N-(3-Arylaminopyridin-4-yl)alkanesulfonamides as pyridine analogs of nimesulide: Cyclooxygenases inhibition, anti-inflammatory studies and insight on metabolism

Article abstract of DOI:10.1016/j.ejmech.2013.12.033

Nimesulide, a COX-2 preferential inhibitor with a favorable gastric and cardiovascular safety profile, was responsible for some cases of acute liver failure attributed to the nitrobenzene ring. A series of analogs of nimesulide resulting from isosteric replacement of the nitrobenzene ring by the pyridine nucleus, was synthesized and their ability to inhibit both cyclooxygenases (COXs) isoforms was evaluated in vitro using a human whole blood model. Compounds 19c, 23b and 23c displayed an important inhibitory activity associated to a COX-2/COX-1 selectivity ratio similar to or higher than that of celecoxib. The anti-inflammatory activity and the ability of several compounds to decrease leukocyte infiltration were further evaluated in vivo in a model of a λ carrageenan-induced pleurisy. Plasma assays were performed on blood samples collected from rats and allowed us to identify the 4-position of the phenyl ring as a major metabolism site explaining the occasionally observed lack of correlation between in vitro and in vivo results.

Full text of DOI:10.1016/j.ejmech.2013.12.033

European Journal of Medicinal Chemistry 74 (2014) 12e22
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
N-(3-Arylaminopyridin-4-yl)alkanesulfonamides as pyridine analogs
of nimesulide: Cyclooxygenases inhibition, anti-inflammatory studies
and insight on metabolism
,
1
Jean-François Renard a, Frédéric Lecomte b, Philippe Hubert b, Xavier de Leval a
,
, ,
1
Bernard Pirotte a
*
a Center for Interdisciplinary Research on Medicines (CIRM), Laboratory of Medicinal Chemistry, University of Liège, Av. de l’Hôpital 1, B-4000 Liège, Belgium
b Center for Interdisciplinary Research on Medicines (CIRM), Laboratory of Analytical Chemistry, University of Liège, Av. de l’Hôpital 1, B-4000 Liège, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Nimesulide, a COX-2 preferential inhibitor with a favorable gastric and cardiovascular safety profile, was
responsible for some cases of acute liver failure attributed to the nitrobenzene ring. A series of analogs of
nimesulide resulting from isosteric replacement of the nitrobenzene ring by the pyridine nucleus, was
synthesized and their ability to inhibit both cyclooxygenases (COXs) isoforms was evaluated in vitro using
a human whole blood model. Compounds 19c, 23b and 23c displayed an important inhibitory activity
associated to a COX-2/COX-1 selectivity ratio similar to or higher than that of celecoxib. The anti-
inflammatory activity and the ability of several compounds to decrease leukocyte infiltration were
Received 19 August 2013
Received in revised form
18 December 2013
Accepted 22 December 2013
Available online 3 January 2014
Keywords:
Nimesulide
COXs inhibitors
Nonsteroidal anti-inflammatory drugs
further evaluated in vivo in a model of a
l carrageenan-induced pleurisy. Plasma assays were performed
on blood samples collected from rats and allowed us to identify the 4-position of the phenyl ring as a
major metabolism site explaining the occasionally observed lack of correlation between in vitro and
in vivo results.
l
carrageenan-induced pleurisy
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
COX-2 enzymes [9,10]. COX-1, the constitutive form of the enzyme is
found in most tissues and is mainly responsible for the physiological
Nonsteroidal anti-inflammatory drugs (NSAIDs) are used in the
management of pain and acute inflammation but also in chronic
inflammation states such as, for example, osteoarthritis and rheu-
matoid arthritis. Beside these current applications, NSAIDs have been
reported to have a protective effect against a wide variety of human
diseases such as atherosclerosis [1], thrombosis, cancer [2e6] and
Alzheimer’s disease [7,8]. The mechanism of action of these thera-
peutic agents is blocking the formation of prostanoids, lipidic medi-
ators deriving from fatty acid metabolism, by inhibiting COX-1 and
production of prostanoids. Among these prostanoids, prostaglandins
(PGs) provide maintenance functions such as cytoprotection in the
stomach [11]. In contrast, COX-2, although constitutively expressed in
brain, kidney and spinal cord, can be mainly considered as an induc-
ible enzyme and its expression is triggered under pathological con-
ditions such as inflammation. The drawback of the traditional NSAIDs
(t-NSAIDs), “equally” inhibiting both cyclooxygenase isoforms, is that
their use is associated with toxic side effects including gastro-
intestinal erosions and bleeding [12]. To avoid these side effects
caused by a COX-1 inhibition, COX-2 selective inhibition appeared as
an attractive approach to manage inflammation in a safer way [13].
Thus, COX-2 selective inhibitors or coxibs were designed and devel-
oped. First, a reduced incidence of gastroduodenal lesions was re-
ported for coxibs [14] but an increased risk of adverse cardiovascular
effect was further demonstrated in clinical trials for some of them
[15e17]. This toxicity led to the withdrawal of rofecoxib (1) [18] and
valdecoxib (2) [19] from the market (Fig. 1). The potential risk for
cardiovascular thrombotic events observed with these COX-2 selec-
tive inhibitors may be due to an imbalance between prostacyclin
(PGI2), a vasodilator and a potent platelet antiaggregator, which is
mainly generated by the constitutively expressed COX-2, and
Abbreviations: NSAIDs, nonsteroidal anti-inflammatory drugs; COX, cyclo-
oxygenase; PGs, prostaglandins; TXA2, thromboxane A2; PGI2, prostacyclin; NO,
nitric oxide; H2S, hydrogen sulfide; SAR, structureeactivity relationship; DMF,
dimethylformamide; DMSO, dimethylsulfoxide; TLC, thin layer chromatography;
RPMI, Roswell Park Memorial Institute culture medium; LPS, lipopolysaccharide;
TXB2, thromboxane B2; PGE2, prostaglandin E2; HPLC, high performance liquid
chromatography; SPE, solid phase extraction.
* Corresponding author. Laboratory of Medicinal Chemistry, University of Liège, 1
av. de l’Hôpital, tour 4 (þ5), Sart-Tilman, B-4000 Liège, Belgium. Tel.: þ32 4 3664365;
fax: þ32 4 3664362.
E-mail address: B.Pirotte@ulg.ac.be (B. Pirotte).
These authors assumed equal supervision.
1
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.033

J.-F. Renard et al. / European Journal of Medicinal Chemistry 74 (2014) 12e22
13
trifluoromethanesulfonamide moiety, a non-substituted phenyl
ring and a secondary amine linkage displayed a strong COX
inhibitory activity (COX-1 and COX-2 IC50 values for compound 6:
0.18 and 0.09 mM, respectively) with a slight COX-2 preferential
profile (COX-2/COX-1 selectivity ratio for 6: 2.00) [38]. In order to
increase this COX-2 selectivity profile, we substituted the ben-
zene ring with various moieties while maintaining the amine
linkage. These modulations allowed us to identify compound 7,
bearing a bromine atom at the 3-position of the benzene ring, as
a promising anti-inflammatory agent with a COX-2 selectivity
ratio similar to that of celecoxib (4, Table 1) [39].
In the present study, we aimed at improving the structureeac-
tivity relationships (SAR) that we previously established. For this
purpose, we have substituted the benzene ring with a halogen
atom or an electron donating or withdrawing group. On the basis of
these SAR, we synthesized disubstituted compounds. After deter-
mination of their COXs inhibitory potency, we evaluated their
in vivo anti-inflammatory activity and the ability of several com-
pounds to reduce leukocyte infiltration. Finally, we determined the
influence of various structural modifications on drug plasma levels.
Fig. 1. Chemical structure of preferential or selective COX-2 inhibitors.
thromboxane A2 (TXA2), a strong pro-aggregant and vasoconstrictor
mediator mainly produced through the COX-1 pathway [20e22]. In
this context, several strategies were investigated to improve the
safety profile of COX inhibitors. One of these strategies consisted of
combining a nitric oxide (NO) or a hydrogen sulfide (H2S) releasing
moiety with a COX inhibitor [23e26]. NO and H2S are messengers
sharing many biological activities such as reduction of neutrophil
adhesion, modulation of inflammatory mediator release, protection
against gastric injury and beneficial effects on cardiovascular system
including vasodilator activity [27,28]. A dual mechanism of action
involvingCOXinhibitionandNOorH2Sreleasemayofferanimproved
safety profile for NSAIDs. Another approach is to limit the selectivity
against COX-2 [29]. Around 10 times selectivity for COX-2 to COX-1
appears to be appropriate for maintaining the balance between PGI2
and TXA2 [30,31]. This strategy uses the concept of COX-2 preferential
inhibitors, among which nimesulide (3, Fig. 1) [32] is the leader [33].
This drug exhibits a good tolerability profile with a lack of demon-
strated cardiovascular side effects suggesting that a “reasonable”
COX-2/COX-1 selectivity ratio is sufficient to provide safe anti-
inflammatory agents. Nevertheless, some cases of nimesulide-
induced hepatotoxicity were reported leading to its withdrawal in
several European countries [34,35]. This hepatotoxicity was found to
beidiosyncratic(predispositionof polymorphismineitherofP450 1A2
and P450 2C19). Several metabolites of nimesulide have been identi-
fied including compounds in which the nitrobenzene group was
reduced. Such amino des-nitro nimesulide derivatives can be further
oxidized by P450 enzyme to diiminoquinone, a highly reactive species
able to alkylate proteins. Modified peptides generated from such
nucleophilic addition can act as antigens and trigger immune
response [36]. Therefore, these data suggest that the mechanism of
the acute liver failure toxicity observed in some patients under
nimesulide therapy is associated with the in vivo metabolism of the
nitrobenzene ring of this NSAID.
2. Chemistry
The synthetic pathway used to prepare compounds 19e24
(Scheme 1, steps iev) was previously reported [39]. It can be sum-
marized as follows: 3-bromopyridine (8) was first oxidized by a
mixture of acetic acid and hydrogen peroxide to afford 3-
bromopyridine N-oxide (9), which was then nitrated at the 4-
position by a nitric and sulfuric acid medium to provide the key
intermediate 3-bromo-4-nitropyridine N-oxide (10). The NH bridge
was achieved by reaction of intermediate 10 with properly
substituted anilines. The 4-aminopyridine intermediates 15e18
were obtained after simultaneous reduction of the nitro and the
pyridine N-oxide moieties using iron in an acetic acid and water
medium. The pyridine analogs of nimesulide 19e24 were obtained
by reaction of the aminopyridine intermediates 15e18 with methyl-
or trifluoromethylsulfonyl chloride in acetonitrile in the presence of
potassium carbonate. The synthesis of N-(cyclopropylsulfonyl)-N-
[(3-phenylamino)pyridin-4-yl]cyclopropanesulfonamide (25) was
performed by reaction between 4-amino-3-phenylaminopyridine
and an excess of cyclopropanesulfonyl chloride in methylene chlo-
ride with triethylamine (Scheme 1, step vi). N-[3-phenylamino)
pyridin-4-yl]cyclopropanesulfonamide (26) was obtained through
hydrolysis of 25 in a 50% aqueous solution of KOH under reflux for
15 min (Scheme 1, step vii).
In order to avoid any cross-reactions with sulfonyl chlorides, the
phenolic compounds 28aeb were obtained through O-demethy-
lation of methoxyaniline derivatives 27 using boron tribromide in
methylene chloride at room temperature for 48 h (Scheme 2).
The substitution of the bromine atom of the key intermediate 10
by 3-aminobenzonitrile did not occur in the previously described
conditions as a probable result of the electron withdrawing effect of
the cyano group that decreases the nucleophilic character of the
aniline. Therefore, N-[3-(3-cyanophenylamino)pyridin-4-yl]tri-
fluoromethanesulfonamide (29) was synthesized from N-[3-(3-
bromophenylamino)pyridin-4-yl]trifluoromethanesulfonamide (7)
through the RosenmundeVon Braun reaction (Scheme 3) [40]
allowing the substitution of the halogen atom of a haloaryl com-
pound bya cyano group using an excess of cuprous cyanide in a polar
and high boiling point solvent such as dimethylformamide (DMF).
Our group is involved for several years in the field of COX
inhibitors and more precisely, in the development of pyridine
analogs of nimesulide [37e39]. In a previous work, we demon-
strated that the isosteric replacement of the nitrobenzene ring of
nimesulide (3) by a pyridine one providing compound 5 (Table 1)
enhanced COX-1 inhibitory activity. Nevertheless, this com-
pounds was almost inactive against COX-2 [37]. Various phar-
macomodulations were considered in order to recover the ability
to inhibit COX-2 and to express a COX-2 preferential profile.
Among the synthesized compounds, N-(3-phenylaminopyridin-
3. Results and discussion
The inhibitory activity of the new compounds against both
4-yl)trifluoromethanesulfonamide (6, Table 1) bearing
a
isoforms of cyclooxygenases was evaluated in vitro using a

14
J.-F. Renard et al. / European Journal of Medicinal Chemistry 74 (2014) 12e22
Table 1
Inhibitory activity of nimesulide, celecoxib, reference compounds (5e7, 30e33), mono- and disubstituted compounds on cyclooxygenases (COX-1 and COX-2) and their
selectivity ratio for the COX-2 enzyme.
O
O
O
X
O
S
S
R
H3C
NH
N
O
Y
R3
R4
N
NO2
R5
nimesulide
compounds 19-26, 28-33
Compounds
X
Y
R
R3
R4
R5
IC50
(m
M)a
Ratiob
COX-1
COX-2
Nimesulide (3)
e
e
e
e
H
H
H
H
H
H
H
H
H
H
H
H
H
e
e
e
e
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3.76 ꢀ 1.02
2.60 ꢀ 0.20
0.41 ꢀ 0.13
0.18 ꢀ 0.06
0.91 ꢀ 0.05
1.15 ꢀ 0.48
1.09 ꢀ 0.16
7.15 ꢀ 3.34
0.44 ꢀ 0.10
0.27 ꢀ 0.03
1.37 ꢀ 0.20
2.09 ꢀ 0.36
1.93 ꢀ 0.10
18.14 ꢀ 4.82
6.65 ꢀ 0.62
19.62 ꢀ 2.63
3.61 ꢀ 1.07
0.99 ꢀ 0.10
4.11 ꢀ 0.49
14.93 ꢀ 0.73
0.71 ꢀ 0.11
3.28 ꢀ 1.16
1.47 ꢀ 0.13
1.40 ꢀ 0.37
2.32 ꢀ 0.16
1.74 ꢀ 0.30
1.35 ꢀ 0.15
4.21 ꢀ 0.38
0.70 ꢀ 0.23
0.35 ꢀ 0.09
>100
5.37
7.46
e
Celecoxib (4)
e
e
e
e
5c
CH3
CF3
CF3
CF3
CF3
CH3
CF3
CF3
CH3
CH3
CF3
CF3
CF3
Cyclopropyl
Cyclopropyl
CF3
CF3
CH3
CF3
CH3
CF3
CF3
O
H
H
Br
H
Cl
Cl
F
F
F
F
I
H
CN
H
H
H
H
Cl
H
H
H
F
H
F
H
OH
H
H
H
H
H
H
6c
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
0.09 ꢀ 0.03
0.12 ꢀ 0. 14
0.41 ꢀ 0.27
0.27 ꢀ 0.11
0.73 ꢀ 0.04
0.24 ꢀ 0.12
0.30 ꢀ 0.07
10.05 ꢀ 3.91
20.68 ꢀ 14.37
0.26 ꢀ 0.11
>100
36.23 ꢀ 4.74
56.76 ꢀ 7.49
35.09 ꢀ 6.58
0.45 ꢀ 0.20
1.04 ꢀ 0.63
>100
0.55 ꢀ 0.22
30.74 ꢀ 1.46
1.96 ꢀ 0.52
0.09 ꢀ 0.03
0.30 ꢀ 0.16
1.57 ꢀ 0.32
2.27 ꢀ 0.65
>100
2.00
7.48
2.81
4.10
9.83
1.83
0.88
0.14
0.10
7.46
e
7c
30c
31c
32c
19a
19b
20a
20b
19c
28a
29
0.18
0.35
0.10
2.19
3.93
e
25
SO2 cyclopropyl
26
H
H
H
H
H
H
H
H
H
H
H
H
H
33c
CH3
CH3
CH3
CH3
CH3
Cl
Br
CH3
Cl
H
21a
22a
21b
22b
23a
23b
23c
24a
24b
28b
CH3
CH3
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
Br
F
1.29
0.11
0.75
15.35
7.67
1.11
0.60
e
CF3
CF3
CF3
CF3
Br
Br
F
OH
H
a
IC50
(
m
M): concentration of drug giving 50% inhibition of the activity of the cyclooxygenase; values expressed in
m
M (mean ꢀ SD; n ꢁ 3).
b
c
Ratio: IC50 COX-1/IC50 COX-2.
Published results: Refs. [37e39].
previously described human whole blood model [41]. The COX-2
selectivity ratio was also calculated using the formula [IC50 COX-1
same positions with other halogen atoms or with an electron-
withdrawing group such as cyano group. New alka-
a
(
m
M)/IC50 COX-2 (
selected for further determination of their in vivo anti-
inflammatory potency in a carrageenan-induced pleurisy model
m
M)]. The most promising compounds were
nesulfonamide moieties were also investigated (Table 1).
The substitution of the benzene ring by a fluorine atom
(compounds 19aeb and 20aeb), which is smaller and less lipo-
philic than a chlorine atom (compounds 30, 31 and 32), led to
compounds displaying drastic COXs inhibitory properties.
Although their COX-2 inhibitory activity was similar, fluoro-
substituted derivatives bearing a trifluoromethanesulfonamide
moiety (compounds 19aeb) exhibited an enhanced COX-1 activity
compared to their corresponding chloro-substituted analogs
(compounds 31a and 30a). As a consequence, compounds 19aeb
did not display a marked selectivity against COX-2. As already
observed with chloro-substituted derivatives, the decrease in the
acidic character of the sulfonamide moiety resulting from the
replacement of the trifluoromethyl group by a methyl group,
decreased COXs inhibitory potency, which was more pronounced
against COX-2. Compounds 20a and 20b must even be classified as
COX-1 preferential inhibitors. Of particular interest was the finding
that an increase of the steric hindrance and the lipophilicity of the
halogen atom at the 3-position were responsible for an increased
selectivity ratio against COX-2 (compounds 19a, 31 and 7). In this
l
in rats [42]. Leukocyte infiltration was also evaluated by counting
the number of leukocytes found in the pleural exudate. At the end
of the in vivo experiments, rat blood samples were also collected in
order to measure drug plasma levels.
3.1. In vitro cyclooxygenase inhibition assays
Nimesulide (3), celecoxib (4) and compound 6 were used as
references. The activity of the original COXs inhibitors was also
compared to that of our published nimesulide analogs (7, 30e33).
Our previous investigation indicated that the replacement of the
methyl group of the methanesulfonamide moiety by
a tri-
fluoromethyl group increased COXs inhibitory potency [39].
Moreover, the introduction of a short alkyl chain or a halogen atom
such as a chlorine or a bromine atom at the 3- or 4-position of the
benzene ring enhanced the selectivity ratio against COX-2 [39].
Therefore, to complete our knowledge of the SAR in this series of
compounds, we synthesized original compounds substituted at the
context, we further evaluated the influence of
a bulkier

J.-F. Renard et al. / European Journal of Medicinal Chemistry 74 (2014) 12e22
15
NO2
NO2
H
N
Br
Br
Br
iii
i
ii
R1
R2
N
O
N
N
O
N
O
8
9
10
11-14
15-18
19-24
iv
O2 O2
O
S
S
S
N
NH2
N
NH
H
N
H
N
O
H
N
vii
vi
R1
R2
R1
R2
R1
R2
N
N
26: R1 = R2 = H
25: R1 = R2 = H
v
O
X
S
NH
O
H
N
R1
R2
N
Scheme 1. Compounds: 11a, R1 ¼ H, R2 ¼ 3-F; 11b, R1 ¼ H, R2 ¼ 4-F; 11c, R1 ¼ H, R2 ¼ 3-I; 12a, R1 ¼ 3-CH3, R2 ¼ 5-CH3; 12b, R1 ¼ 3-CH3, R2 ¼ 4-CH3; 13a, R1 ¼ 3-Cl, R2 ¼ 4-CH3; 13b,
R1 ¼ 3-Br, R2 ¼ 4-CH3; 13c, R1 ¼ 4-Br, R2 ¼ 3-CH3; 14a, R1 ¼ 3-Cl, R2 ¼ 4-F; 14b, R1 ¼ 3-Br, R2 ¼ 4-F; 15a, R1 ¼ H, R2 ¼ 3-F; 15b, R1 ¼ H, R2 ¼ 4-F; 15c, R1 ¼ H, R2 ¼ 3-I; 16a, R1 ¼ 3-CH3,
R2 ¼ 5-CH3; 16b, R1 ¼ 3-CH3, R2 ¼ 4-CH3; 17a, R1 ¼ 3-Cl, R2 ¼ 4-CH3; 17b, R1 ¼ 3-Br, R2 ¼ 4-CH3; 17c, R1 ¼ 4-Br, R2 ¼ 3-CH3; 18a, R1 ¼ 3-Cl, R2 ¼ 4-F; 18b, R1 ¼ 3-Br, R2 ¼ 4-F; 19a,
X ¼ CF3, R1 ¼ H, R2 ¼ 3-F; 19b, X ¼ CF3, R1 ¼ H, R2 ¼ 4-F; 19c, X ¼ CF3, R1 ¼ H, R2 ¼ 3-I; 20a, X ¼ CH3, R1 ¼ H, R2 ¼ 3-F; 20b, X ¼ CH3, R1 ¼ H, R2 ¼ 4-F; 21a, X ¼ CF3, R1 ¼ 3-CH3,
R2 ¼ 5-CH3; 21b, X ¼ CF3, R1 ¼ 3-CH3, R2 ¼ 4-CH3; 22a, X ¼ CH3, R1 ¼ 3-CH3, R2 ¼ 5-CH3; 22b, X ¼ CH3, R1 ¼ 3-CH3, R2 ¼ 4-CH3, 23a, X ¼ CF3, R1 ¼ 3-Cl, R2 ¼ 4-CH3; 23b, X ¼ CF3,
R1 ¼ 3-Br, R2 ¼ 4-CH3; 23c, X ¼ CF3, R1 ¼ 4-Br, R2 ¼ 3-CH3; 24a, X ¼ CF3, R1 ¼ 3-Cl, R2 ¼ 4-F; 24b, X ¼ CF3, R1 ¼ 3-Br, R2 ¼ 4-F. Reagents: (i) H2O2, CH3COOH; (ii) HNO3, H2SO4; (iii)
NH2eC6H4eR1eR2; (iv) Fe, CH3COOH/H2O; (v) R2-SO2Cl, K2CO3, CH3CN; (vi) C3H5SO2Cl, TEA, CH2Cl2; (vii) 50% KOH.
O
O
F3C
F3C
S
S
NH2
NO2
NH
NH
O
O
H
N
H
N
Br
R
R
i
+
R
OCH3
N
O
N
OCH3
N
OH
10
27a-b: R = H, Br
28a-b: R = H, Br
Scheme 2. Synthetic pathway used for 4-hydroxy-substituted compounds. Reagents: (i) BBr3, CH2Cl2, RT 48 h.
substituent at the 3-position of the phenyl ring such as an iodine
atom. Compound 19c exhibited a pronounced COXs inhibitory
activity but slightly weaker than the bromo compound 7. Never-
theless, this small decrease in activity occurred on both COXs
isoforms, resulting for 19c in a selectivity ratio similar to that of
the bromo compound 7 and of celecoxib (4). Looking at the impact
of an electron-withdrawing substituent, the introduction of a
cyano group at the 3-position of 6 (compound 29) was clearly
disappointing with a weak COXs inhibitory activity and a COX-1
preferential profile. The introduction of a hydrophilic group on
the benzene ring was also considered leading to compound 28a
with a hydroxyl group at the para position. Cyclooxygenase inhi-
bition assays revealed a lack of activity against COX-2 while the
activity against COX-1 was very weak. Finally, the increase of the
steric hindrance of the alkanesulfonamide moiety was also
investigated by the replacement of the methyl or the
trifluoromethyl moiety with a cyclopropyl moiety leading to
compounds 25 and 26. Compared to bearing tri-
6
a
fluoromethanesulfonamide group, the increase in the steric hin-
drance of the alkanesulfonamide group drastically decreased COXs
inhibitory properties. This loss of activity was more marked for 25,
which can no longer be ionized. Taken together, these results
confirmed the need of an ionizable sulfonamide function at the
physiological pH of 7.4 to keep strong inhibitory properties against
COXs enzymes. Concerning the nature of the substituent on the
phenyl ring, the introduction of a small halogen atom such as a
fluorine atom provided strong COXs inhibitors with poor selec-
tivity while the bulkier iodine atom improved the selectivity
against COX-2. Nevertheless, the iodo compound 19c did not reach
the efficiency of the bromo compound 7 suggesting that an iodine
atom displayed a greater steric hindrance that could be respon-
sible for the weaker COXs inhibitory activity.
On the basis of the SAR studies established with the mono-
substituted derivatives, we investigated the synthesis and phar-
macological evaluation of disubstituted compounds (Table 1). The
O
O
F3C
F3C
S
S
trifluoromethanesulfonamide compound 33 bearing
a methyl
NH
NH
O
O
H
N
H
N
group at the meta position of the benzene ring was previously
identified as a strong COXs inhibitor with a moderate COX-2 pref-
erential profile [39]. The introduction of a second methyl group on
the phenyl ring (compounds 21aeb and 22aeb) led to a decrease in
the COXs inhibitory activity when compared to 33 except for 21b.
Of interest is the fact that 21a and 21b bearing a strong acidic
Br
CN
i
N
N
7
29
Scheme 3. Synthetic pathway used for 4-hydroxy-substituted compounds. Reagents:
(i) CuCN, DMF, refluxed 24 h.

16
J.-F. Renard et al. / European Journal of Medicinal Chemistry 74 (2014) 12e22
trifluoromethanesulfonamide group were more active than their
corresponding less acidic methanesulfonamide counterpart 22a
and 22b. Regarding the selectivity profile, compound 21a bearing
the methyl groups at the 3- and 5-positions of the phenyl ring
exhibited a selectivity ratio against COX-2 of 3.93. Another struc-
tural modification examined was the substitution of the phenyl ring
by both a halogen atom and a methyl group at the 3- and 4-
positions (compounds 23aec). Compound 23a bearing a chlorine
atom at the meta position of the benzene ring, although active on
COX enzymes, did not exhibit a COX-2 selectivity profile while the
introduction of a bromine atom whatever its position (compounds
23bec) provided potent COX-2 inhibitors with a marked COX-2
selectivity ratio (COX-2 selectivity ratio for 23b and 23c: 15.35
and 7.67, respectively). As observed in the monosubstituted series,
the greater COX-2 inhibitory activity and selectivity were reached
by the compound bearing the bromine atom at the 3-position. Even
with a bromine atom at the 3-position of the phenyl ring, the hy-
droxyl derivative 28b like the non-brominated 28a exhibited no
inhibitory activity against COX-2. Lastly, 24a and 24b bore a fluorine
atom at the 4-position and a chlorine or a bromine atom at the 3-
position of the phenyl ring, respectively. Compared to 23a and
23b, the replacement of the methyl group by a fluorine atom pre-
served COX-1 inhibitory potency while, for 24b, an important loss
of activity against COX-2 was observed.
displayed an in vivo activity similar to that of 7 at the higher dose
investigated (20 mg/kg). Finally, we evaluated compound 23b,
which displayed in vitro a COX-2 inhibitory activity similar to that of
6 and 7 (COX-2 IC50 value for 23b, 6 and 7: 0.09, 0.09 and 0.12 mM,
respectively), but the highest COX-2 selectivity ratio (COX-2 ratio
for 23b: 15.35). The anti-inflammatory profile of 23b was statisti-
cally significant and comparable to that of 6 and 7 at 10 and 20 mg/
kg, but did not reach the efficiency of 7 at 5 mg/kg.
3.3. Leukocytes infiltration
The injection of
l carrageenan in the pleural space of rats in-
duces the appearance of an inflammatory exudate and the migra-
tion of various cells. Among these, leukocyte infiltration represents
a key step in the inflammatory response [43]. In this context, we
performed a count of the number of leukocytes located in the
pleural exudates (Table 3). Nimesulide (3), celecoxib (4) and com-
pound 6 were used as reference drugs. The decrease in the number
of leukocytes was statistically significant at the doses of 10 and
20 mg/kg for 3 and the unsubstituted compound 6 although the
activity of 3 appeared to be more pronounced (% decrease in the
number of leukocytes at the dose of 20 mg/kg for 3 and 6: 72.9 and
51.7%, respectively). 4 exerted a weaker activity, which was only
statistically significant at the higher dose investigated. Moreover, 4
did not exhibit a dose-dependent profile. The substitution of the
phenyl ring of 6 at the 4-position by a chlorine atom (compound 30)
provided a compound that displayed a decrease in the leukocyte
migration similar to that of 3 at 10 and 20 mg/kg. However, at the
dose of 5 mg/kg, 30 presented a statistically significant inhibitory
activity of 44.2%. The introduction of a halogen atom at the 3-
position seemed detrimental to the activity against leukocyte
infiltration. The only representative of the methanesulfonamide
series (compound 32) evaluated failed to prevent the leukocyte
migration at all the doses considered. The COX-2 preferential
compound 7, one of the most active compounds in the in vivo anti-
inflammatory study, exhibited only a weak and not statistically
significant decrease in the number of leukocytes regardless of the
dose administered to the animals. The introduction on the benzene
ring of a methyl moiety at the meta position provided a molecule
(33) sharing the same inhibitory profile than 6 at the dose of 20 mg/
kg while the decrease in the number of leukocytes was slightly
weaker at the lower doses. Concerning the disubstituted de-
rivatives, the introduction of a bromine atom at the 3-position and
a methyl group at the 4-position of the phenyl ring (compound
23b) was responsible for a statistically significant inhibitory activity
3.2. In vivo anti-inflammatory activity
Among the original derivatives evaluated in vitro, three disub-
stituted compounds (23b, 24a and 24b) were selected for further
in vivo studies. The anti-inflammatory activity of these molecules
was determined in the rat
l carrageenan-induced pleurisy model
and compared to nimesulide (3), celecoxib (4) and several previ-
ously reported unsubstituted or monosubstituted compounds (6, 7,
30e33) (Table 2). Compound 31 was found to display an anti-
inflammatory activity similar to that of 4 at the dose of 5 mg/kg
i.p. At 10 and 20 mg/kg i.p., the decrease in the volume of exudate
was higher for 31 than for compound 4 but failed to reach the ef-
ficiency of 3. The introduction of a fluorine atom at the 4-position of
the phenyl ring of 31 providing compound 24a enhanced the anti-
inflammatory activity, which was statistically significant for all
investigated doses (p < 0.01). 24a was even the most active com-
pound at the higher doses evaluated (% inhibition at 10 and 20 mg/
kg for 24a: 67.5 and 73.6%, respectively). The introduction of a
fluorine atom to the brominated derivative 7 leading to 24b did not
increase the anti-inflammatory activity. However, compound 24b
Table 2
Table 3
In vivo anti-inflammatory activity of and selected compounds at 5, 10 and 20 mg/kg
i.p. in the rat carrageenan-induced pleurisy (mean ꢀ SE, n ¼ 3e6 rats/group).
Percentage of reduction in the number of leukocytes found in the pleural space after
i.p. administration of selected compounds at 5, 10 and 20 mg/kg in the rat
carrageenan-induced pleurisy (mean ꢀ SE, n ¼ 3e6 rats/group).
Compounds
% Inhibition of exudate
Compounds
% Decrease in the number of leukocytes
5 mg/kg
10 mg/kg
20 mg/kg
5 mg/kg
10 mg/kg
20 mg/kg
3
4
31.05 ꢀ 7.85**
15.67 ꢀ 5.99
25.41 ꢀ 3.30
42.07 ꢀ 3.74**
39.39 ꢀ 3.65**
16.14 ꢀ 11.42
11.48 ꢀ 6.04
23.50 ꢀ 13.75
19.43 ꢀ 6.36
36.57 ꢀ 4.84**
21.94 ꢀ 8.73
48.44 ꢀ 3.09**
27.32 ꢀ 5.60*
55.68 ꢀ 9.50**
52.16 ꢀ 2.63**
51.41 ꢀ 5.28**
40.03 ꢀ 4.76*
18.31 ꢀ 7.74
61.53 ꢀ 6.75**
42.09 ꢀ 9.70**
72.20 ꢀ 6.28**
64.52 ꢀ 3.21**
68.83 ꢀ 9.95**
52.60 ꢀ 4.17**
25.46 ꢀ 2.50
3
4
6
7
30
31
32
33
23b
24a
24b
23.08 ꢀ 8.35
24.02 ꢀ 5.95
24.82 ꢀ 5.06
27.10 ꢀ 8.17
44.15 ꢀ 3.50**
NI
52.05 ꢀ 15.39*
23.05 ꢀ 7.44
34.83 ꢀ 9.53**
27.07 ꢀ 15.55
54.44 ꢀ 7.04**
NI
72.93 ꢀ 2.53*
28.87 ꢀ 8.04*
51.70 ꢀ 1.60**
28.52 ꢀ 6.80
72.47 ꢀ 2.76**
16.41 ꢀ 5.61
NI
50.18 ꢀ 6.30**
44.06 ꢀ 14.18*
64.71 ꢀ 16.69**
69.78 ꢀ 13.96**
6
7a
30a
31a
32a
33a
23b
24a
24b
50.29 ꢀ 15.04**
57.07 ꢀ 2.65**
67.46 ꢀ 3.79**
42.56 ꢀ 11.64**
69.53 ꢀ 4.17**
71.02 ꢀ 8.86**
73.55 ꢀ 3.91**
66.47 ꢀ 5.73**
NI
NI
17.07 ꢀ 9.73
24.98 ꢀ 7.56*
26.24 ꢀ 14.78
48.23 ꢀ 9.67**
41.22 ꢀ 28.59*
NI
14.74 ꢀ 4.77
11.13 ꢀ 10.08
*p < 0.05, **p < 0.01 vs control, one way ANOVA followed by a Dunnett’s multiple
comparison test.
*p < 0.05. **p < 0.01 vs control, one way ANOVA followed by a Dunnett’s multiple
comparison test.
a
Published results: Ref. [39].

J.-F. Renard et al. / European Journal of Medicinal Chemistry 74 (2014) 12e22
17
Table 4
against leukocyte infiltration only at the dose of 20 mg/kg when
compared to the brominated derivative 7. The fluorinated com-
pounds bearing a chlorine or a bromine atom at the 3-position
(compounds 24a and 24b) displayed a similar marked activity on
leukocytes infiltration. The decrease in the number of leukocytes
was significant at the doses of 10 and 20 mg/kg, and, at these doses,
their activity was comparable to that of 30 and nimesulide (3).
Plasma concentrations determined 4 h 30 after i.p. administration of the selected
compounds at 5, 10 and 20 mg/kg in rats (mean ꢀ SE, n ¼ 3e6).
Compounds
Plasma levels (
mg/mL)
5 mg/kg
10 mg/kg
20 mg/kg
3
4
6
7
30
31
32
33
23b
24a
24b
17.01 ꢀ 4.02
0.62 ꢀ 0.06
0.72 ꢀ 0.17
0.30 ꢀ 0.06
4.61 ꢀ 0.33
0.56 ꢀ 0.18
0.26 ꢀ 0.07
0.10 ꢀ 0.04
0.17 ꢀ 0.02
13.00 ꢀ 1.48
11.41 ꢀ 1.08
41.27 ꢀ 0.83
1.13 ꢀ 0.19
1.54 ꢀ 0.62
0.43 ꢀ 0.04
9.49 ꢀ 0.53
0.68 ꢀ 0.09
0.69 ꢀ 0.20
0.20 ꢀ 0.04
0.18 ꢀ 0.05
30.03 ꢀ 3.26
34.10 ꢀ 6.24
75.15 ꢀ 9.27
2.68 ꢀ 0.32
3.62 ꢀ 1.22
0.82 ꢀ 0.19
27.29 ꢀ 1.34
1.40 ꢀ 0.35
1.86 ꢀ 0.22
0.41 ꢀ 0.05
0.92 ꢀ 0.58
54.64 ꢀ 3.12
59.07 ꢀ 4.07
3.4. Determination of drug plasma levels
Blood samples were collected from rats at the end of the in vivo
experiments (4 h 30 after drug i.p. administration). Drug plasma
levels were measured using a SPE-HPLC method. Fig. 2 shows that
plasma levels of the pyridine analogs of nimesulide and 4 were
drastically lower than those measured with nimesulide (3) what-
ever the dose administered. Interestingly, this decrease in the
plasma levels was not observed with compounds substituted by a
halogen atom at the 4-position (compounds 30, 24a and 24b). The
values measured for each compound were reported in Table 4.
These data confirmed lower plasma concentrations for pyridine
analogs of nimesulide when compared to this latter. Regarding the
kind of substituent at the 3-position of the phenyl ring, a chlorine or
a bromine atom (31 and 7, respectively) did not modify plasma
levels while the amount of drug measured for 33 bearing a methyl
group at the same position was slightly lower. The acidic character
of the sulfonamide moiety also did not affect plasma concentration,
since plasma levels measured for the methanesulfonamide 32 were
similar to that of its trifluoromethanesulfonamide analog 31. On the
other hand, the position of the substituent on the phenyl ring
caused a significant impact on the plasma concentration. In this
way, compound 30 bearing a chlorine atom at the 4-position dis-
played plasma levels approximately 20 times greater than the 3-
chloro-substituted analog 31. Concerning the disubstituted de-
rivatives, it was interesting to observe that the introduction of a
methyl group at the 4-position of the benzene ring (compound
23b) did not alter plasma levels when compared to its mono-
substituted analog 7. However, results obtained with the disubsti-
tuted compounds 24a and 24b were clearly different. Compared to
the other pyridine analogs of nimesulide, these two compounds
exhibited the highest drug plasma levels. Of particular interest is
the fact that 24a and 24b, as observed with 30, bore a halogen atom
at the 4-position of the benzene ring.
second to the compound of interest. In order to check the selectivity
of the method used, we compared the retention time of the pyri-
dine analog of nimesulide 7 to its 4-hydroxylated derivative 28b.
Retention times measured for 7 and pentobarbital were 13.65 and
11.71 min, respectively, while 28b displayed a retention time of
10.34 min.
Taken together, these results indicate that the replacement of
the nitrobenzene ring of nimesulide by a pyridine one decreases the
plasma half-life. Neither the modulation of the acidic character of
the sulfonamide moiety nor the nature of the substituent at the 3-
postion of the phenyl ring can modify drugs plasma levels. How-
ever, the introduction of a halogen atom at the 4-position (com-
pounds 30, 24a and 24b) greatly enhanced the amount of drug
measured in the plasma at time point 4 h 30. These data suggest
that the 4-position of the benzene ring of our pyridine analogs of
nimesulide is the major site of metabolism. The presence of a
halogen atom at this position (see 30, 24a, 24b) blocks the meta-
bolic process and increases the plasma levels of the drugs. The
presence of a methyl group (see 23b) is not sufficient to block the
metabolism since such a group is also known to be sensitive to
metabolic oxidation into the carboxylic acid function [45].
4. Conclusions
In this study, we reported the synthesis of original pyridine an-
alogs of nimesulide. The inhibitory activity against both isoforms of
COXs was evaluated for all these molecules in a human whole blood
model. Results obtained with the monosubstituted derivatives
demonstrated that the highest inhibitoryactivity was observed with
halogen derivatives in the trifluoromethanesulfonamide series.
Among the latter compounds, those bearing a bulky halogen atom
(compounds 7 and 19c) displayed the most important activity (COX-
Invivo, nimesulide is metabolized into five species. The structure
of one of these metabolites was identified as 4-hydroxynimesulide
(OH introduced at the 4-position of the benzene ring) [44]. On the
chromatograms obtained, we observed only two peaks: the first
corresponding to the anesthetic drug used (pentobarbital) and the
2 IC50 values for 7 and 19c: 0.12 and 0.26 mM, respectively) and
selectivity against COX-2 (COX-2 selectivity ratio for 7 and 19c: 7.48
and 7.46, respectively). According to this first set of results, we
selected a halogen atom and a methyl group as substituents on the
phenyl ring to prepare disubstituted compounds. From those, 23b
displayed the highest COX-2 selectivity ratio observed during this
study (COX-2 selectivity ratio for 23b: 15.35).
In a second step, we investigated the ability of three disubsti-
tuted compounds to reduce inflammation in an acute model of
inflammation. All these compounds exhibited a strong anti-
inflammatory activity in vivo at the doses of 10 and 20 mg/kg.
Leukocyte count in the pleural exudate demonstrated that com-
pounds bearing a halogen atom at the 4-position of the phenyl ring
were the most active against leukocyte infiltration. Surprisingly,
some compounds displaying strong COXs inhibitory activity in vitro
and an important anti-inflammatory activity in vivo did not
Fig. 2. Plasma concentrations determined in rats 4 h 30 after i.p. administration of the
selected compounds at 5, 10 and 20 mg/kg (mean, n ¼ 3e6 rats/group).

18
J.-F. Renard et al. / European Journal of Medicinal Chemistry 74 (2014) 12e22
significantly decrease the number of leukocytes located in the
pleural exudate.
5.2.1. 4-Nitro-3-(3-fluorophenylamino)pyridine N-oxide (11a)
Yield: 28%; mp: 199e200 ꢂC; 1H NMR (DMSO-d6):
7.12 (m, 1H,
d
Monosubstituted compounds demonstrated that the replace-
ment of the nitrobenzene ring of nimesulide by a pyridine nu-
cleus drastically decreased drug plasma levels at time point 4 h
30 regardless of the pharmacomodulation considered except for
the substitution of the benzene ring at the 4-position by a
halogen atom (compounds 30, 24a and 24b). An explanation of
the higher plasma levels measured with such compounds is that
the presence of a halogen atom at the 4-position of the phenyl
ring constitutes an obstacle to the metabolic way leading to hy-
droxylation of this position. The increase of metabolic stability
could explain that the in vivo activity of these 4-halo-substituted
compounds was not always correlated with the in vitro COXs
inhibitory potency.
In summary, the synthesis of new pyridine analogs of nimesu-
lide was achieved and provided several compounds with a COX-2
preferential profile. Among them, compound 23b was found to
exhibit a COX-2 selectivity ratio higher than that of nimesulide (3)
and celecoxib (4). According to plasma assays, the para-position of
the phenyl ring of our compounds was identified as a possible
metabolism site (para-hydroxylation), explaining the poor corre-
lation observed with some compounds between in vitro and in vivo
activities.
H-40), 7.26 (m, J ¼ 7.5 Hz, 1H, H-60), 7.32 (d, 1H, H-20), 7.49 (dt,
J ¼ 7.5 Hz/1 Hz,1-H, H-50), 7.70 (d, J ¼ 7.5 Hz,1H, H-6), 7.82 (s, 1H, H-
2), 8.12 (d, J ¼ 7.5 Hz, 1H, H-5), 9.57 (s, 1H, NH). Anal. (C11H8FN3O3)
theoretical: C, 53.02; H, 3.24; N, 16.86. Found: C, 53.34; H, 3.26; N,
16.68.
5.3. General procedure for the synthesis of 4-amino-3-
arylaminopyridines (15e18)
The appropriate 4-nitro-3-arylaminopyridine N-oxide de-
rivatives (11e14, 1.2 g) were dissolved in a mixture of acetic acid
(16 mL) and water (4 mL). The solution was refluxed and iron
powder (6 eq.) was added. The suspension was stirred overnight
under reflux and then filtered on CeliteÒ. The pH of the filtrate was
adjusted to 12 and the suspension was extracted three times with
ethyl acetate (50 mL). The organic layers were dried over magne-
sium sulfate and evaporated under reduced pressure. The 4-amino-
3-arylaminopyridines were obtained as oils and were used without
further purification.
5.3.1. 4-Amino-3-(3-fluorophenylamino)pyridine (15a)
Yield: 92%; 1H NMR (DMSO-d6):
d 5.75 (s, 2H, NH2), 6.27 (m, 1H,
H-20), 6.43 (m, 2H, H-40 þ H-60), 6.65 (d, J ¼ 5.5 Hz,1H, H-5), 7.13 (dt,
J ¼ 8.0 Hz/1 Hz, 1H, H-50), 7.55 (s, 1H, NH), 7.90 (d, J ¼ 5.5 Hz, 1H, H-
6), 7.97 (s, 1H, H-2).
5. Experimental section
5.1. Chemistry
5.4. General procedure for the synthesis of N-(3-arylaminopyridin-
4-yl)alkanesulfonamides (19e24, 27b)
All commercial chemicals (SigmaeAldrich, Belgium, Appolo
Scientific, United Kingdom and Fluorochem, United Kingdom) and
solvents are reagent grade and were used without further puri-
fication. 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 PerkineElmer 1000 FTIR spec-
trophotometer. NMR spectra were recorded on a Bruker Avance
(500 MHz) spectrometer using DMSO-d6 as solvent and tetra-
methylsilane as internal standard; chemical shifts were reported
The appropriate 4-amino-3-arylaminopyridine (15e18, 3 mmol)
was dissolved in dry acetonitrile (20 mL). Anhydrous potassium
carbonate (18 mmol) was added and the suspension was stirred for
5 min. The appropriate alkanesulfonyl chloride (3.6 mmol) was
added to the mixture. At the end of the reaction (0.5e5 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 the resulting suspension was filtered. The
filtrate was adjusted to pH 6 with 6 N HCl and the crude product,
which precipitated, was collected by filtration, and crystallized in
acetone/water or purified by column chromatography on silicagel
using chloroform/methanol mixture (18:2) as eluent to give the N-
(3-arylaminopyridin-4-yl)alkanesulfonamide as a white solid.
in
d values (ppm) relative to internal tetramethylsilane. The
abbreviation s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quadruplet,
m ¼ multiplet and bs ¼ broad signal were used throughout.
Elemental analyses (C, H, N, S) were determined on a Thermo
Flash EA 1112 series elemental analyser and were within ꢀ0.4% of
the theoretical values. All reactions were followed by TLC (silicagel
60F254 Merck) and visualization was accomplished with UV light
(254 or 366 nm).
5.4.1. N-[3-(3-Fluorophenylamino)pyridin-4-yl]
Supplementary data section reports the synthesis of 3-bromo-4-
nitropyridine N-oxide (10) as well as the physicochemical charac-
terization of the 4-nitro-3-arylaminopyridine N-oxides (11e14) and
of the 4-amino-3-arylaminopyridines (15e18). The general syn-
thetic procedures and one example of compound are only given
below.
trifluoromethanesulfonamide (19a)
Yield: 51%; mp: 179e181 ꢂC; 1H NMR (DMSO-d6):
d 6.78 (td,
J ¼ 8.4, 2.1 Hz, 1H, H-40), 7.12 (m, 2H, H-20 þ H-60), 7.31 (dd, J ¼ 15.2,
8.1 Hz, 1H, H-50), 7.64 (m, 2H, H-5 þ NH), 8.02 (d, J ¼ 6.6 Hz, 1H, H-
6), 8.28 (s, 1H, H-2), 13.60 (bs, 1H, NHSO2CF3). 13C NMR (DMSO-d6):
d
163.86, 161.93, 152.20, 143.24, 143.15, 133.65, 133.26, 130.81,
130.74, 124.75, 123.89, 122.16, 119.56, 116.97, 114.68, 113.03, 108.30,
108.13, 105.80, 105.60. Anal. (C12H9F4N3O2S) theoretical: C, 42.99;
H, 2.71; N, 12.53; S, 9.56. Found: C, 42.97; H, 2.74; N, 12.83; S, 9.67.
5.2. General procedure for the synthesis of 4-nitro-3-
arylaminopyridine N-oxides (11e14)
3-Bromo-4-nitropyridine N-oxide (10, 2 g, 9.13 mmol) was dis-
solved in pyridine (8 mL) and the appropriate aniline (27.4 mmol)
was added to the solution (compound was directly dissolved in the
aniline when it was a liquid). The resulting solution was heated at
70 ꢂC under stirring for 16e72 h. The solution was cooled on ice
bath under stirring and ethanol (50 mL) was added to the solution
till precipitation of the desired compound, which was collected by
filtration and washed with ethanol.
5.4.2. N-[3-(4-Fluorophenylamino)pyridin-4-yl]
trifluoromethanesulfonamide (19b)
Yield: 54%; mp: 127e128 ꢂC; 1H NMR (DMSO-d6):
d 7.20 (t,
J ¼ 8.8 Hz, 2H, H-20 þ H-60), 7.33 (m, 2H, H-30 þ H-50), 7.40 (s, 1H,
NH), 7.60 (d, J ¼ 6.5 Hz, 1H, H-5), 7.94 (d, J ¼ 6.5 Hz, 1H, H-6), 7.97 (s,
1H, H-2), 12.50 (bs, 1H, NHSO2CF3). 13C NMR (DMSO-d6):
d 159.06,
157.16, 150.69, 136.79, 136.77, 135.68, 132.15, 124.81, 123.02, 122.95,
122.22, 120.67, 119.62, 117.02, 116.06, 115.88, 112.54. Anal.

J.-F. Renard et al. / European Journal of Medicinal Chemistry 74 (2014) 12e22
19
(C12H9F4N3O2S) theoretical: C, 42.99; H, 2.71; N, 12.53; S, 9.56.
Found: C, 42.88; H, 2.83; N, 12.58; S, 9.60.
54.35; H, 6.19; N,13.58; S,13.79. Found: C, 54.51; H, 6.12; N,13.79; S,
10.66.
5.4.3. N-[3-(3-Iodophenylamino)pyridin-4-yl]
5.4.9. N-[3-(3,4-Dimethylphenylamino)pyridin-4-yl]
trifluoromethanesulfonamide (19c)
methanesulfonamide (22b)
Yield: 77%; mp: 243e244 ꢂC; 1H NMR (DMSO-d6):
d
7.08 (t,
Yield: 35%; mp: 186e188 ꢂC; 1H NMR (DMSO-d6):
d 2.18 (s, 3H,
J ¼ 8.0 Hz, 1H, H-50), 7.28 (d, J ¼ 8.1 Hz, 1H, H-60), 7.32 (d, J ¼ 7. Hz,
30-CH3), 2.20 (s, 3H, 40-CH3), 2.89 (s, 3H, SO2CH3), 6.97 (d, J ¼ 8 Hz,
1H, H-60), 7.07 (m, 3H, H-20 þ H-50 þ NH), 7.19 (d, J ¼ 6.7 Hz, 1H, H-
5), 7.73 (d, J ¼ 6.7, 1H, H-6), 7.82 (s, 1H, H-2), 12.48 (bs, 1H,
1H, H-40), 7.57 (s, 1H, NH), 7.63 (m, 2H, H-20 þ H-5), 7.98 (d,
J ¼ 12.9 Hz, 1H, H-6), 8.20 (s, 1H, H-2), 13.54 (bs, 1H, NHSO2CF3). 13
C
NMR (DMSO-d6):
d
152.09, 142.90, 133.74, 133.37, 131.10, 130.34,
NHSO2CH3). 13C NMR (DMSO-d6):
d 138.59, 137.22 (2C), 132.65,
127.40, 124.76, 123.99, 122.17, 119.57, 118.18, 116.97, 113.03, 95.29.
Anal. (C12H9F3IN3O2S) theoretical: C, 32.52; H, 2.05; N, 9.48; S, 7.23.
Found: C, 32.76; H, 2.20; N, 9.72; S, 7.44.
131.57, 130.24, 129.88, 121.07, 116.97 (2C), 108.43, 41.01, 19.49, 18.64.
Anal. (C14H17N3O2S) theoretical: C, 57.71; H, 5.88; N, 14.42; S, 11.00.
Found: C, 57.99; H, 5.65; N, 14.72; S, 10.97.
5.4.4. N-[3-(3-Fluorophenylamino)pyridin-4-yl]
5.4.10. N-[3-(3-Chloro-4-methylphenylamino)pyridin-4-yl]
methanesulfonamide monohydrate (20a)
trifluoromethanesulfonamide (23a)
Yield: 50%; mp: 202e204 ꢂC; 1H NMR (DMSO-d6):
d
2.89 (s, 3H,
Yield: 32%; mp: 159e160 ꢂC; 1H NMR (DMSO-d6):
d 2.28 (s, 3H,
SO2CH3), 6.71 (td, J ¼ 8.5 Hz, 1H, H-40), 7.04 (m, 2H, H-20 þ H-60),
7.28 (m, 2H, H-50 þ H-5), 7.43 (s, 1H, NH), 7.79 (d, J ¼ 6.7 Hz, 1H, H-
6), 8.03 (s, 1H, H-2), 12.46 (bs, 1H, NHSO2CH3). 13C NMR (DMSO-d6):
CH3), 7.17 (dd, J ¼ 8.2 Hz/2.2 Hz, 1H, H-6’), 7.28 (d, J ¼ 8.3 Hz, 1H, H-
50), 7.36 (d, J ¼ 2.2 Hz, 1H, H-20), 7.49 (s, 1H, NH), 7.60 (d, J ¼ 6.6 Hz,
1H, H-5), 7.97 (d, J ¼ 6.6 Hz, 1H, H-6), 8.13 (s, 1H, H-2), 13.51 (bs, 1H,
d
163.94, 162.02, 143.94, 143.86, 130.79, 130.71, 130.53, 113.61,
NHSO2CF3). 13C NMR (DMSO-d6):
d 151.43, 140.15, 134.51, 133.65,
109.34, 107.23, 107.06, 104.51, 104.31, 99.49, 41.07. Anal.
(C12H14FN3O3S) theoretical: C, 48.15; H, 4.71; N, 14.04; S, 10.71.
Found: C, 47.92; H, 4.97; N, 13.99; S, 11.02.
132.77, 131.62, 128.91, 124.79, 122.50, 122.19, 120.12, 119.60, 118.56,
117.00, 112.81, 18.82. Anal. (C13H11ClF3N3O2S) theoretical: C, 42.69;
H, 3.03; N, 11.49; S, 8.77. Found: C, 42.59; H, 3.33; N, 11.43; S, 8.76.
5.4.5. N-[3-(4-Fluorophenylamino)pyridin-4-yl]
5.4.11. N-[3-(3-Bromo-4-methylphenylamino)pyridin-4-yl]
methanesulfonamide (20b)
trifluoromethanesulfonamide (23b)
Yield: 42%; mp: 197e198 ꢂC; 1H NMR (DMSO-d6):
d
2.89 (s, 3H,
Yield: 38%; mp: 167e169 ꢂC; 1H NMR (DMSO-d6):
d 2.30 (s, 3H,
SO2CH3), 7.14e7.27 (m, 6H, H-20 þ H-30 þ H-50 þ H-60 þ H-5 þ NH),
7.73 (d, J ¼ 6.7 Hz, 1H, H-6), 7.78 (s, 1H, H-2), 12.50 (bs, 1H,
CH3), 7.22 (d, J ¼ 8.2 Hz, 1H, H-60), 7.28 (s, 1H, H-50), 7.52 (m, 2H, H-
20 þ NH), 7.60 (d, J ¼ 6.5 Hz, 1H, H-5), 7.97 (d, J ¼ 6.5 Hz, 1H, H-6),
8.16 (s, 1H, H-2), 13.53 (bs, 1H, NHSO2CF3). 13C NMR (DMSO-d6):
NHSO2CH3). 13C NMR (DMSO-d6):
d 158.38, 156.49, 137.63, 132.56,
132.29, 132.27, 121.49, 121.43, 115.98, 115.80, 108.75, 99.49, 41.03.
Anal. (C12H12FN3O2S) theoretical: C, 51.24; H, 4.30; N,14.94; S,11.40.
Found: C, 51.16; H, 4.41; N, 14.91; S, 11.13.
d 151.38, 140.25, 134.49, 132.88, 131.38, 130.68, 124.80, 124.40,
123.22, 122.20, 119.60, 119.04, 117.01, 112.83, 21.58. Anal.
(C13H11BrF3N3O2S) theoretical: C, 38.06; H, 2.70; N, 10.24; S, 7.82.
Found: C, 37.83; H, 2.79; N, 10.13; S, 7.68.
5.4.6. N-[3-(3,5-Dimethylphenylamino)pyridin-4-yl]
trifluoromethanesulfonamide (21a)
5.4.12. N-[3-(4-Bromo-3-methylphenylamino)pyridin-4-yl]
Yield: 59%; mp: 200e202 ꢂC; 1H NMR (DMSO-d6):
d
2.25 (s, 6H,
trifluoromethanesulfonamide (23c)
CH3), 6.72 (s, 1H, H-40), 6.92 (s, 2H, H-20 þ H-60), 7.22 (s, 1H, NH),
7.59 (d, J ¼ 6.5 Hz, 1H, H-5), 7.95 (d, J ¼ 6.6 Hz, 1H, H-6), 8.12 (s, 1H,
Yield: 35%; mp: 186e188 ꢂC; 1H NMR (DMSO-d6):
d 2.31 (s, 3H,
CH3), 7.07 (dd, J ¼ 8.6 Hz/2.7 Hz, 1H, H-60), 7.29 (d, J ¼ 2.5 Hz, 1H, H-
H-2), 13.46 (bs, 1H, NHSO2CF3). 13C NMR (DMSO-d6):
d 150.67,
20), 7.47 (m, 2H, H-50 þ NH), 7.60 (d, J ¼ 6.6 Hz, 1H, H-5), 7.97 (d,
140.18, 138.61, 134.88, 132.17, 124.78, 124.38, 122.18, 121.17, 119.59,
117.64, 116.99, 112.46, 20.94. Anal. (C14H14F3N3O2S) theoretical: C,
48.69; H, 4.09; N, 12.17; S, 9.28. Found: C, 48.72; H, 4.10; N, 12.20; S,
9.48.
J ¼ 6.6 Hz, 1H, H-6), 8.16 (s, 1H, H-2), 13.53 (bs, 1H, NHSO2CF3). 13
C
NMR (DMSO-d6):
d 151.51, 140.36, 138.11, 134.32, 132.71, 132.63,
124.77, 122.43, 122.16, 119.58, 119.10, 116.99, 116.33, 112.76, 22.44.
Anal. (C13H11BrF3N3O2S) theoretical: C, 38.06; H, 2.70; N, 10.24; S,
7.82. Found: C, 38.44; H, 2.88; N, 10.31; S, 7.43.
5.4.7. N-[3-(3,4-Dimethylphenylamino)pyridin-4-yl]
trifluoromethanesulfonamide (21b)
5.4.13. N-[3-(3-Chloro-4-fluorophenylamino)pyridin-4-yl]
trifluoromethanesulfonamide (24a)
Yield: 44%; mp: 185e186 ꢂC; 1H NMR (DMSO-d6):
d 2.19 (s, 3H,
30-CH3), 2.21 (s, 3H, 40-CH3), 7.03 (d, J ¼ 8.0 Hz, 1H, H-60), 7.11 (m,
2H, H-20 þ H-50), 7.19 (s, 1H, NH), 7.58 (d, J ¼ 6.5 Hz, 1H, H-5), 7.92
(d, J ¼ 6.5 Hz, 1H, H-6), 8.00 (s, 1H, H-2), 13.45 (bs, 1H, NHSO2CF3).
Yield: 67%; mp: 202e203 ꢂC; 1H NMR (DMSO-d6):
d 7.27 (m, 1H,
H-20), 7.35 (t, J ¼ 9 Hz, 1H, H-50), 7.50 (dd, J ¼ 6.5 Hz/2.6 Hz, 1H, H-
20), 7.61 (m, 3H, H-2 þ H-5 þ NH), 7.98 (d, J ¼ 6 Hz, 1H, H-6), 8.12 (s,
13C NMR (DMSO-d6):
d
150.22, 137.75, 137.36, 135.62, 131.80, 131.03,
1H, H-2), 13.53 (bs, 1H, NHSO2CF3). 13C NMR (DMSO-d6):
d 153.71,
130.30, 124.81, 122.22, 122.15, 120.12, 119.62, 118.18, 117.03, 112.34,
19.43, 18.69. Anal. (C14H14F3N3O2S) theoretical: C, 48.69; H, 4.09; N,
12.17; S, 9.28. Found: C, 48.89; H, 4.01; N, 12.45; S, 9.39.
151.82, 151.80, 138.49, 138.47, 134.56, 132.92, 124.76, 122.90, 122.17,
121.76, 120.40, 120.35, 119.82, 119.67, 119.57, 117.29, 117.11, 116.97,
112.89. Anal. (C12H8ClF4N3O2S) theoretical: C, 39.98; H, 2.18; N,
11.37; S, 8.67. Found: C, 39.32; H, 2.39; N, 11.21; S, 8.31.
5.4.8. N-[3-(3,5-Dimethylphenylamino)pyridin-4-yl]
methanesulfonamide monohydrate (22a)
5.4.14. N-[3-(3-Bromo-4-fluorophenylamino)pyridin-4-yl]
trifluoromethanesulfonamide (24b)
Yield: 12%; mp: 163e164 ꢂC; 1H NMR (DMSO-d6):
d 2.25 (s, 6H,
30-CH3 þ 50-CH3), 2.89 (s, 3H, SO2CH3), 6.62 (s, 1H, H-40), 6.82 (s, 2H,
Yield: 42%; mp: 210e211 ꢂC; 1H NMR (DMSO-d6):
d 7.31 (m, 2H,
H-20 þ H-60), 7.10 (s, 1H, NH), 7.21 (d, J ¼ 6.7 Hz, 1H, H-5), 7.74 (d,
H-50 þ H-60), 7.56 (s, 1H, NH), 7.61 (d, J ¼ 6.5 Hz, 2H, H-20 þ H-5),
7.97 (d, J ¼ 6.6 Hz, 1H, H-6), 8.10 (s, 1H, H-2), 13.51 (bs, 1H,
J ¼ 6.7 Hz, 1H, H-6), 7.91 (s, 1H, H-2), 12.40 (bs, 1H, NHSO2CH3). 13
C
NMR (DMSO-d6):
d
141.04, 138.48, 132.23, 131.87, 123.35, 116.52,
NHSO2CF3). 13C NMR (DMSO-d6):
d 154.79, 152.89, 151.73, 138.72,
108.67, 99.50, 41.06, 21.00. Anal. (C14H19N3O3S) theoretical: C,
138.70, 134.62, 132.93, 124.77, 124.58, 122.85, 122.17, 121.07, 121.01,

20
J.-F. Renard et al. / European Journal of Medicinal Chemistry 74 (2014) 12e22
119.57, 117.05, 116.86, 112.88, 108.30, 108.13. Anal. (C12H8BrF4N3O2S)
theoretical: C, 34.80; H, 1.95; N, 10.15; S, 7.74. Found: C, 34.76; H,
2.01; N, 9.91; S, 7.61.
temperature for 5 h. At the end of the reaction, water (100 mL) was
added to the mixture and the solution was neutralized with a
saturated solution of sodium bicarbonate. The suspension was
extracted with diethyl ether (3 ꢃ 15 mL). The organic layers were
dried over magnesium sulfate and 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 6 N HCl
and the crude product, which precipitated, was collected by filtra-
tion. Compound 28a was purified by column chromatography on
silicagel using chloroform/methanol mixture (18.5:1.5) as eluent to
give the product as a white solid. Yield: 73%; mp: 229e231 ꢂC; 1H
NMR (DMSO-d6): 6.77 (d, J ¼ 8.6 Hz, 2H, H-20 þ H-60), 7.05 (s,1H, NH),
7.11 (d, J ¼ 8.6 Hz, 2H, H-30 þ H-50), 7.54 (d, J ¼ 6.5 Hz,1H, H-5), 7.70 (s,
1H, H-2), 7.87 (d, J ¼ 6.5 Hz, 1H, H-6), 9.36 (s, 1H, OH), 13.40 (bs, 1H,
5.4.15. N-[3-(3-Bromo-4-methoxyphenylamino)pyridin-4-yl]
trifluoromethanesulfonamide (27b)
Yield: 63%; mp: 232e234 ꢂC; 1H NMR (DMSO-d6):
d 3.84 (s, 3H,
OCH3), 7.10 (d, J ¼ 8.9 Hz,1H, H-50), 7.30 (dd, J ¼ 8.8 Hz/2.6 Hz,1H, H-
60), 7.34 (s,1H, NH), 7.55 (d, J ¼ 2.6 Hz,1H, H-20), 7.57 (d, J ¼ 6.5 Hz,1H,
H-5), 7.89 (s, 1H, H-2), 7.92 (d, J ¼ 6.6 Hz, 1H, H-6), 13.44 (bs, 1H,
NHSO2CF3). 13C NMR (DMSO-d6):
d 151.72, 150.54, 136.09, 134.33,
131.88, 126.63, 124.82, 122.36, 122.22, 120.31, 119.62, 117.03, 113.21,
112.44, 110.87, 56.39. Anal. (C13H11BrF3N3O3S) theoretical: C, 36.63;
H, 2.60; N, 9.86; S, 7.52. Found: C, 36.23; H, 2.60; N, 9.46; S, 7.23.
NHSO2CF3). 13C NMR (DMSO-d6):
d 154.23, 149.49, 137.37, 131.09,
5.5. N-(Cyclopropylsulfonyl)-N-[3-(phenylamino)pyridin-4-yl]
cyclopropanesulfonamide (25)
130.97, 124.84, 124.53, 122.25, 119.65, 118.59, 117.05, 115.99, 112.10.
Anal. (C12H10F3N3O3S) theoretical: C, 43.24; H, 3.02; N,12.61; S, 9.62.
Found: C, 42.87; H, 3.07; N, 12.51; S, 9.54.
4-Amino-3-phenylaminopyridine (500 mg, 2.70 mmol) [39] was
dissolved in methylene chloride (5 mL) and triethylamine (1.14 mL,
8.10 mmol) was added under stirring. Cyclopropanesulfonyl chlo-
5.8. N-[3-(3-Bromo-4-hydroxyphenylamino)pyridin-4-yl]
trifluoromethanesulfonamide (28b)
ride (687 mL, 6.75 mmol) was added to the mixture and the solution
was stirred for 1 h at room temperature. At the end of reaction, the
solution was washed with 1 N NaOH. The organic layer was dried
over magnesium sulfate and evaporated under reduced pressure.
The residue was taken up with ethyl acetate (20 mL) and filtered. The
filtrate was concentrated under reduced pressure and the residue
was purified by column chromatography on silicagel using ethyl
acetate as eluent to give compound 25 as a pale yellow solid. Yield:
The title compound was obtained following the experimental
procedure described for compound 28a starting from N-[3-(3-
bromo-4-methoxyphenylamino)pyridin-4-yl]tri-
fluoromethanesulfonamide (27b, 300 mg, 0.70 mmol). Yield: 57%;
mp: 253e254 ꢂC; 1H NMR (DMSO-d6): 6.94 (d, J ¼ 8.6 Hz, 1H, H-50),
7.14 (dd, J ¼ 8.6 Hz/2.3 Hz, 1H, H-60), 7.23 (s, 1H, NH), 7.45 (d,
J ¼ 2.1 Hz, 1H, H-20), 7.56 (d, J ¼ 5.6 Hz, 1H, H-5), 7.78 (d, 1H, H-2),
7.89 (d, J ¼ 6.5 Hz, 1H, H-6), 10.09 (s, 1H, OH), 13.49 (bs, 1H,
31%; mp: 152e153 ꢂC; 1H NMR (DMSO-d6):
d 0.53 (m, 4H, Hcyclo-
propyl), 1.06 (m, 4H, Hcyclopropyl), 1.89 (m, 1H, Hcyclopropyl), 2.95 (m, 1H,
NHSO2CF3). 13C NMR (DMSO-d6):
d 150.70, 150.15, 136.71, 132.55,
H
cyclopropyl), 7.01 (t, J ¼ 7.4 Hz, 1H, H-40), 7.16 (d, J ¼ 8.5 Hz, 2H, H-
131.46, 127.18, 124.81, 123.30, 122.22, 119.62, 119.46, 117.02, 116.65,
112.26, 109.36. Anal. (C12H9BrF3N3O3S) theoretical: C, 34.97; H,
2.20; N, 10.19; S, 7.78. Found: C, 35.34; H, 2.45; N, 9.89; S, 7.39.
20 þ H-60), 7.34 (t, J ¼ 8.4Hz, 2H, H-30 þ H-50), 7.71 (d, J ¼ 6.5Hz,1H, H-
5), 8.10 (d, J ¼ 6.6 Hz,1H, H-6), 8.28 (s,1H, H-2).13C NMR (DMSO-d6):
d
141.20, 133.79, 131.62, 129.54, 122.11, 118.57, 111.67, 30.86, 29.00,
5.36, 4.24. Anal. (C17H19N3O4S2) theoretical: C, 51.89; H, 4.87; N,
10.68; S, 16.29. Found: C, 51.52; H, 4.96; N, 10.34; S, 16.22.
5.9. N-[3-(3-Cyanophenylamino)pyridin-4-yl]
trifluoromethanesulfonamide (29)
5.6. N-[3-(Phenylamino)pyridin-4-yl]cyclopropanesulfonamide
N-[3-(3-Bromophenylamino)pyridin-4-yl]tri-
(26)
fluoromethanesulfonamide (7, 300 mg, 0.76 mmol) [39] was dis-
solved in anhydrous dimethylformamide (DMF, 5 mL) under
nitrogen. Copper (I) cyanide (170 mg, 1.89 mmol) was added and
the suspension was refluxed under stirring for 24 h. At the end of
the reaction, DMF (20 mL) was added to the mixture and the sus-
pension was filtered. The filtrate was then evaporated under
reduced pressure. The residue was taken up with a 10% aqueous
solution of NaOH (w/v) and purified with charcoal. After filtration,
the filtrate was neutralized by 6 N HCl. Compound 29 was then
purified by column chromatography on silicagel using ethyl acetate
as eluent to give the title compound as a white solid. Yield: 47%;
mp: 222e223 ꢂC; 1H NMR (DMSO-d6): 7.36 (d, J ¼ 7.6 Hz, 1H, H-40),
7.46 (t, J ¼ 7.9 Hz, 1H, H-50), 7.55 (dd, J ¼ 8.3 Hz/1.4 Hz, 1H, H-60),
7.64 (m, 2H, H-20 þ H-5), 7.82 (s, 1H, NH), 8.05 (d, J ¼ 6.7 Hz, 1H, H-
6), 8.30 (s, 1H, H-2), 13.60 (bs, 1H, NHSO2CF3). 13C NMR (DMSO-d6):
Compound 25 was suspended in a 50% aqueous solution of KOH
(10 mL, w/v). The suspension was refluxed under stirring for
15 min. At the end of the reaction, water (30 mL) was added and the
solution was washed with diethyl ether. The aqueous layer was
adjusted to pH 6 with 6 N HCl and extracted with ethyl acetate
(3 ꢃ 10 mL). The organic layers were dried over magnesium sulfate
and filtered. The volume of the filtrate was reduced under vacuum.
The concentrated solution was then cooled on an ice bath till pre-
cipitation of the title compound as a white solid. Yield: 72%; mp:
197e198 ꢂC; 1H NMR (DMSO-d6):
d 0.87 (m, 4H, Hcyclopropyl), 2.59
(m, 1H, Hcyclopropyl), 6.97 (t, J ¼ 7.3 Hz, 1H, H-40), 7.20 (m, 3H, H-
30 þ H-50 þ NH), 7.32 (m, 3H, H-20 þ H-60 þ H-5), 7.74 (d, J ¼ 6.1 Hz,
1H, H-6), 7.92 (s, 1H, H-2), 12.41 (bs, 1H, NHSO2cyclopropane). 13C NMR
(DMSO-d6):
d
141.28, 133.00, 131.74, 129.39, 121.58, 118.77, 109.28,
d 152.99, 142.70, 133.96, 132.99, 130.43, 125.61, 124.68, 123.41,
99.50, 30.73, 4.57. Anal. (C14H15N3O2S) theoretical: C, 58.11; H, 5.23;
N, 14.52; S, 11.08. Found: C, 58.11; H, 5.28; N, 14.39; S, 10.72.
123.18, 122.11, 120.97, 119.52, 118.82, 116.92, 113.30, 112.01. Anal.
(C13H9F3N4O2S) theoretical: C, 45.61; H, 2.65; N, 16.36; S, 9.37.
Found: C, 45.49; H, 2.62; N, 16.23; S, 9.04.
5.7. N-[3-(3-Hydroxyphenylamino)pyridin-4-yl]
trifluoromethanesulfonamide (28a)
5.10. Pharmacology
N-[3-(4-Methoxyphenylamino)pyridin-4-yl]tri-
fluoromethanesulfonamide (27a, 300 mg, 0.86 mmol) [39] was
5.10.1. In vitro COX-1 and COX-2 inhibitory activity determination
using human whole blood assay
suspended in methylene chloride (60 mL). Boron tribromide (816
8.46 mmol) was added and the suspension was stirred at room
m
L,
5.10.1.1. COX-1 assay. Fresh human blood was collected into hep-
arinized tubes by venipuncture and directly diluted with RPMI

J.-F. Renard et al. / European Journal of Medicinal Chemistry 74 (2014) 12e22
21
(Roswell Park Memorial Institute culture medium) (1:4). 0.25 mL
diluted blood aliquots were transferred into siliconized tubes pre-
centrifugation. The resulting supernatants (1 mL) were loaded onto
Isolute C18 (50 mg, 1 mL) solid phase extraction cartridges from
Biotage AB (Uppsala, Sweden). The SPE cartridges were previously
conditioned with methanol (1 mL) and then with 10 mM ammo-
nium phosphate buffer pH ¼ 3 (1 mL). The washing step was made
with 10 mM ammonium phosphate buffer pH ¼ 3 (1 mL) and then
with water (1 mL). After drying, the compounds were eluted with
loaded with 1
centration ranging from 0.01 to 100
vortexed and incubated at 37 ꢂC under constant agitation for 5 min
and then 10 L of calcium ionophore A-23,187 (0.2 mg/mL) were
m
L vehicle (DMSO) or test compounds at final con-
mmol/L. The tubes were
m
added and the aliquots were incubated at 37 ꢂC under constant
agitation for 15 min. At the end of the incubation, the serum was
obtained by centrifugation (3200 g for 5 min) and was assayed for
thromboxane B2 (TXB2) production using an enzyme immunoassay
kit (Assay design TXB2 Correlate-EIAÔ Kit, Ann Arbor, MI, USA)
according to the manufacturer’s instructions.
acetonitrile (500
mL) and 500 mL of ammonium formate buffer
(pH ¼ 3, 10 mM) was added to the collected fractions. Separation
was performed on a Sunfire C18 column (4.6 mm i.d. ꢃ 150 mm,
3.5 mm, Waters Inc., Milford, MA, USA) preceded by a Sunfire C18
guard column (4.6 mm i.d. ꢃ 10 mm, 3.5
m
m, Waters Inc.) at 30 ꢂC.
The mobile phase was a mixture of ammonium formate buffer
(pH ¼ 3, 10 mM) (A) and methanol (B) which were both degassed
by sparging with helium. The mobile phase was pumped at 1 mL/
min by using a linear gradient program [%B, 40e90% (0e15 min),
90% (15e19 min), 90e40% (19e19.1 min), 40% (19.1e30 min)]. The
5.10.1.2. COX-2 assay. Fresh human blood was collected in hepa-
rinized tubes by venipuncture. 1 mL aliquots of blood were trans-
ferred in heparinized tubes preloaded with either 4
(DMSO) or 4 L of test compound at final concentration ranging
from 0.01 to 100 mol/L. The tubes were vortexed and incubated at
37 ꢂC under constant agitation for 5 min. This first treatment was
followed by incubation of the blood with 20 L lipopolysaccharide
(LPS from Escherichia coli serotype B8) at final concentration of
100
g/mL under constant agitation for 24 h at 37 ꢂC, in order to
induce COX-2 expression. Appropriate controls (LPS replaced by
20 L PBS) were used as blanks. After incubation, the tubes were
mL vehicle
m
m
sample injection volume was set at 20 mL.
m
Acknowledgments
m
This study was supported by Belgian grants of the “Fonds pour la
Recherche dans l’Industrie et l’Agriculture (F.R.I.A.)” and the “Leon
Fredericq Foundation”.
m
centrifugated at 3200 g for 5 min. Plasma was assayed for PGE2
production by a specific enzyme immunoassay (Cayman Chemical
PGE2 EIA Kit, Ann Arbor, MI, USA) according to the manufacturer’s
instructions.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.033.
5.10.2. Carrageenan-induced pleurisy
5.10.2.1. Animals. Male Wistar rats, weighing 230e260 g were
housed in the University of Liège 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 Liège, Belgium.
References
[1] S. Jacob, L. Laury-Kleintop, S. Lanza-Jacoby, The select cyclooxygenase-2 in-
hibitor celecoxib reduced the extent of atherosclerosis in apo Eꢄ/ꢄ mice,
J. Surg. Res. 146 (2008) 135e142.
[2] T. Dassesse, X. de Leval, L. de Leval, B. Pirotte, V. Castronovo, D. Waltregny,
Activation of the thromboxane A2 pathway in human prostate cancer corre-
lates with tumor Gleason score and pathologic stage, Eur. Urol. 50 (2006)
1021e1031 discussion 1031.
[3] Y. Harrak, G. Casula, J. Basset, G. Rosell, S. Plescia, D. Raffa, M.G. Cusimano,
R. Pouplana, M.D. Pujol, Synthesis, anti-inflammatory activity, and in vitro
antitumor effect of a novel class of cyclooxygenase inhibitors: 4-(aryloyl)
phenyl methyl sulfones, J. Med. Chem. 53 (2010) 6560e6571.
[4] X. Liao, P. Lochhead, R. Nishihara, T. Morikawa, A. Kuchiba, M. Yamauchi,
Y. Imamura, Z.R. Qian, Y. Baba, K. Shima, R. Sun, K. Nosho, J.A. Meyerhardt,
E. Giovannucci, C.S. Fuchs, A.T. Chan, S. Ogino, Aspirin use, tumor PIK3CA
mutation, and colorectal-cancer survival, N. Engl. J. Med. 367 (2012) 1596e
1606.
5.10.2.2. Carrageenan-induced pleurisy. Rats were pretreated with
an intraperitoneal (i.p.) 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, i.p.)
and
l carrageenan (0.2 mL, 10 mg/mL) was administered into the
right pleural cavity. Each experimental group contained at least 3
animals. Four hours later, the animals were anaesthetized with
sodium pentobarbital (80 mg/kg, i.p.).
[5] N. Nadda, V. Vaish, S. Setia, S.N. Sanyal, Angiostatic role of the selective
cyclooxygenase-2 inhibitor etoricoxib (MK0663) in experimental lung cancer,
Biomed. Pharmacother. 66 (2012) 474e483.
[6] B. Takkouche, C. Regueira-Mendez, M. Etminan, Breast cancer and use of
nonsteroidal anti-inflammatory drugs: a meta-analysis, J. Natl. Cancer Inst.
100 (2008) 1439e1447.
[7] T. Kukar, T.E. Golde, Possible mechanisms of action of NSAIDs and related
compounds that modulate gamma-secretase cleavage, Curr. Top. Med. Chem.
8 (2008) 47e53.
[8] S. Weggen, M. Rogers, J. Eriksen, NSAIDs: small molecules for prevention of
Alzheimer’s disease or precursors for future drug development? Trends
Pharmacol. Sci. 28 (2007) 536e543.
The chest was carefully opened and the pleural cavity rinsed
with 2.0 mL 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 solution (2.0 mL) from the
total volume harvested. A sample of each exudate was diluted in
phosphate buffer (1:9) and total leukocytes count was performed
using a Horiba ABX Micros 60 OT hematology analyzer (Horiba Ltd.,
Kyoto, Japan).
[9] D.A. Kujubu, B.S. Fletcher, B.C. Varnum, R.W. Lim, H.R. Herschman, TIS10, a
phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes
a novel prostaglandin synthase/cyclooxygenase homologue, J. Biol. Chem. 266
(1991) 12866e12872.
[10] J.R. Vane, Inhibition of prostaglandin synthesis as a mechanism of action for
aspirin-like drugs, Nat. New. Biol. 231 (1971) 232e235.
[11] J.L. Wallace, Pathogenesis of NSAID-induced gastroduodenal mucosal injury,
Best. Pract. Res. Clin. Gastroenterol. 15 (2001) 691e703.
[12] C.J. Hawkey, Nonsteroidal anti-inflammatory drug gastropathy, Gastroenter-
ology 119 (2000) 521e535.
5.10.3. Plasma assays
Blood samples were collected from the abdominal aorta at the
end of the carrageenan-induced pleurisy experimentations (4 h 30
after drug administration). Plasma was separated by centrifugation
and stored frozen until analysis. Evaluated compounds were
extracted from plasma by a solid phase extraction procedure and
quantified by reverse phase HPLC with UV detection (250 nm).
Plasma samples (700
phosphate buffer pH ¼ 3 (700
mixer, the samples were left standing in the dark for 15 min prior to
[13] G.A. FitzGerald, C. Patrono, The coxibs, selective inhibitors of cyclooxygenase-
2, N. Engl. J. Med. 345 (2001) 433e442.
[14] A. Rostom, K. Muir, C. Dube, E. Jolicoeur, M. Boucher, J. Joyce, P. Tugwell,
G.W. Wells, Gastrointestinal safety of cyclooxygenase-2 inhibitors: a cochrane
m
L) were diluted with 50 mM ammonium
mL). After a brief stirring with a vortex

22
J.-F. Renard et al. / European Journal of Medicinal Chemistry 74 (2014) 12e22
collaboration systematic review, Clin. Gastroenterol. Hepatol. 5 (2007) 818e
828, 828 e811ee815; quiz 768.
A. Giordani, P. Anzellotti, P. Patrignani, M. Anzini, Novel ester and acid de-
rivatives of the 1,5-diarylpyrrole scaffold as anti-inflammatory and analgesic
agents. Synthesis and in vitro and in vivo biological evaluation, J. Med. Chem.
53 (2010) 723e733.
[15] C. Bombardier, L. Laine, A. Reicin, D. Shapiro, R. Burgos-Vargas, B. Davis,
R. Day, M.B. Ferraz, C.J. Hawkey, M.C. Hochberg, T.K. Kvien, T.J. Schnitzer,
V.S. Group, Comparison of upper gastrointestinal toxicity of rofecoxib and
naproxen in patients with rheumatoid arthritis. VIGOR Study Group, N. Engl. J.
Med. 343 (2000) 1520e1528, 1522p. following 1528.
[30] Z. Chen, Y. Wu, Y. Liu, S. Yang, Y. Chen, L. Lai, Discovery of dual target in-
hibitors against cyclooxygenases and leukotriene A4 hydrolyase, J. Med.
Chem. 54 (2011) 3650e3660.
[16] R.S. Bresalier, R.S. Sandler, H. Quan, J.A. Bolognese, B. Oxenius, K. Horgan,
C. Lines, R. Riddell, D. Morton, A. Lanas, M.A. Konstam, J.A. Baron,
I. Adenomatous polyp prevention on Vioxx trial, cardiovascular events asso-
ciated with rofecoxib in a colorectal adenoma chemoprevention trial, N. Engl.
J. Med. 352 (2005) 1092e1102.
[31] K. Yang, H. Bai, Q. Ouyang, L. Lai, C. Tang, Finding multiple target optimal
intervention in disease-related molecular network, Mol. Syst. Biol. 4 (2008)
228.
[32] K.F. Swingle, G.G. Moore, T.J. Grant, 4-Nitro-2-phenoxymethanesulfonanilide
(R-805): a chemically novel anti-inflammatory agent, Arch. Int. Pharmacodyn.
Ther. 221 (1976) 132e139.
[33] L. Cullen, L. Kelly, S.O. Connor, D.J. Fitzgerald, Selective cyclooxygenase-2 in-
hibition by nimesulide in man, J. Pharmacol. Exp. Ther. 287 (1998) 578e582.
[34] M.A. Macia, A. Carvajal, J.G. del Pozo, E. Vera, A. del Pino, Hepatotoxicity
associated with nimesulide: data from the Spanish Pharmacovigilance Sys-
tem, Clin. Pharmacol. Ther. 72 (2002) 596e597.
[35] G. Traversa, C. Bianchi, R. Da Cas, I. Abraha, F. Menniti-Ippolito, M. Venegoni,
Cohort study of hepatotoxicity associated with nimesulide and other non-
steroidal anti-inflammatory drugs, Br. Med. J. 327 (2003) 18e22.
[36] F. Li, M.D. Chordia, T. Huang, T.L. Macdonald, In vitro nimesulide studies to-
ward understanding idiosyncratic hepatotoxicity: diiminoquinone formation
and conjugation, Chem. Res. Toxicol. 22 (2009) 72e80.
[17] B.M. Psaty, C.D. Furberg, COX-2 inhibitors-lessons in drug safety, N. Engl. J.
Med. 352 (2005) 1133e1135.
[18] P. Prasit, Z. Wang, C. Brideau, C.C. Chan, S. Charleson, W. Cromlish, D. Ethier,
J.F. Evans, A.W. Ford-Hutchinson, J.Y. Gauthier, R. Gordon, J. Guay, M. Gresser,
S. Kargman, B. Kennedy, Y. Leblanc, S. Leger, J. Mancini, G.P. O’Neill, M. Ouellet,
M.D. Percival, H. Perrier, D. Riendeau, I. Rodger, R. Zamboni, et al., The dis-
covery of rofecoxib, [MK 966, Vioxx, 4-(4’-methylsulfonylphenyl)-3-phenyl-
2(5H)-furanone], an orally active cyclooxygenase-2-inhibitor, Bioorg. Med.
Chem. Lett. 9 (1999) 1773e1778.
[19] J.J. Talley, D.L. Brown, J.S. Carter, M.J. Graneto, C.M. Koboldt, J.L. Masferrer,
W.E. Perkins, R.S. Rogers, A.F. Shaffer, Y.Y. Zhang, B.S. Zweifel, K. Seibert, 4-[5-
Methyl-3-phenylisoxazol-4-yl]-benzenesulfonamide, valdecoxib:
a potent
and selective inhibitor of COX-2, J. Med. Chem. 43 (2000) 775e777.
[37] F. Julemont, X. de Leval, C. Michaux, J. Damas, C. Charlier, F. Durant, B. Pirotte,
J.M. Dogné, Spectral and crystallographic study of pyridinic analogues of
nimesulide: determination of the active form of methanesulfonamides as
COX-2 selective inhibitors, J. Med. Chem. 45 (2002) 5182e5185.
[38] F. Julemont, X. de Leval, C. Michaux, J.F. Renard, J.Y. Winum, J.L. Montero,
J. Damas, J.M. Dogné, B. Pirotte, Design, synthesis, and pharmacological
evaluation of pyridinic analogues of nimesulide as cyclooxygenase-2 selective
inhibitors, J. Med. Chem. 47 (2004) 6749e6759.
[20] X. de Leval, J. Hanson, J.L. David, B. Masereel, B. Pirotte, J.M. Dogné, New de-
velopments on thromboxane and prostacyclin modulators part II: prostacyclin
modulators, Curr. Med. Chem. 11 (2004) 1243e1252.
[21] J.M. Dogné, X. de Leval, J. Hanson, M. Frederich, B. Lambermont, A. Ghuysen,
A. Casini, B. Masereel, K.H. Ruan, B. Pirotte, P. Kolh, New developments on
thromboxane and prostacyclin modulators part I: thromboxane modulators,
Curr. Med. Chem. 11 (2004) 1223e1241.
[22] D. Wang, M. Wang, Y. Cheng, G.A. Fitzgerald, Cardiovascular hazard and non-
steroidal anti-inflammatory drugs, Curr. Opin. Pharmacol. 5 (2005) 204e210.
[23] D. Boschi, C. Cena, A. Di Stilo, B. Rolando, P. Manzini, R. Fruttero, A. Gasco,
Nitrooxymethyl-substituted analogues of rofecoxib: synthesis and pharma-
cological characterization, Chem. Biodivers. 7 (2010) 1173e1182.
[39] J.F. Renard, D. Arslan, N. Garbacki, B. Pirotte, X. de Leval, Pyridine analogues of
nimesulide: design, synthesis, and in vitro and in vivo pharmacological
evaluation as promising cyclooxygenase 1 and 2 inhibitors, J. Med. Chem. 52
(2009) 5864e5871.
[40] K.W. Rosenmund, E. Struck, Das am Ringkohlenstoff gebundene Halogen und
sein Ersatz durch andere substituenten. I. Mitteilung: Ersatz des Halogens
durch die Carboxylgruppe, Ber. Dtsch. Chem. Ges. A and B Ser. 52 (1919)
1749e1756.
[24] M.A. Chowdhury, K.R. Abdellatif, Y. Dong, G. Yu, Z. Huang, M. Rahman, D. Das,
C.A. Velazquez, M.R. Suresh, E.E. Knaus, Celecoxib analogs possessing a N-(4-
nitrooxybutyl)piperidin-4-yl
or
N-(4-nitrooxybutyl)-1,2,3,6-
tetrahydropyridin-4-yl nitric oxide donor moiety: synthesis, biological eval-
uation and nitric oxide release studies, Bioorg. Med. Chem. Lett. 20 (2010)
1324e1329.
[41] X. de Leval, J. Delarge, P. Devel, P. Neven, C. Michaux, B. Masereel, B. Pirotte,
J.L. David, Y. Henrotin, J.M. Dogné, Evaluation of classical NSAIDs and COX-2
selective inhibitors on purified ovine enzymes and human whole blood,
Prostaglandins Leukot. Essent. Fatty Acids 64 (2001) 211e216.
[42] N. Garbacki, M. Tits, L. Angenot, J. Damas, Inhibitory effects of proanthocya-
nidins from Ribes nigrum leaves on carrageenin acute inflammatory reactions
induced in rats, BMC Pharmacol. 4 (2004) 25.
[43] E. Corsini, R. Di Paola, B. Viviani, T. Genovese, E. Mazzon, L. Lucchi,
M. Marinovich, C.L. Galli, S. Cuzzocrea, Increased carrageenan-induced acute
lung inflammation in old rats, Immunology 115 (2005) 253e261.
[44] M. Carini, G. Aldini, R. Stefani, C. Marinello, R.M. Facino, Mass spectrometric
characterization and HPLC determination of the main urinary metabolites of
nimesulide in man, J. Pharm. Biomed. Anal. 18 (1998) 201e211.
[45] V.B. Reddy, G.A. Doss, B.V. Karanam, K. Samuel, T.J. Lanza Jr., L.S. Lin, N.X. Yu,
A.S. Zhang, C.E. Raab, R.A. Stearns, S. Kumar, In vitro and in vivo metabolism of
[25] Z. Huang, C.A. Velazquez, K.R. Abdellatif, M.A. Chowdhury, J.A. Reisz,
J.F. DuMond, S.B. King, E.E. Knaus, Ethanesulfohydroxamic acid ester prodrugs
of nonsteroidal anti-inflammatory drugs (NSAIDs): synthesis, nitric oxide and
nitroxyl release, cyclooxygenase inhibition, anti-inflammatory, and ulcer-
ogenicity index studies, J. Med. Chem. 54 (2011) 1356e1364.
[26] L. Lazzarato, K. Chegaev, E. Marini, B. Rolando, E. Borretto, S. Guglielmo,
S. Joseph, A. Di Stilo, R. Fruttero, A. Gasco, New nitric oxide or hydrogen
sulfide releasing aspirins, J. Med. Chem. 54 (2011) 5478e5484.
[27] R.C. Jin, J. Loscalzo, Vascular nitric oxide: formation and function, J. Blood Med.
2010 (2010) 147e162.
[28] J.L. Wallace, Hydrogen sulfide-releasing anti-inflammatory drugs, Trends
Pharmacol. Sci. 28 (2007) 501e505.
[29] M. Biava, G.C. Porretta, G. Poce, C. Battilocchio, F. Manetti, M. Botta, S. Forli,
L. Sautebin, A. Rossi, C. Pergola, C. Ghelardini, N. Galeotti, F. Makovec,
a
novel cannabinoid-1 receptor inverse agonist, taranabant, in rats and
monkeys, Xenobiotica 40 (2010) 650e662.