Synthesis, in-vitro microbial and cytotoxic studies of new benzimidazole derivatives

Article abstract of DOI:10.1002/ardp.200900022

Several new classes of benzimidazole derivatives were synthesized and evaluated for in-vitro antimicrobial and cytotoxic activities. The results showed that all synthesized compounds exhibited moderate antimicrobial activity, and compounds 2, 4, and 13 displayed cytotoxic activity (as LD₅₀) at the concentration 1 × 10^-3 M against Artemia salina.

Full text of DOI:10.1002/ardp.200900022

412
Arch. Pharm. Chem. Life Sci. 2009, 342, 412–419
Full Paper
Synthesis, in-vitro Microbial and Cytotoxic Studies of New
Benzimidazole Derivatives
Reddy S. Harisha, Kallappa M. Hosamani, and Rangappa S. Keri
P. G. Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad, India
Several new classes of benzimidazole derivatives were synthesized and evaluated for in-vitro anti-
microbial and cytotoxic activities. The results showed that all synthesized compounds exhibited
moderate antimicrobial activity, and compounds 2, 4, and 13 displayed cytotoxic activity (as
LD50) at the concentration 1610– 3 M against Artemia salina.
Keywords: Coumarin / Pyrazole / Thiazolidine / Triazolo-thiadiazole / 2-(Trifluoromethyl)-1H-benzimidazole /
Received: January 31, 2009; accepted: February 27, 2009
DOI 10.1002/ardp.200900022
dazole derivatives were performed and are described in
the present study.
Introduction
The synthesized compounds were tested for their possi-
ble in-vitro antimicrobial and cytotoxic activities. Most of
the tested compounds showed activity against Vancomy-
cin-resistant Enteroccoccus (ATCC-51299), Staphylococcus
aureus (ATCC-29213), Micrococcus (natural isolates), Bacil-
lus subtilis (natural isolates), Shigella dysentery (natural iso-
lates), Escherichia coli (ATCC-25922) – as examples for
Gram-positive and Gram-negative bacteria, and Candida
albicans, Aspergillus niger, Penicillium as representatives of
fungi. The minimum inhibitory concentration (MIC) was
determined for the test compounds as well as for the
reference standards.
Recently, fluorinated heterocycles have attracted atten-
tion due to the ability of fluorine to act as polar hydrogen
or hydroxyl mimic. Substituted 2-(trifluoromethyl)-1H-
benzimidazoles are known as an important class of com-
pounds due to their wide range of biological activity act-
ing as antiviral, antifungal, antibacterial, and anticancer
drugs [1–4]. These compounds also inhibit photosynthe-
sis and, therefore, exhibit appreciable herbicidal activity
[5]. Most recently, antiparasitic activities of this class of
compounds have been reported [6, 7].
A survey of literature revealed that S-triazolo[3,4-b]-
1,3,4-thiadiazole rings, have received much attention
during recent years on possessing a broad spectrum of
biological activities [8–13], in addition, thiazolidines
also exhibit a broad spectrum of pharmacological proper-
ties [14]. Moreover, a triazolo-thiadiazole system may be
viewed as a cyclic analogue of two very important compo-
nents – a thiosemicarbazide [15] and a biguanide [16],
which often display diverse biological activities. Also,
coumarin derivatives condensed with other heterocycles
shows a wide range of pharmacological activities [17–
20]. Therefore, prompted by these observations, synthe-
ses and in-vitro biological evaluations of the new benzimi-
Results and discussion
Chemistry
For the synthesis of the target compounds, the reaction
sequences outlined in Schemes 1, 2, and 3 were followed.
Hydrolysis of 2-(trifluoromethyl)-1H-benzimidazole I in
NaOH / HCl gives the 1H-benzimidazole-2-carboxylic acid
II. Treatment of compound II with thionyl chloride fol-
lowed by hydrazine hydrate gives the desired 1H-benzi-
midazole-2-carboxylic acid hydrazide III in 90% yield.
In the present work, compound III was used as the key
intermediate for the subsequent synthesis. Ethyl acetoa-
cetate, thioglycolic acid, acetyl acetone, and chloroacetyl
chloride were reacted with compound III resulting in the
Correspondence: Kallappa M. Hosamani, P. G. Department of Studies
in Chemistry, Karnatak University, Pavate Nagar, Dharwad-580 003, In-
dia.
E-mail: dr_hosamani@yahoo.com
Fax: +91-836-274 7884 and +91-836-277 1275
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2009, 342, 412–419
New Benzimidazole Derivatives
413
Reactions and conditions: a) 2 N NaOH / 2 N HCl; b) SOCl2 / N2H4 6H2O; c) CH3COOCH2COC2H5, C2H5OH; d) C6H5CHO,
SHCH2COOH, C2H5OH; e) CH3COCH2COCH3, C2H5OH; f) C6H5SCN, ClCH2COCl, C2H5OH.
Scheme 1. Schematic representation of compounds 1–4.
Table 1. Molecular formula, yields, and melting points of the
newly synthesized compounds.
novel substituted 4-bromomethyl coumarins with com-
pound I afforded the corresponding 11, 12, and 13,
respectively (Scheme 3, Table 1).
Compound
Molecular
Formula
Yield
(%)
Melting Point
(8C)
Biological evaluation
Antimicrobial activity
1
2
3
4
5
6
7
8
9
10
11
12
13
C12H10N4O2
C13H12N4O
C17H14N4O2S
C17H13N5O2S
C9H8N6S
C11H7ClN6S
C10H6N6S
C10H6N6S2
73.40
78.70
71.21
85.93
81.14
87.86
65.97
77.01
70.35
73.22
71.33
69.68
77.55
253–255
245–247
284–286
229–231
266–268
254–256
273–275
289–291
230–232
241–243
180–182
225–227
204–206
All the test compounds were assayed for in-vitro antimi-
crobial activity, and the strains used in this study were
maintained at the Department of Botany, Karnatak Uni-
versity, Dharwad, India. The antimicrobial activity was
determined by using disk-diffusion method [21–23] and
MIC (twofold serial dilution method). Streptomycin and
Nystatin were used as reference standards to compare
antibacterial and antifungal activities, respectively. For
determining the antimicrobial activity, the synthesized
compounds were dissolved in dimethyl sulphoxide (stock
solution 1 mg/mL). Further dilutions were prepared at
the required quantities of 100, 50, and 25 lg/mL concen-
trations. In order to ensure that the solvent had no effect
on bacterial growth, a control test was also performed
containing a disc loaded only with DMSO at the same
dilutions used in our experiment. For determining the
MIC of the synthesized compounds, they were diluted at
100, 50, 25, and 12 lg/mL concentrations and expressed
in lM/mL. The MIC of the compounds was defined as the
lowest concentration at which there was 100% inhibition
C11H8N6S
C11H8N6OS
C19H13F3N2O2
C18H10ClF3N2O2
C22H13F3N2O2
corresponding derivatives of pyrazoles and thiazolidines
1–4, respectively (Scheme 1, Table 1). Reacting com-
pound III with carbon disulphide and then with hydra-
zine hydrate (99%) gives 4-amino-5-(1H-benzimidazole-2-
yl)-4H-[1,2,4]triazole-3-thiol 5 (Scheme 2, Table 1). Then,
compound 5 was treated with chloroacetyl chloride, for-
mic acid, carbon disulfide, acetic acid, and chloroacetic
acid to give the corresponding triazolo-thiadiazole deriv-
atives 6–10, respectively. Further condensation of the
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

414
R. S. Harisha et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 412–419
Reactions and conditions: g) CS2 / KOH, C2H5OH; h) N2H46H2O, C2H5OH; i) ClCH2COCl, C2H5OH; j) HCOOH / H2SO4; k) CS2, /
KOH, C2H5OH; l) CH3COOH / POCl3; m) ClCH2COOH / NaOAc, C2H5OH.
Scheme 2. Schematic representation of compounds 5–10.
Table 2. Antibacterial activity: The MIC values of compounds 1–13 (in lM/mL).
Compound
V. r. e.a)
S. a.b)
M.c)
B. s.d)
S. d.e)
E. c.f)
1
2
3
4
5
6
7
8
9
10
11
12
13
SDg)
1.27–2.17
F2.76
0.37–0.84
0.72–1.65
0.34–0.89
1.65–2.37
0.88–1.51
0.34–0.88
f0.28
0.59–1.31
0.75–1.41
F2.21
0.88–2.32
F2.82
1.27–2.27
0.75–1.49
1.36–2.21
0.34–0.78
0.65–1.39
1.43–2.78
1.56–2.35
1.56–2.35
0.23–0.72
1.33–2.53
F2.14
F2.73
1.85–2.87
F2.15
F2.52
0.27–0.77
1.71–2.37
F2.12
F2.82
F2.43
0.41–0.78
0.55–1.39
F2.32
1.67–2.35
F2.29
1.66–2.91
F2.73
0.95–1.72
0.23–0.68
1.56–3.13
F2.29
F2.15
1.45–2.72
f0.88
0.97–2.92
F2.83
F2.31
F2.41
1.56–2.37
0.21–0.79
0.94–2.29
0.33–0.81
F2.31
F3.33
1.62–2.63
f0.88
F2.33
F2.14
0.37–0.91
F2.12
1.31–2.57
F2.82
0.85–2.35
0.23–0.82
0.65–1.42
F2.82
F2.49
F2.82
F2.12
1.43–2.22
0.39–1.71
0.78–1.67
f0.88
1.31–2.57
F2.87
F2.33
1.58–2.33
1.81–3.36
0.88–1.81
0.88–1.81
a)
V. r. e.: Vancomycin-resistant enteroccoccus.
S. a.: Staphylococcus aureus.
M.: Micrococcus.
B. s.: Bacillus subtilis.
S. d.: Shigella dysentery.
E. c.: Escheria coli.
b)
c)
d)
e)
f)
g)
SD: Streptomycin (Standard).
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 412–419
New Benzimidazole Derivatives
415
Scheme 3. Schematic representation of compounds 11–13.
Table 3. Antifungal activity: The MIC values of compounds 1–
13 (in lM/mL).
of growth compared with the growth for a drug-free con-
trol. Every experiment in the antibacterial assay and MIC
was replicated thrice.
Compound
C. a.a)
A. n.b)
P.c)
1
2
3
4
5
6
7
8
9
10
11
12
13
SDd)
0.69–1.33
0.28–0.85
1.44–3.11
0.34–0.78
0.31–0.75
1.72–2.82
f0.33
0.73–1.46
0.33–0.72
0.32–0.79
f0.37
0.82–1.56
f0.35
f1.82
1.33–2.45
0.89–1.85
1.67–2.71
0.81–1.56
f0.27
A2.63
Cytotoxic activity
1.51–3.47
1.65–2.33
1.56–3.13
0.78–1.46
1.51–2.73
0.78–1.65
A2.71
Brine-shrimp (Artemia salina leach) eggs were hatched in
a shallow rectangular plastic dish (22632 cm) filled with
artificial seawater, which was prepared with a commer-
cial salt mixture and double-distilled water. An unequal
partition was made in the plastic dish with the help of a
perforated device. Approximately 50 mg of eggs were
sprinkled into the large compartment, which was dark-
ened while the other compartment was opened to ordi-
nary light. After two days, nauplii were collected by pip-
ette from the lighted side. A sample of the test compound
was prepared by dissolving 20 mg of each compound in
2 mL of DMF. From these stock solutions, 100, 50, and
25 lg/mL were transferred to nine vials (three for each
dilution were used for each test sample, and LD50 is the
mean of three values) and one vial was kept as control
having 2 mL of DMF only. The solvent was allowed to
evaporate overnight. After two days, when the shrimp
larvae were ready, 1 mL of seawater and ten shrimps
were added to each vial (30 shrimps/dilution) and the vol-
ume was adjusted with seawater to 5 mL per vial. After
A2.08
0.37–0.78
1.43–2.71
A2.97
A2.78
0.39–0.91
0.84–1.56
1.52–2.84
f0.29
1.61–3.12
1.66–2.21
0.63–1.39
1.39–3.18
f3.54
f3.54
a)
C. a.: Candida albicans.
A. n.: Aspergillus niger.
P.: Penicillium.
b)
c)
d)
SD: Nystatin (Standard).
24 h, the numbers of survivors were counted [24]. Data
were analyzed by a Finney computer program to deter-
mine the LD50 values [25].
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

416
R. S. Harisha et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 412–419
Table 4. Brine-shrimp bioassay data for compounds 1–13.
tered. The filtrate was acidified with hydrochloric acid (1 N) to
pH 4. The precipitate was filtered off, washed twice with cold
water, and recrystallized from DMSO solvent.
Compound
LD50 (mol/mL)
IR (KBr): m (cm– 1) 3340 br (ArNH), 1665 (C=O), 1592 (C=O), 1580
4.531610– 3
8.742610– 4
6.398610– 3
8.324610– 4
4.054610– 3
5.728610– 3
7.750610– 3
5.431610– 3
6.839610– 3
4.241610– 3
4.733610– 3
5.135610– 3
7.517610– 4
1
1
2
3
4
5
6
7
8
9
10
11
12
13
(C=N); H-NMR (DMSO-d6, 300 MHz,) d (ppm): 4.16 (br, s, 1H, NH-
benzimidazole), 7.16 (m, 2H, Ar-H), 7.59 (s, 2H, Ar-H); 13C-NMR
(DMSO-d6, 75 MHz,) d (ppm): 111.97 (s, C, Ar-C), 137.1 (s, C, Ar-C),
145.03 (s, C, C of imidazole), 171.2 (s, C, C of carboxylic acid);
CIMS m/z: 162.14: Anal. Calcd. for C8H6N2O2: C, 59.26; H, 3.73; N,
17.28; O, 19.73. Found: C, 59.25; H, 3.74; N, 17.29; O, 19.71. M.p.:
170–1728C.
General procedure for the preparation of 1H-
benzimidazole-2-carboxylic acid hydrazide III
1.621 g (0.01 mol) of 1H-benzimidazole-2-carboxylic acid and thi-
onyl chloride 1.78 g (0.01 mol) were refluxed for about 1 h.
Excess thionyl chloride was removed by repeated evaporation in
vacuo. Finally, we get 1H-benzimidazole-2-carboxylic acid chlor-
ide as intermediate product. An appropriate amount of 1H-ben-
zimidazole-2-carboxylic acid chloride (1.803 g, 0.01 mol) and
hydrazine hydrate (1.761 g, 0.01 mol) in 15 mL of dry ethanol
was refluxed for 3–4 h. After completion of the reaction, the sol-
ution was concentrated and the resulting solid was collected,
washed with ether, and recrystallized from DMSO / water.
IR (KBr) m (cm– 1): 3370 (aliphatic NH2), 3356.5 br (ArNH), 2915
(aliphatic NH), 1652 (C=O), 1579 (C=N); 1H-NMR (DMSO-d6,
300 MHz) d (ppm): 2.3 (s, 2H, aliphatic NH2, 3.93 (br, s, 1H, NH-
benzimidazole), 7.21 (s, 2H, Ar-H), 7.63 (d, J = 8.1 Hz, 2H, Ar-H),
8.24 (s, 1H, aliphatic NH); 13C-NMR (DMSO-d6, 75 MHz) d (ppm):
114.0 (s, C, Ar-C), 121.6 (s, C, Ar-C), 139.9 (s, C, Ar-C), 142.7 (s, C, C
of imidazole), 161.04 (s, C, C of C=O); CIMS m/z: 176.17: Anal.
Calcd. for C8H8N4O: C, 54.54; H, 4.58; N, 31.80; O, 9.08. Found: C,
54.55; H, 4.57; N, 31.79; O, 9.09. M.p.: 217–2198C.
Conclusion
This synthetic approach provides convenient access to
new structurally complex, pharmacologically interesting
five- and six-member benzimidazole derivatives in good
purities. The bioassays indicated that all the compounds
showed in-vitro antimicrobial activity against six strains
of bacteria and three strains of fungi. Compounds 2, 4, 7,
and 13 displayed potent cytotoxic activity against Artemia
salina.
This research work is financially supported by the University
Grant Commission (UGC), New Delhi – 110 002 (Ref. No 33-296/
2007 (SR) dated 28-02-2008). Dr. K. M. Hosamani is indebted to
the British Commonwealth Scholarship Commission, UK, (Ref.
No. INCF-2008-68).
2-(1H-Benzimidazole-2-ylcarbonyl)-5-methyl-2,4-
dihydro-3H-pyrazol-3-one 1
The authors have declared no conflict of interest.
A mixture of carbohydrazide (0.01 mol) and ethyl acetoacetate
(0.01 mol) in absolute ethanol (20 mL) was heated at reflux tem-
perature for 3–4 h. The reaction mixture was cooled and the
formed precipitate was filtered off and recrystalized to yield
compound 1.
Experimental
IR (KBr) m (cm– 1): 3310 br (ArNH), 744 (C=O of pyrazolone),
Materials and methods
1
1592 (C=O), 1563 (C=N); H-NMR (DMSO-d6, 300 MHz,) d (ppm):
Thin layer chromatography was used to confirm the completion
of the reaction and the purity of the compounds synthesized.
Melting points were determined with open capillary method
and are uncorrected. IR spectra were recorded on a Nicolet 5700
FT-IR instrument (Nicolet, Madison, WI, USA) as potassium bro-
mide discs. The 1H- and 13C-NMR spectra were measured with a
Bruker-300 (Bruker Bioscience, USA), 300 MHz instrument using
TMS as internal standard and DMSO-d6. All chemical shifts were
reported as d values (in ppm). Mass spectra with ionization
energy maintained at 70 eV using were taken on a Shimadzu
spectrometer (GC-MS) (Shimadzu, Tokyo, Japan).
2.50 (s, 3H, CH3 of pyrazolone), 3.86 (br, s, 1H, NH-benzimida-
zole), 7.33 (d, J = 7.8 Hz, 2H, Ar-H), 7.70 (m, 2H, Ar-H); 13C-NMR
(DMSO-d6, 75 MHz) d (ppm): 20.70 (s, C, CH3 of pyrazolone),
115.67 (s, C, Ar-C), 120.9 (s, C, Ar-C), 168.0 (s, C, C=O), 170.75 (s, C,
C=O of pyrazolone); CIMS m/z: 242.23: Anal. Calcd. for
C12H10N4O2: C, 59.50; H, 4.16; N, 23.13; O, 13.21. Found: C, 59.48;
H, 4.15; N, 23.10; O, 13.20.
N-(4-Oxo-2-phenyl-1,3-thiazolidin-3-yl)-1H-
benzimidazole-2-carboxamide 2
A mixture of carbohydrzide (0.01 mol) and the benzaldehyde
(0.01 mol) was refluxed in absolute ethanol (20 mL) for 5–6 h.
The reaction mixture was concentrated, cooled, and the formed
precipitate was filtered off, dried, and then recrystallized to give
the intermediate Schiff base. A mixture of the above-mentioned
compound (0.005 mol) and thioglycolic acid (0.006 mol) was
refluxed in dry benzene (30 mL) for 6–7 h. The solvent was
Chemistry
General procedure for the preparation of 1H-
benzimidazole-2-carboxylic acid II
1.861 g (0.01 mol) 2-(trifluoromethyl)-1H-benzimidazole was
added to a cooled solution of sodium hydroxide (30 mL, 1 N).
The resulting solution was kept for 1 h and the solution was fil-
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 412–419
New Benzimidazole Derivatives
417
evaporated and the reaction mixture was neutralized with cold
dilute sodium bicarbonate solution, the formed product was fil-
tered off and recrystallized from ethanol / water.
mixture was heated under reflux for 6–7 h, then it was concen-
trated, acidified with dilute HCl, and the resulting solid was col-
lected, washed with water, and recrystallized from a suitable sol-
vent to give the intermediate compound. A mixture of this com-
pound (0.01 mol) and 99% hydrazine hydrate (0.03 mol) in abso-
lute ethanol (20 mL) was refluxed for 6–7 h. The solvent and
excess hydrazine hydrate were removed under reduced pres-
sure, the residue washed with ether, then recrystallized from
DMF / water (1 : 1).
IR (KBr) m (cm– 1): 3240 br (ArNH), 2915 (CH), 2971 m (AlNH),
1723 (C=O of thiazolidinone), 1599 (C=O, CONH), 1552 (C=N); 1H-
NMR (DMSO-d6, 300 MHz) d (ppm): 3.12 (s, 2H, CH2 of thiazoli-
dinyl), 3.52 (br, s, 1H, NH-benzimidazole), 5.52 (s, 1H, CH of thia-
zolidinyl), 6.95 (d, J = 5.7 Hz, 2H, Ar-H), 7.07 (s, 1H, Ar-H), 7.16 (s,
2H, Ar-H), 7.39 (m, 1H, CONH), 7.28 (d, J = 7.4 Hz, 2H, Ar-H), 7.73
(s, 2H, Ar-H); 13C-NMR (DMSO-d6, 75 MHz) d (ppm): 35.6 (s, C, CH2
of thiazolidinyl), 115.32 (s, C, Ar-C), 133.49 (s, C, Ar-C), 163.01 (s,
C, C=O of thiazolidinyl), 167.32 (s, C, CONH); CIMS m/z: 338.38:
Anal. Calcd. for C13H12N4O: C, 64.99; H, 5.03; N, 23.32; O, 6.66.
Found: C, 64.98; H, 5.01; N, 23.30; O, 6.65.
IR (KBr) m (cm– 1): 3300 (NH2), 3145 br (ArNH), 2654 (SH), 1595
1
(C=N); H-NMR (DMSO-d6, 300 MHz) d (ppm): 3.62 (br, s, 1H, NH-
benzimidazole), 7.27 (s, 2H, Ar-H), 7.57 (d, J = 7.9 Hz, 2H, Ar-H),
7.84 (s, 1H, SH), 10.67 (s, 2H, NH2); 13C-NMR (DMSO-d6, 75 MHz) d
(ppm): 110.02 (s, C, Ar-C), 127.9 (s, C, Ar-C), 143.33 (s, C, Ar-C),
151.18 (s, C, Ar-C); CIMS m/z: 232.25: Anal. Calcd. for C9H8N6S: C,
46.54; H, 3.47; N, 36.18; S, 13.81. Found: C, 46.52; H, 3.46; N,
36.15; S, 13.80.
2-[(3,5-Dimethyl-1H-pyrazol-1-yl)carbonyl]-1H-
benzimidazole 3
A mixture of carbohydrazide (0.01 mol) and acetyl acetone
(0.01 mol) in absolute ethanol (20 mL) was heated at reflux tem-
perature for 4–5 h. The reaction mixture was cooled and the
formed precipitate was filtered off and recrystallized from etha-
nol / water.
3-(1H-Benzimidazole-2-yl)-6-chloro-7H-[1,2,4]triazole
[3,4-b] [1,3,4] thiadiazine 6
To a suspension of the corresponding compound 5 (0.01 mol) in
absolute ethanol (15 mL), chloro acetyl chloride (0.01 mol) was
added and the reaction mixture was refluxed for 2–3 h, cooled
to room temperature and then neutralized with ammonia. The
precipitate was filtered off and washed with water. The solid
obtained was recrystallized from DMSO / water (1 : 1).
IR (KBr) m (cm– 1): 3430 br (ArNH), 1595, 1551, 1501 & 1462 (4
IR (KBr) m (cm– 1): 3445 br (ArNH), 1623 (C=O), 1595 (C=N); 1H-
NMR (DMSO-d6, 300 MHz) d (ppm): 2.50 (d, 6H, CH3 of pyrazolyl),
4.13 (br, s, 1H, NH-benzimidazole), 7.33 (s, 1H, CH of pyrazolyl),
7.35 (d, J = 7.2 Hz, 2H, Ar-H), 7.70 (s, 2H, Ar-H); 13C-NMR (DMSO-d6,
75 MHz) d (ppm): 13.8 (s, C, CH3 of pyrazolyl), 122.3 (s, C, Ar-C),
125.3 (s, C, Ar-C), 143.23 (s, C, C=O); CIMS m/z: 240.26: Anal. Calcd.
for C17H14N4O2S: C, 60.34; H, 4.17; N, 16.56; S, 9.48. Found: C,
60.31; H, 4.16; N, 16.54; S, 9.46.
1
C=N); H-NMR (DMSO-d6, 300 MHz) d (ppm): 2.20 (s, 2H, CH2 of
thiadiazine), 4.76 (br, s, 1H, NH-benzimidazole), 7.28 (s, 2H, Ar-
H), 7.90 (d, J = 8.5 Hz, 2H, Ar-H) CIMS m/z: 290.73. Anal. Calcd. for
C11H7ClN6S: C, 45.44; H, 2.43; N, 28.91; S, 11.03; Cl, 12.19. Found:
C, 45.42; H, 2.41; N, 28.90; S, 11.01; Cl, 12.18.
N-[(2 E or Z)-4-Oxo-2-(phenylimino)-1,3-thiazolidin-3-yl]-
1H-benzimidazole-2-carboxamide 4
2-[1,2,4] Triazolo [3,4-b] [1,3,4] thiadizol-3-yl-1H-
benzimidazole 7
To a solution of carbohydrazide (0.01 mol) in ethanol (5 mL) phe-
nyl isothiocyanate (0.01 mol) and sodium hydroxide (0.01 mol, 2
N) were added. The mixture was stirred for 24 h and filtered. The
filtrate was acidified with hydrochloric acid. The precipitate was
filtered and recrystallized from ethanol / water, to give the inter-
mediate compound. A mixture of the above-mentioned com-
pound (0.005 mol) and chloroacetyl chloride (0.005 mol) in
chloroform (30 mL) was refluxed for 6-7 h. The solvent was dis-
tilled off under reduced pressure and the residue was washed
with ethanol, filtered, washed again with water, and recrystal-
lized from DMSO / water, to give compound 4.
A mixture of the corresponding compound 5 (0.01 mol) and for-
mic acid (1 mL) in dry benzene (20 mL) was refluxed for 1 h. After
evaporating the reaction mixture under reduced pressure, an
oily product was obtained. The crude product was crystallized
from a suitable solvent. The obtained product (0.001 mol) was
treated with cold concentrated sulfuric acid (15 mL) and the
formed oily mass was poured into water. The precipitate was
recrystallized from DMSO / water (1 : 1).
IR (KBr) m (cm– 1): 3446 br (ArNH), 1595, 1552, 1500 & 1462 (4
IR (KBr) m (cm– 1): 3244 br (ArNH), 3060 m (AlNH), 1753 (C=O of
thiazolidinyl), 1583 (C=N); 1H-NMR (DMSO-d6, 300 MHz) d (ppm):
3.81 (s, 2H, CH2 of thiazolidinyl), 4.25 (br, s, 1H, NH-benzimida-
zole), 7.28 (d, J = 6.9 Hz, 2H, Ar-H), 7.42 (s, 5H, Ar-H), 7.89 (s, 2H,
Ar-H), 10.51 (m, 1H, CONH); 13C-NMR (DMSO-d6, 75 MHz) d (ppm):
33.20 (s, C, CH2 of thiazolidinyl), 127.01 (s, C, Ar-C), 130.22 (s, C,
Ar-C), 165.33 (s, C, CONH), 173.1 (s, C, C=O of thiazolidinyl); CIMS
m/z: 351.38: Anal. Calcd. for C17H13N5O2S: C, 58.11; H, 3.73; N,
19.93; S, 9.13; O, 9.11. Found: C, 58.10; H, 3.71; N, 19.91; S, 9.12;
O, 9.12.
C=N); H-NMR (DMSO-d6, 300 MHz) d (ppm): 3.45 (br, s, 1H, NH-
1
benzimidazole), 7.27 (d, J = 7.0 Hz, 2H, Ar-H), 7.43 (s, 2H, Ar-H),
8.21 (s, 1H, CH of thiadiazole); CIMS m/z: 242.26: Anal. Calcd. for
C10H6N6S: C, 49.58; H, 2.50; N, 34.69; S, 13.24. Found: C, 49.55; H,
2.47; N, 34.67; S, 13.22.
3-(1H-Benzimidazole-2-yl)-[1,2,4] triazolo [3,4-b] [1,3,4]
thiadiazole-6-thiol 8
To a solution of the corresponding compound 5 (0.01 mol) in
ethanol, KOH (1 g) and carbon disulphide (2 mL) were added and
the reaction content was allowed to reflux for 2–3 h. The solvent
was removed under reduced pressure; then, ice water added
onto the reaction content while stirring. The obtained solid was
washed with water and recrystallized from ethanol / water.
IR (KBr) m (cm– 1): 3401 br (ArNH), 2655 (SH), 1595, 1552, 1500 &
4-Amino-5-(1H-benzimidazole-2-yl)-4H-[1,2,4] triazole-3-
thiol 5
To a mixture of carbohydrazide (0.01 mol) in ethanol (20 mL)
and a solution of potassium hydroxide (0.015 mol) in ethanol
(10 mL) was added followed by carbon di-sulfide (10 mL); the
1
1462 (4 C=N); H-NMR (DMSO-d6, 300 MHz) d (ppm): 3.68 (br, s,
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

418
R. S. Harisha et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 412–419
1H, NH-benzimidazole), 7.27 (d, J = 7.3 Hz, 2H, Ar-H), 7.57 (s, 1H,
SH), 7.89 (d, J = 8.1 Hz, 2H, Ar-H); CIMS m/z: 274.33. Anal. Calcd.
for C10H6N6S2: C, 43.78; H, 2.20; N, 30.64; S, 23.38. Found: C,
43.76; H, 2.20; N, 30.63; S, 23.37.
6-Chloro-4-{[ 2-(trifluoromethyl)-1H-benzimidazol-1-
yl]methyl}-2H-chromen-2-one 12
Compound 12 was prepared according to the procedure
described above for compound 11.
IR (KBr) m (cm– 1): 1715 (C=O), 1601 (C=N), 1147 (C-O), 1117 (CF3),
742 (C-Cl); 1H-NMR (DMSO-d6, 300 MHz) d (ppm): 2.98 (s, 2H, CH2),
5.61 (s, 1H, Ar-H), 5.66 (s, 1H, Ar-H), 7.28 (s, 1H, Ar-H), 7.38 (d, J =
7.5 Hz, 2H, Ar-H), 7.61 (s, 1H, Ar-H), 7.64 (d, J = 8.0 Hz, 2H, Ar-H);
CIMS m/z: 378.73. Anal. Calcd. for C18H10ClF3N2O2: C, 57.08; H,
2.66; N, 7.40; O, 8.45; Cl, 9.36; F, 15.05. Found: C, 57.09; H, 2.68;
N, 7.43; O, 8.44; Cl, 9.37; F, 15.04.
2-(6-Methyl-[1,2,4] triazolo [3,4-b] [1,3,4] thiadiazol-3-yl-)-
1H-benzimidazole 9
To a mixture of the corresponding compound 5 (0.01 mol), phos-
phorus oxychloride (10 mL) was added and the reaction contents
were refluxed for 2–3 h on a water bath. After removing the
excess of phosphorus oxychloride under reduced pressure, ice
water was added to the residue with vigorous stirring. The pre-
cipitate was filtered off and washed with 20% sodium bicarbon-
ate solution and water. This white solid was recrystalized from
DMSO / water (1 : 1).
1-{[2-(Trifluoromethyl)-1H-benzimidazol-1-yl]methyl}-3H-
benzo [f] chromen-3-one 13
Compound 13 was prepared according to the procedure
described above for compound 11.
IR (KBr) m (cm– 1): 3430 br (ArNH), 1621, 1551, 1500 & 1460 (4
C=N); 1H-NMR (DMSO-d6, 300 MHz) d (ppm): 1.62 (s, 3H, CH3), 3.72
(br, s, 1H, NH-benzimidazole), 7.18 (d, J = 7.4 Hz, 2H, Ar-H), 7.61
(s, 2H, Ar-H); CIMS m/z: 256.29. Anal. Calcd. for C11H8N6S: C, 51.55;
H, 3.15; N, 32.79; S, 12.51. Found: C, 51.53; H, 3.14; N, 32.77; S,
12.49.
IR (KBr) m (cm– 1): 1726 (C=O), 1624 (C=N), 1169 (C-O), 1123 (CF3);
1H-NMR (DMSO-d6, 300 MHz) d (ppm): 2.46 (s, 3H, CH3), 4.79 (s, 2H,
CH2), 6.17 (s, 1H, Ar-H), 7.12 (s, 1H, Ar-H), 7.19 (s, 2H, Ar-H), 7.28
(d, J = 7.7 Hz, 2H, Ar-H), 7.48 (s, 1H, Ar-H), 7.61 (s, 2H, Ar-H), 7.72
(d, J = 8.2 Hz, 2H, Ar-H); CIMS m/z: 394.35: Anal. Calcd. for
C22H13F3N2O2: C, 67.01; H, 3.32; N, 7.10; O, 8.11; F, 14.45. Found:
C, 67.00; H, 3.31; N, 7.10; O, 8.10; F, 14.46.
Biological evaluation
3-(1H-Benzimidazol-2-yl)-5H-[1,2,4] triazolo [3,4–
b][1,3,4] thiadiazin-6(7H)-one 10
Antibacterial activity
a-Chloroacetic acid (0.012 mol) and (0.01 mol) compound 5 were
suspended in absolute ethanol (30 mL) along with anhydrous
fused sodium acetate (1 g) and refluxed for 4–6 h on a water
bath. The resulting clear solution was then concentrated,
cooled, and poured into ice water. The separated solid was fil-
tered and recrystalized from DMSO / water to give compound 10.
IR (KBr) m (cm– 1): 3410 br (ArNH), 1718 (C=O), 1595, 1551 &
The antibacterial activity of the benzimidazole derivatives was
tested by the agar disc-diffusion method against Gram-positive
and Gram-negative bacteria. Test-compound solutions were pre-
pared in DMSO, were serially diluted and loaded (10 lL) to sterile
filter-paper discs (6 mm diameter), which finally contained 25,
50, and 100 lg/mL of the compound per disc, respectively.
Impregnated disks were then dried for 1 h and placed on inocu-
lated plates. The seeded plates were incubated at 378C for 16 h.
The radii of inhibition zones (in mm) of triplicate sets were
measured and the standard MIC for streptomycin is Vancomycin-
resistant enteroccoccus (0.88–1.81), Staphylococcus aureus (f0.88),
Micrococcus (f0.88), Bacillus subtilis (0.88–1.81), Shigella dysentery
(1.81–3.36), and Escheria coli (f0.88). Every experiment in the
antibacterial assay was replicated thrice in order to define the
MIC values, as shown in Table 2.
1
1500 (3 C=N); H-NMR (DMSO-d6, 300 MHz) d (ppm): 3.80 (br, s,
1H, NH-benzimidazole), 7.27 (s, 2H, CH2 of thiadiazine), 7.56 (d, J
= 7.6 Hz, 2H, Ar-H), 7.91 (d, J = 8.3 Hz, 2H, Ar-H), 10.67 (br, s, 1H,
NH of thidiazine); CIMS m/z: 272.29. Anal. Calcd. for C11H8N6OS:
C, 48.52; H, 2.96; N, 30.86; S, 11.78; O, 5.88. Found: C, 48.51; H,
2.95; N, 30.83; S, 11.76; O, 5.89.
Among all compounds tested, 2, 4, and 13 exhibited better
antibacterial activity against the Gram-positive Bacillus subtilis
than the antibiotic streptomycin. On the other hand, compound
7 acted against the Gram-negative Escheria coli in a way compara-
ble with the antibiotic streptomycin.
7-Methyl-4-{[2-(trifluoromethyl)-1H-benzimidazol-1-
yl]methyl}-2H-chromen-2-one 11
2-(Trifluoromethyl)-1H-benzimidazole (0.01 mol) and anhydrous
potassium carbonate (0.01 mol) was stirred in dry acetone
(25 mL) for 30 min. 4-Bromomethyl coumarin (0.01 mol) was
added and stirring was continued for 24 h. The reaction mixture
was concentrated to one fourth of the original volume and
poured into ice-cold water. The solid separated was filtered and
washed with 50% HCl to neutralize excess potassium carbonate.
Then, the solid was washed with 100 mL of cold water and with
dilute ethanol. The crude product was dried and recrystallized
from DMF.
Antifungal activity
The synthesized benzimidazole derivatives were tested for their
antifungal activity in vitro in comparison with Nystatin (Nystatin
Ns 100, HIMEDIA, 100 units/disc) as a reference drug using the
standard agar disk diffusion method against three strains of
fungi (Aspergillus niger, Penicillium, and Candida albicans). A spore
suspension in sterile distilled water was prepared from 3 to 5
days-old culture of the test fungi growing on Potato Dextrose
Agar (PDA) media. The final spore concentration was 5610– 4
spores/mL. About 15 mL of the growth medium was placed into
sterilized Petri dishes of 9 cm diameter and inoculated with
100 lL of the spore suspension. Sterile 6-mm filter paper disk
(HiMedia Laboratories Pvt. Ltd, India) was saturated with 10 lL
IR (KBr) m (cm– 1): 1721 (C=O), 1621 (C=N), 1196 (C-O), 1139 (CF3);
1H-NMR (DMSO-d6, 300 MHz) d (ppm): 2.46 (s, 3H, CH3), 4.49 (s, 2H,
CH2), 6.47 (s, 1H, Ar-H), 7.19 (s, 1H, Ar-H), 7.26 (d, J = 7.5 Hz, 2H,
Ar-H), 7.28 (s, 1H, Ar-H), 7.57 (s, 1H, Ar-H), 7.60 (d, J = 7.9 Hz, 2H,
Ar-H); CIMS m/z: 358.31. Anal. Calcd. for C19H13F3N2O2: C, 63.69;
H, 3.66; N, 7.82; O, 8.93; F, 15.91. Found: C, 63.70; H, 3.68; N,
7.84; O, 8.92; F, 15.90.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2009, 342, 412–419
New Benzimidazole Derivatives
419
of the test compound solution. Impregnated disks were then
dried for 1 h and placed on inoculated plates. The seeded plates
were incubated at 278C for four days. The radii of inhibition
zones (in mm) of triplicate sets were measured and the standard
MIC for Nystatin is Aspergillus niger (f3.54), Penicillium (3.54), and
Candida albicans (f1.82) at 100 units, as showed in Table 3.
It is evident from the screening data (MIC shown in Table 3)
that compounds 2 and 4 were more effective against Aspergillus
niger, compared with the standard Nystatin. The remaining com-
pounds showed moderate antifungal activity against all fungal
species with a MIC of 50 lg/mL.
[9] J. T. Witkowski, R. K. Robins, G. P. Khare, R. W. Sidwell, J.
Med. Chem. 1973, 16, 935–937.
[10] V. Mathew, J. Keshavayya, V. P. Vaidya, Eur. J. Med. Chem.
2006, 41, 1048–1058.
[11] B. S. Holla, R. Gonsalvel, S. Shenoy, IL Farmaco 1998, 53,
574–578.
[12] B. S. Holla, B. Sorojini, S. B. Rao, P. M. Akberali, et al., IL
Farmaco 2001, 56, 565–570.
[13] M. Kritsanida, A. Mouroutsw, P. Marakos, N. Pouli, et al., IL
Farmaco 2002, 57, 253–257.
[14] A. R. Surrey, J. Am. Chem. Soc. 1949, 71, 3105–3107; J. Am.
Chem. Soc. 1949, 71, 3354–3356.
Cytotoxic activity
As can be seen from the data recorded in Table 4, only com-
pounds 2, 4, and 13 displayed cytotoxic activity as LD50 at concen-
trations of 1610– 3 M against Artemia salina, while the remain-
ing compounds were almost inactive in this assay.
[15] K. C. Joshi, S. Giri, J. Ind. Chem. Soc. 1963, 40, 42–44.
[16] J. Haglind, Chem. Abstr. 1966, 64, 16509.
[17] P. Laurin, D. Ferroud, L. Schio, M. Klich, et al., Bioorg. Med.
Chem. Lett. 1999, 9, 2875–2880.
[18] I. A. Khan, M. V. Kulkarni, C. M. Sun, Eur. J. Med. Chem.
2005, 40, 1168–1172.
References
[19] D. N. Nicolaides, K. C. Fylaktakidou, K. E. Litinas, D. Hadji-
[1] J. Z. Stefanska, R. Gralewska, B. J. Starosciak, Z. Kazimiere-
pavlou–Litina, Eur. J. Med. Chem. 1998, 33, 715–724.
zuk, Pharmazie 1999, 54, 879–884.
[20] H. H. Adavi, R. A. Kusanur, M. V. Kulkarni, J. Ind. Chem. Soc.
2004, 81, 981–985.
[2] M. Andrzejewska, M. A. Pagano, F. Meggio, A. M. Brunati,
Z. Kazimierezuk, Bioorg. Med. Chem. 2003, 11, 3997–4002.
[21] National Committee for Clinical Laboratory Standards,
NCCLS Approval Standard Document M2–A7. Villanova,
PA, USA, 2000.
[3] M. Andrzejewska, L. Yepez–Mulia, R. Cedillo–Rivera, A.
Tapia, et al., Eur. J. Med. Chem. 2002, 37, 973–978.
[4] Z. Kazimierezuk, J. A. Upcroft, P. Upcroft, A. Gorska, et al.,
Acta Biochim. Pol. 2002, 49, 185–195.
[22] National Committee for Clinical Laboratory Standards,
NCCLS Approval Standard Document M7–A5. Villanova,
PA, USA, 2000.
[5] D. E. Burton, A. J. Lambie, J. C. Ludgate, G. T. Newbold, et
al., Nature 1965, 208, 1166–1169.
[23] H. William, Microbiological assay. An Introduction to Quanti-
tative Principles and Evaluation, Academic Press, New York,
1977.
[6] G. Navarrete–Vazquez, R. Cedillo, A. Hernandez–Campos,
L. Yepez–Mulia, et al., Bioorg. Med. Chem. Lett. 2001, 11,
187–190.
[24] B. N. Meyer, N. R. Ferrigni, J. E. Putnam, L. B. Jacobsen, et
al., Planta Med. 1982, 45, 31–34.
[7] G. Navarrete–Vazquez, L. Yepez, A. Hernandez–Campos,
A. Tapia, et al., Bioorg. Med. Chem. 2003, 11, 4615–4622.
[8] D. H. Boschelli, D. T. Connor, D. A. Barnemeier, J. Med.
Chem. 1993, 36, 1802–1810.
[25] A. W. Bauer, W. M. Kirby, J. C. Sherris, M. Turck, Am. J. Clin.
Pathol. 1966, 45, 493–496.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com