Pyridine analogues of nimesulide: Design, synthesis, and in vitro and in vivo pharmacological evaluation as promising cyclooxygenase 1 and 2 inhibitors

Article abstract of DOI:10.1021/jm900702b

Nonsteroidal anti-inflammatory drugs (NSAIDs) represent one of the most prescribed medications, although the chronic use of such pharmacological agents is commonly associated with numerous side effects. The demonstration that the use of COX-2 selective or

Full text of DOI:10.1021/jm900702b

5864 J. Med. Chem. 2009, 52, 5864–5871
DOI: 10.1021/jm900702b
Pyridine Analogues of Nimesulide: Design, Synthesis, and in Vitro and in Vivo Pharmacological
Evaluation as Promising Cyclooxygenase 1 and 2 Inhibitors
Jean-Franc-ois Renard,*,† Deniz Arslan,† Nancy Garbacki,‡ Bernard Pirotte,†,§ and Xavier de Leval†,§
†
ꢀ
Drug Research Center (CIRM), Laboratory of Medicinal Chemistry, University of Liege, Avenue de l’Hopital 1, B-4000 Liege, Belgium, and
Laboratory of Connective Tissues Biology, GIGA-R, University of Liege, Avenue de l’Hopital 3, B-4000 Liege, Belgium. The two last authors
^
ꢀ
§
‡
ꢀ
^
ꢀ
assumed equal supervision.
Received May 25, 2009
Nonsteroidal anti-inflammatory drugs (NSAIDs) represent one of the most prescribed medications,
although the chronic use of such pharmacological agents is commonly associated with numerous side
effects. The demonstration that the use of COX-2 selective or preferential inhibitors is associated with a
better tolerability opened new horizons in the search of safer drugs for the management of inflamma-
tion. In the present study, we report the synthesis and the pharmacological evaluation of pyridine
analogues of nimesulide, a COX-2 preferential inhibitor. The cyclooxygenases (COXs) inhibitory
activities were evaluated in vitro using a human whole blood model. According to the in vitro results, a
selection of compounds exhibiting moderate to high COX-2/COX-1 selectivity ratio (from weak COX-2
preferential inhibitors to compounds displaying a celecoxib-like selectivity profile) were further
evaluated in vivo in a model of λ carrageenan-induced pleurisy in rats. Some of the selected com-
pounds displayed similar or improved anti-inflammatory properties when compared to nimesulide and
celecoxib.
Introduction
some COXs inhibitors displayed interesting preclinical activ-
ity,8 they failed to demonstrate convincing therapeutic effect
in clinical practice.9,10
Inflammation can be summarized as a complex process
involving the release of several biochemical factors called
inflammation mediators. These mediators can act both di-
rectly or synergistically in order to promote or maintain the
inflammatory process. Among these mediators, prostanoids,
lipidic mediators deriving from fatty acid metabolism, have
been identified as key influencing mediators. Indeed, single
inhibition of cyclooxygenases, the enzymes responsible for
prostanoids biosynthesis, has been demonstrated for a long
time to be a sufficient and an efficient strategy to reduce
inflammation.1 Marketed COXsa inhibitors, whatever their
chemical structures, are grouped together under the denomi-
nation of NSAIDs. They are widely used in the management
of pain and acute inflammation but also in chronic inflamma-
tion states such as, for example, osteoarthritis andrheumatoid
arthritis. Besides these therapeutic indications, the use of
NSAIDs could be of interest in the prevention of several
cancer types such as colon, lung, breast, prostate, and eso-
phagus cancers.2-7 Cyclooxygenases also seem to be involved
in the development of neurodegenerative diseases, especially
Alzheimer’s and Parkinson’s diseases. However, although
Cyclooxygenases exist under two isoforms called COX-1
and COX-2. COX-1, the constitutive form of the enzyme, is
present in the stomach, intestines, kidneys, and platelets. This
form is mainly responsible for the physiological production
of prostanoids. COX-2, although it is also constitutively
expressed in brain, spinal cord,11 and kidneys, is an inducible
form and its expression is triggered under pathological con-
ditions such as inflammation. Most of the first-generation
anti-inflammatory drugs inhibit both cyclooxygenase iso-
forms. This broad inhibition profile induces a decrease of
inflammation but also downregulates the basal or physiolo-
gical prostaglandin production in all tissues. This lack of
selectivity represents the theoretical basis of their well-known
gastric12,13 and renal toxicity.14 In this context, the use of
COX-2 specific inhibitors appears as an attractive approach
to manage inflammation in a safer way.
This promising concept of COX-2 selective inhibition led to
the development of a large number of new molecules. Among
these, the well-known ones are rofecoxib (1),15 celecoxib (2),16
etoricoxib (3),17 and valdecoxib (4)18 (Figure 1). Clinical trials
and clinical use of these agents confirmed that COX-2 specific
inhibitors display anti-inflammatory, antipyretic, and analge-
sic properties with fewer gastrointestinal side effects.19 Never-
theless, an increased risk of cardiovascular events associated
with the use of rofecoxib was demonstrated in two indepen-
dent clinical trials: the Vioxx Gastrointestinal Outcomes
Research (VIGOR)20 and the Adenomatous Polyps Preven-
tion on Vioxx (APPROVe)21 trials. This finding led to the
withdrawal of this compound from the market and reached
*To whom correspondence should be addressed. Phone: þ32-4-
3664381. Fax: þ32-4-3664362. E-mail: jf.renard@ulg.ac.be. University
ꢀ
^
of Liege Laboratory of Medicinal Chemistry 1, Avenue de l’Hopital,
ꢀ
tour 4(þ5), B-4000 Liege, Belgium.
aAbbreviations: NSAID, nonsteroidal anti-inflammatory drug;
COX, cyclooxygenase; PGI2, prostacyclin; TXA2, thromboxane A2;
CAI, carbonic anhydrase inhibitor; hCA, human carbonic anhydrase;
DMSO, dimethylsulfoxide; RPMI, Roswell Park Memorial Institute
culture medium; LPS, lipopolysaccharide; TXB2, thromboxane B2;
PGE2, prostaglandin E2.
r
pubs.acs.org/jmc
Published on Web 09/03/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5865
Figure 1. Chemical structures of COX-2 inhibitors.
Table 1. IC50 COX-1 and IC50 COX-2 of Previous Pyridinic Analogues
of Nimesulide and the Selectivity Ratio (IC50 COX-1/IC50 COX-2)
the question of a rofecoxib-related toxicity or a pharmaco-
logical class associated cardiovascular toxicity.
The reasons for this toxicity are currently not fully under-
stood, but different hypothesis were investigated.22 Among
these, some only concern compound 1 and are rather related
to its particular chemical structure while others concern the
COX-2 selective inhibition. First, a drastic and selective
inhibition of COX-2 is associated with a decrease of prosta-
cyclin (PGI2) levels, an antiaggregant and vasodilator agent
mainly synthesized through the COX-2 catalytic activity with-
out suppressing the thromboxane A2 (TXA2) production,
a strong pro-aggregant and vasoconstrictor mediator mainly
produced through the COX-1 pathway. The modification of
the physiological balance between TXA2/PGI2 may generate
an alteration of the vascular homeostasis.23,24 Regarding
the compound-related toxicity hypothesis, 1 does not bear
a sulfonamidemoietyas do compounds 2 and 4, which possess
an unsubstituted arenesulfonamide group and for which the
clinical use is not associated with an increased incidence of
cardiovascular events. Moreover, this sulfonamide moiety is
a common pattern to many carbonic anhydrase inhibitors
(CAIs). Compounds 2 and 4 have been shown to present
a high carbonic anhydrase inhibitory activity25,26 that was
similar to that of acetazolamide, a human carbonic anhydrase
(hCA) inhibitor marketed for its diuretic and antihypertensive
properties. Crystallographic studies have also confirmed the
ability of 2 to bind hCA-II. Finally, sulfone COX-2 inhibitors
such as 1 and 3 increase the susceptibilityof biological lipids to
oxidative modification through a nonenzymatic process. This
pro-oxidant activity could contribute to the progression of
atherogenesis and could, at least in part, explain the increase
of cardiovascular risk observed with 1.27-29
As a consequence, COX-2 selective inhibitors, despite their
good tolerability and therapeutic activities, may contribute or
promote a cardiovascular toxicity. Our group has been in-
volved for several years in the development of nimesulide30 (5)
derivatives, a NSAID exhibiting a COX-2 preferential pro-
file.31 Of particular interest is the excellent tolerability profile
of this drug with a lack of demonstrated cardiovascular
toxicity, therefore suggesting that a “sufficient” COX-2/
COX-1 selectivity ratio may lead to safe anti-inflammatory
agents. In a previous work, we synthesized a nimesulide
analogue for which the nitrobenzenic ring was replaced by
a pyridinic one (6; Table 1).32 This compound displayed a
better COX-1 inhibitory activity than that of the parent
compound but was almost inactive against COX-2. Several
IC50 (μM)
compd
R
X
COX-1
COX-2
ratio
6
7
8
9
CH3
CF3
CH3
CF3
O
0.41 ( 0.13
0.14 ( 0.01
3.62 ( 0.90
0.18 ( 0.06
>100
O
0.62 ( 0.18
1.19 ( 0.58
0.09 ( 0.03
0.22
3.05
2.00
S
NH
pharmacomodulations were investigated in order to increase
the COX-2 inhibitory properties and to recover a COX-2
preferential profile. The acidic character of the alkanesulfo-
namide moiety was increased by introducing a trifluoro-
methanesulfonamide group, the phenyl ring was substituted
with various halogen atoms or groups, and finally the replace-
ment of the ether linkage by a thioether, a sulfone, and a
secondaryortertiaryamine bridgewasevaluated(compounds
7-9; Table 1). From all of these modifications, N-(3-pheny-
lamino-4-pyridinyl)trifluoromethanesulfonamide (FJ29),33
bearing a trifluoromethanesulfonamide moiety, a nonsubsti-
tuted phenyl ring, and a secondary amine linkage, appeared as
a promising approach. Indeed, 9 (FJ29) presented a strong
inhibitory activity on both cyclooxygenases with a COX-2/
COX-1 selectivity ratio equal to 2 (COX-1 and COX-2 IC50
values for compound 9: 0.18 and 0.09 μM, respectively).33
In the present study, we aimed at increasing the COX-2
preferential profile of compound 9 while retaining its COXs
inhibitory activity. For this purpose, we have substituted the
phenyl ring with various halogen atoms and some alkyl or
alkoxy chains. The benzenic ring was also replaced with a
variety of cycloalkyl rings. Finally, we have kept the possibi-
lity to modulate the acidic character of the sulfonamide
moiety by introducing methane- and trifluoromethanesul-
fonamide moieties. The influence of the length of this group
was also evaluated by increasing the size of the alkyl chain.
Results and Discussion
Chemistry. The synthetic pathway (Scheme 1) used to
prepare the new compounds consists of a five-step process

5866 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Renard et al.
Scheme 1a
a (i) H2O2, CH3COOH; (ii) HNO3, H2SO4; (iii a) NH2-C6H5-R1; (iii b) NH2-CH(CH2)n (n = 4, 5, 6); (iv) Fe, CH3COOH/H2O; (v) R2-SO2Cl,
K2CO3, CH3CN.
starting from 3-bromopyridine (10). The synthesis of the
common intermediate, 3-bromo-4-nitropyridine N-oxide
(12), was achieved in two steps.34 First, 10 was oxidized by
a mixture of acetic acid and hydrogen peroxide in order to
generate in situ peracetic acid. This oxidation was followed
by a nitration of 3-bromopyridine N-oxide (11) at the
4-position. This step was conducted in a nitric and sulfuric
acid medium at 90 °C for 5 h. Compound 12 was obtained as
a yellow solid. The next step consisted in the formation of the
amine linkage by substitution of the bromine atom of 12 with
the appropriate amine (13a-o, 18-20). The fourth step was
the reduction of the nitro and the N-oxide moieties, which
were simultaneously reduced using iron in an acetic acid and
water medium. The corresponding aminopyridines (14a-o,
21-23) were obtained as oily compounds and were used
without further purification. The final compounds were
obtained by reaction between the aminopyridine derivatives
and the suitable sulfonyl chloride.
The inhibitory activity of the synthesized compounds
against COX-1 and COX-2 was evaluated in vitro using a
human whole blood model.35 During these experiments, the
selectivity ratio toward COX-2 was calculated using the
formula [IC50 COX-1 (μM)/IC50 COX-2 (μM)]. These in
vitro studies enabled us to select compounds for further
determination of their anti-inflammatory profile in a
λ carrageenan-induced pleurisy model in rats.36
the cyclooxygenases inhibitory activity was first evaluated.
The results obtained with the newly synthesized compound
bearing a trifluoromethanesulfonamide moiety are pre-
sented in Table 2. Compared to the unsubstituted compound
with a trifluoromethanesulfonamide moiety (9), the substi-
tution of the phenyl ring with a chlorine atom (compound
15a-c) led to compounds displaying drastic COXs inhibi-
tory properties, although slightly weaker when compared to
compound 9. Of particular interest was the finding that this
weaker inhibitory profile mainly concerns the COX-1 acti-
vity when the chlorination is located at the 3- and the
4-position. As a consequence, compounds 15b and 15c ex-
hibited an enhanced selectivity ratio when compared to
compound 9 (COX-2 selectivity ratio of 4.10 and 2.81,
respectively).
The increase of steric hindrance and lipophilicity was
further evaluated by replacing the chlorine atom by a
bromine one (15d and 15e). Substitution of the phenyl ring
with a bromine atom at the 3- and 4-position was investi-
gated while 2-position substitution was, due to excessive
steric hindrance, not achieved using the described synthesis
pathway. Among these two compounds, compound 15d
displayed strong COX-2 inhibitory properties (COX-2 IC50
value for 15d: 0.12 μM) and a COX-2 selectivity similar to
that observed with compound 2 (COX-2 selectivity ratio:
7.48 and 7.46 for compound 15d and 2, respectively).
The substitution of the phenyl ring with short alkyl chains
was also explored. In this context, the hydrogen atom at the
In Vitro COXs Inhibition Studies. The influence of the
nature and the position of substitution on the phenyl ring on

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5867
Table 2. IC50 COX-1 and IC50 COX-2 of Compounds 2, 5, 9, and
15a-n, and the Selectivity Ratio (IC50 COX-1/IC50 COX-2)
Table 3. IC50 COX-1 and IC50 COX-2 of Compounds 2, 5, and 16a-o
and the Selectivity Ratio (IC50 COX-1/IC50 COX-2)
IC50 (μM)
IC50 (μM)
compd
R1
COX-1
COX-2
ratio
compd
R1
COX-1
COX-2
ratio
5
3.76 ( 1.02
2.60 ( 0.20
0.18 ( 0.06
0.19 ( 0.09
1.09 ( 0.16
1.15 ( 0.48
0.91 ( 0.05
1.60 ( 0.15
0.48 ( 0.36
0.99 ( 0.10
0.17 ( 0.05
3.25 ( 0.71
0.69 ( 0.12
1.22 ( 0.29
1.11 ( 0.51
1.00 ( 0.27
0.10 ( 0.01
0.70 ( 0.23
0.35 ( 0.09
0.09 ( 0.03
2.11 ( 1.22
0.27 ( 0.11
0.41 ( 0.27
0.12 ( 0.14
0.91 ( 0.14
2.69 ( 1.16
0.45 ( 0.20
0.46 ( 0.27
7.24 ( 4.53
0.24 ( 0.13
0.91 ( 0.02
2.53 ( 2.05
1.58 ( 0.85
0.16 ( 0.04
5.37
7.46
2.00
0.09
4.10
2.81
7.48
1.77
0.18
2.19
0.38
0.44
2.84
1.35
0.44
0.63
0.60
5
3.76 ( 1.02
2.60 ( 0.20
2.26 ( 0.91
0.48 ( 0.13
7.15 ( 3.34
1.31 ( 0.27
20.02 ( 2.22
2.12 ( 0.59
0.56 ( 0.10
2.57 ( 0.55
1.31 ( 0.84
3.79 ( 0.28
60.39 ( 5.17
2.73 ( 0.92
9.76 ( 0.22
25.24 ( 2.47
0.56 ( 0.08
0.70 ( 0.23
0.35 ( 0.09
2.69 ( 0.62
9.38 ( 6.48
0.73 ( 0.04
5.93 ( 2.23
2.56 ( 0.69
21.9 ( 12.7
18.6 ( 12.43
8.81 ( 0.48
12.03 ( 3.36
>100
5.37
7.46
0.85
0.05
9.83
0.22
7.81
0.10
0.08
0.29
0.11
2
2
9
H
2-Cl
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
16o
H
2-Cl
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
3-Cl
4-Cl
3-Cl
4-Cl
3-Br
4-Br
3-Br
4-Br
2-CH3
3-CH3
4-CH3
2-C2H5
3-C2H5
4-C2H5
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-C2H5
3-C2H5
4-C2H5
2-OCH3
3-OCH3
4-OCH3
>100
8.15 ( 1.90
>100
>100
0.33
0.03
21.61 ( 3.22
2-, 3-, or 4-position of the benzene ring was substituted with a
methyl or an ethyl group. Regarding the methyl-substituted
derivatives (15f-h), we noticed important COXs inhibitory
properties, whatever the methyl substitution position,
although inhibitory properties appeared slightly weaker
when compared to compound 9 profile. Among these three
derivatives, the best selectivity ratio was obtained, as it was
already observed with the chlorinated and brominated deri-
vatives when the methyl substituent is located at the 3-
position (compound 15g, COX-2 selectivity ratio of 2.19).
Regarding now the ethyl derivatives, we found that the
replacement of the methyl group with an ethyl one at the
2- and 4-position (compounds 15i and 15k, respectively) led
to a significant decrease in COXs inhibitory properties with-
out drastic modification of the COX-2 selectivity ratio. On
the contrary, an ethyl group at the 3-position (compound
15j) showed slightly improved COXs inhibitory properties
when compared to those of its methyl analogue (COX-1 and
COX-2 IC50 values for 15g: 0.99 and 0.45 μM; for compound
15j: 0.69 and 0.24 μM, respectively). Moreover, the selectiv-
ity ratio of 15j was improved when compared to 15g (COX-2
selectivity ratio for compounds 15j and 15g: 2.84 and 2.19,
respectively). Taken together, these data demonstrate that
the substitution of the benzene ring with a halogen atom or
an alkyl chain lead to compounds displaying strong inhibi-
tory properties against COXs enzymes. Moreover, the best
selectivity ratios are observed with compounds bearing a
substituent at the 3-position. Regarding the steric hindrance,
the replacement of the methyl substituent of 15g with an
ethyl one (15j) was accompanied with an improved activity,
subsequently suggesting that an ethyl group did not display
sufficient steric hindrance to limit the COXs inhibitory
activities.
displayed COX-2 selectivity ratios below 1 and therefore
appeared more active against COX-1. Among these, it seems
interesting to point out that compound 15n, although it has
COX-1 preferential inhibitory activity, displayed particu-
larly high COXs inhibitory properties (COX-1 and COX-2
IC50 values for 15n: 0.10 and 0.16 μM, respectively).
In a second step, we aimed to evaluate the influence of the
same substitution pattern in the methanesulfonamide series.
Indeed, during our previous work,33 we demonstrated that
the modulation of the sulfonamide acidic character impacted
on both the cyclooxygenases inhibitory activity and the
COX-2 selectivity. As a brief summary of these previous
works on pyridine derivatives of nimesulide bearing an ether
or thioether linkage, we showed that compounds with a
methanesulfonamide moiety, when compared to their tri-
fluoromethanesulfonamide analogues, were characterized
by a weaker COXs inhibitory profile but often an increased
selectivity ratio against COX-2. All the results of the in vitro
pharmacological evaluations performed with these newly
synthesized compounds bearing a methanesulfonamide
moiety are presented in Table 3.
In a first step, the analogue of compound 9, compound
16a, was synthesized and pharmacological results demon-
strated that the replacement of the trifluoromethanesulfo-
namide with a methanesulfonamide moiety led to a drastic
loss of COXs inhibitory properties (COX-1 and COX-2 IC50
values for compound 16a: 2.26 and 2.69 μM, respectively).
Moreover, the COX-2 selectivity ratio was also strongly
decreased when compared to that of compound 9 (COX-2
selectivity ratio for 9 and 16a: 2.00 and 0.85, respectively).
The substitution of the benzene ring with a chlorine atom
(16b-d) did not significantly modify the COX-2 inhibitory
activity. Of particular interest are the results obtained with
16c for which the well-preserved COX-2 inhibitory activity
was accompanied with a decreased COX-1 inhibitory acti-
vity (COX-1 and COX-2 IC50 values for compound 16c:
7.15 and 0.73 μM, respectively), subsequently leading to a
The last modulation investigated in the trifluoromethane-
sulfonamide series was the substitution of the benzene ring of
9 with a methoxy moiety (compounds 15l-n). Globally, all
the methoxy derivatives, whatever the substitution position,

5868 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Renard et al.
Table 4. IC50 COX-1 and IC50 COX-2 of Compounds 9, the Cycloalk-
anes Derivatives, and the Selectivity Ratio (IC50 COX-1/IC50 COX-2)
Table 5. In Vivo Anti-inflammatory Activity of Compounds 2, 5, and
Selected Compounds at 5, 10, and 20 mg/kg ip in the Rat Carrageenan-
Induced Pleurisy (mean ( SE, n = 3-6 Rats/Group)
% inhibition of exudate
compd
5 mg/kg
10 mg/kg
20 mg/kg
5
31.05 ( 7.85*
15.67 ( 5.99
25.41 ( 3.30
16.14 ( 11.42
39.39 ( 3.65**
42.07 ( 3.74**
23.50 ( 13.75
11.48 ( 6.04
48.44 ( 3.09**
27.32 ( 5.60
61.53 ( 6.75**
42.09 ( 9.70**
72.20 ( 6.28**
52.60 ( 4.17**
68.83 ( 9.95**
64.52 ( 3.21**
69.53 ( 4.17**
25.46 ( 2.50
2
9
55.68 ( 9.50**
40.03 ( 4.76*
51.41 ( 5.28**
52.16 ( 2.63**
50.29 ( 15.04*
18.31 ( 7.74
15b
15c
15d
15g
16c
* p < 0.05. ** p < 0.01 vs control, one way ANOVA, and Bonferroni’s
multiple comparison test.
Cycloalkanes used for these investigations bore 5, 6, or 7
carbon atoms. All of these compounds showed a COX-1
preferential profile. Compounds bearing a trifluorometha-
nesulfonamide moiety demonstrated a higher COXs inhibi-
tory potency than their methanesulfonamide analogues.
Moreover, the size of the cycloalkane ring seemed to be
important for the COXs inhibitory activity. Cyclohexane
(compounds 25a and 25b) provided drugs with a strong
COXs inhibitory activity. The increase of the size of the
cycloalkane (26a and 26b) slightly decreased this activity,
while a cyclopentane (compounds 24a and 24b) led to
compounds exhibiting a poor inhibitory activity against
the two cyclooxygenases. Compound 24b bearing a metha-
nesulfonamide moiety was even inactive on COX-2.
Finally, the influence of the length of the sulfonamide
moiety was evaluated by using the 3,3,3-trifluoropro-
panesulfonamide group. The replacement of the trifluoro-
methanesulfonamide moiety of 9 by a 3,3,3-trifluoro-
propanesulfonamide (17) drastically decreased the COXs
inhibitory potency. Furthermore, this decrease was more
important regarding the COXs inhibitory profile and COX-2
selectivity ratio when compared to compound 9.
In Vivo Anti-inflammatory Activity. The in vitro results
allowed us to select compounds exhibiting a high COXs
inhibitory activity and/or an interesting COX-2/COX-1
selectivity ratio for further investigation. Compounds 15b,
15c, 15d, 15g, and 16c were therefore selected and evaluated
for their in vivo anti-inflammatory properties in an animal
model of carrageenan-induced pleurisy (Table 5). Com-
pounds 5, 2, and our lead compound 9 were evaluated in
the same test as reference drugs.
In this test, the inhibition by 5 of the exudate production,
which is the reflection of the anti-inflammatory activity,
was statistically significant whatever the dose administered
(5, 10, and 20 mg/kg) while the anti-inflammatory activity of
2 was found to be only significant at the dose of 20 mg/kg.
Compound 9, our lead compound, displayed a strong anti-
inflammatory activity, reaching 72% inhibition at the dose
of 20 mg/kg. Nevertheless, the decrease of exudate volume
was only statistically significant at the two higher doses
(10 and 20 mg/kg) investigated (p < 0.001). Compound
15c exhibited an inhibition profile similar to that of 9, but its
activity was already statistically significant at the dose of
5 mg/kg (p < 0.01). The anti-inflammatory properties of 15b
were similar to that of 2 at the dose of 5 mg/kg (% of
inhibition: 16.1% and 15.7%, respectively). At higher doses,
15b displayed a stronger anti-inflammatory activity when
compared to 2 but failed to reach the efficiency of compound
compound displaying a selectivity ratio of 9.83. Results
obtained with the brominated derivatives (16e and 16f)
confirmed that the best selectivity profile was obtained when
the substituent is located at the 3-position (COX-2 selectivity
ratio for compound 16e: 7.81), although the COXs inhibitory
properties appeared weaker when compared to the chlori-
nated analogues 16c and 16d. The substitution of the phenyl
ring of 16a with a short chain was also investigated. Among
the methylated derivatives (16g-i), we observed a decrease in
the COX-2 inhibitory property when compared to their
chlorinated analogues, whatever the substitution position.
Moreover, the selectivity ratios measured were below the
unit, identifying these three compounds as COX-1 prefer-
ential inhibitors.
The loss of activity against COXs enzymes was even more
pronounced when evaluating the ethylated derivatives
(16j-l). Indeed, derivatives 16j and k, bearing an ethyl chain
at the 2- and 3-position, were characterized as weak COX-2
inhibitors with IC50 greater than 100 μM. Compound 16l
displayed a slightly better inhibitory profile with an IC50
against COX-2 of 8.15 μM, although its selectivity ratio
remained below the unit. Finally, derivatives bearing a
methoxy moiety (16m-o) behaved in the same manner as
the ethyl derivatives with 2- and 3-methoxy derivatives
showing a COX-2 IC50 greater than 100 μM. As observed
with the ethyl derivatives, the best results in this substitution
pattern were achieved with the 4-methoxy derivative (COX-1
and COX-2 IC50 values for compound 16o: 0.56 and 21.61
μM, respectively).
The third pharmacomodulation investigated was the re-
placement of the benzene ring of 9 with several cycloalkane
rings (Table 4). Indeed, the replacement of the benzene ring
of 5 with a cyclohexane one led to the [N-(2-cyclohexy-
loxy-4-nitrophenyl]methanesulfonamide (NS-398).37 This
compound exhibited an important cyclooxygenase inhi-
bitory potency and an enhanced COX-2 preferential pro-
file in different tests38,39 when compared to compound 5.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 5869
5 even at the highest investigated dose (% inflammation
inhibition at the dose of 20 mg/kg for 15b and 5: 52.6% and
61.5%, respectively). Its methanesulfonamide analogue,
compound 16c, showed a weak and non-statistically signifi-
cant anti-inflammatory activity in vivo (% inhibition at the
dose of 20 mg/kg: 25.5%), leading to the conclusion that the
weaker COXs inhibitory profile of the methanesulfonamide
compound 16c when compared to 15b is associated with a
drastically decrease in anti-inflammatory activity although
this compound displayed a high COX-2 selectivity ratio.
Substitution of the benzene ring of 9 with a methyl group
at the 3-position (15g) did not significantly modify the anti-
inflammatory activity. Indeed, the anti-inflammatory prop-
erties of 15g and 9 were similar at all the doses investigated
with a decrease of the pleural exudate volume statistically
significant at the doses of 10 and 20 mg/kg while no
statistically significant effect was observed at 5 mg/kg.
Finally, compound 15d, which presented a COX-2 in-
hibitory profile similar to 9 (COX-2 IC50 value for 15d and 9:
0.12 and 0.09 μM, respectively), but a COX-2 selectivity ratio
comparable to compound 2 (COX-2 selectivity ratio for 15d
and 2: 7.48 and 7.46, respectively) was evaluated. 15d dis-
played statistically significant anti-inflammatory properties
at the doses of 5, 10, and 20 mg/kg (p < 0.001). The strongest
inhibition of inflammation response at the dose of 5 mg/kg
was obtained with this drug while it exhibited a decrease of
exudate development similar to that of compounds 9, 15c,
and 15g at the doses of 10 and 20 mg/kg, therefore identifying
this compound as the most active drug synthesized during
this study.
displayed weak anti-inflammatory profile in vivo, whatever
their COX-2 selectivity ratio.
Regarding now the safety profile, comparison of nonselec-
tive and COX-2 selective agents evidenced that the use of
COX-2 selective inhibitors such as compound 1 or 2 was
associated with a significantly decreased risk of gastrointest-
inal toxicity when compared to nonselective NSAIDs. Never-
theless, the occurrence of cardiovascular side effects observed
with 1 led to its withdrawal from the market. Although not yet
fully elucidated, this toxicity seems to be related to either
its high COX-2 selectivity ratio or its chemical structure,
more precisely to its methylsulfone moiety. Having obtained
compounds of which the best representatives showed a COX-
2 selectivity ratio in the range of that displayed by 2
(compounds 15d, 16c, and 16e) and characterized by the
presence of a methane- or trifluoromethanesulfonamide moi-
ety instead of a methylsulfone moiety, we can theoretically
expect that the use of our compounds should be associated
with a good safety profile.
Finally, some cases of nimesulide-induced hepatotoxicity
were recently reported and led to the withdrawal of this
compound first in Finland and soon after in Spain. More
recently, 5 was also withdrawn in Belgium for similar hepatic
injuries. This hepatotoxicity was found to be idiosyncratic
(predisposition of polymorphism in either of P450 1A2 and
P450 2C19).40 More precisely, it seems that several nimesulide
metabolites areinvolvedin thishepatotoxicity. Indeed, several
metabolites of this compound have been identified including
4-hydroxynimesulide and amino des-nitro nimesulide in
which the nitrobenzene group has been reduced. Of particular
interest is the ability of amino des-nitro nimesulide to be
further oxidized by P450 enzyme to diiminoquinone, highly
reactive species able to alkylate proteins. In a second step,
modified peptides generated from such nucleophilic addition
can act as antigens and trigger an immune response.40 This
metabolism pathway may represent the mechanism of the
acute liver failure described in some patients under nimesulide
therapy.
Therefore, this occasionally observed toxicity seems to be
associated with the in vivo metabolism of the nitrobenzene
ring of nimesulide (reduction followed by the formation of a
reactive diiminoquinone). Since we performed the replace-
ment of the nitrobenzene ring of nimesulide with a pyridinic
one, we believe that the use of our newly synthesized com-
pounds should not be associated with such toxicity.
To conclude, we have achieved the synthesis of pyridinic
derivatives of nimesulide displaying high COXs inhibi-
tory properties associated, for some of them, with a COX-2
selectivity ratio similar to that of 2. Among these deriva-
tives, compound 15d, displaying a drastic inhibition of COX-2
(COX-2 IC50 value for compound 15d: 0.12 μM) with a COX-
2 selectivity ratio of 7.48 and a strong anti-inflammatory
profile in vivo appeared as the most promising agent.
Conclusion
In the present study, we synthesized 36 original pyridine
derivatives of nimesulide bearing an amino linkage by using a
five-step synthesis pathway. These 36 original compounds can
be classified into two series: the methanesulfonamide and the
trifluoromethanesulfonamide derivatives. All these original
derivatives were evaluated for their ability to inhibit human
COX-1 and COX-2 enzymes in a human whole blood model.
Results obtained demonstrated that, when comparing
methane and trifluoromethanesulfonamide analogues, the
highest inhibitory properties were observed with trifluoro-
methanesulfonamide derivatives while the highest COX-2
selectivityratioswere foundinthe methanesulfonamideseries.
For example, compounds 15d and 15n exhibited the most
important inhibitory properties against COXs enzymes
(COX-1 and COX-2 IC50 values for compound 15d: 0.91
and 0.12 μM; for compound 15n: 0.10 and 0.16 μM, re-
spectively), while compounds 16c and 16e exhibited the high-
est COX-2 selectivity ratios (COX-2 selectivity ratio for
compounds 16c and 16e: 9.83 and 7.81, respectively).
Inasecond step, weselectedseveralsynthesizedcompounds
on the basis of their COXs inhibitory properties and COX-2
selectivity and we investigated their ability to reduce inflam-
mation in an acute inflammation model in rats. In this in vivo
model, all the evaluated compounds, except 16c, displayed
a strong anti-inflammatory activity at the dose of 20 mg/kg.
Of particular interest were the results obtained with com-
pounds 15c and 15d that demonstrated an enhanced anti-
inflammatory activity at the dose of 5 mg/kg when compared
to nimesulide and celecoxib, therefore identifying these
two compounds as promising anti-inflammatory agents.
Moreover, results obtained also evidenced that compounds
with weaker COXs inhibitory properties (compound 16c)
Experimental Section
Chemistry. All commercial chemicals (Sigma-Aldrich,
Belgium and Fluorochem, United Kingdom) and solvents are
reagent grade and were used without further purification.
Melting points were determined on a Stuart SMP3 apparatus
in open capillary tubes and are uncorrected. IR spectra were
recorded as KBr pellets on a Perkin-Elmer 1000 FTIR spectro-
photometer. NMR spectra were recorded on a Bruker Avance
(500 MHz) spectrometer using DMSO-d6 as solvent and tetra-
methylsilane as internal standard; chemical shifts are reported in

5870 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Renard et al.
δ values (ppm) relative to internal tetramethylsilane. The ab-
breviation s = singlet, d = doublet, t = triplet, q = quadruplet,
m = multiplet, and bs = broad signal are used throughout.
Elemental analyses (C, H, N, S) were determined either on a
Carlo-Erba EA 1108 or a Thermo Flash EA 1112 series ele-
mental analyzer and were within (0.4% of the theoretical
values. All reactions were followed by TLC (silica gel 60F254
Merck) and visualization was accomplished with UV light
(254 nm).
blood aliquots were transferred into siliconized tubes preloaded
with 1 μL of vehicle (DMSO) or test compounds at final concen-
tration ranging from 0.01 to 100 μmol/L. The tubes were vor-
texed and incubated at 37 °C under constant agitation for 5 min,
and then 10 μL of calcium ionophore A-23,187 (0.2 mg/mL)
were added and the aliquots were incubated at 37 °C under
constant agitation for 15 min. At the end of the incubation, the
serum was obtained by centrifugation (3200g for 5 min) and was
assayed for thromboxane B2 (TXB2) production using an
enzyme immunoassay kit (Cayman Chemical TXB2 EIA Kit,
Ann Arbor, MI, and Assay design TXB2 Correlate-EIA Kit,
Ann Arbor, MI) according to the manufacturer’s instructions.
COX-2 assay. Fresh human blood was collected in hepar-
inized tubes by venipuncture. One mL aliquots of blood were
transferred in heparinized tubes preloaded with either 4 μL of
vehicle (DMSO) or 4 μL of test compound at a final concentra-
tion ranging from 0.01 to 100 μmol/L. The tubes were vortexed
and incubated at 37 °C under constant agitation for 5 min. This
first treatment was followed by incubation of the blood with
20 μL of lipopolysaccharide (LPS from E. coli serotype B8) at
final concentration of 100 μg/mL under constant agitation for
24 h at 37 °C in order to induce COX-2 expression. Appropriate
controls (LPS replaced by 20 μL of PBS) were used as blanks.
After incubation, the tubes were centrifugated at 3200g for
5 min. Plasma was assayed for PGE2 production by a specific
enzyme immunoassay (Cayman Chemical PGE2 EIA Kit, Ann
Arbor, MI) according to the manufacturer’s instructions.
Carrageenan-Induced Pleurisy. Animals. Male Wistar rats,
Experimental procedures for the preparation of 4-nitropyr-
idine N-oxide (13a-o, 18-20) and 4-aminopyridine derivatives
(14a-o, 21-23) are presented in the Supporting Information.
General Procedure for the Preparation of Alkanesulfonamides
with Alkanesulfonyl Chlorides. The appropriate aminopyridine
(14a-o, 21-23, 5 mmol) was dissolved in dry acetonitrile
(40 mL). Anhydrous potassium carbonate (30 mmol) was
added, and the suspension was stirred for 5 min and the appro-
priate alkanesulfonyl chloride (6-7.5 mmol) was added to the
mixture. When the reaction was finished (1-4 h), the suspension
was filtered and the filtrate was evaporated under reduced
pressure. The residue was taken up with NaOH (10% aqueous
sol. w/v) and then was filtered. The filtrate was neutralized with
1 N HCl, and the crude product was collected by filtration. The
resulting product was crystallized in ethanol/water or purified by
column chromatography on silica gel using a chloroform/metha-
nol mixture (18:2) as eluent to give the product as a white solid.
N-(3-Phenylamino-4-pyridinyl)trifluoromethanesulfonamide
(9). Yield: 52%; mp: 188-189 °C. 1H NMR (DMSO-d6) δ 7.05 (t,
1H, H-40), 7.30 (d, 2H, H-20 þ H-60), 7.35 (t, 2H, H-30 þ H-50),
7.40 (s, 1H, NH), 7.61 (d, 1H, H-5), 7.96 (d, 1H, H-6), 8.13 (s, 1H,
H-2), 13.52 (s, 1H, NHþ). Anal. (C12H10F3N3O2S) C, H, N, S.
N-[3-(3-Chlorophenylamino)-4-pyridinyl]trifluoromethanesul-
fonamide (15b). Yield: 65%; mp: 211-213 °C. 1H NMR
(DMSO-d6) δ 7.00 (d, 1H, H-60), 7.21 (d, 1H, H-40), 7.31 (bm,
2H, H-20 þ H-50), 7.63 (m, 2H, H-5 þ NH), 8.01 (d, 1H, H-6),
8.23 (s, 1H, H-2), 13.55 (s, 1H, NHþ). Anal. (C12H9ClF3N3O2S)
C, H, N, S.
N-[3-(4-Chlorophenylamino)-4-pyridinyl]trifluoromethanesul-
fonamide (15c). Yield: 27%; mp: 203-205 °C. 1H NMR
(DMSO-d6) δ 7.30 (d, 2H, H-20 þ H-60), 7.35 (d, 2H, H-30 þ
H-50), 7.52 (s, 1H, NH), 7.62 (d, 1H, H-5), 7.98 (d, 1H, H-6), 8.13
(s, 1H, H-2), 13.54 (s, 1H, NHþ). Anal. (C12H9ClF3N3O2S) C,
H, N, S.
N-[3-(3-Bromophenylamino)-4-pyridinyl]trifluoromethanesul-
fonamide (15d). Yield:69%; mp:241-242 °C. 1H NMR (DMSO-
d6) δ 7.26 (m, 3H, H-40 þ H-50, H-60), 7.47 (s, 1H, H-20), 7.63 (d,
1H, H-5), 7.64 (s, 1H, NH), 8.01 (d, 1H, H-6), 8.23 (s, 1H, H-2),
13.55 (s, 1H, NHþ). Anal. (C12H9BrF3N3O2S) C, H, N, S.
N-[3-(3-Methylphenylamino)-4-pyridinyl]trifluoromethanesul-
fonamide (15g). Yield: 42%; mp: 167-169 °C. 1H NMR
(DMSO-d6) δ 2.29 (s, 3H, CH3), 6.87 (d, 1H, H-60), 7.09 (d,
1H, H-40), 7.10 (s, 1H, H-20), 7.23 (t, 1H, H-50), 7.31 (s, 1H, NH),
7.60 (d, 1H, H-5), 7.96 (d, 1H, H-6), 8.13 (s, 1H, H-2), 13.50
(s, 1H, NHþ). Anal. (C13H12F3N3O2S) C, H, N, S.
ꢀ
weighing 230-260 g, were housed in the University of Liege
animal facilities in accordance with local guidelines. The
animals were maintained on a standard laboratory diet with
free access to water. The experiments were conducted as
approved by the Animal Ethics Committee of the University
ꢀ
of Liege, Belgium.
Carrageenan-Induced Pleurisy. Rats were pretreated with an
intraperitoneal (ip) injection of saline or drugs (5, 10, or 20 mg/kg)
30 min before the intrapleural injection of carrageenan. They were
then anaesthetized with sodium pentobarbital (40 mg/kg, ip) and
λ carrageenan (0.2mL, 10 mg/mL) was administered into the right
pleural cavity. Each experimental group contained at least three
animals. Four hours later, the animals were anaesthetized with
sodium pentobarbital (80 mg/kg, ip).
The chest was carefully opened and the pleural cavity rinsed
with 2.0 mL of saline solution containing heparin (5 U/mL).
Exudates and washing solutions were collected by aspiration.
Exudates with blood were rejected. The volume of the exudates
was calculated by subtracting the volume of the washing solu-
tion (2.0 mL) from the total volume harvested.
Acknowledgment. This study was supported by Belgian
grants of the “Fonds pour la Recherche dans l’Industrie et
ꢁ ꢁ
l’Agriculture” (FRIA) and the “Leon Fredericq Foundation”.
Supporting Information Available: Experimental procedure
for 4-nitropyridine N-oxide derivatives (13a-o, 18-20), 4-ami-
nopyridine derivatives (14a-o, 21-23), 4-alkylsulfonylamino-
pyridine derivatives (15a, 15e-f, 15h-n, 16a-b, 16d-o, 24a-
b, 25b, 26a-b, 17), and elemental analysis of the compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
N-[3-(3-Chlorophenylamino)-4-pyridinyl]methanesulfonamide
(16c). Yield: 23%; mp: 185-187 °C. 1H NMR (DMSO-d6)
δ 2.89 (s, 3H, SO2CH3), 6.93 (d, 1H, H-60), 7.16 (d, 1H, H-40),
7.28 (bm, 3H, H-5 þ H-20 þ H-50), 7.43 (s, 1H, NH), 7.78 (d,
1H, H-6), 8.00 (s, 1H, H-2), 12.52 (bs, 1H, NHþ). Anal.
(C12H9ClF3N3O2S) C, H, N, S.
N-(3-Cyclohexylamino-4-pyridinyl)trifluoromethanesulfona-
mide hydrochloride (25a). Yield: 71%; mp: 217-218 °C. 1H
NMR (DMSO-d6): δ 1.15-1.95 (bm, 10H, Hcyclohexyl), 3.33 (m,
1H, H-10), 7.44 (bs, 1H, H-6), 7.70 (d, 1H, H-2), 7.80 (d, 1H, H-5),
13.81 (s, 1H, NHþ). Anal. (C12H17ClF3N3O2S) C, H, N, S.
Pharmacology. In Vitro COX-1 and COX-2 Inhibitory Acti-
vity Determination Using Human Whole Blood Assay. COX-1
assay. Fresh human blood was collected into heparinized tubes
by venipuncture and directly diluted with RPMI (Roswell Park
Memorial Institute culture medium) (1:4). Then 0.25 mL diluted
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