Selected aryl thiosemicarbazones as a new class of multi-targeted monoamine oxidase inhibitors

Article abstract of DOI:10.1039/c8md00399h

A series of 13 phenyl substituted thiosemicarbazones (SB1-SB13) were synthesized and evaluated for their inhibitory potential towards human recombinant monoamine oxidase A and B (MAO-A and MAO-B, respectively) and acetylcholinesterase. The solid state structure of SB4 was ascertained by the single X-ray diffraction technique. Compounds SB5 and SB11 were potent for MAO-A (IC₅₀ 1.82 ± 0.14) and MAO-B (IC₅₀ 0.27 ± 0.015 μM), respectively. Furthermore, SB11 showed a high selectivity index (SI > 37.0) for MAO-B. The effects of fluorine orientation revealed that SB11 (m-fluorine) showed 28.2 times higher inhibitory activity than SB12 (o-fluorine) against MAO-B. Furthermore, inhibitions by SB5 and SB11 against MAO-A and MAO-B, respectively, were recovered to near reference levels in reversibility experiments. Both SB5 and SB11 showed competitive inhibition modes, with K_i values of 0.97 ± 0.042 and 0.12 ± 0.006 μM, respectively. These results indicate that SB5 and SB11 are selective, reversible and competitive inhibitors of MAO-A and MAO-B, respectively. Compounds SB5, SB7 and SB11 showed moderate inhibition against acetylcholinesterase with IC₅₀ values of 35.35 ± 0.47, 15.61 ± 0.057 and 26.61 ± 0.338 μM, respectively. Blood-brain barrier (BBB) permeation was studied using the parallel artificial membrane permeation assay (PAMPA) method. Molecular docking studies were carried out using AutoDock 4.2.

Full text of DOI:10.1039/c8md00399h

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Cheol Baek, D. Grace Thomas Parambi, J. Pil Lee, M. Joy, A. Rilda, R. Ranadev, P. nithymol, V. Vijayan, S. t.
inasu, G. E. Mathew, K. K. Lohidhakshan, G. Kumar Krishnan and H. kim, Med. Chem. Commun., 2018,
DOI: 10.1039/C8MD00399H.
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Selected aryl thiosemicarbazones as new class of multiꢀtargeted monoamine oxidase
inhibitors
Bijo Mathew1*#, Seung Cheol Baek2#, Della Grace Thomas Parambi3, Jae Pil Lee2, Monu
Joy4, P.R. Annie Rilda1, Rugma V. Randev1, P. Nithymol1, Vijitha Vijayan,1 Sini. T. Inasu1,
Githa Elizabeth Mathew5, Krishnakumar K. Lohidakshan6, Girish Kumar Krishnan7, Hoon
Kim2*
1
Division of Drug Design and Medicinal Chemistry Research Lab, Department of
Pharmaceutical Chemistry, Ahalia School of Pharmacy, Palakkadꢀ678557, Kerala, India
2 Department of Pharmacy and Research Institute of Life Pharmaceutical Sciences, Sunchon
National University, Suncheonꢀ57922, Republic of Korea.
3 Department of Pharmaceutical Chemistry, Jouf University, Sakaka, Al Joufꢀ2014, Saudi
Arabia.
4 School of Pure & Applied Physics, M.G. University, Kottayam, Kerala, India.
5 Department of Pharmacology, Grace College of Pharmacy, Palakkad, India.
6Department of Pharmaceutical Chemistry, Nirmala college of Health Science, Chalakudyꢀ
680311, India
7 Department of Pharmaceutical Chemistry, College of Pharmaceutical Sciences, Government
Medical College Trivandrum, India
#Authors contributed equally.
*Corresponding Author: Bijo Mathew (B. Mathew) (bijovilaventgu@gmail.com)
Hoon Kim (H. Kim) (hoon@sunchon.ac.kr)

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Abstract
A series of 13 phenyl substituted thiosemicarbazones (SB1ꢀSB13) were synthesized and
evaluated for their inhibitory potential towards human recombinant monoamine oxidase A
and B (MAOꢀA and MAOꢀB, respectively) and acetylcholinesterase. The solid state structure
of SB4 was ascertained by single Xꢀray diffraction technique. Compounds SB5 and SB11
were potent for MAOꢀA (IC50 1.82 ± 0.14) and MAOꢀB (IC50 0.27 ± 0.015 µM), respectively.
Furthermore, SB11 showed high selectivity index (SI > 37.0) for MAOꢀB. The effects of
fluorine orientation revealed that SB11 (mꢀfluorine) showed 28.2 times higher inhibitory
activity than SB12 (oꢀfluorine) against MAOꢀB. Furthermore, inhibitions by SB5 and SB11
against MAOꢀA and MAOꢀB, respectively, were recovered to near reference levels in
reversibility experiments. Both SB5 and SB11 showed competitive inhibition modes, with Ki
values of 0.97 ± 0.042 and 0.12 ± 0.006 µM, respectively. These results indicate that SB5 and
SB11 are selective, reversible and competitive inhibitors of MAOꢀA and MAOꢀB,
respectively. Compounds SB5, SB7 and SB11 show moderate inhibition against acetylcholine
sterase with IC50 values of 35.35 ± 0.47, 15.61 ± 0.057 and 26.61 ± 0.338 µM, respectively.
Bloodꢀbrain barrier (BBB) permeation was studied using parallel artificial membrane
permeation assay (PAMPA) method. Molecular docking studies were carried out by
AutoDock4.2.
Keywords: Thiosemicarbazones, Monoamine oxidase, Acetylcholinesterase, Selective
reversible inhibitor, Molecular docking, PAMPAꢀBBB.

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Introduction
The role of monoamine oxidases (MAOs) in brain neurochemistry is mainly connected with
the oxidative deamination of biogenic amines (BA) [1]. The regulation of amines such as
adrenaline, norꢀadrenaline, melatonin and serotonin are predominantly metabolized by MAOꢀ
A, whereas benzylamine and phenylethylamine are controlled by MAOꢀB. Both types of
MAO isoforms have common substrates such as dopamine and tyramine [2, 3]. The selective
types of MAO inhibitors (MAOIs) have a great impact in treating various psychiatric and
neurodegenerative disorders by depleting MAO levels in the brain [4]. Serotonin
concentration maintained by the inhibitors of MAOꢀA shows superior antidepressant activity
[5]. Conversely, the endꢀproducts, hydrogen peroxide and reactive oxygen species (ROS),
produced during dopamine metabolism by MAOꢀB, generate oxidative stress and apoptosis in
dopamine producing cells. These highly reactive toxic radicals produce neural toxicity which
may be the prime indications for Alzheimer’s and Parkinson’s diseases (AD & PD) [6].
Hence, selective MAOꢀB inhibitors are highly recommended as coꢀadjuvant therapy for
treating AD and PD patients [7, 8].
The current scenario of PD therapy focuses on restoring the level of dopamine in the
brain and thereby curtailing the motor symptoms [9]. This therapy is accelerated by the
administration of dopamine precursors (Lꢀdopa), dopamine agonists, catecholꢀOꢀ
methyltransferase (COMT) and MAOꢀB inhibitors such as selegiline and rasagiline (highly
selective and irreversible) [10]. The molecules which have a closer acetylcholinesterase
(AChE) and MAOꢀB affinities are able to limit the neurotoxicity related with sources of ROS
in age related AD diseases [11]. In 2011, the molecule ladostigil, a dual inhibitor of both
AChE and MAOꢀB designed by Youdim, entered into the phase II clinical trial for the
treatment of AD [12]. Considering the complex pathogenetic factors of various

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neurodegenerative diseases, it is highly appropriate to recommend selective MAOꢀB
inhibitors with cocktail therapy to trigger the multiꢀtargets associated with the diseases.
The MAOꢀB inhibitors currently in use are the irreversible type, having a covalent
bond to the FAD unit of the inhibitor binding cavity (IBC) of the enzyme [13]. Disruption of
target, poor ADME profile and increased duration of action occur due to this irreversible
binding. Hence, the development of reversible MAOꢀB inhibitors has a greater therapeutic
value in treating neurodegenerative diseases [14]. Some of the evidence also document that
mild symptomatic benefits were gained by the administration of moclobemide (a reversible
MAOꢀA inhibitor) when combined with levodopa for the treatment of PD [15, 16]. Recently,
many small molecules like chalcones, coumarins and chromones have a considerable
potential for the development of MAOꢀA/MAOꢀB inhibitors with a highly selective and
reversible mode of inhibition [17ꢀ
From a chemical point of view, thiosemicarbazones are thiourea linked with an
azomethine scaffold, and are the key intermediates for the synthesis of 2ꢀaminoꢀ1,3,4ꢀ
thiadiazoles via
a ring chain tautomerism mechanism [20, 21]. Besides this,
thiosemicarbazones are excellent chelators of transition metals such as zinc (Zn), copper (Cu)
and iron (Fe), which are potent inhibitors of various carcinogenesis inducing pathways [22].
In the past, many efforts have addressed the development of thiosemicarbazones based
MAOIs [23ꢀ29]. Moreover, the cyclized form of thiosemicarbazones from chalcones afforded
Nꢀthiocarbamoyl pyrazolines, which show a remarkable inhibition profile against MAOs [30ꢀ
34]. The presence of hydrazine units in thiosemicarbazide also afforded a number of
hydrazone scaffolds via the acid catalyzed nucleophilic addition mechanism [35]. Numerous
studies recommend the multiꢀpotent MAO and cholinesterase inhibitors for treating AD and
PD [36, 37]. Accordingly, this work describes the synthesis of phenyl substituted

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thiosemicarbazones, the MAO and acetylcholinesterase inhibition studies, and kinetics of the
inhibition mechanism of MAOs using the LineweaverꢀBurk plot, reversibility mode, and
blood ꢀ brain barrier (BBB) permeation assays. Finally, the lead molecules from the in vitro
results were subjected to molecular docking studies to elucidate the binding interactions of
both MAOꢀA and B.
Result and discussion
Chemistry
The target aryl thiosemicarbazones were synthesized as presented in Scheme 1. Synthesis
was accomplished by the single step reaction between commercially available
1
thiosemicarbazide hydrochloride and various substituted benzaldehydes. In the HꢀNMR
spectra, a sharp singlet peak observed between 7.71ꢀ7.89 is ascribed to the azomethine (ꢀ
CH=Nꢀ) proton. The down field of proton of the NH group attached to the thiocarbamoyl unit
is observed in the range between 9.18ꢀ9.88. Two broad singlets were found at 6.23ꢀ7.25 and
7.23ꢀ7.26, corresponding to the terminal NH2 group. 13C NMR spectra displayed
carbothioamide groups for SB1–SB13 between δ180.60–184.30. All spectral
characterizations are in full agreement with previous literatures [38ꢀ40]. The solid state
structure of SB4 was ascertained by the single Xꢀray diffraction technique, and the ORTEP
diagram is depicted in Fig.1. Mass spectra of all fluorinated chalcones showed intensive
molecular ions, assisting the structure of the targeted compounds.

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Fig. 1 ORTEP diagram of compound SB4
Scheme 1 Synthetic route of aryl thiosemicarbazones

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Monoamine oxidase inhibition studies
Inhibition profile of aryl substituted thiosemicarbazones. Six different compounds of
the derivatives showed higher inhibitory activities (more than 50%) against MAOꢀA or
MAOꢀB, while the other compounds were not effective (Table 1). Compounds SB3, SB5, and
SB6 showed efficient inhibition against MAOꢀA with IC50 values of 4.99 ± 0.25, 1.82 ± 0.14,
and 5.98 ± 0.15 µM, respectively. Compounds SB7, SB11, and SB12 were also effectively
inhibitory against MAOꢀB with IC50 values of 1.43 ± 0.014, 0.27 ± 0.015, and 7.61 ± 0.49
µM, respectively. All 6 compounds showed good selectivity, and SB5 and SB11 were the
most potent for MAOꢀA and MAOꢀB, respectively. Furthermore, SB11 showed high
selectivity index (SI > 37.0) for MAOꢀB with a low IC50 value (0.27 µM), suggesting it a
good candidate for selective MAOꢀB inhibition. Considering the structural comparisons, we
concluded that based on their IC50 values, the
(SB11) showed 28.2 times higher inhibitory activity against MAOꢀB than
substitution (SB12) and > 37.0 times potent than ꢀfluorine (SB10) (Table 1). However, the
substituent ꢀNO2 (SB7) was more effective than the ꢀfluorine substitution (SB10).
m
ꢀfluorine substitution of the compounds
o
ꢀfluorine
p
p
p
The potency of SB5 for MAOꢀA (IC50 = 1.82 µM) was lower than IM5 (IC50 = 0.30 µM),
which is a synthesized imidazole bearing chalcone derivative of the eleven series and is the
most potent for MAOꢀA reported by our group recently [41]. However, the SI value of SB5
(0.18) in this study was > 4.2 times than IM5 (0.75). The potency of SB11 for MAOꢀB (IC50
= 0.27 µM) was higher than IM4 (IC50 = 0.32 µM), which is another derivative and the most
potent for MAOꢀB in the IM series. Similar to SB5, the SI value of SB11 (> 37.0) was >11.2
times higher than the IM4 (3.3). Although the potency of SB11 for MAOꢀB was 6.4 times
lower than that of the marketed drug lazabemide for MAOꢀB (IC50 = 0.042 µM), the relatively
low molecular weight of SB11 (MW = 197.2) is comparable to lazabemide (MW = 199.6)
and smaller than IM4 (MW = 288.3); it also has an IC50 value comparable to that of

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lazabemide in the range of nanomolar concentration. The structural features of SB11 were
mainly divided as: (a) halogenated aryl system; (b) side chain with almost similar length; and
(c) terminal amino group at the side chain. Similar type of features are seen in the potent
MAOꢀB inhibitor (lazabemide) and are depicted in the Fig. 2. These structural features are
responsible for the design and development of a new class of MAOꢀB inhibitors.
Fig. 2 Similarity based structure of SB11 and standard MAOꢀB inhibitor
Structure activity relationship (SAR) analysis of MAO inhibition. Changes in the
MAO inhibitory potency of the tested aryl thiosemicarbazones could be correlated to the
effect of various electron donating and withdrawing groups anchored to the phenyl system.
To explore the structure activity relationship of target compounds, we initially focused on
variating substituents at para position of the phenyl system of aryl thiosemicarbazones.
Unsubstituted aryl thiosemicarbazones are less effective against both MAOꢀA and MAOꢀB

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with IC50 > 10.0 µM. Modifications in the position and orientation of substituent on the
phenyl system result in a shift in this trend. The presence of electron donating groups (EDGs)
such as methoxy, dimethylamino and ethyl on the para position of the phenyl system in
compounds SB3, SB5 and SB6 significantly contribute to the activity ratio towards MAOꢀA.
Furthermore, introduction of electron donating groups such as hydroxyl and methyl (SB2 &
SB4) results in a dramatic decrease in the activity. Hence, it is commonly recommended that
EDGs with bulky groups on phenyl system of aryl thiosemicarbazones adapt well in the
hydrophobic pocket of the inhibitor binding cavity (IBC) of MAOꢀA. Shifting of MAOꢀA
selectivity was revealed after the introduction of an electron withdrawing nitro group.
Presence of halogen such as chlorine, bromine and fluorine at the para position of aryl
thiosemicarbazones had no impact on the MAOꢀB inhibition. In particular, shifting of
fluorine atom to the meta position makes to be more potent MAOꢀB inhibition (SB11) with
Ki value of 0.12 ± 0.006 µM. The inhibition constant was found to be better than the standard
hMAOꢀB inhibitor (irreversible type) which is reported previously by our research group [42ꢀ
50].

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Table 1 Inhibition of recombinant human MAO enzymes and acetylcholinesterase by aryl
substituted aryl thiosemicarbazonesa
Residual activity at 10 µM
IC50 (µM)
SIb
(%)
Compounds
MAOꢀA
MAOꢀB
MAOꢀA
MAOꢀB
AChE
83.5 ± 0.7
66.0 ± 1.4
37.5 ± 0.7
56.5 ± 0.7
16.5 ± 2.1
36.0 ± 5.7
75.5 ± 3.5
76.0 ± 1.4
59.5 ± 0.7
84.5 ± 3.5
75.5 ± 2.1
77.5 ± 0.7
92.5 ± 0.7
75.5 ± 0.7
87.5 ± 3.5
82.1 ±1.4
91.5 ± 2.1
82.3 ± 1.4
86.2 ± 1.4
7.5 ± 0.7
> 10.0
> 10.0
> 10.0
> 10.0
> 10.0
> 10.0
> 10.0
> 10.0
> 40.0
> 40.0
SB1
SB2
4.99 ± 0.25
> 10.0
36.14 ± 0.45
> 40.0
< 0.50
< 0.18
SB3
SB4
1.82 ± 0.14
5.98 ± 0.15
> 10.0
35.35 ± 0.47
SB5
26.72 ± 0.006 < 0.60
SB6
1.43 ± 0.014 15.61 ± 0.057 > 6.99
SB7
60.5 ± 2.1
82.5 ± 4.9
86.5 ± 2.1
3.0 ± 1.4
> 10.0
> 10.0
> 10.0
> 40.0
SB8
> 10.0
31.12 ± 0.44
37.93 ± 0.57
26.61 ± 0.34
30.84 ± 0.58
36.23 ± 0.44
SB9
> 10.0
> 10.0
SB10
SB11
SB12
SB13
Toloxatone
Lazabemide
Clorgyline
> 10.0
0.27 ± 0.015
7.61 ± 0.49
> 10.0
> 37.0
> 1.31
33.5 ± 2.1
77.5 ± 0.7
> 10.0
> 10.0
0.92 ± 0.016
> 80
> 80
< 0.012
> 1,900
0.0042
14.4
0.042 ± 0.0010
1.69 ± 0.32
0.091 ± 0.005
0.0071 ± 0.0003
1.31 ± 0.068
Pargyline
Tacrine
0.23 ± 0.014
aResults are expressed as means ± standard errors of duplicate experiments. Inhibitory
activities for reference compounds of MAO and AChE were measured after preincubation
with the enzymes for 30 min and 15 min, respectively.
bSI was expressed for MAOꢀB by dividing IC50 of MAOꢀA by that of MAOꢀB.
Kinetics. The inhibition modes of SB5 and SB11 for MAOꢀA and MAOꢀB, respectively,
were analyzed by Lineweaver–Burk plots. The plots for SB5 and SB11 were linear and
intersecting the yꢀaxis (Fig. 3A & 3C). The Ki values determined by the secondary plot
(slopes of Lineweaver–Burk plots vs. inhibitor concentrations) of MAOꢀA inhibition by SB5
and MAOꢀB inhibition by SB11 were 0.97 ± 0.042 and 0.12 ± 0.006 µM, respectively (Table

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1, Fig. 3B & 3D). These results indicate that SB5 and SB11 are selective and reversible
competitive inhibitors of MAOꢀA and MAOꢀB, respective
(A)
(B)
2000
1500
1000
500
0
Control
1.0 uM
2.0 uM
4.0 uM
-50
0
50
100
150
200
1/[S], mM-1
12
10
8
6
4
2
0
-2
0
2
4
6
[SB5], uM
(C)
(D)

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4000
3500
3000
2500
2000
1500
1000
500
Control
0.16 uM
0.32 uM
0.64 uM
0
-20
-10
0
10
20
30
40
1/[S], mM-1
120
100
80
60
40
20
0
-0.2
0
0.2
0.4
0.6
0.8
[SB11], uM
Fig. 3 Kinetic analyses of inhibitions of MAOꢀA by SB5 (A) and MAOꢀB by SB11 (C)
using LineweaverꢀBurk plots, and their respective secondary plots of slopes vs. inhibitor
concentrations of SB5 (B) and SB11 (D).
Reversibility studies. No changes in the residual activities were observed when SB5 and
SB11 were preincubated with MAOꢀA and MAOꢀB, respectively, for up to 30 min. In
reversibility experiments, the AU and AD values obtained by SB5 for MAOꢀA were 34.7%
and 70.3%, respectively (Fig. 4A). Values for toloxatone (a reversible inhibitor) reference
experiments for MAOꢀA were 30.7% and 74.0%, respectively, and the values for clorgyline

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(an irreversible inhibitor) were 21.4% and 19.8%, respectively. The inhibition by toloxatone
was greatly recovered by dialysis, while inhibition by clorgiline was not recovered. Similar to
these results, the activity by SB5 was recovered close to the reversible reference level. The
enzymatic activity of the MAOꢀB by SB11 revealed AU and AD values of 27.4% and 81.0%,
respectively (Fig. 4B); values for lazabemide were 29.5% and 86.9%, respectively, and for
pargyline were 27.9% and 32.0%, respectively. MAOꢀB inhibition by pargyline was not
recovered by dialysis, whereas the activity by lazabemide was greatly recovered, similar to
the recovery to near reference levels for inhibitory activity by SB11. These results indicate
that analogues SB5 and SB11 are reversible inhibitors for MAOꢀA and MAOꢀB, respectively.

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Fig. 4 Reversibility of MAO enzymes by aryl substituted thiosemicarbazones. MAOꢀA and
MAOꢀB were inhibited at approximately 2 × IC50 by SB5 (A) and SB11 (B), respectively, and
the activities were recovered by dialysis experiments against 100 mM sodium phosphate (pH
7.2) before measuring the residual activities. Concentrations of inhibitors and references used:
SB5, 3.6 µM; toloxatone, 2.0 µM; clorgyline, 0.014 µM; SB11, 0.54 µM; lazabemide, 0.08
µM; pargyline, 0.20 µM.

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Acetylcholinesterase inhibition
As presented in Table 1, it was noted that all the compounds are moderate and less potent
than the reference compound tacrine for AChE inhibition. Considering the type of
substitution at the phenyl ring, the inhibitory potency against AChE clearly favored the
electron withdrawing nitro group at the para position of phenyl system (SB7, IC50 15.61 ±
0.057 µM). According to the data, the presence of chlorine or hydroxyl group is not crucial
for imparting the inhibitory potential against AChE in aryl thiosemicarbazones. Furthermore,
fluorine orientation also showed moderate inhibition in titled compounds for AChE
inhibition. The orthoꢀ and metaꢀsubstituted analogue SB12 and SB11 show slightly higher
AChE inhibitory activities compared to the paraꢀsubstituted analogue SB10, likely being
MAOꢀB inhibition.
Bloodꢀbrain barrier (BBB) permeation assay
An essential requirement for successful CNS drugs is the ability to cross the BBB, Which is
determined using the parallel artificial membrane permeation assay (PAMPA). According to
the limits established by Di et al., the BBB permeation test compounds are classified as
follows: [51].
CNS+ (high BB permeation predicted): Pe (× 10ꢀ6 cmsꢀ1) ꢀ ˃ 4.00
CNSꢀ (high BB permeation predicted): Pe (× 10ꢀ6 cmsꢀ1) ꢀ ˃ 2.00
Table 2 indicates the permeability of PAMPAꢀBBB assay of commercial drugs and the top
ranked 6 aryl thiosemicarbazones. Our results indicate that all the tested thiosemicarbazones
are capable of crossing the BBB to target the MAOꢀA and MAOꢀB enzymes in the central
nervous system (CNS), which is consistent with our design strategy.

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Table 2 PAMPAꢀBBB of aryl thiosemicarbazones and commercial drugs
Bibliography
Experimental
Compoundsa
Prediction
Pe (× 10ꢀ6 cmsꢀ1)b
Pe (× 10ꢀ6 cmsꢀ1)c
ꢀ
ꢀ
11.24 ± 0.44
10.14 ± 0.56
09.44 ± 0.65
12.78 ± 0.45
13.12 ± 0.54
10.33 ± 0.22
17.33 ± 0.12
08.13 ± 0.42
0.21 ± 0.01
1.71 ± 0.02
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
CNSꢀ
CNSꢀ
SB3
SB5
ꢀ
SB6
ꢀ
SB7
ꢀ
SB11
ꢀ
SB12
17.0
9.3
0.2
1.8
Testosterone
Progesterone
Dopamine
Hydrocortisone
a)
Compounds were dissolved in DMSO to a concentration of 5 mg/mL and diluted with
PBS/EtOH (70:30). The final concentration of compound was 100 µg/mL.
b) Taken from [reference 51]
c) Values are expressed as the mean ± SEM of three independent experiments.
Molecular docking
From the in vitro results, it is evident that compounds SB5 and SB11 show a good
inhibitory profile towards MAOꢀA and MAOꢀB, respectively, in the micro molar range. We

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therefore attempted to investigate the hypothetical binding modes of the respective
compounds in the IBC of isoenzymes. Of the 50 runs in docking methodology, we selected
the highest binding energy in the largest cluster for hypothetical binding pose. The binding
mode of SB5 (hMAOꢀA inhibitor) is shown in Fig. 5. The presence of imino nitrogen and
terminal amino group in SB5 contributes significant hydrogen bonding interactions with the
TYR444 and the N5 atom of the flavin adenine dinucleotide (FAD) unit of MAOꢀA,
respectively. SB5 adopts an ‘L’ type configuration in which the aryl thiosemicarbazone unit is
accommodated by the facing FAD unit with a hydrogen bond of distance 2.15 Å surrounded
by the aromatic cage of TYR444 and TYR407. The position and close proximity towards the
FAD unit of SB5 may enhance its binding energy towards MAOꢀA.
The binding mode of potent MAOꢀB inhibitor SB11 is shown in Fig. 6. The entrance
cavity of the MAOꢀB leading to the substrate cavity is hydrophobic in nature [52]. ILE199
and TYR326 are the side chains responsible for the separation and fusion between the
entrance and substrate cavity, depending on the nature of the bound inhibitor [53ꢀ56]. The
meta substituted fluorine of the phenyl system of SB11 is efficiently accommodated in the
entrance cavity of the MAOꢀB. This lipophilic environment enhances the binding affinity of
SB11 towards the IBC of MAOꢀB. The terminal amino group of thiosemicarbazone shows a
significant hydrogen bonding with GLN206 and the electron rich thiocarbamoyl group of
SB11 surrounded by the aromatic cage of TYR398 and TYR435 nearer to the FAD unit.

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Fig. 5 SB5 in the active site of MAOꢀA

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Fig. 6 SB11 in the active site of MAOꢀB
Conclusions
To summarize our results, various aryl thiosemicarbazones with different electron donating
and withdrawing environments were synthesized, characterized and evaluated for their MAO
inhibitory and blood brain barrier permeation potential. The representative compounds SB5
and SB11 were potent for MAOꢀA and MAOꢀB, respectively, with reversible and competitive
mode of inhibition, having IC50 values of 1.82 ± 0.14 and 0.27 ± 0.015 µM, respectively. The
results explicate that nature and orientation of groups on the aryl system of titled scaffold can
bestow significant selectivity profile on both MAOꢀA and MAOꢀB. The SAR revealed that
presence of an electron donating bulky group produces good selectivity towards MAO, and at
the same the time electron withdrawing nitro group in the same position shifts the selectivity

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to MAOꢀB. The presence of halogen at the para position of the phenyl ring has no impact on
MAO inhibition, but shifting of fluorine to the meta position dramatically results in good
MAOꢀB inhibition with a high selectivity index. Compounds SB5, SB7 and SB11 show
moderate inhibition against acetylcholinesterase with IC50 values of 35.35 ± 0.47, 15.61 ±
0.057 and 26.61 ± 0.338 µM, respectively. PAMPA studies revealed that the representative
molecules are able to cross the bloodꢀbrain barrier, which is a preꢀrequisite of CNS drug
design for the treatment of various neurodegenerative and psychiatric disorders. Molecular
modelling studies identified that presence of the imino nitrogen and terminal amino group of
SB5 contributed significant hydrogen bonding interactions with the TYR444 and N5 atom of
flavin adenine dinucleotide (FAD) unit of MAOꢀA, and m-fluorine of the phenyl system of
SB11 efficiently accommodated in the entrance cavity of the MAOꢀB. Also, the terminal
amino group of thiosemicarbazone formed a significant hydrogen bonding with GLN206 of
MAOꢀB.This study has thus provided new insights into the SARs of various aryl
thiosemicarbazones compounds towards MAO inhibition and could possibly afford new
attractive and more promising mutliꢀtargeted ligands for the treatment of AD and PD.
Experimental
Chemistry
A mixture of thiosemicarbazide hydrochloride and substituted benzaldehyde in the presence
of catalytic acetic acid was stirred for 3ꢀ4 hours. The resultant mixture was refluxed for 4ꢀ5
hours and poured onto crushed ice. The formed solid was washed with water until it was free
from the acid, filtered and crystallized with ethanol. The following13 phenyl substituted
thiosemicarbazones (SB1ꢀSB13) were procured:

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(2E)ꢀ2ꢀbenzylidenehydrazineꢀ1ꢀcarbothioamide (SB1): Yellowish white; Yield: 76%;
m.p: 126ꢀ128°C. 1H NMR (400 MHz, CDCl3)δ: 6.40 (s, 1H, NH2), 7.23 (s, 1H, NH2), 7.44ꢀ
7.42 ( m, 3H, J= 8Hz, ArꢀH), 7.66ꢀ7.64 ( d, 2H, J= 8Hz, ArꢀH), 7.86 (s, 1H, ꢀCH=Nꢀ), 9.60 (s,
1H, =NꢀNHꢀC=S). 13CꢀNMR (100 MHz, CDCl3) δ: 182.6 (C=S), 142.8 (CH=N), 134.3 (Arꢀ
C1), 131.0 (ArꢀC4), 129.3 (ArꢀC2 & ArꢀC6), 128.6 (ArꢀC3 & ArꢀC5). ESIꢀMS (m/z):
Calculatedꢀ 179.24, Observedꢀ361.23.
(2E)ꢀ2ꢀ[(4ꢀhydroxyphenyl)methylidene]hydrazineꢀ1ꢀcarbothioamide (SB2): Pale
yellow; Yield: 63%; m.p: 210ꢀ212°C. 1H NMR (400 MHz, CDCl3)δ: 5.21 (s, IH, ArꢀOH) 6.77,
(s, 1H, NH2), 6.94ꢀ6.92 ( d, 2H, J= 8Hz, ArꢀH), 7.28 (s, 1H, NH2), 7.66ꢀ7.64 ( d, 2H, J= 8Hz,
ArꢀH), 7.77 (s, 1H, ꢀCH=Nꢀ), 9.56 (s, 1H, =NꢀNHꢀC=S). 13CꢀNMR (100 MHz, CDCl3) δ:
181.3 (C=S), 160.2 (ArꢀC4), 142.37 (CH=N), 130.3 (ArꢀC2 & ArꢀC6), 125.2 (ArꢀC1), 118.2
(ArꢀC3 & ArꢀC5). ESIꢀMS (m/z): Calculatedꢀ 195.24, Observedꢀ195.23.
(2
E
)ꢀ2ꢀ[(4ꢀmethoxyphenyl)methylidene]hydrazineꢀ1ꢀcarbothioamide
(SB3):
Yellowish; Yield: 78%; m.p: 150ꢀ152°C. 1H NMR (400 MHz, CDCl3)δ: 3.84 (s, 3H, OCH3),
6.37 (s, 1H, NH2), 6.92ꢀ6.90 ( d, 2H, J= 8Hz, ArꢀH), 7.26 (s, 1H, NH2), 7.60ꢀ7.58 ( d, 2H, J=
13
8Hz, ArꢀH), 7.83 (s, 1H, ꢀCH=Nꢀ), 9.66 (s, 1H, =NꢀNHꢀC=S). CꢀNMR (100 MHz, CDCl3)
δ: 181.4 (C=S), 161.9 (ArꢀC4), 145.2 (CH=N), 129.8 (ArꢀC2 & ArꢀC6), 128.9 (ArꢀC1), 119.3
(ArꢀC3 & ArꢀC5), 55.4 (OCH3). ESIꢀMS (m/z): Calculatedꢀ 209.26, Observedꢀ209.25.
(2
E
)ꢀ2ꢀ[(4ꢀmethylphenyl)methylidene]hydrazineꢀ1ꢀcarbothioamide (SB4): White;
1
Yield: 82%; m.p: 155ꢀ157°C. H NMR (400 MHz, CDCl3)δ: 2.38 (s, 3H, CH3), 6.46 (s, 1H,
NH2), 7.21ꢀ7.19 ( d, 2H, J= 8Hz, ArꢀH), 7.26 (s, 1H, NH2), 7.54ꢀ7.52 ( d, 2H, J= 8Hz, ArꢀH),
7.88 (s, 1H, ꢀCH=Nꢀ), 9.88 (s, 1H, =NꢀNHꢀC=S). 13CꢀNMR (100 MHz, CDCl3) δ: 181.6
(C=S), 145.3 (CH=N), 142.1 (ArꢀC4), 132.2 (ArꢀC1), 131.7 (ArꢀC2 & ArꢀC6), 131.4 (ArꢀC3
& ArꢀC5), 21.6 (CH3). ESIꢀMS (m/z): Calculatedꢀ 193.26, Observedꢀ193.25.

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(2
E
)ꢀ2ꢀ{[4ꢀ(dimethylamino)phenyl]methylidene}hydrazineꢀ1ꢀcarbothioamide (SB5):
1
Yellowish; Yield: 81%; m.p: 190ꢀ192°C. H NMR (400 MHz, CDCl3)δ: 3.03 (s, 6H,
N(CH3)2), 6.23 (s, 1H, NH2), 6.68ꢀ6.66 ( d, 2H, J= 12Hz, ArꢀH), 7.26 (s, 1H, NH2), 7.52ꢀ7.49
( d, 2H, J= 12Hz, ArꢀH), 7.71 (s, 1H, ꢀCH=Nꢀ), 9.18 (s, 1H, =NꢀNHꢀC=S). 13CꢀNMR (100
MHz, CDCl3) δ: 181.7 (C=S), 151.8 (ArꢀC4), 141.4 (CH=N), 132.0 (ArꢀC2 & ArꢀC6), 123.5
(ArꢀC1), 114.4 (ArꢀC3 & ArꢀC5), 41.9 (Nꢀ(CH3)2). ESIꢀMS (m/z): Calculatedꢀ 222.30,
Observedꢀ222.29.
(2E)ꢀ2ꢀ[(4ꢀethylphenyl)methylidene]hydrazineꢀ1ꢀcarbothioamide (SB6): White; Yield:
84%; m.p: 125ꢀ126°C. 1H NMR (400 MHz, CDCl3)δ: 1.24ꢀ1.22 (t, 3H, J= 8Hz, CH3), 2.70ꢀ
2.68 (q, 2H, J= 8Hz, CH2), 6.47 (s, 1H, NH2), 7.24ꢀ7.22 ( d, 2H, J= 8Hz, ArꢀH), 7.26 (s, 1H,
NH2), 7.57ꢀ7.55 ( d, 2H, J= 8Hz, ArꢀH), 7.89 (s, 1H, ꢀCH=Nꢀ), 9.92 (s, 1H, =NꢀNHꢀC=S).
13CꢀNMR (100 MHz, CDCl3) δ: 181.2 (C=S), 145.99 (CH=N), 137.3 (ArꢀC4), 130.5 (ArꢀC1),
129.8 (ArꢀC2 & ArꢀC6), 128.8 (ArꢀC3 & ArꢀC5), 33.5 (CH2), 14.8 (CH3). ESIꢀMS (m/z):
Calculatedꢀ 207.29, Observedꢀ207.00.
(2E)ꢀ2ꢀ[(4ꢀnitrophenyl)methylidene]hydrazineꢀ1ꢀcarbothioamide (SB7): Turmeric
yellow; Yield: 82%; m.p: 220ꢀ222°C. 1H NMR (400 MHz, CDCl3)δ: 6.23 (s, 1H, NH2), 6.68ꢀ
6.66 ( d, 2H, J= 8Hz, ArꢀH), 7.26 (s, 1H, NH2), 7.52ꢀ7.49 ( d, 2H, J= 12Hz, ArꢀH), 7.71 (s,
1H, ꢀCH=Nꢀ), 9.20 (s, 1H, =NꢀNHꢀC=S). 13CꢀNMR (100 MHz, CDCl3) δ: 183.9 (C=S),
150.2 (ArꢀC4), 143.4 (CH=N), 136.9 (ArꢀC1), 131.3 (ArꢀC2 & ArꢀC6), 124.5 (ArꢀC3 & Arꢀ
C5). ESIꢀMS (m/z): Calculatedꢀ 224.23, Observedꢀ224.22.
(2E)ꢀ2ꢀ[(4ꢀchlorophenyl)methylidene]hydrazineꢀ1ꢀcarbothioamide (SB8): Pale white;
Yield: 83%; m.p: 175ꢀ177°C. 1H NMR (400 MHz, CDCl3)δ: 7.25 (s, 1H, NH2), 7.40ꢀ7.38 ( d,
2H, J= 8Hz, ArꢀH), 7.26 (s, 1H, NH2), 7.59ꢀ7.57 ( d, 2H, J= 12Hz, ArꢀH), 7.78 (s, 1H, ꢀ
CH=Nꢀ), 9.33 (s, 1H, =NꢀNHꢀC=S). 13CꢀNMR (100 MHz, CDCl3) δ: 184.3 (C=S), 142.9

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(CH=N), 137.3 (ArꢀC4), 131.3 (ArꢀC1), 130.6 (ArꢀC2 & ArꢀC6), 130.5 (ArꢀC3 & ArꢀC5).
ESIꢀMS (m/z): Calculatedꢀ 213.68, Observedꢀ213.67.
(2
E
)ꢀ2ꢀ[(4ꢀbromophenyl)methylidene]hydrazineꢀ1ꢀcarbothioamide (SB9): White;
1
Yield: 85%; m.p: 140ꢀ142°C. H NMR (400 MHz, CDCl3) δ: 6.39 (s, 1H, NH2), 7.26 (s, 1H,
NH2), 7.56ꢀ7.54 (d, 4H, J= 8Hz, ArꢀH), 7.76 (s, 1H, ꢀCH=Nꢀ), 9.29 (s, 1H, =NꢀNHꢀC=S).
13CꢀNMR (100 MHz, CDCl3) δ: 182.0 (C=S), 142.9 (CH=N), 131.2 (ArꢀC1), 130.3 (ArꢀC2 &
ArꢀC6), 130.2 (ArꢀC3 & ArꢀC5), 121.6 (ArꢀC4).
ESIꢀMS (m/z): Calculatedꢀ 258.13, Observedꢀ258.13.
(2E)ꢀ2ꢀ[(4ꢀfluorophenyl)methylidene]hydrazineꢀ1ꢀcarbothioamide (SB10): White;
Yield: 82%; m.p: 120ꢀ122°C. 1H NMR (400 MHz, CDCl3)δ: 6.38 (s, 1H, NH2), 7.40ꢀ7.38 ( d,
2H, J= 8Hz, ArꢀH), 7.26 (s, 1H, NH2), 7.59ꢀ7.57 ( d, 2H, J= 12Hz, ArꢀH), 7.78 (s, 1H, ꢀ
CH=Nꢀ), 9.33 (s, 1H, =NꢀNHꢀC=S). 13CꢀNMR (100 MHz, CDCl3) δ: 180.6 (C=S), 160.3 (Arꢀ
C4), 142.2 (CH=N), 131.3 (ArꢀC1), 130.6 (ArꢀC2 & ArꢀC6), 125.6 (d, JCꢀF = 62 Hz, ArꢀC3 &
ArꢀC5). ESIꢀMS (m/z): Calculatedꢀ 197.23, Observedꢀ197.23.
(2E)ꢀ2ꢀ[(3ꢀfluorophenyl)methylidene]hydrazineꢀ1ꢀcarbothioamide (SB11): Pinkish
white; Yield: 79%; m.p: 155ꢀ157°C. 1H NMR (400 MHz, CDCl3)δ: 6.39 (s, 1H, NH2), 7.26 (s,
1H, NH2),7.56ꢀ7.54 ( m, 4H, J= 8Hz, ArꢀH), 7.76 (s, 1H, ꢀCH=Nꢀ), 9.29 (s, 1H, =NꢀNHꢀC=S).
13CꢀNMR (100 MHz, CDCl3) δ: 182.2 (C=S), 166.2 (ArꢀC3), 140.1 (CH=N), 136.6, (ArꢀC5),
134.3 (ArꢀC1), 125.3 (ArꢀC6), 122.9 (d, JCꢀF = 64 Hz, ArꢀC4), 121.6 (d, JCꢀF = 68 Hz, ArꢀC2).
ESIꢀMS (m/z): Calculatedꢀ 197.23, Observedꢀ197.23.
(2E)ꢀ2ꢀ[(2ꢀfluorophenyl)methylidene]hydrazineꢀ1ꢀcarbothioamide (SB12): Yellowish
1
1
grey; Yield: 82%; m.p: 121ꢀ123°C. H NMR (400 MHz, CDCl3)δ: H NMR (400 MHz,
CDCl3)δ: 6.38 (s, 1H, NH2), 7.12ꢀ7.10 ( m, 2H, J= 8Hz, ArꢀH), 7.26 (s, 1H, NH2), 7.65ꢀ7.63
( m, 2H, J= 8Hz, ArꢀH), 7.85 (s, 1H, ꢀCH=Nꢀ), 9.66 (s, 1H, =NꢀNHꢀC=S). 13CꢀNMR (100
MHz, CDCl3) δ: 182.1 (C=S), 164.1 (ArꢀC2), 143.6 (CH=N), 134.3, (d, JCꢀF = 68 Hz, ArꢀC4),

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130.2 (ArꢀC6), 125.2 (ArꢀC5), 122.6 (ArꢀC1), 121.5 (ArꢀC3). ESIꢀMS (m/z): Calculatedꢀ
197.23, Observedꢀ197.22.
(2E)ꢀ2ꢀ{[4ꢀ(trifluoromethyl)phenyl]methylidene}hydrazineꢀ1ꢀcarbothioamide (SB13):
1
1
Grey; Yield: 79%; m.p: 135ꢀ137°C. H NMR (400 MHz, CDCl3) δ: H NMR (400 MHz,
CDCl3)δ: 6.44 (s, 1H, NH2), 7.11ꢀ7.09 ( d, 2H, J= 8Hz, ArꢀH), 7.26 (s, 1H, NH2), 7.37ꢀ7.35
( d, 2H, J= 12Hz, ArꢀH), 7.87 (s, 1H, ꢀCH=Nꢀ), 9.83 (s, 1H, =NꢀNHꢀC=S). 13CꢀNMR (100
MHz, CDCl3) δ: 181.4 (C=S), 160.3 (ArꢀC4), 142.1 (CH=N), 138.4 (ArꢀC1), 135.6 (ArꢀC4),
129.2 (ArꢀC2 & ArꢀC6), 124.3 (q, JCꢀF = 278 Hz, ArꢀC3 & ArꢀC5), 123.6 (CF3). ESIꢀMS (m/z):
Calculatedꢀ 247.24, Observedꢀ247.23.
Monoamine oxidase inhibition studies
Enzyme assays. Chemicals and enzymes were used as described previously. MAO
activities were assayed by the continuous method using 0.06 mM kynuramine for MAOꢀA
and 0.3 mM benzylamine for MAOꢀB as substrates, and reaction rates were expressed as
absorbance changes per min. The Km values of kynuramine and benzylamine obtained in this
study were 0.040 mM and 0.15 mM, respectively, and the substrate concentrations used were
1.5 × and 2.0 × Km values, respectively [57].
Analysis of inhibitory activities and enzyme kinetics. The inhibitions of MAOꢀA or
MAOꢀB activities by the 13 compounds were primarily analyzed at a concentration of 10 µM.
The IC50 values were then determined for 6 compounds showing more than 50% inhibitory
activity, along with the reference compounds for reversible and irreversible inhibitors. Two
potent compounds, SB5 for MAOꢀA and SB11 for MAOꢀB, were further investigated for
timeꢀdependent inhibition, kinetic studies for assessing the inhibition types, and Ki values of
the compounds, as previously described [58].
Analysis of reversibility of the inhibitors. Reversibility experiments for the potent
inhibitors were performed using the dialysis method, including reference compounds for

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reversible and irreversible inhibitors, as previously described. The experiments were
conducted at 3.6 µM SB5 for MAOꢀA and 0.54 µM SB11 for MAOꢀB in 100 mM sodium
phosphate (pH 7.2) after preincubation for 30 min. Residual activities for undialyzed and
dialyzed experiments were measured, and the relative activities for undialyzed (AU) and
dialyzed (AD) experiments were calculated comparing with each control without inhibitor.
The reversibility pattern was determined by comparing the relative AU and AD values [59].
Acetylcholinesterase inhibition
AChE inhibitory activity was assayed using the method developed by Ellman et al., with
slight modifications. The reaction was assayed for 10 min at 412 nm using 0.2 U/ml of AChE
(Electrophorus electricus, Type VIꢀS, Sigma) in 0.5 ml reaction mixture of 50 mM sodium
phosphate (pH 7.5), in the presence of 0.5 mM 5,5’ꢀdithiobis(2ꢀnitrobenzoic acid) (DTNB)
and 0.5 mM acetylthiocholine iodide (ACTI). For measurement of inhibitory activity, each
inhibitor (and tacrine as a reference) was preincubated for 15 min with the enzyme prior to
addition of DTNB and ACTI [60].
Bloodꢀbrain barrier (BBB) assay
The top ranked 6 synthesized thiosemicarbazones and the known commercial drugs were
dissolved in DMSO at a final concentration of 5 mg/mL, followed by appropriate dilution
with a mixture 70:30 of phosphate buffered saline solution and ethanol (PBS/EtOH) to give a
final concentration of 25 µg/mL. The filter membrane in the donor microplate was coated
with polar brain lipid (PBL) dissolved in docodecane (4 µg/mL, 20 mg/mL). A total of 200
µL of diluted solution and 300 µL of PBS/EtOH (70:30) were added to the donor and the
acceptor wells, respectively. The donor filter plate was carefully placed on the acceptor plate,
And the sandwich system was kept at 25°C for 16 h. The donor plate was carefully removed,

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and the concentrations of the compounds and the commercial drugs in the acceptor, donor
and the reference wells were measured with a UV plate reader [51].
Molecular docking
AUTODOCK4.2 software was employed for molecular docking studies for the lead
molecules [61]. Preparation Wizard of Maestroꢀ8.4 (Schrodinger LLC) was used to prepare
the protein. Crystallographic models 2BXR (hMAOꢀA) and 2BYB (hMAOꢀB) were
downloaded from www.rcsb.org [62]. Ligands were prepared through PRODRG webserver
(http://davapc1.bioch.dundee.ac.uk/cgiꢀbin/prodrg) [63]. Grid preparation and the docking
parameters are prepared on the basis of a reported method [64].
Conflict of Interest
There are no conflicts of interest to declare.
Acknowledgements
This research was supported by the Basic Science Research Program through the National
Research Foundation (NRF) of Korea, which is funded by the Ministry of Education
(2017R1D1A3B03028559) (To H. Kim).
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