N-hydroxypyrazolyl glycine derivatives as selective N-methyl-D-aspartic acid receptor ligands

Article abstract of DOI:10.1021/jm800025e

A series of analogues based on N-hydroxypyrazole as a bioisostere for the distal carboxylate group of aspartate have been designed, synthesized, and pharmacologically characterized. Affinity studies on the major glutamate receptor subgroups show that thes

Full text of DOI:10.1021/jm800025e

J. Med. Chem. 2008, 51, 4179–4187
4179
N-Hydroxypyrazolyl Glycine Derivatives as Selective N-Methyl-D-aspartic Acid Receptor
Ligands
Rasmus P. Clausen,*,† Caspar Christensen,† Kasper B. Hansen,†,‡,| Jeremy R. Greenwood,† Lars Jørgensen,† Nicola Micale,†
Jens Christian Madsen,§ Birgitte Nielsen,† Jan Egebjerg,| Hans Bra¨uner-Osborne,† Stephen F. Traynelis,‡ and
Jesper L. Kristensen†
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, UniVersity of Copenhagen, 2 UniVersitetsparken,
DK-2100 Copenhagen, Denmark, Department of Pharmacology, Emory UniVersity School of Medicine, Rollins Research Center,
Atlanta, Georgia, Department of Medicinal Chemistry and Department of Molecular Neurobiology, H. Lundbeck A/S,
9 OttiliaVej, DK-2500 Valby, Denmark
ReceiVed January 15, 2008
A series of analogues based on N-hydroxypyrazole as a bioisostere for the distal carboxylate group of aspar-
tate have been designed, synthesized, and pharmacologically characterized. Affinity studies on the major
glutamate receptor subgroups show that these 4-substituted N-hydroxypyrazol-5-yl glycine (NHP5G)
derivatives are selectively recognized by N-methyl-D-aspartic acid (NMDA) receptors and that the (R)-
enantiomers are preferred. Moreover, several of the compounds are able to discriminate between individual
subtypes among the NMDA receptors, providing new pharmacological tools. For example, 4-propyl NHP5G
is an antagonist at the NR1/NR2A subtype but an agonist at the NR1/NR2D subtype. Molecular docking
studies indicate that the substituent protrudes into a region that may be further exploited to improve subtype
selectivity, thereby opening up a design strategy for ligands which can differentiate individual NMDA receptor
subtypes.
Introduction
The glutamate receptors (GluRs) are a group of receptors with
a prominent role in neurotransmission because most of the
excitatory signals in the brain are mediated by glutamic acid
(Glu, Figure 1).1–3 These receptors are involved in key processes
of the central nervous system (CNS) such as learning and
memory4 but are also implicated in several neuropathological
conditions such as ischemia, epilepsy, schizophrenia, chronic
pain, and Alzheimer’s disease.5 Therefore, the GluRs have been
the target of extensive research for more than three decades,
and one of the goals has been to develop compounds that can
alter glutamatergic neurotransmission in a selective manner.
Such compounds have been and continue to be important tools
in discovering new therapeutic possibilities and to gain a
functional understanding of this group of receptors.6,a
Two different signaling pathways divide the GluRs: metabo-
tropic glutamate receptors (mGluRs) are G-protein-coupled
receptors that mediate slow modulatory responses, and iono-
tropic glutamate receptors (iGluRs) are cation-conducting ion
channels that mediate fast neurotransmission via depolarization
of the membrane potential. Based on activation by selective
ligands and cDNA homology, the iGluRs are divided into
N-methyl-D-aspartic acid (NMDA), (S)-2-amino-3-(3-hydroxy-
Figure 1. Structures of the excitatory amino acids glutamic acid and
aspartic acid and bioisosteric analogues of these compounds with
activity at the glutamate receptors.
5-methyl-4-isoxazolyl)propionic acid (AMPA), and kainic acid
(KA) receptors. The iGluRs are homo- or heteromeric as-
semblies of four subunits. Seven NMDA subunits (NR1, NR2A-
D, NR3A, NR3B), four AMPA (GluR1-4), and five KA
(GluR5-7 and KA1-2) preferring subunits have been identi-
fied.1 NMDA glutamate receptors are assembled from two NR1
subunits and two NR2 subunits and are activated by the
simultaneous binding of glycine and glutamate to the NR1 and
NR2 subunits, respectively. NR3 subunits also can coassemble
with NMDA receptors, although the stoichiometry is unclear.
The iGluR subunit consists of an intracellular C-terminal
domain, a transmembrane domain that forms the ion channel
pore, and a pair of extracellular domains: the agonist binding
domain (ABD) and the N-terminal domain. Over the past
decade, an increasing number of X-ray crystal structures of the
ABDs of the GluRs have appeared (86 available structures to
* To whom correspondence should be addressed. Phone: +45 35 33 65
66. Fax: +45 35 33 60 40. E-mail: rac@farma.ku.dk.
†
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sci-
ences, University of Copenhagen.
‡
Department of Pharmacology, Emory University School of Medicine,
Rollins Research Center.
§
Department of Medicinal Chemistry, H. Lundbeck A/S.
Department of Molecular Neurobiology, H. Lundbeck A/S.
|
a Abbreviations: NHP5G, 2-(N-hydroxypyrazol-5-yl)glycine, 2-(N-hy-
droxypyrazol-4-yl)glycine; NMDA, N-methyl-D-aspartic acid; GluRs,
glutamate receptors; Glu, glutamate; CNS, central nervous system; mGluRs,
metabotropic glutamate receptors; iGluRs, ionotropic glutamate receptors;
AMPA, (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid;
KA, kainic acid; ABD, agonist binding domain; AMAA, (R,S)-2-amino-
2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid.
10.1021/jm800025e CCC: $40.75
2008 American Chemical Society
Published on Web 06/25/2008

4180 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Clausen et al.
Scheme 1a
date, covering all the major mammalian subtypes as well as
bacterial homologues), providing detailed structural knowledge
of agonist binding and the molecular pharmacology of this group
of receptors.3,7–11 These crystallographic structures have shown
that iGluR ABDs are clamshell-like structures, where the agonist
binds in the cleft formed by two lobes. Upon agonist binding,
the two lobes close around the agonist, whereas binding of
antagonists stabilizes an open domain structure. Furthermore,
the extent to which substituents on AMPA receptor ligands
promote domain closure at the GluR2 ABD correlates with the
degree of receptor activation.12,13 By contrast, crystallographic
structures of the NR1 ABD, which hosts the binding site of the
NMDA receptor coagonist glycine, have revealed that the degree
of ABD closure is the same for partial and full agonists at the
glycine site.14 Nevertheless, a correlation between the steric
requirements of the ligand and the extent of receptor activation
still seems to exist because series of carbocyclic agonists show
decreasing efficacy with the size of the ring (cyclopropylglycine
> cyclobutylglycine > cyclopentylglycine).
a Reagents and conditions: (a) I-Cl; (b) (1) i-PrMgCl, -78°C, THF;
(2) RCdOR′; (c) TFA, Et3SiH; (d) SO2Cl2 (for 10e); (e) (1) n-BuLi, -78°C;
(2) diethyl N-Boc iminomalonate; (f) H2, Pd/C, MeOH; (g) (1) LiOH; (2)
HCl.
We and co-workers have previously reported the synthesis
and pharmacological characterization of a series of 5-substituted
(R,S)-2-(N-hydroxypyrazol-4-yl)glycine ((R,S)-NHP4G, (5) ana-
logues as GluR ligands.15,16 These glutamic acid analogues are
structurally related to the naturally occurring excitotoxin ibotenic
acid (Ibo, 3), but with an N-hydroxypyrazole instead of a
3-hydroxyisoxazole moiety as the bioisosteric substitute for the
distal carboxylic acid of Glu. The unsubstituted analogue (R,S)-
NHP4G displayed activity at several GluRs, but the pharma-
cological profile narrowed upon substitution, limiting the activity
to group II mGluRs and NMDA receptors. Even small substit-
uents at the 5-position of the partial NMDA agonist (R,S)-
NHP4G turned these compounds into antagonists at NMDA
receptors. Recently, we expanded this series17 to include aspartic
acid analogues such as the unsubstituted (R,S)-2-(N-hydroxy-
pyrazol-5-yl)glycine analogue ((R,S)-NHP5G, 6a). This com-
pound (6a) is a selective NMDA receptor partial agonist, like
the structurally related NMDA receptor agonist (R,S)-2-amino-
2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid AMAA (4),
whereas 4-aryl NHP5G analogues are antagonists.
We have extended the NHP5G series and separated enanti-
omers in order to establish the steric requirements for the
pharmacological behavior of these derivatives. Compounds 6a
and 6f from this series have already been applied in an extensive
mutation study18 in order to establish the structural determinants
that could account for the partial agonism at the NR1/NR2B
receptor subtype. The synthesis and pharmacological charac-
terization of a larger 4-substituted NHP5G series will be
presented here.
decarboxylation yields racemic amino acids 6b-f. The enan-
tiomers could be separated by chiral HPLC, thus enabling a
preparative resolvation of the compounds including NHP5G.
Because the mobile phase was diluted aqueous TFA, the
enantiomers were isolated as TFA salts, containing varying
amounts of TFA. The amount of TFA was determined by NMR
spectroscopy using the ERETIC technique,20 where an elec-
tronically generated signal serves as a quantification reference.
For TFA, the ERETIC technique must be applied to 19F NMR
spectroscopy because the absence of nonlabile protons makes
1H NMR unsuitable in this case. However, the compounds
studied here are void of fluorine nuclei, so quantification relative
to TFA necessitates the use of an ERETIC signal in both the
1
1H and the 19F NMR spectra. The relative strength of the H
and 19F ERETIC signals must then be calibrated using a
compound containing both protons and fluorine. For this
purpose, we used a sample of citalopram oxalate. In general,
the content of TFA was less than 1 equivalent.
Determination of Absolute Configuration. We were unable
to obtain crystals suitable for X-ray structure determination of
any of the compounds and therefore we turned to a number of
other methods in order to determine the absolute configuration.
First, the elution order on the chiral HPLC column clearly
indicated the absolute stereochemistry for all the compounds
(Figure 2). Thus, the L-form of R-amino acids generally elutes
before the D-form on a CR(-) Crownpak chiral column,
corresponding to the (S)- and (R)-enantiomer, respectively.21,22
Second, we turned to the electronic circular dichroism (ECD)
spectra. Traditionally, an empirical correlation has been noted
between the S-isomer of aryl amino acids and observation of a
positive Cotton effect in the ECD.23 However, in recent years,
prediction of the ECD spectrum has become tractable from first
principles using methods such as time-dependent density
functional theory (TD-DFT), reducing the reliance on empiri-
cism for interpretation. Thus, to obtain further evidence for the
absolute configuration, CD spectra (Figure 3) of at least one
enantiomer of 6b-6f were therefore recorded and in the case
of (S)-Cl-NHP5G (6f) compared with that calculated using TD-
DFT for eight low energy conformers in dilute acidic aqueous
solution (Figure 3). Although the first major peak according to
the Boltzmann average lies approximately 20 nm lower in
energy than observed, the magnitude was in good agreement
with experiment and indicative of the same correlation as usually
seen. Although the exact shape of the predicted peak depends
Results
Chemistry. The synthesis of the compounds (Scheme 1)
started from O-benzyl protected N-hydroxypyrazole 7, which
is iodinated, yielding 8 as previously described.19 Metal-halogen
exchange converts 8 into a Grignard reagent with isopropyl
magnesium chloride, which then reacts with various aldehydes
and ketones, yielding alcohols 9a-9d. These alcohols are
deoxygenated by treatment with TFA and triethylsilane, giving
4-substituted hydroxypyrazoles 10a-10d. The chloro substituted
compound 10e is obtained directly from 7 by treatment with
sulfuryl chloride. Selective lithiation followed by treatment with
the amino acid synthon diethyl N-Boc iminomalonate yields
protected amino diesters 11a-e, which are debenzylated by
hydrogenation, yielding 12a-e. Ester hydrolysis followed by
acidic removal of the Boc group and concomitant spontaneous

N-Hydroxypyrazolyl Glycines as NMDA Receptor Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4181
Figure 2. Two chiral HPLC chromatograms of (S)- and (R)-6d,
respectively, showing the elution order on an analytical Crownpak
CR(-) column. The order is reversed on a preparative Crownpak CR(+)
column.
on the weighting of the conformers, and is thus highly sensitive
to the accuracy of conformational energies, the small experi-
mental negative peak at around 230 nm for (S)-6f was also
visible in two of the four lowest energy conformers and in
agreement with that previously observed for phenylglycine.23
Comparing (S)-6b with (S)-6f showed that there was no
significant qualitative difference between the calculated spectra
or between any of the experimental spectra, and thus it is
reasonable to expect the correlation to hold throughout this
series.
Figure 3. Electronic circular dichroism spectra (A) of compounds (R)-
and (S)-6f at a concentration of 0.325 mg/mL in H2O compared with
a TD-DFT simulated spectra (B) of a weighted average of eight low
energy conformers as well as a single low energy conformer of (S)-6f
in dilute acid.
Finally, we prepared the Mosher amide of (S)-6c and
compared the NMR spectra of the two diastereomers with the
isotropic magnetic shielding values calculated for the hydrogen
nuclei using DFT. Because these amide derivatives have several
rotatable bonds, spectra were calculated for the hundred lowest
energy conformers of both (S,S) and (R,S), according to a
conformational search with OPLS2005. Although qualitative
agreement was noted, the precise differences in the calculated
shifts between the diastereomers again proved highly sensitive
to conformational energies when using a Boltzmann weighted
average. We therefore calculated regression coefficients between
the shifts in the two experimental proton spectra and the 200
calculated spectra. For a majority of the calculated (R,S)
conformers, the calculated spectrum was a better match for (R,S)
than (S,S), and for a majority of the calculated (S,S) conformers,
the calculated spectrum was a better match for experimental
(S,S). Moreover, for the single conformers of (R,S) and (S,S)
with the highest regression coefficients, the calculated (R,S)
spectrum was a better match for experimental (R,S) spectrum
(R2 ) 0.9993) and vice versa for (S,S) (R2 ) 0.9985). The
absolute configurations are therefore assigned on the basis of
three independent types of experiment, interpreted by a com-
bination of empirical and first-principles correlations.
Pharmacology. First we investigated the affinity of the
compounds for the major iGluR subgroups. As shown in Table
1, all the analogues are selective NMDA receptor ligands with
affinity in the low micromolar range. Furthermore, the affinity
was found to reside with the (R)-enantiomer of the compounds,
the most potent being (R)-6a, (R)-6b, and (R)-6f.
The affinity at NMDA receptors prompted functional char-
acterization at recombinant NMDA receptors expressed in
Xenopus laeVis oocytes to determine whether the compounds
could discriminate between individual subtypes (Table 2, Figures
4 and 5).
Compounds (R,S)-6c, (R,S)-6d, and (R,S)-6f acted as partial
agonists on four NMDA receptor subunit combinations (NR1/
NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D) but with

4182 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Clausen et al.
Table 1. Receptor Binding Affinities of Compounds 6a-6f at Three
In our previous study including compounds (R)-6a and (R)-
6f, ligand variation was matched against corresponding muta-
tions in the receptor, leading to the conclusion that the degree
of agonist efficacy relative to Glu correlates inversely with the
steric bulk introduced into the agonist binding pocket, either
by the ligand or in the receptor by mutation. It was therefore
interesting to see whether this dependence would manifest in
the present series too. The R-enantiomer of compounds 6a-d
and 6f was therefore characterized at NR1/NR2B (Table 3 and
Figure 6; see also Supporting Information). However, the
relative agonist efficacy of the compounds did not show a simple
correlation with the nature of the substituents. Thus, the un-
substituted NHP5G (6a) induced a fraction of 0.61 of the Glu-
induced current and compounds with methyl-(6b) and n-propyl-
substituents (6d) displayed markedly lower relative efficacies
of 0.23 and 0.09, respectively. The chloro substituted compound
(6f) was slightly more efficacious (0.33) than the methyl
substituted compounds, but the ethyl substituted compound (6c)
induced a 0.46 current relative to the Glu current, which is twice
that of the methyl substituted compound and which is notable
because an ethyl substituent is more bulky than a methyl
substituent. The isopropyl substituted compound (6e) was an
antagonist at NR1/NR2B (see Supporting Information).
Major Groups of iGluRs in Rat Cortical Synaptosome Assaya,24
IC50 (µM)
Ki (µM)
compd
R
[3H]AMPA
[3H]KAIN
[3H]CGP39653
Glu
0.34b
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
NDc
0.38b
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ND
0.20b
(R,S)-6a
(R)-6a
(S)-6a
(R,S)-6b
(R)-6b
(S)-6b
(R,S)-6c
(R)-6c
(S)-6c
(R,S)-6d
(R)-6d
(S)-6d
(R,S)-6e
(R)-6e
(S)-6e
H
10 [9, 11]b
2.2 [2.0, 2.4]
>100
Me
Et
22 [20, 24]
2.2 [2.1, 2.4]
>100
13 [11, 15]
4.4 [3.3, 5.7]
>100
14 [14, 15]
6.9 [5.9, 8.2]
>100
20 [19, 21]
13 [11, 16]
>100
n-Pr
i-Pr
Cl
(R,S)-6f
(R)-6f
(S)-6f
2.9 [2.7, 3.2]
2.9 [2.6, 3.2]
25 [24, 26]
>100
>100
a The numbers in brackets [min, max] indicate mean ( SEM according
to a logarithmic distribution. b Ref 16. c ND: not determined.
Table 2. Potency and Efficacy Relative to Glu of Compounds 6a, 6c,
6d, and 6f at Recombinant NR1/NR2A-D Receptors Expressed in
Xenopus Oocytesa,24
Molecular Modeling. To understand the unexpected trend
in the agonist efficacies of this series of 4-substituted NHP5G,
we docked the compounds in a homology model of the ABD
of NR2B using the recently published X-ray structure of the
ABD of NR2A (PDB code 2A5S) as a template.25 In our recent
mutation study, the homology model was based on a NR1
crystallographic structure (PDB code 1PB7), but the new X-ray
structure of NR2A in complex with glutamate enabled the
inclusion of two water molecules in the homology model that
are involved in the binding of the charged groups of glutamate
into the homology model. The compounds were submitted to a
conformational search in Macromodel 9.0 in the tri-ionized state,
and the resulting minimized structures were then docked flexibly
into a homology model of NR2B. This resulted in similar
binding modes for all the compounds, with the amino acid
moiety overlapping with that of Glu as seen in our previous
study18 and the deprotonated N-hydroxy group receiving an
H-bond from the water molecule involved in binding the distal
acidic group of Glu. The substituents point like a wedge toward
residues H486 and V686, which form a crevice between the
upper and lower domains of the clamshell-like ABD structure
(Figure 7A). While the ethyl substituent is accommodated by
the crevice, the n-propyl is in close proximity with residues
Lys485 (Figure 7B) and Tyr731 (not shown). In the X-ray
crystal structure of NR2A, several water molecules are present
in this region and two of them are shown in Figure 7.
Furthermore, the n-propyl substituent reaches a less conserved
region of the ABD. Thus, in the X-ray structure of the ABD of
NR2A, Lys485 is in close proximity to the backbone carbonyl
of Arg711, which is followed by Gly712. This two-peptide
sequence corresponds to Arg712-Gly713 in NR2B, but Arg722-
Ser723 in NR2C and Pro736-Arg737 in NR2D. This sequence
difference could explain the observed difference in pharmacol-
ogy of Pr-NHP5G (6d) and designing ligands with substituents
protruding into this region may lead to NMDA receptor ligands
with increased subtype-selectivity as suggested previously.26
compound
Glub
R
subtype
EC50 (µM)
rel Imax
nHill
NR1/NR2A 2.9
NR1/NR2B 1.8
NR1/NR2C 1.0
NR1/NR2D 0.45
1
1
1
1
1.5
1.7
1.3
1.6
NMDAb
(R,S)-6ac
(R,S)-6c
(R,S)-6d
(R,S)-6f
NR1/NR2A 75
NR1/NR2B 22
NR1/NR2C 23
NR1/NR2D 8.3
NR1/NR2A 82 [75, 90]
NR1/NR2B 48 [47, 49]
NR1/NR2C 54 [50, 57]
NR1/NR2D NDd
0.90 ( 0.04 1.5
0.77 ( 0.01 1.4
0.73 ( 0.02 1.4
0.80 ( 0.02 1.6
0.45 ( 0.03 1.3
0.58 ( 0.02 1.6
0.52 ( 0.01 1.4
H
ND
ND
Et
NR1/NR2A 47 [39, 57]
NR1/NR2B 68 [64, 72]
NR1/NR2C 91 [82, 101]
NR1/NR2D 43 [42, 44]
0.05 ( 0.01 2.4
0.45 ( 0.06 1.2
0.52 ( 0.03 1.3
0.70 ( 0.03 1.5
n-Pr NR1/NR2A NAd
NA
NA
NR1/NR2B 105 [95, 116]
0.06 ( 0.01 1.9
NR1/NR2C 429 [367, 500] 0.22 ( 0.02 1.1
NR1/NR2D 153 [142, 165] 0.37 ( 0.02 1.6
NR1/NR2A 565 [464, 687] 0.07 ( 0.03 3.2
NR1/NR2B 149 [139, 160] 0.29 ( 0.01 1.2
NR1/NR2C 255 [250, 260] 0.55 ( 0.01 1.4
NR1/NR2D 152 [149, 155] 0.73 ( 0.02 1.4
Cl
a Data are from 3-5 oocytes and the numbers in brackets [min, max]
indicate mean ( SEM according to a logarithmic distribution. b Ref 38.
c Ref 16. d NA: no activity; ND: not determined.
highly varying potency and agonist efficacy at the different
subtypes. At NR2A-containing receptors, the ethyl substituted
(R,S)-6c and chloro substituted (R,S)-6f are low-efficacy partial
agonists, but (R,S)-6c is an order of magnitude more potent than
(R,S)-6f. (R,S)-6d displayed antagonist activity at NR1/NR2A
(n ) 5) but was an agonist on all other subtypes (Figure 5) and
is thus the compound showing the largest difference among
subtypes in this series. At NR1/NR2B (R,S)-6c and (R,S)-6f were
approximately equipotent and had higher agonist efficacies than
at NR1/NR2A, but the efficacies varied more. By contrast,
compounds (R,S)-6c and (R,S)-6f showed similar agonist
efficacy relative to Glu around 0.5 at NR2C and 0.7 at NR2D,
and again compound (R,S)-6c was more potent than (R,S)-6f.
Discussion
We have synthesized and pharmacologically characterized a
series of 4-substituted NHP5G derivatives to identify ligands
suitable for investigating the mechanisms governing partial

N-Hydroxypyrazolyl Glycines as NMDA Receptor Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4183
Figure 4. Mean concentration-response curves for racemic compounds Et-(6c), Pr- (6d), and Cl-NHP5G (6f) determined using two-electrode
voltage-clamp recordings on Xenopus oocytes expressing NR1 in combination with NR2A-D. The curves are normalized to the maximal current
response (Imax) in the same recording. Data points are represented as mean ( SEM. All EC50 values are listed in Table 2.
Table 3. Potency and Efficacy Relative to Glu of the (R)-Form of 6a-d
and 6f at Recombinant NR1/NR2B Receptors Expressed in Xenopus
Oocytes.a,24
compd
R
EC50 (µM)
rel Imax
nHill
(R)-6a
(R)-6b
(R)-6c
(R)-6d
(R)-6f
H
Me
Et
n-Pr
Cl
14 [14, 15]
36 [35, 38]
34 [32, 35]
63 [59, 68]
58 [55, 62]
0.61 ( 0.02
0.23 ( 0.01
0.46 ( 0.02
0.09 ( 0.004
0.33 ( 0.01
1.4
1.6
1.6
1.4
1.2
a Data are from 4-6 oocytes and the numbers in brackets [min, max]
indicate mean ( SEM according to a logarithmic distribution.
subtypes both in terms of potency and efficacy. Thus, these
compounds highlight the importance of introducing substituents
in NMDA receptor ligands that can reach less conserved regions
of the receptor. We were able to separate the enantiomers of
the racemic mixtures and have been able to determine to a high
degree of confidence the absolute configuration from several
lines of evidence, including the HPLC elution order, the good
agreement between calculated and measured ECD spectra, and
the similarity between calculated and experimental NMR spectra
of Mosher amide diastereomers of the ethyl compounds. The
separation of enantiomers is important because apparent partial
agonism can arise from mixed agonistic and antagonistic
behavior of opposing enantiomers.27 However, our data showed
that the activity could be attributed to the (R)-enantiomers in
this series. The unsubstituted (R)-6a and chloro substituted (R)-
6f have already been employed in a mutagenic study, where it
was concluded that steric clashes between the substituents and
residues His486 and Val686 were important determinants for
the agonist efficacy of the compounds relative to Glu. These
two residues form a crevice in the ligand binding domain
separating upper and lower domains in the clamshell-like ABD
structure, and introducing steric bulk in this region was expected
to prevent closure of the ligand binding domain and concomitant
receptor activation.
Figure 5. (A) Comparison of maximal currents induced by Et-(6c),
Pr-(6d), and Cl-NHP5G (6f) relative to Glu at NR1/NR2A-D subtypes.
All bars are represented as mean ( SEM. All relative Imax values are
listed in Table 2. (B) Representative current responses obtained from
a two-electrode voltage-clamp recording on a Xenopus oocyte express-
ing NR1/NR2A receptors. Currents were evoked by Glu alone or
coapplication of Glu and Pr-NHP5G at the concentrations indicated
above each response.
agonism at the NMDA receptor. This series of NHP5G
compounds displays several remarkable properties. All of the
compounds are partial agonists at the NMDA receptors with
varying degrees of receptor activation. Furthermore, the com-
pounds show marked variation at the various NMDA receptor

4184 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Clausen et al.
Figure 6. (A) Mean concentration-response curves for compounds
(R)-6a-d,f (R ) H, Me, Et, n-Pr, Cl, respectively) determined using
two-electrode voltage-clamp recordings on Xenopus oocytes expressing
NR1/NR2B receptors. The curves are normalized to the maximal current
response (Imax) to Glu in the same recording. Data points are represented
as mean ( SEM. All EC50 values are listed in Table 3. (B) Bar graph
showing the maximal currents (Imax) of the ligands relative to the
maximal current induced bu Glu. (C) Representative current responses
obtained from a two-electrode voltage-clamp recording on a Xenopus
oocyte expressing NR1/NR2B receptors. Currents were evoked by Glu
alone or coapplication of Glu and (R)-6e at the concentrations indicated
above each response.
Figure 7. (A) Structure of a homology model of the ABD of NR2B
(cyan) containing n-Pr-NHP5G ((R)-6d, blue) showing the residues (red)
lining a crevice to the agonist binding site. Previous studies have shown
these residues to be important determinants of agonist efficacies of
several compounds.18 (B) Et-NHP5G ((R)-6c, pink) and n-Pr-NHP5G
((R)-6d, blue) docked in the homology model. The distances from the
amino group of Lys485 to the n-propyl group and the peptide carbonyl
between Arg712 and Gly713 are indicated in Ångstroms. Water
molecules are shown as red asterisks.
Completion of the entire series now reveals that the com-
pounds display a trend with respect to substituents that was
unexpected on the basis of the mutagenesis study because the
efficacy does not completely follow inversely the steric bulk
added to the ligands. In particular, the ethyl substituted
compound is more efficacious than the methyl substituted com-
pound but still less efficacious compared to the unsubstituted
compound, which is surprising considering the increased steric
bulk imposed by the ethyl group. Docking the compounds into
a homology model of the ABD of NR2B based on the X-ray
crystallographic structure of the highly homologous NR2A ABD
construct shows that the ethyl group reaches a region where
several water molecules are present. Compared with the methyl
and chloro groups, displacement of some of these water
molecules by the ethyl group could thermodynamically favor a
conformation of the ABD that permits receptor activation,
offering a plausible explanation for the unusual trend. By
contrast, n-propyl and isopropyl substituents are in close contact
with residues in the receptor, which again is in agreement with
steric inhibition of the domain closure.
In conclusion, we have identified a series of analogous
selective NMDA receptor ligands that induce various degrees
of receptor activation. These compounds may constitute valuable
pharmacological tools for studying the mechanisms governing
partial agonism at the NMDA receptor. This series shows that
factors other than steric hindrance may determine the degree
of activation of NMDA receptors. In particular, the n-propyl-
substituted compound in this series is interesting because it
significantly differentiates between NMDA receptors containing
different NR2 subunits and therefore offers a promising
pharmacological tool for investigating differences in the func-

N-Hydroxypyrazolyl Glycines as NMDA Receptor Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4185
1-Benzyloxy-4-propylpyrazole (10c). From 9c (2.14 g, 9.2
mmol). FC (PE:EtOAc 6:1) afforded 1.71 g (87%) of the title
compound.
1-Benzyloxy-4-isopropylpyrazole (10d). From 9d (2.32 g, 10.8
mmol). FC (PE:EtOAc 6:1) afforded 1.64 g (82%) of the title
compound.
tional properties of NMDA receptor subtypes at a molecular
and cellular level. This could have relevance in several CNS
disorders, including schizophrenia. Finally, modeling studies
suggest a strategy for designing novel ligands that may be able
to discriminate between NMDA receptor subtypes.
1-Benzyloxy-4-chloropyrazole (10e). 1-Benzyloxypyrazole30 (3
g, 17 mmol) was dissolved in diethyl ether (50 mL) and cooled to
-15 °C. Sulfuryl chloride (5 mL, 62 mmol) was added dropwise
over 30 min, and the resulting solution stirred for an additional 30
min at 0 °C. Water (20 mL) was slowly added, and the aqueous
phase extracted with diethyl ether (3 × 25 mL). The combined
organic phases was washed with NaHCO3 (saturated aq, 3 × 25
mL), brine (1 × 25 mL), dried over MgSO4, and evaporated in
vacuo to yield 3.51 g of the title compound, which crystallized
upon standing in a refrigerator.
General Procedure for the Synthesis of the Protected Amino
Acid. To a stirred solution of the benzyloxypyrazole (10a-e) (1
mmol) in THF (10 mL) at -78 °C, n-BuLi (0.75 mL, 1.6 M in
hexane, 1.2 mmol) was added dropwise over approximately 2 min.
After 5 min, a solution of diethyl N-Boc iminomalonate (382 mg,
1.4 mmol) in THF (1 mL) was added and the resulting mixture
was stirred at -78 °C for 3 h before quenching with water. The
solution was allowed to warm to room temperature, NH4Cl (satrated
aq, 10 mL) was added, the organic phase was separated, and the
water phase was extracted with EtOAc (3 × 10 mL). The combined
organic layers were dried over MgSO4 and evaporated in vacuo to
afford a crude residue, which was purified by FC (PE:EtOAc) to
give the title compound containing minor amounts of coeluting
diethyl N-Boc-iminimalonate. This material was used without
further purification.
Experimental Section
Chemistry. General Methods. All reactions involving air-
sensitive reagents were performed under N2 using syringe-septum
cap technique and all glassware was flame-dried prior to use. Flash
chromatography (FC) was performed using silica Merck 60
(230-400 mesh). 1H (300 MHz), 19F (282 MHz), and 13C (75
MHz) NMR were recorded on a Varian instrument using TMS and
citalopram oxalate as internal standard. HRMS was performed on
a JEOL Hx110/110 mass spectrometer. All solvents and reagents
were analytical grade purchased from Aldrich or Fluka and used
without further purification unless otherwise stated. THF was
distilled from Na/benzophenone under N2. DMF was stored over
3Å molecular sieves. n-Buli28 and i-PrMgCl29 solutions were
titrated prior to use. Preparative chiral HPLC was performed using
a Crownpak CR(+) column (10 mm × 150 mm, Daicel) equipped
with a CR(+) guard column (4.0 mm × 10 mm, Daicel) connected
to a HPLC system consisting of a Jasco 880PU pump, a Rheodyne
7125 injector, a TSP UV100 detector set at 234 nm, and a Merck-
Hitachi D-2000 chromato-integrator. The column was eluted at r.t.
with 1.5 mL/min of aqueous TFA, pH 2. Upon resolvation, the
solutions containing the amino acid was collected and lyophilized.
Chiral HPLC analyses of the resolved stereoisomers were performed
using a Crownpak CR(-) column (4.0 mm × 150 mm, Daicel)
equipped with a water-jacket, and generally the enantiomeric
purities of the isolated amino acids were >95% unless otherwise
indicated. The column was eluted with aqueous HClO4 (pH 1.5 or
pH 2.0) at 0.4 mL/min, and the temperature was controlled by a
Hetofrig thermostat at 20, 10, or 1 °C. A TSP HPLC system
consisting of a P2000 pump, an AS3000 autoinjector, and an
SM5000 PDA detector was used for the chiral HPLC analyses.
Diethyl 2-(1-Benzyloxy-4-methyl-pyrazol-5-yl)-2-tert-butylox-
ycarbonylaminomalonate (11a). From 10a (0.90 g, 4.8 mmol).
FC (PE:EtOAc 7:1) afforded 0.91 g (41%) of 11a as a colorless
oil.
Diethyl 2-(1-Benzyloxy-4-ethyl-pyrazol-5-yl)-2-tert-butyloxy-
carbonylaminomalonate (11b). From 10b (0.92 g, 4.8 mmol). FC
(PE:EtOAc 7:1) afforded 1.08 g 50%) of 11b as a white solid.
Diethyl 2-(1-Benzyloxy-4-propyl-pyrazol-5-yl)-2-tert-butyloxy-
carbonylaminomalonate (11c). From 10c (1.1 g, 5.1 mmol). FC
(PE:EtOAc 4:1) afforded 1.83 g (73%) of 11c as a white solid.
Diethyl 2-(1-Benzyloxy-4-isopropyl-pyrazol-5-yl)-2-tert-buty-
loxycarbonylaminomalonate (11d). From 10d (0.96 g, 4.4 mmol).
FC (PE:EtOAc 4:1) afforded 1.14 g (53%) of 11d as a colorless
oil.
Diethyl 2-(1-Benzyloxy-4-chloro-pyrazol-5-yl)-2-tert-butyloxy-
carbonylaminomalonate (11e). From 10e (1 g, 5.7 mmol). FC (PE:
EtOAc 6:1-4:1) afforded 2.1 g (76%) of 11e as a colorless oil.
General Procedure for Debenzylation. The malonate (11a-e)
(1 mmol) was dissolved in MeOH (10 mL). 10% Pd/C (0.05 mmol)
was added, and the mixture was vigorously stirred under hydrogen
(1 atm) at 0 °C for 30 min, filtered through celite, and evaporated
in vacuo to afford the crude product, which was purified by FC
(PE:EtOAc) to give the title compound.
General Procedure for Metal-Halogen Exchange and Reac-
tion with Aldehyde. 8 (1 mmol) in THF (20 mL) was added to a
flame-dried flask, and the resulting solution was cooled to 0 °C.
Isopropyl magnesium chloride (1.2 mmol, 2 M in THF) was added
dropwise over a period of 5 min, and the resulting mixture was
stirred at 0 °C for 1 h. The aldehyde or ketone (1.2 mmol) dissolved
in THF (2 mL) was added, and the resulting solution was stirred
for an additional 30 min at 0 °C before allowing to warm to r.t.
over 30 min and quenched with NH4Cl (saturated aq, 10 mL). The
aqueous phase was extracted with EtOAc (3 × 20 mL), and the
combined organic phases were dried over MgSO4 and evaporated
in vacuo. FC (PE:EtOAc) afforded the title compound.
1-(1-Benzyloxy-pyrazol-4-yl)-ethanol (9b). From 8 (3.0 g, 10
mmol) and acetaldehyde (0.8 mL, 12 mmol). FC (PE:EtOAc 2:1)
afforded 1.47 g (72%) of the title compound.
1-(1-Benzyloxy-pyrazol-4-yl)-propanol (9c). From 8 (3.0 g, 10
mmol) and propanal (1.1 mL, 12 mmol). FC (PE:EtOAc 2:1-1:1)
afforded 2.13 g (92%) of the title compound.
Diethyl 2-tert-Butyloxycarbonylamino-2-(4-methyl-1-hydrox-
ypyrazol-5-yl)-malonate (12a). From 11a (624 mg, 1.35 mmol).
FC (PE:EtOAc 2:1) afforded 426 mg (85%) of 12a as a yellowish
oil.
2-(1-Benzyloxy-pyrazol-4-yl)-propan-2-ol (9d). From 8 (3.0 g,
10 mmol) and acetone (0.87 mL, 12 mmol). FC (PE:EtOAc 2:1-1:
1) afforded 2.14 g (92%) of the title compound.
Diethyl 2-tert-Butyloxycarbonylamino-2-(4-ethyl-1-hydroxy-
pyrazol-5-yl)-malonate (12b). From 11b (1.0 g, 2.1 mmol). FC
(PE:EtOAc 2:1) afforded 789 mg (97%) of 12b as a yellowish oil.
Diethyl 2-tert-Butyloxycarbonylamino-2-[4-propyl-1-hydroxy-
pyrazol-5-yl]-malonate (12c). From 11c (1.71 g, 3.5 mmol). FC
(PE:EtOAc 2:1) afforded 1.06 g (97%) of 12c as a yellowish oil.
Diethyl 2-tert-Butyloxycarbonylamino-2-[4-isopropyl-1-hy-
droxypyrazol-5-yl]-malonate (12d). From 11d (1.0 g 2.04 mmol).
FC (PE:EtOAc 2:1) afforded 1.06 g (97%) of 12d as a white
semisolid.
General Procedure for Dehydroxylation. To a solution of the
alcohol (9a-9d) (1 mmol) and triethylsilane (2 mmol) in CHCl3
(10 mL), trifluoroacetic acid (5 mL) was added dropwise at room
temperature, and the resulting solution was stirred at reflux for 2 h.
The mixture was cooled, water (10 mL) was added, and the aqueous
phase was extracted with diethyl ether (3 × 10 mL). The combined
organic phases were dried over MgSO4 and evaporated in vacuo.
FC (PE:EtOAc) afforded the title compound.
1-Benzyloxy-4-methylpyrazole (10a). From 9a (2 g, 9.8 mmol).
FC (PE:EtOAc 8:1) afforded 1.45 g (79%) of the title compound.
1-Benzyloxy-4-ethylpyrazole (10b). From 9b (1.3 g, 6 mmol).
FC (PE:EtOAc 8:1) afforded 1.13 g (95%) of the title compound.
Diethyl 2-tert-Butyloxycarbonylamino-2-(4-chloro-1-hydroxy-
pyrazol-5-yl)-malonate (12e). From 11e (1.5 g, 3.11 mmol). FC
(PE:EtOAc 2:1) afforded 1.08 g (88%) of 12e as a colorless oil.

4186 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Clausen et al.
General Procedure for Preparation of the Free Amino Acid.
To a stirred solution of the malonate (12a-e) (1 mmol) in THF
(10 mL), LiOH (2 M, aq, 9 mmol) was added, and the solution
was stirred for 6 h at r.t. The solution was cooled to 0 °C and
neutralized with citric acid (10%, aq.). The mixture was extracted
with EtOAc (3 × 20 mL), and the combined organic phases were
washed with brine (20 mL), dried over MgSO4, and evaporated in
vacuo. The resulting residue was stirred in HCl (2 M, 10 mL) at
50 °C for 30 min, and the resulting solution was evaporated to
dryness. The product was triturated with Et2O or CH3CN to afford
a solid that was more >95% pure according to NMR and HPLC.
Attempts to recrystallize the compounds resulted in degradation of
the products, leading to a less pure material. All the amino acids
were separated on a Crownpack CR(+) column with the (R)-form
eluting first.
Amino-(1-hydroxy-4-methyl-pyrazol-3-yl)-acetic Acid Hy-
drochloride (6b). From 12a (281 mg, 0.76 mmol) afforded 153
mg (97%) of 6b as a white solid.
Amino-(1-hydroxy-4-ethyl-pyrazol-3-yl)-acetic Acid Hydro-
chloride (6c). From 12b (759 mg, 1.97 mmol) afforded 247 mg
(60%) of 6c as a pale-yellow solid.
Amino-(1-hydroxy-4-propyl-pyrazol-3-yl)-acetic Acid Hydro-
chloride (6d). From 12c (1.06 mg, 2.65 mmol) afforded 450 mg
(72%) of 6d as a white solid.
Amino-(1-hydroxy-4-isopropyl-pyrazol-3-yl)-acetic Acid Hy-
drochloride (6e). From 12d (685 mg, 1.71 mmol) afforded 285
mg (71%) of 6e as a white solid.
Amino-(1-hydroxy-4-chloro-pyrazol-3-yl)-acetic Acid Hydro-
chloride (6f). From 12e (1.07 g, 2.7 mmol) afforded 0.536 mg
(86%) of 6f as a white solid.
Molecular Modeling and Ligand-Protein Docking. A homol-
ogy model of the agonized state of NR2B was constructed based
on the soluble NR2A-ABD construct in complex with glutamate
(PDB code 2A5S)11 following the procedure for the NR2B
homology model based on NR1 (1PB7). The crystal structure of
the soluble NR2A-S1S2 (PDB code 2A5S)11 was used as a
template for the ligand binding domain of NR2B. The sequence of
NR2B was aligned with NR2A, truncated, and the GT linker added
to form a virtual NR2B-ABD construct containing residues
404-540 followed by the GT linker and residues 662-802.
Residues are numbered according to the sequence of total wild type
NR2B, including the signal peptide. Using the NR2B construct, a
homology model was created using Prime (Schro¨dinger, Portland,
OR) with standard parameters. Van der Waals and electrostatic grids
within a (14Å + 14Å)3 box around the ligand position were
calculated on this model with the docking code Glide 2.5 (Schro¨-
dinger, Portland, OR); default parameters were used, apart from
the scaling of nonpolar atoms of the receptor, which was set to
0.9. These grids were then used for ligand docking.
The ligands (R)-Et-NHP5G and (R)-Pr-NHP5G were submitted
to Monte Carlo analysis in tri-ionized forms using the MMFFs force
field31,32 including GB-SA treatment of aqueous solvation in
Macromodel 8.1 (Schro¨dinger, Portland, OR). The global minima
of conformations without intramolecular hydrogen bonds of the
compounds were flexibly docked with Glide 3.5 to the agonist
binding site of the NR2B-S1S2 model. Default parameters were
used, apart from the scaling factors of the radii of the nonpolar
receptor and ligand atoms, both set to 0.9.
Computational Prediction of Spectra. Initial geometries for the
low pH monocationic states of (S)-6b and (S)-6f were calculated using
OPLS2005 with GB-SA aqueous solvation, and for the Mosher amide
of (S)-6c with chloroform solvation, in Macromodel 9.0 (Schro¨dinger
2006). For TD-DFT calculations, the conformers were first optimized
at B3LYP/6-311+G(d,p), and excitations calculated at the same level
of theory, with IEF-PCM treatment of aqueous solvation, in Gaussi-
an’03 (Gaussian 2003). Gaussian line broadening with a half-width
of 0.2 eV was used to transform rotatory velocities to simulated spectra.
NMR calculations were conducted on the optimized geometries at
B3LYP/6-311+G(d,p) with solvation by chloroform (PB-SCRF model)
in Jaguar 7.0 (Schro¨dinger 2006), with magnetic shielding tensors
calculated in gas phase at the same level of theory. Correlation
coefficients were calculated between experimental and calculated shifts
of all protons relative to TMS at the same level of theory, neglecting
the five phenyl protons, which are of little value in distinguishing the
diastereomers.
In Vitro Pharmacology. Receptor Binding Assays. Affinities
for native AMPA, KA, and NMDA receptors in rat cortical
synaptosomes were determined using 5 nM [3H]AMPA (45.5 Ci/
mmol),33 5 nM [3H]KA (47.0 Ci/mmol),34 and 2 nM [3H]CGP
39653 (Kd ) 6 nM, 50.0 Ci/mmol),35 respectively, with minor
modifications as previously described.36 Rat brain membrane
preparations used in these receptor binding experiments were
prepared according to a method previously described.37
Two-Electrode Voltage-Clamp Electrophysiology. For expres-
sion in Xenopus oocytes, rat NR1 (GenBank U11418) cDNA was
subcloned into pCI-IRES-neo38 or pGEMHE vectors. The open
reading frames of rat NR2A (GenBank D13211), NR2B (M91562),
NR2C (D13212), and NR2D (D13214) cDNAs were subcloned into
pCI-IRES-bla38 or pCI-neo (NR2A), pBluescript (NR2B), SP6
(NR2C-D) vectors. Xenopus oocytes and cRNA for injection were
prepared as previously described.39 Two-electrode voltage-clamp
recordings were performed essentially as previously described.39
During recordings, the oocytes were voltage-clamped at -60 to
-40 mV and continuously perfused at 23 °C with extracellular
solution containing (in mM) 90 NaCl, 0.5 BaCl2, 1 KCl, 0.01
EDTA, and 10 HEPES (pH 7.6); 20 µM glycine was included in
the extracellular solution at all times.
Agonist concentration-response data for individual oocytes were
fitted to the Hill equation. The log EC50 and nH from the individual
oocytes were used to calculate the mean ( SEM. For graphical
presentation, data sets from individual oocytes were normalized to
the maximal response evoked by glutamate in the same recording.
The mean ( SEM was calculated for each of the normalized data
points. The averaged data points were then fitted to the Hill equation
and plotted together with the resulting curve. Relative Imax was
calculated from a full concentration-response measurement as
Imax,agonist/Imax,glutamate, where Imax,agonist is the fitted value according
to the Hill equation and Imax,glutamate is the maximal response evoked
by glutamate in the same recording.
Acknowledgment. Lars Skov and Michael Gajhede are
acknowledged for their help with CD measurements. We thank
Kimberly Haustein and Phuong Le for excellent technical
assistance. We thank Drs. S. Heinemann (Salk Institute), S.
Nakanishi (Kyoto University), and P. Seeburg (University of
Heidelberg) for sharing cDNA encoding the NMDA receptor
subunits. Tommy Liljefors is acknowledged for fruitful discus-
sions on modeling and design. This work was supported by NIH
(S.F.T.), the Carlsberg Foundation (C.C.), the Alfred Benzon
Foundation (K.B.H.), Villum Kann Rasmussen Foundation
(K.B.H.), the Lundbeck Foundation (K.B.H.), the Drug Research
Academy (N.M.), and the Augustinus Foundation (H.B.O.).
Supporting Information Available: Routine NMR spectra,
routine NMR data, preparation of Mosher amides, CD spectra,
HPLC traces, and table of purity data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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