Pyridine bioisosteres of potent GluN2B subunit containing NMDA receptor antagonists with benzo[7]annulene scaffold

Article abstract of DOI:10.1016/j.ejmech.2018.08.003

It has been reported that benzo [7]annulen-7-amines bearing electron withdrawing substituents such as 3d with a 2-Cl or 3e with a 2-NO₂ moiety show very high affinity towards the ifenprodil binding site of GluN2B subunit containing NMDA receptors. Therefore, bioisosteres of 3 with an electron deficient pyridine ring instead of the chloro- or nitrobenzene ring were envisaged. Starting from pyridine-2,3-dicarboxylic acid (5) a five-step synthesis of the key intermediate, the ketone 10, was developed. Reductive amination with various primary amines and NaBH(OAc)₃ led to the homologous secondary amines 11a-c. Subsequent methylation yielded the tertiary amines 12b and 12c. Receptor binding studies with [³H]ifenprodil revealed K_i-values above 100 nM for the most active phenylpropyl- and phenylbutylamines 11b and 11c. The >100-fold reduced GluN2B affinity of pyridines 11b and 11c compared to the GluN2B affinity of the corresponding chloro- and nitrobenzene derivatives 3d and 3e indicates that the pyridine ring is not tolerated as bioisosteric replacement of the chloro- or nitrobenzene ring in this type of compounds.

Full text of DOI:10.1016/j.ejmech.2018.08.003

Accepted Manuscript
Pyridine bioisosteres of potent GluN2B subunit containing NMDA receptor
antagonists with benzo[7]annulene scaffold
Robert Zscherp, Sören Baumeister, Dirk Schepmann, Bernhard Wünsch
PII:
S0223-5234(18)30658-5
10.1016/j.ejmech.2018.08.003
EJMECH 10614
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 April 2018
Revised Date: 22 May 2018
Accepted Date: 1 August 2018
Please cite this article as: R. Zscherp, Sö. Baumeister, D. Schepmann, B. Wünsch, Pyridine bioisosteres
of potent GluN2B subunit containing NMDA receptor antagonists with benzo[7]annulene scaffold,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.003.
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ACCEPTED MANUSCRIPT
Pyridine bioisosteres of potent GluN2B subunit containing NMDA receptor
antagonists with benzo[7]annulene scaffold
Robert Zscherpa, Sören Baumeister,a Dirk Schepmann,a Bernhard Wünscha,b*
a 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
b Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), Westfälische Wilhelms-
Universität Münster, Germany.
Abstract
It has been reported that benzo[7]annulen-7-amines bearing electron withdrawing
substituents such as 3d with a 2-Cl or 3e with a 2-NO2 moiety show very high affinity
towards the ifenprodil binding site of GluN2B subunit containing NMDA receptors.
Therefore, bioisosteres of 3 with an electron deficient pyridine ring instead of the
chloro- or nitrobenzene ring were envisaged. Starting from pyridine-2,3-dicarboxylic
acid (5) a five-step synthesis of the key intermediate, the ketone 10, was developed.
Reductive amination with various primary amines and NaBH(OAc)3 led to the
homologous secondary amines 11a-c. Subsequent methylation yielded the tertiary
amines 12b and 12c. Receptor binding studies with [3H]ifenprodil revealed Ki-values
above 100 nM for the most active phenylpropyl- and phenylbutylamines 11b and 11c.
The >100-fold reduced GluN2B affinity of pyridines 11b and 11c compared to the
GluN2B affinity of the corresponding chloro- and nitrobenzene derivatives 3d and 3e

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indicates that the pyridine ring is not tolerated as bioisosteric replacement of the
chloro- or nitrobenzene ring in this type of compounds.
Keywords: NMDA receptor, GluN2B antagonists, ifenprodil binding site, affinity,
selectivity, structure-affinity relationships, [7]annuleno[b]pyridin-7-amines; pyridine-
benzene bioisosteres
1. Introduction
Glutamate receptors are transmembrane proteins in neurons binding specifically the
excitatory neurotransmitter (S)-glutamate. They are classified in metabotropic and
ionotropic glutamate receptors. Whereas metabotropic glutamate receptors belong to
the class of G-protein coupled receptors inducing or inhibiting metabolic processes,
ionotropic glutamate receptors are ligand-gated ion channels controlling the flow of
cations (Na+, K+. Ca2+) across the cell membrane. The group of ionotropic glutamate
receptors consists of three receptor types: the 2-amino-3-(3-hydroxy-5-methyl-
isoxazol-4-yl)propanoic acid (AMPA), the kainate and the N-methyl-D-aspartate
(NMDA) receptor. [1-4]
The NMDA receptor represents a ligand-gated and voltage dependent ion channel. In
addition to the presence of the physiological agonist (S)-glutamate, activation of the
NMDA receptor requires the presence of the coagonist glycine and depolarization of
the surrounding membrane to remove the Mg2+ block. This is necessary, since at the
resting potential the NMDA receptor associated ion channel is blocked by Mg2+-ions.

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Depolarization of the surrounding membrane can be achieved by glutamate-induced
opening of AMPA and/or kainate receptors in the membrane. [1-4]
Upon activation of the NMDA receptor, Ca2+- and Na+-ions can penetrate into the
neuron, whilst K+-ions can leave the cell. The high Ca2+-ion permeability represents a
further characteristic feature of the NMDA receptor playing an important role in many
physiological functions of the central nervous system like memory and learning.
However, overactivation of the NMDA receptor leading to an excessive influx of Ca2+-
ions into the neuron is associated with the process of excitotoxicity resulting in
apoptosis of the neuron.[3,4]
The NMDA receptor consists of four subunits, which form a heterotetramer. For
humans, seven subunits are known, which are termed GluN1, GluN2A–D and
GluN3A-B. The (S)-glutamate binding site is localized on GluN2 subunits and the
glycine binding site on GluN1 and GluN3 subunits. Since GluN3 subunits are
predominantly expressed in the embryonic brain, a functional NMDA receptor usually
consists of two GluN1 and two GluN2 subunits. [5-7]
A single subunit of the NMDA receptor is subdivided into four domains. The C-
terminal domain (CTD) is located on the intracellular site of the membrane and
connects the receptor with the cytoskeleton. The transmembrane domain (TMD) is
composed of four α-helices forming the channel pore and anchoring the receptor into
the membrane. The TMD contains the binding site for open channel blockers, which
is termed PCP binding site according to the prototypical ligand phencyclidine (1-(1-
phjenylcyclohexyl)piperidine, PCP). In order to reach this binding site the ion channel
has to be opened. Therefore, compounds interacting with this binding site (e.g.

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ketamine, memantine, amantadine, MK-801) are termed open-channel blockers. [6]
The ligand binding domain (LBD) for the agonists glutamate (GluN2 subunit) and
glycine (GluN1 subunit) is located on the extracellular site. The extracellularly located
N-terminal domain (NTD) far away from the ion channel pore contains binding sites
for several non-competitive positive and negative allosteric modulators including
polyamines, ifenprodil, Zn2+-ions, NO and protons. [6,7] The ifenprodil binding site,
named after the negative allosteric modulator ifenprodil, is found exclusively on the
GluN2B subunit. Therefore, compounds interacting with the ifenprodil binding site can
only modulate the opening state of GluN2B subunit containing NMDA receptors. As a
consequence, high selectivity is achieved in the sense that only those NMDA
receptors containing the GluN2B subunit can be modulated by ifenprodil-like
compounds. [8]
Recently, X-ray crystal structure analyses have shown that the ifenprodil binding site
is located at the interface between the GluN1 and GluN2B subunit. [9-12] Negative
allosteric modulators interacting selectively with the ifenprodil binding site have a
potential for the treatment of neurodegenerative and neurological diseases. In
particular, negative allosteric modulators of GluN2B-NMDA receptors could be
exploited for the treatment of depression, cerebral ischemia, stroke, Parkinson’s,
Alzheimer’s and Huntington’s disease. [7, 13-17]

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Figure 1: Development of pyridine based GluN2B antagonists.
In order to develop novel high-affinity and selective negative allosteric modulators
interacting with the ifenprodil binding site, the phenylpropylamine Ro 25-6981 (1)
[18,19] served as lead compound in this project. (Figure 1) Formal connection of the
methylene moiety adjacent to the N-atom of the piperidine ring with the benzene ring
led to the benzo[7]annulen-7-amines 2 showing high GluN2B affinity (cis: Ki = 16
nM), high selectivity over related receptors, and cytoprotective activity (cis: IC50 = 12
nM). [20] Removal of the benzylic OH moiety and introduction of various substituents
in 2-position resulted in high-affinity GluN2B ligands. Whereas electron releasing OH
(3b) and OCH3 (3c) moiety resulted in compounds with high GluN2B affinity ((3a: Ki =
16 nM; 3b: Ki = 28 nM; 3c: Ki = 10 nM), electron withdrawing substituents were able
to further increase the GluN2B affinity in the low single-digit nanomolar range (3d: Ki
= 2.1 nM; 3e: Ki = 1.6 nM). [21,22] Since the substituents Cl in 3d and NO2 in 3e
have quite different properties, the idea came up that an electron deficient aromatic
system is responsible for the high GluN2B affinity. In order to prove this hypothesis,
the bioisosteric replacement of the benzene ring of 3 by an electron-deficient pyridine

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ring was envisaged. Moreover, a pyridine ring would avoid the NO2 moiety, which
could lead to the formation of toxic metabolites in vivo. Herein, we report on the
synthesis and pharmacological evaluation of pyridine bioisosteric GluN2B ligands of
type 4. (Figure 1)
2. Results and discussion
2.1. Synthesis
For the synthesis of pyridine derivatives of type 4, ketone 10 should serve as key
building block. The synthesis of 10 was accomplished in five steps starting with
quinolinic acid 5. Since initial attempts to reduce the diacid 5 with LiAlH4 failed to
yield the diol 7, the diacid 5 was converted into the dimethyl ester 6 by a Fischer
esterification with methanol and concentrated H2SO4. [23] (Scheme 1) Then, the
diester 6 was reduced with NaBH4 to give the diol 7 in 54 % yield. [23] The dibromide
8 was obtained by reaction of diol 7 with PBr3. In order to avoid decomposition of the
highly reactive and unstable dibromide 8, fast work-up was performed and the
product was directly used in the next reaction step. For the establishment of the
[7]annulene ring in the oxodiester 9, two subsequent SN2 reactions of dimethyl 3-
oxoglutarate and dibromide 8 were performed. [24] After purification, the exact
spectroscopic analysis of oxodiester 9 turned out to be rather complex due to the
existence of a mixture of four enantiomeric pairs of isomers (two pairs of keto-enol
tautomers, two pairs of diastereomers). Therefore, the oxodiester 9 was hydrolyzed
and decarboxylated with HCl to provide the ketone 10. The ketone 10 was purified by
reversed-phase column chromatography, since considerable decomposition was
observed during normal phase silica gel chromatography.

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Scheme 1: Synthesis of the ketone 10. Reagents and reaction conditions: (a) MeOH,
H2SO4, reflux, 16 h, 75 %. (b) NaBH4, EtOH, -15 °C to reflux, 2 h, 54 %. (c) PBr3,
THF, 0 °C to rt, 1 h. (d) Dimethyl 3-oxoglutarate, K2CO3, KI, THF, reflux, 16 h. (e) 6 M
HCl, 95 °C, 4 h, 11 % over 3 steps. (f) Ph(CH2)nNH2, NaBH(OAc)3, CH2Cl2, rt, 3 h,
22-52 %. (g) Formalin, NaBH(OAc)3, CH2Cl2, rt, 3 h, 23-32 %.
Reductive amination of ketone 10 with homologous phenylalkylamines and
NaBH(OAc)3 [25] led to the secondary amines 111a-c. (Scheme 1) The secondary
amines 11b and 11c were converted into the tertiary amines 12b and 12c by
reductive methylation with formaldehyde and NaBH(OAc)3.
Some transformations gave only low yields of products. However, the low yields are
due to purification problems of the rather polar pyridine derivatives and our high
requirements regarding the purity of the compounds for the in vitro binding assays.
2.2. Pharmacological evaluation
2.2.1. GluN2B affinity
In order to determine the affinity of the synthesized compounds towards GluN2B
subunit containing NMDA receptors, competitive binding assays with the radioligand

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[3H]ifenprodil were performed. [26] Cell membrane preparations from stably
transfected mouse fibroblast cells (L(tk-) cells) were used as receptor source. The
results are summarized in Table 1.
Table 1. Receptor affinity of the pyridine derivatives 11 and 12 compared with the
affinity of lead and reference compounds.
Ki ± SEM [nM]
Compound
NR2
GluN2B
28 ± 5.0
2.1 ± 1.2
1.6 ± 0.9
>1000
PCP
0 %
0 %
σ1
123
σ2
32 ± 13
7.4 ± 1.6
11 ± 4
490
3b[22]
3d[22]
3e[22]
11a
18 ± 7.0
18 ± 5
942
3 %
0 %
10 %
11b
302 ± 69
992
276
11c
149 ± 30
8 %
214
68 ± 13
12b
12c
445 ± 123
477 ± 64
10 ± 0.7
0 %
5 %
-
261
263
68 ± 15
115 ± 14
98 ± 34
Ifenprodil
125 ± 24
Data for compounds with Ki values higher than 100 nM in the first test were not
repeated. For very low affinity compounds only inhibition (in %) of radioligand binding
at a test compound concentration of 1 µM (i.e. IC50 > 1 µM) is given.

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The phenylbutylamine 11c reveals the highest affinity towards the ifenprodil binding
site with a Ki value of 149 nM. The GluN2B affinity of 11b with a shorter
phenylpropylamine side chain is slightly reduced (Ki = 302 nM), whereas the
benzylamine 11a with only one methylene moiety between the phenyl ring and the
amino moiety shows considerably lower GluN2B affinity (Ki > 1000 nM). These
results are in good accordance to the structure-affinity relationships obtained for the
benzene analogous lead compounds of type 3 in which a phenylpropyl or phenylbutyl
side chain represented the optimum chain length for high GluN2B affinity. Conversion
of the secondary amines 11b and 11c into tertiary methylamines 12b and 12c
resulted in a slightly reduced GluN2B affinity.
The loss of GluN2B affinity of the pyridine derivatives 11b and 11c compared to the
GluN2B affinity of the corresponding substituted benzene derivatives 3d (2-Cl) and
3e (2-NO2) clearly indicates that despite the reduced electron density the pyridine
ring is not an appropriate bioisosteric replacement of the chlorobenzene (3d) and
nitrobenzene moiety (3e). This result was surprising as the pyridine ring can also
serve as an H-bond acceptor. In the hydrogen bond basicity scale the pyridine ring is
given with a pKBHX value of 1.86. [27] It is speculated that either the wrong position of
the N-atom in the pyridine ring or the rather polar properties of the pyridine ring itself
might be responsible for the reduced GluN2B affinity.
2.2.2. Receptor selectivity
In order to test, whether the pyridine derivatives 11 and 12 also interact with the PCP
binding site within the channel pore of the NMDA receptor, the affinity towards this
binding site was evaluated in radioligand receptor binding studies as well. [28,29]
Table 1 clearly shows that the pyridine derivatives 11 and 12 did not interact with the

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PCP binding site even at the rather high test compound concentration of 1 µM. This
result correlates nicely with the negligible PCP affinity of the corresponding benzene
derivatives 3.
It has been reported that ifenprodil and similar GluN2B ligands also interact with σ1
and σ2 receptors. Therefore, the σ1 and σ2 receptor affinities of 11 and 12 were
recorded in radioligand receptor binding studies as well. [30-32] According to the
data given in Table 1, the σ1 affinity of the pyridine derivatives 11 and 12 is in the
same range as their GluN2B affinity. However, the σ2 affinity often exceeds the
GluN2B affinity indicating a slight preference for binding at σ2 receptors over GluN2B
receptors. The substituted benzo[7]annulene derivatives 3 show also rather high σ2
affinity, but the GluN2B affinity is always higher than the σ2 affinity. Replacement of
the nitrobenzene or chlorobenzene moiety of potent GluN2B ligands 3d and 3e by a
pyridine ring shifts the receptor profile towards preferred σ2 affinity. The changed
ratio of GluN2B : σ2 receptor interaction of the pyridine derivatives 11 and 12 can be
attributed to the considerably reduced GluN2B affinity.
3. Conclusion
In order to prove the hypothesis of beneficial effects of electron deficient aromatic
systems on GluN2B affinity, a set of pyridine annulated [7]annulenes was prepared.
Bioisosteric replacement of the nitrobenzene moiety of 3e by the pyridine ring in 11b
led to 200-fold reduced GluN2B affinity. Therefore, it was concluded that an electron
deficient aromatic system is not alone responsible for the high GluN2B affinity of the
nitrobenzene derivative 3e. Polar interactions of the NO2 moiety with the receptor
protein, in particular the establishment of an H-bond network with the conserved

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water molecule in the binding pocket, is responsible for the very high GluN2B affinity
of 3e.[20] Obviously, a similar H-bond network cannot be formed by the pyridine
analogs 11 and 12.
4. Experimental Part
4.1. Chemistry, General methods
Oxygen and moisture sensitive reactions were carried out under nitrogen, dried with
silica gel with moisture indicator (orange gel, VWR, Darmstadt, Germany) and in
glassware (Schlenk flask or Schlenk tube). Temperatures were controlled with dry ice
and NaCl (-15 °C), ice/water (0 °C) and magnetic stirrer MR 3001 K (Heidolph,
Schwalbach, Germany) or RCT CL (IKA, Staufen, Germany), together with
temperature controller EKT HeiCon (Heidolph) or VT-5 (VWR) and PEG or silicone
bath. All solvents were of analytical or technical grade quality. Demineralized water
was used. CH2Cl2 was distilled from CaH2; THF was distilled from
sodium/benzophenone; MeOH was distilled from magnesium methanolate. Thin layer
chromatography (tlc): tlc silica gel 60 F254 on aluminum sheets (VWR). Flash
chromatography (fc): Silica gel 60, 40–63 µm (VWR), silica 60 RP – 18 (EM
Industries Inc.); parentheses include: diameter of the column (d), length of the
stationary phase (l), fraction size (V) and eluent. Automated flash chromatography:
IsoleraTM Spektra One (Biotage®); parentheses include: cartridge size, flow rate,
eluent, fractions size was always 20 mL. Melting point: Melting point system MP50
(Mettler Toledo, Gießen, Germany), open capillary, uncorrected. MS: MicroTOFQII
mass spectrometer (Bruker Daltonics, Bremen, Germany); deviations of the found
exact masses from the calculated exact masses were 5 mDa or less; the data were
analyzed with DataAnalysis® (Bruker Daltonics). NMR: NMR spectra were recorded
in deuterated solvents on Agilent DD2 400 MHz and 600 MHz spectrometers

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(Agilent, Santa Clara CA, USA); chemical shifts (δ) are reported in parts per million
(ppm) against the reference substance tetramethylsilane and calculated using the
solvent residual peak of the undeuterated solvent; coupling constants are given with
1
0.5 Hz resolution; assignment of H and 13C NMR signals was supported by 2-D
NMR techniques where necessary.IR: FT/IR IR Affinity®-1 spectrometer (Shimadzu,
Düsseldorf, Germany) using ATR technique.
4.2. HPLC method for the determination of the purity
Pump: LPG-3400SD, degasser: DG-1210, autosampler: ACC-3000T, UV-detector:
VWD-3400RS, interface: DIONEX UltiMate 3000, data acquisition: Chromeleon 7
(equipment and software from Thermo Fisher Scientific, Lauenstadt, Germany);
column: LiChrospher® 60 RP-select B (5 µm), LiChroCART® 250-4 mm cartridge;
flow rate: 1.0 mL/min; injection volume: 5.0 µL; detection at λ = 210 nm; solvents: A:
demineralized water with 0.05 % (V/V) trifluoroacetic acid, B: CH3CN 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 %.
4.3. Synthetic procedures
4.3.1. Dimethyl pyridine-2,3-dicarboxylate (6)
Quinolinic acid (5, 15.6 g, 94.0 mmol, 1.00 eq.) was dissolved in MeOH (100 ml) and
concentrated H2SO4 (10 ml) was added dropwise. The reaction mixture was heated
to reflux for 16 h. H2O (80 ml) was added and the mixture was adjusted to pH 8 by
addition of NaOH platelets and extracted with CH2Cl2 (3x100ml). The combined
organic layers were dried (anhydr. Na2SO4) and the solvent was removed in vacuo.
Yellow solid, yield 13.8 g (75 %). C9H9NO4 (195.2 g/mol). Mp = 55 °C. 1H NMR (600

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MHz, CD3OD): δ = 3.96 (s, 3H, CH3), 3.99 (s, 3H, CH3), 7.70 (dd, J = 8.0, 4.9 Hz, 1H,
5-H), 8.35 (dd, J = 8.0, 1.6 Hz, 1H, 4-H), 8.78 (dd, J = 4.9, 1.6 Hz, 1H, 6-H). 13C NMR
(151 MHz, CD3OD): δ = 55.1 (1C, CO2CH3), 55.5 (1C, CO2CH3), 126.7 (1C, 5-C),
127.5 (1C, 4-C), 139.4 (1C, 3-C), 152.0 (1C, 6-C), 152.9 (1C, 2-C), 168.2 (1C, C=O),
+
166.9 (1C, C=O). Exact MS (APCI): m/z = 196.0609 (calcd. 196.0604 for C9H10NO4
[M+H+]).
4.3.2. Pyridine-2,3-dimethanol (7)
Diester 6 (250 mg, 1.3 mmol, 1.0 eq.) was dissolved in EtOH (10 ml) and
NaBH4 (240 mg, 6.40 mmol, 5.00 eq.) was added portion wise at -10 °C. The
resulting slurry was heated to reflux for 2 h and then MeOH (10 ml) and H2O (1 ml)
were added. The mixture was filtered over Celite® and the filtrate was dried
(Na2SO4). The solvent was removed in vacuo and the residue was purified via fc
(ø = 2 cm, l = 20 cm, V = 10 ml, Rf = 0.75, CH2Cl2:MeOH:triethylamine = 15:5:0.1).
Colorless oil, yield 96 mg (54 %). C7H9NO2 (139.2 g/mol). Rf = 0.75
(CH2Cl2:MeOH:triethylamine = 15:5:0.1). 1H NMR (400 MHz, CDCl3): δ = 4.64 (s, 2H,
8-CH2-OH), 4.73 (s, 2H, 7-CH2-OH), 7.22 (dd, J = 7.6, 4.9 Hz, 1H, 5-H), 7.72 (dd, J =
7.6, 4.9 Hz, 1H, 4-H), 8.40 (d, J = 4.9, 1H, 6-H). 13C NMR (151 MHz, CDCl3): δ = 62.7
(1C, 8-CH2-OH), 65.9 (1C, 7-CH2-OH), 121.7 (1C, 5-C), 135.7 (1C, 4-C), 137.7 (1C,
3-C), 145.5(1C, 6-C), 161.5 (1C, 2-C). Exact MS (APCI): m/z = 140.0700 (calcd.
+
140.0706 for C7H10NO2 [M+H+]).
4.3.3. 5,6,8,9-Tetrahydro-7H-[7]annuleno[b]pyridin-7-one (10)
Diol 7 (140 mg, 1.0 mmol, 1.0 eq.) was dissolved in THF (20 ml) and
PBr3 (0.28 ml, 3.0 mmol, 3.0 eq.) was added slowly under ice bath cooling. The
reaction mixture was stirred for 1 h at rt and then saturated NaHCO3 solution was

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added until pH 8 was adjusted. The layers were separated, and the aqueous layer
was extracted with EtOAc (3x20 ml). The combined organic layers were dried over
anhydrous Na2SO4 and the solvent was removed in vacuo. The residue was
dissolved in THF (5 ml) and filtrated over Celite®. The solvent was removed in vacuo.
The resulting dibromide 8 (131 mg, 0.5 mmol, 1.0 eq.) was dissolved in THF (10 ml)
and K2CO3 (205 mg, 1.5 mmol, 3.0 eq.) and KI (4 mg, 0.03 mmol, 0.05 eq.) were
added. Dimethyl 3-oxoglutarate (0.86 ml, 0.8 mmol, 1.2 eq.) was added dropwise and
the reaction mixture was heated to reflux for 16 h. The slurry was filtered over
Celite®. The solvent was removed in vacuo and the residue was purified via fc (ø = 2
cm, l = 27 cm, V = 10 ml, Rf = 0.75, ethyl acetate:triethylamine = 97:3). Oxodiester 9
(75 mg, 0.3 mmol, 1.0 eq.) was dissolved in 6 M HCl (10 ml) and heated up to 95 °C
for 4 h. 6 M NaOH (15 ml) was added to the mixture until pH 8 was adjusted and the
mixture was extracted with EtOAc (3x20 ml). The solvent was removed in vacuo and
the residue was purified via flash column chromatography (ø = 2.5 cm, l = 6 cm, V =
12 ml, H2O : acetonitrile : trifluoroacetic acid = 80:20:0.1). Colorless oil, yield 18 mg
(16 %). C10H11NO (161.2 g/mol). FTIR (neat): ṽ (cm-1): 752 (δAr-H (out of plane)), 1697
1
(vC=N), 1770 (vC=O), 2954 (vC-H). H NMR (400 MHz, CDCl3): δ = 2.63 – 2.66 (m, 2H,
6-CH2), 2.67 – 2.70 (m, 2H, 8-CH2), 2.92 - 2.95 (m, 2H, 5-CH2), 3.15 – 3.19 (m, 2H,
9-CH2), 7.17 (dd, J = 7.6, 4.9 Hz, 1H, 3-H), 7.51 (dd, J = 7.6, 1.6 Hz, 1H, 4-H),
8.43 (dd, J = 4.9, 1.6 Hz, 1H, 2-H). 13C NMR (151 MHz, CDCl3): δ = 32.6 (9-C), 33.3
(1C, 5-C), 42.8 (1C, 6-C), 45.1 (1C, 8-C), 122.5 (1C, 3-C), 135.5 (1C, 4a-C), 137.2
(1C, 4-C), 147.6 (1C, 2-C), 160.1 (1C, 9a-C), 210.5 (1C, CO). Exact MS (APCI): m/z
= 162.0918 (calcd. 162.0913 for C10H12NO+ [M+H+]). Purity (HPLC - Method I): 45.7
% (tR = 8.11 min)
4.3.4. N-Benzyl-6,7,8,9-tetrahydro-5H-[7]annuleno[b]pyridin-7-amine (11a)

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Ketone 10 (34 mg, 0.2 mmol, 1.0 eq.) was dissolved in CH2Cl2 (5 ml) and
Na2SO4 (50 mg) was added. Benzylamine (23 µl, 0.2 mmol, 1.0 eq.) was added and
the mixture was stirred for 5 min. NaBH(OAc)3 (58 mg, 0.3 mmol, 1.3 eq.) was added
and the mixture was stirred for 3 h. H2O (0.5 ml) was added and the slurry was
filtered. The solvent was removed in vacuo and the residue was purified via fc
(ø = 2.5 cm, l = 6 cm, V = 12 ml, H2O : acetonitrile : trifluoroacetic acid = 80:20:0.1).
Yellow oil, 12 mg (22 %). C17H20N2 (252.4 g/mol). FTIR (DMSO): ṽ (cm-1): 705, 675
(δPh-H (out of plane)), 1658 (vC=N), 3473 (vN-H). 1H NMR (400 MHz, CDCl3): δ = 1.42 – 1.49
(m, 2H, 6,8-H), 2.12 - 2.17 (m, 2H, 6,8-H), 2.65 – 2.70 (m, 1H, 9-H), 2.72 – 2.77 (m,
2H, Ph-CH2), 2.85 – 2.91 (m, 3H, 5,9-CH, CH-NH), 3.17 – 3.21 (m, 1H, 5-H),
7.03 (dd, J = 7.5, 4.9 Hz, 1H, 3-H),
7.25
–
7.27
(m,
3H,
3’,4’-H),
7.33 - 7.34 (m, 2H, 2’-H), 7.38 (dd,
J
=
7.5, 1.7 Hz, 1H, 4-H),
13
8.30 (dd, J = 4.9, 1.7 Hz, 1H, 2-H). C NMR (151 MHz, CDCl3): δ = 29.6 (1C, 9-C),
30.7 (1C, 8-C), 32.2 (1C, 6-C), 32.6 (1C, 5-C), 50.6 (1C, Ph-C), 62.7 (1C, CH-N),
121.4 (1C, 3-C), 127.0 (1C, 4’-C), 127.2 (2C, 3’-C), 128.4 (2C, 2’-C), 131.9 (1C, 4-C),
136.3 (1C, 4a-C), 137.1 (1C, 1’-C), 146.4 (1C, 2-C), 162.2 (1C, 9a-C). Exact MS
+
(APCI): m/z = 253.1707 (calcd. 253.1705 for C17H21N2 [M+H+]). Purity (HPLC): 78.8
% (tR = 6.11 min).
4.3.5. N-(3-Phenylpropyl)-6,7,8,9-tetrahydro-5H-[7]annuleno[b]pyridin-7-amine
(11b)
Ketone 10 (51 mg, 0.3 mmol, 1.0 eq.) was dissolved in CH2Cl2 (5 ml) and
Na2SO4 (50 mg) was added. 4-Phenylbutylamine (47 µl, 0.3 mmol, 1.1 eq.) was
added and the mixture was stirred for 5 min. NaBH(OAc)3 (69 mg, 0.3 mmol, 1.1 eq.)
was added and the mixture was stirred for 4 h. H2O (0.5 ml) was added and the slurry
was filtered. The solvent was removed in vacuo and the residue was purified via flash

16
ACCEPTED MANUSCRIPT
column chromatography (ø = 2.5 cm, l = 6 cm, V = 12 ml, H2O : acetonitrile :
trifluoroacetic acid = 80:20:0.1). Green oil, 47 mg (52 %). C19H24N2 (280.4 g/mol).
FTIR (neat): ṽ (cm-1): 744, 698 (δPh-H (out of plane)), 799 (δAr-H (out of plane)), 1685 (vC=N),
1
2924 (vC-H), 3280 (vN-H). H NMR (600 MHz, CDCl3): δ = 1.28 – 1.34 (m, 1H, 6-H),
1.35 – 1.40 (m, 1H, 8-H), 1.80 – 1.86 (m, 2H, Ph-CH2-CH2), 2.10 – 2.13 (m, 2H, 6,8-
H), 2.65 – 2.71 (m, 5H, N-CH2, Ph-CH2, 9-H), 2.78 – 2.83 (m, 2H, 9-H, CH-N), 2.86 –
2.90 (m, 1H, 5-H), 3.12 – 3.16 (m, 1H, 5-H), 7.01 (dd, J = 7.5, 4.9 Hz, 1H, 3-H), 7.16
– 7.19 (m, 3H, 3’,4’-H), 7.26 – 7.29 (m, 2H, 2-H), 7.35 (dd, J = 7.5, 1.6 Hz, 1H, 3-H),
8.28 (dd, J = 4.9, 1.6 Hz, 1H, 2-H). 13C NMR (151 MHz, CDCl3, 300K): δ = 31.4 (1C,
9-C), 32.2 (1C, 8-C), 32.8 (1C, Ph-CH2-CH2), 34.1 (1C, Ph-C), 34.3 (1C, 6-C), 35.3
(1C, 5-C), 46.8 (1C, N-CH2), 61.2 (1C, CH-N), 121.6 (1C, 3-C), 126.1 (1C, 4’-C),
128.7 (2C, 3’-C), 128.7 (2C, 2’-C), 136.6 (1C, 4-C), 137.5 (1C, 4a-C), 142.3 (1C, 1’-
C), 146.7 (1C, 2-C), 162.39 (1C, 9a-C). Exact MS (APCI): m/z = 281.2006 (calcd.
281.2012 for C19H25N2 [M+H+]). Purity (HPLC): 95.6 % (tR = 10.80 min).
+
4.3.6. N-(4-Phenylbutyl)-6,7,8,9-tetrahydro-5H-[7]annuleno[b]pyridin-7-amine
(11c)
Ketone 10 (40 mg, 0.2 mmol, 1.0 eq.) was dissolved in CH2Cl2 (5 ml) and
Na2SO4 (50 mg) was added. 4-Phenylbutylamine (47 µl, 0.2 mmol, 1.0 eq.) was
added and the mixture was stirred for 5 min. NaBH(OAc)3 (67 mg, 0.3 mmol, 1.3 eq.)
was added and the mixture was stirred for 3 h. H2O (0.5 ml) was added and the slurry
was filtered. The solvent was removed in vacuo and the residue was purified via fc
(ø = 2.5 cm, l = 6 cm, V = 12 ml, H2O : acetonitrile : trifluoroacetic acid = 80:20:0.1).
Green oil, 26 mg (36%). C20H26N2 (294.2 g/mol). FTIR (neat): ṽ (cm-1): 744, 698 (δPh-
H (out of plane)), 799 (δAr-H (out of plane)), 1685 (vC=N), 2924 (vC-H), 3270 (vN-H). 1H NMR (600
MHz, CDCl3, 300K): δ = 1.28 – 1.34 (m, 1H, 6-H), 1.36 – 1.40 (m, 1H, 8-H), 1.50 –

17
ACCEPTED MANUSCRIPT
1.55 (m, 2H, N-CH2-CH2), 1.63 – 1.68 (m, 2H, Ph-CH2-CH2), 2.04 – 2.11 (m, 2H, 6,8-
H), 2.60 – 2.63 (m, 2H, Ph-CH2), 2.65 – 2.70 (m, 3H, 5-H, N-CH2), 2.77 – 2.83 (m,
2H, 9-H, CH-NH), 2.86 – 2.90 (m, 1H, 9-H), 3.11 – 3.15 (m, 1H, 5-H), 7.01 (dd, J =
7.5, 4.6 Hz, 1H, 3-H), 7.16 – 7.17 (m, 3H, 3’,4’-H), 7.25 – 7.27 (m, 2H, 2’-H), 7.35
(dd, J = 7.5, 1.6 Hz, 1H, 4-H), 8.28 (dd, J = 4.9, 1.6 Hz, 1H, 2-H). 13C NMR (151
MHz, CDCl3, 300K): δ = 29.6 (1C, Ph-CH2-CH2), 30.3 (1C, N-CH2-CH2), 31.5 (1C, 9-
C), 32.9 (1C, 6-C), 34.4 (1C, 8-C), 35.3 (1C, 5-C), 36.2 (1C, C-Ph), 47.2 (1C, CH2-
NH), 61.3 (1C, C-NH), 123.8 (1C, 3-C), 126.0 (2C, 2’-C), 128.6 (2C, 3’-C), 128.7 (1C,
4’-C), 136.7 (1C, 4-C), 137.5 (1C, 4a-C), 142.7 (1C, 2-C), 146.7 (1C, 1’-C), 162.6
+
(1C, 9a-C). Exact MS (APCI): m/z = 295.2170 (calcd. 295.2169 for C20H27N2
[M+H+]). Purity (HPLC): 98.6 % (tR = 11.67 min).
4.3.7. N-Methyl-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-[7]annuleno[b]pyridin-
7-amine (12b)
3-Phenylpropylamine 11b (40 mg, 0.1 mmol, 1.0) and formalin (37 %, 100 µl, 6.5 eq.)
were dissolved in CH2Cl2 (5 ml) and Na2SO4 (50 mg) was added. NaBH(OAc)3
(75 mg, 0.4 mmol, 2.5 eq.) was added and the mixture was stirred for 4.5 h. The
slurry was filtered, and the solvent was removed in vacuo. The residue was purified
via fc (ø = 2.5 cm, l = 6 cm, V = 12 ml, H2O : acetonitrile : trifluoroacetic acid =
80:20:0.1). Green oil, 13 mg (32 %). C20H26N2 (294.4 g/mol). FTIR (neat): ṽ (cm-1):
748, 698 (δPh-H (out of plane)), 802 (δAr-H (out of plane)), 1678 (vC=N), 2789 (vN-C), 2927 (vC-H).
1H NMR (600 MHz, CDCl3, 300K): δ = 1.33 – 1.48 (m, 2H, 6,8-H), 1.77 – 1.85
(m, 2H, N-CH2-CH2), 2.08 – 2.12 (m, 2H, 6,8-H), 2.26 (s, 3H, CH3), 2.47 – 2.51
(m, 2H, N-CH2), 2.61 – 2.65 (m, 2H, PH-CH2), 2.69 – 2.88 (m, 4H, CH-N, 5,9-H), 3.11
– 3.16 (m, 1H, 5-H), 7.04 (dd, J = 7.5, 4.9 Hz, 1H, 3-H), 7.18 – 7.20 (m, 3H, 3’,4’-H),
7.26 – 7.29 (m, 2H, 2’-H), 7.38 (dd, J = 7.5, 1.5 Hz, 1H, 4-H),

18
ACCEPTED MANUSCRIPT
8.31 (dd, J = 4.9, 1.5 Hz, 1H, 2-H). 13C NMR (151 MHz, CDCl3, 300K): δ = 27.7 (1C,
8-C), 29.9 (1C, 6-C), 30.0 (1C, N-CH2-CH2), 32.5 (1C, 9-C), 33.7 (1C, Ph-C), 36.3
(1C, 5-C), 37.5 (1C, CH3), 53.2 (1C, N-CH2), 67.5 (1C, CH-N), 121.7 (1C, 3-C),
125.9 (1C, 4’-C), 128.5 (2C, 2’-C), 128.52 (2C, 3’-C), 136.5 (1C, 4-C),
137.3 (1C, 4a-C), 142.1 (1C, 1’-C), 146.7 (1C, 2-C), 162.3 (1C, 9a-C). Exact MS
+
(APCI): m/z = 295.2157 (calcd. 295.2174 for C20H27N2 [M+H+]). Purity (HPLC): 92.2
% (tR = 10.96 min).
4.3.8. N-Methyl-N-(4-phenylbutyl)-6,7,8,9-tetrahydro-5H-[7]annuleno[b]pyridin-7-
amine (12c)
4-Phenylbutylamine 11c (53 mg, 0.2 mmol, 1.0) and formalin (37 % aq., 128 µl, 6.5
eq.) were dissolved in CH2Cl2 (5 ml) and Na2SO4 (50 mg) was added. NaBH(OAc)3
(116 mg, 0.5 mmol, 3.0 eq.) was added and the mixture was stirred for 4 h. The slurry
was filtered, and the solvent was removed in vacuo. The residue was purified via fc
(ø = 2.5 cm, l = 6 cm, V = 12 ml, H2O : acetonitrile : trifluoroacetic acid = 80:20:0.1).
Green oil, 13 mg (23 %). C21H28N2 (308.5 g/mol). FTIR (neat): ṽ (cm-1): 744, 698 (δPh-
H (out of plane)), 802 (δAr-H (out of plane)), 1678 (vC=N), 2789 (vN-C), 2927 (vC-H). 1H NMR (600
MHz, CDCl3, 300K): δ = 1.35 – 1.40 (m, 1H, 6-H), 1.41 – 1.46 (m, 1H, 8-H), 1.50 –
1.55 (m, 2H, N-CH2-CH2), 1.61 – 1.66 (m, 2H, Ph-CH2-CH2), 2.09 – 2.12 (m, 2H, 6,8-
H), 2.25 (s, 3H, CH3), 2.46 – 2.48 (m, 2H, N-CH2), 2.61 – 2.64 (m, 2H, Ph-CH2), 2.67
– 2.71 (m, 1H, 9-H), 2.74 – 2.81 (m, 3H, 5,9-H, CH-N), 3.11 – 3.15 (m, 1H, 5-H), 7.05
(dd, J = 7.5, 4.9 Hz, 1H, 3-H), 7.16 – 7.18 (m, 3H, 3’,4’-H), 7.25 – 7.28 (m, 2H, 2’-H),
7.38 (dd, J = 7.5, 1.5 Hz, 1H, 4-H), 8.31 (dd, J = 4.9, 1.5 Hz, 1H, 2-H). 13C NMR (151
MHz, CDCl3, 300K): δ = 27.5 (1C, 8-C), 27.7 (1C, N-CH2-CH2), 29.3 (1C, Ph-CH2-
CH2), 29.9 (1C, 6-C), 32.4 (1C, 9-C), 35.1 (1C, Ph-C), 36.2 (1C, 5-C), 37.6 (1C, N-
CH3), 53.5 (N-CH2), 67.5 (N-CH), 121.7 (1C, 3-C), 125.8 (1C, 4’-C), 128.4 (2C, 3’-C),

19
ACCEPTED MANUSCRIPT
128.5 (2C, C), 136.5 (1C, 4-C), 137.2 (1C, 5a-C), 142.6 (1C, 1’-C), 146.7 (1C, 2-C),
+
162.3 (1C, 9a-C). Exact MS (HRMS): m/z = calc. for C21H29N2 [M+H+] 309.2325,
found 309.2314. Purity (HPLC): 91.4 % (tR = 12.76 min).
4.4. Receptor binding studies
4.4.1. Materials
Guinea pig brains and rat livers were commercially available (Harlan-Winkelmann,
Borchen, Germany). Pig brains were a donation of the local slaughterhouse
(Coesfeld, Germany). The recombinant L(tk-) cells stably expressing the GluN2B
receptor were obtained from Prof. Dr. Dieter Steinhilber (Frankfurt, Germany).
Homogenizers: Elvehjem Potter (B. Braun Biotech International, Melsungen,
Germany) and Soniprep® 150, MSE, London, UK). Centrifuges: 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). 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).
4.4.2. Cell culture and preparation of membrane homogenates from GluN2B
cells
Mouse L(tk-) cells stably transfected with the dexamethasone-inducible eukaryotic
expression vectors pMSG GluN1a, pMSG GluN2B (1:5 ratio) were grown in Modified
Earl’s Medium (MEM) containing 10 % of standardized FCS (Biochrom AG, Berlin,

20
ACCEPTED MANUSCRIPT
Germany). The expression of the NMDA receptor at the cell surface was induced
after the cell density of the adherent growing cells had reached approximately 90 %
of confluency. For the induction, the original growth medium was replaced by growth
medium containing 4 µM dexamethasone and 4 µM ketamine (final concentration).
After 24 h, the cells were rinsed with phosphate buffered saline solution (PBS,
Biochrom AG, Berlin, Germany), harvested by mechanical detachment and pelleted
(10 min, 5,000 x g).
For the binding assay, the cell pellet was resuspended in PBS solution and the
number of cells was determined using a Scepter® cell counter (MERCK Millipore,
Darmstadt, Germany). Subsequently, the cells were lysed by sonication (4 °C,
6 x 10 s cycles with breaks of 10 s). The resulting cell fragments were centrifuged
with a high performance cool centrifuge (23,500 x g, 4 °C). The supernatant was
discarded and the pellet was resuspended in a defined volume of PBS yielding cell
fragments of approximately 500,000 cells/mL. The suspension of membrane
homogenates was sonicated again (4 °C, 2 x 10 s cycles with a break of 10 s) and
stored at -80 °C.
4.4.3. Preparation of membrane homogenates from pig brain cortex
Fresh pig brain cortex was 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
TRIS/EDTA buffer (5 mM TRIS/1 mM EDTA, pH 7.5) and centrifuged again at
31,000 x g (20 min, 4 °C). The final pellet was resuspended in 5-6 volumes of buffer
and frozen (-80 °C) in 1.5 mL portions containing about 0.8 mg protein/mL.

21
ACCEPTED MANUSCRIPT
4.4.4. 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
1,200 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.
4.4.5. 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 rt 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.
4.4.6. General procedures 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 rt before use. All binding

22
ACCEPTED MANUSCRIPT
experiments were carried out in duplicates in the 96 well multiplates. The
concentrations given are the final concentration in the assay. Generally, the assays
were performed by addition of 50 µL of the respective assay buffer, 50 µL of test
compound solution in various concentrations (10-5, 10-6, 10-7, 10-8, 10-9 and 10-
10 mol/L), 50 µL of the 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 rt, the trapped radioactivity in the filtermats was measured with the
scintillation 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.[33] The Ki values are given as mean value
± SEM from three independent experiments.
4.4.7. Affinity for the GluN2B binding site of the NMDA receptor
The competitive binding assay was performed with the radioligand [3H]ifenprodil
(60 Ci/mmol; BIOTREND, Cologne, Germany). The thawed cell membrane
preparation from the transfected L(tk-) cells (about 20 µg protein) was incubated with
various concentrations of test compounds, 5 nM [3H]-ifenprodil, and TRIS/EDTA-

23
ACCEPTED MANUSCRIPT
buffer (5 mM TRIS/1 mM EDTA, pH 7.5) at 37 °C. The non-specific binding was
determined with 10 µM unlabeled ifenprodil. The Kd value of ifenprodil is 7.6 nM.[26]
4.4.8. Affinity for the PCP binding site of the NMDA receptor
The assay was performed with the radioligand [3H]-(+)-MK-801 (22.0 Ci/mmol; Perkin
Elmer). [17,18] The thawed membrane preparation of pig brain cortex (about 100 µg
of the protein) was incubated with various concentrations of test compounds, 2 nM
[3H]-(+)-MK-801, and TRIS/EDTA buffer (5 mM TRIS/1 mM EDTA, pH 7.5) at rt. The
non-specific binding was determined with 10 µM unlabeled (+)-MK-801. The Kd value
of (+)-MK-801 is 2.26 nM. [34]
4.4.9. Affinity for the σ1 receptor
The assay [28-30] was performed with the radioligand [3H]-(+)-pentazocine
(22.0 Ci/mmol; Perkin Elmer). The thawed membrane preparation of guinea pig brain
cortex (about 100 µg of the 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 µM unlabeled (+)-pentazocine. The
Kd value of (+)-pentazocine is 2.9 nM.[35]
4.4.10. Affinity for the σ2 receptor
The assay [28-30] was performed with the radioligand [3H]di-o-tolylguanidine (specific
activity 50 Ci/mmol; ARC, St. Louis, MO, USA). The thawed rat liver membrane
preparation (about 100 µg protein) was incubated with various concentrations of the
test compound, 3 nM [3H]di-o-tolylguanidine and buffer containing (+)-pentazocine
(500 nM (+)-pentazocine in TRIS buffer (50 mM TRIS, pH 8.0)) at rt. The non-specific

24
ACCEPTED MANUSCRIPT
binding was determined with 10 µM non-labeled di-o-tolylguanidine. The Kd value of
di-o-tolylguanidine is 17.9 nM.[36]
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (DFG), which is
gratefully acknowledged. Moreover, we are grateful to Cells-in-Motion (CiM) Cluster
of Excellence for supporting our research.
Supporting Information
The Supporting Information contains purity data of prepared compounds, protein
determination of biological material and NMR spectra.
Conflict of interest
The authors declare no conflict of interest.
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