Isoxazolo[3,4-d]pyridazinones and analogues as Leishmania mexicana PDE inhibitors

Article abstract of DOI:10.1016/S0014-827X(01)01188-0

A series of isoxazolopyridazinones and analogues has been prepared and evaluated as Leishmania mexicana phosphodiesterase (PDE) inhibitors. Some of the synthesized compounds showed a moderate PDE inhibitory activity at 100 μM and preliminary structure-act

Full text of DOI:10.1016/S0014-827X(01)01188-0

Il Farmaco 57 (2002) 89–96
www.elsevier.com/locate/farmac
Isoxazolo[3,4-d]pyridazinones and analogues as Leishmania
mexicana PDE inhibitors
Vittorio Dal Piaz a,*, A. Rasco´n b, M.E. Dubra b, M.P. Giovannoni a, C. Vergelli a,
M.C. Castellana a
a Dipartimento di Scienze Farmaceutiche, Via G. Capponi 9, 50121 Florence, Italy
b Instituto de Biologia Experimental, Uni6ersidad Central de Venezuela, Apartado 47.069, Caracas 1041, Venezuela
Received 22 September 2001; accepted 28 October 2001
Abstract
A series of isoxazolopyridazinones and analogues has been prepared and evaluated as Leishmania mexicana phosphodiesterase
(PDE) inhibitors. Some of the synthesized compounds showed a moderate PDE inhibitory activity at 100 mM and preliminary
´
structure–activity relationships were discussed. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Leishmania mexicana; Phosphodiesterase inhibitors; Isoxazolopyridazinones
1. Introduction
turally different compound, displaying the same activity
is Cibacron blue which today represents the most po-
tent inhibitor of L. mexicana PDE (IC50=8 mM)
[11,12]. Therefore it makes interesting and feasible the
discovery of potent and selective PDE inhibitors, which
could be therapeutically useful to modulate cAMP le-
vels and proliferation in Leishmania, and maybe other
trypanosomatids.
In the tropics and subtropics, haemoflagellate proto-
zoan parasites of genus Leishmania are causative agents
of a variety of diseases representing a major health
problem known as leishmaniasis [1].
The only available drugs (stibogluconate, meglumine
antimonate, pentamidine and allopurinol) are of limited
efficacy and display serious side effects [2–4].
Recently some of us have characterized and purified
for the first time a highly specific cAMP phosphodies-
terase (PDE) from Leishmania mexicana which shows a
low sensitivity towards selective and non-selective PDE
inhibitors [5]. A similar refractoriness to inhibition has
been described for two recently cloned PDEs from
another member of the Trypanosomatidae family, Try-
panosoma brucei (TbPDE2A and TbPDE2B), the cau-
sing agent of sleeping sickness [6,7]. Nevertheless, non-
specific PDE inhibitors (caffeine, theophylline, papaver-
ine, dipyridamole and allopurinol) (Chart 1), although
very weak Leishmania PDE inhibitors, are able to in-
hibit the transformation of amastigotes to promastig-
otes and the proliferation of promastigotes in L.
dono6ani and L. tropica [8–10]. A completely struc-
* Corresponding author.
E-mail address: vittorio.dalpiaz@unifi.it (V. Dal Piaz).
Chart 1
´
0014-827X/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(01)01188-0

90
V. Dal Piaz et al. / Il Farmaco 57 (2002) 89–96
The absence of a useful template for modeling novel
2. Chemistry
agents on one hand and our experience in designing
and synthesizing mammalian PDE inhibitors [13,14]
bearing the pyridazine fragment on the other, led us to
start a research program aimed at the discovery of
novel chemical entities belonging to this chemical class
as Leishmania PDE inhibitors. Thus we evaluated as
inhibitors of this enzyme a group of isoxazolo[3,4-
d]pyridazin-3(2H)-ones, some of which had been pre-
viously described by us, as well as other related com-
pounds (Tables 1–3).
The key intermediates for the synthesis of the novel
compounds 5a–e, 6 and 9a–e were isoxazolo[3,4-
d]pyridazinones of type 4, which have been previously
described [13,15,16] with the exception of that with
R=n-propyl which was prepared as follows: condensa-
tion of ethyl chlorooximido acetate
2 with 1-
phenylexan-1,3-dione [17] 1 afforded the isoxazole 3
(Scheme 1) which in turn was cyclocondensed with
hydrazine to give 4. Alkylation of the appropriate 4
Table 1
Chemical and physical data of isoxazolopyridazinones and their inhibitory activity against Leishmania mexicana PDE
a
Comp.
R1
Y
R
X
Yield (%)
m.p. (°C)
Formula j
% inhibition k
A b
B c
C c
D d
E c
F e
G f
5a
H
CH3
H
CH3
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH2CN
(CH2)2OCH3
CH2COOH
CH2COOC2H5
(CH2)2COOH
(CH2)2COOC2H5
(CH2)3COOH
CH3
CH3
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
m-ClC6H4
p-ClC6H4
4-pyridyl
2-thienyl
C6H5
C6H5
C6H5
C6H5
C6H5
CH3
CH3
CH3
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
15.7
11.0
35.2
11.7
43.0
0
55.4
23.7
48.5
6.4
25.0
0
14.3
35.9
0
CH3
CH3
C2H5
nC3H7
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
60
76
122–124
163–166
C15H15N3O2
C18H10ClN3O2
5b
H g
I h
5c
5d
57
55
72
159–161
140–142
169–172
C11H9ClN3O2S
C14H10N4O2
C15H15N3O3
5e
J i
K i
L i
7.7
32.9
16.8
25.2
17.3
4.8
58.7
IC50=8 mM
M i
Ni
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
O i
6
62
75
187–189
135–137
C13H11N3OS
C13H11N3O2
11
Cibacron
a New compounds were rechrystallized from EtOH, with the exception of 11 which was from cyclohexane.
b Ref. [21].
c Ref. [22].
d Ref. [24].
e Ref. [13].
f Ref. [25].
g Ref. [26].
h Ref. [27].
i Ref. [20].
j C, N, H, analysis within 90.4%.
k All compounds were tested at 100 mM.

V. Dal Piaz et al. / Il Farmaco 57 (2002) 89–96
91
Table 2
Chemical and physical data of isoxazolo[3,4-d]pyridazine and of isoxazolo[4,5-d]pyridazine and their inhibitory activity against Leishmania
mexicana PDE
Comp.
R
Yield (%)
m.p. (°C)
Formula f
% inhibition g
P a
OMe
SMe
5.1
5.5
8 a
9a b
NHMe
NHEt
NHn-Pr
NHn-But
piperidine
Cl
NHMe
NHEt
NHn-Pr
NHn-But
piperidine
60
70
80
67
76
72
81
77
82
69
67
149–152
114–116
130–133
115–117
130–132
184–187
182 dec
165–167
146–149
160–162
130–134
C
13H12N4O
24.1
22.0
28.0
51.8
22.0
NT h
0
9b c
C14H14N4O
C15H16N4O
C16H18N4O
C17H18N4O
9c c
9d c
9e d
12 d
C12H8N3OCl
13a e
13b e
13c e
13d e
13e b
Cibacron
C13H12N4O
C14H14N4O
C15H16N4O
C16H18N4O
C17H18N4O
0
22.0
22.0
26.0
IC50=8 mM
a Ref. [18].
b Crystallized from EtOH–H2O 1:1.
c Purified by column chromatography using cyclohexane–ethyl acetate 1:2 as eluent.
d Crystallized from EtOH.
e Crystallized from cyclohexane.
f C, N, H, analysis within 90.4%.
g All compounds were tested at 100 mM.
h Not tested.
with the required halo derivative afforded the final
compounds 5a–e. When compound 5 with R=R1=
CH3 and Ar=C6H5 was refluxed with P2S5 in toluene,
compound 6 was obtained in good yield. In similar
conditions using Lawesson’s reagent, the intermediate 7
[18] was isolated and converted into the final 9a–e in
two steps: alkylation with iodomethane [18] and nucleo-
philic replacement with the appropriate (cyclo)-
alkylamine.
3. Experimental procedures
3.1. Chemistry
All melting points were determined on a Bu¨chi ap-
paratus and are uncorrected. 1H NMR spectra were
recorded with Varian Gemini 200 instruments. Chemi-
cal shifts are reported in ppm, using the solvent as
internal standard. Extracts were dried over Na2SO4 and
the solvents were removed under reduced pressure. E.
Merck F-254 commercial plates were used for analyti-
cal TLC to follow the course of reaction. Silica gel 60
(Merck 70-230 mesh) was used for column
chromatography.
The synthesis of isoxazolo[4,5-d]pyridazinones 11
and 13a–e is depicted in Scheme 2: treatment of 10 [19]
with POCl3 in the presence of triethylamine afforded
the corresponding 5-chloroderivative 12 which was con-
verted in the final 13a–e in the same reaction condi-
tions described for 9a–e. Compound 11 was prepared
from 10 by alkylation in standard conditions.
Finally the open models 15a,b, reported in Scheme 3,
were easily obtained by treatment of 5-acetyl-4-nitro
pyridazinones 14a,b [20] by nucleophilic displacement
with chlorine using TEBA in CH3CN.
All common reagents were purchased from Aldrich
Chemical and used without purification.
3.1.1. Synthesis of ethyl
4-benzoyl-5-n-propylisoxazolo-3-carboxylate (3)
(R=n-C3H7, Ar=C6H5)
To a cooled and stirred solution of sodium ethoxide
(13 mmol) in anhydrous EtOH (20 ml) was added a
1H NMR spectra of new compounds are shown in
Table 4.

92
V. Dal Piaz et al. / Il Farmaco 57 (2002) 89–96
solution of 1-phenylhexane-1,3-dione [17] (13 mmol) in
the same solvent (70 ml). Then a solution of ethyl
chlorooximido acetate [21] (13.3 mmol) in anhydrous
EtOH (10 ml) was added dropwise over a 1 h period.
After solvent evaporation, the residue was washed with
cold 0.5 N NaOH and water and extracted with CH2Cl2
(3×20 ml): 70% yield; oil (column chromatography,
After dilution with cold water, the crude precipitate
was recovered by suction.
3.1.4. Synthesis of 3,6-dimethyl-4-phenylisoxazolo-
[3,4-d]pyridazin-3(2H)-thione (6)
A
mixture of 3,6-dimethyl-4-phenylisoxazolo[3,4-
d]pyridazin-7(6H)-one [22] (1.6 mmol), P2S5 (10.0
mmol) in toluene (10 ml) was heated at 100 °C for 90
min. After cooling, the residue was filtered off and
toluene was evaporated in vacuo affording 6 as yellow
precipitate.
eluent:
cyclohexane/ethyl
acetate
1:1).
Anal.
(C16H17NO4) C, H, N.
3.1.2. Synthesis of 4-phenyl-3-n-propylisoxazolo-
[3,4-d]pyridazin-7(6H)-one (4) (R=n-C3H7,
Ar=C6H5)
3.1.5. General procedure for the synthesis of
The isoxazole 3 (R=n-C3H7, Ar=C6H5) (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 mix-
ture: 40% yield; m.p. 146–148 °C (EtOH). Anal.
(C14H13N3O2) C, H, N.
isoxazolo[3,4-d]pyridazine 9a-e
7-Methylthiopyridazine 8 [18] (1.5 mmol) and the
appropriate (cyclo)alkylamine (30.0 mmol) in EtOH (2
ml) were heated at 110–120 °C for 4–7 h in a sealed
tube. After cooling the mixture was diluted with cold
water. Compound 9a was recovered by suction, 9b–d
were obtained after extraction with CH2Cl2 (3×20 ml)
and evaporation in vacuo.
3.1.3. General procedure for the synthesis of
isoxazolo[3,4-d]pyridazinones 5a–e and of
For compound 9e the reaction was carried out in
toluene at 130 °C for 7 h. After cooling, the organic
layer was washed with water (10 ml) and the resultant
aqueous layer extracted with ethyl acetate (3×15 ml).
Compound 9e was obtained by the evaporation in
vacuum of the collected organic layers.
3,5-dimethyl-7-phenylisossazolo[4,5-d]pyridazinone (11)
A mixture of isoxazolopyridazinone 4 or 10 (1.6
mmol), anhydrous K2CO3 (13.0 mmol) and the appro-
priate haloderivative (15.0 mmol) in anhydrous DMF
(5 ml) was heated at 70–90 °C for 1–2 h under stirring.
Table 3
Chemical and physical data of pyridazinones and their inhibitory activity against Leishmania mexicana PDE
Comp.
R
R1
Yield (%)
m.p. (°C)
Formula f
% inhibition g
Q a
CH3
CH3
CH3
CH3
CH3
CH3
CH2COOC2H5
(CH2)2COOC2H5
NO2
Cl
Br
I
SCH3
NHC6H5
Cl
0
10.7
39.0
31.1
42.9
0
42.5
51.9
R b
S b
T c
U b
V d
15a e
15b e
Cibacron
93
95
113-115
oil
C16H15ClN2O4
C17H17ClN2O4
Cl
IC50=8 mM
a Ref. [25].
b Ref. [28].
c Ref. [29].
d Ref. [27].
e Compounds were purified by column chromatography using tolune–ethyl acetate 9:1 as eluent.
f C, N, H, analysis within 90.4%.
g All compounds were tested at 100 mM.

V. Dal Piaz et al. / Il Farmaco 57 (2002) 89–96
93
Scheme 1. (a) EtONa, abs. EtOH; (b) hydrazine hydrate, EtOH; (c) Lawesson’s reagent, toluene; (d) RX, DMF, K2CO3; (e) (cyclo)alkylamine;
(f) P2S5, toluene.
3.1.6. Synthesis of 4-chloro-3-methyl-7-
recovered by filtration after heating at 110 °C for 1 h
and the addition of water.
phenylisoxazolo[4,5-d]pyridazinone (12)
Et3N (0.1 mmol) was added to a cooled suspension
of compound 10 [19] (1.2 mmol) in POCl3 (3 ml). Then
the mixture was slowly heated at 100 °C under stirring
for 7 h. The precipitate was recovered by suction after
cooling and neutralization with 6 N NaOH.
3.1.8. General procedure for the synthesis of
5-acetyl-4-chloropyridazinones 15a,b
A mixture of the appropriate 5-acetyl-4-nitroderiva-
tive 14 [20] (0.8 mmol), TEBA (24.0 mmol) in CH3CN
(3 ml) was refluxed for 1–2 h. After concentration, ice
cold water was added and the mixture was extracted
with CH2Cl2 (3×20 ml). Evaporation of the solvent in
vacuo afforded the crude 15a,b which were purified by
column chromatography.
3.1.7. Synthesis of isoxazolo[4,5-d]pyridazines 13a–e
Compounds 13a–e were obtained following the pro-
cedure described above for 9a–d. All products were

94
V. Dal Piaz et al. / Il Farmaco 57 (2002) 89–96
Table 4
3.2. Biology
1H NMR spectra of new compounds
3.2.1. Phosphodiesterase assay
Comp. 1H NMR
PDE activity was assayed at 100 mM [3H]cAMP
([2,8-3H]cAMP, 37 Ci.mmol−1, from Amersham, UK)
according to the method of Kinkaid and Manganiello
[23]. The reactions were performed in a buffer contain-
ing 50 mM Hepes (pH 7.5), 0.1 mM EGTA, 8.3 mM
Mg chloride and 0.2 mg/ml BSA in a final reaction
volume of 300 ml, at 30 °C for 15 min. Hydrolysis of
substrate did not exceed 20% under these conditions
and cAMP–PDE activity was proportional to time and
enzyme concentration. The soluble fraction of L. mexi-
cana was used as a source of enzyme prepared as
described in Rasco´n et al. [5]. Assays were run in
triplicates and results are the average of at least two
independent assays. For inhibition studies, each syn-
thetic compound was dissolved in dimethyl sulfoxide
(DMSO) and serially diluted to a final concentration of
100 mM in the assay. Controls in presence of DMSO
were run parallel to samples. Special care was taken to
avoid a final concentrations of DMSO over 1% in the
assay which otherwise inactivates the enzyme.
3
1.00 (m, 6H, CH2CH2CH3); 1.80 (m, 2H, CH2CH2CH3);
2.80 (t, J=7.8 Hz, 2H, CH2CH2CH3); 7.50 (m, 5H, Ar);
10.0 (exch., br. s., 1H, NH).
0.90 (t, 3H, CH2CH2CH3 and COOCH2CH3); 1.80 (m,
2H, CH2CH2CH3); 2.90 (t, J=7.8 Hz, 2H, CH2CH2CH3);
4.10 (q, J=7.8 Hz, 2H, COOCH2CH3); 7.50 (m, 5H, Ar).
0.80 (t, J=7.8 Hz, 3H, (CH2)2CH3); 1.60 (m, 2H,
CH2CH3); 2.80 (t, J=7.9 Hz, CH2CH2CH3); 3.80 (s, 3H,
NCH3); 7.55 (s, 5H, Ar).
2.60 (s, 3H, CCH3); 3.80 (s, 3H, NCH3); 7.40–7.60 (m,
4H, Ar).
2.85 (s, 3H, CCH3); 3.80 (s, 3H, NCH3); 7.30–7.60 (m,
4H, Ar).
2.60 (s, 3H, CCH3); 5.15 (s, 2H, NCH2); 7.60 (m, 5H,
Ar).
2.55 (s, 3H, CCH3); 3.40 (s, 3H, OCH3); 3.80 (t, J=8.2
Hz, NCH2CH2); 4.45 (t, J=8.1 Hz, 2H, NCH2CH2); 7.55
(s, 5H, Ar).
2.60 (s, 3H, CCH3); 4.20 (s, 3H, NCH3); 7.60 (s, 5H, Ar).
2.65 (s, 3H, CCH3); 3.70 (d, J=6.5 Hz, 2H, NHCH3);
7.55 (s, 5H, Ar); 11.30–11.50 (exch. br. s., 1H, NH).
1.50 (t, J=8.0 Hz, 3H, CH2CH3); 1.80–2.10 (exch. br. s.,
1H, NH); 2.70 (s, 3H, CCH3); 4.10–4.25 (m, 2H,
CH2CH3); 7.55 (s, 5H, Ar).
1.05 (t, J=8.1 Hz, 3H, CH2CH3); 1.70–1.90 (m, 2H,
CH2CH2CH3); 2.65 (s, 3H, CCH3); 3.75 (t, J=8.1 Hz,
2H, CH2NH); 5.40–5.65 (exch. br. s., 1H, NH); 7.45–7.75
(m, 5H, Ar).
4
5a
5b
5c
5d
5e
6
9a
9b
9c
9d
1.00 (t, J=8.0 Hz, 3H, (CH2)3CH3); 1.40–1.60 (m, 2H,
(CH2)2CH2CH3); 1.70–1.85 (m, 2H, CH2CH2CH2CH3);
2.75 (s, 3H, CCH3); 3.75 (t, J=7.9 Hz, 2H, NHCH2);
5.40–5.80 (exch. br. s., 1H, NH); 7.45–7.55 (m, 3H, Ar);
7.60–7.70 (m, 2H, Ar).
9e
2.70–2.85 (m, 6H, piperidine); 2.60 (s, 3H, CCH3);
4.10–4.25 (m, 4H, piperidine); 7.45–7.65 (m, 5H, Ar).
2.75 (s, 3H, CCH3); 3.95 (s, 3H, NCH3); 7.45–7.55 (m,
3H, Ar); 8.10–8.20 (m, 2H, Ar).
11
12
2.85 (s, 3H, CH3); 7.55–7.65 (m, 3H, Ar); 8.25–8.35 (m,
2H, Ar).
13a
2.75 (s, 3H, CCH3); 3.35 (d, J=7.7 Hz, 3H, NHCH3);
4.85–5.00 (exch. br. s., 1H, NH); 7.45–7.60 (m, 3H, Ar);
8.35–8.45 (m, 2H, Ar).
13b
13c
1.45 (t, J=8.0 Hz, 3H, CH2CH3); 2.75 (s, 3H, CCH3);
3.80–3.95 (m, 2H, CH2CH3); 4.85–5.00 (exch. br. s., 1H,
NH); 7.40–7.60 (m, 3H, Ar); 8.35–8.45 (m, 2H, Ar).
1.10 (t, J=8.1 Hz, 3H, CH2CH3); 2.75–2.90 (m, 2H,
CH2CH3); 2.75 (s, 3H, CCH3); 3.75 (q, J=8.1 Hz,
CH2NH); 4.90–5.05 (exch. br. s., 1H, NH); 7.40–7.60 (m,
3H, Ar); 8.35–8.45 (m, 2H, Ar).
Scheme 2. (a) POCl3, Et3N; (b) CH3I, DMF, K2CO3; (c) (cy-
clo)alkylamine.
13d
1.00 (t, J=8.0 Hz, 3H, (CH2)3CH3); 1.20–1.40 (m, 2H,
(CH2)2CH2CH3); 1.70–1.85 (m, 2H, CH2CH2CH2CH3);
2.75 (s, 3H, CCH3); 3.80 (t, J=7.9 Hz, 2H, NHCH2);
4.90–5.10 (exch. br. s., 1H, NH); 7.45–7.60 (m, 3H, Ar);
8.35–8.45 (m, 2H, Ar).
13e
15a
15b
1.60–1.90 (m, 6H, piperidine); 2.75 (s, 3H, CCH3);
3.50–3.65 (m, 4H, piperidine); 7.45–7.60 (m, 3H, Ar);
8.40–8.50 (m, 2H, Ar).
1.35 (t, J=8.1 Hz, 3H, COOCH2CH3); 2.25 (s, 3H,
COCH3); 4.30 (q, J=8.1 Hz, 2H, COOCH2CH3); 5.00 (s,
2H, NCH2); 7.45 (s, 5H, Ar).
1.25 (t, J=8.0 Hz, 3H, COOCH2CH3); 2.20 (s, 3H,
COCH3); 2.95 (t, J=8.0 Hz, 2H, CH2COOCH2CH3); 4.15
(q, J=8.0 Hz, 2H, COOCH2CH3); 4.65 (t, 7.9 Hz, 2H,
NCH2); 7.40–7.55 (m, 5H, Ar).
Scheme 3. (a) TEBA, CH3CN.

V. Dal Piaz et al. / Il Farmaco 57 (2002) 89–96
95
4. Result and discussion
phenylpyridazinones are reported in Table 3. In the
series of compounds Q–V can be observed that the
presence of both oxidated and reduced nitrogen func-
tions is detrimental (Q and V); among the halo-deriva-
tives the 4-bromo was the most potent. Finally, the
presence of a methylthio group in position 4 (U) was
associated with a good level of activity. When in com-
pound R the methyl group at position 2 was replaced
with an ethylacetate (15a) or ethylpropionate (15b)
residue, a significant improvement of activity was
observed.
All compounds were tested at 100 mM concentration
for their ability to inhibit the activity of PDE extracted
from Leishmania mexicana [5]. Cibacron blue was
tested in the same conditions and used as reference
compound. Results are reported in Tables 1–3, where
the data obtained by testing all novel compounds and
several previously synthesized analogues (A–V) [24–29]
are indicated. Although none of the tested compounds
showed a similar level of activity in comparison with
Cibacron blue, some interesting results could be empha-
sized and preliminary structure–activity relationships
could be obtained.
Further studies are in progress in order to obtain
more potent and selective inhibitors.
In the series of isoxazolo[3,4-d]pyridazinones (Table
1) the best results were furnished by compounds E, G
and 5b. The presence of a phenyl group in 4-position of
the bicyclic system plays an important role. In fact
replacement of this group with a methyl (B), 4-pyridyl
(I) or the introduction in para position of a chlorine
(H), was detrimental. The presence of a 2-thienyl group
completely abolished the activity (5c). On the other
hand, moving chlorine in the meta position (5b) com-
pletely restored the activity. It is interesting to observe
that compound 11 which is the isomer of the previously
examined E, was the most potent in this group (58.7%
inhibition), probably indicating that two methyl and a
phenyl group are the best arranged in this bicyclic
system.
In position 3, the length of the carbon chain plays a
fundamental role, the presence of the ethyl group being
associated with the best activity (G, 55.4% inhibition).
Homologation or elimination of this carbon chain re-
sulted in the loss of activity (compounds D and 5a). As
regards the substituent in position 6, the methyl repre-
sents the best group (E), its homologation being very
detrimental (F). Introduction in the same position of a
functionalized alkyl chain gave interesting results for
R1=(CH2)2OCH3 (5e) and (CH2)2COOH (L). It is
interesting to observe that, with the exception of com-
pound K, the carboxylic derivatives are more potent
than the corresponding esters. Finally, replacement of
the carbonyl dipole with CꢀS group led to a dramatic
loss of activity (6), indicating that the hydrogen bond
acceptor properties could play a significant role in the
activity.
Acknowledgements
This work was supported by grants from the
Venezuelan Consejo Nacional de Investigaciones Cien-
t´ıficas y Tecnolo´gicas (20010000654) and the Consejo
de Desarrollo Cient´ıfico y Human´ıstico de la Universi-
dad Central de Venezuela (03.33.4833.01).
References
[1] R.W. Ashford, P. Desjeux, P. De Raad, Estimation of popula-
tion at risk of infection and number of cases of leishmaniasis,
Parasitol. Today 8 (1992) 104–105.
[2] J.D. Berman, Chemotherapy for leishmaniasis: biochemical
mechanism, clinical efficacy and future strategies, Rev. Infect.
Dis. 10 (1988) 560–586.
[3] R.A. Gasser, A.J. Magill, C.N. Oster, E.D. Franke, M. Grogl,
J.D. Berman, Pancreatitis induced by pentavalent antimonial
agents during treatment of leishmaniasis, Clin. Infect. Dis. 18
(1994) 83–90.
[4] L.T. Webster Jr., Drugs used in chemotherapy of protozoal
infections, in: A.G. Gilman, L.S. Goodman (Eds.), The Pharma-
cological Basis of Therapeutics, Macmillan Publishing Company,
New York, 1985, pp. 1058–1065.
[5] A. Rasco´n, M.E. Viloria, L. De-Chiara, M.E. Dubra, Character-
ization of cyclic AMP phosphodiesterases in Leishmania mexi-
cana and purification of
Parasitol. 106 (2000) 283–292.
[6] R. Zoraghi, S. Kunz, K. Gong, T. Seebeck, Characterization of
TbPDE2A, a novel cyclic nucleotide-specific phosphodiesterase
from the protozoan parasite Trypanosoma brucei, J. Biol. Chem.
276 (2001) 11559–11566.
a soluble form, Mol. Biochem.
[7] A. Rasco´n, S. Soderling, J. Schaefer, J. Beavo, Cloning and
characterization
of
cAMP
specific
phosphodiesterase
(TbPDE2B) from Trypanosoma brucei, submitted for
publication.
In Table
2 the data related to isoxazolo[3,4-
d]pyridazines and their [4,5-d]condensed isomers 13a–e
are reported. Obtained data seem to indicate that the
presence of the alkoxy and methylthio groups in posi-
tion 7 of the isoxazolo[3,4-d]pyridazine system is detri-
mental for the activity. Among the different alkyl- and
cycloalkylamino derivatives 9a–e, compound 9d
emerged (51.8% inhibition). On the contrary, in the
isomeric series 13a–e no compounds showed interesting
activity. The data obtained for a group of 5-acetyl-6-
[8] R.D. Walter, E. Buse, F. Ebert, Effect of cAMP on transforma-
tion and proliferation of Leishmania cells, Tropenmed. Parasit.
29 (1987) 439–447.
[9] T. Seebeck, K. Gong, S. Kunz, R. Schaub, T. Shalaby, R.
Zoraghi, cAMP signalling in Trypanosoma brucei, Int. J. Para-
sitol. 31 (2001) 491–498.
[10] S.W. Kamau, R. Nunez, F. Grimm, Flow cytometry analysis of
effect of allopurinol and the dinitroaniline compound (Chloralin)
on the viability and proliferation of Leishmania infantum pro-
mastigotes, BMC Pharmacol. 1 (2001) 1.

96
V. Dal Piaz et al. / Il Farmaco 57 (2002) 89–96
[11] D. Hanggi, P. Carr, Analytical evaluation of the purity of
commercial preparations of Cibacron Blue F3GA and related
dyes, Anal. Biochem. 149 (1985) 91–104.
[12] S.J. Burton, S.S. McLoughlin, C.V. Stead, C.R. Lowe, Design
and applications of biomimetic antraquinone dyes. I. Synthesis
and characterization of terminal ring of C.I. reactive blue 2, J.
Chromatogr. 435 (1988) 127–137.
[13] V. Dal Piaz, M.P. Giovannoni, M.C. Castellana, J.M. Palacios,
J. Beleta, T. Domenech, Novel heterocyclic-fused pyridazinones
as potent and selective phosphodiesterase IV inhibitors, J. Med.
Chem. 40 (1997) 1417–1421.
dazin-7-(6H)-one as a potential substrate for new aldose reduc-
tase inhibitors, J. Med. Chem. 42 (1999) 1894–1900.
[21] G. Renzi, V. Dal Piaz, Investigation on some 4,5-disubstituted-3-
ethoxycarbonyl isoxaloles, Gazz. Chim. Ital. 95 (1965) 1478–
1491.
[22] G. Renzi, S. Pinzauti, New derivatives of isoxazolo[3,4-
d]pyridazin-7(6H)-one system, Farmaco Ed. Sci. 24 (1969) 885–
889.
[23] R.L. Kincaid, V.C. Manganiello, Assay of cyclic nucleotide
phosphodiesterase using radiolabeled and fluorescent substrates,
Adv. Enzymol. 159 (1988) 457–470.
[14] V. Dal Piaz, M.P. Giovannoni, M.C. Castellana, J.M. Palacios,
J. Beleta, T. Domenech, V. Segarra, Heterocyclic-fused pyridazi-
nones as potent and selective PDE IV inhibitors: further struc-
ture–activity relationships and molecular modeling studies, Eur.
J. Med. Chem. 33 (1998) 789–797.
[15] V. Dal Piaz, M.C. Castellana, C. Vergelli, M.P. Giovannoni, A.
Gavalda`, V. Segarra, J. Beleta, H. Ryder, J.M. Palacios, Synthe-
sis and evaluation of some pyrazolo[3,4-d]pyridazinones and
analogues as PDE5 inhibitors potentially useful as peripheral
vasodilators, submitted for publication.
[16] V. Sprio, E. Aiello, A. Mazza, Studies on nitrogen heterocycles.
Hydrogenation of isoxazolo [3,4-d]pyridazin-4-ones and isoxa-
zolo [3,4-d]pyridazin-4,7-diones, Ann. Chim. 57 (1967) 836–845.
[17] R. Levine, J.A. Conroy, J.T. Adams, C.R. Hanser, The acylation
of ketones with esters to form b-diketones by sodium amide
method, J. Am. Chem. Soc. 67 (1945) 1510–1512.
[24] G. Renzi, V. Dal Piaz, C. Musante, Condensation between
hydroxymic chlorides and b-oxobenzoyl derivatives, Gazz. Chim.
Ital. 98 (1968) 656–666.
[25] V. Dal Piaz, G. Ciciani, G. Turco, Oxidative cleavage of 7-oxo-
6,7-dihydroisoxazolo[3,4-d]pyridazines by cerium(IV) diammo-
nium nitrate.
A synthetic approach to new 5-acyl-4-nitro-
3-oxo-2,3-dihydropyridazines, Synthesis (1989) 213–214.
[26] V. Dal Piaz, G. Ciciani, M.P. Giovannoni, Synthesis of 4,5-func-
tionalized-2-methyl-6-(substituted aryl)-3(2H)-pyridazinones:
a
new group of potent platelet aggregation inhibitors, Farmaco 52
(1997) 173–178.
[27] V. Dal Piaz, G. Ciciani, G. Turco, M.P. Giovannoni, M. Miceli,
R. Pirisino, M. Perretti, 5-Acyl-6-ary-4-nitro-3(2H)pyridazinones
and related 4-amino compounds: synthesis and pharmacological
evaluation, J. Pharm. Sci. 80 (1991) 341–348.
[28] V. Dal Piaz, M.P. Giovannoni, R. Laguna, E. Cano, Synthesis
and evaluation of some 4,5-disubstituted-6-phenyl-3(2H)-pyri-
dazinones as hypotensive agents, Eur. J. Med. Chem. 29 (1994)
249–252.
[18] V. Dal Piaz, S. Pieretti, C. Vergelli, M.C. Castellana, M.P.
Giovannoni, 4-Amino-5-vinyo`-3(2H)-pyridazinones and related
compounds: synthesis and evaluation of antinociceptive activity,
J. Med. Chem., in press.
[29] V. Dal Piaz, G. Ciciani, M.P. Giovannoni, F. Franconi, Synthe-
sis and evaluation as platelet aggregation inhibitors of 6-phenyl-
2,4-substituted-3(2H)-pyridazinones and their rigid analogues
benzo[h]cinnolin-3,5-diones, Drug Design Discovery 14 (1996)
53–75.
[19] B. Chantegrel, C. Deshayes, B. Pujol, Z.J. Wie, Synthesis of
isoxazolo[4,5-d]pyridazin-4(5H)-ones and 4-acyl-5-hydroxy-
3(2H)-pyridazinones, J. Heterocyclic Chem. 27 (1990) 927–934.
[20] L. Costantino, G. Rastelli, M.C. Gamberini, M.P. Giovannoni,
V. Dal Piaz, P. Vianello, D. Barlocco, Isoxazolo-[3,4-d]-pyri-