Conventional and microwave-assisted synthesis of benzimidazole derivatives and their in vitro inhibition of human cyclooxygenase

Article abstract of DOI:10.1002/jhet.1058

A large series of 1,2-diaryl-benzimidazole and 2-aryl-1H-benzimidazole derivatives were synthesized with slight differences using both microwave irradiation and conventional heating methods. Usually higher yields and time reactions reduction were obtained

Full text of DOI:10.1002/jhet.1058

September 2012
Conventional and Microwave-Assisted Synthesis of
Benzimidazole Derivatives and Their In Vitro Inhibition of
Human Cyclooxygenase
a
1187
a
a
a
*
Daniela Secci, Adriana Bolasco, Melissa D’Ascenzio, Flavio della Sala,
Matilde Yáñez,b and Simone Carradoria
aDipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La
Sapienza”, P.le A. Moro 5, 00185 Rome, Italy
bDepartamento de Farmacología and Instituto de Farmacia Industrial, Universidad de Santiago de
Compostela, E-15782 Santiago de Compostela (La Coruña), Spain
*
E-mail: daniela.secci@uniroma1.it
Received April 11, 2011
DOI 10.1002/jhet.1058
Published online 26 October 2012 in Wiley Online Library (wileyonlinelibrary.com).
A large series of 1,2-diaryl-benzimidazole and 2-aryl-1H-benzimidazole derivatives were synthesized
with slight differences using both microwave irradiation and conventional heating methods. Usually
higher yields and time reactions reduction were obtained with the former method. All compounds were
assayed for their in vitro ability to inhibit human cyclooxygenases, and most of them showed an encour-
aging inhibitory activity and isoform selectivity in the micromolar range.
J. Heterocyclic Chem., 49, 1187 (2012).
an inducible enzyme responsible for inflammation and
hyperalgesia but devoid of gastroprotective functions
[3], the obvious solution to the gastrointestinal and renal
toxicity of NSAIDs is the development of selective
COX-2 inhibitors (COXIB) [4]. However, after repeated
clinical trials, it was clear that the prolonged administra-
tion of COXIBs could enhance the incidence of cardio-
vascular events such as myocardial infarctions and
strokes [5]. Hence, the search for novel analgesic/anti-
inflammatory agents devoid of severe side effects con-
tinues to be an active area of research in medicinal
chemistry.
Starting from these assumptions, the aim of our work
is to propose the MW-assisted synthesis and biological
evaluation of a large number of 2-monosubstituted and
1,2-disubstituted benzimidazoles as anti-inflammatory
agents. The benzimidazole nucleus, in fact, overlaps
with the structures of some potent traditional NSAIDs
such as sulindac and indometacin (Fig. 1).
Moreover, 1,2-disubstituted benzimidazoles were reported
to exhibit biologically important activities as anti-
inflammatory and analgesic agents [6], and to pos-
sess higher potency than celecoxib and rofecoxib when
tested as COX-2 selective inhibitors [7]. Finally, the
benzimidazole moiety fulfils the minimum structural require-
ments for anti-inflammatory compounds: the pseudoacidic
nature of its nucleus (pKa 5.5) falls within desirable
pKa range of 5.3–7.9 for acidic NSAIDs [6].
INTRODUCTION
Nonsteroidal anti-inflammatory drugs (NSAIDs) are
among the most prescribed drugs due to their high effi-
cacy in the treatment of pain, fever, inflammation, and
rheumatic disorders. The exact mechanism of action is
not fully understood due to the complexity of inflamma-
tion response. However, as many symptoms of inflam-
mation are caused by prostaglandins (PG), the efficacy
of NSAIDs should be in part attributed to their ability to
inhibit PG synthesis and release. This inhibition occurs
through the blockage of the arachidonic acid cascade,
which is catalyzed by two isoforms of the cyclo-
oxygenase (prostaglandin G/H synthase, EC 1.14.99.1):
COX-1 and COX-2 [1]. The major differences between
the two iso-enzymes involve their distribution and expres-
sion. COX-1 is widely distributed in human tissues
and is supposed to be encoded by a “Housekeeping Gene”
which is involved in gastroprotection, platelets aggre-
gation, renal sodium and water balance. Conversely,
COX-2 is almost undetectable under normal physio-
logical conditions, while its expression is dramatical-
ly increased by inflammatory mediators. Traditional
NSAIDs are nonselective COX-inhibitors [2], which are
the most representative and prescribed anti-inflammatory
agents, even though their use is often associated with
the occurrence of a range of hazardous side effects
such as nausea, constipation, dyspepsia, gastrointestinal
erosions, peptic ulcer, overt bleeding and perforation
which frequently induce therapy suspension or a low
compliance. Based on the assumption that COX-2 is
Although some of the reported 1,2-disubstituted-
benzimidazole and 2-substituted-1H-benzimidazole deriva-
tives have already been synthesized by classical thermal
© 2012 HeteroCorporation

1188
D. Secci, A. Bolasco, M. D’Ascenzio, F. della Sala, M. Yáñez, and S. Carradori
Vol 49
the corresponding products 1a–48a and 1b–48b in
good yields. All reactions were performed with the
minimum amounts of solvent (DMF) in vials of 10
mL, confirming that the focused microwave irradiation
is a very effective technique for accelerating thermal
organic reactions. The same products were also pre-
pared by the classical thermal method, using the same
reagents of microwave method in a reflux flask with
about 50 mL of DMF. Classical heating afforded lower
yields for almost all compounds with many difficulties
in separating pure products even by liquid chromatog-
raphy. The most important result of our approach is
the optimization of yields and reaction times using
microwave irradiation. In conclusion, we have devel-
oped a very simple, rapid, and efficient method for
the preparation of 1,2-diaryl-benzimidazole and 2-aryl-
1H-benzimidazole compounds using readily available
and inexpensive reagents.
Figure 1. Indane and indole scaffolds.
methods, which required rather forcing conditions and
reagents, we found out that there was no information
about oxidative cyclization performed by using micro-
wave reactors and only minimal data about household
oven microwave-assisted synthesis [8]. Therefore, aim-
ing at the synthesis of new heterocyclic systems with re-
markable biological importance, we report here on the
detailed synthesis by conventional and microwave-assisted
methods of a large series of 1,2-diaryl-benzimidazoles
and 2-aryl-1H-benzimidazoles.
BIOLOGICAL EVALUATION
Unless otherwise specified, results shown in the text
and tables are expressed as mean ꢁ standard error of the
mean (SEM) from five experiments. Significant differen-
ces between two means (P < 0.05 or P < 0.01) were
determined by one-way analysis of variance (ANOVA)
followed by the Dunnett’s post hoc test. To study the
possible effects of the test drugs (new compounds or
reference inhibitors) on human COX isoform enzymatic
activity, we evaluated the rate of N,N,N0,N0-tetramethyl-p-
phenylenediimine formation, i.e., the increase in absorb-
ance at 600 nm per unit of time (ΔA600 U/min). In these
experiments, the inhibitory activity of the tested drugs
(new compounds and reference inhibitors) is expressed
as IC50, i.e., the concentration of these compounds
required for a 50% reduction of the control COX isoform
enzymatic activity, estimated by least-squares linear
regression, using the program Origin 5.0 (Microcal
Software, Northampton, MA), with X, log of tested
compound molar concentration, and Y, the corresponding
percentage of inhibition of control N,N,N0,N0-tetramethyl-
p-phenylenediimine production obtained with each con-
centration. This regression was performed using data
obtained with four to six different concentrations of each
tested compound that inhibited the control hCOX isoform
enzymatic activity by between 20 and 80%. In addition,
we calculated the corresponding COX-1 selectivity index
(SI): IC50 (COX-2)/IC50 (COX-1) (Table 2).
CHEMISTRY
We synthesized molecules with mono-, bi-, and poly-
substituted 2-aryl groups (1a–42a and 1b–42b) and het-
eroaromatic ring (43a–48a and 43b–48b) based on
N-phenyl-benzimidazole and benzimidazole scaffolds
(Table 1). The design of these derivatives explores the
change in the electronic density and steric hindrance of
the pharmacophoric group. For this reason, we intro-
duced many different substituents, electron-withdrawing
and electron-donor groups, (—CH3, —OH, —F, —Cl,
—Br, —OCH3, —NMe2, —NO2, —SCH3, —CF3, —
SO2CH3, —OCH2Ph) at position 20, 30, 40, 50, and 60
of the phenyl ring. In addition, we changed the aromatic
group at position 2 of the benzimidazole scaffold with
some heteroaromatic rings such as pyrrole, furan, thio-
phene, indole, benzodioxole, and naphtalene. In this study,
96 derivatives have been synthesized by the reaction of N-
phenyl-o-phenylenediamine and 1,2-phenylenediamine
with substituted aldehydes and sodium metabisulfite
under microwave irradiation. Compounds 21a and 21b
were synthesized using oxone as oxidizing agent from
compounds 19a and 19b (Scheme 1).
Solid compounds were purified by chromatography,
and the structure of the pure compounds was established
by spectroscopic and spectrometric data. First, the reac-
tion between 1,2-phenylenediamines and the correspond-
ing aromatic aldehydes was carried out in 10–40 min
(80–100ꢀC) under microwave irradiation and afforded
RESULTS AND DISCUSSION
Most of the tested compounds inhibited human COX-
1 and COX-2 at micromolar concentrations with selec-
tivity index values ranging from <0.02 (47a) to >8.6
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2012
Conventional and Microwave-Assisted Synthesis of Benzimidazole Derivatives
1189
and Their In Vitro Inhibition of Human Cyclooxygenase
Table 1
Conventional and microwave-assisted synthesis conditions of benzimidazole derivatives.
Microwave heating
Conventional heating
Comp.
R
R0
Temperature (ꢀC)
Time (h)
Yield (%)
Temperature (ꢀC)
Time (h)
Yield (%)
1a [9]
2a [10]
3a [10]
4a [10]
5a [11]
6a [10]
7a [10]
8a [12]
9a [10]
10a [10]
11a [10]
12a [10]
13a [10]
14a [10]
15a [13]
16a
17a [10]
18a [10]
19a
20a [10]
21a [14]
22a [15]
23a [16]
24a [17]
25a
26a
27a [18]
28a
29a
30a
31a
32a
33a [19]
34a
35a [10]
36a
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
Ph
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
ND
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
80
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
ND
0.1
0.1
0.1
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.3
0.2
0.2
0.2
0.2
0.2
0.2
0.4
0.4
0.4
0.4
0.4
0.4
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
62
67
60
60
73
66
99
70
61
51
90
99
75
53
80
55
65
85
98
73
ND
65
86
62
85
60
73
69
73
68
71
74
99
75
74
99
76
89
61
55
60
99
77
56
85
62
60
59
90
80
76
82
99
61
68
79
73
62
67
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
51
47
50
43
55
61
65
67
44
39
65
87
67
49
30
31
48
76
85
62
80
60
75
36
42
39
33
49
48
51
60
65
79
60
60
75
70
75
36
39
50
88
65
47
78
58
46
32
75
63
55
58
74
52
53
59
54
48
47
20-CH3-Ph
30-CH3-Ph
40-CH3-Ph
20-OH-Ph
30-OH-Ph
40-OH-Ph
40-F-Ph
20-OCH3-Ph
30-OCH3-Ph
40-OCH3-Ph
20-Cl-Ph
30-Cl-Ph
40-Cl-Ph
40-N(CH3)2-Ph
20-NO2-Ph
30-NO2-Ph
40-NO2-Ph
40-SCH3-Ph
40-CF3-Ph
40-SO2CH3-Ph
20-Br-Ph
30-Br-Ph
40-Br-Ph
(20-OCH2Ph)-Ph
(30-OCH2Ph)-Ph
(40-OCH2Ph)-Ph
20,30-OH-Ph
20,40-OH-Ph
20,50-OH-Ph
20-OH-40-OCH3-Ph
20-OH-50-Cl-Ph
20,40-OCH3-Ph
20,50-OCH3-Ph
30,40-OCH3-Ph
20-OH-50-NO2-Ph
20-OH-50-Br-Ph
20,40,50-OCH3-Ph
30,40,50-OCH3-Ph
20-OH-30,50-Br-Ph
20-OH-30,50-I-Ph
40-OH-30,50-I-Ph
Pyrrol-20-yl
Fur-20-yl
37a
38a
39a [10]
40a
41a
42a
43a
44a [19]
45a [10]
46a
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
Thiophen-20-yl
Indol-30-yl
47a
Benzodioxol-50-yl
Naphtalen-10-yl
Ph
48a [20]
1b [21]
2b [22]
3b [22]
4b [20]
5b [20]
6b [23]
7b [21]
8b [21]
9b [24]
10b [24]
11b [21]
H
H
H
H
H
H
H
H
20-CH3-Ph
30-CH3-Ph
40-CH3-Ph
20-OH-Ph
30-OH-Ph
40-OH-Ph
40-F-Ph
20-OCH3-Ph
30-OCH3-Ph
40-OCH3-Ph
H
H
(Continues)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1190
D. Secci, A. Bolasco, M. D’Ascenzio, F. della Sala, M. Yáñez, and S. Carradori
Vol 49
Table 1
(Continued)
Microwave heating
Conventional heating
Comp.
R
R0
Temperature (ꢀC)
Time (h)
Yield (%)
Temperature (ꢀC)
Time (h)
Yield (%)
12b [25]
13b [26]
14b [21]
15b [27]
16b [25]
17b [27]
18b [21]
19b [28]
20b [22]
21b [29]
22b [30]
23b [27]
24b [21]
25b [8]
26b [24]
27b [31]
28b [32]
29b [23]
30b [23]
31b [33]
32b [34]
33b [26]
34b [27]
35b [21]
36b [35]
37b [26]
38b [8]
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
20-Cl-Ph
30-Cl-Ph
80
80
80
80
80
80
80
80
80
ND
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
ND
0.1
0.1
0.1
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.4
0.4
0.4
0.4
0.4
0.4
69
93
99
82
89
65
90
99
70
ND
88
67
66
80
85
96
87
62
62
62
90
87
75
94
99
73
73
63
92
92
83
95
54
99
99
97
99
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
80
24
24
24
24
24
24
24
24
24
48
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
61
87
85
80
73
55
81
80
47
65
63
49
44
58
55
72
59
46
44
47
75
61
59
78
81
56
62
59
77
68
52
58
43
70
73
68
61
40-Cl-Ph
40-N(CH3)2-Ph
20-NO2-Ph
30-NO2-Ph
40-NO2-Ph
40-SCH3-Ph
40-CF3-Ph
40-SO2CH3-Ph
20-Br-Ph
30-Br-Ph
40-Br-Ph
(20-OCH2Ph)-Ph
(30-OCH2Ph)-Ph
(40-OCH2Ph)-Ph
20,30-OH-Ph
20,40-OH-Ph
20,50-OH-Ph
20-OH-40-OCH3-Ph
20-OH-50-Cl-Ph
20,40-OCH3-Ph
20,50-OCH3-Ph
30,40-OCH3-Ph
20-OH-50-NO2-Ph
20-OH-50-Br-Ph
20,40,50-OCH3-Ph
30,40,50-OCH3-Ph
20-OH-30,50-Br-Ph
20-OH-30,50-I-Ph
40-OH-30,50-I-Ph
Pyrrol-20-yl
39b [21]
40b [34]
41b [36]
42b [37]
43b [38]
44b [21]
45b [21]
46b [39]
47b [31]
48b [22]
Fur-20-yl
Thiophen-20-yl
Indol-30-yl
Benzodioxol-50-yl
Naphtalen-10-yl
ND: not obtained with microwave irradiation.
(22a). With the only exception of compounds 28b and
30b, all the derivatives characterized by the absence of
the phenyl substituent on the N1 (“b” series) were
shown to be inactive. On the other side, compounds
belonging to the “a” series resulted quite active and
selective. In fact, thanks to the presence of the phenyl
ring on the N1, the “a” series is almost superposable to
some well-known anti-inflammatory drugs such as sulin-
dac or indometacin.
With reference to the phenyl ring on the C2 of the
benzimidazole, it is outstanding that monosubstitution
endowed compounds with a greater inhibitory activity
against both hCOX-1 and hCOX-2. However, if the sub-
stituent was an ortho-hydroxy group, disubstitution was
well tolerated (30a–32a, 28b, 30b) and, in the particular
case in which both substituents were OH, it was possible
to reach a quite good activity (11.79 ꢁ 0.26 mM) and
selectivity index (>8.5) against hCOX-1 in the “a” series
(30a), and to find a surprising regain of activity in
the corresponding derivatives of the “b” series (28b and
30b). Concerning about the activity against hCOX-
1, electron withdrawing substituents, especially halogens
(F, Br, Cl), seemed to assure good results when placed in
all the positions of the phenyl ring, while the para-
position could stand the presence of both electron with-
drawing and releasing groups (4a, 11a, 15a, 19a). On
the contrary, the introduction of bulky benzyloxy sub-
stituents on the phenyl ring, especially in ortho (25a)
and meta (26a) position, or heteroaromatic moieties
directly on the C2 of the benzimidazole (46a and 47a),
determined a switch of selectivity toward hCOX-2.
These results could be easily ascribed to the presence of
a second hydrophobic pocket in the active site of
hCOX-2 which is not present in hCOX-1, and to the
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2012
Conventional and Microwave-Assisted Synthesis of Benzimidazole Derivatives
1191
and Their In Vitro Inhibition of Human Cyclooxygenase
Scheme 1. General chemistry of the benzimidazole derivatives.
(DMF) in a 10-mL vial suitable for an automatic single-mode
microwave reactor (2.45-GHz high-frequency microwaves,
power range 0–300 W). The mixture was prestirred for 30 s
and then heated by microwave irradiation for 10–40 min at
80–100ꢀC (irradiation power reaches its maximum at the
beginning of reaction, then it decreases to lower and quite
constant values). The internal vial temperature was controlled
by an IR sensor. After cooling with pressurized air, the
reaction mixture was poured onto ice, filtered, and dried
under vacuum.
establishment of hydrogen bonds between the heteroa-
toms of the compounds and the active site of the
enzyme that are known to contribute to hCOX-2 selec-
tivity [6, 7]. It is also worthwhile to evaluate the MW
effects on kinetic and yields enhancements. In fact,
the same reactions have been carried out with slight
differences under microwave and thermal conditions.
The products obtained by both methodologies were
found to be identical but the superiority of the former
procedure in this scaffold may be ascribed to a concen-
tration effect. The robustness of our protocol derives
from the large synthesized scaffold which ranges from
mono- to poly-substitution on the aromatic ring at C2
of the benzimidazole nucleus with moieties differing
from size, steric hindrance, lipophilicity, and electronic
properties.
General procedure for the synthesis of derivatives 1a-20a,
22a-48a, 1b-20b, and 22b-48b under thermal conditions.
In
a 500-mL flask, N-phenyl-o-phenylenediamine or 1,2-
phenylenediamine (1 mmol), sodium metabisulfite (1 mmol),
and the corresponding aldehyde (1 mmol) were dissolved in 50
mL of N,N-dimethylformamide. The mixture was refluxed for
24 h. The reaction mixture was then poured onto ice and
extracted with CHCl3 (3 ꢂ 50 mL). The organic layer was dried
with anhydrous Na2SO4 and evaporated under reduced pressure.
The crude product was purified by chromatography to obtain
the desired benzimidazole.
EXPERIMENTAL
The compounds, synthesized with the microwave method,
were obtained with a Biotage Initiator™ 2.0. The chemicals,
solvents for synthesis, and spectral grade solvents were pur-
chased from Aldrich (Italy) and used without further purifica-
tion. Melting points (uncorrected) were determined automatically
on an FP62 apparatus (Mettler-Toledo). 1H-NMR spectra
were recorded at 400 MHz on a Bruker spectrometer using
DMSO-d6 or CDCl3 as solvent. Chemical shifts are expressed
as d units (parts per millions) relative to the solvent peak.
Coupling constants J are valued in Hertz (Hz). Elemental anal-
yses for C, H, and N were recorded on a Perkin-Elmer 240 B
microanalyzer and the analytical results were within ꢁ0.4% of
the theoretical values for all compounds. All reactions were
monitored by TLC performed on 0.2-mm thick silica gel plates
(60 F254 Merck).
General procedure for the synthesis of derivatives 21a
and 21b. In a 100-mL flask, compounds 19a or 19b (1 mmol)
was dissolved in 50 mL of methanol. The mixture was refluxed
at 100ꢀC. In a flask, oxone (3 mmol) was dissolved in the
minimum amount of water. Then, the mixture with oxone was
added dropwise to the flask. The mixture was refluxed for 24 h.
The desired benzimidazole was filtered and dried under vacuum.
Chemical-physical data for new compounds. 2-(2-Nitro-
phenyl)-1-phenyl-1H-benzo[d]imidazole (16a).
Yellow solid,
mp 180–181ꢀC. 1H-NMR (deuteriochloroform): d 7.23–7.25
(m, 2H, Ar), 7.34–7.41 (m, 6H, Ar), 7.57–7.61 (m, 1H, Ar),
7.67–7.68 (m, 2H, Ar), 7.90–7.92 (m, 1H, Ar), 7.97–7.99
(m, 1H, Ar).
2-[4-(Methylthio)phenyl]-1-phenyl-1H-benzo[d]imidazole
(19a). White solid, mp 257–258ꢀC. 1H-NMR (deuteriochloroform):
d 2.47 (s, 3H, SCH3), 7.14–7.16 (d, Jo = 8.5 Hz, 2H, Ar),
7.16–7.24 (m, 2H, Ar), 7.32–7.34 (m, 3H, Ar), 7.48–7.52
(m, 5H, Ar), 7.85–7.87 (m, 1H, Ar).
General procedure for the synthesis of derivatives
1a–20a, 22a–48a, 1b–20b, and 22b–48b under MW
irradiation. N-phenyl-o-phenylenediamine or 1,2-phenylenediamine
(1 mmol), sodium metabisulfite (1 mmol), and the corresponding
aldehyde (1 mmol) were dissolved in 2 mL of N,N-dimethylformamide
2-[2-(Benzyloxy)phenyl]-1-phenyl-1H-benzo[d]imidazole
(25a). Green solid, mp 144 –145ꢀC. 1H-NMR (deuteriochloroform):
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1192
D. Secci, A. Bolasco, M. D’Ascenzio, F. della Sala, M. Yáñez, and S. Carradori
Vol 49
Table 2
In vitro inhibition of hCOX-1 and hCOX-2 by compounds 1a–48a and 1b–48b.
“a” series
(N-Ph)
“b” series
(N—H)
hCOX-1
hCOX-2
hCOX-1 (IC50
)
hCOX-2 (IC50
)
SI
(IC50
)
(IC50
)
SI
a
b
b
b
b
b
b
b
b
b
a
b
a
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
23a
24a
25a
26a
27a
28a
29a
30a
31a
32a
33a
34a
35a
36a
37a
38a
39a
40a
41a
42a
43a
44a
45a
46a
47a
13.34 ꢁ 1.06 mM
18.16 ꢁ 1.32 mM
12.12 ꢁ 1.15 mM*
>7.5
1b
2b
3b
4b
5b
6b
7b
8b
9b
b
>5.5***
1.8
a
21.93 ꢁ 1.27 mM
b
b
a
9.15 ꢁ 0.76 mM*
23.85 ꢁ 1.58 mM
2.6
c
c
c
c
c
c
b
a
6.57 ꢁ 0.48 mM*
35.48 ꢁ 0.14 mM
5.4
a
a
26.91 ꢁ 2.57 mM
>3.7***
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
20b
21b
22b
23b
24b
25b
26b
27b
28b
29b
30b
31b
32b
33b
34b
35b
36b
37b
38b
39b
40b
41b
42b
43b
44b
45b
46b
47b
b
b
b
b
b
b
b
b
a
14.63 ꢁ 1.23 mM*
23.54 ꢁ 1.32 mM*
8.02 ꢁ 0.65 mM*
16.71 ꢁ 1.38 mM**
29.03 ꢁ 1.46 mM
36.52 ꢁ 1.90 mM
32.70 ꢁ 1.28 mM
19.61 ꢁ 0.06 mM
38.13 ꢁ 1.72 mM
2.0
1.6
4.1
1.2
9.04 ꢁ 0.35 mM*
4.2
b
18.54 ꢁ 1.23 mM
<0.19***
a
a
b
b
24.26 ꢁ 2.10 mM
39.66 ꢁ 3.15 mM
1.6
b
b
a
b
a
b
b
b
a
a
11.61 ꢁ 0.89 mM
>8.6***
2.1
11.27 ꢁ 0.53 mM*
23.57 ꢁ 2.86 mM
a
b
b
b
b
a
24.77 ꢁ 0.99 mM
<0.25***
<0.20***
b
a
19.67 ꢁ 0.76 mM
b
a
a
a
a
a
c
a
a
a
a
b
a
a
a
a
b
c
b
29.52 ꢁ 2.37 mM
>3.4***
21.87 ꢁ 0.92 mM
26.89 ꢁ 2.13 mM
1.2
2.1
a
a
b
11.79 ꢁ 0.26 mM
27.83 ꢁ 0.69 mM
>8.5***
>3.6***
>3.5***
8.30 ꢁ 0.76 mM
17.63 ꢁ 1.46 mM
b
a
b
a
a
b
b
b
b
a
b
a
b
b
b
b
a
b
b
b
b
b
a
28.60 ꢁ 0.48 mM
c
a
a
b
a
b
a
a
a
a
b
c
b
b
b
b
b
b
b
b
a
23.96 ꢁ 0.32 mM
>4.2***
<0.04***
<0.02***
2.9
b
b
b
3.84 ꢁ 0.19 mM
2.34 ꢁ 0.15 mM
35.20 ꢁ 1.41 mM
b
Indometacin
Diclofenac
FR122047
Nimesulide
DuP 697
12.16 ꢁ 1.16 mM**
18.23 ꢁ 1.73 mM
23.62 ꢁ 1.97 mM
1.3
b
93.80 ꢁ 6.55 nM
>1066***
<0.46***
0.0056
d
231.40 ꢁ 19.84 mM
126.32 ꢁ 7.41 nM
22.61 ꢁ 1.56 mM*
Each IC50 value is the mean ꢁ SEM from five experiments.
a100 mM inhibits hCOX-1 or hCOX-2 activity by ∼40–45%.
bInactive at 100 mM (highest concentration tested).
cInactive at 25 mM (highest concentration tested).
dInactive at 500 mM (highest concentration tested).
Level of statistical significance:
*P < 0.01 versus the corresponding IC50 values obtained against hCOX-2, as determined by ANOVA/Dunnett’s post hoc test.
**P < 0.05 versus the corresponding IC50 values obtained against hCOX-2, as determined by ANOVA/Dunnett’s post hoc test.
***Value obtained under the assumption that the corresponding IC50 against hCOX-1 or hCOX-2 is the highest concentration tested.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2012
Conventional and Microwave-Assisted Synthesis of Benzimidazole Derivatives
1193
and Their In Vitro Inhibition of Human Cyclooxygenase
d 4.69 (s, 2H, OCH2Ar), 6.73–6.75 (d, Jo = 8.1 Hz, 1H, Ar),
6.97–6.98 (m, 2H, Ar), 7.07–7.13 (m, 3H, Ar), 7.22–7.37
(m, 11H, Ar), 7.70–7.72 (d, Jo = 7.7 Hz, 1H, Ar), 7.92–7.94
(d, Jo = 7.4 Hz, 1H, Ar).
2,4-Diiodo-6-(1-phenyl-1H-benzo[d]imidazol-2-yl)phenol
(41a). Yellow solid, mp 180–181ꢀC. 1H-NMR (deuteriochloroform):
d 7.25–7.38 (m, 9H, Ar), 7.58–7.61 (m, 1H, Ar), 7.89–7.90 (s, 1H,
Ar), 12.32 (bs, 1H, OH, D2O exch.).
2-[3-(Benzyloxy)phenyl]-1-phenyl-1H-benzo[d]imidazole
(26a). Grey solid, mp 120–121ꢀC. 1H-NMR (dimethyl sulfoxide
d6): d 4.93 (s, 2H, OCH2Ar), 6.99–7.00 (m, 1H, Ar), 7.11–7.12
(m, 1H, Ar), 7.18–7.38 (m, 13H, Ar), 7.49–7.53 (m, 3H, Ar),
7.90–7.92 (d, Jo = 7.5 Hz, 1H, Ar).
2,6-Diiodo-4-(1-phenyl-1H-benzo[d]imidazol-2-yl)phenol
(42a). Brown solid, mp 96–97ꢀC. 1H-NMR (deuteriochloroform):
d 6.06 (s, 1H, Ar), 6.92–6.94 (m, 1H, Ar), 7.04–7.34 (m, 4H, Ar),
7.47 (s, 1H, Ar), 7.67–7.71 (m, 2H, Ar), 8.11 (s, 1H, Ar), 8.48
(s, 1H, Ar), 14.09 (bs, 1H, OH, D2O exch.).
3-(1-Phenyl-1H-benzo[d]imidazol-2-yl)benzene-1,2-diol
(28a). Orange solid, mp 122–123ꢀC. 1H-NMR (deuteriochloroform):
d 6.36–6.38 (m, 2H, Ar), 6.44–6.46 (d, Jo = 8.0 Hz, 1H, Ar),
6.91–7.45 (m, 6H, Ar), 7.62–7.64 (m, 3H, Ar), 7.81–7.83
(d, Jo = 8.0 Hz, 1H, Ar), 8.62 (bs, 1H, OH, D2O exch.),
9.58 (bs, 1H, OH, D2O exch.).
1-Phenyl-2-(1H-pyrrol-2-yl)-1H-benzo[d]imidazole (43a).
White solid, mp 185–186ꢀC. 1H-NMR (deuteriochloroform):
d 6.12–6.14 (m, 1H, Ar), 6.92–6.94 (m, 1H, Ar), 7.10–7.20
(m, 4H, Ar), 7.30–7.34 (m, 1H, Ar), 7.67–7.71 (m, 2H, Ar),
8.11–8.12 (m, 1H, Ar), 8.47–8.49 (m, 1H, Ar), 14.11 (bs, 1H,
OH, D2O exch.).
4-(1-Phenyl-1H-benzo[d]imidazol-2-yl)benzene-1,3-diol
(29a). Brown solid, mp 167–168ꢀC. 1H-NMR (deuteriochloroform):
6.06–6.08 (d, Jo = 8.2 Hz, 1H, Ar), 6.48–6.71 (m, 3H, Ar), 7.04–7.42
(m, 4H, Ar), 7.57–7.60 (m, 3H, Ar), 7.76–7.78 (d, Jo = 7.6 Hz, 1H,
Ar), 8.53 (bs, 1H, OH, D2O exch.), 9.58 (bs, 1H, OH, D2O exch.).
2-(1-Phenyl-1H-benzo[d]imidazol-2-yl)benzene-1,4-diol
(30a). Grey solid, mp 91–92ꢀC. 1H-NMR (deuteriochloroform):
d 6.34 (s, 1H, Ar), 6.72–6.78 (m, 1H, Ar), 6.96–6.98 (d, Jo = 8.6 Hz,
1H, Ar), 7.07–7.09 (d, Jo = 8.3 Hz, 1H, Ar), 7.26–7.42 (m, 3H, Ar),
7.58–7.60 (m, 3H, Ar), 7.79–7.81 (d, J = 7.7 Hz, 1H, Ar), 7.98 (m,
1H, Ar), 8.51 (bs, 1H, OH, D2O exch.), 9.49 (bs, 1H, OH, D2O
exch.).
5-Methoxy-2-(1-phenyl-1H-benzo[d]imidazol-2-yl)phenol
(31a). Green solid, mp 174–175ꢀC. 1H-NMR (deuteriochloroform):
d 3.78 (s, 3H, OCH3), 6.04–6.12 (m, 1H, Ar), 6.62–6.63 (m,
1H, Ar), 6.72–6.75 (d, Jo = 8.6 Hz, 1H, Ar), 7.06–7.33 (m,
5H, Ar), 7.41–7.43 (m, 3H, Ar), 7.61–7.62 (m, 1H, Ar),
13.41 (bs, 1H, OH, D2O exch.).
4-Chloro-2-(1-phenyl-1H-benzo[d]imidazol-2-yl)phenol
(32a). Yellow solid, mp 109–110ꢀC. 1H-NMR (deuteriochloroform):
d 6.75 (s, 1H, Ar), 7.03–7.43 (m, 8H, Ar), 7.66–7.67 (m,
3H, Ar), 7.82–7.84 (d, Jo = 8.2 Hz, 1H, Ar), 12.83 (bs,
1H, OH, D2O exch.).
2-(2,5-Dimethoxyphenyl)-1-phenyl-1H-benzo[d]imidazole
(34a). Green solid, mp 69–70ꢀC. 1H-NMR (deuteriochloroform):
d 3.25 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 6.65–6.67 (d,
Jo = 8.3 Hz, 1H, Ar), 6.92–6.93 (m, 1H, Ar), 7.25–7.41
(m, 9H, Ar), 7.89–7.91 (d, Jo = 7.7 Hz, 1H, Ar).
4-Nitro-2-(1-phenyl-1H-benzo[d]imidazol-2-yl)phenol (36a).
Yellow solid, mp 179–180ꢀC. 1H-NMR (dimethyl sulfoxide
d6): d 7.07–7.09 (d, Jo = 9.2 Hz, 2H, Ar), 7.25–7.27 (d,
Jo = 7.2 Hz, 2H, Ar), 7.37–7.39 (m, 3H, Ar), 7.55–7.61 (m,
2H, Ar), 7.88–7.89 (d, Jo = 7.0 Hz, 1H, Ar), 8.11–8.12 (m,
1H, Ar), 8.17–8.20 (m, 1H, Ar), 12.05 (bs, 1H, OH, D2O
exch.).
2-(1H-Indol-3-yl)-1-phenyl-1H-benzo[d]imidazole (46a).
Grey solid, mp 223–224ꢀC. 1H-NMR (deuteriochloroform):
d 6.59–6.60 (m, 1H, Ar), 7.01–7.03 (m, 1H, Ar), 7.16–7.25
(m, 4H, Ar), 7.40–7.42 (m, 1H, Ar), 7.52–7.54 (m, 2H, Ar),
7.55–7.67 (m, 3H, Ar), 7.74–7.76 (d, J = 7.8 Hz, 1H, Ar),
8.47–8.49 (d, J = 7.0 Hz, 1H, C2H-indole), 11.41 (bs, 1H, NH,
D2O exch.).
2-(Benzo[d][1,3]dioxol-5-yl)-1-phenyl-1H-benzo[d]imidazole
(47a). Grey solid, mp 122–123ꢀC. 1H-NMR (deuteriochloroform):
d 5.96–5.97 (s, 2H, OCH2O), 6.72–6.74 (d, J = 7.7 Hz,
1H, Ar), 7.05–7.07 (m, 2H, Ar), 7.23–7.26 (m, 2H, Ar),
7.31–7.34 (m, 3H, Ar), 7.50–7.54 (m, 3H, Ar), 7.86–7.87
(d, J = 7.3 Hz, 1H, Ar).
Preparation of microsomes from human platelets. Human
platelets were isolated by centrifugation from buffy coats
obtained from the Centro de Transfusión de Galicia (Santiago
de Compostela, Spain) and prepared as we have previously
described [40]. Briefly, buffy coat was diluted 1:1 with washing
buffer of the following composition (mM): NaCl 120, KCl
5, trisodium citrate 12, glucose 10, sucrose 12.5 (pH 6) and
then centrifuged at 400 g for 8 min in a centrifuge (Omnifuge
2.0 RS, Heraeus Sepatech, Osterade, Germany) at 25ꢀC to
obtain platelet rich plasma. The upper layer obtained in this
centrifugation, containing platelet rich plasma, was gently
removed and centrifuged at 850 g for 20 min at 4ꢀC in a
centrifuge (J2-MI, Beckman Instruments, Palo Alto, CA). The
platelet pellet was recovered, resuspended with washing
buffer, and centrifuged again at 850 g for 20 min at 4ꢀC. To
prepare human platelets microsomes, the resultant platelet
pellet of the above centrifugation was resuspended in 7 mL
of sodium phosphate buffer (10 mM, pH 7.4), sonicated at 50
W for 50 s (5 pulses of 10 s), and centrifuged at 850 g for
20 min at 4ꢀC in a refrigerated centrifuge (J2-MI, Beckman
Instruments, Palo Alto, CA). The pellet was discarded and the
supernatant was subsequently centrifuged at 10,000 g for 10
min at 4ꢀC in the same centrifuge. The pellet obtained in this
centrifugation was discarded and the supernatant was finally
centrifuged at 100,000 g for 1 h at 4ꢀC in a centrifuge
(J2-MI, Beckman Instruments, Palo Alto, CA). The resultant
pellet containing platelet microsomes was resuspended in
1 mL of sodium phosphate buffer (50 mM, pH 7.4) and the
protein concentration in the platelet microsome suspension
(∼2 mg/mL) was measured by the method of Bradford [41]
4-Bromo-2-(1-phenyl-1H-benzo[d]imidazol-2-yl)phenol
(37a). Brown solid, mp 120–121ꢀC. 1H-NMR (deuteriochloroform):
d 6.88–7.43 (m, 8H, Ar), 7.66–7.68 (m, 3H, Ar), 7.82–7.83 (m, 1H,
Ar), 13.36 (bs, 1H, OH, D2O exch.).
1-Phenyl-2-(2,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole
(38a). Green solid, mp 104–105ꢀC. 1H-NMR (deuteriochloroform):
d 3.25 (s, 3H, OCH3), 3.88 (s, 6H, 2 ꢂ OCH3), 6.32 (s, 1H, Ar),
7.22–7.41 (m, 9H, Ar), 7.89–7.91 (m, 1H, Ar).
2,4-Dibromo-6-(1-phenyl-1H-benzo[d]imidazol-2-yl)phenol
(40a). Orange solid, mp 186–187ꢀC. 1H-NMR (deuteriochloroform):
d 6.85 (s, 1H, Ar), 6.86–7.89 (m, 11H, Ar), 13.79 (bs, 1H,
OH, D2O exch.).
using
a
protein assay kit from BioRad Laboratories
(Alcobendas, Spain). Platelet microsome aliquots were stored
at ꢃ80ꢀC for several days (without apparent loss of COX
activity) until use.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1194
D. Secci, A. Bolasco, M. D’Ascenzio, F. della Sala, M. Yáñez, and S. Carradori
Vol 49
In addition, stock solutions of the following drugs were prepared
daily before use: arachidonic acid (30 mM) and indometacin
(10 mM) in ethanol; hematin (0.2 mM) and TMPD (40 mM) in
DMSO. Because of the instability of some chemicals (e.g., DuP
697, arachidonic acid), the corresponding solutions of these che-
micals were maintained in inert atmosphere until use. In all assays,
neither deionized water (Milli-Q, Millipore Ibérica S.A., Madrid,
Spain) nor appropriate dilutions of the vehicle used (DMSO) had
significant pharmacological effects.
Determination of human cyclooxygenase (hCOX) isoform
activity.
The biological evaluation of the test drugs on total
hCOX activity (bisdioxygenase and peroxidase reactions) was
investigated by measuring their effects on the oxidation of N,N,
N0,N0-tetramethyl-p-phenylenediamine (TMPD) to N,N,N0,N0-
tetramethyl-p-phenylenediimine, using araquidonic acid as com-
mon substrate for both hCOX-1 and hCOX-2, microsomal
COX-2 prepared from insect cells (Sf 21 cells) infected
with recombinant baculovirus containing cDNA inserts for
hCOX-2 (Sigma-Aldrich Química S.A., Alcobendas, Spain)
and COX-1 from human platelet microsomes (obtained as
described in the above paragraph since, unlike hCOX-2, hCOX-
1 is not commercially available). The formation of N,N,N0,N0-
tetramethyl-p-phenylenediimine (a colored compound) from N,
N,N0,N0-tetramethyl-p-phenylenediamine (TMPD) catalyzed by
COX can be detected spectrophotometrically at 600 nm. In this
study, hCOX activity was evaluated using the above spectropho-
tometric method following the general procedure described previ-
ously [42] with several modifications. Briefly, 0.1 mL of Tris-HCl
buffer (100 mM, pH 8) containing 1 mM hematin, 100 mM TMPD,
various concentrations of the test drugs (new compounds or refer-
ence inhibitors), and appropriate amounts of hCOX-1 and hCOX-
2 required and adjusted to obtain in our experimental conditions
the same control absorbance increase (0.08 A600 U/min) were in-
cubated for short periods of time (3–5 min) to avoid a notable loss
of COX activity at 37ꢀC in a flat-bottom 96-well microtest plate
(BD Biosciences, Franklin Lakes, NJ) placed in the dark multi-
mode microplate reader chamber. After this incubation period,
the reaction was started by adding (final concentration) 100-mM
arachidonic acid and the formation of N,N,N0,N0-tetramethyl-p-
phenylenediimine from TMPD, i.e., the increase in absorbance
at 600 nm was measured at 37ꢀC in a multimode microplate
reader (Fluostar Optima, BMG Labtech GmbH, Offenburg,
Germany) for 25 s, a period in which the absorbance increased
linearly from the beginning.
Acknowledgments. This work was supported by grants from
FIRB RBI067F9E (Italy).
REFERENCES AND NOTES
[1] Marnett, L. J. Annu Rev Pharmacol Toxicol 2009, 49, 265.
[2] (a) FitzGerald, G. A.; Patrono, C. N Engl J Med 2001, 345;
(b) Radi, Z. A.; Khan, N. K. Exp Toxicol Pathol 2006, 58, 163.
[3] Vane, J. R.; Bakhle, Y. S.; Botting, R. M. Annu Rev Phar-
macol Toxicol 1998, 38, 97.
[4] Dubois, R. W.; Melmed, G. Y.; Henning, J. M.; Laine, L.
Aliment Pharmacol Ther 2004, 19, 197.
[5] Heim, H.; Broich, K. Thromb Haemost 2006, 96, 423.
[6] (a) Paramashivappa, R.; Phani, K. P.; Subba, R. P. V.;
Srinivasa, R. A. Bioorg Med Chem Lett 2003, 13, 657; (b) Le, H.;
Lemaire, I. B.; Gilbert, A.; Jolicoeur, F.; Yang, L.; Leduc, N.; Lemaire,
S. J Pharmacol Exp Ther 2004, 309, 146.
[7] Franke, L.; Byvatov, E.; Werz, O.; Steinhilber, D.; Schneider,
P.; Schneider, G. J Med Chem 2005, 48, 6997.
[8] (a) Navarrete-Vázquez, G.; Moreno-Diaz, H.; Aguirre-Crespo,
F.; León-Rivera, I.; Villalobos-Molina, R.; Munoz-Muniz, O.;
Estrada-Soto, S. Bioorg Med Chem Lett 2006, 16, 4169; (b) Navarrete-
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León-Rivera, I.; Tlahuext, H.; Munoz-Muniz, O.; Torres-Gómez, H.
Synth Commun 2007, 37, 2815; (c) Algul, O.; Kaessler, A.; Apcin, Y.;
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The specific absorbance (used to obtain the results) was cal-
culated after subtraction of the background absorbance generated
in wells containing a blank solution, i.e., all components except
the COX isoforms, which were replaced by a Tris-HCl buffer
solution. In our experimental conditions, this background activity
was practically negligible.
[9] Peng, J.; Ye, M.; Zong, C.; Hu, F.; Feng, L.; Wang, X.;
Wang, Y.; Chen, C. J Org Chem 2011, 76, 716.
[10] Iyengar, S; Muhlhauser, M. A.; Thor, K. B. WO 9733873,
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Control experiments were carried out simultaneously by repla-
cing the test drugs (new compounds and reference inhibitors)
with appropriate dilutions of the vehicles. In addition, the possible
capacity of the above test drugs to modify the absorbance of
the reaction mixture due to nonenzymatic inhibition (e.g., for
directly reacting with TMPD) was determined by adding these
drugs to solutions containing only TMPD in a Tris-HCl buffer
solution.
Drugs and chemicals. The drugs, vehicle, and chemicals used
in the experiments were the new compounds, dimethyl sulfoxide
(DMSO), hematin porcine, N,N,N0,N0-tetramethyl-p-phenylene-
diamine dihydrochloride (TMPD), arachidonic acid sodium salt,
indometacin (purchased from Sigma-Aldrich Química S.A., Alco-
bendas, Spain), diclofenac sodium salt, FR122047 monohy-
drochloride hydrate, nimesulide, and DuP 697 (purchased from
Cayman Chemical, Ann Arbor, MI). Appropriate dilutions of
some of the above drugs were prepared every day immediately be-
fore use in deionized water from the following concentrated stock
solutions kept at ꢃ20ꢀC: the new compounds, diclofenac,
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