Arylpiperazinylalkylpyridazinones and analogues as potent and orally active antinociceptive agents: Synthesis and studies on mechanism of action

Article abstract of DOI:10.1021/jm060743g

A number of arylpiperazinylalkylpyridazinones structurally related to the previously described lead A (5-{[4-(3-chlorophenyl)piperazin-1-yl]-propyl}-3- methyl-7-phenylisossazolo[4,5-d]pyridazin-4-(5H)-one) were synthesized and tested for their analgesic activity. Many of the tested molecules, at the dose of 20 mg kg^-1 p.o., showed high antinociceptive activity, in particular, compounds 5a, 11c, 15a, 21 and 22, which were able to reduce the number of abdominal constrictions by more than 50% in writhing test. The pharmacological investigation of lead A led us to clarify the mechanism of action of this compound, showing that it carries out its analgesic action through the inhibition of reuptake of noradrenaline. The antinociception of some of the most interesting new molecules was completely prevented by pretreatment with α₂-antagonist yohimbine, suggesting the involvement of α₂-adrenoceptors, as with prototype A.

Full text of DOI:10.1021/jm060743g

7826
J. Med. Chem. 2006, 49, 7826-7835
Arylpiperazinylalkylpyridazinones and Analogues as Potent and Orally Active Antinociceptive
Agents: Synthesis and Studies on Mechanism of Action
Nicoletta Cesari,† Claudio Biancalani,† Claudia Vergelli,† Vittorio Dal Piaz,† Alessia Graziano,† Pierfrancesco Biagini,†
Carla Ghelardini,‡ Nicoletta Galeotti,‡ and Maria Paola Giovannoni*,†
Dipartimento di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino Firenze, Italy, and
Dipartimento di Farmacologia Clinica e Preclinica, Viale Pieraccini 6, 50139 Firenze, Italy
ReceiVed June 22, 2006
A number of arylpiperazinylalkylpyridazinones structurally related to the previously described lead A (5-
{[4-(3-chlorophenyl)piperazin-1-yl]-propyl}-3-methyl-7-phenylisossazolo[4,5-d]pyridazin-4-(5H)-one) were
synthesized and tested for their analgesic activity. Many of the tested molecules, at the dose of 20 mg kg-1
p.o., showed high antinociceptive activity, in particular, compounds 5a, 11c, 15a, 21 and 22, which were
able to reduce the number of abdominal constrictions by more than 50% in writhing test. The pharmacological
investigation of lead A led us to clarify the mechanism of action of this compound, showing that it carries
out its analgesic action through the inhibition of reuptake of noradrenaline. The antinociception of some of
the most interesting new molecules was completely prevented by pretreatment with R2-antagonist yohimbine,
suggesting the involvement of R2-adrenoceptors, as with prototype A.
Introduction
The identification of compounds able to treat both acute and
chronic pain states with limited side effects is one of the
prominent goals in biomedical research. In fact, the clinical use
of the two major classes of analgesic drugs, nonsteroidal anti-
inflammatory drugs (NSAIDs) and opioids, is associated with
important side effects, which include gastrointestinal lesions and
nefrotoxicity (NSAIDs),1 respiratory depression, tolerance, and
physical dependence (opioids).2 The identification and charac-
terization of an inducible form of cyclooxygenase (COX-2)3
led to the development of more selective NSAIDs with fewer
unwanted side effects.4 Recent results demonstrated that these
drugs represent a good choice for patients at high risk of serious
gastrointestinal complications, but not for patients suffering from
cardiovascular diseases.5-7
Finally, a particular type of pain, the neuropathic pain, is very
difficult to treat since it is often refractory to conventional
analgesic drugs, with the exception of particular opioids, which
may be used with success in some cases.8 Neuropathic pain is
a very complex phenomenon that involves several mechanisms
both in the peripheral and in the central nervous system, and it
is characterized by burning pain together with hyperalgesia and
allodynia. At present, the first-line treatment options for this
Figure 1. Lead A and general structure of compounds B.
pathology are represented by the so-called “analgesic adjuvants”,
dose; MAD ) minimal analgesic dose).17 The most interest-
such as antidepressants, anticonvulsivants, and local anaesthetics
ing term, the 5-{[4-(3-chlorophenyl)piperazin-1-yl]propyl}-3-
(gabapentin, lidocaine, tramadol, nortriptyline, doxepine).9 Many
methyl-7-phenylisoxazolo[4,5-d]pyridazinon-4-(5H)-one A,
drugs belonging to different therapeutic classes, such as NMDA
showed an MNTD/MAD ratio value of 200 in the hot plate
receptor antagonists, R2-agonists, nicotinic, and adenosine
test, an efficacy of 104% with respect to morphine, and it was
receptor agonists, are in development as promising agents for
able to almost completely abolish abdominal constrictions 15
the treatment of neuropathic pain.10,11
and 30 min after administration at a dose of 20 mg/kg, sc
Previous studies in the field of antinociceptive agents12-16
(writhing test). Because a similar effect was observed after icv
have led us to identify a group of potent antinociceptive
administration, it is reasonable to suppose that its site of action
compounds that are active in the hot-plate test, with an efficacy
comparable to that of morphine in the same test and showing a
very good MNTD/MAD ratio (MNTD ) maximal nontoxic
is within the central nervous system. Moreover, the analgesia
induced by compound A (Figure 1) was completely prevented
by pretreatment with reserpine, indicating that the antino-
ciceptive effect is mediated by a partial or complete activation
of the monoaminergic system.17
* To whom correspondence should be addressed. Tel.: +39-55-4573682.
Fax: +39-55-4573780. E-mail: mariapaola.giovannoni@unifi.it.
On this basis, we designed and synthesized a new series of
derivatives bearing an arylpiperazinyl moiety linked to different
† Dipartimento di Scienze Farmaceutiche.
‡ Dipartimento di Farmacologia Clinica e Preclinica.
10.1021/jm060743g CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/06/2006

AntinociceptiVe Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7827
Scheme 2a
Scheme 1a
a Reagents and conditions: (a) ethyl 3-bromopropionate, K2CO3, anhy-
drous DMF, rt, 15h; (b) 6 N NaOH, EtOH, rt, 15h; (c) SOCl2, NEt3, T )
60 °C, 2 h; (d) chlorophenylpiperazine, anhydrous THF, rt, 2 h.
a Reagents and conditions: (a) Br-alkyl-Br, K2CO3, anhydrous DMF, T
) 60 °C, 1-2 h; (b) H2SO4/HNO3 1:1 v/v, 0 °C, 15 min; (c) SnCl2, concd
HCl, rt, 12h; (d) substituted arylpiperazine, K2CO3, anhydrous DMF, rt-
60 °C, 5-15 h; (e) 1-(3-haloalkyl)-4-(3-chlorophenyl)-piperazine, K2CO3,
anhydrous DMF, rt, 4h.
Scheme 3a
heterocyclic systems through (functionalized)alkyl chains (com-
pounds B) and we evaluated all compounds for their ability to
produce an analgesic effect. To define the role played by the
different structural elements (arylpiperazinyl moiety, linker, and
heterocyclic system) for the analgesic activity, we performed a
relevant number of modifications. Thus, in addition to the
previously investigated isoxazolo[4,5]pyridazinone, at the het-
erocyclic system level we explored phtalazinone, isoindole-1,3-
dione, pyrazolo[3,4-d]pyridazinone, and pyrido[2,3-d]pyridazi-
none systems and, at the spacer level, we performed elongation,
branching, and functionalization.
At the same time, we investigated in greater depth and
elucidated the mechanism of action of lead compound A.
Chemistry
The synthetic pathways followed to obtain the final com-
pounds are depicted in Schemes 1-7.
The synthesis of isoxazolo[4,5-d]pyridazinones 5a-k is
reported in Scheme 1 (for compounds 2 and 5 see Table 1 and
Table 2), and it was performed starting from the precursors 1a,b,
previously described by Chantegrel and co-workers.18 Com-
pound 1b (RdH), is fully characterized in the present work;
a Reagents and conditions: (a) Br-alkyl-Br, K2CO3, anhydrous DMF,
rt, 90-120 min; (b) halophenylpiperazine, K2CO3, anhydrous DMF, rt,
15 h.
1
the above paper reports its identification by H NMR signals,
but not its purification.
with the appropriate substituted arylpiperazine in anhydrous
DMF.
Treatment of 1a with a mixture of H2SO4/HNO3 1:1 v/v at 0
°C afforded a mixture of the intermediates 3a,b, which were
separated by column chromatography; compound 3b was treated
with SnCl2 in concd HCl to give the o-amino derivative 4.
Starting from the above-described intermediates (1, 3, and 4),
the introduction of the alkylpiperazinylaryl fragment was
performed in two different ways: (1) by a direct alkylation with
1-(3-haloalkyl)-4-(3-chlorophenyl)-piperazine, prepared by con-
densing 3-chlorophenylpiperazine with 1-bromo-3-halopropane
(route e for compounds 5g and 5k); and (2) by a two-step
procedure (routes a and d), which consists of the synthesis of
the N-alkylated intermediates 2a-g, followed by condensation
Isoxazolo[4,5-d]pyridazinones 8a,b, bearing a functionalized
chain at N-5, were prepared as reported in Scheme 2, starting
from the precursor 1a and performing an alkylation with ethyl
3-bromopropionate. Intermediate 6 was converted into the
corresponding carboxylic acid (compound 7), which, by treat-
ment with SOCl2, followed by condensation with the appropriate
chlorophenylpiperazine in anhydrous THF at room temperature,
afforded the final 8a,b.
In Scheme 3 is reported the synthesis of isoindole-1,3-dione
derivatives 11a-c, which were prepared starting from com-
mercially available compound 9. This precursor was alkylated

7828 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Cesari et al.
Scheme 4a
Scheme 6a
a Reagents and conditions: (a) 1-(3-bromopropyl)-4-(3-chlorophenyl)-
piperazine, K2CO3, anhydrous DMF, T ) 60-90 °C, 1.5-5 h.
Scheme 7a
a Reagents and conditions: (a) Br(CH2)nBr, K2CO3, anhydrous DMF,
rt, 2 h; (b) piperazine, K2CO3, anhydrous DMF, rt, 15 h; (c) m- and
p-chlorophenylpiperazine, K2CO3, anhydrous DMF, rt, 15 h; (c) m- and
p-chlorophenylpiperazine, K2CO3, anhydrous DMF, rt, 2 h; (d) m-CN-
phenylboronic acid, Cu(AcO)2, anhydrous CH2Cl2, NEt3; (e) appropriate
1-(3-haloalkyl)-4-(chlorophenyl)piperazine, K2CO3, anhydrous DMF, rt, 2
h.
Scheme 5a
a Reagents and conditions: (a) Lawesson’s reagent, toluene; (b) 1-(3-
bromopropyl)-4-(4-fluororophenyl)-piperazine, K2CO3, anhydrous acetone,
rt, 4 h.
Table 1. Legend for Compounds 2
2
R
linker
a
b
c
d
e
f
H
H
(CH2)3
(CH2)4
(CH2)2
(CH2)3
(CH2)4
(CH2)3
(CH2)3
Ph
Ph
Ph
o-NO2Ph
p-NO2Ph
g
by condensation with the appropriate substituted phenylpiper-
azine in anhydrous DMF (routes a and c). To obtain compound
15e, the intermediate 13a was condensed with piperazine leading
compound 14, which, in turn, was treated with m-CN-phenyl-
boronic acid, Cu(AcO)2 in anhydrous CH2Cl2 (routes a, b, and
d).
In Schemes 5 and 6, the synthetic pathways affording products
18, 21, and 22 are described.19-21 These final compounds exhibit
the same propyl-4-(3-chlorophenyl)-piperazine fragment of the
lead A, but they differ from this latter for the heterocondensed
system, which is a pyrido[2,3-d]pyridazin-8-one (compound 18),
an isoxazolo[3,4-d]pyridazin-7(6H)-one (compound 21) or a
pyrazolo[3,4-d]pyridazin-7-one (compound 22). For all com-
pounds, the starting products have been previously described
in literature,19-21 with the exception of intermediate 17, which
a Reagents and conditions: (a) anhydrous acetone, EtONa, abs EtOH, T
) 90 °C, 12 h; (b) 1-(3-bromopropyl)-4-(3-chlorophenyl)-piperazine, K2CO3,
anhydrous DMF, rt, 15 h.
to afford 10a,b which, in turn, were condensed with the
appropriate arylpiperazine in standard conditions to give
11a-c.
The final compounds 15a-e (Scheme 4) were obtained from
the commercially available 2H-phtalazin-1-one 12. For com-
pounds 15a,b, the alkylpiperazinylaryl substructure, prepared
by condensing the appropriate arylpiperazine with the opportune
alkyl halide, was inserted in one step (route e). The synthesis
of compounds 15c-e was performed through the intermediates
13a,b, which were transformed into the final compounds (15c,d)

AntinociceptiVe Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7829
Table 2. Legend for Compounds 5
Table 3. Antinociceptive Effect of Final Compounds in the Writhing
Test and Hot Plate Testa
5
R
linker
(CH2)3
(CH2)4
(CH2)3
(CH2)3
(CH2)3
(CH2)4
CH2CH(CH3)CH2
(CH2)2
X
no. of writhes no. of writhes
a
b
c
d
e
f
g
h
i
H
H
m-ClPh
m-ClPh
p-FPh
in the
in the
dose
absence
of yohimbine of yohimbine
presence
licking
latency
Ph
Ph
Ph
Ph
Ph
Ph
treatment
(mg kg-1
)
2-Py
p-ClPh
m-ClPh
m-ClPh
m-ClPh
m-ClPh
m-ClPh
m-ClPh
CMC
yohimbine
5a
5b
5c
5d
5e
5f
5g
5h
5i
5J
5k
6
8a
8b
11a
11b
11c
15a
15b
15c
15d
15e
18
34.5 ( 2.2
35.9 ( 3.7
15.3 ( 1.7
15.8 ( 1.5
27.7 ( 1.9b
3
20
5
11.9 ( 3.4b
27.2 ( 3.5
39.2 ( 5.0
30.2 ( 5.1
27.1 ( 2.4
30.6 ( 3.6
35.5 ( 2.7
23.6 ( 2.8b
31.7 ( 4.4
26.8 ( 2.5
28.0 ( 3.1
32.7 ( 5.3
22.1 ( 4.2b
23.5 ( 3.4b
22.6 ( 3.2b
34.9 ( 4.4
19.4 ( 3.8b
8.2 ( 2.5b
36.7 ( 4.0
23.9 ( 3.2b
28.5 ( 4.1
17.2 ( 2.9b
18.0 ( 2.6b
16.2 ( 3.4b
10.3 ( 3.9b
28.6 ( 4.6
27.4 ( 3.5
37.2 ( 4.3
o-NO2Ph
p-NO2Ph
o-NH2Ph
(CH2)3
(CH2)3
(CH2)3
30
30
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
10
20
20
20
20
17.6 ( 1.8
15.1 ( 2.1
j
k
was obtained by treatment of 1619 with anhydrous acetone and
EtONa in absolute ethanol in the sense of Friedlander’s
synthesis; the condensation of 17, 19,20 and 2021 with the
piperazinyl substructure was performed in standard conditions
using K2CO3 in anhydrous DMF, as depicted in Scheme 1.
Finally, to define the importance of the lactamic function for
the antinociceptive activity, we synthesized compound 24
(Scheme 7), bearing the alkylpiperazinyl chain linked at posi-
tion 4 of the pyridazine ring. Starting compound 1a was
transformed into the corresponding 4-thione derivative (23),
using Lawesson’s reagent in toluene. Treatment of 23 with 1-(3-
bromopropyl)-4-(fluorophenyl)-piperazine22 regioselectivity af-
forded the final compound 24.
15.4 ( 2.0
16.5 ( 2.2
17.4 ( 1.5
41.3 ( 4.4
45.4 ( 3.8
37.9 ( 4.1
17.2 ( 2.5
16.2 ( 2.0
36.5 ( 3.6b
28.0 ( 2.2b
31.8 ( 4.2
21
22
24
Results and Discussion
In the present study, the potential antinociceptive activity of
the investigated compounds was tested by two different
experimental models: the abdominal constriction test, in which
a painful chemical stimulus was applied, and the hot plate test,
in which an acute thermal stimulus was used.
a All drugs were administered per os 30 min before test. b P < 0.01 versus
CMC treated-mice. Each value represents the mean of at least two
experiments.
Table 4. Effect of Yohimbine, BRL-44408, and ARC-239 on
Compounds 5a, 5h, 8a, 11a, 11c, 15a, 15c, 15e, 18, 21, and
22 were able to reduce the number of abdominal constrictions,
in particular 15a which, tested at 20 mh kg-1 p.o., was the most
potent in this series (Table 3). Compound 15e induced a good
antinociceptive effect at 10 mg kg-1 p.o. but, unfortunately,
when tested at higher doses, it induced a reduced spontaneous
mobility in animals. The same effect was observed for com-
pound 5b, which was tested only at 5 mg kg-1 p.o. Compounds
that showed the highest intensity of antinociceptive activity were
5a, 11c, 15a, 18, 21, and 22 being able to reduce by more than
50% the number of abdominal constrictions. Conversely,
compounds 5b, 5c, 5d, 5e, 5f, 5g, 5i, 5j, 5k, 6, 11b, 15b, 15d,
and 24 were devoid of any effect in the abdominal constriction
test.
Antinociception Induced by Compound A in the Abdominal Constriction
Test
No. of
mice
abdominal
constrictions
treatmenta
saline/CMC
yohimbine + CMC
BRL-44408 + CMC
saline + A
yohimbine + A
BRL-44408 + A
ARC-239 + A
15
12
11
10
12
12
10
31.8 ( 3.2
28.5 ( 3.6
32.9 ( 2.7
11.6 ( 3.3b
27.8 ( 4.1
33.4 ( 4.6
9.6 ( 3.8*
a Yohimbine was tested at 3 mg/kg i.p., BRL-44408 was tested at 1 mg/
kg i.p., ARC-239 was tested at 10 mg/kg i.p., and compound A was tested
at 1 mg/kg s.c. in the abdominal constriction test. b P < 0.05 in comparison
with saline/CMC-treated mice
From the inactive and less-active compounds in the abdominal
constriction test, we choose several products (5c, 5d, 5k, 11b,
6, 5h, 8b), and we also performed the hot plate test to unmask
a potential antinociceptive activity against an acute thermal
stimulus. Compounds 5c, 5d, 5h, 5k, 6, 8b, and 11 were all
unable to increase the pain threshold in the mouse hot plate
test (Table 3). At the same time, to further confirm the
antinociceptive profile of some promising molecules, we also
tested compounds 5a, 11c, and 15c in the hot plate test. They
showed an antinociceptive activity comparable to that observed
in the writhing test.
Previously we demonstrated that the analgesia induced by
compound A, which was the prototype of this series, was
completely prevented by pretreatment with reserpine, indicating
that the antinociceptive effect is mediated by a partial or
complete activation of the monoaminergic system.17 To clarify
whether the noradrenergic and/or serotoninergic systems were
involved in the mechanism of action of compound A, we
pretreated animals with the R2-adrenoceptor antagonist yohim-
bine. The dose of yohimbine employed to prevent the analgesia
induced by the above-reported compounds is the minimal dose
able to antagonize antinociception induced by activation of R2-
adreniceptor since opioid, muscarinic, and GABAergic anti-
nociception is not modified.23 A complete reversal of the
antinociceptive effect of compound A was evidenced in the
mouse abdominal constriction test (Table 4). We demonstrated
that the R2-adrenoceptor subtype involved in the modulation of
pain perception is the R2A, whereas the subtype R2C is devoid
of any effect.23,24 For this reason, we evaluated the effect
produced by the administration of the selective R2A-adrenoceptor
antagonist BRL44408 and of the selective R2C-adrenoceptor
antagonist ARC239 on compound A analgesia. As illustrated
in Table 4, only BRL44408 was able to completely antagonize
compound A antinociception. Pretreatment with the R1-adreno-
ceptor antagonist prazosin did not modify the increased pain

7830 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Cesari et al.
) 1.3 × 10-7 M). These results indicate that the analgesic action
of compound A underlies an indirect activation of the nor-
adrenergic system through an inhibition of the noradrenaline
reuptake.
Because all the new molecules tested in the present study
were derivatives of compound A, we choose as representative
some of the most potent compounds to evaluate if their analgesic
effect was mediated through an activation of the noradrenergic
system. The antinociception of 5a, 5h, 8a, 8b, 11a, and 15a
was completely prevented by pretreatment with the R2-antagonist
yohimbine (3 mg kg-1 i.p.), suggesting the involvement of R2-
adrenoceptors in the mechanism of analgesic action of the
above-mentioned compounds (Table 3).
The highest doses investigated did not produce any alteration
of the animals’ gross behavior nor modify motor coordination,
as revealed by the rota rod test, or spontaneous motility, as
revealed by the hole board test (data not shown).
All these data clearly indicate that a relevant part of the
synthesized compounds are endowed with potent antinociceptive
effect by oral route, with a maximum of activity for 15a, which
was able to reduce by 75% the writhes number at 20 mg/kg.
Moreover, the same compound, tested in the hot plate, exhibited
a licking latency of 83% at the same dose. Thus, this compound
appears to be a very interesting lead for further development,
especially when taking into account the absence of behavioral
effects at the maximum tested doses. Because the therapeutic
use of the amitriptiline, a mechanism-related drug used in the
management of neurophatic pain, is associated with several
unwanted effects, 15a may have some possibilities for further
pharmacological evaluation.
Taking into account the fact that the tested compounds were
administered by oral route and, therefore, that ADME properties
may influence the obtained results for all of them, some SARs
can be inferred. For the series of isoxazolopyridazinones 5a-
k, which are structurally related to the prototype A, the obtained
data indicated that both the three carbon chain of the spacer
and the m-chlorophenyl moiety placed at the end of the side
chain are essential requirements for antinociceptive activity. In
fact, when a phenyl group is appended in position 7 of the
heterocyclic core, as in compound A, elongation of (5f) and
branching (5g) of the spacer are detrimental for the activity.
The same result is associated with some modifications of the
phenyl at position 7, being the ortho-and para-nitro derivatives
5i and 5j and the o-amino derivative 5k devoid of activity.
Moreover, when in the prototype A the chlorine at Ar is shifted
to position para (5e), a reduced activity was observed; further-
more, by replacing the chlorine with a fluorine (5c), a complete
loss of activity was evidenced. Replacement of 3-chlorophenyl
with 3-pyridyl (5d) led to the same result. The only tolerated
modification with respect to A was, quite surprisingly, the
replacement of the 7-phenyl with hydrogen, which led to one
of the most active compounds (5a).
Figure 2. Effect of compound A (i.p.) on noradrenaline extracellular
levels from rat cerebral cortex. The doses are expressed as mg kg-1
.
*P < 0.01 in comparison with saline treated rats. Each point represents
the mean of at least three rats.
threshold produced by compound A, further confirming, among
the alpha subtypes, an R2-mediated mechanism of action. The
selective involvement of the R2A-adrenoceptor subtype was also
evidenced by the complete lack of antagonism exerted by some
other receptor antagonists, like the opioid antagonist naloxone,
the muscarinic antagonist atropine, and the GABA antagonist
CGP35348 (data not shown).
In these experimental conditions, yohimbine, BRL 44408, and
ACR 239 did not modify the pain threshold of mice in
comparison with control animals. The lack of effect of these
antagonists agrees with the results of studies in which these
compounds did not modify the nociceptive threshold against
either thermal (hot plate) or chemical (writhing) noxious
stimuli.25 Therefore, we can exclude the hypothesis that the
prevention of compound A analgesia was due to a hyperalgesic
effect of the R1-adrenoceptor antagonist used.
The antinociceptive doses of compound A did not provoke
any visible change in normal behavior of mice as demonstrated
in rota-rod experiments in which no impairment of mouse rota-
rod performance was observed (data not shown). Moreover,
compound A was endowed with a good potency and efficacy,
comparable to that exhibited by some well-known analgesic
drugs clinically employed, such as diphenhidramine, baclofen,
amitriptiline, and so on.
The R2A-mediated analgesia can be induced by a direct R2A
-
adrenoceptor activation or by an indirect R2A-adrenoceptor
system activation, such as an increase of noradrenaline release
and/or inhibition of noradrenaline reuptake. To elucidate whether
compound A is endowed with a direct or indirect noradrenergic
mechanism, we performed R2 binding experiments. In the rat
cortical membranes, compound A showed a Ki > 1 µM,
indicating the absence of affinity for R2-adrenoceptors and ruling
out a direct noradrenergic mechanism.
When the tertiary amino group of the piperazine was
transformed into an amidic nitrogen, the activity was maintained
(compounds 8a and 8b). This finding suggests that the three-
carbon chain plays an essential role as spacer and that the
piperazine nitrogen does not interact with the biological target
in a protonated form. It is interesting to observe that, in this
series, when moving the chlorine from the position meta to the
position para, the antinociceptive activity remained unmodified.
In the series of phthalimide derivatives, different SARs can
be evidenced, with the m-chlorophenyl derivative (n ) 3; 11b)
being the only inactive term and the p-fluorophenyl analogue
being active both with n ) 2 (11a) and n ) 3 (11c).
Noradrenaline extracellular levels from rat cerebral cortex
were determined after administration of compound A at the
doses of 1 and 20 mg kg-1 i.p.; a dose-dependent increase of
noradrenaline contents was revealed (Figure 2). Also, an
inhibition of the synaptosomal [3H]-noradrenaline uptake in the
presence of concentrations of compound A was observed (IC50

AntinociceptiVe Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7831
5-(3-Bromopropyl)-3-methyl-7-(2-nitrophenyl)-5H-isoxazolo-
[4,5-d]pyridazin-4-one 2f. Yield ) 35%; oil; 1H NMR (CDCl3) δ
2.40-2.50 (m, 2H, CH2CH2CH2), 2.75 (s, 3H, CH3), 3.45 (t, 2H,
CH2CH2Br), 4.45 (t, 2H, CONCH2), 7.75 (m, 1H, Ar), 7.80 (m,
2H, Ar), 8.20 (d, 1H, Ar).
In the series of the phthalazinone derivatives, the classical
SARs are partially restored, with m-chlorophenyl and n ) 3
(15a) being important requirements for the activity. Also, in
this series, the shift of the chlorine to the para position (15c) or
its replacement with a pseudo halogen (15e) maintained the
activity.
5-(3-Bromopropyl)-3-methyl-7-(4-nitrophenyl)-5H-isoxazolo-
1
[4,5-d]pyridazin-4-one 2g. Yield ) 21%; oil; H NMR (CDCl3)
Finally, the interesting antinociceptive activity found for
compounds 18, 21, and 22 seems to confirm that, when the
lactamic nitrogen is substituted with the 3-chlorophenylpiper-
azinylpropyl chain, both the fused system (pyridine, pyrazole,
and isoxazole) and the group linked to the pyridazine (methyl,
2-thienyl, and phenyl) can be extensively modified without
significant loss of activity.
δ 2.45-2.55 (m, 2H, CH2CH2CH2), 2.75 (s, 3H, CH3), 3.50 (t,
2H, CH2CH2Br), 4.55 (t, 2H, CONCH2), 8.40 (m, 4H, Ar).
General Procedure for 3a,b. To a cooled (T ) 0 °C) and stirred
mixture of concd HNO3 (1 mL) and concd H2SO4 (1 mL), 0.7 mmol
of compound 1a was added. The reaction was carried out at room
temperature for 15 min and then the mixture was poured into ice
water (40 mL) drop by drop to afford a mixture of 4-nitrophenyl
and 2-nitrophenyl derivatives. Final compounds 3a and 3b were
separated by column chromatography using WE7 as eluent. (WE7
) EtOH, 4.5 mL; NH4OH, 0.25 mL; CHCl3, 18 mL; Et2O, 18 mL;
and petroleum ether, 45 mL)
3-Methyl-7-(4-nitrophenyl)-5H-isoxazolo[4,5-d]pyridazin-4-
one 3a. Yield ) 22%; mp ) >250 °C (EtOH); 1H NMR (DMSO-
d6) δ 2.65 (s, 3H, CH3), 8.30 (d, 2H, Ar), 8.40 (d, 2H, Ar), 10.15
(exch br s, 1H, NH).
3-Methyl-7-(2-nitrophenyl)-5H-isoxazolo[4,5-d]pyridazin-4-
one 3b. Yield ) 24%; mp ) >250 °C (EtOH); 1H NMR (DMSO-
d6) δ 2.70 (s, 3H, CH3), 7.75 (t, 2H, Ar), 8,25 (d, 1H, Ar), 8.85 (t,
1H, Ar), 10.35 (exch br s, 1H, NH).
Conclusion
The present results indicate the involvement of R2-adreno-
ceptor and, in particular, of the subtype R2A. Compound A
increases the pain threshold, and this effect is imputable to an
amplification of adrenergic neurotransmission due to a inhibition
of noradrenaline uptake.
Experimental Section
Chemistry. All melting points were determined on a Bu¨chi
apparatus and are uncorrected. 1H NMR spectra were recorded with
Avance 400 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 pressure. Merck
F-254 commercial plates were used for analytical TLC to follow
the course of reaction. Silica gel 60 (Merck 70-230 mesh) was
used for column chromatography. Reagents and starting materials
9 and 12 were commercially available.
7-(2-Aminophenyl)-3-methyl-5H-isoxazolo[4,5-d]pyridazin-4-
one 4. To a solution of 3b (0.4 mmol) in concd HCl (0.5 mL),
SnCl2 (1.9 mmol) dissolved in concd HCl (0.5 mL) was added,
and the reaction was carried out at room temperature for 12 h. The
mixture was treated with ice water, basified with 6 N NaOH, and
extracted with CH2Cl2 (3 × 20 mL). Evaporation of the solvent
afforded compound 4, which was purified by column chromatog-
raphy using toluene/ethyl acetate 1:1 as eluent. Yield ) 40%; mp
1
) 246-250 °C; H NMR (CDCl3) δ 2.75 (s, 3H, CH3), 6.85 (d,
3-Methyl-5H-isoxazolo[4,5-d]pyridazin-4-one 1b. To a cooled
and stirred solution of 5-formyl-3-methylisoxazole-4-carboxylic acid
ethyl ester18 (2.7 mmol) in EtOH (3 mL), hydrazine hydrate (26
mmol) was added. The mixture was stirred for 1 h at room
temperature, and the crude precipitate was recovered by suction.
1H, Ar), 6.90 (t, 1H, Ar), 7.30 (m, 1H, Ar), 8.05 (d, 1H, Ar), 10.50
(exch br s, 1H, NH).
General Procedure for 5a-f and 5h-j. A mixture of isoxazolo-
[4,5-d]pyridazinones 2a-g (0.4 mmol), anhydrous K2CO3 (0.8
mmol), and the appropriate (substituted)arylpiperazine (0.8 mmol),
commercially available, in anhydrous DMF (4 mL) was stirred for
3-15 h at rt to 60 °C. After dilution with cold water (20-30 mL),
the mixture was extracted with CH2Cl2 (3 × 15 mL) and the solvent
was evaporated in vacuo to afford final compounds that were
purified by column chromatography using cyclohexane/ethyl acetate
2:1 (5a,h), cyclohexane/ethyl acetate 1:3 (5b,f), cyclohexane/ethyl
acetate 1:2 (5c-e), and cyclohexane/ethyl acetate 1:1 (5i,j) as
eluents.
5-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-3-methyl-5H-
isoxazolo[4,5-d]pyridazin-4-one 5a. Yield ) 64%; mp ) 104-
106 °C (EtOH); 1H NMR (CDCl3) δ 2.05-2.20 (m, 2H,
CH2CH2CH2), 2.55-2.70 (m, 6H: 2H, CH2CH2N; 4H, piperazine),
2.70 (s, 3H, CH3), 3.15-3.30 (m, 4H, piperazine), 4.35 (t, 2H,
CONCH2), 6.75-6.85 (m, 2H, Ar), 6.90 (s, 1H, Ar), 7.20 (t, 1H,
Ar), 8.30 (s, 1H, Ar).
1
Yield ) 53%; mp ) 214-217 °C (EtOH); H NMR (CDCl3) δ
2.70 (s, 3H, CH3), 8.35 (s, 1H, 7-H), 11.3-11.5 (exch br s, 1H,
NH).
General Procedure for 2a-g. A mixture of isoxazolo[4,5-d]-
pyridazinones 1a,b18 or 3a,b (0.1 mmol), anhydrous K2CO3 (0.2
mmol), and the appropriate alkyl dibromide (0.20 mmol) in
anhydrous DMF (2 mL) was heated under stirring for 0.5-2 h at
room temperature. After dilution with cold water (20-30 mL), the
mixture was extracted with CH2Cl2 (3 × 15 mL), and the solvent
was evaporated in vacuo to afford compounds 2a-g (compound
2d was previously described17), which were purified by column
chromatography. (eluents: cyclohexane/ethyl acetate 2:1 for com-
pounds 2a,c,f; cyclohexane/ethyl acetate 1:1 for 2g; and cycloexane/
ethyl acetate 1:3 for 2b).
5-(3-Bromopropyl)-3-methyl-5H-isoxazolo[4,5-d]pyridazin-4-
one 2a. Yield ) 31%; mp ) 59-62 °C (EtOH); 1H NMR (CDCl3)
δ 2.40 (m, 2H, CH2CH2CH2), 2.70 (s, 3H, CH3), 3.45 (t, 2H,
CH2CH2Br), 4.40 (t, 2H, CONCH2), 8.30 (s, 1H, Ar).
5-(4-Bromobutyl)-3-methyl-5H-isoxazolo[4,5-d]pyridazin-4-
one 2b. Yield ) 38%; oil; 1H NMR (CDCl3) δ 1.90-2.10 (m, 4H,
NCH2CH2CH2CH2), 2.70 (s, 3H, CH3), 3.45 (t, 2H, CH2CH2Br),
4.30 (t, 2H, CONCH2), 8.30 (s, 1H, Ar).
5-{4-[4-(3-Chlorophenyl)piperazin-1-yl]butyl}-3-methyl-5H-
isoxazolo[4,5-d]pyridazin-4-one 5b. Yield ) 52%; mp ) 98-
1
100 °C (EtOH); H NMR (CDCl3) δ 1.50-1.70 (m, 2H, NCH2-
CH2CH2CH2), 1.85-1.95 (m, 2H, NCH2CH2CH2CH2), 2.70-2.90
(m, 6H: 2H, CH2CH2N; 4H, piperazine), 2.70 (s, 3H, CH3), 3.20-
3.35 (m, 4H, piperazine), 4.30 (t, 2H, CONCH2), 6.75-6.85 (m,
2H, Ar), 6.85 (s, 1H, Ar), 7.20 (t, 1H, Ar), 8.25 (s, 1H, Ar).
5-{3-[4-(4-Fluorophenyl)piperazin-1-yl]propyl}-3-methyl-7-
phenyl-5H-isoxazolo[4,5-d]pyridazin-4-one 5c. Yield ) 90%; mp
5-(2-Bromoethyl)-3-methyl-7-phenyl-5H-isoxazolo[4,5-d]pyr-
1
idazin-4-one 2c. Yield ) 71%; mp ) 145-147 °C (EtOH); H
1
NMR (CDCl3) δ 2.75 (s, 3H, CH3), 3.85 (t, 2H, CH2CH2Br), 4.75
(t, 2H, CONCH2), 7.50 (m, 3H, Ar), 8.15 (m, 2H, Ar).
) 117-120 °C (EtOH); H NMR (CDCl3) δ 2.10-2.20 (m, 2H,
CH2CH2CH2), 2.60 (m, 2H, CH2N), 2.65 (m, 4H, piperazine), 2.75
(s, 3H, CH3), 3.10-3.20 (m, 4H, piperazine), 4.45 (t, 2H,
CONCH2), 6.80-6.90 (m, 2H, Ar), 6.90-7.00 (m, 2H, Ar), 7.55
(m, 3H, Ar), 8.10-8.20 (m, 2H, Ar).
5-(4-Bromobutyl)-3-methyl-7-phenyl-5H-isoxazolo[4,5-d]pyr-
idazin-4-one 2e. Yield ) 44%; mp ) 82-84 °C (EtOH); 1H NMR
(CDCl3) δ 1.95-2.15 (m, 4H, NCH2CH2CH2CH2), 2.75 (s, 3H,
CH3), 3.50 (t, 2H, CH2CH2Br), 4.40 (t, 2H, CONCH2), 7.50-7.60
(m, 3H, Ar), 8.20 (m, 2H, Ar).
3-Methyl-7-phenyl-5-[3-(4-pyridin-2-yl-piperazin-1-yl)propyl]-
5H-isoxazolo[4,5-d]pyridazin-4-one 5d. Yield ) 80%; mp ) 110-

7832 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Cesari et al.
113 °C (EtOH); 1H NMR (CDCl3) δ 2.20-2.35 (m, 2H,
CH2CH2CH2), 2.55-2.70 (m, 6H: 2H, CH2N; 4H, piperazine), 2.75
(s, 3H, CH3), 3.50-3.70 (m, 4H, piperazine), 4.95 (t, 2H,
CONCH2), 6.60-6.70 (m, 2H, Ar), 7.50-7.60 (m, 4H, Ar), 8.15-
8.25 (m, 3H, Ar).
0.4 mmol of 3-bromopropionic acid ethyl ester were added. After
stirring for 15 h at room temperature, the mixture was diluted with
cold water (15-20 mL) and extracted with CH2Cl2 (3 × 15 mL).
Evaporation of the solvent afforded a crude precipitate, which was
purified by column chromatography using cyclohexane/ethyl acetate
1
2:1 as eluent. Yield ) 70%; mp ) 89-91 °C (EtOH); H NMR
5-{3-[4-(4-Chlorophenyl)piperazin-1-yl]propyl}-3-methyl-7-
phenyl-5H-isoxazolo[4,5-d]pyridazin-4-one 5e. Yield ) 80%; mp
(CDCl3) δ 1.25 (t, 3H, COOCH2CH3), 2.75 (s, 3H, CH3), 2.95 (t,
2H, CH2COO), 4.15 (q, 2H, COOCH2CH3), 4.65 (t, 2H, CONCH2),
7.50-7.60 (m, 3H, Ar), 8.10-8.20 (m, 2H, Ar).
1
) 120-123 °C (EtOH); H NMR (CDCl3) δ 2.15-2.25 (m, 2H,
CH2CH2CH2), 2.60-2.70 (m, 6H: 2H, CH2N; 4H, piperazine), 2.75
(s, 3H, CH3), 3.15-3.25 (m, 4H, piperazine), 4.45 (t, 2H,
CONCH2), 6.80 (d, 2H, Ar), 7.20 (d, 2H, Ar), 7.50-7.60 (m, 3H,
Ar), 8.20 (m, 2H, Ar).
3-(3-Methyl-4-oxo-7-phenyl-4H-isoxazolo[4,5-d]pyridazin-5-
yl)propionic Acid 7. Compound 6 (0.7 mmol) in ethanol (2-3
mL) was treated with 6 N NaOH (8 mL) at room temperature for
15 h, to afford the corresponding carboxylic derivative 7. The
mixture was concentrated, diluted with water (10 mL), and acidified
with 6 N HCl. The crude compound 7 was recovered by suction.
5-{4-[4-(3-Chlorophenyl)piperazin-1-yl]butyl}-3-methyl-7-
phenyl-5H-isoxazolo[4,5-d]pyridazin-4-one 5f. Yield ) 41%; mp
1
) 124-127 °C (EtOH); H NMR (CDCl3) δ 1.65-1.80 (m, 2H,
1
NCH2CH2CH2CH2), 1.90-2.00 (m, 2H, NCH2CH2CH2CH2), 2.50-
2.75 (m, 9H: 3H, CH3; 2H, CH2CH2N; 4H, piperazine), 3.20-
3.40 (m, 4H, piperazine), 4.40 (m, 2H, CONCH2), 6.75-6.90 (m,
3H, Ar), 7.20 (m, 1H, Ar), 7.50-7.60 (m, 3H, Ar), 8.15 (d, 2H,
Ar).
Yield ) 80%; mp ) 195-198 °C (EtOH); H NMR (CDCl3) δ
2.75 (s, 3H, CH3), 3.00 (t, 2H, CH2COOH), 4.65 (t, 2H, CONCH2),
7.40-7.60 (m, 3H, Ar), 8.10-8.20 (m, 2H, Ar).
General Procedure for 8a,b. To a cooled (T ) 0 °C) and stirred
solution of compound 7 (0.4 mmol) in SOCl2 (2 mL), 0.05 mL of
Et3N was slowly added. The mixture was heated to 60 °C and stirred
for 2 h. The excess of SOCl2 was removed in vacuo, and the residue
was dissolved in anhydrous THF (1-2 mL) and added portionwise
to a cooled and stirred solution of appropriate commercially
available piperazine (0.8 mmol) in anhydrous THF (1-2 mL). The
mixture was stirred at room temperature for 2 h. The solid residue
was filtered off, and the solution was concentrated in vacuo, diluted
with cold water (10-15 mL), and extracted with CH2Cl2 (3 × 15
mL). Evaporation of the solvent afforded compounds 8a,b, which
were purified by column chromatography using cyclohexane/ethyl
acetate 1:3 as eluent.
5-{3-[4-(3-Chlorophenyl)piperazin-1-yl]-3-oxopropyl}-3-methyl-
7-phenyl-5H-isoxazolo[4,5-d]pyridazin-4-one 8a. Yield ) 58%;
mp ) 153-156 °C (EtOH); 1H NMR (CDCl3) δ 2.75 (s, 3H, CH3),
3.05 (t, 2H, CH2CON), 3.25 (m, 2H, piperazine), 3.30 (m, 2H,
piperazine), 3.70-3.80 (m, 2H, piperazine), 3.85-3.95 (m, 2H,
piperazine), 4.70 (t, 2H, CONCH2), 6.95 (m, 2H, Ar), 7.00 (s, 1H,
Ar), 7.25 (m, 1H, Ar), 7.55 (m, 3H, Ar), 8.15 (m, 2H, Ar).
5-{3-[4-(4-Chlorophenyl)piperazin-1-yl]-3-oxo-propyl}-3-meth-
yl-7-phenyl-5H-isoxazolo[4,5-d]pyridazin-4-one 8b. Yield ) 57%;
mp ) 160-163 °C (EtOH); 1H NMR (CDCl3) δ 2.75 (s, 3H, CH3),
3.05 (t, 2H, CH2CON), 3.10-3.20 (m, 4H, piperazine), 3.60-3.75
(m, 2H, piperazine), 3.80-3.90 (m, 2H, piperazine), 4.70 (t, 2H,
CONCH2), 6.85-7.00 (m, 2H, Ar), 7.20-7.30 (m, 2H, Ar), 7.55
(m, 3H, Ar), 8.15 (m, 2H, Ar).
General Procedure for 10a,b. To a suspension of the appropri-
ate dibromoalkane (2.0 mmol) and K2CO3 (2.7 mmol) in anhydrous
DMF (2-3 mL), a solution of the phtalimmide 9 commercially
available (1.35 mmol) in anhydrous DMF (0.5 mL) was added
dropwise. The suspension was stirred at room temperature for 90
min. After dilution with cold water, the mixture was extracted with
CH2Cl2 (3 × 15 mL). Evaporation of the solvent afforded
compounds 10a,b, which were purified by column chromatography
using cyclohexane/ethyl acetate 2:1 as eluent.
2-(2-Bromoethyl)isoindole-1,3-dione 10a. Yield ) 73%; mp
) 150-153 °C (EtOH); 1H NMR (CDCl3) δ 3.65 (t, 2H,
CH2CH2Br), 4.15 (t, 2H, NCH2CH2), 7.70-7.80 (m, 2H, Ar), 7.85-
7.95 (m, 2H, Ar).
2-(3-Bromopropyl)isoindole-1,3-dione 10b. Yield ) 45%; mp
) 72-75 °C (EtOH); 1H NMR (CDCl3) δ 2.30 (m, 2H,
CH2CH2CH2), 3.45 (t, 2H, CH2CH2Br), 3.85 (t, 2H, NCH2CH2),
7.75 (m, 2H, Ar), 7.80-7.90 (m, 2H, Ar).
General Procedure for 11a-c. A mixture of derivative 10a,b
(0.6 mmol), anhydrous K2CO3 (1.20 mmol), and opportune pip-
erazine (0.9 mmol) in anhydrous DMF (3-4 mL) was stirred at
room temperature for 15 h. After cooling, ice water was added and
the solution was extracted with CH2Cl2 (3 × 15 mL). Evaporation
of the solvent afforded the final compounds 11a-c, which were
purified by column chromatography using cyclohexane/ethyl acetate
3:1 as eluent for 11a and cycloexane/ethyl acetate 2:1 for 11b,c.
5-{2-[4-(3-Chlorophenyl)piperazin-1-yl]ethyl}-3-methyl-7-
phenyl-5H-isoxazolo[4,5-d]pyridazin-4-one 5h. Yield ) 51%; mp
1
) 155-158 °C (EtOH); H NMR (CDCl3) δ 2.70-2.85 (m, 5H:
3H, CH3; 2H, CH2CH2N), 2.90-3.10 (m, 4H, piperazine), 3.15-
3.30 (m, 4H, piperazine), 4.50-5.60 (m, 2H, CONCH2), 6.75-
6.85 (m, 2H, Ar), 6.90 (s, 1H, Ar), 7.15 (s, 1H, Ar), 7.55 (m, 3H,
Ar), 8.15 (d, 2H, Ar).
5-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-3-methyl-7-
(2-nitrophenyl)-5H-isoxazolo[4,5-d]pyridazin-4-one 5i. Yield )
1
26%; oil; H NMR (CDCl3) δ 2.00-2.15 (m, 2H, CH2CH2CH2),
2.50-2.70 (m, 6H: 2H, CH2N; 4H, piperazine), 2.75 (s, 3H, CH3),
3.10-3.30 (m, 4H, piperazine), 4.40 (t, 2H, CONCH2), 6.75-6.85
(m, 2H, Ar), 6.90 (s, 1H, Ar), 7.20 (t, 1H, Ar), 7.70-7.80 (m, 1H,
Ar), 7.75-7.85 (m, 2H, Ar), 8.15 (d, 1H, Ar).
5-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-3-methyl-7-
(4-nitrophenyl)-5H-isoxazolo[4,5-d]pyridazin-4-one 5j. Yield )
52%; mp ) 142-145 °C (EtOH); 1H NMR (CDCl3) δ 2.15-2.30
(m, 2H, CH2CH2CH2), 2.55-2.75 (m, 6H: 2H, CH2N; 4H,
piperazine), 2.80 (s, 3H, CH3), 3.15-3.25 (m, 4H, piperazine), 4.90
(t, 2H, CONCH2), 6.75-6.85 (m, 2H, Ar), 6.90 (s, 1H, Ar), 7.20
(t, 1H, Ar), 8.35-8.45 (m, 4H, Ar).
General Procedure for 5g,k. The isoxazolopyridazinone 1a or
4 (0.1 mmol) was condensed with the appropriate 1-(3-haloalkyl)-
4-(3-chlorophenyl)piperazine (0.1-0.2 mmol)17,27 in anhydrous
DMF (1-3 mL) and K2CO3 (0.2 mmol). The reaction was carried
out at room temperature for 48 h for compound 4 and at 80 °C for
6 h for compound 1a. After dilution with cold water (10-15 mL),
the mixture was extracted with CH2Cl2 (3 × 15 mL). Evaporation
of the solvent afforded the desiderate 5g and 5k, which were
purified by column chromatography using cyclohexane/ethyl acetate
2:1 as eluent for compound 5g and WE2 for compound 5k (WE2:
EtOH, 1.8 mL; NH4OH, 0.1 mL; CHCl3, 3.6 mL; Et2O, 3.6 mL;
and petroleum ether, 9 mL).
5-{3-[4-(3-Chlorophenyl)piperazin-1-yl]-2-methylpropyl}-3-
methyl-7-phenyl-5H-isoxazolo[4,5-d]pyridazin-4-one 5g. Yield )
1
35%; mp ) 112-113 °C (EtOH); H NMR (CDCl3) δ 1.00 (d,
3H, CH(CH3)), 2.30-2.75 (m, 7H: 1H, CH(CH3); 2H, CHCH2N;
4H, piperazine), 2.75 (s, 3H, CH3), 3.00-3.20 (m, 4H, piperazine),
4.05-4.20 (m, 1H, CONCH2), 4.50-4.65 (m, 1H, CONCH2),
6.70-6.90 (m, 3H, Ar), 7.15 (m, 1H, Ar), 7.50-7.60 (m, 3H, Ar),
8.15 (d, 2H, Ar).
7-(2-Aminophenyl)-5-{3-[4-(3-chlorophenyl)piperazin-1-yl]-
propyl}-3-methyl-5H-isoxazolo[4,5-d]pyridazin-4-one 5k. Yield
) 50%; oil; 1H NMR (CDCl3) δ 2.05-2.15 (m, 2H, CH2CH2CH2),
2.55 (t, 3H, CH2N), 2.55-2.65 (m, 4H, piperazine), 2.75 (s, 3H,
CH3), 3.15 (m, 4H, piperazine), 4.40 (t, 2H, CONCH2), 5.45 (exc
br s, 2H, NH2), 6.75-6.95 (m, 5H, Ar), 6.90 (t, 1H, Ar), 7.20-
7.40 (m, 1H, Ar), 8.05 (d, 1H, Ar).
3-(3-Methyl-4-oxo-7-phenyl-4H-isoxazolo[4,5-d]pyridazin-5-
yl)propionic Acid Ethyl Ester 6. To a solution of compound 1a
(0.4 mmol) in anhydrous DMF (1.5 mL), 0.6 mmol of K2CO3 and

AntinociceptiVe Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7833
2-{3-[4-(4-Chlorophenyl)piperazin-1-yl]propyl}-2H-phthalazin-
2-{2-[4-(4-Fluorophenyl)piperazin-1-yl]ethyl}isoindole-1,3-di-
one 11a. Yield ) 40%; oil; H NMR (CDCl3) δ 2.70-2.90 (m,
1
1
1-one 15c. Yield ) 90%; mp ) 110-113 °C (EtOH); H NMR
6H: 2H, CH2CH2N; 4H piperazine), 3.05-3.20 (m, 4H, piperazine),
3.90 (t, 2H, NCH2CH2), 6.80-6.90 (m, 2H, Ar), 6.90-7.00 (m,
2H, Ar), 7.70-7.80 (m, 2H, Ar), 7.85 (m, 2H, Ar).
2-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}isoindole-1,3-
dione 11b. Yield ) 39%; oil; 1H NMR (CDCl3) δ 2.00-2.10 (m,
2H, CH2CH2CH2), 2.60-2.70 (m, 6H: 2H, CH2CH2N; 4H, pip-
erazine), 3.10-3.20 (m, 4H, piperazine), 3.85 (t, 2H, NCH2CH2),
6.75-6.85 (m, 3H, Ar), 7.15 (t, 1H, Ar), 7.75 (m, 2H, Ar), 7.80-
7.90 (m, 2H, Ar).
2-{3-[4-(4-Fluorophenyl)piperazin-1-yl]propyl}isoindole-1,3-
dione 11c. Yield ) 73%; mp ) 92-94 °C (EtOH); 1H NMR
(CDCl3) δ 1.90-2.05 (m, 2H, CH2CH2CH2), 2.55 (t, 2H, CH2CH2N),
2.60-2.75 (m, 4H, piperazine), 2.95-3.10 (m, 4H, piperazine), 3.85
(t, 2H, NCH2CH2), 6.80 (m, 2H, Ar), 6.90-7.00 (m, 2H, Ar), 7.65-
7.75 (m, 2H, Ar), 7.85 (m, 2H, Ar).
General Procedure for 13a,b. Compounds 13a,b were obtained
from the commercially available 12, following the general procedure
described for 2a-g. For these compounds, the reaction was carried
out at 50 °C for 4 h. The final compounds were purified by column
chromatography using cyclohexane/ethyl acetate 1:1 as eluent.
2-(3-Bromopropyl)-2H-phthalazin-1-one 13a. Yield ) 24%;
oil; 1H NMR (CDCl3) δ 2.45-2.55 (m, 2H, CH2CH2CH2), 3.50 (t,
2H, CH2CH2Br), 4.40 (t, 2H, CONCH2), 7.75 (d, 1H, Ar), 6.85-
7.80 (m, 2H, Ar), 8.20 (s, 1H, Ar), 8.45 (d, 1H, Ar).
(CDCl3) δ 2.20-2.35 (m, 2H, CH2CH2CH2), 2.70-2.90 (m, 6H:
2H, CH2CH2N; 4H, piperazine), 3.25-3.40 (m, 4H, piperazine),
4.35 (t, 2H, CONCH2), 6.85 (d, 2H, Ar), 7.25 (d, 2H, Ar), 7.70 (d,
1H, Ar), 7.75-7.90 (m, 2H, Ar), 8.20 (s, 1H, Ar), 8.45 (d, 1H,
Ar).
2-{4-[4-(3-Chlorophenyl)piperazin-1-yl]butyl}-2H-phthalazin-
1
1-one 15d. Yield ) 41%; mp ) 127-129 °C (EtOH); H NMR
(CDCl3) δ 1.65-1.80 (m, 2H, NCH2CH2CH2CH2), 1.90-2.00 (m,
2H, NCH2CH2CH2CH2), 2.50-2.90 (m, 6H: 2H, CH2CH2N; 4H,
piperazine), 3.20-3.45 (m, 4H, piperazine), 4.30 (t, 2H, CONCH2),
6.80 (m, 2H, Ar), 7.00 (s, 1H, Ar), 7.15 (t, 1H, Ar), 7.70 (d, 1H,
Ar), 7.75-7.85 (m, 2H, Ar), 8.20 (s, 1H, Ar), 8.45 (d, 1H, Ar).
3-{4-[3-(1-Oxo-1H-phthalazin-2-yl)propyl]piperazin-1-yl}-
benzonitrile 15e. Molecular sieves (4Å) were heated under stirring
for 30 min at 200 °C in a N2 atmosphere. After cooling a mixture
of compound 14 (0.4 mmol) in anhydrous CH2Cl2 (8-10 mL),
3-cyanophenylboronic acid (0.8 mmol), Cu(Ac)2 (0.6 mmol), and
Et3N (0.8 mmol) was added to molecular sieves. The reaction was
carried out at room temperature for 15 h. Anhydrous CH2Cl2 (15
mL) was added, the residue was filtered off, and the organic layer
was washed with 33% NH4OH (2 × 15 mL). CH2Cl2 was
evaporated, and the final compound 15b was purified by column
chromatography using cyclohexane/ethyl acetate 1:1 as eluent. Yield
1
) 28%; oil; H NMR (CDCl3) 2.05-2.20 (m, 2H, CH2CH2CH2),
2.55 (t, 2H, CH2CH2N), 2.60-2.70 (m, 4H, piperazine), 3.15-
3.25 (m, 4H, piperazine), 4.35 (t, 2H, CONCH2), 7.10 (m, 2H, Ar),
7.25-7.35 (d, 2H, Ar), 7.70 (d, 1H, Ar), 7.75-7.85 (m, 2H, Ar),
8.20 (s, 1H, Ar), 8.45 (d, 1H, Ar).
2-(4-Bromobutyl)-2H-phthalazin-1-one 13b. Yield ) 32%; mp
1
) 87-88 °C (EtOH); H NMR (CDCl3) δ 1.95-2.10 (m, 4H,
CH2CH2CH2CH2), 3.45-3.55 (m, 2H, CH2CH2Br), 4.25-4.35 (m,
2H, CONCH2), 7.65-7.90 (m, 3H, Ar), 8.15 (s, 1H, Ar), 8.45 (d,
1H, Ar).
2,4-Dimethyl-5-phenyl-7H-pyrido[2,3-d]pyridazin-8-one 17.
To a stirred solution of sodium ethoxide (1 mmol) in anhydrous
ethanol (3 mL), a solution of compound 1619 (0.5 mmol) in
anhydrous acetone (0.8 mL) was added, and the reaction was carried
out at 90 °C for 12 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 compound 17, which
was purified by flash chromatography using as eluent CHCl3/MeOH
2-(3-Piperazin-1-yl-propyl)-2H-phthalazin-1-one 14. A mixture
of 13a (0.5 mmol), anhydrous K2CO3 (0.7 mmol), and piperazine
(0.5 mmol) in anhydrous DMF (3 mL), was stirred for 15 h at room
temperature. After dilution with cold water, the mixture was
extracted with CH2Cl2 (3 × 20 mL). Evaporation of the solvent
afforded 14, which was purified by column chromatography using
CH2Cl2/MeOH 8:2 as eluent. Yield ) 76%; oil; 1H NMR (CDCl3)
δ 2.00-2.10 (m, 2H, CH2CH2CH2), 2.30-2.55 (m, 6H: 2H,
CH2CH2N; 4H, piperazine), 2.80-2.95 (m, 4H, piperazine), 4.30
(t, 2H, CONCH2), 7.70 (d, 1H, Ar), 7.75-7.85 (m, 2H, Ar), 8.20
(s, 1H, Ar), 8.45 (d, 1H, Ar).
1
9:1. Yield ) 22%; mp > 250 °C (EtOH); H NMR (CDCl3) δ
1.95 (s, 3H, CH3), 2.80 (s, 3H, CH3), 7.35 (s, 1H, Ar), 7.40 (m,
2H, Ar), 7.25 (s, 1H, Ar), 7.40 (m, 2H, Ar), 7.50 (m, 3H, Ar),
10.15 (exc br s, 1H, NH).
7-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-2,4-dimethyl-
5-phenyl-7H-pyrido[2,3-d]pyridazin-8-one 18. Compound 18 was
obtained from compound 17, following the general procedure
described for 15c,e. The mixture was stirred at room temperature
for 15 h, and the final compound 18 was purified by flash
chromatography using WE7 as eluent. Yield ) 52%; oil; 1H NMR
(CDCl3) δ 1.90 (s, 3H, CH3), 2.10-2.20 (m, 2H, CH2CH2CH2),
2.55 (t, 2H, CH2CH2N), 2.60 (m, 4H, piperazine), 2.80 (s, 3H, CH3),
3.10-3.20 (m, 4H, piperazine), 4.40 (t, 2H, CONCH2), 6.70-6.80
(m, 2H, Ar), 6.85 (s, 1H, Ar), 7.15 (t, 1H, Ar), 7.30 (s, 1H, Ar),
7.30-7.40 (m, 2H, Ar), 7.45-7.55 (m, 3H, Ar).
General Procedure for 21 and 22. Compounds 21 and 22 were
obtained starting from compounds 1920 and 20,21 following the
general procedure described for 15c,e. The mixture was stirred at
60-80 °C for 2-5 h. After cooling, cold water was added, and
compound 21 was recovered by suction and recrystallized by
ethanol. Compound 22 was recovered after extraction with CH2Cl2
(3 × 15 mL), evaporation in vacuo of the solvent, and it was
purified by column chromatography using cyclohexane/ethyl acetate
1:1 as eluent.
General Procedure for 15a,b. A mixture of 12 (0.6 mmol),
anhydrous K2CO3 (1.2 mmol), and appropriate piperazine17,26 (0.8
mmol) in anhydrous DMF (2-3 mL) was stirred at 70 °C for 4 h.
After cooling, the mixture was diluted with ice water (20 mL) and
extracted with CH2Cl2 (3 × 20 mL). Evaporation of the solvent
afforded the final compounds 15a,b, which were purified by column
chromatography using CHCl3/MeOH 9:1 as eluent for compound
15a and cyclohexane/ethyl acetate 1:2 for compound 15b.
2-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-2H-phthalazin-
1
1-one 15a. Yield ) 40%; mp ) 83-84 °C (EtOH); H NMR
(CDCl3) δ 2.00-2.20 (m, 2H, CH2CH2CH2), 2.55-2.75 (m, 6H:
2H, CH2CH2N; 4H, piperazine), 3.10-3.30 (m, 4H, piperazine),
4.35 (t, 2H, CONCH2), 6.75-6.90 (m, 3H, Ar), 7.20 (t, 1H, Ar),
7.70-7.90 (m, 3H, Ar), 8.20 (s, 1H, Ar), 8.45 (d, 1H, Ar).
2-{3-[4-(3-Chlorophenyl)piperazin-1-yl]-2-methylpropyl}-2H-
phthalazin-1-one 15b. Yield ) 24%; oil; 1H NMR (CDCl3) δ 1.00
(d, 3H, CH(CH3)), 2.30-2.80 (m, 7H: 2H, CH2CH2N; 4H,
piperazine; 1H, CH(CH3)), 3.05-3.20 (m, 4H, piperazine), 3.95-
4.05 (m, 1H, CONCH2), 4.45 (m, 1H, CONCH2), 6.70-6.80 (m,
3H, Ar), 7.15 (t, 1H, Ar), 7.70-7.85 (m, 3H, Ar), 8.20 (s, 1H, Ar),
8.45 (d, 1H, Ar).
6-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-3-methyl-4-
thiophen-2-yl-6H-isoxazolo[3,4-d]pyridazin-7-one 21. Yield )
General Procedure for 15c,d. Compounds 15c and 15d were
obtained from compounds 13a,b, following the procedure described
for 5a-f and 5h-j. For these compounds, dilution with cold water
(20-30 mL) of the reaction mixture afforded a crude precipitate
that was recovered by suction. Purification of the final compounds
was performed by column chromatography using cyclohexane/ethyl
acetate 1:2 as eluent for compound 15c and cyclohexane/ethyl
acetate 1:3 for compound 15d.
1
24%; mp ) 85-87 °C (EtOH); H NMR (CDCl3) δ 2.00-2.30
(m, 2H, CH2CH2CH2), 2.50-2.70 (m, 6H: 2H, CH2CH2N; 4H,
piperazine), 2.75 (s, 3H, CH3), 3.10-3.20 (m, 4H, piperazine), 4.30
(t, 2H, CONCH2), 6.70-6.85 (m, 3H, Ar), 7.10-7.20 (m, 2H, Ar),
7.30-7.50 (m, 2H, Ar).
6-{3-[4-(3-Chlorophenyl)piperazin-1-yl]propyl}-3,4-dimethyl-
2-phenyl-2,6-dihydropyrazolo[3,4-d]pyridazin-7-one 22. Yield )

7834 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Cesari et al.
28%; mp ) 115-118 °C (EtOH); 1H NMR (CDCl3) δ 1.90-2.15
(m, 2H, CH2CH2CH2), 2.45-2.70 (m, 12H: 2H, CH2CH2N; 4H,
piperazine; 3H, 3C-CH3; 3H, 4C-CH3), 3.15 (m, 4H, piperazine),
4.30 (t, 2H, CONCH2), 6.70-6.90 (m, 3H, Ar), 7.15 (t, 1H, Ar),
7.50 (s, 5H, Ar).
The amount of NE in the dialysate samples was determined with
HPLC-ED. Dialysate samples were injected into the HPLC system.
The detection system consisted of an ESA Coulochem II electro-
chemical detector. The baseline value against which drug admin-
istration was compared to was derived from the average of three
samples just prior to manipulation. The neurochemical data were
expressed as the mean ( SEM. All statistics were performed using
StatView software.
3-Methyl-7-phenyl-5H-isoxazolo[4,5-d]pyridazine-4-thione 23.
Isoxazolopyridazinone 1a (0.9 mmol) was treated with Lawesson’s
reagent (0.8 mmol) in toluene (10 mL) and heated under stirring
for 2 h at 110 °C. The precipitate 23 was recovered by suction.
Synaptosomal [3H]-Noradrenaline Uptake. Synaptosomal prepa-
ration brain synaptosomes were isolated according to the method
described by Franceschini et al.30 Rats were decapitated, the brains
rapidly excised, and the frontal cortex dissected out on ice. The
tissue was homogenized in 10 volumes (w/v) of ice-cold 0.32 M
sucrose and then centrifuged at 1800 g for 10 min (4 °C). The
pellet was discarded and the supernatant was centrifuged at 17 000
g for 60 min (4 °C). The pellet was resuspended in a small volume
of 0.32 M sucrose, layered on a 0.8-1.2 M sucrose gradient, and
centrifuged at 37 000 g for 60 min (4 °C). The synaptosomal
fraction at the interface between 0.8 and 1.2 M sucrose was collected
and further centrifuged at 17 000 g for 30 min. The pellet was
resuspended in buffer (composition in mM: NaCl, 115; KCl, 4.97;
CaC12, 1; MgSO4, 1.22; KH2P04, 1.2; NaHC03, 25; and glucose,
11.1, pH 7.4, plus 0.01 mM pargyline) and immediately used.
Synaptosomes were preincubated for 5 min at 37 °C in the absence
or presence of different concentrations of drug. Uptake was started
by addition of [3H]-NE, and incubation continued for 6 min. Control
samples were incubated at 0 °C to evaluate membrane diffusion.
The reaction was stopped by cooling the tubes in ice. The samples
were then filtered through Whatman GF/C glass fiber filters
(Whatman, Inc., Clifton, NJ) and washed twice with 150 mM Tris
HCl, pH 7.4. Filter-bound radioactivity was counted by liquid
scintillation spectrometry with Filter Count (Packard). The differ-
ence in [3H]-NE accumulation at 37 °C and 0 °C was taken as a
measure of active uptake.
1
Yield ) 84%; mp ) 241-243 °C (EtOH); H NMR (CDCl3) δ
2.85 (s, 3H, CH3), 3.85 (exch br s, 1H, NH), 7.50-7.60 (m, 3H,
Ar), 8.10-8.20 (m, 2H, Ar).
4-{3-[4-(4-Fluorophenyl)piperazin-1-yl]propylsulfanyl}-3-
methyl-7-phenylisoxazolo[4,5-d]pyridazine 24. A mixture of
compound 23 (0.8 mmol), anhydrous K2CO3 (1.6 mmol), and 1-(3-
bromopropyl)-4-(4-fluorophenyl)-piperazine22 (0.8 mmol) in an-
hydrous acetone (4 mL) was stirred at room temperature for 4 h.
The mixture was concentrated, diluted with cold water (15 mL),
and extracted with CH2Cl2 (3 × 15 mL). Evaporation of the solvent
afforded the desiderated 24, which was purified by column
chromatography using cyclohexane/ethyl acetate 1:2 as eluent. Yield
1
) 24%; mp ) 111-113 °C (EtOH); H NMR (CDCl3) δ 2.20-
2.40 (m, 2H, CH2CH2CH2), 2.50-2.80 (m, 6H: 4H, piperazine;
2H, CONCH2), 2.85 (s, 3H, CH3), 3.10-3.20 (m, 4H, piperazine),
4.95 (t, 2H, SCH2), 6.80-7.05 (m, 4H, Ar), 7.45-7.60 (m, 3H,
Ar), 8.10-8.25 (m, 2H. Ar).
Biological Assays. Animals. Male Swiss albino mice (23-30
g) from Morini breeding farm (Italy) and male Sprague-Dawley
(200-250) rats from Harlan Laboratories (Italy) were used. Fifteen
mice and four rats were housed per cage. The cages were placed
in the experimental room 24 h before the test for acclimatization.
The animals were kept at 23 ( 1 °C with a 12 h light/dark cycle,
light at 7 a.m., with food and water ad libitum. All experiments
were carried out according to the guidelines of the European
Community Council.
Hot Plate Test. The method adopted was described by
O’Callaghan and Holzman.27 Mice were placed inside a stainless
steel container, thermostatically set at 52.5 ( 0.1 °C in a precision
water-bath from KW Mechanical Workshop, Siena, Italy. Reaction
times (s), were measured with a stop-watch before and at regular
intervals up to a maximum of 60 min after treatment. The endpoint
used was the licking of the fore or hind paws. Those mice scoring
below 12 and over 18 s in the pretest were rejected (30%). An
arbitrary cutoff time of 45 s was adopted.
Abdominal Constriction Test. Mice were injected i.p. with a
0.6% solution of acetic acid (10 mL kg-1) according to Koster et
al.28 The number of stretching movements was counted for 10 min,
starting 5 min after acetic acid injection.
Statistical Analysis. All experimental results are given as the
mean ( S.E.M. Analysis of variance, followed by Fisher’s protected
least significant difference (PLSD) procedure for post-hoc com-
parison, was used to verify the significance between two means.
Data were analyzed with the StatView software for the Macintosh
(1992). P values of less than 0.05 were considered significant.
Drugs. The following drugs were used: yohimbine hydrochlo-
ride, naloxone hydrochloride, atropine sulfate and prazosin (Sigma),
BRL-44408 (Tocris), ARC-239 (Tocris), and CGP-35358 (Ciba
Geigy). Other chemicals were of the highest quality commercially
available. All drugs were dissolved in isotonic (NaCl 0.9%) saline
solution or dispersed in sodium carboxymethylcellulose 1%.
Noradrenaline Release from Rat Cerebral Cortex Evaluated
by Microdialysis Technique. Experiments were performed fol-
lowing the methods described by Oropeza et al.29
Rats were anesthetized with 8% chloral hydrate (400 mg/kg) and
placed in a stereotaxic apparatus with the skull flat. A small burr
hole was made in the skull centered at 3.2 mm anterior and (0.9
mM MgCl2 and 4 mM KCl was continuously perfused through the
probe by a microliter infusion pump. Approximately 18 h following
surgery, dialysate samples were collected every 20 min. Dialisate
samples were stored at -80 °C for subsequent analysis by HPLC-
ED. Vertical concentric microdialysis probes were used.
Supporting Information Available: Elemental analyses for all
target compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Allison, M. C.; Howatson, A. G.; Torrance, C. J.; Lee, F. D.; Russel,
R. I. Gastrointestinal damage associated with the use of nonsteroidal
anti-inflammatory drugs. N. Engl. J. Med. 1992, 327, 749-754.
(2) Walsh, T. D. Prevention of opioid side effects. J. Pain Symptom
Manage. 1990, 5, 362-367.
(3) Vane, J. R.; Bakhle, Y. S.; Botting, R. M. Cyclooxigenases 1 and 2.
Annu. ReV. Pharmacol. Toxicol. 1998, 20, 649-654.
(4) Prasit, P.; Riendeau, D. Selective cyclooxigenase-2 inhibitors. Annu.
Rep. Med. Chem. 1997, 32, 211-220.
(5) Bombardier, C.; Laine, L.; Reicin, A.; Shapiro, D.; Burgos-Vargas,
R.; Davis, B.; Day, R.; Ferraz, M. B.; Hawkey, C. J.; Hochberg, M.
C.; Kvien, T. K.; Schhnitzer, T. J. Comparison of upper gastrointes-
tinal toxicity of rofecoxib and naproxen in patiens with rheumatoid
arthritis. VIGOR Study Group. N. Engl. J. Med. 2000, 343, 1520-
1528.
(6) Juni, P.; Nartey, L.; Reichenbach, S.; Sterchi, R.; Dieppe, P. A.;
Egger, M. Risk of cardiovascular events and rofecoxib: cumulative
meta-analysis. Lancet 2004, 364, 2021-2029.
(7) Stichtenoth, D. O.; Frolich, J. C. The second generation of COX-2
inhibitors: what advantages do the newer offer? Drugs 2003, 63,
33-45.
(8) Rowbotham, M. C.; Twilling, L.; Davies, P. S.; Reisner, L.; Taylor,
K.; Mohr, D. Oral opioid therapy for chronic peripheral and central
neuropathic pain. N. Engl. J. Med. 2003, 348, 1223-1232.
(9) Gordon, D. B.; Love, G. Pharmacologic management of neuropathic
pain. Pain Manage. Nurs. 2004, 5, 19-33.
(10) Wallace, M. S. Emerging drugs for neuropathic pain. Exp. Opin.
Emerging Drugs 2001, 6, 249-259.
(11) Collins, S. D.; Chessell, I. P. Emerging therapies for neuropathic
pain. Exp. Opin. Emerging Drugs 2005, 10, 95-108.
(12) Dal Piaz, V.; Ciciani, G.; Turco, G.; Giovannoni, M. P.; Miceli, M.;
Pirisino, R.; Perretti, M. 5-Acyl-6-aryl-4-nitro-3(2H)-pyridazinones
and related 4-amino compounds: synthesis and pharmacological
evaluation. J. Pharm. Sci. 1991, 80, 341-348.

AntinociceptiVe Agents
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7835
(13) Dal Piaz, V.; Giovannoni, M. P.; Ciciani, G.; Barlocco, D.; Giardina,
G.; Petrone, G.; Clarke, G. D. 4,5-Functionalized 6-phenyl-3(2H)-
pyridazinones: synthesis and evaluation of antinociceptive activity.
Eur. J. Med. Chem. 1996, 31, 65-70.
(22) Aoki, T.; Takaheshi, A.; Sato, H. Eur. Pat. Appl. CODEN EPXXDW,
EP 661266, 1995, p 123.
(23) Ghelardini, C.; Galeotti, N.; Bartolini, A. Antinociception Induced
by Amitriptyline and Imipramine is Mediated by R2-adrenoceptor.
Jpn. J. Pharmacol. 2000, 82, 130-137.
(24) Galeotti, N.; Ghelardini, C.; Vinci, M. C.; Bartolini, A. Role of
potassium channels in the antinociception induced by agonists of R2-
adrenoceptors. Br. J. Pharmacol. 1999, 126, 1214-1220.
(25) Millan, M. J.; Bervoets, K.; Rivet, J. M.; Widdowson, P.; Renouard,
A.; Le Marouille-Girardon, S.; Gobert, A. Multiple R2-adrenergic
receptor subtypes. Evidence for a role of rat RR2A adrenergic receptors
in the control of nociception, motor behaviour, and hippocampal
synthesis of noradrenaline. J. Pharmacol. Exp. Ther. 1994, 270, 958-
972.
(26) Giannangeli, M.; Cazzola, N.; Luparini, M. R.; Magnani, M.; Mabilia,
M.; Picconi, G.; Tommaselli, M.; Baiocchi, L. Effect of modification
of the alkyl piperazine moiety of trazodone on 5HT2A and alpha-1
receptor binding affinity. J. Med. Chem. 1999, 42, 336-345.
(27) O’Callaghan, J. P.; Holtzman, S. G. Quantification of the analgesic
activity of narcotic antagonists by a modified hot-plate procedure.
J. Pharmacol. Exp. Ther. 1975, 192, 497-505.
(28) Koster, R.; Anderson, M.; De Beer, E. J. Acetic acid for analgesic
screening. Fed. Proc. 1959, 18, 412.
(29) Oropeza, V. C.; Page, M. E.; Van Bockstaele, E. J. Systemic
administration of WIN 55,212-2 increases norepinephrine release in
the rat frontal cortex. Brain Res. 2005, 1046, 45-54.
(14) Pieretti, S.; Dal Piaz, V.; Matucci, R.; Giovannoni, M. P.; Galli, A.
Antinociceptive activity of 3-(2H)-pyridazinone derivative in mice.
Life Sci. 1999, 65, 1381-1394.
(15) Dal Piaz, V.; Pieretti, S.; Vergelli, C.; Castellana, M. C.; Giovannoni,
M. P. 4-Amino-5-vinyl-3-(2H)-pyridazinones and related com-
pounds: synthesis and evaluation of antinociceptive activity. J.
Heterocycl. Chem. 2002, 39, 523-533.
(16) Dal Piaz, V.; Vergelli, C.; Giovannoni, M. P.; Scheideler, M. A.;
Petrone, G.; Zaratin, P. 4-Amino-5-vinyl-3(2H)-pyridazinones bearing
arylpiperazinylalkyl groups and related compounds: synthesis and
antinociceptive activity. Farmaco 2003, 58, 1063-1071.
(17) Giovannoni, M. P.; Vergelli, C.; Ghelardini, C.; Galeotti, N.; Bartolini,
A.; Dal Piaz, V. [(3-Chlorophenyl)piperazinylpropyl]pyridazinones
and analogues as potent antinociceptive agents. J. Med. Chem. 2003,
46, 1055-1059.
(18) Chantegrel, B.; Deshajes, C.; Pujol, B.; Wei, Z. J. Synthesis of
isoxazolo[4,5-d]pyridazin-4(5H)-ones and 4-acyl-5-hydroxy-3(2H)-
pyridazinones. J. Heterocycl. Chem. 1990, 27, 927-934.
(19) Sprio, V.; Aiello, E.; Mazza, A. Nitrogen heterocycles. II. Hydro-
genation of isoxazolo[3,4-d]pyridazin-7-ones, isoxazolo[3,4-d]-
pyridazin-4-ones, and isoxazolo[3,4-d]pyridazine-4,7-diones. Ann.
Chim. 1967, 57, 836-845.
(20) Dal Piaz, V.; Graziano, A.; Haider, N.; Holzer, W. Synthesis and
complete 1H, 13C, and 15N NMR assignment of substituted isoxazolo-
[3,4-d]pyridazin-7-(6H)-ones. Magn. Reson. Chem. 2005, 43, 240-
254.
(30) Franceschini, D.; Lipartiti, M.; Giusti, P. Effect of acute and chronic
tramadol on [3H]-norepinefrine uptake in rat cortical synaptosomes.
Prog. Neuro-Psycopharmacol. Biol. Psychiatry 1999, 23, 485-
496.
(21) Hassan, A. A. The regioselectivity of 1,3-dipolar cycloaddition
of R-carbonylformonitrile N-arylimides to benzylidenacetone and
â-diketones. J. Chem. Res. 1996, 7, 316-317.
JM060743G