Design and synthesis of new 1,2-diaryl-4,5,6,7-tetrahydro-1Hbenzo[ d] imidazoles as selective cyclooxygenase (COX-2) inhibitors

Article abstract of DOI:10.1007/s00044-011-9709-y

A new series of 1,2-diaryl-4,5,6,7-tetrahydro- 1H-benzo[d]imidazoles, possessing a methylsulfonyl pharmacophore, were synthesized to evaluate their biological activities as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1 and COX-2 isozyme inhibition studies were carried out to acquire structure-activity relationship data with respect to the point that molecular modeling studies showed that designed compounds bind in the primary binding site such that the SO₂Me substituent at para-position of C-2 phenyl ring inserts into the 2° pocket present in COX-2 enzyme. COX-1 and COX-2 inhibition studies showed that all compounds were selective inhibitors of the COX-2 isozyme with IC50 values in the highly potent 0.34-0.69 μM range, and COX-2 selectivity indexes in the 52.3-163.8 range. 1-(4-Fluorophenyl)-2-(4- (methylsulfonyl) phenyl)-4,5,6,7-tetrahydro-1H-benzo[d] imidazole was identified as the most potent (IC50 = 0.34 μM), and selective (SI = 163.8), COX-2 inhibitor among the synthesized compounds.

Full text of DOI:10.1007/s00044-011-9709-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1869–1875
DOI 10.1007/s00044-011-9709-y
ORIGINAL RESEARCH
Design and synthesis of new 1,2-diaryl-4,5,6,7-tetrahydro-1H-
benzo[d] imidazoles as selective cyclooxygenase (COX-2)
inhibitors
•
A. Zarghi H. Reihanfard S. Arfaei
•
•
•
B. Daraei M. Hedayati
Received: 18 January 2011 / Accepted: 13 June 2011 / Published online: 25 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A new series of 1,2-diaryl-4,5,6,7-tetrahydro-
1H-benzo[d]imidazoles, possessing methylsulfonyl
Introduction
a
pharmacophore, were synthesized to evaluate their bio-
logical activities as selective cyclooxygenase-2 (COX-2)
inhibitors. In vitro COX-1 and COX-2 isozyme inhibition
studies were carried out to acquire structure–activity rela-
tionship data with respect to the point that molecular
modeling studies showed that designed compounds bind in
the primary binding site such that the SO2Me substituent at
para-position of C-2 phenyl ring inserts into the 2° pocket
present in COX-2 enzyme. COX-1 and COX-2 inhibition
studies showed that all compounds were selective inhibi-
tors of the COX-2 isozyme with IC50 values in the highly
potent 0.34–0.69 lM range, and COX-2 selectivity indexes
in the 52.3–163.8 range. 1-(4-Fluorophenyl)-2-(4-(methyl-
sulfonyl)phenyl)-4,5,6,7-tetrahydro-1H-benzo[d] imidaz-
ole was identified as the most potent (IC50 = 0.34 lM),
and selective (SI = 163.8), COX-2 inhibitor among the
synthesized compounds.
Prostaglandins act as potent mediators of pain, fever, and
inflammation. Cyclooxygenase (COX) catalyzes the con-
version of arachidonic acid to the prostaglandin endoper-
oxide PGG2 then PGH2, which serves as the precursor for
the formation of PGs and thromboxanes (Fu et al., 1990).
Nowadays, it is well established that there are at least two
COX isozymes, COX-1 and COX-2 (Vane and Botting,
1998). The constitutive COX-1 isozyme is produced in a
variety of tissues and appears to be important to the
maintenance of physiological functions such as gastro
protection and vascular homeostasis (Smith and Dewitt,
1996). Alternatively, the COX-2 isozyme is induced by
mitogenic and proinflammatory stimuli linking its
involvement to inflammatory processes (Herschman,
1996). Thus, selective inhibition of COX-2 over COX-1 is
useful for the treatment of inflammation and inflammation-
associated disorders with reduced gastrointestinal toxici-
ties. In addition to role of COX-2 in rheumatoid arthritis
and osteoarthritis, it is also implicated in cancer and
angiogenesis (Kawamori et al., 1998; Katori and Majima,
2000; Srinath et al., 2003; Takkouche et al., 2008). Recent
studies have also shown that selective COX-2 inhibitors
may slow the progress of Alzheimer’s disease without
causing gastrointestinal damage (Nivsarkar et al., 2008).
1,2-Diarylheterocycles have been extensively studied as
selective COX-2 inhibitors (Ghodsi et al., 2010; Penning
et al.,1997; Prasit et al., 1999; Riendeau et al., 2002;
Navidpour et al., 2006; Salimi et al., 2007; Zarghi et al.,
2009). All these tricyclic molecules possess 1,2-diaryl
substitution on a central hetero or carbocyclic ring system
(Fig. 1). Celecoxib and rofecoxib are two typical COX-2
inhibitors in this class. However, the recent withdrawal of
some diarylheterocyclic selective COX-2 inhibitors such as
Keywords Imidazoles ꢀ COX-1 ꢀ COX-2
A. Zarghi (&) ꢀ H. Reihanfard ꢀ S. Arfaei
Department of Medicinal Chemistry, School of Pharmacy,
Shahid Beheshti University of Medical Sciences,
P.O. Box: 14155-6153, Tehran, Iran
e-mail: azarghi@yahoo.com
B. Daraei
Department of Toxicology, Faculty of Medical Sciences,
Tarbiat Modarres University, Tehran, Iran
M. Hedayati
Obesity Research Center, Research Institute for Endocrine
Sciences, Shahid Beheshti University of Medical Sciences,
Tehran, Iran
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Med Chem Res (2012) 21:1869–1875
Me
Fig. 1 Some representative
examples of COXIBs (celecoxib
and rofecoxib), 1,3-
SO2NH2
SO2Me
benzthiazine-4-one (a),
quinoline-4-carboxylic acid
(b) lead compounds and our
4,5,6,7-tetrahydro-1H-
N
benzo[d] imidazole scaffolds
N
O
O
F3C
Rofecoxib
Celecoxib
SO2Me
SO2Me
N
HOOC
O
N
S
A
B
X
SO2Me
N
N
Designed compounds
rofecoxib due to its adverse cardiovascular side effects
´
(Dogne et al., 2005; Solomon, 2005) encourages the
Results and discussions
researchers to explore and evaluate new structural ring
templates possessing COX inhibitory activity. In addition,
recent studies have suggested that rofecoxib’s adverse
cardiac events may not be a class effect but rather related to
its particular chemical structure (Dogne et al., 2006). For
this reason novel scaffolds with high COX-2 inhibitory
activity need to be found and evaluated for their biological
activities. Recently, we reported a group of new diaryl-
heterocyclic COX-2 inhibitors possessing a bicyclic central
ring which exhibited highly selectivity for COX-2 inhibi-
tion. (Ghodsi et al., 2010; Zarghi et al., 2009). As part of
our ongoing program to design new types of tricyclic
selective COX-2 inhibitors, we now report the design,
synthesis, cyclooxygenase inhibitory, and some molecular
modeling studies of a new group of 1,2-diarylheterocycles
Chemistry
The target 4,5,6,7-tetrahydro-1H-benzo[d]imidazole deriv-
atives were synthesized via the route outlined in Scheme 1.
Accordingly, 1,2-cyclohexanedione, 4-methylthiobenzal-
dehyde and aniline derivatives in the presence of excess
amount of ammonium acetate were treated in microwave
reactor at 180 watt power to obtain 1,2-diaryl-4,5,6,7-tetra-
hydro-1H-benzo[d]imidazoles (1a–f). Oxidation of the
crude products 1a–f using Oxone in THF/water afforded the
1-(4-substituted-phenyl)-2-(4-(methylsulfonyl)phenyl)-
4,5,6,7-tetrahydro-1H-benzo[d] imidazoles 2a–f (Yields:
12.7–15.5%).
Enzyme inhibitory activity
possessing
a
4,5,6,7-tetrahydro-1H-benzo[d]imidazole
central ring and a COX-2 SO2Me pharmacophore at the
It is well established for the diarylheterocyclic class of
para-position of C-2 phenyl ring.
COX-2 inhibitors that a para-methylsulfone substituent on
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Med Chem Res (2012) 21:1869–1875
1871
O
O
SCH3
+
X
a
+
OHC
H2N
N
N
N
b
SCH3
SO2CH3
N
X
X
1a; X=H
1b; X=F
1c; X=Cl
2a; X=H
2b; X=F
2c; X=Cl
2d; X=Me
1d; X=Me
1e; X= OMe
1d; X=NHCOMe
2e; X= OMe
2f; X= NHCOMe
Scheme 1 Reagents and conditions: a NH4OAc, ACOH, CHCl3, microwave 180 watt, 20 min. b Oxone, THF, water, 25°C, 2 h
one of the phenyl rings is a requirement for good COX-2
potency and selectivity. Accordingly, a group of 1,2-diaryl-
4,5,6,7-tetrahydro-1H-benzo[d]imidazoles (2a–f) contain-
ing a variety of substituents (H, F, Cl, Me, OMe, and
NHCOMe) at the para-position of the N-1 phenyl ring,
were synthesized to investigate the effect of these substit-
uents on COX-2 selectivity and potency. The ability of the
synthesized compounds to inhibit the COX-1 and COX-2
isozymes was determined using chemiluminescent enzyme
assays (Table 1). In vitro COX-1/COX-2 inhibition studies
showed that all compounds were selective inhibitors of the
COX-2 isozyme with IC50 values in the highly potent
0.34–0.69 lM range, and COX-2 selectivity indexes (SI) in
the 61.2–163.8 range. SAR data (IC50 lM values) acquired
by determination of the in vitro ability of the title com-
pounds to inhibit the COX-1 and COX-2 isozymes showed
that the nature and size of substituent attached to N-1
phenyl ring influenced both selectivity and potency for
COX-2 inhibitory activity. Our results indicated that the
introduction of suitable substituents such as F (2b) and
OMe (2e) at the para-position of N-1 phenyl ring increased
both selectivity and potency for COX-2 inhibitory activity.
However, compounds having Cl or NHCOMe substituents
(2c and 2f) showed less potency for COX-2 isozyme that
may be explained by steric parameter. Based upon, com-
pound 2a containing unsubstituted N-1 phenyl ring showed
better COX-2 inhibitory activity compared to compounds
2c and 2f. According to these results, 1-(4-fluorophenyl)-2-
(4-(methylsulfonyl)phenyl)-4,5,6,7-tetrahydro-1H-benzo[d]
imidazole (2b) was the most potent (IC50 = 0.34 lM), and
selective (SI = 163.8), COX-2 inhibitor among the syn-
thesized compounds. However, it was less selective than
Table 1 In vitro COX-1 and COX-2 enzyme inhibition data
N
SO2CH3
N
X
Compound
X
IC50 (lM)a
COX-2 SIb
COX-1
COX-2
2a
H
56.5
55.7
61.6
40.1
42.2
36.1
24.3
0.55
0.34
0.67
0.62
0.37
0.69
0.06
102.7
163.8
91.9
2b
F
2c
Cl
2d
Me
64.6
2e
OMe
NHCOMe
114.0
52.3
2f
Celecoxib
405
a
Values are mean values of two determinations acquired using an
ovine COX-1/COX-2 assay kit, where the deviation from the mean is
\10% of the mean value
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50
)
123

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Med Chem Res (2012) 21:1869–1875
celecoxib (IC50 = 0.06 lM; SI = 405) in terms of COX-2
inhibitory activity. These data suggest that the compound
2b should inhibit the synthesis of inflammatory prosta-
glandins via the cyclooxygenase pathway at sites of
inflammation.
with other analogs. These data also suggest that the com-
pound 2b should inhibit the synthesis of inflammatory
prostaglandins via the cyclooxygenase pathway at sites of
inflammation.
Docking study
Conclusion
The orientation of the highly potent and selective COX-2
inhibitors, 1-(4-fluorophenyl)-2-(4-(methylsulfonyl)phenyl)-
4,5,6,7-tetrahydro-1H-benzo[d] imidazole 2b in the COX-2
active site was examined by a docking experiment (Fig. 2).
This molecular modeling showed that compound 2b binds in
the primary binding site such that the C-2 para-SO2Me
substituent inserts into the 2° pocket present in COX-2. One
This study indicates that (i) the 4,5,6,7-tetrahydro-1H-
benzo[d]imidazole moiety is a suitable scaffold (template)
to design COX-1/-2 inhibitors, (ii) COX-1/-2 inhibition is
sensitive to the type of substituent at C-2 phenyl ring,
and (iii) 1-(4-fluorophenyl)-2-(4-(methylsulfonyl)phenyl)-
4,5,6,7-tetrahydro-1H-benzo[d] imidazole (2b) exhibited
good COX-2 inhibitory potency and selectivity.
of the O-atoms of the SO2Me moiety forms a H-bondwith the
´
NH of Arg513 (distance = 2.6 A), whereas the other O-atom
˚
´
is close to the NH of His90 (distance = 3.8 A). The N-3 atom
Experimental
˚
of the central ring is also close to the NH of Arg120 and can
´
˚
form hydrogen bond (distance = 2.9 A) with this amino
Materials
acid. It was interesting to note that, the para-fluoro sub-
stituent of N-1 phenyl ring was forming a hydrogen bond
´
All reagents purchased from the Aldrich (USA) or Merck
(Germany) Chemical Company and were used without
further purifications.
with hydroxyl group (OH) of Tyr385 (distance \ 5 A which
˚
may explain the higher potency of compound 2b compared
Fig. 2 Compound 2b: 1-(4-
fluorophenyl)-2-(4-
(methylsulfonyl)phenyl)-
4,5,6,7-tetrahydro-1H-
benzo[d] imidazole docked in
the active site of murine COX-2
isozyme. Hydrogen atoms of the
amino acid residues have been
removed to improve clarity
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Med Chem Res (2012) 21:1869–1875
General
1873
4-methylsulfonyl phenyl H3 & H5, J = 8.6 Hz); LC–MS
(ESI) m/z: 353 (M ? 1, 100); Anal. Calcd. for
C20H20N2O2S: C, 68.16; H, 5.72; N, 7.95. Found: C, 68.25;
H, 5.92; N, 8.01.
Melting points (mp) were determined using a Thomas
Hoover capillary apparatus (Philadelphia, USA). Infrared
spectra were acquired on a Perkin-Elmer 1420 ratio
recording spectrometer. A Bruker FT-500 MHz instrument
(Brucker Biosciences, Germany) was used to acquire
1HNMR spectra; chloroform-D used as solvent. Coupling
constant (J) values are estimated in hertz (Hz) and spin
multiples are given as s (singlet), d (double), t (triplet), q
(quartet), m (multiplet), and br (broad). The mass spectral
measurements were performed on a 6410Agilent LCMS
triple quadrupole mass spectrometer (LCMS) with an
electrospray ionization (ESI) interface. Elemental analyses
were carried out with a Perkin-Elmer Model 240-C appa-
ratus (Perkin-Elmer, Norwalk, CT, USA). The results of
the elemental analyses (C, H, N) were within 0.4% of the
calculated amounts.
1-(4-Fluorophenyl)-2-(4-(methylsulfonyl)phenyl)-4,5,6,7-
tetrahydro-1H-benzo[d] imidazole (2b)
Yield, 13.5%; white crystalline powder; mp 117–118°C; IR
(KBr disk): t (cm-1) 1150, 1310 (SO2); HNMR (CDCl3,
1
500 MHz): d 1.87–1.95 (m, 4H, CH2), 2.43 (t, 2H, CH2),
2.78 (t, 2H, CH2), 3.06 (s, 3H, SO2CH3), 7.19–7.22 (m, 4H,
4-fluorophenyl H2–H6), 7.58 (d, 2H, 4-methylsulfonyl-
phenyl H2 & H6, J = 8.6 Hz), 7.80 (d, 2H, 4-meth-
ylsulfonylphenyl H3 & H5, J = 8.6 Hz); LC–MS (ESI) m/z:
371.1 (M ? 1, 100); Anal. Calcd. for C20H19FN2O2S: C,
64.85; H, 5.17; N, 7.56. Found: C, 64.99; H, 5.01; N, 7.66.
1-(4-Chlorophenyl)-2-(4-(methylsulfonyl)phenyl)-4,5,6,7-
tetrahydro-1H-benzo[d] imidazole (2c)
General procedure for preparation of 1-(4-substituted-
phenyl)-2-(4-(methyl sulfonyl) phenyl)-4,5,6,7-
tetrahydro-1H-benzo[d] imidazoles (2a–f)
Yield, 14.7%; white crystalline powder; mp 135–136°C; IR
1
(KBr disk): t (cm-1) 1160, 1320 (SO2); HNMR (CDCl3,
2 mmol 4-methylthiobenzaldehyde and 2.4 mmol of ani-
line derivatives were dissolved in 4 ml CHCl3 and 6 ml
glacial acetic acid and placed in microwave reactor with
180 watt power for 5 min. 2 mmol of 1,2-cyclohexanedi-
one and excess amount of ammonium acetate were added
subsequently and placed in microwave reactor for 15 min
with 180 watt power. After cooling, aqueous ammonia
solution was added to obtain neutral pH and then extraction
with CHCl3 was performed. The organic phase was washed
three times with saturated sodium bicarbonate solution,
dried with sodium sulfate, and evaporated in vacuo (Gelens
et al., 2006). The crude product was dissolved in THF and
excess amount of Oxone in THF/water was added and
stirred at room temperature overnight. THF was evaporated
and the residue was extracted with chloroform. The organic
phase was washed three times with saturated sodium
bicarbonate solution and dried with sodium sulfate and
concentrated in vacuo. The oily residue was purified by
plate chromatography using ethyl acetate–hexane (5:1) as
mobile phase (Yields: 12.7–15.5%).
500 MHz): d 1.87–1.93 (m, 4H, CH2), 2.42 (t, 2H, CH2), 2.77
(t, 2H, CH2), 3.06 (s, 3H, SO2CH3), 7.16 (d, 2H, 4-chloro-
phenyl H2 & H6, J = 8.6 Hz), 7.47 (d, 2H, 4-chlorophenyl
H3 & H5, J = 8.6 Hz), 7.57 (d, 2H, 4-methylsulfonylphenyl
H2 & H6, J = 8.6 Hz), 7.81 (d, 2H, 4-methylsulfonylphenyl
H3 & H5, J = 8.6 Hz); LC–MS (ESI) m/z: 387.4 (M ? 1,
100); Anal. Calcd. for C20H19ClN2O2S: C, 62.09; H, 4.95;
N, 7.24. Found: C, 62.25; H, 5.16; N, 7.22.
1-(4-Methylphenyl)-2-(4-(methylsulfonyl)phenyl)-4,5,6,7-
tetrahydro-1H-benzo[d] imidazole (2d)
Yield, 15.5%; white crystalline powder; mp 146–147°C; IR
1
(KBr disk): t (cm-1) 1150, 1310 (SO2); HNMR (CDCl3,
500 MHz): d 1.85–1.93 (m, 4H, CH2), 2.42 (t, 2H, CH2),
2.77 (t, 2H, CH2), 3.05 (s, 3H, SO2CH3), 7.09 (d, 2H,
4-methylphenyl H3 & H5, J = 8.3 Hz), 7.29 (d, 2H,
4-methylphenyl H2 & H6, J = 8.3 Hz), 7.58 (d, 2H,
4-methylsulfonylphenyl H2 & H6, J = 8.6 Hz), 7.78 (d,
2H, 4-methylsulfonylphenyl H3 & H5, J = 8.6 Hz); LC–
MS (ESI) m/z: 367.1 (M ? 1, 100); Anal. Calcd. for
C21H22N2O2S: C, 68.82; H, 6.05; N, 7.64. Found: C, 68.55;
H, 6.12; N, 7.49.
2-(4-(Methylsulfonyl)phenyl)-1-phenyl-4,5,6,7-tetrahydro-
1H-benzo[d]imidazole (2a)
Yield, 13.5%; white crystalline powder; mp 102–103°C; IR
1-(4-Methoxyphenyl)-2-(4-(methylsulfonyl)phenyl)-4,5,6,7-
tetrahydro-1H-benzo [d] imidazole (2e)
1
(KBr disk): t (cm-1) 1150, 1310 (SO2); HNMR (CDCl3,
500 MHz): d 1.87–1.97 (m, 4H, CH2), 2.40–2.43 (t, 2H,
CH2), 2.77–2.79 (t, 2H, CH2), 3.06 (s, 3H, SO2CH3),
7.20–7.22 (m, 5H, phenyl), 7.58 (d, 2H, 4-methylsulfo-
Yield, 13.3%; white crystalline powder; mp 108–109°C; IR
1
(KBr disk): t (cm-1) 1150, 1300 (SO2); HNMR (CDCl3,
nylphenyl H2
&
H6, J = 8.6 Hz), 7.80 (d, 2H,
500 MHz): d 1.86–1.94 (m, 4H, CH2), 2.42 (t, 2H, CH2),
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Med Chem Res (2012) 21:1869–1875
2.83 (t, 2H, CH2), 3.02 (s, 3H, SO2CH3), 3.91 (s, 3H,
minimized structures obtained initially. The quality of
the docked structures was evaluated by measuring the
intermolecular energy of the ligand–enzyme assembly
(Kurumbail et al., 1996).
OCH3), 7.01 (d, 2H, 4-methoxyphenyl H3
& H5,
J = 8.7 Hz), 7.14 (d, 2H, 4-methoxyphenyl H2 & H6,
J = 8.7 Hz), 7.66 (d, 2H, 4-methylsulfonylphenyl H2 &
H6, J = 8.6 Hz), 7.82 (d, 2H, 4-methylsulfonylphenyl H3
& H5, J = 8.6 Hz); LC–MS (ESI) m/z: 383.1 (M ? 1,
100); Anal. Calcd. for C21H22N2O3S: C, 65.94; H, 5.80; N,
7.32. Found: C, 66.15; H, 5.72; N, 7.65.
Acknowledgment This study was financially supported by
Research Deputy of Shahid Beheshti University of Medical sciences
as part of thesis of H. Reihanfard.
1-(4-Acetamidopheny)-2-(4-(methylsulfonyl)phenyl)-
4,5,6,7-tetrahydro-1H-benzo[d] imidazole (2f)
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Yield, 12.7%; white crystalline powder; mp 100–101°C; IR
1
(KBr disk): t (cm-1) 1150, 1320 (SO2); HNMR (CDCl3,
´
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OCH3), 2.41 (t, 2H, CH2), 2.80 (t, 2H, CH2), 3.06 (s, 3H,
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Docking studies were performed using Autodock software
Version 3.0. The coordinates of the X-ray crystal structure
of the selective COX-2 inhibitor SC-558 bound to the
murine COX-2 enzyme was obtained from the RCSB
Protein Data Bank (1cx2) and hydrogens were added. The
ligand molecules were constructed using the Builder
module and were energy minimized for 1,000 iterations
˚
reaching a convergence of 0.01 kcal/mol A. The energy
minimized ligands were superimposed on SC-558 in the
PDB file 1cx2 after which SC-558 was deleted. The pur-
pose of docking is to search for favorable binding config-
uration between the small flexible ligands and the rigid
˚
protein. Protein residues with atoms greater than 7.5 A
from the docking box were removed for efficiency.
Searching is conducted within a specified 3D docking box
using annealing based on the Monte Carlo method and
MMFF94 molecular mechanics force field for 8,000 itera-
tions. These docked structures were very similar to the
Prasit P, Wang Z, Brideau C, Chan CC, Charlson S, Cromlish W,
Ethier D, Evans JF, Ford-Hutchinson AW, Gauthier JY, Gordon
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Riendeau D, Rodger I, Tagari P, Therien M, Vikers P, Wong E,
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