Synthesis of 3,3′-(1-piperidino)substituted methylene-bis-isoxazoles preventing stimulus-induced leukocytes activation

Article abstract of DOI:10.1016/S0014-827X(02)00008-3

Some 3,3′-(1-piperidino)substituted methylene-bis-isoxazoles were prepared via Mannich base and tested to verify their antiinflammatory-related activity. Human neutrophils stimulated with either PMA and f-MLP were used as the cellular model. The efficiency of eight differently substituted compounds (2-9) was established on their capacity to reduce the O₂^- production by activated human neutrophils. The rising hydrophobicity in the side-chain of methylene-bis-isoxazoles leads to a distinction in the neutrophil response against the two stimuli, favoring the inhibition of the PMA elicited cell activation and leaving inaffected the f-MLP induced cell responses. Compounds 8 and 9 are particularly active and abolish almost completely the neutrophil activation in the presence of PMA stimulus.

Full text of DOI:10.1016/S0014-827X(02)00008-3

Il Farmaco 58 (2003) 121ꢀ127
/
www.elsevier.com/locate/farmac
Synthesis of 3,3?-(1-piperidino)substituted methylene-bis-isoxazoles
preventing stimulus-induced leukocytes activation
M. Mazzei a,*, E. Nieddu a, E. Melloni b, R. Minafra b
a Dipartimento di Scienze Farmaceutiche, Viale Benedetto XV, 3, Genoa 16132, Italy
b Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Viale Benedetto XV, 1-16132 Genoa, Italy
Received 27 May 2002; accepted 20 July 2002
Abstract
Some 3,3?-(1-piperidino)substituted methylene-bis-isoxazoles were prepared via Mannich base and tested to verify their
antiinflammatory-related activity. Human neutrophils stimulated with either PMA and f-MLP were used as the cellular model.
The efficiency of eight differently substituted compounds (2ꢀ
9) was established on their capacity to reduce the O2ꢁ production by
/
activated human neutrophils. The rising hydrophobicity in the side-chain of methylene-bis-isoxazoles leads to a distinction in the
neutrophil response against the two stimuli, favoring the inhibition of the PMA elicited cell activation and leaving inaffected the f-
MLP induced cell responses. Compounds 8 and 9 are particularly active and abolish almost completely the neutrophil activation in
the presence of PMA stimulus.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 3,3?-(1-Piperidino) substituted methylene-bis-isoxazoles; Mannich bases; Superoxide anion; Neutrophils; Ischemia; Reperfusion injuries
1. Introduction
activation of enzyme cascades leading to multiple cell
responses [8]. Activation of these kinase forms take
place following elevation of intracellular calcium, which
promotes translocation and insertion of some PKC
isozymes into the inner face of plasma membranes.
Following this peculiar step, the enzyme acquires the
active conformation by interacting with diacylglicerol
and phosphatidylserine. The quaternary complex is
required to reach the fully functional enzymes [9].
PKC activation occurs also in human neutrophils
stimulated by natural or synthetic agents. Formylated
peptides such as f-MetLeuPhe (f-MLP) are recognized
by serpentine receptors which stimulation leads to the
production of intracellular conditions causing the tran-
sient PKC translocation on the membrane and activa-
tion. The tumor promotor phorbol 12-myristate-13-
acetate (PMA) activates directly PKC isozymes sub-
stituting the diacylglicerol moiety on the enzyme produ-
cing the permanent insertion of the kinase into the
plasma membrane moiety. These differently activated
PKC isoforms may be correlated to the neutrophil
responses elicited by PMA or f-MLP. In fact, PMA is
a potent stimulus of neutrophil oxidase activation and
specific granule exocytosis, without any detectable
A number of evidence are indicating that reperfusion
of an ischemic tissue, such as heart, can cause further
cellular damages [1,2]. This myocardial reperfusion
injury [3,4] is mediated by the production of large
quantity of reactive oxygen free radicals (ROS). This
effect is supported by the reduction of infarct size and an
increased functional recovery obtained in hearts treated
with antioxidant enzymes. The presence of leukocytes in
damaged tissues increases greatly the magnitude and the
duration of ROS generation [5].
It has been suggested that leukocytes, namely neu-
trophils, mediate the reperfusion injury by chemotaxis
and activation leading to the production of ROS and the
release of proteolytic enzymes. Several studies are
indicating that a decrease in the number of neutrophils
can reflect a decrease in the infarct size [6,7].
It is well known that protein kinase C (PKC) isozymes
are involved in many cell functions, through the
* Corresponding author.
E-mail address: mazzei@cba.unige.it (M. Mazzei).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 0 0 0 8 - 3

122
M. Mazzei et al. / Il Farmaco 58 (2003) 121ꢀ127
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cytoplasmatic Ca2ꢂ flux. However, f-MLP mimics the
effects of chemotaxins released by invading bacteria,
through the binding to a G protein associated receptor.
The two pathways, although promoting similar cell
responses, utilize different mediators.
The presence of activated PKC in neutrophils is
correlated to many cell defence responses, including
promotion of ROS and secretion of granule content.
However, neutrophil activation occurring at ischemic
and inflammatory sites can favor a further progress of
the cell damage, through the secretion of lytic enzymes
and toxic ROS.
In previous works, naphthopyran-1-ones and benzo-
pyran-4-ones bearing a dialkylamine in the position
adjacent to the heteroatom were extensively tested as
potential antiinflammatory agents because their ability
to inhibit the PKC dependent signal transduction path-
way [10]. In particular, these studies showed that the
cited activity was prominent when the dialkylamino
substituent was piperidine, diethylamine or bis(2-meth-
oxyethyl)amine. In that context, we showed that some
benzopyran-derived isoxazoles bearing a piperidino
substituent in 3 were endowed with interesting antiin-
flammatory activity when tested against activated hu-
man neutrophils [11].
Following our interest in synthetic substances valu-
able in the antiinflammatory field, we now desire
amplify our study synthesizing some symmetrical meth-
ylene derivatives of 3-(1-piperidino)isoxazoles useful to
restrain the inflammatory response during ischemia/
reperfusion injuries.
hydrochloride in pyridine. Details of these three steps
are given in Section 5.
The methylene-bis-isoxazole 4 was subjected to alky-
lation by action of dimethyl- and diethylsulfate yielding
the alkyloxyderivatives 5 and 6; then, the compound 4
was treated with acetic and propionic anhydride yielding
the acyl derivatives 7 and 8. Finally, by action of 2-
(diethylamino)ethyl chloride hydrochloride in N,N-
dimethylformamide (DMF), the compound 4 was
transformed into the basic derivative 9 (Fig. 1).
All synthesized compounds (2ꢀ9) are white crystals
/
and their structures are in agreement with elemental
analyses and spectral data.
3. Experimental
Melting points (m.p.) were determined using a Elec-
trothermal apparatus and are uncorrected. Microana-
lyses were carried out on a Carlo Erba 1106 elemental
analyzer. The results of elemental analysis were within
9
/
0.3% for C and 9
/
0.1 for H and N of the theoretical
value. H NMR spectra were performed on a Hitachi
PerkinꢀElmer R 600 (60 MHz) spectrometer using TMS
as internal standard (dꢃ0). IR spectra were recorded
on a PerkinꢀElmer 398 spectrophotometer.
1
/
/
/
3.1. N,N?([2-(1-piperidinyl)-7-methoxychromon-3-
yl]methyl)piperazine (2)
To 1.04 g (4.0 mmol) of chromone 1 dissolved in 15
ml of ethanol, 0.26 g (3.0 mmol) of piperazine, 1.5 ml of
40% formaldehyde and 0.06 ml of acetic acid were
added. The resulting mixture was refluxed for 3 h. After
cooling, the precipitate was filtered off and the solid was
The synthetic pathway was already utilized in recent
works to produce unsymmetrical methylene derivatives
via Mannich bases [12ꢀ14]. Whereas in the cited papers
/
some Mannich bases of coumarins, as an example,
reacted in acetic anhydride with indoles forming un-
symmetrical derivatives, for the present purpose, a
Mannich base of a chromone was reacted with the
same chromone to yield a symmetrical methylene dimer,
which in turn was converted into an isoxazole dimer.
Thus, the preparation of symmetrical derivatives is
carried out as a particular case of a more general
pathway yielding unsymmetrical methylene derivatives.
crystallized from acetone obtaining 2; m.p.: 264ꢀ
75.6% yield.
1H NMR (DMSO-d6): d 1.54ꢀ
g-piperidine), 3.35ꢀ
/
265 8C;
/
2.00 (m, 12H, CH2 b,
/
3.78 (m, 16H, CH2 a-piperidine and
piperazine), 3.95 (s, 6H, OCH3), 4.30 (s, 4H, CH2
bridge), 6.95ꢀ
/
7.20 (m, 4H, H arom), 7.86ꢀ8.18 (d, 2H,
/
H arom). IR (KBr) cmꢁ1: 1615, 1590, 1548. Anal.
C36H44N4O6 (C, H, N).
2. Chemistry
3.2. 3,3?-Methylenbis[2-(1-piperidinyl)-7-
methoxychromone] (3)
To obtain the title compounds, we followed the
reaction pattern depicted in Fig. 1, starting from the 7-
methoxy-2-(1-piperidinyl)chromone (1).
The chromone 1 was transformed in a Mannich base
using 40% formaldehyde and piperazine. In turn, the
Mannich base 2 and the chromone 1 were treated with
acetic anhydride at 95 8C to yield the methylene-bis-
derivative 3. Then the compound 3 was converted into
methylene-bis-isoxazole 4 by action of hydroxylamine
To the solution of 1.04 g (4.0 mmol) of chromone 1 in
8 ml of acetic anhydride, 1.26 (2.0 mmol) of Mannich
base 2 were added and the mixture was heated at 95 8C
for 1.5 h. After cooling, the solution was poured onto
crushed ice and the mixture was stirred for 1ꢀ
precipitate was filtered off and the solid was crystallized
from ethyl acetate obtaining 3; m.p.: 176ꢀ177 8C; 70.5%
yield.
/
2 h. The
/

M. Mazzei et al. / Il Farmaco 58 (2003) 121ꢀ
/127
123
Fig. 1.
1H NMR (CDCl3): d 1.20ꢀ
piperidine), 2.95ꢀ3.35 (m, 8H, CH2 a-piperidine), 3.90
/
1.65 (m, 12H, CH2 b, g-
water. The crude product was crystallized from ethanol
obtaining 4; m.p.: 251ꢀ252 8C; 72.5% yield.
1H NMR (CDCl3): d 1.00ꢀ
1.70 (m, 12H, CH2 b, g-
piperidine), 2.73ꢀ3.20 (m, 8H, CH2 a-piperidine), 3.57
(s, 2H, CH2 bridge), 3.81 (s, 6H, OCH3), 6.36ꢀ6.72 (m,
/
/
(s, 6H, OCH3), 4.05 (s, 2H, CH2 bridge), 6.70ꢀ7.05 (m,
4H, H arom), 8.10 (d, 2H, H arom). IR (KBr) cmꢁ1
/
/
:
/
1605, 1547, 1499. Anal. C31H34N2O6 (C, H, N).
/
4H, H arom), 7.30 (d, 2H, H arom), 9.03 (s, 2H, OH).
IR (KBr) cmꢁ1: 3110, 1620, 1510. Anal. C31H36N4O6
(C, H, N).
3.3. 4,4?-Methylenbis[3-(1-piperidinyl)-5-(2-hydroxy-4-
methoxyphenyl)isoxazole] (4)
To a solution of 0.53 g (1.0 mmol) of 3 in 18 ml of
ethanol, 0.5 g of hydroxylamine hydrochloride and 0.7
ml of pyridine were added. The mixture was refluxed for
24 h. The final solution was evaporated under reduced
pressure to leave a pale yellow solid. The solid was
dissolved in a small amount of 2 N NaOH, filtering off
any impurity of the unreacted starting product. Then the
alkaline solution was acidified with 6 M HCl obtaining a
white precipitate which was filtered off and washed with
3.4. 4,4?-Methylenbis[3-(1-piperidinyl)-5-(2, 4-
dimethoxyphenyl)isoxazole] (5)
To 0.28 g (0.5 mmol) of methylene-bis isoxazole 4
dissolved in 3 ml of NaOH 1 N, 0.20 g of dimethylsul-
fate were added and the mixture were heated for 2 h at
70 8C. After cooling, the solution was poured onto
crushed ice and the mixture was stirred for 1ꢀ
extracted with CHCl3. The organic phase was evapo-
/2 h and

124
M. Mazzei et al. / Il Farmaco 58 (2003) 121ꢀ127
/
rated under reduced pressure leaving a solid. The solid
was crystallized from ethyl acetate yielding 5, m.p.: 190ꢀ
191 8C; 74.4% yield.
1H NMR (CDCl3): d 1.28ꢀ
piperidine), 2.78-3.16 (m, 8H, CH2 a-piperidine), 3.44 (s,
2H, CH2 bridge), 3.66 (s, 6H, OCH3-orto), 3.86 (s, 6H,
OCH3-para) 6.32-6.75 (m, 4H, H arom), 7.35 (d, 2H, H
arom). IR (KBr) cmꢁ1: 1635, 1600, 1580. Anal.
C33H40N4O6 (C, H, N).
3.8. 4,4?-Methylenbis[3-(1-piperidinyl)-5-(2-[2-N,N-
diethylaminoethoxy]-4-methoxy-phenyl)isoxazole] (9)
/
/
1.75 (m, 12H, CH2 b, g-
To 1.90 g (3.4 mmol) of methylene-bis isoxazole 4
dissolved in 19 ml of DMF, 1.75 g (10.2 mmol) of 2-
(diethylamino)ethyl chloride hydrochloride and 1.87 g
of anhydrous potassium carbonate were added and the
mixture was heated at 120 8C for 7 h. After cooling, 30
ml of water were added obtaining a precipitate that was
crystallized from diethyl ether/light petroleum ether.
The derivative 9 was obtained; m.p.: 82ꢀ
yield.
1H NMR (CDCl3): d 0.93 (t, 12H, NCH2CH3), 1.31ꢀ
1.70 (m, 12H, CH2 b, g-piperidine), 2.30ꢀ2.72 (m, 12H,
NCH2), 2.80ꢀ3.15 (m, 8H, CH2 a-piperidine), 3.30ꢀ3.96
6.75 (m, 4H,
/
83 8C: 21.7%
3.5. 4,4?-Methylenbis[3-(1-piperidinyl)-5-(2-ethoxy-4-
methoxyphenyl)isoxazole] (6)
/
/
To 0.28 g (0.5 mmol) of methylene-bis isoxazole 4
dissolved in 3 ml of NaOH 1 N, 0.25 g of diethylsulfate
were added and the mixture were heated for 3 h at
90 8C. With the same procedure as above the derivative
/
/
(m, 12H, CH2 bridge, OCH2, OCH3), 6.34ꢀ
/
H arom), 7.19 (d, 2H, H arom). IR (KBr) cmꢁ1: 1638,
1605, 1515. Anal. C43H62N6O6 (C, H, N).
6 was obtained; m.p.: 192ꢀ
1H NMR (CDCl3): d 1.05ꢀ
1.37ꢀ1.72 (m, 12H, CH2 b, g-piperidine), 2.82ꢀ
8H, CH2 a-piperidine), 3.38ꢀ3.65 (q, 4H, CH2CH3),
/
193 8C; 85.3% yield.
1.34 (t, 6H, CH2CH3),
3.20 (m,
/
/
/
4. Biology
/
3.76 (s, 2H, CH2 bridge), 3.86 (s, 6H, OCH3), 6.48ꢀ6.72
/
(m, 4H, H arom), 7.38 (d, 2H, H arom). IR (KBr) cmꢁ1
1638, 1605, 1577. Anal. C35H44N4O6 (C, H, N).
:
4.1. Isolation of neutrophils
Freshly collected heparinized human blood (100 ml)
from healthy donors was treated with 1.6% dextran
3.6. 4,4?-Methylenbis[3-(1-piperidinyl)-5-(2-acetoxy-4-
methoxyphenyl)isoxazole] (7)
(final concentration) and left at 25ꢀ28 8C to sedimentate
/
for 1 h. The supernatants (40 ml) were collected and
layered onto 10 ml of 6% Ficoll 400 solution containing
A 0.28 g quantity (0.5 mmol) of methylene-bis
isoxazole 4 was dissolved in 5 ml of acetic anhydride
and heated for 1.5 h at 95 8C. At the end, after cooling,
the solution was poured onto crushed ice and the
0.17% (v/v) Urovison and centrifuged at 800ꢄg for 20
/
min. The pellets containing mostly neutrophils and
contaminating red cells were resuspended in 10 ml of
hypotonic 0.2% NaCl. After 30 s, 10 ml of hypertonic
1.6% NaCl were added to normalize the osmotic
pressure. This treatment lyses all contaminating red
cells. The white cells were recovered and washed three
times with 0.01 M sodium phosphate (pH 7.4), 5 mM
KCl, 0.12 M NaCl, 24 mM NaHCO3, 5 mM glucose.
Prior to use, the cells were maintained in an ice bath in
mixture was stirred for 1ꢀ
solid was filtered off and crystallized from ethyl acetate
yielding 7; m.p.: 237ꢀ238 8C; 71.1% yield.
1H NMR (CDCl3): d 1.49ꢀ
1.90 (m, 12H, CH2 b, g-
piperidine), 2.37 (s, 6H, COCH3), 3.12ꢀ3.50 (m, 8H,
CH2 a-piperidine), 3.85 (s, 8H, CH2 bridge, OCH3),
/2 h obtaining a solid. The
/
/
/
the same medium at a concentration of 15ꢀ
/
20ꢄ
106 cells
/
6.56ꢀ7.10 (m, 4H, H arom), 7.80 (d, 2H, H arom). IR
/
(KBr) cmꢁ1: 1766, 1618, 1572. Anal. C35H40N4O8 (C,
H, N).
per ml. The cell population obtained consisted of more
than 96% neutrophils, as evaluated by microscopic
examination. The few remaining cells were eosinophils
and monocytes. Cell viability, evaluated by Trypan Blue
dye exclusion, resulted to be more than 97%.
3.7. 4,4?-Methylenbis[3-(1-piperidinyl)-5-(2-propyloxy-
4-methoxyphenyl)isoxazole] (8)
A 0.28 g quantity (0.5 mmol) of methylene-bis-
isoxazole 4 was dissolved in 5 ml of propionic anhy-
dride. With the same procedure as above the derivative 8
4.2. Activation of neutrophils and assay of superoxide
anion (O2ꢁ)
was obtained: m.p.: 232ꢀ
1H NMR (CDCl3): d 1.04 (t, 6H, CH2CH3), 1.42ꢀ
1.73 (m, 12H, CH2 b, g-piperidine), 2.25 (q, 4H,
COCH2), 2.95ꢀ3.25 (m, 8H, CH2 a-piperidine), 3.84
/
233 8C; 71.5% yield.
Cells (106) were diluted in 1 ml of 10 mM HEPES, pH
7.4, containing 0.15 M NaCl, 5.0 mM glucose, 1.0 mM
Ca2ꢂ and 0.625 mg mlꢁ1 of cytochrome C (Fe3ꢂ) and
incubated at 37 8C for 2 min. PMA (100 ng) or f-MLP,
(0.1 mM final concentration) were then added. Super-
oxide production was evaluated by measuring the
superoxide dismutase-inhibitable reduction of ferricyto-
/
/
(s, 8H, CH2 bridge, OCH3), 6.67ꢀ6.98 (m, 4H, H arom),
/
7.69 (d, 2H, H arom). IR (KBr) cmꢁ1: 1768, 1620, 1515.
Anal. C37H44N4O8 (C, H, N).

M. Mazzei et al. / Il Farmaco 58 (2003) 121ꢀ
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125
chrome C followed continuously at 550 nm in an
automatic recording spectrophotometer [15,16]. The
rates of O2ꢁ production (100% values) were 6.2 and
3.7 nmol per 106 cells minꢁ1 for cells activated with
PMA or f-MLP, respectively.
high molecular weight, precipitates better than mor-
pholinomethyl or piperidinomethyl derivatives from
the reaction mixture or from the crystallization
solvent (data not shown).
Following the reaction mechanism proposed for the
unsymmetrical methylene derivatives [12], the yield of
the methylene-bis-derivative 3 was increased adding
the chromone 1 to the Mannich base 2 in molar ratio
2:1. In fact, one molecule of Mannich base 2 produces
two carbocations, which in turn react with two
molecules of chromone 1.
ꢀ
/
4.3. Samples
The compounds were dissolved in DMSO at the
appropriate concentration. When tested on neutrophils,
1 ml of standard solution was added to 1 ml of cell
suspension. As the control, 1 ml of DMSO was added to
the blank sample. Each compound was tested at
concentrations of 2, 20, 200 mM. Only the data obtained
with 20 mM compound were utilized, being more
indicative of their efficiency in inhibiting neutrophil
function. To establish if these compounds can hydro-
phobically interact with the lipid stimulus PMA, cells
were incubated with 100 ng PMA and 20 mM compound
ꢀ
/
The nucleophilic attack of hydroxylamine at the
position 2 of the bis-chromone 3 produces the open-
ing of the chromone ring and the loss of a molecule of
water, leading to bis-isoxazole 4 [18].
To evaluate the antiinflammatory activity of com-
pounds bearing a double piperidine substitution, the
synthesized molecules were tested for their ability to
affect the O2ꢁ production by human neutrophils stimu-
lated with either PMA and f-MLP. The results are
reported in Table 1.
and immediately centrifuged at 2000ꢄg for 1 min.
/
Supernatans were removed and replaced with fresh
medium containing cytochrome C. The amount of O2ꢁ
produced in these conditions was not affected by the
presence of the synthesized compounds in the incubated
mixture, indicating that no subtraction of stimulus
occurred during neutrophil activation. Each determina-
tion was done in triplicate and the results were the
arithmetical mean of the three values.
From these data it is possible to draw the following
conclusions:
ꢀ
/
Compounds retaining the chromone moiety (2 and 3)
are enoughly active in inhibiting the neutrophil
activation and their action is similar to that already
found for analogous substances [11].
ꢀ
/
The methylene-bis-isoxazole 4 having free hydroxy
groups is poorly active.
5. Results
ꢀ
/
The methylene-bis-isoxazoles 5ꢀ8 with the hydroxy
/
groups bearing short alkyl and acyl substituents are
endowed with an interesting behavior. These com-
pounds differently affect the cell response elicited by
the two stimuli. Thus, the substituted bis-isoxazoles
almost completely are devoid of activity against the f-
MLP stimulus whereas they are highly efficient
against PMA stimulation, particularly if the acyl
moiety is present at the position 2?. Maximal
efficiency, in this group of compounds, is observed
In recent works we pointed out our attention at the
mechanism of the reaction between Mannich bases and
acidic hydrogen-containing compounds (in particular
chromones, coumarins and indoles) to obtain unsymme-
trical methylene derivatives [12ꢀ14]. Formerly, we had
/
shown that the formation of symmetrical derivatives
may also occur, although with poor yield, treating a
proper Mannich base with acetic anhydride without the
presence of a acidic hydrogen-containing compound
[17].
Table 1
Effects of compounds 2ꢀ
stimulated with f-MLP and PMA
Now, our results suggest that the formation of
symmetrical methylene-bis-derivatives, following the
route proposed in this paper, must be considered a
special case of the more general pattern leading to the
production of unsymmetrical methylene derivatives. In
fact, our synthetic pathway relies on these considera-
tions:
/
9 on O2ꢁ production by human neutrophils
Comp.
PMA% inhibition
f-MLP% inhibition
2
3
4
5
6
7
8
9
58
76
8
71
62
36
4
40
34
62
94
100
ꢀ
/
As the type of amine is not significant, due to the
removal of the amine in the next step, the piperazine
was preferred. Indeed such amine yields good amount
of base 2, which is manageable more easily than the
corresponding morpholinomethyl or piperidino-
methyl derivatives. In fact, compound 2, having a
2
5
3
0
Neutrophil stimulation was carried out as described in Section 4.
The concentration of each compound was 20 mM.

126
M. Mazzei et al. / Il Farmaco 58 (2003) 121ꢀ127
/
with derivative 8 substituted in 2? by the propionyl
group.
that these kinases undergo translocation to the plasma
membranes, the site at which the tested hydrophobic
compounds are accumulated.
ꢀ
/
The methylene-bis-isoxazoles 9 with the hydroxy
groups bearing a 2-(diethylamino)ethyl group shows
an 100% efficiency against PMA stimulated neutro-
phils whereas is deprived of activity against f-MLP
stimulated neutrophils.
The specificity shown by these compounds could be
attributed to the dimerization of 3-(1-piperidinyl)isox-
azoles found previously active on neutrophils stimulated
with both PMA and f-MLP. The highest degree of
molecular complexity and hydrophobicity of methylene-
bis-isoxazoles 8 and 9, as compared with previously
tested isoxazoles [11], can explain the obtained results.
Chemokines potentially released from damaged cells
or tissues, such as IL-8, can stimulate neutrophils
promoting the secretion of toxic agents, including
ROS, amplifying the injury [29]. This hypothesis is
supported by the fact that treatment with antioxidant
enzymes increases the functional recovery and decreases
the heart infarct size. Furthermore, high number of
leukocytes are present in tissue damaged by ischemia
and reperfusion.
6. Discussion
It has been established that polymorphonuclear
(PMN) leukocytes play a primary role in the setting of
myocardial injury due to ischemia/reperfusion, contri-
buting to microvascular plugging, cardiac contractile
dysfunction, and enhanced cardiomyocyte necrosis [19ꢀ
/
21]. On reperfusion, PMN leukocytes accumulate in the
coronary microvasculature and infiltrate into cardiac
tissue [20,22]. These transmigrated PMN leukocytes can
provoke tissue injury by the release of cytotoxic
substances, including ROS, chemotactic factors and
proteolytic enzymes [20,23]. These activated cells can
contributed also to endothelial dysfunction [24]. There-
fore, it is likely that compounds inhibiting in one site
PMN leukocytes adherence to the vascular endothelium
[25], and on the other site production of ROS and
secretion of toxic agents by activated leukocytes, can
exert tissue-protective effects against the PMN leuko-
cyte induced-injury.
In our experimental design, we have considered the
possibility to counteract neutrophil activation with
specific bis-isoxazoles derivatives as a tool to protect
tissues from further cellular damages. Neutrophil acti-
vation was induced with two different stimuli (PMA and
f-MLP) in order to better classify the action of the
synthesized compounds.
The present results could be interesting in designing
new agents endowed with specific activity against a
particular step in the complex mechanism leading to
neutrophil activation in damaged tissues.
Acknowledgements
This work was supported by grants from Italian
MIUR.
References
[1] E. Braunwald, R.A. Kloner, Myocardial reperfusion: a double-
edged sword, J. Clin. Invest. 76 (1985) 1713ꢀ
[2] M.A. DeWood, R. Berg, The role of surgical reperfusion in
myocardial infarction, Cardiol. Clin. 2 (1984) 113ꢀ122.
/1719.
The chain extension in the bis-isoxazoles 5ꢀ9 is clearly
/
/
addressed in the distinction of the response against the
two stimuli. In fact, whereas the tested bis-isoxazoles
seem do not affect the neutrophil activation induced by
f-MLP, the cell response elicited by PMA is highly
affected. Such biological activity increases from com-
pound 5 to 9, compounds 8 and 9 not only are those
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