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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 [7] 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 :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 in
the presence of 1 mM EGTA. The compounds to be
tested were added where required 10 min later, 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.
in Table 4 confirmed high inhibitory activities for these
compounds: in particular compound 17a was extremely
active (IC50B0.1 mM).
The collagen-induced aggregation made it possible to
evaluate the latency time of aggregation, that is the
time, expressed in seconds, between the addition of the
agonist and the start of aggregation of platelets. This
parameter expresses the delay of platelet aggregation
activation mechanisms.
On the basis of results for this parameter, compared
with a mean basal value of 72–85 s, all the compounds
tested, 11c, 12a, 13a and 17a, delayed the start of
aggregation by 310–360 s, evaluated at a concentration
of 10 mM, and by a time lag ranging from 125 to 300 s
at a concentration of 5 mM, and from 100 to 250 s at a
concentration of 1 mM (Table 5).
To complete the study on the effects of these com-
pounds on platelet aggregation, the dose–effect curve
for compounds 11c, 14c and 17a versus the major
physiological agonist, ADP, was determined. The re-
sults obtained show that compounds 11c, 14c and 17a
(Table 6) exhibited a significant biological activity.
The study of intracellular events due to contact of
intact platelets with the compounds 14c and 17a
showed an increase in c-AMP levels, as shown in Table
7, independently of the activation of adenylate cyclase,
whose activity was, on the contrary, reduced by the
compounds tested (data not shown) and probably me-
diated by inhibition of phosphodiesterase [2,4].
Further studies on the mechanism of action of these
compounds are in progress.
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.
4. Results and discussion
All the substances were subjected to preliminary
screening to evaluate their effects at a fixed concentra-
tion (10 mM) on the platelet aggregation induced by 0.7
mM arachidonate (Table 3). On the basis of these
results, the substances tested were subdivided into two
different groups, in accordance with the inhibition val-
ues observed, which are expressed by a percentage: (A)
from 0% to 50%, from inactive to moderately active;
and (B) from 51% onward, really active.
Only compounds 11c, 12a, 13a, 14c and 17a showed
a significant ability to inhibit the arachidonate- and
collagen-induced aggregation of platelets, whereas sev-
eral compounds were insoluble or showed a poor
activity.
We were unable to determine the efficacy of com-
pounds 4c, 5b, 6b,c, 10b,c, 12b,c and 13b,c, because
they could not be dissolved by the procedures used.
Compounds 4a,b, 5a,c, 6a, 10a, 11a,b, 14a,b, 15a–c,
16a, 17c, 18a,c, 19a,c and 20a,c displayed a poor activ-
ity. The IC50 for the dose-dependent inhibition of
platelet aggregation induced by arachidonate was eval-
uated for compounds 11c, 12a, 13a, 14c and 17a, which
exhibited a considerable activity in the preliminary
screening. As shown in Table 4, all the compounds
revealed a very low IC50, ranging from 0.7 to 10.0 mM
for both parameters measured (inhibition of maximum
aggregation and inhibition of the speed of aggregation);
these values were higher than those of the reference
compounds.
In a previous paper [4] we reported that some 2-
N-cycloamino-3-phenyl-1,8-naphthyridine derivatives
showed an appreciable ability to inhibit in vitro human
platelet aggregation induced by arachidonate, collagen
or ADP, but on the basis of the pharmacological
results, no structure–activity relationship could be de-
duced. Hoping to obtain a structure–activity relation-
ship, we also synthesized and tested this series
of 2,7-di(N-cycloamino)-3-phenyl-1,8-naphthyridine
derivatives.
The pharmacological results obtained for this last
series of compounds have not fully made clear the role
of the various substituents, also because in this series,
many compounds are insoluble.
To draw a conclusion, we analyzed the pharmacolog-
ical results of the 2-N-cycloamino- [4] and 2,7-di(N-cy-
cloamino)-3-phenyl-1,8-naphthyridine derivatives.
The influence of the nitro group in position 6 of the
1,8-naphthyridine nucleus on the inhibition of the ag-
gregation induced by arachidonate in these two series
With the aim of excluding a possible selective inhibi-
tion of the membrane enzyme phospholipase A2, the
inhibition of the aggregation induced by collagen at a
concentration of 2.0 mg/ml was evaluated for com-
pounds 11c, 12a, 13a, 14c and 17a. The results shown
Ferrarini, Pier Luigi
