Aryl-substituted methyleneaminoxymethyl (MAOM) analogues of diarylcyclopentenyl cyclooxygenase-2 inhibitors: effects of some structural modifications on their biological properties.

Article abstract of DOI:10.1016/S0223-5234(02)01385-5

The (E)-[2-(4-aminosulfonylphenyl)-1-cyclopentenyl-1-methyliden]-(arylmethyloxy)amines (6a,b), which are the sulfonamidic analogues of the previously described methylsulfonyl derivatives 5a,b, and their corresponding sulfides (7a,b) and sulfoxides (8a,b) were synthesised in order to obtain information about the role played by these different sulphur-containing groups in the cyclooxygenase-2 inhibitory activity of this class of compounds. In addition, other chemical manipulations concerning the oxime-ether substituent of this type of derivatives were affected by preparing compounds 9a,b, which present a methyl group on the oximic carbon of the oxime-ether chain of 5a,b, and compounds 10 and 11, in which the atomic sequence (C=NOCH(2)) of the MAOMM of 8b and 5b, respectively, is inverted. Compounds 6-11 were tested in vitro for their inhibitory activity towards COX-1 and COX-2 by measuring prostaglandin E2 (PGE2) production in U937 cell lines and activated J774.2 macrophages, respectively. Some of the new compounds showed an appreciable in vitro COX-2 inhibitory activity, with IC(50) values in the microM (7a,b, 8a and 9b) or sub-microM (8b) range. This last compound was also assayed in vivo for its antiinflammatory activity by means of the carrageenan-induced paw edema test in rats. No inhibitory effects were detected up to dose of 30 mg kg(-1) orally administered.

Full text of DOI:10.1016/S0223-5234(02)01385-5

Eur. J. Med. Chem. 37 (2002) 585–594
www.elsevier.com/locate/ejmech
Original article
Aryl-substituted methyleneaminoxymethyl (MAOM) analogues of
diarylcyclopentenyl cyclooxygenase-2 inhibitors: effects of some
structural modifications on their biological properties
Aldo Balsamo a,*, Isabella Coletta b, Angelo Guglielmotti b, Carla Landolfi b,
Annalina Lapucci a, Francesca Mancini b, Claudio Milanese b, Filippo Minutolo a,
Elisabetta Orlandini a, Gabriella Ortore a, Mario Pinza b, Simona Rapposelli a
a Dipartimento di Scienze Farmaceutiche, Facolta` di Farmacia, Uni6ersita` di Pisa, 6ia Bonanno 6, 56126 Pisa, Italy
b ACRAF, Angelini Ricerche, 00040 S. Palomba, Pomezia, Rome, Italy
Received 11 January 2002; accepted 12 April 2002
Abstract
The (E)-[2-(4-aminosulfonylphenyl)-1-cyclopentenyl-1-methyliden]-(arylmethyloxy)amines (6a,b), which are the sulfonamidic
analogues of the previously described methylsulfonyl derivatives 5a,b, and their corresponding sulfides (7a,b) and sulfoxides (8a,b)
were synthesised in order to obtain information about the role played by these different sulphur-containing groups in the
cyclooxygenase-2 inhibitory activity of this class of compounds. In addition, other chemical manipulations concerning the
oxime-ether substituent of this type of derivatives were affected by preparing compounds 9a,b, which present a methyl group on
the oximic carbon of the oxime-ether chain of 5a,b, and compounds 10 and 11, in which the atomic sequence (CꢀNOCH2) of the
MAOMM of 8b and 5b, respectively, is inverted. Compounds 6–11 were tested in vitro for their inhibitory activity towards
COX-1 and COX-2 by measuring prostaglandin E2 (PGE2) production in U937 cell lines and activated J774.2 macrophages,
respectively. Some of the new compounds showed an appreciable in vitro COX-2 inhibitory activity, with IC50 values in the mM
(7a,b, 8a and 9b) or sub-mM (8b) range. This last compound was also assayed in vivo for its antiinflammatory activity by means
of the carrageenan-induced paw edema test in rats. No inhibitory effects were detected up to dose of 30 mg kg−1 orally
´
administered. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Antiinflammatory drug; COX-2 inhibitor; Aryl-substituted methyleneaminoxymethyl moiety; (E)-(2-aryl-1-cyclopentenyl-1-alkyli-
dene)(arylmethyloxy)amine; (E)-[2-aryl-1-cyclopentenylmethyloxy)-(benzylidene)amine
1. Introduction
Most non-steroidal
tors, and which appears to play a major role in the
production of prostaglandins associated with inflamma-
tion responses [3–8].
antiinflammatory
drugs
(NSAIDs) act through the inhibition of cyclooxygenase
(COX) [1,2]. This enzyme exists in two isoforms, one
constitutive (COX-1), which is involved in different
physiological processes, such as gastric cytoprotection,
and the other inducible (COX-2), which is expressed in
the presence of a wide variety of inflammatory media-
The target of an ideal NSAID is therefore that of
inhibiting the production of COX-2 during the inflam-
mation process, without modifying the physiological
levels of constitutive COX-1 [9,10].
In the last decade, new COX-2 selective inhibitors
have been described, in which the common molecular
characteristic consists of two aromatic moieties at-
tached to adjacent atoms in a bridging carbocyclic or
heterocyclic five-membered ring; one of the aromatic
groups is substituted in the para position by a methyl-
sulfonyl (1, 2 and 3) [11,12] or by an aminosulfonyl
Abbre6iations: NSAID, non-steroidal antiinflammatory drug; COX,
cyclooxygenase; MAOMM, methyleneaminoxymethylmoiety; PGE2,
prostaglandin E2.
* Correspondence and reprints
E-mail address: balsamo@farm.unipi.it (A. Balsamo).
´
0223-5234/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0223-5234(02)01385-5

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A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 585–594
Some of the new compounds have been shown to
possess an appreciable in vitro inhibitory activity, with
IC50 values in the mM range, and in the case of the
methylenedioxy derivative (5a), of 0.4, versus 0.02 mM
of celecoxib (4) [16]. Furthermore compound 5a
showed a modest antiinflammatory activity in the in
vivo test of carrageenan-induced paw edema in rats,
only after intraperitoneal administration [16]. Since the
sulfonamidic moiety, with respect to the methylsulfonic
one, is known to provide better bioavailability in other
COX-2 inhibitors structurally related to 4 [8,17], we
decided to synthesise compounds 6a,b, which are the
sulfonamidic analogues of 5a,b. In addition, also the
corresponding sulfides (7a,b) and sulfoxides (8a,b) were
prepared.
This paper also describes further chemical manipula-
tions concerning the oximethereal substituent of some
of these compounds, able to influence the conforma-
tional freedom of this side-chain. In compounds 9a and
9b, which represent superior homologues of 5a and 5b,
the conformational situation of the oximethereal system
should be modified, with respect to that of 5a and 5b,
by the increase in the steric hindrance of the substituent
linked to the oximic carbon, i.e. a methyl group instead
of a hydrogen atom. In compounds 10 and 11, which
can be viewed as isomeric analogues of 8b and 5b,
respectively, in which the atomic sequence CꢀNOCH2
of the MAOMM is transformed into the formally in-
verted one CH2ONꢀC, the oximethereal system should
possess a higher conformational freedom with respect
to 8b and 5b, as a result of the loss of conjugation
between the oximic double bond and the unsaturated
cyclopentenic portion.
Fig. 1. Structures of some representative COX-2 inhibitors containing
a 1,2-diaryl-substituted carbocyclic or heterocyclic five-membered
ring.
group (4) [13–15] (Fig. 1). At present, compounds 2
(rofecoxib) and 4 (celecoxib) are marketed drugs.
In the course of a programme aiming to discover new
molecules with a selective COX-2 inhibitory activity, we
have described the synthesis and the COX-1 and COX-
2 inhibitory activity of a series of compounds of type 5
(Fig. 2) which can be viewed as analogues of type 3
drugs, in which one of the two aryl groups is replaced
by an aryl-substituted methyleneaminoxymethyl moiety
(MAOMM) [16].
Fig. 2. Structures of oximethereal cyclopentene derivatives 5–11.

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 585–594
587
Fig. 3. Synthesis of compounds 6a,b and 8a,b.
hydroxymethylvinylbromide derivative 17 with endo-N-
hydroxy-5-norbornene-2,3-dicarboximide in the pre-
sence of triphenylphosphine and diethylazodicarboxylate
in THF yielded the imide-derivative 18, which by hy-
drazinolysis with H2NNH2·H2O in anhydrous EtOH,
afforded the O-alkyl-hydroxylamine 19. Treatment of
19 with benzaldehyde in a heterogeneous medium
(H2O/CHCl3) gave the (Z)-(20) and (E)-bromooxime
(21) in a ratio of about 20:80; the separation was
performed by column chromatography. The cross-cou-
pling reaction of the vinylbromide 21 with p-methyl-
thiophenylboronic acid in toluene–EtOH solution in the
presence of Pd(PPh3)4 and aqueous Na2CO3 afforded
the sulfide 22, which by oxidation with 1 or 2 equiv. of
oxone in a THF–MeOH mixture gave the sulfoxide
(10) or the sulfone (11) derivatives, respectively.
The E/Z-configuration of the oxime-ethers 14 and 15
(Fig. 4) was assigned bearing in mind that in the
compounds with the E configuration (15a,b) the pro-
tons of the methyl linked to the oximic carbons re-
sonate at field values lower (0.2 ppm) than in the
isomers with the Z configuration (14a,b). The E-
configuration of the intermediate sulfides 16a,b and
final products 9a,b, was made because during the syn-
thetic sequence the configuration around the oximic
double bond should not change. Also the E/Z-configu-
ration of the oxime-ethers 20 and 21 (Fig. 5) was
assigned on the basis of the chemical shift values of the
oximic proton signal, which resonates at lower fields
(0.5 ppm) in the E isomer (21) with respect to the Z one
(20). The E-configuration of the sulfide (22), sulfoxide
(10) and sulfone (11) was made on the basis of
the chemical shift values of their diagnostic oximic
2. Chemistry
The oximethereal sulfonamides 6a and 6b were pre-
pared by a one-step procedure (Fig. 3), treating the
corresponding methylsulfones 5a and 5b with lithium
diisopropylamide (LDA) and triethylborane, and then
with aminoxysulfonic acid in the presence of sodium
acetate.
The sulfoxides 8a and 8b were synthesised by oxida-
tion of the corresponding sulfides (7a and 7b) [16] with
0.6 equiv. amount of potassium peroxymonosulfate
(oxone) in a THF–MeOH–H2O mixture.
The oxime-ethers substituted with a methyl group on
the oximic carbon (9a and 9b) were obtained as out-
lined in Fig. 4, starting from the cyclopentanone 12
[18], which by treatment with acetyl chloride in the
presence of AlCl3 afforded 1-acetyl-2-chlorocyclopen-
tene (13). Reaction of 13 with the appropriate O-
benzylhydroxylamine
hydrochloride
yielded
the
corresponding oxime-ethers with the Z (14) and E (15)
configuration in a ratio of about 20:80 in the case of
14a and 15a, or 38:62 in the case of 14b and 15b; the
separation was performed by column chromatography.
The cross-coupling reaction [19] of the chloroderiva-
tives 15a or 15b with p-methylthio-phenylboronic acid
in anhydrous dioxane in the presence of P(c-C6H11)3,
tris-(dibenzylideneacetone)-dipalladium and Cs2CO3,
afforded the oxime-ether sulfides 16a or 16b, respec-
tively. Oxidation of 16a or 16b with 1.2 equiv. of oxone
gave the corresponding sulfones with the E configura-
tion, 9a or 9b.
The sulfoxide (10) and sulfone (11) oxime-ethers were
synthesised as indicated in Fig. 5. Reaction of the

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A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 585–594
hydrogens, which resulted very similar to that of the
intermediate oxime-ether with the E configuration (21).
tion values ranging from 12 to 20% at a concentration
of 10 mM. The sulfides 7a,b and the sulfoxide 8a
appeared to possess a higher activity, with an IC50
value in the micromolar range, and the sulfoxide 8b
proved to be the most active of the new compounds,
with an activity index of 0.1 mM.
3. Biopharmacological results
Among the superior homologues of 5a and 5b (9a
and 9b), only the unsubstituted (9b) proved to possess
an appreciable activity, with an IC50 value of 1 mM,
while the methylenedioxy-substituted one (9a) showed a
very modest activity.
For compound 8b, which showed the highest in-
hibitory activity towards COX-2 in the in vitro test,
also the in vivo inhibitory activity was evaluated by the
carrageenan-induced paw edema test in rats. In this
experiment, compound 8b was administered per os at
increasing doses ranging from 1 to 30 mg kg−1. No
inhibitory effects were detected in any case.
The in vitro inhibitory activity towards COX-1 and
COX-2 enzymes of compounds 6–11, celecoxib (4),
chosen as a reference compound, and previously stu-
died methylsulfonyl compounds 5a,b is reported in
Table 1.
In regard to the COX-1 enzyme, as previously found
for 5a and 5b, only the sulfide and the sulfoxide ana-
logues of 5a,b (7a,b and 8a,b, respectively) proved to be
slightly active, with percentage inhibition values rang-
ing from 20% of 7a to 29% of 5b at a concentration of
10 mM or IC50 values of 23 and 32 mM for 8a and 8b,
respectively. The other new compounds, i.e. the sul-
fonamidic analogues of 5a,b (6a,b), the superior homo-
logues of 5a,b (9a and 9b) in which the oximic carbon
is substituted by a methyl group, and compounds 10
and 11, in which the atomic sequence of the oximic
side-chain is inverted with respect to 8b and 5b, proved
to be practically inactive.
4. Discussion and conclusions
The primary aim of this work was that of verifying
the effects on the inhibitory activity towards COXs of
the substitution of the methylsulfonyl moiety of type 5
compounds with different sulphur-containing moieties,
such as the aminosulfonilic, methylsulfoxidic or methyl-
sulfidic ones. A second aim was that of evaluating the
In regard to the inhibitory activity towards the COX-
2 enzyme, while compound 11 was completely inactive,
both sulfonamidic derivatives 6a,b and compound 10
showed a very modest activity, with percentage inhibi-
Fig. 4. Synthesis of compounds 9a,b.

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 585–594
589
Fig. 5. Synthesis of compounds 10 and 11.
Overall, the in vitro tests of compounds 5–8 indicate
that among the sulfurated groups examined, only the
sulfonamidic one of 6a,b is completely unable to substi-
tute the methylsulfonyl group effectively in the interac-
tion with COX-2. A possible explanation of this
observation is that some peculiar chemical and physical
characteristics of 6a,b may hinder their approach to the
catalytic COX-2 site; an alternative hypothesis is that
substantial differences may exist between the structure–
activity relationship of oximether compounds, with re-
spect to that of tricyclic drugs.
As for compounds in which the oximether side-chain
has been modified (9–11), the results obtained with
9a,b indicate that the substitution of the oximic hydro-
gen of 5a,b with a methyl group leads to a complete
loss of the activity towards COX-1; towards COX-2,
the same modification, while has a significantly negative
effect in the case of the methylenedioxy derivative 9a,
which is almost inactive, in the case of the unsubsti-
tuted compound 9b approximately doubles the activity.
For compounds 10 and 11, the data in Table 1 show
that the formal inversion of the atomic sequence of the
MAOMM of 8b and 5b has a marked negative effect
towards both types of COX, leading to the complete
loss of activity.
effects on this type of activity of some modifications of
the oxime-ether side-chain of compounds 5, which pre-
sumably influence their conformational situation.
The data in Table 1 indicate that the substitution of
the methylsulfonyl group of 5a and 5b with the sulfona-
midic one of 6a and 6b leads to compounds devoid of
any in vitro inhibitory activity, discouraging their fur-
ther in vivo studies. The substitution of the methylsul-
fonyl group of 5a and 5b with the methylsulfidic one as
in 7a and 7b, does not have any appreciable effect on
the activity towards COX-1, whereas it determines a
decrease in their COX-2 activity of about 1.2 or 0.4
order of magnitude, respectively.
The substitution of the methylsulfonyl group of 5a
and 5b with the methylsulfoxidic one of 8a and 8b leads
to a slight increase in the inhibitory activity towards
COX-1; on the contrary, the same type of substitution
has an opposite effect towards COX-2, depending on
the type of substituent on the aromatic ring of the
oximic side-chain: it reduces the activity by about one
order of magnitude in the case of the methylenedioxy
compound 8a, while it increases the same type of
activity by more than one order of magnitude in the
case of the unsubstituted compound 8b.

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A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 585–594
In order to attempt a rationalisation of these results,
compounds (b, c, and d), together with those obtained
for SC57666 (a). A comparison of the conformational
profile of 5b (b in Fig. 6) with that of 9b (c in Fig. 6)
shows that their common portion (cyclopentene and
a conformational study was carried out on compounds
5b, 9b, and 11. Fig. 6 shows the superimposition of the
most favourable conformations found for each of these
Table 1
Biological data of compounds 5a,b–9a,b and 10,11
Compound
R
R1
R2
In vitro inhibitory activity a
COX-1
COX-2
5a b
5b b
3,4-OCH2O-
H
H
H
SO2Me
SO2Me
27% c
29% c
0.4
1.9
6a
6b
3,4-OCH2O-
H
H
H
SO2NH2
SO2NH2
0
0
12% c
19% c
7a
7b
3,4-OCH2O-
H
H
H
SMe
SMe
20% c
22% c
8.0
4.4
8a
8b
3,4-OCH2O-
H
H
H
SOMe
SOMe
23
32
5.0
0.1
9a
9b
3,4-OCH2O-
H
Me
Me
SO2Me
SO2Me
0
0
20% c
1
10
11
–
–
–
–
SOMe
SO2Me
0
0
20% c
0
4 (Celecoxib)
1.04
0.02
a Data are indicated as IC50 (mM) or as the percentage of inhibition at a concentration of 10 mM.
b See Ref. [16].
c IC50\1 mM.
Fig. 6. Compounds SC57666 (a); 5b (b); 9b (c) and 11 (d). Conformations found through GMMX conformational searching techniques reported
by superimposing the cyclopentene rings.

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 585–594
591
5.1.1. Preparation of the (E)-2-(4-aminosulfonyl-
methyl)-1-(arylmethyloxyiminomethyl)cyclopentene
(6a,b)
aryl substituent) present analogous conformational
preferences; the oximether side-chain, on the other
hand, while in 5b always lies on the side opposite to the
adjacent aryl, in compound 9b may also be found on
the same side as this aryl. The improvement in the
activity of 9b compared with 5b might be attributed to
these conformational differences, assuming that certain
low-energy conformations which are preferred only for
9b make a greater contribution to the activity thanks to
a better ability to interact with the enzyme. The loss of
activity on passing from 9b to its methylenedioxy ana-
logue 9a may be tentatively attributed to this subs-
tituent, which in a structure possessing an additional
methyl group, with respect to that of type 5, may be of
excessive hindrance for the access to the COX-2
enzyme.
A comparison of the conformational profile of 5b (b
in Fig. 6) and 11 (d in Fig. 6) reveals a marked
difference in the oximic side-chain portion: unlike 5b, in
which this chain substantially assumes a single spatial
orientation, in the inactive compound 11 (d in Fig. 6),
it assumes various arrangements, which are all different
from the preferential one of 5b, which might potentially
be the pharmacophoric one.
A cooled (0 °C) and stirred solution of the appropri-
ate methylsulfone 5a,b [16] (1.70 mmol) in anhydrous
THF (0.9 mL), was treated with LDA 2.0 M in hexane
(1.1 mL, 2.2 mmol), and the resulting mixture was
stirred at room temperature (r.t.) for 1 h. The mixture
was cooled at 0 °C, and treated with a 1.0 M solution
of triethylborane in THF (2.8 mL, 2.8 mmol). At the
end of the addition, the reaction was stirred at r.t. for
1 h and then refluxed for 20 h. The mixture was cooled
at 0 °C, treated with AcONa (0.93 g, 1.2 mmol), water
(4 mL) and hydroxylamine-O-sulfonic acid (0.80 g, 7.1
mmol) and then stirred at r.t. for 24 h. The organic
phase was diluted with AcOEt, washed with water and
brine, dried and then evaporated to afford a crude
residue, which was purified by column chromatography
using as the eluent hexane–AcOEt (6:4). 6a (10%):
1H-NMR: l 1.95–2.07 (m, 2H, CH2CH2CH2), 2.77–
2.89 (m, 4H, CH2CH2CH2), 4.82 (s, 2H, NH2), 5.00 (s,
2H, ArCH2O), 5.94 (s, 2H, OCH2O), 6.75–6.87 (m, 3H,
Ar), 7.40 and 7.86 (AA1BB1 system, 4H, J=8.4 Hz,
ArSO2NH2), 8.01 ppm (s, 1H, CHꢀN). Anal.
1
C20H20N2O5S (C, H, N). 6b (5%): H-NMR: l 1.92–
In regard to the lack of in vivo activity of compound
8b in the carrageenan-paw edema test in rats after oral
administration, this should be attributed to its limited
gastrointestinal stability, poor absorption and/or rapid
first pass metabolism.
2.10 (m, 2H, CH2CH2CH2), 2.75–2.92 (m, 4H,
CH2CH2CH2), 4.82 (s, 2H, NH2), 5.10 (s, 2H,
ArCH2O), 7.22–7.38 (m, 5H, ArCH2O), 7.33 and 7.85
(AA1BB1 system, 4H, J=8.8 Hz, ArSO2NH2), 8.00
ppm (s, 1H, CHꢀN). Anal. C19H20N2O3S (C, H, N).
5.1.2. Preparation of the (E)-2-(4-methylsulfinyl-
phenyl)-1-(arylmethyloxyiminomethyl)cyclopentene
(8a,b)
5. Experimental
5.1. Chemistry
A cooled (0 °C) and stirred solution of the appropri-
ate methylsulfide 7a,b [16] (1.47 mmol) in a 1:1
MeOH–THF mixture (8 mL) was treated dropwise
with a solution of oxone (0.86 mmol) in water (4 mL).
At the end of the addition, the reaction was stirred at
r.t. for 12 h. The solvent was evaporated and the crude
residue was taken up in AcOEt, washed with water and
brine, dried and evaporated to afford the crude sulfo-
xide 8a or 8b, which was purified by crystallisation
from (i-Pr)2O. 8a (38%): 1H-NMR: l 1.96–2.04 (m,
2H, CH2CH2CH2), 2.70 (s, 3H, SOCH3), 2.82–2.93 (m,
4H, CH2CH2CH2), 4.98 (s, 2H, ArCH2O), 5.92 (s, 2H,
OCH2O), 6.75–6.85 (m, 3H, Ar), 7.33 and 7.58
(AA1BB1 system, 4H, J=8.4 Hz, ArSOCH3), 8.00 ppm
(s, 1H, CHꢀN). Anal. C21H21NO4S (C, H, N). 8b (76%):
m.p. 128–130 °C; 1H-NMR: l 1.96–2.21 (m, 2H,
CH2CH2CH2), 2.70 (s, 3H, SOCH3), 2.80–2.93 (m, 4H,
CH2CH2CH2), 5.09 (s, 2H, ArCH2O), 7.27 (s, 5H, Ar),
7.31 and 7.58 (AA1BB1 system, 4H, J=8.0 Hz,
ArSOCH3), 8.02 ppm (s, 1H, CHꢀN); MS m/e: 339
(M+). Anal. C20H21NO2S (C, H, N).
Melting points were determined on a Kofler hot-
stage apparatus and are uncorrected. IR spectra for
comparison of compounds were taken as paraffin oil
mulls or as liquid films on a Unicam Mattson 1000
FTIR spectrometer. 1H-NMR spectra of all compounds
were obtained with a Gemini 200 spectrometer opera-
ting 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 chro-
matography was performed using 70–230 mesh silica
gel. Mass spectra were detected with a Hewlett Packard
5988A spectrometer (EI, 70 eV). Evaporation was made
in vacuo (rotating evaporator); the not commercially
available O-arylmethyloxyamines used for the prepara-
tion of the chlorooximethers 14 and 15, were synthe-
sised as described in Ref. [20]. Na2SO4 was always used
as the drying agent. Elemental analyses were performed
in our analytical laboratory and agreed with the theo-
retical values to within 90.4%.

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A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 585–594
5.1.3. 2-Chloro-1-acetylcyclopentene (13)
with Et2O, filtered on celite and then evaporated to
afford a crude residue, which was subjected to column
chromatography to yield pure 16a or 16b. 16a (40%):
1H-NMR: l 1.73 (s, 3H, CH3CꢀN), 1.87–1.98 (m, 2H,
CH2CH2CH2), 2.48 (s, 3H, SCH3), 2.75–2.82 (m, 4H,
CH2CH2CH2), 5.01 (s, 2H, ArCH2O), 5.96 (s, 2H,
OCH2O), 6.75–6.87 (m, 3H, ArCH2O), 7.15 ppm (s,
4H, ArSCH3). MS m/e: 381 (M+). Anal. C22H23NO3S
(C, H, N). 16b (28%): 1H-NMR: l 1.75 (s, 3H,
CH3CꢀN), 1.87–2.01 (m, 2H, CH2CH2CH2), 2.48 (s,
3H, SCH3), 2.74–2.81 (m, 4H, CH2CH2CH2), 5.13 (s,
2H, ArCH2O), 7.15–7.34 ppm (m, 9H, 2Ar). Anal.
C21H23NOS (C, H, N).
A cooled (0 °C) suspension of AlCl3 (40.0 g, 0.30
mol) in CH2Cl2 (75 mL) was treated dropwise with
acetylchloride (23.5 g, 0.30 mol) and then with cy-
clopentanone (12.7 g, 0.15 mol) [18]. After 2 h at r.t.,
the mixture was treated with ice and solid NaHCO3.
The aq. phase was extracted with CHCl3 and the or-
ganic layer was washed with a saturated K2CO3 solu-
tion, filtered and evaporated to yield an oily residue
which was purified by column chromatography using a
9:1 hexane–AcOEt mixture as the eluent. 13 (15%):
1H-NMR: l 1.87–1.95 (m, 2H, CH2CH2CH2), 2.48 (s,
3H, CH3CO), 2.63–2.85 ppm (br, 4H, CH2CH2CH2);
MS m/e: 144 (M+). Anal. C7H9ClO (C, H).
5.1.6. Preparation of the (E)-2-(4-methylsulfonyl-
phenyl)-1-[1-(arylmethyloxyimino)ethyl]cyclopentene
(9a,b)
5.1.4. Preparation of the (E)- and (Z)-2-chloro-1-[1-
(arylmethyloxyimino)ethyl]cyclopentenes (14a,b and
15a,b)
A solution of oxone (0.35 mmol) in water (1 mL) was
added to a cooled (0 °C) solution of the appropriate
methylsulfide 16a or 16b (0.29 mmol) in 1:1 MeOH–
THF (2 mL). The resulting mixture was stirred at r.t.
for 12 h. The solvent was then evaporated and the
residue was dissolved in AcOEt. The organic phase was
washed with water and brine, dried and evaporated to
yield a crude residue, which was purified by crystallisa-
tion from EtOH. 9a (58%): m.p. 134–135 °C; 1H-
NMR: l 1.74 (s, 3H, CH3CꢀN), 1.95–2.04 (m, 2H,
CH2CH2CH2), 2.78–2.85 (m, 4H, CH2CH2CH2), 3.05
(s, 3H, SO2CH3), 4.99 (s, 2H, ArCH2O), 5.98 (s, 2H,
OCH2O), 6.78–6.84 (m, 3H, ArCH2O), 7.44 and 7.79
ppm (AA1BB1 system, 4H, J=8.4 Hz, ArSO2). Anal.
A mixture of 13 (1.0 g, 6.9 mmol) in CHCl3 (45 mL)
and the appropriate O-arylmethyloxyamine (13.8
mmol) in water (10 mL), was stirred at r.t. for 36 h. The
organic phase was separated and the aq. solution was
extracted twice with CHCl3. Evaporation of the dried
and filtered organic extracts afforded a residue consist-
ing almost exclusively of the stereoisomeric mixture of
the chlorooximes in a ratio of 80:20 for (E)-14a and
(Z)-15a, and 60:40 for (E)-14b and (Z)-15b, which were
separated by column chromatography. (E)-14a (38%):
1H-NMR: l 1.80–1.92 (m, 2H, CH2CH2CH2), 2.20 (s,
3H, CH3CꢀN), 2.62–2.71 (m, 4H, CH2CH2CH2), 5.02
(s, 2H, ArCH2O), 5.95 (s, 2H, OCH2O), 6.75–6.88 ppm
(m, 3H, Ar). Anal. C15H16ClNO3 (C, H, N). (Z)-15a
(9%): 1H-NMR: l 1.80–1.92 (m, 2H, CH2CH2CH2),
2.02 (s, 3H, CH3CꢀN), 2.62–2.71 (m, 4H,
CH2CH2CH2), 4.95 (s, 2H, ArCH2O), 5.95 (s, 2H,
OCH2O), 6.75–6.88 ppm (m, 3H, Ar). Anal.
1
C22H23NO5S (C, H, N). 9b (82%): H-NMR: l 1.76 (s,
3H, CH3CꢀN), 1.95–2.04 (m, 2H, CH2CH2CH2), 2.78–
2.85 (m, 4H, CH2CH2CH2), 3.05 (s, 3H, SO2CH3), 5.11
(s, 2H, ArCH2O), 7.33 (s, 5H, ArCH2O), 7.44 and 7.82
ppm (AA1BB1 system, 4H, J=8.3 Hz, ArSO2). MS
m/e: 368 (M+). Anal. C21H23NO3S (C, H, N).
1
C15H16ClNO3 (C, H, N). (E)-14b (30%): H-NMR: l
1.80–1.93 (m, 2H, CH2CH2CH2), 2.22 (s, 3H,
CH3CꢀN), 2.62–2.70 (m, 4H, CH2CH2CH2), 5.13 (s,
2H, ArCH2O), 7.31–7.37 ppm (m, 5H, Ar). Anal.
C14H16ClNO (C, H, N). (Z)-15b: 1H-NMR: l 1.80–
1.92 (m, 2H, CH2CH2CH2), 2.03 (s, 3H, CH3CꢀN),
2.62–2.70 (m, 4H, CH2CH2CH2), 5.08 (s, 2H,
ArCH2O), 7.31–7.37 ppm (m, 5H, Ar). Anal.
C14H16ClNO (C, H, N).
5.1.7. 2-Bromo-1-(hydroxymethyl)cyclopentene (17)
A solution of 2-bromo-1-cyclopentene-carbaldehyde
(4.95 mmol) in EtOH (20 mL) was treated with NaBH4
(11.3 mmol). The resulting mixture was stirred at r.t.
for 24 h. The solvent was then evaporated and the
residue was dissolved in CH2Cl2, washed with water,
10% aq. HCl, and brine, and then filtered and evapo-
rated to give an oily residue consisting of practically
pure 17 (54%): IR: w 3320–3340 cm−1 (OH). 1H-NMR:
l 1.92–2.06 (m, 2H, CH2CH2CH2), 2.38–2.61 (m, 4H,
CH2CH2CH2) and 4.23 ppm (s, 2H, CH2OH). MS m/e:
176 (M+).
5.1.5. Preparation of the (E)-2-(4-methylthiophenyl)-
1-[1-(arylmethyloxyimino)ethyl]cyclopentene (16a,b)
A mixture of Pd2(dba)3 (0.3 mmol), 4-methylthio-
phenylboronic acid (0.30 g, 1.8 mmol) and Cs2CO3
(0.67 g, 2.0 mmol) was treated with the appropriate
1,2-cyclopentenylidene derivative of E configuration
14a or 14b (1.7 mmol) in dioxane (1.4 mL) and then
with a solution of P(Cy)3 10% in C6H5CH3 (100 mL,
0.06 mmol) [19]. The resulting mixture was stirred
under N2 at 80 °C for 12 h. The mixture was diluted
5.1.8. endo-N-(2-Bromo-1-cyclopentenylmethyloxy)-5-
norbornene-2,3-dicarboximide (18)
A solution of 2-bromo-1-(hydroxymethyl)cyclopen-
tene (17) (1.0 g, 5.6 mmol) in anhydrous THF (23 mL)
was treated with endo-N-hydroxy-5-norbornene-2,3-di-

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 585–594
593
carboximide (1.0 g, 5.6 mmol), triphenylphosphine
(1.47 g, 5.6 mmol) and diethyl azodicarboxylate (1.11 g,
6.17 mmol) and the resulting mixture was stirred at r.t.
for 12 h. The solvent was then evaporated and the
crude residue was dissolved in Et2O. The insoluble salts
were filtered and the organic phase was evaporated to
yield a solid residue which was subjected to chromatog-
raphy eluting with 6:3:1 hexane–AcOEt–MeOH mix-
yield a crude residue which was subjected to column
chromatography eluting with a 9.5:0.5 hexane–AcOEt
1
mixture to give pure 22 as a solid. 22 (49%): H-NMR:
l 1.85–2.03 (m, 2H, CH2CH2CH2), 2.47 (s, 3H, SCH3),
2.65–2.82 (m, 4H, CH2CH2CH2), 4.80 (s, 2H, CH2O),
7.23–7.58 (m, 9H, 2Ar) and 8.06 ppm (s, 1H, CHꢀN).
5.1.12. (E)-1-(Benzylideneiminoxymethyl)-2-(4-
methylsulfinylphenyl)cyclopentene (10) and
1
ture to give pure 18 (84%): H-NMR: l 1.35–2.20 (br
m, 4H, CH2CH2CH2 and CH2 norbornene), 2.53–2.71
(m, 4H, CH2CH2CH2), 3.12–3.38 (m, 4H, H1ꢁH4 nor-
bornene), 4.56 (s, 2H, CH2O), 6.13 (s, 2H, H5, H6
norbornene). Anal. C15H16BrNO3 (C, H, N).
(E)-1-(benzylideneiminoxymethyl)-2-(4-methyl-
sulfonylphenyl)cyclopentene (11)
A cooled (0 °C) and stirred solution of 22 (0.22 g,
0.69 mmol) in a 1:1 THF–MeOH mixture (8 mL) was
treated dropwise with a solution of oxone (0.71 mmol)
in water (3 mL). At the end of the addition, the
reaction mixture was stirred at r.t. for 12 h and then
evaporated. The residue was taken up in AcOEt,
washed with brine, dried and evaporated to give the
crude product consisting of sulfoxide 10 and sulfone 11
which were separated by column chromatography using
AcOEt as the eluent. 10 (36%): m.p. 126–127 °C (i-
5.1.9. O-(2-Bromo-1-cyclopentenylmethyl)hydroxyl-
amine hydrochloride (19·HCl)
A solution of hydrazine monohydrate (0.22 g, 4.4
mmol) was added dropwise to a suspension of 18 (1.5 g,
4.4 mmol) in anhydrous EtOH (10 mL) and the mixture
was refluxed for 8 h and then evaporated. The crude
residue was taken up in Et2O and the organic layer was
filtered, cooled (0 °C) and treated with Et2O·HCl. The
solid precipitate was filtered and crystallised from
1
PrOH); H-NMR: l 1.85–2.10 (m, 2H, CH2CH2CH2),
2.72–2.85 (br m, 7H, SOCH3 and CH2CH2CH2), 4.79
(s, 2H, CH2O), 7.22–7.55 (m, 9H, 2Ar) and 8.06 ppm
(s, 1H, CHꢀN). Anal. C20H21NO2S (C, H, N). 11 (40%):
1
MeOH. 19·HCl (62%): H-NMR: l 1.92–2.06 (m, 2H,
CH2CH2CH2), 2.48–2.59 (m, 4H, CH2CH2CH2), 4.27
(s, 2H, CH2O), 5.35 ppm (s, 2H, NH2).
1
m.p. 135–137 °C (EtOH); H-NMR: l 1.85–2.16 (m,
2H, CH2CH2CH2), 2.72–2.86 (br m, 4H, CH2CH2-
CH2), 3.05 (s, 3H, SO2CH3), 4.78 (s, 2H, CH2O),
7.33–7.84 (m, 9H, 2Ar) and 8.06 ppm (s, 1H, CHꢀN).
Anal. C20H21NO3S (C, H, N).
5.1.10. (Z)-(20) and (E)-1-(Benzylideneiminoxymethyl)-
2-bromocyclopentene (21)
A solution of 19·HCl (0.63 g, 2.8 mmol) in water (5
mL) was added to a stirred solution of benzaldehyde
(0.30 g, 2.8 mmol) in CHCl3 (15 mL). The mixture was
vigorously stirred at r.t. for 24 h. The organic phase
was then separated, the aq. solution was extracted twice
with CHCl3 and the collected organic layers were then
filtered and evaporated to dryness. The crude residue
consisting of 20 and 21 in a ratio of about 20:80
was submitted to column chromatography using 6:4
hexane–CHCl3 as the eluent to yield the pure configu-
rational isomers 20 and 21. 20 (2%). 1H-NMR: l
1.74–2.13 (m, 2H, CH2CH2CH2), 2.38–2.63 (m, 4H,
CH2CH2CH2), 4.77 (s, 2H, CH2O), 7.35–7.89 ppm (m,
5.2. Conformational studies
A conformational analysis on compounds SC57666,
5b, 9b and 11 was performed through the Global-MMX
method (GMMX) which is part of the PCMODEL com-
puter program [21]. GMMX uses a conformational
searching technique, which randomly moves torsion
angles selected by the user and cartesian coordinates of
all heavy atoms and then performs an energy minimisa-
tion. In our case the bonds selected to be moved were
those which connect the phenyl rings and the cyclopen-
tene ring to the rest of the molecular structure, the
forcefield was MMFF94, an energy window of 1 kcal
mol−1 was used and a total of 5000 starting conforma-
tions were taken into account. The conformational
searching found 8 conformations for SC57666 16 for
5b, 17 for 9b and 27 for 11. In Fig. 6 all these
conformations were reported.
1
6H, Ar and CHꢀN). 21 (52%): H-NMR: l 1.91–2.10
(m, 2H, CH2CH2CH2), 2.39–2.67 (m, 4H, CH2CH2-
CH2), 4.77 (s, 2H, CH2O), 7.22–7.59 (m, 5H, Ar), 8.04
ppm (s, 1H, CHꢀN). Anal. C13H14BrNO (C, H, N).
5.1.11. (E)-1-(Benzylideneiminoxymethyl)-2-(4-
methylthiophenyl)cyclopentene (22)
A solution of 21 (0.40 g, 1.4 mmol) and methylthio-
phenylboronic acid (0.24 g, 1.4 mmol) in 1:1
C6H5CH3–EtOH mixture (30 mL), was treated with 2.0
M aq. Na2CO3 (3 mL) and Pd(PPh3)4 (40 mg). The
stirred mixture was refluxed under N2 for 12 h and then
evaporated. The crude residue was dissolved in AcOEt,
washed with water and brine, dried and evaporated to
5.3. Biopharmacological methods
5.3.1. Enzyme assays
All compounds 6–11 were tested following the proce-
dure previously described [16] in intact cell assays to
verify their capacity to inhibit prostaglandin E2 (PGE2)

594
A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 585–594
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production, considered as an index of activity on COX-
1 and COX-2 enzymes. For the COX-1 assay, 1.5×106
resting U937 human cells were incubated with the test
compounds for 30 min 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
COX-2 assay was performed in accordance with the
method described by Mitchell et al. [22] with minor
modifications as suggested by Grossman et al. [23].
Murine J774.2 cells were pretreated for 1 h with 300
mM 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 mea-
sured as described above. All compounds were tested in
duplicate. For each product a stock solution was pre-
pared in DMSO at a concentration of 100 mM. Linear
regression was used to calculate IC50 values. SEM were
lower than 10% in all experiments.
5.3.2. Carrageenan-induced paw edema
Compound 8b was tested after oral administration
following a modified version of the method of Winter et
al. [24] previously reported [16]. Paw edema was in-
duced by injecting 0.05 ml of a 1% carrageenan suspen-
sion, prepared in sterile saline, into the subcutaneous
plantar tissue of the right hind paw. Paw volume was
measured using a plethysmometer (Ugo Basile, Italy)
before (basal value) and 3 h after carrageenan injection.
Molecules were suspended in a 0.5% methylcellulose aq.
solution and orally or intraperitoneally administered
(10 ml kg−1 body weight) 30 min before irritant injec-
tion. Indomethacin, used as the standard, was adminis-
tered at 3 mg kg−1 po. Control rats received only the
vehicle. Statistical analysis was performed by the split
plot test followed by Tukey’s test for individual
comparisons.
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