Hydrazones of 4-(Trifluoromethyl)benzohydrazide as new inhibitors of acetyl- and butyrylcholinesterase

Article abstract of DOI:10.3390/molecules26040989

Based on the broad spectrum of biological activity of hydrazide-hydrazones, trifluoromethyl compounds, and clinical usage of cholinesterase inhibitors, we investigated hydrazones obtained from 4-(trifluoromethyl)benzohydrazide and various benzaldehydes or aliphatic ketones as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). They were evaluated using Ellman’s spectrophotometric method. The hydrazide-hydrazones produced a dual inhibition of both cholinesterase enzymes with IC₅₀ values of 46.8-137.7 μM and 19.1-881.1 μM for AChE and BuChE, respectively. The majority of the compounds were stronger inhibitors of AChE; four of them (2-bromobenzaldehyde, 3-(trifluoromethyl)benzaldehyde, cyclohexanone, and camphor-based 2o, 2p, 3c, and 3d, respectively) produced a balanced inhibition of the enzymes and only 2-chloro/trifluoromethyl benzylidene derivatives 2d and 2q were found to be more potent inhibitors of BuChE. 4-(Trifluoromethyl)-N’-[4-(trifluoromethyl)benzylidene]benzohydrazide 2l produced the strongest inhibition of AChE via mixed-type inhibition determined experimentally. Structure-activity relationships were identified. The compounds fit physicochemical space for targeting central nervous systems with no apparent cytotoxicity for eukaryotic cell line together. The study provides new insights into this CF₃-hydrazide-hydrazone scaffol.

Full text of DOI:10.3390/molecules26040989

molecules
Article
Hydrazones of 4-(Trifluoromethyl)benzohydrazide as New
Inhibitors of Acetyl- and Butyrylcholinesterase
Martin Krátký 1,
*
, Katarína Svrcˇková 2 , Quynh Anh Vu 1 , Šárka Šteˇpánková 2 and Jarmila Vinšová 1
1
Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové,
Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic;
vuq@faf.cuni.cz (Q.A.V.); vinsova@faf.cuni.cz (J.V.)
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice,
2
Studentská 573, 532 10 Pardubice, Czech Republic; katarina.svrckova@upce.cz (K.S.);
sarka.stepankova@upce.cz (Š.Š.)
*
Correspondence: martin.kratky@faf.cuni.cz
Abstract: Based on the broad spectrum of biological activity of hydrazide–hydrazones, trifluo-
romethyl compounds, and clinical usage of cholinesterase inhibitors, we investigated hydrazones
obtained from 4-(trifluoromethyl)benzohydrazide and various benzaldehydes or aliphatic ketones as
potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). They were
evaluated using Ellman’s spectrophotometric method. The hydrazide–hydrazones produced a dual
inhibition of both cholinesterase enzymes with IC50 values of 46.8–137.7 µM and 19.1–881.1 µM for
AChE and BuChE, respectively. The majority of the compounds were stronger inhibitors of AChE;
four of them (2-bromobenzaldehyde, 3-(trifluoromethyl)benzaldehyde, cyclohexanone, and camphor-
based 2o, 2p, 3c, and 3d, respectively) produced a balanced inhibition of the enzymes and only
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
2-chloro/trifluoromethyl benzylidene derivatives 2d and 2q were found to be more potent inhibitors
of BuChE. 4-(Trifluoromethyl)-N’-[4-(trifluoromethyl)benzylidene]benzohydrazide 2l produced the
strongest inhibition of AChE via mixed-type inhibition determined experimentally. Structure–activity
relationships were identified. The compounds fit physicochemical space for targeting central nervous
systems with no apparent cytotoxicity for eukaryotic cell line together. The study provides new
insights into this CF3-hydrazide–hydrazone scaffold.
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Krátký, M.; Svrcˇková, K.;
Vu, Q.A.; Šteˇpánková, Š.; Vinšová, J.
Hydrazones of 4-(Trifluoromethyl)
benzohydrazide as New Inhibitors of
Acetyl- and Butyrylcholinesterase.
Molecules 2021, 26, 989. https://
doi.org/10.3390/molecules26040989
Keywords: acetylcholinesterase inhibition; butyrylcholinesterase inhibition; enzyme inhibition;
hydrazides; hydrazones; 4-(trifluoromethyl)benzohydrazide
Academic Editor: Clelia Dallanoce
Received: 10 January 2021
Accepted: 11 February 2021
Published: 13 February 2021
1. Introduction
An increasing interest in fluorinated compounds is due to the favorable effect of
fluorine on pharmacological properties. It can be conservatively estimated that globally
about 20–25% of drugs in the pharmaceutical pipeline contain at least one fluorine atom [
Fluorine is making an impact in pharmaceuticals not only in a fast-growing number of
fluorinated drugs but also in the development of many of the best health care products [ ].
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1].
2
The special properties that make fluorinated motifs very attractive in medicinal chemistry
and chemical biology include small atomic radius, high electronegativity, nuclear spin of
one-half, and low polarizability of the C–F bond [3,4]. Substituting C–H by C–F bonds
clearly benefits from the fact that the size of organofluorine is only slightly larger than the
size and volume of the hydrogen substituent and that consequently no particular steric
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
hindrance is encountered in most H
such as F, CF3, OCF3, CHF2 etc., CF3 is a very familiar pharmacophore [
synthesized group—usually replacing chlorine, methyl, ethyl, or isopropyl group—is a
frequently used bioisostere in medicinal chemistry, dating from 1929 [ ]. This electron
withdrawing functionality increases the biological half-life by impeding the oxidative
→
F replacements [
5
]. Among the fluorinated groups
6]. This abiotically
7
Molecules 2021, 26, 989. https://doi.org/10.3390/molecules26040989
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 989
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metabolism, and its lipophilicity improves cellular membrane permeability, thus increasing
absorption [8,9].
Given together, the CF3 group belongs to common and useful motifs in central nervous
system (CNS)-acting drugs—for example, antipsychotics trifluoperazine (I) and flupentixol
(II), aprepitant (III) used against nausea and vomitus; central anesthetics isoflurane (IV)
and sevoflurane (V); fluoxetine (VI) and fluvoxamine (VII) prescribed as antidepressants;
centrally acting nonsteroidal anti-inflammatory drug celecoxib (VIII); anorectic drug fen-
fluramine (IX); and benzodiazepine anxiolytics, hypnotics, and anticonvulsants such as
triflunordazepam (X) and quazepam (XI) (Figure 1).
Figure 1. Examples of trifluoromethyl motif containing CNS-acting drugs.
Compounds bearing hydrazide–hydrazone scaffold (
−
CO–NH–N=C(R) ; i.e., fused
−
hydrazide and hydrazone moieties) have a substantial beneficial value in the development
of novel antibacterial, mainly antitubercular, antiplatelet, anticancer, anti-inflammatory,
and, with regard to targeting the CNS, also of anticonvulsant, analgesic, and antidepressant
agents [10,11].
4-(Trifluoromethyl)benzohydrazide (
tential antimicrobial agents active against Gram-positive and Gram-negative bacteria,
mycobacteria, yeasts, and molds [12 14]. Moreover, hydrazones of have exhibited
inhibition of various enzymes, i.e., bacterial DNA-gyrase and FabH ( -ketoacyl-acyl car-
1)-based hydrazones have been reported as po-
–
1
β
rier protein synthase III) [15], urease [14], cyanobacterial fructose-1,6-/sedoheptulose-
1,7-bisphosphatase [16] and, more importantly, also acetylcholinesterase (AChE) and bu-
tyrylcholinesterase (BuChE) [17,18]. The enzyme inhibiting properties are not unique
for the CF3 group bearing hydrazide–hydrazones, of course. Illustratively, hydrazones
derived from 4-hydroxybenzohydrazide have been studied as a new class of dual in-
hibitors of thymidine phosphorylase and cancer cell proliferation, which deserves to be
further investigated for anti-cancer drug development [19], as well as indole-based ace-
tohydrazide inhibitors of this enzyme [20]. Also, xanthinoxidase can be inhibited by
hydrazide–hydrazones in free form as well as after their coordination to copper (II) [21].
Laccase is a copper-containing polyphenol oxidase that catalyzes the radical reduction
of atmospheric oxygen to water with simultaneous oxidation of electron-rich aromatic
compounds (polyphenols, anilines). Inhibition of this enzyme might weaken activity of the
plant pathogens that utilize it in various biochemical processes. The relationship between
4-hydroxybenzohydrazide-hydrazones and laccase from Trametes versicolor was studied

Molecules 2021, 26, 989
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and a competitive type of inhibition was identified [22]. In our previous study [23], N-alkyl-
2-[4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides were identified as inhibitors of
both AChE and BuChE. Based on here presented findings, we investigated our trifluo-
romethylated hydrazide–hydrazones 2–3 (Table 1) [12] as potential inhibitors of AChE a
BuChE together with their newly prepared analogues 2n–2u.
Table 1. Inhibition of AChE and BuChE, selectivity and toxicity data of 1–3.
Selectivity
BuChE/AChE
IC50
(HepG2) [12]
Code
R
IC50 AChE (µM)
IC50 BuChE (µM)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
H
4-Cl
3-Cl
75.90 ± 1.6
88.5 ± 1.4
77.1 ± 0.03
128.5 ± 10.0
101.9 ± 3.8
70.4 ± 2.8
47.4 ± 1.8
137.7 ± 3.0
91.4 ± 6.2
108.5 ± 2.2
62.1 ± 0.02
46.8 ± 2.1
60.0 ± 0.4
96.9 ± 1.3
62.6 ± 3.2
49.9 ± 1.4
50.0 ± 1.6
81.3 ± 2.6
59.7 ± 5.8
651.3 ± 31.5
253.8 ± 3.3
260.9 ± 40.7
63.6 ± 0.9
229.2 ± 14.5
249.7 ± 7.5
881.1 ± 8.0
287.2 ± 12.6
253.1 ± 9.6
279.1 ± 2.9
342.3 ± 11.3
175.0 ± 8.3
190.6 ± 1.1
166.3 ± 0.8
54.2 ± 0.1
63.6 ± 2.8
19.1 ± 0.5
132.9 ± 10.2
93.1 ± 4.9
8.7
2.9
3.4
0.5
2.3
3.5
18.6
2.1
2.8
2.6
5.5
3.7
3.2
1.7
0.9
1.3
0.4
1.6
1.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ND
2-Cl
4-OH
3-OH
2-OH
2-OH-5-Cl
4-CH3
4-CH3O
4-Br
4-CF3
4-NO2
3-Br
2-Br
ND
ND
ND
ND
3-CF3
2-CF3
3-NO2
2-NO2
2s
ND
2t
2u
H
CH3
62.9 ± 0.8
75.3 ± 1.5
189.8 ± 1.7
244.9 ± 7.0
3.0
3.3
ND
ND
3a
3b
3c
propan-2-ylidene
cyclopentylidene
cyclohexylidene
1,7,7-
69.7 ± 5.2
69.2 ± 1.0
61.2 ± 1.3
424.3 ± 1.4
199.8 ± 13.2
74.7 ± 2.7
6.1
2.9
1.2
>100
>100
>100
trimethylbicyclo-
[2.2.1]heptan-2-
ylidene
3d
59.3 ± 1.8
57.2 ± 3.4
1.0
>100
1 [23]
-
69.4 ± 1.4
204.0 ± 2.7
2.77 ± 0.15
2.9
1.8
>100
ND
Galantamine
1.54 ± 0.02
IC50 values are expressed as the mean
enzyme are given in bold as well as the most selective inhibitors for both enzymes.
±
SD (n = three independent experiments). ND: not determined. The lowest IC50 values for each

Molecules 2021, 26, 989
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2. Results and Discussion
2.1. Chemistry
The title hydrazide
1
was prepared from 4-(trifluoromethyl)benzoic acid by a two-step
process. First, this acid was esterified by methanol in the presence of a catalytic amount of
sulfuric acid under heating in an excess of the alcohol. Then, the methyl ester was converted
to hydrazide by hydrazinolysis using hydrazine hydrate in boiling ethanol (Scheme 1).
The overall yield was almost quantitative. This procedure was described previously by
our group for 4-iodobenzohydrazide [24]. The hydrazone–hydrazides were synthesized
by the treatment of
methanol for 2 h (Scheme 2); for ketones with acidic catalyst (concentrated sulfuric acid). The
yields of the hydrazide–hydrazones and ranged from 85% to 99% and from 68% to 87%,
respectively. The characterization of the compounds 2a 2m and was reported in depth
1 (1 mmol) with various aldehydes and ketones (1.1 mmol) in boiling
2
3
–
3
by Krátký et al. [12]; the remaining ones were prepared newly on the basis of the results of
biological evaluation for a deeper insight into structure–activity relationships. The group of
novel compounds covers positional isomers of the most effective AChE inhibitors (2k
i.e., the derivatives 2n 2s, as well as hydrazide–hydrazones without 4-CF3-benzohydrazide
moiety (trifluoromethyl was removed, 2t, or replaced by methyl, 2u).
–2m),
–
Scheme 1. Synthesis of the 4-(trifluoromethyl)benzohydrazide 1.
Scheme 2. Synthesis of the hydrazide–hydrazones 2 and 3 [X = CF3 (2a-2s), H (2t), CH3 (2u)].
2.2. Inhibition of Acetyl- and Butyrylcholinesterase
The hydrazide–hydrazones 2 and 3 as well as the parent compound 1 were screened
for their potential to affect the function of AChE isolated from electric eel (EeAChE) and
BuChE obtained from equine serum (EqBuChE) using Ellman’s spectrophotometric method
(Table 1). These enzymes of nonhuman origin were used to screen inhibitory activity and to
compare the results with our previous compounds sharing a certain degree of a chemical
similarity [23,24]. Their efficacy is expressed as the concentration leading to 50% inhibition
of the enzymatic activity (IC50). To quantify the preference to particular acetylcholine
decomposing enzymes, selectivity indexes (SI) were calculated. SI is defined as a ratio of
IC50 for BuChE/IC50 for AChE (Table 1; illustrated also in Figures S1–S6). Values higher
than 1 mean stronger inhibition of AChE and vice versa. Based on analogy with therapeutic
index, SI over the borderline of 10 indicates selective AChE inhibition. In addition to
1,
galantamine was employed as a standard for comparison of IC50 values. Moreover, we

Molecules 2021, 26, 989
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investigated the type of inhibition of the most active AChE inhibitor identified (2l; Figure 2,
Table S1).
Figure 2. Lineweaver–Burk plot for 2l inhibiting eeAChE (ATCh = acetylthiocholine; [I] = concentration of inhibitor).
The original [12] hydrazide–hydrazones 2a
enzymes with IC50 values of 46.8–137.7 M and 63.6–881.1
tively. Focusing on the benzaldehyde derivatives and AChE, 4-(trifluoromethyl)benzylidene
hydrazone 2l was the most in vitro efficient, together with salicylidene hydrazone 2g. The
introduction of unsubstituted benzylidene moiety into the molecule resulted in a comparable
activity ( vs. 2a). The inhibition potency was improved when an electronegative substituent
–2m and
3
exhibited a dual inhibition of both
µ
µM for AChE and BuChE, respec-
2
1
1
was present at the position 4: second CF3 group (–I effect; 2l), nitro group (–I and –M effects;
2m), or bromine (+M and predominant –I effect; 2k). The replacement of bromine by chlorine
led to a mild drop in the activity (2k vs. 2b). On the other hand, electron-donating substituents
were unfavorable (methyl 2i, methoxy 2j). We also evaluated chlorine and hydroxyl group
positional isomers (2b–2d and 2e–2g, respectively); however, there was no clear structure–
activity relationship. Interestingly, the combination of these two groups at their best positions
led to the less potent compound within this study (2h).
IC50 values of
2 for BuChE were clearly higher. 2-Chlorobenzylidene analogue 2d
showed the lowest value, followed by approximately 3-fold less active 4-CF3 (2l) and
4-NO2 (2k) substituted hydrazones. Contrarily, benzylidene (2a), salicylidene (2g), and 4-
bromobenzylidene (2k) substituents were detrimental to inhibition. Remaining derivatives
produced similar IC50 values. The positional isomers of 2d shared significantly lower
inhibitory potency.
Among ketone-derived hydrazones
(59–70 M); bigger ketones (camphor, cyclohexanone) were preferred over smaller ones
(acetone, cyclopentanone). This structure–activity dependence was even more obvious in
3, the inhibition of AChE was virtually uniform
µ
the case of BuChE inhibition (3a→3d, from 424 to 57
µM), favoring again cyclohexanone
and especially camphor as starting scaffolds.
Then, we also evaluated additional derivatives 2n
brominated derivative 2k provided an improved activity against BuChE for both ortho-
and meta-isomers (up to 6.3 2k vs. 2o), although meta-isomer 2n was less effective
–2u. The positional isomers of the
×
,
against AChE. Analogous structure–activity relationships were also found for the novel
nitro derivatives 2r and 2s. The hydrazones based on 2- and 3-CF3-benzaldehyde (2p and

Molecules 2021, 26, 989
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2q) retained activity against AChE but were significantly more effective against BuChE,
especially the ortho-isomer 2q with IC50 of 19.1 µM, i.e., the most active BuChE inhibitor
in this study. In sum, especially 2-substituted benzaldehydes-based hydrazones were
beneficial when compared to their original para-isomers in terms of improved activity
against BuChE along with the same inhibition of AChE. Meta-isomers also led to a more
balanced inhibition of both acetylcholine hydrolyzing enzymes.
Focusing on the removal of CF3 group (2t) and its replacement by CH3 group (2u),
these modifications led to a decreased activity, although these two derivatives were also
capable of inhibiting both AChE and BuChE. The benzohydrazide derivative 2t was a more
potent inhibitor.
The modification of the parent hydrazide
against AChE (2g 2k 2m 2o 2q 2s 2t 3c, and 3d) and 5 others were fully comparable (2a
2f 2u 3a, and 3b). For BuChE, 13 compounds (2d 2l 2m 2t 3b 3d) exhibited lower IC50
than the hydrazide . Then, we compared the results with those obtained for galantamine,
1 led to 11 derivatives that were more active
,
–
,
–
,
,
,
,
,
,
,
,
–
,
–
1
the highly active inhibitor of AChE approved for the treatment of Alzheimer’s disease-
related dementia. However, none of the hydrazide–hydrazones exhibited comparable
IC50 values.
Focusing on selectivity toward cholinesterases, the majority of the hydrazide–hydrazones
2
–
3
inhibited AChE more strongly (selectivity indexes, SI,
≥
2). 2-Hydroxybenzylidene deriva-
tive 2g, the second most potent AChE inhibitor, was the most selective AChE inhibitor
(SI = 18.6), followed by unsubstituted benzylidene 2a (8.7), propan-2-ylidene 3a (6.1), and
4-bromobenzylidene analogue 2k (SI of 5.5). On the other hand, only the presence of 2-
chlorobenzylidene and 2-(trifluoromethyl)benzylidene substituents (2d and 2q) led to a
higher inhibition of BuChE; however, not selectively (SI = 0.5 and 0.4, respectively). Two
larger ketone-based hydrazones obtained from cyclohexanone (3c) and camphor (3d) pro-
duced a balanced inhibition of both acetylcholine hydrolyzing enzymes as well as the re-
maining hydrazones of isomeric 2-/3-bromo- and nitrobenzaldehydes (2n, 2o, 2r, and 2s) and
3-CF3-benzaldehyde (2p).
Moreover, all the original derivatives were without any signs of cytotoxicity for HepG2
cells at 100 µM (Table 1) [12].
Type of Inhibition
The reversible enzyme inhibitors can be classified into four groups: competitive, non-
competitive, uncompetitive, and mixed type of inhibition. The type of inhibition could be
distinguished using a Lineweaver–Burk plot [25] and corresponding comparison of two
kinetic parameters: Michaelis constant (KM) and maximum velocity (Vm) of uninhibited
and inhibited reaction. The Lineweaver–Burk plot of the most potent AChE inhibition
with 2l is depicted in Figure 2. Based on the plot of KM and Vm, this hydrazide–hydrazone
caused a mixed type of inhibition.
2.3. Prediction of BBB Permeability
In addition to their efficacy and low toxicity, compounds intended for treatment of
dementias must be able to cross the blood-brain barrier (BBB) to reach their target site. To
predict this crucial issue in silico, various empirical approaches have been proposed and
validated. They are based on various physicochemical and structural parameters inferred
from known CNS drugs.
Classical selection criteria include molecule size, lipophilicity, number of hydrogen
bond donors and acceptors, aromatic rings, basic groups, heteroatoms, polar surface area,
molar refractivity, molecular flexibility quantified by a number of rotatable single bonds,
pKa, etc. [26–29]. In general, lipophilicity is regarded to be the most important property,
and its increased value often results in an improved in vitro activity [27].
The data (Mw, consensus log P, number of hydrogen bond donors and acceptors,
rotatable bonds, and topological polar surface area (tPSA)) of the derivatives
1–3 (Table 2)
were compared with margins proposed as ideal for CNS drugs [29]. The vast majority

Molecules 2021, 26, 989
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of these parameters were within the suggested ranges; only several compounds (2j
, 2l,
2m, and 2p 2s) had one more rotatable bond. Nevertheless, delivery across the BBB is not
–
bounded sharply. Since it is a consequence of many factors, the cut-offs are not strict, and
they are related to a higher probability of targeting the CNS.
Table 2. Blood-brain barrier (BBB) permeability parameters.
H-Bond
Donors
H-Bond
Acceptors
tPSA
(Å2)
Rotatable
Bonds
BOILED-
Egg
Code
Mw
log P
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
292.26
326.70
326.70
326.70
308.26
308.26
308.26
342.70
306.28
322.28
371.15
360.25
337.25
371.15
371.15
360.25
360.25
337.25
337.25
292.26
306.28
244.21
270.25
284.28
338.37
3.66
4.19
4.20
4.37
3.26
3.26
3.43
4.01
4.00
3.67
4.28
4.72
3.06
4.29
4.45
4.71
4.77
3.05
3.23
3.65
3.91
2.93
3.35
3.69
4.60
1
1
1
1
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
3
3
3
4
4
4
4
3
4
3
3
5
3
3
3
3
5
5
3
3
3
3
3
3
41.46
41.46
41.46
41.46
61.69
61.69
61.69
61.69
41.46
50.69
41.46
41.46
87.28
41.46
41.46
41.46
41.46
87.28
87.28
41.46
41.46
41.46
41.46
41.46
41.46
5
5
5
5
5
5
5
5
5
6
5
6
6
5
5
6
6
6
6
5
5
4
4
4
4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
-
-
ꢀ
ꢀ
-
-
-
2s
2t
2u
3a
3b
3c
3d
-
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1
204.15
1.70
2
3
55.12
3
ꢀ
Breakpoint [29]
≤500
≤6.0
<5
<10
≤90
≤5
-
Mw: molecular weight, tPSA: topological polar surface area.
In addition to this structural multiparameter appraisal, we also involved a newer
BOILED-Egg (Brain Or IntestinaL EstimateD permeation) approach [26] based on lipophilic-
ity and polarity of small molecules to predict penetration through the BBB via passive
diffusion. Passive diffusion usually represents a key entrance route for centrally acting
drugs, and it requires a certain degree of lipophilicity (log P) in combination with a mild
polarity (tPSA). To estimate brain permeability using BOILED-Egg, the SwissADME [30
]
calculator was utilized.
The results are summarized in Table 2. With the exception of 4-CF3 derivative 2l and
their isomers 2p and 2q (due to enhanced lipophilicity) and 4-NO2 compounds (2m 2r, and
,
2s), the remaining hydrazide–hydrazones met the requirements for CNS active molecules.
However, the BOILED-Egg approach-based prediction often failed in the prediction of
BBB permeability of nitro group-containing drugs that are indicated far from the optimal
physicochemical space, even in the case of typical CNS drugs and drugs exhibiting their
pharmacologic effects in the brain (e.g., calcium channel blockers including nitrendipine,
nilvadipine and nimodipine, non-steroidal anti-inflammatory agent nimesulide, tolcapone
used as a levodopa booster, organophosphorus AChE inhibitors such as paraoxon and
parathion, benzodiazepine hypnotics nitrazepam and flunitrazepam, antibiotic chloram-
phenicol, etc.). Thus, the prediction seems to be unreliable for nitro derivatives.

Molecules 2021, 26, 989
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Furthermore, BOILED-Egg was useful for prediction of gastrointestinal absorption
after oral administration based on the identical physicochemical descriptors. Hopefully,
all the derivatives 1–3 meet in silico criteria for high gastrointestinal absorption; only for
bis-CF3 analogues it was predicted to be lower.
3. Materials and Methods
3.1. Chemistry
General. All the reagents and solvents were purchased from Sigma-Aldrich (Darm-
stadt, Germany) and Penta Chemicals (Prague, Czech Republic), and they were used as
received. The progress of the reactions and the purity of the products were monitored by
thin layer chromatography using a mixture of toluene with ethyl acetate and tetrahydro-
furan (4:1:0.3, v/v) as an eluent; plates were coated with 0.2 mm Merck 60 F254 silica gel
(Merck Millipore, Darmstadt, Germany) and were visualized by UV irradiation (254 nm).
Melting points were determined on a Büchi Melting Point B-540 apparatus (BÜCHI, Flawil,
Switzerland) using open capillaries; the reported values are uncorrected.
Elemental analysis (C, H, N) was performed on a Vario MICRO Cube Element Ana-
lyzer (Elementar Analysensysteme, Hanau, Germany). Both calculated and found values
are given as percentages. Infrared spectra (ATR) were recorded on a Nicolet 6700 FT-IR
spectrometer (ThermoFisher Scientific, Waltham, MA, USA) in the range of 600 to 4000
cm−1. The NMR spectra were measured in DMSO-d6 at ambient temperature on a Varian
V NMR S500 instrument (500 MHz for 1H and 125 MHz for 13C; Varian Comp., Palo Alto,
CA, USA). The chemical shifts, δ, are given in ppm with respect to tetramethylsilane via
signals of DMSO-d6 (2.49 for 1H and 39.7 for 13C spectra). The coupling constants (J) are
reported in Hz.
The synthesis and characterization of the compounds 2a
depth by Krátký et al. [12].
–2m and 3 were reported in
The novel derivatives were prepared and purified according to an identical procedure.
Briefly, an appropriate hydrazide (1 mmol) was dissolved in 6 mL of MeOH and then a
substituted benzaldehyde was added in one portion (1.1 of equivalents) under vigorous
stirring. The reaction mixture was stirred at rt for 5 min and then refluxed for 2 h. Resulting
◦
suspension was let to stay overnight at +4 C and then was filtered off. The resulting
crystals were washed with a small volume of cold MeOH and diethyl ether and then dried
to obtain pure hydrazones 2n–2u.
N’-(3-Bromobenzylidene)-4-(trifluoromethyl)benzohydrazide 2n. White solid; yield
96%; mp 193–195 ◦C. IR (ATR): 3018, 1654, 1558, 1327, 1304, 1290, 1164, 1151, 1118, 1108,
1067, 1019, 962, 927, 853, 786, 770, 683 cm−1
.
1H-NMR (500 MHz, DMSO-d6):
δ
12.19
(1H, bs, NH), 8.47 (1H, s, CH=N), 8.12 (2H, d, J = 8.3 Hz, H2, H6), 7.96–7.90 (3H, m, H3,
H5, H2’), 7.75 (1H, dt, J = 7.8, 1.3 Hz, H6 ), 7.64 (1H, dt, J = 8.0, 1.5 Hz, H4 ), 7.43 (1H, t,
J = 7.8 Hz, H5 162.58, 147.23, 137.49, 137.07, 133.25, 132.09
). 13C-NMR (126 MHz, DMSO):
´
´
´
δ
(q, J = 32.0 Hz), 131.50, 129.73, 129.09, 126.79, 125.96 (q, J = 3.8 Hz), 124.33 (q, J = 272.8 Hz),
122.66. Analytically calculated for C15H10BrF3N2O (371.15): C, 48.54; H, 2.72; N, 7.44.
Found: C, 48.52; H, 2.77; N, 7.40.
N’-(2-Bromobenzylidene)-4-(trifluoromethyl)benzohydrazide 2o. White solid; yield
97%; mp 217–219.5 ◦C. 1H-NMR (500 MHz, DMSO-d6):
δ
12.30 (1H, bs, NH), 8.84 (1H, s,
CH=N), 8.15 (2H, d, J = 8.1 Hz, H2, H6), 8.02 (1H, dd, J = 7.9, 1.8 Hz, H6 ), 7.92 (2H, d,
J = 8.2 Hz, H3, H5), 7.70 (1H, dd, J = 8.0, 1.3 Hz, H3 ), 7.50–7.45 (1H, m, H5 ), 7.38 (1H,
td, J = 7.6, 1.8 Hz, H4 162.49, 147.27, 137.40, 133.67,
). 13C-NMR (126 MHz, DMSO):
´
´
´
´
δ
133.36, 132.41, 132.14 (q, J = 32.0 Hz), 129.11, 128.62, 127.79, 125.97 (q, J = 3.8 Hz), 124.33 (q,
J = 272.3 Hz), 124.18. Analytically calculated for C15H10BrF3N2O (371.15): C, 48.54; H, 2.72;
N, 7.44. Found: C, 48.50; H, 2.78; N, 7.48.
4-(Trifluoromethyl)-N’-[3-(trifluoromethyl)benzylidene]benzohydrazide 2p. White
◦
solid; yield 90%; mp 162.5–164.5 C. IR (ATR): 3007, 1649, 1570, 1327, 1306, 1292, 1281, 1217,
1
1167, 1126, 1111, 1068, 1062, 1019, 945, 854, 803, 770, 698, 670 cm−1. H-NMR (500 MHz,
DMSO-d6):
δ 12.27 (1H, bs, NH), 8.55 (1H, s, CH=N), 8.13 (2H, d, J = 8.1 Hz, H2, H6),

Molecules 2021, 26, 989
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8.09–8.03 (2H, m, H2
7.71 (1H, t, J = 7.8 Hz, H5
´
, H6
´
), 7.92 (2H, d, J = 8.1 Hz, H3, H5), 7.80 (1H, d, J = 7.9 Hz, H4
´),
´
). 13C-NMR (126 MHz, DMSO):
δ
162.43, 147.00, 137.20, 135.54,
131.87 (q, J = 31.9 Hz), 131.38, 130.29, 129.89 (q, J = 31.9 Hz), 128.86, 126.72 (q, J = 3.8 Hz),
125.73 (q, J = 3.9 Hz), 124.22 (q, J = 272.3 Hz), 124.07 (q, J = 272.8 Hz), 123.35 (q, J = 3.9 Hz).
Analytically calculated for C16H10F6N2O (360.25): C, 53.34; H, 2.80; N, 7.78. Found: C,
53.32; H, 2.83; N, 7.90.
4-(Trifluoromethyl)-N’-[2-(trifluoromethyl)benzylidene]benzohydrazide 2q. White
◦
solid; yield 95%; mp 182.5–184 C. IR (ATR): 3034, 1651, 1559, 1489, 1316, 1290, 1278,
1
1170, 1160, 1137, 1112, 1104, 1062,1032, 1020, 964, 860, 766, 671 cm−1. H-NMR (500 MHz,
DMSO-d6):
δ
12.36 (1H, bs, NH), 8.85 (1H, q, J = 2.2 Hz, CH=N), 8.25 (1H, d, J = 7.9 Hz,
), 8.14 (2H, d, J = 8.1 Hz, H2, H6), 7.93 (2H, d, J = 8.2 Hz, H3, H5), 7.83–7.76 (2H, m,
, H5 ), 7.65 (1H, t, J = 7.7 Hz, H4 162.62, 144.00,
). 13C-NMR (126 MHz, DMSO):
H6
H3
´
´
´
´
δ
137.29, 133.37, 132.46, 132.21 (q, J = 31.9 Hz), 130.77, 129.13, 127.40, 127.37 (q, J = 32.0 Hz),
126.42 (q, J = 3.9 Hz), 125.98 (q, J = 3.7 Hz), 124.62 (q, J = 272.7 Hz), 124.32 (q, J = 272.3 Hz).
Analytically calculated for C16H10F6N2O (360.25): C, 53.34; H, 2.80; N, 7.78. Found: C,
53.39; H, 2.77; N, 7.72.
N’-(3-Nitrobenzylidene)-4-(trifluoromethyl)benzohydrazide 2r. Yellowish solid; yield
99%; mp 205–207 ◦C. IR (ATR): 3205, 3055, 1665, 1551, 1527, 1343, 1327, 1282, 1179, 1149,
1
1132, 1113, 1066, 1019, 949, 936, 898, 862, 814, 771, 738, 690, 669 cm−1. H-NMR (500 MHz,
DMSO-d6):
8.17 (1H, dd, J = 7.8, 1.4 Hz, H6
H3, H5), 7.76 (1H, t, J = 8.0 Hz, H5
δ
12.31 (1H, bs, NH), 8.58–8.55 (2H, m, CH=N, H2
), 8.13 (2H, d, J = 8.0 Hz, H2, H6), 7.92 (2H, d, J = 8.1 Hz,
). 13C-NMR (126 MHz, DMSO):
162.70, 148.68, 146.59,
´), 8.28–8.25 (1H, m, H4´),
´
´
δ
137.36, 136.46, 133.95, 132.15 (q, J = 31.7 Hz), 130.95, 129.13, 125.98 (q, J = 3.9 Hz), 124.90,
124.32 (q, J = 272.4 Hz), 121.50. Analytically calculated for C15H10F3N3O3 (337.25): C, 52.42;
H, 2.99; N, 12.46. Found: C, 52.43; H, 2.95; N, 12.53.
N’-(2-Nitrobenzylidene)-4-(trifluoromethyl)benzohydrazide 2s. Yellowish solid; yield
◦
98%; mp 215.5–216.5 C. IR (ATR): 3201, 3089, 1651, 1554, 1522, 1365, 1343, 1323, 1292, 1175,
1
1152, 1131, 1113, 1065, 1018, 955, 922, 855, 788, 744, 702, 687 cm−1. H-NMR (500 MHz,
DMSO-d6):
7.89 (2H, d, J = 8.1 Hz, H2, H6), 7.79 (1H, d, J = 7.6 Hz, H5
13C-NMR (126 MHz, DMSO):
162.73, 148.84, 144.44, 137.33, 134.32, 132.29 (q, J = 32.2 Hz),
δ
12.35 (1H, bs, NH), 8.86 (1H, s, CH=N), 8.13–8.04 (4H, m, H3, H5, H3
´
, H6´
),
´
), 7.66 (1H, t, J = 7.8 Hz, H4
´
).
δ
131.41, 129.29, 129.13, 128.51, 126.06 (q, J = 3.8 Hz), 125.30, 124.43 (q, J = 272.3 Hz). Analyti-
cally calculated for C15H10F3N3O3 (337.25): C, 52.42; H, 2.99; N, 12.46. Found: C, 52.37; H,
3.06; N, 12.55.
N’-[4-(Trifluoromethyl)benzylidene]benzohydrazide 2t. White solid; yield 99%; mp
188–189 ◦C. IR (ATR): 3290, 1664, 1548, 1377, 1325, 1305, 1281, 1171, 1156, 1147, 1107, 1067,
1
1018, 953, 856, 839, 718, 692, 670 cm−1. H-NMR (500 MHz, DMSO-d6):
δ
12.06 (1H, bs,
), 7.82 (2H, d, J = 8.0 Hz, H3
), 7.61 (1H, d, J = 7.3 Hz, H4), 7.54 (2H, t, J = 7.5 Hz, H3, H5). 13C-NMR (126 MHz,
DMSO): 163.81, 146.44, 138.79, 133.66, 132.41, 130.15 (q, J = 31.6 Hz), 128.99, 128.16, 128.12,
NH), 8.54 (1H, s, CH=N), 7.98–7.91 (4H, m, H2, H6, H2
´
, H6
´
´,
H5´
δ
126.20 (q, J = 3.9 Hz), 124.57 (q, J = 272.2 Hz). Analytically calculated for C15H11F3N2O
(292.26): C, 61.65; H, 3.79; N, 9.59. Found: C, 61.60; H, 3.85; N, 9.63.
4-Methyl-N’-[4-(trifluoromethyl)benzylidene]benzohydrazide 2u. White solid; yield
94%; mp 212–213.5 ◦C. IR (ATR): 3277, 1661, 1545, 1386, 1369, 1323, 1280, 1159, 1146, 1120,
1
1105, 1066, 1017, 950, 838, 750, 672 cm−1. H-NMR (500 MHz, DMSO-d6):
δ
11.93 (1H, bs,
, H3 , H5
), 7.30 (2H, d, J = 7.9 Hz, H3, H5), 2.34 (3H, s, CH3). 13C-NMR (126 MHz, DMSO):
NH), 8.49 (1H, s, CH=N), 7.90 (2H, d, J = 8.0 Hz, H2, H6), 7.83–7.76 (4H, m, H2
H6
´
´
´,
δ
´
163.69, 146.32, 142.57, 138.95, 130.85, 130.16 (q, J = 31.5 Hz), 129.58, 128.26, 128.15, 126.26
(q, J = 3.8 Hz), 124.66 (q, J = 272.2 Hz), 21.57. Analytically calculated for C16H13F3N2O
(306.29): C, 62.74; H, 4.28; N, 9.15. Found: C, 62.69; H, 4.24; N, 9.12.
3.2. Evaluation of Acetyl- and Butyrylcholinesterase Inhibition
The activities of the investigated compounds
2–3 were measured via Ellman’s spec-
trophotometric method that was modified according to Zdražilová et al. [31] to determine

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the IC50 values. Ellman’s method has been widely applied for the screening of the efficiency
of potential AChE and BuChE inhibitors. The activity of cholinesterases was assessed
indirectly by quantifying the amount of 5-mercapto-2-nitrobenzoic acid ion formed in the
reaction of the thiol reagent 5,50-disulfanediylbis(2-nitrobenzoic acid) with thiocholine,
a product of the hydrolysis of acetyl- (ATCh) or butyrylthiocholine (BTCh) catalyzed by
AChE and BuChE, respectively.
AChE used in this assay was obtained from electric eel (Electrophorus electricus L.)
and BuChE from equine serum. The benzazepine alkaloid drug galantamine was used
as a reference enzyme inhibitor. All the enzymes, the thiol reagent, ATCh, BTCh, and
galantamine were purchased from Sigma-Aldrich (Prague, Czech Republic).
The enzyme activity in final reaction mixture (2 mL) was 0.2 U/mL, the concentration
0
of ATCh or BTCh was 40
µ
M, and the concentration of 5,5 -disulfanediylbis(2-nitrobenzoic
acid) was 0.1 mM for all reactions. The investigated hydrazide–hydrazones were dissolved
in DMSO and then diluted by demineralized water (conductivity 3 S, equipment supplier
µ
BKG Water Treatment, Hradec Králové, Czech Republic) to the concentration of 1 mM.
For all of them as well as galantamine, five different concentrations of inhibitor in final
reaction mixture were used. All measurements were carried out in triplicates, and the
average values of reaction rate (v0-uninhibited reaction, vi-inhibited reaction) were used
for construction of the dependence v0/vi vs. concentration of inhibitor. From the obtained
equation of the regression curve, IC50 values were calculated.
For the determination of the type of inhibition of the most active AChE inhibitor 2l, a
Lineweaver–Burk plot [25] was used, and the measuring procedure was analogous to the de-
termination of IC50. The enzyme activity in the final reaction mixture (2 mL) was 0.2 U/mL,
a concentration of ATCh was 20–80
nitrobenzoic acid) was 0.1 mM. For each of the substrate concentrations, four different con-
centrations of the inhibitor were used (10, 20, 30, and 40 M). The dependence absorbance
µ
M, and a concentration of 5,50-disulfanediylbis(2-
µ
vs. time was observed and the reaction rate was calculated. All measurements were carried
out in duplicate and the average values of reaction rate were used for the construction of
the Lineweaver–Burk plot. Furthermore, the dependencies vs. concentration of ATCh in ab-
sence and presence of inhibitor 2l were analyzed by nonlinear regression using GraphPad
Prism 9.0.1 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com).
From obtained equations of regression curves in the Lineweaver–Burk plot, the values of
Michaelis constant (KM) and maximum velocity (Vm) were calculated. Using nonlinear
regression (chosen panel: enzyme kinetics—inhibition/mixed model inhibition) the values
of KI (54.63 µM) and alpha (1.28) were determined. Based on all obtained results, the type
of inhibition was identified.
3.3. Determination of Parameters for CNS Delivery
The parameters for in silico prediction of CNS delivery (log P as a value of consensus
Log Po/w, number of rotatable bonds, and topological polar surface area, tPSA) as well as
BOILED-Egg [26] method estimation of brain permeability were calculated using a free
web tool SwissADME [30]; http://www.swissadme.ch/index.php.
4. Conclusions
We screened twenty-five hydrazide–hydrazones for their in vitro potency to inhibit
AChE and BuChE. These compounds were derivatives of 4-(trifluoromethyl)benzohydrazide
and various aromatic aldehydes and aliphatic ketones of choice. In addition, we evalu-
ated the influence of various substitution patterns of benzaldehyde. IC50 values found
were in the micro molar range from 46.8 µM for AChE and 19.1 µM for BuChE. Most of
the derivatives were better inhibitors of AChE. 2-/3-/4-(Trifluoromethyl)-, 2-/4-nitro-,
2-/4-bromo-, and 2-hydroxybenzaldehydes together with camphor and cyclohexanone rep-
resented the most successful carbonyl compounds used for the modification of the parent
hydrazide. Focusing on the type of inhibition, the most potent AChE inhibitor 2l derived
from 4-(trifluoromethyl)benzaldehyde exhibited a mixed inhibition of the enzyme. After in

Molecules 2021, 26, 989
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silico evaluation, the hydrazide–hydrazones shared generally convenient properties for
CNS delivery and gastrointestinal absorption.
Supplementary Materials: The following are available online. Table S1: Kinetic parameters for 2l
inhibiting eeAChE calculated from L–B plot; Figure S1: The dependence v0/vi vs. concentration
of 2a inhibiting eeAChE; Figure S2: The dependence v0/vi vs. concentration of 2g inhibiting
eeAChE; Figure S3: The dependence v0/vi vs. concentration of 2k inhibiting eeAChE; Figure S4: The
dependence v0/vi vs. concentration of 3c inhibiting eeAChE; Figure S5: The dependence v0/vi vs.
concentration of 2d inhibiting eqBuChE; Figure S6: The dependence v0/vi vs. concentration of 3d
inhibiting eqBuChE.
Author Contributions: Conceptualization, M.K.; methodology, M.K. and Š.Š.; investigation, M.K.,
K.S., and Q.A.V.; writing—original draft preparation, M.K., Š.Š., and J.V.; writing—review and editing,
K.S. and Š.Š.; supervision, M.K., Š.Š., and J.V. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the Czech Science Foundation, grant number 20-19638Y,SVV
260 547, and the project EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data available on request due to restrictions. The data presented in
this study are available on request from the corresponding author. The data are not publicly available
due to privacy.
Acknowledgments: The authors wish to acknowledge financial support from the University of
Pardubice, Faculty of Chemical Technology.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 1, 2a–2u, and 3a–3d are available from the authors.
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