Synthesis of new benzothiazole derivatives bearing thiadiazole as monoamine oxidase inhibitors

Article abstract of DOI:10.1002/jhet.3942

Monoamine oxidases (MAO) are enzymes that catalyze the oxidative deamination of monoamines such as dopamine, noradrenaline, adrenaline, and serotonin. Recent studies have shown that numerous benzothiazole derivatives exhibit hMAO inhibitory activity in the micromolar concentration range. In this study, a novel series of benzothiazole-thiadiazole (5a-5l) was synthesized and characterized their chemical structures by ¹H-NMR, ¹³C-NMR, and Mass spectroscopy. These compounds were evaluated as inhibitors for types A and B MAO enzymes. Compounds 5f and 5l were the most active derivatives in the series with an IC₅₀ values of 0.107 ± 0.003 and 0.128 ± 0.004, respectively. Furthermore, cytotoxicity of compounds 5f and 5l were investigated and found as non-cytotoxic.

Full text of DOI:10.1002/jhet.3942

Received: 9 December 2019
DOI: 10.1002/jhet.3942
Revised: 30 January 2020
Accepted: 9 February 2020
A R T I C L E
Synthesis of new benzothiazole derivatives bearing
thiadiazole as monoamine oxidase inhibitors
1,2
Ulviye Acar Çevik1,2
Serkan Levent1,2 | Betül K. Çavus¸oglu
Ümide D. Özkay4 | Yusuf Özkay1,2
| Derya Osmaniye1,2
| Begüm N. Saglik
|
ꢀ
| Abdullah B. Karaduman3
|
| Zafer A. Kaplancikli1 | Gülhan Turan1
1,2
ꢀ
1Department of Pharmaceutical
Abstract
Chemistry, Faculty of Pharmacy, Anadolu
University, Eskisehir, Turkey
Monoamine oxidases (MAO) are enzymes that catalyze the oxidative deamina-
tion of monoamines such as dopamine, noradrenaline, adrenaline, and seroto-
nin. Recent studies have shown that numerous benzothiazole derivatives
exhibit hMAO inhibitory activity in the micromolar concentration range. In
this study, a novel series of benzothiazole-thiadiazole (5a-5l) was synthesized
2Anadolu University, Faculty of
Pharmacy, Doping and Narcotic
Compounds Analysis Laboratory,
Eskis¸ehir, Turkey
3Faculty of Pharmacy, Department of
Pharmaceutical Toxicology, Anadolu
University, Eskisehir, Turkey
and characterized their chemical structures by H-NMR, 13C-NMR, and Mass
1
spectroscopy. These compounds were evaluated as inhibitors for types A and B
MAO enzymes. Compounds 5f and 5l were the most active derivatives in the
series with an IC50 values of 0.107 0.003 and 0.128 0.004, respectively. Fur-
thermore, cytotoxicity of compounds 5f and 5l were investigated and found as
non-cytotoxic.
4Department of Pharmacology, Faculty of
Pharmacy, Anadolu University, Eskisehir,
Turkey
Correspondence
Derya Osmaniye, Department of
Pharmaceutical Chemistry, Faculty of
Pharmacy, Anadolu University, Eskisehir,
Turkey.
Email: dosmaniye@anadolu.edu.tr
1 | INTRODUCTION
β-phenylethylamine, benzylamine while MAO-A has sub-
strate specificity for bulkier endogenous amines, for
Monoamine oxidase (MAO) is the FAD-dependent
enzyme and is found in the outer mitochondrial mem-
brane of the glial, neuronal other mammalian cell.[1]
MAO present in the liver and intestinal epithelium and
are abundant in noradrenergic nerve terminals. MAOs
catalyze the oxidative deamination of exogenous and
endogenous monoamines to the conforming hydrogen
peroxide, aldehyde, and ammonia or substituted
amine.[2] This enzymes is crucial for a regular function of
synaptic neurotransmission such as serotonin (5-HT),
norepinephrine (NE), and dopamine (DA).[3]
MAO consist of two isoforms namely, MAO-A and
MAO-B. These isoforms have a sequence similarity of
73% but differ essentially in their inhibitor selectivity,
substrate specificity, and tissue delivery.[4] The MAO-B
isoform has substrate specificity for small amines like
example, serotonin, epinephrine, and NE.[5] Some sub-
strates such as DA and tyramine are available for both
enzyme isoforms.
MAOs, which play an important role in the regulation
of central nervous system activity, are involved in the
pathogenesis of many neurodegenerative and depressive
disorders. Thus, the development of MAO inhibitors
(MAOIs) characterizes a very important and beneficial
approach for the treatment of various neurological and
neuropsychiatric disorders.[2]
MAOIs, the first antidepressant agents to be discov-
ered, had a number of side effects, including irreversible
binding to the enzyme, and lethal food–drug interactions.
New generation MAOIs, which are less toxic, selective
inhibitors, reversible, have been found to be effective
against a variety of neurological diseases. The MAO
J Heterocyclic Chem. 2020;1–9.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

2
ACAR ÇEVIK ET AL.
enzyme is now considered to be an important drug target,
and MAO-A selective inhibitors have been developed as
drug candidates for the management of depression
and anxiety disorders. On the other hand, MAO-B selec-
tive inhibitors have been developed as drug candidates
for treatment of Alzheimer's disease and Parkinson's dis-
ease with a better safety profile than nonselective
MAOIs.
The neuroprotective potential of MAOIs has been the
focus of interest among researchers and neuroscientists.
On the other hand, the mechanisms of action of MAOIs
are not yet fully clear. Attention is now shifting towards
the development of a single drug as a highly targeted
agent, and this is due to the complexity of various neuro-
genic disorders. The expectation of these multi-targeted
drugs is that they bind to different targets at the same
time and therefore help in the treatment of complex neu-
rological diseases. Computational and synthetic chemis-
try are tools to design ligands with properties for
simultaneous inhibition of MAO enzyme and other
receptors/enzymes such as cholinesterase.[6]
Heterocyclic compounds have become progressively
attractive because of their potential applications in many
fields. Benzothiazole belonging to an enormously impor-
tant family of synthetic compounds of heterocyclic sys-
tems is a advantaged bicyclic ring system with multiple
applications. Moreover, 1,3,4-thiadiazole moiety is con-
nected to a broad spectrum of biological activities possi-
bly due to the presence of pharmacophoric (N C S)
moiety. 1,3,4-thiadiazole is the most extensively investi-
gated as the sulfur atom in its ring produces an inductive
effect, resulting in weak basicity and relatively high
aromaticity.[7–10]
The prior studies have demonstrated that ben-
zothiazoles have potent inhibitory activity for MAO
enzymes.[11–14] In this study, a similar strategy was used
for the design of target compounds. The synthesized com-
pounds are designed to have three basic parts: (a) a
2-amino-or mercapto-substituted benzothiazole core;
(b) a heteroaromatic or aromatic ring system; and (iii) a
linker, including H-donor and/or H-acceptor nitrogen,
between the (hetero)aromatic ring system and ben-
zothiazole core (Figure 1).
(Hetero)
Aromatic
Side Ring
Benzothiazole
Core Ring
Design Strategy
Linker
N
N
O
H
R1
N
S
S
N
H
N
Title compounds
S
R2
FIGURE 1 Design of the target compounds
Based on this data and our previous studies,[13,14,17]
new benzothiazole derivatives as MAOIs were designed,
synthesized, and biological activities were evaluated in
this study.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
In this study, we have synthesized new compounds, which
possessed benzothiazole-thidiazole moiety. The synthesis
procedure was carried out via five steps. And synthetic
pathway was outlined in Figure 2. First, suitable isothiocy-
anate and excess of hydrazine hydrate were reacted in
order to obtained compounds 1a-1c. Then, compounds 1a-
1j and carbondisulfide were reacted to gain thiadiazoles
(2a-2c). Then, 5-substituebenzothiazol-2-amine (3a-3d)
was obtained via ring closure reaction. The obtained
benzothiazole-2-amine derivatives (3a-3d) were acetylated
with chloroacetyl chloride. Finally, target compounds (5a-
5l) were obtained with the reaction between compounds
2a-2c and compounds 4a-4d. The final compounds were
purified, and their structures were characterized by spec-
troscopic methods (1H-NMR, 13C-NMR, and LCMSMS)
(see Data S1). The 1H-NMR spectra of compounds showed
signals at 4.03 to 4.20 ppm for methylene proton. Ben-
zothiazole N H had peaks between 12.62 ppm and
13.12 ppm. Thiadiazole NH gave peaks between 7.81 ppm
and 7.93 ppm. In the 13C NMR spectrum, aliphatic peaks
belonging to substituents were observed between
11.79 ppm and 70.37 ppm. Aromatic carbons were gained
between 119.61 ppm and 170.45 ppm. All masses were
accorded with the estimated M + H values.
In addition, the thiosemicarbazides (TSCs) which are
open-chain counterparts of the thiadiazoles also possess
MAO inhibitor activity.[11,12] TSCs, which are small mole-
cules that can cross the blood brain barrier, can be used
as potential drugs for the treatment of neurodegenerative
diseases due to these properties. Moreover, it has been
reported that TSCs have some common features as H-
bond donors, H-bond acceptors, positive ionizable atoms,
hydrophobic substructures, and aromatic rings to interact
with MAO enzymes.[15,16]
2.2 | Biological activity
The synthesized compounds were evaluated for their
hMAO-A and hMAO-B inhibitory activities. Selegiline

ACAR ÇEVIK ET AL.
3
FIGURE 2 The general synthetic pathway of the target compounds (5a-5l)
and moclobemide were evaluated as reference com-
pounds. The results, expressed as % inhibition and IC50
values are summarized in Tables 1 and 2. In the first step,
the synthesized compounds were prepared at concentra-
tions of 10−3 ve 10−4 M and inhibition values of MAO-A
and MAO-B were calculated. The compounds showing
>50% inhibition were selected for the second stage
according to the first step results. In this second step,
inhibition values and IC50 values of the selected com-
pounds at concentrations of 10−5 to 10−9 M were calcu-
lated. Only compounds 5f and 5l passed the second step
hMAO-A enzyme inhibition assay.
drug. When the active compounds are examined, it is
seen that both of them carry nitro groups on the ben-
zothiazole ring. This finding proves the reported MAO
inhibitory effect of the nitro group. However, compounds
5c and 5i did not show high activity even though they
contained nitro group. The effect of this may be related
to the substituent on the thiadiazole ring. In view of the
active compounds, their structures include methoxyethyl
and butyl groups, respectively. So, a 4-membered chain is
attached to the amine of the thiadiazole ring. In this case,
the 4-membered thiadiazole amine-bound group has a
positive effect on activity.
Generally, the compounds showed higher activity
against hMAO-A than hMAO-B. In particular, com-
pounds 5f and 5l have a significant activity value against
hMAO-A. Compounds 5f and 5l showed efficient inhibi-
tion against hMAO-A with IC50 values of 0.128
0.004 μM, 0.107 0.003 μM, respectively. Both of them
displayed more potent activity compared to reference
2.3 | Cytotoxicity test
It is also important that a compound has minimal side
effects and cytotoxicity. For this purpose, the cytotoxic
effects of the most active compounds (5f and 5l) on the

4
ACAR ÇEVIK ET AL.
TABLE 1 % Inhibition of
compounds 5a-5l, moclobemide and
MAO-A Inhibition (%)
MAO-B Inhibition (%)
10−3 10−4
M
Comp.
10−3 M
10−4
M
M
selegiline against MAO-A and MAO-B
5a
28.29 0.95
41.44 0.71
39.55 0.67
41.58 0.80
35.20 0.56
88.03 1.39
45.39 0.99
49.33 0.79
46.98 0.92
23.02 0.58
41.83 0.89
81.20 1.05
94.12 2.76
-
21.53 0.84
32.58 0.55
28.61 0.46
30.29 0.47
30.29 0.49
83.30 1.40
40.21 0.81
39.10 0.88
42.75 0.81
15.95 0.55
36.74 0.71
74.25 1.10
82.14 2.69
-
25.12 0.62
36.65 0.89
48.29 0.97
24.50 0.62
31.28 0.49
39.55 0.77
38.20 0.57
36.79 0.63
43.29 0.85
44.07 0.75
26.33 0.65
29.82 0.63
-
18.11 0.54
28.51 0.60
26.11 0.84
17.55 0.48
20.23 0.44
24.66 0.63
19.67 0.44
31.77 0.59
25.18 0.41
36.99 0.88
20.44 0.53
23.67 0.51
-
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
Moclobemide
Selegiline
98.91 1.28
96.88 1.31
Note: The most effective derivatives are shown in bold.
FIGURE 3 The interacting
mode of compound 5l in the active
region of hMAO-A. The inhibitor,
colored with red, and the important
residues in the active site of the
enzyme are presented by tube model.
The FAD molecule is colored green
with ball and stick model
[18]
NIH3T3 cell line were investigated. Compounds 5f and 5l
displayed non-cytotoxicity. The IC50 value of the 5f was
calculated as 0.316 0.013 μM. And the IC50 of com-
pound 5I was found to be greater than one.
structure of hMAO-A (PDB ID: 2Z5X)
was obtained
from Protein Data Bank server (www.pdb.org). The dock-
ing pose of compound 5l on hMAO-A is presented in
Figure 3.
The compounds 5l adequately binds to amino acid
residues lining the cavity and are located very near the
FAD cofactor. When analyzed docking pose of this com-
pound, it is clearly seen that nitrogen atom of nitro moi-
ety at sixth position of benzothiazole ring is in
interaction with phenyl of Tyr407 by doing cation–π
interaction. A hydrogen bond formation is seen between
carbonyl of amide group near to benzothiazole ring and
amino of Gln215. Also, another hydrogen bond observed
is belonging to amino group between thiadiazole ring
2.3.1 | Molecular docking studies
The compound 5l was found to be the most active and
selective hMAO-A inhibitor as mentioned in MAO inhi-
bition assay. Docking studies were conducted to learn
more about the binding modes of compound 5l and to
evaluate the effects of structural modifications on
the inhibitory activity against hMAO-A. X-ray crystal

ACAR ÇEVIK ET AL.
5
and butyl alkyl chain. This amino group forms a hydro-
gen bond with carbonyl of Ala111. Besides, it is thought
that the presence of extended alkyl chain such as butyl
substituents of this amino group contribute to bind to
active site positively via van der Waals interactions. All
these findings could help to explain the potent in vitro
inhibition profile of compound 5l.
3 | EXPERIMENTAL
3.1 | Chemistry
All chemicals were purchased from Sigma-Aldrich
Chemical Co (Sigma-Aldrich Corp., St. Louis, MO, USA)
and Merck Chemicals (Merck KGaA, Darmstadt, Ger-
many). All reactions were monitored by thin-layer chro-
matography (TLC) using Silica Gel 60 F254 TLC plates
(Merck KGaA, Darmstadt, Germany). All melting points
(m.p.) were determined by MP90 digital melting point
apparatus (Mettler Toledo, Ohio, USA) and were
uncorrected. Spectroscopic data were recorded with the
1
following instruments: H-NMR (nuclear magnetic reso-
nance) Bruker DPX 300 FT-NMR spectrometer; 13C-
NMR, Bruker DPX 75 MHz spectrometer (Bruker Biosci-
ence, Billerica, MA, USA) and M + 1 peaks were deter-
mined by Shimadzu 8040 LC/MS/MS system (Shimadzu,
Tokyo, Japan). Elemental analyses were performed on a
Leco 932 CHNS analyzer (Leco, Michigan, USA).
3.1.1 | Preparation of N-
substitutedhydrazinecarbothioamides
(1a-1c)
A
mixture of suitable isothiocyanate derivatives
(0.02 mol) and hydrazine hydrate (0.04 mol) were stirred
in ethanol (30 mL) for 4 h. After completion of the reac-
tion, the precipitated product was filtered and washed
with cold-ethanol.
3.1.2 | Preparation of
5-(substituteamino)-1,3,4-thiadiazole-
2-thiols (2a-2c)
Carbon disulfide (0.019 mol) was added in a solution of
compounds 1a-1c (0.018 mol) in EtOH in presence of
sodium hydroxide (0.019 mol) and then the mixture was
refluxed for 8 h.
After this period, the solution was cooled and acidi-
fied to pH 4 to 5 with hydrochloric acid solution and crys-
tallized from ethanol.

6
ACAR ÇEVIK ET AL.
3.1.3 | Preparation of
5-substituebenzothiazol-2-amine (3a-3d)
J = 5.3 Hz, NH), 7.99 (d, 1H, J = 7.8 Hz, H-ben-
zothiazole), 12.64 (s, 1H, NH). 13C-NMR (DMSO-d6,
ppm, 75 MHz,): δ = 14.64, 38.00, 39.83, 121.11, 122.24,
124.16, 126.67, 131.91, 148.61, 148.95, 158.21, 167.78,
170.22. ESI-MS [M + H]+: 352.
3-substitueanilines (0.01 mol) and potassium thiocyanate
(0.01 mol) were dissolved in acetic acid. This mixture was
cooled in an ice bath and brome solution (0.012 mol) in
acetic acid was added dropwise with stirring. After com-
pletion of the reaction, the precipitated product was fil-
tered and dissolved with water at 100ꢀC. After cooling,
the heated mixture was neutralized with ammonia. The
precipitated product was filtered and crystallization with
ethanol.
3.1.7 | N-(6-chlorobenzothiazol-2-yl)-
2-((5-(ethylamino)-1,3,4-thiadiazol-2-yl)
thio)acetamide (5b)
Yield: 84%, M.P. = 281.1 to 282.8ꢀC, 1H-NMR (DMSO-d6,
ppm, 300 MHz,): 1.13 (t, 3H, J = 7.1 Hz, CH3), 3.19 to
3.28 (m, 2H, CH2CH3 ), 4.15 (s, 2H, CH2 ), 7.46 (d,
1H, J = 8.6 Hz, H-benzothiazole), 7.75 (d, 1H, J = 8.6 Hz,
H-benzothiazole), 7.81 (br.s., 1H, NH), 8.13 (br.s., 1H,
H-benzothiazole), 12.71 (1H, s, NH). 13C-NMR (DMSO-
d6, ppm, 75 MHz,): δ = 14.63, 37.92, 121.97, 122.35,
127.03, 128.02, 133.61, 147.85, 148.56, 159.01, 167.98,
170.22. ESI-MS [M + H]+: 385.
3.1.4 | Preparation of 2-chloro-N-
(5-substituebenzothiazol-2-yl)acetamide
(4a-4d)
5-Substituebenzothiazol-2-amine (0.01 mol) and triethyl
amine (0.011 mol) were dissolved in tetrahydrofuran.
This mixture was cooled in an ice bath and chloroacetyl
chloride (0.012 mol) in tetrahydrofuran was added
dropwise with stirring. After completion of reaction, tet-
rahydrofuran was removed under reduced pressure, pre-
cipitated product was washed with water in order to
removed obtained salts.
3.1.8 | 2-((5-(ethylamino)-
1,3,4-thiadiazol-2-yl)thio)-N-
(6-nitrobenzothiazol-2-yl)acetamide (5c)
Yield: 79%, M.P. = 282.9 to 283.9ꢀC, 1H-NMR (DMSO-d6,
ppm, 300 MHz,): 1.13 (t, 3H, J = 7.1 Hz, CH3), 3.22 to
3.26 (m, 2H, CH2CH3 ), 4.20 (s, 2H, CH2 ), 7.85 (br.
s., 1H, NH), 7.91 (d, 1H, J = 8.9 Hz, H-benzothiazole),
8.28 (d, 1H, J = 8.8 Hz, H-benzothiazole), 9.06 (br.s., 1H,
H-benzothiazole), 13.04 (s, 1H, NH). 13C-NMR (DMSO-
d6, ppm, 75 MHz,): δ = 14.62, 37.93, 119.61, 121.23,
122.30, 132.66, 143.55, 148.57, 153.84, 163.71, 168.55,
170.21. ESI-MS [M + H]+: 397.
3.1.5 | Preparation of target compounds
(5a-5l)
2-chloro-N-(5-substituebenzothiazol-2-yl)acetamide (4a-
4d) (0.0013 mol), 5-(substituteamino)-1,3,4-thiadiazole-
2-thiols (2a-2c) (0.0013 mol) and potassium carbonate
(0.0013 mol) as catalyst were stirred for 4 h in acetone.
After completion of the reaction, acetone was evaporated
under reduced pressure. The precipitated product was
washed with water in order to removed potassium car-
bonate, dried and recrystallized from EtOH. The synthe-
sized derivatives are presented in Table 3.
3.1.9 | N-(benzothiazol-2-yl)-
2-((5-((2-methoxyethyl)amino)-
1,3,4-thiadiazol-2-yl)thio)acetamide (5d)
Yield: 78%, M.P. = 241.2 to 243.0ꢀC, 1H-NMR (DMSO-d6,
ppm, 300 MHz,): 3.24 (s, 3H, OCH3), 3.39 to 3.47 (m,
4H, CH2CH2 ), 4.16 (s, 2H, CH2 ), 7.31 (td, 1H,
J1 = 1.1 Hz, J2 = 6.9 Hz, H-benzothiazole), 7.44 (td, 1H,
J1 = 1.2 Hz, J2 = 7.3 Hz, H-benzothiazole), 7.76 (d, 1H,
J = 7.9 Hz, H-benzothiazole), 7.93 (t, 1H, J = 5.3 Hz,
NH), 7.99 (d, 1H, J = 7.4 Hz, H-benzothiazole), 12.64
(s, 1H, NH). 13C-NMR (DMSO-d6, ppm, 75 MHz,):
δ = 37.89, 44.40, 58.40, 70.37, 121.14, 122.25, 124.18,
126.68, 131.91, 148.96, 149.03, 158.14, 167.73, 170.16. ESI-
MS [M + H]+: 382.
3.1.6 | N-(benzothiazol-2-yl)-
2-((5-(ethylamino)-1,3,4-thiadiazol-2-yl)
thio)acetamide (5a)
Yield: 81%, M.P. = 242.6 to 244.9ꢀC, 1H-NMR (DMSO-d6,
ppm, 300 MHz,): 1.13 (t, 3H, J = 7.2 Hz, CH3), 3.25 (q,
2H, J = 5.4 Hz, CH2CH3 ), 4.16 (s, 2H, CH2 ), 7.32
(td, 1H, J1 = 0.9 Hz, J2 = 7.0 Hz, H-benzothiazole), 7.45
(td, 1H, J1 = 1.0 Hz, J2 = 7.1 Hz, H-benzothiazole), 7.77
(d, 1H, J = 7.9 Hz, H-benzothiazole), 7.83 (t, 1H,

ACAR ÇEVIK ET AL.
7
benzothiazole), 13.04 (s, 1H, NH). 13C-NMR (DMSO-d6,
ppm, 75 MHz,): δ = 37.99, 44.42, 58.40, 70.38, 119.55,
121.16, 122.27, 132.69, 143.48, 148.91, 153.92, 163.92,
168.68, 170.19. ESI-MS [M + H]+: 427.
TABLE 3 Synthesized compounds (5a-5l)
3.1.12 | N-(benzothiazol-2-yl)-
2-((5-(propylamino)-1,3,4-thiadiazol-2-yl)
thio)acetamide (5g)
Yield: 82%, M.P. = 232.6 to 234.7ꢀC, 1H-NMR (DMSO-d6,
ppm, 300 MHz,): 0.87 (t, 3H, J = 7.3 Hz CH3), 1.53 (q,
2H, J = 7.1 Hz, CH2 ), 3.18 (q, 2H, J = 6.9 Hz,
CH2 ), 4.15 (s, 2H, CH2 ), 7.32 (td, 1H, J1 = 1.1 Hz,
J2 = 6.9 Hz, H-benzothiazole), 7.45 (td, 1H, J1 = 1.2 Hz,
J2 = 7.3 Hz, H-benzothiazole), 7.76 (d, 1H, J = 7.9 Hz, H-
benzothiazole), 7.84 (t, 1H, J = 5.4 Hz, NH), 7.99 (d,
1H, J = 7.4 Hz, H-benzothiazole), 12.62 (s, 1H, NH).
13C-NMR (DMSO-d6, ppm, 75 MHz,): δ = 11.79, 22.19,
37.97, 46.81, 121.14, 122.25, 124.18, 126.68, 143.29,
148.48, 149.05, 158.13, 167.75, 170.45. ESI-MS [M
+ H]+: 366.
Compounds
R1
R2
5a
5b
5c
5d
5e
5f
-Ethyl
H
-Ethyl
Cl
-Ethyl
NO2
H
-Methoxyethyl
-Methoxyethyl
-Methoxyethyl
-Propyl
Cl
NO2
H
5g
5h
5i
-Propyl
Cl
-Propyl
NO2
H
5j
-Butyl
5k
5l
-Butyl
Cl
-Butyl
NO2
3.1.13 | N-(6-chlorobenzothiazol-2-yl)-
2-((5-(propylamino)-1,3,4-thiadiazol-2-yl)
thio)acetamide (5h)
3.1.10 | N-(6-chlorobenzothiazol-2-yl)-
2-((5-((2-methoxyethyl)amino)-
1,3,4-thiadiazol-2-yl)thio)acetamide (5e)
Yield: 78%, M.P. = 265.5 to 267.0ꢀC, 1H-NMR (DMSO-d6,
ppm, 300 MHz,): 0.87 (t, 3H, J = 7.4 Hz CH3), 1.53 (q,
2H, J = 7.2 Hz, CH2 ), 3.17 (q, 2H, J = 6.9 Hz,
CH2 ), 4.15 (s, 2H, CH2 ), 7.46 (dd, 1H, J1 = 2.2 Hz,
J2 = 8.6 Hz, H-benzothiazole), 7.75 (d, 1H, J = 8.6 Hz, H-
benzothiazole), 7.84 (t, 1H, J = 5.3 Hz, NH), 8.13 (d,
1H, J = 2.1 Hz, H-benzothiazole), 12.72 (s, 1H, NH).
13C-NMR (DMSO-d6, ppm, 75 MHz,): δ = 11.79, 22.18,
37.94, 46.81, 121.97, 122.34, 127.02, 128.21, 133.62,
147.85, 148.41, 159.04, 168.01, 170.45. ESI-MS [M
+ H]+: 400.
Yield: 80%, M.P. = 258.9 to 261.5ꢀC, 1H-NMR (DMSO-d6,
ppm, 300 MHz,): 3.24 (s, 3H, -OCH3), 3.39 to 3.45 (m, 4H,
-CH2CH2-), 4.16 (s, 2H,
CH2 ), 7.47 (dd, 1H,
J1 = 2.2 Hz, J2 = 8.6 Hz, H-benzothiazole), 7.76 (d, 1H,
J = 8.6 Hz, H-benzothiazole), 7.93 (t, 1H, J = 5.3 Hz,
NH), 8.14 (d, 1H, J = 2.0 Hz, H-benzothiazole), 12.74
(s, 1H, NH). 13C-NMR (DMSO-d6, ppm, 75 MHz,):
δ = 37.83, 44.39, 58.40, 70.36, 121.99, 122.36, 127.04,
128.21, 133.61, 147.86, 148.96, 159.02, 167.97, 170.16. ESI-
MS [M + H]+: 416.
3.1.14 | N-(6-nitrobenzothiazol-2-yl)-
2-((5-(propylamino)-1,3,4-thiadiazol-2-yl)
thio)acetamide (5i)
3.1.11 | 2-((5-((2-methoxyethyl)amino)-
1,3,4-thiadiazol-2-yl)thio)-N-
(6-nitrobenzothiazol-2-yl)acetamide (5f)
Yield: 75%, M.P. = 265.5 to 267.6ꢀC, 1H-NMR (DMSO-d6,
ppm, 300 MHz,): 0.86 (t, 3H, J = 7.3 Hz CH3), 1.52 (q,
2H, J = 7.2 Hz, CH2 ), 3.17 (q, 2H, J = 6.8 Hz,
CH2 ), 4.19 (s, 2H, CH2 ), 7.84 (t, 1H, J = 5.3 Hz,
NH), 7.91 (d, 1H, J = 8.9 Hz, H-benzothiazole), 8.28
(dd, 1H, J1 = 2.4 Hz, J2 = 8.9 Hz, H-benzothiazole), 9.06
(d, 1H, J = 2.3 Hz, H-benzothiazole), 13.04 (s, 1H, NH).
13C-NMR (DMSO-d6, ppm, 75 MHz,): δ = 11.79, 22.18,
Yield: 77%, M.P. = 263.0 to 264.7ꢀC, 1H-NMR (DMSO-d6,
ppm, 300 MHz,): 3.24 (s, 3H, OCH3), 3.38 to 3.45 (m,
4H, CH2CH2 ), 4.20 (s, 2H, CH2 ), 7.89 to 7.92 (m,
2H, H-benzothiazole NH), 8.28 (dd, 1H, J1 = 2.3 Hz,
J2 = 8.9 Hz, H-benzothiazole), 9.05 (d, 1H, J = 2.3 Hz, H-

8
ACAR ÇEVIK ET AL.
38.03, 46.81, 119.57, 121.17, 122.28, 132.68, 143.50,
148.34, 153.90, 163.88, 168.66, 170.45. ESI-MS [M
+ H]+: 411.
30.96, 38.57, 44.64, 119.35, 120.79, 122.14, 132.79, 143.11,
148.72, 154.33, 165.12, 169.42, 170.28. ESI-MS [M
+ H]+: 425.
3.1.15 | N-(benzothiazol-2-yl)-
2-((5-(butylamino)-1,3,4-thiadiazol-2-yl)
thio)acetamide (5j)
3.2 | MAO-A and MAO-B inhibition
assay
The inhibitory activities of the obtained compounds
against MAO-A and MAO-B enzymes were evaluated in
black-bottom 96-well plates by method as defined in our
previous studies.[17,19,20]
1
Yield: 81%, M.P. = 229.6 to 231.4ꢀC, H-NMR (DMSO-
d6, ppm, 300 MHz,): 0.86 (t, 3H, J = 7.3 Hz CH3), 1.30
(q, 2H, J = 7.6 Hz, CH2 ), 1.44 to 1.54 (m, 2H,
CH2 ), 3.20 (q, 2H, J = 6.9 Hz, CH2 ), 4.15 (s, 2H,
CH2 ), 7.31 (td, 1H, J1 = 1.1 Hz, J2 = 8.1 Hz, H-ben-
zothiazole), 7.44 (td, 1H, J1 = 1.3 Hz, J2 = 7.3 Hz, H-
benzothiazole), 7.76 (d, 1H, J = 7.9 Hz, H-ben-
zothiazole), 7.83 (t, 1H, J = 5.4 Hz, NH), 7.99 (d, 1H,
J = 7.3 Hz, H-benzothiazole), 12.62 (s, 1H, NH). 13C-
NMR (DMSO-d6, ppm, 75 MHz,): δ = 14.07, 19.97,
30.96, 37.99, 44.68, 121.11, 122.25, 124.15, 126.66,
131.91, 148.48, 148.95, 158.23, 167.81, 170.39. ESI-MS
[M + H]+: 380.
3.2.1 | Cytotoxicity test
Metabolic activity of viable cells were measured by MTT
assay based on the reduction of 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium salt to formazan product,
which can be quantified spectrophotometrically to deter-
mine percent of viable cells. The cytotoxicity of com-
pounds 5a and 5l were screened according to the MTT
assay. The MTT assay was performed as previously
described.[21,22] NIH3T3 cell line was used in the MTT
assay.
3.1.16 | 2-((5-(butylamino)-
1,3,4-thiadiazol-2-yl)thio)-N-
(6-chlorobenzothiazol-2-yl)acetamide (5k)
3.2.2 | Molecular docking studies
1
Yield: 79%, M.P. =239.0 to 240.9ꢀC, H-NMR (DMSO-d6,
ppm, 300 MHz,): 0.87 (t, 3H, J = 7.3 Hz CH3), 1.31 (q,
2H, J = 7.8 Hz, CH2 ), 1.46 to 1.53 (m, 2H, CH2 ),
3.20 (q, 2H, J = 6.9 Hz, CH2 ), 4.03 (s, 2H, CH2 ),
7.33 (dd, 1H, J1 = 2.2 Hz, J2 = 8.6 Hz, H-benzothiazole),
7.57 (d, 1H, J = 8.6 Hz, H-benzothiazole), 7.82 (t, 1H,
J = 5.3 Hz, NH), 7.93 (d, 1H, J = 2.1 Hz, H-ben-
zothiazole). 13C-NMR (DMSO-d6, ppm, 75 MHz):
δ = 14.09, 19.99, 30.99, 39.38, 44.64, 121.18, 121.27,
125.95, 126.51, 134.35, 148.89, 149.59, 157.75, 163.44,
170.02. ESI-MS [M + H]+: 414.
A structure based in silico procedure was applied to dis-
cover the binding modes of compound 5l to hMAO-A
enzyme active site. The crystal structures of hMAO-A
(PDB ID: 2Z5X),[18] which was crystallized with harmine,
was retrieved from the Protein Data Bank server (www.
pdb.org).
The structures of ligands were built using the
[23]
Schrödinger Maestro
interface and then were submit-
ted to the Protein Preparation Wizard protocol of the
Schrödinger Suite 2016 Update 2.[24] The ligands were
[25]
prepared by the LigPrep 3.8
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 7.1.[26] The grid box with dimensions of 20 Å x 20 Å
x 20 Å was centered in the vicinity of the flavin (FAD)
N5 atom on the catalytic site of the protein to cover all
binding sites and neighboring residues.[27–29] Flexible
docking runs were performed with single precision dock-
ing mode (SP).
3.1.17 | 2-((5-(butylamino)-
1,3,4-thiadiazol-2-yl)thio)-N-
(6-nitrobenzothiazol-2-yl)acetamide (5l)
Yield: 82%, M.P. = 239.1 to 241.5ꢀC, 1H-NMR (DMSO-d6,
ppm, 300 MHz,): 0.86 (t, 3H, J = 7.3 Hz CH3), 1.30 (q,
2H, J = 7.6 Hz, CH2 ), 1.44 to 1.52 (m, 2H, CH2 ),
3.20 (q, 2H, J = 6.8 Hz, CH2 ), 4.17 (s, 2H, CH2 ),
7.84 to 7.92 (m, 3H, H-benzothiazole, NH), 8.26 (dd,
1H, J1 = 2.3 Hz, J2 = 8.9 Hz, H-benzothiazole), 9.01 (d,
1H, J = 2.3 Hz, H-benzothiazole), 13.12 (s, 1H, NH).
13C-NMR (DMSO-d6, ppm, 75 MHz,): δ = 14.07, 19.98,
ACKNOWLEDGMENTS
This study was carried out with the support of Anadolu
University BAP Project No: 1805S191.

ACAR ÇEVIK ET AL.
9
[15] B. K. Cavusoglu, B. N. Saglik, D. Osmaniye, S. Levent, U. Acar
Cevik, A. B. Karaduman, et al., Molecules 2017, 23(1), 60.
[16] S. Vilar, G. Ferino, E. Quezada, L. Santana, C. Friedman, Curr.
Top Med Chem 2012, 12(20), 2258.
[17] N. O. Can, D. Osmaniye, S. Levent, B. N. Saglik, B. Korkut,
O. Atli, et al., Eur J Med Chem 2018, 144, 68.
[18] S. Y. Son, J. Ma, Y. Kondou, M. Yoshimura, E. Yamashita,
T. Tsukihara, Proc Natl Acad Sci U. S. A. 2008, 105(15), 5739.
[19] O. D. Can, D. Osmaniye, U. Demir Ozkay, B. N. Saglik,
S. Levent, S. Ilgin, et al., Eur J Med Chem 2017, 131, 92.
[20] B. N. Saglik, B. Kaya Cavusoglu, D. Osmaniye, S. Levent,
U. Acar Cevik, S. Ilgin, et al., Bioorg Chem 2019, 85, 97.
[21] U. Demir Ozkay, O. D. Can, B. N. Saglik, U. Acar Cevik, S. Levent,
Y. Ozkay, et al., Bioorg Med Chem Lett 2016, 26(22), 5387.
[22] B. N. Saglik, S. Ilgin, Y. Ozkay, Eur J Med Chem 2016, 124,
1026.
CONFLICT OF INTEREST
Authors have no conflict of interest regarding this study.
ORCID
Ulviye Acar Çevik https://orcid.org/0000-0003-1879-
1034
Derya Osmaniye https://orcid.org/0000-0002-0499-
436X
ꢀ
Betül K. Çavus¸oglu https://orcid.org/0000-0002-1713-
9485
Yusuf Özkay https://orcid.org/0000-0001-8815-153X
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SUPPORTING INFORMATION
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How to cite this article: Acar Çevik U,
ꢀ
Osmaniye D, Saglik BN, et al. Synthesis of new
benzothiazole derivatives bearing thiadiazole as
monoamine oxidase inhibitors. J Heterocyclic
Chem. 2020;1–9. https://doi.org/10.1002/jhet.3942