Investigations on the 1-(2-biphenyl)piperazine motif: Identification of new potent and selective ligands for the serotonin7 (5-HT7) receptor with agonist or antagonist action in vitro or ex vivo

Article abstract of DOI:10.1021/jm3003679

Here we report the design, synthesis, and 5-HT₇ receptor affinity of a set of 1-(3-biphenyl)- and 1-(2-biphenyl)piperazines. The effect on 5-HT₇ affinity of various substituents on the second (distal) phenyl ring was analyzed. Several compounds showed 5-HT₇ affinities in the nanomolar range and >100-fold selectivity over 5-HT_1A and adrenergic α₁ receptors. 1-[2-(4-Methoxyphenyl)phenyl] piperazine (9a) showed 5-HT₇ agonist properties in a guinea pig ileum assay but blocked 5-HT-mediated cAMP accumulation in 5-HT₇- expressing HeLa cells.

Full text of DOI:10.1021/jm3003679

Article
pubs.acs.org/jmc
Investigations on the 1-(2-Biphenyl)piperazine Motif: Identification
of New Potent and Selective Ligands for the Serotonin7 (5-HT7)
Receptor with Agonist or Antagonist Action in Vitro or ex Vivo
Enza Lacivita,† Daniela Patarnello,† Nikolas Stroth,∥ Antonia Caroli,‡ Mauro Niso,†
Marialessandra Contino,† Paola De Giorgio,† Pantaleo Di Pilato,† Nicola A. Colabufo,†
Francesco Berardi,† Roberto Perrone,† Per Svenningsson,∥ Peter B. Hedlund,§ and Marcello Leopoldo*,†
†Dipartimento Farmaco-Chimico, Universita
̀
degli Studi di Bari “A. Moro”, via Orabona, 4, 70125, Bari, Italy
‡Department of Physics, Sapienza University, piazzale A. Moro, 5, 00185, Rome, Italy
§Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, United States
∥Center for Molecular Medicine, Department of Neurology and Clinical Neuroscience, Karolinska Institute and Karolinska University
Hospital, 17176 Stockholm, Sweden
S
* Supporting Information
ABSTRACT: Here we report the design, synthesis, and 5-
HT7 receptor affinity of a set of 1-(3-biphenyl)- and 1-(2-
biphenyl)piperazines. The effect on 5-HT7 affinity of various
substituents on the second (distal) phenyl ring was analyzed.
Several compounds showed 5-HT7 affinities in the nanomolar
range and >100-fold selectivity over 5-HT1A and adrenergic α1
receptors. 1-[2-(4-Methoxyphenyl)phenyl]piperazine (9a)
showed 5-HT7 agonist properties in a guinea pig ileum assay but blocked 5-HT-mediated cAMP accumulation in 5-HT7-
expressing HeLa cells.
INTRODUCTION
■
The serotonin 5-HT7 receptor was identified starting in 1993
by the application of targeted molecular biology techniques. It
has been described in various species and remains the last 5-HT
receptor to be discovered. The 5-HT7 receptor is localized in
discrete areas of the brain and in the periphery. Within the
central nervous system (CNS), high levels of this receptor have
been detected in the hippocampus, thalamus, and hypothal-
amus (especially within the suprachiasmatic nucleus).1 Nearly
two decades after its discovery, much information is available
on the pathophysiological role of 5-HT7 in the CNS.2
Pharmacological blockade of the 5-HT7 receptor by 1 (SB-
269970) (Figure 1) or inactivation of the 5-HT7 receptor gene
leads to an antidepressant-like behavioral profile in rodent
Figure 1. Structures of selective 5-HT7 receptor agents.
forced swim test and tail suspension test. It has also been
suggested that the atypical antipsychotic drug amisulpride
since subcutaneous administration of the selective 5-HT7
receptor agonist 2 (E-55888) increased the analgesic potency
of oral morphine, it has been proposed that 5-HT7 receptor
agonists could be used as adjuvants of opioid analgesia.6
Animal studies of learning and memory have suggested a
potentially important role for the 5-HT7 receptor in cognitive
processes.7 5-HT7 drugs such as 1, 3 (DR-4004), and 4 (AS-
19) (Figure 1) are able to reverse amnesia induced by post-
exerts its antidepressant action through blockade of 5-HT7
receptors.3 Recently, a study showed that the pharmacological
blockade of 5-HT7 receptors produced a faster antidepressant-
like response than the commonly prescribed antidepressant
fluoxetine.4 This is particularly interesting, since antidepressants
with a faster onset of action are an unmet need in depression
therapy.
Several studies have suggested that 5-HT7 receptors are
involved in nociceptive processing. Activation of 5-HT7
receptors exerts antinociceptive effects at the level of the spinal
cord and pronociceptive effects in the periphery.5 Moreover,
Received: March 16, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm3003679 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
training administration of scopolamine (a cholinergic antago-
nist). This supports the hypothesis that cholinergic, gluta-
matergic, and serotonergic systems interact in cognitively
impaired animals. Moreover, it shows that the 5-HT7 receptor
can significantly influence cognitive dysfunction and therefore
represents a potential therapeutic target for the treatment of
memory dysfunction in cognitive disorders (schizophrenia,
Alzheimer’s disease, age-related decline).8 Collectively, the
aforementioned findings underscore the importance of
developing new 5-HT7 receptor drugs.
Our research group has been involved for several years in the
study of structure−activity relationships (SARs) of 4-
substituted 1-arylpiperazine derivatives, the so-called “long-
chain” arylpiperazines.9−13 Our studies have led to the
identification of 5 (LP-211) (Figure 1) which showed high 5-
HT7 receptor affinity (Ki = 0.58 nM, rat cloned receptors), high
selectivity over 5-HT1A and D2 receptors (324- and 245-fold,
respectively), and agonist properties in an ex vivo assay of 5-
HT7 receptor activation. Disposition studies in mice evidenced
that 5 undergoes N-dealkylation to 6a (Table 1). This might be
of relevance because N-unsubstituted 1-arylpiperazines can
reach the brain and may display pharmacological effects
opposite to those of their parent drugs. Therefore, the final
pharmacological effect of such drugs might result from the
interplay between the neurochemical actions of the parent drug
and of its active metabolite.14 For this reason, the affinities for a
range of 5-HT receptors of 6a and 5 were evaluated under the
same experimental conditions. Compound 6a displayed higher
5-HT7 receptor affinity (Ki = 1.4 nM, human cloned receptor)
and overall better selectivity profile than 5 (Ki = 15 nM, human
cloned receptor).15 This was unexpected because the majority
of the long-chain arylpiperazine derivatives described in the
literature display higher 5-HT7 receptor affinity than the N-(4)-
unsubstituted counterparts.11,16,17 Thus, compound 6a at-
tracted our attention as a low molecular weight lead compound
that could deliver a completely new set of selective 5-HT7
receptor ligands.
CHEMISTRY
■
The synthesis of the target 1-arylpiperazine derivatives 6a,b−
12a,b (Table 1) required the key aniline intermediates 13a,b−
17a,b, 20a,b, and 21a,b (Scheme 1). Among these, 13a,b were
a
Scheme 1. Synthesis of Target Compounds 6a,b−12a,b
Table 1. Binding Affinities of Target Compounds
a
Reagents: (A) Pd(dppf)Cl2 and 5 N NaOH or tetrakis-
(triphenylphosphino)palladium(0) and 2 M Na2CO3; (B) ammonium
formate, 10% Pd/C; (C) bis(2-chloroethyl)amine hydrochloride,
K2CO3, KI.
commercially available, whereas the remaining anilines where
prepared according to literature methods by cross-coupling
reaction of 2- or 3-iodoaniline and the appropriate
benzeneboronic acid under Suzuki conditions (14a,b−17a,b)
or through catalytic reduction of nitroderivatives 18a,b and
19a,b (anilines 20a,b and 21a,b). The target compounds 6a,b−
12a,b were obtained by condensing the intermediate anilines
13a,b−17a,b, 20a,b, and 21a,b with bis(2-chloroethyl)amine
hydrochloride.
RESULTS AND DISCUSSION
■
Structure−Activity Relationships for 5-HT7 Receptor.
The notion that N-(4)-unsubstituted 1-arylpiperazines can bind
to the 5-HT7 receptor was reported by Shen et al. in 1993.18 In
particular, it was shown that 1-(1-naphthalenyl)piperazine, 1-
(2-methoxyphenyl)piperazine, and 1-(3-chlorophenyl)-
piperazine can bind at 5-HT7 receptors with moderate to
good affinities (Ki = 83 nM, 243 nM, and 352 nM,
respectively). Over the years, the above-mentioned 1-
arylpiperazines were used as starting points for the develop-
ment of several classes of long-chain arylpiperazine derivatives.
a
Values are the mean SEM from three independent experiments.
b
c
Data taken from ref 15. Displacement of [3H]prazosin from rat
cerebral cortex membranes by a single concentration of test compound
(100 nM). Data are the mean of two independent experiments.
d
Displacement of [3H]-8-OH-DPAT from human cloned 5-HT1A
receptors by a single concentration of test compound (100 nM).
Data are the mean of two independent experiments.
B
dx.doi.org/10.1021/jm3003679 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. (A) Main interactions of 6a (yellow), 7a (cyan), 8a (green), 9a (magenta), and 10a (blue) within the 5-HT7 receptor binding site. The
protonated piperazine N(4) nitrogen forms an ionic interaction with D3.32 at the distance of 2.57 Å. The oxygen of methoxy group of 9a forms
hydrogen-bond contacts with S5.42 and Y5.38 at distances of 3.4 and 3.1 Å, respectively. The same pattern of interactions is shown by 7a and 10a.
(B) Main interactions of 12b within the 5-HT7 receptor binding site. (C) Main interactions of 22a,b and 23a,b within the 5-HT7 receptor binding
site.
In many cases the introduction of a large substituent on the
basic nitrogen of the piperazine ring had a beneficial effect on
the affinity and specificity for the 5-HT7 receptor.11,16,17
Therefore, it was unexpected that removal of the pendant
substituent from the basic nitrogen of 5 afforded the potent and
selective 5-HT7 ligand 6a. Since no studies dealing with N-(4)-
unsubstituted 1-arylpiperazines as 5-HT7 receptor ligands can
be found in the literature, we decided to explore the SARs of a
set of compounds structurally related to 6a. For our study, we
have evaluated the 1-(2-biphenyl)piperazine derivatives 6a−
12a, focusing on the aromatic ring denoted as Ar2 in the general
structure reported in Table 1.
7.1 nM), which has higher affinity than its isomer 12a (Ki = 96
nM).
To rationalize the SARs, we developed a pharmacophore
model (see Supporting Information). Matrix distances between
the pharmacophoric features (i.e., a positively charged nitrogen
atom and two aromatic rings that corresponded to Ar1 and Ar2)
were in general agreement with those reported in the
pharmacophore models by Kolaczkowski et al.19 (the general
“affinity” hypothesis) and by Badarau et al.20 (the “second
hypothesis”). The analysis of aligned target compounds clearly
indicated that the pharmacophoric groups of compounds 6a−
12a matched the features of the pharmacophore model more
closely than those of compounds 6b−12b (Figure SI3,
Supporting Information). Next, docking simulations were
conducted by use of the Autodock4 program21 to analyze the
binding mode of 6a,b−12a,b. Kolaczkowski et al.19 defined the
5-HT7 receptor binding site as constituted by the interaction
point of the positively charged nitrogen atom and two pockets:
one localized between transmembrane helices (TMHs) 4−6 in
a deep cavity and the other one between TMHs 7−3 in the
extracellular exposed area. According to our docking studies,
the compounds reported in Table 1 preserve the crucial ionic
interaction between protonated piperazine N(4) nitrogen with
D3.32 (according to Ballesteros and Weinstein)22 and the
CH−π interaction between the phenyl ring attached to the
piperazine ring (Ar1) with F6.51 (Figure 2A). Moreover, the
compounds with the highest affinities also showed CH−π
interaction between Ar2 and F6.52. The binding mode analysis
suggested that the TMH 4−6 pocket is not large enough to
accommodate the Ar2 group when it is in the 3-position of Ar1,
thus explaining the lower affinity of 6b−11b as compared to
6a−11a. In particular, the conformations proposed for 9b do
not allow the crucial ionic interaction with D3.32. On the other
hand, a different binding scenario could be hypothesized for
compound 12b: the methoxy group formed H-bonding
interactions with Y5.38 and S5.42 that drove Ar2 into a
hydrophobic pocket formed by I3.29, V3.33, and F6.52 (Figure
2B). Consequently, the structure-based conformation of 12b
showed that the orientation and distances of the essential triplet
features were comparable with those observed for the
compounds with highest affinity. To check this further, we
have studied an additional small set of compounds having a 2-
methoxy- or a 2-methyl group on Ar2, with Ar2 in either 2- or 3-
position on Ar1 (compounds 22a,b and 23a,b, Table 1). These
compounds were prepared following the synthetic routes
In particular, we wanted to explore if introduction of
substituent(s) that could modify the electronic properties or
the steric hindrance of the Ar2 group had an effect on 5-HT7
receptor affinity. We also evaluated the affinity for the 5-HT7
receptor of the corresponding 1-(3-biphenyl)piperazine
counterparts 6b−12b. The 1-(4-biphenyl)piperazine frame-
work was not taken into consideration at this stage because a
previous study showed that 1-(4-substituted-phenyl)piperazines
were nearly devoid of 5-HT7 receptor affinity.11 The binding
affinity values of the target compounds at the 5-HT7 receptor
are shown in Table 1. In comparison of the 5-HT7 affinity
values of 6a (Ar2 = phenyl), 7a (Ar2 = 4-fluorophenyl), 8a (Ar2
= 3,4-dimethylphenyl), 9a (Ar2 = 4-methoxyphenyl), and 10a
(Ar2 = 4-nitrophenyl), no great differences can be noted. This
indicates that there is no preference between electron-rich and
electron-poor aromatic rings in that position. Introduction of
an aza group in 6a afforded compound 11a (Ar2 = 2-pyridyl),
causing a >10-fold loss in affinity. The presence of substituents
in different positions of the Ar2 ring had different effects on 5-
HT7 receptor affinity. In particular, 8a (Ar2 = 3,4-
dimethylphenyl) and 9a (Ar2 = 4-methoxyphenyl) displayed
affinities in the same range as 6a, suggesting that the binding
pocket of the receptor is large enough to accommodate such
substituents. By contrast, a marked decrease in affinity was
observed in the case of 12a (Ar2 = 2,6-dimethoxyphenyl). Next,
we evaluated the effect on 5-HT7 receptor affinity of shifting
the Ar2 ring from 2- to 3-position (derivatives 6b−12b). This
modification reduced the 5-HT7 receptor affinity (except for
12b) to various extents: compound 10b showed only half the
affinity of its isomer 10a, whereas 9b was devoid of 5-HT7
affinity (Ki > 5000 nM), contrary to the isomer 9a (Ki = 2.6
nM). A notable exception to this trend was derivative 12b (Ki =
C
dx.doi.org/10.1021/jm3003679 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
depicted in Scheme 1 (see Supporting Information). As in the
case of compounds 12a,b, 1-(3-biphenyl)piperazine derivatives
22b and 23b showed higher 5-HT7 receptor affinity than the 1-
(2-biphenyl) counterparts 22a and 23a, but in this case, a small
difference was shown. Docking analysis suggested that the
methoxy or methyl substituent in 2-position of compounds
22a,b and 23a,b interacts with the pocket created by I3.29,
V3.33, M3.34, T3.37, and I4.37 residues and allows optimal
interaction of Ar1 and Ar2 with F6.51 and F6.52, respectively
(Figure 2C). Therefore, affinity data and docking analysis of
compounds 12a,b, 22a,b, and 23a,b indicate that the
introduction of small substituents in one or both ortho-
positions of Ar2 enables the molecules to adopt the optimal
conformation for interaction through hydrophobic or hydro-
gen-bonding interactions. All in all, the binding mode analysis
evidenced that the interaction of biphenyl piperazines with the
5-HT7 receptor is driven essentially by steric and hydrophobic
requirements. In the case of compound 12b, the hydrogen-
bond formation with Y5.38 and S5.42 is fundamental for the
optimal orientation of Ar2.
Figure 3. Percent relaxation of substance P-mediated contraction
induced by 5-HT7 agonists in isolated guinea pig ileum.
other hand, 7a and 9a were able to induce 5-HT7 receptor-
mediated relaxation at concentrations of 0.5 μM and 1 μM,
because the effects were abolished by 1 (3 μM). Finally, 4
induced relaxation at concentrations of 1 μM and 2 μM but not
at 0.5 μM (data not shown), the effects being reverted by 1 (3
μM). All in all, these data indicate that compounds 7a and 9a
behave as 5-HT7 competitive agonists in this assay with
specificity as good as 4, contrary to 6a and 8b.
Selectivity over 5-HT1A and Adrenergic α1 Receptors.
Target compounds were counterscreened for their affinity
against the serotonin 5-HT1A receptor because it is colocalized
with the 5-HT7 receptor. Also, target compounds were
evaluated for their adrenergic α1 receptor affinity because it
has been reported that the 1-(2-biphenyl)piperazinyl scaffold
might have affinity for this receptor.23 As far as the affinity for
5-HT1A receptors is concerned, all the 2-biphenyl derivatives
displayed poor affinities except 9a (Ar = 4-methoxyphenyl, Ki =
476 nM). On the other hand, the 3-biphenyl derivatives
showed a different trend. Compounds 6b, 7b, 10b, and 11b
displayed good 5-HT1A receptor affinity (72.1 nM < Ki < 14.9
nM), differently from 8b and 9b. With regard to the adrenergic
α1 receptor, all compounds showed weak affinities, except 6a
and 10a (Ki = 59.1 nM and 105 nM, respectively). Taken
together, these data indicated that the 1-(2-biphenyl)piperazine
motif can deliver high-affinity 5-HT7 ligands and that an
appropriate substitution pattern around the Ar2 ring can lead to
selective 5-HT7 receptor ligands.
Activity at 5-HT7 Receptors Stably Expressed in HeLa
Cells. Compounds 6a and 9a were active in the guinea pig
ileum assay, but the effect of 6a appeared not to be specific to
5-HT7 (no blockade with 1). To further characterize their
function, we therefore tested both compounds against 5-HT7(a)
receptors stably expressed in HeLa cells. Neither 6a nor 9a
stimulated cAMP accumulation (data not shown), and both
compounds behaved as antagonists instead. Thus, cAMP
accumulation induced by 1 μM 5-HT was inhibited by 6a
and 9a with IC50 values of 531 and 438 nM, respectively
(Figure 4A). To confirm their inhibitory action at recombinant
Activation of 5-HT7 Receptors in Guinea Pig Ileum. 5-
HT7 receptor activation is known to mediate relaxation of
gastrointestinal smooth muscle. Previous studies have demon-
strated that activation of 5-HT7 receptors inhibits contractions
induced by substance P in the guinea pig ileum.24 We
investigated the effect of compounds 6a−9a, which showed
the highest 5-HT7 receptor affinity and selectivity values among
the newly reported compounds, on substance P-induced
contractions in the guinea pig ileum. The assays were
conducted in the presence of a cocktail of non-5-HT7 receptor
antagonists (see Supporting Information) in order to elucidate
whether or not the relaxation response in this particular tissue is
due to direct activation of the 5-HT7 receptor. For comparative
purposes, we also tested 4, a selective 5-HT7 ligand that
behaves as a potent (EC50 = 9 nM) partial 5-HT7 receptor
agonist (77% maximal effect compared to 5-HT) toward cAMP
accumulation in HEK-293F/h5-HT7 cells.25 As already
reported,11 the 5-HT7 agonist 5-CT was able to reduce 40%
of the contraction induced by substance P, an effect that was
competitively reverted by the antagonist 1. Compounds 6a−9a
were able to induce relaxation of substance P-mediated
contraction (Figure 3). However, the effects elicited by 6a
(0.5 μM and 1 μM) and 8a (2 μM) were not 5-HT7 receptor-
mediated because they were not reverted by 1 (3 μM). On the
Figure 4. Effects of compounds 6a and 9a on (A) 5-HT- and (B)
forskolin-induced cAMP accumulation in vitro.
5-HT7(a), the compounds were compared with established
receptor antagonists regarding their capacity to block forskolin-
induced cAMP. Similar to 1, clozapine, and ketanserin, 6a and
9a significantly inhibited cAMP accumulation, thus providing
further evidence for antagonist effects, at least in the context of
a heterologous system (Figure 4B).
CONCLUSIONS
■
The main aim of the present study was exploration of the SARs
of a set of 1-(2-biphenyl)- and 1-(3-biphenyl)piperazines,
toward the goal of identifying new high-affinity 5-HT7 ligands.
This goal was achieved, as several compounds with Kis in the
low nanomolar range were identified. The SARs showed that
various substituents can be introduced in the distal phenyl ring
of the 1-(2-biphenyl)piperazine scaffold without affecting 5-
HT7 receptor affinity. On the other hand, the 1-(3-biphenyl)-
piperazine scaffold appears to be more sensitive to the presence
and position of substituents. Considering the selectivity over 5-
D
dx.doi.org/10.1021/jm3003679 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Measurement of Intracellular cAMP Accumulation. HeLa
cells stably expressing human 5-HT7(a) (kindly provided by Dr. Mark
Hamblin) were cultured as described32 and seeded into 96-well plates
before treatment. Intracellular cAMP was measured by a commercial
enzyme-linked immunosorbent assay (ELISA; Abnova, Taipei City,
Taiwan).
HT1A and α1 receptors, it is noteworthy that, despite their low
molecular weight, several ligands herein reported showed
affinities 2 orders of magnitude lower than those at 5-HT7
receptors. All in all, affinity and computational data indicate
that both 1-(2-biphenyl)- and 1-(3-biphenyl)piperazine scaf-
folds can deliver high-affinity 5-HT7 ligands or can be starting
points for development of newer long-chain arylpiperazines
capable of binding at 5-HT7 receptors.
ASSOCIATED CONTENT
■
S
* Supporting Information
Investigation of signaling through the 5-HT7 receptor yielded
interesting results. While compounds 9a and 10a induced
relaxation of guinea pig ileum in the same fashion as 5-CT and
the selective 5-HT7 receptor agonist 4, compounds 6a and 9a
failed to stimulate cAMP accumulation in 5-HT7a-expressing
HeLa cells. Instead, inhibition was observed when cells were
cotreated with 5-HT and 6a or 9a. Moreover, similar to
established antagonists of 5-HT7,26 both compounds inhibited
forskolin-stimulated cAMP. Thus, at least in the context of our
heterologous system, 6a and 9a displayed antagonist properties.
It has previously been shown that a given 5-HT receptor
ligand can act as agonist in one and antagonist in another
receptor-expressing system. For example, the 5-HT4 ligand 4-
amino-5-chloro-2-methoxybenzoic acid 2-(1-piperidinyl)ethyl
ester (ML 10302) is a partial agonist in guinea pig ileum but an
antagonist of cAMP accumulation in guinea pig hippocampus.27
Similarly, partial agonist as well as antagonist properties at
central 5-HT1A receptors have been described for 8-{2-[4-
(methoxyphenyl)-1-piperazinyl]ethyl}-8-azaspiro[4.5]decane-
7,9-dione (BMY 7378).28 Our present results demonstrate that
dual agonist/antagonist ligands also exist for the 5-HT7
receptor, exemplified by compound 9a. In light of this finding,
we believe that the reinvestigation of agonists and antagonists
previously published by our laboratory and others will tell us if
the dual agonist/antagonist feature of 9a is shared by other 5-
HT7 ligands. This might ultimately help to explain the observed
inconsistencies in the role of 5-HT7 receptors in the CNS.8
Additional text, three figures, and two tables with synthetic
details and spectral data for 6a,b−12a,b, 15a,b, 17a,b, and
20a,b−23a,b; elemental analysis of 6a,b−12a,b, 22a,b, and
23a,b; matrix distances; molecular modeling and molecular
docking methods; PHASE alignment analysis; training set for
pharmacophore model identification; list of inactive com-
pounds; and biological methods and statistical analysis. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone +39-080-5442798; fax 39-080-5442231; e-mail
leopoldo@farmchim.uniba.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Research was performed as part of the “ADHD-sythe” young-
investigator project (principal investigator, W. Adriani; local
unit, E.L.), assigned by the Italian Ministry of Health, “under-40
call 2007”. Studies by P.S. and N.S. were funded by
Vetenskapsradet (Swedish Research Council). “Fondazione
̊
Roma” is gratefully acknowledged for the financial support to
A.C.
EXPERIMENTAL SECTION
■
REFERENCES
■
General Procedure for the Preparation of Piperazines 6a,b−
12a,b, 22a,b, and 23a,b. A mixture of the appropriate aniline (4.0
mmol), bis(2-chloroethyl)amine hydrochloride (0.71 g, 4.0 mmol),
K2CO3 (0.55 g, 4.0 mmol), and KI (0.66 g, 4.0 mmol) in xylene (30
mL) was refluxed for 48 h. After cooling, the solvent was distilled off in
vacuo and the residue was taken up with AcOEt (30 mL) and 5%
aqueous NaOH solution (30 mL). The organic phase was separated
and then washed with brine. The organic phase was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The
crude product was chromatographed with CHCl3/MeOH, 9:1, to
afford the desired compounds as pale yellow oil. The purity of the
tested compounds 6a,b−12a,b, 22a,b, and 23a,b was assessed by RP-
HPLC and combustion analysis. All compounds showed ≥95% purity.
1-[2-(4-Methoxyphenyl)phenyl]piperazine (9a). Yield 30%. 1H
NMR (CDCl3) δ 1.71 (br s, 1H, D2O exchanged), 3.12 (br s, 8H),
3.84 (s, 3H), 6.94 (d, 2H, J = 8.8 Hz), 7.00 (d, 1H, J = 8.0 Hz), 7.10−
7.15 (m, 1H), 7.22−7.30 (m, 2H), 7.46 (d, 2H, J = 8.8 Hz). GC-MS
m/z 269 (M+ + 1, 11), 268 (M+, 56), 226 (100), 210 (43), 167 (24).
The hydrochloride salt melted at 198−200 °C (from MeOH/Et2O).
Anal. (C17H20N2O·HCl·0.5 H2O) C, H, N.
(1) Hedlund, P. B.; Sutcliffe, J. G. Functional, molecular and
pharmacological advances in 5-HT7 receptor research. Trends
Pharmacol. Sci. 2004, 25, 481−486.
(2) Leopoldo, M.; Lacivita, E.; Berardi, F.; Perrone, R.; Hedlund, P.
B. Serotonin 5-HT7 receptor agents: Structure-activity relationships
and potential therapeutic applications in central nervous system
disorders. Pharmacol. Ther. 2011, 129, 120−148.
(3) Abbas, A. I.; Hedlund, P. B.; Huang, X. P.; Tran, T. B.; Meltzer,
H. Y.; Roth, B. L. Amisulpride is a potent 5-HT7 antagonist: relevance
for antidepressant actions in vivo. Psychopharmacology (Berlin) 2009,
205, 119−128.
(4) Mnie-Filali, O.; Faure, C.; Lambas
Belblidia, H.; Gondard, E.; Etievant, A.; Scarna, H.; Didier, A.; Berod,
A.; Blier, P.; Haddjeri, N. Pharmacological blockade of 5-HT7
receptors as a putative fast acting antidepressant strategy. Neuro-
psychopharmacology 2011, 36, 1275−1288.
(5) Brenchat, A.; Zamanillo, D.; Hamon, M.; Romero, L.; Vela, J. M.
Role of peripheral versus spinal 5-HT7 receptors in the modulation of
pain undersensitizing conditions. Eur. J. Pain 2012, 16, 72−81.
(6) Brenchat, A.; Ejarque, M.; Zamanillo, D.; Vela, J. M.; Romero, L.
Potentiation of morphine analgesia by adjuvant activation of 5-HT7
receptors. J. Pharmacol. Sci. 2011, 116, 388−391.
(7) Roberts, A. J.; Hedlund, P. B. The 5-HT7 receptor in learning and
memory. Hippocampus 2012, 22, 762−771.
(8) Matthys, A.; Haegeman, G.; Van Craenenbroeck, K.;
Vanhoenacker, P. Role of the 5-HT7 receptor in the central nervous
system: from current status to future perspectives. Mol. Neurobiol.
2011, 43, 228−253.
́
-Senas, L.; El Mansari, M.;
̃
́
Radioligand Binding Assays. Binding of [3H]-5-CT at human
cloned 5-HT7 receptor was performed according to Jasper et al.29
Binding of [3H]-8-OH-DPAT at human cloned 5-HT1A receptors was
performed as previously described.30 Binding of [3H]prazosin at α1-
adrenoceptors was performed according to Glossmann and
Hornung.31
Isolated Guinea Pig Ileum Assay. Inhibition of substance P-
induced contraction in guinea pig ileum by 5-HT7 receptor agonists
was performed as previously described.11
E
dx.doi.org/10.1021/jm3003679 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(9) Perrone, R.; Berardi, F.; Colabufo, N. A.; Lacivita, E.; Leopoldo,
M.; Tortorella, V. Synthesis and structure−affinity relationships of 1-
[omega-(4-aryl-1-piperazinyl)alkyl]-1-aryl ketones as 5-HT7 receptor
ligands. J. Med. Chem. 2003, 46, 646−649.
(10) Leopoldo, M.; Berardi, F.; Colabufo, N. A.; Contino, M.;
Lacivita, E.; Perrone, R.; Tortorella, V. Studies on 1-arylpiperazine
derivatives with affinity for rat 5-HT7 and 5-HT1A receptors. J. Pharm.
Pharmacol. 2004, 56, 247−255.
(11) Leopoldo, M.; Berardi, F.; Colabufo, N. A.; Contino, M.;
Lacivita, E.; Niso, M.; Perrone, R.; Tortorella, V. Structure−affinity
relationship study on N-(1,2,3,4-tetrahydronaphthalen-1-yl)-4-aryl-1-
piperazinealkylamides, a new class of 5-hydroxytryptamine7 receptor
agents. J. Med. Chem. 2004, 47, 6616−6624.
(12) Leopoldo, M.; Lacivita, E.; Contino, M.; Colabufo, N. A.;
Berardi, F.; Perrone, R. Structure−activity relationship study on N-
(1,2,3,4-tetrahydronaphthalen-1-yl)-4-aryl-1-piperazinehexanamides, a
class of 5-HT7 receptor agents. 2. J. Med. Chem. 2007, 50, 4214−4221.
(13) Leopoldo, M.; Lacivita, E.; De Giorgio, P.; Fracasso, C.;
Guzzetti, S.; Caccia, S.; Contino, M.; Colabufo, N. A.; Berardi, F.;
Perrone, R. Structural modifications of N-(1,2,3,4-tetrahydronaph-
thalen-1-yl)-4-aryl-1-piperazinehexanamides: influence on lipophilicity
and 5-HT7 receptor activity. Part III. J. Med. Chem. 2008, 51, 5813−
5822.
D.; Vela, J. M. 5-HT7 receptor activation inhibits mechanical
hypersensitivity secondary to capsaicin sensitization in mice. Pain
2009, 141, 239−247.
(26) Klein, M. T.; Teitler, M. Antagonist interaction with the human
5-HT7 receptor mediates the rapid and potent inhibition of non-G-
protein-stimulated adenylate cyclase activity: a novel GPCR effect. Br.
J. Pharmacol. 2011, 162, 1843−1854.
(27) Hegde, S. S.; Eglen, R. M. Peripheral 5-HT4 receptors. FASEB J.
1996, 10, 1398−1407.
(28) Sharp, T.; Backus, L. I.; Hjorth, S.; Bramwell, S. R.; Grahame-
Smith, D. G. Further investigation of the in vivo pharmacological
properties of the putative 5-HT1A antagonist, BMY 7378. Eur. J.
Pharmacol. 1990, 176, 331−340.
(29) Jasper, J. R.; Kosaka, A.; To, Z. P.; Chang, D. J.; Eglen, R. M.
Cloning, expression and pharmacology of a truncated splice variant of
the human 5-HT7 receptor (h5-HT7b). Br. J. Pharmacol. 1997, 122,
126−132.
(30) Fargin, A.; Raymond, J. R.; Regan, J. W.; Cotecchia, S.;
Lefkowitz, R. J.; Caron, M. G. Effector coupling mechanisms of the
cloned 5-HT1A receptor. J. Biol. Chem. 1989, 264, 14848−14852.
(31) Glossmann, H.; Hornung, R. alpha-Adrenoceptors in rat brain:
sodium changes the affinity of agonists for prazosin sites. Eur. J.
Pharmacol. 1980, 61, 407−408.
(32) Sjogren, B.; Hamblin, M. W.; Svenningsson, P. Cholesterol
̈
(14) Caccia, S. N-dealkylation of arylpiperazine derivatives:
disposition and metabolism of the 1-aryl-piperazines formed. Curr.
Drug Metab. 2007, 8, 612−622.
depletion reduces serotonin binding and signaling via human 5-HT7a
receptors. Eur. J. Pharmacol. 2006, 552, 1−10.
(15) Hedlund, P. B.; Leopoldo, M.; Caccia, S.; Sarkisyan, G.;
Fracasso, C.; Martelli, G.; Lacivita, E.; Berardi, F.; Perrone, R. LP-211
is a brain penetrant selective agonist for the serotonin 5-HT7 receptor.
Neurosci. Lett. 2010, 481, 12−16.
(16) Medina, R. A.; Sallander, J.; Benhamu, B.; Porras, E.; Campillo,
́
́
M.; Pardo, L.; Lopez-Rodríguez, M. L. Synthesis of new serotonin 5-
HT7 receptor ligands. Determinants of 5-HT7/5-HT1A receptor
selectivity. J. Med. Chem. 2009, 52, 2384−2392.
(17) Volk, B.; Barkoc
́
zy, J.; Hegedus, E.; Udvari, S.; Gacsal
́
yi, I.;
Mezei, T.; Pallagi, K.; Kompagne, H.; Levay, G.; Egyed, A.; Harsing, L.
́
́
G.; Spedding, M.; Simig, G. (Phenylpiperazinyl-butyl)oxindoles as
selective 5-HT7 receptor antagonists. J. Med. Chem. 2008, 51, 2522−
2532.
(18) Shen, Y.; Monsma, F. J., Jr.; Metcalf, M. A.; Jose, P. A.; Hamblin,
M. W.; Sibley, D. R. Molecular cloning and expression of a 5-
hydroxytryptamine7 serotonin receptor subtype. J. Biol. Chem. 1993,
268, 18200−18204.
(19) Kołaczkowski, M.; Nowak, M.; Pawłowski, M.; Bojarski, A. J.
Receptor-based pharmacophores for serotonin 5-HT7R antagonists
implications to selectivity. J. Med. Chem. 2006, 49, 6732−6741.
(20) Badarau, E.; Bugno, R.; Suzenet, F.; Bojarski, A. J.; Finaru, A. L.;
Guillaumet, G. SAR studies on new bis-aryls 5-HT7 ligands: Synthesis
and molecular modeling. Bioorg. Med. Chem. 2010, 18, 1958−1967.
(21) Goodsell, D. S.; Morris, G. M.; Olson, A. J. Automated docking
of flexible ligands: applications of AutoDock. J. Mol. Recognit. 1996, 9,
1−5.
(22) Ballesteros, J. A.; Weinstein, H. Integrated methods for
modeling G-protein coupled receptors. Methods Neurosci. 1995, 25,
366−428.
(23) Elworthy, T. R.; Ford, A. P.; Bantle, G. W.; Morgans, D. J.;
Ozer, R. S.; Palmer, W. S.; Repke, D. B.; Romero, M.; Sandoval, L.;
́
Sjogren, E. B.; Talamas, F. X.; Vazquez, A.; Wu, H.; Arredondo, N. F.;
Blue, D. R.; De Sousa, A.; Gross, L. M.; Kava, M. S.; Lesnick, J. D.;
Vimont, R. L.; Williams, T. J.; Zhu, Q. M.; Pfister, J. R.; Clarke, D. E.
N-arylpiperazinyl-N′-propylamino derivatives of heteroaryl amides as
functional uroselective alpha 1-adrenoceptor antagonists. J. Med. Chem.
1997, 40, 2674−2687.
(24) Carter, D.; Champney, M.; Hwang, B.; Eglen, R. M.
Characterization of a postjunctional 5-HT receptor mediating
relaxation of guinea-pig isolated ileum. Eur. J. Pharmacol. 1995, 280,
243−250.
(25) Brenchat, A.; Romero, L.; García, M.; Pujol, M.; Burgueno, J.;
̃
Torrens, A.; Hamon, M.; Baeyens, J. M.; Buschmann, H.; Zamanillo,
F
dx.doi.org/10.1021/jm3003679 | J. Med. Chem. XXXX, XXX, XXX−XXX