Synthesis and structure-activity relationships of a new model of arylpiperazines. 8. Computational simulation of ligand-receptor interaction of 5-HT1AR agonists with selectivity over α1- adrenoceptors

Article abstract of DOI:10.1021/jm048999e

We have designed and synthesized a new series of arylpiperazines V exhibiting high 5-HT_1AR affinity and selectivity over α₁-adrenoceptors. The new selective 5-HT_1AR ligands contain a hydantoin (m = 0) or diketopiperazine (m = 1) moiety and an arylpiperazine moiety separated by one methylene unit (n = 1). The aryl substituent of the piperazine moiety (Ar) consists of different benzofused rings mimicking the favorable voluminous substituents at ortho and meta positions predicted by 3D-QSAR analysis in the previously reported series I. In particular, (S)-2-[[4-(naphth-1-yl)piperazin-1-yl]methyl]-1,4- dioxoperhydropyrrolo[1,2-a]pyrazine [(S)-9, CSP-2503] (5-HT_1A, K _i = 4.1 nM; α₁, K_i > 1000 nM) has been pharmacologically characterized as a 5-HT_1AR agonist at somatodendritic and postsynaptic sites, endowed with anxiolytic properties. Ligand (S)-9 is predicted, in computer simulations, to bind Asp^3.32 in TMH 3, Thr^5.39 and Ser^5.42 in TMH 5, and Trp ^6.48 in TMH 6. We propose that agonists modify, by means of an explicit hydrogen bond, the conformation of Trp^6.48 from pointing toward TMH 7, in the inactive gauche+ conformation, to pointing toward the ligand binding site, in the active trans conformation.

Full text of DOI:10.1021/jm048999e

2548
J. Med. Chem. 2005, 48, 2548-2558
Synthesis and Structure-Activity Relationships of a New Model of
Arylpiperazines. 8.1 Computational Simulation of Ligand-Receptor Interaction
of 5-HT1AR Agonists with Selectivity over r1-Adrenoceptors
Mar´ıa L. Lo´pez-Rodr´ıguez,*,§ Maria Jose´ Morcillo,‡ Esther Ferna´ndez,§ Bellinda Benhamu´,§ Ignacio Tejada,§
David Ayala,§ Alma Viso,§ Mercedes Campillo,# Leonardo Pardo,# Mercedes Delgado,| Jorge Manzanares,| and
Jose´ A. Fuentes|
Departamento de Quı´mica Orga´nica I, Facultad de Ciencias Qu´ımicas, Universidad Complutense, E-28040 Madrid, Spain,
Facultad de Ciencias, Universidad Nacional de Educacio´n a Distancia, E-28040 Madrid, Spain, Laboratori de Medicina
Computacional, Unitat de Bioestadı´stica and Institut de Neurocie`ncies, Universitat Auto`noma de Barcelona,
E-08193 Cerdanyola del Valle`s, Barcelona, Spain, and Unidad de Cartograf´ıa Cerebral, Instituto Pluridisciplinar,
Universidad Complutense, E-28040 Madrid, Spain
Received December 9, 2004
We have designed and synthesized a new series of arylpiperazines V exhibiting high 5-HT1AR
affinity and selectivity over R1-adrenoceptors. The new selective 5-HT1AR ligands contain a
hydantoin (m ) 0) or diketopiperazine (m ) 1) moiety and an arylpiperazine moiety separated
by one methylene unit (n ) 1). The aryl substituent of the piperazine moiety (Ar) consists of
different benzofused rings mimicking the favorable voluminous substituents at ortho and meta
positions predicted by 3D-QSAR analysis in the previously reported series I. In particular,
(S)-2-[[4-(naphth-1-yl)piperazin-1-yl]methyl]-1,4-dioxoperhydropyrrolo[1,2-a]pyrazine [(S)-9,
CSP-2503] (5-HT1A, Ki ) 4.1 nM; R1, Ki > 1000 nM) has been pharmacologically characterized
as a 5-HT1AR agonist at somatodendritic and postsynaptic sites, endowed with anxiolytic
properties. Ligand (S)-9 is predicted, in computer simulations, to bind Asp3.32 in TMH 3, Thr5.39
and Ser5.42 in TMH 5, and Trp6.48 in TMH 6. We propose that agonists modify, by means of an
explicit hydrogen bond, the conformation of Trp6.48 from pointing toward TMH 7, in the inactive
gauche+ conformation, to pointing toward the ligand binding site, in the active trans
conformation.
Introduction
sequence, many 5-HT1AR ligands are poorly selective
over R1-adrenergic receptors. In this context, some years
ago our group participated in a wide research program
aimed at developing new high-affinity 5-HT1AR ligands
with selectivity over R1-adrenoceptors.1,17-26 Using as
a starting point a series of arylpiperazines I (n ) 3, 4)
(Chart 1) showing affinity at both receptors,17,18 we have
carried out a systematic study based on the nonpharma-
cophoric and pharmacophoric sites of both receptors to
gain insight into the structural factors that are respon-
sible for 5-HT1A/R1 selectivity.
With respect to the pharmacophore part (arylpiper-
azine), a training set of 32 compounds of general
structure I was used to derive classical QSAR and
neural networks models for both 5-HT1A and R1-adren-
ergic receptors.24 These models provide a significant
correlation of electrostatic, steric, and lipophilic param-
eters with biological affinities and led us to design and
synthesize a new ligand, EF-7412 [X ) -(CH2)3-,
Serotonin (5-hydroxytryptamine, 5-HT) represents a
major target for neurobiological research because of its
involvement in numerous (patho)physiological proc-
esses.2-5 The development of drugs that alter 5-HT
neurotransmission is thus an area of intense research
because of the potential to find new therapeutic agents.
Molecular cloning, pharmacological properties, second
messenger coupling, and amino acid sequence have led
to the identification of 14 serotonin receptor subtypes
that can be classified into seven subfamilies (5-HT1-7).6,7
The 5-HT1A subtype is one of the best studied, its
agonists and partial agonists being effective in the
treatment of anxiety and depression.8-10 Moreover,
recent preclinical studies have suggested that 5-HT1AR
agonists also have neuroprotective properties.11-13
All serotonin receptors, except the 5-HT3 subtype,
belong to the seven transmembrane G-protein-coupled
receptor superfamily (GPCR).14 The GPCR family pos-
sesses highly conserved motifs in the transmembrane
region, which suggests a common transmembrane heli-
cal bundle.15 In particular, the transmembrane amino
acid sequence of the 5-HT1AR presents a high degree of
homology with the R1-adrenergic receptor.16 As a con-
m ) 0, n ) 4, Ar ) m-(ethylsulfonamido)phenyl; 5-HT1A
,
Ki ) 27 nM; R1, Ki > 1000 nM], with a high selectivity
profile for the 5-HT1AR sites.
Regarding the nonpharmacophoric part, the influence
of electronic, steric, and lipophilic factors on the stabi-
lization of receptor-ligand complexes has been inves-
tigated in different series of derivatives II-IV (Chart
1). SAR studies of series II, focusing on the terminal
amide fragment, have revealed a significant influence
of electronic factors on the R1-adrenergic receptor site
without modifying the stabilization of the 5-HT1AR-
* To whom correspondence should be addressed. Phone:
34-91-3944239. Fax: 34-91-3944103. E-mail: mluzlr@quim.ucm.es.
§ Departamento de Qu´ımica Orga´nica I, Universidad Complutense.
‡ Universidad Nacional de Educacio´n a Distancia.
# Universitat Auto`noma de Barcelona.
| Instituto Pluridisciplinar, Universidad Complutense.
10.1021/jm048999e CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/12/2005

Selective Binding of New 5-HT1A Agonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2549
Chart 1. Arylpiperazines of General Structures I-V
Scheme 1a
a Reagents and conditions: (a) Br(CH2)4Br, NaH, DMF, 110 °C,
1-3 h; (b) NEt3, CH3CN, 60 °C, 20 h; (c) (i) HCHO(aq), EtOH,
reflux, 6 h; (ii) for 16, HCl(g), Et2O, room temp, 20 min.
groups of epinephrine with the cluster of Ser/Thr play
a critical role in the proper positioning of TMH 5 in the
active state of the receptor.28 In addition, it has recently
been observed in the structure of metarhodopsin I,
determined by electron crystallography,29 that Trp6.48
undergoes a conformational transition, in the process
of receptor activation, from pointing toward TMH 7, in
the inactive gauche+ conformation, to pointing toward
TMH 5, in the active trans conformation as was previ-
ously suggested by computer simulations.30 The fact
that these residues are also present in the serotoninergic
receptors led us to suggest that the interaction of
agonists of the 5-HT1AR with Asp3.32, Ser5.42, Thr5.43, and
Trp6.48 is probably the key determinant for agonist-
induced receptor activation.
In the present work, we have designed and synthe-
sized a new series of arylpiperazines of general structure
V exhibiting high 5-HT1AR affinity and selectivity over
R1-adrenoceptors. The new selective 5-HT1AR ligands
contain a hydantoin (m ) 0) or diketopiperazine
(m ) 1) moiety in the nonpharmacophoric part, one
methylene unit in the spacer to the pharmacophoric
arylpiperazine (n ) 1), and different benzofused rings
as the aryl substituent of the piperazine (Ar). The
influence of the stereogenic center present in the non-
pharmacophoric part of these derivatives as well as of
a substituent in the piperazine ring on 5-HT1AR affinity
is also discussed. Among these compounds, we
report the pharmacological characterization of (S)-9
(CSP-2503) as a potent 5-HT1AR agonist.
ligand complex.20 Analysis of series III, aiming to study
the steric requirements of the terminal amide portion,
led us to conclude that the nonpharmacophoric pocket
in the 5-HT1AR has less restriction than the correspond-
ing pocket in the R1-adrenergic receptor, suggesting
some differences between the nonpharmacophoric sites
of both 5-HT1A and R1-adrenergic receptors.21 Finally,
the influence of lipophilic factors at the amide fragment
of a new series IV has been studied. Variations of log
P have been carried out by introducing different hydro-
carbonated substituents (R1) at the 7a position of the
bicyclohydantoin. Lipophilicity of the ligands does not
exert a crucial influence on the 5-HT1AR affinity or on
the selectivity over R1-adrenergic receptors.1
We have recently constructed a computer model of the
complexes between arylpiperazines of formula
I
[X ) -(CH2)3-, m ) 0, n ) 4, Ar ) m-(ethylsulfonami-
do)phenyl (EF-7412); X ) -(CH2)3-, m ) 0, n ) 4,
Ar ) m-(acetylamino)phenyl] and a rhodopsin-based 3D
model of the 5-HT1AR transmembrane domain.24,25
These models have permitted us to identify the molec-
ular determinants of the ligand-receptor interaction
that include the ionic interaction between the proto-
nated amine of the ligand and Asp3.32, the hydrogen
bonds between the m-NHSO2Et or m-NHCOCH3 groups
and Asn7.39, and the hydrogen bonds between the
hydantoin moiety and Ser5.42 and Thr5.43. Recently, a
kinetic analysis of the agonist-induced conformational
changes in the â2-adrenergic receptor has shown that
the interaction of the ligand with Asp3.32 and the
Ser/Thr residues in transmembrane helix (TMH) 5 is a
fast component (t1/2 < 5 s) in the process of receptor
activation.27 These interactions of the catechol hydroxyl
Results
Chemistry. The synthesis of new compounds of
general structure V has been performed by using two
different strategies, depending on the length of the
spacer chain (n ) 4 or n ) 1; see Scheme 1). Compounds
1-6 (n ) 4) have been obtained by alkylation of
arylpiperazines 22-25 with 4-bromobutyl derivatives

2550 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Table 1. Binding Data of Compounds Va
Lo´pez-Rodrı´guez et al.
compd
configuration
X
m
n
R
Ar
Ki ( SEM (5-HT1A
)
Ki ( SEM (R1)
1
(7aRS)
(8aRS)
(2R,8aRS)
(2S,8aRS)
(8aRS)
(7aRS)
(7aRS)
(8aRS)
(8aRS)
(8aR)
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)4
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)3
(CH2)4
(CH2)4
(CH2)3
(CH2)3
(CH2)3
0
1
1
1
1
0
0
0
1
1
1
1
1
1
1
0
0
1
1
0
4
4
4
4
4
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
H
H
naphth-1-yl
2.4 ( 0.1
0.5 ( 0.2
4.2 ( 0.9
15.3 ( 1.8
3.1 ( 0.9
1.2 ( 0.02
10.4 ( 0.8
5.6 ( 0.3
6.7 ( 3.0
4.6 ( 0.4
4.1 ( 1.2
9.5 ( 2.2
2.5 ( 0.1
6.3 ( 0.2
6.4 ( 0.1
9.3 ( 0.4
6.1 ( 0.4
11.6 ( 3.7
12.3 ( 2.1
4.1 ( 0.2
64.9 ( 1.5
8.0 ( 1.7
20.1 ( 0.8
34.4 ( 1.2
348 ( 21
19.4 ( 0.6
>1000
2
naphth-1-yl
3
R-Me
S-Me
H
naphth-1-yl
naphth-1-yl
4
5
benzodioxepin-6-yl
benzimidazol-4-yl
naphth-1-yl
6
H
7
H
8
H
naphth-1-yl
>1000
9
H
naphth-1-yl
>1000
(R)-9
(S)-9
(R,R)-10
(S,S)-10
(R,S)-11
(S,R)-11
12
H
naphth-1-yl
>1000
(8aS)
H
naphth-1-yl
>1000
(2R,8aR)
(2S,8aS)
(2R,8aS)
(2S,8aR)
(8aRS)
(8aRS)
(8aRS)
(8aS)
R-Me
S-Me
R-Me
S-Me
H
naphth-1-yl
>1000
naphth-1-yl
>1000
naphth-1-yl
>1000
naphth-1-yl
>1000
benzodioxan-5-yl
benzodioxepin-6-yl
benzodioxepin-6-yl
benzodioxepin-6-yl
benzimidazol-4-yl
>1000
13
14
(S)-14
16
H
>1000
H
>1000
H
>1000
(7aRS)
H
>10000
a Values are the mean of two to four experiments performed in triplicate.
20 and 21, which were obtained as racemic materials
by reaction of bicyclohydantoin 17 and bicyclodiketo-
piperazine 19 with 1,4-dibromobutane, respectively.23
Piperazines 22-24 were prepared according to known
procedures.31-33 Enantiopure (R)-25 and (S)-25
(R ) CH3) were synthesized by Pd(0) amination of
naphthyl triflate using commercially available 2(R)- and
2(S)-methylpiperazines, respectively. Consequently, com-
pounds 3 and 4 were obtained as diastereomeric mix-
tures. The synthesis of derivative 6 was already pub-
lished.33 Compounds 7-15 (n ) 1) were synthesized
from racemic or enantiopure bicyclohydantoins 17, 18,
and bicyclodiketopiperazine 19, and arylpiperazines
22-26 were synthesized by means of a Mannich reac-
tion in the presence of aqueous formaldehyde. Piper-
azine 26 was prepared according to a known proce-
dure.34 8, 12, and 13 were obtained as racemic mixtures
from 18, previously generated from DL-pipecolic acid.35
7 and 15 were synthesized from 17 readily available
from DL-proline.36 Selective removal of the trityl protect-
ing group in 15 was easily achieved simultaneous to the
transformation into the hydrochloride salt by bubbling
HCl(g) through an ethereal solution to afford 16. To
establish the influence of the affinity of a stereogenic
center in the no-pharmacophoric fragment of this family
of ligands, 9 and 14 were synthesized as racemic and
enantiopure materials [(S)-9, (R)-9, and (S)-14] using
racemic or enantiopure bicyclodiketopiperazine 1937 and
arylpiperazines 22 and 23. An additional stereocenter
was introduced into the arylpiperazine fragment by
Mannich reaction of (R)-25 or (S)-25 with either of the
two enantiomers of 18, affording the four optically pure
diastereoisomers [(R,R)- and (S,S)-10; (S,R)- and
(R,S)-11].
tively, in rat cerebral cortex membranes. All the com-
pounds were used in the form of hydrochloride salts and
were water- or methanol-soluble. In the experimental
binding assays, the compounds were first tested at a
fixed dose of 10-6 M, and for only those that in the
screening process presented a displacement of the
radioligand of >55%, the dose-response curves were
determined. The inhibition constant Ki was calculated
from the IC50 by using the Cheng-Prusoff equation.40
The results of these assays are illustrated in Table 1.
All the synthesized compounds V (1-14, 16) exhibited
high or very high 5-HT1AR affinity (Ki ) 0.5-15.3 nM).
Compounds with four methylene units in the spacer also
showed affinity for R1-adrenergic receptors. In contrast,
analogues 7-14 and 16, containing one methylene unit
in the spacer (n ) 1), are selective over R1-adrenergic
receptors. With respect to the stereogenic center of the
no-pharmacophoric part, no influence is observed in
5-HT1AR affinity orselectivity. Thus, racemic and both
enantiomers of 9 show similar binding constants
(6.7, 4.6, and 4.1 nM, respectively), and the same can
be seen in the case of racemic and enantiopure 14
(11.6 and 12.3 nM, respectively). When a substituent
was introduced in the pharmacophoric arylpiperazine
fragment, the noninfluence of an additional stereocenter
in the molecule could be assessed if we compare 3 with
4, (R,R)-10 with (S,S)-10, and (S,R)-11 with (R,S)-11.
Compound (S)-9 was also evaluated for affinity at
serotonin 5-HT2A, 5-HT3, 5-HT4, and 5-HT7 receptors,
at serotonin transporter (5-HTT), dopamine D2 recep-
tors, and benzodiazepine receptors, exhibiting high
affinity, in the nanomolar range, and at 5-HT2A and
5-HT3 receptors (Table 2). The following specific ligands
and tissue sources were used: 5-HT2A, [3H]ketanserin,
rat cerebral frontal cortex membranes;41 5-HT3,
[3H]LY278584, rat cerebral cortex membranes;42 5-HT4,
[3H]GR113808, rat striatum membranes;43 5-HT7,
[3H]-5-CT, rat hypothalamus membranes;44 5-HT trans-
Binding Affinities. Target compounds were assessed
for in vitro binding affinity at serotoninergic 5-HT1A and
R1-adrenergic receptors by radioligand binding assays,
using [3H]-8-OH-DPAT38 and [3H]prazosin,39 respec-

Selective Binding of New 5-HT1A Agonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2551
Table 2. Binding Affinity Data of Compound (S)-9a
receptor
Ki ( SEM (nM)
receptor
Ki ( SEM (nM)
5-HT1A
5-HT2A
5-HT3
5-HT4
5-HT7
4.1 ( 1.2
13.5 ( 2.5
8.9 ( 1.4
>10000
D2
192 ( 20
>1000
976 ( 43
>10000
R1
5-HTT
Bz
101 ( 1
a Values are the mean of two to four experiments performed in
triplicate.
Figure 2. Effect of (S)-9 on cAMP increase induced by
forskolin in 5-HT1A transfected HeLa cells. The cAMP content
was measured by radioimmunoassay. Adenylate ciclase activ-
ity is expressed as the percentage of 5-HT-sensitive forskolin-
stimulated adenylate ciclase activity in HeLa cells in the
presence of vehicle or different concentrations of (S)-9. The
data shown are the mean values ( SEM of duplicate experi-
ments. In each of them, four wells per concentration were
assayed.
was 0.15 µM, and the maximal inhibitory effect was
90.3 ( 1.3%. This negative control of (S)-9 on adenylate
cyclase activity indicates a transduction system coupled
to 5-HT1AR stimulation. As expected for an action
mediated through the activation of the 5-HT1AR, the
inhibition of forskolin-stimulated adenylate cyclase by
(S)-9 was significantly reduced by a 10-8 M concentra-
tion of the 5-HT1AR antagonist WAY100,635.
Figure 1. Dose-response effect of (S)-9 on rectal temperature.
Values represent the mean ( SEM of rectal temperature in
eight to nine mice. The asterisk (/) represents values from
(S)-9 that decrease more than 1.1 °C and are significantly
different (p < 0.05) from their respective basal rectal temper-
ature before drug administration (vehicle alone did not alter
rectal temperature). Statistical significance was assessed by
ANOVA followed by student Newman Keul’s test.
porter, [3H]paroxetine, rat cerebral cortex membranes;45
D2, [3H]raclopride, rat striatum membranes;46 benzodi-
azepine, [3H]flunitrazepam, rat cerebral cortex mem-
branes.47
Pharmacological Characterization of (S)-9. Pre-
synaptic 5-HT1AR activity was assessed by measuring
mouse rectal temperature 60 min after sc administra-
tion of either vehicle (water 4 mL/kg) or different doses
of (S)-9 (2.5, 5, 10, and 20 mg/kg).48 The administration
of (S)-9 provoked a dose-related decrease in mice rectal
temperature (Figure 1). This induced hypothermia
suggests that (S)-9 acts on 5-HT1A somatodendritic
autoreceptors.
The transduction mechanism of (S)-9 was determined
by using HeLa cells expressing human 5-HT1ARs.49
Assays were done 2 days after plating cells at 75 000
cells/well in 1 mL of DMEM containing glutamax, 10%
heat-inactivated calf serum, 5% penicilin/streptomycin,
and geneticin (300 µL/mL). The HeLa cells transfected
with the human 5-HT1AR were pretreated with 1-meth-
yl-3-isobutylxanthine (0.5 mM), forskolin (10 µM), and
(S)-9 (10-9-10-5 M) for 10 min at 37 °C in a 5% CO2
incubator. The cAMP content was measured by radio-
immunoassay. Adenylate cyclase activity is expressed
as the percentage of 5-HT-sensitive forskolin-stimulated
adenylate cyclase activity in HeLa cells in the presence
of vehicle or different concentrations of (S)-9. Ligand
(S)-9 inhibited in a dose-dependent manner the cAMP
increase induced by forskolin (Figure 2). The concentra-
tion that produced the half-maximal effect (EC50)
Functional activity of (S)-9 on 5-HT1ARs was further
assessed by evaluating its ability to decrease hypotha-
lamic 5-HT neuronal activity.50 Mice were injected sc
with vehicle (4 mL/kg water) or different doses of (S)-9
(5, 10, and 20 mg/kg) and killed by decapitation 30 min
later. The 5-hydroxyindoleacetic acid (5-HIAA)/5-HT
ratio in whole hypothalamus of vehicle or drug-treated
mice was measured. The results represented in Figure
3 indicate that the administration of (S)-9 induced a
decrease in the 5-HIAA/5-HT ratio, suggesting its
behavior as a 5-HT1AR agonist acting at the somato-
dendritic site.
Finally, we evaluated the potential anxiolytic activity
of (S)-9 by using a light/dark box test.51 Thirty minutes
after the sc administration of vehicle (4 mL/kg water)
or different doses of (S)-9 (0.5, 5, 10, and 20 mg/kg), the
time that mice spent in the lit area was measured
during a period of 5 min. Indeed, the administration of
(S)-9 (5 mg/kg) caused an increase in the time that mice
spent in the lit area (116.7 ( 9.3 vs 76.4 ( 7.6 s,
p < 0.05), as shown in Figure 4. The 5-HT1AR agonist
8-OH-DPAT was tested in the same test as for the
reference compound, at a dose of 2.5 mg/kg (time spent
in the lit area: 188.8 ( 26 vs 105 ( 14.7 s).
Overall, the pharmacological characterization of (S)-9
indicates that this compound is an agonist of the
5-HT1AR at the somatodendritic and postsynaptic sites,
with anxiolytic potential. The present data suggest that
(S)-9 may be therapeutically useful in the treatment of
anxiety-related disorders.

2552 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Lo´pez-Rodrı´guez et al.
Figure 3. Dose-response effect of (S)-9 on hypothalamic 5-HT
activity. 5-HIAA/5-HT ratios in mouse whole hypothalamus
are shown. The asterisk (/) represents values from (S)-9
treated mice that are significantly different from vehicle group
(p < 0.05, ANOVA followed by student Newman Keul’s test).
Figure 5. Molecular models of the complexes between com-
pounds 2 (a, c) and (S)-9 (b, c) and the 5-HT1AR in a view
parallel to the membrane. The CR traces of TMHs 3 (yellow),
4 (white), 5 (red), 6 (blue), and 7 (purple) are shown.
(a) Compound 2 is predicted to form an ionic interaction
between Asp3.32 and the protonated piperazine ring; hydrogen
bonds are between Ser5.42, Thr5.43, and Trp6.48 and the dike-
topiperazine moiety; and aromatic-aromatic interactions are
between Phe3.28, Trp7.40, and Tyr7.43 and the naphthalene ring.
(b) Compound (S)-9 is predicted to form an ionic interaction
between Asp3.32 and the protonated piperazine ring; hydrogen
bonds are between Thr5.39, Ser5.42, and Trp6.48 and the diketo-
piperazine moiety; and aromatic-aromatic interactions are
between Trp6.48 and Phe6.51 (not shown to clarify) and the
naphthalene ring. (c) Comparison of the modes of binding of
compounds 2 (light green) and (S)-9 (dark green) to the
5-HT1AR.
Figure 4. Dose-response effect of (S)-9 in the light/dark box
test. Values represent the mean values ( SEM of time spent
in the lit area in vehicle or drug treated mice that are
significantly different from water vehicle treated mice control
group (p < 0.05, ANOVA followed by student Newman Keul
test).
Tyr7.43. Importantly, this model considers that agonists
bind Trp6.48 in the active trans conformation.29 This
mode of binding of compound 2 contrasts with the
previously reported models of the complexes between
the 5-HT1AR and arylpiperazines of formula I.24,25 The
difference resides in the interaction of one of the oxygen
atoms of the hydantoin/diketopiperazine group with
either Trp6.48 or Thr3.37. In previous models the confor-
mation of Trp6.48 was modeled as observed in the crystal
structure of rhodopsin,54 in the inactive gauche+ con-
formation and thus far away from the ligand binding
site.
Figure 5b shows compound (S)-9 in the binding pocket
of the 5-HT1AR. (S)-9 cannot expand deep into the
bundle as compound 2 (Figure 5c) because of its shorter
side chain. Therefore, the formation of the ionic interac-
tion between the protonated piperazine and Asp3.32
allows the oxygen atoms of the diketopiperazine group
to interact with Thr5.39 (instead of Thr5.43 for compound
2), Ser5.42, and Trp6.48. The rigidity of (S)-9 also modifies,
relative to compound 2, the binding of the naphthalene
In agreement with binding data, compound (S)-9
(0.63-5.00 mg/kg, sc) prevented the head-twitch re-
sponse induced by the 5-HT2A/2CR agonist 2,5-dimethoxy-
4-iodoamphetamine (DOI) in mice. This would support
the activity of (S)-9 at 5-HT2ARs, since in this animal
species such a behavioral pattern induced by this
hallucinogenic compound (DOI) is considered as a
5-HT2AR-mediated action.52 Additionally, ligand (S)-9
reduced the bradycardia reflex due to 2-methyl-5-HT
administration in anesthetized rats. This may be taken
as an indication of antagonistic activity of (S)-9 at
5-HT3Rs.53
Computational Models of 5-HT1AR-Ligand In-
teraction. Figure 5a shows compound 2 in the binding
pocket of the 5-HT1AR. The protonated piperazine
moiety of the ligand is interacting with Asp3.32; the
oxygen atoms of the diketopiperazine moiety are inter-
acting with Ser5.42, Thr5.43, and Trp6.48; and the naph-
thalene ring is interacting with Phe3.28, Trp7.40, and

Selective Binding of New 5-HT1A Agonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2553
R1-adrenergic receptors is significantly smaller than in
the 5-HT1AR, impeding the binding of arylpiperazine
derivatives with n ) 1 at this site.
Structure-Activity Studies. Molecular Mecha-
nism of Receptor Activation. The structural basis of
5-HT1AR activation by agonists is poorly understood. It
has recently been described that Trp6.48, a partially
conserved side chain among family A of GPCRs (71% of
the receptors),15 undergoes a conformational transition
during receptor activation, from pointing toward
TMH 7, in the inactive gauche+ conformation, to
pointing toward TMH 5, in the active trans conforma-
tion.29,30 We suggest that agonists studied herein, by
means of an explicit hydrogen bond between one oxygen
atom of the diketopiperazine group and Trp6.48, trigger
the experimentally observed active conformation of
Figure 6. Sequence alignments of TMHs 3, 5, 6, and 7 of
5-HT1A and R1-adrenergic receptors. Residues referenced in the
manuscript are boxed.
ring, which expands between TMHs 6 and 7 (instead of
TMHs 3 and 7 for compound 2), interacting with Trp6.48
and Phe6.51 (not shown to offer a better view of the
ligand) (Figure 5).
Trp6.48
.
Discussion
Structure-Affinity Studies. Using the previously
reported computer models of the interaction between the
transmembrane domain of the 5-HT1AR and compounds
I24,25 and 3D-QSAR studies,19 we designed compound
1 [X ) -(CH2)3-, m ) 0, n ) 4, R ) H, Ar )
naphth-1-yl]. An increase of the substituent size of the
aromatic ring at the ortho and meta positions was
predicted to enhance the affinity for the 5-HT1AR.
Moreover, the presence of a cluster of aromatic rings
within TMHs 3 and 7 (Phe3.28, Trp7.40, and Tyr7.43) allows
the large naphthalene ring to optimally interact with
them in a face-to-edge orientation (T-shaped) (Figure
5a).
The influence of the spacer between the arylpiper-
azine and hydantoin moieties was explored in series
I-IV (Chart 1). The length of the spacer is of great
importance for 5-HT1A/R1 affinity. Ligands with n ) 3
or n ) 4 (compounds 1-6) possess the maximum
binding affinity at both receptors, whereas compounds
with n ) 2 are inactive or poorly active.18 Notably,
reduction of the spacer to n ) 1 [X ) -(CH2)3-, m ) 0,
n ) 1, R ) H, Ar ) naphth-1-yl] leads to compounds
7-14 and 16, which maintain high binding affinity for
the 5-HT1AR but are inactive at R1-adrenoceptors (Table
1).
Conclusion
We have designed and synthesized a new series of
arylpiperazines V that exhibit high 5-HT1AR affinity and
selectivity over R1-adrenoceptors. The new selective
5-HT1AR ligands contain a hydantoin (m ) 0) or diketo-
piperazine (m ) 1) moiety in the no-pharmacophoric
part, one methylene unit in the spacer to the pharma-
cophoric arylpiperazine (n ) 1), and different benzofused
rings as the aryl substituent of the piperazine (Ar). Also,
there is not a notable influence on 5-HT1AR affinity of
the stereogenic center present in the no-pharmacophoric
part of these derivatives or of an additional stereocenter
introduced with a substituent in the piperazine ring.
In particular, (S)-2-[[4-(napht-1-yl)piperazin-1-yl]methyl]-
1,4-dioxoperhydropyrrolo[1,2-a]pyrazine [(S)-9] (5-HT1A
,
Ki ) 4.1 nM; R1, Ki > 1000 nM) has been pharmacologi-
cally characterized as a 5-HT1AR agonist at somato-
dendritic and postsynaptic sites and may be therapeu-
tically useful in the treatment of anxiety-related
disorders. Ligand (S)-9 is predicted, in computer simu-
lations, to bind Asp3.32 in TMH 3, Thr5.39 and Ser5.42 in
TMH 5, and Trp6.48 in TMH 6. We propose that agonists
modify, by means of an explicit hydrogen bond, the
conformation of Trp6.48 from pointing toward TMH 7,
in the inactive gauche+ conformation, to pointing
toward the ligand binding site, in the active trans
conformation.
Structure-Selectivity Studies. Figure 6 shows the
sequence alignments of TMHs 3, 5, 6, and 7 of 5-HT1A
and R1-adrenergic receptors. The presence of aromatic
residues at positions 3.28, 7.40, and 7.43, the acidic
Experimental Section
Asp3.32 residue, and the hydrogen bond donor Ser5.42
,
Chemistry. Melting points (uncorrected) were determined
on a Gallenkamp electrothermal apparatus. Infrared (IR)
spectra were obtained on a Perkin-Elmer 781 infrared spectro-
photometer. 1H and 13C NMR spectra were recorded on a
Varian VXR-300S, Bruker Avance 300, or Bruker AC-200
instrument. Chemical shifts (δ) are expressed in parts per
million relative to internal tetramethylsilane used as internal
reference. Coupling constants (J) are in hertz (Hz). The
following abbreviations are used to describe peak patterns
Thr/Ser5.43, and Trp6.48 residues, which are the predicted
anchoring points of arylpiperazine derivatives with
n ) 4 (Figure 5a), in both 5-HT1A and R1-adrenergic
receptors (see boxed residues in Figure 6) explains their
lack of selectivity. In contrast, the predicted anchoring
points of arylpiperazine derivatives with n ) 1 (Figure
5b) are the aromatic Trp6.48 and Phe6.51 residues, the
acidic Asp3.32 residue, and the hydrogen bond donor
Thr5.39, Ser5.42, and Trp6.48 residues. The fact that Thr5.39
is present only in the 5-HT1AR (Figure 6) explains the
observed selectivity of compounds 7-14 and 16 over
R1-adrenergic receptors. The side chain at position 6.55,
close to the ligand binding site (Figure 5b), might also
influence the observed selectivity of these compounds.
While the 5-HT1AR contains the small Ala residue, the
R1-adrenergic receptors contain the bulky Met or Leu
residue. Thus, the cavity present at this locus in the
when appropriate:
s (singlet), d (doublet), t (triplet),
q (quartet), qt (quintet), m (multiplet), br (broad). Elemental
analyses (C, H, N) were carried out on a Perkin-Elmer 2400
apparatus at the UCM’s analysis services and were within
0.4% of the theoretical values. Optical rotation [R] was
measured using a Perkin-Elmer 781 polarimeter. Analytical
thin-layer chromatography (TLC) was run on Merck silica gel
plates (Kieselgel 60 F-254) with detection by UV light, iodine,
acidic vanillin solution, or 10% phosphomolybdic acid solution
in ethanol. For normal pressure and flash chromatography,
Merck silica gel type 60 (size 70-230 and 230-400 mesh,

2554 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Lo´pez-Rodrı´guez et al.
respectively) was used. Unless stated otherwise, starting
materials and reagents used were high-grade commercial
products purchased from Aldrich, Fluka, or Merck. All solvents
were distilled prior to use.
pyrazine (3 and 4). From 21 and (R)-25 was obtained 3 as
an equimolecular mixture of diasteroisomers in 57% yield
(oil, [R]25 -20.0 (c 1.1, CHCl3)). From 21 and (S)-25 was
D
obtained 4 as an equimolecular mixture of diasteroisomers in
35% yield (oil, [R]25 +21.0 (c 1.1, CHCl3)). Chromatography:
D
The following compounds were synthesized according to
described procedures: 2-[4-[4-(benzimidazol-4(7)-yl)piperazin-
chloroform/ethanol, from 20:1 to 12:1.
(6),33
2-[4-[4-(3,4-Dihydro-2H-1,5-benzodioxepin-6-yl)piper-
azin-1-yl]butyl]-1,4-dioxoperhydropyrrolo[1,2-a]pyra-
zine (5). From 21 and 23 was obtained 5 in 52% yield.
Chromatography: chloroform/methanol, from 9.5:0.5 to 9:1;
mp 212-213 °C (dec) (methanol/ethyl ether); IR (CHCl3) 1670,
1590, 1485, 1460 cm-1; 1H NMR (CDCl3) δ 1.43-1.55 (m, 4H),
1.82-2.05 (m, 3H), 2.13 (qt, J ) 5.7, 2H), 2.27-2.31 (m, 1H),
2.35 (t, J ) 7.2, 2H), 2.55 (br s, 4H), 3.00 (br s, 4H), 3.27-3.34
(m, 1H), 3.41-3.57 (m, 3H), 3.72 (d, J ) 16.2, 1H), 4.01
(t, J ) 7.5, 1H), 4.07 (d, J ) 16.5, 1H), 4.14-4.21 (m, 4H),
6.54 (dd, J ) 7.8, 1.5, 1H), 6.59 (dd, J ) 8.2, 1.4, 1H), 6.76
(t, J ) 7.9, 1H); 13C NMR (CDCl3) δ 22.6, 23.9, 25.1, 28.8, 31.5,
45.2, 46.0, 51.0, 51.6, 53.4, 58.0, 59.0, 70.2, 70.3, 112.9, 115.5,
122.5, 144.6, 145.0, 152.1, 163.1, 167.1. Anal. (C24H34N4O4‚
2HCl‚2H2O) C, H, N.
1-yl]butyl]-1,3-dioxoperhydropyrrolo[1,2-c]imidazole
1,3-dioxoperhydropyrrolo[1,2-c]imidazole (17),36 1,3-dioxoper-
hydroimidazo[1,5-a]pyridine (18),35 1,4-dioxoperhydropyrrolo-
[1,2-a]pyrazine (19)37 as racemic and as enantiopure materials,
2-(4-bromobutyl)-1,3-dioxoperhydropyrrolo[1,2-c]imidazole (20),23
2-(4-bromobutyl)-1,4-dioxoperhydropyrrolo[1,2-a]pyrazine (21),23
1-(naphth-1-yl)piperazine (22),31 1-(3,4-dihydro-2H-1,5-benzo-
dioxepin-6-yl)piperazine (23),32 1-(1-tritylbenzimidazol-4-yl)-
piperazine (24),33 1-(2,3-dihydro-1,4-benzodioxan-5-yl)piper-
azine (26).34 Collected data for compounds 1-15 refer to free
bases, and then hydrochloride salts were prepared prior to
melting point determination, elemental analyses, and biologi-
cal assays. Spectroscopic data of all described compounds were
consistent with the proposed structures. For series 1-6 and
7-16 we include the data of compounds 1, 5, 12, 15, and 16.
General Procedure for the Synthesis of Arylpiper-
azines 7-15 (n ) 1). To a suspension of 17-19 (7 mmol) and
formaldehyde (7 mmol from a 35% aqueous solution) in
methanol (15 mL) was added the corresponding arylpiperazine
22-26 (7 mmol). The resultant suspension was refluxed for
2-6 h after complete disappearance of the starting materials
(TLC). The mixture was then cooled to room temperature, and
the solvent was evaporated at reduced pressure. The crude
mixture was diluted in chloroform (75 mL) and washed with
water (3 × 75 mL). The organic layer was dried over anhydrous
Na2SO4, filtered, and evaporated at reduced pressure. The
obtained crude was purified by column chromatography on
silica gel using the appropriate eluent.
2-[[4-(Naphth-1-yl)piperazin-1-yl]methyl]-1,3-dioxo-
perhydropyrrolo[1,2-c]imidazole (7). From 17 and 22 was
obtained 7 in 68% yield. Chromatography: ethyl acetate/
hexane, 1:1; mp 168-170 °C (methanol/ethyl ether). Anal.
(C21H24N4O2‚2HCl) C, H, N.
2-[[4-(Naphth-1-yl)piperazin-1-yl]methyl]-1,3-dioxo-
perhydroimidazo[1,5-a]pyridine (8). From 18 and 22 was
obtained 8 in 77% yield. Chromatography: hexane/ethyl
acetate, 2:8; mp 171-174 °C (dec) (methanol/ethyl ether). Anal.
(C22H26N4O2‚HCl‚3/2H2O) C, H, N.
Synthesis of (R)- and (S)-3-Methyl-1-(naphth-1-yl)-
piperazine (R)-25 and (S)-25. To a solution of 100 mg
(0.35 mmol) of 1-naphthyl trifluoromethanesulfonate in tolu-
ene (10 mL × mmo), under an argon atmosphere, (R)- or (S)-
2-methylpiperazine (2.11 equiv), NaOtBu (0.51 equiv), Pd-
(OAc)2 (0.007 mmol), and (()-BINAP (0.021 mmol) were added.
The mixture was heated at 100 °C (5 h). Then, the mixture
was cooled and filtered through a short column of Celite. The
resulting solution was evaporated to dryness under vacuum.
The crude mixture was purified by column chromatography
on silica gel using dichloromethane/methanol mixtures as
eluent. (R)-25 was obtained in 72% yield ([R]25 +10.9 (c 1.3,
D
CHCl3)), and (S)-25 was obtained in 78% yield ([R]25 -12.4
D
(c 1.2, CHCl3)). IR (CHCl3) 3700-3200, 3020, 1595, 1575, 1510,
1455 cm-1 1H NMR (CDCl3) δ 1.12 (d, J ) 6.3, 3H), 1.89
;
(br s, 1H), 2.47 (ap t, J ) 10.1, 1H), 2.81 (dt, J ) 10.1, 2.7,
1H), 3.10-3.30 (m, 5H), 7,06 (dd, J ) 7.3, 1.0, 1H), 7.39
(t, J ) 7.3, 1H), 7.42-7.48 (m, 2H), 7.53 (d, J ) 8.0, 1H), 7.77-
7.83 (m, 1H), 8.17-8.22 (m, 1H); 13C NMR (CDCl3) δ 19.7, 46.4,
51.0, 53.5, 61.0, 114.7, 123.4, 123.5, 125,3, 125.7, 125.8, 128.3,
128.9, 134.7, 149.8.
General Procedure for the Synthesis of Arylpiper-
azines 1-6 (n ) 4). To a suspension of the bromoalkyl
derivative 20 or 21 (4.5 mmol) and the appropriate arylpiper-
azine 22-25 (7.5 mmol) in dry acetonitrile (10 mL) was added
triethylamine (1.0 mL, 7.5 mmol), and the mixture was
refluxed for 20-24 h. After cooling, the solvent was evaporated
under reduced pressure and the residue was resuspended in
water and extracted with dichloromethane (3 × 50 mL). The
combined organic layers were washed with water and dried
over anhydrous Na2SO4. After evaporation of the solvent the
crude oil was purified by column chromatography in silica gel
using the appropriate eluent.
(RS)-, (R)-, and (S)-2-[[4-(Naphth-1-yl)piperazin-1-yl]-
methyl]-1,4-dioxoperhydropyrrolo[1,2-a]pyrazine [9, (R)-
9, and (S)-9]. From 19 and 22 was obtained 9 in 62% yield.
From (R)-19 and 22 was obtained (R)-9 in 53% yield ([R]25
D
+68.2 (c 1.3, CHCl3)). From (S)-19 and 7 was obtained (S)-9
in 59% yield ([R]25 -68.1 (c 1.4, CHCl3)). Chromatography:
D
from ethyl acetate to ethyl acetate/ethanol, 9:1; mp [(S)-9]
256-259 °C (dec) (acetone). 9: Anal. (C22H26N4O2‚HCl‚3/2H2O).
(R)-9: Anal. (C22H26N4O2‚HCl‚2H2O) C, H, N. (S)-9: Anal.
(C22H26N4O2‚HCl‚3/2H2O) C, H, N.
2-[4-[4-(Naphth-1-yl)piperazin-1-yl]butyl]-1,3-dioxo-
perhydropyrrolo[1,2-c]imidazole (1). From 20 and 22 was
obtained 1 in 60% yield. Chromatography: chloroform/
methanol, from 9.5:0.5 to 9:1; IR (CHCl3) 1770, 1705, 1610,
1590, 1520, 1485 cm-1; 1H NMR (CDCl3) δ 1.50-1.71 (m, 5H),
2.01-2.07 (m, 2H), 2.19-2.24 (m, 1H), 2.44 (t, J ) 7.4, 2H),
2.70 (br s, 4H), 3.18-3.24 (m, 1H), 3.30-3.47 (m, 6H), 3.65-
3.75 (m, 1H), 4.07-4.24 (m, 1H), 7.50 (d, J ) 7.2, 1H), 7.38-
7.46 (m, 3H), 7.53 (d, J ) 7.8, 1H), 7.78-7.82 (m 1H), 8.11-
8.15 (m, 1H); 13C NMR (CDCl3) δ 23.3, 25.8, 27.1, 27.9, 38.5,
45.6, 51.0 (2 C), 53.0 (2 C), 57.5, 63.1, 114.7, 123.6 (2 C), 125.4,
125.9 (2 C), 128.5, 128.8, 134.8, 149.6, 161.5, 175.1. Anal.
(C26H34N4O2‚2HCl) C, H, N.
(2R,8aR)-2-[[4-(Naphth-1-yl)-2-methylpiperazin-1-yl]-
methyl]-1,4-dioxoperhydropyrrolo[1,2-a]pyrazine [(R,R)-
10]. From (R)-19 and (R)-25 was obtained (R,R)-10 in 23%
yield ([R]25D +36.6 (c 1.9, CHCl3)). Chromatography: from ethyl
acetate to ethyl acetate/ethanol, 9:1. Anal. (C23H28N4O2‚HCl‚
5/2H2O) C, H, N.
(2S,8aS)-2-[[4-(Naphth-1-yl)-2-methylpiperazin-1-yl]-
methyl]-1,4-dioxoperhydropyrrolo[1,2-a]pyrazine [(S,S)-
10]. From (S)-19 and (S)-25 was obtained (S,S)-10 in 36% yield
([R]25 -38.0 (c 1.2, CHCl3)). Spectroscopic data are identical
D
to those of (R,R)-10 (see Supporting Information). Anal.
(C23H28N4O2‚HCl‚2/3H2O) C, H, N.
(2R,8aS)-2-[[4-(Naphth-1-yl)-2-methylpiperazin-1-yl]-
methyl]-1,4-dioxoperhydropyrrolo[1,2-a]pyrazine [(R,S)-
11]. From (S)-19 and (R)-25 was obtained (R,S)-11 in 43% yield
2-[4-[4-(Naphth-1-yl)piperazin-1-yl]butyl]-1,4-dioxo-
perhydropyrrolo[1,2-a]pyrazine (2). From 21 and 22 was
obtained 2 in 43% yield. Chromatography: chloroform/
methanol, from 9.5:0.5 to 9:1; mp 277-280 °C (dec) (methanol/
ethyl ether). Anal. (C25H32N4O2‚HCl‚1/2H2O) C, H, N.
([R]25 -53.2 (c 1.4, CHCl3)). Chromatography: from ethyl
D
acetate to ethyl acetate/ethanol, 9:1. Anal. (C23H28N4O2‚HCl‚
H2O) C, H, N.
(2R,8aRS)- and (2S,8aRS)-2-[4-[4-(Naphth-1-yl)-2-meth-
ylpiperazin-1-yl]butyl]-1,4-dioxoperhydropyrrolo[1,2-a]-
(2S,8aR)-2-[[4-(Naphth-1-yl)-2-methylpiperazin-1-yl]-
methyl]-1,4-dioxoperhydropyrrolo[1,2-a]pyrazine [(S,R)-

Selective Binding of New 5-HT1A Agonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2555
11]. From (R)-19 and (S)-25 was obtained (S,R)-11 in 40% yield
absence of the competing drug, in a final volume of 1.1 mL of
assay buffer (50 mM Tris-HCl, 10 nM clonidine, 30 nM
prazosin, pH 7.4 at 37 °C). Nonspecific binding was determined
with 10 µM 5-HT.
([R]25 +53.7 (c 0.9, CHCl3)). Spectroscopic data are identical
D
to those of (R,S)-11 (see Supporting Information). Anal.
(C23H28N4O2‚HCl‚3/2H2O) C, H, N.
2-[[4-(2,3-Dihydro-1,4-benzodioxan-5-yl)piperazin-1-yl]-
methyl]-1,3-dioxoperhydroimidazo[1,5-a]pyridine (12).
From 18 and 26 was obtained 12 in 88% yield. Chromatogra-
phy: hexane/ethyl acetate, 1:9; mp 172-174 °C (chloroform/
5-HT2A Receptor. Binding assays were performed by a
modification of the procedure previously described by Titeler
et al.41 The frontal cortex was homogenized in 60 volumes of
ice-cold buffer (50 mM Tris-HCl, 0.5 mM Na2EDTA, 10 mM
MgSO4, pH 7.4 at 25 °C) and centrifuged at 30000g for 15 min
at 4 °C. The membrane pellet was washed by resuspension
and centrifugation. After the second wash the resuspended
pellet was incubated at 37 °C for 10 min. Membranes were
then collected by centrifugation, and the final pellet was
resuspended in 10 volumes of assay buffer (50 mM Tris-HCl,
0.5 mM Na2EDTA, 10 mM MgSO4, 0.1% ascorbic acid, 10 µM
pargyline, pH 7.4 at 25 °C). Fractions of 100 µL of the final
membrane suspension (about 5 mg/mL of protein) were
incubated at 37 °C for 15 min with 0.4 nM [3H]ketanserin, in
the presence or absence of the competing drug, in a final
volume of 2 mL of assay buffer. Nonspecific binding was
determined with 1 µM cinanserin.
5-HT3 Receptor. Binding assays were performed by a
modification of the procedure previously described by Wong
et al.42 The cerebral cortex was homogenized in 9 volumes of
ice-cold 0.32 M sucrose and centrifuged at 1000g for 10 min
at 4 °C. The supernatant was centrifuged at 17000g for
20 min at 4 °C. The membrane pellet was washed twice by
resuspension in 60 volumes of ice-cold 50 mM Tris-HCl buffer
(pH 7.4 at 25 °C) and centrifugation at 48000g for 10 min at
4 °C. After the second wash the resuspended pellet was
incubated at 37 °C for 10 min and centrifuged at 48000g for
10 min at 4 °C. Membranes were resuspended in 2.75 volumes
of assay buffer (50 mM Tris-HCl, 10 µM pargyline, 0.6 mM
ascorbic acid, and 5 mM CaCl2, pH 7.4 at 25 °C). Fractions of
100 µL of the final membrane suspension (about 2 mg/mL of
protein) were incubated at 25 °C for 30 min with 0.7 nM
[3H]LY 278584, in the presence or absence of the competing
drug, in a final volume of 2 mL of assay buffer. Nonspecific
binding was determined with 10 µM 5-HT.
5-HT4 Receptor. Binding assays were performed by a
modification of the procedure previously described by Gross-
man et al.43 The striatum was homogenized in 15 volumes of
ice-cold 50 mM HEPES buffer (pH 7.4 at 4 °C) and centrifuged
at 48000g for 10 min. The pellet was resuspended in
20 volumes of assay buffer (50 mM HEPES, pH 7.4 at 25 °C).
Fractions of 100 µL (about 5 mg/mL of protein) of the final
membrane suspension were incubated at 37 °C for 30 min with
0.1 nM [3H]GR 113808, in the presence or absence of the
competing drug, in a final volume of 1 mL of assay buffer.
Nonspecific binding was determined with 30 µM 5-HT.
5-HT7 Receptor. Binding assays were performed by a
modification of the procedure previously described by Aguirre
et al.44 The hypothalamus was homogenized in 5 mL of ice-
cold Tris buffer (50 mM Tris-HCl, pH 7.4 at 25 °C) and
centrifuged at 48000g for 10 min. The membrane pellet was
washed by resuspension and centrifugation, and then the
resuspended pellet was incubated at 37 °C for 10 min.
Membranes were then collected by centrifugation, and the final
pellet was resuspended in 100 volumes of ice-cold 50 mM Tris-
HCl, 4 mM CaCl2, 1 mg/mL ascorbic acid, 0.01 mM pargyline,
and 3 µM pindolol55 buffer (pH 7.4 at 25 °C). Fractions of
400 µL of the final membrane suspension were incubated at
23 °C for 120 min with 0.5 nM [3H]-5-CT (88 Ci/mmol), in the
presence or absence of several concentrations of the competing
drug, in a final volume of 0.5 mL of assay buffer (50 mM Tris-
HCl, 4 mM CaCl2, 1 mg/mL ascorbic acid, 0.01 mM pargyline,
and 3 µM pindolol buffer (pH 7.4 at 25 °C)). Nonspecific
binding was determined with 10 µM 5-HT.
ethyl ether); IR (KBr) 1770, 1710, 1600, 1500, 1450 cm-1
;
1H NMR (CDCl3) δ 1.30-1.51 (m, 3H), 1.74 (d, J ) 13.2, 1H),
1.99 (d, J ) 13.5, 1H), 2.20 (d, J ) 13.2, 1H), 2.76-2.84
(m, 5H), 2.97-3.08 (m, 4H), 3.77 (dd, J ) 11.7, 4.2, 1H), 4.15
(dd, J ) 13.5, 3.6, 1H), 4.20-4.30 (m, 4H), 4.53 (s, 2H), 6.50
(dd, J ) 7.8, 1.5, 1H), 6.56 (dd, J ) 8.2, 1.6, 1H), 6.75 (t, J )
8.1, 1H); 13C NMR (CDCl3) δ 22.8, 25.0, 27.9, 39.4, 50.6 (2 C),
50.8 (2 C), 57.5, 60.1, 64.0, 64.4, 110.8, 112.1, 120.7, 136.4,
141.7, 144.1, 155.3, 174.5. Anal. (C20H26N4O4‚2HCl‚1/2H2O) C,
H, N.
2-[[4-(3,4-Dihydro-2H-1,5-benzodioxepin-6-yl)piperazin-
1-yl]methyl]-1,3-dioxoperhydroimidazo[1,5-a]pyridine
(13). From 18 and 23 was obtained 13 in 63% yield. Chroma-
tography: hexane/ethyl acetate, 1:9; mp 183-185 °C (dec)
(methanol/ethyl ether). Anal. (C21H28N4O4‚2HCl‚H2O) C, H, N.
(RS)- and (S)-2-[[4-(3,4-Dihydro-2H-1,5-benzodioxepin-
6-yl)piperazin-1-yl]methyl]-1,4-dioxoperhydropyrrolo-
[1,2-a]pyrazine [14 and (S)-14]. From 19 and 23 was
obtained 14 in 56% yield. From (S)-19 and 23 was obtained
(S)-14 in 41% yield ([R]25 -22.3 (c 0.6, MeOH)); mp [(S)-14]
D
144-145 °C (methanol/ethyl ether). Chromatography: ethyl
acetate/ethanol, 9:1. (S)-14: Anal. (C21H28N4O4‚2HCl‚H2O) C,
H, N.
2-[[4-(1-Tritylbenzimidazol)piperazin-1-yl]methyl]-1,3-
dioxoperhydropyrrolo[1,2-c]imidazole (15). From 17 and
24 was obtained 15 in 86% yield. Chromatography: from
chloroform to chloroform/methanol, 9:1; mp 120-123 °C (hex-
ane/chloroform); IR (CHCl3) 1770, 1710, 1495, 1445, 1430 cm-1
;
1H NMR (CDCl3) δ 1.62-1.83 (m, 1H), 2.00-2.31 (m, 3H), 2.92
(br s, 4H), 3.20-3.33 (m, 1H), 3.51 (br s, 4H), 3.62-3.78
(m, 1H), 4.12 (m, 1H), 4.53 (s, 2H), 6.11 (d, J ) 8.1, 1H), 6.53
(d, J ) 7.8, 1H), 6.77 (t, J ) 8.1, 1H), 7.11-7.19 (m, 5H), 7.27-
7.35 (m, 10H), 7.83 (s, 1H); 13C NMR (CDCl3) δ 26.5, 27.3, 45.2,
49.7 (2 C), 50.1 (2 C), 59.9, 62.8, 74,9, 107.2, 108.4, 118.4, 122.4,
127.5, 127.6, 129.6, 135.5, 136.4, 140.9, 143.2, 160.9, 174.5.
Anal. (C37H36N6O2) C, H, N.
2-[[4-(Benzimidazol-4(7)-yl)piperazin-1-yl]methyl]-1,3-
dioxoperhydropyrrolo[1,2-c]imidazole (16). Removal of
the trityl group in 15 was carried out simultaneous to the
transformation into the hydrochloride salt. HCl (g) (generated
from H2SO4 and anhydrous CaCl2) was bubbled through a
solution of 54 mg of 15 in Et2O (3 mL) for 1 h. Solid 16 as the
hydrochloride salt was separated by filtration, washed with
Et2O, and dried under vacuum (yield 90%). All data of
compound 16 are measured as the hydrochloride salt: mp
>300 °C (dec). Anal. (C18H22N6O2‚2HCl‚H2O) C, H, N.
Radioligand Binding Assays. For all receptor binding
assays, male Sprague-Dawley rats (Rattus norvegicus albinus)
weighing 180-200 g were killed by decapitation, and the
brains were rapidly removed and dissected. Tissues were
stored at -80 °C for subsequent use and homogenized on a
Polytron PT-10 homogenizer. Membrane suspensions were
centrifuged on a Beckman J2-HS instrument.
5-HT1A Receptor. Binding assays were performed by a
modification of the procedure previously described by Clark
et al.38 The cerebral cortex was homogenized in 10 volumes of
ice-cold Tris buffer (50 mM Tris-HCl, pH 7.7 at 25 °C) and
centrifuged at 28000g for 15 min. The membrane pellet was
washed twice by resuspension and centrifugation. After the
second wash the resuspended pellet was incubated at 37 °C
for 10 min. Membranes were then collected by centrifugation,
and the final pellet was resuspended in 50 mM Tris-HCl,
5 mM MgSO4, and 0.5 mM EDTA buffer (pH 7.4 at 37 °C).
Fractions of 100 µL of the final membrane suspension (about
1 mg of protein) were incubated at 37 °C for 15 min with
0.6 nM [3H]-8-OH-DPAT (133 Ci/mmol), in the presence or
5-HT Transporter. Binding assays were performed by a
modification of the procedure previously described by Habert
et al.45 The cerebral cortex was homogenized in 50 volumes of
ice-cold Tris buffer (50 mM Tris-HCl containing 20 mM NaCl
and 5 mM KCl, pH 7.4 at 25 °C) and centrifuged at
20000 rpm for 10 min at 4 °C. The membrane pellet was

2556 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Lo´pez-Rodrı´guez et al.
washed twice by resuspension and centrifugation. The final
pellet was resuspended in assay buffer, and fractions of 1 mL
of the final membrane suspension (about 120 µg of protein)
were incubated at 25 °C for 60 min with 0.2 nM [3H]paroxetine,
in the presence or absence of the competing drug, in a final
volume of 1.2 mL of assay buffer. Nonspecific binding was
determined with 10 µM fluoxetine.
R1 Adrenoceptor. Binding assays were performed by a
modification of the procedure previously described by Ambrosio
et al.39 The cerebral cortex was homogenized in 20 volumes of
ice-cold buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 7.4 at
25 °C) and centrifuged at 30000g for 15 min. Pellets were
washed twice by resuspension and centrifugation. Final pellets
were resuspended in the same buffer. Fractions of the final
membrane suspension (about 250 µg of protein) were incubated
at 25 °C for 30 min with 0.2 nM [3H]prazosin (23 Ci/mmol), in
the presence or absence of six concentrations of the competing
drug, in a final volume of 2 mL of buffer. Nonspecific binding
was determined with 10 µM phentolamine.
D2 Receptor. Binding assays were performed by a modi-
fication of the procedure previously described by Hall et al.46
The striatum was homogenized in 50 mM Tris-HCl (pH 7.7 at
25 °C) and centrifuged at 48000g for 10 min. The pellet was
resuspended and centrifuged as before. The final pellet was
resuspended in 50 mM Tris-HCl (pH 7.7 at 25 °C) containing
120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and
0.1% ascorbic acid. Fractions of the final membrane suspension
(125-150 µg of protein) were incubated at 25 °C for 60 min
with 0.8 nM [3H]raclopride (77 Ci/mmol), in the presence or
absence of six concentrations of the competing drug, in a final
volume of 1.1 mL of the assay buffer (pH 7.4 at 25 °C).
Nonspecific binding was determined with 1 µM (+)-butaclamol.
Benzodiazepine Receptor. Binding assays were per-
formed by a modification of the procedure previously described
by Orensanz et al.47 The tissue was homogenized in 25 mM
potassium phosphate (KPi) buffer (pH 7.4). Homogenized
fractions of 100 µL of the membrane suspension (about
100 µg of protein) were incubated at 0-4 °C for 90 min with
0.25 nM [3H]flunitrazepam (37 Ci/mmol), in the presence or
absence of the competing drug, in a final volume of 1 mL of
assay buffer. Nonspecific binding was determined with 2 µM
diazepam.
For all binding assays, competing drug, nonspecific, total,
and radioligand bindings were defined in triplicate. Incubation
was terminated by rapid vacuum filtration through Whatman
GF/B filters, presoaked in 0.05% poly(ethylenimine), using a
Brandel cell harvester. The filters were then washed with the
assay buffer, dried, and placed in poly(ethylene) vials to which
were added 4 mL of a scintillation cocktail (Aquasol). The
radioactivity bound to the filters was measured by liquid
scintillation spectrometry. The data were analyzed by an
iterative curve-fitting procedure (program Prism, Graph Pad),
which provided IC50, Ki, and r2 values for test compounds,
Ki values being calculated from the Cheng and Prusoff equa-
tion.40 The protein concentrations of the rat cerebral cortex
and the rat striatum were determined by the method of
Lowry,56 using bovine serum albumin as the standard.
Rectal Temperature. Male Swiss albino mice (24-30 g)
were obtained from Interfauna Ibe´rica and maintained in a
temperature- and light-controlled environment (25 ( 1 °C,
light on between 8.00 a.m. and 8.00 p.m.). Food and tap water
were provided ad libitum. All experiments were performed
between 9.00 a.m. and 2.00 p.m. After removal of mice from
their home cages, basal rectal temperature was measured with
a lubricated digital thermistoprobe that was inserted into the
rectum 1.5 cm for 40 s. Rectal temperature was determined
again after the appropriate treatments with (S)-9 or vehicle.
The difference between the temperatures measured before and
60 min after the administration represents an index of
hypothermia. A decrease of more than 1.1 °C from basal rectal
temperature was considered to be a hypothermic response.
receptor (HA 6 cells) were kindly provided by Dr. J. del R´ıo
(Departamento Farmacolog´ıa, Universidad de Navarra) and
grown in 75 mL flasks containing 20 mL of Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal calf
serum, 500 units of penicillin, 500 µg of streptomycin/mL
(P/S), and 0.3 mg/mL geneticin. Forty-eight hours before the
experiment, cells were plated at a density of 75 × 103 cells in
1.5 mL of DMEM-P/S-fetal calf serum-geneticin medium in
12 multiwell plates. On the day of the experiment, cells were
treated with 0.5 mM 1-methyl-3-isobutylxanthine (IBMX),
10 µM forskolin, and vehicle or different concentrations of
(S)-9 in a 37 °C and 5% CO2 incubator. Ten minutes later,
treatment was stopped and cells were lysed with a 65% ethanol
solution for 2 h, and then the ethanol was collected and
evaporated at 55 °C, leaving a pellet with cAMP. For the study
of (S)-9 receptor specificity, cells were preincubated for
20 min with 10-8 M WAY 100,635 prior to adding (S)-9.
Samples were stored at -20 °C and later analyzed using a
commercial radioimmunoassay (RIA) kit ([3H]cAMP assay
system, model TRK 432; Amersham). Protein was measured
by Bradford’s method. Competition binding isotherms were
analyzed by using an iterative curve-fitting procedure (pro-
gram Origin 7.0), which provided EC50 values for test com-
pounds.
Neurochemical Activity. 5-HIAA/ 5-HT Ratios. Follow-
ing appropriate treatments, mice were decapitated and brains
were removed from the skull. Hypothalamus were dissected
on ice and immediately frozen over dry ice. Tissue samples
were placed in 200 µL of 0.1 M phosphate-citrate buffer
(pH 2.5) containing 15% methanol and stored at -80 °C until
assayed. On the day of assay, tissue samples were thawed,
sonicated for 3 s (Vibra Cell, model VC-501, Sonics and
Materials, Danbury, CT), and centrifuged for 60 s in a
Microfuge (IEC, Centra-MP4R, Needham, MA). 5-Hydroxyin-
dolacetic acid (5-HIAA), 5-hydroxytryptamine (5-HT),
3,4-dihydroxyphenylacetic acid (DOPAC), and dopamine con-
centrations in hypothalamus tissue extracts were measured
by high-performance liquid chromatography (HPLC) with
electrochemical detection. A total of 20 µL of the supernatant
was injected onto a Nucleosil 120 5 C18 reverse-phase analyti-
cal column. The HPLC column was coupled to a single
coulometric electrode conditioning cell in series with dual
electrode analytical cells (Coulochem II, ESA, Bedford, MA).
The conditioning electrode potential was set at 100 nA, and
the analytical electrodes were set at +0.12 and -0.31 V
relative to internal Ag reference electrodes. The HPLC mobile
phase consisted of 0.1 M phosphate/citrate buffer (pH 2.8)
containing 0.1 mM ethylenediaminetetracetic acid (EDTA),
0.05% sodium octylsulfate, and 25% methanol. The 5-HIAA
and 5-HT content of each sample was quantified by comparing
peak heights with those peaks of standards assayed the same
day as determined by the Shimadzu integrator. The lower limit
of sensitivity of this assay for these compounds was 2-8 pg
per sample. Tissue pellets were dissolved in 1.0 N NaOH and
assayed for protein.56
Light/Dark Box Test. The light/dark test uses the rodent
natural aversion to bright areas compared with darker ones.57
In a two-compartment box, rodents will prefer the dark areas,
whereas anxiolytics should increase the time spent in the lit
compartment. The apparatus consisted of two methacrylate
boxes (20 cm × 20 cm × 14 cm), one transparent and one black
and opaque separated by an opaque tunnel (5 cm × 7 cm ×
10 cm). A light from a 60 W desk lamp placed 25 cm above
the light box provided the room illumination. Male Swiss
albino mice were housed 5 days before the experiment. On the
day of the experiment drugs were dissolved in distilled water
and sc injected. After 30 min of absorption time mice were
individually tested in 5 min sessions in the apparatus de-
scribed above. The floor of each box was cleaned between
sessions. At the beginning of the session, mice were placed in
the tunnel facing the dark box. The amount of time spent by
mice in the lit area was recorded over 5 min periods. A mouse
whose four paws were in the new box was considered as having
changed boxes.
Cell Culture and Determination of cAMP Levels after
Stimulation of the Adenylate Cyclase Enzyme with
Forskolin. HeLa cells transfected with the human 5-HT1A

Selective Binding of New 5-HT1A Agonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2557
Functional Abnormalities in the Hippocampus in Mice Lacking
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Statistical Analyses. Statistical analyses were performed
using analysis of variance (ANOVA) followed by the Student-
Newman-Keul’s test. Differences were considered significant
if the probability of error was less than 5%.
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Models of 5-HT1AR-Ligand Interaction. The previously
reported 3D model of the 5-HT1AR complexed with aryl-
piperazines of formula I25 was employed in the manuscript.
The conformation of Trp6.48 was modeled in the observed active
trans conformation,29 and thus, Phe6.52 was also modeled in
trans to avoid the steric clash.30 These models were placed in
a rectangular box (∼80 Å × 93 Å × 75 Å in size) containing a
lipid bilayer (∼95 molecules of palmitoyloleoylphospha-
tidylcholine and ∼13000 molecules of water), resulting in a
final density of ∼1.0 g cm-3. The ligand-receptor-lipid bilayer
systems were subjected to 500 iterations of energy minimiza-
tion and then heated to 300 K in 15 ps. This was followed by
an equilibration period (15-250ps) and a production run
(250-500ps) at constant pressure with anisotropic scaling,
using the particle mesh Ewald method to evaluate electrostatic
interaction. During the processes of minimization, heating, and
equilibration a positional restraint of 10 kcal mol-1 Å-2 was
applied to the CR atoms of the receptor structure. Structures
were collected for analysis every 10 ps during the production
run. Parameters for the system were obtained from the ff99
force field58 and the “general Amber force field” for compounds
2 and 9 using RESP point charges.59 The molecular dynamics
simulations were run with the Sander module of AMBER 8.60
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M. J.; Sanz, A. M.; Orensanz, L.; Beneitez, M. E.; Fuentes, J.
A.; Manzanares, J. Synthesis and Structure-Activity Relation-
ships of a New Model of Arylpiperazines. 1. 2-[4-(o-Methoxy-
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pyridine: A Selective 5-HT1A Receptor Agonist. J. Med. Chem.
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M. J.; Ferna´ndez, E.; Schaper, K.-J. Synthesis and Structure-
Activity Relationships of a New Model of Arylpiperazines. 2.
Three-Dimensional Quantitative Structure-Activity Relation-
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Affinity for 5-HT1A and R1 Receptors. A Comparison of CoMFA
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Acknowledgment. This work was supported by
Ministerio de Ciencia y Tecnolog´ıa (Grants BQU2001-
1459, SAF2002-04487, and SAF2004-07103), the Euro-
pean Community (Grant LSHB-CT-2003-503337), Com-
unidad Auto´noma de Madrid (Grant 08.5/0028.1/2003),
and CEPA-SCHWARZPHARMA. E. Ferna´ndez and I.
Tejada are grateful to U.N.E.D. for a predoctoral grant,
and D. Ayala is grateful to Comunidad Auto´noma de
Madrid for a postdoctoral grant.
Supporting Information Available: Spectroscopic data
of final compounds 2-4, 7-11, 13, and 14, and results from
elemental analysis. This material is available free of charge
via the Internet at http://pubs.acs.org.
(20) Lo´pez-Rodr´ıguez, M. L.; Morcillo, M. J.; Ferna´ndez, E.; Porras,
E.; Murcia, M.; Sanz, A. M.; Orensanz, L. Synthesis and
Structure-Activity Relationships of a New Model of Aryl-
piperazines. 3. 2-[ω-(4-Arylpiperazin-1-yl)alkyl]perhydropyrrolo-
[1,2-c]imidazoles and -Perhydroimidazo[1,5-a]pyridines: Study
of the Influence of the Terminal Amide Fragment on 5-HT1A
Affinity/Selectivity. J. Med. Chem. 1997, 40, 2653-2656.
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E.; Vicente, B.; Sanz, A. M.; Herna´ndez, M.; Orensanz, L.
Synthesis and Structure-Activity Relationships of a New Model
of Arylpiperazines. 4. 1-[ω-(4-Arylpiperazin-1-yl)alkyl]-3-di-
phenylmethylene-2,5-pyrrolidinediones and -3-(9H-Fluoren-9-
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of the Terminal Amide Fragment on 5-HT1A Affinity/Selectivity.
J. Med. Chem. 1999, 42, 36-49.
(22) Lo´pez-Rodr´ıguez, M. L.; Viso, A.; Benhamu´, B.; Rominguera, J.
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with Affinity for the 5-HT1A Receptor via Pd(0) Amination of
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(23) Lo´pez-Rodr´ıguez, M. L.; Morcillo, M. J.; Ferna´ndez, E.; Porras,
E.; Orensanz, L.; Beneytez, M. E.; Manzanares, J.; Fuentes, J.
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