Synthesis of N-(6-Arylbenzo[d]thiazole-2-acetamide derivatives and their biological activities: An experimental and computational approach

Article abstract of DOI:10.3390/molecules21030266

A new series of N-(6-arylbenzo[d]thiazol-2-yl)acetamides were synthesized by C-C coupling methodology in the presence of Pd(0) using various aryl boronic pinacol ester/acids. The newly synthesized compounds were evaluated for various biological activities like antioxidant, haemolytic, antibacterial and urease inhibition. In bioassays these compounds were found to have moderate to good activities. Among the tested biological activities screened these compounds displayed the most significant activity for urease inhibition. In urease inhibition, all compounds were found more active than the standard used. The compound N-(6-(p-tolyl)benzo[d]thiazol-2-yl)acetamide was found to be the most active. To understand this urease inhibition, molecular docking studies were performed. The in silico studies showed that these acetamide derivatives bind to the non-metallic active site of the urease enzyme. Structure-activity studies revealed that H-bonding of compounds with the enzyme is important for its inhibition.

Full text of DOI:10.3390/molecules21030266

molecules
Article
Synthesis of N-(6-Arylbenzo[d]thiazole-2-acetamide
Derivatives and Their Biological Activities: An
Experimental and Computational Approach
Yasmeen Gull 1,2, Nasir Rasool *, Mnaza Noreen , Ataf Ali Altaf , Syed Ghulam Musharraf ,
1,
1
3
4
1
5
5
6,
Muhammad Zubair , Faiz-Ul-Hassan Nasim , Asma Yaqoob , Vincenzo DeFeo * and
Muhammad Zia-Ul-Haq 7
1
Department of Chemistry, Faculty of Science and Technology, Government College University Faisalabad,
Faisalabad 38000, Pakistan; yasmeenchem1@yahoo.com (Y.G.); mnazanoreen@yahoo.com (M.N.);
nr_308@hotmail.com (M.Z.)
Department of Chemistry, Faculty of Science, University of Sargodhah, Bhakkar Campus,
2
Bhakkar 30000, Pakistan
3
Department of Chemistry, Faculty of Science, University of Gujrat, Hafiz Hayat Campus, Gujrat 50700,
Pakistan; atafali_altaf@yahoo.com
International Center for Chemical and Biological Sciences, Hussain Ebrahim Jamal Research Institute of
4
Chemistry, University of Karachi, Karachi 75270, Pakistan; musharraf1977@yahoo.com
Department of Chemistry, Faculty of Science, Islamia University of Bahawalpur, Bahawalpur 63000,
5
Pakistan; faiznasim@hotmail.com (F.-H.N.); asmayaqoobctn@gmail.com (A.Y.)
Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Via Ponte don Melillo,
6
Fisciano (Salerno) I-84084, Italy
Offices of Research, Innovation and Commercialization, Lahore College for Women University,
7
Lahore 54600, Pakistan; ahirzia@gmail.com
*
Correspondence: nasirrasool@gcuf.edu.pk (N.R.); defeo@unisa.it (V.D.F.); Tel.: +92-332-749-1790 (N.R.);
39-089-959-751(V.D.F.); Fax: +92-41-920-1032 (N.R.); +39-089-969-602 (V.D.F.)
+
Academic Editor: Maxim L. Kuznetsov
Received: 31 December 2015 ; Accepted: 1 February 2016 ; Published: 25 February 2016
Abstract: A new series of N-(6-arylbenzo[d]thiazol-2-yl)acetamides were synthesized by C-C coupling
methodology in the presence of Pd(0) using various aryl boronic pinacol ester/acids. The newly
synthesized compounds were evaluated for various biological activities like antioxidant, haemolytic,
antibacterial and urease inhibition. In bioassays these compounds were found to have moderate to
good activities. Among the tested biological activities screened these compounds displayed the most
significant activity for urease inhibition. In urease inhibition, all compounds were found more active
than the standard used. The compound N-(6-(p-tolyl)benzo[d]thiazol-2-yl)acetamide was found to be
the most active. To understand this urease inhibition, molecular docking studies were performed.
The in silico studies showed that these acetamide derivatives bind to the non-metallic active site of the
urease enzyme. Structure-activity studies revealed that H-bonding of compounds with the enzyme is
important for its inhibition.
Keywords: Suzuki cross coupling; Pd(0) catalyst; benzothiazole; nitric oxide scavenging activity;
antiurease activity; haemolytic activity
1
. Introduction
Benzothiazoles consist of a benzene ring fused with a thiazole ring. Various benzothiazole
derivatives serve as drugs, dyes and industrial chemicals [1–3]. Benzothiazoleand its derivatives
such as esters have also been reported as active against Gram-positive and Gram-negative bacteria
Molecules 2016, 21, 266; doi:10.3390/molecules21030266
www.mdpi.com/journal/molecules

Molecules 2016, 21, 266
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such as Staphylococcus epidermidis, Escherichia coli, Enterobacter and yeast (Candida albicans) [
Benzothiazole derivatives have also found to possess anticancer, antifungal and antibacterial
activities [ ]. 2-Aminobenzothiazole and a number of other aminobenzothiazole derivatives have
been reported as muscle relaxants [ ]. A literature survey performed for the current study revealed
that 6-substituted-2-aminobenzothiazole derivatives such as 6-methyl, 6-methoxy, 6-ethoxy and
-isopropoxy show antibacterial, anti-inflammatory and analgesic properties [ ]. Various other
derivatives are found to be cytotoxic against various tumors [10,11].
4].
5,6
7,8
6
9
Suzuki cross coupling reactions are remarkable methods for C-C bond formation, utilized for
the synthesis of agrochemicals, advanced materials and pharmaceuticals at both the industrial and
laboratory scale [12–17].
The purpose of this study was to synthesize novel N-(6-arylbenzo[d]thiazol-2-yl)acetamides
employing the Pd(0)-catalyzed Suzuki cross coupling methodology. This article describes our
optimized experiments for the synthesis of N-protected 6-bromobenzothiazoles. The biological
activities of these newly synthesized molecules were studied with the intention to explore their
potential as future drugs. We investigated urease inhibition, nitric oxide scavenging, haemolytic and
antibacterial activities. Molecular docking studies were performed to determine how they bind to the
urease enzyme. To the best of our knowledge, all the studies reported in the current manuscript have
not been reported in the literature to date.
2
. Results and Discussion
2
.1. Chemistry
Majo et al. reported low to moderate yielding one-step Suzuki cross coupling reactions using
various boronic acids/ester with 2-bromobenzothiazole under thermal conditions [18]. We have
previously reported Pd(0)-catalyzed reactions of 2-amino-6-bromobenzothiazole with different
arylboronic pinacol esters/arylboronic acids using Suzuki cross coupling methodology with moderate
yields [19]. We have not been able to achieve better yields as the amino moiety present in the
benzothiazole molecule is basic and nucleophilic. In the current study, we have tried to enhance the
yield of the synthesized molecules. In order to achieve high yields, the amino group was protected via
acylation, which led to substantially enhanced yields of the products 3a
–
3h (Figure 1) compared to
unprotected benzothiazole derivatives as reported in literature [19].
O
O
N
N
S
N
S
Acetic Anhydride
Aryl Boronic Acid/Esters
NH2
NH
NH
o
Acetonitrile, 60 C
o
S
Br
Br
K
3
PO
4
, Pd(PPh
3
)
4
, 90 C, 30 h Ar
1
85%
2
3a - 3h
Figure 1. Synthesis of N-(6-bromobenzo[d]thiazol-2-yl)acetamide (2) and N-(6-aryl-benzo[d]thiazol-
2
-yl)acetamides 3a–3h.
Furthermore, we also optimized other reaction parameters like catalyst loading, solvent,
temperature and base used in the reactions producing 3a 3h. Thus, we tried various solvents like
–
˝
toluene, dimethylformamide (DMF) and 1,4-dioxane at different temperatures (80–100 C). It was
noted (in Table 1) that the solvent has an effect on overall reaction yield. 1,4-Dioxane was found to be
the best solvent because of its better solvation of the reactants. In our studies, these cross coupling
reactions progressed efficiently, even in the presence of known sensitive groups such as CN, to give
the desired products in very good yields. Finally after optimization, we investigated the coupling of
with different arylboronic pinacol esters/acids in the presence of Pd(PPh ) as catalyst.
2
3
4
Product 3a was prepared with 80% yield, when 2 was coupled with phenyl boronic acids under
the set reaction conditions. The highest yield obtained in this series of reactions (85%) corresponded to
product 3b. Product 3h was also obtained in excellent yield (83%) with this cross coupling method.

Molecules 2016, 21, 266
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It was seen that the product 3c with an electron withdrawing moiety showed a high yield (81%). Our
studies showed that overall the acetylated 2-amino-6-bromobenzothiazole (
these coupling reactions.
2) gave good yields in
Table 1. Synthesis of N-(6-arylbenzo[d]thiazol-2-yl)acetamides 3a–3h.
Arylboronic Pinacol
Ester/Arylboronic Acid
Entry
Product
H2O/Solvent (1:4)
Yields %
O
N
S
NH
1
Toluene 1,4-Dioxane
75 80
3a
O
N
NH
2
3
4
S
1,4-Dioxane
1,4-Dioxane
1,4-Dioxane
77
85
81
3a
O
B(OH)2
CH3
N
S
NH
H C
3
3b
O
N
S
NH
F3C
CF3
3c
O
B(OH)2
OCH3
N
S
NH
5
6
1,4-Dioxane
1,4-Dioxane
79
75
3
H CO
3d
O
N
3
H C
S
NH
S
3e
O
N
S
NH
3
F C
7
1,4-Dioxane
77
CN
3f
O
N
NH
S
8
9
1,4-Dioxane
1,4-Dioxane
79
83
F
Cl
3g
O
N
S
NH
3
H C
CH
3
3h

Molecules 2016, 21, 266
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2
.2. Biological Studies
2
.2.1. Urease Inhibitory Activity
Urease is a nickel-containing metalloenzyme that catalyzes the hydrolysis of urea to form ammonia
and carbamate, which further decomposes to yield a second equivalent of ammonia and carbon
dioxide [20]. Bacterial ureases have been reported as an important virulence factor in the development
of many harmful clinical conditions for human and animal health. Urease is directly involved in the
formation of infectious stones and contributes to pathogenesis [21]. It is the major cause of pathogenesis
induced by Helicobacter pylori, which plays an important role in peptic ulcers and may lead to stomach
cancer. In recent years, a number of compounds have been proposed as urease inhibitors [22,23], which
are considered interesting new targets for anti-ulcer drugs and for the treatment of infections caused
by urease producing bacteria [24].
In the current study, urease inhibitory activity assays were performed following a previously
reported protocol [25]. Thiourea was used as standard in the assay with an IC50 value of 23.1
All of the synthesized benzothiazole derivatives were examined for their urease inhibitory activities at
concentration of 15 and 40 g/mL. All of our synthesized compounds exhibit good to excellent urease
µg/mL.
µ
inhibitory activities (Table 2, Figures 2 and 3).
Table 2. Antiurease activity of N-(6-arylbenzo[d]thiazole-2-yl)acetamides 3a–3h at 15µg/mL and
4
0 µg/mL.
Compound
% Inhibition at 15 µg/mL
% Inhibition at 40 µg/mL
IC50 (µg/mL)
3
a
44 ˘ 0.12
47 ˘ 0.11
83.9 ˘ 0.12
90.51 ˘ 0.19
90.07 ˘ 0.20
88.5 ˘ 0.24
87 ˘ 0.26
18.6
16.5
17.2
17
17.2
19.2
18.9
18.4
23.1
3
b
3
c
46.09 ˘ 0.10
46.5 ˘ 0.15
46 ˘ 0.12
3
d
3
3
e
f
42 ˘ 0.12
87.5 ˘ 0.26
87 ˘ 0.26
3
g
42 ˘ 0.14
3
h
43.59 ˘ 0.13
90.33 ˘ 0.20
65 ˘ 0.01
Standard
47 ˘ 0.31
Each values is mean ˘ Standard deviation of three parallel measurements.
Figure 2. The urease percentage inhibition values at 15 µg/mL and 40 µg/mL.
Figure 3. IC50 values of anti-urease activity.

Molecules 2016, 21, 266
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Urease enzyme has an active binding site and it was believed that the newly synthesized
benzothiazole derivatives have the capability to bind to these active sites of urease enzyme. In this
way the hydrolysis of enzyme is stopped and activity of enzyme is inhibited. Compound 3b showed
the highest urease inhibition activity (90.51 ˘ 0.19) with an IC value of 16.5 µg/mL at 40 µg/mL.
50
Compounds 3h and 3c exhibited excellent urease inhibitory activities (90.07
˘
0.20 and
g/mL, respectively. Molecules 3a
3f and 3g exhibited good urease inhibition with IC50 values of 18.6, 17, 17.2, 19.2 and 18.9 g/mL,
9
3
0.33 ˘ 0.20 at 40
µg/mL) with IC50 values of 17.2
µg/mL and 18.4
µ
,
d
,
3e
,
µ
respectively. Notably we observed that the presence of electron donating methyl functional groups
produced high urease inhibition. We also noted that different functional groups are responsible for
variable antiurease activities of the compounds.
2
.2.2. In Silico Studies with Urease
To understand the binding of the synthesized compounds with urease in-silico studies were
performed. Only compounds with IC50 values were analyzed for docking studies using the freely
available software AutoDock 4.2 and others as described in the Experimental Section. All the
compounds were screened at different sites (A & B) of the enzyme. The nickel-containing catalytic site
A is the most commonly tested site in the literature [26], while site B is less commonly targeted [20,27].
Our compounds bind more strongly to Site B than to site A. The docking studies results were compared
with the experimental results and listed in Tables 3 and 4. Binding energy, inhibition constant and
moldock scores are reported in Table 3, whereas the moldock H-binding energy and number of
H-bonding interactions between the test compounds and enzyme are reported in Table 4.
Table 3. Experimental and docking comparative data.
Compound
* IC50 (µg/mL)
Inhibition Constant
Binding Energy
Moldock Score
3
a
18.6
16.5
17.2
17.0
17.2
19.2
18.9
18.4
267.93
232.29
232.56
434.76
278.10
90.570
153.46
150.67
´4.87
´4.96
´4.96
´4.59
´4.85
´5.52
´5.20
´5.21
´83.39
´81.68
´68.81
´69.20
´87.00
´87.90
´79.91
´80.11
3
b
3
c
3
3
d
e
3
f
3
g
3
h
*
Experimentally measured in vitro.
Table 4. H-bonding parameters calculated by Mol-Dock Molegero Molecular Viewer 2.5.
Number of
H-bonds
H-Bonding Type
(K–H–L) *
H-Bond Distance
(K–L) (Å)
Compound
IC50 (µg/mL)
H-Binding Energy
3
a
1
2
1
2
O—H–N
N—H–N
N–H—O
N–H—O
N–H—O
N—H–N
N–H—O
N—H–N
N–H—O
N–H—O
–
2.957
2.837
3.544
2.782
3.023
3.23
2.906
3.388
2.739
2.696
–
18.6
16.5
17.2
17.0
´2.50
´3.45
´2.98
´3.30
3
b
3
c
3
d
3
e
2
17.2
´3.01
´1.67
´1.99
–
3
f
1
1
zero
19.2
18.9
18.4
3
g
3
h
*
K atom from the compound and L atom from the protein residue.
The number of H-bonding interactions was estimated from the most stable complex formed
between the test compound and the enzyme. We found a linear correlation between the experimental
IC50 values and the calculated binding energy, as shown in Figure 3. This linear correlation of results is

Molecules 2016, 21, 266
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in agreement with the data reported in the literature [26
,
28]. More interestingly a better correlation
was found between IC50 values and the moldock H-binding energy (Figure 4).
Figure 4. Correlation between docking predicted energies (in arbitrary unit) and in vitro IC50 values.
These results show that the H-bonding is more important, in urease inhibition mechanism, than
other factors involved in this biological reaction. Compound 3b, with the highest in vitro activity,
is represented here for modeling analysis and its most active conformations are explained in the
following paragraphs. As urease is a nickel-dependent enzyme, the active site A of the enzyme shows
weak hydrophobic interaction with compound 3b therefore, a drug with hydrophobic substituents
would be able to bind strongly as it would project into the hydrophobic grooves of the enzyme and
thus effectively inhibit its activity. The LIGPLOT interaction images show that compound 3b has a
total of seven interactions with enzyme site A (Figure 5a).
Figure 5. LIGPLOT images of compound 3b with urease enzyme (a) at the catalytic site A; and (b) at
the catalytic site B.
The amino acids His593, Met637, Ala636, Gln635, Met588 and Asp494 form hydrophobic
interactions with compound 3b. The hydrophobic interactions favor ligand binding with proteins
having metal ions. Furthermore, the study showed hydrogen bonding (2.66 Å, N–H—O type) between
compound 3b and PO group of the enzyme nickel catalytic site A.
4
Figure 6a represents the most interacting conformation of 3b in the active pocket (site A) of the
enzyme at the electrostatic surface. Figure 6 is generated by Molegro Molecular viewer and analyzed
by moldock score. The moldock analysis shows that there are two H-bonding interactions in both
cases these are also reported in Table 4.

Molecules 2016, 21, 266
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(
a)
(b)
Figure 6. Compound 2c in the Molegro molecular viewer generated electrostatic surface of urease
enzyme ( ) at the catalytic site A; and ( ) at the catalytic site B. The yellow dashed line represents the
H-bonding.
a
b
All of the synthesized compounds 3a–3h show better interaction at site B. The LIGPLOT
interaction diagram of compound 3b illustrates that this inhibitor has better interactions with the
protein. The LIGPLOT interaction images show that the compound 3b has a total of eight interactions
with enzyme site B (Figure 5b). The amino acids Lys208, Asp206, Thr158, Glu254, Phe182, Lys156 and
Asp183 form cationic—π interactions with compound 3b, while Glu252 interacts via hydrogen bonding
(2.84Å, N–H—O type) with compound 3b. Figure 5b presents the most interacting conformation of
compound 3b in the active pocket of the enzyme (site B). The diagram shows that the enzyme provides
enough space for the accommodation of 3b inside the pocket. The backbone dose sterically favors the
3
b molecule to interact with catalytic site.
As shown in Figure 4, a strong correlation with experimental results is found between the IC50
values and H-bonding data calculated by moldock in the Molegero docking software at site B of enzyme.
In the moldock analysis for 3b at site A two H-bonding interactions (N–H—O and N–H—N type)
were observed between 3b and PO 844, His593, respectively, while two strong H-bonding interactions
4
between 3b and enzyme residues Glu252 and Lys156 were observed at site B. The H-bonding distances
and moldock scores for all compounds, at site B, are listed in Table 4. Figure 7 shows the H-bonding
interactions of all of the compounds (except 3h) with the active residue of urease enzyme at site B.
Compound 3h does not show any H-bonding interaction with the active site residue. In the linear
correlation with in vitro IC , stronger H-bonding is found for compounds with lower IC value. All
50
50
the compounds have H-bonding distances in the 2.696 Å–3.544 Å range and H-bonding energies in the
1.674–´3.45 a.u. range.
´
3
a
3b
3
c
3d
Figure 7. Cont.

Molecules 2016, 21, 266
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3
f
3
e
3
g
3
h
Figure 7. Molegro molecular viewer generated sketches between compounds 3a–3h and the active
residue of urease at site B. The blue dashed lines represent the H-bonding.
2
.2.3. Nitric Oxide Scavenging Percentage Assay
The literature contains reports on the antioxidant activities of 6-flourobenzothiazole-substituted
triazoles using DPPH assays [29]. A survey of the literature showed that benzothiazole molecules
along with pyrozoline rings showed the highest antioxidant activities. Having a phenyl ring on
the pyrozoline increased the antioxidant activity in the ferric ion reduction and DPPH solution
methods [30]. Our newly synthesized N-protected benzothiazole derivatives exhibit nitric oxide
scavenging activities. Ascorbic acid was used as a standard in the assay with 38.5
˘
0.16 and 84.1 ˘ 0.12
percent nitric oxide scavenging at 20 g/mL and at 50 g/mL with an IC50 value of 50.43 µ
µ
µ
g/mL.
Synthesized compounds with their calculated IC50 values are listed in Table 5 (Figures 8 and 9).
It was found that acetyl-protected amino group products were more active in the nitric oxide
scavenging assay, than the previously reported non-acetylated compounds [19]. Molecules 3a
3g and 3h were found to be the most active for nitric oxide scavenging activity, with percentage
inhibitions of 57.75 0.12, 69 0.12, 55 0.31, 51.25 0.15 and 60.5 0.1 at 40 g/mL with IC50
, 2b,
3e,
˘
˘
˘
˘
˘
µ
values of 32.7,26.4, 37.1, 39.1 and 32.3, respectively. Compound 3f, however, was found to be inactive
in the nitric oxide scavenging assay. We are unable to account for this inactivity.
Table 5. Nitric oxide scavenging activity of N-(6-arylbenzo[d]thiazole-2-yl)acetamides 3a
0 µg/mL and 40 µg/mL.
–3h at
2
Compound
% Activity at 20 µg/mL
% Activity at 40 µg/mL
IC50 (µg/mL)
3
a
36.25 ˘ 0.12
41 ˘ 0.11
10 ˘ 0.18
14.25 ˘ 0.17
20.25 ˘ 0.15
0
57.75 ˘ 0.12
69 ˘ 0.12
41.75 ˘ 0.20
46 ˘ 0.2
32.7
26.4
NC
NC
37.1
NC
3
b
3
c
3
d
3
3
e
f
55 ˘ 0.31
0
3
g
20.25 ˘ 0.15
33.5 ˘ 0.13
38.5 ˘ 0.16
51.25 ˘ 0.15
60.5 ˘ 0.1
84.1 ˘ 0.12
39.1
32.3
50.43
3
h
Standard
Each value is mean
less activity.
˘
Standard deviation of three parallel measurements. NC stands for not calculated due to

Molecules 2016, 21, 266
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Figure 8. The percentage nitric oxide inhibition at 20 µg/mL and 40 µg/mL.
Figure 9. IC50 values of nitric oxide scavenging activity.
2
.2.4. Haemolytic Activity
The haemolytic activity of benzothiazole derivatives has already been reported. A literature survey
reveals that amino-substituted derivatives of benzothiazole have high cytotoxicites. Benzothiazole
compounds with halogen substitutions show cytotoxicity towards cancer cell lines [31]. The haemolytic
activity of the newly synthesized benzothiazole derivatives were studied against Triton X-100 by a
reported method [32].
The newly synthesized benzothiazole derivatives exhibit moderate to high haemolytic activities
(
see in Table 6, Figure 10). Compound 3c exhibits the highest haemolytic activity (47.089
˘
0.130).
Fluorinated analog 2d also displayed the highest toxicity among all the tested compounds. The
antitumor activity of a compound might be considered as corresponding to the highest haemolytic
activity. It was observed that substitution does not markedly affected the haemolytic activity of these
newly synthesized N-protected benzothiazole derivatives. Compounds 2, 3b and 3d–3g showed
good haemolytic activities. The lowest haemolytic activity was found for compound 3h. It was
concluded that halogen substitution on N-protected benzothiazole molecules promotes haemolytic
activity. These compounds have potential to be used as future anticancer agents.
Table 6. Haemolytic activity of newly synthesized N-(6-bromobenzo[d]thiazol-2-yl)acetamide (
2) and
N-(6-arylbenzo[d]thiazole-2-yl)acetamides (3a–3h).
Entry
2
% lysis of RBC
Entry
% lysis of RBC
44.628 ˘ 0.369
42.123 ˘ 0.479
44.179 ˘ 0.157
47.089 ˘ 0.130
44.078 ˘ 0.279
3e
3f
3g
3h
44.425 ˘ 0.181
44.063 ˘ 0.314
43.614 ˘ 0.157
40.661 ˘ 0.216
3a
3
3
b
c
3d
Standard
99.78 ˘ 0.912
Each value is mean ˘ Standard deviation of three parallel measurements.

Molecules 2016, 21, 266
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Figure 10. Haemolytic activity of the synthesized compounds.
2
.2.5. Antibacterial Activity
The synthesized benzothiazole derivatives were examined for their anti-bacterial activity against
two Gram positive-bacterial strains (Baccilus subtiles, Staphylococcus aureus) and four Gram-negative
strains (Escherichia coli, Psuedomonas aeruginosa, Shigella dysenteriae, Salmonella typhae) at concentrations
of 40 and 80 µg/mL (Tables 7 and 8). It was concluded that the potent antibacterial activities of these
compounds might be due to electron withdrawing groups present on the aryl moiety in N-protected
benzothiazole molecule. Similar observations are also reported by other groups which suggest that the
presence of electron releasing and electron withdrawing groups substantially affects the antibacterial
activity [33].
Table 7. Antibacterial activity (40 µg/mL) of N-(6-arylbenzo[d]thiazole-2-yl)acetamides 2b–2i.
%
Activity at 40 µg/mL
Entry
B. subtilis
S. aureus
P. aeruginosa
S. dysenteriae
S. typhae
E. coli
3
a
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.94 ˘ 0.45
-
-
37.52 ˘ 0.38
34.04 ˘ 0.40
49.05 ˘ 0.32
42.10 ˘ 0.36
37.82 ˘ 0.38
33.53 ˘ 0.41
45.93 ˘ 0.3
22.89 ˘ 0.47
19 ˘ 0.31
3
b
-
3
c
0 ˘ 0.45
2.92 ˘ 0.44
-
2.59 ˘ 0.44
1.45 ˘ 0.44
3.51 ˘ o.43
35 ˘ 0.32
6.0 ˘ 0.47
3
d
-
-
3
3
e
f
-
7.54 ˘ 0.6
1.2 ˘ 0.50
3
g
-
-
-
-
3
h
Ampicillin
23 ˘ 0.1
29 ˘ 0.61
25 ˘ 0.12
29 ˘ 0.61
Each value is mean ˘ standard deviation of three parallel measurements.
Table 8. Antibacterial activity (80 µg/mL) of N-(6-arylbenzo[d]thiazole-2-yl)acetamides 2b–2i.
Activity at 80 µg/mL
%
Entry
B. subtilis
S. aureus
P. aeruginosa
S. dysenteriae
S. typhae
E. coli
3
a
6.08 ˘ 0.571
15.25 ˘ 0.5
-
-
-
-
8.47 ˘ 0.44
6.57 ˘ 0.45
-
18.5 ˘ 0.58
17.7 ˘ 0.5
26.2 ˘ 0.53
28.86 ˘ 0.5
19.2 ˘ 0.5
25 ˘ 0.54
57.97 ˘ 0.25
56.13 ˘ 0.32
50.49 ˘ 0.30
51 ˘ 0.30
3
b
-
-
-
3
c
-
-
3
d
7.31
12.66
4.27 ˘ 0.582
˘
0.5635
0.531
5.92 ˘ 0.46
3
3
e
f
˘
3.75 ˘ 0.65
-
-
50.8 ˘ 0.30
55 ˘ 0.32
8.89 ˘ 0.5
2.96 ˘ 0.6
10.93 ˘ 0.43
11.51 ˘ 0.42
7.48 ˘ 0.45
56 ˘ 0.26
3
g
-
-
-
-
-
21.05 ˘ 0.5
17.43 ˘ 0.5
42.9 ˘ 0.29
57.84 ˘ 0.25
53.96 ˘ 0.40
45.9 ˘ 0.21
3
h
5.67
˘
0.5735
Ampicillin 50.5 ˘ 0.31
52.9 ˘ 0.29
52 ˘ 0.26
Each value is mean ˘ standard deviation of three parallel measurements.
The results show that the benzothiazole compounds 3a–3h exhibit higher activities than the
standard against some species. Functional group changes in the benzothiazole molecule led to
differences in activity. The newly synthesized compounds were found to be inactive against Baccilus

Molecules 2016, 21, 266
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subtilis and Staphylococcus aureus. Only compound 3f showed activity against Psuedomonas aeruginosa
with a very small value (7.54 0.6). These newly synthesized compounds do not exhibit considerable
antibacterial activity and the highest value (3.51 0.43) was observed for compound 3h against
Shigella dysenteriae at concentration of 40 g/mL. These new benzothiazole molecules showed weak
activities against Salmonella typhae at 40 g/mL. It was found that all newly synthesized benzothiazole
compounds gave good to very good activity against E. coli at 40 g/mL. Compounds 3c and 3g
exhibited very good activities against the E. coli strain with values of 49.05 0.32 and 45.93 0.3,
˘
˘
µ
µ
µ
e
˘
˘
respectively. These differences in activities may be attributed to the presence of electron loving
atoms/groups on the aryl moiety of these N-protected benzothiazole derivatives.
The synthesized compounds were also checked for antibacterial activities at 80
compared against ampicillin. It was shown that these compounds showed moderate activities against
Bacillus subtiles with the highest value (12.66 0.531) corresponding to compound 3e. The authors
µg/mL and
˘
concluded that these benzothiazole derivatives showed non-significant activity against Staphylococcus
aureus. In addition, these compounds were to be found active against Shigella dysenteriae and Salmonella
typhae at 80
benzothiazole derivatives were discovered to be the most potent against the E. coli strain. Compounds
3b and 3g proved to be the most potent at the concentration of 80 g/mL with the highest
antibacterial activities with values of 57.97 0.25, 57.84 0.25 and 56.13 0.32, respectively. The
µg/mL. These compounds displayed very good activity against E. coli at 80 µg/mL. The
3
a
,
µ
˘
˘
˘
results of this study revealed that electron withdrawing group substitution on the aryl moiety on the
benzothiazole molecule enhanced the antimicrobial activity of the compounds.
3
. Experimental Section
3
.1. General Information
All reagents and chemicals were brought from Alfa-Aesar Chemical Co. (Ward Hill, MA, USA)
and Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Solvents CDCl and CD OD were used for
3
3
13
1
C-NMR and H-NMR spectra on an Aspect AM-400 instrument at 400/100 MHz (Bruker, Billerica,
MA, USA). The coupling constant was determined in Hz and chemical shift in in ppm. A JMS-HX-110
δ
spectrometer (JEOL, Peabody, MA, USA) was used for EI-MS spectra. Melting points of benzothiazole
compounds were measured on a B-540 melting point apparatus (Büchi, New Castle, DE, USA). Column
chromatography with silica gel (mesh size 70 to 230 and 230 to 400) was used for compound purification.
TLC (silica gel 60 PF 254 cards, Merck, Kenilworth, NJ, USA) was used for reaction monitoring. Plates
were visualized using a UV lamp (254 to 365 nm) (Spectronics Corporation, Westbury, NY, USA).
3
.2. Procedure for the Preparation of N-(6-arylbenzo[d]thiazole-2-yl)acetamides 3a–3h
The preparation of products 3a 3h was carried out under a nitrogen atmosphere. Compound
synthesized by literature reported method [19]) (2.183 mmol) and 5 mol % Pd(PPh ) in 1,4-dioxane
–
2
(
(
3
4
20 mL) was mixed and stirred for 30 min. After 30 min K PO (4.366 mmol), aryl boronic pinacol
3
4
esters/aryl boronic acids (2.401 mmol), and H O (1.5 mL) was added under inert atmospheric
2
˝
conditions. The mixture was stirred for 30 h at 95 C and cooled down to room temperature. Ethyl
acetate was used for work up and the organic layer was separated and dried under vacuum. For
purification purposes, column chromatography was done. The desired product was obtained by
using ethylacetate and n-hexane (20% and 80%respectively) as eluents. The desired products were
characterized by various spectroscopic techniques [34].
˝
1
N-(6-Phenylbenzo[d]thiazol-2-yl)acetamide(3a).m.p. 203–205 C; H-NMR (CDCl + CD OD)
δ
9.46
3
3
13
(
s, 1H), 7.92 (s, 1H), 7.67–7.50 (m, 4H), 7.46–7.31 (m, 3H), 2.31 (s, 3H); C-NMR (CDCl + CD OD)
δ
3
3
1
68.3, 159.2, 151.9, 145.6, 132.4, 130.1, 129.8 (2C), 128.4, 128.4 (2C), 124.2, 121.7, 116.8, 23.7; EIMS (m/z +
+
ion mode): 269.32 [M + H ] 269.08; Anal Calcd for C H N OS: C, 67.13; H, 4.53; N, 10.43; found C,
15
12
2
6
7.23; H, 4.54; N, 10.37.

Molecules 2016, 21, 266
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˝
1
N-(6-(p-Tolyl)benzo[d]thiazol-2-yl)acetamide(3b).m.p. 205–207 C; H-NMR (CDCl + CD OD)
δ
9.33
3
3
(
s, 1H), 8.11 (d, J = 10 Hz, 3H), 7.61 (d, J = 10.4 Hz, 1H), 7.31–7.18 (m, 3H), 2.41 (s, 3H), 2.35 (s, 3H);
13
C-NMR (CDCl + CD OD)
21.2, 116.8, 22.9, 21.6; EIMS (m/z + ion mode): [M + H ] = 283.08; [M
δ 168.2, 159.3, 148.4, 146.8, 133.1, 131.3, 129.6 (2C), 128.4 (2C), 126.1, 124.4,
3
3
+
´
COCH3] = 241.01. Anal
1
´
Calcd for C H N OS: C, 68.07; H, 5.01; N, 9.93 found C, 68.12; H, 5.04; N, 9.81.
16
14
2
˝
1
N-(6-(3,5-bis(Trifluoromethyl)phenyl)benzo[d]thiazol-2-yl)acetamide (3c). m.p. 203–205 C; H-NMR (CDCl
+
3
13
CD OD) δ 9.41 (s, 1H), 7.92 (s, 1H), 7.66–7.61 (m, 2H), 7.55–7.43 (m, 3H), 2.27 (s, 3H); C-NMR
3
(
1
CDCl + CD OD)
δ
168.7, 159.4, 153.7, 146.3, 137.1, 132.1 (2C), 129.3, 128.4 (2C), 124.1 (2C), 123.3, 121.4,
3
3
+
20.12, 118.6, 25.2; EIMS (m/z + ion mode): 405.32, [M + H] ; 405.16. Anal Calcd for C H F N OS: C,
17
10
6
2
5
0.51; H, 2.48; N, 6.92 found C, 50.44; H, 2.52; N, 6.82.
˝
1
N-(6-(4-Methoxyphenyl)benzo[d]thiazol-2-yl)acetamide (3d). m.p.: 218–220 C; H-NMR (CDCl + CD OD)
3
3
δ
9.41 (s, 1H), 8.01 (s, 1H), 7.66 (d, J = 8, 2H), 7.57–7.47 (m, 2H), 7.08 (d, J = 8, 2H), 3.57 (s, 3H), 1.91
(
s, 3H). 13C-NMR (CDCl + CD OD)
δ
168.2, 158.8, 151.3, 149.5, 145.3, 132.2, 131.8 (2C), 129.5, 123.7,
3
3
´
´
=
121.3, 116.6, 115.5 (2C), 49.2, 22.5; EIMS (m/z
´
ion mode): [M
´
H] = 297.25; [M
´
COCH3]
´
2
55.34; [M
´
OCH and COCH ] = 227.01; Anal Calcd for C H N O S : C, 64.42; H, 4.73; N, 9.38
3
3
16 14
2
2 2
found C, 64.51; H, 4.71; N, 9.42.
˝
1
N-(6-(5-Methylthiophen-2-yl)benzo[d]thiazol-2-yl)acetamide (3e). m.p.: 192–194 C; H-NMR (CDCl +
3
13
CD OD)
δ
9.41 (s, 1H), 7.90 (s, 1H), 7.67–7.35 (m, 4H), 2.34 (s, 3H), 2.11 (s, 3H); C-NMR (CDCl +
3
3
CD OD)
δ
166.2, 159.6, 148.7, 145.2, 132.4, 132.3, 129.5, 128.7, 128.3, 123.6, 121.8, 116.8, 22.7, 14.03; EIMS
3
´
´
´
CH3] = 271.02; Anal Calcd for
(m/z
´
ion mode): [M
´
H] = 286.93; [M
´
COCH3] = 224.93; [M
´
C H N OS : C, 58.32; H, 4.20; N, 9.72 found C, 58.41; H, 4.12; N, 9.79.
14
12
2
2
˝
1
N-(6-(3-Cyano-5-(trifluoromethyl)phenyl)benzo[d]thiazol-2-yl)acetamide (3f). m.p.: 212–213 C; H-NMR
(
CDCl + CD OD) δ 9.48 (s, 1H),7.92 (d, J = 1.2, 1H), 7.72–7.41 (m, 4H), 7.36 (s, 1H), 2.27 (s, 3H);
3 3
13
C-NMR (CDCl + CD OD)
23.3, 121.8, 120.3, 117.7, 114.8, 22.5; EIMS (m/z
δ
167.7, 158.8, 148.7, 146.4, 132.3, 132.0, 132.0, 131.8, 129.6, 128.4, 124.2,
3
3
´
1
´
ion mode): 360.33 [M
´
H] 360.01; Anal Calcd for
C H F N OS: C, 56.52; H, 2.78; N, 11.62 found C, 56.60; H, 2.77; N, 11.57.
17
11
3
3
˝
1
N-(6-(3-Chloro-4-fluorophenyl)benzo[d]thiazol-2-yl)acetamide (3g). m.p.: 166–167 C; H-NMR (CDCl +
3
13
CD OD)
δ
9.32 (s, 1H), 8.11 (d, J = 10 Hz, 1H), 7.90 (s, 1H), 7.80–7.41 (m, 4H), 2.30 (s, 3H); C-NMR
3
(
1
CDCl + CD OD) 168.5, 156.3, 151.2, 147.8, 137.3, 135.1, 131.0, 129.7, 129.6, 124,6, 124.0, 121.6, 117.5,
δ
3
3
+
+
16.3, 22.5; EIMS (m/z + ion mode): [M + H] = 321.16; [M
´
Cl and F-benzene and COCH ] = 150.07;
3
Anal Calcd for C H ClFN OS: C, 56.16; H, 3.13; N, 8.72 found C, 56.30; H, 3.15; N, 8.76.
15
12
2
˝
1
N-(6-(3,5-Dimethylphenyl)benzo[d]thiazol-2-yl)acetamide (3h). m.p. 205–207 C; H-NMR (acetone-d )
6
δ
9.41 (s, 1H),8.14 (d, J = 2 Hz, 1H), 7.63 (d, J = 1.5, 1H), 7.72–7.53 (m, 4H), 2.31 (s, 6H), 2.28 (s, 3H);
13
C-NMR (acetone-d6)
δ
167.2, 158.0, 147.7, 140.3 (2C), 133.0, 129.9, 129.5, 128.3, 127.4 (2C), 123.7, 121.3,
´
119.2, 23.1, 22.2 (2C); EIMS (m/z
´
ion mode): [M
´
H] = 294.91; [M
´
COCH3
´
2OCH and
3
´
´
benzene] = 149.24; [M
´
2OCH and benzene] = 190.6; Anal Calcd for C H N OS: C, 68.88; H,
3
17 16
2
5
.43; N, 9.44 found C, 68.83; H, 5.46; N, 9.34.
3
.3. Procedure for Urease Inhibition Activity
Urease inhibitory assay of newly synthesized compounds 3a–3h were determined as follows:
Enzyme (1 unit) in phosphate buffer (200
µ
L, pH 7) was combined with a particular stock solution
L). The solution was incubated
L of urea stock solution (20 mM) was added to the
solution. Calibration solution was synthesized without urea solution. For the action of urease, test
(
20 µL, a test compound or thiourea) and phosphate buffer (230
µ
˝
for 5 min at 25 C. After incubation period 400
µ
˝
tubes were incubated for 10 min at 40 C. The solution of phenol hypochlorite reagent (1150
µL) was
˝
added. These tubes were incubated for 25 min at 56 C. Absorbance of the blue colored compound

Molecules 2016, 21, 266
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was noted at 625 nm after 5 min of cooling. Then percentage urease inhibition was determined. While
EZ-fit kinetic data base was used to obtain IC50 values [25,35].
3
.4. Molecular Docking Study
The PDB structure of 3LA4 was retrieved for docking purposes as a complex co-crystallized with
inhibitor 2-amino-3-(2-(2-hydroxyethyl)disulfanyl)propan-1-ol and (5-amino-6-hydroxyhexyl)carbamic
acid at the nickel-containing catalytic site [36]. Then, the amino acid chain was retained and the
water molecules and co-crystallized ligands were removed and subsequently the missing atom types
were repaired using Modeller 9.11 (University of California San Francisco, San Francisco, CA, USA).
Afterwards, the polar hydrogen was added to the receptor and the resulting protein was subjected
to minimization using OPLS 2005 force field. The prepared protein was saved in pdbqt format
using Autodock Tools 1.5.4 [21
,37–39]. The ligand coordinates were generated using MarvineSketch
5
.8.3, 2012 (ChemAxon LLC, Cambridge, MA, USA) [40], which was converted to 3D structure using
Openbabel version (2.3.1). Finally the pdbqt formats (The input format of docking software) of the
ligands were prepared with Autodock Tools 1.5.4 using default parameters. AutodockVina ver. 1.1.1
(
The Scripps Research Institute, La Jolla CA, USA) was used for docking calculations with default
parameters except for exhaustiveness that was set to 80. For all the docking calculations, a grid box size
of 40 40 40, centered at the geometrical center of co-crystallized ligands was used. Co-crystalized
ˆ
ˆ
ligands were attached at two different sites, one near the nickel catalytic site (A) and the other site
where the inhibitor 2-amino-3-(2-(2-hydroxyethyl)disulfanyl)propan-1-ol was attached (Site B) [20].
The coordinates x, y, z for the center of grid box were (Site A)
´
39.86,
´
45.06, 72.52 and (Site B)
´
75.03,
2
0.84, 81.83 respectively. To validate our docking procedure, the co-crystallized ligands were re-docked
into their respective site of the enzyme and the reasonable RMSD value of 1.947 A was obtained.
Finally, the conformations with the most favorable free energy of binding were selected for analyzing
the interactions between urease and its inhibitor. All of the 3D models are generated using the Molegro
Molecular Viewer 2.5 (CLC bio company, Aarhus N, Denmark) [41] and LigPlot + (The European
Bioinformatics Institute, Hinxton, Cambridge, UK) [42] software.
3
.5. Nitric Oxide Scavenging Activity
The activity of newly synthesized benzothiazole derivatives was determined using the Garrat
method. Griess reported the Garrat method which is followed by a diazotization reaction [43]. Under
acidic conditions, this method utilizes sodium nitroprusside as the source of nitric oxide, sulfanilamide
´
and N-1-naphthylethylenediamine dihydrochloride to detect NO2 , produced at the expense of nitric
oxide. A known amount of tested compounds 3a 3h was dissolved in sodium nitroprusside solution
20 mM, 100 L) and then the volume was made up to 1000 L with phosphate buffer (200 mM, pH 7.4).
This solution was incubated for 2 h at 37 C and Griess reagent (100 L) was added. This solution
–
(
µ
µ
˝
µ
was stored for 20 min at room temperature. At 528 nm, optical density of this colored solution was
observed. For positive control, ascorbic acid was used. Negative control was used to form the standard
curve [43].
3
.6. Haemolytic Activity
Haemolytic activity of newly synthesized benzothiazole derivatives 3a–3h was determined using
a reported method [44]. Solutions of compounds were prepared at concentrations of 1 mg/mL in 10%
DMSO with 90% water Heparinized human fresh blood (3 mL) was used that was homogeneously
mixed and added into a 15 mL sterile Falcon tube. It was centrifuged for 5 min and the supernatant was
˝
removed. Chilled sterile isotonic phosphate buffer saline solution (5 mL, 7.4 pH) at 4 C was used three
times. Washed red blood cells were suspended in chilled RBS (20 mL). A haemacytometer was used
8
for counting erythrocytes. For each assay 7.068
and then diluted blood cells (180
Eppendorf tubes. It was incubated for 35 min at 37 C then the tubes were kept in an ice bath for 5 min
ˆ
10 red blood cells per mL count were maintained
µ
L) were added to the test compound (20 µL) and suspended in
˝

Molecules 2016, 21, 266
14 of 17
and centrifuged again for 5 min. After centrifugation, the obtained supernatant was collected carefully
and diluted with 900 L of chilled PBS. All these tubes were kept in ice bath and solution (200 L) was
µ
µ
added into 96 well plates from each Eppendrof tube. For each essay, Triton X-100 (0.1%) was taken as
positive control. For negative control, phosphate buffer was used. A microplate reader was used for
determining the absorbance at 576 nm [32].
3
.7. Antibacterial Activity
These newly benzothiazole derivatives were tested for their antibacterial activities against two
Gram positive strains (Baccilus subtilis, Staphylococcus aureus) and four Gram negative strains (Escherichia
coli, Psuedomonas aeruginosa, Shigella dysenteriae and Salmonella typhae) using a reported protocol [45].
Streptomycin was used as positive control. The 96 well plate method was optimized for measuring
the antibacterial activities of these compounds. In each well, sterilized broth (175
and inoculated with glycerol stock (5 L) of a specific bacterial strain. The initial absorbance was
observed between 0.12–0.19. The bacteria were allowed to grow in an incubator overnight. After a
certain waiting time (12 h), test sample (20 L) was added to the pre-determined wells. Concentration
of test sample was 20 g/well. Total volume was 200 L in each well. These plates were incubated for
6–24 h at 37 C. Absorbance was observed at 630 nm by using an ELISA plate reader. The difference
µL) was added
µ
µ
µ
µ
˝
1
in absorbance values were observed and were used as an index of bacterial growth. The following
formula was used to calculate percentage inhibition:
Percentage Inhibition “ pO.D of ` ve control ´ O.D of sample ˆ 100q{O.D of ` ve control (1)
4
. Conclusions
This study reports C-C coupling reactions of
acids using palladium catalyst. These new products 3a
2
with various arylboronic pinacol ester/aryl boronic
3h were prepared in moderate to good yields.
–
These Suzuki coupling benzothiazole derivatives were checked for their biological (urease inhibitory,
nitric oxide scavenging, haemolytic and antibacterial) activities. The urease inhibition results showed
that product 3b was an excellent urease inhibitor. Products with electron releasing groups on the
aryl moiety of the benzothiazole molecule showed the highest inhibition of urease activity. Molecular
docking studies of the urease inhibitory activity showed that the H-bonding ability present in these
N-protected benzothiazoles prevents the catalytic activity of the enzyme. Nitric oxide scavenging
assays were done for these compounds. Compound 3b also exhibited highest nitric oxide scavenging
activity. All newly synthesized compounds showed haemolytic activity. It was found that electron
withdrawing substitution on the aryl produced the highest haemolytic activity. The newly synthesized
benzothiazole derivatives 3a–3h showed excellent antibacterial activities against E. coli.
Acknowledgments: This study reported herein is part of Ph.D. thesis work of Yasmeen Gull. Financial assistance
for this study was provided by the Higher Education Commission (HEC), Pakistan and HEC Scholarship awarded
to Ms. Yasmeen Gull.
Author Contributions: Conceived and designed the experiments: Y.G., N.R., M.N., A.A.A. Performed
the experiments: (Synthesis and bioassay—Y.G., M.N.); (Enzyme inhibition—A.Y.); (Docking and other
computational—A.A.A.). Analyzed the data: Y.G., A.A.A., M.N., M.Z., S.G.M. Contributed to manuscript
preparation N.R., F.-H.N., M.Z., V.D.F.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all the newly synthesized compounds are available from the authors.
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(