Novel 1,3,4-thiadiazole compounds as potential MAO-A inhibitors-design, synthesis, biological evaluation and molecular modelling

Article abstract of DOI:10.1039/d0md00150c

Monoamine oxidases (MAOs) are important drug targets for the management of neurological disorders. Herein, a series of new 1,3,4-thiadiazole derivatives bearing various alkyl/arylamine moieties as MAO inhibitors were designed and synthesized. All of the compounds were more selective against hMAO-A than hMAO-B. The half maximal inhibitory concentration (IC50) values of most of the compounds were lower than that of the common drug moclobemide (IC50 = 4.664 μM) and compound 6b was proven to be the most active compound (IC50 = 0.060 μM). Moreover, it was seen that compound 6b showed a similar inhibition profile to that of clorgyline (IC50 = 0.048 μM). The inhibition profile was found to be reversible and competitive for compound 6b with MAO-A selectivity. Molecular modelling studies aided in the understanding of the interaction modes between compound 6b and MAO-A. Furthermore, this compound was predicted to have a good pharmacokinetic profile and high BBB penetration. Therefore, such compounds are of interest towards developing new MAO inhibitors.

Full text of DOI:10.1039/d0md00150c

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RESEARCH ARTICLE
Novel 1,3,4-thiadiazole compounds as potential
MAO-A inhibitors – design, synthesis, biological
evaluation and molecular modelling†
Cite this: DOI: 10.1039/d0md00150c
ab
Begüm Nurpelin Sağlık, ab Betül Kaya Çavuşoğlu,
bc Ulviye Acar Çevik,
*
ab
Derya Osmaniye, ab Serkan Levent,
ab
a
Yusuf Özkay
and Zafer Asım Kaplancıklı
Monoamine oxidases (MAOs) are important drug targets for the management of neurological disorders.
Herein, a series of new 1,3,4-thiadiazole derivatives bearing various alkyl/arylamine moieties as MAO
inhibitors were designed and synthesized. All of the compounds were more selective against hMAO-A than
hMAO-B. The half maximal inhibitory concentration (IC50) values of most of the compounds were lower
than that of the common drug moclobemide (IC50 = 4.664 μM) and compound 6b was proven to be the
most active compound (IC50 = 0.060 μM). Moreover, it was seen that compound 6b showed a similar
inhibition profile to that of clorgyline (IC50 = 0.048 μM). The inhibition profile was found to be reversible
and competitive for compound 6b with MAO-A selectivity. Molecular modelling studies aided in the
understanding of the interaction modes between compound 6b and MAO-A. Furthermore, this compound
was predicted to have a good pharmacokinetic profile and high BBB penetration. Therefore, such
compounds are of interest towards developing new MAO inhibitors.
Received 7th May 2020,
Accepted 24th July 2020
DOI: 10.1039/d0md00150c
rsc.li/medchem
with
depression,
autism,
drug
addiction,
and
1. Introduction
neurodegenerative diseases.13,14
Monoamine oxidase (MAO) is a flavoenzyme that exists in all
mammalian tissues, placed at the outer membrane of the
mitochondria as an integral protein.1–3 The MAO enzyme has
2 isoforms: MAO-A and MAO-B. Their amino acid sequences
have 70% similarity.4,5 However, they are encoded from
separate genes and they have been identified by their
substrate selectivity, inhibitor sensitivity, and 3-dimensional
structures.6–8 Catalysis of the biological amine oxidative
deamination reaction is carried out by MAO isozymes.9 The
inhibition of noradrenaline, adrenaline, and serotonin is
carried out by MAO-A, while the inhibition of
phenylethylamine and benzylamine is carried out by MAO-B.
Both dopamine and tyramine are oxidized by the MAO-A and
MAO-B isoenzymes.10–12 Abnormal activation or a decrease in
levels of the MAO enzymes in humans has been associated
MAO isoenzymes have broad pharmacological importance
due to their roles in the metabolism of certain
neurotransmitters. In major depression-related diseases, a
deficit of monoamines is observed, basically norepinephrine
and serotonin, at critical synapses in the central nervous
system. In Parkinson's disease, the concentration of
dopamine is decreased.15 Selective MAO-A inhibitors are used
clinically as antidepressants and anxiolytics. MAO-B
inhibitors are used to reduce the progression of Parkinson's
disease, and manage symptoms related to Alzheimer's
disease.16–18 MAO-A and MAO-B inhibitors provide the
treatment of these diseases by inhibiting MAO isoenzymes
and thus regulating the reduced levels of the relevant
neurotransmitters. Considering the monoamine deamination
function of MAO, results have shown that MAO inhibitors
play a critical role in the therapy of severe neurological and
psychological disorders.19,20
Research in the area of medicinal chemistry has resulted
in a significant number of derivatives based on a variety of
oxygen-, sulphur-, and nitrogen-containing compounds.21–23
One of the main goals of medicinal chemistry is the discovery
of novel safer and better-tolerated therapeutic agents.
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu
University, Eskişehir, Turkey
b Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy,
Anadolu University, Eskişehir, Turkey
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Bulent Ecevit
University, 67600 Zonguldak, Turkey. E-mail: betul.kcavusoglu@beun.edu.tr;
Fax: +90 (372) 261 02 10; Tel: +90 (372) 261 31 54
Thiadiazoles are
a very important class of heterocyclic
compounds, possessing diverse biological activities, such as
antimicrobial,24 antitumor,25 antihypertensive,26 antifungal,27
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00150c
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antimycobacterial,28 antitubulin,29 antiviral,30 antioxidant,31
the desired compounds were achieved with 62–81% yields.
All of the acquired compounds were purified by
crystallization. The results of the spectral analysis performed
to characterize the structures of the compounds were in
accordance with the assigned structures.
antileishmanial,32
and
carbonic
anhydrase33
and
acetylcholinesterase inhibitory34 activities.
Some studies have indicated that the benzylamine moiety
possesses MAO inhibitory activity.35–37 Previously, our
research group synthesized new compounds and examined
them in terms of their hMAO inhibitory activity, mechanism
of enzyme inhibition and physicochemical properties.9,38–45
In one of our studies, compounds containing a benzothiazole
ring in addition to the benzylamine moiety were synthesized.
Some compounds displayed considerable inhibitory activity
against the MAO-A and MAO-B enzymes.46 In this study, new
compounds were designed and synthesized using the 1,3,4-
thiadiazole ring with a different alkyl/arylamine structure
instead of the benzothiazole ring (Fig. 1). Moreover, the
physicochemical properties and pharmacokinetic profiles of
the obtained compounds were predicted with the help of
computational methods. The in vitro fluorometric method
was conducted to evaluate their inhibition profiles on the
MAO isoenzymes. The mechanism of enzyme inhibition also
was determined by enzyme kinetic studies. Furthermore,
molecular docking studies were carried out to gain structural
insight into the binding modes of the synthesized
compounds in the enzyme active site.
In the 1H NMR spectra, CH2 protons bound to the
carbonyl group and nitrogen were observed at between 3.89–
4.02 ppm and 5.00–5.02 ppm, respectively. The signal
belonging to the NH proton was determined at between 7.70
and 7.94 ppm, in the spectra of compounds 6a–6g, which
had a 1,3,4-thiadiazole ring and an alkyl moiety both bound
to nitrogen. The same signal was detected at 10.35 ppm and
10.25 ppm, respectively, in compounds 6h and 6i, which
possessed 1,3,4-thiadiazole and phenyl rings both bound to
nitrogen. All of the aliphatic and aromatic protons and
carbons were assigned at their expected area. In the mass
spectra of the compounds, the [M + 1] peaks were in good
agreement with their calculated molecular weights (MWs).
2.2. MAO inhibition assay
All of the obtained 1,3,4-thiadiazole derivatives were
investigated for their inhibitory activity against the MAO
isoforms using a previously described in vitro fluorometric
method, which is based on the detection of H2O2 in a
horseradish peroxidase-coupled reaction using 10-acetyl-3,7-
dihydroxyphenoxazine (Amplex red reagent).
The assay was carried out in 2 steps. The first step was
carried out using concentrations of 10−3 and 10−4 M of all of
the synthesized compounds and reference inhibitors, namely
moclobemide, clorgyline, selegiline and safinamide. Next, the
selected compounds that displayed more than 50% inhibitory
activity at a concentration of 10−4 M were further tested,
along with the reference inhibitors, at concentrations of 10−5
to 10−11 M. The inhibition percentages and half maximal
inhibitory concentration (IC50) values of the test compounds
and reference inhibitors are presented in Table 1.
According to the enzyme inhibition results, none of the
synthesized compounds showed significant activity against
the hMAO-B enzyme. All of the obtained compounds
displayed selective inhibition of hMAO-A. At a concentration
of 10−3 M, all of the compounds showed more than 50%
inhibitory activity. Compounds 6a–6g were able to pass the
second step of the enzyme activity assay and their IC50 values
were calculated by performing an enzyme inhibition study at
concentrations of 10−5–10−11 M. It was shown that the
selected compounds displayed a more potent inhibition
2. Results and discussion
2.1. Chemistry
The synthetic route used in this work is outlined in
Scheme 1. The condensation of 3-nitrobenzaldehyde and
4-fluoroaniline at reflux yielded compound 1. Subsequently,
to synthesize compound 2, the imine bond was reduced in
methanol with sodium borohydride in portions. Next,
compound 3 was afforded with the reaction of compound 2
and chloroacetyl chloride in the presence of triethylamine.
Substituted isothiocyanate derivatives were reacted with
hydrazine hydrate to synthesize compounds 4a–4i. Nine
5-substituted amino-1,3,4-thiadiazole-2(3H)-thione derivatives
(5a–5i) were obtained via the ring closure reaction of
compounds 4a–4i with carbon disulfide in the presence of
potassium hydroxide. Finally, compounds 5a–5i were treated
with compound 3 in acetone in the presence of potassium
carbonate to acquire target compounds 6a–6i. Synthesis of
profile than the reference inhibitor moclobemide (IC50
=
4.664 0.235 μM). Moreover, it was seen that compound 6b
showed a similar inhibition profile to that of clorgyline (IC50
= 0.048 0.002 μM). Compound 6b, with the methoxyethyl
group bound to nitrogen at the fifth position of the
thiadiazole ring, appeared as the most potent MAO-A
inhibitor (IC50 = 0.060 0.002 μM), followed by compound
6c, with a propyl substituent (IC50 = 0.241 0.011 μM). The
inhibitory activity of the other compounds was determined in
Fig.
1 Structure of the designed compounds by combining a
substituted 1,3,4-thiadiazole ring (the red rectangle) with an arylamine
(the blue circle).
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Scheme 1 The synthetic pathway of compounds 6a–6i. Reagents and conditions: i: CH3COOH, EtOH, reflux, 8 h; ii: NaBH4, MeOH, room
temperature (r.t.), 10 h; iii: ClCH2COCl, TEA/THF, ice-bath, 5 h; iv: NH2NH2·H2O, EtOH, r.t., 4 h; v: (1) CS2/KOH, EtOH, reflux, 10 h; (2) HCl, pH 4–5;
vi: K2CO3, acetone, r.t., 8 h.
the order 6a (ethyl) > 6e (allyl) > 6d (isopropyl), 6f (butyl) >
6g (isobutyl). The replacement of these aliphatic groups with
aromatic phenyl (6h) or 4-methylphenyl (6i) groups led to
inhibition of this compound, linear Lineweaver–Burk graphs
were used. Substrate velocity curves in the absence and
presence of compound 6b were recorded. The secondary plots
of slope (Km/Vmax) versus varying concentrations of compound
6b (0, IC50/2, IC50, and 2(IC50)) were created to calculate the
Ki (intercept on the x-axis) value of this compound. The
graphical analyses of the steady-state inhibition data for
compound 6b are shown in Fig. 2.
According to the Lineweaver–Burk plots, a graph that
shows parallel lines without any cross-overs is observed for
uncompetitive inhibition. For mixed-type inhibition, a graph
with lines that do not intersect at the x-axis or the y-axis is
formed. Non-competitive inhibition is seen if the lines
intersect on the x-axis, and the slopes and y-intercepts are
different. In contrast, competitive inhibition has the opposite
result, wherein the plotted lines have the same y-intercept,
but there are diverse slopes and x-intercepts. Therefore, as
shown in Fig. 2, compound 6b was a competitive inhibitor
dramatically
diminished
MAO-A
inhibitory
activity.
Introduction of the propyl group (6c) (IC50 = 0.241 0.011
μM), increased the MAO-A inhibitory activity more than that
of the isopropyl group (6d) (IC50 = 1.073
0.051 μM). A
similar situation was observed between the compounds with
butyl (6f) (IC50 = 1.175 0.050 μM) and isobutyl (6g) groups
(IC50 = 1.786 0.079 μM).
2.3. Kinetic studies of enzyme inhibition
Enzyme kinetic studies were performed to determine the
mechanism of hMAO-A inhibition using a procedure similar
to that of the MAO inhibition assay. Compound 6b, which
was found to be the most potent agent, was included in the
enzyme kinetic studies. In order to estimate the type of
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Table 1 Inhibition percentage and IC50 values of the synthesized compounds, moclobemide, clorgyline, selegiline and safinamide against the MAO-A
and MAO-B enzymes
MAO-A %inhibition
Compounds 10−3 10−4
MAO-A
IC50
(μM)
MAO-B %inhibition
10−3 10−4
M
M
10−5
M
10−6
M
10−7
M
10−8
M
10−9
M
10−10
M
10−11
M
M
M
6a
6b
6c
6d
6e
6f
93.618
1.974
94.125
2.884
95.025
1.902
92.525
2.011
92.005
1.809
90.524
2.015
93.491
1.988
89.506
1.618
91.548
1.695
91.102
1.891
89.142
1.618
86.418
1.036
82.308
1.902
84.276
1.632
41.378
0.811
42.659
0.935
83.145
2.311
92.148
1.951
—
74.142
1.296
80.603
1.541
78.344
1.456
86.102
1.903
75.227
1.419
76.015
1.408
72.633
1.408
—
61.895
1.102
72.675
1.324
62.311
0.999
46.039
1.045
56.496
1.110
47.321
0.983
43.189
0.925
—
42.088
0.955
61.082
0.979
45.147
0.933
43.014
0.919
43.161
0.982
33.582
0.991
31.797
0.903
—
30.268
0.991
34.748
0.851
25.133
0.901
23.882
0.902
32.693
0.903
29.963
0.834
25.495
0.809
—
21.028
0.882
26.911
0.805
20.495
0.896
13.603
0.588
20.477
0.872
19.741
0.716
17.331
0.700
—
14.201
0.596
20.074
0.766
12.036
0.512
10.801
0.496
13.529
0.570
11.088
0.402
12.645
0.489
—
10.305
0.411
13.464
0.510
8.497
0.385
6.872
0.297
10.248
0.423
6.943
0.285
6.357
0.296
—
0.336
0.014
0.060
0.002
0.241
0.011
1.073
0.051
0.502
0.023
1.175
0.050
1.786
0.079
—
41.255
0.985
38.625
0.856
42.689
0.966
35.963
0.715
38.125
0.795
40.567
0.825
35.975
0.723
43.065
0.899
37.895
0.735
—
22.352
0.522
25.741
0.618
26.788
0.579
28.455
0.539
29.221
0.750
34.322
0.850
24.162
0.639
20.711
0.845
26.505
0.790
—
6g
6h
6i
89.475
1.465
87.560
1.288
—
—
—
—
—
—
—
—
Moclobemide 95.302
2.658
62.435
1.894
88.547
1.816
—
35.756
1.022
79.115
1.688
—
24.474
0.997
63.941
1.021
—
19.635
0.855
35.259
0.903
—
15.418
0.725
21.718
0.750
—
12.849
0.518
16.995
0.612
—
7.251
0.289
11.332
0.484
—
4.664
0.235
0.048
0.002
—
Clorgyline
Selegiline
Safinamide
96.381
2.055
—
—
—
98.258
1.052
97.375
2.048
96.107
1.165
94.455
1.274
—
—
—
—
—
—
—
—
—
—
with similar inhibition features to those of the substrates.
The Ki value for compound 6b was calculated as 0.033 μM for
the inhibition of hMAO-A.
respectively. These results verify that moclobemide is a
reversible inhibitor and clorgyline is an irreversible inhibitor
of the MAO-A enzyme. It was seen that MAO-A activity was
recovered up to 91.508 1.577% after dialysis for compound
6b and this finding indicated that the inhibition by
compound 6b was almost completely reversible. It was
concluded that this novel reversible inhibitor can have
considerable advantages compared to irreversible inhibitors
which may possess serious pharmacological side effects.
2.4. Reversibility of MAO-A inhibition
The clinical use of irreversible inhibitors has been reported
to be limited by some serious cardiovascular or
gastrointestinal side effects due to MAO inhibition in the
intestine, liver and endothelium.47 Moreover, it is seen that
the use of irreversible inhibitors has a disadvantage that the
turnover rate of the MAO biosynthesis in the brain reaches
normal values at only 40 days, when its use is discontinued.
Therefore, it is very important to develop reversible MAO
inhibitors with higher selectivity for either one of the
isoforms in order to avoid side effects.48 In the present study,
the reversibility of MAO-A inhibition by the most active
derivative, compound 6b, was determined by using a dialysis
method.49–51 Reversible (moclobemide) and irreversible
(clorgyline) MAO-A inhibitors were also incubated with the
MAO-A enzyme. Table 2 presents the results of reversibility
experiments.
2.5. Molecular docking studies
As mentioned in the MAO inhibition assay, compound 6b
was found to be the most active derivative in the series
against the hMAO-A enzyme. Therefore, docking studies were
carried out to evaluate its inhibition capability in silico. Using
the X-ray crystal structure of hMAO-A (PDB ID: 2Z5X),52
docking studies were performed and the binding modes of
compound 6b were assigned. The docking poses of this
compound are presented in Fig. 3–6. According to Fig. 3,
compound 6b adequately bound to amino acid residues
lining the cavity and was located very near the flavin adenine
dinucleotide (FAD) cofactor.
According to Table 2, the remaining MAO-A activities
before dialysis were found as 31.253
0.985% for
Fig. 4 presents the 3-dimensional interacting mode of
compound 6b in the active region of hMAO-A. When the
docking poses of this compound were analyzed, it was clearly
seen that there were many types of interactions, such as π–π
and cation–π interactions, and the formation of hydrogen
moclobemide, 88.315 1.578% for clorgyline and 25.956
0.951% for compound 6b. After dialysis, the remaining MAO-
A activities for moclobemide and clorgyline were calculated
as follows: 90.347
1.895% and 93.419
1.994%,
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Fig. 2 (A) Lineweaver–Burk plots for the inhibition of hMAO-A by compound 6b. [S], substrate concentration (μM); V, reaction velocity (nmol min−1
mg−1 protein). Inhibitor concentrations are shown at the left. (B) Secondary plot for the calculation of the steady-state inhibition constant (Ki) of
compound 6b. Ki was calculated as 0.033 μM.
bonds. Compound 6b had the nitro group at the 3rd position
of the phenyl ring. The nitrogen atom in the nitro group was
interacting with the phenyl of Tyr444 by cation–π interaction.
Similarly, the 4-fluorophenyl ring near the amide group
formed a π–π interaction with the phenyl of Phe352. Another
π–π interaction was observed between the 1,3,4-thiadiazole
ring and the phenyl of Phe208. Moreover, this 1,3,4-
thiadiazole ring created a hydrogen bond via its nitrogen
atom. The nitrogen atom formed a hydrogen bond with the
–SH of Cys323. Furthermore, it was understood from the
docking poses that the methoxyethylamine moiety
substituted at the 2nd position of the 1,3,4-thiadiazole ring
was very essential for polar interactions. The amino group
established a hydrogen bond with the carbonyl of Ala111.
The oxygen atom of the methoxy moiety at the end of the
structure formed another hydrogen bond with the amino of
Thr211. Actually, this interaction between the oxygen atom
and the amino of Thr211 was an indicator to explain the
difference in the enzyme inhibition profiles of the
synthesized compounds. It was thought that the substituents,
which were capable of the formation of a hydrogen bond,
such as the methoxyethyl moiety, strongly contributed to
binding to the active site of the enzyme. Thus, this situation
Table 2 Reversibility of the inhibition of hMAO-A by compound 6b,
moclobemide and clorgyline
Compounds incubated
hMAO-A activity
hMAO-A activity
with hMAO-A
before dialysis (%)
after dialysis (%)
With no inhibitor
Moclobemide
Clorgyline
6b
100 0.00
31.253 0.985
88.315 1.578
25.956 0.951
100 0.00
90.347 1.895
93.419 1.994
91.508 1.577
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interactions with amino acids Tyr69, Leu97, Phe108, Ala111,
Ile180, Asn181, Tyr197, Ile207, Phe208, Ser209, Gln215,
Cys323, Ile325, Ile335, Leu337, Thr339, Met350, Phe352,
Tyr407, Gly443, and Tyr444; displayed in pink and red
(Fig. 5), as described in the Glide user guide.53 Similarly,
promising electrostatic contributions of compound 6b were
determined with amino acids Tyr69, Ala111, Ile180, Asn181,
Tyr197, Ile207, Phe208, Val210, Gln215, Lys305, Cys323,
Ile335, Thr336, and Tyr444 (Fig. 6).
2.6. Molecular properties
Computational studies were used to determine whether a
particular molecule was similar to known drugs in terms of
its physicochemical properties, such as lipophilicity,
molecular size, flexibility, and bioavailability. Thereby, in the
early discovery, therapeutic opportunities expand through
consideration of its suitability to be developed as an oral
drug candidate within large numbers of compounds.
Prediction of the absorption, distribution, metabolism, and
excretion (ADME) properties of compounds 6a–6i was carried
out using QikProp 4.8 software (Schrödinger Inc.),54 which
calculates the violations of Lipinski's rule of five55 and
Jorgensen's rule of three.56 The results are presented in
Table 3.
Fig. 3 The three-dimensional pose of compound 6b in the active
region of hMAO-A (PDB ID: 2Z5X). This compound is represented by a
tube model and colored with maroon.
could explain why compound 6b exhibited
a stronger
inhibition profile than the other compounds, because only
this compound had such mentioned substituents, which
were different from the other compounds in the series.
In order to analyze the contribution of the van der Waals
and electrostatic interactions in binding to the enzyme active
site, the docking studies were detailed using Glide
(Schrödinger Inc., Portland, OR, USA) according to the Per-
Residue Interaction panel. Fig. 5 and 6 present the van der
Waals and electrostatic interactions of compound 6b. It can
be seen that this compound had favorable van der Waals
The computational study for the prediction of the ADME
properties of the molecules was performed by determination
of the molecular weight (MW), number of rotatable bonds
(RB), computed dipole moment (DM), total solvent-accessible
volume (MV), estimated number of hydrogen bond donors
(DHB), estimated number of hydrogen bond acceptors (AHB),
van der Waals surface area of polar nitrogen and oxygen
Fig. 4 The three-dimensional interacting mode of compound 6b in the active region of hMAO-A. The inhibitor and the important residues in the
active site of the enzyme are represented by a tube model. The FAD molecule is colored green with a ball and stick model.
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Fig. 5 The van der Waals interaction of compound 6b with the active region of hMAO-A. The active ligand has a lot of favorable van der Waals
interactions (red and pink).
Fig. 6 The electrostatic interaction of compound 6b with the active region of hMAO-A. The residues are colored (blue, red, and pink) according
to the distance from the ligand by the Per-Residue Interaction panel.
atoms and carbonyl carbon atoms (PSA), predicted octanol/
water partition coefficient (log P), predicted aqueous
solubility (log S), predicted apparent Caco-2 cell permeability
(PCaco), predicted brain/blood partition coefficient (log BB),
predicted apparent MDCK cell permeability (PMDCK),
number of likely metabolic reactions (PM), predicted human
oral absorption percent (% HOA), number of violations of
Lipinski's rule of five (VRF) and number of violations of
Jorgensen's rule of three (VRT). The molecular weights of all
of the compounds were smaller than 500 daltons, except for
that of compound 6i. When considering Lipinski's rule of
five, most of the compounds had log P values smaller than 5;
however, all of the compounds had positive PSA values, DHB
values between 0 and 6, and AHB values between 2 and 20.
Considering Jorgensen's rule of three, all of the compounds
had PCaco values bigger than 22 and PM values between 1
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Table 3 Calculated ADME parameters of compounds 6a–6i
Comp. MW
RB DM
MV
DHB AHB PSA
log P
log S
PCaco
log BB PMDCK PM % HOA VRF VRT
6a
6b
6c
6d
6e
6f
6g
6h
6i
447.501
477.527 11
461.528 10 13.286 1360.3
461.528
459.512 10
475.555 11
475.555 10 14.484 1363.6
495.545
509.572
9
5.539 1305.5
6.217 1398
1
1
1
1
1
1
1
1
1
6.5
8.2
6.5
6.5
6.5
6.5
6.5
6.5
6.5
111.04 4.117 −6.094 165.361 −1.729 246.297
120.47 4.173 −6.138 228.846 −1.708 498.468
4
5
4
4
5
4
4
5
5
90.757
93.612
90.464
90.808
91.927
86.033
92.855
80.813
76.599
0
0
0
0
0
1
0
1
2
1
1
1
1
0
1
1
1
1
114.67 4.427 −6.62
126.056 −1.952 234.915
9
9.849 1328.1
4.477 1271.5
7.351 1420.4
111.46 4.242 −6.164 151.441 −1.716 249.063
116.46 4.004 −4.364 209.254 −1.287 326.433
113.6
5.072 −7.157 232.388 −1.734 542.708
112.64 4.395 −6.126 175.686 −1.782 203.019
118.05 5.065 −7.392 119.309 −1.998 199.191
107.28 5.526 −7.687 259.671 −1.566 529.221
9
9
13.006 1430.2
8.779 1468.8
MW: molecular weight; RB: number of rotatable bonds (recommended value: 0–15); DM: computed dipole moment (recommended value: 1–
12.5); MV: total solvent-accessible volume (recommended value: 500–2000); DHB: estimated number of hydrogen bond donors (recommended
value: 0–6); AHB: estimated number of hydrogen bond acceptors (recommended value: 2–20); PSA: van der Waals surface area of polar nitrogen
and oxygen atoms and carbonyl carbon atoms (recommended value: 7–200); log P: predicted octanol/water partition coefficient (recommended
value: −2–6.5); log S: predicted aqueous solubility (recommended value: −6.5–0.5); PCaco: predicted apparent Caco-2 cell permeability
(recommended value: <25 poor, >500 great); log BB: predicted brain/blood partition coefficient (recommended value: −3–1.2); PMDCK:
predicted apparent MDCK cell permeability (recommended value: <25 poor, >500 great); PM: number of likely metabolic reactions
(recommended value: 1–8); % HOA: predicted human oral absorption percent (recommended value: >80% is high, <25% is poor); VRF:
number of violations of Lipinski's rule of five. The rules are: MW < 500, log P < 5, DHB ≤ 5, AHB ≤ 10, and a positive PSA value. VRT: number
of violations of Jorgensen's rule of three. The three rules are: log S > −5.7, PCaco > 22 nm s−1, and PM < 7.
and 8; however, the log S values were smaller than −5.7,
except for that of compound 6e.
increase the clinical potential of this promising class of
compounds.
The prediction of access to the central nervous system
(CNS) of the drug molecules is often expressed with log BB.
MAO inhibitors regulate local levels of neurotransmitters,
and therefore, may be able to pass through the blood–brain
barrier (BBB). The log BB values of all of the compounds were
within the recommended range of −3 to 1.2. Consequently,
most of the compounds were predicted to have a good
pharmacokinetic profile and high BBB penetration, which
enhanced the biological importance of these compounds in
the therapy of certain CNS disorders.
4. Experimental section
4.1. Chemistry
All chemicals were acquired from Sigma-Aldrich (Sigma-
Aldrich, St. Louis, MO, USA) and Merck Chemicals (Merck
KGaA, Darmstadt, Germany). All melting points (m.p.) were
recorded using an Electrothermal 9100 digital melting point
apparatus (Electrothermal, Essex, UK) and were uncorrected.
All reactions were monitored by thin-layer chromatography
(TLC) using silica gel 60 F254 TLC plates (Merck).
Spectroscopic data were registered with the following
instruments: IR, Shimadzu 8400S spectrophotometer (Tokyo,
Japan); 1H NMR, Bruker DPX 300 NMR spectrometer
(Billerica, MA, USA), and 13C NMR, Bruker DPX 75 NMR
spectrometer (Bruker) in DMSO-d6, using tetramethylsilane
(TMS) as the internal standard; HRMS, Shimadzu LC/MS
ITTOF system (Shimadzu).
3. Conclusion
Herein, 9 novel 1,3,4-thiadiazole compounds were reported
and evaluated for their inhibitory properties towards the
hMAO enzymes. All of the compounds were proven to be
more selective towards hMAO-A than hMAO-B. For the 7
most potent compounds, the IC50 values were determined
against hMAO-A. The IC50 values of the studied compounds
were lower (0.060–1.786 μM) when compared to that of the
reference, moclobemide (IC50 = 4.664 μM), and compound
6b was selected as the most potent compound (IC50 = 0.060
μM). Moreover, it was seen that compound 6b showed a
similar inhibition profile to that of clorgyline (IC50 = 0.048
μM). Further studies revealed that the type of inhibition of
this compound was reversible and competitive, and the
inhibitory activity of compound 6b was supported by its
molecular interactions with hMAO-A. In addition, this
compound was predicted to have a good pharmacokinetic
profile and high BBB penetration. Based on the results, it
was concluded that in the treatment of neurological
diseases, as MAO inhibitors, more studies are warranted to
4.1.1. Synthesis of 4-fluoro-N-(3-nitrobenzylidene)aniline
(1)38. 3-Nitrobenzaldehyde (33 mmol, 5 g) and 4-fluoroaniline
(33 mmol, 4.22 g) were refluxed in ethanol (100 mL) for 8 h
with a catalytic amount of glacial acetic acid (0.5 mL). After
TLC screening, the mixture was cooled, and the precipitated
product was filtered and recrystallized from ethanol. The
structural analysis and yield were consistent with the
literature values.
4.1.2. Synthesis of 4-fluoro-N-(3-nitrobenzyl)aniline (2)38
.
To a methanolic (100 mL) solution of compound 1 (20 mmol,
5.21 g), sodium borohydride was added in 4 portions (4 × 0.5
g) at 15 min intervals. After addition, the reaction mixture
was allowed to stir for 1 h at room temperature. Methanol
was evaporated under reduced pressure, and the crude
product was washed with water, dried and recrystallized from
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ethanol. The structural analysis and yield were consistent
with the literature values.
7.69 (1H, d, J = 7.8 Hz, Ar–H), 7.87 (1H, t, J = 5.3 Hz, NH),
8.07–8.13 (2H, m, Ar–H). 13C NMR (75 MHz, DMSO-d6, ppm)
δ 38.27, 44.40, 52.30, 58.41, 70.40, 117.02 (d, J= 22.8 Hz),
122.80, 123.26, 130.38, 130.90 (d, J = 9.0 Hz), 135.19, 137.73
(d, J = 2.9 Hz), 139.73, 148.25, 149.57, 161.82 (d, J = 244.7
4.1.3. Synthesis of 2-chloro-N-(4-fluorophenyl)-N-(3-
nitrobenzyl)acetamide (3)38. Chloroacetyl chloride (0.024 mol,
1.91 mL) was added dropwise by stirring into a mixture of
compound 2 (0.02 mol, 5.41 g) and triethylamine (0.024 mol,
3.35 mL) in tetrahydrofuran (80 mL) at 0–5 °C. After
completion of the dripping, the reaction mixture was stirred
for additional 1 h at room temperature. The solvent was
evaporated under reduced pressure, and the product was
washed with water, dried and recrystallized from ethanol.
The structural analysis and yield were consistent with the
literature values.
Hz), 167.58, 169.75. HRMS (m/z): [M
C20H20FN5O4S2: 478.1014; found 478.0995.
+
H]+ calcd for
4.1.6.3.
N-(4-Fluorophenyl)-2-[(5-(propylamino)-1,3,4-
thiadiazol-2-yl)thio]-N-(3-nitrobenzyl)acetamide (6c). Yield 79%.
1H NMR (300 MHz, DMSO-d6, ppm) δ 0.88 (3H, t, J = 7.4 Hz,
CH3), 1.48–1.60 (2H, m, CH3–CH_2–CH2–), 3.14–3.20 (2H, m,
CH3–CH2–CH_2–), 3.89 (2H, s, CO–CH2), 5.00 (2H, s, N–CH2),
7.24 (2H, t, J = 8.7 Hz, Ar–H), 7.32–7.37 (2H, m, Ar–H), 7.59
(1H, t, J = 7.9 Hz, Ar–H), 7.69 (1H, d, J = 7.7 Hz, Ar–H), 7.78
(1H, t, J = 5.4 Hz, NH), 8.07–8.13 (2H, m, Ar–H). 13C NMR (75
MHz, DMSO-d6, ppm) δ 11.81, 22.21, 38.34, 46.82, 52.31,
117.01 (d, J = 22.6 Hz), 122.79, 123.26, 130.36, 130.90 (d, J =
8.9 Hz), 135.19, 137.72 (d, J = 2.9 Hz), 139.73, 148.24, 149.05,
161.82 (d, J = 246.0 Hz), 167.60, 170.02. HRMS (m/z): [M + H]+
calcd for C20H20FN5O3S2: 462.1064; found 462.1044.
4.1.4. Synthesis of 4-substituted thiosemicarbazides
(4a–4i)44. A mixture of substituted isothiocyanate (20 mmol)
and hydrazine hydrate (40 mmol) in ethanol (50 mL) was
stirred at room temperature for
4 h. The precipitated
compound was filtered and crystallized from ethanol. The
structural analysis and yields were consistent with the
literature values.
4.1.5. Synthesis of 5-substituted amino-1,3,4-thiadiazole-
2(3H)-thiones (5a–5i)44. Carbon disulfide (27 mmol, 1.6 mL)
4.1.6.4.
N-(4-Fluorophenyl)-2-[(5-(isopropylamino)-1,3,4-
thiadiazol-2-yl)thio]-N-(3-nitrobenzyl)acetamide (6d). Yield 74%.
1H NMR (300 MHz, DMSO-d6, ppm) δ 1.15 (6H, d, J = 6.4 Hz,
2CH3), 3.68–3.79 (1H, m, CH), 3.89 (2H, s, CO–CH2), 5.00
(2H, s, N–CH2), 7.24 (2H, t, J = 8.8 Hz, Ar–H), 7.32–7.37 (2H,
m, Ar–H), 7.59 (1H, t, J = 7.9 Hz, Ar–H), 7.68–7.70 (2H, m,
Ar–H and NH), 8.07–8.13 (2H, m, Ar–H). 13C NMR (75 MHz,
DMSO-d6, ppm) δ 22.56, 38.34, 46.96, 52.29, 117.01 (d, J =
23.2 Hz), 122.79, 123.25, 130.36, 130.90 (d, J = 8.8 Hz),
135.19, 137.71 (d, J = 2.9 Hz), 139.72, 148.24, 149.03, 161.82
(d, J = 245.2 Hz), 167.60, 168.98. HRMS (m/z): [M + H]+ calcd
for C20H20FN5O3S2: 462.1064; found 462.1043.
was added to
a
solution of the 4-substituted
thiosemicarbazides (4a–4i) and potassium hydroxide in
ethanol, and the mixture was refluxed for 10 h. The solution
was cooled and acidified to a pH of 4–5 with a hydrochloric
acid solution and crystallized from ethanol. The structural
analysis and yields were consistent with the literature values.
4.1.6.
Synthesis
of
N-(4-fluorophenyl)-2-[(5-
substitutedamino-1,3,4-thiadiazol-2-yl)thio]-N-(3-nitrobenzyl)
acetamide derivatives (6a–6i). A mixture of 5-substituted
amino-1,3,4-thiadiazole-2(3H)-thione (5a–5i) (4 mmol) and
2-chloro-N-(4-chlorophenyl)-N-(4-nitrobenzyl)acetamide (3) (4
mmol) in acetone (40 mL) was stirred at room temperature
for 8 h in the presence of potassium carbonate (5 mmol, 0.66
g). After evaporation of the acetone, the residue was washed
with water and crystallized from ethanol.57
4.1.6.5.
N-(4-Fluorophenyl)-2-[(5-(allylamino)-1,3,4-
thiadiazol-2-yl)thio]-N-(3-nitrobenzyl)acetamide (6e). Yield 63%.
1H NMR (300 MHz, DMSO-d6, ppm) δ 3.85–3.90 (4H, m,
CH2CH–CH_2 and CO–CH2), 5.00 (2H, s, N–CH2), 5.10–5.25
(2H, m, CH_2CH–CH2), 5.82–5.94 (1H, m, CH2CH_–CH2),
7.24 (2H, t, J = 8.8 Hz, Ar–H), 7.32–7.37 (2H, m, Ar–H), 7.59
(1H, t, J = 7.8 Hz, Ar–H), 7.69 (1H, d, J = 7.7 Hz, Ar–H), 7.94
(1H, t, J = 5.6 Hz, NH), 8.07–8.13 (2H, m, Ar–H). 13C NMR (75
MHz, DMSO-d6, ppm) δ 38.34, 47.11, 52.32, 116.64, 117.02 (d,
J = 22.8 Hz), 122.79, 123.25, 130.37, 130.89 (d, J = 8.9 Hz),
134.76, 135.19, 137.72 (d, J = 2.9 Hz), 139.72, 148.24, 149.76,
161.82 (d, J = 244.8 Hz), 167.57, 168.79. HRMS (m/z): [M + H]+
calcd for C20H18FN5O3S2: 460.0908; found 460.0890.
4.1.6.1.
N-(4-Fluorophenyl)-2-[(5-(ethylamino)-1,3,4-
thiadiazol-2-yl)thio]-N-(3-nitrobenzyl)acetamide (6a). Yield 72%.
1H NMR (300 MHz, DMSO-d6, ppm) δ 1.14 (3H, t, J = 7.3 Hz,
CH2–CH_3), 3.21–3.26 (2H, m, CH_2–CH3), 3.90 (2H, s, CO–
CH2), 5.00 (2H, s, N–CH2), 7.24 (2H, t, J = 8.8 Hz, Ar–H), 7.32–
7.37 (2H, m, Ar–H), 7.59 (1H, t, J = 7.9 Hz, Ar–H), 7.69 (1H, d,
J = 7.9 Hz, Ar–H), 7.76 (1H, t, J = 5.2 Hz, NH), 8.07–8.13 (2H,
m, Ar–H). 13C NMR (75 MHz, DMSO-d6, ppm) δ 14.64, 38.34,
39.85, 52.31, 117.01 (d, J = 22.7 Hz), 122.78, 123.25, 130.35,
130.89 (d, J = 8.8 Hz), 135.18, 137.72 (d, J = 2.8 Hz), 139.72,
148.23, 149.17, 161.82 (d, J = 245.8 Hz), 167.59, 169.81. HRMS
(m/z): [M + H]+ calcd for C19H18FN5O3S2: 448.0908; found
448.0891.
4.1.6.2. N-(4-Fluorophenyl)-2-[(5-((methoxyethyl)amino)-1,3,4-
thiadiazol-2-yl)thio]-N-(3-nitrobenzyl)acetamide (6b). Yield 71%.
1H NMR (300 MHz, DMSO-d6, ppm) δ 3.26 (3H, s, OCH3),
3.41 (2H, t, J = 5.0 Hz, CH2), 3.45–3.49 (2H, m, CH2), 3.89
(2H, s, CO–CH2), 5.00 (2H, s, N–CH2), 7.24 (2H, t, J = 8.7 Hz,
Ar–H), 7.32–7.37 (2H, m, Ar–H), 7.60 (1H, t, J = 7.9 Hz, Ar–H),
4.1.6.6.
N-(4-Fluorophenyl)-2-[(5-(butylamino)-1,3,4-
thiadiazol-2-yl)thio]-N-(3-nitrobenzyl)acetamide (6f). Yield 62%.
1H NMR (300 MHz, DMSO-d6, ppm) δ 0.88 (3H, t, J = 7.4 Hz,
CH3), 1.26–1.38 (2H, m, CH2), 1.46–1.55 (2H, m, CH2), 3.17–
3.24 (2H, m, CH2), 3.89 (2H, s, CO–CH2), 5.00 (2H, s, N–CH2),
7.24 (2H, t, J = 8.8 Hz, Ar–H), 7.32–7.37 (2H, m, Ar–H), 7.59
(1H, t, J = 7.9 Hz, Ar–H), 7.69 (1H, d, J = 7.7 Hz, Ar–H), 7.76
(1H, t, J = 5.4 Hz, NH), 8.07–8.13 (2H, m, Ar–H). 13C NMR (75
MHz, DMSO-d6, ppm) δ 14.08, 19.99, 30.98, 38.34, 44.70,
52.29, 117.01 (d, J = 22.3 Hz), 122.79, 123.26, 130.36, 130.90
(d, J = 8.8 Hz), 135.19, 137.72 (d, J = 2.9 Hz), 139.73, 148.24,
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149.04, 161.82 (d, J = 246.0 Hz), 167.59, 169.97. HRMS (m/z):
The solutions of inhibitors (20 μL per well) and hMAO-A
(100 μL per well) or hMAO-B (100 μL per well) were added to
a black flat-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 (100 μL per well).
The mixture was incubated at 37 °C for 30 min and the
fluorescence (Ex/Em = 535/587 nm) was measured at 5 min
[M + H]+ calcd for C21H22FN5O3S2: 476.1221; found 476.1213.
4.1.6.7.
N-(4-Fluorophenyl)-2-[(5-(isobutylamino)-1,3,4-
thiadiazol-2-yl)thio]-N-(3-nitrobenzyl)acetamide (6g). Yield 81%.
1H NMR (300 MHz, DMSO-d6, ppm) δ 0.88 (6H, d, J = 6.7 Hz,
2CH3), 1.78–1.91 (1H, m, CH), 3.02–3.06 (2H, m, CH2), 3.89
(2H, s, CO–CH2), 5.00 (2H, s, N–CH2), 7.24 (2H, t, J = 8.8 Hz,
Ar–H), 7.32–7.37 (2H, m, Ar–H), 7.59 (1H, t, J = 7.9 Hz, Ar–H),
7.69 (1H, d, J = 7.7 Hz, Ar–H), 7.81 (1H, t, J = 5.6 Hz, NH),
8.07–8.12 (2H, m, Ar–H). 13C NMR (75 MHz, DMSO-d6, ppm)
δ 20.49, 27.94, 38.33, 52.30, 52.63, 117.01 (d, J = 22.7 Hz),
122.78, 123.26, 130.35, 130.89 (d, J = 8.8 Hz), 135.19, 137.72
(d, J = 2.8 Hz), 139.72, 148.24, 149.00, 161.82 (d, J = 246.0
Hz), 167.60, 170.16 (CO). HRMS (m/z): [M + H]+ calcd for
C21H22FN5O3S2: 476.1221; found 476.1210.
intervals.
Control
experiments
were
carried
out
simultaneously by replacing the inhibitor solution with 2%
DMSO (20 μL). To check the probable inhibitory effect of the
inhibitors on horseradish peroxidase, a parallel reading was
performed by replacing the enzyme solutions with a 3% H2O2
solution (20 mM, 100 μL per well). In 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 the inhibitor and
working solutions.
The specific fluorescence emission that was used to obtain
the final results was calculated after subtraction of the
background activity, which was determined from vials
containing all of the components, except the hMAO isoforms,
which were replaced by a phosphate buffer (100 μL per well).
The blank, control, and all of the concentrations of the
inhibitors were analyzed in quadruplicate and the percent of
inhibition was calculated using the following equation:
4.1.6.8.
N-(4-Fluorophenyl)-2-[(5-(phenylamino)-1,3,4-
thiadiazol-2-yl)thio]-N-(3-nitrobenzyl)acetamide (6h). Yield 74%.
1H NMR (300 MHz, DMSO-d6, ppm) δ 4.02 (2H, s, CO–CH2),
5.02 (2H, s, N–CH2), 6.99 (1H, t, J = 7.4 Hz, Ar–H), 7.23–7.41
(6H, m, Ar–H), 7.54–7.62 (3H, m, Ar–H), 7.71 (1H, d, J = 7.7
Hz, Ar–H), 8.09–8.12 (2H, m, Ar–H), 10.35 (1H, s, NH). 13C
NMR (75 MHz, DMSO-d6, ppm) δ 38.18, 52.36, 117.07 (d, J =
22.8 Hz), 117.80, 122.41, 122.81, 123.27, 129.56, 130.35,
130.93 (d, J = 8.9 Hz), 135.20, 137.71 (d, J = 2.8 Hz), 139.71,
140.82, 148.25, 152.57, 161.87 (d, J = 245.8 Hz), 165.15,
167.45 (CO). HRMS (m/z): [M
C23H18FN5O3S2: 496.0908; found 496.0891.
4.1.6.9. N-(4-Fluorophenyl)-2-[(5-(4-methylphenylamino)-
+
H]+ calcd for
ðFCt2 − FCt1Þ − ðFIt2 − FIt1Þ
%Inhibition ¼
× 100
FCt2 − FCt1
1,3,4-thiadiazol-2-yl)thio]-N-(3-nitrobenzyl)acetamide (6i). Yield
67%. H NMR (300 MHz, DMSO-d6, ppm) δ 2.24 (3H, s, CH3),
FCt2: fluorescence of a control well measured at time t2; FCt1:
1
fluorescence of a control well measured at time t2; FIt2:
fluorescence of an inhibitor well measured at time t2; FIt1:
fluorescence of an inhibitor well measured at time t1. 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).
4.01 (2H, s, CO–CH2), 5.02 (2H, s, N–CH2), 7.13 (2H, d, J = 8.3
Hz, Ar–H), 7.26 (2H, t, J = 8.8 Hz, Ar–H), 7.36–7.41 (2H, m,
Ar–H), 7.44 (2H, d, J = 8.5 Hz, Ar–H), 7.59 (2H, t, J = 7.9 Hz,
Ar–H), 7.71 (1H, d, J = 7.7 Hz, Ar–H), 8.08–8.13 (2H, m, Ar–
H), 10.25 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6, ppm) δ
20.82, 38.22, 52.35, 117.06 (d, J = 23.0 Hz), 117.92, 122.81,
123.26, 129.94, 130.35, 130.93 (d, J = 9.0 Hz), 131.39, 135.20,
137.70 (d, J = 2.8 Hz), 138.45, 139.71, 148.24, 152.06, 161.86
(d, J = 246.2 Hz), 165.36, 167.46 (CO). HRMS (m/z): [M + H]+
calcd for C24H20FN5O3S2: 510.1064; found 510.1047.
4.3. Kinetic studies of enzyme inhibition
A protocol similar to that of the MAO inhibition assay was
followed using the same materials. The most active
compound, 6b, was tested at concentrations of IC50/2, IC50,
and 2(IC50). The solutions of compound 6b (20 μL per well)
and the MAO-A (0.5 U mL−1, 100 μL per well) enzyme were
added to a black flat-bottomed 96-well micro test plate, and
incubated at 37 °C for 30 min. Once incubation was
completed, the working solution, comprising various
concentrations (20, 10, 5, 2.5, 1.25, and 0.625 μM) of
substrate (100 μL per 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 an inhibitor.
All of the experiments were performed in quadruplicate. The
experimental data were analysed as Lineweaver–Burk plots
using Microsoft Office Excel 2013 (Microsoft Corp.,
Redmond, WA, USA). The Km/Vmax (slope) values of the
4.2. MAO inhibition assay
The MAO inhibition potency of the synthesized compounds
was determined according to the assay reported in our recent
study.40 In the enzymatic assay, three different daily prepared
solutions were used. I) Inhibitor solutions: Synthesized
compounds and reference agents were prepared in 2% DMSO
in 10−3–10−11
M concentrations. II) Enzyme solutions:
Recombinant hMAO-A (0.5 U mL−1) and recombinant hMAO-
B (0.64 U mL−1) enzymes were dissolved in phosphate buffer
and the final volumes were adjusted to 10 mL. III) Working
solution: Horseradish peroxidase (200 U mL−1, 100 μL),
Ampliflu™ red (20 mM, 200 μL) and tyramine (100 mM, 200
μL) were dissolved in phosphate buffer and the final volume
was adjusted to 10 mL.
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Lineweaver–Burk plots were re-plotted versus the inhibitor Conflicts of interest
concentration to determine the Ki values from the x-axis
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to have influenced the work reported in this paper.
intercept as −Ki.
4.4. Reversibility of MAO-A inhibition
Reversibility of the MAO-A inhibition with compounds References
(moclobemide, clorgyline and compound 6b) was determined
1 D. Chavarria, C. Fernandes, V. Silva, C. Silva, E. Gil-Martins,
P. Soares, T. Silva, R. Silva, F. Remiao, P. J. Oliveira and F.
Borges, Eur. J. Med. Chem., 2020, 185, 111770.
2 D. G. T. Parambi, J. M. Oh, S. C. Baek, J. P. Lee, A. R. Tondo,
O. Nicolotti, H. Kim and B. Mathew, Bioorg. Chem., 2019, 93,
103335.
by
a
dialysis method previously described.49–51 Dialysis
tubing 16 × 25 mm (Sigma, Germany) with a molecular
weight cut-off of 12 000 Da and a sample capacity of 0.5–10
mL was used. Adequate amounts of the recombinant hMAO-A
enzyme (1 U mL−1) and compound 6b at a concentration
equal to four fold the IC50 values, for the inhibition of hMAO-
A, were incubated in potassium phosphate buffer (0.05 M,
pH 7.4, 5% sucrose containing 1% DMSO) for 15 min at 37
°C. Other sets were prepared by preincubation of the same
amount of hMAO-A with the reference inhibitors. Enzyme–
inhibitor mixtures were subsequently dialyzed at 4 °C in 80
mL of dialysis buffer (100 mM potassium phosphate, pH 7.4,
5% sucrose). The dialysis buffer was replaced with fresh
buffer two times during the 24 h of dialysis. After dialysis,
residual MAO-A activities were measured. All reactions were
carried out in triplicate and the residual enzyme activity was
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expressed as mean
mixtures of MAO-A and the inhibitors were included in the
study.
SEM. For comparison, undialyzed
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4.5. Molecular docking studies
A structure-based in silico procedure was applied to discover
the binding modes of compound 6b to the hMAO-A enzyme
active site. The crystal structures of hMAO-A (PDB ID:
2Z5X),52 which was crystallized with harmine, were retrieved
from the Protein Data Bank server (www.pdb.org).
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Schrödinger Maestro58 interface and were then submitted
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Schrödinger Suite 2016 Update 2.59 The ligands were
prepared using LigPrep 3.8 (Schrödinger Inc.)60 to
correctly assign the protonation states at a pH of 7.4
1.0 and the atom types. Bond orders were assigned,
and hydrogen atoms were added to the structures. The
grid generation was performed using Glide 7.1.53 The
grid box, with dimensions of 20 Å × 20 Å × 20 Å, was
cantered in the vicinity of the FAD N5 atom on the
catalytic site of the protein to cover all of the binding
sites and neighbouring residues.61–63 Flexible docking
runs were performed in single-precision docking mode
(SP).
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4.6. Molecular properties
Physicochemical properties of the compounds were predicted
through QikProp 4.8 software.54
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