Synthesis and in-vivo evaluation of carbonyl-amide linkage based new benzimidazole derivatives

Article abstract of DOI:10.1002/ardp.201100068

In search of pharmacologically active potent compounds, a series of carbonyl-amide linkage based new benzimidazole derivatives were synthesized from acid, aldehydes and isocyanide at ambient temperature via Passerini reaction. All the compounds synthesize

Full text of DOI:10.1002/ardp.201100068

Arch. Pharm. Chem. Life Sci. 2012, 345, 65–72
65
Full Paper
Synthesis and In-vivo Evaluation of Carbonyl-amide Linkage
Based New Benzimidazole Derivatives
Irfan N. Shaikh1, Kallappa M. Hosamani1, Harisha R. Seetharamareddy1, and Mallinath H. Hugar2
1 P.G. Department of Studies in Chemistry, Karnatak University, Dharwad, India
2 Rajiv Gandhi University of Health Science, Luqman College of Pharmacy, Gulbarga, India
In search of pharmacologically active potent compounds, a series of carbonyl-amide linkage based
new benzimidazole derivatives were synthesized from acid, aldehydes and isocyanide at ambient
temperature via Passerini reaction. All the compounds synthesized were screened for their potential
anti-inflammatory, antidiabetic and anticonvulsant properties. The results revealed that compounds
2i and 2j were found to be the most potent anti-inflammatory agents, while compounds 2a, 2c, 2e, 2f,
2i and 2j showed increased antidiabetic activity than the reference drugs and 2a, 2g, 2h, 2i and 2j were
found to be the main structural requirement for maintaining anticonvulsant activity.
Keywords: Anticonvulsant property / Antidiabetic property / Anti-inflammatory property / Benzimidazole derivatives /
One-pot synthesis
Received: February 22, 2011; Revised: May 6, 2011; Accepted: May 6, 2011
DOI 10.1002/ardp.201100068
Introduction
that may be of use in designing new, potent and less toxic
anti-inflammatory agents.
Inflammation is a local reaction of the vascular and support-
ing elements of a tissue to injury resulting in the formation
of protein-rich exudates; it is a protective response of the
nonspecific immune system that serves to localize, neutral-
ize, or to destroy an injurious agent in preparation for the
process of healing. The cardinal signs of inflammation are
rubor (redness), calor (heat), dolor (pain), tumor (swelling)
and functio laesa (loss of function). Cause of inflammation
includes physical agents, chemical agents, immunological
reactions and infection by pathogenic organism [1]. Non-
steroidal anti-inflammatory drugs (NSAIDs) are useful tools
in the treatment of acute and chronic inflammation [2], pain
[3] and fever [4]. However, long-term clinical usage of NSAIDs
is associated with significant side effects of gastrointestinal
lesions, bleeding and nephrotoxicity [5, 6]. Therefore, the
discovery of new and safer anti-inflammatory drugs
represents a challenging goal for such a research area [7].
As resistance to anti-inflammatory drugs is widespread, there
is an increasing need for identification of novel structures
Diabetes mellitus is a common endocrine disorder charac-
terized by chronic hyperglycemia, alterations in carbo-
hydrate and lipid metabolism. The hyperglycemia causes
extensive tissue damage appearing in eyes, kidneys, nerves,
heart and blood vessels [8]. The prevalence of diabetes keeps
increasing with age, reaching a plateau at 10–20% in people
over 70 years old. The number of diabetic people is expected
to rise from present estimate of 150 million to 230 million in
2025 [9, 10]. Nowadays, benzimidazoles play a vital role for
the treatment of diabetes in spite of other therapeutic
options.
Epilepsy is one of the most common diseases of the brain
affecting at least 50 million persons worldwide. There is
currently a need of improved agents for the treatment of
seizures, since currently available drugs are effective only in
60–80% of epileptic patients [11]. Polytherapy with antiepi-
leptic drugs (AEDs) is necessary in clinical practice because of
the limited efficacy of monotherapy [12]. During the past
decade several new drugs were approved (Rufinamide,
Retigabine, Pregabaline, Remacemide). However, none of
the available antiepileptic drugs is an ideal one as these
can be associated with chronic and toxic side effects [13].
Thus, the search for the new anti-convulsant drugs
continues to be an active area of investigation in medicinal
chemistry.
Correspondence: Kallappa M. Hosamani, P.G. Department of Studies
in Chemistry, Karnatak University, Pavate Nagar, Dharwad – 580 003,
India.
E-mail: dr_hosamani@yahoo.com
Fax: þ91-836-2771275, þ91-836-2747884
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

66
I. N. Shaikh et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 65–72
Benzimidazole moiety plays an important heterocyclic
CHO
pharmacophore in the pharmaceutical chemistry and drug
discovery. These compounds carrying different substituents
in benzimidazole biomolecules are associated with a wide
range of biological and pharmacological activities including
anticancer [14], antiviral [15], antimicrobial [16], antihelmen-
tic [17], anti-inflammatory [18], antihistaminic [19], antioxi-
dant [20], antihypertensive [21] and anticoagulant [22]
properties. These derivatives also exhibit significant activities
against several viruses such as HIV [23].
N
-
C
+
+
S
N
N
H
COOH
+
R
Ambient
Temperature
3 - 4 h
Stirring
Some of the important benzimidazole derivatives have
been reported as proton pump inhibitor [24], anti-dopamin-
ergic Domperidone [25], anti-psychotic Pimozide [26], thyroid
receptor agonists [27], gonadotropin releasing hormone
receptor antagonists [28] and interestingly alkynylbenzimi-
dazoles as modulators of metabotropic glutamate receptors
[29]. Recently, we have also published some series of biologi-
cally active benzimidazole derivatives [30, 31]. There is a lack
of literature on the synthesis and biological evaluation of
carbonyl-amide linkage based benzimidazole derivatives.
Owing to the immense biological importance of benzimida-
zole libraries, we wish to report synthesis of a new class of
benzimidazole derivatives and their biological activity
screening studies.
R
N
S
O
N
H
O
H
N
O
Where R= 2a) H, 2b) 2-CH3, 2c)4-CH3, 2d)2-OCH3, 2e)4-OCH3, 2f) 4-C2H5,
2g) 2-NO2, 2h) 4-NO2, 2i) 2-Cl, 2j) 4-Cl, 2k)2,4-NO2, 2l) 2,4-Cl
Scheme 1. Schematic representation of compounds 2a–2l.
was excellent and varied according to the substitution pat-
tern of the aromatic aldehydes. Structure of the synthesized
Results and discussion
1
compounds was confirmed on the basis of IR, H-NMR, 13C-
Chemistry
NMR and mass spectral data. The IR spectra of all compounds
(2a–l) have aromatic –NH stretching bands in the range of
3488–3420 cmꢀ1, whereas aliphatic-NH stretching bands
In the present protocol, we are reporting one-pot synthesis of
new benzimidazole derivatives using 1H-benzimidazol-2-yl-
sulfanyl acetic acid, substituted aldehydes and butyl-isocya-
nide (1:1:1) with stirring at ambient temperature in the
presence of water to give desired products as shown in
Table 1 (see also Scheme 1). Aromatic aldehydes substituted
with both electron withdrawing and electron donating
groups could be used successfully. The yield of the products
appear in between 3157–3138 cmꢀ1. While C O stretching
–
–
bands of carboxylic acid appeared from 1688–1632 cmꢀ1 and
–
similarly C O stretching bands of CONH was observed from
–
1
1524-1578 cmꢀ1. In the H-NMR spectra of the target substi-
tuted benzimidazoles, the –NH proton of benzimidazole ring
resonated as a broad singlet in the region d 3.95–4.17 ppm
and NH proton of CONH was observed as singlet in the region
d 7.77–7.96 ppm and was exchangeable on D2O addition con-
firming the formation of carbonyl-amide linkage. A singlet in
Table 1. Physical Data of Synthesized Compounds (2a–2l).
Compound
R
Time
(hrs)
Yielda
(%)
Melting
Point (8C) the range of d 6.43–6.93 can be assigned to O–CH. The other
protons appeared at the expected chemical shifts. 13C-NMR
225–228
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
3.25
3.45
3.55
3.15
3.20
3.35
3.30
3.10
3.40
3.50
3.25
3.40
90
91
83
87
79
85
78
90
82
86
88
84
spectral data for the title compounds most characteristic peak
2-CH3
4-CH3
2-OCH3
4-OCH3
4-C2H5
2-NO2
4-NO2
2-Cl
231–232
around d 169.6–171.9 ppm (CONH) indicated the formation
of carbonyl-amide linkage. Mass spectra showed accurate
molecular ion peak with respect to the targeted compounds.
185–187
218–220
201–202
175–176
242–244
159–161
195–197
212–214
168–169
238–240
Pharmacology
All the compounds prepared herein were screened for their
potential in-vivo biological activities such as anti-inflamma-
tory, antidiabetic and anticonvulsant activities. Anti-inflam-
matory activity was evaluated using carrageenan induced
hind paw edema method [32]. Evaluation of antidiabetic
2j
2k
2l
4-Cl
2,4-NO2
2,4-Cl
a
Isolated yields
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Arch. Pharm. Chem. Life Sci. 2012, 345, 65–72
Carbonyl-amide Linkage Based Benzimidazole Derivatives
67
Table 2. Anti-inflammatory activity of synthesized compounds (2a–2l) by carrageenan-induced hind paws edema method.
Groups
Dose
Paw volume (mean ꢁ SEM)
mg/kg p.o.
0 hr
1 hr
2 hr
3 hr
4 hr
6 hr
Control
Std. drug
Diclofenac
sodium
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
Tween 80 1.356 ꢁ 0.001
200 mg/kg 0.862 ꢁ 0.002
1.303ꢁ0.001
1.332 ꢁ 0.001
0.647 ꢁ 0.001
1.334 ꢁ 0.001
0.588 ꢁ 0.001
1.448 ꢁ 0.002
0.636 ꢁ 0.002
1.493 ꢁ 0.001
0.768 ꢁ 0.001
0.664 ꢁ 0.002
400 mg/kg 1.153 ꢁ 0.002ꢂꢂꢂ 1.285 ꢁ 0.005ꢂꢂ 1.226 ꢁ 0.003ꢂꢂꢂ 1.189 ꢁ 0.002ꢂꢂ 1.343 ꢁ 0.003ꢂꢂ 1.288 ꢁ 0.005ꢂꢂ
400 mg/kg 0.855 ꢁ 0.003
400 mg/kg 0.752 ꢁ 0.004ꢂꢂ
400 mg/kg 1.150 ꢁ 0.002
400 mg/kg 1.252 ꢁ 0.001
400 mg/kg 0.656 ꢁ 0.001
400 mg/kg 1.226 ꢁ 0.002
0.778 ꢁ 0.004
0.623 ꢁ 0.002
0.887 ꢁ 0.001
0.768 ꢁ 0.005
0.987 ꢁ 0.003
0.893 ꢁ 0.005ꢂꢂ 0.778 ꢁ 0.003ꢂꢂ
0.682 ꢁ 0.001ꢂꢂ 0.830 ꢁ 0.005ꢂꢂ 0.785 ꢁ 0.006ꢂꢂ
1.196 ꢁ 0.003
1.293 ꢁ 0.001
0.593 ꢁ 0.001
1.218 ꢁ 0.003
1.328 ꢁ 0.003
1.328 ꢁ 0.001
0.328 ꢁ 0.001
1.208 ꢁ 0.001
1.067 ꢁ 0.005
1.387 ꢁ 0.001
0.458 ꢁ 0.001
1.209 ꢁ 0.001
1.112 ꢁ 0.004
1.442 ꢁ 0.002
0.642 ꢁ 0.002
1.211 ꢁ 0.004
1.406 ꢁ 0.003
1.485 ꢁ 0.001
0.544 ꢁ 0.001
1.216 ꢁ 0.004
400 mg/kg 0.551 ꢁ 0.002ꢂꢂꢂ 0.599 ꢁ 0.003ꢂꢂ 0.457 ꢁ 0.001ꢂꢂ
0.684 ꢁ 0.004ꢂꢂ 0.542 ꢁ 0.004ꢂꢂ 0.583 ꢁ 0.002ꢂꢂꢂ
400 mg/kg 0.848 ꢁ 0.004
400 mg/kg 0.658 ꢁ 0.004ꢂꢂ
0.793 ꢁ 0.005
0.828 ꢁ 0.003
0.687 ꢁ 0.002
0.442 ꢁ 0.001
0.785 ꢁ 0.004
0.892 ꢁ 0.005ꢂꢂ 0.721 ꢁ 0.002ꢂꢂ
0.725 ꢁ 0.002ꢂꢂ 0.842 ꢁ 0.001ꢂꢂ 0.679 ꢁ 0.003ꢂꢂꢂ
400 mg/kg 0.851 ꢁ 0.003ꢂꢂꢂ 0.793 ꢁ 0.002ꢂꢂ 0.628 ꢁ 0.004ꢂꢂꢂ 0.687 ꢁ 0.001ꢂꢂ 0.642 ꢁ 0.005ꢂꢂ 0.685 ꢁ 0.002ꢂꢂ
400 mg/kg 1.151 ꢁ 0.002
1.123 ꢁ 0.001
1.148 ꢁ 0.003
1.237 ꢁ 0.001
1.242 ꢁ 0.004
1.185 ꢁ 0.001
Data was analyzed by unpaired one-way ANOVA test.
Each value represents the mean ꢁ SEM (n ¼ 6).
ꢂP < 0.05, ꢂꢂP < 0.01, ꢂꢂꢂP < 0.001 as compared with control Student’s ‘t’ test.
activity was done by blood glucose test and oral glucose
tolerance test (OGTT) [33, 34]. Male Wister albino rats (150–
200 g) of either sex were selected. The anticonvulsant activi-
ties of the synthesized compounds were tested through an in-
vivo rodent model of convulsions (maxima electro shock, MES
test) [35, 36].
group, a methyl residue (2b, 2c). On the other hand, compar-
ing the activity of the compounds 2d–2h, it was observed that
replacing the hydrogen atom by a methoxy, ethyl and nitro
group reduces the anti-inflammatory activity.
Antidiabetic evaluation
In the acute toxicity study, all the synthesized compounds
showed better toxic effects when observed for the parameters
during 2nd h, then frequently for 4th h and finally for 9th day.
No mortality was observed, the substance is found to be safe
at the tested dose level of 350 mg/kg body weight. The results
of blood glucose of control and all the synthesized com-
pounds are shown in Table 3. Oral anti-diabetic efficiencies
of twelve compounds (2a–2l) were assessed using an oral
glucose tolerance test (OGTT) in normal rat model. The mar-
keted drug Glibenclamide was used as standard drug. The
sugar lowering effect of these compounds is presented in
Table 3. Compounds 2c, 2d, 2h, 2j and 2l showed better
reduction in the blood glucose levels on 9th day compared
with diabetic control rats. Oral administrations of the same
compounds processed good reduction in the glucose levels in
diabetic rats. Compounds 2a, 2c, 2e, 2f, 2i and 2k showed
increased activity compared to the reference drug (P < 0.001)
Glibenclamide at 90 min interval (Table 3). The acute toxicity
study of all the compounds showed no death of these rats
even under high levels indicating the margin of safety. These
results indicate compounds mentioned above showed better
anti-diabetic activity, maybe due to its protective role against
pancreatic injury in diabetic rats.
Anti-inflammatory evaluation
In the acute oral toxicity study, no mortality was noticed
within 48 h by any of the compounds up to 1500 mg/kg p.o.
dose level in the present study. However, some behavioral
changes were noticed depending at the higher dose
(400 mg/kg). The effect of the tested compounds as well as
reference standard were measured before and 1, 2, 3, 4, 5
and 6 h after carrageenan injection. Most of the tested com-
pounds showed a reasonable inhibition of edema size in
comparison with standard drug Diclofenac sodium. From
the obtained results (Table 2), it has been observed that newly
prepared compounds (2a–2l) reveal better anti-inflammatory
properties comparable to that of Diclofenac sodium which
was used as a reference standard. Structure–activity relation-
ships based on the observed results indicated that the type of
group substitution attached to the phenyl ring plays a
controlling role for developing the exhibited pharmacologi-
cal properties. It has been noticed that substitution of
an electron-withdrawing group, a chlorine atom at the
o-position (2i) and p-position (2j) on the phenyl ring, seems
more favorable for constructing an anti-inflammatory active
agent than the case of substitution with an electron-donating
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68
I. N. Shaikh et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 65–72
Table 3. Anti-diabetic activity of synthesized compounds (2a–2l) by oral glucose tolerance test.
Treatment & Groups
Blood glucose level (mg/dL)
Oral glucose tolerance test (OGTT) in non-diabetic rats
Basal
9th day
Fasting
30 min
90 min
Control
73.80 ꢁ 1.36
80.29 ꢁ 0.40
82.12 ꢁ 1.57
–
180.32 ꢁ 3.68
–
136.66 ꢁ 2.85
–
Diabetic control (Alloxan) 330.52 ꢁ 2.64
336.68 ꢁ 1.79
Glibenclamide (SD)
332.20 ꢁ 4.31
328.20 ꢁ 4.24
342.20 ꢁ 12.52
353.23 ꢁ 6.58
326.33 ꢁ 5.13
321.20 ꢁ 7.28
340.34 ꢁ 12.53
342.20 ꢁ 6.54
334.33 ꢁ 5.20
337.17 ꢁ 4.29
323.33 ꢁ 5.10
369.22 ꢁ 6.55
324.33 ꢁ 5.10
140.18 ꢁ 2.64
82.33 ꢁ 3.29
78.32 ꢁ 1.89
72.39 ꢁ 3.20
81.28 ꢁ 2.59
80.83 ꢁ 3.55
79.30 ꢁ 2.81
74.36 ꢁ 3.33
83.28 ꢁ 2.45
82.73 ꢁ 3.52
78.30 ꢁ 1.73
81.86 ꢁ 3.55
88.28 ꢁ 2.57
82.80 ꢁ 3.54
178.40 ꢁ 2.95
170.62 ꢁ 6.34
173.40 ꢁ 5.98
163.80 ꢁ 6.42
166.62 ꢁ 3.03
160.66 ꢁ 4.38
173.42 ꢁ 5.82
162.80 ꢁ 6.44
168.66 ꢁ 3.33
171.62 ꢁ 6.37
163.62 ꢁ 3.09
164.80 ꢁ 6.40
165.62 ꢁ 3.02
104.82 ꢁ 2.23ꢂꢂ
104.43 ꢁ 3.28ꢂꢂ
128.04 ꢁ 4.54
107.25 ꢁ 2.80ꢂꢂ
127.24 ꢁ 2.89ꢂꢂ
106.42 ꢁ 3.27
116.06 ꢁ 4.52ꢂꢂ
128.18 ꢁ 2.71
118.22 ꢁ 2.82
116.43 ꢁ 3.29ꢂꢂ
125.21 ꢁ 2.86
108.24 ꢁ 2.82ꢂꢂ
125.27 ꢁ 2.86
2a
2b
2c
2d
2e
2f
2g
2h
2i
384.68 ꢁ 12.55
373.18 ꢁ 25.62ꢂꢂꢂ
182.20 ꢁ 16.21
166.26 ꢁ 15.20ꢂꢂꢂ
334.62 ꢁ 12.54
371.14 ꢁ 25.68
372.27 ꢁ 16.23
175.26 ꢁ 15.26ꢂꢂꢂ
379.63 ꢁ 12.57
165.25 ꢁ 15.20ꢂꢂꢂ
364.27 ꢁ 16.29
163.25 ꢁ 15.20ꢂꢂꢂ
2j
2k
2l
Data was analyzed by unpaired one-way ANOVA followed by Dunnet’s test.
Each value represents the mean ꢁ SEM.
P > 0.05 is considered as non-significant (ns), P < 0.05 is considered as significant.
ꢂꢂꢂP < 0.001 as compared to normal control group.
ꢂꢂ
P < 0.001 as compared to normal control group.
Control ¼ Normal saline.
Diabetic control ¼ Alloxan.
SD ¼ Standard drug Glibenclamide.
Anticonvulsant evaluation
devoid of toxicity at the dose tested. From the structure
activity relationship, it is evident that the presence of a
chloro- and nitro-function at ortho- and para-position of the
phenyl ring as seen with compounds 2g, 2h, 2i and 2j was
Results of anticonvulsant activity with standard drug
Phenytoin are tabulated in Table 4. The compounds pre-
sented in Table 4 showed good protection and most were
Table 4. Anti-convulsant activity of synthesized compounds (2a–2l) by maximal electroshock model.
Treatment
Dose (mg/kg)
Time (s) in various phases of convulsion (mean ꢁ SEM)
Flexon
Extensor
Clonus
Stupor
Recovery/Death
Control Gum acacia
Phenytoin (SD)
1 % w/w
20
80
84
65
70
84
62
75
70
85
76
72
3.16 ꢁ 0.32
11.07 ꢁ 0.58ꢂꢂ
0.0 ꢁ 0.00
16.78 ꢁ 2.54
4.85 ꢁ 4.29 ns
16.30 ꢁ 2.24ꢂꢂ
1.08 ꢁ 1.27ꢂ
1.35 ꢁ 1.35ꢂ
1.02 ꢁ 1.21ꢂ
1.29 ꢁ 1.22ꢂ
1.35 ꢁ 1.36ꢂ
15.64 ꢁ 2.12ꢂ
15.67 ꢁ 2.13ꢂ
1.83 ꢁ 1.29ꢂ
15.40 ꢁ 2.15ꢂ
14.62 ꢁ 2.17ns
12.65 ꢁ 2.28 ns
219.34 ꢁ 5.79
160.4 ꢁ 20.54ns
213.8 ꢁ 5.68ꢂ
208.6 ꢁ 4.81 ns
214.5 ꢁ 20.54ns
202.1 ꢁ 20.55ns
193.2 ꢁ 13.26ns
214.5 ꢁ 20.54ns
208.7 ꢁ 4.96ꢂ
208.6 ꢁ 4.95ꢂ
209.5 ꢁ 13.42ꢂ
210.7 ꢁ 4.83ꢂ
207.3 ꢁ 4.97 ns
208.4 ꢁ 5.66 ns
–
2.87 ꢁ 0.35 ns
3.30 ꢁ 0.36ns
4.23 ꢁ 1.08 ns
4.57 ꢁ 1.04 ns
3.96 ꢁ 1.16 ns
3.22 ꢁ 0.27 ns
4.55 ꢁ 1.04 ns
3.14 ꢁ 0.30 ns
3.14 ꢁ 0.31 ns
3.08 ꢁ 0.27ꢂ
3.16 ꢁ 0.25ꢂ
3.17 ꢁ 0.38 ns
3.02 ꢁ 0.25 ns
Recovery
Death
Death
Death
Death
Death
Death
Death
Death
Death
Death
Death
Death
2a
2b
2c
2d
2e
2f
2g
2h
2i
4.21 ꢁ 0.39ꢂꢂ
5.33 ꢁ 0.3ꢂ
12.2 ꢁ 0.58ꢂ
3.73 ꢁ 0.42ꢂꢂ
9.24 ꢁ 0.71ꢂꢂ
12.0 ꢁ 0.59ꢂꢂ
4.38 ꢁ 0.44ꢂꢂ
4.38 ꢁ 0.46
3.99 ꢁ 0.45ꢂꢂ
5.67 ꢁ 0.21ꢂꢂ
11.0 ꢁ 0.56ꢂ
9.50 ꢁ 0.49ꢂ
2j
2k
2l
60
Data was analyzed by unpaired one-way ANOVA test.
Each value represents the mean ꢁ SEM (n ¼ 6).
P > 0.05 is considered as non-significant (ns), P < 0.05 is considered as significant.
ꢂꢂ
P < 0.001 as compared to normal control group.
ꢂꢂꢂP < 0.001 as compared to standard drug.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 65–72
Carbonyl-amide Linkage Based Benzimidazole Derivatives
69
found to be the main structural requirement for maintaining Experimental
anti-convulsant activity. The chloro-substitution led to com-
pounds (2i, 2j) with slightly different behavior, but all charac-
terized by a very low toxicity: derivatives 2i and 2j, having a
chlorine on the phenyl ring in the 2 and 4 position, demon-
strated good activity in rats at 85 mg/kg and 76 mg/kg, while
the introduction of methyl and methoxy groups on the
phenyl ring (2b, 2c, 2d and 2e) was detrimental for
the protection against electrical induced seizures in rats.
The nitro-substitution afforded good protection, especially
for the derivatives 2g and 2h. Finally, the introduction of the
ethyl substituent led to a decrease of potency (2f). All the
compounds showed protection against seizures in extensor
phase in both phenytoin and tested compounds with control
group. The extensor phase time was remarkably reduced for
these compounds. Compound 2a showed remarkable change
in the clonus stage, while compounds 2b–j remains moderate
and compounds 2k–l were non-significant. These compounds
exhibited their ability to diminish the magnitude of tonic-
clonus seizures. In flexon and stupor no significant change
was observed. Only five compounds namely 2a, 2g, 2h, 2i and
2j showed rapid and prolonged action in stupor stage. These
compounds showed excellent (P < 0.001) anticonvulsant
activity when compared with standard Phenytoin drug as
tabulated in Table 4. Finally, these compounds represent new
structure that could be further optimized for future develop-
ment of more potent and selective anticonvulsant agents.
Instrumentation
All compounds were routinely checked by thin-layer chromatog-
raphy (TLC) on aluminum-backed silica gel plates. Melting points
were determined with open capillary method and are uncor-
rected. IR spectra were recorded on a Nicolet 5700 FT-IR instru-
1
ment (Nicolet, Madison, WI, USA) as KBr discs. The H-NMR and
13C-NMR spectra were measured with a Bruker-300 (Bruker
Bioscience, USA), 300 MHz instrument using DMSO as solvent
and TMS as internal standard. All chemical shifts were reported
as d values (ppm). Mass spectrometer with ionization energy
maintained at 70 eV using on Shimadzu Mass Spectrometer
(LCMS) and the elemental analysis was carried out by using
Heraus CHN rapid analyzer. The biological activities were carried
out at Luqman College of Pharmacy, Gulbarga, Rajiv Gandhi
University of Health Science, India.
General procedure for the synthesis of compounds
A
mixture of (1H-benzimidazol-2-yl-sulfanyl)-acetic acid
(0.01 mol), butyl-isocyanide (0.01 mol) and substituted aldehyde
(0.01 mol) was given into a round bottom flask. The reaction was
stirred at ambient temperature. After 40–50 min, precipitation
starts forming on the sides of the flask and the stirring should be
continued for further 3–4 h The progress of the reaction is
monitored by TLC using n-hexane/ethyl acetate (80:20) as eluents.
The reaction mixture was filtered to get a desired product and
recrystallized from methanol.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoylphenyl-methyl ester (2a)
Pale yellow solid, yield 1.8774 g; IR (KBr) nmax cmꢀ1: 3422 (br,
arom-NH), 3140 (aliph-NH), 1679 (CO of acid), 1576 (CO of CONH);
1H-NMR (300 MHz, DMSO) d ppm: 0.94 (t, J ¼ 3.8, 6.6 Hz, 3H,
CH3), 1.47 (q, J ¼ 5.0 Hz, 2H, CH2), 1.62 (d, J ¼ 6.6 Hz, CH2), 3.47
(s, 2H, CH2), 3.71 (s, 2H, CH2), 4.15 (br, 1H, NH of imidazole), 6.43
(s, 1H, O–CH), 7.21–7.26 (m, 5H, arom-H), 7.64–7.73 (q, 4H, arom-
H), 7.96 (s, 1H, aliph-NH); 13C-NMR (75 MHz, CDCl3) d ppm: 12.2,
21.6, 114.9, 122.5, 143.3, 170.6, 172.4; MS m/z: 398.02 (Mþ); anal.
calcd. for C21H23N3O3S: C, 63.45; H, 5.83; N 10.57%, Found: C,
63.43; H, 5.85; N, 10.60%.
Conclusion
In present study, we have demonstrated one-pot synthesis
of new benzimidazole derivatives in water at ambient
temperature via Passerini reaction. All the synthesized
compounds were screened for in-vivo anti-inflammatory,
antidiabetic and anticonvulsant activities. From the
results, it can be concluded that compounds 2i and 2j was
found to be the most potent anti-inflammatory compound,
whereas compounds 2b, 2c and 2k showed the moderate
inhibitory effect. 5 compounds (2c, 2d, 2h, 2j and 2l) at the
dose of 20 mg/kg exhibited considerably potent blood glu-
cose lowering activity. The anti-diabetic evaluation against
oral glucose tolerance test (OGTT) indicated that compounds
2a, 2c, 2e, 2f, 2i, and 2k showed increased activity than their
reference drug Glibenclamide. All compounds exhibited pro-
tection in the seizure models, viz., MES and 2a, 2g, 2h, 2i and
2j have emerged as the most active compounds in these
models with no neurotoxicity. SAR studies revealed the
critical role of chloro-function in the target compounds that
showed very promising in-vivo activities. So we conclude that,
further structural modifications of these molecules might
lead to the discovery of more potent in-vivo agents with still
lower neurotoxicity.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoyl-otolyl-methyl ester (2b)
White solid, yield 1.9058 g; IR (KBr) nmax cmꢀ1: 3435 (br, arom-
1
NH), 3142 (aliph-NH), 1632 (CO of acid), 1524 (CO of CONH); H-
NMR (300 MHz, DMSO) d ppm: 0.90 (t, J ¼ 3.3, 5.7 Hz, 3H, CH3),
1.30 (d, J ¼ 10 Hz, 2H, CH2), 1.62 (d, J ¼ 6.6 Hz, 2H, CH2), 2.34 (s,
3H, CH3), 3.16 (s, 2H, CH2), 3.87 (s, 2H, CH2), 3.95 (br, 1H, NH of
imidazole), 6.80 (s, 1H, O–CH), 7.15–7.21 (m, 4H, arom-H), 7.65–
7.68 (q, J ¼ 6.6 Hz, 4H, arom-H), 7.90 (s, 1H, aliph-NH); 13C-NMR
(75 MHz, CDCl3) d ppm: 12.9, 15.6, 21.5, 115.8, 123.3, 142.1, 170.8,
172.6; MS m/z: 410.41 (M); anal. calcd. for C22H25N3O3S: C, 61.21;
H, 6.12; N, 10.21%, Found: C, 61.23; H, 6.10; N, 10.19%.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoyl-ptolyl-methyl ester (2c)
Light brown solid, yield 1.7463 g; IR (KBr) nmax cmꢀ1: 3433 (br,
arom-NH), 3146 (alp-NH), 1679 (CO of acid), 1576 (CO of CONH);
1H-NMR (300 MHz, DMSO) d ppm: 0.92 (d, J ¼ 10 Hz, 3H, CH3),
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I. N. Shaikh et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 65–72
1.29 (s, 2H, CH2), 1.65 (d, J ¼ 6.6 Hz, 2H, CH2), 2.32 (s, 3H, CH3),
3.37 (s, 2H, CH2), 3.87 (s, 2H, CH2), 3.99 (br, 1H, NH of imidazole),
6.87 (s, 1H, O–CH), 7.22–7.27 (m, 4H, arom-H), 7.65–7.71 (m, 4H,
arom-H), 7.80 (s, 1H, aliph-NH); 13C-NMR (75 MHz, CDCl3) d ppm:
13.1, 18.9, 21.6, 116.1, 122.8, 142.9, 170.0, 171.9; MS m/z: 412.06
(Mþ); anal. calcd. for C22H25N3O3S: C, 61.21; H, 6.12; N, 10.21%,
Found: C, 61.19; H, 6.15; N, 10.23%.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoyl-(4-nitro-phenyl) methyl ester (2h)
White solid, yield 1.8741 g; IR (KBr) nmax cmꢀ1: 3424 (br, arom-
1
NH), 3252 (aliph-NH), 1683 (CO of acid), 1578 (CO of CONH); H-
NMR (300 MHz, DMSO) d ppm: 0.89 (t, J ¼ 3.3, 5.6 Hz, 3H, CH3),
1.29 (d, J ¼ 6.6 Hz, 2H, CH2), 1.62 (s, 2H, CH2), 3.29 (s, 2H, CH2),
3.72 (s, 2H, CH2), 3.96 (br, 1H, NH of imidazole), 6.60 (s, 1H, O–CH),
7.20–7.28 (m, 4H, arom-H), 7.56–7.77 (m, 4H, arom-H), 7.79 (s, 1H,
aliph-NH); 13C-NMR (75 MHz, CDCl3) d ppm: 14.1, 20.3, 112.2,
125.5, 143.0, 171.3, 172.8; MS m/z: 440.44 (M); anal. calcd.
for C21H22N4O5S: C, 57.00; H, 5.01; N, 12.66%, Found: C, 57.02;
H, 5.03; N, 12.62%.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoyl (2-methoxy-phenyl) methyl ester (2d)
White solid, yield 1.821 g; IR (KBr) nmax cmꢀ1: 3476 (br, arom-NH),
1
3145 (aliph-NH), 1679 (CO of acid), 1574 (CO of CONH); H-NMR
(300 MHz, DMSO) d ppm: 0.87 (d, J ¼ 10.3 Hz, 3H, CH3), 1.24
(s, 2H, CH2), 1.63 (s, 2H, CH2), 3.19 (s, 2H, CH2), 3.83 (s, 3H,
OCH3), 3.87 (s, 2H, CH2), 4.15 (br, 1H, NH of imidazole), 6.93 (s,
1H, O–CH), 7.04–7.14 (m, 4H, arom-H), 7.46 (q, J ¼ 4.9 Hz, 4H,
arom-H), 7.89 (s, 1H, aliph-NH); 13C-NMR (75 MHz, CDCl3) d ppm:
13.9, 22.1, 56.9, 116.1, 123.1, 144.8, 170.6, 172.8; MS m/z: 426.40
(M); anal. calcd. for C22H25N3O4S: C, 61.81; H, 5.89; N, 9.83%,
Found: C, 61.79; H, 5.91; N, 9.80%.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid butylcarbamoyl
(2-chloro-phenyl) methyl ester (2i)
Yellow solid, yield 1.7146 g, IR (KBr) nmax cmꢀ1: 3488 (br, arom-
NH), 3151 (aliph-NH), 1680 (CO of acid), 1571 (CO of CONH);
1H-NMR (300 MHz, DMSO) d ppm: 0.94 (s, 3H, CH3), 1.39 (s, 2H,
CH2), 1.63 (d, J ¼ 4.9, 2H, CH2), 3.20 (s, 2H, CH2), 3.75 (s, 2H, CH2),
4.17 (br, 1H, NH of imidazole), 6.66 (s, 1H, O–CH), 7.10–7.27 (s, 4H,
arom-H), 7.34–7.67 (s, 4H, arom-H), 7.77 (s, 1H, aliph-NH);
13C-NMR (75 MHz, CDCl3) d ppm: 13.3, 21.8, 112.7, 124.1,
145.5, 169.6, 172.3; MS m/z: 430.52 (M); anal. calcd. for
C21H22ClN3O3S: C, 58.39; H, 5.13, N, 9.73%, Found: C, 58.42; H,
5.10; N, 9.71%.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoyl (4-methoxy-phenyl) methyl ester (2e)
White solid, yield 1.6575 g; IR (KBr) nmax cmꢀ1: 3454 (br, arom-
1
–
NH), 3148 (aliph-NH), 1634 (CO of acid), 1574 (C O of CONH); H-
–
NMR (300 MHz, DMSO) d ppm: 0.93 (t, J ¼ 4.1, 6.8 Hz, 3H, CH3),
1.24 (d, J ¼ 6.6 Hz, 2H, CH2), 1.70 (s, 2H, CH2), 3.17 (s, 2H, CH2),
3.82 (s, 3H, OCH3), 3.87 (s, 2H, CH2), 4.13 (br, 1H, NH of imidazole),
6.91 (s, 1H, O–CH), 7.03–7.13 (m, 4H, arom-H), 7.42–7.45 (m, 4H,
arom-H), 7.91 (s, 1H, aliph-NH); 13C-NMR (75 MHz, CDCl3) d ppm:
14.2, 20.9, 58.3, 115.8, 123.5, 142.4, 171.0, 171.8; MS m/z: 426.38
(M); anal. calcd. for C22H25N3O4S: C, 61.81; H, 5.89; N 9.8%, Found:
C, 61.83; H, 5.87; N, 9.84%.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoyl (4-chloro-phenyl) methyl ester (2j)
White solid, yield 1.8014 g; IR (KBr) nmax cmꢀ1: 3482 (br, arom-
NH), 3138 (aliph-NH), 1684 (CO of acid), 1575 (CO of CONH);
1H-NMR (300 MHz, DMSO) d ppm: 0.99 (t, J ¼ 5.3 Hz, 3H, CH3),
1.23 (d, J ¼ 6.8, 2H, CH2), 1.58 (s, 2H, CH2), 3.23 (s, 2H, CH2), 3.76
(s, 2H, CH2), 4.14 (br, 1H, NH of imidazole), 6.91 (s, 1H, O–CH),
7.02–7.14 (m, 4H, arom-H), 7.43–7.65 (m, 4H, arom-H), 7.89 (s, 1H,
aliph-NH); 13C-NMR (DMSO, 75 MHz): d (ppm) 12.8, 20.9, 113.4,
122.6, 145.8, 168.7, 172.2; MS m/z: 432.02 (Mþ); anal. calcd.
for C21H22ClN3O3S: C, 58.39; H, 5.13; N, 9.73%, Found: C,
58.42; H, 5.11; N, 9.74%.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoyl-(4-ethyl-phenyl) ethyl ester (2f)
White solid, yield 1.7767 g; IR (KBr) nmax cmꢀ1: 3441 (br, arom-
NH), 3151 (aliph-NH), 1634 (CO of acid), 1575 (CO of CONH);
1H-NMR (300 MHz, DMSO) d ppm: 0.97 (t, J ¼ 3.3, 6.6 Hz, 3H,
CH3), 1.30 (d, J ¼ 6.6 Hz, 2H, CH2), 1.46 (s, 3H, ar-CH3), 1.62 (s, 2H,
CH2), 2.40 (s, 2H, ar-CH2), 3.22 (s, 2H, CH2), 3.81 (s, 2H, CH2), 4.16
(br, 1H, NH of imidazole), 6.64 (s, 1H, O–CH), 7.19–7.27 (m, 4H,
arom-H), 7.66 (d, J ¼ 7.1 Hz, 4H, arom-H), 7.90 (s, 1H, aliph-NH);
13C-NMR (75 MHz, CDCl3) d ppm: 14.3, 17.6, 23.3, 35.3, 115.2,
122.6, 144.1, 171.1, 172.6; MS m/z: 426.02 (Mþ); anal. calcd.
for C23H27N3O3S: C, 64.92; H, 6.40; N, 9.87%, Found: C, 62.95;
H, 6.41; N, 9.85%.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoyl-(2,4-dinitro-phenyl) methyl ester (2k)
White solid, yield 1.8387 g; IR (KBr) nmax cmꢀ1: 3466 (br, arom-
1
NH), 3149 (aliph-NH), 1688 (CO of acid), 1574 (CO of CONH); H-
NMR (300 MHz, DMSO) d ppm: 0.98 (s, 3H, CH3), 1.27 (s, 2H, CH2),
1.66 (s, 2H, CH2), 3.18 (s, 2H, CH2), 3.87 (s, 2H, CH2), 4.14 (br, 1H,
NH of imidazole), 6.93 (s, 1H, O–CH), 7.11–7.14 (m, 4H, arom-H),
7.43–7.46 (s, 4H, arom-H), 7.92 (1H, s, aliph-NH); 13C-NMR (DMSO,
75 MHz): d (ppm) 14.5, 20.9, 116.2, 123.8, 144.3, 170.8, 172.3; MS
m/z: 488.12 (Mþ): anal. calcd. for C21H21N5O7S: C, 51.74; H, 4.34; N,
14.37%, Found: C, 51.76; H, 4.37; N, 14.35%.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoyl-(2-nitro-phenyl) methyl ester (2g)
White solid, yield 1.6255 g; IR (KBr) nmax cmꢀ1: 3420 (br, arom-
NH), 3151 (aliph-NH), 1664 (CO of acid), 1578 (CO of CONH);
1H-NMR (300 MHz, DMSO) d ppm: 0.98 (d, J ¼ 6.6 Hz, 3H, CH3),
1.33 (d, J ¼ 10 Hz, 2H, CH2), 1.63 (m, 2H, CH2), 3.18 (s, 2H, CH2),
3.87 (s, 2H, CH2), 4.15 (br, 1H, NH of imidazole), 6.52 (s, 1H, O–CH),
7.23–7.33 (m, 4H, arom-H), 7.44–7.75 (m, 4H, arom-H), 7.96 (s, 1H,
aliph-NH); 13C-NMR (75 MHz, CDCl3) d ppm: 12.6, 22.5, 114.3,
123.8, 142.6, 170.1, 172.5; MS m/z: 440.36 (M); anal. calcd.
for C21H22N4O5S: C, 57.00; H, 5.01; N, 12.66%, Found: C, 56.97;
H, 5.03; N, 12.70%.
(1H-Benzimidazol-2-yl-sulfanyl)-acetic acid
butylcarbamoyl (2,4-dichloro-phenyl) methyl ester (2l)
White solid, yield 1.7531 g; IR (KBr) nmax cmꢀ1: 3488 (br, arom-
NH), 3157 (aliph-NH), 1674 (CO of acid), 1575 (CO of CONH);
1H-NMR (300 MHz, DMSO) d ppm: 0.91 (t, J ¼ 3.8 Hz, 3H, CH3),
1.24 (s, 2H, CH2), 1.72 (s, 2H, CH2), 3.17 (s, 2H, CH2), 3.82 (s, 2H,
CH2), 4.13 (br, 1H, NH of imidazole), 6.88 (s, 1H, O–CH), 7.00–7.13
(m, 4H, arom-H), 7.42–7.45 (s, 4H, arom-H), 7.91 (1H, s, aliph-NH);
13C-NMR (DMSO, 75 MHz): d (ppm) 14.7, 22.1, 115.2, 123.2, 143.6,
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Arch. Pharm. Chem. Life Sci. 2012, 345, 65–72
Carbonyl-amide Linkage Based Benzimidazole Derivatives
71
170.3, 172.2; MS m/z: 464.29 (M); anal. calcd. for C21H21Cl2N3O3S:
C, 54.08; H, 4.54; N, 9.01%, Found: C, 54.11; H, 4.52, N, 8.98%.
compounds 2a–2l, respectively, at a dose of 250 mg/kg
body weight by oral route and group III for Glibenclamide
standard. The rats of all groups were given glucose (2 g/kg
body weight, p.o.) 30 min after administration of the drug.
Blood samples were collected from the tail vein just prior to
glucose administration at 2nd h, 4th h and 9th day after
the glucose loading. Serum was separated and blood glucose
levels were measured immediately by glucose–oxidase
method [38].
Pharmacological assay
Animals used
Albino-Wistar rats (150–200 g) were used for in-vivo activities.
Animals were maintained under standard laboratory con-
ditions (24 ꢁ 28C; relative humidity 60–70%). Study protocol
was approved by the institutional Animal Ethics Committee
(IAEC, Reg.No. 346/CPCSEA: dated 03-01-2001, Department of
Pharmacy, Luqman College of Pharmacy and Research,
Gulbarga) before experiment. The animals were kept in poly-
propylene cages and maintained on balanced ration with free
access to clean drinking water. All experimental procedures
were conducted in accordance with the guide for care and
use of laboratory animals and in accordance with the local
animal care and use committee.
OGTT Screening
Oral glucose tolerance test (OGTT) method was used to evalu-
ate anti-diabetic activity. Albino-Wistar rats of either sex
(150–200 g) were induced diabetic by a single i.p. injection
of 150 mg/kg body weight of alloxan monohydrate in sterile
normal saline. The rats were maintained on 5% glucose
solution for next 24 h to prevent hypoglycemia [39]. Nine
days later blood samples were collected by retro orbital
puncture of each rat under mild anesthesia in 1-mL
Eppendorff tubes containing 50 mL of anticoagulant hepa-
rin. The diabetic rats were divided into three groups, each
containing six animals. Controls rats (group I) were given
normal saline orally, while compounds 2a–2l were given to
group II, respectively, at a dose of 250 mg/kg, orally. Group III
received Glibenclamide at a dose of 10 mg/kg. Blood samples
were collected from the tail vein just prior to 30 min and
90 min after drug administration. The blood glucose level
was determined by glucose–oxidase method.
Acute toxicity study
For testing the acute toxicity potential of the test compounds,
Albino-Wistar rats of either sex, weighing (20–25 g) were
used. The dosage was varied from 100 up to 3000 mg/kg body
weight. The animals were continuously observed for 8 h for
any signs of acute toxicity such as increased-decreased motor
activity, ataxia, tremors, convulsions, sedation, lacrimation,
etc. After 24 h the animals were sacrificed, stomach,
intestine, and liver were inspected under the magnifying
lenses for any ulcer-hemorrhagic spots.
Anti-convulsant activity screening
Anti-inflammatory activity screening
Evaluation of anti-convulsant activity was done by
maxima electroshock model [35, 36] and Albino-Wistar rats
(150–200 g) were used for the above method. In MES con-
vulsions electroshock is applied through the corneal elec-
trode; from this optic stimulation cortical excitation is
produced. The MES convulsions are divided into five phases
such as a) tonic flexion, b) tonic extensor, c) clonic convul-
sions, d) stupor and e) recovery or death. The time (s) spent
by the animal in each phase of the convulsions is noted
down. A substance is known to possess anti-convulsant
property if it reduces or abolishes the extensor phase of
MES convulsions.
Anti-inflammatory activity was evaluated by using carra-
geenan-induced hind paw edema method [32, 37]. Albino-
Wistar rat strains of either sex between 150–200 g were
selected for the studies. The animals were kept on diet and
allowed food and water ad libitum. They were housed in
polypropylene cages maintained under standard condition
(12 h light/12 h dark cycles at 25 ꢁ 38C temperature, 35–60%
humidity). The rats were divided into fourteen groups of six
rats each as described in Table 2. The control group received
Tween-80 (1%) 10 mL/kg p.o. The test groups received
400 mg/kg p.o. of synthetic compounds. Diclofenac sodium
200 mg/kg p.o. served as standard. All the suspensions were
administrated 30 min before the injection of carrageenan
(0.1 mL of 1%). The paw edema volume was measured with
the help of plethysmograph by mercury displacement
method at 0, 1, 2, 3, 4 and 6 h.
Statistical analysis
The experimental results for anti-inflammatory, anti-diabetic
and anti-convulsant activities were expressed as the
mean ꢁ standard error of mean (SEM), evaluated by com-
pared one-way ANOVA test. The statistical significance
was evaluated by using the Student’s ‘t’ and Dunnet’s test.
Values of P < 0.01 were considered as statistically significant
[37, 40].
Antidiabetic activity screening
Effect of blood glucose level
Fasted rats were divided into three groups of six each. Group
I, the control group, received normal saline. Group II received
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72
I. N. Shaikh et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 65–72
This research work is financially supported by the University Grants
Commission (UGC), New Delhi 110 012. (Ref: No. 33-296/2007 (SR) dated
28-02-2008).
[21] K. Kubo, Y. Kohara, Y. Yoshimura, Y. Inada, Y. Shibouta, Y.
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The authors have declared no conflict of interest.
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Townsend, J. Med. Chem. 1998, 41, 1252–1262.
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