Synthesis of new 1,2,3-benzotriazin-4-one-arylpiperazine derivatives as 5-HT(1A) serotonin receptor ligands

Article abstract of DOI:10.1016/S0968-0896(00)00004-3

A series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radioligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5-HT(1A) receptor, some of which were selective with respect 5-HT(2A) and 5-HT(2C) receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected and further evaluated for their binding affinities on D₁, D₂ dopaminergic and α₁-, α₂-adrenergic receptors. A o-OCH₃ derivative (2e) bound at 5-HT(1A) sites with subnanomolar affinity (IC₅₀=0.059 nM) and shows high selectivity over all considered receptors and may offer a new lead for the development of therapeutically efficacious agents. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00004-3

Bioorganic & Medicinal Chemistry 8 (2000) 533±538
Synthesis of New 1,2,3-Benzotriazin-4-one-arylpiperazine
Derivatives as 5-HT1A Serotonin Receptor Ligands
Giuseppe Caliendo, a,* Ferdinando Fiorino, a Paolo Grieco, a Elisa Perissutti, a
Vincenzo Santagada, a Beatrice Severino, a Giancarlo Bruni b
and Maria Rosaria Romeo b
a
Á
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita di Napoli ``Federico II'', Via D. Montesano, 49-80131, Naples, Italy
b
Á
Istituto di Farmacologia, Universita di Siena, Via delle Scotte, 6- 53100 Siena, Italy
Received 10 August 1999; accepted 15 October 1999
AbstractÐA series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radio-
ligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5-HT1A
receptor, some of which were selective with respect 5-HT2A and 5-HT2C receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected
and further evaluated for their binding anities on D1, D2 dopaminergic and a1-, a2-adrenergic receptors. A o-OCH3 derivative (2e)
bound at 5-HT1A sites with subnanomolar anity (IC50=0.059 nM) and shows high selectivity over all considered receptors and may
o€er a new lead for the development of therapeutically ecacious agents. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
region of the 5-HT1A receptor, and the importance of
the nature of the polymethylene chain connecting the
heterocyclic nucleus and piperazine rings, we replaced
the benzoyltriazole nucleus by a 3-hydroxy-1,2,3-ben-
zotriazin-4-one ring also introducing an oxygen atom in
the polymethylene chain.
Serotonin (5-HT) is an important molecule in medicinal
chemistry, and several 5-HT receptor subtypes have
been found by molecular biological methods as well as
by speci®c agonists or antagonists.1,2 Particular atten-
tion, over the last decade, has been focused on 5-HT1A
because this receptor plays an important role in the
central nervous system (CNS) modulating a number of
behaviours such as impulsivity, sexual behaviour and
food intake.3 Moreover, several agonists for this recep-
tor have been shown to possess anxiolytic and anti-
depressant properties in man.4 It is potentially useful to
develop ligands that are selective for the 5-HT1A sub-
type to facilitate the study and characterization of this
receptor. Continuing our study in this ®eld,5±8 recently
we reported the synthesis and the pharmacological
characterization of a new class of benzoyltriazole aryl-
piperazine derivatives,9 which have 5-HT1A receptor
anity while they showed low anity versus two other
considered serotonin receptors (5-HT2A, 5-HT2C). Tak-
ing into account these results, in an attempt to increase
anity and selectivity for the 5-HT1A receptor, in order
to clarify the role played by the heterocyclic nucleus in
the interaction with a corresponding hydrophobic
Substituents used on the arylpiperazine moiety (X=o-Cl,
m-Cl, p-Cl, o-OCH3, p-OCH3, o-F, p-F and m-CF3) are
those previously reported9 except for m-CF3 which had
not been considered in the previous work. The piperazine
ring is connected to the 3-hydroxybenzotriazinone moiety
through an ethylene (series 1) or a propylene (series 2)
bridge.
In this article, we described the synthesis of new 1,2,3-
benzotriazin-4-one arylpiperazines (1a±i and 2a±i), their
anity and selectivity for 5-HT1A receptor, obtained by
radioligand binding studies. Several analogues showing
interesting 5-HT1A binding properties were further
evaluated for their anity versus dopaminergic D1, D2
and adrenergic al, a2 receptors.
Chemistry
The synthesis of the 1,2,3-benzotriazinone derivatives
1a±i and 2a±i is summarized in Scheme 1. The 1-(2-
chloroethyl) and 1-(3-chloropropyl)-4-phenylpiperazine
intermediates 4 and 5, substituted on the aromatic ring,
*Corresponding author. Tel.: +39-81-7486646; fax: +39-81-7486630;
e-mail: caliendo@unina.it
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00004-3

534
G. Caliendo et al. / Bioorg. Med. Chem. 8 (2000) 533±538
Scheme 1. Reagents and conditions: (a) Br(CH2)nCl, toluene, ~; (b) 3-hydroxy-1,2,3-benzotriazin-4(3H)-one, NaOH, C2H5OH, ~.
were prepared by alkylation of 1-bromo-2-chloroethane
or 1-bromo-3-chloropropane, respectively, with various
4-phenylpiperazines 3, commercially available. The
reaction yielded the bis compound 6 as a by-product.
To avoid this, the reagents were dissolved in toluene as
an inert solvent, using a small excess of 1-bromo-2-
chloroethane or 1-bromo-3-chloropropane. To reduce
the production of the bis derivative 6, the substituted
piperazine had to be added when the reaction mixture
was already under re¯ux. Condensation of the obtained
compound 4 or 5, with 3-hydroxy-1,2,3-benzotriazin-
4(3H)-one in ethyl alcohol and in the presence of
sodium hydroxide gave the expected compounds 1a±i
and 2a±i (Table 1) in yields ranging between 40 and
65%. Analytical puri®cation of each product, reported
in Table 1, was obtained by chromatography on silica
gel column and further by crystallization from the
appropriate solvent. All new compounds gave satisfac-
tory elemental analyses (C, H, Cl, F, N) and were char-
Table 1. Physicochemical properties of 1,2,3-benzotriazin-4-one
derivatives 1a±i and 2a±i
Compound
X
n
Formulaa
Mp
(ꢀC)
Cryst. Yieldc
solventb
%
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
o-Cl
m-Cl
p-Cl
o-OCH3
p-OCH3
o-F
p-F
m-CF3
2
2
2
2
2
2
2
2
2
C19H21N5O2 112±114
C19H20N5O2Cl 84±85
a
a+e
b
a
a
40
41
53
45
50
55
50
48
40
C
19H20N5O2Cl 96±97
C19H20N5O2Cl 140±141
C20H23N5O3 68±69
C20H23N5O3 141±142
b
C
19H20N5O2F 137±138
C19H20N5O2F 145±147
C20H21N5O2F3 101±102
a+b
a+b
a+b
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
o-Cl
m-Cl
p-Cl
o-OCH3
p-OCH3
o-F
p-F
m-CF3
3
3
3
3
3
3
3
3
3
C
20H23N5O2 124±125
C20H22N5O2Cl 99±100
20H22N5O2Cl 116±117
C20H22N5O2Cl 119±120
C21H25N5O3 81±82
C21H25N5O3 153±155
a+d
42
63
45
54
48
65
48
62
33
a
a+b
c
a
a
a+b
a+b
b
1
C
acterized by H NMR spectroscopy.
Pharmacology
C
20H22N5O2F 140±141
C20H22N5O2F 135±136
C21H23N5O2F3 118±119
The compounds reported in Table 1 (1±2) were tested
for their in vitro anity on serotonin 5-HT1A, 5-HT2A
and 5-HT2C receptors by radioligand binding assays.
The more active compounds on serotonin receptors have
been selected and evaluated for their anity on dopami-
nergic (D1 and D2) and adrenergic (a1 and a2) receptors.
All the compounds were tested as water soluble, hydro-
chloride salts. The following speci®c radioligands and
tissue sources were used: (a) serotonin 5-HT1A receptors,
[3H]-8-OH-DPAT, rat brain cortex membranes; (b) ser-
otonin 5-HT2A receptors, [3H] ketanserin, rat brain
cortex membranes; (c) serotonin 5-HT2C receptors, [3H]
mesulergine, rat brain cortex membranes; (d) dopamine
D1 receptors, [3H]SCH-23390, rat strial membranes; (e)
dopamine D2 receptors, [3H]spiroperidol, rat strial
membranes; (f) a1-adrenergic receptors, [3H]prazosin, rat
brain cortex membranes; (g) a2-adrenergic receptors,
[3H]yohimbine, rat brain cortex membranes. Concentra-
tions required to inhibit 50% of radioligand speci®c
binding (IC50) were determined through four indepen-
dent experiments with samples in triplicate using seven
aSatisfactory microanalyses obtained: C, H, Cl, F, N values are within
‹0.4% of the theoretical values.
bCryst. solvent: a=diethyl ether; b=ethyl alcohol; c=methyl alcohol;
d=chloroform; e=n/hexane.
cYield related to last step.
to nine di€erent concentrations of the title compound.
Speci®c binding, de®ned as described in the Experi-
mental, represented more than 75% of total binding in
all three assays. The obtained IC50 nM values are listed
in Table 2 and 3.
Results and Discussion
The novel compounds (1a±i and 2a±i) derived from
benzoyltriazole derivatives9 were prepared and pre-
liminarily evaluated in vitro for their receptor binding
anity for 5-HT1A receptors and their selectivity was

G. Caliendo et al. / Bioorg. Med. Chem. 8 (2000) 533±538
535
Table 2. Binding anities and selectivities of 1,2,3-benzotriazin-4-one derivatives 1a±i and 2a±i
IC50 nM (‹ SEM)
Selectivity versus 5-HT1A
receptor IC50 ratio
Compound
X
n
5- HT1A
[3H]8-OH-DPAT
5-HT2A
[3H] ketanserin
5-HT2C
[3H] mesulergine
5-HT2A
5-HT2C
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
o-Cl
m-Cl
p-Cl
o-OCH3
p-OCH3
o-F
p-F
m-CF3
2
2
2
2
2
2
2
2
2
19‹2.0
95‹8.7
38‹3.6
15,000‹1350
9100‹862
6800‹700
9000‹897
>105
8.4‹0.9
490‹52
97‹9.2
1300‹110
1100‹980
860‹82
>105
>105
7400‹720
789
96
179
2.4
>2 103
1
2
0.02
0.05
0.4
5
3
0.3
27
<9 10
>2 102
>13
3
3800‹382
41‹3.8
>105
>105
3
470‹49
7700‹680
2700‹300
990‹95
160‹ 20
130‹15
2a
2b
2c
2d
2e
2f
2g
H
o-Cl
m-Cl
p-Cl
o-OCH3
p-OCH3
o-F
p-F
m-CF3
3
3
3
3
3
3
3
3
3
7.0‹0.5
1.0‹0.1
2.4‹0.3
20‹1.8
0.059‹0.006
770‹74
21‹2.0
160‹18
0.54‹0.04
2.1‹0.3
Ð
7700‹740
1000‹950
530‹55
630‹60
5500‹520
9300‹900
16‹1.8
6.8‹0.7
0.0069‹0.0008
Ð
26‹2.3
130‹11
40‹3.7
47‹4.4
26‹2.5
150‹18
>105
4.2‹0.4
16‹1.4
Ð
1100
1000
221
31
93220
12
0.8
0.04
0.01
Ð
Ð
Ð
4
130
17
2.4
441
0.2
>5 103
0.03
30
Ð
Ð
Ð
Ð
2h
2i
8-OH-DPAT
Ketanserin
Cinanserin
Mesulergine
1.7‹0.3
1.6‹1.0
Ð
Ð
Ð
1.2‹0.2
Ð
Ð
Ð
Table 3. Binding anities for D1, D2, a1 and a2 receptors in compounds 1a, 2a, 2b, 2c, 2e and 2i
Compound
X
n
IC50 nM (SEM)
D1
[3H] SCH-23390
D2
[3H] spiroperidol
a1
a2
[3H] prazosin
[3H] yohimbine
1a
2a
2b
2c
2e
2i
H
H
o-Cl
m-Cl
o-OCH3
m-CF3
2
3
3
3
3
3
>105
>105
3400‹270
1500‹150
4900‹410
2.3‹0.3
3.6‹0.5
>105
4400 350
820‹85
2.5‹0.4
8.4‹0.8
340‹35
17‹1.8
400 38
1600‹180
15‹1.7
9900‹920
1500‹160
5300‹4.50
2600‹270
5200‹490
1100‹110
>105
9700‹890
4.6‹0.7
Spiroperidol
Prazosin
Yohimbine
1.3‹0.7
22‹2
compared to those of two other receptors, 5-HT2A and
5-HT2C. The obtained IC50 (nM) values are reported in
Table 2. The values obtained with 8-OH-DPAT, ketan-
serine, and mesulergine were also reported as reference
The study of the results presented in Table 2 shows that
the length of the alkyl chain plays an important role in
the anity and selectivity for the 5-HT1A receptor.
Among the ethylene series the compounds 1a, 1c and 1e
appear to be good ligands for the 5-HT1A receptor since
they show IC50S values in the range of 15±50 nM.
Moreover, these compounds have appreciable selectivity
for the 5-HT1A versus the 5-HT2A receptor. Notably, 1e
is 1000-fold more potent on 5-HT1A than towards 5-
HT2A. Unfortunately, 5-HT2C/5-HT1A IC50 ratios of 1a,
1c and 1e, are all below 30.
for 5-HT1A-selective, 5-HT2A-selective and 5-HT2C
selective ligands.
-
The products selected on the preliminary binding screen-
ing were tested on an extended binding screening: dopa-
minergic D1, D2 and adrenergic a1, a2 receptors (Table
3). Biological data presented in these tables agree with
our working hypothesis noted in the Introduction. In
fact, most of the considered compounds demonstrated
moderate to high anity for the 5-HT1A receptor.
The propylene derivatives (series 2) showed receptor
anities higher than the corresponding ethylene homo-

536
G. Caliendo et al. / Bioorg. Med. Chem. 8 (2000) 533±538
logues. In fact it should be noted that several com-
pounds (2a, 2b, 2c, 2e and 2i) on 5-HT1A receptor exhi-
bit IC50 values lower than 10 nM. Remarkably, 2e and
2i, exhibit subnanomolar anity on 5-HT1A, IC50 0.059
and 0.54 nM, respectively. These results allowed us to
conclude that the propylene chain was the better to
furnish analogues with a good balance of anity and
selectivity.
and 2 on 5-HT1A (1a, 2a, 2b, 2c, 2e and 2i), were further
evaluated for their anity at dopaminergic and adre-
nergic receptors. Results are summarized in Table 3.
As far as the dopaminergic system was concerned, the
D1 and D2 receptor anity consistently showed IC50
6
values of above 10 M except compound 2i which
exhibited a IC50 value of 2.3 nM at the D1 receptor. The
anities for a1 receptors were in some cases quite con-
siderable (2b, 2c and 2i). In particular 2b and 2c deriva-
tives showed anity for the a1 similar to that shown
concerning the 5-HT1A receptor. In regards to the a-
nity for a2-adrenergic receptors, the IC50 values were
high for all selected compounds with the exception of 2b
(IC50=15 nM).
Subsequently, we attempted the modi®cation of the
nature and the position of the substituents on the aro-
matic ring. Analysis of the structure endowed with
highest 5-HT1A anity suggests that ortho (1e, 2b, 2e and
2g) or meta (1c, 2c and 2i) by Cl, OCH3 or F are favorable
positions for substitution on the phenyl ring. Moving the
substituent to the para position (1f, 2d and 2f) leads to a
reduction of potency, especially in the case of the p-OCH3
derivative (1f and 2f). The same deleterious e€ect on a-
nity was observed in the benzoyltriazole series.9 Note
compounds 1a and 2a with no substituent also bind to the
5-HT1A receptor with high anity.
Our results con®rm that the replacement of the ben-
zoyltriazole ring of previously studied structures,9 by a
3-hydroxy-1,2,3-benzotriazinone ring, proved com-
pounds of series 1 and 2, have led to the same binding
pro®le. In fact these new compounds, appear to own the
physicochemical requirements necessary to insure a
higher anity on the 5-HT1A receptor. In particular
compound 2e, the most potent 5-HT1A ligand, which
showed the best selectivity pro®le versus all considered
receptors, may o€er a new lead for the development of
new ligands.
Compound 2e shows the best anity-selectivity pro®le
for the 5-HT1A receptor (5-HT2A/5-HT1A and 5-HT2C
/
5-HT1A IC50 ratios are about of 100,000 and 500,
respectively). Compounds 2a and 2b are also sig-
ni®cantly selective for 5-HT1A over 5-HT2A receptor (5-
HT2A/5-HT1A IC50 ratios of both compounds are in the
order of 1000).
In conclusion, we have synthesized and evaluated a new
class of 5-HT1A ligands having a 3-hydroxy-1,2,3-ben-
zotriazinone skeleton. Although the compounds exhibit
a good anity for the 5-HT1A receptor, further syn-
thesis and biological in vitro studies are in progress to
evaluate its intrinsic ecacy. The results of this work
will be published in the near future.
As far as 5-HT2A the results of these studies indicate
that the anity for this receptor are usually lower than
the anity for the 5-HT1A receptor. The introduction of
electron withdrawing substituents, such as the F or CF3
group, increase the binding anity for the 5-HT2A
receptor. In fact we obtained very interesting results
with compound 2i, which exhibits pM anity (IC50
value is 6.9 10 3 nM). In addition, highly potent ligands
are 2g and 2h showing IC50 values of 16 and 6.8 nM,
respectively.
Experimental
Chemistry
In particular, compounds 2i and 2g show the best
selectivity for 5-HT2A versus 5-HT2C receptor (5-HT2A
General. Melting points were determined using a Ko¯er
hot-stage apparatus and are uncorrected. Kieselgel 60
was used for column chromatography and kieselgel 60
F254 plates from Merck were used for TLC. Where
analyses are indicated only by the symbols of the ele-
ments, results obtained are within ‹0.4% of the theo-
retical values. The purity of compounds was carefully
assessed using analytical TLC and the structure veri®ed
spectroscopically by proton NMR spectra (CDCl3)
recorded on a Bruker AMX-500 instrument. Chemical
shifts in ppm are referenced to the residue solvent signal
at 7.27 ppm. Reagent grade materials were purchased
from Aldrich. Appropriate aromatically substitued-4-
phenylpiperazines and 3-hydroxy-1,2,3-benzotriazin-
4(3)-one are commercially available from Aldrich.
/
5-HT2C IC50 ratios are 2300 and 6250, respectively) but
they are moderately or scarcely selective versus the
receptor 5-HT1A (5-HT1A/5-HT2C IC50 ratios 78 and 1.3
nM, respectively). Compound 2h is likewise nonselective
for the 5-HT2A receptor since it is only 20-fold more
potent at the 5-HT2A than at the 5-HT1A receptor and
has comparable anity for the 5-HT2C receptor.
As for the 5-HT2C receptor, in addition to the above
mentioned compound 2h (IC50=4.2 nM) the com-
pounds 2a, 2c, 2d, 2e and 2i, exhibit IC50 values in the
range of 10±50 nM. All these compounds are moder-
ately selective on the 5-HT2C versus 5-HT2A receptor
and are not selective versus 5-HT1A
.
1-(2-Chloroethyl)piperazine-4-substituted derivatives (4).
A solution of the appropriate aromatically substituted-
4-phenyl-piperazine (0.1 mol) in toluene (30 mL) was
added dropwise over a 1 h period to a boiling solution of
1-bromo-2-chloroethane (0.15 mol) in 80 mL of toluene.
The reaction mixture was stirred and re¯uxed for 5 h.
As mentioned in the Introduction, to evaluate potential
therapeutic uses it is crucial to check whether high
potency is ¯anked by undesirable anity for other
receptors (e.g. dopaminergic and adrenergic recep-
tors).5±8 Thus, the most active compounds of the series 1

G. Caliendo et al. / Bioorg. Med. Chem. 8 (2000) 533±538
537
Successively, the mixture was cooled and partitioned
between water, alkalinized with 2 N NaOH and chloro-
form. The combined organic layers were washed with
water, dried over anhydrous Na2SO4 and evaporated in
vacuum. The crude residue was puri®ed by silica-gel col-
umn chromatography (eluent: diethyl ether) to obtain
derivatives 4 in a yield ranging between 40 and 55%.
GF/B ®lters. The ®lters were washed twice with 5 mL of
ice-cold Tris±HCl bu€er, and the radioactivity bound to
the ®lters was measured by liquid scintillation spectro-
metry. Speci®c [3H]-8-OH-DPAT binding was de®ned
as the di€erence between binding in the absence and
presence of 5-HT (10 mM).
5-HT2A and 5-HT2C binding assays. Radioligand bind-
ing assays were performed as previously reported by
Herndon et al.11 Brie¯y, frontal cortical regions of male
Sprague±Dawley rats (200±250 g, Charles River) were
dissected on ice and homogenized (1:10 w/v) in ice-cold
bu€er solution (50 mM Tris±HCl, 0.5 mM EDTA and
10 mM MgCl2 at pH 7.4) and centrifuged at 3000 g for 15
min. The pellet was resuspended in bu€er (1:30 w/v),
incubated at 37 ꢀC for 15 min and then centrifuged twice
more at 3000 g for 10 min (with resuspension between
1-(3-Chloropropyl)piperazine-4-substituted derivatives (5).
These compounds were prepared by the same proce-
dure described above, from di€erent 4-substituted-
piperazine (0.1 mol) and 1-bromo-3-chloropropane
(0.15 mol). All derivatives 5 were obtained in a yield
ranging between 45 and 60%.
General procedure for the preparation of 3-{2-[4-(X-phen-
yl)-1-piperazinyl]etoxy]}benzo-triazinone derivatives (1a±i).
To a stirred mixture of absolute ethanol (100 mL) and
sodium hydroxide (0.1 mol) were added 3-hydroxy-1,2,3-
benzotriazin-4(3H)-one (0.1 mol) and appropriate 1-(2-
chloroethyl)piperazine-4-substituted (0.1 mol). The mix-
ture was re¯uxed for 24 h, diluted with H2O, and extracted
with CH2Cl2. The combined organic layers were dried on
anhydrous Na2SO4 and concentrated in vacuo. The resi-
due was puri®ed by column chromatography (eluent:
CH2Cl2:EtOH 9:1 (v/v)). Further puri®cation was per-
formed by crystallization from the appropriate solvent to
give the ®nal products 1a±i in the yields provided in Table
1. NMR spectra for all intemediates and ®nal compounds
were consistent with the proposed structures.
centrifugation). The ®nal pellet was resuspended in
5
bu€er that also contained 0.1% ascorbate and 10
pargyline.
M
Assays were performed in triplicate in a 2.0 mL volume
containing 5 mg wet weight of tissue and 0.4 nM [3H]
ketanserin (76 Ci/mmol; New England Nuclear) for 5-
HT2A receptor assays, and 10 mg wet weight of tissue
and 1 nM [3H] mesulergine (75.8 Ci/mmol; Amersham)
for 5-HT2C receptor assays. Cinanserin (1.0 mM) was
used to de®ne nonspeci®c binding in the 5-HT2A assay.
In the 5-HT2C assays, mianserin (1.0 mM) was used to
de®ne nonspeci®c binding, and 100 nM spiperone was
added to all tubes to block binding to 5-HT2A receptors.
Tubes were incubated for 15 min at 37ꢀC, ®ltered on
Schliecher and Schuell (Keene, NH) glass ®ber ®lters pre-
soaked in poly(ethylene imine), and washed with 10 mL of
ice-cold bu€er. Filters were counted at an eciency of
50%.
General procedure for the preparation of 3 - {3 - [4 - (X -
phenyl)-1-piperazinyl]propoxy} -benzotriazinone deriva-
tives (2a±i). These compounds were prepared by the
same procedure described above from 3-hydroxy-1,2,3-
benzotriazin-4(3H)-one (0.1 mol) and the appropriate
1-(3-chloropropyl)piperazine-4-substituted (0.1 mol).
Compounds 2a±i were obtained in the yields provided in
Table 1. NMR spectra for all intemediates and ®nal
compounds were consistent with the proposed structures.
D1 dopaminergic binding assay. The binding assay for
D1 dopaminergic receptors was that described by Bill-
ard et al.12 Corpora striata were homogenized in 30 vol
(w/v) ice-cold 50 mM Tris±HCl bu€er (pH 7.7 at 25 ꢀC)
using a Polytron PT10 (setting 5 for 20 s). Homogenates
were centrifuged twice for 10 min at 50 000 g with
resuspension of the pellet in fresh bu€er. The ®nal pellet
was resuspended in 50 mM ice-cold Tris±HCl contain-
ing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM
MgCl2, 0.1% ascorbic acid and 10 mM pargyline (pH
7.1 at 37 ꢀC). Each assay tube contained 50 mL
[3H]SCH-23390 to achieve a ®nal concentration of 0.4
nM, and 900 mL resuspended membranes (3 mg fresh
tissue). The tubes were incubated for 15 min at 37 ꢀC
and the incubation was terminated by rapid ®ltration
under vacuum through Whatman GF/B glass ®ber ®l-
ters. The ®lters were washed three times with 5 mL ice-
cold 50 mM Tris±HCl bu€er (pH 7.7 at 25 ꢀC). The
radioactivity bound to the ®lters was measured by a
liquid scintillation counter. Speci®c [3H]SCH-23390
binding was de®ned as the di€erence between binding in
the absence or in the presence of 0.1 mM pi¯utixol.
Pharmacology
5-HT1A binding assay. Radioligand binding assay was
performed following a published procedure.10 Cerebral
cortex from male Sprague±Dawley rats (180±220 g) was
homogenized in 20 volumes of ice-cold Tris±HCl bu€er
(50 mM, pH 7.7 at 22 ꢀC) with a Brinkmann Polytron
(setting 5 for 15 s), and the homogenate was centrifuged
at 50,000 g for 10 min. The resulting pellet was then
resuspended in the same bu€er, incubated for 10 min at
37 ꢀC, and centrifuged at 50,000 g for 10 min. The ®nal
pellet was resuspended in 80 volumes of the Tris±HCl
bu€er containing 10 mM pargyline, 4 mM CaCl2, and
0.1% ascorbate. To each assay tube were added the fol-
lowing: 0.1 mL of the drug dilution (0.1 mL of distilled
water if no competing drug was added), 0.1 mL of [3H]-8-
hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-DPAT)
in bu€er (containing Tris, CaCl2, pargyline and ascor-
bate) to achieve a ®nal assay concentration of 0.1 nM,
and 0.8 mL of resuspended membranes. The tubes
were incubated for 30 min at 37 ꢀC, and the incubations
were terminated by vacuum ®ltration through Whatman
D2 dopaminergic binding assay. The procedure used in
the radioligand binding assay was reported in detail by
Creese et al.13 Corpora striata were homogenized in

538
G. Caliendo et al. / Bioorg. Med. Chem. 8 (2000) 533±538
30 vol (w/v) ice-cold 50 mM Tris±HCl bu€er (pH 7.7 at
25 ꢀC) using a Polytron PT10 (setting 5 for 20 s).
Homogenates were centrifuged twice for 10 min at
50,000 g with resuspension of the pellet in fresh bu€er.
The ®nal pellet was resuspended in 50 mM ice-cold
Tris±HCl containing 120 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 0.1% ascorbic acid and 10 mM
pargyline (pH 7.1 at 37 ꢀC). Each assay tube contained
50 mL [3H]spiroperidol to achieve a ®nal concentration
of 0.4 nM and 900 mL resuspended membranes (3 mg
fresh tissue). The tubes were incubated for 15 min at
37 ꢀC and the incubation was terminated by rapid ®l-
tration under vacuum through Whatman GF/B glass
®ber ®lters. The ®lters were washed three times with
5 mL ice-cold 50 mM Tris±HCl bu€er (pH 7.7 at 25 ꢀC).
The radioactivity bound to the ®lters was measured by a
liquid scintillation counter. Speci®c [3H]spiroperidol
binding was de®ned as the di€erence between binding in
the absence or in the presence of 1 mM (+)-butaclamol.
the incubation was terminated by rapid ®ltration under
vacuum through Whatman GF/B glass ®ber ®lters. The
®lters were washed three times with 5 mL ice-cold 50
mM Tris±HCl, 0.5 mM EDTA bu€er (pH 7.5 at 25 ꢀC).
The radioactivity bound to the ®lters was measured by a
liquid scintillation counter. Speci®c [3H]yohimbine
binding was de®ned as the di€erence between binding in
the absence or in the presence of 10 mM phentolamine.
Acknowledgements
This work was supported by a grant from Regione
Campania ai sensi della L. R. 31 dicembre 1994, No. 41,
art. 3, 1ꢀ comma. The NMR spectral data were pro-
vided by Centro di Ricerca Interdipartimentale di Ana-
lisi Strumentale, Universita degli Studi di Napoli
``Federico II''. The assistance of the sta€ is gratefully
appreciated.
ꢀ1-Adrenergic binding assay. The procedure used in the
radioligand binding assay has been reported in detail by
Greengrass and Bremner.14 Brain cortex was homo-
genized in 30 vol (w/v) ice-cold 50 mM Tris±HCl bu€er,
(pH 7.2 at 25 ꢀC) using a Polytron PT10 (setting 5 for
20 s). Homogenates were centrifuged twice for 10 min at
50,000 g with resuspension of the pellet in fresh bu€er.
The ®nal pellet was resuspended in 50 mM ice-cold
Tris±HCl, (pH 7.4 at 25 ꢀC). Each assay tube contained
50 mL drug solution, 50 mL [3H]prazosin to achieve a
®nal concentration of 0.4 nM, and 900 mL resuspended
membranes (10 mg fresh tissue). The tubes were incu-
bated for 30 min at 25 ꢀC and the incubation was ter-
minated by rapid ®ltration under vacuum through
Whatman GF/B glass ®ber ®lters. The ®lters were
washed three times with 5 mL ice-cold 50 mM Tris±
HCl, bu€er (pH 7.2 at 25 ꢀC). The radioactivity bound
to the ®lters was measured by a liquid scintillation
counter. Speci®c [3H]prazosin binding was de®ned as
the di€erence between binding in the absence or in the
presence of 10 mM phentolamine.
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for 20 s). Homogenates were centrifuged three times for
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fresh bu€er. The ®nal pellet was resuspended in 50 mM
ice-cold Tris±HCl, 0.5 mM EDTA (pH 7.5 at 25 ꢀC).
Each assay tube contained 50 mL drug solution, 50 mL
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