Synthesis and in vitro inhibitory activity on human platelet aggregation of novel properly substituted 4-(1-piperazinyl)coumarins

Article abstract of DOI:10.1016/j.ejmech.2003.12.010

Pursuing our chemical and biological studies in this field, we described the multistep preparation of the new 5-, 6-, or 7-alkoxy and 7-alkoxy-8-methyl substituted 4-(1-piperazinyl)coumarins 5d-v, as well as the in vitro evaluation of their inhibitory activity on human platelet aggregation induced in platelet-rich plasma by ADP, collagen or the Ca²⁺ ionophore A23187. Compounds 5h-j,p,r-u showed notably high activity towards all the platelet aggregation inducers used, and the most active one, 8-methyl-4-(1-piperazinyl)- 7-(3-pyridylmethoxy)coumarin (5t), proved to be a potent in vitro antiplatelet agent.

Full text of DOI:10.1016/j.ejmech.2003.12.010

European Journal of Medicinal Chemistry 39 (2004) 397–409
www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro inhibitory activity on human platelet aggregation
of novel properly substituted 4-(1-piperazinyl)coumarins
Mario Di Braccio a, Giancarlo Grossi a, Giorgio Roma a,*,
Maria Grazia Signorello b, Giuliana Leoncini b
a Dipartimento di Scienze Farmaceutiche, Università di Genova, viale Benedetto XV, 16132 Genoa, Italy
b Dipartimento di Medicina Sperimentale, Sezione Biochimica, Università di Genova, viale Benedetto XV, 16132 Genoa, Italy
Received 11 November 2003; accepted 17 December 2003
Abstract
Pursuing our chemical and biological studies in this field, we described the multistep preparation of the new 5-, 6-, or 7-alkoxy and
7-alkoxy-8-methyl substituted 4-(1-piperazinyl)coumarins 5d-v, as well as the in vitro evaluation of their inhibitory activity on human platelet
aggregation induced in platelet-rich plasma by ADP, collagen or the Ca2+ ionophore A23187. Compounds 5h-j,p,r-u showed notably high
activity towards all the platelet aggregation inducers used, and the most active one, 8-methyl-4-(1-piperazinyl)-7-(3-
pyridylmethoxy)coumarin (5t), proved to be a potent in vitro antiplatelet agent.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Substituted 4-(1-piperazinyl)coumarins; Synthesis; Antiplatelet activity; Structure-activity relationships
1. Introduction
not obtainable, and the few derivatives prepared showed
low or very low activity [6].
In a number of previous papers we described the syntheses
and human platelet aggregation in vitro inhibitory properties
of substituted 2-aminochromones 1 [1–3], their benzo-fused
derivatives 2-4 [3,4], and the corresponding 4-amino-
coumarin derivatives 5-8 [3,4], as well as of the 1,2-fused
pyrimidine derivatives 9-11 [4–7] and 12-14 [6], isosteric
analogues of compounds 1-3 and 5-7, respectively. Also
several compounds 15, whose structures were derived from
that of the very active compound 9a [6] (Table 1) by properly
modifying its pyridine ring and/or 2-substituent, were syn-
thesized and tested for their antiplatelet activity [4,5,7]
(Fig. 1).
Considering the antiplatelet activity data afforded by the
compounds 1-15 tested in the above studies, towards all the
platelet aggregation inducers employed [adenosine diphos-
phate (ADP), collagen, and the Ca2+ ionophore A23187], the
following remarks can be made.
• The impressive activity difference between compounds
9a [6] and 15b [7] (Table 1) seems to confirm the great
importance for the activity, in this structural field, of the
characteristic b-enaminonic moiety.
• Nevertheless, the activity of the b-enaminonic (1-
piperazinyl)derivatives 1-11, 15 is appreciably influ-
enced by the structure of the supporting cyclic system
(Table 1).
• On the whole, the 7,8-disubstituted 4-(1-pipera-
zinyl)coumarin 5a [4] is clearly the most active of all
compounds 1-15 previously studied by us, and the pres-
ence of proper substituents in the benzene ring proved to
be very effective (see Table 1, compounds 5a, 5b and
5c).
In this connection, the antiplatelet activity data previously
shown by the most active compounds 1-11, 15 [i.e. the
(1-piperazinyl) substituted derivatives 1a-11a, 15a] and by
compounds 5b,c and 15b are summarized in Table 1.
Regarding the expected bioisosterism of the 2,3-fused
pyran derivatives 1-3 and 1,2-fused pyrimidine derivatives
9-11, it is interesting to point out that both 2-(diethylamino)-
7-hydroxychromone [8,9], chosen as an example of com-
pounds 1, and 2-(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-
• In each structural class 1-piperazinyl was the most effec-
tive amino substituent among all those used. In the case
of compounds 12-14 the (1-piperazinyl)derivatives were
* Corresponding author.
E-mail address: roma@unige.it (G. Roma).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.12.010

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M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
Table 1
In vitro inhibitory activity of some significant previously describeda compounds 1-11, 15 (Figure 1) on human platelet aggregation induced in PRPb by ADP,
collagen and A23187 (see Introduction)
IC (µM) SD
50
2
3
1
ADP
(5.0 µM)
Collagen
A23187
(20.0 µM)
R
R
Compd
X
Y
N
NRR
(5.0 µg mL-1)c
N
N
N
N
N
NH
NH
NH
NH
NH
1a
2a
3a
4a
5a
H
H
-
-
-
-
-
-
-
-
-
56 13
39 3
60 12
56 16
240 90
201 61
278 15
180 18
1.1 0.2
-
-
-
-
-
-
-
119 40
105 30
1.9 0.2
117 32
159 31
1.8 0.4
-
H
7-OCH
2
8-CH
-
-
-
-
-
-
3
N
N
N
N
N
N
N
N
NH
NH
NH
NH
NH
NH
NH
NH
7-OCH(CH )
3 2
5b
H
H
-
5.6 1.4
49 14
53 15
81 23
24 4
4.4 2.0
36 10
49 17
71 27
31 14
3.6 1.2
15 8
3.8 1.0
250 38
245 65
334 89
32 8
5c
H
-
-
-
-
-
-
-
6a
7a
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CH
8a
-
3
9a
H
-
H
-
O
-
6 1.8
13 4
19 9
10a
11a
15a
28 8
-
-
-
38 10
3.6 1.2
21 9
54 17
13.0 5
O
N
NH
-
-
-
-
-
8.8 5.6
N
Cl
15b
>1000
>1000
>1000
-
-
-
CH
N
NH
2
N
a Compounds 1a, 2a, 3a, 5c, 6a, 7a : ref 3; compounds 4a, 5a,b, 8a, 15a : ref 4; compounds 9a, 10a, 11a : ref 6; compound 15b : ref 7.
b PRP = platelet-rich plasma.
c 10.0 µg/mL for compounds 1a, 2a, 3a, 5c, 6a, 7a.
4-one 9a [6,10] (the most active among all compounds 1-4,
9-11 studied by us) proved to exert their platelet antiaggre-
gating activity by specifically inhibiting the activity of high
affinity cAMP phosphodiesterase and, consequently, increas-
ing the intracellular cAMP concentration.
activity relationships in this structural class and, if possible,
obtain further significantly active compounds.
2. Chemistry
Starting from the results outlined above, we have now
regarded it interesting to synthesize and test in vitro for their
human platelet antiaggregating properties several novel 4-(1-
piperazinyl)coumarins 5 suitably substituted in the benzene
ring, in order to increase our knowledge of the structure-
The intermediates 17d,e,h-v, 18d,e,h-v, 19d-h,l,m,p-t,
and the final 4-(1-piperazinyl)coumarin derivatives 5i-
k,n,o,u,v and 5d-h,l,m,p-t were prepared as illustrated in
Scheme 1.

M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
399
2
R
3
R
NRR1
O
NRR1
NRR1
O
O
NRR1
O
O
R2
4
O
O
O
O
O
O
3
1
2
2
R
3
R
O
O
O
O
O
R2
NRR1
NRR1
NRR1
NRR1
8
5
7
6
NRR1
N
X
NRR1
R2
N
N
O
NRR1
R3
N
N
N
O
9
10
11
N
O
Y
N
N
O
N
O
N
O
15
N
N
N
NRR1
NRR1
NRR1
12
13
14
Fig. 1. Structures of 2-aminochromone and 4-aminocoumarin bicyclic and tricyclic derivatives 1-4 and 5-8, pyrido[1,2-a]pyrimidine bicyclic and tricyclic
derivatives 9-11 and 12-14, and 1,2-fused pyrimidine derivatives 15.
Thus, the treatment of the dihydroxyacetophenones 16a-c
with proper alkyl halides (anhydrous K2CO3 or KOH/
anhydrous K2CO3, dry 2-butanone at reflux) afforded the
corresponding alkoxyderivatives 17d,e,h-v, whose cyclo-
condensation with diethyl carbonate in the presence of Na
(Dowtherm A, 150°C) gave good yields of the substituted
4-hydroxycoumarins 18d,e,h-v. Compounds 18i-k,n,o,u,v
were in turn heated (160°C) with a large excess of piperazine
to give the corresponding 4-(1-piperazinyl)coumarin deriva-
tives 5i-k,n,o,u,v.
On the other hand, in the case of compounds 5d-h,l,m,p-t,
they were more conveniently prepared via the intermediate
4-chloroderivatives 19. Actually, by heating (130°C) the
4-hydroxycoumarins 18d-h,l,m,p-t with excess phosphorus
oxychloride, in the presence of triethylamine, the
4-chloroderivatives 19d-h,l,m,p-t were obtained, which
were then treated with excess piperazine (ethanol, room
temperature) to afford the above mentioned 4-(1-
piperazinyl)coumarins 5. The starting 4-hydroxycoumarin
derivatives 18f and 18g were previously described in the
literature [11].
3. Biological results and discussion
The twenty novel compounds 5d-v and 21 were tested in
vitro for their inhibitory activity on the human platelet aggre-
gation induced in platelet-rich plasma (PRP) by adenosine
diphosphate (ADP), collagen, or the Ca2+ ionophore A23187
(calcimycin) (see Experimental). Acetylsalicylic acid
(ASA), trifluoperazine and propranolol were also tested un-
der the same conditions as reference compounds. The IC50
values obtained are reported in Table 2 and suggest the
following observations on the structure-activity relationships
(SAR) in this structural field, taking the 4-(1-
piperazinyl)coumarin 5c [3] as a lead (see Table 1).
• Concerning the 7-alkoxyderivatives of 5c, it can be
pointed out that, as we previously reported for the
7-ethoxy-4-(1-piperazinyl) [3] and 7-isopropoxy-4-(1-
piperazinyl) [4] substituted coumarins 5 (see Table 1,
compound 5b), also the 7-(sec-butoxy) and 7-benzyloxy
substituted 4-(1-piperazinyl)coumarins 5d and 5g, re-
spectively, (Table 2) have now proved to be differently,
but significantly more active than 4-(1-piperazinyl)
coumarin 5c (Table 1), towards all the platelet aggrega-
tion inducers used (P< 0.0005 for 5d in the presence of
all tested agonists ; P< 0.0005 in the presence of ADP
and A23187, and P< 0.0125 in the presence of collagen,
for 5g). Compound 5b [4] appears to be the most active
one synthesized by us in this class of derivatives.
Finally, 6-methyl-4-(1-piperazinyl)-2H-pyran-2-one (21)
was prepared by treating the corresponding 4-chloro-
derivative 20 [12] with an excess of piperazine (ethanol,
room temperature) (see Scheme 1).
The structures attributed to the compounds described in
this paper are supported by the results of elemental analyses,
as well as by the IR and 1H NMR spectral data (see Experi-
mental) which agree with the ones previously reported by us
for analogous compounds [3,4].
• In this connection, the antiplatelet activity data afforded
by the 5-, 6-, and 7-benzyloxy substituted isomers 5e, 5f
and 5g, respectively, seem to confirm that the 7-alkoxy-
derivative is the most effective one in order to increase

400
M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
3
3
3
R
3'
R
3'
R
1
OC2H5
OC2H5
8
OH
OH
O C
O
O
2'
1'
2'
1'
4'
5'
4'
5'
7
6
Alkyl halide
2
3
2
2
2
R
R'
R'
CH3
CH3
anhydrous K CO or
2
3
, ∆
1) Na, Dowtherm A, ∆
C
O
C
O
6'
6'
4
5
KOH/an K CO
+
2) H O
3
2
3
OH
16a-c
17d,e,h-v
18d,e,h-v
2
3
Compd
R'
R
2
2
or
R3
R
R'
Compounds
4'-OH
16a
16b
16c
H
H
CH3
7-OCH C2H5
6'-OH
4'-OH
H
H
4'- or
17d, 18d, 19d, 5d
17e, 18e, 19e, 5e
CH3
6'- or 5-OCH2
3
3
R
R
6-OCH2
7-OCH2
H
H
O
O
O
N
O
18f, 19f, 5f
HN
NH
2
2
R
R
18g, 19g, 5g
∆
OH
NH
CH3
CH3
7-O(CH2)2CH3
17h, 18h, 19h, 5h
17i, 18i, 5i
4'- or
18i-k,n,o,u,v
5i-k,n,o,u,v
4'- or 7-OCH(CH3)2
4'- or 7-O(CH2)3CH3
CH3
CH3
17j, 18j, 5j
3
3
4'- or
17k, 18k, 5k
7-OCH2
R
R
O
O
O
O
CH3
POCl3
2
2
R
R
CH3
CH3
17l, 18l, 19l, 5l
4'- or
7-OCH2
(C2H5)3N, ∆
CH3
OH
Cl
7-OCH2
7-OCH2
4'- or
4'- or
17m, 18m, 19m, 5m
17n, 18n, 5n
18d-h,l,m,p-t
19d-h,l,m,p-t
OCH3 CH3
17o, 18o, 5o
4'- or 7-OCH2
CH3
Cl
CH3
3
R
HN
NH
O
N
O
CH3
CH3
7-OCH
4'- or
4'- or
4'- or
17p, 18p, 19p, 5p
17q, 18q, 19q, 5q
2
R
7-OCH2CH2
7-OCH2
NH
17r, 18r, 19r, 5r
CH3
CH3
5d-h,l,m,p-t
N
7-OCH2
4'- or
17s, 18s, 19s, 5s
N
CH3
O
O
4'- or 7-OCH2
4'- or
17t, 18t, 19t, 5t
17u, 18u, 5u
CH3
CH3
CH3
CH3
O
O
HN
NH
7-OCH2
N
N
N
NH
Cl
4'- or 7-OCH2
17v, 18v, 5v
20
21
Scheme 1. Synthetic routes to the substituted 4-(1-piperazinyl)coumarins 5d-v and 6-methyl-4-(1-piperazinyl)-2H-pyran-2-one 21.
the activity of the lead compound 5c, whose activity, on
the contrary, can be strongly decreased by the presence
of 5-alkoxy substituent (see 5c and 5e, Tables 1 and 2,
respectively).
• On the other hand, the biological data until now obtained
by us clearly indicate that the introduction of 8-methyl
substituent usually increases, depending on the type of
alkoxy substituent, the antiplatelet activity of 7-alkoxy
derivatives of 5c. For instance, the antiplatelet activity
differences between compounds 5i and 5b [4] and be-
tween compounds 5a [4] and 5g (see Tables 1 and 2)
correspond respectively, on the whole, to the lowest and
the highest increase of activity observed by us between a
7-alkoxy-4-(1-piperazinyl)coumarin and its 8-methyl-
derivative.
• Considering in detail the IC50 values now obtained for
the 7-alkoxy-8-methyl derivatives of 5c (5h-v)
(Table 2), we can observe that both compounds 5h-k,
whose 7-substituents contain only alkyl or cycloalkyl
moieties, and compounds 5l-q, whose 7-substituents
contain benzyl moieties differently substituted either in
the phenyl ring or in the methylene group, generally
show notable and in some cases (5h-j,p) very high
antiplatelet activity, but always lower than that of the
7-benzyloxy-8-methyl-4-(1-piperazinyl)coumarin 5a
(Table 1). The isosteric replacement in compound 5a of

M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
401
Table 2
In vitro inhibitory activity of the new (1-piperazinyl) substituted compounds 5d-v, 21 on platelet aggregation induced in human PRP by ADP, collagen and
A23187
3
R
CH3
O
O
O
N
O
2
R
N
NH
21
5d-v
NH
IC 50 (µM) SD
Collagen (5.0 µg mL-1)
R2
R3
Compd
ADP (5.0 µM)
A23187 (20.0 µM)
CH
3
H
H
9.3 5.4
187 42
18.0 3.1
144 40
29.5 5.1
429 105
5d
5e
7-OCH C H
2
5
5-OCH
6-OCH
7-OCH
2
2
2
23.6 4.7
16.3 4.1
34.2 3.3
24.2 5.1
116 12.3
78.8 10.3
5f
H
H
5g
2.95 0.78
2.4 0.5
2.58 0.46
3.2 0.7
7.77 1.36
4.4 1.8
5h
5i
7-OCH CH CH
CH
3
2
2
3
7-OCH(CH )
CH
3
3
2
2.79 1.30
1.92 0.96
5.03 1.02
5j
7-OCH CH CH CH
CH
3
2
2
2
3
4.9 1.8
7.6 1.4
27.0 6.0
5k
7-OCH
CH
3
2
CH
3
9.8 4.2
11.7 2.6
26.8 8.2
5l
CH
7-OCH
3
2
CH
3
11.1 2.6
3.8 1.2
26.5 5.8
4.3 0.7
3.7 1.4
43.2 12.5
15.2 3.8
41.0 17.5
5m
5n
5o
CH
7-OCH
7-OCH
7-OCH
3
2
2
2
CH
OCH
3
3
13.1 1.5
CH
Cl
3
CH
3
2.2 0.8
4.4 2.6
3.2 0.8
7.9 2.7
11.6 2.9
21.7 6.2
5p
5q
CH
3
7-OCH
CH
7-OCH CH
3
2
2
6.6 1.4
6.3 2.8
6.9 1.0
5r
CH
7-OCH
7-OCH
7-OCH
7-OCH
3
2
2
2
2
N
2.14 0.63
0.98 0.36
2.9 0.7
1.40 0.58
0.51 0.12
2.2 1.0
5.12 1.46
1.42 0.43
7.8 1.0
5s
5t
CH
3
N
CH
3
5u
N
CH
3
5.6 1.5
273 39
6.1 1.7
441 71
29.9 3.8
324 43
5v
21
CH
3
7-OCH
N
2
-
-
>1000
180 48
291 76
183 4
138 41
131 31
>1000
318 61
269 62
ASA
Trifluoperazine
Propranolol
the 7-benzyloxy with the three isomeric 7-(pyridyl-
methoxy) substituents afforded a group of novel very
active antiplatelet agents (compounds 5s-u), among
which the 8-methyl-4-(1-piperazinyl)-7-(3-pyridyl-
methoxy)coumarin 5t, clearly due to the particular elec-
tronic effect of the 3-pyridyl substituent, proved to be
the overall most active in vitro antiplatelet agent until
now synthesized by us, and significantly more active
than 5a in the presence of ADP and collagen
(P< 0.0005).
• With reference to the steric hindrance of 7-alkoxy sub-
stituents of compounds 5l-v, it can be pointed out that

402
M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
compounds 5l-q and the 7-(1-naphthylmethoxy) substi-
tuted compound 5r (with bulkier 7-substituents) are all
less active than their 7-benzyloxy analogue 5a, as well
as the 7-(2-quinolylmethoxy)derivative 5v is less active
than its 7-(2-pyridylmethoxy) analogue 5s.
• Finally, the highly different potencies shown by the
4-(1-piperazinyl)-2H-pyran-2-one derivatives 21
(Table 2) and 5c (Table 1) further confirm the impor-
tance of the supporting cyclic system for the compound
antiplatelet activity in this structural class.
cal values and were performed by the Laboratorio di Mi-
croanalisi, Dipartimento di Scienze Farmaceutiche, Univer-
sity of Genoa.
Thin-layer chromatograms were run on Merck silica gel
60 F254 precoated plastic sheets (layer thickness 0.2 mm).
Column chromatography was performed using Carlo Erba
silica gel (0.05 - 0.20 mm) or Carlo Erba neutral aluminium
oxide (Brockmann activity I).
5.1.1. General procedure for the synthesis of substituted
acetophenones 17d,e,h-v
A mixture of 1-(2,4-dihydroxyphenyl)ethanone 16a
(3.80 g, 25.0 mmol), 1-(2,6-dihydroxyphenyl)ethanone 16b
(3.80 g, 25.0 mmol), or 1-(2,4-dihydroxy-3-methyl-
phenyl)ethanone 16c (4.15 g, 25.0 mmol), 25.0 mmol of the
proper alkyl halide, 1.50 g of finely powdered KOH mixed
with 4.0 g of anhydrous K2CO3 (in the case of preparation of
compounds 17d,h-j only 5.0 g of anhydrous K2CO3 were
used) and 2-butanone (150 mL) was heated at reflux for 6 h,
with stirring. The mixture was then poured into cold water
(500 mL) after which, in many cases , the rough compound
17 separated out as a solid which was filtered, washed with
water and dried: the pure compounds 17o and 17v were
plainly obtained by crystallization of this solid from isopro-
pyl ether. On the contrary, in the case of compounds 17d,h-
j,s-u an emulsion was obtained which was exhaustively ex-
tracted with ethyl ether-ethyl acetate (1:1): the combined
extracts were then washed with water, dried ( anhydrous
Na2SO4), and solvents were removed under reduced pressure
to give an oily or nearly solid residue. Both the solids recov-
ered by filtration (except for 17o and 17v ) and the residues
obtained by extraction were then chromatographed on a
silica gel column to recover pure compound 17, generally
eluting with dichloromethane-petroleum ether (1:1); in the
only case of the (pyridylmethoxy)derivatives 17s-u their elu-
tion was carried out with ethyl acetate, after discarding some
impurities previously eluted with dichloromethane. The pure
compounds 17 were finally crystallized from the proper sol-
vent, or distilled in vacuo (17d). According to this procedure
the following compounds were prepared (1H-NMR and IR
data are reported only for compound 17t, chosen as an
example).
4. Conclusions
The biological results obtained for the compounds 5 de-
scribed in this paper confirm the high effectiveness of
7-alkoxy-8-methyl-4-(1-piperazinyl)coumarins as in vitro
antiplatelet agents, depending on the structure of the
7-substituent.
The now synthesized 8-methyl-4-(1-piperazinyl)-7-(3-
pyridylmethoxy)coumarin 5t proved to be a potent in vitro
inhibitor of human platelet aggregation towards all the plate-
let aggregation inducers used, suggesting proper heteroaryl-
methoxy groups as novel useful 7-alkoxy substituents.
In this connection, further biological studies have been
expressly carried out to investigate the mode of action of 5t
[13]. The results of these experiments have shown that 5t
inhibits human platelet aggregation by increasing intracellu-
lar cAMP levels through the specific inhibition of cAMP
phosphodiesterase [13], as previously reported by us for
2-(diethylamino)-7-hydroxychromone [9] and 2-(1-pipera-
zinyl)-4H-pyrido[1,2-a]pyrimidin-4-one 9a [10], studied as
examples of compounds 1 and 9, respectively (see Introduc-
tion); concerning this, compound 5t has also proved to re-
duce intracellular calcium levels and protein kinase C (PKC)
activation induced by thrombin [13]. In addition, the cou-
marin derivative 5t increases the production of nitric oxide,
well known antiaggregating compound, by directly stimulat-
ing the platelet nitric oxide synthase enzyme [13].
5. Experimental protocols
5.1.1.1. 1-[4-(sec-Butoxy)-2-hydroxyphenyl]ethanone (17d).
Obtained (3.39 g, 65%) from reaction of 16a with
2-iodobutane (4.60 g); colourless liquid, b.p. 125-127°C
(0.3 mm Hg). Anal. (C12H16O3) C, H.
5.1. Chemistry
Melting points were determined using a Fisher-Johns ap-
paratus and are uncorrected. IR spectra were recorded on a
Perkin-Elmer ″Spectrum One″ spectrophotometer (abbrevia-
tions relative to IR bands: br = broad, s = strong, w = weak,
sh = shoulder). 1H-NMR spectra were recorded on a Varian
Gemini 200 (200 MHz) spectrometer and chemical shifts (d)
are reported in ppm using (CH3)4Si as an internal reference
(d = 0). Spin multiplicities are given as follows: s (singlet), d
(doublet), dd (double doublet), t (triplet), q (quartet), m
(multiplet). Analyses of all new compounds, indicated by the
symbols of the elements, were within 0.4% of the theoreti-
5.1.1.2. 1-(6-Benzyloxy-2-hydroxyphenyl)ethanone (17e).
Obtained (4.12 g, 68%) from reaction of 16b with benzyl
chloride (3.16 g); white crystals, m.p. 109-110°C (isopropyl
ether). Anal. (C15H14O3) C, H.
5.1.1.3. 1-(2-Hydroxy-3-methyl-4-propoxyphenyl)ethanone
(17h). Obtained (4.68 g, 90%) from reaction of 16c with
1-iodopropane (4.25 g); white crystals, m.p. 36-37°C (petro-
leum ether). Anal. (C12H16O3) C, H.

M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
403
5.1.1.4. 1-(2-Hydroxy-4-isopropoxy-3-methylphenyl)etha-
none (17i). Obtained (3.49 g, 67%) from reaction of 16c with
2-iodopropane (4.25 g); white crystals, m.p. 60-61°C (petro-
leum ether). Anal. (C12H16O3) C, H.
16c with 2-(chloromethyl)pyridine hydrochloride (4.10 g);
whitish crystals, m.p. 128-129°C (isopropyl ether). Anal.
(C15H15NO3) C, H, N.
5.1.1.15. 1-[2-Hydroxy-3-methyl-4-(3-pyridylmethoxy)phe-
nyl]ethanone (17t). Obtained (2.64 g, 41%) from reaction of
16c with 3-(chloromethyl)pyridine hydrochloride (4.10 g);
5.1.1.5. 1-(4-Butoxy-2-hydroxy-3-methylphenyl)ethanone
(17j). Obtained (3.45 g, 62%) from reaction of 16c with
1-iodobutane (4.60 g); white crystals, m.p. 52-53.5°C (petro-
leum ether). Anal. (C13H18O3) C, H.
1
white crystals, m.p. 122-123°C (isopropyl ether). H-NMR
(CDCl3) d 2.15 (s, 3H, 3-CH3), 2.57 (s, 3H, COCH3), 5.17 (s,
2H, OCH2C5H4N), 6.50 (d of AB q, J = 9 Hz, 1H, H-5), 7.32
(m, 1H, pyridyl H-5’), 7.60 (d of AB q, J = 9 Hz, 1H, H-6),
7.79 (near d, 1H, pyridyl H-4’), 8.60 (near d, 1H, pyridyl
H-6’), 8.70 (near s, 1H, pyridyl H-2’), 12.78 (s, 1H, OH;
disappeared with D2O). IR (KBr): 3130-2520 br, w (chelated
OH), 1630 s (CO), 1594, 1580, 1500 cm–1. Anal.
(C15H15NO3) C, H, N.
5.1.1.6. 1-[4-(Cyclohexylmethoxy)-2-hydroxy-3-methylphe-
nyl]ethanone (17k). Obtained (1.18 g, 18%) from reaction of
16c with (bromomethyl)cyclohexane (4.43 g); white crystals,
m.p. 128-129°C (petroleum ether). Anal. (C16H22O3) C, H.
5.1.1.7. 1-[2-Hydroxy-3-methyl-4-(2-methylbenzyloxy)phe-
nyl]ethanone (17l). Obtained (3.12 g, 46%) from reaction of
16c with 2-methylbenzyl chloride (3.51 g); white crystals,
m.p. 111-112°C (isopropyl ether/petroleum ether). Anal.
(C17H18O3) C, H.
5.1.1.16. 1-[2-Hydroxy-3-methyl-4-(4-pyridylmethoxy)phe-
nyl]ethanone (17u). Obtained (2.78 g, 43%) from reaction of
16c with 4-(chloromethyl)pyridine hydrochloride (4.10 g);
white needles, m.p. 120-121°C (isopropyl ether). Anal.
( C15H15NO3) C, H, N.
5.1.1.8. 1-[2-Hydroxy-3-methyl-4-(3-methylbenzyloxy)phe-
nyl]ethanone (17m). Obtained (3.04 g, 45%) from reaction
of 16c with 3-methylbenzyl chloride (3.51 g); white crystals,
m.p. 118-119°C (isopropyl ether). Anal. (C17H18O3) C, H.
5.1.1.17. 1-[2-Hydroxy-3-methyl-4-(2-quinolylmethoxy)phe-
nyl]ethanone (17v). Obtained (3.46 g, 45%) from reaction of
16c with 2-(chloromethyl)quinoline hydrochloride (5.35 g);
white needles, m.p. 134-135°C (isopropyl ether). Anal.
(C19H17NO3) C, H, N.
5.1.1.9. 1-[2-Hydroxy-4-(4-methoxybenzyloxy)-3-methyl-
phenyl]ethanone (17n). Obtained (4.44 g, 62%) from reac-
tion of 16c with 4-methoxybenzyl chloride (3.91 g); white
crystals, m.p. 128-129°C (isopropyl ether).Anal. (C17H18O4)
C, H.
5.1.2. General procedure for the synthesis of substituted
4-hydroxy-2H-1-benzopyran-2-ones 18d,e,h-v
A mixture of 10.0 mmol of the proper substituted ac-
etophenone 17, sodium (0.58 g, 25.0 mmol), diethyl carbon-
ate (3.54 g, 30.0 mmol) and DowthermA (10 mL) was heated
at 160°C with stirring. Within few minutes, after the sodium
melting, a sudden reaction occurred resulting in the precipi-
tation of an abundant whitish solid. Xylene (30 mL) was
added in order to dilute the mixture and allow the stirring,
then the mixture was further heated at 160°C for 1 h. After
cooling, methanol (10 mL) was added in order to inactivate
residual sodium, then the mixture was poured into cold water
(400 mL). By adding then 2N aqueous NaOH (100 mL) and
ethyl ether (200 mL) and stirring few minutes at room tem-
perature, in most cases the separation of two limpid phases
was obtained. The aqueous phase was collected and the
organic one extracted twice with water, then discarded. The
whole aqueous phase was acidified with 6N aqueous HCl (or
carefully neutralized with 6N aqueous HCl, then acidified
down to pH 4 with citric acid, in the case of the amphoteric
compounds 18s-v): this way compounds 18d,e,h-k,n,o,s-v
separated out as whitish solids that were collected by filtra-
tion, washed with water, dried and crystallized from the
proper solvent. On the other hand, in the case of compounds
18l,m,p-r, the separation of two clear phases did not occur,
therefore the whole resulting mixture was acidified with 6N
aqueous HCl. This way compounds 18l,m,p-r, separated out
5.1.1.10. 1-[4-(4-Chlorobenzyloxy)-2-hydroxy-3-methylphe-
nyl]ethanone (17o). Obtained (4.65 g, 64%) from reaction of
16c with 4-chlorobenzyl chloride (4.03 g); white crystals,
m.p. 144-145°C (isopropyl ether). Anal. (C16H15ClO3) C, H,
Cl.
5.1.1.11. 1-[2-Hydroxy-3-methyl-4-(1-phenylethoxy)phenyl]
ethanone (17p). Obtained (3.72 g, 55%) from reaction of 16c
with (1-bromoethyl)benzene (4.63 g); white crystals, m.p.
94-95°C (petroleum ether). Anal. (C17H18O3) C, H.
5.1.1.12. 1-[2-Hydroxy-3-methyl-4-(2-phenylethoxy)phenyl]
ethanone (17q). Obtained (1.35 g, 20%) from reaction of 16c
with (2-bromoethyl)benzene (4.63 g); white crystals, m.p.
93-94°C (petroleum ether). Anal. (C17H18O3) C, H.
5.1.1.13.
1-[2-Hydroxy-3-methyl-4-(1-naphthylmethoxy)
phenyl]ethanone (17r). Obtained (4.21 g, 55%) from reac-
tion of 16c with 1-(chloromethyl)naphthalene (4.42 g); white
needles, m.p. 135-136°C (isopropyl ether).Anal. (C20H18O3)
C, H.
5.1.1.14. 1-[2-Hydroxy-3-methyl-4-(2-pyridylmethoxy)phe-
nyl]ethanone (17s). Obtained (3.73 g, 58%) from reaction of

404
M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
as insoluble solids, were recovered by filtration, washed with
water, dried, treated with ethyl ether (50 mL) in order to
remove the adsorbed Dowtherm A, and finally crystallized
from the proper solvent.
5.1.2.6. 7-(Cyclohexylmethoxy)-4-hydroxy-8-methyl-2H-1-
benzopyran-2-one (18k). Obtained (2.36 g, 82%) from 17k
(2.62 g); white crystals, m.p. 243-244°C (ethanol). 1H-NMR
(DMSO-d6) d 0.94-1.43 and 1.58-1.95 (2 m, 11H, cyclohexyl
H’s), 2.17 (s, 3H, 8-CH3), 3.89 (d, 2H, OCH2C6H11), 5.44 (s,
1H, H-3), 7.00 (d of AB q, J = 9 Hz, 1H, H-6), 7.64 (d of AB
q, J = 9 Hz, 1H, H-5), 12.28 (broad s, 1H, OH; disappeared
with D2O). IR (KBr): 3200-2570 br, complex (OH), 1673 br
(CO), 1600 s, br, 1562, 1512 cm-1. Anal. (C17H20O4) C, H.
5.1.2.1. 7-(sec-Butoxy)-4-hydroxy-2H-1-benzopyran-2-one
(18d). Obtained (1.76 g, 75%) from 17d (2.08 g); white
crystals, m.p. 182-183°C (ethyl acetate/isopropyl ether). 1H-
NMR (CDCl3) d 0.99 (t, 3H, CH2CH3), 1.34 (d, 3H,
CHCH3), 1.54-1.92 (m, 2H, CHCH2CH3), 4.39 [near q, 1H,
OCH(CH3)CH2CH3], 5.82 (s, 1H, H-3), 6.73-6.89 (m, 2H,
H-6,8), 7.77 (d of AB q, J = 9 Hz, 1H, H-5), 10.60 (broad s,
1H, OH; disappeared with D2O). IR (KBr): 3200-2400 s, br,
complex (OH), 1680 s, br (CO), 1612 s, br, 1546, 1518 cm-1.
Anal. (C13H14O4) C, H.
5.1.2.7. 4-Hydroxy-8-methyl-7-(2-methylbenzyloxy)-2H-1-
benzopyran-2-one (18l). Obtained (2.07 g, 70%) from 17l
(2.70 g); whitish crystals, m.p. 223-223.5°C (ethyl
acetate/isopropyl ether). 1H-NMR (DMSO-d6) d 2.21 (s, 3H,
8-CH3), 2.34 (s, 3H, C6H4CH3), 5.25 (s, 2H,
OCH2C6H4CH3), 5.48 (s, 1H, H-3), 7.15-7.34 and 7.42-7.52
(2 m, 4H + 1H, H-6 + C6H4CH3), 7.70 (d of AB q, J = 9 Hz,
1H, H-5), 12.30 (broad s, 1H, OH; disappeared with D2O).
IR (KBr): 3150-2300 br, complex (OH), 1646 br (CO),
1597 s, 1557, 1513 cm-1. Anal. (C18H16O4) C, H.
5.1.2.2.
5-Benzyloxy-4-hydroxy-2H-1-benzopyran-2-one
(18e). Obtained (1.85 g, 69%) from 17e (2.42 g); white
crystals, m.p. 138-139°C (ethyl acetate). 1H-NMR (CDCl3) d
5.28 (s, 2H, OCH2C6H5), 5.66 (s, 1H, H-3), 6.90 and 7.07
(2d, J = 9 Hz, 1H + 1H, H-6,8), 7.42-7.52 (m, 6H, H-7 +
phenyl H’s), 9.61 (s, 1H, OH; disappeared with D2O). IR
(KBr): 3296 (OH), 1709 s (CO), 1655, 1608, 1569 w cm-1.
Anal. (C16H12O4) C, H.
5.1.2.8. 4-Hydroxy-8-methyl-7-(3-methylbenzyloxy)-2H-1-
benzopyran-2-one (18m). Obtained (2.19 g, 74%) from 17m
(2.70 g); white crystals, m.p. 218-219°C (ethyl acetate).
1H-NMR (DMSO-d6) d 2.22 (s, 3H, 8-CH3), 2.34 (s, 3H,
C6H4CH3), 5.21 (s, 2H, OCH2C6H4CH3), 5.47 (s, 1H, H-3),
7.12 (d of AB q, J = 9 Hz, 1H, H-6), 7.17-7.35 (m, 4H,
C6H4CH3), 7.67 (d ofAB q, J = 9 Hz, 1H, H-5), 12.30 (broad
s, 1H, OH; disappeared with D2O). IR (KBr): 3130-2300 br,
complex (OH), 1662 (CO), 1593 s, 1545, 1514 cm-1. Anal.
(C18H16O4) C, H.
5.1.2.3. 4-Hydroxy-8-methyl-7-propoxy-2H-1-benzopyran-
2-one (18h). Obtained (1.71 g, 73%) from 17h (2.08 g);
white crystals, m.p. 211-212°C (ethanol). 1H-NMR (DMSO-
d6) d 1.00 (t, 3H, CH2CH2CH3), 1.80 (near q, 2H,
CH2CH2CH3), 2.16 (s, 3H, 8-CH3), 4.04 (t, 2H,
OCH2CH2CH3), 5.44 (s, 1H, H-3), 7.00 (d ofAB q, J = 9 Hz,
1H, H-6), 7.64 (d ofAB q, J = 9 Hz, 1H, H-5), 12.30 (broad s,
1H, OH; disappeared with D2O). IR (KBr): 3230-2500 s, br,
complex (OH), 1678 s (CO), 1603 s, br, 1550, 1520 cm-1.
Anal. (C13H14O4) C, H.
5.1.2.9. 4-Hydroxy-7-(4-methoxybenzyloxy)-8-methyl-2H-1-
benzopyran-2-one (18n). Obtained (2.44 g, 78%) from 17n
1
(2.86 g); whitish crystals, m.p. 212-212.5°C (ethanol). H-
NMR (DMSO-d6) d 2.17 (s, 3H, 8-CH3), 3.76 (s, 3H, OCH3),
5.16 (s, 2H, OCH2C6H4OCH3), 5.46 (s, 1H, H-3), 6.77 and
7.41 (AB q, J = 9 Hz, 2H + 2H, C6H4OCH3), 7.12 (d ofAB q,
J = 9 Hz, 1H, H-6), 7.64 (d ofAB q, J = 9 Hz, 1H, H-5), 12.30
(broad s, 1H, OH; disappeared with D2O). IR (KBr): 3200-
2420 br, complex (OH), 1667 s (CO), 1601 s, br, 1569,
1515 cm–1. Anal. (C18H16O5) C, H.
5.1.2.4.
4-Hydroxy-7-isopropoxy-8-methyl-2H-1-benzo-
pyran-2-one (18i). Obtained (1.73 g, 74%) from 17i (2.08 g);
1
white crystals, m.p. 200-200.5°C (ethyl acetate). H-NMR
(DMSO-d6) d 1.32 [d, 6H, CH(CH3)2], 2.16 (s, 3H, 8-CH3),
4.76 [m, 1H, OCH(CH3)2], 5.46 (s, 1H, H-3), 7.04 (d of AB
q, J = 9 Hz, 1H, H-6), 7.64 (d of AB q, J = 9 Hz, 1H, H-5),
12.26 (broad s, 1H, OH; disappeared with D2O). IR (KBr):
3250-2450 br, complex (OH), 1660 (CO), 1595 s, br,1560,
1515 cm–1. Anal (C13H14O4) C, H.
5.1.2.10. 7-(4-Chlorobenzyloxy)-4-hydroxy-8-methyl-2H-1-
benzopyran-2-one (18o). Obtained (2.38 g, 75%) from 17o
1
(2.90 g); whitish crystals, m.p. 273-275°C (ethanol). H-
NMR (DMSO-d6) d 2.11 (s, 3H, 8-CH3), 5.25 (s, 2H,
OCH2C6H4Cl), 5.47 (s, 1H, H-3), 7.09 (d of AB q, J = 9 Hz,
1H, H-6), 7.48 and 7.50 (near AB q, J = 9 Hz, 4H, C6H4Cl),
7.64 (d of AB q, J = 9 Hz, 1H, H-5), 12.30 (broad s, 1H, OH;
disappeared with D2O). IR (KBr): 3230-2550 br, complex
(OH), 1681 s (CO), 1602 s, br, 1568, 1515 w cm-1. Anal.
(C17H13ClO4) C, H, Cl.
5.1.2.5. 7-Butoxy-4-hydroxy-8-methyl-2H-1-benzopyran-2-
one (18j). Obtained (1.99 g, 80%) from 17j (2.22 g); white
crystals, m.p. 232-234°C dec. (ethyl acetate). 1H-NMR
(DMSO-d6) d 0.96 (t, 3H, CH2CH2CH2CH3), 1.39-1.60 and
1.70-1.86 (2 m, 2H + 2H, CH2CH2CH2CH3), 2.18 (s, 3H,
8-CH3), 4.11 (t, 2H, OCH2CH2CH2CH3), 5.45 (s, 1H, H-3),
7.03 (d ofAB q, J = 9 Hz, 1H, H-6), 7.64 (d ofAB q, J = 9 Hz,
1H, H-5), 12.29 (broad s, 1H, OH; disappeared with D2O).
IR (KBr): 3100-2300 br, complex (OH), 1660 (CO), 1590 s,
br, 1543, 1514 cm-1. Anal. (C14H16O4) C, H.
5.1.2.11.
4-Hydroxy-8-methyl-7-(1-phenylethoxy)-2H-1-
benzopyran-2-one (18p). Obtained (1.96 g, 66%) from 17p
(2.70 g); white crystals, m.p. 190-191.5°C (ethyl

M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
405
1
acetate/isopropyl ether). H-NMR (CDCl3) d 1.67 (d, 3H,
1H-NMR (DMSO-d6) d 2.27 (s, 3H, 8-CH3), 5.33 (s, 2H,
OCH2C5H4N), 5.44 (s, 1H, H-3), 7.06 (d of AB q, J = 9 Hz,
1H, H-6), 7.47 (near d, 2H, pyridyl H-3’,5’), 7.65 (d ofAB q,
J = 9 Hz, 1H, H-5), 8.60 (near d, 2H, pyridyl H-2’,6’), 12.30
(broad s, 1H, OH; disappeared with D2O). IR (KBr): 3200-
2400 br, complex (OH), 1702 br (CO), 1608 s, br, 1562,
1512 w cm-1. Anal. (C16H13NO4) C, H, N.
C6H5CHCH3), 2.33 (s, 3H, 8-CH3), 5.37 [q, 1H,
OCH(CH3)C6H5], 5.80 (s, 1H, H-3), 6.50 (d of AB q,
J = 9 Hz, 1H, H-6), 7.23-7.44 (m, 6H, H-5 + phenyl H’s),
11.45 (broad s, 1H, OH; disappeared with D2O). IR (KBr):
3220-2500 s, br, complex (OH), 1704 s and 1680 sh (CO),
1606 s, 1552, 1512 w cm-1. Anal. (C18H16O4) C, H.
5.1.2.12.
4-Hydroxy-8-methyl-7-(2-phenylethoxy)-2H-1-
5.1.2.17. 4-Hydroxy-8-methyl-7-(2-quinolylmethoxy)-2H-1-
benzopyran-2-one (18v). Obtained as monohydrate (18v •
H2O) (2.42 g, 69%) from 17v (3.07 g); whitish crystals, m.p.
benzopyran-2-one (18q). Obtained (2.23 g, 75%) from 17q
(2.70 g); white crystals, m.p. 191-192°C (ethyl
acetate/isopropyl ether). H-NMR (CDCl3) d 2.22 (s, 3H,
1
1
254-256°C dec. (ethanol). H-NMR (DMSO-d6) d 2.30 (s,
8-CH3), 3.14 (t, 2H, OCH2CH2C6H5), 4.22 (t, 2H,
OCH2CH2C6H5), 5.81 (s, 1H, H-3), 6.70 (d ofAB q, J = 9 Hz,
1H, H-6), 7.20-7.42 (m, 5H, phenyl H’s), 7.64 (d of AB q,
J = 9 Hz, 1H, H-5), 11.20 (broad s, 1H, OH; disappeared with
D2O). IR (KBr): 3230-2530 br, complex (OH), 1712 and
1690 sh (CO), 1605 s, br, 1562, 1511 w cm–1. Anal.
(C18H16O4) C, H.
3H, 8-CH3), 5.47 (s, 1H, H-3), 5.52 (s, 2H, OCH2C9H6N),
7.12 (d of AB q, J = 9 Hz, 1H, H-6), 7.57-7.87 (m, 4H, 3H of
C9H6N + H-5), 7.96-8.09 (m, 2H, 2H of C9H6N), 8.44 (d,
J = 8.5 Hz, 1H, 1H of C9H6N), 12.30 (broad s, 1H, OH;
disappeared with D2O). IR (KBr): 3396 (water of crystalliza-
tion), 3220-2400 br, complex (OH), 1678 (CO), 1611 s, br,
1567, 1508 cm-1. Anal. (C20H15NO4 • H2O) C, H, N.
5.1.2.13. 4-Hydroxy-8-methyl-7-(1-naphthylmethoxy)-2H-
1-benzopyran-2-one (18r). Obtained (2.66 g, 80%) from 17r
(3.06 g); white crystals, m.p. 231.5-233°C dec. (ethanol).
1H-NMR (DMSO-d6) d 2.15 (s, 3H, 8-CH3), 5.47 (s, 1H,
H-3), 5.70 (s, 2H, OCH2C10H7), 7.34 (d of AB q, J = 9 Hz,
1H, H-6), 7.48-8.25 (m, 8H, naphthyl H’s + H-5), 12.32 (near
s, 1H, OH; disappeared with D2O). IR (KBr): 3220-2300 s,
br, complex (OH), 1684 s (CO), 1607 s, br, 1563 s,
1511 cm-1. Anal. (C21H16O4) C, H.
5.1.3. General procedure for the synthesis of substituted
4-chloro-2H-1-benzopyran-2-ones 19d-h,l,m,p-t
A mixture of the proper 4-hydroxycoumarin derivative 18
(5.0 mmol), triethylamine (0.51 g, 5.0 mmol) and phospho-
rus oxychloride (5.0 mL) was stirred at 130°C for the time
below reported for each compound. The final reaction mix-
ture was poured onto ice-water and stirred, then exhaustively
extracted with dichloromethane (after careful addition of
sodium bicarbonate until neutral, in the case of the pyridyl-
derivatives 19s,t). The combined extracts, after drying (anhy-
drous Na2SO4) and removal of solvents, afforded a dark oily
or nearly solid residue which was chromatographed on a
silica gel column eluting with the mixture dichloromethane-
petroleum ether (2:1)[dichloromethane-ethyl acetate (1:1) in
the case of compounds 19s,t]. The eluate collected, after
removal of solvents, afforded the desired 4-chlorocoumarin
derivative as a solid which was taken up in a little petroleum
ether, separated by filtration, then crystallized from the
proper solvent.
5.1.2.14. 4-Hydroxy-8-methyl-7-(2-pyridylmethoxy)-2H-1-
benzopyran-2-one (18s). Obtained (1.76 g, 62%) from 17s
(2.57 g); ivory-white crystals, m.p. 229-230°C (ethanol).
1H-NMR (DMSO-d6) d 2.69 (s, 3H, 8-CH3), 5.33 (s, 2H,
OCH2C5H4N), 5.48 (s, 1H, H-3), 7.13 (d of AB q, J = 9 Hz,
1H, H-6), 7.40 (m, 1H, pyridyl H-5’), 7.57 (near d, 1H,
pyridyl H-3’), 7.65 (d of AB q, J = 9 Hz, 1H, H-5), 7.87 (m,
1H, pyridyl H-4’), 8.61 (near d, 1H, pyridyl H-6’), 12.31
(broad s, 1H, OH; disappeared with D2O). IR (KBr): 3170-
2400 br, complex (OH), 1710 (CO), 1659, 1601 s, br,
1561 cm-1. Anal. (C16H13NO4) C, H, N.
5.1.3.1. 7-(sec-Butoxy)-4-chloro-2H-1-benzopyran-2-one
(19d). Obtained (0.49 g, 39%) from the reaction carried out
(30 min) with 18d (1.17 g); whitish crystals, m.p. 70-71°C
(petroleum ether). 1H-NMR (CDCl3) d 0.99 (t, 3H,
CH2CH3), 1.34 (d, 3H, CHCH3), 1.50-1.91 (m, 2H,
CHCH2CH3), 4.40 [near q, 1H, OCH(CH3)CH2CH3], 6.41
(s, 1H, H-3), 6.70-7.05 (m, 2H, H-6,8), 7.75 (d of AB q,
J = 9 Hz, 1H, H-5). IR (KBr): 1743 s (CO), 1603 s, 1550 w,
1500 cm-1. Anal. (C13H13ClO3) C, H, Cl.
5.1.2.15. 4-Hydroxy-8-methyl-7-(3-pyridylmethoxy)-2H-1-
benzopyran-2-one (18t). Obtained (1.93 g, 68%) from 17t
(2.57 g); white crystals, m.p. 235-237°C dec.
(pyridine/petroleum ether). 1H-NMR (DMSO-d6) d 2.21 (s,
3H, 8-CH3), 5.31 (s, 2H, OCH2C5H4N), 5.47 (s, 1H, H-3),
7.16 (d of AB q, J = 9 Hz, 1H, H-6), 7.48 (m, 1H, pyridyl
H-5’), 7.68 (d of AB q, J = 9 Hz, 1H, H-5), 7.94 (m, 1H,
pyridyl H-4’), 8.59 (near d, 1H, pyridyl H-6’), 8.74 (near s,
1H, pyridyl H-2’), 12.30 (broad s, 1H, OH; disappeared with
D2O). IR (KBr): 3300-2340 br, complex (OH), 1709 br (CO),
1607 s, br, 1566, 1508 w cm-1. Anal. (C16H13NO4) C, H, N.
5.1.3.2. 5-Benzyloxy-4-chloro-2H-1-benzopyran-2-one (19e).
Obtained (0.82 g, 57%) from the reaction carried out
(30 min) with 18e (1.34 g); ivory-white crystals, m.p. 128-
1
129°C (isopropyl ether). H-NMR (CDCl3) d 5.21 (s, 2H,
5.1.2.16. 4-Hydroxy-8-methyl-7-(4-pyridylmethoxy)-2H-1-
benzopyran-2-one (18u). Obtained (2.01 g, 71%) from 17u
(2.57 g); whitish crystals, m.p. 282-283°C dec. (methanol).
OCH2C6H5), 6.48 (s, 1H, H-3), 6.89 and 7.00 (2d, J = 9 Hz,
1H + 1H, H-6,8), 7.32-7.55 (m, 6H, H-7 + phenyl H’s). IR

406
M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
(KBr): 1737 and 1717 s (CO), 1602, 1558, 1499 w cm-1.
Anal. (C16H11ClO3) C, H, Cl.
OCH(CH3)C6H5], 6.40 (s, 1H, H-3), 6.73 (d of AB q,
J = 9 Hz, 1H, H-6), 7.24-7.41 (m, 5H, phenyl H’s), 7.54 (d of
AB q, J = 9 Hz, 1H, H-5). IR (KBr): 1728 s (CO), 1609 s,
1601 sh, 1562, 1490 cm-1. Anal. (C18H15ClO3) C, H, Cl.
5.1.3.3. 6-Benzyloxy-4-chloro-2H-1-benzopyran-2-one (19f).
Obtained (1.05 g, 73%) from the reaction carried out (3 h)
with 18f [11] (1.34 g); whitish crystals, m.p. 128-129°C
(isopropyl ether). 1H-NMR (CDCl3) d 5.14 (s, 2H,
OCH2C6H5), 6.62 (s, 1H, H-3), 7.22-7.53 (m, 8H, H-5,7,8 +
phenyl H’s). IR (KBr): 1750 and 1723 s (CO), 1599, 1572,
1489 cm-1. Anal. (C16H11ClO3) C, H, Cl.
5.1.3.9. 4-Chloro-8-methyl-7-(2-phenylethoxy)-2H-1-benzo-
pyran-2-one (19q). Obtained (0.63 g, 40%) from the reaction
carried out (30 min) with 18q (1.48 g); whitish crystals, m.p.
1
132-133°C (isopropyl ether). H-NMR (CDCl3) d 2.28 (s,
3H, 8-CH3), 3.16 (t, 2H, OCH2CH2C6H5), 4.29 (t, 2H,
OCH2CH2C6H5), 6.42 (s, 1H, H-3), 6.87 (d ofAB q, J = 9 Hz,
1H, H-6), 7.22-7.42 (m, 5H, phenyl H’s), 7.66 (d of AB q,
J = 9 Hz, 1H, H-5). IR (KBr): 1724 s, br (CO), 1606 s, 1563,
1494 cm-1. Anal. (C18H15ClO3) C, H, Cl.
5.1.3.4. 7-Benzyloxy-4-chloro-2H-1-benzopyran-2-one (19g).
Obtained (0.76 g, 53%) from the reaction carried out (3 h)
with 18g [11] (1.34 g); whitish crystals, m.p. 127-128°C
(isopropyl ether). 1H-NMR (CDCl3) d 5.16 (s, 2H,
OCH2C6H5), 6.44 (s, 1H, H-3), 6.91 (d, J8,6 = 2.5 Hz, 1H,
H-8), 7.00 (dd, J6,5 = 9 Hz, J6,8 = 2.5 Hz, 1H, H-6), 7.34-7.48
(m, 5H, phenyl H’s), 7.76 (d, J5,6 = 9 Hz, 1H, H-5). IR (KBr):
1734 s and 1715 (CO), 1613 s, 1560 w, 1503 cm-1. Anal.
(C16H11ClO3) C, H, Cl.
5.1.3.10. 4-Chloro-8-methyl-7-(1-naphthylmethoxy)-2H-1-
benzopyran-2-one (19r). Obtained (0.63 g, 36%) from the
reaction carried out (3 h) with 18r (1.66 g); whitish crystals,
1
m.p. 197-198°C (ethyl acetate/isopropyl ether). H-NMR
(CDCl3) d 2.33 (s, 3H, 8-CH3), 5.66 (s, 2H, OCH2C10H7),
6.46 (s, 1H, H-3), 7.12 (d of AB q, J = 9 Hz, 1H, H-6),
7.46-7.68 and 7.85-8.13 (2 m, 4H + 3H, naphthyl H’s), 7.72
(d of AB q, J = 9 Hz, 1H, H-5). IR (KBr): 1716 s (CO),
1603 s, 1558, 1496 cm-1. Anal. (C21H15ClO3) C, H, Cl.
5.1.3.5. 4-Chloro-8-methyl-7-propoxy-2H-1-benzopyran-2-
one (19h). Obtained (0.51 g, 40%) from the reaction carried
out (40 min) with 18h (1.17 g); white crystals, m.p. 107-
1
108°C (isopropyl ether). H-NMR (CDCl3) d 1.08 (t, 3H,
CH2CH2CH3), 1.88 (m, 2H, CH2CH2CH3), 2.30 (s, 3H,
8-CH3), 4.04 (t, 2H, OCH2CH2CH3), 6.40 (s, 1H, H-3), 6.87
(d ofAB q, J = 9 Hz, 1H, H-6), 7.67 (d ofAB q, J = 9 Hz, 1H,
H-5). IR (KBr): 1726 s, br (CO), 1606 s, 1560, 1496 cm-1.
Anal. (C13H13ClO3) C, H, Cl.
5.1.3.11.
4-Chloro-8-methyl-7-(2-pyridylmethoxy)-2H-1-
benzopyran-2-one (19s). Obtained (0.66 g, 44%) from the
reaction carried out (2 h) with 18s (1.42 g); whitish crystals,
m.p. 228.5-229°C (ethyl acetate). 1H-NMR (CDCl3) d 2.42
(s, 3H, 8-CH3), 5.34 (s, 2H, OCH2C5H4N), 6.44 (s, 1H, H-3),
6.95 (d of AB q, J = 9 Hz, 1H, H-6), 7.26 (m, 1H, pyridyl
H-5’), 7.54 (near d, 1H, pyridyl H-3’), 7.67 (d of AB q,
J = 9 Hz, 1H, H-5), 7.78 (m, 1H, pyridyl H-4’), 8.62 (near d,
1H, pyridyl H-6’). IR (KBr): 1725 s (CO), 1608, 1600 sh,
1574, 1498 cm-1. Anal. (C16H12ClNO3) C, H, N, Cl.
5.1.3.6.
4-Chloro-8-methyl-7-(2-methylbenzyloxy)-2H-1-
benzopyran-2-one (19l). Obtained (0.79 g, 50%) from the
reaction carried out (30 min) with 18l (1.48 g); whitish
crystals, m.p. 154-155°C (isopropyl ether). 1H-NMR
(CDCl3) d 2.35 (s, 3H, 8-CH3), 2.39 (s, 3H, C6H4CH3), 5.18
(s, 2H, OCH2C6H4CH3), 6.44 (s, 1H, H-3), 7.00 (d of AB q,
J = 9 Hz, 1H, H-6), 7.14-7.46 (m, 4H, C6H4CH3), 7.69 (d of
AB q, J = 9 Hz, 1H, H-5). IR (KBr): 1724 s (CO), 1609 sh,
1602 s, 1560, 1494 cm-1. Anal. (C18H15ClO3) C, H, Cl.
5.1.3.12. 4-Chloro-8-methyl-7-(3-pyridylmethoxy)-2H-1-
benzopyran-2-one (19t). Obtained (0.85 g, 56%) from the
reaction carried out (20 min) with 18t (1.42 g); whitish
1
crystals, m.p. 222°C (dichloromethane/ethyl acetate). H-
NMR (CDCl3) d 2.35 (s, 3H, 8-CH3), 5.22 (s, 2H,
OCH2C5H4N), 6.45 (s, 1H, H-3), 6.95 (d of AB q, J = 9 Hz,
1H, H-6), 7.39 (m, 1H, pyridyl H-5’), 7.71 (d of AB q,
J = 9 Hz, 1H, H-5), 7.82 (near d, 1H, pyridyl H-4’), 8.64 (near
d, 1H, pyridyl H-6’), 8.72 (near s, 1H, pyridyl H-2’). IR
(KBr): 1727 s (CO), 1602 s, 1562, 1497 cm-1. Anal.
(C16H12ClNO3) C, H, N, Cl.
5.1.3.7.
4-Chloro-8-methyl-7-(3-methylbenzyloxy)-2H-1-
benzopyran-2-one (19m). Obtained (0.79 g, 50%) from the
reaction carried out (30 min) with 18m (1.48 g); whitish
crystals, m.p. 141-142°C (isopropyl ether). 1H-NMR
(CDCl3) d 2.37 and 2.39 (2 s, 3H + 3H, 8-CH3 + C6H4CH3),
5.17 (s, 2H, OCH2C6H4CH3), 6.43 (s, 1H, H-3), 6.94 (d of
AB q, J = 9 Hz, 1H, H-6), 7.13-7.37 (m, 4H, C6H4CH3), 7.67
(d of AB q, J = 9 Hz, 1H, H-5). IR (KBr): 1724 s (CO),
1603 s, 1560, 1497 cm-1. Anal. (C18H15ClO3) C, H, Cl.
5.1.4. General procedures for the synthesis of substituted
4-(1-piperazinyl)-2H-1-benzopyran-2-ones 5
Method A). From the proper 4-hydroxycoumarin deriva-
tives 18 (compounds 5i-k,n,o,u,v)
A mixture of 3.0 mmol of the proper compound 18 and
excess piperazine (7.0 g) was stirred at 160°C for 1 h, then
final hot solution was poured into ice-water. The resulting
solution was exhaustively extracted with chloroform. The
5.1.3.8. 4-Chloro-8-methyl-7-(1-phenylethoxy)-2H-1-benzo-
pyran-2-one (19p). Obtained (0.47 g, 30%) from the reaction
carried out (30 min) with 18p (1.48 g); white crystals, m.p.
137.5-139°C (isopropyl ether). 1H-NMR (CDCl3) d 1.71 (d,
3H, C6H5CHCH3), 2.41 (s, 3H, 8-CH3), 5.47 [q, 1H,

M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
407
combined extracts (dried over anhydrous Na2SO4) were
evaporated to dryness under reduced pressure to give a thick
oil from which (compounds 5k,n,o,u,v), after treatment with
a little ethyl ether and standing, nearly pure compound 5
separated out as a whitish solid which was then crystallized
from the suitable solvent. In the case of compounds 5i,j the
final oily residue was treated with a solution of maleic acid
(0.35 g, 3.0 mmol) in anhydrous ethanol to give the corre-
sponding pure maleates (5i • H4C4O4 or 5j • H4C4O4) as a
white solid which was then crystallized from anhydrous
ethanol. The free amines 5i,j were obtained, as thick oils,
from the analytical samples of these maleates, by treatment
with aqueous sodium bicarbonate, exhaustive extraction with
chloroform and removal of solvent, then were crystallized
from the proper solvents.
5.1.4.3. 6-Benzyloxy-4-(1-piperazinyl)-2H-1-benzopyran-2-
one (5f). Obtained (0.56 g, 55%) from 19f (0.86 g); whitish
crystals, m.p. 104-105°C (ethyl acetate/petroleum ether).
1H-NMR (CDCl3) d 1.82 (s, 1H, NH; disappeared with
D2O), 2.93-3.18 (m, 8H, piperazine CH2’s), 5.14 (s, 2H,
OCH2C6H5), 5.71 (s, 1H, H-3), 6.99-7.47 (m, 8H, H-5,7,8 +
phenyl H’s). IR (KBr): 3343 (NH), 1695 s (CO), 1604 w,
1558, 1487 w cm-1. Anal. (C20H20N2O3) C, H, N.
5.1.4.4. 7-Benzyloxy-4-(1-piperazinyl)-2H-1-benzopyran-2-
one (5g). Obtained (0.79 g, 78%) from 19g (0.86 g); white
crystals, m.p. 149-150°C (ethyl acetate). 1H-NMR (CDCl3) d
1.73 (s, 1H, NH; disappeared with D2O), 2.98-3.30 (m, 8H,
piperazine CH2’s), 5.12 (s, 2H, OCH2C6H5), 5.59 (s, 1H,
H-3), 6.80-6.94 (m, 2H, H-6,8), 7.30-7.56 (m, 6H, H-5 +
phenyl H’s). IR (KBr): 3220 (NH), 1703 s (CO), 1610 s, br,
1548 cm-1. Anal. (C20H20N2O3) C, H, N.
Method B). From 4-chlorocoumarin derivatives 19 (com-
pounds 5d-h,l,m,p-t)
A mixture of 3.0 mmol of the proper chloroderivative 19,
piperazine (2.58 g, 30.0 mmol) and ethanol (100 mL) was
stirred at room temperature for 2 h. The solution obtained
was poured into water (250 mL) and the mixture exhaustively
extracted with chloroform. The combined extracts, after dry-
ing and removal of solvents, afforded an oily or nearly solid
residue which, in most cases, was treated with a little ethyl
ether to give pure compound 5 as a whitish solid which was
then crystallized from the suitable solvent (compounds
5e,g,h,l,m,p-t). Compounds 5d,f were obtained as maleates
by the treatment described in Method A for 5i,j; 5d was
stored as maleate, while 5f was transformed in the corre-
sponding free amine as described above, then crystallized.
According to procedures above described, the following
compounds were prepared.
5.1.4.5. 8-Methyl-4-(1-piperazinyl)-7-propoxy-2H-1-benzo-
pyran-2-one (5h). Obtained (0.59 g, 65%) from 19h (0.76 g);
white crystals, m.p. 150-150.5°C (ethyl acetate). H-NMR
(CDCl3) d 1.07 (t, 3H, CH2CH2CH3), 1.80 (s, 1H, NH;
disappeared with D2O), 1.87 (m, 2H, CH2CH2CH3), 2.30 (s,
3H, 8-CH3), 3.02-3.30 (m, 8H, piperazine CH2’s), 4.00 (t,
2H, OCH2CH2CH3), 5.60 (s, 1H, H-3), 6.79 (d of AB q,
J = 9 Hz, 1H, H-6), 7.43 (d of AB q, J = 9 Hz, 1H, H-5). IR
(KBr): 3322 (NH), 1695 s (CO), 1604 s, 1557, 1501 w cm-1.
Anal. (C17H22N2O3) C, H, N.
1
5.1.4.6. 7-Isopropoxy-8-methyl-4-(1-piperazinyl)-2H-1-ben-
zopyran-2-one (5i). Obtained (0.58 g, 64%) from 18i
(0.70 g); ivory-white crystals, m.p. 139-140°C (ethyl
1
acetate/petroleum ether). H-NMR (CDCl3) d 1.36 [d, 6H,
CH(CH3)2], 2.20 (s, 1H, NH; disappeared with D2O), 2.27 (s,
3H, 8-CH3), 2.96-3.37 (m, 8H, piperazine CH2’s), 4.62 [m,
1H, OCH(CH3)2], 5.60 (s, 1H, H-3), 6.79 (d of AB q,
J = 9 Hz, 1H, H-6), 7.39 (d of AB q, J = 9 Hz, 1H, H-5). IR
(KBr): 3341 w (NH), 1710 s (CO), 1606 s, 1557, 1497 w
cm-1. Anal. (C17H22N2O3) C, H, N.
5.1.4.1. 7-(sec-Butoxy)-4-(1-piperazinyl)-2H-1-benzopyran-
2-one (5d). Obtained as maleate (5d • H4C4O4) (1.19 g,
95%) from 19d (0.76 g); whitish crystals, m.p. 169-170.5°C
(anhydrous ethanol/ethyl ether). The 1H-NMR and IR spec-
tra were recorded on the free amine obtained (thick oil) from
1
the corresponding maleate as above described. H-NMR
(CDCl3) d 0.98 (t, 3H, CH2CH3), 1.33 (d, 3H, CHCH3), 1.72
(m, 2H, CHCH2CH3), 1.79 (s, 1H, NH; disappeared with
D2O), 2.99-3.34 (m, 8H, piperazine CH2’s), 4.37 [m, 1H,
OCH(CH3)CH2CH3], 5.57 (s, 1H, H-3), 6.69-6.84 (m, 2H,
H-6,8), 7.50 (d of AB q, J = 9 Hz, 1H, H-5). IR (film): 3330
(NH), 1709 s, br (CO), 1611 s, br, 1549, 1507 cm-1. Anal.
(C17H22N2O3 • H4C4O4) C, H, N.
5.1.4.7. 7-Butoxy-8-methyl-4-(1-piperazinyl)-2H-1-benzo-
pyran-2-one (5j). Obtained (0.43 g, 45%) from 18j (0.74 g);
ivory-white crystals, m.p. 142-143°C (ethyl acetate). H-
1
NMR (CDCl3) d 1.01 (t, 3H, CH2CH2CH2CH3), 1.54 (m,
2H, CH2CH2CH2CH3), 1.76 (s, 1H, NH; disappeared with
D2O), 1.84 (m, 2H, CH2CH2CH2CH3), 2.32 (s, 3H, 8-CH3),
3.05-3.30 (m, 8H, piperazine CH2’s), 4.07 (t, 2H,
OCH2CH2CH2CH3), 5.62 (s, 1H, H-3), 6.80 (d of AB q,
J = 9 Hz, 1H, H-6), 7.43 (d of AB q, J = 9 Hz, 1H, H-5). IR
(KBr): 3321 (NH), 1693 s (CO), 1605 s, 1557, 1503 w cm-1.
Anal. (C18H24N2O3) C, H, N.
5.1.4.2. 5-Benzyloxy-4-(1-piperazinyl)-2H-1-benzopyran-2-
one (5e). Obtained (0.83 g, 82%) from 19e (0.86 g); ivory-
1
white crystals, m.p. 159-159.5°C (ethyl acetate). H-NMR
(CDCl3) d 1.78 (s, 1H, NH; disappeared with D2O), 2.68-
2.83 and 2.97-3.20 (2 m, 4H + 4H, piperazine CH2’s), 5.14
(s, 2H, OCH2C6H5), 5.60 (s, 1H, H-3), 6.84 and 6.99 (2 d,
J = 9 Hz, 1H + 1H, H-6,8), 7.32-7.50 (m, 6H, H-7 + phenyl
H’s). IR (KBr): 3345 w (NH), 1702 s (CO), 1599 s,
1543 cm-1. Anal. (C20H20N2O3) C, H, N.
5.1.4.8. 7-(Cyclohexylmethoxy)-8-methyl-4-(1-piperazinyl)-
2H-1-benzopyran-2-one (5k). Obtained (0.62 g, 58%) from
18k (0.87 g); whitish crystals, m.p. 177-178°C (ethyl ac-
1
etate). H-NMR (CDCl3) d 1.00-1.46 and 1.66-1.96 (2 m,

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M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
11H, cyclohexyl H’s), 1.77 (s, 1H, NH; disappeared with
D2O), 2.32 (s, 3H, 8-CH3), 3.06-3.28 (m, 8H, piperazine
CH2’s), 3.85 (d, 2H, OCH2C6H11), 5.61 (s, 1H, H-3), 6.78 (d
of AB q, J = 9 Hz, 1H, H-6), 7.42 (d of AB q, J = 9 Hz, 1H,
H-5). IR (KBr): 3320 w (NH), 1709 s and 1690 s (CO),
1607 s, 1551, 1503 w cm-1. Anal. (C21H28N2O3) C, H, N.
acetate/petroleum ether). 1H-NMR (CDCl3) d 1.69 (d + s, 3H
+ 1H, C6H5CHCH3 + NH; d, 3H, after treatment with D2O),
2.40 (s, 3H, 8-CH3), 3.00-3.22 (m, 8H, piperazine CH2’s),
5.40 [q, 1H, OCH(CH3)C6H5], 5.58 (s, 1H, H-3), 6.64 (d of
AB q, J = 9 Hz, 1H, H-6), 7.22-7.40 (m, 6H, phenyl H’s +
H-5). IR (KBr): 3334 w (NH), 1701 s (CO), 1603 s, 1553,
1495 cm-1. Anal. (C22H24N2O3) C, H, N.
5.1.4.9. 8-Methyl-7-(2-methylbenzyloxy)-4-(1-piperazinyl)-
2H-1-benzopyran-2-one (5l). Obtained (0.99 g, 91%) from
19l (0.94 g); whitish crystals, m.p. 162-163°C (ethyl acetate).
1H-NMR (CDCl3) d 1.69 (s, 1H, NH; disappeared with
D2O), 2.34 (s, 3H, 8-CH3), 2.39 (s, 3H, C6H4CH3), 3.04-3.28
(m, 8H, piperazine CH2’s), 5.15 (s, 2H, OCH2C6H4CH3),
5.62 (s, 1H, H-3), 6.89 (d of AB q, J = 9 Hz, 1H, H-6),
7.18-7.32 (m, 3H, 3H of C6H4CH3), 7.40-7.50 (m, 2H, 1H of
C6H4CH3 + H-5). IR (KBr): 3308 w (NH), 1689 s (CO),
1604 s, 1555, 1504 w cm-1. Anal. (C22H24N2O3) C, H, N.
5.1.4.14. 8-Methyl-7-(2-phenylethoxy)-4-(1-piperazinyl)-
2H-1-benzopyran-2-one (5q). Obtained (0.98 g, 90%) from
19q (0.94 g); white crystals, m.p. 144-145°C (ethyl
1
acetate/isopropyl ether). H-NMR (CDCl3) d 1.68 (s, 1H,
NH; disappeared with D2O), 2.28 (s, 3H, 8-CH3), 3.04-3.26
(m, 8H + 2H, piperazine CH2’s + OCH2CH2C6H5), 4.26 (t,
2H, OCH2CH2C6H5), 5.61 (s, 1H, H-3), 6.77 (d of AB q,
J = 9 Hz, 1H, H-6), 7.20-7.45 (m, 6H, phenyl H’s + H-5). IR
(KBr): 3267 w (NH), 1705 s (CO), 1604 s, 1558, 1499 w
cm-1. Anal. (C22H24N2O3) C, H, N.
5.1.4.10. 8-Methyl-7-(3-methylbenzyloxy)-4-(1-piperazinyl)-
2H-1-benzopyran-2-one (5m). Obtained (1.01 g, 92%) from
19m (0.94 g); ivory-white crystals, m.p. 143-144°C (ethyl
5.1.4.15. 8-Methyl-7-(1-naphthylmethoxy)-4-(1-piperazinyl)-
2H-1-benzopyran-2-one (5r). Obtained (0.77 g, 64%) from
19r (1.05 g); whitish crystals, m.p. 196-197°C (ethyl ac-
1
acetate/petroleum ether). H-NMR (CDCl3) d 1.74 (s, 1H,
1
etate). H-NMR (CDCl3) d 1.70 (s, 1H, NH; disappeared
NH; disappeared with D2O), 2.37 and 2.38 (2 s, 3H + 3H,
8-CH3 + C6H4CH3), 3.00-3.29 (m, 8H, piperazine CH2’s),
5.14 (s, 2H, OCH2C6H4CH3), 5.61 (s, 1H, H-3), 6.84 (d of
AB q, J = 9 Hz, 1H, H-6), 7.11-7.37 (m, 4H, C6H4CH3), 7.41
(d of AB q, J = 9 Hz, 1H, H-5). IR (KBr): 3322 (NH), 1698 s
(CO), 1605 s, 1558, 1501 w cm-1. Anal. (C22H24N2O3) C, H,
N.
with D2O), 2.34 (s, 3H, 8-CH3), 3.03-3.28 (m, 8H, piperazine
CH2’s), 5.64 (overlapped singlets, 2H + 1H, OCH2C10H7 +
H-3), 7.00 (d of AB q, J = 9 Hz, 1H, H-6), 7.40-7.65 (m, 5H,
4H of C10H7 + H-5), 7.85-8.12 (m, 3H, 3H of C10H7). IR
(KBr): 3311 w (NH), 1700 s (CO), 1606 s, 1556, 1506 w
cm-1. Anal. (C25H24N2O3) C, H, N.
5.1.4.16. 8-Methyl-4-(1-piperazinyl)-7-(2-pyridylmethoxy)-
2H-1-benzopyran-2-one (5s). Obtained (0.99 g, 94%) from
19s (0.91 g); white crystals, m.p. 217-218°C (ethyl acetate).
1H-NMR (CDCl3) d 1.74 (s, 1H, NH; disappeared with
D2O), 2.41 (s, 3H, 8-CH3), 2.97-3.32 (m, 8H, piperazine
CH2’s), 5.30 (s, 2H, OCH2C5H4N), 5.61 (s, 1H, H-3), 6.84 (d
of AB q, J = 9 Hz, 1H, H-6), 7.26 (m, 1H, pyridyl H-5’), 7.42
(d of AB q, J = 9 Hz, 1H, H-5), 7.55 (near d, 1H, pyridyl
H-3’), 7.76 (m, 1H, pyridyl H-4’), 8.62 (near d, 1H, pyridyl
H-6’). IR (KBr): 3289 w (NH), 1713 s (CO), 1611 s, 1593 sh,
1560, 1500 w cm-1. Anal. (C20H21N3O3) C, H, N.
5.1.4.11. 7-(4-Methoxybenzyloxy)-8-methyl-4-(1-piperazinyl)-
2H-1-benzopyran-2-one (5n). Obtained (0.32 g, 28%) from
18n (0.94 g); ivory-white crystals, m.p. 174.5-175°C (ethyl
acetate). 1H-NMR (CDCl3) d 1.70 (s, 1H, NH; disappeared
with D2O), 2.36 (s, 3H, 8-CH3), 3.05-3.16 and 3.18-3.27
(2 m, 4H + 4H, piperazine CH2’s), 3.84 (s, 3H, OCH3), 5.12
(s, 2H, OCH2C6H4OCH3), 5.63 (s, 1H, H-3), 6.86 (d ofAB q,
J = 9 Hz, 1H, H-6), 6.95 and 7.38 (AB q, J = 9 Hz, 2H + 2H,
C6H4OCH3), 7.43 (d of AB q, J = 9 Hz, 1H, H-5). IR (KBr):
3313 w (NH), 1700 s (CO), 1601 s, 1554, 1514 cm-1. Anal.
(C22H24N2O4) C, H, N.
5.1.4.17. 8-Methyl-4-(1-piperazinyl)-7-(3-pyridylmethoxy)-
2H-1-benzopyran-2-one (5t). Obtained (0.83 g, 79%) from
19t (0.91 g); white crystals, m.p. 197-198°C (ethyl acetate).
1H-NMR (CDCl3) d 1.74 (s, 1H, NH; disappeared with
D2O), 2.34 (s, 3H, 8-CH3), 3.00-3.30 (m, 8H, piperazine
CH2’s), 5.19 (s, 2H, OCH2C5H4N), 5.62 (s, 1H, H-3), 6.84 (d
of AB q, J = 9 Hz, 1H, H-6), 7.37 (m, 1H, pyridyl H-5’), 7.43
(d of AB q, J = 9 Hz, 1H, H-5), 7.80 (near d, 1H, pyridyl
H-4’), 8.62 (near d, 1H, pyridyl H-6’), 8.70 (near s, 1H,
pyridyl H-2’). IR (KBr): 3268 (NH), 1705 s (CO), 1605 s,
1557, 1505 w cm-1. Anal. (C20H21N3O3) C, H, N.
5.1.4.12. 7-(4-Chlorobenzyloxy)-8-methyl-4-(1-piperazinyl)-
2H-1-benzopyran-2-one (5o). Obtained (0.53 g, 46%) from
18o (0.95 g); ivory-white crystals, m.p. 188-189°C (ethyl
acetate). 1H-NMR (CDCl3) d 1.79 (s, 1H, NH; disappeared
with D2O), 2.36 (s, 3H, 8-CH3), 3.03-3.13 and 3.17-3.27
(2 m, 4H + 4H, piperazine CH2’s), 5.15 (s, 2H,
OCH2C6H4Cl), 5.62 (s, 1H, H-3), 6.81 (d of AB q, J = 9 Hz,
1H, H-6), 7.38 (s, 4H, C6H4Cl), 7.43 (d ofAB q, J = 9 Hz, 1H,
H-5). IR (KBr): 3305 w (NH), 1703 s (CO), 1605 s, 1556,
1493 w cm-1. Anal. (C21H21ClN2O3) C, H, N, Cl.
5.1.4.13. 8-Methyl-7-(1-phenylethoxy)-4-(1-piperazinyl)-
2H-1-benzopyran-2-one (5p). Obtained (0.76 g, 69%) from
19p (0.94 g); white crystals, m.p. 156-157°C (ethyl
5.1.4.18. 8-Methyl-4-(1-piperazinyl)-7-(4-pyridylmethoxy)-
2H-1-benzopyran-2-one (5u). Obtained (0.51 g, 48%) from
18u (0.85 g); whitish crystals, m.p. 208-208.5°C (ethyl ac-

M. Di Braccio et al. / European Journal of Medicinal Chemistry 39 (2004) 397–409
409
1
etate). H-NMR (CDCl3) d 1.82 (s, 1H, NH; disappeared
turbidimetric method [14] and quantified by the light trans-
mission reached after 3 min.
with D2O), 2.42 (s, 3H, 8-CH3), 3.03-3.27 (m, 8H, piperazine
CH2’s), 5.22 (s, 2H, OCH2C5H4N), 5.64 (s, 1H, H-3), 6.77 (d
of AB q, J = 9 Hz, 1H, H-6), 7.38 (near d, 2H, pyridyl
H-3’,5’), 7.43 (d of AB q, J = 9 Hz, 1H, H-5), 8.65 (near d,
2H, pyridyl H-2’,6’). IR (KBr): 3335 (NH), 1692 s (CO),
1600 s, 1556 cm-1. Anal. (C20H21N3O3) C, H, N.
5.2.2. Calculation of inhibition
In order to calculate the percentage of inhibition, the
extent of aggregation measured in the presence of the com-
pounds tested was always compared with that measured for a
control sample containing the solvent, in an experiment car-
ried out under the same conditions. From each series of
experiments, in which the inhibitors were tested in at least
five concentrations, a percentage inhibition-concentration
curve was derived. From this curve the IC50 value was calcu-
lated as the concentration of inhibitor causing a 50% inhibi-
tion of the aggregation.
5.1.4.19. 8-Methyl-4-(1-piperazinyl)-7-(2-quinolylmethoxy)-
2H-1-benzopyran-2-one (5v). Obtained (0.53 g, 44%) from
18v (1.05 g); whitish crystals, m.p. 237-238°C
1
(dichloromethane/ethyl acetate). H-NMR (CDCl3) d 1.76
(s, 1H, NH; disappeared with D2O), 2.47 (s, 3H, 8-CH3),
3.02-3.26 (m, 8H, piperazine CH2’s), 5.51 (s, 2H,
OCH2C9H6N), 5.63 (s, 1H, H-3), 6.88 (d of AB q, J = 9 Hz,
1H, H-6), 7.40 (d of AB q, J = 9 Hz, 1H, H-5), 7.51-7.92 (m,
4H, 4H of C9H6N), 8.11 (near d, 1H, 1H of C9H6N), 8.24 (d,
J = 8.5 Hz, 1H, 1H of C9H6N). IR (KBr): 3241 (NH), 1716 s
(CO), 1608 s, 1559, 1500 cm-1. Anal. (C24H23N3O3) C, H, N.
5.2.3. Statistical analysis
The IC50 values reported in Table 2 are averages ( stan-
dard deviation) of those obtained from at least four indepen-
dent determinations carried out on different batches of plate-
lets (usually 5-8 batches). Statistical analysis was performed
using the Student’s t-test and considering significant the
activity difference between two compounds when corre-
sponding to P< 0.05.
5.1.5. 6-Methyl-4-(1-piperazinyl)-2H-pyran-2-one (21). A
mixture of 4-chloro-6-methyl-2H-pyran-2-one 20 [12]
(0.43 g, 3.0 mmol), piperazine (2.58 g, 30.0 mmol) and
ethanol (100 mL) was stirred at room temperature for 1 h.
The resulting solution was poured into water (250 mL) and
the mixture exhaustively extracted with chloroform. The
extracts, after drying and removal of solvents, afforded a
thick oil from which, after treatment with ethyl ether and
standing, pure 21 separated out as whitish solid (0.39 g,
67%); ivory-white crystals, m.p. 144-145°C (ethyl acetate).
1H-NMR (CDCl3) d 1.77 (s, 1H, NH; disappeared with
D2O), 2.18 (s, 3H, CH3), 2.88-3.04 and 3.28-3.43 (2 m, 4H +
4H, piperazine CH2’s), 5.12 (near s, 1H, H-5), 5.85 (near s,
1H, H-3). IR (KBr): 3288 (NH), 1678 s (CO), 1634, 1539 s
cm-1. Anal. (C10H14N2O2) C, H, N.
Acknowledgements
The financial support from MIUR (Rome, Italy) is grate-
fully acknowledged.
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