Further studies on arylpiperazinyl alkyl pyridazinones: Discovery of an exceptionally potent, orally active, antinociceptive agent in thermally induced pain

Article abstract of DOI:10.1021/jm900458r

A number of pyridazinone derivatives bearing an arylpiperazinylalkyl chain were synthesized and tested icv in a model of acute nociception induced by thermal stimuli in mice (tail flick). The most interesting and potent compound in this series was 6a, whi

Full text of DOI:10.1021/jm900458r

J. Med. Chem. 2009, 52, 7397–7409 7397
DOI: 10.1021/jm900458r
Further Studies on Arylpiperazinyl Alkyl Pyridazinones: Discovery of an Exceptionally Potent,
Orally Active, Antinociceptive Agent in Thermally Induced Pain†
Claudio Biancalani,† Maria Paola Giovannoni,*,† Stefano Pieretti,‡ Nicoletta Cesari,† Alessia Graziano,† Claudia Vergelli,†
Agostino Cilibrizzi,† Amalia Di Gianuario,‡ Mariantonella Colucci,‡ Giorgina Mangano,§ Beatrice Garrone,§
Lorenzo Polenzani,§ and Vittorio Dal Piaz†
†Dipartimento di Scienze Farmaceutiche, via Ugo Schiff 6, 50019 Sesto Fiorentino, Firenze, Italy, ‡Department of Therapeutic Research and
Medicine Evaluation, Viale Regina Elena 299, 00161 Roma, Italy, and §Angelini Research Center, Piazzale della Stazione,
00040 S. Palomba-Pomezia, Roma, Italy
Received April 9, 2009
A number of pyridazinone derivatives bearing an arylpiperazinylalkyl chain were synthesized and tested
icv in a model of acute nociception induced by thermal stimuli in mice (tail flick). The most interesting
and potent compound in this series was 6a, which showed an ED50 = 3.5 μg, a value about 3-fold higher
with respect to morphine by the same route of administration. When administered per os, 6a was 4-fold
more potent than morphine in the same test, suggesting a significant bioavailability. The same
compound also showed high potency in the hot plate test. The antinociceptive effect of 6a was
completely reversed by pretreatment with yohimbine both in the hot plate test and in the tail flick test.
This demonstrated the involvement of the adrenergic system, which was confirmed by in vitro
radioligand binding studies.
Introduction
choline, adenosine, and adrenaline, NMDA, ion channels,
and so on).8-10 Thus, at the present, NMDA, mGLU1, Ca2þ
channels, Naþ channels, and TRPV-1 are the most promising
molecular targets for the treatment of neuropathic pain.11
Our work in the field of the analgesic agents has produced a
large number of molecules, with pyridazine scaffold, show-
ing an interesting level of antinociceptive potency.12-21 We
have synthesized two distinct series of compounds: one
series is of 4-amino-5-vinyl-3(2H)-pyridazinones, for which
we performed in-depth structure-activity relationship
studies.12-15,19-21 In this series, modifications at positions
2 and 6 of the pyridazine ring are well-tolerated, while an
amino group or a low alkylamino group at position 4 is an
essential requirement. Substitution of the vinyl group at
position 5 led to inactive compounds, with the exception of
the homologation (propenyl) and the introduction of a bro-
mine at position β of the vinyl. But the most interesting
modification performed at this position was the replacement
of the vinyl group with five-membered heterocycles, which led
topotentcompounds, active per osanddevoid ofthe potential
toxicity related to the transformation of the vinyl group in
epoxidic function in vivo.19 Pretreatment of the above com-
pounds with the R2-antagonist yohimbine induced a complete
reversal of the antinociceptive effect, suggesting involvement
of the adrenergic system in the mechanism of analgesic action.
The second series includes a large number of pyridazinones
and (hetero)condensed pyridazinones substituted with an
arylpiperazinylalkyl moiety.16-18 One of the most interesting
terms showed an efficacy of 104% compared to morphine in
the hot plate test and was able to completely abolish abdom-
inal constrictions in the writhing test at the dose of 20 mg/kg
sca.17 The involvement of the adrenergic system and especially
Although rapid advances in medicinal chemistry research
have greatly increased the options for analgesic therapy,
traditional pain treatment, based on the use of NSAIDs and
opioids, continues to dominate clinical analgesia. As is well-
known, NSAIDs show adverse reactions at the gastrointest-
inal level together with inhibition of platelet aggregation and
renal toxicity. Meanwhile, opioids induce severe side effects
such as sedation, constipation, respiratory depression, and
dependence.1,2 In the 1990s, researchers discovered the two
isoforms of cyclooxygenases, COX-1 and COX-2. It was
initially observed that potent and selective COX-2 inhibitors
had comparable analgesic potency with respect to the classic
NSAIDs but a lower incidence of secondary effects.3 This led
to the development and commercialization of a large number
of these selective inhibitors, collectively called “coxibs”. Un-
fortunately, more recent studies have correlated an elevated
risk of acute myocardial infarction with the use of COX-2
inhibitors4,5 which remain a rational therapeutic approach for
patients athighrisk of serious gastrointestinal complications.6
At present, a third class of analgesics, the “analgesic adju-
vants”, which are drugs with a different primary use, finds
application for particular forms of pain. Neurophatic pain,
for example, is particularly difficult to treat and is often
refractory to conventional analgesics.7 There is an active field
of research into the numerous neuromodulators that are
involved in complex pain-processing pathways (i.e., acetyl-
†We are honored to contribute the present article to the special issue
for the 100th anniversary of the Division of the Medicinal Chemistry
that always worked for the diffusion of the more recent advances in
Medicinal Chemistry.
*To whom correspondence should be addressed. Phone: þ39-055-
4573682. Fax: þ39-055-4573671. E-mail: mariapaola.giovannoni@uni-
fi.it.
a Abbreviations: ip, intra peritoneal; icv, intracerebral ventricular; sc,
sub cutis; po, per os.
r
2009 American Chemical Society
Published on Web 09/29/2009
pubs.acs.org/jmc

7398 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Biancalani et al.
Scheme 1a
Scheme 2a
a (a) K2CO3, anhyd DMF, 60 °C, 3.5 h; (b) NaBH4, MeOH, rt, 15 min;
(c) PPA, 70 °C, 3 h.
Scheme 3a
a (a) K2CO3, anhyd DMF, rt, 5-24 h; (b) Br-(CH2)n-Br, K2CO3
anhyd DMF rt, 2 h; (c) K2CO3, anhyd DMF, rt, 4-16 h; (d) NaBH4,
MeOH, rt, 0.5-3 h; (e) PPA 60-70 °C, 2-8 h.
indirect activation of the noradrenergic system through in-
hibition of noradrenaline reuptake was also demonstrated for
this series.18
In this paper, we describe a new series of pyridazinones
bearing an arylpiperazinylalkyl chain, designed and synthesized
with the aim of completing the structure-activity relationship
studies of this series. Analgesic activity was assessed by thermal
nociceptive tests in mice. In addition, using a prototypical
compound of the series, in vitro radioligand binding studies
were performed on a panel of adrenergic receptors in order to
define the pharmacological profile. These studies led us to
identify compound 6a as an exceptionally potent antinocicep-
tive agent by oral route in the tail flick and hot plate tests.
a (a) Hydrazine hydrate, EtOH, PPA, 70 °C, 30 min; (b) 1-(3-bromo-
propyl)-4-(p-tolyl)piperazine, K2CO3, anhyd DMF, 60°C, 4 h; (c)
hydrazine hydrate, EtOH, 10% Pd/C, reflux, 1 h; (d) NaBH4, MeOH,
rt, 2 h; (e) PPA, 60 °C, 3 h.
Chemistry
The synthetic pathways affording the final compounds
6a-i, 10, 16, 20a,b, 23, 25a-c, 30, and 35 are reported in
Schemes 1-8.
Scheme 1 shows the synthetic procedure, widely used in our
laboratories, that produces the final 5-vinylpyridazinones
6a-i. Compounds 4a-i, which represent the key intermedi-
ates, were obtained starting from the 4-amino-5-acetylpyri-
dazinone 1,22 following two different procedures:
1. A direct alkylation of precursor 1 with the opportune
arylpiperazinylpropyl bromide 2a-f (route a, com-
pounds 4a-f, Table 1). Compounds 2a and 2d were
previously described (2a,16 2d17) while 2b,c,e,f were
prepared by condensing 1,3-dibromopropane with ap-
propriate substituted-arylpiperazine in anhydrous
acetone and K2CO3 at room temperature;
a standard alkylation, followed by condensation with
the appropriate aryl(alkyl)piperazine or piperidine in
anhydrous DMF and K2CO3. Treatment of 4a-i with
sodium borohydride in anhydrous methanol afforded
the secondary alcohols 5a-i, which, in turn, were
transformed into the 5-vinyl derivatives 6a-i by dehy-
dratation with polyphosphoric acid (PPA).
Scheme 2 depicts a similar synthetic route for the final
compound 10. In this case, a basic side-chain was introduced
through a Michael addition reaction using the 1-(4-p-tolylpi-
perazin-1-yl)propenone 7, obtained from 1-p-tolylpiperazine
and 3-chloropropanoyl chloride in THF and triethylamine.
Then the 4-amino-5-acetylderivative 8 was converted into
2. Two steps (route b,c compounds 4g-i, Table 2), which
consist of the synthesis of N-alkylbromide 3a,b through

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7399
Scheme 6a
Scheme 4a
a (a) 1-(3-Bromopropyl)-4-(4-methylphenyl)-piperazine, K2CO3, anhyd
DMF, rt-60 °C, 2-24 h.
Scheme 7a
a (a) Br2, AcOH, HBr, 50 °C, 5 h; (b) thioacetamide, anhyd EtOH,
80 °C, 5 h; (c) 1-(3-bromoalkyl)-4-(4-methylphenyl)-piperazine, K2CO3,
anhyd DMF, rt, 24 h.
Scheme 5a
a (a) Anhyd EtOH, rt, 12 h; (b) NaBH4, MeOH, rt, 20 min; (c) H2SO4
on silica gel, toluene, reflux, 4 h.
a (a) N,N-Dimethylformammide dimethylacetal, 100-110 °C, 3-4 h;
(b) hydrazine hydrate, EtOH, 80 °C, 3 h.
acetic acid afforded product 18, which, by treatment with
thioacetamide in ethanol, gave rise to compound 19. The final
compounds 20a,b were obtained by alkylation in standard
conditions with the appropriate 1-(3-bromoalkyl)-4-(p-tolyl)-
piperazine. The introduction of the pyrazole system at posi-
tion 5 (Scheme 5) was performed by treatment of precursor
2116 with N,N-dimethylformamide dimethylacetal to afford
the 3-dimethylaminovinyl derivative 22, which, with hydra-
zine hydrate, gave rise to the final compound 23.
The synthetic step affording 5 and 4,5-unsubstituted final
compounds (25a-c) is reported in Scheme 6. The reaction was
carriedoutinstandardconditionsstarting fromthepreviously
described24-26 compounds 24a-c.
final compound 10 using the same conditions described in
Scheme 1.
For the synthesis of 4-amino-5-vinylpyridazinone 16
(Scheme 3), it was necessary to synthesize the 3-methyl-
isoxazolo[3,4-d]pyridazin-7(6H)-one 12 starting from the
isoxazole 1123 and hydrazine hydrate in PPA and ethanol.
The intermediate 12 was alkylated with 3-bromopropyl-
4-(p-tolyl)piperazine in standard conditions using K2CO3
in anhydrous DMF. Reductive cleavage with hydrazine
hydrate and Pd/C in ethanol of the intermediate 13 gave
rise to the corresponding 5-acetyl-4-aminopyridazinone 14,
which was transformed into the final 16 following the above-
described two-step procedure.
Finally, with the synthesis of compound 30, we obtained the
shift of the alkylarylpiperazinyl chain from position 2 to position
4 (Scheme 7), whereas in compound 35, we substituted the
pyridazine scaffold with a thiazole ring bearing those fragments
and functionalities important for activity (Scheme 8). Scheme 7
Schemes 4 and 5 show the synthesis of 5-heterocyclic
substituted pyridazinones. The 5-thiazolyl derivatives 20a,b
(Scheme 4) were prepared starting from the previously de-
scribed compound 17.22 Treatment of 17 with bromine in

7400 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Biancalani et al.
Scheme 8a
Table 2. Legend for Compounds 4-6a-i
compd 4-6
X
n
a
b
c
d
e
f
N-C6H4-CH3 (p)
N-C6H4-OCH3 (p)
N-C6H4-Cl (p)
N-C6H4-Cl (m)
N-C6H4-OCH3 (m)
N-C6H4-CH3 (m)
CHCH3
3
3
3
3
3
3
3
3
2
g
h
i
NCH3
N-C6H4-OCH3 (p)
able to exhibit good antinociceptive activity because their
ED50s were less than 10 μg. The antinociceptive effect induced
by these last compounds, although lower than those observed
after morphine administration, was in any case interesting
because they showed potency and efficacy comparable to that
demonstrated by other analgesic drugs clinically employed
such as diphenhydramine, baclofen, amitriptyline, etc.31
Compounds 6b, 6c, 6i, 23, and 25a showed ED50s in the range
of 10-20 μg, and compounds 6d, 16, 20b, and 30 showed
ED50s in the range of 20-25 μg. ED50s calculated for 6g, 10,
and20a werehigher than25μg and weresignificantly different
from the ED50 for 6a.
After the results obtained from the above experiments, the
antinociceptive activity of 6a was further investigated and
demonstrated after oral administration to mice which under-
went the hot plate and tail flick tests (Table 5). These effects
were evident at low doses and the ED50 for 6a was several fold
lower than the morphine ED50 in both thermal nociceptive
tests. These findings clearly indicate that 6a is endowed with
exceptionally potent antinociceptive activity and its bioavail-
ability is significantly better than that of morphine. Previously
we demonstrated that the analgesia induced by structurally
related compounds19 was completely prevented by pretreat-
ment with the R2-antagonist yohimbine. To clarify whether
the R2-adrenoceptor was involved in the mechanism of action
of 6a, we pretreated mice with yohimbine as antagonist. The
dose of yohimbine employed to prevent the analgesia induced
by the above-reported compound is the minimal dose able to
antagonize antinociception induced by activation of R2-adre-
noceptor, as demonstrated by the block exerted on amitripty-
line and imipramine antinociception.32 A significant reversal
of antinociceptive effect of compound 6a by the R2-antagonist
yohimbine (2 mg kg-1 ip) was evidenced both in the hot plate
and in the tail flick test (Table 5). In our experimental
conditions, the R2-antagonist yohimbine did not modify the
pain threshold of mice in comparison with control animals.
The lack of effect of this antagonist agrees with results of
studiesinwhichthis compounddid notmodifythe nociceptive
threshold against both thermal (hot-plate) and chemical
(writhing) noxious stimuli.33 We can therefore exclude that
prevention of the antinociception induced by the assayed
compounds was due to a hyperalgesic effect of the R2-adre-
noceptor antagonist used.
a (a) K2CO3, anhyd DMF, 40 °C, 2 h; (b) NaBH4, MeOH, rt, 5 min; (c)
H2SO4 on silica gel, toluene, reflux, 4 h.
Table 1. Legend for Compounds 2a-i
compd 2
R
X
a
b
c
d
e
f
(CH2)3Br
(CH2)3Br
(CH2)3Br
(CH2)3Br
(CH2)3Br
(CH2)3Br
N-C6H4-CH3 (p)
N-C6H4-OCH3 (p)
N-C6H4-Cl (p)
N-C6H4-Cl (m)
N-C6H4-OCH3 (m)
N-C6H4-CH3 (m)
CHCH3
g
h
i
NCH3
N-C6H4-OCH3 (p)
depicts the synthesis of compound 30 for which the starting
product is represented by the 4-nitropyridazinone 26.27 Nucleo-
philic displacement of the nitro group with the amine 2728
afforded the 4-substituted aminopyridazinone 28, which under-
went the same synthetic procedures reported in the previous
schemes. Similarly, the thiazolone 35 was synthesized following
the three-step procedure (alkylation, reduction, and dehydrata-
tion) reported for all the other final compounds, starting from
product 31.29 In this case, the transformation of hydroxyethyl
derivative 34 into the corresponding 5-vinyl derivatives 35 was
performed using sulfuric acid adsorbed on silica gel following a
method described in the literature.30
Results and Discussion
In the present study, the antinociceptive activity of the
investigated compounds was first evaluated after central
administration in a model of acute nociception induced by
thermal stimuli in mice. All compounds were administered in
the cerebral ventricle, and their activity was compared with
that of morphine (Tables 3 and 4). Compound 6a strongly
increased the nociceptive threshold to thermal stimuli show-
ing a potency in the tail flick test 3-fold lower with respect to
the reference compound morphine, the most important an-
algesic used in humans. However, 6h, 25b, 25c, and 35 were
Our further experiments performed to verify the involve-
ment of R1-receptors evidenced that the R1-antagonist prazo-
sin at both doses (0.1 and 1 mg/kg ip) administered 15 min
before 6a (1 mg/kg po) was able to potentiate 6a-induced
antinociception, increasing latencies to cutoff values from 30
to 120 min after 6a administration. These last results might
suggest that 6a behaves also as an R1-agonist. It was recently
demonstrated, using a genetic and pharmacological ap-
proach, that R1-adrenergic receptor agonist activity might

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7401
Table 3. Antinociceptive Effect of Final Compounds 6a-j, 10, 16 in the Tail Flick Test (icv)
a At least four groups of six mice were used to generate dose-response curves and to estimate ED50 values. ED50s and their significant differences were
calculated with the aid of a computer program.41 Statistical significance was assumed at P < 0.05. * is for a significant difference vs morphine; ∧ is for a
significant difference vs 6a. b Reference 16.
reduce R2-mediated antinociception when the same com-
pound is both an R1 and an R2 agonist.34 Finally, to confirm
the mechanism of 6a action involving adrenergic receptors, we
administered the opioid-antagonist naloxone, the GABAB-
antagonist CGP35348, and the nicotinic antagonist mecamy-
lamine at doses reported to antagonize the antinociception
induced by opioid, GABAB, or nicotinic receptor activation
before 6a.14 Neither naloxone nor CGP35348 or mecamyla-
mine were able to reduce 6a effects on thermal nociceptive
threshold either in the hot plate or in the tail flick test.
panel of adrenergic receptors(Table6). Compound6ashowed
selectivity for alpha adrenergic receptors R1A, R1B, and R2A
,
with IC50 values in the range 56-320 nM, these data confirm-
ing an involvement of the adrenergic system in the analgesia
produced by 6a. On the contrary, evaluation of binding to
subtype R2C, also involved in the modulation of antinocicep-
tion,35 clearly demonstrated that compound 6a showed low
affinity for R2C in comparison with the other examined
subtypes, the inhibition being 71% at 10 μM.
SAR studies were performed by analyzing the results
obtained after icv administration, where differences in phar-
macokinetic properties among different compounds have a
To further verify the pharmacological profile of compound
6a, in vitro radioligand binding studies were performed on a

7402 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Biancalani et al.
Table 4. Antinociceptive Effect of Final Compounds 20a,b, 23, 25a-c, 30, 35 in the Tail Flick Test (icv)
a At least four groups of six mice were used to generate dose-response curves and to estimate ED50 values. ED50s and their significant differences were
calculated with the aid of a computer program.41 Statistical significance was assumed at P < 0.05. * is for a significant difference vs morphine; ∧ is for a
significant difference vs 6a.
Table 5. Effect of Naloxone, CGP, Mecamilamine, and Yohimbine on
Antinociception Induced by Compound 6a in Hot Plate and Tail Flick
Test
reduced impact on the activity. The most potent compound
was 6a (ED50 = 3.5 μg). By comparing 6a with compound 16,
where the methyl group in position 6 of the heterocycle was
hot plate,
ED50 mg/kgb
tail flick,
ED50 mg/kgb
eliminated, we found that the nor derivative had strongly
reduced activity (ED50 = 25.1 μg), suggesting that the pre-
sence of a small lipophilic group in this part of the molecule is
an important structural requirement for antinociceptive ac-
tivity. The nature and length of the arylpiperazinylpropyl side
chain in position 2 also plays a relevant role for the activity.
Thus, replacementof the CH2 attachedatpiperazineN1 witha
CO group (compound 10) was associated with about a 10-fold
reduction in activity. This finding strongly suggests that
nitrogen is an essential requirement of this piperazine, its
abilitybeing tobe protonated ata physiological pH criticalfor
activity. Likewise, replacement of the p-tolylpiperazine with
methylpiperidine (compound 6g) led to similar results, sug-
gesting a fundamental role also for piperazine N4. This
hypothesis was clearly confirmed by examining the behavior
treatmenta
morphine
6a
6.9 (2.7-17.2)
0.5 (0.1-3.3)
1.1 (0.3-5.9)
0.6 (0.1-5.9)
0.7 (0.05-6.7)
8.6 (0.8-91.7)*
3.5 (1.4-8.9)
0.8 (0.1-6.7)
1.2 (0.3-5.8)
0.5 (0.02-13.0)
2.3 (0.3-16.0)
6.1 (0.9-42.6)*
6a þ NAL
6a þ CGP
6a þ MEC
6a þ YOH
a In some experiments, 6a was administered po alone or 30 min after
ip treatment with naloxone (NAL, 1 mg/kg), CGP 35348 (CGP, 50 mg/
kg), mecamylamine (MEC, 2 mg/kg), or yohimbine (YOH, 2 mg/kg) and
the relative ED50 was then calculated. Morphine was administered po.
bAt least four groups of six mice were used to generate dose-response
curves and to estimate ED50 values. ED50s and their significant differ-
ences were calculated with the aid of a computer program.41 Statistical
significance was assumed at P < 0.05. * is for a significant difference vs
6a; ∧ is for a significant difference vs morphine

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7403
Table 6. Binding Experiments Performed on 6a
assay IDa
species
IC50 (nM)
Emax10 μM, %
reference compound
IC50 (nM)
adrenergic R1A
adrenergic R1B
adrenergic R2A
adrenergic R2C
rat
rat
56
101
99
prazosin
prazosin
0.254
0.190
3.67
102
320
ncb
human
human
100
71
yohimbine
yohimbine
27.1
a All the procedures were performed according to standard radioligand binding technique. The R1A-adrenoceptor and R1B-adrenoceptor binding
assays were performed on Wistar rat submaxillary gland and Wistar rat liver membrane extracts, respectively, using 0.25 nM [3H]-prazosin as
radioligand. R2A-adrenoceptor binding assay was performed on human recombinant insect Sf9 cell extracts using 1nM of [3H]-MK-912 as radioligand.
The compound was dissolved in dimethyl sulfoxide (DMSO) and diluted in incubation buffer, then tested in duplicate at 1 μM concentration. b
was not calculated and maximal inhibition effect at the highest tested concentration (Emax10 μM) is reported.
R2C IC50
of 6h, where the piperazine N4 basicity was restored. This
compound’s activity (ED50 = 8.7 μg) was at a level compar-
able to 6a. The nature of the substituent in the phenyl group
was also critical. Thus, replacement of CH3 with Cl was
at the NH2, which might explain the good antinociceptive
effect observed for 25a. Finally, compound 30, where a side
chain similar to that of 6a at position 4 and a methyl group at
position 2 of the pyridazinone were inserted, showed low
activity (ED50 = 24.1 μg).
associated with reduced activity (compound 6c, ED50
=
In conclusion, we have identified the very potent antinoci-
ceptive agent 6a, whose structure is characterized by a
6-methylpyridazinone scaffold substituted with a p-tolylpi-
perazinylpropyl side chain at position 2, a primary amino
group at position 4, and a vinyl group at position 5. This
compound was several times more potent than morphine both
in the tail flick and hot plate test when orally administered to
mice. SAR studiesperformed onthisprototypeshowed thatin
this series the above side chain and NH2 are essential require-
ments for the activity of 6a.
15.4 μg) as well as the substitution with a methoxy group
(6b), which led to a compound 3-fold less potent than 6a.
When the above-mentioned groups (chlorine, methoxy, and
methyl) were inserted at the meta position of the phenyl ring,
we observed reduced activity as demonstrated by ED50 values
which are 23.7 μg for 6d, 72.6 μg for 6e, and 51.5 μg for 6f.
Finally, to evaluate the importance of the length of the alkyl
chain, we tested compound 6j, the lower homologue of 6a,
which we have previously described.16 This compound, which
showed potent activity in the writhing test by sc administra-
tion to mice (ED50 = 2.5 mg/kg)16 when tested in the tail flick
test (icv), is more than 7-fold less potent than 6a. This trend
was not observed for the methoxyderivativebecause the lower
homologue of 6b (compound 6i) showed the same activity.
These data indicate that antinociceptive activity is signifi-
cantly affected by electronic and/or steric properties of the
substituent and the related position on the aromatic ring.
Compounds 20a-b and 23 were synthesized as metaboli-
cally more stable analogues of 6a in which the vinyl group in
position 5 was replaced with five-membered heterocycles as
possible bioisosters. We applied this type of structural mod-
ification successfully to a series of structurally related pyrida-
zinones.19 In this case, we found that the thiazole derivatives
20a and 20b were almost devoid of antinociceptive activity
(ED50 = 48.3 and 25.1 μg, respectively), whereas the pyrazole
23 showed weak activity (ED50 = 16.9 μg). When we analyzed
the role of the amino and the vinyl groups at positions 4 and 5,
interesting results emerged: compound 25b, where the vinyl
group was eliminated, showed antinociceptive activity at a
similar level as 6a (ED50 = 4.9 μg), while when both the
substituents were eliminated (compound 25a), a reduction
in activity was observed (ED50 = 13.8 μg). The analogue
with the pyridine scaffold 25c exhibited comparable activity
(ED50 = 7.6 μg), suggesting that the presence of the NH2 in
position 4 is more crucial to activity than the CHdCH2 group
in position 5. To evaluate the role played by the heterocyclic
scaffold, we synthesized the thiazolone 35 in which the side
chain, and the CHdCH2 of 6a were inserted in a different
heterocycle. This compound showed almost the same potency
as 6a (ED50 = 5.1 μg). This result disagrees with that found in
the pyridazinone series, where the role played by the vinyl
group appeared less important compared with that of NH2.
On the other hand, as previously observed, the side-chain also
contributes to the antinociceptive activity because 25a, lack-
ing both the functional groups, maintains a significant level of
activity (ED50 = 13.8 μg). Better interaction of the thiazolone
system with the biological target counterbalances the absence
Experimental Section
Chemistry. All melting points were determined on a Buchi
apparatus and are uncorrected. 1H NMR spectra were recorded
with Avance 400 instruments (Bruker Biospin version 002 with
SGU). 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. Microanalyses were performed
with a Perkin-Elmer 260 elemental analyzer for C, H, N and the
results were within (0.4% of the theoretical values, unless
otherwise stated. Reagents and starting materials 2e-g were
commercially available.
General procedure for 2b,c,e,f (2a,16 2d17). To a suspension of
1,3-dibromopropane (3.0 mmol) and anhydrous K2CO3 (3.9
mmol) in anhydrous acetone, a solution of appropriate com-
mercially available (substituted)phenylpiperazine (1.9 mmol), in
anhydrous acetone was slowly added. The suspension was
stirred for 3-4 h at room temperature. After evaporation of
the solvent and dilution with cold water (20-30 mL), the
mixture was extracted with CH2Cl2 (3 ꢀ 20 mL) and the solvent
was evaporated in vacuo to afford compounds 2b,c,e,f, which
were purified by flash chromatography using cyclohexane/ethyl
acetate 1:1 as eluent for compound 2b, cyclohexane/ethyl acet-
ate 1:2 for compound 2c,e, and cyclohexane/ethyl acetate 2:1 for
compound 2f.
1-(3-Bromopropyl)-4-(4-methoxyphenyl)piperazine 2b. Yield =
21%; mp = 55-58 °C (EtOH). 1H NMR (CDCl3) δ 2.05 (m, 2H,
BrCH2CH2CH2N), 2.55 (t, 2H, BrCH2CH2CH2N), 2.55-2.65 (m,
4H, piperazine), 3.05-3.15 (m, 4H, piperazine), 3.50 (t, 2H,
BrCH2CH2CH2N), 3.75 (s, 3H, OCH3), 6.85 (d, 2H, Ar), 6.90 (d,
2H, Ar). Anal. (C14H21BrN2O) C, H, N.
1-(3-Bromopropyl)-4-(4-chlorophenyl)piperazine 2c. Yield =
70%; oil. 1H NMR (CDCl3) δ 2.10-2.20 (m, 2H, Br-
CH2CH2CH2N), 2.60-2.70 (m, 2H, BrCH2CH2CH2N), 2.65-
2.80 (m, 4H, piperazine), 3.20-3.30 (m, 4H, piperazine), 3.50 (t,
2H, BrCH2CH2CH2N), 6.85 (d, 2H, Ar), 7.20 (d, 2H, Ar). Anal.
(C13H18BrClN2) C, H, N.

7404 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
1-(3-Bromopropyl)-4-(3-methoxyphenyl)piperazine 2e. Yield =
Biancalani et al.
CONCH2CH2CH2N), 2.65 (s, 3H, COCH3), 2.65-2.75 (m, 4H,
piperazine), 3.15-3.25 (m, 4H, piperazine), 4.20 (t, 2H, CON-
CH2CH2CH2N), 6.75-6.85 (m, 2H, Ar), 6.90 (m, 1H, Ar), 7.20
(m, 1H, Ar), 7.75 (exch br s, 2H, NH2). Anal. (C20H26ClN5O2)
C, H, N.
5-Acetyl-4-amino-2-{3-[4-(3-methoxyphenyl)-piperazin-1-yl]pro-
pyl}-6-methylpyridazin-3(2H)-one 4e. Yield = 75%; mp =
111-113 °C (EtOH). 1H NMR (CDCl3) δ 2.08 (m, 2H, CON-
CH2CH2CH2N), 2.53 (s, 3H, 6-CH3), 2.59 (s, 3H, COCH3),
2.60-2.73 (m, 6H (2H, CONCH2CH2CH2N; 4H, piperazine)),
3.23 (m, 4H, piperazine), 3.81 (s, 3H, OCH3), 4.18 (t, 2H,
CONCH2CH2CH2N), 6.44 (d, 1H, Ar), 6.48 (s, 1H, Ar), 6.55
(d, 1H, Ar), 7.19 (t, 1H, Ar), 7.76 (exch br s, 2H, NH2). Anal.
(C21H29N5O3) C, H, N.
5-Acetyl-4-amino-2-{3-[4-(3-methoxyphenyl)-piperazin-1-yl]-
propyl}-6-methylpyridazin-3(2H)-one 4f. Yield = 85%; mp =
133-134 °C (EtOH). H NMR (CDCl3) δ 2.06-2.20 (m, 2H,
CONCH2CH2CH2N), 2.33 (s, 3H, CH3-Ar), 2.50-2.60 (m,
2H, CONCH2CH2CH2N), 2.53 (s, 3H, 6-CH3), 2.59 (s, 3H,
COCH3), 2.61-2.72 (m, 4H, piperazine), 3.15-3.33 (m, 4H,
piperazine), 4.18 (t, 2H, CONCH2CH2CH2N), 6.68-6.72 (m,
2H, Ar), 6.76 (s, 1H, Ar), 7.17 (t, 1H, Ar), 7.75 (exch br s, 2H,
NH2). Anal. (C21H28N5O2) C, H, N.
1
35%; oil. H NMR (CDCl3) δ 2.11 (m, 2H, BrCH2CH2CH2N),
2.59 (m, 2H, BrCH2CH2CH2N), 2.65 (m, 4H, piperazine), 3.23 (m,
4H, piperazine), 3.51 (t, 2H, BrCH2CH2CH2N), 3.81 (s, 3H,
OCH3), 6.44 (d, 2H, Ar), 6.48 (s, 1H, Ar), 6.55 (s, 1H, Ar), 7.19
(t, 1H, Ar). Anal. (C14H21BrN2O) C, H, N.
1-(3-Bromopropyl)-4-(3-methylphenyl)piperazine 2f. Yield =
35%; oil. 1H NMR (CDCl3) δ 2.10 (quint, 2H, BrCH2CH2CH2N),
2.35 (s, 3H, CH3), 2.57 (t, 2H, BrCH2CH2CH2N), 2.64 (t, 4H,
piperazine), 3.22 (t, 4H, piperazine), 3.52 (t, 2H, BrCH2-
CH2CH2N), 6.68-6.75 (m, 2H, Ar), 6.78 (s, 1H, Ar), 7.19 (t,
1H, Ar). Anal. (C14H21BrN2) C, H, N.
General Procedure for 3a,b. To a suspension of the appro-
priate dibromoalkane (2.6 mmol) and K2CO3 (3.5 mmol) in
anhydrous DMF (2 mL) a solution of 122 (1.7 mmol) in
anhydrous DMF was slowly added. The mixture was stirred at
room temperature for 2 h. After dilution with cold water, the
suspension was extracted with CH2Cl2 (3 ꢀ 15 mL). Evapora-
tion of the solvent afforded the final compounds 3a,b, which
were purified by column chromatography using cyclohexane/
ethyl acetate 1:1 as eluent for 3b and the mixture petroleum
ether/Et2O/CHCl3/abs EtOH/NH4OH 10:4:4:1:0.5 for com-
pound 3a.
1
5-Acetyl-4-amino-2-(2-bromoethyl)-6-methylpyridazin-3(2H)-
General Procedure for 4g-i. To a suspension of 3a,b (0.43
mmol) and K2CO3 (0.86 mmol) in anhydrous DMF, the appro-
priate (substituted)cycloalkylamine 2g-i, commercially avail-
able, (0.43-0.86 mmol) was added. The mixture was stirred for
4-16 h at room temperature. After dilution with cold water
(20-30 mL), the suspension was extracted with CH2Cl2 (3 ꢀ 15
mL) and the solvent was evaporated in vacuo to afford com-
pounds 4g-i, which were purified by column chromatography
(eluents: CHCl3/CH3OH 9:1 for compounds 4g,h and the
mixture petroleum ether/Et2O/CHCl3/abs EtOH/NH4OH
10:4:4:1:0.5 for compound 4i).
1
one 3a. Yield = 25%; mp = 102-104 °C (EtOH). H NMR
(CDCl3) δ 2.50 (s, 3H, 6-CH3), 2.60 (s, 3H, COCH3), 3.70 (t, 2H,
NCH2CH2Br), 4.50 (t, 2H, NCH2CH2Br), 7.80 (exch br s, 2H,
NH2). Anal. (C9H12BrN3O2) C, H, N.
5-Acetyl-4-amino-2-(3-bromopropyl)-6-methylpyridazin-3(2H)-
one 3b. Yield = 20%; mp = 220-223 °C (EtOH). 1H NMR (CD-
Cl3) δ 2.35-2.45 (m, 2H, NCH2CH2CH2Br), 2.55 (s, 3H, 6-CH3),
2.60 (s, 3H, COCH3), 3.45 (t, 2H, NCH2CH2CH2Br), 4.25 (t, 2H,
NCH2CH2CH2Br), 7.80 (exch br s, 2H, NH2). Anal. (C10H14-
BrN3O2) C, H, N.
General Procedure for 4a-f. A mixture of 5-acetyl-4-amino-
pyridazin-3(2H)-one 122 (0.84 mmol), anhydrous K2CO3 (1.7
mmol), and the appropriate 1-(3-bromoalkyl)(substituted)aryl-
piperazine 2a-f, (2a16 and 2d17) (0.84 mmol) in anhydrous DMF
(1-2 mL) was stirred for 5-24 h at room temperature. After
dilution with cold water (20-30 mL), the crude precipitate was
recovered by suction.
5-Acetyl-4-amino-6-methyl-2-[3-(4-p-tolylpiperazin-1-yl)-
propyl]pyridazin-3(2H)-one 4a. Yield = 56%; mp = 138-140 °C
(EtOH). 1H NMR (CDCl3) δ 2.05-2.20 (m, 2H, CONCH2-
CH2CH2N), 2.30 (s, 3H, Ph-CH3), 2.50 (s, 3H, 6-CH3), 2.60 (s,
3H, COCH3), 2.65-2.85 (m, 6H: 2H, CONCH2CH2CH2N and
4H, piperazine), 3.15-3.35 (m, 4H, piperazine), 4.20 (t, 2H, CO-
NCH2CH2CH2N), 6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar), 7.75 (exch br
s, 2H, NH2). Anal. (C21H29N5O2) C, H, N.
5-Acetyl-4-amino-6-methyl-2-[3-(4-methylpiperidin-1-yl)pro-
pyl]pyridazin-3(2H)-one 4g. Yield = 38%; mp = 96-98 °C
(EtOH). 1H NMR (CDCl3) δ 0.90-1.00 (m, 3H, CHCH3),
1.40-1.50 (m, 3H, piperidine), 1.60-1.70 (m, 2H, piperidine),
2.05-2.20 (m, 4H: 2H, CONCH2CH2CH2N and 2H,
piperidine), 2.50 (s, 3H, 6-CH3), 2.50-2.60 (m, 2H, CON-
CH2CH2CH2N), 2.60 (s, 3H, COCH3), 3.00-3.10 (m, 2H,
piperidine), 4.15 (t, 2H, CONCH2CH2CH2N), 7.75 (exch br s,
2H, NH2). Anal. (C16H26N4O2) C, H, N.
5-Acetyl-4-amino-6-methyl-2-[3-(4-methylpiperazin-1-yl)pro-
pyl]pyridazin-3(2H)-one 4h. Yield = 13%; mp = 98-100 °C
(EtOH). 1H NMR (CDCl3) δ 1.95-2.10 (m, 2H, CONCH2-
CH2CH2N), 2.40 (s, 3H, NCH3), 2.50 (s, 3H, 6-CH3), 2.60 (s,
3H, COCH3), 2.60-2.75 (m, 10H: 8H, piperazine and 2H,
CONCH2CH2CH2N), 4.15 (t, 2H, CONCH2CH2CH2N), 7.75
(exch br s, 2H, NH2). Anal. (C15H25N5O2) C, H, N.
5-Acetyl-4-amino-2-{2-[4-(4-methoxyphenyl)piperazin-1-yl]-
ethyl}-6-methylpyridazin-3(2H)-one 4i. Yield = 42%; mp =
135-137 °C (EtOH). 1H NMR (CDCl3) δ 2.50 (s, 3H, 6-CH3),
2.60 (s, 3H, COCH3), 2.70-2.80 (m, 4H, piperazine), 2.80-
2.90(m, 2H, CONCH2CH2N), 3.05-3.15 (m, 4H, piperazine),
3.80 (s, 3H, OCH3), 4.30 (t, 2H, CONCH2CH2N), 6.85 (d, 2H,
Ar), 6.90 (d, 2H, Ar), 7.75 (exch br s, 2H, NH2). Anal. (C20-
H27N5O3) C, H, N.
5-Acetyl-4-amino-2-{3-[4-(4-methoxyphenyl)piperazin-1-yl]-
propyl}-6-methylpyridazin-3(2H)-one 4b. Yield = 65%; mp =
1
142-144 °C (EtOH). H NMR (CDCl3) δ 2.05-2.15 (m, 2H,
CONCH2CH2CH2N), 2.50 (s, 3H, 6-CH3), 2.55 (s, 3H,
COCH3), 2.60 (m, 2H, CONCH2CH2CH2N), 2.65-2.75 (m,
4H, piperazine), 3.10-3.20 (m, 4H, piperazine), 3.80 (s, 3H,
OCH3), 4.20 (t, 2H, CONCH2CH2CH2N), 6.85 (d, 2H, Ar),
6.95 (d, 2H, Ar), 7.80 (exch br s, 2H, NH2). Anal. (C21H29N5O3)
C, H, N.
5-Acetyl-4-amino-2-{3-[4-(4-chlorophenyl)-piperazin-1-yl]pro-
pyl}-6-methylpyridazin-3(2H)-one 4c. Yield = 93%; mp = 159-
161 °C (EtOH). 1H NMR (CDCl3) δ 2.05-2.15 (m, 2H, CON-
CH2CH2CH2N), 2.55 (s, 3H, 6-CH3), 2.60-2.75 (m, 9H: 2H,
CONCH2CH2CH2N and 4H, piperazine and 3H, COCH3),
3.15-3.25 (m, 4H, piperazine), 4.20 (t, 2H, CONCH2-
CH2CH2N), 6.85 (d, 2H, Ar), 7.20 (d, 2H, Ar), 7.75 (exch br s,
2H, NH2). Anal. (C20H26ClN5O2) C, H, N.
General Procedure for 5a-i. Sodium borohydride (2-5.5
mmol) was added portionwise to a stirred solution of appro-
priate 4-amino-5-acetyl derivatives 4a-i (0.37-0.47 mmol) in
CH3OH (5-7 mL). The reaction mixture was stirred for 0.5-3 h
at room temperature. After concentration in vacuo, the residue
was diluted with ice water (20 mL) and extracted with CH2Cl2
(3 ꢀ 20 mL). Evaporation of the solvent afforded final com-
pounds 5a-i.
5-Acetyl-4-amino-2-{3-[4-(3-chlorophenyl)-piperazin-1-yl]pro-
pyl}-6-methylpyridazin-3(2H)-one 4d. Yield = 76%; mp =
4-Amino-5-(1-hydroxyethyl)-6-methyl-2-[3-(4-p-tolylpipera-
zin-1-yl)propyl]pyridazin-3(2H)-one 5a. Yield = 88%; mp =
1
1
138-140 °C (EtOH). H NMR (CDCl3) δ 2.00-2.15 (m, 2H,
176-178 °C (EtOH). H NMR (CDCl3) δ 1.50 (d, 3H, CH-
CONCH2CH2CH2N), 2.50 (s, 3H, 6-CH3), 2.50-2.60 (m, 2H,
(OH)CH3), 2.05-2.15 (m, 2H, CONCH2CH2CH2N), 2.20 (s,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7405
3H, 6-CH3), 2.30 (s, 3H, Ph-CH3), 2.50-2.60 (m, 2H, CON-
CH2CH2CH2N), 2.65-2.75 (m, 4H, piperazine), 2.85 (exch br s,
1H, OH), 3.10-3.25 (m, 4H, piperazine), 4.15 (t, 2H, CON-
CH2CH2CH2N), 5.00 (q, 1H, CH(OH)CH3), 5.80 (exch br s,
2H, NH2), 6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar). Anal. (C21-
H31N5O2) C, H, N.
4-Amino-5-(1-hydroxyethyl)-2-{3-[4-(4-methoxyphenyl)-pi-
perazin-1-yl]propyl}-6-methylpyridazin-3(2H)-one 5b. Yield =
99%; mp = 147-150 °C (EtOH). 1H NMR (CDCl3) δ 1.45 (d,
3H, CH(OH)CH3), 2.10-2.20 (m, 2H, CONCH2CH2CH2N),
2.20 (s, 3H, 6-CH3), 2.60-2.95 (m, 6H: 2H, CONCH2CH2-
CH2N and 4H, piperazine), 3.00 (exch br s, 1H, OH), 3.20-3.30
(m, 4H, piperazine), 3.75 (s, 3H, OCH3), 4.10-4.20 (m, 2H,
CONCH2CH2CH2N), 5.00 (q, 1H, CH(OH)CH3), 5.80 (exch
br s, 2H, NH2), 6.80 (d, 2H, Ar), 7.00 (d, 2H, Ar). Anal. (C21H31-
N5O3) C, H, N.
CH(OH)CH3), 5.80 (exch br s, 2H, NH2). Anal. (C15H27N5O2)
C, H, N.
4-Amino-5-(1-hydroxyethyl)-2-{2-[4-(4-methoxyphenyl)-pi-
perazin-1-yl]ethyl}-6-methylpyridazin-3(2H)-one 5i. Yield =
1
85%; mp = 158-160 °C (EtOH). H NMR (CDCl3) δ 1.50
(d, 3H, CH(OH)CH3), 2.20 (s, 3H, 6-CH3), 2.55 (exch br s, 1H,
OH), 2.75 (m, 4H, piperazine), 2.90 (m, 2H, CONCH2CH2N),
3.05-3.15 (m, 4H, piperazine), 3.75 (s, 3H, OCH3), 4.20-4.30
(m, 2H, CONCH2CH2N), 5.00 (q, 1H, CH(OH)CH3), 5.80
(exch br s, 2H, NH2), 6.85 (d, 2H, Ar), 6.90 (d, 2H, Ar). Anal.
(C20H29N5O3) C, H, N.
General Procedure for 6a-i. The appropriate pyridazinones
5a-i (0.4 mmol) were reacted with PPA (40 mmol) for 2-8 h at
60-70 °C. After treatment with ice water, the mixture was
neutralized with 6N NaOH, extracted with CH2Cl2 (3 ꢀ 15
mL), and the solvent was evaporated in vacuo to afford final
compound 6a-i.
4-Amino-6-methyl-2-[3-(4-p-tolylpiperazin-1-yl)propyl]-5-vinyl-
pyridazin-3(2H)-one 6a. Yield = 98%; mp = 99-101 °C (EtOH).
1H NMR (CDCl3) δ 2.05-2.15 (m, 2H, CONCH2CH2CH2N),
2.20 (s, 3H, 6-CH3), 2.30 (s, 3H, Ph-CH3), 2.50-2.65 (m, 2H,
CONCH2CH2CH2N), 2.65-2.80 (m, 4H, piperazine), 3.15-3.30
(m, 4H, piperazine), 4.20 (t, 2H, CONCH2CH2CH2N), 5.20 (exch
br s, 2H, NH2), 5.60 (d, 1H, CHdCH2), 5.70 (d, 1H, CHdCH2),
6.50 (dd, 1H, CHdCH2), 6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar). Anal.
(C21H29N5O) C, H, N.
4-Amino-2-{3-[4-(4-chlorophenyl)piperazin-1-yl]propyl}-5-(1-
hydroxyethyl)-6-methylpyridazin-3(2H)-one 5c. Yield = 99%;
1
mp = 193-195 °C (EtOH). H NMR (CDCl3) δ 1.50 (d, 3H,
CH(OH)CH3), 2.05-2.15 (m, 2H, CONCH2CH2CH2N), 2.20
(s, 3H, 6-CH3), 2.55-2.80 (m, 6H: 2H, CONCH2CH2CH2N and
4H, piperazine), 2.85 (exch br s, 1H, OH), 3.15-3.30 (m, 4H,
piperazine), 4.20 (t, 2H, CONCH2CH2CH2N), 5.05 (q, 1H, CH-
(OH)CH3), 5.80 (exch br s, 2H, NH2), 6.85 (d, 2H, Ar), 7.20 (d,
2H, Ar). Anal. (C20H28ClN5O2) C, H, N.
4-Amino-2-{3-[4-(3-chlorophenyl)piperazin-1-yl]propyl}-5-(1-
hydroxyethyl)-6-methylpyridazin-3(2H)-one 5d. Yield = 86%;
4-Amino-2-{3-[4-(4-methoxyphenyl)-piperazin-1-yl]propyl}-6-
methyl-5-vinylpyridazin-3(2H)-one 6b. Yield = 73%; mp =
97-99 °C (EtOH). 1H NMR (CDCl3) δ 2.10-2.20 (m, 2H,
CONCH2CH2CH2N), 2.25 (s, 3H, 6-CH3), 2.60-2.80 (m, 6H:
4H, piperazine and 2H, CONCH2CH2CH2N), 3.10-3.25 (m,
4H, piperazine), 3.80 (s, 3H, OCH3), 4.20 (m, 2H, CON-
CH2CH2CH2N), 5.20 (exch br s, 2H, NH2), 5.60 (d, 1H,
CHdCH2), 5.70 (d, 1H, CHdCH2), 6.50 (dd, 1H, CHdCH2),
6.85 (d, 2H, Ar), 6.90 (d, 2H, Ar). Anal. (C21H29N5O2) C, H, N.
4-Amino-2-{3-[4-(4-chlorophenyl)-piperazin-1-yl]propyl}-6-
methyl-5-vinylpyridazin-3(2H)-one 6c. Yield = 70%; mp =
1
mp = 124-126 °C (EtOH). H NMR (CDCl3) δ 1.50 (d, 3H,
CH(OH)CH3), 2.00-2.10 (m, 2H, CONCH2CH2CH2N), 2.20
(s, 3H, 6-CH3), 2.50-2.60 (m, 2H, CONCH2CH2CH2N), 2.60-
2.70 (m, 4H, piperazine), 2.80 (exch br s, 1H, OH), 3.20-3.30
(m, 4H, piperazine), 4.15 (t, 2H, CONCH2CH2CH2N), 5.00-
5.10 (m, 1H, CH(OH)CH3), 5.80 (exch br s, 2H, NH2), 6.75-
6.85 (m, 2H, Ar), 6.90 (m, 1H, Ar), 7.20 (t, 1H, Ar). Anal. (C20-
H28ClN5O2) C, H, N.
4-Amino-5-(1-hydroxyethyl)-2-{3-[4-(3-methoxyphenyl)-pi-
perazin-1-yl]propyl}-6-methylpyridazin-3(2H)-one 5e. Yield =
92%; oil. 1H NMR (CDCl3) δ 1.47 (d, 3H, CH(OH)CH3),
1.97-2.09 (m, 2H, CONCH2CH2CH2N), 2.18 (s, 3H, 6-CH3),
2.50 (t, 2H, CONCH2CH2CH2N), 2.62 (m, 4H, piperazine), 3.20
(m, 4H, piperazine), 3.80 (s, 3H, OCH3), 4.06-4.19 (m, 2H,
CONCH2CH2CH2N), 4.99 (q, 1H, CH(OH)CH3), 5.79 (exch br
s, 2H, NH2), 6.43 (d, 1H, Ar), 6.47 (s, 1H, Ar), 6.54 (s, 1H, Ar),
7.18 (t, 2H, Ar). Anal. (C21H31N5O3) C, H, N.
1
127-129 °C (EtOH). H NMR (CDCl3) δ 2.00-2.10 (m, 2H,
CONCH2CH2CH2N), 2.25 (s, 3H, 6-CH3), 2.50-2.60 (m, 2H,
CONCH2CH2CH2N), 2.60-2.70 (m, 4H, piperazine), 3.10-
3.20 (m, 4H, piperazine), 4.20 (m, 2H, CONCH2CH2CH2N),
4.75 (exch br s, 2H, NH2), 5.60 (d, 1H, CHdCH2), 5.70 (d, 1H,
CHdCH2), 6.50 (dd, 1H, CHdCH2), 6.85 (d, 2H, Ar), 7.20 (d,
2H, Ar). Anal. (C20H26ClN5O) C, H, N.
4-Amino-5-(1-hydroxyethyl)-2-{3-[4-(3-methylphenyl)-pipera-
zin-1-yl]propyl}-6-methylpyridazin-3(2H)-one 5f. Yield = 95%;
mp = 55-57 °C. 1H NMR (CDCl3) δ 1.50 (d, 3H, CH-
(OH)CH3), 2.08 (q, 2H, CONCH2CH2CH2N), 2.20 (s, 3H, 6-
CH3), 2.33 (s, 3H, CH3-Ar), 2.56 (t, 2H, CONCH2CH2CH2N),
2.60-2.70 (m, 4H, piperazine), 3.17-3.28 (m, 4H, piperazine),
4.16 (t, 2H, CONCH2CH2CH2N), 5.02 (q, 1H, CH(OH)CH3),
5.79 (exch br s, 2H, NH2), 6.67-6.71 (m, 2H, Ar), 6.76 (s, 1H,
Ar), 7.17 (t, 1H, Ar). Anal. (C21H31N5O2) C, H, N.
4-Amino-5-(1-hydroxyethyl)-6-methyl-2-[3-(4-methylpiper-
idin-1-yl)propyl]pyridazin-3(2H)-one 5g. Yield = 83%; mp =
77-80 °C (EtOH). 1H NMR (CDCl3) δ 0.90 (d, 3H, CHCH3),
1.15-1.40 (m, 3H, piperidine), 1.45 (d, 3H, CH(OH)CH3),
1.55-1.65 (m, 2H, piperidine), 1.90-2.10 (m, 4H: 2H, piper-
idine and 2H, CONCH2CH2CH2N), 2.20 (s, 3H, 6-CH3),
2.35-2.45 (m, 2H, CONCH2CH2CH2N), 2.80-2.90 (m, 2H,
piperidine), 3.10 (exch br s, 1H, OH), 4.00-4.15 (t, 2H, CON-
CH2CH2CH2N), 4.90-5.00 (m, 1H, CH(OH)CH3), 5.80 (exch
br s, 2H, NH2). Anal. (C16H28N4O2) C, H, N.
4-Amino-2-{3-[4-(3-chlorophenyl)-piperazin-1-yl]propyl}-6-
methyl-5-vinylpyridazin-3(2H)-one 6d. Yield = 56%; mp =
86-88 °C (EtOH). 1H NMR (CDCl3) δ 2.10-2.20 (m, 2H,
CONCH2CH2CH2N), 2.25 (s, 3H, 6-CH3), 2.50-2.66 (m, 2H,
CONCH2CH2CH2N), 2.65-2.75 (m, 4H, piperazine), 3.20-
3.30 (m, 4H, piperazine), 4.20 (m, 2H, CONCH2CH2CH2N),
5.20 (exch br s, 2H, NH2), 5.60 (d, 1H, CHdCH2), 5.70 (d, 1H,
CHdCH2), 6.50 (dd, 1H, CHdCH2), 6.75-6.85 (m, 2H,
Ar), 6.90 (s, 1H, Ar), 7.20 (m, 1H, Ar). Anal. (C20H26ClN5O)
C, H, N.
4-Amino-2-{3-[4-(3-methoxyphenyl)-piperazin-1-yl]propyl}-6-
methyl-5-vinylpyridazin-3(2H)-one 6e. Yield = 40%; oil. 1H
NMR (CDCl3) δ 2.06 (quint, 2H, CONCH2CH2CH2N), 2.23
(s, 3H, 6-CH3), 2.53 (t, 2H, CONCH2CH2CH2N), 2.61-2.66
(m, 4H, piperazine), 3.21 (t, 4H, piperazine), 3.80 (s, 3H, OCH3),
4.19 (t, 2H, CONCH2CH2CH2N), 5.21 (exch br s, 2H, NH2),
5.59 (dd, 1H, CHdCH2), 5.71 (dd, 1H, CHdCH2), 6.40-6.58
(m, 4H: 1H, CHdCH2, and 3H Ar), 7.17 (t, 1H, Ar). Anal.
(C21H29N5O2) C, H, N.
4-Amino-5-(1-hydroxyethyl)-6-methyl-2-[3-(4-methylpipera-
zin-1-yl)propyl]pyridazin-3(2H)-one 5h. Yield = 93%; mp =
103-105 °C (EtOH). H NMR (CDCl3) δ 1.50 (d, 3H, CH-
(OH)CH3), 1.95-2.05 (m, 2H, CONCH2CH2CH2N), 2.20 (s,
3H, 6-CH3), 2.40 (s, 3H, NCH3), 2.40-2.80 (m, 10H: 8H,
piperazine and 2H, CONCH2CH2CH2N), 3.05 (exch br s, 1H,
OH), 4.05-4.20 (m, 2H, CONCH2CH2CH2N), 5.05 (q, 1H,
4-Amino-2-{3-[4-(3-methylphenyl)-piperazin-1-yl]propyl}-6-
methyl-5-vinylpyridazin-3(2H)-one 6f. Yield = 45%; oil. 1H
NMR (CDCl3) δ 2.06 (quint, 2H, CONCH2CH2CH2N), 2.23
(s, 3H, 6-CH3), 2.33 (s, 3H, CH3-Ar), 2.53 (t, 2H, CONCH2-
CH2CH2N), 2.63 (t, 4H, piperazine), 3.20 (t, 4H, piperazine),
4.19 (t, 2H, CONCH2CH2CH2N), 5.21 (exch br s, 2H, NH2),
5.59 (dd, 1H, CHdCH2), 5.71 (dd, 1H, CHdCH2), 6.47 (dd, 1H,
1

7406 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Biancalani et al.
CHdCH2), 6.67-6.73 (m, 2H, Ar), 6.76 (s, 1H, Ar), 7.16 (t, 1H,
Ar). Anal. (C21H29N5O) C, H, N.
Yield = 50%; mp = 129-131 °C (EtOH). 1H NMR (CDCl3)
δ 2.20 (s, 3H, 6-CH3), 2.30 (s, 3H, Ph-CH3), 2.90 (t, 2H,
CONCH2CH2CO), 3.05-3.15 (m, 4H, piperazine), 3.60-3.70
(m, 2H, piperazine), 3.75-3.85 (m, 2H, piperazine), 4.65 (m, 2H,
CONCH2CH2CO), 5.20 (exch br s, 2H, NH2), 5.60 (d, 1H,
CHdCH2), 5.70 (d, 1H, CHdCH2), 6.45 (dd, 1H, CHdCH2),
6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar). Anal. (C21H27N5O2) C, H, N.
3-Methylisoxazolo[3,4-d]pyridazin-7(6H)-one 12. To a cooled
and stirred mixture of 650 mg (3.58 mmol) of 1123 and 3 g of PPA
(30 mmol) in EtOH (5 mL), 390 mg (7.97 mmol) of hydrazine
hydrate were added. The reaction was carried out at 70 °C for 30
min. After cooling, cold water was added and the precipitate was
recovered by suction.
4-Amino-6-methyl-2-[3-(4-methylpiperidin-1-yl)propyl]-5-vinyl-
pyridazin-3(2H)-one 6g. Yield = 64%; mp = 80-83 °C (EtOH).
1H NMR (CDCl3) δ 0.90 (d, 3H, CHCH3), 1.20-1.40 (m, 3H,
piperidine), 1.60-1.70 (m, 2H, piperidine), 1.90-2.00 (m, 2H,
piperidine), 2.00-2.10 (m, 2H, CONCH2CH2CH2N), 2.20 (s,
3H, 6-CH3), 2.40-2.50 (m, 2H, CONCH2CH2CH2N), 2.90-3.00
(m, 2H, piperidine), 4.10 (t, 2H, CONCH2CH2CH2N), 5.20 (exch
br s, 2H, NH2), 5.60 (d, 1H, CHdCH2), 5.70 (d, 1H, CHdCH2),
6.45 (dd, 1H, CHdCH2). Anal. (C16H26N4O) C, H, N.
4-Amino-6-methyl-2-[3-(4-methylpiperazin-1-yl)propyl]-5-vinyl-
pyridazin-3(2H)-one 6h. Yield = 53%; oil. 1H NMR (CDCl3) δ
1.95-2.05 (m, 2H, CONCH2CH2CH2N), 2.20 (s, 3H, 6-CH3),
2.40 (s, 3H, NCH3), 2.40-2.80 (m, 10H: 8H, piperazine and
2H, CONCH2CH2CH2N), 4.15 (t, 2H, CONCH2CH2CH2N),
5.20 (exch br s, 2H, NH2), 5.60 (d, 1H, CHdCH2), 5.80 (d, 1H,
CHdCH2), 6.50 (dd, 1H, CHdCH2). Anal. (C15H25N5O) C, H, N.
4-Amino-2-{2-[4-(4-methoxyphenyl)piperazin-1-yl]ethyl}-6-
methyl-5-vinylpyridazin-3(2H)-one 6i. Yield = 90%; mp =
133-135 °C (EtOH). 1H NMR (CDCl3) δ 2.20 (s, 3H, 6-CH3),
2.75-3.10 (m, 6H: 4H, piperazine and 2H, CONCH2CH2N),
3.10-3.25 (m, 4H, piperazine), 3.80 (s, 3H, OCH3), 4.30-4.40
(m, 2H, CONCH2CH2N), 5.20 (exch br s, 2H, NH2), 5.60 (d,
1H, CHdCH2), 5.70 (d, 1H, CHdCH2), 6.50 (dd, 1H, CHd
CH2), 6.85 (d, 2H, Ar), 6.90 (d, 2H, Ar). Anal. (C20H27N5O2)
C, H, N.
Yield = 60%; mp = 261-263 °C (EtOH). 1H NMR (CDCl3)
δ 2.85 (s, 3H, 3-CH3), 8.05 (s, 1H, 4-H), 9.50 (exch br s, 1H, NH).
Anal. (C6H5N3O2) C, H, N.
3-Methyl-6-[3-(4-p-tolylpiperazin-1-yl)propyl]isoxazolo[3,4-d]-
pyridazin-7(6H)-one 13. Compound 13 was obtained starting
from compound 12 following the procedure described for 4a-d.
For this compound, the reaction was carried out at 60 °C for 4 h.
Yield = 72%; mp = 130-132 °C (EtOH). 1H NMR (CDCl3)
δ 2.15-2.25 (m, 2H, CONCH2CH2CH2N), 2.30 (s, 3H, Ph-
CH3), 2.60-2.85 (m, 9H: 3H, 3-CH3 and 4H, piperazine and 2H,
CONCH2CH2CH2N), 3.15-3.35 (m, 4H, piperazine), 4.30 (t,
2H, CONCH2CH2CH2N), 6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar),
8.05 (s, 1H, 4-H). Anal. (C20H25N5O2) C, H, N.
5-Acetyl-4-amino-2-[3-(4-p-tolylpiperazin-1-yl)propyl]pyridazin-
3(2H)-one 14. To a suspension of 150 mg (0.49 mmol) of com-
pound 13 in EtOH (10 mL) 60 mg (1.20 mmol) of hydrazine
hydrate and 30 mg of 10% Pd/C were added and the mixture was
refluxed for 1 h. Then, after cooling, the catalyst was filtered off
and the solvent was evaporated in vacuo to afford 14.
1-(4-p-Tolylpiperazin-1-yl)-propenone 7. To a mixture of 200
mg (1.14 mmol) of 1-p-tolylpiperazine and 110 mg (1.14 mmol)
of triethylammine in anhydrous THF (3 mL), 110 mg (1.14
mmol) of 3-chloropropionylchloride were slowly added. The
reaction mixture was stirred for 30 min at room temperature.
After concentration in vacuo, it was diluted with water (20 mL)
and extracted with CH2Cl2 (3 ꢀ 20 mL). After evaporation of
the solvent, the residue was dissolved in anhydrous DMF (1
mL), 165 mg (1.19 mmol) of K2CO3 was added, and the mixture
was stirred at 50 °C for 3 h. After cooling, 20 mL of water was
added and the solution was extracted with CH2Cl2 (3 ꢀ 20 mL).
Evaporation of the solvent afforded compound 7.
Yield = 80%; mp = 144-146 °C (EtOH). 1H NMR (CDCl3)
δ 2.10-2.20 (m, 2H, CONCH2CH2CH2N), 2.30 (s, 3H, Ph-
CH3), 2.55 (s, 3H, COCH3), 2.60-2.90 (m, 6H: 4H, piperazine
and 2H, CONCH2CH2CH2N), 3.15-3.35 (m, 4H, piperazine),
4.25 (t, 2H, CONCH2CH2CH2N), 6.80 (exch br s, 1H, NH2),
6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar), 8.00 (s, 1H, 6-H), 9.00 (exch br
s, 1H, NH2). Anal. (C20H27N5O2) C, H, N.
4-Amino-5-(1-hydroxyethyl)-2-[3-(4-p-tolylpiperazin-1-yl)-
propyl]pyridazin-3(2H)-one 15. Compound 15 was obtained
from compound 14 following the procedure described for 5a-i.
Yield = 96%; mp = 148-151 °C (EtOH). 1H NMR (CDCl3)
δ 1.55 (d, 3H, CH(OH)CH3), 2.05-2.15 (m, 2H, CON-
CH2CH2CH2N), 2.30 (s, 3H, Ph-CH3), 2.50-2.60 (m, 2H,
CONCH2CH2CH2N), 2.60-2.75 (m, 4H, piperazine), 2.90
(exch br s, 1H, OH), 3.15-3.25 (m, 4H, piperazine), 4.20 (t,
2H, CONCH2CH2CH2N), 4.85 (q, 1H, CH(OH)CH3), 5.60
(exch br s, 2H, NH2), 6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar), 7.45
(s, 1H, 6-H); Anal. (C20H29N5O2) C, H, N.
1
Yield = 97%; mp = 150-152 °C (cyclohexane). H NMR
(CDCl3) δ 2.30 (s, 3H, CH3), 3.15 (t, 4H, piperazine), 3.70-3.80
(m, 2H, piperazine), 3.85-3.95 (m, 2H, piperazine), 5.75 (d, 1H,
CHdCH2), 6.35 (d, 1H, CHdCH2), 6.65 (dd, 1H, CHdCH2),
6.95 (m, 2H, Ar), 7.15 (m, 2H, Ar). Anal. (C14H18N2O) C, H, N.
5-Acetyl-4-amino-6-methyl-2-[3-oxo-3-(4-p-tolylpiperazin-1-yl)-
propyl]pyridazin-3(2H)-one 8. Compound 8 was obtained from
compound 122 following the procedure described for 4a-d.
For this compound, the reaction was carried out at
60 °C for 4 h.
Yield = 98%; mp = 198-200 °C (EtOH). 1H NMR (CDCl3)
δ 2.30 (s, 3H, Ph-CH3), 2.55 (s, 3H, 6-CH3), 2.60 (s, 3H,
COCH3), 2.90 (t, 2H, CONCH2CH2CO), 3.05-3.20 (m, 4H,
piperazine), 3.60-3.75 (m, 2H, piperazine), 3.75-3.90 (m, 2H,
piperazine), 4.60 (t, 2H, CONCH2CH2CO), 6.80-6.95 (m, 2H,
Ar), 7.15 (d, 2H, Ar), 7.80 (exch br s, 2H, NH2). Anal.
(C21H27N5O3) C, H, N.
4-Amino-2-[3-(4-p-tolylpiperazin-1-yl)propyl]-5-vinylpyrida-
zin-3(2H)-one 16. Compound 16 was obtained from compound
15 following the procedure described for 6a-i.
Yield = 23%; mp = 126-128 °C (EtOH). 1H NMR (CDCl3)
δ 2.15-2.25 (m, 2H, CONCH2CH2CH2N), 2.30 (s, 3H, Ph-
CH3), 2.55-2.85 (m, 6H: 2H, CONCH2CH2CH2N and 4H,
piperazine), 3.15-3.35 (m, 4H, piperazine), 4.25 (t, 2H, CON-
CH2CH2CH2N), 5.15 (exch br s, 2H, NH2), 5.50 (d, 1H,
CHdCH2), 5.70 (d, 1H, CHdCH2), 6.50 (dd, 1H, CHdCH2),
6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar), 7.70 (s, 1H, 6-H). Anal.
(C20H27N5O) C, H, N.
4-Amino-5-(1-hydroxyethyl)-6-methyl-2-[3-oxo-3-(4-p-tolyl-
piperazin-1-yl)propyl]pyridazin-3(2H)-one 9. Compound 9 was
obtained from compound 8 following the procedure described
for 5a-i.
Yield = 90%; mp = 201-204 °C (EtOH). 1H NMR (CDCl3)
δ 1.50 (d, 3H, CH(OH)CH3), 2.20 (s, 3H, 6-CH3), 2.30 (s, 3H,
Ph-CH3), 2.80-2.95 (t, 2H, CONCH2CH2CO), 3.05-3.20 (m,
4H, piperazine), 3.60-3.75 (m, 2H, piperazine), 3.75-3.90 (m,
2H, piperazine), 4.40-4.50 (m, 2H, CONCH2CH2CO), 5.05 (q,
1H, CH(OH)CH3), 5.80 (exch br s, 2H, NH2), 6.85-6.95 (m,
2H, Ar), 7.15 (d, 2H, Ar). Anal. (C21H29N5O3) C, H, N.
4-Amino-6-methyl-2-[3-oxo-3-(4-p-tolylpiperazin-1-yl)pro-
pyl]-5-vinylpyridazin-3(2H)-one 10. Compound 10 was obtained
from compound 9 following the procedure described for 6a-i.
4-Amino-5-(2-bromoacetyl)-6-phenylpyridazin-3(2H)-one 18.
To a stirred suspension of 300 mg (0.13 mmol) of 1722 and a
catalytic amount of HBr in AcOH (1.9 mL), a solution of Br2
(210 mg, 0.13 mmol) in acetic acid (1 mL) was slowly added. The
mixture was heated for 5 h at 50 °C, and after dilution with 30
mL of cold water, the precipitate was recovered by suction.
Yield = 64%; mp = 228-230 °C (EtOH). 1H NMR (CDCl3)
3.50 (s, 2H, CH2Br), 6.80 (exch br s, 2H, NH2), 7.40 (exch br s, 1H,
NH); 7.45-7.60 (m, 5H, Ar). Anal. (C12H10BrN3O2) C, H, N.

Article
4-Amino-5-(2-methylthiazol-4-yl)-6-phenylpyridazin-3(2H)-one
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7407
2.65 (m, 4H, piperazine), 3.15 (m, 4H, piperazine), 4.20 (m, 2H,
CONCH2CH2CH2N), 6.80-6.90 (m, 3H, Ar), 7.00-7.10 (m,
3H, Ar). Anal. (C19H26N4O) C, H, N.
19. A suspension of 18 (160 mg, 0.52 mmol) and 40 mg (0.52
mmol) ofthioacetamide inanhydrousEtOH (2 mL) was heated at
80 °C for 5 h. After cooling, 15 mL of water was added and the
final product was recovered by suction.
4-Amino-6-methyl-2-[3-(4-p-tolylpiperazin-1-yl)propyl]pyridazin-
1
3(2H)-one 25b. Yield = 52%; mp = 165-168 °C (EtOH). H
Yield = 68%; mp = 189-191 °C (EtOH). 1H NMR (CDCl3)
2.80 (s, 3H, thiazole-CH3), 6.25 (s, 1H, Ar), 6.70 (exch br s, 2H,
NH2), 7.30-7.40 (m, 5H, Ar), 7.45 (exch br s, 1H, NH). Anal.
(C14H12N4OS) C, H, N.
NMR (CDCl3) δ2.00-2.20 (m, 2H, CONCH2CH2CH2N), 2.20 (s,
3H, 6-CH3), 2.30 (s, 3H, Ph-CH3), 2.60-3.05 (m, 6H: 4H, piper-
azine and 2H, CONCH2CH2CH2N), 3.20-3.45 (m, 4H, pipera-
zine), 4.20 (m, 2H, CONCH2CH2CH2N), 4.90 (exch br s, 2H,
NH2), 6.15 (s, 1H, Ar), 6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar). Anal.
(C19H27N5O) C, H, N.
General Procedure for 20a,b. Compounds 20a,b were ob-
tained from compound 19 following the general procedure
described for 4a-d. In this case, the final compounds 20a,b
were purified by column chromatography using the mixture
petroleum ether/Et2O/CHCl3/abs EtOH/NH4OH 10:4:4:1:0.5
as eluent.
4-Amino-5-(2-methylthiazol-4-yl)-6-phenyl-2-[3-(4-p-tolylpi-
perazin-1-yl)-propyl]pyridazin-3(2H)-one 20a. Yield = 20%; oil.
1H NMR (CDCl3) δ 2.15-2.25 (m, 2H, CONCH2CH2CH2N),
2.30 (s, 3H, Ph-CH3), 2.70 (m, 2H, CONCH2CH2CH2N), 2.75
(s, 3H, thiazole-CH3), 2.80-2.90 (m, 4H, piperazine), 3.15-3.30
(m, 4H, piperazine), 4.35 (t, 2H, CONCH2CH2CH2N), 6.20 (s,
1H, Ar), 6.60 (exch br s, 2H, NH2), 6.85 (d, 2H, Ar), 7.10 (d, 2H,
Ar), 7.30-7.40 (m, 5H, Ar). Anal. (C28H32N6OS) C, H, N.
4-Amino-5-(2-methylthiazol-4-yl)-6-phenyl-2-[2-(4-p-tolylpi-
5-Amino-1-[3-(4-p-tolylpiperazin-1-yl)-propyl]-1H-[3,40]bipyridin-
yl-6-one 25c. Yield = 30%; mp = 65-67 °C (EtOH). 1H NMR
(CDCl3) δ 2.10-2.20 (m, 2H, CONCH2CH2CH2N), 2.30 (s, 3H,
Ph-CH3), 2.50-2.60 (m, 2H, CONCH2CH2CH2N), 2.70-2.80 (m,
4H, piperazine), 3.20-3.30 (m, 4H, piperazine), 4.20 (t, 2H, CON-
CH2CH2CH2N), 4.40 (exch br s, 2H, NH2), 6.85 (m, 3H, Ar), 7.10
(d,2H,Ar),7.25(s,1H, Ar),7.35(d,2H, Ar),8.60(d,2H, Ar).Anal.
(C24H29N5O) C, H, N.
5-Acetyl-2,6-dimethyl-4-[2-(4-p-tolylpiperazin-1-yl)ethylamino]-
pyridazin-3(2H)-one 28. A mixture of 165 mg (0.77 mmol) of 2627
and 170 mg (0.77 mmol) of 2728 in anhydrous ethanol was stirred
for 12 hat room temperature. After evaporationof the solvent, the
mixture was diluted with cold water (20 mL) and extracted with
CH2Cl2 (3 ꢀ 20 mL). Evaporation of the solvent afforded the final
compound 29, which was purified by column chromatography
using cyclohexane/ethyl acetate 1:1 as eluent.
perazin-1-yl)ethyl]pyridazin-3(2H)-one 20b. Yield
= 15%;
1
mp = 181-183 °C (EtOH). H NMR (CDCl3) δ 2.30 (s, 3H,
CH3Ph), 2.75 (s, 3H, thiazole-CH3), 2.80-2.90 (m, 4H,
piperazine), 2.95-3.05 (m, 2H, CONCH2CH2N), 3.15-3.30
(m, 4H, piperazine), 4.40-4.50 (m, 2H, CONCH2CH2N), 6.20
(s, 1H, Ar), 6.60 (exch br s, 2H, NH2), 6.85 (d, 2H, Ar), 7.10 (d,
2H, Ar), 7.30-7.40 (m, 5H, Ar). Anal. (C27H30N6OS) C, H, N.
3-(2-Dimethylaminovinyl)-4-methyl-6-[2-(4-p-tolyl-piperazin-
1-yl)ethyl]isoxazolo[3,4-d]pyridazin-7(6H)-one 22. A suspension
of 100 mg of compound 2116 (0.27 mmol) in 1.03 mL (7.55
mmol) of N,N-dimethylformammide dimethyl acetal was stirred
at 90 °C for 1 h. After cooling, 15 mL of water were added and
the precipitate was recovered by suction.
Yield = 30%; oil. 1H NMR (CDCl3) δ 2.20 (s, 3H, 6-CH3),
2.30 (s, 3H, Ph-CH3), 2.55 (s, 3H, COCH3), 2.65-2.80 (m, 6H:
4H, piperazine and 2H, NHCH2CH2N), 3.20-3.40 (m, 6H: 4H,
piperazine and 2H, NHCH2CH2N), 3.75 (s, 3H, NCH3), 6.55
(exch br s, 1H, NH), 6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar). Anal.
(C21H29N5O2) C, H, N.
5-(1-Hydroxyethyl)-2,6-dimethyl-4-[2-(4-p-tolylpiperazin-1-
yl)-ethylamino]pyridazin-3(2H)-one 29. Compound 29 was ob-
tained from compound 28 following the procedure described
for 5a-i.
Yield = 61%; mp = 150-152 °C (EtOH). 1H NMR (CDCl3)
δ 2.30 (s, 3H, Ph-CH3), 2.45 (s, 3H, 4-CH3), 2.90-3.60 (m, 16H:
8H, piperazine and 2H, CONCH2CH2N and 6H, N(CH3)2),
4.40-4.60 (m, 2H, CONCH2CH2N), 5.20 (d, 1H, CHdCH-N),
6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar), 7.60 (d, 1H, CHdCH-N).
Anal. (C23H30N6O2) C, H, N.
4-Amino-6-methyl-5-(1H-pyrazol-3-yl)-2-[2-(4-p-tolylpipera-
zin-1-yl)ethyl]pyridazin-3(2H)-one 23. A suspension of com-
pound 22 (70 mg, 0.17 mmol) and 150 mg of hydrazine
hydrate (3 mmol) in ethanol (2-3 mL) was stirred at 70 °C for
3 h. The mixture was concentrated in vacuo, diluted with cold
water, and extracted with CH2Cl2 (3 ꢀ 15 mL). Evaporation of
the solvent afforded the desired 23, which was purified by
column chromatography using cycloexane/ethyl acetate 1:4 as
eluent.
Yield = 90%; mp = 90-92 °C (EtOH). 1H NMR (CDCl3) δ
1.45 (d, 3H, CH(OH)CH3), 2.20 (s, 3H, 6-CH3), 2.30 (s, 3H, Ph-
CH3), 2.65-2.75 (m, 2H, NHCH2CH2N), 2.75-2.90 (m, 4H,
piperazine), 3.25 (m, 4H, piperazine), 3.70 (s, 3H, NCH3),
3.70-3.80 (m, 2H, NHCH2CH2N), 4.20-4.30 (exch br s, 1H,
OH), 5.00 (q, 1H, CH(OH)CH3), 6.50 (exch br s, 1H, NH), 6.55
(d, 2H, Ar), 7.10 (d, 2H, Ar). Anal. (C21H31N5O2) C, H, N.
2,6-Dimethyl-4-[2-(4-p-tolylpiperazin-1-yl)ethylamino]-5-vinyl-
pyridazin-3(2H)-one 30. To a solution of 70 mg (0.182 mmol) of
29 in 3 mL of toluene, 1.5 g of H2SO4 on silica gel, prepared
following the procedure previously described in literature,30 was
added portionwise over 2 h. The mixture was refluxed for 4 h.
After cooling, silica gel was removed by suction and washed with
K2CO3 saturated solution (100 mL) first and then with ethyl
acetate (60 mL). The aqueous layer was eliminated, and the
organic layer was evaporated in vacuo to afford the vinyl
derivative, which was purified by column chromatography using
CH2Cl2/CH3OH 9.5:0.5 as eluent.
Yield = 46%; mp = 172-174 °C (EtOH). 1H NMR (CDCl3)
δ 2.30 (s, 3H, Ph-CH3), 2.35 (s, 3H, 6-CH3), 2.80-3.20 (m, 6H:
4H, piperazine and 2H, NCH2CH2N), 3.25-3.45 (m, 4H,
piperazine), 4.40-4.55 (m, 2H, NCH2CH2N), 6.35 (exch br s,
2H, NH2), 6.55 (d, 1H, Ar), 6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar),
7.75 (d, 1H, Ar). Anal. (C21H27N7O) C, H, N.
Yield = 45%; mp = 114-115 °C (EtOH). 1H NMR (CDCl3)
δ 2.20 (s, 3H, 6-CH3), 2.30 (s, 3H, Ph-CH3), 2.60-2.70 (m, 6H:
4H, piperazine and 2H, NHCH2CH2N), 3.20 (m, 4H,
piperazine), 3.40 (q, 2H, NHCH2CH2N), 3.75 (s, 3H, NCH3),
5.25 (dd, 1H, CHdCH2), 5.65 (dd, 1H, CHdCH2), 6.40 (exch br
s, 1H, NH), 6.65 (dd, 1H, CHdCH2), 6.85 (d, 2H, Ar), 7.10 (d,
2H, Ar). Anal. (C21H29N5O) C, H, N.
General Procedure for 25a-c. Compounds 25a-c were ob-
tained from 24a-c24-26 following the general procedure de-
scribed for 4a-d. For compound 25b, the suspension was heated
for 2 h at 60 °C. Differently from compounds 4a-d, compounds
25a-c were not recovered by suction, but after dilution, the
reaction mixture was extracted with CH2Cl2 (3 ꢀ 15 mL).
Evaporation of the solvent afforded 25a-c, which were purified
by column chromatography using CH2Cl2/MeOH 9:1 as eluent.
6-Methyl-2-[3-(4-p-tolylpiperazin-1-yl)propyl]pyridazin-3(2H)-
5-Acetyl-4-methyl-3-[3-(4-p-tolylpiperazin-1-yl)-propyl]-3H-
thiazol-2-one, 33. Compound 33 was obtained starting from 3129
following the general procedure described for 4a-d. For com-
pound 33, the mixture was stirred at 40 °C for 2 h and the final
compound was purified by column chromatography using
cyclohexane/ethyl acetate 1:2 as eluent.
1
one 25a. Yield = 57%; mp = 93-94 °C (EtOH). H NMR
(CDCl3) δ 2.00-2.10 (m, 2H, CONCH2CH2CH2N), 2.25 (s, 3H,
6-CH3), 2.30 (s, 3H, Ph-CH3), 2.50 (t, 2H, CONCH2CH2CH2N),
Yield = 30%; mp = 106-107 °C (EtOH). 1H NMR (CDCl3)
δ 1.95 (m, 2H, NCH2CH2CH2N), 2.30 (s, 3H, Ph-CH3), 2.35 (s,

7408 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Biancalani et al.
3H, 4-CH3), 2.50 (t, 2H, NCH2CH2CH2N), 2.60-2.70 (m, 7H:
4H, piperazine and 3H, COCH3), 3.20 (m, 4H, piperazine), 3.90
(t, 2H, NCH2CH2CH2N), 6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar).
Anal. (C20H27N3O2S) C, H, N.
were administered in the right cerebral ventricle (icv) and the
mice performed the tail flick test. Then, the relative ED50 were
calculated. In the second series of experiments, morphine hy-
drochloride and the compound 6a showing the lowest ED50, was
administered po in the hot plate and the tail flick test and the
relative ED50 was calculated. Antagonism studies were per-
formed administering the putative antagonist ip 30 min before
6a po administration. The following antagonists were used:
naloxone hydrochloride (1 mg/kg), CGP 35348 (50 mg/kg),
mecamylamine hydrocholoride (2 mg/kg), and yohimbine hy-
drochloride (2 mg/kg).
5-(1-Hydroxyethyl)-4-methyl-3-[3-(4-p-tolylpiperazin-1-yl)-
propyl]-3H-thiazol-2-one 34. To a cooled (0 °C) and stirred
solution of 33 (120 mg, 0.107 mmol) in CH3OH (4 mL), 30 mg
of sodium borohydride (0.214 mmol) were added. The mixture
was stirred for 5 min at room temperature, concentrated in
vacuo, diluted with cold water, and extracted with CHCl3 (3 ꢀ
20 mL). Evaporation of the solvent afforded final compound 34.
Yield = 98%; mp = 116-117 °C (EtOH). 1H NMR (CDCl3)
δ 1.45 (d, 3H, CH(OH)CH3), 1.90-2.00 (m, 2H, NCH2-
CH2CH2N), 2.20 (s, 3H, 4-CH3), 2.30 (s, 3H, Ph-CH3), 2.50-
2.60 (m, 2H, NCH2CH2CH2N), 2.70-2.80 (m, 4H, piperazine),
3.15-3.25 (m, 4H, piperazine), 3.70-3.80 (m, 2H, NCH2-
CH2CH2N), 5.05 (q, 1H, CH(OH)CH3), 6.85 (d, 2H, Ar), 7.10
(d, 2H, Ar). Anal. (C20H29N3O2S) C, H, N.
In some experiments, prazosin (0.1 and 1 mg/kg) was ip
administered 15 min before 6a (administered po at the dose of
1 mg/kg).
Radioligand Binding Assays. All the procedures were per-
formed according to standard radioligand binding technique. In
particular, the R1A-adrenoceptor and R1B-adrenoceptor binding
assays37,38 were performed on Wistar rat submaxillary gland
and Wistar rat liver membrane extracts, respectively, using 0.25
nM [3H]-Prazosin as radioligand. Nonspecific binding was
measured in the presence of 10 μM of phentolamine in incuba-
tion buffer containing 50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA.
4-Methyl-3-[3-(4-p-tolylpiperazin-1-yl)-propyl]-5-vinyl-3H-thia-
zol-2-one 35. Compound 35 was obtained starting from 34
following the procedure30 described for compound 30.
Yield = 15%; mp = 166-168 °C (EtOH). 1H NMR (CDCl3)
δ 1.95 (m, 2H, NCH2CH2CH2N), 2.20 (s, 3H, 4-CH3), 2.30 (s,
3H, Ph-CH3), 2.55 (t, 2H, NCH2CH2CH2N), 2.65-2.75 (m, 4H,
piperazine), 3.20 (m, 4H, piperazine), 3.80 (t, 2H, NCH2-
CH2CH2N), 5.05-5.15 (m, 2H, CHdCH2), 6.65 (dd, 1H,
CHdCH2), 6.85 (d, 2H, Ar), 7.10 (d, 2H, Ar). Anal.
(C20H27N3OS) C, H, N.
R
2A-Adrenoceptor binding assay39 was performed on human
recombinant insect Sf9 cell extracts using 1 nM of [3H]-MK-912
as radioligand. Nonspecific binding was measured in the pre-
sence of 10 μM of WB-4101 in incubation buffer containing
50 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2, 2 mM EDTA
After 60 min of incubation at 25 °C, membranes were
harvested into glass fiber (GF/B) filters (presoaked in 0.5%
polyethylenimine) using Filtermate cell harvester (PerkinElmer).
Subsequently, the filters were washed with 2.5 mL of ice-cold
buffer and dried for 30 min in an oven at 45 °C. Then 50 μM of
Microscint 20 (Packard) were added to each well, incubated
15 min on an orbital shaker, and counted with a TopCount for
1 min/well.
Biological Assays. Animals. Male CD-1 mice (Harlan, Italy)
weighing 25-30 g were used for all experiments. Mice were
housed for at least 1 week before experimental sessions in colony
cages (seven mice in each cage) under standard light (light on
from 7.00 a.m. to 7.00 p.m.), temperature (21 ( 1 °C), relative
humidity (60 ( 10%) with food and water available ad libitum.
The guidelines of the European Community Council (86/609/
EEC) for animal care and use were followed.
The compound was dissolved in dimethyl sulfoxide (DMSO)
and diluted in incubation buffer, then tested in duplicate at 1 μM
concentration (1% vehicle DMSO final concentration).
The IC50 values (concentration causing a half-maximal in-
hibition of control specific binding) were determined by non-
linear regression analysis of five 10-fold serially diluted
concentrations (ranging from 10 to 0.001 μM) in duplicate
competition curves with constant top = 100, bottom = 0, and
Hill coefficients (nH) = 1 using GraphPad 3.0 software.
Statistical Analysis. Data obtained from hot plate and tail
flick experiments were expressed as time course of the percen-
tage of maximum effect (% MPE) = (post drug latency -
baseline latency)/(cutoff time - baseline latency) ꢀ 100. Data
obtained from the hot plate and the tail flick test were first
evaluated with the analysis of variance (one-way ANOVA),
followed by Student’s t-test or Bonferroni’s posthoc compar-
isons using the statistical software SPSS. Then, data collected at
the peak time after compound 6a-25c administration were
made quantal by designating as a positive an increase of %
MPE up to 50%. At least four groups of six mice were used to
generate dose-response curves and to estimate ED50 values.40
ED50s and their significant differences were calculated with the
aid of a computer program.41 Statistical significance was as-
sumed at P < 0.05.
Tail Flick Test. The tail flick latency was obtained using a
commercial unit (Ugo Basile, Italy), consisting of an infrared
radiant light source (100 W, 15 V bulb) focused onto a photocell
utilizing an aluminum parabolic mirror. During the trials, the
mice were gently hand-restrained with a glove. Radiant heat was
focused 3-4 cm from the tip of the tail, and the latency (s) of the
tail withdrawal recorded. The measurement was interrupted if
the latency exceeded the cut off time (15 s at 15 V). Also in this
case, the baseline was calculated as mean of three readings
recorded before testing at intervals of 15 min and the time
course of latency determined at 15, 30, 45, 60, 90, and 120 min
after treatment.36
Hot Plate Test. In the hot plate test, the thermal nociception
was assessed with a commercially available apparatus consisting
in a metal plate 25 cm ꢀ 25 cm (Ugo Basile, Italy) heated to a
constant temperature of 48.5 ( 0.1 °C, on which a plastic
cylinder (20 cm diameter, 18 cm high) was placed. The time of
latency (s) was recorded from the moment the animal was
inserted inside the cylinder up to when it licked its paws or
jerked them off the hot plate or jumped off the hot plate; the
latency exceeded the cutoff time of 60 s. The baseline was
calculated as mean of three readings recorded before testing at
intervals of 15 min. The time course of latency was then
determined at 15, 30, 45, 60, 72, and 90 min after treatment.36
Surgery for Central Injections. Mice were anesthetized with
ketamine-xylazine (80 þ 10 mg kg-1, ip), and a small incision
was made on the scalp to expose the reference point bregma. The
position of the right lateral ventricle was stereotaxically located
at þ2 mm ML and -0.3 mm AP to bregma. Intracerebral
ventricular injection was performed by using a Hamilton micro-
syringe fitted with a 26 gauge needle that was directly inserted
into the skull to a depth of 2 mm.36
Supporting Information Available: Elemental analyses for all
target compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
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