New platelet aggregation inhibitors based on pyridazinone moiety

Article abstract of DOI:10.1016/j.ejmech.2015.02.061

New series of pyridazinone derivatives (4, 5 and 6) were synthesized in good yields following a synthetic strategy based on singlet oxygen oxidation of alkyl furans, in which a suitable β(α)-substituted γ ghydroxybutenolide (10 or 11) or a bicyclic lactone (12 or 13) was the key intermediate. The synthesized compounds were tested in vitro as antiplatelet agents and some of them (compounds 4b, 4d and 5b) exhibited potent inhibitory effects on collagen-induced platelet aggregation with IC₅₀ values in the low μM range. Studies performed with the most active compound of these series (4b) demonstrated its lack of activity as inhibitor of platelet aggregation induced by other agonists as thrombin, ionomycin or U- 46619 suggesting a selective action on the biochemical mechanisms triggered by collagen in the platelets.

Full text of DOI:10.1016/j.ejmech.2015.02.061

European Journal of Medicinal Chemistry 94 (2015) 113e122
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New platelet aggregation inhibitors based on pyridazinone moiety
a
,
b
,
1
a
,
b
,
1, Noemí Vila a b, Pedro Besada a b,
,
,
Tamara Costasc
, María Carmen Costas-Lago
a, b, *
Ernesto Cano , Carmen Ter aꢀ n
Departamento de Química Org aꢀ nica, Universidade de Vigo, 36310 Vigo, Spain
Instituto de Investigaci oꢀ n Biom ꢀe dica (IBI), Universidade de Vigo, 36310 Vigo, Spain
Departamento de Farmacoloxía, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 November 2014
Received in revised form
New series of pyridazinone derivatives (4, 5 and 6) were synthesized in good yields following a synthetic
strategy based on singlet oxygen oxidation of alkyl furans, in which a suitable
b(a)-substituted g-
hydroxybutenolide (10 or 11) or a bicyclic lactone (12 or 13) was the key intermediate. The synthesized
compounds were tested in vitro as antiplatelet agents and some of them (compounds 4b, 4d and 5b)
exhibited potent inhibitory effects on collagen-induced platelet aggregation with IC50 values in the low
27 February 2015
Accepted 28 February 2015
Available online 3 March 2015
m
M range. Studies performed with the most active compound of these series (4b) demonstrated its lack
of activity as inhibitor of platelet aggregation induced by other agonists as thrombin, ionomycin or U-
6619 suggesting a selective action on the biochemical mechanisms triggered by collagen in the
platelets.
Keywords:
Pyridazinone
4
3-Alkylfuran
Singlet oxygen
© 2015 Elsevier Masson SAS. All rights reserved.
Butenolide
Bicyclic lactone
Platelet aggregation inhibitors
1. Introduction
zardaverine, levosimendan and motapizone some characteristic
drugs [5a,6] (Fig. 1).
Cardiovascular diseases (CVDs), in particular coronary heart
The presence of the aryl (heteroaryl) group at C6 was considered
essential when the cardiovascular effects are associated with
phosphodiesterase III (PDE III) inhibition [5a,7]. Recently, several
pyridazinone derivatives, in which the aryl or heteroaryl group at
C6 was removed or replaced (compounds 1 and 2, Fig. 2), were
described as potential antihypertensive agents or platelet aggre-
gation inhibitors whose activity is not related with inhibition of
PDE [8,9].
In relation to cardiovascular drug research based on pyr-
idazinone moiety we have recently reported a series of 2,6-
disubstituted analogs with good antiplatelet properties. These
pyridazinone derivatives have an alkyl chain of varying magnitude
(1, 2, or 3 carbon atoms) functionalized with alcohol or ether groups
in C6, and substituted or not with different lipophilic fragments in
N2 (compounds 3, Fig. 2). Most of the previously designed com-
pounds inhibited platelet aggregation induced by collagen in the
disease and stroke, are a leading cause of mortality in developed
countries. According to the World Health Organization (WHO) 17
million people die every year by CVDs, accounting for almost one
third of deaths worldwide per year [1]. In addition, CVDs are not
only responsible for high morbidity and mortality, but negatively
impact the quality of life of a huge amount of people across the
world. Thrombus formation and hypertension are the most com-
mon risk factors for myocardial infarction and stroke. The pivotal
role played by platelet activation in the physiopathology of
thrombotic and ischemic diseases and the current limitations of
antiplatelet therapy have prompted the search for new antiplatelet
agents acting over novel therapeutic targets [2].
The 6-(aryl or heteroaryl)pyridazinone derivatives show a wide
pharmacological profile that includes interesting properties on
cardiovascular system, such as cardiotonic effects [3], antihyper-
tensive activity [4] and platelet aggregation inhibition [5], being
low
mM range (1.80e69.6), being silyl ethers and N,O-dibenzyl
derivatives the most active compounds. Many of them also
showed a moderate vasorelaxant effect [10].
The interesting antiaggregatory properties showed by the 2,6-
disubstituted pyridazinones 3 led us to continue with these
studies in order to establish structureeactivity relationships.
*
Corresponding author. Departamento de Química Org ꢀa nica, Universidade de
Vigo, 36310 Vigo, Spain.
E-mail address: mcteran@uvigo.es (C. Ter ꢀa n).
T.C. and M.C.C.-L. contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2015.02.061
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

114
T. Costas et al. / European Journal of Medicinal Chemistry 94 (2015) 113e122
N
N
OCH3
OCHF2
CN
CN
N
O
O
NH
O
S
HN
N
HN
N
HN
N
Zardaverine
Levosimendan
Motapizone
Fig. 1. Some cardiovascular drugs with the characteristic structure of 6-(aryl or heteroaryl)pyridazinone.
Fig. 2. Several pyridazinone derivatives with cardiovascular activity devoid of C6-(aryl or heteroaryl) group.
Therefore, we have synthesized and evaluated three new series of
pyridazinone derivatives that have been designed considering two
kinds of structural modifications, a positional change of the alkyl
chain, which in the new analogs was placed at C5 or C4 of the
pyridazinone ring (compounds 4 and 5 respectively, Fig. 3), and a
reduction in conformational flexibility through the alkyl chain
incorporation in different size rings resulting in bicyclic analogs 6
for the regioselective transformation of 3-substituted furans into
a-
substituted or -substituted -hydroxybutenolides, via oxidation
b
g
with singlet oxygen, in which a base-promoted regiocontrol was
observed. In all cases, the oxidation was carried out in methanol or
ꢁ
dichloromethane (DCM) at ꢀ78 C in the presence of the suitable
base. Thus, when the reaction is accomplished with some bulky
bases, such as diisopropylethyl amine (DIPEA, Hünig's base), the
substituted -hydroxybutenolides were the major products as a
result of the sterically most favorable rearrangement [13,14]. The
selective synthesis of the corresponding -substituted -hydrox-
b-
(Fig. 3).
g
a
g
2
. Results and discussion
ybutenolides is also possible by switching the base, for instance by
using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or tetrabuty-
lammonium fluoride (TBAF). However, in this last strategy the re-
ported results are extremely substrate-dependent [14,15]. In our
case, oxidation of the alkylfuran 9 with singlet oxygen in the
presence of the Hüning's base and methanol gave a mixture of
regioisomers 10 and 11 in a 4:1 ratio and quantitative yield (Method
2.1. Chemistry
The pyridazinone derivatives studied in this work (compounds
4
, 5 and 6) were synthesized according to the general procedures
outlined in Schemes 1e3. Thus, the pyridazinone nucleus for
monocyclic derivatives (4 and 5) was built by reaction of the )-
substituted -hydroxybutenolide (10 or 11) with hydrazine or
b(a
A). Moreover, the proportion of the
a-substituted g-hydrox-
g
ybutenolide 11 was increased with the use of DBU in DCM
obtaining the mixture of 10 and 11 in 1:1.25 ratio and excellent
yield (Method B, 96%). Purification by column chromatography
afforded enough quantities of both regioisomers in order to identify
them. Characterization of butenolides 10 and 11 was performed
benzylhydrazine (Scheme 2), whereas for obtaining the fused pyr-
idazinone core (6) a bicyclic lactone (12 or 13) was selected as the
precursor suitable to react with hydrazine (Scheme 3).
The 4-hydroxybutenolides 10 and 11 were prepared in 3 steps
from the commercially available ethyl 3-furoate (7) via oxidation
with singlet oxygen, as shown in Scheme 1. Reduction of ester 7
1
analyzing the differences found in their H NMR data and specif-
ically in the multiplicity of the methylene hydrogen atoms. These
4
with lithium aluminum hydride (LiAlH ) in diethyl ether afforded
protons, which for the
a-substituted butenolide 11 resonate as a
alcohol 8 [11] (93% yield) which was protected as tert-butyldiphe-
nylsilyl ether (compound 9 [12], yield 92%) in order to be submitted
to singlet oxygen oxidation. Several protocols have been reported
multiplet centered at 4.48 ppm, appear fully differentiated for the
b-substituted compound 10 as two doublet of doublets at 4.46 and
4
.60 ppm, (J ¼ 17.8 and 2.0 Hz) respectively. It is noteworthy that in
the b-substituted butenolide (10) the oxygen atom of the side chain
is close to the C5 hydroxyl group allowing the formation of a stable
-membered ring through hydrogen bonding interactions which
6
would explain the multiplicity differences observed.
Treatment of a mixture containing 10 and 11 with hydrazine
monohydrate in ethanol at reflux gave the pyridazinones 4a and 5a
which were easily isolated and purified by column chromatography
(Scheme 2). The structure of pyridazinone isomers 4ae5a was
established by the single crystal X-ray studies (Figs. 4 and 5) [16,17].
Fig. 3. General structure of pyridazinone analogs studied.

T. Costas et al. / European Journal of Medicinal Chemistry 94 (2015) 113e122
115
Scheme 1. Reagents and conditions: (i) LiAlH
4
, Et
2
O, r.t., 45 min, 93%; (ii) TBDPSCl, imidazole, DMF, r.t., 20 h, 92%; (iii) O
2
, h
n
, rose bengal, DIPEA, MeOH, ꢀ78 ꢁC, 5 h, (10 and 11 ratio
4:1, Method A) or DBU, CH
2
Cl
2
, ꢀ78 ꢁC, 2 h (10 and 11 ratio 1:1.5, Method B).
Scheme 2. Reagents and conditions: (i) NH
from 9); (iii) NaH, BnBr, Bu NI, THF, r.t., 24 h (Method D), 68% (4b), 62% (5b), 81% (4d), 56% (5d); (iv) TBAF, THF, r.t., 30 min, 87% (4c), 75% (5c); (v) CBr
4e), 84% (5e).
2
NH
2
, EtOH, reflux, 4 h, 56% (4a from 9), 5% (5a from 9); (ii) BnNHNH
2
$2HCl, Et
3
N, EtOH, reflux, 7 h (Method C), 46% (4b from 9), 7% (5b
4
4
, Ph P, CH Cl , reflux, 1.5 h, 58%
3
2
2
(
2 2 3 2 2 2 2
Scheme 3. Reagents and conditions: (i) NH NH , EtOH, reflux, 5 h, 97% (14), 98% (15); (ii) BF $OEt , CH Cl , rt, 14 h or 1.5 h, 96% (6a), 73% (6b); (iii) MnO , DMF, reflux, 5 h, 47%.
The N-benzylpyridazinones 4be5b were obtained in a similar way
by reaction of the mixture containing 10 and 11 with benzylhy-
drazine dihydrochloride (Method C) and their structures were
determined from NMR spectroscopy by comparison with 4ae5a
data, in particular taking into account the J values of the ring proton
signals (see experimental part). Regiochemistry of compounds
compounds 4be5b by treatment with TBAF provided the hydrox-
ymethyl derivatives 4ce5c, which were transformed into the N,O-
dibenzyl analogs 4de5d using the standard procedure above
mentioned. The bromomethyl derivatives 4ee5e were obtained in
moderate to good yields (58e84%) by refluxing of hydroxymethyl
derivatives IVceVc with carbon tetrabromide and triphenylphos-
phine in DCM [19] (Scheme 2).
4
be5b was further confirmed by benzylation of 4ae5a with benzyl
bromide at room temperature (Method D) [18]. Moreover,
The bicyclic lactones 12 and 13, key intermediates to obtain the

116
T. Costas et al. / European Journal of Medicinal Chemistry 94 (2015) 113e122
ethanol was refluxed with a catalytic amount of p-toluenesulfonic
acid. However, these conditions led to the pyran ring-opening via
the pyridazinone nucleus aromatization, resulting in the mono-
cyclic derivative 6-(3-hydroxypropyl)pyridazin-3(2H)-one instead
of 6b. Finally, oxidation of 6b with manganese dioxide in DMF at
reflux gave rise to the bicyclic unsaturated pyranopyridazinone
analog 6c in moderate yield (47%).
2.2. Antiplatelet activity
Platelet aggregation studies of target compounds were per-
formed in washed human platelets following the Born's turbidi-
metric method [22] and using collagen (2e4 g/mL) as platelet
m
aggregation inducer [23]. A total of thirteen new pyridazinone
derivatives (4aee, 5aee and 6aec) were tested in order to deter-
mine if the new monocyclic and bicyclic analogs show similar an-
tiplatelet activity to that exhibited by the C6-substituted
pyridazinone derivatives (3aec) previously reported [10].
Table 1 shows the IC50 values against collagen for the new an-
alogs and 3aec derivatives [10]. They were obtained from experi-
ments conducted at concentration intervals of 0.5e5
0e100 M, depending on the compound.
The obtained results revealed that five of the new studied de-
mM or
1
m
Fig. 4. X-ray crystal structure of compound 4a. Displacement ellipsoids are shown at
the 20% probability level.
rivatives displayed high potency against collagen-induced platelet
aggregation.
As can be seen from Table 1 data, the N-benzyl silyl ethers
4
be5b and the N,O-dibenzyl derivatives 4de5d exhibited the best
activity, with IC50 values of 0.55 ± 0.08 (4b), 1.31 ± 0.37 (5b),
.75 ± 0.48 (4d) and 17.52 ± 2.53 (5d), which is consistent with the
3
results previously reported for the C6-substituted analogs (com-
pounds 3).
In addition, in the fused-pyridazinone series, the furopyr-
idazinone derivative 6a showed low activity (IC50 value of
70.57 ± 15.55
mM) as compared with the corresponding monocyclic
analogs [10].
It should be noted that in all monocyclic analogs, both the C6-
substituted previously described [10] and the C5 or C4
substituted derivatives now synthesized, the presence of some
lipophilic fragments, such as a tertbutyldiphenylsilyl or a benzyl
group in the alkyl chain and a benzyl group in N2, was favorable for
the activity.
Therefore, the obtained results showed that antiplatelet activity
was unaffected by a positional change of the lateral chain in the
pyridazinone core. However, the activity was significantly dimin-
ished when the alkyl chain was incorporated in a ring.
In order to find any significant information about the molecular
effect of tested pyridazinone derivatives on platelet biology, sub-
sequent pharmacological studies were conducted with 4b, the most
active compound of this series. Firstly, it has been found that a
Fig. 5. X-ray crystal structure of compound 5a. Displacement ellipsoids are shown at
the 20% probability level.
supramaximal dose of 4b (20 mM) was unable to significantly
inhibit platelet aggregation induced by different platelet aggrega-
tory stimulants, such as thrombin, U-46619 (a stable thromboxane
A2 analog) or the calcium ionophore ionomycin (Fig. 6). These re-
sults suggested a selective action of 4b on the biochemical mech-
anisms triggered by collagen in the platelets.
Collagen interacts with platelets through direct and indirect
mechanisms, and although several targets have been involved, two
main surface receptors have been identified, the integrin a2b1, with
a major role in adhesion and platelet anchoring and the Immuno-
globulin (Ig) superfamily member glycoprotein VI (GPVI), princi-
pally responsible for signaling and platelet activation [24]. GPVI-
selective agonists, such as convulxin and collagen-related peptide
fused pyridazinone core analogs 6, were synthesized from the
corresponding g-substituted g-methoxy butenolide by treatment
with TBAF in THF, trough an intramolecular Michael addition, as
previously reported [20,21]. Treatment of 12 or 13 with hydrazine
monohydrate in ethanol at reflux afforded the bicyclic hydrox-
yhydrazides 14 or 15 respectively in excellent yields. Then they
were easily dehydrated by reaction with boron trifluoride diethyl
ꢁ
etherate (BF
3
$EtO
2
) in DCM at 0 C to give the desired furopyr-
idazinone 6a (yield 96%) and pyranopyridazinone 6b (yield 73%)
Scheme 3). Moreover, with the idea of getting a direct trans-
(
(CRP), are strong inducers of platelet aggregation. It has been found
formation of the bicyclic lactones into the fused pyridazinone de-
rivatives 6, a solution of compound 13 and hydrazine hydrate in
that 4b completely inhibits platelet aggregation caused by the
agonist CRP-XL (cross-linked form of CRP; Fig. 6), indicating that

T. Costas et al. / European Journal of Medicinal Chemistry 94 (2015) 113e122
117
Table 1
Antiplatelet activity data against collagen induced platelet aggregation.
0
IC50 m
Ma
Compound
R
R
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
a
b
c
a
b
c
d
e
a
b
c
d
e
a
b
c
Bn
Bn
H
OTBDPS
OBn
OTBDPS
OTBDPS
OTBDPS
OH
OBn
Br
OTBDPS
OTBDPS
OH
4.6 ± 0.4b
b
8.0 ± 0.4
3.87 ± 0.38b
*
H
Bn
Bn
Bn
Bn
H
Bn
Bn
Bn
Bn
e
0.55 ± 0.08
>100
3.75 ± 0.48
>100
*
1.31 ± 0.37
>100
17.52 ± 2.53
>100
70.57 ± 15.55
>100
>100
OBn
Br
e
e
e
e
e
*
Induces platelet aggregation at concentration lower than 100
m
M.
a
Values were expressed as means ± S.E.M (n ¼ 4).
b
Values obtained from ref. [10].
this pyridazinone derivative acts on some of the steps involved in
platelet activation via GPVI pathway. In addition, as the CRP is able
to induce full aggregation in the presence of inhibitors of cyclo-
oxygenase [25,26], our results demonstrate that 4b is not a COX
inhibitor, one of the mechanisms previously suggested for
explaining the activity of this kind of pyridazinone analogs [10].
Stimulation of platelet by any agonist requires activation of the
signaling [29].
Using flow cytometry and PAC-1 [30] (a monoclonal antibody
which recognizes the active conformation of GP IIb/IIIa receptor)
the level of Gp IIb/IIIa activation following platelet stimulation, and
hence the effect of a particular compound on the cellular events
that cause conformational changes of GpIIb/IIIa (i.e. the “inside-
out” signaling), can be measured. Thus, we have found that dose of
4b causing complete inhibition of platelet aggregation only
partially inhibits the GP IIb/IIIa activation (Fig. 7). Since that (i) a
platelet integrin adhesion receptor
aIIbb3 (GP IIb/IIIa) to bind
fibrinogen and link adjacent platelets together in an aggregate [27].
Binding of platelet agonists to their plasma membrane receptors
conformational change in aIIbb3 is crucial for platelet activation
promote cellular responses leading to the change of
a
IIb
b
3 from the
[30], (ii) inhibition of platelet aggregation correlates well with
percentage blockage of GPIIb/IIIa [31] and (iii) total inhibition of
this integrin causes the failure of platelets to aggregate [32], these
results showing a partial inhibition suggest that 4b should act on
both “inside-out” and “outside-in” signaling.
resting state to the activated state via a conformational change in
the extracellular domain, which enhances its affinity for soluble
ligands like fibrinogen; these molecular events are referred to as
“inside-out” signaling [28]. Ligand binding to aIIbb3 becomes
progressively irreversible by integrin clustering, triggering a num-
ber of cellular events that include cytoskeletal reorganization,
changes in enzymatic activities and stabilization of large platelet
aggregates; these molecular events are referred as “outside-in”
Stimulation of GPVI by collagen or CRP induces platelet activa-
tion through a tyrosine kinaseebased signaling pathway. GPVI is
non-covalently associated with the Fc Receptor
g chain (FcRg) [33],
which acts as the signal-transducing subunit of the receptor [34].
Ligand binding causes cross-linking of GPVI leading to tyrosine
phosphorylation of the immunoreceptor tyrosine-based activation
motif (ITAM) within the FcR
g cytoplasmic domain by Src-family
1
00
tyrosine kinases (mainly Lyn and Fyn) bound to the cytoplasmic
domain of GPVI [35]. The ITAM phosphorylation gives rise to
binding and activation of the tyrosine kinase Syk, which phos-
phorylates downstream targets and generates a large signaling
complex involving adapter proteins, Tec family tyrosine kinases,
GTP-exchange factors, small GTPase Rac1, phosphatidylinositol 3-
8
6
4
2
0
0
0
0
0
kinase isoforms and phospholipase C
36].
Therefore, the effect of 4b on the increase of phosphotyrosine
g2 as main effector protein
[
induced by CRP-XL on platelet proteins through an immunodot blot
assay was investigated, using a platelet lysate and the anti-
phosphotyrosine specific antibody 4G10. The obtained results
showed that compound 4b induces a decrease in total amount of
Collagen
CRP
Thrombin
U-46619
Ionomycin
Fig. 6. Effects of supramaximal dose of 4b on platelet aggregation induced by different
platelet activators. Data represent mean ± standard error of the mean (s.e.m) of at least
four experiments in duplicate; for Collagen and CRP s.e.m was 0.

118
T. Costas et al. / European Journal of Medicinal Chemistry 94 (2015) 113e122
Fig. 7. Effect of compound 4b on GpIIb/IIIa activation induced by CRP-XL. A: RGDS treated platelets (non-specific binding of PAC-1). B: non-stimulated platelets (basal conditions). C:
CRP-XL stimulated platelets. D: platelets treated with compound 4b and stimulated with CRP-XL.
singlet oxygen oxidation of alkyl furans, in which a suitable
b(a)-
substituted -hydroxybutenolide or a bicyclic lactone was the key
g
intermediate. Three of these new pyridazinone analogs displayed
high activity against collagen-induced platelet aggregation. Studies
performed with the most active compound of these series (4b)
demonstrated its lack of activity as inhibitor of platelet aggregation
induced by other agonists as thrombin, ionomycin or U-46619.
However, when CRP was used as platelet aggregation inducer,
compound 4b partially decreased the conformational change of
GPIIb/IIIa receptor. In addition, compound 4b also inhibited the
increase in the phosphotyrosine content of platelet proteins caused
by activation of GPVI receptor.
Fig. 8. Effect of compound 4b on protein tyrosine phosphorylation in platelets stim-
ulated by CRP-XL. A: non-stimulated platelets (basal conditions). B: platelets treated
with compound 4b (10
treated with compound 4b (10
tative result from four experiments in duplicate.
m
M). C: platelets stimulated by CRP-XL (5
m
g/mL). D: platelets
mM) and stimulated by CRP-XL (5
mg/mL). Represen-
phosphotyrosine residues of platelet proteins in a dose-dependent
manner, which is well correlated with antiplatelet effect (Fig. 8).
Thus, the decrease on tyrosine phosphorylation displayed by 4b
could explain the effectiveness of this compound against the acti-
vation of platelets by collagen and CRP. Bearing in mind that the
phosphorylation state of any protein is the result of the balance
between phosphorylation/dephosphorylation processes, the
decrease in the level of tyrosine phosphorylation caused by 4b can
be explained both by a decrease in the tyrosineekinase activity and
a stimulation in tyrosineephosphatase activity. Our data provide a
starting point for a new research work in order to know the pro-
teins affected by the action of tested compounds as well as the
different platelet tyrosine kinases or tyrosine phosphatases that
could be involved.
Accordingly, the inhibition of collagen-induced platelet aggre-
gation caused by the target pyridazinone analogs can be due to its
ability to decrease tyrosine phosphorylation of some platelet
proteins.
4. Experimental section
4.1. Chemistry
All starting materials and common laboratory chemicals were
purchased from commercial sources and used without further
purification. All solvents were distilled and dried according to
1
13
standard procedures. H and C NMR spectra were recorded on a
Bruker ARX400 instrument, using TMS as internal standard
3
. Conclusions
[chemical shifts (d) in ppm, J in Hz]. The assignment of the signals
was performed by COSY, DEPT, HSQC experiments. High resolution
mass spectra were recorded using a Bruker microTOF focus spec-
trometer. X-ray analysis was performed on a Bruker SMART 1000
Three new series of pyridazinone derivatives (4, 5 and 6) were
synthesized in good yields following a synthetic strategy based on

T. Costas et al. / European Journal of Medicinal Chemistry 94 (2015) 113e122
119
CCD diffractometer. Silica gel (Merck 60, 230e400 mesh) was used
for flash chromatography (FC). Analytical TLC was performed on
plates precoated with silica gel (Merck 60 F254, 0.25 mm).
1H, H3), 5.99 (s, 1H, H5), 4.69 (br s, 1H, OH), 4.60 (dd, 1H, J ¼ 17.8,
2.0 Hz, CH
2
), 4.46 (dd, 1H, J ¼ 17.8, 2.0 Hz, CH
2
), 1.09 (s, 9H, (CH
3
)
3
).
1
3
C NMR (CDCl ):
3
d
¼ 171.2 (C2), 168.7 (C4), 135.6 (CH-Ph), 132.5 (C-
Ph), 130.3 (CH-Ph), 128.1 (CH-Ph), 117.7 (C3), 97.4 (C5), 60.0 (CH
26.8 ((CH
2
),
þ
4.1.1. 3-Furylmethanol (8)
3
)
3
), 19.3 (C(CH
3
)
3
). HRMS-ESI: m/z [MþH] calcd for
To a solution of ethyl 3-furoate (7) (4.00 g, 28.5 mmol) in Et
2
O
C
21
H
25
O
4
Si: 369.15166, found: 369.15162. Compound 11: R
f
¼ 0.2
¼ 7.66e7.62 (m, 4H,
H-Ph), 7.47e7.36 (m, 6H, H-Ph), 7.15e7.13 (m, 1H, H4), 6.11 (s, 1H,
1
(
0
4
140 mL) was added LiAlH
C. The reaction mixture was stirred at room temperature for
4
(1.62 g, 42.8 mmol) in small portions at
3
(hexane/ethyl acetate, 2:1). H NMR (CDCl ): d
ꢁ
13
5 min, followed by quenching with H
2
O (2 mL), NaOH 1 M (2 mL)
H5), 4.49e4.47 (m, 2H, CH
2 3 3 3
), 1.09 (s, 9H, (CH ) ). C NMR (CDCl ):
ꢁ
and H
2
O (6 mL) at 0 C. The resulting white precipitate was filtered
d
¼ 169.5 (C2), 143.7 (C4), 138.6 (C3), 135.6 (CH-Ph), 132.7 (C-Ph),
off and the solvent was removed under reduced pressure affording
compound 8 (2.60 g, 93%) as a yellowish oil. R
¼ 0.2 (hexane/ethyl
¼ 7.42e7.41 (m, 1H, H2), 7.40e7.39
m, 1H, H5), 6.44e6.42 (m, 1H, H4), 4.55 (s, 2H, CH ), 1.81 (br s, 1H,
130.2 (CH-Ph), 128.1 (CH-Ph), 97.3 (C5), 58.8 (CH
19.3 (C(CH
369.15166, found: 369.15261.
2
), 26.9 ((CH
3
)
3
),
f
3
)
3
). HRMS-ESI: m/z [MþH]þ calcd for C21
25 4
H O
Si:
1
acetate, 4:1). H NMR (CDCl
3
):
d
(
2
13
OH). C NMR (CDCl
C4), 56.1 (CH
found: 98.0371.
3
):
d
¼ 143.3 (C5), 139.9 (C2), 125.1 (C3), 109.9
4.1.4. 5-(tert-Butyldiphenylsilyloxymethyl)pyridazin-3(2H)-one
(4a) and 4-(tert-butyldiphenylsilyloxymethyl)pyridazin-3(2H)-one
(5a)
þ
(
2
). HRMS-EI: m/z [M] calcd for C
5
H
6
O
2
: 98.0368,
To a solution of a mixture containing 10 and 11 (1.09 g) in a 4:1
ratio in ethanol (15 mL) was added hydrazine monohydrate (0.3 mL,
6.18 mmol). After the reaction mixture was stirred under reflux for
4 h, the solvent was removed and the residue was purified by
column chromatography on silica gel (hexane/ethyl acetate, 4:1) to
afford 5a (45 mg, 5% from 9) as a colorless oil and then 4a (508 mg,
4
.1.2. 3-(tert-Butyldiphenylsilyloxymethyl)furan (9)
To a solution of compound 8 (2.60 g, 26.5 mmol) in DMF (30 mL)
was added imidazole (2.49 g, 36.5 mmol) and TBDPSCl (9.5 mL,
6.5 mmol). The reaction mixture was stirred at room temperature
for 20 h, quenched with H O (50 mL) and extracted with Et
SO
3
2
2
O
(
3 ꢂ 50 mL). The combined organic phases were dried over Na
2
4
,
56% from 9) as a white solid. Compound 4a: R
acetate, 2:1). H NMR (CDCl ): d
3
f
¼ 0.2 (hexane/ethyl
¼ 12.91 (br s, 1H, NH), 7.72 (d, 1H,
J ¼ 1.9 Hz, H6), 7.69e7.65 (m, 4H, H-Ph), 7.49e7.38 (m, 6H, H-Ph),
7.09e7.07 (m, 1H, H4), 4.61 (d, 2H, J ¼ 1.3 Hz, CH ), 1.12 (s, 9H,
1
filtered and the solvent was evaporated to dryness. The residue was
purified by column chromatography on silica gel (hexane/ethyl
acetate, 99:1) to obtain compound 9 (8.20 g, 92%) as a yellowish oil.
R
2
1
13
f
¼ 0.6 (hexane/ethyl acetate, 4:1). H NMR (CDCl
3
):
d
¼ 7.72e7.69
(CH
3
)
3
). C NMR (CDCl
3
):
d
¼ 162.8 (C3),147.3 (C5),136.4 (C6),135.6
(
m, 4H, H-Ph), 7.46e7.37 (m, 7H, H-Ph, H5), 7.33e7.32 (m, 1H, H2),
(CH-Ph), 132.4 (C-Ph), 130.3 (CH-Ph), 128.1 (CH-Ph), 124.5 (C4), 62.1
13
þ
6
.35e6.33 (m, 1H, H4), 4.63 (s, 2H, CH
2
), 1.08 (s, 9H, (CH
3
)
3
).
C
(CH
for C21
2
), 26.8 ((CH
3
)
3
), 19.4 (C(CH
3
)
3
). HRMS-ESI: m/z [MþH] calcd
NMR (CDCl
3
):
d
¼ 143.1 (C5), 139.5 (C2), 135.7 (CH-Ph), 133.7 (C-Ph),
25
H N
2
O
2
Si: 365.16798, found: 365.16806. Compound 5a:
1
129.9 (CH-Ph), 127.9 (CH-Ph), 125.5 (C3), 109.7 (C4), 58.4 (CH
2
), 26.9
R
f
¼ 0.3 (hexane/ethyl acetate, 2:1). H NMR (CDCl
3
):
d
¼ 12.32 (br s,
(
(CH
3
)
3
), 19.4 (C(CH
3
)
3
). HRMS-ESI: m/z [MþH]þ calcd for
1H, NH), 7.90 (d, 1H, J ¼ 4.0 Hz, H6), 7.67e7.63 (m, 4H, H-Ph),
21 25 2
C H O Si: 337.16183, found: 337.16241.
7.61e7.58 (m, 1H, H5), 7.46e7.35 (m, 6H, H-Ph), 4.77 (d, 2H,
13
J ¼ 1.7 Hz, CH
2
), 1.14 (s, 9H, (CH
3
)
3
). C NMR (CDCl
3
):
d
¼ 161.1 (C3),
4
.1.3. 4-(tert-Butyldiphenylsilyloxymethyl)-5-hydroxy-5H-furan-2-
143.7 (C4), 137.9 (C6), 135.5 (CH-Ph), 132.7 (C-Ph), 130.2 (CH-Ph),
one (10) and 3-(tert-butyldiphenylsilyloxymethyl)-5-hydroxy-5H-
furan-2-one (11)
Method A: To a solution of compound 9 (3.00 g, 8.92 mmol) in
MeOH (40 mL) was added N,N-diisopropylethylamine (7.1 mL,
128.1 (CH-Ph), 126.5 (C5), 60.5 (CH
2
), 27.0 ((CH
3
)
3
), 19.5 (C(CH ).
3 3
)
þ
HRMS-ESI: m/z [MþH] calcd for C21
25 2 2
H N O
Si: 365.16798, found:
365.16804.
4
1
0.2 mmol), rose bengal (15 mg). Then it was purged with O
2
for
4.1.5. 2-Benzyl-5-(tert-butyldiphenylsilyloxymethyl)pyridazin-
3(2H)-one (4b) and 2-benzyl-4-(tert-butyldiphenylsilyloxymethyl)
pyridazin-3(2H)-one (5b)
Method C: To a solution of a mixture containing 10 and 11
(531 mg) in a 4:1 ratio in ethanol (15 mL) was added benzylhy-
h. The reaction mixture was irradiated with a 200 W lamp and
ꢁ
stirred under oxygen atmosphere for 5 h at ꢀ78 C. The solvent was
evaporated, the residue was dissolved in CH
oxalic acid in H O (350 mL) was added. The mixture was stirred for
min at room temperature and extracted with CH Cl
3 ꢂ 100 mL). The combined organic phases were dried over
Na SO , filtered and the solvent was evaporated to dryness. The
2
Cl
2
(40 mL) and 0.12 M
2
3
(
0
2
2
drazine dihydrochloride (562 mg, 2.88 mmol) and Et
4.30 mmol) and the reaction mixture was stirred under reflux for
7 h. After the solvent was removed, CH Cl (10 mL) and H O (10 mL)
were added and the mixture was extracted with CH Cl
SO
3
N (0.6 mL,
2
4
2
2
2
residue was rapidly passed through a column chromatography on
silica gel (hexane/ethyl acetate, 2:1) to afford compound 10 (56 mg)
as a colorless oil, then a mixture containing 10 and 11 (3.61 g) in a
2
2
(3 ꢂ 10 mL). The combined organic phases were dried over Na
2
4
,
filtered and the solvent was evaporated to dryness. The residue was
purified by column chromatography on silica gel (hexane/ethyl
acetate, 9:1) to afford 5b (43 mg, 7% from 9) as a colorless oil and
then 4b (277 mg, 46% from 9) as a yellowish oil. Method D: A so-
lution of compound 4a or 5a (50 mg, 0.14 mmol) in THF (4 mL) was
4
:1 ratio and finally compound 11 (20 mg) as a colorless oil.
Method B: To a solution of compound 9 (300 mg, 0.89 mmol) in
CH Cl (20 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene
0.27 mL, 1.78 mmol), rose bengal (5 mg). Then it was purged
2
2
(
ꢁ
with O
2
for 1 h. The reaction mixture was irradiated with a 200 W
added, dropwise at 0 C, to a suspension of NaH (9 mg, 0.21 mmol,
ꢁ
lamp and stirred under oxygen atmosphere for 2 h at ꢀ78 C. The
60% dispersion in mineral oil) in THF (4 mL). After the mixture was
stirred at r.t. for 1 h, BnBr (35 mL, 0.29 mmol) and Bu NI (5 mg,
4
ꢁ
mixture was allowed to warm at 0 C, washed with HCl 1 M (15 mL)
and extracted with CH
over Na SO and evaporated to dryness and the residue was puri-
fied by column chromatography on silica gel (hexane/ethyl acetate,
:1) to afford a mixture containing 10 and 11 (299 mg) in a 1:1.25
ratio and then compound 11 (15 mg) as a colorless oil. Compound
2
Cl
2
(2 ꢂ 15 mL). The organic phase was dried
0.01 mmol) were added. The reaction mixture was stirred at r.t. for
24 h, followed by quenching with MeOH. The solvent was evapo-
rated to dryness, and the residue was purified by column chro-
matography on silica gel (hexane/ethyl acetate, 9:1) to afford 4b
2
4
4
(44 mg, 68%) as a yellowish oil or 5b (39 mg, 62%) as a yellowish oil.
1
1
1
0:
R
f
¼
0.3 (hexane/ethyl acetate, 2:1).
H
NMR (CDCl
3
):
Compound 4b: R
(CDCl ):
f
¼ 0.5 (hexane/ethyl acetate, 2:1). H NMR
d
¼ 7.67e7.63 (m, 4H, H-Ph), 7.47e7.38 (m, 6H, H-Ph), 6.18e6.16 (m,
3
d
¼ 7.69e7.64 (m, 5H, H6, H-Ph), 7.49e7.27 (m, 11H, H-Ph),

120
T. Costas et al. / European Journal of Medicinal Chemistry 94 (2015) 113e122
7
CH
.01e6.99 (m, 1H, H4), 5.34 (s, 2H, CH
2
N), 4.57 (d, 2H, J ¼ 1.5 Hz,
66.6 (CH
2
OBn), 55.2 (CH
2
N). HRMS-ESI: m/z [MþH]þ calcd for
13
2
O), 1.12 (s, 9H, (CH
3
)
3
). C NMR (CDCl ):
3
d
¼ 160.6 (C3), 145.6
19 19 2 2
C H N O : 307.14410, found: 307.14494.
(
(
C5), 136.4 (C-Ph), 135.5 (CH-Ph), 135.3 (C6), 132.3 (C-Ph), 130.2
CH-Ph), 128.8 (CH-Ph), 128.7 (CH-Ph), 128.1 (CH-Ph), 128.0 (CH-
4.1.10. 2-Benzyl-5-bromomethylpyridazin-3(2H)-one (4e)
To a solution of compound 4c (83 mg, 0.39 mmol) in CH
Ph), 124.7 (C4), 61.8 (CH
C(CH
2
O), 58.9 (CH
). HRMS-ESI: m/z [MþH] calcd for
2
N), 26.8 ((CH
3
)
3
), 19.3
Si:
¼ 0.6 (hexane/ethyl
¼ 7.85 (d, 1H, J ¼ 4.0 Hz, H6),
2
Cl
(202 mg,
0.77 mmol) and the reaction mixture was refluxed for 1.5 h. After
quenching with NaHCO (sat. aqueous solution, 5 mL) the product
was extracted with CH Cl SO , and the
2
þ
(
3
)
3
C
28
H
31
N
2
O
2
4 3
(5 mL) was added CBr (256 mg, 0.77 mmol), PPh
4
55.21493, found: 455.21473. Compound 5b: R
f
1
acetate, 2:1). H NMR (CDCl
3
):
d
3
7.65e7.61 (m, 4H, H-Ph), 7.52e7.49 (m, 1H, H5), 7.45e7.26 (m, 11H,
2
2
(3 ꢂ 5 mL), dried over Na
2
4
H-Ph), 5.29 (s, 2H, CH
2
N), 4.73 (d, 2H, J ¼ 1.5 Hz, CH
2
O), 1.12 (s, 9H,
solvent was removed under reduced pressure. The residue was
purified by column chromatography on silica gel (dichloro-
methane/methanol, 99.5:0.5) to afford 4e (62 mg, 58%) as a
13
(
CH
3
)
3
). C NMR (CDCl
3
):
d
¼ 159.5 (C3), 143.4 (C4), 136.7 (C6),
136.4 (C-Ph), 135.6 (CH-Ph), 132.9 (C-Ph), 130.1 (CH-Ph), 128.9 (CH-
1
Ph), 128.7 (CH-Ph), 128.0 (2ꢂ(CH-Ph)), 125.3 (C5), 61.0 (CH
2
O), 55.1
yellowish oil. R
(CDCl ):
7.35e7.27 (m, 3H, H-Ph), 6.85 (d, 1H, J ¼ 1.9 Hz, H4), 5.30 (s, 2H,
f
¼ 0.6 (ethyl acetate/methanol, 95:5). H NMR
þ
(
CH
2
N), 27.1 ((CH
H N O Si: 455.21493, found: 455.21371.
31 2 2
3
)
3
), 19.5 (C(CH
3
)
3
). HRMS-ESI: m/z [MþH] calcd
3
d
¼ 7.77 (d, 1H, J ¼ 1.9 Hz, H6), 7.44e7.39 (m, 2H, H-Ph),
for C28
13
CH
2
N), 4.17 (s, 2H, CH
2
Br). C NMR (CDCl
3
):
d
¼ 159.7 (C3), 141.7
4
.1.6. 2-Benzyl-5-hydroxymethylpyridazin-3(2H)-one (4c)
A solution of compound 4b (247 mg, 0.54 mmol) in THF (5 mL)
and TBAF 1 M in THF (0.8 mL, 0.81 mmol) was stirred at r.t. for
0 min. The solvent was evaporated to dryness, and the residue was
(C5), 137.0 (C6), 136.1 (C-Ph), 128.9 (CH-Ph), 128.8 (CH-Ph), 128.2
(CH-Ph), 128.0 (C4), 55.2 (CH
2
2
N), 27.0 (CH Br). HRMS-ESI: m/z
[MþH]þ calcd for C12
H12BrN O: 279.01275, found: 279.01210.
2
3
purified by column chromatography on silica gel (ethyl acetate/
4.1.11. 2-Benzyl-4-bromomethylpyridazin-3(2H)-one (5e)
methanol, 98:2) to afford 4c (102 mg, 87%) as a yellowish oil.
Compound 5e (31 mg, 84%), white solid, was obtained from 5c
(29 mg, 0.13 mmol) following the same procedure as for prepara-
tion of compound 4e from 4c. Compound 5e was purified by col-
1
R
f
¼
0.5 (ethyl acetate/methanol, 95:5).
3
H NMR (CDCl ):
d
¼ 7.66e7.64 (m, 1H, H6), 7.36e7.31 (m, 2H, H-Ph), 7.30e7.24 (m,
H, H-Ph), 6.87e6.84 (s, 1H, H4), 5.26 (s, 2H, CH N), 4.45 (s, 2H,
¼ 160.9 (C3), 146.9 (C5), 136.3 (C6), 136.1
3
CH
2
umn chromatography on silica gel (hexane/ethyl acetate, 6:1).
13
1
2
O). C NMR (CDCl
3
):
d
R
f
¼ 0.2 (hexane/ethyl acetate, 6:1). H NMR (CDCl
3
):
d
¼ 7.77 (d,1H,
(
(
C-Ph), 128.7 (CH-Ph), 128.6 (CH-Ph), 128.1 (CH-Ph), 124.8 (C4), 60.7
CH
J ¼ 4.0 Hz, H6), 7.46e7.41 (m, 2H, H-Ph), 7.36e7.27 (m, 4H, H5, H-
N). HRMS-ESI: m/z [MþH]þ calcd for C12
H
13
N
2
O
2
:
13
2
O), 55.2 (CH
2
2 2 3
Ph), 5.35 (s, 2H, CH N), 4.39 (s, 2H, CH Br). C NMR (CDCl ):
217.09715, found: 217.09782.
d
¼ 159.5 (C3), 138.9 (C4), 136.1 (C6), 136.0 (C-Ph), 129.7 (C5), 129.0
(CH-Ph), 128.8 (CH-Ph), 128.2 (CH-Ph), 55.7 (CH
2
N), 26.6 (CH
2
Br).
þ
4
.1.7. 2-Benzyl-4-hydroxymethylpyridazin-3(2H)-one (5c)
Compound 5c (25 mg, 75%), yellowish oil, was obtained from 5b
70 mg, 0.15 mmol) following the same procedure as for prepara-
tion of compound 4c from 4b. R
¼ 0.6 (ethyl acetate/methanol,
¼ 7.80 (d,1H, J ¼ 4.0 Hz, H6), 7.42e7.38 (m,
H, H-Ph), 7.34e7.27 (m, 3H, H-Ph), 7.21 (dt, 1H, J ¼ 4.0, 1.2 Hz, H5),
HRMS-ESI: m/z [MþH] calcd for C12
2
H12BrN O: 279.01275, found:
279.01337.
(
f
4.1.12. 4,4a,6,7,7a,1-Hexahydro-7a-hydroxyfuro[3,2-c]pyridazin-
3(2H)-one (14)
1
9
2
5
d
3
5:5). H NMR (CDCl ): d
To a solution of compound 12 [20] (30 mg, 0.19 mmol) in ethanol
13
.33 (s, 2H, CH
¼ 160.5 (C3), 142.1 (C4), 136.8 (C6), 136.1 (C-Ph), 128.8 (CH-Ph),
28.7 (CH-Ph), 128.1 (CH-Ph), 126.4 (C5), 60.9 (CH O), 55.3 (CH N).
: 217.09715, found:
2
N), 4.62 (d, 2H, J ¼ 1.2 Hz, CH
2
O). C NMR (CDCl
3
):
(4 mL) was added hydrazine monohydrate (37
reaction mixture was refluxed for 5 h, and the solvent was removed
obtaining 14 (29 mg, 97%) as a white solid. R
¼ 0.1 (ethyl acetate/
¼ 4.11 (d, 1H, J ¼ 6.7 Hz, H4a),
3.99e3.92 (m, 1H, H6), 3.88e3.82 (m, 1H, H6), 2.76 (dd, 1H, J ¼ 18.0,
.7 Hz, H4), 2.45e2.38 (m, 1H, H7), 2.26 (d, 1H, J ¼ 18.0 Hz, H4),
mL, 0.76 mmol). The
1
2
2
f
þ
1
HRMS-ESI: m/z [MþH] calcd for C12
13
H N
2
O
2
3
methanol, 95:5). H NMR (CD OD): d
217.09782.
6
13
4.1.8. 2-Benzyl-5-benzyloxymethylpyridazin-3(2H)-one (4d)
2.13e2.05 (m, 1H, H7). C NMR (CD
3
OD):
d
¼ 173.2 (C3), 100.4
Compound 4d (32 mg, 81%), yellowish oil, was obtained from 4c
(C7a), 80.7 (C4a), 68.4 (C6), 37.1 (C7), 36.0 (C4).
(
28 mg, 0.13 mmol) following the same procedure as for prepara-
tion of compound 4b from 4a (Method D). R
¼ 0.3 (hexane/ethyl
acetate, 2:1). 1H NMR (CDCl
):
¼ 7.74 (d, 1H, J ¼ 2.1 Hz, H6),
.43e7.39 (m, 2H, H-Ph), 7.38e7.26 (m, 8H, H-Ph), 6.90e6.88 (m,
H, H4), 5.31 (s, 2H, CH N), 4.59 (s, 2H, OCH Ph), 4.38 (d, 2H,
J ¼ 1.2 Hz, CH ): ¼ 160.4 (C3), 143.2 (C5),
OBn). 13C NMR (CDCl
37.1 (C-Ph), 136.3 (C-Ph), 136.0 (C6), 128.8 (CH-Ph), 128.7 (CH-Ph),
28.7 (CH-Ph), 128.3 (CH-Ph), 128.1 (CH-Ph), 127.9 (CH-Ph), 126.2
f
4.1.13. 4,4a,6,7,8,8a-Hexahydro-1H-8a-hydroxypyrano[3,2-c]
pyridazin-3(2H)-one (15)
Compound 15 (113 mg, 98%), white solid, was obtained from 13
[20] (115 mg, 0.67 mmol) following the same procedure as for
3
d
7
1
2
2
2
3
d
preparation of compound 14 from 12. R
f
¼ 0.2 (ethyl acetate/
1
1
1
methanol, 95:5). H NMR (CD
3
OD):
d
¼ 3.85 (d, 1H, J ¼ 4.8 Hz, H4a),
3.83e3.74 (m, 1H, H6), 3.46e3.36 (m, 1H, H6), 2.76 (dd, 1H, J ¼ 16.9,
(
[
C4), 73.2 (OCH
MþH] calcd for C19
2
Ph), 67.7 (CH
2
OBn), 54.9 (CH
2
N). HRMS-ESI: m/z
4.8 Hz, H4), 2.59e2.50 (m, 1H, H7), 2.09 (d, 1H, J ¼ 16.9 Hz, H4),
þ
13
H
19
N
2
O
2
: 307.14410, found: 307.14329.
1.74e1.63 (m, 1H, H7), 1.60e1.51 (m, 2H, H8). C NMR (CD
3
OD):
d
¼ 176.5 (C3), 88.3 (C8a), 76.5 (C4a), 66.6 (C6), 38.6 (C4), 30.3 (C7),
þ
4
.1.9. 2-Benzyl-4-benzyloxymethylpyridazin-3(2H)-one (5d)
Compound 5d (12 mg, 56%), yellowish oil, was obtained from 5c
15 mg, 0.07 mmol) following the same procedure as for prepara-
tion of compound 4b from 4a (Method D). R
¼ 0.5 (hexane/ethyl
acetate, 2:1). 1H NMR (CDCl
):
¼ 7.79 (d, 1H, J ¼ 4.0 Hz, H6),
.44e7.40 (m, 2H, H-Ph), 7.37e7.27 (m, 9H, H5, H-Ph), 5.33 (s, 2H,
23.2 (C8). HRMS-ESI: m/z [MþH] calcd for C
7 13 2 3
H N O 173.09207,
found: 173.09227.
(
f
4.1.14. 4,4a,6,7-Tetrahydrofuro[3,2-c]pyridazin-3(2H)-one (6a)
3
d
To a solution of compound 14 (33 mg, 0.21 mmol) in CH
2
Cl
2
ꢁ
7
(4 mL) was added BF
3
$OEt
2
(31
m
L, 0.25 mmol) at 0 C. The reaction
1
3
CH
NMR (CDCl
36.3 (C-Ph), 128.9 (CH-Ph), 128.7 (CH-Ph), 128.7 (CH-Ph), 128.1
CH-Ph), 128.1 (CH-Ph), 127.9 (CH-Ph), 126.0 (C5), 73.5 (OCH Ph),
2
N), 4.66 (s, 2H, OCH
2
Ph), 4.54 (d, 2H, J ¼ 1.7 Hz, CH
2
OBn).
C
mixture was stirred at r.t. for 14 h, quenched with NaHCO
3
(sat.
3
):
d
¼ 159.5 (C3), 141.0 (C4), 137.7 (C-Ph), 136.6 (C6),
aqueous solution, 0.5 mL), dried over Na SO and filtered. After
2
4
1
(
removal of the solvent the residue was purified by column chro-
matography on silica gel (ethyl acetate) to afford 6a (28 mg, 96%) as
2

T. Costas et al. / European Journal of Medicinal Chemistry 94 (2015) 113e122
121
a colorless oil. R
CD OD):
f
¼ 0.5 (ethyl acetate/methanol, 95:5). 1H NMR
added instead of the compounds. The final DMSO concentration
was below 1% (v/v) in all cases.
Results shown in Table 1 are expressed as means ± SEM from at
least four experiments.
(
3
d
¼ 4.49e4.42 (m, 1H, H4a), 4.31e4.25 (m, 1H, H6),
4
.08e4.01 (m, 1H, H6), 2.85e2.67 (m, 2H, H7), 2.81 (dd, 1H, J ¼ 16.1,
13
7.7 Hz, H4), 2.50 (dd, 1H, J ¼ 16.1, 13.9 Hz, H4). C NMR (CD
3
OD):
d
¼ 168.8 (C3),164.3 (C7a), 72.6 (C4a), 69.0 (C6), 34.5 (C4), 30.3 (C7).
þ
HRMS-EI: m/z [M] calcd for C
6
H
8
N
2
O
2
, 140.0586, found: 140.0591.
4.2.1. Effect on Gp IIb/IIIa activation
Expression of activated GpIIb/IIIa on platelets membrane was
analyzed using the specific FITC-labeled monoclonal antibody PAC-
1, which recognizes an epitope on the activated form of glycopro-
tein IIb/IIIa complex of stimulated platelets.
4
(
.1.15. 4,4a,7,8-Tetrahydro-6H-pyrano[3,2-c]pyridazin-3(2H)-one
6b)
To a solution of compound 15 (50 mg, 0.29 mmol) in CH
2
Cl
L, 0.36 mmol) at 0 C. The reaction
(sat.
and filtered. After
2
ꢁ
(
5 mL) was added BF
3
$OEt
2
(44
m
Washed human platelets were pre-incubated with DMSO or test
compounds for 5 min, and then stimulated with CRP-XL in the
presence of FITC conjugated PAC-1 monoclonal antibody for
15 min at room temperature. The samples were then diluted and
analyzed on a Beckman Coulter EPICS XL flow cytometer with
System II software. Platelets were identified by logarithmic signal
amplification for forward and side scatter. For every histogram,
10,000 platelets events were counted to evaluate both PAC-1 mean
fluorescence intensity and the percentage of platelets positive for
PAC-1 expression. All assays included samples to which RGDS
peptide was added in order to determine the specificity of PAC-1
binding.
ꢁ
mixture was stirred at 0 C for 1.5 h, quenched with NaHCO
aqueous solution, 0.5 mL), dried over Na SO
2 4
removal of the solvent the residue was purified by column chro-
matography on silica gel (hexane/ethyl acetate, 7:3) to afford 6b
3
(
33 mg, 73%) as a white solid. R
f
¼ 0.4 (ethyl acetate/methanol,
1
9
1
2
5:5). H NMR (CD
3
OD):
d
¼ 4.37e4.30 (m, 1H, H4a), 4.01e3.95 (m,
H, H6), 3.65e3.57 (m, 1H, H6), 2.82 (dd, 1H, J ¼ 17.4, 9.0 Hz, H4),
.63e2.56 (m, 1H, H8), 2.53 (dd, 1H, J ¼ 17.4, 11.5 Hz, H4), 2.51e2.41
13
(
m, 1H, H8), 1.92e1.86 (m, 2H, H7). C NMR (CD
3
OD):
d
¼ 168.7
(
C3),153.1 (C8a), 70.9 (C4a), 67.4 (C6), 34.5 (C4), 30.3 (C8), 26.9 (C7).
þ
HRMS-ESI: m/z [MþH] calcd for C
7 11 2 2
H N O , 155.08150, found:
155.08147.
4
.2.2. Effect on protein tyrosine phosphorylation
Washed platelet lysates were prepared by addition of SDS/EDTA
4
.1.16. 7,8-Dihydro-6H-pyrano[3,2-c]pyridazin-3(2H)-one (6c)
To a solution of compound 6b (16 mg, 0.10 mmol) in DMF (3 mL)
(2%/1 mM) final concentration) to samples of platelets pre-
was added MnO
2
(169 mg, 1.94 mmol) and the reaction mixture
ꢁ
incubated with DMSO (control) or tested compounds at 37 C for
was refluxed for 5 h. The mixture was filtered and the solvent was
removed in vacuo. The residue was purified by column chroma-
tography on silica gel (ethyl acetate) to afford 6c (7 mg, 47%) as a
5
min and treated with or without (unstimulated samples) platelet
aggregation inducers in the aggregometer as indicated above. The
samples were later boiled for 5 min and the proteins contained in
platelet lysates were transferred onto Polyvinylidene Fluoride
1
white solid. R
CD OD):
f
¼ 0.2 (ethyl acetate/methanol, 95:5). H NMR
(
3
d
¼ 6.12 (s, 1H, H4), 4.28 (t, 2H, J ¼ 5.3 Hz, H6), 2.83 (t, 2H,
(PVDF) membranes using a dot-blot filtration manifold. After
13
J ¼ 6.5 Hz, H8), 2.12e2.05 (m, 2H, H7). C NMR (CD
3
OD):
d
¼ 166.4
blotting, the membranes were blocked overnight in 5% bovine
serum albumin (BSA) in Tris-buffered saline supplemented with
0
for 1 h in the anti-phosphotyrosine 4G10 antibody solution pre-
pared in TBST with 1% bovine serum albumin. After washing with
TBST, the membrane was incubated with horseradish peroxidase-
conjugated secondary antibody for 1 h. After washing with TBST,
protein spots on the membrane were visualized by an enhanced
chemiluminescence Western blotting detection system (Pierce,
Thermo Scientific).
(
(
1
C3), 160.4 (C4a), 140.2 (C8a), 107.5 (C4), 69.1 (C6), 26.6 (C8), 22.6
þ
C7). HRMS-ESI: m/z [MþH] calcd for C
7 9 2 2
H N O ,153.06585, found:
.1% Tween 20 (TBST), washed three times in TBST, and incubated
53.06537.
4.2. Antiplatelet activity studies
Washed human platelets were prepared from blood anti-
coagulated with citrateephosphateedextrose, which was obtained
from Centro de Transfusi oꢀ n de Galicia (Santiago de Compostela.
Spain). Bags containing buffy coat from individual donors were
diluted with the same volume of washing buffer (NaCl, 120 mM;
KCl, 5 mM; trisodium citrate, 12 mM; glucose, 10 mM; sucrose,
Acknowledgments
1
2.5 mM; pH 6) and centrifuged at 400 g for 8 min. The upper layer
We acknowledge the Xunta de Galicia (CN2012/184) for the
financial support. M.C.C.-L. and N.V. thank the Xunta de Galicia and
the Universidade de Vigo for their PhD fellowships.
containing platelets (platelet rich plasma) was removed and
centrifuged at 1100 g for 18 min. The resulting platelet pellet was
recovered, resuspended with washing buffer, and centrifuged again
at 1100 g for 15 min. Finally, the platelet pellet from this step was
resuspended in a modified TyrodeeHEPES buffer (HEPES, 10 mM;
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