Design, synthesis, in vitro and in silico studies of new thiazolylhydrazine-piperazine derivatives as selective MAO-A inhibitors

Article abstract of DOI:10.3390/molecules25184342

Monoamine oxidase (MAO) isoenzymes are very important drug targets among neurological disorders. Herein, novel series of thiazolylhydrazine-piperazine derivatives were designed, synthesized and evaluated for their MAO-A and -B inhibitory activity. The structures of the synthesized compounds were assigned using different spectroscopic techniques such as ¹H-NMR, ¹³C-NMR and HRMS. Moreover, the prediction of ADME (Absorption, Distribution, Metabolism, Elimination) parameters for all of the compounds were performed using in silico method. According to the enzyme inhibition results, the synthesized compounds showed the selectivity against MAO-A enzyme inhibition. Compounds 3c, 3d and 3e displayed significant MAO-A inhibition potencies. Among them, compound 3e was found to be the most effective derivative with an IC₅₀ value of 0.057 ± 0.002 μM. Moreover, it was seen that this compound has a more potent inhibition profile than the reference inhibitors moclobemide (IC₅₀ = 6.061 ± 0.262 μM) and clorgiline (IC₅₀ = 0.062 ± 0.002 μM). In addition, the enzyme kinetics were performed for compound 3e and it was determined that this compound had a competitive and reversible inhibition type. Molecular modeling studies aided in the understanding of the interaction modes between this compound and MAO-A. It was found that compound 3e had significant and important binding property.

Full text of DOI:10.3390/molecules25184342

molecules
Article
Design, Synthesis, In Vitro and In Silico Studies of
New Thiazolylhydrazine-Piperazine Derivatives as
Selective MAO-A Inhibitors
Begüm Nurpelin Sag˘lık 1,2 , Osman Cebeci 1, Ulviye Acar Çevik 1,2, Derya Osmaniye 1,2,*,
Serkan Levent 1,2 , Betül Kaya Çavus¸og˘lu 3, Sinem Ilgın 4, Yusuf Özkay 1,2 and
Zafer Asım Kaplancıklı 1
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University,
Eskis¸ehir 26470, Turkey; bnsaglik@anadolu.edu.tr (B.N.S.); ocebeci@gmail.com (O.C.);
uacar@anadolu.edu.tr (U.A.Ç.); serkanlevent@anadolu.edu.tr (S.L.); yozkay@anadolu.edu.tr (Y.Ö.);
zakaplan@anadolu.edu.tr (Z.A.K.)
2
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University,
Eskis¸ehir 26470, Turkey
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Zonguldak Bülent Ecevit University,
Zonguldak 67600, Turkey; betul.kcavusoglu@beun.edu.tr
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University,
4
Eskis¸ehir 26470, Turkey; silgin@anadolu.edu.tr
*
Correspondence: dosmaniye@anadolu.edu.tr; Tel.: +90-222-335-0580/3778
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Tiziano Tuccinardi
Received: 22 August 2020; Accepted: 20 September 2020; Published: 22 September 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Monoamine oxidase (MAO) isoenzymes are very important drug targets among neurological
disorders. Herein, novel series of thiazolylhydrazine-piperazine derivatives were designed,
synthesized and evaluated for their MAO-A and -B inhibitory activity. The structures of the
synthesized compounds were assigned using different spectroscopic techniques such as 1H-NMR,
13C-NMR and HRMS. Moreover, the prediction of ADME (Absorption, Distribution, Metabolism,
Elimination) parameters for all of the compounds were performed using in silico method. According
to the enzyme inhibition results, the synthesized compounds showed the selectivity against MAO-A
enzyme inhibition. Compounds 3c
Among them, compound 3e was found to be the most effective derivative with an IC50 value of
0.057 0.002 M. Moreover, it was seen that this compound has a more potent inhibition profile than
the reference inhibitors moclobemide (IC50 = 6.061 0.262 M) and clorgiline (IC50 = 0.062 0.002 M).
, 3d and 3e displayed significant MAO-A inhibition potencies.
±
µ
±
µ
±
µ
In addition, the enzyme kinetics were performed for compound 3e and it was determined that this
compound had a competitive and reversible inhibition type. Molecular modeling studies aided in
the understanding of the interaction modes between this compound and MAO-A. It was found that
compound 3e had significant and important binding property.
Keywords: ADME properties; in vitro enzyme inhibition; molecular docking; monoamine oxidases;
thiazolylhydrazine; piperazine
1. Introduction
Monoamine oxidases (MAOs) containing flavin adenine dinucleotide (FAD) are enzymes that
catalyze the oxidative deamination of dietary amines and monoamine neurotransmitters [
two types of MAOs in mammals, MAO-A and MAO-B, defined by the cysteine amino acid (Cys406 in
MAO-A and Cys397 in MAO-B) bound covalently to their co-factor FAD [ ]. MAOs, which are about
70% similar at the amino acid sequence level, also have similar three-dimensional structures in which
1]. There are
2
Molecules 2020, 25, 4342; doi:10.3390/molecules25184342
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4342
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the active sites are highly conserved [3]. MAO-A and MAO-B are distinguished by the main differences,
which contain details of the relevant active regions that explain their differences in substrate and
inhibitor specificity [ ]. Serotonin is a common MAO-A substratum, whereas 2-phenylethylamine
and benzylamine are similar MAO-B substrates [ ]. By knowing these structural differences,
4
5,6
the rationalized drug design of isoform selective MAO inhibitors has been paved. MAO-A targeting
has an antidepressant effect while limited MAO-B antagonists are used to manage Parkinson’s
disease [6].
In many studies, in which our team has been involved in recent years, many thiazolylhydrazine
derivatives have been shown to exhibit MAO inhibitory activity in the micromolar concentration
range [7–15]. This study was carried out in order to further the activities of the compounds synthesized
by our team and showing MAO-A activity in our previous studies. In our previous study [15],
the pyrrole ring in the N1 position of the hydrazine did not contribute to the activity. Activity improved
significantly with the morpholine ring replacing the pyrrole ring. In the docking studies, it was
observed that the oxygen atom in the morpholine ring and the amino group in the Gly67 amino acid in
the active region interact with the hydrogen bond.
In the light of the findings above, new thiazolylhydrazine derivatives were synthesized in this
study to examine the MAO inhibitory activities. When designing the compounds to be synthesized,
based on the active derivatives obtained earlier [15], the piperazine ring was introduced instead of
the morpholine ring (Figure 1). Thus, the activity comparison of the piperazine ring relative to the
morpholine ring can be made. The methyl group was chosen as the substituent of the piperazine ring.
Depending on the contribution of the methyl group to the activity, other substituents may be tried in
later studies. In addition, as the substituents of the phenyl ring in the fourth position of the thiazole
ring, it was preferred activated groups (-NO2, -OCH3). Thus, the effect of these substituents on activity
will be seen.
O
N
S
N
R2
N
N
H
R1
piperazine ring was introduced
instead of the morpholine ring
activated groups added
N
N
S
N
R2
N
N
H
R1
Figure 1. Design of target compounds.

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2. Materials and Methods
2.1. Chemistry
The chemicals used in the synthesis process were obtained from either Merck Chemicals (Merck
KGaA, Darmstadt, Germany) or Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., Louis, MO, USA).
Thin layer chromatography (TLC) on silica gel 60 F254 aluminum sheets acquired from Merck
(Darmstadt, Germany) has identified the reactions and purities of the compounds. The MP90
automated melting point equipment (Mettler Toledo, Ohio, USA) registered melting points of the
1
synthesized compounds and were identified as uncorrected. H NMR and 13C NMR spectra were
reported in DMSO-d6 with a Bruker 300 MHz and a 75 MHz digital FT-NMR spectrometer (Bruker
Bioscience, Billerica, MA, USA). The patterns of splitting were defined as follows in the NMR spectra:
s: singlet; d: doublet; t: triplet; m: multiplet. Coupling constants (J) were reported as Hertz. Mass
spectra were collected using an ESI system on an LCMS-IT-TOF (Shimadzu, Kyoto, Japan).
2.1.1. General Procedure for the Synthesis of the Compounds
Synthesis of 4-(4-Methylpiperazin-1-yl)benzaldehyde (1)
1-Methylpiperazine (0.04 mol, 4.00 g), 4-fluorobenzaldehyde (0.040 mol, 4.96 g) and potassium
carbonate (0.048 mol, 6.62 g) were refluxed in dimethylformamide (10 mL) for 24 h. The mixture was
poured into ice water and filtered. Product 1 was recrystallized from ethanol [16].
1
4-(4-Methylpiperazin-1-yl)benzaldehyde (
1
): Yield: 77%, m.p. = oily. H-NMR (300 MHz, DMSO-d6):
δ
= 2.21 (3H, s, -CH3), 2.41 (4H, t, J = 5.1 Hz, piperazine), 3.36 (4H, t, J = 5.1 Hz, piperazine), 7.03 (2H, d,
J = 8.8 Hz, 1,4-disubstituted benzene), 7.70 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 9.71 (O=C-H).
13C-NMR (75 MHz, DMSO-d6):
δ = 46.15, 46.77, 54.70, 113.73, 126.69, 131.92, 155.19, 190.67. HRMS
(m/z): [M + H]+ calcd for C12H16N2O: 205.1335; found: 205.1328.
Synthesis of 2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinecarbothioamide (2)
Equal amounts of 4-(4-methylpiperazin-1-yl)benzaldehyde (1) (0.030 mol, 6.42 g) and
thiosemicarbazide (0.030 mol, 2.87 g) were refluxed for 3 h in ethanol (80 mL). After completion of
the reaction, the mixture was cooled, and the precipitated compound was filtered and recrystallized
from ethanol.
2-(4-(4-Methylpiperazin-1-yl)benzylidene)hydrazine-1-carbothioamide (
2
): Yield: 85%, m.p. = 227–229 ◦C.
1H-NMR (300 MHz, DMSO-d6):
δ
= 2.20 (3H, s, -CH3), 2.42 (4H, t, J = 4.8 Hz, piperazine), 3.21 (4H,
t, J = 4.7 Hz, piperazine), 6.92 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.60 (2H, d, J = 8.9 Hz,
1,4-Disubstituebenzene), 7.82 (1H, br s., -NH), 7.94 (1H, s, -CH=N-), 8.05 (1H, br s, -NH), 11.23 (1H, s,
-NH). 13C-NMR (75 MHz, DMSO-d6):
δ = 42.22, 47.59, 54.89, 114.87, 124.37, 128.94, 143.27, 152.42, 177.69.
HRMS (m/z): [M + H]+ calcd for C13H19N5S: 278.1434; found: 278.1426.
Synthesis of 4-(2,4-Disubstitutedphenyl)-2-{2-[4-(4-methylpiperazin-1-yl)benzylidene]hydrazinyl}
thiazoles (3a–3l)
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-phenylthiazole (3a): Yield 79%, m.p.
◦
1
254–255 C. H NMR (300 MHz, DMSO-d6, ppm)
δ 2.84 (3H, s, CH3), 3.33 (4H, br s, piperazine),
3.51 (4H, br s, piperazine), 7.05 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.29–7.31 (2H, m,
monosubstituted benzene, thiazole), 7.40 (2H, t, J = 7.3 Hz, 1,4-disubstituted benzene), 7.54 (2H, d,
J = 8.9 Hz, monosubstituted benzene), 7.85 (2H, d, J = 7.2 Hz, monosubstituted benzene), 7.97 (1H, s,
CH=N), 12.01 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6, ppm)
δ 42.59, 45.39, 52.56, 103.74, 115.99,
125.95, 126.17, 127.90, 127.96, 129.07, 135.19, 141.87, 150.59, 150.94, 168.77. HRMS (m/z): [M + H]+ calcd
for C21H23N5S: 378.1747; found: 378.1719.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-(4-methylphenyl)thiazole (3b): Yield 72%,
m.p. 252–254 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm)
δ 2.30 (3H, s, CH3), 2.86 (3H, s, CH3), 3.38

Molecules 2020, 25, 4342
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(4H, br s, piperazine), 3.45 (2H, br s, piperazine), 3.88 (2H, br s, piperazine), 7.05 (2H, d, J = 8.9 Hz,
1,4-disubstituted benzene), 7.19 (2H, d, J = 8.1 Hz, 1,4-disubstituted benzene), 7.20 (1H, s, thiazole),
7.54 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.73 (2H, d, J = 8.1 Hz, 1,4-disubstituted benzene),
7.97 (1H, s, CH=N), 11.98 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6, ppm)
δ 21.28, 42.46, 45.30, 52.47,
102.82, 116.01, 125.91, 126.24, 127.89, 129.63, 132.55, 137.19, 141.79, 150.52, 150.98, 168.68. HRMS (m/z):
[M + H]+ calcd for C22H25N5S: 392.1903; found: 392.1880.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-(4-methoxyphenyl)thiazole (3c): Yield 76%,
m.p. 226–228 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm) δ 2.86 (3H, s, CH3), 3.20 (4H, br s, piperazine),
3.53 (4H, br s, piperazine), 3.77 (3H, s, OCH3), 6.96 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene),
7.05 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.11 (1H, s, thiazole), 7.54 (2H, d, J = 8.8 Hz,
1,4-disubstituted benzene), 7.78 (2H, d, J = 8.8 Hz, 1,4-disubstituted benzene), 7.95 (1H, s, CH=N),
11.97 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6, ppm)
δ 42.48, 45.34, 52.50, 55.59, 101.56, 114.40, 116.02,
126.27, 127.28, 127.88, 128.07, 141.71, 150.51, 150.78, 159.18, 168.65. HRMS (m/z): [M + H]+ calcd for
C22H25N5OS: 408.1853; found: 408.1833.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-(4-cyanophenyl)thiazole (3d): Yield 82%,
m.p. 234–235 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm) δ 2.86 (3H, s, CH3), 3.16 (4H, br s, piperazine),
3.49 (2H, br s, piperazine), 3.94 (2H, br s, piperazine), 7.06 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene),
7.55 (2H, d, J = 8.8 Hz, 1,4-disubstituted benzene), 7.62 (1H, s, thiazole), 7.86 (2H, d, J = 8.5 Hz,
1,4-disubstituted benzene), 7.97 (1H, s, CH=N), 8.02 (2H, d, J = 8.5 Hz, 1,4-disubstituted benzene),
12.09 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6, ppm)
δ 42.49, 45.31, 52.51, 107.66, 109.96, 115.99,
119.49, 126.06, 126.55, 127.99, 133.17, 139.28, 142.33, 149.27, 150.63, 169.08. HRMS (m/z): [M + H]+ calcd
for C22H22N6S: 403.1699; found: 403.1672.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-(4-nitrophenyl)thiazole (3e): Yield 75%, m.p.
260–261 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm)
δ 2.87 (3H, s, CH3), 3.10 (4H, br s, piperazine), 3.44
(2H, br s, piperazine), 3.90 (2H, br s, piperazine), 7.05 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene),
7.54 (2H, d, J = 8.8 Hz, 1,4-disubstituted benzene), 7.68 (1H, s, thiazole), 7.98 (1H, s, CH=N), 8.09 (2H,
d, J = 8.9 Hz, 1,4-disubstituted benzene), 8.25 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 12.12 (1H,
s, NH). 13C NMR (75 MHz, DMSO-d6, ppm)
δ 42.47, 45.27, 52.48, 108.71, 115.97, 124.56, 126.01, 126.76,
127.99, 141.17, 142.42, 146.56, 148.95, 150.64, 169.16. HRMS (m/z): [M + H]+ calcd for C21H22N6O2S:
423.1598; found: 423.1584.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-(4-fluorophenyl)thiazole (3f): Yield 69%, m.p.
247–249 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm)
δ 2.85 (3H, s, CH3), 3.33 (4H, br s, piperazine), 3.53 (4H,
br s, piperazine), 7.06 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.20–7.26 (2H, m, 1,4-disubstituted
benzene), 7.28 (1H, s, thiazole), 7.54 (2H, d, J = 8.8 Hz, 1,4-disubstituted benzene), 7.86–7.91 (2H, m,
1,4-disubstituted benzene), 7.96 (1H, s, CH=N), 12.01 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6, ppm)
δ
42.54, 45.36, 52.53, 103.48, 115.90 (J = 21.1 Hz), 115.99, 126.16, 127.92, 127.93 (J = 6.8 Hz), 131.82
(J = 2.8 Hz), 141.95, 149.91, 150.59, 162.01 (J = 242.7 Hz), 168.86. HRMS (m/z): [M + H]+ calcd for
C21H22FN5S: 396.1653; found: 396.1638.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-(4-chlorophenyl)thiazole (3g): Yield 77%,
m.p. 249–250 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm) δ 2.85 (3H, s, CH3), 3.33 (4H, br s, piperazine),
3.55 (4H, br s, piperazine), 7.06 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.36 (1H, s, thiazole), 7.46
(2H, d, J = 8.6 Hz, 1,4-disubstituted benzene), 7.55 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.86
(2H, d, J = 8.6 Hz, 1,4-disubstituted benzene), 7.96 (1H, s, CH=N), 12.02 (1H, s, NH). 13C NMR (75
MHz, DMSO-d6, ppm)
δ 42.57, 45.38, 52.57, 104.55, 115.99, 126.12, 127.66, 127.93, 129.08, 132.31, 134.05,
142.05, 149.72, 150.61, 168.90. HRMS (m/z): [M + H]+ calcd for C21H22ClN5S: 412.1357; found: 412.1338.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-(4-bromophenyl)thiazole (3h): Yield 85%,
m.p. 253–255 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm) δ 2.86 (3H, s, CH3), 3.19 (4H, br s, piperazine),
3.48 (2H, br s, piperazine), 3.93 (2H, br s, piperazine), 7.05 (2H, d, J = 8.8 Hz, 1,4-disubstituted benzene),
7.36 (1H, s, thiazole), 7.54 (2H, d, J = 8.9 Hz, 1,4-disubstituted benzene), 7.59 (2H, d, J = 8.6 Hz,
1,4-disubstituted benzene), 7.80 (2H, d, J = 8.6 Hz, 1,4-disubstituted benzene), 7.98 (1H, s, CH=N),

Molecules 2020, 25, 4342
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11.98 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6, ppm)
δ
42.51, 45.32, 52.53, 104.64, 116.01, 120.91,
126.19, 127.95, 127.99, 131.97, 134.42, 142.10, 149.79, 150.57, 168.92. HRMS (m/z): [M + H]+ calcd for
C21H22BrN5S: 456.0852; found: 456.0821.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-([1,1’-biphenyl]-4-yl)thiazole (3i): Yield 83%,
m.p. 275–276 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm) δ 2.86 (3H, s, CH3), 3.34 (4H, br s, piperazine),
3.57 (4H, br s, piperazine), 7.06 (2H, d, J = 8.8 Hz, 1,4-disubstituted benzene), 7.34–7.39 (2H, m,
monosubstituted benzene, thiazole), 7.47 (2H, t, J = 7.4 Hz, monosubstituted benzene), 7.56 (2H, d,
J = 8.7 Hz, 1,4-disubstituted benzene), 7.71 (4H, d, J = 8.4 Hz, 1,4-disubstituted benzene), 7.94 (2H,
d, J = 8.3 Hz, monosubstituted benzene), 7.99 (1H, s, CH=N), 12.00 (1H, s, NH). 13C NMR (75 MHz,
DMSO-d6, ppm) δ 42.55, 45.37, 52.56, 103.98, 104.41, 116.02, 126.25, 126.55, 126.92, 127.28, 127.93, 128.49,
129.43, 134.34, 139.45, 140.12, 141.98, 150.57, 168.84. HRMS (m/z): [M + H]+ calcd for C27H27N5S:
454.2060; found: 454.2061.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-(2,4-dimethylphenyl)thiazole (3j): Yield 68%,
m.p. 238–240 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm)
δ 2.28 (3H, s, CH3), 2.41 (3H, s, CH3), 2.85
(3H, s, CH3), 3.31 (4H, br s, piperazine), 3.52 (4H, br s, piperazine), 6.81 (1H, s, 1,2,4-trisubstituted
benzene), 7.01–7.07 (4H, m, 1,4-disubstituted benzene, 1,2,4-trisubstituted benzene, thiazole), 7.48 (1H,
d, J = 7.9 Hz, 1,2,4-trisubstituted benzene), 7.54 (2H, d, J = 8.7 Hz, 1,4-disubstituted benzene), 7.96 (1H,
s, CH=N), 11.84 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6, ppm)
δ 21.13, 21.57, 42.57, 45,41, 52.57,
105.80, 116.05, 126.38, 126.81, 127.86, 129.58, 131.86, 132.58, 135.48, 137.04, 141.69, 150.51, 151.11, 167.78.
HRMS (m/z): [M + H]+ calcd for C23H27N5S: 406.2060; found: 406.2021.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-(2,4-difluorophenyl)thiazole (3k): Yield 70%,
m.p. 250–251 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm) δ 2.79 (3H, s, CH3), 3.25 (4H, br s, piperazine),
3.40–3.45 (2H, m, piperazine), 3.84 (2H, br s, piperazine), 7.04 (2H, d, J = 8.8 Hz, 1,4-disubstituted
benzene), 7.09–7.23 (2H, m, thiazole, 1,2,4-trisubstituted benzene), 7.32 (1H, td, J1 = 2.6 Hz, J2 = 9.3 Hz,
1,2,4-trisubstituted benzene), 7.54 (2H, d, J = 8.8 Hz, 1,4-disubstituted benzene), 7.94–8.07 (2H, m,
CH=N, 1,2,4-trisubstituted benzene,), 12.05 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6, ppm)
δ 42.34,
45.19, 52.26, 104.95 (t, J = 26.2 Hz), 107.80 (d, J = 14.0 Hz), 112.25 (dd, J1 = 3.1 Hz, J2 = 20.9 Hz), 115.96,
119.65 (dd, J1 = 3.8 Hz, J2 = 11.5 Hz), 126.09, 127.95, 130.83 (dd, J1 = 4.8 Hz, J2 = 9.3 Hz), 142.20, 143.83,
150.66, 159.98 (dd, J1 = 12.1 Hz, J2 = 250.2 Hz), 161.61 (dd, J1 = 12.2 Hz, J2 = 245.7 Hz), 168.27. HRMS
(m/z): [M + H]+ calcd for C22H21Cl2N5S: 414.1558; found: 414.1527.
2-{2-[4-(4-Methylpiperazin-1-yl)benzylidene]hydrazinyl}-4-(2,4-chlorophenyl)thiazole (3l): Yield 73%,
m.p. >300 ◦C. 1H NMR (300 MHz, DMSO-d6, ppm)
δ 2.80 (3H, s, CH3), 3.18–3.22 (4H, m, piperazine),
3.40–3.47 (2H, m, piperazine), 3.89–3.93 (2H, m, piperazine), 7.05 (2H, d, J = 8.8 Hz, 1,4-disubstituted
benzene), 7.34 (1H, s, thiazole), 7.49 (1H, dd, J1 = 2.2 Hz, J2 = 8.5 Hz, 1,2,4-trisubstituted benzene), 7.54
(2H, d, J = 8.8 Hz, 1,4-disubstituted benzene), 7.66 (1H, d, J = 2.1 Hz, 1,2,4-trisubstituted benzene), 7.90
(1H, d, J = 8.5 Hz, 1,2,4-trisubstituted benzene), 7.99 (1H, s, CH=N), 12.03 (1H, s, NH). 13C NMR (75
MHz, DMSO-d6, ppm)
δ 42.34, 45.19, 52.26, 109.26, 115.98, 126.10, 127.95, 130.20, 131.98, 132.68, 132.69,
132.85, 142.19, 146.40, 149.92, 150.66, 168.02. HRMS (m/z): [M + H]+ calcd for C22H21Cl2N5S: 446.0967;
found: 446.0936.
2.2. In Vitro MAO-A and MAO-B Inhibition Assay
Enzymatic testing was conducted using the current fluorometric approach declared by our research
community [14–18]. Sigma-Aldrich (Steinheim, Germany) provided the chemicals and reagents used
in the test (AmplifluTM Red (10-Acetyl-3,7-dihydroxyphenoxazine), MAO-A, MAO-B, peroxidase
from horseradish, tyramine hydrochloride, H2O2, moclobemide, clorgiline and selegiline) and they
were maintained by the manufacturer under the specified conditions. All pipetting processes were
performed using a Biotek Precision XS robotic system (BioTek Instruments Inc., Winooski, VT, USA).
Measurements were carried out by a BioTek-Synergy H1 microplate reader (BioTek Instruments Inc.,
Winooski, VT, USA) based on the fluorescence generated (excitation, 535 nm, emission, 587 nm) over a
30 min period, in which the fluorescence increased linearly.

Molecules 2020, 25, 4342
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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 10−3–10−9 M concentrations.
II) Enzyme solutions: recombinant MAO-A (0.5 U/mL) and recombinant MAO-B (0.64 U/mL) enzymes
were dissolved in the phosphate buffer and final volumes were adjusted to 10 mL. III) Working solution:
horseradish peroxidase (200 U/mL, 100
200 µL) were dissolved in the phosphate buffer and the final volume was adjusted to 10 mL.
The solutions of the inhibitor (20 L/well) and MAO-A (100 L/well) or MAO-B (100 L/well) were
added to the flat black bottom 96-well micro test plate, and incubated at 37 ◦C for 30 min. After this
µL), Ampliflu™ Red (20 mM, 200 µL) and tyramine (100 mM,
µ
µ
µ
incubation period, the reaction was started by adding a working solution (100 µL/well). The mixture
was incubated at 37 ◦C for 30 min and the fluorescence (Ex/Em = 535/587 nm) was measured at 5 min
intervals. Control experiments were carried out simultaneously by replacing the inhibitor solution
with 2% DMSO (20
a parallel reading was performed by replacing enzyme solutions with 3% H2O2 solution (20 mM
100 L/well). In addition, the possible capacity of the inhibitors to modify the fluorescence generated
µL). To check the probable inhibitory effect of inhibitors on horseradish peroxidase,
µ
in the reaction mixture due to non-enzymatic inhibition was determined by mixing inhibitor and
working solutions.
The specific fluorescence emission (used to obtain the final results) was calculated after subtraction
of the background activity, which was determined from vials containing all components except
the MAO isoforms, which were replaced by phosphate buffer (100 µL/well). Blank, control and
all concentrations of inhibitors were analyzed in quadruplicate and the inhibition percentage was
calculated by using the following equation:
(FCt2 − FCt1) − (FIt2 − FIt1)
% Inhibition =
× 100
(1)
FCt2 − FCt1
where FCt2 is the fluorescence of a control well measured at t2 time, FCt1 is the fluorescence of a control
well measured at t1 time, FIt2 is the fluorescence of an inhibitor well measured at t2 time and FIt1 is the
fluorescence of an inhibitor well measured at t1 time.
The IC50 values were calculated from a dose-response curve obtained by plotting the percentage
inhibition versus the log concentration with the use of GraphPad ‘PRISM’ software (version 5.0, GraphPad
Software, San Diego, CA, USA). The results were displayed as mean ± standard deviation (SD).
2.3. Enzyme Kinetic Studies
The same materials were used in the MAO inhibition assay. In keeping with the assay given in our
previous research, the most active compound, 3e, defined as a consequence of the MAO inhibition assay,
was experienced in three independent concentrations of IC50/2, IC50 and 2(IC50) [14–18]. All processes
were evaluated in quadruplicate. The results were analyzed by means of Microsoft Office Excel 2013 as
Lineweaver-Burk diagrams. The Vmax values of the Lineweaver-Burk plots were replotted versus the
inhibitor concentration, and the Ki values were determined from the x-axis intercept as Ki.
2.4. Cytotoxicity Assay
The NIH/3T3 mouse embryonic fibroblast cell line (ATCC® CRL-1658
used for cytotoxicity assays. The incubation period of NIH/3T3 cells was based on the supplier’s
recommendation. NIH/3T3 cells were seeded at 1
104 cells into each well of 96-well plates. MTT assay
was carried out in accordance with the standards previously described manner [19 20]. The most
™, London, UK) was
×
,
effective compound 3e was tested between 1 mM and 0.000316 mM concentrations. Inhibition % for
each concentration was calculated according to the following formula and IC50 values were reported
by plotting the % inhibition dose response curve against the compound concentrations tested [19
–21]:
% inhibition = 100 − (mean sample × 100/mean solvent).
(2)

Molecules 2020, 25, 4342
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2.5. Prediction of ADME Parameters
In order to predict the pharmacokinetic profiles of synthesized compounds 3a
–
3l, QikProp 4.8
software (Schrödinger, LLC, New York, NY, USA) [22] was used, and the physicochemical parameters
were calculated via the in silico method.
2.6. Molecular Docking Studies
A structure-based molecular docking protocol was used to reveal the binding mechanisms of
compound 3e to the active site of the MAO-A enzyme. For this purpose, the crystal structure of
MAO-A crystallized with harmine (PDB ID: 2Z5X) [23] was extracted from the Protein Data Bank
database (www.pdb.org).
The ligands’ configurations were designed using the Schrödinger Maestro [24] tool (Schrödinger,
LLC, New York, NY, USA) and submitted to the Schrödinger Suite 2016 Update 2 Protein Preparation
Wizard method. The ligands were processed using LigPrep 3.8 [25] to correctly detect the atom groups
as well as the protonation conditions at a pH of 7.4
atoms were added to the structures. The induced-fit docking (IFD) protocol [26] included in the
Schrödinger Maestro interface was used to perform the IFD.
±
1.0. Bond orders were assigned, and hydrogen
3. Results and Discussion
3.1. Chemistry
The synthesis of the compounds was completed using well established methods [14
The synthetic pathways of target compounds were summarized in Scheme 1 and Table 1. Intermediate
was synthesized by the reaction of 1-methylpiperazine and 4-fluorobenzaldehyde in the presence of
potassium carbonate. For the synthesis of compound , 4-(4-methylpiperazin-1-yl)benzaldehyde (
was reacted with thiosemicarbazide in ethanol. In addition, the target products 3a 3l were gained by the
reaction of the compound and a variety of substituted phenacyl bromide derivatives. The structures
,27].
1
2
1)
–
2
and purities of the thiazole analogues were verified by 1H NMR, 13C NMR and HRMS spectral data as
cited in the Supplementary Materials. The most characteristic signals observed in the 1H NMR spectra
were those of methyl, azomethine, N-H and piperazine protons, which were present as singlets at
2.84–2.87 ppm, 7.94–7.99 ppm, 11.84–12.12 ppm and broad singlet at 3.10–3.94 ppm, respectively. In the
13C NMR spectra, all aliphatic and aromatic carbons were recorded at the expected regions. For all
compounds, HRMS spectra corresponded with the proposed structures.
Table 1. The substituents of synthesized compounds (3a–3l).
Compound
R1
R2
Compound
R1
R2
3a
3b
3c
3d
3e
3f
-H
-H
-H
-H
-H
-H
-H
-CH3
-OCH3
-CN
-NO2
-F
3g
3h
3i
3j
3k
3l
-H
-H
-H
-CH3
-F
-Cl
-Cl
-Br
-Phenyl
-CH3
-F
-Cl

Molecules 2020, 25, 4342
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Scheme 1. The synthetic route of the compounds (3a–3l). Reagents and conditions: (i) DMF, K2CO3,
reflux, 24 h; (ii) EtOH, reflux, 3 h; (iii) EtOH, reflux, 4–8 h.
3.2. MAO Inhibition Assay
All the gained thiazolylhydrazine-piperazine derivatives were evaluated for their inhibition
potency against MAO isoforms using a previously described in vitro fluorometric method by our
research group [14–18]. The enzyme activity protocol was applied in two steps according to the
inhibition percentages and concentrations of the compounds. For all compounds and reference drugs,
namely moclobemide, clorgiline and selegiline, the concentrations of 10−3 and 10−4 M were used in the
first stage of the assay (Table 2). In this step, the reference inhibitors and compounds that showed
more than 50% inhibitory activity at 10−4 M concentration were selected for the second step, and these
compounds in question were prepared in their further concentrations by serial dilutions (ranging from
10−5 M to 10−9 M). Therefore, the half maximal inhibitory concentration (IC50) values of the selected
compounds and reference inhibitors could be calculated, and these results are given in Table 3.
Table 2. Inhibition percentage of the synthesized compounds, moclobemide, clorgiline and selegiline
against MAO-A and MAO-B enzymes.
MAO-A % Inhibition
10−3 10−4
MAO-B % Inhibition
10−3 10−4
M
Compounds
M
M
M
3a
3b
3c
3d
3e
3f
3g
3h
68.137 ± 1.026
70.957 ± 1.114
92.075 ± 2.218
90.192 ± 2.035
95.314 ± 1.895
75.942 ± 1.235
73.661 ± 1.108
70.753 ± 1.317
66.197 ± 0.972
72.308 ± 1.033
79.991 ± 1.299
73.521 ± 1.180
94.121 ± 2.760
96.940 ± 1.250
-
34.297 ± 0.851
31.456 ± 0.732
87.671 ± 1.874
84.369 ± 1.808
90.788 ± 1.728
40.666 ± 0.980
33.503 ± 0.833
37.905 ± 0.796
41.499 ± 0.870
38.122 ± 0.798
35.134 ± 0.833
40.578 ± 0.914
82.143 ± 2.691
91.308 ± 1.305
-
32.258 ± 0.985
34.553 ± 0.886
30.336 ± 0.812
36.648 ± 0.903
39.102 ± 0.810
30.655 ± 0.912
31.250 ± 0.789
34.362 ± 0.824
30.589 ± 0.836
28.143 ± 0.901
29.573 ± 0.782
33.667 ± 0.991
-
25.011 ± 0.721
27.591 ± 0.649
21.474 ± 0.879
26.044 ± 0.836
28.163 ± 0.791
24.718 ± 0.854
23.952 ± 0.623
22.104 ± 0.796
21.475 ± 0.809
20.034 ± 0.792
23.194 ± 0.876
21.373 ± 0.769
-
3i
3j
3k
3l
Moclobemide
Clorgiline
Selegiline
-
-
98.258 ± 1.052
96.107 ± 1.165

Molecules 2020, 25, 4342
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Table 3. IC50 values of 3c, 3d, 3e, moclobemide and clorgiline against MAO-A.
MAO-A % Inhibition
10−6
Compounds
IC50 (µM)
10−3
M
10−4
M
10−5
M
M
10−7
M
10−8
M
10−9
M
3c
3d
3e
92.075
90.192
95.314
±
±
±
±
±
2.218 87.671
2.035 84.369
1.895 90.788
2.760 82.143
1.250 91.308
±
±
±
±
±
1.874 75.428
1.808 81.059
1.728 85.025
2.691 60.458
1.305 87.635
±
±
±
±
±
1.425 70.985
1.937 78.997
1.027 82.367
2.559 36.151
1.456 78.498
±
±
±
±
±
1.095 48.336
1.247 49.589
1.392 63.942
1.984 22.135
1.024 65.235
±
±
±
±
±
0.869 30.910
0.811 30.637
0.893 42.570
0.337 18.166
0.997 34.198
±
±
±
±
±
0.711 21.785
0.699 18.107
0.835 23.018
0.812 14.128
0.841 22.477
±
±
±
±
±
0.638 0.188 ± 0.008
0.593 0.117 ± 0.004
0.715 0.057 ± 0.002
0.725 6.061 ± 0.262
0.736 0.062 ± 0.002
Moclobemide 94.121
Clorgiline 96.940
As seen in Table 2, all of the synthesized compounds demonstrated selectivity in terms of enzyme
inhibitory activity on MAO-A. All of the compounds displayed a more than 50% inhibitory effect at a
concentration of 10−3 M. This was not observed in the MAO-B enzyme inhibition results. The second
stage of the enzyme activity assay was carried out through compounds 3c
values were determined as seen in Table 3. It was understood that compound 3e displayed a more
potent inhibition profile than the reference inhibitors moclobemide (IC50 = 6.061 0.262 M) and
clorgiline (IC50 = 0.062 0.002 M), with an IC50 value of 0.057 0.002 M. Moreover, compound 3e was
, 3d and 3e, and their IC50
±
µ
±
µ
±
µ
followed by compound 3d as the most second active derivative with an IC50 value of 0.117
Similarly, compound 3c showed a significant inhibition potency with an IC50 value of 0.188
Of the compounds effectively observed, 3d and 3e carried cyano and nitro groups as substituents
at the phenyl ring para-position, respectively. According to the findings of the enzyme inhibition,
the electron withdrawing groups such as cyano and nitro moieties were assumed to have contributed
positively to enzyme inhibition ability on MAO-A enzyme.
±
0.004
0.008
µ
M.
M.
±
µ
3.3. Kinetic Studies of Enzyme Inhibition
The MAO-A inhibition mechanism was defined by conducting enzyme kinetics experiments
utilizing a protocol close to that of the MAO inhibition assay. To this end, compound 3e was included
in the enzyme kinetic tests by preparation its concentrations of IC50/2, IC50 and 2(IC50). To estimate
the type of inhibition of this compound, linear Lineweaver-Burk graphs were used. The velocity
curves of the substrates were reported in the absence and presence of compound 3e. In each case,
the initial velocity measurements were collected at various concentrations of substrates (tyramine)
varying from 20 µM to 0.625 µM. To measure the Ki (intercept on the x-axis) value of this compound,
a secondary plot (Dixon plot) of the slope (Km/Vmax obtained from Lineweaver-Burk graph) versus
varying concentrations (0, IC50/2, IC50 and 2(IC50)) were generated. Figure 2 shows graphical study of
the steady-state inhibition results for compound 3e.
According to the Lineweaver-Burk plots, the type of inhibition consists of two general classes:
reversible and irreversible. Mixed-type, uncompetitive, competitive and noncompetitive inhibition
types are included in the reversible inhibition [14–18]. According to Lineweaver–Burk plots, a graph
that shows parallel lines without any cross-overs is observed in the uncompetitive type of inhibition.
Competitive inhibition is seen if the lines intersect on the y-axis, and the slopes and x-intercepts are
different. On the contrary, non-competitive inhibition has the opposite result: the plotted lines have
the same x-intercept but there are diverse slopes and y-intercepts. For mixed-type inhibition, a graph
with lines that do not intersect at the x-axis or the y-axis is formed.
As seen in the Lineweaver-Burk plot of compound 3e (Figure 2), the lines were intersected on the
y-axis, and their slopes and x-intercepts were different. This observation indicated that compound 3e
was a reversible and competitive inhibitor with similar inhibition features as the substrates. The Ki
value of compound 3e was calculated as 0.011 µM with the help of secondary plot.

Molecules 2020, 25, 4342
(A)
10 of 17
(B)
Figure 2.
concentration (
at the left. ( ) Secondary plot (slope, namely Km/Vmax obtained from graph
(
A
) Lineweaver-Burk plots for the inhibition of MAO-A by compound 3e. [S], substrate
M); , reaction velocity (nmol/min/mg protein). Inhibitor concentrations are shown
, versus inhibitor
µ
V
B
A
concentrations) of compound 3e for determining the steady-state inhibition constant (Ki). Ki was
calculated to be 0.011 µM.
It is known that reversible enzyme inhibition has advantages compared with the irreversible
inhibition type. Non-covalent interactions, such as hydrophobic interactions, ionic bonds and

Molecules 2020, 25, 4342
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hydrogen bonds between the substrate and the enzyme, are in question in the reversible inhibition and
these interactions provide the forming rapidly and breaking easily of the enzyme-inhibitor complex.
Reversible inhibitors often have a lower chance of adverse effects than irreversible inhibitors because
of their non-covalent binding ability. Consequently, compound 3e, whose form of inhibition has
been decided to be reversible and competitive, has a therapeutic value in comparison to irreversible
MAO-based hydrazine inhibitors.
3.4. Cytotoxicity Assay
Compound 3e displayed potent MAO-A inhibition profile and was further tested for toxicity
using the MTT assay in the NIH/3T3 cell line; the IC50 value of this compound is shown in Table 4.
Compound 3e showed an IC50 value of >1000
µM against NIH/3T3 cells, which was significantly
higher than its IC50 value (0.057 M) against MAO-A. Consequently, compound 3e was found to be
µ
non-cytotoxic at its effective concentration against MAO-A. This result further increases the biological
importance of this compound.
Table 4. IC50 value of compound 3e against the NIH/3T3 cell line.
Compound
3e
IC50 (µM) NIH/3T3 Cell Line
>1000
IC50 (µM) MAO-A Enzyme
0.057 ± 0.002
3.5. Prediction of ADME Parameters
Inappropriate ADME (absorption, distribution, metabolism and excretion) profiles make
the clinical trials of new drug development studies complex, time-consuming and costly. Thus,
the assessment of pharmacokinetic profiles of new drug candidates is a vital step in the process of drug
development studies [28]. Nowadays, applying in silico ADME screens can provide advantages to pick
out the most promising compounds and minimize the risk of drug election in late stages [29]. Therefore,
in this paper, a large amount of parameters (partition coefficient, aqueous solubility, brain/blood
partition coefficient, central nervous system activity, apparent Caco-2 and MDCK cell permeability,
total solvent-accessible volume, Van der Waals surface area of polar nitrogen and oxygen atoms and
carbonyl carbon atoms, human oral absorption percent and drug likeness score, namely Lipinski’s rule
of five and Jorgensen’s rule of three) were studied via thorough methods of virtual screening using
QikProp 4.8 software [22]. The predicted parameters and their recommended values are presented
in Table 5.
The drug-like quality of the compounds was tested as per the “Rule of Five” by Lipinski and the
“Rule of Three” by Jorgensen [30–33]. Depending on the interaction between pharmacokinetic and
physicochemical parameters, the “Rule of Five” by Lipinski and the “Rule of Three” by Jorgensen
specifies the structural features found in a candidate compound, which may be a pharmaceutical
product [30
,
31]. In Table 5, it is shown that all parameters fall inside the standard ranges. In accordance
3l) were in full compliance with
with the rules of three and five, the compounds collected (3a
–
the parameters set, since they did not cause more than one violation. Additionally, the results
from compounds exhibited good CNS absorption (score of 1 and 2, namely active absorption).
The investigated compounds showed medium to high cell permeability in Caco-2 and MDCK cell lines
range from 98.431 to 1152.894 and 88.255 to 4373.785, respectively. These findings are very important
for CNS-related drugs such as MAO-A inhibitors.
Based on the findings of the ADME parameter trials, the synthesized compounds have good and
promising pharmacokinetic profiles and could be appropriate for clinical usage.

Molecules 2020, 25, 4342
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Table 5. Calculated ADME parameters of compounds 3a–3l.
Compound MW
RB
DM
MV
DHB AHB
PSA
logP
logS
PCaco logBB PMDCK CNS
PM
%HOA VRF VRT
3a
3b
3c
3d
3e
3f
3g
3h
3i
377.506
391.533
407.532
402.516
422.504
395.497
411.951
456.402
453.604
405.560
413.487
446.396
4
4
5
5
5
4
4
4
5
4
4
4
2.979
2.503
3.620
9.298
1263.957
1322.897
1331.741
1330.658
1
1
1
1
1
1
1
1
1
1
1
1
7
7
46.680
46.680
55.161
72.475
95.346
46.684
46.682
46.682
46.680
41.791
45.242
43.269
4.092
4.393
4.126
3.323
3.384
4.326
4.583
4.660
5.687
4.557
4.487
4.759
−5.338 957.697 0.369
−5.895 957.686 0.357
−5.444 957.690 0.301
1032.177
1032.164
1032.168
1
1
1
1
0
2
2
2
1
2
2
2
2
3
3
2
3
2
2
2
2
4
2
2
100
100
100
87.508
82.432
100
100
100
100
100
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
1
0
1
1
1
1
1
1
1
7.750
8.500
−6.273 198.052 −0.544 187.908
12.959 1346.359
8
7
7
7
7
7
7
7
−5.568 98.431 −0.912 88.255
5.390
5.330
5.015
2.742
3.108
4.457
4.564
1280.070
1308.076
1316.988
1496.172
1347.245
1289.708
1316.883
−5.702 957.638 0.480
−6.075 957.661 0.535
−6.190 957.675 0.547
−7.304 957.700 0.269
−5.748 1152.894 0.472
−5.929 994.545 0.573
−5.928 1060.430 0.695
1866.285
2546.410
2737.950
1032.180
1261.337
2862.946
4373.785
3j
3k
3l
100
100
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) logP: Predicted octanol/water partition coefficient
(recommended value:
great) logBB: Predicted brain/blood partition coefficient (recommended value:
great) CNS: Predicted central nervous system activity on a –2 (inactive) to +2 (active) scale (recommended value: –2 (inactive), +2 (active)) 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.
−
2–6.5) logS: Predicted aqueous solubility (recommended value:
−
6.5–0.5) PCaco: Predicted apparent Caco-2 cell permeability (recommended value: <25 poor, >500
−
3–1.2) PMDCK: Predicted apparent MDCK cell permeability (recommended value: <25 poor, >500
The rules are: MW < 500, logP < 5, DHB
< 7.
≤
5, AHB
≤
10, Positive PSA value. VRT: Number of violations of Jorgensen’s rule of three. The three rules are: logS > 5.7, PCaco > 22 nm/s, PM
−

Molecules 2020, 25, 4342
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3.6. Molecular Docking Studies
Compounds 3c, 3d and 3e were determined to be the most effective derivatives in the series
against the MAO-A enzyme as described in the MAO inhibition assay. Hence, docking studies were
conducted to assess their inhibition potentials as in silico. Using the X-ray crystal structure of MAO-A
(PDB ID: 2Z5X) [23], docking studies were performed, and the binding modes of these compounds
were assigned. Figures 3 and 4 demonstrate the docking poses of these compounds. According to
Figure 3, compounds 3c, 3d and 3e were correctly attached to amino acid residues filling the cavity
and were positioned very close to the cofactor of FAD.
Figure 3. The three-dimensional (3D) poses of compounds 3c, 3d and 3e in the active region of MAO-A
(PDB ID: 2Z5X). The compounds are shown in a tube pattern and are yellow, maroon and orange
colored, respectively.
Figure 4 indicates the three-dimensional (3D) interacting modes of compounds 3c
active region of MAO-A. While examining the docking poses of these compounds, it can be obviously
seen that there were many forms of interactions, such as , cation- interactions and hydrogen bond
forming. Moreover, it was detected that there were the same interactions related to these compounds.
A cation- interaction occurred between the methyl substituted N atom of the piperazine ring and the
phenyl of Tyr444 in all these compounds. Additionally, this N atom of compound 3d formed another
cation- interaction with the phenyl of Tyr407. The other common interaction for all these compounds
was observed between the thiazole ring and the phenyl of Phe208 by doing interaction. Moreover,
, 3d and 3e in the
π–
π
π
π
π
π–π
a similar interaction with this amino acid, Phe208, was determined by the phenyl ring near the thiazole
ring in compounds 3c and 3d.

Molecules 2020, 25, 4342
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A
B
C
Figure 4. The 3D interacting modes of compound 3c
model introduce the ligands and the relevant residues in the enzyme’s active site. The flavin adenine
dinucleotide (FAD) molecule is shown in a ball and stick pattern and is blue colored. ( ) Compound 3
is colored = yellow. (B) Compound 3d is colored maroon. (C) Compound 3e is colored orange.
, 3d and 3e in the active region of MAO-A. Tube
A
c
When analyzing the docking pose of compound 3e (Figure 4C), it was seen that the hydrazine
group in the structure was essential for polar interactions. The hydrogen bond formation was detected
between the N atom of the hydrazine group and the amino of Gln215. Moreover, the phenyl ring

Molecules 2020, 25, 4342
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near to the hydrazine group in compound 3e created a
π
–
π
interaction with the phenyl of Phe352.
The mentioned additional two interaction were not observed with compounds 3c and 3d. Therefore,
these determinations were thought to be very important for the explaining of more potent enzyme
inhibitory activity of compound 3e than other compounds.
The principal structural distinction between compound 3e and the other derivatives was the
nitro group at the fourth location of the phenyl ring. The oxygen atom of the nitro group formed a
hydrogen bond with the amino of Phe177. The same interaction with this amino acid was detected
in the cyano group of compound 3d. In this sense, the enzyme inhibition findings were supported
by the molecular docking studies. It was thought that the substituents at this position, which served
as electron withdrawing, such as the nitro and cyamo groups, strongly contributed to binding to the
active site of the enzyme. This condition may also clarify why compounds 3d and 3e showed stronger
inhibition profiles than other compounds.
4. Conclusions
In this paper, 12 novel thiazolylhydrazine-piperazine derivatives were reported as selective
MAO-A inhibitors based on our previous studies and investigated for their inhibitory properties
towards MAO enzymes using in vitro assay. Compounds 3c
inhibition profiles. The IC50 value of compound 3e was lower (0.057
to the references, moclobemide (IC50 = 6.061 0.262 M) and clorgiline (IC50 = 0.062
,
3d and 3e displayed significant MAO-A
0.002 M) when compared
0.002 M).
±
µ
±
µ
±
µ
Therefore, compound 3e was found to be the most active agent in the series. Enzyme kinetic studies
revealed that the type of inhibition of this compound was identified as reversible and competitive.
Moreover, the inhibitory activity on MAO-A enzyme of compound 3e was simulated as in silico by
molecular docking studies. In addition, this compound had a good pharmacokinetic profile and high
BBB (blood brain barrier) penetration. Based on these findings, it was concluded that further research
is needed to improve the therapeutic efficacy of this important class of compounds in the treatment of
neurological disorders as MAO inhibitors. New chemical modifications can be designed based on this
paper so that novel effective derivatives may be subject to future studies. Hence, studies to develop
new candidates that may be effective in depression can be followed rationally.
Supplementary Materials: The following are available online, Figure S1. 1H-NMR spectra of compound
Figure S2. 13C-NMR spectra of compound . Figure S4. 1H-NMR
. Figure S3. HRMS spectra of compound
spectra of compound . Figure S6. HRMS spectra of compound
. Figure S5. 13C-NMR spectra of compound
1.
1
1
2
2
2
. Figure S7. 1H-NMR spectra of compound 3a. Figure S8. 13C-NMR spectra of compound 3a. Figure S9.
HRMS spectra of compound 3a. Figure S10. 1H-NMR spectra of compound 3b. Figure S11. 13C-NMR spectra
of compound 3b. Figure S12. HRMS spectra of compound 3b. Figure S13. 1H-NMR spectra of compound
3c. Figure S14. 13C-NMR spectra of compound 3c. Figure S15. HRMS spectra of compound 3c. Figure S16.
1H-NMR spectra of compound 3d. Figure S17. 13C-NMR spectra of compound 3d. Figure S18. HRMS spectra of
compound 3d. Figure S19. 1H-NMR spectra of compound 3e. Figure S20. 13C-NMR spectra of compound 3e
.
Figure S21. HRMS spectra of compound 3e. Figure S22. 1H-NMR spectra of compound 3f. Figure S23. 13C-NMR
spectra of compound 3f. Figure S24. HRMS spectra of compound 3f. Figure S25. 1H-NMR spectra of compound
3g. Figure S26. 13C-NMR spectra of compound 3g. Figure S27. HRMS spectra of compound 3g. Figure S28.
1H-NMR spectra of compound 3h. Figure S29. 13C-NMR spectra of compound 3h. Figure S30. HRMS spectra
of compound 3h. Figure S31. H-NMR spectra of compound 3i. Figure S32. 13C-NMR spectra of compound 3i
.
1
Figure S33. HRMS spectra of compound 3i. Figure S34. 1H-NMR spectra of compound 3j. Figure S35. 13C-NMR
spectra of compound 3j. Figure S36. HRMS spectra of compound 3j. Figure S37. 1H-NMR spectra of compound
3k. Figure S38. 13C-NMR spectra of compound 3k. Figure S39. HRMS spectra of compound 3k. Figure S40.
1H-NMR spectra of compound 3l. Figure S41. 13C-NMR spectra of compound 3l. Figure S42. HRMS spectra of
compound 3l.
Author Contributions: Y.Ö. and Z.A.K. conceived and designed the experiments; B.N.S. performed the synthesis,
activity tests and molecular docking studies; O.C., D.O., U.A.Ç. and B.K.Ç. performed the synthesis; D.O. and S.L.
performed the analysis studies; S.I. performed the cytotoxicity test; B.N.S., O.C., D.O., U.A.Ç., S.L., B.K.Ç., S.I.,
Y.Ö. and Z.A.K. wrote the paper. All authors have read and agreed to the published version of the manuscript.

Molecules 2020, 25, 4342
16 of 17
Funding: This study was financially supported by Anadolu University Scientific Projects Fund, Project
No: 2005S046.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 3a–3l are available from the authors.
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