4-Substituted 1,5-diarylpyrazole, analogues of celecoxib: Synthesis and preliminary evaluation of biological properties

Article abstract of DOI:10.1016/S0014-827X(03)00136-8

A number of 5-aryl-1-[4-(methylsulfonyl)-phenyl]-1H-pyrazoles and 4-(5-aryl-1H-pyrazol-1-yl)benzenesulfonamides 3, 4, 5, 6, analogues of the COX-2 selective inhibitor celecoxib (celebrex), were synthesized. In order to verify the effects on the biological properties of certain substituents put on position 4 of the pyrazole nucleus, some of these compounds were screened in vivo for their anti-inflammatory and analgesic activities. Moreover, sodium salts of carboxylic acids 4 were tested in vitro for their platelet anti-aggregating properties. The results of these preliminary biological assays showed that new derivatives are not endowed with improved anti-inflammatory and analgesic properties, in comparison with celecoxib. In addition, docking studies were carried out on the most significative compounds to evaluate their interaction mode at the active site of both COX-1 and COX-2. Some remarks about the SAR of this class of COX-inhibitors are drown out.

Full text of DOI:10.1016/S0014-827X(03)00136-8

Il Farmaco 58 (2003) 795ꢀ808
/
www.elsevier.com/locate/farmac
4-Substituted 1,5-diarylpyrazole, analogues of celecoxib: synthesis
and preliminary evaluation of biological properties
Giulia Menozzi a,*, Luisa Merello a, Paola Fossa a, Luisa Mosti a, Antonietta Piana b,
Francesca Mattioli c
a Dipartimento di Scienze Farmaceutiche, Universita` di Genova, Viale Benedetto XV 3, I-16132 Genova, Italy
b Dipartimento di Medicina Interna, Centro di Ricerca sulla Trombosi, Universita` di Genova, Viale Benedetto XV 6, I-16132 Genova, Italy
c Dipartimento di Medicina Interna, Sezione di Farmacologia e Tossicologia Clinica, Universita` di Genova, Viale Benedetto XV 6, I-16132 Genova, Italy
Received 25 November 2002; accepted 29 January 2003
Abstract
A number of 5-aryl-1-[4-(methylsulfonyl)-phenyl]-1H-pyrazoles and 4-(5-aryl-1H-pyrazol-1-yl)benzenesulfonamides 3, 4, 5, 6,
analogues of the COX-2 selective inhibitor celecoxib (celebrex), were synthesized. In order to verify the effects on the biological
properties of certain substituents put on position 4 of the pyrazole nucleus, some of these compounds were screened in vivo for their
anti-inflammatory and analgesic activities. Moreover, sodium salts of carboxylic acids 4 were tested in vitro for their platelet anti-
aggregating properties. The results of these preliminary biological assays showed that new derivatives are not endowed with
improved anti-inflammatory and analgesic properties, in comparison with celecoxib. In addition, docking studies were carried out
on the most significative compounds to evaluate their interaction mode at the active site of both COX-1 and COX-2. Some remarks
about the SAR of this class of COX-inhibitors are drown out.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Pyrazole derivatives; Anti-inflammatory agents; Analgesic agents; COX-inhibitors
1. Introduction
tion. Classical NSAIDs inhibit both the isoforms to
different extents, many with a preferential selectivity for
COX-1. It is believed that it is the inhibition of COX-1
that causes unfavourable side-effects [1].
Nonsteroidal anti-inflammatory drugs (NSAIDs) are
among the most widely used drugs. The research in this
field, in the last years, aimed at discovering new potent
agents without unfavourable side effects characteristic
of traditional drugs, such as gastrointestinal erosion and
renal damage.
It is now known that inhibition of the enzyme
cyclooxygenase (COX) is the principal mechanism for
both the efficacy and the toxicity of NSAIDs. This
enzyme exists as at least two distinct and independently
regulated isoenzymes, COX-1 and COX-2. COX-1 is
constitutive and synthesizes prostaglandins that mediate
normal homeostasis in the gastrointestinal tract, kidneys
and platelets, whereas COX-2 is inducible and produces
mainly prostaglandins that mediate pain and inflamma-
A high level of selective COX-2 inhibition represents,
therefore, a therapeutic strategy to alleviate pain and
inflammation without the untoward gastrointestinal,
renal, antiaggregating effects due to the COX-1 inhibi-
tion associated to non-selective NSAIDs. Moreover,
various epidemiological and laboratory studies have
indicated that COX-2 inhibitors may have important
therapeutic relevance as anticancer agents (against
colorectal and breast cancers, in particular). COX-2
inhibition may also help the prevention of premature
labour and even retard the progression of Alzheimer’s
disease [2ꢀ4].
/
Nevertheless, the therapeutic contribution of COX-2
highly selective blockers has to be more extensively
evaluated, particularly as these agents could delay the
healing of duodenal ulcers and interfere with several
COX-2-induced physiological functions [5,6]. Actually
* Correspondence and reprints.
E-mail address: menozzi@unige.it (G. Menozzi).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00136-8

796
G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ808
/
COX-2 appears to be also expressed under basal
conditions in many organs, suggesting that this iso-
enzyme may play a more complex physiological role
than was expected [7].
a) The conversion of indomethacin (a well known
nonselective NSAID) into ester derivatives gener-
ated highly selective COX-2 inhibitors [16].
b) The presence of a hydroxycarbonyl function in the
molecule of many traditional NSAIDs, on the
contrary, seems to improve their potency against
COX-1 [17] and, consequently, it should enhance
platelet anti-aggregating properties.
c) A metabolite of the diarylisoxazole derivative val-
decoxib, a potent and selective inhibitor of COX-2
recently approved in USA by FDA (Fig. 1), was
reported to be more potent than valdecoxib itself in
the carrageenan paw edema assay [18]; this meta-
bolite has a hydroxymethyl group on the isoxazole
nucleus.
Various classes of selective COX-2 inhibitors have
been identified in last few years: in particular, tricyclic
molecules bearing a methylsulfonyl or sulfamoyl group
(coxibs) have been extensively studied and have proved
to be a fertile area for the discovery of ever-new active
compounds [8,9]. Celecoxib [10] and rofecoxib [11] (Fig.
1) were the first two agents to be approved in selected
markets for the treatment of acute pain and certain
inflammatory conditions (osteoarthritis, rheumatoid
arthritis).
In particular celecoxib, a 3-substituted 1,5-diarylpyr-
azole, aroused our interest, given that the synthesis of 1-
arylpyrazole compounds with potential anti-inflamma-
tory activity has been a topic of our researches for many
years [12].
For the synthesis of the new compounds we followed
a pathway (Fig. 2) employed in our previous researches
which afforded numerous 1-aryl or 1,5-diarylpyrazole
derivatives [19ꢀ21].
/
So, in pursuing our studies and in order to acquire
further structureꢀactivity correlations in the field of this
/
novel class of non-ulcerogenic anti-inflammatory
agents, we planned the synthesis of a number of 4-
substituted 1,5-diarylpyrazoles 3, 4, 5, 6, as illustrated in
Fig. 2.
These derivatives have a 4-sulfamoyl (as celecoxib) or
a 4-methylsulfonyl (as rofecoxib) group on the 1-phenyl
ring; the second aromatic ring is unsubstituted, para-
substituted or meta, para-disubstituted, bearing in mind
2. Chemistry
Benzoylacetonitrile 1a and aroylacetates 1bꢀ
known. Some of them are available on the market
(1a,b,h), the others can be easily synthesized by the
Claisen condensation of the appropriate substituted
benzoic ethyl ester with ethyl acetate in the presence of
/i are all
what suggested by structureꢀ
studies carried out on various 1,2-diarylheterocyclic
inhibitors [10,11,13ꢀ15].
/
activity relationship (SAR)
sodium hydride (1cꢀ
transesterification of the corresponding ethyl ester 1b.
Treatment of 1aꢀi with N,N-dimethylformamide di-
methylacetal provided a-dimethylaminomethylene deri-
vatives (2aꢀi). A survey of the literature revealed that
only two of them (2f,i) are unknown [22ꢀ27]. Enam-
/
g); methyl ester 1i was prepared by
/
/
Among the numerous 1,5-diarylpyrazoles studied as
possible selective COX-2 inhibitors, only few are sub-
stituted at the position 4 of the heterocyclic ring,
whereas most have a substituent at the position 3 [10].
Thus, we prepared a series of 1,5-diarylpyrazoles bear-
ing a cyano, methoxy- or ethoxycarbonyl, hydroxycar-
bonyl, hydroxymethyl or formyl function at the position
4, to investigate the effects of such substitutions on the
biological properties of this class of compounds.
Some remarks guided our choices:
/
/
inones (2a-i) were condensed with [4-(methylsulfo-
nyl)phenyl]hydrazine or 4-hydrazinobenzenesulfona-
mide hydrochlorides to afford pyrazoles 3aꢀ
/
i and 3kꢀ
/
s, respectively, as sole products in agreement with what
1
already observed [19] and as confirmed by H NMR
spectra. Esters (3bꢀ
/
h,lꢀr) were converted to carboxylic
/
acids (4bꢀg,lꢀr) by alkaline hydrolysis (potassium
/
/
Fig. 1. Structures of tricyclic selective COX-2 inhibitors.

G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ
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797
Fig. 2. Synthetic routes to compounds 3ꢀ6.
/
hydroxide in ethanol), followed by acidification. In the
case of 3h, it was not possible to isolate the correspond-
ing carboxylic acid from the reaction mixture. Fisher
esterification of 4g,p with methanol afforded 3j,t.
Lithium aluminum hydride reduction in tetrahydro-
4. Experimental
4.1. Chemistry
Melting points were determined with a Fisher-Johns
apparatus and are uncorrected. IR spectra were regis-
furan of ethyl esters (3lꢀ
xymethyl derivatives (5lꢀ
/
q) gave corresponding hydro-
q); two of them (5m,o) were
/
tered on a PerkinꢀElmer 398 spectrophotometer and are
/
expressed in cmꢁ1. 1H NMR spectra were registered on
a Varian Gemini 200 (200 MHz) spectrometer; chemical
shifts are reported as d (ppm) relative to TMS as
internal standard; coupling constants (J) are expressed
in Hertz (Hz). Microanalyses for C, H, N, S were
performed using a Carlo Erba elemental analyzer model
finally oxidized to the corresponding aldehydes (6m,o)
by pyridinium chlorochromate (PCC) in dichloro-
methane.
EA 1110 and results agree within 9
/
0.3% with calculated
3. Pharmacology
values.
Among the compounds prepared by us, we have
selected esters 3b,e,g,j,o,p,q,t, carboxylic acids 4g,p,
4.1.1. General procedure for compounds 1cꢀg
/
alcohols 5mꢀ
/
o,q, aldehyde 6o, nitriles 3a,k to be
A solution of appropriate substituted benzoic ethyl
ester (10 mmol) in anhydrous benzene (50 ml) was added
to sodium hydride (0.80 g of 60% dispersion in paraffin
oil, 20 mmol) in anhydrous benzene (50 ml). The
mixture was stirred and heated to reflux while ethyl
acetate (10.60 g, 120 mmol) in anhydrous benzene (30
ml) was slowly added. The mixture was then refluxed for
4 h, cooled at 0 8C, treated with absolute ethanol (3 ml)
and stirred for 10 min. After dilution with diethyl ether
(100 ml), the mixture was poured into ice-water/glacial
acetic acid 1:1 (50 ml), the organic layer separated and
evaluated in vivo regarding their anti-inflammatory
activity (carrageenan-induced paw edema test in rats)
(Table 9).
Nitrile 3k and esters 3g,p, the most active as anti-
inflammatory agents, as well as corresponding methyl
esters 3j,t were further assayed in vivo regarding their
analgesic effect (writhing test in mice).
Finally, in vitro tests were performed to evaluate the
platelet antiaggregating properties of sodium salts of
carboxylic acids 4bꢀ
/
g,lꢀr (Table 10).
/

798
G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ808
/
the aqueous layer further extracted with diethyl ether.
The combined organic extracts were washed with
saturated sodium carbonate solution and water, dried
(magnesium sulfate) and evaporated under reduced
pressure to give a liquid residue, which was purified by
bulb-to-bulb distillation in vacuo. IR spectra in CHCl3
of all compounds showed absorption values (nmax) for
both ester and ketone functions, ranging from 1730 to
1738 cmꢁ1 and from 1673 to 1687 cmꢁ1, respectively.
Amide corresponding to 3a:
Yield 10%; m.p. 239ꢀ40 8C (from 95% ethanol); IR
/
(KBr), cmꢁ1: 3450, 3390, 1628; 1H NMR (DMSO-d6) d:
3.31 (s, 3H, CH3), 7.47 (s, 2H, NH2, exchange with
D2O), 7.5ꢀ
aromatic), 7.91 (d, Jꢂ
pyrazole H-3), 8.12 (d, Jꢂ
C17H15N3O3S (C, H, N, S).
Amide corresponding to 3k:
/
7.65 (m, 3H, aromatic), 7.75ꢀ
8.6, 2H, aromatic), 7.92 (s, 1H,
8.6, 2H, aromatic). Anal.
/7.85 (m, 2H,
/
/
Yield 8%; m.p. 212ꢀ213 8C (from 95% ethanol); IR
/
1
4.1.2. General procedure for compounds 2aꢀ
A solution of the corresponding aroylacetate 1aꢀ
/
i
(KBr), cmꢁ1: 3440, 3400, 3360, 3275, 1620; H NMR
(DMSO-d6) d: 7.42 (s, 2H, CONH2, exchange with
/i (20
mmol) in N,N-dimethylformamide dimethyl acetal (2.86
g, 24 mmol) was refluxed for 1 h. The excess acetal was
distilled off under reduced pressure and the residue was
chromatographed on Florisil, using diethyl ether as
eluant. The solid product was then recrystallized from
anhydrous diethyl ether/petroleum ether.
D2O), 7.5ꢀ
change with D2O), 7.75ꢀ
zole H-3), 7.87 (d, Jꢂ8.8, 2H, aromatic), 8.00 (d, Jꢂ
/
7.65 (m, 5H, aromaticꢃ
/
SO2NH2, 2H ex-
/
7.85 (m, 3H, aromaticꢃ
/pyra-
/
/
8.8, 2H, aromatic). Anal. C16H14N4O3S (C, H, N, S).
4.1.4. General procedure for compounds 3j,t
A solution of 4g,p (10 mmol) in anhydrous methanol
(50 ml) added with concentrated sulphuric acid (6 drops)
was refluxed with stirring for 14 h. After cooling to
room temperature, the reaction mixture was diluted with
water (30 ml), concentrated under reduced pressure and
4.1.2.1. Ethyl 3-dimethylamino-2-(4-methoxybenzoyl)-
acrylate 2f. Yield 91%; m.p. 74ꢀ
/
75 8C; IR (CHCl3),
cmꢁ1: 1675, 1625, 1600; H NMR (CDCl3) d: 0.98 (t,
Jꢂ7.2, 3H, ethyl CH3), 2.96 [s, 6H, N(CH3)2], 3.87
(s,3H, CH3O), 4.02 (q, Jꢂ7.2, 2H, ethyl CH2), 6.93 (d,
Jꢂ8.4, 2H, aromatic), 7.70 (s, 1H, ꢀCH), 7.83 (d, Jꢂ
8.4, 2H, aromatic). Anal. C15H19NO4 (C, H, N).
1
/
/
extracted with chloroform (3ꢄ30 ml). The extracts were
/
/
/
/
washed twice with saturated sodium hydrogen carbon-
ate solution and with water, dried (magnesium sulfate),
filtered and evaporated in vacuo, to give a solid residue
which was recrystallized from 95% ethanol.
For the analytical, physical and spectral data of com-
pound 3j see Tables 1 and 3; for 3t see Tables 2 and 4.
4.1.2.2. Methyl 2-benzoyl-3-dimethylaminoacrylate 2i.
Yield 72%; m.p. 83ꢀ
84 8C; IR (CHCl3), cmꢁ1: 1685,
1628, 1600; 1H NMR (CDCl3) d: 2.96 [s, 6H, N(CH3)2],
3.50 (s, 3H, CH3O), 7.25ꢀ7.6 (m, 3H, aromaticꢃ CH),
7.65ꢀ8.0 (m, 3H, aromatic). Anal. C13H15NO3 (C, H,
N).
/
/
/
ꢀ
/
/
4.1.5. General procedure for compounds 4bꢀ
Potassium hydroxide (1.68 g, 30 mmol) dissolved in
95% ethanol (10 ml) was added to a solution of 3bꢀg,lꢀr
/
g,lꢀ
/
r
/
/
4.1.3. General procedure for compounds 3aꢀ
[4-(Methylsulfonyl)phenyl]hydrazine hydrochloride
(2.23 g, 10 mmol) (3aꢀi) or (4-hydrazinobenzenesulfo-
namide hydrochloride (2.24 g, 10 mmol) (3kꢀs) was
added to a stirred solution of 2aꢀi (10 mmol) in absolute
/
i,kꢀ
/
s
(10 mmol) in the same solvent (30 ml). The mixture was
refluxed with stirring for 5 h, the solvent was evaporated
under reduced pressure and the residue dissolved with
water (50 ml). The aqueous solution was acidified with 6
/
/
/
N hydrochloric acid (pH Â1) and the white solid which
/
ethanol (60 ml). The mixture was heated to reflux for 2 h
and evaporated under reduced pressure. The residue was
taken up in chloroform and the organic solution was
washed with water, dried (magnesium sulfate), filtered
and evaporated under reduced pressure. The solid
residue was purified by recrystallization from 95%
ethanol.
separated was collected by filtration and washed with
water. The crude product was then recrystallized from
95% ethanol.
For the analytical, physical and spectral data of
compounds 4bꢀ
/
g,lꢀr see Tables 5 and 6.
/
4.1.6. General procedure for compounds 5lꢀ
/
q
For the analytical, physical and spectral data of
s see Tables
2 and 4.
Nitriles 3a,k were obtained in a mixture with the
corresponding amides from which they were separated
by chromatography on silica gel: nitriles were eluted
A solution of 3lꢀq (10 mmol) in anhydrous tetrahy-
/
compounds 3aꢀ
/
i see Tables 1 and 3; for 3kꢀ
/
drofuran (60 ml) was slowly added to a stirred solution
of lithium aluminum hydride (0.76 g, 20 mmol) in the
same solvent (50 ml). The mixture was refluxed with
stirring for 24 h, cooled at 0 8C, diluted with diethyl
ether (100 ml) and treated in succession with water (2
ml), 10% sodium hydroxide solution (2 ml) and water
(10 ml). The supernatant organic solution was decanted
and the insoluble residue was treated with 3 N hydro-
chloric acid until an acid aqueous suspension was
with ethyl acetate/petroleum ether (b.p. 40ꢀ
/
60 8C) 1:1;
amides, successively, with pure ethyl acetate.
The analytical, physical and spectral data of amides
are below reported.

G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ
/808
799
Table 1
Structures and chemical data of nitrile (3a) and esters (3bꢀ
/j)
Compound
Y
Z
Yield (%)
M.p. (8C) a
Molecular formula b
3a
3b
3c
3d
3e
3f
H
CN
73
90
86
95
97
93
88
97
78
65
213ꢀ
131ꢀ
129ꢀ
156ꢀ
163ꢀ
109ꢀ
142ꢀ
166ꢀ
192ꢀ
188ꢀ
/
14
32
30
57
64
10
43
67
93
89
C17H13N3O2S
C19H18N2O4S
C19H17FN2O4S
C19H17ClN2O4S
C20H20N2O4S
C20H20N2O5S
C21H22N2O6S
C19H17N3O6S
C18H16N2O4S
C20H20N2O6S
H
4-F
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2CH3
CO2CH3
/
/
4-Cl
/
4-CH3
4-OCH3
3,4-(OCH3)2
4-NO2
H
/
/
3g
3h
3i
/
/
/
3j
3,4-(OCH3)2
/
a
From 95% ethanol.
All compounds were analysed for C, H, N, S.
b
obtained (pH Â
/
1). Insoluble white solid was collected
by filtration and washed with water. The crude product
4.1.7. General procedure for compounds 6m,o
A solution of 5m,o (10 mmol) in anhydrous acetoni-
trile (100 ml) was added to a stirred suspension of
pyridinium chlorochromate (PCC) (3.23 g, 15 mmol) in
the same solvent (50 ml). The mixture was stirred at
was then recrystallized from ethyl acetate.
For the analytical, physical and spectral data of
compounds 3lꢀq see Tables 7 and 8.
/
Table 2
Structures and chemical data of nitrile (3k) and esters (3lꢀ
/
t)
Compound
Y
Z
Yield (%)
M.p. (8C) a
Molecular formula b
3k
3l
H
CN
72
93
84
92
96
91
88
94
80
59
192ꢀ
186ꢀ
187ꢀ
195ꢀ
178ꢀ
158ꢀ
223ꢀ
162ꢀ
197ꢀ
214ꢀ
/
93
87
88
96
79
59
24
63
98
15
C16H12N4O2S
C18H17N3O4S
C18H16FN3O4S
C18H16ClN3O4S
C19H19N3O4S
C19H19N3O5S
C20H21N3O6S
C18H16N4O6S
C17H15N3O4S
C18H17N3O5S
H
4-F
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2CH3
CO2CH3
/
3m
3n
3o
3p
3q
3r
3s
3t
/
4-Cl
/
4-CH3
4-OCH3
3,4-(OCH3)2
4-NO2
H
/
/
/
/
/
4-OCH3
/
a
From 95% ethanol.
All compounds were analysed for C, H, N, S.
b

800
G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ808
/
Table 3
Spectral data of compounds 3aꢀj
/
Comp. IR (cmꢁ1) (CHCl3)
1H NMR, d (CDCl3)
3a
3b
3c
2240 (CN); 1323, 1152 (SO2)
3.08 (s, 3H, CH3), 7.3ꢀ
3)
/
7.55 (m, 7H, aromatic), 7.94 (d, Jꢂ8.8, 2H, aromatic), 8.07 (s, 1H, pyrazole H-
/
1705 (CO); 1323, 1152 (SO2)
1713 (CO); 1322, 1155 (SO2)
1.22 (t, Jꢂ
aromatic), 7.85 (d, Jꢂ
1.24 (t, Jꢂ7.1, 3H, ethyl CH3), 3.05 (s, 3H, CH3SO2), 4.22 (q, Jꢂ
aromatic), 7.25ꢀ7.35 (m, 2H, aromatic),, 7.41 (d, Jꢂ8.6, 2H, aromatic), 7.88 (d, Jꢂ
aromatic), 8.21 (s, 1H, pyrazole H-3)
1.25 (t, Jꢂ7.1, 3H, ethyl CH3), 3.05 (s, 3H, CH3SO2), 4.23 (q, Jꢂ
aromatic), 7.38 (d, Jꢂ8.6, 2H, aromatic), 7.42 (d, Jꢂ8.8, 2H, aromatic), 7.89 (d, Jꢂ
aromatic), 8.21 (s, 1H, pyrazole H-3)
1.24 (t, Jꢂ7.1, 3H, ethyl CH3), 2.39 (s, 3H, CH3Ar), 3.04(s, 3H, CH3SO2), 4.22 (q, Jꢂ
7.18 (s, 4H, aromatic), 7.42 (d, Jꢂ8.8, 2H, aromatic), 7.86 (d, Jꢂ8.8, 2H, aromatic), 8.20 (s, 1H,
pyrazole H-3)
1.26 (t, Jꢂ7.1, 3H, ethyl CH3), 3.05 (s, 3H, CH3SO2), 3.84 (s, 3H, CH3O), 4.23 (q, Jꢂ
6.90 (d, Jꢂ8.8, 2H, aromatic), 7.21 (d, Jꢂ8.8, 2H, aromatic), 7.43 (d, Jꢂ8.6, 2H, aromatic), 7.86 (d,
Jꢂ8.6, 2H, aromatic), 8.20 (s, 1H, pyrazole H-3)
1.26 (t, Jꢂ7.1, 3H, ethyl CH3), 3.04 (s, 3H, CH3SO2), 3.76 (s, 3H, CH3O), 3.91 (s, 3H, CH3O), 4.24 (q,
Jꢂ7.1, 2H, CH2), 6.75ꢀ6.9 (m, 3H, aromatic), 7.45 (d, Jꢂ8.6, 2H, aromatic), 7.87 (d, Jꢂ8.6, 2H,
aromatic), 8.20 (s, 1H, pyrazole H-3)
1.26 (t, Jꢂ7.1, 3H, ethyl CH3), 3.06 (s, 3H, CH3SO2), 4.24 (q, Jꢂ
aromatic), 7.52 (d, Jꢂ8.9, 2H, aromatic), 7.91 (d, Jꢂ8.4, 2H, aromatic), 8.25 (s, 1H, pyrazole H-3),
8.26 (d, Jꢂ8.9, 2H, aromatic)
3.09 (s, 3H, CH3SO2), 3.76 (s, 3H, CH3OCO), 7.25ꢀ
aromatic), 8.21 (s, 1H, pyrazole H-3)
3.04 (s, 3H, CH3SO2), 3.77 (s, 6H, 2 CH3), 3.91 (s, 3H, CH3), 6.75ꢀ
8.6, 2H, aromatic), 7.87 (d, Jꢂ8.6, 2H, aromatic), 8.20 (s, 1H, pyrazole H-3)
/
7.1, 3H, ethyl CH3), 3.04 (s, 3H, CH3SO2), 4.21 (q, Jꢂ
8.9, 2H, aromatic), 8.22 (s, 1H, pyrazole H-3)
7.1, 2H, CH2), 7.05ꢀ
8.6, 2H,
/7.1, 2H, CH2), 7.25ꢀ/7.5 (m, 2H,
/
/
/
/7.15 (m, 7H,
/
/
/
3d
3e
3f
1710 (CO); 1320, 1150 (SO2)
1705 (CO); 1320, 1152 (SO2)
1708 (CO); 1320, 1153 (SO2)
1710 (CO); 1322, 1153 (SO2)
/
/
7.1, 2H, CH2), 7.24 (d, Jꢂ
/
8.5, 2H,
/
/
/8.6, 2H,
/
/
7.1, 2H, CH2),
/
/
/
/7.1, 2H, CH2),
/
/
/
/
3g
3h
/
/
/
/
/
1718 (CO); 1525, 1350 (NO2);
1323, 1157 (SO2)
/
/
7.1, 2H, CH2), 7.41 (d, Jꢂ8.5, 2H,
/
/
/
/
3i
3j
1712 (CO); 1320, 1152 (SO2)
1715 (CO); 1318, 1157 (SO2)
/
7.5 (m, 7H, aromatic), 7.85 (d, Jꢂ
/8.5, 2H,
/
6.9 (m, 3H, aromatic), 7.45 (d, Jꢂ
/
/
Table 4
Spectral data of compounds 3kꢀ
/
t
Comp. IR (cmꢁ1) (KBr)
1H NMR, d (DMSO-d6)
3k
3l
3375, 3240 (NH2); 2230 (CN); 1352, 1168 7.35ꢀ
(SO2) (d, Jꢂ
3370, 3240 (NH2); 1700 (CO); 1342, 1168 1.13 (t, Jꢂ
(SO2) NH2, exchange with D2O), 7.80 (d, Jꢂ
3320, 3215 (NH2); 1705 (CO); 1367, 1175 1.15 (t, Jꢂ7.1, 3H, CH3), 4.14 (q, Jꢂ7.1, 2H, CH2), 7.2ꢀ
(SO2) (m, 6H, aromaticꢃNH2, 2H exchange with D2O), 7.82 (d, Jꢂ
pyrazole H-3)
3340, 3225 (NH2); 1705 (CO); 1365, 1172 1.15 (t, Jꢂ7.1, 3H, CH3), 4.15 (q, Jꢂ
(SO2) exchange with D2O), 7.83 (d, Jꢂ8.5, 2H, aromatic), 8.27 (s, 1H, pyrazole H-3)
3370, 3230 (NH2); 1685 (CO); 1342, 1163 1.15 (t, Jꢂ7.1, 3H, ethyl CH3), 2.34 (s, 3H, CH3Ar), 4.14 (q, Jꢂ7.1, 2H, CH2), 7.22 (s, 4H,
(SO2) 8.6, 2H, aromatic), 7.48 (s, 2H, NH2, exchange with D2O), 7.80 (d, Jꢂ
/
7.45 (m, 2H, aromatic), 7.5ꢀ
8.6, 2H, aromatic), 8.51 (s, 1H, pyrazole H-3)
7.1, 3H, CH3), 4.14 (q, Jꢂ7.1, 2H, CH2), 7.3ꢀ
8.5, 2H, aromatic), 8.27 (s, 1H, pyrazole H-3)
7.35 (m, 2H, aromatic), 7.35ꢀ
8.9, 2H, aromatic), 8.28 (s, 1H,
/
7.6 (m, 7H, aromaticꢃNH2, 2H exchange with D2O), 7.87
/
/
/
/
/7.45 (m, 7H, aromatic), 7.48 (s, 2H,
/
3m
/
/
/
/7.5
/
/
3n
3o
/
/
7.1, 2H, CH2), 7.35ꢀ
/
7.55 (m, 8H, aromaticꢃNH2, 2H
/
/
/
/
aromatic), 7.42 (d, Jꢂ
/
/
8.6, 2H, aromatic), 8.25 (s, 1H, pyrazole H-3)
3p
3q
3290, 3215 (NH2); 1700 (CO); 1345, 1172 1.17 (t, Jꢂ
(SO2) 8.9, 2H, aromatic), 7.27 (d, Jꢂ
NH2, exchange with D2O), 7.82 (d, Jꢂ
3360, 3265 (NH2); 1697 (CO); 1340, 1160 1.66 (t, Jꢂ7.1, 3H, ethyl CH3), 3.63 (s, 3H, CH3O), 3.78 (s, 3H, CH3O), 4.16 (q, Jꢂ
(SO2) CH2), 6.82 (dd, J?ꢂ8.8, Jƒꢂ2, 1H, aromatic), 6.9ꢀ7.0 (m, 2H, aromatic), 7.45 (d, Jꢂ
2H, aromatic), 7.49 (s, 2H, NH2, exchange with D2O), 7.82 (d, Jꢂ
1H, pyrazole H-3)
3320, 3250 (NH2); 1727 (CO); 1523, 1335 1.14 (t, Jꢂ7.1, 3H, CH3), 4.15 (q, Jꢂ
(NO2); 1345, 1177 (SO2) 2H, NH2, exchange with D2O), 7.69 (d, Jꢂ
8.27 (d, Jꢂ8.8, 2H, aromatic), 8.34 (s, 1H, pyrazole H-3)
3360, 3265 (NH2); 1700 (CO); 1343, 1167 3.69 (s, 3H, CH3), 7.3ꢀ7.45 (m, 7H, aromatic), 7.49 (s, 2H, NH2, exchange with D2O), 7.80 (d,
(SO2) Jꢂ8.5, 2H, aromatic), 8.29 (s, 1H, pyrazole H-3)
3345, 3220 (NH2); 1708 (CO); 1351, 1170 3.77 (s, 3H, CH3), 3.84 (s, 3H, CH3), 6.91 (d, Jꢂ
(SO2) aromatic), 7.26 (s, 2H, NH2, exchange with D2O), 7.38 (d, Jꢂ
8.6, 2H, aromatic), 8.19 (s, 1H, pyrazole H-3)
/
7.1, 3H, ethyl CH3), 3.79 (s, 3H, CH3O), 4.15 (q, Jꢂ
8.8, 2H, aromatic), 7.43 (d, Jꢂ8.8, 2H, aromatic), 7.49 (s, 2H,
8.6, 2H, aromatic), 8.24 (s, 1H, pyrazole H-3)
7.1, 2H,
8.5,
/
7.1, 2H, CH2), 6.96 (d, Jꢂ
/
/
/
/
/
/
/
/
/
/
/
8.5, 2H, aromatic), 8.24 (s,
3r
/
/7.1, 2H, CH2), 7.47 (d, Jꢂ/8.8, 2H, aromatic), 7.49 (s,
/
8.8, 2H, aromatic), 7.82 (d, Jꢂ8.6, 2H, aromatic),
/
/
3s
3t
/
/
/
8.6, 2H, aromatic), 7.21 (d, Jꢂ/8.7, 2H,
/
8.5, 2H, aromatic), 7.85 (d, Jꢂ
/

G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ
/808
801
Table 5
Structures and chemical data of carboxylic acids (4bꢀ
/
g,lꢀr)
/
Compound
X
Y
Yield (%)
M.p. (8C)
Molecular formula e
4b
4c
4d
4e
4f
SO2CH3
SO2CH3
SO2CH3
SO2CH3
SO2CH3
SO2CH3
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
H
4-F
98
90
83
91
94
91
83
94
94
90
93
97
93
234ꢀ
260ꢀ
253ꢀ
234ꢀ
237ꢀ
253ꢀ
281ꢀ
234ꢀ
255ꢀ
239ꢀ
237ꢀ
279ꢀ
262ꢀ
/
35 a
61 b
54 b
35 b
38 a
54 b
82 b
35 a
56 a
40 c
38 c
80 d
63 d
C17H14N2O4S
C17H13FN2O4S
C17H13ClN2O4S
C18H16N2O4S
C18H16N2O5S
C19H18N2O6S
C16H13N3O4S
C16H12FN3O4S
C16H12ClN3O4S
C17H15N3O4S
C17H15N3O5S
C18H17N3O6S
/
4-Cl
/
4-CH3
4-OCH3
/
/
4g
4l
(3,4-OCH3)2
H
/
/
4m
4n
4o
4p
4q
4r
4-F
4-Cl
/
/
4-CH3
4-OCH3
3,4-(OCH3)2
4-NO2
/
/
/
/
C16H12N4O6S×H2O
/
a
From ethyl acetate.
From 95% ethanol.
b
c
From ethyl acetate/petroleum ether (b.p. 40ꢀ
From anhydrous ethanol.
All compounds were analysed for C, H, N, S.
/
70 8C).
d
e
room temperature for 24 h and, therefore, diluted with
anhydrous diethyl ether (100 ml). The supernatant
organic solution was decanted from a black gum, which
aromatic), 8.26 (s, 1H, pyrazole H-3), 9.74 (s, 1H,
CHO). Anal. C17H15N3O3 S (C, H, N, S).
was washed with anhydrous diethyl ether (3ꢄ30 ml).
/
4.2. Pharmacology
The combined organic solution was filtered on Florisil
and the solvent was removed by distillation. The crude
residue was purified by recrystallization from ethyl
acetate.
All tested compounds were administered orally, at the
initial dose of 200 mg/kg. Compounds, which exhibited
a statistically significant activity at this dose, were
further tested at the dose of 100 mg/kg.
4.1.7.1. 4-[5-(4-Fluorophenyl)-4-formyl-1H-pyrazol-1-
yl]benzenesulfonamide (6m). Yield 81%; m.p. 179ꢀ
/
180 8C; IR (KBr), cmꢁ1: 3305, 3170, 1677, 1345, 1163;
1H NMR (CDCl3) d: 4.92 (s, 2H, NH2, exchange with
4.2.1. Anti-inflammatory activity
The carrageenan-induced paw edema test [28] was
used on groups of five rats. Sixty minutes after admin-
istering the test compound, 0.1 ml of a 1% carrageenan
solution in saline was injected into the plantar surface of
the right hind paw of each rat. Paw volume, as
determined by measuring the amount of water displaced
after immersing the paw up to the level of the lateral
malleolus, was recorded immediately after the carragee-
nan injection and again 2 h later. The difference between
these two values was taken as edema volume. The
percent inhibition of the edema of treated rats with
respect to controls was calculated. Indomethacin (6 mg/
kg p.o.) was used as a reference standard.
D2O), 7.1ꢀ
aromatic), 7.91 (d, Jꢂ
pyrazole H-3), 9.76 (s, 1H, CHO). Anal. C16H12N3O3 S
(C, H, N, S).
/
7.35 (m, 4H, aromatic), 7.41 (d, Jꢂ
/
8.9, 2H,
/8.9, 2H, aromatic), 8.27 (s, 1H,
4.1.7.2. 4-[5-(4-methylphenyl)-4-formyl-1H-pyrazol-1-
yl]benzenesulfonamide (6o). Yield 53%; m.p. 207ꢀ
/
209 8C; IR (KBr), cmꢁ1: 3345, 3290, 1655, 1357, 1168;
1H NMR (CDCl3) d: 2.43 (s, 3H, CH3), 4.89 (s, 2H,
NH2, exchange with D2O), 7.15ꢀ
7.43 (d, Jꢂ8.6, 2H, aromatic), 7.89 (d, Jꢂ
/
7.3 (m, 4H, aromatic),
8.6, 2H,
/
/

802
G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ808
/
Table 6
Spectral data of compounds 4bꢀ
/
g,lꢀ
/
r
Comp. IR (cmꢁ1) (KBr) 1H NMR, d (DMSO-d6)
4b
4c
4d
4e
4f
3200ꢀ
(CO2H)
3200ꢀ2400, 1685
(CO2H)
3200ꢀ2400, 1680
(CO2H)
3200ꢀ2400, 1683
(CO2H)
3200ꢀ2400, 1675
(CO2H)
/
2400, 1675
3.26 (s, 3H, CH3), 7.3ꢀ
1H, pyrazole H-3), Â12.2 (very br s, 1H, CO2H, exchanges with D2O)
3.27 (s, 3H, CH3), 7.2ꢀ7.35 (m, 2H, aromatic), 7.4ꢀ7.55 (m, 4H, aromatic), 7.94 (d, Jꢂ
1H, pyrazole H-3), 12.57 (br s, 1H, CO2H, exchanges with D2O)
3.27 (s, 3H, CH3), 7.35ꢀ7.55 (m, 6H, aromatic), 7.96 (d, Jꢂ8.6, 2H, aromatic), 8.26 (s, 1H, pyrazole H-3), 12.59 (br
s, 1H, CO2H, exchanges with D2O)
2.40 (s, 3H, CH3Ar), 3.05 (s, 3H, CH3SO2), 7.18 (s, 4H, aromatic), 7.41 (d, Jꢂ
2H, aromatic), 8.25 (s, 1H, pyrazole H-3), 12.52 (br s, 1H, CO2H, exchanges with D2O)
3.26 (s, 3H, CH3SO2), 3.79 (s, 3H, CH3O), 6.96 (d, Jꢂ8.8, 2H, aromatic), 7.27 (d, Jꢂ8.8, 2H, aromatic), 7.48 (d,
Jꢂ8.6, 2H, aromatic), 7.94 (d, Jꢂ8.6, 2H, aromatic), 8.22 (s, 1H, pyrazole H-3), Â12.4 (very br s, 1H, CO2H,
exchanges with D2O)
3.26 (s, 3H, CH3SO2), 3.60 (s, 3H, CH3O), 3.78 (s, 3H, CH3O), 6.84 (dd, J?ꢂ
2H, aromatic), 7.50 (d, Jꢂ8.8, 2H, aromatic), 7.94 (d, Jꢂ8.8, 2H, aromatic), 8.22 (s, 1H, pyrazole H-3), Â
(very br s, 1H, CO2H, exchanges with D2O)
7.3ꢀ7.5 (m, 9H, aromaticꢃNH2, 2H exchange with D2O), 7.78 (d, Jꢂ
12.49 (br s, 1H, CO2H, exchanges with D2O)
7.2ꢀ7.3 (m, 2H, aromatic), 7.35ꢀ7.55 (m, 6H, aromaticꢃ
aromatic), 8.23 (s, 1H, pyrazole H-3), Â12.5 (very br s, 1H, CO2H, exchanges with D2O)
7.3ꢀ7.55 (m, 8H, aromaticꢃNH2, 2H exchange with D2O), 7.82 (d, Jꢂ8.7, 2H, aromatic), 8.24 (s, 1H, pyrazole H-
3), Â12.5 (br s, 1H, CO2H, exchanges with D2O)
2.34 (s, 3H, CH3), 7.24 (s, 4H, aromatic), 7.41 (d, Jꢂ
(d, Jꢂ8.7, 2H, aromatic), 8.21 (s, 1H, pyrazole H-3), 12.43 (br s, 1H, CO2H, exchanges with D2O)
3.79 (s, 3H, CH3O), 6.96 (d, Jꢂ8.8, 2H, aromatic),7.26 (d, Jꢂ8.8, 2H, aromatic), 7.42 (d, Jꢂ8.7, 2H, aromatic),
7.48 (s, 2H, NH2, exchange with D2O), 7.81 (d, Jꢂ8.7, 2H, aromatic), 8.20 (s, 1H, pyrazole H-3), 12.44 (br s, 1H,
CO2H, exchanges with D2O)
3.61 (s, 3H, CH3O), 3.77 (s, 3H, CH3O), 6.83 (dd, J?ꢂ
Jꢂ8.5, 2H, aromatic), 7.47 (s, 2H, NH2, exchange with D2O), 7.81 (d, Jꢂ
H-3), Â12.4 (very br s, 1H, CO2H, exchanges with D2O)
7.45 (d, Jꢂ8.8, 2H, aromatic), 7.48 (s, 2H, NH2, exchanges with D2O), 7.68 (d, Jꢂ
8.6, 2H, aromatic), 8.25 (d, Jꢂ8.9, 2H, aromatic), 8.29 (s, 1H, CH-3 pyrazole), Â
exchanges with D2O)
/
7.45 (m, 5H, aromatic), 7.47 (d, Jꢂ
/
8.6, 2H, aromatic), 7.92 (d, Jꢂ
/
8.7, 2H, aromatic), 8.26 (s,
/
/
/
/
/8.6, 2H, aromatic), 8.25 (s,
/
/
/
/
/8.5, 2H, aromatic), 7.87 (d, Jꢂ8.5,
/
/
/
/
/
/
/
4g
3200ꢀ
/
2400, 1688
/
8.5, Jƒꢂ/2, 1H, aromatic), 6.9ꢀ
/7.0 (m,
(CO2H)
/
/
/
12.4
4l
3200ꢀ
(CO2H)
3200ꢀ2400 1689
(CO2H)
3200ꢀ2400, 1678
(CO2H)
3200ꢀ2400, 1688
(CO2H)
3200ꢀ2400, 1694
(CO2H)
/
2400, 1665
/
/
/
8.6, 2H, aromatic), 8.23 (s, 1H, pyrazole H-3),
4m
4n
4o
4p
/
/
/
/
NH2, 2H exchange with D2O), 7.81 (d, Jꢂ8.6, 2H,
/
/
/
/
/
/
/
/
/
8.7, 2H, aromatic), 7.47 (s, 2H, NH2, exchange with D2O), 7.80
/
/
/
/
/
/
4q
4r
3200ꢀ
/
2400, 1681
/
8.5, Jƒꢂ
/
2, 1H, aromatic), 6.9ꢀ
/
7.0 (m, 2H, aromatic), 7.43 (d,
(CO2H)
/
/8.5, 2H, aromatic), 8.19 (s, 1H, pyrazole
/
3200ꢀ
/
2400, 1705
/
/8.9, 2H, aromatic), 7.82 (d, Jꢂ
/
(CO2H)
/
/12.7 (very br s, 1H, CO2H,
4.2.2. Analgesic activity
Platelet count in PRP was maintained at 300.000/
mmc.
The writhing test [29] was used on a group of five
mice. One hour after the administration of the test
compound, 0.01 ml/g of a 0.6% acetic acid solution was
injected intraperitoneally in each mouse. The writhing
movements of each animal were counted for 10 min
(between the 5th and 15th min after the injection of the
irritant). The antinociceptive effect was expressed as the
percentage of protection compared to the control group.
Indomethacin (6 mg/kg p.o.) was used as a reference
standard.
Platelet aggregation, performed in a Aggrecorder II
PA 3220 aggregometer (Menarini, Firenze, Italy) was
measured according to the Born’s turbidimetric method
[30] and quantified by the maximal light transmission
reached 5 min after the addition of the agonist.
A first sample of PRP was pre-incubated at 37 8C for
2 min. After this time, platelet aggregation was induced
adding adenosine diphosphate (ADP; 2 and 5 mM),
collagen (4 and 8 mg/ml) or adrenaline (5 mM). A second
sample of PRP was incubated for 2 min with a solution
of the tested compound, before the addition of the
agonist.
4.2.3. Platelet aggregation
Comparing the maximal light transmission of the
aggregation curves obtained with and without the
addition of the tested compound, the percentage of
inhibition of platelet aggregation was calculated.
Human blood samples from normal subjects were
drawn through a 19-gauge needle, avoiding carefully
prolonged venous stasis. None of these subjects was
treated with any drug known to influence the platelet
function.
4.3. Computational methods
Blood was collected in plastic tubes containing 3.8%
trisodium citrate aqueous solution. Platelet rich plasma
(PRP) was obtained by centrifuging the blood at 100ꢄ
for 20 min. Platelet poor plasma (PPP) was obtained by
centrifuging the remaining blood at 1100ꢄg for 15 min.
/
g
4.3.1. Molecular docking
The crystal structures of murine COX-2 in complex
with SC-558 (1cx2) [17] and ovine COX-1 in complex
/

G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ
/808
803
Table 7
Structures and chemical data of carbinols (5lꢀ
specific portion of the catalytic site of the enzyme, in
particular the system size was reduced for the docking
calculations by including only residues containing atoms
/q)
˚
within 15 A from any ligand atom. The search was
carried out with the Lamarckian Genetic Algorithm:
populations of 250 individuals with a mutation rate of
0.03 have been evolved for 100 generations. Evaluation
of the results was done by sorting the different
complexes with respect to the predicted binding energy,
internal strain energy of the ligand, van der Waals and
electrostatic interaction energies. All selected complexes
underwent to a final energy minimization, before the
model would be achieved. All calculations were per-
formed on an O2 SGI workstation.
Compound
Y
Yield (%) M.p. (8C) Molecular formula c
5l
H
4-F
82
85
88
84
73
58
209ꢀ
204ꢀ
184ꢀ
181ꢀ
167ꢀ
228ꢀ
/
10 a C16H15N3O3S
05 a C16H14FN3O3S
85 b C16H14ClN3O3S
83 b C17H17N3O3S
68 a C17H17N3O4S
30 b C18H19N3O5S
5m
5n
5o
5p
5q
/
4-Cl
/
5. Results and discussion
4-CH3
4-OCH3
3,4-
/
/
/
In the carrageenan-induced paw edema test, at a dose
of 200 mg/kg, only esters 3g,p and nitrile 3k showed a
good anti-inflammatory activity, comparable to that of
celecoxib, but in the writhing test they did not show the
same analgesic effect as the reference drug (Table 9).
Considering the couples of nitriles 3a,k and esters
3e,o, pharmacological results confirmed a better anti-
phlogistic activity in vivo for sulfamoyl group with
respect to methylsulfonyl one, as already observed [10],
but, in the case of esters 3g,q, the biological data did not
match with these remarks.
(OCH3)2
a
From 95% ethanol.
From ethyl acetate.
All compounds were analysed for C, H, N, S.
b
c
with flurbiprofen (1eqh) [31] were obtained from the
Protein Data Bank. The potential of the 3D structures
of COX-2 and COX-1 was assigned according to the
Amber 4.0 force field.
The structural models of selected compounds, gener-
ated and energy minimized within MacroModel [32],
were docked to COX-2 and COX-1 active sites using
AutoDock 3.0 [33]. The region of interest used by
Autodock was defined in such a way to include a
As regards the ethyl esters 3g,p, they appeared most
effective as antiinflammatory agents than the corre-
sponding methyl esters 3j,t, but less active as analgesics.
Conversion of the ester group of 3g,p into the
carboxylic function (compounds 4g,p) produced the
Table 8
Spectral data of compounds 5lꢀq
/
Comp. IR (cmꢁ1
(KBr)
)
1H NMR, d (DMSO-d6)
5l
3480 (OH)
4.33 (d, Jꢂ
aromatic), 7.4ꢀ
H-3)
4.31 (d, Jꢂ
aromatic), 7.45 (s, 2H, NH2, exchange with D2O), 7.81 (d, Jꢂ
4.32 (d, Jꢂ5, 2H, CH2), 5.10 (t, Jꢂ5, 1H, OH, exchanges with D2O), 7.32 (d, Jꢂ
aromatic), 7.46 (s, 2H, NH2, exchange with D2O),7.53 (d, Jꢂ8.6, 2H, aromatic), 7.82 (d, Jꢂ
1H, pyrazole H-3)
2.35 (s, 3H, CH3), 4.31 (d, Jꢂ
(d, Jꢂ8, 2H, aromatic), 7.38 (d, Jꢂ
aromatic), 7.85 (s, 1H, pyrazole H-3)
3.80 (s, 3H, CH3), 4.30 (d, Jꢂ5, 2H, CH2), 5.30 (t, Jꢂ
(d, Jꢂ9, 2H, aromatic), 7.39 (d, Jꢂ8.8, 2H, aromatic), 7.44 (s, 2H, NH2, exchange with D2O), 7.80 (d, Jꢂ
aromatic), 7.84 (s, 1H, pyrazole H-3)
3.61 (s, 3H, CH3), 3.79 (s, 3H, CH3), 4.34 (d, Jꢂ
8.3, Jƒꢂ2, 1H, aromatic), 6.88 (d, Jꢂ2, 1H, aromatic), 7.01 (d, Jꢂ
(s, 2H, NH2, exchange with D2O), 7.81 (d, Jꢂ9, 2H, aromatic), 7.84 (s, 1H, pyrazole H-3)
/
5, 2H, CH2), 5.07 (t, Jꢂ
/
5, 1H, OH, exchanges with D2O), 7.2ꢀ
/
7.3 (m, 3H, aromatic), 7.34 (d, Jꢂ
/
8.8, 2H,
/7.5 (m, 4H, aromaticꢃ
/NH2, 2H exchange with D2O), 7.79 (d, Jꢂ
/
8.8, 2H, aromatic), 7.88 (s, 1H, pyrazole
5m
5n
3525 (OH)
3485 (OH)
/5, 2H, CH2), 5.08 (t, Jꢂ
/
5, 1H, OH, exchanges with D2O), 7.25ꢀ
8.6, 2H, aromatic), 7.87 (s, 1H, pyrazole H-3)
8.6, 2H, aromatic), 7.40 (d, Jꢂ
8.6, 2H, aromatic), 7.88 (s,
/
7.35 (m, 4H, aromatic), 7.39 (d, Jꢂ
/8.6, 2H,
/
/
/
/
/
8.6, 2H,
/
/
5o
5p
5q
3470 (OH)
3465 (OH)
3470 (OH)
/
5, 2H, CH2), 5.05 (t, Jꢂ
/5, 1H, OH, exchanges with D2O), 7.17 (d, Jꢂ
/8, 2H, aromatic), 7.25
/
/
8.5, 2H, aromatic), 7.45 (s, 2H, NH2, exchange with D2O), 7.80 (d, Jꢂ
/
8.5, 2H,
/
/5, 1H, OH, exchanges with D2O), 7.00 (d, Jꢂ
/9, 2H, aromatic), 7.22
/
/
/
8.9, 2H,
/5, 2H, CH2), 5.05 (t, Jꢂ/5, 1H, OH, exchanges with D2O), 6.80 (dd, J?ꢂ
/
/
/
/8.3, 1H, aromatic), 7.41(d, Jꢂ
/
9, 2H, aromatic), 7.44
/

804
G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ808
/
Table 9
Anti-inflammatory and analgesic activities of tested compounds
Compound
Tested dose (mg/kg p.o.)
Anti-inflammatory activity in rat a
Analgesic activity in mice b
Inhibition (%)
Protection (%)
3a
3b
3e
3g
200
200
200
200
100
200
200
100
200
200
100
200
200
100
200
200
200
200
200
200
200
200
6
26
9
0
40**
0
7
3j
3k
24
20
26
46*
30
3o
3p
21
37**
15
23
3q
3t
23
25
37**
31**
4g
4p
0
14
5m
5n
0
20
5o
5q
16
17
6o
24
Celecoxib
Indomethacin
Dipyrone
Morphine
45*
65*
77**
85*
100
15
58**
84*
a
Carrageenan-induced paw edema test (nꢂ
/
5). Statistical significance versus control group (nꢂ
/
25; control value: 2159
/
65) was evaluated by t-
test.
b
Writhing test (nꢂ
* P B0.01.
** P B0.05 versus control.
/
5). Statistical significance versus control group (nꢂ
/
10; control value: 52913) was evaluated by the Mannꢀ
/
/Whitney test.
/
/
total loss or a strong decrease of anti-inflammatory
activity; reduction of esters 3o,q to alcohols 5o,q and
oxidation of 5o to aldehyde 6o did not produce any
improvement in biological properties.
In order to further rationalize these results, a mole-
cular docking study was carried out on compounds 3g,k,
the most active as anti-inflammatory agents, and 4e,
endowed with interesting platelet anti-aggregating prop-
erties. Selected compounds were docked into the active
site of COX-1 and COX-2, using AutoDock procedure.
The ability of this program in predicting a conformation
of the ligand very close to its X-ray structure has been
widely described in literature [15].
Starting point for this study were the X-rays struc-
tures of murine COX-2 complexed with the analogue of
celecoxib SC-558 (Fig. 3) [17] and ovine COX-1
complexed with the non-selective cyclooxygenase inhi-
bitor flurbiprofen (Fig. 3) [31].
Concerning results on binding to COX-2, the main
conformational difference between our 1,5-diarylpyra-
zoles and SC-558 is represented by the two torsional
angles defined by rotation of the two phenyl rings on
positions 1 and 5, respectively. According to our
calculations (Fig. 4), 1,5-diarylpyrazoles are located in
the centre of the COX-2 binding pocket in a conforma-
tion similar to the one adopted by SC-558 in the co-
All carboxylic acids (4bꢀ
/
g,lꢀr), tested in vitro, as
/
sodium salts, in platelet anti-aggregating assays, showed
a strong inhibition of the platelet function (generally
70ꢀ
/
90% at a concentration of 1ꢄ
10ꢁ3 M), when ADP
/
(5 or 2 mM) was used; good inhibition was also produced
in the presence of collagen at a concentration of 1 mg/ml,
whereas weak or no activity appeared when adrenaline
(5 mM) or collagen (4 mg/ml) were employed as agonists
(Table 10). The only isoform detectable in the platelets is
COX-1, responsible of the production of thromboxane
A2 (TXA2), a potent pro-aggregatory substance [34];
therefore, the inhibition of platelet aggregation could be
acquired as an indirect proof of activity versus COX-1.
The lack of anti-inflammatory efficacy together with
good platelet anti-aggregating activity, as found
for compounds 4g,p, seems to confirm that the presence
of a carboxylic function promotes selectivity for COX-1
[18].

G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ
/808
805
Table 10
Platelet anti-aggregating activity of compounds 4bꢀ
plasma
/
g,lꢀr: inhibition (%) of platelet aggregation induced by ADP, collagen and adrenaline in human
/
a
Comp.
Inhibition (%) of platelet aggregation induced by ADP, collagen and adrenaline in human plasma a
ADP (5 mM)
ADP (2 mM)
Collagen (4 mg/ml)
Collagen (1 mg/ml)
Adrenaline (5 mM)
4b
4c
62
79
88
76
80
73
73
81
86
68
78
7
80
82
82
81
80
10
86
78
69
81
70
90
71
45
1
2
40
27
63
84
52
40
67
14
22
76
77
30
25
79
14
0
4d
4e
1
17
56
20
5
9
4f
5
4g
4l
6
12
15
4
17
3
4m
4n
4o
4p
4q
4r
12
13
21
7
18
2
0
77
33
0
60
4
67
ASA
a
Mean value of four determinations at the final concentration of tested compoundsꢂ
/
1ꢄ
10ꢁ3 M.
/
crystallized structure with this enzyme. They share with
SC-558 a common hydrogen bonding interaction,
Arg513, but lack the hydrogen bond with Arg120, being
unsubstituted on position 3. The fluorine atom in the
trifluoromethyl group of SC-558, in fact, acts as an
acceptor to form a H-bond with one of the two NH2
groups of Arg120 side chain, anchoring the inhibitor in
the binding pocket. Compound 4e is, however, able to
form an additional hydrogen bond with the backbone
carbonyl group of Ala527, in agreement with recent
findings of Liu [15]; while 3k shows an additional bond
via a polar interaction between the ciano group on
position 4 and the backbone carbonyl group of the same
residue, Ala527. This interaction with Ala527, however,
does not seem so strong as the one with the residue
Arg120. Interestingly, the binding model obtained with
AutoDock for 3g points out that the substituent on
position 4 should not exceed the ethoxycarbonyl group
in order to maintain the favourable interaction with the
active site of COX-2. In Fig. 4 is displayed the fitting of
ester portion of 3g in a small pocket defined by Ala527,
Pro528, Ser530, Leu 531, Leu384, in agreement with the
above cited study of Liu.
hydrogen bonds with surrounding residues Val119,
Ser121, Glu524, Leu531.
Considering these preliminary partial data from the
biological tests and the computational studies, we could
say therefore that these results are generally in agree-
ment with the information already acquired on the SAR
of diarylpyrazole cyclooxygenase inhibitors.
In particular, our modeling study:
i) confirms that the main interaction of COX-2
inhibitors with the enzyme is the hydrogen bond
with Arg513, made by the sulfonamide or methyl-
sulfone mojety, which has however the possibility of
making an extended net of alternative hydrogen
bonds in the surrounding area, interacting with
residues Gln 192, Leu 352, Ser 353;
ii) suggests that the interaction with Arg120 could
be important not only for COX-1, but also for
COX-2 inhibitors, since our derivatives, which are
In the case of COX-1 complexes, the results of our
calculations suggest that, among the studied molecules,
carboxylic acid 4e is able to interact with the enzyme
active site in a way similar to flurbiprofen, the co-
crystallized ligand for ovine COX-1, since it makes two
hydrogen bonds with Arg120 (Fig. 5). The cyano
derivative 3k shows a common conformational prefer-
ence as 4e, but interacts with Arg120 making only a
polar interaction. For ester 3g, a different docking pose
in the active site of COX-1 has been observed, probably
due to the possibility of establishing a high number of
Fig. 3. Structures of a selective COX-2 inhibitor (SC-558) and a non-
selective COX inhibitor (flurbiprofen).

806
G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ808
/
Fig. 4. Overlay of the docked orientations for 3g,k, 4e and SC-558 in the active site of COX-2. The most significative amino acid residues are
reported and labelled accordingly. Residues involved in hydrogen bonds with the inhibitors are represented as sticks, the dashed lines are the
hydrogen bonds.
not able to make an hydrogen bond with this
residue, result less potent than celecoxib and SSC-
558;
However, none of the substituents we have intro-
duced on position 4 of the pyrazole ring produced
an improvement of the anti-inflammatory activity in
comparison with celecoxib.
iii) confirms that substituents on position 4 are able to
interact with Ala527, but should not exceed a well
defined volume. Among the groups introduced by
us in this position, the ethoxycarbonyl is the best in
fitting the small hydrophobic pocket defined by
residues Ala527, Pro528, Ser530, Leu531, Leu384,.
Further biological and 3D QSAR investigations are in
progress to provide possible guidelines and suggestions
for the rational design of improved COX-1 or COX-2
inhibitors.
Fig. 5. Overlay of the docked orientations for 3k, 4e and flurbiprofen in the active site of COX-1. The most significative amino acid residues are
reported and labelled accordingly. Residues involved in hydrogen bonds with the inhibitors are represented as sticks, the dashed lines are the
hydrogen bonds.

G. Menozzi et al. / Il Farmaco 58 (2003) 795ꢀ
/808
807
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Seibert, P.C. Isakson, Selective cyclooxygenase inhibitors: novel
1,2-diarylcyclopentenes are potent and orally active COX-2
Acknowledgements
We would like to thank the Ministero dell’Universita`
e della Ricerca Scientifica e Tecnologica for the financial
support.
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