Synthesis and biological evaluation of neutrophilic inflammation inhibitors

Article abstract of DOI:10.1016/j.farmac.2003.08.005

In several non-infectious human diseases, such as ulcerous colitis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), the extravasal recruitment of neutrophils plays a crucial role in the development of tissue damage, which, when persist

Full text of DOI:10.1016/j.farmac.2003.08.005

IL FARMACO 59 (2004) 223–235
www.elsevier.com/locate/farmac
Synthesis and biological evaluation of neutrophilic
inflammation inhibitors
Olga Bruno a,*, Chiara Brullo a, Nicoletta Arduino a, Silvia Schenone a, Angelo Ranise a,
Francesco Bondavalli a, Luciano Ottonello b, Patrizia Dapino b, Franco Dallegri b
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Genova, Viale Benedetto XV, Genova 3-16132, Italy
b Dipartimento di Medicina Interna, Università degli Studi di Genova, Viale Benedetto XV, Genova 6-16132, Italy
Received 9 May 2003; accepted 1 August 2003
Abstract
In several non-infectious human diseases, such as ulcerous colitis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), the
extravasal recruitment of neutrophils plays a crucial role in the development of tissue damage, which, when persistent, can lead to the
irreversible organ dysfunction. The neutrophil activation is controlled by a number of intracellular pathways, particularly by a cAMP-
dependent protein kinase A (PKA) which also acts on phosphodiesterase IV (PDE4) gene stimulating the synthesis of this enzyme, able to
transform cAMP to inactive AMP. PDE4 inhibitors enhance intracellular cAMP and decrease inflammatory cell activation. Several
3-cyclopentyloxy-4-methoxybenzaldehyde and 3-cyclopentyloxy-4-methoxybenzoic acid derivatives were synthesized and studied by us to
evaluate their ability to inhibit the superoxide anion production in human neutrophils. These compounds were found able to inhibit the
neutrophil activation and some of them increased the cAMP level on tumor necrosis factor-a-stimulated neutrophils. Moreover, they also
inhibited selectively the human PDE4 enzyme, although they are less potent than the reference compound Rolipram. We report here synthesis,
biological studies and some SAR considerations concerning the above mentioned compounds.
© 2003 Elsevier SAS. All rights reserved.
Keywords: 3-Cyclopentyloxy-4-methoxybenzaldehyde derivatives; 3-Cyclopentyloxy-4-methoxybenzoic acid derivatives; Neutrophilic inflammation; Neutro-
phil inhibitors; PDE4 inhibitors
1. Introduction
In inflamed tissues, neutrophils live in a microenvironment
rich of pro-inflammatory substances, such as cytokines,
chemotactic factors and immuno-complexes, which can ef-
fectively induce a full cell activation [7]. This activation leads
to a rapid and impressive consumption of oxygen by a mem-
brane oxidase, NADPH oxidase, which reduces the oxygen
to superoxide anion (O2–) giving the well-known
“respiratory-burst” [8]. A number of oxidants, such as super-
oxide anion or hypochlorous acid (HOCl), spring up and are
released extracellularly. Neutrophils activation also causes
the release of potential histo-damaging enzymes from cyto-
plasmatic granules [6]. Oxidants, particularly HOCl, and
enzymes provoke a damage of inflamed tissue and enhance
the inflammatory response by their synergic pro-
inflammatory activities.
The hallmark of inflammatory responses is represented by
the extravascular recruitment of inflammatory cells [1,2].
Among these cells, neutrophils predominate in common
forms of inflammation, such as those occurring as a conse-
quence of tissue invasion by microorganisms and those re-
lated to immune disorders or non-specific tissue injury [3,4].
These conditions include many human diseases, such as
ulcerous colitis, rheumatoid arthritis, chronic obstructive
pulmonary disease (COPD), gouty arthritis and also some
dermatites as psoriasis, or some vasculitis. In these non-
infectious diseases, extravasated neutrophils play a crucial
role in the development of tissue damage, which, when
persistent, can lead to the irreversible demolition of normal
tissue architecture with consequent organ dysfunction [5,6].
Intensity and length of inflammatory response [9,10] are
controlled by a cAMP dependent protein kinase A (PKA).
High levels of cAMP activate PKA which phosphorylates
some cytoplasmatic proteins with consequent inhibition of
* Corresponding author.
E-mail address: obruno@unige.it (O. Bruno).
© 2003 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2003.08.005

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O. Bruno et al. / IL FARMACO 59 (2004) 223–235
chemotaxis, degranulation and oxidative neutrophil metabo-
lism [11]. Moreover, PKA also acts on phosphodiesterase IV
(PDE4) gene [9,11] stimulating the synthesis of this enzyme,
able to transform cAMP to inactive AMP. PDE4 inhibitors
enhance intracellular cAMP and decrease inflammatory cells
activation [12–14]. Consequently, in the last years the inter-
est for substances able to inhibit PDE4 activation is substan-
tially increased [15–17].
PDE4, present in the immune and inflammatory cells, is
the most diffuse and, likely, the most important and selective
cAMP hydrolyzing enzyme [18]. Several different structures
have been reported and patented as PDE4 inhibitors [19–22],
the first of which being Rolipram [23], unsuitable for thera-
peutic use owing to its peripheral (nausea, gastric irritation)
and central (strong sedative action) side effects. It was clearly
demonstrated that emesis [24], gastric acid secretion [25],
and psychotropic activity [26], are linked to inhibition of the
high affinity Rolipram binding site (HARBS), and that an
improvement of therapeutic index can be obtained with a low
PDE4 inhibition/Rolipram binding site affinity (RBSA) ra-
tio.
lytic site [21] and present in several “second-generation”
PDE4 inhibitors such as RP-73401 [35], Ariflo (Cilomilast)
[36], CDP-840 [37].
In our compounds some simple non-aromatic nitrogen
heterocycles have been linked to the catechol moiety by
different functions. In particular, in the first two series 1a–c
and 2a–d, the 2-pyrrolidinone, 2-piperidinone, 2-imidazo-
lidinone cycles and some cycloamines, have been bonded to
the aromatic ring by an amide function; in a third group of
compounds, 3a–d and 4a–d, this function has been replaced
by an imine (planar) or amine (non-planar) bond, respec-
tively, in order to verify the role of the carbonyl group, as
well as the disposition of the heterocycle moiety with respect
to the phenyl ring. Finally, we have synthesized compounds
5a–c and 6a–c, in which heterocycle rings were spaced out
from the catechol ring by more long and flexible iminoether
alkyl chains.
2. Chemistry
Compounds 1a–c and 2a–d were prepared starting from
3-cyclopentyloxy-4-methoxy benzoic acid (7) (obtained
from isovanilline by alkylation and subsequent oxidation
[38]) which was transformed into the corresponding acid
chloride 8 with thionyl chloride and then treated with
2-pyrrolidinone, 2-imidazolidinone, d-valerolactame or the
appropriate N-aminocycloamine in CH2Cl2, in the presence
of anhydrous triethylamine, as shown in Scheme 1. In two
cases we obtained a little amount of byproducts (the anhy-
dride 9 and the isomer 10, respectively) which were sepa-
rated by recrystallization. In the case of compound 1b we
used 2-imidazolidinone and acid chloride 8 in the 2:1 ratio,
without triethylamine in order to avoid the formation of the
double acylation product. For compounds 3a–d the
3-cyclopentyloxy-4-methoxybenzaldehyde (11) [38] was
condensed with the appropriate N-aminocycloamines in an-
hydrous toluene using a Dean-Stark apparatus to remove
water as azeotropic mixture (see Scheme 2). We used an
alternative synthetic procedure to prepare the 3a–d reduction
products 4a–d. We carried out the reductive amination of
3-cyclopentyloxy-4-methoxybenzaldehyde (11) with sodium
cyanoborohydride in THF/ethanol mixture, in the presence
of zinc chloride [39]. Then, a further addition of sodium
cyanoborohydride and acidification of the reaction mixture
to pH 3.8 [40] completely reduce the intermediate imino
derivatives 3a–d (quantitatively and easier formed with re-
spect to the previous method, but not isolated in this case).
Compounds 5a–c and 6a–c were prepared starting from
3-cyclopentyloxy-4-methoxybenzaldehyde oxime (12), ob-
tained from 3-cyclopentyloxy-4-methoxybenzaldehyde (11)
and hydroxylamine hydrochloride. This compound, reported
in literature as yellow oil [41] was obtained by us, through
some modifications of the reaction workup, as a white, flaky,
low-melting solid (the structure was confirmed by IR and 1H
NMR spectral data). Oxime 12, as sodium salt, was then
OCH3
OCH3
OCH3
O
O
O
CN
O
NH
N
Cl
Cl
O
OH
N
RP-73401
Ariflo
CDP-840
OCH3
O
O
NH
O
Rolipram
The design of new PDE4 inhibitors remained empirical
for a long time, assisted in part by interesting studies of
molecular modeling aimed at defining the pharmacophoric
model of PDE4 catalytic site [27–30]. Recently, Xu et al. [31]
determined the three-dimensional structure of the catalytic
domain of PDE4B2B providing useful framework for a ratio-
nal design of more selective PDE4 inhibitors and for the
understanding of their mechanism of action.
In the contest of our studies on anti-inflammatory drugs
[32–34], with the aim of selecting new leads for the develop-
ment of compounds active in the neutrophilic inflammation,
we designed and synthesized several series of Rolipram
related structures in which we maintained the catechol ring
with cyclopentyl and methyl substituents, regarded as crucial
part for the interaction with a lipophilic pocket of the cata-

O. Bruno et al. / IL FARMACO 59 (2004) 223–235
225
OCH3
OCH3
OCH3
OCH3
O
O
O
O
H
H
O
N
O
NH
2a-d
N
H
N
N
NH
4a-d
N
1a-c
3a-d
2, 3, 4,
5,6
1
N
N
OCH3
OCH3
N
a
b
c
d
O
O
a
N
N
O
OH
O
N
N
O
N
b
c
H
N
N
H
N
O
O
N
N
O
5a-c
6a-c
NCH3
condensed with the appropriate x-chloroethylamines, to ob-
tain compounds 5a–c. The condensation of the same sodium
salt with epichlorohydrin produced the intermediate epoxide
13, which was reacted, without purification, with the suitable
cycloamines to give compounds 6a–c (see Scheme 3).
presence and in the absence of the substances and Rolipram,
as reference compound. Rolipram, as well as tested com-
pounds, provoked a dose-dependent increase of intracellular
cAMP level as reported in Table 2 and in Fig. 1.
A further study was performed with the aim of investigat-
ing effects and selectivity of the same compounds on the
human phosphodiesterases 3, 4 and 5. The effects of the
tested compounds on the activities of the studied phosphodi-
esterases are reported in Table 3.
3. Biological studies
Moreover, in order to obtain useful indications regarding
the propensity to induce side effects, we evaluated, for com-
pound 6c, the affinity for the HARBS. We report the compe-
tition curve obtained with 6c at the Rolipram receptor in
Fig. 2, the IC50 for RBA and PDE4/RBA ratio in Table 3.
All synthesized compounds were studied to evaluate their
ability to inhibit the superoxide anion production tumor ne-
crosis factor-a (TNFa) induced, in human neutrophils, in
comparison with Rolipram as reference compound. When
placed in fibronectin-coated wells, in absence of stimuli,
neutrophils released detectable amount of superoxide anion
(O2–) up to 0.5 nmol/5 × 104 neutrophils during 3 h of
incubation. If added at the beginning of the incubation, the
addition of TNFa induced a prolonged (1.5–2 h) production
of O2– (3.1 0.2 nmol O2–/5 × 104 neutrophils). When
Rolipram was added to the system, dose-dependent inhibi-
tion of the production of O2– was observed (IC50
1.43 1.32 µM).
The synthesized compounds were investigated for their
ability to interfere with the production of superoxide anion in
the same test. All compounds, with the exception of 3c and
4c, inhibited in a dose-dependent way the production of O2–.
We reported in Table 1 the results, expressed as IC50 (mean of
two or more experiments), for tested compounds and Rolip-
ram.
4. Results and discussion
No compounds, at the concentrations used in the present
work, show cytotoxic action towards the neutrophils. The
percentage of vital cells never results lower than 90%. The
results of the superoxide anion assay (see Table 1) suggest
the following remarks. In all series screened, with the excep-
tion of 6a–c, the products having a five-membered hetero-
cycle linked to the catechol moiety are the most active (1a,
2a, 3a, 4a and 5a), and the activity do not substantially
change if a second heteroatom is present in the ring (see 1a
and 1b). Compounds with six-membered heterocycles ap-
pear less active and show a further decrease of activity if one
more heteroatom is introduced. This is particularly evident in
compounds 3b, 4b and 5b in comparison with 3c–d, 4c–d
and 5c, respectively. Moreover, comparing the series 2, 3, 4,
we can assert that the activity level is independent from the
type of functionality linking the heterocycle to the phenyl
In addition, with the aim of verifying whether the lesser
superoxide anion production was actually related to an inhi-
bition of PDE4, we selected three compounds (1a, 2a and 3a)
and we determined the intracellular level of cAMP, in neu-
trophils adherent to fibronectin and TNFa stimulated, in the

226
O. Bruno et al. / IL FARMACO 59 (2004) 223–235
OCH3
OCH3
O
O
+
O
O
O
N
O
O
O
H3CO
OCH3
1a (48%)
9 (13%)
O
b
O
N
c
OCH3
OCH3
NH
O
O
a
O
1b (44%)
O
OH
O
Cl
e
8
7
OCH3
O
d
OCH3
O
NH N
OCH3
O
O
2a-d (35-87%)
+
O
N
O
N
O
HO
10 (20%)
1c (74%)
aReagents: (a) SOCl2; (b) 2-Pyrrolidinone, CH2Cl2, Et3N; (c) 2-Imidazolidinone 2 equiv., CH2Cl2;
(d)δ -v alerolactame, CH2Cl2, Et3N; (e) N-aminocycloamines, CH2Cl2, Et3N.
Scheme 1. Synthetic route for the preparation of compounds 1a–c and 2a–d.
ring. Thus, neither the carbonyl group of the amide nor the
double bond of imine function play an important role in the
action. Compounds 5 result generally less active than com-
pounds 1, 2, 3 and 4, suggesting that spacing out the hetero-
cyclic ring from the catecholic moiety by an iminoether chain
is detrimental for the activity. Compounds of the series 6a–c
present a different behavior from all the previous ones. In
these compounds the inhibiting activity reverts to the level of
compounds of the series 1, 2, 3 and 4, in spite of the spacer
lengthening; furthermore, in this case, the morpholinyl de-
rivative 6c is the most active, while in the preceding series the
morpholine ring causes a drastic decrease of activity (see
particularly 3c and 4c). Therefore, we can suppose that, in
compound 6c, could develop a conclusive role both the oxy-
gen atom of the morpholine ring and the hydroxy group of
the iminoether chain.
Moreover, compounds 1a, 2a and 3a induce a dose-
dependent increase of intracellular cAMP level in neutro-
phils adherent to fibronectin and TNFa stimulated (see
Table 2). The close correlation between this activity and the
inhibition of the superoxide anion production (IC50), in com-
pounds 1a, 2a and 3a, suggests that they act on the neutro-
phils either by stimulating the cAMP production or inhibit-
ing its hydrolysis (see Fig. 1).
The latter hypothesis seems to be confirmed by the enzy-
matic tests on the human PDE4 for compounds 1a, 2a, 3a
and 6c. All the four tested compounds present a fair good
inhibitory activity on the PDE4 (see Table 3), the most active
being compound 6c with an IC50 (µM) of 1.45
0.35.
Anyway, all tested compounds are less active than the refer-
ence compound Rolipram, but, being almost inactive towards

O. Bruno et al. / IL FARMACO 59 (2004) 223–235
227
OCH3
OCH3
O
O
a
H
N
N
O
H
3a-d (32-98%)
11
b
OCH3
O
c
[ 3a-d ]
H
H
NH
N
4a-d (50-66%)
aReagents: (a) N-aminocycloamines, an. toluene, Dean-Stark's
apparatus; (b) N-aminocycloamines, an. THF, NaBH3CN, ZnCl2;
(c) NaBH3CN, EtOH/HCl.
Scheme 2. Synthetic route for the preparation of compounds 3a–d and 4a–d.
OCH3
OCH3
OCH3
O
O
O
b
a
O
O
H
H
N
OH
c
H
N
N
11
12
5a-c (54-71%)
OCH3
O
OCH3
O
d
OH
O
O
O
N
H
H
N
N
13 (95%)
6a-c (56-76%)
aReagents: (a) NH2OH*HCl, EtOH, NaHCO3; (b) 1: EtONa/EtOH, 2: 2-Chloroethylcycloamines, an.DMF;
(c) 1: EtONa/EtOH, 2: Epichlorohydrin, an. DMF; (d) Cycloamines, an. toluene.
Scheme 3. Synthetic route for the preparation of compounds 5a–c and 6a–c.
PDE3 and PDE5, they could be considered selective PDE4
inhibitors.
superoxide anion production, TNFa induced, in human neu-
trophils. All compounds, except 3c and 4c, were able to
inhibit neutrophils activation, chiefly those with a five-
membered heterocycle linked to the catechol moiety. How-
ever, no correlation seems to be between the activity and the
type of function that links the heterocycle to the phenyl ring.
The most active compounds 1a, 2a and 3a showed also a
good capability of increasing the cAMP level on neutrophils
TNFa stimulated. The same compounds and 6c also inhib-
ited the human PDE4 activity. These evidences allow to
affirm that the activity on the neutrophils of the synthesized
compounds is due to an intracellular cAMP increase, caused
in turn by the PDE4 inhibition. Finally, the most active
compound 6c showed a low PDE4/RBSA ratio, suggesting a
Finally, the selected compound 6c specifically binds to the
Rolipram receptor (see Fig. 2), but it posses about 280 times
weaker affinity than Rolipram for [3H]Rolipram binding site
(IC50 = 0.59 µM vs. 0.0021 µM and Ki = 0.3 µM vs.
0.001 µM), with a low PDE4/RBSA ratio (60-fold lower than
Rolipram).
5. Conclusions
In conclusion, several new Rolipram-related compounds
were synthesized and tested for their ability to inhibit the

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O. Bruno et al. / IL FARMACO 59 (2004) 223–235
Table 1
Compounds 1a–c, 2a–d, 3a–d, 4a–d, 5a–c, 6a–c and their effect on superoxide anion production, in human neutrophils (5 × 104/plate). Results are expressed
as IC50 (mean values of two or more experiments)
OCH3
O
R
Comp
1a
R
Comp.
4a
R
IC50 (∝M)
IC50 (∝M)
C
H
H
12.64 3.20
11.62 3.73
O
N
N
N
C
NH
N
O
C
H
H
1b
1c
2a
14.34 6.12
20.71 3.22
10.28 3.06
4b
4c
4d
22.23 4.68
Inactive
O
C
NH
N
NH
O
C
H
H
O
C
NH
N
O
O
C
H
H
112.71 11.30
O
C
NH
N
NH
N
NCH3
C
C
O
C
2b
2c
2d
23.84=12.18
28.13 9.70
27.71 2.96
5a
5b
5c
52.22 8.29
68.68 5.99
O
N
H
N
NH
NH
N
O
C
O
N
H
N
N
O
C
O
C
295.79 10.17
O
N
H
N
N
NH
N
NCH3
O
OH
C
3a
3b
3c
3d
12.14 6.60
17.23 6.19
Inactive
6a
6b
6c
31.35 13.58
21.20 8.44
16.30 7.39
1.43 1.32
H
N
N
C
O
N
H
OH
C
C
H
H
N N
O
N
H
N
OH
C
O
C
N
N
O
O
N
H
N
C
116.80 12.63 Rolipram
O
H
N
N
NCH3
NH
O
Table 2
6. Experimental section
Effect of compounds 1a, 2a and 3a and Rolipram, as reference compound,
on intracellular cAMP level in neutrophils (106/plate) adherent to fibronec-
tin and TNFa stimulated. The results are expressed as EC50
6.1. Chemistry: materials and methods
All chemicals were obtained from Sigma Aldrich srl (Mi-
lan, Italy). Melting points are uncorrected and were mea-
sured with a Büchi 530 instrument. IR spectra were recorded
with a Perkin-Elmer 398 spectrophotometer. 1H NMR were
recorded on a Varian Gemini 2 (200 MHz) instrument;
chemical shifts are reported as d (ppm) relative to tetrameth-
ylsilane (TMS) as internal standard; signals were character-
ized as s (singlet), d (doublet), t (triplet), m (multiplet), br s
(broad signal); J in Hz. Reactions were followed by TLC on
Kieselgel 60F254 (DC-Alufolien, E. Merck, Darmstadt, Ger-
many).Analyses for C, H, N ( 0.4% of the theoretical value),
were determined with an elemental analyzer EA 1110
(Fison-Instruments, Milan, Italy).
Compound
EC50 (µM)
13.44
11.46
6.17
1a
2a
3a
Rolipram
1.14
possible lack of side effects in vivo. In addition, we must
underline that compound 6c was synthesized and tested as
racemic form. As the hydroxy group, indicated as possible
responsible of the activity, is just bonded to a chiral center in
the iminoether chain, we cannot exclude the existence of an
eutomeric form. Thus, we consider compound 6c an interest-
ing starting point to develop new molecules in which the
rational modification of the aromatic moiety linked to the
morpholinyl–hydroxypropyl–iminoether chain, as well as a
stereoselective synthesis, could give new potent PDE4 in-
hibitors with reduced side effects.
6.1.1. Preparation of 3-(cyclopentyloxy)-4-methoxybenzoyl
chloride (8)
Thionyl chloride (3.6 ml, 50 mmol) was added to
3-(cyclopentyloxy)-4-methoxy-benzoic acid 7 [38], (2.36 g,

O. Bruno et al. / IL FARMACO 59 (2004) 223–235
229
1a
2 a
100
50
0
100
50
0
1
3
10
30
100
1
3
10
30
100
3 a
ROLIPRAM
100
50
0
100
50
0
0,1
0,3
1
3
10
30
100
1
3
10
30
100
Fig. 1. Dose-dependent effect of compounds 1a, 2a, 3a and Rolipram (µM) as % inhibition of superoxide anion production (gray bars) (mean 1 S.D. of two
experiments) and % cAMP accumulation (black bars, results of a single experiment) in TNFa stimulated polymorphonuclear leukocytes.
Table 3
Results of PDE enzyme assay for compounds 1a, 2a, 3a, 6c. The results are expressed as a percent inhibition of control activity (mean values; n = 2) or as IC50
Compound
PDE3
PDE5
PDE4
RBA
IC50
(µM)
ntb
PDE4/RBA
% Inhibition
(10 µM)
% Inhibition
(10 µM)
IC50
(µM)
a
1a
2a
3a
6c
†
†
2.7 0.42
4.65 0.07
2.85 0.07
1.45 0.35
0.32
–
23
†
†
nt
–
†
nt
–
†
22
–
0.59
0.0021
2.46
152
Rolipram
–
a The symbol † indicates an inhibition lesser than 10%.
b nt, not tested.
After evaporation under reduced pressure of the solvent the
obtained crude yellow oil (2.37 g) was purified by flash-
chromatography on Florisil® (100–200 Mesh) with CH2Cl2
as eluent. From the ether solution of the purified oil a by-
product crystallizes as white solid which was recrystallized
from ethyl acetate. It corresponds to 3-(cyclopentyloxy)-4-
10 mmol) and the reaction mixture was stirred at 60–80 °C
for 2 h. The excess thionyl chloride was evaporated under
reduced pressure to give 3-(cyclopentyloxy)-4-methoxy-
benzoyl chloride (8) as a yellow oil which, after washing with
ethyl ether, crystallizes by standing (yield: 1.90 g, 75%).
Compound 8 has been used crude in all the following reac-
tions.
1
methoxybenzoic anhydride (9), as confirmed by IR and H
NMR spectra and elemental analysis. The desired compound
1a was recovered from the ether solution by evaporation of
the solvent and distillation in vacuo of the oily residue (b.p.:
195 °C/0.1 mmHg). Finally, the obtained viscous oil was
crystallized from ethyl ether/petroleum ether (1:1). Yield:
6.1.2. Preparation of 1-[3-(cyclopentyloxy)-4-methoxy-
benzoyl]pyrrolidin-2-one (1a)
A solution of 8 (1.9 g, 7.5 mmol) in CH2Cl2 (25 ml) was
added to a solution of 2-pyrrolidinone (0.68 g, 8 mmol) and
anhydrous triethylamine (1.5 ml) in CH2Cl2 (25 ml). The
reaction mixture was stirred at 60–80 °C for 6 h and, after
cooling, washed with water (20 ml) and dried (MgSO4).
1
1.1 g, 48%, m.p. 58–60 °C. H NMR (CDCl3) d 1.20–2.10
(m, 8H), 2.10–2.40 (m, 2H), 2.40–2.90 (m, 2H), 3.91 (s, 3H),
4.60–5.10 (m, 1H), 6.70–7.10 and 7.20–7.50 (2m, 3H). IR
(CHCl3) cm–1 1745, 1665, 1270. Anal. Calcd. for

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O. Bruno et al. / IL FARMACO 59 (2004) 223–235
phase was dried (MgSO4) and, after evaporation under re-
duced pressure of the solvent, an oil (3.43 g) was obtained
which crystallizes with ethyl ether. The white–pink solid
obtained was then recrystallized from ethyl acetate to get
desired compound 1c. From the filtered ether solution a
further white precipitate, soluble in 1 M NaOH solution and
different from compound 1c [TLC: CHCl3/CH3OH (9:1)]
was obtained. It was recrystallized from ethyl acetate and
resulted to be 1-[3-(cyclopentyloxy)-4-methoxybenzoyl]-
1
1,4,5,6-tetrahydropyridin-2-ol (10), as confirmed by IR, H
NMR spectra and elemental analysis. Compound 1c: yield
1
2.36 g, 48%, m.p. 100–101 °C. H NMR (CDCl3) d 1.46–
2.20 (m, 12H), 2.30–3.80 (m, 2H), 3.59–4.01 (m, 5H),
4.60–5.00 (m, 1H), 6.65–7.00 and 7.10–7.40 (2m, 3H). IR
(CHCl3) cm–1 1708, 1675, 1265. Anal. Calcd. for
C18H23NO4.0.5H2O: C, 66.24; H, 7.41; N, 4.29. Found: C,
66.42; H, 7.79; N, 4.27.
6.1.4.1. 1-[3-(Cyclopentyloxy)-4-methoxybenzoyl]-1,4,5,6-
tetrahydropyridin-2-ol (10). Yield 0.73 g, 31%, m.p. 126–
128 °C. H NMR (CDCl3) d 1.50–2.10 (m, 10H, 4 CH2-
Fig. 2. Competition curve obtained with compound 6c at the Rolipram
receptor.
1
cyclop. + CH2-piper.), 2.43 (t, J = 7.5, 2H, CH2-piper.),
3.40–3.54 (m, 3H, CH2-N-piper. + OH, 1H disappears with
D2O), 3.87 (s, 3H, OCH3), 4.80–4.91 (m, 1H, CH-cyclop.),
6.24–6.37 (m, 1H, CH-piper.), 6.84 (d, J = 8.5, 1H, Ar), 7.27
(d, J = 8.5, 1H, Ar), 7.41 (s, 1H, Ar). IR (CHCl3) cm–1 3460,
3380 (OH), 1710 (C=O), 1645 (C=C–N), 1270 (C–O). Anal.
Calcd. for: C, 64.46; H, 7.51; N, 4.18. Found: C, 64.33; H,
7.50; N, 4.17.
C17H21NO4: C, 67.31; H, 6.98; N, 4.62. Found: C, 66.94; H,
6.86; N, 4.28.
6.1.2.1. 3-(Cyclopentyloxy)-4-methoxybenzoic anhydride
(9). Yield 0.45 g, 13%, m.p. 148–149 °C, 1H NMR (CDCl3)
d 1.20–2.10 (m, 16H, 8CH2 cyclop.), 3.93 (s, 6H, 2OCH3),
4.85 (mc, 2H, 2CH), 6.92 (d, J = 8, 2H, Ar), 7.61 (s, 2H, Ar),
7.74 (d, J = 8, 2H, Ar). IR (CHCl3) cm–1 1710 (C=O), 1775
(C=O), 1265 (C–O). Anal. Calcd. for C26H30O7: C, 68.71; H,
6.65. Found: C, 68.37; H, 6.84.
6.1.5. General procedure for the preparation of 3-
(cyclopentyloxy)-4-methoxy-N-cycloamine-1-ylbenzamides
(2a–d)
6.1.3. Preparation of 1-[3-(cyclopentyloxy)-4-
methoxybenzoyl]imidazolidin-2-one (1b)
A solution of 8 (1.9 g, 7.5 mmol) in CH2Cl2 (25 ml) was
added dropwise to
a solution of the appropriate
A solution of 8 (1.9 g, 7.5 mmol) in CH2Cl2 (25 ml) was
added dropwise to a solution of 2-imidazolidinone (1.29 g,
15 mmol) in CH2Cl2 (25 ml). The reaction mixture was
stirred at 60–80 °C for 6 h and, after cooling, washed with
water (20 ml). The organic phase was dried (MgSO4) and
evaporated under reduced pressure to give an oil which
crystallized with ethyl ether. The crude solid was recrystal-
lized from ethyl acetate to give compound 1b as white pure
solid. Yield 1.0 g, 44%, m.p. 127–129 °C, 1H NMR (CDCl3)
d 1.34–2.44 (m, 8H), 3.49 (t, J = 7.2, 2H), 3.90 (s, 3H), 4.21
(t, J = 7.2, 2H), 4.60–5.00 (m, 1H), 6.32 (s, 1H, disappears
with D2O), 6.74–7.06 and 7.20–7.75 (2m, 3H). IR (CHCl3)
cm–1 3470, 1745, 1660, 1260. Anal. Calcd. for C16H20N2O4:
C, 63.14; H, 6.62; N, 9.20 Found: C,63.28; H, 6.83; N, 9.39.
N-aminocycloamines (8 mmol) and anhydrous triethylamine
(1.5 ml) in CH2Cl2 (25 ml). Only in the case of compound 2a
anhydrous triethylamine (1 ml) was added to a suspension of
1-amino-pyrrolidine hydrochloride (1.48 g, 12 mmol) in
CH2Cl2 (10 ml), the mixture was stirred at room temperature
for 15 min, next a further amount of anhydrous triethylamine
(1 ml) and a solution of 8 (2.31 g, 9 mmol) in CH2Cl2 (25 ml)
were added dropwise.
In all cases the reaction mixture was then stirred at 60–
80 °C for 6 h and, after cooling, washed with water (20 ml).
The organic phase was dried (MgSO4) and evaporated under
reduced pressure to yield white solids which were recrystal-
lized from ethyl acetate or ethyl ether/petroleum ether (1:0.5)
in the case of 2a.
6.1.4. Preparation of 1-[3-(cyclopentyloxy)-4-
methoxybenzoyl]piperidin-2-one (1c)
6.1.5.1. 3-(Cyclopentyloxy)-4-methoxy-N-pyrrolidin-1-yl-
benzamide (2a). Yield 58%; m.p. 131–132 °C. H NMR
1
A solution of 8 (1.9 g, 7.5 mmol) in CH2Cl2 (25 ml) was
added dropwise to a solution of d-valerolactame (0.8 g,
8 mmol) and anhydrous triethylamine (1.5 ml) in CH2Cl2
(25 ml). The reaction mixture was stirred at 60–80 °C for 6 h
and, after cooling, washed with water (20 ml). The organic
(CDCl3) d 1.40–2.30 (m, 12H), 2.80–3.25 (m, 4H), 3.88 (s,
3H), 4.60–5.00 (m, 1H), 6.65–7.60 (m, 4H, 1H disappears
with D2O). IR (CHCl3) cm–1 3000–2760, 1660, 1260. Anal.
Calcd. for C17H24N2O3: C, 67.08; H, 7.95; N, 9.20. Found:
C, 66.82; H, 8.04; N, 9.12.

O. Bruno et al. / IL FARMACO 59 (2004) 223–235
231
6.1.5.2. 3-(Cyclopentyloxy)-4-methoxy-N-piperidin-1-ylben-
zamide (2b). Yield 35%; m.p. 154–156 °C. 1H NMR
(CDCl3) d 1.10–2.20 (m, 14H), 2.75–3.15 (m, 4H), 3.88 (s,
3H), 4.70–5.00 (m, 1H), 6.72–7.05 and 7.25–7.65 (2m, 4H,
1H disappears with D2O). IR (CHCl3) cm–1 3460–3150,
1660, 1260. Anal. Calcd. for C18H26N2O3: C, 67.90; H, 8.23;
N, 8.80. Found: C, 67.71; H, 8.30; N, 8.80.
3.70–4.10 (m, 7H), 4.65–5.10 (m, 1H), 6.70–7.10 and 7.20–
7.80 (2m, 4H). IR (CHCl3) cm–1 2860, 1665, 1260. Anal.
Calcd. for C17H24N2O3: C, 67.08; H, 7.95; N, 9.20. Found:
C, 66.82; H, 7.78; N, 9.25.
6.1.6.4. N-{[3-(cyclopentyloxy)-4-methoxyphenyl]methyle-
ne}-4-methylpiperazin-1-amine (3d). Yield 32%, m.p. 78–
80 °C. 1H NMR (CDCl3) d 1.33–2.15 (m, 8H), 2.36 (s, 3H),
2.46–2.80 (m, 4H), 3.00–3.40 (m, 4H), 3.85 (s, 3H), 4.60–
5.05 (m, 1H), 6.70–7.10 and 7.10–7.65 (2m, 4H). IR (CHCl3)
cm–1 2840, 1660, 1260. Anal. Calcd. for C18H27N3O2: C,
68.11; H, 8.57; N, 13.00. Found: C, 68.40; H, 8.74; N, 13.32.
6.1.5.3. 3-(Cyclopentyloxy)-4-methoxy-N-morpholin-4-yl-
1
benzamide (2c). Yield 87%; m.p. 169–170 °C. H NMR
(CDCl3) d 1.60–2.10 (m, 8H), 2.80–3.11 (m, 4H), 3.80–4.05
(m, 4H), 3.89 (s, 3H), 4.60–5.00 (m, 1H), 6.70–7.10 and
7.20–7.50 (2m, 4H, 1H disappears with D2O). IR (CHCl3)
cm–1 2900–2800, 1670, 1265. Anal. Calcd. for C17H24N2O4:
C, 67.73; H, 7.55; N, 8.74. Found: C, 63.40; H, 7.72; N, 8.91.
6.1.7. General procedure for the preparation of N-[3-(cy-
clopentyloxy)-4-methoxybenzyl]cycloamin-1-amines (4a–d)
The appropriate amine (10 mmol), NaBH3CN (0.63 g,
10 mmol), ZnCl2 (0.5 g, 5 mmol) were added in succession to
a solution of 3-(cyclopentyloxy)-4-methoxybenzaldehyde
(11) (2.2 g, 10 mmol) in anhydrous tetrahydrofuran (THF)
(8 ml) and anhydrous ethanol (2 ml). Only in the case of
compound 4a also anhydrous triethylamine (1.5 ml) was
added to release 1-amino-pyrrolidine from its hydrochloride.
The reaction mixture was stirred at room temperature and
after 1 h the presence of only compounds 3a–d was detected
[TLC: CHCl3]. So, a further amount of NaBH3CN (0.3 g, 5
mmol) was added and the solution was made acidic (pH 3.8)
by adding dropwise a saturated HCl/ethanol solution (color
change of Bromocresol Green from blue to yellow). After
stirring at room temperature for 12 h the solvents were
evaporated under reduced pressure, the residue was dissolved
in water (10 ml), the water solution made alkaline with
NaOH 1 M solution and, finally, extracted three times with
CHCl3 (5 ml). The organic phases were dried (MgSO4) and
evaporated under reduced pressure to give crude oils which
were purified by flash-chromatography (Florisil® 100–
200 Mesh, CHCl3 as eluent) and distillation under high
vacuo.
6.1.5.4. 3-(Cyclopentyloxy)-4-methoxy-N-(4-methylpipera-
1
zin-1-yl)benzamide (2d). Yield 55%; m.p. 176–178 °C. H
NMR (CDCl3) d 1.10–2.15 (m, 8H), 2.32 (s, 3H), 2.45–
2.80 and 2.80–3.25 (2m, 8H), 3.90 (s, 3H), 4.60–5.10 (m,
1H), 6.70–7.10 and 7.20–7.60 (2m, 4H, 1H disappears with
D2O). IR (CHCl3) cm–1 3140–3360, 1665, 1260. Anal.
Calcd. for C18H27N3O3: C, 64.84; H, 8.16; N, 12.60. Found:
C, 64.93; H, 8.12; N, 12.79.
6.1.6. General procedure for the preparation of N-{[3-
(cyclopentyloxy)-4-methoxyphenyl]methylene}cycloamin-
1-amines (3a–d)
A
solution of 3-(cyclopentyloxy)-4-methoxybenzal-
dehyde (11), (2.2 g, 10 mmol) in anhydrous toluene (20 ml)
was added to a solution of the appropriate N-amino-
cycloamines (10 mmol) in anhydrous toluene (10 ml) and the
reaction mixture was refluxed in a Dean-Stark apparatus for
10 h. After cooling, the solvent was evaporated under re-
duced pressure to give crude solids which were purified by
flash-chromatography (Florisil®, 100–200 Mesh or Silica-
gel, CH2Cl2 as eluent) and then recrystallized from ethyl
ether.
6.1.7.1. N-[3-(cyclopentyloxy)-4-methoxybenzyl]pyrrolidin-
1-amine (4a). Yield 63%, b.p. 180/0.8 °C/mmHg. 1H NMR
(CDCl3) d 1.50–2.20 (m, 12 H), 2.65 (bs, 1H, NH, disappears
with D2O), 3.17–3.50 (m, 4H), 3.85 (s, 3H), 3.95 (s, 2H),
4.65–5.10 (m, 1H), 6.80–7.04 and 7.10–7.34 (2m, 3H). Anal.
Calcd. for C17H26N2O2: C, 70.31; H, 9.02; N, 9.65. Found:
C, 70.16; H, 9.14; N, 9.84.
6.1.6.1. N-{[3-(cyclopentyloxy)-4-methoxyphenyl]methyle-
ne}pyrrolidin-1-amine (3a). Yield 98%, m.p. 84 °C. 1H
NMR (CDCl3) d 1.40–2.20 (m, 12H), 3.10–3.50 (m, 4H),
3.84 (s, 3H), 4.63–5.10 (m, 1H), 6.70–7.40 (m, 4H). IR
(CHCl3) cm–1 2870, 1260. Anal. Calcd. for C17H24N2O2: C,
70.80; H, 8.39; N, 9.71. Found: C, 71.13; H, 8.45; N, 9.83.
6.1.6.2. N-{[3-(cyclopentyloxy)-4-methoxyphenyl]methyle-
ne}piperidin-1-amine (3b). Yield 50%, m.p. 65–67 °C. H
6.1.7.2. N-[3-(cyclopentyloxy)-4-methoxybenzyl]piperidin-
1-amine (4b). Yield 50%, b.p. 180/0.4 °C/mmHg. 1H NMR
(CDCl3) d 1.30–2.20 (m, 15H, 1H disappears with D2O),
3.10–3.50 (m, 4H), 3.82 (s, 3H), 4.28 (s, 2H), 4.70–5.00 (m,
1H) and 6.77–7.25 (m, 3H). Anal. Calcd. for
C17H28N2O2.HCl: C, 63.42; H, 8.57; N, 8.22. Found: C,
63.40; H, 8.43; N, 7.98.
1
NMR (CDCl3) d 1.10–2.20 (m, 14H), 2.90–3.30 (m, 4H),
3.84 (s, 3H), 4.65–5.05 (m, 1H), 6.60–7.10 and 7.10–7.62
(2m, 4H). IR (CHCl3) cm–1 2860, 1685, 1260. Anal. Calcd.
for C18H26N2O2: C, 71.49; H, 8.67; N, 9.26. Found: C, 71.34;
H, 8.81; N, 9.40.
6.1.6.3. N-{[3-(cyclopentyloxy)-4-methoxyphenyl]methyle-
ne}morpholin-4-amine (3c). Yield 71%, m.p. 107–108 °C.
1H NMR (CDCl3) d 1.45–2.20 (m, 8H), 3.00–3.32 (m, 4H),
6.1.7.3. N-[3-(cyclopentyloxy)-4-methoxybenzyl]morpholin
-4-amine (4c). Yield 66%, b.p. 190–200/0.4 °C/mmHg. H
NMR (CDCl3) d 1.46–2.10 (m, 8H), 2.54–2.90 (m, 4H),
1

232
O. Bruno et al. / IL FARMACO 59 (2004) 223–235
3.58–4.04 (m, 10H, 1H disappears with D2O), 4.60–4.96 (m,
1H), 6.70–7.10 and 7.15–7.65 (2m, 3H). Anal. Calcd. for
C17H26N2O3: C, 66.64; H, 8.55; N, 9.14. Found: C, 66.30; H,
8.58; N, 9.32.
3H), 8.08 (s, 1H). IR (CHCl3) cm–1 1600, 1260. Anal. Calcd.
for C19H28N2O3: C, 68.65; H, 8.49; N, 8.43. Found: C, 68.26;
H, 8.66; N, 8.70.
6.1.9.2. 3-(Cyclopentyloxy)-4-methoxybenzaldehyde O-(2-
piperidin-1-ylethyl)oxime (5b). Yield 54%, b.p. 190–
195/0.4 °C/mmHg. 1H NMR (CDCl3) d 1.10–2.15 (m, 14 H),
2.50 (t, J = 6, 4H), 2.70 (t, J = 6, 2H), 3.86 (s, 3H), 4.30 (t,
J = 6, 2H), 4.65–5.00 (m, 1H), 6.70–7.06 and 7.11–7.30 (2m,
3H), 8.05 (s, 1H). IR (CHCl3) cm–1 1600, 1260. Anal. Calcd.
for C20H30N2O3: C, 69.33; H, 8.73; N, 8.09. Found: C, 69.26;
H, 9.07; N, 8.06.
6.1.7.4. N-[3-(cyclopentyloxy)-4-methoxybenzyl]-4-methyl-
piperazin-1-amine (4d). Yield 57%, b.p. 190/0.4 °C/mmHg.
1H NMR (CDCl3) d 1.52–2.10 (m, 8H), 2.35 (s, 3H), 2.47–
2.77 (m, 7H, 1H disappears with D2O), 3.06–3.36 (m, 6H),
3.84 (s, 3H), 4.70–4.96 (m, 1H), 6.65–7.05 and 7.22–7.68
(2m, 3H). Anal. Calcd. for C18H29N3O2: C, 67.68; H, 9.15;
N, 13.15. Found: C, 67.35; H, 8.96; N, 13.00.
6.1.8. Preparation of 3-(cyclopentyloxy)-4-methoxybenzal-
dehyde oxime (12)
6.1.9.3. 3-(Cyclopentyloxy)-4-methoxybenzaldehyde O-(2-
morpholin-4-ylethyl)oxime (5c). Yield 57%, b.p. 190–
200/0.4 °C/mmHg. 1H NMR (CDCl3) d 1.59–2.11 (m, 8H),
2.51 (t, J = 4.8, 4H), 2.70 (t, J = 6, 2H), 2.72 (t, J = 4.8, 4H),
3.84 (s, 3H), 4.30 (t, J = 6, 2H), 4.68–4.98 (m, 1H), 6.83–
7.05 and 7.18–7.32 (2m, 3H), 8.05 (s, 1H). IR (CHCl3) cm–1
1600, 1260. Anal. Calcd. for C19H28N2O4: C, 65.49; H, 8.10;
N, 8.04. Found: C, 65.25; H, 8.10; N, 8.20.
A solution of hydroxylamine hydrochloride (7.5 g,
108 mmol) in water (50 ml) was added to a solution of
3-(cyclopentyloxy)-4-methoxybenzaldehyde (11) (12 g,
54 mmol) in ethanol 95% (150 ml). Then, NaHCO3 (9.1 g,
108 mmol), in small portions, and water (50 ml) in succes-
sion were added, and the mixture was stirred for 4 h at room
temperature. Additional water (50 ml) was added and the
mixture allowed to stand at –4 °C for 12 h. Finally, from the
frozen suspension a white solid crystallized, which was fil-
tered and dried at room temperature.
6.1.10. Preparation of 3-(cyclopentyloxy)-4-methoxybenzal-
dehyde O-(oxiran-2-ylmethyl)oxime (13). 3-(Cyclopen-
tyloxy)-4-methoxybenzaldehyde oxime (12) (2.36 g,
10 mmol) was added to a solution of sodium (0.24 g,
10 mmol) in anhydrous ethanol (10 ml) and the mixture was
stirred for 15 min. Then, ethanol was evaporated under re-
duced pressure, the sodium salt of 12 was dissolved in anhy-
drous DMF (10 ml) and a solution of epichlorohydrin
(1.38 g, 15 mmol) was added dropwise. The reaction mixture
was stirred at 40–50 °C for 12 h. After cooling to room
temperature the mixture was poured into water (20 ml),
extracted with CH2Cl2, dried (MgSO4) and the solvent
evaporated under reduced pressure. The pale yellow oil ob-
tained was purified by distillation in high vacuo and used
without characterization in the following reactions. Yield
2.78 g, 95%, b.p. 200–210/0.4 °C/mmHg.
1
Yield 7.87 g, 62%, m.p. 54 °C. H NMR (CDCl3) d
1.50–1.70 and 1.70–2.10 (2m, 8H), 3.87 (s, 3H), 4.75–4.85
(m, 1H), 6.84 (d, J = 8, 1H), 7.02 (dd, J = 8, J = 1, 1H), 7.20
(d, J = 1, 1H), 8.07 (s, 1H) and 8.30 (bs, 1H, disappears with
D2O). IR (CHCl3) cm–1 3580, 3480–3120, 1600, 1260. Anal.
Calcd. for C13H17NO3.H2O: C, 61.64; H, 7.56; N, 5.53.
Found: C, 62.01; H, 7.95; N, 5.64.
6.1.9. General procedure for the preparation of 3-(cyclo-
pentyloxy)-4-methoxybenzaldehyde O-(2-cycloamin-1-yle-
thyl)oximes (5a–c)
3-(Cyclopentyloxy)-4-methoxybenzaldehyde oxime (12)
(1.18 g, 5 mmol) was added to a solution of sodium (0.12 g,
5 mmol) in anhydrous ethanol (5 ml) and the mixture was
stirred for 15 min. Then, ethanol was evaporated under re-
duced pressure, the sodium salt of 12 was dissolved in anhy-
drous DMF (5 ml) and a solution of the appropriate
x-chloroethylamine, previously released from its hydrochlo-
ride, in anhydrous DMF (5 ml) was added dropwise. The
reaction mixture was stirred at 40–50 °C for 12 h. After
cooling to room temperature the mixture was poured into
water (30 ml), extracted with CHCl3 and dried (MgSO4).
After solvent evaporation under reduced pressure yellow oils
were obtained which were purified by distillation in high
vacuo.
6.1.11. General procedure for the preparation of 3-(cyclo-
pentyloxy)-4-methoxybenzaldehyde O-(2-hydroxy-3-cyclo-
amin-1-ylpropyl)oximes (6a–c). A suitable amine (10 ml)
was added to a solution of epoxide 13 (2.91 g, 10 mmol) in
anhydrous ethanol and the mixture stirred at 40–50 °C for
12 h. Then, the cooled solution was washed twice with water
(10 ml) and extracted three times with 1 M HCl solution. The
acid phases were made alkaline with 1 M NaOH solution and
extracted with CHCl3. The organic phase was dried (MgSO4)
and evaporated under reduced pressure to give yellow oils
which were purified by distillation in high vacuo. Compound
6b was characterized as hydrochloride.
6.1.9.1. 3-(Cyclopentyloxy)-4-methoxybenzaldehyde O-(2-
pyrrolidin-1-ylethyl)oxime (5a). Yield 71%, b.p. 195–
200/0.4 °C/mmHg. 1H NMR (CDCl3) d 1.40–2.16 (m, 12H),
2.60 (t, J = 8.4, 4H), 2.80 (t, J = 6, 2H), 3.84 (s, 3H), 4.30 (t,
J = 6, 2H), 4.65–5.00 (m, 1H), 6.68–7.07 and 7.12–7.36 (2m,
6.1.11.1. 3-(Cyclopentyloxy)-4-methoxybenzaldehyde O-(2-
hydroxy-3-pyrrolidin-1-ylpropyl)oxime (6a). Yield 56%, b.p.
220–225/0.4 °C/mmHg. 1H NMR (CDCl3) d 1.38–2.18 (m,
12 H), 2.28–2.88 (m, 6H), 3.76 (s, 1H, disappears with D2O),

O. Bruno et al. / IL FARMACO 59 (2004) 223–235
233
3.87 (s, 3H), 4.10–4.35 (m, 3H), 4.63–5.00 (m, 1H), 6.70–
7.05 and 7.10–7.35 (2m, 3H), 8.10 (s, 1H). IR (CHCl3)
cm–13410, 1600, 1200–1260. Anal. Calcd. for C20H30N2O4:
C, 66.27; H, 8.34; N, 7.73. Found: C, 66.21; H, 8.61; N, 8.01.
with a coverslip and analyzed under ultraviolet light in a dark
field illumination. Neutrophils with intact membrane (i.e.
viable cells) appeared as green fluorescent cells, whereas
neutrophils with damaged and ethidium bromide-permeable
membrane (i.e. necrotic cells) displayed a fluorescent red
nucleus.
6.1.11.2. 3-(Cyclopentyloxy)-4-methoxybenzaldehyde O-(2-
hydroxy-3-piperidin-1-ylpropyl)oxime (6b). Yield 68%, b.p.
225–230/0.4 °C/mmHg. 1H NMR (CDCl3) d 1.45–2.25 (m,
14 H), 3.09–3.39 (m, 4H), 3.53–3.70 (m, 2H), 3.73–4.00 (bs,
1H, disappears with D2O), 3.87 (s, 3H), 4.17–4.38 (m, 3H),
4.55 (s, 1H, H2O, disappears with D2O), 4.56–4.98 (m, 1H),
6.75–7.07 and 7.12–7.30 (2m, 3H), 8.07 (s, 1H), 10.90–
11.10 (bs, 1H, NH+, disappears with D2O). IR (CHCl3) cm–1
3500–3150, 2680–2300, 1600, 1260. Anal. Calcd. for
C21H32N2O4.HCl.0.5H2O: C, 59.78; H, 8.12; N, 6.64.
Found: C, 59.52; H, 7.80; N, 6.86.
6.2.3. Superoxide anion assay (Table 1)
The production of superoxide anion by neutrophils was
measured by the superoxide dismutase (SOD)-inhibitable
cytochrome c reduction, using a microplate reader. The assay
was carried out in 96-well, flat-bottomed, polystyrene plates
[43]. The wells were pretreated with 50 µl of fibronectin
(1 ng/well) by incubation in 5% CO2 at 37 °C for 2 h. After
three washings with normal saline, 25 µl incubation medium
containing 75 µM cytochrome c (final concentration) were
added to each well. Then, TNFa added (10 ng/ml, final
concentration) followed by 50 µl/cells (5 × 104 neutrophils).
Experiments were carried out in triplicate, in the presence
and absence of SOD (150 U/ml). Moreover, experiments
were carried out in absence and presence of Rolipram and
new synthesized compounds at concentration range of
1–300 µM. Each compound was added to wells before the
addition of TNFa. The reduction of cytochrome c was moni-
tored at intervals (5–15 min) by reading the plate at 550 nm.
The amounts of O2– produced by neutrophils were deter-
mined from the OD550 of samples without SOD minus the
OD550 of matched samples with SOD, using an extinction
coefficient e 9500 l mol–1 cm–1, calculated according to
Leslie [44].
6.1.11.3. 3-(Cyclopentyloxy)-4-methoxybenzaldehyde O-(2-
hydroxy-3-morpholin-4-ylpropyl)oxime (6c). Yield 76%,
b.p. 215–220/0.4 °C/mmHg. 1H NMR (CDCl3) d 1.50–2.10
(m, 8H), 2.40–2.80 (m, 6H), 3.55 (s, 1H, disappears with
D2O), 3.76 (t, J = 4.8, 4H), 3.88 (s, 3H), 4.10–4.35 (m, 3H),
4.70–5.00 (m, 1H), 6.85–7.05 and 7.11–7.32 (2m, 3H), 8.10
(s, 1H). IR (CHCl3) cm–1 3600–3200, 1600, 1260. Anal.
Calcd. for C20H30N2O5: C, 63.47; H, 7.99; N, 7.40. Found:
C, 63.50; H, 8.03; N, 7.65.
6.2. Biology: materials and methods
Dulbecco’s PBS and HBSS were purchased from Irvine
Scientific. Heparin was obtained from Roche, Milano, Italy.
Ficoll-Hypaque was purchased from Seromed, Berlin, Ger-
many. Fluorescein diacetate, ethidium bromide, human albu-
min, ferricytochrome c, superoxide dismutase were obtained
from Sigma-Aldrich S.r.l., Milano, Italy. Fibronectin, TNFa,
Rolipram were obtained from ICN Biomedical S.r.l., Milano,
Italy.
6.2.4. Intracellular cAMP assay (Table 2 and Fig. 1)
The intracellular cAMP accumulation was determined by
a radioimmunoassay cAMP [125I] assay system (Amersham
Pharmacia Biotech). The assay was carried out in 16 mm
diameter wells, flat-bottomed, polystyrene plates (Falcon
3047, Becton Dickinson) pretreated with fibronectin. Neu-
trophils (106 in 100 µl) were added to prewarmed incubation
medium containing 10 ng/ml TNFa (final volume 500 µl).
After 30 min incubation the reaction was stopped by boiling
the samples for 4 min.After centrifugation at 600 g for 5 min,
supernatants were stored at –80 °C until cAMP measurement
according to the manufacturer’s protocol.
6.2.1. Neutrophil isolation
Heparinized (heparin 10 U/ml) venous blood was ob-
tained from healthy volunteers after informed consent. Neu-
trophils were isolated by dextran sedimentation, subsequent
centrifugation on a density gradient and removal of contami-
nating erythrocytes by hypotonic lysis. The resulting neutro-
phils were washed three times with incubation medium and
final cell suspensions always containing 97% or more viable
cells.
6.3. Enzyme assay
All the phosphodiesterase enzyme assay were performed
by CEREP (Study Director: Prof. Dupuis P., Celle
L’Evescault, France), using the following standard proce-
dures.
In all the experiments, the reference compounds were
tested at nine concentrations in duplicate to obtain an inhibi-
tion curve in order to validate this experiment.
Results are expressed as a percent inhibition of control
activity obtained in the presence of the test compounds. IC50
values and Hill coefficient (nH) were determined for the
6.2.2. Neutrophil membrane integrity assay
Neutrophil viability measured as integrity of membrane
was assessed according Dankberg and Perdinsky [42] as
previously described. Briefly, cells (4 × 104/100 µl) harvested
from culture tubes were mixed with 50 µl of staining solution
(2 µg/ml fluorescein diacetate, 4 µg/ml ethidium bromide in
HBSS) and incubated 10 min at room temperature. Thereaf-
ter, a drop of cell suspension was placed on a slide, sealed

234
O. Bruno et al. / IL FARMACO 59 (2004) 223–235
reference compounds by non-linear regression analysis of its
inhibition curve. These parameters were obtained by Hill
equation curve fitting. The IC50 values obtained for the ref-
erence compounds are within accepted limits of historic
averages obtained 0.5 log unit.
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