Synthesis, antiplatelet and antithrombotic activities of new 2-substituted benzopyrano[4,3-d]pyrimidin-4-cycloamines and 4-amino/cycloamino-benzopyrano[4, 3-d]pyrimidin-5-ones

Article abstract of DOI:10.1016/j.bmc.2005.07.066

Atherothrombotic coronary artery disease, associated with deep vein thrombosis, is one of the most common causes of death worldwide. Recently, antiplatelet combination therapy using agents with different mechanisms of action, such as aspirin, dipyridamole, and thienopyridines, seems to be an attractive preventive approach. Moreover, several large, randomized clinical trials support combination therapy with aspirin plus warfarin in high-risk patients with atherosclerotic heart disease. Our research on the benzopyrano[4,3-d]pyrimidine system gave rise to the synthesis of a large number of compounds endowed with in vitro anti-aggregating activity. Several SAR considerations suggest that the benzopyranopyrimidine system is an appropriate scaffold to obtain molecules that are able to act simultaneously in different pathways of aggregation. Now, we report the synthesis of new 2-substituted benzopyrano[4,3-d]pyrimidin-4-cycloamines and 4-amino/cycloamino-benzopyrano[4, 3-d]pyrimidin-5-ones and the results of the pharmacological study on haemostasis. Some tested compounds showed a large-spectrum antiplatelet activity in vitro, and are more potent than aspirin as antithrombotics in vivo but, at variance with aspirin, they do not increase bleeding. This paper describes novel antithrombotic compounds with an interesting pharmacological profile and a potentially attractive benefit/risk ratio, with their mechanism of action generally, but not exclusively, dependent on antiplatelet activity, deserving further investigations.

Full text of DOI:10.1016/j.bmc.2005.07.066

Bioorganic & Medicinal Chemistry 14 (2006) 121–130
Synthesis, antiplatelet and antithrombotic activities of new
2-substituted benzopyrano[4,3-d]pyrimidin-4-cycloamines
and 4-amino/cycloamino-benzopyrano[4,3-d]pyrimidin-5-ones
Olga Bruno,a,* Chiara Brullo,a Silvia Schenone,a Francesco Bondavalli,a
Angelo Ranise,a Massimiliano Tognolini,b Mariannina Impicciatore,b
Vigilio Ballabenib and Elisabetta Barocellib
a
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Genova, Viale Benedetto XV, 3-16132 Genova, Italy
b
`
Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate-Universita degli Studi di
Parma-Parco Area delle Scienze 27/A, 43100–Parma, Italy
Received 23 June 2005; revised 27 July 2005; accepted 29 July 2005
Available online 9 September 2005
Abstract—Atherothrombotic coronary artery disease, associated with deep vein thrombosis, is one of the most common causes of
death worldwide. Recently, antiplatelet combination therapy using agents with different mechanisms of action, such as aspirin, dipy-
ridamole, and thienopyridines, seems to be an attractive preventive approach. Moreover, several large, randomized clinical trials
support combination therapy with aspirin plus warfarin in high-risk patients with atherosclerotic heart disease. Our research on
the benzopyrano[4,3-d]pyrimidine system gave rise to the synthesis of a large number of compounds endowed with in vitro anti-ag-
gregating activity. Several SAR considerations suggest that the benzopyranopyrimidine system is an appropriate scaffold to obtain
molecules that are able to act simultaneously in different pathways of aggregation. Now, we report the synthesis of new 2-substituted
benzopyrano[4,3-d]pyrimidin-4-cycloamines and 4-amino/cycloamino-benzopyrano[4,3-d]pyrimidin-5-ones and the results of the
pharmacological study on haemostasis. Some tested compounds showed a large-spectrum antiplatelet activity in vitro, and are more
potent than aspirin as antithrombotics in vivo but, at variance with aspirin, they do not increase bleeding. This paper describes novel
antithrombotic compounds with an interesting pharmacological profile and a potentially attractive benefit/risk ratio, with their
mechanism of action generally, but not exclusively, dependent on antiplatelet activity, deserving further investigations.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Thrombosis may occur when the haemostatic stimulus
becomes unregulated. Important predisposing condi-
tions to thrombosis are disturbed blood flow, hyperco-
agulation, and altered vessel wall.3,4 Arterial thrombi
are predominantly composed of platelets, a small
amount of fibrin and a few red blood cells. It is for this
reason that antiplatelet agents are successfully used in
the treatment and prevention of arterial thrombosis.5
Venous thrombi are mainly composed of red blood cells
in a fibrin mesh, and anticoagulant agents are used in
the treatment of venous thrombosis.
Atherothrombotic coronary artery disease, giving rise to
a number of cardiocirculatory disorders, such as myo-
cardial infarction (MI), unstable angina (UA), or acute
stroke associated with deep vein thrombosis (DVT), is
one of the most common causes of death worldwide
and a growing public health problem.1 To prevent fur-
ther ischaemic events and vascular death ensuing from
thrombosis, primary and secondary prevention strate-
gies are currently adopted for patients with cardiovascu-
lar diseases.2
The relevance of antiplatelet drugs has been firmly
established by clinical trials and experience with drugs,
such as aspirin, dipyridamole and the thienopyri-
dines.6–8 These drugs are the only oral antiplatelet
agents currently approved by the FDA (Food and Drug
Administration) for use in patients. Recently, antiplate-
let combination therapy using agents with different
Keywords: 4-Amino/cycloamino-2-methanesulfonyl/methylthio-benzo-
pyrano[4,3-d]pyrimidines; 4-Amino/cycloamino-2- amino/cycloamino/
methanesulfonyl/methylthio/methoxy-benzopyrano[4,3-d]pyrimidin-5-
ones; Antiplatelet activity; Antithrombotic activity.
*
Corresponding author. Tel.: +39 010 3538367; fax: +39 010 35383;
e-mail: obruno@unige.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.066

122
O. Bruno et al. / Bioorg. Med. Chem. 14 (2006) 121–130
mechanisms of action seems to be an attractive preven-
tive approach, since different signalling pathways con-
tribute to platelet activation.9,10 In this way, several
trials with clopidogrel plus aspirin, including CURE
(Clopidogrel in Unstable angina to prevent Recurrent
Events),11 MATCH (Management of Atherothrombosis
with Clopidogrel in High-Risk Patients with Recent
Transient Ischaemic Attacks or Ischemic Stroke),12
and CHARISMA (Clopidogrel for High Atherothrom-
botic Risk and Ischaemic Stabilization, Management
and Avoidance),13 demonstrated the benefit of the com-
bination therapy.14
induced aggregation.22 These results convinced us that
the benzopyranopyrimidine system is an appropriate
scaffold to obtain molecules that are able to act simulta-
neously in the different intracellular pathways that are
involved in platelet aggregation and to serve as potent
and effective antiplatelet agents.
For the above-mentioned reasons, and according to the
observation that a large spectrum of antiplatelet activity
is obtained when a small, oxygen function is present in
position 5 of the benzopyranopyrimidine system, we
planned the synthesis of the new molecules 2–5 (Fig. 2,
Table 1) in which a carbonyl function has been inserted
in position 5 of the benzopyrano moiety.
Moreover, in spite of reluctance to use oral anticoagu-
lants, several large, randomized clinical trials support
combination therapy with aspirin plus warfarin in
high-risk patients with atherosclerotic heart disease.15,16
The WATCH (Warfarin and Antiplatelet Therapy in
Chronic Heart Failure) trial was undertaken to deter-
mine the optimal antithrombotic therapy for heart fail-
ure patients.17
Furthermore, in these new derivatives we achieved the
fusion of pyrimidine cycle with the benzopyran-2-one
system, well-known as the typical structure of anticoag-
ulants drugs. The anticoagulant activity of coumarins,
blocking the prothrombin biosynthesis by inhibition of
vitamin K-epoxide reductase, is closely dependent on
the presence of a hydroxy group in position 4 and a
highly lipophilic substituent in position 3 of the benzo-
pyrano (see Warfarin). On the other hand, despite the
lack of aforementioned structural requirements, a simi-
lar mechanism of action to coumarin is supposed to take
place for the anticoagulant 1,3-indandione derivatives
(see Miradon).
In the last few years, our research on the benzopyr-
ano[4,3-d]pyrimidine system gave rise to the synthesis
of a large number of compounds (shown in structure
1, Fig. 1) endowed with interesting pharmacological
properties such as antiinflammatory, analgesic and, par-
ticularly, in vitro anti-aggregating activities.18–22
The large number of data obtained in this pharmacolog-
ical screening allowed us to make useful SAR predic-
tions. In fact, several lines of evidence have confirmed
that antiplatelet activity was dependent on concomitant
contribution of both functions inserted in positions 2
and 5 of the heterocyclic system. Inhibition of ADP-in-
duced platelet aggregation, for instance, is present in
2-methylthio-5-amino derivatives or in 2-methoxy-5-alk-
oxy/phenoxy derivatives, but neither in 2-methoxy-5-
amino nor in 2-methylthio-5-alkoxy/phenoxy ones.22
On the contrary, no chemical modification affects anti-
platelet activity against arachidonic acid (AA)-induced
aggregation: in fact, most of compounds 1, apart from
the substituents introduced, are active as antiplatelets,
frequently as potent as acetyl salicylic acid (ASA).
Moreover, the 2,5-cycloamino disubstituted compounds
showed a large spectrum of action, inhibiting ADP-,
AA-, and collagen-induced aggregation. Some of these
were, in vivo, more potent antithrombotics than lysine
acetylsalicylate and showed prohaemorrhagic activity
lower than that of the reference drug.23 The last synthe-
sized 2-methanesulfonyl-5-alkoxy derivatives showed a
remarkable in vitro antiplatelet activity of broad spec-
trum, being active against ADP-, AA-, and U46619-
R'
N
N
R
X
O
2-7
Figure 2. General structure of compounds 2–7.
Table 1. Compounds 2–7
Compound
X
R
R0
2a
2b
2c
2d
3a
3b
3c
3d
3e
3f
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
C=O
CH2
CH2
CH2
CH2
CH2
CH2
H
OCH3
SCH3
H
H
Pyrrolidino
Morpholino
H
H
NH2
NH2
NH2
NH2
OCH3
SCH3
NH2
NH2
Pyrrolidino
Morpholino
SCH3
NH2
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
Pyrrolidino
Piperidino
Morpholino
Pyrrolidino
Piperidino
Morpholino
Pyrrolidino
Piperidino
Morpholino
Pyrrolidino
Piperidino
Morpholino
SCH3
SCH3
SO2CH3
SO2CH3
SO2CH3
SCH3
SCH3
SCH3
SO2CH3
SO2CH3
SO2CH3
Figure 1. General structure of compounds 1.

O. Bruno et al. / Bioorg. Med. Chem. 14 (2006) 121–130
123
Cl
R'
O
R"
O
N
O
N
O
(a)
H
OH
O
O
O
NH
2
OCH
3
CH
3
9
R" = CN
10 R" = COOCH3
O
O
Miradon
Warfarin
3a-f
(b)
SCH
N
SCH
N
SCH
N
3
3
3
(d)
(c)
N
N
N
O
The tricyclic structure of our new benzopyrano[4,3-
d]pyrimidin-5-one derivative could mimic the hemichetal
coumarinÕs active form (see the hemichetal of Warfarin).
OH
Cl
N
O
O
O
O
O
11
12
4a-c
Scheme 2. Synthesis of compounds 3a–f and 4a–c: (a) Compound 9,
amidine or guanidine, EtOH, 40 ꢁC, 4 h; (b) Compound 10, S-
methylisothiourea, EtOH, 60 ꢁC, 3 h; (c) POCl3, 150 ꢁC, 5 h; (d)
Cycloamine, dry toluene, reflux, 3 h.
HO
O
CH
3
O
O
Warfarin: hemichetal structure
SO2CH3
N
O
N
As regards the position 2 of compounds 2–5, we main-
tained the same substituents that were essential for anti-
platelet activity in compounds 1. Moreover, in position
4, which was never substituted in previous compounds,
we inserted NH2 and cycloamines with the aim of
obtaining new information for SAR studies.
(a)
N
4a-c
O
5a-c
(b)
SCH3
N
SO2CH3
N
O
N
N
N
(a)
N
Finally, reduction of carbonyl in position 5 of compounds
4,5 yielded the 2-methylthio- and 2-methanesulfonyl-4-
cycloamino derivatives 6–7, already planned, but never
obtained, by other alternative synthetic ways.22
O
7a-c
6a-c
Scheme 3. Synthesis of compounds 5a–c, 6a–c, and 7a–c: (a) m-
chloroperbenzoic acid, CHCl3, r.t., 12 h; (b) NaBH4, BF3/Et2O;
diglyme, dry THF, reflux, 3 h.
Hence, in this work we describe the synthesis and activ-
ity on haemostatic processes of compounds 2–7.
of 4-chloro-2-oxo-2H-[1]benzopyrano-3-carboxylic acid
1026 reacted with an excess of S-methylisothiourea in
dry ethanol to give compound 11 which, by chlorination
with an excess of POCl3, afforded the intermediate 12.
Then, replacement of chlorine with suitable amine, in
dry toluene, gave the desired compounds 4a–c (see
Scheme 2). Subsequent oxidation of SCH3 group of
compounds 4a–c, with an excess of m-Cl-perbenzoic
acid in CHCl3 at room temperature, yielded 2-metha-
nesulfonyl analogues 5a–c (see Scheme 3).
2. Chemistry
2-Substituted benzopyrano[4,3-d]pyrimidin-5-ones 2a–d
were prepared from the suitable hemiacetal derivatives
8a–d19,21,22 by oxidation with KMnO4 in alkali, as already
described by Petersen and Heitzer24 (see Scheme 1).
Starting from 3-cyano-4-chlorocoumarin 9,25 with an
excess of the suitable amidine or guanidine in dry etha-
nol at 40 ꢁC for 4 h, we obtained the 2-substituted
4-amino-benzopyrano[4,3-d]pyrimidin-5-ones 3a–f (see
Scheme 2). Compounds 2b,c, and 3b, yet synthesized
by Petersen and Heitzer24 and Checchi et al.,25 respec-
tively, were never tested for pharmacological activities.
(2-Methylthio-benzopyrano[4,3-d]pyrimidino)-4-cyclo-
amines 6a–c were prepared from compounds 4a–c by
reduction of the carbonyl with sodium borohydride,
boron trifluoride diethyl etherate, and diglyme in dry
27
THF at reflux (see Scheme 3).
4-Cycloamino-2-methylthio-benzopyrano[4,3-d]pyrim-
idin-5-ones 4a–c were obtained as follows: methyl ester
Finally, by oxidation of compounds 6a–c, following the
same procedure reported above, we obtained the
2-methanesulfonyl derivatives 7a–c (see Scheme 3).
R'
R'
N
O
N
N
O
N
O
3. Pharmacology
(a)
H
OH
The new compounds were studied in vitro towards ara-
chidonic acid- (AA), ADP- and the stable TXA2-recep-
tor agonist U46619-induced platelet aggregation in
rabbit platelet rich plasma.
8a-d
2a-d
Scheme 1. Synthesis of compounds 2a–d: (a) KMnO4, NaOH, H2O,
reflux, 2 h.

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O. Bruno et al. / Bioorg. Med. Chem. 14 (2006) 121–130
Furthermore, they were investigated against thrombin-
induced clot retraction in rat platelet-rich plasma. Clot
retraction is a physiological event that stabilizes the
coagulum. It is triggered by an interaction between
extracellular fibrinogen or fibrin and the activated
fibrinogen receptor aIIbb3, located on the platelet sur-
face and anchored to intracellular actin and myosin
chains. The ensuing contraction of actin/myosin fila-
ments, within the platelet, places tension on the fibrin
clot, causing it to contract.28
system and the simultaneous insertion of any substitu-
ents (except for SO2CH3) in positions 2 and 4 are detri-
mental to antiplatelet activity and for the prevention of
the fibrin clotting retraction (compounds 2a–d, 3a–f,
and 4a–c became completely inactive up to 1 mM con-
centration) (Table 2).
In 2-SCH3 derivatives 6a–c, devoid of the carbonyl in po-
sition 5, translation of the cycloamine groups from posi-
tion 5 to position 4 triggers a loss of antiplatelet activity
against ADP, as a evidenced by comparison with com-
pounds 1 (X = SCH3, Y = NR1R2)19 that have been pre-
viously described as potent and effective anti-aggregant
agents against several aggregating substances. Therefore,
among the tested compounds, only derivatives 6a–c be-
have as selective inhibitors of AA-induced aggregation
and show an ASA-like profile with similar antiplatelet
potency and complete ineffectiveness in clot retraction.
Both inhibition of platelet aggregation and clot retrac-
tion are critical factors for the activity of antithrombotic
drugs. Antiplatelet agents limit the activation of plate-
lets, which are essential elements to prime haemostatic
plug formation; agents inhibiting retraction destabilize
clots making them weaker and more likely to be degrad-
ed by the fibrinolytic system.29
Additionally, we investigated the in vivo antithrombotic
effect produced by subacute treatment using an experi-
mental model of pulmonary acute thromboembolism
produced by collagen plus epinephrine injection in
mice.30
The simultaneous presence of SO2CH3 in position 2 of
benzopyranopyrimidinone nucleus and cycloamine in
position 4 (obtained in derivatives 5a–c) seems to repre-
sent a favourable structural feature to confer to the mol-
ecules a large-spectrum antiplatelet activity. The absence
of carbonyl group in position 5 is well-tolerated only in
the case of 2-pyrrolidinyl derivative (7a), which was as
potent as compounds 5a–c both in inhibiting platelet
aggregation and clot retraction.
Tolerability of the subacute treatment with such com-
pounds was examined by evaluating their adverse
pro-haemorrhagic effect in tail bleeding31 and daily
monitoring the body weight, behaviour, and physical
appearance of the treated mice.
The compounds were studied in vivo by assessing their
ability to prevent the paralysis caused by pulmonary
acute thromboembolism in mice. After subacute intra-
peritoneal treatment, only compounds 2d, 5a–c, 6a,
and 7a significantly protected mice from thromboembo-
lic paralysis and they were more potent than aspirin but
less powerful than the conventional anticoagulant war-
farin (Fig. 3). Among these compounds, the 2-metha-
nesulfonyl derivatives, equipotent in vitro as broad
spectrum antiplatelet agents, displayed a slightly differ-
ent antithrombotic potency in vivo (compound 5c being
three times more potent than compounds 5a, 5b, and
7a). Moreover, a strong protective effect in vivo was also
detected for 2-morpholinyl benzopyranopyrimidinone
2d and 2-methylthio benzopyranopyrimidine 6a, the
former devoid of any antiplatelet activity and the latter
active only towards AA-induced aggregation. Different
explanations can account for the discrepancies between
in vivo antithrombotic activity and in vitro antiplatelet
property observed for the aforementioned compounds.
Indeed, it is well-known that antithrombotic activity
can be independent of the inhibition of platelet aggrega-
tion and that it can ensue from compounds, bioactiva-
tion or involvement of indirect mechanisms occurring
only in vivo. Paradigmatic example is warfarin, which
is an effective antithrombotic agent in vivo, devoid of
any antiplatelet activity in vitro. At doses that are able
to halve the percentage of thrombotic events compared
to control group, the compounds do not produce any
adverse side effects. Indeed, they do not increase bleed-
ing at variance with aspirin (Fig. 4) nor do they affect
the body weight and physical appearance of the treated
mice (data not shown). Furthermore, they do not cause
lethal events, as warfarin does, at antithrombotic doses
4. Results and discussion
Only a minority of new compounds inhibit platelet
aggregation and clot retraction in vitro, as given in Ta-
ble 2. This finding suggests that the presence of the car-
bonyl group in position 5 of the benzopyranopirimidine
Table 2. In vitro antiplatelet potency of compounds against arachi-
donic acid (AA), U46619, and ADP induced aggregation in rabbit
PRP and inhibitory potency against thrombin induced clot retraction
in rat PRP
Inhibition of platelet
aggregation IC50 (lM)
Inhibition of clot
retraction IC50 (lM)
Compounds ADP AA
U46619 Thrombin
20 U/mL
3 lM 50 lM 1 lM
2a–d
3a–f
4a–c
5a
—
—
—
13
18
16
—
—
—
45
—
—
—
15
16
21
45
23
71
14
—
—
—
4
—
—
—
13
27
37
—
—
—
72
—
—
—
—
5b
5c
21
3
6a
6b
—
—
—
24
390
—
0.01
—
6c
7a
7b
7c
>500 377
—
>10
>500
2
SQ 29548
ASA
>500 61
The IC50 values (lM) are calculated from n = 6–8 distinct experiments.

O. Bruno et al. / Bioorg. Med. Chem. 14 (2006) 121–130
125
100
80
60
40
20
0
*
*
*
**
**
*
*
**
Cnt
50 100
1
3
10 30
3
10 30
10 30
1
3
10
10 30
10 30
(mg/kg)
ASA
WAR
2d
5a
5b
5c
6a
7a
Figure 3. Acute pulmonary thromboembolism induced by i.v. shot injection of a mixture of 12 mg/kg collagen and 1 mg/kg epinephrine in mice,
treated intraperitoneally for 5 days with vehicle alone (Cnt), the reference drugs aspirin (ASA) and warfarin (WAR) and the test compounds. Every
column represents the percentage of paralysed animals. *p < 0.05 and **p < 0.01 by v2 test compared to control.
40
30
20
*
10
0
Cnt
50 100
1
3
10 30
3
10 30
10 30
1
3
10
10 30
10 30
(mg/kg)
ASA WAR
2d
5a
5b
5c
6a
7a
Figure 4. Bleeding test in mice treated intraperitoneally for 5 days with vehicle alone (Cnt), the reference drugs aspirin (ASA), and warfarin (WAR)
and the test compounds. The volume of blood lost in 2 min by each mouse is reported as lL/2 min. *p < 0.05 by Mann–Whitney test.
(data not shown). This favourable risk benefit ratio
increases the interest in these compounds whose anti-
thrombotic activity, generally but not exclusively depen-
dent on antiplatelet activity, deserves further
investigations.
5. Experimental protocols
5.1. Chemistry
All chemicals were obtained from Sigma–Aldrich s.r.l.
(Milan, Italy).
In conclusion, we synthesized several new benzopyr-
ano[4,3-d]pyrimidine and benzopyrano[4,3-d]pyrimi-
Melting points are uncorrected and were measured with
a Buchi 540 instrument. IR spectra were recorded with a
¨
Perkin-Elmer 398 spectrophotometer. H NMR spectra
were recorded on a Varian Gemini 200 (200 MHz)
instrument; chemical shifts are reported as d (ppm) rel-
ative to tetramethylsilane (TMS) as internal stardard;
signals were characterized as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet) and br s (broad
signal); J in Hz.
din-5-one derivatives, some of them showing
a
1
large-spectrum antiplatelet activity in vitro and poten-
cy higher than that of aspirin as antithrombotics
in vivo. Interestingly, the most active compounds seem
to possess a potentially good benefit-risk profile; there-
fore, new pharmacological studies have been planned
with the aim of examining their mechanism of action
carefully.

126
O. Bruno et al. / Bioorg. Med. Chem. 14 (2006) 121–130
All compounds were tested for purity by TLC (Kieselgel
60F254 DC-Alufolien, E. Merck, Darmstadt, Germa-
ny). Analyses, indicated by the symbols of the elements
or functions, were within 0.4% of the theoretical values
and were determined with an Elemental Analyzer EA
1110 (Fison-Instruments, Milan, Italy).
5.1.7. 4-Amino-5H-[1]benzopyrano[4,3-d]pyrimidin-5-one
3a. Mp 287–288 ꢁC; yield: 60%. IR (KBr) cmꢀ1: 3397,
1
3280 (NH2), 1704 (CO). H NMR (DMSO-d6) d: 3.36
(s, cryst. H2O), 7.42–7.53 (m, 2H, H7+H8), 7.76 (t,
J = 6, 1H, H9), 8.22 (br s, 1H, NH disappears with
D2O), 8.41 (d, J = 6, 1H, H10), 8.63 (br s, 1H, NH disap-
pears with D2O), 8.72 (s, 1H, H2). Anal. Calcd for
C11H7N3O21/3H2O: C, 60.27; H, 3.53; N, 19.17. Found:
C, 59.96; H, 3.42; N, 19.50.
5.1.1. General procedure for 2-substituted 5H-[1]benzo-
pyrano[4,3-d]pyrimidin-5-ones 2a–d. To
a
suitable
2-substituted 5-hydroxy-5H-[1]benzopyrano[4,3-d]py-
rimidine 8 (10 mmol) were added NaOH (12.5 mmol,
0.5 g), KMnO4 (10 mmol, 1.6 g) and H2O (30 mL) and
the mixture was stirred at reflux for 2 h. After cooling,
the brown reaction mixture was filtered and the solution
was acidified with conc. HCl (1.7 mL). The yellow solid
obtained was filtered and recrystallized from DMF/H2O
(1:1) (compounds 2a–c) or absolute ethanol (compound
2d).
5.1.8. 2,4-Diamino-5H-[1]benzopyrano[4,3-d]pyrimidin-5-
one 3b. Mp 288–290 ꢁC (lit. 280–282); yield: 54%. IR
1
(KBr) cmꢀ1: 3609, 3412, 3300 (NH2), 1688 (CO). H
NMR (DMSO-d6) d: 7.22 (br s, 2H, NH2, disappears
with D2O), 7.36–7.43 (m, 2H, H7+H8), 7.69 (t, J = 6,
1H, H9), 7.83 (br s, 1H, NH, disappears with D2O),
7.92 (br s, 1H, NH disappears with D2O), 8.26 (d,
J = 6, 1H, H10). Anal. Calcd for C11H8N4O2: C, 57.89;
H, 3.53; N, 24.55. Found: C, 57.69; H, 3.79; N, 24.66.
5.1.2. 2-Methoxy-5H-[1]benzopyrano[4,3-d]pyrimidin-5-
one 2a. Mp 164–165 ꢁC; yield: 31%. IR (KBr) cmꢀ1
:
5.1.9. 2-Methoxy-4-amino-5H-[1]benzopyrano[4,3-d]pyr-
imidin-5-one 3c. Mp 277–278 ꢁC; yield: 53%. IR (KBr)
cmꢀ1: 3410, 3340 (NH2), 1710 (CO). 1H NMR
(DMSO-d6) d: 3.34 (s, cryst. H2O), 3.99 (s, 3H,
OCH3), 7.40–7.55 (m, 2H, H7+H8), 7.75 (t, J = 6, 1H,
H9), 8.20 (br s, 1H, NH, disappears with D2O), 8.34
(d, J = 6, 1H, H10), 8.58 (br s, 1H, NH, disappears with
D2O). Anal. Calcd for C12H9N3O31/3H2O: C, 57.83; H,
3.91; N, 16.86. Found: C, 58.02; H, 3.61; N, 16.94.
1734 (CO). 1H NMR (DMSO-d6) d: 4.15 (s, 3H,
OCH3), 7.42–7.56 (m, 2H, H7+H8), 7.81 (t, J = 6, 1H,
H9), 8.42 (d, J = 6, 1H, H10), 9.31 (s, 1H, H4). Anal.
Calcd for C12H8N2O3: C, 63.16; H, 3.53; N, 12.28.
Found: C, 63.07; H, 3.61; N, 12.32.
5.1.3. 2-Methylthio-5H-[1]benzopyrano[4,3-d]pyrimidin-
5-one 2b. Mp 187–188 ꢁC (lit. 187–190); yield: 37% (lit.
1
27%). IR (KBr) cmꢀ1: 1725 (CO). H NMR (DMSO-
d6) d: 2.69 (s, 3H, SCH3), 7.40–7.51 (m, 2H, H7+H8),
7.76 (t, J = 6, 1H, H9), 8.40 (d, J = 6, 1H, H10), 9.20
(s, 1H, H4). Anal. Calcd for C12H8N2O2S: C, 59.01; H,
3.30; N, 11.47. Found: C, 59.25; H, 3.52; N, 11.20.
5.1.10.
2-Methylthio-4-amino-5H-[1]benzopyrano[4,3-
d]pyrimidin-5-one 3d. Mp 278–280 ꢁC; yield: 43%. IR
(KBr) cmꢀ1: 3400, 3270 (NH2), 1710 (CO). H NMR
1
(DMSO-d6) d: 2.60 (s, 3H, SCH3), 3.35 (s, cryst. H2O),
7.41–7.48 (m, 2H, H7+H8), 7.75 (t, J = 6, 1H, H9),
8.17 (br s, 1H, NH, disappears with D2O), 8.36 (d,
J = 6, 1H, H10), 8.58 (br s, 1H, NH, disappears with
D2O). Anal. Calcd for C12H9N3O2S.1/3H2O: C, 54.33;
H, 3.67; N, 15.84. Found: C, 54.25; H, 3.32; 15.80.
5.1.4. 2-Pyrrolidino-5H-[1]benzopyrano[4,3-d]pyrimidin-
5-one 2c. Mp 205–206 ꢁC (lit. 205–207); yield: 45% (lit.
1
45%). IR (KBr) cmꢀ1: 1726 (CO). H NMR (DMSO-
d6) d: 1.91–2.04 (m, 4H, 2CH2 pyrr), 3.30 and 3.65
(2m, 4H, 2CH2N pyrr), 7.31–7.45 (m, 2H, H7+H8),
7.69 (t, J = 6, 1H, H9), 8.31 (d, J = 6, 1H, H10), 8.96
(s, 1H, H4). Anal. Calcd for C15H13N3O2: C, 67.41; H,
4.90; N, 15.72. Found: C, 67.25; H, 4.80; N, 15,83.
5.1.11.
2-Pyrrolidino-4-amino-5H-[1]benzopyrano[4,3-
d]pyrimidin-5-one 3e. Mp 253–254 ꢁC; yield: 59%. IR
(KBr) cmꢀ1: 3501, 3375 (NH2), 1677 (CO). H NMR
1
(DMSO-d6) d: 1.91–2.05 (m, 4H, 2CH2 pyrr), 3.38 (s,
cryst. H2O), 3.63–3.76 (m, 4H, 2CH2N pyrr), 7.33–
7.43 (m, 2H, H7+H8), 7.68 (t, J = 6, 1H, H9), 7.90 (br
s, 2H, NH2, disappears with D2O), 8.30 (d, J = 6, 1H,
H10). Anal. Calcd for C15H14N4O2.1/3H2O: C, 62.49;
H, 5.13; N, 19.43. Found: C, 62.68; H, 4.88; N, 19.73.
5.1.5. 2-Morpholino-5H-[1]benzopyrano[4,3-d]pyrimidin-
:
5-one 2d. Mp 195–196 ꢁC; yield: 42%. IR (KBr) cmꢀ1
1
1726 (CO). H NMR (DMSO-d6) d: 3.69–3.87 (m, 4H,
2CH2N morph), 3.92–4.10 (m, 4H, 2CH2O morph),
7.38–7.45 (m, 2H, H7+H8), 7.76 (t, J = 6, 1H, H9),
8.39 (d, J = 6, 1H, H10), 9.04 (s, 1H, H4). Anal. Calcd
for C15H13N3O3: C, 63.60; H, 4.63; N, 14.83. Found:
C, 63.24; H, 5.03; N, 14.50.
5.1.12.
2-Morpholino-4-amino-5H-[1]benzopyrano[4,3-
d]pyrimidin-5-one 3f. Mp 229–230 ꢁC; yield: 55%. IR
1
(KBr) cmꢀ1: 3438, 3314 (NH2), 1684 (CO). H NMR
5.1.6. General procedure for 2-substituted 4-amino-5H-
[1]benzopyrano[4,3-d]pyrimidin-5-ones 3a–f. To a solu-
tion of 4-chloro-3-cyanocoumarin 9 (5 mmol, 1.03 g)
in absolute ethanol (10 mL) was slowly added a solu-
tion of suitable guanidine or amidine (obtained from
the corresponding hydrochloride or hydrogen sulfate
with equimolar EtONa) (10 mmol) in absolute ethanol
and the mixture was stirred at 40 ꢁC for 4 h. The solid
obtained was filtered and recrystallized from DMF/
H2O (1:1).
(DMSO-d6) d: 3.67–3.71 (m, 4H, 2CH2N morph), 3.36
(s, cryst. H2O), 3.82–4.02 (m, 4H, 2CH2O morph),
7.33–7.43 (m, 2H, H7+H8), 7.69 (t, J = 6, 1H, H9),
7.98 (br s, 2H, NH2, disappears with D2O), 8.32 (d,
J = 6, 1H, H10). Anal. Calcd for C15H14N4O3. H2O: C,
56.96; H, 5.10; N, 17.71. Found: C, 56.60; H, 4.88; N,
17.66.
5.1.13. Preparation of 4-hydroxy-2-methylthio-5H-
[1]benzopyrano[4,3-d]pyrimidin-5-one 11. To a solution

O. Bruno et al. / Bioorg. Med. Chem. 14 (2006) 121–130
127
of EtONa, obtained from Na (10 mmol, 0.23 g) and
absolute ethanol (10 mL), was added S-methylisothiou-
rea sulfate (5 mmol, 1.39 g). The mixture was stirred for
20 min, then Na2SO4 was filtered off and the solution
was slowly added to methyl 4-chloro-2-oxo-2H-[1]benzo-
pyrane-3-carboxylate 10 (5 mmol, 1.19 g) solved in abso-
lute ethanol. The mixture was stirred at 60 ꢁC for 3 h.
The solid was filtered and recrystallized from DMF/
H2O (1:1).
C17H17N3O2S: C, 62.37; H, 5.23; N, 12.83. Found: C,
62.60; H, 5.06; N, 12.89.
5.1.18.
2-Methylthio-4-morpholino-5H-[1]benzopyr-
ano[4,3-d]pyrimidin-5-one 4c. Mp 175–176 ꢁC; yield:
1
83%. IR (KBr) cmꢀ1: 1717 (CO). H NMR (CDCl3) d:
2.61 (s, 3H, SCH3), 3.73–3.82 (m, 4H, 2CH2N morph),
3.84–3.90 (m, 4H, 2CH2O morph), 7.26–7.40 (m, 2H,
H7+H8), 7.62 (t, J = 6, 1H, H9), 8.46 (d, J = 6, 1H,
H10). Anal. Calcd for C16H15N3O3S: C, 58.35; H, 4.59;
N, 12.76. Found: C, 58.07; H, 4.66; N, 12.53.
White solid, mp: 260–261 ꢁC, yield: 40%. IR (KBr)
cmꢀ1: 3651 (OH), 3456 (OH), 1723 (C=O).1H NMR
(DMSO-d6) d: 2.46 (s, 3H, SCH3), 3.37 (m, OH+ cryst.
H2O, disappear with D2O), 7.26–7.38 (m, 2H, H7+H8),
7.62 (t, J = 6, 1H, H9), 8.26 (d, J = 6, 1H, H10). Anal.
5.1.19. General procedure for 2-methanesulfonyl-4-cycloa-
mino-5H-[1]benzopyrano[4,3-d]pyrimidin-5-ones 5a–c. To
an ice-cooled solution of the appropriate 2-methylthio
derivative 4 (3 mmol) in CHCl3 (20 mL), m-chloroper-
benzoic acid (70–75%) (6 mmol, 1.5 g) was added in
small portions. The mixture was stirred at room tem-
perature for 12 h, diluted with CHCl3 (25 mL) and
washed once with Na2S2O5 satd solution (25 mL),
once with NaHCO3 satd solution (25 mL) and once
with water (25 mL). Finally, it was dried (MgSO4)
and evaporated under reduced pressure. Compounds
5a and 5c were purified by flash chromatography on
silica gel with CH2Cl2 or CHCl3/CH3OH (9:1), respec-
tively, as eluents. All the solids obtained were recrys-
tallized from absolute ethanol.
5
Calcd for C12H8N2O3S. /2 H2O: C, 47.21; H, 4.29; N,
9.18. Found: C, 47.15; H, 3.90; N, 8.99.
5.1.14. Preparation of 4-chloro-2-methylthio-5H-[1]ben-
zopyrano[4,3-d]pyrimidin-5-one 12. A mixture of 4-hy-
droxy-2-methylthio-5H-[1]benzopyrano[4,3-d]pyrimidin-
5-one 11 (5 mmol, 1.30 g) and POCl3 (50 mL) was
heated at 150 ꢁC for 5 h. The excess POCl3 was evap-
orated under reduced pressure and the crude product
was treated with ice. The white solid obtained was fil-
tered and recrystallized from absolute ethanol/CHCl3
(9:1).
White solid, mp: 230–231 ꢁC; yield: 71%. IR (CHCl3)
5.1.20. 2-Methanesulfonyl-4-pyrrolidino-5H-[1]benzopyr-
ano[4,3-d]pyrimidin-5-one 5a. Mp 218–219 ꢁC; yield:
1
cmꢀ1: 1744 (C=O). H NMR (CDCl3) d: 2.73 (s, 3H,
1
SCH3), 7.26–7.46 (m, 2H, H7+H8), 7.69 (t, J = 6, 1H,
H9), 8.51 (d, J = 6, 1H, H10). Anal. Calcd for
C12H7N2ClO2S: C, 51.71; H, 2.53; N, 10.05. Found: C,
51.55; H, 2.54; N, 10.09.
51%. IR (KBr) cmꢀ1: 1731 (CO), 1307, 1114 (SO2). H
NMR (CDCl3) d: 1.96–2.17 (m, 4H, 2CH2 pyrr), 3.41
(s, 3H, CH3), 3.55 (t, J = 6.4, 2H, CH2N pyrr), 3.95 (t,
J = 6.4, 2H, CH2N pyrr), 7.32–7.46 (m, 2H, H7+H8),
7.70 (t, J = 6, 1H, H9), 8.32 (d, J = 6, 1H, H10). Anal.
Calcd for C16H15N3O4S: C, 55.64; H, 4.38; N, 12.17.
Found: C, 55.79; H, 4.40; N, 11.96.
5.1.15. General procedure for 2-methylthio-4-cycloamino-
5H-[1]benzopyrano[4,3-d]pyrimidin-5-ones 4a–c. To a
suspension of 4-chloro-2-methylthio-5H-[1]benzopyr-
ano[4,3-d]pyrimidin-5-one 12 (5 mmol, 1.39 g) in anhyd
toluene (50 mL) was added the suitable cycloamine
(20 mmol) and the mixture was stirred at reflux for
3 h. After cooling, the organic solution was washed
twice with water (20 mL), then dried (MgSO4) and evap-
orated under reduced pressure. The crude oil was crys-
tallized by addition of ethyl ether and the white solid
obtained was filtered and recrystallized from absolute
ethanol.
5.1.21. 2-Methanesulfonyl-4-piperidino-5H-[1]benzopyr-
ano[4,3-d]pyrimidin-5-one 5b. Mp 211–212 ꢁC; yield:
1
83%. IR (KBr) cmꢀ1: 1727 (CO), 1313, 1135 (SO2). H
NMR (CDCl3) d: 1.74–1.84 (m, 6H, 3CH2 pip), 3.40
(s, 3H, CH3), 3.60–4.00 (m, 4H, 2CH2N pip), 7.32–
7.45 (m, 2H, H7+H8), 7.68 (t, J = 6, 1H, H9), 8.50 (d,
J = 6, 1H, H10). Anal. Calcd for C17H17N3O4S: C,
56.81; H, 4.77; N, 11.69. Found: C, 56.74; H, 4.83; N,
11.52.
5.1.16. 2-Methylthio-4-pyrrolidino-5H-[1]benzopyrano-
[4,3-d]pyrimidin-5-one 4a. Mp 174–176 ꢁC; yield: 74%.
5.1.22. 2-Methanesulfonyl-4-morpholino-5H-[1]benzopyr-
ano[4,3-d]pyrimidin-5-one 5c. Mp 228–229 ꢁC; yield:
1
1
IR (KBr) cmꢀ1: 1714 (CO). H NMR (CDCl3) d: 1.82–
41%. IR (KBr) cmꢀ1: 1730 (CO), 1310, 1109 (SO2). H
2.12 (m, 4H, 2CH2 pyrr), 2.63 (s, 3H, SCH3), 3.54 and
3.90 (2m, 4H, 2CH2N pyrr), 7.20–7.38 (m, 2H, H7+H8),
7.58 (t, J = 6, 1H, H9), 8.45 (d, J = 6, 1H, H10). Anal.
Calcd for C16H15N3O2S: C, 61.32; H, 4.82; N, 13.41.
Found: C, 61.02; H, 5.00; N, 13.26.
NMR (CDCl3) d: 3.41 (s, 3H, CH3), 3.68–4.03 (m, 8H,
4CH2 morph), 7.28–7.48 (m, 2H, H7+H8), 7.70 (t,
J = 6, 1H, H9), 8.49 (d, J = 6, 1H, H10). Anal. Calcd
for C16H15N3O5S: C, 53.18; H, 4.18; N, 11.63. Found:
C, 52.93; H, 4.24; N, 11.49.
5.1.17.
2-Methylthio-4-piperidino-5H-[1]benzopyrano-
5.1.23. General procedure for (2-methylthio-5H-[1]benzo-
[4,3-d]pyrimidin-5-one 4b. Mp 160–161 ꢁC; yield: 87%.
pyrano[4,3-d]pyrimidino)-4-cycloamines 6a–c. To
cooled mixture of the suitable 4-cycloamino-2-methyl-
thio-5H-[1]benzopyrano[4,3-d]pyrimidin-5-ones 4a–c
(3 mmol) in anhyd. THF (12 mL), boron trifluoride eth-
erate (12 mL), drop to drop, and sodium borohydride
a
1
IR (KBr) cmꢀ1: 1715 (CO). H NMR (CDCl3) d: 1.71–
1.78 (m, 6H, 3CH2 pip), 2.61 (s, 3H, SCH3), 3.65–3.77
(m, 4H, 2CH2N pip), 7.25–7.37 (m, 2H, H7+H8), 7.58 (t,
J = 6, 1H, H9), 8.44 (d, J = 6, 1H, H10). Anal. Calcd for

128
O. Bruno et al. / Bioorg. Med. Chem. 14 (2006) 121–130
(520 mg) in small portions were added. Then, diglyme
(12 mL) was slowly added and the mixture was stirred
at room temperature for 30 min and then refluxed for
3 h. After cooling, water (3 mL) and small portions of
Na2CO3 until pH 6 were added. Aqueous solution was
extracted twice with ethyl ether (20 mL), the organic
phases were washed once with water (20 mL), dried
(MgSO4) and evaporated under reduced pressure. The
crude oil obtained was treated with ethyl ether; the white
solid obtained was filtered and recrystallized from abso-
lute ethanol/CHCl3 (9:1).
for C16H17N3O3S: C, 57.99; H, 5.17; N, 12.68. Found:
C, 57.85; H, 4.91; N, 12.67.
5.1.29. (2-Methanesulfonyl-5H-[1]benzopyrano[4,3-d]pyr-
imidino)-4-piperidine 7b. Mp 202–203 ꢁC; yield: 57%. IR
1
(KBr) cmꢀ1: 1307, 1129 (SO2). H NMR (CDCl3) d:
1.60–1.80 (m, 6H, 3CH2 pip), 3.35 (s, 3H, CH3), 3.38–
3.45 (m, 4H, 2CH2N pip), 5.13 (s, 2H, OCH2), 6.97 (d,
J = 8, 1H, H7), 7.12 (t, J = 8, 1H, H8), 7.40 (t, J = 8,
1H, H9), 8.14 (d, J = 8, 1H, H10). Anal. Calcd for
C17H19N3O3S: C, 59.11; H, 5.54; N, 12.17. Found: C,
59.27; H, 5.41; N, 12.16.
5.1.24. (2-Methylthio-5H-[1]benzopyrano[4,3-d]pyrimidi-
no)-4-pyrrolidine 6a. Mp 163–165 ꢁC; yield: 53%. 1H
NMR (CDCl3) d: 1.92–1.97 (m, 4H, 2CH2 pyrr), 2.58
(s, 3H, SCH3), 3.57–3.72 (m, 4H, 2CH2N pyrr), 5.34
(s, 2H, OCH2), 6.93 (d, J = 6, 1H, H7), 7.10 (t, J = 6,
1H, H8), 7.34 (t, J = 6, 1H, H9), 8.16 (d, J = 6, 1H,
H10). Anal. Calcd for C16H17N3OS: C, 64.19; H, 5.72;
N, 14.04. Found: C, 64.02; H, 5.73; N, 14.11.
5.1.30. (2-Methanesulfonyl-5H-[1]benzopyrano[4,3-d]pyr-
imidino)-4-morpholine 7c. Mp 219–220 ꢁC; yield: 47%.
1
IR (KBr) cmꢀ1: 1308, 1118 (SO2). H NMR (CDCl3)
d: 3.38 (s, 3H, CH3), 3.52 (t, J = 4.4, 4H, 2CH2N
morph), 3.85 (t, J = 4.4, 4H, 2CH2O morph), 5.16 (s,
2H, OCH2), 6.99 (d, J = 8, 1H, H7), 7.14 (t, J = 8, 1H,
H8), 7.42 (t, J = 8, 1H, H9), 8.14 (d, J = 8, 1H, H10).
Anal. Calcd for C16H17N3O4S: C, 55.32; H, 4.93; N,
12.10. Found: C, 55.20; H, 4.97; N, 12.09.
5.1.25. (2-Methylthio-5H-[1]benzopyrano[4,3-d]pyrimidi-
no)-4-piperidine 6b. Mp 129–130 ꢁC; yield: 75%. 1H
NMR (CDCl3) d: 1.63–1.77 (m, 6H, 3CH2 pip), 2.59
(s, 3H, SCH3), 3.23–3.38 (m, 4H, 2CH2N pip), 5.06 (s,
2H, OCH2), 6.95 (d, J = 6, 1H, H7), 7.11 (t, J = 6, 1H,
H8), 7.37 (t, J = 6, 1H, H9), 8.15 (d, J = 6, 1H, H10).
Anal. Calcd for C17H19N3OS: C, 65.15; H, 6.11; N,
13.41. Found: C, 64.96; H, 6.08; N, 13.45.
5.2. Pharmacology
5.2.1. Animals. Male Swiss mice (20–30 g), male Wistar
rats (200–250 g) (Charles River) and male albino rabbits
(3–3.5 kg) fed on a regular diet were used for the exper-
iments. The animals were starved at 20 ꢁC and fasted
18 h with free access to water before the experiments.
All the experiments were performed according to ethical
standard guidelines and were approved by Italian Min-
istry of Health (DL116/92).
5.1.26. (2-Methylthio-5H-[1]benzopyrano[4,3-d]pyrimidi-
no)-4-morpholine 6c. Mp 155–156 ꢁC; yield: 53%. 1H
NMR (CDCl3) d: 2.59 (s, 3H, SCH3), 3.34 (t, J = 5,
4H, 2CH2N morph), 3.82 (t, J = 5, 4H, 2CH2O
morph), 5.07 (s, 2H, OCH2), 6.92 (d, J = 6, 1H, H7),
7.10 (t, J = 6, 1H, H8), 7.25 (t, J = 6, 1H, H9), 8.12
(d, J = 6, 1H, H10). Anal. Calcd for C16H17N3O2S: C,
60.93; H, 5.43; N, 13.32. Found: C, 60.78; H, 5.52;
N, 13.32.
The concentration-dependent inhibition of platelet
aggregation and clot retraction were studied in vitro
on rabbit or rat PRP by adding different concentrations
(from 1 lM to 1 mM) of the test compounds. Acetylsal-
icylic acid (ASA), as well as the TXA2 antagonist SQ
29548, were used as reference drugs. The inhibitory
potency of compounds, in both platelet aggregation
and clot retraction tests, was expressed as IC50 (the con-
centration required to inhibit the response by 50% com-
pared to the control experiment) and it was calculated
by linear regression analysis of the specific concentra-
tion–response curves using the least-squares method.
5.1.27. General procedure for (2-methanesulfonyl-5H-
[1]benzopyrano[4,3-d]pyrimidino)-4-cycloamines 7a–c. To
an ice-cooled solution of the suitable 2-methylthio-
5H-[1]benzopyrano[4,3-d]pyrimidin-4-cycloamine
6
(3 mmol) in CHCl3 (20 mL), m-chloroperbenzoic acid
(70–75%) (6 mmol, 1.5 g) was added in small portions.
The reaction mixture was stirred at room temperature
for 12 h, diluted with CHCl3 (25 mL) and washed once
with Na2S2O5 satd solution (25 mL), once with NaH-
CO3 satd solution (25 mL) and once with water
(25 mL). Finally, it was dried (MgSO4) and evaporated
under reduced pressure. The crude was purified by flash
chromatography on silica gel with CHCl3 as eluent. The
white solid obtained was recrystallized from absolute
ethanol/CHCl3 (9:1).
To study the effects of the compounds on experimental
thrombosis and bleeding time, the drugs or the vehicle
was administered intraperitoneally at different doses
(1–30 mg/kg/day) for 5 days in mice randomly distribut-
ed across treatments (n = 10–14 mice for each group).
AnimalsÕ body weight, behaviour and physical appear-
ance were monitored daily. The experiments were per-
formed 1 h after the last pharmacological treatment.
Acetyl salicylic acid (ASA) (50–100 mg/kg/day i.p.)
and warfarin (1–3 mg/kg/day i.p.) were used as reference
drugs. Dose–response curves of test compounds and ref-
erence drugs were constructed to evaluate antithrombot-
ic and pro-haemorrhagic potency.
5.1.28. (2-Methanesulfonyl-5H-[1]benzopyrano[4,3-d]pyr-
imidino)-4-pyrrolidine 7a. Mp 240–241 ꢁC; yield: 56%.
1
IR (KBr) cmꢀ1: 1306, 1113 (SO2). H NMR (CDCl3)
d: 1.90–2.15 (m, 4H, 2CH2 pyrr), 3.36 (s, 3H, CH3),
3.68–3.78 (m, 4H, 2CH2N pyrr), 5.45 (s, 2H, OCH2),
6.94 (d, J = 8, 1H, H7), 7.12 (t, J = 8, 1H, H8), 7.23–
7.43 (m, 1H, H9), 8.18 (d, J = 8, 1H, H10). Anal. Calcd
5.2.2. In vitro antiplatelet activity. Rabbit blood, antico-
agulated with sodium citrate 3.8% 1 part citrate:9 part

O. Bruno et al. / Bioorg. Med. Chem. 14 (2006) 121–130
129
blood, was obtained by cardiac puncture after CO2
euthanasia and collected in plastic tubes.
5.2.5. Bleeding test. Bleeding test was determined,
according to the method of Dejana et al.31 Tails of
lightly anaesthetized mice were transected at 2 mm
from the tip and immersed in 1 mL of 37 ꢁC saline
for 2 min. Red blood cells were lysed by adding
20 lL triton 5% and absorbance of the solution was
read at 560 nm (LKB, Ultraspec 4050). The amount
of haemorrhage was estimated by linear regression
analysis of a standard curve constructed from known
volumes of mouse blood.
After centrifugation for 15 min at 180 g to obtain
platelet-rich plasma (PRP), the remaining blood was
spun 10 min at 2000 g to obtain platelet poor plasma
(PPP). Platelet aggregation was performed in the
Aggrecorder PA 3220 (Menarini, Firenze) at 37 ꢁC
and in continuous stirring (1000 rpm), following BornÕs
turbidimetric method.32 Aggregation was recorded as
the percent change in light transmission: the baseline
was set using PRP and maximal transmission using
PPP. PRP was preincubated at 37 ꢁC for 5 min with
solvent (dimethyl sulfoxide, at a maximal final concen-
tration of 0.5%), the compounds under study or the
reference drug at various concentrations before addi-
tion of platelet aggregant agents. Maximal platelet
aggregation was induced with 5 lM ADP, 100 lM
AA, or 2 lM U46619. Tests were performed within
3 h from blood collection to avoid platelet inactivation.
The effects of test compounds were assessed as percent
inhibition compared with the control sample. In blank
tests, DMSO 0.5% did not interfere with platelet
aggregation.
5.2.6. Statistical analysis. Data are expressed as mean-
s
SEM. Differences between treated groups and
vehicle-treated control group were analysed by v2 test
for the evaluation of antithrombotic activity and
by Mann–Whitney test for bleeding assay.
Otherwise stated unpaired StudentÕs t test was applied to
perform statistical analysis in the other studies with p
value <0.05 or <0.01 being considered as statistically
significant or highly significant, respectively.
5.2.7. Drugs. Drugs and reagents were obtained as fol-
lows: arachidonic acid. ADP, bovine thrombin
(T3399), acetylsalicylic acid (ASA), calf collagen type
III, epinephrine bitartrate, sodium citrate, dim-
ethylsulpfoxide (DMSO) and Triton were from Sigma
Chemical Co., St. Louis, MO. U46619 and SQ 29548
were from Caymanchem (MI, USA). ASA as lysine ace-
tylsalicylate used for in vivo experiments was from
Sanofi (Synthe Cabo). Drugs were dissolved in saline,
except for collagen, which was suspended in isotonic
glucose buffer according to Momo et al. (1992).34 The
compounds under study were dissolved in DMSO for
in vitro experiments (0.5% DMSO final concentration)
or suspended in saline for in vivo experiments.
5.2.3. Clot retraction assay. Blood, collected through
cardiac puncture from rats euthanized with CO2, was
centrifuged, as previously described, and properly dilut-
ed with Tyrode buffer (137 mM NaCl, 20 mM Hepes,
5.6 mM glucose, 1 mM MgCl2, 2.7 mM KCl and
3.3 mM NaH2PO4, pH 7.4) to obtain PRP with about
200,000 platelets/lL. The assay was performed accord-
ing to Davidson and Henry.33
Briefly, 450 lL aliquots of the above platelet suspension
were added to siliconized glass tubes and incubated for
10 min with 5 lL solvent (DMSO 0.5% final concentra-
tion) or compounds under study at 37 ꢁC. Fibrin clot
retraction was induced by addition of 50 lL thrombin
20 U/mL. Clotting proceeded for 60 min at 37 ꢁC. Clot
formation and subsequent clot retraction were recorded
visually by digital camera after 2 and 60 min from
thrombin addition. Quantification of fibrin clot retrac-
tion was performed measuring by clot area by means
of the NIH Image 1.67e software. Data were expressed
as percentage of retraction = (area t0 ꢀ area t)/area
t0 · 100, wheret0 is the area of the clot 2 min after
thrombin addition and t is the area at the test time
(60 min).
Acknowledgments
Financial support from Italian MIUR (Cofin 2003,
Grant 2003033405_006) and skillful help in the experi-
mental work from Dr G. Domenichini, Dr R. Raggio,
Mr O. Gagliardo, and Mr F. Tuberoni are gratefully
acknowledged.
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