Pyrazolo[1′,5′:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones as selective human A1 adenosine receptor ligands

Article abstract of DOI:10.1016/j.bmc.2010.09.043

A series of pyrazolo[1′,5′:1,6]pyrimido[4,5-d]pyridazin-4(3H)- ones was synthesized and tested in radioligand binding assays to determine their affinities for the human adenosine A₁, A_2A, A _2B and A₃ receptors.

Full text of DOI:10.1016/j.bmc.2010.09.043

Bioorganic & Medicinal Chemistry 18 (2010) 7890–7899
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones as selective human
A1 adenosine receptor ligands
Maria Paola Giovannoni a, , Claudia Vergelli a, Agostino Cilibrizzi a, Letizia Crocetti a, Claudio Biancalani a,
⇑
Alessia Graziano a, Vittorio Dal Piaz a, Maria Isabel Loza b, Maria Isabel Cadavid b, José Luis Díaz c,
Amadeu Gavaldà c
a Dipartimento di Scienze Farmaceutiche, via Ugo Schiff 6, 50019 Sesto Fiorentino Firenze, Italy
b Departamento de Farmacologia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
c Almirall R&D Center, Sant Feliu de Llobregat, Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
A series of pyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones was synthesized and tested in radio-
ligand binding assays to determine their affinities for the human adenosine A1, A2A, A2B and A3 receptors.
Results indicated that this scaffold is appropriate for adenosine receptor subtype A1 ligands and that the
best arranged groups around this scaffold are 3- and 4-pyridinyl at position 1, benzyl at position 3, hydro-
gen at position 6 and 3-thienyl or phenyl at position 9. The most interesting compounds showed Ki for A1
in the nanomolar range and an appreciable selectivity for other receptor subtypes.
Article history:
Received 16 June 2010
Revised 14 September 2010
Accepted 16 September 2010
Available online 22 September 2010
Keywords:
Adenosine
Ó 2010 Elsevier Ltd. All rights reserved.
A1 subtype
Selectivity
Ligands
Pyrazolo[10,50:1,6]pyrimido
[4,5-d]pyridazin-4(3H)-ones
1. Introduction
density in the striatum,13 but also in numerous peripheral tissue
such as platelets, lymphocytes and neutrophils,16–18 are promising
Adenosine is an endogenous and ubiquitous neuromodulator
that elicits its biological functions by interacting with at least four
receptor subtypes, classified as A1, A2A, A2B and A3.1,2 All four aden-
osine receptors belong to the superfamily of seven transmembrane
G-protein-coupled receptors and have been cloned from different
species.3
A1 and A3 receptors are coupled with Gi protein, with a conse-
quent decrease in cellular cAMP level; on the contrary, A2A and
A2B are coupled to GS protein and stimulate adenylate cyclase,
leading to an increase in cellular cAMP levels.4 The A1 receptor is
able to activate different messenger systems such as phospholipase
C, potassium channels and inhibits calcium channels,2 while sub-
type A3 is positively coupled to phospholipase C and D.5,6
The potential therapeutic applications of agonists and antago-
nists of the various receptor subtypes have been widely investi-
gated in these last years.7–12 A1 antagonists have been proposed
for the treatment of central nervous system pathologies, such as
Alzheimer’s disease, as diuretics and as antihypertensives.10,13–15
Selective antagonists of subtype A2A, which is present in high
drugs for the treatment of Parkinson’s disease.19 A2B receptors are
implicated in vascular tone, gene expression, cell growth, mast cell
degranulation and hepatic glucose balance,20–22 and are proposed
as antiasthmatic and antidiabetic drugs.23–25 Finally, antagonists
of A3 receptors, which are particularly abundant in human lung
and liver26 and seem also to be involved in cell survival regula-
tion,27 could be useful in the treatment of inflammatory states,28
asthma,29 chronic obstructive pulmonary disease,30 cancer31 and
glaucoma.32
Looking at the more recent literature about polyheterocyclic
derivatives, numerous series have been developed as potent and
subtype selective adenosine antagonists (Fig. 1). The 3-aryl-
[1,2,4]triazino[4,3-a]-benzimidazol(10H)-4-one derivative A33 is a
selective A1 antagonist, displaying high affinity for the bovine
A1 subtype (Ki = 18 nM) and significant selectivity for A2A
(Ki >10,000 nM) and A3 (Ki >1000 nM). The pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidine B is a very potent and selective A2A
antagonist (hKi = 0.048 nM) and its high affinity for the adenosine
A2A receptor makes it an attractive candidate for PET studies.34
Among the A2B selective antagonists, a significant example is com-
pound C which has a Ki = 20 nM for the human A2B subtype.35
Finally, one of the most potent and selective A3 antagonists
⇑
Corresponding author. Tel.: +39 055 4573682; fax: +39 055 4573780.
E-mail address: mariapaola.giovannoni@unifi.it (M.P. Giovannoni).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.043

M. P. Giovannoni et al. / Bioorg. Med. Chem. 18 (2010) 7890–7899
7891
H
R3
R3
O
bA1 = 18 nM
bA2A> 10000 nM
bA3> 1000 nM
N N
N N
N
N N
a
O
R1
b
O
R1
N
N
O
R1
N
N
N
N
O
2a-g
CH3
O
N
O
CH3
CH=CHR9
3a-k
A
1a-f
c
R3
N N
NH2
hA1 = 1111 nM
hA2A> 0.048 nM
hA2B> 10000 nM
bA3> 10000 nM
R3
N N
N
N
N
N
d
O
R1
O
R1
CH2Ph
N N
O
f
CH3O
N
H2N
N
B
N
HN
R9
O
CH3
N
H
N
R9
4a-k
N
5a-k
e
g
N N
O
N
Ph
R3
N N
7
hA1 = 1100 nM
hA2 = 800 nM
hA2B = 20 nM
bA3 = 300 nM
NH
CH2Ph
N N
for 6p
N
N
N
O
R1
S
N
N
O
N
N
N
N
N
C
R6
N
6a-u
R9
N
H3C
N
Ph
8
Scheme 1. Synthesis of pyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones
5a–k, 6a–u, 7 and 8. Reagents: (a) R3X, DMF, K2CO3; (b) R9CHO, CH3ONa, CH3OH;
(c) NH2NH2, C2H5OH; (d) CH(OC2H5)3, H2SO4, DMF; (e) (R6CO)2O; (f) CH3COOH,
NaNO2; (g) Lawesson’s reagent, toluene.
O
R3
N N
N
N
HN
OCH3
O
CH3
N
D
N
E
N
R9
R6
N
bA1 = 0% (34 mM)
bA2A = 8.7% (34 mM)
hA3 = 2.1 nM
H
H
5a : R3 = CH2Ph, R6 = H, R9 = Ph
N N
a
N N
O
R1
O
R1
9, 10 R1
N
N
Figure 1. Adenosine receptors antagonists.
CH=CHPh
CH3
a
b
4-F-Ph
4-Pyridine
O
O
1c,e
9a,b
b
reported up till now is represented by the 1,2,4-triazolo[1,5-a]qui-
noxalin-4-one (compound D), synthesized by Catarzi and cowork-
ers36 which exhibits a Ki = 2.1 nM for human subtype A3, and a
complete selectivity for hA3 versus bA1 selectivity showing 0% inhi-
H
H
N N
N N
e
O
N
O
R1
for 10b
H2N
N
HN
bition of binding at 34 lM concentration at the letter subtype.
N
Ph
N
H3C
N
Ph
Taking into account the above reported examples and by con-
10a,b
13
sidering the classic structural requirements proposed by Jacobson
c
for 10a
f
et al.13 for adenosine receptor antagonists (planar, aromatic or
p
H
H
electron rich and nitrogen-containing heterocycles) we planned
to test some of the pyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-
4(3H)-ones derivatives of E (Fig. 1) as adenosine receptors ligands.
These compounds, synthesized in recent years in our laboratory,
N N
N N
S
N
O
F
N
N
N
H3C
N
Ph
14
N
H
are potent and selective phosphodiesterase
5 (PDE5) inhibi-
N
Ph
tors,37,38 but formally also comply with the structural require-
ments reported for the binding to the adenosine receptors.13 Our
results have been encouraging, with some substituents showing
submicromolar levels of affinity for A1 subtype. In particular
3-benzyl-1-methyl-9-phenyl-pyrazolo[10,50:1,6]pyrimido[4,5-d]-
pyridazin-4(3H)-one 5a38 was selected as the lead compound in
the design synthesis of adenosine receptor ligands. Preliminarly
we modified position 1, 6 and 9 of the scaffold E maintaining
unchanged the benzyl group at position 3.
11
g
d
N N
N N
PhH2CS
N
Cl
F
N
N
N
N
H3C
N
Ph
H
N
Ph
12
15
Scheme 2. Synthesis of pyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones 12
and 15. Reagents: (a) PhCHO, CH3ONa, CH3OH; (b) NH2NH2, C2H5OH; (c)
CH(OC2H5)3, H2SO4, DMF; (d) POCl3; (e) (CH3CO)2O; (f) Lawesson’s reagent, toluene;
(g) benzylchloride, DMF, K2CO3.
2. Chemistry
All the final compounds were synthesized as reported in
Schemes 1–3, following a common three-step synthetic route pre-
viously described.37,38 Thus isoxazolo[3,4-d]pyridazinones were
condensed with the available arylaldehydes to give the vinyl deriv-
atives which, in turn, were treated with hydrazine hydrate to fur-
nish 5-pyrazolylpyridazinones intermediates. Ring closure to
pyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones was car-
ried out in different conditions depending on the desired substitu-
ent at position 6.
In Scheme 1 the key intermediates 2a–g (2a,38 2c,39 2f40) were
obtained by alkylation of the 2-unsubstituted isoxazolo[3,
4-d]pyridazinones 1a–f with the appropriate halides, in standard
conditions, as reported in literature (1a,42 1b,41 1c,d,44 1e,39 1f43).
The 4-amino-5-pyrazolylderivatives 4a–k (compounds 4a,b and
their precursors 3a,b are reported in Ref. 38) were treated with
triethylorthoformate, catalytic amount of concentrated sulfuric

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M. P. Giovannoni et al. / Bioorg. Med. Chem. 18 (2010) 7890–7899
Table 2
H
H
N N
H H
N N
Key to substituents for compounds 2a–g
N N
a
O
Cl
Cl
O
Compd 2
R1
R3
+
O
O
a
b
c
d
e
f
CH3
CH3
Ph
4-F-Ph
3-F-Ph
4-Pyridyl
3-Pyridyl
CH2Ph
(CH)CH3Ph
CH2Ph
CH2Ph
CH2Ph
N
N
CH3
N
CH3
18
O
O
CH3
16
O
17
b
CH2Ph
N N
CH2Ph
CH2Ph
CH2Ph
CH2Ph
c
g
N N
Cl
N N
O
Cl
O
Cl
O
+
N
N
N
CH3
O
O
CH=CHPh
O
CH3
Table 3
Key to substituents for compounds 3–5a–k
21
20
19
d
Compd 3–5
R1
R3
R9
CH2Ph
CH2Ph
a
b
c
d
e
f
g
h
i
CH3
CH3
CH3
CH3
CH2Ph
CH2Ph
CH2Ph
(CH)CH3Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
Ph
3-Thienyl
N(CH3)2 (for 3) and H (for 4 and 5)
Ph
Ph
Ph
3-Thienyl
Ph
N N
Cl
N N
Cl
O
O
e
NH2
N
Ph
HN
N
Ph
N
N Ph
CH3
4-F-Ph
4-F-Ph
3-F-Ph
3-F-Ph
4-Pyridyl
3-Pyridyl
3-F-Ph
22
23
Scheme 3. Synthesis of pyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones 23.
Reagents: (a) POCl3; (b) benzylchloride, DMF, K2CO3; (c) PhCHO, CH3ONa, CH3OH;
(d) NH2NH2, C2H5OH; (e) (CH3CO)2O.
3-Thienyl
j
k
h
Ph
Ph
Ph
acid in anhydrous DMF, affording the 6-unsubstituted compounds
5a–k (5a38). Definition of compounds 1–5 is reported in Tables 1–3.
When the ring closure of the intermediates of type 4 was per-
formed with the appropriate anhydride under refluxing conditions,
the corresponding 6-substituted tricyclic compounds 6a–u were
recovered (6a37). For compound 6p a further transformation was
performed using Lawesson’s reagent in toluene to afford the final
thioderivative 8. Compound 6 is defined in Table 4.
Table 4
Key to substituents for compounds 6a–u
Compd 6
R1
R3
R6
R9
a37
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
CH3
CH3
CH3
CH3
Ph
Ph
Ph
Ph
4-F-Ph
4-F-Ph
4-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
4-Pyridyl
4-Pyridyl
4-Pyridyl
4-Pyridyl
3-Pyridyl
3-Pyridyl
CH2Ph
CH2Ph
(CH)CH3Ph
(CH)CH3Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH3
CH3
CH3
C2H5
CH3
C2H5
iC3H7
nC3H7
CH3
C2H5
CF3
CH3
C2H5
CH3
C2H5
CH3
C2H5
nC3H7
iC3H7
CH3
Ph
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
3-Thienyl
3-Thienyl
Finally, the hexaaza-cyclopenta[a]naphthalen-6-one 7 was syn-
thesized by using a saturated solution of NaNO2 in glacial acetic
acid.
Scheme 2 reports the synthesis of the final compounds 12 and
15 which were obtained following a procedure similar to that de-
scribed in Scheme 1. After building the tricyclic system (com-
pounds 11 and 13), the final compounds 12 and 15 were
obtained respectively by treatment with POCl3 at reflux (com-
pound 12) and by transformation in the corresponding 4-thione
derivatives with Lawesson’s reagent (14) followed by selective
alkylation at position 4 (compound 15).
In order to insert a chlorine at position 1 (Scheme 3), we re-
peated the same three-step synthesis starting from the isoxazol-
o[3,4-d]pyridazin-4,7-dione 1645 which was treated with POCl3.
As expected, a mixture of the two isomers 17 and 18 was obtained;
the unresolved mixture was reacted with benzyl chloride in anhy-
drous DMF to give the corresponding isomers 19 and 20. After sep-
aration by column chromatography, compound 19 was submitted
to the above described synthetic route.
Ph
Ph
Ph
Ph
Ph
Ph
s
t
u
C2H5
3. Biological results and discussion
Elemental analyses of all new compounds are reported as
Supplementary data.
All final compounds were tested in radioligand binding assays
to determine their affinities for the human adenosine A1, A2A, A2B
and A3 receptors and all compounds which do not inhibit the bind-
ing by 50% or more at 10 lM were considered inactive.
Table 1
SAR analysis was performed by considering 3 series of com-
pounds which differ in the substituent at position 1 (methyl, (flu-
oro)phenyl and pyridine) and the biological results are reported in
Tables 5–7.
Starting the analyses from the sub-class of 1-methyl derivatives
(compounds 5a–d, 6a–d, 7 and 23 Table 5) we can observe that the
lead compound 5a38 showed Ki = 104.3 nM for A1, 73-fold selectiv-
ity versus A2B and no affinity for A2A and A3 (binding <50% at
Key to substituents for compounds
1a–f
Compd 1
R1
a
b
c
d
e
f
CH3
Ph
4-F-Ph
3-F-Ph
4-Pyridyl
3-Pyridyl
10 lM); the replacement of the phenyl at R9 of 5a with a 3-thienyl

M. P. Giovannoni et al. / Bioorg. Med. Chem. 18 (2010) 7890–7899
7893
Table 5
Binding activity at human A1, A2A, A2B and A3 adenosine receptors
CH2Ph
N N
R3
CH2Ph
N N
N N
O
O
Cl
O
CH3
CH3
R9
N
N
N
N
N
N N
H3C
R6
N
N
Ph
N
Ph
5a-d, 6a-d
23
7
a,b
a,c
a,d
a,e
Compd
R3
R6
R9
hA1
hA2A
hA2B
hA3
5a38
5b
5c
CH2Ph
CH2Ph
CH2Ph
(CH)CH3Ph
CH2Ph
CH2Ph
(CH)CH3Ph
(CH)CH3Ph
H
H
H
H
CH3
CH3
CH3
C2H5
Ph
3-Thienyl
H
Ph
Ph
H
Ph
Ph
104.3 4.7
85.3 2.5
<50%
475.2 127.1
3250 114
<50%
3080 1210
2385 225
<50%
< 50%
7365 805
<50%
<50%
<50%
<50%
<50%
<50%
<50%
7560 1640
4650 790
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
<50%
4730 130
<50%
<50%
5d
6a37
6b
6c
6d
7
23
DPCPX
1616 723
3150 660
<50%
<50%
3.2 0.2f
260 18f
51 1.5f
1300 125f
a
b
c
d
e
f
The binding activity is reported as Ki (nM) or percentage of inhibition at 10
Displacement of [3H]DPCPX binding in CHO-A1 cells membranes.
Displacement of specific [3H]ZM241385 in HeLa-A2A cells.
Displacement of [3H]DPCPX from HEK-293-A2B cells.
Displacement of [3H]NECA in HeLa-A3 cells.
lM; values are means DS of four separate assays, each performed in triplicate.
Values are reported as Ki (nM) SEM.
Table 6
Table 7
Binding activity at human A1 adenosine receptor
Binding activity at human A1 and A2A adenosine receptors
CH2Ph
N N
CH2Ph
N N
CH2Ph
N N
N N
N N
Cl
F
O
R1
PhH2CS
N
O
R1
S
N
N
N
N
N
N
N
N
N
N
N
R6
N
Ph
N
R9
H
R6
N
N
Ph
N
Ph
H3C
H3C
Ph
5j,k, 6p-u
15
8
5e-i, 6e-o
12
Compda
R1
R6
hA1
hA2A
b,c
b,d
Compda
R1
R6
R9
hA1
b,c
5j
4-Py
3-Py
4-Py
4-Py
4-Py
4-Py
3-Py
3-Py
H
H
CH3
C2H5
nC3H7
iC3H7
CH3
10.2 2.3
11.4 2.2
43.6 16.8
71.8 8.9
28.3 5.1
192.3 37.4
47.4 5.9
180.2 70.1
267.4 14.3
<50%
67.9 1.9
5e
5f
5g
5h
5i
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
12
DPCPX
Ph
H
H
H
H
Ph
Ph
3-Thienyl
Ph
3-Thienyl
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
3-Thienyl
3-Thienyl
1432 947
252.3 60.1
122.9 52.2
92.7 3.5
188.5 20.5
1595 115
2445 95
<50%
563.5 99.5
<50%
<50%
<50%
3245 115
<50%
5k
6p
6q
6r
6s
6t
6u
8
15
DPCPX
805.1 374.5
501.2 238.3
2490 290
772.5 216.5
5190 2060
2115 1055
<50%
4-F-Ph
4-F-Ph
3-F-Ph
3-F-Ph
Ph
Ph
Ph
Ph
4-F-Ph
4-F-Ph
4-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
3-F-Ph
H
CH3
C2H5
iC3H7
nC3H7
CH3
C2H5
CF3
CH3
C2H5
CH3
C2H5
C2H5
572.1 20.4
<50%
3.2 0.2d
260 18d
a
None of the compounds is able to inhibit the binding by 50% or more at 10
for A2B and A3.
lM
b
The binding activity is reported as Ki (nM) or percentage of inhibition at 10 lM;
<50%
<50%
values are means DS of four separate assays, each performed in triplicate.
Displacement of [3H]DPCPX binding in CHO-A1 cells membranes.
c
Displacement of specific [3H]ZM241385 in HeLa-A2A cells.
d
<50%
3.2 0.2d
e
Values are reported as Ki (nM) SEM.
a
None of the compounds is able to inhibit the binding by 50% or more at 10
lM
for A2A, A2B and A3.
b
The binding activity is reported as Ki (nM) or percentage of inhibition at 10
lM;
respectively). Also for 5b no affinity for A3 was observed. Elimina-
tion of the phenyl group at R9 (compound 5c) led to a dramatic
reduction in affinity for all adenosine subtype receptors, this find-
ing indicating that at position 9 the phenyl group is the optimal
substituent with respect to both potency and selectivity. The intro-
duction of a methyl at position 6 (6a37) in the lead compound 5a
led decreased affinity for the A1 subtype, resulting in a loss of
values are means DS of four separate assays, each performed in triplicate.
c
Displacement of [3H]DPCPX binding in CHO-A1 cells membranes.
d
Values are reported as Ki (nM) SEM.
nucleus (5b) was associated with a small improvement in A1 affin-
ity (Ki = 85.3 nM) but also with a decrease of selectivity for A2A
and A2B subtype receptors (90- and 56-fold versus A2A and A2B
,

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M. P. Giovannoni et al. / Bioorg. Med. Chem. 18 (2010) 7890–7899
potency of one order of magnitude (Ki = 3250 nM), while in com-
pound 6a elimination of the phenyl at position 9 led to an inactive
compound (6b), according to the results above reported. The intro-
duction of a methyl group on the benzyl at position 3 (compound
5d, Ki = 475.2 nM) reduced the A1 affinity by about fourfold with
respect to the benzyl analogue 5a, but increased the selectivity
toward all other receptor subtypes. The introduction at position
6 of 5d of a methyl or an ethyl group (compounds 6c and 6d)
resulted in a loss of affinity of one order of magnitude for A1 and,
surprisingly, the appearance of affinity for the A3 subtype in
compound 6c (Ki = 3080 nM). Finally the isosteric replacement of
CH with N (compound 7) in the pyrimidine nucleus resulted in a
complete loss of A1 affinity with respect to the deaza analogue
5a. The same strategy applied to CH3/Cl replacement which led
to negative effects such as A1 affinity and A2A selectivity (com-
pound 23, A1 Ki = 1616 nM).
Since the pyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-
ones scaffold is an appropriate system also for PDE5 inhibitors, as
above mentioned,37,38 we selected and tested on this enzyme some
representative compounds. Compounds 6p, 6t and 6u belonging to
the 1-pyridyl series and displaying an affinity for A1 subtype in the
nanomolar range (Ki = 43.6–180.2 nM), showed a low or a very low
PDE5 inhibitory activity tested at 2 lM (6p: 5.7%; 6t: 29.7%; 6u:
32.8%). On the contrary, 1-methyl derivatives 5a and 6a, previously
published as PDE5 inhibitors37,38 are active, with different potency,
as A1 ligands and PDE5 inhibitors (5a: Ki(A1) = 104.3 nM and IC50(P-
DE5) = 180 nM; 6a: Ki(A1) = 3250 nM and IC50(PDE5) = 160 nM).
This fact suggests that the nature of the substituent at position 1
of the tricyclic system is important to address the activity to
PDE5 or A1 subtype receptor (data not shown).
4. Conclusions
The biological results of the 1-phenyl and 1-(fluoro)phenyl ser-
ies (compounds 5e–i, 6e–o and 12) are presented in Table 6. Only
data relating to the A1 subtype receptor are reported since none of
the compounds were able to inhibit the binding by 50% or more at
In conclusion, by taking all these data together, it is possible to
delineate the first SAR for pyrazolopyrimidopyridazinone. It
emerged that it is an appropriate system for adenosine receptor
subtype A1 ligands and that the optimal groups around this scaf-
fold are 3- and 4-pyridinyl at position 1, benzyl at position 3,
hydrogen at position 6 and 3-thienyl or phenyl at position 9. In this
preliminary study we identified ligands for hA1 adenosine subtype
receptor endowed with good selectivity versus other adenosine
subtypes. In the series of 1-pyridyl derivatives, compounds 5k
and 6t displayed the best balance between potency and selectivity
as well as compounds 5b and 5h in the series of 1-methyl and 1-
fluorophenyl derivatives, respectively. Further studies are in pro-
gress to improve our knowledge of the SAR.
10 lM for A2A, A2B and A3, thus showing a very good selectivity. In
the phenyl series the A1 affinity was evaluated as a function of the
nature of the alkyl group at position 6. The trend observed (n-
Pr > H and Me > Et > i-Pr) suggested that a linear carbon chain is
a strict requirement in this part of the molecule, 6h (R6 = n-Pr,
Ki = 563.5 nM) being the most active compound and 6g (R6 = i-Pr)
completely devoid of affinity.
In the 1-(4-fluorophenyl) subseries it emerged that keeping
benzyl at position 3 and phenyl at position 9, all 6-alkyl substituted
compounds (6i–k) are inactive (inhibition <50% at 10 lM).
Replacement of the alkyl chain at position 6 with a hydrogen cre-
ated compound 5f, whose Ki was 252.3 nM. The substitution of the
phenyl at position 9 of compound 5f with a 3-thienyl afforded
compound 5g which showed an improved affinity for A1 subtype
receptor (Ki = 122.9 nM), in agreement with the results obtained
for the 1-methyl subseries. Elimination of the benzyl chain and
aromatization of the pyridazine ring led to an inactive compound
(12). An analogue trend was observed for the series of 1-(3-fluoro-
phenyl) derivatives 6l–o and 5h,i, where only the 9-phenyl deriv-
ative 5h and the corresponding 9-(3-thienyl) derivative 5i
showed an appreciable affinity for A1 (Ki = 92.7 and 188.5 nM
respectively). All the other substitutions, with the exception of
the 6-methyl derivative 6l (Ki = 3245 nM) are not able to inhibit
5. Experimental section
5.1. Chemistry
All melting points were determined on a Büchi apparatus and
are uncorrected. 1H NMR spectra were recorded with Avance 400
instruments (Bruker Biospin Version 002 with SGU). Chemical
shifts are reported in ppm, using the solvent as internal standard.
Extracts were dried over Na2SO4 and the solvents were removed
under reduced pressure. Merck F-254 commercial plates were used
for analytical TLC to follow the course of reaction. Silica Gel 60
(Merck 70–230 mesh) was used for column chromatography.
Microanalyses were performed with a Perkin-Elmer 260 elemental
analyzer for C, H, N and the results were within 0.4% of the theo-
retical values, unless otherwise stated. Reagents and starting mate-
rials were commercially available.
the binding by 50% at 10 lM.
Biological data related to 1-pyridinyl derivatives 5j,k, 6p–u, 8
and 15 are shown in Table 7, where also the column of A2A affinity
values is also reported showing that this series has appreciable
binding for this adenosine subtype receptor. None of the com-
pounds is able to inhibit the binding by 50% or more at 10 lM
5.1.1. General procedure for 2b, 2d–e and 2g
for A2B and A3. The analysis of 1-(4-pyridinyl) derivatives demon-
strates that for this series the difference in affinity among the var-
ious 6-alkyl derivatives was significantly reduced, with the Me, Et
and n-Pr derivatives 6p–r showing good values for A1 in the same
nanomolar range (Ki = 28.3–71.8 nM). Analogously to the 1-phenyl
series, the 6-unsubstituted term 5j is the most potent compound
(Ki = 10.2 nM) and the 6-branched alkyl derivative 6s is the less ac-
tive (Ki = 192.3 nM). All these compounds displayed low A1 selec-
tivity, in particular 5j (A2A/A1 = 7). Moreover, the essential role
played by the carbonyl function at position 4 was evidenced by
observing the sixfold reduced affinity for A1 of the sulfur analogue
8. Further evidence of the key role played by the CO was suggested
by the behaviour of compound 15 in which the absence of CO was
associated with the shift of the benzyl group in a neighbouring po-
sition. Also 1-(3-pyridyl) derivatives (Table 7) were active in the
nanomolar range. In particular the 6-unsubstituted 5k displayed
a Ki = 11.4 nM for A1 and good A2A/A1 selectivity (71).
A mixture of isoxazolopyridazinone 1a, 1c–d and 1f (1a,42 1c,44
1d,44 1f43) (1.1 mmol), K2CO3 (2.2 mmol) and the appropriate
alkylhalide (1.7–4.3 mmol) in anhydrous DMF (3 mL) was stirred
at 70–90 °C for 0.5–3 h. After cooling, the mixture was diluted with
cold water and the precipitate recovered by suction. For compound
2b the reaction was carried out at 90 °C for 10 h: after dilution with
cold water the suspension was extracted with CH2Cl2 (3 ꢀ 15 mL)
and the solvent was evaporated in vacuo affording a residue oil
which was purified by column chromatography using cyclohex-
ane/ethyl acetate 1:2 as eluent.
5.1.1.1. 3,4-dimethyl-6-(1-phenylethyl)isoxazolo[3,4-d]pyrida-
zin-7(6H)-one, 2b. Yield = 72%; oil (purified by column chroma-
tography using cyclohexane/ethyl acetate 1:2 as eluent); 1H NMR
(CDCl3) d 1.80 (d, 3H, CHCH3), 2.45 (s, 3H, 4-CCH3), 2.80 (s, 3H, 3-
CCH3), 6.40 (q, 1H, CHCH3), 7.10–7.60 (m, 5H, Ar).

M. P. Giovannoni et al. / Bioorg. Med. Chem. 18 (2010) 7890–7899
7895
5.1.1.2. 6-Benzyl-4-(4-fluorophenyl)-3-methylisoxazolo[3,4-d]
pyridazin-7(6H)-one, 2d. Yield = 85%; mp = 145–147 °C (EtOH);
1H NMR (CDCl3) d 2.45 (s, 3H, CH3), 5.30 (s, 2H, CH2), 7.15–7.60 (m,
9H, Ar).
5.1.2.8. 6-Benzyl-4-pyridin-3-yl-3-styrylisoxazolo[3,4-d]pyrida-
zin-7(6H)-one, 3k. Yield = 53%; mp = 166–169 °C (EtOH); 1H
NMR (CDCl3) d 5.40 (s, 2H, CH2), 6.80 (d, 1H, CH@), 7.30–7.60 (m,
11H, Ar), 7.65 (d, 1H, @CH), 8.00 (m, 1H, Ar), 8.80–8.95 (m, 2H, Ar).
5.1.3. General procedure for 4c–k
5.1.1.3. 6-Benzyl-4-(3-fluorophenyl)-3-methylisoxazolo[3,4-d]
pyridazin-7(6H)-one, 2e. Yield = 85%; mp = 138–140 °C (EtOH);
1H NMR (CDCl3) d 2.60 (s, 3H, CH3), 5.35 (s, 2H, CH2), 7.20–7.50 (m,
9H, Ar).
A suspension of compounds 3c–k (0.3 mmol) in ethanol (2–
3 mL) and hydrazine hydrate (4–9 mmol) was stirred at room tem-
perature for 0.5–7 h (compounds 4c, 4g and 4i for 2–5 h at 50–
90 °C). Then the mixture was concentrated in vacuo and cooled
for 6–12 h: the precipitate was recovered by suction. For com-
pound 4e, after concentration, the mixture was diluted with water
(5 mL) and, after cooling, the solid was isolated by filtration.
5.1.1.4. 6-Benzyl-3-methyl-4-pyridin-3-yl-isoxazolo[3,4-d]pyri-
dazin-7(6H)-one, 2g. Yield = 84%; mp = 142–145 °C (EtOH); 1H
NMR (CDCl3) d 2.60 (s, 3H, CH3), 5.40 (s, 2H, CH2), 7.20–7.40 (m,
6H, Ar), 7.95 (m, 1H, Ar), 8.80 (m, 2H, Ar).
5.1.3.1. 4-Amino-2-benzyl-6-methyl-5-(2H-pyrazol-3-yl)pyrida-
zin-3(2H)-one, 4c. Yield = 43%; mp = 130–132 °C (EtOH); 1H NMR
(CDCl3) d 2.30 (s, 3H, CH3), 5.40 (s, 2H, CH2), 6.30 (exch br s, 2H,
NH2), 6.50 (d, 1H, Ar), 7.25–7.45 (m, 5H, Ar), 7.60 (d, 1H, Ar),
8.70 (exch br s, 1H, NH).
5.1.1.5. 6-Benzyl-3-(2-dimethylaminovinyl)-4-methylisoxazolo
[3,4-d]pyridazin-7(6H)-one, 3c. A suspension of 2a38 (0.4 mmol) in
N,N-dimethylformammide dimethyl acetal (15 mmol) was stirred at
90 °C for 15 min. After cooling the precipitate was recovered by suction.
Yield = 82%; mp = 203–206 °C (MeOH); 1H NMR (CDCl3) d 1.90
(s, 3H, 4-CCH3), 3.00 (s, 6H, N(CH3)2, 5.15 (d, 1H, CH = ), 5.25 (s,
2H, CH2), 7.20–7.35 (m, 5H, Ar), 7.55 (d, 1H, @CH).
5.1.3.2. 4-Amino-6-methyl-2-(1-phenylethyl)-5-(5-phenyl-2H-
pyrazol-3-yl)pyridazin-3(2H)-one, 4d. Yield = 96%; mp = 197–
198 °C (EtOH); 1H NMR (CDCl3) d 1.80 (d, 3H, CH3CH), 2.40 (s,
3H, 6-CCH3), 6.20 (exch br s, 2H, NH2), 6.40 (q, 1H, CH3CH), 6.70
(s, 1H, Ar), 7.20–7.65 (m, 10H, Ar), 7.90 (exch br s, 1H, NH).
5.1.2. General procedure for 3d–k
To a suspension of compounds 2b–g (2c,39 2f40) (0.6 mmol) and
the appropriate arylaldehyde (1.2–3.6 mmol) in anhydrous metha-
nol (2 mL), CH3ONa (1.0–3.0 mmol) was added. The mixture was
refluxed under stirring for 1–40 min. After cooling, the precipitate
was recovered by suction.
5.1.3.3. 4-Amino-2-benzyl-6-phenyl-5-(5-phenyl-2H-pyrazol-3-
yl)pyridazin-3(2H)-one, 4e. Yield = 97%; mp = 118–119 °C
(EtOH); 1H NMR (CDCl3) d 5.40 (s, 2H, CH2), 6.15 (exch br s, 2H,
NH2), 7.25–7.65 (m, 16H, Ar), 8.85 (exch br s, 1H, NH).
5.1.2.1. 4-Methyl-6-(1-phenylethyl)-3-styrylisoxazolo[3,4-d]
pyridazin-7(6H)-one, 3d. Yield = 49%; mp = 189–191 °C (EtOH);
1H NMR (CDCl3) d 1.80 (d, 3H, CH3CH), 2.60 (s, 3H, CH3), 6.40 (q,
1H, CH3CH), 7.20 (d, 1H, CH@), 7.30–7.65 (m, 10H, Ar), 7.80 (d, 1H,
@CH).
5.1.3.4. 4-Amino-2-benzyl-6-(4-fluorophenyl)-5-(5-phenyl-2H-
pyrazol-3-yl)-pyridazin-3(2H)-one, 4f. Yield = 53%; mp = 137–
143 °C (EtOH); 1H NMR (CDCl3) d 5.40 (s, 2H, CH2), 5.75 (s, 1H,
Ar), 6.80 (exch br s, 2H, NH2), 7.00–7.20 (m, 2H, Ar), 7.25–7.60
(m, 12H, Ar), 8.25 (exch br s, 1H, NH).
5.1.2.2. 6-Benzyl-4-phenyl-3-styrylisoxazolo[3,4-d]pyrida-
zin-7(6H)-one, 3e. Yield = 78%; mp = 241–242 °C (EtOH); 1H
NMR (CDCl3) d 5.40 (s, 2H, CH2), 6.80 (d, 1H, CH@), 7.20–7.70 (m,
16H: 10H, Ar; 1H, @CH).
5.1.3.5. 4-Amino-2-benzyl-6-(4-fluorophenyl)-5-(5-thiophen-3-
yl-2H-pyrazol-3-yl)-pyridazin-3(2H)-one, 4g. Yield = 58%; mp =
225–227 °C (EtOH); 1H NMR (CDCl3) d 5.40 (s, 2H, CH2), 5.60 (s, 1H,
Ar), 6.70 (exch br s, 2H, NH2), 7.00–7.15 (m, 3H, Ar), 7.20–7.50 (m,
9H, Ar), 8.80 (exch br s, 1H, NH).
5.1.2.3. 6-Benzyl-4-(4-fluorophenyl)-3-styrylisoxazolo[3,4-d]
pyridazin-7(6H)-one, 3f. Yield = 91%; mp = 197–199 °C (EtOH);
1H NMR (CDCl3) d 5.40 (s, 2H, CH2), 6.80 (d, 1H, CH@), 7.20–7.60
(m, 14H, Ar), 7.70 (d, 1H, @CH).
5.1.3.6. 4-Amino-2-benzyl-6-(3-fluorophenyl)-5-(5-phenyl-2H-
pyrazol-3-yl)pyridazin-3(2H)-one, 4h. Yield = 30%; mp = 167–
169 °C dec. (EtOH); 1H NMR (CDCl3) d 5.35 (s, 2H, CH2), 5.95 (s,
1H, Ar), 6.90 (exch br s, 2H, NH2), 7.00–7.65 (m, 14H, Ar), 9.20
(exch br s, 1H, NH).
5.1.2.4. 6-Benzyl-4-(4-fluorophenyl)-3-(2-thiophen-3-yl-vinyl)
isoxazolo[3,4-d]pyridazin-7(6H)-one, 3g. Yield = 85%; mp = 203–
204 °C (EtOH); 1H NMR (CDCl3) d 5.40 (s, 2H, CH2), 6.60 (d, 1H,
CH@), 7.00 (d, 1H, Ar), 7.15–7.60 (m, 11H, Ar), 7.65 (d, 1H, @CH).
5.1.3.7. 4-Amino-2-benzyl-6-(3-fluorophenyl)-5-(5-thiophen-3-
yl-2H-pyrazol-3-yl)pyridazin-3(2H)-one, 4i. Yield = 39%; mp =
219–222 °C (EtOH); 1H NMR (CDCl3) d 5.35 (s, 2H, CH2), 5.95 (s,
1H, Ar), 6.80–7.50 (m, 12H, Ar), 7.60 (exch br s, 2H, NH2), 9.10
(exch br s, 1H, NH).
5.1.2.5. 6-Benzyl-4-(3-fluorophenyl)-3-styrylisoxazolo[3,4-d]
pyridazin-7(6H)-one, 3h. Yield = 70%; mp = 142–145 °C (EtOH);
1H NMR (CDCl3) d 5.40 (s, 2H, CH2), 6.80 (d, 1H, CH@), 7.20–7.60
(m, 14H, Ar), 7.80 (d, 1H, @CH).
5.1.3.8. 4-Amino-2-benzyl-5-(5-phenyl-2H-pyrazol-3-yl)-6-pyri-
din-4-yl-pyridazin-3(2H)-one, 4j. Yield = 67%; mp = 226–229 °C
(EtOH/H20 1/1); 1H NMR (CDCl3) d 5.45 (s, 2H, CH2), 5.95 (s, 1H,
Ar), 6.40 (exch br s, 2H, NH2), 7.20–7.50 (m, 10H, Ar), 7.70 (m,
2H, Ar), 8.60 (m, 2H, Ar), 9.25 (exch br s, 1H, NH).
5.1.2.6. 6-Benzyl-4-(3-fluorophenyl)-3-(2-thiophen-3-yl-vinyl)
isoxazolo[3,4-d]pyridazin-7(6H)-one, 3i. Yield = 83%; mp = 205–
207 °C (EtOH); 1H NMR (CDCl3) d 5.40 (s, 2H, CH2), 6.60 (d, 1H,
CH@), 7.00 (d, 1H, Ar), 7.25–7.60 (m, 11H, Ar), 7.65 (d, 1H, @CH).
5.1.3.9. 4-Amino-2-benzyl-5-(5-phenyl-2H-pyrazol-3-yl)-6-pyri-
din-3-yl-pyridazin-3(2H)-one, 4k. Yield = 45%; mp = 200–203 °C
(EtOH); 1H NMR (CDCl3) d 5.40 (s, 2H, CH2), 5.70 (s, 1H, Ar), 6.60
(exch br s, 2H, NH2), 7.20–7.50 (m, 11H, Ar), 7.80 (d, 1H, Ar),
7.95 (exch br s, 1H, NH), 8.60 (d, 1H, Ar), 8.70 (s, 1H, Ar).
5.1.2.7. 6-Benzyl-4-pyridin-4-yl-3-styrylisoxazolo[3,4-d]pyrida-
zin-7(6H)-one, 3j. Yield = 65%; mp = 187–189 °C (EtOH); 1H NMR
(CDCl3) d 5.40 (s, 2H, CH2), 6.80 (d, 1H, CH@), 7.30–7.60 (m, 10H,
Ar), 7.80 (d, 1H, @CH), 8.20 (d, 2H, Ar), 9.00 (d, 2H, Ar).

7896
M. P. Giovannoni et al. / Bioorg. Med. Chem. 18 (2010) 7890–7899
5.1.4. General procedure for 5b–k
5.1.4.10. 3-Benzyl-9-phenyl-1-pyridin-3-yl-pyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazin-4(3H)-one, 5k. Yield = 81%; mp
>300 °C dec. (Ether); 1H NMR (CDCl3) d 5.40 (s, 2H, CH2), 6.30
(s, 1H, Ar), 7.40–8.20 (m, 11H, Ar), 8.50 (m, 1H, Ar), 9.10 (m,
2H, Ar), 9.85 (s, 1H, Ar); MS (ESI) m/z 430.46 ([M+H]+). Anal.
(C26H18N6O) C, H, N.
A mixture of compounds 4b–k (4b38) (0.16 mmol), triethylor-
thoformate (12 mmol), anhydrous DMF (1–3 mL) and a catalytic
amount of concentrated sulfuric acid was stirred at room temper-
ature for 5–30 min. After cooling the precipitate was recovered by
suction. For compounds 5h and 5i, the mixture was diluted with
cold water (10 mL) and the solid was filtered off.
5.1.5. General procedure for 6b–d, i, j, l, m, t and u
A mixture of 4-amino-5-pyrazolyl derivatives 4c–d, 4f, 4h
and 4k (0.13 mmol), the appropriate anhydride (7–15 mmol)
and a catalytic amount of concentrated sulfuric acid was stirred
at room temperature for 15–30 min. The mixture was diluted
with cold water (10 mL) and the precipitate was recovered by
suction.
5.1.4.1. 3-Benzyl-1-methyl-9-thiophen-3-yl-pyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazin-4(3H)-one, 5b. Yield = 81%; mp =
288–290 °C (EtOH); 1H NMR (CDCl3) d 2.80 (s, 3H, CH3), 5.40 (s,
2H, CH2), 7.20–7.60 (m, 7H, Ar), 7.70 (m, 1H, Ar), 7.95 (d, 1H, Ar),
9.40 (s, 1H, Ar); MS (ESI) m/z 373.43 ([M+H]+). Anal. (C20H15N5OS)
C, H, N.
5.1.5.1. 3-Benzyl-1,6-dimethylpyrazolo[10,50:1,6]pyrimido[4,5-d]
pyridazin-4(3H)-one, 6b. Yield = 47%; mp = 220–222 °C (EtOH);
1H NMR (CDCl3) d 2.70 (s, 3H, 1-CCH3), 3.00 (s, 3H, 6-CCH3), 5.35
(s, 2H, CH2), 7.30 (m, 1H, Ar), 7.35 (m, 4H, Ar), 8.45 (d, 1H, Ar),
8.50 (d, 1H, Ar). Anal. (C17H15N5O) C, H, N.
5.1.4.2. 3-Benzyl-1-methylpyrazolo[10,50:1,6]pyrimido[4,5-d]
pyridazin-4(3H)-one, 5c. Yield = 68%; mp = 160–163 °C (EtOH);
1H NMR (CDCl3) d 2.80 (s, 3H, CH3), 5.50 (s, 2H, CH2), 7.10 (s, 1H,
Ar), 7.20–7.40 (m, 4H, Ar), 8.20 (m, 2H, Ar), 9.40 (s, 1H, Ar). Anal.
(C16H13N5O) C, H, N.
5.1.5.2. 1,6-Dimethyl-9-phenyl-3-(1-phenylethyl)pyrazolo
[10,50:1,6]pyrimido-[4,5-d]pyridazin-4(3H)-one, 6c. Yield = 75%;
mp = 230–232 °C (EtOH); 1H NMR (CDCl3) d 1.80 (d, 3H, CH3CH),
2.80 (s, 3H, 1-CCH3), 3.20 (s, 3H, 6-CCH3), 6.60 (q, 1H, CH3CH),
7.20–7.60 (m, 9H, Ar), 8.10 (m, 2H, Ar). Anal. (C24H21N5O) C, H, N.
5.1.4.3. 1-Methyl-9-phenyl-3-(1-phenylethyl)pyrazolo[10,50:1,6]
pyrimido-[4,5-d]pyridazin-4(3H)-one, 5d. Yield = 97%; mp =
247–250 °C dec. (EtOH); 1H NMR (CDCl3) d 1.90 (d, 3H, CH3CH),
2.80 (s, 3H, CH3), 6.60 (q, 1H, CH3CH), 7.20–7.60 (m, 9H, Ar), 8.00
(m, 2H, Ar), 9.40 (s, 1H, Ar); MS (ESI) m/z 381.43 ([M+H]+). Anal.
(C23H19N5O) C, H, N.
5.1.5.3. 6-Ethyl-9-phenyl-3-(1-phenylethyl)-1-methylpyrazolo
[10,50:1,6]pyrimido-[4,5-d]pyridazin-4(3H)-one, 6d. Yield = 73%;
mp = 149–152 °C (EtOH); 1H NMR (CDCl3) d 1.60 (t, 3H, CH3CH2),
1.80 (d, 3H, CH3CH), 2.80 (s, 3H, 1-CCH3), 3.60 (q, 2H, CH2CH3),
6.60 (q, 1H, CH3CH), 7.20–7.60 (m, 9H, Ar), 8.10 (m, 2H, Ar). Anal.
(C25H23N5O) C, H, N.
5.1.4.4. 3-Benzyl-1,9-diphenylpyrazolo[10,50:1,6]pyrimido[4,5-d]
pyridazin-4(3H)-one, 5e. Yield = 72%; mp = 230–232 °C (EtOH);
1H NMR (CDCl3) d 5.50 (s, 2H, CH2), 6.20 (s, 1H, Ar), 7.25–7.80
(m, 15H, Ar), 9.45 (s, 1H, Ar); MS (ESI) m/z 429.47 ([M+H]+). Anal.
(C27H19N5O) C, H, N.
5.1.5.4. 3-Benzyl-1-(4-fluorophenyl)-6-methyl-9-phenylpyrazol-
o[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-one, 6i. Yield = 71%;
mp = 263–264 °C (Ether); 1H NMR (CDCl3) d 3.20 (s, 3H, CH3),
5.50 (s, 2H, CH2), 6.10 (s, 1H, Ar), 7.25–7.60 (m, 12H, Ar), 7.85
(m, 2H, Ar). Anal. (C28H20FN5O) C, H, N.
5.1.4.5. 3-Benzyl-1-(4-fluorophenyl)-9-phenylpyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazin-4(3H)-one, 5f. Yield = 82%; mp = 227–
229 °C (EtOH); 1H NMR (CDCl3) d 5.50 (s, 2H, CH2), 6.20 (s, 1H, Ar),
7.25–7.60 (m, 12H, Ar), 7.80 (m, 2H, Ar), 9.40 (s, 1H, Ar); MS (ESI)
m/z 447.46 ([M+H]+). Anal. (C27H18FN5O) C, H, N.
5.1.5.5. 3-Benzyl-6-ethyl-1-(4-fluorophenyl)-9-phenylpyrazol-
o[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-one, 6j. Yield = 80%;
mp = 267–268 °C (Ether); 1H NMR (CDCl3) d 1.60 (t, 3H, CH2CH3),
3.55 (q, 2H, CH2CH3), 5.50 (s, 2H, CH2), 6.20 (s, 1H, Ar), 7.20–7.60
(m, 12H, Ar), 7.80 (m, 2H, Ar). Anal. (C29H22FN5O) C, H, N.
5.1.4.6. 3-Benzyl-1-(4-fluorophenyl)-9-thiophen-3-yl-pyrazol-
o[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-one, 5g. Yield = 78%;
mp = 218–222 °C dec. (EtOH); 1H NMR (CDCl3) d 5.50 (s, 2H, CH2),
6.00 (s, 1H, Ar), 7.20–7.70 (m, 11H, Ar), 8.20 (s, 1H, Ar), 9.40 (s, 1H,
Ar); MS (ESI) m/z 453.49 ([M+H]+). Anal. (C25H16FN5OS) C, H, N.
5.1.4.7. 3-Benzyl-1-(3-fluorophenyl)-9-phenylpyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazin-4(3H)-one, 5h. Yield = 49%; mp =
202–204 °C dec. (Ether); 1H NMR (CDCl3) d 5.50 (s, 2H, CH2), 6.10
(s, 1H, Ar), 7.20–7.60 (m, 12H, Ar), 7.80 (m, 2H, Ar), 9.40 (s, 1H,
Ar); MS (ESI) m/z 447.46 ([M+H]+). Anal. (C27H18FN5O) C, H, N.
5.1.5.6. 3-Benzyl-1-(3-fluorophenyl)-6-methyl-9-phenylpyrazol-
o[10,50:1,6]pyrimido[4,5d]pyridazin-4(3H)-one, 6l. Yield = 68%;
mp = 245–247 °C (Ether); 1H NMR (CDCl3) d 3.20 (s, 3H, CH3),
5.50 (s, 2H, CH2), 6.15 (s, 1H, Ar), 7.25–7.60 (m, 12H, Ar), 7.70
(m, 2H, Ar); MS (ESI) m/z 461.49 ([M+H]+). Anal. (C28H20FN5O) C,
H, N.
5.1.4.8. 3-Benzyl-1-(3-fluorophenyl)-9-thiophen-3-yl-pyrazolo
[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-one, 5i. Yield = 78%;
mp = 206–208 °C dec. (EtOH); 1H NMR (CDCl3) d 5.50 (s, 2H,
CH2), 6.00 (s, 1H, Ar), 7.20–7.75 (m, 12H, Ar), 9.40 (s, 1H, Ar); MS
(ESI) m/z 453.49 ([M+H]+). Anal. (C25H16FN5OS) C, H, N.
5.1.5.7. 3-Benzyl-6-ethyl-1-(3-fluorophenyl)-9-phenylpyrazolo
[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-one, 6m. Yield = 77%;
mp = 254–256 °C (Ether); 1H NMR (CDCl3) d 1.60 (t, 3H, CH2CH3),
3.60 (q, 2H, CH2CH3), 5.40 (s, 2H, CH2), 6.10 (s, 1H, Ar), 7.20–7.40
(m, 10H, Ar), 7.60 (m, 2H, Ar), 7.80 (m, 2H, Ar). Anal. (C29H22FN5O)
C, H, N.
5.1.4.9. 3-Benzyl-9-phenyl-1-pyridin-4-yl-pyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazin-4(3H)-one, 5j. Yield = 82%; mp =
>300 °C (EtOH); 1H NMR (CDCl3) d 5.50 (s, 2H, CH2), 6.20 (s,
1H, Ar), 7.40–7.60 (m, 8H, Ar), 7.70 (m, 2H, Ar), 7.80 (m, 2H,
Ar), 8.80 (d, 2H, Ar), 9.50 (s, 1H, Ar); MS (ESI) m/z 430.46
([M+H]+). Anal. (C26H18N6O) C, H, N.
5.1.5.8. 3-Benzyl-6-methyl-9-phenyl-1-pyridin-3-yl-pyrazolo
[10,50:1,6]pyrimido-[4,5-d]pyridazin-4(3H)-one, 6t. Yield = 81%;
mp = 284–286 °C (EtOH); 1H NMR (CDCl3) d 3.20 (s, 3H, CH3),
5.50 (s, 2H, CH2), 6.20 (s, 1H, Ar), 7.30–7.80 (m, 11H, Ar), 8.00 (d,

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7897
1H, Ar), 8.90 (m, 2H, Ar); MS (ESI) m/z 444.49 ([M+H]+). Anal.
(C27H20N6O) C, H, N.
Ar), 8.40 (d, 2H, Ar), 9.05 (d, 2H, Ar); MS (ESI) m/z 444.49
([M+H]+). Anal. (C27H20N6O) C, H, N.
5.1.5.9. 3-Benzyl-6-ethyl-9-phenyl-1-pyridin-3-yl-pyrazolo
[10,50:1,6]pyrimido-[4,5-d]pyridazin-4(3H)-one, 6u. Yield = 91%;
mp = 288–289 °C (EtOH); 1H NMR (CDCl3) d 1.60 (t, 3H, CH3CH2),
3.60 (q, 2H, CH3CH2), 5.55 (s, 2H, CH2), 6.10 (s, 1H, Ar), 7.20–7.80
(m, 11H, Ar), 8.05 (d, 1H, Ar), 9.00 (m, 2H, Ar); MS (ESI) m/z
458.51 ([M+H]+). Anal. (C28H22N6O) C, H, N.
5.1.6.8. 3-Benzyl-6-ethyl-9-phenyl-1-pyridin-4-yl-pyrazolo
[10,50:1,6]pyrimido-[4,5-d]pyridazin-4(3H)-one, 6q. Yield = 38%;
mp = 269–271 °C (EtOH); 1H NMR (CDCl3) d 1.60 (t, 3H, CH2CH3),
3.60 (q, 2H, CH2CH3), 5.50 (s, 2H, CH2), 6.20 (s, 1H, Ar), 7.30–7.80
(m,12H, Ar), 8.90 (d, 2H, Ar); MS (ESI) m/z 458.51 ([M+H]+). Anal.
(C28H22N6O) C, H, N.
5.1.5.10. 3-Benzyl-1-(4-fluorophenyl)-9-phenyl-6-trifluoro
methylpyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-one,
6k. A solution of 4j (0.12 mmol) in toluene (2.5 mL) and trifluoro-
acetic anhydride (1 mmol) was stirred at 60 °C for 16 h. After cooling
the precipitate was recovered by suction.
5.1.6.9. 3-Benzyl-9-phenyl-6-n-propyl-1-pyridin-4-yl-pyrazolo
[10,50:1,6]pyrimido-[4,5-d]pyridazin-4(3H)-one, 6r. Yield = 30%;
mp = 194–197 °C dec. (EtOH); 1H NMR (CDCl3) d 1.10 (t, 3H,
CH2CH2CH3), 2.10 (m, 2H, CH2CH2CH3), 3.50 (t, 2H, CH2CH2CH3),
5.50 (s, 2H, CH2), 6.20 (s, 1H, Ar), 7.30–7.50 (m,10H, Ar), 7.80 (d,
2H, Ar), 8.95 (d, 2H, Ar); MS (ESI) m/z 472.54 ([M+H]+). Anal.
(C29H24N6O) C, H, N.
Yield = 68%; mp = >300 °C (EtOH); 1H NMR (CDCl3) d 5.50 (s, 2H,
CH2), 6.25 (s, 1H, Ar), 7.20–7.60 (m, 12H, Ar), 7.80 (m, 2H, Ar). Anal.
(C28H17F4N5O) C, H, N.
5.1.6.10. 3-Benzyl-6-isopropyl-9-phenyl-1-pyridin-4-yl-pyrazolo
[10,50:1,6]pyrimido-[4,5-d]pyridazin-4(3H)-one, 6s. Yield = 36%;
mp = 263–265 °C dec. (EtOH); 1H NMR (CDCl3) d 1.60 (d, 6H,
(CH3)2CH), 4.20 (m, 1H, (CH3)2CH), 5.55 (s, 2H, CH2), 6.20 (s, 1H,
Ar), 7.20–7.60 (m,10H, Ar), 7.80 (d, 2H, Ar), 8.90 (d, 2H, Ar); MS
(ESI) m/z 472.54 ([M+H]+). Anal. (C29H24N6O) C, H, N.
5.1.6. General procedure for 6e–h, 6n–s
A suspension of compounds 4e, 4i and 4j (0.2–0.3 mmol) and
the suitable anhydride (9–15 mmol) was refluxed under stirring
for 5–60 min. Then the mixture was cooled and the solid was
recovered by suction.
5.1.6.1. 3-Benzyl-1,9-diphenyl-6-methylpyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazin-4(3H)-one, 6e. Yield = 42%; mp =
265–266 °C (EtOH); 1H NMR (CDCl3) d 3.20 (s, 3H, CH3), 5.55 (s,
2H, CH2), 6.00 (s, 1H, Ar), 7.30–7.80 (m, 15H, Ar); MS (ESI) m/z
443.50 ([M+H]+). Anal. (C28H21N5O) C, H, N.
5.1.7. 3-Benzyl-1-methyl-9-phenylpyrazolo[1,5-c]pyridazino
[4,5-e][1,2,3]triazin-4(3H)-one, 7
To a solution of 4a38 in glacial acetic acid (4 mL) a saturated
aqueous solution of NaNO2 (1.5 mL) was added dropwise under
stirring. After the mixture was cooled and the solid was recovered
by suction.
5.1.6.2. 3-Benzyl-1,9-diphenyl-6-ethylpyrazolo[10,50:1,6]pyrimi-
do[4,5-d]pyridazin-4(3H)-one, 6f. Yield = 52%; mp = 256–257 °C
(EtOH); 1H NMR (CDCl3) d 1.60 (t, 3H, CH2CH3), 3.60 (q, 2H,
CH2CH3), 5.50 (s, 2H, CH2), 6.00 (s, 1H, Ar), 7.25–7.80 (m, 15H,
Ar); MS (ESI) m/z 457.53 ([M+H]+). Anal. (C29H23N5O) C, H, N.
Yield = 97%; mp = 253–255 °C dec. (EtOH); 1H NMR (CDCl3) d
2.80 (s, 3H, CH3), 5.40 (s, 2H, CH2), 6.10 (s, 1H, Ar), 7.20–7.60 (m,
7H, Ar), 8.10 (s, 1H, Ar), 8.30 (m, 2H, Ar). Anal. (C21H16N6O) C, H, N.
5.1.8. 3-Benzyl-6-methyl-9-phenyl-1-pyridin-4-yl-
pyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-thione, 8
5.1.6.3.
3-Benzyl-1,9-diphenyl-6-isopropylpyrazolo[10,50:1,6]
A
mixture of 6p (0.32 mmol) and Lawesson0s reagent
pyrimido[4,5-d]pyridazin-4(3H)-one, 6g. Yield = 59%; mp =
233–235 °C (EtOH); 1H NMR (CDCl3) d 1.60 (d, 6H, (CH3)2CH),
4.25 (m, 1H, (CH3)2CH), 5.50 (s, 2H, CH2), 6.05 (s, 1H, Ar), 7.20–
7.80 (m,15H, Ar). Anal. (C30H25N5O) C, H, N.
(0.74 mmol) in toluene (10 mmol) was heated at 140 °C for 4 h.
After cooling, toluene was evaporated in vacuo affording com-
pound 8 which was purified by column chromatography using
cyclohexane/ethyl acetate 1:2 as eluent.
Yield = 95%; mp = 254–258 °C dec.(EtOH); 1H NMR (CDCl3) d
3.20 (s, 3H, CH3), 6.15 (s, 2H, CH2), 6.40 (s, 1H, Ar), 7.30–7.60 (m,
9H, Ar), 7.80 (m, 2H, Ar), 8.10 (m, 1H, Ar), 9.00 (d, 2H, Ar); MS
(ESI) m/z 460.55 ([M+H]+). Anal. (C27H20N6S) C, H, N.
5.1.6.4. 3-Benzyl-1,9-diphenyl-6-n-propylpyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazin-4(3H)-one, 6h. Yield = 45%; mp =
181–183 °C (EtOH); 1H NMR (CDCl3) d 1.15 (t, 3H, CH2CH2CH3),
2.20 (m, 2H, CH2CH2CH3), 3.50 (t, 2H, CH2CH2CH3), 5.50 (s, 2H,
CH2), 6.00 (s, 1H, Ar), 7.20–7.80 (m,15H, Ar); MS (ESI) m/z
471.55 ([M+H]+). Anal. (C30H25N5O) C, H, N.
5.1.9. General procedure for 9a,b
Compounds 9a,b were obtained from 1c,e (1c,44 1e39) following
the general procedure described for compounds 3d–k.
5.1.6.5. 3-Benzyl-1-(3-fluorophenyl)-6-methyl-9-thiophen-3-yl-
pyrazolo[10,50:1,6]pyrimido-[4,5-d]pyridazin-4(3H)-one,
6n. Yield = 63%; mp = 253–255 °C (EtOH); 1H NMR (CDCl3) d 3.00
(s, 3H, CH3), 5.40 (s, 2H, CH2), 5.95 (s, 1H, Ar), 7.20–7.75 (m, 11H,
Ar), 8.00 (s, 1H, Ar). Anal. (C26H18FN5OS) C, H, N.
5.1.9.1. 4-(4-Fluorophenyl)-3-styrylisoxazolo[3,4-d]pyridazin-
7(6H)-one, 9a. Yield = 92%; mp = 240–243 °C dec. (EtOH); 1H
NMR (DMSO) d 6.80 (d, 1H, CH@), 7.25–7.80 (m, 10H: 9H, Ar; 1H,
@CH), 12.15 (exch br s, 1H, NH).
5.1.6.6. 3-Benzyl-6-ethyl-1-(3-fluorophenyl)-9-thiophen-3-yl-
pyrazolo[10,50:1,6]pyrimido-[4,5-d]pyridazin-4(3H)-one,
60. Yield = 48%; mp = 246–247 °C (EtOH); 1H NMR (CDCl3) d 1.50 (t,
3H, CH2CH3), 3.55 (q, 2H, CH2CH3), 5.45 (s, 2H, CH2), 6.00 (s, 1H, Ar),
7.20–7.80 (m, 12H, Ar). Anal. (C27H20FN5OS) C, H, N.
5.1.9.2. 4-Pyridin-4-yl-3-styrylisoxazolo[3,4-d]pyridazin-7(6H)-
one, 9b. Yield = 93%; mp = 268–271 °C (EtOH); 1H NMR (DMSO)
d 6.90 (d, 1H, CH@), 7.35–7.80 (m, 8H: 7H, Ar; 1H, @CH), 8.85 (s,
2H, Ar), 12.95 (exch br s, 1H, NH).
5.1.10. General procedure for 10a,b
5.1.6.7. 3-Benzyl-6-methyl-9-phenyl-1-pyridin-4-yl-pyrazolo
[10,50:1,6]pyrimido-[4,5-d]pyridazin-4(3H)-one, 6p. Yield = 40%;
mp = >300 °C (MeOH); 1H NMR (CDCl3) d 3.40 (s, 3H, CH3), 5.50
(s, 2H, CH2), 6.40 (s, 1H, Ar), 7.40–7.60 (m, 8H, Ar), 7.85 (m, 2H,
Compounds 10a,b were obtained from 9a,b following the gen-
eral procedure described for compounds 4c–k. For compound
10b the mixture was stirred at 80 °C for 30 min.

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M. P. Giovannoni et al. / Bioorg. Med. Chem. 18 (2010) 7890–7899
5.1.10.1. 4-Amino-6-(4-fluorophenyl)-5-(5-phenyl-2H-pyrazol-
3-yl)-pyridazin-3(2H)-one, 10a. Yield = 60%; mp = 154–157 °C
dec. (EtOH); 1H NMR (CDCl3) d 5.80 (exch br s, 1H, NH), 5.95 (s,
1H, Ar), 6.80 (exch br s, 2H, NH2), 7.05–7.60 (m, 9H, Ar), 13.00
(exch br s, 1H, NH).
5.1.17. 6-Benzyl-4-chloro-3-styrylisoxazolo[3,4-d]pyridazin-
7(6H)-one, 21
Compound 21 was obtained from 19 following the procedure
described for compounds 3d–k. Yield = 56%; mp = 224–227 °C
(EtOH); 1H NMR (CDCl3) d 5.30 (s, 2H, CH2), 7.25–7.65 (m, 11H:
10H, Ar; 1H, CH@), 7.85 (d, 1H, @CH).
5.1.10.2. 4-Amino-5-(5-phenyl-2H-pyrazol-3-yl)-6-pyridin-4-yl-
pyridazin-3(2H)-one, 10b. Yield = 37%; mp = >300 °C (EtOH); 1H
NMR (CDCl3) d 5.95 (s, 1H, Ar), 6.50 (exch br s, 2H, NH2), 7.00
(m, 2H, Ar), 7.10–7.65 (m, 5H, Ar), 8.50 (m, 2H, Ar), 13.00 (exch
br s, 1H, NH), 13.50 (exch br s, 1H, NH).
5.1.18. 4-Amino-2-benzyl-6-chloro-5-(5-phenyl-2H-pyrazol-3-
yl)pyridazin-3(2H)-one, 22
Compound 22 was obtained from 21 following the general pro-
cedure described for compounds 4c–k. For compound 22, the reac-
tion was carried out at 60 °C for 5 h and, after cooling, the crude
precipitate was recovered by suction.
5.1.11. 1-(4-Fluorophenyl)-9-phenylpyrazolo[10,50:1,6]pyrimido
[4,5-d]pyridazin-4(3H)-one, 11
Compound 11 was obtained from 10a following the general
procedure described for compounds 5b–k. Yield = 78%;
mp = >300 °C (EtOH); 1H NMR (DMSO) d 5.90 (s, 1H, Ar), 5.95 (s,
1H, Ar), 7.40–7.60 (m, 9H, Ar), 9.80 (s, 1H, Ar), 13.00 (exch br s,
1H, NH).
Yield = 38%; mp = 200–202 °C (EtOH); 1H NMR (CDCl3) d 5.30 (s,
2H, CH2), 6.95 (s, 1H, Ar), 6.80 (exch br s, 2H, NH2), 7.20–7.70 (m,
10H, Ar), 13.00 (exch br s, 1H, NH).
5.1.19. 3-Benzyl-1-chloro-6-methyl-9-phenylpyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazin-4(3H)-one, 23
5.1.12. 4-Chloro-1-(4-fluorophenyl)-9-phenylpyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazine, 12
Compound 23 was obtained from 22 following the reported
general procedure of 6e–h and 6n–s. In this case the mixture
was stirred at 100 °C for 5 h. Yield = 62%; mp = 221–223 °C (EtOH);
1H NMR (CDCl3) d 3.15 (s, 3H, CH3), 5.40 (s, 2H, CH2), 7.20–7.60 (m,
8H, Ar), 7.85 (s, 1H, Ar), 8.10 (m, 2H, Ar). Anal. (C22H16ClN5O) C, H,
N.
A suspension of 11 (0.28 mmol) in POCl3 (1 mL, 6.5 mmol) was
stirred at 90 °C for 3 h. After cooling, the mixture was diluted with
cold water and neutralized with 6 N NaOH: the final compound 12
was recovered by suction. Yield = 40%; mp = >300 °C (EtOH); 1H
NMR (CDCl3) d 6.40 (s, 1H, Ar), 7.20–7.50 (m, 5H, Ar), 7.70–7.90
(m, 4H, Ar), 9.50 (s, 1H, Ar). Anal. (C20H11ClFN5) C, H, N.
5.2. Biological assays
5.1.13. 6-Methyl-9-phenyl-1-pyridin-4-yl-pyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazin-4(3H)-one, 13
Compound 13 was obtained from 10b following the general
procedure described for compounds 6b–d, 6i–j, 6l–m, 6t–u.
Yield = 65%; mp >300 °C (EtOH); 1H NMR (DMSO) d 3.05 (s, 3H,
6-CCH3), 6.35 (s, 1H, Ar), 7.40 (s, 3H, Ar), 7.80–8.00 (m, 4H, Ar),
9.00 (d, 2H, Ar), 13.50 (exch br s, 1H, NH).
5.2.1. Radioligand binding assays
Radioligand binding competition assays were performed
in vitro using A1, A2A, A2B and A3 human receptors expressed in
transfected CHO (hA1), HeLa (hA2A and hA3) and HEK-293 (hA2B
)
cells as previously described.46 Briefly:
5.2.2. Human A1 receptors
Adenosine A1 receptor competition binding experiments were
carried out in membranes from CHO-A1 cells (Euroscreen, Gosse-
lies, Belgium) labelled with 2 nM [3H]DPCPX. Non-specific binding
was determined in the presence of 10 lM (R)-PIA. The reaction
mixture was incubated at 25 °C for 60 min.
5.1.14. 6-Methyl-9-phenyl-1-pyridin-4-yl-pyrazolo[10,50:1,6]
pyrimido[4,5-d]pyridazin-4(3H)-thione, 14
Compound 14 was obtained from 13 following the procedure
described for compound 8: in this case the mixture was heated
at 140 °C for 24 h: after cooling the precipitate was recovered by
suction.
Yield = 89%; mp = 238–242 °C dec.(DMSO); 1H NMR (CDCl3) d
3.00 (s, 3H, CH3), 5.20 (exch br s, 1H, NH), 6.30 (s, 1H, Ar), 7.00
(m, 2H, Ar), 7.40–7.90 (m, 5H, Ar), 9.00 (m, 2H, Ar).
5.2.3. Human A2A receptors
Adenosine A2A receptor competition binding experiments were
carried out in membranes from HeLa-A2A cells labelled with 3 nM
[3H]ZM241385. Non-specific binding was determined in the pres-
ence of 50 lM NECA. The reaction mixture was incubated at
25 °C for 30 min.
5.1.15. 4-(Benzylthio)-6-methyl-9-phenyl-1-pyridin-4-
ylpyrazolo[10,50:1,6]pyrimido-[4,5-d]pyridazine, 15
Compound 15 was obtained following the procedure described
for compounds 2b, 2d–e and 2g, starting from compound 14.
Yield = 63%; mp = 260–263 °C dec. (EtOH); 1H NMR (CDCl3) d
3.20 (s, 3H, CH3), 4.75 (s, 2H, CH2), 6.40 (s, 1H, Ar), 7.20–7.80 (m,
12H, Ar), 8.95 (d, 2H, Ar). Anal. (C27H20N6S) C, H, N.
5.2.4. Human A2B receptors
Adenosine A2B receptor competition binding experiments were
carried out in membranes from HEK-293-A2B cells (Euroscreen,
Gosselies, Belgium) labelled with 35 nM [3H]DPCPX. Non-specific
binding was determined in the presence of 400 lM NECA. The
reaction mixture was incubated at 25 °C for 30 min.
5.1.16. 6-Benzyl-4-chloro-3-methylisoxazolo[3,4-d]pyridazin-
7(6H)-one, 19
5.2.5. Human A3 receptors
Compound 19 was obtained following the procedure described
for compounds 2b, 2d–e and 2g starting from isoxazolopyridazi-
nones 17 and 18 (the mixture of 17 and 18 intermediates was
not separated): after dilution with water the suspension was ex-
tracted with ethyl acetate (3 ꢀ 15 mL) and the solvent was evapo-
rated in vacuo to afford a crude precipitate which was purified by
column chromatography using cyclohexane/ethyl acetate 2:1.
Yield = 83%; mp = 158–160 °C (EtOH); 1H NMR (CDCl3) d 2.95 (s,
3H, CH3), 5.25 (s, 2H, CH2), 7.30–7.50 (m, 5H, Ar).
Adenosine A3 receptor competition binding experiments were
carried out in membranes from HeLa-A3 cells labelled with
30 nM [3H]NECA. Non-specific binding was determined in the pres-
ence of 100 lM (R)-PIA. The reaction mixture was incubated at
25 °C for 180 min.
After each incubation time, samples were filtered and measured
in
a microplate beta scintillation counter (Microbeta Trilux,
Perkin-Elmer, Madrid, Spain).

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17. Varani, K.; Gessi, S.; Dalpiaz, A.; Borea, P. A. Br. J. Pharmacol. 1997, 122, 386.
18. Varani, K.; Gessi, S.; Dionisotti, S.; Ongini, E.; Borea, P. Br. J. Pharmacol. 1998,
123, 1723.
5.2.6. Purification of phosphodiesterase isoenzymes
PDE5 was purified from human platelets as described by Grist-
wood et al.47 Briefly, the supernatant of the cell lysate from 109
platelets was chromatographed by a Mono-Q ion exchange column
attached to a Pharmacia FPLC system.
19. Müller, C. E. Drugs Future 2000, 25, 1043.
20. Harada, H.; Osamu Asano, O.; Hoshino, Y.; Yoshikawa, S.; Matsukura, M.;
Kabasawa, Y.; Kotake, Y.; Watanabe, N.; Kawata, T.; Inoue, T.; Horizoe, T.;
Yasuda, N.; Minami, H.; Nagata, K.; Murakami, M.; Nagaoca, J.; Kobayashi, S.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.09.043.
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