Novel σ1 antagonists designed for tumor therapy: Structure – activity relationships of aminoethyl substituted cyclohexanes

Article abstract of DOI:10.1016/j.ejmech.2020.112950

Depending on the substitution pattern and stereochemistry, 1,3-dioxanes 1 with an aminoethyl moiety in 4-position represent potent σ₁ receptor antagonists. In order to increase the stability, a cyclohexane ring first replaced the acetalic 1, 3-dioxane ring of 1. A large set of aminoethyl substituted cyclohexane derivatives was prepared in a six-step synthesis. All enantiomers and diastereomers were separated by chiral HPLC at the stage of the primary alcohol 7, and their absolute configuration was determined by CD spectroscopy. Neither the relative nor the absolute configuration had a large impact on the σ₁ affinity. The highest σ₁ affinity was found for cis-configured benzylamines (1R,3S)-11 (K_i = 0.61 nM) and (1S,3R)-11 (K_i = 1.3 nM). Molecular dynamics simulations showed that binding of (1R,3S)-11 at the σ₁ receptor is stabilized by the typical polar interaction of the protonated amino moiety with the carboxy group of E172 which is optimally oriented by an H-bond interaction with Y103. The lipophilic interaction of I124 with the N-substituent also contributes to the high σ₁ affinity of the benzylamines. The antagonistic activity was determined in a Ca²⁺ influx assay in retinal ganglion cells. The enantiomeric cis-configured benzylamines (1R,3S)-11 and (1S,3R)-11 were able to inhibit the growth of DU145 cells, a highly aggressive human prostate tumor cell line. Moreover, cis-11 could also inhibit the growth of further human tumor cells expressing σ₁ receptors. The experimentally determined logD_7.4 value of 3.13 for (1R,3S)-11 is in a promising range regarding membrane penetration. After incubation with mouse liver microsomes and NADPH for 90 min, 43% of the parent (1R,3S)-11 remained unchanged, indicating intermediate metabolic stability. Altogether, nine metabolites including one glutathione adduct were detected by means of LC-MS analysis.

Full text of DOI:10.1016/j.ejmech.2020.112950

European Journal of Medicinal Chemistry 210 (2021) 112950
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel s1 antagonists designed for tumor therapy: Structure e activity
relationships of aminoethyl substituted cyclohexanes
a
a
a
a
€
Nicole Kopp , Catharina Holtschulte , Frederik Borgel , Kirstin Lehmkuhl ,
Kristina Friedland b, Gianluca Civenni c, Erik Laurini d, Carlo V. Catapano c,
Sabrina Pricl d e, Hans-Ulrich Humpf f, Dirk Schepmann a, Bernhard Wünsch a
,
, g, *
a
€
€
Institut für Pharmazeutische und Medizinische Chemie, Westfalische Wilhelms-Universitat Münster, Corrensstraße 48, D-48149 Münster, Germany
b Pharmacology and Toxicology, Pharmacy and Biochemistry, Johannes Gutenberg University, Staudingerweg 5, 55128 Mainz, Germany
c Institute of Oncology Research, Universita Della Svizzera Italiana (USI), Via Vincenzo Vela 6, CH-6500 Bellinzona, Switzerland
d Molecular Biology and Nanotechnology Laboratory (MolBNL@UniTS), DEA, University of Trieste, 34127, Trieste, Italy
e Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland
Institut für Lebensmittelchemie, Westfalische Wilhelms-Universitat Münster, Corrensstraße 45, D-48149 Münster, Germany
Cells-in-Motion Cluster of Excellence (EXC 1003 e CiM), Westfalische Wilhelms-Universitat Münster, Germany
f
€
€
g
€
€
a r t i c l e i n f o
a b s t r a c t
Article history:
Depending on the substitution pattern and stereochemistry, 1,3-dioxanes 1 with an aminoethyl moiety in
4-position represent potent s1 receptor antagonists. In order to increase the stability, a cyclohexane ring
first replaced the acetalic 1, 3-dioxane ring of 1. A large set of aminoethyl substituted cyclohexane de-
rivatives was prepared in a six-step synthesis. All enantiomers and diastereomers were separated by
chiral HPLC at the stage of the primary alcohol 7, and their absolute configuration was determined by CD
spectroscopy. Neither the relative nor the absolute configuration had a large impact on the s1 affinity.
The highest s1 affinity was found for cis-configured benzylamines (1R,3S)-11 (Ki ¼ 0.61 nM) and (1S,3R)-
11 (Ki ¼ 1.3 nM). Molecular dynamics simulations showed that binding of (1R,3S)-11 at the s1 receptor is
stabilized by the typical polar interaction of the protonated amino moiety with the carboxy group of E172
which is optimally oriented by an H-bond interaction with Y103. The lipophilic interaction of I124 with
the N-substituent also contributes to the high s1 affinity of the benzylamines. The antagonistic activity
was determined in a Ca2þ influx assay in retinal ganglion cells. The enantiomeric cis-configured ben-
zylamines (1R,3S)-11 and (1S,3R)-11 were able to inhibit the growth of DU145 cells, a highly aggressive
human prostate tumor cell line. Moreover, cis-11 could also inhibit the growth of further human tumor
cells expressing s1 receptors. The experimentally determined logD7.4 value of 3.13 for (1R,3S)-11 is in a
promising range regarding membrane penetration. After incubation with mouse liver microsomes and
NADPH for 90 min, 43% of the parent (1R,3S)-11 remained unchanged, indicating intermediate metabolic
stability. Altogether, nine metabolites including one glutathione adduct were detected by means of LC-
MS analysis.
Received 23 July 2020
Received in revised form
19 September 2020
Accepted 14 October 2020
Available online 22 October 2020
Keywords:
s
receptors
Aminoethylcyclohexanes
Stereochemistry
Chiral HPLC
CD spectroscopy
s1 receptor affinity
Selectivity
Structure affinity relationships
Molecular interactions
Molecular dynamics simulations
Calculated free energy of binding
per-residue binding free energy
Voltage gated Ca2þ channel
Ca2þ influx assay
© 2020 Elsevier Masson SAS. All rights reserved.
Antagonistic activity
Inhibition of human prostate tumor cell
growth
DU145 tumor cells
Lipophilicity
Biotransformation
1. Introduction
Originally, the
ceptor subtype [1]. Further studies showed that the effects of the
prototypical
receptor agonist (þ)-SKF-10,047 could not be
antagonized by the opioid receptor antagonist naltrexone;
s receptor has been classified as an opioid re-
* Corresponding author. Institut für Pharmazeutische und Medizinische Chemie,
€
€
s
Westfalische Wilhelms-Universitat Münster, Corrensstraße 48, D-48149 Münster,
Germany.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
https://doi.org/10.1016/j.ejmech.2020.112950
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

€
N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
therefore, the
s
receptor was no longer considered as a member of
receptor
affinity towards the PCP binding site (Ki > 10 mM). On the other
hand, the (2R,4R)-configured primary amine 1b derived from
propiophenone exhibited high affinity towards the PCP binding site
the opioid receptor family [2]. In the early 1990s, two
s
subtypes were distinguished based on their different binding pro-
file of benzomorphans and they were termed s1 and s2 receptor,
respectively [3,4]. The s1 receptor subtype was already cloned in
1996 from different tissues (brain, liver, choriocarcinoma cell line)
of different species (rat, mouse, guinea pig, human) [5e9]. The s2
receptor, however, was not characterized until 2017, when it was
isolated from calf liver tissue and identified as the endoplasmic
reticulum (ER)-resident transmembrane protein 97 (TMEM97) [10].
In contrast to the s2 receptor, which is a protein of 176 amino acids
(Mw ~20.8 kDa) whose three-dimensional structure has not been
solved yet, the human s1 receptor (223 amino acids, Mw ~25.3 kDa)
was crystallized recently [11,12].
The s1 receptor is widely expressed in the central nervous
system (CNS) and various peripheral organs including liver, heart,
kidney and the eye [13e16]. It is involved in central nervous system
(CNS) pathologies such as schizophrenia, depression, Alzheimer’s
disease and drug/alcohol dependence [17e22]. Therefore, s1 re-
ceptor ligands can find application as potential therapeutics for the
treatment of various CNS disorders including neuropathic pain
[23,24] and depression.
(Ki ¼ 13 nM), but very low affinity towards s1 receptors (Ki > 10
mM)
[36], The racemic benzylamine 1a (Ki(s1) ¼ 19 nM [37]. showed
very high antiallodynic activity in vivo in the mouse capsaicin assay
[38]. This analgesic activity thus confirmed the s1 antagonistic
activity of racemic benzylamine 1a.
Although the in vitro and even the in vivo (intraperitoneal
application) pharmacological profile of the 1,3-dioxane 1a
appeared to be promising, its acetalic nature limits its practical use.
In particular, fast hydrolysis of the acetal in the stomach after
peroral application is expected. Therefore, the acetalic substructure
of 1a should be replaced by a more stable structural element. In a
first attempt toward this direction, we considered the replacement
of the O-atoms in the 1,3-dioxane by two CH2-moieties, which
resulted in cyclohexane derivatives 2 (Fig.1). Herein, we now report
the synthesis and pharmacological evaluation of cyclohexanes 2
with various amino moieties with a particular emphasis on the
stereochemistry of this novel class of
s ligands.
2. Chemistry
Furthermore, an increased expression of s1 receptors in cancer
cell lines and human tumors has been reported [25,26]. The
increased expression of s1 receptor in human cancer cell lines and
human tumors raises the possibility that s1 receptor modulators
might be therapeutically beneficial also for treating cancer in
addition to neurological disorders [25,26]. Analysis of large public
databases with gene and protein expression data in human cell
lines and tumor samples shows expression of s1 receptors in many
hematological and solid tumor types, including prostate, breast,
bladder, colon, and lung cancer [26]. Indeed, the widespread
expression of s1 receptors across tumor types of different histology
and the heterogeneity of its expression in tumors and cell lines
within the same subtypes suggest that the s1 receptor is an
important modulator of key biological processes in cancer cells
acting in a context-dependent manner [26,27]. Its functions in
cancer cells might be particularly relevant in the context of stress
response, metabolic plasticity and activation of adaptive and sur-
vival mechanisms [28e32]. Importantly, upon treatment with
various s1 receptor modulators reduction of cell proliferation and
survival of tumor cells was observed [32e35]. However, the un-
derlying mechanisms and the specificity of the observed effects
have not been fully understood [26,27].
In this project, novel highly selective s1 receptor ligands will be
developed with a strong potential to target cancer cells. Recently,
we have reported on various 1,3-dioxanes 1 with various amino-
ethyl substituents in 4-position (Fig. 1). Depending on the absolute
configuration, the substitution pattern at the acetalic 2-position
and the amino group, compounds 1 displayed high affinity to-
wards either the s1 receptor or the phencyclidine (PCP) binding site
of the NMDA receptor. As an example, the (2S,4R)-configured
benzylamine 1a derived from benzaldehyde showed very high af-
finity towards the s1 receptor (Ki ¼ 6.0 nM), but only negligible
2.1. Synthesis
The synthesis of the designed cyclohexane derivatives 2 started
with a Rh-catalyzed conjugate addition of phenylboronic acid at
cyclohexenone (3) [39]. (Scheme 1) Using Rh (cod)2BF4 as catalyst
in hot dioxane/KOH provided the addition product 4 in 86% yield.
Wittig reaction of the ketone 4 with the stabilized P-Ylid Ph3P]
CHCO2CH3 led to the
(E)/(Z)-diastereomers. Hydrogenation of the
5 using the catalyst Pd/C afforded cis- and trans-configured satu-
rated esters 6 in the ratio 75 : 25.
The primary alcohol 7 was obtained by LiAlH4 reduction of ester
6. Two different routes were pursued to prepare the amines 10e15
from the primary alcohol 7. Upon treatment with Zn(N3)2 py2, PPh3
and DIAD [40], the alcohol 7 was converted into the azide 8, which
was reduced with H2, Pd/C to give the primary amine 10. Alterna-
tively, the primary alcohol 7 was transformed into the mesylate 9,
which could be substituted by various primary and secondary
amines to yield the amines 11e15 (Scheme 1).
a,
b
-unsaturated ester 5 as 53:47 mixture of
a,b-unsaturated ester
During the hydrogenation of the a,b-unsaturated ester 5 a sec-
ond center of chirality was established. The ester was obtained as
75 : 25 mixture of diastereomers cis-6: trans-6. The same ratio of
diastereomers was found for all products 7e15 derived from ester
6. In the 1H NMR spectra of the compounds 6e15 a quartet-like
signal with three rather large coupling constants of
12.1e12.3 Hz at 1.0e1.2 ppm was found for the axially oriented
proton in 2-position of the major diastereomer. Such a signal
structure caused by a geminal coupling and two 1,2-trans-diaxial
couplings is only possible if both substituents in 1- and 3-position
adopt an equatorial orientation, i.e. cis-configuration.
2.2. Stereochemistry
The amines were obtained as 75 : 25 mixture of diastereomers,
which could not be separated by flash chromatography. Therefore,
the diastereomers were separated by preparative HPLC on a RP18
Phenomenex Gemini 5
mm column on a preparative scale. The
diastereomeric benzylamines 11 and phenylpiperazines 15 were
separated with an eluent mixture of CH3CN, H2O and NEt3 (0.1%),
whereas the pyrrolidine diastereomers cis-14 and trans-14 were
separated using a CH3OH, H2O and NEt3 (0.1%) mobile phase. (HPLC
methods A1-A3).
Fig. 1. Lead compounds 1 for the development of novel potent s1 receptor antagonists
2 based on the cyclohexane ring.
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N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
Scheme 1. Synthesis of amines 10e15. Reagents and reaction conditions: (a) PhB(OH)2, Rh (cod)2BF4, dioxane/1.5 M KOH (3:1), 90 ꢀC, 4.5 h, 86%. (b) Ph3P]CHCO2CH3, toluene,
110 ꢀC, 17 h, 89%. (c) H2 (balloon), Pd/C, CH3OH, rt, 15 min, 83%. (d) LiAlH4, THF, 0 ꢀC, 30 min, then rt, 2 h, 91%. (e) Zn(N3)2 py2, PPh3, DIAD, toluene, rt, 6 h, 73% (8) [40]. (f) CH3SO2Cl,
CH2Cl2, NEt3, rt, 22 h, 85% (9). (g) 8: H2 (balloon), Pd/C, CH3OH, rt, 2 h, 32% (10). (h) 9: R2NH, CH3CN, 82 ꢀC, 15e27 h, or microwave (for 13), 72e89% (11e15). The ratio of di-
astereomers cis: trans ¼ 75 : 25.
Since the diastereomeric racemic benzylamines cis-11
(Ki ¼ 1,8 0.19 nM) and trans-11 (Ki ¼ 2.4 0.17 nM) showed high
s1 affinity, the enantiomers of cis-11 and trans-11 should be
separated and investigated pharmacologically. Separation of the
benzylamine enantiomers 11 turned out to be difficult. Therefore,
the stereoisomeric alcohols 7 were separated by preparative chiral
HPLC using a Daicel Chiralpak IB column (250 mm, 20 mm, HPLC
method B) as stationary phase. The chromatogram displayed in
Fig. 2 shows the separation of all four stereoisomers (1R,3R)-7,
(1R,3S)-7, (1S,3S)-7, and (1S,3R)-7. It should be noted that each pair
of enantiomers ((1R,3R)-7/(1S,3S)-7 and (1R,3S)-7/(1S,3R)-7,
respectively) is well separated, whereas the pairs of diastereomers
((1R, R)-7/(1R,3S)-7 and (1S,3S)-7/(1S,3R)-7, respectively) have
very similar retention times. Therefore, it can be concluded that
separation of these molecules at the diastereomer level appears to
be difficult, while the same process becomes less problematic when
dealing with the corresponding enantiomers.
stereoisomeric benzylamines 11 as described for the mixture of
diastereomers. At first, the alcohols 7 were converted into the
mesylates 9, which were substituted with benzylamine to afford
the four stereoisomerically pure benzylamines (1R,3S)-11, (1S,3R)-
11, (1R,3R)-11, and (1S,3S)-11 (Scheme 2).
In order to produce large amounts of enantiomerically pure
benzylamines 11, the stereoisomers were separated by an alterna-
tive method. At first, chiral HPLC was used to separate the enan-
tiomers of the alcohol 7 without separating the diastereomers; this
separation could be performed on large amounts due to the big
difference in retention times (see Fig. 2). Thus, after performing the
chiral HPLC, enantiomerically pure mixtures of diastereomers
(1R,3S)-7þ(1R,3R)-7 and (1S,3R)-7þ(1S,3S)-7 were obtained (HPLC
method C). The mixtures of diastereomeric alcohols (1R, 3S)-
7þ(1R,3R)-7 and (1S,3R)-7þ(1S,3S)-7 were then converted into
benzylamines (1R,3S)-11þ(1R,3R)-11 and (1S,3S)-11/(1S,3S)-11.
Finally, the pairs of diastereomeric benzylamines could be easily
separated by preparative RP-HPLC to achieve the diastereomeri-
cally and enantiomerically pure benzylamines (1R,3S)-11, (1R,3R)-
11, (1S,3R)-11, and (1S,3S)-11 (HPLC method D).
Since primary amines are of interest as NMDA receptor antag-
onists (compare primary amine 1b in Fig. 1), the stereoisomerically
pure benzylamines 11 should be converted into primary amines 10
upon hydrogenolytic removal of the benzyl moiety. In the first re-
actions methanolic solutions of benzylamines (1R 3S)-11 and
(1S,3R)-11 were treated with H2 in the presence of Pd/C as catalyst.
However, instead of the primary amines (1R,3S)-10 and (1S,3R)-10,
the dimethylamines (1R,3S)-13 and (1S,3R)-13, were formed. The
unexpected methylation was explained by a Pd-catalyzed dehy-
drogenation of CH3OH to afford formaldehyde, which in turn un-
derwent reductive alkylation of primary and secondary amines
[41]. This methylation could be avoided by using the solvent THF
instead of CH3OH. However, in THF higher H2 pressure and longer
reaction times were required to reach complete debenzylation of
benzylamines 11. Thus, the stereoisomeric primary amines (1R,3S)-
10, (1S,3R)-10, (1R,3R)-10, and (1S,3S)-10 were obtained in 30e48%
yields.
After successful separation of the four stereoisomeric alcohols 7,
the stereochemical purity was assessed by chiral analytical HPLC. In
each sample the amount of the desired stereoisomer was higher
than 98.57% indicating high stereochemical purity of the alcohols 7
(see Table S2 (Supporting Information)).
The stereoisomeric alcohols 7 were next transformed into the
In order to analyze the absolute configuration of the stereoiso-
meric benzylamines, circular dichroism (CD) spectra of (1R,3S)-11,
(1S,3R)-11, (1R,3R)-11, and (1S,3S)-11 were recorded. Figs. 3 and 4
show the CD spectra of the enantiomeric cis-configured benzyl-
amines (1R,3S)-11 and (1S,3R)-11 and of the enantiomeric trans-
Fig. 2. HPLC chromatogram of the separation of stereoisomeric alcohols 7. HPLC
method B: Daicel Chiralpak IB 5
m
m, 250 mm, 20 mm, isohexane: isopropanol ¼ 98 : 2,
19 mL/min, detection at
l
¼ 210 nm.
3

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N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
Scheme 2. Synthesis of enantiomerically pure primary amines 10, benzylamines 11 and dimethylamines 13: Reagents and reaction conditions: (a) f) CH3SO2Cl, CH2Cl2, NEt3, rt,
87e92%. (b) BnNH2, CH3CN, 82 ꢀC, 15e23 h, 83e89%. (c) H2 (5 bar), Pd/C, THF, 25e27 h, 40e81%. (d) H2 (balloon), Pd/C, CH3OH, HOAc (2 drops), 6e20 h, 30e48%.
Fig. 4. CD spectra (CH3CN) of enantiomeric trans-configured benzylamines (1S,3S)-11
and (1R,3R)-11.
Fig. 3. CD spectra (CH3CN) of enantiomeric cis-configured benzylamines (1R,3S)-11
and (1S,3R)-11.
Therefore, (3S)-configuration was assigned to the both benzyl-
amines (1R,3S)-11 and (1S,3S)-11, which also experimentally
exhibited a positive Cotton effect at the same wavelength (Fig. 5).
configured benzylamines (1R,3R)-11 and (1S,3S)-11, respectively.
From these images it can be clearly seen that the stereoisomers
with a (3S)-configuration of the center of chirality adjacent to the
phenyl ring show a positive Cotton effect at approx. 210 nm,
whereas the benzylamines with the alternative (3R)-configuration
exhibit mirror-image spectra characterized by a negative Cotton
effect at the same wavelength.
To determine the absolute configuration of the stereoisomeric
benzylamines CD spectra of the model compounds (1R,3S)- and
(1S, S)-1-methyl-3-phenylcyclohexane (1R,3S)-16 and (1S,3S)-16)
were predicted [42e44]. (Fig. 5) In order to reduce the computa-
tional time required for such calculations, the rather flexible ben-
zylaminoethyl side chain of 11 was replaced by the small methyl
moiety. Calculated CD spectra [45] of both (3S)-configured cyclo-
hexanes 16 show a positive Cotton effect at approx. 210 nm.
3. Receptor affinity
The affinity towards s1 and s2 receptors was determined in
competitive receptor binding studies with radioligands, i.e.,
[3H](þ)-pentazocine for s1 assay and [3H]di-o-tolylguanidine for s2
assay, respectively. Membrane preparations from guinea pig brain
(s1 assay) and rat liver (s2 assay) served as receptor material.
Radioactivity emitted by each receptor/radioligand complex was
recorded after incubation with different concentrations of the test
compounds [46e48]. Table 1 summarizes the s1 and s2 receptor
affinities of the prepared amines 10e15 thus obtained.
4

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N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
and a low eudismic ratio of 1.3. The highest eudismic ratio of 14 was
found for the trans-configured primary amines (1S,3S)-10 and
(1R,3R)-10. However, the s1 affinity of the primary amines 10 is
rather low, and the most potent stereoisomer (1R,3R)-10 reveals a
Ki value of 43 nM, which is more than 10-fold higher than the Ki
values of benzylamines 11, phenylbutylamine 12 and pyrrolidines
14.
All compounds show excellent selectivity for the s1 receptor
over the s2 subtype. In particular, the most potent compounds of
this class of ligands, the stereoisomeric benzylamines 11, exhibit a
s1
/s2 selectivity of 35- to 50-fold.
In addition to the s1 and s2 affinity, the affinity towards the PCP
binding site of the NMDA receptor [36,49] was recorded. However,
the compounds were not able to replace the radioligand [3H](þ)-
MK-801 from its binding site up to a concentration of 1 mM. This
result was surprising as the primary amine 1b possesses high PCP
affinity. It was concluded that the missing ethyl moiety in 3-
position of primary amines 10 is responsible for their negligible
PCP affinity. In 1b, this ethyl moiety changes the orientation of the
phenyl moiety at the six-membered core heterocycle, which might
be the reason for the high PCP affinity of 1b compared with primary
amines 10.
Fig. 5. Calculated CD spectra for the model compounds cis-(1R,3S)-16 and trans-
(1S,3S)-16.
The benzylamines 11 reveal the highest s1 affinity with Ki values
in the low nanomolar to subnanomolar range. Extension of the
distance between the terminal phenyl moiety and the basic amino
group by four CH2-moieties as in 12 provided almost the same s1
affinity. Compared with the benzylamines 11, the s1 affinity of the
tertiary dimethylamines 13 is reduced 10- to 15-fold. Whereas the
large 4-phenylpiperazine derivatives 15 display almost the same s1
affinity as the dimethylamines 13, the s1 affinity of the pyrrolidines
14 is slightly increased. The lowest s1 affinity within this novel
compound class was found for the primary amines 10.
In general, the relative configuration does not have a large
impact on the s1 affinity. In all examples, cis- and trans-configured
diastereomers show almost the same s1 affinity. Moreover, the
absolute configuration also does not influence the s1 affinity to a
large extent. The Ki values of the stereoisomeric benzylamines 11
range from 0.61 nM ((1R,3S)-11) to 1.8 nM ((1R,3R)-11). The eud-
ismic ratio of both pairs of enantiomers is 2.2 and 1.3, respectively.
A similar observation can be made for the tertiary dimethylamines
(1R,3S)-13 and (1S,3R)-13, exhibiting almost the same s1 affinity
4. Molecular interactions between the novel ligands and the
s1 receptor
Since (1R,3S)-11 (Kis1 ¼ 0.61 nM) turned out to have the highest
s1 affinity of this new class of ligands, molecular dynamics (MD)
simulations were performed on the corresponding s1 receptor/(1R
3S)-11 complex in order to gain more details on the binding
mechanism of this compound to its biological target. The mem-
brane bound 3D structure of the s1 receptor protein obtained by X-
ray crystal structure analysis (pdb code 5HK1) was used as starting
point for these calculations [11]. Thus, an optimal binding site for
(1R,3S)-11 was initially identified on s1 receptor (Fig. 6A and B)
following a consolidated protocol [50,51]. Next, the corresponding
ligand/protein free energy of binding (DGbind) - along with the
Table 1
Affinity of prepared amines 10e15 towards s1 and s2 receptors as well as towards the PCP binding site of the NMDA receptor.
compd.
-NR2
Ki SEM [nM] (n ¼ 3)
s1
Eudismic ratio
1.2
s2
Eudismic ratio
PCPa)
a)
(1R,3S)-10
(1S,3R)-10
(1S,3S)-10
(1R,3R)-10
cis-11
NH2
167 68
138 44
603 184
43 8,1
1.8 0.19
2.4 0.17
0.61 0.12
1.3 0.18
1.4 0.20
1.8 0.13
3.0 0.68
4.2 0.55
14 1,6
38%
24%
60%
43%
e
e
e
e
11%
14%
31%
15%
11%
31%
14
NHBn
NHBn
130b)
268b)
49 31
47 10
46 11
trans-11
(1R,3S)-11
(1S,3R)-11
(1S,3S)-11
(1R,3R)-11
12 cis:trans ¼ 75:25
13 cis:trans ¼ 75:25
(1R,3S)-13
(1S,3R)-13
cis-14
2.2
1.3
1.0
1.2
57
7
NH(CH2)4Ph
N(CH3)2
N(CH3)2
93 9.7
11%
25%
115b)
1.3
76 15
156 24
61 9.6
33 6.0
2.1
10 4,8
3.6 1.2
2.0 0.98
36%
28%
trans-14
cis-15
trans-15
16 6.8
13 3.8
118 23
144 33
0%
0%
(þ)-pentazocine
haloperidol
di-o-tolylguanidine
5.7 2.2
6.3 1.6
89 29
e
78 2.3
58 18
a)
Values in % describe the inhibition of radioligand binding at a test compound concentration of 1 mM.
Due to low affinity, these values were determined only once.
b)
5

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N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
Fig. 6. (A) Details of compound (1R,3S)-11 in the binding pocket of s1 receptor. (1R,3S)-11 is shown as atom-colored sticks-and-balls (C, grey, N, blue, O, red) while the side chains of
protein residues mainly interacting with (1R,3S)-11 are depicted as colored sticks and labeled. Hydrogen atoms, water molecules, ions, and counterions are omitted for clarity. (B) 2D
schematic representation of the general stabilizing interactions between the new aminoethyl substituted cyclohexanes and s1 receptor (C) Calculated free energy of binding (
DGbind,
red), and enthalpic ( Hbind, brown-red) and entropic (-T Sbind, orange) components for: (1R,3S)-11 complexed with the s1 receptor. (D) Per-residue binding free energy
D
D
decomposition of the main involved amino acids of the complex between (1R,3S)-11 and s1 receptor. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)
enthalpic and entropic components (
D
Hbind and -T
D
Sbind, respec-
cyclohexane-based amines (with the exception of the primary
amines 10) are provided with high affinity toward the s1 receptor
with favorable free energy values lower than ꢁ10 kcal/mol. The
computational analysis substantiated that stereochemistry has only
a small impact on the s1 binding affinity of these compounds.
Indeed, all four stereoisomeric benzylamines 11 bind at the s1 re-
ceptor by following the same pattern of interactions as detailed
above for the lead compound (1R,3S)-11 (Fig. 7AeC, Figure S1).
Finally, the free energy decomposition analysis confirmed the very
small preference of the s1 receptor for the cis-configured benzyl-
amines (1R,3S)-11 and (1S,3R)-11. A similar preference for cis-
configured derivatives was observed in a previous work on spi-
rocyclic benzopyrans[51].
tively) - were calculated via the MM/PBSA (Molecular Mechanics/
Poisson- Boltzmann Surface Area) approach [52], yielding values in
agreement with the experimental affinity (Fig. 6C and Table S3
(Supporting
Hbind ¼ ꢁ20.69 kcal/mol; -T
through a per-residue binding free energy deconvolution (PRBFED)
of the enthalpic terms ( Hres), the binding mechanisms of (1R,3S)-
Information),
D
Gbind
¼
ꢁ11.31
kcal/mol;
D
D
Sbind ¼ 9.38 kcal/mol). Finally,
D
11 to its target protein could be defined precisely as described
below. (Fig. 6D and Table S4 (Supporting Information))
Fig. 6A and B shows in detail the qualitative pattern of the in-
teractions between (1R,3S)-11 and the s1 receptor. Three receptor
hydrophobic regions contribute to host the lipophilic moieties of
(1R,3S)-11: the phenyl group in position 3 of the cyclohexane ring is
encased in a receptor cavity lined by the side chains of residues
Replacement of the benzylamino moiety of 11 by smaller sub-
stituents such as the dimethylamino group in 13 or the pyrrolidine
ring in 14 led to a slight decrease of the corresponding s1 binding
L182, L186, T202 and Y206 of the membrane-proximal
(S
Hres ¼ ꢁ3.57 kcal/mol); the cyclohexane ring is suitably located
within a hydrophobic region made up by residues L105, T181, and
A185 (S
Hres ¼ ꢁ3.03 kcal/mol); and the N-benzyl moiety per-
forms hydrophobic interactions with I124 (
a-helix
D
capacity, quantified by a loss of about 1 kcal/mol in their DGbind
values (Fig. 7DeF, Figure S2). According to the interaction spectra
shown in Fig. 7F, the reduced free energy of binding of the dime-
thylamine (1R,3S)-13 and the pyrrolidine (1R,3S)-14 can be attrib-
uted to a reduced hydrophobic interaction of I124 with the smaller
alkyl groups at the N-atom. Even more dramatic is the decrease in
s1 affinity for the primary amines 10. Although primary amine
(1R,3S)-10 exhibited the least entropic penalty of the entire series,
it paid more than 2 kcal/mol in s1 binding efficiency, due to com-
plete missing of interactions with I124 and the suboptimal polar
D
DHres ¼ ꢁ1.11 kcal/mol).
Furthermore, the basic amino moiety of (1R,3S)-11 is engaged in
the s1-typical polar interaction with the carboxylic group of E172,
oriented in an optimal position by virtue of a hydrogen bond with
Y103 (S
moiety stabilizes the binding through a
the aromatic side chain of F107 (
Hres ¼ ꢁ1.27 kcal/mol).
D
Hres ¼ ꢁ5.69 kcal/mol). Finally, the protonated amino
p
-cation interaction with
D
In order to provide a quantitative SAR analysis, the same
computational approach was extended to all other molecules of
this new series. Accordingly, quantification of the main contribu-
tions of the single protein residues involved in ligand binding
allowed to rationalize and compare the specific interactions of each
tested compound with each amino acid residue in the binding
cavity of the s1 receptor protein. (Figs. 6B and 7 and S1-3, Table S3
and S4 (Supporting Information)).
and
(Table S4).
A larger spacer between the basic amino moiety and the ter-
p-cation interactions with E172 and F107, respectively.
minal phenyl ring as in the phenylbutylamine 12 and the phenyl-
piperazine 15 is well tolerated in the s1 binding cavity only if the
spacer is a flexible alkyl chain (e.g. (1R,3S)-12, Fig. 7GeI, Figure S3).
In (1R,3S)-12 the four CH2 moieties allow an appropriate orienta-
tion of the terminal phenyl moiety to form very similar interaction
spectra compared to (1R,3S)-11. (Table S4 (Supporting
The MM/PBSA estimated values of
DGbind confirmed that all
6

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N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
Fig. 7. Overlay of s1 binding modes of lead compound (1R,3S)-11 (firebrick) with (A) (1S,3R)-11 (red), (1S,3S)-11 (orange red) and (1R,3R)-11 (orange), (D) (1R,3S)-10 (cyan), (1R 3S)-
13 (cornflower blue) and (1R,3S)-14 (medium blue) and (G) (1R,3S)-12 (purple) and (1R,3S)-15 (hot pink).
Comparison of s1 receptor e ligand calculated free energy of binding (
DGbind, red), enthalpic (DHbind, brown-red) and entropic (-TDSbind, orange) components of lead compound
(1R,3S)-11 with (B) (1S,3R)-11, (1S,3S)-11 and (1R,3R)-11 (E) (1R,3S)-10, (1R,3S)-13 and (1R,3S)-14 and (H) (1R,3S)-12 and (1R,3S)-15.
Comparison of s1 receptor e ligand PRBFED analysis of lead compound (1R,3S)-11 with (C) (1S,3R)-11, (1S,3S)-11 and (1R,3R)-11 (F) (1R,3S)-10, (1R,3S)-13 and (1R,3S)-14 and (I)
(1R,3S)-12 and (1R,3S)-15. The color code indicating the contribution of the respective structural elements to the free binding enthalpy is the same as given in Fig. 6C.(For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Information)). On the other hand, the more rigid piperazine spacer
inhibits adopting this favorable phenyl ring orientation in (1R,3S)-
15. Consequently, the lower s1 affinity of the phenylpiperazine
(1R,3S)-15 is due to a globally unfavorable binding pose in the s1
receptor cavity, which, in turn, reflects in substantial decreased
contributions of each amino acid residue to the specific interaction
with the ligand. (Fig. 7GeI).
co-incubated with opipramol (c ¼ 100
mM) and, subsequently, the
Ca2þ influx was stimulated with KCl. With exception of cis-11, both
trans-configured compounds trans-11 and trans-14 reduced the s1
agonistic effect of opipramol almost completely (Fig. 8). Obviously,
both compounds behaved as s1 receptor antagonists in this assay.
The low activity of cis-11, the ligand with the highest s1 affinity,
was unexpected. We assume that a higher concentration of cis-11 is
required to antagonize the opipramol effect in this assay.
5. Antagonistic activity: interaction with voltage-gated Ca2þ
channels in retinal ganglion cells
6. Inhibition of tumor cell growth
The influence of the most promising s1 ligands cis-11
(Ki ¼ 1.8 nM), trans-11 (Ki ¼ 2.4 nM) and trans-14 (Ki ¼ 2.0 nM) on
voltage dependent intracellular Ca2þ levels in retinal ganglion cells
was investigated in a functional assay [53]. The principle of the
assay is as follows: at first, the retinal ganglion cells were incubated
with the Ca2þ sensitive fluorescent dye fura-2-AM and one of the
test compounds cis-11, trans-11 or trans-14 at a concentration of
10 nM. Then, the influx of Ca2þ ions was induced upon treatment of
the cells with KCl and the intracellular fluorescence was recorded
and correlated with the intracellular Ca2þ concentration.
While the s1 agonist opipramol was able to reduce the KCl
induced influx of Ca2þ ions into the retinal ganglion cells signifi-
cantly, the aminoethyl substituted cyclohexanes cis-11, trans-11
and trans-14 could not inhibit the Ca2þ influx. Obviously, these
three compounds do not behave like the s1 agonist opipramol.
The s1 antagonistic effect of the test compounds was next
investigated by reverting the s1 agonistic effect of opipramol on
these cells. For this purpose, the test compounds (c ¼ 10 nM) were
Next, the inhibition of tumor cell growth by stereoisomeric
benzylamines 11 and phenylpiperazines 15 was investigated. The
androgen negative human prostate cancer cell line DU145[54] was
selected to evaluate the antiproliferative properties of the amines
11 and 15. In brief, approximately 2000 cells were seeded in a 96-
well plate and 24 h later, the test compounds were added in a
concentration of 10 mM. After 72 h, the cells were fixed and pro-
liferation/survival of the cells was evaluated by staining with Sul-
forhodamine B.[55].
The racemic cis-configured benzylamine cis-11 as well as its
enantiomers (1R,3S)-11 and (1S,3R)-11 showed significant anti-
proliferative effects on the DU145 prostate cancer cells (Fig. 9A).
The trans-configured benzylamine (1R,3R)-11 and (1S,3S)-11 and
the phenylpiperazines trans-15 and cis-15, could not inhibit the
growth of the tumor cells at the tested concentration of 10 mM.
Thus, cis-11 as well as its enantiomers (1R,3S)-11 and (1S,3R)-11
could represent promising candidates for further development in
therapeutic targeting of human tumor cells.
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European Journal of Medicinal Chemistry 210 (2021) 112950
7.2. Biotransformation of (1R,3S)-11
At first the metabolic stability of (1R,3S)-11 was determined
in vitro. For this purpose, (1R,3S)-11 was incubated with mouse
liver microsomes and NADPH. Mouse liver microsomes were cho-
sen, since it was planned to test the s1 receptor ligand (1R,3S)-11 in
mice. After an incubation period of 90 min at 37 ꢀC, 43 1.6% (SD,
n ¼ 3) of the parent compound (1R,3S)-11 remained unchanged.
Although (1R,3S)-11 is more stable than the reference compound
imipramine (12 1.7% (SD, n ¼ 3) intact parent), more than 50% of
(1R,3S)-11 were transformed after 90 min.
In order to identify metabolic soft spots of the s1 receptor ligand
(1R,3S)-11, metabolite structure elucidation was carried out by
means of different LC-MS methods and with the use of collision-
induced dissociation (CID). After incubation with mouse liver mi-
crosomes and NADPH, nine metabolites were identified (Scheme
3). Oxidation of the phenyl or cyclohexyl moiety led to phenol
11A and cyclohexanol 11B, respectively. Oxidation adjacent to the
amino moiety resulted in the amide 11C and the primary amine 11D
was formed by oxidative N-debenzylation. Further oxidation of
cyclohexanol 11B provided cyclohexanone 11F. The catechol
metabolite 11E and the phenolamide metabolite 11G were formed
by oxidation at two different positions. The most interesting me-
tabolites are the dimers 11H and the glutathione adduct 11K, the
last being formed with and without addition of additional gluta-
thione to the incubation mixture. The structures of the dimers 11H
and the glutathione adduct 11K were elucidated by different ex-
periments (see Section 5 “Biotransformation of (1R,3S)-11” in
Supporting Information).
In order to analyze the pathway of metabolite formation, the
phenol metabolite 11A and the catechol metabolite 11E were iso-
lated and again incubated with mouse liver microsomes and
NADPH. Upon incubation of phenol 11A the metabolites 11E, 11G,
11H, and 11K were formed, but incubation of catechol 11E led only
to 11K (Figure S6). Therefore, the following pathway is postulated:
at first phenol 11A is oxidized to give a reactive epoxide, which is
either trapped by another phenol 11A to form the regioisomeric
dimers 11H or rearranged into catechol 11E. Then, catechol 11E will
be oxidized to form an o-quinone, which is trapped by glutathione
to afford the glutathione adduct 11K (Figure S7).
Fig. 8. Reversion of the effect of the s1 agonist opipramol on the Ca2þ influx in retinal
ganglion cells by the s1 antagonists trans-11, cis-11 and trans-14.
The activity of cis-11 was evaluated in additional cancer cell
lines under the same conditions (Fig. 9B). Both prostate (PC3 and
22RV1) and lung (H358, H441 and H522) cancer cell lines were
sensitive to the antiproliferative effects of cis-11. Interestingly, the
growth inhibitory activity of cis-11 was comparable to that of the
known s1 antagonist NE-100 tested under similar conditions
(Fig. 9C).
8. Conclusion
7. Pharmacokinetic properties
Replacement of the O-atoms of the acid labile acetalic 1,3-
dioxane ring of the potent s1 receptor antagonist 1a by two CH2
moieties led to novel cyclohexane-based s1 receptor ligands 10e15.
The highest s1 receptor affinity was detected for the benzylamines
11, which behaved as s1 antagonists in the Ca2þ influx assay using
retinal ganglion cells. The cis-configured benzylamines (1R,3S)-11
(Ki ¼ 0.61 nM) and (1S,3R)-11 (Ki ¼ 1.3 nM) exhibited even higher
s1 affinity than the 1,3-dioxane 1a (Ki ¼ 6.0 nM). Although the
cyclohexane derivatives 11 are less polar than the 1,3-dioxane de-
rivative 1a, the logD7.4 value experimentally determined for
(1R,3S)-11 (logD7.4 ¼ 3.13) lies in a promising range for penetration
of physiological (i.e., cellular and blood-brain) barriers.
Separation of stereoisomeric primary alcohols 7 by chiral HPLC
provided diastereomerically and enantiomerically pure amines.
However, neither the relative nor the absolute configuration had a
great influence on the s1 receptor affinity and selectivity. Whereas
primary amines 10 and tertiary amines 13 and 15 show low to
moderate s1 affinity, the secondary benzylamines 11 interact with
high affinity with s1 receptors (Ki ¼ 0.61e1.8 nM). Molecular dy-
namics simulations starting with the recently reported X-ray
crystal structure of the s1 receptor showed very small differences in
the free energy of binding for the four stereoisomeric benzylamines
7.1. Determination of the logD7.4 value
The lipophilicity of a drug has a strong impact on various
pharmacokinetic parameters. In particular, it has a strong influence
on solubility [56,57], membrane and blood-brain-barrier penetra-
tion [58,59] and extent of biotransformation [60,61]. The lip-
ophilicity can be described by the distribution coefficient at a
specific pH value (logDpH). In general, the physiological pH value of
7.4 is chosen. Very recently, we have reported on a micro shake
flask method, which requires less than 1 mg of compound for the
determination of logD7.4 values, since the amount of drug in the
buffer layer is determined by MS [62,63]. This method led to an
experimentally determined logD7.4 value of 3.23
0.02 ( SEM,
n ¼ 9) for the most potent s1 receptor ligand (1R,3S)-11 of this set
of compounds. The experimentally determined logD7.4 value of the
trans-configured diastereomer (1S,3S)-11 is in the same range
(logD7.4 ¼ 3.34
0.03 ( SEM, n ¼ 9) showing that the relative
configuration (cis or trans) did only slightly modulate the polarity
of the corresponding molecules. Both logD7.4 values are in a good
range regarding barrier penetration.
8

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European Journal of Medicinal Chemistry 210 (2021) 112950
Fig. 9. A. Growth inhibition of human prostate tumor cells DU145 by stereoisomeric benzylamines 11 and phenylpiperazines 15 compared to DMSO. B. Antiproliferative activity of
cis-11 in various human cancer cells. C. growth inhibition of PC3 cells by NE-100.
11 in agreement with the corresponding experimental evidences.
All compounds form a central polar interaction with E172 and bind
to three receptor hydrophobic pockets. However, the different s1
affinity of aminoethylcyclohexanes with various N-substituents
was attributed to different interactions with I124 forming optimal
interactions with the benzyl moiety of 11 and least interactions
with the primary amino group of 10.
incubation with mouse liver microsomes and NADPH for 90 min,
43% of unchanged parent compound were detected. Altogether,
nine phase I and phase II metabolites were found including the
glutathione adduct 11k.
9. Experimental
At a concentration of 10
mM, the two cis-configured benzyl-
9.1. Chemistry, general
amines (1R,3S)-11 and (1S,3R)-11 as well as the racemic mixture
cis-11 displaying the highest s1 affinity of this series of compounds
reduced the growth of DU145 cells, a highly aggressive prostate
cancer cell line chosen for the initial compounds screening. The
corresponding trans-configured diastereomers (1R,3R)-11 and
(1S,3S)-11 and the phenylpiperazines cis-15 and trans-15 were not
active in this assay. Importantly, the antiproliferative activity of cis-
11 was seen in additional human cancer cell lines, giving confidence
on the reproducibility of the results. Due to the promising results,
the metabolic stability of (1R,3S)-11 was investigated. After
Unless otherwise noted, moisture sensitive reactions were
conducted under dry nitrogen. CH2Cl2 was distilled over CaH2. THF
was distilled over sodium/benzophenone. Et2O and toluene were
dried over molecular sieve 0.4 Å. Thin layer chromatography (tlc):
Silica gel 60 F254 plates (Merck). Flash chromatography (fc): Silica
gel 60, 40e64 mm (Merck); parentheses include: diameter of the
column (d), length of the stationary phase, fraction size (V), eluent.
Melting point: Melting point apparatus Mettler Toledo MP50
Melting Point System, uncorrected. MS: microOTOF-Q II (Bruker
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European Journal of Medicinal Chemistry 210 (2021) 112950
Scheme 3. Postulated and identified structures of phase I and phase II metabolites of s1 receptor antagonist (1R,3S)-11 produced in vitro: (a) mouse liver microsomes and NADPH,
(b) mouse liver microsomes, NADPH and glutathione (GSH).
Daltonics); APCI, atmospheric pressure chemical ionization. IR: FT-
IR spectrophotometer MIRacle 10 (Shimadzu) equipped with ATR
technique. Circular dichroism spectroscopy: JASCO J-600 spec-
235 nm. Different solvents were used to separate different amines.
Method A1 for separation of cis-11 and trans-11:
CH3CN:H2O ¼ 55:45 þ 0.1% NEt3.
tropolarimeter (Jasco, Grob-Umstadt), 0.1 cm cell, solvent CH3CN.
Nuclear magnetic resonance (NMR) spectra were recorded on
Method A2 for separation of cis-15 and trans-15:
CH3CN:H2O ¼ 60:40 þ 0.1% NEt3.
Agilent 600-MR (600 MHz for 1H, 151 MHz for 13C) or Agilent 400-
MR spectrometer (400 MHz for 1H, 101 MHz for 13C);
d
in ppm
Method A3 for separation of cis-14 and trans-14:
CH3OH:H2O ¼ 70:30 þ 0.1% NEt3.
related to tetramethylsilane and measured referring to CHCl3
(
(
(
d
d
d
¼ 7.26 ppm (1H NMR) and
d
¼ 77.2 ppm (13C NMR)), CHD2OD
¼ 3.31 ppm (1H NMR) and
d
¼ 49.0 ppm (13C NMR)) and DMSO‑d6
¼ 2.54 ppm (1H NMR) and
d
¼ 39.5 ppm (13C NMR)); coupling
constants are given with 0.5 Hz resolution; the assignments of 13
C
9.2.3. 9.2.3. HPLC method B (chiral preparative separation of all four
stereoisomers of 7)
and 1H NMR signals were supported by 2-D NMR techniques where
necessary.
Equipment: Set 1; column Daicel Chiralpak IB, 5
mm, 250 mm/
20 mm; guard column Daicel Chiralpak IB, 5 m, 20 mm/10 mm;
m
flow rate 19 mL/min; detection wavelength 210 nm; solvent iso-
hexane: isopropanol ¼ 98 : 2.
9.2. HPLC equipment and methods
9.2.1. HPLC equipment
9.2.4. 9.2.4. HPLC method C (separation of enantiomerically pure
pairs of diastereomeric alcohols 7)
Set 1: Pump L-7150, autosampler L-7200, UV-detector L-7400,
interface D-7000, data transfer
software (all Merck Hitachi).
D-line, data acquisition: HSM-
Equipment Set 1; column: Daicel Chiralpak IB, 5
mm, 250 mm/
20 mm; guard column Daicel Chiralpak IB, 5 m, 20 mm/10 mm;
m
Set 2: Pump L-7100, degasser L-7614, autosampler L-7200, UV-
flow rate 20 mL/min; detection wavelength 210 nm; solvent iso-
hexane: isopropanol ¼ 98 : 2..
detector L-7400, interface D-7000, data transfer
acquisition: HSM-software (all Merck Hitachi).
D-line, data
9.2.5. 9.2.5. HPLC method D (separation of diastereomeric
benzylamines 11)
9.2.2. 9.2.2. HPLC method A (preparative separation of cis/trans-
diastereomers)
Equipment Set 1; column Phenomenex Gemini, 5
mm, C18, 110A,
Equipment Set 1; column Phenomenex Gemini, 5
250 mm/21.2 mm; guard column Phenomenex Gemini, 5
110A, 50 mm/21.2 mm; flow rate 12 mL/min; detection wavelength
m
m, C18, 110A,
250 mm/21.2 mm; guard column Phenomenex Gemini, 5 m
110A, 50 mm/21.2 mm; flow rate 20 mL/min; detection wavelength
210 nm; solvent CH3CN: H2O ¼ 65 : 35 þ 2.0% NH3.
m, C18,
mm, C18,
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European Journal of Medicinal Chemistry 210 (2021) 112950
9.2.6. 9.2.6. HPLC method E (analysis of purity)
cyclohexane:ethyl acetate ¼ 25:1, Rf ¼ 0.22). Colorless oil, yield
1.01 g (83%). Purity (HPLC method E): 95.1% (tR ¼ 22.5 min).
Equipment Set 2; column: LiChrospher® 60 RP-select B, 5 mm,
LiChroCART® 250 mm/4 mm cartridge; guard column: LiChros-
pher® 60 RP-select B, 5 m, LiChroCART® 4 mm/4 mm cartridge,
manu-CART® NT cartridge holder; flow rate 1.0 mL/min; injection
volume 5 L; detection wavelength 210 nm; solvent A: water with
C
15H20O2 (232.3 g/mol). MS (ESI): m/z ¼ 255 (M þ Naþ), 250
m
(M þ NHþ4 ), 233 (M þ Hþ). IR: ṽ (cmꢁ1) ¼ 2922, 2852 (CeH), 1734
(C]O), 755, 698 (C-Harom). 1H NMR (CDCl3):
d
(ppm) ¼ 1.03 (qd,
m
J ¼ 12.5/3.2 Hz, 0.75H, 4-Hax-cyclohexane or 6-Hax-cyclohexane
cis), 1.19 (q, J ¼ 12.1 Hz, 0.75H, 2-Hax-cyclohexane cis), 1.31e2.12 (m,
9 ꢂ 0.25H, 7 ꢂ 0.75H, cyclohexane), 2.25 (dd, J ¼ 14.8/6.9 Hz, 0.75H,
CH2CO2CH3 cis), 2.29 (dd, J ¼ 14.8/7.1 Hz, 0.75H, CH2CO2CH3 cis),
2.50e2.54 (m, 2 ꢂ 0.25H, CH2CO2CH3 trans), 2.60 (tt, J ¼ 12.0/
3.0 Hz, 0.75H, PhCH cis), 2.74e2.81 (tt, J ¼ 10.6/3.8 Hz, 0.25H, PhCH
trans), 3.67 (s, 3 ꢂ 0.75H, OCH3 cis), 3.70 (s, 3 ꢂ 0.25H, OCH3 trans),
7.18e7.33 (m, 5H, arom). Ratio of cis-6:trans-6 ¼ 75:25.
0.05% (v/v) trifluoroacetic acid; solvent B: CH3CN with 0.05% (v/v)
trifluoroacetic acid; gradient elution: (A %): 0e4 min: 90%,
4e29 min: 90 / 0%, 29e31 min: 0%, 31e31.5 min: 0 / 90%,
31.5e40 min: 90%. The purity of all compounds was determined by
this method. The purity of all test compounds is higher than 95%
(unless otherwise noted).
9.3. Synthetic procedures
9.3.4. cis- and trans-2-(3-Phenylcyclohexyl)ethan-1-ol (cis-7 and
trans-7)
9.3.1. 3-Phenylcyclohexanone (4)
A mixture of dioxane/1.5 M KOH ¼ 3/1 was degassed for 30 min
in an ultrasonic bath. Under N2 [Rh (cod)2]BF4 (19 mg, 0.047 mmol)
and phenylboronic acid (288 mg, 2.36 mmol) were suspended in
the dioxane/KOH mixture (5 mL) and the mixture was stirred for
30 min at room temperature. Afterwards cyclohex-2-en-1-one (3)
(0.15 mL, 1.56 mmol) was added dropwise and the mixture was
heated to 90 ꢀC for 4.5 h. After cooling down the reaction mixture
was transferred to a separating funnel, brine (15 mL) was added
and the mixture was extracted with CH2Cl2 (4 x 20 mL). The com-
bined organic layers were dried (Na2SO4), filtered and concentrated
in vacuo. The crude product was purified by flash column chro-
Under N2 the ester cis-6/trans-6 (136 mg, 0.59 mmol) was dis-
solved in THF (20 mL). Under ice cooling a 1 M LiAlH4 solution in
THF (0.61 mL, 0.61 mmol) was added dropwise and the mixture was
stirred for 30 min at 0 ꢀC. The ice cooling was removed and the
reaction mixture was stirred for 2 h at room temperature. H2O was
added under ice cooling till the gas formation stopped and the
mixture was heated to reflux for 30 min. The precipitate was
filtered through Celite® and washed with ethyl acetate. The filtrate
was concentrated in vacuo and the residue was purified by flash
column chromatography (Ø
3 cm, h 16 cm, 10 mL, cyclo-
hexane:ethyl acetate ¼ 3:1, Rf ¼ 0.32). Colorless oil, yield 109 mg
(91%). Purity (HPLC method E): cis-7 71.7% (tR ¼ 20.6 min), trans-7
26.2% (tR ¼ 20.4 min). C14H20O (204.3 g/mol). MS (ESI): m/z ¼ 227
(M þ Naþ). IR: ṽ (cmꢁ1) ¼ 3324 (OeH broad), 2918, 2850 (CeH),
matography (Ø 2.5 cm,
h 18 cm, 10 mL, cyclohexane:ethyl
acetate ¼ 7:1, Rf ¼ 0.35). Pale yellow oil, yield 233 mg (86%).
C
12H14O (174.2 g/mol). MS (ESI): m/z ¼ 371 (2 M þ Naþ), 271
(2 M ꢁ Ph), 197 (M þ Naþ). IR: ṽ (cmꢁ1) ¼ 2938, 2866 (CeH), 1708
753, 697 (C-Harom). 1H NMR (CDCl3):
d
(ppm) ¼ 0.91 (qd, J ¼ 12.5/
(C]O), 753, 698 (C-Harom). 1H NMR (CDCl3):
d
(ppm) ¼ 1.73e1.91
3.0 Hz, 0.75H, 4-Hax-cyclohexane or 6-Hax-cyclohexane cis), 1.06 (q,
J ¼ 12.2 Hz, 0.75H, 2-Hax-cyclohexane cis),1.12e1.98 (m,11 ꢂ 0.25H,
9 ꢂ 0.75H, cyclohexane, CH2CH2OH), 2.47 (tt, J ¼ 11.9/3.2 Hz, 0.75H,
PhCH cis), 2.69 (tt, J ¼ 10.4/4.4 Hz, 0.25H, PhCH trans), 3.64 (t,
J ¼ 6.7 Hz, 2 ꢂ 0.75H, CH2OH cis), 3.64 (t, J ¼ 6.9 Hz, 2 ꢂ 0.25H,
CH2OH trans), 7.08e7.24 (m, 5H, arom). A signal for the OH-group is
not seen in the spectrum. Ratio of cis-7:trans-7 ¼ 75:25.13C NMR
(m, 2H, cyclohexane), 2.06e2.19 (m, 2H, cyclohexane), 2.34e2.63
(m, 4H, CH2COCH2), 3.02 (tt, J ¼ 11.8/3.8 Hz, 1H, PhCH), 7.21e7.26
(m, 3H, arom), 7.31e7.36 (m, 2H, arom).
9.3.2. Methyl (3-phenylcyclohexylidene)acetate (5)
Ph3P]CHCO2CH3 (4.81 g, 14.4 mmol) was added to a solution of
3-phenylcyclohexanone (4, 982 mg, 5.64 mmol) in toluene (8 mL)
and the mixture was heated to reflux for 17 h. The solvent was
removed in vacuo and the residue was purified by flash column
chromatography (Ø 6 cm, h 20 cm, 160 mL, cyclohexane:ethyl
acetate ¼ 10:1, Rf ¼ 0.43). Pale yellow oil, yield 1.15 g (89%). Purity
(HPLC method E): 99.7% (tR ¼ 23.5 min). C15H18O2 (230.3 g/mol).
MS (ESI): m/z ¼ 253 (M þ Naþ), 231 (M þ Hþ). IR: ṽ (cmꢁ1) ¼ 2929
(CeH), 1714 (C]O), 1647 (C]C), 755, 698 (C-Harom). 1H NMR
(CDCl3):
d
(ppm) ¼ 21.4, 29.8, 30.0, 33.9, 35.2, 38.0, 38.2 (7 ꢂ 0.25C,
cyclohexane trans, CH2CH2OH trans), 26.5, 32.8, 34.1, 34.6, 40.3,
41.2, 44.3 (7 ꢂ 0.75C, cyclohexane cis, CH2CH2OH cis), 60.7 (0.75C,
CH2OH cis), 61.6 (0.25C, CH2OH trans), 125.8 (0.25C, C-4 arom
trans), 125.9 (0.75C, C-4 arom cis), 126.8 (2 ꢂ 0.75C, C-2, C-6 arom
cis), 126.9 (2 ꢂ 0.25C, C-2, C-6 arom trans), 128.3 (2 ꢂ 0.25C, C-3, C-
5 arom trans), 128.3 (2 ꢂ 0.75C, C-3, C-5 arom cis), 147.3 (0.25C, C-1
arom trans), 147.6 (0.75C, C-1 arom cis). The stereoisomers (1R,3S)-
7, (1S,3R)-7, (1S,3S)-7 and (1R,3R)-7 were separated by preparative
chiral HPLC (method B).
(CDCl3):
d
(ppm) ¼ 1.45e1.74 (m, 2H, cyclohexane), 1.86e2.10 (m,
3H, cyclohexane), 2.16e2.26 (m, 0.53H, 2-Hax (E)), 2.30e2.49 (m,
1.47H, cyclohexane), 2.64e2.76 (m, 1H, PhCH), 3.67 (s, 3 ꢂ 0.47H,
OCH3), 3.70 (s, 3 ꢂ 0.53H, OCH3), 3.90 (d, broad, J ¼ 13.6 Hz, 0.53H,
6-Heq (E)), 4.02 (ddt, J ¼ 13.3/3.6/1.8 Hz, 0.47H, 2-Heq (Z)), 5.68 (t,
J ¼ 1.5 Hz, 0.53H, C]CH), 5.71 (t, J ¼ 1.7 Hz, 0.47H, C]CH),
7.15e7.36 (m, 5H, arom). The ratio of the two isomers is (E)-5:(Z)-
5 ¼ 53:47.
9.3.5. (1R,3S)-2-(3-Phenylcyclohexyl)ethan-1-ol ((1R,3S)-7)
Pale yellow oil. Purity (HPLC method E): 99.0% (tR ¼ 20.3 min).
Ratio of the stereoisomers: (1R,3S)-7: (1S,3R)-7: (1S,3S)-7: (1R,3R)-
7 ¼ 98.57 : 0.12: 0.02 : 1.29. Retention time: 23.0 min (HPLC method
B). Specific rotation: ½a D20
¼ þ15.7 (c ¼ 1.02; CH2Cl2).
ꢃ
9.3.3. Methyl cis- and trans-2-(3-phenylcyclohexyl)acetate (cis-6
and trans-6)
9.3.6. (1S,3R)-2-(3-Phenylcyclohexyl)ethan-1-ol ((1S,3R)-7)
Pale yellow oil. Purity (HPLC method E): 99.5% (tR ¼ 20.3 min).
Ratio of the stereoisomers: (1R,3S)-7: (1S,3R)-7: (1S,3S)-7: (1R,3R)-
7 ¼ 0.10 : 99.41: 0.42 : 0.07. Retention time: 40.2 min (HPLC method
The a,b-unsaturated ester 5 (1.21 g, 5.25 mmol) was dissolved in
CH3OH (30 mL) and Pd/C (10%, 311 mg) was added. The mixture was
stirred under H2 (balloon) for 3 h 15 min at room temperature. The
mixture was filtered through Celite® and the residue was washed
with small amounts of CH3OH. The filtrate was concentrated in
vacuo and the residue was purified by flash column chromatog-
raphy twice (1. Ø 5 cm, h 14 cm, 30 mL, cyclohexane:ethyl
B). Specific rotation: ½a D20
¼ ꢁ15.7 (c ¼ 1.02; CH2Cl2).
ꢃ
9.3.7. (1S,3S)-2-(3-Phenylcyclohexyl)ethan-1-ol ((1S,3S)-7)
Pale yellow oil. Purity (HPLC method E): 98.7% (tR ¼ 20.0 min).
Ratio of the stereoisomers: (1R,3S)-7: (1S,3R)-7: (1S,3S)-7: (1R,3R)-
acetate
¼
10:1, Rf
¼
0.50, 2. Ø 8 cm, h 12 cm, 65 mL,
11

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N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
7 ¼ 0.35 : 0.42: 99.01 : 0.22. Retention time: 37.7 min (HPLC
Then methanesulfonyl chloride (0.04 mL, 0.52 mmol) was added,
the ice bath was removed and the solution was stirred at room
temperature overnight. The mixture was transferred into a sepa-
rating funnel and washed with 0.5 M NaOH (2 x) and with a
saturated solution of NH4Cl (1 x). The organic layer was dried
(Na2SO4), filtered and the solvent was removed under reduced
pressure. The residue was purified by flash column chromatog-
raphy (Ø 2.5 cm, h 15 cm, 10 mL, cyclohexane:ethyl acetate ¼ 3:1).
Colorless solid, mp 52 ꢀC, yield 91 mg (92%). Purity (HPLC method
method B). Specific rotation: ½a D20
¼ þ2.4 (c ¼ 0.84; CH2Cl2).
ꢃ
9.3.8. (1R,3R)-2-(3-Phenylcyclohexyl)ethan-1-ol ((1R,3R)-7)
Pale yellow oil. Purity (HPLC method E): 99.6% (tR ¼ 20.0 min).
Ratio of the stereoisomers: (1R,3S)-7: (1S,3R)-7: (1S,3S)-7: (1R,3R)-
7 ¼ 0.16 : 0.27: 0.03 : 99.54. Retention time: 21.4 min (HPLC method
B). Specific rotation: ½a D20
¼ ꢁ1.6 (c ¼ 0.84; CH2Cl2).
ꢃ
E): 99.7% (tR ¼ 21.6 min). Specific rotation: ½a D20
¼ þ29.8 (c ¼ 1.45;
ꢃ
9.3.9. cis- and trans-1-(2-Azidoethyl)-3-phenylcyclohexane (cis-8
and trans-8)
CH2Cl2). Further analytical data see diastereomeric mixture cis-9
and trans-9.
Under N2, Zn(N3)2 py2 (154 mg, 0.50 mmol) and PPh3 (359 mg,
1.37 mmol) were dissolved in toluene (dried with molecular sieves
4 Å, 14 mL). The alcohol cis-/trans-7 (134 mg, 0.66 mmol) was also
dissolved in a small amount of toluene (dried with molecular sieves
4 Å) and the solution was added to the reaction mixture. Under ice
cooling diisopropyl azodicarboxylate (DIAD, 0.26 mL, 1.32 mmol)
was added dropwise, the ice bath was removed and the mixture
was stirred at room temperature for 6 h. The solvent was evapo-
rated in vacuo and the residue was purified by flash column chro-
matography (Ø 4.5 cm, h 17 cm, 30 mL, cyclohexane:ethyl
acetate ¼ 15:1, Rf ¼ 0.70). Pale yellow oil, yield 110 mg (73%). Purity
(HPLC method E): cis-8 72.3% (tR ¼ 24.2 min), trans-8 23.3%
(tR ¼ 24.0 min). C14H19N3 (229.3 g/mol). MS (APCI): m/z ¼ 202
(M ꢁ N2 þ Hþ). IR: ṽ (cmꢁ1) ¼ 2921, 2852 (CeH), 2091 (N]N]N),
9.3.12. {2-[(1S,3R)-3-Phenylcyclohexyl]ethyl} methanesulfonate
((1S,3R)-9)
As described for (1R,3S)-9, a solution of (1S,3R)-7 (85 mg,
0.42 mmol), NEt3 (0.17 mL, 1.25 mmol) and methanesulfonyl
chloride (0.05 mL, 0.63 mmol) in CH2Cl2 (10 mL) was stirred at
room temperature overnight. The residue was purified by flash
column chromatography (Ø 2.5 cm, h 19 cm, 10 mL, cyclo-
hexane:ethyl acetate ¼ 3:1). Colorless solid, mp 52 ꢀC, yield 108 mg
(91%). Purity (HPLC method E): 99.8% (tR ¼ 21.6 min). Specific
rotation: ½a 2D0
¼ ꢁ29.7 (c ¼ 1.45; CH2Cl2). Further analytical data
ꢃ
see diastereomeric mixture cis-9 and trans-9.
754, 698 (C-Harom). 1H NMR (CDCl3):
d
(ppm) ¼ 0.85e0.95 (m,
9.3.13. {2-[(1S,3S)-3-Phenylcyclohexyl]ethyl} methanesulfonate
((1S,3S)-9)
0.75H, cyclohexane cis), 1.05 (q, J ¼ 12.3 Hz, 0.75H, 2-Hax-cyclo-
hexane cis), 1.19e1.93 (m, 11 ꢂ 0.25H, 9 ꢂ 0.75H, cyclohexane,
CH2CH2N3), 2.48 (tt, J ¼ 11.9/3.0 Hz, 0.75H, PhCH cis), 2.67 (tt,
J ¼ 10.5/3.9 Hz, 0.25H, PhCH trans), 3.25 (t, J ¼ 6.9 Hz, 2 ꢂ 0.75H,
CH2N3 cis), 3.25 (t, J ¼ 7.2 Hz, 2 ꢂ 0.25H, CH2N3 trans), 7.09e7.25 (m,
5H, arom). Ratio of cis-8:trans-8 ¼ 75:25.
As described for (1R,3S)-9, a solution of (1S,3S)-7 (25 mg,
0.12 mmol), NEt3 (0.05 mL, 0.36 mmol) and methanesulfonyl
chloride (0.01 mL, 0.18 mmol) in CH2Cl2 (5 mL) was stirred at room
temperature for 5 h. The residue was purified by flash column
chromatography (Ø 2.5 cm, h 15 cm, 10 mL, cyclohexane:ethyl
acetate ¼ 3:1). Pale yellow oil, yield 30 mg (87%). Purity (HPLC
method E): 99.4% (tR ¼ 21.8 min). Specific rotation: ½a D20
¼ ꢁ1.4
ꢃ
9.3.10. cis- and trans-[2-(3-Phenylcyclohexyl)ethyl]
methanesulfonate (cis-9 and trans-9)
(c ¼ 1.04; CH2Cl2). Further analytical data see diastereomeric
mixture cis-9 and trans-9.
Under N2 the alcohol cis-7/trans-7 (1.16 g, 5.68 mmol) was
dissolved in CH2Cl2 (20 mL). NEt3 (2.37 mL, 17.03 mmol) was added
and the mixture was stirred for 10 min under ice cooling. Meth-
anesulfonyl chloride (0.66 mL, 8.51 mmol) was added dropwise and
the mixture was stirred for 22 h at room temperature. The mixture
was transferred into a separating funnel and the organic layer was
washed with 0.5 M NaOH (2 x), 2 M NaOH (1 x) and a saturated
solution of NH4Cl (1 x). The combined aqueous layers were
extracted with CH2Cl2 (2 x 20 mL).The combined organic layers
were dried (Na2SO4), filtered and the solvent was removed in
vacuo. The resulting product was purified by flash column chro-
9.3.14. {2-[(1R,3R)-3-Phenylcyclohexyl]ethyl} methanesulfonate
((1R,3R)-9)
As described for (1R,3S)-9, a solution of (1R,3R)-7 (24 mg,
0.12 mmol), NEt3 (0.05 mL, 0.35 mmol) and methanesulfonyl
chloride (0.01 mL, 0.18 mmol) in CH2Cl2 (5 mL) was stirred at room
temperature for 4 h. The residue was purified by flash column
chromatography (Ø 2.5 cm, h 17 cm, 10 mL, cyclohexane:ethyl
acetate ¼ 3:1). Pale yellow oil, yield 31 mg (92%). Purity (HPLC
method E): 99.9% (tR ¼ 21.8 min). Specific rotation: ½a D20
¼ þ1.4
ꢃ
matography (Ø
6 cm, h 17 cm, 30 mL, cyclohexane:ethyl
(c ¼ 1.04; CH2Cl2). Further analytical data see diastereomeric
mixture cis-9 and trans-9.
acetate ¼ 3:1, Rf ¼ 0.42). Pale yellow solid, mp 40 ꢀC, yield 1.36 g
(85%). Purity (HPLC method E): cis-9 75.4% (tR ¼ 22.0 min), trans-9
24.5% (tR ¼ 21.8 min). C15H22O3S (282.4 g/mol). MS (ESI): m/z ¼ 305
(M þ Naþ). IR: ṽ (cmꢁ1) ¼ 2920, 2851 (CeH), 1351, 1171 (O-mesyl),
9.3.15. cis- and trans-2-(3-Phenylcyclohexyl)ethanamine (cis-10
and trans-10)
756, 700 (C-Harom). 1H NMR (CDCl3):
d
(ppm) ¼ 0.85e0.97 (m,
Pd/C (10%,16 mg) was added to a solution of the azide cis-/trans-
8 (53 mg, 0.23 mmol) in CH3OH (12 mL). The mixture was stirred
under H2 (balloon) for 2 h at room temperature. The mixture was
filtered and the solvent removed in vacuo. The crude product was
purified by flash column chromatography twice (1. Ø 2 cm, h 15 cm,
8 mL, CH2Cl2:CH3OH ¼ 1:1, Rf ¼ 0.10, 2. Ø 1 cm, h 15 cm, 5 mL,
CH2Cl2:CH3OH ¼ 1:1). Yellow oil, yield 15 mg (32%). Purity (HPLC
0.75H, cyclohexane cis), 1.08 (q, J ¼ 12.2 Hz, 0.75H, 2-Hax-cyclo-
hexane cis), 1.18e2.00 (m, 11 ꢂ 0.25H, 9 ꢂ 0.75H, cyclohexane,
CH2CH2O), 2.48 (tt, J ¼ 11.9/3.0 Hz, 0.75H, PhCH cis), 2.63e2.71 (m,
0.25H, PhCH trans), 2.92 (s, 3 ꢂ 0.25H, OSO2CH3 trans), 2.93 (s,
3
ꢂ
0.75H, OSO2CH3 cis), 4.20e4.24 (m, 2H, CH2OSO2CH3),
7.10e7.25 (m, 5H, arom). Ratio of cis-9:trans-9 ¼ 75:25.
method E): cis-10 81.4% (tR
¼
16.3 min), trans-10 16.3%
9.3.11. {2-[(1R,3S)-3-Phenylcyclohexyl]ethyl} methanesulfonate
((1R,3S)-9)
The alcohol (1R,3S)-7 (72 mg, 0.35 mmol) was dissolved in
CH2Cl2 (10 mL) under N2. NEt3 (0.15 mL, 1.05 mmol) was added
under ice cooling and the mixture was stirred for 10 min at 0 ꢀC.
(tR ¼ 16.2 min). C14H21N (203.3 g/mol). MS (ESI): m/z ¼ 204
(M þ Hþ). IR: ṽ (cmꢁ1) ¼ 2919, 2848 (CeH), 753, 698 (C-Harom). 1H
NMR (CDCl3):
d
(ppm) ¼ 0.90e1.00 (m, 0.75H, cyclohexane cis), 1.11
(q, J ¼ 12.1 Hz, 0.75H, 2-Hax-cyclohexane cis), 1.25e1.95 (m,
11 ꢂ 0.25H, 9 ꢂ 0.75H, cyclohexane, CH2CH2N), 2.53 (tt, J ¼ 11.8/
12

€
N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
3.1 Hz, 0.75H, PhCH cis), 2.73e2.80 (m, 3 ꢂ 0.25H, 2 ꢂ 0.75H,
CH2NH2, PhCH trans), 7.11e7.31 (m, 5H, arom). Ratio of cis-10:trans-
NaOH (2 x). The combined NaOH-layers were extracted with ethyl
acetate (1 x). The combined organic layers were dried (Na2SO4),
filtered, concentrated in vacuo and the residue was purified by flash
column chromatography (Ø 2.5 cm, h 15 cm, 10 mL, cyclo-
hexane:ethyl acetate ¼ 7:1 þ 1% dimethylethylamine, Rf ¼ 0.27).
Pale yellow oil, yield 112 mg (83%). Purity (HPLC method E): 99.2%
(tR ¼ 19.5 min). C21H27N (293.5 g/mol). MS (ESI): m/z ¼ 294
(M þ Hþ). IR: ṽ (cmꢁ1) ¼ 2917, 2848 (CeH), 731, 696 (C-Harom). The
diastereomers cis-11 and trans-11 were separated by preparative
HPLC (HPLC method A1).
10 ¼ 75:25.13C NMR (CDCl3):
d
(ppm) ¼ 21.4, 30.0, 30.8, 33.8, 36.1,
38.0 (6 ꢂ 0.25C, cyclohexane trans, CH2CH2N trans), 26.5, 32.8, 34.1,
35.6, 39.5, 41.2 (6 ꢂ 0.75C, cyclohexane cis, CH2CH2N cis), 38.1
(0.25C, CH2NH2 trans), 40.5 (0.25C, PhCH trans), 41.2 (0.75C,
CH2NH2 cis), 44.3 (0.75C, PhCH cis), 125.7 (0.25C, C-4 arom trans),
125.8 (0.75C, C-4 arom cis), 126.7, 128.3 (4 ꢂ 0.75C, arom cis), 126.9,
128.2 (4 ꢂ 0.25C, arom trans), 147.4 (0.25C, C-1 arom trans), 147.6
(0.75C, C-1 arom cis).
9.3.16. 2-[(1R,3S)-3-Phenylcyclohexyl]ethanamine ((1R,3S)-10)
A solution of the benzylamine (1R 3S)-11 (40 mg, 0.14 mmol) in
THF (12 mL) was filled in a pressure vessel. Pd/C (10%, 16 mg) was
added and the mixture was placed in a hydrogenation apparatus.
The solution was stirred under H2 under pressure (5 bar) for 27 h.
Then it was filtered through Celite®, the residue was washed with
ethyl acetate and the combined organic layers were concentrated in
vacuo. The product was purified by flash column chromatography
twice (1. Ø 1 cm, h 15 cm, 5 mL, CH2Cl2:CH3OH ¼ 1:1, 2. Ø 1 cm, h
16 cm, 5 mL, CH2Cl2:CH3OH ¼ 1:1). Yellow oil, yield 13 mg (47%).
Purity (HPLC method E): 94.0% (tR ¼ 16.7 min). MS (EM, APCI): m/
z ¼ calculated for C14H22N (M þ Hþ) 204.1747, found 204.1758.
Further analytical data see diastereomeric mixture cis-10 and
trans-10.
9.3.21. cis-N-Benzyl-2-(3-phenylcyclohexyl)ethanamine (cis-11)
After separation by preparative HPLC method A1, the fractions
containing cis-11 were concentrated in vacuo to about 100 mL and
extracted with CH2Cl2 (4 x). The combined organic layers were
dried (Na2SO4), filtered and the solvent was evaporated under
reduced pressure. Colorless oil. Purity (HPLC method E): 99.7%
(tR ¼ 20.2 min). Retention time: 110 min (HPLC method A1). 1H
NMR (CDCl3):
d
(ppm) ¼ 0.87 (qd, J ¼ 12.5/3.0 Hz, 1H, 4-Hax
-
cyclohexane or 6-Hax-cyclohexane), 1.04 (q, J ¼ 12.2 Hz, 1H, 2-Hax
-
cyclohexane), 1.18e1.83 (m, 9H, cyclohexane, CH2CH2N), 2.45 (tt,
J ¼ 11.9/3.0 Hz, 1H, PhCH), 2.60 (t, J ¼ 7.4 Hz, 2H, CH2CH2NH), 3.71
(s, 2H, NHCH2Ph), 7.09e7.27 (m, 10H, arom). 13C NMR (CDCl3):
d
(ppm) ¼ 26.5, 32.9, 34.2, 36.1, 37.8, 41.2 (6C, cyclohexane,
CH2CH2N), 44.3 (1C, PhCH), 47.1 (1C, CH2CH2NH), 54.1 (1C,
NHCH2Ph), 125.8 (1C, C-4 arom(benzyl)), 126.8 (2C, arom), 126.9 (1C,
C-4 arom(benzene)), 128.1, 128.3, 128.4 (6C, arom), 140.4 (1C, C-1
arom(benzyl)), 147.7 (1C, C-1 arom(benzene)).
9.3.17. 2-[(1S,3R)-3-Phenylcyclohexyl]ethanamine ((1S,3R)-10)
As described for (1R,3S)-10, a mixture of the benzylamine
(1S,3R)-11 (24 mg, 0.08 mmol) and Pd/C (10%,11 mg) in THF (12 mL)
was reacted with H2 under pressure (5 bar) for 25.5 h. The residue
was purified by flash column chromatography (Ø 1 cm, h 13 cm,
5 mL, CH2Cl2:CH3OH ¼ 1:1). Pale yellow oil, yield 7 mg (44%). Purity
(HPLC method E): 98.0% (tR ¼ 16.3 min). MS (EM, APCI): m/
z ¼ calculated for C14H22N (M þ Hþ) 204.1747, found 204.1751.
Further analytical data see diastereomeric mixture cis-10 and
trans-10.
9.3.22. trans-N-Benzyl-2-(3-phenylcyclohexyl)ethanamine (trans-
11)
After separation by preparative HPLC method A1, the fractions
containing trans-11 were concentrated in vacuo to about 40 mL and
extracted with CH2Cl2 (4 x). The combined organic layers were
dried (Na2SO4), filtered and the solvent was evaporated under
reduced pressure. Colorless oil. Purity (HPLC method E): 99.4%
(tR ¼ 20.0 min). Retention time: 102 min (HPLC method A1). 1H
9.3.18. 2-[(1S,3S)-3-Phenylcyclohexyl]ethanamine ((1S,3S)-10)
As described for (1R,3S)-10, a mixture of the benzylamine
(1S,3S)-11 (37 mg, 0.13 mmol) and Pd/C (10%, 22 mg) in THF (14 mL)
was reacted with H2 under pressure (5 bar) for 27 h. The residue
was purified by flash column chromatography (Ø 1 cm, h 15 cm,
5 mL, CH2Cl2:CH3OH ¼ 1:1). Yellow oil, yield 21 mg (81%). Purity
(HPLC method E): 92% (tR ¼ 16.4 min). MS (EM, APCI): m/
z ¼ calculated for C14H22N (M þ Hþ) 204.1747, found 204.1748.
Further analytical data see diastereomeric mixture cis-10 and
trans-10.
NMR (CDCl3):
d
(ppm) ¼ 1.18e1.88 (m,11H, cyclohexane, CH2CH2N),
2.60 (t, J ¼ 7.6 Hz, 2H, CH2CH2NH), 2.65e2.72 (m, 1H, PhCH), 3.74 (s,
2H, NHCH2Ph), 7.08e7.26 (m, 10H, arom). 13C NMR (CDCl3):
d
(ppm) ¼ 21.4, 30.1, 31.3, 32.5, 33.9, 38.0 (6C, cyclohexane,
CH2CH2N), 38.1 (1C, PhCH), 47.9 (1C, CH2CH2NH), 54.1 (1C,
NHCH2Ph), 125.7 (1C, C-4 arom(benzyl)), 126.9 (3C, arom), 128.2,
128.3, 128.4 (6C, arom), 140.2 (1C, C-1 arom(benzyl)), 147.4 (1C, C-1
arom(benzene)).
9.3.23. N-benzyl-2-[(1R,3S)-3-phenylcyclohexyl]ethanamine
((1R,3S)-11)
9.3.19. 2-[(1R,3R)-3-Phenylcyclohexyl]ethanamine ((1R,3R)-10)
As described for (1R,3S)-10, a mixture of the benzylamine
(1R,3R)-11 (36 mg, 0.12 mmol) and Pd/C (10%,12 mg) in THF (12 mL)
was reacted with H2 under pressure (5 bar) for 26 h. The residue
was purified by flash column chromatography (Ø 1 cm, h 14 cm,
5 mL, CH2Cl2:CH3OH ¼ 1:1). Yellow oil, yield 10 mg (40%). Purity
(HPLC method E): 91% (tR ¼ 16.3 min). MS (EM, APCI): m/
z ¼ calculated for C14H22N (M þ Hþ) 204.1747, found 204.1749.
Further analytical data see diastereomeric mixture cis-10 and
trans-10.
As described for cis/trans-11, a solution of (1R,3S)-9 (88 mg,
0.31 mmol) and benzylamine (0.10 mL, 0.93 mmol) in CH3CN
(10 mL) was heated to reflux for 23 h. The residue was purified by
flash column chromatography (Ø 2.5 cm, h 17 cm, 10 mL, cyclo-
hexane:ethyl acetate ¼ 7:1 þ1% dimethylethylamine). Colorless oil,
yield 77 mg (85%). Purity (HPLC method E): 99.0% (tR ¼ 19.5 min).
Specific rotation: ½a D20
¼ þ27.4 (c ¼ 0.99; CH2Cl2). Further analytical
ꢃ
data see cis-11.
9.3.24. N-benzyl-2-[(1S,3R)-3-phenylcyclohexyl]ethanamine
((1S,3R)-11)
9.3.20. cis-N-Benzyl-2-(3-phenylcyclohexyl)ethanamine (cis-11)
and trans-N-benzyl-2-(3-phenylcyclohexyl)ethanamine (trans-11)
Benzylamine (0.15 mL, 1.39 mmol) was added to a solution of
cis/trans-9 (131 mg, 0.46 mmol) in CH3CN (10 mL) and the mixture
was heated to reflux for 15 h. The solvent was removed in vacuo.
The residue was dissolved in ethyl acetate and washed with 0.5 M
As described for cis/trans-11, a solution of (1S, 3R)-9 (77 mg,
0.27 mmol) and benzylamine (0.09 mL, 0.81 mmol) in CH3CN
(6 mL) was heated to reflux for 15 h. The residue was purified by
flash column chromatography (Ø 2.5 cm, h 16 cm, 10 mL, cyclo-
hexane:ethyl acetate ¼ 7:1 þ1% dimethylethylamine). Colorless oil,
13

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N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
yield 66 mg (83%). Purity (HPLC method E): 99.3% (tR ¼ 19.5 min).
(max.) 140 ꢀC; reaction time ramp time 5 min, hold time 15 min.
After the microwave irradiation, the solvent was evaporated under
reduced pressure and the crude product was purified by flash
Specific rotation: ½a D20
¼ ꢁ27.3 (c ¼ 0.99; CH2Cl2). Further analytical
ꢃ
data see cis-11.
column chromatography (Ø
2
cm,
h
18 cm,
8
mL,
CH2Cl2:CH3OH ¼ 9.5:0.5 þ 1% NEt3, Rf ¼ 0.33). Pale yellow resin,
9.3.25. N-benzyl-2-[(1S,3S)-3-phenylcyclohexyl]ethanamine
((1S,3S)-11)
yield 22 mg (79%). Purity (HPLC method E): 97.1% (tR ¼ 18.0 min).
C
16H25N (231.4 g/mol). MS (ESI): m/z ¼ 232 (M þ Hþ). MS (EM,
As described for cis/trans-11, a solution of (1S, 3S)-9 (26 mg,
0.09 mmol) and benzylamine (0.03 mL, 0.27 mmol) in CH3CN
(8 mL) was heated to reflux for 23 h. The residue was purified by
flash column chromatography (Ø 2.5 cm, h 15 cm, 10 mL, cyclo-
hexane:ethyl acetate ¼ 7:1 þ1% dimethylethylamine). Colorless oil,
yield 22 mg (85%). Purity (HPLC method E): 97.5% (tR ¼ 19.4 min).
APCI): m/z ¼ calculated for C16H26N (M þ Hþ) 232.2060, found
232.2119. IR: ṽ (cmꢁ1) ¼ 2920, 2852 (CeH), 2813, 2761 (NeCeH),
752, 697 (C-Harom). 1H NMR (CDCl3):
d
(ppm) ¼ 0.85e0.94 (m,
0.75H, cyclohexane cis), 1.05 (q, J ¼ 12.3 Hz, 0.75H, 2-Hax-cyclo-
hexane cis), 1.18e1.85 (m, 11 ꢂ 0.25H, 9 ꢂ 0.75H, cyclohexane,
CH2CH2N), 2.20 (s, 6 ꢂ 0.75H, (tt, J ¼ 11.9/3.0 Hz, 0.75H, PhCH cis),
2.64e2.73 (m, 0.25H, PhCH trans), 7.08e7.24 (m, 5H, arom). Ratio of
Specific rotation: ½a D20
¼ þ8.3 (c ¼ 0.90; CH2Cl2). Further analytical
ꢃ
data see trans-11.
cis-13:trans-13 ¼ 75:25.13C NMR (CDCl3):
(ppm) ¼ 21.4, 29.8, 30.0,
d
31.5, 33.9, 38.0 (6 ꢂ 0.25C, cyclohexane trans, CH2CH2N trans), 26.4,
32.9, 34.1, 35.0, 36.2, 41.2 (6 ꢂ 0.75C, cyclohexane cis, CH2CH2N cis),
38.2 (0.25C, PhCH trans), 44.3 (0.75C, PhCH cis), 45.3 (2 ꢂ 0.75C,
9.3.26. N-benzyl-2-[(1R,3R)-3-phenylcyclohexyl]ethanamine
((1R,3R)-11)
As described for cis/trans-11, a solution of (1R,3R)-9 (24 mg,
0.08 mmol) and benzylamine (0.03 mL, 0.25 mmol) in CH3CN
(8 mL) was heated to reflux for 23 h. The residue was purified by
flash column chromatography (Ø 2.5 cm, h 16 cm, 10 mL, cyclo-
hexane:ethyl acetate ¼ 7:1 þ1% dimethylethylamine). Colorless oil,
yield 21 mg (89%). Purity (HPLC method E): 95.1% (tR ¼ 19.3 min).
N(CH3)2 cis), 45.3 (2
ꢂ
0.25C, N(CH3)2 trans), 57.3 (0.75C,
CH2N(CH3)2 cis), 58.3 (0.25C, CH2N(CH3)2 trans), 125.7 (0.25C, C-4
arom trans), 125.8 (0.75C, C-4 arom cis), 126.8 (2 ꢂ 0.75C, C-2, C-6
arom cis), 126.9 (2 ꢂ 0.25C, C-2, C-6 arom trans), 128.2 (2 ꢂ 0.25C,
C-3, C-5 arom trans), 128.3 (2 ꢂ 0.75C, C-3, C-5 arom cis), 147.4
(0.25C, C-1 arom trans), 147.6 (0.75C,C-1 arom cis).
Specific rotation: ½a D20
¼ ꢁ8.3 (c ¼ 0.90; CH2Cl2). Further analytical
ꢃ
data see trans-11.
9.3.29. N,N-Dimethyl-2-[(1R,3S)-3-phenylcyclohexyl]ethanamine
((1R,3S)-13)
9.3.27. cis- and trans-4-phenyl-N-[2-(3-phenylcyclohexyl)ethyl]
butan-1-amine (cis-12 and trans-12)
Pd/C (10%, 9 mg) was added to a solution of benzylamine
(1R,3S)-11 (37 mg, 0.13 mmol) in CH3OH (15 mL). HOAc (2 drops)
was added and the mixture was stirred under H2 (balloon) for
22.5 h. Additional Pd/C (10%, 9 mg) was added and the solution was
again stirred under H2 (balloon) for 6 h. Then it was filtered, the
solvent was removed in vacuo and the residue was purified by flash
column chromatography twice (1. Ø 1 cm, h 13 cm, 5 mL,
CH2Cl2:CH3OH ¼ 9.5:0.5 þ 1% dimethylethylamine, Rf ¼ 0.33, 2. Ø
1 cm, h 16 cm, 5 mL, CH2Cl2:CH3OH ¼ 1:1, Rf ¼ 0.30). Pale yellow oil,
yield 15 mg (48%). Purity (HPLC method E): 96.8% (tR ¼ 17.0 min).
MS (EM, APCI): m/z ¼ calculated for C16H26N (M þ Hþ) 232.2060,
found 232.2052. Further analytical data see diastereomeric mixture
cis-13 and trans-13.
After dissolving cis-9/trans-9 (51 mg, 0.18 mmol) in CH3CN
(10 mL), 4-phenylbutan-1-amine (0.09 mL, 0.54 mmol) was added
and the mixture was heated to reflux for 22 h. Then the solvent was
evaporated in vacuo and ethyl acetate (15 mL) was added. The
resulting suspension was washed with 0.5 M NaOH (2 x 5 mL). The
organic layer was dried (Na2SO4), filtered, concentrated in vacuo
and the residue was purified by flash column chromatography (Ø
2.5 cm, h 15 cm, 10 mL, CH2Cl2:CH3OH ¼ 4:1, Rf ¼ 0.28). Pale yellow
resin, yield 45 mg (75%). Purity (HPLC method E): 97.3%
(tR ¼ 21.9 min). C24H33N (335.5 g/mol). MS (ESI): m/z ¼ 336
(M þ Hþ). MS (EM, APCI): m/z ¼ calculated for C24H34N (M þ Hþ)
336.2686, found 336.2694. IR: ṽ (cmꢁ1) ¼ 2920, 2852 (CeH), 745,
697 (C-Harom). 1H NMR (CDCl3):
d
(ppm) ¼ 0.89e1.00 (m, 0.75H,
9.3.30. N,N-Dimethyl-2-[(1S,3R)-3-phenylcyclohexyl]ethanamine
((1S,3R)-13)
cyclohexane cis), 1.11 (q, J ¼ 12.3 Hz, 0.75H, 2-Hax-cyclohexane cis),
1.26e1.89 (m, 15 ꢂ 0.25H, 13 ꢂ 0.75H, cyclohexane, CH2CH2N,
PhCH2CH2CH2), 2.52 (tt, J ¼ 11.9/3.1 Hz, 0.75H, PhCH cis), 2.60e2.80
(m, 7.25H, CH2NHCH2, CH2Ph, PhCH trans), 7.15e7.30 (m, 10H,
As described for (1R,3S)-13, a solution of (1S,3R)-11 (37 mg,
0.13 mmol), Pd/C (10%, 10 mg) and HOAc (2 drops) in CH3OH
(15 mL) was stirred under H2 (balloon) for 19.5 h. Addition of
further Pd/C (10%) was not necessary. The residue was purified by
flash column chromatography twice (1. Ø 1 cm, h 18 cm, 5 mL,
CH2Cl2:CH3OH ¼ 9.5:0.5 þ 1% dimethylethylamine, 2. Ø 1 cm, h
15 cm, 5 mL, CH2Cl2:CH3OH ¼ 1:1). Pale yellow oil, yield 9 mg (30%).
Purity (HPLC method E): 96.1% (tR ¼ 17.0 min). MS (EM, APCI): m/
z ¼ calculated for C16H26N (M þ Hþ) 232.2060, found 232.2048.
Further analytical data see diastereomeric mixture cis-13 and
trans-13.
arom). Ratio of cis-12:trans-12
¼
75:25.13
C NMR (CDCl3):
d
(ppm) ¼ 21.4, 29.2, 29.3, 30.1, 31.4, 32.1, 33.9, 35.8 (8 ꢂ 0.25C,
cyclohexane trans, CH2CH2N trans, CH2CH2CH2CH2 trans), 26.5,
29.0, 29.1, 32.8, 34.1, 35.7, 36.1, 37.1 (8 ꢂ 0.75C, cyclohexane cis,
CH2CH2N cis, CH2CH2CH2CH2 cis), 37.9 (0.25C, PhCH trans), 38.1
(0.25C, PhCH2 trans), 41.1 (0.75C, PhCH cis), 44.3 (0.75C, PhCH2 cis),
47.3 (0.75C, NHCH2 cis), 48.3 (0.25C, NHCH2 trans), 49.6 (0.75C,
NHCH2 cis), 49.7 (0.25C, NHCH2 trans), 125.7, 125.8, 125.9, 126.8,
126.9, 128.2, 128.3, 128.4 (10C, arom), 142.2 (0.75C, C-1 arom cis),
142.3 (0.25C, C-1 arom trans), 147.3 (0.25C, C-1 arom trans), 147.6
(0.75C, C-1 arom cis).
9.3.31. cis-1-[2-(3-Phenylcyclohexyl)ethyl]pyrrolidine (cis-14) and
trans-1-[2-(3-Phenylcyclohexyl)ethyl]pyrrolidine (trans-14)
Pyrrolidine (0.18 mL, 2.18 mmol) was added to a solution of
mesylate cis-9/trans-9 (205 mg, 0.73 mmol) in CH3CN (25 mL) and
the mixture was heated to reflux for 25 h CH3CN was removed in
vacuo and the residue was dissolved in ethyl acetate (30 mL). The
organic layer was washed with 0.5 M NaOH (2 x 10 mL). Due to slow
phase separation more ethyl acetate was added. Afterwards the
organic layer was dried (Na2SO4), filtered and concentrated in
vacuo. The residue was purified by flash column chromatography
9.3.28. cis- and trans-N,N-Dimethyl-2-(3-phenylcyclohexyl)
ethanamine (cis-13 and trans-13)
The mesylate cis-9/trans-9 (34 mg, 0.12 mmol) was dissolved in
CH3CN (6 mL) and transferred to a 10-mL microwave reaction
vessel. A 2 M solution of dimethylamine in THF (0.18 mL,
0.36 mmol) was added. The microwave parameters were set as
follows: Power (max.) 200 W; pressure (max.) 8 bar; temperature
14

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N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
(Ø 2 cm, h 16 cm, 10 mL, CH2Cl2:CH3OH ¼ 9:1 þ1% NEt3, Rf ¼ 0.57).
Pale yellow oil, yield 135 mg (72%). Purity (HPLC method E): 97.8%
(tR ¼ 18.6 min). C18H27N (257.4 g/mol). MS (ESI): m/z ¼ 591
(2 M þ Ph), 258 (M þ Hþ). IR: ṽ (cmꢁ1) ¼ 2919, 2850 (CeH), 2782
(NeCeH), 754, 698 (C-Harom). The diastereomers cis-14 and trans-
14 were separated by preparative HPLC (HPLC method A3).
Colorless solid. Purity (HPLC method E): 99.3% (tR ¼ 21.0 min).
Retention time: 139 min (HPLC method A2). MS (EM, APCI): m/
z ¼ calculated for C24H33N2 (M þ Hþ) 349.2638, found 349.2659.1H
NMR (CDCl3):
d
(ppm) ¼ 0.86e0.97 (m, 1H, cyclohexane), 1.07 (q,
J ¼ 12.2 Hz,1H, 2-Hax-cyclohexane),1.19e1.48 (m, 5H, cyclohexane),
1.73e1.87 (m, 4H, cyclohexane, CH2CH2N), 2.37 (t, J ¼ 7.1 Hz, 2H,
CH2CH2N), 2.47 (tt, J ¼ 11.9/3.0 Hz, 1H PhCH), 2.53 (t, J ¼ 5.0 Hz, 4H,
CH2N(CH2CH2)2N), 3.14 (t, J ¼ 5.1 Hz, 4H, N(CH2CH2)2NPh), 6.78 (tt,
J ¼ 7.3/0.8 Hz,1H, arom(phenylpiperazine) para), 6.86 (dd, J ¼ 8.7/0.9 Hz,
2H, arom(phenylpiperazine) ortho), 7.09e7.24 (m, 7H, arom).
9.3.32. cis-1-[2-(3-Phenylcyclohexyl)ethyl]pyrrolidine (cis-14)
After separation by preparative HPLC method A3, the fractions
containing cis-14 were concentrated in vacuo to about 150 mL and
extracted with CH2Cl2 (4 x). The combined organic layers were
dried (Na2SO4), filtered and the solvent was evaporated under
reduced pressure. Pale yellow oil. Purity (HPLC method E): 99.5%
(tR ¼ 18.5 min). Retention time: 129 min (HPLC method A3). MS
(EM, APCI): m/z ¼ calculated for C18H28N (M þ Hþ) 258.2216, found
9.3.36. trans-1-Phenyl-4-[2-(3-phenylcyclohexyl)ethyl]piperazine
(trans-15)
After separation by preparative HPLC, CH3CN of the fractions
containing trans-15 was removed and the residue was extracted
with CH2Cl2 (4 x). The combined organic layers were dried
(Na2SO4), filtered and the solvent was evaporated under reduced
pressure. Pale brown oil. Purity (HPLC method E): 99.0%
(tR ¼ 20.4 min). Retention time: 131 min (HPLC method A2). MS
(EM, APCI): m/z ¼ calculated for C24H33N2 (M þ Hþ) 349.2638,
258.2255.1H NMR (CDCl3):
d
(ppm) ¼ 0.85e0.94 (m, 1H, cyclo-
hexane), 1.06 (q, J ¼ 12.2 Hz, 1H, 2-Hax-cyclohexane), 1.19e1.85 (m,
13H, cyclohexane, CH2CH2N, N(CH2CH2)2), 2.39e2.45 (m, 6H,
CH2N(CH2CH2)2), 2.50 (tt, J ¼ 11.9/3.1 Hz, 1H, PhCH), 7.09e7.24 (m,
5H, arom). 13C NMR (CDCl3):
d
(ppm) ¼ 23.4 (2C, N(CH2CH2)2, 26.5,
33.0, 34.2, 36.7, 41.3 (6C, cyclohexane, CH2CH2N), 44.4 (1C, PhCH),
54.2 (2C, N(CH2CH2)2), 54.3 (1C, CH2N(CH2CH2)2), 125.8 (1C, C-4
arom), 126.8 (2C, C-2, C-6 arom), 128.3 (2C, C-3, C-5 arom), 147.7
(1C, C-1 arom).
found 349.2612.1H NMR (CDCl3):
d
(ppm) ¼ 1.40e1.85 (m, 11H,
cyclohexane, CH2CH2N), 2.36 (t, J ¼ 7.8 Hz, 2H, CH2CH2N), 2.56 (t,
J ¼ 4.8 Hz, 4H, CH2N(CH2CH2)2N), 2.68e2.75 (m, 1H, PhCH), 3.16 (t,
J ¼ 4.9 Hz, 4H, N(CH2CH2)2NPh), 6.79 (t, J ¼ 7.3 Hz, 1H, arom(phe-
nylpiperazine) para), 6.87 (d, J ¼ 8.4 Hz, 2H, arom(phenylpiperazine) ortho),
7.09e7.24 (m, 7H, arom).
9.3.33. trans-1-[2-(3-Phenylcyclohexyl)ethyl]pyrrolidine (trans-14)
After separation by preparative HPLC method A3, the fractions
containing trans-14 were concentrated in vacuo to about 40 mL and
extracted with CH2Cl2 (4 x). The combined organic layers were
dried (Na2SO4), filtered and the solvent was evaporated under
reduced pressure. Pale yellow oil. Purity (HPLC method E): 96.7%
(tR ¼ 18.4 min). Retention time: 118 min (HPLC method A3). MS
(EM, APCI): m/z ¼ calculated for C18H28N (M þ Hþ) 258.2216, found
9.4. Receptor binding studies
Experimental details for recording s1 and s2 receptor affinity
[46e48] are given in the Supporting Information.
9.5. Computational details
258.2259.1H NMR (CDCl3):
hexane, CH2CH2N, N(CH2CH2)2),
CH2N(CH2CH2)2), 2.66e2.74 (m, 1H, PhCH), 7.08e7.24 (m, 5H,
arom). 13C NMR (CDCl3):
d
(ppm) ¼ 0.66e1.83 (m, 15H, cyclo-
2.38e2.47 (m, 6H,
The membrane-bound 3D structure of the s1 receptor was ob-
tained starting from the available Protein Data Bank file (pdb code:
5HK1)11 and following a procedure previously described [50,51].
The optimized structures of the new tested compounds were
docked into each protein identified binding pocket using Autodock
4.2.6/Autodock Tools1.4.61[64] on a win64 platform. The resulting
complex was further energy minimized to convergence. The
intermolecular complex was then solvated by a cubic box of TIP3P
water molecules [65] and energy minimized using a combination of
molecular dynamics (MD) techniques [50,51]. 20 ns molecular dy-
namics (MD) simulations at 298 K were then employed for system
equilibration, and further, 50-ns MD were run for data production.
Following the MM/PBSA approach [52] each binding free energy
d
(ppm) ¼ 21.4, 29.7, 30.2, 31.9, 34.0, 38.0
(6C, cyclohexane, CH2CH2N), 23.4 (2C, N(CH2CH2)2), 38.1 (1C,
PhCH), 54.3 (2C, N(CH2CH2)2), 55.3 (1C, CH2N(CH2CH2)2), 125.7 (1C,
C-4 arom),126.9 (2C, C-2, C-6 arom),128.2 (2C, C-3, C-5 arom),147.4
(1C, C-1 arom).
9.3.34. cis-1-Phenyl-4-[2-(3-phenylcyclohexyl)ethyl]piperazine
(cis-15) and trans-1-phenyl-4-[2-(3-phenylcyclohexyl)ethyl]
piperazine (trans-15)
To a solution of mesylate cis-9/trans-9 (126 mg, 0.45 mmol) in
CH3CN (27 mL), 1-phenylpiperazine (0.20 mL, 1.34 mmol) was
added and the mixture was heated to reflux for 27 h. Then the
solvent was evaporated in vacuo, the residue was dissolved in ethyl
acetate (40 mL) and the organic layer was washed with 0.5 M NaOH
(2 x 10 mL). The organic layer was dried (Na2SO4), filtered, the
solvent was removed in vacuo and the residue was purified by flash
column chromatography (Ø 3.5 cm, h 15.5 cm, 10 mL, cyclo-
hexane:ethyl acetate ¼ 3:1, Rf ¼ 0.61 (ethyl acetate)). Pale yellow
resin, yield 140 mg (89%). C24H32N2 (348.5 g/mol). MS (ESI): m/
z ¼ 349 (M þ Hþ). IR: ṽ (cmꢁ1) ¼ 2919 (CeH), 2815 (NeCeH), 754,
697 (C-Harom). The diastereomers cis-15 and trans-15 were sepa-
rated by preparative HPLC (HPLC method A2).
values (
der Waals, polar solvation, nonpolar solvation,
entropic contributions (T Sbind). The PRBFED analysis was carried
DGbind) were calculated as the sum of the electrostatic, van
(
D
Hbind and
)
D
out using the Molecular Mechanics/Generalized Boltzmann Surface
Area (MM/GBSA) approach [66] and was based on the same snap-
shots used in the binding free energy calculation. All simulations
were carried out using the pmemd and pmemd.CUDA modules of
Amber 18,[67] running on our own CPU/GPU calculation cluster.
Molecular graphics images were produced using the UCSF Chimera
package (v.1.14) [68]. All other graphs were obtained using Graph-
Pad Prism (v. 6.0) [69].
9.3.35. cis-1-Phenyl-4-[2-(3-phenylcyclohexyl)ethyl]piperazine
(cis-15)
After separation by preparative HPLC, CH3CN of the fractions
containing cis-15 was removed and the residue was extracted with
CH2Cl2 (4 x). The combined organic layers were dried (Na2SO4),
filtered and the solvent was evaporated under reduced pressure.
9.6. Ca2þ influx assay, fluorescence measurements with fura-2-AM
Concentrations c (Ca2þi) were measured in single PC12 cells
using the fluorescent indicator fura-2-AM in combination with a
monochromator-based imaging system (FEI today Thermo Fisher
Scientific, SCR_008452) attached to a fluid immersion objective.
15

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N. Kopp, C. Holtschulte, F. Borgel et al.
European Journal of Medicinal Chemistry 210 (2021) 112950
Cells were loaded with 0.5
mM fura-2-AM and 0.01% Pluronic F-127
Supporting Information
for 30 min at 37 ꢀC in a standard solution composed of 138 mM
NaCl, 6 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5.5 mM glucose, and
10 mM HEPES (adjusted to pH 7.4 with NaOH at 37 ꢀC). Cover slips
were then washed in fresh buffer for 30 min and mounted in a
perfusion chamber on the stage of the microscope (Olympus
EX51WI, Hamburg, Germany). For measurements of c (Ca2þi), cells
were excited at 340 and 380 nm and emission was measured at
510 nm. After correction for background fluorescence, the fluores-
cence ratio F340/F380 of the emission was calculated. Fura-2-
signals were calibrated according to the method of Grynkiewicz
et al. [53], using a KD value of 224 nM. 10e20 cells were measured
on slide and at least two replicates/independent experiment were
conducted. At least five independent experiments were measured.
The Supporting Information contains purity data of all com-
pounds, chiral HPLC of the stereoisomeric alcohols 7, experimental
procedures for receptor binding studies, in silico binding thermo-
dynamics and per residue binding enthalpy, details on biotrans-
formation studies, and 1H and 13C NMR spectra of prepared
compounds.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
9.7. Proliferation assay
This work was supported by the Deutsche For-
schungsgemeinschaft (DFG) and the Swiss National Science Foun-
dation (SNSF), which is gratefully acknowledged. We thank Prof. Dr.
S. Grimme for calculating the CD spectra.
Human prostate cancer cell lines (i.e., DU145, PC3 and 22Rv1)
and lung cancer cell lines (i.e, H358, H441 and H522) were pur-
chased from ATCC and maintained in adherent conditions in RPMI
1640 medium (21,875e034, Gibco®) supplemented with 2% (S)-
glutamine, 10% fetal bovine serum (FBS) (10,437e036, Gibco®) and
1% penicillin-streptomycin (15,140e122, Gibco®). For the prolifer-
ation assays, the cells were seeded at a density of 1 ꢂ 103 (lung) or
2 ꢂ 103 (prostate) cells/well in 96-well tissue culture plates. 24 h
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112950.
later, the cells were treated with the compounds (10 mM) for 72 h.
Then, the cells were fixed with 10% (w/v) trichloroacetic acid and
stained with sulforhodamine B for 30 min. The excess of the dye
was removed by washing repeatedly with 1% (v/v) acetic acid and
the protein-bound dye was solubilized with Tris base solution for
optical density (OD) measurement at 510 nm with a microplate
reader. Cell proliferation and viability were expressed as percent-
ages relative to those of DMSO treated cells. *p ꢄ 0.05.
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MOPS (372.5 mg, 8.9 mM) and MOPS sodium salt (513.4 mg,
11.1 mM) were dissolved in H2O (200 mL) to prepare 20 mM MOPS
buffer with pH 7.4. n-octanol and MOPS buffer were saturated with
each other by stirring a two-phase system for 24 h. The final con-
centration of DMSO was kept below 1% in all samples. The logD7.4
values were determined using three different volume ratios of
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