P.L. Ferrarini et al. / Il Farmaco 55 (2000) 603–610
607
3.1.2. General procedure for the preparation of
2-methoxy deri6ati6es 7 and 8
for 10 min at 37°C before the addition of the aggregat-
ing agent. To express the aggregation of platelets, the
transmittance of PRP itself was set at 0% while the
platelet poor plasma (PPP) was set at 100%. The aggre-
gation rate was also evaluated from the slope of the
experimental plot of aggregation as a function of time.
The test substances were dissolved in DMSO and the
stock solution was diluted with water to obtain the
experimental concentration. The DMSO solutions of
compounds 2d, 3d, 5a–c, 6a,c, 7a–c, 8a, and 10b,c were
diluted with water and a few drops of 0.1 M hydrochlo-
ric acid. The pH was then set to 7.4 with sodium
hydrogen carbonate. Compounds 6b, 8b,c and 9b–d,
were insoluble under these experimental conditions.
The final DMSO concentration was 0.5% v/v. Control
aggregation was studied in the presence of DMSO at
the same concentration used for treated platelets.
Both adenylate cyclase and intracellular c-AMP level
were measured by a radioimmunoassay technique using
commercially available tests (Rianen c-AMP 125I-RIA
kit; 32P-ATP; NEN-Du Pont). Adenylate cyclase was
measured in platelet plasma membranes prepared in
accordance with the method described by Kahn and
Sinha [12], while c-AMP levels were measured in intact
platelets. 200 ml of PRP was centrifuged at 200g for 30
min. The supernatant was gently decanted, and the soft
pellet containing intact platelets was suspended in 12 ml
of Tyrode buffer, pH 7.4; the final number of platelets
was adjusted to ca. 108/ml. For each sample, 300 ml
aliquots of this suspension were preincubated at 30°C
with the phosphodiesterase inhibitor 3-isobutyl-1-
methylxanthine (0.5 mM), both in the absence and the
presence of 1 mM EGTA. 10 min later, the compounds
to be tested were added where required to a final
volume of 500 ml. Incubation was stopped after 10 min
by the addition of 1 ml of 3% perchloric acid. Samples
were sonicated and centrifuged at 30 000g for 15 min.
The supernatant was neutralized with an excess (about
100 mg) of CaCO3.
A solution of 10 mmol of freshly prepared sodium
methoxide and 1.0 mmol of the 2-chloro derivatives 5
or 6 in 10 ml of anhydrous methanol was refluxed for 4
h for 5a,b and 6 or 12 h for 5c and the reaction mixture
was evaporated to dryness in vacuo. The crude residue
was treated with water and neutralized with 10% hydro-
chloric acid, and the solid precipitate, collected by
filtration, was purified by crystallization to obtain 7 and
8 (Tables 1 and 2).
3.1.3. General procedure for the preparation of 3-amino
deri6ati6es 9 and 10
A solution of 1.1 mmol of 3-nitro derivatives 4 or 8
in glacial acetic acid was hydrogenated in the presence
of 0.03 g of 10% palladium on charcoal at room
temperature and at atmospheric pressure for 3 h. The
catalyst was filtered and the solvent evaporated to
dryness in vacuo to give compounds 9 and 10, which
were purified by crystallization (Tables 1 and 2).
3.1.4. General procedure for the preparation of
piperazin-7-yl-1,8-naphthyridine deri6ati6es 2d, 3d, 7d,
9d and 10d
A suspension of 1.0 mmol of the appropriate N-
ethoxycarbonylpiperazinyl derivatives 2c [9], 3c [9], 9c,
10c or N-methoxycarbonylpiperazinyl 7c, 15 ml of eth-
anol and 15 ml of 10% sodium hydroxide was refluxed
for 16 h and the organic solvent was evaporated in
vacuo. The aqueous solution was extracted with chloro-
form, dried (MgSO4) and evaporated to dryness in
vacuo to give compounds 2d, 3d, 7d, 9d and 10d, which
were purified by crystallization (Tables 1 and 2).
3.2. Pharmacological methods
Human blood samples were drawn from the anticu-
bital vein and were anticoagulated with 3.8% sodium
citrate (9:1 v/v). Platelet rich plasma (PRP) was pre-
pared in accordance with the method described by
Miceli et al. [10]. The platelet count was adjusted to
about 280 000 cell/ml.
The samples were then centrifuged twice at 30 000g
for 15 min to remove the excess of CaCO3 and 100 ml
aliquots of the supernatant were assayed for their cyclic
AMP. c-AMP was measured in triplicate determina-
tions using the above-mentioned RIA kit.
Platelet aggregation was measured turbidimetrically
in accordance with the method described by Born and
Cross [11], using an aggregometer (Daichii model PA-
3220).
4. Results and discussion
ADP (3.0 mM), arachidonate sodium (0.7 mM) and
collagen (2.0 mg/ml) were used as aggregating agents.
Arachidonate sodium, ADP, papaverine, ASA, ibupro-
fen and indomethacin were provided by Sigma Chemi-
cals, and collagen (from bovine tendon) was provided
by Menarini Diagnostics.
Experiments were conducted by the following proce-
dures. Substances at different concentrations, ranging
from 10 to 0.1 mM, were added to PRP and incubated
To evaluate the antiplatelet properties, the substances
were subjected to a preliminary screening estimating the
effects of a fixed concentration (10 mM) on the platelet
aggregation induced by 0.7 mM arachidonate (Table 3).
It was thus possible to determine the inhibition of the
maximum aggregation due to the agonist, and the speed
of aggregation or ‘slope’ which gives the amount of
platelets aggregating in the time unit, whose levels are
expressed as a percentage.
Ferrarini, Pier Luigi
