Negative allosteric modulators of the GluN2B NMDA receptor with phenylethylamine structure embedded in ring-expanded and ring-contracted scaffolds

Article abstract of DOI:10.1016/j.ejmech.2020.112138

A set of GluN2B NMDA receptor antagonists with conformationally restricted phenylethylamine substructure was prepared and pharmacologically evaluated. The phenylethylamine substructure was embedded in ring expanded 3-benzazocines 4 as well as ring-contracted tetralinamines 6 and indanamines 7. The ligands 4, 6 and 7 were synthesized by reductive alkylation of secondary amine 11, reductive amination of ketones 12 and 16 and nucleophilic substitution of nosylates 14 and 17. The moderate GluN2B affinity of 3-benzazocine 4d (K_i = 32 nM) translated into moderate cytoprotective activity (IC₅₀ = 890 nM) and moderate ion channel inhibition (60% at 10 μM) in two-electrode voltage clamp experiments with GluN1a/GluN2B expressing oocytes. Although some of the tetralinamines 6 and indanamines 7 showed very high GluN2B affinity (e.g. K_i (7f) = 3.2 nM), they could not inhibit glutamate/glycine inducted cytotoxicity. The low cytoprotective activity of 3-benzazocines 4, tetralinamines 6 and indanamines 7 was attributed to the missing OH moiety at the benzene ring and/or in benzylic position. Docking studies showed that the novel GluN2B ligands adopt similar binding poses as Ro 25–6981 with the central H-bond interaction between the protonated amino moiety of the ligands and the carbamoyl moiety of Gln110. However, due to the lack of a second H-bond forming group, the ligands can adopt two binding poses within the ifenprodil binding pocket.

Full text of DOI:10.1016/j.ejmech.2020.112138

European Journal of Medicinal Chemistry 190 (2020) 112138
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Negative allosteric modulators of the GluN2B NMDA receptor with
phenylethylamine structure embedded in ring-expanded and ring-
contracted scaffolds
,
,
b
,
, b
Louisa Temme a b, Elena Bechthold a, Julian A. Schreiber a c, Sandeep Gawaskar a
,
Dirk Schepmann a, Dina Robaa d, Wolfgang Sippl d, Guiscard Seebohm c,
Bernhard Wünsch a
, b, *
a
€
Institut für Pharmazeutische und Medizinische Chemie der Universitat Münster, Corrensstraße 48, D-48149, Münster, Germany
Cells-in-Motion Cluster of Excellence (EXC 1003 e CiM), Westfalische Wilhelms-Universitat, Münster, Germany
b
c
€
€
€
Institut für Pharmazie der Martin-Luther-Universitat Halle-Wittenberg, Wolfgang-Langenbeck-Straße 4, 06120, Halle (Saale), Germany
d Cellular Electrophysiology and Molecular Biology, Institute for Genetics of Heart Diseases (IfGH), Department of Cardiovascular Medicine, University
Hospital Münster, Robert-Koch-Str. 45, D-48149, Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of GluN2B NMDA receptor antagonists with conformationally restricted phenylethylamine sub-
structure was prepared and pharmacologically evaluated. The phenylethylamine substructure was
embedded in ring expanded 3-benzazocines 4 as well as ring-contracted tetralinamines 6 and indan-
amines 7. The ligands 4, 6 and 7 were synthesized by reductive alkylation of secondary amine 11,
reductive amination of ketones 12 and 16 and nucleophilic substitution of nosylates 14 and 17. The
moderate GluN2B affinity of 3-benzazocine 4d (Ki ¼ 32 nM) translated into moderate cytoprotective
Received 9 December 2019
Received in revised form
20 January 2020
Accepted 9 February 2020
Available online 10 February 2020
activity (IC50 ¼ 890 nM) and moderate ion channel inhibition (60% at 10
mM) in two-electrode voltage
Keywords:
clamp experiments with GluN1a/GluN2B expressing oocytes. Although some of the tetralinamines 6 and
indanamines 7 showed very high GluN2B affinity (e.g. Ki (7f) ¼ 3.2 nM), they could not inhibit glutamate/
glycine inducted cytotoxicity. The low cytoprotective activity of 3-benzazocines 4, tetralinamines 6 and
indanamines 7 was attributed to the missing OH moiety at the benzene ring and/or in benzylic position.
Docking studies showed that the novel GluN2B ligands adopt similar binding poses as Ro 25e6981 with
the central H-bond interaction between the protonated amino moiety of the ligands and the carbamoyl
moiety of Gln110. However, due to the lack of a second H-bond forming group, the ligands can adopt two
binding poses within the ifenprodil binding pocket.
NMDA receptor
GluN2B antagonists
Ifenprodil binding site
GluN2B affinity
Selectivity
Cytoprotective activity
Electrophysiology
TEVC
Structure-affinity relationships
Structure-activity relationships
Docking studies
© 2020 Elsevier Masson SAS. All rights reserved.
Ligand-receptor interactions
1. Introduction
for the therapy of these diseases. However, the strong blockade of
the NMDA receptor associated ion channel by open-channel
The N-methyl-
D
-aspartate (NMDA) receptor belongs to the class
blockers, such as phencyclidine (PCP) and (þ)-MK-801 leads to
strong psychotomimetic side effects (e.g. hallucinations, psychosis,
amnesia). On the other hand, open-channel blockers with reduced
affinity towards the PCP binding site of the NMDA receptor are in
clinical use as anti-Parkinson drugs (amantadine, budipine), anti-
Alzheimer drugs (memantine) and as dissociative anesthetics
((S)-ketamine) [1e6].
of ionotropic receptors activated by the excitatory amino acid
neurotransmitter (S)-glutamate. It is associated with neurodegen-
erative disorders including Parkinson’s, Alzheimer’s and Hunting-
ton’s disease and cerebral ischemia. Therefore, the NMDA receptor
represents a promising target for the development of novel drugs
During the last decades, subtype selective NMDA receptor an-
tagonists interacting selectively with NMDA receptors containing
the GluN2B subunit emerged as attractive drug candidates. NMDA
receptors with GluN2B subunit (shortly GluN2B receptors) present
* Corresponding author. Institut für Pharmazeutische und Medizinische Chemie
€
der Universitat Münster, Corrensstraße 48, D-48149, Münster, Germany.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
https://doi.org/10.1016/j.ejmech.2020.112138
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138
an additional binding site at the interface between the GluN1 and
GluN2B subunits, which is addressed by ifenprodil (1) as proto-
typical negative allosteric modulator (NAM). Therefore, this binding
site is referred to as ifenprodil binding site [7e10]. (Fig. 1).
Interestingly, a H-bond interaction between the benzylic OH
moiety of ifenprodil-like compounds and either the carbonyl O-
atom of Ser132 (GluN1) in case of ifenprodil [12] and the terminal
carbamoyl moiety of Gln110 (GluN2B) [12,13] or the backbone NH
group of Leu135 (GluN1) in case of Ro 25-698113 was found.
According to X-ray crystal structures of the GluN2B receptor
together with ifenprodil (1), the phenolic OH moiety interacts
with a conserved water molecule and Glu236 (GluN2B subunit).
The benzylic OH moiety and the protonated amino moiety form
H-bonds with the carbonyl O-atom of Ser132 (GluN1) [12] and
the amide of Gln110 (GluN2B) [12,13]. Ifenprodil is further sta-
bilized in the allosteric binding site by lipophilic interactions
formed by the benzylpiperidine part, the methyl moiety and the
phenol [12,13].
Fig. 2. Design of novel GluN2B selective ligands without benzylic and phenolic OH
moieties. In the hexahydro-3-benzazocines 4 the N-heterocycle is expanded and in the
tetralines 6 and indanes 7 the carbocycle is contracted compared to the lead com-
pounds 3 and 5, respectively.
2. Synthesis
In a previous study, ifenprodil (1) was reorganized into 2,3,4,5-
tetrahydro-1H-3-benzazepine-1,7-diols bearing various side
chains at the N-atom. 3-Benzazepine WMS-1410 (2) with the
phenylbutyl side chain at the N-atom imitating the benzylpiper-
idine substructure of ifenprodil showed high GluN2B affinity
(Ki ¼ 84 nM), high cytoprotective activity (IC50 ¼ 18.4 nM) and
high activity in two-electrode voltage clamp experiments
(IC50 ¼ 116 nM) [11,14]. The impact of the OH moieties in 1- and 7-
position of WMS-1410 (2) was analyzed by their stepwise removal.
The 3-benzazepine without phenolic OH moiety showed a Ki value
of 73 nM15 and even the 3-benzazepine 3d (R ¼ 4-phenylbutyl)
without both OH moieties displayed a GluN2B affinity of 230 nM
[15,16]. Obviously, the contribution of the phenolic and benzylic
OH moieties to the interaction with the ifenprodil binding site is
limited. Therefore, it was planned to synthesize and pharmaco-
logically evaluate further analogs of 3-benzazepines 3 without OH
moieties.
Herein, we wish to report the synthesis and pharmacological
evaluation of 3-benzazocines of type 4 and tetralinamines and
indanamines of type 6 and 7, respectively (Fig. 2). Compared to 3-
benzazepines 3, 3-benzazocines 4 have a larger (8-membered) N-
heterocycle. In contrast, the tetralinamines 6 and indanamines 7
represent ring-contracted analogs of benzo [7]annulen-7-amines 5,
which also show high GluN2B affinity [17,18]. The common feature
of the ring-expanded and ring-contracted analogs 4, 6, and 7 is a
phenylethylamine substructure. The phenylethylamine substruc-
ture is a crucial structural element of typical GluN2B selective
NAMs such as ifenprodil (1), WMS-1410 (2), eliprodil and trax-
oprodil (Fig. 1).
The synthesis of 3-benzazocines 4 started with a Schmidt
rearrangement of ketone 8 (Scheme 1). Unexpectedly, treatment of
ketone 8 with NaN3 and H2SO4/HOAc led exclusively to tetrazole 10,
the desired lactam 9 could not be detected. Formation of tetrazole
10 is due to trapping of intermediate nitrilium ion by Nꢀ3 in a 1,3-
dipolar cycloaddition. However, using Cl3CCO2H instead of H2SO4
at 65 ꢁC provided the lactam 9 in 84% yield without formation of
tetrazole 10 [19,20]. Reduction of lactam 9 with LiAlH4 afforded the
secondary amine 11, which was reductively alkylated with 3-
phenylpropionaldehyde or 4-phenylbutyraldehyde and NaB-
H(OAc)3 to give the tertiary amines 4c and 4d (Scheme 1).
Reductive amination of
b-tetralone (12) with homologous
phenylalkylamines and NaBH(OAc)3 led to the homologous phe-
nylalkylamines 6a-d in 80e95% yield. In addition to phenyl-
alkylamino groups at the tetraline ring system, 4-phenyl- and 4-
benzylpiperidino groups should be introduced, since these struc-
tural elements are found in the GluN2B NAMs ifenprodil, trax-
oprodil and Ro 25e6981. Since reductive amination of ketone 12
with 4-phenyl- and 4-benzylpiperidine did not lead to amines 6e
and 6f, a nucleophilic substitution was envisaged. After reduction
of b-tetralone 12 with NaBH4 and activation of the resulting alcohol
13 with 4-nitrobenzenesulfonyl chloride (nosyl chloride), SN2
substitution with 4-phenyl- and 4-benzylpiperidine provided the
desired amines 6e and 6f in moderate yields of 33% and 39%,
respectively (Scheme 2).
Indanamines 7a and 7b were obtained upon PCC oxidation of
commercially available indanol 15 and subsequent reductive ami-
nation of the resulting indanone 16 with benzylamine or 2-
phenylethan-1-amine and NaBH(OAc)3. Alternatively, indanol 15
was reacted with nosyl chloride to afford the nosylate 17, which
underwent nucleophilic substitution with 3-phenylpropan-1-
amine and 4-phenylbutan-1-amine to yield the homologous phe-
nylalkylamines 7c and 7d. The piperidine derivatives 7e and 7f
were also obtained by SN2 substitution of the nosylate 17
employing 4-phenyl- and 4-benzylpiperidine, respectively. In case
of the indanyl nosylate 17 nucleophilic substitution with primary
and secondary amines led to moderate to high yields of the sub-
stitution products (Scheme 3).
3. Pharmacological evaluation
Affinity towards GluN2B subunit containing NMDA receptors
and related receptors.
In the GluN2B assay, test compounds were competing with the
radioligand [3H]ifenprodil for the ifenprodil binding site of NMDA
receptors. Standardized membrane fragments from L (tk-)cells
Fig. 1. Ifenprodil (1), WMS-1410 (2) [11], eliprodil and traxoprodil
negative allosteric modulators of GluN2B subunit containing NMDA receptors.
- prototypical

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138
3
Scheme 1. Synthesis of hexahydro-3-benzazocines 4. Reagents and reaction conditions: (a) NaN3, H2SO4, HOAc, rt, 9: 0%; 10: 41%. (b) NaN3, Cl3CCO2H, 65 ꢁC, 9: 84%; 10: 0%. (c)
LiAlH4, THF, 75 ꢁC, 16 h then rt, 5 d, 40%. (d) Ph(CH2)2CH]O or Ph(CH2)3CH]O, NaBH(OAc)3, CH2Cl2, Na2SO4, rt, 14 h, 4c: 24%; 4d: 57%.
Scheme 2. Synthesis of tetrahydronaphthalenamines 6. Reagents and reaction conditions: (a) Ph(CH2)nNH2, CH2Cl2, NaBH(OAc)3, rt, 14 h, 6a: 80%; 6b: 95%, 6c: 84%; 6d: 87%. (b)
NaBH4, CH3OH, 0 ꢁC, 2 h, 98%. (c) nosyl chloride, CH2Cl2, NEt3, DMAP, rt, 16 h, 64%. (d) 4-phenylpiperidine or 4-benzylpiperidine, CH3CN, 75 ꢁC, 48 h, 6e: 33%; 6f: 39%.
Scheme 3. Synthesis of indanamines 7. Reagents and reaction conditions: (a) PCC, CH2Cl2, rt, 4 h, 68%. (b) Ph(CH2)nNH2, CH2Cl2, NaBH(OAc)3, rt, 14 h, 7a: 50%; 5b: 35%. (c) nosyl
chloride, CH2Cl2, NEt3, rt, 14 h, 70%. (d) Ph(CH2)nNH2, 4-phenylpiperidine or 4-benzylpiperidine, CH3CN, 60 ꢁC, 16e20 h, 7c: 59%; 7d: 45%, 7e: 59%; 7f: 68%.
stably expressing recombinant human GluN1a and GluN2B sub-
units of the NMDA receptor were employed as receptor material.
Unspecific binding was determined with an excess of ifenprodil
(100 nM) [21].
In addition to the GluN2B affinity, the interaction with s1 and s2
receptors was recorded in receptor binding studies [22e24]. In
general, we always include the
s affinity in our GluN2B projects and
vice versa, since the lead compound ifenprodil shows high affinity

4
L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138
Table 1
Affinity towards the ifenprodil binding site of GluN2B subunit containing NMDA receptors and
s receptors.
compd.
n
m
Ki SEM [nM] (n ¼ 3)
GluN2B
s1
s2
211,14
e
4
3
4
3
4
1
2
3
4
e
e
2
3
4
e
e
e
e
e
e
e
e
e
e
e
e
0
84 18
230
190
10
17
>10 mM
35 13
178
3d15,16
8
3
8
4c
36
32
16
17
3
1
4
2
4d
22
50
27 12
55
65 15
1
5c17 or 18
150
215
285
51 14
59 16
46 10
1.9 0.6
6.0 1.4
168
5d17 or 18
7
6a
6b
6c
6d
6e
6f
7b
7c
7d
7e
7f
ifenprodil
eliprodil
4000
237
163 51
266
8.4 3.4
8.0 4.0
455
70
29
4
9
50 19
2.9 0.6
2.1 0.3
694
1
e
e
e
0
117 15
58 13
38
9
66
8
31
44
51
125 24
e
7
9
9
150
25
28
4.5 1.5
3.2 0.8
10 0.7
13 2.0
7
2
1
98 34
e
haloperidol
di-o-tolylguanidine
6.3 1.6
78 2.3
e
e
89 29
57 18
The affinity of compounds with Ki values > 170 nM was recorded only once (n ¼ 1).
Due to low purity, compound 7a was not evaluated pharmacologically.
3c, 3d: see Fig. 2, R ¼ (CH2)3Ph (3c), R ¼ (CH2)4Ph (3d).
5c, 5d: see Fig. 2, NR2 ¼ NH(CH2)3Ph (5c); NR2 ¼ NH(CH2)4Ph (5d).
towards both
s
receptor subtypes (see Table 1). Moreover, the
3.1. Functional activity
pharmacophores for potent s1 ligands [25] and negative allosteric
modulators at the ifenprodil binding site [26] are very similar. The
The cytoprotective activity of GluN2B ligands with various ring
sizes was determined in an assay recording the released amount of
lactate dehydrogenase (LDH) after stimulation of cells with (S)-
glutamate and glycine. The same mouse fibroblast L (tk-) cells
expressing only GluN1a and GluN2B subunits as described in the
receptor binding assay were employed in this assay. The amount of
released LDH correlates with cell damage and/or death. A reduced
release of LDH upon treatment with the GluN2B ligands is regarded
as cytoprotective effect [32].
In order to get information about the influence of the ring size
on the cytoprotective activity, compounds with phenylpropyl
moiety (c-series) were investigated systematically. In case of 3-
benzazocines 4 with the basic amino moiety located within the
cross reactivity at
s receptors and GluN2B-NMDA receptors has
been shown in several projects [27e29].
The GluN2B, s1 and s2 affinities of the 3-benzazocines 4, tet-
ralinamines 6 and indanamines 7 together with the GluN2B affinity
of some lead and reference compounds are summarized in Table 1.
Expansion of the 3-benzazepine ring (3d) to a 3-benzazocine
ring led to almost 10-fold increased GluN2B affinity of 4c and 4d.
Moreover, the affinity towards both s1 and s2 receptors is reduced
resulting in an increased selectivity over these receptors. In
particular, the low s2 affinity renders the phenylpropyl derivative
4c an interesting GluN2B ligand with 5-fold selectivity over the s2
receptor. Up to a concentration of 10 mM both 3-benzazocines 4c
and 4d did not interact with the PCP binding site [30,31] of the
NMDA receptor.
Table 2
Phenylpropyl- or phenylbutylamino substituents seem to be
favorable for high GluN2B affinity of ring-contracted tetralinamines
6 and indanamines 7. However, neither the tetralinamines 6c,d nor
the indanamines 7c,d reach the GluN2B affinity of the benzo[7]
annulenamines 5c,d. Introduction of the 4-phenyl- or 4-
benzylpiperidino moiety led to very potent GluN2B ligands with
very low Ki values (e.g. 6e: Ki ¼ 8.4 nM; 7f: Ki ¼ 3.2 nM). Unfortu-
nately, the s1 and s2 receptor affinities of all tetralinamines 6a-f are
higher than their GluN2B affinity, indicating preference for these
receptors instead for the ifenprodil binding site of the NMDA re-
ceptor. For the indanamines 7c and 7d with a phenylalkyl side chain
a similar result was obtained. However, the 4-phenylpiperidine 7e
and the 4-benzylpiperidine 7f show a considerable selectivity for
Cytoprotective effects of GluN2B ligands with various ring sizes and reference
compound ifenprodil correlated with their GluN2B affinity.
compd.
n
GluN2B affinity
cytoprotective activity
Ki SEM (nM) (n ¼ 3)
IC50 SEM (
mM) (n ¼ 3)
4c
4d
5c
6c
7c
7f
3
4
3
3
3
36
32
16
163 51
117 15
3.2 0.8
10 0.7
3
1
4
7.8 6.3
0.89 0.38
29%[a]
30%[a]
31[b]
7%
0.59 0.2
Ifenprodil
e
a values in % indicate the cytoprotective effect at a test compound concentration of
10 M.
b due to low activity, the IC50 value was recorded only once.
m
GluN2B receptors over both
s receptor subtypes.

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138
5
water molecule, and (3) benzylic OH moiety and the backbone N-
atom of Leu135 (GluN1b). The benzyl moiety interacts with a hy-
drophobic pocket formed by the residues of Ile111 (GluN2B),
Phe114 (GluN2B) and Tyr109 (GluN1b) via hydrophobic and aro-
matic interactions. In addition to the H-bond interactions, the
phenol of Ro 25e6981 undergoes hydrophobic and aromatic in-
teractions with Phe176 (GluN2B) and Arg115 (GluN1b). (Fig. 4F).
In order to prove the reliability of the docking results, the co-
crystallized ligand Ro 25e6981 was removed from its binding site
and the binding pose after re-docking of Ro 25e6981 into the
binding pocket was compared with the original pose in the crystal
structure. The RMSD value was lower than 1 Å indicating a reliable
docking procedure. All docking studies were performed with the
€
docking software Glide® version 2014 (Schrodinger®) [34e36].
In contrast to Ro 25e6981, the benzazocine derivatives 4c and
4d as well as the indanamines 7e and 7f contain only one polar
functional group, which is able to form H-bonds. Thus, the docking
studies show a conserved central H-bond interaction between the
protonated amino moiety of the ligands and the amide-carbonyl
group of Gln110. However, the H-bond donating phenol and
benzylic alcohol of Ro 25e6981 are missing in the new ligands,
which hampers their clear orientation in the binding pocket. Thus,
all ligands can adopt two poses in the binding pocket with the
phenylalkyl moiety embedded either in the right hydrophobic
binding pocket with Phe114 or alternatively in the left hydrophobic
binding pocket with Phe176 and Arg115. This is illustrated for the
phenylpropyl derivative 4c in Fig. 4A and B as example.
The hydrophobic pocket on the right hand side (Ile111, Phe114,
Tyr109) can accommodate either the terminal phenyl moiety of the
phenylalkyl substituent (Fig. 4A and C) or the annulated benzene
ring of the ligands (Fig. 4B, D, 4E). In particular, the piper-
idinylindanes 7e and 7f show an opposite orientation, with the
indane ring located in the right hydrophobic pocket (Fig. 4D and E).
Fig. 3. Inhibition (in %) of ion currents evoked by 10
mM glycine and 10 mM (S)-
glutamate in presence of 1 M and 10 M 4d and 10 M ifenprodil at GluN1a/GluN2B
m
m
m
expressing oocytes (n ¼ 5 per concentration). Significance of mean differences was
evaluated by one-way-ANOVA and posthoc mean comparison Tukey test (*** indicate
p < 0.001).
eight-membered ring, the 4-phenylbutyl derivative 4d was also
investigated, because the exocyclic 4-phenylbutyl moiety of 4d
correlates nicely with the exocyclic 3-phenylpropylamino moiety of
benzo[7]annulene 5c, tetraline 6c and indane 7c.
Data in Table 2 clearly show that the eight-membered 3-
benzazocines 4c and 4d display considerable protection against
cell damaging agents (S)-glutamate and glycine. Particularly high
activity was found for the 4-phenylbutyl derivative 4d with an IC50
-
value of 890 nM. Shortening of the side chain (4c) and contraction
of the eight-membered ring to a smaller seven-, six- or five-
membered ring led to reduced or loss of cytoprotective activity.
For the most protective compound 4d the inhibition of GluN2B
receptors was evaluated directly in two-electrode voltage clamp
(TEVC) measurements with GluN1a/GluN2B expressing oocytes
[33] and compared to the activity of ifenprodil. The results are
shown in Fig. 3.
5. Conclusion
Herein, novel GluN2B receptor antagonists with a conforma-
tionally constrained 2-phenylethylamine substructure are re-
ported. The phenylethylamine structure is incorporated in larger
(3-benzazocine ring, compounds 4) or smaller rings (tetralin-2-
amines (6), indan-2-amines (7)).
The results of TEVC measurements clearly confirm 4d as GluN2B
receptor inhibitor. Increasing the concentration from 1
of 4d led to a significantly increased inhibition of the ion current
from 8% (n ¼ 5) to 60% (n ¼ 5). However, 10 M ifenprodil resulted
in significantly higher ion current inhibition (95%, n ¼ 5) than
10 M 4d.
In general, the cytoprotective activity of the test compounds in
mM to 10 mM
An expanded eight-membered 3-benzazocine ring increased
considerably the GluN2B affinity (e.g. Ki (4d) ¼ 32 nM. 3-
Benzazocine 4d was identified as the most active antagonist of
this series of compounds showing moderate cytoprotective activity
(IC50 (4d) ¼ 890 nM) and moderate ion current inhibition (60% at
m
m
Table 2 correlates nicely with their GluN2B affinity in receptor
binding studies. However, the 3-benzazepine 5c and the indan-
amine 7f represent exceptions of this correlation, as 5c and 7f
display high GluN2B affinity (Ki ¼ 16 nM and 3.2 nM), but negligible
cytoprotective activity. Recently it was observed that affinity and
activity of 3-benzazepines are also not correlating completely
indicating a mechanism of inhibition, which is not only based on
the binding of the compounds [33].
10 mM) in TEVC measurements.
Ring-contraction of the [7]annulene ring of benzo [7]annulen-
amines 5 resulted in reduced GluN2B affinity of tetralinamines 6
and indanamines 7. Phenylpropyl- and phenylbutylamino sub-
stituents appear to be favorable for GluN2B affinity, but 4-phenyl-
and 4-benzylpiperidino groups led to high-affinity GluN2B ligands
(e.g. Ki (7f) ¼ 3.2 nM). Despite their high GluN2B affinity, the tet-
ralinamine 6c and the indanamines 7c and 7f do not show cyto-
protective activity. It was hypothesized that at least one hydroxy
moiety either at the benzene ring (phenol) or in benzyl position has
to be present in order to induce a conformational change and thus a
closure of the NMDA receptor associated ion channel. Moreover, a
recent study showed, that both hydroxy moieties contribute to the
inhibitory activity in an additive manner [33].
The novel GluN2B ligands 4, 6 and 7 contain only one functional
group for the establishment of H-bonds. This protonated amino
moiety is located in the middle of the molecule and undergoes a H-
bond interaction with the carbonyl moiety of Gln110 in the ifen-
prodil binding site. However, due to rather similar structural
4. Docking studies
The docking studies were carried out using the X-ray crystal
structure of the GluN1b/GluN2B dimer co-crystallized with the
prototypical negative allosteric modulator Ro 25e6981 (PDB-ID
3QEM) [13]. Three important H-bonds are responsible for the
orientation of Ro 25e6981 in the ifenprodil binding pocket: H-
bonds between (1) protonated piperidine N-atom and the O-atom
of the carbonyl moiety of Gln110 (GluN2B), (2) phenolic OH group
and the carboxylate group of Glu236 (GluN2B) and a conserved

6
L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138
Fig. 4. Binding modes of the 3-benzazocine derivative 4c (A and B) and 4d (C), and indanamines 7e (D) and 7f (E) compared with the binding mode of Ro 25e6981 (F) co-
crystallized in the ifenprodil binding pocket of the NMDA receptor (PDB-ID 3QEM). For the phenylpropyl derivative 4c two poses with the phenylpropyl moiety located either
in the right (A) or left (B) hydrophobic binding pocket are shown.
elements on the central amino moiety two possible orientations in
the binding site were found. The hydrophobic binding pocket
formed by Ile111, Phe114 and Tyr109 can accommodate either the
terminal phenyl moiety of the phenylalkyl side chain or the ben-
zene ring of the annulated ring system.
found exact masses from the calculated exact masses were 5 mDa
or less; the data were analyzed with DataAnalysis® (Bruker Dal-
tonics). NMR: NMR spectra were recorded in deuterated solvents
on Agilent DD2 400 MHz and 600 MHz spectrometers (Agilent,
Santa Clara CA, USA); chemical shifts (d) 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 0.5 Hz resolution;
assignment of 1H and 13C NMR signals was supported by 2-D NMR
techniques where necessary. IR: FT/IR Affinity®-1 spectrometer
(Shimadzu, Düsseldorf, Germany) using ATR technique.
6. Experimental part
6.1. 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 dry glassware (Schlenk flask or
Schlenk tube). Temperatures were controlled with dry ice/acetone
(ꢀ78 ꢁC), ice/water (0 ꢁC), Cryostat (Julabo TC100E-F, Seelbach,
Germany), magnetic stirrer MR 3001 K (Heidolph, Schwalbach,
Germany) or RCT CL (IKA, Staufen, Germany), together with tem-
perature 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 chro-
6.2. HPLC methods for the determination of the purity
In method 1 equipment 1 and in method 2 equipment 2 were
used. All other parameters are the same for both methods.
Equipment 1: Pump: L-7100, degasser: L-7614, autosampler: L-
7200, UV detector: L-7400, interface: D-7000, data transfer: D-line,
data acquisition: HSM-Software (all from LaChrom, Merck Hitachi).
Equipment 2: Pump: LPG-3400SD, degasser: DG-1210, auto-
sampler: ACC-3000T, UV-detector: VWD-3400RS, interface: DIO-
NEX UltiMate 3000, data acquisition: Chromeleon 7 (Thermo Fisher
Scientific, Lauenstadt, Germany).
matography (fc): Silica gel 60, 40e63
m
m (VWR); parentheses
Column: LiChrospher® 60 RP-select B (5
250-4 mm cartridge; flow rate: 1.0 mL/min; injection volume:
5.0 L; detection at
with 0.05% (V/V) trifluoroacetic acid, B: CH3CN with 0.05% (V/V)
trifluoroacetic acid; gradient elution (A %): 0e4 min: 90%;
4e29 min: gradient from 90% to 0%; 29e31 min: 0%; 31e31.5 min:
gradient from 0% to 90%; 31.5e40 min: 90%. The purity of all
compounds was determined by one of these methods.
mm), LiChroCART®
include: diameter of the column (d), length of the stationary phase
(l), fraction size (V) and eluent. Automated flash chromatography:
Isolera™ Spektra One (Biotage®); parentheses include: cartridge
size, flow rate, eluent, fractions size was always 20 mL. Melting
point: Melting point system MP50 (Mettler Toledo, Gieben, Ger-
many), open capillary, uncorrected. MS: MicroTOFQII mass spec-
trometer (Bruker Daltonics, Bremen, Germany); deviations of the
m
l
¼ 210 nm; solvents: A: demineralized water

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138
7
6.3. Synthetic procedures
2phenyl, C-6phenyl), 129.4 (2C, C-3phenyl, C-5phenyl), 129.8 (1C, C-7),
130.1 (1C, C-10), 141.68 (1C, C-6a), 141.71 (1C, C-10a), 143.7 (1C, C-
6.3.1. 3-(3-Phenylpropyl)-1,2,3,4,5,6-hexahydro-3-benzazocine (4c)
1
C
phenyl). Exact mass (APCI): m/z ¼ 294.2241 (calcd. 294.2216 for
21H28N [MþH]þ). Purity (HPLC, method 2): 99.0% (tR ¼ 19.77 min).
Under N2 atmosphere, 3-phenylpropanal (57 mL, 0.43 mmol, 1.1
eq.) and Na2SO4 (approx. 400 mg) were added to a solution of
secondary amine 11 (69 mg, 0.39 mmol, 1 eq.) in CH2Cl2 (3 mL). The
solution was stirred for 5 h at rt. NaBH(OAc)3 (126 mg, 0.59 mmol,
1.5 eq.) was added and the reaction mixture was stirred for 14 h at
rt. After treatment with saturated aqueous solution of NaHCO3
(10 mL) and water (10 mL), the organic layer was separated and the
aqueous phase was washed with CH2Cl2 (3 ꢂ 30 mL). The combined
organic layers were dried (Na2SO4), filtered and the solvent was
removed in vacuo. The crude product was purified by fc (d ¼ 2 cm,
l ¼ 16 cm, V ¼ 10 mL, gradient elution: cyclohexane/ethyl acetate
99:1 / 95:5 / 90:10 þ 1% N,N-dimethylethylamine / 80:20 þ 1%
6.3.3. N-(3-phenylpropyl)-1,2,3,4-tetrahydronaphthalen-2-amine
(6c)
A solution of
b-tetralone (12, 30
mL, 33.18 mg, 0.23 mmol, 1 eq.),
3-phenylpropan-1-amine (66
m
L, 62.5 mg, 0.46 mmol, 2 eq.) and
NaBH(OAc)3 (98.9 mg, 0.47 mmol, 2 eq.) in CH2Cl2 (4 mL) was
vigorously stirred for 14 h at rt. A saturated solution of NaHCO3
(6 mL) was added and the reaction mixture was extracted with
CH2Cl2 (3 ꢂ 5 mL). The CH2Cl2 layer was dried (Na2SO4), filtered and
the solvent was evaporated in vacuo. The crude product was pu-
rified by fc (d ¼ 3 cm, l ¼ 12 cm, v ¼ 10 mL, CH2Cl2:CH3OH 98:2 þ 1%
N,N-dimethylethylamine). Yellow oil, yield 27 mg (24%). C20H25
N
NH3, Rf ¼ 0.24) to obtain a yellow oil, yield 50.8 mg (84%). C19H23
N
(279.4 g/mol). TLC: Rf ¼ 0.67 (cyclohexane/ethyl acetate/N,N-
(265.4 g/mol). FT-IR (neat):
n
(cmꢀ1) ¼ 2920 (CeH), 740 and 698
dimethylethylamine 82:9:9). FT-IR:
H
H
n
[cmꢀ1] ¼ 3059, 3021 (C-
(1,2-disubst. arom.). 1H NMR (600 MHz, CD3OD):
d
(ppm) ¼ 1.51
~
arom.); 2924, 2855 (C-Haliph.); 1601, 1493 (C¼Carom.); 756, 698 (Ar-
(dtd, J ¼ 12.6/10.9/5.8 Hz, 1H, 3-H), 1.85 (tt, J ¼ 9.1/6.8 Hz, 2H,
PhCH2CH2CH2), 2.01e2.08 (m, 1H, 3-H), 2.53 (dd, J ¼ 15.9/10.0 Hz,
1H, 1-H), 2.63e2.72 (m, 4H, PhCH2CH2CH2), 2.73e2.88 (m, 3H, 2-H,
4-H), 2.93e3.00 (m, 1H, 1-H), 6.96e7.08 (m, 4H, AreH), 7.13e7.17
(m, 1H, Ph-H), 7.18e7.21 (m, 2H, Ph-H), 7.23e7.27 (m, 2H, Ph-H). A
signal for the NH proton is not observed. 13C NMR (151 MHz,
out of plane). 1H NMR (400 MHz, CDCl3):
d
[ppm] ¼ 1.60e1.77 (m,
4H, 5-CH2, PhCH2CH2CH2), 2.32e2.41 (m, 2H, 4-CH2), 2.46e2.57 (m,
4H, PhCH2CH2CH2), 2.71e2.79 (m, 2H, 2-CH2), 2.79e2.92 (m, 4H, 1-
CH2, 6-CH2), 7.06e7.21 (m, 7H, AreH,1-Hphenyl, 3-Hphenyl, 5-Hphenyl),
7.26 (m, 2H, 2-Hphenyl, 6-Hphenyl). 13C NMR (101 MHz, CDCl3):
d
[ppm] ¼ 29.9 (1C, PhCH2CH2CH2), 31.6 (1C, C-6), 32.7 (1C, C-5), 33.3
(1C, PhCH2CH2CH2), 35.6 (1C, C-1), 52.8 (1C, C-4), 58.2 (1C,
PhCH2CH2CH2), 59.2 (1C, C-2),125.7 (1C, C-4phenyl),126.3 (1C, C-8 or
C-9),126.6 (1C, C-9 or C-8),128.3 (2C, C-2phenyl, C-6phenyl), 128.6 (2C,
C-3phenyl, C-5phenyl), 129.0 (1C, C-7), 129.2 (1C, C-10), 140.8 (1C, C-6a
or C-10a), 141.2 (1C, C-10a or C-6a), 142.6 (1C, C-1phenyl). Exact mass
(APCI): m/z ¼ 280.2046 (calcd. 280.2060 for C20H26N [MþH]þ).
Purity (HPLC, method 2): 98.1% (tR ¼ 18.76 min).
CD3OD):
d
(ppm) ¼ 29.2 (1C, C-4), 30.1 (1C, C-3), 32.4 (1C,
PhCH2CH2CH2), 34.7 (1C, PhCH2CH2CH2), 36.8 (1C, C-1), 47.3 (1C,
PhCH2CH2CH2), 55.0 (1C, C-2), 126.7 (1C, Carom), 126.9 (1C, CPh),
126.9 (1C, Carom), 129.4 (4C, CPh), 129.6 (1C, Carom), 130.2 (1C, Carom),
136.0 (1C, Cqarom), 137.1 (1C, Cqarom), 143.2 (1C, CPqh). Exact Mass
(APCI): m/z ¼ 266.1933 (calcd. 266.1903 for C19H24N [M þ Hþ]).
Purity (HPLC, method 2): 96.3% (tR ¼ 19.51 min).
6.3.4. 4-Phenyl-1-(1,2,3,4-tetrahydronaphthalen-2-yl)piperidine
(6e)
6.3.2. 3-(4-Phenylbutyl)-1,2,3,4,5,6-hexahydro-3-benzazocine (4d)
Under N2 atmosphere, 4-phenylbutanal (93
m
L, 0.63 mmol, 1.1
A solution of nosylate 14 (50.5 mg, 0.15 mmol, 1 eq.) and 4-
phenylpiperidine (116.7 mg, 0.72 mmol, 4.8 eq.) in CH3CN (5 mL)
was stirred vigorously for 48 h at 75 ꢁC. The reaction mixture was
concentrated in vacuo and the residue was purified by fc (d ¼ 2 cm,
l ¼ 16 cm, v ¼ 10 mL, CH2Cl2:CH3OH 98:2 þ 1% NH3, Rf ¼ 0.24) to
eq.) was added to a mixture of secondary amine 11 (100 mg,
0.57 mmol, 1 eq.) and Na2SO4 (approx. 800 mg) in dry CH2Cl2
(4.5 mL). After stirring for 3 h at rt, NaBH(OAc)3 (184 mg,
0.87 mmol, 1.5 eq.) was added and the reaction mixture was stirred
for additional 14 h at rt. Sequentially a saturated aqueous solution
of NaHCO3 (10 mL) and water (10 mL) were added. The organic
layer was separated and the water layer was washed with CH2Cl2
(3 ꢂ 30 mL). The combined organic layers were dried (Na2SO4),
filtered and concentrated in vacuo. The crude product was purified
by fc (d ¼ 2 cm, l ¼ 16 cm, V ¼ 10 mL, gradient elution: cyclohexane/
ethyl acetate 99:1 / 90:10 / 80:20 / 50:50, 1% N,N-dimethy-
lethylamine was added to every solvent mixture). The product
(121 mg) was further purified by a second fc (d ¼ 2 cm, l ¼ 16 cm,
V ¼ 10 mL, cyclohexane/ethyl acetate 99:1 þ 1% N,N-dimethyle-
thylamine) and a third fc (d ¼ 2 cm, l ¼ 16 cm, V ¼ 10 mL, cyclo-
obtain a yellow solid, mp 104 ꢁC, yield 14.6 mg (33%). C21H25
N
(291.4 g/mol). FT-IR (neat):
(1,2-disubst. arom.).
n
1H
(cmꢀ1) ¼ 2931 (CeH), 744 and 698
NMR
(600
MHz,
CD3OD):
d
(ppm) ¼ 1.64e1.72 (m, 1H, 3-H), 1.78e1.87 (m, 2H, 3-CH2pip, 5-
CH2pip), 1.87e1.92 (m, 2H, 3-CH2pip, 5-CH2pip), 2.18e2.25 (m, 1H,
3-H), 2.45e2.55 (m, 2H, 2-CH2pip, 6-CH2pip), 2.59 (tt, J ¼ 12.1/4.1 Hz,
1H, 4-CHpip), 2.79e2.90 (m, 3H, 2-H, 4-H), 2.90e2.96 (m, 1H, 1-H),
2.99e3.05 (m, 1H, 1-H), 3.14e3.24 (m, 2H, 2-CH2pip, 6-CH2pip),
7.01e7.12 (m, 4H, AreH), 7.15e7.20 (m, 1H, Ph-H), 7.21e7.32 (m, 4H,
Ph-H). 13C NMR (151 MHz, CD3OD):
d
(ppm) ¼ 26.9 (1C, C-4), 30.4
(1C, C-3), 32.4 (1C, C-1), 34.4 (1C, C-3pip), 34.4 (1C, C-5pip), 43.9 (1C,
C-4pip), 50.7 (1C, C-2pip), 51.2 (1C, C-6pip), 62.4 (1C, C-2), 126.8 (1C,
Carom), 126.9 (1C, Carom), 127.2 (1C, CPh), 127.8 (2C, CPh), 129.4 (1C,
Carom), 129.5 (2C, CPh), 130.4 (1C, Carom), 136.6 (1C, Caqrom), 137.2 (1C,
Caqrom), 147.4 (1C, CPqh). Exact Mass (APCI): m/z ¼ 292.2096 (calcd.
292.2060 for C21H26N [M þ Hþ]). Purity (HPLC, method 2): 93.2%
(tR ¼ 18.93 min).
hexane/ethyl acetate 99:1). Yellow oil, yield 95 mg (57%). C21H27
N
(293.5 g/mol). TLC: Rf ¼ 0.20 (cyclohexane/ethyl acetate 90:10). FT-
~
IR:
n
[cmꢀ1] ¼ 3283 (NeH); 2955, 2916, 2847 (C-Haliph.); 1605, 1493
(C¼Carom.); 752, 729, 698 (Ar-Hout
plane). 1H NMR (600 MHz,
of
CD3OD):
d
[ppm] ¼ 1.40e1.47 (m, 2H, PhCH2CH2CH2CH2), 1.49e1.55
(m, 2H, PhCH2CH2CH2CH2), 1.59e1.64 (m, 2H, 5-CH2), 2.27e2.31 (m,
2H, 4-CH2), 2.48e2.52 (m, 2H, PhCH2CH2CH2CH2), 2.56 (t, J ¼ 7.5 Hz,
2H, PhCH2CH2CH2CH2), 2.71e2.75 (m, 2H, 2-CH2), 2.77e2.81 (m,
2H, 6-CH2), 2.81e2.85 (m, 2H, 1-CH2), 7.05e7.15 (m, 7H, AreH, 1-
6.3.5. 4-Benzyl-1-(1,2,3,4-tetrahydronaphthalen-2-yl)piperidine
(6f)
Hphenyl
H
,
3-Hphenyl
,
5-Hphenyl), 7.21e7.25 (m, 2H, 2-Hphenyl
,
6-
A solution of nosylate 14 (50.5 mg, 0.15 mmol, 1 eq.) and 4-
benzylpiperidine (126.7 mg, 0.72 mmol, 4.8 eq.) in CH3CN (6 mL)
was stirred vigorously for 48 h at 75 ꢁC. The reaction mixture was
concentrated in vacuo and the residue was purified by fc (d ¼ 2 cm,
l ¼ 14 cm, v ¼ 10 mL, CH2Cl2:CH3OH 98:2 þ 1% NH3, Rf ¼ 0.22) to
obtain a yellow oil, yield 18.2 mg (39%). C22H27N (305.5 g/mol). FT-
phenyl). 13C NMR (151 MHz, CD3OD):
d
[ppm] ¼ 27.9 (1C,
PhCH2CH2CH2CH2), 30.2 (1C, PhCH2CH2CH2CH2), 32.0 (1C, C-6),
33.3 (1C, C-5), 35.7 (1C, C-1), 36.7 (1C, PhCH2CH2CH2CH2), 53.5 (1C,
C-4), 59.9 (1C, PhCH2CH2CH2CH2), 60.5 (1C, C-2), 126.7 (1C, C-
4
phenyl), 127.4 (1C, C-8 or C-9), 127.7 (1C, C-9 or C-8), 129.2 (2C, C-

8
L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138
IR (neat):
n
(cmꢀ1) ¼ 2916 (CeH), 740 and 698 (1,2-disubst. arom.).
was concentrated in vacuo and the residue was purified by fc
1H NMR (600 MHz, CD3OD):
d
(ppm) ¼ 1.30e1.38 (m, 2H, 3-H, 4-
(d ¼ 2 cm, l ¼ 11 cm, v ¼ 10 mL, CH2Cl2:CH3OH 98:2 þ 1% NH3,
Hpip), 1.57e1.66 (m, 2H, 3-CH2pip, 5-CH2pip), 1.67e1.73 (m, 2H, 3-
Rf ¼ 0.22) to obtain a colorless solid, mp 102 ꢁC, yield 26.6 mg (68%).
CH2pip
,
5-CH2pip), 2.14 (ddt,
J
¼
12.6/5.5/2.8 Hz, 1H, 3-H),
C
21H25N (291.4 g/mol). FT-IR (neat):
n
(cmꢀ1) ¼ 2924 (CeH), 732
2.27e2.36 (m, 2H, 2-CH2pip, 6-CH2pip), 2.56 (d, J ¼ 7.2 Hz, 2H,
PhCH2), 2.72e2.83 (m, 3H, 2-H, 4-H), 2.85e2.92 (m, 1H, 1-H),
2.93e2.98 (m, 1H, 1-H), 3.01e3.10 (m, 2H, 2-CH2pip, 6-CH2pip),
7.02e7.07 (m, 4H, AreH), 7.14e7.18 (m, 3H, Ph-H), 7.23e7.28 (m, 2H,
and 698 (1,2-disubst. arom.). 1H NMR (300 MHz, CD3OD):
d
(ppm) ¼ 1.26e1.42 (m, 2H, 3-CH2pip, 5-CH2pip), 1.53e1.64 (m, 1H,
4-CHpip), 1.64e1.74 (m, 2H, 3-CH2pip, 5-CH2pip), 1.98e2.13 (m, 2H, 2-
CH2pip, 6-CH2pip), 2.56 (d, J ¼ 6.8 Hz, 2H, PhCH2), 2.77e2.92 (m, 2H,
2-CH2pip, 6-CH2pip), 3.01e3.17 (m, 5H, 1-H, 2-H, 3-H), 7.06e7.31 (m,
Ph-H). 13C NMR (151 MHz, CD3OD):
d
(ppm) ¼ 26.8 (1C, C-4), 30.4
(1C, C-3), 32.3 (1C, C-1), 32.9 (2C, C-3pip, C-5pip), 39.3 (1C, C-4pip),
44.0 (1C, PhCH2), 50.3 (1C, C-2pip), 50.7 (1C, C-6pip), 62.4 (1C, C-2),
126.8 (1C, Carom), 126.9 (1C, Carom), 126.9 (1C, CPh), 129.2 (2C, CPh),
129.4 (1C, Carom), 130.1 (2C, CPh), 130.3 (1C, Carom), 136.5 (1C, Caqrom),
137.2 (1C, Caqrom), 141.7 (1C, CPqh). Exact Mass (APCI): m/z ¼ 306.2227
(calcd. 306.2216 for C22H28N [M þ Hþ]). Purity (HPLC, method 2):
87.2% (tR ¼ 19.88 min).
9H, AreH, Ph-H). 13C NMR (101 MHz, CDCl3):
d
(ppm) ¼ 32.1 (2C, C-
3
pip, C-5pip), 37.3 (2C, C-1, C-3), 37.9 (1C, C-4pip), 43.3 (1C, PhCH2),
52.2 (2C, C-2pip, C-6pip), 67.3 (1C, C-2), 124.5 (2C, Carom), 125.9 (1C,
CPh),126.5 (2C, Carom),128.3 (2C, CPh),129.2 (2C, CPh),140.7 (1C, CPqh),
141.7 (2C, Caqrom). Exact Mass (APCI): m/z ¼ 292.2061 (calcd.
292.2060 for C21H26N [M þ Hþ]). Purity (HPLC, method 2): 98.3%
(tR ¼ 18.15 min).
6.3.6. N-(4-phenylbutyl)indan-2-amine (7d)
6.4. Receptor binding studies
A solution of nosylate 17 (57.1 mg, 0.18 mmol, 1 eq.) and 4-
phenylbutan-1-amine (142
m
L, 134.3 mg, 0.9 mmol, 5 eq.) in
Performance of receptor binding studies is described in the
Supporting Information and in Refs. [21e24] and references [30,31].
CH3CN (4 mL) was stirred vigorously for 16 h at 60 ꢁC. The reaction
mixture was concentrated in vacuo and the residue was purified by
fc (d ¼ 2 cm, l ¼ 11 cm, v ¼ 10 mL, CH2Cl2:CH3OH 99:1 þ 1% NH3,
6.5. Cytoprotection
Rf ¼ 0.17) to obtain an orange oil, yield 21.3 mg (45%). C19H23
N
(265.4 g/mol). FT-IR (neat):
n
(cmꢀ1) ¼ 2927 (CeH), 740 and 698
Mouse L (tk-) cells stably transfected with the dexamethasone-
inducible eukaryotic expression vectors pMSG NR1-1a, pMSG NR2B
(1:5 ratio) were grown and harvested as described in chapter 4.4.2.
The assay was based on reference [32].
(1,2-disubst. arom.).
1H
NMR
(400
MHz,
CD3OD):
d
(ppm) ¼ 1.52e1.62 (m, 2H, PhCH2CH2CH2CH2), 1.63e1.73 (m, 2H,
PhCH2CH2CH2CH2), 2.60e2.69 (m, 4H, PhCH2CH2CH2CH2), 2.75 (dd,
J ¼ 15.6/7.2 Hz, 2H, 1-H, 3-H), 3.14 (dd, J ¼ 15.6/7.3 Hz, 2H,1-H, 3-H),
3.56 (quint, J ¼ 7.2 Hz, 1H, 2-H), 7.06e7.12 (m, 2H, AreH), 7.12e7.21
(m, 5H, AreH), 7.21e7.28 (m, 2H, AreH). A signal for the NH proton
After careful removal of the medium the plate was blocked twice
with 200
mL DMEM containing 1% BSA and rinsed once with 200 mL
DMEM. Then the cells in the inner wells (B2-G11) were incubated
is not observed. 13C NMR (101 MHz, CD3OD):
d
(ppm) ¼ 30.2 (1C,
with 50 mL DMEM and 50 mL of the respective test compound in 5
PhCH2CH2CH2CH2), 30.5 (1C, PhCH2CH2CH2CH2), 36.8 (1C,
PhCH2CH2CH2CH2), 40.2 (2C, C-1, C-3), 60.7 (1C, C-2), 125.5 (2C,
Carom), 126.7 (1C, CPh), 127.5 (2C, Carom), 129.3 (2C, CPh), 129.4 (2C,
CPh), 142.6 (2C, Cqarom), 143.6 (1C, CqPh). The NHCH2 signal cannot be
assigned due to interference with the methanol signal. However,
the presence of the signal is confirmed by 2D-HSQC NMR spec-
troscopy. Exact Mass (APCI): m/z ¼ 266.1903 (calcd. 266.1903 for
different concentrations (e.g. 4 ꢂ 10ꢀ5, 10ꢀ6, 10ꢀ7, 10ꢀ8, 10ꢀ9 mol/L)
and 100 mL DMEM was added to the remaining outer wells. Each
concentration of the test compound was incubated at least in
triplicates for 30 min at 37 ꢁC. Afterwards 100
L of a glutamate/
M) were added and the cells were
m
glycine solution (each 20
incubated for 6 h at 37 ꢁC.
m
Subsequently, 50 mL of the supernatant were transferred into a
C
19H24N [M þ Hþ]). Purity (HPLC, method 2): 90.0% (tR ¼ 19.17 min).
96-well plate and incubated with 50 mL of the LDH-assay mixture (1
U/mL diaphorase, 1% Na lactate, 0.1% NADþ, 0.08% BSA, 0.4% iodo-
nitrotetrazolium chloride in 75 mM PBS) at 37 ꢁC for 45 min. The UV
6.3.7. 1-(Indan-2-yl)-4-phenylpiperidine (7e)
A solution of nosylate 17 (55.0 mg, 0.17 mmol, 1 eq.) and 4-
phenylpiperidine (135.4 mg, 0.84 mmol, 4.9 eq.) in CH3CN (3 mL)
was stirred vigorously for 20 h at 60 ꢁC. The reaction mixture was
concentrated in vacuo and the residue was purified by fc (d ¼ 1 cm,
l ¼ 12 cm, v ¼ 10 mL, cyclohexane:EtOAc 7:3 þ 1% NH3, Rf ¼ 0.28) to
absorption was measured at
l
¼ 485 nm in a plate reader (TECAN,
Crailsheim, Germany). Total LDH activity was determined with
buffer instead of ligand solution and a solution of (S)-ketamine with
a concentration of 4 ꢂ 10ꢀ5 mol/L served as positive control.
obtain a colorless solid, mp 119 ꢁC, yield 28.2 mg (59%), C20H23
N
6.6. Molecular biology and two-electrode voltage clamp (TEVC)
(277.4 g/mol). FT-IR (neat):
(1,2-disubst. arom.).
n
1H
(cmꢀ1) ¼ 2927 (CeH), 740 and 698
NMR
(400
MHz,
CD3OD):
Two-electrode voltage clamp experiments were carried out as
previously described [14,33]. Xenopus laevis oocytes were pur-
chased from EcoCyte Bioscience (Dortmund, Germany) and injec-
ted with 0.8 ng GluN1a and 0.8 ng GluN2B cRNA. cRNAs were
generated by in vitro transcription with the T7 mMessage mMa-
chine kit (Life Technologies, Darmstadt, Germany) from linearized
cDNA templates (wildtype rat). Injected oocytes were stored for
5 d at 18 ꢁC in Bath’s solution containing (mmol/L): 88 NaCl, 1 KCl,
0.4 CaCl2, 0.33 Ca(NO3)2, 0.6 MgSO4, 5 TRIS-HCl, 2.4 NaHCO3. Barth’s
solution was supplemented with 80 mg/L theophylline, 63 mg/L
benzylpenicillin, 40 mg/L streptomycin and 100 mg/L gentamycin.
Ifenprodil (þ)-tartrate was purchased from Sigma Aldrich. Com-
pound 4d and ifenprodil were used as 10 mM solutions in DMSO,
which were diluted with agonist solution (Ba2þ-Ringer supple-
d
(ppm) ¼ 1.76e1.95 (m, 4H, 3-CH2pip, 5-CH2pip), 2.20e2.32 (m, 2H,
2-CH2pip, 6-CH2pip), 2.54e2.65 (m, 1H, 4-CHpip), 2.86e2.98 (m, 2H,
2. CH2pip, 6-CH2pip), 3.10e3.26 (m, 5H, 1-H, 2-H, 3-H), 7.06e7.37 (m,
9H, AreH, Ph-H). 13C NMR (101 MHz, CD3OD):
d
(ppm) ¼ 34.1 (2C,
C-3pip, C-5pip), 38.1 (2C, C-1, C-3), 43.5 (1C, C-4pip), 53.7 (2C, C-2pip
,
C-6pip), 68.5 (1C, C-2), 125.4 (2C, Carom), 127.3 (1C, CPh), 127.7 (2C,
Carom), 127.8 (2C, CPh), 129.5 (2C, CPh), 142.3 (2C, Caqrom), 147.3 (1C,
CPqh). Exact Mass (APCI): m/z ¼ 278.1927 (calcd. 278.1903 for
C
20H24N
[M
þ
Hþ]). Purity (HPLC, method 2): 98.3%
(tR ¼ 17.07 min).
6.3.8. 4-Benzyl-1-(indan-2-yl)piperidine (7f)
A solution of nosylate 17 (42.6 mg, 0.13 mmol, 1 eq.) and 4-
benzylpiperidine (115
m
L, 114.7 mg, 0.65 mmol, 5 eq.) in CH3CN
mented with 10
mM glycine and 10 mM (S)-glutamate) to obtain the
(3 mL) was stirred vigorously for 20 h at 60 ꢁC. The reaction mixture
desired concentration of compound and constant concentration of

L. Temme et al. / European Journal of Medicinal Chemistry 190 (2020) 112138
9
agonists. All test solutions were adjusted to equal DMSO concen-
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
trations of 0.1%. Ion current inhibition of compounds was tested by
two-electrode voltage clamp (TEVC) in Xenopus laevis oocytes at
room temperature. For measurements, oocytes were constantly
superfused with Ba2þ Ringer (pH 7.4) containing (mmol/L): 10
HEPES, 90 NaCl, 1 KCl,1.5 BaCl2. Measurements were performed at a
holding potential of ꢀ70 mV and recording pipettes (0.5e1.5 M
U)
We thank Mark Worrmann for his assistance during laboratory
work. This work was supported by the Deutsche For-
schungsgemeinschaft (DFG), which is gratefully acknowledged.
Moreover, we are grateful to Cells-in-Motion (CiM) Cluster of Excel-
lence for funding a lab visit at the University of Halle-Wittenberg to
learn molecular modelling.
were backfilled with 3 M KCl. Each compound concentration was
tested at five different oocytes.
6.7. Data analysis and statistics
Data recording and data analysis was carried out like previously
described [14,33]. Electrophysiological data were recorded with
GePulse (Dr. Michael Pusch, Genova, Italy; http://users.ge.ibf.cnr.it/
pusch/) and analyzed with Ana (Dr. Michael Pusch, Genova, Italy;
http://users.ge.ibf.cnr.it/pusch/) and OriginPro 2019 (OriginLab
Corporation, Northampton, MA, USA). Inhibitory activity was
calculated as previously described by the following equation:
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112138.
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