Synthesis of heteroaromatic analogues of (2-aryl-1-cyclopentenyl-1-alkylidene)-(arylmethyloxy)amine COX-2 inhibitors: Effects on the inhibitory activity of the replacement of the cyclopentene central core with pyrazole, thiophene or isoxazole ring

Article abstract of DOI:10.1016/S0223-5234(02)01448-4

Several heteroaromatic analogues of (2-aryl-1-cyclopentenyl-1-alkylidene)-(arylmethyloxy)amine COX-2 inhibitors, in which the cyclopentene moiety was replaced by pyrazole, thiophene or isoxazole ring, were synthesized, in order to verify the influence of the different nature of the central core on the COX inhibitory properties of these kinds of molecules. Among the compounds tested, only the 3-(p-methylsulfonylphenyl) substituted thiophene derivatives 17 and 22, showed a certain COX-2 inhibitory activity, accompanied by an appreciable COX-2 versus COX-1 selectivity. Only one of the 1-(p-methylsulfonylphenyl)pyrazole compounds (16) displayed a modest inhibitory activity towards both type of isoenzymes, while the pyrazole 1-(p-aminosulfonylphenyl) substituted 12 proved to be significantly active only towards COX-1. All the isoxazole derivatives were inactive on both COX isoforms.

Full text of DOI:10.1016/S0223-5234(02)01448-4

European Journal of Medicinal Chemistry 38 (2003) 157ꢀ168
/
www.elsevier.com/locate/ejmech
Original article
Synthesis of heteroaromatic analogues of (2-aryl-1-cyclopentenyl-1-
alkylidene)-(arylmethyloxy)amine COX-2 inhibitors: effects on the
inhibitory activity of the replacement of the cyclopentene central core
with pyrazole, thiophene or isoxazole ring
Aldo Balsamo a,*, Isabella Coletta b, Angelo Guglielmotti b, Carla Landolfi b,
Francesca Mancini b, Adriano Martinelli a, Claudio Milanese b, Filippo Minutolo a,
Susanna Nencetti a, Elisabetta Orlandini a, Mario Pinza b, Simona Rapposelli a,
Armando Rossello a
a Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, via Bonanno 6, 56126 Pisa, Italy
b ACRAF *
/
Angelini Ricerche, 00040 S. Palombaꢀ
/
Pomezia, Rome, Italy
Received 6 May 2002; received in revised form 11 October 2002; accepted 12 November 2002
Abstract
Several heteroaromatic analogues of (2-aryl-1-cyclopentenyl-1-alkylidene)-(arylmethyloxy)amine COX-2 inhibitors, in which the
cyclopentene moiety was replaced by pyrazole, thiophene or isoxazole ring, were synthesized, in order to verify the influence of the
different nature of the central core on the COX inhibitory properties of these kinds of molecules. Among the compounds tested, only
the 3-(p-methylsulfonylphenyl) substituted thiophene derivatives 17 and 22, showed a certain COX-2 inhibitory activity,
accompanied by an appreciable COX-2 versus COX-1 selectivity. Only one of the 1-(p-methylsulfonylphenyl)pyrazole compounds
(16) displayed a modest inhibitory activity towards both type of isoenzymes, while the pyrazole 1-(p-aminosulfonylphenyl)
substituted 12 proved to be significantly active only towards COX-1. All the isoxazole derivatives were inactive on both COX
isoforms.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Antiinflammatory drug; COX-2 inhibitor; Aryl-substituted methyleneaminoxymethyl moiety; Heteroaromatic COX-2 inhibitor
1. Introduction
blood vessel dilation, and vascular permeability, causing
the typical redness, heat, and swelling phenomena
involved in inflammation. Moreover, they promote
pain transmission from nociceptors to the brain by
increasing the sensitivity of the nerve endings. However,
prostaglandins also play a cytoprotective role in the
gastrointestinal tract, and they are necessary for normal
platelet aggregation and renal function. Therefore, a
reduction in the production of circulating prostaglan-
dins leads to numerous side effects, the most important
being gastrointestinal ulcers [3].
Non-steroidal antiinflammatory drugs (NSAIDs) are
mainly used in the treatment of pain and inflammation
related to a large variety of pathologies [1]. Their
mechanism of action involves the inhibition of the
production of prostaglandins by the enzyme cycloox-
ygenase (COX) [2]. Prostaglandins are among the most
important mediators of inflammation. They promote
Abbreviations: NSAID, non-steroidal antiinflammatory drug;
COX, cyclooxygenase; MAOMM, methyleneaminoxymethylmoiety;
PGE2, prostaglandin E2; TMEDA, tetramethylethylenediamine;
DMF, dimethylformamide; THF, tetrahydrofuran.
The double nature (i.e. inflammation reduction vs.
gastrolesivity) of the effects caused by early NSAIDs
acting as COX inhibitors was explained by the discovery
of two isoforms of the COX enzyme [4,5]. One isoform
(COX-1) is constitutive and regularly expressed, produ-
* Corresponding author.
E-mail address: balsamo@farm.unipi.it (A. Balsamo).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(02)01448-4

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A. Balsamo et al. / European Journal of Medicinal Chemistry 38 (2003) 157ꢀ168
/
cing prostaglandins involved in the cytoprotection of the
gastrointestinal tract. The other isoform (COX-2) is
associated with inflammatory states and is generally
absent from all tissues, unless induced by inflammation
mediators. Therefore, the inducible isoform COX-2
constitutes the real target for antiinflammatory drugs.
In fact, molecules which are capable of being selective
COX-2 inhibitors, without affecting the constitutive
isoform (COX-1), have proved to be excellent drugs in
the treatment of inflammatory pathologies and, most
importantly, are devoid of side effects which are typical
of earlier non-selective NSAIDs.
A common structural backbone of most COX-2
selective inhibitors consists of two aryl groups linked
to adjacent atoms of a central ring which can be
homocyclic or heteroaromatic, one of which is substi-
tuted in the para position with either an aminosulfonyl
(-SO2NH2) or a methylsulfonyl (-SO2Me) group. There
are also examples of potent COX-2 inhibitors that
possess cycloalkyl [6], alkoxy [7] or phenoxy [7,8]
moieties in the non-sulfonyl containing ‘aryl’ region.
The central rings most commonly found within this class
of molecules are cyclopentene (1, Fig. 1) [6], thiophene
(2) [9], pyrazole (3) [10,11], furanone (4) [12] and
isoxazole (5) [13]. The first examples of selective COX-
2 inhibitors which entered the market were celecoxib (3)
and rofecoxib (4) [10ꢀ12].
/
We have previously described the synthesis and
antiinflammatory activity of a series of analogues of
the 1,2-diarylcyclopentene COX-2 inhibitor 1, in which
the para-fluoro substituted aryl has been replaced by
variously substituted oxime-ether groups (6, Fig. 1)
[14,15], on the basis of the fact that the oxime motif
can provide a similar enzyme or receptor-binding
affinity as noted for an aryl moiety in several drug
Fig. 1. Structures of some COX-2 selective antiinflammatory drugs
characterized by diaryl-substituted carbocyclic or heterocyclic 5-
membered ring (1ꢀ5), together with the general structure (6) of the
/
previously studied MAOM analogues of type 4 drugs.
commercially available pyrazole-based COX-2 inhibitor
celecoxib (3, Fig. 1).
pharmacophores [16ꢀ19].
/
Some of these new arylcyclopentene oxime-ethers
showed COX-2 inhibitory properties, with satisfactory
levels of activity and COX-2 vs COX-1 selectivity.
Among the new compounds, the benzyl-substituted
one containing the methylsulfonyl moiety (7, Fig. 2)
and its methylenedioxybenzyl analogue (8) appeared to
be the most active of the series (Table 1) [14,15]; on the
contrary, their aminosulfonyl analogues (9 and 10)
proved to be devoid of any activity. These data, together
with the observation that most of the already known
2. Chemistry
The synthesis of the sulfonamidic 3-trifluoromethyl-
substituted pyrazoles 11 and 12 (Fig. 3) started from the
a,b-unsaturated ketone 26 [20] which, by condensation
with 4-(aminosulfonyl)phenylhydrazine hydrochloride
[21] in refluxing ethanol, afforded the regioisomeric
mixture of the two pyrazoles 27 and 28. The isomer 27
was separated by column chromatography, subjected to
a-lithiation with n-BuLi/TMEDA [22], and quenched,
firstly with DMF, and then with a saturated aqueous
solution of ammonium chloride, affording the a-for-
mylated pyrazole 29. Condensation of 29 with the
appropriate O-benzyl hydroxylamine hydrochloride
afforded compounds 11 and 12.
tricyclic COX-2 inhibitors (2ꢀ5, Fig. 1) possess a central
/
ring of a heteroaromatic nature, prompted us to study in
depth this type of oxime-ether COX-2 inhibitors,
synthesizing some heteroaromatic analogues of methyl-
sulfonyl compounds of type 6 (Fig. 1), in which the
central cyclopentene ring is replaced by a pyrazole (14ꢀ
16), a thiophene (17ꢀ22), or an isoxazole (23ꢀ25) ring
(Fig. 2). In addition, we prepared the p-aminosulfonyl-
phenyl-substituted pyrazole derivatives 11ꢀ13 (Fig. 2),
since this sulfonamidic moiety was typical of the
/
/
/
/
For the preparation of the 3-methyl-substituted
pyrazole 13 (Fig. 3), the b-diketone 30 was condensed

A. Balsamo et al. / European Journal of Medicinal Chemistry 38 (2003) 157ꢀ
/168
159
Fig. 2. Structures of the more active methylsulfonyl-MAOM compounds of type 6 (7, 8) and of their aminosulfonyl analogues (9, 10) previously
described, together with the structures of the heteroaromatic analogues of type 6 compounds, described in this work (11ꢀ25).
/
with 4-(aminosulfonyl)phenylhydrazine hydrochloride
[21] in refluxing ethanol [23], to give the pyrazole
derivative 31 as the only regioisomer. Further condensa-
tion of 31 with 3,4-(methylenedioxy)benzyloxyamine
hydrochloride afforded the oxime-ether derivative 13
with the E configuration.
which was then condensed with the appropriate aryl-
methyloxy- or phenoxy-amine hydrochloride to afford
the corresponding oximes 14ꢀ16. After purification,
compound 14 was still obtained as a 1:1 E/Z mixture
and was tested as such, whereas compounds 15 and 16
were obtained and tested as pure E diastereoisomers.
/
Pyrazole derivatives lacking substituents in the 3
position but possessing a methylsulfonyl group in the
Thiophene derivatives 17ꢀ22 were prepared as re-
/
ported in Fig. 5. The cross-coupling reaction of 3-
bromothiophene with 4-(methylthio)phenylboronic acid
under Suzuki conditions [26] gave 3-[4-(methylthio)phe-
nyl]thiophene 36 [27] which constitutes the branch point
of the synthetic scheme, since it was the common
intermediate of different pathways for the synthesis of
para position of the aromatic substituent (14ꢀ16), were
/
synthesized as shown in Fig. 4. Condensation of 4-
(methylthio)phenylhydrazine 32 [24] with the bis-di-
methylacetale of malonaldehyde afforded the N-aryl-
substituted pyrazole 33. Regioselective a-lithiation of 33
with n-BuLi/DMF [25], followed by sequential quench-
ing with a solution of HCl, afforded the a-formylated
pyrazole 34. Selective oxidation of the methylthio
moiety to a methylsulfonyl group was obtained using
oxone as the oxidant, to give pyrazole derivative 35,
both aldoximes 17ꢀ
/
19 and ketoximes 20ꢀ22. Vilsmeier
/
reaction of 36 with DMF/POCl3, afforded the 2-
formylated thiophene compound 37. Subsequent oxida-
tion with oxone of the methylthio group of 37 to a
methylsulfonyl one gave compound 38, which was

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A. Balsamo et al. / European Journal of Medicinal Chemistry 38 (2003) 157ꢀ168
/
Table 1
COX-1 and COX-2 inhibitory activity of compounds 7ꢀ25
/
a
b
c
Compound
M.p. (8C)
Crystn solvent
Formula
In vitro inhibitory activity
COX-1
COX-2
d
7
29%
27%
0
1.9
0.41
19%
12%
17%
7%
d
8
e
9
10
e
0
f
11
12
ꢀ/
ꢀ
A
/
C18H15N4O3SF3
C19H15N4O5SF3
C19H18N4O5S
C18H17N3O3S
C19H17N3O5S
C17H15N3O3S
C19H17NO3S2
C20H17NO5S2
C19H17NO3S2
C20H19NO3S2
C21H19NO5S2
C19H17NO3S2
C18H16N2O4S
C19H16N2O6S
C17H14N2O4S
21%
2.4
20%
0
96ꢀ
166ꢀ
103ꢀ
137ꢀ
124ꢀ
139ꢀ
149ꢀ
131ꢀ
93ꢀ
98ꢀ100
140ꢀ
128ꢀ
153ꢀ
138ꢀ
/
98
13
14
/
168
105
139
125
141
151
133
B
20%
5%
/
C
D
E
15
16
/
22%
112
18%
11%
0
20%
43
/
17
18
/
D
D
D
D
D
A
D
C
B
1.7
/
11%
12%
18%
28%
11
19
/
20
21
/
95
10%
0
/
22
23
/
142
130
155
140
0
/
4%
4%
0
0
24
25
/
21%
0
/
3 (celecoxib)
1.04
0.02
a
Aꢁ
/
hexaneꢀ
/
Et2O, Bꢁi
All compounds were analysed for C, H, and N.
/
PrOH, Cꢁ
/
MeOH, Dꢁ
/
EtOH, EꢁEt2O.
/
b
c
Data are indicated as IC50 (mM) for the more active compounds or as inhibition percentage (%) at a concentration of 10 mM for compounds
showing IC50 values ꢀ
/1 mM.
d
e
f
See Ref. [12].
See Ref. [13].
Vitreous solid.
Fig. 3. Synthesis of the aminosulfonyl type 3-trifluoromethyl (11, 12) and 3-methyl (13) pyrazoles.

A. Balsamo et al. / European Journal of Medicinal Chemistry 38 (2003) 157ꢀ
/168
161
Fig. 4. Synthesis of methylsulfonyl type pirazoles (14ꢀ16).
/
condensed with O-benzylhydroxylamine hydrochloride
to afford aldoxime 17 with the E configuration, or with
40 with the appropriate O-substituted arylhydroxyla-
mine hydrochloride afforded, after purification, ketox-
imes 20-22 with the Z configuration.
O-(3,4-methylenedioxybenzyl)hydroxylamine
hydro-
The synthesis of isoxazole derivatives 23ꢀ25 started
/
chloride to give a mixture of the aldoximes 18 and 19
with the E and Z configuration, respectively, which
were separated by crystallisation. Acetylation of inter-
mediate 36 with acetyl chloride in the presence of tin(IV)
chloride [28], afforded the methyl ketone derivative 39,
which was then submitted to oxone oxidation to yield
the methylsulfonyl compound 40. Final condensation of
with the oxime 41, obtained by oximation of 4-
(methylthio)benzaldehyde [29] (Fig. 6), which was sub-
mitted to oxidation/chlorination [30] in the presence of
oxone and aqueous HCl. The chlorooximate 42 thus
obtained was then treated with triethylamine and 3-
(dimethylamino)acrolein [31], affording the 4-formyl
Fig. 5. Synthesis of thiophene derivatives 17ꢀ22.
/

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A. Balsamo et al. / European Journal of Medicinal Chemistry 38 (2003) 157ꢀ168
/
Fig. 6. Synthesis of isoxazole derivatives 23ꢀ25.
/
isoxazole derivative 43. Final condensation of 43 with
the appropriate O-substituted hydroxylamines gave the
appreciable activity, with an IC50 value of 1.7 mM,
which is very close to the one previously found for the
cyclopentenyl analogue 7 [14]. Compounds 16 and 22
proved to be slightly active with IC50 values lower than
50 mM, whereas the other derivatives proved to be either
only scarcely active (13, 15, 21, 24) or practically
inactive (11, 12, 14, 18, 19, 20, 23, 25).
corresponding oximic derivatives 23ꢀ25 as a single Z-
/
isomer (for 23) or diastereomeric E/Z mixtures (for 24
and 25) which were purified as such.
The configuration around the oximic double bond of
11ꢀ
values of the aldoximic (for 11ꢀ
methylketoximic (20ꢀ
22) proton signal in their 1H-
/25 was assigned on the basis of the chemical shift
/
19 and 23ꢀ25) or
/
From the results obtained at the COX-2 level with the
/
new methylsulfonyl derivatives 14ꢀ25, it appeared that,
/
NMR spectra. In fact, in compounds with the E
configuration this signal, due to the paramagnetic effect
of the more proximal oxygen, lies at lower fields than in
the corresponding Z-isomers [14,15].
among the heteroaromatic rings utilized in constructing
the new compounds, only the pyrazolic and the thio-
phenic ones, as in the cases of 16 and 17, 22,
respectively, are able to replace, more or less effectively,
the cyclopentenic system of compounds of type 6 (Fig.
1). Furthermore, for the thiophenic compounds (17, 22),
the activity appears to be linked to the type of oxime-
ether side chain, which allows a good interaction with
the COX-2 active site only when it is of the unsubsti-
tuted benzyl aldoximic type with the E configuration, as
in compound 17. The introduction of a methylenedioxy
substituent on the aromatic ring of the oximic side chain
of 17 causes a dramatic reduction in the inhibitory
activity, independently of the type of configuration
around its double bond (see 18 and 19). Incidentally,
also thiophene compound 22, which may be considered
as structurally related to compound 19 as far as the Z-
configuration is concerned, but possesses an oximic side
chain which is less bulky, even though substituted with a
methyl group on the oximic carbon atom, is still capable
of exerting a certain inhibitory action. An influence of
the steric factors on the activity results also for pyrazole
3. Biopharmacology
The in vitro inhibitory activity of heteroaromatic
compounds 11ꢀ25 towards COX-1 and COX-2 was
/
evaluated by measuring prostaglandin E2 (PGE2)
production in U937 cell lines for COX-1 and activated
J774.2 macrophages for COX-2. The results are re-
ported in Table 1, together with those obtained in the
same types of tests with celecoxib (3), chosen as the
reference compound, and with the previously studied
cyclopentenyl analogues 7ꢀ10 [14,15].
/
4. Results and discussion
As far as COX-1 inhibitory properties are concerned,
only the 4-(aminosulfonylphenyl) pyrazole derivative 12
and the 4-(methylsulfonylphenyl) pyrazole 16 showed
IC50 values in the micromolar range (2.42 and 112 mM,
respectively, vs. 1.04 mM of celecoxib 3). All the other
compounds proved to be practically devoid of any
COX-1 inhibitory activity. A modest activity or a
complete inactivity on the same enzyme had already
derivatives 14ꢀ16 for which only the one less hindered
/
on the oximic side-chain (16) possesses a certain activity
on both types of COX-enzymes.
As for compounds 11ꢀ13, which may be considered
/
as related to the inactive cyclopentenyl derivatives 9 and
10, since they possess the aminosulfonyl substituent on
the non-oximic aromatic ring, it appears that the
replacement of the cyclopentene ring of 9,10 with the
heteroaromatic system (i.e. the pyrazole) present in one
of the better known aminosulfonyl COX-2 inhibitors,
been found also for compounds 7ꢀ10 [14,15].
/
As far as COX-2 inhibitory properties are concerned,
only thiophene derivative 17 proved to possess an

A. Balsamo et al. / European Journal of Medicinal Chemistry 38 (2003) 157ꢀ
/168
163
Fig. 7. Compounds 7, 14 and 17 docked into the catalytic site of COX2. The most important residues are labeled.
celecoxib 3, arouses a modest inhibitory activity only
towards the COX-1 enzyme, and only in the case in
which the phenyl ring linked to the oximic side chain
presents the methylenedioxy substituent (see 12 vs. 11).
A possible explanation for the different effects due to
the various central cores of the oximic compounds
studied, can be found in a study of the docking with
the active site of the COX-2 enzyme of the series of
benzyloximic derivatives to which most of the active
compounds (7, IC50 1.9 mM and 17, IC50 1.7mM) belong.
Fig. 7 indicates that O-benzyl oximes with the E
configuration (7, 14, 17) interact with the active site of
the enzyme in an analogous spatial orientation. It
appears that in the case of the more active compounds
(7 and 17), the enzyme-inhibitor complex is stabilised by
an interaction between the sulfonic group and the
determining the biological activity. The results reported
here indicate that, among the heteroaromatic systems
tested, the thiophene ring is the one which gives results
of a certain interest. Furthermore, among the thiophene
and pyrazole derivatives, the steric factors related to the
size of the oximic side-chain also seems to have a
significant effect on the COX inhibition process. As
regards the benzyloximic compounds with the E-con-
figuration which are active towards COX-2 (7, 17), the
docking studies seem to explain the inhibitory properties
in terms of particular interactions with specific aminoa-
cidic residues in the catalytic site of the enzyme.
6. Experimental protocols
˚
Arg513 (distance 3.19 and 3.13 A, respectively) and by
6.1. Chemistry
a hydrophobic interaction between the benzylic group
and the Tyr385; this interaction is strengthened by the
Phe381 in the case of 7, and by the Phe518 in the case of
Melting points were determined on a Kofler hot-stage
apparatus and are uncorrected. IR spectra for compar-
ison of compounds were taken as paraffin oil mulls or as
liquid films on a Unicam Mattson 1000 FT-IR spectro-
meter. 1H-NMR spectra of all compounds were ob-
tained with a Gemini 200 spectrometer operating 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 chromatography was
17. As regards the inactive compound 14, the N2 of the
˚
N 3.62 A)
and this modifies the orientation of the inhibitor in the
enzyme active site, thus disturbing the interaction with
pyrazole ring interacts with the Arg120 (Nꢀ
/
˚
the Arg513 (3.49 A) and preventing the hydrophobic
stabilisation of the aromatic ring with the Tyr385.
As regards the isoxazole derivative 23 and the Z-
isomer of pyrazole, which takes part of the mixture of 14
(1:1), the study of docking indicates their inability to fit
in the active site of the enzyme without producing
important deformations of the molecules.
performed using 70ꢀ230 mesh silica gel. Mass spectra
/
were detected with a Hewlett Packard 5988A spectro-
meter. Evaporation was made in vacuo (rotating eva-
porator); the O-arylmethyloxyamines to be used for the
preparation of the oxime-ethers 11ꢀ25, not commer-
/
5. Conclusions
cially available, were prepared following the synthetic
method described in Ref. [32]. Na2SO4 was always used
as the drying agent. Elemental analyses were performed
in our analytical laboratory and agreed with the
These results indicate that in the class of COX-2
inhibitors of the oxime-ether type, the pentatomic
central system plays an important role on its own in
theoretical values to within 90.4%.
/

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A. Balsamo et al. / European Journal of Medicinal Chemistry 38 (2003) 157ꢀ168
/
6.1.1. Synthesis of 4-[3-(trifluoromethyl)-1-
pyrazolyl]benzenesulfonamide (27)
6.1.4. General procedure for the preparation of 4-{5-
[(E)-((arylmethyloxy)imino)methyl]-3-
A suspension of 4-sulfonylamidophenylhydrazine×
HCl (2.63 g, 11.8 mmol) in anhydrous EtOH (60 mL)
was treated with a solution of 4-ethoxy-1,1,1-trifluoro-3-
buten-2-one 26 (2.0 g, 12 mmol) in anhydrous EtOH (10
mL). The resulting solution was stirred under reflux for
4h and cooled at room temperature (r.t.), and the
solvent was then evaporated to dryness to yield a crude
residue consisting of a mixture of regioisomers 27 and 4-
[5-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfona-
mide (28) in a ratio of 60:40, which were separated by
/
(trifluoromethyl)-1-pyrazolyl}benzenesulfonamides (11,
12) and 4-{5-[(E)-((3,4-methylenedioxy-
phenylmethyloxy)imino)methyl]-3-(methyl)-1-
pyrazolyl}benzenesulfonamide (13)
A two-phase mixture of aldehyde 29 or 31 (1.5 mmol)
in CHCl3 (15 mL) and the appropriate O-arylmethylox-
yamine hydrochloride (1.51 mmol) in H2O (5 mL), was
stirred at r.t. for 16 h. The organic phase was then
separated and the aqueous solution was extracted twice
with CHCl3. Evaporation of the dried and filtered
combined organic extracts afforded a residue consisting
almost exclusively of the corresponding E oxime-ethers
11, 12 and 13 which were purified by column chromato-
column chromatography eluting with a 4:6 hexaneꢀ
AcOEt mixture. 27 (40%): m.p. 177ꢀ179 8C (CHCl3);
1H-NMR d 6.43 (br, 2H, SO2NH2), 6.67 and 8.0 (2d,
2H, Jꢁ2.6 Hz, H4 and H5 pyrazole), 7.76 and 7.96 ppm
(2d, 4H, Jꢁ8.6 Hz, ArSO2NH2); MS m/zꢁ
291 (Mꢂ).
C10H8F3N3O2S (C, H, N). 28 (20%): H-NMR d 6.12
(br, 2H, SO2NH2), 6.83 and 7.71 (2d, 2H, Jꢁ1.5 Hz, H4
and H5 pyrazole), 7.60 and 8.03 ppm (2d, 4H, Jꢁ8.2
/
/
/
graphy eluting with a 95:5 CHCl3ꢀMeOH mixture, for
/
/
/
11 and 12 and by crystallisation for 13. (E)-11 (36%):
1H-NMR d 4.95 (br, 2H, SO2NH2), 5.17 (s, 2H,
1
/
PhCH2O), 7.06 (s, 1H, H4 pyrazole), 7.35ꢀ
5H, Ph), 7.60 and 8.05 (2d, 4H, Jꢁ8.9 Hz, ArSO2NH2),
N). (E)-12 (35%): H-NMR d
/7.68 (m,
/
/
1
Hz, ArSO2NH2).
8.04 ppm (s, 1H, CHÄ
/
4.93 (br, 2H, SO2NH2), 5.03 (s, 2H, ArCH2O), 5.98 (s,
2H, OCH2O), 6.81 (m, 3H, Ar), 7.04 (s, 1H, H4
6.1.2. Synthesis of 4-[5-formyl-3-(trifluoromethyl)-1-
pyrazolyl]benzenesulfonamide (29)
pyrazole), 7.60 and 8.04 (2d, 4H, Jꢁ/8.7 Hz, Ar-
A solution of n-BuLi 1.6M in hexane (6.4 mL, 10
mmol) was added dropwise under argon to a stirred and
SO2NH2), 8.02 ppm (s, 1H, CHÄN); MS (FAB) m/
/
1
zꢁ
/
469 (Mꢂ
Hꢂ). (E)-13 (30%): H-NMR d 2.36 (s,
3H, Me), 5.02 (s, 2H, ArCH2O), 5.88 (s, 2H, OCH2O),
6.60 (s, 1H, H4 pyrazole), 6.81 (s, 3H, Ar), 7.57 and 7.96
/
cooled solution (ꢃ
/
78 8C) of 27 (1.0 g, 3.4 mmol) and
TMEDA (10 mmol) in anhydrous THF (50 mL). The
resulting mixture was stirred at 0 8C for 30 min and then
(d, 2H, Jꢁ
/
7.9 Hz, ArSO2NH2), 8.04 ppm (s, 1H, CHÄ
/
cooled to ꢃ
/
78 8C and treated with DMF (1.1 mL).
N); MS m/zꢁ
/
414 (Mꢂ).
After 6 h at r.t., the resulting suspension was quenched
with 300 mL of saturated NH4Cl solution. The aqueous
phase was extracted three times with AcOEt, and the
combined organic phases were washed with water and
brine, dried and evaporated to dryness. The crude
residue was subjected to column chromatography elut-
6.1.5. Synthesis of 1-[4-(methylthio)phenyl]-pyrazole
(33)
A solution of tetramethoxypropane (7.40g, 45 mmol)
and 4-methylthiophenylhydrazine×HCl (8.60g, 45.2
/
mmol) in anhydrous EtOH (70 mL) was refluxed for 2
h. The solvent was evaporated and the residue was
suspended in a 5% aqueous solution of NaOH and
extracted three times with AcOEt. Combined organic
extracts were dried, filtered and evaporated to yield a
crude product which was crystallised from hexane to
ing with a 95:5 CH2Cl2ꢀ
(38%): m.p. 122ꢀ124 8C (Et2Oꢀ
5.32 (br, 2H, SO2NH2), 7.38 (s, 1H, H4 pyrazole), 7.68
and 8.09 (2d, 4H, Jꢁ8.7 Hz, ArSO2NH2), 9.90 ppm (s,
1H, CHO); MS m/zꢁ
319 (Mꢂ).
/
MeOH mixture to yield pure 29
/
/
hexane); 1H-NMR d
/
/
give pure 33 (54%): m.p. 72ꢀ
2.51 (s, 3H, SMe), 6.46 (br, 1H, H4 pyrazole), 7.33 and
7.61 (2d, 4H, Jꢁ8.2 Hz, Ar), 7.72 (br, 1H, H3
pyrazole), 7.88 ppm (d, 1H, Jꢁ2.4 Hz, H5 pyrazole);
MS m/zꢁ
190 (Mꢂ). C10H10N2S (C, H, N).
/
73 8C (hexane); 1H-NMR d
6.1.3. Synthesis of 4-(5-formyl-3-methyl-1-
pyrazolyl)benzenesulfonamide (31)
/
A cooled (10 8C) solution of 4-sulfonylamidophenyl-
hydrazine hydrochloride (1.0 g, 4.5 mmol) in MeOH
(110 mL) was treated with a solution of 1,1-diethoxy-
acethylacetone 30 (1.20g, 6.4 mmol) in MeOH (30 mL).
After the addition was complete, the mixture was
refluxed for 1 h and cooled at r.t., and the solid
precipitate was filtered off. Evaporation of the solvent
yielded 31 as a crude product, which was used in the
subsequent reaction without any further purification.
/
/
6.1.6. Synthesis of 1-[4-(methylthio)phenyl]-pyrazole-5-
carbaldehyde (34)
A 1.6 M solution of n-BuLi in hexane (6.6 mL) was
slowly added under argon to a solution of 33 (2.0 g, 10.5
mmol) in dry THF (20 mL). The suspension was stirred
mechanically for 2 h at r.t. and then treated with DMF
(0.80 mL, 11 mmol). The mixture was stirred for an
additional hour, poured into 1 N aqueous HCl (10 mL)
and the organic solvent was evaporated. The resulting
1
31(37%); H-NMR d 2.40 (s, 3H, Me), 6.90 (s, 1H, H4
9.6 Hz, Ar), 9.80
pyrazole), 7.68 and 7.98 (2d, 4H, Jꢁ
/
ppm (s, 1H, CHO); MS m/zꢁ
/
265 (Mꢂ).

A. Balsamo et al. / European Journal of Medicinal Chemistry 38 (2003) 157ꢀ
/168
165
aqueous mixture was basified to pH 8 with 1N aqueous
NaOH and extracted three times with AcOEt. Com-
bined organic extracts were filtered and evaporated to
yield a viscous oil which was subjected to column
6.1.9. Synthesis of 3-[4-(methylthio)phenyl]thiophene
(36)
Compound 36 was prepared as reported previously
[27]. A mixture of 3-bromothiophene (5.15 g, 25 mmol)
and 4-methylthiophenylboronic acid (4.12 g, 24.5
chromatography eluting with 8:2 hexaneꢀ
/
AcOEt mix-
ture to give pure 34 (38%): H-NMR d 2.54 (s, 3H,
SCH3), 7.10 and 7.75 (2d, 2H, Jꢁ2.0 Hz, H4 and H3
pyrazole), 7.33ꢀ7.44 (m, 4H, Ar), 9.87 ppm (s, 1H,
CHO); MS m/zꢁ
218 (Mꢂ). C11H10N2OS (C, H, N).
1
mmol), in a 1:1 tolueneꢀEtOH mixture (80 mL) and
/
/
aqueous 2 M Na2CO3 (18 mL, 36 mmol) was treated at
r.t. with Pd(PPh3)4 (0.4 g) and then refluxed under argon
with stirring for 12 h. Evaporation of the organic phase
gave an aqueous residue which was taken up in AcOEt.
The organic phase was washed with H2O, filtered and
evaporated to afford a crude residue which was crystal-
/
/
6.1.7. Synthesis of 1-[4-(methylsulfonyl)phenyl]-
pyrazole-5-carbaldehyde (35)
A cooled (0 8C) solution of 34 (1.0g, 4.6 mmol) in a
1
lised from EtOH to yield pure 36 (49%): H-NMR d
2.48 (s, 3H, SMe), 7.18ꢀ7.53 (m, 7H, Ar).
/
1:1 THFꢀMeOH mixture (28 mL), was treated with a
/
solution of oxone (3.4 g, 5.5 mmol) in H2O (15 mL).
After 6h of vigorous stirring at r.t., the mixture was
poured into AcOEt and the organic phase was washed
with H2O and brine, filtered and evaporated to afford
the crude methylsulfonyl derivative 35 which was
purified by crystallisation from i-PrOH (41%): m.p.
6.1.10. Synthesis of 3-[4-(methylthio)phenyl]thiophene-
2-carbaldehyde (37)
A cooled (0 8C) and stirred mixture of DMF (1.65
mL, 21.2 mmol) and anhydrous CH2Cl2 (30 mL) was
treated dropwise with POCl3 (10 mL, 11 mmol). After 1
h at 0 8C, a solution of 36 (1.45 g, 7.0 mmol) in
anhydrous CH2Cl2 (20 mL) was added. After 2h at
r.t., the mixture was treated with ice and the pH was
adjusted to 5 with solid AcONa. The aqueous phase was
extracted with Et2O and the organic extracts were dried
and evaporated to yield an oily residue which was
purified by column chromatography eluting with a 1:9
142ꢀ
1H, Jꢁ
Jꢁ8.4 Hz, Ar), 7.84 (d, 1H, Jꢁ
9.94 ppm (s, 1H, CHO); MS m/zꢁ
C11H10N2O3S (C, H, N).
/
144 8C; 1H-NMR d 3.12 (s, 3H, SO2CH3), 7.20 (d,
1.3 Hz, H4 pyrazole), 7.75 and 8.10 (2d, 4H,
1.3 Hz, H3 pyrazole),
250 (Mꢂ).
/
/
/
/
6.1.8. General procedure for the preparation of 1-[4-
(methylsulfonyl)phenyl]-pyrazole-5-carbaldehyde O-
arylmethyl- (14, 15) and O-phenyl-oximes (16)
petroleum etherꢀ
1H-NMR d 2.52 (s, 3H, SMe), 7.16 and 7.71 (2d, 2H,
Jꢁ4.9 Hz, H4 and H5 thiophene), 7.33 (s, 4H, Ar), 9.83
/
AcOEt mixture to give pure 37 (60%):
/
A solution of 35 (0.4 g, 1.6 mmol) and the appropriate
arylmethyloxyamine or phenylmethyloxyamine (1.6
mmol), as hydrochlorides, in anhydrous EtOH (20
mL) was heated for 2 h at reflux temperature, the
solvent was then evaporated and the residue was
crystallised from the appropriate solvent to yield pure
oxime-ethers as single E-isomers 15,16 or a 1:1 E/Z
mixture 14. (E/Z)-14 (58%): 1H-NMR (E isomer) d 3.09
(s, 3H, SO2CH3), 5.16 (s, 2H, OCH2Ph), 6.83 and 7.73
ppm (s, 1H, CHO). C12H10OS2 (C, H, N).
6.1.11. 3-[4-(Methylsulfonyl)phenyl]thiophene-2-
carbaldehyde (38)
Compound 38 was synthesized from 37 following the
same procedure described above for the preparation of
35. Compound 38 (71%): m.p. 152ꢀ
NMR d 3.12 (s, 3H, SO2CH3), 7.25 and 7.81 (2d, 2H,
Jꢁ4.9 Hz, H4 and H5 thiophene), 7.68 and 8.07 (2d,
4H, Jꢁ8.0 Hz, Ar), 9.86 ppm (s, 1H, CHO).
/
154 8C (EtOH); 1H-
/
(2d, 2H, Jꢁ
/
1.6 Hz, H4 and H3 pyrazole), 7.31ꢀ
8.6 Hz, ArSO2CH3),
N). H-NMR (Z isomer) d 3.11
(s, 3H, SO2CH3), 5.31 (s, 2H, OCH2Ph), 7.31 and 7.76
(2d, 2H, Jꢁ1.6 Hz, H4 and H3 pyrazole), 7.32ꢀ7.43 (m,
6H, Ph and CHÄN), 7.69 (d, 2H, Jꢁ8.6 Hz, Ar-
SO2CH3), 8.09 (d, 2H, Jꢁ8.6 Hz, ArSO2CH3). For the
E/Z mixture of 14: MS m/zꢁ
355 (Mꢂ). (E)-15 (49%):
1H-NMR d 3.09 (s, 3H, SO2CH3), 5.04 (s, 2H,
OCH2Ph), 5.97 (s, 2H, OCH2O), 6.80ꢀ7.25 (m, 4H,
ArCH2O and H4 pyrazole), 7.64 and 8.05 (2d, 4H, Jꢁ
8.6 Hz, ArSO2), 7.74 (d, 1H, Jꢁ1.8 Hz, H3 pyrazole),
8.09 ppm (s, 1H, CHÄN). (E)-16 (21%): H-NMR d
3.12 (s, 3H, SO2CH3), 6.98 and 7.81 (2d, 2H, Jꢁ1.8 Hz,
H4 and H3 pyrazole), 7.07ꢀ7.43 (m, 5H, Ph), 7.74 and
8.13 (2d, 4H, Jꢁ8.4 Hz, ArSO2), 8.41 ppm (s, 1H,
CHÄN).
/
7.43 (m,
/
5H, Ph), 7.64 and 8.03 (2d, 4H, Jꢁ
/
C12H10O3S2 (C, H, N).
1
8.11 ppm (s, 1H, CHÄ
/
6.1.12. General procedure for the synthesis of 3-[4-
(methylsulfonyl)phenyl]thiophene-2-carbaldehyde O-
/
/
/
/
arylmethyl oximes (17ꢀ
/
19)
/
Oxime-ethers 17ꢀ19 were synthesized from 38 and the
/
/
appropriate arylmethyloxyamine hydrochloride follow-
ing the same procedure described above for the pre-
/
paration of 14ꢀ16. While 17 was purified by
/
/
crystallisation of the crude reaction product, 18 and 19
/
were separated by column chromatography. (E)-17
1
1
/
(43%): m.p. 137ꢀ
/
139 8C (EtOH); H-NMR d 3.08 (s,
/
3H, SO2CH3), 5.18 (s, 2H, PhCH2), 7.13 (d, 1H, Jꢁ
Hz, H4 thiophene), 7.07ꢀ7.43 (m, 5H, Ph), 7.37ꢀ7.62
(m, 6H, PhCH2 and H5 thiophene), 7.78 and 8.01 (2d,
4H, Jꢁ8.4 Hz, ArSO2CH3), 8.21 ppm (s, 1H, CHÄN).
/5.1
/
/
/
/
/
/
/

166
A. Balsamo et al. / European Journal of Medicinal Chemistry 38 (2003) 157ꢀ168
/
1
(E)ꢀ
(s, 2H, ArCH2), 5.93 (s, 2H, OCH2O), 6.80ꢀ
7H, H4 and H5 thiophene and ArCH2 and ArSO2), 7.95
(d, 2H, Jꢁ8.0 Hz, ArSO2CH3), 8.15 ppm (s, 1H, CHÄ
N). (Z)-19 (36%): H-NMR d 3.08 (s, 3H, SO2CH3),
5.20 (s, 2H, ArCH2), 5.91 (s, 2H, OCH2O), 6.80ꢀ7.11
(m, 4H, H4 thiophene and ArCH2), 7.49ꢀ7.60 (m, 3H,
H5 thiophene and ArSO2), 7.56 (s, 1H, CHÄN), 7.98
ppm (d, 2H, Jꢁ8.0 Hz, ArSO2CH3).
/
18 (34%): H-NMR d 3.08 (s, 3H, SO2CH3), 5.04
6.1.16. Synthesis of 4-(methylthio)benzaldehyde oxime
(41)
/7.54 (m,
A solution of hydroxylamine hydrochloride (5.48 g,
78.8 mmol) in H2O (30 mL) was added dropwise to a
cooled (0 8C) and stirred solution of NaOH (4.89 g, 122
mmol) in H2O (30 mL). The resulting mixture was then
treated with 4-thiomethylbenzaldehyde (10 g, 150 mmol)
in EtOH (60 mL). The reaction was followed by TLC
until the disappearance of the aldehyde. The mixture
was concentrated and the pH was adjusted to 4 with
10% aqueous HCl. The aqueous phase was extracted
with Et2O. Combined organic extracts were dried and
evaporated to afford a crude residue which was purified
/
/
1
/
/
/
/
6.1.13. Synthesis of 2-acetyl-3[4-
(methylthio)phenyl]thiophene (39)
A cooled (0 8C) and stirred mixture of 36 (1.0 g, 4.8
mmol) and AcCl (0.38 g, 4.8 mmol) in anhydrous C6H6
(20 mL) was treated dropwise with SnCl4 (1.26 g, 4.84
mmol). After 2h at r.t., the mixture was quenched with
5% aqueous HCl (10 mL). The organic phase was
separated, washed with water, dried and evaporated to
give a residue which was crystallised from hexane-
AcOEt to yield pure 39 (35%): m.p.: 79ꢀ
NMR d 2.20 (s, 3H, CH3CO), 2.53 (s, 3H, SCH3),
7.05 and 7.54 (2d, 2H, Jꢁ5.1 Hz, H4 and H5
thiophene), 7.31 (s, 4H, Ar); MS m/zꢁ
248 (Mꢂ).
by crystallisation from hexane. Compound 41 (46%):
1
m.p. 115ꢀ
/
117 8C; IR nmax (nujol) nCÄN 1591; H-NMR
d 2.51 (s, 3H, SCH3), 7.24 and 7.50 (2d, 4H, Jꢁ
/8.4 Hz,
Ar), 8.10 ppm (s, 1H, CHÄN). C8H9NOS (C, H, N).
/
6.1.17. Synthesis of 4-(methylsulfonyl)benzhydroximic
acid chloride (42)
/
82 8C;1H-
Oxone (6.5 g, 11 mmol) was added at 0 8C to a stirred
solution of 41 (1 g, 6 mmol) in a 0.5 N HCl (0.25 mL)
aqueous solution and DMF (10 mL), and the resulting
mixture was stirred at r.t. for 30 min. The reaction
mixture was poured into cold water (25 mL) and
extracted with CHCl3. The organic layers were washed
with brine, dried and evaporated. Removal of CHCl3
gave a crude residue which was purified by crystal-
/
/
C13H12OS2 (C, H).
6.1.14. Synthesis of 2-acetyl-3[4-
(methylsulfonyl)phenyl]thiophene (40)
Compound 40 was synthesized from 39 following the
same procedure described above for the preparation of
35. Compound 40 (40%): m.p.: 141ꢀ
NMR d 2.34 (s, 3H, CH3CO), 3.12 (s, 3H, SO2CH3),
7.08 (d, 1H, Jꢁ4.9 Hz, H4 thiophene), 7.58ꢀ7.63 (m,
3H, Ar and H5 thiophene), 8.0 ppm (d, 2H, Jꢁ7.8 Hz,
Ar). C13H12O3S2 (C, H).
lisation from AcOEt. Compound 42 (39%): m.p. 183ꢀ
185 8C; 1H-NMR d 3.08 (s, 3H, SO2CH3), 7.98 and 8.06
ppm (2d, 4H, Jꢁ8.6 Hz, Ar). C8H8NO3SCl (C, H, N).
/
/
143 8C (EtOH); 1H-
/
/
/
/
6.1.18. Synthesis of 3-[4-
(methylsulfonyl)phenyl]isoxazole-4-carbaldehyde (43)
A solution of 42 (500 mg, 2.14 mmol) was added
dropwise to a stirred solution of 3-dimethylaminoacro-
lein (0.11 mL, 1.1 mmol) and triethylamine (0.29 mL,
2.1 mmol) in THF (13 mL). The reaction mixture was
stirred for 2 h and then evaporated. The crude residue
was taken up with CHCl3. The organic layer was washed
with brine, filtered and evaporated, and the residue was
purified by column chromatography using CHCl3 as the
6.1.15. General procedure for the preparation of (Z)-2-
acetyl-3[4-(methylsulfonyl)phenyl] thiophene O-
arylmethyl- (20, 21) and O-phenyl-oximes (22)
Compounds 20ꢀ22 were synthesized from 40 and the
/
appropriate arylmethyloxyamine hydrochlorides (for
the preparation of 20 and 21) or phenoxyamine hydro-
chloride (for the preparation of 22) following the same
procedure described above for the preparation of 14ꢀ
/
16.
(Z)-20 (45%): H-NMR d 1.92 (s, 3H, CH3), 3.08 (s,
3H, SO2CH3), 5.18 (s, 2H, ArCH2), 7.08 (d, 1H, Jꢁ5.1
Hz, H4 thiophene), 7.30ꢀ7.36 (m, 6H, H5 thiophene
8.2 Hz,
eluant. Compound 43 (45%): m.p. 135ꢀ138 8C (EtOH);
/
1H-NMR d 3.11 (s, 3H, SO2CH3), 8.09 (s, 4H, Ar), 9.17
(s, 1H, H5 isoxazole), 10.08 ppm (s, 1H, CHO); MS m/
1
/
/
zꢁ
251 (Mꢂ). C11H9NO4S (C, H, N).
/
and PhCH2), 7.55 and 7.90 ppm (2d, 4H, Jꢁ
/
ArSO2); (Z)-21 (47%):1H-NMR d 1.90 (s, 3H, CH3),
3.09 (s, 3H, SO2CH3), 5.06 (s, 2H, ArCH2), 5.98 (s, 2H,
6.1.19. General procedure for the preparation of 3-[4-
(methylsulfonyl)phenyl]isoxazole-4-carbaldehyde O-
arylmethyl- (23, 24) and O-phenyl-oximes (25)
OCH2O), 6.81ꢀ
2H, Jꢁ5.3 Hz, H4 and H5 thiophene), 7.58 and 7.95
ppm (2d, 4H, Jꢁ
NMR d 2.15 (s, 3H, CH3), 3.09 (s, 3H, SO2CH3), 7.01ꢀ
7.32 (m, 6H, H4 thiophene and Ph), 7.42 (d, 1H, Jꢁ5.1
Hz, H5 thiophene), 7.62 and 8.0 ppm (2d, 4H, Jꢁ8.1
Hz, ArSO2CH3).
/6.87 (m, 3H, ArCH2), 7.09 and 7.32 (2d,
/
Oximes 23ꢀ
appropriate aryloxyamine hydrochloride following the
same procedure described above for 14ꢀ16. (Z)-23
(45%): H-NMR d 3.11 (s, 3H, SO2CH3), 5.34 (s, 2H,
PhCH2), 7.28 (s, 1H, CHÄN), 7.40 (s, 5H, PhCH2), 7.82
and 8.11 (2d, 4H, Jꢁ8.1 Hz, ArSO2), 9.37 ppm (s, 1H,
/
25 were synthesized from 43 and the
1
/
8.2 Hz, ArSO2). (Z)-22 (50%): H-
/
/
1
/
/
/
/

A. Balsamo et al. / European Journal of Medicinal Chemistry 38 (2003) 157ꢀ
/168
167
1
H5 isoxazole). (E/Z)-24 (50%): H-NMR (Z isomer) d
3.12 (s, 3H, SO2CH3), 5.22 (s, 2H, ArCH2), 5.98 (s, 2H,
score which indicates how good the interaction is.
During this search the protein molecule is maintained
rigid, while some torsion angles of the ligand are allowed
to rotate [36,37].
OCH2O), 6.80ꢀ
N), 7.82 and 8.11 (2d, 4H, Jꢁ
(s, 1H, H5 isoxazole). H-NMR (E isomer) d 3.10 (s,
3H, SO2CH3), 4.99 (s, 2H, ArCH2), 5.98 (s, 2H,
/
6.90 (m, 3H, ArCH2), 7.27 (s, 1H, CHÄ
/
/
8.2 Hz, ArSO2), 9.35 ppm
1
The COX2-inhibitor complexes were subsequently
minimised, using MacroModel program [38] to solve
any conflicts due to the rigidity of aminoacids of the site
during docking. The procedure used to refine the
complex involved 100 ps of molecular dynamics with a
constraint of 0,02 kcal on the protein backbone while
the ligand and the residues of the binding site were free.
The simulation temperature was 300 K, the timestep 1.5
fs. the molecular dynamics simulation was followed by a
minimization in which no kind of restraint was applied.
The calculation was performed using AMBER forcefield
with distance dependent electrostatic treatment and
dielectric constant 4.0.
OCH2O), 6.80ꢀ
(2d, 4H, Jꢁ8.2 Hz, ArSO2), 8.05 (s, 1H, CHÄ
ppm (s, 1H, H5 isoxazole). (E/Z)-25 (56%): H-NMR
(Z isomer) d 3.13 (s, 3H, SO2CH3), 7.13ꢀ7.43 (m, 5H,
PhO), 7.53 (s, 1H, CHÄN), 7.87 and 8.15 (2d, 4H, Jꢁ
8.6 Hz, ArSO2), 9.59 ppm (s, 1H, H5 isoxazole). H-
NMR (E isomer) d 3.13 (s, 3H, SO2CH3), 7.13ꢀ7.40 (m,
5H, PhO), 7.84 and 8.12 (2d, 4H, Jꢁ8.6 Hz, ArSO2),
8.37 (s, 1H, CHÄN), 8.93 ppm (s, 1H, H5 isoxazole).
/
6.90 (m, 3H, ArCH2), 7.82 and 8.11
/
/
N), 8.75
1
/
/
/
1
/
/
/
6.2. Biopharmacological methods
6.2.1. Enzyme assays
All compounds 11ꢀ25 were tested following the
/
References
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assays to verify their capacity to inhibit PGE2 produc-
tion, considered as an index of activity on COX-1 and
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Ruddon, A.G. Gilman (Eds.), Goodman and Gilsman’s The
Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill,
COX-2 enzymes. For the COX-1 assay, 1.5ꢄ
106 resting
/
U937 human cells were incubated with the test com-
pounds for 30 minutes in the presence of 10 mM
arachidonic acid. Tubes were then centrifuged and the
PGE2 content in the supernatant was measured by a
commercial immunoenzymatic assay (Amersham). The
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sured as described above. All compounds were tested in
duplicate. For each product a stock solution was
prepared in DMSO at a concentration of 100 mM.
Linear regression was used to calculate IC50 values.
SEM were lower than 10% in all experiments.
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