Inhibitions of monoamine oxidases and acetylcholinesterase by 1-methyl, 5-phenyl substituted thiosemicarbazones: Synthesis, biochemical, and computational investigations

Article abstract of DOI:10.1016/j.procbio.2020.05.016

A series of eleven 1-methyl, 5-phenyl substituted thiosemicarbazones (MT1–MT11) with the phenyl ring substitutions were prepared and investigated for their inhibitory activities against monoamine oxidases (MAOs) and acetylcholinesterase (AChE). [4-(dimeth

Full text of DOI:10.1016/j.procbio.2020.05.016

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Inhibitions of monoamine oxidases and acetylcholinesterase by 1-methyl,
5-phenyl substituted thiosemicarbazones: Synthesis, biochemical, and
computational investigations
Githa Elizabeth Mathew, Jong Min Oh, Kumar Mohan, Kumudhavalli
Suresh Kumar, Sivaraman Jayanthi, Hoon Kim, Bijo Mathew
PII:
S1359-5113(20)30457-8
DOI:
https://doi.org/10.1016/j.procbio.2020.05.016
PRBI 12034
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Inhibitions of monoamine oxidases and acetylcholinesterase by 1-methyl, 5-
phenyl substituted thiosemicarbazones: Synthesis, biochemical, and
computational investigations
Githa Elizabeth Mathewa, c#, Jong Min Ohb#, Kumar Mohanc*, Kumudhavalli Suresh Kumarc,
Sivaraman Jayanthid, Hoon Kimb* Bijo Mathewd*
a Department of Pharmacology, Grace College of Pharmacy, Palakkad-678004, Kerala, India.
b Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon
National University, Suncheon 57922, Republic of Korea.
c
Department of Pharmaceutical Chemistry, Vinayaka Mission’s College of Pharmacy,
Vinayaka Mission’s Research Foundation (Deemed to be University) Salem 636308,
Tamilnadu, India.
d Computational Drug Design Lab, School of Bio Sciences and Technology, Vellore Institute
of Technology, Vellore, India
e
Department of Pharmaceutical Chemistry, Ahalia School of Pharmacy, Palakkad-678557,
Kerala, India.
#Authors contributed equally.
*Corresponding Authors: Kumar Mohan (kumarvmcp@yahoo.co.in)
Hoon Kim (H. Kim) (hoon@sunchon.ac.kr)
Bijo Mathew (B. Mathew) (bijovilaventgu@gmail.com) (bijo.mathew@ahalia.ac.in)
1

Graphical abstract
Highlights



Eleven methylthiosemicarbazones were prepared and characterised.
Inhibitory activities against monoamine oxidase (MAO) and acetylcholinesterase were evaluated.
Compound MT5 was non-toxic, and reversible competitive inhibitor for MAO-B (IC50 = 8.77 µM).

MT5 binding was stabilized by hydrogen bonding to Cys172 and π–π hydrophobic
interaction with Tyr326 of MAO-B.

MT5 may be considered a potential compound for the treatment of neurodegenerative
disorders.
2

ABSTRACT
A series of eleven 1-methyl, 5-phenyl substituted thiosemicarbazones (MT1-MT11) with the
phenyl ring substitutions were prepared and investigated for their inhibitory activities against
monoamine oxidases (MAOs) and acetylcholinesterase (AChE). [4-(dimethylamino)
phenyl]methylidene}-N-methylhydrazine-1-carbothioamide (MT5) inhibited MAO-B
potently with an IC50 of 8.77 µM. Potencies for MAO-B increased in the order –N(CH3)2 in
MT5 > -OCH3 in MT3 > -Br in MT9. Most of the 11 compounds weakly inhibited AChE by
< 30% at 10 µM. MT5 competitively inhibited MAO-B and Ki value was 6.58 ± 0.064 µM.
Reversibility experiments showed MT5 also reversibly inhibited MAO-B. MTT assays
revealed that MT5 and MT3 were non-toxic to normal VERO cell lines with IC50 values of
191.96 and 187.04 µg/mL, respectively. From the molecular docking, MT5 binding was found
to be stabilized by hydrogen bonding to the non-bonding electron of the terminal N-methyl
group with Cys172 (binding energy = -7.01 kcal/mol) of MAO-B. The molecular dynamics
further predicted that MT5 had a major π–π hydrophobic interaction with Tyr326 of MAO-B,
suggesting that it plays an important role in the stabilization of protein-ligand interaction.These
results documents that MT5 is a moderately selective, reversible, and competitive inhibitor of
MAO-B with low cytotoxic profile.
Keywords: Imines, Monoamine oxidase-B, Monoamine oxidase-A, Acetylcholinesterase,
Kinetics, Reversibility
3

1. Introduction
Monoamine oxidase (MAO) A and B are flavoenzymes containing flavin adenine
dinucleotide (FAD) and are primarily responsible for the degradation of biogenic amines [1].
This degradation involves oxidative deamination into corresponding aldehydes, hydrogen
peroxide, and ammonia using molecular oxygen as electron acceptor [2]. Non-physiologic
levels of biogenic amines can lead to diverse conditions such as depression, anxiety, and
migraine and neurodegenerative diseases like Alzheimer’s disease (AD) and Parkinson’s
disease (PD) [3-6]. Therefore, the medicinal chemists have been prompted to design selective
MAO isoform-based inhibitors that are effective for a wide range of central nervous system
(CNS) pathologies. MAO-A inhibitors like moclobemide and clorgyline are currently
considered a third-line therapeutic defense, particularly for the treatment of mental disorders,
like depression and anxiety [7-9]. On the other hand, MAO-B inhibitors like selegiline,
safinamide and lazabemide are considered adjuvant treatments for AD and PD (Fig. 1) [10].
Recently, many different scaffolds, such as chalcones, coumarins, chromones, isatin
derivatives, thiazolidindiones, xanthines, thiazoles base hydrazones, and analogues of food and
drugs administration (FDA) approved drugs have been shown to significantly inhibit MAOs
[11-18], and considerable research attempts have been tried on the discovery and development
of hydrazone/thiosemicarbazone-based MAO inhibitors (MAOIs) [19-24]. Recently, our group
synthesized a series of 5-phenyl substituted thiosemicarbazones and found that the majority
4

potently inhibited MAOs in the micromolar range and had moderate acetylcholinesterase
inhibitory profiles [25]. In the present study, we tried to enhance the lipophilicity of 5-phenyl
substituted thiosemicarbazones by introducing a methyl group on the terminal amino of the
thiosemicarbazone group and subsequently evaluated the possible use of these new adducts for
the treatments of neurodegenerative diseases. Here, we describe the synthesis of eleven 1-
methyl, 5-phenyl thiosemicarbazones, their MAO and acetylcholinesterase inhibitory activities,
the kinetics of their inhibitions using Lineweaver–Burk (LB) plots, reversibilities, and
cytotoxic profiles on normal cell lines. Finally, the lead molecule as determined using in vitro
results, was investigated to molecular docking analysis to investigate the nature of its
interaction with MAO-B.
2. Materials and methods
2.1. Synthesis
Methylthiosemicarbazide (0.01 M) and an appropriate substituted benzaldehyde (0.01 M)
in 1-2 mL acetic acid was mixed with stirring for 2-3 h. The resultant was refluxed for 6–7 h
and treated on crushed ice. Solid obtained was thoroughly washed with water until the acid
catalyst was free, filtered, and crystallized from ethanol [26].
2.2 Enzyme assays
MAO activities were determined by continuous methods as described previously [27, 28].
Shortly, recombinant human MAO-A or B were used, and kynuramine (0.06 mM) or
benzylamine (0.3 mM), respectively, as substrates. Substrate concentrations were 1.5 × and 1.4
× Km, respectively, and their Km values were 0.041 mM and 0.21 mM, respectively. AChE
5

activities were measured using and Electrophorus electricus Type VI-S in a mixture of 0.5 mM
5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) as a color development reagent and 0.5 mM
acetylthiocholine iodide (ACTI) as a substrate [29].
2.3. Mode of inhibitions and kinetics
The inhibitory potencies of MT1-11 on MAO-A, MAO-B, and AChE were assayed at a
concentration of 10 µM, and then IC50 values were determined for compounds that inhibited
MAO-B by >50%, by measuring residual activities at 7 concentrations between ~ 0.1 × and 10
× IC50. The values were calculated using Graph pad PRISM 5 (Graphpad, San Diego, CA) with
duplicated data. MAO-A inhibitory activities were then assayed [30, 31]. Reversibilities were
performed by the dialysis method, and kinetic parameters were analyzed at five substrates and
three inhibitor concentrations using Lineweaver-Burk plots on [4-(dimethylamino)
phenyl]methylidene}-N-methylhydrazine-1-carbothioamide (MT5), which most potently
inhibited MAO-B, as previously described [32, 33].
2.4. Reversibility studies
The inhibitory reversibilities of compound MT5 were determined by dialysis after
preincubating it with MAO-B for 30 min [34, 35]. The concentration used was ; MT5 18.0
µM, lazabemide as a reversible MAO-B inhibitor (positive control) at 0.12 µM, and pargyline
as an irreversible MAO-B inhibitor (negative control) at 0.040 µM. The relative activities of
dialyzed (AD) and non-dialyzed (AU) samples were compared to determine reversibility
patterns [36].
2.5. Cytotoxicity
Tubes containing VERO cells (African green monkey kidney cells) were centrifuged and
6

adjusted to 1.0 X 105 cells/ml. Cultured cells were then diluted using Dulbecco’s modified
eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Diluted suspensions (100
µl) were added to the wells of 96-well microplates. After incubation for 24 h, cells were
centrifuged and pellets were diluted with 100 µl of maintenance medium. Plates were then
incubated in a CO2 atmosphere (5%) for 48 h at 37°C, during which cells were examined every
24 h under a microscope. Cells were then treated with 20 µl of 3-(4,5-dimethylthiazolyl-2)-2,5-
diphenyltetrazolium bromide (MTT) (2 mg/ml). The formazan crystals formed were dissolved
by gently adding 100 µl of a DMSO/isopropanol mixture per well and absorbances were
immediately measured using a microplate reader at 540 nm. Dose-response curves were used
to determine the concentrations required to inhibit cell growth by 50% [37].
2.6. Molecular docking
The molecular docking study of MT5 was carried out with the AUTODOCK4.2 version
[38]. Energy minimization of the edited protein 2V5Z (hMAO-B) was performed using Swiss-
Pdb viewer (4.1.0) [39, 40]. The energy minimization of the ligand was prepared using the
PRODRG webserver [41]. The protocols of grid and docking parameters were as previously
described [42-45].
2.7. Molecular dynamics
The molecular dynamics (MD) simulations were carried out using Desmond simulation
package of Schrodinger LLC. The lead molecule MT5 against MAO-B using the aqueous
solvent system was initially prepared for the protein-ligand complex using the Desmond system
builder panel [46]. The simulation conditions were 50 ns at 300 K, and 1.01325 bar pressure
7

with 1000 frames for entire protein-ligand simulations, for protein-ligand interactions and
stability trajectory analysis.
3. Results
3.1. Chemistry
The target compounds were synthesized by acid catalyzed nucleophilic addition between
various phenyl substituted aldehydes and methylthiosemicarbazide in the presence of ethanol
[47]. The synthetic route is outlined in the scheme 1. The synthesized candidates were
1
1
ascertained by H-NMR and Mass spectroscopy. In H-NMR spectra, a sharp singlet upfield
observed between 3.24-3.38 is represented to the terminal N-CH3 group. The singlet peaks
observed between 7.64-7.95 are represented to the azomethine (-CH=N-) proton. The
secondary amino (-NH-) group nearer to the thiocarbamoyl unit (HN-N-C=S) is observed in
the range between 9.06–10.54. The mass spectra of all 1-methyl, 5-phenyl substituted
thiosemicarbazones indicated intensive molecular ions, supporting the targeted compound
structures. The spectral characterization is deposited in the supplementary material.
3.2. Inhibitory activities on MAO enzymes and AChE
MT5 inhibited MAO-B potently with an IC50 value of 8.77 µM, and whereas the other 10
compounds had IC50 values of > 10 µM (Table 1). Potencies for MAO-B increased in the order
–N(CH3)2 in MT5 > -OCH3 in MT3 > -Br in MT9. Other substituents had slight effects on
MAO-B. The weak inhibition was observed for MT1, which contained an unsubstituted phenyl.
Most of the compounds weakly inhibited AChE by < 30% at 10 µM.
8

3.3. Kinetics of MAO-B inhibition
LB plots showed that MT5 competitively inhibited MAO-B (Fig. 2A). Ki value was
determined to be 6.58 ± 0.064 µM from the secondary plots (Fig. 2B). These results suggested
MT5 competed with the substrate at the active site of the enzyme and that it selectively and
competitively inhibited MAO-B.
3.4. Reversibility studies
Inhibition of MAO-B by MT5 recovered from 21.6 (AU) to 96.4% (AD) after dialysis (Fig.
3), which was comparable to that standard lazabemide, a reversible inhibitor, from 19.3 to
94.6%. On the other hand, MAO-B inhibition by pargyline, an irreversible inhibitor, was not
recovered by dialysis (from 37.8 to 36.8%). These results showed MT5 reversibly inhibited
MAO-B.
3.5. Cytotoxicity evaluation
The biocompatibilities of MT5 and MT3 were evaluated using an MTT assay and normal
VERO cell lines. Both compounds achieved > 89% cell viability after treatment at 100 µg/mL.
MTT assays revealed MT5 and MT3 were relatively non-toxic to normal VERO cell lines with
IC50 values of 191.96 and 187.04 µg/mL, respectively.
3.6. Molecular docking
Molecular docking study was performed to investigate the interaction between MT5 and
hMAO-B co-crystallized with native ligand safinamide (PDB code: 2V5Z). The experimental
inhibitory binding constant (Ki) 6.58 µM was found to agree well with the calculated inhibitory
binding constant of 7.32 µM, obtained from 50 molecular docking simulations. Energies
associated with the binding of MT5 in the inhibitor binding cavity (IBC) of MAO-B are
9

provided in Table 2. Binding of MT5 to MAO-B was found to be stabilized by hydrogen
bonding with the non-bonding electrons of terminal N-methyl group with Cys172 (2.012Å).
The binding pose of MT5 is depicted in the Fig. 4.
3.7. Molecular dynamics
The simulation study of MT5 was followed by MD simulations using Desmond package.
As per RMSD (root mean square deviations) analysis, the protein C-alpha and ligand were
stable in the range 1.7–2.4 Å for a long duration of simulation 50 ns time without any major
fluctuations. This suggested that the complexes were energetically compatible (Fig. 6A). The
interaction patterns between the MT5 and protein was categorized as hydrogen bonds,
hydrophobic, ionic and water bridges (Fig. 6B). The stacked bar charts were normalized over
the course of the trajectory. The results documented that the interaction fraction value with
Tyr326 was 0.6. This clearly recommended that 60% simulation time from the 50 ns was
maintained by the Tyr326 hydrophobic interaction with the ligand. The other hydrophobic
networks were with amino acids such as Phe168, Tyr188, Ile199, Ile316, Tyr 398 and Tyr435.
4. Discussion
In-line with structure activity relations (SARs) regarding the effect of electron-accepting
or -donating groups at the para position of the phenyl of 1-methyl, 5-phenyl
thiosemicarbazones, inhibitory activities improved when electron donating groups such as
methoxyl, hydroxyl, dimethylamino, methyl, or ethyl were introduced on the para position of
the phenyl ring. Several previous studies have concluded that the presence of electron donating
groups on the phenyl group promotes selective MAO-B inhibition [48-51]. As regards halogen
10

substitution, chlorine or bromine improved MAO-B inhibitory activity more than fluorine
substitution (residual activities were 63% and 62% at 10.0 μM, respectively). Fluorine
substitution resulted in weak MAO-A and MAO-B inhibitions with residual activities of 96%
and 78% at 10.0 μM, respectively. Interestingly, the substitution of three fluorine atoms in
MT11 at para position of phenyl ring (trifluoromethyl) moderately inhibited MAO-B (residual
activity 63% at 10.0 μM).
MT1-11 weakly inhibited MAO-A (all IC50 values > 10.0 µM), which concurs with
previous studies on aryl thiosemicarbazones that methyl substitution on the terminal amino
group has little impact on MAO-A or B inhibitors. These results suggest that the presence of a
small lipophilic unit on the terminal amino group reduces inhibitory activities for MAO-A, B,
and AChE, and that the presence of lipophilic groups on the phenyl system of 1-methyl, 5-
phenyl thiosemicarbazones greatly influences on MAO-A and MAO-B inhibitions. The
common SAR principles of aryl methylthiosemicarbazones /thiosemicarbazones, as elucidated
by our previous studies, are summarized in Fig. 6. Interestingly, of the 11 synthesized
compounds, MT7, which possessed an electron withdrawing nitro group inhibited AChE most
(residual activity 63% at 10.0 μM).
Kinetic studies were carried out on MT5, which inhibited most potently MAO-B in the
present study. A set of LB plots was created in the presence or absence of compound MT5 at
four concentrations totally. The plots showed that lines were linear and intersected the y-axis,
indicating MT5 interacts competitively with the active site of hMAO-B. The reversibility
studies of MAO–B inhibition by MT5 was inspected by dialysis, and compared with the
reversibilities of a reversible and an irreversible MAO-B inhibitor, i.e., lazabemide and
paragyline, respectively. Percent recoveries suggested that MT5 is a better reversible MAO-B
11

inhibitor than lazabemide. Reversible inhibitors have considerable therapeutic advantages over
irreversible inhibitors due to less target disruption, shorter action durations, and better
pharmacokinetic properties [52, 53].
The binding mode of MT5 was investigated by AutoDock4.2 simulation. Architecturally
MAO-B has two cavities, that is, an “entrance cavity” and a “substrate cavity” [54]. These two
cavities are fused or separated by Ile199, which acts as a gating residue [55], and the fusing
and binding of cavities normally depends on the nature of the inhibitor [56-58]. Docking studies
showed that the side chain thiomethylsemicarbazone unit was anchored towards the FAD unit
of the MAO-B substrate cavity and that the para substituted dimethylamino group on the
phenyl group was efficiently positioned in the entrance cavity of MAO-B (Fig. 7). The electron
donating dimethyl amino group enhances lipophilic character and might enhance binding
between MT5 and the MAO-B. The MD further predicted that the lead molecule MT5 had a
major π–π hydrophobic interaction with Tyr326, suggesting that it plays an important role in
the stabilization of protein-ligand interaction.
5. Conclusions
Eleven 1-methyl, 5-phenyl thiosemicarbazones were synthesized and investigated for their
MAO and AChE inhibitory profiles. Inhibitions were dependent on the natures of substituents
on the phenyl ring at para substitution. Substituents with an electron-donating group such as
the dimethylamino group effectively inhibited MAO-B. Our kinetic and reversibility studies
revealed that MT5 competitively and reversibly inhibited MAO-B, and our cytotoxicity study
showed MT5 is non-toxic to normal VERO cell lines. Reassuringly, calculated inhibition by
docking simulation and experimentally determined inhibition agreed well. The hypothetical
12

binding mode of the MT5 in the IBC of MAO-B is also well established. Summarizing, the
study shows MT5 effectively, competitively and reversibly inhibits MAO-B and has a low
cytotoxic profile and suggests MT5 may be the forerunner of a new class of compounds with
therapeutic potential for the treatment of various neurodegenerative disorders.
Author statement
The manuscript is not submitted anywhere and the revision is submitted
conflict of intrest
No conflict of intrest
Acknowledgments
This research was supported by the National Research Foundation of Korea (NRF) grant
funded by the Republic of Korea government (NRF-2019R1A2C1088967) (to H. Kim). The
authors also thank Mr. Vinod Devaraji for MD study.
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21

Scheme 1. Synthetic route used to produce 1-methyl, 5-phenyl thiosemicarbazones (MT1-
MT11).
22

Fig. 1. Structures of known MAO-A and -B inhibitors and their uses.
23

(A)
4000
3500
3000
2500
2000
1500
1000
500
0
-10
0
10
20
30
40
1/[S], mM-1
(B)
24

120
100
80
60
40
20
0
-10
-5
0
5
10
15
20
25
[MT5], uM
Fig. 2. Lineweaver-Burk plots of MAO-B inhibition by [4-(dimethylamino)
phenyl]methylidene}-N-methylhydrazine-1-carbothioamide (MT5) (A), and a secondary plot
(B) of slopes vs. inhibitor concentrations. [MT5]: 5, 10, and 20 µM.
Undialyzed
Dialyzed
100
80
60
40
20
0
Fig. 3. Reversibility tests of MAO-B inhibition MT5 as determined by dialysis method.
25

Fig. 4. Hypothetical binding mode of MT5 in the active site of MAO-B. A hydrogen bonding
with the non-bonding electrons of terminal N-methyl group with Cys172 (2.012Å) was shown.
26

Fig. 5 (A) Protein-ligand root mean square deviation (RMSD); (B) Desmond-molecular
dynamics (MD) calculated protein−ligand contacts.
27

Fig. 6. Molecular recognition and structure activity relationships (SARs) of the aryl
methylthiosemicarbazones/thiosemicarbazones at the active site of MAO-B.
28

Fig. 7. A schematic of ligand atom interactions with protein residues. Simulation time in the
selected trajectory: 50 ns.
29