[(3-Chlorophenyl)piperazinylpropyl]pyridazinones and analogues as potent antinociceptive agents

Article abstract of DOI:10.1021/jm021057u

A number of [(3-chlorophenyl)piperazinylpropyl]pyridazinones and the corresponding isoxazolopyridazinones, showing the arylpiperazinyl substructure present in very potent antinociceptive agents reported in the literature, were synthesized and tested for t

Full text of DOI:10.1021/jm021057u

J . Med. Chem. 2003, 46, 1055-1059
1055
[(3-Ch lor op h en yl)p ip er a zin ylp r op yl]p yr id a zin on es a n d An a logu es a s P oten t
An tin ocicep tive Agen ts
Maria Paola Giovannoni,*,† Claudia Vergelli,† Carla Ghelardini,‡ Nicoletta Galeotti,‡ Alessandro Bartolini,‡ and
Vittorio Dal Piaz†
Dipartimento di Scienze Farmaceutiche, Universita` di Firenze, via Gino Capponi 9, 50121 Firenze, Italy, and
Dipartimento di Farmacologia Clinica e Preclinica, Universita` di Firenze, viale Pieraccini 6, 50139 Firenze, Italy
Received October 2, 2002
A number of [(3-chlorophenyl)piperazinylpropyl]pyridazinones and the corresponding isoxazolo-
pyridazinones, showing the arylpiperazinyl substructure present in very potent antinociceptive
agents reported in the literature, were synthesized and tested for their analgesic activity. The
investigated compounds showed antinociceptive properties in the mouse hot-plate test (thermal
nociceptive stimulus) after systemic administration with an efficacy similar to that exerted by
morphine. The increase of the pain threshold induced by the compounds labeled 5a , 7, 8, and
11 was prevented by reserpine, suggesting the involvement of the noradrenergic and/or
serotoninergic system in their mechanism of action. Among them, 7 and 11 showed the highest
analgesic potency and efficacy together with a good ratio (133 and 200, respectively) of the
minimal nontoxic dose (MNTD) to the minimal analgesic dose (MAD). Furthermore, they were
also active after icv administration and in the presence of a chemical, painful stimulus
(abdominal constriction test).
In tr od u ction
to take into account that pain is a very complex process
in which many different neuromodulators are involved,
such as glutamate, acetylcholine, GABA, adrenaline,
and so on.8,10-12
Two major groups of drugs are currently used for
treatment of pain: the traditional non-steroidal anti-
inflammatory drugs (NSAIDs) and opioids. The first
class, whose effects are mediated by the peripheral
inhibition of cyclooxygenase (COX), the enzyme respon-
sible for prostaglandins synthesis,1 is generally used in
the treatment of mild to moderate pain,2 and the
common side effects include gastrointestinal lesions,
such as ulcerations and perforation, nephrotoxicity, and
inhibition of platelet aggregation.3 After the discovery
of the two isoforms (COX1 and COX2),1 together with
the evidence that COX2 is induced by proinflammatory
mediators,4 potent and selective COX2 inhibitors were
developed, showing that these compounds have compa-
rable analgesic activity with respect to traditional
NSAIDs but with very low incidence of unwanted side
effects.5
The class of opioid drugs, which includes morphine
and congeners, is used in moderate to severe pain and
operates by activating central G-protein-coupled recep-
tors µ, δ, and κ.6 Side effects associated with the clinical
use of opioids are very strong and use-limiting, the most
important being respiratory depression, tolerance, physi-
cal dependence, and constipation.7
Our experience in the pyridazine field,13-16 together
with the observation that some pyridazine derivatives
such as emorfazone (A, Chart 1),17 on the market in
J apan, and compound B,18 bearing an alkylpiperazinyl
alkyl moiety, show interesting antinociceptive activity
not related to effects on prostaglandins or opioid system,
led us to design and synthesize a series of pyridazinone
derivatives as potential analgesic drugs. In this study
we identified compound C14 as a promising lead, show-
ing good antinociceptive activity in mouse abdominal
constrictions model with an ED50 of 14.9 mg kg-1, sc
(quantal protection of 100% at 100 mg kg-1), resulting
in 7-fold more potent with respect to emorfazone. In the
same investigation, another interesting compound (D)14
emerged, showing 60% inhibition of abdominal constric-
tion and 40% of quantal protection at 100 mg kg-1
.
Taking into account these results, our research was
focused on the synthesis of 4-amino-5-vinyl- and 4-amino-
5-acetylpyridazinones bearing an arylpiperazinyl moiety
at position 2 of the pyridazine ring. Very encouraging
results were obtained by combining the vinyl and the
arylpiperazinyl functions, in particular for compound
E19 which showed a dramatic increase of activity in the
writhing test with respect to our previous lead C (ED50
) 2.5 mg kg-1, sc and 100% of quantal protection at 100
mg kg-1).
Finally, it is possible to define a third class of anal-
gesic drugs, the so-called “analgesic adjuvants”,8 which
are drugs with a different primary use, for example,
antidepressants, anticonvulsants, and anesthetics, but
able to control neuropathic pain.8,9
Current research in pain therapy looks at the discov-
ery of new potent drugs devoid of the limiting side
effects of the above-mentioned classes. It is necessary
In the present paper, we report the effect of the
elongation of the methylenic spacer in compound E and
that of insertion of a chlorine at the meta position of
the arylpiperazinyl moiety; in this way, we inserted into
our nucleus a substructure present in very potent
antinociceptive agents, like compound F .20 At the same
time, we synthesized and tested bicyclic derivatives
* To whom correspondence should be addressed. Phone: +39-55-
2757296. Fax: +39-55-240776. E-mail: mariapaola.giovannoni@unifi.it.
† Dipartimento di Scienze Farmaceutiche.
‡ Dipartimento di Farmacologia Clinica e Preclinica.
10.1021/jm021057u CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/30/2003

1056 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Giovannoni et al.
Ch a r t 1
Sch em e 1a
bearing the same fragment, in which the pyridazinone
ring is [4,5-d]- and [3,4-d]-fused with an isoxazole.
a
(a) EtOH, EtONa; (b) hydrazine hydrate, EtOH, room temp;
(c) 1-(3-bromopropyl)-4-(3-chlorophenyl)piperazine, DMF, anhy-
drous K2CO3, 60-70 °C; (d) ammonium formate, 10% Pd/C, EtOH,
reflux; (e) NaBH4, MeOH, room temp; (f) PPA, room temp.
Ch em istr y
The synthetic pathway affording the final 5-8 is
depicted in Scheme 1. With the exception of 3c and 4c,
the isoxazoles 3 and the isoxazolo[3,4-d]pyridazinones
4 were previously described.21-23 1-(3-Bromopropyl)-4-
(3-chlorophenyl)piperazine was prepared by condensing
3-chlorophenylpiperazine with 1,3-dibromopropane in
DMF and in the presence of K2CO3 at room tempera-
ture. The crude reaction product was reacted with
compounds 4 under the same reaction conditions to
afford 5. Compound 5c was transformed into the cor-
responding 5-acetyl-4-amino derivative 6 by reductive
cleavage with ammonium formate in ethanol and then
in the corresponding 5-hydroxyethyl 7 by using sodium
borohydride in anhydrous methanol. Finally, the dehy-
dratation of 7 with polyphosphoric acid (PPA) afforded
the vinyl derivative 8.
Sch em e 2a
Under the same conditions described above for 5,
compound 11 was obtained starting from 924 (Scheme
2) through the bromoderivative 10.
a
(a) 1,3-Dibromopropane, K2CO3, anhydrous DMF, 60 °C; (b)
3-chlorophenylpiperazine, K2CO3, anhydrous DMF, 70 °C.
minimal analgesic dose (MAD). The range of MNTD/
MAD values is from 4 (5a , 5c) to 200 (6, 8, 11). All the
compounds, at the highest effective doses, neither
produced any alteration of the animals’ gross behavior
nor modified the spontaneous motility and inspection
activity, as revealed by the hole board test (data not
shown). The analgesia induced by 5a , 5b, 7, and 11 was
completely prevented by pretreatment with the monoam-
ine store depletor reserpine (2 mg kg-1 ip, administered
48 and 24 h before the test), 8 was partially prevented,
whereas 5c and 6 were unmodified by reserpine pre-
treatment (Table 2). The dose and administration
schedule of reserpine was able to selectively prevent
antinociception induced by the antidepressant drugs
Resu lts a n d Discu ssion
Subcutaneous injection of 5a (20 mg kg-1), 11 (20 mg
kg-1), 6 (3 mg kg-1), 7 (6 mg kg-1), 8 (1 mg kg-1), and
5c (20 mg kg-1) and per os administration of 5b (10 mg
kg-1) induced antinociception in the mouse hot-plate
test. The effect appeared 15 min after administration,
peaked after 30 min, and then slowly diminished (Table
1). Analgesic efficacy of the investigated compounds was
comparable to that exerted by morphine (8 mg kg-1),
used as a reference drug, since 71-75% of the morphine
effect was reproduced by the less active compounds
5a -c of the series (Table 2). All compounds showed a
good ratio of the minimal nontoxic dose (MNTD) to the

New Antinociceptive Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1057
Ta ble 1. Antinociceptive Effect of Compounds 5a -c, 6-8, and 11 in the Mouse Hot-Plate Test
licking latency (s)
after treatment
treatment
before treatment
15 min
30 min
45 min
60 min
saline, 10 mL kg-1, sc
vehicle, 10 mL kg-1, sc
morphine, 8 mg kg-1, sc
5a , 20 mg kg-1, sc
5b, 10 mg kg-1, po
5c, 20 mg kg-1, sc
6, 3 mg kg-1, sc
14.1 ( 0.9
14.2 ( 1.2
14.6 ( 1.3
16.6 ( 0.7
15.1 ( 1.3
16.0 ( 0.9
15.5 ( 0.8
14.5 ( 0.9
14.2 ( 0.8
14.6 ( 0.9
15.7 ( 2.4
14.9 ( 1.9
24.5 ( 1.3b
18.0 ( 2.0
20.8 ( 1.7a
22.2 ( 1.4b
23.7 ( 1.4b
22.4 ( 1.8b
21.3 ( 1.5a
19.6 ( 1.8
14.4 ( 1.0
15.3 ( 1.5
28.7 ( 1.9b
24.0 ( 1.6b
25.1 ( 2.0b
23.8 ( 2.6b
33.2 ( 2.0b
33.7 ( 1.9b
33.5 ( 2.3b
34.5 ( 1.6b
15.5 ( 1.9
16.0 ( 2.1
26.4 ( 1.8b
22.6 ( 1.5b
19.1 ( 1.8a
23.2 ( 2.1b
34.3 ( 2.4b
29.3 ( 1.8b
28.2 ( 2.5b
31.2 ( 1.6b
14.7 ( 1.6
15.3 ( 1.8
23.8 ( 1.6b
17.6 ( 1.9
16.6 ( 1.8
17.8 ( 2.0
32.7 ( 2.3b
23.3 ( 1.5b
22.6 ( 2.1b
29.3 ( 1.6b
7, 6 mg kg-1, sc
8, 1 mg kg-1, sc
11, 20 mg kg-1, sc
a
b
P < 0.05 in comparison with vehicle-treated animals. Vehicle is represented by saline + DMSO 2:1. P < 0.01 in comparison with
vehicle-treated animals. Vehicle is represented by saline + DMSO 2:1.
Ta ble 2. Comparison among the Minimal Analgesic Dose
(MAD), the Maximal Nontoxic Dose (MNTD), and the Efficacy
of the Tested Compounds and Morphine and Effect of
Reserpine on Antinociception Induced by the Same Compounds
% analgesic
efficacy
% of
antagonism
exerted by
reserpined
MAD,a
MNTD,b
mg kg-1
compared to
compd
mg kg-1
morphinec
morphine
0.2 sc
10 sc
1 po
10 sc
0.1 sc
0.3 sc
0.1 sc
0.3 sc
30 sc
40 sc
50 sc
40 sc
20 sc
40 sc
20 sc
60 sc
100
72
75
0
92
94
0
5a
5b
5c
6
7
8
71
100
101
100
104
0
100
61
100
11
a
Antinociceptive effect was evaluated on the mouse hot-plate
test. The percent of analgesic efficacy was evaluated at the
maximal analgesic dose. The maximal analgesic effect of morphine
b
is indicated as 100%. Minimal dose able to induce a statistically
significant increase of the pain threshold. c Tested at 8 mg kg-1
.
F igu r e 1. Antinociceptive effect of 7 (6 mg kg-1, sc) and 11
(20 mg kg-1, sc) in the mouse abdominal constriction test. Both
compounds were administered 30 min before the test. Vertical
lines show SEM, and the asterisk (/) represents P < 0.01 in
comparison with vehicle controls. Each point represents the
mean of at least eight mice.
d
Tested at 2 mg kg-1
.
clomipramine and amitriptyline,25 indicating that it is
ideal for preventing the increase of pain threshold
induced by activation of the catecholaminergic and
serotoninergic systems. On the basis of these data, we
can exclude that 5c and 6 exert their antinociceptive
effect through a catecholaminergic mechanism. Among
the compounds completely prevented by reserpine, 7
and 11 were further investigated to elucidate their
pharmacological profile because they showed the best
MNTD/MAD ratio. 7 (6 mg kg-1 sc) and 11 (20 mg kg-1
sc) also showed antinociceptive properties in the ab-
dominal constriction test in which a chemical nocicep-
tive stimulus was applied (Figure 1). Both compounds
were able to almost completely abolish the number of
abdominal constriction 15 and 30 min after administra-
tion. Their analgesic activity persisted almost un-
changed up to 60-75 min, and then it progressively
diminished, disappearing at 120 min. Furthermore, 7
and 11 induced analgesia after icv administration with
an efficacy and time course similar to those observed
after systemic administration (Figure 2). These results
indicate that their site of action is within the central
nervous system. It was, in fact, possible to reach the
same intensity of analgesia by injecting directly into the
cerebral ventricles doses of both compounds that were
lower than those needed parenterally. That the anti-
nociception depends on a retrodiffusion of the drug from
the cerebral ventricles to the periphery can thus be
ruled out. In conclusion, all the investigated compounds
F igu r e 2. Antinociceptive effect of 7 (10 µg per mouse icv)
and 11 (20 µg per mouse icv) in the mouse hot-plate test.
Vertical lines give SEM. Each point is the mean of at least 12
mice. The asterisk (/) represents P < 0.01 in comparison with
saline-treated mice.
are endowed with good analgesic activity, comparable
to that exerted by morphine. Furthermore, with the
exception of 5c and 6, these derivatives exert their

1058 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Giovannoni et al.
(m, 2H, CONCH2CH2CH2N), 2.55 (s, 3H, 4-CH3), 2.60 (m, 6H,
piperazine), 3.20 (m, 4H, CONCH2CH2CH2N and 2H pipera-
zine), 4.30 (t, 2H, CONCH2CH2CH2N), 6.80 (m, 3H, Ar), 7.25
(m, 1H, Ar), 7.60 (s, 5H, Ar). Anal. (C25H26N5O2Cl) C, H, N.
analgesic activity through a partial or complete activa-
tion of the monoaminergic system.
For the structure-activity relationships (SARs), the
data obtained in the hot-plate test clearly suggest that
the activity of this series does not depend on the
presence of an isoxazolo[3,4-d]- (5a -c) or [4,5-d]-fused
system (11). Likewise, when the substructure arylpip-
erazinylpropyl is linked to a functionalized pyridazi-
none, good results were obtained with different func-
tional groups at position 5 (compounds 6-8).
Eth yl 6-{[4-(3-ch lor op h en yl)p ip er a zin -1-yl]p r op yl}-3-
m et h ylisoxa zolo[3,4-d ]p yr id a zin -7-(6H )-on e-4-ca r b oxy-
la te, 5c: yield ) 72%; mp ) 98-100 °C (EtOH); 1H NMR
(CDCl3) δ 1.40 (t, 3H, CH2CH3), 2.15 (m, 2H, CONCH2CH2-
CH2N), 2.55 (m, 6H, piperazine), 3.05 (s, 3H, CCH3), 3.10 (m,
4H, CONCH2CH2CH2N and 2H piperazine), 4.30 (t, 2H,
CONCH2CH2CH2N), 4.50 (q, 2H, CH2CH3), 6.80 (m, 3H, Ar),
7.20 (m, 1H, Ar). Anal. (C22H26N5O4Cl) C, H, N.
Thus, it seems that the side chain plays a prominent
role in determining the antinociceptive activity of the
present series. Since compound F is the most potent
antinociceptive agent in a large series of isothiazolo-
[5,4-b]pyridines, it seems to confirm this hypothesis.
In conclusion, in this study we identified a group of
potent antinociceptive agents that are active in the hot-
plate test with an efficacy comparable to or higher than
that of morphine. Interestingly, these compounds showed
a good MNTA/MAD ratio, which in some cases achieved
a value of 200.
5-Acetyl-4-a m in o-2-{[4-(3-ch lor op h en yl)p ip er a zin -1-yl]-
p r op yl}-6-m eth ylp yr id a zin -3(2H)-on e, 6. A mixture of 5a
(0.4 mmol), 10% Pd/C (80 mg), and ammonium formate (2
mmol) in EtOH (3 mL) was refluxed for 1 h. After addition of
CH2Cl2 (4 mL) and filtration of charcoal, crude 6 was recovered
1
by suction. Yield ) 75%; mp ) 125-126 °C (EtOH); H NMR
(CDCl3) δ 2.10 (m, 2H, CONCH2CH2CH2N), 2.50 (s, 3H, CCH3),
2.55 (s, 3H, COCH3), 2.70 (m, 4H, piperazine), 3.25 (m, 6H,
CONCH2CH2CH2N and 4H piperazine), 4.15 (t, 2H, CONCH2-
CH2CH2N), 6.90 (m, 2H, Ar), 7.25 (m, 2H, Ar). Anal. (C20H26
N5O2Cl) C, H, N.
-
4-Am in o-2-{[4-(3-ch lor op h en yl)p ip er a zin -1-yl]p r op yl}-
5-h yd r oxyeth yl-6-m eth ylp yr id a zin -3(2H)-on e, 7. To a so-
lution of 6 (0.2 mmol) in methanol (3 mL), sodium borohydride
(1.6 mmol) was added portionwise under stirring at room
temperature. After 1 h, the mixture was concentrated, diluted
with water (15 mL), and extracted with CH2Cl2 (3 × 15 mL).
Evaporation of the solvent afforded the desiderated 7. Yield
) 57%; mp ) 132-134 °C (EtOH); 1H NMR (CDCl3) δ 1.55 (d,
3H, CH(OH)CH3), 2.10 (m, 2H, CONCH2CH2CH2N), 2.20 (s,
3H, CCH3), 2.50 (t, 2H, CONCH2CH2CH2N), 2.60 (m, 4H,
piperazine), 3.20 (m, 4H, piperazine), 4.15 (t, 2H, CONCH2-
CH2CH2N), 5.10 (q, 1H, CH(OH)CH3), 5.85 (exch br s, 1H, OH),
6.50 (m, 3H, Ar), 7.30 (m, 1H, Ar). Anal. (C20H28N5O2Cl) C, H,
N.
Exp er im en ta l Section
Ch em istr y. All melting points were determined on a Bu¨chi
apparatus and are uncorrected. 1H NMR spectra were recorded
with Varian Gemini 200 instruments. Chemical shifts are
reported in ppm, using the solvent as internal standard.
Extracts were dried over Na2SO4, and the solvents were
removed under reduced 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
1a -c and 2 were commercially available.
Eth yl 4-eth yloxoa ceta te-5-m eth ylisoxa zole-3-ca r boxy-
la te, 3c. To a cooled (0 °C) and stirred solution of sodium
ethoxide, obtained from sodium (15 mmol) and anhydrous
ethanol (30 mL), a solution of ethyl 2,4-dioxopentanoate 1c
(15 mmol) in the same solvent (15 mL) was slowly added. A
solution of ethyl chloro(hydroximino)acetate 2 (15 mmol) in
anhydrous EtOH (10 mL) was added dropwise. The mixture,
neutralized with 6 N HCl, was evaporated to afford 3c, which
was purified by column chromatography using cyclohexane/
ethyl acetate 1:2 as eluent. Yield ) 73%; oil; 1H NMR (CDCl3)
δ 1.40 (t, 6H, 2CH2CH3), 2.70 (s, 3H, CCH3), 4.40 (q, 4H, 2CH2-
CH3).
Eth yl [(6,7-d ih yd r o-3-m eth yl-7-oxoisoxa zole[3,4-d ]p y-
r id a zin yl]-4-ca r boxyla te, 4c. To a solution of 3c (0.2 mmol)
in EtOH (2 mL), hydrazine hydrate (0.4 mmol) was added, and
the mixture was stirred at room temperature for 10 min. The
precipitate 4c was recovered by suction. Yield ) 65%; mp )
189-190°C (EtOH); 1H NMR (CDCl3) δ 1.50 (t, 3H, CH2CH3),
3.05 (s, 3H, CCH3), 4.50 (q, 2H, CH2CH3).
Gen er a l P r oced u r e for 5a -c. A mixture of isoxazolopy-
ridazinones 4a -c22,23 (0.1 mmol), anhydrous K2CO3 (0.5 mmol),
and 1-(3-bromopropyl)-4-(3-chlorophenyl)piperazine (0.4 mmol)
in anhydrous DMF (2 mL) was heated under stirring for 2-6
h at 60-70 °C. After dilution with cold water (20-30 mL),
compounds 5a ,b were recovered by suction. For compound 5a ,
the suspension was extracted with CH2Cl2 (3 × 15 mL) and
the solvent was evaporated in vacuo to afford a crude precipi-
tate.
4-Am in o-2-{[4-(3-ch lor op h en yl)p ip er a zin -1-yl]p r op yl}-
6-m eth yl-5-vin ylp yr id a zin -3(2H)-on e, 8. Compound 7 (0.5
mmol) was treated with PPA (50 mmol) at room temperature
for 4 h. After dilution with water, the mixture was neutralized
with 6 N NaOH and extracted with CH2Cl2 (3 × 20 mL). The
residue was purified by column chromatography using CHCl3/
MeOH 9:1 as eluent.
1
Yield ) 55%; mp ) 86-88 °C; H NMR (CDCl3) δ 2.15 (m,
2H, CONCH2CH2CH2N), 2.30 (s, 3H, CCH3), 2.55 (s, 3H,
COCH3), 2.50 (t, 2H, CONCH2CH2CH2N), 2.80 (m, 4H, pip-
erazine), 3.20 (m, 4H, piperazine), 4.20 (t, 2H, CONCH2CH2-
CH2N), 5.60 (d, 1H, J ) 17.9 Hz, CHdCH2), 5.70 (d, 1H, J )
12.1 Hz, CHdCH2), 6.55 (dd, 1H, J ) 12.1 Hz, J ) 17.9 Hz,
CHdCH2), 6.90 (m, 2H, Ar), 7.30 (m, 2H, Ar). Anal. (C20H26N5-
OCl) C, H, N.
5-(3-Br om op r op yl)-3-m eth yl-7-p h en ylisoxa zolo[4,5-d ]-
p yr id a zin -4(5H)-on e, 10. A mixture of 9 (1.5 mmol), K2CO3
(7.2 mmol), and 1,3-dibromopropane (2.0 mmol) in anhydrous
DMF (1 mL) was stirred at 60 °C for 1 h. After the mixture
was cooled, water was added and the mixture was extracted
with CH2Cl2 (3 × 20 mL). Evaporation of the solvent afforded
10. Yield ) 72%; mp ) 108-110 °C (EtOH); 1H NMR (CDCl3)
δ 2.45 (m, 2H, CONCH2CH2CH2N), 2.75 (s, 3H, CCH3), 3.50
(t, 2H, CONCH2CH2CH2Br), 4.50 (t, 2H, CONCH2CH2CH2N),
7.50 (m, 3H, Ar), 8.20 (m, 2H, Ar).
5-{[4-(3-Ch lor op h en yl)p ip er a zin -1-yl]p r op yl}-3-m eth -
yl-7-p h en ylisoxa zolo[4,5-d ]p yr id a zin -4-(5H)-on e, 11. A
mixture of 10 (0.25 mmol), K2CO3 (1.3 mmol), and 3-chlo-
rophenylpiperazine (0.6 mmol) in anhydrous DMF (1 mL) was
stirred at 70 °C for 3 h. After dilution with cold water, the
precipitate was recovered by suction and purified by column
chromatography using cyclohexane/ethyl acetate 1:2 as eluent.
Yield ) 85%; mp ) 102-103 °C (acetone); 1H NMR (CDCl3) δ
2.15 (m, 2H, CONCH2CH2CH2N), 2.60 (m, 6H, CONCH2-
CH2CH2N and 4H piperazine), 2.75 (s, 3H, CCH3), 3.20 (m,
4H, piperazine), 4.55 (t, 2H, CONCH2CH2CH2N), 6.80 (m, 3H,
6-{[4-(3-Ch lor op h en yl)p ip er a zin -1-yl]p r op yl}-3,4-d i-
m eth ylisoxa zolo[3,4-d ]p yr id a zin -7-(6H)-on e, 5a : yield )
1
51%; mp ) 100-102 °C (EtOH); H NMR (CDCl3) δ 2.15 (m,
2H, CONCH2CH2CH2N), 2.55 (s, 3H, 4-CH3), 2.65 (m, 6H,
CONCH2CH2CH2N and 4H piperazine), 2.85 (s, 3H, 3-CH3),
3.15 (m, 4H, piperazine), 4.20 (t, 2H, CONCH2CH2CH2N), 6.80
(m, 3H, Ar), 7.15 (m, 1H, Ar). Anal. (C20H24N5O2Cl) C, H, N.
6-{[4-(3-Ch lor op h en yl)p ip er a zin -1-yl]p r op yl}-3-m eth -
yl-4-p h en ylisoxa zolo[3,4-d ]p yr id a zin -7-(6H)-on e, 5b: yield
) 88%; mp ) 112-115 °C (EtOH); 1H NMR (CDCl3) δ 2.15

New Antinociceptive Agents
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1059
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(C25H26N5O2Cl) C, H, N.
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Biologica l Assa ys. An im a ls. Male Swiss albino mice (23-
30 g) from Morini (San Polo d’Enza, Italy) breeding farm were
used. Fifteen mice 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
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guidelines of the European Community Council.
Hot-P la te Test. The method adopted was described by
O’Callaghan and Holzman.26 Mice were placed inside a stain-
less 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 stopwatch
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
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Dr u gs. The following drugs were used: reserpine (Ciba-
Geigy), morphine hydrochloride (SALARS). Other chemicals
were of the highest quality commercially available. All drugs
were dissolved in isotonic (0.9% NaCl) saline solution or
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solution of ascorbic acid. Drug concentrations were prepared
in such a way that the necessary dose could be administered
in a volume of 10 mL kg-1 by the subcutaneous (sc) or per os
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described by Haley and McCormick.28 Briefly, during anaes-
thesia, mice were grasped firmly by the loose skin behind the
head. A 0.4 mm external diameter hypodermic needle attached
to a 10 µL syringe was inserted perpendicularly through the
skull at a depth of no more than 2 mm into the brain of the
mouse where an amount of 5 µL was then administered. The
injection site was 1.5 mm from either side of the midline on a
line drawn to the anterior base of the ears. To ascertain that
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