Synthesis, Molecular Modeling, and Evaluation of Novel Sulfonylhydrazones as Acetylcholinesterase Inhibitors for Alzheimer's Disease

Article abstract of DOI:10.1002/ardp.201700163

Alzheimer's disease (AD) is the most common type of dementia and related to the degeneration of hippocampal cholinergic neurons, which dramatically affects cognitive ability. Acetylcholinesterase (AChE) inhibitors are employed as drugs for AD therapy. Three series of sulfonylhydrazone compounds were designed, and their ability to inhibit AChE was evaluated. Fifteen compounds were synthesized and twelve of them had IC₅₀ values of 0.64–51.09 μM. The preliminary structure–activity relationships indicated that the methylcatechol moiety and arylsulfonyl substituents generated better compounds than both the benzodioxole and alkylsulfonyl chains. Molecular dynamics studies of compound 6d showed that the interaction with the peripheral binding site of AChE was similar to donepezil, which may explain its low IC₅₀ (0.64 μM). Furthermore, the drug-likeness of 6d suggests that the compound may have appropriate oral absorption and brain penetration. Compound 6d also presented antiradical activity and was not cytotoxic to LL24 cells, suggesting that this compound might be considered safe. Our findings indicate that arylsulfonylhydrazones may be a promising scaffold for the design of new drug candidates for the treatment of AD.

Full text of DOI:10.1002/ardp.201700163

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Full Paper
Synthesis, Molecular Modeling, and Evaluation of Novel
Sulfonylhydrazones as Acetylcholinesterase Inhibitors for
Alzheimer’s Disease
1
Thais B. Fernandes
ꢀ, Micael R. Cunha1ꢀ, Renata P. Sakata2, Thalita M. Candido1,
1
1
3
2
1
ꢀ
ꢀ
ꢀ
Andre R. Baby , Maurıcio T. Tavares , Euzebio G. Barbosa , Wanda P. Almeida , and Roberto Parise-Filho
1
~
~
Faculty of Pharmaceutical Sciences, Department of Pharmacy, University of Sao Paulo, Sao Paulo, Brazil
Faculty of Pharmaceutical Sciences, Department of Pharmacy, University of Campinas, Campinas, Brazil
Health Sciences Centre, Department of Pharmacy, Federal University of Rio Grande do Norte, Natal, Brazil
2
3
Alzheimer’s disease (AD) is the most common type of dementia and related to the degeneration of
hippocampal cholinergic neurons, which dramatically affects cognitive ability. Acetylcholinesterase
(AChE) inhibitors are employed as drugs for AD therapy. Three series of sulfonylhydrazone
compounds were designed, and their ability to inhibit AChE was evaluated. Fifteen compounds were
synthesized and twelve of them had IC50 values of 0.64–51.09 mM. The preliminary structure–activity
relationships indicated that the methylcatechol moiety and arylsulfonyl substituents generated better
compounds than both the benzodioxole and alkylsulfonyl chains. Molecular dynamics studies of
compound 6d showed that the interaction with the peripheral binding site of AChE was similar to
donepezil, which may explain its low IC50 (0.64 mM). Furthermore, the drug-likeness of 6d suggests
that the compound may have appropriate oral absorption and brain penetration. Compound 6d also
presented antiradical activity and was not cytotoxic to LL24 cells, suggesting that this compound
might be considered safe. Our findings indicate that arylsulfonylhydrazones may be a promising
scaffold for the design of new drug candidates for the treatment of AD.
Keywords: Acetylcholinesterase / Alzheimer’s disease / Drug design / Molecular modeling /
Sulfonylhydrazone
Received: June 5, 2017; Revised: September 1, 2017; Accepted: September 5, 2017
DOI 10.1002/ardp.201700163
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
cases. AD is associated with several clinical symptoms,
including memory loss, mental confusion, difficulty
Introduction
Dementia is a neurocognitive disorder that negatively affects
the cognitive function and performance of everyday activities.
More than 46 million people worldwide suffered from
dementia in 2015. Alzheimer’s disease (AD) is the most
common type of dementia, corresponding to 60–80% of
performing everyday tasks, poor judgment, apathy, and
depression [1, 2].
The pathophysiological factors that are related to AD
include the aggregation of b-amyloid (Ab) protein (plaques)
outside neurons in the brain and twisted strands of tau (t)
protein (tangles) inside neurons, which may damage them.
This neurodegeneration is most pronounced in hippocampal
cholinergic neurons, which are related to cognitive func-
tion [1, 3].
Correspondence: Prof. Roberto Parise-Filho, Faculty
of Pharmaceutical Sciences, Department of Pharmacy, University
~
of Sao Paulo, Av. Prof. Lineu Prestes Avenue, 580, Cidade
~
Universitaria, Sao Paulo, SP 05508-000, Brazil.
E-mail: roberto.parise@usp.br
Fax: þ55-11-30913793
ꢀThese authors contributed equally to this work.
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The cholinergic hypothesis suggests molecules that target and
inhibit acetylcholinesterase (AChE), such as rivastigmine,
galantamine, and donepezil, are useful for AD treatment.
They inhibit the AChE enzyme and avoid the hydrolysis of
acetylcholine (ACh), thus increasing ACh concentrations.
These drugs are mainly the only available therapeutic
treatments for AD, in addition to recently approved
N-methyl-D-aspartate receptor antagonist memantine [4–8].
The current agents for AD treatment have been questionable,
once there is no evidence that any of these drugs are superior
in terms of efficacy [9]. Thus, new drugs are still needed for
the treatment of AD [10–14].
Donepezil is the most widely employed AChE inhibitor and
has been used as the prototype for several other AChE
inhibitors [15]. N0-Benzylidene-2-(4-benzylpiperazin-1-yl)acyl-
hydrazones were derived from donepezil (Fig. 1) and reported
to moderately inhibit AChE, butyrylcholinesterase (BuChE),
and Ab aggregation [16]. These derivatives retain the
aromatic ring in region A of donepezil, which interacts with
aromatic residues of AChE’s binding site. Region C presents a
benzylpiperidine or benzylpiperazine. In region B, the cyclo-
pentanone ring is replaced by acylhydrazone, which has been
found in the structures of other AChE inhibitors [17, 18].
Da Silva and co-workers [17] synthesized several acylhy-
drazone-4-quinolone derivatives (Fig. 1) that exerted a good
inhibitory effect on AChE (IC50 ¼ 1.2–17.0 mM). These com-
pounds also had a notable inhibitory effect on the formation
of Ab42 fibrils.
strategy [19] was employed at region A, resulting in a
benzodioxole bicyclic ring that is observed in compounds that
are able to inhibit both AChE and BuChE [20]. Bioisosteric
substitution was performed in region B of the prototypes,
replacing the acylhydrazone moiety with a sulfonylhydrazone
moiety because of its described high biological activity
[21–27]. Region C was originated from the 4-quinolone
prototype, which has an aryl group that is bound to region B.
For series I, a phenyl group was retained in region C, and
several substituents (R) with different hydrophobic and
electronic properties were introduced at the para posi-
tion [28]. Series II present alkyl groups in the region C for
better exploitation of the catalytic pocket. Series III was
designed with methylcatechol in region A aiming to explore
the antiradical properties related to phenol groups, as in
methylcatechol moiety, which are important for neuro-
protection [29, 30]. For series III, the aromatic ring was
retained in region C, based on our preliminary data on the
ability of this scaffold to inhibit AChE.
The ability of the compounds to inhibit AChE was evaluated
by Ellman’s colorimetric method. Physicochemical properties
were evaluated to provide insights into structure–activity
relationships (SARs) and verify drug-likeness properties.
Molecular dynamics simulations were performed for the
most active compound, 6d, to propose a preliminary modus
interandi for AChE. The antiradical potential was also
evaluated by the DPPH assay because this is a relevant
property in AD therapy. The cytotoxicity was evaluated by the
MTT assay, thus providing additional information about its
safety.
To identify novel AChE inhibitors, we have designed three
series of sulfonylhydrazones by merging the previous
acylhydrazone and 4-quinolone analogues into
a new
sulfonylhydrazone hybrid scaffold (Fig. 1). The aromatic
characteristic of region A was retained, which was observed
for acylhydrazone and donepezil. For series I, a ring closure
Results and discussion
Chemistry
The method for preparing the sulfonylhydrazone analogues
was based on a condensation reaction from sulfonylhydra-
zide and an appropriate aldehyde (piperonal or vanillin) as
shown in Fig. 2 [31]. Vanillin was commercially obtained.
Piperonal was prepared by the simple oxidation of piperonyl
alcohol in the presence of MnO2 with good yield (75%) [32].
Sulfonylhydrazides were generated from the reaction of
hydrazine hydrate and sulfonyl chloride with a good yield
(60–96%) [33].
Fifteen sulfonylhydrazone analogues were successfully
synthesized, and their yields (23–85%) varied based on the
substituents. All of the final products were obtained as white/
yellow solids.
Biological evaluation
AChE inhibition
Twelve sulfonylhydrazones were tested with regard to their
ability to inhibit AChE from Electrophorus electricus (electric
eel), using Ellman’s colorimetric method, presenting IC50
values of 0.64–51.09 mM (Table 1). Three compounds (5b, 5c,
and 5l) had solubility problems and could not be tested.
Figure 1. Design of sulfonylhydrazone analogues that inhibit
AChE.
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Figure 2. Synthesis of sulfonylhydrazones from sulfonylhydrazide and aldehyde.
For series I, compounds 5a (methyl) and 5d (methoxyl) had
good AChE inhibitory activities (IC50 ¼ 7.12 and 1.84 mM,
respectively). Methyl and methoxyl substituents present
similar electronic properties (s ¼ ꢁ0.17 and ꢁ0.27, respec-
tively), although large differences are observed when
compared hydrophobicity properties (p ¼ 0.60 and ꢁ0.03,
respectively). This might partially explain differences in
activity between compounds 5a and 5d. To date, the crystal
structure of donepezil complexed with hAChE (PDB code:
4EY7) reveals water-mediated hydrogen bounds between the
two methoxyl moieties and the backbone of Trp286 [34]. In
this regard, the replacement of methyl for methoxyl provides
the insertion of a hydrogen bond acceptor (HBA) that might
be related to the greater activity of compound 5d [35]. This
information may also support the excellent activity observed
for compound 6d from series III.
Alkyl analogues (5g–l) from series II presented good activity
profile with IC50 ranging from 5.12 to 51.09 mM. The literature
provides some information regarding the insertion of alkyl
chain in AChE inhibitors [36] and, as observed for this series,
IC50 tend to decrease according to the number of carbons in
the alkyl chain and Clog P-values. However, for propyl
compounds (5h and 5i) this pattern changed a little which
may be related to linearity of the chain. Furthermore, a sharp
drop in activity (IC50 ¼ 51.09 mM) was observed for compound
5k (Clog P ¼ 3.81), suggesting that long alkyl chain may cause
steric hindrance imparing interaction between micro- and
macromolecules and/or solubility problems due to its high
lipophilicity. Compound 5l, with eight carbons, was insoluble
and could not be tested as well.
Although physical–chemical properties of the ligands might
assist drug-receptor interactions, for series III, we could not
correlate the electronic (s) and hydrophobic (p) properties of
substituents with AChE inhibitory activity, as well as total
polar surface area (tPSA), and CLogP of the molecules.
Comparisons of two equivalent molecules from series I and
III showed that the methylcatechol compounds are better
inhibitors. For compounds that presented the same group in
region C, the presence of methylcatechol implied an increase
in potency. Briefly, compound 6a presented
a 5.1-fold
increase in activity compared with 5a, and compound 6d
presented a 4.5-fold increase in activity compared with 5d.
These results suggest that methylcatechol might enhance the
interaction with AChE, but also increase hydrosolubility. The
main difference between both moieties is that benzodioxole
carries two HBAs, whereas methylcatechol presents both
HBAs and HBDs, which correspond to the phenol hydroxyl.
The insertion of a hydrogen bond donating group contrib-
uted to greater activity and might explain the lower IC50
values for series III.
Antiradical activity
Phenol groups have been associated with the antiradical
activity of several compounds, such as eugenol, quercetin,
curcumin, and capsaicin [37–40]. This is an interesting
property for medicinal purposes. Previous studies have shown
that induced oxidative stress can contribute to neurodegen-
eration and consequently neurological diseases [41–43].
Although antiradical substances do not improve cognition
per se, they may exert a neuroprotective effect and retard the
evolution of AD [44–46].
In the context of AD, the impact of oxidative damage
has been especially emphasized. Deposits of Ab in the
brain are a pathophysiological factor in AD. The accumu-
lation of Ab increases oxidative stress, promotes the
hyperphosphorylation of t protein, and results in toxic
effects on synapses and mitochondrial dysfunction.
Oxidative stress can also aggravate the production and
For series III, benzodioxole ring was replaced by methyl-
catechol in region A aiming to optimize the AChE inhibitory
activity, to measure the antiradical activity and to enhance the
solubility. This molecular modification resulted in optimized
compounds with IC50 values of 0.64–6.25 mM, which also
allowed the t-butyl derivative (6c) to be tested. Compound 6d
was the most active compound from this series, followed by 6e
and 6f.
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Table 1. AChE inhibitory activity of sulfonylhydrazones and their physicochemical parameters. CLogP and tPSA refer to
the molecule, and p and s refer to the substituent group attached to the phenyl ring in region C.
50
Entry
5a
Structure
CLogP
tPSA
p
s
IC (mM)
3.57
76.99
0.60
ꢁ0.17
7.12 ꢂ 1.02
5b
5c
2.81
4.90
128.8
76.99
0.22
1.88
0.78
n.d.
n.d.
ꢁ0.20
5d
5g
2.99
1.69
86.22
76.99
ꢁ0.03
ꢁ0.27
1.84 ꢂ 0.26
9.69 ꢂ 1.39
–
–
5h
5i
2.22
2.00
76.99
76.99
–
–
–
–
6.72 ꢂ 0.96
5.12 ꢂ 0.73
5j
2.75
3.81
76.99
76.99
–
–
–
–
4.35 ꢂ 0.62
5k
51.09 ꢂ 7.32
5l
4.86
76.99
–
–
n.d.
6a
6c
2.79
4.11
87.99
87.99
0.60
1.88
ꢁ0.17
ꢁ0.20
1.39 ꢂ 0.19
6.25 ꢂ 0.89
(Continued)
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Table 1. (Continued)
50
Entry
Structure
CLogP
tPSA
p
s
IC (mM)
0.64 ꢂ 0.09
0.95 ꢂ 0.14
6d
2.21
97.22
ꢁ0.03
ꢁ0.27
6e
6f
3.00
1.31
87.99
0.73
0.23
0.00
117.09
ꢁ0.56
1.24 ꢂ 0.18
Donepezil
–
–
–
–
0.002
n.d. ¼ not determined (insoluble).
aggregation of Ab, which induces a vicious cycle of
pathogenesis in AD [47, 48].
metabolism, and excretion (ADME), because of the risk of
unwanted adverse effects or poor bioavailability when
assayed in vivo [49, 50]. This is one of the main reasons for
the frequent failure to develop drug candidates [51]. Many
ADME virtual platforms have been developed, but medicinal
chemists can execute in silico analyses to reduce the cost of
molecules that would not present desirable pharmacokinetic
or pharmacodynamic profiles [52].
The most active compound, 6d, was evaluated regarding its
ability to scavenge reactive oxygen species (ROS), particularly
the 1,10-diphenyl-2-picrylhydrazyl (DPPH) radical, because it
carries a phenol group. To confirm that the antiradical activity
of 6d is related to the phenol hydroxyl, we also tested its
benzodioxole analogue, 5d. The results indicated that 6d
presented antiradical activity with an IC50 of 216.2 mM,
whereas 5d had no antiradical activity at 5000.0 mM. These
results indicate the antiradical activity of 6d is related to the
phenol hydroxyl. These findings demonstrate that compound
6d may be beneficial for the treatment of AD with regard to
both AChE inhibition and antiradical activity.
The most active compound, 6d, appeared to be absorved
through gastrointestinal (GI) system by passive difusion, as
well as its analogue 5d (Table 2). Although the SwissADME did
not predict these molecules would cross the blood–brain
barrier passively, Lipinski’s rule for central nervous system
(CNS) drugs was applied, based on drugs that are known to
have CNS activity [6, 53]. Lipinski’s rule states that most “drug-
like” compounds that are orally active in the human CNS must
have a molecular weight ꢃ400 Da, number of HBDs ꢃ3,
number of HBAs ꢃ7, and a calculated partition coefficient
(CLogP) ꢃ5 [54]. Among the CNS drugs that presented GI
absorption in Fernandes et al. [6], three of them (tolcapone,
entacapone, and temozolomide) were found to be quite
similar to 6d and 5d with regard to Lipinski parameters: a
small hydrophilic scaffold, HBA ¼ 7–9, HBD ¼ 2, molecular
weight ¼ 194.15–305.29 Da, and CLogP ¼ ꢁ0.28 to 3.38. Based
on these parameters, compounds 6d and 5d are likely active in
the CNS. Other authors have studied why some outliers based
on Lipinski’s rule for CNS drugs are still active, suggesting the
involvement of intramolecular hydrogen bonds and carrier
proteins [55]. The in silico analysis revealed that 6d may not be
removed from the CNS by P-glycoprotein (P-gp), an important
membrane transporter that is responsible for the efflux of
different molecules and thus influences drug absorption,
Cytotoxicity
Compound 6d and its analogue, 5d, were evaluated in the
MTT assay to investigate their cytotoxic effects and safety
profile. The MTT tetrazolium salt assay allows the measure-
ment of cell viability. The level of formazan that is produced
by MTT metabolism in cells is proportional to the number of
viable cells. Human lung fibroblast cells (LL24) were treated
with compounds 6d and 5d (100 mM) and for both com-
pounds, cells presented cellular viability up to 50%, indicating
that the compounds might be considered safe at 100 mM
when tested against LL24 cells.
Molecular modeling
In silico absorption, distribution, metabolism, and
excretion
The development of drug candidates involves the assessment
of several parameters, such as absorption, distribution,
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Table 2. ADME results for 5d, 6d, and donepezil.
2 a)
MWa)
HBDa)
HBAa)
CLogPa)
PSA (A )
̊
Entry
5d
6d
334.35
336.37
380.51
1
2
0
8
8
4
2.62
2.32
3.66
80.55
89.15
39.91
Donepezil
Entry
Water solubilityb)
GI absorptionb)
BBB permeantb)
P-gp substrateb)
CYP inhibitionb)
5d
6d
Soluble
Soluble
Poorly
High
High
High
No
No
Yes
No
No
Yes
1A2; 2C19; 2C9
2C9
Donepezil
2D6; 3A4
Drug-likenessb)
Entry
Lipinski
Ghose
Verber
Egan
Muegge
5d
6d
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Donepezil
MW, molecular weight; HBD, hydrogen bond donor; HBA, hydrogen bond acceptor; PSA, polar surface area; CLogP, calculated
LogP; GI, gastrointestinal absorption; BBB, blood–brain barrier permeant; P-gp, P-glycoprotein substrate; CYP, cytochrome P450
inhibition.
distribution, and excretion. In the context of AD treatment, a
non-substrate of P-gp is desirable because it would favor
permanence of the drug in the CNS at therapeutic concen-
trations [56, 57].
structures of hAChE (PDB code: 4EY7) and eeAChE, it is
noteworthy that eeAChE does not present any co-crystallized
waters, known to be participative in water-mediated
hydrogen bonds between important ligands and the
backbone of residues of AChE, for instance, territrem B;
dihydrotanshinoni I; huperzine A; galantamine and
donepezil [71–73]. Other important aspect that limited the
use of crystallographic data of eeAChE is based on the fact
that these three examples do not present ligands co-
crystallized, also known as apo protein [74]. The literature
highlighted that apo proteins differ from that with ligands
(holo) because some amino acids residues or even entire
protein chains can be displaced by the presence of a
inhibitor [75–80]. The presence of co-crystallized ligands
facilitates the identification of most adequate mode of
interaction in docking studies [81]. However, we analyzed
the homology between those enzymes, aiming to determine
if the eeAChE may be modeled to carry the molecular
docking and dynamics (MD). For this purpose, BLAST (Basic
Local Alignment Search Tool) was performed comparing
each eeAChE (1C2B; 1C2O; 1EEA) with the hAChE (4EY7), in
the NCBI website (https://blast.ncbi.nlm.nih.gov/BlastAlign.
cgi) [82, 83]. Interestingly, 1C2B and 1C2O presented 81% of
identity with 4EY7, while 1EEA only 59%. Moreover, the PAS,
gorge and CAS seem to be conserved in the position and
nature of amino acids (Fig. 3). In this context, we considered
eeAChE acceptable for the subsequent homology studies.
The structure of eeAChE was further modeled with
SWISS-MODEL fully automated protein structure homology-
modeling server [84, 85] using as template hAChE complexed
with the donepezil inhibitor (PDB code: 4EY7). AutoDock
Vina [86] was used to obtain two different orientations for the
In agreement with our findings, the literature indicates that
donepezil is highly absorbed by the GI system after oral
administration [58] and is metabolized by CYP3A4 and
CYP2D6 [59, 60]. Moreover, Ishiwata et al. [61] highlighted
the ability of P-gp to efflux donepezil from the CNS to the
blood [62]. These results corroborate the ability of SwissADME
to predict the pharmacokinetics of small molecules.
Although sulfonylhydrazones and N-acylhydrazones are
associated to toxicity in vivo, the literature describes some
examples of bioactive compounds possessing this moiety
which proved to be stable in vivo and in vitro [63–66].
Moreover, nitrogen accompanied by an electronegative
group tends to be more stable since it may participate in
the conjugation of the imine double bond. This conjugation
decreases the dþ charge on the imine carbon and increases
the energy of the LUMO, making it less susceptible to be
hydrolyzed [67].
Finally, five other relevant strategies for selecting molecules
with a high drug-like profile were evaluated for SwissADME.
All of these strategies – Lipinski, Ghose, Verber, Egan, and
Muegge – indicate that 6d and 5d are drug candidates
because they possesses important functional groups, high
probability of GI absorption, and bioavailability.
Molecular dynamics
The Protein Data Bank (PDB) has only three entries of
E. electricus AChE (PDB codes: 1C2B; 1C2O; 1EEA), all of them
̊
with low resolution (>4.0 A) [68–70]. Comparing the 3D
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natural substrate (ACh) [97, 98]. Noteworthy, the structure of
5d has a cyclic and more rigid region A, which does not allow
the interaction with a second water molecule as observed in
6d, thus preventing the direct interactions with other amino
acids from CAS (Fig. 4a and c). Additionally, 5d is also involved
in polar interactions with residues of PAS by water-mediated
̊
̊
hydrogen bound with Phe295 (2.0 A) and Tyr124 (2.0 A).
On the other hand, the hydroxyl moiety of 6d was found to
interact with important amino acids involved in the process of
catalysis of ACh. As observed in Fig. 4b and d, the hydroxyl
reaches the CAS through two water-mediated hydrogen
̊
̊
bounds with Tyr337 (1.6 A), Gly121 (1.8 A), and Glu202 (2.3
̊
and 1.8 A). According to the literature [99, 100], Gly121 is
believed to stabilize the transition state between ACh and
AChE prior to the acylation of Ser203. The insertion of 6d into
the catalytic pocket of AChE and the stabilization of those
residues may explain its good inhibitory activity. Moreover, 6d
is anchored into the entrance of PAS due to hydrogen bonds
between the backbone of Tyr341 (2.3 A) and Phe295 (2.0 A)
with NH and the SO2 of sulfonylhydrazone moiety,
respectively.
Figure 3. Superimposition of acetylcholinesterases from
Electrophorus electricus [(yellow; 1C2B), (blue; 1C2O) (green;
1EEA)] and Homo sapiens (magenta; 4EY7). Protein chains are
depicted as cartoon. Amino acids from PAS, gorge, and CAS are
highlighted as sticks. The PAS is represented by Trp286, Tyr124,
and Tyr72. The gorge is represented by Phe338 and Tyr337. The
CAS is represented by His447, Glu334, Ser203, and Trp86.
Hydrogens were removed to improve clarity. Image was
generated with PyMol 1.3 software.
̊
̊
The bioisosteric substitution of cycloketone and acylhy-
drazone by the sulfonylhydrazone moiety has proved to be an
accurate choice [101–104] due to its ability to mimic the
carbonyl (e.g., donepezil) involved in hydrogen bonding with
the backbone of Phe295, as observed for 5d and 6d.
Figure 5 shows the binding mode of donepezil with
hAChE. The two aromatic rings that are present in donepezil
mediate hydrophobic interactions (p stacking) with Trp286
and Trp86, respectively, present in the PAS and CAS. The
intermediary region, which comprises the carbonyl group of
the 2,3-dihydro-inden-1-one moiety, is responsible for
anchoring the scaffold of donepezil to the gorge by a
most active compound (6d) and for the benzodioxole
analogue (5d) within the binding pocket. These orientations
were used as starting point for MD simulations.
Based on the simulations, we could determine the
molecular interactions between protein and ligand, which
could explain the observed activity. The active pocket of
eeAChE has two distinct binding sites: peripheral binding site
(PAS) and catalytic active site (CAS). The PAS is located at
the entrance of a deep and hydrophobic gorge that connects
the PAS to the CAS in the bottom of the active site [87, 88]. The
PAS comprises Trp286, Tyr124, and Tyr72. The gorge
comprises Phe338 and Tyr337. The CAS comprises the catalytic
triad His447, Glu334 and Ser203, and Trp86. Good hAChE
inhibitors are able to bind both sites [36, 89–96].
Our MD simulations for compounds 5d and 6d (Fig. 4)
revealed that these molecules presented a very similar
binding mode compared with the crystallographic data for
donepezil (Fig. 5). Among the desired profile of an AChE
inhibitor, both molecules interacted deeply with the PAS and
gorge. Inversely, the methylcatechol and benzodioxole
moieties were inserted into the gorge, reaching the begin-
ning of CAS where Trp86 is located. However, the variation of
the IC50 between 5d and 6d (1.84 and 0.64 mM, respectively)
could be explained by several differences in their structures.
In one hand, 5d seems to interact with the backbone of
His447 through a water-mediated hydrogen bound, which
can explain why this molecule displayed an inhibitory activity,
once His447 is involved in the mechanism of catalysis of the
̊
hydrogen bond (2.9 A) with the backbone of Phe295 [34].
Water-mediated hydrogen bonds are observed between the
protonated nitrogen of piperidine ring and hydroxyls of
̊
̊
Tyr337 (2.5 A) and Tyr341 (2.8 A). Beyond these interactions
some authors [96] highlighted the ability the 5,6-dimethoxy
moiety to interact with the backbone of Trp286 by two water
molecules.
The observed binding mode of 6d is comparable to
donepezil in terms of interactions with the PAS and gorge
of AChE. This could explain why the compound inhibited the
enzyme with a low IC50. Although compound 6d could
properly anchor to the PAS and gorge sites, the CAS was still
weakly explored. This fact presents interesting perspectives
for the design of enhanced inhibitors that are able to explore
both binding sites. However, all regions of AChE can be
targets of inhibitors, and the PAS has been attributed to non-
cholinergic function, such as adhesion and the deposition of
~
b-amyloid plaques [105]. This fact was investigated by Munoz-
Ruiz et al. [106]. These authors synthesized indole-tacrine
analogues that were able to interact with the PAS and CAS,
inhibit AChE at picomolar concentrations, and inhibit Ab
aggregation whereas donepezil was found to partially inhibit
Ab aggregation (22%) [107].
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̊
Figure 4. Hydrogen bond and hydrophobic interactions in the binding pocket of eeAChE and the distance (A) of compounds 6d (a)
and 5d (b). Molecular dynamics studies were performed with GROMACS 5 software. Hydrogens were removed to improve clarity.
Hydrogen bond interactions are depicted as yellow-dashed lines. Water molecules involved in hydrogen bounds are displayed as
sticks. Two-dimensional interactions for 6d (c) and 5d (d) were generated by LigPlotþ v.1.4.5 [126]. Water molecules are depicted as
light blue spheres. Hydrogen bond interactions are depicted as green-dashed lines. Hydrophobic interactions are depicted as red-
dashed semicircles.
̊
Figure 5. Hydrogen bond and hydrophobic interactions in the binding pocket of hAChE (4EY7) and distance (A) of donepezil (a).
Hydrogens were removed to improve clarity. Hydrogen bond interactions are depicted as yellow-dashed lines. Two-dimensional
interactions of donepezil (b) were generated by LigPlotþ v.1.4.5 [126]. Water molecules are depicted as light blue spheres. Hydrogen
bond interactions are depicted as green-dashed lines. Hydrophobic interactions are depicted as red-dashed semicircles.
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using a Buchi M-565 digital melting point apparatus and were
uncorrected. High resolution mass spectrometry (HRMS) were
measured with a MicroToF Bruker Daltonics spectrometer
with an ESI source coupled to a Waters HPLC system.
Elemental analyses were performed on a Perkin Elmer 2400
CHN analyzer.
The NMR spectra as well as the InChI codes of the
investigated compounds, together with some biological
activity data, are provided as Supporting Information.
Conclusion
To develop new AChE inhibitors, appropriate modifications in
the structures of acylhydrazone compounds generated three
series of new sulfonylhydrazone analogues. Twelve sulfonyl-
hydrazone analogues were synthesized in three reaction steps
and evaluated regarding their ability to inhibit AChE. Our
results showed that the sulfonylhydrazone compounds
inhibited AChE within the micromolar range (0.64 and
51.09 mM). In general, the most active compounds presented
aryl group in region C. Additionally, SAR studies demon-
strated that methylcatechol derivatives were more active than
benzodioxole. The molecular dynamic simulations showed
that the most active compound, 6d, was able to interact with
the PAS of AChE in a similar mode to donepezil. However,
interactions with the CAS were less significant and should be
further investigated to design new inhibitors. The ability of 6d
to interact with the PAS is noteworthy because the literature
indicates that such blockade is associated with the inhibition
of Ab aggregation. Molecular modeling also revealed that 6d
presented physicochemical properties based on Lipinski’s rule
for CNS drugs, suggesting that this compound may present
good CNS penetration and GI absorption, which are desirable
properties of anti-AD drugs. Complementary studies of 6d
revealed that the compound scavenged ROS, which is also an
interesting property for neuroprotection and AD treatment.
Furthermore, the MTT assay showed that 6d might be safe
under the tested conditions, in which it was not cytotoxic at
100 mM in a human lung cell line. Therefore, arylsulfonylhy-
drazones may represent a molecular framework that should
be explored as a prototype for the design of new analogues,
aiming to optimize their AChE inhibitory activity, to access the
in vivo kinetic profile (such as stability and toxicity), and to
explore others molecular targets, which could enhance the
multifactorial aspects of AD therapy.
General procedure for the synthesis of sulfonylhydrazides
2a–l
Hydrazine hydrate (10 mmol) was dissolved in anhydrous
tetrahydrofuran (2 mL) and stirred at 5°C. Sulfonyl chloride
(1 mmol) was dissolved in tetrahydrofuran (1 mL) and added
dropwise to the solution of hydrazine. The mixture was stirred
at 5°C until complete conversion of the sulfonyl chloride (TLC,
0.5–1 h). The mixture was then extracted with ethyl acetate
(5 ꢄ 5 mL) and concentrated under vacuum. The residue was
dissolved in CHCl3 and precipitated in hexane.
4-Methylbenzenesulfonohydrazide (2a)
Yield, 74%. M.p., 107–109°C. 1H NMR (300 MHz, CDCl3,
d ¼ ppm): 1.96 (1H, s), 2.45 (3H, s), 5.69 (1H, s), 7.35 (2H, d,
J ¼ 8.2 Hz), 7.80 (2H, d, J ¼ 8.2 Hz). 13C NMR (75 MHz, CDCl3,
d ¼ ppm): 21.5, 128.2, 129.9, 133.2, 144.5.
4-Nitrobenzenesulfonohydrazide (2b)
Yield, 83%. M.p., 144–145°C. 1H NMR (300 MHz, CDCl3,
d ¼ ppm): 8.05 (2H, dt, J ¼ 8.9, 2.4 Hz), 8.42 (2H, dt, J ¼ 8.9,
2.4 Hz), 8.74 (1H, s). 13C NMR (75 MHz, DMSO-d6, d ¼ ppm):
124.1, 129.2, 144.1, 149.7.
4-(tert-Butyl)benzenesulfonohydrazide (2c)
Yield, 77%. M.p., 92–94°C. 1H NMR (300 MHz, CDCl3, d ¼ ppm):
1.35 (9H, s), 7.57 (2H, dt, J ¼ 10.8, 2 Hz), 7.83 (2H, dt, J ¼ 10.8,
2 Hz). 13C NMR (75 MHz, CDCl3, d ¼ ppm): 31.0, 35.2, 126.3,
128.1, 133.1, 157.5.
Experimental
Chemistry
4-Methoxybenzenesulfonohydrazide (2d)
General
Yield, 84%. M.p., 101–103°C. 1H NMR (300 MHz, CDCl3,
d ¼ ppm): 1.96 (1H, s), 3.88 (3H, t, J ¼ 3.5, 7.1 Hz), 5.60 (1H,
s), 7.02 (2H, dt, J ¼ 2.7, 9 Hz), 7.84 (2H, dt, J ¼ 2.7, 9 Hz).
13C NMR (75 MHz, DMSO-d6, d ¼ ppm): 55.6, 114.1, 129.5,
129.7, 162.3.
Reagents and solvents were purchased from Sigma–Aldrich,
and were used without further purification. Thin-layer
chromatography (TLC) was carried out using silica gel plates
(SiGel 60 F254 aluminum sheets, Merck) and the spots were
visualized under UV light at 254 and 280 nm. Flash chroma-
tography was performed for purification of the compounds
on silica gel 60 (SiGel 60, 220–240 mesh, Sigma–Aldrich).
1H and 13C nuclear magnetic resonance (NMR) spectra were
recorded using a Bruker Advanced DPX-300 spectrometer at
300 and 75 MHz, respectively. Chemical shifts were expressed
as d units, using tetramethylsilane (TMS) as internal standard,
and CDCl3, DMSO-d6 or acetone-d6 as solvents. The spectral
splitting patterns are described as follows: s, singlet; d,
doublet; dd, double doublet; q, quartet; t, triplet; m, multiplet
peak. Melting points were determined in open capillary tubes
4-Chlorobenzenesulfonohydrazide (2e)
Yield, 94%. M.p., 110–112°C. 1H NMR (300 MHz, CDCl3,
d ¼ ppm): 1.96 (1H, s), 5.74 (1H, s), 7.53 (2H, d, J ¼ 8.5 Hz),
7.86 (2H, d, J ¼ 8.5 Hz). 13C NMR (75 MHz, DMSO-d6, d ¼ ppm):
129.0, 129.5, 137.0, 137.5.
N-(4-(Hydrazinylsulfonyl)phenyl)acetamide (2f)
Yield, 69%. M.p., 177°C (decomposition). 1H NMR (300 MHz,
DMSO-d6, d ¼ ppm): 2.09 (3H, s), 4.04 (1H, s), 7.73 (2H, d,
J ¼ 9.0 Hz), 7.77 (2H, d, J ¼ 9.0 Hz), 8.21 (1H, s), 10.29 (1H, s).
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13C NMR (75 MHz, DMSO-d6, d ¼ ppm): 24.0, 118.4, 128.7,
General procedure for the synthesis of sulfonylhydrazones
5a–l and 6a–f
131.5, 142.9, 169.9.
A mixture of sulfonylhydrazide (1 mmol) and corresponding
aldehyde (1 mmol) was dissolved in methanol. The mixture
was stirred at reflux until complete conversion of the starting
materials (TLC, 2–9 h). The final product was filtered and
purified by crystallization or a chromatographic column
(hexane/ethyl acetate, 1:1).
Ethanesulfonohydrazide (2g)
Yield, 55%. Colorless oil. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 1.16 (3H, t, J ¼ 7.4 Hz), 3.04 (2H, q, J ¼ 7.4 Hz), 4.17
(2H, s), 7.77 (1H, s). 13C NMR (75 MHz, DMSO-d6, d ¼ ppm):
41.5, 7.7.
Propane-1-sulfonohydrazide (2h)
(E)-N-(Benzo[d][1,3]dioxol-5-ylmethylene)-4-
methylbenzenesulfonohydrazone (5a)
Yield, 56%. Colorless oil. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 0.97 (3H, t, J ¼ 7.4 Hz), 1.64 (2H, se, J ¼ 7.4 Hz),
3.01 (2H, t, J ¼ 7.4 Hz), 7.69 (1H, s), 4.26 (2H, s). 13C NMR
(75 MHz, DMSO-d6, d ¼ ppm): 12.8, 16.6, 48.8.
Yield, 77%. M.p., 136–137°C. 1H NMR (300 MHz, CDCl3,
d ¼ ppm): 2.40 (3H, s), 5.97 (2H, s), 6.75 (1H, d, J ¼ 8 Hz), 6.91
(1H, dd, J ¼ 1.5, 8 Hz), 7.19 (1H, d, J ¼ 1.5 Hz), 7.30 (3H, d,
J ¼ 8 Hz), 7.67 (1H, s), 7.85 (2H, d, J ¼ 8 Hz), 7.90 (1H, s).
13C NMR (75 MHz, CDCl3, d ¼ ppm): 21.5, 101.4, 105.7, 108.0,
123.7, 127.8, 127.9, 129.6, 135.3, 144.2, 148.0, 148.2, 149.7. %
CHN (found): C ¼ 56.61, H ¼ 4.56, N ¼ 8.69. % CHN (calcd.):
C ¼ 56.59, H ¼ 4.43, N ¼ 8.80.
Propane-2-sulfonohydrazide (2i)
Yield, 14%. Colorless oil. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 1.20 (6H, d, J ¼ 6.8 Hz), 3.43 (1H, hep, J ¼ 13.7,
J ¼ 6.8 Hz), 4.25 (2H, s), 7.77 (1H, s). 13C NMR (75 MHz, DMSO-
d6, d ¼ ppm): 15.9, 46.9.
(E)-N-(Benzo[d][1,3]dioxol-5-ylmethylene)-4-
Butane-1-sulfonohydrazide (2j)
nitrobenzenesulfonohydrazone (5b)
Yield, 70%. Colorless oil. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 0.88 (3H, t, J ¼ 7.3 Hz), 1.38 (2H, se, J ¼ 14.4,
7.3 Hz), 1.60 (2H, qi, J ¼ 7.3 Hz), 3.03 (2H, t, J ¼ 7.6 Hz), 7.72
(1H, s), 4.26 (2H, s). 13C NMR (75 MHz, DMSO-d6, d ¼ ppm):
13.5, 21.0, 24.9, 46.9.
Yield, 53%. M.p., 189–190°C. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 6.06 (2H, s), 6.93 (1H, d, J ¼ 8 Hz), 7.06 (1H, dd,
J ¼ 1.5, 8 Hz), 7.14 (1H, d, J ¼ 1.5 Hz), 7.87 (1H, s), 8.14 (2H, dt,
J ¼ 2, 8.8 Hz), 8.43 (2H, dt, J ¼ 2, 8.8 Hz), 11.66 (1H, s). 13C NMR
(75 MHz, DMSO-d6, d ¼ ppm): 101.5, 104.9, 108.3, 123.3, 124.4,
127.6, 128.8, 144.2, 147.8, 148.1, 149.2, 149.9. % CHN (found):
C ¼ 48.07, H ¼ 3.27, N ¼ 12.05. % CHN (calcd.): C ¼ 48.14,
H ¼ 3.17, N ¼ 12.03.
Hexane-1-sulfonohydrazide (2k)
Yield, 82%. White solid. 1H NMR (300 MHz, CDCl3, d ¼ ppm):
0.88 (3H, t, J ¼ 6.0 Hz), 1.42–1.30 (6H, m), 1.79 (2H, qi, J ¼ 8.1),
3.09 (2H, t, J ¼ 7.9 Hz), 3.82 (2H, s), 5.79 (1H, s). 13C NMR
(75 MHz, CDCl3, d ¼ ppm): 14.0, 22.4, 23.2, 28.0, 31.3, 49.4.
(E)-N-(Benzo[d][1,3]dioxol-5-ylmethylene)-4-(tert-butyl)-
benzenesulfonohydrazone (5c)
Yield, 65%. M.p., 133–134°C. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 1.28 (9H, s), 6.05 (2H, s), 6.92 (1H, d, J ¼ 8 Hz), 7.04
(1H, dd, J ¼ 1.2, 8 Hz), 7.11 (1H, d, J ¼ 1.2 Hz), 7.63 (2H, d,
J ¼ 8.5 Hz), 7.80 (2H, d, J ¼ 8.5 Hz), 7.83 (1H, s), 11.29 (1H, s).
13C NMR (75 MHz, DMSO-d6, d ¼ ppm): 30.7, 34.8, 101.4, 104.8,
108.3, 122.9, 126.0, 127.0, 128.0, 136.3, 146.7, 147.8, 149.0,
155.9. % CHN (found): C ¼ 60.15, H ¼ 5.73, N ¼ 7.77. % CHN
(calcd.): C ¼ 59.98, H ¼ 5.59, N ¼ 7.77.
Octane-1-sulfonohydrazide (2l)
Yield, 60%. White solid, m.p.: 52.0–53.3°C. 1H NMR (300 MHz,
CDCl3, d ¼ ppm): 0.86 (3H, t, J ¼ 5.7 Hz), 1.39–1.26 (10H, m),
1.78 (2H, qi, J ¼ 7.5 Hz), 3.09 (2H, t, J ¼ 7.5 Hz), 3.78 (1H, s), 6.07
(1H, s). 13C NMR (75 MHz, CDCl3, d ¼ ppm): 14.1, 22.6, 23.2,
29.0, 29.1, 31.8, 49.2.
General procedure for the synthesis of piperonal (3)
Piperonyl alcohol (1 mmol) was dissolved in CH2Cl2 (5 mL),
and MnO2 (15 mmol) was added to the solution. The mixture
was stirred at room temperature until complete conversion
of the starting material (TLC, 4 h). The mixture was filtered in
celite and treated with activated charcoal. The solution was
filtered and concentrated under vacuum. The residue was
purified by a chromatographic column (hexane/ethyl ace-
tate, 1:1).
(E)-N-(Benzo[d][1,3]dioxol-5-ylmethylene)-4-
methoxybenzenesulfonohydrazone (5d)
Yield, 28%. M.p., 122–124°C. 1H NMR (300 MHz, CDCl3,
d ¼ ppm): 3.85 (3H, s), 5.98 (2H, s), 6.76 (1H, d, J ¼ 7.9 Hz), 6.91
(1H,d, J ¼ 7.9 Hz),6.97(2H, d,J ¼ 8.5 Hz),7.19(1H,s), 7.67(1H,s),
7.70 (1H, s), 7.90 (2H, d, J ¼ 8.5 Hz). 13C NMR (75 MHz, DMSO-d6,
d ¼ ppm): 55.6, 101.4, 104.8, 108.3, 114.3, 122.8, 128.1, 129.4,
130.6, 146.7, 147.8, 148.9, 162.5. % CHN (found): C ¼ 54.36,
H ¼ 4.46, N ¼ 8.28. % CHN (calcd.): C ¼ 53.88, H ¼ 4.22, N ¼ 8.38.
Benzo[d][1,3]dioxole-5-carbaldehyde (3)
Yield, 75%. M.p., 37–38°C. 1H NMR (300 MHz, CDCl3, d ¼ ppm):
6.07 (1H, s), 6.92 (1H, d, J ¼ 7.8 Hz), 7.32 (1H, s), 7.40 (1H, d,
J ¼ 7.8 Hz), 9.81 (1H, s). 13C NMR (75 MHz, CDCl3) d ¼ ppm:
102.2, 106.2, 108.5, 128.4, 131.4, 148.3, 152.7, 190.8.
(E)-N0-(Benzo[d][1,3]dioxol-5-ylmethylene)-
ethanesulfonohydrazide (5g)
Yield, 34%. M.p., 123.4–125.0°C. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 1,20 (3H, t, J ¼ 7.0 Hz), 3.21 (2H, q, J ¼ 7.0 Hz), 6.07
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(2H, s), 6.95 (1H, d, J ¼ 7.9), 7.10 (1H, d, J ¼ 7.0 Hz), 7.21 (1H, s),
7.90 (1H, s), 10.96 (1H, s). 13C NMR (75 MHz, DMSO-d6,
d ¼ ppm): 7.7, 44.5, 101.5, 105.0, 108.3, 122.9, 128.2, 146.2,
147.9, 148.9. % CHN (found): C ¼ 46.87, H ¼ 4.72, N ¼ 10.93. %
CHN (calcd.): C ¼ 46.36, H ¼ 4.75, N ¼ 10.52.
J2 ¼ 8.1 Hz), 7.27 (1H, d, J ¼ 1.5 Hz), 8.14 (1H, s), 7.76 (1H, s).
13C NMR (75 MHz, CDCl3, d ¼ ppm): 14.0, 22.5, 23.1, 28.2, 28.9,
29.0, 31.7, 51.1, 101.5, 105.8, 108.2, 123.7, 127.7, 147.6, 148.3,
149.8. % CHN (found): C ¼ 56.45, H ¼ 7.11, N ¼ 8.23. % CHN
(calcd.): C ¼ 56.22, H ¼ 6.56, N ¼ 8.25.
(E)-N0-(Benzo[d][1,3]dioxol-5-ylmethylene)propane-1-
sulfonohydrazide (5h)
(E)-N-(4-Hydroxy-3-methoxybenzylidene)-4-
methylbenzenesulfonohydrazone (6a)
Yield, 25%. M.p., 123.4–125.2°C. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 0.98 (3H, t, J ¼ 7.4 Hz), 1.69 (2H, sex, J ¼ 7.5 Hz), 3.17
(2H, t, J ¼ 7.5 Hz), 6.07 (2H, s), 6.96 (1H, d, J ¼ 8.0 Hz), 7.10 (1H,
d, J ¼ 8.0 Hz), 7.22 (1H, s), 7.89 (1H, s), 10.95 (1H, s). 13C NMR
(75 MHz, DMSO-d6, d ¼ ppm): 12.2, 16.1, 51.4, 101.1, 104.7,
108.0, 122.5, 127.9, 145.8, 147.5, 148.6. % CHN (found):
C ¼ 48.88, H ¼ 5.22, N ¼ 10.36. % CHN (calcd.): C ¼ 49.57,
H ¼ 5.29, N ¼ 9.94.
Yield, 82%. M.p., 100–101°C. 1H NMR (300 MHz, (CD3)2CO,
d ¼ ppm): 2.40 (3H, s), 3.887 (3H, s), 6.83 (1H, d, J ¼ 8.1 Hz), 7.04
(1H, dd, J ¼ 1.5, 8.1 Hz), 7.27 (1H, d, J ¼ 1.5 Hz), 7.39 (2H, d,
J ¼ 8.1 Hz), 7.84 (d, 2H, J ¼ 8.1 Hz), 7.89 (1H, s), 9.79 (1H, s).
13C NMR (75 MHz, (CD3)2CO, d ¼ ppm): 21.4, 56.2, 109.8, 115.8,
122.6, 127.0, 128.6, 130.2, 137.6, 144.5, 148.6, 148.7, 149.8. %
CHN (found): C ¼ 55.87, H ¼ 5.21, N ¼ 8.43. % CHN (calcd.):
C ¼ 56.24, H ¼ 5.03, N ¼ 8.74.
(E)-N0-(Benzo[d][1,3]dioxol-5-ylmethylene)propane-2-
sulfonohydrazide (5i)
(E)-4-(tert-Butyl)-N0-(4-hydroxy-3-methoxybenzylidene)-
benzenesulfonohydrazone (6c)
Yield, 29%. M.p., 109.5–111.6°C. 1H NMR (300 MHz, CDCl3,
d ¼ ppm): 1.44 (3H, s), 1.46 (3H, s), 3.59 (H, sep, J ¼ 6.9 Hz), 5.99
(2H, s), 6.80 (1H, d, J ¼ 8.0 Hz), 7.08 (1H, dd, J1 ¼ 1.6 Hz,
J2 ¼ 8.0 Hz), 7.26 (1H, s), 7.74 (1H, s), 7.82 (1H, s). 13C NMR
(75 MHz, DMSO-d6, d ¼ ppm): 16.3, 52.3, 101.5, 105.8, 108.2,
123.6, 127.8, 147.0, 148.3, 149.7. % CHN (found): C ¼ 48.88,
Yield, 86%. M.p., 74–76°C. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 1.28 (9H, s), 3.78 (3H, s), 6.77 (1H, d, J ¼ 8.1 Hz), 6.98
(1H, dd, J ¼ 1.5, 8.1 Hz), 7.10 (1H, d, J ¼ 1.5 Hz), 7.62 (2H, d,
J ¼ 8.4 Hz), 7.80 (2H, d, J ¼ 8.4 Hz), 7.80 (1H, s), 9.46 (1H, s),
11.16 (1H, s). 13C NMR (75 MHz, DMSO-d6, d ¼ ppm): 31.2, 35.6,
56.2, 109.8, 115.8, 122.6, 126.7, 127.0, 128.4, 137.7, 148.6,
148.7, 149.8, 157.2. % CHN (found): C ¼ 59.34, H ¼ 6.24,
N ¼ 7.48. % CHN (calcd.): C ¼ 59.65, H ¼ 6.12, N ¼ 7.73.
H ¼ 5.22, N ¼ 10.36.
N ¼ 10.07.
%
CHN (calcd.): C ¼ 48.65, H ¼ 5.19,
(E)-N0-(Benzo[d][1,3]dioxol-5-ylmethylene)butane-1-
sulfonohydrazide (5j)
(E)-N0-(4-Hydroxy-3-methoxybenzylidene)-4-
methoxybenzenesulfonohydrazone (6d)
Yield, 55%. M.p., 89.8–90.4°C. 1H NMR (300 MHz, CDCl3,
d ¼ ppm): 0.94 (3H, t, J ¼ 7.2 Hz), 1.47 (2H, se, J ¼ 7.5 Hz), 1.84
(2H, qi, J ¼ 7.5 Hz), 3.29 (2H, t, J ¼ 7.8 Hz), 5.99 (2H, s), 6.79 (1H,
d, J ¼ 8.0 Hz), 7.00 (1H, d, J ¼ 8.0 Hz), 7.76 (1H, s), 8.22 (1H, s).
13C NMR (75 MHz, CDCl3, d ¼ ppm): 13.5, 25.1, 21.5, 50.8, 101.5,
105.8, 108.2, 123.7, 127.7, 147.6, 148.3, 149.8. % CHN (found):
Yield, 65%. M.p., 139–141°C. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 3.88 (3H, s), 3.88 (3H, s), 6.83 (1H, d, J ¼ 8.1 Hz), 7.04
(1H, dd, J ¼ 1.7, 8.1 Hz), 7.09 (2H, dd, J ¼ 1.9, 7.0 Hz), 7.27 (1H,
d, J ¼ 1.7 Hz), 7.88–7.91 (3H, m, 7-CH), 9.72 (1H, s). 13C NMR
(75 MHz, (CD3)2CO, d ¼ ppm): 56.0, 56.2, 109.7, 114.8, 115.8,
122.6, 127.1, 130.7, 132.1, 148.5, 148.7, 149.7, 164.0. % CHN
(found): C ¼ 53.87, H ¼ 4.94, N ¼ 8.33. % CHN (calcd.): C
¼ 53.56, H ¼ 4.79, N ¼ 8.33.
C ¼ 50.69, H ¼ 5.67, N ¼ 9.85.
%
CHN (calcd.): C ¼ 50.97,
H ¼ 5.70, N ¼ 9.60.
(E)-N0-(Benzo[d][1,3]dioxol-5-ylmethylene)hexane-1-
sulfonohydrazide (5k)
(E)-4-Chloro-N0-(4-hydroxy-3-methoxybenzylidene)-
benzenesulfonohydrazone (6e)
Yield, 57%. M.p., 65.4–67.5°C. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 0.87 (3H, t, J ¼ 6.5 Hz), 1.85 (4H, qi, J ¼ 7.8 Hz), 1.45–
1.39 (2H, m), 1.90–1.80 (2H, m), 3.28 (2H, t, J ¼ 7.9 Hz), 5.99
(2H, s), 6.79 (1H, d, J ¼ 8.0 Hz), 7.00 (1H, d, J ¼ 8.0 Hz), 7.76 (1H,
s), 8.09 (1H, s). 13C NMR (75 MHz, DMSO-d6, d ¼ ppm): 13.9,
22.3, 23.1, 27.9, 31.1, 51.1, 101.5, 105.8, 108.2, 123.7, 127.7,
147.6, 148.3, 149.8. % CHN (found): C ¼ 53.83, H ¼ 6.45,
N ¼ 8.97. % CHN (calcd.): C ¼ 51.83, H ¼ 5.89, N ¼ 8.39.
Yield, 64%. M.p., 117–119°C. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 3.78 (3H, s), 6.78 (1H, d, J ¼ 8.1 Hz), 6.98 (1H, dd,
J ¼ 1.8, 8.1 Hz), 7.11 (1H, d, J ¼ 1.8 Hz), 7.69 (2H, dt, J ¼ 2,
8.7 Hz), 7.81 (1H, s), 7.89 (2H, dt, J ¼ 2, 8.7 Hz), 9.49 (1H, s),
11.27 (1H, s). 13C NMR (75 MHz, DMSO-d6, d ¼ ppm): 55.5,
109.6, 115.4, 121.2, 124.9, 129.1, 129.2, 137.8, 147.8, 148.3,
149.0. % CHN (found): C ¼ 49.23, H ¼ 3.83, N ¼ 8.14. % CHN
(calcd.): C ¼ 49.34, H ¼ 3.85, N ¼ 8.22.
(E)-N0-(Benzo[d][1,3]dioxol-5-ylmethylene)octane-1-
sulfonohydrazide (5l)
(E)-N-(4-((2-(4-Hydroxy-3-methoxybenzylidene)-
hydrazinyl)sulfonyl)phenyl)acetamide (6f)
Yield, 37%. M.p., 67.6–67.9°C. 1H NMR (300 MHz, CDCl3,
d ¼ ppm): 0.86 (3H, t, J ¼ 6.7 Hz), 1.26 (8H, t), 1.41 (2H, q,
J ¼ 6.9 Hz), 1.85 (2H, qi, J ¼ 7.6 Hz), 3.28 (2H, t, J ¼ 7.9 Hz), 5.99
(2H, s), 6.79 (1H, d, J ¼ 8.0 Hz), 7.01–6.98 (1H, dd, J1 ¼ 1.4 Hz,
Yield, 46%. M.p., 173–175°C. 1H NMR (300 MHz, DMSO-d6,
d ¼ ppm): 2.07 (3H, s), 3.78 (3H, s), 6.77 (1H, d, J ¼ 8.1 Hz), 6.97
(1H, d, J ¼ 8.1 Hz), 7.10 (1H, s), 7.76 (2H, d, J ¼ 9 Hz), 7.79 (1H, s),
7.81 (2H, d, J ¼ 9 Hz), 9.45 (1H, s), 10.29 (1H, s), 11.04 (1H, s).
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13C NMR (75 MHz, DMSO-d6, d ¼ ppm): 24.0, 55.5, 109.5, 115.4,
118.4, 121.0, 125.1, 128.4, 132.5, 143.1, 147.5, 147.8, 148.3,
169.9. % CHN (found): C ¼ 52.80, H ¼ 4.87, N ¼ 11.37. % CHN
(calcd.): C ¼ 52.88, H ¼ 4.72, N ¼ 11.56.
streptomycin. Cells were grown at 37°C in 5% CO2, 95%
air, and complete humidity. All of the experiments were
performed using cells in the exponential growth phase
[110, 111]. The LL24 cell line was purchase from the American
Type Culture Collection.
LL24 cells were plated at a concentration of 104 cells per
well in 96-well plates. Cells were incubated for 24 h with
solutions of compounds (100 mM). Following incubation,
10 mL of MTT solution (1 mg/mL) was added to each well,
and the plate was further incubated for 2 h. The supernatant
was removed, and 100 mM DMSO was added and incubated
for 5 min at room temperature. The absorbance of formazan
was read at 540 nm in a Thermoplate TP plate reader [112].
Each experiment was performed using four replicates for each
concentration and was repeated in triplicate.
Biological assays
AChE inhibition
Ellman’s colorimetric method [108] was used to evaluate the
inhibitory effect of the synthesized compounds against AChE
from E. electricus (electric eel, EC Number: 3.1.1.7), Type VI-S,
lyophilized powder (200–1000 units/mg protein). The enzyme
was purchased from Sigma–Aldrich as a tetramer composed of
four equal subunits of 70 kDa each. The molecular weight is
280 kDa (gel filtration); isoelectric point: 5.51; extinction
coefficient: E1% ¼ 18.0 (280 nm).
The AChE was resuspended in Tris/HCl, 50 mmol/L, at pH 8.
The resulting solution was diluted to a final concentration of
1 U/mL. Activity was measured using 125 mL of a 10 mM
solution of 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB, also
known as Ellman’s reagent), 50 mL of Milli-Q H2O, and 25 mL of
AChE at 1 U/mL in a 96-well microplate. After 15 min of
incubation at 25°C with 30 mL of a solution of each test
compound (0.1, 0.5, 1, 10, and 100 mM), 30 mL of a 0.8 mM
solution of the substrate (acetylthiocholine iodide [ATC]) was
added. The AChE inhibitor tacrine (0.0025 mM) was used as a
positive control. Absorbance was read at 415 nm every 30 s for
5 min in a plate reader (Microplate Reader Model 680, Bio-Rad
Laboratories, UK). All of the experiments were performed in
triplicate. The minimum inhibitory concentration that re-
duced enzyme activity by 50% (IC50) was calculated using
Molecular modeling
In silico ADME
The in silico ADME profile was computed based on Lipinski’s
rule for CNS drugs and other pharmacokinetic properties of
compounds 5d, 6d, and donepezil, including computed
aqueous solubility (CLogS), polar surface area (PSA), GI
absorption, blood–brain barrier penetration, CYP inhibition,
parameters of drug-likeness, and other medicinal chemistry
tools using the free SwissADME website (http://www.
swissadme.ch/) of the Swiss Institute of Bioinformatics
[113–115] and Spartan 14 software [116].
The structures of 5d, 6d, and donepezil were built and
energy-minimized at MMFF94 [117] and PM6 [118] using
Spartan 14 software [116]. For SwissADME, the structures
were constructed and converted into SMILES format.
Among the strategies for determining drug-likeness, we
used the Ghose (presence of a benzene ring, tertiary aliphatic
amines, carboxamides, and alcohols) [119], Verber (routable
bond and PSA) [120], Egan (PSA and AlogP98) [121], and
Muegge (number of pharmacophore points) [122] models.
GraphPad Prism software and
method.
a non-linear regression
DPPH assay
The ROS scavenger activity of the test compounds was
determined using DPPH• as a stable free radical. Sulfonylhy-
drazones 5d and 6d were diluted in dimethylsulfoxide (DMSO)
at 10–4000 mg/mL. BHT (IC50 ¼ 17 mM) and rutine (IC50 ¼ 0.94
mM) were used as controls. DMSO DPPH• (100 mM, 3 mL) was
added to previously sample solutions (1 mL). After 1 h
incubation at room temperature while protected from light,
Molecular dynamics
The structure of eeAChE was modeled with SWISS-MODEL
fully automated protein structure homology-modeling
server [84, 85] using as template AChE complexed with the
donepezil inhibitor (PDB code: 4EY7). AutoDock Vina [86] was
used to obtain two different orientations for the most active
compound (6d) and for the benzodioxole analogue (5d)
within the binding pocket. These orientations were used as
starting point for MD simulations.
absorbance was read at 515 nm in
(1 cm) [109]. All of the experiments were performed in
triplicate. Absorbance values were transformed into
a quartz cuvette
a
percentage (equation below), and IC50 values were calculated
from calibration curves using Microsoft Excel software.
Every simulation was performed with the GROMOS96 54a7
force field. The ligand topology, for compounds 6d and 5d,
was obtained from the automated topology builder and
repository (ATB) service [123]. The MD simulations were
carried out using GROMACS 5 [124] package. The model was
solvated in a box with TIP3P water molecules, keeping the
ðAc ꢁ AaÞ ꢄ 100
% scavenging of free radicals ¼
Ac
where Aa is sample absorbance and Ac is negative control
absorbance.
̊
MTT assay
boundary of box at least 12 A away from any protein atoms.
The human lung fibroblast cell line LL24 was cultured in
media that consisted of RPMI-1640 supplemented with 10%
fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL
The whole system was energetically minimized by the
steepest-descent method, and the minimized systems submit-
ted to nVT and nPT equilibration runs keeping fixed the
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€
protein structure. The MD simulations were then performed
in the nPT ensemble with periodic boundary conditions.
Electrostatic interactions were calculated using the particle
mesh Ewald (PME) algorithm and van der Waals interactions
[16] E. Ozturan Ozer, O. Unsal Tan, K. Ozadali,
€ €
T. KucSukkilincS, A. Balkan, G. UcSar, Bioorg. Med. Chem.
Lett. 2013, 23, 440–443.
ꢀ
[17] G. S. Da-Silva, M. Figueiro, C. F. Tormena, F. Coelho,
̊
were accounted for with a cutoff distance of 11 A. All
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3rd ed., Academic Press, New York 2008.
simulations were carried out for 10 ns using a 2 fs time step,
and snapshots for analysis were saved every 20 ps. The most
representative conformation of the ligand bound to the
active site was obtained by a cluster analysis using Molecular
Dynamics Simulation analysis module from UCSF
chimera [125].
€
ꢁ
ꢀ
ꢀ
ꢀ
[20] J. Chlebek, Z. Novak, D. Kassemova, M. Safratova,
ꢀ
ꢀ
ꢁꢀ
The authors are grateful to Faculty of Pharmacy of University
J. Kostelnık, L. Maly, M. Locarek, L. Opletal,
ꢁ ꢀ ꢀ ꢀ ꢁ ꢀ
A. Host’alkova, M. Hrabinova, J. Kunes, P. Novotna,
~
of Sao Paulo, which allowed the development of this work,
ꢀ ꢀ ꢀ ꢀ ꢀ ꢀ
M. Urbanova, L. Novakova, K. Macakova, D. Hulcova,
and to CNPq (134669/2013-4), CAPES and FAPESP (2013/19311-
ꢀ
ꢀ
ꢀ
ꢀ
6) for financial support.
P. Solich, C. Perez Martın, D. Jun, L. Cahlıkova, Chem.
Biodivers. 2016, 13, 91–99.
The authors have declared no conflict of interest.
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