Structural changes of benzylether derivatives of vesamicol and their influence on the binding selectivity to the vesicular acetylcholine transporter

Article abstract of DOI:10.1016/j.ejmech.2005.06.007

¹⁸F labelled vesamicol analogues, which bind to the vesicular acetylcholine transporter (VAChT) in central cholinergic nerve terminals, are expected to be potential radioligands for the visualisation of cholinergic transmission deficits via pos

Full text of DOI:10.1016/j.ejmech.2005.06.007

European Journal of Medicinal Chemistry 40 (2005) 1197–1205
www.elsevier.com/locate/ejmech
Original article
Structural changes of benzylether derivatives of vesamicol
and their influence on the binding selectivity
to the vesicular acetylcholine transporter
Barbara Wenzel a,*, Dietlind Sorger b, Katrin Heinitz b, Matthias Scheunemann a,
Reinhard Schliebs c, Jörg Steinbach a, Osama Sabri b
a Institut für Interdisziplinäre Isotopenforschung, Permoserstr. 15, 04318 Leipzig, Germany
b Klinik und Poliklinik für Nuklearmedizin der Universität Leipzig, Stephanstr. 11, 04103 Leipzig, Germany
c Paul-Flechsig-Institut für Hirnforschung der Universität Leipzig, Jahnallee 59, 04109 Leipzig, Germany
Received 3 May 2005; accepted 20 June 2005
Available online 10 August 2005
Abstract
18F labelled vesamicol analogues, which bind to the vesicular acetylcholine transporter (VAChT) in central cholinergic nerve terminals, are
expected to be potential radioligands for the visualisation of cholinergic transmission deficits via positron emission tomography (PET). In this
report the regioselective synthesis of five novel vesamicol analogues as well as their in vitro binding properties to the VAChT are described.
Beside having the 4-fluorobenzylether-substitution at the cyclohexyl ring as an unique structural feature, the new compounds are additionally
modified at the phenyl and piperidine moiety of the vesamicol skeleton. The affinity and selectivity to the VAChT were analysed by competi-
tive binding studies using tritium labelled radioligands. The VAChT affinities (Ki-values) of the novel compounds were estimated ranging
between 7.8 3.5 nM and 161.6 17.3 nM, thus some of them are binding with higher affinity to the transporter than vesamicol. However, the
compounds tested demonstrated also affinities to the sigma receptors r1 and r2 ranging between 4.1 1.5 nM and 327.5 75.9 nM. Never-
theless, these data provide the basis for future structure-binding-studies and further underline the potential and usefulness of vesamicol
analogues for imaging of the VAChT.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Vesamicol analogue; VAChT; PET; 18
F
1. Introduction
terminals [8,9], is responsible for the transport of acetylcho-
line into presynaptic vesicles [10,11]. Therefore, radioli-
gands which bind specifically to the VAChT, are assumed to
provide a tool for studying the density of central cholinergic
neurons using PET (positron emission tomography) or SPECT
(single photon emission computed tomography). Such sub-
stances afford an opportunity to investigate neurodegenera-
tive processes in vivo and to follow up the efficiency of thera-
peutic strategies.
The cholinergic system in the brain is known to be involved
in cognitive function and memory. Neurodegenerative disor-
ders likeAlzheimer’s disease as well as physiological ageing
processes are characterised by a profound loss of central cho-
linergic neurons which is accompanied by deficiencies in cho-
linergic neurotransmission [1–7]. The vesicular acetylcho-
line transporter (VAChT), localised in cholinergic nerve
All of the previously described VAChT ligands are based
on the structure of vesamicol, a drug that binds with high
affinity to the vesamicol binding site of the transporter,
whereby it non-competitively inhibits the transport of acetyl-
choline into the vesicles [12–15]. However, vesamicol dem-
onstrates considerable affinity to sigma receptors [16], being
also present in cholinergic brain regions, and which makes it
Abbreviations: VAChT, vesicular acetylcholine transporter; PET, posi-
tron emission tomography; DTG, 1,3-(di-o-tolyl)-guanidine.
* Corresponding author. Dr. Barbara Wenzel, Institut für Interdiszi-
plinäre Isotopenforschung, Permoserstraße 15, D-04318 Leipzig, Ger-
many, Tel.: +49/341 235 4015, fax: +49/341 235 2731.
E-mail address: wenzel@iif-leipzig.de (B. Wenzel).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.06.007

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B. Wenzel et al. / European Journal of Medicinal Chemistry 40 (2005) 1197–1205
Fig. 1. Selected ligands for the vesicular acetylcholine transporter (VAChT).
less useful as a selective radiotracer for imaging cholinergic
deficits. Therefore, in the past numerous efforts have been
undertaken to improve the selectivity by chemical modifica-
tions of the vesamicol structure. A number of compounds are
structurally derived from benzovesamicol including ABV
[17], [18F]NEFA [18], [18F]FEOBV [19,20] or [125I]IBVM
[21,22] and from trozamicol e.g. [18F]FBT [23–26] or
In this paper we report on selected structural modifica-
tions of 1 in order to decrease the affinity to sigma receptors,
while the high binding affinity toVAChT is not affected. Sub-
stitutions on the ring B (piperidine moiety) and ring C (phe-
nyl moiety) of the vesamicol skeletal structure resulted in the
five new analogues 12a,b-15 (Fig. 3). To get reliable infor-
mation on the binding properties of these derivatives, com-
petitive in vitro binding assays were performed using rat brain
tissue.
[
123/125I]MIBT [27–29] (Fig. 1).Although most of them were
described as promising radiotracers because of their in vivo
evaluation in rats, rodents or nonhuman primates, only
[
123I]IBVM and [18F]NEFA were used for human studies
[30–32]. But so far none of these ligands is really accepted
for clinical application due to undesired side effects. For
instance the extremely high lipophilicity of the iodinated mol-
ecule [123I]IBVM causes a high accumulation of the com-
pound in the intestine and liver which resulted in an unac-
ceptable radiation exposure of the gastrointestinum.
With the aim to evaluate a potential 18F labelled ligand,
that binds with high affinity and selectivity to the VAChT, we
developed the 4-O- and 5-O-substituted fluorobenzylether
derivatives 1 and 2 (Fig. 2). In 2004, we reported on the syn-
thesis and in vitro evaluation of this novel class of vesamicol
analogues [33].
Especially the 4-O-fluorobenzylether derivative 1 was char-
acterised by its high binding affinity to the VAChT
(Ki = 10.7 1.7 nM). However, it also exhibited a consider-
able affinity to both sigma receptors r1 and r2
(Ki = 18.5 6.9 nM).
2. Chemistry
The new vesamicol analogues 12a,b-15 and their required
key precursors, the amines 6a,b, 7, 10, 11 were synthesised
as outlined in Fig. 4 and Fig. 5.
The preparation of amines 6a and 6b (Fig. 4) based on
procedures partially described in the literature [34–36]. Com-
mercially available 1-benzyl-piperidine-4-one was reacted
with p-fluorophenylmagnesium bromide in THF at 0 °C to
give the tertiary alcohol 4a. Elimination of water under acidic
conditions delivered the olefine 5a. Subsequent palladium
catalysed hydrogenation by simultaneous debenzylation of
5a in a one-step procedure yielded amine 6a. The analogue
bromo derivative 6b, so far not described in the literature,
was prepared by the same method using p-bromo-
phenylmagnesium bromide as starting material.
The synthesis of amine 10 was already reported [37], but
modifications of the known synthetic pathway resulted in an
increased yield in our study. Thereby, 10 was prepared by
methylation of 1-benzyl-piperidine-4-one using the commer-
cially available Grignard reagent methylmagnesium chloride
followed by Friedel-Crafts reaction with benzene and subse-
quent palladium catalysed debenzylation.
In general, the synthesised amines were converted into their
oxalates.
4-Morpholinopiperidine 7 and 4-phenylpiperazine 11 for
the synthesis of 14 and 15 are commercially available.
Applying a regioselective ring opening reaction of the cis-
epoxide precursor 3 (Fig. 5) a convenient approach to the
Fig. 2. 4-O- and 5-O-substituted fluorobenzylether derivatives 1 and 2.

B. Wenzel et al. / European Journal of Medicinal Chemistry 40 (2005) 1197–1205
1199
Fig. 3. Novel 4-O-fluorobenzylether derivatives of vesamicol.
Fig. 4. Synthesis of amines 6a, 6b and 10.
Fig. 5. Synthesis of 12a,b-15.
4-substituted diasteriomerically pure aminoalcohols 12a,b-15
was achieved. Due to the favoured axial attack of a nucleo-
philic amine on C-1 of 3 only the 4-substituted regioisomeres
are formed under normal reaction conditions [38]. This high
regiochemical control was observed for all of the used amines
6a, 6b, 7, 10 and 11. In general, the ring opening reactions
were carried out in ethanol at 70 °C under stirring for 4 days
to provide crystalline products.

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B. Wenzel et al. / European Journal of Medicinal Chemistry 40 (2005) 1197–1205
Cis-epoxide 3 was prepared according to methods de-
formationally restricted spirovesamicol showed a significant
decrease in its binding affinity to sigma receptors when it was
brominated on the phenyl group of the spiro ring system
(adequate to the ring C fragment of vesamicol). Based on this
result it was the aim to investigate whether the halogenation
of the para-position of the phenyl group of 1 effects also a
reduction of the sigma binding. Fluorination of 1 to get 12a
resulted in a decreased binding affinity to VAChT
(Ki = 36.8 2.4 nM) compared to 1 (Ki = 10.7 1.7 nM),
whilst the affinity to sigma receptors was unaffected. On the
other hand, the bromination of 1 did not impair the binding
affinity toVAChT, which is reflected by comparable Ki-values
of 12b and 1. In contrast to the results of Efange et al. [16]
who reported a decreased sigma receptor binding for the bro-
minated spirovesamicol, the sigma affinity of 12b increased
remarkably (Ki = 4.1 1.5 nM) compared to vesamicol
(Ki = 183.4 11.4 nM) and also marginal compared to 1.
Apparently, the introduction of a halogen atom into the para-
position of the phenyl ring of the 4-fluorobenzylether deriva-
tive 1 led to a significant decrease of the selectivity factor
scribed by Scheunemann et al. [33].
3. In vitro binding studies
To evaluate the binding affinity to VAChT, the fluoroben-
zylether derivatives 12a,b-15 (Fig. 3) were investigated in
competitive binding assays on rat brain membranes using
(-)-[3H]vesamicol as high affinity VAChT radioligand
(Kd = 23 nM, [39,40]). To determine the selectivity of bind-
ing to VAChT the compounds were investigated with regard
to their affinity to sigma receptors. The sigma binding com-
petition assays were performed using rat liver membranes
which are known to be rich in r1 and r2 receptors. [3H]DTG,
one of the commonly used potent and selective sigma ligands
(r1 and r2), was applied as radiotracer.
The potency of the new compounds to successfully com-
pete with the binding of (-)-[3H]vesamicol to the VAChT is
expressed by their Ki-values determined according to the
method of Cheng and Prussoff [41]. For comparison,
(-)-vesamicol was analysed in the same assay run. The
Ki-values measured in sigma receptor binding assays were
compared to DTG as reference substance.
KiDTG/KiVes = 0.5 for 12a and 12b compared to
KiDTG/KiVes = 4.8 for vesamicol and also compared to
KiDTG/KiVes = 1.7 for 1.
In compound 13, the phenyl ring was completely replaced
by a morpholine ring, which is characterised by two hydro-
philic hetero atoms and additionally by the chair conforma-
tion of the six membered ring compared to the planar aro-
matic phenyl group of 1. It was the aim to study the influence
of this completely different structural moiety on the one hand,
and on the other hand to lower the lipophilicity of such com-
pounds. However, the in vitro binding data showed clearly
that these strong structural and electronic changes in the ring
C fragment resulted in a dramatic decrease of the binding
affinity of 13 to the VAChT (Ki about 9 000 nM). Rogers et
al. [44] could show that the substitution of the phenyl ring in
the vesamicol structure by a cyclohexyl ring, which has the
same conformation as a morpholine ring, did not influence
the capability of this ligand to inhibit the vesicular transport
of [3H]acetylcholine (the activity to block the acetylcholine
storage of most of the known VAChT ligands is comparable
to their affinity to the allosteric vesamicol binding site). There-
fore it has to be assumed that the hydrophilic hetero atoms
are responsible for the decreased binding affinity of 13 to
VAChT. For instance multiple hydrogen bonds may cause this
alteration of binding properties.
4. Results and discussion
This study continues our efforts to synthesise a highly spe-
cific 18F labelled radioligand with the potency to visualise
cholinergic dysfunction in vivo via the vesamicol binding site
of the vesicular acetylcholine transporter in central neurons.
In a previous work [33] we reported on the development of
novel benzylether and fluorobenzylether derivatives of vesa-
micol either 4- or 5-substituted at the cyclohexyl ring on the
basis of two different regioselective ring opening reactions.
The 4-substituted analogues showed significantly higher bind-
ing affinities to the VAChT than the corresponding 5-O-ether
derivatives. However, all isomers were characterised by their
low selectivity as shown by nanomolar binding affinities to
sigma receptors. Maintaining the 4-O-fluorobenzylether struc-
ture in the cyclohexyl ring of the vesamicol skeleton it was of
fundamental interest to modify the two ring systems B and C
of 1 in order to investigate the influence of structural changes
on the binding selectivity. Inspired by modification strategies
described for ring B and C in the literature [16,42] on the one
hand, and aiming at a well balanced lipophilicity of the 4-O-
fluorobenzylether derivatives on the other hand, the follow-
ing chemical modifications were made: a) the phenyl ring
(ring C) was fluorinated (12a), brominated (12b) and replaced
by a morpholine ring (13), respectively and b) into the
4-position of the piperidine ring (ring B) a methyl group was
introduced (14) and the piperidine ring was replaced by a
piperazine ring (15) (Fig. 3).
Modifications of ring B (14 and 15):
The replacement of the piperidine ring by a piperazine ring
in compound 15 is based on the results of Bando et al. [42].
The piperazine analogue of iodobenzovesamicol (DRC140)
has been shown to combine a high affine binding to the
VAChT with a low affine binding to sigma receptors. In our
study the introduction of the piperazine ring in 1 caused also
a significant decrease of the sigma binding affinity
(Ki = 327.5 75.9 nM) compared to 1 (Ki = 18.5 6.9 nM)
and vesamicol (Ki = 183.4 11.4 nM). However, this find-
ing was accompanied by an unacceptable reduction of bind-
ing to the transporter (Ki = 161.6 17.3 nM).
Modifications of ring C (12a, 12b, 13):
Efange et al. reported in 1995 on vesamicol analogues con-
cerning their sigma binding affinity [16]. Especially the con-

B. Wenzel et al. / European Journal of Medicinal Chemistry 40 (2005) 1197–1205
1201
Table 1
Inhibition constants (Ki) of (-)-vesamicol, 1, 12a, 12b, 13, 14 and 15 for their binding to VAChT sites and to sigma receptor sites estimated from competition
experiments in rat brain and rat liver membranes, respectively
Compound
VAChT ((-)-[3H]vesamicol)a
Nc
r1, r2 ([3H]DTG)b
Selectivity factor (KiDTG/KiVes)
Ki (nM)
Ki (nM)
Nc
2
(-)-vesamicol
38.5 4.2
10.7 1.7
36.8 2.4
7.8 3.5
12
6
183.4 11.4
18.5 6.9
19.3 1.5
4.1 1.5
4.8
1.7
0.5
0.5
ND
0.4
2.0
1
3
12a
12b
13
14
15
4
6
4
3
8 900
2
NDd
ND
6
158.2 37.1
161.6 17.3
4
57.1 13.8
327.5 75.9
6
5
Ki-values were determined in competitive binding studies using different tritium labelled radioligands in the presence of the compounds tested at concentrations
ranging from 10−11 M to 10−5 M. Ki-values are expressed as mean SD (nM).
a Binding to rat brain membranes, (-)-[3H]vesamicol [6.1 1.3 nM]; Kd rat brain = 23 nM [39,40]. Nonspecific binding was defined as the binding of
(-)-[3H]vesamicol in the presence of 10−5 M unlabelled (-)-vesamicol hydrochloride.
b Binding to rat liver membranes, [3H]DTG [4.9 0.5 nM]; Kd rat liver = 17.9 nM [43]. Nonspecific binding was defined as the binding of [3H]DTG in the
presence of 10−5 M unlabelled DTG.
c N, number of experiments performed.
d ND, not determined.
The introduction of a methyl group into the 4-position of
the piperidine ring in compound 14 yielded in results con-
trary to those expected. Thus, a decrease in the binding affin-
ity to the transporter (Ki = 158.2 37.1 nM) was observed.
The sigma binding affinity of 14 with a Ki = 57.1 13.8 nM
ranges between the Ki-values of 1 and vesamicol.
(AVANCE). Melting points were determined using a Lin-
ström apparatus and are uncorrected. Mass spectra were
recorded on a Mariner Biospectrometry workstation (Applied
Biosystems) (MS-TOF) using direct inlet system and Elec-
tronSpray Ionisation (ESI). Analytical TLC was performed
on silica gel coated plates (Merck Kieselgel 60 F254
,
These findings indicate that chemical modifications of the
4-position of the piperidyl moiety result in structural changes
which mainly impair the binding affinity to VAChT.
0.25 mm). The spots were identified using a UV lamp or by
dipping into a solution of 2% ninhydrine in EtOH/MeOH 1:1
(especially for amines). Elemental analyses were performed
in our laboratory using a CHN-O-Rapid analyser (Foss Her-
aeus) and agreed with the theoretical values within 0.4%.
Following abbreviations are used: EE for ethyl acetate,
MTBE for methyl-tert-butylether, MeOH for methanol, EtOH
for ethanol, THF for tetrahydrofuran, H for n-hexane.
Compounds 4a, 5a, 6a, 4b and 5b are described in the
literature [34–36]. Since the authors used different reaction
pathways or working up methods and most of these com-
pounds are insufficient characterised the procedures and ana-
lytical data are listed again.
5. Conclusion
It was the aim of this study to evaluate novel structurally
modified 4-O-fluorobenzylether derivatives of vesamicol with
improved binding properties to the VAChT in comparison to
compound 1 and to vesamicol itself, respectively. Both, vesa-
micol and 1, were revealed to bind with high affinity but low
selectivity to the transporter. The modifications of ring C and
ring B fragments of the 4-fluorobenzylether derivative 1, pre-
sented here, did not result in compounds potential and suit-
able for imaging cholinergic deficiencies as demonstrated by
their in vitro binding data given in Table 1. Therefore, we
suggest that the selectivity of vesamicol derivatives cannot
be improved by chemical modifications of ring B or C. Nev-
ertheless, the binding data obtained contribute to a better
understanding of structural requirements for a selective bind-
ing to the VAChT and provide the basis for future structure-
binding-studies.
6.1.2. Synthetic procedures
6.1.2.1. 1-Benzyl-4-(4-fluoro-phenyl)-piperidin-4-ol (4a). To
a mixture of a Grignard reagent prepared from magnesium
turnings (2.20 g, 0.091 mol) and p-bromo-fluoro-benzene
(10 mL, 0.091 mol) in 80 mL anhydrous THF a solution of
1-benzyl-piperidin-4-one (16.20 mL, 0.091 mol) in 20 mL
THF was slowly added at 4 °C over a period of 120 min. The
reaction mixture was stirred overnight and the resultant white
slurry was hydrolysed using 4 M aqueous NH4Cl (40 mL).
The organic material was extracted with THF/MTBE (1:1)
and dried over Na2CO3. Concentration of the solution yielded
a brown oil, that was dissolved in 75 mL CHCl3 followed by
extraction of the basic products using aqueous citric acid 10%
(3 × 30 mL). 6 M aqueous NaOH was added to get 4a as free
base and the organic material was re-extracted with CHCl3
(4 × 40 mL).After drying over Na2CO3 the solution was con-
centrated to give 21.2 g (81%) of 4a as orange oil. Rf = 0.38
6. Experimental
6.1. Chemistry
6.1.1. General
NMR spectra were recorded on Gemini 200 and Gemini
300 spectrometer and in one case on a Bruker DRX-600

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B. Wenzel et al. / European Journal of Medicinal Chemistry 40 (2005) 1197–1205
1
(EE/H 1:1). H NMR (CDCl3, 200.14 MHz): d (ppm) 7.46
(m, 2H, Ar), 7.32 (m, 2H, Ar), 7.01 (m, 2H, Ar), 3.58 (s, 2H,
N-CH2Ph), 2.74 (bm, 2H), 2.47 (bm, 3H), 2.13 (m, 2H) 1.74
(bm, 2H) (OH / Pip H2a, H2b, H3a, H3b). 13C NMR (CDCl3,
for a few seconds and filled with hydrogen. After stirring for
24 h at room temperature the catalyst was separated, the etha-
nolic solution was treated with aqueous 6 M NaOH and
organic material was extracted using CHCl3 (5 × 15 mL). Dry-
ing over Na2CO3 and concentration of the solution yielded in
0.90 g (77%) of nearly colourless oil. Rf = 0.73
(CHCl3/MeOH/NH3 2:1:0.2). 1H NMR (CDCl3,
300.06 MHz): d (ppm) 7.20 (m, 2H, Ar), 7.01 (m, 2H, Ar),
3.22 (bm, 2H) and 2.76 (dt, 2H, JHH = 12.3 Hz, JHH = 2.2 Hz)
(Pip H2a, H2b), 2.63 (tt, 1H, JHH = 12.3 Hz, JHH = 3.8 Hz,
Pip H4), 1.86-1.82 (bm, 2H) and 1.64 (dq, 2H, JHH = 12.8 Hz,
1
50.32 MHz): d (ppm) 162.0 (d, JCF = 245.0 Hz), 144.5 (d,
4JCF = 3.0 Hz), 138.4, 129.4, 128.4, 127.3, 126.6 (d,
3JCF = 7.6 Hz), 115.0 (d, 2JCF = 21.1 Hz) (Ar), 71.2 (Pip C4),
63.4 (N-CH2Ph), 49.6 (Pip C2), 38.7 (Pip C3).
6.1.2.2. 1-Benzyl-4-(4-bromo-phenyl)-piperidin-4-ol (4b). The
synthesis of 4b was carried out according to the same proce-
dure described for 4a. 19.80 g (83%) of 4b was obtained as
brownish oil. Rf = 0.50 (EE). 1H NMR (CDCl3, 199.97 MHz):
d (ppm) 7.44-7.28 (bm, 9H, Ar), 3.59 (s, 2H, N-CH2Ph), 2.75
(bm, 2H), 2.45 (bm, 3H), 2.11 (m, 2H) 1.74 (bm, 2H) (OH /
Pip H2a, H2b, H3a, H3b). 13C NMR (CDCl3, 50.28 MHz): d
(ppm) 171.1, 147.7, 138.2, 129.2, 128.2, 127.1, 126.6, 124.6
(Ar), 71.1 (Pip C4), 63.1 (N-CH2Ph), 49.3 (Pip C2), 38.3 (Pip
C3).
J
HH = 3.6 Hz) (Pip H3a, H3b). 13C NMR (CDCl3,
1
50.28 MHz): d (ppm) 161.4 (d, JCF = 243.9 Hz), 142.2 (d,
4JCF = 3.0 Hz), 128.1, (d, JCF = 7.5 Hz), 115.2 (d,
3
2JCF = 20.8 Hz) (Ar), 46.8 (Pip C2), 42.2 (Pip C4), 34.2 (Pip
C3).
The amine was converted into its oxalate (M.p.: 158-
160 °C) using an ethanolic solution of oxalic acid 10%.
6.1.2.6. 4-(4-Bromo-phenyl)-piperidine (6b). 0.81 g (80%)
of 6b was obtained as nearly colourless oil according to the
same procedure described for 6a. 1.36 g (4.16 mmol) of 5b,
2 mL acetic acid and 1.50 g PdC were used. Rf = 0.80
(CHCl3/MeOH/NH3 2:1:0.2). 1H NMR (CDCl3,
300.06 MHz): d (ppm) 7.23 (m, 3H, Ar), 7.30 (m, 2H, Ar),
3.39 (bd, 2H, JHH = 12.6 Hz, Pip H2a), 2.91-2.82 (bm, 2H,
Pip H2b), 2.68 (m, 1H, Pip H4), 1.95-1.89 (bm, 4H, Pip H3a,
H3b), 13C NMR (CDCl3, 75.45 MHz): d (ppm) 145.2, 128.6,
126.8, 126.6 (Ar), 45.9 (Pip C2), 41.9 (Pip C4), 32.3 (Pip
C3).
6.1.2.3. 1-Benzyl-4-(4-fluoro-phenyl)-1,2,3,6-tetrahydro-
pyridine (5a). A solution of 4a (5.20 g, 0.018 mol) and
p-toluenesulfonic acid monohydrate (4.71 g, 0.025 mol) in
80 mL toluene was refluxed for 7 h using a dean stark trap to
remove water. Concentration of the reaction mixture pro-
vides a brown residue, that was treated with 6 M aqueous
NaOH. Extraction using CHCl3, drying over Na2CO3 and con-
centration yielded a brown oil, that was purified via column
chromatography (silica gel, EE/H 1:1) to get 2.91 g (62%) of
1
5a as yellow oil. Rf = 0.71 (EE/H 1:1). H NMR (CDCl3,
A part of the product could be crystallised as free amine
(M.p.: 144-146 °C). As described above the residual oil was
converted into its oxalate (M.p. > 220 °C).
200.14 MHz): d (ppm) 7.47-7.20 (overlapping m, 7H, Ar),
7.03 (m, 2H,Ar), 6.04 (bs, 1H, Py H5), 3.68 (s, 2H, N-CH2Ph),
3.20 (bm, 2H, Py H6), 2.75 (m, 2H, Py H2), 2.56 (bm, 2H, Py
H3). 13C NMR (CDCl3, 50.33 MHz): d (ppm) 162.2 (d,
4
1JCF = 245.6 Hz), 138.5, 137.2 (d, JCF = 3.8 Hz), 134.2,
6.1.2.7. ( )-2-[4-(4-Fluoro-phenyl)-piperidin-1-yl]-4-(4-
fluoro-benzyloxy)-cyclohexanol (12a). To a solution of
epoxide 3 (0.66 g, 3.00 mmol) in 10 mL ethanol amine 6a
(0.4 g, 2.25 mmol) in 5 mL ethanol was added and stirred at
70 °C for 5 days. At 4 °C a mixture of 12a and amine 6a
crystallised as white solid. The precipitate was purified via
column chromatography (silica gel, EE to EE/MeOH 1:1) to
give 0.45 g (55%) of nearly colourless ductile oil that partly
crystallised from ethanol. M.p.: 91-92 °C. Rf = 0.41 (EE/H
1:1). 1H NMR (CDCl3, 200.14 MHz): d (ppm) 7.32 (m, 2H,
Ar), 7.17 (m, 2H, Ar), 7.00 (m, 4H, Ar), 4.48 (AB, 2H,
3
129.4, 128.5, 127.4, 126.6 (d, JCF = 8.5 Hz), 122.1, 115.3
2
(d, JCF = 20.5 Hz) (Ar, Py C4), 65.4 (N-CH2Ph), 53.5 and
50.1 (Py C2, C6), 28.4 (Py C3).
6.1.2.4. 1-Benzyl-4-(4-bromo-phenyl)-1,2,3,6-tetrahydro-
pyridine (5b). 5b was prepared using the same method
described for 5a. 4.49 g (0.013 mol) of 4b and 3.38 g
(0.018 mol) of p-toluenesulfonic acid monohydrate were used.
The purification was carried out via column chromatography
(silica gel, EE/H 3.5:1) to get 2.34 g (55%) of 5b as orange
oil. Rf = 0.87 (EE). 1H NMR (CDCl3, 199.97 MHz): d (ppm)
7.45-7.22 (overlapping m, 9H,Ar), 6.07 (m, 1H, Py H5), 3.65
(s, 2H, N-CH2Ph), 3.18 (bm, 2H, Py H6), 2.73 (m, 2H, Py
H2), 2.54 (bm, 2H, Py H3). 13C NMR (CDCl3, 50.33 MHz):
d (ppm) 139.7, 137.9, 134.0, 131.4, 129.3, 128.3, 127.3, 126.5,
122.5, 119.8 (Ar, Py C4), 62.7 (N-CH2Ph), 53.2 and 49.8 (Py
C2, C6), 27.9 (Py C3).
J
HH = 12.0 Hz, O-CH2PhF), 4.02 (bs, 1H, OH), 3.84 (bs, 1H,
Cy H1), 3.47 (m, 1H, Cy H4), 2.95 (bd, 1H, Pip H2a), 2.84-
2.64 (bm, 3H, Cy H2, Pip H6a, H6b), 2.48 (m, 1H, Pip H4),
2.27-1.25 (overlapping m, 11H, Pip H2b, H3a, H3b, H5a,
H5b, Cy H3a, H3b, H5a, H5b, H6a, H6b). 13C NMR (CDCl3,
50.28 MHz, APT): d (ppm) 162.2 (d, 1JCF = 244.8 Hz), 161.4
1
4
(d, JCF = 243.3 Hz), 141.9 (d, JCF = 3.0 Hz), 134.8 (d,
4JCF = 3.5 Hz), 129.0 (d, JCF = 8.0 Hz), 128.0 (d,
3
2
6.1.2.5. 4-(4-Fluoro-phenyl)-piperidine (6a). To a solution
of 5a (1.74 g, 6.50 mmol) in 20 mL ethanol were added 2 mL
acetic acid and 1.50 g PdC (10%). The flask was evacuated
3JCF = 7.5 Hz), 115.2 (d, JCF = 21.6 Hz), 115.1 (d,
2JCF = 20.7 Hz) (Ar), 73.4 (Cy C1), 69.3 (O-CH2PhF), 68.6
(Cy C4), 64.7 (Cy C2), 53.2 (Pip C6), 45.3 (Pip C2), 42.3

B. Wenzel et al. / European Journal of Medicinal Chemistry 40 (2005) 1197–1205
1203
(Pip C4), 34.4 and 34.1 (Pip C3, C5), 27.7, 27.6, 26.3 (Cy
C3, C5, C6). MS: m/z 402.2 (M+1). Anal. C24H29F2NO2 (C,
H, N).
(Pip C6), 49.9 (Mor C2), 44.2 (Pip C2), 29.1 and 28.8 (Pip
C3, C5), 27.69, 27.60, 26.3 (Cy C3, C5, C6). MS: m/z 393.2
(M+1). Anal. C22H33FN2O3 (C, H, N).
6.1.2.10. 1-Benzyl-4-methyl-piperidin-4-ol (8). To a solution
of methylmagnesium chloride (20% in THF, 23 mL,
0.063 mol) at 4 °C under nitrogen a solution of 1-benzyl-
piperidin-4-one (10 g, 0.053 mol) in 75 mL anhydrous THF
was added. The reaction mixture was stirred overnight at room
temperature, 4 M aqueous NH4Cl (30 mL) was added and
organic material was extracted with MTBE (3 × 15 mL). The
combined organic solutions were dried over MgSO4 and con-
centrated to yield 10.40 g (96%) of yellow oil. Rf = 0.15
(EE/H 1:1). 1H and 13C NMR spectra are in agreement with
the literature [37].
6.1.2.8. ( )-2-[4-(4-Bromo-phenyl)-piperidin-1-yl]-4-(4-
fluoro-benzyloxy)-cyclohexanol (12b). To a solution of
epoxide 3 (0.63 g, 2.85 mmol) in 10 mL ethanol amine 6b
(0.67 g, 2.85 mmol) in 5 mL ethanol was added and the reac-
tion mixture was stirred at 70 °C for 5 days.At 4 °C a mixture
of 12b and amine 6b crystallised as white solid. The precipi-
tate was recrystallised from EtOH/CHCl3 1:3 to get 0.62 g
(47%) of 12b as white needles. M.p.: 121-122 °C. Rf = 0.33
1
(EE/H 1:2). H NMR (CDCl3, 199.97 MHz): d (ppm) 7.29
(m, 6H, Ar), 7.04 (m, 2H, Ar), 4.47 (AB, 2H, JHH = 12.0 Hz,
O-CH2PhF), 4.06 (bs, 1H, OH), 3.85 (bm, 1H, Cy H1), 3.47
(m, 1H, Cy H4), 2.95 (bd, 1H, Pip H2a), 2.85-2.63 (bm, 3H,
Cy H2, Pip H6a, H6b), 2.48 (m, 1H, Pip H4), 2.27-1.24 (over-
lapping m, 11H, Pip H2b, H3a, H3b, H5a, H5b, Cy H3a, H3b,
H5a, H5b, H6a, H6b). 13C NMR (CDCl3, 50.28 MHz): d
6.1.2.11. 1-Benzyl-4-methyl-4-phenyl-piperidine (9). The
reaction procedure was adopted from literature [37] and the
analytical data are in agreement with the data reported.
1
(ppm) 162.2 (d, JCF = 245.1 Hz), 146.3, 134.8 (d,
4JCF = 3.0 Hz), 129.0 (d, 3JCF = 8.1 Hz), 128.4, 126.8, 126.2,
6.1.2.12. 4-Methyl-4-phenyl-piperidine (10). To a solution
of 9 (3.75 g, 0.014 mol) in 30 mL ethanol were added 2.5 mL
acetic acid and 1 g PdC. The flask was evacuated for a few
seconds and filled with hydrogen. After stirring for 24 h at
room temperature the reduction was finished and the catalyst
was separated. The ethanolic solution was treated with 6 M
aqueous NaOH and organic material was extracted using
CHCl3 (5 × 15 mL) . Drying over Na2CO3 and concentration
of the solution yielded 2.11 g (85%) of nearly colourless oil.
By dissolving of the oily product in MTBE a part of com-
pound 10 could be isolated as a white powder at 4 °C. M.p.:
2
115.2 (d, JCF = 21.5 Hz) (Ar), 73.5 (Cy C1), 69.2 (O-
CH2PhF), 68.6 (Cy C4), 64.6 (Cy C2), 53.3 (Pip C6), 45.3
(Pip C2), 43.1 (Pip C4), 34.3 and 34.0 (Pip C3, C5), 27.75,
27.70, 26.2 (Cy C3, C5, C6). MS: m/z 384.3 (M-Br+1). Anal.
C24H29BrFNO2 (C, H, N).
6.1.2.9. ( )-4-(4-Fluoro-benzyloxy)-2-(4-morpholin-4-yl-
piperidin-1-yl)-cyclohexanol (13). A mixture of epoxide 3
(0.43 g, 1.80 mmol) in 10 mL ethanol and
4-morpholinopiperidine 7 (0.30 g, 1.70 mmol) in 5 mL etha-
nol was stirred at 70 °C for 5 days. The solution was concen-
trated to give a yellow oil, that was dissolved in 10 mL CH2Cl2
followed by extraction of the basic products using aqueous
citric acid 10% (3 × 10 mL). 6 M aqueous NaOH was added
to the water layer to get 13 as free base, that was re-extracted
with CH2Cl2 (4 × 20 mL).After drying over Na2CO3 the solu-
tion was concentrated and the nearly colourless oily residue
was purified via column chromatography (silica gel,
EE/MeOH 1:1). After dissolving of the product in EtOH
0.32 g (34%) of 13 was obtained as white needles at 4 °C.
M.p.: 128-129 °C. Rf = 0.54 (CHCl3/MeOH/NH3 10:1:0.1).
1H NMR (CDCl3, 600.13 MHz, HH Cosy): d (ppm) 7.29 (m,
2H, Ar), 7.02 (m, 2H, Ar), 4.43 (AB, 2H, O-CH2PhF), 3.92
(bs, 1H, OH), 3.80 (bm, 1H, Cy H1), 3.72 (m, 4H, Mor H3),
3.40 (dt, 1H, JHH = 10.4 Hz, JHH = 4.3 Hz, Cy H4), 2.87 (bd,
1H, Pip H2a), 2.71 (bm, 2H, Cy H2, Pip H6a), 2.55 (m, 5H,
Mor H2, Pip H6b), 2.14 (bm, 1H, Pip H4), 2.06-2.01 (bm,
2H, Pip H2b, Cy H3a), 1.99-1.91 (bm, 2H, Cy H5a, H6a),
1.86 (bd, 2H, Pip H3a, H5a), 1.67 (bq, 1H), 1.34 (bt, 1H),
1.24 (dt, 1H, JHH = 13.0 Hz, JHH = 2.4 Hz) (Cy H3b, H5b,
H6b), 1.56 (bq, 1H), 1.34 (bq, 1H) (Pip H3b, H5b). 13C NMR
(CDCl3, 150.91 MHz, HETCOR, APT): d (ppm) 162.2 (d,
1JCF = 245.3 Hz), 134.7, 129.0 (d, 3JCF = 8.0 Hz), 115.2 (d,
2JCF = 21,0 Hz) (Ar), 73.4 (Cy C1), 69.3 (O-CH2PhF), 68.6
(Cy C4), 67.6 (Mor C3), 64.3 (Cy C2), 62.3 (Pip C4), 51.8
69-72 °C. Rf = 0.69 (CHCl3/MeOH/NH3 2:1:0.2). 1H and 13
NMR spectra are in agreement with the literature [37].
C
For storage the rest of the amine was converted into its
oxalate (M.p.: 180-185 °C) using an ethanolic solution of
oxalic acid 10%.
6.1.2.13. ( )-4-(4-Fluoro-benzyloxy)-2-(4-methyl-4-phenyl-
piperidin-1-yl)-cyclohexanol (14). To a solution of the epoxide
3 (0.47 g, 2.10 mmol) in 10 mL ethanol was added amine 10
(0.37 g, 2.10 mmol) in 5 mL ethanol. The reaction mixture
was stirred at 70 °C for 5 days. After cooling the solvent was
evaporated and the residue was dissolved in CHCl3 (15 mL).
After extracting of the basic products with 10% citric acid
(4 × 5 mL), to the combined aqueous phases 6 M aqueous
NaOH was added and organic material was re-extracted with
CHCl3 (4 × 15 mL). Following drying over Na2CO3 and
evaporation of the solvent yielded an orange ductile oil, which
was purified by column chromatography (silica gel, EE to
EE/MeOH 1:1) to give 0.23 g (28%) of nearly colourless oil.
Rf = 0.55 (EE). 1H NMR (CDCl3, 199.97 MHz): d (ppm) 7.37-
7.21 (m, 7H, Ar), 7.07 (m, 2H, Ar), 4.44 (AB, 2H,
JHH = 12.1 Hz, O-CH2PhF), 3.80 (bm, 1H, Cy H1), 3.45 (m,
1H, Cy H4), 2.83-2.63 (overlapping m, 3H), 2.53-2.31 (bm,
2H), 2.24-1.57 (overlapping m, 8H), 1.28 (m, 2H) (Cy, Pip),
1.25 (s, 3H, CH3). 13C NMR (CDCl3, 50.28 MHz, APT): d

1204
B. Wenzel et al. / European Journal of Medicinal Chemistry 40 (2005) 1197–1205
1
(ppm) 162.1 (d, JCF = 245.4 Hz), 149.1, 134.7 (d,
4JCF = 3.0 Hz), 129.0 (d, 3JCF = 8.1 Hz), 128.4, 125.7, 125.6,
membranes for drug competition assays were obtained by cen-
trifugation of the brain homogenate at 15 000 x g for 15 min
at 4 °C, washed twice (once with 0.25 M sucrose solution,
once with distilled water) and re-suspended to a concentra-
tion of 200 mg/mL wet tissue in assay buffer (50 mM Tris
pH 7.4 containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2,
1 mM MgCl2).
Rat liver (for analysing sigma receptor binding) was pre-
pared in the same way. The liver lobes were minced before
homogenisation.
2
115.1 (d, JCF = 21.2 Hz) (Ar), 73.4 (Cy C1), 69.2 (O-
CH2PhF), 68.5 (Cy C4), 64.4 (Cy C2), 45.3 and 45.0 (Pip
C2, C6), 37.7 and 37.6 (Pip C3, C5), 36.5 (CH3), 29.4 (Pip
C4), 27.7, 27.6, 26.2 (Cy C3, C5, C6). MS: m/z 398.3 (M+1).
To get 14 as a solid the oily product was converted into its
oxalate using an ethanolic solution of oxalic acid 10%. M.p.:
214-215 °C. Anal. C27H34FNO6 (C, H, N).
6.1.2.14. ( )-4-(4-Fluoro-benzyloxy)-2-(4-phenyl-piperazin-
1-yl)-cyclohexanol (15). A mixture of epoxide 3 (0.75 g,
3.37 mmol) in 10 mL ethanol and 4-phenylpiperazine 11
(0.36 g, 2.25 mmol) in 5 mL ethanol was stirred at 70 °C for
5 days. The solution was concentrated to give a brown oil,
that was dissolved in 10 mL CH2Cl2 followed by extraction
of the basic products using aqueous citric acid 10%
(4 × 10 mL). 6 M aqueous NaOH was added to the water
layer to get 15 as free base that was re-extracted with CH2Cl2
(3 × 20 mL).After drying over Na2CO3 the solution was con-
centrated and the nearly colourless oily residue was dis-
solved in EtOH. At 4 °C 0.43 g (49%) of 15 was obtained as
6.2.3. Determination of binding data
Various concentrations (10−11 to 10−5 M) of the synthe-
sised compounds were incubated in duplicates with mem-
branes (600 µg protein) for 2 h at 25 °C during agitation
(200 U min−1) in a total volume of 1 mL of assay buffer in the
presence of one of the different radiotracers: (-)-[3H]vesamicol
(AH 5183) [6.1 1.3 nM] and [3H]DTG (r1 and r2 ligand,
[4.9 0.5 nM]), respectively. Unlabelled (-)-vesamicol and
DTG were tested for comparison in parallel experiments.After
incubation membrane bound radiotracer was separated from
free radioligand by rapid vacuum filtration through What-
man GF/B glas-fiber filters presoaked for 3 h in 0.5% poly-
ethyleneimine using a Brandel cell harvester (Gaithersburg,
MD). The filters were washed 3 times with 4 mL ice-cold
buffer - dried, soaked in 10 mL Rotiszint eco plus cocktail
overnight and counted in a TriCarb Liquid Scintillation
Counter (Tri-Carb2900TR, Packard, Meriden, USA) at 70%
counting efficiency.
1
white needles. M.p.: 105-106 °C. Rf = 0.73 (EE). H NMR
(CDCl3, 199.97 MHz): d (ppm) 7.32 (m, 4H, Ar), 7.05-6.84
(m, 5H, Ar), 4.47 (AB, 2H, O-CH2PhF), 3.92 (s, 1H, OH),
3.85 (bs, 1H, Cy H1), 3.52 (dt, 1H, JHH = 10.4 Hz,
J
HH = 4.4 Hz, Cy H4), 3.30-3.12 (bm, 4H, Pip H3a, H3b),
2.92-2.75 (bm, 3H, Pip H2a, Cy H2), 2.62-2.51 (bm, 2H, Pip
H2b), 2.16-1.97 (bm, 3H), 1.77 (m, 1H), 1.35 (m, 2H) (Cy
H3a, H3b, H5a, H5b, H6a, H6b). 13C NMR (CDCl3,
50.28 MHz, HETCOR): d (ppm) 167.1 (d, 1JCF = 245.2 Hz),
151.3, 134.7 (4JCF = 3,1 Hz), 129.1 (d, 3JCF = 8.1 Hz), 128.7,
6.2.4. Data analysis
Inhibitory concentration at 50% specific binding of the
radioligand (IC50-value) was determined by a nonlinear curve
fitting method, using Graphpad Prism,Vers. 3 (GraphPad Soft-
ware, Inc., San Diego, USA). Specific binding of radioli-
gands was defined as total binding minus nonspecific bind-
ing. Nonspecific binding was determined in parallel
experiments in the presence of 10−5 M of the corresponding
unlabelled ligand. Ki-values were derived from IC50-values
according to the equation: Ki=IC50/(1+C/Kd) [41] whereby
C is the concentration of the radioligand and Kd is the disso-
ciation constant of the radioligand. Kd-values were taken from
the literature as indicated.
2
119.9, 116.2, 115.2 (d, JCF = 21,3 Hz) (Ar), 73.4 (Cy C1),
69.3 (O-CH2PhF), 68.5 (Cy C4), 64.3 (Cy C2), 49.9 (Pip C3),
48.2 (Pip C2), 27.6, 27.5, 26.3 (Cy C3, C5, C6). MS: m/z
385.2 (M+1). Anal. C23H29FN2O2 (C, H, N).
6.2. Radioligand binding and competition experiments
6.2.1. Radioligands and drugs
(-)-[3H]Vesamicol
cyclohexanol, AH
([3H]2-(4-phenylpiperidinyl)-
5183, specific radioactivity
1258 GBq/mmol resp. 34 Ci/mmol) and [3H]DTG ([3H]1,3-
(di-o-tolyl)-guanidine, specific radioactivity 1110 GBq/mmol
resp. 30 Ci/mmol) were obtained from Perkin Elmer Life Sci-
ences, Boston, MA, USA). (-)-Vesamicol hydrochloride and
1,3-(di-o-tolyl)-guanidine (DTG) were purchased from
TOCRIS, BIOTREND Chemikalien GmbH, Köln.
Acknowledgments
This work has been supported by a grant from Sächsis-
ches Ministerium für Wissenschaft und Kunst, contract no.
7531.50-03-0361-01/6.
6.2.2. Tissue preparation
Female Sprague Dawley rats (150–200 g) were anaesthe-
tised with ether and decapitated. Their brains were rapidly
removed from the skull. The brain tissue minus cerebellum
was homogenised in 10 volumes (w/v) of ice-cold 0.25 M
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