Enantioselective σ1 receptor binding and biotransformation of the spirocyclic PET tracer 1′-benzyl-3-(3-fluoropropyl)-3H-spiro[[2] benzofuran-1,4′-piperidine]

Article abstract of DOI:10.1002/chir.20893

It was shown that racemic (±)-2 [1′-benzyl-3-(3-fluoropropyl)- 3H-spiro[[2]benzofuran-1,4′-piperidine], WMS-1813] represents a promising positron emission tomography (PET) tracer for the investigation of centrally located σ₁ receptors. To study the pharmacological activity of the enantiomers of 2, a preparative HPLC separation of (R)-2 and (S)-2 was performed. The absolute configuration of the enantiomers was determined by CD-spectroscopy together with theoretical calculations of the CD-spectrum of a model compound. In receptor binding studies with the radioligand [ ³H]-(+)-pentazocine, (S)-2 was thrice more potent than its (R)-configured enantiomer (R)-2. The metabolic degradation of the more potent (S)-enantiomer was considerably slower than the metabolism of (R)-2. The structures of the main metabolites of both enantiomers were elucidated by determination of the exact mass using an Orbitrap-LC-MS system. These experiments showed a stereoselective biotransformation of the enantiomers of 2. Copyright

Full text of DOI:10.1002/chir.20893

CHIRALITY 23:148–154 (2011)
Enantioselective r1 Receptor Binding and Biotransformation of the
Spirocyclic PET Tracer 10-Benzyl-3-(3-fluoropropyl)-3H-
spiro[[2]benzofuran-1,40-piperidine]
CHRISTIAN WIESE,1 EVA GROßE MAESTRUP,1 DIRK SCHEPMANN,1 STEFAN GRIMME,2 HANS-ULRICH HUMPF,3
1
PETER BRUST,4 AND BERNHARD WUNSCH
¨
*
1Institut fu¨r Pharmazeutische und Medizinische Chemie der Universita¨t Mu¨nster, Hittorfstraße 58-62, Mu¨nster, Germany
2Organisch-Chemisches Institut der Universita¨t Mu¨nster, Corrensstraße 40, Mu¨nster, Germany
3Institut fu¨r Lebensmittelchemie der Universita¨t Mu¨nster, Corrensstraße 45, Mu¨nster, Germany
4Institut fu¨r Interdisziplina¨re Isotopenforschung, Permoserstraße 15, Leipzig, Germany
ABSTRACT
It was shown that racemic (6)-2 [10-benzyl-3-(3-fluoropropyl)-3H-spiro[[2]ben-
zofuran-1,40-piperidine], WMS-1813] represents a promising positron emission tomography
(PET) tracer for the investigation of centrally located r1 receptors. To study the pharmacologi-
cal activity of the enantiomers of 2, a preparative HPLC separation of (R)-2 and (S)-2 was per-
formed. The absolute configuration of the enantiomers was determined by CD-spectroscopy to-
gether with theoretical calculations of the CD-spectrum of a model compound. In receptor bind-
ing studies with the radioligand [3H]-(1)-pentazocine, (S)-2 was thrice more potent than its (R)-
configured enantiomer (R)-2. The metabolic degradation of the more potent (S)-enantiomer
was considerably slower than the metabolism of (R)-2. The structures of the main metabolites
of both enantiomers were elucidated by determination of the exact mass using an Orbitrap-LC-
MS system. These experiments showed a stereoselective biotransformation of the enantiomers
C
VV
of 2. Chirality 23:148–154, 2011.
2010 Wiley-Liss, Inc.
KEY WORDS: r1 receptor ligands; PET tracer; resolution; CD spectroscopy; enantioselective
receptor binding; enantioselective metabolism
INTRODUCTION
nous ligands.12 In 2009, the hallucinogenic N,N-dimethyl-
tryptamine (Ki 5 15 lM) was described as endogenous r1
After the original classification of r receptors as opioid re-
ceptor subtype has been discarded, the class of r receptors
is now recognized as an independent receptor family with an
own binding profile and characteristic distribution in the cen-
tral nervous system (CNS) as well as in peripheral tissues,
like kidney, liver lung, and heart.1
The r receptor family contains two subtypes, which are
termed r1 and r2 receptor. The r2 subtype has not been
cloned so far and its molecular weight was only estimated by
photoaffinity labeling. In contrast to the r2 subtype, the r1
receptor is much better characterized. It was cloned from
various tissues and species, like guinea pig liver, rat brain,
and human placental choriocarcinoma cells.2–4 The sequence
homology between r1 receptors of different species is quite
high (e.g., 93% between human and guinea pig receptors)
and the similarity is even higher (>96%).4 Surprisingly, a
considerable similarity of the r1 receptor protein with
another mammalian protein was not found.
r1 receptors are involved in the modulation of various neu-
rotransmitter systems, in particular glutamatergic,5 dopami-
nergic,6 and cholinergic7 neurons are affected. Moreover, it
was shown that r1 receptor ligands inhibit voltage-dependent
K1-ion channels8 and the influx of Ca21 ions into neurons of
rats.9 However, the r1 receptor itself is neither a ligand-gated
ion channel nor a G-protein coupled receptor.10
Endogenous ligands for the r1 receptor are nont unequivo-
cally identified. After two steroid binding site like domains
(SBDL-I and SBDL-II) on the r1-receptor protein have been
discovered,11 neurosteroids like progesterone and dehydroe-
piandrosterone (DHEA) were postulated to be the endoge-
receptor ligand.13
Noninvasive in vivo targeting of cerebral r1 receptors by
neuroimaging approaches such as positron emission tomog-
raphy (PET) is considered to be of value for (1) the develop-
ment of novel drugs by target validation (e.g., correlation of
r1 receptor occupancy with efficacy of a new potential
drug)14,15 and (2) basic and clinically applied research by
quantifying and comparison of r1 receptor expression in the
healthy and diseased brain. Additionally, PET studies with
selective r1 ligands may also provide (3) important new in-
formation about the role of r1 receptors in the complex neu-
rotransmitter balance in the CNS, which can lead to new
strategies for the treatment of several CNS diseases.
During the last 10 yr, we have developed a novel class of
spirocyclic r1 receptor ligands showing nanomolar affinity at
the r1 receptor subtype and excellent selectivity against the
r2 subtype.16–19 One of the most promising ligands of this
type is the spiro[2-benzofuran-1,40-piperidine] 1 (Fig. 1) with
very high r1 receptor affinity (Ki 5 1.1 nM) and excellent se-
lectivity against the r2 subtype (>1000-fold) and more than
60 other receptors, ion channels (including the hERG chan-
nel) and transporters.20
Contract grant sponsor: IRTG, Deutsche Forschungsgemeinschaft
*Correspondence to: Bernhard Wu¨nsch, Institut fu¨r Pharmazeutische und
Medizinische Chemie der Universita¨t Mu¨nster, Hittorfstraße 58-62, D-48149
Mu¨nster, Germany. E-mail: wuensch@uni-muenster.de
Received for publication 14 January 2010; Accepted 18 May 2010
DOI: 10.1002/chir.20893
Published online 15 September 2010 in Wiley Online Library
(wileyonlinelibrary.com).
C
VV
2010 Wiley-Liss, Inc.

ENANTIOSELECTIVE SIGMA-1 RECEPTOR BINDING AND BIOTRANSFORMATION
149
Fig. 1. Structure of the spirocyclic lead compound 1 and the developed
fluoropropyl derivative 2 (WMS-1813).
Therefore, the spirocyclic piperidine 1 was used as lead
compound for the development of the PET-tracer 2 (WMS-
1813) with a 3-fluoropropyl side chain in position 3 (Fig. 1).
Very recently, the synthesis, r1 receptor affinity and selectiv-
ity, in vitro and in vivo metabolism of the 3-fluoropropyl sub-
stituted spirocyclic compound 2 (WMS-1813) and the
radiosynthesis of [18F]2 including the first evaluation of its
pharmacokinetics in CD-1 mice was reported.21
So far all spirocyclic r1 receptor ligands were developed
and pharmacologically evaluated as racemic mixtures.
Because of the excellent pharmacological properties of the 3-
fluoropropyl derivative 2 and the suitability of [18F]2 as a
PET tracer, we were interested in the properties of the single
enantiomers of 2. In contrast to the lead compound 1 with
an acetalic chiral centre, the fluoropropyl derivative 2 is con-
figurationally stable.
Fig. 2. Chromatogram of the separation of (R)-2 and (S)-2 on an analyti-
cal column. Chiralpak1 IB (5 lm, 250 mm 3 4.6 mm); isohexane/isopropa-
nol 97.5:2.5; 1.0 ml/min; k 5 210 nm. (R)-2: tR 5 5.4 min; (S)-2: tR 5 6.1
min.
(isohexane/isopropanol 5 97.5:2.5). A flow rate of 9.0 ml/
min resulted in the HPLC chromatogram shown in Fig. 3.
Under these conditions, a complete baseline separation was
not achieved due to the tailing of the peaks, which was
caused by overloading of the column. Therefore, a careful
selection of the respective fractions was required during the
collection of the pure enantiomers.
The enantiomeric purity of the separated enantiomers was
determined with the described analytical HPLC method.
Because the enantiomeric purity was not sufficient after the
first separation process, a second semipreparative HPLC sep-
aration was performed with the enriched samples. After the
second separation, the first fraction contained (R)-2 and (S)-
2 in the ratio 95.5:4.5, and the second fraction was composed
of (R)-2 and (S)-2 in the ratio 4:96. These samples were
used for further experiments.
Herein, we report on the resolution of the racemic mixture
2, elucidation of the absolute configuration of the enantiom-
ers (R)-2 and (S)-2, as well as the pharmacological proper-
ties and the metabolism of the single enantiomers.
Separation of the Enantiomers of 2
At first, an analytical method for the separation of the
enantiomeric spiro derivatives (R)-2 and (S)-2 was devel-
oped. The separation was carried out on a Chiralpak1 IB col-
umn, because a corresponding semipreparative column for
the subsequent preparative separation was available in the
group. In this chiral stationary phase, the chiral selector, a
cellulose derivative bearing three (3,5-dimethylphenylcarba-
moyl)-residues at the free OH-moieties, is covalently bound
to silica gel.
For the separation of (R)-2 and (S)-2, isohexane (C6H14
isomers with maximal 3% of n-hexane) with a small amount
of isopropanol as a polar modifier was used as eluent. Addi-
tion of acid or base was avoided with respect to the prepara-
tive separation.
The best mixture turned out to be isohexane/isopropanol
97.5:2.5. At a flow rate of 1.0 ml/min, both enantiomers were
baseline separated and eluted within 8 min. The compounds
were detected by an UV-detector at a wavelength of 210 nm
(Fig. 2).
The successful analytical separation of the enantiomers
(R)-2 and (S)-2 was transferred onto a semipreparative Chir-
alpak1 IB column (Ø20 mm) using the same mobile phase
Fig. 3. Chromatogram of the separation of (R)-2 and (S)-2 on a semipre-
parative column. Chiralpak1 IB (5 lm, 250 3 20 mm) isohexane/isopropanol
97.5:2.5; 9.0 ml/min; k 5 210 nm. (R)-2: tR 5 12.4 min; (S)-2: tR 5 15.4 min.
Chirality DOI 10.1002/chir

150
WIESE ET AL.
radioligand. The nonspecific binding was determined in the
presence of an excess of nontritiated (1)-pentazocine.19
In this assay, a Ki-value of 0.59 6 0.06 nM (SEM) was
determined for (S)-2. The corresponding (R)-enantiomer
(R)-2 was considerably less active (Ki 5 1.8 6 0.13 nM
standard error of the mean (SEM)). With respect to r1 re-
ceptor affinity, (S)-2 represents the eutomer with an eudis-
mic ratio of 3. The racemic mixture (6)-2 showed a r1 re-
ceptor affinity of 1.4 6 0.26 nM (SEM).21
Metabolic Stability of the Enantiomers (R)-2 and (S)-2
To monitor the metabolic stability, several incubations of
both enantiomers were started at the same time. They were
stopped after 5, 10, 30, 40, 60, and 90 min by addition of cold
acetonitrile. Directly after termination of the biotransforma-
tion, praziquantel was added as an internal standard. The
remaining parent compound was analyzed using a matrix cal-
ibration. The samples were analyzed by HPLC using UV
detection at 210 nm. All experiments were carried out in
duplicate.
Figure 6 shows the metabolic degradation of the enantio-
meric spirocyclic r1 ligands (R)-2 and (S)-2. The (R)-enan-
tiomer was metabolized faster than the (S)-enantiomer. After
an incubation period of 30 min, only 28% of (R)-2 was left,
whereas 54% of (S)-2 remained unchanged. Apparently, the
cytochrome P450 enzymes in the microsomal matrix showed
high enantioselectivity in the biotransformation of the enan-
tiomers (R)-2 and (S)-2.
To identify the different metabolic pathways of the enan-
tiomers (R)-2 and (S)-2, in separate experiments, each enan-
tiomer was incubated with rat liver microsomes for 90 min
and the structures of the respective metabolites (Fig. 7) were
determined by liquid chromatography-mass spectrometry
(LC-MS) techniques in electrospray ionisation (ESI)1 mode
using MSn fragmentations.
Generally, oxidation by P450 enzymes in the microsomal
preparation took place at the a-position of the N-atom
(debenzylation), at the phenyl moiety of the benzyl group
and at the 3-fluoropropyl side chain of 2. Oxidation products
of the 2-benzofuran system were not detected. The same
metabolic pathways (N-debenzylation, benzyl hydroxylation,
Fig. 4. Superposed CD-spectra of the enantiomers (R)-2 and (S)-2
recorded in methanol.
Elucidation of the Absolute Configuration of the
Enantiomers
To determine the absolute configuration circular dichro-
ism (CD) spectra of the separated enantiomers (R)-2 and
(S)-2 were recorded. For this purpose, methanolic solutions
of the isolated enantiomers were measured.
In Figure 4, the resulting CD-spectra are shown. For the
observation of a circulardichroism, the presence of a chromo-
phore is essential. Because chromophoric systems are planar
and achiral structures, the chiral information should be
located close to the chromophoric system. This situation
leads to dissymmetric perturbance on orbital level and finally
to the different absorption of left and right circularly polar-
ized light.22
There are empiric, semiempiric, and theoretical
approaches for the interpretation of CD-spectra. In case of
the enantiomers (R)-2 and (S)-2, an empiric interpretation of
the CD-spectra did not lead to an unequivocal assignment of
the configuration, because only one chromophore is located
in the neighborhood of the chiral centre. Therefore, the CD-
spectrum of the model compound (R)-3 was calculated by
time-dependent density functional theory (DFT)23,24 using
the TURBOMOLE25 suite of programs (for details see below
and Ref. 26). The neglected side chains are expected to have
no impact on the calculated CD spectrum but would compli-
cate matters considerably due to the presence of a huge
number of different conformers.
The calculated CD-spectrum for the (R)-configured 2-ben-
zofuran (R)-3 is shown in Figure 5. The most important La
(ꢀ220 nm) and the Lb bands (ꢀ260 nm) of the benzene unit
are clearly separated and have a negative sign. These fea-
tures are in good agreement with the lower experimental CD-
curve in Figure 4. Therefore, (R)-configuration can definitely
be assigned to this enantiomer. The former (splitted) E1u
states of the benzene chromophore gives rise to two strong
bands below 210 nm with opposite sign, which are not fully
resolved in the experiment due to instrumental limitations.
r1 Receptor Binding Studies
The r1 receptor affinities were determined in competition
experiments with radioligands. Guinea pig brain preparations
were used as receptor material and [3H]-(1)-pentazocine as
Fig. 5. Calculated CD-spectrum of the model compound (R)-3.
Chirality DOI 10.1002/chir

ENANTIOSELECTIVE SIGMA-1 RECEPTOR BINDING AND BIOTRANSFORMATION
151
Fig. 8. HPLC MS chromatograms of samples resulting upon incubation of
the enantiomers (R)-2) and (S)-2 with rat liver microsomes for 90 min.
LiChrospher1 RP Select B column (5 lm, 250 mm 3 4.0 mm), gradient sys-
tem, 1.0 ml/min.
Fig. 6. Metabolic degradation of (R)-2 and (S)-2 over a period of 90 min;
each data point was determined as duplicate.
experiments did not allow the exact localization of the OH-
group in the side chain. However, the influence of the abso-
lute configuration in position 3 on the amount of formed
metabolites also indicates hydroxylation close to the chiral
center (Fig. 8).
side chain hydroxylation) were observed for the racemic
mixture 2.21 However, here additional minor metabolites
were analyzed in more detail.
The N-debenzylated metabolite 7 was unequivocally iden-
tified by its exact mass and comparison of its properties with
a reference compound.21
The side chain oxidation products 9 (tR 5 15.1 min and
16.9 min, Fig. 8) were clearly distinguished from the hydrox-
ybenzyl metabolites 10 (tR 5 18.3 min and 18.6 min), which
lost the hydroxybenzyl substituent during fragmentation
(MH–107 (CH2C6H4OH)). There are two peaks for 9 in the
chromatogram. The identical exact masses (m/z 5 356.2016
for C22H27FNO2) and fragmentation patterns of the com-
pounds referring to these peaks indicate two diastereomeric
pairs of enantiomers due to hydroxylation of the 3-fluoro-
propyl side chain near the chiral center. Fragmentation
Oxidation of the side chain of 7 and/or N-debenzylation of
9 led to the very polar metabolite 4.
The compounds of both peaks marked with 10 in the
chromatogram lose the fragment CH2C6H4OH as described
above. Therefore, it is assumed that the peaks were gener-
ated from two regioisomeric hydroxybenzyl derivatives. Fur-
ther oxidation of the hydroxybenzyl metabolite 10 generated
the di- and tri-hydroxylated metabolites 6 and 5. The second
hydroxy group of the metabolite 6 is located in the 3-fluoro-
propyl side chain, because after loss of water (218) a
hydroxybenzyl moiety is cleaved off leading to a fragment
Fig. 7. Structure of the metabolites identified after incubation with rat liver microsomes.
Chirality DOI 10.1002/chir

152
WIESE ET AL.
ters for methanol.29 The CD spectrum has been computed at the double-
hybrid time-dependent (TD)DFT level using the B2PLYP functional.30–32
Five electronic states have been computed, which were red-shifted by
0.25 eV to account for remaining solvent and basis set incompleteness
effects. The spectra were simulated by using the computed excitation
energies and transition moments (dipole length formalism) and overlap-
ping Gaussian band shape functions with a half-width at 1/e height of 0.5
eV.
with the exact mass of 248.1445 (M–H2O–CH2C6H4OH). The
third OH-moiety of the trihydroxy metabolite 5 (tR 5 11.3
min) has to be in the N-benzyl group, because the main frag-
ment of 5 is at m/z 5 266.1546, which is caused by the loss
of a dihydroxybenzyl group (CH2C6H3(OH)2). The trihy-
droxylated metabolite 5 is formed only in traces from both
enantiomers.
The minor metabolite 8 (tR 5 14.4 min) was produced
from both enantiomers in rather low amounts. The exact
mass of m/z 5 352.1905 indicates two additional O-atoms
and the loss of the fluorine atom. The oxidation had taken
place in the side chain and not at the benzyl moiety, because
a benzyl group together with parts of the piperidine ring (M–
CH2C6H5–N(CH2)3) were cleaved off in the first fragmenta-
tion process. The terminal epoxide 8 represents a possible
structure for this metabolite.
After incubation of both enantiomers with rat liver micro-
somes for 90 min, HPLC analyses of the resulting product
mixtures using UV and MS detection methods were per-
formed. The HPLC-MS chromatogram in Figure 8 clearly
demonstrates the different development of metabolites dur-
ing the incubation process. In case of the more stable (S)-
enantiomer, only low amounts of the secondary amine 7 (tR
5 12.6 min) were generated, whereas large amounts of the
side chain hydroxylated product 9 (tR 5 16.9 min) were
observed. On the contrary, the incubation mixture of the less
stable enantiomer (R)-2 contained only minor amounts of
the hydroxylated product 9 but large amounts of the hydrox-
ybenzyl metabolite 10 (tR 5 18.6 min) and the secondary
amine 7 (tR 5 12.6 min). The very polar secondary amine 4
(tR 5 8.1 min) was formed in small amounts starting from
both enantiomers.
Receptor Binding Studies
Materials and general procedures. Guinea pig brains were com-
mercially available (Harlan-Winkelmann, Germany). Homogenizer:
Elvehjem Potter (B. Braun Biotech International). Centrifuge: High-
speed cooling centrifuge model Sorvall RC-5C plus (Thermo Finnigan).
Filter: Printed Filtermat Type A (Perkin Elmer), presoaked in 0.5% aque-
ous polyethylenimine for 2 h at room temperature before use. The filtra-
tion was carried out with a MicroBeta FilterMate-96 Harvester (Perkin
Elmer). The scintillation analysis was performed using Meltilex (Type
A) solid scintillator (Perkin Elmer). The solid scintillator was melted on
the filtermat at a temperature of 958C for 5 min. After solidification of the
scintillator at room temperature, the scintillation was measured using a
MicroBeta Trilux scintillation analyzer (Perkin Elmer). The overall
counting efficiency was 20%.
Performance of the r1 Assay
Five guinea pig brains were homogenized with the potter (500–800
rpm, 10 up-and-down strokes) in six volumes of cold 0.32 M sucrose.
The suspension was centrifuged at 1200g for 10 min at 48C. The superna-
tant was separated and centrifuged at 23,000g for 20 min at 48C. The pel-
let was resuspended in five to six volumes of buffer (50 mM TRIS, pH
7.4) and centrifuged again at 23,000g (20 min, 48C). This procedure was
repeated twice. The final pellet was resuspended in 5–6 volumes of
buffer, the protein concentration was determined according to the
method of Bradford33 using bovine serum albumin as standard, and sub-
sequently, the preparation was frozen (2808C) in 1.5 ml portions con-
taining about 1.5 mg protein/ml.
The test was performed with the radioligand [3H]-(1)-pentazocine
(42.5 Ci/mmol; Perkin Elmer). The thawed membrane preparation
(about 75 lg of the protein) was incubated with various concentrations
of test compounds, 2 nM [3H]-(1)-pentazocine, and buffer (50 mM
TRIS, pH 7.4) in a total volume of 200 ll for 180 min at 378C. The incuba-
tion was terminated by rapid filtration through the presoaked filtermats
by using the cell harvester. After washing each well five times with 300
ll of water, the filtermats were dried at 958C. The bound radioactivity
trapped on the filters was counted in the scintillation analyzer. The non-
specific binding was determined with 10 lM unlabeled (1)-pentazocine.
The Kd-value of the radioligand [3H]-(1)-pentazocine is 2.9 nM. All
experiments were carried out in triplicates using standard 96-well-multi-
plates (Diagonal). The IC50-values were determined in competition
experiments with six concentrations of the test compounds and were cal-
culated with the program GraphPad Prism1 3.0 (GraphPad Software) by
nonlinear regression analysis. The Ki-values were calculated according
to Cheng and Prusoff.34 The Ki-values are given as mean values 6 SEM
from three independent experiments.
EXPERIMENTAL
Analytical HPLC Separation
The HPLC analysis was carried out with a Merck Hitachi equipment
consisting of UV detector: L-7400 and pump: L-6200A. The separation
was carried out with a Chiralpak1 IB column (5 lm, 250 mm 3 4.6
mm). The eluent consisted of isohexane/isopropanol 97.5:2.5 at a flow
rate of 1 ml/min. UV-detection wavelength was 210 nm. About 1 mg of
the racemate of 2 was dissolved in 1 ml of the eluent, and a 20 ll volume
was applied onto the column. (R)-2: tR 5 5.4 min; (S)-2: tR 5 6.1 min.
Semipreparative HPLC Separation
The analytical method was afterward transferred to a Chiralpak1 IB
column (5 lm, 250 mm 3 20 mm). Because of limitations of the analyti-
cal pump, the flow rate was set only up to 9 ml/min. About 40 mg of the
racemic mixture was dissolved in 1.5 ml of eluent. A total of 100 ll each
was injected onto the column. (R)-2: tR 5 12.4 min; (S)-2: tR 5 15.4 min.
The fractions containing the corresponding enantiomers were collected
manually. The separation was carried out twice. The mobile phase was
afterward concentrated in vacuo leading to 10.3 mg of the (R)-enan-
tiomer [(R)-2:(S)-2 5 95.5:4.5] and 11.7 mg of the (S)-enantiomer [(S)-
2:(R)-2 5 96:4], both as colorless oils.
Preparation of Rat Liver Microsomes
Frozen rat livers from Wistar rats were thawed in phosphate buffer pH
7.4 with 0.25 M sucrose and 5 mM ethylenediaminetetraacetic acid
(EDTA), cut into pieces, and homogenized with a Potter (Elvehjem Pot-
ter, B. Braun Biotech International). The complete process was carried
out at 48C. The resulting suspension was centrifuged (high-speed cool-
ing centrifuge model Sorvall, RC-5C plus, Thermo Finnigan) at 10,000g
for 15 min at 48C. Fat was removed by absorption with cellulose. The
pellet was resuspended in buffer, the mixture centrifuged again, and
afterward both supernatants were combined. This suspension was trans-
ferred into an ultracentrifuge (Beckmann L8-M with rotor Ti 70.1) and
centrifuged for 60 min at 100,000g and 48C. The resulting supernatant
(cytosol) was discarded, the pellet carefully washed with buffer, resus-
pended, and the centrifugation repeated. Finally, the supernatant was
Recording of the CD Spectra
The CD spectra were recorded on a JASCO J-600 polarimeter in a 1-
cm cell. The solvent was methanol and the spectra were recorded at
room temperature. Each spectrum was recorded four times and the
resulting data display the mean.
Calculation of the CD Spectrum
The molecular structure has been fully energy optimized at the DFT-
B97-D/def2-TZVP level of theory.27,28 Bulk solvent effects have been
included by the COSMO continuum solvation model using the parame-
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ENANTIOSELECTIVE SIGMA-1 RECEPTOR BINDING AND BIOTRANSFORMATION
153
removed, the pellet resuspended in a small amount of phosphate buffer
pH 7.4, and the microsome suspension was stored at 2808C. The protein
concentration was determined according to the method of Bradford33
using bovine serum albumin as standard.
Metabolite 7. HPLC: tR 5 12.6 min. MS1 (ESI1 exact mass): m/z
calcd. for [C15H20FNO 1 H] 250.1602, found 250.1602. MS2 (ESI1 exact
mass): m/z calcd. for [C15H18FN 1 H] 232.1496, found 232.1493 (M–H2O).
Metabolite 8. HPLC: tR 5 14.4 min. MS1 (ESI1 exact mass): m/z
calcd. for [C22H25NO3 1 H] 352.1907, found 352.1905. MS2 (ESI1 exact
mass): m/z calcd. for [C12H12O3 1 H] 205.0857, found 205.0865 (M–
CH2C6H5–N(CH2)3).
Metabolic Stability
Six incubations of the enantiomers were carried out in phosphate
buffer pH 7.4 at room temperature in a circular shaker containing rat
liver microsomes (1.5 mg/ml protein), 0.86 mM MgCl2, and 2.6 mM
NADPH/H1. The enantiomer concentrations were 320 lM in a final vol-
ume of 0.9 ml. After 5, 10, 30, 40, 60, and 90 min, incubation was stopped
by addition of cold acetonitrile (2208C). A total of 200 ll of a 0.6 mg/ml
praziquantel solution were added as internal standard resulting in a final
concentration of 220 lM in a total volume of 1.045 ml. All incubations
were carried out as duplicates.
The samples were kept at 2208C for 30 min to complete protein pre-
cipitation. After thawing, the samples were centrifuged at 10,000g for 10
min, then the supernatant was decanted, filtered with a 0.45 lm (pore
size) syringe filter made from regenerated cellulose (Macherey-Nagel,
Du¨ren, Germany), and analyzed. A calibration was carried out with the
same microsomal matrix except NADPH/H1. All calibration standards
were treated in the same way (90 min on the shaker, protein precipita-
tion with acetonitrile, centrifugation, etc.).
The HPLC analysis was carried out with a Merck Hitachi equipment
consisting of UV detector: L-7400; autosampler: L-7200; pump: L-7150;
interface: D-7000. A total of 20 ll of the prepared incubation solutions
were injected onto a Phenomenex Gemini1 C18 column (5 lm, 250 mm
3 4.6 mm) at a flow rate of 1.0 ml/min and UV detection wavelength of
210 nm. The mobile phase was composed of (A) 15% acetonitrile in water
and (B) 60% acetonitrile in water. Trifluoroacetic acid (0.05%) was added
to both components. The following gradient was applied (A%): 0 min:
100%, 20 min: 0%, 23 min: 0%, 24 min: 100%, 30 min: 100%.
Metabolite 9. HPLC: tR 5 15.1 min and 16.9 min. MS1 (ESI1 exact
mass): m/z calcd. for [C22H26FNO2 1 H] 356.2020, found 356.2016. MS2
(ESI1 exact mass): m/z calcd. for [C12H13FO2 1 H] 209.0972, found
209.0969 (M–CH2C6H5–N(CH2)3).
Metabolite 10. HPLC: tR 5 18.3 min and 18.6 min. MS1 (ESI1
exact mass): m/z calcd. for [C22H26FNO2
1 H] 356.2020, found
356.2016. MS2 (ESI1 exact mass): m/z calcd. for [C15H20FNO 1 H]
250.1602, found 250.1598 (M–CH2C6H4OH).
CONCLUSIONS
After HPLC separation and determination of the absolute
configuration of the enantiomers (S)-2 and (R)-2, the biologi-
cal activities of the single enantiomers were investigated.
The (S)-configured enantiomer (S)-2 showed a threefold
higher r1 receptor affinity than (R)-2. Moreover, the euto-
mer (S)-2 was metabolically more stable than (R)-2, which
qualifies the (S)-enantiomer for further development. The
biotransformation with rat liver microsomes was highly ste-
reoselective, because the N-debenzylated secondary amine 7
and the hydroxbenyzl metabolite 10 were formed from (R)-
2, whereas (S)-2 was predominantly converted into the side
chain hydroxylated product 9 as main metabolite.
LC-MS
The LC-MS System consisted of a LTQ Orbitrap1 XL with Accela1
HPLC pump and autosampler (Thermo Fisher). A total of 20 ll of the
prepared incubation solutions were injected onto a LiChrospher1 RP
Select B column (5 lm, 250 mm 3 4.0 mm) with guard column LiChro-
CART1 RP Select B (4 mm 3 4.0 mm) at a flow rate of 1.0 ml/min The
mobile phase was composed of (A) 15% acetonitrile in water and (B) 60%
acetonitrile in water. Formic acid (0.1%) was added to both components.
The following gradient was applied (A%): 0 min: 100%, 20 min: 0%, 23
min: 0%, 24 min: 100%, 30 min: 100%.
ACKNOWLEDGMENTS
This work was performed within the framework of the
International Research Training Group ‘Complex Functional
Systems in Chemistry: Design, Synthesis and Applications’
in collaboration with Nagoya University.
LITERATURE CITED
The flow rate from the HPLC analysis (1.0 ml/min) was reduced to
0.250 ml/min for the MS analysis by a post column splitter (Acurate, LC
Packings, Dionex). The MS parameters were as follows: ion spray volt-
age: 4 kV in positive mode, sheath gas flow: 40 arbitrary units, aux gas
flow: 15 arbitrary units, sweep gas flow: 10 arbitrary units, capillary tem-
perature: 2758C, capillary: voltage 40.5 V.
First, a total ion current spectrum was recorded. To elucidate the
structures of the metabolites, collision-induced dissociation fragmenta-
tion experiments were carried out. The Orbitrap MS-system generated
data with fragmentations up to MS.3
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