Synthesis and Pharmacological Evaluation of Enantiomerically Pure GluN2B Selective NMDA Receptor Antagonists

Article abstract of DOI:10.1002/cmdc.201800214

To determine the eutomers of potent GluN2B-selective N-methyl-d-aspartate (NMDA) receptor antagonists with a 3-benzazepine scaffold, 7-benzyloxy-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepin-1-ols (S)-2 and (R)-2 were separated by chiral HPLC. Hydrogenolysis and subsequent methylation of the enantiomerically pure benzyl ethers of (S)-2 and (R)-2 provided the enantiomeric phenols (S)-3 and (R)-3 [3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol] and methyl ethers (S)-4 and (R)-4. All enantiomers were obtained with high enantiomeric purity (≥99.7 % ee). The absolute configurations were determined by CD spectroscopy. R-configured enantiomers turned out to be the eutomers in receptor binding studies and two-electrode voltage clamp experiments. The most promising ligand of this compound series is the R-configured phenol (R)-3, displaying high GluN2B affinity (K_i=30 nm), high inhibition of ion flux (IC₅₀=61 nm), and high cytoprotective activity (IC₅₀=93 nm). Whereas the eudismic ratio in the receptor binding assay is 25, the eudismic ratio in the electrophysiological experiment is 3.

Full text of DOI:10.1002/cmdc.201800214

Accepted Article
Title: Synthesis and pharmacological evaluation of enantiomerically
pure GluN2B selective NMDA receptor antagonists
Authors: Bernhard Wünsch, Frederik Börgel, Marina Szermerski, Julian
A. Schreiber, Louisa Temme, Nathalie Strutz-Seebohm,
Kirstin Lehmkuhl, Dirk Schepmann, Simon M. Ametamey,
Guiscard Seebohm, and Thomas J. Schmidt
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To be cited as: ChemMedChem 10.1002/cmdc.201800214
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Synthesis and pharmacological evaluation of enantiomerically
pure GluN2B selective NMDA receptor antagonists
Frederik Börgel,[a] Dr. Marina Szermerski,[a] Julian A. Schreiber,[a] Dr. Louisa Temme,[a] Dr. Nathalie
Strutz-Seebohm,[b] Kirstin Lehmkuhl,[a] Dr. Dirk Schepmann,[a,e] Prof. Dr. Simon M. Ametamey,[c] Prof.
Dr. Guiscard Seebohm,[b] Prof. Dr. Thomas J. Schmidt,[d] Prof. Dr. Bernhard Wünsch[a,e]
[a]
Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Corrensstraße 48, D-48149 Münster, Germany
Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail: wuensch@uni-muenster.de
[b]
Myocellular Electrophysiology and Molecular Biology, Institute for Genetics of Heart Diseases (IfGH), Department of Cardiovascular Medicine, University
Hospital Muenster, D-48149 Muenster, Germany
[c]
[d]
[e]
Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland
Institut für Pharmazeutische Biologie und Phytochemie der Universität Münster, Corrensstraße 48, D-48149 Münster, Germany
Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), Westfälische Wilhelms-Universität Münster, Germany
Abstract: In order to determine the eutomers of potent GluN2B
selective NMDA receptor antagonists with 3-benzazepine scaffold,
7-benzyloxy-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepin-
1-ols (S)-2 and (R)-2 were separated by chiral HPLC.
Hydrogenolysis and subsequent methylation of the enantiomerically
pure benzyl ethers (S)-2 and (R)-2 provided the enantiomeric
phenols (S)-3 and (R)-3 (3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-
benzazepin-1,7-dool) and methyl ethers (S)-4 and (R)-4. All
enantiomers were obtained with high enantiomeric purity (ee ≥
99.7 %). The absolute configuration was determined by CD-
spectroscopy. (R)-configured enantiomers turned out to be the
eutomers in receptor binding studies and two-electrode voltage
clamp experiments. The most promising ligand of this series of
compounds is the (R)-configured phenol (R)-3 displaying high
Heterotetrameric NMDA receptors containing GluN2B subunits
(here GluN2B receptors) are of particular interest, since GluN2B
subunits transfer an additional binding site for negative allosteric
modulators (NAM) and extend the drugability of receptor
complexes.[6] Recent X-ray crystal structure analyses of the
whole NMDA receptor and the dimer of the amino terminal part
together with various ligands have shown that this binding site is
located at the interface between the GluN1 and the GluN2B
subunits.[7][8][9][10] Targeting this additional so-called ifenprodil
binding site allows selective subtype-specific modulation of
GluN2B receptors. Inhibition of GluN2B subunit containing
NMDA receptors could be exploited for the treatment of
neurodegenerative
diseases
such
as
Parkinson’s,[11]
Alzheimer’s[11] and Huntington’s disease[12] as well as
neuropathic pain.[13]
GluN2B affinity (Ki = 30 n
M
), high inhibition of ion flux (IC50 = 61 n
M)
and high cytoprotective activity (IC50 = 93 ± 39 n
M
). Whereas the
eudismic ratio in the receptor binding assay is 25, the eudismic ratio
in the electrophysiological experiment is 3.
Recently, racemic 3-benzazepin-1-ols rac-2-4 with moderate to
high GluN2B affinity were reported (see also Table 1). Both the
phenol rac-3 and the methyl ether rac-4 revealed high selectivity
against a panel of selected receptors, ion channels and
enzymes.[14][15][16] In in vivo experiments with mice racemic
phenol rac-3 was well tolerated, and showed analgesic activity in
the formalin assay.[15] Furthermore, promising properties of
phenol rac-3 were found during treatment of immune-mediated
and inflammatory CNS diseases.[17] The racemic methyl ether
rac-[11C]4 has been successfully developed as PET tracer for
imaging of GluN2B receptors in the CNS.[18] For the synthesis of
carbon-11 labeled methyl ether rac-[11C]4 the phenol precursor
rac-3 was alkylated with [11C]CH3I.[19]
Introduction
The N-methyl-d-aspartate (NMDA) receptor belongs to the
ionotropic glutamate receptors together with the 2-amino-3-(3-
hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and the
kainate receptor. These ligand-gated ion channels are essential
components of the neurotransmitter system.[1] The NMDA
receptor is highly expressed in the central nervous system
(CNS)[2] and additionally in some tissues in the periphery like
pancreas[3] and heart[4]. Seven different proteins are known
which are able to form the heterotetrameric ion channel of the
NMDA receptor. These proteins are termed GluN1 (with eight
splice variants a-h), GluN2A-D and GluN3A-B subunits. A
functional NMDA receptor is composed of at least one GluN1
and one GluN2 subunit, usually two GluN1 and two GluN2
subunits are present.[5]
Motivated by the promising in vitro and in vivo activities of
racemic phenol rac-3 and methyl ether rac-4 we here address
the pharmacological properties of the pure enantiomers. In
particular the eutomers of both GluN2B antagonists should be
identified. Herein, we report the resolution of the three 3-
benzazepines rac-2-4 and the pharmacological and
physicochemical characterization of the respective pure
enantiomers.
1
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Results and Discussion
Synthesis
HO
HO
HO
a)
(c)
(d)
N R
N R
N R
BnO
HO
H3CO
2
rac-
(b)
(R)-2
3
(R)-
4
(R)-
2
(S)-
(S)-3
(S)-4
R = (CH2)4C6H5
Figure 1. Synthesis of enantiomerically pure 3-benzazepines 2-4. Reagents and reaction conditions: (a) 1-chloro-4phenyl-butane, TBAI, K2CO3, CH3CN, reflux, 72 h,
87 % (rac-2). (b) preparative chiral HPLC, 43 % (R)-2, 43 % (S)-2. (c) H2, Pd/C, CH3OH, rt, 1 h, 94 % (R)-3, 97 % (S)-3. (d) CH3I, Cs2CO3, DMF, rt, 16 h, 57 % (R)-
4, 44 % (S)-4.
Table 1. Enantiomeric purity of the 3-benzazepines (S)-2-4 and (R)-2-4 after
The desired enantiomers of 3-benzazepin-1-ols 2-4 were
synthesis (first line) and after incubation with murine S9 fraction and NADPH
for 120 min at 37 °C (second line).
synthesized from racemic secondary amine rac-1,[20] which was
alkylated with 1-chloro-4phenylbutane in the presence of
tetrabutylammonium iodide (TBAI) to give rac-2. The enantiomers
compd.
(R)-2
99.8
99.4
(S)-2
99.7
(R)-3
(S)-3
99.7
99.2
(R)-4
99.8
(S)-4
99.7
(R)-2 and (S)-2 were separated by preparative chiral HPLC using
the Daicel Chiralpak® IA chiral column. Afterwards, the benzyl
ethers of (R)-2 and (S)-2 were cleaved with H2 and Pd/C affording
the phenols (R)-3 and (S)-3. Finally, the enantiomerically pure
phenols (R)-3 and (S)-3 were methylated with methyl iodide
yielding methyl ethers (R)-4 and (S)-4. (Figure 1)
ee [%]
after
synthesis
ee [%]
after
≥99.8a
≥99.8a
≥99.7a
≥99.8a
≥99.7a
incubation
[a] a A peak for the corresponding enantiomer could not be found.
The enantiomeric purity of the three pairs of enantiomers was
determined by analytical chiral HPLC using the same stationary
phase (Daicel Chiralpak® IA). In Figure
2
the HPLC
Absolute configuration
chromatogram of the enantiomerically pure phenol (S)-3 is shown
exemplarily. After expansion of the small peak, less than 0.15 %
of (R)-3 could be detected (Figure 2). The enantiomeric purity of
the six enantiomers is summarized in Table 1.
The absolute configuration of the phenols (R)-3 and (S)-3 was
analyzed by CD spectroscopy. Thus, the CD spectra of both
enantiomers were recorded and the CD-spectrum of model
compound (S)-5 was simulated using Gaussian 03W (TD-DFT
B3LYP/6-31G(d,p)). In the model compound (S)-5 the rather
flexible 4-phenylbutyl side chain of 3 was replaced by a small
methyl moiety in order to reduce the time required for the
0,6
0.4
0.2
0.0
0.6
0,5
0.5
(R)-3
(S)-3
0,4
0.4
calculation of the circulardichroism of
a large number of
conformers. The CD spectrum calculated for (S)-5 (black curve)
is in good agreement with the red CD curve. Thus, (S)-
configuration was assigned to the compound leading to the red
curve. (Figure 3) As a consequence (S)-configuration was
assigned to the benzyl ether (S)-2 leading to (S)-3 and the methyl
ether (S)-4 prepared from (S)-3.
0,3
0.3
22
18 20
24
0,2
0.2
0,1
0.1
Moreover, X-ray crystal structure analysis of the enantiomeric
methyl ethers (S)-4 and (R)-4 confirmed the assignment of
absolute configuration.[27]
0
0
.
,
0
0
0
0
5
5
10
10
15
15
20
20
25
25
30
30
Retention Ttiimme (emin/)min
Retention
Figure 2. Chiral HPLC-UV chromatogram of (S)-3. Daicel Chiralpak® IA,
isohexane:ethanol 90:10, 1 mL/min, detection at 254 nm.
2
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Table 2. Affinities towards the GluN2B subunit of the NMDA receptor of
enantiomerically pure 3-benzazepin-1-ols (S)-2-4 and (R)-2-4 compared with
the affinities of the corresponding racemates.
30
20
compd.
rac-2 [12]
(R)-2
R
OBn
OBn
OBn
OH
Ki ± SEM [n
M
] (n = 3)a
eudismic ratio
(R)-3
(S)-5
10
0
505 ± 170
298 ± 70
557 ± 162
84 ± 18
2
(S)-2
rac-3 [12]
(R)-3
–10
–20
–30
(S)-3
OH
30 ± 8
25
(S)-5
(S)-3
OH
740 ± 61
706 ± 100
542 ± 79
720 ± 68
10 ± 0.7
13 ± 2.0
rac-4 [12]
(R)-4
OCH3
OCH3
OCH3
--
220
300
200
240 260
280
320 340
1.3
(S)-4
Wavelength / nm
ifenprodil
eliprodil
Figure 3. Recorded CD spectra of enantiomerically pure phenols (R)-3, (S)-3
and calculated CD spectrum of model compound (S)-5.
--
a The Ki values were recorded three times (n = 3). SEM: standard error of the
mean.
Configurational stability
The data in Table 2 clearly indicate that the (R)-configured
enantiomers show higher GluN2B affinity than the corresponding
(S)-configured enantiomers. This difference is lower for the low-
affinity ethers 2 and 4 but very high for the high-affinity phenol 3.
(R)-3 represents the eutomer with an eudismic ratio of 25.
The center of chirality of the enantiomerically pure 3-
benzazepines 2-4 is located in benzylic position. In order to prove
the configurational stability of the enantiomerically pure test
compounds, (S)-2-4 and (R)-2-4 were incubated with murine S9
fraction and NADPH at 37 °C for 120 min. The ratio of
enantiomers was determined by chiral HPLC as described above
(Table 1). The data clearly indicate that racemization had not
occurred during incubation with murine S9 fraction and NADPH.
Inhibition of ion flux in two electrode voltage clamp
experiments
The channel blocking activity of the enantiomerically pure phenols
(R)-2 and (S)-2 was investigated by two-electrode voltage clamp
(TEVC) measurements. For this purpose, Xenopus laevis oocytes
were injected with cRNAs encoding GluN1a / GluN2B and
incubated for 5 to 6 days to express the functional receptors. Ion
A possible racemization pathway is the oxidation of the benzylic
alcohol resulting in the respective ketone. This oxidation and the
following reduction of the resulting ketone can be catalyzed by
alcohol dehydrogenase (ADH). Therefore, the enantiomerically
pure alcohols (S)-2-4 and (R)-2-4 were incubated with the
cofactor NAD+ and murine S9 fraction as ADH is known to be
present in the S9 fraction.[21] After work-up, the reaction mixtures
were analyzed by LC-MS. Benzylic ketones could not be detected
confirming high configurational stability of the enantiomerically
pure 3-benzazepin-1-ols (S)-2-4 and (R)-2-4.
flux was stimulated by addition of 10 µM (S)-glutamate and 10 µM
glycine. The inhibition of opened NMDA receptors by different test
compound concentrations was determined in the presence of the
two enantiomers to obtain dose response curves. (Table 3)
Table 3. Inhibition of glutamate/glycine stimulated ion flux in TEVC.
GluN2B affinity
Inhibition of ion flux
eudismic
ratio
compd.
Ki ± SEM [n
M
84 ± 18
30 ± 8
] (n = 3)a
IC50 ± SD [n
M]
Receptor affinity
rac-3
116 ± 7
The GluN2B affinity of the enantiomerically pure 3-benzazepin-1-
ols (S)-2-4 and (R)-2-4 was determined in a competitive
radioligand receptor binding assay. [3H]Ifenprodil was used as
radioligand.[22] Selectivity of the assay towards GluN2B subunit
containing NMDA receptors was achieved by using membrane
preparations from a recombinant cell line stably expressing the
human GluN1a splice variant and the human GluN2B subunit.
The GluN2B assay was slightly modified compared to the original
reported procedure[22] resulting in slightly different Ki values for
the racemates rac-2-4. (Table 2).
(R)-3
(S)-3
61 ± 4
3
740 ± 61
190 ± 40
The two-electrode voltage clamp experiment showed that the high
GluN2B affinity of the phenols translated into an inhibitory effect.
The corresponding inhibition curves are depicted in Figure 4.
Moreover, a similar trend in enantioselectivity was detected.
Whereas (R)-configured phenol (R)-3 resulted in higher inhibitory
activity than the racemate rac-3, the (S)-configured enantiomer
(S)-3 revealed lower activity. However, the eudismic ratio
between the two enantiomers is only 3 in this assay, and thus
considerably lower than the eudismic ratio determined in the
receptor binding studies.
3
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inhibition of glutamate/glycine induced ion flux in two-electrode
voltage
clamp
experiments
and
cytoprotection
in
glutamate/glycine induced cytotoxicity. It is highly likely that the
phenolic OH moiety is a structural key component inducing a
conformational switch in the ligand gated ion channel leading to
inhibition of the ion flux.
Experimental Section
Chemistry, General Methods
Oxygen and moisture sensitive reactions were carried out under
nitrogen, dried with silica gel with moisture indicator (orange gel,
Merck) and in dry glassware (Schlenk flask or Schlenk tube).
Temperatures were controlled with dry ice/acetone (-78 °C),
ice/water (0 °C), Cryostat (Julabo FT 901 or Huber TC100E-F),
magnetic stirrer MR 3001 K (Heidolph) or RCT CL (IKA®),
together with temperature controller EKT HeiCon (Heidolph) or
VT-5 (VWR) and PEG or silicone bath. All solvents were of
analytical grade quality. Demineralized water was used. THF was
distilled from sodium/benzophenone. Methanol was distilled from
magnesium methanolate. CH3CN and ethanol abs. were dried
with molecular sieves (3 Å); DMF, ethyl acetate and toluene were
dried with molecular sieves (4 Å). Thin layer chromatography (tlc):
tlc silica gel 60 F254 on aluminum sheets (Merck). Flash
chromatography (fc): Silica gel 60, 40–63 µm (Merck);
parentheses include: diameter of the column (∅), length of the
stationary phase (l), fraction size (v) and eluent. Melting point:
Melting point system MP50 (Mettler Toledo), open capillary,
uncorrected. MS: MicroTOFQII mass spectrometer (Bruker
Daltonics); deviations of the found exact masses from the
calculated exact masses were 5 ppm or less; the data were
analyzed with DataAnalysis (Bruker). NMR: NMR spectra were
recorded on Agilent DD2 400 MHz and 600 MHz spectrometers;
chemical shifts (δ) are reported in parts per million (ppm) against
the reference substance tetramethylsilane and calculated using
the solvent residual peak of the undeuterated solvent. IR: FT/IR
IRAffinity-1 IR spectrometer (Shimadzu) using ATR technique.
Optical rotation: Polarimeter 341 (Perkin Elmer); 1.0 dm tube;
concentration c in g/100 mL; T = 20 °C; wavelength 589 nm (D-
Figure 4: Inhibition curves of (R)-3 (blue), rac-3 (black), (S)-3. Each data point
represents the mean of n = 3 experiments ± SEM.
Although the methyl ether rac-4 revealed a similar GluN2B affinity
as the distomer (S)-3 of the phenols (Ki = 706 / 740 n ), inhibition
M
of opened NMDA receptors in TEVC measurements was much
lower. It is postulated that the methyl ether rac-4 and its
enantiomers are able to interact with the ifenprodil binding site of
the GluN2B receptor, but a phenolic OH moiety is required for the
high inhibitory activity.
Cytoprotection
It has been shown that the (R)-configured enantiomer (R)-3
interacts with low nanomolar affinity with the ifenprodil binding site
and is able to inhibit the glutamate/glycine induced ion flux into
oocytes. In the next step it should be investigated, whether these
properties can translate into protection of cells against
glutamate/glycine induced excitotoxicity. For this purpose, mouse
fibroblast cells stably expressing NMDA receptors with GluN1a
and GluN2B subunits were used. In a lactate dehydrogenase
assay the amount of released lactate dehydrogenase after
stimulation with (S)-glutamate and glycine in the presence or
absence of a test compound was determined.
T
line of Na light); the unit of the specific rotation ([]D
grad.mL.dm-1.g-1) is omitted for clarity.
In this assay (R)-3 resulted in cytoprotection of the cells. With an
IC50 value of 93 ± 39 n
cytotoxic effects of (S)-glutamate and glycine. The cytoprotective
activity of the (R)-configured phenol is higher than those of the
lead compound ifenprodil (250 ± 56 n
potency of Ro 25-6981 (IC50: 13 ± 2.9 n
M
the cells were protected against the
HPLC method for the determination of the purity
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,
autosampler: ACC-3000T, UV-detector: VWD-3400RS, interface:
M). However, the high
M), another negative
allosteric modulator of GluN2B subunit containing NMDA
receptors besides ifenprodil, was not achieved.[23]
DIONEX UltiMate 3000, data acquisition: Chromeleon
7
(Thermo Fisher Scientific); column: LiChropher® 60 RP-select B
(5 µm), LiChroCART® 250-4 mm cartridge; flow rate: 1.0 mL/min;
injection volume: 5.0 µL; detection at λ = 210 nm; solvents: A:
demineralized water with 0.05 % (V/V) trifluoroacetic acid, B:
acetonitrile with 0.05 % (V/V) trifluoroacetic acid; gradient elution
(% A): 0 - 4 min: 90 %; 4 - 29 min: gradient from 90 % to 0 %;
29 - 31 min: 0 %; 31 - 31.5min: gradient from 0 % to 90 %;
31.5 - 40 min: 90 %.
Conclusions
A set of enantiomerically pure 3-benzazepin-1-ols (S)-2-4 and
(R)-2-4 was prepared by resolution of racemic benzyl ether rac-2
using chiral HPLC. In receptor binding studies with [3H]ifenprodil,
(R)-configured enantiomers represent the eutomers with an
eudismic ratio of 25 for the enantiomeric phenols (R)-3 and (S)-3.
The high GluN2B affinity of (R)-3 (Ki = 30 nM) translates into
4
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CD spectroscopy
The CD spectra were recorded with
(S)-7-Benzyloxy-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-
benzazepin-1-ol ((S)-2)
a
J-815 CD
spectropolarimeter (Jasco). The samples were measured at a
concentration of 0.1 mg/mL in CH3OH. A background correction
was performed by subtracting a spectrum of pure CH3OH. For the
simulation of the CD spectrum of (S)-5 the structure was built and
geometry optimized with the Molecular Operations Environment
(MOE[24]) using the MMFF94x force field and a low-mode-
dynamics conformational search. The quantum mechanical
calculations were performed with the lowest energy conformer
using Gaussian 03W[25]. Energy minimization was performed with
the B3LYP density functional and the 6-31G(d,p) basis set. A time
dependent DFT calculation was then performed using the same
functional and basis set, taking into account the first 30 electronic
transitions. The resulting electronic transition vectors (r, length
[cgs]) and their respective energy values (eV) were transformed
into simulated ECD spectra as reported earlier,[26] using a band
width at 1/e height of 0.1 eV for the Gaussian functions. The
resulting CD spectrum as shown in Figure 3 was obtained without
the necessity for intensity scaling or energy shift.
The enantiomers of rac-2 were separated by chiral HPLC. A total
amount of 400 mg rac-2 was separated. Colorless oil, yield
ꢀ ꢂꢄꢅ
99.7 % (tR = 25.28 min). Purity (HPLC equipment 2): 98.0 % (tR =
21.34 min).
170 mg (43 %). ꢁ
= –48.2 (c = 0.26, CH2Cl2). ee (chiral HPLC):
ꢃ
Spectroscopic data of rac-2, (R)-2 and (S)-2
C27H31NO2 (401.6 g/mol). Rf (Cy:EtOAc 2:1 + 1Vol.-% N,N-
dimethylethylamine): 0.44. Exact Mass (APCI): m/z = 402.2469
(calc. 402.2428 for C27H32NO2 [M+H]+). IR: ꢆꢇ [cm-1] = 3348 (O-H),
3059, 3024 (C-Harom.), 2931, 2858, 2820 (C-Haliph.), 1678, 1605,
1582, 1497 (C=Carom.), 1242 (C-OH). 1H NMR (400 MHz, CDCl3):
δ [ppm] = 1.56 – 1.63 (m, 2H, N-CH2-CH2-CH2-CH2-Ph), 1.63 –
1.70 (m, 2H, N-CH2-CH2-CH2-CH2-Ph), 2.45 – 2.56 (m, 1H, 4-H),
2.62 (d, 1H, 2-H), 2.62 – 2.67 (m, 5H, 5-H, N-CH2-CH2-CH2-CH2-
Ph), 3.02 (t, J = 9.2 Hz, 1H, 4-H), 3.19 (s, 1H, 2-H), 3.29 (t, J =
13.4 Hz, 1H, 5-H), 4.66 (s, 1H, 1-H), 5.03 (s, 2H, Ph-CH2-O), 6.72
– 6.75 (m, 2H, 6-H, 8-H), 7.12 (d, J = 7.9 Hz, 1H, 9-H), 7.16 – 7.21
(m, 3H, CHarom.), 7.27 – 7.34 (m, 3H, CHarom.), 7.36 – 7.43 (m, 4H,
CHarom.). 13C NMR (101 MHz, CDCl3): δ [ppm] = 26.4 (1C, N-CH2-
CH2-CH2-CH2-Ph), 29.1 (1C, N-CH2-CH2-CH2-CH2-Ph), 35.7 (1C,
N-CH2-CH2-CH2-CH2-Ph), 36.4 (1C, C-5), 55.9 (1C, C-4), 59.6
(1C, N-CH2-CH2-CH2-CH2-Ph), 60.7 (1C, C-2), 69.9 (1C, Ph-CH2-
O), 72.1 (1C, C-1), 111.3 (1C, C-8), 117.4 (1C, C-6), 125.8, 127.5,
127.6, 127.9, 128.3, 128.4, (10C, CHarom.), 129.6 (1C, C-9), 135.6
(1C, C-9a), 137.0 (1C, C-1benzyl), 141.0 (1C, C-1phenyl), 142.2 (1C,
C-5a), 158.2 (1C, C-7).
HPLC method for the determination of the enantiomeric
purity
Equipment: UV/vis detector: L-7455, pump: L-6200A Intelligent
Pump, manual injection, data acquisition: HSM-software
(LaChrom, Merck-Hitachi); column: Daicel Chiralpack® IA (250 x
4.6 mm, 5 μm particle size); flow rate: 1.0 mL/min; injection
volume: 10 µL; detection at λ = 210 nm; solvent: i-hexane:ethanol
9:1 + 1 % triethylamine, isocratic.
HPLC method for the preparative separation of the
enantiomers of 2
(R)-3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepin-
1,7-diol ((R)-3)
Equipment: UV/vis detector: L-7400, pump: L-7150, autosampler:
L-7200 (LaChrom, Merck-Hitachi); column: Daicel Chiralpack® IA
semi-prep (250 x 20 mm, 5 μm particle size); flow rate: 18.0
mL/min; injection volume: 400 µL; detection at λ = 210 nm;
solvent: i-hexane:ethanol 9:1 + 1 % triethylamine, isocratic.
(R)-2 (90.0 mg, 0.22 mmol, 1.0 eq.) was dissolved in abs. CH3OH
and Pd/C (27.0 mg, 10 %) was added. The reaction mixture was
stirred for 1 h under 3 bar H2 atmosphere at rt. Afterwards the
catalyst was filtered off over Celite®. The solvent was removed in
vacuo and the residue was purified via flash column
chromatography
(Cy:EtOAc
1:2
+
1Vol.-%
N,N-
Synthetic procedures
dimethylethylamine, d = 2 cm, V = 10 mL, h = 15 cm) to afford (R)-
ꢀ ꢂꢄꢅ
3. Yellow solid, mp 129 °C, yield 64.6 mg (94 %). ꢁ
= +38.4 (c
ꢃ
7-Benzyloxy-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-
benzazepin-1-ol (rac-2)
= 0.23, CH2Cl2). ee (chiral HPLC): 99.8 % (tR = 19.89 min). Purity
(HPLC equipment 1): 97.2 % (tR = 16.05 min).
Secondary amine 1 (150 mg, 0.56 mmol, 1.0 eq.), 1-chloro-4-
phenylbutane (0.14 mL, 0.84 mmol, 1.5 eq.), TBAI (206 mg,
0.56 mmol, 1.0 eq.), K2CO3 (616 mg, 4.46 mmol, 8.0 eq.) and
CH3CN (80 mL) were stirred and heated to reflux for 72 h. After
cooling to rt, the salt was filtered off and washed with CH3CN. The
solvent was removed under reduced pressure and the residue
was purified via flash column chromatography (Cy:EtOAc 2:1 +
1Vol.-% N,N-dimethylethylamine, d = 4 cm, V = 10 mL, h = 20 cm)
to afford rac-2. Colorless oil, yield 195 mg (0.25 mmol, 87 %).
(S)-3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepin-
1,7-diol ((S)-3)
The synthetic procedure for the synthesis of (R)-3 was adapted.
(S)-2 (105 mg, 0.26 mmol, 1.0 eq.) was transformed into (S)-3.
ꢀ ꢂꢄꢅ
Yellow solid, mp 129 °C, yield 78.6 mg (97 %). ꢁ
= –38.4 (c =
ꢃ
0.27, CH2Cl2). ee (chiral HPLC): 99.7 % (tR = 24.74 min). Purity
(HPLC equipment 1): 97.2 % (tR = 15.99 min).
Spectroscopic data of (R)-3 and (S)-3
(R)-7-Benzyloxy-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-
benzazepin-1-ol ((R)-2)
C20H25NO2 (311.4 g/mol). Rf (Cy:EtOAc 1:2 + 1Vol.-% N,N-
dimethylethylamine): 0.14. Exact Mass (APCI): m/z = 312.1950
(calc. 312.1958 for C20H26NO2 [M+H]+). IR: ꢆꢇ [cm-1] = 3255 (O-H),
3024 (C-Harom.), 2932, 2854, 2824 (C-Haliph.), 1701, 1609, 1585,
1493 (C=Carom.), 1234 (C-OH). 1H NMR (400 MHz, CDCl3): δ
[ppm] = 1.53 – 1.63 (m, 2H, N-CH2-CH2-CH2-CH2-Ph), 1.62 – 1.70
(m, 2H, N-CH2-CH2-CH2-CH2-Ph), 2.45 (t, J = 11.9 Hz, 1H, 4-H),
2.57 (d, J = 11.9 Hz, 1H, 2-H), 2.60 – 2.68 (m, 5H, N-CH2-CH2-
CH2-CH2-Ph, 5-H), 2.95 – 3.04 (m, 1H, 4-H), 3.11 – 3.29 (m, 2H,
The enantiomers of rac-2 were separated by chiral HPLC. A total
amount of 400 mg rac-2 was separated. Colorless oil, yield
ꢀ ꢂꢄꢅ
= +48.4 (c = 0.26, CH2Cl2). ee (chiral
ꢃ
170 mg (43 %).
ꢁ
HPLC): 99.8 % (tR = 21.45 min). Purity (HPLC equipment 2):
97.2 % (tR = 21.33 min).
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800214
ChemMedChem
FULL PAPER
2-H, 5-H), 4.61 (d, J = 6.7 Hz, 1H, 1-H), 6.52 – 6.60 (m, 2H, 6-H,
Configurational stability
Incubation in rat liver S9 fraction with NADPH - chiral HPLC
10 µL of a methanol stock solution (10 m ) of the respective
compound ((R)-2-4 and (S)-2-4) was added to an Eppendorf cup.
The solvent was removed under reduced pressure. Afterwards
50 µL NADPH solution (2 mg/mL in PBS), 114 µL PBS (0.1 M, pH
7.4) and 36 µL S9 fraction (rat liver, 18 mg/mL protein) were
added. The suspension was mixed vigorously and incubated
8-H), 7.00 (d, J = 7.9 Hz, 1H, 9-H), 7.15 – 7.21 (m, 3H, 2-Hphenyl
,
4-Hphenyl, 6-Hphenyl), 7.27 – 7.31 (m, 2H, 3-Hphenyl, 5-Hphenyl). The
signal for the OH proton is not seen in the spectrum. 13C NMR
(101 MHz, CDCl3): δ [ppm] = 26.2 (1C, N-CH2-CH2-CH2-CH2-Ph),
29.0 (1C, N-CH2-CH2-CH2-CH2-Ph), 35.7 (1C, N-CH2-CH2-CH2-
CH2-Ph), 35.9 (1C, C-5), 55.9 (1C, C-4), 59.5 (1C, N-CH2-CH2-
CH2-CH2-Ph), 60.41 (1C, C-2), 71.9 (1C, C-1), 112.5 (1C, C-8),
117.43 (1C, C-6), 125.8 (1C, C-4phenyl), 128.32, 128.35 (4C, C-
2phenyl, C-3phenyl, C-5phenyl, C-6phenyl), 129.8 (1C, C-9), 134.7 (1C, C-
9a), 141.0 (1C, C-5a), 142.1 (1C, C-1phenyl), 155.3 (1C, C-7).
M
(120 min,
800 rpm,
37 °C).
Afterwards
400 µL
of
acetonitril:methanol 1:1 was added to the suspension to stop the
incubation. The Eppendorf cups were cooled down to 0 °C for
10 min using a water/ice bath. The precipitated proteins were
separated via centrifugation (15 min, 16000 rpm, 4 °C) and the
supernatant was evaporated under reduced pressure. 100 µL
ethanol were added to the residue and the suspension was
centrifuged (5 min, 16000 rpm, 4 °C). Subsequently 50 µL of the
supernatant was analyzed via chiral HPLC.
(R)-7-Methoxy-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-
benzazepin-1-ol ((R)-4)
(R)-3 (40.0 mg, 0.13 mmol, 1.0 eq.) was dissolved in abs. N,N-
dimethylformamide (10 mL). Cs2CO3 (168 mg, 0.51 mmol,
4.0 eq.) was added and the mixture was stirred for 10 min at rt.
Afterwards CH3I (12.0 µL, 0.19 mmol, 1.5 eq.) was added and the
reaction was stirred for 16 h at rt. Subsequently the salt was
filtered off and the solvent was removed under reduced pressure.
The precipitated salt was filtered off and the solvent was removed
in vacuo. The residue was purified via flash column
Incubation in rat liver S9 fraction and NAD+ - LC/MS
50 µL NAD+ solution (2 mg/mL in PBS), 113 µL PBS (0.1
M
, pH
7.4), 36 µL S9 fraction (rat liver, 18 mg/mL protein) and 1 µL
DMSO stock solution (10 m ) of the respective compound ((R)-
M
chromatography
dimethylethylamine, d = 2 cm, V = 10 mL, h = 10 cm) to yield (R)-
4. Colorless solid, mp 58 °C, yield 23.6 mg (57 %). ꢁ
(c = 0.32, CH2Cl2). ee (chiral HPLC): 99.8 % (tR = 13.84 min).
Purity (HPLC equipment 1): 97.3 % (tR = 18.49 min).
(Cy:EtOAc
1:1
+
1Vol.-%
N,N-
2-4 and (S)-2-4) were added to an Eppendorf cup. The
suspension was mixed vigorously and incubated (90 min,
800 rpm, 37 °C). Afterwards 400 µL of acetonitril:methanol 1:1
was added to the suspension to stop the incubation. The
Eppendorf cups were cooled down to 0 °C for 10 min using a
water/ice bath. The precipitated proteins were separated via
centrifugation (15 min, 16000 rpm, 0 °C) and the supernatant was
analyzed via LC-MS. With the same procedure the empty value
(without stock solution) and the blank value (without NAD+) were
prepared. The MS was used in scan mode (m/z 80 - 1000) as well
as in SIM mode with the m/z of the respective benzylic ketones
(m/z 400 (2), m/z 310 (3), m/z 324 (4)).
ꢀ ꢂꢄꢅ
= +40.6
ꢃ
(S)-7-Methoxy-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-
benzazepin-1-ol ((S)-4)
The synthetic procedure for the synthesis of (R)-4 was adapted.
(S)-3 (43.0 mg, 0.14 mmol, 1.0 eq.) was transformed into (S)-4.
ꢀ ꢂꢄꢅ
Colorless solid, mp 58 °C, yield 19.6 mg (44 %). ꢁ
= –40.1 (c
ꢃ
= 0.26, CH2Cl2). ee (chiral HPLC): 99.7 % (tR = 16.30 min). Purity
(HPLC equipment 1): 99.0 % (tR = 18.48 min).
LC-MS method
Spectroscopic data of (R)-4 and (S)-4
Equipment: UPLC-UV/MS (Agilent Technologies): Degasser:
1260 HiP (G4225A), Pump: 1260 Bin Pump (G1212B),
Autosampler: 1260 HiP ALS (G1367E), Column Oven: 1290 TCC
(G1316C), MS-Detector: 6120 Quadrulpol LC/MS (G1978B). MS
Source: Multimode source; Precolumn: Zorbax Eclipse Plus-C18
(2.1 x 12.5 mm, 5 μm particle size); Main column: Zorbax SB-C18
(2.1 x 50 mm, 1.8 μm particle size); flow rate: 0.4 mL/min;
injection volume: 1 µL; solvents: A: bidist. H2O:CH3CN 95:5 with
0.1 Vol.-% formic acid, B: bidist. H2O:CH3CN 5:95 with 0.1 Vol.-%
formic acid; gradient elution (% A): 0 – 2.5 min: gradient from
100 % to 0 %; 2.5 - 3.5 min: 0 %; 29 - 31 min: 0 %; 3.5 – 4.0 min:
gradient from 0 % to 100 %; 4.0 – 8.0 min: 100 %.
C21H27NO2 (325.5 g/mol). Rf (Cy:EtOAc 1:2 + 1Vol.-% N,N-
dimethylethylamine): 0.27. Exact Mass (APCI): m/z = 326.2121
(calc. 326.2115 for C21H28NO2 [M+H]+). IR: ꢆꢇ [cm-1] = 2932, 2855,
2831 (C-Haliph.), 1609, 1582, 1497 (C=Carom.), 1250 (C-OH). 1H
NMR (400 MHz, CDCl3): δ [ppm] = 1.58 – 1.62 (m, 2H, N-CH2-
CH2-CH2-CH2-Ph), 1.65 – 1.70 (m, 2H, N-CH2-CH2-CH2-CH2-Ph),
2.45 – 2.55 (m, 1H, 4-H), 2.62 – 2.69 (m, 6H, 2-H, N-CH2-CH2-
CH2-CH2-Ph, 5-H), 2.98 – 3.07 (m, 1H, 4-H), 3.14 – 3.25 (m, 1H,
2-H), 3.29 (t, J = 13.5 Hz, 1H, 5-H), 3.78 (s, 3H, O-CH3), 4.62 –
4.71 (m, 1H, 1-H), 6.63 (d, J = 2.7 Hz, 1H, 6-H), 6.67 (dd, J =
2.7/8.1 Hz, 1H, 8-H), 7.13 (d, J = 8.1 Hz, 1H, 9-H), 7.15 – 7.22 (m,
3H, 2-Hphenyl, 4-Hphenyl, 6-Hphenyl), 7.24 – 7.33 (m, 2H, 3-Hphenyl, 5-
H
phenyl). The signal for the OH proton is not seen in the spectrum.
Receptor binding studies
13C NMR (101 MHz, CDCl3): δ [ppm] = 26.4 (1C, N-CH2-CH2-CH2-
CH2-Ph), 29.2 (1C, N-CH2-CH2-CH2-CH2-Ph), 35.8 (1C, N-CH2-
CH2-CH2-CH2-Ph), 36.5 (1C, C-5), 55.4 (1C, O-CH3), 56.1 (1C, C-
4), 59.7 (1C, N-CH2-CH2-CH2-CH2-Ph), 60.8 (1C, C-2), 72.0 (1C,
C-1), 110.5 (1C, C-8), 116.7 (1C, C-6), 126.0 (1C, C-4phenyl),
128.49 (1C, C-3phenyl, C-5phenyl), 128.52 (1C, C-2phenyl, C-6phenyl),
129.7 (1C, C-9), 135.4 (1C, C-9a), 141.0 (1C, C-5a), 142.3 (1C,
C-1phenyl), 159.1 (1C, C-7).
Materials
The recombinant L(tk-) cells stably expressing the GluN2B
receptor were obtained from Prof. Dr. Dieter Steinhilber (Frankfurt,
Germany). Homogenizers: Elvehjem Potter (B. Braun Biotech
International, Melsungen, Germany) and Soniprep® 150, MSE,
London, UK). Centrifuges: Cooling centrifuge model Rotina® 35R
(Hettich, Tuttlingen, Germany) and High-speed cooling centrifuge
model Sorvall® RC-5C plus (Thermo Fisher Scientific,
Langenselbold, Germany). Multiplates: standard 96 well
multiplates (Diagonal, Muenster, Germany). Shaker: self-made
device with adjustable temperature and tumbling speed (scientific
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800214
ChemMedChem
FULL PAPER
workshop of the institute). Harvester: MicroBeta® FilterMate 96
Harvester. Filter: Printed Filtermat Typ A and B. Scintillator:
Meltilex® (Typ A or B) solid state scintillator. Scintillation analyzer:
MicroBeta® Trilux (all Perkin Elmer LAS, Rodgau-Jügesheim,
Germany).
counting efficiency was 20 %. The IC50 values were calculated
with the program GraphPad Prism® 3.0 (GraphPad Software, San
Diego, CA, USA) by non-linear regression analysis. Subsequently,
the IC50 values were transformed into Ki values using the equation
of Cheng and Prusoff.3 The Ki values are given as mean value ±
SEM from three independent experiments.
Cell culture and preparation of membrane homogenates from
GluN2B cells
Performance of the GluN2B binding assays
Mouse L(tk-) cells stably transfected with the dexamethasone-
inducible eukaryotic expression vectors pMSG GluN1a, pMSG
GluN2B (1:5 ratio) were grown in Modified Earl’s Medium (MEM)
containing 10 % of standardized FCS (Biochrom AG, Berlin,
Germany). The expression of the NMDA receptor at the cell
surface was induced after the cell density of the adherent growing
cells had reached approximately 90 % of confluency. For the
induction, the original growth medium was replaced by growth
The competitive binding assay was performed with the
radioligand [3H]ifenprodil (60 Ci/mmol; BIOTREND, Cologne,
Germany). The thawed cell membrane preparation from the
transfected L(tk-) cells (about 20 μg protein) was incubated with
various concentrations of test compounds, 5 n
M
[3H]-ifenprodil,
and TRIS/EDTA-buffer (5 m TRIS/1 m EDTA, pH 7.5) at 37 °C.
M
M
The non-specific binding was determined with 10 μM unlabeled
[22]
ifenprodil. The Kd value of ifenprodil is 7.6 n .
M
medium containing 4 µ
M dexamethasone and 4 µM ketamine
(final concentration). After 24 h, the cells were rinsed with
phosphate buffered saline solution (PBS, Biochrom AG, Berlin,
Germany), harvested by mechanical detachment and pelleted
(10 min, 5000 rpm).
Electrophysiological activity evaluation
Xenopus laevis oocytes were commercially obtained from
EcoCyte Bioscience (Castrop-Rauxel, Germany). Each oocyte
was injected with 0.8 ng GluN1a and 0.8 ng GluN2B cRNA using
nanoliter injector 2000 (WPI, Berlin, Germany). CRNA was
generated by in vitro transcription with Ambion T7 mMessage
mMachine kit (Life Technologies, Darmstadt, Germany) from
linearized cDNA templates (wildtype rat, subloned into pSGEM
vector, linearized with restriction enzyme PacI). Injected oocytes
were stored for 5 to 6 days 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 and supplemented with 80 mg/L
theophylline, 63 mg/L penicillin, 40 mg/L streptomycin and 100
mg/L gentamycin. After incubation time for expression of
functional NMDA receptors oocytes were used for activity testing
by TEVC at room temperature using Turbo Tec 10CX amplifier
(NPI electronic, Tamm, Germany), NI USB 6221 DA/AD Interface
(National Instruments, Austin, USA) and GePulse Software (Dr.
Michael Pusch, Genova, Italy). For Measurements and
preparation of compound solutions Ba2+-Ringer containing 10
HEPES, 90 NaCl, 1 KCl, 1.5 BaCl2 (mmol/L) was used and pH
was adjusted with 1 M NaOH to 7.4. Agonist and compound
For the binding assay, the cell pellet was resuspended in PBS
solution and the number of cells was determined using a Scepter®
cell counter (MERCK Millipore, Darmstadt, Germany).
Subsequently, the cells were lysed by sonication (4 °C, 6 x 10 s
cycles with breaks of 10 s). The resulting cell fragments were
centrifuged with a high performance cool centrifuge (23500 x g,
4 °C). The supernatant was discarded and the pellet was
resuspended in a defined volume of PBS yielding cell fragments
of approximately 500,000 cells/mL. The suspension of membrane
homogenates was sonicated again (4 °C, 2 x 10 s cycles with a
break of 10 s) and stored at -80 °C.
General procedures for the binding assays
The test compound solutions were prepared by dissolving
approximately 10 µmol (usually 2-4 mg) of test compound in
DMSO so that a 10 mM stock solution was obtained. To obtain the
required test solutions for the assay, the DMSO stock solution
was diluted with the respective assay buffer. The filtermats were
presoaked in 0.5 % aqueous polyethylenimine solution for 2 h at
rt before use. All binding experiments were carried out in
duplicates in the 96 well multiplates. The concentrations given are
the final concentration in the assay. Generally, the assays were
performed by addition of 50 µL of the respective assay buffer,
50 µL of test compound solution in various concentrations (10-5,
10-6, 10-7, 10-8, 10-9 and 10-10 mol/L), 50 µL of the corresponding
radioligand solution and 50 µL of the respective receptor
preparation into each well of the multiplate (total volume 200 µL).
The receptor preparation was always added last. During the
solutions containing 10 µM glycine and 10 µM (S)-glutamate were
freshly prepared for each measurement. Measurements were
performed at a holding potential of -70 mV and recording pipettes
were backfilled with
3 M KCl. Data analysis of TEVC
measurements were performed with Ana (Dr. Michael Pusch,
Genova, Italy) and OriginPro 2016 (OriginLab Corporation,
Northampton, USA).
Cytotoxicity assay
10 mM Stock solutions in DMSO were diluted with Dulbecco
Modified Earl’s Medium (DMEM) to afford the test solutions. Cell
culture see part 4.8.2 “Cell culture and preparation of membrane
homogenates from GluN2B cells”.
incubation, the multiplates were shaken at
a speed of
500-600 rpm at the specified temperature. Unless otherwise
noted, the assays were terminated after 120 min by rapid filtration
using the harvester. During the filtration, each well was washed
five times with 300 µL of water. Subsequently, the filtermats were
dried at 95 °C. The solid scintillator was melted on the dried
filtermats at a temperature of 95 °C for 5 min. After solidifying of
the scintillator at rt, the trapped radioactivity in the filtermats was
measured with the scintillation analyzer. Each position on the
filtermat corresponding to one well of the multiplate was
measured for 5 min with the [3H]-counting protocol. The overall
The assay was modified based on previous publications.[14][15]
After careful removal of the medium the plate was blocked twice
with 200 µL DMEM containing 1 % BSA and rinsed once with
200 µL DMEM. Then the cells in the inner wells (B2-G11) were
incubated with 50 µL DMEM and 50 µL of the respective test
compound in 5 different concentrations (e.g. 4 x 10-5, 10-6, 10-7,
10-8, 10-9 mol/L) and 100 µL DMEM was added to the remaining
outer wells. Each concentration of the test compound was
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800214
ChemMedChem
FULL PAPER
incubated at least in triplicates for 30 min at 37 °C. Afterwards
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Glun2B
enantiomerically pure 3-benzazepines, chiral HPLC, CD-
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10.1002/cmdc.201800214
ChemMedChem
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Corresponding author
Bernhard Wünsch*
Institute of Pharmaceutical and Medicinal Chemistry, University of
Münster, Corrensstr. 48, D-48149 Münster, Germany
Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail:
wuensch@uni-muenster.de
Conflict of interest
There is no conflict of interest.
9
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10.1002/cmdc.201800214
ChemMedChem
FULL
PAPER
Table of Contents graphic
0,5
0.5
chiral HPLC
ee > 99.7 %
0,4
0.4
GluN2B affinity: Ki = 30 n
M
0,3
0.3
eudismic ratio: 25
inhibition of ion flux: IC50 = 61 n
cytoprotection: IC50 = 93 n
M
0,2
0.2
M
0,1
0.1
0,0
0.0
0
2
4
6
8
10
12
16
20
24
28
0
4
8
12 14 16 18 20 22 24 26 28
Retention time / min
Enantiomerically pure tetrahydro-3-benzazepin-1-ols were
obtained with high enantiomeric purity (ee > 99.2 %) by
separation of enantiomers using chiral HPLC. The absolute
configuration was determined by CD spectroscopy and DFT
calculations. (R)-configured enantiomers represent the eutomers.
(R)-3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-
diol shows high affinity towards GluN2B subunit containing NMDA
receptors, high inhibition of ion flux in two-electrode voltage clamp
experiments and high cytoprotective activity.
10
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