New pyrazolo[1′,5′:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones as potent and selective PDE5 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.02.090

A series of potent PDE5 inhibitors with high selectivity versus PDE6 isoenzymes was identified.

Full text of DOI:10.1016/j.bmcl.2005.02.090

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2381–2384
New pyrazolo[10,50:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones
as potent and selective PDE5 inhibitors
b
Joan Feixas, Maria Paola Giovannoni, Claudia Vergelli, Amadeu Gavalda,
a
a
b
`
Nicoletta Cesari,a Alessia Grazianoa and Vittorio Dal Piaza,*
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Firenze,Via U. Schiff 6, Sesto Fiorentino 50019, Firenze, Italy
bAlmirall Prodesfarma Research Center, Cardener 68–74, 08024 Barcelona, Spain
Received 6 October 2004; revised 21 February 2005; accepted 28 February 2005
Abstract—A series of potent PDE5 inhibitors with high selectivity versus PDE6 isoenzymes was identified.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
potent compounds with reduced secondary effects such
as Vardenafil 213 and Tadalafil 3.14 The latter was
launched (CialisTM) for treatment of MED at the begin-
ning of 2003 showing a longer half-life (>17 h) with re-
spect to Sildenafil and the absence of colour vision
disturbances and cardiovascular effects.15,16 Very prom-
ising results are coming from Bristol–Myers research
which has identified some substituted pyrazolopyridopy-
ridazines of type 4 as potent (IC50 = 0.03–0.3 nM) and
selective PDE5 inhibitors, orally bioavailable, poten-
tially useful in the treatment of ED.17
Phosphodiesterases 5 (PDE5) are enzymes responsible
for the hydrolysis of cyclic guanosine-30,50-phosphate
(cGMP) and are mainly distributed in kidney, spleen,
lung and smooth muscle tissue.1 PDE5 inhibitors, ele-
vating cGMP levels, represent very attractive agents
for the treatment of cardiovascular pathologies and
erectile dysfunction (ED).2,3 This latter is a common
problem in men over 40, and in the United States alone
more than 30 million men suffer from ED.4–6 Penile
smooth muscle relaxation is mainly mediated by nitric
oxide (NO) released from vascular endothelium and
parasympatic nerve ending. NO stimulates the soluble
guanilate cyclase to increase cGMP levels which, acting
as an intracellular second messenger, induces the relaxa-
tion of smooth muscle cells of corpus cavernosus.7
PDE5 inhibitors, stopping the process of hydrolysis of
cGMP, enhance these effects and related erectile
function.
Our studies in the field of PDE inhibitors18–21 led us to
design and develop a new series of pyrazolopyrimidopy-
ridazinones (compounds 10–42), structurally related to
compound 4, showing a good inhibitory activity on
PDE5 and a fair selectivity towards PDE6.
2. Chemistry
Compounds 10–42 were prepared by following the gen-
eral procedure reported in Scheme 1. Isoxazolo[3,4-d]-
pyridazin-7(6H)-one 6 was obtained by treatment with
hydrazine of isoxazole 5, following the synthetic proce-
dure reported in the literature.22 Intermediate 6 was con-
densed with the opportune arylaldehydes (or pivalic
aldehyde) to give the vinyl derivatives 7.23 Treatment
of compounds 7 with hydrazine in ethanol afforded, in
very good yields, the pyrazole intermediates 8 by opening
the isoxazole ring and at the same time closure to pyraz-
ole.24 Compounds 9 were obtained by treatment of 4-
amino-5-(1H-pyrazol-5-yl)pyridazin-3(2H)-ones 8 with
The first PDE5 inhibitor used for treating male erectile
dysfunction (MED) was Sildenafil (ViagraTM) 18–10 in
1998 (see Fig. 1). This drug, despite its potency, showed
many notable side effects such as nausea, headache,
cutaneous flushing and retinal effects (bluish haze, or in-
creased light sensitivity) due to inhibition of PDE6 iso-
form.11,12 The research in this field, aimed at
discovering more selective PDE5 inhibitors, led to very
*
Corresponding author. Tel.: +39 55 4573681; fax: +39 55 4573671;
e-mail: vittorio.dalpiaz@unifi.it
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.090

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J. Feixas et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2381–2384
C2H5O
The first term of this series was compound 10 (R1 = Ph,
C2H5O
R2 = Me, R3 = Bn), for which an appreciable activity
and selectivity was found (IC50 = 160 nM for PDE5
and 25% inhibition at 2 lM for PDE6). Keeping the
same tricyclic skeleton, structural modifications were
performed at R1, R2 and R3 and related results are sum-
marized in Table 1.
H
N
H
N
S
N
N CH3
S
N
N
C2H5
O
O
N
O
O
O
N
O
N
N
N
CH3
CH3
N
CH3
CH3
Sildenafil 1
Vardenafil 2
As regards substituents R1 at position 9 of the tricyclic
system, aryl, substituted phenyl as well as t-butyl groups
were inserted. Data showed a good activity associated
with the presence of a 2-pyridyl ring (compounds 18,
IC50 = 270 nM) comparable to 10. Introduction of an
amino group in para position of the phenyl (compound
20) reduced the activity (IC50 = 920 nM), but increased
the selectivity towards PDE6 isoenzymes (4.5% inhibi-
tion at 2 lM) with respect to the more active compounds
10 and 18. On the other hand, a complete loss of po-
tency was found for the 3,4-methylenedioxy and the 4-
nitrophenyl derivatives 16 and 19, indicating that steric
and electronic factors may limit the tolerance in this po-
sition. Finally, also the t-butyl derivative resulted com-
pletely inactive, suggesting that the aromatic system in
this part of the molecule could play a significant role
in determining PDE5 inhibitory activity.
CH3
O
O
N
H3CHNOC
N
N
N
N
N
N
NHCH2
N
OCH3
Cl
N
H
O
N
O
C2H5
4
Tadalafil 3
Figure 1.
H
N
H
N
N
N
O
O
CH3
CH3
O
CH3
EtOOC
CH
3 a
b
N
N
N
CH=CH R1
CH3
O
O
O
7
Modifications of substituent R2 at position 6 gave
interesting results, achieving for some compounds
IC50s in the 34–81 nM range. Starting from the analysis
of aromatic and aralkyl substituents, it seems that ex-
act requirements were necessary. In fact, when a pyr-
idyl system was inserted at 6, different results were
obtained depending on the linked position. The 2-pyr-
idyl derivative (compound 24) is the most interesting
compound, showing a very good balance of potency
and selectivity versus PDE5 (IC50 = 81 nM for PDE5
and 21.3% at 2 lM for PDE6). The potency, together
with the selectivity, decreased when a 3-pyridyl ring
was inserted (compound 25), but a complete loss of
activity was observed for the 4-pyridyl derivative (26),
suggesting that the nitrogen could be involved in a spe-
cific bond. On the other hand, activity and selectivity
were restored by inserting a methylenic spacer (com-
pound 22). The 6-phenyl derivative 21 showed an anal-
ogous potency (IC50 = 81 nM) to compound 24, but
lower selectivity. Introduction of an alkyl spacer be-
tween the phenyl ring and the tricyclic system led to
different results: the benzyl derivative 23 was found
completely devoid of activity, whereas the insertion of
a phenylethyl residue partially restored the inhibitory
activity (compound 27, IC50 = 980 nM). A good level
of potency and selectivity was found for compound
29, in which a m-tolyl was inserted; on the contrary,
the isomer o-tolyl led to an inactive product indicating
that steric factors could play an important role for
interaction with the biological target. Similarly, com-
pounds 30 and 31, in which a cyclohexylmethyl and a
cyclopentylmethyl residue respectively were inserted,
are completely inactive. Very interesting results were
obtained by the introduction of alkyl chains and func-
tionalized alkyl chains. For this type of substituent a
higher tolerance seems to be allowed since the IC50s
of all tested compounds are in the 430–434 nM range.
6
5
c
H
N
R3
N
H
N N
N
N
N
O
CH3
O
CH3
O
CH3
f
d or e
H2N
N
N
N
HN
R1
N
R2
N
R2
N
R1
R1
8
9
10-42
Scheme 1. Reagents and conditions: (a) hydrazine hydrate, EtOH, rt;
(b) R1CHO, MeONa, abs MeOH, reflux; (c) hydrazine hydrate, EtOH,
rt; (d) (R2CO)2O, reflux; (e) R2COOH, dichloromethane, anhydrous
DMF, DMAP, EDC, reflux; (f) R3X, NaH, DMSO, rt.
the opportune anhydride under refluxing conditions
without solvent. When acids were used, the condensation
was performed at room temperature using 1-[3-(dimeth-
ylamino)propyl]-3-ethylcarbodiimide hydrochloride as
coupling agent and the resulting amides were then ther-
mally cyclized. Finally, alkylation of the pyrazolopyri-
midopyridazinones 9 with a variety of halides in
standard conditions afforded the final compounds 10–42.
3. Results and discussion
All compounds were evaluated for their ability to inhibit
PDE5 and PDE6. PDE5 was purified from human
platelets as described by Gristwood et al.25 PDE6 was
purified from bovine retinas as described by Beavo
et al.26 Cyclic nucleotide phosphodiesterase activities
were measured using a two-step procedure according
to Thompson and Strada.27 Sildenafil was used as
reference drug.

J. Feixas et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2381–2384
Table 1. PDE5 and PDE6 inhibitory activity of compounds 10–42
2383
R3
N
N
N
O
CH3
N
R2
N
R1
10-42
Compd
R1
R2
R3
Bn
PDE5 IC50 or % inhibition (lM)b PDE6 IC50 or % inhibition (lM)b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Sildenafil
Ph
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
0.16
25(2)
11
Cyclohexylmethyl 3.1
Ph
Ph
p-Nitrobenzyl
p-Aminobenzyl
Ph
65.9(20)
1.1
11.9(0.2)
10.4(2)
2.0
Ph
Ph
Aa
3.7(0.2)
28.6(0.2)
78
m-(H2PO4)benzyl 0.23
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
13.0
31.0(2)
0.27
t-Butyl
2-Pyridyl
4-Nitrophenyl
ꢀ20.9(2)
10
65.5(20)
0.92
14.4(0.2)
4.5(2)
2.8
4-Aminophenyl Me
Ph
Ph
Ph
0.081
0.45
4-Pyridylmethyl
Bn
20.4(2)
13.6(2)
21.3(2)
17.7(2)
ꢀ4.8(2)
30
Ph
Ph
22.5(2)
0.081
0.18
2-Pyridyl
3-Pyridyl
4-Pyridyl
Phenylethyl
o-Tolyl
Ph
Ph
69.0(2)
0.98
Ph
Ph
55.1(2)
0.075
33.6(2)
18.2(2)
0.1
ꢀ16.6(2)
11.5(2)
19.2(2)
4.2(2)
53.3(2)
45(2)
Ph
Ph
m-Tolyl
Cyclohexylmethyl Bn
Cyclopentylmethyl Bn
Ph
Ph
CH2OCH3
CH2OCH2CH3
CH2SCH3
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Ph
Ph
0.35
0.1
38.1(2)
0.59
0.58
Ph
Ph
CH2CH2COOH
CH@CHCOOH
(CH2)3COOH
(CH2)4COOH
CH(C2H5)2
0.43
0.32
Ph
Ph
0.03
0.09
0.11
0.063
Ph
Ph
0.067
0.034
CH(CH3)CH2CH3 m-(H2PO4)benzyl 0.059
37.4(2)
42.5(2)
66.9(2)
34.6(2)
0.040
CH(CH3)CH2CH3 Bn
Ph
2-Pyridyl
CH(CH3)CH2CH3 Bn
0.039
0.020
a A = 3,4-methylendioxyphenyl.
b The IC50 were obtained from dose–response curves at three or four different concentrations (n = 2–3).
Ether derivatives (32 and 33) and the corresponding
thioether 34, as well as the carboxylic acids 35 and
36, showed a good level of activity and an appreciable
selectivity with the exception of compounds 35 and 36
which demonstrated the same affinity for PDE5 and
PDE6 isoenzymes. Homologation of carboxylic acid
35 (compounds 37 and 38) resulted in an increase of
potency, but, as the above compounds, in the complete
absence of selectivity (IC50 = 30, 90 nM for PDE5 and
IC50 = 110, 63 nM for PDE6). The best results were
associated with the introduction of branched chains
(compounds 39 and 40) which showed a significant
improvement of the potency with respect to the corre-
sponding 6-methyl derivative 10. These compounds ap-
peared particularly interesting because of their high
activity towards PDE5 associated to very good selecti-
vity (IC50 = 67 and 34 nM for PDE5; 37.4% and 42.5%
inhibition at 2 lM for PDE6).
Finally, some modifications were performed at position
3 by inserting substituents on the benzyl group as well as
by removing the methylenic spacer with the introduction
of a phenyl ring. The best result is related to the intro-
duction of a phosphate group at meta position of the
benzyl (compound 15), the activity being comparable,
but not improved with respect to the 3-unsubstituted
benzyl derivative 10.
The best substituents selected for position 9, 6 and 3 (2-
pyridyl, CH(CH3)CH2CH3 and m-(H2PO4)-benzyl,
respectively) were finally combined in compounds 41
and 42 in order to verify if hybridization is allowed. In

2384
J. Feixas et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2381–2384
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improvement of potency was found, suggesting that the
combining approach is not effective.
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13. Vardenafil Drugs Future 2001, 26, 141.
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15. Gresser, U.; Gleiter, C. H. Eur. J. Med. Res. 2002, 7, 435–
446.
In conclusion we have identified a novel series of potent
and selective PDE5 inhibitors potentially useful as
peripheral vasodilators. The most interesting com-
pounds showed values of PDE5 inhibitory activity in
the same nanomolar range of Sildenafil but with a much
better selectivity versus PDE6; this last aspect could
make them good candidates to be used as MED treating
drugs devoid of some well-known unwanted effects.
16. Porst, H. Int. J. Impot. Res. 2002 (Suppl 1), S57–
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Humprey, W. G.; Krupinski, J.; Macor, J. E. J. Med.
Chem. 2003, 46, 457–460.
Further modifications at positions 9, 6, 3 and 1 are in
progress to completely define structure–activity relation-
ships and in order to improve the potency and selectivity
profile of the present series.
18. Dal Piaz, V.; Giovannoni, M. P.; Castellana, M. C.;
`
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Acknowledgements
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21. Dal Piaz, V.; Castellana, M. C.; Vergelli, C.; Giovannoni,
Support of this work by MURST (40%, Italy) is grate-
fully acknowledged.
`
M. P.; Gavalda, A.; Segarra, V.; Beleta, J.; Ryder, H.;
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