Synthesis and COX-2 inhibitory properties of N-phenyl- and N-benzyl-substituted amides of 2-(4-methylsulfonylphenyl)cyclopent-1-ene-1- carboxylic acid and of their pyrazole, thiophene and isoxazole analogs

Article abstract of DOI:10.1016/j.farmac.2003.09.003

Some N-phenyl- (7a-10a) and N-benzyl-substituted (7b-10b) amido analogs of cyclooxygenase (COX-2) selective tricyclic non-steroidal anti-inflammatory drugs have been synthesized with the aim to obtain information on the structural requirements for the COX

Full text of DOI:10.1016/j.farmac.2003.09.003

IL FARMACO 59 (2004) 25–31
www.elsevier.com/locate/farmac
Synthesis and COX-2 inhibitory properties of N-phenyl-
and N-benzyl-substituted amides
of 2-(4-methylsulfonylphenyl)cyclopent-1-ene-1-carboxylic acid
and of their pyrazole, thiophene and isoxazole analogs>
Simona Rapposelli a, Annalina Lapucci a, Filippo Minutolo a, Elisabetta Orlandini a,
Gabriella Ortore a, Mario Pinza b, Aldo Balsamo a,*
a Dipartimento di Scienze Farmaceutiche, Facoltà di Farmacia, Università di Pisa, Via Bonanno, 6, 56100 Pisa, Italy
b ACRAF, Angelini Ricerche, 00040 S. Palomba-Pomezia, Rome, Italy
Received 1 July 2003; accepted 5 September 2003
Abstract
Some N-phenyl- (7a–10a) and N-benzyl-substituted (7b–10b) amido analogs of cyclooxygenase (COX-2) selective tricyclic non-steroidal
anti-inflammatory drugs have been synthesized with the aim to obtain information on the structural requirements for the COX-inhibitory
activity. Compounds 7–10 were tested in vitro for their inhibitory properties only towards COX-2 enzyme by measuring prostaglandin E2
(PGE2) production on activated J774.2 macrophages. Some of the new compounds (7a, 8a, 9a and 9b) showed a modest activity, with
percentage inhibition values near 30% at a concentration of 10 µM. These data have been tentatively explained by a conformational study
which indicates that at least the N-phenyl-substituted amides 7a–9a present steric hindrances which may prevent a good interaction with
COX-2 active site.
© 2003 Elsevier SAS. All rights reserved.
Keywords: Anti-inflammatory drug; COX-2 inhibitor; NSAID
1. Introduction
constitutively expressed and is involved in the synthesis and
supply of the necessary arachidonic acid metabolites for a
maintenance of gastric and renal functions as well as for an
adequate vascular homeostasis, COX-2 is expressed only
after an inflammatory stimulus, releasing metabolites that are
used to induce inflammation and pain [4–6]. Most of the
non-steroidal anti-inflammatory drugs (NSAIDs) act by re-
ducing prostaglandin biosynthesis through the inhibition of
the COX reaction [7,8]. In spite of their beneficial action,
their activity is associated with deleterious side effects, and
continuous administration of these drugs leads to nefrotoxic-
ity and gastric ulcerations [9,10]. The therapeutic anti-
inflammatory action of NSAIDs is produced by inhibition of
COX-2, while the unwanted side effects arise from the inhi-
bition of COX-1 activity. Recently, some new NSAIDs pos-
sessing a good selectivity towards COX-2 have been de-
scribed. The common structural backbone of these COX-2
selective inhibitors (A, Fig. 1) consists of two aryl groups
linked to adjacent atoms of a central ring (X) which may be
Prostaglandins are endogenous substances involved in dif-
ferent processes of physiological nature, and are potent me-
diators of inflammation. Prostaglandins are produced, to-
gether with other prostanoids, in the arachidonic acid
metabolism, whose first step, consisting of the oxidative
conversion of arachidonic acid into prostaglandin H2, is
catalyzed by cyclooxygenase (COX) [1,2]. This enzyme ex-
ists at least as two isoforms, one constitutive (COX-1) and
the other inducible (COX-2) [2,3]. Thus, while COX-1 is
Abbreviations: NSAID, non-steroidal anti-inflammatory drug; COX,
cyclooxygenase; MAOMM, methyleneaminoxymethyl moiety.
> Part of this work was presented at the XVI Convegno Nazionale della
Divisione di Chimica Farmaceutica della Società Chimica Italiana, Sor-
rento, Italy, 18–22 September 2002.
* Corresponding author.
E-mail address: balsamo@farm.unipi.it (A. Balsamo).
© 2003 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2003.09.003

26
S. Rapposelli et al. / IL FARMACO 59 (2004) 25–31
SO2Y
R
R
R
O
R
O
N
HN
X
a
b
c
A
Fig. 3. Schematic representation of the possible bioisosterism between aryl
(a) and methyleneaminoxymethyl (MAOM) (b) or amidic group (c).
F
SO2Me
F
SO2Me
in Fig. 3) in many other classes of drugs in which this
aromatic system is considered important for the activity
[21–27]. Some of the new oxime-ethers of type B in which
X = cyclopentene (5) or thiophene (6) showed COX-2 inhibi-
tory properties, with satisfactory level of activity and COX-2
vs. COX-1 selectivity [18,20].
S
Br
1
2
(SC57666)
(DuP697)
These results suggested to widen the study of the effects
on the inhibitory activity towards COX enzymes due to a
further chemical manipulation at level of the non-sulfurated
aromatic ring of compounds of type A. The amido group (c in
Fig. 3) seemed to be suitable for this purpose since, as an aryl
ring, it presents a nearly planar geometry; furthermore, the
amido-moiety possess a p-system which may partially simu-
late the electronic system of an aryl.
Me
SO2NH2
SO2NH2
N
N
N
Me
O
CF3
4
3
On the basis of these considerations, we have designed
and synthesized some compounds of type C (Fig. 2) in which
the central core may be the cyclopentene (7) as in SC57666
(1), the thiophene (8) as in Dup697 (2), the pyrazole (9) as in
celecoxib (3) or the isoxazole (10) as in valdecoxib (4)
(Fig. 4). As R substituent we have chosen the phenyl (a) or
the benzyl (b) group which have been resulted important for
the activity as substituents of the oximic oxygen in type B
compounds (Fig. 2).
(valdecoxib)
(celecoxib)
Fig. 1. General formula (A) and structures of some COX-2 selective anti-
inflammatory drugs (1–4).
homocyclic or heteroaromatic, one of which is substituted in
the p-position with either an aminosulfonyl (Y = NH2) or a
methylsulfonyl (Y = Me) group. There are also examples of
potent COX-2 inhibitors that possess cycloalkyl [11], alkoxy
[12] or phenoxy [12,13] moieties in the non-sulfonyl con-
taining ‘aryl’region. The central rings most commonly found
within this class of molecules are cyclopentene (1) [11],
thiophene (2) [14], pyrazole (3) [15,16] and isoxazole (4)
[17] (Fig. 1).
The limited chemical diversity of these molecules
prompted us to search for new structures which, even though
different from that of the COX-2 tricyclic inhibitors of type
A, may possess analogs biopharmacological properties
[18–21]. Therefore, some type B compounds were synthe-
sized which may be viewed as analogs of 1,2-
diarylsubstituted NSAIDs of type A (Fig. 2) in which the
non-sulfurated aromatic ring is replaced with a methylene-
aminoxymethyl moiety (MAOMM). This kind of group (b in
Fig. 3) was previously described as bioequivalent of aryls (a
SO2Me
SO2Me
O
O
R
R
N
H
N
H
S
7a,b
8a,b
SO2Me
SO2Me
O
O
R
R
N
H
N
H
N
N
N
O
SO2Y
SO2Y
SO2Me
10a,b
9a,b
R
O
Ar
O
R
N
N
H
X
X
a: R = Ph
b: R = PhCH2
X
B
C
A
Fig. 4. Structures of the N-phenyl and N-benzyl-substituted amides of 2-[4-
(methylsulfonyl)phenyl]cyclopent-1-ene-1-carboxylic acid (7) and of their
pyrazole, thiophene and isoxazole analogs (8–10).
Fig. 2. General formulas of the tricyclic drugs (A) and of their MAOM (B)
and amidic (C) analogs.

S. Rapposelli et al. / IL FARMACO 59 (2004) 25–31
27
SMe
and a solution of NaH2PO4 (for the preparation of 17 and 19)
or by oxone® in THF–MeOH (for the preparation of 18). The
reaction of the carboxylic acids 17–19 with aniline or benzy-
lamine in the presence of N-hydroxybenzotriazole (HBT)
and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC)
yielded the corresponding amides 8–10 of type a or b, re-
spectively.
SO2Me
Br
HOOC
O
O
i
ii
13
iii
11
12
7a,b
3. Biological results and conclusion
Scheme 1. Synthesis of N-phenyl- (7a) and N-benzyl-substituted (7b) ami-
des of 2-[4-(methylsulfonyl)phenyl]cyclopent-1-ene-1-carboxylic acid. i:
4-(MeS)PhB(OH)2, Pd(PPh3)4, Na2CO3 2 M, EtOH–toluene, reflux; ii:
NaClO2, H2O2, NaH2PO4, CH3CN; iii: PhNH2 or PhCH2NH2, HBT, EDC,
THF, r.t., 24 h.
For the new compounds 7–10 the inhibitory activity to-
wards COX-2, which constitutes the ideal target of an anti-
inflammatory drug, was evaluated in vitro by measuring the
PGE2 production on activated J774.2 macrophages. The re-
sults were reported in Table 1 together with those obtained in
the same type of test with celecoxib (3) and with the previ-
ously studied cyclopentenyl and thienyl oxime-ethers 5 and
6. As it can be seen, only compounds 7a, 8a, 9a and 9b, at a
concentration of 10 µM showed a modest activity, with per-
centage inhibition values close to 30%, while the other ana-
logs resulted practically inactive. In the same experimental
conditions, the benzyloxyimino-ethers 5 and 6 showed IC50
values in the µM range.
These data indicate that in the field of the analogs of the
tricyclic anti-inflammatory drugs, an amidic group, either
N-phenyl- or N-benzyl-substituted, as the ones present in
type C compounds 7–10, is less effective than MAOMM in
replacing the aryl lacking of sulfurated moiety of compounds
of type A.
2. Chemistry
As far as the N-phenyl- (7a) and the N-benzyl-substituted
(7b) cyclopentene amides are concerned, they were prepared
as reported in Scheme 1. The cross-coupling reaction be-
tween the 2-bromocyclopent-1-ene-1-carbaldehyde 11 [18]
with p-thiomethylphenylboronic acid in toluene–EtOH solu-
tion in the presence of Pd(PPh3)4 and aqueous Na2CO3 af-
forded the 2-[4-(methylthio)phenyl]cyclopent-1-ene-1-
carbaldehyde 12. Compound 12 was oxidized in CH3CN by a
one-step procedure both at level of methylthio- and aldehy-
dic groups with NaClO2–H2O2 and aqueous solution of
NaH2PO4 to the methylsulfonyl-carboxylic acid 13 which
was then condensed with aniline or benzylamine to give the
amides 7a and 7b, respectively.
In order to attempt a possible rationalization of these
results, a conformational study was carried out on com-
pounds of the homogeneous series of the N-phenyl-
substituted amides 7a, 8a and 9a, which includes the larger
number of amidic compounds able to prove a certain ability
to interact with COX-2 enzyme. Fig. 5 shows the superimpo-
sition of the most favorable conformation found for each of
these compounds, together with those obtained for oxime-
ether derivative 5 and for celecoxib 3. From this figure it
results clear that for compounds 7a–9a the practically planar
amidic group occupies a spatial region which roughly corre-
sponds to the one which hosts both the aryl of celecoxib (3)
and the methyleneaminoxy portion of the MAOMM of com-
pound 5. Nevertheless, amidic compounds 7a–9a present an
additional steric hindrance, due to N-phenyl substituent, in a
spatial region, which is free in the more active compounds 3
and 5. Therefore, the lower activity of the amidic compounds
7–9, with respect to that of celecoxib (3) and the oxime-
ethers 5 and 6, may be tentatively attributed to this additional
steric hindrance, which may at least partially prevent their
good fit with the COX-2 active site.
The heteroaromatics phenyl- and benzyl amides 8–10
were prepared as shown in Scheme 2. The methylthiophenyl
carbaldehydes of thiophene (14), pyrazole (15) and isoxazole
(16) [20] were oxidized by a reaction with NaClO2–H2O2
SMe
SO2Me
HOOC
O
i
iii
8a,b
S
S
17
14
SMe
SO2Me
HOOC
O
N
N
ii
iii
9a,b
N
N
15
18
SO2Me
SO2Me
HOOC
O
i
iii
10a,b
N
N
4. Experimental procedures
O
O
16
19
4.1. Chemistry
Scheme 2. Synthesis of the thiophene (8a,b), pyrazole (9a,b) and isoxazole
(10a,b) analogs of 7a,b. i: NaClO2, H2O2, NaH2PO4, CH3CN; ii: oxone,
THF–MeOH, r.t., 6 h; iii: PhNH2 or PhCH2NH2, HBT, EDC, THF, r.t., 24 h.
Melting points were determined on a Kofler hot-stage
apparatus and are uncorrected. 1H NMR spectra of all com-

28
S. Rapposelli et al. / IL FARMACO 59 (2004) 25–31
Table 1
Chemical and biological data for compounds 7a,b–10a,b
SO2Me
SO2Me
O
R
O
N
H
N
X
X
5: X =
7a,b: X =
9a,b: X =
S
8a,b: X =
N
N
6: X =
S
10a,b: X =
N
O
Compound
R
m.p. (°C)
Recrystallizing
Formula b
In vitro inhibitory activity c COX-2
solvent a
7a
7b
5 d
PhNH
188–190
120–122
A
B
C
19H19NO3S
20H21NO3S
24%
7%
1.9
PhCH2NH
–
C
8a
8b
6 e
PhNH
209–211
164–166
B
C
C
18H15NO3S2
19H17NO3S2
28%
4%
1.7
PhCH2NH
–
C
9a
9b
10a
10b
PhNH
211–213
180–182
243–245
146–148
B
B
B
D
C17H15N3O3S
C18H17N3O3S
C17H14N2O4S
C18H16N2O4S
34%
26%
0
PhCH2NH
PhNH
PhCH2NH
0
3 (celecoxib)
0.02
a A, i-PrOH; B, EtOH; C, CHCl3–hexane; D, MeOH.
b All compounds were analyzed for C, H and N.
c Data are indicated as IC50 (µM) (for compounds 5, 6) or as percentage of inhibition at a concentration of 10µM.
d See Ref. [18].
e See Ref. [20].
pounds were obtained with a Gemini 200 spectrometer oper-
ating at 200 MHz, in a ca. 2% solution of CDCl3. Analytical
TLCs were carried out on 0.25 mm layer silica gel plates
containing a fluorescent indicator; spots were detected under
UV light (254 nm). Column chromatographies were per-
formed using 70–230 mesh silica gel. Mass spectra were
detected with a Hewlett Packard 5988A spectrometer.
Evaporations were made in vacuo (rotating evaporator).
Na2SO4 was always used as the drying agent. Elemental
analyzes were performed in our analytical laboratory and
agreed with the theoretical values to within 0.4%.
4.1.1. Synthesis of 2-[(4-methylthio)phenyl]cyclopent-1-
ene-1-carbaldehyde 12
A mixture of 2-bromocyclopent-1-ene-1-carbaldehyde 11
(1.0 g, 5.72 mmol) and 4-methylthiophenylboronic acid
(0.96 g, 5.72 mmol) in a 1:1 toluene–EtOH mixture (32 ml)
and aqueous 2 M Na2CO3 (12 ml) was treated with Pd-
(PPh3)4 (0.2 g) and then refluxed under argon with stirring for
12 h. The organic phase was then evaporated and the aqueous
residue was taken up in AcOEt, washed with H2O, filtered
and evaporated to afford a crude residue which was subjected
to column chromatography eluting with 2:8 hexane–AcOEt
5
1
mixture to yield pure 12 (70%): H NMR: d 2.00 (q, 2H,
J = 7.6 Hz, CH2); 2.51 (s, 3H, SMe); 2.75–2.97 (m, 4H,
CH2); 7.27 (s, 4H, Ar); 9.83 (s, 1H, CHO).
celecoxib
7a
8a
9a
4.1.2. Synthesis of 2-(4-methylsulfonylphenyl)cyclopent-1-
ene-1-carboxylic acid 13
A solution of 2-(4-methylthiophenyl)cyclopent-1-ene-1-
carbaldehyde 12 (0.10 g, 0.46 mmol) in CH3CN (0.5 ml) was
treated with NaH2PO4 (15 mg) in H2O (0.2 ml) and with a
Fig. 5. Superimposition of the N-phenyl-substituted amides 7a–9a, with
celecoxib (3) and cyclopentene MAOM derivative 5, in their lowest energy
conformations.

S. Rapposelli et al. / IL FARMACO 59 (2004) 25–31
29
solution of H2O2 30% (0.2 ml). The resulting solution was
then treated dropwise with NaClO2 (0.03 g, 0.33 mmol) in
H2O (1 ml). After 5 h of vigorous stirring at r.t., the mixture
was diluted with H2O and neutralized with aqueous NaHCO3
and extracted three times with CHCl3. The aqueous phase
was then acidified to pH 4 with a solution of HCl 10% and the
solid residue which precipitated was then collected and puri-
fied by crystallization from i-PrOH (25%): m.p. 184–186 °C;
1H NMR: d 2.04 (m, 2H, CH2); 2.80–2.91 (m, 4H, CH2); 3.07
(s, 3H, SO2Me); 7.48 and 7.89 (2d, 4H, J = 8.4 Hz,
ArSO2Me). Anal. C13H14O4S (C, H).
AcOEt, and washed with aqueous solutions of NaOH 10%
and HCl 10%. The organic phase was then dried and evapo-
rated to yield a residue, which was purified by crystallization
from the appropriate solvent. 7a (30%) 1H NMR: d 2.23 (m,
2H, CH2); 3.11 (s, 3H, SO2Me); 2.90–3.32 (m, 4H, CH2);
7.36–7.56 (m, 5H, Ph); 7.61 and 8.02 (2d, 4H, J = 8.2 Hz,
1
ArSO2). 7b (75%) H NMR: d 2.25 (m, 2H, CH2); 3.10 (s,
3H, SO2Me); 2.92–3.35 (m, 4H, CH2); 4.42 (d, 2H,
J = 5.6 Hz, PhCH2); 6.83 (bs, NH); 7.12–7.34 (m, 5H, Ph);
7.59 and 7.88 (2d, 4H, J = 8.2 Hz, ArSO2).
4.1.7. Synthesis of 3-(4-methylsulfonylphenyl)thiophene-2-
carboxylic acid phenylamide and 3-(4-methylsulfonylphe-
nyl)thiophene-2-carboxylic acid benzylamide 8a,b
4.1.3. Synthesis of 3-(4-methylsulfonylphenyl)thiophene-2-
carboxylic acid 17
Compound 17 was synthesized from 14 [20] following the
same procedure described above for the preparation of 13.
Compound 17 (50%): m.p. 163–165 °C (i-PrOH); 1H NMR:
d 3.11 (s, 3H, SO2Me); 7.09 (s, 1H, H4 thiophene); 7.62–7.68
(m, 3H, H5 thiophene and ArSO2); 7.97 (d, 2H, J = 8.4 Hz,
ArSO2). Anal. C12H10O4S2 (C, H).
Compounds 8a,b were synthesized from 17 following the
same procedure described above for the preparation of 7a,b.
Compound 8a (38%) H NMR: d 3.11 (s, 3H, SO2Me);
7.12 and 7.57 (2d, 2H, J = 5.1 Hz, H4 and H5 thiophene);
7.20–7.32 (m, 5H, Ph); 7.74 and 8.05 (2d, 4H, J = 8.4 Hz,
ArSO2). 8b (30%) 1H NMR: d 3.03 (s, 3H, SO2Me); 4.46 (d,
2H, J = 5.7 Hz, PhCH2); 5.65 (bs, NH); 7.06–7.50 (2d, 2H,
J = 5.1 Hz, H4 and H5 thiophene); 7.11–7.31 (m, 5H, Ph);
7.59 and 7.88 (2d, 4H, J = 8.2 Hz, ArSO2).
1
4.1.4. Synthesis of 1-(4-methylsulfonylphenyl)pyrazole-5-
carboxylic acid 18
A
cooled (0 °C) and stirred solution of 1-(4-
4.1.8. Synthesis of 3-(4-methylsulfonylphenyl)pyrazole-5-
carboxylic acid phenylamide and 3-(4-methylsulfonylphe-
nyl)pyrazole-5-carboxylic acid benzylamide 9a,b
methylthiophenyl)pyrazole-5-carbaldehyde 15 (0.67 g,
3.04 mmol) [20] in a 1:1 THF–MeOH mixture (18 ml) was
treated dropwise with a solution of oxone® (4.12 g,
6.70 mmol) in H2O (17 ml). The resulting mixture was
vigorously stirred for 6 h, and then diluted with AcOEt and
washed with water. The organic phase was extracted with a
solution of NaHCO3 and the aqueous extracts were com-
bined and acidified to pH 1 and extracted with AcOEt.
Evaporation of the organic phase gave a crude residue which
was purified by crystallization from CHCl3. Compound 18
(65%): m.p. 240–242 °C; 1H NMR (DMSO): d 3.31 (s, 3H,
SO2Me); 7.09 and 7.87 (2d, 2H, J = 2.7 Hz, H3 and H4
pyrazole); 7.78 and 8.03 (2d, 4H, J = 8.4 Hz, ArSO2Me).
Anal. C11H10N2O4S (C, H, N).
Compounds 9a,b were synthesized from 18 following the
same procedure described above for the preparation of 7a,b.
1
Compound 9a (72%) H NMR: d 3.08 (s, 3H, SO2Me);
6.88 and 7.78 (2d, 2H, J = 2.0 Hz, H3 and H4 pyrazole);
7.19–7.56 (m, 5H, Ph); 7.81 (bs, 1H, NH), 7.74 and 7.99 (2d,
1
4H, J = 8.6 Hz, ArSO2). MS (m/z) 341 (M+). 9b (38%) H
NMR: d 3.08 (s, 3H, SO2Me); 4.57 (d, 2H, J = 5.9 Hz,
PhCH2); 6.39 (bs, NH); 6.74 (d, 1H, J = 1.8 Hz, H4 pyra-
zole); 7.29–7.41 (m, 5H, Ph); 7.71–7.77 (m, 3H, ArSO2 and
H3 pyrazole); 7.99 (d, 2H, J = 8.6 Hz, ArSO2). MS (m/z) 355
(M+).
4.1.9. Synthesis of 3-(4-methylsulfonylphenyl)isoxazole-4-
carboxylic acid phenylamide and 3-(4-methylsulfonylphe-
nyl)isoxazole-4-carboxylic acid benzylamide 10a,b
Compounds 10a,b were synthesized from 19 following
the same procedure described above for the preparation of
7a,b. Compound 10a (38%) 1H NMR: d 3.10 (s, 3H,
SO2Me); 7.31–7.48 (m, 5H, Ph); 8.02–8.07 (m, 4H, SO2Me);
9.02 (s, 1H, H5 isoxazole). MS (m/z) 342 (M+). 10b (20%)
1H NMR: d 3.06 (s, 3H, SO2Me); 4.54 (d, 2H, J = 5.7 Hz,
PhCH2); 7.23–7.37 (m, 5H, Ph); 7.92 and 7.98 (2d, 4H,
J = 8.6 Hz,ArSO2); 8.88 (s, 1H, H5 isoxazole). MS (m/z) 356
(M+).
4.1.5. Synthesis of 3-(4-methylsulfonylphenyl)isoxazole-4-
carboxylic acid 19
Compound 19 was synthesized from 16 [20] following the
same procedure described above for the preparation of 13.
Compound 19 (47%): m.p. 219–221 °C (i-PrOH); 1H NMR:
d 3.11 (s, 3H, SO2Me); 8.05 (s, 4H, Ar); 9.14 (s, 1H, H5
isoxazole). Anal. C11H9NO5S (C, H, N).
4.1.6. Synthesis of 2-(4-methylsulfonylphenyl)cyclopent-1-
ene-1-carboxylic acid phenylamide and 2-(4-methylsulfo-
nylphenyl)cyclopent-1-ene-1-carboxylic acid benzylamide
7a,b
A solution of 13 (0.33 g, 1.25 mmol) in THF (17 ml) was
treated with the appropriate amine (1.25 mmol), HBT
(0.25 g, 1.87 mmol) and EDC (0.42 g, 2.20 mmol). The
resulting mixture was stirred at r.t. for 24 h. Evaporation of
the solvent afforded to a crude residue which was taken up in
4.2. Conformational studies
A conformational optimization on compounds celecoxib
(3), oxime-ether 5 and anilides 7a, 8a and 9a was performed

30
S. Rapposelli et al. / IL FARMACO 59 (2004) 25–31
[7] J.R. Vane, Inhibition of prostaglandin synthesis as a mechanism of
action for aspirin-like drugs, Nature 231 (1971) 232–235.
through the Gaussian 98 program [28] atAM1 semiempirical
level. A superimposition of the five-membered rings of these
compounds was then made and the results are shown in
Fig. 5.
[8] G. Lombardino, Nonsteroidal Antiinflammatory Drugs, John Wiley
and Sons, Wiley Interscience Eds, New York, 1985.
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4.3. Enzyme assays
[10] H.R. Herschman, Prostaglandin synthase 2, Biochim. Biophys. Acta.
1299 (1996) 125–140.
Compounds 7–10 were tested following the procedure
previously described [18] in intact cell assays to verify their
capacity to inhibit PGE2 production, considered as an index
of activity on COX-2 enzymes. The COX-2 assay was per-
formed in accordance with the method described by Mitchell
et al. [29] with minor modifications, as suggested by Gross-
man et al. [30]. Murine J774.2 cells were pretreated for 1 h
with 300 µM aspirin to inactivate endogenous constitutive
COX-1 and were then stimulated with LPS to induce COX-2
expression. After overnight incubation, cells were treated for
45 min with the different test compounds. Supernatants were
then collected and PGE2 was measured by a commercial
immunoenzymatic assay (Amersham). All compounds were
tested in duplicate. For each product, a stock solution was
prepared in DMSO at a concentration of 100 mM.
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