Exploration of in vitro time point quantitative evaluation of newly synthesized benzimidazole and benzothiazole derivatives as potential antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2011.10.034

Present communication deals with the in vitro time point quantitative antibacterial evaluation of newly synthesized 1,2-disubstituted benzimidazoles (3a-p) and 2-substituted benzothiazoles (5a-h) against Gram-positive bacteria Staphylococcus aureus, Bacil

Full text of DOI:10.1016/j.bmcl.2011.10.034

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7306–7309
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploration of in vitro time point quantitative evaluation of newly
synthesized benzimidazole and benzothiazole derivatives as potential
antibacterial agents
a
a
a
a
b
Prabal Bandyopadhyay , Manisha Sathe , S. Ponmariappan , Arti Sharma , Pratibha Sharma ,
a
a,
⇑
A.K. Srivastava , M.P. Kaushik
Discovery Centre, Process Technology Development Division, Defence R&D Establishment, Jhansi Road, Gwalior 474 002, MP, India
School of Chemical Sciences, Devi Ahilya University, Khandwa Road, Indore 452 001, MP, India
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2011
Revised 17 September 2011
Accepted 11 October 2011
Available online 18 October 2011
Present communication deals with the in vitro time point quantitative antibacterial evaluation of newly
synthesized 1,2-disubstituted benzimidazoles (3a–p) and 2-substituted benzothiazoles (5a–h) against
Gram-positive bacteria Staphylococcus aureus, Bacillus cereus, and Gram-negative bacteria Vibrio cholerae,
Shigella dysenteriae and Escherichia coli. These compounds were synthesized under mild reaction condi-
2 3 2 3
tions using Al O –Fe O nanocrystals as heterogeneous catalyst. Bio-evaluation studies revealed that,
compounds 3a, 5a and 5d exhibited moderate to good antibacterial activity against all the tested bacterial
stains. The compounds 3a, 3f and 5a have shown enhanced inhibitory activity compared with standard
antibacterial drug ciprofloxacin against V. cholerae, B. cereus, and S. dysenteriae, respectively. Additionally,
the compounds 3a, 3e, 3f, 3h and 5b displayed complete bactericidal activity within 24 h, whereas cip-
rofloxacin took 48 h to kill those bacteria completely.
Keywords:
Antibacterial activity
Quantitative assay
Benzimidazole
Benzothiazole
In vitro
Ó 2011 Elsevier Ltd. All rights reserved.
Infectious as well as highly contagious microbial diseases are
increasing with course of time round the world due to the emer-
gence of new multidrug resistant bacteria which are resistant to a
number of antimicrobial agents due to the development of mutage-
acids and proteins inside the bacterial cell wall.21,22 The therapeutic
importance of these class of compounds inspired us to develop a ser-
ies of 1,2-disubstituted benzimidazoles and 2-substituted benzo-
thiazoles in order to study their antibacterial activities. Recently,
we explored the use of mesoporous mixed metal oxide nanocrystals
as heterogeneous catalysts for the synthesis of novel series of 1,2-
1
nicity. One way to battle with this challenge is the conscious usage
of the currently marketed antibiotics and the other is to develop and
2
23
screen new chemical entities for antimicrobial activities. In view of
disubstituted benzimidazoles and 2-substituted benzothiazoles.
this, it is imperative to discover new chemotherapeutic agents to
prevent the emergence of resistance and ideally shorten the
duration of therapy.
In continuation of our studies on bioactive compounds,24 herein
we report the in vitro qualitative as well as time point quantitative
antibacterial activity evaluation of the synthesized benzimidazole
and benzothiazole derivatives bearing various functional groups of
electron-withdrawing and electron-donating properties against
the representative genera of Gram-positive and Gram-negative
bacteria.
Benzimidazoles and benzothiazoles are the important pharma-
cophore and privileged sub-structures in medicinal chemistry ow-
ing to their involvement as a key component for various biological
3
–8
activities.
The high profile of biological applications displayed
by compounds associated with these nuclei have prompted wide
The synthetic pathways leading to 1,2-disubstituted benzimi-
dazoles (3a–p) and 2-substituted benzothiazoles (5a–h) via one-
pot condensation reaction between aromatic and aliphatic alde-
hydes 2 with 1,2-phenylenediamine 1 and 2-aminothiophenol 4,
studies for their synthesis.9
–20
Extensive biochemical and pharma-
cological studies have confirmed that benzimidazole and benzothi-
azole derivatives are effective against various strains of microor
ganisms. Due to the structural similarity with purine, antibacterial
ability of these compounds manifested their competition with pur-
ines resulting in the distinct inhibition of the synthesis of nucleic
2 3 2 3
respectively, using Al O –Fe O nanocrystals are depicted in
Scheme 1. The structures of the synthesized compounds were con-
1
13
firmed by H and C NMR, ESI-MS spectral data, and elemental
1
analysis. The H NMR spectra of all substances displayed multiplets
in the aromatic region indicating the presence of the benzimid-
azole and benzothiazole ring. The mass spectra (ESI-MS) of the
⇑
Corresponding author. Tel.: +91 751 2343972; fax: +91 751 2340042.
E-mail address: mpkaushik@rediffmail.com (M.P. Kaushik).
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.034

P. Bandyopadhyay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7306–7309
7307
NH2
NH2
N
N
Table 2
Al O -Fe O
2
3
2
3
2
R-CHO
R
2 3 2 3
Synthesis of 2-substituted benzothiazoles (5a–h) using Al O –Fe O nanocatalyst
6
0
º
C
0
Timea (min)
Yieldb (ꢀ)
Entry
R
Product
solvent or solvent-free
R
1
2
3a-p
1
2
3
4
5
6
7
8
2-EtC
4-HOC
6
H
4
5a
5b
5c
5d
5e
5f
22
16
33
15
24
28
17
23
85
84
83
85
83
89
87
88
6
H
4
NH2
SH
3,4,5-(MeO)
4-EtOC
4-EtO-3-MeOC
5-Br-2-MeOC
4-(Me) HCC
4-NO
3 6 2
C H
Al O -Fe O
N
S
2
3
2
3
6 4
H
R'-CHO
R'
60 C
º
6 3
H
solvent or solvent-free
6 3
H
4
2
5a-h
6
H
5g
5h
2
4
2
C
6
H
4
Scheme 1. Synthesis of 1,2-disubstituted benzimidazoles (3a–p) and 2-substituted
benzothiazoles (5a–h) using Al –Fe nanocatalyst.
a
All the reactions monitored by TLC.
Isolated yield.
2
O
3
2 3
O
b
compounds showed (M+H)+ peaks, in agreement with their
molecular weight. Elemental analysis results for C, H and N ele-
ments were satisfactory within ±0.4ꢀ calculated values of the
compounds.
lent yields (81–89ꢀ). Furfural (Table 1, entry 10) and cinnamyl
aldehyde (Table 1, entry 11) also afforded the desired products in
excellent yields (84–90ꢀ). Furthermore, this synthetic approach
was extended to aliphatic aldehydes, such as heptanal to get the
desired 1,2-disubstituted benzimidazole (Table 1, entry 12) with
reasonably good yield (82ꢀ).
In order to study the catalytic activity, nanocrystalline Al
2 3
O –
Fe O was synthesized by aerogel technique and studied as hetero-
2 3
geneous catalyst for the preparation of a series of 1,2-disubstituted
benzimidazoles and 2-substituted benzothiazoles. When the reac-
tion was carried out with 1,2-phenylendiamine and aldehyde in
The antibacterial activity (zone of growth inhibition) of the
tested 1,2-disubstituted benzimidazoles (3a–p) and 2-substituted
benzothiazoles (5a–h) in comparison with that of control drugs
ciprofloxacin was determined by Kirby–Bauer disc diffusion meth-
1
2 3 2 3
:1 molar ratio in presence of Al O –Fe O (5 wt ꢀ of 1,2-phenylene-
diamine), then a mixture of mono and di-substituted product were
formed. On the other hand, when 1:2 molar ratio was used then
26
od on Mueller–Hinton agar according to the guidelines of Clinical
Laboratory Standards Institute, 2007, USA (Table 3). Among all the
tested compounds, many compounds exhibited moderate to excel-
lent activity. The compound 3n showed moderate activity (zone of
growth inhibition 22 mm) against Escherichia coli MTCC 1610 as
compared to the standard drug (32 mm). Compounds 3a exhibited
excellent efficacy (33 mm) against tested Vibrio cholerae MTCC
2
5
the desired 1,2-disubstituted benzimidazole was formed with
excellent selectivity and yields. However, trace amount of monosub-
stituted product was also formed in few cases, which was separated
by column chromatography. To demonstrate the synthetic potential
of this protocol the same procedure was extended for one-pot syn-
thesis of benzothiazole25 class of compounds. An overview of syn-
thetic highlights delineating the synthesis of 1,2-disubstituted
benzimidazoles and 2-substituted benzothiazoles is depicted in Ta-
bles 1 and 2, respectively.
3
3
904, while the zone of growth inhibition for compounds 3d and
h (21 and 23 mm) was nearly equal to the control drug ciproflox-
acin (24 mm). Likewise, against Shigella dysenteriae NICED, com-
pound 5a displayed highest inhibitory activity profile (35 mm)
and compounds 3a, 3l, 3n and 5c showed enhanced growth inhibi-
tion (19–23 mm) as compared to the standard drug. Only two com-
pounds 3e and 3n demonstrated better activity profile (zone of
growth inhibition up to 22–23 mm) against Gram-positive bacteria
Staphylococcus aureus MTCC 3160. Interestingly, in case of Bacillus
cereus MTCC 430, large number of compounds (3a, 3f, 3g, 5a, 5b,
It is evident from Tables 1 and 2 that, the proposed synthetic
procedure works well with mono, di (Table 1, entries 5, 6 and 9
and Table 2, entries 5, 6) and even tri-substituted (Table 2, entry
3
) aryl aldehydes. The effect of substitution present on aromatic
aldehyde on the reaction rate and the overall yield was also stud-
ied. As shown, a variety of benzaldehydes bearing electron-donat-
ing (Table 1, entries 2–6 and Table 2, entries 1–7) and electron-
withdrawing substituents (Table 1, entries 13, 14 and Table 2,
entry 8) were successfully employed to prepare the corresponding
benzimidazoles and benzothiazoles derivatives in good to excel-
5
d, 5f and 5g) displayed inhibitory activity (18–33 mm) higher
than that of ciprofloxacin (14 mm). Among them compound 3f
exhibited the maximum inhibition (33 mm). It is evident from
the above table that, compounds 3a, 5a and 5d exhibited moderate
to good antibacterial activity against both Gram-positive as well as
Gram-negative bacteria. It is noteworthy to mention that, com-
pound 5c was found to be selectively active against shigellosis
causing bacteria S. dysenteriae NICED.
Table 1
Synthesis of 1,2-disubstituted benzimidazoles (3a–p) using Al
2
O
3
–Fe
2
O
3
nanocatalyst
Yieldb (ꢀ)
a
Further, upon close inspection of results depicted in Table 3, it
has been inferred that, the Gram-positive bacteria were more sus-
ceptible towards the newly synthesized series of compounds as
compared to the Gram-negative bacteria. This may be due to the
absence of a unique outer membrane in Gram-positive bacteria
and hence, the wall of Gram-positive bacteria is permeable to these
derivatives. Generally, the Gram-positive bacteria are more sus-
ceptible having only an outer peptidoglycan layer which is not
Entry
R
C
Product
Time (min)
1
2
3
4
5
6
7
8
9
6
H
5
3a
3b
3c
3d
3e
3f
12
27
45
22
35
27
21
25
14
12
37
24
10
34
24
10
84
85
83
81
89
85
88
84
85
90
84
82
87
83
81
82
6 4
4-MeC H
4-(Me)
4-(Me)
2
NC
CC
6
H
4
3
6
H
4
3-EtO-4-HOC
5-Br-2-HOC
4-FC
4-F COC
6
H
3
H
3
6
6
H
4
3g
3h
3i
3
6 4
H
2
7
3,4-Cl
Furyl
2
C
H
6 3
an effective permeability barrier whereas the Gram-negative
bacteria possess an outer phospholipidic membrane carrying the
structural lipopolysaccharide components. This makes the cell wall
10
11
12
13
14
15
16
3j
C
CH
6
H
5
CH=CH
-(CH2) -CH
3-NO
4-F CC
4-BrC
4-HOC
3k
3l
3m
3n
3o
3p
3
)
4
2
2
8
impermeable to drug constituents.
2
C
6
H
4
H
4
The results of the quantitative assay for the antibacterial activ-
ity of the selected synthesized compounds against B. cereus MTCC
3
6
6
H
4
6
H
4
4
30, S. aureus MTCC 3160, V. cholerae MTCC 3904, E. coli MTCC
a
All the reactions monitored by TLC.
Isolated yield.
1610 and S. dysenteriae NICED at different time point are graphi-
b
29
cally represented in Figure 1 [(i)–(v)]. The time point assay

7
308
P. Bandyopadhyay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7306–7309
Table 3
Antibacterial activity of compounds: diameter of zone of growth inhibition (mm) against five different bacterial strains based on Kirby–Bauer method
0
Compounds
R (R )
Gram-negative bacteria
Gram-positive bacteria
S. aureus MTCC 3160 B. cereus MTCC 430
E. coli MTCC 1610
V. cholerae MTCC 3904
S. dysentriae NICED
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
C
6
H
5
19
—
—
17
14
17
—
—
—
—
—
—
—
22
—
—
11
—
—
12
—
—
33
18
17
21
—
—
—
23
—
13
11
16
13
13
16
—
16
18
—
15
16
17
—
—
24
23
13
16
13
—
10
12
11
—
23
—
10
10
—
10
—
12
10
22
—
—
13
—
22
—
—
12
—
33
18
—
—
—
14
15
—
—
15
—
21
19
—
22
—
21
20
14
14
6 4
4-MeC H
4-(Me)
4-(Me)
3-EtO-4-HOC
5-Br-2-HOC
4-FC
4-F COC
3,4-Cl
Furyl
2
NC
CC
6
H
4
3
6
H
4
6
H
3
6
H
3
—
—
6
H
4
3
6
H
4
11
11
12
—
21
—
19
13
11
35
—
23
16
15
—
2
C
6
H
3
j
k
l
C
6
H
5
CH@CH
-(CH2) -CH
3-NO
4-F CC
4-BrC
4-HOC
2-EtC
4-HOC
CH
3
)
4
2
m
n
o
p
a
b
c
d
e
f
2
C
6
H
4
3
6
H
4
6
H
4
6
H
4
6
H
4
6
H
4
3,4,5-(MeO)
4-EtOC
4-EtO-3-MeOC
5-Br-2-MeOC
4-(Me) HCC
3
C
6
H
2
—
6
H
4
16
12
12
13
—
6
H
3
6
H
3
g
h
2
6
H
4
—
—
32
15
13
14
4-NO
—
2
C
6
H
4
a
Ciprofloxacin
15
a
Antibacterial activity of the synthesized compounds was compared with the standard antibacterial drug ciprofloxacin.
Bacillus cereus MTCC 430
Staphylococcus aureus MTCC 3160
9
8
7
6
5
4
3
2
1
0
8
7
6
5
4
3
2
1
0
control
3a
control
3e
3c
3h
3f
3j
5
a
3l
5
b
3n
5d
g
5d
5
5g
0
20
40
60
80
CIP
0
20
40
60
80
CIP
Time (h)
Time (h)
(i)
(ii)
Vibrio cholerae MTCC 3904
Shigella dysenteriae NICED
Escherichia coli MTCC 1610
8
7
6
5
4
3
2
1
0
control
3a
9
8
7
6
5
4
3
2
1
0
9
control
3a
3d
3f
3n
5b
5d
CIP
8
7
6
5
4
3
2
1
0
Control
3b
3
3
a
c
3c
3d
3d
3h
3
5
i
a
5b
5e
CIP
CIP
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
Time (h)
Time (h)
Time (h)
(
iii)
(iv)
(v)
Figure 1. Graphical representation of quantitative antibacterial activity study against (i) B. cereus MTCC 430; (ii) S. aureus MTCC 3160; (iii) V. cholerae MTCC 3904; (iv) E. coli
MTCC 1610 and (v) S. dysenteriae NICED.
results revealed that, the compounds 3f and 5b exhibited complete
bactericidal effect on B. cereus MTCC 430 within 24 h, whereas the
compounds 3a, 5a, 5d, 5g diminished approximately 3–4 log val-
ues of the bacterial concentration within 48 h of incubation. In
the case of S. aureus MTCC 3160, only the compound 3e completely
exterminated the bacteria within 24 h, but compounds 3h, 3j, 5d
and 5g were able to prevent the bacteria completely within 48 h.
Against Gram-negative bacteria V. cholerae MTCC 3904, the com-
pounds 3a and 3h killed the bacteria completely within 24 h,
whereas against E. coli MTCC 1610, only compound 3n able to
eliminate the bacteria completely within 48 h. Further, against S.
dyesteriae NICED, the compound 5a took 72 h to inhibit the growth
of the bacteria completely. The positive control ciprofloxacin (CIP)
took 48 h to kill the tested bacteria completely except S. dyesteriae
NICED. It completely exterminated S. dyesteriae within 72 h.
In conclusion, a simple, efficient and environmentally benign
method was developed for the synthesis of 1,2-disubstituted benz-
imidazoles and 2-substituted benzothiazoles by using Al O –Fe O
2 3 2 3
nanomaterials having high surface area and high catalytic activity.
The results based on disc diffusion method showed that, com-

P. Bandyopadhyay et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7306–7309
7309
pounds 3a, 5a and 5d exhibited moderate to good antibacterial
activity against all the tested bacterial stains. In addition, com-
pound 5c was selectively active against shigellosis causing bacteria
S. dysenteriae. Moreover, the compounds 3a, 3f and 5a displayed
significant inhibitory activity compared to standard antibacterial
drug ciprofloxacin against V. cholerae, B. cereus and S. dysenteriae,
respectively. The time point assay results revealed that, the com-
pounds 3a, 3e, 3f, 3h and 5b demonstrated complete bactericidal
activity within 24 h, whereas ciprofloxacin took 48 h to kill those
bacteria completely. These antibacterial results indicated that
structural factors, such as type of substitution in the ring and water
solubility of the target compounds could further enhance their
inhibitory activities.
15. Jacob, R. G.; Dutra, L. G.; Radatz, C. S.; Mendes, S. R.; Perin, G.; Lenardão, E. J.
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Acknowledgments
25. To a mixture of 1,2-phenylenediamine (0.54 g, 5 mmol) and aromatic/aliphatic
aldehyde (10 mmol), catalytic amount (0.0108 g, 5 wt ꢀ of 1,2-
We thank Director, DRDE, Gwalior for his keen interest and
encouragement. We are also thankful to Mr. Ajay Pratap for record-
ing NMR spectra. P.B. is thankful to DRDO, New Delhi, for financial
support.
2 3 2 3
phenylenediamine) of Al O –Fe O nanocrystals was added using 5 mL of
acetonitrile as solvent at room temperature under stirring. The reaction
progress was monitored by TLC. After stirring for 10–45 min, under heating
condition at 60 °C, the reaction mixture was cooled to room temperature and it
was dissolved in ethanol (20 mL) and then poured into ice-water (40 mL). Ethyl
acetate (50 mL) was added and the catalyst was separated out by filtration
from the extraction mixture. The organic extract was dried over anhydrous
sodium sulfate and excess of solvent was removed under reduced pressure so
as to obtain the product. It was then purified by column chromatography over
silica gel using hexanes/EtOAc as the eluting solvent system, yielding the pure
products. An identical procedure for the synthesis of 2-substituted
benzothiazoles was employed using 2-aminothiophenol (0.53 mL, 5 mmol)
and aromatic/aliphatic aldehyde (5 mmol) in the presence of catalytic amount
Supplementary data
Supplementary data (experimental informations and spectral
1
13
data ( H, C NMR, ESI-MS and elemental analysis)) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.10.034.
(
5 wt ꢀ of 2-aminothiophenol) of Al
2 3 2 3
O –Fe O nanocrystals under heating
condition (60 °C).
2
6. Clinical and Laboratory Standard Institute, Performance standards for
antimicrobial susceptibility testing; Seventeenth Informational Supplement:
CLSI document M100-S17, Clinical and Laboratory Standard Institute, 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA, 2007.
7. Scherrer, R.; Gerhardt, P. J. Bacteriol. 1971, 107, 718.
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9. The newly synthesized compounds which showed antibacterial activity in agar
diffusion method were selected for further screening though quantitative assay
at different time point. In this method, minimum time required to kill the
bacteria was estimated by pour plate method at different time point. Mueller–
Hinton broths (5 mL) were prepared in a test tube, sterilized and cooled to
room temperature and incubated at room temperature for overnight to check
the sterility of the media. A loop full of respective bacterial cultures (B. cereus
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