Efficient synthesis of 2-amino-6-arylbenzothiazoles via Pd(0) Suzuki cross coupling reactions: Potent urease enzyme inhibition and nitric oxide scavenging activities of the products

Article abstract of DOI:10.3390/molecules18088845

In general, benzothiazole derivatives have attracted great interest due to their pharmaceutical and biological importance. New 2-amino-6-arylbenzothiazoles were synthesized in moderate to excellent yields via Suzuki cross coupling reactions using various aryl boronic acids and aryl boronic acid pinacol esters and the antiurease and nitric oxide (NO) scavenging activity of the products were also examined. The most active compound concerning urease enzyme inhibition was 6-phenylbenzo[d]thiazole-2-amine 3e, with an IC₅₀ value of 26.35 μg/mL. Compound 3c, 6-(4-methoxyphenyl) benzo[d]thiazole-2-amine, exhibited the highest nitric oxide percentage scavenging at 100 μg/mL.

Full text of DOI:10.3390/molecules18088845

Molecules 2013, 18, 8845-8857; doi:10.3390/molecules18088845
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Efficient Synthesis of 2-Amino-6-Arylbenzothiazoles via
Pd(0) Suzuki Cross Coupling Reactions: Potent Urease
Enzyme Inhibition and Nitric Oxide Scavenging Activities
of the Products
Yasmeen Gull 1, Nasir Rasool 1,*, Mnaza Noreen 1, Faiz-ul-Hassan Nasim 2, Asma Yaqoob 2,
Shazia Kousar 1, Umer Rashid 3,*, Iftikhar Hussain Bukhari 1, Muhammad Zubair 1 and
Md. Saiful Islam 4
1
Department of Chemistry, Governament College University, Faisalabad 38000, Pakistan;
E-Mails: yasmeenchem1@yahoo.com (Y.G.); mnazanoreen@yahoo.com (M.N.);
shazia_hej@hotmail.com (S.K.); pdiftikhar@yahoo.com (I.H.B); zubairmkn@yahoo.com (M.Z.)
2
Department of Chemistry, the Islamia University of Bahawalpur, Bahawalpur 63000, Pakistan;
E-Mail: faiznasim@hotmail.com (F.N.); asmayaqoobctn@gmail.com (A.Y.)
3
Institute of Advanced Technology, Universiti Putra Malaysia, UPM Serdang 43400,
Selangor, Malaysia
4
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, UPM Serdang 43400,
Selangor, Malaysia; E-Mail: mdsaiful@science.upm.edu.my
* Authors to whom correspondence should be addressed; E-Mails: nasirhej@yahoo.co.uk (N.R.);
umer.rashid@yahoo.com (U.R.); Tel.: +92-332-749-1790 (N.R.); +60-38-946-7393 (U.R.);
Fax: +92-41-920-1032 (N.R.); +60-38-946-7004 (U.R.).
Received: 21 May 2013; in revised form: 24 June 2013 / Accepted: 9 July 2013 /
Published: 25 July 2013
Abstract: In general, benzothiazole derivatives have attracted great interest due to their
pharmaceutical and biological importance. New 2-amino-6-arylbenzothiazoles were
synthesized in moderate to excellent yields via Suzuki cross coupling reactions using
various aryl boronic acids and aryl boronic acid pinacol esters and the antiurease and nitric
oxide (NO) scavenging activity of the products were also examined. The most active
compound concerning urease enzyme inhibition was 6-phenylbenzo[d]thiazole-2-amine 3e,
with an IC50 value of 26.35 µg/mL. Compound 3c, 6-(4-methoxyphenyl) benzo[d]thiazole-
2-amine, exhibited the highest nitric oxide percentage scavenging at 100 µg/mL.

Molecules 2013, 18
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Keywords: Suzuki cross coupling; benzothiazoles; urease activity; nitric oxide
scavenging activity
1. Introduction
Nowadays the benzothiazole moiety has great scientific importance as it is a weak base heterocyclic
compound having numerous biological activities such as anti-inflammatory [1], analgesics [2],
antitumor [3], antibacterial [4], antihistamines [5], suchistosomicidal [6] and antivirus properties [7].
Benzothiazoles are mostly found in medicinal and bio-organic chemistry, with multiple applications in
drug discovery [8]. 2-Aminobenzothiazoles are used as reaction intermediates and reactants since the
endocyclic N functions and NH2 are ideally situated to enable reactions with common bis-electrophilic
reagents to make different types of fused heterocyclic compounds [9]. Priyanka and co-workers have
recently reported the antimicrobial activity of benzothiazoles and their derivatives against Gram
positive and negative bacteria. A number of 2-aminobenzothiazoles have intensely investigated as
central muscle relaxant [10]. A series of N-bis-benzothiazole derivatives have been synthesized by
Kumbhare and tested for cytotoxicity studies against a mouse melanama cell line (B16-F10) and
human monocyte cell line (U937,THP-1) [11]. Some 2-aminobenzothiazole derivatives have
cytotoxicities towards tumor cells which are comparable to that of cisplatin [12].
Vicendo and coworkers have synthesized new class of thiol and aminothiol compounds derived
from benzothiazole and thiadiazole and checked their ability to scavenge free radical (ABTS.+, .OH,
DPPH.). Thiol derivatives with a thiadiazole structure are the most active compounds in scavenging
ABTS.+ and DPPH free radicals. On the basis of QSAR and DFT studies of thiazole and benzothiazole
molecules, it has been observed that these compounds are potential antioxidant agents and free radical
scavengers [13]. Amtul et al reported that urease enzyme inhibition is an important area of drug
research that has led to the discovery of vast variety of drugs that useful in a number of diseases.
Urease inhibitors have been considered as goals for new antiulcer drugs [14]. Moreover bacterial
urease has been shown to be an important toxic determinant in the pathogenesis of many clinical
conditions in animals and humans. Urease is responsible for development of kidney stones and
contributes to the pathogenesis of pyelonephritis, urolithiasis, hepatic and ammonia encephalopathy,
urinary catheter encrustation and hepatic coma. Urea is the major reason of pathologies induced by
Helicobacter pylori (HP), which permits HP to survive during colonization at the low pH of the
stomach [15].
The Suzuki cross coupling reaction has developed as a powerful method in organic synthesis [16].
The main advantage of choosing Suzuki cross coupling reactions as compared to other coupling
methods [17] is the availability of different aryl boronic acids that are environmentally safer and the
milder reaction conditions than those needed for other organometallic reagents [18]. For the synthesis
of biaryl systems the palladium-catalysed Suzuki cross coupling reaction represents one of the best
methods [19]. Piscitelli and coworker reported the treatment of 4-substituated anilines with potassium
thiocyanate in the presence of bromine in acetic acid for the synthesis of 6-substituted 2-amino-
benzothiazoles and further their Suzuki cross coupling reactions in the presence of Pd(PPh3)4 under

Molecules 2013, 18
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microwave conditions to yield phenyl-substituted 2-aminobenzothiazoles [20]. After reviewing the
literature it has been found that oxadiazoles/thiadiazoles and triazoles class of compounds are the
novel competitive inhibitors of urease enzyme [21]. Similarly thiazole compounds were found to
present moderate to potent activity in a NO scavenging protocol [22]. The main focus of the present
research work is directed to the novel synthesis of 2-amino-6-arylbenzothiazoles by utilizing the basic
Suzuki cross coupling chemistry and to investigate the antiurease, NO activity of the products on the
basis of previously studied hetroaryl molecules as strong urease inhibitors and NO scavengers.
2. Results and Discussion
2.1. Chemistry
Majo and coworkers have been reported Suzuki cross coupling reactions of 2-bromobenzothiazole
with aryl boronic acids to provide the corresponding 2-arylbenzothiazole compounds [23]. We have
investigated the Suzuki cross coupling reactions of 2-amino-6-bromobenzothiazole (2) with various
aryl boronic acids and esters under optimized heating conditions. There is a very little work on the
synthesis of 2-amino-6-aryl benzothiazoles 3a–e so far and limited information has been published on
the urease enzyme inhibition and NO scavenging activities of 2-amino-6-arylbenzothiazole
derivatives. 2-Amino-6-bromobenzothiazole (2) was prepared stirring by 4-bromoaniline with acetic
acid (0.011 mol) and potassium thiocynate (0.044 mol) in solution of bromine (0.011 mol) in AcOH
(7 mL) at room temperature (Scheme 1) [24].
Scheme 1. Synthesis of Benzothiazoles 2, 4, 3a–e.
O
N
NH
S
Br
NH2
(ii) 4
N
S
AcOH/Br2
KSCN
NH2
Br
Br
(i) 2
N
S
1
NH2
Ar
3a-3e
(iii)
(1) Synthesis of 2-amino-6-bromobenzothiazole (2), Synthesis of N-(6-bromobenzo[d]thiazol-2-yl)acetamide
(4) and 2-amino-6-arylbenzothiazole (3a–e). Reagents and conditions: (i): 1 (0.011 mol), acetic acid
(0.011 mol), potassium thiocynate (0.044 mol), bromine (0.011 mol); (ii) 2 (0.002 mol), acetic anhydride
(0.0031 mol), acetonitrile (5 ml); (iii) 2 (2.183 mmol), aryl boronic acids and aryl boronic acid pinacol esters
(2.401 mmol), K3PO4 (4.3 mmol), Pd(PPh3)4 (5 mol %), solvent / H2O (4:1), (see Table 1), 95 °C, 31 h.
Firstly, we studied the optimization of the reaction solvent. Thus, the effect of solvents on the
coupling reactions between aryl boronic acids/esters and 2-amino-6-bromobenzothiazole (2) under
thermal conditions at 90–100 °C was evaluated as shown in Scheme 1. We next focused our attention
on the Suzuki cross coupling reaction of several varieties of aryl boronic acids/esters with 2-amino-6-

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bromobenzothiazole (2). Results of the Suzuki cross coupling reactions under optimized condition are
summarized in Table 1.
When 2-amino-6-bromobenzothiazole (2) (2.183 mmol) was coupled with tolyl boronic acids
(2.401 mmol) using a 4:1 toluene/water ratio in the presence of 5 mol% Pd(0) catalyst using K3PO4
(4.366 mmol) as base, product 3a was obtained with moderate yield. However, when 1,4 dioxane was
used in the the reaction solvent, 6-tolybenzo-[d]thiazole-2-amine (3a) was obtained after 31 h heating
(Table the Suzuki cross coupling of 2-amino-6-bromobenzothiazole (2) with 4-methoxyphenyl boronic
acid (4.366 mmol) in refluxing with DMF using 5 mol% of Pd(0) catalyst this resulted in the
conversion into 6-(4methoxyphenyl)benzo[d]thiazol-2-amine (3c) in 64% isolated yield) (Scheme 1,
Table 1).
Table 1. Synthesis of 2-amino-6-arylbenzothiazoles.
Aryl Boronic
Solvent/H2O
(4:1)
Yield%
[a]
Entry
Acid/Aryl Boronic
Pinacol Ester
Product
1
Toluene
65
H3C
(HO)2B
CH3
N
S
NH2
3a
2
Dioxane
75
(HO)2B
CH3
H3C
N
S
NH2
3a
3b
3c
3c
3
4
5
6
Toluene
DMF
61
64
71
67
(HO)2B
Cl
Cl
N
S
NH2
H3CO
H3CO
(HO)2B
(HO)2B
H3C
OCH3
OCH3
N
N
S
S
NH2
Dioxane
Dioxane
NH2
CH3CH3
CH3
F3C
F3C
O
O
N
B
S
NH2
3d
F3C
H3C
CF3
CH3 CH3
CH3
7
Dioxane
78
N
O
O
B
S
NH2
3e
[a] isolated yield Conditions: (95 °C, 31 h).

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Moreover, 6-bromo-2-amino benzothiazole (2) was smoothly coupled with 1,3-trifluorobenzene
boronic ester in 1,4-dioxane under heating conditions with conversion into the corresponding 6-(3,5
bis(triflouromethyl)phenyl)benzo[d]thiazol-2-amine (3d) in high yield (Table 1).
Another reaction was also carried at the same time out to obtain N-(6-bromobenzo[d]thiazol-2-yl)
acetamide (4) in excellent 85% isolated yield. The reaction was carried out by the acylation of
2-amino-6-bromobenzothiazole (2) using a previously reported method [25] Further Suzuki cross
coupling reactions of acylated 6-bromo-2-aminobenzothiazole 2 are currentlybeing studied.
2.2. Pharmacology
2.2.1. Antiurease Activity
Hetroaryl (thiazole, triazole) pharmacophores exhibit excellent inhibitory action against bacterial as
well as plant urease. Pentacyclic hetroaryl oxygen, sulphur, and nitrogen (OSN) molecules exhibit
variable antiurease activity and these derivatives are planned to have these structural features: (i) a
primary amine group which should compete with substrate at the acyl enzyme intermediate by means
of nucleophilic substitutions during reactions (ii) a hydrophilic C- terminal to promote better diffusion
into cells and to increaes affinity of inhibitor for enzyme (iii) an oxide or azole (thiazole, imidazole,
pyrazole, oxazole and isoxazole) or sulfide group to enhance solubility in isotonic media [21].
The newly synthesized compounds 3a–e were examined for their urease inhibition activities in vitro
at 25 and 50 μg/mL concentrations. It is supposed that all these synthesized compounds have the
ability to bind with the enzyme active site. Thus, enzyme stops catalyzing hydrolysis and the activity
of the enzyme is inhibited. Thiourea was used as standard inhibitor, having a percentage inhibition
activity of 77 ± 0.01 with an IC50 value of 34.65 µg/mL and the percentage urease inhibition activity was
calculated according to literature protocols [26,27]. The results are presented in Table 2 (Figures 1 and 2).
Table 2. Urease inhibition studies of benzothiazoles.
Percentage inhibition at
25 µg/mL
Percentage inhibition at
50 µg/mL
Compound
IC50 (µg/mL)
2
43 ± 0.0034
39 ± 0.001
95 ± 0.005
28.4
30.5
4
3a
89 ± 0.009
46 ± 0.005
90.49 ± 0.002
88.19 ± 0.007
86.24 ± 0.001
88.6 ± 0.002
85 ± 0.002
27.27
30.6
3b
39 ± 0.0012
44 ± 0.005
3c
28.57
28.88
26.35
34.65
3d
43 ± 0.007
3e
48 ± 0.005
Standard
33 ± 0.04
77 ± 0.01
Values are mean ± SD of three parallel measurements.
All synthesized compounds showed very good to excellent urease inhibition activity. The
compounds 2-amino-6-bromobenzothiazole (2) and 6-p-tolylbenzo[d]thiazol-2-amine (3a) proved to
be the most potent urease inhibitors, with activities of 95 ± 0.005 and 90.49 ± 0.002 at 50 μg/mL with
IC50 values of 28.4 and 27.27, respectively.

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8850
N-(6-bromobenzo[d]thiazol-2-yl)acetamide (4) also showed comparatively good activity, with a
percentage inhibition of 39 ± 0.001 at 25 μg/mL and 89 ± 0.009 at 50 μg/mL with an IC50 value of
30.5. Munakata et al. showed that carbon chain and branch chain structure of the aryl residues
apparently have no effect on activity. The additional fall in inhibitory potency may be attributed to less
interaction of the surrounding residues of urease active site with the non- aromatic cyclohexane
functional group [28].
Figure 1. The urease inhibition activity graph.
25ug/mL
50ug/mL
100
80
60
40
20
0
2
4
3a
3b
3c
3d
3e
Standard
Compounds
Figure 2. IC50 values of anti-urease activity.
40
35
30
25
20
15
10
5
0
Compounds
Compounds 3a,b,d exhibited significant urease inhibition activities (90.49 ± 0.002, 88.19 ± 0.007,
88.60 ± 0.002) with IC50 values ranging between 27.27, 30.6 and 28.88, respectively. Kobashi and
coworkers reported [29] that a terminal hydroxamic acid functionality (O=C-NHOH) was evaluated
against microbial and plant source urease. Fishbein and Carbone concluded that inhibitory potential of
compounds was reduced with a decrease in alkyl chain length [30].
Urease inhibitions of 86.24 ± 0.001 and 85 ± 0.002 percent were seen at 50 μg/mL for
6-(4-methoxyphenyl)benzo[d]thiazol-2-amine (3c) and 6-phenylbenzo[d]thiazol-2-amine (3e). The
IC50 values were 28.57 and 26.35, respectively. Contribution of various functional groups present on
2-amino-6-bromobenzothiazole (2) in urease inhibition was also investigated. In general, compounds

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containing electron-donating functional groups exhibited high anti-urease activity. 2-Amino-6-
bromobenzothiazole (2) and compound 3a containing an electron-donating methyl functional group
exhibited high urease activity, whereas compound 3c containing methoxy functional group also
showed good uease anti-urease activity. These observations are in agreement with the expected results.
Since urease needs Ni ions as a cofactor, compounds containing electron donating groups would be
expected, though neighbouring group participation to facilitate substituent amino or imino groups to
chelate these ions thereby inactivating the urease enzyme.
2.2.2. Nitric Acid (NO) Scavenging Percentage (Antioxidant Activity)
Nitric acid (NO) is a free radical causing cancer [31] and inflammatory disorders [32] in the human
body. As reported in previous literature, 6-flourobenzothiazole-substituated triazole compounds
showed excellent antioxidant activity when measured by the DPPH method [33]. Similarly pyrozoline
rings along with benzothiazole molecules exhibited very good antioxidant activity and phenyl ring
present on pyrozoline enhanced the antioxidant activity against DPPH and in the ferric ion reduction
method [34].
The newly synthesized compounds showed very good to excellent ability to scavenge NO
radical [35,36]. Ascorbic acid was taken as standard with 40.5 ± 0.01 percent NO scavenging at
25 μg/mL, 70.50 ± 0.91 at 50 μg/mL and 99.5 ± 0.91 at 100 μg/mL. NO scavenging percentage
activity was studied at 25, 50 and 100 μg/mL concentration, respectively. NO scavenging activities of
our synthetic compounds and their corresponding IC50 values are shown in Table 3 (Figures 3 and 4).
Table 3. Antioxidant activity of synthesized compounds by nitric oxide scavenging activity.
Nitric Oxide
Percentage
Scavenging Activity
at 25 µg/mL
20 ± 0.004
Nitric Oxide
Percentage
Scavenging Activity
at 50 µg/mL
48 ± 0.0007
54 ± 0.002
Nitric Oxide
Percentage
IC50
Compound
Scavenging Activity (µg/mL)
at 100 µg/mL
1
2
2
90 ± 0.0007
92 ± 0.002
95 ± 0.001
66 ± 0.005
98 ± 0.002
63 ± 0.009
52 ± 0.001
99.5 ± 0.91
57.8
46.5
4
25 ± 0.001
3
3a
3b
3c
3d
3e
24 ± 0.003
56 ± 0.001
68.3
4
5
6
7
17 ± 0.006
34 ± 0.005
75
31 ± 0.022
67 ± 0.002
38.19
83.75
96.66
66.66
10 ± 0.01
23 ± 0.009
9 ± 0.007
22 ± 0.001
Standard
40.5 ± 0.01
70.5 ± 0.91
Values are mean ± SD of three parallel measurements.
The compounds 6-(4-methoxyphenyl)benzo[d]thiazol-2-amine (3c) and 6-p-tolylbenzo[d]thiazol-2-
amine (3a) were found to be the most potent NO scavengers with percentage activities of 67 ± 0.002
and 67 ± 0.009 at 50 μg/mL with IC50 values of 68.3 and 38.19 μg/mL each, respectively. N-(6-
bromobenzo[d]thiazol-2-yl) acetamide (4) also exhibited comparatively good activity showing a
percentage NO scavenging of 54 ± 0.002 at 50 μg/mL and 92 ± 0.002 at 100 μg/mL, with an IC50 of
46.5. Compounds 3a, 3c, 4 presented significant percentages of NO scavenging (56 ± 0.001, 67 ± 0.002,
54 ± 0.002 at 50 μg/mL) and IC50 values were 68.3, 38.9 and 46.5, respectively. 2-Amino-6-

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bromobenzothiazole (2) also exhibited good NO scavenging activity, 48 ± 0.0007 at 50 μg/mL and
90 ± 0.007 at 100 μg/mL with an IC50 of 57.8. Moderate NO scavenging percentages of 34 ± 0.005,
23 ± 0.009 and 22 ± 0.001 at 50 μg/mL were observed for 6-(4-chlorophenyl)benzo[d]thiazol-2-amine
(3b), 6-(3,5-bis(triflouromethyl)phenyl)benzo[d]thiazol-2-amine (3d) and 6-phenylbenzo[d]thiazol-2-
amine (3e) with IC50 values of 75, 83.75 and 96.66, respectively.
Figure 3. Percent nitric oxide scavenging graph.
25ug/mL
50ug/mL
100ug/mL
120
100
80
60
40
20
0
2
4
3a
3b
3c
3d
3e Standard
Compounds
Figure 4. Nitric oxide scavenging activity IC50 values of compounds 2,3a–e,4.
120
100
80
60
40
20
0
Compounds
3. Experimental
3.1. General
Melting points were determined using a Buchi Melting Point B-540 apparatus. All reagents were
purchased from Alfa Aesar and Sigma Aldrich company. 1H-NMR and C-NMR spectra was measured
in CDCl3 and CD3OD on Bruker Aspect AM-400 at 400 MHz.The chemical shift was given in δ in

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ppm and coupling constant in Hz. EI-MS spectras were recorded on a JMS-HX-110 spectrometer, with
a data system. For column chromatography technique, silica gel (70–230 mesh) and silica gel
(230–400 mesh) were used. The reaction were monitored on TLC, using Merk Silica gel 60PF254 cards.
The compound were visualized by UV lamp (254, 365).
3.2. General Procedure for the Synthesis of 2-Amino-6-arylbenzothiazoles
For the synthesis of 2-amino-6-arylbenzothiazole 3a–e, to 2-amino-6-bromobenzothiazole (2,
2.183 mmol), under a nitrogen atmosphere Pd(PPh3)4 (5 mol%) was added and the resulting mixture
stirred for 30 min with the addition of the appropriate solvent (3 mL). After 30 min the aryl boronic
acid (2.401 mmol), and K3PO4 (4.366 mmol) were added, following the addition of water (1.5 mL). The
solution was stirred for 31 h at 95 °C, removed from heating and cooles to room temperature. After
work up with ethyl acetate, the organic layer was separated, dried with magnesium sulfate and the
solvent was removed under vacuum. The crude solid residue obtained was purified by column
chromatography using a 50% ratio of ethyl-acetate and n-hexane to give the desired products, which
were further analyzed by using different spectroscopic techniques [37].
3.3. Preparation of N-(6-bromobenzo[d]thiazol-2-yl)acetamide (4)
Under a nitrogen atmosphere a solution of 2-amino-6-brombromobenzothiazole (2, 0.00213 mol),
acetonitrile (5 mL), and acetic anhydride (0.003 mol) was stirred at 60 °C for 40 min with the addition
of few drops of conc. H2SO4, After 40 min distilled water (15–20 mL) was added to form a precipitate
and stirring was continued for one hour at room temperature. The solution was filtered and washed
with water and the product further analyzed by spectroscopic techniques [25].
3.4. Characterization Data
1
6-Bromobenzo[d]thiazole-2-amine (2). M.p. 215–217 °C; H-NMR (400 MHz, CDCl3 + CD3OD):
13
δ = 7.80 (d, J = 1.8, 1H-Ar), 7.67–7.42 (m, 2H-Ar), 7.30 (s, 2H-NH2). C-NMR (100 MHz, CDCl3
+CD3OD): δ = 119.86, 120.35, 123.58, 124.45, 129.78, 154.0, 167.0; EIMS (m/z -ion mode): 228.92
[M−H]; [M-bromine and 2H fragment]− = 147.00. Anal. Calcd for C7H5 N2S: C, 36.70; H, 2.20; N,
12.24. Found: C, 36.63; H, 2.15; N, 12.20.
1
N-(6-Bromobenzo[d]thiazol-2-yl)acetamide (4). M.p. 193–195 °C; H-NMR (400 MHz, CDCl3 +
CD3OD): δ = 7.94 (d, J = 1.6, 1H-Ar), 7.61–7.53 (m, 2H-Ar), 12.20 (s, 1H-NH), 2.31 (s, 3H-CH3).
13C-NMR (100 MHz, CDCl3 + CD3OD): δ = 23.50, 117.12, 121.62, 124.17, 129.95, 146.22, 148.71,
159.20, 168.37; EIMS (m/z -ion mode): 271.08 [M−H−]; [M−Acetyl fragment]− = 229.00. Anal. Calcd
for C9H7Br N2OS: C, 39.88; H, 2.59; N, 10.34. Found: C, 39.56; H, 2.43; N, 10.29.
1
6-p-Tolylbenzo[d]thiazole-2-amine (3a). M.p. 184–186 °C; H-NMR (400 MHz, CDCl3 +CD3OD):
13
δ = 8.11 (d, J = 7.6, 2H-Ar), 7.7–7.30 (m, 6H-Ar), 7.25 (s, 2H-NH2). C-NMR (100 MHz, CDCl3 +
CD3OD): δ = 21.46, 116.01, 119.20, 128.57 (2C), 128.69, 132.09, 132.81 (2C), 134.0, 135.72, 140.81,
145.96, 167.85; EIMS (m/z -ion mode): 239.08 [M−H−]; [M-2H fragment]− = 237.08; [M-benzene-CH3

Molecules 2013, 18
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fragment]− = 149.17; [M−CH3 fragment and 2-H]− = 221.25. Anal. Calcd for C14H12 N2S: C, 69.97; H,
5.03; N, 11.66. Found: C, 69.75; H, 4.95; N, 11.46.
1
6-(4-Chlorophenyl)benzo[d]thiazole-2-amine (3b). M.P, 194–196 °C; H-NMR (400 MHz, CDCl3
+CD3OD): δ = 7.59 (d, J = 2, 1H-Ar), 7.43–7.26 (m, 6H-Ar). 13C-NMR (100 MHz, CDCl3 + CD3OD):
δ = 114.65, 119.27, 122.96, 123.42 (2C), 128.51, 128.91, 129.32 (2C), 132.03, 134.96, 150.02, 167.48;
EIMS (m/z -ion mode): 259.08 [M-H−]; [M-NH2 fragment]− = 243.00; [M-benzene and Cl-fragment]− =
149.08 Anal. Calcd for C13H9Cl N2S: C, 59.88; H, 3.48; N, 10.74. Found: C, 59.78; H, 3.33; N, 10.69.
1
6-(4-Methoxyphenyl)benzo[d]thiazole-2-amine (3c). M.p. 165–167 °C; H-NMR (400 MHz, CDCl3
+CD3OD): δ = 7.56–7.40 (m, 3H-Ar), 7.38 (d, J = 3.2, 1H-Ar), 7.25 (2H-NH2), 3.48 (S,3H-OCH3).
13C-NMR (100 MHz, CDCl3 + CD3OD): δ = 49.57, 114.51, 118.13, 122.96, 123.37 (2C), 129.24 (2C),
131.88, 132.28, 149.38, 167.73; EIMS (m/z -ion mode): 255.08 [M−H−]; [M−OCH3 fragment]− =
237.17; [M-CH3 and N fragment]− = 227.08; [M-NH fragment]− =242.0. Anal. Calcd for C14H12N2OS:
C, 65.60; H, 4.72; N, 10.93. Found: C, 65.54; H, 4.57; N, 10.79.
6-(3,5-bis(Triflouromethyl)phenyl)benzo[d]thiazole-2-amine (3d). M.p. 180–182 °C; 1H-NMR
(400 MHz, CDCl3 + CD3OD): δ = 8.22 (s, 1H-Ar), 8.08 (s, 2H-Ar), 7.86–7.55 (m, 3H-Ar), 7.26 (s,
13
2H-NH2). C-NMR (100 MHz, CDCl3 +CD3OD): δ = 116.6, 117.5, 122.6, 123.5, 124.72 (2C), 128.5,
129.73, 132.03 (2c), 133.5, 134.50, 152.02, 168.50; EIMS (m/z -ion mode): 361.08 [M−H−]; [M-NH2
fragment]− = 346.2; [M-2-aminobenzothiazole fragment]− = 214.11. Anal. Calcd for C15H8 F6N2S: C,
49.73; H, 2.23; N, 7.73. Found: C, 99.35; H, 2.19; N, 7.69.
6-Phenylbenzo[d]thiazole-2-amine (3e). M.p. 175–177 °C; 1H-NMR (400 MHz, CDCl3 +CD3OD): δ =
7.59 (d, J = 1.6, 1H-Ar), 7.57–7.32 (m, 7H-Ar), 7.27 (s,2H-NH2). 13C-NMR (100 MHz, CDCl3 +CD3OD):
δ = 115.01, 118.70, 123.57, 128.56, 128.68 (2C), 129.57 (2C), 131.91, 132.24, 132.01, 147.61, 167.85;
EIMS (m/z -ion mode): 225.08 [M−H−]; [M-benzene and 2 H fragment]− = 147.08; [M-benzene and
NH2 fragment]− = 135.17. Anal. Calcd for C13H10N2S: C, 69.43; H, 4.45; N, 12.38. Found: C, 69.39;
H, 4.43; N, 12.36.
3.5. NO Scavenging Activity
The antioxidant activity of 2-aminobenzothiazole and it derivatives was determined by assaying
Nitric oxide (NO) scavenging activity suing a test adopted from the Garrat method based on the
diazotization reaction described by Griess. The assay uses sodium nitroprusside as the source of NO
and sulfanilamide and N-1-naphthylethylenediamine dihydrochloride under acidic conditions to detect
−
NO2 generated at the expense of NO by the antioxidant system. Briefly, a known quantity of the test
compounds 2, 3a–e, 4 in solution form was mixed with 20 mM sodium nitroprusside solution
(100 µL). Total volume was made up to 1,000 µL with 200 mM Phosphate buffer, pH 7.4. The
contents were mixed well and incubated at 37 °C for 2 h followed by addition of Griess reagent
(100 µL). The mixture was kept at room temperature for 20 min. Optical density (OD) of the colored
solution formed was measured at 528 nm. Ascorbic acid was used as the positive control while the
negative control was used to develop the standard curve [36].

Molecules 2013, 18
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3.6. Antiurease Activity
The urease inhibitory activity of the synthesized compounds 2, 3a–e, 4 was determined as follows:
phosphate buffer (pH 7, 200 µL) containing 1 unit of enzyme was mixed with phosphate buffer
(230 µL) and of a particular stock solution (20 µL, thiourea or the test compound). The reaction
mixture was incubated at 25 °C for 5 min. After the incubation period urea stock solution (400 µL,
20 mM) was added. Calibration mixture was prepared with no urea solution. All test tubes were further
incubated at 40 °C for the action of urease for 10 min. This was followed by the addition of phenol
hypochlorite reagent (1150 µL). Tubes were again incubated at 56 °C for 25 min. After 5 min of
cooling absorbance of the blue coloured complex thus formed was noted at 625 nm and percentage
inhibition was calculated. The IC50 values were determined using the EZ-fit kinetic data base [26,27].
4. Conclusions
In summary we have reported an efficient synthesis of 2-amino-6-arylbenzothiazoles and
N-(6-bromobenzo[d]thiazol-2-yl)acetamide starting from 2-amino-6-bromobenzothiazole. A palladium
catalyst was used in Suzuki coupling reactions in the presence of different boronic acids/esters. The
results of this study reveal that 2-aminobenzothiazole derivatives can be used explored as anti-urease
and antioxidant molecules.
Acknowledgments
The financial support for this work was granted by HEC Pakistan (HEC Indigenous Scholarship for
Yasmeen Gull) and special thanks of HEJ Research Institute of Chemistry, Karachi University,
Pakistan for NMR techniques.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available.
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