Novel heterocyclic-fused pyridazinones as potent and selective phosphodiesterase IV inhibitors

Article abstract of DOI:10.1021/jm970105l

A series of 6-aryl-4,5-heterocyclic-fused pyridazinones were designed and synthesized as selective phosphodiesterase (PDE) IV inhibitors. Biological evaluation of these compounds demonstrated a good selectivity profile toward the PDE IV family and greatly

Full text of DOI:10.1021/jm970105l

J . Med. Chem. 1997, 40, 1417-1421
1417
Novel Heter ocyclic-F u sed P yr id a zin on es a s P oten t a n d Selective
P h osp h od iester a se IV In h ibitor s
Vittorio Dal Piaz,* Maria Paola Giovannoni, and Carla Castellana
Dipartimento di Scienze Farmaceutiche, via Gino Capponi 9, 50121 Firenze, Italy
J ose´ Maria Palacios, J orge Beleta, Teresa Dome´nech, and Victor Segarra
Laboratorios Almirall, S.A., Research Center, Cardener, 68-74, 08024 Barcelona, Spain
Received February 21, 1997X
A series of 6-aryl-4,5-heterocyclic-fused pyridazinones were designed and synthesized as
selective phosphodiesterase (PDE) IV inhibitors. Biological evaluation of these compounds
demonstrated a good selectivity profile toward the PDE IV family and greatly attenuated affinity
for the Rolipram high-affinity binding site that seems to be responsible for undesiderable side
effects. Structure-activity relationships (SARs) studies showed that the presence of an ethyl
group at pyridazine N-2 is associated with the best potency and selectivity profile.
Ch a r t 1
In tr od u ction
Phosphodiesterases (PDEs) are enzymes responsible
for the hydrolysis of cyclic adenosine monophosphate
(c-AMP) and cyclic guanosine monophosphate (c-GMP),
which are second messengers involved in the regulation
of important cell functions, such as secretion, contrac-
tion, metabolism, and growth.1 At least seven different
families of PDEs are known at present, characterized
by different tissue distribution as well as by different
functional roles.2,3
Presently the interest in therapeutic utility of PDEs
inhibitors (PDEIs) is mostly focused on new agents
selectively inhibiting the PDE IV family,4 one of the
c-AMP specific PDEs. These inhibitors are promising
drugs in the treatment of asthma, inflammation, and
several CNS pathologies: in fact PDE IV isoenzyme is
particulary abundant in brain5 and immunocompetent
cells such as neutrophiles,6 T-lymphocytes,7 macro-
phages,8 and eosinophiles,9 where PDE IV inhibitors
reduce the synthesis and release of proinflammatory
mediators, cytokines, and active oxygen species.2 More-
over they modulate “in vitro” the tone of bronchial
smooth muscle tissue and have “in vivo” bronchodilatory
effects.10 Since inflammation is generally accepted as
an important component in the pathogenesis of asthma,11
these data seem to confirm the therapeutic utility of
PDE IV inhibitors in the bifunctional treatment of
asthma.
Rolipram (1), which is the best known PDE IV inhib-
itor, was clinically tested as an antidepressant several
years before the discovery of its potent and selective
PDE IV inhibitory activity.12 Rolipram and its ana-
logs,13,14 as well as Rolipram structurally unrelated PDE
IV inhibitors, like Nitraquazone (2)15,16 and congeners
3,17 exhibit Rolipram binding site affinity in the nano-
molar range for preparation of animal and human brain
tissue homogenate. Very recently the high-affinity
Rolipram binding site has been associated with emesis
and nausea, which are common side effects of PDE IV
inhibitors. Thus it appears particularly important to
dissociate affinity for this binding site from PDE IV
inhibitory activity in order to obtain more selective
agents potentially useful as antiasthmatics. This goal
was recently reached with several Rolipram analogs.18,19
In connection with our studies on the chemistry and
pharmacology of pyridazine derivatives,20-22 we syn-
thesized a group of heterocyclic-fused pyridazinones 4
structurally related to 2 and 3. Compounds 4 still
retain the pyridazione ring, present in 3, from which
they significantly differ in the condensed system, which
was modified with the aim to possibly have potent and
selective PDE IV inhibitors with attenuated affinity for
Rolipram binding site.
Ch em istr y
Compounds 5a -c were synthesized by 1,3-dipolar
cycloaddition between the appropriate nitriloxide23 and
the suitable 1,3-diketone.24 Ring closure of 5 with
hydrazine, followed by treatment with the appropriate
alkyl bromide, afforded 7a -d . Following a procedure
previously reported by us,25 oxidative cleavage of the
isoxazole ring with cerium ammonium nitrate (CAN)
gave the key intermediates 8a -d (Scheme 1).
X Abstract published in Advance ACS Abstracts, April 15, 1997.
S0022-2623(97)00105-2 CCC: $14.00 © 1997 American Chemical Society

1418 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Dal Piaz et al.
Sch em e 1a
selectivity profile (PDE III/PDE IV > 30). Moreover this
compound, as well as 9c,d , 10a ,c, and 12, which also
emerged for their good balance of potency and selectivity
versus PDE IV, displayed an affinity for Rolipram
binding site by 2 orders of magnitude lower than that
of compound 3 (X ) N, R ) Cl, R1 ) 4-pyridyl) and
Rolipram. It should be highlighted that for the majority
of our compounds a remarkable better balance between
potency and selectivity was evidenced, with respect to
the reference drugs, as clearly indicated by low value
of PDE IV/[3H]Rolipram ratio.
Due to their structural resemblance with xanthines
and analogs, the synthesized compounds were also pre-
liminarly evaluated for their affinity toward A1 and A2
adenosine receptors. The absence of significant affinity
(data not shown) confirmed the interesting profile of
these derivatives as potential antiasthmatic and anti-
inflammatory agents, devoided of CNS and cardiovas-
cular side effects.
In conclusion these data seem to demonstrate that
for compounds structurally related to 2 and 3 it is
possible to dissociate the affinity for [3H]Rolipram
binding site and PDE IV inhibitory activity.
Further structure-activity relationships studies as
well as the in vivo evaluation of some selected com-
pounds are in progress.
a
(a) NH2NH2; (b) alkyl bromide; (c) CAN.
Compounds 5a , 6a , 7b, and 8b were previously
described.26-28
Exp er im en ta l Section
Ch em istr y. All melting points were determined with Buchi
510 melting point apparatus and are uncorrected. Infrared
spectra (IR) were recorded as Nujol mulls with a Perkin-Elmer
spectrometer. Nuclear magnetic resonance (1H-NMR) spectra
were obtained using Gemini 200 spectrometer in CDCl3.
Chemical shift values are reported in ppm (δ). Analyses
indicated by the symbols of the elements or function were
within (0.4% of the theoretical values. Analytical TLC using
E. Merck F-254 commercial plates was used to follow the
course of reaction. Silica gel 60 (Merck 70-230 mesh) was
used for column chromatography. Extracts were dried over
sodium sulfate, and solvents were removed under reduced
pressure.
Treatment of 8a -d with sodium ethyl thioglycolate
in absolute ethanol at room temperature afforded the
thienopyridazinones 9a -d in moderate yields. Follow-
ing similar reaction conditions pyrazolopyridazinones
10a -d were synthesized using hydrazine or methylhy-
drazine. Finally, the synthesis of pyrrolopyridazinones
11a ,b and pyridopyridazinone 12 was performed, gener-
ally in good yield, in two steps: treatment with sar-
cosine ethyl ester and N-methyl-â-alaninenitrile, re-
spectively, in ethanol at room temperature, followed by
short heating with sodium ethoxide in ethanol.
N,N-Dimethylformamide (DMF) was purchased from Ald-
rich Chemical Co. and dried over 4 Å molecular sives before
use.
Biologica l Resu lts a n d Discu ssion
All of the final compounds were tested as PDE III and
PDE IV inhibitors and evaluated for their ability to
displace [3H]Rolipram from its binding site. Rolipram,
Milrinone,29 and Syntex compound 3 (X ) N, R ) Cl,
R1 ) 4-pyridyl)17 were tested as reference substances
(see Table 1).
Unlike compounds 3, all compounds substituted at
pyridazine N-2 with an ethyl group, with the exception
of only 10c, were 12-50-fold more potent as PDE IV
inhibitors with respect to the corresponding benzyl
analogs, which also showed lower selectivity. In the
series of pyrazolopyridazinones, replacement of hydro-
gen in compound 10a with a methyl group (10b) brought
about a 2.5-fold reduction of activity. The same struc-
tural modification performed on 10c led to a 60-fold
reduction of potency (10d ).
In the thienopyridazinones series, compound 9a dis-
played high potency but low selectivity; introduction at
the meta position of the phenyl ring of a nitro (9c) or a
chlorine substituent (9d ) left the potency versus PDE
IV unchanged, but strongly enhanced the selectivity.
The pyrrolopyridazinone 11a was the most interesting
compound, having a IC50 for PDE IV in the same sub-
micromolar range of Rolipram and showing a good
Eth yl 5-Meth yl-4-(3-n itr oben zoyl)isoxa zole-3-ca r boxy-
la te (5b). To a cooled and stirred solution of sodium ethoxide
(0.3 g, 13 mmol) in anhydrous EtOH (20 mL) was added a
solution of 1-(3-nitrophenyl)butane-1,3-dione24 (2.8 g, 13 mmol)
in the same solvent (70 mL). Then a solution of ethyl chloro-
(hydroximino)acetate25 (2.0 g, 13.3 mmol) in anhydrous EtOH
(10 mL) was added dropwise over 1 h period. After solvent
evaporation, the residue was washed with cold 0.5 N NaOH
and water and recovered by suction: 67% yield; mp 105-106
1
°C (EtOH); IR (cm-1) 1740 (CO), 1670 (CO), 1350 (NO2); H-
NMR 1.15 (t, 3H, J ) 7.2 Hz, CH2CH3), 2.60 (s, 3H, CH3), 4.15
(q, 2H, J ) 7.2 Hz, CH2CH3), 7.70 (t, 1H, J ) 8.1 Hz, Ar), 8.10
(d, 1H, J ) 8.0 Hz) Ar), 8.40-8.55 (m, 2H, Ar). Anal.
(C14H12N2O6) C, H, N.
E t h yl 4-(3-Ch lor ob en zoyl)-5-m et h ylisoxa zole-3-ca r -
boxyla te (5c). Compound 5c was synthesized following the
procedure reported for 5b, using the same molar ratio: 51%
yield; mp 67-68 °C (EtOH); IR (cm-1) 1740 (CO), 1670 (CO);
1H-NMR 1.15 (t, 3H, J ) 7.2 Hz, CH2CH3), 2.60 (s, 3H, CH3),
4.20 (q, 2H, J ) 7.2 Hz, CH2CH3), 7.40-7.65 (m, 3H, Ar), 7.75
(s, 1H, Ar). Anal. (C14H12ClNO4) C, H, N.
3-Met h yl-4-(3-n it r op h en yl)isoxa zolo[3,4-d ]p yr id a zin -
7(6H)-on e (6b). The isoxazole 5b (0.15 g, 0.5 mmol) was
dissolved in EtOH (5 mL), and then 0.15 mL of hydrazine
hydrate was added. After 1 h the crude product was collected
by suction from the cooled mixture: 87% yield; mp 255-256
1
°C (EtOH); IR (cm-1) 3280 (NH), 1670 (CO), 1530 (NO2); H-

Pyridazinones as Phosphodiesterase IV Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1419
Sch em e 2a
a
(a) NaSCH2COOC2H5; (b) NH2NH2 or CH3NHNH2; (c) (1) NH2CH2COOC2H5; (2) EtONa; (d) (1) CH3NH(CH2)2CN; (2) EtONa.
Ta ble 1. Effects of Heterocyclic-Fused Pyridazinones 9-12 on PDE III and PDE IV Isoenzymes and Displacement of [3H]Rolipram
from Its Binding Site
compd
9a
9b
9c
9d
10a
10b
10c
10d
11a
11b
12
milrinone
rolipram
3d
PDE IIIa,b
5.9 ( 1.4
PDE IVa,b
0.9 ( 0.2
[3H]ROLa,c
PDE IV/[3H]ROL
1.8 ( 0.2
0.50
0.50
3.25
0.50
2.00
-
20.0 ( 2.8 (20 µM)
25.0 ( 1.0 (20 µM)
56.0 ( 8.0 (20 µM)
35.0 ( 2.0 (20 µM)
35.0 ( 4.0 (20 µM)
60.0 ( 26.0 (200 µM)
30.0 ( 6.0 (200 µM)
30.0 ( 3.0 (20 µM)
33.0 ( 9.0 (20 µM)
51.0 ( 0.3 (20 µM)
0.73 ( 0.03
11.0 ( 1.0
1.3 ( 0.3
22.0 ( 2.0
0.4 ( 0.1
0.8 ( 0.4
1.6 ( 0.2
2.0 ( 0.3
1.0 ( 0.2
7.4 ( 0.7
46.0% ( 3.0 (10 µM)
54.0% ( 7.0 (10 µM)
11.0% ( 6.0 (10 µM)
2.0 ( 1.0
3.1 ( 0.4
-
-
45.0 ( 9.0 (200 µM)
0.6 ( 0.1
0.30
-
31.0 ( 9.0
1.1 ( 0.4
28.0% ( 2.0 (10 µM)
1.0 ( 0.4
1.10
242 ( 11.0
5.1 ( 2.0
0.32 ( 0.09
0.056 ( 0.01
0.006 ( 0.004
0.0048 ( 0.001
53.33
11.67
a
b
Data are indicated as IC50 (µM) ( SEM or inhibition percentage ( SEM at indicated concentration (µM) (n ) 3). PDE III and PDE
IV were obtained from guinea pig ventricular tissue30 and dosed following the procedure of Thompson et al. (ref 31). c [3H]Rolipram tests
were performed using brain membranes according to ref 13. X ) N, R ) Cl, R1 ) 4-pyridyl (ref 17).
d
NMR (DMSO-d6) 2.55 (s, 3H, CH3), 7.85 (t, 1H, J ) 8.3 Hz,
Ar), 8.15 (d, 1H, J ) 8.3 Hz, Ar), 8.35-8.50 (m, 2H, Ar). Anal.
(C12H8N4O4) C, H, N.
4-(3-Ch lor op h en yl)-3-m eth ylisoxa zolo[3,4-d ]p yr id a zin -
7(6H)-on e (6c). Compound 6c was synthesized following the
same procedure reported for 6b: 85% yield; mp 252-253 °C
(EtOH); IR (cm-1) 3290 (NH), 1670 (CO); 1H-NMR (DMSO-
d6) 2.50 (s, 3H, CH3), 7.55-7.70 (m, 4H, Ar). Anal. (C12H8-
ClN3O2) C, H, N.
following the procedure reported for 7a , using the same molar
ratio: 84% yield; mp 138-140 °C (EtOH); IR (cm-1) 1665 (CO);
1H-NMR 1.40 (t, 3H, J ) 6.9 Hz, CH2CH3), 2.55 (s, 3H, CH3),
4.25 (q, 2H, J ) 6.9 Hz, CH2CH3), 7.40-7.60 (m, 4H, Ar). Anal.
(C14H12ClN3O2) C, H, N.
5-Acetyl-2-eth yl-4-n itr o-6-p h en yl-3(2H)-p yr id a zin on e
(8a ). To a stirred suspension of isoxazolo[3,4-d]pyridazinone
7a (0.3 g, 1.2 mmol) in a mixture of 50% AcOH (10 mL) and
14.4 M HNO3 (2 mL) was added portionwise cerium am-
monium nitrate (CAN) (4.5 g, 8 mmol) at 55 °C during 30 min.
Addition of ice-water (50 mL) furnished a precipitate which
was recovered by suction: 45% yield; mp 107-108 °C (EtOH);
IR (cm-1) 1720 (CO), 1680 (CO), 1550 and 1350 (NO2); 1H-NMR
1.50 (t, 3H, J ) 6.9 Hz, CH2CH3), 2.15 (s, 3H, CH3), 4.40 (q,
2H, J ) 6.9 Hz, CH2CH3), 7.40-7.55 (m, 4H, Ar). Anal.
(C14H13N3O4) C, H, N.
5-Ace t yl-2-e t h yl-4-n it r o-6-(3-n it r op h e n yl)-3(2H )-p y-
r id a zin on e (8c). Compound 8c was synthesized following the
procedure reported for 8a , using the same molar ratio: 60%
yield; mp 118-119 °C (EtOH); IR (cm-1) 1710 (CO), 1680 (CO),
1530 and 1350 (NO2); 1H-NMR 1.50 (t, 3H, J ) 7.0 Hz,
CH2CH3), 2.25 (s, 3H, CH3), 4.45 (q, 2H, J ) 7.0 Hz, CH2CH3),
7.65-7.75 (m, 2H, Ar), 8.30-8.45 (m, 2H, Ar). Anal.
(C14H12N4O6) C, H, N.
6-E t h yl-3-m et h yl-4-p h en ylisoxa zolo[3,4-d ]p yr id a zin -
7(6H)-on e (7a ). A mixture of isoxazolopyridazinone 6a 27 (0.5
g, 2.2 mmol), anhydrous K2CO3 (1.8 g, 13 mmol) and ethyl
bromide (1.9 g, 15 mmol) in anhydrous DMF (6 mL) was heated
at 60 °C with stirring for 30 min. After dilution with cold
water (80-100 mL), the suspension was extracted with ethyl
acetate (3 × 60 mL). Removal of the solvent afforded com-
pound 7a : 59% yield; mp 132-135 °C (EtOH); IR (cm-1) 1680
1
(CO); H-NMR 1.40 (t, 3H, J ) 7.0 Hz, CH2CH3), 2.50 (s, 3H,
CH3), 4.25 (q, 2H, J ) 7.0 Hz, CH2CH3), 7.55 (s, 5H, Ar). Anal.
(C14H13N3O2) C, H, N.
6-Eth yl-3-m eth yl-4-(3-n itr op h en yl)isoxa zolo[3,4-d ]p y-
r id a zin -7(6H)-on e (7c). Compound 7c was synthesized
following the procedure reported for 7a , using the same molar
ratio: 73% yield; mp 132-134 °C (EtOH); IR (cm-1) 1660 (CO),
1
1350 (NO2); H-NMR 1.40 (t, 3H, J ) 7.0 Hz, CH2CH3), 2.60
5-Acet yl-6-(3-ch lor op h en yl)-2-et h yl-4-n it r o-3(2H )-p y-
r id a zin on e (8d ). Compound 8d was synthesized following
the procedure reported for 8a , using the same molar ratio: 42%
yield; mp 104-105 °C (EtOH); IR (cm-1) 1710 (CO), 1680 (CO),
(s, 3H, CH3), 4.30 (q, 2H, J ) 7.0 Hz, CH2CH3), 7.80 (s, 2H,
Ar), 8.50 (s, 2H, Ar). Anal. (C14H12N4O4) C, H, N.
4-(3-Ch lor op h en yl)-6-et h yl-3-m et h ylisoxa zolo[3,4-d ]-
p yr id a zin -7(6H)-on e (7d ). Compound 7d was synthesized
1
1540 (NO2); H-NMR 1.50 (t, 3H, J ) 7.0 Hz, CH2CH3), 2.20

1420 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Dal Piaz et al.
(s, 3H, CH3), 4.40 (q, 2H, J ) 7.0 Hz, CH2CH3), 7.40-7.60 (m,
Eth yl 6,7-Dih yd r o-6-eth yl-3-m eth yl-4-p h en yl-1H-p yr -
r olo[2,3-d ]p yr id a zin e-2-ca r boxyla te (11a ). A mixture of
8a (0.1 g, 0.3 mmol) and sarcosine ethyl ester (0.07 g, 0.6
mmol) in EtOH (3-4 mL) was stirred at room temperature.
for 20 min. The suspension was diluted with ice-water and
4-N-ethylglycinate recovered by suction. The crude product
was then added to EtONa prepared from Na (0.06 g, 2.6 mmol)
and absolute EtOH (4 mL), and the mixture was heated at 50
°C for 30 min. After dilution with water, the mixture was
acidified with 6 N HCl and the final product was isolated by
suction: 85% yield; mp 184-186 °C (EtOH); IR (cm-1) 3200
4H, Ar). Anal. (C14H12ClN3O4) C, H, N.
E t h yl 6,7-Dih yd r o-6-et h yl-3-m et h yl-7-oxo-4-p h en yl-
th ien o[2,3-d ]p yr id a zin e-2-ca r boxyla te (9a ). To a solution
of sodium ethyl thioglycolate, prepared from EtONa (0.35 g, 5
mmol) and ethyl thioglycolate (0.6 g, 5 mmol) in anhydrous
EtOH (10 mL), was added pyridazinone 8a (0.24 g, 0.8 mmol)
dissolved in the same solvent (2 mL). The mixture was stirred
at room temperature for 30 min, after it was diluted with ice-
water and the crude precipitate recovered by suction: 46%
yield; mp 151-152 °C (EtOH); IR (cm-1) 1730 (CO), 1660 (CO);
1H-NMR 1.35-1.55 (m, 6H, 2 × CH2CH3), 2.15 (s, 3H, CH3),
4.30-4.45 (m, 4H, 2 × CH2CH3), 7.35-7.60 (m, 5H, Ar). Anal.
(C18H18N2O3S) C, H, N.
1
(NH), 1720 (CO), 1660 (CO); H-NMR 1.35-1.50 (m, 6H, 2 ×
CH2CH3), 2.10 (s, 3H, CH3), 4.30-4.50 (m, 4H, 2 × CH2CH3),
7.50 (s, 5H, Ar). Anal. (C18H19N3O3) C, H, N.
Eth yl 6-Ben zyl-6,7-d ih yd r o-3-m eth yl-4-p h en yl-1H-p yr -
r olo[2,3-d ]p yr id a zin e-2-ca r boxyla te (11b). Compound 11b
was synthesized following the procedure reported for 11a ,
using the same molar ratio: 84% yield; mp 153-154 °C
(EtOH); IR (cm-1) 3180 (NH), 1720 (CO), 1660 (CO); 1H-NMR
1.40 (t, 3H, J ) 7.1 Hz, CH2CH3), 2.15 (s, 3H, CCH3), 4.40 (q,
2H, J ) 7.1 Hz, CH2CH3), 5.50 (s, 2H, CH2), 7.20-7.55 (m,
10H, 2Ar). Anal. (C23H21N3O3) C, H, N.
E t h yl 6-Ben zyl-6,7-d ih yd r o-3-m et h yl-7-oxo-4-p h en yl-
th ien o[2,3-d ]p yr id a zin e-2-ca r boxyla te (9b). Compound
9b was synthesized following the procedure reported for 9a ,
using the same molar ratio: 40% yield; mp 141 °C (EtOH); IR
(cm-1) 1700 (CO), 1650 (CO); 1H-NMR 1.40 (t, 3H, J ) 7.0 Hz,
CH2CH3), 2.15 (s, 3H, CH3), 4.40 (q, 2H, J ) 7.0 Hz, CH2CH3),
5.45 (s, 2H, CH2), 7.25-7.55 (m, 10H, 2Ar). Anal.
(C23H20N2O3S) C, H, N.
3-Cya n o-1,4-d im et h yl-7-et h yl-8-oxo-5-p h en yl-1,2,7,8-
tetr a h yd r op yr id o[2,3-d ]p yr id a zin e (12). A mixture of 8a
(0.15 g, 0.5 mmol) and N-methyl-â-alaninenitrile (0.1 g, 1.2
mmol) in EtOH (5 mL) was stirred at room temperature for
20 min. The suspension was diluted with water (40 mL) and
extracted with CH2Cl2 (3 × 20 mL). Evaporation of the solvent
afforded a brown oil which was dissolved in EtOH (3 mL) and
added to a solution of EtONa prepared from Na (0.11 g, 2.5
mmol) and absolute EtOH (5 mL). The mixture was heated
at 50-60 °C for 90 min. Then it was diluted with ice-water,
and the final product was recovered by suction: 45% yield;
Eth yl 6,7-Dih yd r o-6-eth yl-3-m eth yl-4-(3-n itr op h en yl)-
7-oxoth ien o[2,3-d ]p yr id a zin e-2-ca r boxyla te (9c). Com-
pound 9c was synthesized following the procedure reported
for 9a , using the same molar ratio: 61% yield; mp 183-184
1
°C (EtOH); IR (cm-1) 1720 (CO), 1660 (CO); H-NMR 1.35-
1.55 (m, 6H, 2 × CH2CH3), 2.15 (s, 3H, CH3), 4.30-4.45 (m,
4H, 2 × CH2CH3), 7.35-7.60 (m, 4H, Ar). Anal. (C18H17N3O5S)
C, H, N.
Eth yl 4-(3-Ch lor op h en yl)-6,7-d ih yd r o-6-eth yl-3-m eth -
yl-7-oxoth ien o[2,3-d ]p yr ida zin e-2-ca r boxyla te (9d ). Com-
pound 9d was synthesized following the procedure reported
for 9a , using the same molar ratio: 38% yield; mp 158-159
1
mp 119-121 °C (EtOH); IR (cm-1) 2200 (CN), 1640 (CO); H-
NMR 1.40 (t, 3H, J ) 7.0 Hz, CH2CH3), 1.65 (s, 3H, CCH3),
3.75 (s, 3H, NCH3), 3.90 (s, 2H, CH2), 4.20 (q, 2H, J ) 7.0 Hz,
CH2CH3), 7.40 (s, 5H, 2Ar). Anal. (C18H18N4O) C, H, N.
Biology. Dr u gs a n d Rea gen ts. [8-3H]Adenosine 3′:5′
cyclic monophosphate was from Amersham (Bucks, UK).
Benzamidine, cAMP, calmodulin, and PMSF (phenyl meth-
anesulfonyl fluoride) were obtained from Sigma-Aldrich Quim-
ica S.A. (Madrid, Spain). Milrinone was obtained from IMPEX
Quimica (Barcelona, Spain).
1
°C (EtOH); IR (cm-1) 1720 (CO), 1660 (CO); H-NMR 1.35-
1.55 (m, 6H, 2 × CH2CH3), 2.20 (s, 3H, CH3), 4.20-4.45 (m,
4H, 2 × CH2CH3), 7.25-7.55 (m, 4H, Ar). Anal. (C18H17
-
ClN2O3S) C, H, N.
6-E t h yl-3-m et h yl-4-p h en ylp yr a zolo[3,4-d ]p yr id a zin -
7(6H)-on e (10a ). To a suspension of nitro derivative 8a (0.1
g, 0.3 mmol) in EtOH (5 mL) was added an excess of hydrate
hydrazine (1.5 mmol), and the mixture was stirred at room
temperature for 10 min. Compound 10a was directly recov-
Rolipram was synthesized in the Department of Chemical
Synthesis, Laboratorios Almirall, S.A., Spain.
ered by suction: 57% yield; mp 233-234 °C (EtOH); IR (cm-1
)
3150 (NH), 1650 (CO); 1H-NMR 1.50 (t, 3H, J ) 7.0 Hz,
CH2CH3), 2.25 (s, 3H, CH3), 4.45 (q, 2H, J ) 7.0 Hz, CH2CH3),
7.45-7.65 (m, 5H, Ar). Anal. (C14H14N4O) C, H, N.
P u r ifica tion of P h osp h od iester a se Isoen zym es. Cyclic
nucleotide phosphodiesterases III and IV were obtained from
guinea pig ventricular tissue following the procedure described
by Gristwood et al.30 Briefly, the tissue was homogenized in
20 mM Bis-Tris pH 6.5 buffer, containing 50 mM sodium
acetate, 2 mM benzamidine, 2 mM EDTA, 5 mM â-mercapto-
ethanol, and 50 µM PMSF using an Ultraturrax homogenizer.
The sample was centrifuged at 40000g for 20 min, and the
supernatant was filtered through a 0.22 µm filter. The clean
sample was chromatographed on a 1 mL ion-exchange MONO-Q
column equilibrated with the same buffer using an FPLC
system. The column was developed at a flow rate of 1 mL/
min using a linear gradient of sodium acetate from 50 to 1000
mM in a total volume of 25 mL. Fractions of 500 µL were
collected.
1,3-Dim eth yl-6-eth yl-4-ph en ylpyr azolo[3,4-d]pyr idazin -
7(6H)-on e (10b). Compound 10b was synthesized following
the procedure reported for 10a using methylhydrazine in the
same molar ratio of hydrazine. In this case the mixture was
diluted with ice-water and the product recovered by suction:
1
60% yield; mp 147-148 °C (EtOH); IR (cm-1) 1670 (CO); H-
NMR 1.45 (t, 3H, J ) 7.1 Hz, CH2CH3), 2.15 (s, 3H, CCH3),
4.30 (q, 2H, J ) 7.1 Hz, CH2CH3), 4.40 (s, 3H, NCH3), 7.50 (s,
5H, Ar). Anal. (C15H16N4O) C, H, N.
6-Ben zyl-3-m eth yl-4-p h en ylp yr a zolo[3,4-d ]p yr id a zin -
7(6H)-on e (10c). Compound 10c was synthesized following
the procedure reported for 10a starting from 8b and using the
same molar ratio. In this case the reaction mixture was
diluted with water and extracted with ethyl acetate. The crude
product was purified by column chromatography (eluent:
cyclohexane/ethyl acetate, 1:2): 85% yield; mp 167-168 °C
(EtOH); IR (cm-1) 3140 (NH), 1650 (CO); 1H-NMR 2.30 (s, 3H,
CH3), 5.55 (s, 2H, CH2), 7.25-7.40 (m, 3H, Ar), 7.45-7.60 (m,
7H, Ar). Anal. (C19H16N4O) C, H, N.
The isoenzymes were characterized prior to use in terms of
substrate selectivity and affinity and by the effect of calcium
ions (10 µM) plus calmodulin (1.2 µM) and the selective
inhibitors Rolipram, SK&F 94120. Active fractions were
pooled and kept frozen at -20 °C in presence of 1 g/L bovine
serum albumin until used.
P h osp h od iester a se Assa y. Cyclic nucleotide phosphodi-
esterases were assayed following the procedure of Thompson
and Strada (1984).31 Inhibition assays were run in duplicate
at a substrate concentration of 0.25 µM. Substrate was cAMP
for PDE III and IV. IC50 values were obtained by nonlinear
regression using the program InPlot from GraphPad Software.
Drugs were dissolved in DMSO, and the effects of this solvent
were taken into consideration in the calculations.
6-Ben zyl-1,3-d im et h yl-4-p h en ylp yr a zolo[3,4-d ]p yr i-
d a zin -7(6H)-on e (10d ). Compound 10d was synthesized
from 8b and methylhydrazine following the procedure reported
for 10c and using the same molar ratio. The crude product
was purified by column chromatography (eluent: cyclohexane/
ethyl acetate, 1:1): 88% yield; mp 166 °C (EtOH); IR (cm-1
1670 (CO); H-NMR 2.15 (s, 3H, CCH3), 4.35 (s, 3H, NCH3),
5.45 (s, 2H, CH2), 7.25-7.40 (m, 3H, Ar), 7.45-7.60 (m, 7H,
Ar). Anal. (C20H18N4O) C, H, N.
)
1
[3H]Rolip r a m Disp la cem en t. The binding of [3H]Rolip-
ram to rat brain membranes was performed according to

Pyridazinones as Phosphodiesterase IV Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1421
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