Pyridazines. Part XXIX: Synthesis and platelet aggregation inhibition activity of 5-substituted-6-phenyl-3(2H)-pyridazinones. Novel aspects of their biological actions

Article abstract of DOI:10.1016/S0968-0896(02)00146-3

A series of 6-phenyl-3(2H)-pyridazinones with a diverse range of substituents in the 5-position have been prepared and evaluated in the search for new antiplatelet agents. A significant dependence of the substituent on the inhibitory effect has been observed. The pharmacological study of these compounds confirms that modification of the chemical group at position 5 of the 6-phenyl-3(2H)-pyridazinone system influences both variations in the antiplatelet activity and the mechanism of action.

Full text of DOI:10.1016/S0968-0896(02)00146-3

Bioorganic & Medicinal Chemistry 10 (2002) 2873–2882
Pyridazines. Part XXIX: Synthesis and Platelet Aggregation
Inhibition Activity of 5-Substituted-6-phenyl-3(2H)-pyridazinones.
Novel Aspects of Their Biological Actionsy
Eddy Sotelo,a Nuria Fraiz,b Matilde Yanez,b Vicente Terrades,a Reyes Laguna,b
Ernesto Canob and Enrique Ravinaa,*
a
´
Laboratorio de Quımica Farmaceutica, Departamento de Quımica Organica, Facultad de Farmacia, Universidad de Santiago de
´
´
´
Compostela, 15782 Santiago de Compostela, Spain
b
´
Departamento de Farmacologıa, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
Received 26 February 2002; accepted 7 May 2002
Abstract—A series of 6-phenyl-3(2H)-pyridazinones with a diverse range of substituents in the 5-position have been prepared and
evaluated in the search for new antiplatelet agents. A significant dependence of the substituent on the inhibitory effect has been
observed. The pharmacological study of these compounds confirms that modification of the chemical group at position 5 of the 6-
phenyl-3(2H)-pyridazinone system influences both variations in the antiplatelet activity and the mechanism of action. # 2002
Elsevier Science Ltd. All rights reserved.
Under physiological conditions, the adhesion of plate-
lets to the subendothelium of damaged vessel walls and
subsequent platelet aggregation are critical events in
haemostasis.2,3 Nevertheless, the uncontrolled deposi-
tion of platelets on the thrombogenic surface, such as
ruptured atherosclerotic plaques, followed by formation
of larger aggregates are the key steps in the development
of various acute vasoocclusive diseases including unstable
angina, myocardial infarction, transient ischemic attacks,
stroke or reocclusion following thrombolytic therapy or
angioplasty.4
platelet inhibitors, and thus inhibit some but not all
agonist-induced pathways of platelet activation and
recruitment. Indeed, up to one third of patients respond
in a limited manner to aspirin therapy, as determined by
circulating platelet aggregate assay.5
Several of the reasons discussed above have driven the
search for new compounds that act on a common
feature of platelet aggregation induced by different
agonists, such as the platelet fibrinogen receptor
antagonists that have become a target in the search for
more efficient antiplatelet drugs.6 Despite extensive
research in the area of GPIIb/IIIa inhibitors, only limited
progress has been made in the search for orally active
anti-thrombotic drugs and, recently, several bleeding
complications have been reported during the use of
these compounds in clinical trials.7 Thus, new agents
having a broad mechanism of action are of great inter-
est not only for use as drugs but also because they are
pharmacological tools that provide important informa-
tion about platelet function.
Aspirin, the primary antiplatelet therapy in use today,
has been shown to reduce the risk of arterial thrombosis
in placebo-controlled clinical trials. Despite the proven
efficacy of aspirin, there are reasons to believe that sub-
stantial improvements in antiplatelet therapy can be
made. Among these is the fact that aspirin and the other
currently available orally administered antiplatelet
agents, such as ticlopidine and clopidogrel, are selective
yPart of this work was presented at the 7th International Symposium
on the Chemistry and Pharmacology of Pyridazines, Santiago de
Compostela, Spain, September, 2000. For the previous paper in this
series, see ref 1.
*Corresponding author. Tel.: +34-981-594628; fax: +34-981-594912;
e-mail: qofara@usc.es
In recent years, the 6-phenyl-3(2H)-pyridazinone system
has aroused a great deal of attention due to its struc-
tural relationship to the 5-aryl-2(1H)-pyridones and, in
particular, to milrinone and amrinone, the prototypes of
a series of non-glycoside, non-catecholamine-based
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00146-3

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E. Sotelo et al. / Bioorg. Med. Chem. 10 (2002) 2873–2882
drugs that have mixed ionotropic/vasodilator activity.
3(2H)-Pyridazinones, 2(1H)-pyridones and related com-
pounds also show antiplatelet activity,8 the inhibition of
the cyclic adenosine monophosphate (c-AMP) phos-
phodiesterase (PDE III in cardiac muscle and platelets)
being assumed to be the primary source of these activ-
ities. However, the exact mechanism of their antiplatelet
effect has not yet been completely elucidated and some
differences in biological effects between milrinone and
amrinone (Chart 1) have been reported.9
3(2H)-pyridazinones, which seem to inhibit platelet
activation due to their ability to suppress the passage of
calcium through the cytoplasmic membrane.19
Prompted by these results we describe here the synthesis
and new pharmacological studies of a series of 5-sub-
stituted-6-phenyl-3(2H)-pyridazinones. Our aim was to
establish more detailed structure–activity relationship in
this series and evaluate the modification of the
pharmacological profile induced by the substituents
in the 5-position of the 6-phenyl-3(2H)-pyridazinone
system.
The intensive study of these compounds has allowed the
establishment of a five-point pharmacophore model for
selective PDE III inhibitors.10 The following character-
istics have a special relevance in terms of this model: a
strong dipole (carbonyl group) in the azine system
adjacent to an NH group, a small lipophilic group in the
5-position, a flat topography and a hydrogen bonding
region at the end of the molecule. There are several
characteristic 3(2H)-pyridazinones that conform to this
model and these include imazodan, zardaverine, pimo-
bendan and bemoradan (Chart 1).
Chemistry
The chemistry used in the construction of the 6-phenyl-
3(2H)-pyridazinone core and the subsequent elabora-
tion to the target compounds is represented in Schemes
1–3. Some of the compounds studied here as antiplatelet
agents have been previously described in papers pub-
lished by our group.16,18 5-Oxygenated-3(2H)-pyri-
dazinones were obtained from 5-hydroxymethyl-6-
phenyl-4,5-dihydro-3(2H)-pyridazinone (2)13 (Scheme
1). Selective aromatization of this compound using
copper(II) chloride in acetonitrile gave alcohol 5 in 55%
yield. Treatment of compound 2 with Jones’ reagent led
to carboxylic acid 4 in a troublesome procedure that
involved the aromatization of the heterocycle and oxi-
dation of the hydroxymethyl group. The low stability of
compound 2 under acidic conditions allows the methyl
derivative 3 (90%) to be obtained by refluxing 2 in
hydrochloric acid. Oxidation or acetylation of alcohol 5
gave aldehyde 618 (80%) or acetate 718 (96%), respec-
tively. Bromination of 5 using carbon tetrabromide and
triphenylphosphine in methylene chloride afforded the
synthetically useful bromomethyl derivative 8,16 which
was the starting material in the preparation of the ethers
916 (57%) and 1016 (67%) and the thioether 11 (85%)
(Scheme 1).
As a continuation of our studies on the chemistry and
pharmacology of 6-aryl-5-substituted-3(2H)-pyridazin-
ones,11À16 and as part of an ongoing project aimed at
the synthesis of new and selective PDE III inhibitors to
be used as antiplatelet agents, we recently prepared sev-
eral 6-aryl-3(2H)-pyridazinones having the general
structure shown in Chart 2. The results of these
studies have confirmed the critical effect of the group
in the 5-position and the hydrogen-bonding region at
the end of the phenyl ring on the inhibition of platelet
aggregation.17
The pharmacological evaluation of these compounds
allows us to describe the antiplatelet activity of a range
of 5-substituted-6-phenyl-3(2H)-pyridazinones that act
through a non-cAMPC PDE III-based mechanism, since
the tested compounds did not increase intracellular
levels of cAMP.18,21,22 On the basis of the experimental
results, which show that this family of compounds inhi-
bits platelet aggregation induced by diverse stimuli, it
was suggested that they were disrupting a common
event in the activation pathways leading to aggregation.
As part of our detailed pharmacological study of these
compounds we have recently reported26 several
new findings regarding the mechanism of action of the
6-phenyl- and 6-thienyl-5-hydroxymethyl-4,5-dihydro-
Condensation of aldehyde
6 with hydroxylamine
hydrochloride in pyridine gave oxime 12,16 which was
efficiently converted to nitrile 1316 (88%) by dehydra-
tion employing trifluoroacetic acid (Scheme 2). Ester 15
was prepared by oxidation of aldehyde 6 using pyri-
dinium dichromate in methanol. Subsequent ammono-
lysis of compound 15 afforded amide 1616 in high yield
(90%).
Chart 1.

E. Sotelo et al. / Bioorg. Med. Chem. 10 (2002) 2873–2882
2875
Disappointingly, all our attempts to obtain compound
18 by direct addition of methyllithium to aldehyde 6
were unsuccessful (Scheme 2). We therefore prepared
the methoxymethyl derivative 14 and reacted this with
methyllithium to afford alcohol 17 in moderate yield
(65%). Removal of the protecting group was accom-
plished by treatment of 17 with hydrochloric acid to
give the desired alcohol 18 (50%). Finally, oxidation of
18 with manganese dioxide in tetrahydrofuran afforded
methylketone 19 in high yield (89%). 6-Phenyl-5-halo-
3(2H)-pyridazinones 20 and 21 were obtained by hydra-
zinolysis of the corresponding 2,3-dihalo-4-phenylcroto-
nolactones.13
5-Thio and 5-nitrogenated derivatives were prepared
starting from 5-bromo-6-phenyl-3(2H)-pyridazinone
(21) (Scheme 3). In contrast to our previous studies
involving nitrogenated nucleophiles, compound 21
showed a high reactivity in the nucleophilic substitution
reactions with mercaptans, to give the corresponding
sulfur compounds in excellent yields. Thus, reaction of
21 with methylisothiourea in a mixture of methanol/
ammonia afforded the methylthio derivative 25 in
almost quantitative yield (96%), even in the presence of
other nucleophiles such as the amidine or ammonia.
Oxidation of the phenyl- or methylthio- derivatives 22
and 25, using 3-chloroperbenzoic acid in methylene
chloride, gave sulfones 26 and 33 in high yield. The
ethers 27 and 28 and the phenylamine 30 were prepared
in excellent yields by nucleophilic substitution of the
methylsulfonyl group in 26. Reaction of bromo deriva-
tive 21 with hydrazine hydrate or sodium azide gave
compounds 23 and 30, respectively. Direct amination of
21 using ammonium chloride/ammonia in a Parr reactor
gave only moderate yields of the amine 31,16 and we
therefore obtained this compound in an efficient manner
by catalytic hydrogenation of azide 30 (Scheme 3).
Chart 2.
Scheme 1. Reagents: (a) CuCl2/acetonitrile; (b) 10% HCl; (c) CrO3/
+; (d) CuCl2/acetonitrile; (e) MnO2/THF; (f) (CH3CO)2/pyridine;
.
Scheme 2. Reagents: (a) NH2OH HCl/pyridine; (b) CF3COOH; (c)
H
(g) CBr4/PPh3/CH2Cl2; (h) EtONa/EtOH; (i) PhONa/EtOH; (j)
PhSH/EtOH.
ClCH2OMe/DMAP/CH2Cl2; (d) PDC/MeOH; (e) NH3; (f): CH3Li/
THF À78 ꢀC; (g) HCl 10%; (h) MnO2/THF; (i) NH2NH2/EtOH.

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E. Sotelo et al. / Bioorg. Med. Chem. 10 (2002) 2873–2882
The chemical structures of all compounds were con-
firmed by spectroscopic (IR, NMR, MS) and analytical
data (see Experimental).
to the ether (9 and 10) or ester (7) produces a slight or
marked increase in the antiplatelet activity, respectively.
A sharp increase in potency (IC50=15 mM) was observed
for compound 22 (X=SPh) but, in contrast, the homo-
logue 11 (X=CH2SPh) is completely inactive. If we
consider the oxygenated analogues 10 (X=CH2OPh)
and 28 (X=OPh), it can be seen that the most potent
compound is 10 while compound 28 induces platelet
aggregation.
Results and Discussion
The platelet aggregation inhibitory activity of the
3(2H)-pyridazinone derivatives, obtained as described
above, was examined on washed human platelets using
thrombin as inducer of platelet aggregation. The results
of these experiments are summarized in Table 1.
After considering the results indicated in Table 1, which
illustrates the important effect that the chemical group
in the 5-position of the 6-phenyl-3(2H)-pyridazinone
system has on the antiplatelet activity, and in order to
collect biological information important for ours SAR
studies, several 3(2H)-pyridazinones were selected for
further pharmacological evaluation and a detailed study
of their mechanism of action. Initially, the anti-PDE III
activity of several representative compounds (6, 7, 10,
13 and 22) was determined in purified preparations
obtained from vague of guinea pigs according to a pre-
viously reported protocol.20 All compounds studied
were found not to inhibit PDE III; thus, in agreement
with our previous results,19 these experiments confirm
that the inhibition of the platelet function shown by
3(2H)-pyridazinones is not a c-AMP-based activity.
Many of the compounds studied inhibit platelet aggre-
gation in a dose-dependent manner. Compound 22
shows the highest efficacy as a platelet aggregation
inhibitor and has an IC50 value in the micromolar range
(15 mM). Some of the pyridazinones 1–33 showed an
interesting antiplatelet activity, although lower than the
reference compound milrinone.
Several modifications of the oxygenated function in
compound 5 reveal that, for example, oxidation to
aldehyde 6 produces an increase in potency—in con-
trast, the acid 4 is inactive—whereas carboxylic acid
derivatives 13 (X=CN), 14 (X=COOMe) and 16
(X=CONH2) are only weakly actives. Conversion of 5
Scheme 3. Reagents: (a) PhSH/K2CO3; (b) NH2NH2; (c) CH3COCH3; (d) (NH2–C¼NH)SCH3/NH3; (e) NaN3,/DMF; (f) H2/Pd/AcOEt;
(g) (CH3CO)2O/pyridine; (h) m-CPBA/CH2Cl2; (i) MeOH; (j) PhONa/THF; (k) PhNH2/THF.

E. Sotelo et al. / Bioorg. Med. Chem. 10 (2002) 2873–2882
2877
The subsequent pharmacological studies were con-
ducted with the most active derivative in the series, that
is compound 22. This substance was found to inhibit
platelet aggregation induced by different stimuli,
including collagen and the calcium ionophore ionomy-
cin (results not shown).
solic calcium levels, caused both by thrombin and
ionomycin in human platelets.19 It is therefore feasible
that similar inhibiting properties could be associated
with compound 22. Related to second effect it should
be considered that in cellular functions the phosphoryl-
ation of proteins has a different outcome depending on
whether the phosphate group is incorporated in serine/
threonine or tyrosine residues. For this reason, we next
investigated the effects on protein phosphorylation.
Stimuli that induce platelet aggregation differ funda-
mentally in the mechanism by which the process
starts—generally by either the activation of a specific
receptor or the massive entry of external calcium into
the platelet (as occurs with ionomycin). Both possibi-
lities have in common: an increase in the level of cyto-
solic calcium (with the associated initiation of processes
that depend on the presence of this ion), the binding of
fibrinogen to its receptor and the phosphorylation of
certain proteins. These points lead us to believe that the
mode of action of compound 22 centers on one of these
key steps in platelet aggregation, a possibility that
would certainly explain the effectiveness of this com-
pound against the variety of stimuli employed.
The results obtained show that compound 22 plays a
role in both processes outlined above and, as such, has
the ability to inhibit increases in cytosolic calcium levels
caused by thrombin as well as promoting an increase in
the phosphotyrosine level of certain platelet proteins
(changes in the level of phosphorylation of serine/
threonine residues were not observed). Both effects were
found to be dose-dependent (Figs 1 and 2).
Bearing in mind the fundamental role played by both
phosphorylation processes and by the increase in cyto-
solic calcium levels in the development of the aggrega-
tion response, it is feasible that the action of compound
22 on one of these processes alone could explain the
anti-aggregatory effect observed. In addition, if we con-
sider the relationship between all the processes taking
place in platelet activation, the effect of compound 22
on the phosphorylation may be the reason why cytosolic
calcium levels do not increase or, conversely, the less
marked increase in the calcium levels induced by the
stimulus, due to inhibition by compound 22, could
explain the increase in protein phosphorylation. How-
ever, for the following reasons this situation does not
seem to be probable:
The absence in compound 22 of the structural elements
required to block the fibrinogen receptor21À25 allows us
to doubt any action in this regard. However, both an
influence on the cytosolic calcium levels and on the
phosphorylation state of the platelet proteins could be
responsible for the anti-aggregatory effect observed for
this compound and, for this reason, both possibilities
were investigated.
As far as the first effect is concerned, we have recently
reported substances that are structurally related to
compound 22 and are able to inhibit increases in cyto-
1. The effect of compound 22 on the phosphoryl-
ation is more marked than its effect on the
increase in cytosolic calcium, as evidenced by the
fact that the increase in phosphorylation of pro-
teins can be observed at concentrations where
there is no effect on cytosolic calcium levels.
Thus, doses lower than 5 mM caused a marked
change on the tyrosine phosphorylation profile
of several proteins whereas no changes on cyto-
solic calcium levels could be appreciated.
2. The anti-aggregatory effect shown by compound
22 appears to be more consistent with an inhibi-
tion of the increase in calcium levels than with
the phosphorylation of proteins. This view is
supported by the fact that concentrations at
which the phosphorylation levels are modified,
but calcium levels are not affected, do not lead to
any noticeable anti-aggregatory effects. Indeed,
doses lower than 5 mM did not induce platelet
aggregation inhibition.
Table 1. Antiplatelet activity of the 5-substituted-6-phenyl-3(2H)-
pyridazinones 1–33
Compound
X
IC50 Compound
(mM)
X
IC50
(mM)
1
3
4
5
6
7
8
9
10
11
12
13
15
16
18
H
CH3
COOH
CH2OH
CHO
0.57
1.20
—
1.50
0.31
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
COCH3
Cl
Br
2.29
—
—
0.015
—
—
—
1.20
SPh
NHNH2
NHN¼C(CH3)2
SCH3
CH2OCOCH3 0.19
CH2Br
CH2OEt
CH2OPh
CH2SPh
CH¼NOH
CN
COOMe
CONH2
CH(OH)CH3
—
0.67
0.50
—
*
SO2CH3
OMe
Oph
NHPh
N3
NH2
—
*
3. Other compounds from this and other related
series, such as 2 and 5, which have shown anti-
aggregatory activity are also capable of inhibiting
the rise in cytosolic calcium levels caused by
various stimuli in platelets without modifying
the phosphorylation profile (ref 19 and results
not shown).
—
0.90
—
—
0.70
1.00
2.00
1.00
—
NHCOCH3
SO2Ph
— Inactive; * induces platelet aggregation.
Milrinone IC50=0.0047 mM.

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E. Sotelo et al. / Bioorg. Med. Chem. 10 (2002) 2873–2882
Figure 1. Effect of tested compound 22 on 0.25 U/mL thrombin-induced intracellular calcium increase in washed human platelets: (a) vehicle
(DMSO); (b) 10 mM compound 22; (c) 15 mM compound 22; (d) 20 mM compound 22.
phatase activity. In an attempt to understand the
mechanism by which compound 22 acts, and in the
absence of specific tests to elucidate the possible modes
of action of this compound on the aforementioned
enzymatic activities, we carried out a pharmacological
study in which we evaluated its anti-aggregatory effect
in the presence of an inhibitor of platelet tyrosine-kinase
activity (tyrphostin B56, a representative member of the
tyrphostin family). We found that neither the anti-
aggregatory effect nor the increase in phosphorylation
diminished in the presence of the inhibitor. For this
reason, it is probable that compound 22 acts as a tyr-
osine-phosphatase inhibitor. This activity would par-
tially explain the anti-aggregatory effect, because other
compounds that are capable of promoting tyrosine-
phosphorylation (by inhibiting the tyrosine-phosphatase
activity) have also shown interesting properties as pla-
telet aggregation inhibitors.27,28
Figure 2. Anti-phosphotyrosine (B1) and anti-phosphoserine/threo-
In light of these results and other obtained with struc-
turally-related compounds,19 we can clearly conclude
that the substituent in the 5-position of the 6-phenyl-
3(2H)-pyridazinone system not only determines the
antiplatelet activity of this series (quantitative aspect)
but also the mechanism of action (qualitative aspect).
This fact must be taken into account when SAR studies
are undertaken with this type of compounds in order to
avoid erroneous conclusions.
nine (B2) immunoblottings obtained from platelet lysates after SDS-
PAGE and western blot, as described in Materials and Methods:
(a) vehicle (DMSO); (b) 10 mM compound 22; (c) 15 mM compound 22;
(d) 20 mM compound 22.
For these reasons, the effects shown by compound 22 on
protein tyrosine phosphorylation and on the increase in
cytosolic calcium levels can be considered due to different
cellular actions. In addition, the overall result is a synergy
of the two effects on the basis that the anti-aggregatory
activity of compound 22 is far greater than that of other
analogues that affect only the calcium levels (e.g., 5).
Experimental
Pharmacology
The phosphorylation state of any protein is the result of
the balance between phosphorylation/dephosphorylation
processes. Bearing this point in mind, the increase in the
level of tyrosine phosphorylation, caused by compound
22, can only be explained by an increase in the tyrosine-
kinase activity or by an inhibition in the tyrosine phos-
Preparation of washed platelets. Human platelet con-
centrates from blood anti-coagulated with citrate–phos-
phate–dextrose were obtained from the Centro de
Transfusion de Galicia (Santiago de Compostela,

E. Sotelo et al. / Bioorg. Med. Chem. 10 (2002) 2873–2882
2879
Spain). Platelets were purified by sedimentation through
a discontinuous metrizamide gradient. For this purpose,
8 mL of platelet concentrate was layered onto a 10%/
20% (1 mL/1 mL) metrizamide gradient and centrifuged
at 1000g for 20 min. The resulting platelet band was
recovered, diluted to 8 mL with washing buffer (NaCl,
140 mM; KCl, 5 mM; trisodium citrate, 12 mM; glucose,
10 mM; sucrose, 12.5 mM; pH 6) and centrifuged again
at 1000 g for 20 min. Finally, the platelet band recovered
from this step was resuspended in a modified Tyrode-
HEPES buffer (HEPES, 10 mM; NaCl, 140 mM; KCl,
3 mM; MgCl, 0.5 mM; NaHCO3, 5 mM; glucose,
10 mM; pH 7.4) affording a concentration of 3–3.5
10À8 platelets/mL. The calcium concentration in the
extracellular medium was 2 mM.
(IR) were recorded on a Perkin-Elmer 1640 FTIR spec-
trophotometer. 1H NMR spectra were obtained on
Bruker WM250 and AM300 Hz spectrometers using
tetramethylsilane as the internal standard (chemical
shifts are in d values, J in Hz). Mass spectra were
determined on a Varian MAT-711 instrument. Ele-
mental analyses were performed on a Perkin-Elmer
240B apparatus at the Microanalysis Service of the
University of Santiago de Compostela. The progress of
the reactions was monitored by thin layer chromato-
graphy with 2.5 mm Merck silica gel GF 254 strips, and
the purified compounds each showed a single spot;
unless otherwise stated iodine vapor and/or UV light
were used for detection. Chromatographic separations
were performed on silica gel columns by flash chroma-
tography (Kieselgel 40, 0.040–0.063 mm).
Platelet aggregation studies. Platelet aggregation was
measured using a dual channel aggregometer (Chrono-
log, Havertown, PA, USA). Each test compound was
incubated with washed platelets at 37 ꢀC for 5 min.
Stimulus was then added to induce platelet aggregation,
and the light transmission was monitored over a 5-min
period. Platelet aggregation is expressed as the maxi-
mum change in light transmission during this period,
with a 100% value being obtained when only stimulus,
and not compound, was added.
6-Phenyl-3(2H)-pyridazinone (1). To a solution of the 4,5-
dihydro-6-phenyl-3(2H)-pyridazinone (3.0 g, 16.3 mmol)
in acetonitrile (25 mL) copper(II) chloride (4.38g,
32 mmol) was added. The reaction mixture was refluxed
during 50 min and then added to ice. The resulting solid
was filtered off and recrystallized from isopropanol to give
2.81 g (95%) of 2. Mp 203.0–205.0ꢀC, IR (KBr) cmÀ1
:
3950 (NH), 1647 (CO). 1H NMR (dimethyl sulfoxide-d6): d
13.18 (s, 1H, NH), 8.00 (d, J=9.9Hz, 1H, CH), 7.84 (m,
2H, Ph), 7.46 (m, 3H, Ph), 6.98 (d, J=9.9 Hz, 1H,
CHCO). Anal. calcd for C10H8N2O: C, 69.76; H, 4.68;
N, 16.27. Found: C, 69.80; H, 4.70; N, 16.34.
Cytosolic free calcium measurements. Platelets recovered
from the first washing step were incubated at 37 ꢀC for
45 min with 3 mM fura-2 acetoxymethyl ester and then
pelleted and resuspended as indicated above. Fura-2
fluorescence was monitored using a Shimadzu RF-5001
PC spectrofluorimeter. Excitation wavelengths were 340
and 380 nm and the emission wavelength was 485 nm.
Cytosolic calcium concentration was calculated from
the ratio of these two fluorescence intensities according
to the method of Grynkiewicz et al.29 with a dissocia-
5-Hydroxymethyl-6-phenyl-3(2H)-pyridazinone (5). To a
solution of 4,5-dihydro-6-phenyl-3(2H)-pyridazinone
(3.5 g, 17.1 mmol) in acetonitrile (25 mL) copper(II)
chloride (4.61 g, 35 mmol) was added. The reaction
mixture was refluxed during 35 min and then added to
ice. The resulting residue was filtered off, purified by
column chromatography (ethyl acetate/hexane 1:1) and
then recrystallized from ethanol to provide alcohol 5 as
colorless needles (1.9 g, 55%). Mp 198–201 ꢀC, IR (KBr)
cmÀ1: 3200–2900 (NH), 1650 (CO pyridazinone), 1590
(C¼C aromatics). 1H NMR (dimethyl sulfoxide-d6):
13.07 (s, 1H, NH, deuterium oxide exchangeable), 7.46–
7.44 (m, 5H, aromatics), 6.93 (s, 1H, CHCO), 5.62 (s,
1H, OH), 4.26 (s, 2H, CH2OH). Anal. calcd for
C11H10N2O2: C, 65.34; H, 4.98; N, 13.85. Found: C,
65.38; H, 4.89; N, 13.85.
tion constant (Kd) of 224 nM for Ca2+
.
Protein phosphorylation. After stimulation, human
platelets were lysated with SDS/EDTA (2%/1 mM) in
order to solubilize proteins and treated according to the
Laemmli method.30 The solubilized proteins were sepa-
rated on SDS-polyacrylamide gel by electrophoresis
(SDS-PAGE) using a 10% gel; known molecular weight
proteins were used as standards. After SDS-PAGE,
proteins were transferred to a PVDF membrane, which
was then incubated for 2 h with appropriate monoclonal
antibodies. In order to test selective phosphorylation
sites (tyrosine or serine/threonine), each protein sample
was run in two separate electrophoresis gels, which were
transfered to PVDF membranes. One sample was incu-
bated with 4G10 anti-phosphotyrosine antibody and the
other with a mixture of anti-phosphoserine (1C8, 4A3,
4A9, 16B4) and anti-phosphothreonine (1E11, 4D11,
14B3) antibodies. Phosphoproteins were visualized by
enhanced chemiluminiscence (ECL) using a goat anti-
mouse antibody coupled to peroxidase.
6-Phenyl-5-phenylthiomethyl-3(2H)-pyridazinone (11). To
a
solution of bromomethyl derivative 9 (0.15 g,
0.56 mmol) in ethanol (20 mL) mercaptobenzene
(0.15 mL, 1.5 mmol) was added and the reaction mixture
was refluxed during 1 h. The reaction mixture was con-
centrated under reduced pressure and the residue was
purified by column chromatography (ethyl acetate/hex-
ane 3:2) and then recrystallized from ethanol to provide
11 as colorless needles (0.14 g, 85%). Mp 171.9–
173.1 ꢀC, IR (KBr) cmÀ1: 3050–2805 (NH), 1681 (CO),
1
1694 (aromatics). H NMR (dimethyl sulfoxide-d6): d
13.13 (s, 1H, NH, deuterium oxide exchangeable), 7.51–
7.44 (m, 5H, Ph), 7.39–7.18 (m, 5H, aromatics), 6.71 (s,
1H, CH–CO), 3.99 (s, 2H, CH2–S). Anal. calcd for
C17H14N2OS: C, 69.36; H, 4.79; N, 9.52; S, 10.89.
Found: C, 69.39; H, 4.81; N, 9.52; S, 10.90.
Chemistry
Melting points were measured on a Gallenkamp melting
point apparatus and are uncorrected. Infrared spectra

2880
E. Sotelo et al. / Bioorg. Med. Chem. 10 (2002) 2873–2882
5-Formyl-2-methoxymethyl-6-phenyl-3(2H)-pyridazinone
(14). To a mixture of aldehyde 6 (1.5 g 7.5 mmol), di-
isopropylethylamine (1.82 mL, 11.5 mmol) and a cataly-
tic amount of 4-dimethylaminopyridine in anhydrous
methylene chloride (25 mL) at 0 ꢀC under an argon
atmosphere methoxymethyl chloride (1.8 mL) was
added slowly. The reaction mixture was stirred during
3 h and the solvent was concentrated under reduced
pressure. The residue was poured into water and
extracted with ethyl acetate. The organic layer was dried
(Na2SO4) and the solvent removed under reduced pres-
sure to give a solid, which was purified by column
chromatography (ethyl acetate/hexane 1:1) to afford
aldehyde 14 (1.65 g, 85%). Mp 79.3–81.0 ꢀC, IR (KBr)
cmÀ1: 1710 (CHO), 1680 (CO), 1590 (aromatics). 1H
NMR (dimethyl sulfoxide-d6): d 9.82 (s, 1H, CHO),
7.55–7.47 (m, 5H, aromatics), 7.38 (s, 1H, H4), 5.39 (s,
2H, –CH2), 3.38 (s, 3H, CH3). Anal. calcd for
C13H12N2O3: C, 63.93; H, 4.95; N, 11.47. Found: C,
64.00; H, 4.96; N, 11.50.
tonitrile to afford 18 (0.20 g, 50%) as a white solid. Mp
186.0–188.0 ꢀC, IR (KBr) cmÀ1: 3255 (OH), 1680 (CO),
1590 (aromatics). H NMR (dimethyl sulfoxide-d6): d
13.00 (s, 1H, NH, deuterium oxide exchangeable), 7.45
(m, 5H, aromatics), 6.95 (s, 1H, H4), 5.43 (m, 1H, OH),
4.58 (m, 1H, CH), 1.01 (m, 3H, CH3). Anal. calcd for
C12H12N2O2: C, 66.65; H, 5.59; N, 12.96. Found: C,
66.69; H, 5.62; N, 12.98.
1
5-Acetyl-6-phenyl-3(2H)-pyridazinone (19). A mixture of
alcohol 18 (0.15 g, 0.69 mmol) and manganese dioxide
(0.60 g, 69 mmol) in tetrahydrofuran (30 mL) was stirred
at room temperature during 48 h. The mixture was fil-
tered through Celite and the solvent removed in vacuo
to give ketone 29, which was recrystallized from iso-
propanol (0.13 g, 89%). Mp 188.2–189.0 ꢀC, IR (KBr):
1
3100–2900 (NH), 1702 (COCH3), 1674 (CO). H NMR
(deuterochloroform): d 12.64 (bs, 1H, NH, deuterium
oxide exchangeable), 7. 44 (m, 5H, aromatics), 7.43 (s,
1H, H4), 2.14 (s, 3H, CH3). Anal. calcd for
C12H10N2O2: C, 67.28; H, 4.71; N, 13.08. Found: C,
67.28; H, 4.70; N, 13.14.
5-Methoxycarbonyl-6-phenyl-3(2H)-pyridazinone (15).
To a solution of aldehyde 6 (0.3 g, 1.5 mmol) in metha-
nol (10 mL) pyridinium dichromate (0.58 g, 2 mmol) was
added slowly and the mixture was stirred at room tem-
perature during 6 h. The solution was poured into ice/
water and the resulting solid was filtered off and recrys-
tallized from methanol to give ester 10 (0.25 g, 70%).
Mp 175.5–177.3 ꢀC, IR (KBr) cmÀ1: 3180–3050 (NH),
5-Chloro-6-phenyl-3(2H)-pyridazinone (20). To a solu-
tion of 2,3-dichloro-4-phenylcrotonolactone (1.5 g,
6.6 mmol) in ethanol (10 mL) hydrazine hydrate
(1.7 mL, 32 mmol) was added slowly and the mixture
was heated under reflux during 3 h. The reaction mix-
ture was allowed to cool and the solid that precipitated
was filtered off and recrystallized from isopropanol
(0.85 g, 63%). Mp 229.8–230.5 ꢀC, IR (KBr): 3000–2900
1
1650 (CO), 1730 (COOCH3). H NMR (dimethyl sulf-
oxide-d6): d 12.40 (s, 1H, NH), 7.89 (s, 1H, CH–CO),
7.50–7.39 (m, 5H, aromatics), 3.73 (s, 3H, OCH3). Anal.
calcd for C12H10N2O3: C, 62.61; H, 4.38; N, 12.17.
Found: C, 62.65; H, 4.38; N, 12.21.
1
(NH), 1680 (CO). H NMR (dimethyl sulfoxide-d6): d
13.85 (bs, 1H, NH, deuterium oxide exchangeable),
7.58–7.50 (m, 5H, aromatics), 7.44 (s, 1H, H4). Anal.
calcd for C10H7ClN2O: C, 58.13; H, 3.41; N, 13.56.
Found: C, 58.18; H, 3.42; N, 13.58.
0
5-(1 -Hydroxyethyl)-2-methoxymethyl-6-phenyl-3(2H)-
pyridazinone (17). To a solution of aldehyde 14 (0.5 g,
2.0 mmol) in dry tetrahydrofuran (25 mL) at À78 ꢀC
under an argon atmosphere was added slowly 1 M
solution of methyllithium (2 mL). The reaction mixture
was stirred for 3 h and then left at room temperature for
24 h. The reaction mixture was treated with water
(25 mL) and the solution extracted with methylene
chloride. The organic layer was dried (Na2SO4) and
concentrated to give a colorless oil, which was purified
by column chromatography (ethyl acetate/hexane 1:2)
to afford a white solid (0.17 g, 40%) identified as 17. Mp
117.3–119.0 ꢀC, IR (KBr) cmÀ1: 3235 (OH), 1680 (CO),
5-Bromo-6-phenyl-3(2H)-pyridazinone (21). To a solu-
tion of 2,3-dibromo-4-phenylcrotonolactone (1.5 g,
4.7 mmol) in ethanol (10 mL) hydrazine hydrate
(1.2 mL, 23 mmol) was added slowly and the mixture
was heated under reflux during 2 h. The reaction mix-
ture was allowed to cool and the solid that precipitated
was filtered off and recrystallized from isopropanol
(0.71 g, 60%). Mp 231.0–232.0 ꢀC, IR (KBr): 3000–2900
1
(NH), 1680 (CO). H NMR (dimethyl sulfoxide-d6): d
13.78 (bs, 1H, NH, deuterium oxide exchangeable), 7.51
(m, 5H, aromatics), 7.45 (s, 1H, H4). Anal. calcd for
C10H7BrN2O: C, 47.84; H, 2.81; N, 11.16. Found: C,
47.88; H, 2.83; N, 11.21.
1
1580 (aromatics), 1150 (C–O–C). H NMR (dimethyl
sulfoxide-d6): d 7.53 (s, 5H, aromatics), 7.10 (s, 1H, H4),
5.54 (m, 1H, CH), 5.38 (s, 2H, N–CH2–O), 4.66 (s, 1H,
OH), 3.41 (s, 3H, OCH3), 1.03 (m, 3H, CH3). Anal.
calcd for C14H16N2O3: C, 64.20; H, 6.20; N, 10.76.
Found: C, 64.24; H, 6.24; N, 10.78.
6-Phenyl-5-phenylthio-3(2H)-pyridazinone (22). To
a
solution of bromo derivative 21 (0.5 g, 2.0 mmol) in
ethanol (25 mL) potassium carbonate (0.15 g, 1.0 mmol)
and mercaptobenzene (0.5 mL, 5.0 mmol) was added.
The reaction mixture was heated under reflux during 6 h
and the solvent was removed in vacuo. The resulting
residue was added to ice to give a solid, which was fil-
tered off and recrystallized from methanol (0.66 g,
88%). Mp 162.4–164.1 ꢀC, IR (KBr): 3007 (NH), 1658
(CO). 1H NMR (dimethyl sulfoxide-d6): d 13.08 (bs, 1H,
NH, deuterium oxide exchangeable), 7.59–7.50 (m, 5H,
aromatics), 7.39–7.25 (m, 5H, aromatics), 5.76 (s, 1H,
5-(10-Hydroxyethyl)-6-phenyl-3(2H)-pyridazinone (18).
To a solution of alcohol 17 (0.35 g, 1.3 mmol) in
methanol (20 mL) 6 M hydrochloric acid (20 mL) was
added and the mixture was stirred at 50 ꢀC during 15 h.
The reaction mixture was poured into ice and then
extracted with ethyl acetate. The organic extracts were
dried (Na2SO4) and concentrated under reduced pres-
sure to give a solid, which was recrystallized from ace-

E. Sotelo et al. / Bioorg. Med. Chem. 10 (2002) 2873–2882
2881
H4). Anal. calcd for C16H12N2OS: C, 68.55; H, 4.31; N,
9.99; S, 11.44. Found: C, 68.60; H, 4.32; N, 10.04; S, 11.47.
5-Methoxy-6-phenyl-3(2H)-pyridazinone (27). A solu-
tion of sulfone 26 (1.05 g, 42 mmol) in methanol (25 mL)
containing a catalytic amount of potassium carbonate
was heated under reflux during 48 h. The solvent was
removed under reduced pressure, the residue was puri-
fied by column chromatography (ethyl acetate/hexane
2:1) and then recrystallized from ethanol to give 27
(0.17 g, 40%). Mp 129.2–130.1 ꢀC, IR (KBr) cmÀ1: 2862
(NH), 1693 (CO), 1598 (aromatics). 1H NMR (dimethyl
sulfoxide-d6): d 12.82 (s, 1H, NH, deuterium oxide
exchangeable), 7.59 (m, 2H, Ph), 7.14 (m, 3H, Ph), 6.31
(s, 1H, CHCO), 3.80 (s, 3H, OCH3). Anal. calcd for
C11H10N2O2: C, 65.34; H, 4.98; N, 13.85. Found: C,
65.34; H, 5.01; N, 13.87.
5-Hydrazino-6-phenyl-3(2H)-pyridazinone (23). A mix-
ture of bromo derivative 21 (0.5 g, 2 mmol) and hydra-
zine hydrate (1 mL, 20 mmol) was heated under reflux
during 5 h. The reaction mixture was allowed to cool
and the solid that precipitated was filtered off and
recrystallized from ethanol (0.71 g, 60%). Mp 194.0–
195.2 ꢀC, IR (KBr): 3305–2935 (NH, NHNH2), 1624
(CO). 1H NMR (dimethyl sulfoxide-d6): d 12.10 (bs, 1H,
NH, deuterium oxide exchangeable), 7.44 (m, 5H, aro-
matics), 6.97 (s, 1H, H4). Anal. calcd C10H10N4O: C,
59.40; H, 4.98; N, 27.71. Found: C, 58.44; H, 5.02; N,
27.76.
5-Phenoxy-6-phenyl-3(2H)-pyridazinone (28). To a solu-
tion of sodium phenoxide (0.14 g, 1.2 mmol) in tetra-
hydrofuran sulfone, 26 (0.30 g, 1.2 mmol) was added
and the mixture was heated under reflux during 12 h.
The solvent was removed under reduced pressure and
the residue was poured into ice. The resulting solid was
purified by recrystallization from ethanol to give 28
5-Isopropylidenehydrazino-6-phenyl-3(2H)-pyridazinone
(24). A solution of hydrazine 23 (0.25 g, 1.2 mmol) in
acetone (10 mL) was heated under reflux during 2 h. The
solvent was removed under reduced pressure to give a
solid, which was recrystallized from methanol to afford
a white solid (0.30 g, 95%) identified as 24. Mp 247.7–
248.2 ꢀC, IR (KBr) cmÀ1: 3025 (NH), 1680 (CO), 1590
(0.26 g, 83%). Mp 202.8–204.1 ꢀC, IR (KBr) cmÀ1
:
1
(aromatics). H NMR (dimethyl sulfoxide-d6): d 12.97
3500–3000 (NH), 1654 (CO), 1596 (aromatics). 1H
NMR (dimethyl sulfoxide-d6): d 13.05 (s, 1H, NH, deu-
terium oxide exchangeable), 7.73 (m, 2H, Ph), 7.54 (m,
6H, Ph), 7.31 (m, 2H, Ph), 6.92 (s, 1H, CHCO). Anal.
calcd for C16H12N2O2: C, 72.72; H, 4.58; N, 10.60.
Found: C, 72.88; H, 4.79; N, 10.56.
(s, 1H, NH, deuterium oxide exchangeable), 8.70 (s, 1H,
NH), 7.80 (m, 2H, aromatics), 7.43 (m, 3H, aromatics),
7.09 (s, 1H, H4), 2.04 (s, 3H, CH3), 1.98 (s, 3H, CH3).
Anal. calcd for C13H14N4O: C, 64.45; H, 5.82; N, 23.13.
Found: C, 64.50; H, 5.82; N, 23.18.
5-Methylthio-6-phenyl-3(2H)-pyridazinone (25). To a
suspension of bromo derivative 21 (0.50 g, 2.0 mmol)
and s-methylisothiourea (0.60 g, 4.0 mmol) in ethanol
(20 mL) aqueous ammonia (37%, 30 mL) was added.
The reaction mixture was heated under reflux during
1 h. The solvent was removed under reduced pressure
and the residue was poured into ice/water to give a
solid, which was recrystallized from ethanol to afford 25
(0.41 g, 96%). Mp 244.3–246.0 ꢀC, IR (KBr) cmÀ1: 3440
(NH), 1666 (CO), 1580 (aromatics). 1H NMR (dimethyl
sulfoxide-d6): d 12.93 (s, 1H, NH, deuterium oxide
exchangeable), 7.48 (m, 5H, Ph), 6.59 (s, 1H, CHCO),
2.39 (s, 3H, SCH3). Anal. calcd for C11H10N2OS: C,
60.53; H, 4.62; N, 12.83, S, 14.69. Found: C, 60.58; H,
4.61; N, 12.89; S, 14.74.
5-Phenylamino-6-phenyl-3(2H)-pyridazinone (29).
A
mixture of sulfone 27 (0.20 g, 0.8 mmol), 4-dimethyl-
aminopyridine (50 mg) and aniline (0.14 g, 1.6 mmol) in
tetrahydrofuran was heated under reflux during 8 h. The
solvent was removed under reduced pressure and the
residue was poured into ice. The resulting solid was
purified by recrystallization from ethanol to give 29
(0.16 g, 78%). Mp 217.6–219.0 ꢀC, IR (KBr) cmÀ1
:
3500–3200 (NH), 1669 (CO), 1578 (aromatics). 1H
NMR (dimethyl sulfoxide-d6): d 12.90 (s, 1H, NH, deu-
terium oxide exchangeable), 7.35 (m, 2H, Ph), 7.20 (m,
3H, Ph), 7.05 (m, 5H, Ph), 6.71 (s, 1H, CHCO), 3.63 (s,
1H, NH, deuterium oxide exchangeable). Anal. calcd
for C16H13N3O: C, 72.99; H, 4.98; N, 15.96. Found: C,
73.11; H, 5.07; N, 115.99.
5-Methylsulfonyl-6-phenyl-3(2H)-pyridazinone (26). To a
cooled (0 ꢀC) solution of methylthio derivative 25
(0.25 g, 4.5 mmol) in methylene chloride (45 mL) m-
chloroperbenzoic acid 77% (1.02 g, 8 mmol) was added
slowly. The reaction mixture was stirred under these
conditions for 1 h and then at room temperature for
12 h. The solvent was removed under reduced pressure
and the residue was purified by column chromato-
graphy (ethyl acetate/hexane 3:1) and then recrystallized
from isopropanol to give sulfone 26 (0.26 g, 90%). Mp
247.0–248.8 ꢀC, IR (KBr) cmÀ1: 3065–2925 (NH), 1695
5-Azido-6-phenyl-3(2H)-pyridazinone (30). A solution of
21 (1.05 g, 42 mmol) in DMF (15 mL) was treated with
sodium azide (0.54 g, 82 mmol) and the mixture was
stirred at room temperature during 24 h. The mixture
was poured into ice/water and the resulting solid was
filtered off and recrystallized from ethanol to give azide
30 (0.80 g). Mp 163.8–165.0 ꢀC, IR (KBr) cmÀ1: 2844
1
(NH), 2177 (N3), 1692 (CO). H NMR (dimethyl sulf-
oxide-d6): d 13.16 (s, 1H, NH, deuterium oxide
exchangeable), 7.58 (m, 2H, Ph), 7.45 (m, 3H, Ph), 6.80
(s, 1H, CH–CO). Anal. calcd for C10H7N5O: C, 56.34;
H, 3.31; N, 32.85. Found: C, 56.39; H, 3.37; N, 33.01.
1
(CO), 1575 (aromatics). H NMR (dimethyl sulfoxide-
d6): d 13.76 (s, 1H, NH, deuterium oxide exchangeable),
7.88 (s, 1H, CHCO), 7.55–7.49 (m, 5H, Ph), 2.93 (s, 3H,
CH3). Anal. calcd for C11H10N2O3S: C, 52.79; H, 4.03;
N, 11.19; S, 12.81. Found: C, 52.81; H, 4.05; N, 11.24;
S, 12.87.
6-Phenyl-5-phenylsulfonyl-3(2H)-pyridazinone (33). To a
cooled (0 ꢀC) solution of phenylthio derivative 22
(0.35 g, 1.2 mmol) in methylene chloride (50 mL) m-
chloroperbenzoic acid 77% (0.37 g, 2.5 mmol) was

2882
E. Sotelo et al. / Bioorg. Med. Chem. 10 (2002) 2873–2882
added slowly. The reaction mixture was stirred under
these conditions for 1 h and then at room temperature
for 12 h. The solvent was removed under reduced pres-
sure, the residue was purified by column chromato-
graphy (ethyl acetate/hexane 2:1) and then recrystallized
from isopropanol to give sulfone 33 (0.31 g, 81%). Mp
172.0 ꢀC (dec), IR (KBr) cmÀ1: 3100–2850 (NH), 1683
11. Ravina, E.; Garcıa-Mera, G.; Santana, L.; Orallo, F.;
Calleja, J. M. Eur. J. Med. Chem. Chim Ther. 1985, 20, 475.
12. Teran, C.; Ravina, E.; Santana, L.; Garcıa-Domınguez,
N.; Garcıa-Mera, G.; Fontela, J. A.; Orallo, F.; Calleja, J. M.
Arch. Pharm. (Weinheim) 1989, 322, 331.
13. Ravina, E.; Teran, C.; Santana, L.; Garcıa-Domınguez,
N.; Estevez, I. Heterocycles 1990, 20, 1967.
14. Laguna, R.; Montero, A.; Cano, E.; Ravina, E.; Sotelo,
E.; Estevez, I. Acta Pharm. Hungarica 1996, 66, S43; Chem.
Abstr. 1997, 126, 165993.
1
(CO), 1576 (aromatics). H NMR (dimethyl sulfoxide-
d6): d 13.74 (s, 1H, NH, deuterium oxide exchangeable),
7.89 (s, 1H, CHCO), 7.70–7.25 (m, 10H, Ph). Anal.
calcd for C16H12N2O3S: C, 61.53; H, 3.87; N, 8.97; S,
10.27. Found: C, 61.61; H, 3.96; N, 9.01; S, 10.34.
15. Estevez, I.; Ravina, E.; Sotelo, E. J. Heterocyclic Chem.
1998, 35, 1421.
16. Sotelo, E.; Ravina, E.; Estevez, I. J. Heterocyclic Chem.
1999, 36, 985.
17. Estevez, I. Doctoral Thesis, University of Santiago de
Compostela, Spain, 1995.
18. Laguna, R.; Rodriguez-Linares, B.; Cano, E.; Estevez, I.;
Ravina, E.; Sotelo, E. Chem. Pharm. Bull. 1997, 45, 1151, and
references cited therein.
19. Montero-Lastres, A.; Fraiz, N.; Laguna, R.; Cano, E.;
Estevez, I.; Ravina, E. Biol. Pharm. Bull. 1999, 22, 1376.
20. Dal Piaz, V.; Giovannoni, M. P.; Castellana, C.; Palacios,
J. M.; Beleta, J.; Domenech, T.; Segarra, V. Eur. J. Med.
Chem. 1998, 33, 789.
Acknowledgements
Financial support of this work by the Xunta de Galicia
(Project XUGA 8151389) is gratefully acknowledged.
´
We thank also to the Spanish Instituto de Cooperacion
Iberoamericana (ICI) for Eddy Sotelo’s Doctoral
Fellowship.
21. Callahan, J. F.; Bean, J. W.; Burguess, J. L.; Eggleston,
D. S.; Hwang, S. M.; Kopple, K. D.; Koster, P. F.; Nichols,
A.; Peishoff, C. E.; Samanen, J. M.; Vasko, J. A.; Wong, A.;
Huffman, W. F. J. Med. Chem. 1992, 35, 3970.
22. Callahan, J. F.; Newlander, K. A.; Burguess, J. L.; Eggle-
ston, D. S.; Nichols, A.; Wong, A.; Huffman, W. F. Tetra-
hedron 1993, 49, 3479.
References and Notes
1. Sotelo, E.; Fraiz, N., Yanez, M., Laguna, R., Cano, E.,
Brea, J. M., Ravina, E. Bioorg. Med. Chem. Lett. 2002, 12,
1575.
2. Leonard, E. F.; Turitto, V. T.; Vroman, L. (Eds.). In Blood
in Contact with Natural and Artificial Surfaces. Annals of the
New York Academy of Sciences, 1987; Vol. 516, pp 1–688.
3. Coller, B. S. New Engl. J. Med. 1990, 322, 33.
4. Falk, E. Circulation 1985, 71, 699.
23. Ku, T. W.; Ali, F. E.; Barton, L. S.; Bean, J. W.; Bondi-
nell, W. E.; Burguess, J. L.; Callahan, J. F.; Calvo, R. R.;
Chen, L.; Eggleston, D. S.; Gleason, J. G.; Huffman, W. F.;
Hwang, S. M.; Jakas, D. R.; Karash, C. B.; Keenan, R. M.;
Kopple, K. D.; Miller, W. H.; Newlander, K. A.; Nichols, A.;
Parker, M. F.; Peishoff, C. E.; Samanen, J. M.; Uzinskas, I.;
Venslavsky, J. W. J. Am. Chem. Soc. 1993, 115, 8861.
24. Alig, L.; Edenhofer, A.; Hadvary, P.; Hurzeler, M.;
Knopp, D.; Muller, M.; Steiner, B.; Trzeciak, A.; Weller, T. J.
Med. Chem. 1992, 35, 4393.
5. Grotemeyer, K. H.; Scharafinski, H. W.; Husstedt, I. W.
Thromb. Res. 1993, 71, 397.
6. Fujita, T.; Nasahide, N.; Inove, Y.; Shimohigushi, Y.
Bioorg. Med. Chem. Lett. 1999, 9, 1351. Liverton, N. J.;
Armstrong, D. J.; Claremon, D.; Remy, C.; Baldwin, J. J.;
Lynch, R. J.; Zhang, G.; Gould, R. J. Bioorg. Med. Chem.
Lett. 1998, 8, 483.
25. Hartman, G. D.; Egberton, M. S.; Halczenko, W.; Las-
well, W. L.; Duggan, M. E.; Smith, R. L.; Naylor, A. M.;
Manno, P. D.; Lynch, R. J.; Zhang, G.; Chang, T. C. C.;
Gould, R. J. J. Med. Chem. 1992, 35, 4640.
7. Iqbal, O.; Welanga, J. M.; Lewis, B. E.; Bakhos, M. Drugs
Today 2000, 36, 8.
8. Thyes, M.; Lehman, H. D.; Gries, J.; Konig, H.; Kretssch-
mar, R.; Kunze, J.; Lebkucher, R.; Lenke, D. J. Med. Chem.
1983, 26, 800. Robertson, D. W.; Jones, N. D.; Krushinski,
J. H.; Pollock, G. D.; Swartzendruber, J. K. J. Med. Chem.
1987, 30, 623.
26. Dillon, A. M. R.; Heath, M. F. Biochem. Biophys. Res.
Commun. 1995, 212, 595.
27. Bachelot, C.; Cano, E.; Grelac, F.; Saleun, S.; Druker,
B. J.; Levy-Toledano, S.; Fischer, S.; Rendu, F. Biochem. J.
1992, 284, 923.
9. Cody, V.; Wojtzack, A.; Davis, F. B.; Blas, S. J. Med.
Chem. 1995, 38, 1990.
28. Villar, R.; Alvarino, M. T.; Flores, R. Biochim. Biophys.
Acta 1997, 1337, 233.
10. Moos, W. H.; Humbet, C. C.; Sircar, I.; Rithner, C.;
Weishar, R. E.; Bristol, J. A.; McPhail, A. J. Med. Chem.
1987, 30, 1963, and references cited therein.
29. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem.
1985, 260, 3440.
30. Laemmli, U. K. Nature (London) 1970, 227, 680.