Design, synthesis and biological assessment of new thiazolylhydrazine derivatives as selective and reversible hMAO-A inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.12.013

In the recent works, it was shown that numerous thiazolylhydrazine derivatives display hMAO inhibitory activity in the range of micromolar concentration. Hence, in the present study a new series of new thiazole-hydrazines (3a-3n) were designed, synthesized, characterized and screened for their hMAO-A and hMAO-B inhibitory activity by an in vitro flurometric method. The enzyme inhibition assay revealed that most of the synthesized compounds have selective inhibition potency against hMAO-A. The compounds 3f and 3h showed promising hMAO-A inhibition with an IC₅₀ values of 0.012 μM and 0.011 μM and significant selectivity indexes of 1214 and 1601 towards hMAO-A, respectively. The mechanism of hMAO-A inhibition of compounds 3f and 3h was investigated by Lineweaver-Burk graphics and reversible-competitive inhibition of hMAO-A was determined. Cytotoxicity and genotoxicity studies were carried out and the compound 3h was found as non-cytotoxic and non-genotoxic. Theoretical calculation of ADME properties suggested that synthesized compounds may have a good pharmacokinetic profile. The docking study of compound 3f and 3h revealed that there is a strong interaction between the active sites of hMAO-A and analyzed compound.

Full text of DOI:10.1016/j.ejmech.2017.12.013

Accepted Manuscript
Design, synthesis and biological assessment of new thiazolylhydrazine derivatives as
selective and reversible hMAO-A inhibitors
Nafiz Öncü Can, Derya Osmaniye, Serkan Levent, Begüm Nurpelin Sağlık, Büşra
Korkut, Özlem Atlı, Yusuf Özkay, Zafer Asım Kaplancıklı
PII:
S0223-5234(17)31019-X
10.1016/j.ejmech.2017.12.013
EJMECH 9991
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 July 2017
Revised Date: 20 October 2017
Accepted Date: 4 December 2017
Please cite this article as: Nafiz.Öü. Can, D. Osmaniye, S. Levent, Begü.Nurpelin. Sağlık, Büş.
Korkut, Ö. Atlı, Y. Özkay, Zafer.Ası. Kaplancıklı, Design, synthesis and biological assessment of new
thiazolylhydrazine derivatives as selective and reversible hMAO-A inhibitors, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.013.
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ACCEPTED MANUSCRIPT
Competitive, Reversible and Selective hMAO-A Inhibitor
IC50= 0.011 µM against hMAO-A; IC50: 17.615 µM
Compound 3h
Possess Good Predicted ADME Profile
Non-cytotoxic (NIH3T3 IC50> 1000 µM)
&
&
Non-genotoxic
Notable interactions in the hMAO-A active site

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Design, Synthesis and Biological Assessment of New Thiazolylhydrazine Derivatives as
Selective and Reversible hMAO-A Inhibitors
Nafiz Öncü Cana,c, Derya Osmaniyeb,c, Serkan Leventb,c, Begüm Nurpelin Sağlıkb,c, Büşra
Korkutd, Özlem Atlıd, Yusuf Özkayb,c,*, Zafer Asım Kaplancıklıb
aDepartment of Analytical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eski
şehir, Turkey
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eski
Turkey
ş
ehir,
cDoping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University, 26470
Eski ehir, Turkey
ş
dDepartment of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, 26470 Eski
şehir,
Turkey
* Corresponding author.
E-mail address: yozkay@anadolu.edu.tr (Y. Özkay).
Tel: +90-222-3350580/3779 Fax: +90-222-3350750.
Address: Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470,
Eski ehir, Turkey.
ş

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Abstract
In the recent works, it was shown that numerous thiazolylhydrazine derivatives display hMAO
inhibitory activity in the range of micromolar concentration. Hence, in the present study a new
series of new thiazole-hydrazines (3a-3n) were designed, synthesized, characterized and screened
for their hMAO-A and hMAO-B inhibitory activity by an in vitro flurometric method. The
enzyme inhibition assay revealed that most of the synthesized compounds have selective
inhibition potency against hMAO-A. The compounds 3f and 3h showed promising hMAO-A
inhibition with an IC50 values of 0.012 µM and 0.011 µM and significant selectivity indexes of
1214 and 1601 towards hMAO-A, respectively. The mechanism of hMAO-A inhibition of
compounds 3f and 3h was investigated by Lineweaver-Burk graphics and reversible-competitive
inhibition of hMAO-A was determined. Cytotoxicity and genotoxicity studies were carried out
and the compound 3h was found as non-cytotoxic and non-genotoxic. Theoretical calculation of
ADME properties suggested that synthesized compounds may have a good pharmacokinetic
profile. The docking study of compound 3f and 3h revealed that there is a strong interaction
between the active sites of hMAO-A and analyzed compound.
Keywords: Thiazole; Hydrazine; hMAO enzymes; Enzyme inhibition; Docking.

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1. Introduction
Monoamine oxidase (MAO) is an outer mitochondrial membrane bound flavin containing
enzyme with a molecular weight of about 60 kDa. It plays an important role in the metabolism of
endogenous and exogenous amines [1, 2]. Intraneuronal MAO terminates the action of
endogenous amine neurotransmitters, regulates intraneuronal amine stores, and protects cells
from dietary amines (false transmitters) [3]. This enzyme exists in two isoforms MAO-A and
MAO-B, sharing about 70% sequence identity and representing separate X chromosome related
gene products [4]. These isoforms have different substrate specificities in that MAO-A catalyzes
the oxidation of serotonin (5-HT), while MAO-B catalyzes the oxidation of both benzylamine
and 2-phenylethylamine. Other substrates like dopamine, epinephrine, norepinephrine, tyramine
and tryptamine are oxidized by both isoforms [5]. In humans MAO-B inhibitors are useful in the
treatment of Parkinson’s disease (PD) and Alzheimer’s disease (AD) [6, 7], while MAO-A
inhibitors are antidepressant and antianxiety agents [8-10]. Since they metabolize the principal
biogenic amines, MAO-A and MAO-B play an important role in the regulation of their
concentrations mainly in the central nervous system, where abnormal values have been involved
in psychiatric and neurodegenerative disorders such as AD and PD. Therefore, selective and
reversible inhibitors of MAO-A or MAO-B may be useful therapeutic agents devoid of unwanted
side effects such as the so-called “cheese effect” [11, 12]. The structure of the two isoforms of
human MAO (hMAO) has provided relevant information about the recognition mechanism
underlying the selective interactions between these proteins and their ligands, in order to probe
the catalytic mechanism and gain an understanding of the pharmacophoric requirements needed
for the rational design of potent and selective new agents [12].

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The most important structural difference between these two isozymes is that human MAO-B
crystallizes as a dimer, while human MAO-A crystallizes as a monomer [13]. It was suggested
that in human MAO-A, a Glu-151 is replaced by a Lys residue. This selective mutation at the
dimeric interface may be responsible for destabilization of the dimeric form in human MAO-A
[14]. Other significant differences are also present between human MAO-B and human MAO-A
regarding active site shape and volume. The shape of active site in human MAO-B is longer and
narrower than that of human MAO-A. This difference in active site shape and volume may be in
part as a result of variation in the conformation of cavity shaping loop, this six residue loop is
present in a more compact conformation in human MAO-B as compared with human MAO-A
[14, 15]. A common structural feature of substrates and inhibitors is an amino or imino group,
which is assumed to play an essential role in orientation and complex formation at the active site
of the enzyme. A general model for the interaction between MAO and substrates or inhibitors
has been proposed. This model describes three binding sites on the enzyme: an amine binding
site, a hydrophobic site and a nucleophilic site. Variation in the physicochemical properties of
substituents are expected to influence the binding of inhibitors to the enzyme surface [16-18].
The classical period of the MAO inhibitors started with hydrazine derivatives. They were
originally proposed as tuberculostatic agents, their prototype, iproniazid, was the first modern
antidepressant. Subsequently, research has been directed towards the preparation of heterocyclic
hydrazines and hydrazides and their potential use as therapeutic agents for the treatment of CNS
depression. Biological studies proved that arylalkylhydrazines inhibition of MAO was
irreversible and non-selective as a result of an alkylation of the flavin cofactor. Because of the
serious side effects of iproniazid with irreversible and non-selective properties, a large number of
substituted hydrazines have been studied as hMAO inhibitors to discover reversible and safer
agents [19, 20]. In these studies, many series of (hetero)arylidene-(4-substituted-thiazol-2-

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yl)hydrazine derivatives was designed by joining the hydrazine part of iproniazid and thiazole
moiety of glitazones [12, 20-21].
In the recent works, it was shown that numerous thiazolylhydrazine derivatives display hMAO
inhibitory activity in the range of micromolar concentration. Moreover, benzylidene-
hydrazinothiazole derivatives were reported to be the most active, with their activity being further
enhanced by the substitution at position 4 of the thiazole nucleus [22-25]. 3D-QSAR studies
helped to rationalize experimental results and to provide robust structure-activity relationships
(Figure 1): (i) the aryl group at C4 of the thiazole is responsible for the correct orientation of the
inhibitor inside the active site of enzymeclose to the catalytic FAD (aromatic cage); (ii) in order
to obtain productive interactions within the aromatic cage, the aryl moiety has to be ortho and/or
para substituted with an electron-withdrawing group for selective MAO-B inhibition and electron
donating group for selective MAO-A inhibition, (iii) 5 or 6-membered cycloaliphatic and
aromatic rings are preferred on the N1 of the hydrazine to display selective MAO-B and MAO-A
inhibition, respectively [26,27]. The choice of different aliphatic, cycloaliphatic, heterocyclic,
and bicyclic moieties on the N1 of the hydrazine allowed a better comprehension of the steric and
electronic influence on the enzyme-inhibitor interaction and a modulation of the chemical
structure of these derivatives. The above studies elucidated some factors responsible for
selectivity against the A and B isoforms, such as the lipophilicity of the inhibitor that is important
for achieving effective binding to MAO-B and the presence of electron-rich aromatic moieties,
typical of selective MAO-A inhibitors [12, 19-23, 26, 28-33].
Prompted from above information and previously reported 3D-QSAR studies, in this paper we
designed and synthesized new thiazolylhydrazine derivatives to gain potent and selective MAO-
A inhibitors.

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2. Result and Discussion
2.1. Chemistry
The compounds 3a-3p were synthesized as summarized in Scheme 1. Initially, 4-fluoro
benzaldehyde and pyrrolidine or morpholine were reacted in order to obtained 4-substituted
benzaldehydes (1a, 1b). In the second step, 4-substituted thiosemicarbazones (2a, 2b) were
prepared by the reaction of 4-substituted benzaldehydes (1a, 1b) and thiosemicarbazide. In the
last step, the target compounds (3a-3p) were synthesized by means of ring closure reaction using
thiosemicarbazones (2a, 2b) and appropriate 2-bromoacetophenone.
Structures of the final compounds were proved by IR, 1H-NMR, 13C-NMR, and HRMS analysis.
In the IR spectra, N-H bond of the hydrazide groups had band over the 3047 cm-1. The stretching
absorption at about 1174-1240 cm-1 were registered for C-N single bonds. Also, triple bond
between carbon and nitrogen in the compound 3c and 3k had signal at 2223 and 2218 cm-1,
1
respectively. In H-NMR spectra, morpholine protons on the nitrogen side of morpholine were
recorded between 3.17 ppm and 3.19 ppm and those on the oxygen side were recorded 3.73 ppm
or 3.74 ppm. Two types of pyrrolidine protons were recorded at 1.93-1.99 ppm and 3.24-3.30
ppm. Proton of azomethine group had a singlet peak between 7.93 ppm and 7.96 ppm. Hydrogens
of aromatic rings were observed between 6.56 ppm and 8.07 ppm. Proton of hydrazide nitrogen
13
was recorded over 11.80 ppm as a singlet peak. In C-NMR, aliphatic carbons on morpholine,
pyrrolidine and methyl of tolyl group were recorded between 21.21 ppm and 66.43 ppm. All
aromatic carbons gave peaks from 102.65 ppm to 161.66 ppm. Carbonyl carbon was observed at
168.03 ppm and over. In compound 3f, 3h, 3n, and 3p, carbon-fluorine splitting was observed. In
high resolution mass spectrum, all masses were matched with the expected M+H values.

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2.2. MAO Inhibition
The newly synthesized 4-(substitutedphenyl)-2-(2-(4-substitutedbenzylidene)hydrazinyl)thiazole
derivatives (3a-3p) were investigated for their hMAO-A and hMAO-B inhibitory activity by a
previously described in vitro fluorometric method, which is based on the detection of H2O2 in a
horseradish peroxidase coupled reaction using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex
Red reagent) [34].
The assay was performed in two steps. First, compounds 3a-3p were tested at 10-3 and 10-4 M
concentrations. Second step was performed by using 10-5-10-9 M concentrations of selected
compounds that indicate more than 50% inhibitory activity at initial concentrations. Table 1
presents the hMAO-A and hMAO-B inhibitory activity of compounds 3a-3p. Compounds 3c, 3e-
3h, 3k, and 3m-3p could pass to the second step because they provided the prescribed condition
against hMAO-B enzyme. Furthermore, all compounds were evaluated in the second step
hMAO-A assay because of higher than 50% inhibition potencies at initial concentrations. The
most active compounds 3f and 3h displayed IC50 values of 0.012 µM and 0.011 µM against
hMAO-A, whereas reference drug moclobemide had an IC50 of 6.061 µM. These findings
revealed that compounds 3f and 3h have very significant potency to inhibit hMAO-A compared
to reference agent moclobemide. On the other hand, none of the compounds could show the same
potency against hMAO-B compared to reference agent selegeline. Selectivity indexes (SI) were
expressed as IC50(MAO-B) / IC50(MAO-A). Selectivity towards MAO-A increases as the corresponding
SI increases, while selectivity towards MAO-B isoform increases as the corresponding SI
decreases. It was determined that synthesized compounds have selective inhibition potency
against hMAO-A. Compound 3f and 3h indicated very significant SIs of 1214 and 1601
respectively.

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2.3. Kinetic Studies of Enzyme Inhibition
The mechanism of hMAO-A inhibition was investigated by enzyme kinetics, following the
similar procedure of the MAO inhibition assay. The linear Lineweaver-Burk graphics were used
to estimate the type of inhibition. Enzyme kinetics were analyzed by recording substrate velocity
curves in absence and presence of the most potent compounds 3f and 3h, which were prepared at
concentrations of IC50/2, IC50 and 2 x IC50. In each case, the initial velocity measurements were
gained at different substrate (tyramine) concentrations ranging from 20 µM to 0.625 µM. The Ki
(intercept on the x-axis) value of compounds 3f and 3h were determined from the secondary plot
of the Km/Vmax (slope) versus varying concentrations. The graphical analysis of steady-state
inhibition data for compounds 3f and 3h are shown in Figures 2 and 3.
The type of inhibition can be determined by the Lineweaver-Burk plot as mixed-type,
uncompetitive, competitive, or noncompetitive, which are the indicators of a reversible inhibitor.
It is known that in the uncompetitive type inhibition, a graphic including the parallel lines
without any cross is observed. If the lines cross neither the x- nor the y-axis at the same point the
inhibition type is called mixed-type. Noncompetitive inhibition is seen if the plots intersect on the
x-axis. There are different slopes and intercepts on y-axis. On the other hand, competitive
inhibition has the opposite situation. This type of inhibition has plots with the same intercept on
y-axis but there are diverse slopes and intercepts on x-axis, which are observed in Figures 2 and
3. Therefore, this pattern indicates that the compound 3f and 3h are reversible and competitive
inhibitors, namely have similar inhibition features with substrate. Ki values for compounds 3f and
3h were calculated as 0.0066 and 0.0044 µM, respectively, for the inhibition of hMAO-A.
Reversible inhibitors bind to enzymes by non-covalent interactions as hydrophobic interactions,
ionic bonds, and hydrogen bonds without forming any chemical bonds or reactions with the

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enzyme. These interactions are formed rapidly and can be easily removed; hence the enzyme and
inhibitor complex is quickly dissociated opposite to irreversible inhibition. Due to reversible
binding ability to biomolecules such inhibitors carry a lower risk of side effects compared to
irreversible inhibitors. As a result, reversible-competitive inhibition potency of compounds 3f and
3h has enhanced their biological importance in contrast to irreversible hydrazine type MAO
inhibitors.
2.4. Cytotoxicity
The cytotoxic activitiy of the compounds 3f and 3h were evaluated against healthy NIH/3T3
mouse embryonic fibroblast cell lines (ATCC CRL1658), which is suggested for preliminary
cytotoxicity screening by ISO (10993-5, 2009) [35]. The IC50 values of the compounds are
represented in Table 2. Both compounds displayed an IC50 value of ≥1000 µM against NIH/3T3
cells, which is significantly higher than their IC50 values (0.012 and 0.0011) against hMAO-A.
This result reveals that compounds 3f and 3h are not cytotoxic at their effective concentration
against hMAO-A and improves the biological importance of these compounds.
2.5. Genotoxicity
Identification of mutagenic properties of new compounds is essential for safety and thus new
drug candidates need to be examined in models of genotoxicity such as Ames test [36]. In this
study Ames MPF assay was performed to investigate the genotoxicity of the compounds 3f and
3h. In this assay, more than 25 positive wells were observed with our positive controls. Negative
control wells also showed less than 8 positive wells in the presence and absence of S9 with TA98
and TA100, which complied with the requirements for the validation of the test as described in
previous studies [37]. Our results were presented in Table 3.

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Compound 3f showed a baseline of 4.82 in the absence of S9 and 3.73 in the presence of S9 with
TA98. Fold inductions over baseline were more than 1.5 in 0.3125 and 1.25 mg/ml concentration
of the compound against TA98 without S9. But, adjacent concentrations were not found to reach
mentioned fold increases and a highest concentration level was not observed significant fold
increases. Therefore, this compound was classified as non-mutagenic against TA98 in the
absence of metabolic activation (S9). Otherwise, fold inductions over baseline were more than
2.5 in all adjacent doses of the compound against TA 98 with S9. Hence, compound 3f was
classified as a mutagen against TA98 with metabolic activation. Compound 3f had a baseline of
6.00 in the absence of S9 and 6.24 in the presence of S9 with TA100. Fold inductions over
baseline were less than 1.5 in each concentrations of the compound against TA100 without S9.
Also, fold inductions over baseline did not reach the mentioned values above the baseline with
TA100 in the presence of S9. As a result, 3f was not genotoxic against TA100 with/without
metabolic activation (Figure 4).
Compound 3h had a baselines of 6.73 and 3.73 in the absence and presence of S9 with TA98,
respectively. Fold inductions over baseline were less than 1.5 in each concentrations of the
compound against TA98 without S9. Therefore, compound 3h was not genotoxic against TA98
without metabolic activation. Besides, fold induction over baseline was more than 1.5 in 1.25
mg/ml concentration of the compound against TA98 with S9. However, adjacent concentrations
were not found to reach mentioned fold increases and a highest concentration level did not
display significant fold increases. Thus, this compound was not genotoxic against TA98 with
metabolic activation (Figure 5).Compound 3h was found to show a baseline of 5.98 and 5.21
with/without S9 against TA100. Mentioned-fold increases over the baseline according to the
criteria were not determined against TA100 without S9. Also, fold inductions over baseline did

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not reach values more 1.5 in each concentrations of the compound against TA100 with S9.
Hence, compound 3h was found to be non-mutagenic against TA100 in the absence of metabolic
activation and in the presence of metabolic activation (Figure 5).
As a result, findings of Ames MPF assay increase the importance of compound 3h as hMAO-A
inhibitor.
2.6. Prediction of ADME Parameters and BBB Permeability
Most of new drug candidates fail in clinical trials owing to their reduced ADME (absorption,
distribution, metabolism, and excretion) properties. The late-stage failures significantly cause to
growing cost of new drug development. The capability to identify problematic issues early can
intensely reduce the amount of missed time and funds, and rationalize the overall development
progression. Therefore, pharmacokinetic properties of new drug candidates are very vital and
should be assessed as early as possible in the drug development process. ADME prediction can
be used to focus lead optimization to improve the preferred features of a compound [38]. Hence,
predictions of ADME parameters of synthesized compounds (3a-3p) were carried out by QikProp
4.8 software [39]. This software calculates the violations of Lipinski’s rule of five [40] and
Jorgensen’s rule of three [41], which evaluate the ADME properties of new drug candidates, and
are essential for the optimization of a biologically active compound. Molecular weight (MW),
number of rotatable bonds (RB), dipol moment (DM), molecular volume (MV), number of
hydrogen donors (DHB), number of hydrogen acceptors (AHB), polar surface area (PSA),
octanol/water partition coefficient (log P), aqueous solubility (log S), apparent Caco-2 cell
permeability (PCaco), number of likely primer metabolic reactions (PM), and percent of human
oral absorption (%HOA) are presented in Table 4 along with the violations of rules of three

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(VRT) and five (VRF). According to Lipinski’s rule of five and Jorgensen’s rule of three all
compounds (3a-3p) are in accordance with the rule by causing no more than one violation.
Drugs that specifically target the CNS must first pass the blood-brain barrier (BBB). Although
the BBB is protective in nature, the inability for drug molecules to permeate the BBB is a
significant impediment for CNS drug candidates and should be addressed early in the drug
discovery process. Thus, the task of predicting the BBB permeability of new compounds is of a
great importance [45]. From this point of view, BBB permeability of the synthesized compounds
(3a-3p) was also predicted by QikProp 4.8 software [39]. Apparent MDCK cell permeability
(PMDCK), brain/blood partition coefficient (log BB), and central nerveous sytem activity scores
(CNSAS) were calculated for this purpose. MDCK cells are considered to be a good mimic for
the blood brain barrier. According to software predictions, the PMDCK values of <25 and >500
nm/sec are recommended as poor and great for non-active transport of compound. So as to pass
BBB, Log BB and CNSAS are the other important parameters, which have recommended values
of -3 - +1.2 and -2 - +2, respectively. In Table 4, parameters of PMDCK, logBB, and CNSAS
are in the recommended ranges. Thus it can be concluded that synthesized compounds are able to
cross through BBB, which is essential for CNS related drugs.
As a result of ADME and BBB Permeability predictions, it can be suggested that all synthesized
compounds may possess a good pharmacokinetic profile.
2.7. Molecular Docking
The compounds 3f and 3h were found to be the most active and selective hMAO-A inhibitors as
mentioned in MAO inhibition assay. Docking studies were performed in order to gain more
insight into the binding modes of compounds 3f and 3h, and to evaluate the effects of structural

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modifications on the inhibitory activity against hMAO-A. X-ray crystal structure of hMAO-A
(PDB ID: 2Z5X) [42] was obtained from Protein Data Bank server (www.pdb.org). The docking
poses of compounds 3f and 3h on hMAO-A are presented in Figures 6-10.
The compounds 3f and 3h adequately bind to amino acid residues lining the cavity by
overlapping at the same site, and are located very near the FAD cofactor. The docking poses on
hMAO-A reveals that there are five common interactions between the compounds 3f and 3h. The
nitrogen atoms of hydrazine, the phenyl groups, and oxygen atom of morpholine show same
interactions in the docking studies of both compounds. There are two π-π interaction belonging to
aromatic parts of the compounds. Namely, the benzylidene substructure creates a π-π interaction
with phenyl of Tyr407 in the aromatic cage, while the other π-π interaction is seen between
phenyl at 4th position of thiazole and phenyl of Phe208. The hydrazine moiety is essential for
polar interactions. This group have two nitrogen atoms capable of forming two hydrogen bonds.
The first hydrogen bond is formed between the amino nitrogen and carbonyl of Asn181. The
other hydrogen bond is seen between the imine nitrogen and hydroxyl of Tyr444. Furthermore,
the oxygen atoms of morpholine contributes to binding ability to active region by forming a
hydrogen bond. This interaction is created with amino of Gly67.
The other important factors of binding to the active site of enzyme are the van der Waals forces
and electrostatic interactions. Thus, probable interactions of these types in the active site of
hMAO-A were evaluated for compound 3f and 3h (Figures 9 and 10). Both compounds have
favorable van der waals interactions with Try407, Try444, Gln215, Phe208, Ile180, and Ile335,
displayed with pink and red colors as described in the user guide of Glide [43] (Figures 9A and
10A). Compounds also display promising electrostatic interactions between Arg51 and Tyr69,
Tyr407, Asn181, Ile180, Phe208, and Thr336, respectively (Figures 10A and 10B).

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In terms of structure activity relationships, it is found out that the compounds 3a-3h, which carry
morpholin-4-yl group at 4th position of benyzlidene have greater hMAO-A inhibitory activity
than compounds 3i-3p including pyrrolydine-1-yl at the same position. This finding reveals that
morpholine ring enhances the potency of the compounds 3a-3h. This approach is also supported
by docking studies visualizing the hydrogen bonding between the oxygen atom of morpholine
and amino of Gly67 amino acid. Besides, presence of fluoro substituents in compounds 3f and 3h
increases the hMAO-A inhibitory potency compared to bromo or chloro substituted compounds
in the series. It is known that fluorine substitution enhances the binding affinity of a compound to
a target protein. This can occur by direct interaction of fluorine with the protein or indirectly by
influencing the polarity of other groups of the compound which interact with the protein [44, 45].
Thus, it can be declared that fluoro substitution improves the binding capacity to enzyme active
site and enhances the hMAO-A inhibiton.
3. Conclusion
Irreversible MAO inhibitors are not preferable in the treatment of neurological disorders in
connection with their side effects. It may be possible to eliminate such side effects by the
development of a new class of compounds with reversible enzyme inhibition. From this point of
view, in present study 16 new thiazolylhydrazine compounds were designed, synthesized and
tested against hMAO isoforms. Moreover, enzyme kinetics, preliminary toxicological screening,
ADME prediction and docking evaluations of obtained compounds (3a-3p) were undertaken in
the current study. Enzymatic studies revealed the potency of the compound 3h as a selective,
reversible and competitive hMAO-A inhibitor. Toxicological and ADME studies enhanced the
biological significance of this compound. Docking studies clearly indicated interactions between
compound 3h and hMAO-A. Consequently, all these data may create an effect on medicinal

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chemists to design and synthesis more potent and safer hMAO-A inhibitors, which may be
beneficial for the treatment of patients with depression.
4. Experimental
4.1. Chemistry
All reagents were purchased from commercial suppliers and were used without further
purification. Melting points (M.P.) were determined on Mettler Toledo-MP90 Melting Point
System and were uncorrected. IR spectra was recorded on an IR Affinity-1S Infrared
spectrophotometer (Shimadzu, Tokyo, Japan). 1H NMR and 13C NMR spectra was recorded on a
Bruker Fourier 300 (Bruker Bioscience, Billerica, MA, USA) respectively, in DMSO-d6.
(Bruker, USA), respectively. Mass spectra were recorded on an LCMS-IT-TOF (Shimadzu,
Kyoto, Japan) by using ESI method.
4.1.1. General procedure for the synthesis of the compounds
4.1.1.1. Synthesis of 4-substituted benzaldehydes (1a, 1b)
4-Fluoro benzaldehyde (2.036 mL, 0.019 mol), pyrrolidine or morpholine (0.019 mol), and
potassium carbonate (K2CO3) (2.622 g, 0.019 mol) were refluxed in DMF (20 mL) for 24 h. After
completion of the reaction, the mixture was poured into ice-water (50 mL), precipitated product
was filtered, washed with deionised water, dried, and recrystallized from EtOH. For compound
1a; yield: 85 %, measured M.P. = 59.8 °C, reported M.P.= 62-63 °C [46]. For compound 1b;
yield: 82 %, measured M.P. = 75.3 °C, reported M.P.= 78-79 °C [47].

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4.1.1.2. Synthesis of 4-substituted thiosemicarbazones (2a, 2b)
A mixture of appropriate 4-substituted benzaldehyde (1a or 1b) (0.016 mol) and
thiosemicarbazide (1.456 g, 0.016 mol) were refluxed in EtOH (100 mL) for 3h. After
completion of the reaction the mixture was cooled in an ice-bath, precipitated product was
filtered, dried, and recrystallized from EtOH. For compound 2a; yield: 80 %, M.P. = 208.5 °C
1
(decomposed), FTIR (ATR, cm-1): 3332 (N-H), 3157 (C-H), 1230 (C-N), 875, 823. H-NMR
(300 MHz, DMSO-d6): δ = 3.18 (4H, t, J=4.60 Hz, morpholine CH2), 3.72 (4H, t, J=4.60 Hz,
morpholine CH2), 6.93 (2H, d, J=8.60 Hz, disubstituted benzene CH), 7.82 (1H, s, -NH2), 7.95
(1H, s, N=CH), 8.04 (1H, s, -NH2), 11.22 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6): δ =
47.97, 66.42, 114.73, 124.87, 128.94, 143.21, 152.53, 177.79. ESI-MS: 265 (100 %), 266 (14 %),
267 (5 %). For compound 2b; yield: 74 %, Oily [48].
4.1.1.3. General procedures of target compounds (3a-3p)
Corresponding 4-substituted thiosemicarbazone (2a or 2b) (0.001 mol) and appropriate 2-
bromoacetophenone derivative (0.001 mol) in EtOH (40 mL) were refluxed for 4h. The mixture
was cooled in an ice-bath, precipitated product was filtered, dried, and recrystallized from EtOH.
4-(4-((2-(4-phenylthiazol-2-yl)hydrazineylidene)methyl)phenyl)morpholine (3a)
Yield: 74 %, M.P. = 263.4 °C, FTIR (ATR, cm-1): 3182 (N-H), 2970 (C-H), 1228 (C-N), 815,
715. 1H-NMR (300 MHz, DMSO-d6): δ = 3.19 (4H, t, J=4.80 Hz, morpholine CH2), 3.74 (4H, t,
J=4.80 Hz, morpholine CH2), 6.98 (2H, d, J=8.85 Hz, disubstituted benzene CH), 7.26-7.31 (2H,
m, thiazole CH, monosubstituted benzene H4), 7.40 (2H, t, J=7.50 Hz, monosubstituted benzene
H3,3’), 7.51 (2H, d, J=8.85 Hz, disubstituted benzene CH), 7.85 (2H, d, J=7.50 Hz,
monosubstituted benzene H2,2’), 7.94 (1H, s, N=CH), 11.93 (1H, s, NH). 13C-NMR (75 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6): δ = 48.10, 66.43, 103.63, 114.72, 115.04, 125.33, 125.96, 127.85, 127.92, 129.05,
135.25, 142.17, 152.14, 168.82. HRMS (m/z): [M+H]+ calcd for C20H20N4OS: 365.1431; found:
365.1439.
4-(4-((2-(4-(p-tolyl)thiazol-2-yl)hydrazineylidene)methyl)phenyl)morpholine (3b)
Yield: 82 %, M.P. = 270.3 °C, FTIR (ATR, cm-1): 3182 (N-H), 2972 (C-H), 1228 (C-N), 819,
727. 1H-NMR (300 MHz, DMSO-d6): δ = 2.32 (3H, s, -CH3), 3.19 (4H, t, J=4.70 Hz, morpholine
CH2), 3.74 (4H, t, J=4.70 Hz, morpholine CH2), 6.98 (2H, d, J=8.88 Hz, disubstituted benzene
CH), 7.19-7.22 (3H, m, thiazole CH, Methylphenyl CH), 7.51 (2H, d, J=8.82 Hz, disubstituted
benzene CH), 7.73 (2H, d, J= 8.10 Hz, Methylphenyl CH), 7.93 (1H, s, N=CH), 11.90 (1H, s,
13
NH). C-NMR (75 MHz, DMSO-d6): δ = 21.28, 48.11, 66.42, 102.71, 114.74, 115.06, 125.35,
125.91, 127.85, 129.61, 132.53, 137.20, 142.19, 152.11, 168.72. HRMS (m/z): [M+H]+ calcd for
C21H22N4OS: 379.1587; found: 379.1592.
4-(2-(2-(4-morpholinobenzylidene)hydrazineyl)thiazol-4-yl)benzonitrile (3c)
Yield: 84 %, M.P. = 274.4 °C, FTIR (ATR, cm-1): 3184 (N-H), 2968 (C-H), 2218 (C≡N), 1228
1
(C-N), 821, 742. H-NMR (300 MHz, DMSO-d6): δ 3.17 (4H, t, J=4.77 Hz, morpholine CH2),
3.73 (4H, t, J=4.77 Hz, morpholine CH2), 6.97 (2H, d, J=8.94 Hz, disubstituted benzene CH),
7.51 (2H, d, J=8.88 Hz, disubstituted benzene CH), 7.59 (1H, s, thiazole CH), 7.85 (2H, d, J=
8.58 Hz, Cyanophenyl CH), 7.95 (1H, s, N=CH), 8.02 (2H, d, J=8.55 Hz, disubstituted benzene
CH), 12.01 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6): δ = 48.06, 66.41, 107.55, 109.96,
115.01, 119.48, 125.16, 126.55, 127.93, 133.13, 139.33, 142.65, 149.27, 152.21, 169.14. HRMS
(m/z): [M+H]+ calcd for C21H19N5OS: 390.1383; found: 390.1380.
4-(4-((2-(4-(4-bromophenyl)thiazol-2-yl)hydrazineylidene)methyl)phenyl)morpholine (3d)

ACCEPTED MANUSCRIPT
Yield: 83 %, M.P. = 276.4 °C, FTIR (ATR, cm-1): 3178 (N-H), 2956 (C-H), 1226 (C-N), 819,
1
729. H-NMR (300 MHz, DMSO-d6): δ 3.18 (4H, br. s, morpholine CH2), 3.74 (4H, m,
morpholine CH2), 6.99 (2H, d, J=8.85 Hz, disubstituted benzene CH), 7.36 (1H, s, thiazole CH),
7.51 (2H, d, J=8.76 Hz, disubstituted benzene CH), 7.59 (2H, d, J= 8.55 Hz, Bromophenyl CH),
13
7.80 (2H, d, J= 8.52 Hz, Bromophenyl CH), 7.94 (1H, s, N=CH), 11.94 (1H, s, NH). C-NMR
(75 MHz, DMSO-d6): δ = 48.08, 66.42, 104.55, 114.72, 115.03, 120.88, 125.24, 127.88, 127.98,
128.94, 131.97, 142.38, 152.18, 169.02. HRMS (m/z): [M+H]+ calcd for C20H19BrN4OS:
443.0536; found: 443.0531.
4-(4-((2-(4-(4-chlorophenyl)thiazol-2-yl)hydrazineylidene)methyl)phenyl)morpholine (3e)
Yield: 87 %, M.P. = 273.9 °C, FTIR (ATR, cm-1): 3176 (N-H), 2958 (C-H), 1226 (C-N), 817,
1
731. H-NMR (300 MHz, DMSO-d6): δ 3.18 (4H, t, J=4.72 Hz, morpholine CH2), 3.74 (4H, t,
J=4.72 Hz, morpholine CH2), 6.98 (2H, d, J=8.94 Hz, Disubstituted benzene CH), 7.35 (1H, s,
Thiazole CH), 7.45 (2H, d, J=8.64 Hz, Chlorophenyl CH), 7.51 (2H, d, J=8.85 Hz, Disubstituted
benzene CH), 7.85 (2H, d, J= 8.58 Hz, Chlorophenyl CH), 7.94 (1H, s, N=CH), 11.94 (1H, s,
NH). 13C-NMR (75 MHz, DMSO-d6): δ = 48.08, 66.42, 104.46, 114.72, 115.03, 125.24, 127.66,
127.88, 128.93, 129.06, 132.29, 142.36, 152.18, 168.95. HRMS (m/z): [M+H]+ calcd for
C20H19ClN4OS: 399.1041; found: 399.1031.
4-(4-((2-(4-(4-fluorophenyl)thiazol-2-yl)hydrazineylidene)methyl)phenyl)morpholine (3f)
Yield: 78 %, M.P. = 267.1 °C, FTIR (ATR, cm-1): 3165 (N-H), 2974 (C-H), 1222 (C-N), 817,
1
732. H-NMR (300 MHz, DMSO-d6): δ 3.18 (4H, t, J=4.74 Hz, morpholine CH2), 3.74 (4H, t,
J=4.74 Hz, morpholine CH2), 6.99 (2H, d, J=8.88 Hz, Disubtituted benzene CH), 7.22 (2H, d,
J=8.94 Hz, Fluorophenyl CH), 7.26 (1H, s, thiazole CH), 7.51 (2H, d, J=8.82 Hz, Disubtituted

ACCEPTED MANUSCRIPT
benzene CH), 7.86-7.90 (2H, m, Fluorophenyl CH), 7.94 (1H, s, N=CH), 11.92 (1H, s, NH). 13C-
NMR (75 MHz, DMSO-d6): δ = 48.11, 66.43, 103.37, 114.73, 115.04, 115.86 (d, J=21.3 Hz),
125.30, 127.87, 127.92 (d, J=8.1 Hz), 131.89 (d, J=2.8 Hz), 142.29, 152.16, 162.02 (d, J=242.8
Hz), 168.93. HRMS (m/z): [M+H]+ calcd for C20H19FN4OS: 383.1336; found: 383.1338.
4-(4-((2-(4-(2,4-dichlorophenyl)thiazol-2-yl)hydrazineylidene)methyl)phenyl)morpholine (3g)
Yield: 78 %, M.P. = 243.0 °C, FTIR (ATR, cm-1): 3182 (N-H), 2985 (C-H), 1222 (C-N), 815,
1
709. H-NMR (300 MHz, DMSO-d6): δ 3.18 (4H, t, J=4.77 Hz, morpholine CH2), 3.74 (4H, t,
J=4.76 Hz, morpholine CH2), 6.99 (2H, d, J=8.88 Hz, Disubtituted benzene CH), 7.36 (1H, s,
thiazole CH), 7.50 (1H, dd, J=8.49 Hz- 2.22, Dichlorophenyl H5), 7.52 (2H, d, J=8.82 Hz,
Disubtituted benzene CH), 7.69 (1H, d, J=2.22 Hz, Dichlorophenyl H3), 7.90 (1H, d, J=8.52 Hz,
Dichlorophenyl H3), 7.94 (1H, s, N=CH), 11.94 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6): δ =
48.09, 66.42, 109.20, 115.03, 125.23, 127.89, 127.94, 128.93, 130.22, 131.97, 132.65, 132.70,
132.84, 142.42, 152.20, 168.03. HRMS (m/z): [M+H]+ calcd for C20H18Cl2N4OS: 433.0651;
found: 433.0637.
4-(4-((2-(4-(2,4-difluorophenyl)thiazol-2-yl)hydrazineylidene)methyl)phenyl)morpholine (3h)
Yield: 75 %, M.P. = 245.8 °C, FTIR (ATR, cm-1): 3180 (N-H), 2987 (C-H), 1226 (C-N), 817,
1
729. H-NMR (300 MHz, DMSO-d6): δ 3.17 (4H, t, J=4.77 Hz, morpholine CH2), 3.73 (4H, t,
J=4.76 Hz, morpholine CH2), 6.97 (2H, d, J=8.91 Hz, Disubtituted benzene CH), 7.13-7.20 (2H,
m, thiazole CH, Difluorophenyl CH), 7.29-7.37 (1H, m, Difluorophenyl CH), 7.51 (2H, d,
J=8.85 Hz, Disubtituted benzene CH), 7.95 (1H, s, N=CH), 7.99-8.07 (1H, m), 11.96 (1H, s,
NH). 13C-NMR (75 MHz, DMSO-d6): δ = 48.08, 66.42, 104.97 (t, J=26.2 Hz), 107.76 (dd,
J=14.02 Hz), 112.25 (dd, J1= 3.1 Hz, J2= 22.0 Hz), 115.01, 119.67 (dd, J1=3.5 Hz, J2=11.3 Hz),

ACCEPTED MANUSCRIPT
125.23, 127.89, 130.83 (dd, J1=4.8 Hz, J2=9.5 Hz), 142.44, 143.81, 152.18, 159.99 (dd, J1=12.0
Hz, J2=250.0 Hz), 161.61 (dd, J1=12.4 Hz, J2=245.8 Hz), 168.29. HRMS (m/z): [M+H]+ calcd
for C20H18F2N4OS: 401.1242; found: 401.1240.
4-phenyl-2-(2-(4-(pyrrolidin-1-yl)benzylidene)hydrazinyl)thiazole (3i)
Yield: 81 %, M.P. = 241.1 °C, FTIR (ATR, cm-1): 3564 (N-H), 2956 (C-H), 1176(C-N), 810,
1
740. H-NMR (300 MHz, DMSO-d6): δ = 1.94-1.99 (4H, m, pyrrolidin CH2), 3.26-3.30 (4H, m,
pyrrolidin CH2), 6.59 (2H, d, J=8.79 Hz, Disubstituted benzene CH), 7.27-7.33 (2H, m, thiazole
CH, monosubstituted benzene H4), 7.41 (2H, t, J=7.43 Hz, monosubstituted benzene H3,3’), 7.48
(2H, d, J=8.70 Hz, Disubstituted benzene CH), 7.83 (2H, d, J=7.14 Hz, monosubstituted benzene
13
H2,2’), 7.94 (1H, s, N=CH). C-NMR (75 MHz, DMSO-d6): δ = 25.38, 47.88, 103.66, 112.28,
121.45, 126.15, 128.33, 128.49, 129.11, 134.10, 144.04, 148.90, 148.94, 168.84. HRMS (m/z):
[M+H]+ calcd for C20H20N4S: 349.1481; found: 349.1470.
2-(2-(4-(pyrrolidin-1-yl)benzylidene)hydrazinyl)-4-(p-tolyl)thiazole (3j)
Yield: 77 %, M.P. = 233.3 °C, FTIR (ATR, cm-1): 3045 (N-H), 2835 (C-H), 1178 (C-N), 815,
752. 1H-NMR (300 MHz, DMSO-d6): δ = 1.94-1.99 (4H, m, pyrrolidin CH2), 2.32 (3H, s, -CH3),
3.26-3.30 (4H, m, pyrrolidin CH2), 6.59 (2H, d, J=8.79 Hz, Disubstituted benzene CH), 7.18
(1H, s, thiazole CH), 7.22 (2H, d, J=8.07 Hz, Methylphenyl CH), 7.50 (2H, d, J=8.73 Hz,
Disubstituted benzene CH), 7.70 (2H, d, J=8.10 Hz, Methylphenyl CH), 7.95 (1H, s, N=CH) 13C-
NMR (75 MHz, DMSO-d6): δ = 21.29, 25.40, 47.80, 102.65, 112.19, 121.48, 126.04, 128.39,
129.51, 129.64, 131.77, 137.51, 144.22, 148.99, 168.76. HRMS (m/z): [M+H]+ calcd for
C21H22N4S: 363.1638; found: 363.1639.
4-(2-(2-(4-(pyrrolidin-1-yl)benzylidene)hydrazinyl)thiazol-4-yl)benzonitrile (3k)

ACCEPTED MANUSCRIPT
Yield: 76 %, M.P. = 221.3 °C, FTIR (ATR, cm-1): 3344 (N-H), 2945 (C-H), 2223 (C≡N),
1
1174(C-N), 813, 734. H-NMR (300 MHz, DMSO-d6): δ = 1.93-1.98 (4H, m, pyrrolidin CH2),
3.25-3.29 (4H, m, pyrrolidin CH2), 6.58 (2H, d, J=8.79 Hz, Disubstituted benzene CH), 7.46 (2H,
d, J=8.73 Hz, Disubstituted benzene CH), 7.57 (1H, s, thiazole CH), 7.85 (2H, d, J=8.55 Hz,
Cyanophenyl CH), 7.93 (1H, s, N=CH), 8.02 (2H, d, J=8.55 Hz, Cyanophenyl CH), 11,90 (1H,
13
br.s., NH) C-NMR (75 MHz, DMSO-d6): δ = 25.38, 47.86, 107.32, 109.96, 112.26, 119.47,
121.65, 126.57, 128.30, 133.12, 139.19, 143.74, 148.85, 148.89, 169.18. HRMS (m/z): [M+H]+
calcd for C21H19N5S: 374.1434; found: 374.1440.
4-(4-bromophenyl)-2-(2-(4-(pyrrolidin-1-yl)benzylidene)hydrazinyl)thiazole (3l)
Yield: 79 %, M.P. = 270.4 °C, FTIR (ATR, cm-1): 3047 (N-H), 2835 (C-H), 1180 (C-N), 829,
1
740. H-NMR (300 MHz, DMSO-d6): δ = 1.94-1.99 (4H, m, pyrrolidin CH2), 3.26-3.30 (4H, m,
pyrrolidin CH2), 6.58 (2H, d, J=8.79 Hz, Disubstituted benzene CH), 7.33 (1H, s, thiazole CH)
7.46 (2H, d, J=8.70 Hz, Disubstituted benzene CH), 7.59 (2H, d, J=8.58 Hz, Bromophenyl CH),
13
7.79 (2H, d, J=8.55 Hz, Bromophenyl CH), 7.92 (1H, s, N=CH). C-NMR (75 MHz, DMSO-
d6): δ = 25.40, 47.79, 104.33, 112.19, 120.95, 121.60, 128.02, 128.28, 131.96, 134.22, 143.66,
148.91, 149.23, 169.02. HRMS (m/z): [M+H]+ calcd for C20H19BrN4S: 427.0587; found:
427.0564.
4-(4-chlorophenyl)-2-(2-(4-(pyrrolidin-1-yl)benzylidene)hydrazinyl)thiazole (3m)
Yield: 74 %, M.P. = 213.2 °C, FTIR (ATR, cm-1): 3280 (N-H), 2835 (C-H), 1176 (C-N), 833,
1
748. H-NMR (300 MHz, DMSO-d6): δ = 1.93-1.98 (4H, m, pyrrolidin CH2), 3.25-3.30 (4H, m,
pyrrolidin CH2), 6.60 (2H, d, J=8.79 Hz, Disubstituted benzene CH), 7.33 (1H, s, thiazole CH),
7.46 (2H, d, J=8.61 Hz, Chlorophenyl CH), 7.47 (2H, d, J=8.73 Hz, Disubstituted benzene CH),

ACCEPTED MANUSCRIPT
13
7.85 (2H, d, J=8.61 Hz, Chlorophenyl CH), 7.96 (1H, s, N=CH). C-NMR (75 MHz, DMSO-
d6): δ = 25.37, 47.95, 104.30, 112.37, 121.76, 127.74, 128.33, 129.07, 132.46, 133.60, 143.94,
148.74, 148.80, 168,97. HRMS (m/z): [M+H]+ calcd for C20H19ClN4S: 383.1092; found:
383.1093.
4-(4-fluorophenyl)-2-(2-(4-(pyrrolidin-1-yl)benzylidene)hydrazinyl)thiazole (3n)
Yield: 80 %, M.P. = 186.1 °C, FTIR (ATR, cm-1): 3286 (N-H), 2837 (C-H), 1240 (C-N), 839,
1
742. H-NMR (300 MHz, DMSO-d6): δ = 1.94-1.99 (4H, m, pyrrolidin CH2), 3.26-3.30 (4H, m,
pyrrolidin CH2), 6.59 (2H, d, J=8.82 Hz, Disubstituted benzene CH), 7.20-7.26 (2H, m, thiazole
CH, Fluorophenyl CH), 7.47 (2H, d, J=8.76 Hz, Disubstituted benzene CH), 7.85-7.90 (2H, m,
Fluorophenyl CH), 7.95 (1H, s, N=CH). 13C-NMR (75 MHz, DMSO-d6): δ = 25.36, 48.03,
103.37, 112.46, 115.93 (d, JCF-2= 21.4 Hz), 121.77, 128.14 (d, JCF-3= 8.2 Hz), 128.42, 130.89,
144.44, 148.13, 148.81, 162.17 (JCF-1= 242.96 Hz), 168,90. HRMS (m/z): [M+H]+ calcd for
C20H19FN4S: 367.1387; found: 367.1382.
4-(2,4-dichlorophenyl)-2-(2-(4-(pyrrolidin-1-yl)benzylidene)hydrazinyl)thiazole (3o)
Yield: 78 %, M.P. =217.2 °C, FTIR (ATR, cm-1): 3170 (N-H), 2854 (C-H), 1174 (C-N), 817,
1
738. H-NMR (300 MHz, DMSO-d6): δ = 1.93-1.97 (4H, m, pyrrolidin CH2), 3,24-3.28 (4H, m,
pyrrolidin CH2), 6.56 (2H, d, J=8.82 Hz, Disubstituted benzene CH), 7.33 (1H, s, thiazole CH),
7.46 (2H, d, J=8.88 Hz, Disubstituted benzene CH), 7.49 (1H, dd, J=8.52 Hz-2.19 Hz,
Dichlorophenyl H5), 7.67 (1H, d, J=2.19 Hz, Dichlorophenyl H3), 7.90 (1H, s, N=CH), 7.49 (1H,
13
d, J=8.52 Hz, Dichlorophenyl H6), 11.80 (1H, br.s, NH). C-NMR (75 MHz, DMSO-d6): δ =
25.42, 47.70, 108.89, 112.08, 121.55, 127.92, 128.21, 130.21, 131.95, 132.64, 132.75, 132.77,

ACCEPTED MANUSCRIPT
143.29, 146.34, 148.91, 168.11. HRMS (m/z): [M+H]+ calcd for C20H18Cl2N4S: 417.0702; found:
417.0709.
4-(2,4-difluorophenyl)-2-(2-(4-(pyrrolidin-1-yl)benzylidene)hydrazinyl)thiazole (3p)
Yield: 79 %, M.P. = 156.9 °C, FTIR (ATR, cm-1): 3292 (N-H), 2966 (C-H), 1178 (C-N), 812,
1
734. H-NMR (300 MHz, DMSO-d6): δ = 1.93-1.98 (4H, m, pyrrolidin CH2), 3,24-3.29 (4H, m,
pyrrolidin CH2), 6.56 (2H, d, J=8.79 Hz, Disubstituted benzene CH), 7.14-7.20 (2H, m, thiazole
CH, Difluorophenyl CH), 7.29-7.37 (1H, m, Difluorophenyl CH), 7.46 (2H, d, J=8.76 Hz,
Disubstituted benzene CH), 7.91 (1H, s, N=CH), 7.98-8.07 (1H, m, Difluorophenyl CH), 11.82
(1H, s, NH). 13C-NMR (75 MHz, DMSO-d6): δ = 25.41, 47.70, 104.96 (t, JCF= 26.4 Hz), 107.47
(d, JCF= 14.45 Hz), 112.09, 112.38 (d, J= 3.2 Hz), 119.71 (dd, J1=3.7 Hz, J2= 11.1 Hz), 121.55,
128.21, 130.83 (dd, J1= 4.8 Hz, J2=9.4 Hz), 143.34, 143.75, 148.91, 160.06 (dd, J1=11.8 Hz, J2=
250.16 Hz), 161.66 (dd, J1= 11.8 Hz, J2= 250.16 Hz), 168.36. HRMS (m/z): [M+H]+ calcd for
C20H18F2N4S: 385.1293; found: 385.1292.
4.2. MAO-A and MAO-B Inhibition Assay
Ampliflu™ Red (10-Acetyl-3,7-dihydroxyphenoxazine), peroxidase from horseradish, hMAO-A,
hMAO-B, H2O2, tyramine hydrochloride, selegiline, and moclobemide were purchased from
Sigma-Aldrich (Steinheim, Germany) and retained under the suggested conditions by supplier.
All pipetting processes were performed using a Biotek Precision XS robotic system (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.

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Enzymatic assay was carried out according to recent method described by our research group
[34]. Blank, control and all concentrations of inhibitors were analyzed in quadruplicate and
inhibition percent was calculated by using following equation:
ꢄ ꢀꢁ ) ꢄ (ꢀꢆ ꢄ ꢀꢆ )
ꢂꢅ
% Inhibition = (ꢀꢁ
ꢇ 100
ꢂꢃ
ꢂꢅ
ꢂꢃ
ꢀꢁ ꢄ ꢀꢁ
ꢂꢃ
ꢂꢅ
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).
The results were displayed as mean ±standard deviation (SD). Selectivity index (SI) was
calculated as IC50(hMAO-A)/ IC50(hMAO-B).
4.3. Enzyme Kinetic Studies
The same materials were used in the MAO inhibition assay. The most active compounds 3f and
3h were tested at three different concentrations of IC50/2, IC50, and 2 x IC50 according to assay
described in the our recent study [34]. All processes were assayed in quadruplicate. The results
were analyzed as Lineweaver-Burk plots using Microsoft Office Excel 2013. The Vmax values of
the Lineweaver-Burk plots were replotted versus the inhibitor concentration, and the Ki values
were determined from the x-axis intercept as -Ki.
4.4. Cytotoxicity Assay
In cytotoxicity test by using NIH/3T3 mouse embryonic fibroblast cell line (ATCC® CRL-1658™,
London, UK), firstly NIH/3T3 cells were incubated to the Dulbecco's Modified Eagle's Medium

ACCEPTED MANUSCRIPT
(DMEM). NIH/3T3 cells were plated on 96-well culture plates as 10000 cells per well and were
then treated with the compounds at concentrations ranging from 1 mM to 0.000316 mM (1, 0.316,
0.1, 0.0316, 0.01, 0.00316, 0.001, 0.000316 mM). MTT assay was performed as previously
described [34, 49, 50]. Dose-response curves were plotted against compound concentrations
applied to determine IC50 values. The following formula was used to calculate the inhibition
percentage for each concentration.
% inhibition = 100 - (mean sample x 100 / mean solvent)
4.5. Genotoxicity Assay
The genotoxicity of the compounds was determined by Ames assay as previously described using
the Ames MPF 98/100 mutagenicity test sample kit (Xenometrix AG, Switzerland). This test was
performed with Salmonella typhimurium strains TA98 (frameshift mutations) and TA100 (base-
pair substitutions). Compounds was prepared in six different concentrations (5, 2.5, 1.25, 0.625,
0.3125, 0.156 mg/mL) which was between 16 and 5000 µg/mL according to the previous
guidelines in DMSO. In order to detect mutagenic potential, test carried out with and without
Aroclor™-1254 induced male Sprague–Dawley rat liver microsomal enzyme (S9) mix
(Xenometrix AG, Switzerland). While positive control for TA 98 without S9 mix was 2-
Nitrofluorene (2 µg/mL), 1 µg/mL of 2-aminoanthracene was positive controls with S9 against
TA 98. For TA100, 4-nitroquinoline N-oxide (0.1 µg/mL) was positive control without S9 mix
and 2.5 µg/mL of 2-aminoanthracene was positive control. 4% DMSO solution was the solvent
control. At the end of the experiment, revertant bacteria decreased the pH of solution and
indicator medium colour was changed to yellow. Yellow wells were counted as positive and
compared with the negative control. Fold induction over the negative control and fold induction
over the baseline were calculated. Fold induction over the negative control is the ratio of the

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mean number of positive wells for the dose concentration divided by the mean number of positive
wells for the zero dose (negative) control. Fold induction over the baseline is the ratio of the mean
number of positive wells for the dose concentration divided by zero dose baseline. The zero dose
baseline is obtained by adding one standard deviation to the mean number of positive wells of the
zero dose control [34, 49, 50]. Mutagenity was determined according to the criteria from previous
studies [37]. For a baseline value ≤ 3, significant increases between 2 and 3-fold the baseline were
classified as weak mutagen, increases ≥ 3-fold the baseline were classified as mutagen. For a
baseline was > 3, significant increases between 1.5 and 2.5-fold the baseline were classified as
weak mutagen, and increases ≥ 2.5-fold the baseline, were classified as mutagen. In principle, for a
mutagenic compound, at least two adjacent doses with significant increase or a significant increase
at the highest dose level should be observed. All doses were compared according to Student’s t-test
at p < 0.05 for statistically. When compounds did not have any of the properties referred above,
they were classified as a non-mutagenic compound.
4.6. Prediction of ADME Parameters and BBB Permeability
Physicochemical parameters were analysed by QikProp 4.8 software [39] to predict
pharmacokinetic profiles and BBB permeability of compounds (3a-3p).
4.7. Molecular Docking
A structure based in silico procedure was applied to discover the binding modes of compounds 3f
and 3h to hMAO-A enzyme active site. The crystal structures of hMAO-A (PDB ID: 2Z5X) [42],
which was crystallized with the reversible inhibitor harmine, was retrieved from the Protein Data
Bank server (www.pdb.org).

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The structures of ligands were built using the Schrödinger Maestro [51] interface and then were
submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2
[52]. The ligands were prepared by LigPrep 3.8 [53] to assign the protonation states at pH
7.4±1.0 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 [43]. The grid box with
dimensions of 20 Å x 20 Å x 20 Å was centered in the vicinity of the flavin (FAD) N5 atom on
the catalytic site of the protein to cover all binding sites and neighboring residues [54-56].
Flexible docking runs were performed with single precision docking mode (SP). Electrostatic and
van der Waalls interactions in the active site of h-MAOA were evaluated by a per-residue
interactions panel of Glide 7.1 [53].
References
[1] W. Weyler, Y. P. Hsu, X. O. Breakefield, Pharmacol Ther. 47 (1990) 391-417.
[2] J. C. Shih, K. Chen, M. J. Ridd, Annu. Rev. Neurosci 22 (1999) 197-217.
[3] M. B. H. Youdim, D. Edmondson, K.F. Tipton, Nat. Rev. Neurosci 7 (2006) 295-309.
[4] A. W. Bach, N. C. Lan, D. L. Johnson, C. W. Abell, M. E. Bembenek, S. W. Kwan, P. H.
Seeburg, J. C. Shih, Proc Natl Acad 85 (1998) 4934-4938.
[5] Youdim MBH, Finberg JPM, Tipton KF. In: Trendelenburg U, Weiner N, editors.
Catecholamine II. Handbook of Experimental Pharmacology. Berlin: Springer; 1988. p 127-199
[6] S. Distinto, R. Meleddu, M. Yanez, R. Cirilli, E. Maccioni, Eur. J. Med. Chem. 108 (2016)
542-552.
[7] M.Y. Wu, G. Esteban, S. Brogi, M. Shionoya, J. Marco-Contelles, Eur. J. Med. Chem. 121
(2016) 864-879.

ACCEPTED MANUSCRIPT
[8] L. Ferrazzano, A. Viola, E. Lonati, A. Bulbarelli, A.Tolomelli, Eur. J. Med. Chem. 124
(2016) 906-909.
[9] M. Strolin-Benedetti, P. Dostert, Biochem. Pharmacol. 38 (1989) 555-561.
[10] L. Emilsson, P. Saetre, J. Balciuniene, A. Castensson, N. Cairns, E. E. Jazin, Neurosci. Lett.
326 (2002) 56-60.
[11] M. Strolin-Benedetti, P. L. Dostert, Adv. Drug Res. 23 (1992) 65-125.
[12] F. Chimenti, E. Maccioni, D. Secci, D. Bolasco, P. Chimenti, A. Granese, S. Carradori, S.
Alcaro, F. Ortuso, M. Yanez, F. Orallo, R. Cirilli, R. Ferretti, F. La Torre, J. Med. Chem. 51
(2008) 4874-4880.
[13] L. De Colibus, M. Li, C. Binda, A. Lustig, D. E. Edmondson, A. Mattevi, Proc. Natl. Acad.
102 (2005) 12684-12689.
[14] A. M. Andres, M. Soldevila, A. Navarro, K. K. Kidd, B. Oliva, J. Bertranpetit, Hum Genet,
115 (2004) 377-386.
[15] D. E. Edmondson, C. Binda, A. Mattevi, Arch. Biochem. Biophys. 464 (2007) 269-276.
[16] M. Husain, D. E. Edmondson, T. P. Singer, Biochemistry 21 (1982) 595-600.
[17] L. B. Pearce, J. A. Roth, Biochemistry 24 (1985) 1821-1826.
[18] D. E. Edmondson, A. K. Bhattacharyya, M. C. Walker, Biochemistry 32 (1993) 5196-5202.
[19] O. Pravin, Patil, B. Sanjay, Bari, Arab. J. Chem. 7 (2014) 857-884.
[20] D. Secci, A. Bolasco, S. Carradori, M. D’Ascenzio, R. Nescatelli, M. Yáñez, Eur J Med
Chem 58 (2012) 405-417.
[21] B. K. Sheetal, A. K. Manthab, V. Kumar, RSC Adv. 6 (2016) 42660-42683.
[22] R. Pignatello, S. Mazzone, F. Castelli, P. Mazzone, G. Raciti, G. Mazzone, Pharmazie 49
(1994) 272-276.