Novel pyrazolopyrimidopyridazinones with potent and selective phosphodiesterase 5 (PDE5) inhibitory activity as potential agents for treatment of erectile dysfunction

Article abstract of DOI:10.1021/jm060265+

Pyrazolo[1′,5′:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones and their analogues, potentially useful for the treatment of erectile dysfunction, were synthesized and evaluated as inhibitors of phosphodiesterase 5 (PDE5). Several compounds showed IC₅₀

Full text of DOI:10.1021/jm060265+

J. Med. Chem. 2006, 49, 5363-5371
5363
Novel Pyrazolopyrimidopyridazinones with Potent and Selective Phosphodiesterase 5 (PDE5)
Inhibitory Activity as Potential Agents for Treatment of Erectile Dysfunction
Maria Paola Giovannoni,*,† Claudia Vergelli,† Claudio Biancalani,† Nicoletta Cesari,† Alessia Graziano,† Pierfrancesco Biagini,†
Jordi Gracia,‡ Amadeu Gavalda`,‡ and Vittorio Dal Piaz†
Dipartimento di Scienze Farmaceutiche, UniVersita` di Firenze, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Firenze, Italy, and
Almirall Prodesfarma Research Center, Cardener 68-74, 08024 Barcelona, Spain
ReceiVed March 9, 2006
Pyrazolo[1′,5′:1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones and their analogues, potentially useful for the
treatment of erectile dysfunction, were synthesized and evaluated as inhibitors of phosphodiesterase 5 (PDE5).
Several compounds showed IC50 values in the low nanomolar range, and in particular, compound 5r,
displaying high potency toward PDE5 (IC50 ) 8.3 nM) and high selectivity versus PDE6 (240-fold) appeared
to be a very promising new lead both in comparison with the potent but not selective sildenafil and in
comparison with some analogues previously reported by us. SAR studies in this triheterocyclic scaffold led
us to conclude that the best arranged groups are a methyl in position 1, a benzyl in position 3, a phenyl in
position 9, and a linear four-carbon chain in position 6.
Introduction
in 1998, showed a lot of side effects such as increased light
sensitivity, nausea, and headache due to its low selectivity
toward PDE6 isoform; tadalafil, B16,17 (Cialis), which pre-
sents small secondary effects and a longer half-life with re-
spect to sildenafil; vardenafil, C18,19 (Levitra), which shows a
positive efficacy-security profile and was launched by Bayer
Pharmaceutical and GlaxoSmithKline at the beginning of 2004
(Figure 1).
The therapeutic effect of PDE5 inhibitors is related to the
increase of cyclic guanosine monophosphate (cGMP) levels,
which are responsible for the relaxation of smooth muscle cells
and vasodilation in the corpus cavernosum, thereby potentiating
penile erection.20 At present, the primary objective of academics
and pharmaceutical companies in this field is to identify new
potent compounds with a very significant selectivity profile, in
particular toward isoform 6, which is responsible for the most
common side effects. The efforts expended in this area have
led to excellent products with different chemical structures
showing an activity in the (sub)nanomolar range and a very
attractive specificity toward PDE5 isoenzyme (to 600-fold).21-24
Our previous studies and experience in the field of PDE
inhibitors25-28 together with the interesting results coming from
Bristol-Myers on the identification of potent, selective, and
orally bioavailable pyrazolopyridopyridazines D29 led us to
synthesize new pyrazolopyrimidopyridazinones of type E, which
showed good inhibitory activity on PDE5 and a fair selectivity
toward PDE630 (Figure 1).
Erectile dysfunction (ED) is a very common pathology that
affects millions of men worldwide, and it appears to be directly
proportional to aging.1,2 The most important epidemiological
studies in this field from the Massachusetts Male Aging Study
(MMAS) demonstrated that about one-third of men over 40
suffer from moderate to complete ED.3 Recent studies suggest
that ED, rather than being an inevitable consequence of aging,
depends not only on a series of elements such as heart disease,
hypertension, diabetes, and psychogenic causes such as anxiety
and depression but also on education.4,5 Finally, it was found
that some drugs induced ED as an undesired effect, in particular
antipsychotic drugs and also products belonging to other classes
such as antihypertensives, diuretics, antilipidemics, and nar-
cotics.5,6
The therapy for erectile dysfunction includes R-adrenoceptor
antagonists such as moxisylyte (Thymoxamine),7 synthetic
prostaglandin E1 (Alprostadil),8 and opioids such as papaverine9
(which are all used for intracavernosal, intraurethral, or topical
administration) and apomorphine, a dopamine D1 and D2
receptor agonist, which has been recently approved for market-
ing in Europe.10 Its use (sublingual via) for moderate ED is
limited by notable side effects such as nausea, orthostatic
hypertension, and vomiting. However,the most widely prescribed
drugs for erectile dysfunction are the orally active and remark-
ably potent phosphodiesterase 5 (PDE5) inhibitors.11
PDE5 belongs to a superfamily of enzymes that catalyzes
the hydrolysis of cyclic nucleotides cAMP and cGMP to the
corresponding 5-nucleoside monophosphate. Currently, 11 dif-
ferent isoforms of phosphodiesterases (PDE1-PDE11) and their
subtypes are known, distinguished by substrate specificities and
tissue concentration.12,13 In particular, PDE5 is localized in cells
of the corpus cavernosum, in vascular smooth muscle, and in
platelets.
We report here the development of the above project with
the synthesis and biological evaluation of a wide series of novel
pyrazolopyrimidopyridazinones as PDE5 inhibitors.
Chemistry
The final compounds were prepared following the synthetic
routes reported in Schemes 1-5.
Currently, three PDE5 inhibitors are commercially available
with excellent expected results and somewhat unique profiles:
the archetypal sildenafil, A14,15 (Viagra), which was launched
30,31
We previously described
the synthesis of the tricyclic
system that is based on three common steps: (a) condensation
of the starting isoxazolo[3,4-d]pyridazinones with the appropri-
ate arylaldehydes to give the vinyl derivatives (3a-n, 15, 17,
28a,b, 33a,b); (b) treatment of the vinyl derivatives with
hydrazine to obtain the pyrazolylpyridazinone intermediates by
isoxazole ring opening (4a-n, 16, 18, 29a,b, 34a,b); (c) ring
* To whom correspondence should be addressed. Phone and fax: +30-
055-4573682. E-mail: mariapaola.giovannoni@unifi.it.
† Universita` di Firenze.
‡ Almirall Prodesfarma Research Center.
10.1021/jm060265+ CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/02/2006

5364 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
GioVannoni et al.
Scheme 1a
a (a) RX, DMF, K2CO3; (b) R1CHO, CH3ONa, CH3OH; (c) NH2NH2,
C2H5OH; (d) (R2CO)2O or R2COOH, DMF, CH2Cl2, DMAP, EDC; (e)
H2O2, CH3COOH; (f) NaOH/C2H5OH.
Scheme 2a
Figure 1. PDE5 inhibitors.
closure to pyrazolo[1′,5′:1,6]pyrimido[4,5-d]pyridazin-4(3H)-
ones with the appropriate anhydride under refluxing or with
the opportune carboxylic acid at room temperature in the
presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (5a-t, 19, 20a,b, 30a,b, 35a,b). The alkylation
under standard conditions with the appropriate halides, which
allowed us to obtain a variety of 3-substituted final compounds,
was carried out on the isoxazolo[3,4-d]pyridazinones or on the
tricyclic system.
Scheme 1 shows the alkylation performed on the precursor
132 to give the corresponding N-alkyl derivatives. In this syn-
thetic pathway the tricyclic compounds 5e and 5j were further
elaborated through an alkaline hydrolysis and through an
oxidative reaction with H2O2 in acetic acid, respectively, to
afford compounds 5u and 5v.
Scheme 2 depicts the synthesis of the final compounds
7a-d, which was performed by alkylation of pyrazolopyrimi-
dopyridazin-4(3H)-ones 6a,b.30,31 Moreover, compound 6b,
treated with POCl3 followed by the appropriate amine, furnished
the corresponding pyrazolopyrimido[4,5-d]pyridazines 9a,b.
Schemes 3-5 report the synthetic routes for the introduction
of different substituents at position 1 of the tricyclic system.
The difunctionalized isoxazoles 12a,b, 22, and 23 were prepared
from the appropriate diketones 10a,b and 21a,b33-36 and the
ethyl (chlorohydroximino)acetate 11, which is commercially
available. 1,3-Dipolar cycloaddition is not a regioselective
reaction in the presence of the above diketones and led to a
mixture of the two isomers (4-COCH3 and 4-COR4 derivatives),
which were separated by column chromatography (Scheme 3)
a (a) POCl3; (b) R3NH2, C2H5OH; (c) RX, DMF, K2CO3.
or subjected as a mixture to the usual train of reactions (Scheme
4). In fact, when the mixture of 26 and 27 was treated with

Pyrazolopyrimidopyridazinones as PDE5 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5365
Scheme 4a
Scheme 3a
a (a) C2H5ONa/C2H5OH; (b) NH2NH2, C2H5OH; (c) PhCH2Br, DMF,
K2CO3; (d) PhCHO, CH3ONa, CH3OH; (e) NH2NH2, C2H5OH; (f)
(CH3CO)2O.
a (a) C2H5ONa/C2H5OH; (b) NH2NH2, C2H5OH; (c) PhCH2Br, DMF,
K2CO3; (d) PhCHO, CH3ONa, CH3OH; (e) NH2NH2, C2H5OH; (f)
(CH3CO)2O.
Scheme 5a
benzaldehyde in the presence of CH3ONa, only the isomer 26
reacted37 affording the 3-arylvinylderivatives 28a,b, which were
collected as precipitates from the reaction mixtures. Compounds
28a,b were converted into the final 30a,b by using the same
two-step procedure.
Finally, compounds 35a,b were prepared starting from
precursor 14, which, when treated with HBr in acetic acid,
afforded the corresponding 4-bromomethyl derivatives as good
substrates for a nucleophilic displacement (Scheme 5).
Physical and chemical data of compounds 2a-i, 3a-n,
4a-n, and 5a-v are reported in Tables 1-4.
Results and Discussion
All the final compounds were evaluated for their PDE5 and
PDE6 inhibitory activity.
First, we extensively explored the role played by the
substituent at position 3, keeping methyl groups at positions 1
and 6 and a phenyl at position 9 (Table 5). Indeed, the presence
of linear (5a, 5c, and 7c) or branched alkyls (5b) is generally
associated with an almost complete absence of activity (PDE5
inhibition of e54.6% at 2 µM). Compounds 5d and 5u, bearing
a propargyl and an acetic acid residue, respectively, were tested
at 0.2 µM, showing low inhibitory activity. For the methyl
derivative 7b, a dose response curve was determined and an
IC50 ) 1.5 µM was found, but this compound was completely
devoid of selectivity. It is interesting to observe that all the
derivatives bearing linear or branched alkyls at position 3 were
demonstrated to have very similar levels of activity, ranging
from 50% at 1.5 µM for 7b to 44.7% at 2 µM for 5b. Thus, the
a (a) HBr, CH3COOH; (b) dimethylamine or 4-methylpiperazine, C2H5OH;
(c) PhCHO, CH3ONa; (d) NH2NH2, C2H5OH; (e) (CH3CO)2O.
length and the steric hindrance of the carbon chain do not play
any role in determining PDE5 inhibitory activity. This finding
lends strong support to the hypothesis that these groups do not

5366 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Table 1. Physical and Chemical Data of Compounds 2a-i
GioVannoni et al.
Table 3. Physical and Chemical Data of Compounds 4a-n
compd
R
R1
yield (%) mp (°C)a
compd
R
yield (%)
mp (°C)a
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
Bn
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
86
60
47
85
50
43
53
20
81
71
96
40
60
58
192-193
182-183
188-190
142-145
192-194
204-208
138-141
143-146
177-180
258-259
200-203
209-210
239-241
220-222
2a
2b
2c
2d
2e
2f
2g
2h
2i
Bn
89
88
40
50
74
72
90
64
70
143-146
83-85
110-113
54-56
121-123
110-112
126-129
oilb
n-C3H7
i-C3H7
n-C4H9
n-C3H7
i-C3H7
n-C4H9
CH2CtCH
CH2CtCH
CH2COOMe
3-OCH3-Bn
CH2-4-pyridyl
(CH2)2-4-piperidyl
CH2COOCH3
3-OCH3-Bn
CH2-4-pyridyl
(CH2)2-4-piperidyl Ph
84-87
Bn
Bn
Bn
Bn
Bn
4-F-Ph
4-S-CH3-Ph
3-pyridyl
3-thienyl
3-furyl
a All solid compounds were purified by crystallization from EtOH.
b Purified by column chromatography using 1:2 cyclohexane/ethyl acetate.
Table 2. Physical and Chemical Data of Compounds 3a-n
a All compounds were purified by crystallization from EtOH.
Table 4. Physical and Chemical Data of Compounds 5a-v
yield
(%)
mp
(°C)
cryst
solvent
compd
R
R1
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
Bn
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
80
78
86
75
80
49
70
90
61
95
85
63
66
98
215-217
181-183
225-226
173-175
238-240
204-207
188-190
193-196
216, dec
231-233
257-259
205-207
209-212
MeOH
MeOH
MeOH
MeOH
MeOH
EtOH
EtOH
EtOH
EtOH
EtOH
MeOH
EtOH
EtOH
n-C3H7
i-C3H7
n-C4H9
yield
compd
R
R1
R2
(%) mp (°C)a
5a
5b
5c
5d
5e
5f
5g
5h
5i
n-C3H7
i-C3H7
n-C4H9
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
63 189-190
53 196-198
44 183-185
45 255-258
41 >300
CH2CtCH
CH2COOCH3
3-OCH3-Bn
CH2-4-pyridyl
CH2CtCH
CH2COOCH3
3-OCH3-Bn
CH2-4-pyridyl
(CH2)2-4-piperidyl Ph
35 187-190
66 231-234
46 143-145
47 280-284
48 270-273
54 235-237
92 230-232
78 233, dec
64 267-269
89 220-222
72 191-192
45 234-237
33 225-227
Bn
Bn
Bn
Bn
Bn
4-F-Ph
4-S-CH3-Ph
3-pyridyl
3-thienyl
3-furyl
(CH2)2-4-piperidyl Ph
Bn
Bn
Bn
Bn
4-F-Ph
4-SCH3-Ph CH3
3-pyridyl
3-thienyl
3-furyl
Ph
5j
5k
5l
197-199 dec EtOH
CH3
CH3
CH3
H
5m Bn
interact with any amino acidic residue of the protein, suggesting
that in these derivatives the carbon chain is probably directed
outside the catalytic pocket. In contrast, we found that an aryl
group separated from the tricyclic core by a methylenic spacer
is an important feature. In agreement with the result obtained
with the previously reported 3-benzyl derivative,30 the analogues
5f, 5g, and 7d showed submicromolar activity coupled with a
certain degree of selectivity versus PDE6. The best compound
proved to be the 3-methoxybenzyl derivative 5f, which showed
good potency at PDE5 (IC50 ) 100 nM) and at least 20-fold
selectivity versus PDE6.
By examining the results obtained from keeping a methyl at
position 1 and a benzyl at position 3 and from varying R1 and
R2 (Table 6), we observe that several compounds are endowed
with affinity versus PDE5 in the same order of magnitude as
the corresponding 9-phenyl derivative.30 Thus, with CH3 at R2,
the introduction of a chlorine in the para position of the phenyl
(compound 7a) or its replacement with different heterocyclic
systems (3-furyl, 3-thienyl, 3-pyridyl) gave compounds with IC50
in the range 0.05-0.47 µM for PDE5 inhibition, but only the
5n
5o
5p
5q
5r
5s
5t
5u
5v
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Ph
Ph
Ph
Ph
Ph
Ph
Ph
C2H5
n-C3H7
i-C3H7
(CH2)2COCH3
(CH2)2COOCH3 42 215-218
(CH2)4COOCH3 39 205-207
CH2COOH
Bn
CH3
63 >300
36 248-251
4-SO2CH3Ph CH3
a All compounds were purified by crystallization from EtOH.
3-thienyl derivative 5l retained an appreciable level of selectivity
(16) versus PDE6. In contrast, the introduction of a fluorine
(compound 5i) or a methylsulfonyl (compound 5v) in the para
position of the phenyl was very detrimental, since a complete
loss of activity was observed. This finding strongly suggests
that both steric and electronic features play an important role
in this part of the molecule because the presence of the small
and strongly electronegative F (π ) 0.14, σp ) 0.06, MR )
0.092)38 and the large electron-withdrawing CH3SO2 group (π
) -1.63, σp ) 0.72, MR ) 1.35) is associated with absence of

Pyrazolopyrimidopyridazinones as PDE5 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5367
Table 5. PDE5 and PDE6 Inhibitory Activity of
Table 7. PDE5 and PDE6 Inhibitory Activity of
Pyrazolopyrimidopyridazinones
Pyrazolopyrimidopyridazinones
compd
9a
9b
20a
20b
30a
30b
35a
R3
R4
PDE5e
PDE6e
compd
5a
5b
5c
5d
5f
5g
5h
R
PDE5c
PDE6c
Bn
Ab
52.4 (2 µM)
56.8 (2 µM)
0.071
14.3 (2 µM)
0.22
NTd
NTd
n-C3H7
i-C3H7
n-C4H9
44.9 (2 µM)
44.7 (2 µM)
54.6 (2 µM)
19.7 (0.2 µM)
0.1
6.9 (2 µM)
1.2 (2 µM)
32.5 (2 µM)
0.3 (0.2 µM)
3.8 (2 µM)
39.4 (2 µM)
26.6 (2 µM)
11.2 (0.2 µM)
2.6
53.4 (0.2 µM)
7 (2 µM)
25 (2 µM)
0.040
CH2OC2H5
cyclopentyl
C2H5
i-C3H7
CH2-N(CH3)2
0.33
12.3 (2 µM)
21.8 (2 µM)
59.8 (2 µM)
1.0
30.0 (0.2 µM)
25 (2 µM)
0.040
CH2CtCH
3-OCH3-Bn
CH2-4-pyridyl
(CH2)2-4-piperidyl
CH2COOH
CH3
0.1
1.4
0.24
35b
Bc
CH3
23.0 (0.2 µM)
0.16
0.020
67.7 (2 µM)
7.6 (0.2 µM)
1.5
49.3 (2 µM)
0.39
Ea
5u
7b
7c
sildenafil
C2H5
a See ref 30. b A ) 3,4-methylendioxyphenylmethyl. c B ) N-methyl-
piperazinylmethyl. d NT ) not tested. e Data are indicated as IC50 (µM) or
inhibition percentage at indicated concentration (µM). The IC50 values were
obtained from dose response curves at three or four different concentrations
(n ) 2-3).
7d
Ab
Ea
Bn
0.16
0.020
sildenafil
a See ref 30. b A ) 3,4-methylendioxyphenylmethyl. c Data are indicated
as IC50 (µM) or inhibition percentage at indicated concentration (µM). The
IC50 values were obtained from dose response curves at three or four
different concentrations (n ) 2-3).
of a branched chain (5q) was associated with the best result in
this subseries of compounds. The best balance of activity and
selectivity was that of the n-propyl derivative 5p (PDE5 IC50
) 40 nM, PDE6/PDE5 ) 15). When position 6 is decorated
with an oxygenated carbon chain, both the activity and the
selectivity were dramatically improved. Thus, compound 5r,
bearing the 2-oxobutyryl group in this position, was character-
ized by high PDE5 affinity (IC50 ) 8.3 nM) and very high
selectivity (>240). Compared with sildenafil, compound 5r was
2.4-fold more potent and at least 200-fold more selective. When
the oxygenated carbon chain of 5r was replaced by a methyl
butyrate residue (5s), an approximately 10-fold reduction of
activity was observed, without modification of the selectivity.
Elongation of the methylenic carbon chain of 5s gave compound
5t, which showed the same activity and selectivity profile.
Compounds 5s and 5t showed potency at the same level as the
analogues with ethyl, n-propyl, and isopropyl at R2, but in terms
of the selectivity issue, the presence of a carbonyl dipole in the
carbon chain seems to be an essential requirement. Taken
together, these results suggest that the presence of a hydrogen
bond acceptor in the carbon chain at R2 plays an important role
in the interaction with the biological target, also allowing us to
nicely discriminate between PDE5 and PDE6. Moreover, the
position of the carbonyl group inside the carbon chain and/or
the nature of the functional group plays a very critical role in
the affinity at PDE5.
We also evaluated a series of analogues modified at position
1 (Table 7); thus, the methyl group of our previous prototype
(compound E, R4 ) CH3)30 was elongated, branched, function-
alized, and replaced by cycloalkyl groups. All these manipula-
tions did not improve the activity. In this subseries the best
compound proved to be 20a, bearing an ethoxymethyl group
(PDE5 IC50 ) 71 nM), but a complete absence of selectivity
was found for this molecule. Low linear and branched alkyls
(compounds 30a,b) were better than cyclopentyl (20b); amino
and cycloalkylamino groups linked to the tricyclic system
through a methylenic spacer (compounds 35a,b) proved to be
almost devoid of activity.
Table 6. PDE5 and PDE6 Inhibitory Activity of
Pyrazolopyrimidopyridazinones
compd
5i
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5v
R1
4-F-Ph
3-pyridyl
3-thienyl
3-furyl
Ph
R2
PDE5b
PDE6b
CH3
CH3
CH3
CH3
H
38.8 (2 µM) 4.5 (2 µM)
0.47
0.14
0.05
0.18
0.038
0.04
0.023
0.0083
0.28
2.2
0.33
0.86
0.55
0.61
0.062
22.8 (2 µM)
16.9 (2 µM)
1.1 (2 µM)
2.8 (2 µM)
4.7
Ph
Ph
Ph
Ph
Ph
Ph
C2H5
n-C3H7
i-C3H7
(CH2)2COCH3
(CH2)2COOCH3 0.079
(CH2)4COOCH3 0.095
4-SO2CH3-Ph CH3
3.8 (2 µM)
0.37
0.16
7a
4-Cl-Ph
CH3
CH3
Ea
Ph
25 (2 µM)
0.040
sildenafil
0.020
a See ref 30. b Data are indicated as IC50 (µM) or inhibition percentage
at indicated concentration (µM). The IC50 values were obtained from dose
response curves at three or four different concentrations (n ) 2-3).
activity in comparison with the active chloro derivative 7a,
where the substituent is characterized by an electronegative
effect and limited steric hindrance (π ) 0.71, σp ) 0.23, MR
) 0.603). Studying the effect due to the modification of the
substituent at position 6, with R1 ) Ph, we affirm that, moving
from H (compound 5n) to low alkyls (5o-p), the activity is
significantly improved (at least 5-fold); moreover, the presence

5368 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
GioVannoni et al.
(CDCl3) δ 1.30 (d, 6H, CH(CH3)2), 2.50 (s, 3H, 4-CCH3), 2.85 (s,
3H, 3-CCH3), 5.25-5.35 (m, 1H, CH(CH3)2).
Finally we synthesized some examples of compounds in
which the (substituted) benzyl group was shifted from position
3 to position 4 through aromatization of the pyridazinone system.
Both compounds 9a and 9b, bearing a (alkoxy)benzylamino
fragment similar to that of Bristol-Myers compound D, proved
to be inactive. This result seems to indicate that the presence
of the carbonyl dipole together with the benzyl group at the
adjacent nitrogen in our series is an essential feature for the
interaction with the protein. This finding is in agreement with
the SAR found for compounds such as sildenafil and vardenafil
targeting PDE5 and also with agents targeting PDE3 and PDE4
isoenzymes, which show a remarkable degree of homology in
the catalytic site with PDE5.39 Compound D probably interacts
with this site with a different orientation with respect to both
the above drugs and our compounds.
General Procedure for Compounds 3a-n. To a suspension
of isoxazolopyridazinone 2a-i (1 mmol) and the appropriate aryl-
aldehyde (2-3 mmol) in anhydrous methanol, CH3ONa (1-2.5
mmol) was added. The mixture was refluxed under stirring for 1-45
min. After the mixture was cooled, the solid was recovered by suc-
tion. For compond 3h the mixture was concentrated and diluted
with ice-water (10 mL) and the precipitate was isolated by
filtration.
6-Benzyl-4-methyl-3-styrylisoxazolo[3,4-d]pyridazin-7(6H)-
1
one, 3a. Yield ) 80%; mp ) 215-217 °C (MeOH); H NMR
(CDCl3) δ 2.60 (s, 3H, 4-CCH3), 5.30 (s, 2H, CH2Ar), 7.20 (d,
1H, CHd), 7.30-7.60 (m, 10H, Ar), 7.80 (d, 1H, dCH).
6-Isopropyl-4-methyl-3-styrylisoxazolo[3,4-d]pyridazin-7(6H)-
1
one, 3c. Yield ) 86%; mp ) 225-226 °C (MeOH); H NMR
(CDCl3) δ 1.35 (d, 6H, CH(CH3)2), 2.65 (s, 3H, 4-CCH3), 5.30-
5.40 (m, 1H, CH(CH3)2), 7.25 (d, 1H, CHd), 7.45-7.65 (m, 5H,
Ar), 7.80 (d, 1H, dCH).
In conclusion, exploration of the space around the pyrazol-
opyrimidopyridazinone system allowed us to identify compound
5r displaying PDE5 inhibitory activity in the low nanomolar
range and 240-fold selectivity versus PDE6. This in vitro profile
suggests 5r as a very promising new lead both in comparison
with the potent but not selective sildenafil and in comparison
with previously reported analogues.30 In fact, optimization of
these compounds allowed us to improve potency at PDE5 by
more than 1 order of magnitude; moreover, selectivity versus
PDE6 was increased from 66- to 240-fold.
General Procedure for Compounds 4a-n. A suspension of
compounds 3a-n (0.3 mmol) in ethanol (2-3 mL) and hydrazine
hydrate (2-9 mmol) was stirred at room temperature for 0.5-5 h
(compounds 4d and 4k for 0.5-1 h at 60 °C). Then the mixture
was concentrated in vacuo and cooled for 1-2 h and the precipitate
was recovered by suction. For compound 4h, after concentration,
the mixture was diluted with water (5 mL) and after the mixture
was cooled, the solid was filtered off.
4-Amino-2-benzyl-6-methyl-5-(5′-phenyl-1H-pyrazol-3-yl)py-
ridazin-3(2H)-one, 4a. Yield ) 86%; mp ) 192-193 °C (EtOH);
1H NMR (CDCl3) δ 2.30 (s, 3H, 6-CCH3), 5.30 (s, 2H, CH2Ar),
6.00 (exch br s, 2H, NH2), 6.70 (s, 1H, Ar), 7.30-7.70 (m, 10H,
Ar).
4-Amino-2-isopropyl-6-methyl-5-(5′-phenyl-1H-pyrazol-3-yl)-
pyridazin-3(2H)-one, 4c. Yield ) 47%; mp ) 188-190 °C
(EtOH); 1H NMR (CDCl3) δ 1.30 (d, 6H, CH(CH3)2), 2.20 (s, 3H,
6-CCH3), 5.20-5.30 (m, 1H, CH(CH3)2), 6.60 (s, 1H, Ar), 7.35-
7.80 (m, 5H, Ar).
SAR studies in this triheterocyclic scaffold led us to conclude
that the best arranged groups are a methyl at R4, benzyl at R,
and a phenyl at R1. At R2 the best substituent was a linear four-
carbon chain bearing a carbonyl dipole.
Taking into account these findings, further modifications are
in progress to improve the in vitro profile of the present series.
In the meantime, compound 5r is under evaluation in some
experimental models of penile dysfunction.
General Procedure for Compounds 5a-m,o-q. A mixture of
4-amino-5-pyrazolyl derivatives 4a-n (0.2-0.3 mmol) and the
suitable anhydride (10-15 mmol) was refluxed under stirring for
5 min to 1 h. After the mixture was cooled, the solid that separated
was filtered and washed with water. For compound 5h, the mixture
was diluted with cold water (15 mL) and neutralized with 6 N
NaOH and the precipitate was recovered by suction. Compound
5k was recovered by suction after dilution of the reaction mixture
with ice-water (10 mL).
Experimental Section
Chemistry. All melting points were determined on a Bu¨chi
apparatus and are uncorrected. 1H NMR spectra were recorded with
Varian Gemini 200 instruments. Chemical shifts are reported in
ppm, using the solvent as internal standard. Extracts were dried
over Na2SO4, and the solvents were removed under reduced pres-
sure. 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. Reagent 11 is commercially
available.
General Procedure for Compounds 2a-i. A mixture of
isoxazolopyridazinone 132 (2.2 mmol), K2CO3 (4.4 mmol), and the
appropriate alkyl halide (3.0-4.5 mmol) in anhydrous DMF (6 mL)
was stirred at 70-110 °C for 0.5-3 h. For compound 2h the
reaction was carried out at 110 °C for 16 h. After cooling, the mix-
ture was diluted with cold water, and the precipitate was recovered
by suction. For compounds 2e,f,h, after dilution with water, the
suspension was extracted with CH2Cl2 (3 × 15 mL). Then the
solvent was evaporated in vacuo to afford a crude precipitate.
1,6-Dimethyl-3-isopropyl-9-phenylpyrazolo[1′,5′:1,6]pyrimido-
[4,5-d]pyridazin-4(3H)-one, 5b. Yield ) 53%; mp ) 196-198
1
°C (EtOH); H NMR (CDCl3) δ 1.40 (d, 6H, CH(CH3)2), 2.80 (s,
3H, 1-CCH3), 3.20 (s, 3H, 6-CCH3), 5.45-5.55 (m, 1H, CH(CH3)2),
7.35-7.60 (m, 4H Ar), 8.10 (d, 2H, Ar).
3-Benzyl-1-methyl-9-phenylpyrazolo[1′,5′:1,6]pyrimido[4,5-
d]pyridazin-4(3H)-one, 5n. A mixture of 4a (0.17 mmol), triethyl-
orthoformate (12 mmol), anhydrous DMF (4 mL), and a catalytic
amount of concentrated sulfuric acid was stirred at room tempera-
ture for 30 min. The mixture was diluted with cold water (10 mL),
and the precipitate was recovered by suction. Yield ) 64%; mp )
1
Compound 2i was synthesized starting from 6-(2-bromoethyl)-
3,4-dimethylisoxazole[3,4-d]pyridazin-7(6H)-one40 (0.73 mmol),
which was suspended in anhydrous DMF (4 mL). K2CO3 (1.16
mmol) and piperidine (1.2 mmol) were added, and the mixture was
stirred at 70 °C for 1 h and 30 min. After the mixture was cooled,
cold water was added and the suspension was extracted with
CH2Cl2 (3 × 20 mL). Evaporation of the solvent afforded 2i.
267-269 °C (EtOH); H NMR (CDCl3) δ 2.80 (s, 3H, 1-CCH3),
5.40 (s, 2H, CH2Ar), 7.25-7.60 (m, 9H, Ar), 8.05 (m, 2H, Ar),
9.45 (s, 1H, Ar).
General Procedure for Compounds 5r-t. A suspension of
4a (0.42 mmol) in anhydrous CH2Cl2 (13 mL), anhydrous DMF
(1-1.5 mL), 0.52 mmol of suitable acid or ester (levulinic acid,
acetoxypropionic acid, methyladipate), 0.51 mmol of 4-(dimethyl-
amino)pyridine, and 0.6 mmol of 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride was stirred at room temperature
for 6 h and then refluxed for 20 h. After the mixture was cooled,
the solid was filtered off and dissolved in CH2Cl2. The organic
layer was washed with 2 N HCl and with 2 N NaOH solution se-
quentially and then evaporated in vacuo to afford a crude precipitate.
6-Benzyl-3,4-dimethylisoxazolo[3,4-d]pyridazin-7(6H)-one, 2a.
1
Yield ) 89%; mp ) 143-146 °C (EtOH); H NMR (CDCl3) δ
2.50 (s, 3H, 4-CCH3), 2.80 (s, 3H, 3-CCH3), 5.25 (s, 2H, CH2Ar),
7.20-7.50 (m, 5H, Ar).
3,4-Dimethyl-6-isopropylisoxazolo[3,4-d]pyridazin-7(6H)-
one, 2c. Yield ) 40%; mp ) 110-113 °C (EtOH); 1H NMR

Pyrazolopyrimidopyridazinones as PDE5 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5369
3-Benzyl-1-methyl-6-(3-oxobutyl)-9-phenylpyrazolo[1′,5′:1,6]-
pyrimido[4,5-d]pyridazin-4(3H)-one, 5r. Yield ) 33%; mp )
225-227 °C (EtOH); 1H NMR (DMSO-d6) δ 2.30 (s, 3H, COCH3),
2.80 (s, 3H, 1-CCH3), 3.20 (m, 2H, COCH2CH2), 3.65 (m, 2H,
COCH2CH2), 5.35 (s, 2H, CH2Ar), 7.35-8.20 (m, 11H, Ar).
Methyl 3-Benzyl-1-methyl-4-oxo-9-phenyl-3,4-dihydropyra-
zolo[1′,5′:1,6]pyrimido[4,5-d]pyridazin-6-yl)propanoate, 5s. Yield
) 42%; mp ) 215-218 °C (EtOH); 1H NMR (DMSO-d6) δ 2.80
(s, 3H, 1-CCH3), 3.00 (m, 2H, CH2CH2COO), 3.60 (s, 3H, OCH3),
3.65-3.75 (m, 2H, CH2CH2COO), 5.40 (s, 2H, CH2Ar), 7.30-
8.20 (m, 11H, Ar).
1,6-Dimethyl-4-oxo-9-phenyl-3,4-diidropyrazolo[1′,5′:1,6]py-
rimido[4,5-d]pyridazin-3-acetic Acid, 5u. A mixture of 5e (0.22
mmol), ethanol (1-1.5 mL), and 6 N NaOH (2 mL) was stirred at
room temperature for 1 h. After acidification with 6 N HCl, the
final compound 5u was recovered by suction. Yield ) 63%; mp >
300 °C (EtOH); 1H NMR (DMSO-d6) δ 2.80 (s, 3H, 1-CCH3), 3.10
(s, 3H, 6-CCH3), 4.85 (s, 2H, CH2COO), 7.55 (m, 3H, Ar), 7.80
(s, 1H, Ar), 8.20 (m, 2H, Ph).
General Procedure for Compounds 12a,b. To a cooled (0 °C)
and stirred solution of sodium ethoxide (15 mmol) and anhydrous
ethanol (20 mL), the appropriate diketone (8 mmol) 10a,b33,34
dissolved in the same solvent (10 mL) was slowly added. After
the mixture was cooled at -5 °C, a solution of ethyl (chlorohy-
droximino)acetate 11 (10 mmol) in anhydrous ethanol (10 mL) was
added dropwise. The mixture, neutralized with 6 N HCl, was evap-
orated, and cold water was added. The aqueous layer was extracted
with CH2Cl2 (3 × 20 mL), and evaporation of the solvent afforded
compounds 12a,b, which were purified by column chromatography
using 2:1 cyclohexane/ethyl acetate as eluent for compound 12a
and 1:2 cyclohexane/ethyl acetate for compound 12b.
Ethyl 4-Ethoxymethylcarbonyl-5-methylisoxazole-3-carboxy-
1
late, 12a. Yield ) 68.7%; oil; H NMR (CDCl3) δ 1.25 (t, 3H,
CH3CH2OCO), 1.40 (t, 3H, CH3CH2O), 2.65 (s, 3H, C-CH3), 3.60
(q, 2H, CH3CH2O), 4.30 (s, 2H, CO-CH2-O), 4.40 (q, 2H,
CH3CH2OCO).
Ethyl 4-Cyclopentancarbonyl-5-methylisoxazole-3-carboxy-
1
late, 12b. Yield ) 52%; oil; H NMR (CDCl3) δ 1.25 (t, 3H,
3-Benzyl-1,6-dimethyl-9-(4-methylsulfonylphenyl)pyrazolo-
[1′,5′:1,6]pyrimido[4,5-d]pyridazin-4(3H)-one, 5v. A suspension
of compound 5j (0.19 mmol) in anhydrous acetic acid (2 mL) and
H2O2 (30%, 2.5 mL) was stirred at 90 °C for 1 h and 30 min. After
the mixture was cooled, the precipitate was recovered by suction.
CH3CH2OCO), 1.60-1.75 (m, 9H, cC5H9), 2.65 (s, 3H, C-CH3),
4.40 (q, 2H, CH3CH2OCO).
General Procedure for Compounds 13a,b. To a solution of
the isoxazole derivative 12a,b (2 mmol) in EtOH (2 mL), hydrazine
hydrate (3-4 mmol) was added. The mixture was stirred at room
temprature for 1-3 h. The mixture was concentrated in vacuo,
diluted with water (10-15 mL), and extracted with CH2Cl2 (3 ×
15 mL). Evaporation of the solvent afforded the 13a,b.
4-Ethoxymethyl-3-methylisoxazolo[3,4-d]pyridazin-7(6H)-
1
Yield ) 36%; mp ) 248-251 °C (EtOH); H NMR (CDCl3) δ
2.10 (s, 3H, CH3-SO2), 2.80 (s, 3H, 1-CCH3), 3.15 (s, 3H,
6-CCH3), 5.50 (s, 2H, CH2Ar), 7.30-7.60 (m, 6H, Ar), 8.10 (d,
2H, Ar), 8.30 (d, 2H, Ar).
General Procedure for Compounds 7a-d. Compounds 7a-d
were obtained starting from 6a,b following the general procedure
described for 2a-i. For compound 7c the reaction was carried out
at 60 °C for 5h. The residue was purified by column chromatog-
raphy using 9:1 CHCl3/MeOH as eluent.
1
one, 13a. Yield ) 73%; mp ) 155-157 °C (EtOH); H NMR
(CDCl3) δ 1.20 (t, 3H, CH3CH2O), 2.85 (s, 3H, C-CH3), 3.60 (q,
2H, CH3CH2O), 4.60 (s, 2H, CH2OEt), 9.80 (exch br s, 1H, NH).
4-Cyclopentyl-3-methylisoxazolo[3,4-d]pyridazin-7(6H)-
1
one, 13b. Yield ) 30%; mp ) 154-157 °C (EtOH); H NMR
3-Benzyl-9-(4-chlorophenyl)-1,6-dimethylpyrazolo[1′,5′:1,6]-
pyrimido[4,5-d]pyridazin-4(3H)-one, 7a. Yield ) 59%; mp )
(CDCl3) δ 1.75-1.90 (m, 9H, cC5H9), 2.90 (s, 3H, C-CH3).
6-Benzyl-4-ethoxymethyl-3-methylisoxazolo[3,4-d]pyridazin-
7(6H)-one, 14. 14 was obtained from 13a following the reported
general procedure to obtain 2a-i. The mixture was diluted with
cold water and was extracted with CH2Cl2 (3 × 15 mL). After
evaporation of solvent, a crude precipitate was obtained. Yield
1
266-269 °C (EtOH); H NMR (CDCl3) δ 2.80 (s, 3H, 1-CCH3),
3.20 (s, 3H, 6-CCH3), 5.45 (s, 2H, CH2Ar), 7.30-7.65 (m, 8H,
Ar), 8.00 (d, 2H, Ar).
1,6-Dimethyl-3-ethyl-9-phenylpyrazolo[1′,5′:1,6]pyrimido[4,5-
d]pyridazin-4(3H)-one, 7c. Yield ) 65%; mp ) 218-220 °C
1
) 48%; oil; H NMR (CDCl3) δ 1.20 (t, 3H, CH3CH2O), 2.80 (s,
1
(EtOH); H NMR (CDCl3) δ 1.45 (t, 3H, CH2CH3), 2.80 (s, 3H,
3H, C-CH3), 3.60 (q, 2H, CH3CH2O), 4.55 (s, 2H,
CH2OEt), 5.20 (s, 2H, CH2Ar), 7.30-7.40 (m, 5H, Ar).
6-Benzyl-4-ethoxymethyl-3-styrylisoxazolo[3,4-d]pyridazin-
7(6H)-one, 15. 15 was obtained from compound 14 following the
general procedure described for 3a-n. Yield ) 53%; mp ) 127-
130 °C (MeOH); 1H NMR (CDCl3) δ 1.25 (t, 3H, CH3CH2O), 3.60
(q, 2H, CH3CH2O), 4.80 (s, 2H, CH2OEt), 5.30 (s, 2H, CH2Ar),
7.20-7.60 (m, 11H, 10H Ar and 1H CHdCH), 7.80 (d, 1H,
CHdCH).
1-CCH3), 3.20 (s, 3H, 6-CCH3), 4.35 (q, 2H, CH2CH3), 7.30-7.60
(m, 4H, Ar), 8.15 (d, 2H, Ar).
4-Chloro-1,6-dimethyl-9-phenylpyrazolo[1′,5′:1,6]pyrimido-
[4,5-d]pyridazine, 8. A suspension of 6b (0.52 mmol) in POCl3
(1.5 mL, 9.5 mmol) was stirred at 130 °C for 2 h. After cooling,
the mixture was treated with cold water (10-15 mL) and the
precipitate was recovered by suction and washed with water. Yield
) 87%; mp ) 253-256 °C (EtOH); 1H NMR (DMSO-d6) δ 2.45
(s, 3H, 1-CCH3), 3.10 (s, 3H, 6-CCH3), 7.60 (m, 3H, Ar), 8.10 (s,
1H, Ar), 8.30 (m, 2H, Ar).
General Procedure for Compounds 9a,b. A suspension of 8
(0.2 mmol), the appropriate amine (benzylamine, methylendioxy-
benzylamine, 10-20 mmol), anhydrous ethanol (1 mL), and
triethylamine (0.05 mL) was stirred at 140 °C for 6 h in a sealed
tube. After cooling, the mixture was diluted with cold water and
compound 9a was recovered by suction. For compound 9b, the
mixture was evaporated and the residue was treated with hot
cyclohexane (3 × 10 mL). The final compound 9b was recrystal-
lized with ethanol.
4-Amino-2-benzyl-6-ethoxymethyl-5-(5′-phenyl-1H-pyrazol-
3-yl)pyridazin-3(2H)-one, 16. Compound 16 was obtained from
compound 15 following the general procedure described for
4a-n. Yield ) 77%; mp ) 197-199 °C (EtOH); 1H NMR (CDCl3)
δ 1.20 (t, 3H, CH3CH2O), 3.45 (q, 2H, CH3CH2O), 4.30 (s, 2H,
CH2OEt), 5.30 (s, 2H, CH2Ar), 7.00 (s, 1H, Ar), 7.20-7.60 (m,
8H, Ar), 8.00 (m, 2H, Ar).
4-Cyclopentyl-3-styrylisoxazolo[3,4-d]pyridazin-7(6H)-one, 17.
17 was obtained from 13b following the reported general pro-
cedure to obtain 3a-n. Yield ) 71%; mp ) 280-282 °C (EtOH);
1H NMR (CDCl3) δ 1.65-2.20 (m, 9H, cC5H9), 7.20 (d, 1H,
CHdCH), 7.45-7.65 (m, 5H, Ar), 7.85 (d, 1H, CHdCH).
4-Amino-6-cyclopentyl-5-(5′-phenyl-1H-pyrazol-3-yl)pyridazin-
3(2H)-one, 18. 18 was obtained from 17 following the general
procedure to obtain 4a-n. Yield ) 67%; mp ) 154-157 °C, dec
4-Benzyl-1,6-dimethyl-9-phenylpyrazolo[1′,5′:1,6]pyrimido-
[4,5-d]pyridazin-4-amine, 9a. Yield ) 96%; mp ) 231-233 °C
1
(EtOH); H NMR (CDCl3) δ 2.70 (s, 3H, 1-CCH3), 3.25 (s, 3H,
6-CCH3), 5.60 (s, 2H, CH2Ar), 7.20-7.53 (m, 10H, Ar), 7.95 (m,
1H, Ar).
1
(EtOH); H NMR (DMSO-d6) δ 1.45-1.90 (m, 9H, cC5H9), 6.90
4-(3,4-Methylendioxybenzyl)-1,6-dimethyl-2-phenylpyrazolo-
[1′,5′:1,6]pyrimido[4,5-d]pyridazin-4-amine, 9b. Yield ) 56%;
mp ) 250-253 °C (EtOH); 1H NMR (CDCl3) δ 2.70 (s, 3H,
1-CCH3), 3.40 (s, 3H, 6-CCH3), 5.50 (s, 2H, CH2Ar), 5.95 (s, 2H,
O-CH2-O), 6.75-6.85 (m, 3H, Ar), 7.20-7.65 (m, 4H, Ar), 7.90
(d, 2H, Ar).
(s, 1H, Ar), 7.25-7.75 (m, 7H, Ar), 7.50 (exch br s, 2H, NH2).
1-Cyclopentyl-6-methyl-9-phenylpyrazolo[1′,5′:1,6]pyrimido-
[4,5-d]pyridazin-4(3H)-one, 19. Compound 19 was obtained from
compound 18 following the general procedure described for
5a-m. Yield ) 46%; mp ) 216-220 °C (EtOH); 1H NMR

5370 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
GioVannoni et al.
(CDCl3) δ 1.80-2.20 (m, 9H, cC5H9), 3.20 (s, 3H, 6-CCH3), 7.40
(s, 1H, Ar), 7.45-7.60 (m, 3H, Ar), 8.05 (m, 2H, Ar).
mL), and the appropriate amine (0.5 mmol) (dimethylamine or
methylpiperazine) was stirred at room temperature for 2 h. For
compound 32a, after dilution with water, the solution was acidified
with 2 N HCl and extracted with CH2Cl2. The aqueous layer was
then was made alkaline with 6 N NaOH and extracted again with
CH2Cl2. Evaporation of the solvent afforded final compound 32a.
Compound 32b was isolated by dilution of the reaction mixture
with water, extraction with CH2Cl2, and evaporation of the solvent.
6-Benzyl-4-[(dimethylamino)methyl]-3-methylisoxazolo[3,4-
d]pyridazin-7(6H)-one, 32a. Yield ) 57%; mp ) 185-188 °C
3-Benzyl-1-ethoxymethyl-6-methyl-9-phenylpyrazolo[1′,5′:1,6]-
pyrimido[4,5-d]pyridazin-4(3H)-one, 20a. Compound 20a was
obtained from compound 16 following the general procedure
described for 5a-m. Yield ) 47%; mp ) 154-157 °C (EtOH);
1H NMR (CDCl3) δ 1.20 (t, 3H, CH3CH2O), 3.20 (s, 3H, 6-CCH3),
3.65 (q, 2H, CH3CH2O), 4.80 (s, 2H, CH2OEt), 5.50 (s, 2H,
CH2Ar), 7.20-7.60 (m, 9H, Ar), 8.10 (m, 2H, Ar).
3-Benzyl-1-cyclopentyl-6-methyl-2-phenylpyrazolo[1′,5′:1,6]-
pyrimido[4,5-d]pyridazin-4(3H)-one, 20b. Compound 20b was
obtained from compound 19 following the general procedure
described for 2a-i. In this case the mixture was stirred at 60 °C
for 5 h. Yield ) 43%; mp ) 205-207 °C (EtOH); 1H NMR
(CDCl3) δ 1.80-2.20 (m, 9H, cC5H9), 3.20 (s, 3H, 6-CCH3), 5.25-
7.60 (s, 2H, CH2Ar), 7.40 (m, 9H, Ar), 8.10 (m, 2H, Ar).
General Procedure for Compounds 28a,b. 28a,b were obtained
following the procedure described for compound 15, starting from
1,3-diketones 21a,b35,36 and compound 11. The mixture of the
isoxazole intermediates 22a,b and 23a,b as well as the isoxazol-
opyridazinones 24a,b-27a,b were not separated until the final
condensation with benzaldehyde. From this reaction the pure 28a,b
were isolated as precipitates.
1
(EtOH); H NMR (CDCl3) δ 2.30 (s, 6H, (CH3)2N), 2.85 (s, 3H,
3-CCH3), 3.60 (s, 2H, CH2N), 5.25 (s, 2H, CH2Ar), 7.20-7.45 (m,
5H, Ar).
6-Benzyl-4-[(4-methylpiperazin-1-yl)methyl]-3-methylisoxazolo-
[3,4-d]pyridazin-7(6H)-one, 32b. Yield ) 62%; mp ) 153-154
1
°C (EtOH); H NMR (CDCl3) δ 2.60 (s, 3H, CH3N), 2.70-2.85
(m, 8H, piperazine), 2.85 (s, 3H, 3-CCH3), 3.60 (s, 2H, CH2N),
5.30 (s, 2H, CH2Ar), 7.20-7.45 (m, 5H, Ar).
General Procedure for Compounds 33a,b. 33a,b were obtained
following the general procedure of 3a-n.
6-Benzyl-4-[(dimethylamino)methyl]-3-styrylisoxazolo[3,4-d]-
pyridazin-7(6H)-one, 33a. Yield ) 34%; mp > 300 °C, dec
1
(MeOH); H NMR (CDCl3) δ 2.40 (s, 6H, (CH3)2N), 3.60 (s, 2H,
CH2N), 5.30 (s, 2H, CH2Ar), 7.20-7.80 (m, 12H, 10H Ar and 2H
6-Benzyl-4-ethyl-3-styrylisoxazolo[3,4-d]pyridazin-7(6H)-
1
CHdCH).
one, 28a. Yield ) 25%; mp ) 176-178 °C (EtOH); H NMR
(CDCl3) δ 1.35 (t, 3H, CH3CH2), 2.90 (q, 2H, CH3CH2), 5.30 (s,
2H, CH2Ar), 7.20 (d, 1H, CHdCH), 7.25-7.60 (m, 10H, Ar), 7.80
(d, 1H, CHdCH).
6-Benzyl-4-[(4-methylpiperazin-1-yl)methyl]-3-styrylisoxazolo-
[3,4-d]pyridazin-7(6H)-one, 33b. Yield ) 43%; mp ) 178-181
1
°C (MeOH); H NMR (CDCl3) δ 2.20 (s, 3H, CH3N), 2.30-2.80
(m, 8H, piperazine), 3.65 (s, 2H, CH2N), 5.30 (s, 2H, CH2Ar),
7.20-7.80 (m, 12H, 10H Ar and 2H CHdCH).
6-Benzyl-4-isopropyl-3-styrylisoxazolo[3,4-d]pyridazin-7(6H)-
1
one, 28b. Yield ) 24%; mp ) 163-164 °C (EtOH); H NMR
(CDCl3) δ 1.40 (d, 6H, (CH3)2CH), 3.15-3.25 (m, 1H, CH(CH3)2),
5.30 (s, 2H, CH2Ar), 7.20 (d, 1H, CHdCH), 7.25-7.65 (m, 10H,
Ar), 7.80 (d, 1H, CHdCH).
General Procedure for Compounds 34a,b. 34a,b were obtained
following the general procedure of 4a-n.
4-Amino-2-benzyl-6-[(dimethylamino)methyl]-5-(5′-phenyl-
1H-pyrazol-3-yl)pyridazin-3(2H)-one, 34a. Yield ) 29%; mp )
4-Amino-2-benzyl-6-ethyl-5-(5′-phenyl-1H-pyrazol-3-yl)py-
ridazin-3(2H)-one, 29a. Compound 29a was obtained from
compound 28a following the general procedure described for
4a-n. Yield ) 73%; mp ) 189-190 °C (EtOH); 1H NMR (CDCl3)
δ 1.20 (t, 3H, CH3CH2), 2.65 (q, 2H, CH3CH2), 5.40 (s, 2H,
CH2Ar), 6.70 (s, 1H, Ar), 7.25-7.80 (m, 10H, Ar).
1
235-238 °C (EtOH); H NMR (CDCl3) δ 2.45 (s, 6H, (CH3)2N),
3.45 (s, 2H, CH2N), 5.40 (s, 2H, CH2Ar), 5.60 (exch br s, 2H,
NH2), 6.80 (s, 1H, Ar), 720-7.55 (m, 8H, Ar), 7.80 (m, 2H, Ar).
4-Amino-2-benzyl-6-[(4-methylpiperazin-1-yl)methyl]-5-(5′-
phenyl-1H-pyrazol-3-yl)pyridazin-3(2H)-one, 34b. Yield ) 67%;
mp ) 186-188 °C (EtOH); 1H NMR (CDCl3) δ 2.75 (s, 3H,
CH3N), 2.80-3.20 (m, 8H, piperazine), 3.55 (s, 2H, CH2N), 5.35
(s, 2H, CH2Ar), 5.70 (exch br s, 2H, NH2), 6.80 (s, 1H, Ar), 7.20-
7.50 (m, 8H, Ar), 7.80 (m, 2H, Ar).
4-Amino-2-benzyl-6-isopropyl-5-(5′-phenyl-1H-pyrazol-3-yl)-
pyridazin-3(2H)-one, 29b. Compound 29b was obtained from
compound 28b following the general procedure described for
4a-n. After dilution with water (10 mL), the mixture was extracted
with CH2Cl2 (3 × 15 mL). Evaporation of the solvent afforded
compound 29b. Yield ) 84%; mp ) 151-154 °C (EtOH); 1H NMR
(CDCl3) δ 1.10 (d, 6H, (CH3)2CH), 3.05-3.15 (m, 1H, CH(CH3)2),
5.40 (s, 2H, CH2Ar), 6.70 (s, 1H, Ar), 7.20-7.70 (m, 10H, Ar).
3-Benzyl-1-ethyl-6-methyl-9-phenylpyrazolo[1′,5′:1,6]pyrimido-
[4,5-d]pyridazin-4(3H)-one, 30a. 30a was obtained from 29a
following the general procedure to obtain 5a-m and 5o-q. Yield
General Procedure for Compounds 35a,b. 35a,b were obtained
following the general procedure of 5a-m and 5o-q.
3-Benzyl-1-[(dimethylamino)methyl]-6-methyl-9-phenylpyra-
zolo[1′5′:1,6]pyrimido[4,5-d]pyridazin-4(3H)-one, 35a. Yield )
1
52%; mp ) 197-200 °C (EtOH); H NMR (CDCl3) δ 2.40 (s,
6H, (CH3)2N), 3.20 (s, 3H, C-CH3), 3.80 (s, 2H, CH2N), 5.50 (s,
2H, CH2Ar), 7.20-7.55 (m, 9H, Ar), 8.00 (m, 2H, Ar).
3-Benzyl-1-[(4-methylpiperazin-1-yl)methyl-6-methyl-9-
phenylpyrazolo[1′5′:1,6]pyrimido[4,5-d]pyridazin-4(3H)-one, 35b.
Yield ) 58%; mp > 300 °C (EtOH); 1H NMR (CDCl3) δ 2.30 (s,
3H, CH3N), 2.40-2.80 (m, 8H, piperazine), 3.20 (s, 3H, 6-CCH3),
3.85 (s, 2H, CH2N), 5.45 (s, 2H, CH2Ar), 7.25-7.60 (m, 9H, Ar),
7.95-8.05 (m, 2H, Ar).
1
) 38%; mp > 300 °C (EtOH); H NMR (CDCl3) δ 1.40 (t, 3H,
CH3CH2), 3.10-3.25 (m, 5H, 2H CH3CH2 and 3H 6-CCH3), 5.55
(s, 2H, CH2Ar), 7.25-7.60 (m, 9H, Ar), 8.10 (m, 2H, Ar).
3-Benzyl-1-isopropyl-6-methyl-9-phenylpyrazolo[1′,5′:1,6]py-
rimido[4,5-d]pyridazin-4(3H)-one, 30b. 30b was obtained from
29b following the general procedure to obtain 5a-m and 5o-q.
1
Yield ) 57%; mp ) 202-204 °C (EtOH); H NMR (CDCl3) δ
Biological Assays. Inhibition of 3’:5’cGMP Phosphodiester-
ases. Purification of Phosphodiesterase Isoenzymes. PDE5 was
purified from human platelets as described by Gristwood et al.41
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.
1.40 (d, 6H, (CH3)2CH), 3.20 (s, 3H, 6-CCH3), 3.55-3.65 (m, 1H,
CH(CH3)2), 5.40 (s, 2H, CH2Ar), 7.20-7.55 (m, 9H, Ar), 8.10 (m,
2H, Ar).
6-Benzyl-4-bromomethyl-3-methylisoxazolo[3,4-d]pyridazin-
7(6H)-one, 31. A mixture of 14 (0.7 mmol) and HBr (1 mmol) in
acetic acid was stirred at 140 °C for 8 h in a sealed tube. After the
mixture was cooled and diluted with cold water (10 mL), a crude
precipitate was recovered by suction and purified by column
chromatography using 8:2 toluene/ethyl acetate as eluent. Yield )
PDE6 was purified from bovine retinas as described by Gillespie
and Beavo.42
The isoenzymes were characterized according to Beavo et al.43
by selectivity and affinity and by effect of calcium ions (10 µM)
plus calmodulin (1.2 µM), cyclic GMP (5 µM), and the selective
inhibitor sildenafil. PDE5 was kept frozen at -80 °C in the presence
of 1 g/L bovine serum albumin until use.
1
60%; mp ) 185-188 °C (EtOH); H NMR (CDCl3) δ 2.90 (s,
3H, 3-CCH3), 4.65 (s, 2H, CH2Br), 5.30 (s, 2H, CH2Ar), 7.20-
7.58 (m, 5H, Ar).
General Procedure for Compounds 32a,b. A mixture of
compound 31 (0.5 mmol), K2CO3 (1.1 mmol), anhydrous DMF (3
Phosphodiesterase Assay. Cyclic nucleotide phosphodiesterase
activities were measured using a two-step procedure according to

Pyrazolopyrimidopyridazinones as PDE5 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5371
Thompson and Strada.44 PDE5 and PDE6 (activated by 250
µg/mL trypsin) were assayed using 0.25 µM [3H]cGMP as substrate.
IC50 values were obtained by nonlinear regression using the Prism
program by GraphPad Software. The reported values are the average
of at least three independent assays. Sildenafil was used as the
reference substance. The drugs were dissolved in DMSO at 10-3
M. The effect of the solvent was taken into consideration in the
calculations.
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Supporting Information Available: 1H NMR spectral data for
derivatives 2b,d-i, 3b,d-n, 4b,e-n, 5a,c-q,t, and 7b,d and
elemental analysis results for all target compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
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