4-(4-Fluorobenzoyl)-1-[2-(4-iodo-2,5-dimethoxyphenyl)ethyl]piperidine and its derivatives: Synthesis and affinity at 5-HT(2A), 5-HT(2B) and 5- HT(2C) serotonin receptors

Article abstract of DOI:10.1016/S0223-5234(99)00208-1

4-(4-Fluorobenzoyl)-1-[2-(4-iodo-2,5-dimethoxyphenyl)ethyl]piperidine (7) and its derivatives modified at the carbonyl group of the fluorobenzoyl moiety were prepared and evaluated for affinity at 5-HT(2A), 5-HT(2C) (rat cortex) and 5-HT(2B) (rat stomach fundus) serotonin receptors. Compound 7 bound the 5-HT(2A) sites with higher affinity (K(i) = 8.2 nM) than the 5- HT(2B) (K(b) = 1 290 nM) and 5-HT(2C) ones (K(i) = 54.2 nM). Modification of the benzoyl carbonyl group decreased the 5-HT(2A) and 5-HT(2C) affinities but did not significantly influence 5-HT(2B) affinity. This suggests that the carbonyl group is the determinant for the interaction with 5-HT(2A) and 5-HT(2C) receptor subtypes. Compound 7 was found to be a 5-HT(2A) receptor antagonist.

Full text of DOI:10.1016/S0223-5234(99)00208-1

Eur. J. Med. Chem. 34 (1999) 843−852
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
843
Preliminary communication
4-(4-Fluorobenzoyl)-1-[2-(4-iodo-2,5-dimethoxyphenyl)ethyl]piperidine
and its derivatives: synthesis and affinity at 5-HT2A, 5-HT2B and 5-HT2C
serotonin receptors
Francesco Claudia*, Loredana Scocciaa, Gianfabio Giorgionia, Beatrice Accorronia,
Gabriella Maruccia, Stefania Gessib, Anna Siniscalchib, Pier Andrea Boreab
aDepartment of Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino, Italy
bDepartment of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara, Via Fossato di Mortara 17–19,
44100 Ferrara, Italy
(Received 24 July 1998; accepted 19 May 1999)
Abstract – 4-(4-Fluorobenzoyl)-1-[2-(4-iodo-2,5-dimethoxyphenyl)ethyl]piperidine (7) and its derivatives modified at the carbonyl group of
the fluorobenzoyl moiety were prepared and evaluated for affinity at 5-HT2A, 5-HT2C (rat cortex) and 5-HT2B (rat stomach fundus) serotonin
receptors. Compound 7 bound the 5-HT2A sites with higher affinity (Ki = 8.2 nM) than the 5-HT2B (Kb = 1 290 nM) and 5-HT2C ones (Ki
= 54.2 nM). Modification of the benzoyl carbonyl group decreased the 5-HT2A and 5-HT2C affinities but did not significantly influence 5-HT2B
affinity. This suggests that the carbonyl group is the determinant for the interaction with 5-HT2A and 5-HT2C receptor subtypes. Compound
7 was found to be a 5-HT2A receptor antagonist. © 1999 Éditions scientifiques et médicales Elsevier SAS
4-(4-Fluorobenzoyl)-1-[2-(4-iodo-2,5-dimethoxyphenyl)ethyl]piperidine derivatives / synthesis / 5-HT2A, 5-HT2B, and 5-HT2C seroto-
nin receptor affinity / 5-HT2A antagonistic activity
1. Introduction
migraine [6]. The 5-HT2C receptor was initially charac-
terized in the choroid plexus, is widely distributed in the
brain [2], and has been suggested as playing a role in
migraine, obsessive compulsive disorders, and anxi-
ety [7].
Serotonin (5-hydroxytryptamine, 5-HT) mediates a
number of neuronal processes both in the central nervous
system and peripheral tissues. During the last decade
multiple 5-HT receptor subtypes have been characterized
and grouped in seven classes (5-HT1–5-HT7) [1]. The
5-HT2 class includes the subtypes 5-HT2A, 5-HT2B, and
5-HT2C which are grouped together considering their
high degree of transmembrane sequence homology and
second messenger coupling system. The 5-HT2A subtype
is present in the brain (cortical regions) [2] and periphery
(gastrointestinal tract, cardiovascular system) [3], and is
involved in various cardiovascular and mental disorders,
such as depression and schizophrenia [4]. The 5-HT2B
receptor is expressed in rat stomach fundus, where it
mediates a contractile response to 5-HT. Its mRNA
transcript is present in the human brain [5], and it has
been suggested to be involved in the pathophysiology of
To date, few antagonists discriminating between the
5-HT2 subtypes are available [8–14]. With the lack of
selective agents has come the realization that many
pharmacological and physiological effects once attributed
to 5-HT2A receptors (previously 5-HT2) may, in fact,
involve more than one receptor subtype. Hence, there is a
need to discover new agents able to bind with high
selectivity at one of the three subtypes.
The 3-{2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl}-
2,4(1H,3H)-quinazolinedione (ketanserin, 1, figure 1) is a
prototypical 5-HT2A antagonist reported to bind with as
little as 15-fold [9] to as much as 140-fold selectivity for
5-HT2A versus 5-HT2C receptors [10]. It was used to
explain the molecular structural prerequisites for the
5-HT2A antagonist binding [9, 10], and to build the
*Correspondence and reprints

844
Some time ago, Ariens suggested that an agonist can be
turned into a competitive antagonist by appending to the
agonist structure a hydrophobic bulky group able to bind
the accessory binding sites of the receptor [22].
On the basis of the Ariens strategy and of the above
mentioned observations, we decided to connect the 4-(4-
fluorobenzoyl)piperidine with the 2-(4-iodo-2,5-dimetho-
xyphenyl)ethyl fragment of 4 to obtain 4-(4-fluoro-
benzoyl)-1-[2-(4-iodo-2,5-dimethoxyphenyl)ethyl]pipe-
ridine 7 (figure 2). This compound was chosen in order to
obtain a new 5-HT2 antagonist and to investigate how a
modified tail tied to 4-(4-fluorobenzoyl)piperidine could
influence selectivity for the 5-HT2A, 5-HT2B, and 5-HT2C
subtypes.
Studies with ketanserin and related 5-HT2 antagonists
have suggested that the carbonyl group of 4-(4-
fluorobenzoyl)piperidine is involved in a hydrogen-
bonding interaction with the 5-HT2A/2C receptors [23]
and may have a prominent role in anchoring ketanserin to
5-HT2A receptors [10, 15]. Thus, in order to obtain
further information about the role of the carbonyl group
in the affinity and selectivity for the three 5-HT2 sub-
types, two series of compounds were synthesized. The
first series keeps an sp2 carbon atom placed between the
piperidine and the fluorophenyl ring (8–11, figure 3),
while in the other (12–16, figure 4) the same carbon has
an sp3 hybridization.
Figure 1. Structures of ketanserin, 4-(4-fluorobenzoyl)
piperidine (2), DOI, and 1-(4-iodo-2,5-dimethoxyphenyl)-2-
aminoethane (4).
three-dimensional models of 5-HT2A and 5-HT2C recep-
tors [15–17].
Several studies on ketanserin analogues showed that
the 4-(4-fluorobenzoyl)piperidine (2) fragment is en-
dowed with 5-HT2A antagonistic activity and seems to be
essential for binding at the 5-HT2A receptor [9, 18].
2. Chemistry
The desired 4-(4-fluorobenzoyl)-1-[2-(4-iodo-2,5-
dimethoxyphenyl)ethyl]piperidine 7 was synthesized by
condensation of 4-(4-fluorobenzoyl)piperidine 2 with the
tosyl ester of 2-(4-iodo-2,5-dimethoxyphenyl)ethanol 6
(figure 2). Iodination of 2-(2,5-dimethoxyphenyl)ethanol
with iodine in the presence of silver trifluoroacetate gave
the 2-(4-iodo-2,5-dimethoxyphenyl)ethanol 5 which was
esterified with tosyl chloride. Oxime 8 and the
O-alkyloximes 10 and 11 were obtained by reaction of the
ketone 7 in pyridine and absolute ethanol with hydroxyl-
amine or O-ethylhydroxylamine or O-benzylhydroxyl-
amine (figure 3). The oxime acetate 9 was obtained from
8 by reaction with acetic anhydride. Oxime isomers were
not separated. Ketone 7 was also reduced by sodium
borohydride to alcohol 12 or transformed into the diox-
olane 13. From 12, by reaction with acetic anhydride or
benzoyl chloride, the esters 14 and 15 were obtained
(figure 4).
Among the 5-HT2 agonists, the most extensively
studied is the 1-(4-iodo-2,5-dimethoxyphenyl)-2-amino-
propane (3 R(-)-DOI), currently reported as a 5-HT2C/2A
agonist [19]. Investigations on 1-(2,5-dimethoxyphenyl)-
2-aminopropane derivatives have established that the
methyl group α to the amine in 3 does not influence the
in vitro receptor affinity. Thus, the 1-(4-iodo-2,5-
dimethoxyphenyl)-2-aminoethane 4 binds with high af-
finity (Ki = 1.53 nM) the 5-HT2 receptors labelled by
R-[125I]-DOI [20], and has been reported to act as agonist
at 5-HT2 receptors [21].
Site-directed mutagenesis and molecular modelling
studies indicate that the piperidine nitrogen atom of 1 and
the amino group of 3 have a strong elecrostatic interac-
tion with the carboxylate anion of the aspartate residue
Asp 155 in transmembrane helix 3 (TM 3) of the 5-HT2A
receptor [15, 17]. Therefore, it would appear that Asp-
155 is a common binding site for agonists 3 and 4, and for
antagonist 1.
In order to obtain the derivative 16 with a methylene
replacing the carbonyl group, the tosylate ester 6 was
reacted with 4-(4-fluorobenzyl)piperidine.

845
Figure 2. Synthesis of 7.
3. Pharmacology
beginning of the experiment, while the 5-HT2C antagonist
mesulergine was unable to counteract it, except at
10 µmol, a high concentration able to block 5-HT2A
receptors as well.
The affinity of compounds for 5-HT2A and 5-HT2C
receptors was assessed in vitro in the cerebral cortex
preparations. [3H]Ketanserin as radiolabelled ligand for
the 5-HT2A receptors, and [3H]mesulergine for the
5-HT2C receptors were used. The antagonistic affinity at
the 5-HT2B receptors was determined by the inhibition of
5-HT-induced contractions of rat stomach fundus. The
results are reported in table I. The 5-HT2B receptor
affinities were obtained from functional data; however,
caution should be exercised when comparing them with
data from binding assays.
Derivatives 7, 9, 10, and 13 were evaluated for their
antagonist activity at central 5-HT2A receptors by testing
their ability to antagonize the facilitatory effect of 5-HT
on basal acetylcholine release from guinea-pig striatal
slices [24]. Acetylcholine release induced by 5-HT could
be attributed to 5-HT2A receptor activation. In fact, it was
concentration-dependently antagonized by 5-HT2A an-
tagonists added to the superfusion medium from the
4. Results and discussion
The data in table I indicate that the 4-(4-fluoro-
benzoyl)piperidine 2 binds with the same affinity at
5-HT2A and 5-HT2C sites. Moreover, 2-(4-iodo-2,5-
dimethoxyphenyl)ethylamine 4 binds with lower affinity
to the 5-HT2A than to the 5-HT2C sites (Ki = 300 ± 33 nM
vs. 2.5 ± 0.14 nM). Accordingly, in the assays of
acetylcholine release from guinea-pig striatal slices, its
5-HT2A agonistic action is negligible (net [3H]choline
efflux increase at 30 µM = 0.11 ± 0.03%, n = 5), as is the
antagonistic one (5 HT 30 µM-induced net [3H]choline
efflux increase in the presence of 4 = 0.55 ± 0.06%, not
significantly different from 5-HT 30 µM alone (0.89 ±
0.11%). Functional assays on rat stomach fundus indicate
that 4 acts as a full agonist (pD2 = 6.54 ± 0.12, less potent

846
Figure 3. Synthesis of compounds 9–11.
than 5-HT: pD2 = 8.20 ± 0.09). Introduction on the
piperidine nitrogen of the 2-(4-iodo-2,5-dimethoxy-
phenyl)ethyl fragment increases the affinity for all 5-HT2
receptor subtypes, and this effect is more pronounced for
5-HT2A sites (80-fold) than for 5-HT2B and 5-HT2C
(13-fold) ones. On the other hand, when the amino group
of 4 is substituted by the 4-(4-fluorobenzoyl)piperidine
moiety, the 5-HT2A and 5-HT2B affinities increase and the
5-HT2C affinity decreases. Compound 7 binds the 5-HT2A
sites with lower affinity than does the reference com-

847
Figure 4. Synthesis of compounds 12–16.
pound 1 (8.2 nM vs. 0.24 nM) and, under our assay
conditions, displays the same selectivity for 5-HT2A
versus 5-HT2C receptors (about 7-fold). Compound 7 is
also 157-fold more selective for 5-HT2A versus 5-HT2B
receptors. These results suggest that the 2-(4-iodo-2,5-
dimethoxyphenyl)ethyl fragment enhances to a greater
extent the affinity for 5-HT2A than that for 5-HT2B or
5-HT2C receptors.

848
Table I. 5-HT2A, 5-HT2C (rat cortex) receptor binding affinities, and 5-HT2B (rat stomach fundus) affinity of compounds 1, 2, 4, 7–16a.
Compound
Ki (nM)b
Kb (nM)c
5-HT2A
5-HT2C
5-HT2B
1
2
4
7
8
9
10
11
12
13
14
15
16
0.24 ± 0.01
667 ± 44
300 ± 33
8.2 ± 0.4
666 ± 44
100 ± 6
1.76 ± 0.05
710 ± 59
8 660 ± 810
> 10 000
> 10 000d
1 290 ± 60
1 300 ± 150
1 350 ± 170
1 000 ± 130
NDe
807 ± 115
1 080 ± 170
ND
7 140 ± 980
ND
2.5 ± 0.14
54.2 ± 2.1
2 270 ± 176
1 450 ± 87
887 ± 59
598 ± 37
1 220 ± 78
432 ± 19
150 ± 6
275 ± 14
616 ± 60
118 ± 14
325 ± 14
275 ± 14
238 ± 7
687 ± 59
1 660 ± 137
497 ± 20
b
aAll values represent means ± SEM; n ≥ 3 determinations. Binding affinity (rat cortex; 5-HT2A [3H]keranserin, 5-HT2C [3H]mesulergine).
cApparent antagonist dissociation constant, rat stomach fundus. The compounds were tested at 10–5 M and behave as competitive antagonists.
e
dAffinity constant (Kb) determined as reported in [28]. Not determined.
The modification of the carbonyl group in 7 always
decreases the 5-HT2A and 5-HT2C affinities and does not
affect significantly the 5-HT2B affinity. In the compounds
having an sp2 carbon atom (8–11), the carbonyl substitu-
tion with the oxime group decreases the 5-HT2A and
5-HT2C affinities. Both acetylation and etherification of
the oxime hydroxyl group increases the affinities. These
are influenced in an opposite way: the 5-HT2A affinity
increases and 5-HT2C affinity decreases following the
order 9, 10, 11. This could suggest a different interaction
of 9–11 with the 5-HT2A and 5-HT2C sites. It can also be
seen from table I that the replacement of the carbonyl
group with an sp3 carbon atom (compounds 12–16)
decreases the 5-HT2A and 5-HT2C affinities. In the series
with an sp3 carbon atom between the piperidine and the
fluorophenyl ring, only the dioxolane 13 presents mod-
erate 5-HT2A affinity. The highest decrease in affinity is
observed when the carbonyl group is reduced to alcohol
12. Of importance is that the 5-HT2A and 5-HT2C
affinities decrease to a greater extent in derivatives 8 and
12 bearing a hydroxyl group. The results indicate that the
carbonyl oxygen participates in a key binding interaction
and that a hydrogen-bond acceptor seems to be required
by the 5-HT2A and 5-HT2C receptors. Moreover, it should
be observed that also the carbonyl group reduction to
methylene (compound 16) decreases the affinities. With
regard to the 5-HT2B receptor, it is evident that the
carbonyl group modification does not significantly influ-
ence the affinity.
acetylcholine induced by 5-HT (figure 5). They behave as
competitive antagonists and are less active than the
standard 1. The potency order was 1 > 7 > 9 > 10 > 13,
in good agreement with the binding data. All the antago-
nists, when tested at the highest concentration, proved to
Figure 5. Basal tritium efflux from guinea-pig striatal slices
prelabelled with [3H]choline. Relationship between 5-HT2A
antagonist concentrations (µmol, abscisses, log scale) and
inhibition of the facilitatory effect of 5-HT 30 µmol (%,
ordinate). Points represent the means ± SEM of 4–9 experi-
ments. ■ Ketonserin, m 7, . 9, ◆ 10, ★ 13.
Derivatives 7, 9, 10, and 13 show antagonist activity at
central 5-HT2A receptors counteracting the release of

849
be devoid of intrinsic activity, except 13, which at 30 µM
evoked a net increase in [3H]choline efflux of 0.67 ±
0.11% (n = 3).
precipitate. This was filtered and crystallized from cyclo-
hexane, m.p.: 110–112 °C; yield 82%. 1H-NMR (CDCl3)
δ 2.46 (3H, s, CH3), 2.89 (2H, t, J = 6.3 Hz, ArCH2), 3.63
(3H, s, OCH3), 3.88 (3H, s, OCH3), 4.22 (2H, t, J = 6.3
Hz, CH2O), 6.57 (1H, s, ArH), 7.08 (1H, s, ArH), 7.23
(2H, d, J = 8.5 Hz, ArH), 7.58 (2H, d, J = 8.5 Hz, ArH).
Anal. C17H19IO5S (C, H).
In conclusion, compound 7 represents a successful
application of the Ariens strategy in the design of a
5-HT2A antagonist by substituting the amino group of
2-(4-iodo-2,5-dimethoxyphenyl)ethylamine with the
bulky lipophilic moiety of 4-(4-fluorobenzoyl)piperidine.
Comparing 1 with 7, it is evident that the 3-ethyl-2,4-
quinazolinedione moiety gives higher 5-HT2A and
5-HT2C affinities than does the 2-(4-iodo-2,5-dimethoxy-
phenyl)ethyl fragment.
5.1.3. 4-(4-Fluorobenzoyl)-1-[2-(4-iodo-2,5-dimethoxy-
phenyl)ethyl]piperidine hydrochloride 7
A
mixture of
6
(3 g, 6.5 mmol), 4-fluoro-
benzoylpiperidine (1.35 g, 6.5 mmol) and K2CO3 (1.28 g,
9.3 mmol) in acetone (30 mL) was warmed to reflux for
20 h. The solvent was removed by evaporation, and the
residue was partitioned between H2O and CHCl3. The
organic extracts were dried (Na2SO4) and evaporated.
The residue was purified by chromatography eluting with
CHCl3:acetone:MeOH (6.7:3:0.3, v/v) and crystallized
5. Experimental protocols
5.1. Chemistry
1
Melting points were determined on a Buchi 510
apparatus and are uncorrected. Microanalyses were per-
formed on a 1106 Carlo Erba CHN Analyzer, and the
from isopropyl ether; m.p.: 98–100 °C; yield 84%. H-
NMR (CDCl3) δ 1.88 (4H, m, H3,5pip), 2.21 (2H, m,
H
2,6ax-pip), 2.58 (2H, m, ArCH2), 2.80 (2H, m, NCH2),
1
results were within ± 0.4% of the theoretical values. H
3.08 (2H, m, H2,6eq-pip), 3.21 (1H, m, H4pip), 3.78 (3H, s,
OCH3), 3.83 (3H, s, OCH3), 6.70 (1H, s, ArH), 7.14 (2H,
m, ArH), 7.20 (1H, s, ArH), 7.98 (2H, m, ArH). Hydro-
chloride: crystallized from absolute EtOH, m.p.:
232–234 °C. Anal. C22H25FINO3.HCl (C, H, N).
NMR spectra were recorded on a Varian VXR 200 MHz
spectrometer. Chemical shifts are reported in parts per
million (δ) downfield from the internal standard tetram-
ethylsilane (Me4Si). The identity of all new compounds
was confirmed both by elemental analysis and NMR data;
homogeneity was confirmed by TLC on silica gel Merck
60 F254. Chromatographic purifications were performed
by Merck-60 silica gel columns 70–230 mesh ASTM
from Merck with a reported solvent.
5.1.4.
(4-Fluorophenyl)-{1-[2-(4-iodo-2,5-dimethoxy-
phenyl)ethyl]piperidin-4-yl}methanone oxime hydro-
chloride 8
A mixture of 7 (1 g, 2 mmol), hydroxylamine hydro-
chloride (0.35 g, 5 mmol), and pyridine (2 mL) in abso-
lute EtOH (5 mL) was warmed to reflux for 3 h. After
evaporation of the solvent, water was added. The precipi-
tate was filtered and crystallized from absolute EtOH;
5.1.1. 2-(4-Iodo-2,5-dimethoxyphenyl)ethanol 5
A solution of iodine (0.92 g, 3.6 mmol) in chloroform
(20 mL) was added dropwise, under stirring at room
temperature, to a slurry of silver trifluoroacetate (0.89 g,
3.6 mmol) and 2-(2,5-dimethoxyphenyl)ethanol (0.66 g,
3.6 mmol) in chloroform (5 mL). The mixture was stirred
for 30 min. The insoluble material was filtered. The
filtrate was washed with aqueous 10% Na2SO3, brine and
dried (Na2SO4). The solvent was evaporated and the
residue was crystallized from isopropyl ether, m.p.:
1
m.p.: 239–241 °C; yield 92%. H-NMR (DMSO-d6) δ
1.88 (4H, m, H3,5pip), 2.23 (1H, m, H4pip), 2.96 (4H, m,
ArCH2, H2,6ax-pip), 3.11 (2H, m, NCH2), 3.55 (2H, m,
H
2,6eq-pip), 3.75 (6H, s, OCH3), 6.93 (1H, s, ArH), 7.38
(5H, m, ArH), 10.24 (1H, bs, NH+), 10.90 e 11.45 (1H,
2s, OH). Anal. C22H26FIN2O3.HCl (C, H, N).
1
5.1.5.
(4-Fluorophenyl)-{1-[2-(4-iodo-2,5-dimethoxy-
93–94 °C; yield 76%. H-NMR (CDCl3) δ 1.59 (1H, bs,
phenyl)ethyl]piperidin-4-yl}methanone O-acetyloxime
hydrochloride 9
OH), 2.88 (2H, t, J = 6.3 Hz, ArCH2), 3.80 (8H, m,
OCH3, CH2O), 6.68 (1H, s, ArH), 7.21 (1H, s, ArH).
Anal. C10H13IO3 (C, H).
A mixture of 8 (0.8 g, 1.6 mmol) and acetic anhydride
(2 mL) was stirred at 60 °C for 1.5 h. The mixture was
cooled and partitioned between 5% aqueous Na2CO3 and
Et2O. The organic extracts were washed with H2O, dried
(Na2SO4) and evaporated. The residue was crystallized
from isopropyl ether; m.p.: 78–80 °C, yield 77%. The
residue was dissolved in Et2O and treated with ethereal
HCl. The solid obtained was crystallized from acetone;
5.1.2. 2-(4-Iodo-2,5-dimethoxyphenyl)ethyl-p-toluensul-
fonate 6
p-Toluenesulfonyl chloride (0.63 g, 3.3 mmol) was
added portionwise to a solution of 5 (0.92 g, 3 mmol) in
dry pyridine (1 mL). After stirring for 5 h at room
temperature, 2 N HCl (9 mL) was added to yield a

850
m.p.: 168–170 °C. NMR (DMSO-d6) δ 2.0 (7H, m,
H3,5pip, CH3), 3.11 (7H, m, ArCH2, H4pip, H2,6ax-pip), 3.61
(2H, m, H2,6eq-pip), 3.78 (6H, s, OCH3), 6.96 (1H, m,
ArH), 7.42 (5H, m, ArH), 10.35 (1H, bs, NH+). Anal.
C24H28FIN2O4.HCl (C, H, N).
was purified by chromatography eluting with
CHCl3:acetone:MeOH (6.7:3:0.3, v/v) to give an uncrys-
tallizable oil; yield 84%. H-NMR (CDCl3) δ 1.43 (4H,
1
m, H3,5pip), 2.0 (4H, m, OH, H4pip, H2,6ax-pip), 2.49 (2H,
m, ArCH2), 2.74 (2H, m, NCH2), 3.02 (2H, m, H2,6eq-pip),
3.70 (3H, s, OCH3), 3.78 (3H, s, OCH3), 4.35 (1H, m,
HCO), 6.65 (1H, s, ArH), 7.01 (2H, m, ArH), 7.18 (1H, s,
ArH), 7.25 (2H, m, ArH). The oil was dissolved in
absolute EtOH and treated with a solution of fumaric acid
in absolute EtOH. The fumarate was filtered and crystal-
lized from absolute EtOH, m.p.: 189–191 °C. Anal.
C22H27FINO3.C4H4O4 (C, H, N).
5.1.6. (4-Fluorophenyl)-{1-[2-(4-iodo-2,5-dimethoxyphe-
nyl)ethyl]piperidin-4-yl}methanone-O-ethyloxime hydro-
chloride 10
This was made in the same way as 8 from 7 and
O-ethylhydroxylamine hydrochloride. The precipitate ob-
tained after addition of water was filtered and treated with
a saturated solution of Na2CO3. The mixture was ex-
tracted with Et2O. The organic extracts were dried
(Na2SO4) and evaporated. The residue was dissolved in
absolute EtOH, and EtOH saturated with HCl gas was
added. The solvent was evaporated and the residue was
crystallized from 2-propanol; m.p.: 203–205 °C, yield
5.1.9. 4-[2-(4-Fluorophenyl)-[1,3]dioxolan-2-yl]-1-[2-
(4-iodo-2,5-dimethoxyphenyl)ethyl]piperidine
hydro-
chloride 13
A mixture of ketone 7 (0.5 g, 1 mmol) and ethylene
glycol (3 mL) was saturated with HCl gas and heated at
90 °C for 1 h. The resulting solid was collected and
crystallized from absolute EtOH; m.p.: 263–265 °C, yield
93%. 1H-NMR (DMSO-d6) δ 1.64 (4H, m, H3,5pip), 2.09
(1H, m, H4pip), 2.88 (4H, m, ArCH2, H2,6ax-pip), 3.11 (2H,
m, NCH2), 3.50 (2H, m, H2,6eq-pip), 3.70 (8H, m, OCH3,
OCH2), 3.98 (2H, m, OCH2), 6.92 (1H, s, ArH), 7.21 (2H,
m, ArH), 7.38 (3H, m, ArH), 9.93 (1H, bs, NH+). Anal.
C24H29FINO4.HCl (C, H, N).
1
75%. H-NMR (CDCl3) δ 1.1 (3H, t, J = 6.7 Hz, CH3),
2.0 (2H, m, H3,5ax-pip), 2.39 (2H, m, H3,5eq-pip), 2.68 (3H,
m, H2,6ax-pip, H4pip), 3.19 (4H, m, ArCH2, NCH2), 3.66
(2H, m, H2,6eq-pip), 3.78 (3H, s, OCH3), 3.80 (3H, s,
OCH3), 4.08 (2H, q, J = 6.7 Hz, OCH2), 6.82 (1H, s,
ArH), 7.10 (2H, m, ArH), 7.25 (3H, m, ArH), 12.42 (1H,
bs, NH+). Anal. C24H30FIN2O3.HCl (C, H, N).
5.1.7.
(4-Fluorophenyl)-{1-[2-(4-iodo-2,5-dimethoxy-
phenyl)ethyl]piperidin-4-yl}methanone O-benzyloxime
hydrochloride 11
5.1.10. (4-Fluorophenyl)-{1-[2-(4-iodo-2,5-dimethoxy-
phenyl)ethyl]piperidin-4-yl}methylacetate fumarate 14
A mixture of 12 (0.5 g, 1 mmol), dry pyridine (5 mL)
and acetic anhydride (0.47 mL, 5 mmol) was stirred at
room temperature for 12 h, and then poured into ice.
EtOH (2 × 30 mL) was added and the mixture was
partially evaporated. The aqueous concentrate was made
basic with 2 N NaOH, and the mixture was extracted with
Et2O. The organic extracts were dried (Na2SO4) and
evaporated to give an uncrystallizable oil; yield 76%.
1H-NMR (CDCl3) δ 1.40 (3H, m, H3,5ax-pip, H4pip), 1.85
(4H, m, H2,6ax-pip, H3,5eq-pip), 2.07 (3H, s, CH3), 2.52 (2H,
m, ArCH2), 2.77 (2H, m, NCH2), 3.01 (2H, m, H2,6eq-pip),
3.75 (3H, s, OCH3), 3.81 (3H, s, OCH3), 5.48 (1H, d, J =
8.8 Hz, OCH), 6.68 (1H, s, ArH), 7.0 (2H, m, ArH), 7.18
(1H, s, ArH), 7.25 (2H, m, ArH). The oil was dissolved in
absolute EtOH and treated with a solution of fumaric acid
in absolute EtOH. The fumarate was filtered and crystal-
lized from absolute EtOH, m.p.: 188–190 °C. Anal.
C24H29FINO4.C4H4O4 (C, H, N).
This was made in the same way as 8 from 7 and
O-benzylhydroxylamine hydrochloride. The precipitate
obtained after addition of water was filtered and treated
with a saturated solution of Na2CO3. The mixture was
extracted with Et2O. The organic extracts were dried
(Na2SO4) and evaporated. The residue was dissolved in
absolute EtOH, and EtOH saturated with HCl gas was
added. The solvent was evaporated and the residue was
crystallized from 2-propanol; m.p.: 145–147 °C, yield
1
71%. H-NMR (DMSO-d6) δ 1.82 (3H, m, H3,5ax-pip
,
H4pip), 2.94 (6H, m, ArCH2, H2,6ax-pip, H3,5eq-pip), 3.12
(2H, m, NCH2), 3.45 (2H, m, H2,6eq-pip), 3.76 (6H, s,
OCH3), 5.02 (2H, s, OCH2), 6,92 (1H, s, ArH), 7.30
(10H, m, ArH), 9.97 (1H, bs, NH+). Anal.
C29H32FIN2O3.HCl (C, H, N).
5.1.8.
(4-Fluorophenyl)-{1-[2-(4-iodo-2,5-dimethoxy-
phenyl)ethyl]piperidin-4-yl}methanol fumarate 12
To a solution of the ketone 7 (0.5 g, 1 mmol) in
CH3OH (12 mL), stirred at room temperature, NaBH4
(0.038 g, 1 mmol) was added. The mixture was stirred for
12 h. The solvent was evaporated and the residue was
partitioned between H2O and CH2Cl2. The organic ex-
tracts were dried (Na2SO4) and evaporated. The residue
5.1.11. (4-Fluorophenyl)-{1-[2-(4-iodo-2,5-dimethoxy-
phenyl)ethyl]piperidin-4-yl}methylbenzoate fumarate 15
A mixture of 12 (0.5 g, 1 mmol), dry pyridine (5 mL)
and acetic anhydride (0.52 mL, 4.5 mmol) was stirred at
room temperature for 12 h, and then poured into ice.

851
EtOH (2 × 30 mL) was added and the mixture was
partially evaporated. The aqueous concentrate was made
basic with 2 N NaOH and the mixture was extracted with
Et2O. The organic extracts were dried (Na2SO4) and
evaporated to give an uncrystallizable oil; yield 74%.
1H-NMR (CDCl3) δ 1.51 (3H, m, H3,5ax-pip, H4pip), 2.0
(4H, m, H2,6ax-pip, H3,5eq-pip), 2.57 (2H, m, ArCH2), 2.79
(2H, m, NCH2), 3.07 (2H, m, H2,6eq-pip), 3.75 (3H, s,
OCH3), 3.81 (3H, s, OCH3), 5.78 (1H, d, J = 8.1 Hz,
OCH), 6.70 (1H, s, ArH), 7.04 (2H, m, ArH), 7.19 (1H, s,
ArH), 7.42 (5H, m, ArH), 8.08 (2H, m, ArH). The oil was
dissolved in absolute EtOH and treated with a solution of
fumaric acid in absolute EtOH. The fumarate was filtered
and crystallized from absolute EtOH, m.p.: 181–183 °C.
Anal. C29H31FINO4.C4H4O4 (C, H, N).
purchased from Nossan (Varese, Italy). Sections of stom-
ach fundus were obtained from male CD Outbred rats
(Charles River, Calco, Italy) weighing 125–150 g.
5.2.3. Binding assays
Cerebral cortices of male Wistar rats (150–200 g) were
dissected on ice. The tissue was homogenized in 50 mmol
Tris-HCl buffer (pH = 7.7 at 25 °C). The homogenate was
centrifuged at 40 000 g for 10 min. The supernatant was
discarded and the pellet was resuspended in the same
volume of Tris-HCl buffer and incubated at 37 °C for
10 min prior to a second centrifugation. Binding experi-
ments [23] with [3H]ketanserin (64.1 Ci/mmol) and
[3H]mesulergine (76 Ci/mmol) were performed in 250 µL
of buffer, which contained 1 nmol [3H]ketanserin or
[3H]mesulergine, membranes from 10 mg (wet weight) of
tissue and the compounds to be tested. After 30 min of
incubation at 25 °C, separation of bound from free
radioligand was performed by rapid filtration through
Whatman GF/B glass fibre filters, which were washed
three times with ice-cold buffer, dried and counted in
5 mL of Aquassure (Packard, Downers Grove, USA).
Non-specific binding was measured in the presence of
10 µmol 5-HT for 5-HT2A sites and 10 µmol cinanserin
for 5-HT2C sites with specific binding defined as the total
binding minus the non-specific binding. Ki values were
calculated from the Cheng-Prusoff equation [25] Ki =
IC50/1 + (ligand/Kd), where Kd = 0.8 nmol/L for [3H]ket-
anserin and Kd = 1.9 nmol/L for [3H]mesulergine [26].
5.1.12. 4-(4-Fluorobenzyl)-1-[2-(4-iodo-2,5-dimethoxy-
phenyl)ethyl]piperidine hydrochloride 16
A
mixture of
6
(0.46 g, 1 mmol), 4-fluoro-
benzoylpiperidine (0.19 g, 1 mmol) and K2CO3 (0.2 g,
1.5 mmol) in acetone (10 mL) was warmed to reflux for
20 h. The precipitate was filtered and the solution was
evaporated. The residue was partitioned between H2O
and CHCl3. The organic extracts were dried (Na2SO4)
and evaporated. The residue was purified by chromatog-
raphy eluting with CHCl3:acetone:MeOH (6.7:3:0.3, v/v)
and crystallized from acetone; m.p.: 96–98 °C; yield
1
66%. H-NMR (CDCl3) δ 1.47 (5H, m, H3,5pip, H4pip),
1.97 (2H, m, H2,6ax-pip), 2.50 (4H, m, H2,6eq-pip, ArCH2),
2.75 (2H, m, ArCH2), 2.98 (2H, m, NCH2), 3.77 (3H, s,
OCH3), 3.82 (3H, s, OCH3), 6.70 (1H, s, ArH), 6.98 (2H,
m, ArH), 7.10 (2H, m, ArH), 7.20 (1H, s, ArH). The
purified residue was dissolved in Et2O and treated with
ethereal HCl. The solid obtained was crystallized from
5.2.4. Determination of apparent 5-HT2B receptor
antagonist dissociation constant
Experiments were performed as described by Nozulak
et al. [27]. Male CD Outbred rats were sacrificed by CO2,
and longitudinal sections of the stomach fundus were
prepared for in vitro examination. Strips were set up in
organ baths of 10 mL containing Krebs solution (compo-
sition in mmol: NaCl, 118; KCl, 4.7; CaCl2, 1.25;
KH2PO4, 1.2; MgSO4, 1.2; glucose, 11; NaHCO3, 25)
constantly bubbled with 5% CO2 in oxygen. Contractions
were measured isotonically under a resting tension of 1 g.
Prior to testing, the strips were allowed to equilibrate for
1 h, during which time the bath was replaced every
15 min.
After control cumulative contractile responses to sero-
tonin were obtained in the stomach fundus, the tissues
were incubated with an appropriate concentration of
antagonist for 1 h. Contractile responses to serotonin
were then repeated in the presence of the antagonist. Only
one antagonist concentration was examined in each
tissue. Apparent dissociation constants (Kb) were deter-
absolute
EtOH;
m.p.:
204–205 °C.
Anal.
C22H27FINO2.HCl (C, H, N).
5.2. Pharmacology
5.2.1. Materials
Ketanserin tartrate was purchased from Research Bio-
chemicals International (Natick, MA, USA). [3H]Ket-
anserin (64.1 Ci/mmol) was purchased from New En-
gland Nuclear, Boston, Mass., USA. [3H]Mesulergine (76
Ci/mmol) and [3H]choline (81 Ci/mmol) were purchased
from Amersham Radiochemical Centre (Buckingham-
shire, UK). All substances employed in the binding
assays were dissolved in distilled water.
5.2.2. Animals
In the radioligand-binding studies rat cortex was ob-
tained from male Wistar rats (250–300 g body weight)

852
[5] Kursar J.D., Nelson D.L., Wainscott D.B., Baez M., Mol. Pharma-
col. 46 (1994) 227–234.
mined for each concentration of antagonist according to
the following equation:
[6] Fozard J.R., Kalkman H.O., Naunyn-Schmiedeberg’s Arch. Pharma-
col. 350 (1994) 225–229.
Kb = [B]/(dose ratio – 1)
[7] Curzon G., Kenneth G.A., Trends Pharmacol. Sci. 11 (1990)
181–182.
where [B] is the concentration of the antagonist, and the
dose ratio is the ED50 of the agonist in the presence of the
antagonist divided by the control ED50.
[8] Kennett G.A., Curr. Opin. Invest. Drugs 2 (1993) 317–362.
[9] Herndon J.L., Ismaiel A., Ingher S.P., Teitler M., Glennon R.A., J.
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38 (1995) 1196–1202.
5.2.5. Inhibition of acetylcholine release
Inhibition of the facilitatory effect of serotonin on basal
acetylcholine release from guinea-pig striatal slices was
determined as previously described [24]. Caudate nucleus
slices (400 µm thick) were incubated with 0.1 µmol
[3H]choline (81 Ci/mmol) for 30 min and superfused at
0.25 mL/min with Krebs solution (compositon in mmol:
NaCl, 118.5; KCl, 4.8; CaCl2, 2.5; KH2PO4, 1.2; MgSO4,
1.2; NaHCO3 25, glucose, 11; hemicholinium-3, 0.01),
bubbled with 95% O2 and 5% CO2. The radioactivity of
the 5 min superfusate samples was determined by liquid
scintillation. The effect of 5-HT, both in absence and in
presence of antagonists, was quantified as the net increase
of tritium efflux over the basal one, calculated as frac-
tional rate (FR), i.e. as percent of tissue tritium content.
The net increase of [3H]choline efflux, induced by 5-HT
30 µM, added to the Krebs solution from the 45th min of
superfusion, was 0.89 ± 0.11% (n = 11).
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Acknowledgements
This work was supported by Consiglio Nazionale delle
Ricerche (CNR, Rome) grant 97.02771.CT03, and by the
University of Camerino.
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