NMDA receptor affinities of 1,2-diphenylethylamine and 1-(1,2-diphenylethyl)piperidine enantiomers and of related compounds

Article abstract of DOI:10.1016/j.bmc.2009.03.025

We resolved 1,2-diphenylethylamine (DPEA) into its (S)- and (R)-enantiomer and used them as precursors for synthesis of (S)- and (R)-1-(1,2-diphenylethyl)piperidine, flexible homeomorphs of the NMDA channel blocker MK-801. We also describe the synthesis o

Full text of DOI:10.1016/j.bmc.2009.03.025

Bioorganic & Medicinal Chemistry 17 (2009) 3456–3462
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
NMDA receptor affinities of 1,2-diphenylethylamine and 1-(1,2-diphenyl-
ethyl)piperidine enantiomers and of related compounds
b
a
b
Michael L. Berger a, , Anna Schweifer , Patrick Rebernik , Friedrich Hammerschmidt
*
a Center for Brain Research, Medical University of Vienna, Spitalgasse 4, Vienna A-1090, Austria
b Institute of Organic Chemistry, University of Vienna, Währingerstraße 38, Vienna A-1090, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
We resolved 1,2-diphenylethylamine (DPEA) into its (S)- and (R)-enantiomer and used them as precur-
sors for synthesis of (S)- and (R)-1-(1,2-diphenylethyl)piperidine, flexible homeomorphs of the NMDA
channel blocker MK-801. We also describe the synthesis of the dicyclohexyl analogues of DPEA. These
and related compounds were tested as inhibitors of [3H]MK-801 binding to rat brain membranes. Stereo-
specificity ranged between factors of 0.5 and 50. Some blockers exhibited stereospecific sensitivity to the
modulator spermine. Our results may help to elucidate in more detail the NMDA channel
pharmacophore.
Received 13 February 2009
Accepted 13 March 2009
Available online 19 March 2009
Keywords:
NMDA receptor
[3H]MK-801 binding
1,2-Diphenylethylamine
1-(1,2-Diphenylethyl)piperidine
Spermine
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
The polyamines spermine and spermidine increase frequency
and burst length of NMDA-induced currents in rat hippocampal
The N-methyl-
D
-aspartate (NMDA) receptor is the most abun-
neurons, by relieve from a partial block by protons, even at physi-
ological pH.6 As a consequence, the channel is more readily acces-
sible not only for the cations constituting its current, but also for
the channel ligand MK-801 (as a secondary amine positively
charged at physiological pH), thus increasing the affinity of the
radioligand [3H]MK-801. Channel blockers that act similarly to
MK-801 profit from increased channel accessibility to a similar ex-
tent.7 Here we demonstrate that the affinity of some of the studied
dant receptor for the principal excitatory neurotransmitter gluta-
mate and consists of 4 protein subunits surrounding a central
channel, with various binding sites at the extracellular domain
regulating permeability to Na+ and Ca2+ 1
Its involvement in
.
learning and memory, but also in pathologic conditions such as
stroke, epilepsy and neurodegenerative diseases makes this recep-
tor an attractive target of drug development.2 Analogues of the
agonist glutamate itself suffer from their poor ability to cross the
blood brain barrier, because they have to mimic an acidic amino
acid with 3 charged functional groups. A direct attack of the ion
channel by more lipophilic channel blockers appears more promis-
ing, however the best known representatives of this class are
Ketamine (1b) and Phencyclidine (2), two widely abused com-
pounds inducing psychotic side effects, and the experimental high
affinity channel ligand (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-
dibenzo[a,d]cyclohepten-5,10-imine, (+)-MK-801 [(+)-3].3 Exam-
ples for moderate potency NMDA channel blockers tolerated with
much less psychotic side effects are 3,5-dimethyladamantan-1-
amine (Memantine)4 and Dextromethorphan [(+)-4b]. To learn
more about SARs at this channel binding site, we resolved racemic
1,2-diphenylethylamine (DPEA, 5a) and used its enantiomers to
prepare (R)- and (S)-1-(1,2-diphenylethyl)piperidine (DEP, 6).5 Un-
til now, both 5a and 6 had only been studied as racemic mixtures.5
R
NH
H
N
N
10
H
O
B
Cl
5
A
A
A
(
S
)-1a (R = H)
)-1b (R = Me)
2
(+)-3
(S
R
N
NH2
R
N
H
13
14
H
B
B
B
H
O
9
A
A
(
(
S
)-5a (R = H)
4a (R = H)
(+)-4b (R = Me)
(S)-6
S)-5b (R = Me)
* Corresponding author. Tel.: +43 1 4277 62892; fax: +43 1 4277 62899.
E-mail address: michael.berger@meduniwien.ac.at (M.L. Berger).
Chart 1. Phenyl rings in NMDA receptor channel blockers.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.025

M. L. Berger et al. / Bioorg. Med. Chem. 17 (2009) 3456–3462
3457
F3C OMe
O
OH
Br
2
NH2
N
a
OH
NH2
O
+
1'
Br
7
(R)-13
(S)-5a or (
R
)-5a
(S)-6 or (R)-6
(2R,1'S)-10
a
Scheme 1. Reagents and conditions: (a) K2CO3, CH3CN; 64%/54%.
d
g
S
stereoisomers was not influenced in the same way by spermine
than that of the radioligand, suggesting that they acted in a way
different from MK-801.
O
O
OH
N3
b
c
2. Results
(S)-9
8
(R)-11
2.1. Chemistry
e
Commercially available ( )-5a was resolved by the method of
Shinohara et al.,8 which was modified, into (+)- and (ꢀ)-enantio-
mers (S)- and (R)-5a, respectively. The piperidine analogues 6 were
prepared from the respective 5a enantiomers with 98% ee by dou-
ble alkylation9 with 1,5-dibromopentane (Scheme 1). Under opti-
mised conditions (3 equiv of alkylating reagent, 6 equiv of K2CO3)
in dry acetonitrile at ambient temperature for 3 days, the yields
for the (+)-isomer (S)-6 and the (ꢀ)-isomer (R)-6 were 64% and
54%, respectively. The oily bases were converted to the hydrochlo-
rides, which crystallised as monohydrates (the racemic hydrochlo-
ride has been described, without crystal water).10
NO2
O
N3
OH
O
f
c
(R)-9
12
(S)-11
g
F3C OMe
d
O
2
Synthesis of the dicyclohexyl analogues 13 was started from
(R,R)-1,2-dicyclohexylethane-1,2-diol (7, Scheme 2). The diol was
converted to the thiocarbonate 8 in high yield (89%) using thiocar-
bonyl diimidazole in refluxing toluene.11 The crystalline product
was deoxygenated to (S)-9 with 60% yield using Bu3SnH/AIBN in
refluxing toluene. With tris(trimethylsilyl)silane instead of the
tin hydride, yield dropped to 18%.12 To determine the ee, a sample
was converted to the (R)-Mosher ester (2R,10S)-10;13 its 19F NMR
spectrum indicated ee >98%. The alcohol (S)-9 was transformed
into the azide (R)-11 smoothly using the Mitsunobu reaction
(SN2 mechanism),14 but subsequent reduction to the amine was
not trivial. Neither catalytic hydrogenation (Pd–C, H2 in EtOH con-
taining some HCl, 3 atm) nor hydrolysis of the iminophosphorane,
which formed very slowly and incompletely from the azide with
triphenylphosphine in a Staudinger reaction, gave the desired
amine. However, after a moderately successful attempt with Li-
BEt3H (yield only 40%), reduction15 with LiAlH4 gave the amine
(R)-13 as an oil with 88% yield, which was finally converted to
the crystalline hydrochloride.
O
NH2
1'
(S)-13
(2R,1'R)-10
Scheme 2. Reagents and conditions: (a) thiocarbonyl diimidazole, toluene, reflux,
89%; (b) Bu3SnH, AIBN, toluene, reflux, 60%; (c) Ph3P, DIAD, HN3, toluene, rt, 87%; (d)
LiAlH4, 88%; (e) 4-nitrobenzoic acid, DIAD, Ph3P, toluene, 45%; (f) 0.2 M MeONa,
MeOH, 87%; (g) (S)-MTPA-Cl, CH2Cl2, pyridine; in grey: synthesised for analytic
reasons only.
distinguished Ph rings A and B according to their distance from
the amino group (Chart 1). Although not all rings A (respectively
B) can be expected to interact with the same amino acid residue(s)
of the receptor protein, this approach allows the exploration of a
common pharmacophore starting from simpler precursors. Already
benzylamine (14) and phenylethylamine (15) inhibited [3H]MK-
801 binding with Ki 1.45 and 0.75 mM, respectively (Table 1).
Replacing an aliphatic hydrogen atom of 14 by a methyl group re-
sults in (S)- and (R)-1-phenylethylamine (16), and similar substitu-
tion of 15 in (R)- and (S)-Amphetamine (17). This modification
increased the potency of 14 by a factor of two, without any stereo-
selectivity. In the case of 15, an increase in potency (also by a factor
For synthesis of (R)-9, the configuration of alcohol (S)-9 was in-
verted by esterification with Ph3P, 4-nitrobenzoic acid and diiso-
propyl azodicarboxylate (DIAD) (Mitsunobu reaction) (Scheme 2,
e).14 Although the starting material was consumed, the yield of
the 4-nitrobenzoate 12 was low (45%) and could not be improved.
Elimination with formation of isomeric alkenes predominated, be-
cause the stereogenic centre is encumbered, which retarded nucle-
ophilic substitution. The yield dropped to 22%, when the less acidic
benzoic acid was used instead of 4-nitrobenzoic acid. Transesteri-
fication of 12 with MeONa in MeOH furnished the alcohol (R)-9 [ee
of two) was only observed for (S)-17 (D-Amphetamine), not for (R)-
17 (as already described).16 Incorporation of the methyl group of
Amphetamine into a cyclopropyl ring leads to the antidepressant
and MAO inhibitor Tranylcypromine. We obtained both trans iso-
mers (three times more potent as MAO inhibitors than the cis iso-
mers) (1R,2S)-(ꢀ)- and (1S,2R)-(+)-2-phenylcyclopropylamine
(18); (+)-18 is more potent than (ꢀ)-18 as MAO inhibitor by a fac-
tor of 4.17 These isomers of Tranylcypromine were slightly more
potent than the respective isomers of Amphetamine, and also their
inhibition was stereoselective (Table 1). On the other hand, intro-
duction of a methoxycarbonyl instead of a methyl group into 14
and 15, giving (R)- and (S)-phenylglycine methylesters (19), and
19
>96%; by F NMR spectroscopy of (R)-Mosher ester (2R,10R)-10],
which was transformed into amine (S)-13 and its hydrochloride
as described above.
2.2. Pharmacology
In search for common features of NMDA channel blockers as
diverse as MK-801, Phencyclidine and Dextromethorphan, we

3458
M. L. Berger et al. / Bioorg. Med. Chem. 17 (2009) 3456–3462
Table 1
Inhibition of [3H]MK-801 binding by structural analogues of benzylamine (14) and phenylethylamine (15)
R2
R2
NH2
H
H2N
H
NH2
H
H2N
H
NH2
H
H2N
H
X
X
R
R
R1
R1 R2
(S)-5a CH Ph
(+)-5c CH Ph NH2
(+)-5d CH Ph OH
R1
X
X
R1
R2
H
H
(R)-5a CH Ph
(-)-5c CH Ph NH2
(-)-5d CH Ph OH
(S)-13
(R)-13
R
H
R
H
H
14
NH2
H2N
(S)-5e
N
Ph
H
H
H
(R)-5e
N
Ph
H
(S)-16 Me
15 CH
(R)-16 Me
(S)-19 CO2Me
H
H
(R)-19 CO2Me (R)-17 CH Me
H
(S)-17 CH Me
(R)-20 CH CO2Me H
H
21 Ph
(S)-20 CH CO2Me H
22 CH
H
Ph
(-)-18
(+)-18
Nr
Config
Ki (lM)
n
Ki (spm)/Ki
n
Stereoselectivity
n
Nr
Config
Ki (lM)
n
Ki (spm)/Ki
n
(S)-1a
(S)-1b
2
(+)-3
4a
(+)-4b
(S)-5a
(S)-5b
(+)-5c
(+)-5d
(S)-5e
(S)-6
(S)-13
14
S
S
4.18, 4.46
0.96 0.21
0.27 0.01
2
5
3
2.91, 2.82
2.70 0.09**
0.78 0.06
0.53, 0.51
0.85, 1.15
0.70 0.21
2.59 0.43**
2.24 0.96*
2.07 0.60*
2.13 0.77*
1.50 0.49
0.83, 0.81
0.81 0.23
1.09, 1.17
1.45, 1.19
1.24, 0.86
1.61 0.20
1.26 0.33
1.07 0.15
1.94 0.43*
1.22 0.14
1.09, 1.21
2
4
3
2
2
4
3
3
3
3
3
2
3
2
2
2
3
3
4
4
3
2
14.4, 12.3
5.6 0.5
2
4
(R)-1a
(R)-1b
R
R
60, 55
5.68 1.52
2
4
1.29, 1.57
1.02 0.10
2
4
10R
9S
9S
S
0.009, 0.012
1.87, 1.70
4.54 1.42
0.70 0.21
0.79 0.14
4.12 1.75
15.7 6.5
14.0 5.8
0.13, 0.12
271 31
1830, 1063
753 152
659, 622
625 158
544 164
1372 921
701 452
64 17
2
2
5
5
4
3
4
3
2
3
2
3
2
4
3
4
5
3
2
5.32, 4.81
2
(ꢀ)-3
10S
9R
9R
R
0.043 0.011
n.a.
3.36 0.50
25.1 3.4
8.08 0.47
4
0.54 0.29
4
0.65 0.28
42.6 15.8
9.6 0.7
15.4 10.7
11.4 1.42
6.3 1.24
40.3, 57.4
0.49, 0.79
3
4
3
3
3
3
2
2
(ꢀ)-4b
(R)-5a
(R)-5b
(ꢀ)-5c
(ꢀ)-5d
(R)-5e
(R)-6
3
4
3
3
3
3
2
3
0.66 0.19
1.93, 1.45
0.58, 0.37
1.61 0.28
1.25, 1.10
0.85 0.13
1.49, 0.99
0.47, 1.15
3
2
2
3
2
3
2
2
S
R
1R
2R
S
S
S
1S
2S
R
R
R
51
6
142
2
84 15
5.25, 7.02
138 51
(R)-13
15
(S)-16
(R)-17
(ꢀ)-18
(R)-19
(S)-20
21
S
R
1R
R
S
1.00, 1.08
0.58, 0.61
0.49 0.08
1.66 0.76
1.23 0.13
2
2
3
4
5
(R)-16
(S)-17
(+)-18
(S)-19
(R)-20
R
S
1S
S
R
659, 674
300 65
269 98
1978 846
855 517
2
4
4
4
5
1.28, 0.74
1.47 0.36
1.78 0.67
1.25 0.12
1.48 0.35
2
4
4
4
4
22
152, 145
Compounds isosterica to (S)-5a on the left, isosteric to (R)-5a on the right side of the table; Ki SD (or both values if n = 2). See also Chart 1 for structures.
Config, configuration at the carbon atom bearing the amino group; n, number of experiments; Ki (spm), Ki in presence of 100 M spermine; stereoselectivity is the mean of Ki
l
ratios obtained in individual experiments (not necessarily identical to the ratio of the mean Ki values); n.a., the (R)-isomer was not available.
a
Depending on the priority of the carbon atoms surrounding the chiral centre(s), compounds sharing the same steric configuration do not necessarily share the same
stereochemical descriptor.
*
Significantly higher than values ꢁ0.85.
**
Significantly higher than values ꢁ1.26 (ANOVA, Newman-Keuls).
(S)- and (R)-phenylalanine methylesters (20), respectively, was
without influence.
5a for a pyridinyl ring resulted in (S)-(+)- and (R)-(ꢀ)-1-phenyl-
2-(pyridin-2-yl)ethylamines (5e). (S)-5e is an experimental drug
known as AR-R15896AR, with blocking properties similar to those
of Ketamine and Memantine.19 Potency was reduced by this Ph/
pyridinyl exchange by a factor of 20, and also stereoselectivity
was strongly reduced (from 43 to 6; Table 1). Incorporation of
the nitrogen atom of the primary amino group of (S)-5a into a
piperidine ring resulted in the highly potent NMDA receptor chan-
nel blocker (S)-6, five times more potent than (S)-5a, with compa-
rable stereoselectivity (factor 49). Finally, changing in 5a Ph to
cyclohexyl resulted in (S)- and (R)-1,2-dicyclohexylethylamines
(13), with potency reduced by a factor of 385 and stereoselectivity
totally lost.
The potency of 14 was increased more than 20-fold by introduc-
tion of a second Ph substituent (aminodiphenylmethane, 21). Into
15, a second Ph residue can be introduced in three different ways:
Introduction in vicinity to the first Ph group resulted in the (achi-
ral) 2,2-diphenylethylamine (22) 5 times more potent than 2. In
vicinity to the primary amino group, two stereoisomers are ob-
tained, (S)- and (R)-DPEA (5a), the first one 1000 times, the second
one 30 times more potent than 15. (S)-(+)- and (R)-(ꢀ)-1,2-Diphe-
nyl-2-propylamines (5b) are 1-methyl derivatives of 5a and des-
glycine metabolites of ( )-Remacemide. They act as NMDA channel
blockers in whole cell voltage-clamp recordings from cultured rat
hippocampal neurons and in binding studies with [3H]MK-801 in
rat forebrain membranes.18 (S)-5b was equipotent to (S)-5a, but
the high stereospecificity of the pair (S)-5a/(R)-5a (stereo-factor
43) was not reproduced in the pair (S)-5b/(R)-5b (factor 10; Table 1).
Potency was reduced by introduction of an amino or a hydroxyl
group at position 2: (R,R)-(+)-1,2-diphenylethylenediamine [(+)-
5c] was six times weaker, and (1S,2R)-(+)-2-amino-1,2-diphenyl-
ethanol [(+)-5d] was 22 times weaker than (S)-5a; their (S,S)-(ꢀ)-
and (1R,2S)-(ꢀ)-isomers (ꢀ)-5c and (ꢀ)-5d revealed stereo-factors
of 15 and 11, respectively; thus, in spite of reduced potency, ste-
reospecificity was not reduced further. Exchange of Ph ring B in
Inhibition of [3H]MK-801 binding by the stereoisomers of Keta-
mine (1b) and its main metabolite Norketamine (1a) has been de-
scribed.20 In agreement with the published data, we found (S)-1b
moderately more potent than the (R)-isomer (factor 5.6), and its
demethylated variant (S)-1a by a factor of 4.5 less potent than
(S)-1b, with somewhat higher stereospecificity (factor 13). The
potency of (S)-1b was similar to that of (S)-5a. Phencyclidine (2,
an achiral compound) with its amino group incorporated into a
piperidine ring (as in DEP) was 3.5 times more potent than
N-methylated (S)-1b, and by a factor of 16 in comparison with
the primary amine (S)-1a (Table 1). Other differences (carbonyl

M. L. Berger et al. / Bioorg. Med. Chem. 17 (2009) 3456–3462
3459
group, Cl substituent) may have contributed to this change.
(ꢀ)-MK-801 [(ꢀ)-3], the enantiomer of (+)-3, was five times less
potent than (+)-3, in agreement with the literature.3 In strict terms,
in Table 1 the more potent isomer (+)-3 should be listed on the
right side, since the orientation of its amino group appears isosteric
to (R)-5a rather than to (S)-5a (see Chart 1). Other structural fea-
tures than amino group orientation might be responsible for the
(moderate) stereoselectivity of MK-801. Finally, the NMDA channel
blocker Dextromethorphan [(+)-4b] blocked [3H]MK-801 binding
with slightly weaker potency than (S)-5a and (S)-1b; here, demeth-
ylation [to (+)-4a] resulted in a moderate increase in potency [not
in a decrease as after demethylation of (S)-1b]. Levomethorphan
[(ꢀ)-4b] yielded values similar to Dextromethorphan (although
100 times more potent at opiate receptors).21
Sensitivity of NMDA channels to inhibition by MK-801 is in-
creased eight times by splicing in ‘exon 5’ into the N-terminus of
subunit NR1, but sensitivity to inhibition by Ketamine only two
times.32 Exon 5 is rich in positively charged amino acid residues
and acts as an intrinsic positive modulator via the polyamine reg-
ulatory site. Most NMDA receptors in adult tissue (as used in our
study) do not contain this exon,33 but respond to spermine in a
similar way. Here we show that accessibility of the channel is
changed by spermine to the disadvantage of (S)-Ketamine as com-
pared to MK-801, in agreement with data obtained with exon 5
containing receptors. The mechanism by which polycationic exon
5 and spermine increase opening frequency and burst length of
NMDA channels is still not fully understood. Although most
researchers agree that this mechanism includes the relieve from
a block by ambient H3O+ ions (‘proton block’), it is not exactly clear
how this is accomplished. Site-directed mutagenesis studies point
to the extracellular N-terminal domain of the NR1 subunit (har-
bouring the splice site for exon 5) as the major mediator of this
stimulatory effect. However, a high number of amino acid residues
lining the inner wall of the channel appear to be involved as
well,34,35 either as a direct target or mediating proton block down
to the narrow constriction of the channel. Detailed SAR studies
with several lipophilic amines have shown that spermine sensitivity
of inhibition of [3H]MK-801 binding depended on a primary amino
group36—with the notable exception of the secondary amine (S)-Ket-
amine. It may, thus, be speculated that spermine and certain channel
blocking (mostly) primary amines share common targets at the in-
ner channel wall.
Inhibition of [3H]MK-801 binding by these compounds was esti-
mated also in the presence of the polyamine modulator spermine.
As a concomitant of increased accessibility of the NMDA receptor
channel, the affinity of [3H]MK-801 to rat brain membranes was
strengthened in the presence of 100
lM
spermine from
14.3 2.6 nM (KD) to 4.70 1.24 nM (mean SD, n = 12). The Ki of
several test compounds was also reduced by spermine [2, (R)-5b,
(+)-4b, (ꢀ)-4b, ratio Ki (spm)/Ki <1, Table 1]. On the other side,
many Ki values were considerably increased by spermine, espe-
cially those of (S)-1a (by a factor of 2.87), (S)-1b (2.70), and
(S)-5a (2.59; Table 1). Similar results had been obtained for
Memantine22 and for tryptamine.16 These influences of spermine
were often stereoselective: for example, the potency of (S)-5b
was decreased, but that of (R)-5b was increased by spermine,
and inhibitions by (R)-1a and (R)-1b were not influenced by
spermine, in contrast to the results obtained with their respective
(S)-enantiomers (Table 1).
Although (S)-Ketamine and Memantine have similar effects on
[3H]MK-801 binding (Ki, sensitivity to spermine), their clinical
properties differ: the former is narcotic and psychotomimetic,
the latter neuroprotective without severe side-effects. It has been
suggested that the block by Memantine is more easily reversible
because it binds to a second site located at a higher level and es-
capes more readily than Ketamine from being trapped in the chan-
nel.37 The special binding mode of the adamantane derivative
Memantine might be related to its non-aromatic nature. Fluoro-
substitution of aromatic residues seems to mitigate psychotic side
effects as exemplified by 3,3-bis(3-fluorophenyl)-propylamine,38
3. Discussion
Stereoselectivity is a characteristic feature of drug interaction
with a target in a geometrically restricted micro-environment.
The high stereo-factors (>30) of DPEA (5a) and DEP (6) in blocking
[3H]MK-801 binding as demonstrated in this article strongly sug-
gest the interaction with such a restricted target. Similar enantio-
selectivity has been reported for several 1-aryl-1,2,3,4-
tetrahydroisoquinoline derivatives.23 NMR spectroscopy of (S)-
5a ꢂ HCl in D2O has shown that the Ph residues preferred an anti-
periplanar conformation pointing into opposite directions, but also
that the central ethane linkage rotated freely.24 The absolute con-
figurations of (S)-Ketamine [(S)-1b]25 and of its metabolite (S)-
Norketamine [(S]-1a]26 have been revealed by X-ray analysis.
However, the target of NMDA channel blockers has not yet been
precisely defined. The extracellular part of the NMDA receptor
complex, with its agonist and regulatory sites, has been crystallised
and subjected to X-ray structural analysis down to 1.35 Å resolu-
tion,27 but for the channel itself only tentative models have been
proposed based on site-directed mutagenesis and accessibility
studies. Nevertheless, evidence is accumulating that not all chan-
nel blockers interact with the channel in the same way. Combina-
tions of NR1 with NR2C or NR2D responded weaker to MK-801
than combinations of NR1 with NR2A or NR2B; for Phencyclidine,
these differences were smaller, whereas Ketamine did not differen-
tiate at all.28 The potency of all channel blockers is affected by
mutating asparagine residues at the selectivity filter in the middle
of the pore.29 But an alanine residue on subunit NR1 slightly above
this position seems to be more important for inhibition by MK-801
than by Phencyclidine,30 and plays also a role for inhibition by
Memantine.31 On the other hand, the aromatic residue W563 still
higher on NR1, if mutated to a non-aromatic residue, weakened
the affinity of MK-801 at least 20-fold, without affecting that of
Memantine.31
possibly due to lowering the density of
p-electrons in the aromatic
nuclei. NMDA receptor inhibition potencies of inhaled aromatic
drugs of abuse correlate with their abilities to engage in cation–
p
interactions.39 Our first attempt to create a channel blocker with
similar favourable properties as Memantine failed, because our
non-aromatic dicyclohexyl analogues 13 were virtually inactive.
4. Conclusion
Resolution of the NMDA channel blocker ( )-DPEA, and stereo-
specific synthesis of (+)- and (ꢀ)-DEP, resulted in enantiomers with
highly differing potencies (stereo selectivity >30). The more potent
DEP-isomer (S)-6 behaved similarly to the high affinity channel li-
gand MK-801, whereas the more potent DPEA-isomer (S)-5a exhib-
ited, like (S)-Ketamine and various primary amine channel
blockers, a high degree of spermine sensitivity. Our detailed SAR
study on enantiomers may help at refining a pharmacophore for
clinically favourable NMDA receptor channel blockers.
5. Experimental
5.1. General procedures
1H, 13C (J modulated), and 19F NMR spectra were measured in
CDCl3 on a Bruker Avance DRX 400, at 400.13, 100.63, and
376.5 MHz, respectively. Chemical shifts were referenced to

3460
M. L. Berger et al. / Bioorg. Med. Chem. 17 (2009) 3456–3462
residual CHCl3 (dH = 7.24) and CDCl3 (dC = 77.00). IR spectra were
run on a Perkin–Elmer 1600 FT-IR spectrometer; liquid samples
were measured as films on a silicon disc.40 Optical rotations were
measured at 20 °C on a Perkin–Elmer 351 polarimeter in a 10 cm
cell. TLC was carried out on 0.25 mm Silica Gel 60 F254 plates
(Merck). Flash (column) chromatography was performed with
Merck Silica Gel 60 (230–400 mesh). Spots were visualised by UV
and/or dipping the plate into a solution of (NH4)6Mo7O24 ꢂ 4H2O
(23.0 g) and Ce(SO4)2 ꢂ 4H2O (1.0 g) in 10% aqueous H2SO4
(500 mL), followed by heating with a heat gun. Melting points were
3.13 (dd, J = 13.4, 9.4 Hz, 1H, CH2CH), 2.56 (m, 4H, Hpip), 1.69 (m,
4H, Hpip), 1.49 (m, 2H, Hpip); 13C NMR: d = 140.0, 139.4, 129.4
(2C), 128.9 (2C), 127.8 (2C), 127.6 (2C), 126.8, 125.6, 72.3, 51.4
(2C), 39.2, 26.3 (2C), 24.6. Anal. Calcd for C19H23N (265.97): C,
85.99; H, 8.74; N, 5.28. Found for (R)-6: C, 86.49; H, 8.73; N, 5.12.
5.5. Hydrochlorides of (R)- and (S)-6
Concd HCl (0.4 mL) was added to a warm (40 °C) mixture of (R)-
6 (0.200 g, 0.754 mmol) and water (5 mL). On slowly cooling to
+4 °C crystals formed, which were collected and recrystallised from
a small amount of water containing HCl. The warm solution was al-
lowed to cool slowly from 40 °C to +4 °C after seeding. The colour-
less crystals were collected and dried to give (R)-6 ꢂ HCl ꢂ H2O
(0.205 g, 85%); mp 220–221 °C (phase transitions at 100 °C and
determined on
uncorrected.
a Reichert Thermovar instrument and were
5.2. Compound sources
[3H]MK-801 was obtained from New England Nuclear (Vienna);
unlabelled (+)-MK-801 [(+)-3] and (ꢀ)-MK-801 [(ꢀ)-3] from Tocris
Cookson Ltd (Bristol, UK). (R)- and (S)-Amphetamine (17) were ob-
tained from Smith, Kline & French; (1R,2S)-(ꢀ)- and (1S,2R)-(+)-
Tranylcypromine (18) from Procter & Gamble Germany (Darms-
tadt); (S)-(+)- and (R)-(ꢀ)-1,2-diphenyl-2-propylamine (5b), and
(S)-(+)- and (R)-(ꢀ)-1-phenyl-2-(pyridin-2-yl)ethylamine (5e)
from Astra Charnwood; (S)-(+)- and (R)-(ꢀ)-Ketamine (1b), (+)-
and (ꢀ)-Norketamine (1a) from Gödecke, Parke-Davis; and
(9S,13S,14S)-(+)-3-Methoxymorphinan (4a) and (9R,13R,14R)-(ꢀ)-
Levomethorphan (ꢀ)-4b) from Hoffmann-La Roche (Basel). (R,R)-
1,2-Dicyclohexylethane-1,2-diol (7) from Aldrich was crystallised
from 1,2-dichloroethane to ee >98%. All other compounds were
used as obtained either from Aldrich or from Sigma.
160 °C); ½a 2D0
¼ ꢀ83:5 (c 0.86, EtOH). Similarly, (S)-6 (0.110 g,
ꢃ
0.414 mmol) was converted to (S)-6 ꢂ HCl ꢂ H2O (0.110 g, 83%);
mp 222–224 °C; ½a D20
¼ þ82:6 (c 0.82, EtOH). We also obtained
ꢃ
the racemic piperidine derivative prepared in analogy to the enan-
tiomers in 50% yield: ( )-6 ꢂ HCl ꢂ H2O, mp 207–209 °C (phase
transitions at 100 °C and 155 °C) (lit.43 207 °C, without crystal
water; lit.10 207–209 °C, without crystal water).
1H NMR: d = 12.40 (br s, 1H, NHCl), 7.35 (br s, 5H, Har), 7.10–
6.97 (m, 5H, Har), 4.18 (ddd, J = 12.1, 4.1, 3.3 Hz, 1H, CHN), 3.99
(dd, J = 12.1, 3.0 Hz, 1H, CH2CN), 3.64–3.46 (m, 2H, Hpip), 3.42
(t, J = 12.1 Hz, 1H, CH2CN), 2.60–2.37 (m, 3H, Hpip), 2.34–2.21 (m,
1H, Hpip), 1.87–1.73 (m, 3H, Hpip), 1.78 (s, 2H, H2O), 1.30–1.17
(m, 1H, Hpip); 13C NMR: d = 135.8, 131.1, 130.2 (2C), 130.0, 129.3
(2C), 129.2 (2C), 128.4 (2C), 126.8, 72.9, 53.4, 48.8, 36.8, 22.7,
22.7, 22.3. Anal. Calcd for C19H24ClN ꢂ H2O (319.87): C, 71.34; H,
8.19; N, 4.38. Found for (R)-6 ꢂ HCl ꢂ H2O: C, 71.32; H, 8.22; N,
4.36; found for ( )-6 ꢂ HCl ꢂ H2O: C, 71.20; H, 8.17; N, 4.54.
5.3. Resolution of ( )-DPEA into its (S)-(+)- and (R)-(ꢀ)-
enantiomers [(S)-5a and (R)-5a]
The dextrorotary salt formed from (ꢀ)-tartaric acid and (S)-5a
was obtained8 by allowing a warm (35 °C) solution of (ꢀ)-tartaric
acid (4.03 g, 26.9 mmol) and ( )-5a (5.3 g, 26.9 mmol) in dry MeOH
(40 mL) to cool very slowly down to +4 °C. It was recrystallised
(again with very slow cooling) twice from dry MeOH, treated with
2 N NaOH, and the free (S)-(+)-amine was extracted with CH2Cl2
and bulb to bulb distilled (82 °C, 0.06 mbar) to give a colourless
5.6. (R,R)-(+)-4,5-Dicyclohexyl-[1,3]dioxolane-2-thione (8)
A mixture of (R,R)-1,2-dicyclohexylethane-1,2-diol (7) (1.223 g,
5.40 mmol, ee >98%) and thiocarbonyl diimidazole (1.29 g,
7.29 mmol, 1.35 equiv) in dry toluene (31.9 mL) was refluxed for
3 h, then allowed to cool and concentrated in vacuo. Water
(15 mL) was added and the mixture was extracted with EtOAc
(3 ꢂ 20 mL). The combined organic layers were dried (Na2SO4)
and concentrated under reduced pressure. The residue was puri-
fied by flash chromatography (hexane/EtOAc = 5/1; Rf = 0.80, hex-
oil; ½a 2D0
ꢃ
¼ þ10:6 (c 1.45, CHCl3), lit.8
½
a 2D0
ꢃ
¼ þ10:7 (c 1.05, CHCl3);
ee >99%; ½a 2D0
¼ þ47:7 (c 1.58, EtOH). The mother liquors were
ꢃ
concentrated, treated with 2 N NaOH and the recovered amine
was resolved with (+)-tartaric acid to give the (R)-(ꢀ)-5a;
ane/EtOAc = 3/1) to give thiocarbonate 8 (1.289 g, 89%) as
½
a 2D0
ꢃ
¼ ꢀ10:4 (c 1.40, CHCl3), lit.8
½
a 2D0
ꢃ
¼ ꢀ11:1 (c 1.03, CHCl3); ee
colourless crystals; mp 161–162 (EtOAc, hexane), ½a D20
¼ þ75:0
ꢃ
>99%; ½a 2D0
ꢃ
¼ ꢀ45:5 (c 1.48, EtOH), lit.41: ½a D20
ꢃ
¼ ꢀ45 (c 2.0, EtOH).
(c 0.90, acetone).
The ee of 98% for both samples was determined by 1H NMR spec-
troscopy using (R)-(ꢀ)-O-acetylmandelic acid as a chiral solvating
agent (molar ratio of amine/chiral solvating agent 1/3).42
IR (Si): mmax = 2926, 2852, 1444, 1348, 1297, 1259, 1197,
1174 cmꢀ1 1H NMR: d = 4.36 (m, 2H, 2CHO), 1.82–1.54 (m, 13H,
;
Hchex), 1.28–0.96 (m, 11H, Hchex); 13C NMR: d = 191.7, 88.0 (2C),
41.3 (2C), 27.7 (2C), 27.0 (2C), 25.9 (2C), 25.5 (2C), 25.3 (2C). Anal.
Calcd for C15H24O2S (268.41): C, 67.12; H, 9.01. Found: C, 67.38; H,
9.10.
5.4. (R)-(ꢀ)- and (S)-(+)-DEP [(R)-6 and (S)-6]
1,5-Dibromopentane (1.083 g, 0.64 mL, 4.71 mmol, 3.0 equiv)
was added dropwise to a solution of (R)-5a (0.309 g, 1.57 mmol)
and K2CO3 (1.30 g, 9.42 mmol, 6.0 equiv) in dry CH3CN (12.1 mL)
under argon. Stirring was continued for 3 days at room tempera-
ture (TLC: hexane/EtOAc = 5/1, Rf = 0.27). The potassium salts were
removed by filtration and washed with CH3CN (3 ꢂ 10 mL). The fil-
trate was concentrated under reduced pressure and the residue
was purified by flash chromatography (hexane/EtOAc = 5/1) to give
5.7. (S)-(ꢀ)-1,2-Dicyclohexylethanol [(S)-9]
A solution of thiocarbonate 8 (0.746 g, 2.78 mmol), Bu3SnH
(1.618 g, 1.5 mL, 5.56 mmol, 2 equiv), and AIBN (0.033 g,
0.20 mmol) in dry toluene (30 mL) was added dropwise to reflux-
ing toluene (54.5 mL) under argon during 40 min. More Bu3SnH
(2 ꢂ 0.75 mL) and AIBN (2 ꢂ 0.033 g) were added after 2 and 4 h.
After 6.5 h the reaction mixture was cooled and treated with aque-
ous NaOH (10%, 27.5 mL) at 40 °C for 12 h. The organic layer was
separated and the aqueous one was extracted with Et2O
(3 ꢂ 30 mL). The combined organic layers were washed with
water, dried (MgSO4) and concentrated under reduced pressure.
The crude product was purified by flash chromatography
(R)-(ꢀ)-6 (0.265 g, 64%) as a colourless oil; ½a D20
¼ ꢀ87:3 (c 0.92,
ꢃ
EtOH). Similarly, (S)-5a (0.174 g, 0.882 mmol) was converted to
(S)-(+)-6 (0.127 g, 54%); ½a D20
¼ þ87:6 (c 0.93, EtOH).
ꢃ
IR (Si):
m ;
max = 3027, 2932, 2793, 1495, 1452, 1114 cmꢀ1 1H
NMR: d = 7.39–7.18 (m, 8H, Har), 7.12 (m, 2H, Har), 3.72 (dd,
J = 9.4, 5.3 Hz, 1H, CHN), 3.44 (dd, J = 13.4, 5.3 Hz, 1H, CH2CN),

M. L. Berger et al. / Bioorg. Med. Chem. 17 (2009) 3456–3462
3461
(hexane/EtOAc = 10/1; Rf = 0.33) to give alcohol (S)-9 (0.353 g, 60%)
15 min at 0 °C and 1 h at room temperature (TLC, for azide: hexane,
for precursor alcohol: hexane/EtOAc = 10/1). The solvent was evap-
orated under reduced pressure and the residue was purified by
flash chromatography (hexane; Rf = 0.75) to give azide (R)-11
as colourless crystals; mp 82–83 °C (hexane), ½a D20
ꢃ
¼ ꢀ33:9 (c 1.03,
acetone).
IR (Si):
m
max = 3278, 2917, 2851, 1450 cmꢀ1 1H NMR: d = 3.45
;
(m, 1H, CHO), 1.83–1.58 (m, 10H, Hchex and CH2), 1.43 (m, 1H,
Hchex), 1.36–0.75 (m, 14H, Hchex); 13C NMR: d = 73.4, 44.1, 42.1,
34.6, 34.2, 32.7, 29.2, 27.6, 26.7, 26.6, 26.4, 26.4, 26.2, 26.2. Anal.
Calcd for C14H26O (210.36): C, 79.94; H, 12.46. Found: C, 80.14;
H, 12.52.
(0.228 g, 87%) as a colourless liquid; ½a D20
¼ þ31:9 (c 0.98, acetone).
ꢃ
Similarly, alcohol (R)-9 (0.210 g, 1.0 mmol) was converted to azide
(S)-11 (0.191 g, 81%); ½a D20
¼ ꢀ32:3 (c 1.11, acetone).
ꢃ
IR (Si):
m ;
max = 2925, 2853, 2099, 1449, 1334, 1260 cmꢀ1 1H
NMR: d = 3.15 (ddd, J = 9.5, 4.9, 4.1 Hz, 1H, CHN3), 1.81–1.59 (m,
10H, Hchex and CH2), 1.46–0.76 (m, 14H, Hchex); 13C NMR:
d = 65.7, 42.8, 39.0, 34.6, 34.1, 32.5, 30.0, 28.5, 26.6, 26.4, 26.3,
26.2, 26.1, 26.1. Anal. Calcd for C14H25N3 (235.37): C, 71.44; H,
10.71; N, 17.85. Found for (R)-11: C, 72.00; H, 11.02; N, 17.59.
5.8. (R)-Mosher esters (2R,10S)-10 and (2R,10R)-10
(S)-MTPA-Cl [0.3 mL, 0.5 M solution in dry CH2Cl2; (S)-
a-meth-
oxy- -trifluoromethyl-phenylacetyl chloride, (S)-Mosher chloride]
a
was added to a solution of alcohol (S)-9 (21 mg, 0.1 mmol) in dry
pyridine (1 mL) and dry CH2Cl2 (0.5 mL). After stirring for 18 h at
room temperature, water and HCl (2 M) were added. The mixture
was extracted with CH2Cl2 (3 ꢂ 10 mL). The combinded organic
layers were washed with a saturated solution of NaHCO3 and
water, dried (Na2SO4) and concentrated under reduced pressure.
The residue was purified by flash chromatography (hexane/
EtOAc = 15/1) to give (R)-Mosher ester (2R,10S)-10 (42 mg, quanti-
tative). The ester of (R)-9 was prepared similarly. 19F NMR spectra
[(2R,10S)-10: ꢀ71.38 ppm; (2R,10R)-10: ꢀ71.57 ppm] indicated ee
>98% for (2R,10S)-10 and >96% for (2R,10R)-10.
5.12. (R)-(+)- and (S)-(ꢀ)-1,2-Dicyclohexylethylamine [(R)-13
and (S)-13]
LiAlH4 (1.73 mL, 0.865 mmol, 0.5 M in dry Et2O, 1.5 equiv) was
added dropwise to a solution of azide (R)-11 (0.136 g, 0.578 mmol)
in dry Et2O (2.3 mL) and stirring was continued for 1.5 h at room
temperature (TLC, for amine: hexane/EtOAc = 1/5, for azide: hex-
ane). The mixture was cooled at 0 °C, aqueous NaOH (0.2 mL,
2 M) was added dropwise and stirring was continued for 30 min
at room temperature. The solvent was decanted and the residue
was extracted with Et2O (3 ꢂ 10 mL). The combined organic layers
were dried (MgSO4) and concentrated under reduced pressure. The
crude product was purified by flash chromatography (hexane/
EtOAc = 1/3; Rf = 0.33) to give (R)-(+)-13 (0.106 g, 88%) as a colour-
5.9. (R)-(+)-1,2-Dicyclohexylethyl 4-nitrobenzoate (12)
Alcohol (S)-9 (0.621 g, 2.95 mmol), triphenylphosphine (1.0 g,
4.13 mmol, 1.4 equiv) and 4-nitrobenzoic acid (0.690 g, 4.13 mmol,
1.4 equiv) in dry toluene (8.6 mL) was cooled at 0 °C under argon.
DIAD (0.81 g, 4.13 mmol, 1.4 equiv) was added and stirring was
continued for 20 h at room temperature (TLC: hexane/
EtOAc = 10/1, Rf = 0.79). The solvent was evaporated under reduced
pressure and the residue was purified by flash chromatography
(hexane/EtOAc = 15/1) to give 4-nitrobenzoate 12 (0.477 g, 45%)
less oil;
(0.177 g, 0.752 mmol) was converted to (S)-(ꢀ)-13 (0.130 g, 83%);
¼ ꢀ27:7 (c 1.2, EtOH).
IR (Si):
max = 2922, 2851, 1449 cmꢀ1
½
a 2D0
ꢃ
¼ þ27:8 (c 0.79, EtOH). Similarly, azide (S)-11
½ ꢃ
a 2D0
m
;
1H NMR: d = 2.57 (dt,
J = 8.9, 4.1 Hz, 1H, CHN), 1.78–1.69 (m, 3H, CH2), 1.69–1.57 (m,
7H, NH2 and Hchex), 1.40–0.72 (m, 17H, Hchex); 13C NMR: d = 52.8,
44.3, 42.8, 34.6, 34.5, 32.7, 29.7, 27.7, 26.7, 26.7, 26.6, 26.5, 26.5,
26.2.
as needles; mp 69–70 °C (hexane); ½a D20
¼ þ31:5 (c 1.06, acetone).
ꢃ
IR (Si):
m
max = 2926, 2853, 1721, 1529, 1450, 1349, 1279 cmꢀ1
;
5.13. Hydrochlorides of 13
1H NMR: d = 8.23 (m, 4H, Har), 5.15 (ddd, J = 9.1, 5.1, 3.8 Hz, 1H,
CHO), 1.87–1.43 (m, 14H, CH2 and Hchex), 1.30–0.79 (m, 10H,
Hchex); 13C NMR: d = 164.4, 150.4, 136.2, 130.7 (2C), 123.5 (2C),
77.8, 42.0, 38.9, 34.2, 34.1, 32.8, 29.2, 28.0, 26.4, 26.4, 26.2, 26.1,
26.1, 26.1. Anal. Calcd for C21H29NO4 (359.46): C, 70.17; H, 8.13;
N, 3.90. Found: C, 70.15; H, 8.39; N, 3.74.
A solution of HCl (0.25 mL, 8 M) in dry Et2O was added drop-
wise to a solution of (R)-13 (0.141 g, 0.673 mmol) in CHCl3
(0.25 mL) and iPr2O (3 mL). The mixture was cooled from 20 °C
to ꢀ18 °C. The colourless crystals were collected, washed with
iPr2O and dried to give hydrochloride (R)-13 ꢂ HCl (0.138 g, 88%);
mp 187–188 °C, ½a D20
¼ þ19:5 (c 1.01, H2O). Similarly, amine (S)-
ꢃ
5.10. (R)-(+)-1,2-Dicycohexylethanol [(R)-9]
13 (0.130 g, 0.621 mmol) was converted to (S)-13 ꢂ HCl (0.130 g,
85%); mp 188–189 °C (lit.44 188–189 °C); ½a 2D0
¼ ꢀ20:0 (c 0.80,
ꢃ
MeONa in MeOH (0.2 M, 4.4 mL) was added dropwise to a stir-
red solution of the 4-nitrobenzoate 12 (0.269 g, 0.748 mmol) in dry
THF (1.1 mL). The reaction mixture was stirred for 12 h at room
temperature. Water (5 mL) was added and the mixture was ex-
tracted with EtOAc (3 ꢂ 10 mL). The combined organic layers were
dried (Na2SO4) and concentrated under reduced pressure. The
crude product was purified by flash chromatography (hexane/
EtOAc = 10/1; Rf = 0.28) to give alcohol (R)-9 (0.137 g, 87%) as col-
H2O) [lit.44 ꢀ22.7 (c 1% in H2O)].
1H NMR: d = 8.28 (br s, 3H, NH3), 3.09 (br s, 1H, CHN), 1.80–1.41
(m, 14H, CH2 and Hchex), 1.36–1.04 (m, 8H, Hchex), 0.97–0.76 (m,
2H, Hchex); 13C NMR: d = 54.6, 40.1, 37.5, 33.6, 33.4, 32.8, 28.9,
27.7, 26.4, 26.2, 26.1, 26.1 (2C), 25.8. Anal. Calcd for C14H28ClN
(245.83): C, 68.40; H, 11.48; N, 5.70. Found for (R)-13 ꢂ HCl: C,
68.02; H, 11.62; N, 5.53.
ourless crystals; mp 81–82 °C (hexane); ½a D20
ꢃ
¼ þ31:1 (c 1.05,
5.14. Binding of [3H]MK-801 to rat neuronal membranes
CHCl3). The spectroscopic data were identical with those of (S)-9.
Whole cell membranes were prepared from adult rat cerebral
cortex and CA1 and dentate gyrus part of hippocampus as de-
scribed.16 Binding assays were conducted in 50 mM Tris-acetate
5.11. (R)-(+)- and (S)-(ꢀ)-1-Azido-1,2-dicyclohexylethane [(R)-
11 and (S)-11]
(pH 7.0) for 2 h at 23 °C, with 5 nM [3H]MK-801, 10
l
M glutamate,
glycine. Under these conditions, equilibrium is
reached.45 For nonspecific binding, glutamate and glycine were re-
placed by 100 -2-amino-5-phosphono valeric acid and 10
A solution of alcohol (S)-9 (0.243 g, 1.155 mmol) and triphenyl-
phosphine (0.393 g, 1.50 mmol, 1.3 equiv) in dry toluene (6.85 mL)
was cooled in ice bath under argon. DIAD (0.304 g, 0.296 mL,
1.50 mmol, 1.3 equiv) and HN3 in toluene (1.06 mL, 1.50 mmol,
1.4 M, 1.3 equiv) were added and stirring was continued for
and 10 lM
lM
D
lM
5,7-dichlorokynurenic acid (Tocris Cookson Ltd, Bristol, UK). Non-
specific binding amounted to 10–20% of total binding. Membranes

3462
M. L. Berger et al. / Bioorg. Med. Chem. 17 (2009) 3456–3462
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with bound radioligand were collected by rapid filtration over
polyethylenimine-soaked glass fibre filters with a 48-places har-
vester (Brandel, Gaithersburg, USA). Filters were immersed in tol-
uene with PPO and POPOP, agitated for 20 min, and radioactivity
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ated by analysis of variance, with post hoc Newman–Keuls test.
Acknowledgements
For parts of this study, M.L.B. received financial support from
Merck KGaA (Darmstadt, Germany). Kerstin Schöringhumer, Man-
uel Nagl and Tanja Werle contributed to the binding data. We
thank S. Felsinger for recording the NMR spectra.
31. Kashiwagi, K.; Masuko, T.; Nguyen, C. D.; Kuno, T.; Tanaka, I.; Igarashi, K.;
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