Ifenprodil Stereoisomers: Synthesis, Absolute Configuration, and Correlation with Biological Activity

Article abstract of DOI:10.1021/acs.jmedchem.0c01912

Ifenprodil (1) is a potent GluN2B-selective N-methyl-d-aspartate (NMDA) receptor antagonist that is used as a cerebral vasodilator and has been examined in clinical trials for the treatment of drug addiction, idiopathic pulmonary fibrosis, and COVID-19. To correlate biological data with configuration, all four ifenprodil stereoisomers were prepared by diastereoselective reduction and subsequent separation of enantiomers by chiral HPLC. The absolute configuration of ifenprodil stereoisomers was determined by X-ray crystal structure analysis of (1R,2S)-1a and (1S,2S)-1d. GluN2B affinity, ion channel inhibitory activity, and selectivity over α, σ, and 5-HT receptors were evaluated. (1R,2R)-Ifenprodil ((1R,2R)-1c) showed the highest affinity toward GluN2B-NMDA receptors (Ki = 5.8 nM) and high inhibition of ion flux in two-electrode voltage clamp experiments (IC50 = 223 nM). Whereas the configuration did not influence considerably the GluN2B-NMDA receptor binding, (1R)-configuration is crucial for elevated inhibitory activity. (1R,2R)-Configured ifenprodil (1R,2R)-1c exhibited high selectivity for GluN2B-NMDA receptors over adrenergic, serotonergic, and σ1 receptors.

Full text of DOI:10.1021/acs.jmedchem.0c01912

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Article
Ifenprodil Stereoisomers: Synthesis, Absolute Configuration, and
Correlation with Biological Activity
Elena Bechthold, Julian A. Schreiber, Kirstin Lehmkuhl, Bastian Frehland, Dirk Schepmann,
́
́
Freddy A. Bernal, Constantin Daniliuc, Ines Alvarez, Cristina Val Garcia, Thomas J. Schmidt,
̈
Guiscard Seebohm, and Bernhard Wunsch*
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ABSTRACT: Ifenprodil (1) is a potent GluN2B-selective N-methyl-D-aspartate (NMDA) receptor antagonist that is used as a
cerebral vasodilator and has been examined in clinical trials for the treatment of drug addiction, idiopathic pulmonary fibrosis, and
COVID-19. To correlate biological data with configuration, all four ifenprodil stereoisomers were prepared by diastereoselective
reduction and subsequent separation of enantiomers by chiral HPLC. The absolute configuration of ifenprodil stereoisomers was
determined by X-ray crystal structure analysis of (1R,2S)-1a and (1S,2S)-1d. GluN2B affinity, ion channel inhibitory activity, and
selectivity over α, σ, and 5-HT receptors were evaluated. (1R,2R)-Ifenprodil ((1R,2R)-1c) showed the highest affinity toward
GluN2B-NMDA receptors (Ki = 5.8 nM) and high inhibition of ion flux in two-electrode voltage clamp experiments (IC50 = 223
nM). Whereas the configuration did not influence considerably the GluN2B-NMDA receptor binding, (1R)-configuration is crucial
for elevated inhibitory activity. (1R,2R)-Configured ifenprodil (1R,2R)-1c exhibited high selectivity for GluN2B-NMDA receptors
over adrenergic, serotonergic, and σ1 receptors.
encoded by four different genes and show different expression
levels throughout the central nervous system.8 The residual
genes encode the remaining GluN3A and GluN3B subunits. In
general, a functional NMDA receptor contains two GluN1 and
two GluN2 subunits, and the GluN3 subunits are expressed
predominantly during the prenatal phase.9 The channel
properties and characteristics as well as their association with
different diseases are mainly driven by the expressed GluN2
subunits.10
NMDA receptor antagonists that selectively target the
GluN2B subunit are promising drug candidates for the
treatment of neurodegenerative diseases due to less side
effects compared to nonselective open-channel blockers such
as ketamine or phencyclidine.11
1. INTRODUCTION
The N-methyl-D-aspartate (NMDA) receptor plays a key role
in excitatory neurotransmission in the mammalian brain.1 It is
a hetero-tetrameric protein complex that belongs to the family
of ionotropic glutamate receptors.2 The NMDA receptor
contributes to long-term potentiation (LTP), a special form of
synaptic plasticity, and is involved in brain functions such as
learning and memory. Depolarization of the cell membrane
combined with simultaneous binding of (S)-glutamate and
glycine results in opening of the ion channel and influx of Ca2+
and Na+ ions as well as efflux of K+ ions.
Overstimulation of NMDA receptors leads to excessive
influx of Ca2+ ions and subsequent cell death via apoptosis.
This pathophysiological process termed excitotoxicity is
involved in a variety of acute (e.g., stroke) and chronic (e.g.,
Parkinson’s disease, Alzheimer’s disease, Huntington’s disease)
neurological disorders.3−5
Received: November 4, 2020
Published: January 11, 2021
Seven genes encode different subunits, which are able to
form the individual hetero-tetrameric ion channel receptor.
The GluN1 subunit is encoded by a single gene, but post-
translational modifications lead to eight splice variants termed
GluN1a-h.6,7 The four GluN2 subunits GluN2A−GluN2D are
https://dx.doi.org/10.1021/acs.jmedchem.0c01912
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Figure 1. Ifenprodil stereoisomers.
a
Scheme 1. Synthesis of Ifenprodil Stereoisomers
a
Reagents and reaction conditions: (a) 2-bromopropionyl bromide, TfOH, neat, 0 °C, 5 min, then rt, 1 h, 69%. (b) 4-Benzylpiperidine, NEt3,
CH2Cl2, rt, 24 h, 86%. (c) KBH4, HOAc, EtOH, 0 °C to rt, 16 h, 65%. (d) LiAlH4, THF, −78 °C to rt, 16 h, 87%. (e) chiral HPLC: Daicel
Chiralpak IA, 5 μm, 250 mm/20 mm; flow rate: 0−0.5 min 5 mL/min, 0.5−130 min 15 mL/min; injection volume: 400 μL (isohexane/iPrOH);
detection λ = 275 nm; eluent: isohexane/iPrOH/MeOH = 93/5/2 + 0.1% Et2NH. (f) chiral HPLC: Daicel Chiralpak IA, 5 μm, 250 mm/20 mm;
flow rate: 0−0.5 min 5 mL/min, 0.5−130 min 15 mL/min; injection volume: 400 μL (isohexane/MeOH); detection λ = 275 nm; eluent:
isohexane/MeOH = 97/3 + 0.1% Et2NH.
Ifenprodil possesses two centers of chirality leading to four
possible stereoisomers. The Fischer projection visualizes the
stereodescriptors erythro and threo. Stereoisomers with the
same or different stereodescriptors for the two centers of
chirality are termed like (l) and unlike (u), respectively.12
In the literature, 2-(4-benzylpiperidin-1-yl)-1-(4-
hydroxyphenyl)propan-1-ol is commonly named ifenprodil
(1, Figure 1). Ifenprodil was initially developed as an α1
receptor antagonist for the treatment of hypertension.13 Later,
it was found that 1 is a potent negative allosteric modulator
selectively inhibiting GluN2B subunit-containing NMDA
receptors.14−16 Commercially available ifenprodil (unlike
racemate, Cerocral®), which is used in Japan as cerebral
vasodilator, showed agonistic activity at σ1 receptors as
well.17,18 Clinical trials are currently evaluating the therapeutic
potential of 1 for the treatment of drug addiction, idiopathic
pulmonary fibrosis (IPF), and COVID-19.19−22 Furthermore,
1 showed positive effects in animal models of Alzheimer’s
disease and neuropathic pain.23−25 However, the selectivity of
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Figure 2. X-ray crystal structure of (1R,2S)-ifenprodil ((1R,2S)-1a). Thermal ellipsoids are shown at 30% probability. Isomer (1R,2S)-1a
crystallized in the chiral space group P212121. CCDC-2041093.
Figure 3. X- ray crystal structure of (1S,2S)-ifenprodil ((1S,2S)-1d). Thermal ellipsoids are shown at 30% probability. Isomer (1S,2S)-1d
crystallized in the chiral space group P21. CCDC-2041094.
1 over other receptors such as σ1, σ2, α1, 5-HT1A, and 5-HT2 is
rather low26−29 and depends on the configuration.27
reaction of α-bromoketone 3 with 4-benzylpiperidine afforded
racemic α-piperidinylketone 4 in 86% yield. Treatment of
ketone 4 with KBH4 in ethanol led stereoselectively to unlike-
ifenprodil (1ab).27 Alternatively, ketone 4 was reduced with
LiAlH4 in THF according to the procedure of Chenard et al.27
to provide a mixture of like-ifenprodil (1cd) and unlike-
ifenprodil (1ab) in the ratio 90:10. (Scheme 1). The relative
The stereodescriptors erythro and threo are widely used to
specify the ifenprodil diastereomers. However, the literature
often lacks information about absolute configuration, or given
information is misleading; e.g., the stereodescriptor erythro is
sometimes used for like-ifenprodil.30 The name ifenprodil
without specifying stereodescriptor is often used for racemic
threo-ifenprodil31−33 but also for the mixture of all four
stereoisomers.34 Biological data are generally correlated with
the specific optical rotation of ifenprodil stereoisomers, e.g.,
(+)-threo- or (−)-threo-ifenprodil, rather than providing the
absolute configuration.27,32,35
GluN2B receptor affinity, inhibitory activity, and affinity to
NMDA, non-NMDA, and related receptors are only reported
for mixtures of diastereomers or without information about the
absolute configuration of used ifenprodil stereoisomers.
Herein, we wish to report a systematic study on the relative
and absolute configuration of ifenprodil stereoisomers and the
unequivocal correlation of the different stereoisomers with
their pharmacological activity.
1
configuration was determined by H NMR spectroscopy and
comparison of the spectra with spectra reported in the
literature.27
The enantiomers of the racemic mixtures 1ab and 1cd were
separated by preparative chiral HPLC. The unlike-enantiomers
1a and 1b were separated by Daicel Chiralpak IA using
isohexane/iPrOH/MeOH = 93/5/2 with addition of 0.1%
Et2NH as eluent (see Figure S1). For the like-enantiomers 1c
and 1d, the same chiral stationary phase, but isohexane/
MeOH = 97/3 + 0.1% Et2NH as the mobile phase, was used
(see Figure S2). All four ifenprodil stereoisomers were isolated
with high enantiomeric excess (ee = 99.4−99.8%, see Figures
S1 and S2). During the preparative chiral HPLC, a small
amount of N-oxides of the enantiomerically pure ifenprodil-
stereoisomers 1a−d was formed. Thus, 1a−d were purified by
a second preparative HPLC using a RP-18 stationary phase
(see Figures S3 and S4).
2. RESULTS AND DISCUSSION
2.1. Chemistry. As reported in the literature,27 the
synthesis of ifenprodil stereoisomers started with acylation of
phenol (2) with racemic 2-bromopropionyl bromide in triflic
acid. After Fries rearrangement of the intermediate ester, 2-
bromopropiophenone 3 was isolated in 69% yield. The SN2
Previously, an X-ray crystal structure of racemic like-
ifenprodil (1cd) has been reported.36 Furthermore, the
absolute configuration of (1S,2S)-configured ifenprodil
((1S,2S)-1d) has been determined by chiral pool synthesis.37
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Table 1. Interaction of Ifenprodil Stereoisomers with NMDA Receptors Containing the GluN2B Subunit
GluN2B affinity
Ki SEM [nM]
TEVC activity at GluN2B-NMDA
a
b
c
compd
IC50 SE [nM]
A2 SE [%]
hill slope SE
eudismic ratio
(1R,2S)-1a
(1S,2R)-1b
(1R,2R)-1c
(1S,2S)-1d
14.4 3.3
7.8 0.2
5.8 1.3
200 28
698 46
218 16
525 32
91
84
93
91
3
2
2
2
1.1 0.2
1.3 0.1
1.3 0.1
1.97 0.18
3.5
d
2.5
13.5 2.8
a
b
Inhibition of (S) glutamate/glycine induced ion flux determined in TEVC experiments. A2 is the maximum inhibition of ion flux in TEVC
c
d
experiments at c(1a−d) = 30 μM. The eudismic ratio refers to the inhibition of ion flux in TEVC experiments. One-way ANOVA with post hoc
Newman Keuls multiple comparison test was used to evaluate the significance of mean differences for GluN2B affinity. The differences of GluN2B
affinity for (1R,2R)-1c vs (1S,2S)-1d and (1R,2R)-1c vs (1R,2S)-1a (p < 0.05) are significant, and all other differences are not significant.
In order to determine the absolute configuration of all
ifenprodil-isomers, an enantiomer of each diastereomer (1a
and 1d) was crystallized, and the structures were determined
by X-ray crystal structure analysis (Figures 2 and 3).
eudismic ratio of approximately 2. The GluN2B affinity of the
enantiomers (1R,2S)-1a and (1S,2R)-1b does not differ
significantly.
Next the inhibition of the (S)-glutamate and glycine
stimulated ion flux by the ifenprodil stereoisomers was
determined by two-electrode voltage clamp (TEVC) measure-
ments on GluN1a/GluN2B expressing Xenopus laevis oo-
cytes.39 Addition of 10 μM (S)-glutamate and 10 μM glycine
led to ion channel opening. Subsequent application of different
concentrations of enantiomerically pure ifenprodil stereo-
isomers resulted in reduced ion flow. The recorded dose
response curves allowed us to evaluate the activity of the test
compounds (Table 1 and Figure 4).
Two crystallographically independent molecules identified as
two conformers (named with suffixes A and B) were found in
the asymmetric unit (see Supporting Information). The
essential difference between the conformers A and B is the
orientation of the phenyl group of the terminal benzyl moiety:
in conformer A, a dihedral angle C11−C12−C15−C16 of
68.5(7)° and for conformer B a dihedral angle of 171.3(5)
were found. Only conformer A will be further discussed.
The structures of both ifenprodil isomers (1R,2S)-1a and
(1S,2S)-1d show a chair conformation for the piperidine ring.
Both substituents at the piperidine ring adopt the energetically
favored equatorial position, respectively. However, the
orientation of the substituents at the ethylene bridge in the
structure of both diastereomers differs considerably. Whereas
the dihedral angle between the OH and CH3 moieties (O1−
C1−C2−C3) of (1R,2S)-1a is 39.3(4)°, the dihedral angle of
the same substituents of the diastereomer (1S,2S)-1d is
176.3(5)°. Moreover, the relative orientation of the hydroxy-
phenyl ring and the piperidine ring is quite different. Regarding
the orientation of these moieties, the dihedral angle (C4−C1−
C2−N1) is 146.4(3)° for (1R,2S)-1a and 170.0(5)° for
(1S,2S)-1d.
Additionally, CD spectroscopy was used to characterize the
four stereoisomers, allowing us to confirm the absolute
configuration of (1S,2R)-1b and (1R,2R)-1c, based on their
respective Cotton effects (see Figures S9 and S10).
2.2. Pharmacological Evaluation. At first, the relation-
ship between the absolute configuration and the affinity toward
the NMDA receptor containing the GluN2B subunit should be
investigated by competitive receptor binding assays. In the
assay, racemic [3H]-unlike-ifenprodil (1ab) was used as a
radioligand. Membrane fragments of L(tk-)cells stably
expressing recombinant human GluN1a and GluN2B subunits
of the NMDA receptor were employed as receptor material.38
With Ki values between 5.8 nM and 14.4 nM, all four
ifenprodil stereoisomers bind with very high affinity to the
GluN2B subunit containing NMDA receptors (Table 1).
Nevertheless, the ifenprodil isomer (1R,2R)-1c (Ki = 5.8 nM)
shows significantly higher GluN2B affinity than its enantiomer
(1S,2S)-1d (Ki = 13.5 nM) and its diastereomer (1R,2S)-1a
(Ki = 14.4 nM). Thus, (1R,2R)-1c is the eutomer with a
Figure 4. Inhibition curves of (1R,2S)-1a (light blue), (1S,2R)-1b
(green), (1R,2R)-1c (dark blue), and (1S,2S)-1d (red). Each data
point represents the mean SEM of three independent experiments
(n = 3).
In TEVC experiments, isomers with (1R)-configuration, i.e.,
(R)-configuration at the chiral center with the benzylic OH
moiety, showed a stronger inhibitory activity than the
stereoisomers with (1S)-configuration (Table 1, Figure 4). A
similar result was obtained previously for other GluN2B
antagonists derived from ifenprodil.39,40 The increase of
activity associated with (1R)-configuration is not affected by
the configuration of the adjacent center of chirality. A
correlation between like- and unlike-configured ifenprodil
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Table 2. Affinity of Ifenprodil Stereoisomers Toward the GluN2B Subunit Containing NMDA Receptors, α Receptors, and σ
Receptors
a
Ki SEM [nM]
compd
GluN2B
α1A
α2A
σ1
σ2
(1R,2S)-1a
(1S,2R)-1b
(1R,2R)-1c
(1S,2S)-1d
14.4 3.3
7.8 0.16
5.8 1.3
13.5 2.8
140 27
27 7.2
370 150
530 150
1600 67
22
3
4.6 2.3
2300 590
5500 1600
6100 780
75 29
96 24
278 43
15
2
6.5 0.8
8.6 2.1
a
Ki values SEM values represent the mean of three experiments (n = 3).
stereoisomers and ion channel blocking activity could not be
observed. Altogether, the enantiomers (1R,2S)-1a and
(1R,2R)-1c represent the eutomers, respectively. However,
the eudismic ratios for both pairs of enantiomers are rather
low.
Table 3. Affinity of Ifenprodil Stereoisomers Toward
Different Types of 5-HT Receptors
a
Ki [nM]
5-
compd
5-HT1A HT2A 5-HT2B
5-HT2C
748
5-HT6
>1000
5-HT7
513
Since ifenprodil had originally been developed as an α1
receptor antagonist for the treatment of hypertension, the
affinity toward α1A and α2A receptors was examined in
radioligand receptor binding studies. In the α1A and α2A
assays, the radioligands [3H]prazosin and [3H]RX821002
were used, respectively.41,42 Isomer (1S,2R)-1b displays high
affinity toward the α1A receptor (Ki = 27 nM). Its enantiomer
(1R,2S)-1a has considerably lower α1A affinity resulting in
higher selectivity for the GluN2B binding site (Table 2). The
α1A receptor affinity of the like-configured enantiomers
(1R,2R)-1c and (1S,2S)-1d was even lower. Thus, (1R,2R)-
1c represents the ifenprodil stereoisomer with the highest
selectivity (64-fold) for GluN2B-NMDA receptors over α1A
receptors. All four ifenprodil stereoisomers exhibit very high
selectivity over the α2A receptor, since the Ki(α2A) values are
generally higher than 1 μM (Table 2).
As high σ receptor affinity has been reported for ifenprodil,
the σ1 and σ2 receptor affinities of the ifenprodil stereoisomers
were recorded as previously reported.43−45 In brief, the
ifenprodil stereoisomers competed with the radioligands
[3H](+)-pentazocine and [3H]di-o-tolylguanidine for σ1 and
σ2 receptors in guinea pig brain and rat liver membrane
preparations, respectively. Since [3H]-di-o-tolylguanidine also
binds to σ1 receptors, an excess of nonradioactive (+)-pent-az-
ocine was added in the σ2 assay to mask σ1 receptors.
Stereoisomer (1R,2S)-1a interacts with high affinity with σ1
receptors (Ki = 22 nM) and σ2 receptors (Ki = 4.6 nM). Thus,
it can be concluded that the unlike-configured ifenprodil
stereoisomer (1R,2S)-1a does not bind selectively to GluN2B
subunit-containing NMDA receptors (Table 2). Moreover, all
ifenprodil isomers bind with high affinity to the σ2 receptor.
However, ifenprodil stereoisomers (1R,2R)-1c and (1S,2S)-1d
are selective for the GluN2B binding site over the σ1 receptor.
It has been previously reported that ifenprodil binds to the
5-HT1A and 5-HT2 receptors.27 Drugs acting as 5-HT1A
receptor agonist (e.g., buspirone) or 5-HT2B receptor
antagonist (e.g., SB204741) have shown antihypertensive
effects.46 Moreover, several 5-HT receptor subtypes influence
NMDA receptor signaling.47−51 (1S,2R)-1b shows high affinity
for the 5-HT1A and the 5-HT2B receptor, whereas its
enantiomer (1R,2S)-1a only exhibits high-affinity binding for
the 5-HT2B receptor. (1R,2R)-1c and (1S,2S)-1d bind with
(1R,2S)-
1a
(1S,2R)-
1b
(1R,2R)-
1c
(1S,2S)-
1d
>1000
421
510
338
369
95
46
66
>10000
>10000
>10000
>1000
>10000
>10000
>10000
>1000
>1000
708
>1000
>10000
>10000
a
Ki values represent the mean of two experiments (n = 2 with
duplicate points).
moderate affinity to the 5-HT2A receptor (Ki = 338 nM and
369 nM, respectively) but can be considered to be selective for
the GluN2B receptor over all tested serotonin receptor
subtypes.
3. CONCLUSION
After determination of the absolute configuration by X-ray
structure analysis and CD spectroscopy, the GluN2B affinity,
selectivity, and inhibitory activity of all enantiomerically pure
ifenprodil stereoisomers were evaluated. In receptor binding
studies using [3H]ifenprodil as a competing radioligand, the
GluN2B affinity of the four stereoisomers is very similar (Ki =
5.8−14.4 nM). It can be concluded that the configuration of
the two centers of chirality does not influence considerably the
binding to the ifenprodil binding site of GluN2B subunit
containing NMDA receptors. However, NMDA receptor
inhibitors have been shown to not always present a strict
affinity−activity relationship.38
Stereoisomers with (1R)-configuration of the center of
chirality in benzylic position showed higher inhibitory activity
in TEVC experiments than the corresponding (1S)-configured
stereoisomers. Thus, it can be concluded that (R)-config-
uration at the benzylic center of chirality is important for
elevated inhibitory activity. On the contrary, the configuration
at the 2-position is less important for ion channel inhibition.
The ifenprodil stereoisomers only weakly interact with α2A
receptors. A considerable α1A receptor affinity was found only
for (1S,2R)-1b displaying a Ki value of 27 nM. However, this
stereoisomer inhibited only weakly the NMDA receptor
associated ion channel (IC50 = 698 nM) indicating a preference
for the α1A receptor over the GluN2B receptor. On the
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4.1.2. HPLC Method 1 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 Merck Hitachi, Darmstadt,
Germany); Equipment 2: Pump: LPG-3400SD, degasser: DG-1210,
autosampler: ACC-3000T, UV-detector: VWD-3400RS, interface:
DIONEX UltiMate 3000, data acquisition: Chromeleon 7 (equipment
and software from Thermo Fisher Scientific, Lauenstadt, Germany);
column: LiChrospher 60 RP-select B (5 μm), LiChroCART 250−4
mm cartridge; flow rate: 1.0 mL/min; injection volume: 5.0 μL;
detection at λ = 210 nm; solvents: A: demineralized water with 0.05%
(V/V) trifluoroacetic acid, B: CH3CN with 0.05% (V/V) trifluoro-
acetic acid; gradient elution (% A): 0−4 min: 90%; 4−29 min:
gradient from 90% to 0%; 29−31 min: 0%; 31−31.5 min: gradient
from 0% to 90%; 31.5−40 min: 90%. Unless otherwise mentioned, the
purity of all test compounds is greater than 95%.
4.1.3. Preparative HPLC Method 2A for Separation of the
Enantiomers (1R,2S)-1a and (1S,2R)-1b. Merck Hitachi equipment;
UV detector: L-7400; interface D-7000, autosampler: L-7200; pump:
L-7150A; data acquisition: HSM-software; guard column: Daicel
Chiralpak IA; column: 5 μm, 10 mm/20 mm, Daicel Chiralpak IA, 5
μm, 250 mm/20 mm; flow rate: 0−0.5 min 5 mL/min, 0.5−130 min
15 mL/min; injection: volume: 400 μL (isohexane/iPrOH);
detection λ = 275 nm; eluent: isohexane/iPrOH/MeOH = 93/5/2
+ 0.1% Et2NH.
contrary, the most active GluN2B antagonist (1R,2R)-1c
shows high selectivity over α1A (64-fold) and α2 receptors
(∼1000-fold). Thus, the GluN2B inhibitory activity and the α1
receptor affinity can be separated by variation of the
stereochemistry, an effect to be considered for the clinical
development of ifenprodil.
With exception of (1R,2S)-1a, the ifenprodil stereoisomers
exhibit 10−20-fold selectivity for GluN2B receptors over σ1
receptors. However, all stereoisomers possess low nanomolar
σ2 affinity. It can be concluded that the GluN2B:σ1 receptor
selectivity can be controlled by the stereochemistry, but the
affinity toward σ2 receptors cannot be eliminated or reduced by
changing the configuration. This interesting finding indicates
that a NMDA receptor inhibitor with high σ1 affinity is
available. Thus, (1R,2S)-1a might have a pharmacological
profile beneficial in the context of an antiflashback therapy of
post-traumatic stress disorder (PTSD).17,52 On the other hand,
a higher NMDA receptor selectivity without reduced σ1
receptor affinity may be beneficial in the context of an
antiapoptotic therapy counteracting excitotoxicity in stroke,
Parkinson’s disease, Alzheimer’s disease, and Huntington’s
disease.3−5
During testing of serotonin receptor affinity (5-HT1A, 5-
HT2A, 5-HT2B, 5-HT2C, 5-HT6, 5-HT7) moderate 5-HT1A and
5-HT2B affinity was detected only for both unlike-configured
enantiomers (1R,2S)-1a and (1S,2R)-1b. The like-configured
stereoisomers (1R,2R)-1c and (1S,2S)-1d showed only
negligible affinity toward the tested serotonin receptors. It
can be concluded that the 5-HT-affinity of the ifenprodil
stereoisomers is rather low, but appropriate configuration can
further increase the selectivity for the GluN2B receptor over
the 5-HT receptors.
Altogether, with respect to GluN2B affinity and inhibitory
activity, (1R,2R)-1c appears to be the most promising
ifenprodil stereoisomer. In addition to high GluN2B affinity
and inhibitory activity, (1R,2R)-1c shows high selectivity over
α1A, α2A, σ1, and six 5-HT receptors. Only the cross reactivity
at σ2 receptors could not be eliminated or reduced by changing
the stereochemistry. Thus, (1R,2R)-1c selectively targeting
GluN2B subunit-containing NMDA receptors could be
beneficial in antiapoptotic therapy resulting in fewer side
effects. Additionally, the NMDA receptor inhibitor (1R,2S)-1a
with high σ1 affinity could be beneficial in the treatment of
PTSD.
4.1.4. Preparative HPLC Method 2B for Separation of the
Enantiomers of (1R,2R)-1c and (1S,2S)-1d. Merck Hitachi equip-
ment; UV detector: L-7400; interface D-7000, autosampler: L-7200;
pump: L-7150A; data acquisition: HSM-software; guard column:
Daicel Chiralpak IA; column: 5 μm, 10 mm/20 mm, Daicel Chiralpak
IA, 5 μm, 250 mm/20 mm; flow rate: 0−0.5 min 5 mL/min, 0.5−130
min 15 mL/min; injection: volume: 400 μL (isohexane/MeOH);
detection λ = 275 nm; eluent: isohexane/MeOH = 97/3 + 0.1%
Et2NH.
4.1.5. Chiral HPLC Method 3A to Determine the Enantiomeric
Purity of (1R,2S)-1a and (1S,2R)-1b. Merck Hitachi equipment;
DAD detector: L-7455; interface D-7000, Rheodyne 7725i; pump: L-
6200A; data acquisition: HSM-software; Daicel Chiralpak IA, 5 μm,
10 mm/4 mm; column: Daicel Chiralpak IA, 5 μm, 250 mm/4.6 mm;
flow rate: 1.00 mL/min; injection: volume: 5.0 μL; detection λ = 275
nm; eluent: isohexane/iPrOH/MeOH = 93/5/2 + 0.1% Et2NH.
4.1.6. Chiral HPLC Method 3B to Determine the Enantiomeric
Purity of (1R,2R)-1c and (1S,2S)-1d. Merck Hitachi equipment; DAD
detector: L-7455; interface D-7000, Rheodyne 7725i; pump: L-
6200A; data acquisition: HSM-software; Daicel Chiralpak IA, 5 μm,
10 mm/4 mm; column: Daicel Chiralpak IA, 5 μm, 250 mm/4.6 mm;
flow rate: 1.00 mL/min; injection: volume: 5.0 μL; detection λ = 275
nm; eluent: isohexane/MeOH = 97/3 + 0.1% Et2NH.
4.1.7. Preparative HPLC Method 4A for Separation of the
Enantiomers (1R,2S)-1a and (1S,2R)-1b from Their N-Oxides.
Merck Hitachi equipment; UV detector: L-7400; interface D-7000;
autosampler: L-7200; pump: L-7100; degasser: L-7614; column:
Phenomenex Gemini C18 110 Å, 250−21.2 mm; 15−21.2 mm
security guard; flow rate: 9 mL/min; injection: volume: 100 μL;
detection λ = 210 nm; eluent: acetonitrile/H2O 7/3 + 0.1% ammonia.
4.1.8. Preparative HPLC Method 4B for Separation of the
Enantiomers of (1R,2R)-1c and (1S,2S)-1d from Their N-Oxides.
Merck Hitachi equipment; UV detector: L-7400; interface D-7000;
autosampler: L-7200; pump: L-7100; degasser: L-7614; column:
Phenomenex Gemini C18 110 Å, 250−21.2 mm; 15−21.2 mm
security guard; flow rate: 9 mL/min; injection: volume: 100 μL;
detection λ = 210 nm; eluent: acetonitrile/H2O 9/1 + 0.1% ammonia.
4.1.9. (1R,2S)- and (1S,2R)-2-(4-benzylpiperidin-1-yl)-1-(4-
hydroxyphenyl)propan-1-ol ((1R,2S)-1a and (1S,2R)-1b): Separa-
tion by Chiral HPLC. The two enantiomers were separated by chiral
preparative HPLC (HPLC method 2A). (1S,2R)-1b: tR = 20.8 min;
(1R,2S)-1a: tR = 24.2 min. The solvent was removed in vacuo,
respectively. The single enantiomers were separated from their N-
oxides by preparative HPLC method 4A. N-Oxide: tR = 3.1 min,
(1R,2S)-1a/(1S,2R)-1b: tR = 7.7 min. The solvent was removed in
vacuo, respectively.
In summary, we systematically correlated the absolute
configuration of all four ifenprodil stereoisomers with their
pharmacological properties. Two ifenprodil stereoisomers with
unique pharmacological profiles were identified, which may be
beneficial in different specific clinical contexts.
4. EXPERIMENTAL SECTION
4.1. Chemistry. 4.1.1. General Methods. Thin layer chromatog-
raphy (tlc): tlc silica gel 60 F254 on aluminum sheets (VWR). MS:
MicroTOFQII mass spectrometer (Bruker Daltonics, Bremen,
Germany); deviations of the found exact masses from the calculated
exact masses were 5 ppm or less; the data were analyzed with
DataAnalysis (Bruker Daltonics). NMR: NMR spectra were recorded
in deuterated solvents on Agilent DD2 400 and 600 MHz
spectrometers (Agilent, Santa Clara CA, USA); chemical shifts (δ)
are reported in parts per million (ppm) against the reference
substance tetramethylsilane and calculated using the solvent residual
peak of the undeuterated solvent; coupling constants are given with
0.5 Hz resolution; assignment of 1H and 13C NMR signals was
supported by 2-D NMR techniques where necessary.
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Figure 5. Representative current trace of GluN1−1a/GluN2B expressing oocytes activated with 10 μM (S)-glutamate and 10 μM glycine (black
bar) and treated subsequently with (1R,2R)-1c (red bar). Current traces of all stereoisomers 1a−d (c = 300 nM) are shown in the Supporting
Information (see Figure S3).
(1R,2S)-1a: HPLC (method 1): tR = 16.4 min, purity 99.2%.
HPLC (method 3A): tR = 24.2 min, ratio of enantiomers 99.8:0.2
(99.6% ee).
(1S,2R)-1b: HPLC (method 1): tR = 16.5 min, purity 98.0%.
HPLC (method 3A): tR = 20.8 min, ratio of enantiomers 99.6:0.4
(99.2% ee).
4.1.10. (1R,2R)- and (1S,2S)-2-(4-benzylpiperidin-1-yl)-1-(4-
hydroxyphenyl)propan-1-ol ((1R,2R)-1c and (1S,2S)-1d): Separa-
tion by Chiral HPLC. The two enantiomers were separated by chiral
preparative HPLC (HPLC method 2B) (1R,2R)-1c: tR = 39.0 min;
(1S,2S)-1d: tR = 42.9 min). The solvent was removed in vacuo,
respectively. The single enantiomers were separated from their N-
oxides by preparative HPLC method 4B. N-oxide: tR = 3.0 min;
(1R,2R)-1c/(1S,2S)-1d: tR = 11.8 min. The solvent was removed in
vacuo, respectively.
(1R,2R)-1c: HPLC (method 1): tR = 16.6 min, purity 97.6%.
HPLC (method 3B): tR = 39.0 min, ratio of enantiomers 99.4:0.6
(98.8% ee).
(1S,2S)-1d: HPLC (method 1): tR = 16.5 min, purity 98.3%.
HPLC (method 3B): tR = 42.9 min, ratio of enantiomers 99.5:0.5
(99.0% ee).
4.1.11. Synthetic Procedures. Synthetic procedures and parts of
the spectroscopic data have been previously reported by Chenard et
al.27 and can be found in the Supporting Information.
subunit (GluN1a/GluN2B) in vitro transcribed from linearized cDNA
templates with Ambion T7 mMessage mMachine kit (Life
Technologies, Darmstadt, Germany) using nanoliter injector 2000
(WPI, Berlin, Germany). Injected oocytes were stored for 5−6 days at
16 °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.
TEVC recordings were conducted at room temperature using a
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). The
oocytes were perfused with Ba2+-Ringer solution containing (mmol/
L): 10 HEPES, 90 NaCl, 1 KCl, 1.5 BaCl2 (pH was adjusted to 7.4
with 1 M NaOH) during measurements. The agonist solution
contained 10 μM each of glycine and (S)-glutamate, which was added
from 100 mM stock solutions of glycine and (S)-glutamate prior
experimentation. The test compound solutions were freshly prepared
from 10 mM DMSO stock solutions. The holding potential was set to
−70 mV, and recording pipettes backfilled with 3 M KCl had
resistances in the range of 0.5−1.5 MΩ.
4.3.4. Data Analysis and Statistics. The data were analyzed using
custom software Ana (Dr. Michael Pusch, Genova, Italy) and
OriginPro 2020. Inhibition was calculated by the following equation:
4.2. X-ray Crystallography. CCDC-2041093 for (1R,2S)-1a and
-2041094 for (1S,2S)-1d contain the supplementary crystallographic
data for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.
uk/data_request/cif.
4.3. Pharmacological Evaluation. 4.3.1. Radioligand Receptor
Binding Studies. The affinity toward the ifenprodil binding site of
GluN2B subunit containing NMDA receptors was recorded as
described in refs 38 and 53. Performance of the σ1 and σ2 assays is
reported in refs 43−45. The corresponding procedures are given in
the Supporting Information.
4.3.2. GluN2B Binding Assay. 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 of protein)
was incubated with various concentrations of test compounds, 5 nM
[3H]ifenprodil, and TRIS/EDTA-buffer (5 mM TRIS/1 mM EDTA,
pH 7.5) at 37 °C. The nonspecific binding was determined with 10
μM unlabeled ifenprodil. The Kd value of ifenprodil is 7.6 nM.38
4.3.3. Molecular Biology and TEVC Experiments. Molecular
biology and TEVC experiments were conducted as described before
by Schreiber et al.39,40 In brief, stage V defoliated Xenopus laevis
oocytes were obtained from EcoCyte Bioscience (Dortmund,
Germany), and oocytes were injected with 0.8 ng of cRNA of each
Ic − Ib
inhibition = 1 −
Ia − Ib
where Ic is the current in the presence of the compound solution, Ib is
the holding current before adding agonist solution and Ia is the
current after adding agonist solution.
Data Analysis was done using Origin Pro 2020 (OriginLab
Corporation, Northampton, MA, USA). Dose−response curves
were fitted to the logistic sigmoid equation:
A1 − A2
y =
+ A2
p
x
1 +
(x )
0
A1 and A2 represent the minimal and maximal inhibition of ion flux
by a compound. A1 was determined as A1 = 0%, whereas A2 was kept
flexible. x0 is the concentration at half-maximum inhibition, and x is
the examined concentration. p is the slope of the curve.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01912.
■
sı
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Article
Münster, D-48149 Münster, Germany; orcid.org/0000-
0003-2634-9705
Purity data of all test compounds, synthesis of the
ketones 3 and 4, chiral HPLC chromatograms of all four
enantiomerically pure ifenprodil stereoisomers, enantio-
meric purity data, experimental procedures of receptor
binding studies, X-ray crystallographic data of 1a and 1d,
1H and 13C NMR spectra, CD spectra and HPLC
chromatograms of all four ifenprodil stereoisomers
(PDF)
Guiscard Seebohm − GRK 2515, Chemical Biology of Ion
Channels (Chembion), Westfälische Wilhelms-Universität
Münster, D-48149 Münster, Germany; Cellular
Electrophysiology and Molecular Biology, Institute for
Genetics of Heart Diseases (IfGH), Department of
Cardiovascular Medicine, University Hospital Münster, D-
48149 Münster, Germany; Grupo de Investigación Biofarma.
Departamento de Farmacología, Farmacia y Tecnología
Farmacéutica. Centro de Investigación CIMUS, Universidad
de Santiago de Compostela, 15782 Santiago de Compostella,
Spain
Molecular formula strings (CSV)
Accession Codes
Authors will release the atomic coordinates and experimental
data upon article publication. PDB IDs have been provided in
Figures 2 and 3, in section 4.2. X-ray crystallography and in the
Supporting Information. (1R,2S)-1a: CCDC-2041093;
(1S,2S)-1d: CCDC-2041094.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01912
Notes
The authors declare no competing financial interest.
AUTHOR INFORMATION
■
ACKNOWLEDGMENTS
Corresponding Author
■
Bernhard Wunsch − GRK 2515, Chemical Biology of Ion
̈
This work was supported by the Research Training Group
“Chemical biology of ion channels (Chembion)” funded by the
Deutsche Forschungsgemeinschaft (DFG), which is gratefully
acknowledged. We thank Prof. Dr. Peter Gmeiner and Dr.
Channels (Chembion) and Institut für Pharmazeutische und
Medizinische Chemie, Westfälische Wilhelms-Universität
Münster, D-48149 Münster, Germany; orcid.org/0000-
0002-9030-8417; Phone: +49-251-8333311;
̈
Harald Hubner, Friedrich-Alexander-Universitat Erlangen, for
̈
recording the α receptor affinity. Furthermore, we thank Dr.
Email: wuensch@uni-muenster.de; Fax: +49-8332144
̈
Jens Kohler for his help with NMR spectroscopy and Luca
Blicker and Marvin Korff for critical proofreading.
Authors
Elena Bechthold − GRK 2515, Chemical Biology of Ion
Channels (Chembion) and Institut für Pharmazeutische und
Medizinische Chemie, Westfälische Wilhelms-Universität
Münster, D-48149 Münster, Germany
Julian A. Schreiber − Institut für Pharmazeutische und
Medizinische Chemie, Westfälische Wilhelms-Universität
Münster, D-48149 Münster, Germany; Cellular
Electrophysiology and Molecular Biology, Institute for
Genetics of Heart Diseases (IfGH), Department of
Cardiovascular Medicine, University Hospital Münster, D-
48149 Münster, Germany
Kirstin Lehmkuhl − Institut für Pharmazeutische und
Medizinische Chemie, Westfälische Wilhelms-Universität
Münster, D-48149 Münster, Germany
Bastian Frehland − Institut für Pharmazeutische und
Medizinische Chemie, Westfälische Wilhelms-Universität
Münster, D-48149 Münster, Germany
Dirk Schepmann − Institut für Pharmazeutische und
Medizinische Chemie, Westfälische Wilhelms-Universität
Münster, D-48149 Münster, Germany
Freddy A. Bernal − Institut für Pharmazeutische Biologie und
Phytochemie, Westfälische Wilhelms-Universität Münster, D-
48149 Münster, Germany
Constantin Daniliuc − Organisch-chemisches Institut,
Westfälische Wilhelms-Universität Münster, D-48149
Münster, Germany; orcid.org/0000-0002-6709-3673
ABBREVIATIONS USED
■
CD, circular dichroism; 5-HT, 5-hydroxytryptamine (=
serotonin); IPF, idiopathic pulmonary fibrosis; l, like; LTP,
long-term potentiation; NMDA, N-methyl-D-aspartate; PTSD,
post-traumatic stress disorder; TEVC, two electrode voltage
clamp; u, unlike
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