N,N-Dialkylaminosubstituted chromones and isoxazoles as potential anti-inflammatory agents

Article abstract of DOI:10.1016/S0014-827X(99)00051-8

The ability of some N,N-dialkylaminosubstituted chromones and isoxazoles to inhibit the protein kinase C (PKC) dependent signal transduction pathway was tested. As a cellular model, human neutrophils stimulated with either phorbol myristate acetate (PMA)

Full text of DOI:10.1016/S0014-827X(99)00051-8

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 452–460
N,N-Dialkylaminosubstituted chromones and isoxazoles as
potential anti-inflammatory agents
Mauro Mazzei a,*, Enzo Sottofattori a, Ramona Dondero a, Munjed Ibrahim a,
Edon Melloni b, Mauro Michetti b
a Dipartimento di Scienze Farmaceutiche, Viale Benedetto XV, 3-16132 Genoa, Italy
b Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Viale Benedetto XV, 1-16132 Genoa, Italy
Accepted 23 April 1999
Abstract
The ability of some N,N-dialkylaminosubstituted chromones and isoxazoles to inhibit the protein kinase C (PKC) dependent
signal transduction pathway was tested. As a cellular model, human neutrophils stimulated with either phorbol myristate acetate
(PMA) or formylmethionine–leucine–phenylalanine (f-MLF) were used. The efficiency of the compounds was established by their
capacity to reduce the O2− production by activated human neutrophils. Compounds carrying a 3-bis(2-methoxyethyl)amino group,
a substituent found active in previously tested tricyclic compounds, do not show significant anti-PKC activity in this study. On
the other hand, substitution with a 1-piperidinyl group leads all tested compounds to a high biological activity against stimulated
neutrophils. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: Anti-PKC activity; Superoxide anion; Neutrophils; 1-Benzopyran-4-ones; 5-Phenylisoxazoles; Anti-inflammatory agents
pare several 3-(dialkylamino)phenylisoxazoles (C)
which exhibited specific anti-rhinovirus activity [11,12].
1. Introduction
Previous work has shown that tricyclic compounds,
such as 3-(dialkylamino)naphtho[2,1-b]pyran-1-ones
(A), and bicyclic compounds, such as 2-(dialkylamino)-
chromones (B), are interesting heterocycles endowed
with various pharmacological activities. In fact, it was
pointed out that by variation of these substituents,
anti-depressive [1,2], anti-allergic [3,4] and anti-platelet
[5–8] activities could be observed. In addition, com-
pounds derived from the above-mentioned benzo- and
naphthopyrans retain interesting pharmacological
properties. In this connection, some Mannich bases
were prepared from parent compounds in order to
modify their original biological activity on the CNS [9].
Some heteropolycycles, obtained from compounds B by
nucleophilic displacement of the dialkylamine, also pos-
sessed significant anti-allergic properties [10]. Further-
more, by opening the chromone ring with
hydroxylamine hydrochloride, it was possible to pre-
Due to the fact that many of the postulated biologi-
cal activities of these compounds are confined to the
membrane level and probably affect signal transduction
pathways, we have analysed in detail these properties
using human neutrophils stimulated with either phorbol
myristate acetate (PMA) or formylmethionine–
leucine–phenylalanine (f-MLF), as a cell model.
It is well known that these external stimuli induce a
cell response through a protein kinase C (PKC)-depen-
dent enzyme cascade. PKC is the collective name of a
number of isozymes [13], most of which are localised in
the cytosol fraction of the resting cells in inactive
enzyme forms. Activation is accomplished by transloca-
tion of the kinase from the soluble fraction to the inner
surface of plasma membranes. The signal triggering the
change in PKC distribution is an increase in the intra-
* Corresponding author. Tel.: +39-010-353 8371.
E-mail address: mazzei@ermes.cba.unige.it (M. Mazzei)
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00051-8

M. Mazzei et al. / Il Farmaco 54 (1999) 452–460
453
cellular concentration of free Ca2+, normally induced
by the second messenger IP3 produced by G-protein
activated phospholipase C. In the membrane-associated
form, the calcium/PKC complex interacts with phos-
pholipids and diacylglycerol, forming the active enzyme
form and inducing a phosphorylation cascade, as cellu-
lar response. Activation of PKC has been reported as a
limiting step in many biochemical processes, such as
proliferation, differentiation, secretion, etc. [14].
In neutrophils, active PKC phosphorilates a mem-
brane associated NADPH oxidase component, inducing
reorganisation of the enzyme which acquires the active
conformation. Detection of O2− produced by NADPH
oxidase in a neutrophil suspension is in turn an indica-
tion of PKC activation. Consequently, the rate of the
reaction catalysed by NADPH oxidase can be used to
evaluate the efficiency of the intracellular phosphorylat-
ing capacity of PKC.
Scheme 1.
general to a single product [1–3,6]. In our case, the
expected benzopyran-4-one is accompanied with by-
products generated from the secondary reaction of
phosphorus oxychloride on the methoxy groups, as
shown by silica gel TLC (ethanol/ethyl acetate 1:1) of
the crude product in which there are three spots. Then,
the recovery of 2-[bis(2-methoxyethyl)amino]-7-meth-
oxychromone (3a) is subjected to column chromatogra-
phy to separate the monochloro derivative 3b and the
dichloro derivative 3c. To minimise the formation of 3b
and 3c, the amount of phosphorus oxychloride is low-
ered with respect to the amount used in the Refs.
[1–3,6].
Using 3-ethoxyphenol (1b) as the starting product, the
reaction pattern was similar: in fact, the TLC of the final
mixture presented three spots in the same sequence as
the previous reaction but the higher spot, attributable to
the dichloro derivative, was in this case present only in
trace amounts. Consequently, it was possible to isolate,
by column chromatography, only the products which
were quantitatively more abundant, namely the
dimethoxy derivative 3d and the monochloro derivative
3e.
Under specific conditions, O2− radicals are believed to
participate in the generation of tissue damage, charac-
teristic of inflammatory states [15]. To prevent these
unfavourable events and to reduce the negative effects
of O2− radicals in target tissues, the inhibition of the
limiting step in its production must be obtained.
In this regard, compounds in which the anti-inflam-
matory activity is due to the inhibition of PKC activa-
tion are extensively searched. Until now, the lead
compounds in this type of action have been stau-
rosporine and its derivatives [16]. Recently, we have
obtained evidence that some naphtho[2,1-b]pyran
derivatives displayed PKC inhibitory activity. Particu-
larly, maximal activity was obtained when the dialkyl-
amine substituent was bis(2-methoxyethyl)amine,
though diethylamine and piperidine also had a relevant
action [17].
Following our interest on synthetic substances useful
as anti-inflammatory agents, we have now analysed the
PKC inhibitory activity of some 2-(dialkylamino)-
chromones and 3-(dialkylamino)isoxazoles, pointing
our attention to a new series of chromones and isoxa-
zoles bearing the bis(2-methoxyethyl)amine or other
amines derived from it; in fact, we are interested in
testing whether the relevance of this amino group in
lowering the neutrophil activation is peculiar of tricyclic
systems or may be extended to mono- or bicyclic
systems.
Compound 3a was selected to obtain some modified
chromones and to transform chromones into isoxazole
derivatives.
When compound 3a was submitted to the action of
57% hydriodic acid, it was possible to obtain different
products depending on the reaction temperature (see
Scheme 2). In fact, when mild conditions are used
(90°C) it is possible to recover a compound (4a) only
2. Chemistry
As depicted in the Scheme 1, the reaction between
3-methoxyphenol (1a) and the malonamic acid ethyl
ester 2 in the presence of phosphorus oxychloride in
1,2-dichloroethane (DCE) gives rise to the formation of
the mixture of benzopyrans 3a–c. The reaction condi-
tions have already been extensively studied and lead in
Scheme 2.

454
M. Mazzei et al. / Il Farmaco 54 (1999) 452–460
3. Experimental
Melting points were determined using a Fisher–
Johns apparatus and are uncorrected. Microanalyses
were carried out on a Carlo Erba 1106 elemental analy-
ser. The results of elemental analysis were within
90.3% for C and 90.1 for H and N of the theoret-
ical values. 1H NMR spectra were performed on a
Hitachi Perkin–Elmer R 600 (60 MHz) spectro-
meter using TMS as internal standard (l=0). IR
spectra were recorded on a Perkin–Elmer 398 spectro-
photometer.
Scheme 3.
demethylated in the aliphatic chain; on the other hand,
when the reaction is refluxed, the demethylation also
occurs in the aromatic moiety whereas, as previously
found in a similar case [18], the aliphatic hydroxy
groups are substituted by iodine to yield 4b. Then, by
treating 3a with morpholine in the presence of 40%
formaldehyde, it is possible to recover the correspond-
ing Mannich base 5 in good yield. As is known for
these dialkylaminopyrans, the substitution involves po-
sition-3 [9,19]. Other substitutions occur only if the
benzene ring bears a hydroxy group [19].
When the chromone 3a was treated with hydroxyl-
amine hydrochloride in ethanol and pyridine, nucleo-
philic attack of the hydroxylamine leads to the opening
of the chromone ring and subsequently to the forma-
tion of an isoxazole ring [11,12].
3.1. 2-bis(2-Methoxyethyl)amino-7-methoxy-4H-benzo-
pyran-4-one (3a), 2-(2-chloroethyl-2-methoxyethyl)-
amino-7-methoxy-4H-benzopyran-4-one (3b) and 2-bis-
(2-chloroethyl)amino-7-methoxy-4H-benzopyran-4-one
(3c)
In an ice cooled flask, protected from moisture with
a calcium chloride drying tube, 7.0 ml (76.5 mmol) of
phosphorus oxychloride were added dropwise under
stirring to 13.6 g (55.0 mmol) of 3-bis(2-methoxy-
ethyl)amino-3-oxo-propanoic acid ethyl ester 1 [18].
After the addition, the mixture was removed from the
ice bath and maintained at room temperature for 0.5 h.
To the resulting yellow mixture, a solution of 6.2 g
(50.0 mmol) of 6-methoxyphenol in 40 ml of DCE was
added slowly under stirring. The reaction mixture was
then heated for 5 h at reflux. After cooling, a solution
of 68 g of sodium acetate trihydrate in 200 ml of water
was added and the mixture was then heated for 1.5 h at
70°C. After cooling, the organic phase was discarded
and the aqueous one was extracted several times with
chloroform. The pooled organic extracts were washed
with water, dried and evaporated under reduced pres-
sure to give a dark red oil. The oil was stirred at
room temperature for 2 h with 200 ml of 2 N NaOH
and 50 ml of light petroleum ether. The resulting solid
was filtered and washed with water. The solid was
chromatographed on a silica gel column with ethyl
acetate as eluent. In the first 200 ml of eluent the
dichloro derivative 3c was recovered; then in the second
amount of eluent (200 ml) the derivative 3b was recov-
ered; finally, changing the solvent to ethyl acetate/
ethanol (1:1) the dimethoxy derivative 3a was
recovered. The three compounds were crystallised from
ethyl acetate obtaining 3a (m.p. 95–96°C, 34% yield),
3b (m.p. 110–111°C, 22% yield) and 3c (m.p. 138–
139°C, 14% yield).
In turn, the isoxazole 6 was easily demethylated by
means of 57% hydriodic acid in mild conditions to give
7 (see Scheme 3).
The isoxazole 6 was treated with some alkylating and
acylating reagents. The use of dimethyl and diethylsul-
fate in alkaline medium yielded 8a and 8b, respectively.
To obtain a longer aliphatic chain (8c), 1-bromohexane
was used in the presence of acetone and anhydrous
potassium carbonate. The esters 9a–c were prepared by
treating 6 with suitable acyl chlorides in anhydrous
pyridine (see Scheme 4). All esterification reactions
were performed at 100°C for a few minutes. Yields
ranged between 90 and 95%.
All compounds described herein are white crystals
whose structures are in agreement with elemental analy-
ses and spectral data (see Section 3).
1
3a: H NMR (CDCl3), l: 3.28 (s, 6H, CH2OCH3),
3.55 (near s, 8H, CH2), 3.77 (s, 3H, 7-OCH3), 5.29 (s,
1H, H-3), 6.58–6.95 (m, 2H, H-6, 8), 7.96 (d, 1H, H-5).
IR (KBr)
w
(cm−1): 1610, 1590, 1550. Anal.
Scheme 4.
C16H21NO5: C, H, N.

M. Mazzei et al. / Il Farmaco 54 (1999) 452–460
455
1
3b: H NMR (CDCl3), l: 3.30 (s, 3H, CH2OCH3),
(KBr) w (cm−1): 3400 (broad), 1610, 1580, 1545. Anal.
C14H17NO5: C, H, N.
3.55 (s, 3H, CH2OCH3), 3.55 (near s, 4H, NCH2CH2O),
3.70 (near s, 4H, NCH2CH2Cl), 3.79 (s, 3H, 7-OCH3),
5.28 (s, 1H, H-3), 6.60–6.98 (m, 2H, H-6, 8), 7.98 (d,
1H, H-5). IR (KBr) w (cm−1): 1612, 1592, 1555. Anal.
C15H18ClNO4: C, H, N.
4b: 1H NMR (DMSO-d6), l: 3.35–4.10 (m, 8H,
CH2), 5.49 (s, 1H, H-3), 6.60–6.98 (m, 2H, H-6, 8), 7.75
(d, 1H, H-5), 11.30 (s, 1H, OH). IR (KBr) w (cm−1):
1619, 1600, 1565. Anal. C13H13I2NO3: C, H, N.
1
3c: H NMR (CDCl3), l: 3.70–4.10 (m, 11H, CH2,
CH3), 5.38 (s, 1H, H-3), 6.68–7.07 (m, 2H, H-6, 8), 8.06
(d, 1H, H-5). IR (KBr) w (cm−1): 1612, 1594, 1555.
Anal. C14H15Cl2NO3: C, H, N.
3.4. 2-bis(2-Methoxyethyl)amino-7-methoxy-4-morpho-
linomethyl-4H-benzopyran-4-one (5)
To 0.82 g (2.68 mmol) of 3a dissolved in 15 ml of
ethanol, 0.80 g (9.2 mmol) of morpholine, 1.28 ml (17.0
mmol) of 40% formaldehyde and 0.26 ml (4.6 mmol) of
acetic acid were added. The resulting mixture was
refluxed for 24 h. After cooling, the solvent was evapo-
rated under reduced pressure and the obtained pale
yellow oil was chromatographed through a silica gel
column using ethyl acetate/cyclohexane (2:1) as eluent.
After discarding the first 100 ml of eluate, a second
fraction of 70 ml was collected. The removal of the
solvent left a white solid (0.63 g, 55.7% yield) which,
after recrystallisation from ethyl acetate, yielded 5 (m.p.
87–88°C).
3.2. 2-bis(2-Methoxyethyl)amino-7-ethoxy-4H-benzo-
pyran-4-one (3d) and 2-(2-chloroethyl-2-methoxyethyl)-
amino-7-ethoxy-4H-benzopyran-4-one (3e)
Following the procedure described for 3a–c, but
using 6.9 g (50.0 mmol) of 3-ethoxyphenol, a solid was
obtained which was chromatographed on a silica gel
column with ethyl acetate as eluent. In the first 300 ml
of eluent the monochloro derivative 3e was recovered;
then, on changing the solvent to ethyl acetate/ethanol
(1:1), the dimethoxy derivative 3d was recovered. The
two compounds were crystallised from ethyl acetate
obtaining 3d (m.p. 125–126°C, 23% yield) and 3e (m.p.
122–123°C, 18% yield).
1H NMR (CDCl3), l: 2.37–2.70 (m, 4H, b-morpho-
line CH2), 3.39 (s, 6H, CH2OCH3), 3.42–3.80 (m, 12H,
a-morpholine CH2+NCH2CH2OCH3), 3.91 (s, 3H, 7-
OCH3), 4.02 (s, 2H, CH2 bridge), 6.73–7.12 (m, 2H,
H-6, 8), 8.13 (d, 1H, H-5). IR (KBr) w (cm−1): 1615,
1590, 1545. Anal. C21H30N2O6: C, H, N.
1
3d: H NMR (CDCl3), l: 1.30 (t, 3H, CH2CH3), 3.26
(s, 6H, CH2OCH3), 3.53 (near s, 8H, CH2), 3.97 (q, 2H,
OCH2), 5.26 (s, 1H, H-3), 6.56–6.91 (m, 2H, H-6, 8),
7.95 (d, 1H, H-5). IR (KBr) w (cm−1): 1610, 1590, 1545.
Anal. C17H23NO5: C, H, N.
1
3e: H NMR (CDCl3), l: 1.45 (t, 3H, CH2CH3), 3.38
(s, 3H, OCH3), 3.65 (near s, 4H, NCH2CH2O), 3.80
(near s, 4H, NCH2CH2Cl), 4.15 (q, 2H, OCH2CH3),
5.42 (s, 1H, H-3), 6.68–7.10 (m, 2H, H-6, 8), 8.09 (d,
1H, H-5). IR (KBr) w (cm−1): 1610, 1590, 1550. Anal.
C16H20ClNO4: C, H, N.
3.5. 3-bis(2-Methoxyethyl)amino-5-(2-hydroxy-4-
methoxyphenyl)isoxazole (6)
A total of 1 g of hydroxylamine hydrochloride and
1.5 ml of pyridine were added to a solution of 1.2 g
(3.90 mmol) of 3a in 40 ml of ethanol. The mixture was
refluxed for 24 h. The final solution was evaporated
under reduced pressure to yield a solid. The latter was
dissolved in a little amount of 2 N NaOH, filtering off
any impurity or unreacted starting product. The alka-
line solution was then acidified with 6 N HCl obtaining
a white precipitate, which was filtered and washed with
water. Compound 6 was obtained after recrystallisation
from ethanol (m.p. 151–152°C, 75.2% yield).
3.3. 3-bis(2-Hydroxyethyl)amino-7-methoxy-4H-benzo-
pyran-4-one (4a) and 3-bis(2-iodoethyl)amino-7-
hydroxy-4H-benzopyran-4-one (4b)
A solution containing 1 g of 2-bis(2-methoxyethyl)-
amino-7-methoxy-4H-benzopyran-4-one (3a) in 15 ml
of 57% HI was heated at 95°C for 0.5 h. After cooling,
the final mixture was treated with a saturated solution
of NaHCO3 until neutrality and the obtained precipi-
tate was filtered, washed with water and recrystallised
from ethanol; the dihydroxy derivative 4a was obtained
(m.p. 178–179°C, 25% yield). The neutral aqueous
solution was acidified with hydrochloric acid (1:1) ob-
taining a second precipitate, which was crystallised
from ethanol, obtaining the diiodo derivative 4b (m.p.
257–258°C, 18% yield).
1H NMR (CDCl3), l: 3.36 (s, 6H, CH2OCH3), 3.61
(m, 8H, CH2), 3.80 (s, 3H, 4%-OCH3), 6.30 (s, 1H, H-4),
6.40–6.72 (m, 2H, H-3%, 5%), 7.65 (d, 1H, H-6%), 8.00
(broad s, 1H, OH). IR (KBr) w (cm−1): 2920 (broad),
1620, 1590, 1546. Anal. C16H22N2O5: C, H, N.
3.6. 3-bis(2-Hydroxyethyl)amino-5-(2-hydroxy-4-
methoxyphenyl)isoxazole (7)
1
4a: H NMR (DMSO-d6), l: 3.81 (m, 8H, CH2), 3.97
(s, 3H, OCH3), 5.68 (broad s, 2H, OH), 6.22 (s, 1H,
H-3), 7.06–7.55 (m, 2H, H-6,8), 7.97 (d, 1H, H-5). IR
A solution of 1 g (3.10 mmol) of 3-bis(2-methoxy-
ethyl)amino-5-(2-hydroxy-4-methoxyphenyl)-isoxazole

456
M. Mazzei et al. / Il Farmaco 54 (1999) 452–460
(6) in 15 ml of 57% HI was heated for 0.5 h at 90°C.
After cooling, the final mixture was treated with a
saturated solution of NaHCO3 until neutrality and the
obtained precipitate was filtered, washed with water
and recrystallised from ethanol/ethyl acetate (m.p. 167–
168°C, 65.0% yield).
the solvent was distilled under reduced pressure and the
pale yellow oil was stirred with 40 ml of 2 N sodium
hydroxide. The solid (8c) which separated out was
filtered and recrystallised from cyclohexane (m.p. 57–
58°C, 86.2% yield).
1H NMR (CDCl3), l: 0.95 (t, 3H, CH2CH3), 1.15–
2.12 (m, 8H, (CH2)4CH3), 3.37 (s, 6H, CH2OCH3), 3.60
(m, 8H, NCH2CH2O), 3.85 (s, 3H, 4%-OCH3), 4.10 (t,
2H, OCH2), 6.40 (s, 1H, H-4), 6.49–6.78 (m, 2H, H-3%,
5%), 7.90 (d, 1H, H-6%). IR (KBr) w (cm−1): 1622, 1572,
1535. Anal. C22H34N2O5: C, H, N.
1H NMR (CDCl3), l: 3.25–3.70 (m, 8H, CH2), 3.81
(s, 3H, OCH6), 4.07 (broad s, 2H, CH2OH), 6.30–6.75
(m, 3H, H-4, H-3%–5%), 7.68 (d, 1H, H-6%), 10.52 (broad
s, 1H, OH). IR (KBr) w (cm−1): 3300 (broad), 1615,
1595, 1535. Anal. C14H18N2O5: C, H, N.
3.7. 3-bis(2-Methoxyethyl)amino-5-(2,4-dimethoxy-
phenyl)isoxazole (8a)
3.10. 3-bis(2-Methoxyethyl)amino-5-(2-m-nitrobenzoyl-
oxy-4-methoxy)isoxazole (9a)
To 1 g (3.10 mmol) of 6 dissolved in 20 ml of water
containing 0.17 g (3.1 mmol) of KOH, 0.39 g (3.1
mmol) of dimethyl sulfate was added dropwise under
stirring. The solution was stirred for 2 h at 40°C. The
resulting oil was extracted three times with chloroform.
The pooled organic extracts were washed with water,
dried and evaporated under reduced pressure. The re-
sulting solid was recrystallised from ethyl acetate/cyclo-
hexane (1:1) (m.p. 73–75°C, 84.8% yield).
To a solution of 0.60 g (1.86 mmol) of 6 in 4 ml of
anhydrous pyridine, 0.4 g (2.28 mmol) of 3-nitroben-
zoyl chloride were added and the mixture was heated at
100°C for 7 min under stirring. The final solution was
then poured into 15 ml of water and the mixture was
stirred for 30 min, obtaining an oil which was extracted
many times with chloroform. The pooled organic ex-
tracts were washed with water, dried and evaporated
under reduced pressure. The resulting solid was crys-
tallised from ethyl acetate/cyclohexane (1:3) obtaining
9a (m.p. 86–87°C, 88.3% yield).
1H NMR (CDCl3), l: 3.37 (s, 6H, CH2OCH3), 3.60
(m, 8H, CH2), 3.85 (s, 3H, OCH3), 3.92 (s, 3H, OCH3),
6.32 (s, 1H, H-4), 6.43–6.76 (m, 2H, H-3%, 5%), 7.84
(d, 1H, H-6%). IR (KBr) w (cm−1): 1620, 1580, 1545.
Anal. C17H24N2O5: C, H, N.
1H NMR (CDCl3), l: 3.24 (s, 6H, CH2OCH3), 3.42
(m, 8H, CH2), 3.89 (s, 3H, 4%-OCH3), 6.07 (s, 1H, H-4),
6.85–9.22 (m, 7H, H arom.). IR (KBr) w (cm−1): 1745,
1620, 1604. Anal. C23H25N3O8: C, H, N.
3.8. 3-bis(2-Methoxyethyl)amino-5-(2-ethoxy-4-
methoxyphenyl)isoxazole (8b)
3.11. 3-bis(2-Methoxyethyl)amino-5-(2-p-nitrobenzoyl-
oxy-4-methoxy)isoxazole (9b)
To 1 g (3.10 mmol) of 6 dissolved in 20 ml of water
containing 0.17 g (3.1 mmol) of KOH, 0.48 g (3.1
mmol) of diethyl sulfate was added dropwise under
stirring. The solution was stirred for 2 h at 75°C. The
resulting oil was extracted three times with chloroform.
The pooled organic extracts were washed with water,
dried and evaporated under reduced pressure. The re-
sulting solid was recrystallised from ethyl acetate/cyclo-
hexane (1:1) (m.p. 54–55°C, 78.6% yield).
Following the procedure described for 9a but using
0.4 g of p-nitrobenzoyl chloride, a solid was obtained
which was crystallised from ethyl acetate/cyclohexane
(1:3) obtaining 9b (m.p. 74–75°C, 86.0% yield).
1H NMR (CDCl3), l: 3.25 (s, 6H, CH2OCH3), 3.44
(m, 8H, CH2), 3.89 (s, 3H, 4%-OCH3), 6.08 (s, 1H, H-4),
6.85–9.24 (m, 7H, H arom.). IR (KBr) w (cm−1): 1750,
1623, 1604. Anal. C23H25N3O8: C, H, N.
1H NMR (CDCl3), l: 1.43 (t, 3H, CH2CH3), 3.30
(s, 6H, CH2OCH3), 3.51 (m, 8H, CH2), 3.77 (s, 3H,
4%-OCH3), 4.10 (q, 2H, CH2CH3), 6.31 (s, 1H, H-4),
6.40–6.68 (m, 2H, H-3%, 5%), 7.80 (d, 1H, H-6%). IR
(KBr) w (cm−1): 1620, 1578, 1546. Anal. C18H26N2O5:
C, H, N.
3.12. 3-bis(2-Methoxyethyl)amino-5-(2-p-
chlorobenzoyloxy-4-methoxy)isoxazole (9c)
Following the procedure described for 9a but using
0.4 g of p-chlorobenzoyl chloride, a solid was obtained
which was crystallised from ethyl acetate cyclohexane
(1:3) obtaining 9c (m.p. 96–97°C, 86.7% yield).
1H NMR (CDCl3), l: 3.24 (s, 6H, CH2OCH3), 3.40
(m, 8H, CH2), 3.86 (s, 3H, 4%-OCH3), 6.02 (s, 1H, H-4),
6.82–8.45 (m, 7H, H arom.). IR (KBr) w (cm−1): 1746,
1621, 1602. Anal. C23H25ClN2O6: C, H, N.
3.9. 3-bis(2-Methoxyethyl)amino-5-(2-n-hexyloxy-4-
methoxyphenyl)isoxazole (8c)
To 1.2 g (3.72 mmol) of 6 dissolved in 30 ml of
acetone, 0.43 g (7.27 mmol) of 1-bromohexane and 0.27
g of anhydrous potassium carbonate were added with
stirring. The mixture was refluxed for 24 h. At the end

M. Mazzei et al. / Il Farmaco 54 (1999) 452–460
457
Table 1
4. Biology
Effect of the chromones B1–12 on O−2 production by human neu-
trophils stimulated with f-MLF or PMA a
4.1. Isolation of neutrophils
Freshly collected heparinised human blood (100 ml)
from healthy donors was treated with 1.6% dextran
(final concentration) and left at 25–28°C to sedimen-
tate for 1 h. The supernatants (40 ml) were collected
and layered onto 10 ml of 6% Ficoll 400 solution
containing 0.17% (v/v) Urovison and centrifuged at
800×g for 20 min. The pellets containing mostly neu-
trophils 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 normalise the
osmotic pressure. This treatment lyses all contaminat-
ing 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
and 5 mM glucose. Prior to use, the cells were main-
tained in an ice bath in the same medium at a concen-
tration of 15–20×106 cells/ml. The cell population
obtained consisted of more than 96% neutrophils, as
evaluated by microscopic examination. The remaining
percentages consisted of eosinophils and monocytes.
Comp.
NR2
R%
f-MLF
PMA
B1
B2
B3
B4
B5
B6
B7
B8
NMe2
NMe2
NHEt
NEt2
NEt2
NEt2
NEtAc
NEtAr
1-pyrrolidinyl
1-pyrrolidinyl
1-piperidinyl
1-piperidinyl
H
CH3
H
H
CH3
CH2CH2CH3
CH3
CH3
H
CH3
H
2
3
20
26
5
4
48
3
35
44
39
18
25
38
43
34
40
66
2
100
29
37
100
100
B9
B10
B11
B12
CH3
a Neutrophils (106 cells/ml) were stimulated with 100 ng/ml PMA
or with 0.1 mmol f-MLF, as described in Section 4, in the presence of
the incubated compounds at the conc. of 20 mmol. Data refer to the
% inhibition of O−2 production with respect to controls. The assays
were carried out in triplicate. Values are the arithmetical mean of
three determinations.
4.2. Acti6ation of neutrophils and assay of a superoxide
−
anion (O2
)
ability to affect the O2− production by human neu-
trophils stimulated with either PMA or f-MLF.
As shown in Table 1, in the absence of substituents,
the chromones express very low inhibitory activity on
O2− production. However, the substitution of methyl
residue (B5) with propyl residue (B6) promotes a sig-
nificant increase in inhibition of O2− production by
neutrophils stimulated with f-MLF. In this context,
the activity is very high when an aromatic substituent
is present on the amino group (B7) but unfortunately
there is only one such substitution in our series. Fur-
thermore, chromones having a pyrrolidino residue at
the NR2 position (B9, B10) show good inhibitory effi-
ciency on neutrophils stimulated with both PMA and
f-MLF.
A total of 106 cells were diluted in 1 ml of 10 mM
HEPES, pH 7.4, containing 0.15 M NaCl, 5.0 mM
glucose, 1.0 mM Ca2+ and 0.625 mg/ml of Cy-
tochrome C (Fe3+) and incubated at 37°C for 2 min.
PMA (100 mg) or f-MLF (0.1 mM final concentration)
were then added. The absorbance at 550 nm was
continuously monitored for 10 min. The amount of
O2− produced was calculated by the difference in
absorbance at zero time and at the end of the reac-
tion.
4.3. Samples
The compounds were diluted in DMSO at standard
concentration of 20 mM. When tested on neutrophils,
1 ml of standard solution was added to 1 ml of cell
suspension. As a control, 1 ml of DMSO was added to
a blank sample.
The introduction of a piperidino group (B11, B12)
largely increases the inhibitory effect of f-MLF stimu-
lated neutrophils, without affecting PMA-stimulated
neutrophils.
Taken together, these findings indicate that an in-
crease in hydrophobicity of the substituents is corre-
lated with the ability of the compounds to inhibit
neutrophil activation. In fact, replacement of dimethyl-
amino (B1, B2) with diethylamino groups (B4–6) pro-
motes the appearance of biological activity of the
compounds, which becomes maximal when the substi-
tution is carried out with the N-arylamino and pipe-
ridino groups.
5. Results and discussion
5.1. Effects of substituted chromones on neutrophil
acti6ation
To understand the role of dialkylamino substituents
in mono- (isoxazoles) and bicyclic (chromones) com-
pounds better, these molecules were tested for their

458
M. Mazzei et al. / Il Farmaco 54 (1999) 452–460
5.2. Effects of substituted isoxazoles on neutrophil acti-
6ation
highest inhibitory activity against PMA and f-MLF
activated neutrophils.
As depicted in Table 2, compound C2 shows in-
hibitory activity against both PMA and f-MLF acti-
vated neutrophils. However, the presence of a methyl
group on the R%% position (C3, C4) significantly reduces
the inhibitory efficiency of the resulting compound on
PMA stimulated neutrophils. When R%% is a methyl
residue, the activity against PMA is generally low, while
the activity against f-MLF is maintained at a good
level, particularly when R%% is a large substituent, e.g. in
benzoyl derivatives (C6, C9–13, C15, C18). When a
heterocycle is present in the R%% position (C16, C17),
activity is maintained. Low efficiency against f-MLF
stimulated neutrophils has been observed with com-
pounds having charged substituting groups at position
R% (C11, C14). The presence of a large hydrocarbon
tail, such as n-octyl (C19) and oleyl (C20) chains,
reduces the biological activity of the compounds. As
already shown for the chromones (see Table 1), com-
pounds having a piperidino residue at R%% position, even
in the absence of substituents at R% and in the presence
of short alkyl groups at R%% (C22, C23), show the
5.3. Effects of bis(2-substituted-ethyl)amino chromones
and isoxazoles on neutrophil acti6ation
We have previously shown that tricyclic compounds
having
a bis(2-methoxyethyl)amino group as the
substituent were very efficient in preventing neutrophil
activation by PMA and f-MLF [17]. In order to
establish whether the introduction of this substitution in
mono and bicyclic compounds could promote the
appearance of biological activity against human neutro-
phils, we synthesised some new derivatives.
As depicted in Table 3, in contrast with those
previously tested, it seems possible with these com-
pounds to discriminate the activation by PMA from
that of f-MLF. Since the two stimuli utilise different
signal transduction pathways [20], including the step
involved in the membrane crossing, it can be postulated
that changes in substituent groups might affect the
biological specificity of the compounds. In fact, in the
chromone series, while the more lypophilic compounds
3a–d are inactive, compounds 4a and 4b carrying one
Table 2
Effect of the isoxazoles C1–23 on O−2 production by human neutrophils stimulated with f-MLF or PMA a
Comp.
NR2
R%
R%%
f-MLF
PMA
C1
C2
C3
C4
C5
C6
C7
C8
NMe2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
COCH3
H
CH2CHꢀCH2
CH2CꢀCH
benzyl
COCH3
H
ND
100
70
89
ND
90
45
ND
86
100
16
88
95
47
100
100
95
75
80
38
21
42
31
10
51
49
66
2
13
80
35
16
77
66
30
62
70
100
95
100
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
benzoyl
phenoxyacetyl
p-chlorobenzoyl
m-chlorobenzoyl
p-nitrobenzoyl
p-aminobenzoyl
p-acetamidobenzoyl
m-nitrobenzoyl
m-aminobenzoyl
m-acetamidobenzoyl
2-furoyl
2-thiophenecarbonyl
3,4,5-trimethoxybenzoyl
n-octyl
oleoyl
H
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
NEt2
NEt2
NEt2
1-pyrrolidinyl
1-piperidinyl
1-piperidinyl
84
ND
ND
71
82
100
H
H
a See Table 1; ND, not determined.

M. Mazzei et al. / Il Farmaco 54 (1999) 452–460
459
Table 3
Effect of the chromones 3–5 and isoxazoles 6–9 on O−2 production by human neutrophils stimulated with f-MLF or PMA a
Comp.
R
R%
R%%
X
f-MLF
PMA
3a
3b
3c
3d
4a
4b
5
OCH3
OCH3
Cl
OCH3
OH
OCH3
Cl
Cl
OCH3
OH
I
OCH3
H
CH3
CH3
CH3
C2H5
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
22
6
4
7
3
28
3
2
12
90
91
75
53
100
92
32
35
71
18
100
100
25
45
30
12
9
18
45
14
23
I
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
morpholinomethyl
6
7
H
CH3
8a
8b
8c
9a
9b
9c
CH2CH3
(CH2)5CH3
m-nitrobenzoyl
p-nitrobenzoyl
p-chlorobenzoyl
a See Table 1.
Acknowledgements
and two hydroxy groups, respectively, are very effective
against f-MLF stimulated neutrophils. On the other
hand, in the isoxazoles series, compounds 6–8 are more
active in PMA stimulated neutrophils. The maximum
of inhibitory efficiency is achieved when R% is a n-hexyl
substituent (8c). As in previous C isoxazoles, the intro-
duction of benzoyl groups (9a–c) does not produce a
positive effect against the f-MLF stimulus; however, the
resulting compounds show some inhibitory activities
against the PMA stimulus (especially for 9a).
This work was supported by grants from MURST
(PRIN 97) and from CNR (P.F. Biotechnology).
References
[1] A. Ermili, G. Roma, M. Mazzei, A. Balbi, A. Cuttica, N.
Passerini, Ricerche chimiche e farmacologiche su derivati del
1H-nafto[2,1-b]pirano. Nota I. Sintesi di 1-oxo-3-dialchilam-
mino-1H-nafto[2,1-b]pirani, Farmaco Ed. Sci. 29 (1974) 225–
236.
6. Conclusions
[2] A. Ermili, M. Mazzei, G. Roma, A. Cuttica, N. Passerini,
Ricerche chimiche e farmacologiche su derivati del 1H-nafto-
[2,1-b]pirano. Nota IV. 1-Oxo-3-dialchilammino-1H-nafto[2,1-b]-
pirani sostituiti, Farmaco Ed. Sci. 30 (1975) 870–883.
[3] M. Mazzei, A. Balbi, A. Ermili, E. Sottofattori, G. Roma, P.
Schiantarelli, S. Cadel, Ricerche chimiche e farmacologiche su
derivati piranici. Nota XVI. Derivati del 2-(dialchilammino)-7-
metossicromone ad attivita` antiallergica, Farmaco Ed. Sci. 40
(1985) 895–908.
[4] M. Mazzei, A. Ermili, A. Balbi, M. Di Braccio, P. Schiantarelli,
S. Cadel, Ricerche chimiche e farmacologiche su derivati pi-
ranici. Nota XVII. Sintesi dei 2-(dialchilammino)-5-idro-
ssicromoni e loro trasformazione in derivati del 2H-pirano[4,3,2-
d,e]-1-benzopirano, Farmaco Ed. Sci. 41 (1986) 611–621.
[5] M. Mazzei, A. Balbi, G. Roma, M. Di Braccio, G. Leoncini, E.
Buzzi, M. Maresca, Synthesis and anti-platelet activity of some
2-(dialkylamino)chromones, Eur. J. Med. Chem. 23 (1988) 237–
242.
A comparison of the results obtained with the com-
pounds tested in this paper (compare Tables 1–3)
shows that the introduction of a piperidino group in all
compounds always leads to derivatives with high bio-
logical activity against stimulated neutrophils. On the
contrary, substitution with the bis(2-methoxy-
ethyl)amino group produces derivatives almost com-
pletely lacking this biological activity.
Taken together, these data indicate that the pipe-
ridino group improves the inhibitory efficiency of the
chromones and isoxazoles tested in this paper, as well
as the naphtho[2,1-b]pyrans previously reported [17].
In conclusion, it can be suggested that piperidino
substituted derivatives, both in the chromone and isox-
azole series, might be the base for the development of
drugs controlling inflammatory diseases.
[6] M. Mazzei, E. Sottofattori, M. Di Braccio, A. Balbi, G.
Leoncini, E. Buzzi, M. Maresca, Synthesis and antiplatelet activ-
ity of 2-(diethylamino)-7-ethoxychromone and related com-
pounds, Eur. J. Med. Chem. 25 (1990) 617–622.

460
M. Mazzei et al. / Il Farmaco 54 (1999) 452–460
[7] G. Leoncini, M. Maresca, C. Colao, E. Buzzi, M. Mazzei, A.
[13] A.C. Newton, Protein kinase C: structure, function, and regula-
tion, J. Biol. Chem. 270 (1995) 28495–28498.
[14] M.J. Berridge, Inositol triphosphate and calcium signalling, Na-
ture 361 (1993) 315–325.
Balbi, Antiplatelet effect of (2-diethylamino)-7-hydroxy-
chromone, Pharmacol. Res. 23 (1991) 139–148.
[8] G. Leoncini, M. Maresca, C. Colao, E. Buzzi, M. Mazzei, Mode
of action of (2-diethylamino)-7-ethoxychromone on human
platelets, Cell Biochem. Funct. 9 (1991) 79–85.
[9] A. Ermili, G. Roma, M. Mazzei, A. Ambrosini, N. Passerini,
Ricerche chimiche e farmacologiche su derivati del 1H-nafto-
[2,1-b]pirano. Nota II. Sintesi di 1-oxo-2-dialchilamminometil-3-
dialchilammino-1H-nafto[2,1-b]pirani, Farmaco Ed. Sci. 29
(1974) 237–246.
[10] A. Balbi, G. Roma, M. Mazzei, E. Sottofattori, S. Cadel, P.
Schiantarelli, Ricerche chimiche e farmacologiche su derivati
piranici. Nota XVIII. Sintesi di 2-(dialchilammino)-3-
formilcromoni sostituiti e di loro derivati triciclici, Farmaco 44
(1989) 565–577.
[11] M. Mazzei, A. Balbi, E. Sottofattori, R. Garzoglio, A. De
Montis, S. Corrias, P. La Colla, Synthesis of new 3,5-disubsti-
tuted isoxazoles with specific anti-group B rhinovirus activity in
vitro, Eur. J. Med. Chem. 28 (1993) 669–674.
[12] M. Mazzei, R. Garzoglio, A. Balbi, T. Grandi, A. De Montis, S.
Corrias, P. La Colla, Synthesis and antirhinovirus activity of
3-(diethylamino)-5-phenylisoxazole derivatives, Farmaco 51
(1996) 351–359.
[15] J.A. Leff, J.E. Repine, in: J.S. Abramson, J.G. Wheeler (Eds.),
The Neutrophils, IRL Press, New York, 1993, pp. 231–262.
[16] J. Budworth, A. Gescher, Differential inhibition of cytosolic and
membrane-derived protein kinase C activity by staurosporine
and other kinase inhibitors, FEBS Lett. 362 (1995) 139–142.
[17] M. Mazzei, R. Garzoglio, E. Sottofattori, E. Melloni, M.
Michetti, Anti-PKC activity of some 3-(dialkylamino)-1H-naph-
tho[2,1-b]pyran-1-ones, Farmaco 52 (1997) 539–545.
[18] M. Mazzei, G. Roma, A. Ermili, Ricerche chimiche e farmaco-
logiche su derivati piranici. Nota XII. Cromoni e benzocromoni
bis-(b-cloroetil)ammino sostituiti, Farmaco Ed. Sci. 34 (1979)
52–61.
[19] A. Ermili, M. Mazzei, G. Roma, C. Cacciatore, Ricerche
chimiche e farmacologiche su derivati piranici. Nota VIII. Sintesi
di 2-dialchilammino-7-metossicromoni e derivati, Farmaco Ed.
Sci. 32 (1977) 375–387.
[20] L.T. Tsao, J.P. Wang, Translocation of protein kinase C iso-
forms in rat neutrophils, Biochem. Biophys. Res. Commun. 234
(1997) 412–418.
.