Synthesis, in vitro enzyme activity and molecular docking studies of new benzylamine-sulfonamide derivatives as selective MAO-B inhibitors

Article abstract of DOI:10.1080/14756366.2020.1784892

Many studies have been conducted on the selective inhibition of human monoamine oxidase B (hMAO-B) enzyme using benzylamine-sulphonamide derivatives. Using various chemical modifications on BB-4h, which was reported previously by our team and showed a significant level of MAO-B inhibition, novel benzylamine-sulphonamide derivatives were designed, synthesised, and their MAO inhibition potentials were evaluated. Among the tested derivatives, compounds 4i and 4t achieved IC₅₀ values of 0.041 ± 0.001 μM and 0.065 ± 0.002 μM, respectively. The mechanism of hMAO-B inhibition by compounds 4i and 4t was studied using Lineweaver–Burk plot. The nature of inhibition was also determined to be non-competitive. Cytotoxicity tests were conducted and compounds 4i and 4t were found to be non-toxic. Molecular docking studies were also carried out for compound 4i, which was found as the most potent agent, within hMAO-B catalytic site.

Full text of DOI:10.1080/14756366.2020.1784892

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis, in vitro enzyme activity and
molecular docking studies of new benzylamine-
sulfonamide derivatives as selective MAO-B
inhibitors
Begüm Nurpelin Sağlık, Derya Osmaniye, Ulviye Acar Çevik, Serkan Levent,
Betül Kaya Çavuşoğlu, Özlem Atlı Eklioğlu, Yusuf Özkay, Ali Savaş Koparal &
Zafer Asım Kaplancıklı
To cite this article: Begüm Nurpelin Sağlık, Derya Osmaniye, Ulviye Acar Çevik, Serkan Levent,
Betül Kaya Çavuşoğlu, Özlem Atlı Eklioğlu, Yusuf Özkay, Ali Savaş Koparal & Zafer Asım
Kaplancıklı (2020) Synthesis, in vitro enzyme activity and molecular docking studies of new
benzylamine-sulfonamide derivatives as selective MAO-B inhibitors, Journal of Enzyme Inhibition
and Medicinal Chemistry, 35:1, 1422-1432, DOI: 10.1080/14756366.2020.1784892
To link to this article: https://doi.org/10.1080/14756366.2020.1784892
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1422–1432
https://doi.org/10.1080/14756366.2020.1784892
RESEARCH PAPER
Synthesis, in vitro enzyme activity and molecular docking studies of new
benzylamine-sulfonamide derivatives as selective MAO-B inhibitors
a,b
€
€
ꢀ
Begum Nurpelin Saglık
, Derya Osmaniyea,b , Ulviye Acar C¸evika,b , Serkan Leventa,b
,
c
d
a,b
Betul Kaya C¸avus¸oglu
, Ozlem Atlı Eklioglu
, Yusuf Ozkay
, Ali Savas¸ Koparale
and
€
€
ꢀ
ꢀ
Zafer Asım Kaplancıklıa
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir, Turkey; Doping and Narcotic Compounds
c
Analysis Laboratory, Faculty of Pharmacy, Anadolu University, Eskis¸ehir, Turkey; Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
d
€
Zonguldak Bulent Ecevit University, Zonguldak, Turkey; Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University,
e
Eskis¸ehir, Turkey; Open Education Faculty, Anadolu University, Eskis¸ehir, Turkey
ABSTRACT
ARTICLE HISTORY
Received 21 April 2020
Revised 16 May 2020
Accepted 18 May 2020
Many studies have been conducted on the selective inhibition of human monoamine oxidase B (hMAO-B)
enzyme using benzylamine-sulphonamide derivatives. Using various chemical modifications on BB-4h, which
was reported previously by our team and showed a significant level of MAO-B inhibition, novel benzylamine-
sulphonamide derivatives were designed, synthesised, and their MAO inhibition potentials were evaluated.
Among the tested derivatives, compounds 4i and 4t achieved IC50 values of 0.041 0.001 mM and
0.065 0.002 mM, respectively. The mechanism of hMAO-B inhibition by compounds 4i and 4t was studied
using Lineweaver–Burk plot. The nature of inhibition was also determined to be non-competitive. Cytotoxicity
tests were conducted and compounds 4i and 4t were found to be non-toxic. Molecular docking studies were
also carried out for compound 4i, which was found as the most potent agent, within hMAO-B catalytic site.
KEYWORDS
Benzylamine; enzyme
inhibition; heterocyclic ring;
MAO enzymes;
molecular docking
1. Introduction
MAO inhibitors are thought to act as neuroprotective agents in
degenerative processes6,7
.
Monoamine oxidase (MAO) is the enzyme responsible for catalys-
ing the oxidative deamination of intracellular amines and mono-
amine neurotransmitters, which contributes to the regulation of
the concentrations of these chemicals in the brain and in periph-
eral tissues1,2. MAOs, which are flavin adenine dinucleotide (FAD)-
containing enzymes, are localised in the outer mitochondrial
membranes of glial, neuronal, and other types of cells; they are
particularly abundant in the liver and the brain. MAOs have two
different isoforms, MAO-A and MAO-B, with 70% homology. The
genes that code for the two isoforms are linked in opposite orien-
tation on the X chromosome, differ in the specificity of their sub-
strates and the selectivity of their inhibitors3. For example, MAO-B
is selectively inhibited by selegiline, and utilises phenylethylamine
and benzylamine as substrates. On the contrary, MAO-A is select-
ively inhibited by clorgiline, and utilises adrenaline, noradrenaline
and serotonin as substrates. However, both isoforms may also act
on the same substrates such as dopamine and tyramine4.
MAOs are of extensive pharmacological importance due to
their roles in the metabolism of certain neurotransmitters.
Selective MAO-A inhibitors are used clinically as antidepressants
and anxiolytics, while MAO-B inhibitors are used to reduce the
progression of Parkinson’s disease, and manage symptoms related
to Alzheimer’s disease5. Moreover, MAO-catalyzed deamination
reactions produce hydrogen peroxide as a byproduct, which may
Parkinson’s disease (PD), which affects more than 5 million people
worldwide, is the second most common disease after Alzheimer’s
disease. Considering the loss of nigrostriatal dopaminergic cells as a
pathological hallmark of PD, therapeutic strategies have been estab-
lished to boost the levels of dopamine in the brain8–10. Although
dopamine is metabolised by both MAO isoforms, MAO-B is the more
common isoform present in the basal ganglia and is therefore
responsible for dopamine metabolism in this region11.
Currently, the Protein Data Bank contains more than 40 crystal
structures of MAO (most of them MAO-B) in complex with different
reversible and irreversible inhibitors, as observed through X-ray dif-
fraction at refinements of 3.0–1.7 Å. Additionally, MAO-A shows a
markedly different monopartite cavity (ꢀ550 Å) compared to the
bipartite cavity (290 Å) found in MAO-B. The “aromatic cage”—a
hydrophobic binding pocket containing the FAD cofactor—is con-
sidered the active region4,7. The FAD is covalently attached to the
cysteine residue of the protein, and the 8a-thioether linkage pro-
vides this connection. It is believed that the catalytic activity of the
two tyrosine residues, Tyr398 and Tyr435, found in the hMAO-B
structure is due to the polarisation of the amine N pair of the sub-
strate12. Therefore, in designing a new inhibitor compound, it is
desirable to have the amine group in the structure.
In light of the above-mentioned information, this study was
conducted to develop new and potent MAO inhibitors. It has
been thought that the proven MAO inhibition of benzylamine
typically contribute to the oxidative stress state. In this context, derivatives may provide MAO-B inhibitory activity due to strong
€
ꢀ
CONTACT Begum Nurpelin Saglık
bnsaglik@anadolu.edu.tr
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Anadolu University, Eskis¸ehir
26470, Turkey
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1423
Figure 1. Design of the target compounds from compound BB-4h.
interactions on the enzyme active side13–15. In our recent study2, (1.427ml, 0.014 mol) or 4-methylbenzaldehyde (1.680 g, 0.014 mol)
we reported a new benzothiazole-benzylamine hybrid compound,
2-((5-chlorobenzothiazol-2-yl)thio)-N-(4-fluorophenyl)-N-(3-nitroben-
zyl)acetamide (BB-4h), as shown in Figure 1, with significant IC50
(2.95 0.09 mM) against MAO-B. Moreover, sulphonamides and
various heterocyclic ring systems have been identified as inhibi-
tors of MAO in previous studies3,16–19. Therefore, we considered
the compound (BB-4h) as a lead compound, and we performed
some modifications, such as removing nitro and fluoro groups,
introducing a sulphonamide group, and changing heterocyclic
rings in order to improve biological activity. Subsequently, 20 ben-
zylamine derivatives containing a sulphonamide moiety and differ-
ent heterocyclic ring moieties were synthesised, and their MAO
inhibitory activities were evaluated in this study.
were refluxed in EtOH (50 ml) for 48 h. Acetic acid was used as
catalyst in this reaction. After completion of the reaction, the mix-
ture was cooled, precipitated product was filtered and dried.
2.1.1.2. Synthesis of 4-(benzylamino)benzenesulfonamide and
4-((4-methylbenzyl)amino)benzenesulfonamide
(Benzylideneamino)benzenesulfonamide (1a) (3.328 g, 0.0128 mol)
or 4-((4-methylbenzylidene)amino)benzenesulfonamide (1b)
(2a,
2b).
4-
(3.507 g, 0.0128 mol) was dissolved in MeOH. Sodium borohydride
was added to the reaction medium in portions of 0.5 moles.
It was observed by controlling the end of the reaction with TLC
that the reaction was complete when the total amount of
sodium borohydride reached 1.5 moles. After completion of the
reaction, the MeOH was removed by a rotavapor. The precipitated
product was washed with deionised water to remove the
excess of the sodium borohydride, dried, and recrystallized
from EtOH.
2. Experimental
2.1. Chemistry
All chemicals used in the synthesis studies were obtained from
Merck Chemicals (Merck KGaA, Darmstadt, Germany) or Sigma-
Aldrich Chemicals (Sigma-Aldrich Corp., St. Louis, MO). MP90
digital melting point apparatus (Mettler Toledo, Ohio) was used
to determine the melting points of the resulting compounds and
was presented uncorrected. A Bruker 300 MHz and 75 MHz digital
FT-NMR spectrometer (Bruker Bioscience, Billerica, MA) in DMSO-
d6, respectively recorded 1H NMR and 13 C NMR spectra. In the
NMR spectra, splitting patterns were determined recognised as
follows: s: singlet; d: doublet; t: triplet; dd: double doublet; td: tri-
ple doublet; br.s.: bronsted singlet; m: multiplet. Coupling con-
stants (J) are reported in units of Hertz (Hz). IRAffinity-1S Fourier
transform IR (FTIR) spectrometer (Shimadzu, Tokyo, Japan) was
used to record the IR spectra of the compounds. Mass spectra
were recorded on an LCMS-IT-TOF (Shimadzu, Kyoto, Japan) by
means of ESI method. Silica gel 60 F254 by TLC (Merck KGaA,
Darmstadt, Germany) was used to control the purity of the
obtained compounds.
2.1.1.3. Synthesis of N-Benzyl-2-chloro-N-(4-sulfamoylphenyl)aceta-
mide and 2-chloro-N-(4-methylbenzyl)-N-(4-sulfamoylphenyl)aceta-
mide (3a, 3b). 4-(Benzylamino)benzenesulfonamide (2a) (3.013 g,
0.0115 mol) or 4-((4-methylbenzyl)amino)benzenesulfonamide
(2b) (3.174 g, 0.0115 mol) and triethylamine (TEA) (1.605 ml,
0.0115 mol) were dissolved in DMF (20 ml) and the reaction mix-
ture was taken up in ice bath. The solution of chloroacetyl chlor-
ide (1.004 ml, 0.0126 mol) in DMF (10 ml) was added dropwise to
the reaction mixture. After completion of the reaction, the mix-
ture was poured into ice-water (50 ml), precipitated product was
filtered, washed with deionised water, dried and recrystallized
from EtOH.
2.1.1.4. General procedure for the synthesis of target compounds
(4a–4u). N-Benzyl-2-chloro-N-(4-sulfamoylphenyl)acetamide (3a)
(0.305 g, 0.0009 mol) or 2-chloro-N-(4-methylbenzyl)-N-(4-sulfa-
moylphenyl)acetamide (3b) (0.317 g, 0.0009 mol), heterocyclic sub-
stituted thiol derivatives (0.0009 mol) and sufficient quantity of
potassium carbonate (K2CO3) (0.193 g, 0.0014 mol) were reacted in
acetone for 3 h. After completion of the reaction, acetone was
removed under reduced pressure, the residue was washed with
2.1.1. General procedure for the synthesis of the compounds
2.1.1.1. Synthesis of 4-(benzylideneamino)benzenesulfonamide
and 4-((4-methylbenzylidene)amino)benzenesulfonamide (1a, 1b).
4-Aminobenzenesulfonamide (2.408 g, 0.014 mol) and benzaldehyde water, dried, and recrystallized from EtOH.

1424
B. N. SAĞLIK ET AL.
N-Benzyl-2-((1-methyl-1H-imidazol-2-yl)thio)-N-(4-sulfamoylphenyl)a-
cetamide (4a). Yield: 85%, M.P. ¼ 137–139 ꢁC, FTIR (ATR, cmꢂ1):
Pyridine CH), 7.21–7.31 (6H, m, Monosubstitutedbenzene, Pyridine
CH), 7.41 (2H, s, –SO2NH2), 7.52 (2H, d, J ¼ 8.4 Hz, 1,4-
Disubstitutedbenzene), 7.62 (1H, t, J ¼ 8.5, Pyridine CH), 7.83 (2H,
1
3340 (N–H), 2941 (C–H), 1666 (C ¼ O), 709, 850. H-NMR (300 MHz,
DMSO-d6): d ¼ 3.55 (3H, s, –CH3), 3.81 (2H, s, –CH2–), 4.92 (2H, s, d, J ¼ 8.4 Hz, 1,4-Disubstitutedbenzene), 8.36 (1H, d, J ¼ 4.3 Hz,
Pyridine CH). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 33.4, 52.9, 120.4,
-CH2), 6.90 (1H, s, Imidazole CH), 7.17–7.31 (6H, m, Imidazole CH,
Monosubstitutedbenzene), 7.39 (2H, d, J ¼ 8.3 Hz, 1,4-
122.0, 127.4, 127.7, 128.2, 128.9, 129.1, 137.1, 137.5, 143.6, 145.2,
149.7, 157.5, 168.1. HRMS (m/z): [M þ H]þ calcd for C20H19N3O3S2:
Disubstitutedbenzene), 7.78 (2H, d, J ¼ 8.3 Hz, 1,4-disubstituted-
benzene). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 33.4, 37.9, 52.8, 123.8,
127.3, 127.7, 128.2, 128.8, 128.9, 129.1, 137.2, 139.7, 144.2, 144.5,
167.6. HRMS (m/z): [M þ H]þ calcd for C19H20N4O3S2: 417.1055;
found: 417.1040
414.0941; found: 414.0950
2-(Benzoxazol-2-ylthio)-N-benzyl-N-(4-sulfamoylphenyl)acetamide (4g).
Yield: 80%, M.P. ¼ 82–84 ꢁC, FTIR (ATR, cmꢂ1): 3363 (N-H), 2985
(C-H), 1651 (C ¼ O), 704, 744, 846. 1H-NMR (300 MHz, DMSO-d6):
d ¼ 4.25 (2H, s, –CH2–), 4.99 (2H, s, –CH2–), 7.26–7.29 (5H, m,
Monosubstitutedbenzene), 7.31–7.35 (2H, m, Benzoxazole CH),
7.45 (2H, s, –SO2NH2), 7.58–7.64 (4H, m, 1,4-Disubstitutedbenzene,
Benzoxazole CH), 7.88 (2H, d, J ¼ 8.4, 1,4-Disubstitutedbenzene),
13 C-NMR (75 MHz, DMSO-d6): d ¼ 36.9, 53.2, 110.7, 118.7, 124.8,
125.1, 127.6, 127.8, 128.2, 128.9, 129.2, 137.1, 141.6, 144.0, 144.6,
151.7, 164.3, 166.6. HRMS (m/z): [M þ H]þ calcd for C22H19N3O4S2:
454.0890; found: 454.0880
N-Benzyl-2-((4-methyl-4H-1,2,4-triazol-3-yl)thio)-N-(4-sulfamoylphenyl)
acetamide (4b). Yield: 88%, M.P. ¼ 201–203 ꢁC, FTIR (ATR, cmꢂ1):
3296 (N-H), 2976 (C–H), 1654 (C ¼ O), 709, 852. 1H-NMR (300 MHz,
DMSO-d6): d ¼ 3.55 (3H, s, –CH3), 4.01 (2H, s, –CH2–), 4.94 (2H, s,
–CH2–), 7.18–7.31 (5H, m, Monosubstitutedbenzene), 7.42 (2H, s,
–SO2NH2), 7.49 (2H, d, J ¼ 8.5 Hz, 1,4-Disubstitutedbenzene), 7.81
(2H, d, J ¼ 8.5 Hz, 1,4-Disubstitutedbenzene), 8.50 (1H, s, Triazole
CH). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 31.2, 37.8, 52.9, 127.4, 127.8,
128.3, 128.9, 129.1, 137.1, 143.8, 144.5, 146.5, 149.1, 167.0. HRMS
(m/z): [M þ H]þ calcd for C18H19N5O3S2: 418.1002; found: 418.1001
2-(Benzothiazol-2-ylthio)-N-benzyl-N-(4-sulfamoylphenyl)acetamide
(4h). Yield: 79%, M.P. ¼ 88–90 ꢁC, FTIR (ATR, cmꢂ1): 3352 (N–H),
N-Benzyl-2-((5-methyl-1,3,4-thiadiazol-2-yl)thio)-N-(4-sulfamoylphenyl)
acetamide (4c). Yield: 82%, M.P. ¼ 164–166 ꢁC, FTIR (ATR, cmꢂ1):
3334 (N-H), 3047 (C-H), 1654 (C ¼ O), 698, 740, 856. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 2.66 (3H, s, –CH3), 4.18 (2H, s, –CH2–),
4.97 (2H, s, –CH2–), 7.21–7.32 (5H, m, Monosubstitutedbenzene),
7.42 (2H, s, –SO2NH2), 7.53 (2H, d, J ¼ 8.5 Hz, 1,4-
1
2941 (C–H), 1651 (C ¼ O), 702, 758, 846. H-NMR (300 MHz, DMSO-
d6): d ¼ 4.24 (2H, s, –CH2–), 4.99 (2H, s, –CH2–), 7.24 (5H, br.s.,
Monosubstitutedbenzene), 7.37 (1H, td, J1¼1.0 Hz, J2¼7.7 Hz,
Benzothiazole CH) 7.44 (2H, s, -SO2NH2), 7.49 (1H, td, J1¼1.1 Hz,
J2¼7.7 Hz, Benzothiazole CH), 7.62 (2H, d, J ¼ 8.3 Hz, 1,4-
Disubstitutedbenzene), 7.83 (1H, d, J ¼ 8.0, Benzothiazole CH), 7.88
(2H, d, J ¼ 8.4 Hz, 1,4-Disubstitutedbenzene), 8.01 (1H, d, J ¼ 7.6 Hz,
Benzothiazole CH). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 37.2, 53.1,
121.6, 122.4, 125.0, 126.8, 127.5, 127.8, 128.3, 128.9, 129.1, 135.2,
137.2, 143.8, 144.8, 152.9, 166.3, 166.8. HRMS (m/z): [M þ H]þ calcd
for C22H19N3O3S3: 470.0661; found: 470.0652
Disubstitutedbenzene),
7.83
(2H,
d,
J ¼ 8.5 Hz,
1,4-
Disubstitutedbenzene). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 15.6, 38.3,
53.0, 127.4, 127.8, 128.3, 128.9, 129.1, 137.1, 143.9, 144.5, 164.6,
166.0, 166.6. HRMS (m/z): [M þ H]þ calcd for C18H18N4O3S3:
435.0614; found: 435.0622
N-Benzyl-2-((1-methyl-1H-tetrazol-5-yl)thio)-N-(4-sulfamoylphenyl)ace-
tamide (4d). Yield: 86%, M.P. ¼ 124–127 ꢁC, FTIR (ATR, cmꢂ1): 3305
(N-H), 2931 (C–H), 1651 (C ¼ O), 702, 734, 848. 1H-NMR (300 MHz,
DMSO-d6): d ¼ 3.95 (3H, s, –CH3), 4.19 (2H, s, –CH2–), 4.96 (2H, s,
–CH2–), 7.20–7.32 (7H, m, Monosubstitutedbenzene, –SO2NH2),
7.51 (2H, d, J ¼ 8.4 Hz, 1,4-Disubstitutedbenzene), 7.84 (2H, d,
J ¼ 8.4 Hz, 1,4-Disubstitutedbenzene). 13 C-NMR (75 MHz, DMSO-d6):
d ¼ 34.1, 38.3, 53.0, 127.5, 127.8, 128.3, 128.9, 129.0, 137.0, 144.1,
144.6, 153.8, 166.5. HRMS (m/z): [M þ H]þ calcd for C17H18N6O3S2:
419.0955; found: 419.0956
N-Benzyl-2-((5-chlorobenzothiazol-2-yl)thio)-N-(4-sulfamoylphenyl)a-
cetamide (4i). Yield: 85%, M.P. ¼ 114–116 ꢁC, FTIR (ATR, cmꢂ1):
3473 (N-H), 2995 (C–H), 1645 (C ¼ O), 702, 732, 846. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 4.22 (2H, s, –CH2–), 4.98 (2H, s, –CH2–),
7.24 (5H, br.s., Monosubstitutedbenzene), 7.41 (1H, d, J ¼ 1.8 Hz,
Benzothiazole CH) 7.44 (2H, s, –SO2NH2), 7.62 (2H, d, J ¼ 8.3 Hz,
1,4-Disubstitutedbenzene), 7.88–7.90 (3H, m, Benzothiazole CH,
1,4-Disubstitutedbenzene), 8.04 (1H, d, J ¼ 8.6 Hz, Benzothiazole
CH). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 37.4, 53.1, 121.0, 123.8,
125.0, 127.6, 127.8, 128.3, 128.8, 129.2, 131.7, 134.1, 137.3, 143.9,
144.7, 153.7, 166.6, 169.2. HRMS (m/z): [M þ H]þ calcd for C22H18
ClN3O3S3: 504.0272; found: 504.0250
N-Benzyl-2-((1-phenyl-1H-tetrazol-5-yl)thio)-N-(4-sulfamoylphenyl)ace-
tamide (4e). Yield: 81%, M.P. ¼ 138–140 ꢁC, FTIR (ATR, cmꢂ1): 3356
(N-H), 2949 (C–H), 1651 (C ¼ O), 734, 759, 848. 1H-NMR (300 MHz,
DMSO-d6): d ¼ 4.27 (2H, s, –CH2–), 4.95 (2H, s, –CH2–), 7.21–7.32
(5H, m, Monosubstitutedbenzene), 7.51 (2H, d, J ¼ 8.4 Hz, 1,4-
Disubstitutedbenzene), 7.67 (7H, br.s., Monosubstitutedbenzene,
N-Benzyl-2-((5-methoxybenzothiazol-2-yl)thio)-N-(4-sulfamoylphenyl)
acetamide (4j). Yield: 80%, M.P. ¼ 170–172 ꢁC, FTIR (ATR, cmꢂ1):
3483 (N-H), 2939 (C-H), 1651 (C ¼ O), 702, 734, 842. 1H-NMR
-SO2NH2), 7.84 (2H, d, J ¼ 8.4 Hz, 1,4-Disubstitutedbenzene). 13 C- (300 MHz, DMSO-d6): d ¼ 3.84 (3H, s, –OCH3), 4.23 (2H, s, –CH2–),
NMR (75 MHz, DMSO-d6): d ¼ 38.5, 53.0, 124.9, 124.9, 127.5, 127.8,
128.3, 128.9, 130.4, 130.6, 130.7, 131.2, 133.5, 137.0, 154.3, 166.3.
HRMS (m/z): [M þ H]þ calcd for C22H20N6O3S2: 481.1111;
found: 481.1096
4.99 (2H, s, –CH2–), 7.01 (1H, dd, J1¼2.5 Hz, J2¼8.8 Hz,
Benzothiazole CH), 7.25 (5H, br.s., Monosubstitutedbenzene), 7.33
(1H, d, J ¼ 2.5 Hz, Benzothiazole CH) 7.43 (2H, br.s, -SO2NH2), 7.59
(2H, d, J ¼ 8.4 Hz, 1,4-Disubstitutedbenzene), 7.86 (1H, d, J ¼ 8.7,
Benzothiazole
CH),
7.88
(2H,
d,
J ¼ 8.2 Hz,
1,4-
Disubstitutedbenzene). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 37.3, 53.1,
56.0, 105.0, 114.2, 122.6, 126.8, 127.5, 127.8, 128.2, 128.9, 129.1,
137.2, 143.9, 144.7, 154.2, 159.2, 166.8, 167.2. HRMS (m/z):
N-Benzyl-2-(pyridin-2-ylthio)-N-(4-sulfamoylphenyl)acetamide
(4f).
Yield: 87%, M.P. ¼ 106–108 ꢁC, FTIR (ATR, cmꢂ1): 3402 (N-H), 2929
(C-H), 1651 (C ¼ O), 698, 763, 854. 1H-NMR (300 MHz, DMSO-d6):
d ¼ 3.94 (2H, s, –CH2–), 4.96 (2H, s, –CH2–), 7.11 (1H, t, J ¼ 5.9 Hz, [M þ H]þ calcd for C23H21N3O4S3: 500.0767; found: 500.0761

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1425
2-((1-Methyl-1H-imidazol-2-yl)thio)-N-(4-methylbenzyl)-N-(4-sulfa-
moylphenyl)acetamide (4k). Yield: 86%, M.P. ¼ 153–155 ꢁC, FTIR
(ATR, cmꢂ1): 3313 (N–H), 2920 (C–H), 1651 (C ¼ O), 840. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 2.25 (3H, s, –CH3), 3.56 (3H, s, –CH3), 3.80
(2H, s, –CH2–), 4.87 (2H, s, –CH2–), 6.90 (1H, d, J ¼ 1.1 Hz, Imidazole
CH), 7.04–7.10 (4H, m, Methylbenzene), 7.21 (1H, d, J ¼ 1.1 Hz,
N-(4-Methylbenzyl)-2-(pyridin-2-ylthio)-N-(4-sulfamoylphenyl)aceta-
mide (4p). Yield: 77%, M.P. ¼ 121–123 ꢁC, FTIR (ATR, cmꢂ1): 3408
(N-H), 2918 (C-H), 1645 (C ¼ O), 848. 1H-NMR (300 MHz, DMSO-d6):
d ¼ 2.25 (3H, s, –CH3), 3.93 (2H, s, -CH2-), 4.91 (2H, s, –CH2–),
7.09–7.13 (5H, m, Methylbenzene, Pyridine CH), 7.29 (1H, d,
J ¼ 8.1 Hz, Pyridine CH), 7.50 (2H, d, J ¼ 8.4 Hz, 1,4-
Disubstitutedbenzene), 7.62 (1H, td, J1¼1.7 Hz, J2¼7.7 Hz, Pyridine
CH), 7.83 (2H, d, J ¼ 8.4 Hz, 1,4-Disubstitutedbenzene). 8.36 (1H, d,
J ¼ 4.3 Hz, Pyridine CH). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 21.1,
33.5, 52.6, 120.4, 122.0, 127.3, 128.2, 129.1, 129.4, 134.4, 136.8,
137.1, 143.8, 145.1, 149.7, 157.5, 168.0. HRMS (m/z): [M þ H]þ calcd
for C21H21N3O3S2: 428.1097; found: 428.1092
Imidazole
CH),
7.37–7.41
7.79
(4H,
(2H,
m,
d,
–SO2NH2,
J ¼ 8.5 Hz,
1,4-
1,4-
Disubstitutedbenzene),
Disubstitutedbenzene). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 21.1, 33.4,
37.9, 52.5, 123.8, 127.3, 128.3, 128.9, 129.0, 129.44, 134.1, 136.9,
140.0, 143.6, 144.7, 167.5. HRMS (m/z): [M þ H]þ calcd for
C20H22N4O3S2: 431.1206; found: 431.1199
2-(Benzoxazol-2-ylthio)-N-(4-methylbenzyl)-N-(4-sulfamoylphenyl)ace-
tamide (4r). Yield: 85%, M.P. ¼ 148–150 ꢁC, FTIR (ATR, cmꢂ1): 3388
2-((4-Methyl-4H-1,2,4-triazol-3-yl)thio)-N-(4-methylbenzyl)-N-(4-sulfa-
moylphenyl)acetamide (4l). Yield: 84%, M.P. ¼ 229–231 ꢁC, FTIR
(ATR, cmꢂ1): 3302 (N-H), 3049 (C–H), 1654 (C ¼ O), 852. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 2.25 (3H, s, -CH3), 3.55 (3H, s, –CH3), 4.00
(2H, s, –CH2–), 4.90 (2H, s, -CH2-), 7.08 (4H, br.s., Methylbenzene),
7.35 (2H, br.s, -SO2NH2), 7.46 (2H, d, J ¼ 8.5 Hz, 1,4-
1
(N-H), 2933 (C–H), 1666 (C ¼ O), 856. H-NMR (300 MHz, DMSO-d6):
d ¼ 2.25 (3H, s, –CH3), 4.24 (2H, s, –CH2–), 4.93 (2H, s, –CH2–),
7.07–7.11 (4H, m, Methylbenzene), 7.32–7.35 (2H, m, Benzoxazole
CH), 7.55 (2H, d, J ¼ 8.3 Hz, 1,4-Disubstitutedbenzene), 7.61–7.64
(2H, m, Benzoxazole CH), 7.88 (2H, d, J ¼ 8.3 Hz, 1,4-
Disubstitutedbenzene). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 21.1, 37.0,
52.9, 110.7, 118.7, 124.8, 125.1, 127.5, 128.3, 129.0, 129.5, 134.1,
136.9, 141.6, 144.1, 145.0, 151.7, 164.3, 166.5. HRMS (m/z):
[M þ H]þ calcd for C23H21N3O4S2: 468.1046; found: 468.1030
Disubstitutedbenzene),
7.81
(2H,
d,
J ¼ 8.5 Hz,
1,4-
Disubstitutedbenzene), 8.51 (1H, s, Triazole CH). 13 C-NMR (75 MHz,
DMSO-d6): d ¼ 21.1, 31.2, 37.9, 52.6, 127.4, 128.3, 129.0, 129.5,
134.0, 136.9, 144.1, 144.4, 146.5, 149.1, 167.0. HRMS (m/z):
[M þ H]þ calcd for C19H21N5O3S2: 432.1159; found: 432.1161
2-(Benzothiazol-2-ylthio)-N-(4-methylbenzyl)-N-(4-sulfamoylphenyl)a-
cetamide (4s). Yield: 88%, M.P. ¼ 170–172 ꢁC, FTIR (ATR, cmꢂ1):
3238 (N-H), 2918 (C-H), 1651 (C ¼ O), 850. 1H-NMR (300 MHz,
DMSO-d6): d ¼ 2.24 (3H, s, –CH3), 4.22 (2H, s, –CH2–), 4.93 (2H, s,
–CH2–), 7.04–7.12 (4H, m, Methylbenzene), 7.37 (1H, td, J1¼1.0 Hz,
J2¼7.7 Hz, Benzothiazole CH), 7.48 (1H, td, J1¼1.1 Hz, J2¼7.7 Hz,
2-((5-Methyl-1,3,4-thiadiazol-2-yl)thio)-N-(4-methylbenzyl)-N-(4-sulfa-
moylphenyl)acetamide (4m). Yield: 79%, M.P. ¼ 178–180 ꢁC, FTIR
(ATR, cmꢂ1): 3288 (N-H), 3070 (C-H), 1651 (C ¼ O), 850. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 2.25 (3H, s, –CH3), 2.66 (3H, s, –CH3), 4.16
(2H, s, –CH2–), 4.92 (2H, s, –CH2–), 7.09 (4H, br.s., Methylbenzene),
7.43 (2H, s, –SO2NH2), 7.51 (2H, d, J ¼ 8.5 Hz, 1,4-
Benzothiazole
CH),
7.56
(2H,
d,
J ¼ 8.4 Hz,
1,4-
Disubstitutedbenzene), 7.82 (1H, d, J ¼ 8.0 Hz, Benzothiazole CH),
7.87 (2H, d, J ¼ 8.4 Hz, 1,4-Disubstitutedbenzene), 8.00 (1H, d,
J ¼ 7.9 Hz, Benzothiazole CH). 13 C-NMR (75 MHz, DMSO-d6):
d ¼ 21.1, 37.3, 52.8, 121.6, 122.3, 125.0, 126.8, 127.4, 128.2, 129.1,
129.4, 134.2, 135.2, 136.9, 144.4, 144.5, 152.9, 166.3, 166.7. HRMS
(m/z): [M þ H]þ calcd for C23H21N3O3S3: 484.0818; found: 484.0796
Disubstitutedbenzene),
7.83
(2H,
d,
J ¼ 8.5 Hz,
1,4-
Disubstitutedbenzene). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 15.6, 21.1,
38.3, 52.7, 127.4, 128.3, 129.1, 129.5, 134.0, 136.9, 143.9, 144.5,
164.6, 166.0, 166.6. HRMS (m/z): [M þ H]þ calcd for C19H20N4O3S3:
449.0770; found: 449.0750
2-((1-Methyl-1H-tetrazol-5-yl)thio)-N-(4-methylbenzyl)-N-(4-sulfamoyl-
phenyl)acetamide (4n). Yield: 80%, M.P. ¼ 154–156 ꢁC, FTIR (ATR,
cmꢂ1): 3338 (N-H), 2947 (C–H), 1649 (C ¼ O), 850. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 2.25 (3H, s, –CH3), 3.95 (3H, s, –CH3), 4.19
(2H, s, –CH2–), 4.91 (2H, s, –CH2–), 7.09 (4H, br.s., Methylbenzene),
7.33 (2H, br.s, –SO2NH2), 7.50 (2H, d, J ¼ 8.4 Hz, 1,4-
2-((5-Chlorobenzothiazol-2-yl)thio)-N-(4-methylbenzyl)-N-(4-sulfamoyl-
phenyl)acetamide (4t). Yield: 79%, M.P. ¼ 109–111 ꢁC, FTIR (ATR,
cmꢂ1): 3360 (N-H), 2927 (C-H), 1664 (C ¼ O), 850. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 2.25 (3H, s, –CH3), 4.20 (2H, s, –CH2–),
4.94 (2H, s, –CH2–), 7.04–7.11 (4H, m, Methylbenzene), 7.40–7.43
(3H, m, -SO2NH2, Benzothiazole CH) 7.60 (2H, d, J ¼ 8.3 Hz, 1,4-
Disubstitutedbenzene), 7.85 (1H, d, J ¼ 2.0 Hz, Benzothiazole CH),
7.90 (2H, d, J ¼ 8.4 Hz, 1,4-Disubstitutedbenzene), 8.03 (1H, d,
J ¼ 8.6, Benzothiazole CH). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 21.1,
37.4, 52.8, 121.1, 123.7, 125.0, 127.6, 128.3, 129.3, 129.4, 131.7,
134.1, 134.2, 136.9, 144.0, 144.7, 153.7, 166.5, 169.2. HRMS (m/z):
[M þ H]þ calcd for C23H20 ClN3O3S3: 518.0428; found: 518.0393
Disubstitutedbenzene),
7.84
(2H,
d,
J ¼ 8.4 Hz,
1,4-
Disubstitutedbenzene). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 21.1, 34.1,
38.4, 52.7, 127.5, 128.3, 129.1, 129.5, 133.9, 137.0, 144.1, 144.4,
153.8, 166.4. HRMS (m/z): [M þ H]þ calcd for C18H20N6O3S2:
433.1111; found: 433.1106
N-(4-Methylbenzyl)-2-((1-phenyl-1H-tetrazol-5-yl)thio)-N-(4-sulfamoyl-
phenyl)acetamide (4o). Yield: 83%, M.P. ¼ 87–90 ꢁC, FTIR (ATR,
cmꢂ1): 3294 (N-H), 2924 (C–H), 1651 (C ¼ O), 848. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 2.25 (3H, s, –CH3), 4.27 (2H, s, –CH2–),
4.92 (2H, s, –CH2–), 7.09 (4H, br.s., Methylbenzene), 7.46 (2H, br.s.,
–SO2NH2), 7.53 (2H, d, J ¼ 8.4 Hz, 1,4-Disubstitutedbenzene), 7.67
(5H, br.s., Monosubstitutedbenzene), 7.86 (2H, d, J ¼ 8.4 Hz, 1,4-
Disubstitutedbenzene). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 21.1, 38.5,
52.7, 124.9, 127.5, 128.3, 129.2, 129.5, 130.6, 131.1, 133.5, 133.9,
137.0, 144.0, 144.3, 154.3, 166.2. HRMS (m/z): [M þ H]þ calcd for
C23H22N6O3S2: 495.1268; found: 495.1243
2-((5-Methoxybenzothiazol-2-yl)thio)-N-(4-methylbenzyl)-N-(4-sulfa-
moylphenyl)acetamide (4u). Yield: 78%, M.P. ¼ 82–84 ꢁC, FTIR
(ATR, cmꢂ1): 3344 (N-H), 2933 (C-H), 1651 (C ¼ O), 844. 1H-NMR
(300 MHz, DMSO-d6): d ¼ 2.25 (3H, s, –CH3), 3.84 (3H, s, –OCH3),
3.84 (3H, s, –OCH3), 4.21 (2H, s, -CH2-), 4.94 (2H, s, -CH2-), 7.01 (1H,
dd, J1¼2.5 Hz, J2¼8.8 Hz, Benzothiazole CH), 7.05–7.12 (4H, m,
Methylbenzene), 7.32 (1H, d, J ¼ 2.4 Hz, Benzothiazole CH), 7.43
(2H,
br.s,
–SO2NH2),
7.57
(2H,
d,
J ¼ 8.4 Hz,
1,4-
Disubstitutedbenzene), 7.84–7.89 (3H, m, Benzothiazole CH, 1,4-
Disubstitutedbenzene). 13 C-NMR (75 MHz, DMSO-d6): d ¼ 21.1, 37.3,

1426
B. N. SAĞLIK ET AL.
52.8, 56.0, 105.0, 114.2, 122.6, 126.8, 127.5, 128.3, 129.2, 129.5, 2.6. Molecular docking
134.2, 136.9, 143.8, 144.7, 154.2, 159.1, 166.7, 167.2. HRMS (m/z):
A structure based in silico procedure was applied to discover the
[M þ H]þ calcd for C24H23N3O4S3: 514.0923; found: 514.0897.
binding modes of compound 4i to hMAO-B enzyme active site.
The crystal structures of hMAO-B (PDB ID: 2V5Z)27, which was crys-
tallised with safinamide, were retrieved from the Protein Data
Bank server (www.pdb.org).
2.2. MAO-A and MAO-B inhibition assay
AmplifluTM Red (10-Acetyl-3,7-dihydroxyphenoxazine), hMAO-A,
hMAO-B, peroxidase from horseradish, tyramine hydrochloride,
H2O2, clorgiline and selegiline were acquired from Sigma-Aldrich
(Steinheim, Germany) and retained under the proposed conditions
by supplier. A Biotek Precision XS robotic system (USA) was used
for all pipetting operations. Measurements were performed with
the use of BioTek-Synergy H1 microplate reader (USA) based upon
the fluorescence generated (excitation, 535 nm, emission, 587 nm)
over a 30 min period, in which the fluorescence increased linearly.
Enzymatic assay was performed according to recent method
pronounced by our research group17,20–22. Control, blank and all
concentrations of obtained compounds were tested in quadrupli-
cate and inhibition percent was calculated with following equation:
€
The structures of ligands were built using the Schrodinger
Maestro28 interface and then were submitted to the Protein
€
Preparation Wizard protocol of the Schrodinger Suite 2016 Update
229. The ligands were prepared by the LigPrep 3.830 to assign the
protonation states at pH 7.4 1.0 and the atom types, correctly.
Bond orders were assigned, and hydrogen atoms were added to
the structures. The grid generation was formed using Glide 7.131.
The grid box with dimensions of 20 Å ꢃ 20 Å ꢃ 20 Å was centred
in the vicinity of the flavin (FAD) N5 atom on the catalytic site of
the protein to cover all binding sites and neighbouring resi-
dues32–34. Flexible docking runs were performed with single preci-
sion docking mode (SP).
ðFCt2 ꢂ FCt1Þ ꢂ ðFIt2 ꢂ FIt1Þ
% Inhibition ¼
ꢃ 100
3. Result and discussion
FCt2 ꢂ FCt1
3.1. Chemistry
FCt2: Fluorescence of a control well measured at t2 time, FCt1:
Fluorescence of control well measured at t2 time, FIt2:
a
Various compounds, labelled 4a to 4u, were synthesised as out-
lined in Scheme 1. Initially, Schiff bases were prepared through
the reaction of benzaldehyde (or 4-methylbenzaldehyde) and
sulphanilamide. Then, benzylamine derivatives (2a, 2b) were
obtained by a reduction reaction. Acetylation of the benzylamine
derivatives (2a, 2b) gave compounds 3a and 3b. Finally, the tar-
get compounds (4a–4u) were obtained through a substitution
reaction using acetylated benzylamine (3a, 3b) and corresponding
heterocyclic thiols.
Fluorescence of an inhibitor well measured at t2 time, FIt1:
Fluorescence of an inhibitor well measured at t1 time,
The IC50 values were calculated using a dose-response curve
achieved by plotting the percentage inhibition versus the log con-
centration using GraphPad ‘PRISM’ software (version 5.0). The
results were showed as mean SD.
2.3. Enzyme kinetic studies
The synthesised compounds were elucidated by instrumental
analyses such as infra-red spectroscopy (IR), mass spectrometry
(MS), and nuclear magnetic resonance (NMR). The N-H bond of
the sulphonamide group appeared as IR bands between 3238 and
3483 cmꢂ1, and as a singlet between 7.30 and 7.60 ppm on the
1H-NMR spectrum. The presence of the carbonyl group was shown
by IR bands between 1645 cmꢂ1 and 1666 cmꢂ1, and a 13 C-NMR
peak over 160 ppm. The CH2 group bound to the nitrogen atom
gave a singlet 1H-NMR peak around 4.90 ppm, and a 13 C-NMR
peak over 50 ppm. The other CH2 group between the carbonyl
and sulphur groups was recorded in 1H-NMR peak around
4.20 ppm and a 13 C-NMR peak between 33.4 and 38.5 ppm. The
carbons of aromatic groups were observed from 105.0 to
166.7 ppm in the 13 C-NMR spectrum, and the protons of the same
The same materials were used in the MAO inhibition assay. The
most active compounds 4i and 4t determined according to the
result of the MAO inhibition assay were experienced in three dif-
ferent concentrations of IC50/2, IC50 and 2(IC50) in accordance with
the assay assigned in our final study.17,20–22. All processes were
evaluated in quadruplicate. The results were analysed as
Lineweaver–Burk plots by means of Microsoft Office Excel 2013.
The Vmax values of the Lineweaver–Burk plots were replotted ver-
sus the inhibitor concentration, and the Ki values were determined
from the x-axis intercept as ꢂKi.
2.4. Cytotoxicity assay
R
The NIH/3T3 mouse embryonic fibroblast cell line (ATCCVCRL-1658
1
TM, London, UK) was 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 ꢃ 104 cells into each well of 96-well
plates. MTT assay was carried out in accordance with the standards
groups were between 6.90 and 8.51 ppm in the H-NMR spectrum.
In mass spectroscopy, the masses were found to differ by at most
5 ppm from the expected masses.
previously described manner23,24
. The compounds were tested
3.2. MAO inhibition
between 1 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
All the obtained benzylamine-sulphonamide derivatives 4a–4u
were investigated for their inhibitory activity against MAO iso-
forms using a previously described in vitro fluorometric method,
which is based on the detection of H2O2 in a horseradish peroxid-
ase-coupled reaction using 10-acetyl-3,7-dihydroxyphenoxazine
curve against the compound concentrations tested.23–25
.
% inhibition ¼ 100ꢂðmean sample ꢃ 100=mean solventÞ
(Amplex Red reagent)17,20–22
.
The assay was carried out in two steps. The first step was car-
2.5. Prediction of ADME parameters and BBB permeability
ried out using 10ꢂ3 and 10ꢂ4 M concentrations of all synthesised
Physicochemical parameters were performed with the use of compounds and reference agents, namely selegiline and clorgiline.
QikProp 4.8 software26 to predict pharmacokinetic profiles and The enzyme activity results of first step are presented in Table 1.
BBB permeability of obtained compounds (4a–4u).
Then, the selected compounds that displayed more than 50%

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1427
Compounds
Ar
R
4a
4b
4c
4d
4e
4f
1-Methyl-1H-imidazol-2-yl
4-Methyl-4H-1,2,4-triazol-3-yl
5-Methyl-1,3,4-thiadiazol-2-yl
1-Methyl-1H-tetrazol-5-yl
1-Phenyl-1H-tetrazol-5-yl
Pyridin-2-ylthio
-H
H
-H
-H
-H
-H
4g
4h
4i
Benzoxazol-2-yl
-H
Benzothiazol-2-yl
-H
5-Chlorobenzothiazol-2-yl
5-Methoxybenzothiazol-2-yl
1-Methyl-1H-imidazol-2-yl
4-Methyl-4H-1,2,4-triazol-3-yl
5-Methyl-1,3,4-thiadiazol-2-yl
1-Methyl-1H-tetrazol-5-yl
1-Phenyl-1H-tetrazol-5-yl
Pyridin-2-ylthio
-H
4j
H
4k
4l
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
4m
4n
4o
4p
4r
4s
Benzoxazol-2-yl
Benzothiazol-2-yl
4t
5-Chlorobenzothiazol-2-yl
5-Methoxybenzothiazol-2-yl
4u
Scheme 1. Synthesis way of the compounds 4a–4u.
inhibitory activity at 10ꢂ3 and 10ꢂ4 M concentrations were further chlorobenzothiazole exhibited more potent inhibitory activity than
tested, along with reference agents, at concentrations of 10ꢂ5 to
the other obtained compounds as in the previously synthesised
10ꢂ9 M. The IC50 values of the test compounds and reference
and reported BB-4h derivative, which has a 5-chlorobenzothiazole
agents are presented in Table 2.
ring. Moreover, the increased inhibitory activity of the synthesised
compounds, compared to that of BB-4h, is likely due to the con-
tribution of the sulphonamide group, which displaced the fluorine
group, and the removal of the nitro group from the structure.
According to the enzyme inhibition results, none of the syn-
thesised compounds showed a significant activity against hMAO-A
enzyme. All of the obtained compounds displayed selective inhib-
ition on hMAO-B. At 1 0 ꢂ3 M concentration, all of the compounds
showed more than 50% inhibitory activity. Compounds 4b, 4d, 4f,
4i and 4t could pass the second step of enzyme activity assay
and the IC50 values of them were calculated by performing
enzyme inhibition study at 10ꢂ5–10ꢂ9 M concentration. The most
active compounds, 4i and 4t, exhibited IC50 values of
0.041 0.001 mM and 0.065 0.002 mM, respectively, against hMAO-
B, while the reference agent, selegiline, had an IC50
of 0.037 0.001 mM.
3.3. Kinetic studies of enzyme inhibition
Enzyme kinetics studies were performed to determine the mech-
anism of hMAO-B inhibition by using a procedure similar to that
of the MAO inhibition assay. Compounds 4i and 4t, which were
found to be the most potent agents, were included in these stud-
ies. In order to estimate the type of inhibition of these com-
pounds, linear Lineweaver-Burk graphs were used. Substrate
These findings from the screening of inhibitory activities
against hMAO-B revealed that the compounds containing 5- velocity curves in the absence and presence of compounds 4i and

1428
B. N. SAĞLIK ET AL.
4t were recorded. These compounds were prepared at concentra- bonds, and hydrogen bonds involved in reversible inhibition. In
tions of IC50/2, IC50, and 2(IC50) for enzyme kinetic studies. In each this type of inhibition, inhibitors bind to the enzymes without
case, the initial velocity measurements were obtained at different forming any chemical bonds; thus, the enzyme-inhibitor complex
substrate (tyramine) concentrations ranging from 20 lM to
0.625 lM. The secondary plots of slope (Km/Vmax) versus varying
concentrations (0, IC50/2, IC50, and 2(IC50)) were created to calcu-
late the Ki (intercept on the x-axis) value of these compounds.
The graphical analyses of steady-state inhibition data for com-
pounds 4i and 4t are shown in Figures 2 and 3.
could be separated quickly because non-covalent interactions can
form rapidly and break easily. Furthermore, reversible inhibitors
have a lower risk of side effects than irreversible inhibitors owing
to their non-covalent binding ability. Consequently, compounds 4i
and 4t, whose inhibition types were determined to be reversible
and non-competitive, have pharmaceutical importance in contrast
to irreversible hydrazine-type MAO inhibitors.
The type of inhibition can be determined as either reversible
or irreversible by using the Lineweaver-Burk plots. The reversible
inhibition type can be classified as mixed-type, uncompetitive,
non-competitive17,20–22
.
According
to
3.4. Cytotoxicity
competitive,
or
Lineweaver–Burk plots, a graph that shows parallel lines without
any cross-overs is observed in the uncompetitive type of inhib-
ition. For mixed-type inhibition, a graph with lines that do not
intersect at the x-axis or the y-axis is formed. Competitive inhib-
ition is seen if the lines intersect on the y-axis, and the slopes and
x-intercepts are different. On the contrary, non-competitive inhib-
ition has the opposite result: the plotted lines have the same x-
intercept but there are diverse slopes and y-intercepts. Therefore,
as shown in Figures 2 and 3, compounds 4i and 4t are reversible
and non-competitive inhibitors with similar inhibition features as
the substrates. Ki values for compounds 4i and 4t were calculated
as 0.036 and 0.055 lM, respectively, for the inhibition of hMAO-B.
Irreversible enzymatic inhibition involves covalent interactions
between the substrate and the enzyme. In contrast, there are
non-covalent interactions such as hydrophobic interactions, ionic
Compounds 4i and 4t displayed potent hMAO-B inhibition profiles
and were further tested for toxicity using the MTT assay in the
NIH3T3 cell line; the IC50 values of compounds 4i and 4t are
shown in Table 3. Both compounds showed an IC50 value of
>1000 mM against NIH3T3 cells, which was significantly higher
(A)
0.015
IC50 / 2 (0.021 µM)
IC50 (0.041 µM)
2 x IC50 (0.082 µM)
0.010
0.005
0.000
Control
Table 1. % Inhibition of the synthesized compounds, clorgiline and selegiline
against MAO-A and MAO-B enzymes.
-3.5
-2.5
-1.5
-0.5
0.5
1.5
1/[S] µM-1
2.5
3.5
MAO-A % Inhibition
10ꢂ3 10ꢂ4
MAO-B % Inhibition
10ꢂ3 10ꢂ4
M
Compounds
M
M
M
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
35.123 0.989 26.505 0.856 92.298 1.108 47.022 0.958
37.588 0.714 20.125 0.621 90.256 1.304 86.301 1.294
40.577 0.749 24.110 0.610 91.102 1.250 48.301 0.855
39.180 0.650 29.515 0.489 91.578 1.247 79.679 1.459
41.528 0.899 28.016 0.714 90.120 1.008 48.363 0.721
46.022 0.863 30.114 0.627 90.585 1.388 83.327 1.259
47.199 0.979 32.233 0.621 94.662 1.345 47.338 0.963
39.108 0.821 20.332 0.608 96.205 1.105 48.755 0.879
56.321 0.996 40.456 0.782 94.859 1.405 91.755 1.258
48.177 0.825 37.362 0.679 90.839 1.245 45.097 0.958
39.347 0.701 30.327 0.582 79.521 0.957 39.011 0.702
42.299 0.630 25.208 0.712 74.204 1.052 43.798 0.882
37.356 0.850 31.088 0.729 76.308 1.004 40.112 0.799
40.158 0.970 21.525 0.632 72.158 0.959 38.254 0.638
44.158 0.877 28.654 0.509 68.332 0.824 30.255 0.721
45.203 0.971 24.854 0.792 70.501 1.071 35.619 0.622
42.388 0.821 32.749 0.697 67.550 0.957 33.126 0.798
38.216 0.734 32.997 0.697 72.651 1.002 40.299 0.835
52.628 0.987 38.320 0.503 92.588 1.129 88.565 1.204
36.775 0.678 29.859 0.539 65.127 0.985 35.956 0.726
-0.005
(B)
0.005
0.004
0.003
0.002
0.001
0
-0.06
-0.03
0
0.03
0.06
Compound 4i (µM)
0.09
-0.001
4r
4s
4t
4u
Clorgiline
Selegiline
Figure 2. (A) Lineweaver–Burk plots for the inhibition of hMAO-B by compound
4i. [S], substrate concentration (mM); V, reaction velocity (nmol/min/mg protein).
Inhibitor concentrations are shown at the left. (B) Secondary plot for the calcula-
tion of the steady-state inhibition constant (Ki) of compound 4i. Ki was calculated
as 0.036 mM.
99.411 1.955 98.257 1.824
–
–
–
–
98.258 1.052 96.107 1.165
Table 2. IC50 values of 4b, 4d, 4f, 4i, 4t and selegiline against MAO-B.
MAO-B % Inhibition
10ꢂ6
Compounds
10ꢂ3
M
10ꢂ4
M
10ꢂ5
M
M
10ꢂ7
M
10ꢂ8
M
10ꢂ9
M
IC50 (mm)
4b
4d
4f
4i
90.256 1.304
91.578 1.247
90.585 1.388
94.859 1.405
92.588 1.129
99.387 1.385
86.301 1.294
79.679 1.459
83.327 1.259
91.755 1.258
88.565 1.204
95.629 1.456
82.456 1.107
76.245 1.057
76.311 1.114
78.629 1.106
80.497 1.157
86.205 1.200
76.901 1.266
70.896 1.006
73.203 1.058
72.455 1.115
74.698 1.009
78.324 1.108
45.325 0.974
47.498 0.957
48.645 0.957
65.167 0.985
60.755 1.164
66.871 1.056
32.608 0.728
29.670 0.723
32.499 0.721
46.508 0.892
36.131 0.862
42.875 0.865
20.417 0.683
15.157 0.602
20.874 0.623
24.475 0.627
20.971 0.594
16.748 0.596
0.188 0.008
0.127 0.005
0.146 0.006
0.041 0.001
0.065 0.002
0.037 0.001
4t
Selegiline

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1429
parameters, including molecular weight (MW), number of rotatable
bonds (RB), dipole moment (DM), molecular volume (MV), number
of hydrogen donors (DHB), number of hydrogen acceptors (AHB),
polar surface area (PSA), octanol/water partition coefficient (log P),
aqueous solubility (log S), apparent Caco-2 cell permeability
(PCaco), number of likely primer metabolic reactions (PM), percent
of human oral absorption (%HOA), and the violations of the rules
of three (VRT) and five (VRF) are presented in Table 4. In keeping
with Jorgensen’s “Rule of Three” and Lipinski’s rule of five, the
obtained compounds (4a–4u) are in accordance with the set
parameters as they did not cause more than one violation.
Drugs that specifically target the CNS must first pass the
blood–brain barrier (BBB). Although the BBB is protective in
nature, the use of drug candidates with CNS effects in a clinical
setting is unlikely if such drug molecules are unable to penetrate
it. Therefore, this feature should be examined earlier on in the
drug discovery process. Accordingly, predicting the BBB perme-
ability of new compounds is of great significance41. Thereby, the
BBB permeability of the obtained compounds (4a–4u) was also
evaluated using QikProp 4.8 software26. Brain/blood partition coef-
ficient (logBB) and apparent MDCK cell permeability (PMDCK)
were calculated for this purpose. In keeping with the software
estimates, the PMDCK values of <25 and >500 nm/sec are advised
as poor and great for non-active transport of compounds. In order
to assess for a compound’s capacity to pass through the BBB,
logBB is the other significant parameter to consider, with recom-
mended values between ꢂ3 and þ1.2. The PMDCK and logBB val-
ues of the synthesised compounds are within the advised ranges
as shown in Table 4. Thus, it can be postulated that the obtained
compounds are capable of crossing the BBB, which is crucial for
CNS-associated drugs.
(A)
0.015
0.010
0.005
0.000
IC50 / 2 (0.033 µM)
IC50 (0.065 µM)
2 x IC50 (0.130 µM)
Control
-3.5
-2.5
-1.5
-0.5
0.5
1.5
1/[S] µM-1
2.5
3.5
-0.005
(B)
0.003
0.002
0.001
0
-0.1
-0.05
0
0.05
0.1
0.15
-0.001
Compound 4t (µM)
Figure 3. (A) Lineweaver–Burk plots for the inhibition of hMAO-B by compound
4t. [S], substrate concentration (mM); V, reaction velocity (nmol/min/mg protein).
Inhibitor concentrations are shown at the left. (B) Secondary plot for the calcula-
tion of the steady-state inhibition constant (Ki) of compound 4t. Ki was calculated
as 0.055mM.
Considering the results of the ADME and BBB permeability
studies, the synthesised compounds have pharmacokinetic profiles
that may be appropriate for clinical use.
Table 3. The IC50 value of the compounds 4i and 4t against NIH/3T3 cell line.
Compounds
IC50 (mM) NIH/3T3 cell line
IC50 (mM) MAO-B enzyme
4i
4t
>1000
>1000
0.041 0.001
0.065 0.002
3.6. Molecular docking
As observed in the MAO inhibition assay studies, compounds 4i
and 4t were found to be the most active derivatives in the
hMAO-B enzyme inhibition series. Furthermore, compound 4i was
determined to be the most potent agent with an IC50 value of
0.041 0.001 mM; hence, docking studies were carried out to
evaluate its inhibition capability in silico. Using the X-ray crystal
structure of hMAO-B (PDB ID: 2V5Z)27, docking studies were per-
formed and the binding modes of compound 4i were assigned.
Also, this compound was subjected to the molecular docking pro-
cedure with the X-ray crystal structure of hMAO-A (PDB ID: 2Z5X)
to compare its binding modes on hMAO-A and hMAO-B enzymes.
Unfortunately, it was determined that compound 4i did not be
settle down to the active site of hMAO-A enzyme (data not
shown). Thus, no important and significant interactions were
observed. Actually, this evidence is consistent with in vitro enzyme
inhibition assay and supports the selective effect of compound 4i
and the other derivatives in the series on hMAO-B enzyme.
than their IC50 values (0.041 and 0.065 mM) against hMAO-B.
Consequently, compounds 4i and 4t were found to be non-cyto-
toxic at their effective concentrations against hMAO-B. This result
further increases the biological importance of the compounds.
3.5. Prediction of ADME parameters and BBB permeability
Intrinsic pharmacological activity and low toxicological effects are
not sufficient for a compound to become a drug nominee35. Most
new drug nominees fail in clinical trials due to their reduced
absorption, distribution, metabolism, and excretion (ADME) prop-
erties. These late-stage failures result in increased drug develop-
ment costs36. The ability to identify problematic issues early can
dramatically reduce the amount of wasted time and funds, and
can streamline the overall development process. Therefore, the
pharmacokinetic properties of new drug candidates are very
important, and it is beneficial to assess them as early as possible
in the drug development process37. ADME estimation can be used
to focus on precursor compound optimisation thereby improving
the preferred properties of a compound38. Predictions of ADME
parameters of the obtained compounds (4a–4u) were performed
The docking poses of this compound are presented in Figures
4–7. Compound 4i adequately binds to the amino acid residues
lining the cavity of hMAO-B enzyme and is located very near the
FAD cofactor. When analysing the docking poses of this com-
pound, it is clear that there is a p-p interaction, formation of three
using QikProp 4.8 software26. The violations of Jorgensen’s “Rule hydrogen bonds, and formation of a halogen bond. Compound 4i
of Three”39 and Lipinski’s rule of five40, which assess the ADME has a sulphonamide group at the 4th position of the phenyl ring.
properties of new drug nominees, are crucial for the optimisation This group is essential for polar interactions. The amino moiety of
of
a biologically active compound. The calculated ADME sulphonamide forms a hydrogen bond with the carbonyl of

1430
B. N. SAĞLIK ET AL.
Table 4. Calculated ADME parameters of compounds 4a–4u.
Comp.
MW
RB
DM
MV
DHB
AHB
PSA
logP
logS
PCaco
logBB
PMDCK
PM
%HOA
VRF
VRT
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4r
4s
4t
416.51
417.5
8
8
8
8
8
8
8
8
8
9
8
8
8
8
8
8
8
8
8
9
9.694
12.125
7.1
11.923
9.94
1233.2
1219.7
1198.4
1207.8
1382
1177.5
1311
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
9
107.701
125.131
113.396
143.808
139.593
95.543
115.869
104.573
104.572
112.58
107.701
125.131
120.374
143.808
139.593
95.543
115.869
104.573
104.572
112.58
2.369
1.753
2.082
0.987
2.334
2.553
2.718
3.385
3.865
3.472
2.651
2.032
2.234
1.261
2.617
2.831
3.004
3.673
4.155
3.76
ꢂ4.323
ꢂ4.058
ꢂ3.755
ꢂ3.669
ꢂ4.976
ꢂ3.681
ꢂ5.106
ꢂ5.787
ꢂ6.5
141.478
57.916
148.509
26.232
40.763
271.256
117.016
158.686
158.573
187.797
141.478
57.916
75.534
26.232
40.763
271.256
117.016
158.686
158.573
187.797
ꢂ1.784
ꢂ2.181
ꢂ1.482
ꢂ2.537
ꢂ2.509
ꢂ1.295
ꢂ2.012
ꢂ1.785
ꢂ1.651
ꢂ1.785
ꢂ1.84
90.865
34.702
183.53
14.803
21.026
217.606
81.775
199.449
491.011
246.266
90.865
34.702
64.7
14.803
21.026
217.606
81.775
199.449
491.011
246.266
2
2
4
2
2
3
3
3
3
4
3
3
5
3
3
4
4
4
4
5
79.311
68.759
78.006
58.119
69.43
85.444
79.88
86.149
75.996
87.968
80.962
70.393
73.642
59.725
71.09
87.072
81.551
87.839
77.697
76.7
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
1
1
1
0
0
0
0
0
0
0
1
1
1
9.5
9.5
10.5
10.5
8.5
9.5
9
9
9.75
9
9.5
9.5
10.5
10.5
8.5
9.5
9
434.55
418.49
480.56
413.51
453.53
469.59
504.04
499.62
430.54
431.53
448.57
432.51
494.59
427.54
467.56
483.62
518.06
513.64
6.179
5.536
6.263
4.786
5.132
10.106
12.578
10.406
12.376
9.925
6.246
5.601
6.255
4.857
4.95
1346.1
1390
1411.6
1292.1
1278.7
1288.9
1266.8
1440.9
1236.4
1369.9
1405
ꢂ5.883
ꢂ4.837
ꢂ4.565
ꢂ4.621
ꢂ4.166
ꢂ5.493
ꢂ4.186
ꢂ5.628
ꢂ6.316
ꢂ7.033
ꢂ6.411
ꢂ2.248
ꢂ2.01
ꢂ2.615
ꢂ2.579
ꢂ1.344
ꢂ2.067
ꢂ1.836
ꢂ1.702
ꢂ1.834
1448.9
1470.6
9
9.75
4u
MW: Molecular weight RB: Number of rotatable bonds DM: Computed dipole moment MV: Total solvent-accessible volume DHB: Estimated number of hydrogen
bond donors AHB: Estimated number of hydrogen bond acceptors PSA: Van der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms
logP: Predicted octanol/water partition coefficient logS: Predicted aqueous solubility PCaco: Predicted apparent Caco-2 cell permeability logBB: Predicted brain/blood
partition coefficient PMDCK: Predicted apparent MDCK cell permeability PM: Number of likely metabolic reactions %HOA: Predicted human oral absorption percent
VRF: Number of violations of Lipinski’s rule of five. The rules are: MW < 500, logP < 5, DHB ꢄ 5, AHB ꢄ 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 < 7.
Figure 5. The three-dimensional interacting mode of compound 4i in the active
region of hMAO-B. The inhibitor and the important residues in the active site of
the enzyme are presented by tube model. The FAD molecule is coloured dark
green with tube model.
Figure 4. The three-dimensional pose of compound 4i in the active region of
hMAO-B (PDB ID: 2V5Z). The important residues in the active site and this com-
pound are presented by tube model and coloured with grey and red,
respectively.
Pro102. In addition, there is another hydrogen bond between the
oxygen atom of sulphonamide and the amino group of Thr201. As
mentioned above, one of the main structural modifications of BB-
4b, as previously reported by our research group2, is the substitu-
Figure 6. The van der Waals interaction of compound 4i with active region of
hMAO-B. The active ligand has a lot of favourable van der Waals interactions (red
and pink).
tion of the fluorine atom with a sulphonamide group (Figure 1).
All the detected interactions of the sulphonamide group prove
that the structural modification of compound BB-4b is a suitable
approach. The addition of the sulphonamide group made a posi-
tive contribution to the MAO enzyme inhibition profile.
group of Leu171. Compound 4i has a benzothiazole ring as a het-
erocyclic ring. The benzene on the benzothiazole ring interacts
Another formation of hydrogen bond is observed between the with the phenyl of Thr398. Interaction with the Thr398 amino acid
carbonyl of the amide group in the structure and the amino is very important in terms of catalytic activity, and the binding of

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1431
Disclosure statement
The authors declare no conflicts of interest.
Funding
This study was financially supported by Anadolu University
Scientific Projects Fund, Project No: 2001E005.
ORCID
€
ꢀ
Begum Nurpelin Saglık
http://orcid.org/0000-0002-0151-6266
_
Figure 7. The electrostatic interaction of compound 4I with active region of
Derya Osmaniye
Ulviye Acar C¸evik
Serkan Levent
http://orcid.org/0000-0002-0499-436X
http://orcid.org/0000-0003-1879-1034
http://orcid.org/0000-0003-3692-163X
hMAO-B. The residues are coloured (blue, red, and pink) according to the dis-
tance from ligand by Per-Residue Interaction panel.
€
ꢀ
Betul Kaya C¸avus¸oglu
http://orcid.org/0000-0001-8270-4949
http://orcid.org/0000-0002-6131-3399
€
ꢀ
Ozlem Atlı Eklioglu
€
Yusuf Ozkay
Ali Savas¸ Koparal
http://orcid.org/0000-0001-8815-153X
http://orcid.org/0000-0002-6894-5604
inhibitor candidates in the substrate cavity of the MAO-B enzyme.
This finding indicates that compound 4i binds very effectively to
the MAO-B enzyme active site.
Zafer Asım Kaplancıklı
http://orcid.org/0000-0003-2252-0923
The main structural difference between compound 4i and the
other compounds in the series is that it carries a chlorine atom at
the 5th position of the benzothiazole ring. It is clearly observed in
Figure 5 that this halogen atom establishes a halogen bond with
the hydrogen of the hydroxyl group of Tyr188. This additional
interaction ensures that it binds more strongly to the active site.
Furthermore, all these interactions explain why compound 4i
exhibits a stronger inhibition profile than the other compounds.
In order to analyse the contribution of van der Waals and elec-
trostatic interactions in binding to the enzyme active site, docking
studies were performed using Glide, according to the Per-Residue
Interaction panel. Figures 6 and 7 present the van der Waals and
electrostatic interactions of compound 4i. As shown in the figures,
this compound has favourable van der Waals interactions with
Leu88, Phe99, Phe103, Pro104, Tyr119, Leu167, Phe168, Leu171,
Cys172, Tyr188, Ile198, Ile199, Ser200, Gln206, Ile316, Tyr326,
Phe343, Tyr398 and Tyr435, which are displayed in pink and red
colours as described in the user guide of Glide31. Similarly, promis-
ing electrostatic contributions of compound 4i have been deter-
mined with Pro102, Phe168, Leu171, Ile199, Ser200 and Ile316
amino acids.
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