Synthesis, p38 kinase inhibitory and anti-inflammatory activity of new substituted benzimidazole derivatives

Article abstract of DOI:10.2174/157340613804488440

P38 mitogen activated protein kinases have been found to involve in the production and release of unwarranted levels of pro-inflammatory cytokines including TNFα and IL-1β in numerous inflammatory diseases. A new series of molecules, 5-substituted benzoylamino-2-substituted phenylbezimidazoles has been synthesized from 4-nitro-1, 2-diaminobenzene. The synthesized compounds were characterized by FTIR, ¹HNMR and Mass. The final compounds were screened for in vitro p38 kinase inhibitory and in vivo anti-inflammatory activity. Three compounds from the series demonstrated nearly 50percent inhibition of p38 kinase in the in vitro screening method at 10 μM concentration and two molecules exhibited greater than 75percent inhibition of paw oedema volume during the first hour. The docking study of synthesized molecule revealed a new binding pose in ATP binding pocket.

Full text of DOI:10.2174/157340613804488440

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Medicinal Chemistry, 2013, 9, 91-99
91
Synthesis, p38 Kinase Inhibitory and Anti-inflammatory Activity of New
Substituted Benzimidazole Derivatives
Ravindra G. Kulkarnia,d*, Stefan A. Lauferb, Chandrashekhar V. Mc and Achaiah Garlapatid*
aSVERI’s College of Pharmacy, Gopalpur Road, Pandharpur- 413304, Maharashtra, India
bInstitute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-University, Tubingen, Auf
der Morgenstelle, 8, 72076, Germany
cBVVS’S Hanagal Shri Kumareshwar College of Pharmacy, BVVS Campus, Bagalkot, Karnataka, 587 101, India
dUniversity College of Pharmaceutical Sciences, Kakatiya University, Warangal, Andhra Pradesh, India
Abstract: P38 mitogen activated protein kinases have been found to involve in the production and release of unwarranted
levels of pro-inflammatory cytokines including TNFꢀ and IL-1ꢁ in numerous inflammatory diseases. A new series of
molecules, 5-substituted benzoylamino-2-substituted phenylbezimidazoles has been synthesized from 4-nitro-1, 2-
diaminobenzene. The synthesized compounds were characterized by FTIR, 1HNMR and Mass. The final compounds were
screened for in vitro p38 kinase inhibitory and in vivo anti-inflammatory activity. Three compounds from the series dem-
onstrated nearly 50% inhibition of p38 kinase in the in vitro screening method at 10 ꢀM concentration and two molecules
exhibited greater than 75% inhibition of paw oedema volume during the first hour. The docking study of synthesized
molecule revealed a new binding pose in ATP binding pocket.
Keywords: p38 kinase inhibitors, benzimidazoles, anti-inflammatory activity.
1. INTRODUCTION
highly desired in inflammatory diseases. Low molecular
weight p38 kinase inhibitors show same therapeutic benefits
like biological anti-cytokines but offer advantages in terms
of oral dosage and affordable cost [11-13].
The elevated levels of pro-inflammatory cytokines like
tumor necrosis factor-ꢀ (TNFꢀ) and interleukin-1ꢁ (IL-1ꢁ)
play a vital role in numerous inflammatory diseases
including rheumatoid arthritis. Several peptide based anti-
cytokine therapies have been implemented in the clinical
treatment of rheumatoid arthritis to suppress such pro-
inflammatory cytokines [1-4]. Despite being potent
inhibitors of pro-inflammatory cytokines, the protein based
anti-cytokines were reported to exhibit severe adverse effects
including microbial infections and are quite exorbitant too
[5]. However several alternative strategies have been
contemplated and quite a few new validated targets were
identified for the search of effective anti-inflammatory
agents [6]. Studies involving mitogen activated protein
kinases (MAPK, proline directed serine-threonine kinases)
revealed that MAPKs are involved in the cellular responses
to extracellular signals including microbial endotoxins and
cytokines [7]. Four isoforms of p38 kinase are known till
date viz., p38ꢀ, p38ꢁ, p38ꢂ and p38ꢃ which are activated by
phosphorylation in the activation loop [8-10]. Activated p38
kinase plays a key role in signal transduction pathway
leading to production and release of elevated levels of pro-
inflammatory cytokines TNFꢀ and IL-1ꢁ. Due to this vital
role being played by p38 kinase, inhibition of p38 kinase is
The seminal discovery of first generation p38 kinase
inhibitors exemplified by SB203580 [14] has opened a new
prospect for the development of next generation agents for
inflammatory diseases and pharmaceutical industries
pursuing aggressively towards the development of new anti-
inflammatory agents by targeting p38 kinase. Further
discovery of allosteric binding, noncompetitive N, Nꢀ-
diarylurea derivatives typified by clinical compound
BIRB796 (Doramapimod) has led to unearthing of several
new lead molecules and some of them are in various stages
of clinical trials [15-17].
Compounds possessing benzimidazole scaffold reported
to possess wide range of pharmacological activities
including anti-inflammatory and interleukin-1 inhibitory
activity [18-22] and also displayed a variety of kinase inhibi-
tory activities [23-27]. Substituted benzimidazoles have
demonstrated potent Raf kinase inhibitory activity which
were designed from theꢄꢅꢆ ꢇꢈꢇꢉꢊꢋꢇꢆꢌꢄof most advanced Raf
kinase inhibitor BAY43-9006 [28] and few p38 kinase
inhibitors were derived from benzimidazole scaffold [29-33].
In continuation to our previous studies on the development
of allosteric binding p38 kinase inhibitors, we designed new
benzimidazoles (7a-j) from our in house urea derivatives
[34] (Fig. 1). In this communication, we report the synthesis
of a series of 2-substituted phenylbenzimidazole derivatives
and their evaluation for p38 kinase inhibitory and anti-
inflammatory activities.
*Address correspondence to this author at the SVERI’s College of
Pharmacy, Gopalpur Road, Pandharpur- 413304, Maharashtra, India; Tel:
+91-94406 01057, +91-8446765426; Fax: +91 2186 225082;
E-mails: ravigk2000@gmail.com; achaiah_g@yahoo.com
1875-6638/13 $58.00+.00
© 2013 Bentham Science Publishers

92 Medicinal Chemistry, 2013, Vol. 9, No. 1
Kulkarni et al.
F
N
O
O
O
N
N
H
N
N
H
N
N
H
O
N
S
SB203580
BIRB796
O
CF3
Cl
O
NHCH3
O
N
N
H
N
H
BAY43-9006
ꢄ
Structures of important p38 kinase inhibitors
R
N
H
N
N
H
R
X
O
Y
X
Y
N
H
N
H
R
X=S, O, NHCO
Y= N, CH
R= H, F, Cl, NO2, OCH3
R1
X
X
N
N
R
N
H
Y
N
H
N
H
7a-j
ꢄ
Fig. (1). Design of benzimidazole p38 kinase inhibitors.
MATERIAL AND METHOD
dried to obtain compounds 2a-c in ~80% yield and were
used without purification in the succeeding step [35].
All the reactions were carried out on six stage reaction
station of Radleys discovery Technologies, Germany.
Melting points were recorded in open glass capillaries on
Polmon melting point apparatus and are uncorrected.
Infrared spectra were recorded on Shimadzu FTIR
spectrophotometer using KBr and mass spectra obtained on
VG-7070H mass spectromoter. 1HNMR spectra were
Synthesis of 2-substituted Phenyl-5-nitrobenzimidazoles
(4a-c)
A mixture of 2a-c (0.00317 mol) and 4-nitro-1, 2-
benzenediamine (3, 0.00314 mol) were heated at 130 °C for
4 h in DMF (20 mL). The reaction mixture was then cooled
to room temperature and poured into water. The precipitate
thus obtained was filtered, washed with plenty of cold water
to remove inorganic salts and dried. The crude compounds
were recrystallized from 70% alcohol to get pure 4a-c.
recorded at 300 MHz on
a Bruker Avance NMR
spectrometer in CDCl3 (ꢀ 7.26) or DMSO- d6 (ꢀ 2.49). Thin
layer chromatography was performed on pre-coated silica gel
F254 (Merck) and column chromatography was performed
using silica gel 60-120 mesh.
Synthesis of 5-amino-2-substituted Phenylbenzimidazoles
(5a-c)
Synthetic Methodology
The Reduction of Nitro Group was Carried by two
Different Methods
Synthesis of Substituted Sodium Metabisulfite Adducts of
Benzaldehydes (2a-c)
Method A: Stannous chloride dihydrate (0.0126 mol)
was added into appropriate solution of 4a-c (0.0028 mol) in
absolute ethanol and heated at 70 ˚C for 4 h. Cooled reaction
mixture was poured over ice, then made alkaline (~pH 8)
with 10% aqueous sodium hydroxide and extracted with
ethyl acetate [36]. The combined organic phase was washed
To the solution of benzaldehydes (1a-c, 0.015 mol) in 50
ml absolute ethanol, sodium metabisulfite (1.6 g) in 10 mL
of water was added drop wise. The reaction mixture was
stirred vigorously, more absolute ethanol (20 mL) was added
and the reaction mixture was kept in a refrigerator for several
hours. The precipitate obtained was separated by filtration,

Synthesis, p38 Kinase Inhibitory
Medicinal Chemistry, 2013, Vol. 9, No. 1 93
with brine and dried over anhydrous sodium sulphate. The
crude product thus obtained was purified by passing through
silica gel using dichloromethane: acetone as eluent to get
respective pure compounds 5a-c.
1:1400 in BB). Then 100 ꢀL of 4-NPP was pipetted in each
well after a final washing step and color development was
measured 1.5-2
h
later with an enzyme linked
immunosorbent assay reader equipped with the SOFTmax
PRO software at 405 nm. After adapting all solution
volumes, this assay was performed automatically with the
robotic liquid handling system [38].
Method B: A suspension of an appropriate 4a-c (0.003
mol) and activated zinc (0.004 mol) in methanol (10 ml) was
stirred with ammonium formate (0.5 g) at room temperature
[37]. The reaction was monitored by TLC and upon
completion of the reaction, the reaction mixture was filtered
to remove unreacted zinc. The volatilities were removed by
evaporation and the residue was dissolved in dichloro-
methane and washed with saturated brine. Evaporation of
dichloromethane resulted in crude amines which were
purified over silica using dichloromethane: acetone (8:2) as
eluent to afford 5a-c.
Carrageenan Induced Paw Oedema
The anti-inflammatory activity of the test compounds
was evaluated as described by Winter et al. One hour after
the oral administration of test compounds, rats in all groups
were challenged with carrageenan (1% prepared in 0.9%
NaCl) in the sub-plantar region of right hind paw. The paw
volume was measured at different intervals of time (1, 2, 3,
and 4 h) using digital plethysmometer (UGO Basil, Italy).
The percentage inhibition of paw volume for each test group
is calculated using following equation [39-42].
Preparation
of
Benzoylated
5-amino-2-substituted
Phenylbenzimidazoles (7a-j)
Appropriate amines (5a-c, 0.001 mol) were stirred in
tetrahydrofuran (15 mL) for 10 minutes to yield uniform
solution and to which triethyl amine (0.002 mol) was added.
Solution of substituted benzoyl chlorides (6a-d, 0.0011 mol)
in 5 mL tetrahydrofuran was added drop wise and the
reaction mixture was stirred for 5 h at room temperature.
Upon completion of the reaction as indicated by TLC, the
volatilities were removed. Then dichloromethane (25 mL)
was added, washed with 2-5% sodium bicarbonate and later
with brine. The organic component was removed by
evaporation and the crude products were subjected to
purification by passing through silica gel using
dichlromethane: acetone (8: 2) as eluent to afford pure
compound (7a-j).
Percentage of inhibition (%)
= [1-Volume (test
compound) / Volume (control)] x100
MOLECULAR DOCKING
The most effective way to understand the interaction of
small molecule with active site is described by docking
studies. Previously we integrated both molecular docking
and 3D QSAR studies and disclosed the probable strategy to
design new p38 kinase inhibitors [43-45]. GOLD 3.5 a
genetic algorithm docking program was employed to reveal
the binding poses of benzimidazoles in the active site. From
the crystal structure 1bl7, the ligand and water molecules
were deleted and hydrogen added. To refine the protein
structure from undesired contact, local minimization was
carried by applying Kollmann partial charges and default
parameters were used: 100 population size, 1.1 for selection,
5 number of islands, 100000 number of genetic operations
and 2 for the niche size. 10 ꢁ radius from Met109 was
assigned to create active site and GOLD score was selected
to rank order the docked conformations.
In Vitro p38 Kinase Assay
Nonradioactive immunosorbent p38 kinase activity assay
method has been followed which is applicable for routine
screening of small molecule p38 kinase inhibitors. In the
method ATF-2 was used as a substrate for phosphorylation,
which showed linearity in 6-24 ng/well range, based on this
12 ng/well and 60 minute incubation period was optimized.
Microtiter plates were coated with 50 ꢀL/well of the p38
kinase substrate ATF-2 (10 ꢀg/mL in TBS) for 1.5 h at 37
˚C. After washing three times with bidistilled water,
remaining open binding sites were blocked with blocking
buffer (BB; 0.05% Tween 20, 0.25% BSA, 0.02% NaN3 in
TBS) for 30 min at room temperature. Plates were washed
again, 50 ꢀL of the respective test solution was filled into the
wells and the plates were incubated for 1 h at 37 ˚C. Test
solutions containing 12 ng/well p38 MAPK were diluted in
kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 10 mM
ꢀ-glycerophosphate, 100 ꢀg/mL BSA, 1mM dithiothretol,
0.1 mM Na3VO4, 100 ꢀM rATP) with or without test
substance (10-4 to 10-8 M). Test substances were dissolved in
dimethyl sulfoxide to form stock solution of 10-2 M, all
further dilutions were carried out in kinase buffer. After
subsequent washing, plates were blocked again with BB for
15 min followed by a fourth washing step. Wells were filled
with 50 ꢀL of the specific anti-bis-(Thr69/71)-phospho-ATF-2
(1. AB, 1:500 in BB) and incubated for 1 h at 37 ˚C followed
by washing and consecutive incubation with 50 ꢀL of the
secondary antibody (2. AB (alkaline phospatase conjugated),
RESULTS AND DISCUSSIONS
Chemistry
The title compounds (7a-j) were synthesized as outlined
in Scheme 1. The key intermediates, 5-nitro-2-
phenylbenzimidazoles (4a-c) were prepared by reaction
between 4-nitro-1, 2-benzenediamine (3) and benzaldehydes
(1a-c). Numerous methods are available for the synthesis of
2-phenylbenzimidazole derivatives, however we followed
the method in which sodium metabisulfite adducts of
substituted benzaldehydes (2a-c) were refluxed with 3 to
afford 4a-c in better yields. The nitro derivatives (4a-c) were
subsequently reduced either by stannous chloride dihydrate
or zinc ammonium formate to result 5a-c in 65-80% yields.
Further compounds 5a-c were benzoylated using appropriate
benzoyl chlorides (6a-d) in anhydrous tetrahydrofuran to
afford the final desired compounds 7a-j in 60-75% yield.
The 1HNMR spectra of compounds 4a-c were recorded in
CDCl3 and the peak assignment has been mentioned in
characterization section. All the three molecules exhibited a
broad singlet peak around 10 delta value which is attributed

94 Medicinal Chemistry, 2013, Vol. 9, No. 1
Kulkarni et al.
OH
SO3Na NO2
NH2
NH2
CHO
O2N
N
Na2S2O3
R
N
H
4a-c
3, DMF
R
R
1a-c
2a-c
4a: R=H
4b: R=4F
4c:R=4OMe
1a: R=H
1b: R=4F
1c:R=4OMe
2a: R=H
2b: R=4F
2c:R=4OMe
Zn/Ammonium
formate/MeOH
or
SnCl2. 2H2O/
EtOH
H
TEA, DCM
COCl
N
N
H2N
N
R1
R
R
O
N
H
N
H
R1
5a-c
7a-j
7a: R=H, R1=H
7b: R=H, R1=4NO2 7f:R=4F, R1=4NO2
5a: R=H
5b: R=4F
5c:R=4OMe
7e:R=4F, R1=H
6a-d
6a: R1=H
7c: R=H, R1=4Cl 7g:R=4F, R1=4Cl
6b: R1=4NO2
6c: R1=4Cl
6d: R1=4OMe
7d: R=H, R1=4OMe 7h: R=4F, R1=4OMe
7i: R=4OMe R1=H 7j: R=4OMe R1=4NO2
ꢄ
Scheme 1. Synthesis of 7a-j.
to resonance of NH proton of benzimidazole. C4, C6 and C7
protons of benzimidazole appeared between 8.1 ppm and 8.5
ppm, C4 was singlet while C6 and C7 were in doublets.
Protons of aromatic ring C'3, C'4 and C'5 appeared as triplet in
4a, whereas C'2, C'6 and C'3, C'5 resonated as doublet in 4b
and 4c. All the three molecules showed the corresponding
M+1 peaks (100%) conforming respective molecular
formula. The FTIR spectrum of amines showed two
absorption bands at 3400-3200 cm-1 which are due to NH2
stretching and a band at 1580 cm-1 indicated NH bending.
1HNMR spectra of compound 5a, a broad singlet at 3.5 ppm
is responsible for protons of primary amine excitation
indicating the reduction of nitro group to amine. Protons of
C4, C6 and C7 of benzimidazole were located slightly upfield,
two doublets were found at 7.9 and 8.0 representing C6 and
C7 protons of benzimidazole respectively. A triplet peak was
noticed at delta 7.1 integrated for three protons due to C'3, C'4
and C'5 protons. A peak at m/z 210 (M+1, 100%) in the mass
spectrum of compound 5a which is agreeing the molecular
formula C13H11N3.
Pharmacology
All the newly synthesized compounds (7a-j) were
screened for in vitro p38 kinase inhibitory and in vivo anti-
inflammatory activity. The p38 kinase inhibitory activity was
determined by robust, nonradioactive and immunosorbent
assay method. The antiinflammatory activity was performed
using carrageenan in which inflammation response is quanti-
fied by increase in paw size (oedema).
In Vitro p38 Kinase Inhibitory Activity
The p38 kinase inhibitory activity of compounds 7a-j is
summarized in Table 1. Unsubstituted compound 7a
exhibited 30.43% enzyme inhibitory activity at 10 ꢁM.
Substitution on either R or R1 led to increase in p38 kinase
inhibitory activity except 7h. Substitution at R1 (NO2, Cl
and OCH3) as in 7b, 7c and 7d enhanced the potency
drastically and compound 7c (R=H, R1=Cl) found to be
potent among the study compounds with 51.86% activity.
Insertion of groups over 4th position of 2-phenyl ring resulted
in reduction in the in vitro activity however compounds 7e
and 7i found to be superior when compared with 7a. Both
nitro compounds 7f and 7j (R=F and OCH3 respectively)
were less potent than 7b (R=H) and similarly chloro
derivative 7g (R=F, R1= Cl) exhibited lower potency when
compared with 7c. The above discussion gives the clue that
substitution at R is not primarily essential which is
exceptional to compound 7j (42.2% kinase inhibitory
activity). The initial activity profile suggests that irrespective
of nature of groups, bulky substitution at R1 is essential for
better activity. New compounds with wide range of bulky
substitution at R1 may strengthen our results.
FTIR spectrum of 7a did not show absorption band for
primary amine however it showed an absorption band at
3200 cm-1 that is responsible for NH stretching and carbonyl
group stretching could be assigned for a peak present at 1720
1
cm-1. HNMR of 7a recorded in DMSO d6 depicted a broad
singlet peak at ꢀ 12.8 indicating amide NH excitation and
another broad singlet peak at ꢀ 10.3 was found which could
be attributed to benzimidazole NH proton. In the ESI mass
spectrum, compound 7a showed peak at m/z 314.2 (M +1,
100%), which matches with its molecular formula
C20H15N3O. FT IR, 1H NMR and Mass spectral results are in
concurrence with the projected structures.

Synthesis, p38 Kinase Inhibitory
Medicinal Chemistry, 2013, Vol. 9, No. 1 95
Table 1. In vitro p38 Kinase Inhibitory Activity of 5-Substituted Benzoylamino, 2-Substituted Phenylbenzimidazole Derivatives (7a-j)
R1
H
N
N
R
O
N
H
GOLD
Codeꢀ
Rꢀ
R1ꢀ
% Inhibition at 10 ꢁMꢀ
Scoreꢀ
7aꢄ
7bꢄ
7cꢄ
7dꢄ
7eꢄ
7fꢄ
7gꢄ
7hꢄ
7iꢄ
Hꢄ
Hꢄ
Hꢄ
NO2ꢄ
Clꢄ
30.43ꢄ
51.55ꢄ
51.86ꢄ
49.84ꢄ
39.75ꢄ
35.49ꢄ
38.89ꢄ
22.68ꢄ
33.33ꢄ
42.29ꢄ
48.50ꢄ
45.20ꢄ
50.91ꢄ
47. 05ꢄ
48.26ꢄ
44.40ꢄ
45.66ꢄ
44.32ꢄ
45.48ꢄ
41.44ꢄ
Hꢄ
Hꢄ
OCH3ꢄ
Hꢄ
Fꢄ
Fꢄ
NO2ꢄ
Clꢄ
Fꢄ
Fꢄ
OCH3ꢄ
Hꢄ
OCH3ꢄ
OCH3ꢄ
7jꢄ
NO2ꢄ
0.041
SB 203580ꢄ
BIRB 796ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
ꢄ
(IC50)ꢄ
0.026
(IC50)ꢄ
In vivo Anti-inflammatory Activity
usual hydrogen bond interaction with NH of Met109
however benzimidazole derivatives did not show such
hydrogen bond interaction, some of the compounds
demonstrated two different hydrogen bond interactions. N3
of benzimidazole revealed a new hydrogen bond interaction
with Asn159 (2.64 ꢁ), N1 of benzimidazole showed
hydrogen bond interaction with Glu163 (2.402 ꢂ), both of
which seemed to be rare in aryl imidazole derivatives as
shown in Fig. (2a). The CO and NH group of Met109 were
away by ~5.5 ꢁ from amide group of benzimidazoles and the
benzimidazole scaffold of all molecules was positioned away
from the imidazole group of phenoxypyrimidinyl imidazole
derivatives by ~8 ꢁ. The 2-substituted phenyl ring of
benzimidazoles was in hydrophobic area surrounded by the
backbones of Ala157, Leu164 and Lys165. The
benzimidazole nucleus was surrounded by Met109, Asn159
and Glu163 (Fig. 2b). The methoxy groups of 7d and 7h
(R1=4-OCH3) were found exposed into unfavorable
hydrophilic environment (Solvent), comparatively 7h
observed to be more exposed to hydrophilic interactions due
to displaced orientation than 7d.
In the in vivo evaluation, compounds 7a-j did not display
any toxicity till 300 mg/kg dose in acute toxicity studies in
mice hence a dose of 50 mg/kg was selected for all the
compounds to observe antiinflammatory activity. All the
compounds (7a-j) exhibited lower anti-inflammatory activity
in rats when compared to our library of diarylureas. All the
molecules have demonstrated maximum protection during
first hour of carrageenan challenge and the activity was
found decreasing in the later stage of observation as depicted
in Table 2. Substitution at R1 position of benzimidazole ring
augmented the anti-inflammatory activity as like in vitro
activity, whereas substitution on 2-phenyl ring showed
reduced activity, however compound 7j (R=OCH3 and
R1=NO2) exhibited greater protection at the first hour of
carrageenan injection (81.26%). Compound 7c (R=H;
R1=Cl, 77.7%) exhibited superior activity over 7b (R=H;
R1=NO2, 63.3%).
Molecular Docking Studies
During the design, it was speculated that benzimidazole
derivatives might bind to ATP binding site of p38 kinase. To
ascertain this, all the inhibitors were docked in the ATP site
and analyzed for the binding interactions which were found
to be different from triarylimidazole derivatives exemplified
by SB203580 [44]. The imidazole derivatives exhibited
Characterization Data of Synthesized Compounds
5-Nitro-2-Phenylbenzimidazole (4a)
Yield: 84.0 %, Melting Point: 138-142˚C. 1H NMR
(CDCl3) ꢂ (ppm): 10.1 (br. s, 1H, NH), 8.5 (s, 1H, C4,

96 Medicinal Chemistry, 2013, Vol. 9, No. 1
Kulkarni et al.
Table 2. Anti-inflammatory Activity of 7a-j in Carrageenan Induced Paw Oedema in Rats a, b
.
Oedema Volume in ml ( Mean % Inhibition)ꢀ
Groupsꢀ
1 hrꢀ
2 hrꢀ
3 hrꢀ
4 hrꢀ
Controlꢄ
0.427 0.211ꢄ
0.495 0.232ꢄ
0.568 0.277ꢄ
0.608 0.3ꢄ
0.290 0.036***
0.207 0.047***
0.212 0.069***
0.216 0.140***
Indomethacinꢄ
(35.59 4.6)ꢄ
(58.18 9.2)ꢄ
(62.67 9.8)ꢄ
(64.47 10.0)ꢄ
0.199 0.126***
0.288 0.152**
0.434 0.48*
0.505 0.455*
7aꢄ
7bꢄ
7cꢄ
7dꢄ
7eꢄ
7fꢄ
7gꢄ
7hꢄ
7iꢄ
(53.4 7.2)ꢄ
(32.6 3.3)ꢄ
(23.6 3.1)ꢄ
(16.9 2.4)ꢄ
0.160 0.212***
0.235 0.2***
0.375 0.32**
0.432 0.310**
(62.5 5.8)ꢄ
(52.5 5.3)ꢄ
(33.9 2.4)ꢄ
(29.0 2.1)ꢄ
0.112 0.092***
0.318 0.112**
0.492 0.368*
0.562 0.33
(73.8 6.9)ꢄ
(35.35 5.1)ꢄ
(13.9 2.9)ꢄ
(07.6 1.3)ꢄ
0.236 0.21***
0.318 0.112***
0.445 0.32**
0.507 0.29**
(44.7 5.7)ꢄ
(35.75 3.2)ꢄ
(21.6 3.0)ꢄ
(16.63 2.7)ꢄ
0.220 0.162***
0.345 0.117**
0.398 0.14*
0.456 0.131**
(48.5 3.5)ꢄ
(30.3 3.1)ꢄ
(29.9 3.8)ꢄ
(25.0 2.8)ꢄ
0.152 0.166***
0.33 0.2**
0.498 0.428*
0.543 0.36*
(64.4 3.7)ꢄ
(33.34 2.6)ꢄ
(12.5 1.8)ꢄ
(10.7 0.9)ꢄ
0.224 0.221***
0.332 0.121**
0.415 0.511*
0.462 0.5**
(47.54 7.0)ꢄ
(32.9 6.6)ꢄ
(26.93 5.5)ꢄ
(24.0 5.1)ꢄ
0.212 0.112***
0.365 0.11**
0.472 ).413**
0.522 0.4**
(50.35 6.7)ꢄ
(26.7 3.5)ꢄ
(16.9 3.1)ꢄ
(14.1 2.7)ꢄ
0.265 0.212**
0.37 0.08*
0.495 0.36*
0.558 0.338
(37.93 7.0)ꢄ
(25.3 5.8)ꢄ
(12.9 4.1)ꢄ
(8.22 3.6)ꢄ
0.089 0.092***
0.156 0.142***
0.307 0.363***
0.437 0.27***
7jꢄ
(79.15 10.0)ꢄ
(63.5 7.6)ꢄ
(45.95 5.8)ꢄ
(28.1 3.0)ꢄ
a Results expressed in mean SEM (n=6), ANOVA followed by Dunnett’s multiple comparison test. *<0.05, **p<0.01, *** p<0.001 as compared to control group.
b Compounds 7a-j tested at 50 mg/kg dose and indomethacin at 10 mg/kg dose
benzimidazole), 8.25 (d, 1H, C6 benzimidazole), 8.22 (d, 1H,
C7 benzimidazole), 7.5-7.6 (t, 3H, aromatic C'3, C'4 and C'5)
and 7.3 (d, 2H, aromatic C'2 and C'6). ESI-MS (m/z): 240.1
[M+H] +.
5-Amino-2-Phenylbenzimidazole (5a)
Yield: 65.5 %, IR (KBr, ꢀmax cm-1): 3326, 3250 (–NH),
3020 (CH aromatic), 1517 and 1460 (aromatic C=C), and
1
1598 (C-N bnd). H NMR (CDCl3) ꢁ (ppm): 9.8 (br. s, 1H,
NH), 8.0 (d, 1H, C7, benzimidazole), 7.9 (d, 1H C6
benzimidazole), 7.7 (s, 1H, C4, benzimidazole), 7.31-7.4 (t,
3H, aromatic C'3, C'4 and C'5) and 7.1 (d, 2H, aromatic C'2
and C'6) and 3.5-3.8 (b, 2H, NH2). ESI-MS (m/z): 210.2
[M+H] +.
2-(4-Fluorophenyl)-5-nitrobenzimidazole (4b)
Yield: 79.8 %, Melting Point: >240 ˚C. 1H NMR
(CDCl3) ꢀ (ppm): 10.3 (br. s, 1H, NH), 8.3 (s, 1H, C4,
benzimidazole), 8.20 (d, 1H, C6 benzimidazole), 8.16 (d, 1H,
C7 benzimidazole), 7.5 (d, 2H, aromatic C'3, and C'5) and 7.4
(d, 2H, aromatic C'2 and C'6). ESI-MS (m/z): 258.0 [M+H] +.
5-N-benzoylamino-2-Phenyl-benzimidazole (7a)
Yield: 75.5%, Melting Point: 214-16 ˚C. IR (KBr):
~3200 cm-1 (ꢀ NH str), ~2990 cm-1 (ꢀ –CH- aromatic, str),
2-(4-methoxyphenyl)-5-nitrobenzimidazole (4c)
1
~1720 cm-1 (ꢀ CO str), 1600 cm-1 (ꢀ NH bend). H NMR
1
Yield: 79.8 %, Melting Point: 228-230 °C. H NMR
(DMSO d6) ꢁ (ppm): 12.8 (br. s, 1H, amide NH), 10.3 (br. s,
(CDCl3) ꢀ (ppm): 10.2 (br. s, 1H, NH), 8.4 (s, 1H, C4,
benzimidazole), 8.20 (d, 1H, C6 benzimidazole), 8.11 (d, 1H,
C7 benzimidazole), 7.3 (d, 2H, aromatic C'2 and C'6) and 7.2
(d, 2H, aromatic C'3, and C'5), 3.7 (s, 3H, OCH3). ESI-MS
(m/z): 270.1 [M+H] +.
ꢀ
ꢀ
1H, NH benzimidazole), 8.25 (d, 2H, C , C aromatic), 8.1-
2
8.2 (t, 3H C , C 4, C 5 aromatic), 7.94-8.06 (d, 2H C6, C7
ꢀ
ꢀ
ꢀ
3
benzimidazole), 7.45-7.64 (m, 6H aromatic and C4
benzimidiazole). ESI-MS (m/z): 314.2 [M+H] +.

Synthesis, p38 Kinase Inhibitory
Medicinal Chemistry, 2013, Vol. 9, No. 1 97
ꢁ
ꢁ
2H, C 2, C , aromatic), 7.7 (d, 2H, C6, C7 benzimidazole),
6
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
7.45 (d, 2H, C 2, C 6 aromatic), 7.3 (t, 3H, C 3, C 4 and C 5
aromatic), 3.8 (s, 3H, OCH3). ESI-MS (m/z): 344 [M+H] +.
2-(4ꢁ-Fluorophenyl)-5-N-benzoylaminobenzimidazole (7e)
Melting Point: 224-26 ˚C. IR (KBr): ~3200 cm-1 (ꢀ NH
str), ~3000 cm-1 (ꢀ –CH- aromatic, str), ~1680 cm-1 (ꢀ CO
1
str), 1600 cm-1 (ꢀ NH bend). H NMR (DMSO d6) ꢁ (ppm):
12.1 (br. s, 1H, amide NH), 9.8 (br. s, 1H,.1 NH
benzimidazole), 8.2 (d, 2H, Cꢁ2 and Cꢁ6 aromatic), 8.05- (t,
ꢁ
ꢁ
ꢁ
3H C 3,
C
C 5 aromatic), 7.9 (d, 2H, C6, C7,
4,
benzimidazole), 7.8 (s, 1H, C4 benzimidazole), 7.6 (d, 2H,
Cꢀ3, Cꢀ5 aromatic), 7.4 (d, 2H, Cꢀ2, Cꢀ6 aromatic), 7.1 (t, 3H,
Cꢁ3, Cꢁ4 and Cꢁ5 aromatic). ESI-MS (m/z): 333 [M+H] +.
2-(4ꢁ-Fluorophenyl)-5-N-(4ꢀ-
Fig. (2a). The binding pose of 7a (capped stick) and triaryl
imidazole (ball stick).
nitrobenzoyl)aminobenzimidazole (7f)
Yield: 67.7 % Melting Point: 295-97 ˚C, IR (KBr):
~3250 cm-1 (ꢀ NH str), ~3000 cm-1 (ꢀ –CH aromatic, str),
~1750 cm-1 (ꢀ CO str), 1600 cm-1 (ꢀ NH bend). H NMR
1
(DMSO d6) ꢁ (ppm): 12.8 (br. s, 1H, amide NH), 10.3 (br. s,
ꢁ
ꢁ
1H, NH benzimidazole), 8.35 (d, 2H, C 3, C 5 aromatic), 8.15
ꢁ
ꢁ
(d, 2H, C , C 6 aromatic), 8.0 (s, 1H, C4 benzimidazole), 7.8
2
ꢀ
ꢀ
(d, 2H, C6, C7 benzimidazole), 7.45 (d, 2H, C 3, C 5,
ꢀ
ꢀ
aromatic), 7.25 (d, 2H, C 2, C 6,aromatic). ESI-MS (m/z):
377 [M+H] + .
2-(4ꢀ-Fluorophenyl)-5-N-(4ꢁ-
chlorobenzoyl)aminobenzimidazole (7g)
Yield: 66.9%, Melting Point: 298-300 ˚C, IR (KBr):
~3200 cm-1 (ꢀ NH str), ~2990 cm-1 (ꢀ –CH- aromatic, str),
~1750 cm-1 (ꢀ CO str), 1600 cm-1 (ꢀ NH bend). H NMR
1
Fig. (2b). The binding pose of 7a (ash colour) and 7f (pink colour).
2-Phenyl-5-N-(4ꢀ-Nitrobenzoyl)aminobenzimidazole (7b)
(DMSO d6) ꢁ (ppm): 12.28 (br. s, 1H, amide NH), 9.8 (br. s,
ꢁ
ꢁ
1H, NH benzimidazole), 8.15 (d, 2H, C 2, C 6 aromatic), 8.04
ꢁ
ꢁ
(d, 2H, C 3, C aromatic), 7.87 (s, 1H, C4 benzimidazole),
5
ꢀ
ꢀ
Yield: 68.8 %, Melting Point: >300 ˚C, IR (KBr): ~3250
cm-1 (ꢀ NH str), ~2990 cm-1 (ꢀ –CH- aromatic, str), ~1700
7.75 (d, 2H, C6, C7 benzimidazole), 7.5 (d, 2H, C 2, C 6
ꢀ
ꢀ
aromatic), 7.3 (d, 2H, C 3, C 5 aromatic). ESI-MS (m/z):
1
cm-1 (ꢀ CO str), 1600 cm-1 (ꢀ NH bend). H NMR (DMSO
366.5 [M+H] + .
d6) ꢁ (ppm): 12.7 (br. s, 1H, amide NH), 10.1 (br. s, 1H, NH
ꢁ
ꢁ
2-(4ꢁ-Fluorophenyl)-5-N-(4ꢀ-
methoxybenzoyl)aminobenzimidazole (7h)
benzimidazole), 8.3 (d, 2H C 3, C 5 aromatic), 8.20 (d, 2H,
ꢁ
ꢁ
C 2, C aromatic), 8.0 (d, 2H, C6, C7 benzimidazole), 7.8 (s,
6
1H, C4 benzimidazole), 7.2-7.5 (m, 5H, aromatic). ESI-MS
Yield: 59.0%, Melting Point: 232-34 ˚C. IR (KBr):
~3200 cm-1 (ꢀ NH str), ~2990 cm-1 (ꢀ –CH- aromatic, str),
(m/z): 359.4 [M+H] +.
1
~1680 cm-1 (ꢀ CO str), 1600 cm-1 (ꢀ NH bend). H NMR
2-Phenyl-5-N-(4ꢀ-Chlorobenzoyl)aminobenzimidazole (7c)
(DMSO d6) ꢁ (ppm): 12.2 (br. s, 1H, amide NH), 10.1 (br. s,
ꢁ
ꢁ
Yield: 62.3 %, Melting Point: >300 ˚C, IR (KBr): ~3180
cm-1 (ꢀ NH str), ~3000 cm-1 (ꢀ –CH- aromatic, str), ~1750
1H, NH benzimidazole), 8.04 (d, 2H, C 2, C 6 aromatic), 7.9
ꢁ
ꢁ
(s, 1H, C4 benzimidazole), 7.89 (d, 2H, C 3, C , aromatic),
5
1
cm-1 (ꢀ CO str). H NMR (DMSO d6) ꢁ (ppm): 12.4 (br. s,
ꢀ
ꢀ
7.77 (d, 2H, C6, C7 benzimidazole), 7.55 (d, 2H, C 2, C 6
ꢀ
ꢀ
1H, amide NH), 9.8 (br. s, 1H, NH benzimidazole), 8.2 (d,
aromatic), 7.32 (d, 2H, C 3, C 5 aromatic), 3.85 (s, 3H,
ꢁ
ꢁ
ꢁ
ꢁ
2H, C , C 6 aromatic), 8.1 (d, 2H, C 3, C aromatic), 7.9 (s,
OCH3). ESI-MS (m/z): 362 [M+H] +.
5
1H, C42 benzimidazole), 7.8 (d, 2H, C6, C7 benzimidazole),
2-(4ꢁ-Methoxyphenyl)-5-N-benzoylaminobenzimidazole (7i)
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
7.4 (d, 2H, C 2, C 6 aromatic), 7.3 (t, 3H, C 3, C 4 and C 5
aromatic). ESI-MS (m/z): 348.4 [M+H] +.
Yield: 66.0%, Melting Point: 198-200 ˚C. IR (KBr):
~3000 cm-1 (ꢀ –CH- aromatic, str), ~2930 (ꢀ, -CH3, str),
~1730 cm-1 (ꢀ CO str), 1610 cm-1 (ꢀ NH bend). ESI-MS
(m/z): 344.7 [M+H] +
2-Phenyl-5-N-(4ꢀ-methoxybenzoyl)aminobenzimidazole
(7d)
Yield: 60.5 %, Melting Point: 156-58 ˚C, IR KBr):
2-(4ꢁ-Methoxyphenyl)-5-N-(4ꢀ-nitrobenzoyl)
~3300 cm-1 (ꢀ NH str), ~1650 cm-1 (ꢀ –CO- str), 1600 cm-1 (ꢀ
aminobenzimidazole (7j)
1
NH bend). H NMR (DMSO d6) ꢁ (ppm): 12.1 (br. s, 1H,
Yield: 61.0 %, Melting Point: 258-60 ˚C. IR (KBr):
~3200 cm-1 (ꢀ NH str), ~3000 cm-1 (ꢀ –CH- aromatic, str),
amide NH), 10.0 (br. s, 1H, NH benzimidazole), 8.0 (d, 2H,
ꢁ
ꢁ
C 2, C aromatic), 7.85 (s, 1H, C4 benzimidazole), 7.8 (d,
6

98 Medicinal Chemistry, 2013, Vol. 9, No. 1
Kulkarni et al.
~2930 (ꢂ, -CH3, str), ~1760 cm-1 (ꢂ CO str), 1600 cm-1 (ꢂ NH
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bend). 1H NMR (DMSO d6) ꢁ (ppm): 11.8 (br. s, 1H, amide
ꢁ
NH), 10.0 (br. s, 1H, NH benzimidazole), 8.25 (d, 2H, C ,
3
ꢁ
ꢁ
ꢁ
C 5 aromatic), 8.17 (d, 2H, C 2, C 6, aromatic), 7.85 (s, 1H,
C4 benzimidazole), 7.7 (d, 2H, C6, C7 benzimidazole), 7.33
ꢀ
ꢀ
ꢀ
ꢀ
Cuenda, A.; Rouse, J.; Doza, Y. N.; Meier, R.; Cohen, P.;
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(d, 2H, C 2, C 6, aromatic), 7.1 (d, 2H, C 3, C 5,aromatic), 3.75
(s, 3H, OCH3). ESI-MS (m/z): 389 [M+H] +.
[15]
[16]
CONCLUSION
In summary, we have identified 5-substituted benzoyl-
amino-2-substituted phenylbenzimidazoles as new p38
kinase inhibitors. The initial SAR reveals that compounds 7b
and 7c were found to be moderately active in both models.
Substitution at both R and R1 may not be effective however
compound 7j exhibited activity which contradicts our
explanation. New molecules with diverse substitution at R1
and methoxy group at R may help to understand incites of
SAR.
[17]
[18]
[19]
CONFLICT OF INTEREST
Author RG thank AICTE for granting fellowship for
carrying this project under quality improvement program.
ACKNOWLEDGEMENTS
The authors acknowledge Dr. G Narahari Sastry,
Scientist, Molecular Modelling Group, and P. Srivani, re-
search scholar, IICT, Molecular Modelling Group Hydera-
bad for their help in molecular docking studies. RGK express
thanks to Dr. V J Rao Scientist, Organic III, IICT, Hydera-
bad, for constructive ideas in solving the difficulties during
the synthesis of molecules.
cell
lines,
by
amine-substituted
2-arylbenzimidazole-4-
carboxamide PARP-1 inhibitors. Bioorg. Med. Chem. Lett., 2004,
14, 2433-37.
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Kazimierczuk, Z.; Andrzejewska, M.; Kaustova, J.; Klimesova, V.
Synthesis and anti-mycobacterial activity of 2-substituted
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Sondhi, S.; Singh, N.; Kumar, A.; Lozach, O.; Meijer, L. Synthesis,
anti-inflammatory, analgesic and kinase (CDK-1, CDK-5 and
GSK-3)
inhibition
activity
evaluation
of
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Received: November 16, 2011
Revised: May 10, 2012
Accepted: July 29, 2012