In-vivo analgesic and anti-inflammatory activities of newly synthesized benzimidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.01.029

A series of 2-methylaminobenzimidazole derivatives (1-11) were synthesized by the reaction of 2-(chloromethyl)-1H-benzimidazole derivatives with primary aromatic amines. All these compounds were characterized by IR, ¹H NMR, ¹³C NMR,

Full text of DOI:10.1016/j.ejmech.2010.01.029

European Journal of Medicinal Chemistry 45 (2010) 2048–2054
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
In-vivo analgesic and anti-inflammatory activities of newly synthesized
benzimidazole derivatives
*
Kavitha C.S. Achar, Kallappa M. Hosamani , Harisha R. Seetharamareddy
P. G. Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad, Karnataka 580 003, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-methylaminobenzimidazole derivatives (1–11) were synthesized by the reaction of
2-(chloromethyl)-1H-benzimidazole derivatives with primary aromatic amines. All these compounds
were characterized by IR, 1H NMR, 13C NMR, GC-MS and elemental analysis. The newly synthesized
compounds were screened for analgesic and anti-inflammatory activities on acetic acid induced writhing
in mice and carrageenan induced paw oedema in rats. Compounds (7) and (2) showed a potent analgesic
(89% at 100 mg/kg b.w) and anti-inflammatory (100% at 100 mg/kg b.w) activities compared with
standard drug Nimesulide (100% at 50 mg/kg b.w) respectively. The other compounds showed good
analgesic and anti-inflammatory activities.
Received 26 September 2009
Received in revised form
11 January 2010
Accepted 14 January 2010
Available online 21 January 2010
Keywords:
2-(chloromethyl)-1H-benzimidazole
derivatives
Ó 2010 Elsevier Masson SAS. All rights reserved.
Analgesic
Anti-inflammatory
Benzimidazole derivatives
1. Introduction
everyday life [3]. Classical NSAIDs exhibit their action by restricting
the biosynthesis of prostaglandin, some of which are pro-inflam-
Inflammation is a local reaction of the vascular and supporting
elements of a tissue to injury resulting in the formation of
a protein-rich exudates; it is a protective response of the nonspe-
cific immune system that serves to localize, neutralize, 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].
Inflammation is divided into acute and chronic patterns. The
characteristics of acute inflammation are the exudation of fluid and
plasma proteins (oedema) and the emigration of leukocytes,
predominantly neutrophils. Chronic inflammation is considered to
be inflammation of prolonged duration (weeks or months) in which
active inflammation, tissue destruction, and attempts at repair are
proceeding simultaneously. Chronic inflammation includes some of
the most common and disabling human diseases, such as rheu-
matoid arthritis, atherosclerosis, tuberculosis, and chronic lung
diseases [2].
matory. This is essentially brought about by inhibiting the rate
limiting cyclooxygenase (COX) enzyme involved in the inflamma-
tory cascade [4]. Among different types of NSAIDs, imidazole and
fused imidazole with six-membered rings [5], occupy central
position among those compounds that are used as analgesic and
anti-inflammatory agents.
Fused imidazole derivatives have occupied a prominent place in
medicinal chemistry because of their significant properties as
therapeutics in clinical applications [6–9]. Thus, benzimidazole is
being explored in the pharmaceutical industries and the
substituted benzimidazole derivatives have also been found in the
diverse therapeutic applications [10,11]. Because of the versatile
core contained in several substances of benzimidazole derivatives
are possess a broad spectrum of pharmacological activities [12–15].
In particular, it has been an important pharmacophore and privi-
leged structure in medicinal chemistry [16,17], encompassing
a diverse range of biological activities including anti-arrhythmic,
HIV-RT inhibitor [18], anti-cancer, pesticide, anti-ulcer, anti-
inflammatory, anthelmintical, inotropic, antihistamin, anti-micro-
bial, anti-viral and cytotoxicity [19–22]. Therefore, the optimization
of benzimidazole derivatives based on their structures have resul-
ted various potent drugs that are now being currently practiced in
the market, amongst Albendazole (inhibitor of Encephalitozoon
Intestinalis infection in AIDS patients), Omeprazole (proton
pump inhibitor), Pimobendan (ionodilator), and Mebendazole
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used
for the choice treatment in various inflammatory diseases such as
arthritis, rheumatisms as well as to relieve the aches and pain of
* Corresponding author. Tel.: þ91 836 2215286; fax: þ91 836 2771275/2747884.
E-mail address: dr_hosamani@yahoo.com (K.M. Hosamani).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.029

K.C.S. Achar et al. / European Journal of Medicinal Chemistry 45 (2010) 2048–2054
2049
(anthelmintic). Therefore, preparation of benzimidazole has
attracted considerable attention in recent years.
method [31]. The maintenance of optimum temperature (80 ꢀC) is
very important as it led to the crystalline products. The corre-
sponding yields (70–89%) were obtained in good agreement. The
postulated structures of newly synthesized targeted benzimidazole
derivatives are in accordance with the elemental analysis, IR, 1H
NMR, 13C NMR and GC-MS. IR spectra of all the compounds showed
stretching bands at 3448–3424 cmꢁ1 and 3327–3308 cmꢁ1 of
aromatic and aliphatic –NH respectively. Further (C]N) stretching
bands appeared at 1596–1619 cmꢁ1 respectively. The 1H NMR
The aforesaid numerous pharmacological activities of benz-
imidazoles prompted us to study the in-vivo analgesic and
anti-inflammatory activities of some important benzimidazole
derivatives. Owing to the importance and in continuation of our
ongoing research project on benzimidazole derivatives [23–26],
now we wish to explore simple and the novel approach towards the
synthesis of 2-methylaminobenzimidazole derivatives by readily
obtainable materials [27–29] such as 2-(chloromethyl)-1H-benz-
imidazole derivatives [30,31] and in-vivo screening of the resultant
compounds by analgesic and anti-inflammatory activities.
spectral broad singlet signals were observed at
d 3.5–4.3 NH
protons of aliphatic and NH protons of imidazole are merged with
aromatic and which are D2O exchangeable. In 13C NMR spectra, the
peaks at
d 115,116,122,129,137 and 141 confirm the formation of
benzimidazole moiety. GC-MS spectra showed the corresponding
molecular ion peaks for all the compounds. All the spectral results
of 2-methylaminobenzimidazole derivatives (1–11) are tabulated in
Table 2.
2. Chemistry
The synthetic strategy has been explored to obtain the title
compounds in excellent yield as shown in Scheme 1. Condensation
of 2-(chloromethyl)-1H-benzimidazole derivatives [32,33] with
various substituted aromatic amines by refluxing in ethanol
containing KI/KOH for 6 h gave 2-methylaminobenzimidazole
derivatives [31] (1–11). Elemental analysis, yield and melting points
were given in the Table 1. This protocol is very significant because of
its specific generation of crystalline products. The synthesized
compounds were characterized by IR, 1H NMR, 13C NMR, GC-MS and
elemental analysis.
4.2. Analgesic activity
The analgesic activity of the synthesized compounds was assessed
by the acetic acid-induced writhing method. According to the struc-
ture–activity relationship (SAR) studies, almost all the compounds
have shown very potent analgesic activity when compared with
standard nimesulide drug. Among the tested compounds N-[(5-
bromo-1H-benzimidazol-2-yl) methyl]-3-chloroaniline (7) showed
pronounced analgesic activity (89%, 100 mg/kg b.w). The remaining
compounds 2, 4, 6, 7, 8, 9, 10 and 11 have also shown good activity
because of bromo and nitro substituted benzimidazole with bromo,
chloro, methyl and methoxysubstituted aryl of N-[(5-substituted-1H-
benzimidazol-2-yl)methyl]-substituted anilines. Within the same
ring systemi.e., 2-methylaminobenzimidazole, itwasnoticed that the
introductionofbromineandnitrogroupinbenzimidazoleandbromo,
chloro, methyl and methoxy in the aniline at different positions
enhanced the analgesic activity. The results are summarized inTable 3
and Fig. 1.
3. Pharmacological evaluation
All the compounds prepared herein were screened for their
pharmacological activities such as in-vivo analgesic and anti-
inflammatory activities. These activities were carried out by
measuring the physiological responses of animals to the thermal
and chemical stimuli. For analgesic activity, acetic acid induced
writhing in mice [34,35] at 50 mg/kg body weight (b.w) was
performed. The percent of protection was calculated compared to
standard drug nimesulide. All compounds showed potent activity
compared with Nimesulide. For anti-inflammatory activity, carra-
geenan induced inflammation on rat hind paw oedema method of
Winter et al. [36], in mice at 50 mg/kg b.w. was performed. The
percent of inhibition was determined for synthesized compounds
as well as standard drug nimesulide.
4.3. Anti-inflammatory activity
The results of tested compounds as well as reference standard
were measured before administration the carrageenan. After the
administration of carrageenan inflammation was induced in rats,
the effect was measured in the intervals of 30, 60, 120 and 180 min.
The percent oedema inhibition was calculated as a regard to saline
control group, as depicted in Table 4, Figs. 2 and 3. Most of the
tested compounds have shown good results in comparison with
standard nimesulide standard drug. Amongst all the compounds,
compound (1) and (2) have shown potent anti-inflammatory
activity. From a view of structure–activity relationship (SAR)
studies, the unsubstituted benzimidazoles and chloro substituted
4. Results and discussion
4.1. Chemistry
Condensation of 2-(chloromethyl)-1H-benzimidazole deriva-
tives with various substituted aromatic amines by refluxing in
ethanol containing KI/KOH for 6 h yielded substituted 2-methyl-
aminobenzimidazole derivatives (1–11) according to literature
NH2
N
N
KI, EtOH
+
Cl
KOH
N
H
N
H
HN
R'
R
°
80 C
R
R'
Where R = H, Br, NO2
R'= H, Cl, Br, CH3, OCH3
Scheme 1. Schematic representation of 2-methylamino-1H-benzimidazole derivatives.

2050
K.C.S. Achar et al. / European Journal of Medicinal Chemistry 45 (2010) 2048–2054
Table 1
Physical data of synthesized compounds.
Compound
R
R0
Molecular
formula
Yielda (%)
m.p. (ꢀC)
C (%)
H (%)
Calcd
N (%)
Calcd
Calcd
Found
Found
Found
1
2
3
4
5
6
7
8
H
H
H
H
Br
Br
Br
Br
Br
Br
NO2
H
Cl
Br
OMe
H
Cl
Cl
Br
C14H13N3
85
84
84
80
82
81
80
80
79
81
82
138–140
143–145
203–205
153–155
198–200
195–198
192–195
186–188
188–190
193–195
186–188
75.31
65.25
55.65
71.13
55.65
49.95
49.94
44.13
54.23
56.98
48.43
75.37
65.05
55.46
71.07
55.60
49.85
49.98
44.02
54.14
56.39
48.35
5.87
4.69
4.00
5.97
4.10
3.29
3.29
2.91
4.25
4.46
3.19
5.79
4.54
3.86
5.99
4.00
3.15
3.15
2.82
4.16
4.32
3.05
18.82
16.30
13.91
16.59
13.91
12.48
12.49
11.03
12.65
13.29
16.14
18.68
16.17
13.78
16.45
13.79
12.35
12.38
10.06
12.51
13.16
16.04
C14H12ClN3
C14H12BrN3
C15H15N3O
C14H12BrN3
C14H11ClN3
C14H11ClN3
C14H11Br2N3
C15H14BrN3O
C15H14BrN3
C14H11BrN4O2
9
10
11
OMe
Me
Br
a
Isolated yield.
aniline have shown potent anti-inflammatory activity when
compared with other substituted benzimidazole (–Br and –NO2)
with standard drug nimesulide. This has resulted new path in the
synthesis of new class of benzimidazole derivatives.
6.2. General procedure for synthesis of 2-
Arylaminomethylbenzimidazoles (1–11)
A mixture of 2-(chloromethyl)-1H-benzimidazole derivatives
(10 mmol), substituted aniline (10 mmol) and KI (10 mmol) in
50 mL of ethanol was heated under reflux. After 6 h, KOH (10 mmol
in 5 mL of water) was added with continuous stirring for 2 h. Finally
the reaction mixture was left aside at r.t. and then poured into
crushed ice water. The solid products that precipitated were filtered
off and recrystallized from ethanol [31].
5. Conclusion
In conclusion, we have described simple and efficient protocol
for the preparation of 2-methylaminobenzimidazole derivatives
with excellent yields. All the synthesized compounds have been
screened for their in-vivo analgesic and anti-inflammatory activities.
In the newly synthesized compounds, it is cleared that the highest
analgesic activity in compound (7) and anti-inflammatory activity in
compound (1) and (2) were observed. Apart from compound (7),
remaining compounds have shown good analgesic activity almost
equal to standard nimesulide drug. Compounds 3, 4, 5, 6, 7, 8, 9, 10
and 11 have shown moderate anti-inflammatory activity. The
preliminary in-vivo studies of these compounds evidenced that,
the chloro group in the meta position in aniline ring enhances the
analgesic as well as anti-inflammatory activities, which might serve
as new templates in the synthesis and development of potent
therapeutics. Therefore, it can be concluded that such compounds
exert their pharmacological effects. This has resulted good impact on
chemists and biochemists for further investigations in the field of
medicinal chemistry for search of analgesic and anti-inflammatory
agents containing halo and methyl functional groups.
6.2.1. N-(1H-benzimidazol-2-ylmethyl) aniline (1)
Yellow crystal: mp. 138–140 ꢀC (85%), IR (KBr):
n
(cmꢁ1), 3429
(Ar-NH) and 3321(aliph-NH), 2919 (aliph-CH) and 1602 (C]N); 1H
NMR, (300 MHz, CDCl3, ppm): 2.19(s, 2H, CH2), 4.3(s, 1H, aliph-
NH), 6.5(s, 1H, imidazole-NH), 6.3–7.56(m, 9H, Ar-H); 13C NMR
d
(75 MHz, CDCl3, d ppm): 52, 112, 115, 116, 122, 129, 138,141, 143; MS:
224.09 [M þ 1].
6.2.2. N-(1H-benzimidazol-2-ylmethyl)-3-chloroaniline (2)
Yellow crystal: m.p. 143–145 ꢀC (85%), IR (KBr)
3437(Ar-NH) and 3318(aliph-NH), 2927 (aliph-CH) and 1596
(C]N); 1H NMR, (300 MHz, CDCl3, d ppm): 2.19(s, 2H, CH2), 3.75(s,
1H, aliph-NH), 6.6(s, 1H, imidazole-NH), 6.4–7.7(m, 7H, Ar-H); 13C
n
(cmꢁ1):
NMR (75 MHz, CDCl3,
d ppm): 52, 110, 112, 115, 117, 122, 130, 134,
137, 141, 145; MS: 258.1 [M þ 1].
6.2.3. N-(1H-benzimidazol-2-ylmethyl)-3-bromoaniline (3)
Yellow crystal, m.p. 203–205 ꢀC (85%), IR (KBr)
n
(cmꢁ1): 3448
6. Experimental protocols
(Ar-NH) and 3321(aliph-NH), 2921 (aliph-CH) and 1612 (C]N); 1H
NMR, (300 MHz, CDCl3, d ppm): 2.18(s, 2H, CH2), 4(s, 1H, aliph-NH),
6.62(s, 1H, imidazole-NH), 6.4–7.7(m, 7H, Ar-H); 13C NMR (75 MHz,
6.1. Materials and methods
CDCl3,
d ppm): 52, 110, 112, 115, 117, 122, 130, 134, 137, 144; MS:
The melting points of the products were determined by open
capillaries on a Buchi apparatus and are uncorrected. The IR spectra
were recorded on a Nicolet Impact 410 FT-IR Spectrophotometer,
using KBr pellets. 1H NMR and 13C NMR spectra were recorded on
a Bruker Avance-300F 300 MHz spectrometer in CDCl3 using TMS as
an internal standard with 1H resonant frequency of 300 MHz and
13C resonant frequency of 75 MHz. D2O exchange was applied to
confirm the assignment of the signals of NH protons. The Mass
spectra were recorded on a GC-2010 Shimadzu Mass Spectrometer
(GCMS). The elemental analysis was carried out by using Heraus
CHN rapid analyzer. All the compounds gave C, H and N analysis
within ꢂ0.5% of the theoretical values. The homogeneity of the
compounds was described by TLC on aluminum silica gel 60 F254
(Merck) detected by U.V light (254 nm) and iodine vapors. All
reagents were analytical graded or chemically pure.
303.08 [M þ 1].
6.2.4. N-(1H-benzimidazol-2-ylmethyl)-4-methoxyaniline (4)
Yellow crystal: m.p. 153–155 ꢀC (85%), IR (KBr),
n
(cmꢁ1): 3427
(Ar-NH) and 3325(aliph-NH), 2936 (aliph-CH) and 1617 (C]N); 1H
NMR, (300 MHz, CDCl3, ppm): 2.17(s, 2H, CH2), 4.3(s, 1H, aliph-
NH), 6.61(s, 1H, imidazole-NH), 6.4–7.9(m, 7H, Ar-H); 13C NMR
(75 MHz, CDCl3, ppm): 52, 56, 113, 114, 117, 118, 126, 135, 136, 140,
d
d
141, 150; MS: 254.11 [M þ 1].
6.2.5. N-[(5-bromo-1H-benzimidazol-2-yl) methyl] aniline (5)
Light buff crystal: m.p. 198–200 ꢀC (85%), IR (KBr)
n
(cmꢁ1),
3433(Ar-NH) and 3308(aliph-NH), 2925 (aliph-CH) and 1619
(C]N); 1H NMR, (300 MHz, CDCl3, d ppm): 2.2 (s, 2H, CH2), 4(s, 1H,
aliph-NH), 6.7(s, 1H, imidazole-NH), 6.4–7.8(m, 7H, Ar-H); 13C NMR

K.C.S. Achar et al. / European Journal of Medicinal Chemistry 45 (2010) 2048–2054
2051
Table 2
Spectral analysis.
Sl.No.
Compounds and Structures
Infrared cmꢁ1 (KBr pallets)
1H Nuclear Magnetic
13C NMR
Mass
Resonance Values in ppm
Values in ppm
At m/z (M þ 1)
1
2
3
4
5
6
3429 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3321 cmꢁ1 –NH bending
for secondary amide
2.19 (s, 2H, CH2)
4.3 (s, 1H, NH, disappeared
on D2O addition)
6.5 (s, 1H, NH, disappeared
on D2O addition)
6.3–7.56 (m, Ar-H)
H
N
52, (Methylene carbon)
112–138 (Ar-Carbons)
141, (C]N carbon)
143, (N–C carbon)
N
N
1.
2.
224.09
258.1
1602 cmꢁ1 C]N stretching,
for imidazole ring
3437 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3318 cmꢁ1 –NH bending
for secondary amide
2.19 (s, 2H, CH2)
3.75 (s, 1H, NH, disappeared
on D2O addition)
6.6 (s, 1H, NH, disappeared
on D2O addition)
6.4–7.7 (m, Ar-H)
Cl
52, (Methylene carbon)
110–137 (Ar-Carbons)
141, (C]N carbon)
145, (N–C carbon)
H
H
N
N
N
1596 cmꢁ1 C]N stretching,
for imidazole ring
3448 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3321 cmꢁ1 –NH bending
for secondary amide
2.18 (s, 2H, CH2)
4.0 (s, 1H, NH, disappeared
on D2O addition)
6.62 (s, 1H, NH, disappeared
on D2O addition)
6.4–7.7 (m, Ar-H)
H
N
H
52, (Methylene carbon)
110–134 (Ar-Carbons)
137, (C]N carbon)
144, (N–C carbon)
Br
N
3.
4.
303.08
254.11
N
1612 cmꢁ1 C]N stretching,
for imidazole ring
3427 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3325 cmꢁ1 –NH bending
for secondary amide
2.17 (s, 2H, CH2)
4.3 (s, 1H, NH, disappeared
on D2O addition)
6.61 (s, 1H, NH, disappeared
on D2O addition)
6.4–7.9(m, Ar-H)
H
H
52, (Methylene carbon)
56, (Methoxy carbon)
113–140 (Ar-Carbons)
141, (C]N carbon)
150, (N–C carbon)
N
OMe
N
N
1617 cmꢁ1 C]N stretching,
for imidazole ring
3433 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3308 cmꢁ1 –NH bending
for secondary amide
2.2 (s, 2H, CH2)
4.0 (s, 1H, NH, disappeared
on D2O addition)
6.7 (s, 1H, NH, disappeared
on D2O addition)
6.4–7.8 (m, Ar-H)
H
N
H
N
Br
52, (Methylene carbon)
112–140(Ar-Carbons)
141, (C]N carbon)
143, (N–C carbon)
5.
6.
7.
302.09
336.89
337.04
1619 cmꢁ1 C]N stretching,
for imidazole ring
N
Cl
3429 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3321 cmꢁ1 –NH bending
for secondary amide
2.2 (s, 2H, CH2)
4.3 (s, 1H, NH, disappeared
on D2O addition)
6.72 (s, 1H, NH, disappeared
on D2O addition)
6.7–8.0 (m, Ar-H)
51, (Methylene carbon)
114–140(Ar-Carbons)
142, (C]N carbon)
144, (N–C carbon)
H
N
H
N
Br
1616 cmꢁ1 C]N stretching,
for imidazole ring
N
Cl
3429 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3327 cmꢁ1 –NH bending
for secondary amide
2.18 (s, 2H, CH2)
4.1 (s, 1H, NH, disappeared
on D2O addition)
6.6 (s, 1H, NH, disappeared
on D2O addition)
6.2–7.9 (m, Ar-H)
52, (Methylene carbon)
111–140 (Ar-Carbons)
141, (C]N carbon)
143, (N–C carbon)
H
N
H
Br
N
1616 cmꢁ1 C]N stretching,
for imidazole ring
N
3430 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3325 cmꢁ1 –NH bending
for secondary amide
2.18 (s, 2H, CH2)
4.1 (s, 1H, NH, disappeared
on D2O addition)
6.8 (s, 1H, NH, disappeared
on D2O addition)
6.32–7.87 (m, Ar-H)
H
N
H
Br
Br
52, (Methylene carbon)
111–140 (Ar-Carbons)
141, (C]N carbon)
142, (N-C carbon)
N
8.
382.07
332.97
316.92
N
1617 cmꢁ1 C]N stretching,
for imidazole ring
3424 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3323 cmꢁ1 –NH bending
for secondary amide
2.19 (s, 2H, CH2)
4.0 (s, 1H, NH, disappeared
on D2O addition)
6.7 (s, 1H, NH, disappeared
on D2O addition)
6.4–7.7 (m, Ar-H)
H
H
52, (Methylene carbon)
56, (Methoxy Carbon)
113–140(Ar-Carbons)
141, (C]N carbon)
148, (N–C carbon)
Br
Br
N
OMe
Me
N
9.
N
1602 cmꢁ1 C]N stretching,
for imidazole ring
3428 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3324 cmꢁ1 –NH bending
for secondary amide
2.18 (s, 2H, CH2)
4.0 (s, 1H, NH, disappeared
on D2O addition)
6.7 (s, 1H, NH, disappeared
on D2O addition)
6.4–7.7 (m, Ar-H)
H
H
20, (Metyl Carbon)
52, (Methylene carbon)
112–136(Ar-Carbons)
140, (C]N carbon)
141, (N–C carbon)
N
N
10.
N
1616 cmꢁ1 C]N stretching,
for imidazole ring
(continued on next page)

2052
Table 2 (continued)
K.C.S. Achar et al. / European Journal of Medicinal Chemistry 45 (2010) 2048–2054
Sl.No.
Compounds and Structures
Infrared cmꢁ1 (KBr pallets)
1H Nuclear Magnetic
13C NMR
Mass
Resonance Values in ppm
Values in ppm
At m/z (M þ 1)
1
2
3
4
5
6
3430 cmꢁ1 –NH stretching,
for benzimidazole ring NH
3321 cmꢁ1 –NH bending
for secondary amide
2.19 (s, 2H, CH2)
4.2 (s, 1H, NH, disappeared
on D2O addition)
6.8 (s, 1H, NH, disappeared
on D2O addition)
6.32–8.63 (m, Ar-H)
H
N
H
N
O N
Br
52, (Methylene carbon)
110–141(Ar-Carbons)
142, (C]N carbon)
144, (N–C carbon)
2
11.
348.04
N
1612 cmꢁ1 C]N stretching,
for imidazole ring
(75 MHz, CDCl3,
d
ppm): 52, 112, 116, 117, 118, 126, 129, 136, 140, 141,
6.2.9. N-[(5-bromo-1H-benzimidazol-2-yl) methyl]-4-
143; MS: 302.09 [M þ 1].
methoxyaniline (9)
Buff crystal: m.p.189–190 ꢀC (85%), IR (KBr)
n
(cmꢁ1), 3424(Ar-
6.2.6. N-[(5-bromo-1H-benzimidazol-2-yl) methyl]-2-
NH) and 3323(aliph-NH), 2924 (aliph-CH) and 1617 (C]N); 1H
NMR, (300 MHz, CDCl3, d ppm) : 2.19(s, 2H, CH2), 4(s, 1H, aliph-NH),
6.7(s, 1H, imidazole-NH), 6.4–7.7(m, 7H, Ar-H); 13C NMR (75 MHz,
CDCl3, d ppm) : 52, 56, 113, 114, 117, 118, 126, 135, 136, 140, 141, 148;
MS: 332.97 [M þ 1].
chloroaniline (6)
Buff crystal: m.p. 195–198 ꢀC (85%), IR (KBr)
n
(cmꢁ1), 3429 (Ar-
NH) and 3321(aliph-NH), 2923 (aliph-CH) and 1616 (C]N); 1H
NMR, (300 MHz, CDCl3, ppm) : 2.2(s, 2H, CH2), 4.3(s, 1H, aliph-
NH), 6.7(s, 1H, imidazole-NH), 6.7–8(m, 7H, Ar-H); 13C NMR
(75 MHz, CDCl3, ppm) : 51, 114, 117, 118, 119.2, 127, 130, 137, 140,
d
d
6.2.10. N-[(5-bromo-1H-benzimidazol-2-yl) methyl]-4-
142, 144; MS: 336.89 [M þ 1].
methylaniline (10)
Buff crystal: m.p.193–195 ꢀC (85%), IR (KBr)
n
(cmꢁ1), 3428 (Ar-
6.2.7. N-[(5-bromo-1H-benzimidazol-2-yl) methyl]-3-
NH) and 3324(aliph-NH), 2921 (aliph-CH) and 1616 (C]N); 1H
NMR, (300 MHz, CDCl3, ppm) : 2.18(S, 2H, CH2), 4(s,1H, aliph-NH),
6.7(s, 1H, imidazole-NH), 6.4–7.7(m, 7H, Ar-H); 13C NMR (75 MHz,
CDCl3, ppm) : 20, 52, 112, 117, 118, 126, 130, 136, 140, 141; MS:
chloroaniline (7)
d
Buff crystal: m.p. 192–195 ꢀC (85%), IR (KBr)
n
(cmꢁ1), 3429 (Ar-
NH) and 3327(aliph-NH), 2932 (aliph-CH) and 1616 (C]N); 1H
NMR, (300 MHz, CDCl3, ppm): 2.18 (s, 2H, CH2), 4.1(s, 1H, aliph-
NH), 6.6(s, 1H, imidazole-NH), 6.2–7.9(m, 7H, Ar-H); 13C NMR
(75 MHz, CDCl3, ppm) : 52, 111, 113, 114, 117, 118, 119, 126, 130, 134,
d
d
316.92 [M þ 1].
d
6.2.11. 4-bromo-N-[(5-nitro-1H-benzimidazol-2-yl) methyl]
136, 140, 141, 143; MS: 337.04 [M þ 1].
aniline (11)
Buff crystal: m.p.186–188 ꢀC (85%), IR (KBr)
n
(cmꢁ1), 3430 (Ar-
6.2.8. 3-bromo-N-[(5-bromo-1H-benzimidazol-2-yl) methyl]
NH) and 3321(aliph- NH), 2927 (aliph-CH) and 1612 (C]N); 1H
NMR, (300 MHz, CDCl3, ppm) : 2.19(s, 2H, CH2), 4.2(s, 1H, aliph-
NH), 6.8(s, 1H, imidazole-NH), 6.32–8.63(m, 7H, Ar-H); 13C NMR
(75 MHz, CDCl3, ppm): 52, 110, 111, 114, 116, 118, 132, 138, 141, 142,
aniline (8)
d
Black crystal: m.p. 186–188 ꢀC (85%), IR (KBr) (cmꢁ1), 3430 (Ar-
n
NH) and 3325(aliph-NH), 2921(aliph-CH) and 1617 (C]N); 1H
NMR, (300 MHz, CDCl3, ppm) : 2.18(s, 2H, CH2), 4.1(s, 1H, aliph-
NH), 6.8(s, 1H, imidazole-NH), 6.32–7.87(m, 7H, Ar-H); 13C NMR
d
d
144; MS: 348.04 [M þ 1].
(75 MHz, CDCl3, d ppm) : 52, 111, 114, 117, 118,126, 136, 140, 141, 142;
6.3. Pharmacological assay
MS: 382.07 [Mþ].
6.3.1. Animals used
Table 3
Adult Swiss albino mice (20–25 g) and albino rats weighing
(150–200 g) of either sex were used for studying acute toxicity. In
each group six animals were housed individually in polypropylene
cages with paddy husk bed. Animals were maintained at 25–27 ꢀC
and 30–70% relative humidity. Study protocol was approved by the
Analgesic activity of tested compounds (100 mg/kg b.w) and Nimesulide (50 mg/kg
b.w).
Compound
Mean writhing
(X ꢂ SE)
Protection
(%)
Control
1
2
3
4
5
6
7
8
9
10
11
Nim
30.0 ꢂ 1.55**
8.3 ꢂ 3.33**
5.6 ꢂ 1.85**
8.3 ꢂ 1.45**
5.0 ꢂ 2.51**
6.0 ꢂ 2.00**
6.0 ꢂ 0.57**
3.3 ꢂ 1.66**
4.3 ꢂ 2.02**
5.0 ꢂ 2.08**
3.6 ꢂ 2.33**
6.3 ꢂ 1.45**
–
–
72.33
81.33
70.00
83.33
80.00
80.00
89.00
85.66
83.33
88.00
79.00
100.00
100
80
60
40
20
0
Data represent mean values ꢂ SE of six mice per group, shown at the final value for
each group (saline, nimesulide and tested compounds) after 3 h.
Data were analyzed using one-way ANOVA followed by Turkey–Krammer Multiple
comparison test **p < 0.01.
Percentage change was calculated from basal (pre-drug) values and post-drug
values.
1
2
3
4
5
6
7
8
9
10 11 Nim
Tested Compounds
Protection was calculated as regards the percentage change of the Nimesulide.
SE, standard error; Nim., Nimesulide.
Fig. 1. Analgesic activity of tested compounds (100 mg/kg b.w) and standard drug
The active compounds are marked in bold letters.
Nimesulide (50 mg/kg b.w.).

K.C.S. Achar et al. / European Journal of Medicinal Chemistry 45 (2010) 2048–2054
2053
Table 4
The anti-inflammatory activity of the tested compounds (100 mg/kg b.w) and Nimesulide (50 mg/kg b.w).
Compound
Paw oedema thickness (mm)
30 m
(X ꢂ SE)
% oedema
inhibition
60 m
(X ꢂ SE)
% oedema
Inhibition
120 m
(X ꢂ SE)
% oedema
inhibition
180 m
(X ꢂ SE)
% oedema
Inhibition
Control
1
2
3
4
5
6
7
8
9
10
11
Nim
1.3 ꢂ 0.05
1.2 ꢂ 0.03
1.1 ꢂ 0.03
1.2 ꢂ 0.05
1.2 ꢂ 0.06
1.2 ꢂ 0.03
1.3 ꢂ 0.05
1.2 ꢂ 0.03
1.4 ꢂ 0.00
1.4 ꢂ 0.00
1.2 ꢂ 0.05
1.1 ꢂ 0.03
1.1 ꢂ 0.05
–
1.5 ꢂ 0.03
–
1.7 ꢂ 0.03
–
1.8 ꢂ 0.03
–
7.6
15.3
7.6
7.6
7.6
–
7.6
–
–
1.1 ꢂ 0.00**
1.1 ꢂ 0.00**
1.3 ꢂ 0.03**
1.1 ꢂ 0.05**
1.1 ꢂ 0.03**
1.2 ꢂ 0.05**
1.1 ꢂ 0.06**
1.2 ꢂ 0.03**
1.3 ꢂ 0.00**
1.2 ꢂ 0.05**
1.2 ꢂ 0.03**
1.1 ꢂ 0.00**
26.6
26.6
13.3
26.6
26.6
20.0
26.6
20.0
13.3
20.0
20.0
26.6
1.1 ꢂ 0.03**
1.1 ꢂ 0.03**
1.3 ꢂ 0.03**
1.2 ꢂ 0.03**
1.2 ꢂ 0.05**
1.3 ꢂ 0.08**
1.4 ꢂ 0.03**
1.3 ꢂ 0.10**
1.4 ꢂ 0.00**
1.3 ꢂ 0.06**
1.3 ꢂ 0.05**
1.0 ꢂ 0.00**
41.1
41.1
23.5
29.4
29.4
23.5
17.6
23.5
17.6
23.5
23.5
41.1
1.1 ꢂ 0.05**
1.0 ꢂ 0.03**
1.3 ꢂ 0.06**
1.2 ꢂ 0.06**
1.4 ꢂ 0.05**
1.4 ꢂ 0.05**
1.5 ꢂ 0.05**
1.5 ꢂ 0.05**
1.6 ꢂ 0.06**
1.5 ꢂ 0.11**
1.4 ꢂ 0.03**
1.1 ꢂ 0.00**
38.8
44.4
29.4
33.3
22.2
22.2
16.6
16.6
12.5
16.6
22.2
44.4
7.6
15.3
15.3
Data represent mean values ꢂ SE of six mice per group and the percent changes versus 30, 60, 120 and 180 m post-carrageenan injection.
Data were analyzed using one-way ANOVA followed by Turkey–Krammer Multiple comparison test **p < 0.01.
Percent oedema inhibition was calculated as regards saline control group.
** Significant difference from the control value at p < 0.01.
SE, standard error; Nim., Nimesulide.
The active compounds are marked in bold letters.
institutional Animal Ethics Committee (IACE, Reg. No. 346/CPCSEA:
Dated. 03-01-2001) before experiment.
was analyzed using one way ANOVA followed by Turkey–Krammer
Multiple comparison test and p < 0.01 was considered significant.
6.3.2. Analgesic activity screening
6.3.3. Anti-inflammatory activity screening
Acetic acid induced writhing model was used to evaluate anal-
gesic activity of the synthesized compounds. Five groups of six
Swiss albino mice, each 20–25 g b.w, were used. 0.6% acetic acid
(dose ¼ 10 ml/Kg) was injected intra-peritoneally. The numbers of
writhes were counted for 20 min, after 5 min of injection of acetic
acid into each mice. This reading was taken as a control. Next day,
same groups of mice were used for evaluating analgesic activity.
Each group was administered orally with the synthesized
compounds. The dose of 100 mg/kg of animal was given 1 hour
before injection of acetic acid. After 5 min of acetic acid injection,
mice were observed for the number of writhings for the duration of
20 min. The mean value for each group was calculated and
compared with control. Nimesulide was used as a standard drug for
comparison of analgesic activity. Percent protection was calculated
using the following formula:
All the synthesized compounds were tested for their anti-
inflammatory activity using carrageenan induced rat hind paw
oedema method of Winter et al. [36]. The oedema hind paw was
induced by injection of 0.1 mL of 1% carrageenan solution into sub-
planter region of right hind paw. The volume of the paw was
measured plethysmographically immediately and 180 m after the
injection of the irritant. The difference in volume gave the amount
of oedema developed. Percent inhibition of the oedema between
control group and the compound treated group was calculated and
compared with the group receiving standard drug at 50 mg/kg b.w.
The results are tabulated in Table 4.
6.3.4. Statistical analysis
In analgesic and anti-inflammatory study, data are expressed as
Means ꢂ SE. Differences between vehicle control and treatment
groups were tested using one-way ANOVA followed by Turkey–
Krammer Multiple comparison test. A probability value less than
0.01 was considered as statistically significant.
ð1 ꢁ Vc=VtÞ ꢃ 100
where Vt ¼ Mean number of writhing in test animals and
Vc ¼ Mean number of writhing in control. Statistical significance
50
40
30
20
10
0
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
Nim
30 m
60 m
120 m
180 m
1
2
3
4
5
6
7
8
9
10 11 Nim
Tested Compounds in 180 m
Time (m)
Fig. 2. The inhibition of anti-inflammatory activity of the tested compounds
Fig. 3. The percentage of inhibition of anti-inflammatory activity of the tested
(100 mg/kg b.w.) and standard drug Nimesulide (50 mg/kg b.w.).
compounds (100 mg/kg b.w) and standard drug Nimesulide (50 mg/kg b.w) in 180 m.

2054
K.C.S. Achar et al. / European Journal of Medicinal Chemistry 45 (2010) 2048–2054
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This research work is financially supported by University Grants
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dated 28-02-2008). One of the author Kavitha. C. S Achar thanks
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3236 dated 08-03-2007).
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