New combination of pharmacophoric elements of potent σ1 ligands: Design, synthesis and σ receptor affinity of aminoethyl substituted tetrahydrobenzothiophenes

Article abstract of DOI:10.1016/j.ejmech.2013.09.006

The aminoethyl substituted tetrahydrobenzothiophenes 4 resulted from combination of the pharmacophoric elements of the potent σ₁ ligands 2 and 3. The aminoethyl substituted tetrahydrobenzothiophenes 4 were prepared in an 8-step synthesis starting with thiophene. Whereas the σ₁ affinity of the N-benzyl derivative 4a is in the medium nanomolar range (K_i = 49 nM), the analogous N-cyclohexylmethyl derivative 4d exhibits low nanomolar affinity (K_i = 5.0 nM). The reduced σ₁ affinity and σ₂/σ₁ selectivity of tetrahydrobenzothiophenes 4 compared to analogous spirocyclic piperidines 3 is attributed to the increased conformational flexibility of the aminoethyl side chain.

Full text of DOI:10.1016/j.ejmech.2013.09.006

Accepted Manuscript
New combination of pharmacophoric elements of potent σ ligands: Design,
1
synthesis and σ receptor affinity of aminoethyl substituted tetrahydrobenzothiophenes
Dipak Harel, Dirk Schepmann, Bernhard Wünsch
PII:
S0223-5234(13)00578-3
10.1016/j.ejmech.2013.09.006
EJMECH 6408
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 June 2013
Revised Date: 4 September 2013
Accepted Date: 5 September 2013
Please cite this article as: D. Harel, D. Schepmann, B. Wünsch, New combination of pharmacophoric
elements of potent σ ligands: Design, synthesis and σ receptor affinity of aminoethyl substituted
1
tetrahydrobenzothiophenes, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.09.006.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
1
New combination of pharmacophoric elements of potent σ1 ligands: Design, synthesis and σ
receptor affinity of aminoethyl substituted tetrahydrobenzothiophenes
Dipak Harel, Dirk Schepmann, Bernhard Wünsch*
Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität
Münster, Corrensstraße 48, D-48149 Münster, Germany
Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail: wuensch@uni-muenster.de
Abstract
The aminoethyl substituted tetrahydrobenzothiophenes 4 resulted from combination of the
pharmacophoric elements of the potent σ1 ligands 2 and 3. The aminoethyl substituted
tetrahydrobenzothiophenes 4 were prepared in an 8-step synthesis starting with thiophene.
Whereas the σ1 affinity of the N-benzyl derivative 4a is in the medium nanomolar range (Ki = 49
nM), the analogous N-cyclohexylmethyl derivative 4d exhibits low nanomolar affinity (Ki = 5.0
nM). The reduced σ1 affinity and σ2/σ1 selectivity of tetrahydrobenzothiophenes 4 compared to
analogous spirocyclic piperidines 3 is attributed to the increased conformational flexibility of the
aminoethyl side chain.
Key words
σ1 ligands; conformational flexibility; combination of pharmacophoric elements;
tetrahydrobenzothiophenes; Horner-Wittig reaction

ACCEPTED MANUSCRIPT
2
1. Introduction
It is well established that σ receptors represent a unique, non-opioid, non-phencyclidine but
haloperidol sensitive receptor family. It consists of two subtypes known as σ1 and σ2 receptor.
They are widely expressed in the central nervous system, but they are also found in various
tissues and organs in the periphery (e.g. heart, liver, lung, and kidney) [1,2]. The gene encoding
the σ1 receptor protein was determined by successful cloning. The protein contains 223 amino
acids and has a molecular weight 25.3 kDa. The membrane-bound σ1 receptor contains two
transmembrane domains located at the mitochondrial-associated endoplasmic reticulum. This
unique structure of the σ1 receptor is different from typical G-protein coupled receptors, ion
channel receptors and tyrosine kinase receptors. Moreover, a similarity to any mammalian
protein on the level of amino acid sequence could not be detected. However, a 30 % homology to
the yeast enzyme sterol ∆8/7-isomerase was found [3-7]. The amino acid sequence of the σ2
receptor subtype is not known and therefore this subtype is less characterized. Very recently, it
has been reported that the σ2 receptor might be identical to the progesterone receptor membrane
component 1 (pgrmc1), which has already been cloned [8,9].
It has been shown that σ1 receptor antagonists have a potential for the treatment of acute and
chronic neurological disorders including schizophrenia, neuropathic pain as well as alcohol and
cocaine abuse, whereas σ1 receptor agonists can be used for the treatment of depression, memory
disorders, including Alzheimer’s disease and stroke [10,11]. The σ1 receptor antagonist S1RA
(1) is currently investigated in phase 2 clinical trials for the treatment of neuropathic pain.
Therefore σ1 antagonists possess a high potential as innovative analgesics with reduced side
effects [12,13]. Due to the high expression of σ1 and σ2 receptors in several tumor cell lines (e.g.

ACCEPTED MANUSCRIPT
3
breast, lung, and prostate cancer cell lines), both receptor subtypes represent interesting targets
for the development of novel biomarkers for tumor diagnosis and antitumor drugs [14,15].
Figure 1: Development of novel σ1 receptor ligands 4 by combination of S1RA (1),
aminoethylindazoles 2 and spirocyclic thienopyrans 3.
R'
S
N
N
O
CH3
N
N
R
N
NR2
O
3
2
N
S
O
S1RA (1)
NR2
4
Our interest has been focused on the development of novel ligands with high affinity towards σ1
receptors and selectivity over related receptors, in particular the σ2 subtype, but also the
phencyclidine binding site of the NMDA receptor. The class of tetrahydroindazoles 2 with an
aminoethyl side chain shows high affinity towards the σ1 receptor. (Figure 1) In particular
compound 2a (R’ = CH3, NR2 = 4-phenylpiperidin-1-yl) represents a very potent and selective σ1
receptor antagonist (Ki (σ1) = 7.0 nM, Ki (σ2) = 39.7 nM) with analgesic activity in the
neuropathic pain model [16,17]. The indazoles 2 represent the lead compounds for the
development of S1RA (1). Bioisosteric replacement of the pyrazole ring of 2 with a thiophene
ring and conformational restriction of the flexible aminoethyl side chain led to the spirocyclic
thienopyrans 3 with very high σ1 affinity and selectivity over the σ2 subtype [18,19]. Therefore

ACCEPTED MANUSCRIPT
4
we planned to merge the aminoethylindazole system 2 with the spirocyclic thienopyran system 3
to form aminoethyl-substituted tetrahydrobenzothiophenes of type 4. In order to fit into the
common pharmacophore models [20] arylalkyl residues should be attached to the basic amino
moiety.
2. Chemistry
For the synthesis of the aminoethyl substituted tetrahydrobenzothiophenes 4 the ketone 9 was
required as key intermediate. According to literature [21,22] ketone 9 was synthesized in three
steps starting with thiophene (5). (Scheme 1)
Scheme 1: Synthesis of the α,β-unsaturated ester 10 starting with thiophene.
o
Reagents and reaction conditions: (a) AlCl3, CH2Cl2, 0 C, 65 %. (b) NH2NH2, KOH,
diethylene glycol, reflux, 72 %. (c) (i) SOCl2, CH2Cl2, reflux; (ii) SnCl2, EtOAc, rt, 62 %.
(d) EtO2CCH2PO(OEt)2, NaOEt, THF, rt, 60 %.

ACCEPTED MANUSCRIPT
5
Reaction of thiophene (5) with succinic anhydride (6) and AlCl3 provided the γ-ketoacid 7 in 65
% yield. The Huang-Minlong variation of the Wolff-Kishner reduction transformed the γ-
ketoacid 7 into the butyric acid 8. For this purpose a solution of γ-ketoacid 7, NH2NH2
monohydrate and KOH in diethylene glycol was heated to reflux to obtain the butyric acid 8 in
72 % yield. An intramolecular Friedel-Crafts acylation after conversion of the acid 8 into its acid
chloride provided the ketone 9 in 62 % yield.
The synthesis of the α,β-unsaturated ester 10 was achieved by a Horner-Wittig reaction of the
ketone 9 with triethyl phosphonoacetate. In the first attempt NaH was used for the deprotonation
of EtO2CCH2PO(OEt)2 leading to 40 % of a 60:40 mixture of (E)-10 and (Z)-10. The same ratio
of diastereomers was obtained after deprotonation of the phosphonate with NaOEt and
conducting the transformation at room temperature. However, performing the condensation of
o
the ketone 9 with the phosphonate at 0 C led almost exclusively to the (E)-configured
diastereomer (E)-10.
The ratio of (E):(Z) diastereomers and the configuration of isomers of 10 were determined by 1H
NMR spectroscopy. The proton in 3-position of the thiophene ring of (Z)-10 resonates as
characteristic doublet at 7.79 ppm. In case of (E)-10 the signal for the 3-CH proton appears at
6.99 ppm. The downfield shift of the 3-CH signal of (Z)-10 is due to the anisotropic effect of the
carbonyl moiety of the ester group. Only in case of the (Z)-configured isomer (Z)-10 the carbonyl
moiety of the planar thiophenylacrylic ester is close to 3-CH of the thiophene ring. This
anisotropic effect is not possible for (E)-10, since the CO2Et group is oriented opposite to 3-CH.

ACCEPTED MANUSCRIPT
6
Scheme 2: Synthesis of aminoethyl tetrahydrobenzothiophenes 4.
Reagents and reaction conditions: (a) H2,Pd/C, rt, 24 h, 70 %. (b) LiOH, THF:H2O, rt, 83
%. (c) RNH2. CDI, THF, 0 oC. (d) LiAlH4, THF, reflux; definition of residues R see Table.
Hydrogenation of the double bond of the α,β-unsaturated ester 10 was performed with H2 and
Pd/C. Since both diastereomers (E)-10 and (Z)-10 were transformed into the same saturated ester
11, separation of the diastereomeric esters (E)-10 and (Z)-10 was not necessary. The saturated
ester 11 was hydrolyzed with LiOH to obtain the acid 12 in 83 % yield. Coupling of acid 12 with
various amines using carbonyl-1,1’-diimidazole (CDI) provided the amides 13a-d. Finally
LiAlH4 reduction of the amides 13a-d afforded the aminoethyl substituted
tetrahydrobenzothiophenes 4a-d. Since at least two hydrophobic substituents are postulated by
the pharmacophore models to achieve high σ1 affinity, amines containing an additional lipophilic
aryl or cyclohexyl moiety were selected for the coupling reactions. (see Table 1)

ACCEPTED MANUSCRIPT
7
3. σ Receptor affinity
The σ1 and σ2 receptor affinities of the aminoethyl substituted tetrahydrobenzothiophenes 4a-d
were determined in competition experiments with the radioligands [3H]-(+)-pentazocine (σ1
assay) and [3H] ditolylguanidine (σ2 assay). Membrane preparations of guinea pig brains and rat
livers were used as receptor material in the σ1 and σ2 assay, respectively. Since ditolylguanidine
also interacts with σ1 receptors, an excess of the selective σ1 receptor ligand (+)-pentazocine was
added in the σ2 assay to mask the σ1 receptors [23,24].
Table 1: σ1 and σ2 receptor affinities of aminoethyl substituted tetrahydrobenzothiophenes 4 and
reference compounds.
Ki ± SEM [nM]
selectivity
R
σ2/σ1
σ1
σ2
3a18
PhCH2
C6H11CH2
PhCH2
0.31 ± 0.06
0.66 ± 0.16
49 ± 2.0
126 ± 73
132
13 ± 2.5
3.3 ± 0.3
149
42
5
3d18
3
4a
PhCH2CH2
PhCH2CH2CH2
C6H11CH2
-
129 ± 20
166
1
4b
1
4c
5.0 ± 2.0
5.7 ± 2.2
10 ± 1.0
-
2
4d
(+)-pentazocine
--
haloperidol
-
-
6.3 ± 1.6
89 ± 29
78 ± 2.3
58 ± 18
12
di-o-tolylguanidine
0.7

ACCEPTED MANUSCRIPT
8
In Table
1
the σ1 and σ2 receptor affinities of the aminoethyl substituted
tetrahydrobenzothiophenes 4 are summarized. The benzylamine 4a reveals a Ki-value of 49 nM
(σ1), which is considerably higher than the Ki-value of the benzyl substituted piperidine 3a (Ki =
0.31 nM), although the structural elements and distances (e.g. the distance between the basic
amino group and the thiophene moiety) are identical in 4a and 3a. The reduced σ1 receptor
affinity of the aminoethyl derivative 4a is explained by entropic factors, i.e. by the increased
conformational flexibility of the aminoethyl side chain of 4a compared to the conformationally
restricted piperidine 3a.
Increasing of the distance between the basic amino moiety and the phenyl ring in the side chain
from benzyl (4a) over 2-phenylethyl (4b) to 3-phenylpropyl (4c) led to a reduced σ1 receptor
affinity. This trend correlates nicely with the reduced σ1 affinities of spirocyclic thienopyrans 3
with the same series of N-substituents. Introduction of the saturated cyclohexylmethyl residue at
the N-atom resulted in an increased σ1 affinity of 4d. The positive effect of the cyclohexylmethyl
moiety on the σ1 receptor affinity has already been observed for spirocyclic piperidines [18].
In general the selectivity of the σ1 ligands 4 over the σ2 receptor subtype is rather low. Even the
cyclohexylmethyl derivative 4d binding in the low nanomolar range to σ1 receptors shows also
high affinity towards the σ2 receptor (Ki = 10 nM). The reduced subtype selectivity of the
aminoethyl derivatives 4 can be attributed to the high conformational flexibility allowing the 2-
aminoethyl side chain adapt to different receptor subtypes. However it should be noted that a
rather low σ2/σ1 selectivity was also observed for spirocyclic piperidines bearing a
cyclohexylmethyl moiety at the N-atom.

ACCEPTED MANUSCRIPT
9
4. Conclusion
Combining the structural elements of two promising σ1 ligands (i.e. pyrazoles 2 and spirocyclic
piperidines 3) led to the novel σ1 ligands 4. The reduced σ1 affinity and subtype selectivity of the
aminoethyl substituted tetrahydrobenzothiophenes 4 compared to analogous spirocyclic
piperidines 3 is explained by increased conformational flexibility of the aminoethyl side chain of
4. Nevertheless, low nanomolar σ1 affinity was achieved by introduction of a cyclohexylmethyl
moiety at the N-atom (4d, Ki (σ1) = 5.0 nM).
5. Experimental part
5.1. General, chemistry
Unless otherwise mentioned, THF was dried with sodium/benzophenone and was freshly
distilled before use. Thin layer chromatography (tlc): Silica gel 60 F254 plates (Merck). Flash
chromatography (fc): Silica gel 60, 40–64 µm (Merck); parentheses include: diameter of the
column, length of column, fraction size, eluent, Rf value. Melting point: Melting point apparatus
SMP 3 (Stuart Scientific), uncorrected. IR: IR spectrophotometer 480Plus FT-ATR-IR (Jasco).
1H NMR (400 MHz), 13C NMR (100 MHz): Mercury plus 400 spectrometer (Varian); δ in ppm
related to tetramethylsilane; coupling constants are given with 0.5 Hz resolution. Where
1
13
necessary, the assignment of the signals in the H NMR and C NMR spectra was performed
1
1
using H-1H and H-13C COSEY NMR spectra. MS: EI = electron impact, ESI = electro spray
ionization: MicroTof (Bruker Daltronics, Bremen), Calibration with sodium formate clusters
before measurement. HPLC method for determination of the product purity: Merck Hitachi
Equipment; UV detector: L-7400; autosampler: L-7200; pump: L-7100; degasser: L-7614;
Method: column: LiChrospher® 60 RP-select B (5 µm), 250-4 mm cartridge; flow rate: 1.00

ACCEPTED MANUSCRIPT
10
mL/min; injection volume: 5.0 µL; detection at λ = 210 nm; solvents: A: water with 0.05 % (v/v)
trifluoroacetic acid; B: acetonitrile with 0.05 % (v/v) trifluoroacetic acid: gradient elution: (A
%): 0-4 min: 90 % , 4-29 min: gradient from 90 % to 0 %, 29-31 min: 0 %, 31-31.5 min: gradient
from 0 % to 90 %, 31.5-40 min: 90 %.
5.2. General procedures
5.2.1. General procedure A for the synthesis of benzothiophenacetamides 13
Under N2 acid 12 (150 mg, 0.77 mmol) was dissolved in dry THF (5 mL). CDI (137 mg, 0.84
mmol) was added slowly at 0 oC and the reaction mixture was stirred for 10 min. Then a solution
of primary amine (0.77 mmol) was added dropwise and the reaction mixture was stirred at rt for
4 h. Water was added and the solution was extracted with ethyl acetate (3 x 10 mL). The organic
layer was dried (Na2SO4), concentrated in vacuum and the residue was purified by fc.
5.2.2. General procedure B for the synthesis of amines 4a-d
Under N2 amide 13 (1.0 equiv.) was dissolved in dry THF (5 mL). Then a solution of 1M LiAlH4
o
(2.2 equiv.) was added dropwise at 0 C and the reaction mixture was heated to reflux for 12 h.
Water was added and the mixture was extracted with EtOAc (3x50 mL). The organic layer was
dried (Na2SO4), concentrated in vacuum and the residue was purified by fc.
5.3. Synthetic procedures
5.3.1. 4-Oxo-4-(thiophen-2-yl)butanoic acid (7) [21,22]
Under N2 succinic anhydride (6) (11.9 g, 119 mmol) was dissolved in dry CH2Cl2 (550 mL).
Anhydrous AlCl3 (19.0 g, 142 mmol) was added at 0 oC and the reaction mixture was stirred for
30 min. Then thiophene (5) (10.0 g, 119 mmol) dissolved in CH2Cl2 (100 mL) was added over a

ACCEPTED MANUSCRIPT
11
period of 60 min and the reaction mixture was stirred for 4 h at rt. Then water was added and
mixture was extracted with CH2Cl2 (3 x 300 mL). The organic layer was dried (Na2SO4) and
concentrated in vacuum, (dichloromethane/MeOH = 85/15, Rf = 0.18). Pale yellow solid, mp 72-
82 oC, yield 14.0 g, (65 %). Crude purity: 92 %, tR = 10.7 min. FT-IR (neat): ν (cm-1) = 3100 (O-
H), 1694 (C=O), 1655 (HOC=O). Exact mass (ESI): m/z = calcd. for (C8H8O3S)H 185.0292,
found 185.0298. 1H NMR (DMSO-d6): δ (ppm) = 2.56 (t, J = 6.7 Hz, 2H, COCH2), 3.18 (t, J =
6.7 Hz, 2H, CH2CO2H), 7.23 (t, J = 4.8 Hz, 1H, 4-CH), 7.68 (dd, J = 4.8/0.9 Hz, 1H, 3-CH), 7.72
(dd, J = 4.8/0.9 Hz, 1H, 5-CH), 12.2 (bs, 1H, CO2H). 13C NMR (DMSO-d6): δ (ppm) = 27.7 (1C,
COCH2), 32.4 (1C, CH2CO2H), 128.7 (1C, C-4), 133.1 (1C, C-3), 134.5 (1C, C-5), 143.3 (1C, C-
2), 173.6 (1C, CO2H), 191.6 (1C, C=O).
5.3.2. 4-(Thiophen-2-yl)butanoic acid (8) [21,22]
Compound 7 (12.0 g, 65.2 mmol) was dissolved in diethylene glycol (250 mL). Hydrazine
o
monohydrate (7.18 g, 143 mmol) and KOH (8.05 g, 143 mmol) were added at 0 C and the
reaction mixture was heated to reflux for 4 h. The reaction mixture was cooled to rt, acidified to
pH 2 by addition of 10 M HCl and extracted with CH2Cl2 (3x500 mL). The organic layer was
dried (Na2SO4), concentrated in vacuum and the residue was purified by fc
(dichloromethane/MeOH = 98/2, ø = 5 cm, h = 30 cm, Rf = 0.32). Colorless liquid, yield 8.0 g,
(72 %). Purity: 94 %, tR = 14.3 min. FT-IR (neat): ν (cm-1) = 2936 (O-H), 1701 (HOC=O). Exact
1
mass (ESI): m/z = calcd. for (C8H10O2S)H 171.0402, found 171.0408. H NMR (CDCl3): δ
(ppm) = 1.95 (quint, J = 7.4 Hz, 2H, CH2CH2CH2CO2H), 2.34 (t, J = 7.4 Hz, 2H,
CH2CH2CH2CO2H), 2.82 (t, J = 7.4 Hz, 2H, CH2CH2CH2CO2H), 6.72 (dd, J = 3.5/1.1 Hz, 1H, 3-
CH), 6.84 (dd, J = 5.1/3.5 Hz, 1H, 4-CH), 7.05 (dd, J = 5.1/1.1 Hz, 1H, 5-CH), 10.7 (bs, 1H,
CO2H). 13C NMR (CDCl3): δ (ppm) = 26.5 (1C, CH2CH2CH2CO2H), 29.0 (1C,

ACCEPTED MANUSCRIPT
12
CH2CH2CH2CO2H), 33.1 (1C, CH2CH2CH2CO2H), 123.4 (1C, C-3), 124.6 (1C, C-4), 126.8 (1C,
C-5), 143.8 (1C, C-2), 179.9 (1C, CO2H).
5.3.3. 6,7-Dihydrobenzo[b]thiophen-4(5H)-one (9) [21,22]
Under N2 compound 8 (5.0 g, 29 mmol) was dissolved in dry CH2Cl2 (150 mL). Thionyl chloride
(3.8 g, 31.9 mmol) was added dropwise at 0 oC and the reaction mixture was heated to reflux for
45 min. The reaction mixture was concentrated in vacuum, the residue was dissolved in EtOAc
(100 mL) and anhydrous SnCl2 (6.6 g, 35 mmol) was added over a period of 30 min at 0 oC. The
reaction mixture was stirred for 3 h at rt. Water was added and the solution was extracted with
EtOAc (3x150 mL). The organic layer was dried (Na2SO4), concentrated in vacuum and the
product was purified by fc (petroleum ether/EtOAc = 95/5, ø = 3 cm, h = 20 cm, Rf = 0.26).
Colorless solid, mp 42 oC, yield 2.8 g, (62 %). Purity: 98 %, tR = 10.7 min. FT-IR (neat): ν (cm-1)
= 2944 (C-H), 1666 (C=O). Exact mass (ESI): m/z = calcd. for (C8H8OS)H 153.0296, found
153.0298. 1H NMR (CDCl3): δ (ppm) = 2.21 (quint, J = 6.5 Hz, 2H, 6-CH2), 2.56 (t, J = 6.5 Hz,
2H, 5-CH2), 2.98 (t, J = 6.5 Hz, 2H, 7-CH2), 7.05 (d, J = 5.3 Hz, 1H, 3-CH), 7.33 (d, J = 5.3 Hz,
13
1H, 2-CH). C NMR (CDCl3): δ (ppm) = 24.5 (1C, C-6), 25.4 (1C, C-5), 37.8 (1C, C-7), 122.9
(1C, C-3), 124.7 (1C, C-3a), 137.1 (1C, C-2), 155.8 (1C, C-7a), 193.1 (1C, C-4).
5.3.4. (E)-Ethyl 2-(6,7-dihydrobenzo[b]thiophen-4(5H)-ylidene)acetate (10)
o
Under N2 EtO2CCH2PO(OEt)2 (3.2 g, 14.5 mmol) was dissolved in dry THF (25 mL). At 0 C
NaH (0.37g, 15.8 mmol) was added and the reaction mixture was stirred for 15 min at 0 oC. Then
compound 9 (2.0 g, 13.2 mmol) dissolved in THF (10 mL) was added dropwise and the reaction
mixture was stirred at rt for 3 h. Water was added to the reaction mixture and the mixture was
extracted with EtOAc (3x150 mL). The organic layer was dried (Na2SO4), concentrated in

ACCEPTED MANUSCRIPT
13
vacuum and the residue was purified by fc (petroleum ether/EtOAc = 90/10, ø = 2.5 cm, h = 15
cm, Rf = 0.29). Colorless liquid, yield 1.9 g, (65 %). Exact mass (ESI): m/z = calcd. for
(C12H14O2S)H 223.0715, found 223.0709. 1H NMR (CDCl3): δ (ppm) = 1.24 (t, J = 7.1 Hz, 3H,
OCH2CH3), 1.88 (quint, J = 6.4 Hz, 2H, 6-CH2), 2.81 (t, J = 6.4 Hz, 5-CH2), 3.07 (t, J = 6.5 Hz,
2H, 7-CH2), 4.12 (q, J = 7.1 Hz, 2H, OCH2CH3), 6.04 (s, 1H, C=CH(CO2Et)), 6.99 (d, J = 5.4
Hz, 1H, 3-CH), 7.13 (d, J = 5.4 Hz, 1H, 2-CH). 13C NMR (CDCl3): δ (ppm) = 14.4 (1C,
OCH2CH3), 23.7 (1C, C-6), 25.5 (1C, C-5), 26.3 (1C, C-7), 59.7 (1C, OCH2CH3), 110.4 (1C,
C=CHCO2CH2CH3), 122.7 (1C, C-3), 123.4 (1C, C-2), 134.9 (1C, C-3a), 144.9 (1C, C-7a),
150.6 (1C, C-4), 167.4 (1C, EtOC=O).
5.3.5. Ethyl 2-(4,5,6,7-tetrahydrobenzo[b]thiophen-4-yl)acetate (11)
Compound 10 (500 mg, 2.25 mmol) was dissolved in EtOH (10 mL), Pd/C (50 %) was added
and the mixture was stirred at rt under H2 atmosphere (balloon) for 24 - 40 h. The mixture was
filtered through Celite® bed and the solvent was removed under reduced pressure to obtain a
residue which was purified by fc (petroleum ether/EtOAc = 80/20, ø = 2.0 cm, h = 10 cm, Rf =
0.28). Colorless liquid, yield 354 mg, (70 %). Exact mass (ESI): m/z = calcd. for (C12H16O2S)H
225.0875, found 225.0869. 1H NMR (CDCl3): δ (ppm) = 1.21 (t, J = 7.1 Hz, 3H, OCH2CH3),
1.43 – 1.49 (m, 1H, 6-CH2), 1.67 – 1.97 (m, 3H, 6-CH2/5-CH2), 2.33 (dd, J = 15.4/8.2Hz, 1H,
CH2CO2Et), 2.62 (dd, J = 15.4/8.2 Hz, 1H, CH2CO2Et), 2.70 (t, J = 6.1 Hz, 2H, 7-CH2), 3.10 –
3.27 (m, 1H, 4-CH), 4.10 (q, J = 7.1 Hz, 2H, OCH2CH3), 6.74 (d, J = 5.2 Hz, 1H, 3-CH), 6.98 (d,
J = 5.2 Hz, 1H, 2-CH).

ACCEPTED MANUSCRIPT
14
5.3.6. 2-(4,5,6,7-Tetrahydrobenzo[b]thiophen-4-yl)acetic acid (12)
Compound 11 (250 mg, 1.12 mmol) was dissolved in THF (3 mL), a solution of LiOH (80 mg,
1.68 mmol) in water (5 mL) was added at 0 oC and the reaction mixture was stirred at rt for 12 h.
The reaction mixture was concentrated in vacuum, the residue was acidified to pH 2 by addition
of 1M HCl and the mixture was extracted with EtOAc (3x50 mL). The organic layer was dried
o
(Na2SO4) and concentrated in vacuum. Colorless solid, mp 89 – 93 C, yield 182 mg, (83 %).
Exact mass (ESI): m/z = calcd. for (C10H12O2S)H 197.0558, found 197.0552. 1H NMR (CDCl3):
δ (ppm) = 1.45 – 1.61 (m, 1H, 6-CH2), 1.67 – 2.02 (m, 3H, 5-CH2/6-CH2), 2.41 (dd, J =
15.2/8.1Hz, 1H, CH2CO2H), 2.68 (dd, J = 15.2/8.1 Hz, 1H, CH2CO2H), 2.72 (t, J = 5.9 Hz, 2H,
7-CH2), 2.98 - 3.26 (m, 1H, 4-CH), 6.77 (d, J = 5.2 Hz, 1H, 3-CH), 6.99 (d, J = 5.2 Hz, 1H, 2-
CH).
5.3.7. N-Benzyl-2-(4,5,6,7-tetrahydrobenzo[b]thiophen-4-yl)acetamide (13a)
According to general procedure A compound 12 (150 mg, 0.77 mmol) was reacted with
benzylamine (83 mg, 0.77 mmol) and CDI (137 mg, 0.84 mmol). The product was purified by fc
(dichloromethane/MeOH = 95/5, ø = 2.0 cm, h = 10 cm, Rf = 0.26). Colorless solid, mp 118 –
122 oC, yield 150 mg, (69 %). Purity: 99.1 %, tR = 21.1 min. Exact mass (ESI): m/z = calcd. for
(C17H19NOS)H 286.1298, found 286.1301. 1H NMR (CDCl3): δ (ppm) = 1.41 – 1.53 (m, 1H, 6-
CH2), 1.99 – 1.64 (m, 3H, 6-CH2/5-CH2), 2.23 (dd, J = 14.2/8.2 Hz, 1H, CH2CONHCH2Ph), 2.53
(dd, J = 14.2/8.2 Hz, 1H, CH2CONHCH2Ph), 2.67 (t, J = 5.9 Hz, 2H, 7-CH2), 2.89 – 3.29 (m,
1H, 4-CH), 4.36 (dd, J = 14.7/5.7 Hz, 1H, PhCH2NHCO), 4.41 (dd, J = 14.7/5.7 Hz, 1H,
PhCH2NHCO), 5.63 (d, J = 5.6 Hz, 1H, PhCH2NHCO), 6.74 (d, J = 5.2 Hz, 1H, 3-CH), 6.95 (d,
13
J = 5.2 Hz, 1H, 2-CH), 7.30 – 7.14 (m, 5H, Ph). C NMR (CDCl3): δ (ppm) = 21.5 (1C, C-6),
25.1 (1C, C-5), 28.7 (1C, CH2CONHCH2Ph), 32.9 (1C, C-7), 43.4 (1C, C-4), 43.7 (1C,

ACCEPTED MANUSCRIPT
15
PhCH2NHCO), 122.1 (1C, C-3), 126.4 (1C, C-3a), 127.5 (1C, Ph), 127.9 (2C, o-Ph), 128.7 (2C,
m-Ph), 136.6 (1C, C-2), 137.7 (1C, C-7a), 138.2 (1C, Ph), 171.6 (1C, NHC=O).
5.3.8. N-(2-Phenylethyl)-2-(4,5,6,7-tetrahydrobenzo[b]thiophen-4-yl)acetamide (13b)
According to general procedure A compound 12 (150 mg, 0.77 mmol) was reacted with 2-
phenylethylamine (93 mg, 0.77 mmol) and CDI (137 mg, 0.84 mmol). The product was purified
by fc (dichloromethane/MeOH = 95/5, ø = 2.0 cm, h = 10 cm, Rf = 0.27). Yellow solid, mp 98 –
103 oC, yield 151 mg, (66 %). Purity: 97.2 %, tR = 19.9 min. Exact mass (ESI): m/z = calcd. for
1
(C18H21NOS)H 300.1338, found 300.1326. H NMR (CDCl3): δ (ppm) = 1.33 – 1.47 (m, 1H, 6-
CH2), 1.61 – 1.92 (m, 3H, 6-CH2/5-CH2), 2.11 (dd, J = 14.2/8.2 Hz, 1H, CH2CONHC2H4Ph),
2.44 (dd, J = 14.2/8.3 Hz, 1H, CH2CONHC2H4Ph), 2.66 (t, J = 5.9 Hz, 2H, 7-CH2), 2.73 (t, J =
6.7 Hz, 2H, PhCH2CH2NHCO), 3.11 – 3.27 (m, 1H, 4-CH), 3.45 (dt, J = 7.8/6.7 Hz, 1H,
PhCH2CH2NHCO), 3.49 (dt, J = 7.8/6.7 Hz, 1H, PhCH2CH2NHCO), 5.41 (t, J = 6.7 Hz, 1H,
NHCO), 6.71 (d, J = 5.2 Hz, 1H, 3-CH), 6.94 (d, J = 5.2 Hz, 1H, 2-CH), 7.06 – 7.27 (m, 5H, Ph).
13C NMR (CDCl3): δ (ppm) = 21.4 (1C, C-6), 25.1 (1C, C-5), 28.6 (1C, HNCOCH2), 32.8 (1C,
C-7), 35.7 (1C, NHCH2CH2Ph), 40.5 (1C, NHCH2CH2Ph), 43.5 (1C, C-4), 121.9 (1C, C-3),
126.4 (1C, C-3a), 126.5 (1C, Ph), 128.6 (2C, o-Ph), 128.7 (2C, m-Ph), 136.5 (1C, C-2), 137.8
(1C, C-7a), 138.8 (1C, Ph), 171.7 (1C, NHC=O).
5.3.9. N-[(3-Phenylpropyl)-2-(4,5,6,7-tetrahydrobenzo[b]thiophen-4-yl)]acetamide (13c)
According to general procedure A compound 12 (150 mg, 0.77 mmol) was reacted with 3-
phenylpropylamine (104 mg, 0.77 mmol) and CDI (137 mg, 0.84 mmol). The product was
purified by fc (dichloromethane/MeOH = 97/3, ø = 2.0 cm, h = 10 cm, Rf = 0.29). Colorless
solid, mp 127 – 131 oC, yield 164 mg, (68 %). Purity: 95.3 %, tR = 18.7 min. Exact mass (ESI):

ACCEPTED MANUSCRIPT
16
1
m/z = calcd. for (C19H23NOS)H 315.1498, found 315.1502. H NMR (DMSO-d6): δ (ppm) =
1.38 – 1.50 (m, 1H, 6-CH2), 1.62 – 1.77 (m, 3H, 6-CH2/5-CH2), 1.76 – 1.89 (m, 2H,
PhCH2CH2CH2NH), 2.12 (dd, J = 14.2, 7.8 Hz, 1H, CH2CONH), 2.50 (dd, J = 14.2, 7.8 Hz, 1H,
CH2CONH), 2.58 (t, J = 7.6 Hz, 2H, PhCH2CH2CH2NH), 2.68 (t, J = 6.2 Hz, 2H, 7-CH2), 3.16 –
2.99 (m, 3H, PhCH2CH2CH2NH/4-CH), 6.86 (d, J = 5.2 Hz, 1H, 3-CH), 7.22 – 7.06 (m, 5H, Ph),
7.28 (d, J = 5.2 Hz, 1H, 2-CH), 7.91 (t, J = 6.7 Hz, 1H, HNCO).13C NMR (DMSO-d6): δ (ppm) =
20.9 (1C, C-6), 24.5 (1C, C-5), 28.0 (1C, NHCH2CH2CH2Ph), 30.9 (1C, NHCOCH2), 32.4 (1C,
NHCH2CH2CH2Ph), 32.5 (1C, C-7), 38.0 (1C, NHCH2CH2CH2Ph), 40.5 (1C, C-4), 121.9 (1C,
C-3), 125.7 (1C, C-3a), 126.6 (1C, Ph), 128.1 (2C, o-Ph), 128.2 (2C, m-Ph), 135.1 (1C, C-2),
138.4 (1C, C-7a), 141.7 (1C, Ph), 170.9 (1C, NHC=O).
5.3.10. N-(Cyclohexylmethyl)-2-(4,5,6,7-tetrahydrobenzo[b]thiophen-4-yl)acetamide (13d)
According to general procedure A compound 12 (150 mg, 0.77 mmol) was reacted with
cyclohexylmethylamine (87 mg, 0.77 mmol) and CDI (137 mg, 0.84 mmol). The product was
purified by fc (dichloromethane/MeOH = 95/5, ø = 2.0 cm, h = 10 cm, Rf = 0.28). Colorless
solid, mp 87 – 91 oC, yield 145 mg, (65 %). Purity: 97.8 %, tR = 19.6 min. Exact mass (ESI): m/z
= calcd. for (C17H25NOS)H 292.1658, found 292.1662. 1H NMR (DMSO-d6): δ (ppm) = 0.79 –
0.94 (m, 2H, C6H11), 1.07 – 1.26 (m, 3H, C6H11), 1.32 – 1.50 (m, 2H, C6H11), 1.54 – 1.75 (m, 6H,
C6H11, 6-CH2), 1.91 – 1.72 (m, 2H, 5-CH2), 2.12 (dd, J = 14.2/8.2 Hz, 1H,
CH2CONHCH2C6H11), 2.46 (dd, J = 14.2/8.2 Hz, 1H, CH2CONHCH2C6H11), 2.68 (t, J = 6.2 Hz,
2H, 7-CH2), 2.90 (dt, J = 12.6/7.9 Hz, 1H, COHNCH2C6H11), 2.93 (dt, J = 12.9/7.9 Hz, 2H,
COHNCH2C6H11), 3.15 – 3.07 (m, 1H, 4-CH), 6.85 (d, J = 5.2 Hz, 1H, 3-CH), 7.19 (d, J = 5.2
13
Hz, 1H, 2-CH), 7.81 (t, J = 7.9 Hz, 1H, NHCO). C NMR (DMSO-d6): δ (ppm) = 20.9 (1C,
CH2C6H11), 24.5 (1C, C-6), 25.3 (1C, CH2C6H11), 26.01, 26.02 (2C, CH2C6H11), 28.0 (1C, C-5),

ACCEPTED MANUSCRIPT
17
30.3 (1C, CH2C6H11), 30.4 (1C, CHC6H11), 32.4 (1C, C-7), 37.3, (1C, COHNCH2C6H11), 42.1
(1C, NHCOCH2), 44.7 (1C, C-4), 121.8 (1C, C-3), 126.7 (1C, C-3a), 135.1 (1C, C-2), 138.4 (1C,
C-7a), 170.9 (1C, NHC=O).
5.3.11. N-Benzyl-2-(4,5,6,7-tetrahydrobenzo[b]thiophen-4-yl)ethanamine (4a)
According to general procedure B compound 13a (150 mg, 0.77 mmol) was reacted with LiAlH4
(73 mg, 1.93 mmol) and the product was purified by fc (dichloromethane/MeOH = 95/5, ø = 1.5
cm, h = 10 cm, Rf = 0.27). Colorless oil, yield 90 mg, (63 %). Purity: 99.1 %, tR = 21.1 min.
Exact mass (ESI): m/z = calcd. for (C17H21NS)H 272.1598, found 272.1504. 1H NMR (CDCl3): δ
(ppm) = 1.35 – 1.47 (m, 1H, 5-CH2), 1.53 – 1.72 (m, 2H, CHCH2CH2NH), 1.72 – 1.97 (m, 3H,
6-CH2/5-CH2), 2.60 – 2.69 (m, 4H, 7-CH2/CHCH2CH2NH), 2.70 – 2.77 (m, 1H, 4-CH), 3.74 (d,
J = 13.2 Hz, 1H, HNCH2Ph), 3.76 (d, J = 13.2 Hz, 1H, HNCH2Ph), 6.76 (d, J = 5.2 Hz, 1H, 3-
CH), 6.95 (d, J = 5.2 Hz, 1H, 2-CH), 7.28 – 7.14 (m, 5H, Ph). 13C NMR (CDCl3): δ (ppm) = 21.7
(1C, C-6), 25.2 (1C, C-5), 28.3 (1C, C-7), 33.8 (1C, CHCH2CH2NH), 36.2 (1C, C-4), 47.2 (1C,
CHCH2CH2NH), 54.1 (1C, PhCH2NH), 121.5 (1C, C-3), 126.7 (1C, C-3a), 126.9 (1C, Ph), 128.2
(2C, o-Ph), 128.4 (2C, m-Ph), 135.8 (1C, C-2), 139.1 (1C, C-7a), 140.3 (1C, Ph).
5.3.12. N-(2-Phenylethyl)-2-(4,5,6,7-tetrahydrobenzo[b]thiophen-4-yl)ethanamine (4b)
According to general procedure B compound 13b (150 mg, 0.50 mmol) was reacted with LiAlH4
(47.0 mg, 1.25 mmol) and the product was purified by fc (dichloromethane/MeOH = 95/5, ø =
1.5 cm, h = 10 cm, Rf = 0.26). Colorless oil, yield 45 mg, (31 %). Purity: 96.2 %, tR = 20.5 min.
Exact mass (ESI): m/z = calcd. for (C18H23NS)H 286.1682, found 286.1680. 1H NMR (CDCl3): δ
(ppm) = 1.35 – 1.48 (m, 1H, 5-CH2), 1.54 – 1.70 (m, 2H, CHCH2CH2NH), 1.74 – 1.94 (m, 3H,
6-CH2/5-CH2), 2.48 – 2.56 (m, 1H, 4-CH), 2.59 – 2.76 (m, 4H, 7-CH2/ PhCH2CH2NH), 2.73 –

ACCEPTED MANUSCRIPT
18
2.93 (m, 4H, PhCH2CH2NH/NHCH2), 6.75 (d, J = 5.2 Hz, 1H, 3-CH), 6.95 (d, J = 5.2 Hz, 1H, 2-
CH), 7.16 – 7.10 (m, 3H, Ph), 7.26 – 7.19 (m, 2H, Ph). 13C NMR (CDCl3): δ (ppm) = 21.7 (1C,
C-6), 25.1 (1C, C-5), 33.9 (1C, CHCH2CH2NH), 34.1 (1C, C-7), 35.9 (1C, PhCH2CH2NH), 36.1
(1C, C-4), 47.6 (1C, CHCH2CH2NH), 51.2 (1C, PhCH2CH2NH), 121.6 (1C, C-3), 126.2 (1C,
Ph), 126.7 (1C, C-3a), 128.5 (2C, o-Ph), 128.7 (2C, m-Ph), 135.9 (1C, C-2), 138.9 (1C, C-7a),
139.8 (1C, Ph).
5.3.13. 3-Phenyl-N-[2-(4,5,6,7-tetrahydrobenzo[b]thiophen-4-yl)ethyl]propan-1-amine (4c)
According to general procedure B compound 13c (120 mg, 0.38 mmol) was reacted with LiAlH4
(35 mg, 0.95 mmol) and the product was purified by fc (dichloromethane/MeOH = 95/5, ø = 1.5
cm, h = 10 cm, Rf = 0.28). Colorless liquid, yield 48 mg, (42 %). Purity: 97.2 %, tR = 19.4 min.
Exact mass (ESI): m/z = calcd. for (C19H25NS)H 300.1841, found 300.1839. 1H NMR (CDCl3): δ
(ppm) = 1.33 – 1.48 (m, 1H, 6-CH2), 1.54 – 1.72 (m, 2H, 5-CH2), 1.74 – 1.97 (m, 5H, 6-
CH2/CHCH2CH2NH/PhCH2CH2CH2NH), 2.21 (bs, 1H, NH), 2.55 – 2.63 (m, 5H, 4-CH/7-
CH2/PhCH2CH2CH2NH), 2.64 – 2.73 (m, 4H, CH2CH2NH/CH2NH), 6.77 (d, J = 5.2 Hz, 1H, 3-
CH), 6.95 (d, J = 5.2 Hz, 1H, 2-CH), 7.01 – 7.14 (m, 3H, Ph), 7.15 – 7.25 (m, 2H, Ph). 13C NMR
(CDCl3): δ (ppm) = 21.8 (1C, C-6), 25.2 (1C, C-5), 28.3 (1C, PhCH2CH2CH2NH), 31.4 (1C, C-
7), 33.7 (1C, CHCH2CH2NH), 33.9 (1C, PhCH2CH2CH2NH), 35.9 (1C, 4-CH), 47.7 (1C,
CHCH2CH2NH), 49.5 (1C, PhCH2CH2CH2NH), 121.6 (1C, C-3), 125.8 (1C, Ph), 126.7 (1C, 3a),
128.3 (2C, o-Ph), 128.4 (2C, m-Ph), 135.9 (1C, C-2), 138.9 (1C, 7a), 141.9 (1C, Ph).
5.3.14. N-(Cyclohexylmethyl)-2-(4,5,6,7-tetrahydrobenzo[b]thiophen-4-yl)ethanamine (4d)
According to general procedure B compound 13d (140 mg, 0.48 mmol) was reacted with LiAlH4
(43 mg, 1.20 mmol) and the product was purified by fc (dichloromethane/MeOH = 97/3, ø = 1.5

ACCEPTED MANUSCRIPT
19
cm, h = 10 cm, Rf = 0.27). Colorless liquid, yield 56 mg, (42 %). Purity: 95.6 %, tR = 19.6 min.
Exact mass (ESI): m/z = calcd. for (C17H27NS)H 278.1953, found 278.1967. 1H NMR (CDCl3): δ
(ppm) = 0.77 – 0.91 (m, 2H, C6H11), 1.02 – 1.25 (m, 4H, C6H11), 1.33 – 1.50 (m, 2H, 6-CH2),
1.53 – 1.73 (m, 7H, 5-CH2/C6H11), 1.78 – 1.94 (m, 3H, CHCH2CH2NH/C6H11), 2.28 – 2.35 (m,
1H, HNCH2C6H11), 2.38 – 2.45 (m, 1H, HNCH2C6H11), 2.63 (dt, 2H, J = 12.1/6.1 Hz, 1H,
CHCH2CH2NH), 2.66 – 2.70 (m, 2H, 7-CH2), 2.72 – 2.78 (m, 1H, 4-CH), 2.65 (t, J = 6.5 Hz, 2H,
13
CH-5), 6.78 (d, J = 5.2 Hz, 1H, CH-2), 6.96 (d, J = 5.2 Hz, 1H, CH-2). C NMR (CDCl3): δ
(ppm) = 21.7 (1C, C-6), 25.2 (1C, C-5), 25.8, 26.1, 26.7, 28.3, 29.7, 31.5, (6C, C6H11), 33.9 (1C,
CHCH2CH2NH), 36.4 (1C, C-7), 38.0 (1C, CHCH2CH2NH), 48.1 (1C, C-4), 57.0 (1C,
HNCH2C6H11), 121.5 (1C, C-3), 126.8 (1C, C-3a), 135.8 (1C, C-2), 139.2 (1C, C-7a).
5.4. Receptor binding studies
5.4.1. Materials
The guinea pig brains and rat liver for the σ1 and σ2 receptor binding assays were commercially
available (Harlan-Winkelmann, Borchen, Germany). Homogenizer: Elvehjem Potter (B. Braun
Biotech International, Melsungen, Germany). Cooling centrifuge model Rotina 35R (Hettich,
Tuttlingen, Germany) and High-speed cooling centrifuge model Sorvall RC-5C plus (Thermo
Fisher Scientific, Langenselbold, Germany). Multiplates: standard 96-well multiplates (Diagonal,
Muenster, Germany). Shaker: self-made device with adjustable temperature and tumbling speed
(scientific workshop of the institute). Vortexer: Vortex Genie 2 (Thermo Fisher Scientific,
Langenselbold, Germany). Harvester: MicroBeta FilterMate-96 Harvester. Filter: Printed
Filtermat Typ A and B. Scintillator: Meltilex (Typ A or B) solid state scintillator. Scintillation
analyzer: MicroBeta Trilux (all Perkin Elmer LAS, Rodgau-Jügesheim, Germany). Chemicals
and reagents were purchased from different commercial sources and of analytical grade.

ACCEPTED MANUSCRIPT
20
5.4.2. Preparation of membrane homogenates from guinea pig brain
5 guinea pig brains were homogenized with the potter (500-800 rpm, 10 up-and-down strokes) in
6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at 1200 x g for 10 min at 4
°C. The supernatant was separated and centrifuged at 23,500 x g for 20 min at 4 °C. The pellet
was resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 7.4) and centrifuged again at
23,500 x g (20 min, 4 °C). This procedure was repeated twice. The final pellet was resuspended
in 5-6 volumes of buffer and frozen (−80 °C) in 1.5 mL portions containing about 1.5 mg
protein/mL.
5.4.3. Preparation of membrane homogenates from rat liver
Two rat livers were cut into small pieces and homogenized with the potter (500-800 rpm, 10 up-
and-down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at
1,200 x g for 10 min at 4 °C. The supernatant was separated and centrifuged at 31,000 x g for 20
min at 4 °C. The pellet was resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 8.0) and
incubated at room temperature for 30 min. After the incubation, the suspension was centrifuged
again at 31,000 x g for 20 min at 4 °C. The final pellet was resuspended in 5-6 volumes of buffer
and stored at -80,°C in 1.5 mL portions containing about 2 mg protein/mL.
5.4.4. Protein determination
The protein concentration was determined by the method of Bradford [25], modified by
Stoscheck [26]. The Bradford solution was prepared by dissolving 5 mg of Coomassie Brilliant
Blue G 250 in 2.5 mL of EtOH (95 %, v/v). 10 mL deionized H2O and 5 mL phosphoric acid
(85%, m/v) were added to this solution, the mixture was stirred and filled to a total volume of

ACCEPTED MANUSCRIPT
21
50.0 mL with deionized water. The calibration was carried out using bovine serum albumin as a
standard in 9 concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 and 4.0 mg /mL). In a 96-well
standard multiplate, 10 µL of the calibration solution or 10 µL of the membrane receptor
preparation were mixed with 190 µL of the Bradford solution, respectively. After 5 min, the UV
absorption of the protein-dye complex at λ = 595 nm was measured with a platereader (Tecan
Genios, Tecan, Crailsheim, Germany).
5.4.5. General protocol for the binding assays
The test compound solutions were prepared by dissolving approximately 10 µmol (usually 2-4
mg) of test compound in DMSO so that a 10 mM stock solution was obtained. To obtain the
required test solutions for the assay, the DMSO stock solution was diluted with the respective
assay buffer. The filtermats were presoaked in 0.5% aqueous polyethylenimine solution for 2 h at
room temperature before use. All binding experiments were carried out in duplicates in 96-well
multiplates. The concentrations given are the final concentrations in the assay. Generally, the
assays were performed by addition of 50 µL of the respective assay buffer, 50 µL test compound
solution in various concentrations (10-5, 10-6, 10-7, 10-8, 10-9 and 10-10 mol/L), 50 µL of
corresponding radioligand solution and 50 µL of the respective receptor preparation into each
well of the multiplate (total volume 200 µL). The receptor preparation was always added last.
During the incubation, the multiplates were shaken at a speed of 500-600 rpm at the specified
temperature. Unless otherwise noted, the assays were terminated after 120 min by rapid filtration
using the harvester. During the filtration each well was washed five times with 300 µL of water.
Subsequently, the filtermats were dried at 95 °C. The solid scintillator was melted on the dried
filtermats at a temperature of 95 °C for 5 min. After solidifying of the scintillator at room
temperature, the trapped radioactivity in the filtermats was measured with the scintillation

ACCEPTED MANUSCRIPT
22
analyzer. Each position on the filtermat corresponding to one well of the multiplate was
measured for 5 min with the [3H]-counting protocol. The overall counting efficiency was 20%.
The IC50-values were calculated with the program GraphPad Prism® 3.0 (GraphPad Software,
San Diego, CA, USA) by non-linear regression analysis. Subsequently, the IC50 values were
transformed into Ki-values using the equation of Cheng and Prusoff [26]. The Ki-values are
given as mean value ± SEM from three independent experiments.
5.4.6. Protocol of the σ1 receptor binding assay
The assay was performed with the radioligand [3H]-(+)-pentazocine (22.0 Ci/mmol; Perkin
Elmer). The thawed membrane preparation of guinea pig brain cortex (about 100 ꢀg of protein)
was incubated with various concentrations of test compounds, 2 nM [3H]-(+)-pentazocine, and
TRIS buffer (50 mM, pH 7.4) at 37 °C. The non-specific binding was determined with 10 ꢀM
unlabeled (+)-Pentazocine. The Kd-value of (+)-pentazocine is 2.9 nM [28].
5.4.7. Protocol of the σ2 receptor binding assay
The assays were performed with the radioligand [3H]DTG (specific activity 50 Ci/mmol; ARC,
St. Louis, MO, USA). The thawed membrane preparation of rat liver (about 100 µg of protein)
was incubated with various concentrations of the test compound, 3 nM [3H]DTG and buffer
containing (+)-pentazocine (500 nM (+)-pentazocine in 50 mM TRIS, pH 8.0) at room
temperature. The non-specific binding was determined with 10 ꢀM non-labeled DTG. The Kd
value of [3H]DTG is 17.9 nM [29].

ACCEPTED MANUSCRIPT
23
Acknowledgement
This work was done within the framework of the NRW International Graduate School of
Chemistry, Münster, which is funded by the Government of the state Nordrhein-Westfalen and
Westfälische Wilhelms-University Münster. The financial support is deeply acknowledged.
References
[1]
[2]
C. Kaiser, J. Pontecorvo, R. E. Mewshaw, Neurotransm., 7 (1991) 1–5.
J. M. Walker, W. D. Bowen, F. O. Walker, R. R. Matsumoto, B. De Costa, K. C. Rice,
Pharmacol. Rev. 42 (1990) 353–402.
[3]
[4]
[5]
[6]
M. Hanner, F. F. Moebius, A. Flandorfer, H. G. Knaus, J. Striessnig, E. Kempner, H.
Glossmann, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 8072-8077.
R. Kekuda, P. D. Prasad, Y. J. Fei, F. H. Leibach, V. Ganapathy, Biochem. Biophys. Res.
Commun. 229 (1996) 553-558.
P. Seth, Y. J. Fei, H. W. Li, W. Huang, F. H. Leibach, V. Ganapathy, J. Neurochem. 70
(1998) 922-931.
Y. X. Pan, J. Mei, J. Xu, B.-. Wan, A. Zuckerman, G. W. Pasternak, J. Neurochem. 70
(1998) 2279-2285.
[7]
[8]
P. Seth, F. H. Leibach, V. Ganapathy, The FASEB journal 12 (1998) A161-A161.
E. Falkenstein, C. Meyer, C. Eisen, P. C. Scriba, M. Wehling, Biochem. Biophys. Res.
Comm. 229 (1996) 86–89.
[9]
J. Xu, C. Zeng, W. Chu, F. Pan, J. M. Rothfuss, F. Zhang, Z. Tu, D. Zhou, D. Zeng, S.
Vangveravong, F. Johnston, D. Spitzer, K. C. Chang, R. S. Hotchkiss, W. G. Hawkins, K.
T. Wheeler, R. H. Mach, Nature Comm. 2 (2011) article number 380.
[10] T. Maurice, T.-P. Su, Pharmacol. Ther. 124 (2009) 195-206.

ACCEPTED MANUSCRIPT
24
[11] M. Ishikawa, K. Hashimoto, J. Recept. Lig. Chan. Res. 3 (2010) 25-36.
[12] J. L. Diaz, R. Cuberes, J. Berrocal, M. Contijoch, U. Christmann, A. Fernández, A. Port,
J. Holenz, H. Buschmann, C. Laggner, M. T. Serafini, J. Burgeno, D. Zamanillo, M.
Merlos, J. M. Vela, C. Almansa, J. Med. Chem. 55 (2012) 8211-8224.
[13] B. Wünsch. J. Med. Chem. 55 (2012) 8209-8210.
[14] S.-R. Choia, B. Yange, K. Ploessla, S. Chumpradita, S.-P. Weya, P. D. Actona, K.
Wheelerc, R. H. Mach, H. F. Kunga, Nucl. Med. Biol. 28 (2001) 657–666.
[15] Z. Tu, J. Xu, L. A. Jones, S. Li, C. Dumstorff, S. Vangveravong, D. L. Chen, K. T.
Wheeler, M. J. Welch, R. H. Mach, J. Med. Chem. 50 (2007) 3194–3204.
[16] A. J. Corbera, D. Vano, D. Martinez, J. M. Vela, D. Zamanillo, A. Dordal, F. Andreu, E.
Hernandez, R. Perez, M. Escriche, L. Salgado, S. Yeste, N. Cano, H. Buschmann, J.
Holenz, Chem. Med. Chem. 1 (2006) 140-154.
[17] J. L. Diaz, D. Zamanillo, J. Corbera, J. M. Baeyens, R. Maldonado, M. A. Pericas, J. M.
Vela, A. Torrens, Cent. Nerv. Syst. Agents Med. Chem. 9 (2009) 172-183.
[18] C. Oberdorf, D. Schepmann, J. M. Vela, H. Buschmann, J. Holenz, B. Wünsch, J. Med.
Chem. 55 (2012) 5350-5360.
[19] C. Meyer, D. Schepmann, S. Yanagisawa, J. Yamaguchi, V. Dal Col, E. Laurini, K.
Itami, S. Pricl, B. Wünsch, J. Med. Chem. 55 (2012) 8047-8065.
[20] B. Wünsch, Curr. Pharm. Design 18 (2012) 930-937.
[21] L. F. Fieser, G. R. Kennelly, J. Am. Chem. Soc. 57 (1935) 1611-1616.
[22] P. P. Yadav, P. Gupta, A. K. Chaturvedi, P. K. Shukla, R. Maurya, Bioorg. Med. Chem.
13 (2005) 1497-1505.
[23] R. Holl, D. Schepmann, R. Fröhlich, R. Grünert, P. J. Bednarski, B. Wünsch, J. Med.
Chem. 52 (2009) 2126–2137.

ACCEPTED MANUSCRIPT
25
[24] C. Meyer, B. Neue, D. Schepmann, S. Yanagisawa, J. Yamaguchi, E.-U. Würthwein, K.
Itami, B. Wünsch, Bioorg. Med. Chem. 21 (2013) 1844-1856.
[25] M. M. Bradford, Anal. Biochem. 72 (1976) 248-254.
[26] C. Stoscheck, Methods in Enzymology 182 (1990) 50-68.
[27] Y. C. Cheng, W. H. Prusoff, Biochem. Pharmacol. 22 (1973) 3099-3108.
[28] D. L. DeHaven-Hudkins, L. C. Fleissner, F. Y. Ford-Rice, Eur. J. Pharmacol. Mol.
Pharmacol. Sect. 227 (1992) 371-378.
[29] H. Mach, C. R. Smith, S. R. Childers Life Sci. 57 (1995) PL-57-62.

ACCEPTED MANUSCRIPT
26
List of captions of Figures, Schemes and Tables
Figure 1
Development of novel σ1 receptor ligands 4 by combination of S1RA (1), aminoethylindazoles 2
and spirocyclic thienopyrans 3.
Scheme 1
Synthesis of the α,β-unsaturated ester 10 starting with thiophene.
Scheme 2
Synthesis of aminoethyl tetrahydrobenzothiophenes 4.
Table 1
σ1 and σ2 receptor affinities of aminoethyl substituted tetrahydrobenzothiophenes 4 and reference
compounds.

ACCEPTED MANUSCRIPT
27
Graphical Abstract

ACCEPTED MANUSCRIPT
Institut für
Pharmazeutische
und Medizinische
Chemie
Prof. Dr. B. Wünsch
|Institut für Pharmazeutische und Medizinische Chemie | Corrensstr. 48
|
48149 Münster
Corrensstr. 48
48149 Münster
Bearbeiter
Datum
Tel. +49 251 83-33310
Fax +49 251 83-32144
wuensch@uni-
muenster.de
06.06.2013
Research Highlights
> Pharmacophoric elements of different compounds were combined.
> Benzothiophenes with a flexible aminoethyl side chain were prepared.
> The most potent compound showed low nanomolar σ1 affinity.
> Relationships between the structure and the σ1 affinity were elaborated.

ACCEPTED MANUSCRIPT
Supporting Information
New combination of pharmacophoric elements of potent σ1 ligands: Design, synthesis and σ
receptor affinity of aminoethyl substituted benzothiophenes
Dipak Harel, Dirk Schepmann, Bernhard Wünsch*
Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität Münster,
Correnstraße 48, D-48149 Münster, Germany
Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail: wuensch@uni-muenster.de
1H NMR and 13C NMR data