Design, synthesis and pharmacological evaluation of omeprazole-like agents with anti-inflammatory activity

Article abstract of DOI:10.1016/j.bmc.2013.01.070

A new series of novel benzimidazole derivatives containing substituted pyrid-2-yl moiety and polyhydroxy sugar conjugated to the N-benzimidazole moiety has been synthesized and evaluated as orally bioavailable anti-inflammatory agents with anti-ulcerogenic activity. The anti-inflammatory and anti-ulcerogenic activities of these compounds were compared to diclofenac and omeprazole, respectively. In carrageenan-induced paw oedema assay, 2-methyl-N-((3,4-dimethoxypyridin-2-yl)methyl)-1H-benzimidazol-5-amine (12d) and 1-(1,2,3,5-tetrahydroxy-α-d-mannofuranose)-5-(((3,4-dimethoxypyridin-2yl) methyl)amino)-2-methyl-1H-benzimidazole (15d) displayed dose-dependent anti-inflammatory activities by decreasing the inflammation by 62% and 72%, respectively which is comparable to that of diclofenac (73%). In contrast to diclofenac, the anti-inflammatory activity of these compounds was not only free from any side effects on the gastric mucosa but also showed significant anti-ulcerogenic activity in rat pyloric ligation and ethanol-induced gastric ulcer models similar to that of omeprazole. Together, these findings suggest that 12d and 15d are potent anti-inflammatory agents with concurrent anti-ulcerogenic activity and support its clinical promise as a component of therapeutic strategies for inflammation, for which the gastric side effects are always a major limitation.

Full text of DOI:10.1016/j.bmc.2013.01.070

Bioorganic & Medicinal Chemistry 21 (2013) 1661–1670
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and pharmacological evaluation of omeprazole-like
agents with anti-inflammatory activity
Ahmed O. H. El-Nezhawy a,c, , Ayman R. Biuomy d,e, Fatma S. Hassan d, Ayman K. Ismaiel d, Hany A. Omar b,f
⇑
a Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Saudi Arabia
b Department of Pharmacology, College of Pharmacy, Taif University, Saudi Arabia
c Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki, Cairo, Egypt
d Faculty of Medicine and Medical Sciences, Taif University, Saudi Arabia
e Pharmacology Department, National Research Center, Dokki, Cairo, Egypt
f Department of Pharmacology, College of Pharmacy, Beni-Suef University, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of novel benzimidazole derivatives containing substituted pyrid-2-yl moiety and polyhy-
droxy sugar conjugated to the N-benzimidazole moiety has been synthesized and evaluated as orally bio-
available anti-inflammatory agents with anti-ulcerogenic activity. The anti-inflammatory and anti-
ulcerogenic activities of these compounds were compared to diclofenac and omeprazole, respectively.
In carrageenan-induced paw oedema assay, 2-methyl-N-((3,4-dimethoxypyridin-2-yl)methyl)-1H-ben-
Received 19 November 2012
Revised 19 January 2013
Accepted 28 January 2013
Available online 11 February 2013
zimidazol-5-amine (12d) and 1-(1,2,3,5-tetrahydroxy-a-D-mannofuranose)-5-(((3,4-dimethoxypyridin-
Keywords:
2yl)methyl)amino)-2-methyl-1H-benzimidazole (15d) displayed dose-dependent anti-inflammatory
activities by decreasing the inflammation by 62% and 72%, respectively which is comparable to that of
diclofenac (73%). In contrast to diclofenac, the anti-inflammatory activity of these compounds was not
only free from any side effects on the gastric mucosa but also showed significant anti-ulcerogenic activity
in rat pyloric ligation and ethanol-induced gastric ulcer models similar to that of omeprazole. Together,
these findings suggest that 12d and 15d are potent anti-inflammatory agents with concurrent anti-
ulcerogenic activity and support its clinical promise as a component of therapeutic strategies for inflam-
mation, for which the gastric side effects are always a major limitation.
Omeprazole
2-Methyl-1H-benzimidazole
Cyclic sulfate
Anti-inflammatory activity
Anti-ulcerogenic activity
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
with gastric proton pump enzyme (H+/K+-ATPase) inhibitors repre-
sents a major approach to minimize such adverse effects.4,5 Many
Gastrointestinal toxicity is the most common adverse effect of
the currently available non-steroidal anti-inflammatory drugs
(NSAIDs) such as ibuprofen, indomethacin, and naproxen. Such ad-
verse effects are manifested by dyspepsia, ulcers, or bleeding.1 The
gastrointestinal damage from NSAIDs is generally attributed to two
factors; first, the local irritation by carboxylic acid group present in
many NSAIDs (topical effect); second, the decreased tissue prosta-
glandin production, which undermines the physiological role of
cytoprotective prostaglandins in maintaining gastrointestinal
integrity and homeostasis.2 The pharmacology of NSAIDs is linked
to the inhibition of prostaglandin biosynthesis from arachidonic
acid by inhibiting cyclooxygenases.3 Therefore, patients treated
with NSAIDs for long periods of time, may suffer from noticeable
gastrointestinal toxicity. Consequently, synthetic approaches
based on chemical mimicking NSAIDs have been taken with the
aim of improving its safety profile. The concurrent use of NSAIDs
pharmaceutical companies have spent considerable efforts in the
identification of irreversible and reversible inhibitors of the H+/
K+-ATPase. Substances belonging to the class of irreversible inhib-
itors are called proton pump inhibitors (PPIs)6–8 such as omepra-
zole, lansoprazole, pantoprazole, rabeprazole and esomeprazole.
PPIs are widely used as acid inhibitory agents for the treatment
of disorders related to gastric acid secretion.9,10
Many of PPIs are benzimidazole derivatives11,12 which consist of
two fragments of benzimidazole and pyridine. These PPIs act as
prodrugs owing to protonation of the pyridine ring under the gas-
trointestinal acid environment, resulting in a chemical rearrange-
ment which forms sulfenic acid then sulfonamide by
dehydration. The active enzyme inhibitor is either the sulfenic acid
or the sulfonamide which reacts with cysteine residues of the H+/
K+-ATP enzyme (Fig. 1).13–15 Omeprazole, a benzimidazole proton
pump inhibitor, has anti-inflammatory and antioxidant properties
besides its ability to stimulate gastric mucus secretion.16 Benz-
imidazole and its derivatives represent one of the most biologically
active classes of compounds, possessing a wide spectrum of activ-
ities including anti-inflammatory and analgesic.17 The unique
⇑
Corresponding author. Tel.: +966 550840062; fax: +20 2 3337 0931.
E-mail address: elnezhawy@yahoo.com (A.O.H. El-Nezhawy).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.070

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R2
R3
R4
R2
R3
H
N
N+
O
N
H+
R1
S
R1
R4
N
H
S
O
N
H
N
b
R2
R2
N
H2O
N
N
R1
N+
R3
R1
N+
R3
N
H
-H2O
R4
S
R4
S
HO
c
d
-H2O
R2
Enz-SH
Enz-SH
N
R1
N+
R3
N
H
R4
S
S
Enz
Figure 1. Mechanism of acid transformation of 2-[(2-pyridylmethyl)sulfinyl]benzimidazole derivatives. First, the gastric acidity causes protonation of the pyridine ring into
intermediate, b. Then, a chemical rearrangement forms sulfenic acid c, which forms sulfonamide d by dehydration. The active enzyme inhibitor is either the sulfenic acid c or
the sulfonamide d which reacts with cysteine of the H+/K+-ATP enzyme.13–15
structural features and pharmaceutical activities of benzimidaz-
oles have encouraged us to synthesize novel orally bioavailable
2-methyl-N-substituted benzimidazole sugar conjugates and study
its anti-inflammatory and anti-ulcerogenic activities.
mide and sodium hydride afforded a mixture of anomers 2
(6.8:1), which was separated by column chromatography. Only
a:2b
the
a-isomer was isolated by crystallization using ethyl acetate/
hexane, the b-anomer was not considered.21,22
Selective acid catalyzed hydrolysis of 2 gave diol 3.21 Cyclic sul-
fate 4 was obtained by the treatment of compound 3 with thionyl
chloride, RuCl3ꢀ3H2O and NaIO4 in anhyd CCl4. All products were
purified by column chromatography and the structure of the syn-
thesized compounds was determined by 1H, 13C NMR (Scheme 1).
Cyclic sulfate 4 is at least as reactive as epoxides and its reactivity
is the result of its ring strain which may be due to angle strain and
the partial double bond character between the ring oxygen and
sulfur atom. In addition, the use of cyclic sulfate in the synthesis
2. Results and discussion
2.1. Chemistry
2,3:5,6-Di-O-isopropylidene-
pared by the reaction of -mannose with acetone in the presence
of catalytic amount of concentrated sulfuric acid.18–20 Benzylation
D-mannofuranose (1) was pre-
D
of 2,3:5,6-di-O-isopropylidene-
D
-mannose (1) using benzyl bro-
O
O
a
b
O
O
O
O
D-mannose
O
O
O
O
OH
OBn
2
1
HO
O
S
O
c
d
O
O
O
O
O
O
O
HO
O
OBn
OBn
4
3
Scheme 1. Preparation of cyclic sulfate. Reagents and conditions: (a) acetone, H2SO4, Na2CO3, rt, 75%; (b) NaH, THF, benzyl bromide, rt, 86%; (c) 80% acetic acid, reflux, 89%;
(d) thionyl chloride, CCl4, RuCl3ꢀ3H2O, NaIO4, rt, 91%.

A. O. H. El-Nezhawy et al. / Bioorg. Med. Chem. 21 (2013) 1661–1670
1663
NH2
NH2
O2N
N
N
a
b
CH3
CH3
N
H
N
H
6
7
5
O2N
H2N
N
N
c
d
N
CH3
CH3
N
O
O
O
O
9
8
Scheme 2. Preparation of cyclic N-Boc benzimidazole derivative. Reagents and conditions: (a) 4 N HCl, CH3COOH, reflux, 68%; (b) H2SO4/HNO3, ammonia, rt, 58%; (c) Boc
anhydride, NaHCO3, methanol, rt, 78%; (d) Pd/C, acetic acid, methanol, H2 gas, rt, 75%.
R2
R3
R1
N
R2
N
H2N
N
N
a
R1
R3
HN
CH3
N
N
CH3
Cl
O
O
O
O
10
a. R1 = R2 = R3 = H
9
b. R1 = CH3, R2 = OCH3, R3 = H
c. R1 = CH3, R2 = OCH3, R3 = CH3
d. R1 = R2 = OCH3, R3 = H
11
a. R1 = R2 = R3 = H
b. R1 = CH3, R2 = OCH3, R3 = H
c. R1 = CH3, R2 = OCH3, R3 = CH3
d. R1 = R2 = OCH3, R3 = H
R2
R3
R1
b
N
HN
N
CH3
N
H
12
a. R1 = R2 = R3 = H
b. R1 = CH3, R2 = OCH3, R3 = H
c. R1 = CH3, R2 = OCH3, R3 = CH3
d. R1 = R2 = OCH3, R3 = H
Scheme 3. Preparation of deprotected benzimidazole derivatives carrying substituted pyridine moiety. Reagents and conditions: (a) K2CO3, KI, acetone, ethanol, reflux, 11a:
45%, 11b: 52%, 11c: 42%, 11d: 59%; (b) trifluoroacetic acid, CH2Cl2, rt, 12a: 98%, 12b: 97%, 12c: 98%, 12d: 98%.
produced only a single substituted product. 2-Methyl-1H-benz-
imidazole (6) and 2-methyl-5-nitrobenzimidazole (7) were pre-
pared as reported before.23–25
about 8 h yielded 11a–d (Scheme 3). The structure of newly syn-
thesized benzimidazole derivatives 11a–d was confirmed by IR,
1H NMR, 13C NMR, MS and the elemental analysis. The desired
deprotected benzimidazole derivatives (12a–d) were obtained by
the treatment of t-Boc protected benzimidazole derivatives 11a–
d with trifluoroacetic acid in CH2Cl2 and the mixture was stirred
at room temperature for 18 h (see spectral results in Section 4).
The ring opening of cyclic sulfate 4 with deprotected benzimid-
azole derivatives 12a–d was performed in tetrahydrofuran (THF)
using NaH as a base. The obtained 2-sulfate ester derivatives
13a–d (not isolated) were subsequently hydrolyzed to the hydroxy
compounds 14a–d by the treatment with concentrated sulfuric
acid and water. Hydrogenolysis of 14a–d with palladium on carbon
N-Boc-protected 2-methyl-5-nitro-1H-benzimidazole (8) was
obtained by the reaction of compound 7 with Boc anhydride in
the presence of sodium bicarbonate in methanol. The hydrogena-
tion of the 5-nitro group in compound 8 using Pd on charcoal, ace-
tic acid and methanol gave 1-(tert-butoxycarbonyl)-2-methyl-5-
amino-1H-benzimidazole (9) in 75% yield (Scheme 2). The struc-
ture of 8 and 9 were confirmed by NMR and mass spectra. Conden-
sation of 2-chloromethyl pyridine hydrochloride (10a–d) with 1-
(tert-butoxycarbonyl)-2-methyl-5-amino-1H-benzimidazole
(9)
by stirring in acetone/ethanol containing K2CO3/KI at 60 °C for

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A. O. H. El-Nezhawy et al. / Bioorg. Med. Chem. 21 (2013) 1661–1670
R2
N
R3
R1
a
O
S
O
O
O
O
O
HN
O
N
CH3
OBn
N
H
4
12 a. R1 = R2 = R3 = H
b. R1 = CH3, R2 = OCH3, R3 = H
c. R1 = CH3, R2 = OCH3, R3 = CH3
d. R1 = R2 = OCH3, R3 = H
R2
N
R2
R3
R1
R3
R1
N
HN
N
N
b
HN
N
N
CH3
CH3
O
O
O
HO
O
O
-O3SO
O
Na+
OBn
OBn
14 a. R1 = R2 = R3 = H
b. R1 = CH3, R2 = OCH3, R3 = H
13 a. R1 = R2 = R3 = H
b. R1 = CH3, R2 = OCH3, R3 = H
c. R1 = CH3, R2 = OCH3, R3 = CH3
d. R1 = R2 = OCH3, R3 = H
c. R1 = CH3, R2 = OCH3, R3 = CH3
d. R1 = R2 = OCH3, R3 = H
R2
N
R3
R1
c
HN
N
N
CH3
HO
OH
O
HO
OH
15 a. R1 = R2 = R3 = H
b. R1 = CH3, R2 = OCH3, R3 = H
c. R1 = CH3, R2 = OCH3, R3 = CH3
d. R1 = R2 = OCH3, R3 = H
Scheme 4. Preparation of target compounds. Reagents and conditions: (a) NaH, THF, rt; (b) H2SO4, water, rt, NaHCO3, 14a: 58%, 14b: 72%, 14c: 60%, 14d: 56%; (c) Pd/C, acetic
acid, ethyl acetate/methanol, H2, trifluoroacetic acid/water, rt, 15a: 67%, 15b: 60%, 15c: 73%, 15d: 76%.
in aqueous acetic acid followed by treatment with TFA/water mix-
ture produced 15a–d in a good yield (Scheme 4). The structures of
15a–d were confirmed by IR, 1H NMR, 13C NMR, MS and the ele-
mental analysis data which revealed the absence of both isopropyl-
idene protons and the benzyl group (see spectral results in
Section 4).
ferent doses; 10, 20 and 30 mg/kg by oral gavage. Most of the test
compounds reduced paw oedema in a dose- and time-dependent
manners. Compounds 12a–d exhibited moderate anti-inflamma-
tory activity compared to diclofenac (Table 1).
Compound 12d, at 20 and 30 mg/kg, resulted in a dose-depen-
dent reduction of carrageenan-induced oedema by 57% and 61%,
respectively after 3 h of inflammation induction. Compounds 12a,
12b, 12c at the same doses showed a moderate anti-inflammatory
activity. The relatively high anti-inflammatory activity of com-
pound 12d may be due to the presence of the electron-donating
methoxy group at 3- and 4-positions in pyrid-2-yl compounds
while bulky substituents such as methyl and methoxy groups at
the 3-, 5- and 4-positions in pyridine reduce the activity. At the
2.2. Biological evaluation
2.2.1. Anti-inflammatory activity
The anti-inflammatory activity of all the newly synthesized
compounds were tested in vivo using carrageenan-induced paw
oedema model in rats. Rats received test compounds in three dif-

A. O. H. El-Nezhawy et al. / Bioorg. Med. Chem. 21 (2013) 1661–1670
1665
Table 1
Effect of compounds 12a–d on carrageenan-induced paw oedema in rats
Treatment
Dose (mg/kg)
Oedema (ml) (mean SEM)
120 min
% Inhibition after 3 h
60 min
180 min
Control
12a
—
10
20
30
10
20
30
10
20
30
10
20
30
20
0.90 0.03
0.97 0.01
0.90 0.01
0.87 0.01
1.13 0.04
1.08 0.08
1.08 0.08
1.03 0.05
0.76 0.03
0.69 0.01
0.52 0.08*
0.42 0.04**
0.68 0.01
0.37 0.03**
0.34 0.01**
0.69 0.01
0.39 0.04**
0.36 0.02**
0.65 0.01*
0.33 0.03**
0.30 0.01**
0.20 0.02**
—
10
32
45
11
52
55
10
49
53
15
57
61
74
12b
0.70 0.01**
0.69 0.01**
0.64 0.01**
0.70 0.02**
0.69 0.01**
0.64 0.01**
0.70 0.02**
0.70 0.01**
0.65 0.01**
0.38 0.01**
0.90 0.02**
0.69 0.03**
0.65 0.03**
0.88 0.02**
0.67 0.03**
0.62 0.02**
0.88 0.02**
0.69 0.03**
0.65 0.03**
0.35 0.01**
12c
12d
Diclofenac
Each value represents mean SEM (n = 6).
*
p <0.05 when compared to control.
p <0.01 when compared to control.
**
Table 2
Effect of compounds 15a–d on carrageenan-induced paw oedema in rats
Treatment
Dose (mg/kg)
Oedema (ml) (mean SEM)
120 min
% Inhibition after 3 h
60 min
180 min
Control
15a
—
10
20
30
10
20
30
10
20
30
10
20
30
20
0.90 0.03
0.69 0.02*
0.68 0.03*
0.63 0.02*
0.68 0.02*
0.68 0.03*
0.63 0.01*
0.61 0.01*
0.50 0.01*
0.49 0.01*
0.53 0.01*
0.44 0.01*
0.41 0.01*
0.38 0.01*
1.13 0.04
0.89 0.02*
0.69 0.03*
0.64 0.03*
0.88 0.06*
0.68 0.01*
0.64 0.01*
0.64 0.01*
0.48 0.01*
0.43 0.01*
0.52 0.01*
0.40 0.01*
0.38 0.01*
0.35 0.01*
0.76 0.03
0.70 0.04
0.40 0.03*
0.37 0.01*
0.60 0.01*
0.36 0.03*
0.32 0.03*
0.49 0.01*
0.38 0.01*
0.34 0.01*
0.45 0.01*
0.29 0.01*
0.21 0.03*
0.20 0.02*
—
8
47
52
22
53
58
36
50
56
41
62
72
73
15b
15c
15d
Diclofenac
Each value represents mean SEM (n = 6).
*
p <0.01 when compared to control.
same time, methyl and methoxy groups at the 3- and 4-positions
can improve the activity.
mechanism for the induction of gastric ulcer. Most of the used
drugs for the treatment of gastric ulcer inhibit the acid secretion,
protect the mucosa, and inhibit the Helicobacter pylori.
Compounds 15a–d, at 10, 20 and 30 mg/kg doses, displayed
more significant anti-inflammatory activity than compounds
15a–d (Table 2). Among the 15a–d series, compound 15d exhibited
the most potent anti-inflammatory activity with 62% and 72% inhi-
bition of inflammation at 20 and 30 mg/kg doses, respectively
which was comparable to that of diclofenac sodium (73%). On
the other hand, compound 15a showed relatively poor anti-inflam-
matory activity. The high potency of the compound 15d could be
attributed to the presence of electron-donating methoxy groups
at 3- and 4-positions of pyrid-2-yl moiety and it could be also
dependent on the polyhydroxy sugar conjugated to the N-benz-
imidazole moiety. To conclude, the present pharmacological inves-
tigation showed that compounds 12d and 15d possess a potential
anti-inflammatory activity, which was evidenced by significant
reduction of carrageenan-induced paw oedema in rats.
Here we used two different experimental models to investigate
the protective effect of test compounds on gastric ulcer namely,
alcohol-induced gastric ulcer in mice and pylorus ligation-induced
gastric ulcer in rats. Alcohol can cause the lesion of gastric mucosa
by reinforcement of the aggressive factors while weaken the pro-
tective factors. Similarly, pylorus ligation leads to the accumula-
tion of gastric juice and enzymes in the stomach causing mucosal
injury. In both models of gastric ulcer, the newly synthesized com-
pounds showed dose-dependent preventive and therapeutic ac-
tions on the gastric mucosa as indicated by decreased ulcer size
(Table 3). The new compounds significantly decreased the gastric
acid secretion and inhibited H+/K+-ATPase in a profile similar to
that of omeprazole (Table 4).
In alcohol induced gastric ulcer, polyhydroxy sugar conjugated
to the N-benzimidazole (15a–d) produced a significant reduction
of the length of gastric lesions compared to control group while
compound 12d exhibited mild anti-ulcer effect. This weaker activ-
ity of 12d may be due to the absence of sugar moiety attached to
benzimidazole. Compounds 15b–d were more effective than 15a
which could be due to the presence of electron donating group in
the pyridine moiety (Table 3).
2.2.2. Anti-ulcerogenic activity
Gastrointestinal ulcer is a common disorder that affects millions
of people worldwide. The imbalance between aggressive factors
(gastric juice and pepsin) and protective factors (mucosal blood
flow, bicarbonate secretion, mucosal integrality, cell regeneration,
prostaglandins and other hormones) is considered the major

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A. O. H. El-Nezhawy et al. / Bioorg. Med. Chem. 21 (2013) 1661–1670
Table 3
ulcer induced by alcohol. However, 15a–d exhibited remarkable
Effect of test compounds on the ulcer index
anti-ulcer effect on ulcer induced by alcohol which may be due
to the sugar moiety attached with benzimidazole. Compounds
15b–d were more effective than 15a which may be due to the pres-
ence of electron donating group in the pyridine moiety and polyhy-
droxy sugar conjugated to the N-benzimidazole. In conclusion, our
results suggest that 12d and 15d have potent anti-inflammatory
activity comparable to that of diclofenac and this activity is not
only free from gastrointestinal toxicity, but also exhibits a potent
anti-ulcerogenic effects comparable to that of omeprazole. These
findings support clinical promise of both 12d and 15d as compo-
nents of therapeutic strategies for inflammation, for which the gas-
tric side effects are always a major limitation.
Treatment
Dose (mg/kg)
Length of lesion (mm)
Inhibition ratio (%)
Control
12d
—
126.0 2.5
88 4.5*
67 4.5*
68 4.5*
58 2.9*
53 4.7*
42 4.5*
47 3.9*
30 2.5*
49 4.9*
34 4.6*
40 5.0*
—
3.6
7.2
3.6
7.2
3.6
7.2
3.6
7.2
3.6
7.2
3.6
30.0
46.8
46.0
54.0
57.9
66.6
62.6
76.2
61.0
73.0
68.3
15a
15b
15c
15d
Omeprazole
Values are expressed as mean SD (n = 6).
p <0.01 compared to control group.
4. Experimental
4.1. Chemistry
*
In pylorus ligation-induced gastric ulcer model, compound 12d
exhibited a mild inhibition of the activity of both H+/K+-ATPase and
pepsin and a mild reduction of the volume of gastric juice. This rel-
atively weak anti-ulcer activity may be due to the absence of sugar
moiety attached with benzimidazole. In the contrary, the com-
pounds with polyhydroxy sugar conjugated to the N-benzimid-
azole (15a–d) displayed potent inhibition of the activity of H+/K+-
ATPase and pepsin in addition to potent inhibition of the gastric
juice production in a manner similar to that of omeprazole
(Table 4).
All chemicals were purchased from common commercial sup-
pliers and used without further purification. Melting points (mp)
were determined on a Gallenkamp melting point apparatus and
were uncorrected; IR spectra (KBr disks) were recorded on Bruker
Vector 22 instrument. 1H and 13C NMR spectra were recorded on a
Jeol ECX-spectrometer at 300 MHz, respectively, in CDCl3 as a sol-
vent and TMS as an internal standard at the National Research Cen-
ter. Mass spectra were recorded on Thermo Finnigan LCQ
Advantage spectrometer in ESI mode, I Spray Voltage 4.8 kV.
Microanalyses were performed at the Micro analytical Center of
Cairo University. All reactions were performed under positive pres-
sure of inert atmosphere. Thin layer chromatography (TLC) was
performed on silica gel 60 F254 plastic plates (E. Merck, layer
thickness 0.2 mm). Detection was achieved by treatment either
with a solution of 20 g of ammonium molybdate and 0.4 g of cer-
ium (IV) sulfate in 400 ml of 10% H2SO4 or with 15% H2SO4, and
heating at 150 °C.
3. Conclusions
In this work, we have prepared a series of benzimidazole sugar
conjugates carrying substituted pyrid-2-yl moiety, 15a–d, from 2-
methyl-N-(substituted-pyridin-2-ylmethyl)-1H-benzimidazol-5-
amine, 12a–d, and cyclic sulphate benzyl 2,3-O-isopropylidene-
5,6-O-sulfuryl-a-D-mannofuranoside 4. The newly synthesized N-
benzimidazole derivatives were found to possess potent anti-
inflammatory and anti-ulcerogenic activities. The results showed
that compounds 12d and 15d possess a potential anti-inflamma-
tory effect, which was evidenced by the significant reduction of
the inflammation in paw oedema model. The potency of the com-
pound 15d is attributed to the presence of the electron-donating
methoxy groups at 3- and 4-positions in pyrid-2-yl moiety also
the potent anti-inflammatory activity of compound 15d is depen-
dent on the linked-position of the polyhydroxy sugar conjugated
to the N-benzimidazole moiety. In addition, the results showed
that newly synthesized compound 12d which lacks the sugar moi-
ety attached to benzimidazole exhibited mild anti-ulcer effect on
4.1.1. 2,3:5,6-Di-O-isopropylidene-
a-
D
-mannofuranose (1)18–20
-man-
Concd sulfuric acid (14 ml) was added to a solution of
D
nose (20 g, 0.11 mol) in anhydrous acetone (30 ml) and the mix-
ture was stirred at rt for 4 h then neutralized with satd Na2CO3
solution. The reaction mixture was filtered and the filtrate was ex-
tracted using ethyl acetate (3 ꢁ 100 ml). The organic layers were
evaporated and concentrated to dryness under reduced pressure
gave 1 (21.6 g, 75%) as white crystals mp 121–122 °C. 1H NMR
(300 MHz, CDCl3 d 1.31 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.44 (s, 3H,
CH3), 1.45 (s, 3H, CH3), 2.90 (br s, 1H), 4.05 (m, 2H), 4.16 (dd,
Table 4
Effect of test compounds on pylorus ligation-induced gastric ulcer
Treatment
Dose (mg/
kg)
The activity of H+/K+-ATPase (
prot)
l
mol Pi/h/mg Peptic activity
ml/4 h
lg tyrosine/
The volume of gastric juice
(ml)
The pH value of gastric
juice
Sham
Control
12d
—
—
5.60 0.5**
11.44 1.5**
7.91 1.0**
6.88 1.15**
6.91 1.0**
6.02 1.15**
6.78 1.2**
6.00 1.12**
7.01 1.14**
6.62 1.00**
6.88 1.21**
6.34 1.25**
7.10 1.02**
2.60 0.75*
5.70 2.40
5.02 1.11
4.74 1.15
4.72 1.11
4.44 1.15
4.62 1.11
4.34 1.15
4.80 1.01
4.56 1.20
4.79 1.24
4.54 1.05
4.92 1.22
1.10 0.64**
5.96 0.80
3.88 0.42*
2.60 0.55
3.6
7.2
3.6
7.2
3.6
7.2
3.6
7.2
3.6
7.2
3.6
2.98 0.42**
2.60 0.51**
2.40 0.42**
2.30 0.51**
2.31 0.40**
2.24 0.31**
2.48 0.66**
2.38 0.50**
2.56 0.20**
2.39 0.41**
2.64 1.00**
3.64 1.12*
4.01 1.75**
4.04 1.12*
4.41 1.75**
4.10 1.12*
4.31 1.75**
4.14 1.62*
4.39 1.21**
4.53 1.32*
4.73 1.35**
4.26 0.92**
15a
15b
15c
15d
Omeprazole
Values are expressed as mean SD (n = 6).
*
p <0.05 compared to control group.
p <0.01 compared to control group.
**

A. O. H. El-Nezhawy et al. / Bioorg. Med. Chem. 21 (2013) 1661–1670
1667
J = 3.6, 7.1 Hz, 1H), 4.39 (ddd, J = 5.4, 5.5, 7.1 Hz, 1H), 4.59 (d,
J = 5.9 Hz, 1H), 4.79 (dd, J = 3.6, 5.9 Hz, 1H), 5.36 (d, J = 3.6 Hz, 1H).
1H NMR (300 MHz, CDCl3 d 2.57 (s, 3H, CH3), 4.71 (s, 1H, NH),
7.51 (d, J = 8.8 Hz, 1H, H7), 8.09 (d, J = 8.8 Hz, 1H, H6), 8.24 (s,
1H, H4).
4.1.2. Benzyl 2,3:5,6-di-O-isopropylidene-
a-
D-mannofuranose
(2)21,22
4.1.7. 1-(tert-Butoxycarbonyl)-2-methyl-5-nitro-1H-
To a solution of 2,3:5,6-di-O-isopropylidene-
a
-D
-mannofura-
benzimidazole (8)
nose (11 g, 0.042 mmol) in THF (5 ml), a suspension of 60% NaH
(0.24 g, 10 mmol) was added at 0 °C, and the mixture was stirred
for 40 min. To the mixture, benzyl bromide (0.51 g, 3 mmol) was
added and the reaction mixture was stirred overnight and concen-
trated by evaporation. A mixture of MeOH (30 ml) and water
(100 ml) was added to the residue. The aqueous phase was ex-
tracted with EtOAc and dried (Na2SO4). The organic layer was con-
centrated under reduced pressure and the residue was purified by
A
mixture of 2-methyl-5-nitro-1H-benzimidazole (0.7 g,
4 mmol), (Boc)2O (4.4 mol) and NaHCO3 (4.4 mol) in methanol
(30 ml) was stirred at rt for 24 h. The solid was filtered then the
solvent was evaporated and purified by column chromatography
(petroleum ether/ethyl acetate, 10:1) gave 8 (0.86 g, 78%) as white
solid, mp 134–136 °C. 1H NMR (300 MHz, CDCl3 d 1.43 (s, 9H,
3CH3), 2.50 (s, 3H, CH3), 7.61 (d, J = 9 Hz, 1H, H7), 8.02 (d,
J = 9 Hz, 1H, H6), 8.34 (s, 1H, H4). 13C NMR (75 MHz, CDCl3
d
crystallization using ethyl acetate/hexane to provide 2
a
(12.7 g,
14.9, 28.6, 84.1, 84.3, 85.7, 113.3, 116.8, 119.0, 137.0, 138.2,
140.1, 145.0, 148.1 ppm. MS (EI) m/z (%): 277.11 (20, M+), 176
(100, M-101). Anal. Calcd for C13H15N3O4: C, 56.31; H, 5.45; N,
15.15. Found: C, 56.50; H, 5.27; N, 15.10.
86%) as white crystals, mp 81–82 °C. 1H NMR (300 MHz, CDCl3
d
1.34 (s, 3H, CH3), 1.42 (s, 3H, CH3), 1.50 (s, 3H, CH3), 1.51 (s, 3H,
CH3), 3.93 (m, 1H), 4.18–4.47 (m, 3H), 4.50–4.65 (2d, J = 12.0 Hz,
2H, OCH2Ph), 4.75–4.88 (m, 2H), 5.11 (s, 1H, H-l), 7.26 (m, 5H, Ar).
4.1.8. 1-(tert-Butoxycarbonyl)-2-methyl-5-amino-1H-
benzimidazole (9)
4.1.3. Benzyl 2,3-O-isopropylidene-
a-
D
-mannofuranose (3)21
A solution of 2 (1.2 g, 4.3 mmol) was dissolved in 80% acetic
acid (100 ml), and the reaction mixture was refluxed for 3 h. The
solution was concentrated under reduced pressure, and the residue
was purified by silica gel column chromatography (ethyl acetate/
methanol, 20:1) to provide 3 (0.94 g, 89%) as white crystals, mp
81–82 °C. 1H NMR (300 MHz, CDCl3 d 1.36 (s, 3H, CH3), 1.50 (s,
3H, CH3), 2.90–3.40 (br s, 2H, 2OH), 3.65 (m, 2H), 3.80 (d,
J = 7.0 Hz, 2H), 4.00 (m, 2H), 4.49–4.65 (2d, J = 11.0 Hz, 2H,
OCH2Ph), 5.10 (s, 1H, H-l), 7.30 (m, 5H, Ar).
Pd on charcoal 10% (8 mg) was added to a solution of 1-(tert-
butoxycarbonyl)-2-methyl-5-nitro-1H-benzimidazole (8) (0.55 g,
2 mmol) and acetic acid (0.2 ml) in methanol (20 ml) then the mix-
ture was hydrogenated at 3.4 bar and stirred at rt for 24 h. The
reaction mixture was filtered on Celite. All organic layers were con-
centrated to dryness under reduced pressure. The residue was
purified by column chromatography (petroleum ether/ethyl ace-
tate, 1:1) gave 9 (0.37 g, 75%) as colorless oil. 1H NMR (300 MHz,
CDCl3 d 1.36 (s, 9H, 3 CH3), 2.59 (s, 3H, CH3), 5.71 (br s, 2H), 7.51
(d, J = 8.2 Hz, 1H, H7), 7.92 (d, J = 8.2 Hz, 1H, H6), 8.20 (s, 1H,
H4). 13C NMR (75 MHz, CDCl3 d 14.5, 28.0, 83.0, 97.9, 113.0,
117.8, 119.8, 139.1, 140.8, 145.1, 150.1 ppm. MS (EI) m/z (%):
247.13 (14, M+), 146 (100, M-101). Anal. Calcd for C13H17N3O2: C,
63.14; H, 6.93; N, 16.99. Found: C, 63.00; H, 6.81; N, 16.78.
4.1.4. Benzyl 2,3-O-isopropylidene-5,6-O-sulfuryl-a-D-
mannofuranoside (4)
The diol 3 (15.4 g, 50 mmol) was dissolved in anhyd CCl4
(50 ml) then thionyl chloride (4.4 ml, 60 mmol) was added. The
mixture was refluxed for 30 min then the solution was cooled to
0 °C and diluted with MeCN (50 ml). RuCl3ꢀ3H2O (8 mg,
0.03 mmol), NaIO4 (16 g, 75 mmol) and H2O (50 ml) were added
to the mixture. This mixture was kept at rt for 1 h and then Et2O
(400 ml) was added. The organic phase was separated, washed suc-
cessively with H2O (200 ml), satd NaHCO3 (2 ꢁ 200 ml), dried
(MgSO4) and concentrated. The product was purified by column
chromatography using (petroleum ether/diethyl ether, 9:1) gave
4 (16.8 g, 91%) as white solid, mp 106 °C. 1H NMR (300 MHz, CDCl3
d 1.20 (s, 3H, CH3), 1.41 (s, 3H, CH3), 4.22 (dd, J = 9.6, 3.5 Hz, 1H),
4.40–4.51 (m, 4H), 4.61–4.81 (m, 3H), 5.06 (s, 1H, H-l), 7.26 (m,
5H, Ar). 13C NMR (75 MHz, CDCl3 d 26.6, 69.9, 72.8, 79.3, 81.1,
84.3, 85.7, 105.3, 120.0, 128.2, 128.1, 129.0, 138.1 ppm.
4.1.9. General procedure for the synthesis of (11a–d)
2-Chloromethyl pyridine hydrochloride (0.8 g, 5.0 mmol), 2-
chloromethyl-4-methoxy-3-methylpyridine hydrochloride (1.0 g,
5.0 mmol),
2-chloromethyl-4-methoxy-3,5-dimethylpyridine
hydrochloride (1.1 g, 5.0 mmol), 2-chloromethyl-3,4-dimethoxy-
pyridine hydrochloride (1.12 g, 5.0 mmol), K2CO3 (1.1 g, 8.0 mmol)
and KI (1.32 g, 8 mmol) were added to a magnetically stirred solu-
tion of 9 (1.24 g, 5.0 mmol) in a mixture of acetone (30 ml) and eth-
anol (30 ml). The reaction mixture was stirred constantly at 60 °C for
about 8 h. When the reaction was finished, as monitored by TLC,
water (100 ml) was added. Then the reaction mixture was extracted
withDCM(30 ml ꢁ 4). Theorganicphasewascombinedandconcen-
trated under reduced pressure gave crude compounds 11a–d.
4.1.5. 2-Methyl-1H-benzimidazole (6)23–25
A
mixture of o-phenylenediamine dihydrochloride (3.24 g,
4.1.9.1.
ylmethyl)amino)-1H-benzimidazole (11a).
lid, (0.76 g, 45%); mp: 276–279 °C. FT-IR
1-(tert-Butoxycarbonyl)-2-methyl-5-((pyridine-2-
0.03 mol) in (20 ml) 4 N HCl and (0.09 mol) of acetic acid was re-
fluxed for 4 h. The cooled reaction mixture was rendered distinctly
basic by gradual addition of concd ammonia solution. The precip-
itate was collected and recrystallized from 10% aqueous ethanol
gave 6 (2.69 g, 68%) as white solid, mp 173–174 °C. 1H NMR
(300 MHz, CDCl3 d 2.51 (s, 3H, CH3), 5.01 (s, 1H, NH), 7.22–7.59
(m, 4H, Ar-H).
Pale yellow so-
m
(cmꢂ1): 3394 (NH),
1689 (C@O), 1624 (C@N). 1H NMR (300 MHz, CDCl3 d 1.36 (s, 9H,
3 CH3), 2.59 (s, 3H, CH3), 4.20 (s, 1H, NH), 4.61 (d, J = 10.1 Hz,
1H), 4.76 (d, J = 10.1 Hz, 1H), 7.48–7.94 (m, 5H), 8.20 (s, 1H), 8.27
(d, 1H). 13C NMR (75 MHz, CDCl3 d 15.2, 29.4, 48.7, 82.7, 102.9,
116.2, 117.4, 120.6, 121.5, 123.1, 138.9, 140.5, 141.7, 143.6,
150.6, 153.9, 162.9 ppm. MS (EI) m/z (%): 338.17 (20, M+), 237
(100, M-101), 145 (80, M-193). Anal. Calcd for C19H22N4O2: C,
67.44; H, 6.55; N, 16.56. Found: C, 67.24; H, 6.65; N, 16.60.
4.1.6. 2-Methyl-5-nitro-1H-benzimidazole (7)23–25
Concd sulfuric acid (10 ml) was added with stirring to 2-
methyl-1H-benzimidazole (0.66 g, 0.005 mol) then a nitrating mix-
ture made from HNO3 (0.36 ml) and concd sulfuric acid (0.4 ml)
was cautiously added dropwise. After stirring for 2 h, compound
7 was isolated by pouring on ice-water then neutralizing with
aqueous ammonia (0.51 g, 58%) as white solid, mp 223–225 °C.
4.1.9.2.
methylpyridin-2-yl)methyl)amino)-1H-benzimidazole
(11b). Yellow solid, (1.0 g, 52%); mp: 231–233 °C. FT-IR
(cmꢂ1): 3400 (NH), 1682 (C@O), 1625 (C@N). 1H NMR (300 MHz,
1-(tert-Butoxycarbonyl)-2-methyl-5-(((4-methoxy-3-
m

1668
A. O. H. El-Nezhawy et al. / Bioorg. Med. Chem. 21 (2013) 1661–1670
CDCl3 d 1.36 (s, 9H, 3 CH3), 2.34 (s, 3H), 2.51 (s, 3H, CH3), 3.75 (s,
3H, CH3), 4.29 (s, 1H, NH), 4.64 (d, J = 10.3 Hz, 1H), 4.78 (d,
J = 10.3 Hz, 1H), 7.50–7.75 (m, 3H), 8.23 (s, 1H), 8.37 (d, 1H). 13C
NMR (75 MHz, CDCl3 d 11.4, 15.7, 28.8, 46.8, 56.9, 82.9, 101.9,
105.4112.5, 116.8, 118.1, 119.9, 121.5, 137.8, 139.3, 143.6, 153.9,
160.8, 165.1 ppm. MS (EI) m/z (%): 382.20 (12, M+), 281 (100, M-
101), 145 (56, M-237). Anal. Calcd for C21H26N4O3: C, 65.95; H,
6.85; N, 14.65. Found: C, 66.01; H, 6.78; N, 14.55.
137). Anal. Calcd for C16H18N4O: C, 68.06; H, 6.43; N, 19.84. Found:
C, 68.16; H, 6.33; N, 19.81.
4.1.10.3.
yl)methyl)-1H-benzimidazol-5-amine (12c).
(2.6 g, 98%). FT-IR
(cmꢂ1): 3394 (NH), 1634 (C@N). 1H NMR
2-Methyl-N-((4-methoxy-3,5-dimethylpyridin-2-
Colorless oil,
m
(300 MHz, CDCl3 d 2.31 (s, 3H, CH3), 2.44 (s, 3H, CH3), 2.50 (s, 3H,
CH3), 3.80 (s, 3H, OCH3), 4.68 (d, J = 10.2 Hz, 1H), 4.73 (s, 1H,
NH), 4.82 (d, J = 10.2 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.86 (d,
J = 8.6 Hz, 1H), 8.26 (s, 1H), 8.34 (s, 1H), 12.20 (s, 1H, NH). 13C
NMR (75 MHz, CDCl3 d 11.0, 14.6, 15.8, 45.6, 60.4, 101.2, 111.0,
115.8, 116.2, 116.8, 123.1, 131.8, 137.9, 148.2, 152.8, 158.1,
165.9 ppm. MS (EI) m/z (%): 296.16 (100, M+), 145 (52, M-151).
Anal. Calcd for C17H20N4O: C, 68.89; H, 6.80; N, 18.90. Found: C,
68.79; H, 6.70; N, 18.98.
4.1.9.3. 1-(tert-Butoxycarbonyl)-2-methyl-5-(((4-methoxy-3,5-
dimethylpyridin-2-yl)methyl)amino)-1H-benzimidazole
(11c).
White solid, (0.83 g, 42%); mp: 289–291 °C. FT-IR m
(cmꢂ1): 3396 (NH), 1694 (C@O), 1635 (C@N). 1H NMR (300 MHz,
CDCl3 d 1.35 (s, 9H, 3 CH3), 2.41 (s, 3H, CH3), 2.49 (s, 3H, CH3),
2.55 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 4.35 (s, 1H, NH), 4.69 (d,
J = 11.3 Hz, 1H), 4.79 (d, J = 11.3 Hz, 1H), 7.50 (d, J = 8.1 Hz, 1H),
7.96 (d, J = 8.1 Hz, 1H), 8.27 (s, 1H), 8.35 (s, 1H). 13C NMR
(75 MHz, CDCl3 d 11.6, 14.8, 15.8, 28.3, 45.9, 60.8, 83.1, 102.2,
111.5, 115.0, 115.7, 116.5, 119.1, 136.9, 140.1, 141.6, 148.5,
150.9, 159.1, 164.9 ppm. MS (EI) m/z (%): 396.22 (25, M+), 295
(100, M-101), 145 (72, M-237). Anal. Calcd for C22H28N4O3: C,
66.64; H, 7.12; N, 14.13. Found: C, 66.54; H, 7.31; N, 14.11.
4.1.10.4. 2-Methyl-N-((3,4-dimethoxypyridin-2-yl)methyl)-1H-
benzimidazol-5-amine (12d).
m
Yellow oil (2.6 g, 98%). FT-IR
(cmꢂ1): 3423 (NH), 1630 (C@N). 1H NMR (300 MHz, CDCl3
d
2.28 (s, 3H, CH3), 3.60 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 4.75 (d,
J = 10.2 Hz, 1H), 4.80 (s, 1H, NH), 4.85 (d, J = 10.2 Hz, 1H), 7.56 (d,
J = 8.2 Hz, 1H), 7.66 (d, J = 6.8 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H),
8.27 (s, 1H), 8.32 (d, J = 6.8 Hz 1H), 11.90 (s, 1H, NH). 13C NMR
(75 MHz, CDCl3 d 14.0, 42.9, 56.3, 60.8, 101.9, 105.8, 115.6, 116.8,
123.8, 131.8, 137.8, 141.3, 142.8, 143.8, 152.6, 157.6 ppm. MS
(EI) m/z (%): 298.14 (100, M+), 145 (60, M-153). Anal. Calcd for
4.1.9.4. 1-(tert-Butoxycarbonyl)-2-methyl-5-(((3,4-dimethoxy-
pyridin-2-yl)methyl)amino)-1H-benzimidazole
(11d).
White solid, (1.18 g, 59%); mp 239–241 °C. FT-IR m
(cmꢂ1): 3390 (NH), 1687 (C@O), 1632 (C@N). 1H NMR (300 MHz,
CDCl3 d 1.36 (s, 9H, 3 CH3), 2.39 (s, 3H, CH3), 3.65 (s, 3H, OCH3),
3.68 (s, 3H, OCH3), 4.39 (s, 1H, NH), 4.79 (d, J = 11.0 Hz, 1H), 4.82
(d, J = 11.0 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 6.5 Hz, 1H),
7.96 (d, J = 8.1 Hz, 1H), 8.27 (s, 1H), 8.35 (d, J = 6.5 Hz 1H). 13C
NMR (75 MHz, CDCl3 d 14.6, 28.9, 43.9, 57.7, 60.9, 83.8, 102.7,
106.7, 115.9, 116.4, 119.2, 137.9, 139.1, 141.9, 142.5, 143.1,
144.2, 150.8, 159.2 ppm. MS (EI) m/z (%): 398.20 (12, M+), 297
(100, M-101), 145 (60, M-253). Anal. Calcd for C21H26N4O4: C,
63.30; H, 6.58; N, 14.06. Found: C, 63.44; H, 6.39; N, 14.12.
C16H18N4O2: C, 64.41; H, 6.08; N, 18.78. Found: C, 64.33; H, 6.19;
N, 18.70.
4.1.11. General procedure for the synthesis of (14a–d)
To an ice-cold suspension of 60% NaH (65 mmol) in THF
(130 ml), benzimidazole derivatives 12a (1.5 g, 6.5 mmol), 12b
(1.8 g, 6.5 mmol), 12c (1.9 g, 6.5 mmol) or 12d (1.92 g, 6.5 mmol)
was added dropwise. The reaction mixture was stirred for 1 h then
cyclic sulfate 4 (2.8 g, 7.5 mmol) was added. The reaction mixture
was stirred overnight. Cleavage was carried out by the addition of
concd H2SO4 (3.6 ml) and water (1.3 ml). After stirring for 1 h at
room temperature, the reaction mixture was neutralized by adding
saturated NaHCO3, and washed with CHCl3 (2 ꢁ 20 ml). The organ-
ic layers were collected, dried with anhydrous Na2SO4, and evapo-
rated. The oil obtained was purified by column chromatography
gave 14a–d.
4.1.10. General procedure for the synthesis of (12a–d)
The t-Boc protected benzimidazole derivatives 11a (4.0 g,
9.0 mmol), 11b (3.4 g, 9.0 mmol), 11c (3.6 g, 9.0 mmol) or 11d
(3.58 g, 9.0 mmol) and trifluoroacetic acid (0.46 g, 4 mmol) in
CH2Cl2 (50 ml) were stirred at room temperature for 18 h. The sol-
vents were removed by rotary evaporation and the residue was
recrystallized from acetonitrile to provide (98%) of the desired
deprotected benzimidazole derivatives (12a–d).
4.1.11.1. 1-(5-Hydroxy benzyl-2,3-O-isopropylidene-
nofuranose)-5-((pyridin-2-ylmethyl)amino)-2-methyl-1H-
benzimidazole (14a). Colorless oil, (2.0 g, 58%); FT-IR
a-D-man-
m
4.1.10.1. 2-Methyl-N-(pyridin-2-ylmethyl)-1H-benzimidazol-5-
(cmꢂ1): 3450 (OH), 3390 (NH), 1630 (C@N). 1H NMR (300 MHz,
CDCl3 d 1.29 (s, 3H, CH3), 1.31 (s, 3H, CH3), 2.30 (s, 3H, CH3), 3.30
(br s, 1H, OH) 4.20 (m, 1H), 4.47 (d, J = 11.8 Hz, 2H), 4.58 (s, 1H,
NH), 4.61–4.68 (m, 4H),4.71 (d, J = 8.9 Hz, 1H), 4.77 (d, J = 8.9 Hz,
1H), 4.92 (dd, J = 3.6, 3.6 Hz, 1H), 5.09 (s, 1H), 7.28 (m, 5H), 7.48–
7.94 (m, 4H), 8.20 (s, 1H), 8.15–8.24 (m, 2H). 13C NMR (75 MHz,
CDCl3 d 14.7, 26.5, 47.6, 47.8, 69.0, 71.4, 80.2, 80.4, 84.7, 101.8,
104.9, 113.4, 115.8, 120.9, 121.1, 121.4, 127.5, 127.7, 128.0,
128.4, 128.7, 131.8, 133.4, 137.3, 137.9, 139.4, 148.8, 149.3,
158.7 ppm. Anal. Calcd for C30H34N4O5: C, 67.91; H, 6.46; N,
10.56. Found: C, 67.78; H, 6.36; N, 10.50.
amine (12a).
White solid, (2.76 g, 98%); mp: 296–298 °C.
FT-IR
m
(cmꢂ1): 3410 (NH), 1624 (C@N). 1H NMR (300 MHz, CDCl3
d 2.30 (s, 3H, CH3), 4.60 (s, 1H, NH), 4.68 (d, J = 8.9 Hz, 1H), 4.77 (d,
J = 8.9 Hz, 1H), 7.48–7.94 (m, 5H), 8.20 (s, 1H), 8.15–8.24 (m, 2H),
11.7 (s, 1H, NH). 13C NMR (75 MHz, CDCl3 d 14.2, 47.7, 101.8,
115.8, 117.6, 120.6, 121.4, 123.9131.8, 137.9, 140.4, 149.6, 152.9,
158.9 ppm. MS (EI) m/z (%): 338.12 (100, M+), 145 (60, M-193).
Anal. Calcd for C14H14N4: C, 70.57; H, 5.92; N, 23.51. Found: C,
70.52; H, 5.82; N, 23.41.
4.1.10.2.
yl)methyl)-1H-benzimidazol-5-amine (12b).
(2.4 g, 97%). FT-IR
(cmꢂ1): 3410 (NH), 1628 (C@N). 1H NMR
(300 MHz, CDCl3 d 2.36 (s, 3H, CH3), 2.41 (s, 3H, CH3), 3.72 (s, 3H,
OCH3), 4.59 (s, 1H, NH), 4.74 (d, J = 9.3 Hz, 1H), 4.79 (d, J = 9.3 Hz,
1H), 7.51–7.70 (m, 3H), 8.22 (s, 1H), 8.37 (d, 1H), 12.11 (s, 1H,
NH). 13C NMR (75 MHz, CDCl3 d 10.8, 14.7, 46.3, 56.6, 101.7,
104.8, 111.5, 115.8, 117.1, 123.5, 131.4, 137.8, 147.6, 152.9,
161.8, 164.8 ppm. MS (EI) m/z (%): 282.15 (100, M+), 145 (70, M-
2-Methyl-N-((4-methoxy-3-methylpyridin-2-
Yellow oil,
4.1.11.2. 1-(5-Hydroxy benzyl-2,3-O-isopropylidene-
nofuranose)-5-(((4-methoxy-3-methylpyridin-2-yl)methyl)a-
mino)-2-methyl-1H-benzimidazole (14b). Colorless oil,
(2.7 g, 72%); FT-IR
(cmꢂ1): 3435 (OH), 3384 (NH), 1635 (C@N).
a-D-man-
m
m
1H NMR (300 MHz, CDCl3 d 1.29 (s, 3H, CH3), 1.31 (s, 3H, CH3),
2.26 (s, 3H, CH3), 2.38 (s, 3H, CH3), 3.36 (br s, 1H, OH), 3.68 (s,
3H, OCH3), 4.22 (m, 1H), 4.44 (d, J = 11.3 Hz, 2H), 4.50 (s, 1H,
NH), 4.58–4.69 (m, 4H), 4.73 (d, J = 8.3 Hz, 1H), 4.80 (d, J = 8.3 Hz,

A. O. H. El-Nezhawy et al. / Bioorg. Med. Chem. 21 (2013) 1661–1670
1669
1H), 4.90 (dd, J = 3.6, 3.6 Hz, 1H), 5.13 (s, 1H), 7.32 (m, 5H), 7.44–
7.74 (m, 3H), 8.20 (s, 1H), 8.37 (d, J = 5.8, 1H). 13C NMR (75 MHz,
CDCl3 d 10.5, 14.2, 27.2, 46.0, 49.8, 57.0, 67.6, 71.0, 80.6, 83.4,
84.9, 102.0, 103.8, 104.6, 111.6, 113.7, 115.6, 121.2, 121.4,
127.5127.7, 128.2, 128.4, 131.5, 133.2, 137.9, 138.1, 148.0, 149.6,
159.7, 165.1 ppm. Anal. Calcd for C32H38N4O6: C, 66.88; H, 6.67;
N, 9.75. Found: C, 66.77; H, 6.80; N, 9.65.
4.1.12.2. 1-(1,2,3,5-Tetrahydroxy-
methoxy-3-methylpyridin-2-yl)methyl)amino)-2-methyl-1H-
benzimidazole (15b). Yellow oil, (0.53 g, 60%); FT-IR
a
-
D
-mannofuranose)-5-(((4-
m
(cmꢂ1):
3455–3410 (OH), 3395 (NH), 1630 (C@N). 1H NMR (300 MHz, CDCl3
d 2.41 (s, 3H, CH3), 2.51 (s, 3H, CH3), 3.60 (s, 3H, OCH3), 3.65 (m, 3H),
3.80 (m, 3H), 3.98 (m, 2H), 4.39 (m, 1H), 4.81 (m, 2H), 5.42 (s, 1H),
5.66 (d, J = 11.8 Hz, 1H), 5.78 (d, J = 11.8 Hz, 1H), 7.56 (d, J = 8.2 Hz,
1H), 7.66 (d, J = 6.8 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 8.27 (s, 1H),
8.32 (d, J = 6.8 Hz 1H). 13C NMR (75 MHz, CDCl3 d 11.0, 14.8, 45.8,
51.0, 56.0, 66.4, 75.2, 77.0, 84.9, 101.9, 103.0, 104.6, 111.8, 113.0,
115.0, 131.3, 133.0, 137.3, 148.0, 149.3, 160.0, 162.9 ppm. Anal.
Calcd for C22H28N4O6: C, 59.45; H, 6.35; N, 12.60. Found: C, 59.40;
H, 6.25; N, 12.69.
4.1.11.3. 1-(5-Hydroxy benzyl-2,3-O-isopropylidene-
nofuranose)-5-(((4-methoxy-3,5-dimethylpyridin-2-yl)methy-
l)amino)-2-methyl-1H-benzimidazole (14c). Colorless oil,
(2.3 g, 60%); FT-IR
(cmꢂ1): 3420 (OH), 3380 (NH), 1630 (C@N).
a-D-man-
m
1H NMR (300 MHz, CDCl3 d 1.28 (s, 3H, CH3), 1.31 (s, 3H, CH3),
2.31 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.51 (s, 3H, CH3), 3.41 (br s,
1H, OH), 3.81 (s, 3H, OCH3), 4.20 (m, 1H), 4.44 (d, J = 11.6 Hz,
2H), 4.53 (s, 1H, NH), 4.58–4.69 (m, 4H), 4.74 (d, J = 8.5 Hz, 1H),
4.81 (d, J = 8.5 Hz, 1H), 4.94 (dd, J = 3.6, 3.6 Hz, 1H), 5.15 (s, 1H),
7.30 (m, 5H), 7.60 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 8.22
(s, 1H), 8.31 (s, 1H). 13C NMR (75 MHz, CDCl3 d 10.8, 14.6, 15.8,
26.2, 45.8, 49.8, 50.6, 60.1, 67.3, 71.9, 80.2, 83.4, 85.2, 101.9,
104.8, 111.5, 113.6, 115.2, 115.9, 121.0, 127.9, 128.1, 128.2,
128.4, 131.9, 133.6, 137.9, 138.4, 148.2, 149.6, 159.7, 164.1 ppm.
Anal. Calcd for C33H34N4O6: C, 67.33; H, 6.85; N, 9.52. Found: C,
67.47; H, 6.80; N, 9.40.
4.1.12.3. 1-(1,2,3,5-Tetrahydroxy-
methoxy-3,5-dimethylpyridin-2-yl)methyl)amino)-2-methyl-
1H-benzimidazole (15c). Pale yellow oil, (0.66 g, 73%); FT-IR
(cmꢂ1): 3450–3415 (OH), 3400 (NH), 1631 (C@N). 1H NMR
a-D-mannofuranose)-5-(((4-
m
(300 MHz, CDCl3 d 2.30 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.50 (s, 3H,
CH3), 3.70 (s, 3H, OCH3), 3.75 (m, 3H), 3.81 (m, 3H), 3.98 (m, 2H),
4.40 (m, 1H), 4.80 (m, 2H), 5.40 (s, 1H), 5.56 (d, J = 10.7 Hz, 1H),
5.77 (d, J = 10.7 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 8.3 Hz,
1H), 8.23 (s, 1H), 8.30 (s, 1H). 13C NMR (75 MHz, CDCl3 d 10.7,
14.9, 15.4, 45.0, 50.0, 60.1, 65.4, 74.8, 78.1, 85.9, 102.8, 104.3,
112.7, 114.1, 116.8, 117.0, 132.7, 136.9, 147.5, 150.9, 156.7,
160.1 ppm. Anal. Calcd for C23H30N4O6: C, 60.25; H, 6.59; N,
12.22. Found: C, 60.37; H, 6.70; N, 12.12.
4.1.11.4. 1-(5-Hydroxy benzyl-2,3-O-isopropylidene-
nofuranose)-5-(((3,4,dimethoxypyridin-2-yl)methyl)amino)-2-
methyl-1H-benzimidazole (14d). Colorless oil, (2.1 g, 56%);
FT-IR
(cmꢂ1): 3428 (OH), 3386 (NH), 1635 (C@N). 1H NMR
a-D-man-
m
4.1.12.4. 1-(1,2,3,5-Tetrahydroxy-
dimethoxypyridin-2-yl)methyl)amino)-2-methyl-1H-benzimid-
azole (15d). Pale yellow oil, (0.7 g, 76%); FT-IR
(cmꢂ1):
a-D-mannofuranose)-5-(((3,4-
(300 MHz, CDCl3 d 1.29 (s, 3H, CH3), 1.32 (s, 3H, CH3), 2.30 (s, 3H,
CH3), 3.49 (br s, 1H, OH), 3.61 (s, 3H, OCH3), 3.78 (s, 3H, OCH3),
4.16 (m, 1H), 4.40 (d, J = 11.0 Hz, 2H), 4.60 (s, 1H, NH), 4.63–4.70
(m, 4H), 4.79 (d, J = 8.4 Hz, 1H), 4.86 (d, J = 8.4 Hz, 1H), 4.90 (dd,
J = 3.5 Hz, 1H), 5.10 (s, 1H), 7.35 (m, 5H), 7.58 (d, J = 8.6 Hz, 1H),
7.62 (d, J = 6.2 Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 8.23 (s, 1H), 8.30
(d, J = 6.2 Hz 1H). 13C NMR (75 MHz, CDCl3 d 14.9, 26.9, 42.8,
50.4, 57.1, 61.4, 67.6, 72.9, 80.2, 83.7, 85.2, 101.6, 105.6, 105.9,
113.0, 115.9, 122.0, 128.9, 129.0, 129.1, 129.2, 129.4, 132.9,
134.6, 137.8, 138.9, 142.1, 143.0, 143.9, 149.2, 157.2 ppm. Anal.
Calcd for C32H38N4O7: C, 65.07; H, 6.48; N, 9.49. Found: C, 65.19;
H, 6.28; N, 9.40.
m
3458–3410 (OH), 3405 (NH), 1633 (C@N). 1H NMR (300 MHz,
CDCl3 d 2.41 (s, 3H, CH3), 3.60 (s, 3H, OCH3), 3.68 (s, 3H, OCH3),
3.70 (m, 3H), 3.80 (m, 3H), 3.93 (m, 2H), 4.42 (m, 1H), 4.69 (m,
2H), 5.36 (s, 1H), 5.60 (d, J = 11.7 Hz, 1H), 5.76 (d, J = 11.7 Hz,
1H), 7.50 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 6.0 Hz, 1H), 7.77 (d,
J = 8.1 Hz, 1H), 8.20 (s, 1H), 8.31 (d, J = 6.0 Hz 1H). 13C NMR
(75 MHz, CDCl3 d 15.9, 43.0, 52.0, 57.5, 61.1, 67.4, 76.7, 78.6,
86.3, 101.0, 104.9, 105.8, 113.7, 115.1, 132.0, 134.0, 138.9, 142.8,
144,0, 145.1, 151.5, 155.8 ppm. Anal. Calcd for C22H28N4O7: C,
57.38; H, 6.13; N, 12.17. Found: C, 57.51; H, 6.03; N, 12.10.
4.1.12. General procedure for the synthesis of (15a–d)
Pd on charcoal 10% (10 mg) was added to a solution of 14a
(1.1 g, 2.0 mmol), 14b (1.15 g, 2.0 mmol), 14c (1.17 g, 2.0 mmol)
or 14d (1.18 g, 2.0 mmol) and acetic acid (0.2 ml) in ethyl ace-
tate/methanol mixture, 4:1 (20 ml), then the mixture was hydroge-
nated and stirred at rt for 2 days. The reaction mixture was filtered
on Celite. The organic layer was concentrated to dryness under re-
duced pressure. Then, 1:1 mixture of trifluoroacetic acid/water
(5 ml) was added with stirring at room temperature for 24 h. The
solvents were evaporated and the residue was evaporated to afford
15a–d.
5. Pharmacological assay
5.1. Animals
Sprague-Dawley strain rats (120–130 g) or Swiss albino mice (20–
25 g)obtainedfromtheAnimalHouseColonyoftheNationalResearch
Centre, Cairo, Egypt and group-housed under conditions of constant
photoperiod (12-h light/dark cycle) with ad libitum access to food
and water. All animal procedures were performed after approval from
the Ethics Committee of the National Research Centre and in accor-
dance with the recommendations for the proper care and use of labo-
ratory animals (NIH publication No. 85-23, revised 1985).
4.1.12.1. 1-(1,2,3,5-Tetrahydroxy-
a-
D-mannofuranose)-5-((pyri-
din-2ylmethyl)amino)-2-methyl-1H-benzimidazole
5.2. Anti-inflammatory activity
(15a).
Colorless oil, (0.56 g, 67%); FT-IR m
(cmꢂ1): 3450–3400
(OH), 3380 (NH), 1635 (C@N). 1H NMR (300 MHz, CDCl3 d 2.50 (s,
3H, CH3), 3.64 (m, 1H), 3.84 (m, 2H), 3.91 (m, 1H), 4.29 (m, 1H),
4.71 (m, 2H), 5.41 (s, 1H), 5.60 (d, J = 11.8 Hz, 1H), 5.73 (d,
J = 11.8 Hz, 1H), 7.20 (m, 2H), 7.30–7.52 (m, 2H), 7.48–7.94 (m,
The newly synthesized compounds were screened for anti-
inflammatory activity using the carageenan-induced paw oedema
assay in rats. Their activity was compared with diclofenac sodium.
Rates were randomized into groups (n = 6) that received test com-
pounds at doses of 10, 20 or 30 mg/kg or diclofenac sodium
(20 mg/kg) and control groups received saline by oral gavage.
Thirty minutes post treatment, paw oedema was induced by sub
4H), 8.20 (s, 1H), 8.15–8.24 (m, 2H). 13C NMR (75 MHz, CDCl3
d
14.2, 47.8, 51.8, 67.0, 75.4, 77.2, 84.9, 101.7, 103.1, 113.8, 115.0,
121.0, 121.8, 131.6, 133.8, 138.3, 139.4, 148.8, 151.3, 156.7 ppm.
Anal. Calcd for C20H24N4O5: C, 59.99; H, 6.04; N, 13.99. Found: C,
60.09; H, 6.17; N, 13.10.
plantar injection of 100 ll of 1% sterile carageenan in saline into

1670
A. O. H. El-Nezhawy et al. / Bioorg. Med. Chem. 21 (2013) 1661–1670
the right hind paw as mentioned before.26 Contralateral paw re-
ceived an equal volume of saline. Paw volume was determined
using a plethysmometer (Ugo Basile, Milan, Italy) before and at 1,
2, and 3 h after carageenan injection. The difference between the
initial and subsequent paw volume represents the actual oedema
volume. The percent inhibition of inflammation was calculated as
following:
expressed in
lM of inorganic phosphorus liberated/min/mg of pro-
tein ( mol Pi/h/mg prot).
l
5.4. Acute toxicity
The median lethal doses (LD50) of all compounds were investi-
gated according to the method of Smith.32 All the compounds were
studied for acute toxicity; LD50 values were found to be >1000 mg/
kg po.
Percentage of inhibition of oedema ¼ ð1 ꢂ Vt=VcÞ100
where Vt and Vc are the oedema volume in the drug-treated and
control groups, respectively.
5.5. Statistical analysis
5.3. Anti-ulcerogenic activity
Results are expressed as mean SEM or mean SD. Statistical
analysis was carried out using One-Way ANOVA followed by Dun-
nett’s test. Results were considered highly significant if p <0.01 and
less significant if p <0.05.
5.3.1. Ethanol-induced gastric ulcers
Ethanol-induced gastric ulcers model was performed as de-
scribed before.27 Briefly, mice were randomized into groups
(n = 6) that received test compounds (3.6 and 7.2 mg/kg), omepra-
zole (3.6 mg/kg) or vehicle (carboxy methyl cellulose solution
0.25%, w/v) for 3 days. Animals were fasted for 24 h before the
intragastric administration of 90% alcohol (10 ml/kg). The animals
were sacrificed by cervical dislocation under ether anesthesia 1 h
post alcohol administration. The stomach was taken out and cut
open along the greater curvature, length of each lesion was mea-
sured under anatomical microscopic.
Acknowledgment
The authors gratefully acknowledge the financial support pro-
vided by Taif University, KSA, through Project (No. 1361/432/1).
References and notes
1. Motawi, T. K.; Abd Elgawad, H. M.; Shahin, N. N. J. Biochem. Mol. Toxicol. 2007,
21, 280.
2. Nakano, Y.; Kuroda, E.; Kito, T.; Uematsu, S.; Akira, S.; Yokota, A.; Nishizawa, S.;
Yamashita, U. J. Neurosurg. 2008, 108, 311.
3. Singh, P.; Mittal, A. Mini Rev. Med. Chem. 2008, 8, 73.
4. Abraham, N. S.; Hartman, C.; Castillo, D.; Richardson, P.; Smalley, W. Am. J.
Gastroenterol. 2008, 103, 323.
5.3.2. Pylorus ligation-induced gastric ulcers
Pylorus ligation-induced gastric ulcers model was induced
according to the procedure mentioned before.28 In summary, rats
were randomized into groups (n = 6) that received test compounds
(3.6 and 7.2 mg/kg), omeprazole (3.6 mg/kg) or vehicle (carboxy
methyl cellulose solution 0.25%, w/v) for 3 days by oral gavage.
On the third day and after 24 h of animal fasting with free access
to water, animals were anaesthetized using pentobarbitone
(35 mg/kg, ip), then the abdomen was opened and pylorus ligation
was done without causing any damage to its blood supply, the
stomach was placed carefully and the abdomen wall was closed
in two layers with interrupted sutures. Negative control group ani-
mals were subjected to the same procedure without pylorus liga-
tion. Five hours after pylorus ligation, animals were sacrificed
and stomachs were dissected out and cut open along the greater
curvature. The volume and pH value of gastric juice were detected
according to reported methods.28 Briefly, gastric content was col-
lected, measured, centrifuged and the pH was measured using
pH meter (Jenway pH meter 3505).
5. Vonkeman, H. E.; Fernandes, R. W.; van der Palen, J.; van Roon, E. N.; van de
Laar, M. A. Arthritis Res. Ther. 2007, 9, R52.
6. Parsons, M. E.; Keeling, D. J. Expert Opin. Investig. Drugs 2005, 14, 411.
7. Fass, R.; Shapiro, M.; Dekel, R.; Sewell, J. Aliment. Pharmacol. Ther. 2005, 22, 79.
8. Vakil, N. Aliment. Pharmacol. Ther. 2004, 19, 1041.
9. Li, X. Q.; Andersson, T. B.; Ahlstrom, M.; Weidolf, L. Drug Metab. Dispos. 2004,
32, 821.
10. Larsson, H.; Carlsson, E.; Junggren, U.; Olbe, L.; Sjostrand, S. E.; Skanberg, I.;
Sundell, G. Am. J. Gastroenterol. 1983, 85, 900.
11. Sjostrom, J. E.; Fryklund, J.; Kuhler, T.; Larsson, H. Antimicrob. Agents Chemother.
1996, 40, 621.
12. Nagata, K.; Satoh, H.; Iwahi, T.; Shimoyama, T.; Tamura, T. Antimicrob. Agents
Chemother. 1993, 37, 769.
13. Lorentzon, P.; Jackson, R.; Wallmark, B.; Sachs, G. Biochim. Biophys. Acta 1987,
897, 41.
14. Shin, J. M.; Cho, Y. M.; Sachs, G. J. Am. Chem. Soc. 2004, 126, 7800.
15. Jain, K. S.; Shah, A. K.; Bariwal, J.; Shelke, S. M.; Kale, A. P.; Jagtap, J. R.; Bhosale,
A. V. Bioorg. Med. Chem. 2007, 15, 1181.
16. Kobayashi, T.; Ohta, Y.; Inui, K.; Yoshino, J.; Nakazawa, S. Pharmacol. Res. 2002,
46, 75.
17. El-Nezhawy, A. O.; Gaballah, S. T.; Radwan, M. A.; Baiuomy, A. R.; Abdel-Salam,
O. M. Med. Chem. 2009, 5, 558.
18. de Belder, A. N. In Advances in Carbohydrate Chemistry and Biochemistry; Tipson,
R. S., Derek, H., Eds.; Academic Press, 1977; Vol. 34, p 179.
19. de Belder, A. N. In Advances in Carbohydrate Chemistry; Melville, L. W., Ed.;
Academic Press, 1965; Vol. 20, p 219.
20. Schmidt, O. T. Methods Carbohydr. Chem. 1963, 2, 318.
21. Oña, N.; Romero, A.; Assiego, C.; Bello, C.; Vogel, P.; Pino-González, M. S.
Tetrahedron: Asymmetry 2010, 21, 2092.
22. Chung, S. K.; Moon, S. H. Carbohydr. Res. 1994, 260, 39.
23. Phillips, A. M. J. Chem. Soc. 1928, 3134.
Peptic activity was determined using hemoglobin as substrate
and was expressed as l
g of tyrosine/ml.29 Acid hemoglobin was pre-
pared by dissolving hemoglobin in 0.15 N NaCl to a final concentra-
tion of 2.5 g/100 ml and mixing with 0.16 N HCl (4:1) to pH 2.2. The
reaction mixture consisted of 2.9 ml of acid hemoglobin and 0.1 ml
of sample. After incubation for 30 min at 37 °C, peptic activity was
stopped by adding 5 ml of ice cold 0.6 M trichloracetic acid. The mix-
ture was centrifuged and the liberated amino acids in the superna-
tant were determined by the method of Lowry.30
24. Ramla, M. M.; Omar, M. A.; El-Khamry, A. M.; El-Diwani, H. I. Bioorg. Med. Chem.
2006, 14, 7324.
25. Ramla, M. M.; Omar, M. A.; Tokuda, H.; El-Diwani, H. I. Bioorg. Med. Chem. 2007,
15, 6489.
5.3.3. Estimation of H+/K+-ATPase activity
The H+/K+-ATPase was determined as described before.31 In
summary, the isolated stomach was scrapped then the scrapped
contents were homogenized in 20 mM Tris–HCl (pH 7.4) and cen-
trifuged for 20 min at 5000g. The supernatant was used to deter-
mine H+/K+-ATPase activity. One milliter of the reaction mixture
contained an aliquot of enzyme in Tris–HCl (20 mM, pH 7.4), MgCl2
(2 mM) and KCl (2 mM). Reaction was started with the addition of
ATP (2 mM, Sigma–Aldrich) then incubated at 37 °C for 30 min. To
stop the reaction, 1 ml of ammonium molybdate and trichloroace-
tic acid mixture was added. The specific activity of the enzyme was
26. Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544.
27. Germano, M. P.; D’Angelo, V.; Biasini, T.; Miano, T. C.; Braca, A.; De Leo, M.; De
Pasquale, R.; Sanogo, R. J. Ethnopharmacol. 2008, 115, 271.
28. Al-Mofleh, I. A.; Alhaider, A. A.; Mossa, J. S.; Al-Sohaibani, M. O.; Rafatullah, S.;
Qureshi, S. Environ. Toxicol. Pharmacol. 2006, 22, 64.
29. Debnath, P. K.; Gode, K. D.; Das, D. G.; Sanyal, A. K. Br. J. Pharmacol. 1974, 51,
213.
30. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193,
265.
31. Reyes-Chilpa, R.; Baggio, C. H.; Alavez-Solano, D.; Estrada-Muniz, E.; Kauffman,
F. C.; Sanchez, R. I.; Mesia-Vela, S. J. Ethnopharmacol. 2006, 105, 167.
32. Smith, W. G. Prog. Med. Chem. 1961, 1, 1.