Synthesis and biodistribution of (R,S)-[O-methyl-11C]-1[3-(5-methoxy- 1,2,3,4-tetrahydro-1-naphtalenyl)propyl]-4-phenylpiperazine (PNU-157760), a putative radioligand for 5-HT(1A) receptors

Article abstract of DOI:10.1006/bioo.1998.1089

Racemic 1-[3-(5-methoxy-1,2,3,4-tetrahydro-1-naphtalenyl) propyl]-4- phenylpiperazine (PNU-157760) was labeled with carbon-11 (t( 1/4 ) = 20.4 min) as a putative radioligand for the noninvasive assessment of 5-HT(1A) receptors in vivo with positron emission tomography (PET). The radiochemical synthesis consisted of O-methylation of desmethyl precursor with [¹¹C]methyl iodide in the presence of potassium tert-butoxide in DMF. The desmethyl precursor for the radiosynthesis of [¹¹C]PNU-157760, was prepared by a convenient one-step demethylation of PNU-157760 with boron tribromide. (R,S)-[O-Methyl-¹¹C]-1-[3-(5-methoxy-1,2,3,4-tetrahydro-1- naphtalenyl)propyl]-4-phenylpiperazine with >99% radiochemical purity was obtained in 30 min with a radiochemical yield of 10 ± 5% (EOS, nondecay corrected) and a specific radioactivity of 2.5 ± 1 Ci/μmol. Biodistribution studies in rats showed that [¹¹C]PNU-157760 readily crosses the blood- brain barrier with a maximum of brain uptake at 30 min after injection; however, the low specific-to-nonspecific binding ratio in vivo as evidenced by the low hippocampus/cerebellum uptake ratio (1.17 at 60 min postinjection) does not make [¹¹C]PNU-157760 a promising radioligand for serotonin 5- HT(1A) receptors.

Full text of DOI:10.1006/bioo.1998.1089

BIOORGANIC CHEMISTRY 26, 91–102 (1998)
ARTICLE NO. BH981089
Synthesis and Biodistribution of (R,S)-[O-Methyl-11C]-1-
[3-(5-Methoxy-1,2,3,4-tetrahydro-1-naphtalenyl)propyl]-4-
Phenylpiperazine (PNU-157760), A Putative
Radioligand for 5-HT1A Receptors
Mario Matarrese,* Dmitrij V. Soloviev,* Rosa M. Moresco,* Valentino Ferri,*
Pasquale Simonelli,* Fulvio Magni,† Diego Colombo,† Sergio Todde,†
Assunta Carpinelli,* Ferruccio Fazio,* and Marzia Galli Kienle†
*CNR INB, Institute H. S. Raffaele; and †Department of Medical Chemistry and Biochemistry,
University of Milan, Milan, Italy
Received December 2, 1997
Racemic 1-[3-(5-methoxy-1,2,3,4-tetrahydro-1-naphtalenyl)propyl]-4-phenylpiperazine
(PNU-157760) was labeled with carbon-11 (t1/2 ϭ 20.4 min) as a putative radioligand for the
noninvasive assessment of 5-HT1A receptors in vivo with positron emission tomography (PET).
The radiochemical synthesis consisted of O-methylation of desmethyl precursor with
[11C]methyl iodide in the presence of potassium tert-butoxide in DMF. The desmethyl precursor
for the radiosynthesis of [11C]PNU-157760, was prepared by
a convenient one-step
demethylation of PNU-157760 with boron tribromide. (R,S)-[O-Methyl-11C]-1-[3-(5-methoxy-
1,2,3,4-tetrahydro-1-naphtalenyl)propyl]-4-phenylpiperazine with Ͼ99% radiochemical purity
was obtained in 30 min with a radiochemical yield of 10 Ϯ 5% (EOS, nondecay corrected)
and a specific radioactivity of 2.5 Ϯ 1 Ci/Ȑmol. Biodistribution studies in rats showed that
[11C]PNU-157760 readily crosses the blood–brain barrier with a maximum of brain uptake
at 30 min after injection; however, the low specific-to-nonspecific binding ratio in vivo as
evidenced by the low hippocampus/cerebellum uptake ratio (1.17 at 60 min postinjection) does
not make [11C]PNU-157760 a promising radioligand for serotonin 5-HT1A receptors.
1998
Academic Press
Key Words: [11C]PNU-157760; 5-HT1A antagonist; rats; biodistribution; radiosynthesis;
serotonin receptor; PET.
INTRODUCTION
Recent studies of the serotoninergic system implicate the involvement of the
5-HT1A serotonin receptor in several neurological and psychiatric disorders such
as depression, anxiety, schizophrenia, and Alzheimer’s disease (1–3). Development
of a radioligand for the in vivo measurement of 5-HT1A receptor occupancy by
positron emission tomography (PET) (for reviews see (4, 5)) would help to unravel
the function of these receptors and their role in various physiological and pathologi-
cal states.
Among the variety of 5-HT1A agonists and antagonists labeled with PET and
SPECT isotopes (6–11) only WAY-100635, [O-methyl-11C] and [N-carbonyl-11C],
has proven to be a potential radioligand for delineation of serotonin receptors in
91
0045-2068/98 $25.00
Copyright 1998 by Academic Press
All rights of reproduction in any form reserved.

92
MATARRESE ET AL.
FIG. 1. WAY-100635 and its analogues.
the living human brain (12, 13). Nevertheless, the metabolic pattern of [11C]WAY-
100635 complicates quantification of PET imaging data (14) and might be responsi-
ble for the failure of [O-methyl-11C]WAY-100635 to demonstrate changes in 5-HT1A
receptor concentrations (10). WAY-100635 is a silent high affinity 5-HT1A receptor
antagonist (Kd ϭ 0.4 nM, rat hippocampal membrane homogenates) (15). This
ligand is rapidly metabolized by human liver (16), the main metabolic route being
decarbonylation to give WAY-100634 (Fig. 1), which in turn is rapidly converted into
more polar metabolites, contributing to both specific and nonspecific radioactivity
binding (14, 17). Published data do not exclude the possibility of dealkylation on
the phenolic moiety, leading to formation of desmethyl-WAY-100635, which was
shown to have a performance comparable to that of [carbonyl-11C]WAY-100635
(18).
A number of fluoroalkyl, fluoroaryl, and iodoaryl analogues of WAY-100635
have been synthesized in search of a more in vivo stable 5-HT1A receptor radioligand
(9, 11, 16, 19–22). SPECT imaging with the p-iodophenyl derivative of WAY-
100635, p-[123I]MPPI, failed to visualize 5-HT1A receptors in humans, inspite of
encouraging results achieved on rats and nonhuman primates (hippocampus/cere-
bellum radioactivity ratio maximum was 3 at 50 min postinjection in cynomolgus
monkey (7). The p-fluorobenzoyl derivative of WAY-100635, p-MPPF (Kd ϭ 1.2 nM
in biogenic amine assay (23), Ki ϭ 3.3 nM against 8-OH-PIPAT in rat hippocampal

PUTATIVE RADIOLIGAND FOR 5-HT1A RECEPTOR
93
homogenates) was labeled with 18F-fluoride (24). This ligand was able to develop
a specific signal in the rat and cynomolgus monkey brain, observed hyppocampus/
cerebellum ratio at 30 min postinjection being 4 in rats (21) and 3 in cynomolgus
monkey (25), though the degree of specific binding was much greater for [11C]WAY-
100635 (hyppocampus/cerebellum ratio was 16 in rats (15) and 5.5 in macaca monkey
(26)). Comparison between 11C-carbonyl WAY-100635 and p-[18F]MPPF showed
that the brain uptake in rats at 30 min postinjection was 5 times greater for WAY-
100635, the blood clearance and the rate of metabolism was fast and similar for
both ligands (at 30 min postinjection approximately 40% of radioactivity was found
as a parent compound) (20). Analysis of arterial plasma in monkey showed that
only 20% of radioactivity remained as the parent compound at 30 min postinjection
(25). The animal biodistribution data reveal that the pharmacological profile of
p-[18F]MPPF is similar to that of p-[123I]MPPI, thus leaving a minor hope for its
possible use as a radioligand for studies in humans due to the drastic metabolism
difference between species.
It was shown by in vitro metabolic assay with human liver cytosol and microsomal
preparations that metabolically stable analogues of WAY-100635 pertaining high
affinity for the serotonin receptor could be obtained by the substitution of cyclohexyl
moiety for the bicycloalkyl group (16). Recently images of the 5-HT1A receptor-
rich areas in the living human brain were obtained with [O-methyl-11C]CPC-222
(27). No decarbonylation metabolite, [O-methyl-11C]WAY-100634, was found in
the plasma of healthy volunteers and a good brain penetration with the peak cortical
concentration corresponding to 2% of the injected dose was reported. The degree
of nonspecific binding with this tracer was higher than that observed with [carbonyl-
11C]WAY-100635 (the ratio of radioactivity in temporal cortex to that of the cerebel-
lum reached a plateau of 3 by 45 min after injection of [O-methyl-11C]CPC-222 as
compared to 25 at 60 min for [carbonyl-11C]WAY) (13, 27). This disappointing
result could be explained partially by the higher lipophilicity of the CPC-222,
calculated logP ϭ 5.3, and its lower affinity for the serotonin receptor, IC50 ϭ 8.1
nM versus [3H]8-OH-DPAT on rat hippocampal homogenates, as compared to
WAY-100635, IC50 ϭ 6.0 nM and logP ϭ 4.7 (16).
We considered the possibility to label an alternative compound, which would
have affinity and selectivity to 5-HT1A receptors comparable to that of WAY-
100635, but would not be susceptible to decarbonylation. The compound, PNU-
157760 (1) (see Fig. 2), was chosen from series of 1-aryl-4-((1-tetralinyl)alkyl)pipera-
zines; it exhibited in vitro high affinity and selectivity toward 5-HT1A receptors
(racemate, IC50 ϭ 0.50 nM, [3H]8-OH-DPAT in rat hippocampus homogenates,
ratios receptor IC50/IC50 5-HT1A : 5-HT2 ϭ 460, D2 ϭ 220, Ͱ1 ϭ 86, Ͱ2 ϭ 520) (21).
The core structure of PNU-157760 is 1-phenylpiperazine to which a 5-methoxytet-
ralinyl ring is attached via a three-membered alkyl chain (28). The molecule does
not contain the carboxamide-moiety susceptible to metabolic cleavage and could
be easily labeled by O-methylation of the phenol group of the desmethyl analogue.
In this article we report the preparation of the des-methyl precursor, radiolabeling
with [11C]methyl iodide and preliminary biodistribution experiments in rats of
the putative 5-HT1A receptor radioligand (R,S)-[O-methyl-11C]-1-[3-(5-methoxy-
1,2,3,4-tetrahydro-1-naphtalenyl)propyl]-4-phenylpiperazine (PNU-157760).

94
MATARRESE ET AL.
FIG. 2. Synthesis of [O-methyl-11C]PNU-157760.
RESULTS AND DISCUSSION
Chemistry
Des-methyl precursor was prepared conveniently from racemic (R,S)-[O-methyl-
11C]-1-[3-(5-methoxy-1,2,3,4-tetrahydro-1-naphtalenyl)propyl]-4-phenylpiperazine,
a free-base of PNU-157760, by O-demethylation of phenol ether with boron tribro-
mide as described by McOmie (29).
Radiolabeling with [11C]methyl iodide in the presence of strong bases is accompa-
nied by the formation of [11C]methanol. Nucleophiles present in the methylation
mixture, the precursor phenolate and hydroxide anion are competing for the
[11C]methyl iodide. Thus optimal temperature and substrate/base concentration
should be determined in order to maximize incorporation of [11C] into the final
product.
In Table 1 the yields of (R,S)-[O-methyl-11C]-1-[3-(5-methoxy-1,2,3,4-tetrahydro-
1-naphtalenyl)propyl]-4-phenylpiperazine and [11C]methanol are reported in rela-
tion to the amount of precursor/base used at different temperatures. The reaction
was carried out in 100 Ȑl of DMF and [11C]-incorporation yields were assessed as
percentage of the total radioactivity in the reaction mixture by integration of HPLC
Ͳ-radioactivity chromatogram.
We observed that partition of the [11C] label between methanol and the product
is the subject of both kinetic and thermodynamic control (Table 1). The first three
data points reflect the situation when [11C]methyl iodide was trapped in the reaction
mixture at Ϫ15ЊC, followed by 1.5 min heating at 100ЊC. Lowering the temperature
preferentially decreased the rate of methylation, thus 15–30% of [11C]methanol was
found in the reaction mixture. Methylation reaction was fast at room temperature;
no unreacted [11C]methyl iodide was left when precursor concentration was 10 mg/
ml and the hydrolysis to [11C]methanol was negligible (5%). Use of the lower
substrate amount (0.5 mg) resulted in the decrease of the methylation rate but did
not affect the formation of [11C]methanol, thus [11C]methanol/product ratio was
higher and 20% of [11C]methyl iodide remained unreacted. Decrease of the base

PUTATIVE RADIOLIGAND FOR 5-HT1A RECEPTOR
95
TABLE 1
Radiolabeling of Des-methyl PNU-157760 with [11C]Methyl Iodide in 100 Ȑl of DMF, 1.5-Min
Reaction Time (Percentage of Radioactivity, Mean of Three Experiments, ϮSD)
Prec.
(mg)
Base
(mg)
Temperature
11C-MeOH
11C-CH3I
(%)
11C-PNU
(%)
(ЊC)
(%)
t-BuOK
1
1
1
1
1
1
1
0.5
1
Ϫ15/100
Ϫ15/100
Ϫ15/100
0
100
40
26
26
27 Ϯ 8
20 Ϯ 5
14
14 Ϯ 6
5 Ϯ 3
2 Ϯ 1
5 Ϯ 3
31 Ϯ 5
0
1 Ϯ 1
15 Ϯ 10
5 Ϯ 4
0
59 Ϯ 1
58a
0.7
0.1
1
1
1
30a
70 Ϯ 8
82 Ϯ 2
85 Ϯ 4
78 Ϯ 4
40a
0
0
1
1
20 Ϯ 10
TBAH (Ȑl)b
40
1
2
26
0
45a
a Mean of two experiments.
b Used as a 60% aqueous solution
concentration (0.1 mg) reduced the rate of both competing reactions. We considered
also tetrabutylammonium hydroxide (60% solution in water) as an alternative base
catalyst. In this case significant hydrolysis to [11C]methanol (40% of [11C]radioactiv-
ity) was observed due to the presence of water.
Under the optimized conditions in the presence of potassium tert-butoxide only
2 Ϯ 1% of [11C]methanol was formed, thus other alternative bases for the phenolate
preparation were not considered. Trapping of [11C]methyl iodide at 40ЊC in 100 Ȑl
of DMF containing 1 mg of precursor and 1 mg of potassium tert-butoxide proceeded
almost quantitatively, as measured by monitoring the radioactivity of the ascarite/
molecular sieves/activated carbon trap placed at the reactor outlet. At the end
of [11C]methyl iodide transfer under these conditions 85% of radioactivity was
incorporated into [11C]PNU-157760, 13% of activity was randomly distributed in
five minor radioactive side-products, which were not identified, but could likely
derive from the N-methylation and hydrolysis of the final compound.
The identity of (R,S)-[O-methyl-11C]-1-[3-(5-methoxy-1,2,3,4-tetrahydro-1-naph-
talenyl]-4-phenylpiperazine was confirmed by HPLC coinjection of the radioactive
product with authentic PNU-157760 using four different conditions. The structure
of the labeled product was confirmed by the mass spectrum of the product fraction,
collected after HPLC purification of a carrier-added synthesis (see Experimental).
EI-LC-MS of the methanolic solution showed the molecular ion at m/z 364 as
expected for 1-[3-(5-methoxy-1,2,3,4-tetrahydro-1-naphtalenyl)propyl]-4-phenyl-
piperazine; moreover the relative intensity of the ion at m/z 365 perfectly corre-
sponded to that expected on the basis of natural isotope abundance for the molecular
formula C24H32N2O (27.9% of ion at m/z 364), thus excluding the presence in the
collected fraction of N-methylated products with MW 365 Da.

96
MATARRESE ET AL.
TABLE 2
Tissue Distribution of [11C]PNU-157760 in Rats (Percentage Injected Dose/g of Tissue;
Average of Three Rats Ϯ SD)
Organ
10 min
30 min
60 min
90 min
Blood
Plasma
Heart
Lung
Liver
Adrenal gland
Kidney
Spleen
Intestine
Muscle
Brain
0.02 Ϯ 0.006
0.02 Ϯ 0.01
0.10 Ϯ 0.04
0.40 Ϯ 0.20
0.30 Ϯ 0.07
0.40 Ϯ 0.06
0.30 Ϯ 0.10
0.20 Ϯ 0.04
0.10 Ϯ 0.05
0.10 Ϯ 0.02
0.10 Ϯ 0.03
0.04 Ϯ 0.02
0.05 Ϯ 0.03
0.16 Ϯ 0.03
0.40 Ϯ 0.20
1.30 Ϯ 0.60
2.40 Ϯ 0.70
0.70 Ϯ 0.20
0.50 Ϯ 0.20
0.40 Ϯ 0.20
0.20 Ϯ 0.10
0.30 Ϯ 0.10
0.02 Ϯ 0.003
0.03 Ϯ 0.01
0.06 Ϯ 0.01
0.16 Ϯ 0.04
0.48 Ϯ 0.06
0.80 Ϯ 0.20
0.24 Ϯ 0.04
0.23 Ϯ 0.04
0.73 Ϯ 0.02
0.10 Ϯ 0.07
0.10 Ϯ 0.02
0.012 Ϯ 0.004
0.016 Ϯ 0.003
0.03 Ϯ 0.003
0.10 Ϯ 0.01
0.40 Ϯ 0.02
0.90 Ϯ 0.30
0.20 Ϯ 0.09
0.17 Ϯ 0.05
0.16 Ϯ 0.06
0.05 Ϯ 0.02
0.07 Ϯ 0.02
In a typical experiment starting from 600 mCi of [11C]carbon dioxide 100 mCi
of [11C]PNU-157760 ready for injection were obtained in 30 min with radiochemical
purity greater than 99% and specific radioactivity of 2 Ci/Ȑmol (EOS). No detectable
amounts of nonradioactive impurities were found in the final preparation after the
HPLC purification on semipreparative column.
Biodistribution Studies
The biodistribution of radioactivity after [11C]PNU injection in rats is reported
in Table 2. Concentration of [11C]PNU-157760 in blood was stable during the entire
period of observation ranging between 0.012 and 0.04% of injected dose per gram of
tissue (% ID/g). The tracer preferentially accumulated in plasma where radioactivity
concentration was approximately 1.3 times higher than that in blood.
Organs with higher degree of [11C]PNU-157760 accumulation were intestine,
liver, lung, spleen, and kidney. Radioactivity uptake reached a peak at 30 min after
tracer injection in liver (1.3 Ϯ 0.6, % ID/g) and at 60 min in intensine (0.73 Ϯ 0.02),
suggesting metabolism of the compound to occur mainly in gastrointestinal tract.
[11C]PNU-157760 uptake was maximum in adrenal gland (2.4 Ϯ 0.7% ID/g, 30 min
postinjection), which is known to be rich in Ͱ-adrenergic receptors (30). Radioactiv-
ity appeared in the gland early (0.4 Ϯ 0.06% ID/g, 10 min postinjection) and
was persistent (at 90 min after injection adrenal gland/plasma concentration ratio
achieved 56.2). In vitro PNU-157760 exhibited a moderate affinity to Ͱ1 adrenergic
receptor, IC50 ϭ 43 nM, [3H]prazosin (31). On the other hand, remarkable high
uptakes in the adrenal gland were also found for several fluorine-18-labeled 5-
hydroxy-2-aminotetralin derivatives that are potential dopamine agonists (32). Ad-
renal gland is rich in catecholamine and steroid synthesizing enzymes (33, 34). It
was shown that 7,12-dimethylbenzanthracene and 7-hydroxymethyl-12-methylbenz-
anthracene, but not benzopyrene, selectively produce necrosis in the two inner
zones of the rat adrenal cortex and are toxic to cultured rat adrenocortical cells.

PUTATIVE RADIOLIGAND FOR 5-HT1A RECEPTOR
TABLE 3
97
Regional Brain Distribution of [11C]PNU-157760 in Rats (Ratio of Tissue Radioactivity to That in
Cerebellum, Average of Four Rats ϮSD) and Relative Radioactivity Uptake by the Whole Brain
Brain area
5 min
10 min
30 min
60 min
90 min
Hipothalamus
Hippocampus
Frontal Cortex
Striatum
0.7 Ϯ 0.3
0.7 Ϯ 0.3
0.8 Ϯ 0.4
1.0 Ϯ 0.4
1.82
0.9 Ϯ 0.4
0.9 Ϯ 0.2
1.1 Ϯ 0.1
1.1 Ϯ 0.3
2.95
0.9 Ϯ 0.3
0.8 Ϯ 0.1
1.0 Ϯ 0.1
1.0 Ϯ 0.3
5.88
1.6 Ϯ 0.9
1.2 Ϯ 0.5
1.0 Ϯ 0.6
1.3 Ϯ 0.5
3.21
1.1 Ϯ 0.6
0.8 Ϯ 0.3
1.0 Ϯ 0.4
0.9 Ϯ 0.4
2.83
Whole brain/plasma
Implication of prostaglandin metabolism in the necrotic mechanism of antracene
derivatives in rat adrenal cortex was proposed (35). These findings suggest that
tetralin derivatives might be metabolized by adrenal enzymes. Nevertheless further
experiments will be needed to explain the high adrenal uptake of the tracer.
[11C]PNU-157760 readily permeates the blood brain barrier and accumulates in
the rat brain where the maximum uptake at 30 min after injection was 0.3 Ϯ 0.1%
ID/g (Table 2), brain to plasma radioactivity ratio reaching a maximum of 5.88 at
this time. Regional brain uptake of the tracer (Table 3) was not homogeneous but
did not completely follow the known pattern of 5-HT1A receptor distribution within
the rat brain. High concentrations of 5-HT1A receptors are found on the cell bodies
of serotonine neurones in the raphe nuclei (autoreceptors) and at postsynaptic
locations in the hippocampus, amygdala, and certain hypothalamic nuclei (36).
Hippocampal areas contain mostly 5-HT1A sites with maximal density of 2860
fmol/mg of protein (37). The lowest densities of 5-HT1A receptors were found in
cerebellum (320 fmol/mg) and striatum (225 fmol/mg) (37). Due to the low 5-HT
receptor densities, radioactivity uptake in cerebellum is often used as a reference
for nonspecific binding. Specific binding of [11C]PNU-157760 to 5-HT1A receptor,
expressed as hippocampus/cerebellum uptake ratio, reached a maximum of 1.2 at
1 h after injection. This value was found to be very low, as compared to the specific
binding of [O-methyl-11C]WAY-100635 (hippocampus/cerebellum 16 at 1 h) (15),
thus indicating poor characteristics of [11C]PNU-157760 as 5-HT1A radioligand for
in vivo studies. Specific uptake of radioactivity strictly depends upon lipophilicity
and affinity of the radioligand as demonstrated by Kessler et al. for dopamine
antagonists (38). Thus one of the reasons for a high nonspecific uptake of [11C]PNU-
157760 could be its high lipophilicity as results from the calculated water/octanol
partition constant logP ϭ 5.89 Ϯ 0.41 (ACD/logP, Advanced Chemistry Develop-
ment, Toronto, Canada) higher than that calculated for WAY-100635, logP ϭ 4.7
(16). The low specific uptake might be explained also by an in vivo interaction of
the ligand with sigma sites (IC50 ϭ 198 nM, [3H]DTG), which are mainly expressed
in cerebellum Ͼ cortex Ͼ striatum (39, 40).
The highest accumulation and retention of [11C]PNU-157760 among different
brain areas was found in hypothalamus. The maximum relative uptake at 60 min
postinjection was 1.6 (hypothalamus/cerebellum, Table 3). The high variability of

98
MATARRESE ET AL.
the obtained data did not allow a proper statistic evaluation, though a two-sample
Student’s t test revealed that the tracer accumulation in hypothalamus at 1 h signifi-
cantly differed from initial uptake at 5 min after injection (P Ͻ 0.05). Hypothalamus
is known to present high concentration of 5-HT1B receptors (1520 fmol/mg of protein
in ventromedial hypothalamic nucleus) (37). No data were previously reported on
the binding selectivity of PNU-157760 to 5-HT1B receptor subtype. These considera-
tions, as well as the absence of metabolites in the rat brain at 1 h after injection
(data not shown) indicate that the biodistribution of [11C]PNU-157760 does not
follow the known distribution of 5-HT1A receptor within rat brain but rather may
suggest its binding to different receptor subtypes.
Conclusion
Racemic (R,S)-[O-methyl-11C]-1-[3-(5-methoxy-1,2,3,4-tetrahydro-1-naphtalen-
yl)propyl]-4-phenylpiperazine was prepared by a simple O-methylation of the des-
methyl precursor in sufficient quantity and with high specific radioactivity for studies
by positron emission tomography. Results reported here imply a limited value of
[11C]PNU-157760 as a radioligand for delineation of serotonin 5-HT1A receptor
subtypes. Nevertheless the observed biodistribution in rats suggest that further
biological studies may be of interest to elucidate the selectivity of [11C]PNU-157760
binding in vivo.
EXPERIMENTAL
General Methods
Reagents and solvents were obtained from Aldrich Italia S.p.A. (Milan, Italy)
and used without further purification unless otherwise noted. An authentic sample
of PNU 157760 dihydrochloride was a kind gift of Pharmacia & Upjohn (Nerviano
(MI), Italy).
Radiochemical syntheses were performed on the fully automated synthesis mod-
ule for [11C]-methylation (PET Tracer Synthesizer, Nuclear Interface Datentechnik
GmbH, Munster, Germany). [11C]Carbon dioxide was produced by the 14N(p,Ͱ)11C
¨
reaction on a CTI-Siemens RDS-112 cyclotron, using 9.5 MeV proton beams at
currents 10–30 ȐA. [11C]Methyl iodide was synthesized by the standard one-pot
method (41) as described elsewhere in details (42).
NMR spectra, referenced to tetramethylsilane as external standard, were regis-
tered on a Bruker AC-200 spectrometer. MS (EI) electron impact spectra were
recorded at 70 eV either by direct inlet with a Finnigan MAT95 double-focusing
mass spectrometer or by LC particle beam introduction with a HP 5988 instrument.
TLC was performed with Silica gel 60 F254 glass plates (Merck). HPLC was per-
formed on a reverse phase analytical HPLC column (Shandon Hypersil BDS C-18,
5 mm, 250 ϫ 4.6 mm) with Waters Millennium system equipped with both uv
(254 nm) and flow radioactivity detectors. GC was performed to measure the
[11C]methanol and [11C]methyl iodide in the methylation mixture using a Carlo

PUTATIVE RADIOLIGAND FOR 5-HT1A RECEPTOR
99
Erba gas chromatograph, equipped with TCD and flow radioactivity detector on
Porapak-Q column, 1000 ϫ 10 mm, 170ЊC, He 50 ml/min.
The course of [11C]-methylation and quality of the final product were controlled
by radio HPLC with acetonitrile—0.05 M sodium dihydrogen orthophosphate (3/2
v/v) at 1.3 ml/min. Retention times under these conditions were: [11C]methanol,
2.3 min: [11C]methyl iodide, 3.8 min; [11C]PNU-157760, 6.3 min.
Synthesis of the Radiolabeling Precursor
(R/S)-1-[3-(5-Hydroxy-1,2,3,4-tetrahydro-1-naphtalenyl)propyl]-4-phenylpipera-
zine (2). The aqueous solution of (R/S)-1-[3-(5-methoxy-1,2,3,4-tetrahydro-1-naph-
talenyl)propyl]-4-phenylpiperazine dihydrochloride (350 mg; 0.8 mmol; 15 ml) was
titrated with aqueous ammonium hydroxide until pH ϭ 12. After extraction with
ethyl acetate (3 ϫ 10 ml), washing with water (3 ϫ 10 ml), and drying over magne-
sium sulfate, the solvent was evaporated and 250 mg (0.68 mmol, 85% yield) of
PNU-157760 free base (1) was obtained as a clear oil.
To 100 mg (0.285 mmol) of PNU-157760 free base (1) dissolved in 4 ml of
anhydrous dichloromethane, boron tribromide (0.052 ml, 0.57 mmol) in 2 ml of
dichloromethane was added at Ϫ78ЊC under argon atmosphere. The course of the
reaction was monitored by TLC (dichloromethane/methanol 95 : 5). After standing
overnight at room temperature the reaction was complete and 5 ml of cold water
was added to destroy the excess of BBr3 . The solution was filtered, neutralized with
ammonium hydroxide, and extracted with dichloromethane (3 ϫ 20 ml). Combined
extracts were added to the filtered powder, washed with water (3 ϫ 20 ml) and
dried over anhydrous magnesium sulfate. The product was purified by means of a
silica gel flash chromatography (dichloromethane/methanol 99 : 1) to give 55 mg
(0.152 mmol, 54% yield, 99% purity) of white crystals.
1H NMR (CDCl3 , 200 MHz): ͳ 1.90 (m, 8H, 4 CH2), 2.41 (m, 2H, CH2), 2.52–2.86
(m, 6H, 3 NCH2), 2.77 (m, 1H, CH), 3.20 (m, 4H, 2 NCH2), 4.80 (br s, 1H, OH)
6.57 (d, 1H, J ϭ 8.0 Hz, Ph), 6.75 (d, 1H, J ϭ 8.0 Hz, Ph), 6.83 (dd, 1H, J ϭ 8.0
and J ϭ 8.0 Hz, Ph), 6.91 (d, 2H, J ϭ 8.0 Hz, Ph), 6.98 (dd, 1H, Ph), 7.24 (dd, 2H,
Ph). EI MS m/z: 350 (Mϩ, 30%), 175 (100%).
Radiochemical Synthesis
[11C]PNU-157760 (3). Into the closed reaction vial containing 1 mg of precursor
(2) and 1 mg of potassium tert-butoxide in DMF (100 Ȑl) [11C]methyl iodide was
transported by a stream of argon (5 ml/min) at 40ЊC. At the end of trapping (2
min) the reaction mixture was diluted with 0.9 ml of mobile phase prior to injection
into the HPLC. Semipreparative HPLC on a reversed phase column (Shandon
Hypersil BDS C-18, 5 mm 250 ϫ 10 mm i.d.) was used for purification. Product
was eluted with acetonitrile—0.05 M sodium dihydrogen orthophosphate (3/2; v/v;
pH 5.5) at 5 ml/min. Retention times were confirmed by comparison with authentic
standards (4.5 min, desmethyl precursor (2); 8.3 min, PNU-157760 (3)).
The fraction corresponding to [11C]PNU-157760 was collected in 40 ml of sterile
water, and the product was recovered by solid phase extraction on preactivated
Sep-Pak C-18 cartridge (Millipore) and formulated as a neutral 10% ethanolic

100
MATARRESE ET AL.
solution in saline after sterile filtration on 0.22 Ȑm filter (Gelman Acrodisc), total
volume 6 ml.
Specific radioactivity was determined in the final solution which was previously
assayed for total radioactivity. The amount of the nonradioactive target compound
was calculated from the uv peak area on a quality control chromatogram of a 20-Ȑl
aliquot by means of the external standard calibration plot (minimal detectable
concentration was 0.1 nmol/ml).
Carrier-Added Synthesis
Synthesis of [11C]PNU-157760 with a low specific radioactivity was carried out
under the conditions described for the preparation of the [11C]-labeled compound
except for the addition of 10 Ȑl of methanol as carrier in the hydriodic acid used
for [11C]methyl iodide formation. HPLC on a semipreparative column showed both
on the uv and radioactivity chromatograms a relevant peak at the retention time
of authentic PNU-157760 (8.3 min) which was collected. The recovered fraction
corresponding to PNU-157760 was eluted from Sep-Pak C-18 cartridge with 2 ml
of methanol and analyzed by HPLC in four different mobile phases and in two
different analytical columns.
Shandon Hypersil BDS C-18, 5 Ȑm, 250 ϫ 4.6 mm:
AcN/0.02M Na2HPO4 ; 3/2; v/v; 2 ml/min; pH 7.1; Rt ϭ 13.6 min
AcN/0.05M NaH2PO4 ; 3/2; v/v; 1.5 ml/min; pH 5,5; Rt ϭ 6.3 min
AcN/0.05M NaH2PO4 ; 2/3; v/v; 1.3 ml/min; pH 3; Rt ϭ 15.0 min
Shandon Hypersil Silica, 5 Ȑm, 250 ϫ 4.6 mm:
n-hexane/i-propanol; 99.5/0.5; v/v; 1.5 ml/min; Rt ϭ 5.8 min
When authentic PNU-157760 was coinjected with the labeled compound the ex-
pected increase of peak area on the uv chromatogram was observed.
After evaporation of methanol the product was identified by EI-LC-MS, the
mass spectrum was superimposable to that of authentic 1-[3-(5-methoxy-1,2,3,4-
tetrahydro-1-naphtalenyl)propyl]-4-phenylpiperazine, showing intense ions at m/z
364 (Mϩ, 31%), 175 (C12H15Oϩ, 100%), and 162 (C10H13N2Hϩ, 34%).
Animal Studies
Biodistribution studies in rats were performed in conformity with the demands
of the Laboratory Animal Ethical Committee (IACUC) of the Scientific Institute
H S Raffaele (Milan, Italy). Albino male CD rats aged 1–2 months and weighing
225–250 g were obtained from Charles River (Italy).
Animals were injected in the tail vein with [11C]PNU-157760 in 300 Ȑl of saline,
injected dose was 580 Ϯ 180 ȐCi per animal with specific radioactivity 1.9 Ϯ
0.2 Ci/Ȑmol. Immediately after decapitation blood sample was collected into a
heparinized tube, plasma and blood proteins were separated by centrifugation.
Regions of interest (heart, lung, kidney, intestine, liver, spleen, muscle, and brain)
were removed, placed into preweighed tubes, and assayed for the radioactivity by
an automated well-counter (LKB Compugamma CS 1282). The uptake of radioac-
tivity was calculated as a percentage of the injected dose per gram of tissue
(%I.D./g tissue).

PUTATIVE RADIOLIGAND FOR 5-HT1A RECEPTOR
101
ACKNOWLEDGMENTS
The authors thank Dr. M. Varasi and Dr. S. Mantegani (Pharmacia Upjohn S.p.A., Nerviano (MI),
Italy) for kindly supplying the batch of authentic PNU-157760 dihydrochloride.
REFERENCES
1. Fletcher, A., Cliffe, I. A., Metzler, C. W., Tada, K., and Jacobs, B. L. (1994) Trends Pharmacol.
Sci. 14, 441–448.
2. Glennon, R. A., Wastkaemper, R. B., and Bartzyer, P. (1991) Serotonin Receptor Subtypes. Peroutka,
S. J., Ed.), Wiley-Liss, New York.
3. Peroutka, S. J. (1993) J. Neurochem. 60, 408–416.
4. Crouzel, C., Guillaume, M., Barre, L., Lemaire, C., and Pike, V. W. (1992) Nucl. Med. Biol. 19,
´
857–870.
5. Pike, V. W. (1995) Nucl. Med. Biol. 22, 1011–1018.
6. Zhuang, Z.-P., Kung, M.-P., and Kung, H. F. (1993) J. Med. Chem. 36, 3161–3165.
7. Kung, H. F., Frederick, D., Kim, H.-J., McElgin, W., Kung, M.-P., Mu, M., Mozley, P. D., Vessotskie,
J. M. Stevenson, D. A., Kushner, S. A., and Zhuang, Z. P. (1996) Synapse 24, 273–281.
8. Halldin, C., Wikstrom, H., Swahn, C.-G., Sedvall, G., Stjernlof, P., and Farde, L. (1993) J. Labelled
¨
Compd. Radiopharm. 35, 494–495.
9. Mathis, C. A., Mahmood, K., Simpson, N. R., Huang, Y., Gerdes, J. M., Price, J. C., and Blastos,
B. P. (1995) in XIth International Symposium on Radiopharmaceutical Chemistry, Vancouver,
Canada. J. Labelled Compd. Radiopharm. 37, 292–294.
10. Visser, G. M., van Waarde, A., Medema, J., Vaalburg, W., Barf, T., Mensonides, M. M., Wikstrom,
¨
H., Postema, F., Korf, J., Korte, S. M., Bohus, B., Leysen, D. (1995) in XIth International Symposium
on Radiopharmaceutical Chemistry, Vancouver, Canada. J. Labelled Compd. Radiopharm. 37,
pp. 280–282.
11. Wilson, A. A., DaSilva, J. N., and Houle, S. (1995) in XIth International Symposium on Radiopharma-
ceutical Chemistry, Vancouver, Canada. J. Labelled Compd. Radiopharm. 37, 295–296.
12. Pike, V. W., McCarron, J. A., Lammerstma, A. A., Hume, S. P., Poole, K., Grasby, P. M., Malizia,
A., Cliffe, I. A., Fletcher, A., and Bench, C. J. (1995) Eur. J. Pharmacol. 283, R1–3.
13. Pike, V. W., McCarron, J. A., Lammertsma, A. A., Osman, S., Hume, S. P., Sargent, P. A., Bench,
C. J., Cliffe, I. A., Fletcher, A., and Grasby, P. M. (1996) Eur. J. Pharmacol. 301, R5–7.
14. Osman, S., Lundkvist, C., Pike, V. W., Halldin, C., McCarron, J. A., Swahn, C.-G., Ginovart, N.,
Luthra, S. K., Farde, L., and Grasby, P. M. (1997) in XIIth International Symposium on Radiopharma-
ceutical Chemistry, Uppsala, Sweden. J. Label. Compd. Radiopharm. 40, 173–175.
15. Pike, V. W., McCarron, J. A., Hume, S. P., Asworth, S., Opacka-Juffry, J., Osman, S., Lammerstma,
A., Poole, K. G., Fletcher, A., White, A. C., and Cliffe, I. A. (1994) Med. Chem. Res. 5, 208–227.
16. Wilson, A. A., DaSilva, J. N., Inaba, T., Fischer, N., and Houle, S. (1997) in XIIth International
Symposium on Radiopharmaceutical Chemistry, Uppsala, Sweden. J. Label. Compd. Radiopharm.
40, 531–533.
17. Osman, S., Lundkvist, C., Pike, V. W., Halldin, C., McCaron, J. A., Swahn, C.-G., Ginovart, N.,
Luthra, S. K., Bench, C. J., Grasby, P. M., Wikstrom, H., Barf. T. (1996) Nucl. Biol. 23, 627–
¨
634.
18. Pike, V. W., McCarron, J. A., Lundkvist, C., Halldin, C., Hume, S. P., Swahn, C.-G., Osman, S., Olsson,
H., Karlsson, P., Hall, H., Wikstrom, H., Poole, K. G. (1997) in XIIth International Symposium on
¨
Radiopharmaceutical Chemistry, Uppsala, Sweden. J. Label. Compd. Radiopharm. 40, 568–570.
19. McCarron, J. A., Lundvist, C., Pike, V. W., Halldin, C., Swahn, C.-G., Wikstrom, H., Barf, T., Cliffe,
¨
I. A., and Fletcher, A. (1995) J. Label. Compd. Radiopharm. 37, 289–291.
20. Lang, L., Schmall, B., Jagoda, E., Carson, R. E., and Eckelman, W. C. (1997) in XIIth International
Symposium on Radiopharmaceutical Chemistry, Uppsala, Sweden. J. Label. Compd. Radiopharm.
40, 534–535.

102
MATARRESE ET AL.
21. Plenevaux, A., Aerts, J., Lemaire, C., Brihaye, C., Lebars, D., Mekhsian, P., Weissmann, D., Pujol,
J. F., Comar, D., and Luxen, A. (1997) in XIIth International Simposium on Radiopharmaceutical
Chemistry, Uppsala, Sweden. J. Label. Compd. Radiopharm. 40, 148–150.
22. Zuang, Z.-P., Kung, M.-P., Chumpradit, S., Mu, M., and Kung, H. F. (1994) J. Med. Chem. 37, 4572–
4575.
23. Schmall, B., Lang, L., Jagoda, E., Channing, M., Sassaman, M., and Eckelman, W. C. (1996) in 43rd
Annual Meeting of the Society of Nuclear Medicine, Denver, CO. J. Nucl. Med. 37, 204P.
24. Zhuang, Z. P., Kung, M. P., and Kung, H. F. (1994) J. Med. Chem. 37, 1406–1497.
25. Shiue, C. Y., Shiue, G. G., Mozley, P. D., Kung, M. P., Zhuang, Z. P., Kim, H. J., and Kung, H. F.
(1997) Synapse 25, 147–154.
26. Mathis, C. A., Simpson, N. R., Mahmood, K., Kinahan, P. E., and Mintun, M. A. (1994) Life Sci.
55, L403–407.
27. Houle, S., Wilson, A. A., Inaba, T., Fisher, N., and DaSilva, J. N. (1997) Nucl. Med. Commun.
18, 1130–1134.
28. Perrone, R., Berardi, F., Leopoldo, M., Tortorella, V., Fornaretto, M. G., Caccia, C., and McArthur,
R. A. (1996) J. Med. Chem. 39, 3195–3202.
29. McOmie, J. F. C., Watts, M. L., and West, D. E. (1968) Tetrahedron 24, 2289–2292.
30. Hieble, P. J., Bondinell, W. E., and Ruffolo, R. R. (1995) J. Med. Chem. 38, 3415–3444.
31. Perrone, R., Berardi, F., Colabufo, N. A., Leopoldo, M., Tortorello, V., Fornaretto, M. G., Caccia,
C., and McArthur, R. A. (1996) J. Med. Chem. 39, 4928–4934.
32. Zijlstra, S., Elsinga, P. H., Oosterhuis, E. Z., Visser, G. M., Korf, J., and Vaalburg, W. (1993) Appl.
Radiat. Isot. 44, 473–480.
33. Kapas, S., Purbrick, A., and Hinson, J. P. (1995) Biochem. J. 305(Pt 2), 433–438.
34. Jahng, J. W., Houpt, T. A., Joh, T. H. and Wessel, T. C. (1997) J. Mol. Neurosci. 8, 45–52.
35. Hallberg, E., and Rydstrom, J. (1990) Xenobiotica 20, 1311–1318.
36. Cowen, P. J. (1991) Br. J. Psych. 159, 7–14.
37. Sanders-Bush, E. (1988) The Serotonin Receptors, in The Receptors (D. B. Bylung, Ed.), 388, The
Humana Press: Clifton, NJ.
38. Kessler, R. M., Ansari, M. S., de Paulis, T., Schmidt, D. E., Clanton, J. A., Smith, H. E., Manning,
R. G., Gillespie, D., and Ebert, M. H. (1991) J. Nucl. Med. 32, 1593–1600.
39. Perrone, R., Berardi, F., Colabufo, N. A., Leopoldo, M., Tortorella, V., Fiorentini, F., Olgiati, V.,
Ghiglieri, A., and Govoni, S. (1995) J. Med. Chem. 38, 942–949.
40. Walker, J. M., Bowen, W. D., Walker, F. O., Matsumoto, R. R., De Costa, B., and Rice, K. C.
(1990) Pharmacol. Rev. 42, 355–402.
41. Langstrom, B., and Lundqvist, H. (1976). Int. J. Appl. Radiat. Isot. 27, pp. 357–363.
˚
¨
42. Matarrese, M., Soloviev, D., Todde, S., Magni, F., Colombo, D., Galli Kienle, M., and Fazio, F.
(1997) Int. J. Appl. Radiat. Isot. 48, 749–754.