4-Amino-5-vinyl-3(2H)-pyridazinones and analogues as potent antinociceptive agents: Synthesis, SARs, and preliminary studies on the mechanism of action

Article abstract of DOI:10.1016/j.bmc.2007.05.035

A series of 4-amino-5-vinyl-3(2H)-pyridazinones and analogues were synthesized and their antinociceptive effect was evaluated in the mouse abdominal constriction model. Several of the novel compounds showed ED₅₀ values in the range 6-20 mg/kg/s

Full text of DOI:10.1016/j.bmc.2007.05.035

Bioorganic & Medicinal Chemistry 15 (2007) 5563–5575
4-Amino-5-vinyl-3(2H)-pyridazinones and analogues as potent
antinociceptive agents: Synthesis, SARs, and preliminary studies
on the mechanism of action
Claudia Vergelli,a Maria Paola Giovannoni,a,* Stefano Pieretti,b Amalia Di Giannuario,b
Vittorio Dal Piaz,a Pierfrancesco Biagini,a Claudio Biancalani,a
Alessia Grazianoa and Nicoletta Cesaria
aDipartimento di Scienze Farmaceutiche, via Ugo Schiff 6, Sesto Fiorentino, 50019 Firenze, Italy
b
`
Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Roma, Italy
Received 31 August 2006; revised 10 May 2007; accepted 15 May 2007
Available online 18 May 2007
Abstract—A series of 4-amino-5-vinyl-3(2H)-pyridazinones and analogues were synthesized and their antinociceptive effect was
evaluated in the mouse abdominal constriction model. Several of the novel compounds showed ED50 values in the range 6–
20 mg/kg/sc and demonstrated to be able to completely protect all the treated animals from the effect of the noxious stimulus at
30 mg/kg/sc. SAR studies confirmed the essential role played by an amino or substituted amino function at position 4 and by a vinyl
group at position 5 of the diazine system.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
and local anesthetic drugs have been reported to amelio-
rate these pain conditions.4
Current research in pain therapy concerns development
of new potent antinociceptive agents endowed with the
efficacy of morphine, but devoid of its adverse and
use-limiting side-effects, such as respiratory depression,
constipation, tolerance, and physical dependence liabil-
ity. However, the second class of clinically dominant
antinociceptive drugs, that of non-steroidal anti-inflam-
matory drugs (NSAIDS), whose effects are mediated by
the peripheral inhibition of prostaglandin synthesis, suf-
fers from strong limitations due to the induction of gas-
tro-intestinal lesions and nephrotoxicity.1 Development
of selective COX-2 inhibitors only partially solved the
problem because of their severe cardiovascular second-
ary effects.2,3
Thus, development of non-anti-inflammatory, non-opi-
oid agents which are able to control pain from a broad
range of causes represents a primary objective in analge-
sic drug research.
Pyridazine derivatives displaying antinociceptive activ-
ity, like compounds 1a–b,5 have been reported in the lit-
erature since the early 1950s (Fig. 1). Among this type of
compounds, Emorfazone 2,6 which is characterized by
the absence of either effects on the prostaglandin system
or affinity for opioid receptors,7,8 emerged and was
launched in Japan.
More recently, a series of 4-carbamoyl-5-aryl-3(2H)-
pyridazinones 3 were also reported as compounds
displaying relevant analgesic activity both in writhing
and hot plate test.9 Compound 4 was patented for the
therapy of mild to moderate pain5 by ASTA Medica
as a peripherally acting analgesic displaying low acute
toxicity.10
Current available drugs are able to control nociceptive
and inflammatory pain, whereas neurogenic and psycho-
genic pain disorders lack an appropriate pharmacological
treatment since only antidepressant, anticonvulsivant,
Keywords: Pain; Antinociception; Vinylpyridazinones; SARs.
*
In addition, very recently Couquelet et al.11–13 described
some series of pyridazines and their 1,2-oxazine
Corresponding author. Tel.: +39 55 4573682; fax: +39 55 4573671;
e-mail: mariapaola.giovannoni@unifi.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.035

5564
C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
R1
N
CH3
N
N
N
O
CH3
O
CH3
X
R2
C2H5O
N
O
2
1
R1 R2
C6H5
X
a
H
NH2
b
CH3 CH3 CN
R1
N
N
N
N
C6H5
O
O
CH3
R3R2NOC
C6H5
3
4
CH3
N
O
N
N
O
CH3
O
C6H5
R1
N
CH2
C6H5
N
H2N
CH=CH2
5
6
Figure 1. Pyridazine derivatives and analogues displaying alalgesic activity.
bioisosteres 5, bearing arylpiperazine moieties, which
display significant antinociceptive effects in animal
models.
variety of amino groups. Moreover, we performed some
modifications at positions 2 and 6, as well as the aroma-
tization of the pyridazinonic ring in order to investigate
the importance of the lactamic function.
As a part of our efforts to discover new therapeutically
useful antinociceptive agents bearing a pyridazine
moiety, we previously described a series of 4,5-func-
tionalized-6-phenyl-3(2H)-pyridazinones14,15 structur-
ally related to some of compounds 1–4 and we
extensively studied the mechanism of action of the
2. Chemistry
The synthetic pathways affording the final 5-vinylpyrid-
azinones 12a–s are depicted in Scheme 1. With the
exception of 7i and 7k, the isoxazolo[3,4-d]pyridazinon-
es (Table 1), which represent the starting material, were
previously described.20–25 Compound 7i was prepared
by alkylation of the 3-methyl-4-phenylisoxazolo[3,4-
d]pyridazin-3(2H)-one (7a) with 3-trifluorobenzyl chlo-
ride in acetone in the presence of K2CO3. Likewise,
the 4-nitropyridazinones 8a, b (8a, R2 = CH3, 8b,
R2 = Ph), which originate from 7b and 7c by oxidative
cleavage with CAN, are known compounds.22 In the ser-
ies of 4-amino (9a–j) and substituted amino analogues
(10a–i), compounds 9a,20 9g26, and 10d14 were previ-
ously described (Table 2). The other compounds of type
9 were obtained in good yields from the appropriate pre-
cursor by reductive cleavage with ammonium formate
and Pd/C. In these reaction conditions the 4-chlorophe-
nyl derivative 7e underwent a concomitant dehalogen-
ation; thus the desired 9d was prepared by treatment
of 7e with Mo(CO)6 in CH3CN. The pyridazinthione
7k, obtained by treatment of 7a with Lawesson’s re-
agent, afforded 9j following the above procedure. The
representative compound
6
(ED50 = 14.9 mg/kg/sc,
quantal protection = 100% at 100 mg/kg) which seven
times more potent than Emorfazone in the mouse
abdominal constriction model.16
Further studies in this field led us to identify a group of
pyridazine derivatives, bearing an arylpyperazinyl moi-
ety, which showed very potent antinociceptive activ-
ity.17–19
In the present paper, we report our continued work on
lead 6. Extensive structure–activity relationship (SAR)
studies were performed, taking into account that com-
pound 6 is characterized by a very low degree of toler-
ance as regards the substitution at positions 4 and 5:
replacement of CH@CH2 with a variety of different func-
tionalities (COCH3, CN, COOCH3, CH(OC2H5)CH3),
as well as the saturation of the double bond, caused a
dramatic impairment of activity.15 On this basis, we pri-
marily focused our attention on position 4 by inserting a

C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
5565
R1
N
N
O
R2
N
a
b
CH2R3
CH3
N
O
7a-j
for 7a
HN
N
N
N
S
C6H5
O
R2
O2N
COCH3
8a,b
CH3
c
O
7k
d
c
R1
N
CH3
N
N
N
Z
R2
O
R2
H2N
COCH2R3
X
COCH3
9a-j
10a-i
e
f
R1
N
R1
N
N
N
Z
R2
Z
R2
Y
CH=CHR3
Y
CH(OH)CH2R3
12a-s
11a-s
Scheme 1. Synthetic procedure for compounds 12a–s. Reagents and conditions: (a) Lawesson’s reagent, toluene, 80 ꢁC, 90 min; (b) CAN, 50%
AcOH, 65% HNO3, 60 ꢁC, 30–60 min; (c) HCOONH4, Pd/C, EtOH, reflux, 1–2 h; Mo(CO)6, CH3CN for 9d; (d) appropriate nucleophile, EtOH, rt,
10–30 min; (e) NaBH4, CH3OH, rt, 10–90 min; (f) method A: PPA, rt À40 ꢁC, 1–48 h; method B: H2SO4 on silica gel, toluene, 45–60 ꢁC, 11–30 h.
methanol at room temperature afforded the correspond-
ing secondary alcohols 11a–s in moderate yield (Table 3)
which, in turn, were transformed into the final 5-vinyl
derivatives 12a–s in moderate yield by treatment with
PPA (Method A) or with sulfuric acid on silica gel27
(Method B) (Table 3).
Table 1.
Compound
R1
R2
R3
Z
7aa
7bb
7cb
7dc
7ed
7fe
7gf
7hb
7i
H
Ph
Me
Ph
Ph
Bh
H
H
H
Me
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
S
Me
Me
Me
Me
n-But
Ph
Scheme 2 depicts the synthesis of compounds 12t–v from
their precursor 6 and 12e. Compound 12t was obtained
by heating 6 with AcOH in pyridine; products 12u, v
were prepared by treatment of the appropriate precursor
with bromine, followed by treatment of the intermedi-
ates 13a, b with DBU in anhydrous THF.
Ph
Me
Ph
Ph
Ph
Ph
Ph
Ag
7je
COOEt
H
7k
a Ref. 20.
Finally, the synthesis of 5-vinylpyridazines 18a, b is
shown in Scheme 3. Treatment of 7k with iodomethane
in acetone in the presence of K2CO3 gave regioselec-
tively28 the methylthio derivative 14 which, in turn,
was transformed into the corresponding 3-methoxy ana-
logue 15 by nucleophilic replacement with sodium meth-
oxide. Treatment of the same precursor 14 with
hydrogen in the presence of 10% Pd/C afforded a mix-
ture of the corresponding open analogues 16a and 16b
in which a concomitant desulfuration took place. Com-
pounds 18a, b were obtained from 16a and 16c following
the synthetic procedure described in Scheme 1.
b Ref. 21.
c Ref. 22.
d Ref. 23.
e Ref. 24.
f Ref. 25.
g A = 3-trifluoromethylbenzyl.
h B = 4-chlorophenyl.
nitroderivatives 8a, b were treated with the appropriate
nucleophile (amines or phenoxide) to give 10a–i. Reduc-
tion of these intermediates with sodium borohydride in

5566
C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
Table 2.
of the substituents at positions 2, 4, and 6; (ii) introduc-
tion of substituents on the vinyl group; (iii) modification
of the carbonyl dipole at position 3.
Compound
R1
R2
R3
Z
X
9aa
9b
9c
9d
9e
9f
9gb
9h
9i
H
Ph
Me
Ph
Be
H
H
Me
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
S
Me
Me
Me
n-But
Ph
The obtained data show that the methyl group at posi-
tion 2 of the reference compound 6 (ED50 = 14.9 mg/
kg/sc) can be replaced by other alkyl (12e), aryl (12g),
or arylalkyl (12h) substituents, as well as by functional
groups (12i), without significant reduction of activity.
Among these analogues, compounds 12g and 12i exhib-
ited a level of antinociceptive activity comparable with
that of 6. On the contrary, the 2-unsubstituted deriva-
tives 12a and 12j proved to be significantly less potent.
Derivative 12e showed a slightly improved activity
(ED50 = 12.3 mg/kg) but, interestingly, it was able to
completely protect all the treated animals from the nox-
ious stimulus (quantal protection = 100%) at a dose
(30 mg/kg) 3-fold lower than that of 6.15
Ph
Me
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Me
Ph
Ph
Ph
Ph
Ph
Ad
COOEt
H
9j
10a
NHMe
10b
10c
10dc
10e
10f
NHn-But
N(Me)2
NHPh
NHPh
NHBz
10g
10h
10i
NH(CH2)2Ph
NHC6H11
OPh
Looking at the structural modifications of the primary
amino group in compound 6, the best results were ob-
a Ref. 20.
b Ref. 26.
c Ref. 14.
d A = 3-trifluoromethylbenzyl.
e B = 4-chlorophenyl.
tained with the methylamino derivative (12k) (ED50
=
6.5 mg/kg). The butylamino analogue (12l) showed the
same activity as 6. The same level of activity of 12l was dis-
played by the acetylamino analogue (12t), suggesting that
the basic properties of the nitrogen function do not play
an important role in eliciting the pharmacological effect.
On the other hand, the 4-dimethylamino derivative
(12m) proved 3-fold less potent than 6.
Table 3.
Compound
R1
R2
R3
Z
Y
11, 12aa
11, 12b
11, 12c
11, 12d
11, 12e
11, 12f
11, 12g
11, 12h
11, 12i
11, 12j
11, 12k
11, 12l
11, 12m
11, 12n
11, 12o
11, 12p
11, 12q
11, 12r
11, 12s
12t
H
Ph
Me
Ph
Bc
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
O
O
O
O
O
O
O
O
O
S
NH2
NH2
The presence of an anilino residue at position 4 (12p)
was associated with a considerable reduction of activity
(ED50 = 55.5 mg/kg). Introduction in this compound of
a methylenic spacer between the phenyl group and the 4-
amino residue (12r) left the activity almost unchanged,
confirming that the basic properties of the nitrogen
function are not related to the antinociceptive activity.
The insertion of a further CH2 (12s) was associated with
a relevant impairment of activity (ED50 = 89.4 mg/kg)
and a similar weak level of activity was demonstrated
by the 4-cyclohexylamino derivative (12n). These data
clearly suggest that there are steric limitations for the
substituent linked to position 4 of the pyridazine system.
Moreover, the very weak activity of the dimethylamino
derivative 12m strongly suggests that the substituent at
position 4 works as a hydrogen bond donor. This
hypothesis is confirmed by the relevant loss of activity
observed when the aromatic amino group in 12p is re-
placed by the isosteric phenoxy group (12o).
Me
Me
Me
n-But
Ph
NH2
NH2
Ph
Me
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Me
Ph
Ph
Ph
Ph
Ph
NH2
NH2
Ph
Ab
NH2
NH2
COOEt
H
NH2
NH2
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
n-But
O
O
O
O
O
O
O
O
O
O
O
O
NHMe
NHBut(n)
N(Me)2
NHC6H11(c)
OPh
NHPh
NHPh
NHBn
NH(CH2)2Ph
NHCOMe
NH2
12u
12v
NH2
a For compound 11a, see Ref. 20.
b A = 3-trifluoromethylbenzyl.
c B = 4-chlorophenyl.
It should be noted that the contemporary presence of an
anilino group at position 4 and a methyl at position 6
(12q) provokes a complete loss of activity. This effect
cannot be attributed to the introduction of the methyl
instead of the phenyl in position 6, because the 2,6-di-
methyl analogue 12b is one of the most potent in the ser-
ies (ED50 = 8.2 mg/kg/sc). Furthermore, compound 12f,
in which the substituents in positions 2 and 6 are inter-
changed, is also endowed with a good level of activity.
Probably a different pharmacokinetic or metabolism
could be invoked to explain the very different behavior
of 12q. Finally, the insertion of a chlorine in the para po-
sition of the phenyl ring (12d) resulted detrimental.
3. Results and discussion
The novel compounds were evaluated for their antinoci-
ceptive effect in the mouse abdominal constriction test
and the results are reported in Table 4.
In order to identify the structural requirements capable
of improving the antinociceptive effect of 6, the SAR
studies were carried out as a function of: (i) the nature

C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
5567
R1
R1
N
N
N
N
O
C6H5
O
C6H5
a
H2N
CHBrCH2Br
H2N
CH=CH2
13a,b
6, 12e
c
for 6
b
R1
CH3
N
N
N
N
O
C6H5
O
C6H5
H2N
CH=CHBr
H3COCHN
CH=CH2
12u,v
12t
R1
CH3
n-But
6, 12u, 13a
12e, 12v, 13b
Scheme 2. Synthetic procedure for compounds 12t–v. Reagents and conditions: (a) Br2, CCl4, rt, 5 min; (b) DBU, THF, rt À40 ꢁC, 10 min À4 h; (c)
Ac2O, pyridine, 140 ꢁC, 3 h.
The effects of some structural modifications of the vinyl
group were also studied: substitution of a hydrogen at
the b-carbon with a methyl (12c) or the introduction
of a bromine (12u) in the same position left the activity
almost unchanged (ED50 = 20.6 and 10.3 mg/kg, respec-
tively). Since, as mentioned, the presence of a n-butyla-
mino group at position 2 is associated with a good level
of activity, we synthesized a compound bearing this
structural feature at position 2 and a bromine at the
b-carbon of the vinyl group (12v). This compound
proved to be significantly less potent both with respect
to 12u and 12e.
at doses lower than ibuprofen. In the hot plate test
12e did not change the response to thermal stimuli
(Fig. 3), whereas when the thermal stimuli were ap-
plied to the mouse tail, 12e was able to raise the
nociceptive threshold (Fig. 4). These effects were ob-
served at doses that per se did not change the loco-
motor activity (Fig. 5) or nor motor coordination
(Fig. 6) of the mice. Regarding the mechanism of ac-
tion, binding studies showed the absence of affinity
(Ki > 10À5 M) for the opioid l, k and d receptors.
Mechanisms based on the adenosine system were also
ruled out on the basis of the absence of affinity for
A1 and A2 receptors. A possible interaction with
the central cholinergic system was also excluded
(Ki > 10À5 M), as well as interactions with GABA-B,
a2 adrenergic, 5-HT2, and dopaminergic systems (data
not shown).
Finally, the isosteric substitution of the oxygen at the
amidic group of lead 6 with sulfur (12j) appears to be
detrimental (ED50 = 40.9 mg/kg), suggesting a possible
involvement of the oxygen in a hydrogen bond. The re-
moval of the carbonyl dipole afforded conflicting data,
since only methylthiopyridazine 18a showed a level of
antinociceptive potency (ED50 = 14.2 mg/kg) compara-
ble to lead 6.
In conclusion, several of the novel molecules exhibited
greater antinociceptive activity than lead 6. The present
SAR studies revealed that modifications at positions 2
and 6, as well as at the b-carbon of the vinyl group,
are tolerated, while for position 4 maximum activity is
associated with amino or low alkylamino groups, being
more hindered aryl-, arylalkyl-, and cycloalkylamino
groups not tolerated.
Among the most interesting compounds, the 4-amino-
2-n-butyl-6-phenyl-5-vinyl-3(2H)pyridazinone 12e was
selected as representative molecule and submitted to
a series of studies in order to investigate its ability
to modify the nociceptive threshold also in other tests
and its effects on animal locomotor behavior; the
mechanism of action of 12e was also investigated.
The results obtained in the formalin test (Fig. 2)
demonstrated that 12e was able to reduce the licking
activity induced by formalin both in the first and sec-
ond phase. It should be noted that 12e was able to
reduce the response to chemical nociceptive stimuli
For some of the tested compounds like 12b and 12k,
IC50 values in the mouse abdominal constriction test
were found to be 2-fold lower with respect to the previ-
ous lead 6. Moreover for compound 12e, which also
showed activity in different animal models like tail flick
and formalin test, a good balance between potency and
behavioral effects was evidenced.

5568
C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
N
N
N
N
H3CO
C6H5
H3CS
C6H5
a
b
7k
N
N
CH3
CH3
O
O
15
14
c
c
N
N
N
N
N
N
H3CS
C6H5
C6H5
H3CO
C6H5
+
H2N
COCH3
H2N
COCH3
H2N
COCH3
16c
16a
16b
d
N
N
R
C6H5
H2N
CH(OH)CH3
17a,b
e
N
N
17, 18
R
R
C6H5
a
b
SCH3
OCH3
H2N
CH=CH2
18a,b
Scheme 3. Synthetic procedure for compounds 18a, b. Reagents and conditions: (a) CH3I, acetone, K2CO3, rt, 15 min; (b) CH3ONa, CH3OH, rt, 3 h;
(c) H2, Pd/C, EtOH, rt, 30–60 min; (d) NaBH4, CH3OH, rt, 15–30 min; (e) PPA, rt, 4–8 h.
Further studies on the mechanism of action of this novel
lead are in progress.
heated under stirring at 40 ꢁC for 6 h. After the mixture
cooled, water (15 mL) was added and the final product
7i was recovered by suction.
4. Experimental
4.1. Chemistry
Yield = 65%; mp = 146–148 ꢁC (EtOH); 1H NMR
(CDCl3) 2.55 (s, 3H, CH3), 5.40 (s, 2H, CH2), 7.40–
7.80 (m, 9H, 2Ar).
All melting points were determined on a Buchi appara-
¨
4.1.2. 3-Methyl-4-phenylisoxazolo[3,4-d]pyridazin-7(6H)-
thione (7k). A mixture of 7a (0.6 mmol) and Lawesson’s
reagent (0.6 mmol) in toluene (8 mL) was heated at
90 ꢁC for 90 min. After cooling the crude yellow precip-
itate was recovered by suction.
tus and are uncorrected. 1H NMR spectra were recorded
with Varian Gemini 200 instruments. Chemical shifts
are reported in ppm, using the solvent as internal stan-
dard. Extracts were dried over Na2SO4 and the solvents
were removed under reduced pressure. E. Merck F-254
commercial plates were used for analytical TLC to fol-
low the course of reaction. Silica gel 60 (Merck 70–
230 mesh) was used for column chromatography.
1
Yield = 93%; mp = 188 ꢁC (EtOH); H NMR (CDCl3)
2.55 (s, 3H, CH3), 7.55 (s, 5H, Ar), 11.0 (exch. br s,
1H, NH).
4.1.1. 6-(m-Trifluoro)-benzyl-3-methyl-4-phenylisoxazol-
o[3,4-d]pyridazin-7(6H)-one (7i). A mixture of 3-methy-
lisoxazolo[3,4-d]pyridazin-3(2H)-one 7a (0.6 mmol),
(m-trifluoromethyl)benzyl chloride (1.2 mmol), and
potassium carbonate (4 mmol) in acetone (3 mL) was
4.1.3. General procedure for compounds 9. The required
isoxazolo[3,4-d]pyridazin-7(6H)-ones 7a–d and 7f–k
(0.7 mmol) were suspended in EtOH (3.5 mL) and then
HCOONH4 (17 mmol) and 10% Pd/C (10 mg) were
added. The mixture was refluxed for 1–2 h and after

C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
5569
Table 4.
R1
N
N
N
N
R
C6H5
Z
R2
H2N
CH=CH2
Y
CH=CHR3
12a-v
18a,b
Z/R
Compound
R1
R2
R3
Y
ED50(mg/kg/sc)c
12a
12b
12c
12d
12e
12f
12g
12h
12i
H
Ph
Me
Ph
Bb
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
O
O
O
O
O
O
O
O
O
S
NH2
NH2
28.8 (11.2–74.0)
8.2 (4.0–16.7)
Me
Me
Me
n-But
Ph
NH2
NH2
20.6 (13.6–31.2)
32.7 (20.2–53.1)
12.3 (1.4–20.3)
23.9 (14.1–40.7)
14.0 (9.2–21.1)
22.2 (12.8–38.3)
18.2 (12.2–27.2)
40.9 (19.1–87.0)
6.5 (4.0–10.6)
Ph
Me
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Me
Ph
Ph
Ph
Ph
Ph
NH2
NH2
Ph
Aa
NH2
NH2
COOEt
H
NH2
NH2
12j
12k
12l
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
n-But
O
O
O
O
O
O
O
O
O
O
O
O
NHMe
NHn-But
N(Me)2
NHC6H11
OPh
15.6 (10.5–23.2)
45.7 (24.4–85.8)
79.4 (46.0–137.1)
145.5 (58.1–364.6)
55.5 (30.5–100.7)
400 (120–1331)
47.2 (23.2–96.2)
89.4 (47.5–168.3)
16.0 (11.3–22.7)
10.3 (6.6–16.1)
61.0 (36.4–102.1)
14.2 (9.2–20.8)
54.7 (23.8–125.3)
14.9
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
18a
18b
6
NHPh
NHPh
NHBn
NH(CH2)Ph
NHCOMe
NH2
NH2
SCH3
OCH3
O
Me
Ph
H
NH2
a A = 3-trifluoromethylbenzyl.
b B = 4-chlorophenyl.
c Dose–response curves were constructed defining as significant an individual reaction lower than 3 SD of the control mean reaction time. Six to eight
animals were used for each dose and at least 18 animals were used in each experiments. The parallel line assay of Finney31 was used to estimate the
ED50 values and their 95% confidence intervals with the aid of a computer.
cooling, CH2Cl2 (5 mL) was added. The solution was
stirred for 5 min, then the catalyst was filtered off and
the solvent was evaporated in vacuo.
4.1.3.1. 5-Acetyl-4-amino-2,6-dimethylpyridazin-3(2H)-
one (9b). Yield = 92%; mp = 123–125 ꢁC (cyclohexane);
1H NMR (CDCl3) d 2.45 (s, 3H, CH3), 2.55 (s, 3H,
70
first phase
second phase
60
pre treatment
post treatment
35
30
25
20
15
10
5
50
40
30
**
**
20
**
10
**
0
CON
VEH
MOR
IBU
12e 1
12e 10
0
Treatments
CON
VEH
12e 3
12e 10
12e 50
Figure 2. Effect of 12e in the formalin test. CON, animals treated with
formalin alone; VEH, animals treated with DMSO:saline 1:4 v/v;
MOR, animals treated with morphine, 5 mg/kg; IBU, animals treated
with ibuprofen 100 mg/kg; 12e, animals treated with 12e at 1 or 10 mg/
kg. **p < 0.01 versus VEH. N = 10.
Treatments
Figure 3. Effect of 12e in the hot plate test. CON, non-treated animals;
VEH, animals treated with DMSO:saline 1:4 v/v; 12e, animals treated
with 12e at 3, 10 or 50 mg/kg. N = 6.

5570
C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
(4 mL) was heated at 100 ꢁC for 2 h. After cooling the
16
14
12
10
8
pre treatment
post treatment
solution was diluted with CH3CN (2 mL) and 10 mg
of charcoal was added. After suction, the solvent was re-
moved in vacuo and the residue, treated with water,
afforded compound 9d.
**
**
Yield = 66%; mp = 235–236 ꢁC (EtOH); 1H NMR
(CDCl3) d 1.80 (s, 3H, COCH3), 3.80 (s, 3H, NCH3),
7.50–7.35 (m, 4H, Ar).
6
4
2
4.1.4. General procedure for compounds 10a–h. A mix-
ture of the required 5-acetyl-2-methyl-4-nitropyridazi-
none 8a or b (0.2 mmol), the appropriate amine
(0.5 mmol) in EtOH (2 mL) was stirred at room temper-
ature for 10–30 min. Compounds 10b and 10d–h were
directly recovered by suction; dilution with ice-water
of reaction mixture afforded compounds 10a and 10c.
0
CON
VEH
12e 3
12e 10 12e 20 12e 50
Treatments
Figure 4. Effect of 12e in the tail flick test. CON, non-treated animals;
VEH, animals treated with DMSO:saline 1:4 v/v; 12e, animals treated
with 12e at 3, 10, 20, and 50 mg/kg. **p < 0.01 versus VEH. N = 10.
4.1.4.1. 5-Acetyl-2-methyl-4-methylamino-6-phenylpy-
ridazin-3(2H)-one (10a). Yield = 73%; mp = 163–164 ꢁC
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
1
(EtOH); H NMR (CDCl3) d 1.60 (m, 1H, NH), 1.90
(s, 3H, COCH3), 2.95 (d, 3H, NHCH3), 3.80 (s, 3H,
NCH3), 7.40 (m, 5H, Ar).
4.1.4.2. 5-Acetyl-2-methyl-4-phenoxy-6-phenylpyrida-
zin-3(2H)-one (10i). To a solution of 8b (0.5 mmol) in
ethyl ether (6 mL) sodium phenoxide (1 mmol) was
added. After stirring for 90 min at room temperature
ice-water was added (20 mL) and the mixture was ex-
tracted with CH2Cl2 (3· 15 mL). The organic layer
was washed with 0.5 N NaOH (2· 5 mL), dried on
Na2SO4, and evaporated in vacuo to furnish compound
10i.
0
CON
VEH
12e 3
Treatments
12e 10
12e 50
Figure 5. Effect of 12e in the activity cage test. CON, non-treated
animals; VEH, animals treated with DMSO:saline 1:4 v/v; 12e, animals
treated with 12e at 3, 10, and 50 mg/kg. N = 8.
Yield = 79%; mp = 78–80 ꢁC (EtOH); 1H NMR
(CDCl3) d 2.30 (s, 3H, COCH3), 3.85 (s, 3H, NCH3),
7.00–7.40 (m, 5H, Ar), 7.45 (s, 5H, Ar).
4.5
**
15
30
60
4
3.5
3
4.1.5. General procedure for compounds 11a–s and 17a, b.
Sodium borohydride (2.2 mmol) was added portionwise
to a stirred solution of 9a–j and 10a–i (0.35 mmol) in
CH3OH (2 mL) for 10–90 min at root temperature.
The reaction mixture was diluted with ice-water
(8 mL). Compounds were collected by filtration, with
the exception of 11d, 11m, and 11o which were obtained
by extraction with CH2Cl2 (3· 15 mL) and evaporation
of the solvent.
**
2.5
2
1.5
1
0.5
0
4.1.5.1. 4-Amino-2,6-dimethyl-5-(1-hydroxyethyl)pyri-
dazin-3(2H)-one (11b). Yield = 95%; mp = 93–94 ꢁC
(cyclohexane); 1H NMR (CDCl3) d 1.45 (d, 3H,
CH(OH)CH3), 3.65 (s, 3H, NCH3), 4.95 (q, 1H,
CH(OH)CH3), 5.70 (exch. br s, 1H, OH).
CON
VEH
12e 3
12e 10
12e 50
Treatments
Figure 6. Effect of 12e in the rotarod test. CON, non-treated animals;
VEH, animals treated with DMSO:saline 1:4 v/v; 12e, animals treated
with 12e at 3, 10, and 50 mg/kg. **p < 0.01 versus VEH. N = 6.
4.1.6. General procedure for compounds 12a–s and 18a, b.
Method A. The appropriate 5-hydroxyalkylpyridazine
11a–o and 17a, b (0.4 mmol) were treated with PPA
(40 mmol) at root temperature for 1–48 h. After treat-
ment with ice-water (20 mL) the crude precipitate of
the desired compound was collected by suction.
COACH3), 3.70 (s, 3H, NACH3), 7.65 (exch. br s, 2H,
NH2).
4.1.3.2. 5-Acetyl-4-amino-6-(p-chlorophenyl)-2-meth-
ylpyridazin-3(2H)-one (9d). A mixture of 7e (0.2 mmol),
Mo(CO)6 (0.25 mmol), water (0.1 mL), and CH3CN
4.1.6.1. 4-Amino-6-phenyl-5-vinylpyridazin-3(2H)one
(12a). Yield = 44%; mp = 277–278 ꢁC (EtOH); 1H

C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
5571
NMR (CDCl3) 5.30 (m, 2H, CH@CH2), 6.25 (m, 1H,
CH@CH2), 7.40 (s, 5H, Ar). Anal. Calcd for
C12H11N3O (213,09): C, 67.59; H, 5.20; N, 19.71.
Found: C, 67.83; H, 5.34; N, 19.47.
6.25 (dd, J = 17.7 Hz, J = 11.4 Hz, 1H, CH@CH2),
7.50 (m, 5H, Ar). Anal. Calcd for C20H16F3N3O
(371,36): C, 64.69; H, 4.34; N, 11.32. Found: C, 65.03,
H, 4.59; N, 11.58.
4.1.6.2. 4-Amino-2,6-dimethyl-5-vinylpyridazin-3(2H)-
one (12b). Yield = 64%; mp = 112–113 ꢁC (cyclohexane);
1H NMR (CDCl3) d 2.20 (s, 3H, CH3), 3.70 (s, 3H,
NCH3), 5.20 (exch. br s, 2H, NH2), 5.65 (dd,
J = 9.6 Hz, J = 16.7 Hz, 2H, CH@CH2), 6.45 (dd,
J = 9.6 Hz, J = 16.7 Hz, 1H, CH@CH2). Anal. Calcd
for C8H11N3O (165,19): C, 58.17; H, 6.71; N, 25.44.
Found: C, 58.01; H, 6.66; N, 25.25.
4.1.6.9. 5-Amino-6-oxo-3-phenyl-4-vinyl-6H-pyrida-
zine-1-carboxylic acid ethyl ester (12i). Yield = 62%;
mp = 102–104 ꢁC (EtOH); H NMR (CDCl3) d 1.40 (t,
3H, OCH2CH3), 4.25 (q, 2H, OCH2CH3), 5.40 (exch.
br s, 2H, NH2) 5.50–5.65 (dd, J = 16.7 Hz,
J = 11.0 Hz, 2H, CH@CH2), 6.25 (dd, J = 16.7 Hz,
J = 11.0 Hz, 1H, CH@CH2), 7.35–7.50 (m, 5H, Ar).
Anal. Calcd for C15H15N3O3 (285,30): C, 63.15; H,
5.30; N, 14.73. Found: C, 62.93; H, 4.97; N, 15.08.
1
4.1.6.3. 4-Amino-2-methyl-6-phenyl-5-propenylpyrida-
zin-3(2H)-one (12c). Yield = 81%; mp = 151–153 ꢁC
(cyclohexane); 1H NMR (CDCl3) d 1.80 (d, 3H,
CH@CHACH3), 3.80 (s, 3H, NCH3), 5.25 (m, 1H,
CH@CHACH3), 5.95 (m, 1H, CH@CHACH3), 7.40
(m, 5H, Ar). Anal. Calcd for C14H15N3O (241,29): C,
69.69; H, 6.27; N, 17.41. Found: C, 70.00; H, 5.99; N,
17.63.
4.1.6.10. 4-Amino-6-phenyl-5-vinylpyridazine-3(2H)-
thione (12j). Yield = 79%; mp = >300 ꢁC dec. (cyclohex-
1
ane); yield = 62%; mp = 102–104 ꢁC (EtOH); H NMR
(CDCl3) d 5.30 (d, J = 17 Hz, 1H, CH@CH2), 5.50 (d,
J = 12 Hz, 1H, CH@CH2), 6.30–6.50 (m, 1H,
CH@CH2), 6.90 (exch. br s, 2H, NH2), 7.30–7.55 (m,
5H, Ar). Anal. Calcd for C12H11N3S (229,30): C,
62.86; H, 4.84; N, 18.33. Found: C, 63.04; H, 4.97; N,
15.08.
4.1.6.4. 4-Amino-6-(4-chlorophenyl)-2-methyl-5-vinyl-
pyridazin-3(2H)-one (12d). Yield = 80%; mp = 109–
1
111 ꢁC (cyclohexane); H NMR (CDCl3) d 3.80 (s, 3H,
4.1.6.11. 2-Methyl-4-methylamino-6-phenyl-5-vinylpy-
ridazin-3(2H)-one (12k). Yield = 71%; mp = 114–116 ꢁC
(cyclohexane); 1H NMR (CDCl3) d 2.95 (s, 3H,
NHCH3), 3.80 (s, 3H, NCH3), 4.95 (d, J = 14.9 Hz,
1H, CH@CH2), 5.45 (d, J = 9.0 Hz, 1H, CH@CH2),
6.45 (dd, J = 15.0 Hz, J = 9.0 Hz, 1H, CH@CH2), 7.35
(s, 5H, Ar). Anal. Calcd for C14H15N3O (241,29): C,
69.69; H, 6.27; N, 17.41. Found: C, 69.78; H, 6.39; N,
17.77.
NCH3), 5.35–5.55 (m, 4H, 2H, NH2; 2H CH@CH2),
6.25 (dd, J = 16.0 Hz, J = 10.0 Hz, 1H, CH@CH2),
7.40 (s, 4H, Ar). Anal. Calcd for C13H12ClN3O
(261,71): C, 59.66; H, 4.62; N, 16.06. Found: C, 59.28;
H, 4.44; N, 15.91.
4.1.6.5. 4-Amino-2-butyl-6-phenyl-5-vinylpyridazin-
3(2H)-one (12e). Yield = 64%; mp = 74–78 ꢁC (EtOH);
1H NMR (CDCl3) d 0.90 (t, 3H, CH2CH3), 1.30–1.65
(m, 4H, CH2CH2CH2CH3), 3.25 (t, 2H, NHCH2), 3.80
(s, 3H, NCH3), 4.95 (d, J = 14.9 Hz, 1H, CH@CH2),
5.40 (d, J = 9.0 Hz, 1H, CH@CH2), 6.00 (exch. br s,
1H, NH), 6.45 (dd, J = 15.0 Hz, J = 9.0 Hz, 1H,
CH@CH2), 7.40 (s, 5H, Ar). Anal. Calcd for
C16H19N3O (269,34): C, 71.35; H, 7.11; N, 15.60.
Found: C, 71.60; H, 6.88; N, 15.84.
4.1.6.12. 4-Butylamino-2-methyl-6-phenyl-5-vinylpyri-
dazin-3(2H)-one (12l). Yield = 71%; mp = 56–58 ꢁC
(EtOH/H2O 1:1); 1H NMR (CDCl3) d 0.95 (t, 3H,
CH2CH3), 1.25–1.70 (m, 4H, CH2CH2CH3), 3.15–3.30
(m, 2H, NHCH2), 3.80 (s, 3H, NCH3), 4.95 (d,
J = 15.0 Hz, 1H, CH@CH2), 5.40 (d, J = 9.0 Hz, 1H,
CH@CH2), 6.00 (exch. br s, 2H, NH2), 6.45 (dd,
J = 15.0 Hz, J = 9.0 Hz, 1H, CH@CH2), 7.40 (s, 5H,
Ar). Anal. Calcd for C17H21N3O (283,37): C, 72.06; H,
7.47; N, 14.83. Found: C, 71.85; H, 7.71; N, 15.08.
4.1.6.6. 4-Amino-6-methyl-2-phenyl-5-vinylpyridazin-
3(2H)-one (12f). Yield = 62%; mp = 104–105 ꢁC (cyclo-
1
hexane); H NMR (CDCl3) d 2.30 (s, 3H, CH3), 5.30
(exch. br s, 2H, NH2), 5.70 (m, 2H, CH@CH2), 6.55
(dd, J = 17.4 Hz, J = 11.0 Hz, 1H, CH@CH2), 7.50 (m,
5H, Ar). Anal. Calcd for C13H13N3O (227,26): C,
68.70; H, 5.77; N, 18.49. Found: C, 69.04; H, 6.01; N,
18.31.
4.1.6.13. 4-Dimethylamino-2-methyl-6-phenyl-5-vinyl-
pyridazin-3(2H)-one (12m). Yield = 88%; mp = 57–60 ꢁC
(cyclohexane); 1H NMR (CDCl3) d 2.95 (s, 6H,
N(CH3)2), 3.80 (s, 3H, NCH3), 4.85 (d, J = 17.7 Hz,
1H, CH@CH2), 5.40 (d, J = 11.4 Hz, 1H, CH@CH2),
6.35 (dd, J = 17.7 Hz, J = 11.4 Hz, 1H, CH@CH2),
7.40 (s, 5H, Ar). Anal. Calcd for C15H17N3O (255,31):
C, 70.56; H, 6.71; N, 16.46. Found: C, 70.90; H, 6.96;
N, 16.73.
4.1.6.7. 4-Amino-2,6-diphenyl-5-vinylpyridazin-3(2H)-
1
one (12g). Yield = 90%; mp = 142–143 ꢁC (EtOH); H
NMR (CDCl3) d 5.65 (m, 2H, CH@CH2), 6.35 (m,
1H, CH@CH2), 7.30–7.70 (m, 10H, 2Ar). Anal. Calcd
for C18H15N3O (289,33): C, 74.72; H, 5.23; N, 14.52.
Found: C, 74.55; H, 5.48; N, 14.39.
4.1.6.14. 4-Cyclohexylamino-2-methyl-6-phenyl-5-
vinylpyridazin-3(2H)-one (12n). Yield = 49%; mp = 79–
81 ꢁC (EtOH); 1H NMR (CDCl3) d 1.10–1.90 (m,
11H, NHC6H11), 3.80 (s, 3H, NCH3), 5.00 (d,
J = 16.0 Hz, 1H, CH@CH2), 5.40 (d, J = 10.0 Hz, 1H,
CH@CH2), 6.45 (dd, J = 16.0 Hz, J = 10.0 Hz, 1H,
CH@CH2), 7.40 (s, 5H, Ar). Anal. Calcd for
4.1.6.8. 4-Amino-6-phenyl-2-(3-trifluoromethylbenzyl)-
5-vinylpyridazin-3(2H)-one (12h). Yield = 84%; mp =
1
124–127 ꢁC (cyclohexane); H NMR (CDCl3) d 5.35–
5.65 (m, 6H: 2H, CH2Ph; 2H, NH2; 2H, CH@CH2),

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C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
C19H23N3O (309,41): C, 73.76; H, 7.49; N, 13.58.
Found: C, 74.04; H, 7.80; N, 13.78.
4.1.6.20. 4-Benzylamino-2-methyl-6-phenyl-5-vinylpy-
ridazin-3(2H)-one (12r). Yield = 34%; mp = 123 ꢁC
1
(EtOH); H NMR (CDCl3) d 3.85 (s, 3H, NCH3), 4.50
4.1.6.15. 2-Methyl-4-phenoxy-6-phenyl-5-vinylpyrida-
zin-3(2H)-one (12o). Yield = 78%; mp = 97–100 ꢁC
(s, 2H, CH2), 5.05 (d, J = 17.2 Hz, 1H, CH@CH2),
5.40 (d, J = 9.1 Hz, 1H, CH@CH2), 6.35 (dd,
J = 17.2 Hz, J = 9.1 Hz, 1H, CH@CH2), 7.25–7.40 (m,
2H, Ar). Anal. Calcd for C20H19N3O (317,38): C,
75.69; H, 6.03; N, 13.24. Found: C, 75.99; H, 5.73; N,
12.91.
1
(EtOH); H NMR (CDCl3) d 3.80 (s, 3H, NCH3), 5.55
(d, J = 9.2 Hz, 1H, CH@CH2), 5.90 (d, J = 15.3 Hz,
1H, CH@CH2), 6.30 (dd, J = 15.3 Hz, J = 9.2 Hz, 1H,
CH@CH2), 6.95–7.40 (m, 5H, Ar), 7.45 (s, 5H, Ar).
Anal. Calcd for C19H16N2O2 (304,34): C, 74.98; H,
5.30; N, 9.20. Found: C, 75.23; H, 5.49; N, 8.87.
4.1.6.21. 2-Methyl-4-phenethylamino-6-phenyl-5-vinyl-
pyridazin-3(2H)-one (12s). Yield = 53%; mp = 116–
118 ꢁC (EtOH); 1H NMR (CDCl3) d 2.85 (t, 2H,
CH2Ph), 3.55 (q, 2H, CH2NH), 3.85 (s, 3H, NCH3),
5.00 (d, J = 15.0 Hz, 1H, CH@CH2), 5.40 (d,
J = 9.1 Hz, 1H, CH@CH2), 6.1 (exch. br s, 1H, NH),
6.45 (dd, J = 15.0 Hz, J = 9.1 Hz, 1H, CH@CH2),
7.15–7.40 (m, 10H, 2Ar). Anal. Calcd for C21H21N3O
(331,41): C, 76.11; H, 6.39; N, 12.68. Found: C, 75.89;
H, 6.71; N, 12.88.
4.1.6.16. 3-Methylsulfanyl-6-phenyl-5-vinyl-pyridazin-
4-ylamine (18a). Yield = 70%; mp = 170–172 ꢁC (cyclo-
1
hexane); H NMR (CDCl3) d 2.80 (s, 3H, SCH3), 4.75
(exch. br s, 2H, NH2), 5.65 (d, J = 17.0 Hz, 1H,
CH@CH2), 5.65 (d, J = 12.0 Hz, 1H, CH@CH2), 6.40
(dd, J = 17.0 Hz, J = 12.0 Hz, 1H, CH@CH2), 7.35–
7.65 (m, 5H, Ar). Anal. Calcd for C13H13N3S (243,33):
C, 64.17; H, 5.39; N, 17.27. Found: C, 64.00; H, 5.12;
N, 17.04.
4.1.7. 4-Acetamide-2-methyl-6-phenyl-3-oxo-5-vinylpy-
ridazine (12t). A mixture of compound 6 (0.2 mmol), pyr-
idine (5 mL), and acetic anhydride (20 mmol) was stirred
at 140 ꢁC for 3 h and 30 min. After cooling, dilution with
water, and acidification with 2 N HCl, the solution was
extracted with CH2Cl2 (3· 15 mL). After the solvent
was dried and evaporated in vacuo, treatment of the res-
idue with EtOH/H2O 1:1 afforded compound 12t.
4.1.6.17. 3-Methoxy-6-phenyl-5-vinyl-pyridazin-4-yla-
mine (18b). Yield = 68%; mp = 110–112 ꢁC (cyclohex-
ane); 1H NMR (CDCl3) d 4.20 (s, 3H, OCH3), 4.85
(exch. br s, 2H, NH2), 5.60 (d, J = 17.9 Hz, 1H,
CH@CH2), 5.65 (d, J = 12.3 Hz, 1H, CH@CH2), 6.35
(dd, J = 17.9 Hz, J = 12.3 Hz, 1H, CH@CH2), 7.35–
7.55 (m, 5H, Ar). Anal. Calcd for C13H13N3O
(227,26): C, 68.70; H, 5.77; N, 18.49. Found: C, 69.04;
H, 6.03; N, 18.24.
Yield = 56%; mp = 84–86 ꢁC (cyclohexane); 1H NMR
(CDCl3) d 2.35 (s, 3H, COCH3), 3.85 (s, 3H, NCH3),
5.50 (m, J = 17.6 Hz, J = 11.5 Hz, 2H, CH@CH2), 6.20
(dd, J = 17.6 Hz, J = 11.5 Hz, 1H, CH@CH2), 7.55–
7.35 (m, 5H, Ar). Anal. Calcd for C15H15N3O2
(269,30): C, 66.90; H, 5.61; N, 15.60. Found: C, 67.13;
H, 5.90; N, 15.83.
Method B: The appropriate 5-hydroxyalkylpyridazinon-
es 11p–s (0.4 mmol) dissolved in toluene (4.5 mL) and
SiO2/H2SO4 (1 g) prepared following the procedure
described in Ref. 23 was added portionwise over 3 h.
The mixture was heated at 45–60 ꢁC for 11–30 h and
after cooling acetone (20 mL) was added. Silica gel
was removed by suction and washed with acetone
(3· 15 mL). The organic layers were collected and
evaporated in vacuo to afford the vinyl derivatives
12p–s.
4.1.8. General procedure for 12u, v. The appropriate 2-al-
kyl-4-amino-6-phenyl-5-vinylpyridazinones 6 and 12e
(0.3 mmol) were suspended in CCl4 (3.5 mL) and, after
cooling at À5 ꢁC, bromine (1 mmol) was added and
the mixture was stirred for 10 min. The crude precipitate
2-alkyl-4-amino-5-(1,2-dibromoethyl)-6-phenylpyridazi-
none (13a, b) was recovered by suction and washed with
ethyl acetate. The dried product was dissolved in anhy-
drous THF (7.5 mL), DBU (10 mmol) was added, and
the reaction was carried out under nitrogen for 10 min
at 20 ꢁC (12u) and for 4 h at 40 ꢁC (12v). After evapora-
tion of the solvent, the residue was treated with ice-
water and acidified with 2 N HCl. The crude precipitate
12u and 12v was recovered by suction.
4.1.6.18. 2-Methyl-6-phenyl-4-phenylamino-5-vinylpy-
ridazin-3(2H)-one (12p). Yield = 99%; mp = 127–130 ꢁC
1
(EtOH); H NMR (CDCl3) d 3.90 (s, 3H, NCH3), 4.75
(d, J = 17.7 Hz, 1H, CH@CH2), 5.05 (d, J = 11.4 Hz,
1H, CH@CH2), 6.15 (dd, J = 17.7 Hz, J = 11.4 Hz,
1H, CH@CH2), 6.85–7.10 (m, 3H, Ar), 7.20–7.45 (m,
7H, Ar), 7.80 (exch. br s, 1H, NH). Anal. Calcd for
C19H17N3O (303,36): C, 75.23; H, 5.65; N, 13.85.
Found: C, 74.87; H, 5.49; N, 14.07.
4.1.6.19. 2,6-Dimethyl-4-phenylamino-5-vinylpyrida-
zin-3(2H)-one (12q). Yield = 38%; mp = 100–112 ꢁC
4.1.8.1. 4-Amino-5-(2-bromovinyl)-2-methyl-6-phen-
ylpyridazin-3(2H)-one (12u). Yield = 50%; mp = 148–
150 ꢁC (EtOH/H2O 1:1); H NMR (CDCl3) d 3.70 (s,
1
1
(EtOH); H NMR (CDCl3) d 2.25 (s, 3H, CH3), 3.80
(s, 3H, NCH3), 5.00 (d, J = 17.2 Hz, 1H, CH@CH2),
5.20 (d, J = 10.3 Hz, 1H, CH@CH2), 6.20 (dd,
J = 17.2 Hz, J = 10.3 Hz, 1H, CH@CH2), 6.80–6.90
(m, 2H, Ar), 6.95–7.05 (m, 1H, Ar), 7.20–7.30 (m,
2H, Ar). Anal. Calcd for C14H15N3O (241,29): C,
69.69; H, 6.27; N, 17.41. Found: C, 70.08; H, 5.95;
N, 17.20.
3H, NCH3), 6.35 (d, 1H, CH@CHBr), 6.80 (d, 1H,
CH@CHBr), 7.40 (m, 5H, Ar). Anal. Calcd for
C13H12BrN3O (306,16): C, 51.00; H, 3.95; N, 13.72.
Found: C, 50.75; H, 4.18; N, 14.03.
4.1.8.2. 4-Amino-5-(2-bromovinyl)-2-butyl-6-phenylpy-
ridazin-3(2H)-one (12v). Yield = 74%; mp = 103–105 ꢁC

C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
5573
1
(EtOH); H NMR (CDCl3) d 0.95 (t, 3H, CH2CH3),
4.2.2. Hot plate test. The hot plate test was performed as
previously reported29 with some modifications. The de-
vice (Socrel Mod. DS-37, Ugo Basile, Italy) consisted
of a metal plate (25 · 25 cm) heated to a constant tem-
perature (50 0.1ꢁC) on which a plastic cylinder
(20 cm diameter, 16 cm high) was placed. The time of la-
tency in second was recorded between the moment the
animal was placed on the hot plate surface and when
it licked its paw or jerked it strongly and then lifted it
off. The measurement was terminated if the latency ex-
ceeded the cut-off time (40 s) or if the mouse jumped
off the hot plate. Baseline latency was of 15–17 s. Hot
plate data were recorded as latency to respond in sec
both 30 and 15 min before the treatment for baseline
determination and six more times—15, 30, 45, 60, 75,
and 90 min, after treatment.
1.30–1.50 (m, 2H, CH2CH2CH3), 1.65–1.90 (m, 2H,
CH2CH2CH3), 4.20 (t, 2H, NCH2), 5.35 (exch. br s,
2H, NH2), 6.55 (d, J = 16.7 Hz, 1H, CH@CHBr),
6.70 (d, J = 16.7 Hz, 1H, CH@CHBr), 7.45 (s, 5H,
Ar). Anal. Calcd for C16H18BrN3O (348,24): C,
55.18; H, 5.21; N, 12.07. Found: C, 54.81; H, 4.99;
N, 11.83.
4.1.9. 3-Methyl-7-methylthio-4-phenylisoxazolo[3,4-d]-
pyridazine (14). A mixture of compound 7k (0.2 mmol),
acetone (3 mL), anhydrous K2CO3 (20 mmol), and
iodomethane (4 mmol) was stirred at room temperature
for 15 min. After dilution with ice-water the crude pre-
cipitate was recovered by suction.
Yield = 74%; mp = 188–190 ꢁC (EtOH); 1H NMR
(CDCl3) d 2.70 (s, 3H, CACH3), 2.80 (s, 3H, SCH3),
7.55 (s, 5H, Ar).
4.2.3. Tail flick test. Tail flick latency29 was obtained
using a tail flick unit (Socrel Mod DS-20, Ugo Basile,
Italy) consisting of an infrared source, radiant light
(100 W bulb) with adjustable intensity which was fo-
cused by an aluminized parabolic mirror on a photocell.
Radiant heat was focused 1–2 cm from the tip of the tail,
and the latency time in second until the mouse flicked its
tail was recorded. The measurement was terminated if
the latency exceeded the cut-off time (15 s). Beam inten-
sity was adjusted to produce a tail flick latency of 3–4 s
in the baseline determination. The animals were re-
strained during trials by means of plastic cylinder 4 cm
in diameter and 8 cm long. Tail flick data were recorded
as latency to respond in sec both 30 and 15 min before
the treatment for baseline determination and six more
times—15, 30, 45, 60, 75, and 90 min, after treatment.
4.1.10. 3-Methyl-7-methoxy-4-phenylisoxazolo[3,4-d]pyr-
idazine (15). CH3ONa (0.4 mmol) in abs CH3OH
(10 mL) was added portionwise to a solution of 14
(0.2 mmol) in CH3OH (10 mL) and the reaction mixture
was stirred at room temperature for 3 h. After neutral-
ization with 2 N HCl, the mixture was concentrated
and after cooling, the precipitate was recovered by
suction.
Yield = 58%; mp = 187–189 ꢁC (MeOH); 1H NMR
(CDCl3) d 2.65 (s, 3H, CACH3), 4.40 (s, 3H, OCH3),
7.45–7.75 (m, 5H, Ar).
4.1.11. General procedure for 16a–c. A mixture of the
appropriate 3-methyl-4-phenylisoxazolo[3,4-d]pyrida-
zine 14 and 15 (0.5 mmol), 10% Pd/C (30 mg), and
EtOH (25 mL) was shaken under hydrogen at room
temperature and 2 bar for 30–120 min. The catalyst
was filtered off and the solvent was evaporated in vacuo.
4.2.4. Writhing test. Writhing test was performed as pre-
viously reported.30 Mice were given an intraperitoneal
(ip) injection of 0.6% v/v acetic acid in a volume of
10 mL/kg. Acetic acid induces a series of writhes, con-
sisting in abdominal contraction and hind limb exten-
sion and the number of writhes was recorded over a
10-min period beginning 5 min after acetic acid injec-
tion. Compounds were subucutaneously (sc) adminis-
tered 30 min before acetic acid injection.
Reduction of 14 in this experimental condition afforded
a mixture of 16a and 16b, which were separated by col-
umn chromatography.
4.2.5. Formalin test. In the formalin test30 20 lL of a
0.5% solution of formalin in saline was injected subcuta-
neously (sc) into the dorsal surface of the right hind paw
of the mouse using a microsyringe with a 27-gauge nee-
dle. The formalin injection produced a distinct biphasic
response consisting of licking or biting the paw. The first
phase occurred from 0 to 5 min after formalin injection
and the second phase from 15–20 to 30–40 min. The
4.1.11.1. 5-Acetyl-4-amino-3-methylthio-6-phenylpy-
ridazine (16a). Yield = 83%; mp = 113–115 ꢁC (cyclo-
hexane); 1H NMR (CDCl3) d 1.90 (s, 3H, COCH3),
2.80 (s, 3H, SCH3), 6.20 (exch. br s, 2H, NH2), 7.40–
7.65 (m, 5H, Ar).
4.2. Biological assays
mouse was then put into
a
plexiglas cage
4.2.1. Animals. Male CD-1 mice (Charles River, Italy)
weighing 25–30 g were used for all experiments. The ani-
mals were housed in colony cages (5 mice each) under
standard light (alight from 7.00 a.m. to 7.00 p.m.), tem-
perature (22 1ꢁC), and relative humidity (60 10%)
conditions for at least 1 week before the experimental
sessions. Food and water were available ad libitum.
All experiments were carried out according to the guide-
lines of the European Community Council for Experi-
mental Animal Care (86/609/EEC).
(30 · 14 · 12 cm) which served as an observation cham-
ber, and the total amount of time in second the animal
spent licking or biting the paw after formalin injection
was recorded from 0 to 5 (first phase) min and from
20 to 40 (second phase) min. Compounds were intraper-
itoneally (ip) administered 30 min before formalin
injections.
4.2.6. Motor coordination. Motor coordination was eval-
uated as previously reported,16 by using a rotarod

5574
C. Vergelli et al. / Bioorg. Med. Chem. 15 (2007) 5563–5575
apparatus (Ugo Basile, Italy) consisting of a bar with a
diameter of 3.0 cm, subdivided into five compartments
by a disk of 24 cm in diameter. The bar rotated at a con-
stant speed of 16 rev/min. A preliminary selection of
mice was made on the day of experiment excluding those
that did not remain on the rotarod bar for two consec-
utive periods of 45 s each. The integrity of motor coor-
dination was assessed on the basis of the number of falls
from the rod in 45 s. The performance time was mea-
sured before and 15, 30, and 60 min after ip treatments.
means of the total time in second the animal spent lick-
ing or biting the injected paw both in the first and in sec-
ond phase after formalin injection and are reported as
means SE. Data obtained in the rotarod experiments
were analyzed as means of the number of falls from
the rotating rod in 45 s measured before and 15, 30,
and 60 min after 18a treatment and are reported as
means SE. Locomotor activity data were analysed as
means of the cumulative counts recorded 2 h after 18a
administration and are reported as means SE. Data
obtained in the experiments were statistically analyzed
by using analysis of variance followed by Newman–
Keul’s procedure for verifying significance between
two means. Statistical significance was always assumed
at least at 5% level.
4.2.7. Locomotor activity. In the locomotor activity eval-
uation16 mice were randomly assigned to one experimen-
tal group and tested in an activity cage (Ugo Basile, Italy)
in groups of four. The cage consisted of three parts: the
animal cage, the electric unit, and the counter and recor-
der which were incorporated into the electric unit. The
dimensions of the cage were 28 · 32 · 40 cm. The sides
were opaque and the ceiling transparent to allow observa-
tion. The floor consisted of stainless steel bars 3 mm in
diameter and 11 mm apart. The odd bars were grounded
and the even bars were active so that movements of the
animals were recorded continuously on a counter as well
as on paper. Temperature, sound, and light conditions
were maintained constant during the course of the exper-
iments. Measurements of locomotor activity following
treatments were carried out at 10-min intervals and cumu-
lative counts were recorded after 2 h. The animals were
placed in the activity cage for at least 30 min before receiv-
ing the treatment so as to reduce the possible contribution
of exploratory activity to the total count to a minimum.
Supplementary data
1H NMR spectral data for derivatives 9c–f, 9h–j, 10b, c
and 10e–h, 11b–f, 11h–s, 12b–o, 12q–s, 16b, c, 17a, b,
18a, b. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.bmc.2007.05.035.
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