(E)-[2-(4-Methylsulphonylphenyl)-1-cyclopentenyl-1-methyliden] (arylmethyloxy)amines. Methyleneaminoxymethyl (MAOM) analogues of diarylcyclopentenyl cyclooxygenase-2 inhibitors: Synthesis and biological properties

Article abstract of DOI:10.1016/S0223-5234(02)01359-4

The (E)-[2-(4-Methylsulphonylphenyl)-1-cyclopentenyl-1-methyliden] (methyloxy)amine (5) and (arylmethyloxy)amines (6-12) were designed in order to verify the effects on the biological properties of the substitution of an aryl of selective diarylcyclopentenyl cyclooxygenase-2 (COX-2) inhibitors of type 3 with a methyleneaminoxymethyl moiety (MAOMM). Compounds 5-12 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. The compound with the highest in vitro activity towards COX-2 (9) was also assayed in vivo for its antiinflammatory activity by means of the carrageenan-induced paw edema test in rats. Some of the new compounds showed an appreciable in vitro COX-2 inhibitory activity, with IC₅₀ values in the μM (6,7,9,10,11) range. Compound 9 also exhibited an appreciable in vivo activity (29% inhibition at a dose of 30 mg kg^-1) when administered intraperitoneally. The structural parameters of 9 were determined by X-ray crystallographic analysis.

Full text of DOI:10.1016/S0223-5234(02)01359-4

Eur. J. Med. Chem. 37 (2002) 391–398
www.elsevier.com/locate/ejmech
Original article
(E)-[2-(4-Methylsulphonylphenyl)-1-cyclopentenyl-1-methyliden]-
(arylmethyloxy)amines. Methyleneaminoxymethyl (MAOM)
analogues of diarylcyclopentenyl cyclooxygenase-2 inhibitors:
synthesis and biological properties
Aldo Balsamo a,*, Isabella Coletta c, Paolo Domiano b, Angelo Guglielmotti c,
Carla Landolfi c, Francesca Mancini c, Claudio Milanese c, Elisabetta Orlandini a,
Simona Rapposelli a, Mario Pinza c, Bruno Macchia a
a Dipartimento di Scienze Farmaceutiche, Uni6ersita` di Pisa, 6ia Bonanno 6, 56126 Pisa, Italy
b Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Uni6ersita` di Parma, 43100 Parma, Italy
c ACRAF-Angelini Ricerche, 00040 S. Palomba, Pomezia, Rome, Italy
Received 30 July 2001; received in revised form 7 December 2001; accepted 22 February 2002
Abstract
The (E)-[2-(4-Methylsulphonylphenyl)-1-cyclopentenyl-1-methyliden](methyloxy)amine (5) and (arylmethyloxy)amines (6–12)
were designed in order to verify the effects on the biological properties of the substitution of an aryl of selective diarylcyclopen-
tenyl cyclooxygenase-2 (COX-2) inhibitors of type 3 with a methyleneaminoxymethyl moiety (MAOMM). Compounds 5–12 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. The compound with the highest in vitro activity towards COX-2 (9) was
also assayed in vivo for its antiinflammatory activity by means of the carrageenan-induced paw edema test in rats. Some of the
new compounds showed an appreciable in vitro COX-2 inhibitory activity, with IC50 values in the mM (6,7,9,10,11) range.
Compound 9 also exhibited an appreciable in vivo activity (29% inhibition at a dose of 30 mg kg−1) when administered
´
intraperitoneally. The structural parameters of 9 were determined by X-ray crystallographic analysis. © 2002 Editions scien-
tifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Antiinflammatory drug; COX-2 inhibitor; Methyleneaminoxymethyl moiety; (E)-[2-(4-methylsulphonylphenyl)-1-cyclopentenyl-1-meth-
ylidene](arylmethyloxy)amines
1. Introduction
COX-1, and an inducible form COX-2 [3,4]. The COX-
1 isoform is constitutively expressed in most tissues and
is involved in the physiological production of
prostaglandins, which are responsible for gastric cyto-
protection. In contrast, the COX-2 isoform is induced
by inflammation mediators such as cytokines, mitogens
and endotoxins [5–7]. The most common side-effects
shown by the classic NSAIDs consist of ulceration,
perforation and haemorrhage of the gastrointestinal
tract, usually attributed to COX-1 inhibition [2,8]. On
the basis of this knowledge, new inhibitors possessing
an appreciable selectivity for COX-2 have been de-
signed and synthesised, with the aim of obtaining drugs
with reduced gastrolesive properties [9–11]. Among
A great many of the non-steroideal antiinflammatory
drugs (NSAIDs) most commonly used in the therapy of
inflammatory conditions belong to the chemical class of
arylacetic and arylpropionic acids. These compounds,
as well as other types of NSAIDs, act through the
inhibition of the cyclooxygenase (COX) enzyme [1,2].
This enzyme exists as two isoforms, a constitutive form
Abbre6iations: COX, cyclooxygenase; MAOMM, methyle-
neaminoxymethylmoiety; NSAID, non-steroidal antiinflammatory
drug; 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)01359-4

392
A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 391–398
For some time our research group has been inte-
rested in studying the effect of substituting selected
aromatic rings with alternative non-aromatic moieties
[18–24]. In particular, we have been interested in
whether the biological activity can be preserved by
isosteric replacement of a key aromatic ring with a
non-aromatic group. In the present study, we sought to
determine if the para-fluorophenyl ring of SC-57666 (3)
could be replaced with a non-aromatic ring with
maintenance of in vitro and in vivo activity. Specially,
our plan called for the preparation of compounds 5–
12, and to determine the biological consequences of
incorporation of the methyleneaminoxymethyl moiety
(MAOMM). Previously, we showed that this aromatic
replacement strategy could be successfully applied to
other classes of drugs [18,21–23].
Using SC-57666 (3) as a template known to possess
selective inhibitory activity against COX-2, our studies
commenced with replacement of the para-fluorophenyl
ring with the MAOMM (5). The MAOMM was then
further substituted on the methyl carbon with an un-
substituted (6) or substituted aromatic moiety (7–12)
(Table 1). Since the active site of COX-2 is known to
possess a greater volume than COX-1 [25], the addi-
tional molecular size of the aryl substituted MAOMM
derivatives 6–12 relative to 3 should contribute to
decreased affinity towards COX-1 and thus greater
selectivity for COX-2.
Fig. 1. Structures of some COX-2 selective antiinflammatory drugs
characterised by a diaryl-substituted carbocyclic or heterocyclic 5-
membered ring: Dup697 (1), rofecoxib (2), SC-57666 (3) and cele-
coxib (4).
these, the most widely studied class is that of the
compounds (Fig. 1) in which the pharmacophore is
characterised by two aromatic moieties attached to
adjacent atoms in a bridging carbocyclic or heterocyclic
5-membered ring [12–17]. For optimal COX-2 in-
hibitory activity one of the two aromatic rings is substi-
tuted in the para-position by a methylsulphonyl (1–3)
[12,14] or by a sulfonamide group (4) [13,15].
Table 1
Chemical and biological data of compounds 5–12
Compound
R
m.p. (°C)
Formula a
In vitro inhibitory activity b
COX1
COX2
5
6
7
8
9
10
11
12
H
C6H5
135–137
98–100
C
14H17NO3S
0
23%
1.9
5.8
46%
0.41
3.1
1.8
0
0.02
C20H21NO3S
C21H23NO3S
C21H23NO4S
C21H21NO5S
C20H20FNO3S
29%
33%
45%
27%
32%
43%
33%
1.04
4-Me-C6H4
4-MeO-C6H4
3,4-OCH2O-C6H3
4-F-C6H4
4-Cl-C6H4
2-Cl,6-F-C6H3
146–148
114–116
130–132
136–138
175–177
128–130
C
20H20ClNO3S
C20H19ClFNO3S
4 (celecoxib)
a All compounds were analysed for C, H and N.
b Data are indicated as IC50 (mM) or as percentage of inhibition at a concentration of 10 mM.

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 391–398
393
a THF–MeOH–H2O mixture, afforded the final pro-
ducts with the E configuration 5–12.
The structure and configuration of the intermediate
compounds (15–30) and final products (5–12) were
1
assigned on the basis of a comparison of the H-NMR
spectral data of all compounds, and by the crystallo-
graphic analysis of one of the final products (9). The
X-ray structure of 9 is shown in Fig. 3, together with
the crystallographic atom numbering scheme. Atomic
coordinates are shown in Table 2. In compound 9 the
configuration around the oximic double bond is E. The
atoms of the cyclopentenic ring (C10ꢀC11ꢀC12ꢀC13ꢀ
C14) are practically coplanar, and the corresponding
plane forms a dihedral angle of about 23° with the
plane of the aromatic C15ꢀC16ꢀC17ꢀC18ꢀC19ꢀC20
atoms. Also the MAOM portion atoms (C8ꢀO3ꢀ
N1ꢀC9) are practically coplanar, and define a plane
which forms an angle of 15° with the cyclopentenic one.
The configuration of the oxime 9 was determined to
be (E) by X-ray crystallography. Based on the configu-
ration of 9 it was possible to assign the same (E)
configuration to compounds 5–8 and 10–12. The (E)
configuration assignment was made on the basis of the
similar values of the chemical shift of the signal of the
hydrogen atom linked to the oximic unsaturated car-
bon. During the synthetic sequence the configuration of
Fig. 2. Synthesis of methyl-(5) and arylmethyl-oximethers (6–12).
Table 2
Atomic coordinates of compound 9
Atom
X/a
Y/b
Z/c
S1
O1
O2
O3
O4
O5
N1
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
H9
0.69040 (4)
0.56575 (18)
0.21124 (56)
0.28154 (56)
−0.19910 (48)
0.72804 (46)
0.69545 (47)
−0.30054 (57)
0.35752 (83)
0.09534 (73)
−0.03822 (70)
−0.22571 (70)
−0.26931 (78)
−0.12906 (76)
0.05044 (78)
−0.37472 (73)
−0.16736 (80)
−0.22731 (64)
−0.41545 (72)
−0.43211 (70)
−0.22187 (67)
−0.12965 (59)
0.04662 (62)
0.18861 (70)
0.35113 (69)
0.37413 (58)
0.23411 (76)
0.06851 (77)
0.32619 (68)
−0.02388 (617)
−0.07185 (4)
0.41839 (12)
0.45399 (11)
0.26663 (10)
−0.08816 (10)
−0.06096 (10)
0.25231 (12)
0.46140 (17)
0.40773 (15)
0.38409 (14)
0.33767 (14)
0.31777 (16)
0.34189 (16)
0.38710 (16)
0.31107 (14)
0.20881 (16)
0.18949 (14)
0.22857 (14)
0.19507 (13)
0.14413 (14)
0.14201 (13)
0.09136 (12)
0.05756 (14)
0.00869 (14)
−0.00709 (13)
0.02459 (15)
0.07316 (15)
−0.12965 (14)
0.19318 (129)
1.12537 (13)
1.00480 (13)
0.84128 (12)
0.62426 (11)
0.76328 (11)
0.76798 (14)
1.08334 (20)
1.00109 (18)
0.93836 (18)
0.94883 (17)
1.02075 (18)
1.08430 (19)
1.07222 (18)
0.88042 (18)
0.73465 (20)
0.65614 (16)
0.61149 (17)
0.53239 (17)
0.53141 (17)
0.61331 (16)
0.63512 (15)
0.57877 (18)
0.59651 (17)
0.67023 (16)
0.72709 (18)
0.70963 (18)
0.69972 (19)
0.76196 (155)
Fig. 3. ORTEP plot (50% probability level for thermal ellipsoids) of the
X-ray structure of 9, showing the crystallographic atom numbering
scheme.
2. Chemistry
The methyl- (5) and the arylmethyl-oximethers (6–
12) were prepared as described in Fig. 2, starting from
cyclopentanone 13, which was treated with PBr3 in
DMF to give the 1-bromo-2-formylcyclopentene 14
[26]. Reaction of bromoaldehyde 14 with the
methoxime or the appropriate O-arylmethyloxyamine
hydrochloride in a CHCl3–H2O mixture afforded ex-
clusively the corresponding (2-bromo-1-cyclopentenyl-
1-methylidene)methoxyamine (15) or (2-bromo-
1-cyclopentenyl - 1 - methylidene)(arylmethyloxy)amines
(16–22) with the E configuration around the oximic
double bond. The cross-coupling reaction [27] of the
bromo-oxime ethers (15–22) with p-methylthiophenyl-
boronic acid in an EtOH–toluene mixture, in the pre-
sence of Pd(PPh3)4 and aqueous Na2CO3, yielded the
corresponding methylsulphides (23–30) which, by oxi-
dation with potassium peroxymonosulphate (oxone) in

394
A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 391–398
Table 3
ducted again administering compound 9 intraperi-
toneally at the same increasing doses. In analogous
experiments carried out injecting compound 9 intraperi-
toneally at a dose of 30 mg kg−1 in na¨ıve animals, no
signs of local irritation were observed. Results are
shown in Table 3, together with the value obtained for
indomethacin taken as reference drug and administered
per os at a dose of 3 mg kg−1. While at the lowest
doses (1 and 3 mg kg−1) compound 9 appeared to be
practically inactive, at the highest doses (10 and 30 mg
kg−1), the compound showed percentage inhibitory
indices of 23% and 29%, respectively; this last result is
not much lower than that obtained for indomethacin at
3 mg kg−1 po.
Antiinflammatory effect of compound 9 on carrageenan-induced paw
edema in rats
Treatment
Dose (mg
kg−1 i.p.)
Paw volume
%
Inhibition
Basal
3 h
Control
Indomethacin 3 (po)
1
–
0.7990.040 1.3190.023
–
0.7990.036 1.1290.041* 36
0.7990.042 1.2590.087 11
0.7990.026 1.2590.122 11
0.7990.046 1.1990.087* 23
0.7990.033 1.1690.140* 29
9
3
10
30
*, pB0.01 vs. control, split plot test.
the oximic double bond should not change, therefore, it
was possible to assign the (E) configuration to sul-
phides 23–30 and to the bromoderivatives 15–22.
4. Discussion and Conclusions
This work was planned in order to verify the effects
on the biological properties of the substitution with
MAOMM of one of the two aryls of COX-inhibitor
drugs such as the 1,2-diarylcyclopentenes (3). Some of
the new compounds showed a more or less marked
ability to interact with the two types of cyclooxyge-
nases. With regard to activity towards COX-1, all the
compounds in which the MAOM moiety was substi-
tuted by an aryl possessed similar inhibitory properties,
with inhibition percentages at a concentration of 10 mM
ranging from about 30% to 40%. On the contrary, the
compound in which the MAOM was completely
aliphatic, appeared to be inactive. In the same experi-
mental conditions, celecoxib showed an IC50 value of
about 1 mM. These data indicate that, on the whole, the
new compounds weakly inhibit COX-1, regardless of
the nature of the substituent present on the aromatic
ring of 6–12. The inactivity of compound 5, in which
the MAOM moiety is completely aliphatic, would seem
to indicate that, in accordance with preceding findings
[21,22], the presence of an aryl group on the methyl
carbon of the MAOMM is essential for an appreciable
interaction with the enzyme. An examination of the
biological data reported in Table 1 shows that most of
the new compounds substituted on the MAOMM with
an aryl are able to inhibit the COX-2 enzyme, with IC50
values lower than 10 mM. Among these compounds, the
3,4-methylenedioxy-substituted analogue (9) exhibited
an appreciable activity index in the sub-micromolar
range (0.41 mM), whereas unsubstituted on the phenyl
analogue (6) and the 4-chlorophenyl derivative (11)
showed activity indices of about 2 mM. In contrast, the
compounds with the completely aliphatic MAOM side-
chain (5) or linked to an aryl in turn substituted by
chlorine and fluorine atoms in the two ortho-positions
(12) showed little or nor activity. These results suggest
that an aryl group is required for COX-2 inhibitory
activity in this series. Additional analogues will need to
3. Biopharmacological results
The inhibitory activity of compounds 5–12 towards
COX-1 and COX-2 was evaluated in vitro by measu-
ring prostaglandin E2 (PGE2) production in U937 cell
lines for COX-1 and activated J774.2 macrophages for
COX-2. The results obtained are reported in Table 1,
together with those obtained in the same tests for
celecoxib (4), chosen as a reference compound between
the tricyclic COX-2 selective inhibitors because of its
current therapeutic interest. The compound substituted
with a completely aliphatic aminoethereal chain (5)
exhibited at a high concentration a very low inhibitory
activity, directed only against COX-2. Among the O-
arylmethyl oximethers, the most active compound at
the COX-2 level proved to be the methylenedioxy-sub-
stituted derivative (9), which showed an IC50 value of
0.41 mM, versus IC50 value of celecoxib (4) of 0.02 mM.
On the contrary, it appeared to possess a very low
activity towards COX-1. In addition, 9 showed very
low activity against COX-1; 27% at 10 mM. The 2,6-di-
substituted analogue 12 showed very little or nor activ-
ity against COX-1 and COX-2, respectively. Other
substitutions on the MAOMM aromatic ring afforded
compounds that had some inhibitory activity against
COX-1 at 10 mM, but considerably greater inhibitory
activity against COX-2 (see Table 1).
For compound 9, which showed the highest in-
hibitory activity towards COX-2 in the in vitro test, the
in vivo activity was evaluated using the carrageenan-in-
duced paw edema test in rats. In a first set of experi-
ments, compound 9 was administered per os at
increasing doses ranging from 1 to 30 mg kg−1. No
anti-inflammatory activity was observed with 9 over a
wide dose range. The same test was, therefore, con-

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 391–398
395
trointestinal stability, poor absorption, and/or rapid
first pass metabolism.
be prepared to identify the optimal aromatic substitu-
tion pattern. From an examination of the data reported
in Table 1 it may also be noted that all new MAOM-
derivatives, with the exception of 8 and 12, possess
better activity against COX-2 than COX-1. However,
the absolute values of their selectivity should be inter-
preted with caution, both because their extrapolability
to the in vivo human condition has not been assessed,
and because in our in vitro tests the reference com-
pound celecoxib (4) showed a COX-2 selectivity signifi-
cantly different from that determined by other
experimental procedures [28].
The presence of an appreciable inhibitory activity in
some of the new MAOM-derivatives indicate the exis-
tence of a bioisosteric relationship between aryls and
the MAOMM also in the context of COX-2 inhibitors.
In order to rationalise this finding in terms of confor-
mational and steric analogies, it is possible to make a
comparison of some structural parameters of com-
pound 9 and of an analogue of celecoxib (i.e. SC 558,
31, Fig. 4) [3], obtained from a drug-murine COX-2
complex. In this last compound, the two aromatic rings,
one substituted by a sulphonamido group and the other
one bromo-substituted are not coplanar with the pyra-
zole ring, making dihedral angles of 37° and 74°, re-
spectively. This type of molecular geometry seems to be
optimal for a positive drug-receptor interaction. In
compound 9, the plane of the para-methylsulpho-
nylphenyl moiety and the plane of the MAOMM are
not coplanar with the cyclopentenyl ring, even if they
form dihedral angles of lesser amplitude (23° and 15°,
respectively). From an observation of the structures of
9 and 31, it may also be noted that the 3,4-methylene-
dioxyphenyl-substituted MAOMM of 9 presents a
higher steric hindrance with respect to the aryl group of
31; this makes it possible to hypothesise that the active
site of the enzyme contains a lipophilic pocket able to
receive groups of a larger size than that of a simple aryl
group.
5. Experimental protocols
5.1. Chemistry
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
FT-IR spectrometer. 1H-NMR spectra of all com-
pounds were obtained with a Varian CFT 20 instru-
ment or a Gemini 200 spectrometer operating at 80 or
200 MHz, respectively, in 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–Pack-
ard 5988A spectrometer. Evaporation was performed in
vacuo (rotating evaporator); the O-arylmethyl-
oxyamines to be used for the preparation of the bro-
moximethers 15–22, not commercially available, were
prepared following the synthetic method described in
ref [29]. Na2SO4 was always used as the drying agent.
Elemental analyses were performed in our analytical
laboratory and agreed with the theoretical values to
within 90.4%.
5.1.1. Synthesis of 2-bromo-1-cyclopenten-1-car-
boxyaldehyde (14)
Compound 14 was prepared following the reaction
indicated in ref. [26]. A cooled (0 °C) and stirred
mixture of DMF (11.6 mL, 0.15 mol) and anhydrous
CHCl3 (40 mL) was treated dropwise with PBr3 (11.8
mL, 0.125 mol) and then with a solution of cyclopen-
tanone (4.43 mL, 0.05 mol) in anhydrous CHCl3 (20
mL). After 12 h at room temperature, the solvent was
evaporated and the residue was treated with ice and
solid NaHCO3. The aqueous phase was extracted with
Et2O and the organic solvent was washed (5% K2CO3
and H2O), dried and evaporated to yield an oily residue
which by distillation at reduced pressure gave pure 14
As regards the in vivo properties of the MAOMM
derivatives studied, the results obtained for compound
9 administered intraperitoneally showed modest anti-
inflammatory activity. The lack of activity of 9 after
oral administration may be the result of limited gas-
1
(90%): b.p. 60–65 °C (0.3 mm Hg); H-NMR l 1.91–
2.18 (m, 2H, CH2CH2CH2), 2.41–2.63 (m, 2H,
CH2CCHO), 2.78–2.99 (m, 2H, CH2CBr), 9.85 ppm (s,
1H, CHO); Lit. [26]: unreported data.
5.1.2. General procedure for the preparation of the
(E)-(2-bromo-1-cyclopentenyl-1-methylidene)-
(methyloxy)amine (15) and -(arylmethyloxy)amines
(16–22)
A mixture of 14 (1.0 g, 5 mmol) in CHCl3 (36 mL)
and the appropriate O-methyl or O-arylmethyloxy-
Fig. 4. Structure of the analogue (31) of celecoxib (4) previously
utilised in a crystallographic study of drug-murine COX-2 complex.

396
A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 391–398
1
lamine (5 mmol) in H2O (5 mL), was stirred at room
temperature for 24 h. The organic phase was separated
and the aqueous solution was extracted twice with
CHCl3. Evaporation of the dried and filtered organic
extracts afforded a residue consisting almost exclusively
of the corresponding bromooximes 15–22, which with-
out further purification were used in the following
from EtOH. H-NMR data for SO2CH3, for the sin-
glets attributable to MeO of 5 or ArCH2 of 6–12 and
to their NꢁCH protons: 5 (80%) l 3.08 (3H, SO2CH3),
3.92 (3H, MeO), 7.98 ppm (1H); 6 (50%) l 3.03 (3H),
5.1 (2H), 8.0 ppm (1H); 7 (35%) l 3.02 (3H), 5.05 (2H),
7.98 ppm (1H); 8 (82%) l 3.02 (3H), 5.02 (2H), 7.97
ppm (1H); 9 (50%) l 3.03 (3H), 4.99 (2H), 7.97 ppm
(1H); 10 (24%) l 3.03 (3H), 5.05 (2H), 7.97 ppm (1H);
11 (37%) l 3.04 (3H), 5.06 (2H), 7.98 ppm (1H); 12
(43%) l 3.02 (3H), 5.2 (2H), 7.94 ppm (1H). MS (m/e):
5, [M+] 279; 6, [M+] 355; 7, [M+] 369; 8, [M+] 385; 9,
[M+] 399; 10, [M+] 373; 11, [M+] 389; 12, [M+] 407.
For the analytical and physical data of 5–12 see Table
1.
1
reaction. H-NMR data for the singlets attributable to
MeO of 15 or ArCH2 of 16–22 and to their NꢁCH
protons: 15 (86%) l 3.88 (3H) and 7.97 ppm (1H); 16
(74%) l 5.1 (2H), 8.04 ppm (1H); 17 (65%) l 5.06 (2H),
8.02 ppm (1H); 18 (84%) l 5.06 (2H), 8.06 ppm (1H);
19 (91%) l 4.92 (2H), 7.94 ppm (1H); 20 (47%) l 5.06
(2H), 8.02 ppm (1H); 21 (43%) l 5.0 (2H), 8.0 ppm
(1H); 22 (36%) l 5.2 (2H), 7.98 ppm (1H).
5.2. Crystal-structure determination
5.1.3. General procedure for the preparation of the
(E)-[2-(4-methylthiophenyl)-1-cyclopentenyl-1-methyl-
idene](methyloxy)amine (23) and
Crystals of the E oximether 9, suitable for X-ray
analysis, mp 130–131 °C, were obtained by slow crys-
tallisation from EtOH: C21H21NO5S, M=399.461,
monoclinic, space group P21/c, a=17.522 (3) A, b=
4.947 (5) A, c=21.997 (2) A, b=93.40 (3), V=1903.4
(2.0) A3, Z=4, Dcalc=1.394 g cm−3. A Siemens AED
diffractometer, equipped with an Mo Ka source (using
the r/2r scan mode, scan speed 3/12 deg min−1, scan
width 1.20+0.34 tan r, r in the range 3–30°) was used
to obtain intensity data. The structure was solved by
direct methods using SIR97 [30] and refined using
SHELX96 [31] by full matrix least squares on Fo with
anisotropic thermal parameters, isotropic for the hydro-
gens. The final R indexes were, respectively: R1=
0.0457 for 1591 reflections with Fo\4| (Fo),
R2=0.1434 for all the 4161 reflections wR2=0.1282.
-(arylmethyloxy)amines (24–30)
A mixture of the appropriate 1,2-disubstituted cy-
clopentenylidene derivative (15–22) (3.18 mmol),
Na2CO3 2 M (4 mL) and 4-methylthiophenylboronic
acid (0.54 g, 3.19 mmol), in a 50% toluene–EtOH
mixture (20 mL), was treated with Pd(PPh3)4 (0.1 g)
and the resulting mixture was refluxed under N2 with
stirring for 12 h. Evaporation of the organic phase gave
a residue which was taken up in AcOEt, washed with
H2O, dried and evaporated to afford a crude residue
which was subjected to column chromatography, to
yield pure 23–30. 1H-NMR data for the singlets at-
tributable to MeO of 23 or ArCH2 of 24–30 and to
their SCH3 and NꢁCH protons: 23 (62%) l 2.49 (3H,
SCH3), 3.88 (3H, MeO), 8.04 ppm (1H); 24 (51%) l
2.46 (3H), 5.09 (2H), 8.08 ppm (1H); 25 (53%) l 2.46
(3H), 5.04 (2H), 8.06 ppm (1H); 26 (29%) l 2.46 (3H),
5.01 (2H), 8.05 ppm (1H); 27 (48%) l 2.45 (3H), 5.0
(2H), 7.98 ppm (1H); 28 (55%) l 2.47 (3H), 5.04 (2H),
8.06 ppm (1H); 29 (42%) l 2.46 (3H), 5.04 (2H), 8.06
ppm (1H); 30 (70%) l 2.45 (3H), 5.2 (2H), 8.02 ppm
(1H).
5.3. Biopharmacological methods
5.3.1. Enzyme assays
All the compounds were tested in intact cell assays to
verify their capacity to inhibit PGE2 production, con-
sidered 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 are then centrifuged and the PGE2 content in the
supernatant was measured by a commercial immunoen-
zymatic assay (Amersham). The COX-2 assay was per-
formed in accordance with the method described by
Mitchell et al. [32] with minor modifications as sug-
gested by Grossman et al. [33]. Murine J774.2 cells were
pretreated for 1 h with 300 mM aspirin to inactivate
endogenous constitutive COX-1 and 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 col-
lected and PGE2 measured as described above. All
compounds were tested in duplicate. For each product
5.1.4. General procedure for the preparation of the
(E)-[2-(4-methylsulphonylphenyl)-1-cyclopentenyl-
1-methylidene]-(methyloxy)amine (5) and
-(arylmethyloxy)amines (6–12)
A cooled (0 °C) and stirred solution of the appropria-
te methylsulphide derivative 23–30 in 1:1 MeOH–THF
mixture (15 mL), was treated dropwise with a solution
of oxone (2KHSO5·KHSO4·K2SO4) (1.35 mmol) in
H2O (7 mL). At the end of the addition, the reaction
was stirred at room temperature for 12 h. The solvent
was evaporated and the crude residue was taken up in
AcOEt, washed with a saturated solution of NaCl,
dried and evaporated to give the corresponding crude
sulphone (5–12), which was purified by crystallisation

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 391–398
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6. Supplementary material
Crystallographic data (excluding structure factors)
for the structure in this paper have been deposited at
the Cambridge Crystallographic Data Centre, as sup-
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may be obtained free of charge from The Director,
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