
mole fractions of tested compounds.
Ar-H); 7.47 (m, 1H, Ar-H). 13C NMR (100 MHz, DMSO‑d6) δ: 161.94,
152.96, 150.41, 149.63, 149.24, 140.25, 136.99, 124.70, 121.59,
120.13. HRMS (ESI) m/z calcd for C12H10N4O [M + H]: 227.234,
found: 227.0935.
4.2.15. N’-(1-H-indol-3lmethylene) hydrazide (1o)
white solid, yield 95%, 98.5% HPLC purity, MP: 241–244 °C. 1H
NMR (500 MHz, DMSO‑d6) δ: 11.76 (s, 1H, NH), 11.65 (s, 1H, NH),
8.79 (d, 2H, J = 6.0 Hz, Ar-H), 8.64 (s, 1H, CH = N), 8.31 (d, 1H,
J = 7.5 Hz, Ar-H), 7.88 (d, 1H, J = 2.5 Hz, Ar-H), 7.85 (d, 2H,
J = 5.5 Hz, Ar-H); 7.47 (d, 1H, J = 8.0 Hz, Ar-H), 7.24 (dt, 2H, Ar-H).
HRMS (ESI) m/z calcd for C15H12N4O [M + H]: 265.2820, found:
265.1091.
4.5. Molecular docking studies
The acylhydrazone derivatives 1a-o were built in Avogadro
v.1.1.132 and molecules were energy-optimized using the PM3 method
in Gaussian 03 W33. GOLD software v.6.0.34 was used in docking stu-
dies. Ten GA runs were performed for each molecule. The parameters
for docking have been determined by pose analysis obtained by re-
docking of the co-crystallized ligands with MPO and AChE, available in
the Protein Data Bank35 with ID codes 4C1M36 and 4M0E37 respectively
(both at a 2.0 Å resolution), using Goldscore, Chemscore and ChemPLP
scoring functions. ChemPLP function with a 10 Å binding site radius
exhibited an RMSD value of 0.43 Å for MPO and 0.22 Å for AChE,
reflecting the capacity of the software to find the binding poses of the
co-crystallized ligands and, therefore, was chosen as the best scoring
function for this study. The amino acid residues selected as the binding
site to perform docking studies were determined by a distance of 10 Å
from Ne2 (ID 745) and O (ID 2239) of the side chains of the catalytic
residues His95 and Ser293 for MPO and AChE, respectively. Water
molecules contained in the PDB file have been removed. Visual in-
spection of intermolecular interactions was performed with Pymol,
v.1.8 and Discovery Studio, v.16.1.0.15350.
4.3. Biological assays
4.3.1. AChE inhibition experiments
AChE inhibitory activity was performed in 96-well plates using a
spectrophotometric method.25 The compounds 1a-o were dissolved in
DMSO. In a final volume of 250 µL, Ellman’s reagent (acid 5′,5′-dithio-
bis-(2-nitrobenzoate) (60 μM), phosphate buffer pH 8 0.05 M, inhibitors
(100 μM), and AChE from E. electricus (0.25 U/mL) were incubated by
30 min, then acetylthiocholine iodide (1.5 mM) was added to start the
reaction. The assay was carried out in three experiments in triplicate
and absorbance was measured at 412 nm by 10 min. The percentage of
inhibition was calculated by comparing rates of enzyme reaction of
samples in relation to control group. Rivastigmine was used as the
positive control.
Declaration of Competing Interest
4.3.2. MPO inhibition experiments
MPO enzyme was obtained from rat bone marrow homogenate su-
pernatant.24 The determination of MPO inhibitory capacity of the
compounds was achieved by the measurement of HOCl production by
monitoring formation of N-mono-chlorotaurine.25 Compounds 1a-o
were dissolved in DMSO. The experiment was performed in a 96-well
plate with a final volume of 125 μL, which included taurine (10 μM),
NaCl (100 μM), bone marrow homogenate supernatant, phosphate
buffer (0.05 M, pH 7.4) and inhibitors (0.01–30 μM). The reaction was
initiated by addition of H2O2 (100 μM) and, after 20 min, it was stopped
by catalase addition (40 μg/mL). Then, 25 μL of a colorimetric reagent
(3,3′,5,5′-Tetramethylbenzidine (TMB)/potassium iodide 100 μM) was
added to detect N-mono-chlorotaurine formed. After the appearance of
blue color, 100 μL of H2SO4 (1 M) was added and the oxidized TMB was
measured at 450 nm.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The authors would like to thank the following Brazilian funding
agencies for financial support: CAPES, CNPq (fellowship, Nelilma C.
Romeiro) and FAPERJ. The authors thank also FIOCRUZ for the HRMS
and NMR spectra data.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115470.
4.3.3. Antioxidant activity assay
The DPPH radical scavenging effect was evaluated using the method
reported earlier by Blois with suitable modifications.31 The reduction of
DPPH was followed by monitoring its decrease in absorbance at a
characteristic wavelength during the reaction. In its radical form, DPPH
absorbs at 517 nm, but after reduction by an antioxidant or a radical
species, the absorption peak disappears. One hundred microliters of
derivatives methanol solution (3–600 μM) was added to 100 μL of
DPPH methanol solution (600 μM). After gentle mixing and allowing it
to stand at room temperature for 30 min the remaining radical DPPH
was measured at 517 nm. Quercetin was used as the positive control.
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