Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Non-steroidal anti-inflammatory drugs (NSAID) are
among the most widely used prescriptions, primarily for the
treatment of pain, bronchial asthma, allergy, and
inflammation[1]. Although modifications of established non-selective
agents, such as the lengthening of the carboxyl side chain of
indomethacin[2] have been strategies for the design of
cyclooxygenase-2 (COX-2) selective
inhibitors[3], the main effort has been addressed to the diarylheterocycle
class[4]. Many lead compounds reported to have selective COX-2
inhibitory activity have been clinically introduced to reduce
inflammation with very little gastrointestinal GI side effects,
namely, celecoxib[5],
rofecoxib[6], valdecoxib[7], lumiracoxib,
etoricoxib[8], and nimesulide. Also, recent studies have
shown that selective COX-2 inhibitors can induce apoptosis
in the colon, stomach, prostate, and breast cancer cell
lines[9_13].
Overall, these selective COX-2 inhibitors have fulfilled
the hope of exhibiting a reduced risk in gastrointestinal
events[14]; however, the increased incidence of
non-gastro-intestinal, serious, adverse events with the COX-2 selective
inhibitors as compared with non-selective NSAID in the
Celecoxib Long-term Arthritis Safety Study and the Vioxx
Gastrointestinal Outcomes Research study, remains a major
concern[15]. With all these aspects considered, developing
drugs that preferentially inhibit COX-2 with moderate
potency and selectivity was of interest, since the currently used,
very selective COX-2 inhibitors cause unwanted side effects
in a significant amount of people[16].
Flumizole, an early known 4,5-diarylimidazole (Figure 1),
and 4,5-diaryl-2-substituted thioimidazole, have been
reported to exhibit anti-inflammatory
activity[14,17]. As a part of our ongoing research to design novel selective COX-2
inhibitors[18_23], we describe herein, the design and
biological evaluation of
4-[2-alkylthio-5(4)-(4-substitutedphenyl)imidazole-4(5)-yl]benzenesulfonamides as COX-2 inhibitors with
anti-inflammatory activities.
Materials and methods
Animals Male Sprague Dawley rats, weighing 150_200 g
(n=6), were supplied by Razi Institute, Tehran,
Iran. All the animals were housed in Plexiglas cages on a 12/12 h
light/dark cycle in temperature- and humidity-controlled
rooms. Food was withheld 24 h before the experiments, but with free
access to water. All the experiments confirmed to the
guidelines of the committee on animal experiments at Tehran
University of Medical Sciences (Tehran, Iran)
Chemicals and reagents All of the chemicals and
reagents were purchased from Merck (KGaA, Darmstadt,
Germany) and Sigma-Aldrich (St Louis, MO, USA).
Molecular modeling and chemistry Docking studies
were performed using the Autodock 3.05 Package (Scripps
Research Institute, La Jolla,
California)[24_26]. The coordinates of the X-ray crystal structure of selective COX-2
inhibitor S-58701 (B) bound to the murine COX-2 enzyme
was obtained from the Protein Data Bank (www.rcsb.org)
code 1CX2 and hydrogens were added. The ligand
molecules were constructed using Chem-3D (CambridgeSoft,
Cambridge, MA) and were minimized for 500 iterations,
reaching a convergence of 0.01 kcal/mol Å.
The compound 4 was docked using Lamarckian genetic algorithm (LGA), where
the number of GA=10, the population size=50, and the
maximum number of energy evaluations is 250000.
The result was analyzed using root mean square deviation (RMSD),
estimated inhibition constant
(Ki), and estimated free energy of binding
(ΔG). The best resulting ΔG=-11.5 kcal/mol was
for compound 4c with RMSD 2.115 Å. The
Ki was 3.72×10-9.
The 4,5-diarylimidazole-2-thiones (compound 2), with
substituents at the para position of one of the phenyl rings,
was prepared in high yield (80%_90%) using ammonium
thiocyanate and 2-oxo-1,2-diphenylethyl benzoates (compound
1) in amyl alcohol at 150_160 ºC. Subsequent alkylation of
compound 2 with alkyl iodide in methanol in the presence of
triethylamine afforded 2-alkylthio-4,5-diarylimidazoles
(compound 3, 24%_81%). The sequential chlorosulfonation
of compound 3 with chlorosulfonic acid, followed by
ammonia gave 4-[2-alkylthio-5(4)-(4-substitutedphenyl)imidazole-
4(5)yl]benzenesulfonamides (compound 4). The structure
of compound 4 was confirmed by infrared, by proton nuclear
magnetic resonance and Mass
spectrometry[23].
Biological assays
In vitro COX inhibition assay COX activity was
determined by using arachidonic acid (AA) as substrate and
N,N,N,N-tetramethylphenylenediamine (TMPD) as the
cosub-strate , as previously
described[18,19,27]. The reaction mixture
(200 µL) contained 0.5 µmol/L heme, 0.05 mmol/L TMPD,
0.1 mmol/LAA, and 36 units of the COX-2 enzyme (57 units
for COX-1) in 0.1 mol/L Tris/HCl (pH 8.1).
The oxidation of the substrate, the starter of the reaction, was measured at
25 ºC by monitoring the increase of absorbance at 630
nm. The inhibition of the studied compound
4[23] was determined after pre-incubation for 5 min with the enzyme in the
presence of heme, and the reaction was started by adding AA
and TMPD. This mixture was incubated for another 5 min
and the absorbance was measured on a strip
reader. For synthesized compound 4, 10 µL of scalar dilutions of the
inhibitors in DMSO was added. Celecoxib, a potent and
selective COX-2 inhibitor, was used as a reference
drug. The average absorbance of all of the samples was
determined. The absorbance of the test wells was normalized with
background and calculated as the percentage of total activity: %
test inhibition=100 (1-test abs/total activity abs) where test
abs=absorbance in the test well and total activity abs=
absorbance in the well without any inhibitor.
The percentage of inhibition was used to calculate the inhibition
concentration IC50 of the compound (concen-tration at which
there was 50% inhibition).
In vivo methods The method of carrageenan-induced
paw edema in rats[28] was used to evaluate the
anti-inflammatory activity. The treatment was performed 30 min before the
injection of 50 µL carrageenan 1% into the rat paw plantar
surface. The foot volume was measured using a
plethysmo-meter[29] at 1 h intervals after the carrageenan injection for
3 h, but the activity was acknowledged only for the third
hour, in which maximum edema occurred. The inflammation
index was calculated as the difference between the final
volume of the carrageenan injected paw
(Vt) and the initial volume of the same paw before injection
(Vo), that is, inflammation index
(Ii)=Vt_V
o. The edema inhibition (%) was
calculated as the percentage of the difference of
Ii according to the following formula: % inhibition=([pre-drug
Ii]-[post-drug
Ii]/[pre-drug
Ii])×100.
In order to evaluate the anti-inflammatory effect of
compound 4a_4f, 3 doses were used. The 50% inhibition of the
compound (by definition, the dose required to reduce the
carrageenan-induced paw edema to 50% of the control
group) was calculated. The compound was injected
intraperitoneally (ip) using the following doses: 5.1, 7.6, and 11.4
mg/kg for celecoxib (reference drug); 4.7, 7.2, and 10.7 mg/kg
for compound 4a; 4.5, 6.4, and 10.3 mg/kg for compound 4b;
5.0, 7.5, and 11.3 mg/kg for compound 4c; 4.8, 7.2, and 10.9
mg/kg for compound 4d; 5.2, 7.8, and 11.8 mg/kg for
compound 4e; and 5.1, 7.6, and 11.4 mg/kg for compound
4f. The rats of the control group received the same volume of DMSO
according to their weight.
Statistical analysis The data were expressed as mean±
SEM. One-way ANOVA with Tukey's
post-hoc test was used, and P<0.05 was considered statistically
significant. The IC50 was calculated using the non-linear regression with
cubic spline method.
Results
Molecular modeling and chemistry The docking
study showed that compound 4 bound to the primary
binding site of COX-2 with the sulfonamide
SO2NH2 moiety interacting with the secondary pocket amino acid residues
Phe518, His90, and
Val523, which is comparable to S-58701 (B) (Figure
2). One of the O-atoms of the
SO2NH2 substituent forms a hydrogen bond with the amide hydrogen of
Phe518 (2.5 Å). The N-atom of the
SO2NH2 forms a hydrogen bond with
His90 (2.5 Å). The substituted phenyl ring
lies in a hydrophobic cavity lined by
Val349. The ethyl sulfide (EtS) substituent is oriented in the direction of the
polar amino acid Arg120, and
Tyr355 and forms a weak hydrogen bond with them (4
Å). It is located in a hydrophobic region formed by
Val116 and Leu531. Also, the amino (NH) of
imidazole forms another hydrogen bond with
Tyr355 (3 Å) (Figure 2). Considering the molecular modeling information,
the synthetic reaction used for the synthesis of
4-[2-alkylthio-5(4)-(4-substitutedphenyl)imidazole-4(5)-yl]benzenesulfo-
namides (4a_4j) are outlined in Figure
3[23].
In vitro assay The ability of compound 4a_4j to inhibit
ovine COX-1 and COX-2 (IC50 values, nmol/L) was
determined using a colorimetric COX (ovine) inhibitor screening
assay. In this regard, compound 4a_4j exhibited a broad
range of COX-2 inhibitory potency (Table 1).
In vivo evaluation The potent and selective COX-2
inhibitors emerging from the in vitro studies were evaluated in
the acute carrageenan-induced rat paw edema.
Pretreatment with celecoxib (0.02 mmol/kg) intraperitoneally resulted in a
marked decrease in paw inflammation when compared to
control group (P<0.001). Repeated experiments with the same
dose of compound 4a_4e also showed significant differences
(P<0.001) in anti-inflammatory effects on carrageenan hind
paw edema (Figure 4). Compound 4a_4f (0.02 mmol/kg)
induced protection against carrageenan-induced paw edema
(Figure 5). The 50% inhibition (by definition, the dose
required to reduce the carrageenan-induced paw edema to
50% of that of the control) for compound 4a_4f ranged from
1.58_4.3 mg/kg, while the 50% inhibition for the reference
drug celecoxib was 2.90 mg/kg (Table 2).
Discussion
In this diarylheterocyclic class of COX-2 inhibitors, the
initial modification was the insertion of sulfonamide at the
para position of one of the phenyl ring and was held
constant throughout of the structure-activity relationship (SAR)
studies. Within the sulfonamide analogues (compound 4),
modification at the alkylthio group at the C-2 position of the
imidazole ring gave variable results. In the presence of smaller
C-4 substituents (H, F; 4a_4d), increasing the size of alkylthio
did not significantly affect the COX-2 inhibitory
potency. However, in the presence of C-4 phenyl chloro substituent
(4e, 4f), the size of C-2 alkylthio had an effect on COX-2
inhibition and increasing the size led to an increase in
COX-2 potency.
There was, however, some sensitivity to the electronic
property at the 4-position of this aromatic ring, particularly
with regards to COX-2 potency. In the EtS-substituted
compound, 4a, 4c, and 4e, the analogs with an
electron-withdrawing group (4c, 4e), tended to increase COX-2
potency. In contrast, electron-donating group had poor COX-2
activity. Therefore, methyl (4g, 4h) and methoxy (4i, 4j)
substituents all worked poorly in this regard.
These results suggested that the electronic property of the substituents at
the para position of the phenyl ring can influence the
COX-2 inhibitory activity.
In the in vivo studies, the fluorine and hydrogen analogs
(4a_4d) showed a good inhibition on edema (Table
2). The other derivative, compound 4e and 4f, despite good COX-2
potency, was moderately active in vivo. In general,
in vivo data prove our SAR of compound
4a_4j. Its potency is greatly influenced by the substitution pattern and shows that para
fluorine or hydrogen besides the
SO2NH2 pharmacophore represents a series of anti-inflammatory agents, which
preferentially inhibit COX-2 with moderate potency and
selectivity.
The potent in vitro and in vivo COX-2 inhibition
exhibited by compound 4c is consistent with observations from a
molecular modeling experiment where compound 4c was
docked in the active site of the COX-2 enzyme.
Molecular modeling studies show that the critical difference between
the binding sites for COX-1 and COX-2 is at position 523
where COX-2 has the amino acid residue Val in place of the
bulkier Ile in COX-1. This difference produces a secondary
pocket extending off the primary binding site in COX-2 that
is absent in COX-1. Consequently, the combined volume of
the primary binding site and the secondary pocket in COX-2
is about 25% larger than the volume of the COX-1 binding
site[30,31]. This difference in volume can be exploited to
manipulate COX-2 selectivity of the diarylheterocyclic class of
COX-2 inhibitors by varying the volume of the drug and the
appropriate placement of substituents with varying electronic
and steric properties[32]. Designed compound 4c binds in
the center of the active site with the phenylsulfonamide
moiety oriented toward the secondary pocket region where
it can undergo H-bonding via one of its
SO2 oxygen atoms and NH2 group of the sulfonamide moiety with
Phe518 and His90. Interestingly, the C-2 EtS substituent is located in a
hydrophobic region, with the S-atom forming a weak
hydrogen bond with the Tyr355 and
Arg120. This shows the importance of the C-2 substituent in orienting the molecule such
that the sulfonamide moiety inserts into the secondary pocket
of COX-2. The ring N-atom of the central imidazole is
oriented in the direction of the polar amino acid
Tyr355, where this N-atom is about 3 Å away from the
NH2 of Tyr355. This interaction may disrupt the salt bridge between
His90, Arg120, and
Tyr355 at the mouth of the COX-2 active site (Figure
2). The similarity between the
Ki (S-8701 B) as the reference drug and the
Ki which was calculated for compound 4c, shows
that changing the structure from S-8701 B to compound 4c
does not decrease the binding for the COX-2
enzyme. How-ever, introducing the imidazole ring, ethyl sulfide SEt
substituent, and SO2NH2 pharmacophore improves the
binding, which is a result of the hydrogen
bonds. These observations confirm the suggested SAR for COX-2
inhibitory activity.
Our observations provide a good explanation for these
results: (i) compound 4 with good COX-2 inhibitory
potency and selectivity can be designed by the appropriate
placement of the para
P-SO2NH2 pharmacophore on the C-4
phenyl ring, in which the 2-alkylthio imidazole ring serves as
a suitable central ring template; and (ii) COX-2 inhibitory
potency and selectivity is sensitive to substituent electronic
property at the para position of the phenyl ring where
compound 4b exhibits the best combination of potency and
selectivity and compound 4c exhibits better potency on
COX-2, but lower selectivity compared to compound 4b.
References
1 Hansch C, Sammes PG, Taylor JB. The rational design,
mechanistic study and therapeutic application of chemical
compounds. Comprehensive medicinal chemistry; v 6. Oxford: Pergamon
Press; 1990.
2 Leblanc Y, Black WC, Chan CC, Charleson S, Delorme D, Denis
D, et al. Synthesis and biological evaluation of both enantiomers
of L-761,000 as inhibitors of cyclooxygenase. Bioorg Med Chem
Lett 1996; 6: 731_6.
3 Kalgutkar AS. Selective cyclooxygenase-2 inhibitors as
non-ulcerogenic anti-inflammatory agents. Exp Opin Ther Pat 1999;
9: 831_49.
4 Reitz DB, Isakson PC. Cyclooxygenase-2
inhibitors. Curr Pharm Des 1995; 1: 211_20.
5 Penning T, Talley J, Bertenshaw S, Carter J, Collins P, Docter S,
et al. Synthesis and biological evaluation of 1,5-diarylpyrazole
class of cyclooxygenase-2 inhibitors: Identification of
4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H
-pyrazole-1-yl] benzene-sulfonamide (SC-58635,
Celecoxib). J Med Chem 1997; 40: 1347_65.
6 Prasit P, Wang Z, Brideau C, Chan CC, Charleson S, Cromlish W,
et al. The discovery of rofecoxib, [MK 966, Vioxx (R),
4-(4'-methylsulfonylphenyl)-3-phenyl-2(5H)-furanone], an orally
active cyclooxygenase-2 inhibitor. Bioorg Med Chem Lett 1999;
9: 1773_8.
7 Talley JJ, Brown DL, Carter JS, Graneto MJ, Koboldt CM,
Masferrer JL, et al.
4-[5-methyl-3-phenylisoxazol-4-yl]-ben-zenesulfonamide, valdecoxib: a potent and selective inhibitor of
COX-2. J Med Chem 2000; 43: 775_7.
8 Riendeau D, Percival MD, Brideau C, Charleson S, Dube D, Ethier
D, et al. Preclinical profile and comparison with other agents
that selectively inhibit cyclooxygenase-2.
J Pharmacol Exp Ther 2001; 296: 558_66.
9 Arico S, Pattingre S, Baury C, Gane P, Barbat A, Codogno P,
et al. Celecoxib induces apoptosis by inhibiting 3-phosphoinositide
dependent protein-kinase-1 activity in the human colon
cancer. J Biol Chem 2002; 277: 27 613_21.
10 Davies G, Martin LA, Sacks N, Dowsett
M. Cyclooxygenase-2 (COX-2), aromatase and breast cancer: a possible role for
COX-2 inhibitors in breast cancer
chemoprevention. Ann Oncol 2002; 13: 669_78.
12 Liu HX, Kirschenbaum A, Yao S, Lee R, Holland FJ, Levine
CAJ. Inhibition of cyclooxygenase-2 suppresses angiogenesis and the
growth of prostate cancer in vivo. J Urol 2000; 164: 820_5.
13 Sawaoka H, Kawano S, Tsuji S, Tsuji M, Gunawan ES, Takei Y,
et al. Cyclooxygenase-2 inhibitors suppress the growth of gastric
cancer xenografts via induction of apoptosis in nude
mice. Am J Physiol 1998; 274: G1061_7.
14 Khanna IK, Weier RM, Yu Y, Xu XD, Koszyk FJ, Collins PW,
et al. 1,2-Diarylimidazoles as potent, cycloxygenase-2 selective,
and orally active anti-inflammatory agents.
J Med Chem 1997; 40: 1634_47.
15 Wright JM. The double-edged sword of COX-2 selective
NSAIDs. CMAJ 2002; 167: 1131_7.
16 Kontogiorgis CA, Hadjipavlou-Litina
DJ. Synthesis and anti-inflammatory activity of coumarin
derivatives. J Med Chem 2005; 48: 6400_8.
17 Niedballa U, Bottcher I. Antiinflammatory
4,5-diphenyl-2-substituted-thio-imidazoles and their corresponding sulfoxides and
sulfones. US patent 4 440 776. 1984 Apr 03.
18 Navidpour L, Shafaroodi H, Abdi KH, Amini M, Ghahremani
MH, Dehpour AR, et al. Design, synthesis, and biological
evaluation of substituted 3-alkylthio-4,5-diaryl-4H-1,2,4-triazoles as
selective COX-2 inhibitors. Biorg Med Chem 2006; 14:
2507_17.
19 Navidpour L, Amini M, Shafaroodi H, Abdi KH, Ghahremani
MH, Dehpour AR, et al. Design and synthesis of new water-
soluble tetrazolide derivatives of celecoxib and rofecoxib as
selective cyclooxygenase-2 (COX-2) inhibitors.
Biorg Med Chem Lett 2006; 16: 4483_7.
20 Navidpour L, Karimi L, Amini M, Vosooghi M, Shafiee
A. Syntheses of
5-alkylthio-1,3-diaryl-1,2,4-triazoles. J Heterocyclic
Chem 2004; 41: 201_4.
21 Karimi L, Navidpour L, Amini M, Shafiee
A. Synthesis of
4,5-Diaryl-1,2,3-thiadiazoles. Phosphorus Sulfur and Silicon and the
Related Elements 2005; 180: 1593_600.
22 Johari Daha F, Matloubi H, Tabatabai SA, Shafiee B, Shafiee
A. Synthesis of 1-(4-methylsulfonylphenyl)-5-aryl-1,2,3-triazoles
and 1-(4-aminosulfonylphenyl)-5-aryl-1,2,3-triazoles
. J Heterocyclic Chem 2005; 42: 33_7.
23 Salimi M, Amini M, Shafiee A. Syntheses of
2-alkylthio-(4,5-diaryl) imidazoles. Phosphorus Sulfur Silicon 2005; 180:
1587_92.
24 Goodsell DS, Olson AJ. Automated docking of substrates to
proteins by simulate annealing. Proteins: structure function and
genetics 1990; 8: 195_202.
25 Morris GM, Goodsell DS, Huey R, Olson
AJ. Distributed automated docking of flexible ligands to proteins: Parallel
applications of autodock 2.4. J Computre-aided Mol Design 1996; 10:
293_304.
26 Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew
RK, et al. Automated docking using Lamarckian genetic
algorithm and an empirical binding free energy
function. J Comp Chem 1998; 19: 1639_62.
27 Kulmacz RJ, Lands WEM. Requirements for hydroperoxide by
the cyclooxygenase and peroxidase activities of prostaglandin H
synthase. Prostaglandins 1983; 25: 531_40.
28 Winter CA, Rislet EA, Nuss GW. Carrageenan-induced edema in
hind paw of the rat as an assay for anti-inflammatory
drugs. Proc Soc Exp Biol Med 1962; 111: 544_7.
29 Ahmadiani A, Fereidoni M, Semnanian S, Kamalinejad M, Saremi
S. Antinociceptive and anti-inflammatory effects of sambucus
ebulus rhysome extracts in rats. J Ethnopharmacol 1998; 61:
229_35.
30 Luong F, Miller A, Barnett J, Chow J, Ramesha C, Browner
MF. Flexibility of the NSAIDs binding site in the structure of human
cyclooxygenase-2. Nat Struct Biol 1996; 3: 927_33.
31 Gierse JK, McDonald JJ, Hauser SD, Rangwala SH, Koboldt CM,
Seibert K. A single amino acid difference between
cyclooxygenase-1 (COX-1) and 2- (COX-2) reverse the selectivity of COX-2
specific inhibitors. J Biol Chem 1996; 271: 15810_4.
32 Kurumbail RG, Stevens AM, Gierse JK, McDonald JJ, Stegman
RA, Pak JY, et al. Structural basis for selective inhibition of
cyclooxygenase-2 by anti-inflammatory agents.
Nature 1996; 384: 644_8.
|
