Synthesis, GluN2B affinity and selectivity of benzo[7]annulen-7-amines

Article abstract of DOI:10.1016/j.bmc.2014.10.004

Due to their beneficial side effect profile, NMDA receptor antagonists interacting selectively with the allosteric ifenprodil binding site of the GluN2B subunit are of major interest for the treatment of neurological and neurodegenerative disorders. A series of benzo[7]annulen-7-amines 6 was designed by conformational restriction of ifenprodil (1). At first the benzo[7]annulen-7-one 11 was prepared in a three-step synthesis comprising of a double Knoevenagel condensation of phthalaldehyde (7) with dimethyl 3-oxoglutarate (8), hydrogenation of 9 and saponification/decarboxylation of 10. Reductive amination of the ketone 11 with primary amines and NaBH(OAc)₃ led to the secondary amines 6a-d, cis-6h and trans-6i. The tertiary amines 6e-g were obtained by S_N2-substitution of the nosylate 13. Although H-bond forming substituents in 2- and 5-position are missing, the amines 6 exhibit high affinity towards GluN2B containing NMDA receptors. A distance of four to five bond lengths between the basic amino moiety and the phenyl ring in the side chain appears to be optimal for high GluN2B affinity. The phenylcyclohexylamine cis-6h and the 4-benzylpiperidine 6g show the highest GluN2B affinities (K_i = 2.3 nM and 2.9 nM, respectively). With respect to selectivity against the PCP binding site, σ₁ and σ₂ receptors the phenylpiperazine 6f is the most promising GluN2B antagonist.

Full text of DOI:10.1016/j.bmc.2014.10.004

Accepted Manuscript
Synthesis, GluN2B affinity and selectivity of benzo[7]annulen-7-amines
Sandeep Gawaskar, Dirk Schepmann, Alessandro Bonifazi, Bernhard Wünsch
PII:
S0968-0896(14)00717-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.10.004
BMC 11850
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
13 August 2014
30 September 2014
6 October 2014
Please cite this article as: Gawaskar, S., Schepmann, D., Bonifazi, A., Wünsch, B., Synthesis, GluN2B affinity and
selectivity of benzo[7]annulen-7-amines, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/
j.bmc.2014.10.004
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Synthesis, GluN2B affinity and selectivity of benzo[7]annulen-7-amines
Sandeep Gawaskar,[a,b] Dirk Schepmann,[a] Alessandro Bonifazi,[c] Bernhard Wünsch*[a,d]
[a] Sandeep Gawaskar, Dr. D. Schepmann, Prof. Dr. B. Wünsch
Institut für Pharmazeutische und Medizinische Chemie der Universität Münster,
Corrensstraße 48, 48149 Münster, Germany.
Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail: wuensch@uni-muenster.de
[b] NRW Graduate School of Chemistry
Wilhelm-Klemm Str. 10, 48149 Münster, Germany.
[c] A. Bonifazi
Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, Via S.
Agostino, 1, 62032 Camerino, Italy.
[d]
Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), University of Münster,
Germany.
1

Abstract
Due to their beneficial side effect profile, NMDA receptor antagonists interacting selectively
with the allosteric ifenprodil binding site of the GluN2B subunit are of major interest for the
treatment of neurological and neurodegenerative disorders. A series of benzo[7]annulen-7-
amines 6 was designed by conformational restriction of ifenprodil (1). At first the
benzo[7]annulen-7-one 11 was prepared in a three-step synthesis comprising of a double
Knoevenagel condensation of phthalaldeyhde (7) with dimethyl 3-oxoglutarate (8),
hydrogenation of 9 and saponification/decarboxylation of 10. Reductive amination of the
ketone 11 with primary amines and NaBH(OAc)3 led to the secondary amines 6a-d, cis-6h
and trans-6i. The tertiary amines 6e-g were obtained by SN2-substitution of the nosylate 13.
Although H-bond forming substituents in 2- and 5-position are missing, the amines 6 exhibit
high affinity towards GluN2B containing NMDA receptors. A distance of four to five bond
lengths between the basic amino moiety and the phenyl ring in the side chain appears to be
optimal for high GluN2B affinity. The phenylcyclohexylamine cis-6h and the 4-
benzylpiperidine 6g show the highest GluN2B affinities (Ki = 2.3 nM and 2.9 nM,
respectively). With respect to selectivity against the PCP binding site, σ1 and σ2 receptors the
phenylpiperazine 6f is the most promising GluN2B antagonist.
Keywords
NMDA receptor; GluN2B antagonists; benzo[7]annulen-7-amines; structure affinity
relationships; selectivity; conformational restriction
2

1. Introduction
Ionotropic glutamate receptors (iGluRs) are ligand gated ion channels which are activated by
the neurotransmitter (S)-glutamate. There exist three subtypes named according to the
glutamate derivative that results in selective activation. These are N-methyl-D-aspartate
(NMDA), 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and kainate
receptors.1–3 The NMDA receptor is one of the pharmacologically most investigated iGluRs
due to its unique properties. It is considered an important target for the development of
therapies for a range of pathological conditions like Parkinson’s disease,4 epilepsy5 and
cerebral ischemia.6
Composed of four subunits,7 which together form the ion channel, the NMDA receptor
requires the simultaneous binding of two agonists (S)-glutamate and glycine. The subunit
composition varies distinctly in different regions of the central nervous system. The subunits
are encoded by seven different genes which are classified into three types. The GluN1 subunit
has eight splice variants termed GluN1a-h, the GluN2 and GluN3 subunits are encoded by
four and two genes termed GluN2A-D and GluN3A-B, respectively. These different subunits
assemble in various combinations to form a heterotetrameric NMDA receptor. However, it
has been shown that a functional NMDA receptor needs to have at least one GluN1 and one
GluN2 subunit containing the binding sites for glycine and (S)-glutamate, respectively. Each
subunit is composed of an extracellular amino terminal domain (ATD), a ligand binding
domain (LBD), a trans-membrane domain (TMD) and an intracellular carboxy terminal
domain (CTD).8,9
One of the interesting aspects of the NMDA receptor is the variety of binding sites which
allows the modulation of the opening state of the ion channel. There exist agonist binding
sites for (S)-glutamate and glycine located on the GluN2 and GluN1 subunit, respectively.
3

Binding sites for phencyclidine (1-(1-phenylcyclohexyl)piperidine), PCP) and Mg2+-ions are
located within the channel pore, whereas the ATD presents binding sites for Zn2+, H+,
polyamines and ifenprodil. Since the ifenprodil binding site is only present on the GluN2B
subunit, ligands interacting with this binding site lead to a selective modulation of NMDA
receptors bearing the GluN2B subunit. The inhibition of GluN2B subunit containing NMDA
receptors occurs by a negative allosteric effect, which prevents (S)-glutamate from binding at
its orthosteric site on the GluN2B subunit, consequently inhibiting ion channel opening.10
Overactivation of the NMDA receptor caused by an excess of (S)-glutamate leads to an
increased influx of Ca2+-ions resulting in neuronal cell death. This phenomenon is termed
excitotoxicity. The expression of the GluN2B subunit is not as widespread as that of the
GluN2A subunit. The GluN2B subunit is typically localized in the cortex, hippocampus and
the striatum of the central nervous system (CNS). The negative allosteric modulation of the
GluN2B subunit by ifenprodil-like ligands interacting with a defined binding site at the ATD
represents a valuable therapeutic avenue. NMDA receptor antagonists interacting with the
ifenprodil binding site of the GluN2B subunit lead to an improved side-effect profile
compared with channel blockers (PCP binding site) or competitive glutamate and glycine
antagonists, which address all NMDA receptor subtypes.11,12
Ifenprodil (1, Vadilex®, Figure 1)13 represents the lead compound for the design of allosteric
GluN2B selective NMDA antagonists interacting with the ifenprodil binding site. Originally
ifenprodil has been developed as a α1 receptor antagonist, but its neuroprotective properties
were soon discovered. Later, the high affinity of ifenprodil at a special binding site of the
NMDA receptor (IC50 = 13.3 nM) was recognized.14 However, the clinical development of 1
was terminated due to undesired side effects and low bioavailability.15 Additionally, lack of
selectivity of ifenprodil interacting with 5-HT1A, 5-HT2, α1, σ1, and σ2 receptors was observed.
4

Subsequently, various efforts were undertaken to discover selective GluN2B antagonists with
high affinity for the ifenprodil binding site but without the drawbacks mentioned above.
In 2011, the X-ray crystal structure of ifenprodil (1) and Ro 25-6981 in complex with a dimer
of the ATD of GluN1b and GluN2B subunits was solved. The crystal structure shows the
ligand binding site at the interface of the two subunits GluN1b and GluN2B. Two H-bonds
were found between the phenol of 1 and Glu236 as well as a water molecule. Furthermore
Gln110 forms two hydrogen bonds with the protonated central piperidine N-atom and the
benzylic OH moiety. In addition to these polar interactions, there also exist substantial
hydrophobic interactions mainly between the lipophilic part of 1 and surrounding hydrophobic
residues, e.g. Phe114 (GluN2B), Ile 111 (GluN2B) and Tyr109 (GluN1b).16
Figure 1: Development of conformationally restricted antagonists of GluN2B subunit
containing NMDA receptors.
Recently, tetrahydro-3-benzazepines 2 and 3 were synthesized as ifenprodil-like compounds
following a conformational restriction approach. (Figure 1) 3-Benzazepines 2 and 3 were
found to have low nanomolar affinity in radioligand binding assays with Ki values of 14 nM
5

and 5.4 nM, respectively. Moreover antagonistic activity (IC50 (2) = 18.4 nM, IC50 (3) = 360
nM) in functional studies on GluN2B-containing NMDA receptors was observed.17,18
Next the N-atom in the 3-position of the 3-benzazepine 3 was transferred outside the seven
membered ring to afford the benzo[7]annulen-7-amines 4 and 5. The conformationally
restricted benzo[7]annulen-7-amines 4 and 5 resemble more closely the clinical candidate Ro
25-698119 in terms of the distance between the substituted aromatic ring and the basic
functional group (4 bond lengths). The phenylpropylamine 4 with a methoxy moiety at the 2-
position of the benzo[7]annulene ring shows high GluN2B affinity (Ki = 15 nM).
Unexpectedly, benzo[7]annulenamine 5 without a benzylic hydroxy moiety led to a slightly
increased GluN2B affinity (Ki = 7.9 nM).20
Stimulated by the high GluN2B affinity of 5, the pharmacological properties of various
benzo[7]annulenamines 6 without a benzylic OH-moiety should be investigated.21 Moreover,
in the first series of ligands 6 the methoxy group in 2-position of 4 and 5 should be removed
to analyze the contribution of this substituent to the GluN2B affinity and selectivity. Herein
we wish to report on the synthesis, GluN2B affinity and selectivity of benzo[7]annulenes 6
bearing aliphatic, cyclic and heterocyclic N-substituents in 7-position.
2. Synthesis
For the synthesis of benzo[7]annulen-7-amines 6 without further substituents the ketone 11
was selected as the key intermediate. The literature provides useful hints for the synthesis of
ketone 11, which were considered during the development and optimization of its synthesis.22–
24
6

Scheme 1: Synthesis of benzo[7]annulen-7-one 11.
o
Reagents and reaction conditions: (a) piperidine, CH3CO2H, toluene, 95 C, 14 h,
70 %; (b) H2, Pd/C, CH3CO2H, 24 h, 93 %; (c) 6 M HCl, CH3OH, 90 oC, 81 %.
Knoevenagel condensation of phthalaldehyde (7) and dimethyl 3-oxopentanedioate (8)
provided the benzo[7]annulenone 9 in 70 % yield, which is comparable with the yield
reported in literature (73 %). (Scheme 1) Catalytic hydrogenation of 9 using Pd/C in the
1
presence of HOAc led to the diester 10. The H NMR spectrum of the diester 10 is
complicated due to the existence of two diastereomeric ketones and one enol tautomer.
o
Treatment of 10 with 6 M HCl and a small amount of CH3OH at 95 C led to hydrolysis and
1
decarboxylation, resulting in the ketone 11 in 81 % yield. The H NMR spectrum of the
ketone 11 shows the expected signals and the high yield confirmed the structure of precursor
10 unequivocally.
Reductive amination of ketone 11 with primary amines and NaBH(OAc)3 provided the
secondary amines 6a-d in 20-92 % yields. (Scheme 2) However, the reductive amination of
the ketone 11 with secondary amines including piperidine and piperazine derivatives failed to
give tertiary amines. Various reaction conditions led either to very low yields or no
conversion was observed (tlc monitoring). It is assumed that the formation of the iminium ion,
which is required for the subsequent hydride transfer, is sterically hindered by the coplanar
orientation of the four CH2-moieties adjacent to the iminium ion.
7

Scheme 2: Synthesis of benzo[7]annulen-7-amines 6.
Reagents and reaction conditions: (a) H2N(CH2)nPh (1.5 – 2 eq.), NaBH(OAc)3
(1.5 – 2.0 eq.) CH2Cl2, 2 – 14 h, 20 – 92 %; (b) NaBH4 (3 eq.), CH3OH, 0 oC, 1 h,
94 %; (c) 4-nitrobenzenesulfonyl chloride (2 eq.), DMAP (0.4 eq.), NEt3 (3 eq.),
CH2Cl2, 16 h, 28 %; (d) 4-phenylpiperidine (5.5 eq.) or 1-phenylpiperazine (5
o
eq.), CH3CN, 75 C, 14 – 16 h, 26 % (6e), 27 % (6f); (e) 4-benzylpiperidine (4.7
eq.), CH3CN, 75 oC, 16 h, 37 %.
In order to prepare tertiary amines, a SN2 substitution instead of reductive amination was
envisaged. The ketone 11 was reduced with NaBH4 to afford the alcohol 12. The nosyloxy (4-
nitrophenylsulfonyloxy) group was selected as a suitable leaving group. Thus the alcohol 12
was transformed into the nosylate 13 with 4-nitrobenzenesulfonyl chloride, NEt3 and DMAP.
SN2 reaction of nosylate 13 with secondary amines provided the tertiary amines 6e-g in 26-37
% yields.
8

Scheme 3: Synthesis of diastereomeric phenylcyclohexyl derivatives cis-6h and trans-6i.
Reagents and reaction conditions: (a) Benzylamine (2 eq.), NaBH(OAc)3 (2 eq.),
dist. CH2Cl2, 16 h, cis-15 (71 %), trans-15 (26 %); (b) H2, Pd/C, CH3OH, cat.
CH3CO2H, 24 h, cis-16 (63 %), trans-16 (17 %); (c) 11 (1 eq.), cis-16/trans-16
(ratio 70:30, 2.2 eq.), NaBH(OAc)3 (2.1 eq.), dist. CH2Cl2, 20 h, 31 % (cis-6h), 9
% (trans-6i).
Compared with the phenylalkyl substituted secondary amines 6a-d, the side chain of the
piperidine and piperazine derivatives 6e-g is conformationally restricted due to its
incorporation into a six-membered heterocycle. However, the N-phenyl distances of 6e,f and
6g correlate with the N-phenyl distances in 6c and 6d, respectively.
Next, the conformationally restricted phenylcyclohexyl derivatives cis-6h and trans-6i were
envisaged showing the same N-phenyl distances as 6d and 6g, but representing secondary
amines instead of tertiary amines. For the synthesis of the secondary amines cis-6h and trans-
6i by reductive amination of ketone 11, the primary amine 16 had to be prepared. (Scheme 3)
For this purpose 4-phenylcyclohexanone (14) was reacted with benzylamine and
NaBH(OAc)3 to yield the secondary amine 15, which was debenzylated by H2 and Pd/C to
provide the primary amine 16.25 Since the mixture of diastereomers cis-6h and trans-6i could
be separated readily by flash column chromatography, the ketone 11 was treated with a
mixture of the diastereomeric primary amines cis-16 and trans-16 (ratio 70:30). The resulting
9

mixture of diastereomers was separated to afford the diastereomerically pure secondary
amines cis-6h and trans-6i in 31 % and 9 % yields, respectively. In order to unequivocally
characterize the intermediates cis-15, trans-15 and cis-16 and trans-16, the diastereomers
were separated and characterized, respectively.
3. Pharmacological Evaluation
Affinity towards the ifenprodil binding site of GluN2B subunit containing NMDA receptors
In order to test the GluN2B affinity of the benzo[7]annulen-7-amines 6 a competitive
radioligand receptor binding assay was employed.14 Membrane preparations of L(tk-) cells
stably expressing recombinant human GluN1a and GluN2B subunits were employed as
receptor material. The membrane preparations were obtained by ultrasonic irradiation.
Addition of dexamethasone during the growth of the cells induced the production of
functional NMDA receptors, which were blocked by an excess of ketamine. Tritium labeled
[3H]ifenprodil ([3H]1) served as radioligand. The high number of NMDA receptors in the
membrane preparation is responsible for the high GluN2B selectivity of this assay.
Table 1: Receptor affinity of the test compounds 6 compared with the affinity data of
reference compounds.
Ki SEM [nM] (n=3)
compd.
NR2
GluN2B
14 1.5
5.4 0.4
PCP
35 %
22 %
12 %
0 %
σ1
194
σ2
218
317
420
520
>10 µM
554
182
16
3
4
132
9.2 2.1
15 5
10
27 7
10

57
20
16
17
6.9
12
6
7
4
2
1
3
16 %
0 %
107
22 7
150
203
198
6a
6b
6c
6d
6e
6f
0 %
27 12
55 7
18 3
200
2 %
216
11 %
5 %
22 6
182
6
2.9 2.5
2.3 1.0
30 %
5.7 0.2 5.4 3.2
6g
cis-6h
43 %
14 2
20 5
3.0 0.7
14 7
trans-6i
5.7 2.2
30 %
ifenprodil (1)
eliprodil
10 0.7
-
125 24 98.3 34
13 2.5
-
-
-
-
-
dexoxadrol
haloperidol
di-o-tolylguanidine
-
-
-
32 7.4
-
-
6.3 1.6 78.1 2.3
89 29 57.5 18
Note: For low-affinity compounds only the inhibition of the radioligand binding (in %) at
a test compound concentration of 1 µM is given.
Table 1 summarizes the affinity of the test and reference compounds towards GluN2B
receptors and some related receptors.
11

Unexpectedly, the secondary amines 6a-d without a substituent at the 2-position have
moderate to high GluN2B affinity. Increasing of the chain length leads to an increase in
GluN2B affinity. The phenylpropylamine 6c (Ki = 16 nM), which is almost four times more
active than the benzylamine 6a (Ki = 57 nM), exhibits the same GluN2B affinity as the
phenylbutylamine 6d (Ki = 17 nM).
The cyclic tertiary amines 6e-g display very high GluN2B affinity. The phenylpiperidine
derivative 6e (Ki = 6.9 nM) is approximately twice as active as its open-chain phenylpropyl
counterpart 6c. The homologous benzylpiperidine 6g (N-phenyl distance of five bond lengths,
Ki = 2.9 nM) has a higher GluN2B affinity than the phenylpiperidine derivative 6e (N-phenyl
distance of four bond lengths). The introduction of a second N-atom into the ring system as
shown for the phenylpiperazine 6f maintains considerable GluN2B affinity (Ki = 12 nM). The
high GluN2B affinity of the tertiary amines 6e-6g indicates the favorable effect of the
conformational restriction of the hydrophobic side chain on GluN2B affinity.
The GluN2B affinities of the phenylcyclohexyl derivatives cis-6h and trans-6i and the
benzylpiperidine 6g, which can be regarded as structural isomers, are very similar.
Nevertheless, the cis-configured phenylcyclohexyl derivative cis-6h shows slightly higher
GluN2B affinity than its trans-configured diastereomer trans-6i. The slightly higher GluN2B
affinity of cis-6h is in accordance with previous observations for analogous phenylcyclohexyl
derivatives in the series of tetrahydro-3-benzazepines 3.17
The established pharmacophore model26 for ifenprodil-like ligands recommends a substituent
at the 2-position of the benzo[7]annulen-7-amines 6. An OH moiety is generally found to be a
favorable substituent, but methoxy and benzyloxy groups are also well tolerated. The
investigated amines 6 lack a substituent in 2-position, which according to the solved crystal
12

structure is involved in H-bond interactions with Glu236 and a water molecule. The high
GluN2B affinity of the benzo[7]annulen-7-amines 6 indicates that the H-bond formed by the
central protonated amine with Gln110 contributes predominantly to the free binding enthalpy
during binding of these compounds. The enthalpic penalty resulting from removal of the H-
bond acceptor in 2-position is probably well compensated by an entropic gain in the binding
site at the GluN2B subunit.
Selectivity over selected receptors
To determine the selectivity, the affinities of the amines 6a-6i to the PCP binding site of the
NMDA receptor and to the σ1 and σ2 receptors were recorded. The radioligand [3H](+)-MK-
801 was used for determining the affinity to the PCP binding site located within the channel
pore.27,28 The benzo[7]annulen-7-amines 6 do not interact significantly with the PCP binding
site of the NMDA receptor. (Table 1)
The affinity of the synthesized ligands 6 towards σ1 and σ2 receptors was recorded using the
radioligands [3H](+)-pentazocine and [3H]di-tolylguanidine ([3H]DTG), respectively.29–31 The
Ki-values for both receptor subtypes do not show any recognizable trends. (Table 1) In
general, the ligands 6 interact with both σ1 and σ2 receptors. The benzylpiperidine 6g shows
high σ1 affinity (Ki(σ1) = 5.7 nM) along with high GluN2B affinity (Ki(GluN2B) = 2.9 nM)
indicating low GluN2B selectivity. The flexible phenylpropyl- and phenylbutylamines 6c and
6d are more selective for the GluN2B subunit over the σ1 receptor. However, both ligands 6c
and 6d show moderate affinity for the σ2 receptor. (6c: Ki(σ2) = 27 nM, 6d: Ki(σ2) = 55 nM).
The diastereomeric phenylcyclohexylamines cis-6h and trans-6i exhibit low selectivity for the
GluN2B receptor, since both compounds interact considerably with σ1 and σ2 receptors. In
terms of selectivity, the most promising ligand in this series is the phenylpiperazine 6f
13

possessing high GluN2B affinity (Ki = 12 nM), but considerably lower σ1 and σ2 affinity
(Ki(σ1) = 182 nM, Ki(σ2) = 200 nM).
4. Conclusion
A series of benzo[7]annulen-7-amines 6a-6i without further substituents in the ring system
was synthesized and the GluN2B affinity and selectivity over the PCP binding site of the
NMDA receptor and σ1 and σ2 receptors were evaluated. Despite lacking of the postulated
substituents in 2-position, the benzo[7]annulen-7-amines show moderate to high GluN2B
affinity. A distance of four to five bond lengths between the basic amino moiety and the
phenyl ring in the flexible side chain led to high GluN2B affinity. Conformational restriction
of the side chain even increases the GluN2B affinity. The presence of a second N-atom in the
side chain substituent (6f) is well tolerated. With respect to selectivity over σ1 and σ2 receptors
the phenylpiperazine 6f represents the most promising GluN2B ligand of this series of
compounds.
5. Experimental
5.1. Chemistry
5.1.1 General
Unless otherwise noted, moisture sensitive reactions were conducted under dry nitrogen.
Acetonitrile was dried over molecular sieves. CH2Cl2 was distilled over CaH2. 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 (d), length (l), fraction
size (v), eluent, Rf value. Melting point: Melting point apparatus Mettler Toledo MP50
Melting Point System, uncorrected. MS: microOTOF-Q II (Bruker Daltronics); APCI,
atmospheric pressure chemical ionization. IR: FT-IR spectrophotometer MIRacle 10
14

(Shimadzu) equipped with ATR technique. Nuclear magnetic resonance (NMR) spectra were
1
recorded on Agilent 600-MR (600 MHz for H, 151 MHz for 13C) or Agilent 400-MR
1
13
spectrometer (400 MHz for H, 101 MHz for C); δ in ppm related to tetramethylsilane and
measured referring to CHCl3 (δ = 7.26 ppm (1H NMR) and δ = 77.2 ppm (13C NMR)) and
CHD2OD (δ = 3.31 ppm (1H NMR) and δ = 49.0 ppm (13C NMR)); coupling constants are
1
given with 0.5 Hz resolution; the assignments of 13C and H NMR signals were supported by
2-D NMR techniques where necessary. HPLC: Merck Hitachi Equipment; UV detector: L-
7400; autosampler: L-7200; pump: L-7100; degasser: L-7614; 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: 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: 90 → 0%, 29-31 min: 0%, 31-31.5 min: 0 → 90 %, 31.5-40 min: 90%. The
purity of all compounds was determined by this method. The purity of all test compounds is
higher than 95 %.
5.1.2. Dimethyl 7-oxo-7H-benzo[7]annulene-6,8-dicarboxylate (9)
Phthalaldehyde (7, 4.00 g, 29.87 mmol, 1 eq.) was dissolved in toluene (60 mL). Dimethyl 3-
oxoglutarate (8, 5.12 mL, 6.08 g, 34.91 mmol, 1.2 eq.), piperidine (0.4 mL) and acetic acid
o
(1.2 mL) were added and the mixture was stirred for 14 h at 95 C using a Dean Stark
apparatus. Most of the toluene was removed under vacuum and the remaining residue was
filtered under reduced pressure and repeatedly washed with ethanol. The washed product was
o
dried in vacuo to obtain a yellow solid, mp 188 C, yield 5.7 g (70 %). C15H12O5 (272.3
g/mol). FT-IR (neat): ν (cm-1) = 2951 (C-H), 1694 (C=O), 783 and 760 (1,2-disubst. arom.).
1H NMR (DMSO): δ (ppm) = 3.82 (s, 6H, CO2CH3), 7.78 – 7.85 (m, 2H, Ar-H), 8.06 – 8.14
13
(m, 2H, Ar-H), 8.25 (s, 2H, 5-H, 9-H). C NMR (DMSO): δ (ppm) = 52.7 (2C, CO2CH3),
15

132.3 (2C, Carom), 133.8 (2C, C-6, C-8), 135.3 (2C, Carom), 136.3 (2C, Cqarom), 141.5 (2C, C-5,
C-9), 166.6 (2C, CO2CH3), 182.0 (1C, C=O).
The synthetic procedure was modified based on references 22-24.
5.1.3. Dimethyl 7-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulene-6,8-dicarboxylate (10)
A mixture of 9 (5.96 g, 21.9 mmol, 1eq.) and Pd/C (714.3 mg) in CH3CO2H (200 mL) was
stirred for 48 h under H2 atmosphere. The mixture was filtered through Celite®. A saturated
solution of NaHCO3 (100 mL) was added and the mixture was extracted with CH2Cl2 (3 x 50
mL). The organic layer was collected and washed with saturated NaHCO3 solution (2 x 50
mL) and then with water (50 mL). The organic layer was dried (Na2SO4), filtered,
concentrated in vacuo and the residue purified by fc (d = 8 cm, l = 15 cm, v = 100 mL,
o
cyclohexane:EtOAc 8:2, Rf = 0.51) to obtain a colorless solid, mp 98 C, yield 5.62 g (93 %).
1
C15H16O5 (276.3 g/mol). H NMR (CDCl3): δ (ppm) = 3.18 – 3.37 (m, 4H, ArCH2CHtrans
,
ArCH2CHcis, ArCH2C=Cenol), 3.45 (dd, J = 14.1/3.8 Hz, 0.2H, ArCH2CHenol), 3.62 – 3.67 (m,
2 x 0.3H, ArCH2CHcis), 3.8 (s, 6 x 0.5H, 2 x CO2CH3trans), 3.8 (s, 3 x 0.2H, CO2CH3enol), 3.9
(s, 6 x 0.3H, CO2CH3cis), 3.9 (s, 3 x 0.2H, CO2CH3enol), 4.02 (dd, J = 8.9/3.5 Hz, 2 x 0.5H,
ArCH2CH), 7.23 – 7.36 (4H, CHarom), 13.04 – 13.04 (s, broad, 0.2H C-OHenol). The ratio of
trans-10: cis-10: enol-10 is 50:30:20.
5.1.4. 5,6,8,9-Tetrahydro-benzo[7]annulen-7-one (11)
6 M HCl was added to a solution of diester 10 (100.4 mg, 0.37 mmol, 1 eq.) dissolved in
o
CH3OH (3 mL) and the mixture was stirred at 90 C for 12 h. The mixture was extracted with
EtOAc (3 x 5 mL), the EtOAc layer was dried (Na2SO4), filtered and the solvent was
evaporated in vacuo. The crude product was purified by fc (d = 1.5 cm, l = 15 cm, v = 10 mL,
o
cyclohexane:EtOAc 8:2, Rf = 0.27) to obtain a colorless solid, mp 42 C, yield 47.4 mg (81
%). C11H12O (160.2 g/mol). FT-IR (neat): ν (cm-1) = 2936 (C-H), 1694 (C=O), 748 (1,2-
16

1
disubst. arom.). H NMR (CDCl3): δ (ppm) = 2.59 – 2.65 (m, 4H, 2x6-H, 2x8-H), 2.89 – 2.95
(m, 4H, 2x5-H, 2x9-H), 7.22 (s, 4H. Ar-H). 13C NMR (CDCl3): δ (ppm) = 30.7 (2C, C-5, C-9)
44.8 (2C, C-6, C-8), 127.3 (2C, Carom), 129.3 (2C, Carom), 140.7 (2C, Cqarom), 211.4 (1C, C=O).
5.1.5. 6,7,8,9-Tetrahydro-5H-benzo[7]annulen-7-ol (12)
NaBH4 (64.1 mg, 1.69 mmol, 3.1 eq.) was added to a solution of ketone 11 (88.7 mg, 0.55
o
mmol, 1 eq.) in CH3OH (3 mL) at 0 C and the reaction mixture was stirred vigorously for 30
min at 0 oC. The reaction mixture was concentrated in vacuo. Water was added to the residue
and the mixture was extracted with Et2O (3 x 5 mL). The Et2O layer was dried (Na2SO4),
filtered and the solvent was evaporated in vacuo. The crude product was purified by fc (d = 2
cm, l = 11 cm, v = 10 mL, cyclohexane:EtOAc 8:2, Rf = 0.27) to obtain a colorless oil, yield
84.7 mg (94 %). C11H14O (162.2 g/mol). FT-IR (neat): ν (cm-1) = 3360 (OH), 2924 (C-H), 748
(1,2-disubst. arom.). 1H NMR (CDCl3): δ (ppm) = 1.48 – 1.62 (m, 2H, 6-H, 8-H), 1.66 (s, 1H,
broad, CHOH), 2.08 (t, J = 11.4 Hz, 2H, 6-H, 8-H), 2.66 (ddd, J = 14.7/10.8/1.3 Hz, 2H, 5-H,
9-H), 2.90 (dd, J = 14.8/8.5 Hz, 2H, 5-H, 9-H), 3.91 – 4.02 (m, 1H, 7-H), 7.10 – 7.15 (broad,
4H, Ar-H). 13C NMR (CDCl3): δ (ppm) = 30.4 (2C, C-5, C-9), 36.5 (2C, C-6, C-8), 73.7 (1C,
C-7), 126.5 (2C, Carom), 129.1 (2C, Carom), 142.3 (2C, Cqarom).
5.1.6. [(6,7,8,9-Tetrahydro-5H-benzo[7]annulen-7-yl) 4-nitrobenzenesulfonyl chloride
(13)
A solution of alcohol 12 (60.1 mg, 0.37 mmol, 1 eq.), 4-nitrobenzenesulfonyl chloride (165.6
mg, 0.75 mmol, 2 eq.), DMAP (16.8 mg, 0.14 mmol, 0.4 eq.) and Et3N (0.15 mL, 115 mg,
1.14 mmol, 3 eq.) in CH2Cl2 (5 mL) was stirred vigorously for 16 h at rt. A solution of 2 M
NaOH (8 mL) was added and the mixture was extracted with CH2Cl2 (3 x 5 mL), the CH2Cl2
layer was dried (Na2SO4), filtered and was concentrated in vacuo. The crude product was
purified by fc (d = 2.5 cm, l = 9 cm, v = 10 mL, cyclohexane:EtOAc 8:2 Rf = 0.41) to obtain a
17

o
colorless solid, mp 97 C, yield 35.9 mg (28 %). C17H17NO5S (347.4 g/mol). FT-IR (neat): ν
(cm-1) = 2924 (C-H), 1528 and 1350 (NO2), 895 (p-disubst. arom.), 748 (1,2-disubst. arom.).
1H NMR (CDCl3): δ (ppm) = 1.79 – 1.93 (m, 2H, 6-H, 8-H), 2.00 (t, J = 11.9 Hz, 2H, 6-H, 8-
H), 2.59 (ddd, J = 14.9/9.5/1.7 Hz, 2H, 5-H, 9-H), 2.95 (dd, J = 14.9/9.7 Hz, 2H, 5-H, 9-H),
4.98 (tt, J = 8.0/3.8 Hz, 1H, 7-H), 7.06 – 7.16 (m, 4H, Ar-H), 8.13 – 8.19 (m, 2H, Ph-H), 8.39
– 8.45 (m, 2H, Ph-H). 13C NMR (CDCl3): δ (ppm) = 29.6 (2C, C-5, C-9), 33.9 (2C, C-6, C-8),
85.9 (1C, C-7), 124.6 (2C, CPh), 126.9 (2C, Carom), 129.0 (2C, CPh), 129.2 (Carom), 141.1 (1C,
Ph
Ph
Cq ), 143.6 (2C, Cqarom), 150.7 (1C, Cq ).
5.1.7. cis- and trans-N-Benzyl-4-phenylcyclohexan-1-amine (cis-15 and trans-15)
A solution of 4-phenylcyclohexanone (14, 54.9 mg, 0.32 mmol, 1 eq.) and benzylamine (72
µL, 70.63 mg, 0.66 mmol, 2.1 eq.) in CH2Cl2 (3.5 mL) was stirred vigorously for 30 min at rt.
NaBH(OAc)3 (143.5 mg, 0.68 mmol, 2.1 eq.) was added and the reaction mixture was stirred
vigorously for 16 h. A saturated solution of NaHCO3 (5 mL) was added and the mixture was
extracted with CH2Cl2 (3 x 5 mL), the CH2Cl2 layer was dried (Na2SO4), filtered and the
solvent was evaporated in vacuo. The crude product was purified by fc (d = 2 cm, l = 13 cm, v
= 8 mL, CH2Cl2:CH3OH 98:2 + 1% NH3)
cis-15 (Rf = 0.24): Yellow oil, yield 58.7 mg (71 %), C19H23N (265.4 g/mol). FT-IR (neat): ν
(cm-1) = 2924 (C-H valence), 736 and 694 (1,2-disubst. arom.).1H NMR (CD3OD): δ (ppm) =
1.57 – 1.68 (m, 4H, 2-H, 3-H, 5-H, 6-H), 1.80 – 1.95 (m, 4H, 2-H, 3-H, 5-H, 6-H), 2.57 (tt, J
= 10.4/3.4 1H, 4-H), 2.85 (quint, J = 3.9 Hz, 1H, 1-H), 3.76 (s, 2H, PhCH2), 7.12 (m, 1H, Ph-
H), 7.20 – 7.29 (m, 5H, Ph-H), 7.29 – 7.39 (m, 4H, Ph-H). A signal for the NH proton is not
observed. 13C NMR (CD3OD): δ (ppm) = 29.3 (2C, C-3, C-5), 30.8 (2C, C-2, C-6), 44.7 (1C,
C-4), 51.9 (1C, PhCH2), 52.3 (1C, C-1), 126.8 (1C, CBn), 128.0 (2C, CPh), 128.0 (1C, CPh),
129.3 (2C, CBn), 129.4 (2C, CBn), 129.5 (2C, CPh), 141.2 (1C, Cq ), 148.4 (1C, CqBn).
Ph
18

trans-15 (Rf = 0.18): Yellow oil, yield 21.9 mg (26 %), C19H23N (265.4 g/mol). FT-IR (neat):
ν (cm-1) = 2924 (C-H), 736 and 694 (1,2-disubst. arom.). 1H NMR (CD3OD): δ (ppm) = 1.27 –
1.39 (m, 2H, 2-H. 6-H), 1.50 (tdd, J = 13.1/11.9/3.0 Hz, 2H, 3-H, 5-H), 1.84 – 1.93 (m, 2H, 3-
H, 5-H), 2.06 – 2.15 (m, 2H, 2-H, 6-H), 2.44 – 2.62 (m, 2H, 1-H, 4-H), 3.81 (s, 2H, PhCH2),
7.09 – 7.39 (m, 10H, Ph-H). A signal for the NH proton is not observed. 13C NMR (CD3OD):
δ (ppm) = 33.8 (2C, C-2, C-6), 34.1 (2C, C-3, C-5), 45.5 (1C, 4-C), 51.5 (1C, PhCH2), 56.8
(1C, 1-C), 127.0 (1C, CBn), 127.8 (2C, CPh), 128.2 (1C, CPh), 129.3 (2C, CBn), 129.5 (2C, CBn),
129.5 (2C, CPh), 140.8 (1C, Cq ), 148.2 (1C, CqBn).
Ph
5.1.8. cis-4-Phenylcyclohexan-1-amine (cis-16)
A solution of cis-15 (50.4 mg, 0.19 mmol, 1 eq.), Pd/C (10.2 mg) and two drops of HOAc
were added to CH3OH (5 mL). The reaction mixture was stirred vigorously for 24 h at rt
under H2 atmosphere. The reaction mixture was filtered through Celite® and concentrated in
vacuo. The crude product was purified by fc (d = 2 cm, l = 4 cm, v = 8 mL, CH2Cl2:CH3OH
1
92:8 + 1% NH3, Rf = 0.19). Yellow oil, yield 20.9 mg (63 %), C12H17N (175.3 g/mol). H
NMR (600 MHz, CD3OD): δ (ppm) = 1.71 – 1.80 (m, 4H, 2-H, 3-H, 5-H, 6-H), 1.92 – 2.06
(m, 4H, 2-H, 3-H, 5-H, 6-H), 2.69 – 2.71 (m, 1H, 4-H), 2.81 (tt, J = 8.4/4.1 Hz, 1H, 1-H), 7.14
– 7.18 (m, 1H, Ph-H), 7.26 – 7.30 (m, 2H, Ph-H), 7.31 – 7.34 (m, 2H, Ph-H). A signal for the
NH2 protons is not observed.
5.1.9. trans-4-Phenylcyclohexan-1-amine (trans-16)
A solution of trans-15 (50.4 mg, 0.19 mmol, 1 eq.), Pd/C (10 mg) and two drops of HOAc
were added to CH3OH (5 mL). The reaction mixture was stirred vigorously for 24 h at rt
under H2 atmosphere. The reaction mixture was filtered through Celite® and concentrated in
vacuo. The crude product was purified by fc (d = 2 cm, l = 3 cm, v = 8 mL, CH2Cl2:CH3OH
1
92:8 + 1% NH3, Rf = 0.15). Colorless oil, yield 2.2 mg (17 %), C12H17N (175.3 g/mol). H
19

NMR (600 MHz, CD3OD): δ (ppm) = 1.30 (tdd, J = 12.7/11.3/3.5 Hz, 2H, 2-H, 6-H), 1.52 –
1.60 (m, 2H, 3-H, 5-H), 1.90 – 1.95 (m, 2H, 3-H, 5-H), 2.08 – 2.14 (m, 2H, 2-H, 6-H), 2.45 (s,
broad, 2H, NH2), 2.47 – 2.58 (m, 2H, 1-H, 4-H), 7.12 – 7.16 (m, 1H, Ph-H), 7.20 (dt, J =
8.2/1.7 Hz, 2H, Ph-H), 7.22 – 7.27 (m, 2H, Ph-H).
5.1.10. N-Benzyl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine (6a)
A solution of ketone 11 (41.3 mg, 0.26 mmol, 1 eq.) and benzylamine (57 µL, 55.92 mg, 0.52
mmol, 2 eq.) in CH2Cl2 (3.5 mL) was stirred vigorously for 30 min at rt. Then NaBH(OAc)3
(106.4 mg, 0.50 mmol, 1.9 eq.) was added and the reaction mixture was stirred vigorously for
14 h. A saturated solution of NaHCO3 (5 mL) was added and the reaction mixture was
extracted with CH2Cl2 (3 x 5 mL), the CH2Cl2 layer was dried (Na2SO4), filtered and the
solvent was evaporated in vacuo. The crude product was purified by fc (d = 2 cm, l = 12 cm, v
= 8 mL, CH2Cl2:CH3OH 98:2 + 1% NH3, Rf = 0.21) to obtain a colorless oil, yield 59.3 mg
(92 %), C18H21N (251.4 g/mol). FT-IR (neat): ν (cm-1) = 2924 (C-H), 748 and 698 (1,2-
disubst. arom.). 1H NMR (CD3OD): δ (ppm) = 1.25 (dtd, J = 13.2/10.7/2.3 Hz, 2H, 6-H, 8-H),
2.11 – 2.21 (m, 2H, 6-H, 8-H), 2.65 – 2.84 (m, 5H, 2x4-H, 2x9-H, 7-H), 3.80 (s, 2H,
PhCH2NH), 7.03 – 7.09 (m, 4H, Ar-H), 7.22 – 7.38 (m, 5H, Ph-H). A signal for the NH
proton is not observed. 13C NMR (CD3OD): δ (ppm) = 33.2 (2C, C-5, C-9), 34.9 (2C, C-6, C-
8), 51.4 (1C, PhCH2), 61.8 (1C, C-7), 127.3 (2C, Carom), 128.2 (1C, CPh), 129.5 (2C, CPh),
129.5 (2C, CPh), 129.8 (2C, Carom), 140.8 (2C, Cqarom), 143.5 (1C, Cq ). Exact mass (APCI):
Ph
m/z = 252.1729 (calcd. 252.1747 for C18H22N [M+H+]). Purity (HPLC): 89.4 % (tR = 16.00
min).
5.1.11. N-(2-Phenylethyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine (6b)
A solution of ketone 11 (49.9 mg, 0.31 mmol, 1 eq.) and 2-phenylethan-1-amine (78 µL,
75.04 mg, 0.62 mmol, 2 eq.) in CH2Cl2 (3 mL) was stirred vigorously for 30 min at rt. Then
20

NaBH(OAc)3 (132.0 mg, 0.62 mmol, 2 eq.) was added and the reaction mixture was stirred
vigorously for 14 h. A saturated solution of NaHCO3 (5 mL) was added and the reaction
mixture was extracted with CH2Cl2 (3 x 5 mL), the CH2Cl2 layer was dried (Na2SO4), filtered
and the solvent was evaporated in vacuo. The crude product was purified by fc (d = 2 cm, l =
11 cm, v = 8 mL, CH2Cl2:CH3OH 96:4 + 1% NH3, Rf = 0.25) to obtain a yellow oil, yield 76.2
mg (92 %), C19H23N (265.4 g/mol). FT-IR (neat): ν (cm-1) = 2924 (C-H), 748 and 698 (1,2-
1
disubst. arom.) H NMR (CD3OD): δ (ppm) = 1.11 – 1.24 (m, 2H, 6-H, 8-H), 2.06 (ddd, J =
12.8/6.6/2.9 Hz, 2H, 6-H, 8-H), 2.69 – 2.82 (m, 7H, 2x5-H, 2x9-H, 7-H PhCH2), 2.86 (m, 2H,
NHCH2CH2Ph), 7.04 (s, broad, 4H, Ar-H), 7.15 – 7.24 (m, 3H, Ph-H), 7.25 – 7.31 (m, 2H,
13
Ph-H). A signal for the NH proton is not observed. C NMR (CD3OD): δ (ppm) = 33.2 (2C,
C-5, C-9) , 35.0 (2C, C-6, C-8), 36.9 (1C, PhCH2), 49.3 (1C, NHCH2CH2), 62.7 (1C, C-7),
127.3 (2C, Carom), 127.3 (1C, CPh), 129.6 (2C, CPh), 129.7 (2C, CPh), 129.8 (2C, Carom), 141.0
(2C, Cqarom), 143.5 (1C, Cq ). Exact mass (APCI): m/z = 266.1901 (calcd. 266.1903 for
Ph
C19H24N [M+H+]). Purity (HPLC): 96.2 % (tR = 17.15 min).
5.1.12. N-(3-Phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine (6c)
A solution of ketone 11 (27.5 mg, 0.17 mmol, 1 eq.) and 3-phenylpropan-1-amine (37 µL,
35.0 mg, 0.26 mmol, 1.53 eq.) in CH2Cl2 (3 mL) was stirred vigorously for 30 min at rt. Then
NaBH(OAc)3 (52.1 mg, 0.25 mmol, 1.47 eq.) was added and the reaction mixture was stirred
vigorously for 12 h. A saturated solution of NaHCO3 (5 mL) was added and the mixture was
extracted with CH2Cl2 (3 x 5 mL), the CH2Cl2 layer was dried (Na2SO4), filtered and the
solvent was evaporated in vacuo. The crude product was purified by fc (d = 1 cm, l = 10 cm, v
o
= 8 mL, CH2Cl2:CH3OH 96:4 + 1% NH3, Rf = 0.41) to obtain a colorless solid, mp 159 C,
yield 9.5 mg (20 %), C20H25N (279.4 g/mol). FT-IR (neat): ν (cm-1) = 2924 (C-H), 748 and
1
698 (1,2-disubst. arom.). H NMR (CD3OD): δ (ppm) = 1.28 (ddt, J = 16.3/12.7/5.7 Hz, 2H,
6-H, 8-H), 1.89 – 1.95 (m, 2H, CH2CH2CH2), 2.17 – 2.27 (m, 2H, 6-H, 8-H), 2.70 (t, J = 7.6
21

Hz, 2H, NHCH2CH2), 2.77 – 2.90 (m, 6H, 2x5-H, 2x9-H, PhCH2), 3.05 (tt, J = 11.2/3.5 Hz,
1H, 7-H), 7.07 – 7.13 (m, 4H, Ar-H), 7.15 – 7.25 (m, 3H, Ph-H), 7.25 – 7.32 (m, 2H, Ph-H).
A signal for the NH proton is not observed. 13C NMR (CD3OD): δ (ppm) = 31.0 (1C,
CH2CH2CH2), 32.8 (2C, C-6, C-8) , 33.5 (2C C-5, C-9), 34.2 (1C CH2Ph), 46.5 (1C, NHCH2),
62.8 (1C, C-7), 127.2 (1C, CPh), 127.6 (2C, Carom), 129.4 (2C, CPh), 129.5 (2C, CPh), 129.9
(2C, Carom), 142.5 (2C, Cqarom), 142.9 (1C, Cq ). Exact mass (APCI): m/z = 280.2095 (calcd.
Ph
280.2060 for C20H26N [M+H+]). Purity (HPLC): 97.6 % (tR = 18.13 min).
5.1.13. N-(4-Phenylbutyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine (6d)
A solution of ketone 11 (40.4 mg, 0.25 mmol, 1 eq.) and 4-phenylbutan-1-amine (60 µL, 56.6
mg, 0.38 mmol, 1.5 eq.) in CH2Cl2 (4 mL) was stirred vigorously for 30 min at rt. Then
NaBH(OAc)3 (82.7 mg, 0.39 mmol, 1.6 eq.) added and the reaction mixture was stirred
vigorously for 12 h. A saturated solution of NaHCO3 (5 mL) was added and the reaction
mixture was extracted with CH2Cl2 (3 x 5 mL), the CH2Cl2 layer dried (Na2SO4), filtered and
the solvent evaporated in vacuo. The crude product was purified by fc (d = 2 cm, l = 12 cm, v
o
= 8 mL, CH2Cl2:CH3OH 96:4 + 1% NH3, Rf = 0.35) to obtain a colorless solid, mp 169 C,
yield 24.9 mg (34 %), C21H27N (293.5 g/mol). FT-IR (neat): ν (cm-1) = 2931 cm-1 (C-H
1
valence), 752 cm-1 and 698 cm-1 (1,2-disubst. arom.). H NMR (CD3OD): δ (ppm) = 1.33 –
1.48 (m, 2H, 6-H, 8-H), 1.60 – 1.79 (m, 4H, CH2CH2CH2CH2), 2.35 (dtd, J = 11.4/3.9/1.9 Hz,
2H, 6-H, 8-H), 2.69 (t, J = 6.9 Hz, 2H, PhCH2), 2.81 – 2.89 (m, 4H, 2x5-H, 2x9-H), 3.04 (t, J
= 7.5 Hz, 2H, NHCH2), 3.36 (tt, J = 11.7/3.6 Hz, 1H, 7-H), 7.09 – 7.14 (m, 4H, Ar-H), 7.14 –
7.24 (m, 3H, Ph-H), 7.24 – 7.31 (m, 2H, Ph-H). A signal for the NH proton is not observed.
13C NMR (CD3OD): δ (ppm) = 27.0 (1C CH2CH2CH2Ph), 29.4 (1C, CH2CH2Ph), 31.8 (2C, C-
6, C-8), 32.3 (2C, C-5, C-9), 36.2 (1C PhCH2), 45.8 (1C, NHCH2), 62.5 (1C, C-7), 127.0 (1C,
CPh), 127.9 (2C, Carom), 129.5 (2C, CPh), 129.5 (2C, CPh), 130.0 (2C, Carom), 142.3 (2C, Cqarom),
22

142.8 (1C, Cq ). Exact mass (APCI): m/z = 294.2157 (calcd. 294.2216 for C21H28N [M+H+]).
Ph
Purity (HPLC): 98.5 % (tR = 19.16 min).
5.1.14. 4-Phenyl-1-(6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-yl)piperidine (6e)
A solution of nosylate 13 (51.2 mg, 0.15 mmol, 1 eq.) and 4-phenylpiperidine (132.0 mg, 0.82
o
mmol, 5.5 eq.) in CH3CN (3 mL) was stirred vigorously for 14 h at 75 C. The reaction
mixture was concentrated in vacuo and the residue was purified by fc (d = 2 cm, l = 15 cm, v
o
= 8 mL, CH2Cl2:CH3OH 98:2 + 1% NH3, Rf = 0.21) to obtain a colorless solid, mp 134 C,
yield 11.6 mg (26 %). C22H27N (305.5 g/mol). FT-IR (neat): ν (cm-1) = 2927 (C-H), 748 and
698 (1,2-disubst. arom.) 1H NMR (CD3OD): δ (ppm) = 1.38 (dtd, J = 13.3/11.4/2.0 Hz, 2H, 6-
H, 8-H), 1.70 – 1.85 (m, 4H, 3-CH2pip, 5-CH2pip), 2.21 (ddt, J = 13.5/7.3/2.3 Hz, 2H, 6-H, 8-
H), 2.43 – 2.56 (m, 3H, 2-CH2pip, 6-CH2pip, 4-CHpip), 2.70 – 2.87 (m, 5H, 5-H, 9-H, 7-H), 2.87
– 2.95 (m, 2H, 2-CH2pip, 6-CH2pip), 7.05 – 7.12 (m, 4H, Ar-H), 7.12 – 7.18 (m, 1H, Ph-H),
7.18 – 7.29 (m, 4H, Ph-H). 13C NMR (CD3OD): δ (ppm) = 30.9 (2C, C-6, C-8), 34.3 (2C, C-5,
C-9), 34.6 (2C, C-3pip, C-5pip), 44.1 (1C, C-4pip), 50.4 (2C, C-2pip, C-6pip), 70.2 (1C, C-7),
127.2 (1C, CPh), 127.3 (2C, Carom), 127.8 (2C, CPh), 129.4 (2C, CPh), 129.8 (2C, Carom), 143.6
(2C, Cqarom), 147.5 (1C, Cq ). Exact mass (APCI): m/z = 306.2206 (calcd. 306.2216 for
Ph
C22H28N [M+H+]). Purity (HPLC): 97.4 % (tR = 18.45 min).
5.1.15. 1-Phenyl-4-(6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-yl)piperazine (6f)
A solution of nosylate 13 (78.4 mg, 0.23 mmol, 1 eq.) and 1-phenylpiperazine (178 µL 189.0
o
mg, 1.17 mmol, 5.1 eq.) in CH3CN (5 mL) was stirred vigorously for 16 h at 75 C. The
reaction mixture was concentrated in vacuo and the residue was purified by fc (d = 2 cm, l =
12 cm, v = 8 mL, CH2Cl2:CH3OH 98:2 + 1% NH3, Rf = 0.45) to obtain a colorless solid, mp
o
141 C, yield 18.5 mg (27 %). C21H26N2 (306.5 g/mol). FT-IR (neat): ν (cm-1) = 2924 (C-H),
1
748 and 698 (1,2-disubst. arom.). H NMR (CD3OD): δ (ppm) = 1.35 – 1.46 (m, 2H, 6-H, 8-
23

H), 2.14 – 2.25 (m, 2H, 6-H, 8-H), 2.70 – 2.90 (m, 9H, 5-H, 9-H, 7-H, N(CH2CH2)2NPh), 3.14
– 3.21 (m, 4H, N(CH2CH2)NPh), 6.83 (tt, J = 7.4/1.0 Hz, 1H, Ph-H), 6.93 – 6.98 (m, 2H, Ph-
13
H), 7.05 – 7.13 (m, 4H, Ar-H), 7.18 – 7.25 (m, 2H, Ph-H). C NMR (CD3OD): δ (ppm) =
31.0 (2C, C-6, C-8), 34.0 (2C, C-5, C-9), 49.6 (2C, N(CH2CH2)2NPh), 50.8 (2C,
N(CH2CH2)2NPh), 69.5 (1C, C-7), 117.5 (2C, CPh), 121.1 (1C, CPh), 127.3 (2C, Carom), 129.8
(2C, Carom), 130.0 (2C, CPh), 143.6 (2C, Cqarom), 152.7 (1C, Cq ). Exact mass (APCI): m/z =
Ph
307.2160 (calcd. 307.2169 for C21H27N2 [M+H+]). Purity (HPLC): 97.9 % (tR = 16.97 min).
5.1.16. 4-Benzyl-1-(6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-yl)piperidine (6g)
A solution of nosylate 13 (81.0 mg, 0.23 mmol, 1 eq.) and 4-benzylpiperidine (189.9 mg, 1.08
o
mmol, 4.7 eq.) in CH3CN (5 mL) was stirred vigorously for 16 h at 75 C. The reaction
mixture was concentrated in vacuo and the residue was purified by fc (d = 2 cm, l = 15 cm, v
= 8 mL, CH2Cl2:CH3OH 98:2 + 1% NH3, Rf = 0.41) to obtain an orange oil, yield 27.8 mg (37
%), C23H29N (319.5 g/mol). FT-IR (neat): ν (cm-1) = 2920 (C-H), 744 and 698 (1,2-disubst.
1
arom.). H NMR (CD3OD): δ (ppm) = 1.23 – 1.35 (m, 4H, 6-H, 8-H, 3-Hpip, 5-Hpip), 1.50 (ttt,
J = 10.9/7.2/3.7 Hz, 1H, 4-Hpip), 1.57 – 1.65 (m, 2H, 3-Hpip, 5-Hpip), 2.07 – 2.18 (m, 2H, 6-H,
8-H), 2.28 (td, J = 11.8/2.5 Hz, 2H, 2-Hpip, 6-Hpip), 2.51 (d, J = 7.1 Hz, 2H, PhCH2), 2.62 –
2.83 (m, 7H, 5-H, 9-H 2-Hpip, 6-Hpip, 7-H), 7.02 – 7.09 (m, 4H, Ar-H), 7.09 – 7.16 (m, 3H,
13
Ph-H), 7.19 – 7.27 (m, 2H, Ph-H). C NMR (CD3OD): δ (ppm) = 30.8 (2C, C-6, C-8), 33.2
(2C, C-3pip, C-5pip), 34.3 (2C, C-5, C-9), 39.5 (1C, C-4pip), 44.1 (1C, PhCH2), 50.0 (2C, C-2pip,
C-6pip), 70.1 (1C, C-7), 126.8 (1C, CPh), 127.3 (2C, Carom), 129.2 (2C, CPh), 129.8 (2C, CPh),
130.1 (2C, Carom), 141.7 (2C, Cqarom), 143.6 (1C, Cq ). Exact mass (APCI): m/z = 320.2403
Ph
(calcd. 320.2373 for C23H30N [M+H+]). Purity (HPLC): 95.7 % (tR = 19.07 min).
24

5.1.17. cis-N-(4-Phenylcyclohexyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine (cis-
6h) and trans-N-(4-Phenylcyclohexyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-
amine (trans-6i)
A solution of ketone 11 (45.8 mg, 0.29 mmol, 1 eq.) and 4-phenylcyclohexan-1-amine (cis-
16:trans-16 = 70:30, 112.7 mg, 0.64 mmol, 2.2 eq.) in dist. CH2Cl2 (4 mL) was stirred
vigorously for 30 min at rt. Then NaBH(OAc)3 (129.9 mg, 0.61 mmol, 2.1 eq.) added and the
reaction mixture was stirred vigorously for 12 h. A saturated solution of NaHCO3 (5 mL) was
added and the mixture was extracted with CH2Cl2 (3 x 5 mL), the CH2Cl2 layer was dried
(Na2SO4), filtered and the solvent was evaporated in vacuo. The crude product was purified
by fc (d = 2 cm, l = 15 cm, v = 12 mL, CH2Cl2:CH3OH 98:2 + 1% NH3).
cis-6h (Rf = 0.48): Colorless oil, yield 28.2 mg (31 %), C23H29N (319.5 g/mol). FT-IR (neat):
ν (cm-1) = 2924 (C-H), 752 and 694 (1,2-disubst. arom.). 1H NMR (CD3OD): δ (ppm) = 1.16 –
1.32 (m, 2H, 6-H, 8-H), 1.62 – 1.75 (m, 4H, 2-H, 3-H, 5-H, 6-Hcyclohexyl), 1.75 – 1.89 (m, 4H,
2-H, 3-H, 5-H, 6-Hcyclohexyl), 2.07 – 2.18 (m, 2H, 6-H, 8-H), 2.61 (tt, J = 10.3/3.6 Hz, 1H, 4-
Hcyclohexyl), 2.78 (dd, J = 7.9/3.4 Hz, 4H, 5-H, 9-H), 2.88 (tt, J = 10.7/3.5 Hz, 1H, 7-H), 3.03
(quint, J = 3.9 Hz, 1H, 1-Hcyclohexyl), 7.03 – 7.10 (m, 4H, Ar-H), 7.14 (ddd, J = 8.5/5.3/3.3 Hz,
13
1H, Ph-H), 7.23 – 7.29 (m, 4H, Ph-H). A signal for the NH proton is not observed. C NMR
(CD3OD): δ (ppm) = 29.4 (2C, C-3/C-5cyclohexyl), 30.9 (2C, C-2/C-6cyclohexyl), 33.5 (2C, C-5, C-
9), 35.5 (2C, C-6, C-8), 44.2 (C-4cyclohexyl), 49.6 (1C, C-1cycloyhexyl), 59.6 (1C, C-7), 126.8 (1C,
CPh), 127.3 (2C, Carom), 128.0 (2C, CPh), 129.3 (2C, CPh), 129.8 (2C, Carom), 143.6 (2C, Cqarom),
148.1 (1C, Cq ). Exact mass (APCI): m/z = 320.2427 (calcd. 320.2373 for C23H30N [M+H+]).
Ph
Purity (HPLC): 98.5 % (tR = 20.65 min).
o
trans-6i (Rf = 0.39): Colorless solid, mp 56 C, yield 8.4 mg (9 %). C23H29N (319.5 g/mol).
FT-IR (neat): ν (cm-1) = 2920 (C-H), 756 and 698 (1,2-disubst. arom.). 1H NMR (CD3OD): δ
(ppm) = 1.13 – 1.37 (m, 4H, 6-H, 8-H, 2-H/6-Hcyclohexyl), 1.57 (qd, J = 13.2/3.2 Hz, 2H, 3-H/5-
Hcyclohexyl), 1.86 – 1.96 (m, 2H, 3-H/5-Hcyclohexyl), 2.03 – 2.12 (m, 2H, 2-H/6-Hcyclohexyl), 2.13 –
25

2.22 (m, 2H, 6-H, 8-H), 2.52 (tt, J = 12.2/3.5 Hz, 1H, 4-Hcyclohexyl), 2.70 – 2.87 (m, 5H, 5-H,
9-H, 1-Hcyclohexyl), 2.99 (tt, J = 10.7/3.6 Hz, 1H, 7-H), 7.04 – 7.11 (m, 4H, Ar-H), 7.11 – 7.17
13
(m, 1H, Ph-H), 7.18 – 7.29 (m, 4H, Ph-H). A signal for the NH proton is not observed. C
NMR (CD3OD): δ (ppm) = 33.4 (2C, C-5, C-9), 34.4 (2C, C-2/C-6cyclohexyl), 34.4 (2C, C-3/C-
5cyclohexyl), 35.5 (2C, C-6, C-8), 45.5 (1C, C-4cyclohexyl), 53.9 (1C, C-1cyclohexyl), 58.9 (1C, C-7),
127.0 (1C, CPh), 127.3 (2C, Carom), 127.8 (2C, CPh), 129.3 (2C, CPh), 129.8 (Carom), 143.59
(2C, Cqarom), 148.3 (1C, Cq ). Exact mass (APCI): m/z = 320.2427 (calcd. 320.2373 for
Ph
C23H30N [M+H+]). Purity (HPLC): 96.2 % (tR = 20.66 min).
5.2. Receptor binding studies
5.2.1. Materials
The recombinant L(tk-) cells stably expressing the GluN2B receptor were a generous donation
of Prof. Steinhilber (Frankfurt, Germany). Cell incubator: Heracell 120 (Thermo Fisher
Scientific, Langenselbold, 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).
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.
26

5.2.2. Cell culture and preparation of membrane homogenates for the GluN2B assay
Mouse L(tk-) cells stably transfected with the dexamethasone-inducible eukaryotic
expression vectors pMSG NR1-1a, pMSG GluN2B (1:5 ratio) were grown in Modified Earl’s
Medium (MEM) containing 10% of standardized FCS (Biochrom AG, Berlin, 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 a growth medium containing 4 µM dexamethasone
and 4 µM ketamine (final concentration). After 24 h the cells were harvested by trypsination
and pelleted (10 min, 5000 x g).
For the binding assay, the cell pellet was resuspended in PBS buffer and the number of cells
was determined using an improved Neubauer’s counting chamber (VWR, 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 (20,000 x g, 4 °C). The supernatant was discarded and the pellet resuspended in a
defined volume of phosphate buffer saline (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 breaks of 10 s) and stored at -80 °C.
5.2.3. Protein determination
The protein concentration was determined by the method of Bradford,32 modified by
Stoscheck.33 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
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-
27

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).
5.2.4. General protocol for the binding assay
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 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
28

analysis. Subsequently, the IC50 values were transformed into Ki-values using the equation of
Cheng and Prusoff.34 The Ki-values are given as mean value SEM from three independent
experiments.
5.2.5. Performing of the GluN2B assay
The competitive binding assay was performed with the radioligand [3H]-ifenprodil (60
Ci/mmol; Perkin Elmer). 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-buffer (5 mM/1 mM, 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.
5.2.6 Affinity toward σ1 and σ2 receptors and the PCP binding site of the NMDA
receptor
The affinity toward the PCP binding site of the NMDA receptor27,28 and the affinity toward
the σ1 and σ2 receptors29–31 were recorded as previously described.
Acknowledgements
The authors are grateful to Prof. Dr. Steinhilber (Department of Pharmacy, University of
Frankfurt) for donating L(tk-) cells stably expressing GluN1a/GluN2B containing NMDA
receptors. The authors also thank the NRW Graduate School of Chemistry for a PhD
scholarship to SG, which is funded by the Government of the state of Nordrhein-Westfalen
and the Westfälische Wilhelms-Universität Münster.
29