New systematically modified vesamicol analogs and their affinity and selectivity for the vesicular acetylcholine transporter - A critical examination of the lead structure

Article abstract of DOI:10.1016/j.ejmech.2015.05.033

To verify vesamicol as lead structure in the development of radioligands for imaging of VAChT in the brain by PET, we systematically modified this molecule and investigated four different groups of derivatives. Structural changes were conducted in all three ring systems A, B, and C resulting in a library of different vesamicol analogs. Based on their in vitro binding affinity toward VAChT as well as σ₁ and σ₂ receptors, we performed a structure-affinity relationship (SAR) study regarding both affinity and selectivity. The compounds possessed VAChT affinities in the range of 1.32 nM (benzovesamicol) to >10 mM and selectivity factors from 0.1 to 73 regarding σ₁ and σ₂ receptors, respectively. We could confirm the exceptional position of benzovesamicols as most affine VAChT ligands. However, we also observed that most of the compounds with high VAChT affinity demonstrated considerable affinity in particular to the σ₁ receptor. Finally, none of the various vesamicol analogs in all four groups showed an in vitro binding profile suitable for specific VAChT imaging in the brain.

Full text of DOI:10.1016/j.ejmech.2015.05.033

European Journal of Medicinal Chemistry 100 (2015) 50e67
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
New systematically modified vesamicol analogs and their affinity and
selectivity for the vesicular acetylcholine transporter e A critical
examination of the lead structure
Claudia Barthel a, Dietlind Sorger b, Winnie Deuther-Conrad a, Matthias Scheunemann a,
a
a
c
a
d
€
€
Stephanie Schweiger , Petra Jackel , Ali Roghani , Jorg Steinbach , Gerrit Schüürmann ,
Osama Sabri b, Peter Brust a, Barbara Wenzel a
,
*
a Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Research Site Leipzig, Germany
b Department of Nuclear Medicine, University of Leipzig, Germany
c Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, USA
d UFZ Department of Ecological Chemistry, Helmholtz Centre for Environmental Research, Leipzig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
To verify vesamicol as lead structure in the development of radioligands for imaging of VAChT in the
brain by PET, we systematically modified this molecule and investigated four different groups of de-
rivatives. Structural changes were conducted in all three ring systems A, B, and C resulting in a library of
different vesamicol analogs. Based on their in vitro binding affinity toward VAChT as well as s1 and s2
receptors, we performed a structure-affinity relationship (SAR) study regarding both affinity and
selectivity. The compounds possessed VAChT affinities in the range of 1.32 nM (benzovesamicol) to
Received 6 February 2015
Received in revised form
20 May 2015
Accepted 21 May 2015
Available online 22 May 2015
>10
mM and selectivity factors from 0.1 to 73 regarding s1 and s2 receptors, respectively. We could
Keywords:
VAChT
Vesamicol
PET
confirm the exceptional position of benzovesamicols as most affine VAChT ligands. However, we also
observed that most of the compounds with high VAChT affinity demonstrated considerable affinity in
particular to the s1 receptor. Finally, none of the various vesamicol analogs in all four groups showed an
in vitro binding profile suitable for specific VAChT imaging in the brain.
Sigma receptors
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
emission computed tomography) is regarded as useful tool for
studying changes of cholinergic function in normal and diseased
The vesicular acetylcholine transporter (VAChT) is a trans-
membrane protein located on the presynaptic vesicular membrane
of cholinergic neurons. It is responsible for the accumulation of
acetylcholine inside the vesicles and thus plays an important role in
cholinergic neurotransmission. In the last decade this transporter
was highly regarded as potential target to investigate functional
alterations observed in degenerative processes of the cholinergic
system [1,2]. Cholinergic deficits in brain are discussed as early
pathological feature of the cognitive decline occurring in neuro-
degenerative disorders such as Alzheimer's Disease (AD) [3e8].
Noninvasive in vivo imaging based on radiolabeled VAChT ligands
using PET (positron emission tomography) or SPECT (single-photon
brain. By contrast to other potential targets available for imaging of
the cholinergic system by PET, VAChT is expressed solely presyn-
aptically and thus might support early diagnostics of AD by exclu-
sive visualization of cholinergic neurons. Imaging of VAChT in
human brain was already performed with SPECT [9] as well as very
recently with PET [10] ligands both showing advantages and dis-
advantages. Nevertheless, PET, with fluorine-18 as the currently
most widely used radionuclide, is superior regarding its detection
efficiency, spatial resolution, and quantification.
The previous development of VAChT radiotracers was based on
the single known chemical lead compound vesamicol (trans-2-(4-
phenylpiperidino)cyclohexanol), that binds with high affinity to
an allosteric site of the transporter [11e13]. However, vesamicol
also demonstrates substantial affinity to the sigma receptors s1 and
s2, which are distributed in cholinergic brain regions, thereby
impairing the possibility of specific in vivo imaging [14,15].
* Corresponding author.
E-mail address: b.wenzel@hzdr.de (B. Wenzel).
http://dx.doi.org/10.1016/j.ejmech.2015.05.033
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
51
Therefore, the development of VAChT ligands is focused on struc-
tural modifications of vesamicol with the aim to keep or improve
the VAChT affinity and to reduce the affinity for sigma receptors. In
1989, Rogers et al. synthesized about 80 vesamicol derivatives and
determined their potential to inhibit the [3H]ACh transport into
vesicles [16]. These data have been the basis for most of the VAChT
ligand developments and as a result, several classes of vesamicol
analogs have arisen. Noteworthy are the benzovesamicols with the
most potential candidate [18F]FEOBV [17e21] with promising
in vivo imaging properties in rodents and monkeys (see Fig. 1). In
2014, the first human study using this tracer has been reported [10].
However, so far the selectivity of this tracer is not sufficiently
verified in vivo. Besides FEOBV, some of the recently published
carbonyl group containing benzovesamicols [22e25] seem to be
appropriate candidates for further in vivo evaluation. Another
important class is the group of trozamicols, with [18F]FBT [26] as
representative. However, in particular these derivatives suffer from
low selectivity [14].
Based on the qualitative structure-activity observations re-
ported by Rogers et al. [16], we started in 2004 with the develop-
ment of 4-O- and 5-O-substituted fluorobenzyl ether derivatives of
vesamicol with the aim of improving the selectivity [27,28]. In
particular, the 4-O-derivatives (Fig.1) showed good VAChT affinities
with Ki values in the low nanomolar range; however the affinity for
sigma receptors was high as well. Based on the idea of a ring fusion
as made with benzovesamicols, structurally related compounds
were developed. Two candidates of the class of morpholinovesa-
micols were 18F-radiolabeled and studied in vivo in rats and pigs
[29e31]. In particular, the 4-fluoro-benzoylated derivative [18F]
FBMV showed a typical accumulation in VAChT-containing brain
regions and a significant reduction of cortical binding after a spe-
cific cholinergic lesion. However, to confirm the high in vitro
selectivity of this tracer, further studies in vivo have to be per-
formed using appropriate sigma receptor ligands. In parallel to the
morpholinovesamicols we developed the azaspirovesamicols [32]
and the recently published novel group of pyrrolovesamicols [33].
In particular, the latter showed unexpectedly low in vitro affinities
for the VAChT and both classes are not appropriate for in vivo
evaluation. The numerous literature data and our own intensive
work in developing VAChT ligands demonstrate the difficulty to
predict properties of a newly synthesized compound regarding
VAChT affinity and selectivity. As for many promising VAChT li-
gands observed, high VAChT affinity is often accompanied by high
sigma receptor binding. However, beside an appropriate target af-
finity, a high selectivity is essential for the development of a specific
VAChT PET tracer. So with our study we wanted to put some light in
particular on this fact and we determined the affinity to the s1 and
s2 receptors. Therefore, in this paper we report on the synthesis of a
library of mainly novel vesamicol analogs and their binding profile
regarding both VAChT and s1 s2 receptors. Systematic modifica-
tions in ring B and C of the vesamicol skeleton were performed and
combined with different modifications in ring A (vesamicol can be
subdivided into the three rings A (cyclohexanol), B (piperidine), and
C (phenyl), see Fig. 1).
The in vitro binding affinities for VAChT and the sigma receptors
s1 and s2 were determined by competitive radioligand binding
assays. In particular, for the determination of VAChT affinities
different protocols are reported resulting in considerable affinity
differences for identical compounds, e.g. vesamicol. Rogers and
Parsons et al. used an assay with synaptic vesicles from the electric
organ of Torpedo californica (Ki(vesamicol) ¼ 1.0 nM) [34], whereas
cerebral membranes of rats were used by the group of K. Shiba
(Ki(vesamicol) ¼ 33.9 nM) [35,36] and a cell line transfected with
human VAChT by Tu et al. [37]. In our study, we investigated rat
brain membranes and a cell line stably transfected with rat VAChT
as possible target materials to develop a standardized test setup.
We have chosen rat material in order to avoid possible species
dependent differences when comparing the VAChT affinity data
/
with the data obtained for the s1/s2 receptor binding. A few of
known vesamicol analogs were re-synthesized to compare their
VAChT affinity data reported with the data obtained in our assay.
2. Results and discussion
2.1. Chemistry
A very convenient approach to synthesize vesamicol derivatives
is the nucleophilic ring opening of an epoxide precursor using a
secondary amine. By varying the epoxide precursor, structural
modifications in ring A of the vesamicol skeleton can be realized.
Due to an elaborate selection of different amines, ring B and C can
be sterically or electronically modified. Therefore, we selected
several amines which are shown in Fig. 2. Amines b-d differ in the
constitution of ring B compared to 4-phenylpiperidine (a), the
amine used for most of the known vesamicol analogs. 4-
Phenylpiperazine (b) contains a further ionizable nitrogen atom
resulting in electronic changes. Considering amine c, the typical
chair conformation of piperidine is hampered due to the double
bond and in amine d the piperidine ring is replaced by an open-
chain amine. In amines eeg, the 4-phenylpiperidine skeleton is
maintained and different sized groups are additionally substituted
at 4-position of the piperidine ring B. Amines hem are character-
ized by spatial and electronic changes in ring C. While in 4-
benzylpiperidine (h) the space between the aromatic and the
piperidine ring is increased, in amine i the benzene ring is directly
annulated to ring B. 4-(piperidin-4-yl)pyridine (m) and 1,40-
Fig. 1. Vesamicol and selected VAChT ligands.

52
C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
Fig. 2. Structurally modified amines aem: a: basic structure; bed: modifications in ring B; eeg: modifications between ring B and C; hem: modifications in ring C.
bipiperidine (j) contain a nitrogen atom at different positions in
ring C, whereas in the latter one ring C is nonaromatic, resulting
additionally in structural changes. With the p-fluorobenzoyl moi-
ety, amine k consists of a carbonyl functionality providing high
electron density.
To investigate the influence of these different modifications of
ring B and C, the group of vesamicols (1be1m) without modifica-
tions in ring A were synthesized (A in Scheme 1). The combination
of the ring B and C moieties with ring A modifications was achieved
by the introduction of (i) a p-fluorobenzyl ether functionality
(3ae3m and 4ae4m, B in Scheme 1) and (ii) the fusion of different
ring systems (benzovesamicols 9be9m and aminobenzovesamicols
11ae12h, C and D in Scheme 1).
The racemic vesamicol derivatives 1be1m were synthesized as
shown in Scheme 1(A) by reaction of cyclohexene oxide with the
corresponding amine. The synthesis of derivatives 1b, 1c, 1h, 1i, and
1k is already described in literature [16,22].
Regioselective nucleophilic ring opening reaction of the epoxide
precursor 2 using two different reaction conditions provided the
racemic 4-O- and 5-O- substituted p-fluorobenzyl ether derivatives
3ae3m and 4ae4m (B in Scheme 1). The principles of these re-
actions as well as the synthesis of the epoxide precursor 2 were
already described in detail by our group [27] and others [38,39].
Regioisomeric purity of all p-fluorobenzyl ether derivatives was
confirmed with HPLC. The 3-fluoropropyl ether derivatives 6a and
7a were synthesized in the same way as the p-fluorobenzyl ether
derivatives. Briefly, to get the epoxide precursor 5, we started with
the elimination of water from cyclohexane-1,4-diol to obtain
cyclohex-3-enol [40] which was epoxidized diasterioselectively by
using tert-butyl hydroperoxide and Mo(CO)6 [41]. Subsequent O-3-
fluoropropylation with 3-fluoropropyl tosylate resulted in the cis-
epoxide 5 which reacted regioselectively with 4-phenylpiperidine
(a) to the isomers 6a and 7a (reaction conditions as described in
B in Scheme 1). To obtain 3-fluoropropyl tosylate, 3-chloropropanol
was converted into 3-fluoropropanol [42] and subsequently tosy-
lated with p-toluenesulfonyl chloride under basic conditions [43].
For the synthesis of racemic benzovesamicols 9be9m, BV and
FBBV (C in Fig. 3) 1,4-dihydronaphthalene was epoxidized to get 8
Scheme 1. Synthesis of A) vesamicols 1be1m, B) 4-O- and 5-O-ether vesamicols 3ae3m, 4ae4m, 6a, and 7a, C) benzovesamicols 9b, 9c, 9e, 9h, 9j, and 9m, and D) amino-
benzovesamicols 11a, 11h, 12a, and 12h.

C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
53
Fig. 3. Molecular structures of synthesized vesamicol derivatives; * means these derivatives are already described by our group or others [16,22,27,37].

54
C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
as precursor for the reaction with the different amines under
conditions as reported earlier [16,37].
present in rVAChT-PC12 cells. This was confirmed by in-house ex-
periments demonstrating that the binding of (ꢀ)-[3H]vesamicol
The structurally related aminobenzovesamicols 11ae12h (D in
Scheme 1) were synthesized on the basis of epoxide 10 which was
obtained in three steps starting from naphthalene-1-amine. Briefly,
the latter was reduced under BIRCH conditions using sodium as
reducing agent in liquid ammonia and the resulting dihydro
product was converted into 10 after N-protection with p-fluo-
robenzoyl chloride and subsequent epoxidation using m-chlor-
operbenzoic acid. The reaction of the racemic epoxide 10 with
amines a and h, as described in D in Scheme 1, resulted in a mixture
of both regioisomers with a ratio of ~1:1 in each case. According to
the FÜRST-PLATTNER rule, there seems to be no preferred conformer of
10 resulting in a non-regioselective attack of the amine and the
equal formation of both products. The resulting pairs of
regioisomers were separated using semi-preparative HPLC (see
experimental section) and, afterwards, their regioisomeric purity
was confirmed by analytical HPLC.
Dehydrovesamicol (DeHVes, A in Fig. 3) was synthesized by
reaction of 4-phenylpiperidine with the monoepoxide of 1,4-
cyclohexadiene as already described [34]. The two regioisomers
of aminobenzovesamicol (5-/8-ABV, D in Fig. 3) were also synthe-
sized according to known synthetic procedures [16].
All synthesized new and reference compounds (shown in Fig. 3)
were well characterized using HRMS, 1H, 13C, APT, and two-
dimensional NMR. Additionally, 19F NMR spectra were recorded
for derivatives containing at least one fluorine atom. Molecular
structures are drawn as enantiomers to illustrate the trans-position
of tertiary amine and hydroxyl group and do not display the actual
stereochemistry. All compounds were yielded as racemic mixtures
and were used without chiral resolution for biological testing.
toward rVAChT-PC12 cells was not significantly affected by the s1/
s2 specific ligand 1,3-di-o-tolylguanidine (DTG) at 50, 100, and
200 nM. The corresponding binding curves obtained with DTG
were similar to those without DTG (see Fig. 4). Based on these re-
sults, we consistently used PC12 cells stably transfected with rat
VAChT and (ꢀ)-[3H]vesamicol as standard method for the deter-
mination of VAChT binding affinity of the new vesamicol de-
rivatives (data in Table 1).
The affinity of the new compounds for s1 and s2 receptors was
determined, in order to estimate their selectivity toward rat VAChT.
Using homogenates obtained from rat cortex and rat liver, respec-
tively, enables a comparison of binding data of the three targets
considered within the same species. (þ)-[3H]Pentazocine and [3H]
DTG were used as radioligands for s1 and s2, respectively [45]. To
block competitive binding of [3H]DTG on s1 receptor, the s2 re-
ceptor assays were performed using 1
dextrallorphan.
mM s1 receptor-specific
When comparing our VAChT affinity data for vesamicol,
DeHVes, BV, and 5-ABV with the data obtained by using vesicles of
the electric organ of Torpedo californica [34], considerable differ-
ences become apparent. In general, Ki values obtained with vesicles
of the torpedo fish are 20- to 50-fold lower than the herein reported
values obtained with rat VAChT-PC12 cells. A particularly strong
deviation shows 5-ABV with a Ki value of 0.013 nM reported for
Torpedo californica compared to 25.1 nM for rat VAChT in our assay.
Also when comparing data obtained with human VAChT trans-
fected PC12 cells slight differences can be observed e.g. for the
previously developed FBBV with a Ki of 2.7 nM for human VAChT
[37] compared to 24.4 nM for rat VAChT. Obviously, the differences
between rVAChT and hVAChT transfected cell lines are not as
pronounced as observed for vesicles of the torpedo fish. Altogether,
these findings show that an interpretation or comparison of in vitro
binding data obtained with different species material should be
avoided.
2.2. In vitro binding studies
With the aim to develop a standard protocol for the determi-
nation of VAChT affinities of the compounds, we investigated rat
brain homogenates and neuroendocrine PC12 cells stably trans-
fected with a cDNA of rat VAChT [44] (rVAChT-PC12) as different
biological targets and (ꢀ)-[3H]vesamicol as radioligand. Briefly, the
brain material was prepared by dissection and homogenization of
the brain of Sprague Dawley rats (without cerebellum) and stored
at ꢀ30 ꢁC in a sucrose solution. Crude membranes for the compe-
tition assay were obtained after thawing, dilution, removal of cell
debris by centrifugation, and homogenization of the pellets in assay
buffer. The stably transfected rVAChT-PC12 cells were grown in
Dulbecco's modified Eagle Medium (see exp. part), harvested, pel-
leted and washed, and stored at ꢀ30 ꢁC in assay buffer. For binding
assay cells were thawed, diluted with test buffer, and homogenized.
When comparing the affinity data of several vesamicol de-
rivatives with these two target systems, considerable differences of
Ki values became apparent. For example for ( )-benzovesamicol
(BV) Ki values of 1.3 nM with rVAChT-PC12 cells and 23.4 nM with
rat brain membranes were obtained. Also, for ( )-vesamicol a
higher binding affinity with rVAChT-PC12 cells (Ki ¼ 47.4 nM) was
observed compared to a Ki ¼ 84.2 nM with rat brain. Since the two
VAChT binding targets investigated are of rat origin, comparable
affinity data of the vesamicol derivatives should be expected. Dif-
ferences between brain and rVAChT-PC12 cell homogenates are
mainly (i) the much lower concentration of VAChT and (ii) the
presence of several other proteins in the brain material, which
becomes evident by a higher nonspecific binding of the (ꢀ)-[3H]
vesamicol of >20% in brain membranes compared to <5% using
rVAChT-PC12 cells. Furthermore, it has to be considered that
(ꢀ)-[3H]vesamicol is not selective due to its high affinity to sigma
receptors which are widely distributed in the brain but rarely
To estimate the biological significance of the selectivity of this
series of vesamicol analogs, the expression densities of VAChT, s1
,
and s2 receptors in brain have to be considered. Regarding VAChT,
Custers et al. reported on Bmax data in rat brain showing values
ranging from about 140 (striatum) to 70 fmol/mg tissue (hippo-
campus) by co-incubation of (ꢀ)-[3H]vesamicol and DTG to block
s
receptors [14]. Selecting the hippocampus as a region of relatively
low VAChT expression, the Bmax value of vesamicol binding sites is
comparable to the Bmax value of about 50 fmol/mg tissue for the s1
receptor sites in rats determined by Bouchard et al. [49]. Besides,
although the densities of s2 receptors are generally lower than that
of s1 receptors in rat brain, with a Bmax of about 20 fmol/mg tissue
(hippocampus) this potential binding site also has to be considered
[49]. Following the rule of thumb that the binding potential
(BP ¼ Bmax/KD) should be at least > 2 for a good PET radioligand
[50], the Ki value for VAChT binding should be lower than ~35 nM.
By comparison, already a Ki value of ~25 nM would be sufficient to
fulfill the minimal binding potential criteria for the s1 receptor sites
in the hippocampus. Moreover, to achieve a good signal to noise
ratio in the VAChT imaging process, the ratio of the BPs of the ligand
to the
s receptors versus VAChT should be at least 1/100. For the
hippocampus this would result in Ki values of >2500 nM and
>1000 nM for the s1 and s2 receptor sites, respectively. In other
words, selectivity factors (Ki(s)/Ki(VAChT)) which were calculated
for each vesamicol analog (Table 1) should be >70 for s1 and >30
for s2. Based on our in vitro data we have to assume that obviously
none of the investigated derivatives fullfils the requirements for a
PET radioligand specifically targeting the VAChT in brain. Best
selectivity factors regarding s1 are in the range of 10e40 and were

C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
55
Fig. 4. Concentration-dependent displacement of (ꢀ)-[3H]vesamicol by (ꢀ)-vesamicol on rat VAChT transfected PC12 cells; A) without DTG, B) with 50 nM DTG, and C) with 200 nM
DTG added as s1/s2 specific blocking agent.
estimated for the aminobenzovesamicol derivative 11a (41.2), BV
(19.7), (ꢀ)-FEOBV (10.7), and FBBV (8.8). Although rather low,
these values might be sufficient for imaging of the brain regions
with high VAChT densities and low s1 receptor expression [14,49],
such as the striatum. Actually, in vivo studies in rats with (ꢀ)-[18F]
FEOBV [17] and [18F]FBBV [37] showed the highest accumulation in
striatal regions followed by the cortex and the hippocampus. In
contrast to our in vitro findings, the pretreatment of (þ)-3-(3-
hydroxyphenyl)-N-propyl piperidine ((þ)-3-PPP) and haloperidol,
two more or less specific s1 receptor ligands, did not result in
significantly diminished tracer accumulation in these three brain
regions, which was interpreted by the authors as high specificity of
(ꢀ)-[18F]FEOBV. For the striatum, this may be plausible, however in
particular for brain regions with similar expression densities of
VAChT and s1 receptors as the hippocampus, the in vivo selectivity
of this most promising VAChT PET tracer should be re-investigated
by using highly affine and selective brain permeable sigma ligands.
a loss in VAChT affinity which is in accordance to 1j of the vesamicol
group. By contrast, the tetrahydroisoquinoline substituted deriva-
tive 3i exhibits similar low affinity for VAChT as 1i but ten-fold
higher affinities for both s1 and s2 receptor. This finding shows
the diversity in the shift of
s receptor affinities when considered as
function of VAChT affinities within the group of 4-O-ether
vesamicols.
When the p-fluorobenzyl ether functionality is inserted in 5-
position of the cyclohexanol ring A, the VAChT affinities decrease
considerably. However, for the open chain derivative 4d and the
diphenyl derivative 4g an increase of VAChT affinity compared to
the corresponding vesamicol derivatives 1d and 1g can be
observed. By contrast, there is no tendency for the sigma receptor
affinities; some derivatives show higher and other lower Ki values
compared to the corresponding vesamicols. In this group the 4-
benzylpiperine-substituted compound 4h, with a Ki value of
9.2 nM, shows a s1 receptor affinity, which is in the range of other
known s1 receptor ligands.
2.3. Structure-affinity considerations
2.3.1.3. Benzovesamicols. In the group of benzovesamicols, the af-
finities toward the transporter increase consistently in comparison
to the vesamicol derivatives 1ae1m. Obviously, ring B and C
modifications which led to a loss of affinity within the vesamicol
group, are well tolerated within the benzovesamicol group. With a
Ki of 1.32 nM, the already described ( )-benzovesamicol (BV) is the
compound with the highest affinity toward VAChT in our study.
Besides, although with a relatively high affinity to s1 receptor, a
selectivity factor of around 20 can be estimated.
The analog 9b, with the piperazine ring B, demonstrates lowest
affinity toward both sigma receptors within this group (Ki
values > 460 nM). This observation is comparable to findings of
Bando et al. [51] for the iodine derivative DRC140 also bearing the
piperazine moiety. The benzovesamicol derivative 9m, with the
substitution of a pyridine ring instead of the phenyl ring C, dem-
onstrates the highest affinity to the s1 receptor (Ki ¼ 8.9 nM).
Interestingly, when we compare the VAChT affinity of this deriva-
tive (Ki ¼ 19.3 nM) to the correspondent vesamicol (1m) and
benzylether analogs (3m, 4m) all exhibiting micromolar binding
affinities for VAChT, the outstanding position of benzovesamicols as
VAChT ligands becomes evident. However, it also demonstrates the
often observed parallel high affinity to both targets, VAChT and s1
receptor.
2.3.1. Alternating ring A, B and C moieties
2.3.1.1. Vesamicols. Considering the group of vesamicols (1ce1m)
which differ regarding their structures in ring B or C, we can
observe considerably decreased binding affinities to VAChT
compared to vesamicol as a reference. As the best derivative within
this group, 1c with a Ki value of 439 nM shows a ten-fold lower
affinity for VAChT than vesamicol. The use of the open chain amine
in 1d and the tetrahydroisoquinoline in 1i results in a complete loss
of VAChT affinity. Interestingly, the introduction of a nitrogen atom
in the phenyl moiety in 1m also reduces the VAChT affinity
drastically.
For most of the derivatives also a reduction of the s1 and s2
receptor affinities can be observed. Only for 1c a higher s1 receptor
affinity (Ki ¼ 9.7 nM) is found compared to vesamicol.
The introduction of a double bond at 4- and 5-positions in ring A
(DeHVes) leads to an increase of affinities for all three investigated
targets, resulting in a low selectivity.
2.3.1.2. 4-O- and 5-O-Ether vesamicols. The insertion of the p-flu-
orobenzyl ether functionality in the 4-position of the cyclohexanol
ring A (derivatives 3ae3m) results in an increase of VAChT affinity
compared to the corresponding vesamicol derivatives 1be1m. 3a,
with a Ki value of 25.1 nM, shows a two-fold higher affinity than
vesamicol and is comparable to the known derivatives FBBV and 5-
ABV. However, the s1 receptor affinity is in the same range as for
vesamicol resulting in low selectivity [27]. Besides 3a, the 4-
benzylpiperidine derivative 3h, with a Ki value of 110 nM, shows
the best affinity within this group. Interestingly, the open chain
compound 3d binds with a very high affinity to the s1 receptor
(Ki ¼ 10.5 nM). The lowest s1 affinity is found for 3j accompanied by
2.3.1.4. Aminobenzovesamicols. When extending the rigid benzyl
ring with a primary amine function in 5-position of BV (5-ABV), the
affinities for both VAChT (Ki ¼ 25.1 nM) and
s
receptors (Ki ¼ 119
and 474 nM, resp.) slightly decrease compared to BV. Substitution
in 8-position (8-ABV) results in a loss of affinity for VAChT
(Ki ¼ 339 nM), however, accompanied by an increase in affinity
toward s1 receptor (Ki ¼ 55.3 nM). Rogers et al. also observed the

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C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
Table 1
Binding affinity data of vesamicol analogs and reference compounds.
Compound
Selectivity factor
Ki (VAChT)a
b
c
Ki (s1
)
Ki (s2
)
Ki (s1)/Ki (VAChT)
Ki (s2)/Ki (VAChT)
A) Vesamicols
(
(
(
(
(
(
(
(
(
(
(
(
(
)-vesamicol
)-1b [16]
)-1c [16]
)-1d
)-1e
)-1f
47.4 6.3 (1.0 0.50)d
838 98
439 30
>10 000
1000 108
1460 311
6708 904
647 62
>10 000
20.4 4.7
83.9 57.1
9.7 5.6
112 36
695 135
158 64
0.4
0.1
<0.1
1.6
0.8
0.4
115
6
652 171
1994 548
164 58
1829 731
108 55
809 145
4710 2880
730 210 (112 8)g
3317 1340
77.1 5.9
454 301
22.5 21.4
43.2 25.7
20.1 21.3
54.3 16.3
>1000
0.4
<0.1
<0.1
<0.1
/
1.9
0.1
0.3
0.2
/
)-1g
)-1h [16]
)-1i [16]
)-1j
)-1k [22]
)-1m
5106 516
/
/
3239 469 (423 2)d
3380 859
60.9 44.9 (302 25)f
>1000
5.8 0.9
<0.1
/
0.2
/
)-DeHVes
18.4 2.3 (0.34 0.04)d
0.3
4.2
B) 4-O- and 5-O-Ether vesamicols
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
)-3a
)-3b
)-3c
)-3d
)-3e
)-3f
)-3g
)-3h
)-3i
25.1 4.4
697 127
310 23
1605 198
316 29
260 15
603 104
110 22
4672 149
4789 210
1026 156
1351 11
2387 370
6194 752
2825 43
1931 192
>10 000
16.6 2.7
103 24
53.1 7.8
167 46
46.9 6.7
109 13
0.7
0.2
<0.1
/
2.1
0.24
0.2
<0.1
0.4
<0.1
3.9
0.6
<0.1
0.1
0.6
<0.1
<0.1
0.2
<0.1
0.1
/
29.9 1.8
10.5 0.01
47.3 9.6
63.1 3.8
456 22
134
2
0.2
0.2
0.8
0.2
<0.1
0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
/
18.8 1.9
2363 735
61.8 0.2
45.9 0.3
543 113
640 30
85.8 28.6
67.1 15.6
1041 510
55.5 1.3
280 21
25.2 15.2
59.0 11.1
)-3j
553
5
)-3k
)-3m
)-4a
)-4b
)-4c
)-4d
)-4e
)-4f
59.4 9.7
26.4 0.9
16.8 4.3
189 25
15.9 1.1
19.7 1.7
31.0 29.2
254 47
>10 000
120
736
5
1
103
5537 2316
207
0
/
/
)-4g
)-4h
)-4i
2733 288
1298 170
>10 000
>10 000
5843 1392
>10 000
0.3
<0.1
/
2.0
0.2
/
9.2 0.09
23.1 5.1
300 105
65.4 14.5
41.1 3.6
25.9 6.0
97.6 0.8
6
45.6 2.4
374 31
1996 381
164 51
88.2 20.9
717 81
)-4j
/
/
)-4k
)-4m
)-6a
)-7a
<0.1
/
0.5
1.6
0.3
/
1.8
12.0
47.8 9.6
59.6 5.5
C) Benzovesamicols
(
(
(
(
(
(
(
(
)-BV [16]
)-9b
)-9c [16]
)-9e
)-9h
)-9j
1.32 0.25 (0.055 0.01)d
56.0 18.2
7.3 1.3
11.5 2.5
23.2 6.7
26.1 4.5
466 67
96.4 22.3
1275 261
166 60
549 73
60 22
19.7
8.3
3.3
5.8
2.8
2.2
8.8
0.5
10.7
73.0
22.7
22.7
47.7
2.6
24
2
67 12
65 29
248 11
112.3 9.3
161
2
1.4
)-FBBV [37]
)-9m
24.4 2.8 (2.70 0.40)e
19.3 0.9
215 132 (191 57)f
8.9 0.4
284 6 (251 39)g
258 57
11.6
13.4
n.c.
(ꢀ)-FEOBV [17,46]
19.6 1.1
209 94
n.d.
D) Aminobenzovesamicols
(
(
(
(
(
(
)-5-ABV [16]
)-8-ABV [16]
)-11a
)-11h
)-12a
25.1 0.5 (0.013 0.001)d
339 17.6
156 26
527 62
188 29
119
6
474 57
4.7
0.2
41.2
3.4
3.3
/
18.9
2.1
23.2
3.9
2.2
/
55.3 27.1
6431 1851
1773 51
626 396
296 202
726 388.6
3623 901
2079 442
1381 724
2112 41
)-12h
>10 000
Ki values in nM (mean S.D., n ꢂ 2) were derived from IC50 values according to the Cheng-Prusoff equation: Ki ¼ IC50/(1 þ C/KD), where C is the concentration of the radioligand
and KD the dissociation constant of the corresponding radioligand. Italics were used to better differenciate between own and reported data.
a
(ꢀ)-[3H]vesamicol: KD ¼ 25.6 nM to VAChT on PC12 cells transfected with rat VAChT cDNA.
b
[3H]pentazocine: KD ¼ 6.9 nM to s1 receptors on rat cortex homogenates.
c
[3H]DTG with blocking of s1 receptors using dextrallorphan: KD ¼ 29.0 nM to s2 receptors on rat liver homogenates.
d
Reported Ki values obtained with (ꢀ)-[3H]vesamicol and vesicles from the electric organ of Torpedo californica [22,34,47].
e
Reported Ki values obtained with (ꢀ)-[3H]vesamicol and PC12 cells stably expressing human VAChT [37,48].
f
Reported Ki values obtained with (þ)-[3H]pentazocine and guinea pig brain membrane homogenates [22,37,48].
g
Reported Ki values obtained with [3H]DTG and (þ)-pentazocine and rat liver homogenates [22,37,47,48].
higher potency of 5-ABV to block the [3H]ACh transport compared
to 8-ABV, however in a less pronounced manner (IC50(5-ABV) ¼ 100
versus IC50(8-ABV) ¼ 300 nM) [16].
The N-substitution of 5-ABV and 8-ABV with the p-fluo-
robenzoyl group results in a high diversity in affinity data. It rea-
ches from moderate VAChT affinity (Ki ¼ 156 nM) and relative high

C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
57
selectivity over
affinity for 12h with a Ki for VAChT over 10
s
receptors (>23) for 11a until complete loss of
M. As general tendency
selectivity profile due to lower affinities toward
s
receptors. So, it
m
can be stated that alkyl ether chains are able to improve the
selectivity by reducing the affinity to the receptors, which is in
for the aminobenzovesamicols, we conclude that the substitution
of this bulky amide functionality in particular in combination with
variations at ring C is obviously not tolerated by the VAChT binding
site.
Regarding the s1 and s2 receptor affinities also a decrease of
binding is observed compared to the corresponding vesamicol
derivative 1h, which is more pronounced than the shift observed
for VAChT binding.
s
contrast to the corresponding p-fluorobenzyl derivative 4a. More-
over, for this derivative the most intense loss of affinity for VAChT
compared to vesamicol was observed, obviously caused by the
substitution of a sterically demanding benzyl ether group in 5-
position of the cyclohexyl ring A.
In summary, a substitution or ring fusion at the 4-position of
ring A of the vesamicol skeleton lead to higher binding affinities
toward VAChT but depends in a complex manner of the steric de-
mand of the substituents. This finding is in accordance with the
results of our CoMFA study based on ligands taken from the liter-
ature [52]. We could demonstrate herein that an increase of steric
bulk in the region of cyclohexyl ring A diagonally opposite to the
hydroxyl group favors binding to VAChT.
2.3.2. Alternating ring A and fixed ring B and C moieties
To investigate the influence of changes in ring A on the affinities
to VAChT, s1, and s2 receptors independently from other modifi-
cations, derivatives of identical ring B and C were compared.
Therefore, the affinities of all derivatives bearing amine a (4-
phenylpiperidine) were considered in relation to the values of
vesamicol to obtain a relative affinity value defined as Ki(target;
vesamicol)/Ki(target; compound). To simplify matters, we visual-
ized our findings in a bar graph (see Fig. 5), which shows the
relative affinity values in logarithmic scale, whereas vesamicol was
set as x-coordinate with a log(rel. aff.) ¼ 0. Thus, all bars above this
coordinate represent a higher affinity than vesamicol, whereas bars
in the negative range indicate decreased binding affinities toward
the correspondent target.
Regarding
s receptor binding, the bar graph also illustrates that
the desired decrease of s1 and s2 receptor affinities is often
accompanied by a decrease in VAChT affinity. In the same way
structural modifications effecting an increase in VAChT affinity (3a
and DeHVes) led to an increase of affinity to at least one of the
sigma receptors. By contrast, a more or less pronounced decrease of
s
receptor binding of BV and 5-ABV was observed with increasing
VAChT affinity. To explain this behavior, we investigated the 3-
dimensional structures of several derivatives and compared them
with known pharmacophore models published in particular for li-
gands of the s1 receptor [53,54]. According to the first and most
common model reported by Glennon et al. [53], a potent s1 re-
ceptor ligand should comprise an amino group flanked by two
hydrophobic substituents with a distance ranging between 6 and
10 Å for the “Primary” and 2.5e3.9 Å for the “Secondary Hydro-
phobic Binding site”. Generally, vesamicol analogues with the
central piperidine ring exhibit this arrangement and are therefore
potential ligands for this target. In particular, the derivative DeHVes
(Ki( ¼ 5.8 nM) with distances of 3.6 for the “Primary” and 7.1 Å for
Four of the ten examined compounds (3a, DeHVes, BV, and 5-
ABV) show higher VAChT affinity than vesamicol. All these de-
rivatives are characterized by a substitution or ring fusion in 4-
position of ring A of the vesamicol skeleton. Only two of them,
BV and 5-ABV, exhibit a favorable selectivity profile compared to
vesamicol with more or less decreased affinities for both
s re-
ceptors. Interestingly, considering compound 11a, the substitution
of a fluorobenzylamide function at 5-position of the benzovesa-
micol structure resulted in a 6-fold decrease of VAChT affinity
compared to the corresponding 5-ABV.
The 3-fluoropropyl ether vesamicols 6a and 7a show compara-
ble VAChT affinities as vesamicol itself, whereas 7a with the sub-
stituent in 5-position of the cyclohexyl ring A exhibits a better
s1)
“Secondary Hydrophobic Site” corresponds to ligands of this
pharmacophore model. Also BV shows distances being within the
range of the model which is reflected by its binding affinity toward
Fig. 5. Relative affinity of all derivatives bearing 4-phenylpiperidine (amine a) in relation to vesamicol (set as x-coordinate); green: VAChT, red: s1, blue: s2. (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this article.)

58
C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
the s1 receptor (Ki( ¼ 26.1 nM). Interestingly, despite the similar
Ultra 10.0. Three-dimensional structures were generated by
Chem3D Pro12.0 and energy-minimized on MM2 level.
Racemic vesamicol was purchased as hydrochloride from BIO-
TREND Chemikalien GmbH (Germany). Amines b, c, and eem were
purchased from several providers. N-Ethyl-3-phenylpropan-1-
amine (d) was synthesized according to known procedures [55].
Vesamicol hydrochloride was purchased by BIOTREND Chemikalien
GmbH, Germany and cyclohexene oxide by SigmaeAldrich,
Germany.
s
1)
structural behavior, the corresponding 5-ABV shows significantly
decreased binding to this receptor (Ki( ¼ 119 nM), which could be
s
1)
caused by the polar ionizable amino group at the hydrophobic re-
gion. This could also explain the low
s receptor binding of the other
aminobenzovesamicol derivatives 11a, 12a, and 8-ABV. Probably, a
substitution of ionic or polar functionalities at the annelated benzyl
ring of benzovesamicols could be an alternative to achieve
improved selectivity of vesamicol based ligands.
3. Conclusion
4.1.2. Syntheses and analytical data
4.1.2.1. General procedure for vesamicol derivatives ( )-1be( )-1m.
Cyclohexene oxide (10 mmol) and the amine (10.5 mmol) were
dissolved in 5e10 mL EtOH and stirred at 65e70 ꢁC for 48 h. The
crude product was purified by column chromatography (silica gel
60) using mixtures of EtOAc/n-hexane/NH3. All derivatives were
obtained as colorless solids with yields of 50e70%.
To our knowledge, for the first time a SAR study was performed
which considered the VAChT affinity and selectivity of derivatives
belonging to four different structural classes of vesamicol. Based on
the in vitro binding data obtained with this series of vesamicol
analogs systematically modified on the three ring systems (A, B,
and C) we can conclude: (i) modifications in ring B and C do not
improve the VAChT binding affinity, (ii) modifications in ring A, in
particular via ring fusion or insertion of less bulky functionalities in
4-position, may increase VAChT affinity, (iii) the influence of the
ring A, B, and C modifications on the affinity to s1 and s2 receptors
is of high diversity without obvious tendency, and (iv) in most
cases, an increase of VAChT affinity is accompanied by an increase
in s1 and/or s2 receptor affinity. On the other hand, high sigma
receptor affinities do not implicate necessarily high VAChT affin-
ities, indicating that the three target affinities are not completely
dependent on each other.
Finally, we have to assume that obviously none of the investi-
gated derivatives fulfill the requirements for a PET radiotracer
specifically targeting the VAChT in the brain. Moreover, this
structure-affinity-selectivity relationship study illustrates the
challenge in the development of VAChT imaging ligands based on
vesamicol as lead structure. This is mainly caused by the vesamicol
skeletal structure which corresponds to structural requirements of
ligands for s1 receptors.
( )-1b
(there are no detailed NMR data in the reference): 1H NMR
(300 MHz, CDCl3) in ppm: 7.27 (m, 2H), 6.94 (m, 2H), 6.88 (m, 1H),
( )-2-(4-phenyl-piperazin-1-yl)-cyclohexanol
[16]
d
3.94 (s, 1H, CHeOH), 3.41 (m, 1H, CHeOH), 3.26e3.13 (om, 4H, CH2
piperazine), 2.93 (m, 2H, CH2 piperazine), 2.62 (m, 2H, CH2 piper-
azine), 2.32 (m, 1H, CHeN), 2.18 (m, 1H, CH2 cyclohexane),
1.87e1.71 (om, 3H, CH2 cyclohexane), 1.30e1.16 (om, 4H, CH2
cyclohexane); 13C NMR (75.4 MHz, CDCl3)
d in ppm: 151.3 (s),
129.0119.8 (s), 116.1 (s), 70.2 (s, CHeN), 68.5 (s, CHeOH), 49.8 and
48.2 (s, CH2 piperazine), 33.1, 25.4, 24.0, and 22.2 (s, CH2 cyclo-
hexane); HRMS (ESI): m/z ¼ 261.19605 (M þ Hþ) calc. 261.19614; m/
z ¼ 283.17792 (M þ Naþ) calc. 283.17808.
( )-1c
( )-2-(4-phenyl-5,6-dihydropyridin-1(2H)-yl)cyclo-
hexanol [16] (there are no detailed NMR data in the reference): 1H
NMR (400 MHz, CDCl3) in ppm: 7.40e7.34 (om, 2H), 7.32 (m, 2H),
d
7.24 (m, 1H), 6.10 (bm, 1H), 3.51e3.41 (om, 2H, CHeOH, CH2eN),
3.22 (m, 1H, CH2eN), 3.00 (m, 1H, CH2eN), 2.62e2.55 (om, 3H,
2 ꢃ CH2CPh, CH2eN), 2.36 (m, 1H, CHeN), 2.16 (m, 1H, CH2 cyclo-
hexane), 1.87e1.81 (om, 2H, CH2 cyclohexane), 1.74 (m, 1H, CH2
cyclohexane), 1.35e1.18 (om, 4H, CH2 cyclohexane); 13C NMR
4. Materials and methods
(100 MHz, CDCl3) d in ppm: 140.8 (s), 135.2 (s), 128.4 (s), 127.1 (s),
124.9 (s), 122.4 (s), 69.6 (s, CHeN), 68.9 (s, CHeOH), 48.8 and 45.1
(s, CH2eN), 33.4 (s, CH2 cyclohexane), 28.9 (s, CH2CPh), 25.7, 24.2,
and 22.1 (s, CH2 cyclohexane); HRMS (ESI): m/z ¼ 258.18530
(M þ Hþ) calc. 258.18524; m/z ¼ 537.34520 (2M þ Naþ) calc.
537.34515.
4.1. Chemical part
4.1.1. Instruments and materials
Analysis of all final compounds was performed by HR-MS, HPLC,
TLC, and NMR spectroscopy.
( )-1d ( )-2-(ethyl(3-phenylpropyl)amino)cyclohexanol: 1H
High-resolution mass spectra were recorded on a FT-ICR APEX II
(Bruker Daltonics) using ElectroSpray Ionization (ESI). NMR spectra
(1H, 13C, 13C-APT, 19F, H,H-COSY, HSQC, HMBC) were recorded on
spectrometers from Varian. Splitting patterns have been designated
as follows: s: singlet, d: doublet, bs: broad singlet, bd: broad
doublet, bm: broad multiplet, om: overlapping multiplet, m:
multiplet, t: triplet, td: triplet of doublet; dt: doublet of triplet.
Analytical thin-layer chromatography (TLC) was performed on
silica gel coated plates (Machery-Nagel, ALUGRAM SIL G/UV254).
The spots were identified using a UV lamp or by dipping into a
solution of 2% ninhydrine in EtOH/MeOH 1:1.
NMR (300 MHz, CDCl3) d in ppm: 7.28 (m, 2H), 7.20e7.16 (om,
3H), 4.01 (bs, 1H, OH), 3.33 (m, 1H, CHeOH), 2.68e2.54 (om, 4H,
3 ꢃ CH2eN, CH2Ph), 2.45e2.28 (om, 3H, CH2eN, CH2Ph, CHeN),
2.15 (m, 1H, CH2 cyclohexane), 1.85e1.71 (om, 5H, 2 ꢃ CH2CH2Ph,
3 ꢃ CH2 cyclohexane), 1.27e1.12 (om, 4H, CH2 cyclohexane), 1.04
3
(t, JHH ¼ 7.0 Hz, 3H, CH3); 13C NMR (75.4 MHz, CDCl3)
d in ppm:
142.3 (s), 128.4 (s), 125.8 (s), 69.2 (s, CHeN), 66.7 (s, CHeOH),
49.3 (s, CH2Ph), 43.8 and 33.8 (s, CH2eN), 33.3 (s, CH2 cyclo-
hexane), 31.0 (s, CH2CH2Ph), 25.8, 24.3, and 22.8 (s, CH2 cyclo-
hexane), 14.8 (s, CH3); HRMS (ESI): m/z ¼ 262.21652 (M þ Hþ)
calc. 262.21654.
The chemical purity of all compounds is >97% and was
controlled by HPLC using a 250 ꢃ 4.6 mm Reprosil-Pur Basic HD e
( )-1e ( )-1-(2-hydroxy-cyclohexyl)-4-phenyl-piperidin-4-ol:
1H NMR (400 MHz, CDCl3)
d in ppm: 7.51 (m, 2H), 7.36 (m, 2H), 7.25
5
m
m (Dr. Maisch GmbH, Germany) or a LiChrospher 100 CN e 5
mm
(m, 1H), 4.08 (bs, 1H, CHeOH), 3.40 (m, 1H, CHeOH), 3.09 (m, 1H,
CH2 pip), 2.70 (m, 1H, CH2 pip), 2.62 (m, 1H, CH2 pip), 2.54 (m, 1H,
CH2 pip), 2.26 (m, 1H, CHeN), 2.19e2.16 (om, 2H, CH2 pip), 2.02 (m,
1H, CH2 cyclohexane), 1.92e1.68 (om, 6H, 3 ꢃ CH2 cyclohexane,
2 ꢃ CH2 pip, OH), 1.30e1.20 (om, 4H, CH2 cyclohexane); 13C NMR
column (Merck KGaA, Germany). These analytical chromatographic
separations were performed on a JASCO LC-2000 system, incor-
porating a PU-2080Plus pump, AS-2055Plus auto injector (100 mL
sample loop), and a MD-2010 dioden-array-detector (monitoring
from 195 to 600 nm). Mixtures of ACN and aqueous 20 mM NH4OAc
were used as eluents.
(100 MHz, CDCl3)
d in ppm: 148.4 (s), 128.5 (s), 127.2 (s), 124.6 (s),
71.7 (s, PhCOH), 70.7 (s, CHeN), 68.8 (s, CHeOH), 48.6 and 41.2 (s,
Chemical names of compounds were generated by ChemDraw
CH2 pip), 39.4 and 39.1 (s, CH2 pip), 33.4, 25.7, 24.2, and 22.5 (s, CH2

C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
59
cyclohexane); HRMS (ESI): m/z ¼ 276.19575 (M þ Hþ) calc.
data): 1H NMR (400 MHz, CDCl3)
d
in ppm: 7.95 (m, 2H), 7.13 (m,
276.1958; m/z ¼ 298.17770 (M þ Naþ) calc. 298.17775.
2H), 3.94 (bs, OH), 3.38 (m, 1H, CHeOH), 3.21 (bm, 1H, CH-COPhF),
2.95 (m, 1H, CH2 pip), 2.76e2.70 (om, 2H, CH2 pip), 2.30e2.21 (om,
2H, CH2 pip, CHeN), 2.12 (m, 1H, CH2 cyclohexane), 1.88e1.70 (om,
7H, 4 ꢃ CH2 pip, 3 ꢃ CH2 cyclohexane), 1.30e1.16 (om, 4H, CH2
( )-1f ( )-1-[1-(2-hydroxy-cyclohexyl)-4-phenyl-piperidin-4-
yl]-ethanone: 1H NMR (300 MHz, CDCl3)
d in ppm: 7.36e7.21
(om, 5H), 3.95 (s, OH), 3.33 (m, 1H, CHeOH), 2.83e2.69 (om, 2H,
CH2 pip), 2.58e2.46 (om, 3H, CH2 pip, 2.30 (m, 1H, CHeN),
2.18e1.93 (om, 4H, 3 ꢃ CH2 pip, 1 ꢃ CH2 cyclohexane), 1.88 (s, 3H,
CH3), 1.73e1.66 (om, 3H, CH2 cyclohexane), 1.24e1.05 (om, 4H, CH2
cyclohexane); 13C NMR (100 MHz, CDCl3)
d in ppm: 201.0 (s, C]O),
1
4
165.7 (d, JCF ¼ 245.7 Hz), 132.5 (d, JCF ¼ 3.0 Hz), 130.9 (d,
2
3JCF ¼ 9.2 Hz), 115.8 (d, JCF ¼ 21.8 Hz), 70.8 (s, CHeN), 68.7 (s,
cyclohexane). 13C NMR (75.4 MHz, CDCl3)
d
in ppm: 209.5 (s, C]O),
CHeOH), 51.9 (s, CH2 pip), 45.1 (s, CH-COPhF), 43.9 (s, CH2 pip), 33.3
141.8 (s), 128.9 (s), 127.2 (s), 126.4 (s), 70.5 (s, CHeN), 68.7 (s,
CHeOH), 55.1 (s, PhCC ¼ O), 48.4 and 43.9 (s, CH2 pip), 33.8 (s, CH2
cyclohexane), 33.5 and 33.3 (s, CH2 pip), 25.7 (s, CH3), 25.6, 24.1, and
22.4 (s, CH2 cyclohexane); HRMS (ESI): m/z ¼ 302.21131 (M þ Hþ)
calc. 302.21146.
(s, CH2 cyclohexane), 29.6 and 29.2 (s, CH2 pip), 25.6, 24.2, and 22.4
(s, CH2 cyclohexane); 19F NMR (376.3 MHz, CDCl3)
d in ppm: e 105.8
(bs); HRMS (ESI): m/z ¼ 306.18626 (M þ Hþ) calc. 306.18638; m/
z ¼ 633.34774 (2M þ Naþ) calc. 633.34744.
( )-1m ( )-2-(4-(pyridin-4-yl)piperidin-1-yl)cyclohexanol: 1H
( )-1g ( )-2-(4,4-diphenylpiperidin-1-yl)cyclohexanol: 1H
NMR (400 MHz, CDCl3) d in ppm: 8.50 (m, 2H), 7.12 (m, 2H), 3.99
NMR (400 MHz, CDCl3)
d
in ppm: 7.27 (bm, 8H), 7.13 (m, 2H), 4.11
(bs, OH), 3.39 (m, 1H, CHeOH), 2.95 (m, 1H, CH2 pip), 2.79e2.70
(om, 2H, CH2 pip, CHeN), 2.48 (m, 1H, CH pip), 2.24 (m, 2H, CH2
pip), 2.12 (m, 1H, CH2 cyclohexane), 1.90e1.59 (om, 7H, 4 ꢃ CH2 pip,
3 ꢃ CH2 cyclohexane), 1.31e1.16 (om, 4H, CH2 cyclohexane); 13C
(bs, OH), 3.35 (m, 1H, CHeOH), 2.82e2.76 (om, 2H, 2 ꢃ CH2 pip),
2.47e2.44 (om, 6H, 6 ꢃ CH2 pip), 2.20e2.10 (om, 2H, 1 ꢃ CHeN,
1 ꢃ CH2 cyclohexane), 1.70e1.60 (om, 3H, 3 ꢃ CH2 cyclohexane),
1.26e1.12 (om, 3H, 3 ꢃ CH2 cyclohexane), 1.06e1.00 (m, 1H, 1 ꢃ CH2
NMR (100 MHz, CDCl3)
d in ppm: 154.9 (s), 150.0 (s), 122.4 (s), 70.9
cyclohexane). 13C NMR (100.58 MHz, CDCl3)
d
in ppm: 147.7 (s),
(s, CHeN), 68.7 (s, CHeOH), 53.1 (s, CH2 pip), 45.3 (s, CH pip), 42.3
(s, CH2 pip), 33.5 (s, CH2 cyclohexane), 33.4 and 33.1 (s, CH2 pip),
25.6, 24.2, and 22.4 (s, CH2 cyclohexane); HRMS (ESI): m/
z ¼ 261.19610 (M þ Hþ) calc. 261.19618; m/z ¼ 543.36722
(2M þ Naþ) calc. 543.36695.
128.4 (s), 127.2 (s), 125.8 (s), 70.4 (s, CHeN), 68.7 (s, CHeOH), 45.7
(bs, CH2 pip), 45.0 (s, CH2 pip), 37.1 (s, CH2 pip), 33.4, 25.6, 24.2, and
22.4 (s, CH2 cyclohexane); HRMS (ESI): m/z ¼ 336.23199 (M þ Hþ)
calc. 336.23219.
( )-1h ( )-2-(4-benzyl-piperidin-1-yl)-cyclohexanol [16] (there
are no detailed NMR data in the reference): 1H NMR (300 MHz,
4.1.2.2. General procedure for 4-O- and 5-O-p-fluorobenzyl ether
vesamicol derivatives ( )-3a-( )-3m and ( )-4a-( )-4m. The cis-
epoxide precursor ( )-2 was synthesized as already described [27].
In brief, elimination of water from cyclohexane-1,4-diol gave
cyclohex-3-enol [40] which was epoxidized diasterioselectively by
using tert-butyl hydroperoxide and Mo(CO)6 [41] to obtain the
racemic epoxy alcohol. Subsequent O-p-fluorobenzylation resulted
in the cis-epoxide ( )-2.
To synthesize the 4-O-p-fluorobenzyl ether vesamicol de-
rivatives ( )-3ae( )-3m, the amine (1.41 mmol) and ( )-2
(1.12 mmol) were dissolved in 5 mL EtOH and stirred at 65e70 ꢁC
for 5 days. The crude product was purified by column chromatog-
raphy (silica gel 60) or by crystallization (details described for each
compound).
To synthesize the 5-O-p-fluorobenzyl ether vesamicol de-
rivatives ( )-4ae( )-4m, the cis-epoxide ( )-2 (1.12 mmol) and
LiClO4 (1.97 mmol) were dissolved in 3 mL CH3CN and stirred for
10 min. Subsequently, the amine (1.41 mmol) was added and the
reaction mixture was stirred at rt for 24 h. Products were purified
by column chromatography (silica gel 60) or by crystallization
(details described for each compound). The regioisomeric purity
was checked by HPLC.
CDCl3) d in ppm: 7.27 (m, 2H), 7.20e7.12 (om, 3H), 4.08 (s, OH), 3.33
(m, 1H, CHeOH), 2.79 (m, 1H, CH2 pip), 2.64e2.48 (om, 4H, 2 ꢃ CH2
pip, CH2Ph), 2.20e1.99 (om, 3H, CHeN, CH2 pip, CH2 cyclohexane),
1.76e1.62 (om, 5H, 2 ꢃ CH2 pip, 3 ꢃ CH2 cyclohexane), 1.51 (m, 1H,
CHeCH2Ph), 1.37e1.09 (om, 6H, 2 ꢃ CH2 pip, 4 ꢃ CH2 cyclohexane).
13C NMR (75.4 MHz, CDCl3)
d in ppm: 140.7 (s), 129.0 (s), 128.1 (s),
125.7 (s), 70.5 (s, CHeN), 68.8 (s, CHeOH), 52.7 (s, CH2Ph), 45.1 and
43.2 (s, CH2 pip), 38.3 (s, CH pip), 33.2 and 33.0 (s, CH2 pip), 32.8
25.6, 24.0, and 22.1 (s, CH2 cyclohexane); HRMS (ESI): m/
z ¼ 274.21640 (M þ Hþ) calc. 274.21654; m/z ¼ 296.19843
(M þ Naþ) calc. 296.19849.
( )-1i
( )-2-(3,4-dihydro-1H-isoquinolin-2-yl)-cyclohexanol
[16] (there are no detailed NMR data in the reference): 1H NMR
(300 MHz, CDCl3) in ppm: 7.12 (bm, 3H), 7.02 (m, 1H), 3.97 (bs,
d
2
OH), 3.93 and 3.66 (AB, JHH ¼ 15 Hz, 2H, CH2 pip), 3.49 (m, 1H,
CHeOH), 3.04 (m, 1H, CH2 pip), 2.90 (m, 2H, CH2 pip), 2.62 (m, 1H,
CH2 pip), 2.41 (m, 1H, CHeN), 2.16 (bm, 1H, CH2 cyclohexane),
1.91e1.75 (om, 3H, CH2 cyclohexane), 1.28 (bm, 4H, CH2 cyclo-
hexane). 13C NMR (75.4 MHz, CDCl3)
d in ppm: 135.4 (s), 134.7 (s),
128.9 (s), 126.6 (s), 126.2 (s), 125.7 (s), 70.1 (s, CHeN), 68.9 (s,
CHeOH), 51.7 (s, CH2 pip), 46.0 (s, CH2 pip), 33.4 (s, CH2 cyclo-
hexane), 30.2 (s, CH2 pip), 25.6, 24.2, and 22.1 (s, CH2 cyclohexane);
HRMS (ESI): m/z ¼ 232.16949 (M þ Hþ) calc. 232.16959; m/
z ¼ 485.31355 (2M þ Naþ) calc. 485.31385.
( )-3a, ( )-3b, and ( )-4a are already described by our group
[27,28].
( )-3c ( )-4-(4-fluorobenzyloxy)-2-(4-phenyl-5,6-dihydropyri-
din-1(2H)-yl)cyclohexanol: The product crystallized in the reac-
tion mixture, was isolated and recrystallized from EtOH at 4 ꢁC to
give 149 mg (35%) of colorless crystals. 1H NMR (300 MHz, CDCl3)
( )-1j ( )-2-[1,40]bipiperidinyl-10-yl-cyclohexanol: 1H NMR
(300 MHz, CDCl3)
d in ppm: 3.99 (s, 1H, OH), 3.30 (m, 1H, CHeOH),
2.84 (m, 1H, CH2 pip), 2.63 (m, 1H, CHeN), 2.69e2.46 (om, 5H, CH2
pip), 2.18e2.05 (om, 4H, CHeN, 2 ꢃ CH2 pip, 1 ꢃ CH2 cyclohexane),
1.83e1.33 (om, 13H, 10 ꢃ CH2 pip, 3 ꢃ CH2 cyclohexane), 1.25e1.09
d
in ppm: 7.40e7.30 (om, 6H), 7.24 (m, 1H), 7.04 (m, 2H), 6.09 (bm,
2
1H), 4.51 and 4.43 (AB, JHH ¼ 11.9 Hz, 2H, OCH2Ph), 3.85 (m, 1H,
CHeOCH2Ph), 3.54 (m, 1H, CHeOH), 3.40 (m, 1H, CH2eN), 3.20 (m,
1H, CH2eN), 3.00e2.84 (om, 2H, CH2eN, CHeN), 2.61e2.56 (om,
3H, CH2eN, 2 ꢃ CH2CPh), 2.13e1.95 (om, 3H, CH2 cyclohexane), 1.76
(om, 4H, 4 ꢃ CH2 cyclohexane); 13C NMR (75.4 MHz, CDCl3)
d in
ppm: 70.2 (s, CHeN), 68.7 (s, CHeOH), 62.8 (s, CHeN), 52.5 (s, CH2
pip), 50.2 (s, 2 ꢃ CH2 pip), 44.4 (s, CH2 pip), 33.2 (s, CH2 cyclo-
hexane), 28.9 and 28.6 (s, CH2 pip), 26.4 (s, 2 ꢃ CH2 pip), 25.5 and
24.7 (s, CH2 cyclohexane), 24.0 (s, CH2 pip), 22.1 (s, CH2 cyclo-
hexane); HRMS (ESI): m/z ¼ 267.24316 (M þ Hþ) calc. 267.24309;
m/z ¼ 289.22501 (M þ Naþ) calc. 289.22503.
(m, 1H, CH2 cyclohexane), 1.45e1.30 (om, 2H, CH2 cyclohexane); 13
C
NMR (75.4 MHz, CDCl3)
d
in ppm: 162.3 (d, 1JCF ¼ 245.3 Hz), 140.7
(s), 135.2 (s), 134.8 (d, 4JCF ¼ 3.1 Hz),129.1 (d, 3JCF ¼ 8.0 Hz),128.4 (s),
2
127.1 (s), 124.9 (s), 122.2 (s), 115.2 (d, JCF ¼ 21.4 Hz), 73.5 (s,
( )-1k ( )-(4-fluoro-phenyl)-[1-(2-hydroxy-cyclohexyl)-piper-
idin-4-yl]-methanone [22] (reference provides exclusively 1H NMR
COCH2Ph), 69.3 (s, OCH2Ph), 68.8 (s, CHeOH), 63.6 (s, CHeN), 48.6
and 44.9 (s, CH2eN), 28.8 (s, CH2CPh), 27.8, 27.7, and 26.1 (s, CH2

60
C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
cyclohexane); 19F NMR (282.3 MHz, CDCl3)
d
in ppm: e 115.6 (m);
collected fractions to a smaller volume and keeping a few days at 4 ꢁC
resulted in the formation of 216 mg (42%) of a colorless solid.1H NMR
HRMS (ESI): m/z ¼ 382.21765 (M þ Hþ) calc. 382.21768; m/
z ¼ 785.41022 (2M þ Naþ) cal. 785.41004.
(400 MHz, CDCl3) d inppm: 7.27e7.22 (om,10H), 7.12 (m, 2H), 6.98(m,
( )-3d ( )-2-(ethyl(3-phenylpropyl)amino)-4-(4-fluorobenzyloxy)
cyclohexanol: For this derivative 1.5 mmol of cis-epoxide ( )-4 was
used and the reaction was performed at 85 ꢁC for 8 days. The crude
product was purified by column chromatography starting with
EtOAc/n-hexane/NH3 (1/2/0.01) and ending with EtOAc/NH3 (1/0.01).
120 mg (28%) of ( )-5d were obtained as nearly colorless oil. 1H NMR
2H), 4.39 and 4.32 (AB, 2JHH ¼ 11.6 Hz, 2H, OCH2Ph), 4.04 (bs, OH), 3.71
(bm, 1H, CHeOCH2Ph), 3.40 (m, 1H, CHeOH), 2.75e2.64 (om, 3H,
2 ꢃ CH2 pip, CHeN), 2.49e2.41 (om, 6H, 6 ꢃ CH2 pip),1.94e1.82 (om,
3H, 3 ꢃ CH2 cyclohexane),1.66 (m,1H, CH2 cyclohexane),1.29 (m,1H,
CH2 cyclohexane),1.11 (m, 1H, CH2 cyclohexane); 13C NMR (100 MHz,
1
CDCl3)
d
in ppm: 162.3 (d, JCF ¼ 244.0 Hz),147.6 (bs), 134.8 (d,
(300 MHz, CDCl3)
d
in ppm: 7.30e7.24 (om, 4H), 7.19 (m, 3H), 7.01 (m,
4JCF ¼ 3.0 Hz),129.1 (d, 3JCF ¼ 7.4 Hz),128.5 (s),127.3 (s),125.9 (s),115.3
2
2
2H), 4.46 and 4.40 (AB, JHH ¼ 11.9 Hz, 2H, OCH2Ph), 3.79 (m, 1H,
CHeOCH2Ph), 3.38 (m, 1H, CHeOH), 2.86 (m, 1H, CHeN), 2.73e2.51
(om, 4H, 3 ꢃ CH2eN, CH2Ph), 2.37 (bm, 2H, CH2eN, CH2Ph), 2.04e1.40
(om, 6H, 2 ꢃ CH2CH2Ph, 4 ꢃ CH2 cyclohexane), 1.40e1.20 (om, 2H, CH2
cyclohexane),1.06(t, 3JHH ¼ 7.0 Hz, 3H, CH3); 13C NMR (75.4 MHz, CDCl3)
(d, JCF ¼ 22.1 Hz), 73.5 (s, COCH2Ph), 69.3 (s, OCH2Ph), 68.5 (s,
CHeOH), 64.4 (s, CHeN), 45.5 (bs, C-(Ph)2), 45.0 (s, 2 ꢃ CH2 pip), 37.0
(s, 2 ꢃ CH2 pip), 27.7, 27.6, and 26.3 (s, CH2 cyclohexane); 19F NMR
(376.3 MHz, CDCl3)
d in ppm: e 115.7 (bs); HRMS (ESI): m/
z ¼ 460.26428 (M þ Hþ) calc. 460.26463.
d
inppm: 162.3 (d,1JCF ¼ 245.3 Hz),142.1 (s),134.8 (d, 4JCF ¼ 3.1Hz),129.5
( )-3h ( )-2-(4-benzylpiperidin-1-yl)-4-(4-fluorobenzyloxy)
cyclohexanol: The crude product was purified by column chroma-
tography using EtOAc/n-hexane/NH3 (1/1/0.01). By dissolving of the
obtained oil in MTBE and keeping at 4 ꢁC for 24 h, 151 mg (34%) of a
colorless crystalline solid could be isolated. 1H NMR (400 MHz,
(d, 3JCF ¼ 8.1 Hz), 128.5 (s), 128.4 (s), 125.9 (s), 115.3 (d, 2JCF ¼ 21.4 Hz),
73.5 (s, COCH2Ph), 69.2 (s, OCH2Ph), 69.0 (s, CHeOH), 60.8 (s, CHeN),
49.2(s,CH2Ph), 43.8 and 33.7 (s, CH2eN), 30.7 (bs, CH2CH2Ph), 27.8, 27.7,
and 26.9 (s, CH2 cyclohexane),14.6 (s, CH3); 19F NMR (282.3 MHz, CDCl3)
d
in ppm: e 115.7 (bs); HRMS (ESI): m/z ¼ 386.24892 (M þ Hþ) calc.
CDCl3) d in ppm: 7.31e7.27 (om, 4H), 7.19 (m, 1H), 7.14 (m, 2H), 7.01
386.24898.
(m, 2H), 4.45 and 4.40 (AB, 2JHH ¼ 11.6 Hz, 2H, OCH2Ph), 4.04 (bs,1H,
OH), 3.79 (m, 1H, CHeOCH2Ph), 3.42 (m, 1H, CHeOH), 2.82e2.47
(om, 7H, CHeN, 4 ꢃ CH2 pip, CH2Ph), 2.01e1.92 (om, 4H, 2 ꢃ CH2
pip, 2 ꢃ CH2 cyclohexane), 1.73e1.63 (om, 3H, 1 ꢃ CH2 pip, 2 ꢃ CH2
cyclohexane), 1.51 (m, 1H, CHeCH2Ph), 1.38e1.20 (om, 3H, 1 ꢃ CH2
( )-3e ( )-1-(5-(4-fluorobenzyloxy)-2-hydroxycyclohexyl)-4-
phenylpiperidin-4-ol: The crude product was purified by column
chromatography using CHCl3/MeOH/NH3 (10/1/0.1). 239 mg (49%)
of colorless hydrochloride of ( )-3e were obtained by adding
ethanolic 10% HCl to an ethanolic solution of ( )-3e. 1H NMR
pip, 2 ꢃ CH2 cyclohexane); 13C NMR (100.0 MHz, CDCl3)
d in ppm:
(300 MHz, CDCl3)
d
in ppm: 7.52 (m, 2H), 7.39e7.27 (om, 5H), 7.03
162.2 (d, 1JCF ¼ 244.0 Hz),140.7 (s), 134.7 (d, 4JCF ¼ 3.0 Hz), 129.2 (s),
129.1 (d, 3JCF ¼ 8.0 Hz), 128.3 (s), 125.9 (s), 115.3 (d, 2JCF ¼ 21.0 Hz),
73.4 (s, COCH2Ph), 69.3 (s, OCH2Ph), 68.6 (s, CHeOH), 64.6 (s,
CHeN), 52.7 (s, CH2Ph), 45.2 (s, CH pip), 43.3, 38.4, 33.1, and 32.7 (s,
CH2 pip), 27.8, 27.7, and 26.2 (s, CH2 cyclohexane); 19F NMR
2
(m, 2H), 4.48 and 4.43 (AB, JHH ¼ 12.0 Hz, 2H, OCH2Ph), 4.05 (bs,
1H, CHeOH), 3.83 (m, 1H, CHeOCH2Ph), 3.49 (m, 1H, CHeOH), 3.06
(m, 1H, CH2 pip), 2.84e2.55 (om, 4H, 3 ꢃ CH2 pip, CHeN), 2.23e1.91
(om, 5H, 2 ꢃ CH2 pip, 3 ꢃ CH2 cyclohexane), 1.81 (s, 1H, OH),
1.77e1.64 (m, 3H, 2 ꢃ CH2 pip, 1 ꢃ CH2 cyclohexane), 1.42e1.29
(376.3 MHz, CDCl3)
d in ppm: e 115.6 (bs); HRMS (ESI): m/
(om, 2H, CH2 cyclohexane); 13C NMR (75.4 MHz, CDCl3)
d
in ppm:
z ¼ 398.24867 (M þ Hþ) calc. 398.24898.
162.6 (d, 1JCF ¼ 245.2 Hz), 148.3 (s), 134.8 (d, 4JCF ¼ 3.3 Hz), 129.1 (d,
3JCF ¼ 7.7 Hz), 128.5 (s), 127.2 (s), 124.6 (s), 115.3 (d, 2JCF ¼ 21.4 Hz),
73.4 (s, COCH2Ph), 71.6 (s, OCH2Ph), 69.2 (s, PhCOH), 68.6 (s,
CHeOH), 64.7 (s, CHeN), 48.4 and 40.9 (s, CH2 pip), 39.2 and 38.9 (s,
CH2 pip), 27.8, 27.7, and 26.4 (s, CH2 cyclohexane); 19F NMR
( )-3i ( )-2-(3,4-dihydroisoquinolin-2(1H)-yl)-4-(4-fluoroben-
zyloxy)cyclohexanol: The crude product was purified by column
chromatography using EtOAc/n-hexane/NH3 (1.5/1/0.01). 126 mg
(28%) of colorless hydrochloride of ( )-3i were obtained by adding of
ethanolic 10% HCl to an ethanolic solution of ( )-3i. 1H NMR
(282.3 MHz, CDCl3)
d
in ppm: e 115.6 (m); HRMS (ESI): m/
(300 MHz, CDCl3) d in ppm: 7.34 (m, 2H), 7.16e7.01 (om, 6H), 4.53
2
z ¼ 400.22797 (M þ Hþ) calc. 400.22825; m/z ¼ 422.21050
and 4.45 (AB, JHH ¼ 12 Hz, 2H, OCH2Ph), 3.91e3.86 (om, 3H, OH,
CHeOCH2Ph, CH2 pip), 3.66e3.53 (om, 2H, CHeOH, CH2 pip),
3.03e2.88 (om, 4H, CHeN, CH2 pip, 2 ꢃ CH2 pip), 2.60 (m, 1H, CH2
pip), 2.17e1.96 (om, 3H, CH2 cyclohexane), 1.78 (m, 1H, CH2 cyclo-
(M þ Naþ) cal. 422.21019.
( )-3f ( )-1-(1-(5-(4-fluorobenzyloxy)-2-hydroxycyclohexyl)-
4-phenylpiperidin-4-yl)ethanone: The crude product was purified
by column chromatography using EtOAc/n-hexane/NH3 (1/1/0.01).
159 mg (33%) of colorless hydrochloride of ( )-3f were obtained by
adding of ethanolic 10% HCl to an ethanolic solution of ( )-3f. 1H
hexane), 1.46e1.33 (om, 2H, CH2 cyclohexane). 13C NMR (75.4 MHz,
1
CDCl3)
d
in ppm: 162.3 (d, JCF ¼ 243.0 Hz),135.1 (s), 134.8 (d,
4JCF ¼ 3.0 Hz),134.6 (s), 129.1 (d, JCF ¼ 10.0 Hz),128.8 (s), 126.6 (s),
126.2 (s),125.7 (s),115.3 (d, 2JCF ¼ 21.0 Hz), 73.5 (s, COCH2Ph), 69.4 (s,
OCH2Ph), 66.7 (s, CHeOH) 64.1 (s, CHeN), 51.5, 45.6, and 30.1(s, CH2
pip), 27.7, 27.6, and 26.2 (s, CH2 cyclohexane); 19F NMR (282.3 MHz,
3
NMR (300 MHz, CDCl3) d in ppm: 7.38e7.28 (om, 7H), 7.01 (m, 2H),
2
4.45 and 4.39 (AB, JHH ¼ 11.9 Hz, 2H, OCH2Ph), 3.92 (bs, 1H, OH),
3.78 (m, 1H, CHeOCH2Ph), 3.43 (m, 1H, CHeOH), 2.82e2.65 (om,
3H, 2 ꢃ CH2 pip, CHeN), 2.58e2.47 (om, 3H, CH2 pip), 2.30 (m, 1H,
CH2 pip), 2.13e1.90 (om, 8H, 2 ꢃ CH2 pip, 3 ꢃ CH2 cyclohexane,
CH3), 1.68 (m, 1H, CH2 cyclohexane), 1.34 (m, 1H, CH2 cyclohexane),
CDCl3)
d
in ppm: e 115.5 (m); HRMS (ESI): m/z ¼ 356.20190
(M þ Hþ) calc. 356.20203; m/z ¼ 733.37859 (2M þ Naþ) calc.
733.37874.
1.21 (m, 1H, CH2 cyclohexane); 13C NMR (75.4 MHz, CDCl3)
d
in
( )-3j ( )-2-(1,40-bipiperidin-10-yl)-4-(4-fluorobenzyloxy)cyclo-
hexanol: The crude product was purified by column chromatography
using CHCl3/MeOH/NH3 (10/1/0.1). The solvent of collected fractions
was removed and the residual oil was dissolved in MTBE. Keeping
the MTBE solution a few days at 4 ꢁC, resulted in the formation of
ppm: 209.6 (s, C]O), 162.4 (d, 1JCF ¼ 244.7 Hz), 141.7 (s), 134.8 (d,
4JCF ¼ 3.3 Hz), 129.1 (d, JCF ¼ 8.2 Hz), 129.0 (s), 127.3 (s), 126.4 (s),
3
115.2 (d, 2JCF ¼ 21.4 Hz), 73.3 (s, COCH2Ph), 69.3 (s, OCH2Ph), 68.5 (s,
CHeOH), 64.5 (s, CHeN), 55.1 (s, PhCC ¼ O), 48.2 and 43.8 (s, CH2
pip), 33,6 and 33.4 (s, CH2 pip), 27.8, 27.7, and 26.2 (s, CH2 cyclo-
97 mg (22%) of colorless needles. 1H NMR (300 MHz, CDCl3)
d in
hexane), 25.8 (s, CH3); 19F NMR (282.3 MHz, CDCl3)
d
in ppm: e
ppm: 7.27 (m, 2H), 7.00 (m, 2H), 4.45 and 4.39 (AB, 2JHH ¼ 11.9 Hz, 2H,
OCH2Ph), 3.92 (bs, OH), 3.78 (bm, 1H, CHeOCH2Ph), 3.38 (m, 1H,
CHeOH), 2.85 (m, 1H, CH2 pip), 2.69e2.65 (om, 2H, CH2 pip, CHeN),
2.55e2.47 (om, 5H, CH2 pip), 2.19 (m,1H, CHeN), 2.05e1.79 (om, 6H,
3 ꢃ CH2 pip, 3 ꢃ CH2 cyclohexane), 1.72e1.17 (om, 11H, 8 ꢃ CH2 pip,
115.6 (m); HRMS (ESI): m/z ¼ 426.24351 (M þ Hþ) calc. 426.24390;
m/z ¼ 448.22606 (M þ Naþ) cal. 448.22584.
( )-3g ( )-2-(4,4-diphenylpiperidin-1-yl)-4-(4-fluorobenzyloxy)
cyclohexanol: The crude product was purified by column chroma-
tography using CHCl3/MeOH/NH3 (10/1/0.1). Concentration of the
3 ꢃ CH2 cyclohexane); 13C NMR (75.4 MHz, CDCl3)
d in ppm: 162.2 (d,

C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
61
1JCF ¼ 245.2 Hz), 134.8 (d, 4JCF ¼ 3.3 Hz), 129.0 (d, 3JCF ¼ 8.0 Hz), 115.2
( )-4c
( )-5-(4-fluorobenzyloxy)-2-(4-phenyl-5,6-
2
(d, JCF ¼ 21.4 Hz), 73.5 (s, COCH2Ph), 69.2 (s, OCH2Ph), 68.6 (s,
dihydropyridin-1(2H)-yl)cyclohexanol: The compound crystal-
lized in the reaction mixture. The white precipitate was isolated
and washed with water, isopropanol, and n-hexane to obtain
CHeOH), 64.2 (s, CHeN), 63.0 (s, CHeN), 52.5 (s, CH2 pip), 50.3 (s,
2 ꢃ CH2 pip), 44.3 (s, CH2 pip), 28.9 and 28.6 (s, CH2 pip), 27.6 (s,
2 ꢃ CH2 cyclohexane), 26.4 (s, 2 ꢃ CH2 pip), 26.1 (s, CH2 cyclohexane),
24.9 (s, CH2 pip); HRMS (ESI): m/z ¼ 391.27540 (M þ Hþ) calc.
391.27553.
255 mg (59%) of ( )-4c. 1H NMR (400 MHz, CDCl3)
d in ppm:
7.44e7.33 (om, 6H), 7.28 (m, 1H), 7.06 (m, 2H), 6.12 (bm, 1H), 4.59
2
and 4.54 (AB, JHH ¼ 11.8 Hz, 2H, OCH2Ph), 3.55 (m, 1H, CHeOH),
( )-3k ( )-(1-(5-(4-fluorobenzyloxy)-2-hydroxycyclohexyl)pip-
eridin-4-yl) (4-fluorophenyl)methanone: The crude product was
purified bycolumn chromatography using EtOAc/n-hexane/NH3 (1/1/
0.01). Removal of the solvent led to 197 mg (41%) of a slightly yellow
3.48e3.39 (om, 2H, CHeOCH2Ph, CH2eN), 3.25 (m, 1H, CH2eN),
2.99 (m, 1H, CH2eN), 2.67e2.59 (om, 4H, CH2eN, 2 ꢃ CH2CPh, CH2
cyclohexane), 2.44 (m, 1H, CHeN), 2.22 (m, 1H, CH2 cyclohexane),
1.91 (m, 1H, CH2 cyclohexane), 1.45e1.22 (om, 3H, CH2 cyclo-
oil.1H NMR (400 MHz, CDCl3)
d
inppm: 7.95 (m, 2H), 7.29 (m, 2H), 7.11
hexane); 13C NMR (100 MHz, CDCl3)
d in ppm: 162.4 (d,
(m, 2H), 7.02 (m, 2H), 4.48 and 4.40 (AB, 2JHH ¼ 11.6 Hz, 2H, OCH2Ph),
3.82 (bm, 1H, CHeOCH2Ph), 3.44 (m, 1H, CHeOH), 3.21 (bm, 1H, CH-
COPhF), 2.95(m,1H, CH2 pip), 2.79e2.64 (om, 3H, 2ꢃ CH2 pip,CHeN),
2.23 (bm,1H, CH2 pip), 2.08 (m,1H, CH2 cyclohexane), 2.01e1.64 (om,
7H, 4 ꢃ CH2 pip, 3 ꢃ CH2 cyclohexane), 1.39e1.26 (om, 2H, CH2
1JCF ¼ 245.3 Hz), 140.7 (s), 135.2 (s), 134.5 (d, 4JCF ¼ 3.0 Hz), 129.4 (d,
3JCF ¼ 8.1 Hz), 128.4 (s), 127.2 (s), 124.9 (s), 122.1 (s), 115.3 (d,
2JCF ¼ 21.4 Hz), 75.0 (s, COCH2Ph), 69.7 (s, OCH2Ph), 68.8 (s, CHeN),
66.5 (s, CHeOH), 48.8 and 45.2 (s, CH2eN), 38.9 (s, CH2 cyclo-
hexane), 31.4 (s, CH2CPh), 28.8 and 18.4 (s, CH2 cyclohexane); 19F
cyclohexane); 13C NMR (100 MHz, CDCl3)
d
in ppm: 201.0 (s, C]O),
NMR (376.3 MHz, CDCl3) d in ppm: e 115.4 (m); HRMS (ESI): m/
1
1
165.7 (d, JCF ¼ 253.0 Hz),162.8 (d, JCF ¼ 244.0 Hz), 134.8 (d,
4JCF ¼ 3.0 Hz), 132.5 (d, 4JCF ¼ 3.0 Hz), 130.9 (d, 3JCF ¼ 9.0 Hz), 129.1 (d,
3JCF ¼ 8.0 Hz),115.9 (d, 2JCF ¼ 21.0 Hz),115.3 (d, 2JCF ¼ 21.0 Hz), 73.4 (s,
COCH2Ph), 69.3 (s, OCH2Ph), 68.5 (s, CHeOH), 64.8 (s, CHeN), 51.7 (s,
CH2 pip), 45.0 (s, CH-COPhF), 43.8 (s, CH2 pip), 29.5 and 29.2 (s, CH2
pip), 27.7, 27.5, and 26.4 (s, CH2 cyclohexane); 19F NMR (376.3 MHz,
z ¼ 382.21778 (M þ Hþ) calc. 382.21768.
( )-4d ( )-2-(ethyl(3-phenylpropyl)amino)-5-(4-fluorobenzy-
loxy)cyclohexanol: The crude product was purified by column
chromatography using EtOAc/n-hexane/NH3 (1/1/0.01). 215 mg
(41%) of ( )-4d were obtained as nearly colorless oil. 1H NMR
(300 MHz, CDCl3) d in ppm: 7.33e7.26 (om, 4H), 7.21e7.18 (om, 3H),
2
CDCl3)
d
in ppm: e 115.6 (bs), ꢀ105.8 (bs); HRMS (ESI): m/
7.03 (m, 2H), 4.50 and 4.48 (AB, JHH ¼ 11.8 Hz, 2H, OCH2Ph),
3.39e3.29 (om, 2H, CHeOCH2Ph, CHeOH), 2.73e2.52 (om, 5H,
3 ꢃ CH2eN, CH2Ph, CH2 cyclohexane), 2.46e2.32 (om, 3H, CHeN,
CH2Ph, CH2eN), 2.13 (m, 1H, CH2 cyclohexane), 1.86e1.74 (om, 3H,
2 ꢃ CH2CH2Ph, 1 ꢃ CH2 cyclohexane), 1.41e1.12 (om, 3H, CH2
z ¼ 430.21847 (M þ Hþ) calc. 430.21883; m/z ¼ 452.20075 (M þ Naþ)
cal. 452.20077; m/z ¼ 881.41217 (2M þ Hþ) calc. 881.41232.
( )-3m
( )-4-(4-fluorobenzyloxy)-2-(4-(pyridin-4-yl)piper-
idin-1-yl)cyclohexanol: The crude product was purified by column
chromatography using CHCl3/MeOH/NH3 (10/1/0.1). Adding of n-
hexane to a concentrated CHCl3 solution of ( )-3m and keeping a
few days at 4 ꢁC resulted in the formation of 220 mg (59%) of a
cyclohexane), 1.05 (t, JHH ¼ 7.1 Hz, 3H, CH3); 13C NMR (75.4 MHz,
3
1
CDCl3)
d
in ppm: 162.4 (d, JCF ¼ 245.0 Hz), 142.0 (s), 134.5 (d,
4JCF ¼ 3.2 Hz), 129.4 (d, JCF ¼ 7.8 Hz), 128.5 (s), 128.4 (s), 125.9 (s),
115.3 (d, 2JCF ¼ 21.5 Hz), 76.7 (s, COCH2Ph), 69.6 (s, OCH2Ph), 66.7 (s,
CHeOH), 66.1 (s, CHeN), 49.3 (s, CH2Ph), 43.9 (s, CH2eN), 38.7 (s,
CH2 cyclohexane), 33.7 (s, CH2eN), 31.6 (s, CH2 cyclohexane), 30.8
(bs, CH2CH2Ph), 19.3 (s, CH2 cyclohexane), 14.6 (s, CH3); 19F NMR
3
colorless solid. 1H NMR (400 MHz, CDCl3)
d in ppm: 8.50 (m, 2H),
7.30 (m, 2H), 7.12 (m, 2H), 7.01 (m, 2H), 4.48 and 4.41 (AB,
2JHH ¼ 11.9 Hz, 2H, OCH2Ph), 3.82 (bm, 1H, CHeOCH2Ph), 3.44 (m,
1H, CHeOH), 2.95 (m, 1H, CH2 pip), 2.79e2.73 (om, 2H, CH2 pip),
2.67 (m, 1H, CHeN), 2.47 (m, 1H, CH pip), 2.20 (m, 1H, CH2 pip), 2.09
(m, 1H, CH2 cyclohexane), 2.01e1.64 (om, 7H, 4 ꢃ CH2 pip, 3 ꢃ CH2
(282.3 MHz, CDCl3)
d in ppm: e 115.4 (m); HRMS (ESI): m/
z ¼ 386.24899 (M þ Hþ) calc. 386.24898.
cyclohexane), 1.40e1.25 (om, 2H, 2 ꢃ CH2 cyclohexane); 13C NMR
( )-4e ( )-1-(4-(4-fluorobenzyloxy)-2-hydroxycyclohexyl)-4-
phenylpiperidin-4-ol: The compound crystallized in the reaction
mixture. The white precipitate was isolated and washed with water,
isopropanol, and MTBE to obtain 292 mg (73%) of ( )-4e. 1H NMR
1
(100.0 MHz, CDCl3)
d
in ppm: 162.2 (d, JCF ¼ 245.2 Hz),154.8 (s),
4
3
149.9 (s), 134.7 (d, JCF ¼ 3.2 Hz), 129.0 (d, JCF ¼ 8.1 Hz), 122.3 (s),
115.2 (d, 2JCF ¼ 21.3 Hz), 73.4 (s, COCH2Ph), 69.3 (s, OCH2Ph), 68.6 (s,
CHeOH), 64.7 (s, CHeN), 52.9 (s, CH2 pip), 45.2 (s, CH pip), 42.8,
33.4, and 33.1 (s, CH2 pip), 27.7, 27.5, and 26.4 (s, CH2 cyclohexane);
(300 MHz, CDCl3)
d in ppm: 7.51 (m, 2H), 7.39e7.27 (om, 5H), 7.03
(m, 2H), 4.54 and 4.49 (AB, 2JHH ¼ 11.8 Hz, 2H, OCH2Ph), 3.89 (bs,1H,
CHeOH), 3.47e3.36 (om, 2H, CHeOH, CHeOCH2Ph), 3.11 (m, 1H,
CH2 pip), 2.66e2.52 (om, 4H, 3 ꢃ CH2 pip, 1 ꢃ CH2 cyclohexane),
2.32 (m, 1H, CHeN), 2.22e2.14 (om, 2H, CH2 pip), 2.08e1.98 (m, 1H,
CH2 cyclohexane), 1.91 (m, 1H, CH2 cyclohexane), 1.83e1.76 (om,
2H, CH2 pip), 1.66 (bs, 1H, OH), 1.42e1.15 (om, 3H, CH2 cyclo-
19F NMR (376.3 MHz, CDCl3)
d in ppm: e 115.6 (m); HRMS (ESI): m/
z ¼ 385.22834 (M þ Hþ) calc. 385.22858; m/z ¼ 791.43224
(2M þ Hþ) calc. 791.43183.
( )-4b ( )-5-(4-fluorobenzyloxy)-2-(4-phenylpiperazin-1-yl)
cyclohexanol: The compound crystallized in the reaction mixture.
The white precipitate was isolated and washed with water, iso-
propanol, and n-hexane to obtain 215 mg (49%) of ( )-4b. 1H NMR
hexane); 13C NMR (75.4 MHz, CDCl3)
d in ppm: 162.4 (d,
1JCF ¼ 245.2 Hz), 148.2 (s), 134.5 (d, JCF ¼ 3.3 Hz), 129.4 (d,
3JCF ¼ 8.2 Hz), 128.5 (s), 127.3 (s), 124.6 (s), 115.3 (d, 2JCF ¼ 21.8 Hz),
75.0 (s, COCH2Ph), 71.6 (s, OCH2Ph), 70.0 (s, PhC-OH), 69.6 (s,
CHeN), 66.4 (s, CHeOH), 48.8 and 41.0 (s, CH2 pip), 39.2 (s, CH2
cyclohexane), 38.9 and 38.8 (s, CH2 pip), 31.5 (s, CH2 cyclohexane),
4
(400 MHz, CDCl3)
d in ppm: 7.33e7.42 (om, 4H), 7.02 (m, 2H),
6.93e6.85 (om, 3H), 4.55 and 4.54 (AB, 2JHH ¼ 11.8 Hz, 2H, OCH2Ph),
3.83 (bs, 1H, CHeOH), 3.48e3.33 (om, 2H, CHeOH, CHeOCH2Ph),
3.26e3.15 (om, 4H, CH2 piperazine), 2.90e2.85 (om, 2H, CH2
piperazine), 2.63e2.54 (om, 3H, 2 ꢃ CH2 piperazine, 1 ꢃ CH2
cyclohexane), 2.35 (m, 1H, CHeN), 2.17 (m, 1H, CH2 cyclohexane),
1.87 (m, 1H, CH2 cyclohexane), 1.43e1.15 (om, 3H, CH2 cyclo-
18.9 (s, CH2 cyclohexane); 19F NMR (282.3 MHz, CDCl3)
d in ppm: e
115.4 (m); HRMS (ESI): m/z ¼ 400.22822 (M þ Hþ) calc. 400.22825;
m/z ¼ 422.21046 (M þ Naþ) cal. 422.21019.
hexane); 13C NMR (100 MHz, CDCl3)
d
in ppm: 162.4 (d,
( )-4f ( )-1-(1-(4-(4-fluorobenzyloxy)-2-hydroxycyclohexyl)-
4-phenylpiperidin-4-yl)ethanone: The crude product was purified
by column chromatography using EtOAc/n-hexane/NH3 (1/1/0.01).
By dissolving of the obtained oil in MTBE and cooling to 4 ꢁC for
24 h, a colorless precipitate could be obtained. 176 mg (37%) of
( )-4f were isolated as colorless solid. 1H NMR (300 MHz, CDCl3)
1JCF ¼ 244.0 Hz), 151.3 (s), 134.4 (d, JCF ¼ 3.0 Hz), 129.4 (d,
3JCF ¼ 8.1 Hz), 129.2 (s), 120.1 (s), 116.3 (s), 115.3 (d, 2JCF ¼ 21.2 Hz),
74.9 (s, COCH2Ph), 69.7 (s, OCH2Ph), 69.6 (s, CHeN), 66.3 (s,
CHeOH), 49.8 and 48.5 (s, CH2 piperazine), 38.8, 31.3, and 18.8 (s,
4
CH2 cyclohexane); 19F NMR (376.3 MHz, CDCl3)
115.3 (m).
d in ppm: e
d
in ppm: 7.37e7.25 (om, 7H), 7.01 (m, 2H), 4.52 and 4.47 (AB,

62
C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
2JHH ¼ 11.8 Hz, 2H, OCH2Ph), 3.84 (bs, 1H, OH), 3.42e3.28 (om, 2H,
CHeOH, CHeOCH2Ph), 2.79e2.70 (om, 2H, CH2 pip), 2.62e2.47
(om, 4H, 3 ꢃ CH2 pip,1 ꢃ CH2 cyclohexane), 2.38e2.22 (om, 2H, CH2
pip, CHeN), 2.14e1.94 (om, 3H, 2 ꢃ CH2 pip, 1 ꢃ CH2 cyclohexane),
1.89 (s, 3H, CH3), 1.76 (m, 1H, CH2 cyclohexane), 1.40e1.03 (om, 3H,
CH2 pip), 2.90 (m, 2H, CH2 pip), 2.68e2.57 (om, 2H, CH2 pip, CH2
cyclohexane), 2.48 (m, 1H, CHeN), 2.19 (m, 1H, CH2 cyclohexane),
1.91 (m, 1H, CH2 cyclohexane), 1.46e1.22 (om, 3H, CH2 cyclohexane).
13C NMR (100 MHz, CDCl3)
d
in ppm: 162.4 (d, 1JCF ¼ 244.0 Hz),134.9
(s), 134.5 (s), 134.4 (d, 4JCF ¼ 3.0 Hz), 129.4 (d, 3JCF ¼ 8.0 Hz),128.9 (s),
2
CH2 cyclohexane); 13C NMR (75.4 MHz, CDCl3)
d
in ppm: 209.6 (s,
126.6 (s), 126.3 (s), 125.8 (s), 115.3 (d, JCF ¼ 17.0 Hz), 75.0 (s,
4
C]O), 162.4 (d, 1JCF ¼ 245.4 Hz), 141.6 (s), 134.5 (d, JCF ¼ 3.3 Hz),
COCH2Ph), 69.7 (s, OCH2Ph), 69.4 (s, CHeN), 66.6 (s, CHeOH), 51.7
3
129.4 (d, JCF ¼ 8.2 Hz), 129.0 (s), 127.3 (s), 126.3 (s), 115.3 (d,
and 46.2 (s, CH2 pip), 38.9 and 31.4 (s, CH2 cyclohexane), 30.0 (s, CH2
2JCF ¼ 21.2 Hz), 74.9 (s, COCH2Ph), 69.8 (s, OCH2Ph), 69.6 (s, CHeN),
66.3 (s, CHeOH), 55.1 (s, PhCC ¼ O), 48.6 and 44.1 (s, CH2 pip), 38.9
(s, CH2 cyclohexane), 33,6 and 33.4 (s, CH2 pip), 31.3 (s, CH2
cyclohexane), 25.7 (s, CH3), 18.8 (s, CH2 cyclohexane); 19F NMR
pip), 18.5 (s, CH2 cyclohexane); 19F NMR (376.3 MHz, CDCl3)
d in
ppm: e 115.4 (m); HRMS (ESI): m/z ¼ 356.20183 (M þ Hþ) calc.
356.20203.
( )-4j ( )-2-(1,40-bipiperidin-10-yl)-5-(4-fluorobenzyloxy)cyclo-
hexanol: The compound crystallized in the reaction mixture. The
white precipitate was isolated and washed with water, isopropanol,
and MTBE to obtain 153 mg (35%) of ( )-4j. 1H NMR (400 MHz,
(282.3 MHz, CDCl3)
d in ppm: e 115.4 (m); HRMS (ESI): m/
z ¼ 426.24388 (M þ Hþ) calc. 426.24390; m/z ¼ 448.22580
(M þ Naþ) cal. 448.22584.
( )-4g ( )-2-(4,4-diphenylpiperidin-1-yl)-5-(4-fluorobenzyloxy)
cyclohexanol: The crude product was purified by column chroma-
tography using CHCl3/MeOH/NH3 (10/1/0.1). Concentration of the
collected fractions to a smaller volume and keeping a few days at 4 ꢁC
resulted in the formation of 335 mg (65%) of a colorless solid.1H NMR
CDCl3) d in ppm: 7.29 (m, 2H), 7.01 (m, 2H), 4.52 and 4.47 (AB,
2JHH ¼ 11.8 Hz, 2H, OCH2Ph), 3.82 (bs, OH), 3.37e3.30 (om, 2H,
CHeOCH2Ph, CHeOH), 2.8 (m, 1H, CH2 pip), 2.72 (m, 1H, CH2
cyclohexane), 2.61e2.48 (om, 6H, 6 ꢃ CH2 pip), 2.26e2.19 (om, 2H,
2 ꢃ CHeN), 2.13e2.03 (om, 2H, 1 ꢃ CH2 pip, 1 ꢃ CH2 cyclohexane),
1.87e1.76 (om, 3H, 2 ꢃ CH2 pip, 1 ꢃ CH2 cyclohexane), 1.62e1.56
(om, 5H, 5 ꢃ CH2 pip), 1.46e1.25 (om, 5H, 3 ꢃ CH2 pip, 2 ꢃ CH2
(400 MHz, CDCl3)
d in ppm: 7.30e7.24 (om, 10H), 7.15 (m, 2H), 7.01
(m, 2H), 4.51 and 4.46 (AB, 2JHH ¼ 11.6 Hz, 2H, OCH2Ph), 3.97 (bs, OH),
3.41e2.27 (om, 2H, CHeOCH2Ph, CHeOH), 2.78 (m, 2H, CH2 pip),
2.56e2.48 (om, 7H, 6 ꢃ CH2 pip, CH2 cyclohexane), 2.29 (m, 1H,
CHeN), 2.05 (m, 1H, CH2 cyclohexane), 1.63 (m, 1H, CH2 cyclo-
hexane), 1.38e1.21 (om, 2H, CH2 cyclohexane), 1.04 (m, 1H, CH2
cyclohexane), 1.11 (m, 1H, 1 ꢃ CH2 cyclohexane); 13C NMR (100 MHz,
4
CDCl3)
d
in ppm: 162.3 (d, 1JCF ¼ 245.4 Hz), 134.5 (d, JCF ¼ 3.2 Hz),
3
2
129.3 (d, JCF ¼ 8.1 Hz), 115.3 (d, JCF ¼ 21.4 Hz), 75.0 (s, COCH2Ph),
69.6 (s, OCH2Ph, CHeN), 66.4 (s, CHeN), 63.0 (s, CHeOH), 52.8 (s, CH2
pip), 50.4 (s, 2 ꢃ CH2 pip), 44.5 (s, CH2 pip), 38.6 and 31.5 (s, CH2
cyclohexane), 28.9 and 28.7 (s, CH2 pip), 26.4 (s, 2 ꢃ CH2 pip), 24.8 (s,
cyclohexane); 13C NMR (100 MHz, CDCl3)
d in ppm: 162.4 (d,
1JCF ¼ 246.0 Hz),147.5 (bs), 134.5 (d, JCF ¼ 3.0 Hz), 129.3 (d,
4
3JCF ¼ 8.8 Hz),128.5 (s), 127.3 (s), 125.9 (s), 115.4 (d, JCF ¼ 22.1 Hz),
CH2 pip), 18.6 (s, CH2 cyclohexane); 19F NMR (376.3 MHz, CDCl3)
d in
2
75.0 (s, COCH2Ph), 69.8 (s, CHeN), 69.6 (s, OCH2Ph), 66.3 (s, CHeOH),
45.8 (bs, C-(Ph)2), 45.0 (s, 2 ꢃ CH2 pip), 38.8 (s, CH2 cyclohexane), 37.1
(s, 2 ꢃ CH2 pip), 31.4 and 18.8 (s, CH2 cyclohexane); 19F NMR
ppm: e 115.5 (m); HRMS (ESI): m/z ¼ 391.27531 (M þ Hþ) calc.
391.27553.
( )-4k
( )-(1-(4-(4-fluorobenzyloxy)-2-hydroxycyclohexyl)
(376.3 MHz, CDCl3)
d
in ppm: e 115.5 (m); HRMS (ESI): m/
piperidin-4-yl) (4-fluorophenyl)methanone: The crude product
was purified by column chromatography using CHCl3/MeOH/NH3
(10/1/0.1). Adding of n-hexane to a concentrated CHCl3 solution of
( )-4k and keeping a few days at 4 ꢁC resulted in the formation of
z ¼ 460.26431 (M þ Hþ) calc. 460.26463; m/z ¼ 941.50377
(2M þ Naþ) calc. 941.50394.
( )-4h ( )-2-(4-benzylpiperidin-1-yl)-5-(4-fluorobenzyloxy)
cyclohexanol: After the reaction, the solvent was removed and the
residual oil dissolved in CHCl3. This organic phase was washed with
small amounts of water, dried with MgSO4 and concentrated to a
smaller volume. Adding of MTBE led to the formation of 232 mg
(52%) of a white precipitate of ( )-4h. 1H NMR (300 MHz, CDCl3)
323 mg (67%) of a colorless solid. 1H NMR (400 MHz, CDCl3)
d in
ppm: 7.95 (m, 2H), 7.29 (m, 2H), 7.13 (m, 2H), 7.01 (m, 2H), 4.53 and
4.48 (AB, 2JHH ¼ 11.6 Hz, 2H, OCH2Ph), 3.78 (bs, OH), 3.43e3.22 (om,
2H, CHeOCH2Ph, CHeOH), 3.22 (bm, 1H, CH-COPhF), 2.87 (m, 1H,
CH2 pip), 2.79e2.71 (om, 2H, CH2 pip), 2.54 (m, 1H, CH2 cyclo-
hexane), 2.28e2.24 (om, 2H, CH2 pip, CH2 cyclohexane), 2.14 (m,
1H, CH2 cyclohexane), 1.88e1.70 (om, 5H, 4 ꢃ CH2 pip, 1 ꢃ CH2
cyclohexane), 1.39e1.31 (om, 2H, CH2 cyclohexane) 1.14 (m, 1H, CH2
d
in ppm: 7.32e7.27 (om, 4H), 7.21e7.12 (om, 3H), 7.01 (m, 2H), 4.53
2
and 4.47 (AB, JHH ¼ 11.6 Hz, 2H, OCH2Ph), 3.94 (bs, 1H, OH),
3.40e3.30 (om, 2H, CHeOCH2Ph, CHeOH), 2.74 (m, 1H, CH2 pip),
2.68e2.52 (om, 5H, 2 ꢃ CH2 pip, CH2Ph, 1 ꢃ CH2 cyclohexane), 2.19
(m, 1H, CHeN), 2.04e1.99 (om, 2H, CH2 pip, CH2 cyclohexane), 1.77
(m, 1H, CH2 cyclohexane), 1.69e1.62 (om, 2H, 2 ꢃ CH2 pip), 1.52 (m,
1H, CHeCH2Ph), 1.36e1.05 (om, 5H, 2 ꢃ CH2 pip, 3 ꢃ CH2 cyclo-
cyclohexane); 13C NMR (100 MHz, CDCl3)
d in ppm: 200.9 (s, C]O),
1
1
165.8 (d, JCF ¼ 253.0 Hz),162.4 (d, JCF ¼ 244.0 Hz), 134.5 (d,
4JCF ¼ 3.0 Hz), 132.5 (d, 4JCF ¼ 3.0 Hz), 130.9 (d, 3JCF ¼ 9.0 Hz), 129.4
(d, 3JCF ¼ 8.0 Hz), 115.9 (d, 2JCF ¼ 22.0 Hz), 115.3 (d, JCF ¼ 21.0 Hz),
2
hexane); 13C NMR (75.4 MHz, CDCl3)
d
in ppm: 162.6 (d,
74.9 (s, COCH2Ph), 70.0 (s, CHeN), 69.6 (s, OCH2Ph), 66.3 (s,
CHeOH), 52.1 (s, CH2 pip), 45.1 (s, CH-COPhF), 43.8 (s, CH2 pip), 38.8
and 31.8 (s, CH2 cyclohexane), 29.6 and 29.2 (s, CH2 pip), 18.8 (s, CH2
1JCF ¼ 243.0 Hz),140.7 (s), 134.5 (d, JCF ¼ 3.0 Hz), 129.4 (d,
3JCF ¼ 8.0 Hz),129.2 (s), 128.3 (s), 125.9 (s), 115.3 (d, 2JCF ¼ 21.0 Hz),
75.0 (s, COCH2Ph), 69.9 (s, OCH2Ph), 69.6 (s, CHeN) 66.3 (s,
CHeOH), 53.1 (s, CH2Ph), 45.3 and 43.3 (s, CH2 pip), 38.9 (s, CH2
cyclohexane), 38.4 (s, CH, pip), 33.1 and 32.8 (s, CH2 pip), 31.5 and
4
cyclohexane); 19F NMR (376.3 MHz, CDCl3)
d in ppm: e 115.4 (m);
HRMS (ESI): m/z ¼ 430.21853 (M þ Hþ) calc. 430.21883; m/
z ¼ 452.20076 (M þ Naþ) cal. 452.20077; m/z ¼ 881.41196
(2M þ Hþ) calc. 881.41232.
18.6 (s, CH2 cyclohexane); 19F NMR (282.3 MHz, CDCl3)
d in ppm: e
115.5 (m); HRMS (ESI): m/z ¼ 398.24880 (M þ Hþ) calc. 398.24898.
( )-4i ( )-2-(3,4-dihydroisoquinolin-2(1H)-yl)-5-(4-fluorobenz-
yloxy)cyclohexanol: The crude product was purified by column
chromatography using EtOAc/n-hexane/NH3 (1/1/0.01). Adding of n-
hexane to a concentrated EtOAc solution of ( )-4i resulted in the
formation of 185 mg (46%) of colorless crystals after a few days at
( )-4m
( )-5-(4-fluorobenzyloxy)-2-(4-(pyridin-4-yl)piper-
idin-1-yl)cyclohexanol: The crude product was purified by column
chromatography using CHCl3/MeOH/NH3 (10/1/0.1). Adding of n-
hexane to a concentrated CHCl3 solution of ( )-4m and keeping a
few days at 4 ꢁC resulted in the formation of 263 mg (61%) of a
colorless solid. 1H NMR (400 MHz, CDCl3)
d in ppm: 8.50 (m, 2H),
4 ꢁC.1H NMR (400 MHz, CDCl3)
d
in ppm: 7.32 (m, 2H), 7.16e7.10 (om,
7.30 (m, 2H), 7.12 (m, 2H), 7.02 (m, 2H), 4.53 and 4.48 (AB,
2JHH ¼ 11.8 Hz, 2H, OCH2Ph), 3.85 (bs, 1H, OH), 3.45e3.32 (om, 2H,
CHeOCH2Ph, CHeOH), 2.90 (m, 1H, CH2 pip), 2.83e2.71 (om, 2H,
CH2 pip), 2.58e2.46 (om, 2H, CH pip, 1 ꢃ CH2 cyclohexane),
2
3H), 7.06e7.01 (om, 3H), 4.54 and 4.42 (AB, JHH ¼ 11.6 Hz, 2H,
2
OCH2Ph), 3.93 and 3.66 (AB, JHH ¼ 14.4 Hz, 2H, CH2 pip), 3.83 (bs,
OH), 3.53 (m, 1H, CHeOH), 3.40 (m, 1H, CHeOCH2Ph), 3.00 (m, 1H,

C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
63
2.33e2.22 (om, 2H, CHeN, CH2 pip), 2.14 (m, 1H, CH2 cyclohexane),
1.91e1.77 (om, 4H, CH2 pip), 1.64 (m, 1H, CH2 cyclohexane),
1.41e1.28 (om, 2H, CH2 cyclohexane), 1.17 (m, 1H, CH2 cyclo-
ppm: 7.35e7.25 (om, 5H), 4.55 (td, 2JHF ¼ 47.1 Hz, 3JHH ¼ 1.5 Hz, 2H,
CH2CH2F), 4.0 (bs, OH), 3.63 (m, 2H, OeCH2CH2CH2F), 3.48 (m, 1H,
CHeOH), 3.31 (m, 1H, CHeOCH2CH2CH2F), 2.94 (m, 1H, CH2 pip),
2.83e2.75 (om, 2H, CH2 pip), 2.58e2.51 (om, 2H, CH pip, CH2
cyclohexane), 2.36e2.16 (om, 3H, CH2 pip, CHeN, CH2 cyclo-
hexane), 2.02e1.66 (om, 7H, 4 ꢃ CH2 pip, 1 ꢃ CH2 cyclohexane,
CH2CH2CH2F), 1.35e1.17 (om, 3H, CH2 cyclohexane); 13C NMR
hexane); 13C NMR (100 MHz, CDCl3)
d in ppm: 162.4 (d,
1JCF ¼ 245.3 Hz),154.7 (s), 150.0 (s), 134.4 (d, 4JCF ¼ 3.2 Hz), 129.4 (d,
3JCF ¼ 8.1 Hz), 122.3 (s), 115.3 (d, 2JCF ¼ 21.4 Hz), 74.9 (s, COCH2Ph),
70.0 (s, CHeN), 69.7 (s, OCH2Ph), 66.3 (s, CHeOH), 53.4 (s, CH2 pip),
45.3 (s, CH pip), 42.2 (s, CH2 pip), 38.9 (s, CH2 cyclohexane), 33.4
and 33.0 (s, CH2 pip), 31.5 and 18.8 (s, CH2 cyclohexane); 19F NMR
(75.4 MHz, CDCl3)
d in ppm: 146.3 (s), 128.7 (s), 127.1 (s), 126.4 (s),
81.2 (d, 1JCF ¼ 163.7 Hz), 76.1 (s, CHeOCH2CH2CH2F), 70.2 (s, CHeN),
66.5 (s, CHeOH), 64.1 (d, 3JCF ¼ 5.7 Hz), 53.9 and 45.7 (s, CH2 pip),
43.1 (s, CH pip), 39.1 (s, CH2 cyclohexane), 34.5, and 34.1 (s, CH2
pip), 31.5e31.3 (os, CH2CH2CH2F, CH2 cyclohexane), 18.8 (s, CH2
(376.3 MHz, CDCl3)
d in ppm: e 115.4 (m); HRMS (ESI): m/
z ¼ 385.22827 (M þ Hþ) calc. 385.22858; m/z ¼ 407.21074
(M þ Naþ) cal. 407.21053; m/z ¼ 791.43210 (2M þ Hþ) calc.
791.43183.
cyclohexane); 19F NMR (282.3 MHz, CDCl3)
d
in ppm: ꢀ44.5 (m).
( )-5 3-(3-fluoropropoxy)-7-oxabicyclo[4.1.0]heptane: To a so-
lution of the epoxy alcohol 7-oxabicyclo[4.1.0]heptan-3-ol [40,41]
(2 g, 17.5 mmol) in 30 mL dried THF, NaH (1.2 g of 60% suspen-
sion in mineral oil, 30 mmol) was added and stirred for 90 min. This
suspension was added via a cannula to a mixture of 3-fluoropropyl
tosylate (4.4 g, 19 mmol) and KI (0.3 g, 1.75 mmol) in 80 mL dried
THF at 0 ꢁC, then stirred for 10 min at rt followed by stirring for 6 h
at 60 ꢁC. When the reaction was finished, the mixture was
concentrated to a smaller volume, treated with water and extracted
with MTBE (4 ꢃ 20 mL). After drying of the organic phase with
Na2SO4, the solvent was removed resulting in brown oil which was
purified by column chromatography (silica gel, EtOAc/n-hexane 2/
1) to give 0.75g (25%) of 5 as slightly yellow oil. 1H NMR (300 MHz,
4.1.2.3. General procedure for benzovesamicol derivatives ( )-9b-
( )-9m. The cis-epoxide precursor ( )-8 was synthesized as
already described [16]. The amine (1.5 mmol) and ( )-8 (1.4 mmol)
were dissolved in 5e10 mL EtOH and stirred at 65e70 ꢁC for 48 h.
The crude product was purified by column chromatography (silica
gel 60) or by crystallization (details described for each compound).
( )-9b ( )-3-(4-phenylpiperazin-1-yl)-1,2,3,4-tetrahydronaph-
thalen-2-ol: The crude product was purified by column chroma-
tography (silica gel 60, EtOAc/n-hexane/NH3 3:1:0.1) to give 78 mg
(19%) of a colorless solid. 1H NMR (400 MHz, CDCl3)
d in ppm:
7.32e7.24 (m, 2H), 7.17e7.07 (m, 4H), 6.96 (m, 2H), 6.89 (m, 1H), 4.21
(s, 1H, OH), 3.93 (m, 1H, CHeOH), 3.39e3.21 (m, 5H, piperazine),
3.05e2.79 (m, 6H, CHeN, CH2 piperazine, cyclohexane), 2.78e2.68
CDCl3)
d
in ppm: 4.45 (td, 2JHF ¼ 47.1 Hz, 3JHH ¼ 1.5 Hz, 2H, CH2CH2F),
3.45 (dt, 3JHH ¼ 1.5 Hz, 4JHF ¼ 0.5 Hz, 2H, CH2CH2CH2F), 3.15 (m, 1H,
CHeOCH2CH2CH2F), 3.02 (om, 2H, 2 ꢃ CHeO), 2.35e2.23 (m, 1H,
CH2 cyclohexane), 2.16e2.10 (m, 1H, CH2 cyclohexane), 1.91e1.63
(om, 4H, 2 ꢃ CH2 cyclohexane, CH2CH2CH2F), 1.58e1.53 (m, 1H, CH2
(m, 2H, CH2 cyclohexane); 13C NMR (101 MHz, CDCl3)
d in ppm: 150.2
(s), 133.4 (s), 132.8 (s), 128.2 (s), 128.1 (s), 128.1 (s), 125.3 (s), 125.1 (s),
119.0 (s), 115.3 (s), 65.2 (s, CHeN), 64.7 (s, CHeOH), 48.8 and 47.2 (s,
CH2 piperazine), 36.8 and 25.2 (s, CH2 cyclohexane); HRMS (ESI): m/
z ¼ 309.19615 (M þ Hþ) calc. 309.19614; m/z ¼ 331.17820 (M þ Naþ)
calc. 331.17808.
( )-9c ( )-3-(4-phenyl-5,6-dihydropyridin-1(2H)-yl)-1,2,3,4-
tetrahydronaphthalen-2-ol: The product crystallized in the reac-
tion mixture. The precipitate was washed with water, isopropanol,
and MTBE to obtain 79 mg (19%) of a colorless solid. 1H NMR
cyclohexane), 1.31 (m, 1H, CH2 cyclohexane); 13C NMR (75.4 MHz,
1
CDCl3)
d
in ppm: 81.0 (d, JCF
¼
163.7 Hz), 73.8 (s,
CHeOCH2CH2CH2F), 63.4 (d, 3JCF ¼ 5.6 Hz), 51.8 and 50.8 (s, CHeO),
31.0 (d, 2JCF ¼ 20.4 Hz), 30.7 and 23.7 (s, 3 ꢃ CH2 cyclohexane).
( )-6a 4-(3-fluoropropoxy)-2-(4-phenylpiperidin-1-yl)cyclo-
hexanol: To synthesize the 4-O-3-fluoropropyl ether vesamicol
derivative ( )-6a, 4-phenylpiperidine 370 mg (2.29 mmol) and
( )-5 (400 mg, 2.29 mmol) were dissolved in 5 mL EtOH and stirred
at 65e70 ꢁC for 4 days. The crude product was purified by column
chromatography (silica gel 60, EtOAc) and 348 mg (46%) of ( )-6a
crystallized as colorless needles from a solution of EtOAc/n-hexane
(300 MHz, CDCl3) d in ppm: 7.51e7.06 (om, 9H), 6.15 (s, 1H, CHCPh),
4.31 (bs, 1H, OH), 4.05e3.88 (m, 1H, CHeOH), 3.65e2.54 (m, 11H,
2xCH2-N, CH2CPh, CHeN, 2xCH2 cyclohexane); 13C NMR (75 MHz,
CDCl3) d in ppm: 140.7 (s),135.3 (s, CPh),134.8 (s),134.1 (s),129.4(s),
129.3 (s), 128.5 (s), 127.3 (s), 126.3 (s), 126.2 (s), 125.0 (s), 122.0
(s,CHCPh), 66.0 (s, CHeOH), 65.5 (s, CHeN), 48.7 and 45.0 (s,
CH2eN), 38.0 (s, CH2 cyclohexane), 28.8 (s, CH2CPh), 26.0 (s, CH2,
cyclohexane); HRMS (ESI): m/z ¼ 306.18515 (M þ Hþ) calc.
306.18524.
( )-9e ( )-1-(3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)-4-
phenylpiperidin-4-ol: The product crystallized in the reaction
mixture. The precipitate was washed with water, isopropanol, and
n-hexane to obtain 133 mg (30%) of a white solid. 1H NMR
at 4 ꢁC. 1H NMR (300 MHz, CDCl3)
d in ppm: 7.33e7.17 (om, 5H),
2
3
4.55 (td, JHF ¼ 47.1 Hz, JHH ¼ 1.5 Hz, 2H, CH2CH2F), 4.0 (bs, OH),
3.72 (m, 1H, CHeOCH2CH2CH2F), 3.52e3.41 (om, 3H, CHeOH,
OeCH2CH2CH2F), 2.95 (m,1H, CH2 pip), 2.79e2.67 (om, 3H, CH2 pip,
CHeN), 2.49 (m, 1H, CH pip), 2.18 (m, 1H, CH2 pip), 2.08 (m, 1H, CH2
cyclohexane), 2.01e1.56 (om, 9H, 4 ꢃ CH2 pip, 3 ꢃ CH2 cyclohexane,
CH2CH2CH2F), 1.40e1.22 (om, 2H, 2 ꢃ CH2 cyclohexane); 13C NMR
(100.0 MHz, CDCl3) d in ppm: 146.4 (s), 128.7 (s), 127.1 (s), 126.4 (s),
1
81.1 (d, JCF ¼ 163.7 Hz), 74.07 (s, CHeOCH2CH2CH2F), 68.7 (s,
CHeOH), 64.8 (s, CHeN), 63.4 (d, 3JCF ¼ 5.7 Hz), 53.5 and 45.6 (s, CH2
pip), 43.2 (s, CH pip), 34.4, and 34.1 (s, CH2 pip), 31.4 (d,
2JCF ¼ 19.9 Hz), 27.9, 27.8, and 26.2 (s, CH2 cyclohexane); 19F NMR
(300 MHz, (CD3)2SO) d in ppm: 7.56e7.44 (m, 2H), 7.34e7.26 (m,
2H), 7.22e7.17 (m, 1H), 7.13e7.02 (m, 4H), 4.76 (s, 1H, PhCH-OH),
4.45 (bs, 1H, CHeOH), 3.91e3.80 (m, 1H, CHeOH), 3.13e2.51 (om,
9H, 4xCH2 pip, CHeN), 2.07e1.88 (m, 2H, 2xCHeq pip), 1.69e1.52
(282.3 MHz, CDCl3)
d
in ppm: 33.15 (m).
(m, 2H, 2xCHax pip).; 13C NMR (75 MHz, (CD3)2SO)
d in ppm: 150.9
( )-7a 5-(3-fluoropropoxy)-2-(4-phenylpiperidin-1-yl)cyclo-
hexanol: To synthesize the 5-O-3-fluoropropyl ether vesamicol
derivative ( )-7a, the cis-epoxide ( )-5 (487 mg, 2.8 mmol) and
LiClO4 (424 mg, 4.0 mmol) were dissolved in 10 mL CH3CN and
stirred for 10 min. Subsequently, 4-phenylpiperidine (496 mg,
3.1 mmol) was added and the reaction mixture was stirred at rt for
24 h. After removal of the solvent, the crude product was purified
by column chromatography (silica gel 60, CHCl3/MeOH/NH3 (10/1/
0.1) and 310 mg (33%) of ( )-7a crystallized as colorless needles
(s), 136.3 (s), 135.1 (s), 129.4 (s), 129.3 (s), 128.4 (s), 126.8 (s), 126.4
(s), 125.5 (s), 70.8 (s, PhCH-OH), 66.4 (s, CHeN), 66.3 (s, CHeOH),
47.9 and 42.5 (s, CH2 pip), 39.5 and 39.1 (s, CH2 pip), 38.5 (s, CH2
cyclohexane), 27.6 (s, CH2 cyclohexane); HRMS (ESI): m/
z ¼ 324.19567 [MþH]þcalc 324.19581.
( )-9h ( )-3-(4-benzylpiperidin-1-yl)-1,2,3,4-tetrahydronaph-
thalen-2-ol: The product crystallized in the reaction mixture, was
isolated and recrystallized from MTBE/n-hexane at 4 ꢁC to give 65 mg
(15%) of colorless crystals. 1H NMR (300 MHz, CDCl3)
d
in ppm:
from a solution of ethanol at 4 ꢁC. 1H NMR (300 MHz, CDCl3)
d in
7.32e7.22 (m, 5H), 7.19e7.03 (m, 4H), 4.52 (bs, 1H, CHeOH),

64
C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
4.02e3.86 (m, 1H, CHeOH), 3.35e3.23 (m, 1H, CH2 cyclohexene),
2.98e2.76 (m, 5H, CH2 pip, CH2 cyclohexene, CHeN), 2.72e2.60 (m,
2H, CH2 pip, CH2 cyclohexene), 2.58e2.52 (m, 2H, CH2Ph), 2.30e2.22
(m, 1H, CH2 pip), 1.92e1.40 (m, 5H, 2xCH2 pip, CHCH2Ph); 13C NMR
3.11e2.93 (m, 2H, CH2 pip), 2.92e2.78 (m, 2H, CH2 pip), 2.71e2.54
(m, 3H, CHeN, CH2 pip, cyclohexene), 2.44e2.36 (m, 3H, CH-Ph,
CH2 pip, cyclohexene), 1.80e1.56 (m, 4H, 2xCH2 pip); 13C NMR
(75 MHz, (CD3)2SO)
d in ppm: 163.6 (C]O), 162.7 (d,
(75 MHz, CDCl3)
d
in ppm: 140.5 (s), 135.5 (s), 134.5 (s), 129.3 (s),
1JCF ¼ 242.1 Hz), 154.1 (s), 147.3 (s), 141.5 (d, 4JCF ¼ 2.6 Hz), 133.5 (s),
130.6 (d, 3JCF ¼ 8.0 Hz), 129.1 (s), 128.9 (s), 127.4 (s), 126.6 (s), 125.3
(s), 121.3 (s), 118.5 (s), 113.7 (d, 2JCF ¼ 20.5 Hz), 67.5 (s, CHeN), 66.9
(s, CHeOH), 52.1 (s) and 47.5 (s, CH2 pip), 43.1 (s, CH-Ph), 39.8 (s,
CH2CHeOH), 34.6 and 34.4 (s, CH2 pip), 24.1 (s, CH2CHeN); HRMS
(ESI): m/z ¼ 445.22823 (M þ Hþ) calc. 445.22858, m/z ¼ 467.21023
(M þ Naþ) calc. 467.21053.
128.7 (s), 128.5 (s), 128.4 (s), 127.0 (s), 126.5 (s), 125.8 (s), 66.2 (s,
CHeN), 66.0 (s, CHeOH), 47.3 and 42.5 (s, CH2 pip), 43.4 (s, CH2Ph),
38.5 (s, CHCH2Ph), 35.6 (s, CH2 cyclohexene), 32.5 and 32.4 (s, CH2
pip), 26.8 (s, CH2 cyclohexene); HRMS (ESI): m/z ¼ 322.21631
[MþH]þcalc 322.21654.
( )-9j
( )-3-([1,40-bipiperidin]-10-yl)-1,2,3,4-tetrahydronaph-
thalen-2-ol: The product crystallized in the reaction mixture, was
( )-12a ( )-(4-fluoro-N-(-7-hydroxy-6-(4-phenylpiperidin-1-
yl)-5,6,7,8-tetrahydronaphthalen-1-yl)benzamide): See descrip-
tion above, 12a was yielded as the polar isomer (54 mg, 6%). 1H
isolated and recrystallized from EtOAc at 4 ꢁC to give 67 mg (16%) of
colorless crystals. 1H NMR (300 MHz, CDCl3)
d in ppm 7.17e7.00 (m,
4H), 4.29 (s, 1H, OH), 3.89e3.75 (m, 1H, CHeOH), 3.35e3.23 (m, 1H,
CH2 cyclohexene), 2.96e2.63 (m, 7H, CH2 cyclohexene, CHeq pip, CH2
cyclohexene, CHeN cyclohexene, CH2 pip), 2.57e2.42 (m, 4H, 2xCH2
pip), 2.34e2.12 (m, 2H, CH2 pip, CHeN), 1.92e1.82 (m, 2H, CH2 pip),
NMR (400 MHz, (CD3)2SO) d in ppm: 9.86 (bs, 1H, NH), 8.15e7.97
(m, 2H), 7.41e7.33 (m, 2H), 7.32e7.23 (m, 4H), 7.21e7.12 (m, 3H),
7.09e7.02 (m, 1H), 4.46 (s, 1H, OH), 3.93e3.80 (m, 1H, CHeOH), 3.08
(dd, J ¼ 16.6, 5.6 Hz, 1H, CH2CHeOH), 3.00e2.78 (m, 4H, CH2 pip,
CH2CHeN), 2.74e2.61 (m, 2H, CHeN, CH2 pip), 2.61e2.41 (m, 3H,
CH2 pip, CH-Ph), 1.83e1.54 (m, 4H, 2xCH2 pip). 13C NMR (101 MHz,
1.72e1.34 (m, 8H, 4xCH2 pip); 13C NMR (101 MHz, CDCl3)
d in ppm
135.1 (s), 134.2 (s), 129.5 (s), 129.4 (s), 126.3 (s), 126.2 (s), 66.4 (s,
CHeN cyclohexane), 66.0 (s, CHeOH), 63.1 (s, CHeN pip), 52.9 (s, CH2
pip), 50.5 (s, 2xCH2-N pip), 44.3 (s, CH2 pip), 38.1 (s, CH2 cyclo-
hexane), 29.2 (s, CH2 pip), 28.8 (s, CH2 pip), 26.6 (s, 2xCH2 pip), 26.3
(s, CH2 cyclohexane), 25.0 (s, CH2 pip), HRMS (ESI): m/z ¼ 315.24323
(M þ Hþ) calc. 315.24309 m/z ¼ 337.22531 (M þ Naþ) calc.
337.22503.
(CD3)2SO)
d
in ppm: 164.2 (s, C]O), 164.0 (d, 1JCF ¼ 248.8 Hz), 146.5
4
(s), 136.6 (s), 135.8 (s), 131.0 (s), 130.9 (d, JCF ¼ 2.8 Hz), 130.3 (d,
3JCF ¼ 9.0 Hz), 128.3 (s), 126.7 (s), 125.9 (s), 125.7 (s), 124.4 (s), 115.3
(d, 2JCF ¼ 21.8 Hz), 66.0 (s, CHeOH), 65.2 (s, CHeN), 51.1 and 47.4 (s,
CH2 pip), 42.4 (s, CH-Ph), 33.9 and 33.8 (s, CH2 pip), 33.6 (s,
CH2CHeOH), 27.7 (s, CH2CHeN); HRMS (ESI): m/z ¼ 445.22869
(M þ Hþ) calc. 445.22858.
( )-9m ( )-3-(4-(pyridin-4-yl)piperidin-1-yl)-1,2,3,4-tetrahy-
dronaphthalen-2-ol: The product crystallized in the reaction
mixture, was isolated and recrystallized from EtOH at 4 ꢁC to give
( )-11h ( )-(N-(7-(4-benzylpiperidin-1-yl)-6-hydroxy-5,6,7,8-
tetrahydronaphthalen-1-yl)-4-fluorobenzamide):
Amine
h
67 mg (16%) of colorless crystals. 1H NMR (400 MHz, CDCl3)
d
in
(2.45 mmol) was added dropwise to the epoxide (1.78 mmol) in
EtOH and the mixture was stirred under reflux for seven days. After
evaporation of the solvent, the crude dark oil was extracted with
20% citric acid solution (5 ꢃ 3 ml). The combined aqueous phase
was treated with 6 M NaOH solution and extracted with CHCl3
(5 ꢃ 4 ml). After evaporation of the solvent, the resulting orange oil
was purified by column chromatography (silica 60, CHCl3/MeOH/
NH3 10:1:0.1) and the regioisomers were separated by semi-
preparative HPLC (ReprosilBasic C-18 HD, MeOH/40 mM NH4OA-
2
ppm: 8.53 (d, JHH ¼ 6 Hz, 2H), 7.20e7.05 (m, 6H), 4.33 (bs, 1H,
CHeOH), 3.96 e 3.89 (m, 1H, CHeOH), 3.36 e 3.30 (m, 1H, CH2
cyclohexane), 3.07e2.77 (m, 7H, CHeN, 2xCH2 cyclohexane, CH2
pip), 2.64e2.38 (m, 2H, CH2 pip), 2.01e1.64 (m, 4H, 2xCH2 pip); 13
C
NMR (101 MHz, CDCl3) d in ppm: 154.8 (s), 150.1 (s), 134.9 (s), 134.2
(s), 129.5 (s), 129.3 (s), 126.4 (s), 126.3 (s), 122.5 (s), 66.8 (s, CHeOH),
65.9 (s, CHeN), 53.4 and 45.1 (s, 2xCH2 pip), 42.4 (s, CH-Ph), 38.1 (s,
CH2 cyclohexane), 33.6 and 33.2 (s, 2xCH2 pip), 26.4 (s, CH2 cyclo-
hexane); HRMS (ESI): m/z ¼ 309.19632 (M þ Hþ) calc. 309.19614.
c
aq 70:30 v/v, flow rate 5 ml/min). 11h was yielded as the less polar
isomer (21 mg, 3%). 1H NMR (300 MHz, CDCl3)
d
in ppm: 7.96e7.82
1
4.1.2.4. General procedure for aminobenzovesamicol derivatives
( )-11a-( )-12h. To synthesize the epoxide precursor, 1-
naphthylamine was selectively reduced under BIRCH conditions in
liquid ammonia using sodium and tert-butanol resulting in 5,8-
dihydro naphthylamine which was protected with p-fluo-
robenzoyl chloride using standard conditions to obtain the p-flu-
orobenzamide. Epoxidation with m-chloroperbenzoic acid resulted
in the epoxide ( )-10. The aminobenzovesamicol derivatives were
synthesized nonregioselectively, stirring the amine (1.4 eq) with
epoxide in EtOH under reflux for several days. After evaporation of
the solvent, the crude product was purified by column chroma-
tography and the regioisomers were separated by semi-preparative
HPLC as described in detail for each derivative.
(m, 2H), 7.50 (d, JHH ¼ 7.8 Hz, 1H), 7.32e7.08 (m, 8H), 7.02 (d,
1JHH ¼ 7.5 Hz, 1H), 3.94e3.80 (m, 1H, CHeOH), 3.31 (dd, 1JHH ¼ 16.2,
5.4 Hz, 1H, CH2CHeOH), 2.95e2.58 (m, 7H, 2xCH2 pip, CHeN,
1
CH2CHeOH, CH2CHeN), 2.55 (d, JHH ¼ 6.8 Hz, 2H, CH2Ph),
1.82e1.16 (m, 5H, 2xCH2 pip, CHCH2Ph). 13C NMR (75 MHz, CDCl3)
d
in ppm: 165.2 (d, 1JCF ¼ 253.1 Hz), 165.1 (s, C]O), 140.5 (s), 135.5
(d, 4JCF ¼ 3.3 Hz),129.8 (d, 3JCF ¼ 8.9 Hz),129.3 (s),128.4 (s),127.5 (s),
127.1 (s), 126.1 (s), 122.7 (s), 116.2 (d, 2JCF ¼ 22.0 Hz), 66.5 (s, CHeN),
65.3 (s, CHeOH), 53.0 and 45.0 (s, CH2 pip), 43.3 (s, CH2Ph), 38.4 (s,
CHCH2Ph), 38.2 (s, CH2CHeOH) 33.1 and 32.6 (s, CH2 pip), 21.9 (s,
CH2CHeN); HRMS (ESI): m/z
¼
459.24391 (M
þ
Hþ) calc.
459.24423, m/z ¼ 481.22580 (M þ Naþ) calc. 481.22618.
( )-12h ( )-(N-(6-(4-benzylpiperidin-1-yl)-7-hydroxy-5,6,7,8-
tetrahydronaphthalen-1-yl)-4-fluorobenzamide): See description
above, 12h was yielded as the polar isomer (22 mg, 3%). 1H NMR
( )-11a ( )-(4-fluoro-N-(-6-hydroxy-7-(4-phenylpiperidin-1-
yl)-5,6,7,8-tetrahydronaphthalen-1-yl)benzamide):
4-
Phenylpiperidine (a) was added dropwise to the epoxide in EtOH
and the mixture was stirred under reflux for six days. After evap-
oration of the solvent, the crude product was purified by column
chromatography (silica 60, CHCl3/MeOH/NH3 10:1:0.1) and the
regioisomers were separated by semi-preparative HPLC (Reprosil
Basic C-18 HD, MeOH/40 mM NH4OAc 65:35 v/v, flow rate 5 ml/
min). 11a was yielded as the less polar isomer (37 mg, 4%). 1H NMR
(300 MHz, CDCl3)
d
in ppm: 7.92e7.85 (m, 2H), 7.79 (d, 1JHH ¼ 7.8 Hz,
1H), 7.64 (s, 1H, NH), 7.31e7.14 (m, 8H), 6.95 (d, 1JHH ¼ 7.5 Hz, 1H),
3.89 (d, 1JHH ¼ 6.2 Hz, 1H, CHeOH), 3.32e3.20 (m, 1H, CH2CHeOH),
2.98e2.59 (m, 7H, CH2CHeOH, CH2CHeN), 2.58 (d, 1JHH ¼ 6.8 Hz,
2H, CH2Ph), 2.33e2.19 (m, 1H, CH2 pip), 1.81e1.30 (m, 5H, 2xCH2
pip, CHCH2Ph). 13C NMR (75 MHz, CDCl3)
d in ppm: 165.2 (d,
1JCF ¼ 252.9 Hz), 164.9 (s, C]O), 140.5 (s), 135.7 (s), 131.1 (d,
3
(300 MHz, (CD3)2SO)
d
in ppm: 8.16e8.06 (m, 2H), 7.30e7.11 (m,
4JCF ¼ 3.1 Hz), 129.7 (d, JCF ¼ 9.0 Hz), 129.3 (s), 128.5 (s), 127.1 (s),
1
6H), 7.06e6.97 (m, 2H), 6.78 (dd, JHH ¼ 7.5 Hz, 1H), 6.34 (d,
126.6 (s), 126.2 (s), 121.6 (s), 116.1 (d, 2JCF ¼ 22.0 Hz), 66.1 (s, CHeN),
66.0 (s, CHeOH), 53.1 and 45.4 (s, CH2 pip), 43.23 (s, CH2Ph), 38.33
1JHH ¼ 7.5 Hz, 1H), 3.76 (td, 1JHH ¼ 9.9, 1JHH ¼ 5.4 Hz, 1H, CHeOH),

C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
65
(s, CHCH2Ph), 33.84 (s, CH2CHeOH), 32.5 and 32.4 (s, CH2 pip), 26.8
(s, CH2CHeN); HRMS (ESI): m/z ¼ 459.24422 (M þ Hþ) calc.
459.24423, m/z ¼ 481.22650 (M þ Naþ) calc. 481.22618, m/
z ¼ 939.46324 (2M þ Naþ) calc. 939.46313.
10 min at 4 ꢁC, and the sediments were re-suspended and washed
as described before, and aliquots stored at ꢀ30 ꢁC until use. After
thawing, homogenates were washed twice as described before and
finally re-suspended in assay buffer (50 mM TRIS-HCl, pH 7.4 at
21 ꢁC, containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM
MgCl2).
DeHVes [56] ( )-6-(4-phenylpiperidin-1-yl)cyclohex-3-enol:
The synthesis of DeHVes was accomplished according to the
reference, however the authors do not provide NMR data in this
report: 1H NMR (300 MHz, CDCl3)
d
in ppm: 7.34e7.29 (om, 2H),
4.2.4. VAChT binding
7.25e7.18 (om, 3H), 5.64 (m, 1H, ethylene), 5.56 (m, 1H, ethylene),
4.34 (bs, OH), 3.75 (m, 1H, CHeOH), 2.93 (m, 1H, CH2 pip),
2.86e2.47 (om, 5H, 2 ꢃ CH2 pip, 1 ꢃ CH2 cyclohexane, CHeN, CH
pip), 2.31 (m, 1H, CH2 pip), 2.19e2.04 (om, 3H, 2 ꢃ CH2 pip, CH2
cyclohexane), 1.92e1.65 (om, 4H, 2 ꢃ CH2 pip, 2 ꢃ CH2 cyclo-
For the VAChT standard equilibrium assay 1.5 ꢃ 106 VAChT-PC12
cells (250 30
m
g protein) together with 1.5 pmol (ꢀ)-[3H]vesa-
micol ([3H]2-(4-phenylpiperidinyl cyclohexanol, specific radioac-
tivity 1258 GBq/mmol, PerkinElmer Life Sciences, Boston, MA, USA)
and the relevant compound (concentrations between 10ꢀ11 and
10ꢀ5 mol/L) were incubated in a volume of 1 mL in duplicate for 2 h
at 25 ꢁC under agitation (200 minꢀ1). Nonspecific binding was
hexane); 13C NMR (75.4 MHz, CDCl3)
d in ppm: 146.2 (s), 128.5 (s),
126.9 (s), 126.3 (s), 125.3 and 124.6 (s, ethylene), 66.3 (s, CHeOH),
65.5 (s, CHeN), 53.7 (s, CH2 pip), 45.1 (s, CH pip), 43.0 (s, CH2 pip),
34.4 (s, CH2 cyclohexane), 34.3 and 33.9 (s, CH2 pip), 22.2 (s, CH2
cyclohexane); HRMS (ESI): m/z ¼ 258.18507 (M þ Hþ) calc.
258.18524.
determined in the presence of 10 mM ( )-vesamicol.
After incubation, bound and free radioligand were separated by
a rapid vacuum filtration system (cell harvester Brandel Gaithers-
burg, MD, USA) through Whatman GF/B glass-fiber filters pre-
soaked in 0.5% polyethylenimine for 3 h. The filters were washed 3
times with 4 mL ice-cold Tris buffer (0.05 mol/L, pH 7.4). The dried
filters were slightly shaken in 4 mL Rotiscint eco plus cocktail (Carl
Roth GmbH, Karlsruhe, Germany) for about 30 min, maintained in
the dark for at least 8 h and the radioactivity was measured in a Tri-
Carb2900TR Liquid Scintillation Counter (PerkinElmer Life Sciences
Boston, MA, USA) with a counting efficiency of 70%.
5-ABV (5-Aminobenzovesamicol) [16] (( )-5-amino-3-(4-
phenylpiperidin-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ol),
8-ABV
(8-Aminobenzovesamicol) [16] (( )-8-amino-3-(4-phenylpiperidin-
1-yl)-1,2,3,4-tetrahydronaphthalen-2-ol), BV (Benzovesamicol) [16]
(( )-3-(4-phenylpiperidin-1-yl)-1,2,3,4-tetrahydronaphthalen-2-ol),
FBT (Fluorobenzyltrozamicol) [57] (( )-10-(4-fluorobenzyl)-4-
phenyl-[1,30-bipiperidin]-40-ol), and FBBV (p-Fluorobenzoylbenzo-
vesamicol) [37] (( )-4-fluorophenyl) (1-(3-hydroxy-1,2,3,4-tetrahy-
dronaphthalen-2-yl)piperidin-4-yl)methanone) were synthesized
according to references and verified with NMR and HRMS.
4.2.5. s1 receptor and s2 receptor binding
Affinity toward s1 receptors was estimated using homogenates
of rat cortical membranes and (ꢀ)-[3H]pentazocine (specific ac-
tivity: 1070 GBq/mmol, PerkinElmer Life Sciences, Boston, USA).
4.2. Competitive binding assays
Nonspecific binding was determined in the presence of 10
mM
4.2.1. Preparation of rVAChT-PC12 cells
haloperidol.
rVAChT-PC12 cells were grown in Dulbecco's modified Eagle
Medium with 4.5 g/L D-glucose and supplemented with 5% cosmic
Affinity toward s2 receptors was estimated using homogenates
of rat liver membranes and [3H]DTG (specific activity: 1110 GBq/
mmol, PerkinElmer Life Sciences, Boston, USA) in the presence of
calf serum, 10% equine serum, 100 U/mL penicillin-streptomycin
and the neomycin analog G-418 (0.125 g/L) to confluence at incu-
bator conditions (37 ꢁC, 5% CO2). Cells were harvested by scraping,
pelleted and washed with PBS. After resuspension in assay buffer
(50 mM TriseHCl pH 7.4 with 120 mM NaCl, 50 mM KCl, 1 mM
MgCl2, 2 mM CaCl2) they were counted and stored at ꢀ30 ꢁC. For
the assay aliquots were thawed and suspended with test buffer to
get cells resp. cell debris in a concentration of 1 ꢃ 107 VAChT-PC12
per mL. After intensive homogenization they were ready for use.
1 m
M dextrallorphan (Roche) to block the binding of [3H]DTG to-
ward s1 receptors. Nonspecific binding was determined in the
presence of 10 M haloperidol. All procedures were the same as
m
those described for VAChT binding assay above.
IC50 values were calculated from competitive binding curves by
a
nonlinear curve fitting, using Graphpad Prism, version 3
(GraphPad Software, Inc., San Diego, USA). Specific binding of the
radioligand was defined as total binding minus nonspecific binding.
The apparent inhibition constant (Ki) was derived from IC50 values
according to the CHENGePRUSOFF equation: Ki ¼ IC50/(1 þ C/KD) where
C is the concentration of the radioligand and KD is the dissociation
constant of the radioligand (KD ((ꢀ)-[3H]vesamicol) ¼ 25.6 nM to
VAChT on rVAChT-PC12 cells, KD ((ꢀ)-[3H]pentazocine) ¼ 6.9 nM to
s1 receptors on rat cortical membranes, KD ([3H]DTG) ¼ 29 nM for
s2 receptors on rat liver membranes with blocking of s1 receptors
using dextrallorphan (unpublished data).
4.2.2. Preparation of rat brain membranes
Female Sprague Dawley rats (150e200 g) were anaesthetized
with ether and decapitated. Their brains were rapidly removed
from the skull. The brain tissue without cerebellum was homoge-
nized in 10 volumes (w/v) of ice-cold 250 mM sucrose solution
using a Teflon-glass homogenizer (50 strokes). Aliquots were stored
at ꢀ30 ꢁC until use. After thawing homogenate was diluted to
50 mL water and intensively homogenized. Cell debris were
removed by rotation (1000 g, 5 min). Crude cell membranes for
drug competition assays were obtained by centrifugation of the
supernatant at 15 000 g for 15 min at 4 ꢁC, washed with 250 mM
sucrose solution and distilled water and re-suspended in assay
buffer (50 mM Tris pH 7.4 containing 120 mM NaCl, 5 mM KCl,
2 mM CaCl2, 1 mM MgCl2).
Acknowledgments
The authors thank Dr. L. Hennig from the University of Leipzig
€
for measuring the NMR spectra and Tina Spalholz, Nicole Schroder,
and Katleen Müller for their lab assistance.
The project was funded by the German Research Foundation,
DFG (WE 2927/4-1).
4.2.3. Preparation of rat liver
Female Sprague Dawley rats (150e200 g) were anaesthetized
and decapitated. Liver was isolated and homogenized in 10 vol-
umes (w/v) of 50 mM TRIS-HCl, pH 7.4 at 4 ꢁC, using a Teflon-glass
homogenizer. Homogenates were centrifuged at 15.000 rpm for
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.05.033.

66
C. Barthel et al. / European Journal of Medicinal Chemistry 100 (2015) 50e67
acetylcholine receptors: design and evaluation of the potent radioligand [18F]
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