Cinnamoyl-N-Acylhydrazone-Donepezil Hybrids: Synthesis and Evaluation of Novel Multifunctional Ligands Against Neurodegenerative Diseases

Neurochemical Research

https://doi.org/10.1007/s11064-020-03148-2

ORIGINAL PAPER

Cinnamoyl‑N‑Acylhydrazone‑Donepezil Hybrids: Synthesis

and Evaluation of Novel Multifunctional Ligands Against

Neurodegenerative Diseases

Cindy Juliet Cristancho Ortiz¹· Caio Miranda Damasio¹· Letizia Pruccoli²· Nathália Fonseca Nadur³·

Luciana Luiza de Azevedo³· Isabella Alvim Guedes⁴· Laurent Emmanuel Dardenne⁴· Arthur Eugen Kümmerle³·

Andrea Tarozzi²· Claudio Viegas Jr.¹

Received: 23 July 2020 / Revised: 25 September 2020 / Accepted: 7 October 2020

Abstract

A new series of ten multifunctional Cinnamoyl-N-acylhydrazone-donepezil hybrids was synthesized and evaluated as multi-

functional ligands against neurodegenerative diseases. The molecular hybridization approach was based on the combination

of 1-benzyl-4-piperidine fragment from the anti-Alzheimer AChE inhibitor donepezil (1) and the cinnamoyl subunit from

curcumin (2), a natural product with remarkable antioxidant, neuroprotective and anti-infammatory properties, using a

N-acylhydrazone fragment as a spacer subunit. Compounds 4a and 4d showed moderate inhibitory activity towards AChE

with IC₅₀values of 13.04 and 9.1 µM, respectively. In addition, compound 4a and 4d showed a similar predicted binding

mode to that observed for donepezil in the molecular docking studies. On the other hand, compounds 4a and 4c exhibited

signifcant radical scavenging activity, showing the best efects on the DPPH test and also exhibited a signifcant protective

neuronal cell viability exposed to t-BuOOH and against 6-OHDA insult to prevent the oxidative stress in Parkinson’s disease.

Similarly, compound 4c was capable to prevent the ROS formation, with indirect antioxidant activity increasing intracel-

lular GSH levels and the ability to counteract the neurotoxicity induced by both OAβ1-42 and 3-NP. In addition, ADMET in

silico prediction indicated that both compounds 4a and 4c did not show relevant toxic efects. Due to their above-mentioned

biological properties, compounds 4a and 4c could be explored as lead compounds in search of more efective and low toxic

small molecules with multiple neuroprotective efects for neurodegenerative diseases.

Graphic Abstract

Electronic supplementary material The online version of this

article (https://doi.org/10.1007/s11064-020-03148-2) contains

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Keywords Cinnamoyl-N-acylhydrazone-donepezil hybrids MTDLs · Multifunctional ligands · Neurodegenerative

diseases · Molecular hybridization

lack in efcacy and the multifactorial complexity related

to the pathophysiology of NDs, it is urgent the adoption

of other therapeutic approaches such as multifunctional or

multi-target directed ligands (MTDLs) strategy [9]. Based

on polypharmacology concept, the MTDLs approach advo-

cates that better therapeutical results could be achieved by

a single molecule that could concomitantly modulate more

than one molecular target associated with NDs pathophysi-

ology. Despite better pharmacodynamics, the use of mul-

tifunctional drugs could avoid adverse efects, deleterious

drug–drug interactions, and metabolic stress. The rationale

behind the structural design of these new multifunctional

chemical entities uses the molecular hybridization as its

main tool, combining diferent structural patterns or struc-

tural fragments from two or more known bioactive molecules

into a new single molecular skeleton capable of reproducing

the original properties of the prototype molecules [10]. The

MTDLs approach represents an interesting strategy for the

development of drug candidates that have antioxidant and

neuroprotective efects, enzyme inhibitory properties and

can efectively penetrate the CNS barrier and restore cogni-

tion, memory functions and brain homeostasis [11].

Introduction

Neurodegenerative diseases (NDs), including Alzheimer’s

disease (AD), Parkinson’s disease (PD), Huntington’s dis-

ease (HD), and amyotrophic lateral sclerosis, are a hetero-

geneous group of neurological disorders caused by the pro-

gressive death of neurons in diferent regions of the nervous

system, leading to locomotion and behavior impairments,

cognitive decline and dementia, that afect millions of peo-

ple worldwide. In particular, AD is characterized by the for-

mation of senile plaques and neurofbrillary tangles in the

cerebral cortex as well as neuron and synapse loss. Similarly,

PD is associated with dopaminergic neuronal death and for-

mation of Lewy bodies due to the deposition of the protein

α-synuclein that leads to dysfunction in the regulation of

major brain structures involved in the movement control.

Although the available treatments may help to alleviate pro-

gression and some symptoms associated with NDs, such as

donepezil, rivastigmine, galanthamine and memantine for

AD [1, 2] or

Lev odopa to PD [3], there is currently no way to efective

blockade disease progression and cure [4 ]. Ageing is the

major risk factor for neurodegeneration [5]. Nonetheless,

there are some intrinsic factors such as genetic, epigenetic

and brain damage associated with neuroinfammation, oxi-

dative stress, as well as lifestyle factors, including diets rich

in sugar and fat foods, alcohol and tobacco dependence [6].

Neurodegeneration is also correlated with an imbalanced

production of free radicals from enzyme activity, mitochon-

drial impairment, and decreased activity of the antioxidant

system. The excessive production of reactive oxygen spe-

cies (ROS) causes oxidative stress, increasing the risk of

developing NDs. Such radical species may react with dif-

ferent functional groups from endogen molecules, including

bases in nucleic acids, amino acid side chains in proteins

and double bonds in unsaturated fatty acids, which can dam-

age DNA, RNA, proteins and lipids [7]. Antioxidant com-

pounds such as curcumin can decrease the oxidative damage

directly, via reacting with free radicals, or indirectly, exert-

ing their antioxidant efects by inducing cytoprotective phase

2 genes and improving intracellular antioxidant enzymes [8].

Currently, the available treatments for NDs, especially

for AD and PD, are only restricted to symptoms alleviation

and curb disease progression and severity, with drugs act-

ing specifcally over single molecular targets. In the case of

AD, galanthamine, donepezil and rivastigmine are AChE

inhibitors and memantine acts as glutamate-related NMDA

receptor antagonist, whereas PD has been treated by tenta-

tive restoring of the dopaminergic system. Considering the

In a previous work [12], we reported the synthesis

and biological evaluation of a series of feruloyl-done-

pezil hybrid compounds designed as multitarget drug

candidates for AD treatment. Some of those compounds

showed significant neuroprotective properties in human

neuronal cells against oxidative damage, anti-inflamma-

tory properties, and potent AChE inhibitory activity. On

the other hand, the use of N-acylhydrazones (NAHs) and

their derivatives has been reported in the literature as

important privileged structures, with important biologi-

cal potential in bioactive ligands of different pharmaco-

logical profiles [13]. NAHs are characterized by being

able to contribute to molecular recognition for a wide

number of biological targets, whose structural modifica-

tion of their substituents can result in compounds with

great biological potential. In addition, NAHs could also

contribute for improving pharmacokinetic properties due

to its higher polarity, with donor and acceptor hydrogen

bond sites, allowing additional molecular interactions

[14]. Considering the promising results evidenced by our

group in earlier studies, exploring the pharmacophoric

contribution of the NAH substructure, the anticholinest-

erase properties of the N-benzylpiperidine fragment from

donepezil (1), we designed another series of donepezil-

based N-acylhydrazone hybrids (4a-j, Fig. 1) based on

the combination of 1-benzyl-4-piperidine system and the

cinnamoyl subunit from curcumin (2) and ferulic acid

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Fig. 1 Design of a new series

of cinnamic-N-acylhydrazone-

donepezil hybrids (4a-j)

Fig. 2 Synthetic route for the

preparation of the cinnamoyl-

N-acylhydrazone-donepezil

hybrids 4a-j

(3), two known natural products with antioxidant, neu-

roprotective and anti-inflammatory properties, using a

N-acylhydrazone subunit as spacer. By this proposal, the

N-benzyl-4-piperidine subunit was elected as a suitable

mimic of the AChE inhibitor pharmacophore N-benzylpi-

peridine fragment from 1, conserving the Nitrogen atom

as a protonable site and inserting a relative conforma-

tional restriction imposed by the exocyclic double bond

at the hydrazone functionality. Moreover, different sub-

stituents at the cinnamoyl fragment could be interesting

in a SAR study and their role in the desired antioxidant

and neuroprotective properties, leading to the identifica-

tion of new multifunctional ligands with an innovative

scaffold, suitable for pharmaceutical development for AD

treatment.

Table 1 Experimental data for compounds 4a-j

Compound R₁R₂

Yield (%) MP (°C)^†Purity (%)

OCH₃46

263 (4a)

264 (4b)

265 (4c)

266 (4d)

267 (4e)

268 (4f)

269 (4 g)

270 (4 h)

OH

OCH₃OH

OH OH

203

192

140

185

>300

226

188

90

100

37

19

100

98,81

100

OCH₃OCH₃55

OCH₃

OH

CF₃

H

29

39

57

56

99,33

100

99,99

100

271 (4i)

272 (4j)

Cl

H

73

11

209

183

100

The synthesis of the target-compounds 4a-j was based

on the preparation of the key-hydrazide intermediates 6a-

j from cinnamic acid derivatives 5a-j (Fig. 2). The start-

ing carboxylic acids were reacted with hydrazine hydrate

†: Melting point

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in the presence of HOBT and EDC to provide the desired

compounds 4a-j. Then, these hydrazide intermediates were

coupled to 1-benzyl-4-piperidone, leading to the desired cin-

namoyl-donepezil hybrids 4a-j in global yields of 11–73%

(Table 1). All compounds were characterized by IR, NMR

and HRMS techniques. The purity of compounds was deter-

mined by HPLC.

BuChE inhibitor, followed by PQM-271 (4i) and PQM-268

(4f), with IC₅₀values of 14.44 and 14.42 µM, respectively.

It is interesting to note that only compound PQM-271 (4i)

showed signifcant inhibition of both enzymes, whereas the

most potent and selective AChE inhibitors did not showed

activity against BuChE and vice versa. This selective profle

in ChE inhibition could be useful in the development of drug

candidates for AD treatment, since in the most advanced

stages of AD, ACh is mainly hydrolyzed by BuChE, that

is indicative of an increase in BuChE activity, leading to a

decrease in ACh level in the late stage of AD. Hence, the

development of selective BuChE inhibitors, such as PQM-

272 (4j) may represent an interesting therapeutic strategy

[16]. In fact, almost all drugs approved for AD manage-

ment worldwide are selective AChE inhibitors, with riv-

astigmine being the only one dual AChE/BuChE inhibitor

currently FDA-approved for the treatment of mild, moder-

ate and severe AD and mild to moderate PD, demonstrating

improvement in the cognitive condition of patients [17]. In

contrast, dual AChE/BuChE inhibitors such as PQM-271

(4i) may also be considered as a therapeutic advantage for

cortical dementias, such as AD and PD, benefting cogni-

tion and treating behavioral symptoms. A SAR analysis evi-

denced that most potent AChE inhibitors were compounds

with 3,4-di-oxygenated substituents at the cinnamoyl subu-

nit, whereas the best BuChE inhibitors have no O-substituent

on that fragment. Moreover, the replacement of the 3,4-di-

hydroxyl groups in PQM-265 (4c) for a 3,4-di-methoxyl

groups in PQM-266 (4d) signifcantly increased the inhibi-

tory potency and selectivity against AChE. However, the

infuence of the substituents at the cinnamoyl fragment

on BuChE activity was the opposite, suggesting that the

absence of substituents increases both inhibition potency

In Vitro Inhibition of AChE and BuChE

Cholinesterase (ChE) inhibitors are efective in improv-

ing behavior, memory, well-being and reducing cognitive

decline in patients with dementia. Thus, the modifed Ell-

man’s method [15] was used to determine the inhibitory

profle of the cinnamoyl-N-acylhydrazone-donepezil hybrids

against AChE and BuChE enzymes. Preliminary results

showed that compounds 4a-j were capable to inhibit AChE

in a range of 47–76% at 30 µM. Additionally, at the same

concentration, some compounds could also inhibit BuChE

in a range of 62–82% (Table 2).

Most of the target-compounds showed moderate inhibi-

tory activity towards AChE or BuChE, indicating that the

introduction of N-benzyl-piperidine fragment was deter-

minant for inhibition of ChEs, which results are shown in

Table 2. Compounds with ChE inhibition higher than 45%

have their IC₅₀values determined, highlighting compound

PQM-266 (4d) as the most potent and selective AChE inhib-

itor with IC₅₀=9.1 µM, followed by compounds PQM-263

(4a), PQM-264 (4b) and PQM-270 (4 h), with IC₅₀values

of 13.04, 17.04 and 19.44 µM, respectively, with no sig-

nifcant activity over BuChE. Conversely, compound PQM-

272 (4j) with IC₅₀=11.94 µM showed to be the most potent

Table 2 Experimental data of in vitro inhibition of AChE and BuChE, DPPH radical scavenging activity and cell viability for compounds 4a-j

Compounds

EeAChE

eqBuChE

% Inhibition of

DPPH radical

(at 80 µM)

% cell viabil-

ity (at 80 µM)^†

% inhibition (at IC₅₀SD (30 µM)

30 µM)

% Inhibition (at IC₅₀SD (30 µM)

30 µM)

PQM-263 (4a)

PQM-264 (4b)

PQM-265 (4c)

PQM-266 (4d)

PQM-267 (4e)

PQM-268 (4f)

PQM-269 (4 g)

PQM-270 (4 h)

PQM-271 (4i)

PQM-272 (4j)

47.1

55.9

43.0

75.6

10.5

32.5

49.4

47.3

58.4

23.2

13.04 0.41

17.40 0.32

NA

9.10 0.68

NA

29.3

23.9

62.0

3.4

NA

21.99 1.58

NA

14.42 0.48

NA

13.44 0.83

11.94 0.65

53.37

6.84

45.05

− 1.41

2.59

99.88

92.43

63.85

80.70

114.31

73.59

127.28

92.48

81.84

77.33

1.8

NA

69.9

43.0

27.5

64.5

82.5

2.33

30.07 0.63

19.44 1.28

31.52 2.28

NA

13.70

− 3.05

8.78

− 2.56

IC₅₀: compound concentration required to produce the 50% of inhibition, data were shown in mean SD of triplicate of independent experiments

NA No active, inhibition 45% at 30 µM

^aCell viability percentage at the maximum concentration by MTT assay

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Fig. 3 Lineweaver-Burk plots for a AChE inhibition by PQM-263 (4a) and b BuChE inhibition by compound PQM-272 (4j)

Table 3 Kinetic parameters

of compound PQM-263 (4a)

against AChE

Concentration V_maxSD^a(µM/min)

(µM)

K_mSD^b(µM)

K_i(µM) SD^c

19.04 0.823

K_i’(µM) SD^d

40.31 1.628

0

7.15 0.495

5.88 0.207

6.39 0.276

133.90 1.41

148.35 2.47

228.00 13.0

11

15

^aMaximum velocity of the enzyme

^bMichaelis constant

^cCompetitive constant

^dNon-competitive constant; data were shown in mean SD of triplicate of independent experiments

Table 4 Kinetic parameters

of compound PQM-272 (4j)

against BuChE

Concentration V_maxSD^a(µM/min)

(µM)

K_mSD^b(µM)

K_i(µM) SD^c

1.62 0.020

K_i’(µM) SD^d

3.35 0.039

0

16.09 0.292

5.17 0.288

3.14 0.136

152.93 0.28

191.75 3.18

247.75 1.34

10

13

^aMaximum velocity of the enzyme

^bMichaelis constant

^cCompetitive constant

^dNon-competitive constant; data were shown in mean SD of triplicate of independent experiments

and selectivity for BuChE as evidenced by compound PQM-

272 (4j), which has no substituents on the aromatic ring.

In order to gain insight into the mechanism of AChE and

BuChE inhibition, the compounds PQM-263 (4a) and PQM-

272 (4j) were selected for kinetic studies for the AChE and

BuChE, respectively. The Lineweaver–Burk plots (Fig. 3) evi-

denced a mixed-type mechanism of inhibition by both com-

pounds tested, suggesting that the inhibitors are capable of

binding both to the catalytic (competitive inhibition) and to the

allosteric (non-competitive inhibition) sites of both enzymes.

The inhibition constants were calculated, and kinetic param-

eters are shown in Tables 3 and 4.

Table 5 Docking results for the compounds 4a-4j against AChE

(PDB code 4EY7) and BuChE (PDB code 6I0C)

Compound

AChE score (kcal/

mol)

BuChE score

(kcal/mol)

PQM-263 (4a)

PQM− 264 (4b)

PQM− 265 (4c)

PQM− 266 (4d)

PQM− 267 (4e)

PQM− 268 (4f)

PQM-269 (4 g)

PQM− 270 (4 h)

PQM− 271 (4i)

PQM− 272 (4j)

Donepezil

− 9.7

− 8.9

− 8.8

− 10.26

− 8.8

− 11.0

− 9.6

− 9.2

− 9.1

− 8.5

− 12.2

− 7.3

− 7.0

− 7.3

− 7.0

− 7.1

− 7.2

− 6.6

− 6.9

− 6.8

− 8.1

− 7.6

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between the methoxy at the meta-position with the Trp286

and Tyr72 side chains from the PAS (Fig. 4b, PQM-266 not

shown). According to the docking results, the dimethoxy-

benzene and hydroxymethoxybenzene groups from these

compounds are not able to perform the optimal stacking

interactions with Trp279 observed for donepezil, probably

due to the slightly longer linker moiety of the NAH deriva-

tive. Furthermore, the methoxy group of these compounds

are more exposed to the solvent than in the donepezil exper-

imental binding mode. However, since the PAS region is

highly fexible, it is possible that the AChE can adjust this

region to accommodate the compounds PQM-263 (4a) and

PQM-266 (4d) through an induced-ft mechanism, provid-

ing optimal hydrophobic and stacking interaction with PAS.

For the BuChE, the compound with the best predicted

afnity was PQM-272 (4j), which was also experimentally

active against the enzyme. The superposition of the pre-

dicted binding mode PQM-272 with the cognate ligand of

the complex 6I0C exhibited some similar interactions that

may justify its potency, being characterized by: (i) π-stacking

Molecular Docking Study with AChE

The docking results for the AChE and BuChE are shown in

Table 5. The compounds PQM-268 (4f), PQM-266 (4d) and

PQM-263 (4a) were predicted as the most potent inhibitors

against AChE, whereas the compound 4j was the only com-

pound predicted to inhibit the BuChE with binding afnity

better than – 8 kcal/mol. From these results, only the com-

pound PQM-268 (4f) did not exhibit in vitro activity against

the respective enzyme.

The predicted binding modes for PQM-263 (4a) and

PQM-266 (4d) are similar to that observed for donepezil

(Fig. 4a), being characterized by: (i) π-stacking interaction

between the phenyl ring with the Trp86 indole ring, (ii)

cation-π interaction of the piperidine group with the Tyr337

side chain, (iii) hydrogen bond between the carbonyl oxy-

gen of the NAH group with the Tyr121 side chain, and (iii)

hydrophobic interactions between the 1,2-dimethoxyben-

zene and the PAS region, and (iv) hydrophobic interactions

Fig. 4 Top-energy predicted binding mode of a PQM-263 (4a) super-

imposed with donepezil, b PQM-263 (4a) interacting with the AChE

binding site (PDB code 4EY7), c cinnamoyl derivatives superim-

posed with the co-crystallized ligand of the BuChE structure 6I0C,

and d PQM-272 (4j) interacting with the BuChE binding site (PDB

code 6I0C). Hydrogen bonds are represented as yellow dashes and

BuChE Trp231 is highlighted as dots. The co-crystallized ligands are

colored green, PQM-263 (4a) is colored cyan and PQM-272 (4j) is

colored pink

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interaction between the phenyl group of the N-benzyl-piper-

idine moiety with the Trp82 indole ring, (ii) hydrogen bond

between the positively charged nitrogen from the piperidine

ring with the Try332 side chain, and (iii) T-stacking interac-

tion between the phenyl ring from the cinnamoyl fragment

and the Trp231 side chain (Fig. 4c, d). It is important to

highlight that the Trp231 is only accessible in the BuChE

due to the substitution of Phe295 in AChE to Val288, pro-

viding enough room to aromatic fragments interacting with

this hydrophobic pocket [16, 18–22]. This hypothesis is rein-

forced by the SAR of our com pounds, which demonstrated

that substitutions at the phenyl ring from the cinnamoyl frag-

ment led to reduced potency and selectivity.

that can be stabilized through conjugation extension. When

the radical is located at the meta-position, the oxygen atom

is unable to share the charge, afecting antioxidant capacity,

which could be evidenced in the mono-hydroxycinnamic

derivative PQM-268 (4f) that exhibited very weak antiradi-

cal activity, but methoxy group improved t he activity as evi-

denced for compound PQM-263 (4a) [12, 26].

Evaluation of Cytotoxicity and Antioxidant

Properties by Cell‑Based Assay

The new compounds have been designed to have a multi-

functional profle using fragments of donepezil and cur-

cumin. By contrast to curcumin, donepezil is commercial

drug with a known pharmacological mechanism. Therefore,

we used donepezil in various experiments with cells. The

donepezil did not show the ability to counteract the oxidative

stress confrming its selective action only on acetylcholinest-

erase activity. We added this information in (Figs. 5 and 6).

At this point, with the current level of drug discovery, we

evaluated only some aspects of pharmacokinetic and toxicity

by ADME studies. The previous information was reported

in Table 6.

In fact, only the compound PQM-272 (4j) from the

cinnamoyl derivatives was able to interact with Trp231

(Fig. 4c). These fndings provide useful insights for further

molecular optimizations of the cinnamoyl derivatives to

obtain more potent and selective compounds.

In Vitro Antioxidant Activity Evaluation

Oxidative stress is a common feature in NDs, which

demands an abnormal increase in free radical generation in

the brain, along with cytotoxicity, leading to mitochondrial

dysfunction, neuronal cell death and altered synaptic func-

tion [23]. There is a number of evidences in the literature

that curcumin and its derivatives, such as ferulic acid, pro-

tect neurons from injury by preventing free radical-mediated

neuroinfammation implicated in the pat hology of NDs and

contribute to memory improvement [24, 25].

Subsequently, we evaluated the neurotoxicity of all target-

compounds in human neuronal (SH-SY5Y) cells by MTT

assay. After testing compounds 4a-j at eight diferent con-

centrations from 2.5 to 80 µM, in triplicate, no signifcant

cytotoxicity was observed at the maximum concentration

of 80 µM (Table 2). In parallel, we selected the best dual

ChE inhibitors and antioxidants PQM-263 (4a), PQM-264

(4b), PQM-265 (4c) and PQM-266 (4d) for evaluating their

ability to inhibit the intracellular ROS formation induced

by t-BOOH in SH-SY5Y cells. Initially, the evaluation was

performed by a co-treatment approach, that is, the target-

substances and t-BOOH were administered at the same

time. In this regard, this approach allows to evaluate the

direct antioxidant activity of the compounds in SH-SY5Y

cells. In Fig. 5a, the results are shown in percentage inhi-

bition of ROS formation in relation to exposed cells only

with t-BOOH, and except for 4d, all other target-compounds

showed signifcant antioxidant efects against oxidative dam-

age induced by t-BOOH at 2.5, 5 and 10 μM. These results

corroborated the signifcant radical scavenging activity of

PQM-265 (4c).

Considering the oxidative damage associated with NDs,

compounds 4a-j were evaluated for their in vitro antioxi-

dant activity by DPPH assay, in diferent concentrations

(1.56–200 µM) and in triplicate. DPPH (2,2-diphenyl-1-pic-

rylhydrazyl) radicals can be used in preliminary screening

of scavenging reactive oxygen species (ROS). The radical

scavenging capacity was represented as a percentage of the

DPPH radical scavenging activity at 80 µM of concentration

and the results are shown in Table 2. The ferulic acid-based

hybrid PQM-263 (4a) was more efective than the iso-ferulic

derivative PQM-264 (4b) and the strongest antioxidant in the

series, inhibiting 53.37% of DPPH radicals at 80 µM, fol-

lowed by PQM-265 (4c) that inhibited 45.05% of DPPH rad-

icals at the same concentration. These results evidenced that

all compounds without a phenolic hydroxyl group lack in

antioxidant activity, which indicates that radical scavenging

activity depends on the number and position of hydroxy and

methoxy substituents attached to the aromatic ring and that

the para-hydr oxy substituent is determinant for antioxidant

efcacy [12, 26]. Therefore, the ferulic acid pattern is the

most adequate pattern for cinnamic the strongest antioxidant

ability, probably due to the formation of a phenolic radical

Interestingly, the compound PQM-265 (4c) also showed

non-concentration-dependent antioxidant effects with a

considerable decrease of ROS formation at 2.5 µM. Similar

behavior was observed for 4b, which showed good activ-

ity against t-BuOOH in physiological—water medium, but

with no signifcant activity against DPPH radical in etha-

nol solution. This suggests that the reaction environment

conditions the ability of 4b to counteract radical species.

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Fig. 6 Efects of compounds PQM-263 (4a), PQM-264 (4b), PQM-

265 (4c) and PQM-266 (4d) on GSH levels in SH-SY5Y cells.

Cells were incubated with the studied compounds (2.5–10 µM) for

24 h. At the end of incubation, GSH levels were detected using the

fuorescent probe MCB, as described in the experimental section.

Data are expressed as percentage increase of GSH and reported

as mean SEM of three independent experiments (*p<0.05 and

**p<0.01 versus untreated cells at one-way ANOVA with Dunnett

post hoc test)

pre-treatment approach. By this experimental approach, the

cells are chronically treated with the test-compound for 24 h

and then exposed to t-BuOOH for 30 min. In particular, by

this approach it is possible to verify the ability of the com-

pounds to enter into the cells and increase the endogenous

antioxidant defenses (i.e. indirect antioxidant activity) [27].

As reported in Fig. 5b both PQM-263 (4a) and PQM-265

(4c) were capable to prevent the ROS formation, with the

maximum indirect antioxidant activity of 44 and 75% at

10 µM, respectively.

Fig. 5 Ability of compounds PQM-263 (4a), PQM-264 (4b), PQM-

265 (4c) and PQM-266 (4d), to counteract (a, direct antioxidant

activity) or prevent (b, indirect antioxidant activity) the intracellular

ROS formation induced by t-BOOH in SH-SY5Y cells. a Cells were

incubated with the studied compounds (2.5–10 µM) and t-BuOOH

(100 µM) for 30 min; b Cells were incubated with the studied com-

pounds (2.5–10 µM) for 24 h and then treated with t-BuOOH

(100 µM) for 30 min. At the end of incubation, intracellular ROS

formation was detected using the fuorescent probe H₂DCF-DA, as

described in the experimental section. Data are expressed in terms

of % of ROS inhibition and reported as mean SEM of three inde-

pendent experiments (*p<0.05, **p<0.01 and ***p<0.001 versus

untreated cells at one-way ANOVA with Dunnett post hoc test)

By recording the reduction of ROS levels following a

long treatment of the PQM-263 (4a) and PQM-265 (4c)

before the treatment with t-BuOOH, we hypothesized that

the antioxidant efect might likely result from an increase in

levels of glutathione (GSH), an endogenous antioxidant for

which key roles in protecting cells against oxidative stress

damage have been recently demonstrated [28]. Thus, the

intracellular GSH levels were analysed by employing the

same experimental conditions used to evaluate the indirect

antioxidant efect. Treatment of SH-SY5Y cells with PQM-

263 (4a), PQM-264 (4b), PQM-265 (4c) and PQM-266 (4d)

showed an increase in intracellular GSH levels at 10 µM

only for 4a (34%) and 4c (33%) (Fig. 6).

Finally, compound PQM-263 (4a), that showed the best

efect in the DPPH test, also exhibited a signifcant anti-

oxidant activity at neuronal cell level exposed to t-BuOOH

using co-treatment test. These results reinforce our hypoth-

esis that the ferulic acid pattern of compound 4a positively

contributes to neutralize the ROS formation and to protect

the SH-SY5Y cell line from the oxidative damage. Then,

the ability of these substances to prevent the formation of

ROS induced by t-BuOOH was evaluated according to the

These outcomes led us to choose compound 4a and 4c

for further evaluation of its ability to activate Nrf2 bind-

ing to ARE at the nuclear level, an essential event for the

induction of GSH production. The Nrf2 is the main regulator

of phase II antioxidant response that is held in the cytosol

by the repressor protein Kelch ECH associated protein 1

(Keap1) [27]. In the presence of oxidative stress, Nrf2 is

released from keap1 and translocated into the nucleus, and

1 3

Neurochemical Research

Table 6 ADMET predictions

of the 4a-j derivatives and the

reference compound donepezil

in the neutral form performed

with the QikProp tool from

Maestro

Compound

Ro5 MW HBD^aHBA^aQPlogPo/w HOA^bQPlogS QPlogHERG QPPMDCK

PQM-263 (4a)

PQM-264 (4b)

PQM-265 (4c)

PQM-266 (4d)

PQM-267 (4e)

PQM-268 (4f)

PQM-269 (4 g)

PQM-270 (4 h)

PQM-271 (4i)

PQM-272 (4j)

donepezil

0

1

0

1

0

379

365

393

363

349

401

377

368

333

379

3

4

2

3

2

1

4

3

2

4

2

3

3.9

3.0

4.7

4.6

3.7

5.5

4.0

5.0

4.5

4.1

87

74

100

86

93

97

90

100

− 5.2

− 5.1

− 4.5

− 5.7

− 5.4

− 4.9

− 6.7

− 4.7

− 6.0

− 5.2

− 4.2

− 7.6

− 7.5

− 7.6

− 7.7

− 7.3

− 7.7

− 7.8

− 6.4

> -5

55.7

59.4

18.0

198.8

199.0

55.2

879.7

199.2

492.6

199.8

439.6

> 500 great

Reference value 0–2 <500 ≤ 5

≤ 10 ≤ 5

> 80 − 6.5 -

0.5

^aProperties obtained for the protonated state (pH 7.0)

^bGiven in percentage

once inside, it binds to the electrophile response element

sequences to stimulate the expression of phase II antioxi-

dant and anti-infammatory genes [29]. The increased Nrf2

protein level in the cell nucleus by Keap1-dependent Nrf2

activation requires strong electrophiles disrupting of the

Keap1-Nrf2 complex by modifying Keap1 at cysteine resi-

dues through the Michael reaction [30]. However, our results

did not show the ability of both 4a and 4c in Nrf2 activation

at 10 μM (data not shown). This result suggests that other

mechanisms may be responsible for the induction of GSH

expression, once the rate of glutathione biosynthesis is con-

trolled by γ-glutamylcysteine synthetase, a typical cytopro-

tective enzyme that is upregulated by inducers coordinately

with many other cytoprotective phase 2 genes [8].

In Vitro Model of AD

Soluble Aβ_1-42oligomers (OAβ_1-42) are responsible for neu-

rotoxic efects, among them neuronal cells death, and repre-

sents a key event in AD pathogenesis [32]. SH-SY5Y cells

were incubated with OAβ_1-42(10 μM) and compounds 4a, 4b

and 4c (10 μM) for 4 h (Fig. 7a). Compound 4c, but not 4a

and 4b, signifcantly reduced OAβ_1-42-induced neurotoxicity.

In Vitro Model of PD

The neurotoxin 6-hydroxydopamine (6-OHDA), by enhanc-

ing oxidative damage and neuroinfammation, provides an

in vitro lesion model suitable for assessing the neuroprotec-

tive potential of PD therapeutics [33]. SH-SY5Y cells was

treated with 6-OHDA (100 μM) and compounds 4a, 4b and

4c (10 μM) for 2 h and starved in complete medium for 22 h

(Fig. 7b). The results showed that compound 4a, but not

4b and 4c, the ability to signifcantly counteract 6-OHDA-

induced neurotoxicity.

Evaluation of Neuroprotective Activity

Profle by Cell‑Based Assay

The neuroprotective ability of compounds in DNs is gener-

ally linked with their antioxidant activity. The most impor-

tant pathological mechanisms in the brain injury process

include oxidative stress due to an imbalanced and excessive

production of ROS that damage diferent cell structures,

induce neuroinfammation and lead to neuronal death. To

avoid the toxic efects of these reactive species, antioxidant

compounds are thought to be used for controlling and neu-

tralizing excessive free radicals and, in turn, protect diferent

neuronal structures [31].

In Vitro Model of HD

The neurotoxin 3-nitropropionic acid (3-NP) generates in

animals behavioural, biochemical and morphologic changes

similar to those occurring in HD, through several mecha-

nisms including the irreversible inhibition of succinate dehy-

drogenase at mitochondrial level [34]. SH-SY5Y cells were

incubated with 10 μM of PQM-263 (4a), PQM-264 (4b)

and PQM-265 (4c) and 5 mM of 3-NP for 24 h (Fig. 7c). As

in in vitro model of AD, only 4c signifcantly reduced the

neurotoxicity evoked by 3-NP.

The above results clearly underline a specifc neuropro-

tective efect of 4a against 6-OHDA insult suggesting a

1 3

Neurochemical Research

We evaluated the some relevant ADMET properties of

the 4a-j derivatives and the reference compound donepezil

in the neutral form through the QikProp tool from Maestro

(Schrödinger Release 2018-4: QikProp, Schrödinger, LLC,

New York, NY, 2018) (Table 6). The Lipinski’s “Rule-of-

fve” (Ro5) comprises a set of molecular descriptors com-

monly used to evaluate the oral availability of small mol-

ecules: as: molecular weight (MW)≤500 Da, lipophilicity

assessed by the QPlogPo/w ≤ 5 (the predicted partition

coefcient between octanol and water), number of hydrogen

bond donors (HBD) and acceptors (HBA), respectively≤5

and≤10 [35]. We also evaluated the percentage of human

oral absorption (HOA), water solubility (QPlogS), block-

age of mammalian HERG K⁺channels (QPlogHERG), and

permeability in Madin-Darby Canine Kidney (MDCK) cells

(QPPMDCK). MDCK cells are considered to be a good

mimic for the blood–brain barrier (BBB) and evaluates the

BBB permeability for non-active transport. According to the

QikProp predictions, all the compounds are drug-like mole-

cules with no violations of the Ro5 rules (except compounds

4 g and 4i that slighly violate that ClogP criterion) with

high human oral absorption (>80%). For the most promis-

ing compounds (i.e., 4a and 4d as AChE inhibitors and 4j as

BuChE inhibitor), the ADMET predictions indicate that the

BBB permeability and water solubility should be improved

and the blockage mammalian HERG K⁺channels avoided

in new derivatives of this class of compounds.

Among all ten synthetized compounds, most of them

showed moderate inhibitory activity towards AChE or

BuChE, with compound 4d showing the best selective

inhibition of AChE with IC₅₀=9.1 µM. In addition, kinetic

assays evidenced a mixed-type mechanism of inhibition

for 4d and showed a similar binding mode to that observed

for donepezil. Considering the oxidative damage associ-

ated with NDs, all compounds were evaluated for their

antioxidant activity, which results highlighted compound

4a was the strongest antioxidant ligand, inhibiting 53.37%

of DPPH radicals at 80 µM, followed by compound 4c with

45.05% of inhibition at the same concentration. Moreo-

ver, the ability to inhibit the intracellular ROS formation

induced by t-BOOH in SH-SY5Y cells corroborated the

signifcant radical scavenging activity of 4a and 4c. The

indirect antioxidant activity was determined through the

ability of compounds 4a and 4c to induce glutathione

release, showing an increase of 33 and 34% in the intra-

cellular GSH levels, respectively. In vitro models of AD,

PD and HD evidenced the neuroprotective efect of 4a

against 6-OHDA damage suggesting as a promising ligand

to prevent the oxidative stress in PD. In addition, 4c show

the neuroprotective efect against mitochondrial dysfunc-

tion induced by both OAβ_1-42and 3-NP. Overall, consid-

ering the best multi-target profle of action of compounds

4a and 4c, being capable to selective inhibit AChE and

Fig. 7 Efects of compounds PQM-263 (4a), PQM-264 (4b) and

PQM-265 (4c) on OAβ_1-42, 6-OHDA and 3-NP induced neurotoxicity

in SH-SY5Y cells. a Cells were incubated with compounds (10 µM)

and OAβ_1-42(10 µ) for 4 h; (B) cells were incubated with compounds

(10 µM) and 6-OHDA (100 µM) for 2 h and starved in complete

medium for 22 h; c cells were incubated with compounds (10 µM)

an3-NP (5 mM) for 24 h. The neurotoxicity was measured by MTT

assay, as described in experimental section. Data are expressed as

percentages of neurotoxicity versus cells treated with OAβ_1-42or

6-OHDA and reported as mean SEM of at least three independent

experiments (* p<0.05, ** p<0.01 and *** p<0.001 versus cells

treated with neurotoxin at one-way ANOVA with Dunnett post hoc

test)

potential usefulness as a promising ligand to prevent the

oxidative stress in PD. Interestingly, 4c show the ability to

counteract the neurotoxicity induced by both OAβ_1-42and

3-NP, two neurotoxins that sharing the ability to induce

mitochondrial dysfunction, a common mechanism in sev-

eral neurodegenerative diseases. Therefore, both compound

4a and 4c could be explored as lead compound in pursuit

of more efective molecules with multiple neuroprotective

efects for DNs.

1 3

Neurochemical Research

modulate neuronal oxidative damage, both ligands could

be considered as promising lead compounds to be explored

in pursuit of more efective molecules with multiple neu-

roprotective efects for DNs.

DMSO-d₆) δ 9.16 (s, 2H, CONHNH₂), 7.32 (d, J=15.75 Hz,

1H, HC=CH), 7.09 (d, J=1.89 Hz, 1H, Ar–H), 6.97 (dd,

J = 1.89, 8.13 Hz, 1H, Ar–H), 6.77 (d, J = 8.13 Hz, 1H,

Ar–H), 6.34 (d, J=15.75 Hz, 1H, HC=CH), 4.37 (s, 2H,

CONHNH₂) and 3.78 (s, 3H, CH₃). ¹³C NMR (75 MHz,

DMSO-d₆) δ 165.3, 148.7, 148.3, 139.1, 126.9, 121.8, 117.4,

116.1, 111.3 and 56.0.

Experimental Section

(E)-3-(3-hydroxy-4-methoxyphenyl)acrylohydrazide

(6b). Yield 78%, white solid. IR (ATR): ν 3334, 1650, 1598

and 1494 cm⁻¹. ¹H NMR (300 MHz, DMSO-d₆) δ 9.24 (s,

2H, CONHNH₂), 9.19 (s, 1H, OH), 7.30 (d, J=15.74 Hz,

1H, HC=CH), 6.99 (d, J=1.56 Hz, 1H, Ar–H), 6.95-6.93

(m, 1H, Ar–H), 6.93 (d, J = 8.13 Hz, 1H, Ar–H), 6.32 (d,

J=15.74 Hz, 1H, HC=CH), 4.42 (s, 2H, CONHNH₂) and

3.79 (s, 3H, CH₃). ¹³C NMR (75 MHz, DMSO-d₆) δ 164.7,

148.9, 146.5, 138.1, 127.6, 120.0, 117.3, 113.2, 111.9 and

55.4.

1

NMR spectra of H and ¹³C were obtained on a Bruker

AC-300 spectrometer operating at 300 MHz for ¹H NMR

and 75 MHz for ¹³C NMR in Nuclear Magnetic Resonance

Laboratory at the Federal University of Alfenas (UNIFAL-

MG). The samples were solubilized in DMSO-d₆using

TMS (tetramethylsilane) as the internal reference. Infrared

(IR) spectra have been generated in an infrared spectrom-

eter Thermo Scientifc USA (Nicolet iS50 model) coupled

to Pike Gladi ATR technologies in analysis and characteri-

zation of drugs laboratory (LACFar) at the UNIFAL-MG.

Mass spectrometric analyzes were acquired in a range of

m/z of 80–1000 in the BRUKER COMPASS mass spec-

trometer by electrospray ionization. The purity of com-

pounds was determined by High Performance Liquid

Chromatography (HPLC) Shimadzu. Thin layer chroma-

tography experiments were performed on silica gel sheet

60 F254, Merck and purifcation by chromatography col-

umn was performed on fash silica gel (220–440 mesh,

0.035 mm–0.075 mm), Sigma-Aldrich. The visualization

of the substances was done in UV chamber (λ = 254 or

365 nm). Melting point were made on Mars equipment

(PFM II) with crushed sample and packaged in capil-

lary tube. All spectra are available in the supplementary

material.

(E)-3-(3,4-dihydroxyphenyl)acrylohydrazide (6c). Yield

38%, Yellow solid. IR (ATR): ν 3443, 3340, 3264, 3027,

1

1686, 1639, 1591 and 1524 cm⁻¹. H NMR (300 MHz,

DMSO-d₆) δ 9.24 (s, 2H, CONHNH₂), 7.27 (d, J=15.72 Hz,

1H, HC=CH), 6.95 (d, J=1.96 Hz, 1H, Ar–H), 6.84 (dd,

J = 1.96, 8.10 Hz, 1H, Ar–H), 6.74 (d, J = 8.10 Hz, 1H,

Ar–H), 6.26 (d, J = 15.72 Hz, 1H, HC = CH) and 4.43 (s,

2H, CONHNH₂). ¹³C NMR (75 MHz, DMSO-d₆) δ 165.6,

147.8, 146.0, 139.2, 126.8, 120.9, 116.9, 116.2 and 114.0.

(E)-3-(3,4-dimethoxyphenyl)acrylohydrazide (6d). Yield

84%, white solid. IR (ATR): ν 3231, 3034, 2962, 1652,

1

1616, 1582, 1510 and 1263 cm⁻¹. H NMR (300 MHz,

DMSO-d₆) δ 9.19 (s, 2H, CONHNH₂), 7.37 (d, J=15.77 Hz,

1H, HC=CH), 7.12 (d, J=1.84 Hz, 1H, Ar–H), 6.97 (dd,

J = 1.84, 8.25 Hz, 1H, Ar–H), 6.95 (d, J = 8.25 Hz, 1H,

Ar–H), 7.09 (d, J=15.77 Hz, 1H, HC=CH), 4.39 (s, 2H,

CONHNH₂), 3.78 (s, 3H, CH₃) and 3.76 (s, 3H, CH₃). ¹³

C

General Procedure for the Synthesis of Hydrazide

Intermediates 6a‑j

NMR (75 MHz, DMSO-d₆) δ 164.8, 150.0, 148.8, 138.2,

127.6, 121.1, 117.8, 111.7, 110.0 and 55.5.

(E)-3-(4-methoxyphenyl)acrylohydrazide (6e). Yield

58%, white solid. IR (ATR): ν 3278, 3013, 1655, 1600

To a suspension of trans-cinnamic acid derivatives 5a-j

(1 eq 1.51 mmol) in 15 mL of acetonitrile at room tem-

perature, was added 1.2 eq. (1.81 mmol) of HOBT and

1.2 eq. (1.81 mmol) of EDC. The reaction was stirred for

2 hat room temperature (25 °C). The resulting mixture

was then slowly added to a solution of hydrazine hydrate

(10 eq.) in 10 mL of acetonitrile and stirred in a second

ice-bath fask, kept between 0 and 10 °C. Then, the solvent

was removed under low pressure and dried under vacuum.

Finally, the resultant solid was re-suspended in 4 mL of 5%

saturated NaHCO₃and stirred to allow formation of the pre-

cipitate. The solid was separated by fltration and washed

with cold water to obtain the hydrazide intermediates 6a-j.

(E)-3-(4-hydroxy-3-methylphenyl)acrylohydrazide (6a).

Yield 96%, white solid. IR (ATR): ν 3401, 3306, 3143, 2945,

1656, 1580, 1514, 1278 and 1043 cm⁻¹. ¹H NMR (300 MHz,

1

and 1507 cm⁻¹. H NMR (300 MHz, DMSO-d₆) δ 9.26

(s, 2H, CONHNH₂), 7.47 (d, J=8.57 Hz, 2H, Ar–H), 7.37

(d, J=15.80 Hz, 1H, HC=CH), 6.94 (d, J=8.57 Hz, 2H,

Ar–H), 6.38 (d, J=15.80 Hz, 1H, HC=CH), 4.45 (s, 2H,

CONHNH₂) and 3.75 (s, 3H, CH₃). ¹³C NMR (75 MHz,

DMSO-d₆) δ 165.4, 160.7, 138.3, 129.5, 127.9, 118.2, 114.8

and 55.7.

(E)-3-(4-hydroxyphenyl)acrylohydrazide (6f). Yield 79%,

Light yelow solid. IR (ATR): ν 3269, 3195, 3013, 2906,

1

1632, 1606, 1587 and 1511 cm⁻¹. H NMR (300 MHz,

DMSO-d₆) δ 9.18 (s, 2H, CONHNH₂), 7.36 (d, J=8.58 Hz,

2H, Ar–H), 7.33 (d, J=15.81 Hz, 1H, HC=CH), 6.77 (d,

J=8.58 Hz, 2H, Ar–H), 6.31 (d, J=15.81 Hz, 1H, HC=CH)

and 4.36 (s, 2H, CONHNH₂). ¹³C NMR (75 MHz, DMSO-

d₆) δ 165.6, 159.3, 138.8, 129.8, 126.4, 117.1 and 116.2.

1 3

Neurochemical Research

(E)-3-(4-(trifuoromethyl)phenyl)acrylohydrazide (6 g).

(d, J=16.5 and 15.6 Hz, 1H, HC=CH), 7.17 and 7.08 (d,

J=8.0 and 7.9 Hz, 1H, Ar–H), 7.17 and 7.03 (d, J=8.0 and

7.9 Hz, 1H, Ar–H), 6.81 (d, J=7.9 Hz, 2H, Ar–H),6.66 (d,

J=8.1 Hz, 1H, HC=CH), 3.81 (s, 6H, OCH₃), 3.54 (s, 4H,

CH₂) and 2.37 (t, J=5.3 Hz, 2H, CH₂). ¹³C NMR (75 MHz,

DMSO-d₆) δ 162.0, 157.3, 152.1, 148.7, 148.4, 147.7, 142.0,

140.2, 138.2, 128.6, 128.1, 126.9, 126.3, 121.8, 117.2,

115.6, 114.2, 111.4, 110.6, 61.3, 55.4, 53.2, 51.8, 34.2 and

27.2. HR-MS (ESI) m/z: Calcd for C₂₂H₂₅N₃O₃[M+H]^+:

380.1974, found: 380.1978.

Yield 76%, white solid. IR (ATR): ν 3317, 3219, 3028, 1649,

1

1609 and 1528 cm⁻¹. H NMR (300 MHz, DMSO-d₆) δ

9.45 (s, 2H, CONHNH₂), 7.77-7.70 (m, 4H, Ar–H), 6.66 (d,

J=15.87 Hz, 1H, HC=CH) and 4.49 (s, 2H, CONHNH₂).

¹³C NMR (75 MHz, DMSO-d₆) δ 164.2, 139.5, 136.9, 129.6

(d, J = 31.88 Hz), 128.5, 126.2 (d, J= 3.53 Hz), 124.6 (d,

J=271.85 Hz) and 123.6.

(E)-3-(benzo[d] [1, 3] dioxol-5-yl)acrylohydrazide (6 h).

Yield 66%, Light bro wn solid. IR (ATR): ν 3342, 3191,

3031, 1660, 1625, 1552 and 1493 cm⁻¹. ¹H NMR (300 MHz,

DMSO-d₆) δ 9.21 (s, 2H, CONHNH₂), 7.34 (d, J=15.76 Hz,

1H, HC=CH), 7.04 (dd, J=1.50, 8.01 Hz, 1H, Ar–H), 7.10

(d, J=1.50 Hz, 1H, Ar–H), 6.91 (d, J=8.01 Hz, 1H, Ar–H),

6.03 (s, 2H, CH₂), 6.36 (d, J=15.76 Hz, 1H, HC=CH) and

4.39 (s, 2H, CONHNH₂). ¹³C NMR (75 MHz, DMSO-d₆)

δ 165.2, 148.9, 148.4, 138.5, 129.8, 123.5, 118.8, 109.0,

106.6 and 101.9.

E)-N’-(1-benzylpiperidin-4-ylidene)-3-(3-hydroxy-

4-methoxyphenyl)acrylohydrazide (4b). White solid (yield

37%), m.p. 192 °C. IR (ATR): ν 3246, 3026, 2901, 1659,

1

1646 and 1586 cm⁻¹. H NMR (300 MHz, DMSO- d₆) δ

10.39 and 10.36 (s, 1H, NH), 9.22 (s, 2H, OH), 7.48 and

7.39 (d, J = 15.7 and 15.6 Hz, 1H, HC = CH), 7.34 (d,

J=4.4 Hz, 8H, Ar–H), 7.31-7.22 (m, 2H, Ar–H), 7.28 and

6.61 (d, J=15.7 and 15.6 Hz, 1H, HC=CH), 7.09 and 7.02

(s, 1H, Ar–H), 7.01 and 6.98 (d, J = 8.4 and 7.8 Hz, 1H,

Ar–H), 6.95 (d, J=7.8 Hz, 2H, Ar–H), 3.80 (s, 6H, OCH₃),

3.54 (s, 4H, CH₂) and 2.36 (t, J = 5.5 Hz, 2H, CH₂). ¹³C

NMR (75 MHz, DMSO-d₆) δ 166.7, 162.0, 157.7, 152.5,

149.6, 149.4 146.8, 141.8, 140.1, 137.9, 128.8, 128.3, 127.9,

127.0, 121.1, 120.5, 118.1, 115.0, 113.5, 112.1, 61.4, 55.7,

53.28, 52.0, 34.4 and 27.4. HR-MS (ESI) m/z: Calcd for

C₂₂H₂₅N₃O₃[M+H]⁺: 380.1974, found: 380.1978.

(E)-3-(4-chlorophenyl)acrylohydrazide (6i). Yield 82%,

white solid. IR (ATR): ν 3311, 3247, 3032, 1650, 1608,

1

1519 and 1036 cm⁻¹. H NMR (300 MHz, DMSO-d₆) δ

9.40 (s, 2H, CONHNH₂), 7.58 (d, J=8.53 Hz, 2H, Ar–H),

7.46 (d, J=8.53 Hz, 2H, Ar–H), 7.43 (d, J=15.87 Hz, 1H,

HC=CH), 6.54 (d, J=15.87 Hz, 1H, HC=CH) and 4.49 (s,

2H, CONHNH₂). ¹³C NMR (75 MHz, DMSO-d₆) δ 164.7,

137.3, 134.3, 134.3, 129.6, 129.4 and 121.5.

Cinnamoylhydrazide (6j). Yield 28%, Light yellow solid.

(E)-N’-(1-benzylpiperidin-4-ylidene)-3-(3,4-dihydroxy-

phenyl)acrylohydrazide (4c). Yellow solid (yield 19%),

m.p. 140 °C. IR (ATR): ν 3243, 2796, 1650, 1597, 1535

1

IR (ATR): ν 3285, 3025, 1651, 1612 and 1522 cm⁻¹. H

NMR (300 MHz, DMSO-d₆) δ 9.36 (s, 2H, CONHNH₂),

7.54 (d, J=6.73 Hz, 2H, Ar–H), 7.43 (d, J=15.84 Hz, 1H,

HC=CH), 7.40-7.31 (m, 3H, Ar–H), 6.54 (d, J=15.84 Hz,

1H, HC = CH) and 4.46 (s, 2H, CONHNH₂). ¹³C NMR

(75 MHz, DMSO-d₆) δ 164.9, 138.6, 135.4, 129.9, 129.4,

127.9 and 120.7.

1

and 1516 cm⁻¹. H NMR (300 MHz, DMSO- d₆) δ 10.32

(s, 2H, NH), 9.24 (s, 2H, OH), 7.42 and 7.36 (d, J = 15.4

and 15.6 Hz, 1H, HC=CH), 7.34-7.27 (m, 8H, Ar–H), 7.17

and 6.65 (d, J=15.4 and 15.6 Hz, 1H, HC=CH), 7.03 and

6.96 (s, 1H, Ar–H), 6.90 and 6.85 (d, J = 7.0 and 8.2 Hz,

1H, Ar–H), 6.74 (d, J = 8.2 Hz, 2H, Ar–H), 3.51 (s, 4H,

CH₂) and 2.33 (t, J=5.3 Hz, 2H, CH₂) ¹³C NMR (75 MHz,

DMSO-d₆) δ 166.7, 162.0, 157.3, 152.2, 147.7, 147.5,

145.5, 142.0, 138.2, 128.6, 128.1, 126.9, 126.3, 121.1,

120.5, 116.8, 115.7, 113.8, 113.8, 61.2, 53.1, 51.8, 34.2 and

27.2. HR-MS (ESI) m/z: Calcd for C₂₁H₂₃N₃O₃[M+H]⁺:

366.1818, found: 366.1803.

General procedure for the coupling reaction between the

hydrazide intermediates 6a-j with 1-benzyl-4-piperidone for

the preparation of compounds 4a-j

To a solution 1.2 eq of 1-benzyl-4-piperidone in 10 mL

of ethanol, was added 0.96 mmol of the corresponding

hydrazide intermediates 6a-j. The mixture was stirred at

room temperature until total consumption of the correspond-

ing hydrazide visualized by TLC using a mixture of hexane/

ethyl acetate (3: 7) as eluent. Then, the solvent was removed

under reduced pressure and the product was purifed by fash

chromatography column (CC) and a mixtures of hexane/

Ethyl acetate in gradient concentration or by recrystalliza-

tion from ethanol/dichloromethane.

(E)-N’-(1-benzylpiperidin-4-ylidene)-3-(3,4-dimeth-

oxyphenyl)acrylohydrazide (4d). White solid (yield 55%),

m.p. 185 °C. IR (ATR): ν 3231, 3034, 2795, 1652, 1616,

1

1536 and 1510 cm⁻¹. H NMR (300 MHz, DMSO- d₆) δ

10.36 (s, 2H, NH), 7.55 and 7.46 (d, J=15.6 and 15.5 Hz,

1H, HC = CH), 7.33–7.30 (m, 8H, Ar–H), 7.27-7.19 (m,

2H, Ar–H), 7.30 and 6.70 (d, J = 15.6 and 15.5 Hz, 1H,

HC=CH), 7.20 and 7.15 (s, 1H, Ar–H), 7.13 (d, J=8.0 Hz,

1H, Ar–H), 6.97 (d, J = 8.0 Hz, 2H, Ar–H), 3.78 (s, 6H,

OCH₃), 3.76 (s, 6H, OCH₃), 3.51 (s, 4H, CH₂) and 2.38-

2.31 (m, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-d₆) δ 166.4,

161.6, 157.2, 152.1, 150.2,150.0, 148.6, 141.5, 139.6, 138.1,

(E)-N’-(1-benzylpiperidin-4-ylidene)-3-(4-hydroxy-

3-methoxyphenyl)acrylohydrazide (4a). White solid (yield

46%), m.p. 203 °C. IR (ATR): ν 3330, 3029, 2945, 1682,

1628, 1588 and 1530 cm⁻¹. ¹H NMR (300 MHz, DMSO- d₆)

δ 10.36 and 10.33 (s, 1H, NH), 9.51 (s, 2H, OH), 7.39-7.25

(m, 8H, Ar–H), 7.30-7.23 (m, 2H, Ar–H), 7.53 and 7.45

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128.4, 127.9, 127.4, 126.7, 121.6, 121.3, 118.0, 115.0,

111.5, 110.4, 109.6, 61.0, 55.3, 55.3, 53.0, 51.6, 34.0 and

27.0.HR-MS (ESI) m/z: Calcd for C₂₃H₂₇N₃O₃[M + H]⁺:

394.2131, found: 394.2124.

and 15.7 Hz, 1H, HC=CH), 7.25 and 7.12 (s, 2H, Ar–H),

7.32 (d, J=4.3 Hz, 8H, Ar–H),), 7.32 and 6.67 (d, J=15.9

and 15.7 Hz, 1H, HC=CH), 7.28-7.21 (m, 2H, Ar–H), 7.13

and 7.09 (d, J = 8.1 Hz, 4H, Ar–H), 6.94 (d, J = 8.1 Hz,

2H, Ar–H), 6.05 (s, 4H, CH₂), 3.52 (s, 4H, CH₂) and 2.34

(t, J = 5.1 Hz 2H, CH₂). ¹³C NMR (75 MHz, DMSO-d₆)

δ 167.1, 162.3, 158.2, 153.0, 149.1, 148.4, 148.3, 141.8,

140.1, 138.8, 129.8, 129.2, 128.7, 127.5, 124.4, 123.9,

119.2, 116.3, 109.1, 107.2, 106.6, 102.0, 61.8, 53.7, 52.4,

34.7 and 27.8. HR-MS (ESI) m/z: Calcd for C₂₂H₂₃N₃O₃

[M+H]⁺: 378.1818, found: 378.1801.

(E)-N’-(1-benzylpiperidin-4-ylidene)-3-(4-methoxy-

phenyl)acrylohydrazide (4e). White solid (yield 29%),

m.p. > 300 °C. IR (ATR): ν 3096, 1655, 1625, 1542 and

1

1511 cm⁻¹. H NMR (300 MHz, DMSO- d₆) δ 10.45 and

10.41 (s, 2H, NH), 7.60 and 7.54 (d, J = 8.3 and 8.7 Hz,

8H, Ar–H), 7.55 and 7.49 (d, J = 15.7 and 15.5 Hz, 1H,

HC = CH), 7.34 (d, J = 4.3 Hz, 8H, Ar–H), 7.30-7.23 (m,

2H, Ar–H), 7.28 and 6.73 (d, J = 15.7 and 15.5 Hz, 1H,

HC = CH), 6.99 (d, J = 8.7 Hz, 4H, Ar–H), 3.79 (s, 6H,

OCH₃), 3.53 (s, 4H, CH₂) and 2.36 (t, J=5.4 Hz, 2H, CH₂).

¹³C NMR (75 MHz, DMSO-d₆) δ 167.1, 162.4, 161.1, 160.9,

158.1, 153.0, 141.8, 140.0, 138.8, 130.2, 130.2, 129.2,

128.7, 128.0, 127.5, 118.7, 115.6, 114.9, 114.9, 61.8, 55.7,

53.7, 52.4, 34.4 and 27.4. HR-MS (ESI) m/z: Calcd for

C₂₂H₂₅N₃O₂[M+H]⁺: 364.2025, found: 364.2014.

(E)-N’-(1-benzylpiperidin-4-ylidene)-3-(4-chlorophenyl)

acrylohydrazide (4i). White solid (yield 73%), m.p. 209 °C.

IR (ATR): ν 3164, 3051, 2905, 1662, 1615 and 1490 cm⁻¹.

¹H 9NMR (300 MHz, DMSO- d₆) δ 10.51 and 10.34 (s, 2H,

NH), 7.68-7.59 (m, 4H, Ar–H), 7.54 and 7.49 (d, J =16.7

and 15.7 Hz, 1H, HC = CH), 7.49-7.44 (m, 4H, Ar–H),

7.31 (d, J=4.3 Hz, 8H, Ar–H), 7.28-7.20 (m, 2H, Ar–H),

7.27 and 6.81 (d, J=16.7 and 15.7 Hz, 1H, HC=CH) and

2.49-2.47 (m, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-d₆)

δ 161.9, 159.2, 139.0, 138.6, 134.6, 134.3, 129.8, 129.8,

129.5, 129.3, 128.7, 127.5, 121.9, 61.7, 53.6, 52.3, 34.6 and

27.8. HR-MS (ESI) m/z: Calcd for C₂₁H₂₂ClN₃O [M+H]⁺:

368.1530, found: 368.1525.

(E)-N’-(1-benzylpiperidin-4-ylidene)-3-(4-hydroxy-

phenyl)acrylohydrazide (4f). Yellow solid (yield 39%),

m.p. 223 °C. IR (ATR): ν 3215, 2812, 1650, 1598, 1550

1

and 1514 cm⁻¹. H NMR (300 MHz, DMSO- d₆) δ 10.34

and 10.30 (s, 2H, NH), 9.90 (s,1H, OH), 7.50 and 7.41 (d,

J = 16.4 and 15.6 Hz, 1H, HC = CH), 7.40 (d, J = 7.8 Hz,

4H, Ar–H), 7.31 (d, J=4.2 Hz, 8H, Ar–H), 7.28-7.21 (m,

2H, Ar–H), 7.23 and 6.61 (d, J = 16.4 and 15.6 Hz, 1H,

HC=CH), 6.78 (d, J=7.8 Hz, 4H, Ar–H), 3.51 (s, 4H, CH₂)

and 2.36-2.31 (m, 2H, CH₂). ¹³C NMR (75 MHz, DMSO-

d₆) δ 166.7, 162.0, 159.2, 158.9, 157.4, 152.2, 141.6, 139.9,

138.2, 129.3, 129.3, 128.6, 128.1, 126.9, 125.9, 117.0,

115.7, 115.7, 113.9, 61.2, 53.1, 51.8, 34.2 and 27.2. HR-MS

(ESI) m/z: Calcd for C₂₁H₂₃N₃O₂[M + H]⁺: 350.1869,

found: 350.1858.

N’-(1-benzylpiperidin-4-ylidene)cinnamohydrazide

(4j). Light yellow solid (yield 11%), m.p. 183 °C. IR

(ATR): ν 3231, 3028, 2790, 1673, 1655, 1538 and

1494 cm⁻¹. 10.48 (s, 2H, NH), 7.62 and 7.55 (d, J = 15.6

and 15.7 Hz, 1H, HC = CH), 7.59 (d, J = 6.7 Hz, 4H,

Ar–H), 7.45-7.40 (m, 2H, Ar–H), 7.42 (d, J = 6.7 Hz,

4H, Ar–H), 7.33 (d, J = 3.5 Hz, 8H, Ar–H), 7.30-7.23 (m,

2H, Ar–H), 7.43 and 6.87 (d, J = 15.6 and 15.7 Hz, 1H,

HC = CH), 3.54 (s, 4H, CH₂) and 2.40-2.36 (m, 2H, CH₂).

¹³C NMR (75 MHz, DMSO-d₆) δ 166.8, 162.0, 158.7,

153.7, 141.9, 140.2, 138.8, 130.8, 130.3, 130.1, 129.4,

129.2, 128.7, 128.1, 127.4, 121.3, 118.3, 61.7, 53.7, 52.4,

34.8 and 27.9. HR-MS (ESI) m/z: Calcd for C₂₁H₂₃N₃O

[M + H]⁺: 334.1919, found: 334.1919.

(E)-N’-(1-benzylpiperidin-4-ylidene)-3-(4-

(trifuoromethyl)phenyl)acrylohydrazide (4 g). White solid

(yield 57%), m.p. 188 °C. IR (ATR): ν 3243, 3027, 1649,

1

1609 and 1528 cm⁻¹. H NMR (300 MHz, Pyridine-d₅) δ

10.61 and 10.56 (s, 2H, NH), 8.14 and 8.06 (d, J=16.0 and

15.7 Hz, 1H, HC=CH), 7.71 (d, J=8.1 Hz, 4H, Ar–H), 7.60

(d, J=8.1 Hz, 4H, Ar–H), 7.45-7.27 (m, 10H, Ar–H), 7.38

and 7.05 (d, J=16.0 and 15.7 Hz, 1H, HC=CH), 3.48 (s,

4H, CH₂) and 2.85–2.42 (m, 16H, CH₂). ¹³C NMR (75 MHz,

DMSO-d₆) δ 166.40 161.5, 159.1, 153.8, 140.1, 139.4,

138.5, 130.0, 129.2, 129.1, 128.7, 127.5, 126.4, 126.4,

124.2, 122.8, 121.2, 61.7, 53.7, 52.4, 34.8 and 27.9. HR-MS

(ESI) m/z: Calcd for C₂₂H₂₂FN₃O [M + H]⁺: 402.1793,

found: 402.1783.

Molecular Docking Study with AChE

In this work, we evaluated the possible binding modes

of the compounds 4a-4j against the AChE through the

molecular docking strategy. Due to the similarity of the

derivatives synthesized in this work with donepezil, we

performed the docking experiments in the structure 4EY7

(human AChE solved at 2.35 Å in complex with done-

pezil) [36]. Conserved waters were identifed through

the superposition of the AChE structures and considered

explicitly during the docking experiments [37]. The four

water molecules W729, W722, W731 and W737 were

(E)-3-(benzo[d] [1, 3] dioxol-5-yl)-N’-(1-benzylpiperi-

din-4-ylidene)acryloh ydrazide (4 h). Light brown solid

(yield 56%), m.p. 90 °C. IR (ATR): ν 3220, 3024, 2949,

1

1656, 1600, 1527 and 1488 cm⁻¹. H NMR (300 MHz,

DMSO- d₆) δ 10.39 (s, 2H, NH), 7.51 and 7.43 (d, J=15.9

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Anti‑cholinesterase Activity Assays

Anticholinesterase activity was determined according to Ell-

man’s method [15] modifed for 96-well plates as previously

described [42 ]. All solutions were prepared in tris–HCl

bufer (0.02 M, pH 7.5) and stock solutions of the test com-

pounds were prepared in DMSO (50 mM). In 96-well plates

were added solutions with the inhibitor compound at 30 µM

fnal concentration. The vehicle control (DMSO-fnal con-

centration 0.2% v/v for AChE) was used as reference (nega-

tive control) and the reagent 5,5′-Dithiobis-2-nitrobenzoic

acid (DTNB) was added to electric eel acetylcholinesterase

(EeAChE) or equine serum butyrylcholinesterase (EqBu-

ChE) in presence of bovine serum albumin (BSA). Absorb-

ance was recorded using an iMark plate reader (Bio-Rad)

equipped with a light flter of=415 nm and this measure-

ment used as a blank reference. After 10 min incubation

at room temperature, acetylthiocholine iodide (ACTI) or

S-butyrylthiocholine iodide (BCTI) was added and absorb-

ance was recorded after 10 min of incubation at room tem-

perature at λ = 415 nm for 3 times within 30 s. Enzyme

activity was calculated as a percentage of the mean absorb-

ance values measured for the DMSO-treated control, dis-

counted from the mean blank reference values. Assays were

performed in triplicate (for standard deviation calculation).

Inhibition values were calculated using the Excel program.

Fig. 8 Rotatable bonds kept fxed during the docking experiments as

highlighted as arrows. Dihedrals not highlighted in this picture were

defned as free to rotate, except amide bonds

extracted from the structure of the AChE complexed with

donepezil (PDB code 4EY7). For BuChE, we used the

recently solved structure complexed with a large chloro-

tacrine-based inhibitor (PDB code 6I0C, solved at 2.65

Å) [38]. The receptor structures were prepared with Pro-

tein Pr eparation Wizard tool from the Schrödinger Suite

2018-4 [39] and the protonation states of the amino acid

residues were predicted using PROPKA with pH = 7.

Finally, the optimization of the hydrogen bond network

of the protein–ligand complexes was performed to adjust

the orientation of the hydrogen atoms, followed by energy

minimization of the hydrogen atoms.

Enzyme Kinetic Assay

The compounds were designed and prepared with Lig-

Prep from Maestro to set up the isomers, protonation states

and tautomers with Epik [40] at pH 7.0 0.4. We applied

torsional constraints to some rotatable bonds to keep the

planarity observed for the compounds during the docking

experiments (Fig. 8). The rotatable bond from the amide

group was kept fxed to the trans conformation.

Enzymatic kinetic was determined according to Ellman’s

method [15 ] modifed for 96-well plates as previously

described [42, 43]. All solutions were prepared in tris–HCl

bufer (0.02 M, pH 7.5) and the stock solutions of the test

compounds were prepared in DMSO (2 mM). In 96-well

plates were added 150 μL inhibitor compound solution

of compound 4j at two diferent concentrations (10 and

13 μM) distributed in eight sets of triplicates each. Eight

sets of DMSO-treated untreated triplicates (fnal concen-

tration 0.18% v/v of BuChE) were used as negative con-

trol. Subsequently, was added 60 μL of DTNB (Ellman’s

reagent) to 1.1 mM and 30 μL of equine serum butyryl-

cholinesterase (EqBChE) at 0.20 U/mL in the presence

of 1 mg/mL bovine serum albumin (BSA). Absorbance

was then recorded using an iMark plate reader (Bio-Rad)

equipped with λ = 415 nm light flter and this measure-

ment used as a blank reference. After 10 min incubation at

25 °C, 24 μL of substrate (BCTI) at eight serially diluted

concentrations (factor = 1.3) of 2.75–0 44 mM (fnal con-

centration: 0.25–0.04 mM) were added to the respective

wells and the absorbance recorded after incubation for

10 min at 25 °C at λ = 415 nm. The Lineweaver–Burk

The docking experiments were performed with the

molecular docking program Glide from Maestro in the

SP precision mode [41]. All the AChE structures and the

BuChE structure were aligned to the 1ZGC conformation

using the super tool from Pymol. The receptor grids were

centred on the native ligand present in the aligned 1Q84

complex, which contains a large inhibitor at both CAS and

PAS binding sites (X: 98.06, Y: 53.14 and Z: 22.06). We

also redocked the co-crystallized ligands into their respec-

tive structures to validate the docking protocol. The top-

energy docked pose of each ligand was selected according

to the lowest Glide Emodel. The predicted binding afnity

was provided as the “docking score”, consisting of the

GlideScore and the Epik penalization due to the selected

protonation state.

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Neurochemical Research

reciprocal plots were obtained by plotting a 1/velocity ver-

sus 1/[substrate] and two diferent inhibitor concentrations

for untreated control. The linear regression of each data-set

shows a convergent behavior, in ways the region to where

the curves converge determine the type of inhibition. The

values of K_i, K_i’(competitive and non-competitive inhibi-

tion constants respectively), K_m(Michaelis–Menten con-

stant) and V_max(maximum speed) were calculated using

Graphpad Prism 7.0 using nonlinear regression models for

kinetics. enzymatic - inhibition and enzymatic kinetics-

substrate versus velocity.

(HBSS) (0.5 mg/mL) for 2 h at 37 °C in 5% CO₂. After

washing with HBSS, formazan crystals were dissolved

in isopropanol. The amount of formazan was measured

(570 nm, reference flter 690 nm) using the multilabel plate

reader VICTOR™ X3 (PerkinElmer, Waltham, MA, USA).

The quantity of formazan was directly proportional to the

number of viable cells.

Determination of ROS Formation Induced

by t‑BuOOH

The intracellular antioxidant activity of the studied com-

pounds induced by t-BuOOH was evaluated in SH-SY5Y

cells as previously described [46]. The ROS formation was

determined using a 2′-7′ dichlorodihydrofuorescein diac-

etate fuorescent probe (DCFH-DA), as previously reported

by Tarozzi, 2007 [46]. Firstly, SH-SY5Y cells were seeded

in a 96-well plate at 2 × 10⁴cells/well and incubated for

24 h at 37 °C in 5% CO₂. The treatment medium was then

removed and 100 µL of DCFH-DA probe (10 µg/mL) was

added to each well. After 30 min of incubation at room

temperature, DCFH-DA solution was replaced with 100 µL

of t-BuOOH (100 µM) and 100 µL of the test compounds at

diferent concentrations (2.5–10 µM) for 30 min. In parallel,

the SH-SY5Y cells were also treated with compounds for

24 h before the treatment with t-BuOOH. The ROS forma-

tion was measured (excitation at 485 nm and emission at

535 nm) using a multilabel plate reader (VICTOR™ X3,

PerkinElmer). The antioxidant activity in terms of inhibi-

tion percentage in ROS formation induced by t-BuOOH, is

calculated using the following formula:

DPPH Scavenging Activity

The ability of compounds PQM 263–272 to scavenge

DPPH free radicals was evaluated according to the

method described by Gontijo 2012 [44] in our laboratory

(PeQuiM). The compounds were evaluated at concentra-

tions of 200, 100, 50, 25, 12.5, 6.25, 3.13 and 1.56 μM in

ethanol. A 4 mL aliquot of sample was mixed with 1 mL

of DPPH (0.5 mM in ethanol). The solution was vigor-

ously stirred at room temperature and after 30 min the

absorbance was measured at 517 nm in a UV–vis spec-

trophotometer (Shimadzu). A low absorbance value indi-

cates efective free radical scavenging. Each solution was

analyzed in triplicate and the mean values were plotted

to obtain the EC₅₀against DPPH by linear regression.

Antioxidants like ascorbic acid and trolox were used as a

standard over the same range of concentrations. The rad-

ical-scavenging activity was evaluated as the percentage

of inhibition according to the following equation: %inhi-

bition = [(absorbance of control − absorbance of sample)/

absorbance of control)] × 100.

(

)

Ptc × 100

% of inhibition = 100 −

100

where Ptc = % of increase in ROS formation induced

by t-BuOOH in the presence of the studied com-

pounds; Pt = % of increase in ROS formation induced

by t-BuOOH.

Determination of Neurotoxicity

Human neuronal (SH-SY5Y) cells were routinely grown in

Dulbecco’s modifed Eagle’s Medium supplemented with

10% fetal bovine serum, 2 mM l-glutamine, 50 U/mL peni-

cillin and 50 µg/mL streptomycin at 37 °C in a humidifed

incubator with 5% CO₂.

Determination of Intracellular Glutathione

Levels

Cells viability, in terms of mitochondrial metabolic func-

tion, was evaluated by the reduction of 3-(4,5-dimethyl-2-

thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to

its insoluble formazan, as previously described [45]. SH-

SY5Y cells were seeded in a 96-well plate at 2×10⁴cells/

well, incubated for 24 h and subsequently treated with dif-

ferent concentrations of test compounds 4a-j (2.5–80 µM)

for 24 h at 37 °C in 5% CO₂. The treatment medium was

then replaced with MTT in Hank’s Balanced Salt Solution

Glutathione (GSH) levels were measured by using the fuo-

rescent probe monochlorobimane (MCB), as previously

described [47]. SH-SY5Y cells were seeded in a black

96-well plate at 2 × 10⁴cells/well, incubated for 24 h and

subsequently treated with test compounds at diferent con-

centrations (2.5–10 µM) for 24 h at 37 °C in 5% CO₂. At the

end of incubation, the treatment medium was removed and

100 µL of MCB was added to each well. After 30 min of

incubation at 37 °C in 5% CO₂, GSH levels were measured

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Neurochemical Research

(excitation at 355 nm and emission at 460 nm) using the

multilabel plate reader VICTOR™ X3 (PerkinElmer).

Results are expressed as a fold increase of control cells.

Data are expressed as percentage of neurotoxicity versus

untreated cells.

Neuroprotective Activity Toward 6‑OHDA

Nuclear Extraction and Nrf2 Binding Activity

Assay

SH-SY5Y cells were seeded in a 96-well plate at 2 × 10⁴

cells/well, incubated for 24 h and subsequently treated with

test compounds (10 μM) and 6-OHDA (100 µM) for 2 h and

starved in complete medium for 22 h. The neuroprotective

activity was measured by using the MTT assay as previously

described [50]. Data are expressed as percentage of neuro-

toxicity versus untreated cells.

SH-SY5Y cells were seeded in 60 mm dishes at 2×10⁶cells/

dish, incubated for 24 h and subsequently treated with test

compounds at 10 μM for 1, 3 and 6 h at 37 °C in 5% CO₂. At

the end of incubation, cytosolic and nuclear extraction for

Nrf2 nuclear translocation were performed by using Nuclear

Extract Kit (Active Motif, Carlsbad, CA, USA), according

to the manufacturer’s guidelines. Nuclear extracts (10 µg)

obtained were then used to determine the active Nrf2 protein

level by using the TransAM Nrf2 Kit (Active Motif), accord-

ing to the manufacturer’s guidelines. The TransAM Nrf2 Kit

is a DNA-binding ELISA able to determine the active Nrf2

protein level in nuclear extract. The primary antibody of the

kit is able to recognize an epitope on Nrf2 protein upon ARE

binding. The active Nrf2 protein levels in the treated cells

are expressed as fold increase with respect to corresponding

untreated cells.

Neuroprotective Activity Toward 3‑NP

SH-SY5Y cells were seeded in a 96-well plate at 2 × 10⁴

cells/well, incubated for 24 h and subsequently treated with

test compounds (10 μM) and 3-NP (5 mM) for 24 h. The

neuroprotective activity was measured by using the MTT

assay. Data are expressed as percentage of neurotoxicity

versus untreated cells.

Aβ_1–42Oligomers Preparation

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Publisher’s Note Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional afliations.

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t.2007.07.017

Afliations

Cindy Juliet Cristancho Ortiz¹· Caio Miranda Damasio¹· Letizia Pruccoli²· Nathália Fonseca Nadur³·

Luciana Luiza de Azevedo³· Isabella Alvim Guedes⁴· Laurent Emmanuel Dardenne⁴· Arthur Eugen Kümmerle³·

Andrea Tarozzi²· Claudio Viegas Jr.¹

2

* Andrea Tarozzi

Department for Life Quality Studies, Alma Mater

Studiorum-University of Bologna, Corso d’Augusto 237,

47921 Rimini, Italy

andrea.tarozzi@unibo.it

* Claudio Viegas Jr.

3

4

cvjviegas@gmail.com

Laboratory of Molecular Pharmacology, Institute

of Biomedical Sciences, Federal University of Rio de Janeiro,

Rio De Janeiro, RJ 21941-902, Brazil

1

PeQuiM-Laboratory of Research in Medicinal Chemistry,

Federal University of Alfenas, Jovino Fernandes Sales

Avenue 2600, Alfenas, MG 37130-840, Brazil

National Laboratory of Scientifc Computation, Petrópolis,

RJ 25651-075, Brazil

1 3

Article Doi

Cinnamoyl-N-Acylhydrazone-Donepezil Hybrids: Synthesis and Evaluation of Novel Multifunctional Ligands Against Neurodegenerative Diseases

DOI: 10.1007/s11064-020-03148-2

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Full text of DOI:10.1007/s11064-020-03148-2

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