Synthesis of new piperazine-pyridazinone derivatives and their binding affinity toward α1-, α2-adrenergic and 5-HT1A serotoninergic receptors

Article abstract of DOI:10.1016/j.bmc.2005.12.009

We report the design and synthesis of a new class of piperazine- pyridazinone analogues. The arylpiperazine moiety, the length of the spacer, and the terminal molecular fragment were varied to evaluate their influence in determining the affinity of the new compounds toward the α₁- adrenergic receptor (α₁-AR), α₂-adrenergic receptor (α₂-AR), and the 5-HT_1A serotoninergic receptor (5-HT_1AR). Biological data showed that most of the compounds have an α₁-AR affinity in the nanomolar or subnanomolar range, while affinity toward the other two receptors was lower in most cases. However, several of the tested compounds also showed very good (in the nanomolar range) or moderate affinity toward the 5-HT_1AR subtype.

Full text of DOI:10.1016/j.bmc.2005.12.009

Bioorganic & Medicinal Chemistry 14 (2006) 2828–2836
Synthesis of new piperazine–pyridazinone derivatives and
their binding affinity toward a1-, a2-adrenergic and
5-HT1A serotoninergic receptors
Laura Betti,a Marco Zanelli,b Gino Giannaccini,a Fabrizio Manetti,c,*
Silvia Schenoned and Giovannella Strappaghettib,*
a
`
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita di Pisa, Via Bonanno 6, I-56126 Pisa, Italy
b
`
Dipartimento di Chimica e Tecnologia del Farmaco, Universita di Perugia, Via del Liceo 1, I-06123 Perugia, Italy
`
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Genova, Viale Benedetto XV, I-16132, Genova, Italy
c
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy
d
Received 5 August 2005; revised 25 November 2005; accepted 2 December 2005
Available online 20 December 2005
Abstract—We report the design and synthesis of a new class of piperazine-pyridazinone analogues. The arylpiperazine moiety, the
length of the spacer, and the terminal molecular fragment were varied to evaluate their influence in determining the affinity of the
new compounds toward the a1-adrenergic receptor (a1-AR), a2-adrenergic receptor (a2-AR), and the 5-HT1A serotoninergic recep-
tor (5-HT1AR). Biological data showed that most of the compounds have an a1-AR affinity in the nanomolar or subnanomolar
range, while affinity toward the other two receptors was lower in most cases. However, several of the tested compounds also showed
very good (in the nanomolar range) or moderate affinity toward the 5-HT1AR subtype.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
macophore-based database search3 or the analysis of the
superposition mode of many a1-AR antagonists into the
pharmacophoric model contributed to the optimization
of the arylpiperazinylalkylpyridazinone scaffold to ob-
tain several new compounds with affinity values in the
Although in the past decade, pharmaceutical research
has focused the attention on the development of subtype
selective ligands, comparable efforts have been made to
discover ligands selective for a1-AR over other related
receptors, such as a2-AR, and the 5-HT1AR (showing
45% of structural similarity). Among compounds that
showed high affinity toward a1-AR, relevant attention
has been devoted to molecules containing an arylpiper-
azinyl moiety as an essential pharmacophoric portion.1,2
Our efforts in the field of a1-AR antagonists led recently
to the identification of a pharmacophoric model (Fig. 1)
characterized by five chemical features corresponding to
three hydrophobic substituents (HY1–3), one hydrogen
bond acceptor moiety (HBA), and a positively ionizable
group (PI).3 In addition, suggestions from either a phar-
Figure 1. Superposition pathway of compound 16 into the five feature
pharmacophoric model for a1-AR antagonists. The o-ethoxyphenyl
group of the arylpiperazine moiety is embedded into the HY1–HY2
hydrophobic system, while the most basic nitrogen atom of the
piperazine ring corresponds to the positively ionizable feature, PI. On
the other hand, the hydrogen bond acceptor group HBA is represented
by the carbonyl of the pyridazinone nucleus, while the hydrophobic
region HY3 is filled by the ethoxy group of the terminal molecular
portion. Features are color coded: blue for hydrophobics (HY), red for
positively ionizable groups (PI), and green for hydrogen bond acceptor
groups (HBA).
Keywords: a-Adrenergic receptor ligands; Piperazine–pyridazinones;
Arylalkylpiperazines; 5-HT1A receptor ligands.
*
Corresponding authors. Tel.: +39 0577 234330; fax: +39 0577 234333
(F.M); tel.: +39 075 5855136; fax: +39 075 5855161 (G.S); e-mail
addresses: manettif@unisi.it; noemi@unipg.it
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.009

L. Betti et al. / Bioorg. Med. Chem. 14 (2006) 2828–2836
2829
subnanomolar range and appreciable selectivity toward
2. Chemistry
both a2-AR and 5-HT1AR.4 Moreover, the analysis of
the superposition mode of arylpiperazine derivatives (a
highly populated class of a1-AR antagonists) reported
by our and other research groups suggested that the
pharmacophoric model can be divided in two parts,1–3
constituted by the arylpiperazine moiety and by a termi-
nal heterocyclic molecular fragment, held at the appro-
priate distance by an alkylen chain that serves as a
spacer. The HY1 and HY2 features were usually found
to be filled by the substituted phenyl ring of the arylpip-
erazine moiety, while the latter satisfied the positively
ionizable group of the pharmacophore (PI) with its
more basic nitrogen atom. Since the polymethylene
spacer did not contact any of the pharmacophoric fea-
tures, it could be thought of as a geometric constraint
able to appropriately separate the arylpiperazine moiety
from the terminal group of a1-AR antagonists. The lat-
ter is required to possess a hydrogen bond acceptor moi-
ety (often represented by a carbonyl group) and a
hydrophobic terminus at the edge of the molecule oppo-
site to the arylpiperazine group, matching the HBA
and HY3 features of the pharmacophoric model,
respectively.
Compounds 5–19 were synthesized as shown in Schemes
1 and 2.
4-Chloro-5-[4-(2-furoyl)piperazin-1-yl]pyridazin-3(2H)-
one (20)3 and 4-chloro-5-[4-(2-ethoxyphenyl)piperazin-
1-yl]pyridazin-3(2H)-one (21) were obtained by con-
densation of 4,5-dichloropyridazin-3(2H)-one with 1-
(2-furoyl)piperazine and 1-(2-ethoxyphenyl)piperazine,
respectively, in ethanol and Et3N.
Condensation of 20 with 1,4-dibromobutane led to 223
that was in turn treated with an appropriate piperazine
(1-(2-isopropoxyphenyl)piperazine,6
1-(2-ethoxyphe-
nyl)piperazine, 1-(2,3-dihydrobenzo[1,4]dioxin-2-yl-meth-
yl)piperazine,7 2-piperazin-1-yl-pyrimidine, and 1-(3-
chlorophenyl)piperazine) in acetonitrile, in the presence
of dry potassium carbonate at reflux (Method A), to give
the final products 5–9. Following the same procedure,
compound 10 was obtained with 1-(3-trifluoromethylphe-
nyl)piperazine using isoamyl alcohol to increase the final
yield.
In a similar way, alkylation of 21 with 1,4-dibromobu-
tane or 1,7-dibromoheptane in acetone, in the presence
of dry potassium carbonate, gave intermediates 23 and
24, respectively, which in turn were converted by reac-
tion with the appropriate piperazine into the final com-
pounds 16–17 and 18–19, following Method A, in
isoamyl alcohol, acetone or acetonitrile, respectively.
With the aim of further investigating the relationships
between structure and affinity of arylpiperazine congen-
ers, we report here the design and synthesis of new com-
pounds, along with their affinity data toward a1-AR, a2-
AR, and 5-HT1AR. Such compounds were designed on
the basis of a previous SAR analysis suggesting that
(i) ortho alkoxy substituents at the phenyl ring attached
to the piperazine nucleus of the arylpiperazine moiety
were optimal for affinity, and meta substituents were
also tolerated, with a drop in affinity. Regarding the size
of such an ortho substituent, bulkier groups led to im-
proved affinity.5 (ii) A linear (unbranched) polymethyl-
ene spacer with four- or seven-carbon atoms was
found to be the best spacer to bring the arylpiperazine
moiety at the due distance from the terminal heterocy-
clic group.5 (iii) A phenylpiperazinylpyridazinone moie-
ty was inserted as the terminal fragment instead of the
previously investigated phenoxyethylpiperazinylpyridaz-
inone group characterized by an ꢀextra-sizeꢁ portion.3
Compounds 11–14 have been synthesized by alkylation of
20 in acetone, in the presence of dry potassium carbonate
at reflux, with 2-(4-bromobutyl)phthalazin-1(2H)-one
(25), 1-(3-bromopropyl)-4-(3-chlorophenyl)piperazine (26),
1-(3-chloropropyl)-4-(3-trifluoromethylphenyl)piperazine
(27), and 1-(3-chloropropyl)-4-pyridin-2-yl-piperazine
(28), respectively (Method B). Compounds 26–28 were in
turn prepared according to the procedure reported by
Bourdais.8
Condensation of 4-chloro-5-(1,4-diazepin-1-yl)pyrida-
zin-3(2H)-one 29 with furoyl chloride in chloroform
and sodium bicarbonate led to 4-chloro-5-[4-(2-furoyl)-
[1,4-diazepin-1-yl]pyridazin-3(2H)-one (30), that was in
turn alkylated with 1,4-dibromobutane to prepare inter-
mediate 31 (Scheme 2). Reaction of 31 with 1-(2-iso-
propoxyphenyl)piperazine in acetonitrile in the
presence of dry potassium carbonate gave the final com-
pound 15.
Based on the suggestions reported above, compounds 5–
19 were designed. In principle, all the new compounds
were characterized by chemical structures able to fill
the five features of the pharmacophoric model. Regard-
ing their orientation with respect to the pharmacophore,
the new compounds showed a great similarity with aryl-
piperazines used to generate the pharmacophore itself.
In detail, the arylpiperazine moiety of the ligands
matched the HY1–HY2–PI system of the pharmaco-
phore, as depicted in Figure 1 for compound 16, taken
as a representative example for the whole set of new
compounds. Moreover, the carbonyl group of the pyrid-
azinone moiety usually corresponded to the hydrogen
bond acceptor group. However, because of a 180 degree
rotation of the pyridazinone nucleus, the N1 nitrogen
atom was sometimes allowed to interact with HBA.
Finally, different chemical substituents were found to
be able to fill the HY3 feature.
3. Pharmacology
The pharmacological activity profile of compounds 5–19
was evaluated for their affinity toward a1-AR, a2-AR,
and 5-HT1AR by determining for each compound the
ability to diplace [3H]prazosin, [3H]rauwolscine, and
[3H]8-OH-DPAT, respectively, from specific binding
sites on rat cerebral cortex. Ki values, reported in Table
1, were determined on the basis of three competition
binding experiments in which seven drug concentra-

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L. Betti et al. / Bioorg. Med. Chem. 14 (2006) 2828–2836
Scheme1. Compounds20, R = 2-furoyl; 21, R = 2-ethoxyphenyl; 22, n = 4, R = 2-furoyl;23, n = 4, R = 2-ethoxyphenyl; 24, n = 7, R = 2-ethoxyphenyl.
Reagents and conditions: (a) 1-(2-furoyl)piperazine or 1-(2-ethoxyphenyl)piperazine, EtOH, Et3N; (b) 1,4-dibromobutane or 1,7-dibromoheptane,
acetone, K2CO3 or Na2CO3, reflux; (c) acetonitrile or acetone or isoamyl alcohol, K2CO3, reflux; (d) 25, 26, 27, or 28, acetone, K2CO3, reflux.
Scheme 2. Reagents and conditions: (a) furoyl chloride, CHCl3, Na2CO3; (b) 1,4-dibromobutane; (c) 1-(2-isopropoxyphenyl)piperazine, acetonitrile,
K2CO3, reflux.
tions, run in triplicate, were used. Further details are
reported in Section 6.
At the a1 receptor, the selected compounds displayed no
significant GTP shift, suggesting that they elicited an
antagonist profile as prazosin.
Moreover, to determine the intrinsic activity of com-
pounds with the best affinity profile toward a1-AR (name-
ly, 5 and 17), competition studies were performed in the
presence and in the absence of 1 mM GTP using the radi-
olabeled antagonist [3H]prazosin. In Table 2, the GTP
shift values of the selected compounds and the antagonist
reference compound prazosin were reported.
4. Results and discussion
Several of the new compounds were obtained keeping
fixed the terminal heterocyclic moiety, while the length
of the alkylen spacer, the substituents, and the substitu-

L. Betti et al. / Bioorg. Med. Chem. 14 (2006) 2828–2836
Table 1. a1-AR, a2-AR, and 5-HT1A binding affinities for compounds 5–19
2831
Compound
Ki (nM)a
a2/a1 ratio
5-HT1A/a1 ratio
a1-AR
a2-AR
5-HT1A
5
0.35 0.03
4.8 1.1
223 55
71
45
6
7
1.3 0.45
11
203
9.4
3.6
2.3
1.0
8.7c
1.2
1.1c
6
4
7
65 15
2793 320
70 10
233 35
209 40
3.4b
1.5
8
1819 760
14
9
10
2
17
1
5.0
1.1b
146 43
9461 750
9.4 2.3
137 31
40%d
137 19
20%d
11
12
201 40
341 38
182 37
123 34
1.5 0.2
31 5.8
6.8 1.0
618 150
118 35
1153 225
2.0 0.6
5.0 0.5
3.2 0.4
3.9 0.3
7.2 2.1
21
9.7
6.3
132
3.3
111
9.7
9.0
66
3.4
40
2.1
11
11
5.6
3.6
13
14
35 16
29 15
15
16
0.93 0.04
0.45 0.02
0.28 0.02
0.70 0.18
2.0 0.6
17
18
19
18
3
Prazosin
Rauwolscine
OH-DPAT
0.24 0.05
4.0 0.3
2.0 0.2
a Ki binding data were calculated as described in Section 6. Ki values are means SD of separate series assay, each performed in triplicate. They were
calculated according to the Cheng and Prusoff equation:14 Ki = IC50/[1 + ([L]/Kd)], where [L] is the ligand concentration and Kd its dissociation
constant. Kd of [3H]prazosin binding to rat cortex membranes was 0.24 nM (a1-AR), Kd of [3H]rauwolscine binding to rat cortex membranes was
4 nM (a2-AR), and Kd of [3H]8-OH-DPAT binding to rat cortex membranes was 2 nM (5-HT1A).
b As a2/a1 ratio.
c As a1/5-HT1A ratio.
d Percent inhibition at the 10 lM dose.
Table 2. Intrinsic Activity of Compounds 5 and 17 to a1-AR
than a methoxy substitutent influenced positively affinity
of the higher homologues.5 Superposition of such com-
pounds to the pharmacophoric model for a1-AR antag-
onists showed very similar orientations and accounted
for the different affinity on the basis of the fact that larg-
er alkoxy substituents were able to fill one of the features
of the HY1–HY2 hydrophobic system better than a
smaller group (such as MeO).
a
Compound
Ki toward a1
GTP shift
ÀGTP (nM)
+GTP (nM)
5
17
0.21 0.05
0.37 0.06
0.23 0.06
0.28 0.04
0.40 0.05
0.28 0.05
1.33
1.08
1.22
Prazosin
a Displacement of [3H]prazosin from rat cerebral cortex membranes in
the absence and in the presence of 1 nM GTP. Values are taken from
three experiments, expressed as means SEM.
Moreover, enlargement of the piperazine ring consti-
tuting a portion of the terminal molecular fragment
to a diazepino nucleus (15) led to an affinity less than
3-fold lower with respect to the corresponding pipe-
razino derivative 5 (0.93 nM vs 0.35 nM), suggesting
that the size of such a ring is not a crucial key in
determining interactions with the receptor. This result
was in agreement with the fact that the piperazino and
the diazepino rings are both able to match the termi-
nal hydrophobic feature (HY3) of the model, thus
contributing in a similar way to define affinity of such
derivatives.
tion pattern on the arylpiperazine moiety were varied. In
detail, with the alkylen chain as a four-carbon atom
spacer, the alkoxy group at the phenylpiperazine moiety
was increased to an ethoxy (6) and isopropoxy (5) sub-
stituent, leading to an affinity enhancement of about
one and two orders of magnitude, respectively, with re-
spect to the parent o-methoxy derivative 13 (Chart 1)
(4.8, 0.35 nM vs 33.5 nM), in agreement with previous
experimental results showing that alkoxy groups bulkier
SAR considerations reported in the literature for aryl-
piperazines also suggested that a chloro substituent (in-
stead of an alkoxy group) at the ortho position of the
phenyl ring of the arylpiperazine moiety was also suit-
able for affinity toward a1-AR.3 Moreover, although
the ortho position is the optimum for affinity toward
a1-AR, a variation in the substitution pattern (a meta
substituent instead of an ortho group) was interesting
to find out whether changing the position of the substi-
tuent in the aryl moiety significantly changes the affinity.
In agreement with this experimental evidence, shifting
the chloro susbtituent of compound 23 (Chart 1) into
Chart 1.

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L. Betti et al. / Bioorg. Med. Chem. 14 (2006) 2828–2836
the meta position (9), no significant variation in affinity
was observed (19 nM vs 14 nM, respectively). In a sim-
ilar way, shortening the alkylen spacer of a methylene
unit led to compound 12 with an affinity value compara-
ble to that of 9 (9 nM vs 14 nM). On the other hand,
when the m-chloro substituent of 9 was replaced by a
bulkier CF3 group (10), affinity underwent a remarkable
decrease of about one order of magnitude, from 14 nM
to 146 nM, probably due to a bad steric effect of the
larger substituent. A less pronounced reduction of affin-
ity was also found in compounds with a propylen spacer
after transformation of the m-Cl derivative 12 into the
corresponding CF3 analogue 13 (9 nM vs 35 nM, respec-
tively). These results suggested that the length of the
spacer and the size of the meta substituent at the arylpip-
erazine moiety contributed at the same time to define
affinity values. In particular, while the m-CF3 group
was always disfavored with respect to the corresponding
Cl substitution, the difference in affinity was much more
evident in derivatives with a butylen spacer than with re-
spect to those with a propylen spacer. This was probably
due to the fact that lengthening the alkylen chain of a
methylene unit could cause steric clashes involving the
m-CF3 group to become more marked, leading to a deep
reduction of affinity.
compound 19 was found to be about 8-fold less active
with respect to 410 (Chart 1) (2.0 vs 0.26 nM). Moreover,
when the length of the alkylen spacer was reduced from
a heptylen to a butylen chain, affinity underwent a slight
decrease for ethoxy derivatives 17 and 16 (0.28 vs
0.45 nM, respectively), while it increased from 2.0 to
0.7 nM for isopropoxy derivatives 19 and 18,
respectively.
While most of the new compounds showed low
(2793 nM) to moderate (45 nM) affinity toward a2-AR,
the remaining compounds 16–19 were characterized by
interesting biological data (from 1.5 to 31 nM). More-
over, 17 also showed an appreciable a1 selectivity (a2/
a1 ratio of 111), similar to 15 and 5 with a2/a1 ratio of
132 and 203, respectively.
As expected on the basis of the fact that an o-alkoxy-
phenylpiperazinyl group as the arylpiperazine moiety11
and/or a propylen or butylen spacer represented opti-
mum chemical features for inducing high affinity for
both a1-AR and 5-HT1AR,12 affinity values of the new
compounds toward 5-HT1AR followed the same trend
already found for affinity toward a1-AR, with a few
exceptions. In fact, the ethoxy derivative 6 showed an
affinity of 11 nM, lower than that of the corresponding
isopropoxy analogue 5 whose affinity of 1.3 nM was
comparable with that of compound 15 (2.0 nM), bear-
ing the same ortho substituent at the phenylpiperazine
moiety and a diazepino ring on the terminal molecular
fragment, instead of a piperazino nucleus. Similarly,
affinity of compounds 9 and 10 (17 and 137 nM, respec-
tively) was comparable to that found toward a1-AR (14
and 146 nM, respectively). On the contrary, affinity of
compounds 12–14 (618, 118, and 1153 nM, respectively)
was found to be significantly lower with respect to affin-
ity toward a1-AR (9.4, 35, and 29 nM, respectively).
Moreover, compounds 16–19 showed affinity value to-
ward the 5-HT1AR in the nanomolar range (5.0, 3.2,
3.9, and 7.2 nM, respectively). As a consequence of
the statement that affinity values toward a1-AR and 5-
HT1AR of compounds belonging to this structural class
are generally comparable, no appreciable selectivity was
found for the new compounds among the two receptors.
The highest 5-HT1AR/a1-AR ratio values are 66 and 40,
associated to compounds 12 and 14, respectively.
Compound 11 was found to be inactive toward all the
receptors tested, being 9461 nM its affinity value against
a1-AR, while only a 40% and 20% of specific [3H]prazo-
sin and [3H]8-OH-DPAT binding was found at the test
dose (10 lM) toward a1-AR and 5-HT1AR, respectively.
Finally, four of the new compounds (namely, 7, 8, 11,
and 14) were appropriately designed to confirm the
hypothesis that the replacement of the substituted phe-
nylpiperazine ring (constituting the arylpiperazine moie-
ty) with different substituents led in most cases to a
marked drop in affinity toward a1-AR. In particular, 14
retained appreciable affinity toward a1-AR (29 nM),
while compounds 7, 8, and 11 were characterized by
unsignificant affinity toward the same receptor (223,
1819, and 9461 nM, respectively), in agreement with the
experimental evidence that such a moiety plays a crucial
role in defining the affinity of phenylpiperazinylalkylpy-
ridazinone derivatives and related compounds.1c
Compounds 16–19 were designed and synthesized,
according to previous findings suggesting that a butylen
or heptylen chain is the optimal spacer and that an alk-
oxy group (at the ortho position on the phenyl ring of
the arylpiperazine moiety) larger than a methoxy substi-
tuent led to enhanced affinity.5 In addition, based on the
observation that phenoxyethylpiperazinylpyridazinone
moiety as the terminal molecular fragment was charac-
terized by an ꢀextra-sizeꢁ portion exceeding the pharma-
cophoric features,3 such a large substituent was replaced
by a smaller o-ethoxyphenylpiperazinylpyridazinone, to
verify the hypothesis9 that a reduced size of the terminal
fragment could lead to compounds retaining good a1-
AR affinity. As a result, compound 17 was characterized
by an affinity value of 0.28 nM, slightly lower than that
of the corresponding derivative 310 (Chart 1), bearing
the extended terminal fragment (0.43 nM). Differently,
Finally, in addition to both an o-alkoxyphenylpiperazinyl
group and a spacer of a given length, a furoylpiperazinylpy-
ridazinone and an o-ethoxyphenylpiperazinylpyridazinone
moiety could be considered as terminal fragments well
tolerated for ligand-5-HT1AR interaction, on the basis
of the nanomolar affinity found for compounds 5, 6, 9,
15, and 16–19, respectively.
5. Conclusions
Among the new arylpiperazine–pyridazinone deriva-
tives, compounds 5, 15, and 17 emerge because of their
subnanomolar a1-AR affinity (0.35, 0.93, and 0.28 nM,
respectively) and significant selectivity over a2-AR
(203, 132, and 111, respectively). Such derivatives have

L. Betti et al. / Bioorg. Med. Chem. 14 (2006) 2828–2836
2833
been selected for further studies in order to investigate
their affinity toward the a1-AR subtypes.
(0.31 g, 2.25 mmol) in 20 mL of acetone was stirred un-
der reflux for 7 h. After evaporation under reduced pres-
sure, the compound was purified by chromatography on
a silica gel column, eluting with a EtOH/CH2Cl2 mixture
(2:98) to give 50% yield of a yellow oil.
6. Experimental
6.1. Chemistry
1H NMR (CDCl3) d: 1.23–1.58 (m, 9H, CH2–CH3,
3CH2); 1.74–1.84 (m, 4H, 2CH2); 3.20–3.35 (m, 4H,
H-pip.); 3.41 (t, 2H, CH2); 3.55–3.60 (m, 4H, H-pip.);
3.99–4.17 (m, 4H, CH2–CH3; CH2); 6.84–7.00 (m, 4H,
H-arom); 7.76 (s, 1H, H-pyrid.).
Starting materials were purchased from Aldrich-Italia
(Milan). Melting points were determined with a Kofler
hot-stage apparatus and are uncorrected. 1H NMR spec-
tra were recorded on a Bruker AC 200 MHz instrument in
the solvent indicated below. Chemical shift values (parts
per million, ppm) are relative to tetramethylsilane used
as an internal reference standard. Elemental analyses
are within 0.4% of theoretical values. Precoated Kiesel-
gel 60 F254 plates (Merck) were used for TLC. The corre-
sponding hydrochlorides were prepared by bubbling dry
HCl into the dry solution of the compound.
6.6. 2-(4-Bromobutyl)-phthalazin-1(2H)-one (25)
A mixture of phthalazin-1(2H)-one (0.80 g, 5.50 mmol),
1,4-dibromobutane (1.80 g, 8.20 mmol), and dry potas-
sium carbonate (1.13 g, 8.20 mmol) in 30 mL of acetone
was stirred under reflux for 24 h. The mixture was evap-
orated under reduced pressure and the compound was
purified by chromatography on a silica gel column, elut-
ing with CH2Cl2 to give 50% yield of an oil.
6.2. Syntheses
Specific examples presented below illustrate general syn-
thetic methods A–B.
1H NMR (CDCl3) d: 1.83–1.97 (m, 4H, 2CH2); 3.38 (t,
2H, J = 7 Hz, CH2); 4.20 (t, 2H, J = 7 Hz, CH2); 7.59–
7.73 (m, 3H, H-arom); 8.06 (s, 1H, H-phthal.); 8.29–
8.31 (m, 1H, H-arom).
6.3. 4-Chloro-5-[4-(2-ethoxyphenyl)piperazin-1-yl)pyrida-
zin-3(2H)-one (21)
6.7. 1-(3-Bromopropyl)-4-(3-chlorophenyl)piperazine (26)
A mixture of 1-(2-ethoxyphenyl)piperazine hydrochlo-
ride (2.60 g, 11.0 mmol), 4,5-dichloro-pyridazin-3(2H)-
one (1.80 g, 11.0 mmol) in ethanol and Et3N was stirred
under reflux for 20 h. The mixture was evaporated under
reduced pressure and the compound was purified by
crystallization with EtOH to give 60% yield of a white
solid: mp 190–191 °C.
Using the method reported by Bourdais,8 a mixture of
1-(3-chlorophenyl)piperazine hydrochloride (1.50 g,
9.10 mmol), 1,3-dibromo-propane (2.80 g, 13.0 mmol),
and dry potassium carbonate (1.90 g, 14.0 mmol) in
20 mL of dry dimethylformamide was stirred for
24 h at room temperature (rt). The compound was
purified by chromatography on a silica gel column,
eluting with ethyl acetate to give 25% yield of a yellow
dense oil.
1H NMR (CDCl3) d: 1.47 (t, 3H, CH2-CH3); 3.22–3.25
(m, 4H, H-pip.); 3.63–3.65 (m, 4H, H-pip.); 4.10 (quart.,
2H, CH2–CH3); 6.84–6.94 (m, 4H, H-arom); 7.61 (s, 1H,
H-pyrid.); 11.42 (s, 1H, NH).
1H NMR (CDCl3) d: 1.97–2.08 (m, 2H, CH2); 2.49–2.59
(m, 6H, CH2, 4H-pip.); 3.48(t, 2H, J = 7 Hz, CH2);
3.79–3.85 (m, 4H, H-pip.); 6.73–6.85 (m, 3H, H-arom);
7.15 (m, 1H, H-arom).
6.4. 2-(4-Bromobutyl)-4-chloro-5-[4-(2-ethoxyphenyl)pip-
erazin-1-yl)pyridazin-3(2H)-one (23)
A mixture of 21 (0.40 g, 1.30 mmol), 1,4-dibromo-bu-
tane (0.43 g, 2.00 mmol), and dry potassium carbonate
(0.28 g, 2.00 mmol) in 20 mL of acetone was stirred un-
der reflux for 6 h. After evaporation under reduced pres-
sure, the compound was purified by chromatography on
a silica gel column, eluting with a EtOH/CH2Cl2 mixture
(2:98) to give 60% yield of a yellow dense oil.
6.8. 4-Chloro-5-[4-(2-furoyl)-[1,4]diazepan-1-yl]pyridazin-
3(2H)-one (30)
A mixture of 4,5-dichloro-pyridazin-3(2H)-one (2.00 g,
12.0 mmol), homopiperazine (1.80 g, 18.0 mmol) in eth-
anol and Et3N was stirred under reflux for 5 h. The mix-
ture was evaporated under reduced pressure and the
compound was purified by crystallization with methanol
to give a 50% yield of homopiperazine derivate 29 as a
yellow solid: mp 167–170 °C
1H NMR (CDCl3) d: 1.46 (t, 3H, CH2–CH3); 1.88–1.96
(m, 4H, 2CH2); 3.19–3.24 (m, 4H, H-pip.); 3.43 (t, 2H,
CH2); 3.54–3.59 (m, 4H, H-pip.); 4.02–4.20 (m, 4H,
CH2–CH3, CH2); 6.84–7.00 (m, 4H, H-arom); 7.76 (s,
1H, H-pyrid.).
Subsequently a mixture of 29 (1.00 g, 4.40 mmol), fur-
oyl chloride (0.58 g, 4.46 mmol), and sodium bicar-
bonate (0.56 g, 6.60 mmol) in 40 mL of chloroform
was stirred for 20 h, rt. After evaporation under re-
duced pressure, 30 was purified by chromatography
on a silica gel column, eluting with a EtOH/CH2Cl2
mixture (6:94) to give 55% yield of a white solid:
mp 184–186 °C.
6.5. 2-(7-Bromoheptyl)-4-chloro-5-[4-(2-ethoxyphe-
nyl)piperazin-1-yl)pyridazin-3(2H)-one (24)
A mixture of 21 (0.50 g, 1.50 mmol), 1,7-dibromo-hep-
tane (0.58 g, 2.25 mmol), and dry potassium carbonate

2834
L. Betti et al. / Bioorg. Med. Chem. 14 (2006) 2828–2836
1H NMR (CDCl3) d: 2.16–2.18 (m, 2H, CH2-homopip.);
3.63–3.90 (m, 8H, H-homopip.); 6.42–6.45 (m, 1H, H-
fur); 7.07–7.09 (m, 1H, H-fur); 7.46–7.48 (m, 1H, H-
fur); 7.68 (s, 1H, H-pyrid.); 12.20 (s, 1H, NH).
1H NMR (CDCl3) d: 1.84–1.88 (m, 4H, 2CH2); 3.40–
3.43 (m, 4H, H-pip.); 3.94–3.96 (m, 4H, H-pip.); 4.19–
4.25 (m, 4H, 2CH2); 6.48–6.50 (d.d.,1H, J = 1.7 and
3.4 Hz, H-fur); 7.04–7.24 (d.d., J = 0.8 and 3.4 Hz, 1H,
H-fur); 7.48–7.77 (m, 5H, 3H-arom, 1H-fur, 1H-pyrid.);
8.12 (s, 1H, H-pyrid.); 8.36 (d, 1H, H-arom).
6.9. 2-(4-Bromobutyl)-4-chloro-5-[4-(2-furoyl)-[1,4]diaze-
pan-1-yl]pyridazin-3(2H)-one (31)
6.12. Biological evaluation
A mixture of 4-chloro-5-[4-(2-furoyl)-[1,4]diazepan-1-
yl]pyridazin-3(2H)-one (30) (0.28 g, 0.87 mmol), 1,4-di-
bromo-butane (0.28 g, 1.3 mmol) and dry potassium
carbonate (0.18 g, 1.3 mmol) in acetone was stirred un-
der reflux for 10 h. After evaporation under reduced
pressure, the compound was purified by chromatogra-
phy on a silica gel column, eluting with a EtOH/CH2Cl2
mixture (4:96) to give 60% yield of a yellow oil.
5-HT1A receptor binding: Rat cerebral cortex was
homogenized in 10 volumes of ice-cold 50 mM Tris–
HCl buffer at pH 7.4 in an ultraturrax homogenizer.
The homogenate was centrifuged at 48,000g for 15 min
at 4 °C. The pellet was suspended in 35 volumes of
50 mM Tris–HCl buffer, incubated at 37 °C for 10 min
to remove endogenous 5-HT, and centrifuged at
48,000g for 15 min at 4 °C. The resulting pellet was fro-
zen at À 80 °C until the time of assay. The pellet was
suspended in 20 volumes of ice-cold 50 mM Tris–HCl
buffer at pH 7.4, and the 5-HT1AR binding assay was
performed in triplicate by incubating at 37 °C for
15 min in 1 mL of buffer containing aliquots of the
membrane fraction (0.2–0.3 mg of protein) and 1 nM
[3H]8-OH-DPAT in the absence or presence of unla-
beled 0 lM 8-OH-DPAT. The binding reaction was con-
cluded by filtration through Whatman GF/C glass fiber
filters under reduced pressure. Filtrates were washed
twice with 5 mL aliquots of ice-cold buffer and placed
in scintillation vials. The level of specific binding was ob-
tained by subtracting the level of non-specific binding
from the total level of binding and was approximated
to be 85–90% of the total level of binding.
1H NMR (CDCl3) d: 1.83–1.95 (m, 4H, 2CH2); 2.14–
2.16 (m, 2H, CH2-homopip.); 3.65–4.15 (m, 10H, 8H-
homopip., CH2); 4.45 (m, 2H, CH2); 6.47–6.49 (m, 1H,
H-fur); 7.07–7.09 (m, 1H, H-fur); 7.46–7.48 (m, 1H,
H-fur); 7.68 (s, 1H, H-pyrid.).
6.10. Method A Example. 4-Chloro-5-[4-(2-furoyl)pip-
erazin-1-yl]-2-{4-[4-(2-isopropoxyphenyl)piperazin-1-
yl]butyl}pyridazin-3(2H)-one (5)
A
mixture of 2-(4-bromobutyl)-4-chloro-5-[4-(2-
furoyl)piperazin-1-yl]pyridazin- 3(2H)-one (22)3 (0.48 g,
1.08 mmol), 1-(2-isopropoxyphenyl)piperazine6 (0.24 g,
1.08 mmol) in 30 mL of acetonitrile in the presence of
dry potassium carbonate (0.18 g, 1.30 mmol) was stirred
under reflux for 2 h. After evaporation under reduced
pressure, the residue was purified by chromatography
on a silica gel column, eluting with a EtOH/CH2Cl2
mixture (6:94) to give 60% yield of a dense oil.
a1-receptor binding: Rat cerebral cortex was homoge-
nized in 20 volumes of ice-cold 50 mM Tris–HCl buffer
at pH 7.7 containing 5 mM EDTA (buffer T1) in an
ultraturrax homogenizer. The homogenate was centri-
fuged at 48,000g for 15 min at 4 °C. The pellet was sus-
pended in 20 volumes of ice-cold buffer T1. It was then
homogenized and centrifuged at 48,000g for 15 min at
4 °C. The resulting pellet (P2) was frozen at À 80 °C un-
til the time of assay.
1H NMR (CDCl3) d: 1.30 (d, 6H, 2 CH3); 1.59–1.61 (m,
2H, CH2); 1.81–1.83 (m, 2H CH2); 2.42–2.43 (m, 2H,
CH2); 2.62–2.65 (m, 4H, H-pip.); 3.10–3.20 (m, 4H,
H-pip.); 3.40–3.45 (m, 4H, H-pip.); 3.96–3.98 (m, 4H,
H-pip.); 4.19 (t, 2H, J = 7 Hz, CH2); 4.58 (sept., 1H,
CH–(CH3)2); 6.45 (d.d., J = 1.7 and 3.4 Hz, 1H, H-
fur); 6.84–6.89 (m, 4H, H-arom); 7.06 (d.d., J = 0.8
and 3.4 Hz, 1H, H-fur); 7.49–7.50 (m, 1H, H-fur); 7.60
(s, 1H, H-pyrid.). For the corresponding hydrochloride:
mp 90–93 °C Anal. (C30H39N6O4ClÆ2HClÆ3H2O) C, H,
N.
Pellet P2 was suspended in 20 volumes of ice-cold
50 mM Tris–HCl buffer at pH 7.7 (T2 buffer), and the
a1 binding assay was performed in triplicate by incubat-
ing at 25 °C for 60 min in 1 mL T2 buffer containing ali-
quots of the membrane fraction (0.2–0.3 mg of protein)
and 0.1 nM [3H]prazosin in the absence or presence of
unlabeled 1 lM prazosin. The binding reaction was ter-
minated by filtering through Whatman GF/C glass fiber
filters under reduced pressure and washing twice with
5 mL of ice-cold Tris buffer. The filtrates were placed
in scintillation vials, and 4 mL of Ultima Gold MN
Cocktail-Packard solvent scintillation fluid was added.
The radioactivity was assessed with a Packard 1600
TR scintillation counter. The level of specific binding
was obtained by subtracting the level of non-specific
binding from the total level of binding and was approx-
imated to be 85–90% of the total level of binding.
6.11. Method B Example. 2-{4-[5-Chloro-4-[4-(2-
furoyl)piperazin-1-yl]-6-oxopyridazin-1(6H)-yl]bu-
tyl}phthalazin-1(2H)-one (11)
A mixture of 2-(4-bromobutyl)phthalazin-1(2H)-one
(25) (0.34 g, 1.20 mmol), 4-chloro-5-[4-(2-furoyl)pipera-
zin-1-yl]pyridazin-3(2H)-one (20) (0.30 g, 0.970 mmol)
in 20 mL of acetone in the presence of dry potassium
carbonate (0.16 g, 1.20 mmol) was refluxed under stir-
ring for 11 h. After evaporation under reduced pres-
sure, the residue was purified by chromatography on
a silica gel column eluting with a EtOH/CH2Cl2 mix-
ture (4:96) to give 70% yield of a white solid: mp
57–60 °C.
a2-receptor binding: Cerebral cortex was dissected from
rat brain, and the tissue was homogenized in 20 volumes

L. Betti et al. / Bioorg. Med. Chem. 14 (2006) 2828–2836
2835
of ice-cold 50 mM Tris–HCl buffer at pH 7.7 containing
5 mM EDTA, as reported above (buffer T1). The
homogenate was centrifuged at 48,000g for 15 min at
4 °C. The resulting pellet was diluted in 20 volumes of
50 mM Tris–HCl buffer at pH 7.7 and used in the bind-
ing assay.
F.M. wishes to thank the Divisione di Chimica Farma-
`
ceutica della Societa Chimica Italiana and Farmindu-
stria for the ꢀPremio Farmindustria 2004ꢁ award.
Supplementary data
The binding assay was performed in triplicate, by incu-
bating aliquots of the membrane fraction (0.2–0.3 mg of
protein) in Tris–HCl buffer at pH 7.7 with approximate-
ly 2 nM [3H]rauwolscine in a final volume of 1 mL. Incu-
bation was carried out at 25 °C for 60 min. Non-specific
binding was defined in the presence of 10 lM rauwols-
cine. The binding reaction was concluded by filtration
through Whatman GF/C glass fiber filters under re-
duced pressure. Filters were washed four times with
5 mL aliquots of ice-cold buffer and placed in scintilla-
tion vials. The level of specific binding was obtained
by subtracting the level of non-specific binding from
the total level of binding and approximated to be 85–
90% of the total level of binding. The receptor-bound
radioactivity was assessed as described above.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2005.12.009.
References and notes
1. (a) Lopez-Rodriguez, M. L.; Morcillo, M. J.; Fernandez,
E.; Benhamu, B.; Tejada, I.; Ayala, D.; Viso, A.; Cam-
pillo, M.; Pardo, L.; Delgado, M.; Manzanares, J.;
Fuentes, J. A. J. Med. Chem. 2005, 48, 2548; (b)
Giannangeli, M.; Cazzolla, N.; Luparini, M. R.; Magnani,
M.; Mabilia, M.; Picconi, G.; Tomaselli, M.; Baiocchi, L.
J. Med. Chem. 1999, 42, 336; (c) Mokrosz, M. J.;
Paluchowska, M. H.; Charakchieva-Minol, S.; Bien, S.
Arch. Pharm. Pharm. Med. Chem. 1997, 330, 177; (d)
`
`
Patane, E.; Pittala, V.; Guerrera, F.; Salerno, L.; Romeo,
G.; Siracusa, M. A.; Russo, F.; Manetti, F.; Botta, M.;
Mereghetti, I.; Cagnotto, A.; Mennini, T. J. Med. Chem.
2005, 48, 2420; (e) Manetti, F.; Corelli, F.; Strappaghetti,
G.; Botta, M. Curr. Med. Chem. 2002, 9, 1303.
Compounds were dissolved in buffer or DMSO (2%
buffer concentration) and added to the assay mixture.
A blank experiment was carried out to determine the ef-
fect of the solvent on binding. Protein estimation was
based on a reported method,13 after solubilization with
0.75 N sodium hydroxide, using bovine serum albumin
as a standard.
2. In the text, we use the following notation. The term
ꢀarylpiperazine moietyꢁ is in general referred to the
molecular portion constituted by a substituted phenylpip-
erazine moiety directly linked to the polymethylene spacer.
As an example, the o-isopropoxyphenylpiperazine group
of compound 18 is termed as the ꢀarylpiperazine moiety,ꢁ
while its o-ethoxyphenylpiperazine substituent bound to
the pyridazinone nucleus constitutes the so-called ꢀtermi-
nal heterocyclic moiety.ꢁ Such a notation derives from the
superposition mode of the new compounds to the phar-
macophoric model for a1-AR antagonists, showing their
phenylpiperazine group (bound to the spacer) onto the
HY1–HY2 system of the model, while the piperazine ring
linked to the pyridazinone moiety lies within a region of
space between HBA and HY3 (see Fig. 1).
The concentration of tested compound that produces
50% inhibition of specific [3H]prazosin or [3H]rauwols-
cine, or [3H]8-OH-DPAT binding (IC50) was determined
by log-probit analysis with seven concentrations of the
displacer, each performed in triplicate. Inhibition con-
stants (Ki) were calculated according to the Cheng–Prus-
off equation.14 Kd of [3H]prazosin binding to cortex
membranes was 0.24 nM (a1), Kd for [3H]rauwolscine
binding to cortex membranes was 4 nM (a2), and Kd
of [3H]8-OH-DPAT binding to cortex membranes was
2 nM (5-HT1AR).
3. Barbaro, R.; Betti, L.; Botta, M.; Corelli, F.; Giannaccini,
G.; Maccari, L.; Manetti, F.; Strappaghetti, G.; Corsano,
S. J. Med. Chem. 2001, 44, 2118.
6.12.1. Molecular modeling details. All calculations and
graphic manipulations were performed on an SGI Ori-
gin300 server and an Octane 12K workstation by means
of the Catalyst (version 4.9) software package.15 All the
compounds used in this study were built using the 2D-
3D sketcher of Catalyst. A representative family of con-
formations was generated for each molecule using the
poling algorithm and the ꢀbest quality conformational
analysisꢁ method. Conformational diversity was empha-
sized by selection of the conformers that fell within
20 kcal/mol above the lowest energy conformation
found. The Compare/Fit command has been used to
superpose the studied compounds into the pharmaco-
phoric model for a1-AR antagonists.
4. Betti, L.; Floridi, M.; Giannaccini, G.; Manetti, F.;
Paparelli, C.; Strappaghetti, G.; Botta, M. Bioorg. Med.
Chem. 2004, 12, 1527.
5. Betti, L.; Corelli, F.; Floridi, M.; Giannaccini, G.;
Maccari, L.; Manetti, F.; Strappaghetti, G.; Botta, M.
J. Med. Chem. 2003, 46, 3555.
6. Martin, G. E.; Elgin, R. J., Jr.; Mathiasen, J. R.; Davis, C.
B.; Kesslick, J. M.; Baldy, W. J.; Shank, R. P.; Di Stefano,
D. L.; Fedde, C. L.; Scott, M. K. J. Med. Chem. 1989, 32,
1052.
7. Nelson, W. L.; Wennerstrom, J. E.; Dyer, D. C.; Engel, J.
E. J. Med. Chem. 1977, 20, 880.
8. Bourdais, J. Bull. Soc. Chim. Fr. 1968, 8, 3246.
9. Betti, L.; Botta, M.; Corelli, F.; Floridi, M.; Giannaccini,
G.; Maccari, L.; Manetti, F.; Strappaghetti, G.; Tafi, A.;
Corsano, S. J. Med. Chem. 2002, 45, 3603.
10. Betti, L.; Floridi, M.; Giannaccini, G.; Manetti, F.;
Strappaghetti, G.; Tafi, A.; Botta, M. Bioorg. Med. Chem.
Lett. 2003, 13, 171.
Acknowledgments
M.B. thanks the Merk Research Laboratories (2004
Academic Development Program Chemistry Award).
11. Barlocco, D.; Cignarella, G.; Montesano, F.; Leonardi,
A.; Mella, M.; Toma, L. J. Med. Chem. 1999, 42, 173.

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L. Betti et al. / Bioorg. Med. Chem. 14 (2006) 2828–2836
12. Lopez-Rodriguez, M. L.; Morcillo, M. J.; Fernandez, E.;
Porras, E.; Orensanz, L.; Beneytez, M. E.; Manzanares, J.;
Fuentes, J. A. J. Med. Chem. 2001, 44, 186.
13. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall,
R. J. J. Biol. Chem. 1951, 193, 265.
14. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
15. Catalyst (version 4.9) software is distributed by Accelrys,
Scranton Road, San Diego, CA.