Novel substituted benzothiazole and Imidazo[2,1-b][1,3,4]Thiadiazole derivatives: Synthesis, characterization, molecular docking study, and investigation of their in vitro antileishmanial and antibacterial activities

Article abstract of DOI:10.1016/j.molstruc.2019.05.104

In this study, we synthesized new imidazo[2,1-b][1,3,4]thiadiazole derivatives containing benzothiazole group. To this end, we firstly obtained the benzo[d]thiazol-2-ylthio/oxy acetonitrile compounds (3a,b), the starting materials, in high yields (82% and 87%, respectively). Then, we synthesized the 2-amino-1, 3,4-thiadiazole derivatives (4a,b) from the reaction of these nitrile derivatives (3a,b) with thiosemicarbazide in trifluoroacetic acid (TFA) (in yields of 83% and 84%). Finally, we synthesized the imidazo[2,1-b][1,3,4]thiadiazole derivatives (5-24) containing benzothiazole group, which are the target compounds, from reactions of 2-amino-1,3,4-thiadiazole derivatives (4a,b) with phenacyl bromide derivatives (in yields of 53%–73%). All of the compounds synthesized were characterized with ¹H NMR, ¹³C NMR, FT-IR, elemental analysis, and mass spectroscopy. Antileishmanial and antibacterial activity tests were applied to the compounds synthesized in the study. It was observed that compound 8 had the highest antileishmanial activity (MIC = 10 000 μg/mL). Also, compounds 7 and 17 were found to be effective at the highest concentration studied (MIC = 20 000 μg/mL). In terms of antibacterial activity, compounds 4b and 7 were found to be the most effective compounds against Escherichia coli (MIC = 625 μg/mL). Theoretical calculations were performed to support the experimental results. To this end, we performed Molecular Docking studies to determine whether or not the compounds (4a, 4b, 7 and 13) optimized with Gaussian09 using the DFT/B3LYP/6-31G(d,p) theory, which is a quantum chemical calculation, could be an inhibitor agent for the 2eg7 Escherichia coli protein structure. Also, we investigated the relationship between the calculated HOMO values of these four ligands and docking studies.

Full text of DOI:10.1016/j.molstruc.2019.05.104

Accepted Manuscript
Novel substituted benzothiazole and Imidazo[2,1-b][1,3,4]Thiadiazole derivatives:
Synthesis, characterization, molecular docking study, and investigation of their in vitro
antileishmanial and antibacterial activities
Mustafa Er, Arif Özer, Şahin Direkel, Tuncay Karakurt, Hakan Tahtaci
PII:
S0022-2860(19)30676-3
DOI:
https://doi.org/10.1016/j.molstruc.2019.05.104
MOLSTR 26611
Reference:
To appear in: Journal of Molecular Structure
Received Date: 2 April 2019
Revised Date: 23 May 2019
Accepted Date: 25 May 2019
Please cite this article as: M. Er, A. Özer, Ş. Direkel, T. Karakurt, H. Tahtaci, Novel substituted
benzothiazole and Imidazo[2,1-b][1,3,4]Thiadiazole derivatives: Synthesis, characterization, molecular
docking study, and investigation of their in vitro antileishmanial and antibacterial activities, Journal of
Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.05.104.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel Substituted Benzothiazole and Imidazo[2,1-b][1,3,4]Thiadiazole Derivatives:
Synthesis, Characterization, Molecular Docking Study, and Investigation of
Their In Vitro Antileishmanial and Antibacterial Activities
a*
a
b
c
a
Mustafa Er , Arif Özer , Şahin Direkel , Tuncay Karakurt , Hakan Tahtaci
a
Department of Chemistry, Faculty of Science, Karabuk University, 78050, Karabuk, Turkey.
b
Department of Medical Microbiology, Faculty of Medicine, Giresun University, Giresun,
2
8100, Turkey
c
Department of Chemical Engineering, Faculty of Engineering and Architecture, Ahi Evran
University, 40100, Kırşehir, Turkey.
*
Corresponding author
a*
Department of Chemistry, Faculty of Science, Karabuk University, 78050, Karabuk, Turkey.
Tel: +90 370 418 72 60, Fax: +90 370 418 72 62
E-mail address: mustafaer@karabuk.edu.tr

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Abstract
In this study, we synthesized new imidazo[2,1-b][1,3,4]thiadiazole derivatives containing
benzothiazole group. To this end, we firstly obtained the benzo[d]thiazol-2-ylthio/oxy
acetonitrile compounds (3a,b), the starting materials, in high yields (82% and 87%,
respectively). Then, we synthesized the 2-amino-1, 3,4-thiadiazole derivatives (4a,b) from the
reaction of these nitrile derivatives (3a,b) with thiosemicarbazide in trifluoroacetic acid
(TFA) (in yields of 83% and 84%). Finally, we synthesized the imidazo[2,1-
b][1,3,4]thiadiazole derivatives (5-24) containing benzothiazole group, which are the target
compounds, from reactions of 2-amino-1,3,4-thiadiazole derivatives (4a,b) with phenacyl
bromide derivatives (in yields of 53% to 73%).
1
13
All of the compounds synthesized were characterized with H NMR, C NMR, FT-IR,
elemental analysis, and mass spectroscopy.
Antileishmanial and antibacterial activity tests were applied to the compounds synthesized in
the study. It was observed that compound 8 had the highest antileishmanial activity (MIC=10
0
00 ꢀg/mL). Also, compounds 7 and 17 were found to be effective at the highest
concentration studied (MIC=20 000 ꢀg/mL). In terms of antibacterial activity, compounds 4b
and 7 were found to be the most effective compounds against Escherichia coli (MIC = 625
ꢀ
g/mL).
Theoretical calculations were performed to support the experimental results. To this end, we
performed Molecular Docking studies to determine whether or not the compounds (4a, 4b, 7
and 13) optimized with Gaussian09 using the DFT/B3LYP/6-31G(d,p) theory, which is a
quantum chemical calculation, could be an inhibitor agent for the 2eg7 Escherichia coli
protein structure. Also, we investigated the relationship between the calculated HOMO values
of these four ligands and docking studies.

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Keywords: Benzothiazole; Imidazo[2,1-b][1,3,4]thiadiazole; Biological activity; Molecular
docking; HOMO.
1
. Introduction
Antibacterial drugs (antibiotics) kill bacteria and prevent them from reproducing. However,
the inappropriate and excessive use of antibiotics causes these microorganisms to develop
resistance against these substances. Also, the constant use of antibiotics transforms health
bacteria into harmful microorganisms over time. Various active ingredients that exhibit
antibacterial activity is being developed as technology advances, leading to the availability of
more drugs that are able to treat various bacterial infections [1].
Leishmania, or more commonly known as oriental sores, is a serious health problem.
Antileishmanial drugs kill Leishmania protozoa and prevent their fatal disease.
Benzothiazoles, thiadiazoles, and imidazo[2,1-b][1,3,4]thiadiazoles constitute an important
class in heterocyclic chemistry due to their wide spectrum of biological activities. Various
active ingredients containing these compounds were synthesized in a laboratory environment
and were eventually introduced to be used to treat various diseases [1,2].
A number of studies have shown that imidazo[2,1-b][1,3,4]thiadiazole derivatives have
various antimicrobial, antifungal, anticancer, antituberculosis, anti-inflammatory,
anticonvulsant, analgesic, anesthetic, antihyperlipidemic, and diuretic activities [3-10].
Numerous chemical and medical studies were conducted on imidazo[2,1-b][1,3,4]thiadiazole
derivatives. These compounds have become a matter of interest in pharmaceutical industry
[
11-17].
Levamisole is used to regulate and strengthen the immune system. Levamisole and
Dexamisole are both enantiomers of Levamisole [18,19], imidazo[2,1-b][1,3,4]thiadiazole
derivatives, and have attracted the attention of researchers studying anticancer activities [20-

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2
6]. Moreover, imidazo[2,1-b][1,3,4]thiadiazoles and their heterocyclic derivatives were
reported to be used in a variety of industrial purposes such as paint production [27].
In light of the aforementioned data, this study aims to synthesize compounds containing
benzothiazole and imidazo[2,1-b][1,3,4]thiadiazole derivatives together (5-24), characterize
them, and investigate their antileishmanial and antibacterial activities.
This study also aims to find the optimal structures of the selected compounds (4a, 4b, 7, and
1
3) using the DFT (B3LYP) theory, with the 6-31G(d,p) basis set [28,29] on Gaussian09 [30]
in order to determine whether or not these compounds might be possible inhibitor agents for
the 2eg7 Escherichia coli protein structure using molecular docking simulation.
The synthetic route used to synthesize compounds containing benzothiazole and imidazo[2,1-
b][1,3,4]thiadiazole derivatives together (5-24) is shown in Scheme 1.

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Scheme 1. Synthetic route for the synthesis of target compounds (5-24).
2
2
. Experimental Section
.1. General Methods
1
13
The H NMR and C NMR spectra of the compounds were assessed using an Agilent Annual
Refill (400 MHz) device. The ESI (+) method and a Thermo TSQ Quantum Access device
were used to determine mass spectrum. The IR spectra of the original compounds were
calculated using ATR and a PerkinElmer Infrared Spectroscopy (FT-IR) device. Elemental
analyses were performed using a LECO 932 CHNS (Leco-932, St. Joseph, MI, USA)

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instrument, and the results were within ± 0.4% of the theoretical values. The melting points of
the compounds were determined using a Thermo Scientific IA9000 device.
2
.2. Synthesis
2
.2.1. General procedure for the synthesis of 2-(benzo[d]thiazol-2-ylthio or -2yl-
oxy)acetonitrile (3a,b)
Benzo[d]thiazol-2-thiol/ol (1) (16.72 g/15.12 g; 0.10 mol) and potassium hydroxide (5.61 g,
0
.10 mol) were dissolved within ethyl alcohol in a two-necked flask. 2-chloroacetonitrile
7.55 g; 0.10 mol) was added into the reaction mixture. The reaction mixture was refluxed for
hours. The resulting product was filtered through a sintered funnel. The filtrate was
crystallized using an appropriate solvent and the pure substance was dried with P O in A
(
5
2
5
vacuum oven. Finally, structure of the synthesized compound was characterized with FT-IR,
1
13
H NMR, C NMR, elemental analysis, and mass spectroscopy.
2
.2.1.1. 2-(Benzo[d]thiazol-2-ylthio)acetonitrile (3a)
-
Light yellow solid, yield:16.97 g (82%), m.p. 74-75 ºC (from DMF-EtOH, 1:5). IR (ATR, cm
1
1
)
: 3055 (Ar-CH), 2970 (Aliph. CH), 2249 (CN), 1617 (C=N). H NMR (400 MHz, DMSO-
d ) δ (ppm): 4.54 (s, 2H, -CH ), Ar-H [8.06 (d, J=8.0 Hz, 1H), 7.93 (d, J=8.4 Hz, 1H), 7.50 (t,
6
2
1
3
J=6.8, 8.0 Hz, 1H), 7.41 (t, J=7.2, 7.6 Hz, 1H)]. C NMR (100 MHz, DMSO-d , δ ppm):
6
1
1
8.55 (-CH ), Ar-C [122.00 (CH), 122.61 (CH), 125.49 (CH), 127.08 (CH), 135.62 (C),
2
52.67 (C), 163.74 (C)], 117.83 (CN); MS: m/z 209.90 (M+3, 39). Anal. Calcd. for
(
C H N S ): C, 52.40; H, 2.93; N, 13.58. Found: C, 52.43; H, 2.89; N, 13.63.
9 6 2 2

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2
.2.1.2. 2-(Benzo[d]thiazol-2yl-oxy)acetonitrile (3b)
-1
Yellowish solid, yield:16.59 g (87%), m.p. 69-70 ºC (DMF-EtOH, 1:6). IR (ATR, cm ): 3034
1
(
Ar-CH), 2981 (Aliph. CH), 2263 (CN), 1665 (C=N). H NMR (400 MHz, DMSO-d ) δ
6
1
3
(
ppm): 4.83 (s, 2H, -CH ), Ar-H [7.66 (d, J=8.0 Hz, 1H), 7.31 (m, 2H), 7.20 (m, 1H)]. C
2
NMR (100 MHz, DMSO-d , δ ppm): 61.84 (-CH ), Ar-C [121.55 (CH), 122.61 (CH), 125.56
6
2
(CH), 127.13 (CH), 135.62 (C), 157.82 (C), 170.43 (C)], 115.69 (CN); MS: m/z 192.12 (M+2,
5
1
). Anal. Calcd. for (C H N OS): C, 56.83; H, 3.18; N, 14.73. Found: C, 56.86; H, 3.20; N,
9 6 2
4.68.
2
.2.2. General procedure for the synthesis of 5-((benzo[d]thiazol-2-ylthio or -2yl-oxy)methyl)-
1
,3,4-thiadiazol-2-amine (4a,b)
The 3a or 3b compound (0.075 mol) was dissolved within 40 ml trifluoroacetic acid in a
round-bottom flask and then thiosemicarbazide (0.100 mol) was added. The mixture was
refluxed for 4 hours at 60 °C. The reaction mixture was then poured on 200 ml ice-water
mixture and neutralized with diluted ammonia. The resulting substance was filtered through a
funnel. The solid substance obtained was washed with water, ethyl alcohol, and diethyl ether,
respectively. Afterwards, the substance was purified by crystallization with an appropriate
solvent or solvent pair. The pure substance was dried in a P O vacuum oven. Finally,
2
5
1
13
structures of the synthesized compounds were characterized with FT-IR, H NMR, C NMR,
elemental analysis, and mass spectroscopy.
2
.2.2.1. 5-((Benzo[d]tiyazol-2-iltiyo)metil)-1,3,4-tiyadiazol-2-amin (4a)
Light brown solid, yield: 17.39 g (83%), m.p. 199-201 ºC (from DMF-EtOH, 1:3). IR (ATR,
-1
1
cm ): 3254-3089 (-NH ), 3067 (Ar-CH), 2958 (Aliph. CH), 1626 (C=N). H NMR (400 MHz,
2
DMSO-d ) δ (ppm): 4.81 (s, 2H, -CH ), Ar-H [8.03 (d, J=8.0 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H),
6
2

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13
7
.48 (t, J=7.6, 7.6 Hz, 1H), 7.38 (t, J=7.2, 7.6 Hz, 1H)], 7.14 (s, 2H, NH ). C NMR (100
2
MHz, DMSO-d , δ ppm): 31.75 (-CH ), Ar-C [121.74 (CH), 122.46 (CH), 125.22 (CH),
6
2
1
26.96 (CH), 135.48 (C), 152.76 (C), 170.08 (C)], Thiadiazole-C [154.48 (C), 165.49 (C)];
MS: m/z 280.79 (M+2, 34). Anal. Calcd. for (C H N S ): C, 42.84; H, 2.88; N, 19.98.
10
8
4 3
Found: C, 42.81; H, 2.86; N, 19.96.
2
.2.2.2. 5-((Benzo[d]tiyazol-2-iloksi)metil)-1,3,4-tiyadiazol-2-amin (4b)
Light brown crystal, yield: 16.55 g (84%), m.p. 238-240 ºC (from DMF-EtOH, 1:2). IR
-1
1
(
ATR, cm ): 3271-3158 (-NH ), 3035 (Ar-CH), 2979 (Aliph. CH), 1667 (C=N). H NMR
2
(
400 MHz, DMSO-d ) δ (ppm): 5.32 (s, 2H, -CH ), Ar-H [7.66 (d, J=8.4 Hz, 1H), 7.37 (m,
6
2
1
3
2
H), 7.20 (m, 2H)], 7.24 (s, 2H, NH ). C NMR (100 MHz, DMSO-d , δ ppm): 41.13 (-CH ),
2 6 2
Ar-C [112.13 (CH), 121.77 (CH), 123.51 (CH), 124.04 (CH), 127.15 (C), 136.60 (C), 170.15
(C)], Thiadiazole-C [152.14 (C), 169.24 (C)]; MS: m/z 261.04 (M-3, 224). Anal. Calcd. for
(
C H N OS ): C, 45.44; H, 3.05; N, 21.20. Found: C, 45.49; H, 3.02; N, 21.26.
1
0
8
4
2
2
.2.3. General procedure for the synthesis of imidazo[2,1-b][1,3,4]thiadiazole derivatives (5-
2
4)
Substituted 2-amino-1,3,4-thiadiazole derivatives (4a,b) (5 mmol) and 2-bromacetophenone
derivatives (5 mmol) were dissolved within absolute ethyl alcohol in a two-necked flask. The
mixture was refluxed for 12 hours, and the progress of the reaction was checked using thin
layer chromatography (TLC). Once the reaction was completed, the mixture was evaporated
until almost dry using a rotary evaporator. Then, the reaction mixture was alkalized with
diluted Na CO solution. The mixture was filtered and washed with water. The substance was
2
3
purified by crystallized with an appropriate solvent or solvent pair. The pure substance was
dried in a P O vacuum oven. Lastly, the structures of the synthesized compounds were
2
5

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1
13
characterized with FT-IR, H NMR, C NMR, mass spectroscopy, and elemental analysis.
The physical properties and the spectral data of the products are listed below.
2
.2.3.1. 2-((6-Phenylimidazo[2,1-b][1,3,4]thiadiazol-2-yl)methylthio)benzo[d]thiazole (5)
Light brown solid, yield: 1.29 g (68%), m.p.169-170 ºC (from DMF-EtOH, 1:2); IR (ATR,
-1
1
cm ): 3085 (Ar-CH), 2968 (Aliph. CH), 1601 (C=N); H NMR (400 MHz, DMSO-d , δ
6
ppm): 5.08 (s, 2H, -CH ), Ar-H [8.02 (d, J=8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.82 (d,
2
J=6.8 Hz, 2H), 7.48 (t, J=8.4 Hz, 1H), 7.37 (m, 3H), 7.24 (t, J=7.6, 7.2 Hz, 1H)], Imidazole-H
13
[
8.64 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 32.94 (-CH ), Ar-C [121.84 (CH),
6 2
1
22.55 (CH), 125.13 (CH), 125.38 (CH), 127.05 (CH), 127.90 (CH), 129.09 (CH), 134.20
(C), 135.59 (C), 152.61 (C), 164.91 (C)], Imidazole-C [110.72 (CH), 145.73 (C)],
Thiadiazole-C [145.60 (C), 162.40 (C)]; MS: m/z 381.07 (M+1, 100). Anal. Calcd. for
(
C H N S ): C, 56.82; H, 3.18; N, 14.72. Found: C, 56.78; H, 3.21; N, 14.74.
18 12 4 3
2
.2.3.2. 2-((6-(4-Fluorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methylthio)
benzo[d]thiazole (6)
-1
Light brown solid, yield: 1.18 g (62%), m.p. 184-185 ºC (from Acetone); IR (ATR, cm ):
1
3
058 (Ar-CH), 2972 (Aliph. CH), 1599 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 5.08
6
(
s, 2H, -CH ), Ar-H [8.03 (d, J=8.0 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.85 (t, J=5.6, 5.6 Hz,
2
2
H), 7.48 (t, J=6.4, 7.6 Hz, 1H), 7.38 (t, J=7.6, 7.2 Hz, 1H), 7.21 (t, J=8.8, 8.0 Hz, 2H)],
13
Imidazole-H [8.63 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 32.91 (-CH ), Ar-C
6
2
[
(
115.88 (CH), 121.84 (CH), 122.56 (CH), 125.38 (CH), 127.05 (CH), 130.73 (CH), 135.58
C), 152.60 (C), 160.77 (C), 162.48 (C), 164.92 (C)], Imidazole-C [110.58 (CH), 145.60 (C)],
+
Thiadiazole-C [144.79(C), 163.20 (C)]; MS: m/z 398.72 (M , 15). Anal. Calcd. for
(
C H FN S ): C, 54.25; H, 2.78; N, 14.06. Found: C, 54.31; H, 2.81; N, 14.09.
18 11 4 3

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2
.2.3.3. 2-((6-(4-Chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methylthio)
benzo[d]thiazole (7)
-1
White solid, yield: 1,22 g (59%), m.p. 225-226 ºC (from DMF-EtOH, 1:2); IR (ATR, cm ):
1
3
104 (Ar-CH), 2969 (Aliph. CH), 1623 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 4.94
6
(
s, 2H, -CH ), Ar-H [8.07 (d, J=8.0 Hz, 1H), 8.01 (d, J=8.0 Hz, 2H), 7.93 (d, J=8.0 Hz, 1H),
2
7
.69 (d, J=8.0 Hz, 2H), 7.52 (t, J=7.6, 7.2 Hz, 1H), 7.42 (t, J=7.2, 7.6 Hz, 1H)], Imidazole-H
13
[
8.81 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 31.43 (-CH ), Ar-C [113.83 (CH),
6 2
1
1
21.75 (CH), 122.72 (CH), 125.53 (CH), 127.12 (CH), 129.57 (CH), 130.79 (C), 132.76 (C),
35.88 (C), 155.47 (C), 169.17 (C)], Imidazole-C [111.93 (CH), 152.80 (C)], Thiadiazole-C
+
[
139.88 (C), 165.17 (C)]; MS: m/z 432.81 (M + H O, 100). Anal. Calcd. for (C H ClN S ):
2
18 11
4 3
C, 52.10; H, 2.67; N, 13.50. Found: C, 52.16; H, 2.71; N, 13.54.
2
.2.3.4. 2-((6-(4-Bromophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methylthio)
benzo[d]thiazole (8)
-1
White solid, yield: 1.53 g (67%), m.p. 234-235 ºC (from DMF-EtOH, 1:3); IR (ATR, cm ):
1
3
089 (Ar-CH), 2873 (Aliph. CH), 1626 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 4.93
6
(
s, 2H, -CH ), Ar-H [8.07 (d, J=7.6 Hz, 1H), 7.93 (d, J=7.6 Hz, 3H), 7.84 (d, J=8.0 Hz, 2H),
2
13
7
.53 (t, J=7.6, 7.2 Hz, 1H), 7.42 (t, J=7.6, 7.2 Hz, 1H)], Imidazole-H [8.91 (s, 1H)]; C NMR
(
100 MHz, DMSO-d , δ ppm): 31.40 (-CH ), Ar-C [112.45 (CH), 121.73 (CH), 122.74 (C),
6 2
1
1
25.54 (CH), 127.12 (CH), 129.17 (CH), 130.82 (C), 132.53 (CH), 133.06 (C), 152.20 (C),
69.20 (C)], Imidazole-C [111.33 (CH), 148.26 (C)], Thiadiazole-C [135.33 (C), 160.19 (C)];
MS: m/z 478.72 (M+1+H O, 100). Anal. Calcd. for (C H BrN S ): C, 47.06; H, 2.41; N,
2
18 11
4 3
1
2.20. Found: C, 47.11; H, 2.39; N, 12.22.

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2
.2.3.5. 2-((6-(4-Methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methylthio)
benzo[d]thiazole (9)
-1
Light yellow solid, yield:1.11 g (54%), m.p. 176-177 ºC (from Acetone); IR (ATR, cm ):
1
3
136 (Ar-CH), 2976 (Aliph. CH), 1599 (C=N), 1241-1172 (-OCH ); H NMR (400 MHz,
3
DMSO-d , δ ppm): 5.08 (s, 2H, -CH ), 3.75 (s, 3H, -OCH ), Ar-H [8.06 (d, J=7.6 Hz, 1H),
6
2
3
7
.90 (d, J=8.0 Hz, 1H), 7.74 (d, J=8.0 Hz, 2H), 7.50 (m, 1H), 7.40 (m, 1H)], Imidazole-H
1
3
[
[
(
(
8.51 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 32.91 (-CH ), 55.58 (-OCH ), Ar-C
6 2 3
115.83 (CH), 121.83 (CH), 122.56 (CH), 125.78 (CH), 127.00 (C), 130.73 (CH), 135.58
CH), 152.40 (C), 160.89 (C), 162.48 (C) 164.92 (C)], Imidazole-C [110.58 (CH), 145.64
C)], Thiadiazole-C [144.76 (C), 163.32 (C)]; MS: m/z 411.04 (M+1, 55), 429.10 (M+1+H O,
2
1
00). Anal. Calcd. for (C H N OS ): C, 55.59; H, 3.44; N, 13.65. Found: C, 55.64; H, 3.47;
19 14 4 3
N, 13.61.
2
.2.3.6. 2-((6-(4-Nitrophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methylthio)
benzo[d]thiazole (10)
-1
Yellow solid, yield:1.47 g (69%), m.p. 209-211 ºC (from DMF-EtOH, 1: 1); IR (ATR, cm ):
1
3
064 (Ar-CH), 2975 (Aliph. CH), 1599 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 5.10
6
(
s, 2H, -CH ), Ar-H [8.24 (d, J=8.4 Hz, 2H), 8.07 (d, J=8.8 Hz, 2H), 8.04 (d, J=8.0 Hz, 1H),
2
7
.90 (d, J=8.0 Hz, 1H), 7.48 (t, J=7.6, 7.2 Hz, 1H), 7.38 (t, J=8.0, 7.2 Hz, 1H)], Imidazole-H
13
[
8.94 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 32.94 (-CH ), Ar-C [121.85 (CH),
6 2
1
1
22.59 (CH), 124.67 (CH), 125.41 (CH), 125.75 (CH), 127.07 (CH), 135.60 (C), 140.74 (C),
46.71 (C), 152.58 (C), 164.90 (C)], Imidazole-C [113.47 (CH), 146.59 (C)], Thiadiazole-C
[
143.46 (C), 163.81 (C)]; MS: m/z 425.79 (M+1, 67). Anal. Calcd. for (C H N O S ): C,
18 11 5 2 3
5
0.81; H, 2.61; N, 16.46. Found: C, 50.77; H, 2.59; N, 16.50.

ACCEPTED MANUSCRIPT
2
.2.3.7. 4-(2-((Benzo[d]thiazol-2-ylthio)methyl)imidazo[2,1-b][1,3,4]thiadiazol-6-yl)
benzonitrile (11)
-1
White solid, yield:1.34 g (66%), m.p. 207-208 ºC (from Acetone); IR (ATR, cm ): 3068 (Ar-
1
CH), 2998 (Aliph. CH), 2219 (CN), 1608 (C=N); H NMR (400 MHz, DMSO-d , δ ppm):
6
5
1
.10 (s, 2H, -CH ), Ar-H [8.03 (d, J=8.4 Hz, 1H), 8.00 (d, J=7.2 Hz, 2H), 7.90 (d, J=8.0 Hz,
2
H), 7.84 (d, J=8.0 Hz, 2H), 7.49 (t, J=7.2, 7.6 Hz, 1H), 7.39 (t, J=7.2, 7.6 Hz, 1H)],
13
Imidazole-H [8.88 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 32.93 (-CH ), Ar-C
6
2
[109.83 (C), 121.85 (CH), 122.59 (CH), 125.40 (CH), 125.58 (CH), 127.07 (CH), 133.21
(CH), 135.59 (C), 138.70 (C), 152.58 (C), 164.90 (C)], Imidazole-C [112.91 (CH), 146.18
(C)], Thiadiazole-C [143.84 (C), 163.56 (C)], 119.63 (CN); MS: m/z 405.70 (M+1, 100).
Anal. Calcd. for (C H N S ): C, 56.27; H, 2.73; N, 17.27. Found: C, 56.32; H, 2.75; N,
1
9
11
5 3
1
7.31.
2
.2.3.8. 2-((6-(3,4-Dichlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methylthio)
benzo[d]thiazole (12)
-1
Whitish solid, yield:1.59 g (71%), m.p. 197-199 ºC (from DMF-EtOH, 1:2); IR (ATR, cm ):
1
3
125 (Ar-CH), 2968 (Aliph. CH), 1599 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 5.09
6
(
s, 2H, -CH ), Ar-H [8.03 (d, J=7.6 Hz, 2H), 7.90 (d, J=8.0 Hz, 1H), 7.80 (d, J=8.4 Hz, 1H),
2
7
.63 (d, J=8.8 Hz, 1H), 7.48 (t, J=7.6, 8.0 Hz, 1H), 7.38 (t, J=8.4, 8.0 Hz, 1H)], Imidazole-H
13
[
8.79 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 32.92 (-CH ), Ar-C [121.84 (CH),
6 2
1
22.58 (CH), 125.11 (CH), 125.40 (CH), 126.60 (CH), 127.07 (CH), 129.88 (CH), 131.40
(
C), 131.99 (C), 134.94 (C), 135.60 (C), 152.59 (C), 164.90 (C)], Imidazole-C [112.00 (CH),
45.73 (C)], Thiadiazole-C [143.18 (C), 163.23 (C)]; MS: m/z 448.94 (M+1, 87), 451.00
M+3, 100). Anal. Calcd. for (C H Cl N S ): C, 48.11; H, 2.24; N, 12.48. Found: C, 48.14;
1
(
1
8
10
2
4 3
H, 2.21; N, 12.53.

ACCEPTED MANUSCRIPT
2
.2.3.9. 2-((6-(4-Phenylphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methylthio)
benzo[d]thiazole (13)
-1
Whitish solid, yield: 1.32 g (58%), m.p. 216-219 ºC (from DMF-EtOH, 1:2); IR (ATR, cm ):
1
3
102 (Ar-CH), 2947 (Aliph. CH), 1610 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 5.10
6
(
s, 2H, -CH ), Ar-H [8.04 (d, J=7.2 Hz, 1H), 7.91 (d, J=7.2 Hz, 3H), 7.69 (d, J=6.0 Hz, 4H),
2
13
7
.41 (m, 5H)], Imidazole-H [8.71 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 32.87 (-
6
CH ), Ar-C [121.85 (CH), 122.59 (CH), 125.39 (CH), 125.66 (CH), 126.90 (CH), 127.07
2
(CH), 127.36 (CH), 127.87 (CH), 129.40 (CH), 133.47 (C), 134.21 (C), 134.79 (C), 135.58
(C), 152.61 (C), 164.92 (C)], Imidazole-C [110.93 (CH), 146.13 (C)], Thiadiazole-C [140.75
+
(
C), 163.10 (C)]; MS: m/z 456.80 (M , 84). Anal. Calcd. for (C H N S ): C, 63.13; H, 3.53;
2
4
16
4 3
N, 12.27. Found: C, 63.16; H, 3.49; N, 12.32.
2
.2.3.10. 2-((6-(Naphthalen-2-yl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methylthio)
benzo[d]thiazole (14)
-1
Whitish solid, yield: 1.31 g (61%), m.p. 203-205 ºC (from DMF-EtOH, 1:2); IR (ATR, cm ):
1
3
050 (Ar-CH), 2974 (Aliph. CH), 1598 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 5.11
6
(
s, 2H, -CH ), Ar-H [8.36 (s, 1H), 8.04 (d, J=7.6 Hz, 1H), 7.97 (d, J=7.6 Hz, 1H), 7.91 (m,
2
13
4
H), 7.48 (m, 3H), 7.38 (t, J=8.4, 6.8 Hz, 1H)], Imidazole-H [8.78 (s, 1H)]; C NMR (100
MHz, DMSO-d , δ ppm): 32.96 (-CH ), Ar-C [121.86 (CH), 122.59 (CH), 123.24 (CH),
6
2
1
23.85 (CH), 125.40 (CH), 126.28 (CH), 127.08 (CH), 128.09 (CH), 128.38 (CH), 128.64
C), 131.68 (C), 132.84 (C), 135.69 (C), 152.61 (C), 164.95 (C)], Imidazole-C [111.30 (CH),
45.87 (C)], Thiadiazole-C [133.64 (C), 162.57 (C)]; MS: m/z 430.85 (M+1, 100). Anal.
Calcd. for (C H N S ): C, 61.37; H, 3.28; N, 13.01. Found: C, 61.39; H, 3.32; N, 12.98.
(
1
2
2
14
4 3

ACCEPTED MANUSCRIPT
2
.2.3.11. 2-((6-Phenylimidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy) benzo[d]thiazole (15)
-
Yellowish solid, yield: 1.31 g (72%), m.p. 201-203 ºC (from DMF-EtOH, 1:3); IR (ATR, cm
1
1
)
: 3044 (Ar-CH), 2983 (Aliph. CH), 1601 (C=N); H NMR (400 MHz, DMSO-d , δ ppm):
6
5
1
.63 (s, 2H, -CH ), Ar-H [7.81 (d, J=7.6 Hz, 2H), 7.69 (d, J=7.6 Hz, 1H), 7.46 (d, J=7.6 Hz,
2
13
H), 7.37 (m, 3H), 7.24 (m, 2H)], Imidazole-H [8.64 (s, 1H)]; C NMR (100 MHz, DMSO-
d , δ ppm): 42.24 (-CH ), Ar-C [111.99 (CH), 121.78 (CH), 123.69 (CH), 124.27 (CH),
6
2
1
25.12 (CH), 127.34 (CH), 127.84 (CH), 129.10 (C), 134.15 (C), 136.48 (C), 169.62 (C)],
Imidazole-C [110.96 (CH), 145.88 (C)], Thiadiazole-C [145.19 (C), 159.51 (C)]; MS: m/z
+
3
64.03 (M , 37). Anal. Calcd. for (C H N OS ): C, 59.32; H, 3.32; N, 15.37. Found: C,
1
8
12
4
2
5
9.28; H, 3.35; N, 15.41.
2
.2.3.12. 2-((6-(4-Fluorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)
benzo[d]thiazole (16)
-1
White solid, yield: 1.32 g (69%), m.p. 186-188 ºC (from DMF-EtOH, 1:3); IR (ATR, cm ):
1
3
105 (Ar-CH), 2973 (Aliph. CH), 1595 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 5.63
6
(
s, 2H, -CH ), Ar-H [7.83 (m, 2H), 7.70 (d, J=8.0 Hz, 1H), 7.45 (d, J=7.6 Hz, 1H), 7.38 (t,
2
13
J=7.6, 7.6 Hz, 1H), 7.23 (m, 3H)], Imidazole-H [8.64 (s, 1H)]; C NMR (100 MHz, DMSO-
d , δ ppm): 42.24 (-CH ), Ar-C [111.99 (CH), 116.11 (CH), 121.77 (CH), 123.71 (CH),
6
2
1
27.01 (CH), 127.09 (C), 127.35 (CH), 130.72 (C), 136.48 (C), 160.80 (C), 169.62 (C)],
Imidazole-C [110.83 (CH), 145.25 (C)], Thiadiazole-C [144.96(C), 163.23 (C)]; MS: m/z
3
5
82.95 (M+1, 83). Anal. Calcd. for (C H FN OS ): C, 56.53; H, 2.90; N, 14.65. Found: C,
18 11 4 2
6.57; H, 2.88; N, 14.69.

ACCEPTED MANUSCRIPT
2
.2.3.13. 2-((6-(4-Chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)
benzo[d]thiazole (17)
-1
White solid, yield: 1.29 g (65%), m.p. 227-229 °C (from DMF-EtOH, 1:3); IR (ATR, cm ):
1
3
131 (Ar-CH), 2987 (Aliph. CH), 1588 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 5.63
6
(
s, 2H, -CH ), Ar-H [7.81 (d, J=8.4 Hz, 2H), 7.70 (d, J=8.0 Hz, 1H), 7.42 (m, 4H), 7.23 (t,
2
1
3
J=7.6, 7.6 Hz, 1H)], Imidazole-H [8.70 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm):
6
4
1
2.24 (-CH ), Ar-C [111.99 (CH), 121.77 (CH), 123.71 (CH), 124.28 (CH), 126.77 (CH),
2
27.35 (CH), 129.14 (CH),132.21 (C), 133.07 (C), 136.48 (C), 169.63 (C)], Imidazole-C
[
111.42 (CH), 145.44 (C)], Thiadiazole-C [144.69 (C), 159.83 (C)]; MS: m/z 399.05 (M+1,
1
2
00). Anal. Calcd. for (C H ClN OS ): C, 54.20; H, 2.78; N, 14.04. Found: C, 54.17; H,
18 11 4 2
.81; N, 14.10.
2
.2.3.14. 2-((6-(4-Bromophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)
benzo[d]thiazole (18)
Light yellow solid, yield: 1.42 g (64%), m.p. 241-243 ºC (from DMF-EtOH, 1:3); IR (ATR,
-1
1
cm ): 3128 (Ar-CH), 2975 (Aliph. CH), 1587 (C=N); H NMR (400 MHz, DMSO-d , δ
6
ppm): 5.63 (s, 2H, -CH ), Ar-H [7.75 (d, J=8.8 Hz, 2H), 7.71 (d, J=8.0 Hz, 1H), 7.57 (d,
2
J=8.8 Hz, 2H), 7.46 (d, J=7.6 Hz, 1H), 7.39 (t, J=7.6, 8.0 Hz, 1H), 7.25 (t, J=7.6, 7.6 Hz,
1
3
1
H)], Imidazole-H [8.71 (s, 1H)]; C NMR (400 MHz, DMSO-d , δ ppm): 42.25 (-CH ), Ar-
6 2
C [112.01 (CH), 120.74 (CH), 121.77 (C), 123.72 (CH), 124.29 (CH), 127.08 (CH), 127.37
(
(
(
C), 132.06 (CH), 133.43 (C), 136.48 (C), 169.63 (C)], Imidazole-C [111.43 (CH), 145.45
C)], Thiadiazole-C [144.69 (C), 159.90 (C)]; MS: m/z 444.91 (M+2, 100). Anal. Calcd. for
C H BrN OS ): C, 48.76; H, 2.50; N, 12.64. Found: C, 48.81; H, 2.47; N, 12.69.
1
8
11
4
2

ACCEPTED MANUSCRIPT
2
.2.3.15. 2-((6-(4-Methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)
benzo[d]thiazole (19)
-
Yellowish solid, yield: 1.05 g (53%), m.p. 238-241 ºC (from DMF-EtOH, 1:3); IR (ATR, cm
1
1
)
: 3097 (Ar-CH), 2978 (Aliph. CH), 1597 (C=N), 1169-1099 (-OCH ); H NMR (400 MHz,
3
DMSO-d , δ ppm): 3.74 (s, 3H, -OCH ), 5.64 (s, 2H, -CH ), Ar-H [8.05 (d, J=7.6 Hz, 1H),
6
3
2
7
6
4
1
.90 (d, J=8.0 Hz, 1H), 7.74 (d, J=8.4 Hz, 2H), 7.48 (t, J=7.6, 8.0 Hz, 1H), 7.38 (m, 1H),
13
.94 (d, J=8.8 Hz, 2H)], Imidazole-H [8.56 (s, 1H)]; C NMR (400 MHz, DMSO-d , δ ppm):
6
2.28 (-CH ), 55.58 (-OCH ), Ar-C [115.76 (CH), 121.64 (CH), 122.65 (CH), 125.43 (CH),
2
3
27.02 (C), 130.77 (CH), 135.58 (CH), 152.42 (C), 160.89 (C), 162.48 (C), 164.97 (C)],
Imidazole-C [109.89 (CH), 145.64 (C)], Thiadiazole-C [144.78 (C), 163.34 (C)]; MS: m/z
3
5
96.48 (M+2, 51). Anal. Calcd. for (C H N O S ): C, 57.85; H, 3.58; N, 14.20. Found: C,
19 14 4 2 2
7.89; H, 2.62; N, 14.23.
2
.2.3.16. 2-((6-(4-Nitrophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)
benzo[d]thiazole (20)
-
Brownish solid, yield: 1.37 g (67%), m.p. 246-248 ºC (from DMF-EtOH, 1:3); IR (ATR, cm
1
1
)
: 3152 (Ar-CH), 2928 (Aliph. CH), 1600 (C=N); H NMR (400 MHz, DMSO-d , δ ppm):
6
5
1
.64 (s, 2H, -CH ), Ar-H [8.25 (d, J=8.8 Hz, 2H), 8.07 (d, J=8.8 Hz, 2H), 8.04 (d, J=8.4 Hz,
2
H), 7.90 (d, J=8.0 Hz, 1H), 7.48 (t, J=7.2, 7.2 Hz, 1H), 7.39 (t, J=7.6, 7.2 Hz, 1H)],
13
Imidazole-H [8.94 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 42.38 (-CH ), Ar-C
6
2
[
121.87 (CH), 122.60 (CH), 124.68 (CH), 125.41 (CH), 125.75 (CH), 127.07 (CH), 135.59
C), 140.74 (C), 146.73 (C), 152.58 (C), 164.92 (C)], Imidazole-C [113.58 (CH), 146.59 (C)],
(
Thiadiazole-C [143.48 (C), 163.85 (C)]; MS: m/z 410.19 (M41, 62). Anal. Calcd. for
C H N O S ): C, 52.80; H, 2.71; N, 17.10. Found: C, 52.78; H, 2.74; N, 17.09.
(
1
8
11
5
3 2

ACCEPTED MANUSCRIPT
2
.2.3.17. 4-(2-((Benzo[d]thiazol-2-yloxy)methyl)imidazo[2,1-b][1,3,4]thiadiazol-6-
yl)benzonitrile (21)
-1
White solid, yield:1.42 g (73%), m.p. 221-222 ºC (from DMF-EtOH, 1:2); IR (ATR, cm ):
1
3
142 (Ar-CH), 29 89 (Aliph. CH), 2225 (CN), 1596 (C=N); H NMR (400 MHz, DMSO-d , δ
6
ppm): 5.65 (s, 2H, -CH ), Ar-H [7.98 (d, J=6.8 Hz, 2H), 7.84 (d, J=6.8 Hz, 2H), 7.71 (d,
2
J=8.0 Hz, 1H), 7.46 (d, J=8.0 Hz, 1H), 7.38 (t, J=7.6, 7.6 Hz, 1H), 7.24 (t, J=7.6, 7.6 Hz,
1
3
1
H)], Imidazole-H [8.88 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 42.28 (-CH ), Ar-
6 2
C [109.89 (C), 113.10 (CH), 121.76 (CH), 123.72 (CH), 124.30 (CH), 125.58 (CH), 127.37
(CH), 133.23 (CH), 136.48 (C), 138.65 (C), 169.64 (C)], Imidazole-C [112.02 (CH), 146.07
(C)], Thiadiazole-C [143.97 (C), 160.63 (C)], 119.43 (CN); MS: m/z 389.99 (M+1, 49). Anal.
Calcd. for (C H N OS ): C, 58.60; H, 2.85; N, 17.98. Found: C, 58.58; H, 2.88; N, 18.03.
1
9
11
5
2
2
.2.3.18. 2-((6-(3,4-Dichlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)
benzo[d]thiazole (22)
-1
Light red solid, yield:1.49 g (69%), m.p. 230-232 ºC (from DMF-EtOH, 1:2); IR (ATR, cm ):
1
3
123 (Ar-CH), 2977 (Aliph. CH), 1590 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 5.63
6
(
s, 2H, -CH ), Ar-H [8.00 (d, J=2.0 Hz, 2H), 7.76 (dd, J=2.0, 2.0 Hz, 1H), 7.69 (d, J=7.6 Hz,
2
1
H), 7.61 (d, J=8.4 Hz, 1H), 7.45 (d, J=8.4 Hz, 1H), 7.38 (t, J=7.2, 8.0 Hz, 1H), 7.22 (t,
1
3
J=8.0, 7.6 Hz, 1H)], Imidazole-H [8.77 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm):
6
2
1
1
2.25 (-CH ), Ar-C [112.17 (CH), 121.77 (CH), 123.72 (CH), 124.29 (CH), 125.10 (CH),
2
26.58 (CH), 127.35 (CH), 129.94 (C), 131.37 (C), 131.99 (C), 134.86 (C), 136.45 (C),
69.64 (C)], Imidazole-C [111.99 (CH), 145.69 (C)], Thiadiazole-C [143.31 (C), 160.28 (C)];
MS: m/z 432.02 (M+1, 34). Anal. Calcd. for (C H Cl N OS ): C, 49.89; H, 2.33; N, 12.93.
1
8
10
2
4
2
Found: C, 49.93; H, 2.34; N, 12.89.

ACCEPTED MANUSCRIPT
2
.2.3.19. 2-((6-(4-Phenylphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)
benzo[d]thiazole (23)
-1
Whitish solid, yield:1.26 g (57%), m.p. 258-261 ºC (from DMF-EtOH, 1:3); IR (ATR, cm ):
1
3
127 (Ar-CH), 2979 (Aliph. CH), 1589 (C=N); H NMR (400 MHz, DMSO-d , δ ppm): 5.64
6
(
s, 2H, -CH ), Ar-H [7.90 (d, J=8.4 Hz, 2H), 7.70 (m, 4H), 7.41 (m, 4H), 7.24 (t, J=7.2, 7.6
2
13
Hz, 1H)], Imidazole-H [8.72 (s, 1H)]; C NMR (100 MHz, DMSO-d , δ ppm): 42.26 (-CH ),
6
2
Ar-C [112.03 (CH), 121.78 (CH), 123.72 (CH), 124.29 (CH), 125.66 (CH), 126.91 (CH),
1
1
27.37 (CH), 127.88 (CH), 129.38 (CH), 133.30 (C), 136.51 (C), 139.46 (C), 140.14 (C),
69.64 (C)], Imidazole-C [111.15 (CH), 146.53 (C)], Thiadiazole-C [145.34 (C), 159.61 (C)];
+
MS: m/z 440.58 (M , 67). Anal. Calcd. for (C H N OS ): C, 65.43; H, 3.66; N, 12.72.
2
4
16
4
2
Found: C, 65.46; H, 3.67; N, 12.70.
2
.2.3.20. 2-((6-(Naphthalen-2-yl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)
benzo[d]thiazole (24)
-
Brownish solid, yield: 1.14 g (55%), m.p. 221-223 ºC (from DMF-EtOH, 1:3); IR (ATR, cm
1
1
)
: 3054 (Ar-CH), 2983 (Aliph. CH), 1592 (C=N); H NMR (400 MHz, DMSO-d , δ ppm):
6
5
7
.65 (s, 2H, -CH ), Ar-H [8.36 (s, 1H), 7.91 (m, 4H), 7.71 (d, J=8.0 Hz, 1H), 7.46 (m, 4H),
2
1
3
.39 (t, J=7.6, 8.0 Hz, 1H), 7.23 (t, J=7.6, 7.6 Hz, 1H)], Imidazole-H [8.78 (s, 1H)]; C
NMR (100 MHz, DMSO-d , δ ppm): 42.28 (-CH ), Ar-C [112.00 (CH), 121.90 (CH), 123.29
6
2
(
CH), 123.82 (CH), 124.29 (CH), 126.29 (CH), 126.91 (CH), 127.37 (CH), 128.07 (CH),
28.39 (C), 128.65 (C), 131.63 (C), 132.86 (C), 136.49 (C), 169.65 (C)], Imidazole-C [111.52
CH), 145.86 (C)], Thiadiazole-C [133.63 (C), 159.62 (C)]; MS: m/z 414.83 (M+1, 83). Anal.
Calcd. for (C H N OS ): C, 63.75; H, 3.40; N, 13.52. Found: C, 63.72; H, 3.39; N, 13.55.
1
(
2
2
14
4
2

ACCEPTED MANUSCRIPT
2
.3. Determination of in vitro Antileishmanial and Antibacterial Activities
2
.3.1. Antileishmanial Activity
In this study, we set out to determine in vitro antileishmanial activities of the compounds
against Leishmania infantum promastigote isolates using the microdilution Alamar blue assay
method.
2
.3.1.1. Preparation of Leishmania Infantum Promastigotes
Axenic standard MON-183 Leishmania infantum promastigote isolates were used in a study
on antiparasitic activity. Standard isolates were grown in RPMI-1640 (Roswell Park
Memorial Institute) (R8758 Sigma Aldrich, USA) medium which was added 10% Fetal
Bovine Serum (FBS F4135 Sigma-Aldrich, USA), 1% Penicillin (P3032 Sigma-Aldrich,
USA), and Streptomycin (S9137 Sigma-Aldrich, USA) (100,000 units of penicillin and 10 mg
streptomycin) and passaged to ensure durability.
2
0 ml of standard promastigote isolate was taken from the medium, transferred into sterile
falcon tubes, and centrifuged for 10 min at 1,000 g. The supernatant was removed, and the
isolates were washed 3 times with sterile PBS (Phosphate Buffered Saline). Then, the parasite
7
count was adjusted to approximately 2.5x10 promastigotes/mL by performing a count in the
RPMI-1640 medium and hemocytometer.
2
.3.1.2. In vitro Antileishmanial Activity Test
The synthesized compounds were dissolved in Dimethyl Sulfoxide (DMSO)/H O (10%).
2
Stock solutions of the compounds were prepared at concentration of 40 mg/mL by adding
RPMI-1640 medium containing heat-inactivated 10% FBS and sterilized by filtering it
through a sterile membrane filter at a porosity of 0.45-ꢀm (Millipore, USA). Sterile 96-well
microplates were used to test antileishmanial activity. The dilution ratios in the wells were set

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to between 20,000 ꢀg/mL and 625 ꢀg/mL. The microdilution test with Alamar blue was
th
performed as described above [31]. The 7 well was added only 100 µl stock solution as
th
negative control, while the 8 well was added only 100 µL standard parasite isolate as
positive control. After the microplates were incubated for 20 hours on a refrigerated incubator
set to 27 °C, they were added to 20 µL of Alamar blue (Resazurin sodium salt R7017 Sigma
Aldrich, USA) (prepared at 0.1 mg/ml and sterilized by filtration). The microplates were
th
th
nd
incubated for another 4 hours at 27 °C and visually assessed at the 24 , 48 , and 72 hour.
Also, a fresh preparation was made between the slide and cover glass by taking a 30 µL
sample from each well in order to observe the vitality of promastigotes. The results of this
were confirmed visually. Amphotericin B was used as the standard control drug. The test was
repeated twice for each compound. The conversion of Alamar blue to its pink derivative was
interpreted as continuing parasite growth in wells. No change in color was interpreted as that
parasite growth had stopped.
2
.3.2. Antibacterial Activity
In this study, in vitro antibacterial activities of the compounds against five selected bacteria
isolates (Gram-negative) were determined using the microdilution Alamar blue assay.
2
.3.2.1. Preparation of Standard Bacterial Isolates
The purpose was to assess minimal inhibitory concentrations (MIC) of the compounds tested
for antibacterial activity against five different standard Gram-negative bacteria using the
microdilution Alamar blue assay method (Clinical and Laboratory Standards Institute:
Performance Standards for Antimicrobial Susceptibility Testing). The standard Escherichia
coli ATCC 25922, Yersinia enterocolitica ATCC 9610, Salmonella typhimurium ATCC
1
4028, Klebsiella pneumoniae ATCC 700603, and Proteus mirabilis ATCC 25933 isolates

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obtained from the American Type Culture Collection (ATCC) were used. The standard
bacteria isolates were cultured using blood agar (Merck) and Eosin Methylene Blue Agar
(EMB, Merck), vitalized by incubation at 37 °C overnight, and were checked for purity. The
8
standard bacteria isolates were prepared at approximately 1.5x10 cfu/mL bacteria count by
suspending in sterile physiological saline solution according to the 0.5 McFarland turbidity
standard.
2
.3.2.2. In vitro Antibacterial Activity Test
The antibacterial activity test for the stock solutions of the compounds was performed using
sterile 96-well microplates. Stock solutions of the compounds were prepared at a
concentration of 40 mg/mL by adding Mueller Hinton Broth (MHB, Merck) medium, and
were sterilized by filtering through a sterile membrane filter with a porosity of 0.45-ꢀm
(
Millipore, USA). Firstly, 100 µl of MHB medium was added to each well. As described
above, the dilution ratios of the wells were set to between 20,000 ꢀg/mL and 625 ꢀg/mL [32].
00 µL of standard bacteria suspension set to 0.5 McFarland turbidity standard were then
1
added to these wells. Negative and positive control wells were added and incubated at 37 °C.
The microplates were incubated for 20 hours, 20 µL of Alamar blue was then added, and then
everything incubated again for another 4 hours. The microplates were visually assessed for
color change at the 24th and 48th hour. Samples were taken from each well using a loop for
growth control in order to see whether there was growth or not in the blood agar medium. The
antibacterial activity test was repeated twice for each compound; Amikacin was used as
control drug.
2
.4. Computational Study

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Molecular docking studies were performed by using Autodock Vina [27] and Discover Studio
Visualizer 4.5 [33]. Optimized structures of ligands (4a, 4b, 7, and 13) were calculated using
the 6-31G(d,p) base set DFT (B3LYP) theory on Gaussian 09.
3
3
. Results and discussion
.1. Chemistry
In this study, we firstly synthesized benzo[d]thiazol-2-ylthio/oxy)acetonitrile compounds
3a,b). The nitrile derivatives were obtained from the reaction of benzo[d]thiazole-2-thiol/ol
(
compounds (1a,b) with chloroacetonitrile in high yields (82% and 87%).
In the second part of the study, we synthesized 2-amino-1,3,4-thiadiazole derivatives (4a,b)
from the reaction of compounds 3a,b with thiosemicarbazide in trifluoroacetic acid (TFA) in
high yields (83% and 84%). Compounds 3a,b and 4a,b, which served as the starting
compounds, were obtained as specified in the literature [24,34].
In this reaction, an addition occurs as a result of the thiosemicarbazide’s nucleophilic attack
on the positively-charged iminium carbon, which forms under the catalytic effect of TFA. It is
believed that the elimination of the ammonia ion and the sulfur’s nucleophilic attack on the
carbon atom results in heterocyclization, which leads to the formation of 2-amino-1,3,4-
thiadiazole derivatives (4a,b) [25,35]. The formation mechanism of these compounds can be
seen in Scheme 2.

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Scheme 2. The mechanism of the formation of 2-amino-1,3,4-thiadiazole derivatives (4a,b).
-1
Sharp absorption bands belonging to the -C N group observed at 2249 and 2263 cm in the
IR spectra of compounds 3a,b disappeared. Symmetric and asymmetric absorption bands
-1
belonging to the -NH group emerged in the 3271-3089 cm range, which is the most
2
important evidence for the formation of compounds 4a,b.
1
Structures of compounds 4a,b were confirmed using H NMR spectroscopy. The -NH group
2
1
proton signals of these compounds bonded to 1,3,4-thiadiazole ring from C position in H
2
NMR spectra were observed as a singlet corresponding to 2 protons in the 7.14-7.24 ppm
range.
Proton peaks belonging to the -NH group of these compounds (4a,b) disappeared as a result
2
of proton-deuterium exchange performed with D O.
2
1
3
Structures of compounds 4a,b were confirmed by C NMR spectrum. C carbon signals
2
belonging to the thiadiazole ring in these compounds appeared at 154.48 and 152.14 ppm. C5
carbon signals of the same ring were recorded as 165.49 and 169.24 ppm. Other spectral data
belonging to the carbon skeleton of the molecule fully support the suggested structures. The

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1
3
C NMR spectra of these compounds were observed to be highly compatible with this type of
compounds in the literature [3,25,35].
In the third part of the study, we synthesized the imidazo[2,1-b][1,3,4]thiadiazole derivatives
(5-24), the target compounds, from reactions of compounds 4a,b with phenacyl bromide
derivatives (in yields of 53% to 73%). Scheme 3 shows the formation mechanism of the target
compounds.
Scheme 3. Formation mechanism of target compounds (5-24).

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In IR spectra of compounds 4a,b, -NH group symmetric and asymmetric absorption bands
2
-1
found in the initial compounds and observed in the 3271-3089 cm range disappeared, which
is the most important evidence for the formation of the target compounds.
1
Also, -NH group proton signals observed at 7.14 and 7.24 ppm in the H NMR of the starting
2
compounds (4a,b) disappeared in these compounds and instead observed as a singlet
corresponding to 1 proton in the 8.94-8.51 ppm range of the C -H signals in the imidazo[2,1-
5
b][1,3,4]thiadiazole derivatives (5-24), this is the most important evidence for the formation
1
of these compounds. This is consistent with the literature [35]. Other H NMR data of these
compounds is given in the experimental part of this study in detail.
Signals that appeared in the 113.58-109.89 ppm range and the 152.80-145.25 ppm range in
1
3
C NMR spectrum of these compounds is important evidence for ring cyclization. These
signals correspond to C and C carbons of imidazo[2,1-b][1,3,4]thiadiazole derivatives (5-24)
5
6
1
3
or the target compounds. Other C NMR spectrum data of these compounds is given in the
experimental part in detail.
1
13
The FT-IR, H NMR, C NMR, elemental analysis, and mass spectroscopy of all compounds
synthesized in the study can be found in the experimental section. All of the spectra are given
in the Supplementary Material Section in detail.
3
.2. In vitro Antileishmanial Activity Studies
Figure 1 shows the images of 21 compounds and Table 1 shows the minimum inhibitory
concentration (MIC) values obtained as a result of the assessment. Positive and negative
control wells were observed to work properly.

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Figure 1. The antileishmanial activity results against axenic standard MON-183 Leishmania
infantum promastigotes; compounds 4a,b and 5-24, Control drug Amphotericin B.
Dilution concentrations 20,000 µg/ml - 625 µg/ml.
N.C.: Negative Control; P.C.: Positive Control.

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Table 1. Minimum inhibitory concentration (MIC) values of the compounds against
Leishmania infantum promastigotes.
MIC values
Compounds
(
µg/ml)
4
a
>20 000
>20 000
>20 000
>20 000
20 000
4b
5
6
7
8
9
10 000
>20 000
>20 000
>20 000
>20 000
>20 000
>20 000
>20 000
>20 000
20 000
1
0
1
2
3
4
5
6
7
8
0
1
2
3
4
1
1
1
1
1
1
1
1
2
2
2
2
2
>20 000
>20 000
>20 000
>20 000
>20 000
>20 000
<625
Amphotericin B
The antileishmanial activity study showed that compound 8 (MIC 10 000 ꢀg/mL) was the
most effective compound. Also, compounds 7 and 17 were found to be effective at the highest
concentration studied (MIC 20 000 ꢀg/mL). Other compounds had no antileishmanial activity
at the studied concentrations.
Amphotericin B, or the standard drug, was found to be effective at a concentration <625
µg/ml.

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3
.3. In vitro Antibacterial Activity Studies
While some of the synthesized compounds were observed to have antibacterial activity
against standard bacteria isolates at different concentrations, some compounds were
ineffective at the studied concentrations.
Bacterial activity images of some compounds can be seen in Figures 2 and 3. Minimum
inhibitory concentration (MIC) values of the compounds can be seen in Table 2.
As it was found that the synthesized compounds were effective against five different standard
bacteria isolates at varying degrees. The compounds were most effective against Escherichia
coli. All compounds except for compound 17 were found to be effective at different
concentrations (MIC=625-10 000 ꢀg/mL). Compounds 4b and 7 were found to be the most
effective compounds against Escherichia coli (MIC=625 ꢀg/mL). The second bacteria on
which the compounds were effective was Yersinia enterocolitica (MIC= 1 250-20 000
ꢀ
g/mL). Compound 4b was once again observed to be the most effective compound against
Yersinia enterocolitica (MIC=1 250 ꢀg/mL) and compounds 4b and 24 were found to be
effective against Salmonella typhimurium (MIC= 2 500 and 5 000). Except for compound 24
(
MIC=10 000 ꢀg/mL), none of the compounds was effective against Klebsiella pneumoniae.
None of the compounds was effective against Proteus mirabilis at the studied concentrations
MIC >20 000).
(