Bivalent ligand approach on 4-[2-(3-methoxyphenyl)ethyl]-1-(2-methoxyphenyl)piperazine: Synthesis and binding affinities for 5-HT7 and 5-HT1A receptors

Article abstract of DOI:10.1016/j.bmc.2007.05.010

We here report on the synthesis and binding properties at 5-HT₇ and 5-HT_1A receptors of ligands 3-12, that were designed according to the 'bivalent ligand' approach. Two moieties of the 5-HT₇/5-HT_1A ligand 4-[2-(3-methoxyphenyl)ethyl]-1-(2-methoxyphenyl)piperazine (1) were linked through their 3-methoxy substituent by polymethylene chains of variable length, with the aim to increase the affinity for 5-HT₇ receptor and the selectivity over 5-HT_1A receptors. In the best cases, the dimers showed affinities for 5-HT₇ receptors as high as the monomer with no improvement in selectivity. Some dimers displayed 5-HT_1A receptor affinities slightly higher than monomer 1.

Full text of DOI:10.1016/j.bmc.2007.05.010

Bioorganic & Medicinal Chemistry 15 (2007) 5316–5321
Bivalent ligand approach on 4-[2-(3-methoxyphenyl)ethyl]-
1-(2-methoxyphenyl)piperazine: Synthesis and
binding affinities for 5-HT7 and 5-HT1A receptors
Marcello Leopoldo,* Enza Lacivita, Nicola A. Colabufo,
Mauro Niso, Francesco Berardi and Roberto Perrone
`
Universita degli Studi di Bari, Dipartimento Farmaco-Chimico, via Orabona, 4, 70125 Bari, Italy
Received 6 March 2007; revised 23 April 2007; accepted 2 May 2007
Available online 6 May 2007
Abstract—We here report on the synthesis and binding properties at 5-HT7 and 5-HT1A receptors of ligands 3–12, that were
designed according to the ‘bivalent ligand’ approach. Two moieties of the 5-HT7/5-HT1A ligand 4-[2-(3-methoxyphenyl)ethyl]-1-
(2-methoxyphenyl)piperazine (1) were linked through their 3-methoxy substituent by polymethylene chains of variable length, with
the aim to increase the affinity for 5-HT7 receptor and the selectivity over 5-HT1A receptors. In the best cases, the dimers showed
affinities for 5-HT7 receptors as high as the monomer with no improvement in selectivity. Some dimers displayed 5-HT1A receptor
affinities slightly higher than monomer 1.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
binding studies in rat suggested that chronic antidepres-
sant treatment results in a functional downregulation of
5-HT7-like binding sites in the hypothalamus.6 New
insights concerning the role of 5-HT7 receptors come
from studies performed with selective 5-HT7 receptor
antagonists or 5-HT7 receptor knockout (KO) mice. 5-
HT7 receptor KO mice displayed behavioral and sleep
patterns consistent with an antidepressant-like profile.7
Structural, functional, and pharmacological characteris-
tics identify seven major classes of 5-HT receptors. The
5-HT7 receptor has been cloned from mouse, rat, gui-
nea-pig, human, and pig. The receptor binding profile
is consistent across species and between cloned and na-
tive 5-HT7 receptors. 5-HT7 receptors are defined
pharmacologically by their high affinity for 5-CT,
5-HT, 5-MeOT, and methiothepin, moderate affinity
for 8-OH-DPAT and ritanserin, and low affinity for pin-
dolol, sumatriptan, and buspirone.1 5-HT7 receptors
couple positively to adenylyl cyclase when expressed in
cell lines.2,3 The distribution of 5-HT7 receptor in the
central nervous system and initial pharmacological stud-
ies using nonselective ligands have suggested that 5-HT7
receptors may modulate 5-HT-induced effects on sup-
rachiasmatic nucleus neuronal activity.4 Thus, 5-HT7
receptors may be implicated in the regulation of mam-
malian circadian rhythms5 and, as such, be associated
with sleep disorders and depression. In support of a role
for 5-HT7 receptors in depression, in vitro radioligand
O
O
O
N
N
S
S
N
O
OH
N
SB-269970
SB-258719
N
O
N
H
Keywords: 5-HT7 receptors; 5-HT1A receptors; Arylpiperazine; Struc-
ture–affinity relationships; Bivalent ligands.
DR 4004
Figure 1. Structures of 5-HT7 receptor antagonists.
*
Corresponding author. Tel.: +39 080 5442798; fax: +39 080
5442231; e-mail: leopoldo@farmchim.uniba.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.010

M. Leopoldo et al. / Bioorg. Med. Chem. 15 (2007) 5316–5321
5317
The same robust antidepressant-like effects can be gener-
ated in wild type mice by the 5-HT7 receptor antagonists
SB-258719 and SB-269970 (Fig. 1).8–10 The involvement
of 5-HT7 receptors in other pathophysiological mecha-
nisms has been highlighted by various studies. Adminis-
structure (Fig. 2, general structures I, II, and III). A
major issue of the arylpiperazine scaffold was the affinity
also for other monoaminergic receptors. In particular,
the 5-HT1A receptor affinity is here relevant because it
may interfere with the evaluation of pharmacological
actions mediated by 5-HT7 receptors.20 Suitable struc-
tural modifications on structures I and II have led us
to identify potent 5-HT7 receptor ligands endowed with
about 200-fold selectivity over 5-HT1A receptor. By con-
trast, we failed to increase the selectivity over 5-HT1A
receptor of the compounds with structure III. Among
the different methods currently available for medicinal
chemists to design potent and selective receptor subtype
ligands, the ‘bivalent ligand’ approach appears very
promising. A bivalent ligand is defined as a molecule
that contains two pharmacophores linked through a
spacer. The rationale for employing the bivalent ligand
approach stems from the possibility that dimeric struc-
tures may be capable of bridging independent recogni-
tion sites (i.e., two recognition sites on a receptor
dimer or one receptor and an accessory site) resulting
in a thermodinamically more favorable binding interac-
tion than a monovalent binding of two molecules,21–23
thus giving enhanced activity. Portoghese and cowork-
ers applied first this approach in the field of opioid re-
search, obtaining excellent results in terms of affinity
and selectivity among opioid receptor subtypes.24–27
This concept has been applied also to 5-HT1B/1D ago-
nists,28 5-HT4 ligands,29 serotonin reuptake inhibitors,30
muscarinic agonists,31 and melatonergic ligands.32
Therefore, with the aim to enhance the specificity for
the 5-HT7 receptor of compounds with general structure
III, we decided to apply the bivalent ligand approach.
To the best of our knowledge, this is the first time that
this approach has been applied to 5-HT7 receptor
ligands. For our purpose, we have selected as pharmaco-
phore the 5-HT7/5-HT1A ligand 4-[2-(3-methoxyphe-
nyl)ethyl]-1-(2-methoxyphenyl)piperazine (1) (Fig. 2) in
order to design selective 5-HT7 receptor ligands.
tration of DR4004,
a
selective 5-HT7 receptor
antagonist (Fig. 1), significantly inhibited the explor-
atory behavior of mice, thus suggesting that 5-HT7
receptor blockade might produce changes in some com-
ponent of emotionality in a novel environment.11 5-HT7
receptors are present in microglial cells that represent
the resident immune cells of the brain. As such, they
are involved in neuroinflammatory processes and can
release inflammatory products like interleukin-6 (IL-6).
It was found that activation of 5-HT7 receptor stimu-
lated the expression of IL-6 mRNA in MC-3 cells. This
effect was abolished by the selective 5-HT7 receptor
antagonist SB-269970.12 An immunocytochemical study
of 5-HT7 receptor distribution at the lumbar level of the
spinal cord evidenced immunolabeling mainly localized
in the two superficial laminae of the dorsal horn and
in small and medium-sized dorsal root ganglion cells.
Such localization is consistent with a predominant role
in nociception.13 Finally, analysis of 5-HT7, receptors
mRNA expression combined with selective stimulation
or blockage of 5-HT7 receptors demonstrated an associ-
ation between 5-HT7 receptors, mRNA expression,
memory consolidation, amnesia, or recovery from
amnesia, supporting the notion that 5-HT7 receptors
play a role in normal and impaired memory.14,15 There-
fore, it results clear that the 5-HT7 receptor could serve
as a putative target for novel drug development.
During the last decade a number of structurally diverse
5-HT7 receptor ligands have been disclosed.16 We have
pointed our attention to arylpiperazine derivatives.17–19
In particular, we have reported on the synthesis and ini-
tial pharmacological characterization of three distinct
classes of 5-HT7 receptor ligands with arylpiperazine
Ar
O
N
Ar
N
N
( )n
HN
N
O
H3CO
(CH2)n
H3CO
N
N Ar
R
I
II
III
N
N
OCH3
OCH3
1
5-HT7: Ki = 24.5 nM
5-HT1A: Ki = 2.37 nM
Figure 2. Structures of arylpiperazine-based 5-HT7 receptor ligands.

5318
M. Leopoldo et al. / Bioorg. Med. Chem. 15 (2007) 5316–5321
cine. We have selected a polymethylene spacer because
3, 4
of the ease of coupling of the pharmacophores to the
spacer and the possibility to find the appropriate linker
length by small increase. The final compounds origi-
nated by linking two moieties of pharmacophore 1
through their 3-methoxy rather than their 2-methoxy
substituent, because structural modifications on the aryl
linked to the piperazine nitrogen can result in a dramatic
loss in 5-HT7 receptor affinity.19
5-10
a
b
N
N
OCH3
OH
2
The chemical structures and binding affinities at 5-HT7
and 5-HT1A receptors of the dimers 3–12 are reported
in Table 1. First, with the aim to find the appropriate
linker length we evaluated the compounds 3, 5, 7–10
bearing a spacer of 2, 4, 6, 8, 10, and 12 methylene units,
respectively. The 5-HT7 affinity values revealed that
dimerization of compound 1 was tolerated. In fact, di-
mers 3, 5, and 7 (n = 2, 4, and 6, respectively) displayed
Ki values at 5-HT7 receptor in the same range as 1. By
contrast, the higher homologues 8–10 proved to be con-
siderably less potent than 1. Because the best affinity val-
ues were displayed by the dimers with a shorter linker,
we wanted to complete this series by preparing also
the dimers with 3 and 5 methylene units (compounds 4
and 6, respectively). Elongation of the spacer from 2
to 3 methylene units afforded compound 4 which was
the most potent 5-HT7 ligand within this series. Com-
pound 6 (n = 5, Ki = 120 nM) was less potent than either
the lower analogue 5 (n = 4, Ki = 28.5 nM) and the
higher analogue 7 (Ki = 42.6 nM). Clearly, the 5-HT7
affinity of dimers 3–10 decreased by increasing the spacer
c
d
11
12
Scheme 1. Synthesis of compounds 3–12. Reagents and conditions: (a)
1,2-dibromoethane, or 1,3-dibromopropane KOH, 18-crown-6, tolu-
ene, reflux, overnight; (b) Br(CH2)nBr, NaH, anhydrous toluene, room
temperature, 48 h; (c) diethylene glycol, triphenylphosphine, diethyl
azodicarboxylate, anhydrous THF, room temperature 48 h; (d)
diglycolyl chloride, Et3N, CH2Cl2, room temperature, 48 h.
2. Chemistry
All the final compounds were prepared from the key
derivative 4-[2-(3-hydroxyphenyl)ethyl]-1-(2-methoxy-
phenyl)piperazine (2), according to Scheme 1. The
ligands 5–10, containing from 4 to 12 methylene units,
were prepared by coupling 2 equiv of 2 with the appro-
priate a,x-dibromoalkane in the presence of sodium
hydride in DMF, according to Neumayer et al.33 This
synthetic route was not useful for the preparation of
the compounds with 2 or 3 methylene units (derivatives
3 and 4, respectively). These compounds were obtained
by the nucleophilic substitution reaction of 2 equiv of
Table 1. Binding affinities of the target compounds 3–12
N
N
N
N
2
with the appropriate a,x-dibromoalkane under
OCH3
H3CO
phase-transfer catalysis. The dimer 11, bearing the alkyl-
oxy linker, was synthesized from phenol 2 and
diethylene glycol in the presence of triphenylphosphine
and diethyl diazodicarboxylate, under Mitsunobu
conditions. Finally, compound 13 was obtained from
the condensation of 2 equiv of 2 with diglycolyl chloride,
previously prepared from commercially available
diglycolic acid by means of thionyl chloride.
O
linker
O
Compound
Linker
Ki (nM) SEMa
5-HT7
5-HT1A
3
4
(CH2)2
(CH2)3
(CH2)4
(CH2)5
(CH2)6
(CH2)8
(CH2)10
(CH2)12
41.1 2.6
2.2 0.35
5.6 0.7
25.0 1.3
28.5 6.4
120 36
42.6 2.4
573 17
258 34
(11%)b
5
0.90 0.02
1.74 0.80
1.6 0.34
4.8 0.50
28.3 0.24
253 27
6
7
8
3. Results and discussion
9
10
Two potential bridging mechanisms have been proposed
to rationalize bivalent ligand activity and selectivity.26
First, if the spacer is of sufficient length, it may be pos-
sible for both pharmacophores in a bivalent ligand to
occupy two recognition sites on a receptor dimer; a
second possible mechanism involves the bridging of
the second pharmacophore of a bivalent ligand to an
accessory site adjacent to the receptor site. Under such
circumstances, it is reasonable that the bridging of
neighboring sites by a bivalent ligand would be depen-
dent on the length and/or flexibility of the spacer. Sev-
eral spacers have been used for dimer formation,
including polymethylene, polyalkyloxy ether, polygly-
11
O
O
37.0 1.4
0.62 0.15
O
O
12
43.0 2.3
2.74 0.12
5-CT
5-HT
0.51 0.01
7.33 0.25
a The values are means SEM from three independent experiments in
triplicate (P < 0.001). Individual difference between the various
compounds has been examined using Tukey’s post hoc test
(P < 0.001). Difference in the Ki values between the receptors for each
compound has been analyzed using the Mann–Whitney U test
(P = 0.007, U = 8.000).
b Full Ki not obtained. Percentage of inhibition measured at 10 lM.

M. Leopoldo et al. / Bioorg. Med. Chem. 15 (2007) 5316–5321
5319
length, but not linearly. At this point, we wondered if
the hydrophobic nature of the spacer negatively affected
the affinity for 5-HT7 receptor, therefore we prepared
compounds 11 and 12 which showed a hydrophilic lin-
ker. The impact on 5-HT7 receptor affinity of a hydro-
philic spacer was modest. In fact, Ki values of 11 and
12 (Ki = 37.0 and 43.0 nM, respectively) were compara-
ble to those of 5 and 6, which possessed a hydrophobic
linker with comparable length as 11 and 12. Taken to-
gether, the 5-HT7 receptor affinity values of dimers 3–
12 indicated that the two pharmacophores did not bind
at two neighboring binding sites because none of the di-
mers showed higher HT7 receptor affinity than that of
the pharmacophore 1. Therefore, on the basis of the
above data, it can be supposed that both the linker
and one pharmacophore are bound to the receptor in
a region of steric tolerance.
in 0.4% of the theoretical values for the formula given.
1H NMR spectra were recorded at 300 MHz on a
Varian Mercury-VX spectrometer. All spectra were re-
corded on free bases. All chemical shift values are
reported in ppm (d). ESI+/MS/MS analyses were
performed with an Agilent 1100 Series LC-MSD trap
System VL workstation. All spectra were in accordance
with the assigned structures. The purity of new com-
pounds that was essential to the conclusions drawn in
the text was determined by HPLC on a Perkin-Elmer
series 200 LC instrument using a Phenomenex Prodigy
ODS-3 RP-18 column, (250 · 4.6 mm, 5 lm particle
size) and equipped with a Perkin-Elmer 785A UV/vis
detector setting k = 254 nm. All compounds were eluted
with CH3CN/H2O/Et3N, 9:1:0.01, v/v at a flow rate of
1 mL/min. A standard procedure was used to transform
final compounds into their hydrochloride salts. 4-[2-(3-
Hydroxyphenyl)ethyl]-1-(2-methoxyphenyl)piperazine (2)
was prepared as previously reported.19
Considering the affinities for 5-HT1A receptor, dimers 3–
8, bearing a linker with 2–8 methylene units, displayed
affinity comparable to that of the monomer 1 (Ki values
ranging between 0.9 and 4.8 nM). Compound 9 dis-
played nearly the same 5-HT1A affinity as 1, whereas
further linker elongation resulted in compound 10 which
was 10-fold less potent than 1. Finally, the 5-HT1A
receptor affinities of the hydrophilic linker bearing com-
pounds 11 and 12 (Ki = 0.62 and 2.74 nM, respectively)
were in the same range as 5 and 6 which were character-
ized by a polymethylene linker. Although the affinities of
compounds 5 and 11 were slightly higher than that of
the monomer 1 (2.6- and 3.8-fold, respectively), the ob-
served increase cannot be accounted for by the interac-
tion of 5 or 11 with two independent recognition sites.
Similar results were obtained by Halazy et al. when
studying dimers of 5-HT.28 On the basis of affinity data
for 5-HT7 and 5-HT1A receptors of dimers 3–12, it is
clear that dimerization of compound 1 did not lead to
any improvement in specificity for 5-HT7 receptor.
5.1. 1,2-Bis-[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-
ethyl]phenoxy]ethane (3)
A mixture of phenol 2 (0.25 g, 0.8 mmol), 1,2-dibromo-
ethane (0.15 g, 0.8 mmol), powdered KOH (0.45 g,
8.0 mmol), and 18-crown-6 (0.085 g, 0.3 mmol) in toluene
(15 mL) was vigorously stirred under reflux overnight.
After cooling, the reaction mixture was concentrated
and the residue was partitioned between H2O (30 mL)
and CHCl3 (30 mL). The organic layer was separated,
dried over Na2SO4, and then concentrated in vacuo.
The crude residue was chromatographed (CHCl3/AcOEt,
1:1 as eluent) to give the target compound (0.16 g, 31%
1
yield). H NMR: d 2.66–2.71 (m, 4H), 2.76 (br s, 8H),
2.82–2.87 (m, 4H), 3.14 (br s, 8H), 3.87 (s, 6H), 4.32 (s,
4H), 6.79–6.90 (m, 8H), 6.92–7.04 (m. 6H), 7.22 (t, 2H,
J = 8.0 Hz). ESI+/MS m/z 651.4 (MH+). ESI+/MS/MS
m/z 459 (100), 205 (59). The hydrochloride salt melted at
226 ꢁC (from CH3OH/diethyl ether). Anal (C40H50N4O4Æ
4HClÆH2O) C, H, N.
4. Conclusion
5.2. 1,3-Bis-[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-
ethyl]phenoxy]propane (4)
We have synthesized the ligands 3–12 that were designed
according to the ‘bivalent ligand’ approach by linking
two moieties of the 5-HT7/5-HT1A ligand 4-[2-(3-
methoxyphenyl)ethyl]-1-(2-methoxyphenyl)piperazine (1)
through their 3-methoxy substituent by polymethylene
chains of variable length, with the aim to increase affinity
and specificity for 5-HT7 receptor. The dimers did not
show 5-HT7 receptor affinities higher than that of the
pharmacophore 1 and did retain high 5-HT1A receptor
affinity. Therefore, the bivalent ligand approach failed,
in this case, to achieve more selective compounds.
As described above, the final compound was prepared
from 2 and 1,3-dibromopropane in 6% yield. 1H NMR: d
2.25 (quintet, 2H, J = 6.0 Hz), 2.98–3.28 (m, 16H), 3.46
(br s, 8H), 3.86 (s, 6H), 4.16 (t, 4H, J = 6.0 Hz), 6.79–
7.09 (m, 14H), 7.23 (t, 2H, J = 8.2 Hz). ESI+/MS m/z
665.4 (MH+). ESI+/MS/MS m/z 473 (100), 205 (54). The
hydrochloride salt melted at 174–177 ꢁC (from CH3OH/
diethyl ether). Anal (C41H52N4O4Æ4HCl) C, H, N.
5.3. General procedure for the preparation of dimers 5–10
5. Experimental
To a solution of 2 (0.80 mmol) in anhydrous DMF
(3 mL), NaH powder (0.96 mmol) was carefully added.
The mixture was stirred at room temperature for 1 h.
Then, a solution of the appropriate dibromoalkane
(0.53 mmol) in anhydrous DMF (1 mL) was added drop-
wise. The resulting mixture was stirred for 2 days at room
temperature. Then, the mixture was evaporated to dry-
ness and the residue was partitioned between H2O
Column chromatography was performed with 1:30
Merck silica gel 60A (63–200 lm) as the stationary
phase. Melting points were determined in open capillar-
ies on a Gallenkamp electrothermal apparatus. Elemen-
tal analyses (C, H, N) were performed on Eurovector
Euro EA 3000 analyzer; the analytical results were with-

5320
M. Leopoldo et al. / Bioorg. Med. Chem. 15 (2007) 5316–5321
(30 mL) and CHCl3 (30 mL). The organic phase was sep-
arated, dried over Na2SO4, and evaporated in vacuo. The
crude residue was chromatographed (CHCl3/AcOEt, 1:1
as eluent) to give the final compounds as pale yellow oils.
5.4. Di-2-[[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-
ethyl]phenoxy]ethyl] ether (11)
To a solution of 2 (0.50 g, 1.6 mmol), triphenylphos-
phine (0.42 g, 1.6 mmol), and diethylen glycol (0.10 g,
0.94 mmol) in anhydrous THF (20 mL) under stirring
was added diethyl azodicarboxylate (0.28 g, 1.6 mmol).
The resulting mixture was stirred at room temperature
under anhydrous condition for 2 days. Then, the solvent
was evaporated under reduced pressure and the residue
was chromatographed (CHCl3/AcOEt, 1:1 as eluent) to
give a mixture of target compound and monosubstituted
2. This mixture was then chromatographed with CHCl3/
MeOH, 49:1 as eluent, to afford pure 11 as a pale yellow
5.3.1. 1,4-Bis-[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-
1
ethyl]phenoxy]butane (5). Yield 14%. H NMR: d 1.98–
2.02 (m, 4H), 2.80–2.86 (m, 4H), 2.88–2.99 (m, 12H),
3.21 (br s, 8H), 3.87 (s, 6H), 4.03 (br t, 4H), 6.74–7.05
(m, 14H), 7.21 (t, 2H, J = 8.0 Hz). ESI+/MS m/z 679.5
(MH+). ESI+/MS/MS m/z 487 (100), 205 (38). The hydro-
chloride salt melted at 250 ꢁC dec (from CH3OH/diethyl
ether). Anal (C42H54N4O4Æ4HClÆH2O) C, H, N.
1
5.3.2. 1,5-Bis-[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-
ethyl]phenoxy]pentane (6). Yield 12%. 1H NMR: d 1.62–
1.70 (m, 2H), 1.82–1.91 (m, 4H), 2.66–2.72 (m, 4H), 2.76
(br s, 8H), 2.80–2.86 (m, 4H), 3.14 (br s, 8H), 3.86 (s,
6H), 3.98 (t, 4H, J = 6.3 Hz), 6.65–7.04 (m, 14H), 7.13–
7.22 (m, 2H). ESI+/MS m/z 693.6 (MH+). ESI+/MS/MS
m/z 501 (100), 205 (38). The hydrochloride salt melted at
170 ꢁC dec (from CH3OH/diethyl ether). Anal
(C43H56N4O4Æ4HCl) C, H, N.
oil (0.15 g, 14% yield). H NMR: d 2.66–2.72 (m, 4H),
2.77 (br s, 8H), 2.82–2.87 (m, 4H), 3.15 (br s, 8H),
3.86 (s, 6H), 3.94 (t, 4H, J = 5.2 Hz), 4.16 (t, 4H,
J = 5.0 Hz), 6.75–7.04 (m, 14H), 7.20 (t, 2H,
J = 8.0 Hz). ESI+/MS m/z 695.1 (MH+). ESI+/MS/MS
m/z 503 (100), 205 (31). The hydrochloride salt melted
at 168–169 ꢁC (from CH3OH/diethyl ether). Anal
(C42H54N4O5Æ4HCl) C, H, N.
5.5. Bis[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-
phenyl] diglycolate (12)
5.3.3. 1,6-Bis-[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-
ethyl]phenoxy]hexane (7). Yield 38%. 1H NMR: d
1.52–1.58 (m, 4H), 1.78–1.82 (m, 4H), 2.66–2.71 (m,
4H), 2.76 (br s, 8H), 2.81–2.86 (m, 4H), 3.14 (br s,
8H), 3.86 (s, 6H), 3.96 (t, 4H, J = 6.3 Hz), 6.73–7.03
(m, 14H), 7.20 (t, 2H, J = 7.7 Hz). ESI+/MS m/z 707.5
(MH+). ESI+/MS/MS m/z 515 (100), 205 (31). The hydro-
chloride salt melted at 238 ꢁC dec (from CH3OH/diethyl
ether). Anal (C44H58N4O4Æ4HCl) C, H, N.
To an ice-cooled mixture containing
2 (0.30 g,
0.96 mmol), Et3N (0.5 mL) in CH2Cl2 (30 mL) was added
dropwise under vigorous stirring a solution of diglycolyl
chloride, prepared by refluxing the corresponding acid
(0.075 g, 0.56 mmol) and SOCl2 (3 mL). Then, the cooling
bath was removed and the mixture was stirred for 2 days
at room temperature. Then, the reaction mixture was
washed with H2O. The separated organic was dried over
Na2SO4 and evaporated to dryness under reduced pres-
sure. The crude residue was chromatographed (CHCl3/
MeOH, 19:1 as eluent) to give target ester as a pale yellow
liquid (0.15 g, 37% yield). 1H NMR: d 2.68–2.73 (m, 4H),
2.77 (br s, 8H), 2.86–2.91 (m, 4H), 3.15 (br s, 8H), 3.86 (s,
6H), 4.58 (s, 4H), 6.85–7.03 (m, 12H), 7.13 (d, 2H,
J = 7.7 Hz), 7.32 (t, 2H, J = 7.7 Hz). ESI+/MS m/z 723.0
(MH+). ESI+/MS/MS m/z 531 (100). The hydrochloride
salt melted at 191 ꢁC (from CH3OH/diethyl ether). Anal
(C42H50N4O7Æ4HClÆH2O) C, H, N.
5.3.4.
1,8-Bis-[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-
1
ethyl]phenoxy]octane (8). Yield 27%. H NMR: d 1.41–
1.51 (m, 8H), 1.74–1.83 (m, 4H), 2.66–2.71 (m, 4H),
2.75 (br s, 8H), 2.80–2.86 (m, 4H), 3.14 (br s, 8H), 3.86
(s, 6H), 3.94 (t, 4H, J = 6.6 Hz), 6.72–7.03 (m, 14H),
7.21 (t, 2H, J = 7.7 Hz). ESI+/MS m/z 735.5 (MH+).
ESI+/MS/MS m/z 543 (100), 205 (21). The hydrochloride
salt melted at 198–204 ꢁC (from CH3OH/diethyl ether).
Anal (C46H62N4O4Æ4HClÆH2O) C, H, N.
5.3.5. 1,10-Bis-[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-
1
ethyl]phenoxy]decane (9). Yield 29%. H NMR: d 1.32
5.6. Pharmacology
(br s, 8H), 1.40–1.45 (m, 4H), 1.72–1.82 (m, 4H), 2.65–
2.70 (m, 4H), 2.75 (br s, 8H), 2.80–2.85 (m, 4H), 3.13
(br s, 8H), 3.86 (s, 6H), 3.93 (t, 4H, J = 6.6 Hz), 6.72–
7.03 (m, 14H), 7.19 (t, 2H, J = 7.7 Hz). ESI+/MS m/z
763.6 (MH+). ESI+/MS/MS m/z 571 (100). The hydro-
chloride salt melted at 196–212 ꢁC (from CH3OH/
diethyl ether). Anal (C48H66N4O4Æ4HCl) C, H, N.
Rat recombinant serotonin 5-HT7 receptor expressed in
HEK-293 cells, [3H]LSD, and [3H]-8-OH-DPAT were
obtained from Perkin-Elmer (Zaventem, Belgium).
5-CT was purchased from Tocris Cookson Ltd (Bristol,
UK). 5-HT was from Sigma–Aldrich RBI (Milan, Italy).
For receptor binding studies, compounds 3–12 were dis-
solved in absolute ethanol.
5.3.6. 1,12-Bis-[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]-
ethyl]phenoxy]dodecane (10). Yield 33%. 1H NMR: d 1.29
(br s, 12H), 1.40–1.45 (m, 4H), 1.73–1.82 (m, 4H), 2.67–
2.72 (m, 4H), 2.77 (br s, 8H), 2.81–2.88 (m, 4H), 3.15 (br
s, 8H), 3.86 (s, 6H), 3.94 (t, 4H, J = 6.6 Hz), 6.73–7.04
(m, 14H), 7.19 (t, 2H, J = 7.7 Hz). ESI+/MS m/z 791.6
(MH+). ESI+/MS/MS m/z 599 (100). The hydrochloride
salt melted at 186–193 ꢁC (from CH3OH/diethyl ether).
Anal (C50H70N4O4Æ4HClÆH2O) C, H, N.
5.6.1. Radioligand binding assay at rat cloned 5-HT7
receptor. Binding of [3H]LSD at rat cloned 5-HT7 recep-
tor was performed according to Jasper et al.34 with min-
or modifications. In 1 mL of incubation buffer (50 mM
Tris, 10 mM MgCl2, and 0.5 mM EDTA, pH 7.4) were
suspended 30 lg of membranes, 2.5 nM [3H]LSD, and
the drugs or reference compound (6–9 concentrations).
The samples were incubated for 60 min at 37 ꢁC.

M. Leopoldo et al. / Bioorg. Med. Chem. 15 (2007) 5316–5321
5321
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The incubation was stopped by rapid filtration on GF/A
glass fiber filters (pre-soaked in 0.5% polyethylenimine
for 30 min). The filters were washed with 3 · 3 mL of
ice-cold buffer (50 mM Tris, pH 7.4). Nonspecific bind-
ing was determined in the presence of 10 lM 5-CT.
Approximately 90% of specific binding was determined
under these conditions.
´
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bly expressed in HEK-293 cells were radiolabeled with
1.0 nM [3H]-8-OH-DPAT.35 Samples containing 40 lg
of membrane protein, different concentrations of each
compound ranging from 0.1 nM to 10 lM were incu-
bated in a final volume of 500 lL of 50 mM Tris–HCl,
pH 7.4, 5 mM MgSO4 for 120 min at 37 ꢁC. After this
incubation time, samples were filtered through GF/C
pre-soaked in polyethylenimine 0.5% for at least
30 min prior to use. The filters were washed twice with
1 mL of ice-cold buffer (50 mM Tris–HCl, pH 7.4).
Nonspecific binding was determined in the presence of
10 lM 5-HT.
´
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5.7. Statistical methods
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