Inhibition of neutrophil O2- production by unsymmetrical methylene derivatives of benzopyrans: Their use as potential antiinflammatory agents

Article abstract of DOI:10.1016/S0223-5234(01)01279-X

Some unsymmetrical derivatives of benzopyrans 9 were synthesized and tested to verify their PKC inhibitory activity. For this purpose, the Mannich bases of 7-hydroxycoumarins 6 were treated with 2-(dialkylamino)benzopyran-4-ones or 3-(dialkylamino)naphtho[2,1-b]pyran-1-ones 8 in the presence of acetic or propionic anhydride, yielding compounds 9. Human neutrophils stimulated with either PMA and f-MLF were used as the cellular model. The efficiency of the compounds 9 was established on their capacity to reduce the O₂^- production by activated human neutrophils. Compounds 9d and 9f, bearing an acetoxy group in position 7 of the chromone moiety, seem to counteract the neutrophil activation efficiently.

Full text of DOI:10.1016/S0223-5234(01)01279-X

Eur. J. Med. Chem. 36 (2001) 851–861
´
©
2001 Editions scientifiques et m e´ dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01279-X/FLA
Original article
−
2
Inhibition of neutrophil O production by unsymmetrical methylene derivatives
of benzopyrans: their use as potential antiinflammatory agents
a,
a
a
b
b
Mauro Mazzei *, Ramona Dondero , Enzo Sottofattori , Edon Melloni , Roberto Minafra
a
Dipartimento di Scienze Farmaceutiche, Viale Benedetto XV, 3-16132 Genova, Italy
b
Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Viale Benedetto XV, 1-16132 Genova, Italy
Received 15 February 2001; revised 6 September 2001; accepted 14 September 2001
Abstract – Some unsymmetrical derivatives of benzopyrans 9 were synthesized and tested to verify their PKC inhibitory activity. For
this purpose, the Mannich bases of 7-hydroxycoumarins 6 were treated with 2-(dialkylamino)benzopyran-4-ones or 3-(dialkyl-
amino)naphtho[2,1-b]pyran-1-ones 8 in the presence of acetic or propionic anhydride, yielding compounds 9. Human neutrophils
stimulated with either PMA and f-MLF were used as the cellular model. The efficiency of the compounds 9 was established on their
−
capacity to reduce the O production by activated human neutrophils. Compounds 9d and 9f, bearing an acetoxy group in position
2
´
7
of the chromone moiety, seem to counteract the neutrophil activation efficiently. © 2001 Editions scientifiques et m e´ dicales
Elsevier SAS
Mannich bases / superoxide anion / neutrophils / 1-benzopyran-4-ones / antiinflammatory agents
1
. Introduction
7
-hydroxycoumarins, under certain experimental con-
ditions, may improve our understanding of the reac-
tion mechanism.
In previous works we showed that benzopyran-4-
ones (or chromones) and naphthopyran-1-ones bear-
ing a dialkylamine in the position adjacent to the
heteroatom are potentially useful as antiinflammatory
agents because their ability to inhibit the protein
kinase C (PKC) dependent signal transduction path-
way. In particular, these studies showed that the cited
activity was prominent when the dialkylamino sub-
stituent was piperidine, diethylamine or bis(2-
methoxyethyl)amine [1, 2].
Following our interest in synthetic substances that
could be useful as antiinflammatory agents, we have
now analyzed the PKC inhibitory activity of some of
those active compounds when they are linked by
means of a methylene bridge to 7-hydroxycoumarins.
This type of synthesis is an application of the use of
Mannich bases in acetic or propionic anhydride to
form unsymmetrical methylene derivatives. In this
context, the recovery of diacetyloxy derivatives of
2
. Chemistry
It is known that the treatment of Mannich base
derivatives with acetic anhydride usually leads to ace-
toxy derivatives [3]. On the contrary, we did not find
any acetoxy derivative when Mannich bases of ben-
zopyran-4-ones or naphthopyran-1-ones were treated
with acetic anhydride but we did find methylene-bis-
derivatives or unsymmetrical methylene derivatives
such as the example shown in figure 1. The formation
of such derivatives was determined by a balance be-
tween deamination and deaminomethylation of the
Mannich bases [4]. Briefly, the Mannich base 1, in the
presence of a substrate different from that coming
from its deaminomethylation but structurally similar,
as the case of naphtho[2,1-b]pyran-1-one 2, yields, in
acetic anhydride, the unsymmetrical methylene
derivative 3. On the contrary, in the absence of a
*
Correspondence and reprints
E-mail address: mazzei@cba.unige.it (M. Mazzei).

8
52
M. Mazzei et al. / European Journal of Medicinal Chemistry 36 (2001) 851–861
substrate or when the substrate was the starting com-
pound of the Mannich base 1, such as the chromone
Following our studies on unsymmetrical methylene
derivatives of biologically active compounds in the
benzopyran series, our interest was focused to the
Mannich bases of 7-hydroxycoumarins 6. Interest-
ingly, the treatment of such bases 6 with acetic anhy-
dride follows the typical route and produces the
diacetyl derivatives 7 (see figure 2).
4
, the symmetrical methylene derivative 5 was ob-
tained. Further details of these reactions are given in
Section 4.
This preliminary observation permitted us to as-
sume that the Mannich bases 6 are less disposed to
deaminomethylation than the previously investigated
Mannich bases on benzopyran-4-ones and naph-
tho[2,1-b]pyran-1-ones. Therefore, the compounds 6
seemed very interesting for the formation and the
reaction study of unsymmetrical methylene
derivatives.
When compounds 6 were treated with benzopyrans
or naphthopyrans 8 in acetic anhydride it was possi-
ble to obtain the unsymmetrical methylene derivatives
9
in good yield (see figure 3). The use of propionic
anhydride gave similar results to acetic anhydride. In
order to counteract the formation of diacetyl deriva-
tives 7 it is preferable to combine the anhydride and
the chromonic compounds 8 first, wait for it to dis-
solve and than add the Mannich bases 6.
All compounds described herein are white crystals
and their structures are in agreement with elemental
analyses and spectral data (see Section 5). In particu-
1
lar, the H-NMR spectra of unsymmetrical deriva-
tives in which the naphtho[2,1-b]pyran-1-one moiety
is present retain the characteristic deshielding signal
(l=10.05) of such angular structures [5].
3
. Biology
3.1. Isolation of neutrophils
Figure 1. Methylene-bis derivatives.
Freshly collected heparinized 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
8
00×g for 20 min. The pellets containing mostly neu-
trophils and contaminating red cells were resuspended in
0 mL of hypotonic 0.2% NaCl. After 30 s, 10 mL of
1
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
Figure 2. Diacetyl derivative.

M. Mazzei et al. / European Journal of Medicinal Chemistry 36 (2001) 851–861
853
mM KCl, 0.12 M NaCl, 24 mM NaHCO , 5 mM
glucose. Prior to use, the cells were maintained in an ice
3.2. Activation of neutrophils and assay of superoxide
anion (O )
2
3
−
bath in the same medium at a concentration of 15–20×
6
6
10 cells per mL. The cell population obtained consisted
About 10 cells were diluted in 1 mL of 10 mM
HEPES, pH 7.4, containing 0.15 M NaCl, 5.0 mM
of more than 96% neutrophils, as evaluated by micro-
scopic examination. The few remaining cells were
eosinophils and monocytes.
2
+
−1
glucose, 1.0 mM Ca
tochrome C (Fe ) and incubated at 37 °C for 2 min.
and 0.625 mg mL
of cy-
3
+
Figure 3. Unsymmetrical methylene derivatives.

8
54
M. Mazzei et al. / European Journal of Medicinal Chemistry 36 (2001) 851–861
Figure 3. (Continued)
Phorbol myristate acetate (PMA, 100 mg) or formylme-
thionine–leucine–phenylalanine (f-MLF, 0.1 mM final
concentration) were then added. The absorbance at 550
4. Results and discussion
In the previous work regarding the synthesis of
unsymmetrical methylene derivatives from benzopy-
ran-4-ones it was shown that, apart from the presence
of the unsymmetrical derivatives, there were small
amounts of methylene-bis-derivatives of the com-
pounds bearing the Mannich base as well as the
compounds without the Mannich base. This fact was
easily explained on the basis of the reversibility of the
Mannich reaction [3]. On the contrary, in the present
work we did not find any methylene-bis-derivatives
and this absence provides further support for the
scarcity of the deaminomethylation process when the
Mannich bases 6 are in the presence of acetic or
propionic anhydride.
nm was continuously monitored for 10 min. The
−
2
amount of O produced was calculated by the difference
in absorbance at zero time and at the end of the
reaction.
3.3. Samples
The compounds were diluted in dimethyl sulfooxide
(
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 the blank sample.
Moreover, it is possible to indicate that the recov-
ery of the diacetyl derivatives 7 or the unsymmetrical
derivatives 9 is dependent on the presence of a nucle-
ophylic reagent. This evidence permits us to give
deeper insight into the reaction mechanism (see figure
4). The first attack of the anhydride presumably leads
to the intermediate A which may evolve in two ways.
The first one occurs in the absence of a nucleophylic
3
.4. PKC purification
2
+
Ca and phospholipid dependent protein kinase C
PKC) were purified from human neutrophils as de-
scribed by Pontremoli et al. [6]. PKC activity was as-
(
2
+
sayed using Istone type IIIS, as substrate, Ca ,
phosphatidylserine, diacylglycerol and P-ATP, as de-
scribed previously [6].
32

M. Mazzei et al. / European Journal of Medicinal Chemistry 36 (2001) 851–861
855
Figure 4. Proposed mechanism for the formation of acetyloxy derivative C (arrow 1) or carbocation B (arrow 2) by treatment of
Mannich bases with acetic anhydride. The carbocation B may evolve in the symmetrical or unsymmetrical derivatives, according to
the reaction conditions.
reagent or if the nucleophylic reagent is weak and
leads to the acetoxy derivative C (see figure 4, arrow
Another interesting observation comes from the
behavior of the 7-hydroxychromone 8c in the reaction
with the Mannich bases 6. It is known that when
7-hydroxychromones are subjected to Mannich reac-
tion the aminomethylation occurs in position 8 (as
already seen for 7-hydroxycoumarin) [7]. But in our
case, the attack of the coumarin intermediate occurs
in position 3 of the chromone ring, yielding 9d and 9f,
as the acylation on the 7-hydroxy group causes the
deactivation of position 8 of the chromone.
This fact means that changing the insertion of the
aminomethyl group from the 7-hydroxycoumarin to
the chromone is not allowed because the 7-acetyloxy-
coumarin cannot receive the nucleophilic attack. On
the contrary, in the case of the previously studied
unsymmetrical derivative 3 in figure 1, the Mannich
base can indifferently be on the bicyclic- or tricyclic
compound [4]; in fact the benzopyran-4-ones and
naphtho[2,1-b]pyran-1-ones, lacking hydroxy groups,
are inert to treatment with acetic anhydride and inac-
tivations due to acetylation are not possible.
Another interesting consequence of the reaction
between the 7-hydroxychromone 8c and the Mannich
bases 6 is the formation of 7-acetyloxy derivatives. In
fact, as far as we can see, 9d and 9f are the first
examples of derivatives of 2-(diethylamino)-7-hydrox-
ychromone which are acylated at the 7-hydroxy
group. This observation will be a source of further
investigations as the 2-(diethylamino)-7-hydroxy-
chromone, a substance endowed with notable and
diverse pharmacological properties, is very resistant
to the acylation [8]. On the other hand, the acylation
of 7-hydroxicoumarin is very easy.
1
). The second one occurs when the nucleophylic
attack is strong enough to lead to the removal of
dialkylamine from the Mannich base with the forma-
tion of a stabilized carbocation B (see figure 4, arrow
2
). The latter may react with position 3 of benzopy-
ran-4-ones (or position 2 of naphthopyran-1-ones)
thus forming the unsymmetrical derivatives 9.
Interestingly, a non acylated hydroxy group is
present in the formula A. In fact, the recovery of
4
some compounds 9 having the R substituent acylated
or not, indicates that the above reaction runs quickly
and its rate, in the case of a hydroxy group ortho to a
dialkylaminomethyl group, is faster than the acyla-
tion reaction.
In fact, when the reaction leads to a derivative 9 in
which the presence of the hydroxy group of the cou-
marin coupled with a particular dialkylamine (espe-
cially pyrrolidine and piperidine) on the benzopyran-
4
-one confers to the unsymmetrical dimer a high
melting point, a precipitate separates out from the
reaction medium in a few minutes before acylation
can occur. It is sometimes possible to recover acylated
and non acylated derivatives in the same reaction. On
the contrary, if the products coming from the primary
attack are soluble in the anhydride, the final unsym-
metrical derivatives are acylated.
A further piece of evidence for the proposed reac-
tion mechanism was the extraction, after the recovery
of the unsymmetrical derivative 9, of N-acetylpipe-
ridine. A solution containing this compound was ex-
amined by GLC–MS, the main chromatographic
peak presented a molecular peak at 127 and the
fragmentation pattern was consistent with that of
N-acetylpiperidine.
All the compounds listed in table I were tested for
−
2
their ability to affect the production of O in human

8
56
M. Mazzei et al. / European Journal of Medicinal Chemistry 36 (2001) 851–861
neutrophils stimulated by chemotactic peptide f-MLF
or by PMA. Some of these compounds were effective
−
2
in inhibiting O production by stimulated neutrophils.
Regarding the action against the f-MLF stimulus,
the tested compounds may be divided into three cate-
gories. The first one is composed of the two com-
pounds endowed with the best activity (9d and 9f); the
second one comprises the 7-methoxychromone
derivative 9g and the naphthopyran derivative 9r; the
remaining part of the tested compounds comprises the
third category. It is important to stress that the two
best compounds (9d and 9f) are the unique com-
pounds with the 7-acetoxy group as substituent in the
chromone ring.
The two compounds which are in the intermediate
zone of action (9g, 9r) show some mutual structural
motifs, such as the coumarin moiety without the
methyl group in position 4, the propionyl group as
−
Figure 5. Effect of compounds 9d, 9f and 9g on O production
2
by human neutrophils stimulated with f-MLF. Neutrophils
were stimulated with 0.1 mM as described in Section 3, in the
presence of the indicated amounts of benzopyran derivatives,
dissolved in 1 mL of DMSO. O2 production by stimulated
human neutrophils in the presence of 1 mL of DMSO was
used as a control and taken to be 100%.
−
Table I. Effects of benzopyran derivatives 9 on O −2 produc-
tion by human neutrophils stimulated with f-MLF and PMA.
Compound
Inhibition (%)
f-MLF
the acylating group of the 7-hydroxycoumarin, and
the diethylamine in the chromone moiety.
PMA
In conclusion, from a structure–activity analysis of
the two above-mentioned categories, we can conclude
that in order to express biological action against the
f-MLF stimulus, it is essential that the dialkylamino
group on the g-pyrone moiety is diethylamine, the
g-pyrone participates in a bicyclic ring instead of a
tricyclic ring, and the a-pyrone is preferentially with-
out the methyl group in position 4.
We cannot draw conclusions about the acyl group
as we do not have data to evaluate the efficiency of
the propionyl group in position 7 of the chromone.
Regarding the other stimulus (PMA), the results
are not easy to interpret as there are not sharp clus-
ters of activity, it is interesting to observe that in this
case the more active compounds are 9g and 9r, the
intermediate group with respect to f-MLF. Instead,
the intermediate group with respect to PMA is com-
prised of compounds 9d and 9f, along with several
others derivatives.
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
0
0
0
100
0
100
89
0
0
0
0
0
0
0
0
0
0
58
0
0
0
0
77
12
63
74
56
92
0
0
0
0
0
50
66
30
0
0
94
46
8
0
44
0
16
0
45
s
t
u
v
w
x
y
z
0
0
0
0
As the compounds 9d, 9f and 9g are on the whole
the more effective ones, their biological action has
been analyzed in more detail (9r was left out due to its
poor solubility).
0
As shown in figure 5, the 7-acetoxychromone
Neutrophil stimulation was carried out as described in Section
. The concentration of each compound was 20 mM. The
values refer to the percentage of the observed inhibition.
−
derivative 9d almost completely inhibits O produc-
3
2
tion at concentration below 5 mM, with a K05 of ca.

M. Mazzei et al. / European Journal of Medicinal Chemistry 36 (2001) 851–861
857
1
7
.0 mM. The 7-methoxychromone derivative 9g and the
-acetoxychromone derivative 9f are two times less
5. Experimental protocols
effective, with K05 values around 3.0 and 6.0 mM,
respectively.
Melting points (m.p.) were determined using a Elec-
trothermal apparatus and are uncorrected. Micro-
analyses were carried out on a Carlo Erba 1106
elemental analyzer. The results of elemental analysis
were within ±0.3% for C and ±0.1% for H and N of
None of these compounds showing the highest inhi-
−
2
bition of O production was effective on purified PKC
activity (data not shown).
1
Taken together these results suggest a putative site
the theoretical value. H-NMR spectra were per-
of action for these compounds. As shown in figure 6,
formed on a Hitachi Perkin–Elmer R 600 (60 MHz)
spectrometer using TMS as internal standard (l=0).
IR spectra were recorded on a Perkin–Elmer 398
spectrophotometer.
−
inhibition of O production by human neutrophils
2
could be preferentially obtained by interfering at three
different levels with (1) a component of the NADPH
oxidase complex [9, 10]; (2) the f-MLF receptor [11,
1
1
2]; and (3) membrane associated PKC isozyme(s) [13,
4].
5.1. Mannich bases 6
The last two possibilities (2 and 3) can be excluded
5.1.1. General method
because the neutrophil responses elicited by both f-
MLF and PMA require an active membrane associ-
ated PKC [13, 14], and compounds 9d, 9f and 9g do
not show PKC inhibiting activity. Activation of
NADPH oxidase in stimulated neutrophils require the
aggregation of different protein components through a
number of events, including phosphorylation and
translocation from cytosol to plasma membrane [9,
To 20.0 mmol of 7-hydroxycoumarin (or 4-methyl-7-
hydroxycoumarin) dissolved in 50 mL of ethanol, 20.0
mmol of piperidine and 2.0 mL of 40% formaldehyde
were added. The resulting mixture was refluxed for 6 h.
After cooling, the solvent was evaporated under reduced
pressure. The pale yellow oil obtained was treated with
cool acetone, leaving a white solid which was crystallized
from acetone obtaining, 8-(piperidinomethyl)-7-hydroxy-
coumarin (6a), m.p. 130–131 °C, 56.4% yield; 8-(pipe-
ridinomethyl)-7-hydroxy-4-methylcoumarin (6b), m.p.
169–70 °C, 62.0% yield.
1
0]. It seems likely that the biological activity such
benzopyran derivatives can interfere with one of the
processes undergoing active NADPH oxidase complex
formation, on the outer surface plasma membrane [15].
The relevance of the biological effects shown by
these active derivatives is due to the fact that activated
neutrophils are involved, at least in part, in tissue
damage characteristic of a number of human patholo-
gies, including inflammation, ischemia and reperfu-
sion. Further analyses are in progress to collect more
data on chromone derivatives bearing different acyl
substitutions on the hydroxy group in position 7.
−
1
6a: IR (KBr) w (cm ): 2600 (broad), 1720, 1595, 1580.
1
H-NMR (l, CDCl ): 1.86–1.31 (m, 6H, b+g-pipe-
3
ridine CH ), 2.87–2.29 (m, 4H, a-piperidine CH ), 4.02
(s, 2H, CH bridge), 6.19 (d, 1H, H-3), 6.75 (d, 1H, H-6),
2
2
2
7.30 (d, 1H, H-5), 7.64 (d, 1H, H-4), 12.09 (s, 1H, OH).
−
1
6b: IR (KBr) w (cm ): 2600 (broad), 1722, 1600, 1580.
1
H-NMR (l, CDCl ): 1.96–1.37 (m, 6H, b+g-pipe-
3
ridine CH ), 2.85–2.27 (m, 7H, a-piperidine CH +4-
CH ), 4.05 (s, 2H, CH bridge), 6.09 (s, 1H, H-3), 6.77
2
2
3
2
(
d, 1H, H-6), 7.46 (d, 1H, H-5), 12.38 (s, 1H, OH).
5.2. Diacetoxy derivatives 7
5.2.1. General method
In a 100 mL flask, protected from moisture with a
calcium chloride tube, 1.0 mmol of 8-(piperidi-
nomethyl)-7-hydroxycoumarin (6a) or 8-(piperidi-
nomethyl)-7-hydroxy-4-methylcoumarin
(6b)
was
dissolved in 5 mL of freshly distilled acetic anhydride
and heated at 95 °C for 1.5 h, with stirring. At the end,
the cooled solution was poured onto crushed ice and
water, and stirred for 1 h obtaining a white solid, which
was filtered off and crystallized from ethyl acetate.
Fig. 6. Possible site of action of benzopyran derivatives on
stimulated human neutrophils. f-MLF-R refers to the receptor
2
+
for the chemotactic peptide f-MLF, PKC refers to Ca lipid
dependent protein kinase C. Further details are given in the
text.

8
58
M. Mazzei et al. / European Journal of Medicinal Chemistry 36 (2001) 851–861
The following compounds were so obtained: 8-acetyl-
oxymethyl-7-acetoxycoumarin (7a), m.p. 121–122 °C
81.3% yield) and 8-acetyloxymethyl-7-acetoxy-4-
methylcoumarin (7b), m.p. 138–139 °C (86.2% yield).
B, C or D) followed for each synthesised compound and
the crystallization solvent are reported in brackets.
(
5.3.1.1. 8-[2%-(Dimethylamino)-7%-methoxychromon-
3%-yl]methyl-7-acetoxycoumarin (9a)
[6a, 8a; 1; C; chloroform–ethanol 1:1], m.p. 216–
−
1
7
a: IR (KBr) w (cm ): 1770, 1730, 1610.
1
H-NMR (l, CDCl ): 2.05 (s, 3H, CH COOCH ),
3
3
2
2
1
.38 (s, 3H, CH CO), 5.38 (s, 2H, CH bridge), 6.43 (d,
217 °C, 68.0% yield.
3
2
−
1
H, H-3), 7.11 (d, 1H, H-6), 7.41–7.90 (m, 2H, H-4, 5).
IR (KBr) w (cm ): 1760, 1740, 1605, 1505.
−
1
1
7
b: IR (KBr) w (cm ): 1760, 1730, 1605.
H-NMR (l, CF OOD): 2.60 (s, 3H, CH CO), 3.75
3
3
1
H-NMR (l, CDCl ): 2.06 (s, 3H, CH COOCH ),
(s, 6H, NCH ), 4.03 (s, 3H, OCH ), 4.48 (s, 2H, CH
3
3
2
3 3 2
2
.37 (s, 3H, CH CO), 2.46 (s, 3H, 4-CH ), 5.39 (s, 2H,
bridge), 6.64 (d, 1H, H-3), 7.85–7.10 (m, 5H, H arom.),
8.07 (d, 1H, H-5%).
3
3
CH bridge), 6.34 (s, 1H, H-3), 7.14 (d, 1H, H-6), 7.71
2
(
d, 1H, H-5).
5
3
.3.1.2. 8-[2%-(Dimethylamino)-7%-methoxychromon-
%-yl]methyl-7-acetoxy-4-methylcoumarin (9b)
5.3. Unsymmetrical methylene derivatives 9
[6b, 8a; 1; A; ethyl acetate], m.p. 178–179 °C, 67.2%
yield.
−
1
5.3.1. General method
IR (KBr) w (cm ): 1760, 1735, 1605, 1550.
1
In a 100 mL flask, protected from moisture with a
H-NMR (l, CDCl ): 2.15 (s, 3H, 4-CH ), 2.42 (s,
3
3
calcium chloride tube, 1.0 mmol of 2-(dialkylamino)-
benzopyran-1-one (8a–f) or 3-(dialkylamino)-1H-
naphto[2,1-b]pyran-1-one (8g–k) was dissolved in 5 mL
of freshly distilled acetic or propionic anhydride. Then
3H, CH CO), 3.01 (s, 6H, NCH ), 3.89 (s, 3H, OCH ),
3 3 3
4.16 (s, 2H, CH bridge), 6.28 (s, 1H, H-3), 7.61–6.68
2
(m, 4H, H arom.), 8.06 (d, 1H, H-5%).
1
.0 mmol of Mannich base (6a or 6b) was added, and
5.3.1.3. 8-[2%-(Diethylamino)-7%-methoxychromon-
3%-yl]methyl-7-acetoxycoumarin (9c)
[6a, 8b; 1; A; ethyl acetate], m.p. 159–160 °C, 54.5%
yield.
the mixture was heated at 95 °C for 1.5 h, with stirring.
At the end, the cooled solution was poured onto crushed
ice and water, and stirred for 2 h obtaining solid (case
A) or oily (case B) residues. Sometimes, the solid was
already obtained during the heating in the anhydride
−
1
IR (KBr) w (cm ): 1770, 1730, 1605, 1560.
1
H-NMR (l, CDCl ): 0.88 (t, 6H, CH CH ), 2.08 (s,
3
2
3
(
case C) or two products, with different solubility, were
3H, CH CO), 3.35 (q, 4H, CH CH ), 3.90 (s, 3H,
3 2 3
isolated (case D).
OCH ), 4.20 (s, 2H, CH bridge), 6.40 (d, 1H, H-3),
3 2
In case A, the solid was collected by filtration, washed
with water and crystallized from suitable solvent.
In case B, the acidic solution was alkalinized with
sodium hydroxide pellets and the resulting solution was
extracted three times with chloroform. The pooled or-
ganic extracts were washed with water, dried over anhy-
drous sodium sulfate and evaporated under reduced
pressure. The oily residue obtained was treated with a
small amount of ethyl acetate and the crystals which
separated out were filtered and washed with cold
solvent.
7.96–6.66 (m, 5H, H-6, 8, 4%, 5%, 6%), 8.16 (d, 1H, H-5%).
5.3.1.4. 8-[2%-(Diethylamino)-7%-acetoxychromon-
3’-yl]methyl-7-acetoxycoumarin (9d)
[6a, 8c; 1; A; ethyl acetate], m.p. 177–178 °C, 56.3%
yield.
−
1
IR (KBr) w (cm ): 1760, 1730, 1620, 1570.
1
H-NMR (l, CDCl ): 0.92 (t, 6H, CH CH N), 2.12 (s,
3
3
2
3H, 7-CH CO), 2.35 (s, 3H, 7%-CH CO), 3.31 (q, 4H,
3
3
CH CH N), 4.18 (s, 2H, CH bridge), 6.42 (d, 1H, H-3),
3
2
2
7.51–6.82 (m, 4H, H-5, 6, 6%, 8%), 7.74 (d, 1H, H-4), 8.25
(d, 1H, H-5%).
In case C, the final mixture was cooled and the solid
obtained was filtered off.
In case D, the final mixture was cooled and a first
solid was filtered off. Then the filtered solution was
poured onto crushed ice and water, and stirred for 2 h
obtaining a second solid.
The starting products, the anhydride used (1 for acetic
anhydride, and 2 for propionic anhydride), the case (A,
5.3.1.5. 8-[2%-(Diethylamino)-7%-methoxychromon-
3%-yl]methyl-7-hydroxy-4-methylcoumarin (9e)
[6b, 8b; 1; A; ethyl acetate], m.p. 179–180 °C, 58.0%
yield.
−
1
IR (KBr) w (cm ): 1765, 1730, 1610, 1590, 1560.
1
H-NMR (l, CDCl ): 1.34 (t, 6H, CH CH ), 2.33 (s,
3
2
3

M. Mazzei et al. / European Journal of Medicinal Chemistry 36 (2001) 851–861
859
3
H, 4-CH ), 3.96–3.53 (m, 7H, OCH +CH CH ), 4.03
9j: [6b, 8d; 1; D; ethyl acetate], m.p. 208–209 °C,
3
3
2
3
(
3
1
s, 2H, CH bridge), 6.03 (s, 1H, H-3), 7.05–6.64 (m,
H, H-6, 8, 6%), 7.36 (d, 1H, H-5%), 8.06 (d, 1H, H-5%),
1.94 (s, 1H, OH).
45.3% yield.
2
−
1
IR (KBr) w (cm ): 1770, 1735, 1630, 1610, 1590.
1
H-NMR (l, CDCl ): 2.18–1.77 (m, 4H, b-pyrrolidine
2
3
CH ), 2.28 (s, 3H, CH CO), 2.37 (s, 3H, 4-CH ), 4.02–
3
3
3
.63 (m, 7H, CH O+a-pyrrolidine CH ), 4.19 (s, 2H,
5
3
.3.1.6. 8-[2%-(Diethylamino)-7%-acetoxychromon-
%-yl]methyl-7-acetoxy-4-methylcoumarin (9f)
3
2
CH bridge), 6.19 (s, 1H, H-3), 7.57–6.60 (m, 4H, H-5,
2
6
, 6%, 8%), 8.02 (d, 1H, H-5%).
[
6b, 8c; 1; A; ethyl acetate], m.p. 209–210 °C, 73.8%
yield.
−
1
IR (KBr) w (cm ): 1760, 1730, 1610, 1570.
5
3
.3.1.10. 8-[2%-(1-Piperidinyl)-7%-methoxychromon-
%-yl]methyl-7-acetoxycoumarin (9k)
1
H-NMR (l, CDCl ): 0.93 (t, 6H, CH CH ), 2.10 (s,
3
2
3
3
H, 4-CH ), 2.54–2.25 (m, 6H, CH CO), 3.30 (q, 4H,
3
3
[
6a, 8e; 1; A; ethyl acetate], m.p. 223–224 °C, 61.0%
CH CH ), 4.14 (s, 2H, CH bridge), 6.25 (s, 1H, H-3),
7
2
3
2
yield.
.63–6.76 (m, 4H, H-5, 6, 6%, 8%), 8.22 (m, 1H, H-5%).
−1
IR (KBr) w (cm ): 1760, 1735, 1625, 1605, 1568.
1
H-NMR (l, CF COOD): 2.39–1.78 (m, 6H, b+a-
3
5
3
.3.1.7. 8-[2%-(Diethylamino)-7%-methoxychromon-
%-yl]methyl-7-propionyloxy-4-methylcoumarin (9g)
piperidine CH ), 2.56 (s, 3H, CH CO), 4.53–3.62 (m,
H, OCH +a-pyrrolidine CH +CH bridge), 6.62 (d,
H, H-3), 8.19–7.04 (m, 6H, H arom.).
2
3
9
1
3
2
2
[
6b, 8b; 2; B; ethyl acetate], m.p. 159–160 °C, 54.7%
yield.
−
1
IR (KBr) w (cm ): 1763, 1722, 1610, 1560.
1
5
3
8
7
.3.1.11. 8-[2%-(1-Piperidinyl)-7%-methoxychromon-
%-yl]methyl-7-hydroxy-4-methylcoumarin (9l) and
-[2%-(1-piperidinyl)-7%-methoxychromon-3%-yl]methyl-
-acetoxy-4-methylcoumarin (9m)
H-NMR (l, CDCl ): 1.09–0.75 (m, 9H, NCH CH +
3
2
3
CH CH CO), 2.57–2.15 (m, 5H, 4-CH +CH CH CO),
3
2
3
3
2
3
2
.24 (q, 4H, NCH CH ), 3.88 (s, 3H, OCH ), 4.17 (s,
2 3 3
H, CH bridge), 6.27 (s, 1H, H-3), 7.63–6.69 (m, 4H,
2
9
l: [6b, 8e; 1; D; chloroform–ethanol 1:1], m.p. 238–
H-5, 6, 6%, 8%), 8.13 (d, 1H, H-5%).
2
1
39 °C, 49.6% yield.
−
1
IR (KBr) w (cm ): 2910 (broad), 1720, 1630, 1600,
580.
5
3
.3.1.8. 8-[2%-(1-Pyrrolidinyl)-7%-methoxychromon-
%-yl]methyl-7-hydroxycoumarin (9h)
1
H-NMR (l, CF COOD): 2.28–1.78 (m, 6H,
3
[
6a, 8d; 1; C; chloroform–ethanol 1:1], m.p. 253–
b+g-piperidine CH ), 2.54 (s, 3H, 4-CH ), 4.50–3.65 (m,
9H, OCH
H-3), 8.18–6.88 (m, 5H, H arom.).
9m:[6b, 8e; 1; D; ethyl acetate], m.p. 215–216 °C,
2
3
2
1
54 °C, 64.5% yield.
−
1
3
+a-piperidine CH
2
+CH
2
bridge), 6.45 (s, 1H,
IR (KBr) w (cm ): 3100 (broad), 1729, 1630, 1605,
580, 1505.
1
H-NMR (l, CF OOD): 2.65–2.21 (m, 4H, b-pyrro-
3
3
8.1% yield.
lidine CH ), 4.03 (s, 3H, OCH ), 4.71–4.22 (m, 6H, CH
bridge+a-pyrrolidine CH ), 6.55 (d, 1H, H-3), 7.76–
7
2
3
2
1
H-NMR (l, CDCl ): 1.70–1.33 (m, 6H, b+g-pipe-
3
2
ridine CH ), 2.07 (s, 3H, CH CO), 2.40 (s, 3H, 4-CH ),
.38–3.02 (m, 4H, a-piperidine CH ), 3.88 (s, 3H,
OCH ), 4.09 (s, 2H, CH bridge), 6.25 (s, 1H, H-3),
2
3
3
.01 (m, 5H, H-4, 5, 6, 6%, 8%), 8.06 (d, 1H, H-5%).
3
2
3
2
5.3.1.9. 8-[2%-(1-Pyrrolidinyl)-7%-methoxychromon-
3%-yl]methyl-7-hydroxy-4-methylcoumarin (9i) and
8-[2%-(1-pyrrolidinyl)-7%-methoxychromon-3%-yl]methyl-
7-acetoxy-4-methylcoumarin (9j)
7
.60–6.67 (m, 4H, H-5, 6, 6%, 8%), 8.03 (d, 1H, H-5%).
5.3.1.12. 8-[2%-bis(2-Methoxyethylamino)-
7%-methoxycrhomon-3%-yl]methyl-7-acetoxycoumarin (9n)
[6a, 8f; 1; A; ethyl acetate], m.p. 104–105 °C, 54.5%
yield.
9i: [6b, 8d; 1; D; chloroform–ethanol 1:1], m. p.
2
1
61–262 °C, 38.5% yield.
IR (KBr) w (cm ): 2910 (broad),1726, 1624, 1605,
566.
−
1
−
1
IR (KBr) w (cm ): 1755, 1740, 1610, 1601, 1565.
1
1
H-NMR (l, CF COOD): 2.67–2.17 (m, 7H, 4-CH +
H-NMR (l, CDCl ): 2.05 (s, 3H, CH CO), 3.70–2.97
3
3
3
3
b-pyrrolidine CH ), 4.01 (s, 3H, CH O), 4.69–4.22 (m,
(m, 14H, NCH CH OCH ), 3.90 (s, 3H, OCH ), 4.21 (s,
2
3
2 2 3 6 3
6
H, CH bridge+a-pyrrolidine CH ), 6.45 (s, 1H, H-3),
2H, CH bridge), 6.41 (d, 1H, H-3), 7.94–6.83 (m, 5H,
2
2
2
8.25–7.02 (m, 5H, H arom.).
H-4, 5, 6, 6%, 8%), 8.12 (d, 1H, H-5%).

8
60
M. Mazzei et al. / European Journal of Medicinal Chemistry 36 (2001) 851–861
5.3.1.13. 8-[2%-bis(2-Methoxyethylamino)-7%-
1.98 (s, 3H, 4-CH ), 2.42 (s, 3H, CH CO), 3.27 (q, 4H,
3
3
methoxychromon-3%-yl]methyl-7-acetoxy-4-
methylcoumarin (9o)
NCH CH ), 4.31 (s, 2H, CH bridge), 6.31 (s, 1H, H-3),
8.20–6.85 (m, 7H, H arom), 10.22 (d, 1H, H-10%).
2 3 2
[
6b, 8f; 1; A; ethyl acetate], m.p. 140–141 °C, 61.0%
yield.
−
1
IR (KBr) w (cm ): 1760, 1740, 1630, 1605, 1580.
5.3.1.18. 8-[3%-(Diethylamino)-1%H-naphtho[2%,1%-b]-
pyran-1%-one-2%-yl]methyl-7-propionyloxy-4-
methylcoumarin (9t)
1
H-NMR (l, CDCl ): 2.07 (s, 3H, 4-CH ), 2.43 (s,
3
3
3
3
1
H, CH CO), 3.70–3.07 (m, 14H, NCH CH OCH ),
3 2 2 3
.90 (s, 3H, 7-OCH ), 4.21 (s, 2H, CH bridge), 6.31 (s,
[6b, 8h; 2; A; ethyl acetate], m.p. 175–176 °C, 90.4%
3
2
H, H-3), 7.73–6.67 (m, 4H, H-5, 6, 6%, 8%), 8.13 (d, 1H,
yield.
−
1
H-5%).
IR (KBr) w (cm ): 1770, 1730, 1630, 1603, 1570.
1
H-NMR (l, CDCl ): 1.07–0.58 (m, 9H, NCH CH +
3
2
3
5.3.1.14. 8-[3%-(Dimethylamino)-1%H-naphtho[2%,1%-b]-
COCH CH ), 2.54–2.08 (m, 5H, 4-CH +CH CO), 3.26
2 3 3 2
pyran-1%-one-2%-yl]methyl-7-hydroxy-4-methylcoumarin
(q, 4H, NCH CH ), 4.32 (s, 2H, CH bridge), 6.29 (s,
2
3
2
(
9p)
1H, H-3), 8.21–6.70 (m, 7H, H arom.), 10.27 (d, 1H,
H-10%).
[
6b, 8g; 2; C; chloroform–ethanol 1:1], m.p. 256–
2
57 °C, 56.8% yield.
IR (KBr) w (cm ): 3100 (broad), 1730, 1630, 1605,
−
1
1
580, 1530.
5.3.1.19. 8-[3%-(1-Pyrrolidinyl)-1%H-naphtho[2%,1%-b]-
pyran-1%-one-2%-yl]methyl-7-hydroxycoumarin (9u) and
8-[3%-(1-pyrrolidinyl)-1%H-naphtho[2%,1%-b]pyran-1%-
one-2%-yl]methyl-7-acetoxycoumarin (9v)
1
H-NMR (l, CF OOD): 2.53 (s, 3H, 4-CH ), 3.87 (s,
3
3
6
8
1
H, CH N), 4.68 (s, 2H, CH bridge), 6.49 (s, 1H, H-3),
3 2
.05–7.20 (m, 6H, H arom.), 8.34 (d, 1H, H-5%), 9.31 (d,
H, H-10%).
9u:[6a, 8i; 1; D; chloroform–ethanol 1:1], m.p. 278–
2
80 °C, 42.5% yield.
−
1
5
.3.1.15. 8-[3%-(Dimethylamino)-1%H-naphtho[2%,1%-b]-
IR (KBr) w (cm ): 3100 (broad), 1730, 1630, 1605,
507.
pyran-1%-one-2%-yl]methyl-7-acetoxycoumarin (9q)
6a, 8g; 1; A; ethyl acetate], m.p. 189–190 °C, 79.5%
yield.
IR (KBr) w (cm ): 1755, 1705, 1630, 1595, 1506.
1
1
[
H-NMR (l, CF COOD): 2.70–2.35 (m, 4H, b-pyrro-
3
lidine CH ), 4.82–4.19 (m, 6H, CH bridge+a-pyrro-
lidine CH ), 6.58 (d, 1H, H-3), 8.48–7.15 (m, 8H,
H-4+H arom.), 9.40 (d, 1H, H-10%).
2
2
−
1
2
1
H-NMR (l, CDCl ): 2.08 (s, 3H, CH CO), 3.00 (s,
3
3
6H, NCH ), 4.28 (s, 2H, CH bridge), 6.36 (d, 1H, H-3),
3 2
9v:[6a, 8i; 1; D; ethyl acetate], m.p. 128–129 °C,
8
.20–6.83 (m, 8H, H-4+H arom.), 10.24 (d, 1H, H-10%).
.3.1.16. 8-[3%-(Diethylamino)-1%H-naphtho[2%,1%-b]-
4
3.2% yield.
−
1
IR (KBr) w (cm ): 1763, 1729, 1629, 1605, 1590.
5
1
H-NMR (l, CDCl ): 2.15–1.81 (m, 4H, b-pyrrolidine
3
pyran-1%-one-2%-yl]methyl-7-propionyloxycoumarin (9r)
6a, 8h; 2; A; ethyl acetate], m.p. 172–173 °C, 89.1%
yield.
CH ), 2.20 (s, 3H, CH CO), 4.01–3.62 (m, 4H, a-pyrro-
lidine CH ), 4.28 (s, 2H, CH bridge), 6.30 (d, 1H, H-3),
8
2
3
[
2
2
.11–6.84 (m, 8H, H arom.), 10.15 (d, 1H, H-10%).
−
1
IR (KBr) w (cm ): 1760, 1730, 1630, 1610, 1570.
1
H-NMR (l, CDCl ): 1.10–0.51 (m, 9H, NCH CH +
3
2
3
CH CH CO), 2.29 (q, 2H, CH CH CO), 3.25 (q, 4H,
3
2
3
2
5.3.1.20. 8-[3%-(1-Pyrrolidinyl)-1%H-naphtho[2%,1%-b]-
pyran-1%-one-2%-yl]methyl-7-hydroxy-4-methylcoumarin
NCH CH ), 4.32 (s, 2H, CH bridge), 6.40 (d, 1H, H-3),
2
3
2
8.31-6.74 (m, 8H, H-4+H arom.), 10.16 (d, 1H, H-10%).
(
9w)
6b, 8i; 1; C; chloroform–ethanol 1:1], m.p. 273–
274 °C, 74.1% yield.
[
5.3.1.17. 8-[3%-(Diethylamino)-1%H-naphtho[2%,1%-b]-
−
1
pyran-1%-one-2%-yl]methyl-7-acetoxy-4-methylcoumarin
9s)
6b, 8h; 1; C; ethyl acetate], m.p. 211–212 °C, 72.4%
yield.
IR (KBr) w (cm ): 3100 (broad), 1730, 1630, 1605,
(
1570, 1520.
1
[
H-NMR (l, CF OOD): 2.70–2.26 (m, 7H, 4-CH +
3
3
b-pyrrolidine CH ), 4.86–4.23 (m, 6H, a-pyrrolidine
2
−
1
IR (KBr) w (cm ): 1760, 1730, 1620, 1600, 1590.
CH +CH bridge), 6.47 (s, 1H, H-3), 8.49–7.18 (m, 7H,
2
2
1
H-NMR (l, CDCl3): 1.08–0.72 (t, 6H, NCH CH ),
H arom.), 9.35 (d, 1H, H-10%).
2
3

M. Mazzei et al. / European Journal of Medicinal Chemistry 36 (2001) 851–861
861
5.3.1.21. 8-[3%-(1-Piperidinyl)-1%H-naphtho[2%,1%-b]-
Acknowledgements
pyran-1%-one-2%-yl]methyl-7-acetoxycoumarin (9x)
[
6a, 8j; 1; C; chloroform–ethanol 1:1], m.p. 199–
This work was supported by grants from Italian
M.U.R.S.T.
2
00 °C, 70.1% yield.
−
1
IR (KBr) w (cm ): 1760, 1729, 1644, 1602.
1
H-NMR (l, CDCl ): 1.68–1.31 (m, 6H, b+g-pipe-
3
ridine CH ), 2.00 (s, 3H, CH CO), 3.38–3.06 (m, 4H,
a-piperidine CH ), 4.25 (s, 2H, CH bridge), 6.40 (d, 1H,
H-3), 8.20–6.85 (m, 8H, H arom), 10.23 (d, 1H, H-10%).
2
3
References
2
2
[1] Mazzei M., Garzoglio R., Sottofattori E., Melloni E., Michetti
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[
[
[
2] Mazzei M., Sottofattori E., Dondero R., Ibrahim M., Melloni
5
.3.1.22. 8-[3%-(1-Piperidinyl)-1%H-naphtho[2%,1%-b]-
E., Michetti M., Il Farmaco 54 (1999) 452–460.
pyran-1%-one-2%-yl]methyl-7-acetoxy-4-methylcoumarin
3] Tramontini M., Angiolini L., in: Rees C.W. (Ed.), Mannich
Bases: Chemistry and Uses, CRC Press, Boca Raton, FL, 1994.
(
9y)
[
6b, 8j; 1; C; chloroform–ethanol 1:1], m.p. 236–
4] Mazzei M., Dondero R., Balbi A., Bonora G.M., J. Heterocycl.
2
37 °C, 76.4% yield.
Chem. 38 (2001) 499–501.
−
1
IR (KBr) w (cm ): 1758, 1729, 1632, 1607.
[5] Ermili A., Roma G., Mazzei M., Balbi A., Cuttica A., Passerini
1
N., Il Farmaco Edn. Sci. 29 (1974) 225–236.
H-NMR (l, CDCl ): 1.66–1.30 (m, 6H, b+g-pipe-
3
[
6] Pontremoli S., Melloni E., Sparatore B., Michetti M., Salamino
ridine CH ), 1.99 (s, 3H, CH CO), 2.40 (s, 3H, 4-CH ),
3
bridge), 6.25 (s, 1H, H-3), 8.12–6.89 (m, 7H, H arom.),
2
3
3
F., Horecker B.L., J. Biol. Chem. 265 (1990) 706–712.
.39–3..02 (m, 4H, a-piperidine CH ), 4.23 (s, 2H, CH2
2
[
7] Ermili A., Mazzei M., Roma G., Cacciatore C., Il Farmaco
Edn. Sci. 32 (1977) 375–387.
10.15 (d, 1H, H-10%).
[
[
8] Mazzei M., Unpublished data.
9] Segal A.W., Abo A., Trends Biochem. Sci. 18 (1993) 43–
5.3.1.23. 8-[3%-Bis(2-methoxyethylamino)-1%H-
47.
naphtho[2%,1%-b]pyran-1%-one-2%-yl]methyl-7-acetoxy-4-
methylcoumarin (9z)
[10] Burnette W.N., Anal. Biochem. 112 (1981) 195–203.
[
11] Schepers T.M., Brier M.E., McLeish K.R., J. Biol. Chem. 267
1992) 784–789.
12] Holmes P.M., Tiffany H.L., Science 253 (1991) 1280–1283.
(
[
6b, 8k; 1; A; ethyl acetate], m.p. 166–167 °C, 91.1%
[
yield.
−
1
[13] Blobe G.C., Stribling S., Obeid L.M., Hannun Y.A., Cancer
IR (KBr) w (cm ): 1760, 1720, 1630, 1600,1505.
Surv. 27 (1996) 213–248.
1
H-NMR (l, CDCl ): 1.94 (s, 3H, 4-CH ), 2.43 (s,
3
3
[
14] Newton A.C., Curr. Opin. Cell Biol. 9 (1997) 161–167.
3
H, CH CO), 3.80–2.87 (m, 14H, NCH CH OCH ),
3 2 2 3
[
15] Dempsey E.C., Newton A.C., Mochly-Rosen D., Fields A.P.,
Reyland M.E., Insel P.A., Messing R.O., Am. J. Physiol Lung
Cell. Mol. Physiol. 279 (2000) 429–438.
4
.33 (s, 2H, CH bridge), 6.30 (s, 1H, H-3), 8.23–6.79
2
(
m, 7H, H arom.), 10.20 (d, 1H, H-10%).