Synthesis and biological evaluation of new thiosemicarbazone derivative schiff bases as monoamine oxidase inhibitory agents

Article abstract of DOI:10.3390/molecules23010060

Twenty-six novel thiosemicarbazone derivative B1–B26 were synthesized via condensation reactions between the corresponding thiosemicarbazides and aldehydes. The chemical characterization of the compounds was carried out by infrared (IR), mass (MS), proton and carbon nuclear magnetic resonance (¹H- and ¹³C-NMR) spectroscopic analyses. The compounds were investigated for their monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) inhibitory activity and most of them were more potent against MAO-A enzyme when compared with MAO-B enzyme. N-Cyclohexyl-2-[4-[(4-chlorophenyl)thio]benzylidene]hydrazine-1-carbothioamide (B24) was the most active compound against MAO-A. The enzyme kinetics study revealed that compound B24 has a reversible and competitive mode of binding. Interaction modes between compound B24 and MAO-A were clarified by docking studies. In addition, the favourable absorption, distribution, metabolism, and excretion (ADME) properties and non-toxic nature of compound B24 make this compound a promising MAO-A inhibitor.

Full text of DOI:10.3390/molecules23010060

molecules
Article
Synthesis and Biological Evaluation of New
Thiosemicarbazone Derivative Schiff Bases as
Monoamine Oxidase Inhibitory Agents
Betül Kaya Çavus¸og˘lu 1, Begüm Nurpelin Sag˘lık 1,2, Derya Osmaniye 1,2, Serkan Levent 1,2
Ulviye Acar Çevik 1,2, Abdullah Burak Karaduman 3, Yusuf Özkay 1,2,* and
,
Zafer Asım Kaplancıklı 1,2
ID
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskis¸ehir, Turkey;
betulkaya@anadolu.edu.tr (B.K.Ç.); bnsaglik@anadolu.edu.tr (B.N.S.); dosmaniye@anadolu.edu.tr (D.O.);
serkanlevent@anadolu.edu.tr (S.L.); uacar@anadolu.edu.tr (U.A.Ç.); zakaplan@anadolu.edu.tr (Z.A.K.)
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University,
26470 Eskis¸ehir, Turkey
2
3
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University,
26470 Eskis¸ehir, Turkey; abkaraduman@anadolu.edu.tr
*
Correspondence: yozkay@anadolu.edu.tr; Tel.: +90-222-335-0580 (ext. 3603)
Received: 15 November 2017; Accepted: 21 December 2017; Published: 28 December 2017
Abstract: Twenty-six novel thiosemicarbazone derivative B1 B26 were synthesized via condensation
–
reactions between the corresponding thiosemicarbazides and aldehydes. The chemical characterization
of the compounds was carried out by infrared (IR), mass (MS), proton and carbon nuclear magnetic
resonance (1H- and 13C-NMR) spectroscopic analyses. The compounds were investigated for
their monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) inhibitory activity and
most of them were more potent against MAO-A enzyme when compared with MAO-B enzyme.
N-Cyclohexyl-2-[4-[(4-chlorophenyl)thio]benzylidene]hydrazine-1-carbothioamide (B24) was the
most active compound against MAO-A. The enzyme kinetics study revealed that compound B24
has a reversible and competitive mode of binding. Interaction modes between compound B24 and
MAO-A were clarified by docking studies. In addition, the favourable absorption, distribution,
metabolism, and excretion (ADME) properties and non-toxic nature of compound B24 make this
compound a promising MAO-A inhibitor.
Keywords: thiosemicarbazone; MAO-A; MAO-B; docking; MTT; enzyme kinetic study; ADME
1. Introduction
The process of synaptic neurotransmission is ensured by the correct functioning of enzymes.
Monoamine oxidases (MAOs) are flavoproteins bound to the outer membranes of mitochondria
throughout the brain, catalysing the oxidative deamination of monoamine neurotransmitters, thereby
participating in this mechanism by modulating the levels of neurotransmitters [
of MAO exist in mammals, namely MAO-A and MAO-B, varying in substrate specificity, sensitivity
to inhibitors and amino acid sequences [ ]. MAO-A preferentially oxidizes norepinephrine and
serotonin and is selectively inhibited by clorgyline, while MAO-B preferentially deaminates dopamine
and is irreversibly inhibited by L-deprenyl [ ]. Both isoforms are significant drug targets in the
therapy of neuropsychiatric and neurodegenerative disorders. Inhibitors of MAO-A are employed
as antidepressants and anxiolytics [ ] while MAO-B inhibitors are clinically used for individuals
suffering from Parkinson’s disease and Alzheimer’s disease [ ]. Adverse effects of irreversible and
1,2]. Two subtypes
3,4
5,6
7,8
9
nonselective MAO inhibitors [10] have led to great interest in the discovery of novel and selective
drug candidates.
Molecules 2018, 23, 60; doi:10.3390/molecules23010060
www.mdpi.com/journal/molecules

Molecules 2018, 23, 60
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Excessive levels of metal ions accumulate in brain with aging, leading to neurodegeneration.
Metal-induced neurotoxicity is assumed to be connected with several neurological diseases [11].
In recent years, chelation therapy has become a useful treatment for the symptoms associated
with the central nervous system [12]. Thiosemicarbazones (TSCs) are a class of sulphur- and
nitrogen- containing compounds that are able to form complexes with metal ions present in biological
systems [13]. Due to the strong reactivity of the hydrazine nitrogen (C=N) and thioketone (C=S)
groups, TCS compounds represent a versatile class of Schiff bases and are important in medicinal
chemistry [14]. Recently, there has been a concentrated research on Schiff bases and their complexes due
to their range of biological activity including antifungal, antibacterial, antimalarial, antiviral, anticancer,
anti-inflammatory, sedative-hypnotic, antidepressant, analgesic, cytotoxic, anticonvulsant [15–22].
TSCs are small molecules that can cross the blood brain barrier and hence be used as potential
drugs in the treatment of neurodegenerative diseases. Furthermore, it has been reported that TSCs
have some common features as H-bond acceptors, H-bond donors, hydrophobic substructures, positive
ionizable atoms, and aromatic rings to interact with MAO enzymes [23].
Taking these facts into account, in this study, we prepared a series of thiosemicarbazide derivatives,
which were substituted with a various aliphatic and aromatic groups at the N(1) and N(4) atoms.
The newly synthesized compounds were examined for their inhibitory activities against MAO-A and
MAO-B. Enzyme kinetic and docking studies were also carried out to clarify their inhibition profiles
and mechanism(s) of action.
2. Results and Discussion
2.1. Chemistry
The synthetic pathways and structural features of the compounds B1
In the first step, phenyl- or cyclohexylisothiocyanate and hydrazine hydrate were reacted to obtain
N-phenyl/cyclohexylhydrazinecarbothioamides . In the second step, in order to synthesize
aldehyde derivatives 16, suitable secondary amines or 4-substituted phenol/thiophenols and
4-fluorophenylbenzaldehyde were refluxed in dimethylformamide. In the final step, the condensation
–B26 are outlined in Scheme 1.
1,2
3
–
in ethanol of N-phenyl/cyclohexylhydrazinecarbothioamides
benzaldehyde derivatives 3–16 gave the title compounds.
1
,
2
and the synthesized 4-substituted
The structures of all final compounds were elucidated by infrared, mass, H- and 13C-NMR
spectroscopy experiments. In the infrared (IR) spectra, N-H, aromatic and aromatic C-Hs, C=N,
C=C, C-N and C-O bands were observed. N-H bands were recorded around 3280 cm−1. C-H bands
for aromatic and aliphatic examples were observed between 3157 cm−1 and 2929 cm−1. C=N and
C=C groups were recorded at 1600 cm−1–1442 cm−1. C-N and C-O bands were observed between
1
1
1287–1124 cm−1. In the H-NMR spectra, alkyl group protons had peaks between 0.92 ppm and
4.37 ppm. The chemical shift values of aromatic ring protons were between 6.81 ppm and 7.82 ppm,
depending on the substituent groups. Imine protons appeared between 7.83 ppm and 8.14 ppm, while
N-H protons were seen at 7.91 ppm
carbons were observed between 19.65 ppm
Thiocarbonyl carbons were observed between 175.53 ppm and 176.44 ppm. In High Resolution Mass
(HRMS) analysis, the calculated and expected masses were matched at the ppm level. The IR, 1H-NMR,
13C-NMR, and HRMS spectra of compound B24 are presented in supplementary file.
−
11.85 ppm. In the 13C-NMR spectra, aliphatic and aromatic
−
55.90 ppm and 112.14 ppm 163.11 ppm, respectively.
−

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Secondary
Amine
Secondary
Amine
Compound
A
X
R
Compound
A
X
R
B1
B2
B3
B4
phenyl
phenyl
phenyl
phenyl
-
-
-
-
piperidine
2-metilpiperidine
4-metilpiperidine
4-phenylpiperazine
-
-
-
-
B14
B15
B16
B17
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
-
-
-
-
piperidine
2-metilpiperidine
4-metilpiperidine
4-phenylpiperazine
-
-
-
-
(4-methoxyphenyl)
(4-methoxyphenyl)
B5
phenyl
-
-
B18
cyclohexyl
-
-
piperazine
piperazine
B6
B7
B8
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
O
S
O
S
O
S
O
S
-
-
-
-
-
-
-
-
CH3
CH3
OCH3
OCH3
Cl
Cl
F
F
B19
B20
B21
B22
B23
B24
B25
B26
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
O
S
O
S
O
S
O
S
-
-
-
-
-
-
-
-
CH3
CH3
OCH3
OCH3
Cl
>Cl
F
B9
B10
B11
B12
B13
F
Scheme 1. The synthetic route of the compounds B1–B26. Reagents and conditions: (i) ethanol, ice bath,
2 h; (ii) N,N-dimethylformamide (DMF), K2CO3, reflux, 24 h; (iii) ethanol, reflux, 4–10 h.
2.2. Monoamine oxidase Inhibition Assay
The novel series of thiosemicarbazone derivatives B1–B26 were investigated for their MAO-A and
MAO-B inhibitory −ac3tivity by a two-step fluorometric method. In the first step, compounds B1
–B26
−4
were screened at 10 and 10 M concentrations. Compounds that indicate more than 50% inhibition
−9
at first step, were tested at 10−5–10 M concentrations. As seen in Table 1, compounds B19
, B21–B26
showed 50% inhibitory activity against MAO-A at 10−3 concentration. Except for compound B24
,
these compounds did not display 50% inhibition at 10−4 concentration, thus only compound B24 was
selected for the second step activity assay. The result of this step is presented in Figure 1. The second
screening of compound B24 against MAO-A revealed that this compound displayed a half-maximal
inhibitory concentration (IC50) of 0.662
2.179 M. None of the compounds showed inhibitory effect towards MAO-B at tested concentrations.
These findings suggest that compounds are more selective inhibitors against MAO-A than MAO-B.
µM, whereas reference drug moclobemide had an IC50 of
µ

Molecules 2018, 23, 60
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Table 1. Initial screening results to determine inhibition potency of compounds B1–B26, moclobemide
and selegiline against monoam−in4e oxidase A (MAO-A) and monoamine oxidase B (MAO-B) at
−3
concentrations of 10 M and 10 M.
MAO-A Inhibition %
MAO-B Inhibition %
Compound
−3
−4
−3
−4
10 M
10
M
10
M
10
M
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
21.08 ± 0.34
30.68 ± 0.85
20.77 ± 0.55
30.85 ± 1.07
32.61 ± 1.03
26.98 ± 0.77
30.10 ± 1.20
34.83 ± 0.79
24.56 ± 0.89
40.79 ± 1.23
35.90 ± 0.22
32.06 ± 0.68
36.65 ± 1.13
41.08 ± 1.26
39.48 ± 1.05
37.96 ± 1.00
48.83 ± 1.02
39.40 ± 0.62
53.56 ± 1.08
41.33 ± 0.39
58.52 ± 1.36
50.88 ± 1.09
64.85 ± 1.04
83.31 ± 1.08
57.14 ± 1.17
51.33 ± 0.38
13.77 ± 0.29
16.28 ± 0.97
15.51 ± 0.50
26.77 ± 0.79
16.63 ± 0.51
11.79 ± 0.28
24.08 ± 0.81
19.76 ± 0.71
18.80 ± 0.62
32.88 ± 0.77
30.01 ± 0.45
26.33 ± 0.57
27.19 ± 0.84
27.09 ± 0.67
20.06 ± 0.97
29.88 ± 0.98
39.77 ± 1.26
31.28 ± 0.57
40.57 ± 1.12
35.66 ± 0.27
35.75 ± 0.91
38.29 ± 0.97
46.10 ± 0.77
70.07 ± 1.12
32.38 ± 0.68
36.09 ± 0.22
15.80 ± 0.30
27.08 ± 0.46
17.85 ± 0.28
20.32 ± 0.67
29.88 ± 0.88
22.58 ± 0.62
23.45 ± 0.16
25.17 ± 0.82
18.62 ± 0.40
20.43 ± 0.58
31.34 ± 0.74
29.66 ± 1.02
27.49 ± 0.66
23.47 ± 0.48
22.17 ± 0.88
24.63 ± 0.48
30.75 ± 0.78
26.79 ± 0.49
32.06 ± 0.81
28.20 ± 0.66
37.03 ± 0.40
27.88 ± 0.45
24.55 ± 0.68
14.43 ± 0.26
30.40 ± 0.60
27.99 ± 0.19
10.21 ± 0.14
18.83 ± 0.37
11.41 ± 0.33
16.10 ± 0.62
14.43 ± 0.41
16.77 ± 0.44
18.79 ± 0.27
20.25 ± 0.80
14.11 ± 0.36
16.02 ± 0.69
27.85 ± 0.61
16.88 ± 0.47
20.66 ± 0.73
18.65 ± 0.33
14.20 ± 0.41
21.74 ± 0.36
17.49 ± 0.77
22.62 ± 0.40
27.05 ± 0.69
21.63 ± 0.50
30.49 ± 0.38
20.22 ± 0.41
19.62 ± 0.44
11.79 ± 0.30
24.61 ± 0.43
19.89 ± 0.11
Moclobemide
Selegiline
91.42 ± 4.60
77.86 ± 3.71
-
-
-
-
97.69 ± 4.16
94.42 ± 3.89
Figure 1. Further screening to determine inhibition potency and a half-maximal inhibitory
concentration (IC50) values of B24 and moclobemide against MAO-A.
Generally, compounds B14
Therefore, it can be concluded that a cyclohexyl ring at the N(1) position is more favourable
for the MAO-A inhibitory activity. Furthermore, between compounds B14 B26, compounds
containing 4-substituted phenyl ring linked by an oxygen or sulphur atom were indicated as more
–B26 have been found to more active than compounds B1–B13.
–

Molecules 2018, 23, 60
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potent. As the most active inhibitory agent is N-cyclohexyl-2-[4-[(4-chlorophenyl)thio]benzylidene]
hydrazine-1-carbothioamide (B24), a 4-chlorophenyl group linked with a sulphur atom was demonstrated
to be beneficial for MAO-A inhibitory activity.
2.3. Enzyme Kinetic Studies
The mechanism of MAO-A inhibition was examined by enzyme kinetics, following a similar
procedure to the MAO inhibition assay. The linear Lineweaver-Burk graphics were used to determine
the type of inhibition. Enzyme kinetics were analyzed by recording substrate velocity curves in
the absence and presence of the most potent compound B24, which was prepared at IC50/2, IC50
and 2
×
IC50 concentrations. The initial velocity measurements were gained at different substrate
M to 0.625 M. The steady-state inhibition constant (Ki)
(tyramine) concentrations ranging from 20
µ
µ
values of compound B24 was determined from the secondary plot slope versus varying concentrations.
The graphical analysis of steady-state inhibition data for compound B24 is presented in Figure 2.
(A)
(B)
Figure 2.
concentration (
at the left. Michaelis-Menten constant (Km) values from IC50/2 to control; 3.303, 6.455, 10.656
and 0.933 ( M). Vmax value of the competitive inhibition; 111.015 2.557 (nmol/min/mg protein);
) Secondary plot for calculation of steady-state inhibition constant (Ki) of compound B24. Ki was
calculated as 0.142 µM.
(A
) Lineweaver–Burk plots for the inhibition of MAO-A by compound B24. [S], substrate
µM); V, reaction velocity (nmol/min/mg protein). Inhibitor concentrations are shown
µ
±
(
B

Molecules 2018, 23, 60
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Enzyme inhibitors can be classified as reversible or irreversible relative to the type of interaction.
Uncompetitive, competitive, noncompetitive, and mixed inhibitions are the subtypes of reversible
inhibitors than can be determined by Lineweaver-Burk graphics [24]. It is known that uncompetitive
type inhibition is observed with a graph, including the parallel lines of inhibitor and control without
any cross. If there is a line crossing at the same point but not on the x-axis or y-axis the inhibition
type is called mixed-type. Non-competitive inhibitors display the same intercept on the x-axis but
with diverse slopes and intercepts on the y-axis between the two data sets. Oppositely, competitive
inhibitors give plots with the same intercept on the y-axis but different slopes and intercepts on the
x-axis as observed in Figure 2. Therefore, it can be stated that compound B24 is a reversible and
competitive inhibitor, showing similar inhibition features as the substrate. The Ki value for compound
B24 was calculated as 0.142 µM for the inhibition of MAO-A.
2.4. Cytotoxicity Test
After the preliminary enzyme inhibition assay, compound B24 was selected for the second step,
and besides its toxicity was measured because the cytotoxicity of a compound is a crucial parameter in
medicinal chemistry to obtain a safe drug candidate. A methylthiazolyl tetrazolium (MTT) assay [25
]
was applied to screen the cytotoxic effects of compound B24 on healthy NIH/3T3 mouse embryonic
fibroblast cell lines (ATCC CRL1658, London, UK). It has been determined that compound B24 had
an IC50 value of 24.6 µM against NIH/3T3 cells, while its IC50 value was 0.662 µM against MAO-A,
thus proving that compound B24 is non-toxic at its effective concentration towards MAO-A.
2.5. Molecular Docking Studies
As stated in MAO inhibition assay, compound B24 was found to be most active and selective
derivative against MAO-A. Thus, to assess in silico its activity and observe the binding modes between
B24 and MAO-A, docking studies were carried out. The X-ray crystal structure of MAO-A (PDB ID:
2Z5X) [26] was obtained from the Protein Data Bank server (www.pdb.org). The docking poses of
compound B24 on MAO-A are presented in Figure 3.
By looking at the interactions described in the literature for harmine, it was seen that harmine is
positioned in the active site of the enzyme MAO-A. It interacts with Tyr69, Asn181, Phe208, Val210,
Gln215, Cys323, Ile325, Ile335, Leu337, Phe352, Tyr407, Tyr444 and flavin adenine dinucleotide (FAD)
molecule. The amide group of the Gln215 side chain interacts tightly with harmine by a
Also, the pyridine ring is stabilized with Tyr407 and Tyr444 by forming a interaction with phenyl
rings [26 27]. When the docking pose of compound B24 was analysed, both similar and additional
interactions were observed. Compound B24 closely binds to some amino acid residues lining the
π–π interaction.
π-π
,
cavity, and is located very near the FAD cofactor. According to the docking pose (Figure 3), two
interactions are observed.
π-π
The 4-chlorophenyl substructure is in an interaction with the phenyl of Phe208, whereas the
benzylidene substructure establishes a interaction with the phenyl of Phe352. Due to its hydrogen
π-π
acceptor and donor atoms thiosemicarbazide moiety is essential for polar interactions. Two nitrogen
atoms (N3 and N4) of this group have ability to form two different hydrogen bonds. The first hydrogen
bond is observed between the amino nitrogen and the carbonyl of Asn181. The other one is seen
between the imine nitrogen and hydroxyl of Tyr407. These interactions support the approach, which
reveals that amino acid side chains, coating the cavity, are very favorable to interact with the amine
moieties [28–31]. The last interaction in the structure is seen as a halogen bond. The chloro atom at the
C4 position of the phenyl ring forms a halogen bond with Ala111. The appearance of this interaction
explains why compound B24 displays stronger binding to MAO-A.
When the chemical structures of the synthesized compounds are examined in general, it would
be expected that compounds B10 and B11 should display similar activity to compound B24. However,
their low inhibition rates may be due to the presence of the phenyl ring in place of a cyclohexyl. It is
thought that the phenyl ring makes the structure more stable and leads a a rigid conformation. As a

Molecules 2018, 23, 60
7 of 18
result, for compounds B10 and B11 it could be suggested that the presence of a phenyl on the N1 atom
leads to less binding to the enzyme active site, which reduces their enzyme inhibition potency.
Figure 3. The interacting mode of compound B24 in the active region of MAO-A. The inhibitor and the
important residues in the active site of the enzyme are presented by tube model. The flavin adenine
dinucleotide (FAD) molecule is colored green with ball and stick model. After superposing X-ray
ligands Root Mean Square Deviation (RMSD) value is 0.029 Å. Docking score, glide gscore and glide
emodel for compound B24 were found as: −11.869, −11.869 and −96.645, respectively.
2.6. Theoretical Determination of Absorption, Distribution, Metabolism, and Excretion (ADME) Properties
The physicochemical properties of a compound are important for its suitability to be developed
as an oral drug candidate. Absorption, distribution, metabolism, and excretion (ADME) studies
were performed using the QikProp 4.8 software [32]. This software calculates any violations of
Lipinski’s rule of five [33] and Jorgensen’s rule of three [34]. The results are exhibited in Table 2.
Considering Lipkinski’s rule of five, compounds with a number of violations of not more than 1 show
good bioavailability. Molecular weights (MW) of the all compounds are smaller than 500 Dalton.
Their hydrogen bond donors (DHB), hydrogen bond acceptors (AHB) and polar surface area (PSA)
values are within the range. Some of the compounds have an octanol/water partition coefficient (log P)
value bigger than five. Compounds B1–B24 are in general accordance with the rule by causing no more
than one violation. In accordance with Jorgensen’s rule of three, all apparent Caco-2 cell permeability
(PCaco) values are very high and number of likely primer metabolic reactions (PM) are within the
limits. The aqueous solubility (log S) of the compounds are smaller than
from this rule.
−
5.7 and the only violation
MAO inhibitors target to have effects on central nervous system (CNS), thus should able to pass
the blood-brain barrier (BBB). The blood/brain partition coefficient (logBB), apparent Madin-Darby
canine kidney (MDCK) cell permeability (PMDCK) are useful parameters to assign the penetration
capacity of a compound from blood-brain barrier. The logBB values of all compounds are within
recommended
CNS value of compound B24 is 1, while this value is 0 for the rest of the compounds. Consequently,
compounds B1 B26 are determined to have good pharmacokinetic profiles and high BBB permeability,
which enhances the biological importance of these compounds as potential CNS drug candidates.
−
3–+1.2 range. The PMDCK value of all compounds are higher than 500. The predicted
–

Molecules 2018, 23, 60
8 of 18
Table 2. Calculated Absorption, Distribution, Metabolism, and Excretion (ADME) parameters of compounds B1–B26.
Compound
MW
DHB
AHB
PSA
logP
logS
PCaco
PM
PMDCK
logBB
CNS
VRF VRT
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
338.470
352.496
352.496
415.555
445.581
361.461
377.521
377.460
393.521
381.879
397.939
365.424
381.485
344.517
358.544
358.544
421.602
451.629
367.508
383.569
383.507
399.568
387.926
403.987
371.472
387.532
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
5
5
5
49.573
49.147
49.575
55.441
63.922
52.430
44.334
60.912
52.815
52.431
44.334
52.431
44.334
47.948
47.521
47.989
54.099
62.782
50.803
42.707
59.285
51.188
50.804
42.707
50.804
42.707
4.334
4.574
4.660
5.471
5.519
4.937
5.540
4.696
5.296
5.129
5.730
4.873
5.473
4.598
4.838
4.898
5.652
5.827
5.204
5.810
4.959
5.562
5.395
6
−6.156
−6.362
−6.598
−7.686
−7.822
−6.253
−7.067
−5.858
−6.666
−6.434
−7.247
−6.063
-6.872
−6.731
−6.938
−7.145
−8.096
−8.356
−6.835
−7.656
−6.433
−7.249
−7.015
−7.835
−6.642
−7.459
2663.609
2679.674
2663.218
2584.097
2584.100
2720.949
2720.949
2720.951
2720.951
2720.949
2720.949
2720.949
2720.949
3310.935
3331.393
3204.223
2974.126
3504.952
3382.919
3382.919
3382.922
3382.922
3382.919
3382.919
3382.919
3382.919
2
2
2
2
3
3
3
3
3
2
2
2
2
0
0
0
0
1
1
1
1
1
0
0
0
0
2461.245
2477.294
2460.854
2381.928
2381.931
2518.564
4269.676
2518.566
4269.729
6213.625
10,000
4554.137
7721.149
3266.539
3288.360
3153.046
2908.896
3665.702
3343.532
5668.228
3343.534
5668.299
8248.927
10,000
−0.222
−0.212
−0.231
−0.281
−0.352
−0.333
−0.249
−0.388
−0.304
−0.154
−0.070
−0.207
−0.123
−0.115
−0.106
−0.138
−0.199
−0.174
−0.226
−0.139
−0.282
−0.194
−0.048
-0.040
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
0
1
0
1
1
1
0
1
0
0
0
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
6.75
4.5
4
5.25
4.75
4.5
4
4.5
4
5
5
5
6
6.75
4.5
4
5.25
4.75
4.5
4
4.5
4
5.138
5.741
6045.866
10,000
−0.102
−0.015
MW: Molecular weight; DHB: Estimated number of hydrogen bond donors; AHB: Estimated number of hydrogen bond acceptors; PSA: Van der Waals surface area of polar nitrogen and
oxygen atoms and carbonyl carbon atoms; logP: Predicted octanol/water partition coefficient; logS: Predicted aqueous solubility; PCaco: Predicted apparent Caco-2 cell permeability;
PM: Number of likely metabolic reactions; PMDCK: Predicted apparent Madin-Darby canine kidney (MDCK) cell permeability; logBB: Predicted brain/blood partition coefficient;
CNS: Predicted central nervous system activity on a
−
2 (inactive) to +2 (active) scale (recommended value:
10, Positive PSA value; VRT: Number of violations of Jorgensen’s rule of three. The three rules are: logS >
−
2 (inactive), +2 (active));VRF: Number of violations of Lipinski’s rule of five.
5.7, PCaco > 22 nm/s,
The rules are: MW < 500, logP < 5, DHB
PM < 7.
≤
5, AHB
≤
−

Molecules 2018, 23, 60
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3. Materials and Methods
3.1. General Information
All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and Merck
KGaA (Darmstadt, Germany). All melting points (m.p.) were determined on an Electrothermal 9100
digital melting point apparatus (Electrothermal, Essex, UK) and are uncorrected. All reactions were
monitored by thin-layer chromatography (TLC) using Silica Gel 60 F254 TLC plates (Merck KGaA).
Spectroscopic data were recorded with the following instruments: IR: 8400S spectrophotometer
(Shimadzu, Tokyo, Japan); 1H-NMR: DPX 300 NMR spectrometer (Bruker Bioscience, Billerica, MA,
USA), 13C-NMR: Bruker DPX 75 NMR spectrometer in dimethyl sulfoxide (DMSO)-d6, using TMS
as internal standard; HRMS: Shimadzu, Liquid Chromatography/Mass Spectrometer Ion-Trap and
Time-of-Flight (LC/MS ITTOF) system.
3.2. Chemistry
3.2.1. Synthesis of N-Phenyl/Cyclohexylhydrazinecarbothioamides 1,2
Phenyl- or cyclohexylisothiocyanate (25 mmol) was dissolved in ethanol (200 mL). Hydrazine
hydrate (50 mmol) was placed in an addition funnel and diluted with ethanol (20 mL). The diluted
hydrazine hydrate was added to the mixture in an ice bath. After the completion of the addition, the
resulting solid product was filtered and washed with cold ethanol. The products were recrystallized
from ethanol.
3.2.2. Synthesis of 4-Substituted Benzaldehyde Derivatives 3–16
Appropriate seconder amines (5 mmol) or 4-substituted phenol/thiophenol derivatives (5 mmol)
were refluxed with 4-fluorobenzaldehyde (5 mmol, 0.62 g) in dimethylformamide (10 mL) with the
presence of potassium carbonate (6 mmol, 0.83 g). After TLC screening, the mixture was poured into
ice water and filtered. The products were recrystallized from ethanol.
3.2.3. Synthesis of 2-(4-Substituted Benzylidene)-N-phenyl/cyclohexylhydrazine-1-carbothioamide
Derivatives B1–B26
Equal amounts of N-phenyl/cyclohexylhydrazinecarbothioamides
4-substituted benzaldehyde (2 mmol) derivatives 16 were refluxed in ethanol (40 mL). After
completion of the reaction, the product was filtered and recrystallized from ethanol.
1,2 (2 mmol) and the synthesized
3–
◦
N-Phenyl-2-[4-(piperidin-1-yl)benzylidene]hydrazine-1-carbothioamide (B1): Yield 68–70%, m.p. 182–184 C.
Fourier transform IR (FTIR) (Attenuated total reflection (ATR), cm−1): 3300 (N-H), 3134 (aromatic
C-H), 2929 (aliphatic C-H), 1591–1444 (C=N and C=C), 1124 (C-N). 1H-NMR (300 MHz, DMSO-d6,
ppm)
δ 1.58 (6H, s, piperidine-H), 3.26 (4H, s, piperidine-H), 6.93 (2H, d, J = 8.9 Hz, disubstituted
phenyl-H), 7.19 (1H, t, J = 7.4 Hz, phenyl-H), 7.36 (2H, t, J = 7.8 Hz, phenyl-H), 7.59 (2H, d, J = 7.9 Hz,
phenyl-H), 7.69 (2H, d, J = 8.8 Hz, disubstituted phenyl-H), 8.05 (1H, s, N=CH), 9.95 (1H, s, NH), 11.60
(1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 24.37, 25.43, 48.85, 114.89, 123.39, 125.54, 126.03,
128.47, 129.43, 139.61, 144.10, 152.90, 175.55. HRMS (m/z): [M + H]+ calcd. for C19H22N4S: 339.1638;
found 339.1626.
N-Pheny◦l-2-[4-(2-methylpiperidin-1-yl)benzylidene]hydrazine-1-carbothioamide (B2): Yield 68–70%, m.p.
178–180 C. FTIR (ATR, cm−1): 3300 (N-H), 3132 (aromatic C-H), 2976 (aliphatic C-H), 1591–1444 (C=N
and C=C), 1182 (C-N). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 0.91 (3H, d, J = 6.6 Hz, CH3), 1.06–1.14
(1H, m, piperidine-H), 1.44–1.79 (4H, m, piperidine-H), 2.37–2.45 (1H, m, piperidine-H), 2.67–2.76 (1H,
m, piperidine-H), 3.70–3.80 (2H, m, piperidine-H), 6.94 (2H, d, J = 8.9 Hz, disubstituted phenyl-H),
7.19 (1H, t, J = 7.3 Hz, phenyl-H), 7.36 (2H, t, J = 8.1 Hz, phenyl-H), 7.59 (2H, d, J = 7.5 Hz, phenyl-H),
7.69 (2H, d, J = 8.9 Hz, disubstituted phenyl-H), 8.04 (1H, s, N=CH), 9.94 (1H, s, NH), 11.59 (1H, s, NH).

Molecules 2018, 23, 60
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13C-NMR (75 MHz, DMSO-d6, ppm)
δ
19.65, 24.87, 30.54, 32.96, 48.25, 55.66, 114.82, 123.25, 125.54,
126.03, 128.47, 129.46, 139.62, 144.13, 152.68, 175.53. HRMS (m/z): [M + H]+ calcd. for C20H24N4S:
353.1794; found 353.1809.
N-Pheny◦l-2-[4-(4-methylpiperidin-1-yl)benzylidene]hydrazine-1-carbothioamide (B3): Yield 68–70%, m.p.
156–159 C. FTIR (ATR, cm−1): 3280 (N-H), 3134 (aromatic C-H), 2943 (aliphatic C-H), 1591–1442 (C=N
and C=C), 1132 (C-N). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 0.92 (3H, d, J = 6.5 Hz, CH3), 1.11–1.24
(2H, m, piperidine-H), 1.47–1.60 (1H, m, piperidine-H), 1.66–1.70 (2H, m, piperidine-H), 2.70–2.79 (2H,
m, piperidine-H), 3.79–3.83 (2H, m, piperidine-H), 6.93 (2H, d, J = 9.0 Hz, disubstituted phenyl-H),
7.19 (1H, t, J = 7.3 Hz, phenyl-H), 7.36 (2H, t, J = 7.9 Hz, phenyl-H), 7.59 (2H, d, J = 7.5 Hz, phenyl-H),
7.69 (2H, d, J = 8.9 Hz, disubstituted phenyl-H), 8.04 (1H, s, N=CH), 9.94 (1H, s, NH), 11.60 (1H, s,
NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 22.22, 30.70, 33.68, 48.18, 114.90, 123.37, 125.54, 126.03,
128.47, 129.44, 139.61, 144.11, 152.68, 175.54. HRMS (m/z): [M + H]+ calcd. for C20H24N4S: 353.1794;
found 353.1793.
N-Pheny◦l-2-[4-(4-phenylpiperazin-1-yl)benzylidene]hydrazine-1-carbothioamide (B4): Yield 68–70%, m.p.
214–217 C. FTIR (ATR, cm−1): 3344 (N-H), 3118 (aromatic C-H), 2966 (aliphatic C-H), 1593–1444 (C=N
and C=C), 1207 (C-N). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 3.25–3.29 (4H, m, piperazine-H), 3.42
(4H, s, piperazine-H), 6.81 (1H, t, J = 7.2 Hz, phenyl-H), 7.01 (4H, m, disubstituted phenyl-H), 7.17–7.27
(3H, m, phenyl-H), 7.36 (2H, t, J = 8.1 Hz, phenyl-H), 7.58 (2H, d, J = 7.5 Hz, phenyl-H), 7.75 (2H, d,
J = 8.9 Hz, disubstituted phenyl-H), 8.07 (1H, s, N=CH), 9.98 (1H, s, NH), 11.63 (1H, s, NH). 13C-NMR
(75 MHz, DMSO-d6, ppm) δ 47.71, 48.62, 115.05, 116.14, 119.68, 124.45, 125.60, 126.14, 128.48, 129.41,
129.48, 139.61, 143.92, 151.29, 152.47, 175.70. HRMS (m/z): [M + H]+ calcd. for C24H25N5S: 416.1903;
found 416.1892.
N-Phenyl-2-[4-[4-(4-methoxyphenyl)piperazin-1-yl]benzylidene]hydrazine-1-carbothioamide (B5): Yield
◦
68–70%, m.p. 212–215 C. FTIR (ATR, cm−1): 3334 (N-H), 3122 (aromatic C-H), 2974 (aliphatic
C-H), 1591–1446 (C=N and C=C), 1269–1028 (C-N and C-O). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ
3.12–3.16 (4H, m, piperazine-H), 3.38 (4H, s, piperazine-H), 3.69 (3H, s, OCH3), 6.84 (2H, d, J = 9.1 Hz,
methoxyphenyl-H), 6.95 (2H, d, J = 9.0 Hz, methoxyphenyl-H), 7.02 (2H, d, J = 8.9 Hz, disubstituted
phenyl-H), 7.19 (1H, t, J = 7.5 Hz, phenyl-H), 7.36 (2H, t, J = 7.9 Hz, phenyl-H), 7.58 (2H, d, J = 7.5 Hz,
phenyl-H), 7.75 (2H, d, J = 8.8 Hz, disubstituted phenyl-H), 8.07 (1H, s, N=CH), 9.98 (1H, s, NH), 11.64
(1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 47.86, 50.12, 55.66, 114.77, 115.05, 118.24, 124.43,
125.58, 126.14, 128.47, 129.40, 139.63, 143.90, 145.67, 152.51, 153.66, 175.70. HRMS (m/z): [M + H]+
calcd. for C25H27N5OS: 446.2009; found 446.1992.
N-Pheny◦l-2-[4-(4-methylphenoxy)benzylidene]hydrazine-1-carbothioamide (B6): Yield 68–70%, m.p.
175–178 C. FTIR (ATR, cm−1): 3318 (N-H), 3113 (aromatic C-H), 2970 (aliphatic C-H), 1593–1449 (C=N
and C=C), 1238–1157 (C-N and C-O). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 2.31 (3H, s, CH3), 6.97–6.99
(4H, m, methylphenyl-H, disubstituted phenyl-H), 7.17–7.24 (3H, m, methylphenyl-H, phenyl-H), 7.37
(2H, t, J = 7.8 Hz, phenyl-H), 7.57 (2H, d, J = 7.8 Hz, phenyl-H), 7.90 (2H, d, J = 8.7 Hz, disubstituted
phenyl-H), 8.13 (1H, s, N=CH), 10.08 (1H, s, NH), 11.78 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6,
ppm)
δ 20.77, 118.09, 119.92, 125.73, 126.30, 128.50, 129.19, 129.97, 131.02, 133.83, 139.54, 142.75, 153.80,
159.46, 176.24. HRMS (m/z): [M + H]+ calcd. for C21H19N3OS: 362.1322; found 362.1315.
N-Phenyl-2-[4-[(4-methylphenyl)thio]benzylidene]hydrazine-1-carbothioamide (B7): Yield 68–70%, m.p.
◦
162–165 C. FTIR (ATR, cm−1): 3420 (N-H), 3136 (aromatic C-H), 2978 (aliphatic C-H), 1591–1447
(C=N and C=C), 1263 (C-N). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 2.32 (3H, s, CH3), 7.18 (2H, d,
J = 8.4 Hz, disubstituted phenyl-H), 7.22–7.24 (3H, m, methylphenyl-H, phenyl-H), 7.33–7.39 (4H, m,
methylphenyl-H, phenyl-H), 7.55 (2H, d, J = 7.7 Hz, phenyl-H), 7.84 (2H, d, J = 8.4 Hz, disubstituted
phenyl-H), 8.09 (1H, s, N=CH), 10.08 (1H, s, NH), 11.82 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6,
ppm)
δ 21.19, 125.82, 126.30, 128.53, 128.61, 128.84, 129.32, 130.98, 132.50, 133.46, 138.87, 139.45, 139.75,
142.51, 176.33. HRMS (m/z): [M + H]+ calcd. for C21H19N3S2: 378.1093; found 378.1086.

Molecules 2018, 23, 60
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N-Phenyl-2-[4-(4-methoxyphenoxy)benzylidene]hydrazine-1-carbothioamide (B8): Yield 68–70%, m.p.
◦
189–192 C. FTIR (ATR, cm−1): 3306 (N-H), 3129 (aromatic C-H), 2968 (aliphatic C-H), 1595–1447
(C=N and C=C), 1254–1194 (C-N and C-O). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ
3.75 (3H, s, OCH3),
6.93 (2H, d, J = 8.8 Hz, disubstituted phenyl-H), 6.99 (2H, d, J = 9.2 Hz, methoxyphenyl-H), 7.06 (2H, d,
J = 9.1 Hz, methoxyphenyl-H), 7.20 (1H, t, J = 7.4 Hz, phenyl-H), 7.36 (2H, t, J = 7.7 Hz, phenyl-H),
7.55 (2H, d, J = 7.3 Hz, phenyl-H), 7.81 (2H, d, J = 8.5 Hz, disubstituted phenyl-H), 8.09 (1H, s, N=CH),
10.08 (1H, s, NH), 11.82 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 55.90, 115.65, 117.40,
121.67, 125.76, 126.28, 128.51, 128.78, 129.94, 139.51, 142.86, 149.01, 156.47, 160.21, 176.20. HRMS (m/z):
[M + H]+ calcd. for C21H19N3SO2: 378.1271; found 378.1263.
N-Pheny◦l-2-[4-[(4-methoxyphenyl)thio]benzylidene]hydrazine-1-carbothioamide (B9): Yield 68–70%, m.p.
175–178 C. FTIR (ATR, cm−1): 3289 (N-H), 3157 (aromatic C-H), 2974 (aliphatic C-H), 1591–1456 (C=N
and C=C), 1287–1190 (C-N and C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 3.80 (3H, s, OCH3), 7.05
(2H, d, J = 8.9 Hz, methoxyphenyl-H), 7.09 (2H, d, J = 8.4 Hz, disubstituted phenyl-H), 7.20 (1H, t,
J = 7.4 Hz, phenyl-H), 7.36 (2H, t, J = 7.8 Hz, phenyl-H), 7.47 (2H, d, J = 8.8 Hz, methoxyphenyl-H),
7.56 (2H, d, J = 7.7 Hz, phenyl-H), 7.88 (2H, d, J = 8.8 Hz, disubstituted phenyl-H), 8.11 (1H, s, N=CH),
10.05 (1H, s, NH), 11.75 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 55.82, 116.01, 122.14,
125.77, 126.28, 127.20, 128.50, 128.76, 131.94, 136.46, 139.49, 141.29, 142.53, 160.54, 176.28. HRMS (m/z):
[M + H]+ calcd. for C21H19N3S2O: 394.1042; found 394.1034.
N-Phenyl-2-[4-(4-chlorophenoxy)benzylidene]hydrazine-1-carbothioamide (B10): Yield 68–70%, m.p.
167–170 C. FTIR (ATR, cm−1): 3318 (N-H), 3111 (aromatic C-H), 2966 (aliphatic C-H), 1589–1447
◦
(C=N and C=C), 1236–1194 (C-N and C-O). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 7.06 (2H, d, J = 8.8
Hz, disubstituted phenyl-H), 7.10 (2H, d, J = 8.9 Hz, chlorophenyl-H), 7.20 (1H, t, J = 7.4 Hz, phenyl-H),
7.37 (2H, t, J = 7.8 Hz, phenyl-H), 7.47 (2H, d, J = 8.9 Hz, chlorophenyl-H), 7.56 (2H, d, J = 7.4 Hz,
phenyl-H), 7.95 (2H, d, J = 8.8 Hz, disubstituted phenyl-H), 8.14 (1H, s, N=CH), 10.10 (1H, s, NH), 11.81
(1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 118.94, 121.29, 125.77, 126.34, 128.25, 128.50, 129.49,
130.10, 130.50, 139.53, 142.57, 155.40, 158.39, 176.32. HRMS (m/z): [M + H]+ calcd. for C20H16N3SOCl:
382.0775; found 382.0764.
N-Pheny◦l-2-[4-[(4-chlorophenyl)thio]benzylidene]hydrazine-1-carbothioamide (B11): Yield 68–70%, m.p.
175–177 C. FTIR (ATR, cm−1): 3329 (N-H), 3127 (aromatic C-H), 2972 (aliphatic C-H), 1591–1447 (C=N
and C=C), 1269–1203 (C-N and C-S). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 7.20 (1H, t, J = 7.4 Hz,
phenyl-H), 7.31 (2H, d, J = 8.4 Hz, disubstituted phenyl-H), 7.37–7.41 (4H, m, chlorophenyl-H,
phenyl-H), 7.47 (2H, d, J = 8.7 Hz, chlorophenyl-H), 7.54 (2H, d, J = 7.4 Hz, phenyl-H), 7.90 (2H,
d, J = 8.4 Hz, disubstituted phenyl-H), 8.12 (1H, s, N=CH), 10.12 (1H, s, NH), 11.85 (1H, s, NH).
13C-NMR (75 MHz, DMSO-d6, ppm)
δ 125.86, 126.36, 128.54, 129.09, 130.16, 130.49, 133.19, 133.38,
133.51, 133.55, 137.31, 139.45, 142.32, 176.44. HRMS (m/z): [M + H]+ calcd. for C20H16N3S2Cl: 398.0547;
found 398.0533.
N-Pheny◦l-2-[4-(4-fluorophenoxy)benzylidene]hydrazine-1-carbothioamide (B12): Yield 68–70%, m.p.
135–136 C. FTIR (ATR, cm−1): 3285 (N-H), 3126 (aromatic C-H), 2974 (aliphatic C-H), 1591–1495 (C=N
and C=C), 1271–1192 (C-N and C-O). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 7.00 (2H, d, J = 8.8 Hz,
disubstituted phenyl-H), 7.11–7.14 (2H, m, fluorophenyl-H), 7.20 (1H, t, J = 7.3 Hz, phenyl-H), 7.24–7.20
(2H, m, fluorophenyl -H), 7.36 (2H, t, J = 7.8 Hz, phenyl-H), 7.54 (2H, d, J = 7.4 Hz, phenyl-H), 7.91
(2H, d, J = 8.8 Hz, disubstituted phenyl-H), 8.12 (1H, s, N=CH), 10.08 (1H, s, NH), 11.77 (1H, s, NH).
13C-NMR (75 MHz, DMSO-d6, ppm)
δ 117.23 (d, J = 23.3 Hz), 118.16, 121.82 (d, J = 8.3 Hz), 125.79,
126.31, 128.52, 129.02 (d, J = 3.1 Hz), 129.47, 130.04, 139.50, 142.71, 159.29, 163.11 (d, J = 242.3 Hz),
176.27. HRMS (m/z): [M + H]+ calcd. for C20H16N3SOF: 366.1071; found 366.1061.
N-Phenyl-2-[4-[(4-fluorophenyl)thio]benzylidene]hydrazine-1-carbothioamide (B13): Yield 68–70%, m.p.
◦
168–169 C. FTIR (ATR, cm−1): 3331 (N-H), 3127 (aromatic C-H), 2974 (aliphatic C-H), 1591–1447
(C=N and C=C), 1271–1153 (C-N and C-S). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 7.20–7.23 (2H, m,

Molecules 2018, 23, 60
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disubstituted phenyl-H, phenyl-H), 7.27–7.39 (2H, m, fluorophenyl-H, phenyl-H), 7.50–7.57 (2H, m,
fluorophenyl-H, phenyl-H), 7.87 (2H, d, J = 8.5 Hz, disubstituted phenyl-H), 8.10 (1H, s, N=CH), 10.10
(1H, s, NH), 11.85 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 117.43 (d, J = 21.8 Hz), 125.81,
126.34, 128.51, 128.81, 128.98 (d, J = 3.0 Hz), 132.89, 135.62 (d, J = 8.4 Hz), 139.03, 139.48, 142.36, 162.68
(d, J = 244.5 Hz), 176.39. HRMS (m/z): [M + H]+ calcd. for C20H16N3S2F: 382.0842; found 382.0831.
N-Cyclohexyl-2-[4-(piperidin-1-yl)benzylidene]hydrazine-1-carbothioamide (B14): Yield 66–68%, m.p.
204–205 ◦C. FTIR (ATR, cm−1): 3310 (N-H), 3061 (aromatic C-H), 2987 (aliphatic C-H), 1600 (C=N),
1
1449 (C=C), 1269 (C-N). H-NMR (300 MHz, DMSO-d6, ppm)
δ 1.08–1.88 (16H, m, cyclohexyl-H,
piperidine-H), 3.25 (4H, br s, cyclohexyl-H, piperidine-H), 4.11–4.22 (1H, m, cyclohexyl-H,
piperidine-H), 6.92 (2H, d, J = 8.9 Hz, disubstituted phenyl-H), 7.57 (2H, d, J = 8.8 Hz, disubstituted
phenyl-H), 7.84 (1H, d, J = 8.6 Hz, N=CH), 7.94 (1H, s, NH), 11.19 (1H, s, NH). 13C-NMR (75 MHz,
DMSO-d6, ppm)
δ 24.38, 25.38, 25.44, 25.62, 32.39, 48.92, 52.85, 115.02, 123.61, 129.03, 143.27, 152.77,
178.58. HRMS (m/z): [M + H]+ calcd. for C19H28N4S: 345.2107; found 345.2091.
N-Cyclohexyl-2-[4-(2-methylpiperidin-1-yl)benzylidene]hydrazine-1-carbothioamide (B15): Yield 68–70%,
m.p. 212–213 C. FTIR (ATR, cm−1): 3306 (N-H), 3051 (aromatic C-H), 2986 (aliphatic C-H), 1599 (C=N),
◦
1452 (C=C), 1219 (C-N). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 0.92 (3H, d, J = 6.5 Hz, CH3), 1.12–1.88
(16H, m, cyclohexyl-H, piperidine-H), 2.69–2.78 (2H, m, cyclohexyl-H, piperidine-H), 3.77–3.81 (2H, m,
cyclohexyl-H, piperidine-H), 4.11–4.22 (1H, m, cyclohexyl-H, piperidine-H), 6.92 (2H, d, J = 8.9 Hz,
disubstituted phenyl -H), 7.57 (2H, d, J = 8.9 Hz, disubstituted phenyl -H), 7.83 (1H, d, J = 8.6 Hz,
N=CH), 7.94 (1H, s, NH), 11.18 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 22.22, 25.38, 25.62,
30.70, 32.39, 33.69, 48.25, 52.85, 115.02, 123.59, 129.04, 143.27, 152.55, 175.58. HRMS (m/z): [M + H]+
calcd. for C20H30N4S: 359.2264; found 359.2252.
N-Cyclohexyl-2◦-[4-(4-methylpiperidin-1-yl)benzylidene]hydrazine-1-carbothioamide (B16): Yield 68–70%,
m.p. 200–201 C. FTIR (ATR, cm−1): 3310 (N-H), 3067 (aromatic C-H), 2976 (aliphatic C-H), 1599
1
(C=N), 1503 (C=C), 1218 (C-N). H-NMR (300 MHz, DMSO-d6, ppm)
δ 0.91 (3H, d, J = 6.6 Hz,
CH3), 1.02–1.89 (16H, m, cyclohexyl-H, piperidine-H), 2.40–2.44 (1H, m, cyclohexyl-H, piperidine-H),
2.65–2.74 (1H, m, cyclohexyl-H, piperidine-H), 3.68–3.76 (2H, m, cyclohexyl-H, piperidine-H), 4.16–4.18
(1H, m, cyclohexyl-H, piperidine-H), 6.92 (2H, d, J = 8.9 Hz, disubstituted phenyl -H), 7.56 (2H, d,
J = 8.9 Hz, disubstituted phenyl-H), 7.83 (1H, d, J = 8.6 Hz, N=CH), 7.94 (1H, s, NH), 11.19 (1H, s, NH).
13C-NMR (75 MHz, DMSO-d6, ppm)
δ 19.67, 24.87, 25.37, 25.62, 30.53, 32.39, 32.97, 48.33, 52.83, 55.75,
114.94, 123.46, 129.05, 143.23, 152.55, 175.57. HRMS (m/z): [M + H]+ calcd. for C20H30N4S: 359.2264;
found 359.2262.
N-Cyclohexyl-2◦-[4-(4-phenylpiperazin-1-yl)benzylidene]hydrazine-1-carbothioamide (B17): Yield 68–70%,
m.p. 227–228 C. FTIR (ATR, cm−1): 3300 (N-H), 3061 (aromatic C-H), 2972 (aliphatic C-H), 1597
(C=N),1493 (C=C), 1229 (C-N). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 1.03–1.89 (10H, m, cyclohexyl-H),
3.26–3.29 (4H, m, piperazine-H), 3.38–3.41 (4H, m, piperazine-H), 4.12–4.23 (1H, m, cyclohexyl-H),
6.81 (1H, t, J = 7.3 Hz, phenyl-H), 6.98–7.03 (4H, m, phenyl-H, disubstituted phenyl-H), 7.24 (2H, t,
J = 8.0 Hz, phenyl-H), 7.64 (2H, d, J = 8.9 Hz, disubstituted phenyl-H), 7.88 (1H, d, J = 8.6 Hz, N=CH),
7.91 (1H, s, NH), 11.23 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 25.40, 25.63, 32.38, 47.79,
48.62, 52.92, 115.16, 116.14, 119.66, 124.64, 129.02, 129.46, 143.10, 151.31, 152.33, 175.66. HRMS (m/z):
[M + H]+ calcd. for C24H31N5S: 422.2373; found 422.2357.
N-Cyclohexyl-2-[4-[4-(4-methoxyphenyl)piperazin-1-yl]benzylidene]hydrazine-1-carbothioamide (B18): Yield
68–70%, m.p. 238–239 ◦C. FTIR (ATR, cm−1): 3333 (N-H), 3052 (aromatic C-H), 2974 (aliphatic C-H),
1599 (C=N), 1489 (C=C), 1227–1107 (C-N and C-O). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 1.12–1.89
(10H, m, cyclohexyl-H), 3.14 (4H, br s, piperazine-H), 3.37 (4H, br s, piperazine-H), 3.69 (3H, s, OCH3),
4.17–4.19 (1H, m, cyclohexyl-H), 6.84 (2H, d, J = 8.0 Hz, methoxyphenyl-H), 6.95 (2H, d, J = 8.1 Hz,
methoxyphenyl-H), 7.01 (2H, d, J = 7.9 Hz, disubstituted phenyl-H), 7.63 (2H, d, J = 7.7 Hz, phenyl-H),
7.88 (1H, d, J = 8.1 Hz, N=CH), 7.97 (1H, s, NH), 11.23 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6,

Molecules 2018, 23, 60
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ppm)
δ 25.40, 25.63, 32.38, 47.93, 50.12, 52.91, 55.66, 114.76, 115.15, 118.22, 124.61, 129.01, 143.10, 145.67,
152.37, 153.66, 175.66. HRMS (m/z): [M + H]+ calcd. for C25H33N5OS: 452.2479; found 452.2460.
N-Cyclohexyl-2-[4-(4-methylphenoxy)benzylidene]hydrazine-1-carbothioamide (B19): Yield 68–70%, m.p.
169–170 ◦C. FTIR (ATR, cm−1): 3312 (N-H), 3057 (aromatic C-H), 2988 (aliphatic C-H), 1595 (C=N),
1
1497 (C=C), 1269–1076 (C-N and C-O). H-NMR (300 MHz, DMSO-d6, ppm)
δ 1.08–1.94 (10H, m,
cyclohexyl-H), 2.30 (3H, s, CH3), 4.12–4.24 (1H, m, cyclohexyl-H), 6.96–6.98 (4H, m, disubstituted
phenyl-H, methylphenyl-H), 7.23 (2H, d, J = 8.4 Hz, methylphenyl-H), 7.78 (2H, d, J = 8.7 Hz,
disubstituted phenyl-H), 7.97 (1H, d, J = 8.6 Hz, N=CH), 8.03 (1H, s, NH), 11.36 (1H, s, NH). 13C-NMR
(75 MHz, DMSO-d6, ppm)
δ 20.77 (CH3), 25.40, 25.62, 32.30, 53.05, 118.16, 119.85, 129.29, 129.62,
131.00, 133.78, 142.00, 153.84, 159.25, 176.01. HRMS (m/z): [M + H]+ calcd. for C21H25N3OS: 368.1791;
found 368.1784.
N-Cyclohexyl-2-[4-[(4-methylphenyl)thio]benzylidene]hydrazine-1-carbothioamide (B20): Yield 68–70%, m.p.
176–177 ◦C. FTIR (ATR, cm−1): 3306 (N-H), 3067 (aromatic C-H), 2986 (aliphatic C-H), 1593 (C=N),
1491 (C=C), 1209 (C-N). 1H-NMR (300 MHz, DMSO-d6, ppm)
δ 1.03–1.87 (10H, m, cyclohexyl-H), 2.33
(3H, s, CH3), 4.11–4.24 (1H, m, cyclohexyl-H), 7.17 (2H, d, J = 8.4 Hz, disubstituted phenyl-H), 7.26
(2H, d, J = 8.1 Hz, methylphenyl-H), 7.35 (2H, d, J = 8.1 Hz, methylphenyl-H), 7.72 (2H, d, J = 8.4 Hz,
disubstituted phenyl-H), 7.99 (1H, d, J = 8.6 Hz, N=CH), 7.99 (1H, s, NH), 11.42 (1H, s, NH). 13C-NMR
(75 MHz, DMSO-d6, ppm) δ 21.18, 25.40, 25.62, 32.26, 53.10, 128.54, 128.68, 129.40, 130.96, 132.62, 133.42,
138.82, 139.41, 141.69, 176.07. HRMS (m/z): [M + H]+ calcd. for C21H25N3S2: 384.1563; found 384.1545.
N-Cyclohexyl-2-[4-(4-methoxyphenoxy)benzylidene]hydrazine-1-carbothioamide (B21): Yield 68–70%, m.p.
159–160 ◦C. FTIR (ATR, cm−1): 3312 (N-H), 3051 (aromatic C-H), 2986 (aliphatic C-H), 1535 (C=N),
1
1495 (C=C), 1229–1076 (C-N and C-O). H-NMR (300 MHz, DMSO-d6, ppm)
δ 1.03–1.88 (10H, m,
cyclohexyl-H), 3.76 (3H, s, OCH3), 4.13–4.23 (1H, m, cyclohexyl-H), 6.93 (2H, d, J = 8.7 Hz, disubstituted
phenyl-H), 6.98 (2H, d, J = 9.0 Hz, methoxyphenyl-H), 7.05 (2H, d, J = 9.1 Hz, methoxyphenyl-H), 7.76
(2H, d, J = 8.7 Hz, disubstituted phenyl-H), 7.96 (1H, d, J = 8.6 Hz, N=CH), 8.02 (1H, s, NH), 11.36 (1H,
s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 25.40, 25.62, 32.31, 53.03, 55.90, 115.64, 117.44, 121.64,
128.90, 129.58, 142.05, 149.05, 156.46, 160.01, 176.00. HRMS (m/z): [M + H]+ calcd. for C21H25N3O2S:
384.1740; found 384.1736.
N-Cyclohexyl-2-[4-[(4-methoxyphenyl)thio]benzylidene]hydrazine-1-carbothioamide (B22): Yield 68–70%,
◦
m.p. 146–147 C. FTIR (ATR, cm−1): 3308 (N-H), 3049 (aromatic C-H), 2974 (aliphatic C-H), 1591 (C=N),
1
1493 (C=C), 1242 (C-N). H-NMR (300 MHz, DMSO-d6, ppm)
δ 1.09–1.87 (10H, m, cyclohexyl-H),
3.80 (3H, s, OCH3), 4.11–4.30 (1H, m, cyclohexyl-H), 7.46 (2H, d, J = 8.9 Hz, methoxyphenyl-H), 7.08
(2H, d, J = 8.3 Hz, disubstituted phenyl-H), 7.46 (2H, d, J = 8.7 Hz, methoxyphenyl-H), 7.69 (2H,
d, J = 8.4 Hz, disubstituted phenyl-H), 7.95–7.98 (2H, m, N=CH, NH), 11.41 (1H, s, NH). 13C-NMR
(75 MHz, DMSO-d6, ppm) δ 25.40, 25.62, 32.27, 53.07, 55.84, 116.00, 122.19, 127.27, 128.45, 130.96, 132.04,
136.42, 141.00, 141.77, 160.53, 176.04. HRMS (m/z): [M + H]+ calcd. for C21H25N3OS2: 400.1512; found
400.1506.
N-Cyclohexyl-2-[4-(4-chlorophenoxy)benzylidene]hydrazine-1-carbothioamide (B23): Yield 68–70%, m.p.
176–177 ◦C. FTIR (ATR, cm−1): 3308 (N-H), 3063 (aromatic C-H), 2988 (aliphatic C-H), 1589 (C=N),
1
1483 (C=C), 1234–1076 (C-N and C-O). H-NMR (300 MHz, DMSO-d6, ppm)
δ 1.06–1.88 (10H, m,
cyclohexyl-H), 4.14–4.24 (1H, m, cyclohexyl-H), 7.05 (2H, d, J = 8.7 Hz, disubstituted phenyl-H), 7.09
(2H, d, J = 8.9 Hz, chlorophenyl-H), 7.46 (2H, d, J = 8.9 Hz, chlorophenyl-H), 7.82 (2H, d, J = 8.8 Hz,
disubstituted phenyl-H), 8.00 (1H, d, J = 8.6 Hz, N=CH), 8.04 (1H, s, NH), 11.39 (1H, s, NH). 13C-NMR
(75 MHz, DMSO-d6, ppm)
δ 25.25, 25.41, 25.62, 32.29, 53.08, 119.01, 121.23, 128.20, 129.75, 130.14,
130.49, 141.82, 155.43, 158.18, 176.06. HRMS (m/z): [M + H]+ calcd. for C20H22ClN3OS: 388.1245;
found 388.1228.
N-Cyclohexyl-2-[4-[(4-chlorophenyl)thio]benzylidene]hydrazine-1-carbothioamide (B24): Yield 68–70%, m.p.
200–201 ◦C. FTIR (ATR, cm−1): 3306 (N-H), 3051 (aromatic C-H), 2980 (aliphatic C-H), 1593 (C=N),

Molecules 2018, 23, 60
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1
1474 (C=C), 1209 (C-N). H-NMR (300 MHz, DMSO-d6, ppm)
δ
1.10–1.88 (10H, m, cyclohexyl-H),
4.12–4.25 (1H, m, cyclohexyl-H), 7.31 (2H, d, J = 8.4 Hz, disubstituted phenyl-H), 7.38 (2H, d, J = 8.7 Hz,
chlorophenyl-H), 7.47 (2H, d, J = 8.7 Hz, chlorophenyl-H), 7.79 (2H, d, J = 8.4 Hz, disubstituted
phenyl-H), 8.01–8.05 (2H, m, N=CH, NH), 11.45 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ
25.41, 25.62, 32.25, 53.14, 128.79, 130.15, 130.61, 133.12, 133.40, 133.49, 133.69, 136.95, 141.51, 176.13.
HRMS (m/z): [M + H]+ calcd. for C20H22ClN3S2: 404.1016; found 404.1001.
N-Cyclohexyl-2-[4-(4-fluorophenoxy)benzylidene]hydrazine-1-carbothioamide (B25): Yield 68–70%, m.p.
175–176 ◦C. FTIR (ATR, cm−1): 3314 (N-H), 3063 (aromatic C-H), 2976 (aliphatic C-H), 1593 (C=N),
1
1476 (C=C), 1248–1088 (C-N and C-O). H-NMR (300 MHz, DMSO-d6, ppm)
δ 1.05–1.91 (10H, m,
cyclohexyl-H), 4.12–4.37 (1H, m, cyclohexyl-H), 6.99 (2H, d, J = 8.8 Hz, disubstituted phenyl-H),
7.11–7.15 (2H, m, fluorophenyl-H), 7.23–7.29 (2H, m, fluorophenyl-H), 7.80 (2H, d, J = 8.8 Hz,
disubstituted phenyl-H), 7.98 (1H, d, J = 8.6 Hz, N=CH), 8.03 (1H, s, NH), 11.38 (1H, s, NH). 13C-NMR
(75 MHz, DMSO-d6, ppm) δ 25.40, 25.62, 32.30, 53.06, 112.14, 117.21 (d, J = 23.3 Hz), 118.22, 121.75 (d,
J = 8.3 Hz), 129.64 (d, J = 6.0 Hz), 141.90, 152.26, 159.01 (d, J = 238.5 Hz), 159.08, 176.03. HRMS (m/z):
[M + H]+ calcd. for C20H22FN3OS: 372.1540; found 372.1534.
N-Cyclohexyl-2-[4-[(4-fluorophenyl)thio]benzylidene]hydrazine-1-carbothioamide (B26): Yield 68–70%, m.p.
173–174 ◦C. FTIR (ATR, cm−1): 3308 (N-H), 3067 (aromatic C-H), 2987 (aliphatic C-H), 1591 (C=N),
1
1487 (C=C), 1225 (C-N). H-NMR (300 MHz, DMSO-d6, ppm)
δ 1.05–1.87 (10H, m, cyclohexyl-H),
4.12–4.24 (1H, m, cyclohexyl-H), 7.21 (2H, d, J = 8.3 Hz, disubstituted phenyl-H), 7.27–7.33 (2H, m,
fluorophenyl-H), 7.48–7.53 (2H, m, fluorophenyl-H), 7.75 (2H, d, J = 8.4 Hz, disubstituted phenyl-H),
7.99–8.02 (2H, m, N=CH and NH),11.43 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm)
δ 25.41,
25.62, 32.26, 53.12, 117.42 (d, J = 21.8 Hz), 128.65, 128.86, 129.08, 132.99, 135.56 (d, J = 8.3 Hz), 138.72,
141.61, 162.66 (d, J = 244.5 Hz), 176.09. HRMS (m/z): [M + H]+ calcd. for C20H22FN3S2: 388.1312;
found 388.1300.
3.3. MAO Inhibition Assay
MAO inhibition assay was applied in concert with our recent study [35]. Ampliflu Red
(10-Acetyl-3,7-dihydroxyphenoxazine), peroxidase from horseradish, MAO-A, MAO-B, H2O2,
tyramine hydrochloride, moclobemide and selegiline were purchased from Sigma-Aldrich Chemical
Co. (St. Louis, MO, USA) and retained under the suggested conditions by supplier. All pipetting
processes were performed using a BioTek Precision XS robotic system (BioTek Instruments, Winooski,
VT, USA). Measurements were carried out by a BioTek-Synergy H1 microplate reader (USA) based on
the fluorescence generated (excitation, 535 nm, emission, 587 nm) over a 30 min period, in which the
fluorescence increased linearly.
In the enzymatic assay, three different daily prepared solutions, consisting of inhibitor solutions,
enzyme solutions and working solution, were used. Synthesized compounds and reference agents
−9
were prepared in 2% DMSO in 10−3–10 M concentrations (10 mL for each concentration) in order to
gain inhibitor solution. Enzyme solutions were prepared using recombinant MAO-A (0.5 U/mL) and
recombinant MAO-B (0.64 U/mL). These enzymes were dissolved in the phosphate buffer and final
volumes were adjusted to 10 mL. Working solution consist of three main components: Horseradish
peroxidase (200 U/mL, 100
These components were dissolved in the phosphate buffer and final volume was adjusted to 10 mL.
The solutions of inhibitor (20 L/well) and MAO-A (100 L/well) or MAO-B (100 L/well) were
added to the flat black bottom 96-well micro test plate, and incubated at 37 ◦C for 30 min. After this
µL), Ampliflu Red (20 mM, 200 µL) and tyramine (100 mM, 200 µL).
µ
µ
µ
incubation period, working solution (100
The mixture was incubated at 37 C for 30 min and the fluorescence (Excitation/emission = 535/587 nm)
µ
L/well) were added into each well to start the reaction.
◦
was measured at 5 min intervals. For control experiments, 2% DMSO (20
µL) were used simultaneously
by replacing the inhibitor solution. In addition, the possible capacity of the inhibitors to modify the

Molecules 2018, 23, 60
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fluorescence generated in the reaction mixture due to non-enzymatic inhibition was determined by
mixing inhibitor and working solutions.
The specific fluorescence emission that used to obtain the final results was calculated after
subtraction of the background activity, which was determined from vials containing all components
except the MAO isoforms, which were replaced by phosphate buffer (100 µL/well). Blank, control and
all concentrations of inhibitors were analyzed in quadruplicate and inhibition percent was calculated
by using following equation:
(FCt2 − FCt1) − (FIt2 − FIt1)
%Inhibition =
× 100
FCt2 − FCt1
FCt2: Fluorescence of a control well measured at t2 time, FCt1: Fluorescence of a control well
measured at t2 time, FIt2: Fluorescence of an inhibitor well measured at t2 time, FIt1: Fluorescence of
an inhibitor well measured at t1 time, The IC50 values were calculated from a dose-response curve
obtained by plotting the percentage inhibition versus the log concentration with the use of GraphPad
PRISM software (version 5.0, GraphPad Software Inc., La Jolla, CA, USA). The results were displayed
as mean ± standard deviation (SD).
3.4. Enzyme Kinetic Studies
The similar protocol of MAO inhibition assay was followed by using the same materials. The most
active compound B24 was tested at the concentrations of IC50/2, IC50, and 2
of compound B24 (20 L/well) and MAO-B enzyme were added to the flat black bottom 96-well
micro test plate, and incubated at 37 ◦C for 30 min. Once incubation was completed, the working
solution, including various concentrations (20, 10, 5, 2.5, 1.25 and 0.625 M) of substrate (100 L/well)
×
IC50. The solutions
µ
µ
µ
was added. The increase of the fluorescence (Excitation/emission = 535/587 nm) was recorded for
30 min. A parallel experiment was carried out without inhibitor. All experiments were performed
in quadruplicate. The experimental data were analyzed as Lineweaver-Burk plots using Microsoft
Office Excel 2013. The Km/Vmax (slope) values of the Lineweaver-Burk plots were replotted versus the
inhibitor concentration to determine Ki values from the x-axis intercept as -Ki.
3.5. Cytotoxicity Test
The cytotoxic potency of the most active compound B24 was measured by MTT method [25
]
using the NIH/3T3 mouse embryonic fibroblast cell line (ATCC CRL-1658, London, UK) which is
recommended for cytotoxicity screening by ISO (10993-5, 2009) [36]. NIH/3T3 cells were incubated
according to the supplier’s recommendations. NIH/3T3 cells were seeded at 1
well of 96-well plates. After this process, cells were treated with the compounds at concentrations
ranging from 1000 M to 0.316 M. The IC50 value was determined by plotting a dose-response curve
×
104 cells into each
µ
µ
of inhibition % versus compound concentrations applied [37].
3.6. Molecular Docking Studies
A structure based in silico procedure was applied to discover the interaction modes between
compound B24 and MAO-A enzyme active site. The crystal structures of MAO-A (PDB ID: 2Z5X) [26],
which was crystallized with the reversible inhibitor harmine, was retrieved from the Protein Data Bank
server (www.pdb.org).
The structures of ligands were built using the Schrödinger Maestro [38] interface and then were
submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2 [39].
The ligands were prepared by the LigPrep 3.8 [40] to assign the protonation states at pH 7.4
and the atom types, correctly. Bond orders were assigned, and hydrogen atoms were added to the
structures. The grid generation was formed using Glide 7.1 [41]. The grid box with dimensions of
±
1.0
20 Å
×
20 Å
×
20 Å was centered in the vicinity of the flavin (FAD) N5 atom on the catalytic site

Molecules 2018, 23, 60
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of the protein to cover all binding sites and neighboring residues [
performed with single precision docking mode (SP).
1
,
42]. Flexible docking runs were
3.7. Theoretical Determination of ADME Properties
Physicochemical properties and BBB permeability of the compounds were predicted via QikProp
4.8 software [32].
4. Conclusions
In this paper, we focused on the synthesis and biological evaluation of some novel thiosemicarbazones
as MAO inhibitors. According to an activity assay, compound B24 was determined as the most active
molecule amongst all prepared compounds. Enzyme kinetics and docking studies clarified the type of
interaction and binding modes between the enzyme and compounds. Compound B24 was found to be
a reversible and competitive inhibitor of MAO-A, that additionally shows molecular interactions with
the active site of MAO-A in a modelling study. Cytotoxicity and ADME studies further indicated the
potential biological importance of this compound. Based on these results, new molecules derived from
compound B24, targeting MAO isoforms selectively, could possibly afford more promising drugs for
the treatment of neurological diseases.
Supplementary Materials: The Supplementary Materials are available online, Figure S1: Compound B24 IR
spectrum, Figure S2: Compound B24 mass spectrum, Figure S3: Compound B24 1H-NMR spectrum, Figure S4:
Compound B24 13C-NMR spectrum.
Acknowledgments: This study was financially supported by Anadolu University Scientific Projects Fund,
Project No.: 1705S183.
Author Contributions: Y.O. and Z.A.K. conceived and designed the experiments; B.K.Ç., D.O. and U.A.Ç.
performed the synthesis; S.L. performed analysis studies; A.B.K. performed the cytotoxicity test; B.N.S. performed
docking, enzyme kinetic and ADME studies; Z.A.K., Y.O., U.A.Ç., B.N.S., S.L., B.K.Ç. and D.O. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds B1–B24 are available from the authors.
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