Evaluation of spirocyclic 3-(3-fluoropropyl)-2-benzofurans as σ1 receptor ligands for neuroimaging with positron emission tomography

Article abstract of DOI:10.1021/jm900909e

A series of various N-substituted 3-(3-fluoropropyl)-3H-spiro[[2] benzofuran-1,4′-piperidines] (7) has been synthesized. In receptor binding studies, the N-benzyl derivative 7a (WMS-1813) revealed extraordinarily high σ₁ receptor affinity (K

Full text of DOI:10.1021/jm900909e

6062 J. Med. Chem. 2009, 52, 6062–6072
DOI: 10.1021/jm900909e
Evaluation of Spirocyclic 3-(3-Fluoropropyl)-2-benzofurans as σ1 Receptor Ligands for Neuroimaging
with Positron Emission Tomography
Eva Große Maestrup,†,§ Steffen Fischer,‡,§ Christian Wiese,† Dirk Schepmann,† Achim Hiller,‡ Winnie Deuther-Conrad,‡
‡
Jorg Steinbach, Bernhard Wunsch,*,† and Peter Brust*,‡
€
€
†Institut fu€r Pharmazeutische und Medizinische Chemie der Westfa€lischen Wilhelms-Universita€t Mu€nster, Hittorfstraße 58-62, D-48149 Mu€nster,
Germany, and ‡Institut fu€r Interdisziplina€re Isotopenforschung, Permoserstraße 15, D-04318 Leipzig, Germany. § Both authors contributed
equally.
Received June 19, 2009
A series of various N-substituted 3-(3-fluoropropyl)-3H-spiro[[2]benzofuran-1,40-piperidines] (7) has
been synthesized. In receptor binding studies, the N-benzyl derivative 7a (WMS-1813) revealed
extraordinarily high σ1 receptor affinity (Ki =1.4 nM) and excellent σ1/σ2 selectivity (>600fold). In
vitro biotransformation of 7a with rat liver microsomes led to three main metabolites. N-Debenzylation
was inhibited by introduction of an N-phenylethyl residue (7g). The PET tracer [18F]7a was synthesized
by nucleophilic substitution of the tosylate 13 with K[18F]F-K222-carbonate complex. The decay
corrected radiochemical yield of [18F]7a was 35-48% with a radiochemical purity of >99.5% and a
specific activity of 150-238 GBq/μmol. The radiotracer properties were evaluated in female CD-1 mice
by organ distribution and ex vivo brain autoradiography. The radiotracer uptake in the brain was fast
and sufficient, with values of ∼4% injected dose per gram. Target specificity of [18F]7a was validated in
blocking studies by preapplication of haloperidol, and significant reduction in the uptake of radio-
activity was observed in the brain and peripheral organs expressing σ1 receptors.
Introduction
long-known endogenous hallucinogen N,N-dimethyltrypta-
mine, has recently been verified10 and proposed to enhance
Ca2þ signaling from the ER into mitochondria via σ1 receptor
binding.11
Since sigma (σ) receptors were initially postulated as sub-
type of opioid receptors by Martin and colleagues,1 they have
been characterized as specific, nonopioid, nonphencyclidine
but haloperidol sensitive binding structures.2 Two distinct
classes of σ receptors, named σ1 and σ2, are postulated,3 but
only σ1 receptor protein has been identified up to now. The
human σ1 receptor is a unique protein consisting of 223 amino
acids, which was first cloned and functionally expressed by
Kekuda and colleagues.4 The corresponding gene is located
on human chromosome 9, band p13, a region known to be
associated with different psychiatric disorders.5 The σ1 recep-
tor is distributed in the neuronal, immune, and endocrine
systems with especially high expression in limbic and motor
brain structures as well as in peripheral organs such as heart,
lung, liver, pancreas, and sexual and immune glands.4,6 By
electron microscope studies in mouse and rat brain σ1 receptor
immunoreactivity has been shown to be related to neuronal
cell bodies and dendrites with particular accumulation within
mitochondrial membranes, the endoplasmatic reticulum
(ER), and postsynaptic membranous thickening.7,8 As these
subcellular compartments are known to be involved into the
regulation of intracellular Ca2þ concentration, these ultra-
structural together with pharmacological data on σ1 receptor
activation9 indicate σ1 receptor-mediated modulation of vari-
ous neurotransmitter systems via Ca2þ-dependent cell signal-
ing cascades. An endogenous agonist for the σ1 receptor, the
Also, a wide range of synthetic σ ligands has been identified.
These are chemically unrelated drugs like the psychotomi-
metic benzomorphan pentazocine, cocaine, and cocaine re-
lated psychostimulants and neuroleptics such as haloperidol.9
Because of substantial antidepressive, antiamnesic, anti-
anxiety, neuroprotective, and cognition enhancing effects of
σ1 receptor activation,8,12 the σ1 receptor became an impor-
tant target in drug development.9,13-15 However, regardless
the pharmacologic efficacy of σ1 receptor agonists, measure-
ments of physiologic and pathologic σ1 receptor protein
expression revealed contrary data. While, e.g., in mice the σ1
receptor activation ameliorates cognitive deficits associated
with physiological aging despite an unchanged and age-
independent σ1 receptor density,8 an aging-related increase in
the number of σ1 receptor sites was observed in rat and monkey
brain.16,17 By contrast, several brain-related pathologies
in humans are characterized by diminished σ1 receptor
sites, as it has been demonstrated in Parkinson’s disease and
Alzheimer’s disease by postmortem [3H]-1,3-di(o-tolyl)guanidine
binding18 and in vivo [11C]SA4503 imaging studies.19,20
Thus, noninvasive in vivo targeting of cerebral σ1 receptor
protein by neuroimaging approaches such as positron emis-
sion tomography (PETa) is considered to be of value for (i)
*Corresponding author. For B.W.: phone, þ49-251-8333311; fax,
þ49-251-8332144; E-mail: wuensch@uni-muenster.de. For P. B.:
phone, þ49-341-2354013; fax, þ49-341-2352731; E-mail: brust@
iif-leipzig.de.
a Abbreviations: PET, positron emission tomography; CNS, central
nervous system; pi, post injection; ID, injected dose; fc, flash chromato-
graphy; ip, intraperitoneal; cps, counts per second; RCY, radiochemical
yield; rt, room temperature.
r
pubs.acs.org/jmc
Published on Web 09/11/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6063
drug-development by target validation via correlation of σ1
receptor occupancy with efficacy of potential therapeutics21,22
and (ii) basic and clinically applied research by quantifying σ1
receptor expression in the healthy and diseased brain. More-
over, (iii) it may also provide important new information
about the role of σ1 receptors in the complex neurotransmitter
balance in the CNS, which can lead to new strategies for the
treatment of several CNS diseases.
The most widely investigated σ1 receptor ligands for PET
imaging belong to the piperidine structure such as [18F]FPS,23
[18F]FBFPA,24 [18F]FM-SA4503,25 and [11C]SA450326 aswell
as [18F]-labeled benzamides.27 However, while experimental
data support the suitability of, e.g., [18F]FPS28 or [18F]FM-
SA4503,25 in vivo evaluation of central σ1 receptors with
18F-labeled ligands is hampered by difficulties such as slow
kinetics29 or the permeation of radiometabolites into the
brain.25 Thus, in the present, the feasibility of clinical
studies on σ1 receptor imaging in neurology is limited to the
11C-labeled PET radiotracer [11C]SA4503 demanding on-site
availability of a cyclotron. The development of 18F-labeled
radiotracers, which will extent the application to sites away
(“satellite concept”), requires further experimental attempts.
The group of high affinity σ1 ligands comprises several
structurally differing compounds (Figure 1).30 Nevertheless,
most of these ligands contain a phenyl ring and a basic amino
group.31 They include butyrophenone neuroleptics (e.g. ha-
loperidol, 1), guanidines(e.g. 1,3-di(o-tolyl)guanidine, 2), dex-
trorotatory benzomorphans (e.g. (þ)-pentazocine, 3), cis-
configured cyclohexane-1,2-diamines (e.g. BD737, 4), and
cocaine and cocaine related psychostimulants.32,33 The halo-
peridol related piperazine derivative BMY-14802, which inter-
acts unselectively with σ1 and σ2 receptors, has been investigated
in clinical trials (phase II) as atypical antipsychotic.34
Figure 1. Structurally diverse σ receptor ligands.
Figure 2. Spirocyclic σ1 receptor ligands. Because of the promising
pharmacological properties, compound 5 was selected as lead
compound for the development of a PET tracer for the central
nervous system.
the optimized radiosynthesis of [18F]7a, including the evalua-
tion of its pharmacokinetics in CD-1 mice.
Synthesis. The synthesis started with the 3-methoxy-2-ben-
zofuran 5. Upon treatment with allyltrimethylsilane and
BF3 OEt2, the acetalic methoxy group of 5 was exchanged
During the last 10 years we have developed a novel class of
σ1 ligands with a spirocyclic scaffold. These spirocyclic piperi-
dines show nanomolar affinity at the σ1 receptor subtype and
excellent selectivity against the σ2 receptor subtype.35-38 The
spiro[2-benzofuran-1,40-piperidine] 5 represents one of the
most promising σ1 ligands of this series. (Figure 2) It is an
extraordinarily potent σ1 receptor antagonist (Ki = 1.1 nM)
with high selectivity against the σ2 subtype (>1000fold) and
more than 60 other receptors, ion channels (including the
hERG channel), and transporters. In the capsaicin assay, 5
was able to reduce the neuropathic pain reaction of mice.39
The excellent pharmacological properties of 5 prompted us to
develop a fluorinated PET tracer based on this spirocyclic
system.
In the first approach, the fluorine atom was introduced into
the N-benzyl moiety. Different substituents and functional
groups were introduced in position 3 of the benzofuran
system. Indeed, the spirocyclic piperidine 6 demonstrated high
σ1 receptor affinity and selectivity over the σ2 subtype.
Unfortunately, the metabolic stability of the para-fluoroben-
zyl derivative 6 was not satisfying andthe preparationof[18F]6
proved to be complicated. However, these investigations
revealed that the spirocyclic compound 6a with an allyl moiety
in position 3 was well tolerated by the σ1 receptor protein (6a,
allyl instead of OCH3, Ki=1.4 nM (σ1), Ki=971 nM (σ2)).40
Therefore, in the second approach, the fluorine atom
should be introduced into the C-3 side chain of the spirocyclic
benzofuran system. Herein we report on the synthesis, σ1
receptor affinity and selectivity, structure-affinity relation-
ships, in vitro and in vivo metabolism of 3-fluoropropyl
substituted spirocyclic compounds of type 7 as well as on
3
for an allyl group to form 8. Hydroboration of the allyl
substituent with 9-borabicyclononane (9-BBN) and subse-
quent oxidative workup with NaOH/H2O2 led to the propanol
derivative 9. The hydroxy moiety of 9 was directly substituted
by diethylaminosulfur trifluoride (DAST) to provide the
3-fluoropropyl derivative 7a. After hydrogenolytic cleavage
of the N-benzyl moiety of 7a, the resulting secondary amine
7b was alkylated or reductively alkylated with alkyl halides or
aldehydes and NaBH(OAc)3 to provide variously substituted
spirocyclic piperidines 7c-7k. (Scheme 1)
According to our experience with spirocyclic σ1 ligands35-38
various N-residues were selected in order to get compounds
with high σ1 affinity and selectivity (7c,h-k). Because we have
shown that N-debenzylation is a predominant metabolic reac-
tion of 5 and 6,39,40 further residues, in which the labile
N-benzyl moiety of 7a was shielded by ortho-substituents
at the benzene ring (7d, 7e) or by additional substituents at
the methylene moiety (7f, 7g), were selected.
To get insight into the effects of the fluorine atom in the
side chain of 7 on the σ1 receptor affinity, the corresponding
propyl derivative 10 was prepared by hydrogenation of the
allylic side chain (Scheme 2). In addition to the propyl
derivative 10 the allyl derivative 8, the related alcohol 9 as
well as the tosylate 13 (see Scheme 4) were included into the
receptor binding studies.
Receptor Affinity. The σ receptor affinities of the spirocyclic
compounds were determined in competition experiments with
radioligands. Homogenates of guinea pig brains were used as

6064 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Große Maestrup et al.
Scheme 1. Synthesis of Spirocyclic σ1 Receptor Ligands with a 3-Fluoropropyl Residue in Position 3a
a Reagents and conditions: (a) allyltrimethylsilane, BF3 OEt2, CH2Cl2, -25 °C, 82%. (b) (1) 9-BBN, THF, rt; (2) H2O2, NaOH, -25 °C, 92%.
3
(c) diethylaminosulfur trifluoride (DAST), CH2Cl2, -78 °C to rt, 53%. (d) NH4HCO2, Pd/C, CH3OH, reflux, 82%. (e) RX, K2CO3, CH3CN, or
R0CHdO, NaBH(OAc)3, CH2Cl2, 63-81% (exception 7d, yield 6%).
Scheme 2. Synthesis of the 3-Propyl Derivative 10a
According to our experience with spirocyclic σ receptor
ligands,37,38 the N-benzyl residue of 7a was also replaced by
N-alkyl and N-alkenyl residues. Generally, the N-alkyl deri-
vatives 7h-7k represent very potent σ1 receptor ligands with
a reduced σ1/σ2-selectivity. In particular the N-cyclohexyl-
methyl derivative 7k, which is the most potent σ1 ligand of
this series (Ki=0.57 nM), has also a very high affinity toward
the σ2 receptor (Ki=26 nM).
To show the influence of the fluorine atom in the side chain
on the σ1 receptor affinity and σ1/σ2-selectivity, the corre-
sponding propyl derivative 10 without a fluorine substituent
a Reagents and conditions: (a) H2, Pd/C, methanol, 86%.
in the propyl side chain as well as the allyl derivative 8 and the
receptor material in the σ1 assay and the σ1 selective ligand
[3H]-(þ)-pentazocine was employed as radioligand. The non-
alcohol 9 were pharmacologically evaluated. The σ1 receptor
affinities of the four derivatives 7a, 8, 9, and 10 are very
similar (Ki = 1.4-3.3 nM). Obviously, the fluorine atom of
7a can be replaced bioisosterically by an OH moiety (9), a
of rat liver served as source for σ2 receptors. The nonselective
specific binding was determined in the presence of a large excess
of nontritiated (þ)-pentazocine. In the σ2 assay homogenates
radioligand [3H]-1,3-di(o-tolyl)guanidine was employed in the
presence of an excess of nontritiated (þ)-pentazocine for
selective occupation of σ1 receptors. The nonspecific binding
of the radioligand was determined by performing the σ2 assay in
the presence of an excess of nontritiated 1,3-di(o-tolyl)-
guanidine.35,40
The receptor binding data in Table 1 clearly indicate that
the benzyl derivative 7a (WMS-1813) is a very potent σ1
receptor ligand with a Ki value of 1.4 nM. The σ1 affinity of
7a is comparable with the σ1 affinity of the lead compound 5,
indicating that the acetalic methoxy moiety of 5 can be
replaced by a 3-fluoropropyl residue without loss of affinity.
In addition to the high σ1 affinity, the selectivity of 7a against
the σ2 subtype (Ki = 837 nM) is in the same range as the
selectivity of 5.
To reduce the N-debenzylation during the biotransforma-
tion, the N-benzyl moiety of 7a was replaced by sterically
demanding benzyl residues. However, the introduction of a
CF3 moiety (7e) or two methyl groups (7d) in the ortho
positions of the phenyl moiety led to a drastic decrease or loss
of affinity. Whereas the benzhydryl derivative 7f is not tolerated
by the σ1 receptor, the corresponding phenylethyl derivative 7g
interacts with considerable affinity (Ki=7.3 nM) with the σ1
receptor protein. Moreover, the σ1/σ2-selectivity of 7g is very
high, with only 19% inhibition of σ2 receptor binding at 1 μM.
proton (10), or a double bond (8) without loss of σ1 affinity.
The four related compounds 7a, 8, 9, and 10 also reveal
similar low σ2 receptor affinities, indicating excellent σ1/σ2-
selectivity for these derivatives.
The high σ1 receptor affinity of the tosylate 13 (Ki = 8.2
nM) was very surprising because it was not expected that the
σ1 receptor protein would accept a compound with such a
large substituent in position 3 of the benzofuran system.35,36
Moreover, the strong alkylating properties of the tosylate 13
could be exploited for the development of an irreversible σ1
receptor ligand.
With respect to σ1 affinity and σ1/σ2-selectivity, the most
promising compound of this series is the N-benzyl derivative
7a. Therefore, the affinity of 7a toward the phencyclidine
binding site of the NMDA receptor and toward the three
classical opioid receptors, μ, δ, and κ receptors, has been
investigated in receptor binding studies. At a test compound
concentration of 1 μM, 7a did not compete considerably with
the radioligand [3H]-MK-801, indicating high selectivity
against the NMDA receptor. Moderate affinities toward
the μ (Ki=201 nM), δ (Ki = 308 nM), and κ opioid receptors
(Ki = 344 nM), were found. Nevertheless, the selecti-
vity factors with respect to σ1 affinity (Ki = 1.4 nM) are
always higher than 140, indicating a sufficient σ1/opioid-
selectivity.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6065
Table 1. Affinities of 3-Fluoropropyl Substituted and Related Spiropiperidines towards σ1 and σ2 Receptors
Ki ( SEM (nM)a
σ1/σ2 selectivity
compd
R
σ2 ([3H]-(þ)-pentazocine) (σ1)
σ1 ([3H]-di-o-tolylguanidine) (σ2)
535
7a (WMS-1813)
1.1 ( 0.12
1.4 ( 0.26
1.9 ( 0.55
1280 ( 79
837
1164
620
C6H5CH2-
4-F-C6H4CH2-
2,6-Me2C6H3CH2-
2-CF3C6H4CH2-
Ph2CH-
Ph(CH3)CH-
(H3C)2CdCHCH2-
n-Bu
(n=1)
(n=1)
(n=1)
(n=1)
(n=1)
(n=1)
(n=1)
(n=1)
7c
7d
742
59% b
0% b
0% b
19% b
376
215
397
17% b
0% b
1110
(n=1)
(n=1)
(n=1)
7e
7f
7g
7.3 ( 0.64
2.0 ( 0.82
4.5 ( 0.58
37 ( 11
7h
7i
191
48
3
7j
7k
n-Oct
109 ( 17
26 ( 2.4
836
34% b
1110
C6H11CH2-
C6H5CH2-
C6H5CH2-
C6H5CH2-
C6H5CH2-
0.57 ( 0.14
1.8 ( 0.25
3.3 ( 0.61
3.2 ( 0.54
8.2 ( 0.68
3.9 ( 1.5
61 ( 18
45
464
8
(n=1)
(n=1)
9
10
346
13
haloperidol
di-o-tolylguanidine
22% b
78 ( 2.0
42 ( 15
(n=5)
(n=5)
21
(n=7)
1.4
a The Ki values were determined in three independent experiments (n = 3) unless otherwise noted. b Inhibition at 1 μM.
Scheme 3. Metabolites Formed by Incubation of 7a and 7g with
Rat Liver Microsomes
Figure 3. Metabolic degradation of the 3-fluoropropyl derivatives
7a and 7g.
benzylation is inhibited by the branched N-residue of 7g,
which results in a reduced metabolic degradation.
To determine the metabolically labile structural elements
in the σ1 ligands 7a and 7g, the structure of the metabolites
was elucidated with LC-MS using an ion trap as detector,
which allows diverse fragmentation experiments (MSn).
It was shown that both σ1 ligands formed comparable
metabolites (Scheme 3). Oxidative debenzylation of 7a and
7g led to the secondary amine 7b, which was unequivocally
identified by comparison with the synthesized reference
compound (see Scheme 1). However, in the case of the
phenylethyl derivative 7g, only small amounts of the second-
ary amine 7b were formed, proving the hypothesis that the
reduced oxidative debenzylation process is responsible for
the increased metabolic stability of 7g. In addition to the
N-debenzylation, an aromatic hydroxylation of the benzyl
moiety took place to afford 11a and 11b, respectively.
Although the position of the hydroxy moiety could not be
determined unequivocally, hydroxylation in para-position is
assumed. A third main metabolic pathway is the hydroxyla-
tion in the 3-fluoropropyl side chain (12a,b). MSn fragmen-
tation experiments revealed clearly a hydroxy moiety in this
part of the molecule but did not allow the determination
of the exact position of the hydroxy group. Nevertheless,
it is assumed that the hydroxylation had taken place in the
In Vitro Metabolism in Rat Liver Microsomes. Because the
3-fluoropropyl derivative 7a was designed for PET studies in
vivo, its metabolic stability was investigated in vitro with rat
liver microsomes. Additionally, the most active of the
branched derivatives, the N-phenylethyl derivative 7g, was
included into this study (cf. Table 1). These experiments were
performed with the non-radioactive compounds bearing the
naturally occurring isotope 19F.
At first the rate of the metabolic degradation was inves-
tigated. For this purpose, eight incubations were started at
the same time and stopped with a time shift of 10 min each to
observe the decomposition. Directly after termination of the
metabolic process, the internal standard praziquantel was
added to quantify the remaining parent compound by HPLC
analysis. A matrix calibration was carried out for both
spirocyclic σ1 ligands. Each data point was recorded once.
In Figure 3, the metabolic degradation of the N-benzyl
derivative 7a and the N-phenylethyl derivative 7g is com-
pared showing that the phenylethyl derivative 7g was meta-
bolically more stable than 7a. Whereas after an incubation
period of 30 min, only 46% of the parent compound 7a
were left more than 80% of the N-phenylethyl derivative 7g
remained unchanged. It is assumed that the oxidative de-

6066 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Scheme 4. Synthesis of the Radiotracer [18F]7aa
Große Maestrup et al.
a (a) TosCl, NEt3, DMAP, CH2Cl2, rt, 76%. (b) K[18F]F/K222,
MeCN, 85 °C, 30 min, 60-70%.
(ω-1)-position, leading to 3-fluoro-2-hydroxypropyl deriva-
tives 12a,b. Because a new center of chirality was created
upon hydroxylation in the side chain, two diastereomers
were formed, which are represented by two similar peaks in
the HPLC analysis.
These investigations demonstrate clearly that the phenyl-
ethyl derivative 7g is metabolically more stable than the
benzyl derivative 7a. However, because of the two centers
of chirality, the phenylethyl derivative 7g represents a mix-
ture of four stereoisomers, which might complicate in vivo
PET studies. The higher σ1 receptor affinity together with the
sufficient metabolic stability in vitro prompted us to develop
the benzyl derivative 7a as PET tracer for the investigation of
σ1 receptors in the CNS. Subsequently, the 18F-labeled
spiropiperidine 7a should be investigated to image and
quantify the σ1 receptor protein by PET.
Figure 4. Radio- and UV-chromatograms from the semiprepara-
tive HPLC separation used in the radiosynthesis of [18F]7a. See
Experimental Section for chromatographic conditions.
Crippen’s fragmentation), 3.74 (ChemDraw, Viswanadhan’s
fragmentation), and 3.47 (ACD/LogD, version 4.56/27) are
higher than the experimentally determined values, the data
suggest that [18F]7a has sufficient lipophilic properties to
pass the blood-brain barrier.
Ex Vivo Evaluation of [18F]7a in Mice. The time-activity
data on the organ distribution as well as on the brain
permeation of the radiotracer acquired in female CD-1 mice
at 5, 30, 60, and 120 min after intravenous administration of
400-600 kBq [18F]7a are summarized in Table 2. Radio-
tracer accumulation in brain and cerebellum was highest at
5 min pi and reached values of 3.66 and 4.62% ID/g wet
weight, respectively. At 120 min pi, 2.14 and 2.41% ID/g
remained in the brain and the cerebellum. Cerebellum to
plasma ratios of 2.49, 3.86, 5.75, and 5.48 at 5, 30, 60, and
120 min pi, respectively, were determined, while cerebellum
to brain ratios remained nearly constant over time. The
time-activity data of bladder and urine (2.89 ( 1.18 and
49.2 ( 15.1% ID/g at 5 and 120 min pi, respectively) point to
renal elimination as a major excretory pathway of the radio-
tracer. Continuous gastrointestinal accumulation of the
radiotracer resulted in an intestine uptake of 9.03% ID/g
at 120 min pi. The high initial uptake in the lung and the heart
was followed by a rapid washout (16.2 and 6.52 vs 3.37 and
1.52% ID/g at 5 and 120 min pi, respectively), while uptake
in the adrenals, pancreas, spleen, and thymus possessed
nearly stable levels (>70% of the initial value at 120 min
pi). Accumulation of radioactivity in the femur, 2.17% ID/g
observed already at 5 min pi and steadily increased up to
6.64% ID/g during the course of the experiment, points to
defluorination, contrary to the below mentioned observa-
tions of [18F]7a metabolism in vivo.
Radiochemistry. [18F]7a was synthesized as shown in
Scheme 4 starting from the tosylate precursor 13 because
the tosylate moiety is known to be a good leaving group for
nucleophilic radiofluorination.41 The precursor synthesis
was conducted by transformation of the alcohol 8 into the
tosylate 13 upon treatment with para-toluenesulfonyl chlor-
ide in the presence of dimethylaminopyridine (DMAP) and
triethylamine.
[18F]fluoride was transferred into the K[18F]F-K222-
carbonate complex using a 1:1 ratio of K2CO3/Kryptofix
K222. The reaction conditions were optimized and labeling
yields of 60-70% were obtained in acetonitrile by heating at
85 °C for 30 min, whereas in the solvents DMF and DMSO,
significantly lower labeling yields of 10% at most were
achieved (n = 11). According to radio-HPLC and radio-
TLC data, only a limited number of radioactive and
nonradioactive byproduct occurred in the crude labeling
mixture. Hence, the crude product was directly applied to
an isocratic semipreparative HPLC for purification
(Figure 4). Within a synthesis time of 90-120 min, [18F]7a
was obtained with a decay-corrected radiochemical yield of
35-48%, a radiochemical purity of >99.5%, and a specific
activity of 150-238 GBq/μmol (n=7).
To investigate the radiotracer accumulation in different
brain regions, brain sections were evaluated by ex vivo
autoradiography at 45 min after iv injection of [18F]7a
(Figure 5). The spatial radiotracer distribution reflects accu-
mulation of [18F]7a in brain areas known to be rich in σ1
receptors such as cerebellum, dorsolateral and lateral peria-
queductal gray, the dorsomedial hypothalamic area, and the
area of the facial nucleus. Comparably low radiotracer
accumulation was found in thalamic nuclei, cortical areas,
and striatum.
[18F]7a was chemically stable in 0.9% NaCl solution,
Sorensen’s phosphate buffer (50 mM, pH 7.2), Dulbecco’s
e
phosphate buffered saline (pH 7.2), and TRIS-HCl (30 mM,
pH 7.2) at 40 °C for up to 120 min, and according to radio-TLC
and analytical radio-HPLC, no defluorination occurred.
Consistent values of apparent distribution coefficients
were determined experimentally in the three buffered solu-
tions with log D = 3.03 ( 0.19 (n = 50; shake-flask method).
This value was consistent with the log D values calculated
from radio-HPLC retention times: log D = 3.32 ( 0.19
(Multisorb C18) and log D=3.09 ( 0.23 (Supelcosil C18).
Although the calculated log D values of 3.81 (ChemDraw,
We proved the target specificity of [18F]7a accumulation
by preinjection of the σ1/σ2 receptor antagonist haloperidol
(1 mg/kg). Organ distribution of the radiotracer at 60 min pi
of the tracer (Table 2) shows a significant reduction in
radioactivity uptake in cerebellum (-79%), brain (-76%),

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6067
Table 2. [18F]7a Biodistribution in Female CD-1 Mice without (control) and with ip Preadministration of 1 mg Haloperidol/kg (blocking)a
radioactivity uptake (% ID/g wet weight)
control
blocking
60 min pi (n = 5)
organ
blood
5 min pi (n = 4)
30 min pi (n = 4)
60 min pi (n = 5)
120 min pi (n = 4)
% blocking
pb
1.44 ( 0.39
1.85 ( 0.32
4.62 ( 0.57
3.66 ( 0.43
6.52 ( 0.97
16.2 ( 1.48
3.39 ( 0.17
4.35 ( 0.36
5.54 ( 1.38
12.1 ( 1.33
2.89 ( 1.18
4.51 ( 0.42
3.98 ( 1.16
8.76 ( 1.80
10.6 ( 1.88
3.40 ( 0.90
2.18 ( 0.27
2.17 ( 0.31
0.56 ( 0.15
0.96 ( 0.13
3.71 ( 0.56
3.18 ( 0.78
3.75 ( 0.68
7.01 ( 1.23
5.13 ( 1.39
6.81 ( 0.86
4.28 ( 1.55
7.49 ( 1.09
24.3 ( 13.9
5.86 ( 1.17
3.26 ( 0.46
12.8 ( 3.06
7.63 ( 1.70
4.11 ( 1.33
1.80 ( 0.30
3.79 ( 2.00
0.37 ( 0.08
0.52 ( 0.11
2.99 ( 0.94
2.27 ( 0.71
2.46 ( 0.59
4.64 ( 0.59
3.17 ( 1.31
6.59 ( 2.41
3.24 ( 0.97
5.14 ( 1.11
7.84 ( 6.70
5.56 ( 1.15
2.87 ( 0.87
8.92 ( 2.86
7.78 ( 2.79
4.22 ( 1.36
0.97 ( 0.27
3.48 ( 1.55
0.48 ( 0.08
0.44 ( 0.07
2.41 ( 0.59
2.14 ( 0.38
1.52 ( 0.22
3.37 ( 0.24
3.12 ( 1.08
9.03 ( 2.91
2.93 ( 1.33
3.68 ( 0.37
49.2 ( 15.1
4.65 ( 0.51
2.86 ( 0.27
10.5 ( 0.90
5.86 ( 1.06
4.16 ( 1.36
0.97 ( 0.25
6.64 ( 3.63
0.56 ( 0.13
0.63 ( 0.16
0.63 ( 0.11
0.54 ( 0.17
1.09 ( 0.35
2.11 ( 0.79
5.88 ( 2.38
6.60 ( 2.65
3.89 ( 0.95
3.42 ( 0.81
22.5 ( 19.8
1.89 ( 0.33
2.36 ( 1.02
3.05 ( 0.25
5.72 ( 1.89
4.92 ( 1.10
0.90 ( 0.33
4.27 ( 1.37
(þ53)
(þ22)
79
0.010
0.114
plasma
cerebellum
brain
<0.001
<0.001
0.001
76
56
heart
lung
54
(þ86)
0
<0.001
0.028
0.496
stomach
intestine
liver
(þ20)
33
0.159
0.012
0.077
kidney
bladder/urine
spleen
(þ189)
66
18
<0.001
0.211
0.001
thymus
pancreas
adrenal
gonad
66
26
0.104
0.200
(þ16)
7
muscle
femur
0.367
0.209
(þ23)
a Means ( SD. b p-Values for the control vs blocking group at 60 min pi calculated by Student’s t test (independent, two-tailed).
Figure 5. Ex vivo autoradiography of a sagittal brain slice of a CD-
1 mouse obtained at 45 min after iv injection of 68 MBq [18F]7a.
spleen (-66%), pancreas (-66%), heart (-56%), and lung
Figure 6. HPLC- and TLC-radiochromatograms of radioactive
compounds in mouse liver homogenate at 45 min after intravenous
injection of [18F]7a. Radio-HPLC (Multisorb RP 18-7): radio-
metabolites with tR ∼17 and 26 min; radio-TLC: samples in
triplicate (1-3), nonmetabolized [18F]7a accounts for ∼57% of
total radioactivity. See Experimental Section for detailed chromato-
graphic conditions.
(-54%). The plasma concentration of [18F]7a was margi-
nally affected by haloperidol (þ22%, P=0.114), and there-
fore the cerebellum to plasma ratio decreased to 1.0.
We examined the in vivo metabolic stability of [18F]7a
using mouse plasma, urine, brain, and liver samples at 45 and
60 min pi. Extraction efficiency of 63-75% indicated high
recovery of the radiotracer from the tissue homogenates. The
supernatants of each sample were analyzed by radio-TLC
and radio-HPLC and quantified by online peak integration
and γ-counting of the isolated HPLC fractions, respectively.
Respective TLC and HPLC chromatograms of liver samples
and urine (TLC not shown), obtained at 45 min pi, are shown
in Figures 6 and 7. Three hydrophilic radiometabolites
accounted for 45% of total radioactivity in samples of crude
liver homogenates. It is assumed that the three metabolites
detected in vivo correlate with the metabolites 7b, 11a, and 12a,
which were identified after incubation of 7a with rat liver
microsomes. By analysis of urine samples, two hydrophilic
and one lipophilic radiometabolites of [18F]7a were detected,
representing ∼98% of the total radioactivity. For plasma
samples and brain tissue, an alternative HPLC system with
improved chromatographic separation (Supelcosil C18 column
with gradient elution; details are given in Experimental Section)
was applied. In plasma samples, >85% radiotracer remained
stable and only one minor radiometabolite peak was detectable.
In brain, the fraction of the nonmetabolized tracer accounted
Figure 7. Radiochromatogram from the analytical HPLC separa-
tion of radioactive compounds in a mouse urine sample at 45 min
after iv injection of [18F]7a. See Experimental Section for chromato-
graphic conditions.
for g95% of the total radioactivity at 60 min pi, in good
agreement with radio-TLC data (Figure 8).

6068 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Große Maestrup et al.
Figure 8. HPLC-(Supelcosil C18) and TLC-radiochromatograms of radioactive compounds in mouse brain homogenate at 60 min after iv
injection of [18F]7a. Radio-TLC: samples in triplicate (1-3), nonmetabolized [18F]7a accounts for >95% of total radioactivity according to
radio-HPLC and -TLC. See Experimental Section for detailed chromatographic conditions.
Discussion
distribution revealed a high initial uptake of radioactivity in
most organs known to express σ1 receptors including brain,
heart, lung, kidney, liver, and organs of the immune and
endocrine system.43,44 In addition, in terms of organ specifi-
city and amount of radioactivity, the estimated uptake of
[18F]7a is comparable with data reported previously with 18F-
and 11C-labeled radiotracers targeting σ1 receptor.23,25,45
During the course of the experiment, the uptake of radio-
activity remained high in pancreas, gonads, spleen, and
thymus at a level of ∼100% to 70% of the initial uptake,
whereas a pronounced washout was observed in the adrenals,
heart, and lung. However, the radioactivity uptake in all these
organs except liver and gonads was sensitive for haloperidol
and is therefore assumed to reflect target specific binding to
some extent despite differences in the washout kinetics.
To evaluate the metabolic stability of [18F]7a in vivo,
plasma, urine, liver, and brain samples were analyzed by
radio-HPLC and radio-TLC. The percentage of unmetabo-
lized [18F]7a at 60 min pi accounted for >85% and >95% of
total plasma and brain radioactivity, respectively. Although
[18F]F- was not detectable in plasma samples, neither by
radio-TLC nor radio-HPLC, the increase of radioactivity
concentration in bone seems to indicate in vivo defluorination
of [18F]7a. Also incubation of 7a with rat liver microsomes in
vitro did not lead to defluorinated metabolites. However, a
thorough evaluation of our experimental data revealed a link
between the age of the mice and the radioactivity uptake in the
bone so that accumulation was higher in younger than older
animals (∼8 weeks vs ∼12 weeks). Therefore, the high stan-
dard deviation estimated for the respectiveraw data (∼55% of
the mean at 120 min pi) could be caused by experimental
reasons due to age-specific differences in bone matrix synth-
esis46 or hematopoietic potential.47 Thus, a binding of [18F]7a
to biological components linked with proliferative processes
might be assumed to contribute at least in part to the radio-
activity uptake in bone in our study as well as to the recently
reported uptake of [18F]FM-SA4503- and [11C]SA5845-
related radioactivity into the bone and vertebrae of mice
and rhesus monkey;22,25 however, the interpretation of the
experimental data remains to be carefully verified.
Neuroimaging of σ1 receptors by PET is currently per-
formed by the application of [11C]SA4503 and thus restricted
to the on-site availability of a cyclotron because the imple-
mentation of experimentally favorable 18F-labeled radiotra-
cers failed so far. Therefore, the aims of our study were to
design and biologically evaluate F-substituted spirocyclic σ1
receptor ligands and to perform 18F-labeling and in vivo
investigation of the pharmacodynamic and pharmacokinetic
properties of 7a, the most suitable compound.
A structure-affinity relationship study has shown that the
spiro[[2]benzofuran-1,40-piperidine] 7a with a 3-fluoropropyl
residue in position 3 and a benzyl residue at the piperidine
N-atom possesses high σ1 affinity and σ1/σ2 selectivity. The in
vitro metabolic stability of 7a with rat liver microsomes was
sufficient. The radiotracer [18F]7a was obtained with high
specific activity and high radiochemical purity in an opti-
mized, rapid, and reliable one-step radiosynthesis by nucleo-
philic substitution of the tosylate 13 with [18F]F-. Despite
differences between the experimentally determined log D
values of [18F]7a and the estimated and calculated values
(3.03 vs 3.32-3.81, respectively), a good blood-brain pene-
tration of [18F]7a was expected because the log D value
obtained via partitioning studies was 3.02 ( 0.19 and lies
withintherangethatis generally consideredasfavorablevalue
for CNS targeting drugs.42
A high uptake and a long time retention of radioactivity in
the brain (∼4% ID/g at 5 min pi and >50% of the initial
uptake at 120 min pi, respectively) indicate favorable brain
penetration as well as specific binding of [18F]7a. In particular,
because of a high σ1 receptor density in cerebellum in mice,8
theconstant increase ofthe cerebellumtoplasmaratios during
the washout phase of the radiotracer (up to 5.8 at 60 min pi) is
considered to reflect target specific binding rather than blood
flow dependent distribution. Furthermore, it indicates that
[18F]7a reaches the pseudoequilibrium of receptor binding
after about 60 min, which is in an acceptable range for a
suitablePET tracer. Thekineticsofthe uptake of radioactivity
in the renal system with a high initial uptake in the kidney
decreasing over time and an increasing amount of radio-
activityin the bladder andurine pointstoa fast renalexcretion
of parent and metabolized radiotracer. Examination of organ
A blocking study was performed to examine the in vivo
target specificity of [18F]7a by using the neuroleptic haloperidol

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6069
(1 mg/kg, ip, 10 min prior to [18F]7a administration). Signifi-
cant reduction in the uptake of radioactivity (P value < 0.005)
was observed in the σ1 receptor expressing organs brain, spleen,
pancreas, heart, and lung. This result is consistent with data
obtained by other groups on the evaluation of potential σ1
receptor radiotracers.23,43,48 Regarding the herein evaluated
spiropiperidine [18F]7a in particular, the high amount of
haloperidol-sensitive binding in the brain (∼80% of total
binding) indicates highly specific σ1 receptor-mediated accu-
mulation of the radiotracer. By contrast, the uptake of [18F]7a
by the liver of CD-1 mice was not affected by haloperidol
pretreatment. This haloperidol-insensitive binding might be
related to a species dependency of σ1 receptor expression49 or
reflect the difficulties to observe specific radiotracer binding in
metabolic active tissue.23
9.72 mmol) and BF3 Et2O (0.25 mL, 1.94 mmol) were added.
3
After stirring for 20 min at -25 °C and for 4 h at ice-cooling,
the reaction mixture was allowed to warm to rt. Then methanol
(2 mL) was added, and the solution was made alkaline with 2 M
NaOH. The organic layer was separated and washed with brine.
The aqueous layer was extracted with CH2Cl2 (4ꢀ). The com-
bined organic layers were dried (Na2SO4), filtered, and the
solvent was removed in vacuo. The residue was purified by fc
(4 cm, cyclohexane/ethyl acetate 7:3, 20 mL, Rf 0.25). Pale-
1
yellow oil, yield 0.441 g (82%). C22H25NO (319.4). H NMR
(CDCl3): δ (ppm) = 1.71 (ddd, J = 13.7/6.1/2.8 Hz, 1H, N-
(CH2CH2)2), 1.92 (td, J = 13.1/4.5 Hz, 1H, N(CH2CH2)2), 2.07
(td, J = 13.1/4.5 Hz, 1H, N(CH2CH2)2), 2.46-2.51 (m, 2H,
N(CH2CH2)2), 2.52-2.61 (m, 2H, CH2-CHdCH2), 2.82 (d, J=
11.1 Hz, 2H, N(CH2CH2)2), 3.59 (s, 2H, NCH2Ph), 5.06-5.15
(m, 2H, CH2-CHdCH2), 5.26 (t, J=5.8 Hz, 1H, Ar-CH-O),
5.81-5.92 (m, 1H, CH2-CHdCH2), 7.12-7.17 (m, 2H, aromat
H), 7.23-7.39 (m, 7H, aromat. H).
For mapping the radioactivity distribution of [18F]7a in the
brain, we performed ex vivo autoradiography at 45 min pi.
Because in the brain no radiometabolites were detected and
the haloperidol-insensitive nonspecific binding accounted for
only ∼20% of total radioactivity accumulation, the radio-
activity distribution is assumed to reflect mainly specific
[18F]7a target sites. The [18F]7a distribution pattern with
strong labeling in the cerebellum, the periaqueductal gray,
the hypothalamic area, and the area of the facial nucleus
corresponds to the σ1 receptor expression pattern derived
from immunohistochemical and in situ hybridization studies
as well as from target-specific radioligand binding in the adult
mouse and rat brain.7,8,50-52
3-(10-Benzyl-3H-spiro[[2]benzofuran-1,40-piperidine-3-yl]propan-
1-ol (9). Under N2, the unsaturated compound 8 (1.095 g, 3.43
mmol) was dissolved in THF (14 mL). 9-Borabicyclo[3.3.1]nonane
(9-BBN, 0.5 M in THF, 14 mL, 7.0 mmol) was added slowly. The
mixture was stirred at rt overnight, and then a further amount of
9-BBN (0.5 M in THF, 5 mL, 2.5 mmol) was added. Then 1.5 h
later, the mixture was cooled to -25 °C and methanol (1 mL) was
added. After 15 min, 2 M NaOH (5.5 mL, 11.0 mmol) and after a
further 15 min H2O2 (30%, 4.2 mL, ∼40 mmol) were added under
vigorous stirring. After 45 min, it was warmed to rt for 1 h and
finally warmed to 40 °C. The mixture was extracted with CH2Cl2
(4ꢀ), the combined organic layers were dried (Na2SO4), filtered,
and the solvent was removed in vacuo. The residue was purified by
fc (5 cm, cyclohexane:ethyl acetate 5:5, 50 mL, Rf 0.15). Colorless
oil, yield 1.073 g (92%). C22H27NO2 (337.5). 1H NMR (CDCl3): δ
(ppm)=1.65-1.93 (m, 6H, N(CH2CH2)2 (3H), CH2CH2CH2OH
(2H), CH2CH2CH2OH (1H)), 2.09-2.19 (m, 2H, N(CH2CH2)2
(1H), CH2CH2CH2OH (1H)), 2.38 (td, J = 11.5/2.5 Hz, 1H,
N(CH2CH2)2), 2.43 (td, J=11.5/2.5 Hz, 1H, N(CH2CH2)2), 2.85
(t, broad, J=11.4 Hz, 2H, N(CH2CH2)2), 3.58 (s, 2H, NCH2Ph),
3.66-3.79 (m, 2H, CH2CH2CH2OH), 5.22 (dd, J=8.2/2.7 Hz, 1H,
Ar-CH-O), 7.11-7.15 (m, 2H, aromat. H), 7.24-7.38 (m, 7H,
aromat. H). A signal for the proton of the OH-group is not visible.
10-Benzyl-3-(3-fluoropropyl)-3H-spiro[[2]benzofuran-1,40-piperi-
dine] (7a). Under N2, diethylaminosulfur trifluoride (DAST, 0.73
mL, 5.96 mmol) was slowly added to CH2Cl2 (25 mL) at -78 °C.
The alcohol 9 (0.911 g, 2.70 mmol) dissolved in CH2Cl2 (3 mL) was
slowly added. After 30 min, the mixture was warmed to rt and
stirred for further 17 h. Under ice-cooling and vigorous stirring,
the reaction was stopped by addition of 2 M NaOH and a small
amount of CaCl2. The mixture was transferred into a separating
funnel and extracted with CH2Cl2 (4ꢀ). The combined organic
layers were dried (Na2SO4), filtered, and the solvent was removed
in vacuo. The residue was purified by fc (5 cm, cyclohexane:ethyl
acetate 8:2, 50 mL, Rf 0.25). Colorless oil, yield 0.486 g (53%).
C22H26FNO (339.5). 1H NMR (CDCl3): δ (ppm)=1.63-1.94 (m,
6H, N(CH2CH2)2 (3H), CH2CH2CH2F (2H), CH2CH2CH2F
(1H)), 2.00-2.13 (m, 2H, N(CH2CH2)2 (1H), CH2CH2CH2F
(1H)), 2.40-2.49 (m, 2H, N(CH2CH2)2), 2.79-2.85 (m, 2H,
N(CH2CH2)2), 3.58 (s, 2H, NCH2Ph), 4.49 (ddt, J=47.2/9.0/6.0
Hz, 1H, CH2CH2CH2F), 4.53 (ddt, J = 47.3/8.9/6.1 Hz, 1H,
CH2CH2CH2F), 5.24 (dd, J = 7.0/3.8 Hz, 1H, Ar-CH-O),
7.08-7.17 (m, 2H, aromat H), 7.24-7.38 (m, 7H, aromat H).
Anal. (C22H26FNO) C, H, N.
Conclusion
In the reported study, a series of spirocyclic piperidines was
synthesized and evaluated. It was shown that the σ1 receptor
protein tolerates a linear propyl chain in position 3 of the
2-benzofuran system. With respect to its high σ1 receptor
affinity, high σ1/σ2 selectivity, and sufficient in vitro metabolic
stability. The 10-benzyl-3-(3-fluoropropyl)-3H-spiro[[2]ben-
zofuran-1,40-piperidine] (7a) represents the most promising
compound. Therefore, the 3-fluoropropyl derivative 7a was
selected as candidate for a 18F-labeled radiotracer applicable
for noninvasive imaging of central σ1 receptor protein by PET
independent from an on-site cyclotron. [18F]7a was success-
fully synthesized. and initial pharmacodynamic and pharma-
cokinetic parameters were estimated in female CD-1 mice.
Target specificity of the radiotracer uptake was supported by
a blocking study as well as by comparing the radiotracer
distribution in ex vivo brain autoradiography with σ1 receptor
expression pattern derived from other methods. Altogether,
our results on [18F]7a synthesis and first in vivo evaluation
provide evidence for the suitability of 3-fluoropropyl substi-
tuted spiropyridines for neuroimaging of central σ1 receptors.
Experimental Section
General Synthesis. Unless otherwise noted, moisture sensitive
reactions were conducted under dry nitrogen. Flash chroma-
tography (fc): Silica Gel 60, 40-64 μm (Merck); parentheses
include: diameter of the column, eluent, fraction size, Rf value.
1H NMR (400 MHz), 13C NMR (100 MHz): Unity Mercury
Plus 400 spectrometer (Varian); δ in ppm related to tetramethyl-
silane; coupling constants are given with 0.5 Hz resolution.
According to HPLC analysis, the purity of all test compounds
exceeds 95%.
3-(3-Fluoropropyl)-3H-spiro[[2]benzofuran-1,40-piperidine] (7b).
The N-benzyl derivative 7a (0.450 g, 1.33 mmol) was dissolved in
CH3OH (25 mL) and Pd/C (10% m/m, 0.089 g) and dried
ammonium formate (0.409 g, 6.49 mmol) were added. The result-
ing suspension was heated to reflux for 4.5 h. After 2 and 3 h,
additional amounts of ammonium formate (0.4 g each) were
added. After the mixture had been cooled down, it was filtrated
over celite. The solvent was removed in vacuo, and the residue was
3-Allyl-10-benzyl-3H-spiro[[2]benzofuran-1,40-piperidine] (8).
Under N2, a solution of acetal 5 (0.520 g, 1.62 mmol) in CH2Cl2
(18 mL) was cooled to -25 °C and allyltrimethylsilane (1.54 mL,

6070 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Große Maestrup et al.
purified by fc (3 cm, ethyl acetate:methanol:ammonia 90:10:2,
20 mL, Rf 0.03). Colorless oil, yield 0.270 g (82%). C15H20FNO
(249.3). 1H NMR (CDCl3): δ (ppm) = 1.65 - 2.09 (m, 8H,
N(CH2CH2)2 (4H), CH2CH2CH2F (4H)), 3.00-3.13 (m, 4H,
N(CH2CH2)2), 4.48(ddt, J=47.3/9.0/6.0 Hz, 1H, CH2CH2CH2F),
4.55 (ddt, J=47.3/8.9/6.1 Hz, 1H, CH2CH2CH2F), 5.25 (dd, J =
7.2/3.8 Hz, 1H, Ar-CH-O), 7.12-7.16 (m, 2H, aromat H),
7.25-7.29 (m, 2H, aromat H). A signal for the NH-proton is not
visible.
pH 7.2), Dulbecco’s phosphate buffered saline (pH 7.2), or
TRIS-HCl (30 mM, pH 7.2) by conventional shake-flask meth-
od (multiple distribution in duplicates) and calculated from RP-
HPLC retention times54 on a Multisorb C18 (250 mm ꢀ 4 mm,
particle size 7 μm; CS-Chromatographie Service, Germany) and
a Supelcosil ABZþPlus (250 mm ꢀ 4 mm, 5 μm particle size;
Sigma-Aldrich, Germany) column using reference compounds
and a protocol according to the EU guideline 67/548/EWG.
Animal Studies. All animal experiments were approved by the
respective State Animal Care and Use Committee and con-
ducted in accordance with the German Law for the Protection of
Animals.
[3-(10-Benzyl-3H-spiro[[2]benzofuran-1,40-piperidin]-3-yl)propyl]
p-toluenesulfonate (13). Under N2, the alcohol 9 (150 mg, 0.45
mmol), 4-dimethylaminopyridine (DMAP, 16 mg, 0.13 mmol),
and triethylamine (0.3 mL, 2.23 mmol) were dissolved in CH2Cl2 .
The mixture was cooled to -25 °C, and p-toluenesulfonyl chloride
(170 mg, 0.89 mmol), dissolved in CH2Cl2 (3 mL), was added.
After 15 min, the mixture was warmed to rt and stirred for further
21.5 h. After addition of diluted NaOH, the aqueous layer was
separated and extracted with CH2Cl2 (4ꢀ). The combined organic
layers were washed with brine (2ꢀ), dried (Na2SO4), and concen-
trated in vacuo. The residue was purified by fc (2 cm, cyclohexane:
ethyl acetate 7:3, 10 mL, Rf 0.09). Colorless oil, yield 165 mg
(76%). C29H33NO4S (491.6). 1H NMR (CDCl3): δ (ppm) =
1.58-1.98 (m, 7H, N(CH2CH2)2 (3H), CH2CH2CH2Tos
(2H), CH2CH2CH2Tos (2H)), 2.05 (td, J = 12.1/4.5 Hz, 1H, 1
N(CH2CH2)2), 2.38 (t, J=12.1 Hz, 2H, N(CH2CH2)2), 2.43 (s, 3H,
ArCH3), 2.79 (d, broad, J=11.1 Hz, 2H, N(CH2CH2)2), 3.56 (s,
2H, NCH2Ph), 4.07 (dt, J=9.7/6.4 Hz, 1H, CH2CH2CH2Tos),
4.13 (dt, J = 9.7/6.5 Hz, 1H, CH2CH2CH2Tos), 5.14 (dd, J=7.1/
3.7 Hz, 1H, Ar-CH-O), 7.04-7.12 (m, 2H, aromat H), 7.24-7.38
(m, 9H, aromat H), 7.78 (td, J=8.3/1.9 Hz, 2H, aromat. H).
Receptor Binding Studies. The receptor binding studies were
performed as previously reported.35,53
All chemical reagents and solvents were purchased from
commercial suppliers and used without further purification.
Details on the investigations about radiotracer metabolism
are given in the Supporting Information.
In vivo biodistribution studies were carried out in female
CD-1 mice (∼8-12 weeks old, 20-28 g; Medizinisch-Experi-
€
mentelles Zentrum, Universitat Leipzig). The animals were
habituated for at least 48 h and deprived of food for 24 h prior
to the experiment with free access to tap water.
For the organ distribution studies, 400-600 kBq [18F]7a
(specific activity >150 GBq/μmol at the end of the synthesis)
in 200 μL of saline was injected via the tail vein into the
conscious mice. At 5, 30, 60, and 120 min post injection (pi) of
the radiotracer, the animals (n=4-5 per time) were anesthe-
tized, blood and urine samples were taken, the animals were
euthanized by luxation of the cervical spine, and samples of
organs of interest were removed. All samples were weighed, and
radioactivity was measured in a calibrated γ-counter. After
counting, the percentage of the injected dose per gram of wet
tissue (% ID/g) was calculated.
In Vitro Metabolism. Details on the metabolism investiga-
tions are given in the Supporting Information.
Blocking studies were conducted by intraperitoneal preinjec-
tion of 1 mg/kg haloperidol dissolved in saline at 10 min before
the radiotracer (n=5). All animals were sacrificed at 60 min after
injection of the radiotracer, and the radioactivity uptake was
determined as described above.
For ex vivo autoradiography, sagittal brain sections were
obtained at 45 min after iv injection of 68 MBq [18F]7a.
The brain was removed, hemispheres were frozen in isopentane
(-35 °C), 12 μm sagittal sections were cut on a cryostat micro-
tome (Microm, Germany), mounted onto microscope slides,
dried in a stream of cold air for 10 min, and exposed to
18F-sensitive storage phosphor screens (Fuji Film, Japan). The
image plates were analyzed using a BAS-1800II system bioima-
ging analyzer (Fuji Film, Japan), and scan data were visualized
and processed by computer assisted microdensitometry (Aida
2.31, Raytest, Germany). Brain structures were confirmed by
Nissl staining of the exposed sections.
At 45 and 60 min after tail vein injection of 200-300 MBq
[18F]7a (dissolved in 200 μL 0.9% saline), blood and urine
samples, brain, and liver of the mice (n = 2 per time) were
isolated. Plasma samples were obtained by centrifugation of
blood (2000g, 4 °C, 10 min), and brain and liver samples by
homogenization in ice-cold 50 mM TRIS-HCl, pH 7.4. Ice-cold
MeCN was added to brain, plasma, urine, and liver samples,
protein precipitates were sedimented by centrifugation (4000g,
4 °C, 15 min), and the supernatants were analyzed by gradient
radio-HPLC and radio-TLC as described above. The percentages
of parent and metabolized radiotracer were determined by mea-
suring the radioactivity of the respective HPLC fractions using a
calibrated γ-counter (Wallac WIZARD, Perkin-Elmer, USA).
Radiosynthesis of [18F]7a. Proton irradiated [18O]H2O
(PETtrace 16.5 MeV cyclotron, GE) containing [18F]fluoride
was transferred onto an anion exchange cartridge (SPE car-
tridge Chromafix 30-PS-HCO3, Macherey-Nagel, Germany),
and [18F]fluoride was isolated by elution with a solution
of aqueous K2CO3 (3.6 mg, 150 μL) and Kryptofix222 (11 mg,
200 μL MeCN). No-carrier-added aliphatic substitution was
performed by reacting the dried K[18F]F-K222 complex with 13
dissolved in 1 mL of anhydrous MeCN at 85 °C for 30 min with
labeling efficiencies of 60-70% (n = 11). [18F]7a was isolated on
a semipreparative radio-HPLC system consisting of a S1021
pump (SYKAM Chromatographie, Germany), UV detector
(Well-ChromK-2001; KNAUER, Germany), NaI(TI)-counter,
and data acquisition by an automated system (NINA, Nuclear
Interface, Germany) on a Multisorb 120 RP-18 AQ column
(150 mm ꢀ 10 mm, particle size 5 μm; CS Chromatographie
Service, Germany) with a mobile phase of 50% MeCN in 20 mM
NH4OAc. At a flow rate of 4 mL/min, [18F]7a eluted at ∼23 min.
The separated fractions were combined, diluted with water,
passed through a Sep-Pak C18 cartridge, and purified [18F]7a
was eluted with 2 mL MeOH. For in vivo experiments, the
solvent was thoroughly removed under reduced pressure and
[18F]7a redissolved in sterile saline. As analyzed by HPLC and
TLC, the final product was obtained with a RCY of 35-48%, a
radiochemical purity of >99.5%, and a specific activity of
150-238 GBq/μmol within a synthesis time of 90-120 min
(n=7).
Stability of [18F]7a. The chemical stability of [18F]7a was
investigated in 0.9% NaCl, Sorensen’s phosphate buffer
e
Acknowledgment. This work was supported by a grant of
the Deutsche Forschungsgemeinschaft, which is gratefully
acknowledged. Parts of this work were performed within the
International Research Training Group “Complex Functional
Systems in Chemistry: Design, Synthesis and Applications” in
collaboration with the University of Nagoya. We also thank the
Deutsche Forschungsgemeinschaft for funding of this IRTG.
(50 mM, pH 7.2), Dulbecco’s phosphate buffered saline (pH
7.2), and TRIS-HCl (30 mM, pH 7.2) at 40 °C for up to 120 min.
At various times, aliquots were analyzed by radio-TLC and
analytical radio-HPLC.
Determination of log D Value. The log D value of [18F]7a was
determined by measuring the distribution of the radiotracer
between n-octanol and Sorensen’s phosphate buffer (50 mM,
e

Article
Supporting Information Available: Purity data of the prepared
compounds, experimental conditions of syntheses, receptor
binding studies, and investigation of metabolism. This material
is available free of charge via the Internet at http://pubs.acs.org.
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6071
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