MAO enzymes inhibitory activity of new benzimidazole derivatives including hydrazone and propargyl side chains

Article abstract of DOI:10.1016/j.ejmech.2017.03.009

In the present work, 15 new N’-(arylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)benzohydrazide (4a-4o) were designed and synthesized. The structures of the synthesized compounds were elucidated using FT-IR, ¹H-NMR, ¹³C-NMR, and HRMS spectral data. The inhibitory activity of the compounds 4a-4o against hMAO-A and hMAO-B enzymes was evaluated by using in?vitro Amlex Red reagent based fluorometric method. Due to lots of high-cost kits including this assay, we determined the ingredients of the kits from the data sheets of several suppliers, and adjusted a protocol by working with various concentrations and volumes of these ingredients. As a result, a fast and sensitive assay was applied as in the commercially available MAO kits with lower costs and clearer ingredients than those of the kits. The enzyme inhibition assay revealed that synthesized compounds have selective inhibition potency against hMAO-B. The compound 4e and 4f displayed IC₅₀ values of 0.075?μM and 0.136?μM against hMAO-B, respectively. The reference drugs selegiline (IC₅₀?=?0.040?μM) and rasagiline (IC₅₀?=?0.066?μM) also displayed a significant inhibition against hMAO-B. The enzyme kinetic study was performed in order to observe the effect of the most active compound 4e on substrate-enzyme relationship and non-competitive inhibition of hMAO-B was determined. Cytotoxicity and genotoxicity studies were carried out and the compound 4e was found as non-cytotoxic and non-genotixic. Theoretical calculation of ADME properties suggested that compound 4e may have a good pharmacokinetic profile. The docking study of compound 4e revealed that there is a strong interaction between the active sites of hMAO-B and analyzed compound.

Full text of DOI:10.1016/j.ejmech.2017.03.009

Accepted Manuscript
MAO enzymes inhibitory activity of new benzimidazole derivatives including
hydrazone and propargyl side chains
Özgür Devrim Can, Derya Osmaniye, Ümide Demir Özkay, Begüm Nurpelin Sağlık,
Serkan Levent, Sinem Ilgın, Merve Baysal, Yusuf Özkay, Zafer Asım Kaplancıklı
PII:
S0223-5234(17)30158-7
10.1016/j.ejmech.2017.03.009
EJMECH 9269
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 November 2016
Revised Date: 22 February 2017
Accepted Date: 4 March 2017
Please cite this article as: E.D. Can, D. Osmaniye, E. Demir Özkay, B.N. Sağlık, S. Levent, S. Ilgın, M.
Baysal, Y. Özkay, Z.A. Kaplancıklı, MAO enzymes inhibitory activity of new benzimidazole derivatives
including hydrazone and propargyl side chains, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.03.009.
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ACCEPTED MANUSCRIPT
MAO Enzymes Inhibitory Activity of New Benzimidazole Derivatives Including
Hydrazone and Propargyl Side Chains
Fifteen novel benzimidazole compounds were synthesized in order to investigate their
inhibitory potency against hMAO-A and hMAO-B enzymes. The compound 4e displayed
significant hMAO-B inhibition, notable interactions in the active site, non-cytotoxicity, non-
genotoxicity and a good predicted ADME profile.
Significant
hMAO-B
inhibition
Non-genotoxic
Non-cytotoxic
NIHI3T3A
Good ADME
profile
prediction
IC = 0.075 µM
50
IC = 316 µM
50
O
N
N
N
NH
Compound 4e
N
Notable interactions in the active site

ACCEPTED MANUSCRIPT
MAO Enzymes Inhibitory Activity of New Benzimidazole Derivatives Including
Hydrazone and Propargyl Side Chains
a
b
a
b,c
Özgür Devrim Can , Derya Osmaniye , Ümide Demir Özkay , Begüm Nurpelin Sağlık
,
b,c
d
d
* b,c
b
Serkan Levent , Sinem Ilgın , Merve Baysal , Yusuf Özkay , Zafer Asım Kaplancıklı
a
Department of Pharmacology, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir,
Turkey
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470
Eskişehir, Turkey
c
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu
University, 26470 Eskişehir, Turkey
d
Department of Toxicology, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir,
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, Eskiꢀehir, Turkey.
1

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Abstract
In the present work, 15 new N’-(arylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-
yl)benzohydrazide (4a-4o) were designed and synthesized. The structures of the synthesized
1
13
compounds were elucidated using FT-IR, H-NMR, C-NMR, and HRMS spectral data. The
inhibitory activity of the compounds 4a-4o against hMAO-A and hMAO-B enzymes was
®
evaluated by using in vitro Amlex Red reagent based fluorometric method. Due to lots of
high-cost kits including this assay, we determined the ingredients of the kits from the data
sheets of several suppliers, and adjusted a protocol by working with various concentrations
and volumes of these ingredients. As a result, a fast and sensitive assay was applied as in the
commercially available MAO kits with lower costs and clearer ingredients than those of the
kits. The enzyme inhibition assay revealed that synthesized compounds have selective
inhibition potency against hMAO-B. The compound 4e and 4f displayed IC50 values of
0
.0075 µM and 0.136 µM against hMAO-B, respectively. The reference drugs selegiline
(
IC =0.040 µM) and rasagiline (IC =0.066 µM) also displayed a significant inhibition
5
0
50
against hMAO-B. The enzyme kinetic study was performed in order to observe the effect of
the most active compound 4e on substrate-enzyme relationship and non-competitive
inhibition of hMAO-B was determined. Cytotoxicity and genotoxicity studies were carried
out and the compound 4e was found as non-cytotoxic and non-genotixic. Theoretical
calculation of ADME properties suggested that compound 4e may have a good
pharmacokinetic profile. The docking study of compound 4e revealed that there is a strong
interaction between the active sites of hMAO-B and analyzed compound.
Keywords: MAO-A; MAO-B; amplex red; benzimidazole; propargyl; hydrazone; docking.
2

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1
. Introduction
Monoamine oxidases (MAOs) are the enzymes catalyzing oxidative deamination of
monoamines such as dopamine, noradrenaline, adrenaline, and serotonin [1]. MAOs are
placed in the mitochondrial outer membrane of neuronal, glial, and other cells [2]. This
enzyme exists in two forms, MAO-A and MAO-B. These two isoforms share almost 70%
sequence identity at the amino acid level and were identified based on their substrate
selectivity and inhibitor sensitivities. MAO-A preferentially breakdown serotonin,
noradrenaline, and adrenaline, and is irreversibly inhibited by clorgyline. On the other hand,
MAO-B preferentially breakdown dopamine, β-phenetylamine, and benzylamine, and is
irreversibly inhibited by selegiline [3,4].
Since inhibition of the enzyme activity prevents degradation of monoamine neurotransmitters,
and induces an increase in the levels of these neurotransmitters at the synaptic cleft, inhibitors
of MAO enzymes have the notable therapeutic potential for the treatment of disorders caused
by deficiencies in monoaminergic neurotransmission of the brain [5]. For instance, reversible
and selective MAO-A inhibitor moclobemide as well as non-selective and irreversible MAO
inhibitor phenelzine, which induce a notable increase in the levels of noradrenaline and
serotonin levels at the central synapses, are among the well-known antidepressant drugs. In
addition to major depressive disorder, numerous clinical studies have exhibited the
therapeutic potential of MAO inhibitors against further emotional disorders such as atypical
depression [6], dysthymic disorder [7], bipolar depression [8], bulimia [9] and also of some
anxiety disorders such as panic disorder [1] social phobia [10,11] and post-traumatic stress
disorder [12]. Monoamine oxidase B (MAO-B) is also a well-known target for
neuroprotective drugs as well as antidepressants [13, 14]. Irreversible and selective MAO-B
inhibitory drug selegiline, which raises dopamine levels in the synapses of basal ganglions, is
known to be beneficial for the palliative treatment of patients with Parkinson disease. Besides,
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MAO-B inhibitors are also used alone or in combination to treat Alzheimer’s disease due to
their protective effects on both vascular and neuronal tissues [15,16].
Although there is a wide therapeutic potential of MAO inhibitors for central nervous system
(CNS) disorders, these drugs have some early phase (such as orthostatic hypotension,
dizziness, drowsiness, insomnia, and nausea) and late phase (such as hepatotoxicity, weight
gain, edema, muscle pains, myoclonus, paresthesias, and sexual dysfunction) adverse effects.
Moreover, risks of drug-drug (serotonin syndrome) or drug-diet (cheese reaction) interaction
induced by some of the MAO inhibitors are seriously limited use of these drugs in psychiatry
clinics [17]. Therefore, discovery and development of new, more potent and safer MAO
inhibitors are among the current scientific targets of the medicinal chemists.
In terms of crystal structure, MAO isoforms differ in nature and volume of the catalytic site,
consenting to discover selective binders [13,18]. The hMAO-B enzyme has two cavities
connected by the amino acid ILE199 that works as a ‘gate’. The entrance cavity has a volume
3
of 290 Å and, possesses very hydrophobic character. The second cavity, with a volume of
3
3
90 Å , includes the substrate binding site. The coenzyme FAD is located at the end of this
cavity. Crystallographic data of the substrate cavity has indicated that the amino acid side
chains, coating the cavity, are very hydrophobic and favorable to interact with an amine
moiety. The FAD and closely two parallel tyrosyl residues (398 and 435) constitute an
3
‘
aromatic cage’. In contrast hMAO-B, hMAO-A has only a single cavity of 550 Å , in which
FAD and two closely parallel tyrosyl residues (407 and 444) also form an ‘aromatic cage’.
The substrate binding sites of both hMAO-A and hMAO-B have quite hydrophobic nature
[
19-23]. All these information simplify the understanding of the selective interactions
between these isoforms and their ligands. Hence, it is possible to observe the catalytic
mechanism and identify the pharmacophoric requirements for the rational design of new
inhibitors.
4

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In the light of above information, in this study we planned to synthesize some novel MAO
inhibitors. Based on the previous literature [2,24-26], demonstrating the inhibitory efficacy of
benzimidazole compounds against MAO enzyme, we decided to synthesize some novel
benzimidazole derivatives as potential MAO inhibitor candidates. Further, we designed the
side chains by using two other pharmacophore groups, which also have notable MAO enzyme
inhibitory capacity. The first pharmacophore group was selected as “hydrazone”, because
substrates and inhibitors of MAO enzyme usually carry an amino or imino group, which
seems to play an essential role in the orientation and complex formation at the active site of
the enzyme. Iproniazide and isocarboxazid were the MAO inhibitors, bearing hydrazone
moiety (Figure 1). Based on this potential, numerous substituted hydrazones and hydrazides
have been reported as MAO inhibitors [27-31]. The second pharmacophore group was chosen
as “propargyl” due to the inhibitory potential of this moiety against MAO-A and/or MAO-B
enzymes [32-34]. Some of the clinically prescribed MAO inhibitors such as rasagiline and
selegiline, are already known to carry the propargyl group in their chemical structures (Figure
1
) [35,36]. Moreover, neuroprotective capacity of the compounds, carrying a propargyl group,
increases the importance of this moiety in drug design studies [32-34].
In the current work, a novel series of benzimidazole derivatives bearing “hydrazone” and
“
propargyl” side chains were synthesized to examine their activity against MAO enzyme
isotypes, with the goal of developing safer and potent MAO-A and MAO-B inhibitors
Figure 1).
(
2
. Result and Discussion
2
.1. Chemistry
The compounds 4a-4o were synthesized as outlined in Scheme 1. Methyl 4-(1H-
benzo[d]imidazol-2-yl)benzoate (1) was prepared under microwave irradiation by reacting
1
,2-phenylenediamine and methyl 4-formylbenzoate. Substitution of compound 1 by
5

ACCEPTED MANUSCRIPT
propargyl bromide gave the methyl 4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)benzoate
(2). Reaction of the compound 2 and hydrazine hydrate afforded 4-(1-(prop-2-yn-1-yl)-1H-
benzo[d]imidazol-2-yl)benzohydrazide (3), which was treated with corresponding aldehyde to
gain the compounds 4a-4o.
Structural elucidation of the synthesized compounds (4a-4o) was performed by spectral
analyses. In the IR spectra, N-H, CC and C=O stretching bands were observed between
-1
-1
-1
1
3
410-3213 cm , 2126-2091 cm and 1672-1641 cm , respectively. In the H-NMR spectra,
acetylene proton recorded as singlet between 3.50 and 3.54 ppm. Methylene bridge protons
between benzimidazole and acetylene group were recorded as a singlet at 5.22-5.23 ppm.
1
3
Amide proton gave a singlet between 11.70 and 12.37 ppm. In the C-NMR spectra, peaks of
methylene and acetylene carbons were observed about 35 ppm, and 77-79 ppm, respectively.
Carbonyl carbon gave a peak about 160-165 ppm. All the other aromatic hydrogens and
1
13
carbons were recorded at expected areas in the both H-NMR and C-NMR spectra. All
measured mass and isotope ratios were compatible with theoretical values in HRMS spectra.
2
.2. MAO Inhibition
The newly synthesized N’-(arylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-
yl)benzohydrazide derivatives (4a-4o) were investigated for their hMAO-A and hMAO-B
inhibitory activity by an in vitro fluorometric method, being able to sensitively detect
monoamine oxidase (MAO) activity. The assay is based on the detection of H O in a
2
2
horseradish peroxidase coupled reaction using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex
Red reagent). There are lots of commercially available MAO-A and MAO-B kits including
this assay. The advantage of the kits is a fast and sensitive protocol implementation. However,
only limited numbers of inhibitor candidates could be studied using a kit, and several numbers
of kits are required to screen potency of a series of inhibitor candidates. Thus, using a kit
causes high costs for researchers. Furthermore, in user guide of many kits, there is only
6

ACCEPTED MANUSCRIPT
experimental procedure to carry out protocol that does not give any information about
concentration of some of the contents. From this point, we determined the ingredients of the
assay by examining various kits, and applied a protocol by working with different
concentrations and volumes of these ingredients. As a result, a fast and a sensitive assay was
performed as in the commercially available MAO kits with lower costs and clearer
information of ingredients than those of the kits.
-3
-4
The assay was performed in two steps. First, compounds 4a-4o were tested at 10 and 10 M
-5
-9
concentrations. Second step was performed by using 10 -10 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 4a-4o. The compounds
4
a, 4c, 4j and 4l could not pass the first step test due to lower inhibitory activity against both
MAO isoforms. In addition, compounds 4d, 4g, 4k, and 4n were not evaluated in the second
step hMAO-A assay because of lower than 50% inhibition potencies. Against hMAO-A, the
most active compound 4f displayed an IC of 0.851 µM, whereas reference drug clorgiline
5
0
had an IC50 of 0.0054 µM. On the other hand, compound 4f showed an IC50 of 0.136 µM
against hMAO-B. Compound 4e displayed an important inhibitory activity against hMAO-B.
It was the most active derivative against hMAO-B with an IC of 0.075 µM. The reference
5
0
drugs selegiline (IC =0.040 µM) and rasagiline (IC =0.066 µM) also displayed a significant
5
0
50
inhibition against hMAO-B. Selectivity indexes (SI) were expressed as IC50(MAO-A) / IC50(MAO-
B). Selectivity towards MAO-A increases as the corresponding SI decreases while selectivity
towards MAO-B isoform increases as the corresponding SI increases. It was observed that
synthesized compounds have selective inhibition potency against hMAO-B. Compound 4e
displayed very significant SI of 127.813.
7

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2
.3. Kinetic Studies of Enzyme Inhibition
The mechanism of hMAO-B 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 kinetic was analysed by recording substrate-
velocity curves in absence and presence of the most potent compound 4e, which was prepared
at concentrations of IC /4 (0.01875 µM), IC /2 (0.0375 µM), IC (0.075 µM), 2xIC (0.150
5
0
50
50
50
µM), and 4xIC (0.300 µM). In each case, the initial velocity measurements were gained at
5
0
different substrate (tyramine) concentrations ranging from 20 µM to 0.625 µM. The Ki
intercept on the x-axis) value of compound 4e was determined from the secondary plot of the
/V versus varying concentrations.
(
1
The graphical analysis of steady-state inhibition data for compound 4e is shown in Figure 1.
The Lineweaver–Burk plot introduces the inhibition type as mixed-type, competitive or non-
competitive. In the mix-typed inhibition, the lines cross neither x- nor y-axis at the same
point. Competitive inhibitors possess the same intercept on y-axis but there are diverse slopes
and intercepts on x-axis between the two data sets. Non-competitive inhibition has plots with
the same intercept on x-axis but there are different slopes and intercepts on y-axis, which is
observed in Figure 1. Therefore, this pattern indicates that the mechanism of hMAO-B
inhibition of 4e is non-competitive, explaining that the inhibitor can bind to either the free
enzyme or the enzyme–substrate complex. K value for compound 4e was calculated as 0.151
i
µM for the inhibition of hMAO-B.
2
.4. Cytotoxicity
Toxicity of compounds 4e and 4f was investigated by MTT assay, which is based on the
reduction of yellow MTT dye by metabolically active eukaryotic and prokaryotic cells to form
the purple formazan product. This assay is mainly preferred to form a view about cell viability
and to observe the growth of cell culture [37,38]. MTT assay was carried out using healthy
8

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NIH/3T3 mouse embryonic fibroblast cell lines (ATCC CRL1658), which is recommended
for cytotoxicity screening by ISO (10993-5, 2009) [39]. As seen in Table 2, the IC values of
5
0
4
e and 4f against NIH/3T3 cells were found to be 316 µM and ≥1000 µM, which are
significantly higher than the IC values of compounds (0.075 µM and 0.136 µM) against
5
0
hMAO-B. Thus, it can be stated that these compounds are nontoxic at their effective
concentrations against hMAO-B.
2
.5. Genotoxicity
Ames assay was performed to investigate the genotoxicity of compounds 4e and 4f. In the
MPF
Ames
assay, more than 25 positive wells were observed with positive controls, and
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
MPF
Ames
and also as described in previous studies [40]. The results are presented in Table 3.
Compound 4e showed a baseline of 5.85 and 1.91 against TA98, in the absence and presence
of S9, respectively. Fold inductions over baseline did not reach the mentioned values of the
baseline and also did not show any dose-response tendency. Therefore, compound 4e was
classified as non-mutagenic against TA98 in the presence/absence of metabolic activation
(S9) (Figure 2). Compound 4e had a baseline of 6.73 against TA100 in the absence of S9 and
baseline of 5.00 in the presence of S9. Furthermore, fold inductions over baseline were less
than 1.5 in each concentrations of the compound and also did not show a dose-response
tendency. Therefore, 4e was not genotoxic against TA100 with/without metabolic activation
(Figure 3).
Compound 4f showed a baseline of 5.85 and 2.82 against TA98 with and without S9,
respectively. Fold inductions over baseline did not reach values more than 1.5 and,
statistically different results did not reveal a dose-response tendency against TA98 without
S9. However, fold inductions over baseline were more than 2.5 and 5 mg/ml concentrations of
9

ACCEPTED MANUSCRIPT
compound 4f against TA 98 with S9. Also, adjacent doses were found to reach mentioned fold
increases with a dose-response tendency. Therefore, 4f was classified as a mutagen against
TA98 with metabolic activation (Figure 4). Compound 4f was found to show a baseline of
6
.73 and 5.49 against TA100 with and without S9, respectively. Mentioned-fold increases
over the baseline according to the criteria were not determined with 4f against TA100 without
S9. However, 2.5 and 5 mg/ml concentrations of 4f reached more than 1.5 fold-increase over
baseline with a statistical significance and a dose-response tendency. Thus, compound 4f was
found to be non-mutagenic against TA100 in the absence of metabolic activation, whereas it
may also be classified as a weak mutagen against TA100 in the presence of metabolic
activation (Figure 5).
MPF
Assuming the results of the Ames
assay, compound 4e was classified as non-mutagenic.
This result enhances the importance of compound 4e as a hMAO-B inhibitor. However,
compound 4f was found to be a weak mutagen, which avoids it to become a drug candidate.
2
.6. Prediction of ADME Parameters and BBB Permeability
An intrinsic pharmacological activity and low toxicological effects are not enough for a
compound to become a drug candidate. A good pharmacokinetics profile is also very
important for the new drug candidates that should be evaluated earlier in the process of drug
development. Thus, pharmacokinetics profiles of the new drug candidates should be evaluated
as early as possible in the drug development process. In recent years, combinatorial chemistry
has significantly increased the number of compounds, for which early data on absorption,
distribution, metabolism and excretion (ADME) are needed [41]. Hence, predictions of
ADME properties of the synthesized compounds (4a-4o) were performed by online
Molinspiration property program [42]. This program applies the Lipinski’s rule of five, which
evaluates the ADME properties of drug like compounds, and is important for the optimization
1
0

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of a biologically active compound. According to this rule, an orally effective drug does not
possess more than one violation [43].
The theoretical calculations of ADME parameters (molecular weight, log P, topological polar
surface area (tPSA), number of hydrogen donors (nON), number of hydrogen acceptors
(nOHNH) and molecular volume (MV)) are presented in Table 3 along with the violations of
Lipinski’s rule. According to these data, all compounds (4a-4o) follow Lipinski’s rule by
causing no more than one violation. Thus, it can be suggested that synthesized compounds
may possess a good pharmacokinetic profile, increasing their pharmacological importance.
Drug-likeness score (DLS) was calculated for compounds (4a-4o) and reference agents
selegiline and clorgiline based on the Molsoft's chemical fingerprints mode consisting of five
thousand of marketed drugs from World Drug Index (positives) and ten thousand of carefully
selected non-drug compounds (negatives) [44]. In the software, DLS was found to be between
0
and 2, suggesting good pharmacokinetics for drug candidates. In the Table 3, it is seen that
only compound 4c has a negative DLS. On the other hand, DLSs of the other compounds are
between 0.26-1.07. Furthermore, DLS of the most active compound 4e (1.02) is very close to
that of selegiline (1.07). As a result, DLS calculation confirms that synthesized compounds
may have a good pharmacokinetics.
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 (4a-4o) was calculated by a CBLigand-BBB prediction server [46]. This predictor
uses two different algorithms as AdaBoost and Support Vector Machine (SVM), combining
with four different fingerprints, employed to predict if a compound can pass (+) or cannot
1
1

ACCEPTED MANUSCRIPT
pass (-) the BBB. In each case, predictor scores higher than 0, if the compound can pass the
BBB. As presented in Table 3, all calculations for synthesized compounds resulted as BBB
(+), which is necessary for MAO inhibitors.
2
.7. Molecular Docking
As stated in MAO inhibition section, compound 4e was found to be the most active and
selective hMAO-B inhibitor. Although, compounds 4c and 4d had a structural similarity to
4
e, they displayed poorer activity than 4e. Hence, docking studies were performed in order to
gain more insight into the binding modes of compounds 4c, 4d and 4e, and to evaluate the
effects of structural modifications on the inhibitory activity against hMAO-A and hMAO-B
enzymes. In the docking studies, X-ray crystal structure of hMAO-A (PDB ID: 2BXR) [47]
and hMAO-B (PDB ID: 1S2Q) [48] were obtained from Protein Data Bank server
(www.pdb.org). The docking poses of compounds 4c, 4d and 4e on hMAO-A and the poses
of compounds 4c and 4d on hMAO-B enzyme are presented in Figures 5-7 and Figures 8, 9,
respectively. The docking poses of the most active compound 4e are presented in Figures
1
0A and 10B, including general view of 4e at the active site and the binding modes of 4e to
amino acid residues. The poses displaying van der Waals interactions and electrostatic
interactions are presented in Figures 11A and 11B.
The docking poses on hMAO-A reveals that there are two main interactions for compounds
4
c, 4d and 4e (Figures 5-7). These are π-π interaction between phenyl of the compounds and
phenyl of Tyr444, and formation of a hydrogen bond between the carbonyl of the compounds
and amino of Met445. Additionally, it is seen that nitrogen of pyridine in the compound 4e
forms a hydrogen bond with the amino group of Thr52 (Figure 7). Disappearance of this
interaction (Figures 5 and 6) in compounds 4c and 4d explains that compound 4e displays
stronger binding to hMAO-A, and thus it has higher activity against hMAO-A than
compounds 4c and 4d.
1
2

ACCEPTED MANUSCRIPT
In the poses of hMAO-B docking studies, there is a π-π interaction between phenyl of Tyr398
and phenyl of compounds 4c and 4d (Figures 8 and 9). The amino group of hydrazone moiety
in 4c forms a hydrogen bond with carbonyl of Gly434. The benzimidazole of 4d displays
cation-π interaction with the phenyl of Tyr435. The carbonyl of 4d creates a hydrogen bond
with the amino group of Ser59. The compound 4e snugly binds to amino acid residues lining
the cavity, and is located very near the FAD cofactor (Figure 10B). The benzimidazole ring
of this compound interacts with phenyl of Tyr435 by a π-π interaction in the aromatic cage of
the active site. There is a hydrogen bond between the carbonyl of compound 4e and the amino
group of Ile14. In addition, two neighboring nitrogens in the guanidine moiety of Arg42
establish two different hydrogen bonds with the imino group of hydrazone and nitrogen of
pyridine (Figure 10B). These interactions support the approach, which reveals that amino
acid side chains, coating the cavity, are very favorable to interact with the amine moieties [19-
2
3].
As stated above, comparing with compounds 4c and 4d, there are more interactions between
compound 4e and the amino acid residues. Particularly, it may be suggested that pyridine ring
in the compound 4e is very important in terms of high hMAO-B inhibition because the main
difference between compound 4e and other derivatives is the presence of this ring.
Compounds 4c and 4d carry pyrrole instead of pyridine in 4e. At first sight, it can be thought
that nitrogens of pyrrole and pyridine can form similar interactions with the amino acid
residues. However, it is well known that characters of pyrrole and pyridine nitrogens are quite
different [49]. The lone pair of nitrogen in pyrrole is in resonance and cannot be donated,
whereas the lone pair of nitrogen in pyridine is localized and ready for donation. Thus,
pyridine nitrogen acts as hydrogen acceptor and can interact with donor sites of amino acid
residues in an enzyme active site. On the other hand, pyrrole nitrogen acts as hydrogen donor
and can interact with acceptor sites of amino acid residues. This difference influences binding
1
3

ACCEPTED MANUSCRIPT
capability of the compounds 4c, 4d and 4e to enzyme active site and enzyme inhibition
potency.
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-B were evaluated for compound 4e (Figure 11A and 11B). Compound 4e has
favorable van der waals interactions, displayed with pink and red colors as described in the
user guide of Glide [50] (Figure 11A). Particularly, the propargyl and carbonyl moieties of
compound 4e are in the strongest van der waals interactions between Gly58 and Arg42,
respectively. Pyridine and carbonyl moieties of 4e are also in an electrostatic interaction
between Arg42 and Arg36, respectively (Figure 11B).
3
. Conclusion
Synthesis of novel compounds including the significant pharmacophore groups of existing
drugs is an important approach in the development of novel agents. Based on this strategy, in
the present study, 15 new benzimidazole compounds that incorporate hydrazone and
propargyl pharmacophores were tested againts MAO isoforms. Pharmacological studies
revealed the potency of the compound 4e as hMAO-B inhibitor. Toxicological and ADME
studies enhanced the biological importance of this compound. The docking studies clearly
explained the molecular interactions between the compound 4e and hMAO-B. Consequently,
all this information may create an influence on medicinal chemists to synthesize more potent
and safer hMAO-B inhibitors, which may be beneficial for the treatment of patients with
Parkinson and Alzheimer’s diseases.
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
1
4

ACCEPTED MANUSCRIPT
System and were uncorrected. IR spectra was recorded on an IR Affinity-1S Infrared
1
13
spectrophotometer (Shimadzu, Tokyo, Japan). H NMR and C 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
4
.1.1. General procedure for the synthesis of the compounds
.1.1.1. Methyl 4-(1H-benzo[d]imidazol-2-yl)benzoate (1)
Methyl-4-formylbenzoate (2) (4.8 g, 0.03 mol), sodium disulfite (5.7 g, 0.03 mol) and DMF
(5 mL) were added into a vial (30 mL) of microwave synthesis reactor (Anton-Paar,
o
Monowave 300, Austria). The reaction mixture, was heated under conditions of 240 C and 10
bar for 5 min. The mixture was cooled down, 1,2-phenylenediamine (3.24 g, 0.03 mol) was
added and then the final mixture was kept under the same reaction conditions in the
microwave reactor. After cooling, the mixture was poured into the iced-water, precipitated
product was washed with water, dried, and recrystallized from ethanol.
4
.1.1.2. Methyl 4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)benzoate (2)
Methyl 4-(1H-benzo[d]imidazol-2-yl)benzoate (1) (5.04 g, 0.02 mol) was dissolved in DMF
10 mL) and sodium hydride (0.576g, 0.024 mol) was added. The mixture was shaken until
(
hydrogen output completed. Propargyl bromide (2.596 g, 0.11 mol) was added and then the
mixture was stirred for 20 h at room temperature. The reaction content was poured into the
iced-water, precipitated product was washed with water, filtered, dried, and recrystallized
from EtOH.
4
.1.1.3. 4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)benzohydrazide (3)
Methyl 4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)benzoate (2) (2.90 g, 0.01 mol) in
ethanol (15 mL) and excess of hydrazine hydrate (3 mL) were put into a vial (30 mL) of
1
5

ACCEPTED MANUSCRIPT
microwave synthesis reactor (Anton-Paar Monowave 300, Austria). The reaction mixture was
o
kept under the conditions of 150 C and 10 bar for 10 min. After cooling, the mixture was
poured into the iced-water, precipitated product was washed with water, dried, and
recrystallized from ethanol.
4
.1.1.4.
benzo[d]imidazol-2-yl)benzohydrazide (4a-4o)
-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)benzohydrazide (0.29 g, 0.001 mol)
General
procedure
for
the
N’-(arylidene)-4-(1-(prop-2-yn-1-yl)-1H-
4
appropriate aldehyde (0.01 mol) and catalytic amount of glacial acetic acid were refluxed in
ethanol for 2 h. The mixture was cooled, precipitated product was filtered, dried, and
recrystallized from ethanol.
4
.1.1.4.1.
N'-[(thiophen-2-yl)methylene]-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4a)
-1
Yield: 78 %, M.P. = 154.9 °C, FTIR (ATR, cm ): 3229 (N-H), 2122 (CC), 1647 (C=O),
1
8
7
–
62, 704. H-NMR (300 MHz, DMSO-d6): δ = 3.50 (1H, s, CCH), 5.22 (2H, s, –CH -),
2
.16 (1H, s, thiophene –CH-), 7.29-7.40 (2H, m, benzimidazole –CH-), 7.50 (1H, s, thiophene
CH-), 7.69-7.76 (3H, m, benzimidazole –CH-, thiophene –CH-), 8.02 (2H, d, J= 7.75 Hz,
disubstituted benzene –CH-), 8.11 (2H, d, J= 7.75 Hz, disubstituted benzene –CH-), 8.69 (1H,
1
3
s, imine –CH-), 11.99 (1H, s,–NH-). C-NMR (75 MHz, DMSO-d6): δ = 34.85 (–CH -),
2
7
6.75 (CCH), 78.95 (CCH), 111.42 (benzimidazole –CH-), 119.91 (benzimidazole –
CH-), 123.19 (benzimidazole –CH-), 123.69 (benzimidazole –CH-), 128.41 (imine –CH-),
28.58 (Ar-CH), 129.53 (Ar-CH), 129.63 (Ar-CH), 131.67 (Ar-CH), 133.08 (Ar-CH), 134.87
1
(Ar-CH), 135.95 (benzimidazole –CH-), 139.46 (Ar-CH), 142.86 (benzimidazole –CH-),
+
1
43.77 (Ar-CH), 151.96 (benzimidazole –CH-), 162.85 (C=O). HRMS (m/z): [M+H] calcd
for C H N OS: 385.1118; found 385.1105.
2
2
16
4
1
6

ACCEPTED MANUSCRIPT
4
.1.1.4.2. N'-[(5-nitrothiophen-2-yl)methylene)]-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-
2
-yl) benzohydrazide (4b)
-1
Yield: 82 %, M.P. = 167.8 °C, FTIR (ATR, cm ): 3283 (N-H), 2091 (CC), 1672 (C=O),
1
1
555, 1339 (NO ), 864. H-NMR (300 MHz, DMSO-d6): δ = 3.53 (1H, s, CCH), 5.23
2
(
2H, s, –CH -), 7.29-7.40 (2H, m, benzimidazole –CH-), 7.61 (1H, d, J= 4.20 Hz, thiophene
2
–
CH-), 7.72-7.76 (2H, m, benzimidazole –CH-), 8.04 (2H, d, J= 8.20 Hz, disubstituted
benzene –CH-), 8.12-8.14 (3H, m, disubstituted benzene –CH-, thiophene –CH-), 8.71 (1H, s,
1
3
imine –CH-), 12.39 (1H, s,–NH-). C-NMR (75 MHz, DMSO-d6): δ = 34.86 (–CH -),
2
7
6.78 (CCH), 78.97 (CCH), 111.44 (benzimidazole –CH-), 119.94 (benzimidazole –
CH-), 123.17 (benzimidazole –CH-), 123.69 (benzimidazole –CH-), 128.75 (imine –CH-),
29.59 (Ar-CH), 130.36 (Ar-CH), 130.99 (Ar-CH), 132.84 (Ar-CH), 133.50 (Ar-CH), 134.32
1
(Ar-CH), 136.00 (benzimidazole –CH-), 142.01 (Ar-CH), 142.90 (benzimidazole –CH-),
+
1
47.06 (Ar-CH), 151.87 (benzimidazole –CH-), 160.24 (C=O). HRMS (m/z): [M+H] calcd
for C H N O S: 430.0968; found 430.0954.
2
2
15
5
3
4
.1.1.4.3. N'-[(1H-pyrrol-2-yl)methylene]-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4c)
-
1
Yield: 80 %, M.P. = 105.4 °C, FTIR (ATR, cm ): 3377, 3221 (N-H), 2120 (CC), 1653
1
(
C=O), 860, 743. H-NMR (300 MHz, DMSO-d6): δ = 3.51 (1H, s, CCH), 5.22 (2H, s,
–
7
7
8
CH -), 6.16 (1H, s, pyrrol –CH-), 6.52 (1H, s, pyrrol –CH-), 6.94 (1H, s, pyrrol –CH-), 7.29-
2
.40 (2H, m, benzimidazole –CH-), 7.74 (2H, br. s, benzimidazole –CH-), 8.01 (2H, d, J=
.75 Hz, disubstituted benzene –CH-), 8.12 (2H, d, J= 7.75 Hz, disubstituted benzene –CH-),
13
.32 (1H, s, imine –CH-), 11.58 (1H, s,–NH-). C-NMR (75 MHz, DMSO-d6): δ = 34.85 (–
CH -), 76.75 (CCH), 78.98 (CCH), 109.79 (Ar-CH), 111.41 (benzimidazole –CH-),
2
1
13.99 (Ar-CH), 115.29 (Ar-CH), 119.90 (benzimidazole –CH-), 123.14 (benzimidazole –
CH-), 123.63 (benzimidazole –CH-), 127.47 (Ar-CH), 128.51 (Ar-CH), 129.45 (Ar-CH),
1
7

ACCEPTED MANUSCRIPT
1
1
32.83 (Ar-CH), 135.30 (Ar-CH), 135.98 (benzimidazole –CH-), 141.69 (imine –CH-),
42.91 (benzimidazole –CH-), 152.04 (benzimidazole –CH-), 162.42 (C=O). HRMS (m/z):
+
[
M+H] calcd for C H N O: 368.1506; found 368.1488.
2
2
17
5
4
.1.1.4.4.
N'-[1-methyl-1H-pyrrol-2-yl)methylene]-4-(1-(prop-2-yn-1-yl)-1H-
benzo[d]imidazol-2-yl)benzohydrazide (4d)
-1
Yield: 82 %, M.P. = 115.3 °C, FTIR (ATR, cm ): 3267 (N-H), 2120 (CC), 1668 (C=O),
1
8
5
–
8
56, 741. H-NMR (300 MHz, DMSO-d6): δ = 3.52 (1H, s, CCH), 3.90 (3H, s, N-CH3),
.22 (2H, s, –CH -), 6.13 (1H, s, pyrrol–CH-), 6.54 (1H, s, pyrrol –CH-), 7.00 (1H, s, pyrrol
2
CH-), 7.29-7.40 (2H, m, benzimidazole –CH-), 7.72-7.76 (2H, m, benzimidazole –CH-),
.02 (2H, d, J= 7.90 Hz, disubstituted benzene –CH-), 8.11 (2H, d, J= 7.90 Hz, disubstituted
1
3
benzene –CH-), 8.42 (1H, s, imine –CH-), 11.70 (1H, s,–NH-). C-NMR (75 MHz, DMSO-
d6): δ = 34.86 (–CH -), 36.40 (-CH ), 76.77 (CCH), 78.99 (CCH), 108.89 (Ar-CH),
2
3
1
11.41 (benzimidazole –CH-), 115.11 (Ar-CH), 119.90 (benzimidazole –CH-), 123.14
benzimidazole –CH-), 123.63 (benzimidazole –CH-), 127.51 (Ar-CH), 128.44 (Ar-CH),
28.63 (Ar-CH), 129.48 (Ar-CH), 132.86 (Ar-CH), 135.23 (Ar-CH), 135.98 (benzimidazole –
(
1
CH-), 141.78 (imine –CH-), 142.91 (benzimidazole –CH-), 152.03 (benzimidazole –CH-),
+
1
62.33 (C=O). HRMS (m/z): [M+H] calcd for C H N O: 382.1662; found 382.1677.
23
19
5
4
.1.1.4.5. N'-[(pyridin-3-yl)methylene]-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4e)
-1
Yield: 79 %, M.P. = 208.5 °C, FTIR (ATR, cm ): 3410 (N-H), 2106 (CC), 1651 (C=O),
1
8
53, 735. H-NMR (300 MHz, DMSO-d6): δ = 3.52 (1H, s, CCH), 5.23 (2H, s, –CH -),
2
7
.29-7.40 (2H, m, benzimidazole –CH-), 7.51 (1H, br. s, pyridine –CH-), 7.73-7.76 (2H, m,
benzimidazole –CH-), 8.04 (2H, d, J= 7.70 Hz, disubstituted benzene –CH-), 8.13-8.19 (3H,
m, disubstituted benzene –CH-, pyridine –CH-), 8.55 (1H, s, imine –CH-), 8.63 (1H, br. s,
13
pyridine –CH-), 8.90 (1H, s, pyridine –CH-), 12.19 (1H, s,–NH-). C-NMR (75 MHz,
1
8

ACCEPTED MANUSCRIPT
DMSO-d6): δ = 34.86 (–CH -), 76.78 (CCH), 78.98 (CCH), 111.43 (benzimidazole –
2
CH-), 119.83 (benzimidazole –CH-), 123.16 (benzimidazole –CH-), 123.67 (benzimidazole –
CH-), 124.52 (Ar-CH), 128.69 (Ar-CH), 129.55 (Ar-CH), 130.64 (Ar-CH), 133.26 (Ar-CH),
1
34.00 (Ar-CH), 134.70 (Ar-CH), 135.99 (benzimidazole –CH-), 142.90 (benzimidazole –
CH-), 145.91 (Ar-CH), 149.30 (imine –CH-), 151.29, 151.94 (benzimidazole –CH-), 163.09
+
(
C=O). HRMS (m/z): [M+H] calcd for C H N O: 380.1506; found 380.1484.
2
3
17
5
4
.1.1.4.6.
N'-(4-fluorobenzylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4f)
-1
Yield: 85 %, M.P. = 153.3 °C, FTIR (ATR, cm ): 3300 (N-H), 2126 (CC), 1644 (C=O),
1
8
41, 733. H-NMR (300 MHz, DMSO-d6): δ = 3.52 (1H, s, CCH), 5.23 (2H, s, –CH -),
2
7
.30-7.40 (4H, m, benzimidazole –CH-, fluorophenyl –CH-), 7.74 (2H, br. s, benzimidazole –
CH-), 7.82 (2H, br. s, fluorophenyl –CH-), 8.04 (2H, d, J= 7.65 Hz, disubstituted benzene –
CH-), 8.13 (2H, d, J= 7.65 Hz, disubstituted benzene –CH-), 8.50 (1H, s, imine –CH-), 12.04
1
3
(
1H, s,–NH-). C-NMR (75 MHz, DMSO-d6): δ = 34.86 (–CH -), 76.78 (CCH), 78.98
2
(CCH), 111.43 (benzimidazole –CH-), 116.43 (JCF-2=21.8 Hz) (Ar-CH), 119.92
(benzimidazole –CH-), 123.15 (benzimidazole –CH-), 123.66 (benzimidazole –CH-), 128.62
(Ar-CH), 129.52 (Ar-CH), 129.82 (JCF-3=8.3 Hz) (Ar-CH), 131.35 (JCF-4=3.1 Hz) (Ar-CH),
1
33.15 (Ar-CH), 134.87 (Ar-CH), 135.99 (benzimidazole –CH-), 142.91 (benzimidazole –
CH-), 147.49 (imine –CH-), 151.97 (benzimidazole –CH-), 162.96 (C=O), 163.66 (JCF-
+
1=
245.7 Hz) (Ar-CH). HRMS (m/z): [M+H] calcd for C H N OF: 397.1459; found
24
17
4
3
97.1455.
.1.1.4.7.
4
N'-(4-chlorobenzylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4g)
-1
Yield: 82 %, M.P. = 222.9 °C, FTIR (ATR, cm ): 3279 (N-H), 2124 (CC), 1643 (C=O),
1
8
37, 733. H-NMR (300 MHz, DMSO-d6): δ = 3.52 (1H, s, CCH), 5.23 (2H, s, –CH -),
2
1
9

ACCEPTED MANUSCRIPT
7
7
.29-7.40 (2H, m, benzimidazole –CH-), 7.55 (2H, d, J= 7.80 Hz, chlorophenyl –CH-), 7.74-
.80 (4H, m, benzimidazole –CH-, chlorophenyl –CH-), 8.04 (2H, d, J= 7.50 Hz,
disubstituted benzene –CH-), 8.14 (2H, d, J= 7.50 Hz, disubstituted benzene –CH-), 8.49 (1H,
1
3
s, imine –CH-), 12.09 (1H, s,–NH-). C-NMR (75 MHz, DMSO-d6): δ = 34.86 (–CH -),
2
7
6.78 (CCH), 78.98 (CCH), 111.43 (benzimidazole –CH-), 119.92 (benzimidazole –
CH-), 123.15 (benzimidazole –CH-), 123.66 (benzimidazole –CH-), 128.65 (Ar-CH), 129.27
Ar-CH), 129.46 (Ar-CH), 129.52 (Ar-CH), 133.21 (Ar-CH), 133.68 (Ar-CH), 134.80 (Ar-
(
CH), 135.08 (Ar-CH), 135.99 (benzimidazole –CH-), 142.90 (benzimidazole –CH-), 147.28
+
(
imine –CH-), 151.95 (benzimidazole –CH-), 163.01 (C=O). HRMS (m/z): [M+H] calcd for
C H N OCl: 413.1164; found 413.1145.
24
17
4
4
.1.1.4.8.
N'-(4-hydroxybenzylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4h)
-1
Yield: 83 %, M.P. = 151.8 °C, FTIR (ATR, cm ): 3385 (O-H), 3238 (N-H), 2120 (CC),
1
1
5
643 (C=O), 862, 831, 734. H-NMR (300 MHz, DMSO-d6): δ = 3.52 (1H, s, CCH),
.23 (2H, s, –CH -), 6.86 (2H, d, J= 7.85 Hz, hydroxyphenyl –CH-), 7.29-7.40 (2H, m,
2
benzimidazole –CH-), 7.59 (2H, d, J= 7.85 Hz, hydroxyphenyl –CH-), 7.74 (2H, br. s,
benzimidazole –CH-), 8.02 (2H, d, J= 7.75 Hz, disubstituted benzene –CH-), 8.12 (2H, d, J=
7
.75 Hz, disubstituted benzene –CH-), 8.39 (1H, s, imine –CH-), 9.96 (1H, s, -OH), 11.82
1
3
(
1H, s,–NH-). C-NMR (75 MHz, DMSO-d6): δ = 34.86 (–CH -), 76.78 (CCH), 78.99
2
(CCH), 111.42 (benzimidazole –CH-), 116.22 (Ar-CH), 119.91 (benzimidazole –CH-),
1
23.14 (benzimidazole –CH-), 123.64 (benzimidazole –CH-), 125.67 (Ar-CH), 128.54 (Ar-
CH), 129.43 (Ar-CH), 129.48 (Ar-CH), 132.95 (Ar-CH), 135.15 (Ar-CH), 135.98
(benzimidazole –CH-), 142.90 (benzimidazole –CH-), 148.96 (imine –CH-), 152.01
+
(
benzimidazole –CH-), 160.02 (Ar-CH), 162.64 (C=O). HRMS (m/z): [M+H] calcd for
C H N O : 395.1503; found 395.1487.
24
18
4
2
2
0

ACCEPTED MANUSCRIPT
4
.1.1.4.9.
N'-(4-methoxybenzylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-
yl)benzohydrazide (4i)
-1
Yield: 89 %, M.P. = 201.0 °C, FTIR (ATR, cm ): 3256 (N-H), 2124 (CC), 1647 (C=O),
1
8
5
58, 837. H-NMR (300 MHz, DMSO-d6): δ = 3.52 (1H, s, CCH), 3.82 (3H, s, -OCH3),
.23 (2H, s, –CH -), 7.04 (2H, d, J= 8.00 Hz, methoxyphenyl –CH-), 7.29-7.40 (2H, m,
2
benzimidazole –CH-), 7.69-7.80 (4H, m, benzimidazole –CH-, methoxyphenyl –CH-), 8.02
2H, d, J= 7.90 Hz, disubstituted benzene –CH-), 8.14 (2H, d, J= 7.90 Hz, disubstituted
(
1
3
benzene –CH-), 8.46 (1H, s, imine –CH-), 12.02 (1H, s,–NH-). C-NMR (75 MHz, DMSO-
d6): δ = 34.86 (–CH -), 55.80 (-OCH ), 76.78 (CCH), 78.99 (CCH), 111.42
2
3
(
benzimidazole –CH-), 112.80 (Ar-CH), 114.83 (Ar-CH), 119.91 (benzimidazole –CH-),
23.14 (benzimidazole –CH-), 123.64 (benzimidazole –CH-), 127.31 (Ar-CH), 128.60 (Ar-
CH), 129.25 (Ar-CH), 129.47 (Ar-CH), 135.09 (Ar-CH), 135.98 (benzimidazole –CH-),
1
1
42.91 (benzimidazole –CH-), 148.59 (imine –CH-), 152.01 (benzimidazole –CH-), 161.36
+
(
Ar-CH), 162.79 (C=O). HRMS (m/z): [M+H] calcd for C H N O : 409.1659; found
2
5
20
4
2
4
4
09.1648.
.1.1.4.10.
N'-(2-methoxybenzylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4j)
-1
Yield: 81 %, M.P. = 127.7 °C, FTIR (ATR, cm ): 3275 (N-H), 2120 (CC), 1672 (C=O),
1
8
5
46, 746. H-NMR (300 MHz, DMSO-d6): δ = 3.53 (1H, s, CCH), 3.88 (3H, s, -OCH3),
.23 (2H, s, –CH -), 7.02-7.13 (2H, m, methoxyphenyl –CH-), 7.29-7.46 (3H, m,
2
benzimidazole –CH-, methoxyphenyl –CH-), 7.73-7.77 (2H, m, benzimidazole –CH-), 7.92
1H, d, J= 7.50 Hz, methoxyphenyl –CH-), 8.03 (2H, d, J= 7.80 Hz, disubstituted benzene –
CH-), 8.16 (2H, d, J= 7.60 Hz, disubstituted benzene –CH-), 8.87 (1H, s, imine –CH-), 12.03
(
1
3
(
1H, s,–NH-). C-NMR (75 MHz, DMSO-d6): δ = 34.87 (–CH -), 56.18 (-OCH ),
2 3
7
6.78(CCH), 78.98 (CCH), 111.42 (benzimidazole –CH-), 112.34 (Ar-CH), 119.92
2
1

ACCEPTED MANUSCRIPT
(benzimidazole –CH-), 121.25 (Ar-CH), 122.71 (Ar-CH), 123.15 (benzimidazole –CH-),
1
23.65 (benzimidazole –CH-), 126.02 (Ar-CH), 128.62 (Ar-CH), 129.49 (Ar-CH), 132.17
(Ar-CH), 133.08 (Ar-CH), 134.89 (Ar-CH), 135.98 (benzimidazole –CH-), 142.92
(benzimidazole –CH-), 144.12 (imine –CH-), 152.00 (benzimidazole –CH-), 158.29 (Ar-CH),
+
1
62.75 (C=O). HRMS (m/z): [M+H] calcd for C H N O : 409.1659; found 409.1652.
25
20
4
2
4
.1.1.4.11.
N'-(3-methoxybenzylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4k)
-1
Yield: 80 %, M.P. = 224.7 °C, FTIR (ATR, cm ): 3213 (N-H), 2120 (CC), 1667 (C=O),
1
8
5
60, 744. H-NMR (300 MHz, DMSO-d6): δ = 3.53 (1H, s, CCH), 3.83 (3H, s, -OCH3),
.23 (2H, s, –CH -), 7.03 (1H, d, J= 7.60 Hz, methoxyphenyl –CH-), 7.32-7.40 (5H, m,
2
benzimidazole –CH-, methoxyphenyl –CH-), 7.73 (2H, br. s, benzimidazole –CH-), 8.04 (2H,
d, J= 7.60 Hz, disubstituted benzene –CH-), 8.14 (2H, d, J= 7.60 Hz, disubstituted benzene –
1
3
CH-), 8.48 (1H, s, imine –CH-), 12.04 (1H, s,–NH-). C-NMR (75 MHz, DMSO-d6): δ =
3
4.86 (–CH -), 55.68 (-OCH ), 76.79 (CCH), 78.98 (CCH), 111.42 (benzimidazole –
2
3
CH-), 111.71 (Ar-CH), 116.83 (Ar-CH), 119.93 (benzimidazole –CH-), 120.62 (Ar-CH),
23.15 (benzimidazole –CH-), 123.66 (benzimidazole –CH-), 128.64 (Ar-CH), 129.52 (Ar-
CH), 130.47 (Ar-CH), 133.16 (Ar-CH), 134.90 (Ar-CH), 135.99 (benzimidazole –CH-),
1
1
–
4
4
36.16 (Ar-CH), 142.92 (benzimidazole –CH-), 148.55 (imine –CH-), 151.97 (benzimidazole
+
CH-), 160.04 (Ar-CH), 162.99 (C=O). HRMS (m/z): [M+H] calcd for C H N O :
25
20
4
2
09.1659; found 409.1649.
.1.1.4.12. N'-(3-hydroxybenzylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4l)
-1
Yield: 85 %, M.P. = 137.2 °C, FTIR (ATR, cm ): 3244 (N-H), 3192 (O-H), 2126 (CC),
1
1
5
641 (C=O), 860, 769, 738. H-NMR (300 MHz, DMSO-d6): δ = 3.52 (1H, s, CCH),
.23 (2H, s, –CH -), 6.86 (1H, d, J= 7.85 Hz, hydroxyphenyl –CH-), 6.96 (1H, d, J= 7.85
2
2
2

ACCEPTED MANUSCRIPT
Hz, hydroxyphenyl –CH-), 7.27-7.40 (4H, m, benzimidazole –CH-, hydroxyphenyl –CH-),
7
.74 (2H, br. s, benzimidazole –CH-), 8.02 (2H, d, J= 7.80 Hz, disubstituted benzene –CH-),
.12 (2H, d, J= 7.75 Hz, disubstituted benzene –CH-), 8.30 (1H, s, imine –CH-), 11.77 (1H,
8
1
3
s,–NH-). C-NMR (75 MHz, DMSO-d6): δ = 34.86 (–CH -), 76.78 (CCH), 78.97
2
(
CCH), 111.42 (benzimidazole –CH-), 113.16 (Ar-CH), 118.04 (Ar-CH), 119.10 (Ar-CH),
19.37 (Ar-CH), 119.92 (benzimidazole –CH-), 123.16 (benzimidazole –CH-), 123.67
benzimidazole –CH-), 128.63 (Ar-CH), 129.52 (Ar-CH), 130.39 (Ar-CH), 133.11 (Ar-CH),
1
(
1
–
34.93 (Ar-CH), 135.98 (benzimidazole –CH-), 142.90 (benzimidazole –CH-), 148.76 (imine
+
CH-), 151.98 (benzimidazole –CH-), 158.18 (Ar-CH), 162.93 (C=O). HRMS (m/z): [M+H]
calcd for C H N O : 395.1503; found 395.1509.
2
4
18
4
2
4
.1.1.4.13. N'-(3,5-dihydroxybenzylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4m)
-1
Yield: 86 %, M.P. = 163.2 °C, FTIR (ATR, cm ): 3275 (N-H), 3202 (O-H), 2126 (CC),
1
1
5
649 (C=O), 856, 769, 740. H-NMR (300 MHz, DMSO-d6): δ = 3.52 (1H, s, CCH),
.22 (2H, s, –CH -), 6.80 (1H, d, J= 7.85 Hz, dihydroxyphenyl –CH-), 6.96 (1H, d, J= 7.85
2
Hz, dihydroxyphenyl –CH-), 7.27-7.40 (3H, m, benzimidazole –CH-, dihydroxyphenyl –CH-
)
, 7.74 (2H, br. s, benzimidazole –CH-), 8.02 (2H, d, J= 7.80 Hz, disubstituted benzene –CH-
, 8.12 (2H, d, J= 7.75 Hz, disubstituted benzene –CH-), 8.30 (1H, s, imine –CH-), 11.77 (1H,
)
1
3
s,–NH-). C-NMR (75 MHz, DMSO-d6): δ = 34.86 (–CH -), 76.76 (CCH), 78.99
2
(CCH), 111.42 (benzimidazole –CH-), 113.18 (Ar-CH), 116.06 (Ar-CH), 119.91
(benzimidazole –CH-), 121.19 (Ar-CH), 123.14 (benzimidazole –CH-), 123.64
(benzimidazole –CH-), 126.13 (Ar-CH), 128.53 (Ar-CH), 129.47 (Ar-CH), 132.93 (Ar-CH),
1
–
35.17 (Ar-CH), 135.98 (benzimidazole –CH-), 142.90 (benzimidazole –CH-), 147.18 (imine
+
CH-), 152.04 (benzimidazole –CH-), 162.59 (C=O). HRMS (m/z): [M+H] calcd for
C H N O : 411.1452; found 411.1434.
24
18
4
3
2
3

ACCEPTED MANUSCRIPT
4
.1.1.4.14. N'-(3,5-dimethoxybenzylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4n)
-1
Yield: 86 %, M.P. = 124.5 °C, FTIR (ATR, cm ): 3219 (N-H), 2118 (CC), 1653 (C=O),
1
7
3
7
62, 745. H-NMR (300 MHz, DMSO-d6): δ = 3.52 (1H, s, CCH), 3.82 (3H, s, -OCH3),
.84 (3H, s, -OCH ), 5.23 (2H, s, –CH -), 7.03-7.06 (1H, m, dimethoxyphenyl –CH-), 7.22-
3
2
.25 (1H, m, dimethoxyphenyl –CH-), 7.29-7.40 (2H, m, benzimidazole –CH-), 7.61 (1H, s,
dimethoxyphenyl –CH-), 7.74 (2H, br. s, benzimidazole –CH-), 8.04 (2H, d, J= 7.45 Hz,
disubstituted benzene –CH-), 8.13 (2H, d, J= 7.45 Hz, disubstituted benzene –CH-), 8.70 (1H,
1
3
s, imine –CH-), 12.37 (1H, s,–NH-). C-NMR (75 MHz, DMSO-d6): δ = 34.85 (–CH -),
2
5
5.95 (-OCH ), 56.04 (-OCH ), 76.77 (CCH), 78.99 (CCH), 108.71 (Ar-CH), 111.42
3
3
(
benzimidazole –CH-), 111.95 (Ar-CH), 119.91 (benzimidazole –CH-), 122.49 (Ar-CH),
23.15 (benzimidazole –CH-), 123.65 (benzimidazole –CH-), 127.41 (Ar-CH), 128.57 (Ar-
CH), 129.50 (Ar-CH), 133.02 (Ar-CH), 135.08 (Ar-CH), 135.98 (benzimidazole –CH-),
1
1
1
4
4
42.91 (benzimidazole –CH-), 148.85 (imine –CH-), 149.56 (Ar-CH), 151.32 (Ar-CH),
+
52.00 (benzimidazole –CH-), 162.79 (C=O). HRMS (m/z): [M+H] calcd for C H N O :
2
6
22
4
3
39.1765; found 439.1753.
.1.1.4.15. N'-(2,3-dimethoxybenzylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)
benzohydrazide (4o)
-1
Yield: 76 %, M.P. = 99.3 °C, FTIR (ATR, cm ): 3219 (N-H), 2120 (CC), 1643 (C=O),
1
7
92, 763. H-NMR (300 MHz, DMSO-d6): δ = 3.52 (1H, s, CCH), 3.82 (3H, s, -OCH3),
3
.85 (3H, s, -OCH ), 5.23 (2H, s, –CH -), 7.14 (2H, br. s, dimethoxyphenyl –CH-), 7.29-7.40
3
2
(2H, m, benzimidazole –CH-), 7.50-7.52 (1H, m, dimethoxyphenyl –CH-), 7.72-7.77 (2H, m,
benzimidazole –CH-), 8.04 (2H, d, J= 7.95 Hz, disubstituted benzene –CH-), 8.16 (2H, d, J=
1
3
7
.95 Hz, disubstituted benzene –CH-), 8.79 (1H, s, imine –CH-), 12.06 (1H, s,–NH-). C-
NMR (75 MHz, DMSO-d6): δ = 34.86 (–CH -), 56.25 (-OCH ), 61.73 (-OCH ), 76.77
2
3
3
2
4

ACCEPTED MANUSCRIPT
(CCH), 78.98 (CCH), 111.42 (benzimidazole –CH-), 114.82 (Ar-CH), 117.50 (Ar-CH),
1
1
19.92 (benzimidazole –CH-), 123.16 (benzimidazole –CH-), 123.67 (benzimidazole –CH-),
24.90 (Ar-CH), 128.16 (Ar-CH), 128.63 (Ar-CH), 129.50 (Ar-CH), 133.13 (Ar-CH), 134.84
(Ar-CH), 135.98 (benzimidazole –CH-), 142.90 (benzimidazole –CH-), 144.26 (Ar-CH),
1
48.55 (imine –CH-), 151.98 (benzimidazole –CH-), 153.16 (Ar-CH), 162.83 (C=O). HRMS
+
(
m/z): [M+H] calcd for C H N O : 439.1765; found 439.1749.
26
22
4
3
4
.2. MAO-A and MAO-B Inhibition Assay
Ampliflu™ Red (10-Acetyl-3,7-dihydroxyphenoxazine), peroxidase from horseradish,
hMAO-A, hMAO-B, H O , tyramine hydrochloride, selegiline, rasagiline and clorgiline were
2
2
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.
In the enzymatic assay, three different daily prepared solutions were used. I) Inhibitor
-3
solutions: Synthesized compounds and reference agents were prepared in 2% DMSO in 10 -
-9
1
0 M concentrations (10 mL for each concentration). II) Enzyme solutions: Recombinant
hMAO-A (0.5 U/mL) and recombinant hMAO-B (0.64 U/mL) enzymes were dissolved in the
phosphate buffer and final volumes were adjusted to 10 mL. III) Working solution:
Horseradish peroxidase (200 U/mL, 100 µL), Ampliflu™ Red (20 mM, 200 µL) and tyramine
(
100 mM, 200 µL) were dissolved in the phosphate buffer and final volume was adjusted to
0 mL.
The solutions of inhibitor (20 ꢁL/well) and hMAO-A (100 ꢁL/well) or hMAO-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 incubation period, the reaction was started by adding a working solution
1
ꢁ
2
5

ACCEPTED MANUSCRIPT
(100 ꢁL/well). The mixture was incubated at 37°C for 30 min and the fluorescence (Ex/Em =
5
35/587 nm) was measured at 5 min intervals. Control experiments were carried out
simultaneously by replacing the inhibitor solution with 2% DMSO (20 µL). To check the
probable inhibitory effect of inhibitors on horseradish peroxidase, a parallel reading was
performed by replacing enzyme solutions with %3 H O solution (20 mM 100 ꢁL/well). In
2
2
addition, the possible capacity of the inhibitors to modify the fluorescence generated in the
reaction mixture due to non-enzymatic inhibition was determined by mixing inhibitor and
working solutions.
The specific fluorescence emission (used to obtain the final results) was calculated after
subtraction of the background activity, which was determined from vials containing all
components except the hMAO isoforms, which were replaced by phosphate buffer (100
ꢁ
L/well). Blank, control and all concentrations of inhibitors were analyzed in quadruplicate
and inhibition percent was calculated by using following equation:
(
ꢁꢂꢃꢄꢀꢅꢀꢁꢂꢃꢆ)ꢀꢅꢀ(ꢁꢇꢀꢃꢄꢅꢀꢁꢇꢃꢆ)
%
ꢀInhibition =
ꢀꢈꢀ100
ꢁ
ꢂꢃꢄꢀꢅꢀꢁꢂꢃꢆ
FCt : Fluorescence of a control well measured at t time, FCt : Fluorescence of a control well
2
2
1
measured at t time, FIt : Fluorescence of an inhibitor well measured at t time, FIt :
2
2
2
1
Fluorescence of an inhibitor well measured at t time,
1
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 IC (hMAO-A)/ IC (hMAO-B).
5
0
50
4
.3. Enzyme Kinetic Studies
The same materials were used in the MAO inhibition assay. The most active compound 4e
was tested at five different concentrations (IC /4, IC /2, IC , 2 x IC and 4 x IC /4). The
5
0
50
50
50
50
2
6

ACCEPTED MANUSCRIPT
solutions of inhibitor (20 ꢁL/well) and enzyme were added to the flat black bottom 96-well
micro test plate, and incubated at 37°C for 30 min. After incubation period, the working
solution, including various concentrations (20, 10, 5, 2.5, 1.25, and 0.625µM) of tyramine
(100 ꢁL/well) was added. The increase of the fluorescence (Ex/Em = 535/587 nm) was
recorded for 30 min. A parallel experiment was carried out without inhibitor. 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 K values were determined from the x-axis
i
intercept as -Ki.
4
.4. Cytotoxicity Assay
Cytotoxicity of compounds 4e and 4f was tested using the NIH/3T3 mouse embryonic
fibroblast cell line (ATCC® CRL-1658™, London, UK). NIH/3T3 cells were incubated
4
according to the supplier’s recommendations. NIH/3T3 cells were seeded at 1x10 cells into
each well of 96-well plates. MTT assay was performed as previously described [51, 52]. The
compounds were tested between 0.0316-1000 µM concentrations. The IC50 values were
determined by plotting a dose-response curve of inhibition % versus compound
concentrations tested [53].
4
.5. Genotoxicity Assay
The genotoxicity of the most effective compounds (4e and 4f) was determined by Ames assay
MPF
using Ames
98/100 mutagenicity assay sample kit (Xenometrix AG, Switzerland) as
previously described [54]. Salmonella typhimurium strains, TA98 (frameshift mutations) and
TA100 (base-pair substitutions), are used in this assay. Concentration range of the compounds
was between 16 and 5000 µg/mL according to the previous guidelines [40]. Compounds were
prepared in six different concentrations (5, 2.5, 1.25, 0.625, 0.3125, 0.156 mg/mL) in DMSO.
Mutagenic potential was determined in absence and presence of Aroclor™-1254 induced
2
7

ACCEPTED MANUSCRIPT
male Sprague–Dawley rat liver microsomal enzyme (S9) mix (Xenometrix AG, Switzerland).
Positive controls without S9 mix were 2-Nitrofluorene (2 ꢁg/mL) and 4-nitroquinoline N-
oxide (0.1 ꢁg/mL) whereas 1 ꢁg/mL and 2.5 ꢁg/mL of 2-aminoanthracene solutions were
used as positive controls with S9 against TA 98 and TA100, respectively. Solvent control was
prepared with 4% DMSO. At the end of the experiment, revertant bacteria drops 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 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. Mutagenicity was determined
according to the criteria from previous studies [55]. For a baseline value ≤ 3, significant
increases between 2 and 3-fold the baseline were classified as weak mutagen, and 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. As a rule, at least two adjacent doses with
significant increases or a significant increase at the highest dose level should be observed for
a mutagenic compound. All doses were compared according to Student’s t-test at p < 0.05 for
statistical significance. The compounds were classified as a non-mutagenic if they did not
have any of the properties mentioned above.
2
8