Synthesis of some benzothiazoles by developing a new protocol using urea nitrate as a catalyst and their antimicrobial activities

Article abstract of DOI:10.1080/17415993.2017.1334781

The present communication demonstrates the development of urea nitrate as an effective and efficient catalyst for the synthesis of some 2-substituted benzothiazoles. Instant progress of reaction at room temperature under solvent-free condition, very high catalytic activity, inexpensive, clean reaction profile, operational simplicity, large-scale synthesis and appreciable yields are the main advantages of this protocol. These synthesized benzothiazoles have been evaluated for their antibacterial and antifungal activities against Gram-positive bacterium (Bacillus subtilis MTCC 121); two Gram-negative bacteria (Escherichia coli MTCC 1652 and Pseudomonas aeruginosa MTCC 741) and two fungi (Candida albicans MTCC 3017 and Saccharomyces cerevisiae MTCC 170). Compound 3n was found the most active against all the tested microbes.

Full text of DOI:10.1080/17415993.2017.1334781

Journal of Sulfur Chemistry
ISSN: 1741-5993 (Print) 1741-6000 (Online) Journal homepage: http://www.tandfonline.com/loi/gsrp20
Synthesis of some benzothiazoles by developing a
new protocol using urea nitrate as a catalyst and
their antimicrobial activities
Parvin Kumar , Rimpy Bhatia, Radhika Khanna, Aarti Dalal, Dinesh Kumar,
Parveen Surain & Ramesh C. Kamboj
To cite this article: Parvin Kumar , Rimpy Bhatia, Radhika Khanna, Aarti Dalal, Dinesh Kumar,
Parveen Surain & Ramesh C. Kamboj (2017): Synthesis of some benzothiazoles by developing
a new protocol using urea nitrate as a catalyst and their antimicrobial activities, Journal of Sulfur
Chemistry, DOI: 10.1080/17415993.2017.1334781
To link to this article: http://dx.doi.org/10.1080/17415993.2017.1334781
Published online: 07 Jun 2017.
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Date: 07 June 2017, At: 22:27

JOURNAL OF SULFUR CHEMISTRY, 2017
https://doi.org/10.1080/17415993.2017.1334781
SHORT COMMUNICATION
Synthesis of some benzothiazoles by developing a new
protocol using urea nitrate as a catalyst and their
antimicrobial activities
Parvin Kumar a, Rimpy Bhatiaa, Radhika Khannaa, Aarti Dalala, Dinesh Kumarb,
Parveen Surainc and Ramesh C. Kamboja
aDepartment of Chemistry, Kurukshetra University, Kurukshetra, Haryana, India; bDepartment of Chemistry,
Hindu College, University of Delhi, Delhi, India; cDepartment of Microbiology, Kurukshetra University,
Kurukshetra, Haryana, India
ABSTRACT
ARTICLE HISTORY
Received 4 March 2017
Accepted 21 May 2017
The present communication demonstrates the development of urea
nitrate as an effective and efficient catalyst for the synthesis of some
2-substituted benzothiazoles. Instant progress of reaction at room
temperature under solvent-free condition, very high catalytic activ-
ity, inexpensive, clean reaction profile, operational simplicity, large-
scalesynthesisandappreciableyieldsarethemainadvantagesofthis
protocol. These synthesized benzothiazoles have been evaluated
for their antibacterial and antifungal activities against Gram-positive
bacterium (Bacillus subtilis MTCC 121); two Gram-negative bacteria
(Escherichia coli MTCC 1652 and Pseudomonas aeruginosa MTCC 741)
and two fungi (Candidaalbicans MTCC 3017 and Saccharomycescere-
visiae MTCC 170). Compound 3n was found the most active against
all the tested microbes.
KEYWORDS
Benzothiazoles; urea nitrate;
antimicrobial activity;
solvent-free synthesis;
catalyst
1. Introduction
Benzothiazole is the important structural motif present in many molecules of medicinal
importance. The benzothiazole nucleus has gained tremendous attraction not only in the
CONTACT Parvin Kumar
parvinchem@kuk.ac.in
Department of Chemistry, Kurukshetra University,
Kurukshetra-136119, Haryana, India
© 2017 Informa UK Limited, trading as Taylor & Francis Group

2
P. KUMAR ET AL.
realm of pharmacology [1–15], but has also gained importance in textile dyeing [16], in
leather industry [17], as vulcanization accelerators [18], as chemiluminescent agents [19],
as dopant in light emitting electroluminescent device [20] and as photosensitizers [21].
A wide range of methods are available for synthesizing benzothiazole nucleus and its
derivatives which involve direct condensation of (i) 2-aminothiophenol with aryl alde-
hydes using MnO2/SiO2 [22], SiO2/MW [23], Bi(NO3)3 [24], HClO4 [25], H2O/Reflux
[26], CAN [27], Co(NO3)2 [28], p-TsOH/Graphite [29], I2/DMF [30], SiO2-H2SO4 [31,32]
(ii) 2-aminothiophenol with esters, acids and acid chlorides using AlMe3 [33], P2O5-
MeSO3H [34], Me3SiOH/SiO2 [35] and other methods using dimethylsulphoxide (DMSO)
[36], Pd(OAc)2/Cs2CO3 [37], I2/t-BuOK [38], CuI/1,10-Phen [39], CAN [40], DDQ [41],
Mn(OAc)3 [42], T3P/DMSO [43] and by cyclization of thiobenzanilides using potassium
ferricynide (Jacobson method) [44]. Most of these methods involve toxic organic solvents,
expensive catalyst, reduced yields, drastic reaction conditions like prolonged reaction time
and high reaction temperature, tedious and multistep procedure. In the framework of
our research program initiated in our laboratory [45] for the development of new meth-
ods, the synthesis of the benzothiazoles was carried out simply by mixing of various
aryl/heteroaryl/styryl/alkyl aldehydes and 2-aminothiophenol under clean reaction condi-
tions in the presence of catalytic amount of urea nitrate at room temperature. Urea nitrate
is an inexpensive catalyst, highly soluble, easy to prepare [46] and is known to possess high
efficiency in organic transformations [47–56] such as nitration, deprotection, Diels–Alder
reaction and oxidation.
2. Results and discussion
2.1. Chemistry
We started our investigation on 2-aminothiophenol 1 and 4-hydroxybenzaldehyde (2h)
in a 1:1 molar ratio as substrate with urea nitrate (1.5 wt% of aldehyde) (Entry 2, Table 1)
which offered the desired product simply on shaking for 2 min at room temperature. When
we used HNO3 as the catalyst (10 mol%) under parallel conditions, the reaction was abnor-
mally slow with lower yield (Entry 3; Table 1). Similarly, using urea alone also furnished the
target compound in low yield with longer reaction time (Entry 4, Table 1). Comparing the
results obtained by using urea nitrate as the catalyst with commercially available reagents
(Table 1), our method exhibited promising results in terms of both rate as well as the yield
of product formation (Scheme 1).
The catalytic efficiency of urea nitrate was further explored in terms of temperature,
catalyst ratio and solvent effect (Table 2). It is evident from the results given in Table 2
that urea nitrate under solvent-free conditions (1.5 wt% of aldehyde) at room temperature
proved to be a very effective and efficient catalyst for the synthesis of benzothiazole 3h.
The synthetic potential of urea nitrate was further established by the reaction of vari-
ously substituted (with electron releasing and withdrawing groups) aldehydes (Table 3). In
all cases, the transformation proceeded in high yields with no effect of substituent what-
soever. Furthermore, the reaction proceeded well and gave similar results with heteroaryl
(Table 3, Entries 19–22), cinnamyl and alkyl aldehydes (Table 3, Entries 1–3) (Scheme 2).
To express the practical application of this protocol on a large scale, we carried out reac-
tions of 2-aminothiophenol (1) with 4-hydroxybenzaldehyde, 4-methoxybenzaldehyde,

JOURNAL OF SULFUR CHEMISTRY
3
Table 1. Reaction condition.
Entry
Catalyst
Time (min)
% Yield of 3a
1
2
3
4
5
6
8
9
Without catalyst
NH2CONH2.HNO3
540
2
180
Overnight
55
120
180
30
300
14
90
76
60
75
79
75
83
67
b
c
Conc. HNO3
c
NH2CONH2
Co(NO3)2.6H2O [28]
Bi(NO3)2 [24]
Ni(NO3)2.6H2O [31,32]
CAN [31,32]
10
Conc. H2SO4 [31,32]
aIsolated product.
bReaction conditions: 1 (2 mmol), 2 h (2 mmol), catalyst (1.5 wt% to aldehyde 2 h).
cReaction conditions: 1 (2 mmol), 2 h (2 mmol), catalyst (10 wt% to aldehyde 2 h).
NH2
N
conditions
+
OHC
OH
OH
S
SH
3h
2h
1
Scheme 1. Reaction of 2-aminothiophenol with 4-hydroxybenzaldehyde.
Table 2. Effect of temperature, catalyst ratio and solvent on yield.
Catalyst (in wt%
of aldehyde)
Reaction time
(min)
Entry
1.
Solvent/temperature
Solvent free/0°C
% Yielda
0.5
1.0
1.5
2.0
0.5
1.0
1.5
2.0
0.5
1.0
1.5
2.0
0.5
1.0
1.5
2.0
0.5
1.0
1.5
2.0
0.5
1.0
1.5
2.0
110
90
60
60
30
15
2
2
240
200
180
180
30
20
13
10
15
10
2
2
240
200
180
150
80
86
88
76
82
85
90
78
70
75
82
78
78
80
85
75
80
82
83
78
75
79
82
70
2.
3.
4.
5.
6.
Solvent free/rt
Water/reflux
MeOH/rt
CH3CN/rt
CH2Cl2/rt
aThe products were isolated by column chromatography.
3,4,5-trimethoxybenzaldehyde, 4-fluorobenzaldehyde and 4-nitrobenzaldehyde individu-
ally on grams’ scale (20 mmol). The reactions proceeded smoothly as per expectations to
provide target compounds in high yield as obtained in the reaction on milligrams’ scale

4
P. KUMAR ET AL.
Table 3. Synthesis of 2-aryl/heteroaryl/styryl/alkylbenzothiazolesa.
Entry
Compound
R
%Yield
Mp °C (Lit. mp)
Reference
1
2
3
4
3a
3b
3c
3d
2-(1-phenylprop-1-en- 2yl)
87
89
86
90
72–73(70–72)
Oil
93–94 (92–94)
112–113 (112–113)
[45]
[23]
[26]
[27]
2-ethyl
2-styryl
5
6
7
3e
3f
89
86
88
84–85 (85–86)
[35]
[27]
[26]
H3C
118–119 (119–120)
136–137 (136–138)
H3CO
H3CO
3g
H3CO
H3CO
8
9
3h
3i
90
88
227–228 (228–229)
162–163 (161–163)
[35]
[34]
HO
HO
10
3j
86
129–130 (131)
[35]
OH
11
12
3k
3l
89
88
100–102 (101–102)
93–94 (92–94)
[35]
[45]
F
F
F
F
13
14
3m
3n
85
84
99–100 (100–102)
76–78
[45]
F
F
F
15
16
17
18
19
20
3o
3p
3q
3r
86
86
85
87
88
90
156–158 (159–160)
224–225 (224–225)
113–114 (114–115)
130–131 (131–132)
123–124 (124–125)
122–123 (123–124)
[38]
[27]
F3C
O2N
Cl
[35]
[35]
Br
3s
3t
[31,32]
[31,32]
N
N
21
22
3u
3v
89
86
132–133 (133–134)
97–98 (97–99)
[31,32]
[27]
N
S
aReaction conditions: 2-aminothiophenol (2 mmol), aldehyde (2 mmol), NH2CONH2 HNO3 catalyst (1.5 wt% to aldehydes).
.

JOURNAL OF SULFUR CHEMISTRY
5
.
Scheme 2. Synthesis of 2-substitued benzothiazoles 3(a–v) using catalyst NH2CONH2 HNO3.
Scheme 3. The proposed mechanism for the synthesis of benzothiazoles.
(Entries 6–8, 11 and 16, respectively, Table 3), which expressed and established the practical
utility of this method.
The synthesis of benzothiazoles have been envisioned to involve the initial condensation
of amino group (–NH2) of 2-aminothiophenol with aldehyde giving the Schiff base in situ
which on the intramolecular nucleophilic attack by the –SH forms benzothiazoline which
are then converted to the target compounds by aerial oxidation (Scheme 3).
2.2. Recovery and reuse of urea
One property of the catalyst is solubility with water. Thus, recovery of the urea is very
convenient. After reaction, the reaction mixture was poured directly into 10 ml ice cold
water. The reaction content was stirred for 30 min and the solid thus formed was filtered to
remove urea and nitric acid from reaction mass. Then water was evaporated under vacuum
to recover urea which was reused for making urea nitrate.

6
P. KUMAR ET AL.
2.3. Biological screening
All the synthesized benzothiazoles derivatives were screened for their in vitro antimicro-
bial (antibacterial and antifungal) activities. For testing minimum inhibitory concentra-
tion (MIC) values, modified agar well diffusion method was chosen [57–59]. Of all the
tested 22 compounds, many compounds revealed moderate to excellent activity against
gram-positive and gram-negative bacteria and fungi. As compared to reference drug
ciprofloxacin, compounds 3i (20.3 mm, MIC = 25 μg/ml) and 3j (20.6 mm, 25 μg/ml)
showed moderate and compound 3n (25.6 mm, MIC = 6.25 μg/ml) excellent activity
against Bacillus subtilis. The compounds 3a (21.0 mm, MIC = 12.5 μg/ml), 3c (20.6 mm.
MIC = 25 μg/ml) and 3n (20.6 mm, MIC = 12.5 μg/ml) showed moderate activity against
E. coli, and the compound 3a (20.6 mm, MIC = 25 μg/ml) showed moderate and com-
pounds 3n, 3s and 3t (34.6 mm, MIC = 3.125 μg/ml; 25.6 mm, MIC = 12.5 μg/ml;
27.6 mm, MIC = 12.5 μg/ml) showed excellent activity against Pseudomonas aeruginosa.
The compounds 3d (17.6 mm, MIC = 50 μg/ml) and 3j (18.6 mm, MIC = 25 μg/ml)
showed moderate and compounds 3i (19.3 mm, MIC = 25 μg/ml) and 3n (21.3 mm,
MIC = 12.5 μg/ml) showed excellent activity against Candida albicans. The Compounds
3e and 3n (20.6, MIC = 25 μg/ml and 25.6 mm, MIC = 12.5 μg/ml) showed excellent
activity against Saccharomyces cerevisiae as compared to standard amphotericin-B. Thus,
the compound 3n promisingly was found to be most effective against all the tested microbes
(Tables 4 and 5 and Figure 1).
Table 4. In vitro antimicrobial activity of synthesized benzothiazoles through agar well diffusion
method.
Diameter of growth of inhibition zone (mm)a
Compound
B. subtilis
E. coli
P. aeruginosa
S. cerevisiae
C. albicans
3a
3b
3c
3d
3e
3f
3g
18.0
11
13.6
10
11
11
21.0
16.6
20.6
11
11
10
20.6
13.6
15.6
10
11
11
11
10
11
17.6
11
11
9
11
17.3
20.6
15.3
10
11
10
10
10
10
3h
10
10
10
10
10
3i
3j
3k
3l
20.3
20.6
12.3
10
17.6
10
10
11
10
10
10
19.3
10
10
15.3
10
12.6
10
10
10
3m
3n
3o
15.6
25.6
10
11
20.6
10
11
34.6
10
12.6
21.3
10
11
25.6
10
3p
10
10
10
10
10
3q
10
10
10
10
10
3r
10
10
10
10
10
3s
3t
3u
3v
15.3
15.6
11
17.6
24.0
-
17.3
17.6
11
11
22.0
-
25.6
27.3
11
11
22.0
-
15.3
15.6
12.0
11
11
11
10
11
Ciprofloxacin
Amphotericin-B
-
-
19.3
16.6
aValues, including diameter of the well (8 mm), are means of three replicates.

JOURNAL OF SULFUR CHEMISTRY
7
Table 5. MIC (in μg/ml) of compounds by using modified agar well diffusion method.
Compound No.
B. subtilis
E. coli
P. aeruginosa
S. cerevisiae
C. albicans
3a
3b
3c
3d
3e
3f
3i
3j
3k
3m
3n
3s
3t
3u
3v
25
200
100
200
200
200
25
25
100
50
6.25
50
25
200
50
6.25
Nt
12.5
50
25
25
50
50
200
200
200
50
200
200
25
200
200
200
50
25
50
200
200
200
50
200
200
200
12.5
50
200
200
200
200
200
200
100
3.125
12.5
12.5
200
200
12.5
Nt
50
50
25
200
200
12.5
50
100
200
12.5
200
200
200
200
Nt
50
25
200
200
6.25
Nt
200
200
Nt
Ciprofloxacin
Amphotericin-B
12.5
12.5
Note: Nt means not tested.
Figure 1. Representation of benzothiazoles with zone of inhibition (mm).
3. Conclusion
A simple, cleaner, mild, robust, cheaper and practical method has been developed to obtain
a wide range of benzothiazoles in appreciable yields. Also, high efficiency and easy prepa-
ration of the catalyst are the salient features of the present methodology. All the synthesized
benzothiazoles have been evaluated for their antibacterial and antifungal activities against
Gram-positive bacterium (B. subtilis MTCC 121); two Gram-negative bacteria (E. coli
MTCC 1652 and P. aeruginosa MTCC 741) and two fungi (C. albicans MTCC 3017 and
S. cerevisiae MTCC 170) and compound 3n was found most active against these microbes.

8
P. KUMAR ET AL.
4. Experimental design
4.1. General
All the chemicals used in the experiments were of highest purity available commercial
products. Melting points reports are uncorrected and determined in open capillaries. The
1H NMR spectra were recorded on 400 and 300 MHz using TMS as an internal standard. IR
spectra were recorded on a MB3000 FT-IR spectrophotometer. For TLC analysis, the plates
were coated with silica gel G (suspended in pet. ether-ethyl acetate) and iodine vapors were
used as visualizing agent.
4.2. Preparation of catalyst
In a conical flask, minimum amount of conc. HNO3 was added to 5 g urea so that urea
dissolved completely on heating. The solution was allowed to stand undisturbed. Crystals
of urea nitrate separated out. The crystals were filtered and stored in air tight containers.
4.3. Synthesis of benzothiazoles
2-Aminothiophenol 1 (2.0 mmol) and carbaldehyde 2 (2.0 mmol) were mixed in a conical
.
flask and NH2CONH2 HNO3 (1.5 wt% of aldehydes) was added to it. This mixture was
shaken at room temperature. An instant exothermic reaction occurred with completion
of reaction (TLC) and gave yellowish orange solid that was directly chromatographed over
silica gel column to afford pure 2-aryl/heteroaryl/styryl/alkylbenzothiazole3 (Table 3). The
structures of synthesized compounds were confirmed by the overlapping 1H, 13C NMR and
melting points data. The spectral data of some of the synthesised compounds are given
below.
4.3.1. 2-(1-Phenylprop-1-en- 2yl)benzothiazole 3a
1H NMR (400 MHz, CDCl3): δ (ppm) 8.02 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H),
7.55 (s, 1H), 2.51 (s, 3H).13C NMR (100 MHz, CDCl3): δ (ppm) 171.51, 153.84, 136.32,
C
134.63, 134.12, 132.12, 129.57, 128.40, 127.87, 126.12, 125.18, 123.03, 121.36. Anal. Calcd
For C16H13NS: C, 76.46; H, 5.21; N, 5.57. Found: C, 76.40; H, 5.17; N, 5.52.
4.3.2. 2-(4-Hydroxyphenyl)benzothiazole 3h
1H NMR (400 MHz, CDCl3): δ (ppm) 9.87 (s, 1H), 7.89–7.95 (m, 4H), 7.43–7.47 (m, 1H),
7.33–7.37 (m, 1H), 6.91–6.93 (m, 2H); 13C NMR (100 MHz, CDCl3): δ (ppm) 167.96,
160.25, 153.66, 139.89, 128.66, 125.82, 124.35, 124.14, 122.03, 121.31, 115.71. Anal. Calcd
For C13H9NOS: C, 68.70; H, 3.99; N, 6.16. Found: C, 68.64; H, 3.91; N, 6.11.
4.3.3. 2-(3-Hydroxyphenyl)benzothiazole 3i
1H NMR (400 MHz, CDCl3): δ (ppm) 8.06 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H),
7.00–6.91(m, 1H) 13C NMR (100 MHz, CDCl3): δ (ppm) 168.22, 156.44, 153.69, 134.69,
130.43, 126.49, 125.38, 123.08, 121.67, 120.18, 118.46, 114.05, 99.98. Anal. Calcd For
C13H9NOS: C, 68.70; H, 3.99; N, 6.16. Found: C, 68.62; H, 3.92; N, 6.10.

JOURNAL OF SULFUR CHEMISTRY
9
4.3.4. 2-(2,4-Difluorophenyl)benzothiazole 3l
1H NMR (500 MHz, CDCl3): δ (ppm) 8.41–8.49 (m, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.94
(d, J = 8.0 Hz, 1H), 7.51–7.54 (m,1H), 7.40–7.43 (m, 1H), 6.97–7.07 (m, 2H); 13C NMR
(125 MHz CDCl3,): δ (ppm) 165.47 (d, J = 10 Hz), 162.01–163.00 (m), 159.46–160.17
(m), 152.39 (s), 135.42 (d, J = 8 Hz), 131.05–131.18 (m), 126.37 (s), 125.34 (s), 123.19
(s), 121.46 (s), 112.28–112.49 (m), 104.65 (t, J = 25 Hz). Anal. Calcd For C13H7F2NS: C,
63.15; H, 2.85; N, 5.66. Found: C, 63.09; H, 2.80; N, 5.60.
4.3.5. 2-(3,4-Difluorophenyl)benzothiazole 3n
1H NMR (400 MHz, CDCl3): δ (ppm) 8.06 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H),
7.53–7.48 (m, 1H), 7.42–7.38 (m, 1H), 7.31–7.24 (m, 1H), 6.71–6.69 (m, 1H), 6.60–6.56
(m, 1H); 13C NMR (100 MHz, CDCl3): δ (ppm) 153.94, 148.60, 136.85, 131.64, 126.65,
125.63, 123.41, 121.72, 118.81, 118.30, 118.10, 117.92, 115.30. MS (70 eV): m/z 247 (M+)
Anal. Calcd. For C13H7F2NS: C, 63.15; H, 2.85; N, 5.66. Found: C, 63.10; H, 2.81; N, 5.62.
4.3.6. 2-(Pyridin-3-yl)benzothiazole 3t
1H NMR (400 MHz, CDCl3) : δ (ppm) 9.29 (d, J = 2.16 Hz, 1H), 8.70 (m, 1H), 8.35–8.38
(m, 1H), 8.09 (d, J = 8.28 Hz, 1H), 7.91–7.93 (m, 1H), 7.50–7.54 (m, 1H), 7.40–7.44 (m,
2H); 13C NMR (100 MHz, CDCl3): δ (ppm) 169.5, 153.9, 151.5, 148.5, 134.9, 134.5, 129.6,
126.6, 125.7, 123.8, 121.7. Anal. Calcd For C12H8N2S: C, 67.90; H, 3.80; N, 13.20. Found:
C, 67.95; H, 3.74; N, 13.14.
4.4. Procedure for biological activities
The agar-well diffusion method [57–59] was used for the evaluation of the antimicrobial
activity of the synthesized benzothiazoles derivatives 3a–3v. The microbial culture for all
the test strains was adjusted to 0.5 McFarland standard, which is visually similar to a micro-
bial suspension of 1.5 × 108 cfu/ml (approx.). Agar medium (20 ml) was transferred into
each Petri plate and plates were swabbed with 100 μl inocula of the test microorganisms
and kept for 15 min for adsorption. Wells were created into the seeded agar plates by using
sterile cork borer of 8 mm diameter which were loaded with a 100 μl volume with con-
centration of 8.0 mg/ml of each compound in the DMSO. All the plates were incubated
at 37°C for 24 h. Antimicrobial activity of benzothiazole derivative was assessed by mea-
suring the zone of growth inhibition against the test organisms with a zone reader (Hi
Antibiotic zone scale). DMSO was used as a negative control whereas ciprofloxacin was
used as positive control (reference drug) for bacteria and amphotericin-B (reference drug)
for fungi, respectively. This procedure was performed in triplicate for each microorgan-
ism. MIC of the all the synthesized compounds was tested through a modified agar well
diffusion method [57–59]. In this method, a two-fold serial dilution of each benzothiazole
derivative was prepared by first dissolving the compound in DMSO followed by dilution
in sterile distilled water to achieve a decreasing concentration range of 50–0.39 μg/ml. A
100 μl volume of each dilution was introduced into wells (in triplicate) in the agar plates
already seeded with 100 μl of standardized inoculum (106 cfu/ml) of the test microbial
strain. All test plates were incubated aerobically at 37°C for 24 h and observed for the inhi-
bition zones. MIC, the lowest concentration of the compound that completely inhibited

10
P. KUMAR ET AL.
the growth of the microbe, showed by a clear zone of inhibition, was documented for each
test microorganism.
Acknowledgements
Financial support as junior research fellowship (JRF) by the Haryana State Council of Science and
Technology is gratefully acknowledged for accomplishing this work.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Parvin Kumar http://orcid.org/0000-0002-2635-6465
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