Efficient microwave combinatorial synthesis of novel indolic arylpiperazine derivatives as serotoninergic ligands

Article abstract of DOI:10.1016/j.ejmech.2009.11.023

An easy and convenient microwave-assisted synthesis of a small library of indolic arylpiperazine derivatives is described. Parallel and mixed pool combinatorial methods are reported and compared. The described reactions are nucleophilic substitutions of several aromatic piperazines in presence of K₂CO₃. Good yields and short reaction times are the main aspect of these procedures. Binding assays shed additional light on the influence of the LCAPs on the 5-HT_1A, 5-HT_2A and 5-HT_2C receptors affinity and allowed to disclose three interesting compounds as 5-HT_2C, mixed 5-HT_2A/5-HT_2C and 5-HT_1A/5-HT_2C ligands (4i, 4l and 4d, respectively), with potential antiepileptic, anxiolytic or atypical antipsychotic agent therapeutical profiles.

Full text of DOI:10.1016/j.ejmech.2009.11.023

European Journal of Medicinal Chemistry 45 (2010) 752–759
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Efficient microwave combinatorial synthesis of novel indolic arylpiperazine
derivatives as serotoninergic ligands
Francesco Frecentese a, Ferdinando Fiorino a, Elisa Perissutti a, Beatrice Severino a, Elisa Magli a,
Antonella Esposito a, Francesca De Angelis a, Paola Massarelli b, Cristina Nencini b, Barbara Viti b,
,
a
Vincenzo Santagada a , Giuseppe Caliendo
*
a
`
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita di Napoli «Federico II», Via D. Montesano, 49, 80131 Naples, Italy
Dipartimento di Farmacologia ‘‘G. Segre’’ Universita di Siena, Via delle Scotte, 6, 53100 Siena, Italy
b
`
a r t i c l e i n f o
a b s t r a c t
Article history:
An easy and convenient microwave-assisted synthesis of a small library of indolic arylpiperazine
derivatives is described. Parallel and mixed pool combinatorial methods are reported and compared. The
described reactions are nucleophilic substitutions of several aromatic piperazines in presence of K2CO3.
Good yields and short reaction times are the main aspect of these procedures. Binding assays shed
additional light on the influence of the LCAPs on the 5-HT1A, 5-HT2A and 5-HT2C receptors affinity and
allowed to disclose three interesting compounds as 5-HT2C, mixed 5-HT2A/5-HT2C and 5-HT1A/5-HT2C
ligands (4i, 4l and 4d, respectively), with potential antiepileptic, anxiolytic or atypical antipsychotic
agent therapeutical profiles.
Received 25 July 2009
Received in revised form
6 November 2009
Accepted 12 November 2009
Available online 1 December 2009
Keywords:
Arylpiperazines
Microwave irradiation
Combinatorial chemistry
Serotonin
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
and anxiety. Moreover, 5-HT2A receptor is known to play a key role
in the action of psychedelics as well as being a therapeutic target for
Over the last decade, microwave-assisted chemistry has become
an important tool that can accelerate drug discovery within
industry and academia. Shifting the focus from screening mixtures
to a single compound libraries, it proved to be a powerful technique
for medicinal chemists for either hit identification or lead refine-
ment [1]. Long-chain arylpiperazines (LCAPs) are a class of mole-
cules of considerable pharmaceutical interest. They bind to many
classes of G-protein-coupled receptors (serotoninergic, dopami-
nergic, adrenergic) and produce a variety of pharmacological
responses. In the past years our outmost attention was focused on
5-HT1A receptor [2], since its role in the pathology of such mental
disorders as anxiety or depression has been well-established [3]. In
particular, 5-HT1A receptor, belonging to the superfamily of
G-protein-coupled receptors and negatively coupled to adenylyl
cyclase, was found in high concentration in the limbic system,
where it is thought to play a role in emotional processes and
represents a major target for research and drug development due to
its implication in the pathophysiology and treatment of major
neuropsychiatric disorders, including depression, schizophrenia
the treatment of schizophrenia [4]; finally 5-HT2C receptor is
considered to be an attractive target for the design of novel drugs
for treatment of CNS-related diseases such as obesity, obsessive
compulsive disorder, and sexual dysfunction, even if few 5-HT2C
selective agonists are known so far [5].
Structure-activity relationship (SAR) studies, performed with
numerous generations of arylpiperazine derivatives, showed that
CNS activity and receptor affinity and selectivity depend on the
N-1-aryl substituent, the terminal fragment and the alkyl spacer
length. To get direct and easy access to this class of molecules, we
developed and optimized
a microwave-assisted synthesis of
a 12-members arylpiperazine small library characterized by an
indolic nucleus. The indole substructure is a basic element for
a number of biologically active natural and synthetic products. In
fact, until today, there have been more than 400 drugs and 3000
patents in which the indole motif has been present; the range of
applications for these therapeutically relevant compounds includes
anti-inflammatory drugs, protein kinase C inhibitors, 5-HT agonists
and antagonists, melatonin agonists and glucocorticoid receptor
modulators [6].
In order to explore its influence on the serotoninergic activity,
5-hydroxy-1H-indole-2-carboxylic acid has been linked to some of
* Corresponding author. Tel.: þ39 81 678648; fax: þ39 81 678649.
E-mail address: santagad@unina.it (V. Santagada).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.023

F. Frecentese et al. / European Journal of Medicinal Chemistry 45 (2010) 752–759
753
the most thoroughly studied aryl-substituted piperazines via
a three methylene spacing unit. In recent years our interest has
been devoted to the application of microwave in the syntheses of
pharmacologically active peptidomimetic [7] or heterocyclic
derivatives [8]. In this paper we describe the application of this
technology for the fast generation of a small library of indolic
arylpiperazines in parallel and mixed pool combinatorial methods.
The application of microwave energy to organic compounds in
order to perform synthetic reactions at highly accelerated rates has
become a well-known technique [9]. Microwave technology,
indeed, has become very popular among synthetic organic chem-
ists both to improve classical organic reactions, shortening reaction
times and/or improving yields, as well as to promote novel efficient
synthetic routes. The developed procedure, particularly, in the last
step, allows the obtaining of the complete arylpiperazines library
with a single reaction and purification step and this can facilitate
the quick screening of a greater number of synthetic analogues in
the identification of a lead structure.
decomposition of the reagents. The aryl-substituted piperazines
(a–l) have been selected in order to consider different steric,
hydrophobic and electronic features useful to develop a small
library of indolic arylpiperazine derivatives with different physi-
cochemical properties. The structures of the employed arylpiper-
azines are depicted in Fig. 1.
The main advantage of this synthetic route is that a short irra-
diation time of the reaction mixtures provided the compounds 4a–l
as the major products.
As starting point for a combinatorial mixed pool procedure,
a small amount of pure previously obtained compounds 4a–l was
mixed and several analytical RP-HPLC elution gradients were
evaluated to optimize a complete separation. The best condition
for analytical determination was found to be the following one,
carried out by two solvent systems: A: 0.05% TFA (v/v) in aceto-
nitrile; B: 0.05% TFA in H2O (linear gradient from 20% to 40% A
over 60 min, UV detection at 254 nm, flow rate 1 mL/min). The
obtained chromatographic profile showed sufficient resolution
between the compounds to undergo preparative purification
(Fig. 2).
2. Chemistry
On the basis of the obtained results, a mixed pool synthetic
procedure was developed as a one-pot reaction: the chloro-alkyl
ethyl ester 3 was reacted with the twelve arylpiperazines a–l in
acetonitrile in presence of K2CO3 and NaI, heating the mixture by
microwave irradiation with 300 W at 100 ꢀC, for a total event time
of 90 min. The reaction mixture was filtered to remove the potas-
sium carbonate, subjected to an extraction with brine and was
purified by preparative RP-HPLC applying the same gradient used
for the analytical determinations. The crude analytical HPLC chro-
matographic profile was similar to the previously obtained (Fig. 3)
with sufficient separations of the final compounds. High-reaction-
temperature conditions achieved by microwave irradiation in
acetonitrile allowed the synthesis to be carried out in one-pot to
obtain the desired compounds 4a–l.
The synthesis of compounds 4a–l is summarized in Scheme 1.
5-Hydroxy-1H-indole-2-carboxylic acid 1 was converted to the
corresponding ethyl ester 2 by treatment with thionyl chloride in
ethanol under reflux; the reaction with 1-bromo-3-chloropropane,
in presence of K2CO3 in acetonitrile, gave the corresponding
chloro-alkyl indolic derivative 3. These reactions were carried out
using a microwave oven (ETHOS 1600, MilestoneÒ) especially
designed for organic synthesis placing reagents and solvents in
a sealed reactor specific for high pressure reactions. The subse-
quent condensation of compound
3 with the opportune
substituted arylpiperazine, performed in acetonitrile in presence
of K2CO3 and NaI, was carried out in parallel using sealed tubes
fitted in the 36 positions of a multiPREP rotor (MilestoneÒ); the
remaining tubes were filled with the same amount of the reaction
solvent (acetonitrile). The synthetic procedure was performed
following a microwave program which was composed by appro-
priate ramping and holding steps. The temperature of the stirred
reaction mixture was monitored by an IR probe and rotation of the
rotor, irradiation time and power were monitored with the
‘‘easyWAVE’’ software package. The reactions provided the final
compounds 4a–l.
3. Results and discussion
Results relative to the synthesis of the compounds 4a–l> are
summarized in Table 1 and show that there is not a significant
difference between yields obtained in parallel or mixed pool
method; a small reduction in yields is evidenced in mixed pool
synthesis, but it can be addressed to the more complexity of the
crude mixture in which the piperazine reactivity plays a dominant
role.
On the basis of these results it’s possible to state that the
application of microwave irradiation and the simultaneous pres-
ence of K2CO3 and NaI improve the yields and significantly reduce
reaction times in the synthesis of arylpiperazine derivatives, either
in parallel or mixed pool combinatorial methods and this procedure
could be applied in future for the generation of larger libraries of
arilpiperazine derivatives to be evaluated for their biological
properties.
The piperazine coupling reaction was performed at 100 ꢀC with
300 W, in acetonitrile for a total time of 1 h. This condition was
found to be the optimized one because higher temperatures, times
or power gave no increase in the obtained yields or resulted in
HO
HO
OH
O
SOCl2
O
O
EtOH abs 80°C MW
N
N
H
H
2
1
The twelve piperazine derivatives (4a–l) were evaluated for
their activity and selectivity towards 5-HT1A, 5-HT2A and 5-HT2C
receptors and some of the molecules showed affinity in nanomolar
range towards these receptors (Table 2). Moreover, the multi-
receptor profiles of promising derivatives were also evaluated in
terms of binding affinities for dopaminergic (D1, D2) and adrenergic
R
Cl
O
O
O
HN
N
Cl
Br
N
H
K2CO3 / CH3CN
100°C MW
K2CO3 / NaI / CH3CN
100°C MW
3
(
a1
,
a2) receptors (Table 3).
R
The outstanding 5-HT1A receptor affinity of compound 4g
N
(Ki ¼ 35.8 nM), that showed a good nanomolar affinity on the
5-HT1A receptor in conjunction with a good selectivity on the
5-HT2A and 5-HT2C receptors, is of particular interest; compound
4i (Ki ¼ 50.4 nM) showed the most interesting selectivity profile
with a good affinity towards 5-HT2C receptor, whereas compound
N
O
O
N
O
4a-l
H
Scheme 1. Synthetic procedure for the synthesis of 4a–l.

754
F. Frecentese et al. / European Journal of Medicinal Chemistry 45 (2010) 752–759
N
NH
N
NH
N
NH
N
NH
Cl
Cl
CH3
CN
a
d
c
b
Cl
N
NH
Cl
N
NH
N
NH O2N
N
NH
g
h
e
f
Cl
F3C
O
N
NH
N
H3CO
N
NH
N
NH
O
O
NH
N
i
k
l
j
O
Fig. 1. Arylpiperazines used for the generation of the library.
4l presented a mixed 5-HT2A/5-HT2C affinity (Ki ¼ 26.1 and
43.6 nM, respectively) and 4d a mixed 5-HT1A/5-HT2C affinity
(Ki ¼ 39 and 1.28 nM, respectively). Successively, the affinity of
the most active compounds (4i, 4d and 4l) on several other
receptors (a1 and a2 adrenergic and D1 and D2 dopaminergic
receptors) was examined in order to verify their selectivity.
Results are summarized in Table 3. All the compounds proved
highly selective against dopaminergic receptors, with Ki values of
above 104 nM or no affinity except for compound 4d, which
exhibited an high Ki value of 1070 nM on the D1 receptor.
Regarding a1 and a2 receptors, only compound 4d showed quite
moderate affinity (32 nM) towards a1 receptors. These results
further support the choice of the indolic nucleus not only for the
preparation of serotoninergic ligands endowed with a 5-HT1A
4. Conclusion
Microwave combinatorial chemistry was adopted in the
synthesis of a new series of N-4-substituted piperazines linked to
an indolic nucleus via three methylene spacing unit and, even in
this case, has shown its potential as technological tool able to
improve on the reaction condition allowing the synthesis of the
final compounds 4a–l with high yields in both the combinatorial
approaches.
The binding data presented in this paper shed additional light
on the influence of the LCAPs on the 5-HT1A, 5-HT2A and 5-HT2C
receptors affinity and selectivity. In fact, the selected
N-4-substituted piperazines are not tolerated, even when indolic
nucleus has been used as terminal fragment, in order to obtain
derivatives with a good affinity/selectivity profile on 5-HT1A
receptor with the only exception represented by compound 4g
(Ki ¼ 35.8 nM), where a moderate nanomolar affinity on the 5-HT1A
affinity, but also with a mixed 5-HT2A/5-HT2C and 5-HT1A/5-HT2C
/
a1 activity, as well as compounds with an interesting 5-HT2C
affinity and selectivity.
The high differences in binding results observed between the
derivatives 4a–l, in accordance to the previously described series of
LCAPs derivatives [2b–2c], demonstrate that the heterocyclic frag-
ment as the terminal part of the LCAPs and the substituents on the
N-4 of the piperazine moiety represent critical structural features
in determining 5-HT1A, 5-HT2A and 5-HT2C receptors affinity and
selectivity.
The introduction of substituents characterized by different
steric, hydrophobic and electronic features on the N-4 position of
the piperazine moiety allowed us to evaluate the influence of the
nature and the position of these on the aromatic ring. Analysis of
the results suggests, in accordance to data already reported in
literature [10], that m-Cl and m-CF3 are favorable substituents in
order to obtain compounds with nanomolar affinity on the 5-HT1A
receptor.
In summary, ethyl 5-(3-(4-(4-methoxyphenyl)piperazin-1-
yl)propoxy)-1H-indole-2-carboxylate (4i, Ki ¼ 50.4 nM) was the
most active and selective derivative for the 5-HT2C receptor; this
result is particularly interesting even because of the few evidences
regarding the interaction of the LCAPs structures on the 5-HT2C
receptor. In fact, only some piperazine analogues were disclosed as
potent and selective 5-HT2C agonists, but unfortunately no sup-
porting in vivo data were reported [11]. Nanomolar interesting
affinity on the 5-HT2C receptor was showed by compounds 4c, 4e
and 4l too, but with lower affinity/selectivity profile. Additionally,
the mixed 5-HT2A/5-HT2C affinity (Ki values of 7.48/5.16 nM
respectively) and the mixed 5-HT1A/5-HT2C/a1 affinity (Ki values of
39/1.28/32.9 nM respectively) showed by compounds 4l and 4d are
of particular interest and outline a potential atypical antipsychotic
profile for these derivatives.
Fig. 2. – HPLC records of separation of compounds obtained by parallel method 4a–l.

F. Frecentese et al. / European Journal of Medicinal Chemistry 45 (2010) 752–759
755
Table 2
Affinities of compounds 4a-n for 5-HT1A, 5-HT2A and 5-HT2C receptors.
X
N
N
O
O
O
N
H
Substituents
X
Receptor affinity Ki ꢁ SD (nM)a
Compd
5-HT1A
5-HT2A
5-HT2C
[3H]8OH-DPAT [3H]Ketanserin [3H]Mesulergine
H3C
4a
>104
No affinity
No affinity
NC
Cl
4b
4c
>104
>104
No affinity
No affinity
537 ꢁ 11
39.7 ꢁ 5.2
Fig. 3. – HPLC records of the crude mixed pool reaction mixture.
Cl
receptor is associated to a concomitant selectivity on the 5-HT2A
and 5-HT2C receptors. Simultaneously, we have disclosed three
4d
4e
4f
39.0 ꢁ 0.8
14.0 ꢁ 0.9
>104
>104
1.28 ꢁ 0.1
45.1 ꢁ 1.7
>104
interesting compounds as 5-HT2C
, mixed 5-HT2A/5-HT2C and
5-HT1A/5-HT2C a1 ligands (4i, 4l and 4d respectively), with
/
a potential therapeutical profile as antiepileptic, anxiolytic or
atypical antipsychotic agents.
Cl
>104
Cl
5. Experimental
Cl
No affinity
5.1. Synthesis
5.1.1. General procedures
CF3
All synthetic reagents were purchased from Aldrich (Milan,
Italy) and were used without further purification. Solvents were
analytical reagent grade or higher purity and were used as supplied.
All reactions were performed, either in parallel or in mixed pool
procedure, using a microwave oven (ETHOS 1600, MilestoneÒ)
especially designed for organic synthesis. Thin layer chromatog-
raphy was performed on Macherey–Nagel DURASIL silica gel plates
4g
35.8 ꢁ 1.2
3610 ꢁ 192
No affinity
NO2
4h
4i
No affinity
>104
>104
247 ꢁ 47
OCH3
>104
50.4 ꢁ 2.4
Table 1
Indolic arylpiperazine derivatives by combinatorial microwave irradiation.
Entry
Parallel synthesis
Yielda(%)
Mixed pool synthesis
Yielda(%)
N
4j
No affinity
No affinity
>104
344 ꢁ 51
452 ꢁ 27
26.1 ꢁ 3.3
104 ꢁ 19
309 ꢁ 16
43.6 ꢁ 55
4a
4b
4c
4d
4e
4f
4g
4h
4i
51
49
59
62
56
48
53
51
51
78
71
83
48
47
55
58
56
45
49
50
50
73
69
79
O
4k
O
O
O
4l
4j
4k
4l
a
For purpose of comparison, 8-OH-DPAT, Ketanserine and Mesulergine bind
5-HT1A, 5-HT2A and 5-HT2C receptors with values of 0.80, 0.85 and 1.90 nM,
respectively, under these assay conditions.
a
All the reactions were performed 3 times and the reaction yields given are the
average values.

756
F. Frecentese et al. / European Journal of Medicinal Chemistry 45 (2010) 752–759
Table 3
d
8.86 (bs, NH), 7.31 (d, 1H, J ¼ 8.8 Hz), 7.13 (s, 1H), 7.09 (s, 1H), 6.99
Affinities of compounds 4i, 4l and 4d for D1, D2, a1 and a2 receptors.
(d, 1H, J ¼ 8.8 Hz), 4.40 (q, 2H, J ¼ 7.2 Hz), 4.15 (t, 2H, J ¼ 6 Hz), 3.78
(t, 2H, J ¼ 6 Hz), 2.26 (quint., 2H, J ¼ 6 Hz), 1.41 (t, 3H, J ¼ 7.2 Hz). 13C
Compd
Receptor affinity Ki ꢁ SD (nM)
NMR (CDCl3):
d 162.1, 154.0, 151.6, 131.2, 128.1, 117.4, 113.0, 108.4,
D1
D2
a1
a2
104.1, 65.1, 61.2, 41.9, 32.6, 14.6. ESI-MS: 282 (MHþ), 304 (M-Na),
320 (M-K). Anal. (C14H16ClNO3) C, H, N, O. m.p. 177–179 ꢀC.
[3H]SCH-23390
[3H]spiperone
[3H]prazosin
[3H]yohimbine
4d
4i
4l
1070 ꢁ 122
No affinity
>104
No affinity
No affinity
No affinity
32.9 ꢁ 1.69
514 ꢁ 9.2
1350 ꢁ 169
6480 ꢁ 533
231 ꢁ 14.8
5.1.4. General parallel synthetic procedure for compounds 4a–l
Using a multiPREP rotor (MilestoneÒ) with 36 reaction tubes,
ethyl 5-(3-chloropropoxy)-1H-indole-2-carboxylate 3 (1.78 mmol,
500 mg) and the appropriate substituted arylpiperazine
(1.78 mmol) were added to each reaction tube and were dissolved
in acetonitrile. K2CO3 (2.67 mmol, 615 mg) and NaI (2.67 mmol,
400 mg) were added and then the tubes were closed. The reactions
were heated in a Milestone microwave oven at 100 ꢀC using 300 W
power for 1 h. After 3 min of ventilation, the tubes were opened and
each reaction was filtered to remove the excess of K2CO3. Each
reaction mixture was then transferred into a separating funnel and
washed with brine. The organic phases were collected, dried over
anhydrous Na2SO4 and evaporated to dryness. Purification by silica
gel column chromatography afforded the pure compounds 4a–l.
338 ꢁ 14.6
with fluorescent indicator and the plates were visualized with UV
light (254 nm). Kieselgel 60 was used for column chromatography.
Melting points, determined using a Buchi Melting Point B-540
instrument, are uncorrected and represent values obtained on
recrystallized or chromatographically purified material. Elemental
analyses were carried out on a Carlo Erba model 1106; analyses
indicated by the symbols of the elements were within ꢁ0.4% of the
theoretical values. A Buchi R-114 rotavapor was utilized for the
removal of the solvents in vacuo. Analytical RP-HPLC determina-
tions were performed with a Phenomenex C18-column (5
mm,
4.6 ꢂ 150 mm, spherical). Preparative RP-HPLC purifications were
performed on a Waters Delta Prep 4000 system equipped with
a Waters 484 multi-wavelength detector on a Vydac C-18 column
5.1.5. General mixed pool synthetic procedure for compounds 4a–l
(15–20
m
m, 22 ꢂ 5000 mm) adopting the same gradient used for
Ethyl 5-(3-chloropropoxy)-1H-indole-2-carboxylate
3 (21.36
the analytical determinations. The operational flow rate was 30 mL/
min. Structures were verified spectroscopically by proton 1H NMR,
13C NMR and ESI-MS. NMR spectra were recorded on Varian
Mercury Plus 400 instrument. Chemical shifts are referred to Me4Si
as internal standard. Mass spectra of the final products were per-
formed on Applied Biosystem API 2000 triple-quadrupole mass
spectrometer.
mmol, g) and 12 commercially available arylpiperazines
6
(1.78 mmol each) were dissolved in a two neck flask in acetonitrile.
K2CO3 (32.04 mmol, 7.38 g) and NaI (32.04 mmol, 4.8 g) were added
and then the reaction was transferred in the microwave oven. The
reaction was heated and stirred for 90 min at 100 ꢀC using 300 W of
power. After 3 min of ventilation, the reaction was filtered to
remove the excess of K2CO3 and washed with brine. The crude
mixture was dried over anhydrous Na2SO4 and evaporated to
dryness. Purification by preparative RP-HPLC afforded the pure
compounds 4a–l.
5.1.2. Ethyl 5-hydroxy-1H-indole-2-carboxylate (2)
1.9 mL of SOCl2 were added dropwise to a solution of 5-hydroxy-
1H-indole-2-carboxylic acid 1 (16.9 mmol, 3 g) in 20 mL of anhy-
drous ethanol. A gentle reflux occurred spontaneously and was
maintained transferring the reaction in a Milestone microwave
oven at 80 ꢀC using 150 W power for 30 min. The mixture was then
allowed to cool to room temperature; the solvent was removed and
the crude mixture taken up in ethyl acetate was extracted with a 5%
NaHCO3 aqueous solution and with brine (twice each). The organic
phase was dried over anhydrous Na2SO4, filtered and evaporated to
dryness to afford 2.3 g of pure compound 2 as a white powder (68%
5.1.6. Ethyl 5-(3-(4-o-tolylpiperazin-1-yl)propoxy)-1H-indole-2-
carboxylate (4a)
1H NMR (400 MHz, CDCl3):
d
8.77 (bs, NH), 7.30 (d,1H, J ¼ 8.8 Hz),
7.25 (t,1H, J ¼ 7.8 Hz), 7.14–7.16 (m, 3H), 7.12 (s,1H), 7.09 (s,1H), 7.00
(d, 1H, J ¼ 8.8 Hz), 4.39 (q, 2H, J ¼ 7.2 Hz), 4.07 (t, 2H, J ¼ 6 Hz),
2.95–2.98 (m, 6H), 2.62–2.66 (m, 4H), 2.30 (s, 3H), 2.04 (quint., 2H,
J ¼ 6 Hz),1.39 (t, 3H, J ¼ 7.2 Hz).13C NMR (CDCl3):
d 160.9,153.3,147.3,
132.1, 129.9, 127.2, 126.7, 125.3, 119.2, 114.2, 111.7, 109.7, 108.4, 102.9,
66.8, 61.1, 55.5, 53.3, 48.8, 27.1, 15.8, 14.6. ESI-MS: 422 (MHþ), 444
(M-Na), 460 (M-K). Anal. (C25H31N3O3) C, H, N, O. m.p. 130–132 ꢀC.
yield). 1H NMR (400 MHz, CDCl3):
d
8.86 (bs, NH), 7.31 (d, 1H, J ¼ 8.8
Hz), 7.13 (s, 1H), 7.09 (s, 1H), 6.99 (d, 1H, J ¼ 8.8 Hz), 4.40 (q, 2H,
J ¼ 7.2 Hz), 1.41 (t, 3H, J ¼ 7.2 Hz). 13C NMR (CDCl3): 162.1, 154.0,
151.6, 131.2, 128.1, 117.4, 113.0, 108.4, 104.1, 61.2, 14.6. ESI-MS: 206
(MHþ), 228 (M ꢃ Na), 244 (M ꢃ K). Anal. (C11H11NO3) C, H, N, O. m.p.
146–148 ꢀC.
5.1.7. Ethyl 5-(3-(4-(2-cyanophenyl)piperazin-1-yl)propoxy)-1H-
indole-2-carboxylate (4b)
1H NMR (400 MHz, CDCl3):
d
8.76 (bs, NH), 7.55 (t,1H, J ¼ 7.8 Hz),
7.47 (d, 1H, J ¼ 7.8 Hz), 7.30 (d,1H, J ¼ 8.8 Hz), 7.27 (d, 1H, J ¼ 7.8 Hz),
7.23 (s, 1H), 7.12 (s, 1H), 7.08 (s, 1H), 7.00 (d, 1H, J ¼ 8.8 Hz), 4.39 (q,
2H, J ¼ 7.2 Hz), 4.07 (t, 2H, J ¼ 6 Hz), 3.23–3.25 (m, 6H), 2.63–2.71
(m, 4H), 2.03 (quint., 2H, J ¼ 6 Hz), 1.40 (t, 3H, J ¼ 7.2 Hz). 13C NMR
5.1.3. Ethyl 5-(3-chloropropoxy)-1H-indole-2-carboxylate (3)
In a two neck flask, K2CO3 (1.57 mmol, 216 mg) was suspended
in acetonitrile and heated by microwave under stirring at 70 ꢀC.
Ethyl 5-hydroxy-1H-indole-2-carboxylate 2 (1.57 mmol, 323 mg)
was then added followed, after 10 min, by 1-bromo-3-chlor-
opropane and the reaction mixture was further stirred at 70 ꢀC for
1 h. Afterwards the mixture was cooled to room temperature,
concentrated to dryness and the residue diluted in water (40 mL).
The solution was extracted several times with CH2Cl2. The
combined organic layers were dried on anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by column chro-
matography (dichloromethane/methyl alcohol 95:5 (v/v)). The
combined and evaporated product fractions were crystallized from
diethyl ether/hexane, affording 312 mg (71% yield) of the desired
compound 3 as a yellow pale solid. 1H NMR (400 MHz, CDCl3):
(CDCl3):
d 160.8, 153.1, 150.2, 134.0, 133.1, 132.3, 129.7, 125.4, 119.2,
116.0,115.3,111.7,109.7,108.4, 102.9, 96.5, 66.7, 61.0, 55.5, 53.3, 48.8,
27.0, 14.5. ESI-MS: 433 (MHþ), 455 (M-Na), 471 (M-K). Anal.
(C25H28N4O3) C, H, N, O. m.p. 172–173 ꢀC.
5.1.8. Ethyl 5-(3-(4-(2-chlorophenyl)piperazin-1-yl)propoxy)-1H-
indole-2-carboxylate (4c)
1H NMR (400 MHz, CDCl3):
d
8.82 (bs, NH), 7.35 (d, 1H, J ¼ 7.6
Hz), 7.30 (d, 1H, J ¼ 8.8 Hz), 7.26 (d, 1H, J ¼ 7.6 Hz), 7.20 (t, 1H,
J ¼ 7.6 Hz), 7.13 (s, 1H), 7.09 (s, 1H), 7.00 (d, 1H, J ¼ 8.8 Hz), 6.95 (t,
1H, J ¼ 7.6 Hz), 4.39 (q, 2H, J ¼ 7.2 Hz), 4.07 (t, 2H, J ¼ 6 Hz),
3.20–3.23 (m, 6H), 2.60–2.64 (m, 4H), 2.04 (quint., 2H, J ¼ 6 Hz),1.41

F. Frecentese et al. / European Journal of Medicinal Chemistry 45 (2010) 752–759
757
(t, 3H, J ¼ 7.2 Hz). 13C NMR (CDCl3):
d
162.1, 154.2, 151.4, 132.5, 129.8,
J ¼ 8.0 Hz), 6.84 (d, 2H, J ¼ 8.0 Hz), 4.39 (q, 2H, J ¼ 7.2 Hz), 4.07
(t, 2H, J ¼ 6 Hz), 3.77 (s, 3H, –OCH3), 3.10–3.12 (m, 6H), 2.62–2.66
(m, 4H), 2.04 (quint., 2H, J ¼ 6 Hz), 1.41 (t, 3H, J ¼ 7.2 Hz). 13C NMR
128.3, 127.8, 123.4, 119.7, 115.7, 112.9, 111.7, 109.7, 108.4, 103.9, 66.9,
61.2, 55.5, 53.3, 48.8, 27.1,14.6. ESI-MS: 442 (MHþ), 464 (M-Na), 480
(M-K). Anal. (C24H28ClN3O3) C, H, N, O. m.p. 133–134 ꢀC.
(CDCl3):
d 162.2, 154.1, 151.6, 132.7, 129.7, 128.2, 119.2, 117.3, 116.0,
115.3, 112.7, 108.4, 103.9, 66.9, 61.2, 57.6, 55.5, 53.3, 48.8, 27.1, 14.6.
ESI-MS: 438 (MHþ), 460 (M-Na), 476 (M-K). Anal. (C25H31N3O4) C,
H, N, O. m.p. 165–167 ꢀC.
5.1.9. Ethyl 5-(3-(4-(3-chlorophenyl)piperazin-1-yl)propoxy)-1H-
indole-2-carboxylate (4d)
1H NMR (400 MHz, CDCl3):
d
8.81 (bs, NH), 7.31 (d,1H, J ¼ 8.8 Hz),
7.16 (t,1H, J ¼ 8.0 Hz), 7.12 (s,1H), 7.08 (s,1H), 7.05 (s,1H), 7.00 (d,1H,
J ¼ 8.8 Hz), 6.79 (m, 2H), 4.39 (q, 2H, J ¼ 7.2 Hz), 4.07 (t, 2H, J ¼ 6 Hz),
3.20–3.23 (m, 6H), 2.60–2.64 (m, 4H), 2.04 (quint., 2H, J ¼ 6 Hz),1.41
5.1.15. Ethyl 5-(3-(4-(pyridin-2-yl)piperazin-1-yl)propoxy)-1H-
indole-2-carboxylate (4j)
1H NMR (400 MHz, CDCl3):
d
8.77 (bs, NH), 8.19 (d, 1H, J ¼ 8.00),
(t, 3H, J ¼ 7.2 Hz). 13C NMR (CDCl3):
d
162.1, 154.2, 151.4, 132.5, 129.9,
7.48 (t, 1H, J ¼ 8.00), 7.30 (d, 1H, J ¼ 8.8 Hz), 7.12 (s, 1H), 7.09 (s, 1H),
6.99 (d, 1H, J ¼ 8.8 Hz), 6.55 (d, 1H, J ¼ 8.0 Hz), 6.18 (d, 1H,
J ¼ 8.0 Hz), 4.39 (q, 2H, J ¼ 7.2 Hz), 4.07 (t, 2H, J ¼ 6 Hz), 3.53–3.57
(m, 6H), 2.62–2.66 (m, 4H), 2.06 (quint., 2H, J ¼ 6 Hz), 1.41 (t, 3H,
128.3, 128.1, 119.4, 117.5, 115.9, 112.9, 111.7, 109.7, 108.4, 103.9, 66.9,
61.2, 55.5, 53.3, 48.8, 27.1,14.6. ESI-MS: 442 (MHþ), 464 (M-Na), 480
(M-K). Anal. (C24H28ClN3O3) C, H, N, O. m.p. 140–142 ꢀC.
J ¼ 7.2 Hz). 13C NMR (CDCl3):
d 159.9, 154.2, 152.5, 148.2, 138.3,
5.1.10. Ethyl 5-(3-(4-(4-chlorophenyl)piperazin-1-yl)propoxy)-1H-
indole-2-carboxylate (4e)
132.0, 130.8, 125.2, 113.3, 111.7, 109.9, 109.7, 108.4, 102.1, 66.8, 60.9,
52.5, 50.5, 48.7, 27.9, 14.2. ESI-MS: 409 (MHþ), 431 (M-Na), 447 (M-
K). Anal. (C23H28N4O3) C, H, N, O. m.p. 155–157 ꢀC.
1H NMR (400 MHz, CDCl3):
d
8.80 (bs, NH), 7.30 (d, 1H, J ¼ 8.8
Hz), 7.20 (d, 2H, J ¼ 8.0 Hz), 7.13 (s, 1H), 7.09 (s, 1H), 7.00 (d, 1H,
J ¼ 8.8 Hz), 6.84 (d, 2H, J ¼ 8.0 Hz), 4.39 (q, 2H, J ¼ 7.2 Hz), 4.07
(t, 2H, J ¼ 6 Hz), 3.20–3.23 (m, 6H), 2.60–2.64 (m, 4H), 2.04 (quint.,
5.1.16. Ethyl 5-(3-(4-(2-furoyl)piperazin-1-yl)propoxy)-1H-indole-
2-carboxylate (4k)
2H, J ¼ 6 Hz), 1.41 (t, 3H, J ¼ 7.2 Hz). 13C NMR (CDCl3):
d
162.3, 154.1,
1H NMR (400 MHz, CDCl3):
d 9.00 (bs, NH), 7.41 (s, 1H), 7.30 (d,
151.6, 132.1, 128.5, 123.2, 119.5, 115.5, 112.7, 111.5, 110.0, 108.5, 103.9,
66.9, 61.2, 55.4, 53.3, 48.8, 27.2, 14.7. ESI-MS: 442 (MHþ), 464 (M-
Na), 480 (M-K). Anal. (C24H28ClN3O3) C, H, N, O. m.p. 128–130 ꢀC.
1H, J ¼ 8.8 Hz), 7.12 (s, 1H), 7.07 (s, 1H), 7.00 (d, 1H, J ¼ 8.8 Hz), 6.98
(t, 1H, J ¼ 8.0 Hz), 6.46 (s, 1H), 4.39 (q, 2H, J ¼ 7.2 Hz), 4.05 (t, 2H,
J ¼ 6 Hz), 3.82 (t, 2H, J ¼ 7.2 Hz), 2.53–2.58 (m, 8H), 2.00 (quint., 2H,
J ¼ 6 Hz), 1.40 (t, 3H, J ¼ 7.2 Hz). 13C NMR (CDCl3):
d 159.3, 154.1,
5.1.11. Ethy-5-(3-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)
152.4, 147.1, 146.0, 132.4, 128.0, 117.4, 116.6, 113.0, 111.5, 108.4, 103.8,
66.7, 61.2, 55.4, 53.6, 50.4, 47.9, 27.0, 14.7. ESI-MS: 426 (MHþ), 448
(M-Na), 464 (M-K). Anal. (C23H27N3O5) C, H, N, O. m.p. 129–131 ꢀC.
propoxy)-1H-indole-2-carboxylate (4f)
1H NMR (400 MHz, CDCl3):
d
8.95 (bs, NH), 7.36 (t, 1H, J ¼ 8.0
Hz), 7.31 (d, 1H, J ¼ 8.8 Hz), 7.12 (m, 2H), 7.07 (m, 2H), 7.05 (s, 1H),
6.97 (d, 1H, J ¼ 8.8 Hz), 4.39 (q, 2H, J ¼ 7.2 Hz), 4.08 (t, 2H, J ¼ 6 Hz),
3.36–3.39 (m, 6H), 2.89–2.93 (m, 4H), 2.15 (quint., 2H, J ¼ 6 Hz), 1.41
(t, 3H, J ¼ 7.2 Hz). 13C NMR (CDCl3): 162.1, 153.9, 151.5, 132.5, 131.9,
129.9, 128.3, 128.1, 124.2, 119.3, 117.3, 116.9, 113.0, 110.1, 108.4, 104.0,
66.4, 61.2, 55.4, 52.8, 48.1, 26.0, 14.6. ESI-MS: 476 (MHþ), 498 (M-
Na), 514 (M-K). Anal. (C25H28F3N3O3) C, H, N, O. m.p. 131–132 ꢀC.
5.1.17. Ethyl 5-(3-(4-((benzo[d][1,3]dioxol-5-yl)methyl)piperazin-
1-yl)propoxy)-1H-indole-2-carboxylate (4l)
1H NMR (400 MHz, CDCl3):
d
8.76 (bs, NH), 7.30 (d, 1H, J ¼ 8.8
Hz), 7.12 (s, 1H), 7.06 (s,1H), 6.97 (d,1H, J ¼ 8.8 Hz), 6.85 (s,1H), 6.84
(d, 2H, J ¼ 8.0 Hz), 6.74 (s, 1H), 5.93 (s, 2H), 4.39 (q, 2H, J ¼ 7.2 Hz),
4.04 (t, 2H, J ¼ 6 Hz), 3.45 (s, 2H), 2.57–2.63 (m, 6H), 2.05 (quint.,
2H, J ¼ 6 Hz), 1.40 (t, 3H, J ¼ 7.2 Hz). 13C NMR (CDCl3):
d 159.9, 154.1,
5.1.12. Ethyl 5-(3-(4-(3,4-dichlorophenyl)piperazin-1-yl)propoxy)-
1H-indole-2-carboxylate (4g)
148.5, 147.3, 138.3, 132.4, 131.6, 128.1, 117.5, 112.9, 109.8, 108.4, 108.1,
103.8, 101.2, 66.8, 62.8, 61.2, 55.4, 53.2, 52.6, 26.7, 14.7. ESI-MS: 466
(MHþ), 488 (M-Na), 504 (M-K). Anal. (C25H29N3O5) C, H, N, O. m.p.
106–108 ꢀC.
1H NMR (400 MHz, CDCl3):
d
8.77 (bs, NH), 7.30 (d, 1H, J ¼ 8.8
Hz), 7.12 (s, 1H), 7.09 (s, 1H), 7.00 (d, 1H, J ¼ 8.8 Hz), 6.95 (s, 1H), 6.75
(d, 1H, J ¼ 8.0 Hz), 6.73 (d, 1H, J ¼ 8.0 Hz), 4.38 (q, 2H, J ¼ 7.2 Hz),
4.07 (t, 2H, J ¼ 6 Hz), 3.18–3.20 (m, 6H), 2.62–2.66 (m, 4H), 2.04
(quint., 2H, J ¼ 6 Hz), 1.41 (t, 3H, J ¼ 7.2 Hz). 13C NMR (CDCl3):
5.2. Pharmacology
d
161.2, 152.1, 151.6, 134.3, 132.7, 129.7, 128.2, 125.3, 122.9, 116.1,
5.2.1. General procedures
115.3, 112.7, 109.7, 108.4, 103.9, 66.7, 61.1, 55.5, 53.3, 48.8, 27.1, 14.6.
ESI-MS: 477 (MHþ), 499 (M-Na), 515 (M-K). Anal. (C24H27Cl2N3O3)
C, H, N, O. m.p. 154–156 ꢀC.
The newly synthesized compounds were tested for in vitro
affinity for serotonin 5-HT1A, 5-HT2A and 5-HT2C receptors by radi-
oligand binding assays. All the compounds were dissolved in ethanol
or in 5% DMSO. The following specific radioligands and tissue
sources were used: (a) serotonin 5-HT1A receptor, [3H]-8-OH-DPAT,
rat brain cortex; (b) serotonin 5-HT2A receptor, [3H]ketanserin, rat
brain cortex; (c) serotonin 5-HT2C receptor, [3H]mesulergine, rat
brain cortex; (d) dopamine D1 receptor [3H]SCH-23390, rat stria-
tum; (e) dopamine D2 receptor [3H]spiperone, rat striatum; (f)
a1adrenergic receptor [3H]prazosin, rat brain cortex; (g) a2 adren-
ergic receptor [3H]yohimbine, rat brain cortex.
Non-specific binding was determined as described in the
experimental section, and specific binding as the difference
between total and non-specific binding. Blank experiments were
carried out to determine the effect of 5% DMSO on the binding and
no effects were observed. Competition experiments were analyzed
by the ‘‘Easy Fit’’ program [12] to obtain the concentration of
unlabeled drug that caused 50% inhibition of ligand binding (IC50),
with six concentrations of test compounds, each performed in
5.1.13. Ethyl 5-(3-(4-(4-nitrophenyl)piperazin-1-yl)propoxy)-1H-
indole-2-carboxylate (4h)
1H NMR (400 MHz, CDCl3):
d
8.79 (bs, NH), 8.12 (d, 2H, J ¼ 7.6
Hz), 7.31 (d, 1H, J ¼ 8.8 Hz), 7.12 (s, 1H), 7.09 (s, 1H), 6.99 (d, 1H,
J ¼ 8.8 Hz), 6.83 (d, 2H, J ¼ 7.6 Hz), 4.39 (q, 2H, J ¼ 7.2 Hz), 4.08 (t,
2H, J ¼ 6 Hz), 3.42–3.45 (m, 6H), 2.62–2.66 (m, 4H), 2.04 (quint., 2H,
J ¼ 6 Hz), 1.41 (t, 3H, J ¼ 7.2 Hz). 13C NMR (CDCl3):
d 159.9, 155.7,
152.4,137.9,132.0,130.8,125.2,122.0,115.2,111.7,109.7,108.5,102.2,
66.6, 60.9, 52.6, 50.4, 48.8, 27.8, 14.1. MS: 453 (MHþ), 475 (M-Na),
491 (M-K). Anal. (C24H28N4O5) C, H, N, O. m.p. 183–185 ꢀC.
5.1.14. Ethyl 5-(3-(4-(4-methoxyphenyl)piperazin-1-yl)propoxy)-
1H-indole-2-carboxylate (4i)
1H NMR (400 MHz, CDCl3):
Hz), 7.12 (s, 1H), 7.09 (s, 1H), 7.00 (d, 1H, J ¼ 8.8 Hz), 6.91 (d, 2H,
d
8.81 (bs, NH), 7.30 (d, 1H, J ¼ 8.8

758
F. Frecentese et al. / European Journal of Medicinal Chemistry 45 (2010) 752–759
triplicate. The IC50 values obtained were used to calculate apparent
inhibition constants (Ki) by the method of Cheng and Prussoff [13],
from the following equation: Ki ¼ IC50/(1 þ S/KD) where S repre-
sents the concentration of the hot ligand used and KD its receptor
dissociation constant (KD values, obtained by Scatchard analysis
[14], were calculated for each labeled ligand).
(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 buffer. The final 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
pargyline (pH 7.1 at 37 ꢀC). Each assay tube contained 50
mM
mL
[3H]SCH-23390 (85.0 Ci/mmol, Perkin Elmer Life Sciences, Boston,
5.2.2. 5-HT1A binding assay
MA, USA) to achieve a final concentration of 0.4 nM, and 900 mL
Radioligand binding assays were performed following a pub-
lished procedure [15]. Cerebral cortex from male Sprague–Dawley
rats (180–220 g) was homogenized in 20 volumes of ice-cold Tris–
HCl buffer (50 mM, pH 7.7 at 22 ꢀC) with a Polytron PT10,
Brinkmann Instruments (setting 5 for 15 s), and the homogenate
was centrifuged at 50,000 g for 10 min at 0 ꢀC. The resulting pellet
was then resuspended in the same buffer, incubated for 10 min at
37 ꢀC, and centrifuged at 50,000 g for 10 min. The final pellet was
resuspended membranes (3 mg fresh tissue). The tubes were
incubated for 15 min at 37 ꢀC and the incubation was terminated by
rapid filtration under vacuum through Whatman GF/B glass fibre
filters. The filters were washed three times with 5 mL ice-cold
50 mM Tris–HCl buffer (pH 7.7 at 25 ꢀC). The radioactivity bound to
the filters was measured by a liquid scintillation counter. Specific
[3H]SCH-23390 binding was defined as the difference between
binding in the absence or in the presence of 0.1 mM piflutixol.
resuspended in 80 volumes of the Tris–HCl buffer containing 10 mM
pargyline, 4 mM CaCl2, and 0.1% ascorbate. To each assay tube was
added the following: 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) (170.0 Ci/mmol,
Perkin Elmer Life Sciences, Boston, MA, USA) in the same buffer as
above to achieve a final 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 filtration
through Whatman GF/B filters (Brandel Biomedical Research and
Laboratories Inc., Gaithersburg, MD, USA). The filters were washed
twice with 5 mL of ice-cold Tris–HCl buffer, and the radioactivity
bound to the filters was measured by liquid scintillation spec-
trometer (Packard TRI-CARBÒ 2000CA - Packard BioScience s.r.l.,
Pero, Milan, Italy). Specific [3H]-8-OH-DPAT binding was defined as
the difference between binding in the absence and presence of 5-
5.2.5. D2 dopaminergic binding assay
The procedure used in the radioligand binding assay was
reported in detail by Creese et al. (Creese et al., 1977) [18] Corpora
striata were homogenized in 30 vol. (w/v) ice-cold 50 mM Tris–HCl
buffer (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 buffer. The final 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
pargyline (pH 7.1 at 37 ꢀC). Each assay tube contained 50
mM
mL
[3H]spiperone (15.7 Ci/mmol, Perkin Elmer Life Sciences, Boston,
MA, USA) to achieve a final 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 filtration under vacuum through Whatman GF/B glass fibre
filters. The filters were washed three times with 5 mL ice-cold
50 mM Tris–HCl buffer (pH 7.7 at 25 ꢀC). The radioactivity bound to
the filters was measured by a liquid scintillation counter. Specific
[3H]spiperone binding was defined as the difference between
HT (10 mM).
5.2.3. 5-HT2A and 5-HT2C binding assays
Radioligand binding assays were performed as previously
reported by Herndon et al. [16]. Briefly, frontal cortical regions of
male Sprague–Dawley rats (180–220 g) were dissected on ice and
homogenized (1:10 w/v) in ice-cold buffer solution (50 mM Tris
HCl, 0.5 mM EDTA, and 10 mM MgCl2 at pH 7.4) with a Polytron
PT10 (setting 5 for 15 s) and centrifuged at 3000 g for 15 min. The
pellet was resuspended in buffer (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 centrifugations). The final pellet was
resuspended in buffer that also contained 0.1% ascorbate and
10ꢃ5 M pargyline.
Assays were performed in triplicate in a 2.0 mL volume con-
taining 5 mg wet weight of tissue and 0.4 nM [3H]ketanserin
hydrochloride (88.0 Ci/mmol; Perkin Elmer Life Sciences, Boston,
MA, USA) for 5-HT2A receptor assays, and 10 mg wet weight of
tissue and 1 nM [3H]mesulergine (87.0 Ci/mmol; Amersham
Biosciences Europe GmbH) for 5-HT2C receptor assays. Cinanserin
binding in the absence or in the presence of 1
m
M (þ)-butaclamol.
5.2.6. a1 Adrenergic binding assay
The procedure used in the radioligand binding assay has been
reported in detail by Greengrass and Bremner [19]. Brain cortex was
homogenized in 30 vol. (w/v) ice-cold 50 mM Tris–HCl buffer,
(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 buffer. The final pellet was
resuspended in 50 mM ice-cold Tris–HCl, (pH 7.4 at 25 ꢀC). Each
assay tube contained 50
(80.5 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA, USA) to
achieve a final concentration of 0.4 nM, and 900 L resuspended
mL drug solution, 50 m
L [3H]prazosin
m
membranes (10 mg fresh tissue). The tubes were incubated for
30 min at 25 ꢀC and the incubation was terminated by rapid
filtration under vacuum through Whatman GF/B glass fibre filters.
The filters were washed three times with 5 mL ice-cold 50 mM
Tris–HCl, buffer (pH 7.2 at 25 ꢀC). The radioactivity bound to the
filters was measured by a liquid scintillation counter. Specific
[3H]prazosin binding was defined as the difference between
(1.0
mM) was used to define non-specific binding in the 5-HT2A
assay. In the 5-HT2C assays, mianserin (1.0
mM) was used to define
non-specific binding, and 100 nM spiperone was added to all tubes
to block binding to 5-HT2A receptors. Tubes were incubated for
15 min at 37 ꢀC, filtered on Schliecher and Schuell (Keene, NH, USA)
glass fibre filters presoaked in polyethylene imine, and washed
with 10 mL of ice-cold buffer. Filters were counted at an efficiency
of 50%.
binding in the absence or in the presence of 10 mM phentolamine.
5.2.7. a2 Adrenergic binding assay
The procedure used in the radioligand binding assay was
reported in detail by Perry and U’Prichard [20]. Brain cortex was
homogenized in 30 vol. (w/v) ice-cold 5 mM Tris–HCl, 5 mM EDTA
buffer (pH 7.3 at 25 ꢀC) using a Polytron PT10 (setting 5 for 20 s).
Homogenates were centrifuged three times for 10 min at 50,000 g
with resuspension of the pellet in fresh buffer. The final pellet was
5.2.4. D1 dopaminergic binding assay
The binding assay for D1 dopaminergic receptors was that
described by Billard et al. (Billard et al., 1985) [17] Corpora striata
were homogenized in 30 vol. (w/v) ice-cold 50 mM Tris–HCl buffer

F. Frecentese et al. / European Journal of Medicinal Chemistry 45 (2010) 752–759
759
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resuspended in 50 mM ice-cold Tris–HCl, 0.5 mM EDTA (pH 7.5 at
25 ꢀC). Each assay tube contained 50
L drug solution, 50
[3H]yohimbine (80.5 Ci/mmol, Perkin Elmer Life Sciences, Boston,
MA, USA) to achieve a final concentration of 1 nM, and 900
resuspended membranes (10 mg fresh tissue). The tubes were
incubated for 30 min at 25 ꢀC and the incubation was terminated by
rapid filtration under vacuum through Whatman GF/B glass fibre
filters. The filters were washed three times with 5 mL ice-cold
50 mM Tris–HCl, 0.5 mM EDTA buffer (pH 7.5 at 25 ꢀC). The
radioactivity bound to the filters was measured by a liquid scintil-
lation counter. Specific [3H]yohimbine binding was defined as the
difference between binding in the absence or in the presence of
m
mL
mL
10 mM phentolamine.
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The NMR spectral data were provided by Centro di Ricerca
`
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