Synthesis and pharmacology of N1-substituted piperazine-2,3-dicarboxylic acid derivatives acting as NMDA receptor antagonists

Article abstract of DOI:10.1021/jm0492498

The binding site for competitive NMDA receptor antagonists is on the NR2 subunit, of which there are four types (NR2A-D). Typical antagonists such as (R)-AP5 have a subunit selectivity of NR2A > NR2B > NR2C > NR2D. The competitive NMDA receptor antagonist

Full text of DOI:10.1021/jm0492498

J. Med. Chem. 2005, 48, 2627-2637
2627
Synthesis and Pharmacology of N1-Substituted Piperazine-2,3-dicarboxylic Acid
Derivatives Acting as NMDA Receptor Antagonists
Richard M. Morley,† Heong-Wai Tse,† Bihua Feng,‡ Jacqueline C. Miller,† Daniel T. Monaghan,‡ and
David E. Jane*,†
Department of Pharmacology, MRC Centre for Synaptic Plasticity, School of Medical Sciences, University Walk,
University of Bristol, Bristol, BS8 1TD, UK, and Department of Pharmacology, University of Nebraska Medical Center,
Omaha, Nebraska 68198-6260
Received September 15, 2004
The binding site for competitive NMDA receptor antagonists is on the NR2 subunit, of which
there are four types (NR2A-D). Typical antagonists such as (R)-AP5 have a subunit selectivity
of NR2A > NR2B > NR2C > NR2D. The competitive NMDA receptor antagonist (2R*,3S*)-
(1-biphenylyl-4-carbonyl)piperazine-2,3-dicarboxylic acid (PBPD, 16b) displays an unusual
selectivity with improved relative affinity for NR2C and NR2D vs NR2A and NR2B. Analogues
of 16b bearing aroyl or aryl substituents attached to the N1 position of piperazine-2,3-
dicarboxylic acid have been synthesized to probe the structural requirements for NR2C/NR2D
selectivity. A phenanthrenyl-2-carbonyl analogue, 16e, had >60-fold higher affinity for NR2C
and NR2D and showed 3-5-fold selectivity for NR2C/NR2D vs NR2A/NR2B. The phenan-
threnyl-3-carbonyl analogue (16f) was less potent but more selective, having 5- and 7-fold
selectivity for NR2D vs NR2A and NR2B, respectively. Thus, antagonists bearing bulky
hydrophobic residues have a different NR2 subunit selectivity than that of typical antagonists.
Introduction
Ionotropic glutamate receptors are glutamate-gated
ion channels that have been classified into three main
types, the N-methyl-D-aspartate (NMDA, 1, Figure 1),
AMPA, and kainate receptors based on their pharma-
cology and more recently their amino acid sequence
homology.1,2 The NMDA receptors play an important
role in synaptic transmission and in particular in
neuronal synaptic plasticity, which is thought to under-
lie learning and memory. In addition, NMDA receptors
are involved in a variety of pathological conditions,
including epilepsy, pain-related disorders, and disorders
arising from neuronal cell death following stroke or head
Figure 1. Structures of NMDA and previously reported
competitive NMDA receptor antagonists.
injury as well as psychiatric disorders such as schizo-
phrenia.3
NMDA receptors are composed of subunits from the
NR1a-h and NR2A-D families of subunits. The NR3A
subunit may play a regulatory role in the functioning
of some NMDA receptors likely controlling Ca2+ ion
influx through the channel.4 The glutamate recognition
site is located on the NR2 subunit,5 while the glycine
coagonist binding site is located on the NR1 subunit.6
It has been proposed that the different NR2 subunits
may be responsible for the distinct glutamate binding
site pharmacologies of NR1/NR2 combinations7-9 and
that this may underlie the diverse NMDA receptor
pharmacologies in native rat brain regions.8,10,11 The
first potent and selective antagonists, such as (R)-AP5
(2) and (R)-AP7 (3) described by Watkins and co-
workers12 and those described later based on the
structure of these lead compounds (such as CPP, 4),13
had very similar patterns of selectivity when tested on
native or recombinant NMDA receptors8,14,15 (see Figure
1 for structures). In general, the rank order of potency
of the competitive antagonists that have been tested on
recombinant NMDA receptors is NR2A > NR2B >
NR2C > NR2D. The only subunit selective antagonists
are selective for the polyamine binding site(s) on the
NR2B subunit, which is distinct from the glutamate
binding site.2 Recently, a competitive antagonist based
on the quinoxalinedione nucleus, PEAQX (5, Figure 1),
has been reported to be selective for NR2A vs NR2B;16
however, this compound has little selectivity for NR2A
vs NR2C or NR2D.17 In our search for subunit selective
NMDA receptor antagonists we have identified PBPD
(16b, Figure 1), which has an unusual selectivity profile,
NR2D > NR2B > NR2C > NR2A.9 We have hypoth-
esized that the moderately potent antagonist activity
exhibited by 16b is probably due to the interaction of
the hydrophobic biphenyl residue with hydrophobic
amino acid residues within the receptor and that this
interaction may be responsible for the unique NR2
subunit selectivity of 16b. We report herein the syn-
* To whom correspondence should be addressed. Phone: +44 (0)117
9546451. Fax: +44 (0)117 9250168. E-mail: david.jane@bristol.ac.uk.
† University of Bristol.
‡ University of Nebraska Medical Center.
10.1021/jm0492498 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/18/2005

2628 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Morley et al.
Scheme 1a
a Reagents and conditions: (a) (i) KOH (aq), 10% Pd/C, H2, (ii)
HNO3, (iii) DOWEX 50WX8-400 H+ form ion-exchange resin.
Scheme 2a
Figure 2. Molecular structure of 10 illustrating the trans
arrangement of the ethyl carboxylate groups. Ethyl and benzyl
hydrogen atoms have been omitted for clarity.
which the benzyl protected diamine (9) was reacted with
8 gave the protected piperazine (10) in reasonable yield.
This gave the required (2R*,3R*)-isomer (2) upon
catalytic hydrogenation to remove the benzyl protecting
groups and hydrolysis of the resulting ester (11) in
ethanolic sodium hydroxide. Compound 10 proved to
have the trans-(2R*,3R*)-configuration when analyzed
using X-ray crystallography (Figure 2) and by implica-
tion 12 must have the (2R*,3R*)-configuration. The 1H
NMR spectra of 12 and 7 were found to be different and
therefore the isomer produced by catalytic hydrogena-
tion of 6 must have the cis-(2R*,3S*)-configuration.
a Reagents and conditions: (a) Et3N, C6H6; (b) 10% Pd/C, H2,
EtOH; (c) (i) NaOH, EtOH (ii) HNO3 (aq), (iii) DOWEX 50WX8-
400 H+ form ion-exchange resin.
thesis of 16b and a range of new analogues with the
aim of probing the hydrophobic binding site and inves-
tigating how modification of the hydrophobic substituent
influences NR2 subunit selectivity. The compounds have
been evaluated as antagonists of currents coactivated
by (S)-glutamate and glycine in Xenopus oocytes ex-
pressing rat recombinant NR1a/NR2 subunits.
Results
A series of N-substituted analogues (16a-n) of 7
including the lead compound 16b were prepared using
modified Schotten-Baumen conditions by reacting the
acid chlorides (15a-n) with the cis-(2R*,3S*)-isomer 7
in the presence of sodium hydroxide in aqueous dioxane
(Scheme 3). The acid chlorides (15a-n) were prepared
from the corresponding carboxylic acids (14a-n) by
treatment with excess thionyl chloride. The carboxylic
acids (14a-n) were either commercially available or
were synthesized by standard methods. The final prod-
ucts (16a-n) were conveniently isolated by acidification
of the reaction mixture and could be readily purified by
redissolving in aqueous sodium hydroxide, acidification
with hydrochloric acid, and washing the resulting
precipitate with dioxane to remove contaminating car-
boxylic acid starting material. Crystallization from hot
water was avoided, as this led to hydrolysis of the amide
back to the carboxylic acid. The low yield (10-30%)
observed for a number of the products is likely due to
hydrolysis of the acid chloride in the aqueous basic
conditions and possibly also due to formation of N1,N4-
disubstituted compounds that were not isolated using
the workup conditions described. The 1H NMR spectra
of 16a-n have two sets of doublets for the methine
protons on the piperazine ring, suggesting that two
rotamers are present on the NMR time scale due to
restricted rotation around the amide bond.
Chemistry. The starting material for most of the
compounds synthesized was the meso-isomer (2R*,3S*)-
piperazine-2,3-dicarboxylic acid (7). This could conven-
iently be prepared in good yield by palladium-catalyzed
hydrogenation of the corresponding pyrazine (6) (Scheme
1). It has been previously reported that this method
gives the (2R*,3R*)-isomer (12), as the product could
be resolved to give the two enantiomers.18 It is possible
that previous workers detected a small rotation ([R]D )
+ or - 0.3) in their “resolved product” due to contami-
nation from the trans-isomer, which may have been
produced via epimerization during the course of the
resolution procedure. In support of this theory, the
rotation values for the separate enantiomers were
obtained from a solution of 20 g/100 mL, and therefore,
even a low percentage contamination of the resolved
trans-isomer would be enough to give a small rotation.
To determine whether hydrogenation of 6 led to the cis-
or trans-isomer, an independent synthesis of 12 was
undertaken. A method reported to give 12 in good yield19
involving reaction of 820 (Scheme 2) with N,N-bis-p-
tolylsulfonylethylene-1,2-diamine was attempted, but in
our hands this method gave only poor yields of the
required compound and suffered from the disadvantage
of having to remove the p-tolylsulfonamide groups,
which proved difficult. A modification of this method in

N1-Substituted Piperazine-2,3-dicarboxylic Acids
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2629
Scheme 3a
Scheme 4a
a Reagents and conditions: (a) (i) LiAlH4, THF, (ii) NH4Cl (aq);
(b) KI, BF3·Et2O, dioxane; (c) H2 10% Pd/C; (d) (i) 20, Et3N, THF,
0 °C, 2 h and then room temp, 12 h; (e) (i) LiOH, THF/H2O (2:1),
(ii) 2 M HCl (aq).
of 13 was accomplished by acylation of 12 with acid
chloride (15e) under modified Schotten-Baumen condi-
tions.
Pharmacology. Activity at Recombinant NMDA
Receptors. The antagonist effects of compounds were
tested on each of the four different NR2 subunits
coexpressed with NR1a in Xenopus oocytes using an
electrophysiological assay. Variations in NR1 subunits
splice forms were not examined because they do not
have the glutamate binding site and because there is
no evidence that the splice forms alter glutamate-site
antagonist specificity. Each of the four NR2 subunits
was examined, since these represent the four genetically
distinct glutamate binding sites.8,10,11 These four binding
sites account for essentially all of the known variations
in glutamate-site antagonist specificity at the NMDA
receptors from both physiological and radioligand bind-
ing experiments.8,9,14,15 Antagonist activity was mea-
sured as IC50 values for the depression of currents
evoked by stimulation with (S)-glutamate (10 µM) and
glycine (10 µM). These IC50 values were converted to Ki
values to take into account differences in agonist
potency for the different NR2 subunits.
The Ki values of all of the new compounds and four
reference substances (2-5) are presented in Table 1,
while Table 2 contains the data normalized to the Ki
values for NR2C to gain a better appreciation of the NR2
subunit selectivity of the compounds. Unlike the refer-
ence substances 2-4, which display a rank order of
potency of NR2A> NR2B > NR2C > NR2D, 16b
displayed an unusual subunit selectivity with the rank
order of potency being NR2D > NR2B > NR2C > NR2A
though the difference in Ki values was only 3-fold at the
most. An analogue of 16b with only one benzene ring
attached to the carbonyl group (16a) was inactive at all
NR2 subunits up to a concentration of 100 µM. The 2′-
bromobiphenylyl-substituted derivative (16c) had ap-
proximately the same affinity for NR2C but much lower
affinity for the other NR2 subunits than 16b and
displayed selectivity for NR2C. In contrast, the 4′-
fluorobiphenylyl-substituted derivative (16d) had ap-
proximately 2-fold greater affinity for NR2A compared
to 16b and as a result had less than a 2-fold difference
in affinity across all four NR2 subunits. When the first
aromatic ring of 16b was replaced with an (E)-ethenyl
a Reagents and conditions: (a) SOCl2, C6H6, 80 °C, 12 h; (b) (i)
7, NaOH, dioxane/H2O (2:1), 0 °C, 2 h and then rt, 12 h, (ii) 2 M
HCl (aq).
To investigate the role of the amide group on NMDA
receptor antagonist activity, compounds were synthe-
sized that had a sulfonyl (17, Scheme 3) or a methylene
(23) rather than a carbonyl group between the N1 atom
of 7 and the aryl moiety. The sulfonyl derivative (17)
was synthesized under similar conditions to those used
for the synthesis of 16a-n by reacting commercially
available biphenylsulfonyl choride with 7. The methyl-
ene derivative (23) was synthesized by alkylation of 21
with the iodomethylphenanthrene (19) and hydrolysis
of the intermediate (22) with lithium hydroxide in THF/
H2O. The synthesis of 19 was achieved by reduction of
carboxylic acid (14e) with lithium aluminum hydride
and treatment of the resulting alcohol (18)21 with
potassium iodide and boron trifluoride etherate in
dioxane (Scheme 4).22 The piperazine (21) was obtained
by palladium-catalyzed hydrogenation of pyrazine (20).
The coupling constants for the methine protons on the
piperazine ring of 23 were very similar to those for the
cis-isomer 16e and larger than those for the trans-
isomer 13, suggesting that the carboxylic acid groups
have the cis-configuration in compound 23. There was
no evidence for the presence of rotamers in the 1H NMR
spectrum of 23, backing up the theory that the presence
of two sets of doublets for the methine protons on the
carbon atom R to the carboxylic acid groups on the
piperazine ring in 16a-n is due to rotamers caused by
restricted rotation around the amide bond.
To determine whether a cis or trans arrangement of
the carboxylic acid groups on the piperazine ring was
necessary for activity at NMDA receptors, the trans-
(2R*,3R*)-isomer (13) was synthesized. The synthesis

2630 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Morley et al.
Table 1. Antagonist Ki Values (µM) for Inhibiting the Responses of Recombinant Rat NMDA Receptors Expressed in Xenopus
Oocytes (means ( SEM (n))
NR1a/NR2A
0.28 ( 0.02
NR1a/NR2B
0.46 ( 0.14
NR1a/NR2C
1.64 ( 0.14
NR1a/NR2D
3.71 ( 0.67
2, (R)-AP5a
3, (R)-AP7
0.49 ( 0.18 (5)
0.041 ( 0.003 (6)
0.0054 ( 0.0004 (5)
>100
4.14 ( 0.36 (5)
0.27 ( 0.02 (5)
0.067 ( 0.003 (5)
>100
6.42 ( 1.08 (6)
0.63 ( 0.05 (5)
0.0116 ( 0.0009 (5)
>100
17.10 ( 0.65 (5)
1.99 ( 0.20 (5)
0.037 ( 0.004 (4)
>100
4, (R)-CPP
5, PEAQX
16a, UBP130
16b, PBPDa
16c, UBP101
16d, UBP102
16e, PPDA
16f, UBP141
16g, UBP142
16h, UBP135
16i, UBP137
16j, UBP132
16k, UBP106
16l, UBP112
16m, UBP131
16n, UBP129
17, UBP124
23, UBP140
13, UBP138
15.79 ( 0.43
5.01 ( 0.25
8.98 ( 0.18
4.29 ( 0.11
42.9 ( 5.3 (4)
8.78 ( 0.88 (5)
0.55 ( 0.15 (3)
14.19 ( 1.11 (5)
0.3200 ( 0.0164 (5)
1.54 ( 0.11 (6)
3.88 ( 0.19 (5)
4.43 ( 0.44 (6)
7.97 ( 0.77 (4)
13.2 ( 0.89 (5)
9.6 ( 1.1 (5)
22.1 ( 5.2 (5)
13.4 ( 0.8 (5)
0.31 ( 0.02 (5)
19.29 ( 1.38 (4)
0.2500 ( 0.0268 (5)
0.76 ( 0.11 (5)
2.15 ( 0.31 (5)
3.25 ( 0.21 (6)
9.36 ( 1.41 (6)
18.0 ( 2.4 (4)
18.2 ( 1.3 (5)
0.3 ( 0.06 (4)
16.2 ( 4.1 (5)
10.25 ( 1.87 (4)
9.08 ( 1.02 (3)
8.49 ( 0.72 (4)
7.38 ( 0.38 (5)
0.096 ( 0.006 (4)
4.22 ( 0.52 (6)
0.0994 ( 0.0043 (5)
0.29 ( 0.02 (5)
0.66 ( 0.07 (4)
1.17 ( 0.06 (5)
4.42 ( 0.40 (6)
27.0 ( 3.1 (5)
3.89 ( 0.09 (5)
0.85 ( 0.19 (5)
8.67 ( 0.62 (5)
2.67 ( 0.13 (4)
4.88 ( 0.57 (5)
12.9 ( 1.1 (4)
8.02 ( 0.31 (5)
0.125 ( 0.035 (5)
2.78 ( 0.16 (5)
0.1500 ( 0.0095 (5)
0.57 ( 0.03 (5)
1.03 ( 0.09 (5)
2.17 ( 0.32 (6)
6.45 ( 0.40 (5)
59.7 ( 8.5 (6)
9.26 ( 0.43 (6)
1.94 ( 0.14 (5)
10.88 ( 0.45 (5)
3.18 ( 0.19 (4)
6.03 ( 2.01 (3)
0.84 ( 0.08 (10)
13.6 ( 1.8 (5)
11.18 ( 1.29 (4)
10.48 ( 1.23 (5)
a Values for 2 and 16b represent n ) 4-6 from ref 9.
Table 2. Potency Relative to Activity on NR1a/NR2C (Ki
NR2/Ki NR2C)
and NR2D vs NR2A and NR2B, which is the opposite
of “typical” competitive NMDA receptor antagonists
such as 2. A positional isomer of 16e with the carbonyl
linker attached to the 3-position of the phenanthrene
ring (16f) had lower affinity across all of the NR2
subunits but showed a 5- and 7-fold selectivity for NR2D
vs NR2A and NR2B, respectively. The 7-bromo analogue
16g of 16e had marginally less affinity than 16e for
NR2C and NR2D but had slightly higher affinity for
NR2A and NR2B and was therefore less selective for
NR2C/NR2D overall. A saturated ethylene bridge be-
tween the two phenyl rings was not as well tolerated,
as 16h had lower affinity than 16e across all four NR2B
subunits, though it did show a similar pattern of NR2
subunit selectivity to 16e. The 7-bromo analogue 16i
had ∼2-fold lower affinity across the NR2 subunits than
16h but showed almost 6-fold selectivity for NR2C vs
NR2A, making it the most NR2C selective antagonist
tested. Replacement of the ethylene bridge with either
a methylene (16j) or keto (16k) group spacer resulted
in a much decreased affinity across all NR2 subunits
compared to 16e and also a decrease in the NR2C/NR2D
vs NR2A/NR2B selectivity. Compound 16m in which the
last aromatic ring of 16e is missing had much lower
affinity for NR2C and NR2D than 16e but showed
almost 5-fold selectivity for NR2C vs NR2B. In agree-
ment with the observed AP5-like pattern of activity of
16l, an analogue of 16e with an ethenyl linker instead
of the first phenyl ring 16n had higher affinity for NR2B
than NR2C and NR2D but unlike 2 had similar affinity
for NR2A and NR2C.
NR1a/NR2A
NR1a/NR2B
NR1a/NR2D
2, (R)-AP5
0.17
0.076
0.065
0.47
1.76
5.05
1.19
5.73
3.36
3.2
0.28
0.64
0.43
5.78
0.56
2.6
1.81
3.23
4.57
2.5
2.62
3.26
2.78
2.12
0.66
4.68
0.35
1.87
3.84
1.86
2.26
2.66
3.16
3.19
0.48
1.52
1.09
1.3
0.66
1.51
1.97
1.56
1.85
1.46
2.2
3, (R)-AP7
4, (R)-CPP
5, PEAQX
16b, PBPD
16c, UBP101
16d, UBP102
16e, PPDA
16f, UBP141
16g, UBP142
16h, UBP135
16i, UBP137
16j, UBP132
16k, UBP106
16l, UBP112
16m, UBP131
16n, UBP129
17, UBP124
23, UBP140
13, UBP138
5.3
5.88
3.79
1.8
0.49
2.47
0.99
1.57
4.19
2.15
2.38
2.28
1.25
1.19
1.24
linker to give 16l, there was a 3-10-fold loss of affinity
across the NR2B-D subunits but a slight increase in
affinity on NR2A. Compound 16l therefore had a NR2
subunit selectivity similar to typical competitive an-
tagonists such as 2. When the carbonyl group linking
the biphenyl ring of 16b to the piperazine ring was
replaced with a sulfonyl linker to give 17, there was a
decrease in affinity for NR2B and NR2D which resulted
in the NR2 selectivity of 17 being less than 2-fold across
the four NR2 subunits.
Replacement of the carbonyl linker in 16e with a
methylene group to give compound 23 resulted in a 20-
30-fold lowering of affinity across all four subunits, but
the pattern of NR2 subunit selectivity was similar to
that of 16e. Changing from a cis- to a trans-configura-
tion of the carboxylic acid groups on the piperazine ring
of 16e also resulted in a lowering of affinity across all
four subunits. In this case compound 13 had an ∼50-
fold lower affinity on NR2C and NR2D compared to
16e and a lower selectivity for NR2C vs NR2A and
NR2B.
A dramatic increase in potency was observed when
the two phenyl rings of 16b were joined together by an
additional phenyl ring, thereby making the aromatic
rings coplanar. The resulting compound 16e had much
higher affinity across all of the NR2 subunits than the
parent compound 16b with Ki values of 96 ( 6 and 125
( 35 nM on NR2C and NR2D (an ANOVA with
Newman-Keuls multiple comparison test suggests that
the Ki values for the NR2 subunits were significantly
different from each other except between NR2C and
NR2D). In addition, 16e showed selectivity for NR2C

N1-Substituted Piperazine-2,3-dicarboxylic Acids
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2631
515) display a higher affinity for NR2B than for NR2A-
containing receptors and have an improved relative
affinity for NR2C- and NR2D-containing receptors.9
Thus, bulky hydrophobic substituents alter the subunit
specificity of NMDA receptor antagonists. It has been
proposed that the biphenyl group of EAB-515 binds to
a hydrophobic binding pocket25,26 presumably corre-
sponding to the “allowed” region described in antagonist
modeling studies.23
A conformationally restricted analogue (16b) of 1 has
been previously shown to be an NMDA receptor antago-
nist.9 Normally competitive NMDA receptor antagonists
have five or seven bonds between the proximal and
distal acidic groups. Unusually, although 16b is an
antagonist, it has a short interacidic group chain length
that is more commonly observed with agonists such as
1.23 It is therefore likely that the amino acid end of 16b
is binding to the same sites in the receptor as 1 and
that it is the large bulky biphenyl ring substituent that
is responsible for the antagonist activity. We have
shown that 16b displays a higher affinity for NR2B than
for NR2A and has an improved relative affinity for
NR2C- and NR2D-containing vs NR2A- and NR2B-
containing receptors, and it is likely that the bulky
biphenyl residue is responsible for this unusual NR2
subunit selectivity.9
Though the difference in affinity between NR2D and
NR2A was only 3-fold, 16b represents a lead for the
development of NR2D selective antagonists. A series of
compounds based on the structure of 16b was designed
to investigate the effect on NR2 subunit selectivity of
adding substituents to the aryl ring (16c,d,g,i), the
position of substitution of the carbonyl linker on the
aromatic ring (16f), changing the aromatic ring (16a,
e,h,j-n), the nature of the group linking the aryl ring
to the piperazine ring (17 and 23), and the configuration
of the carboxylic acid groups attached to the piperazine
ring (16e and 13).
In general, structural modifications of 16b did not
lead to antagonists with high selectivity for individual
NR2 subunits (see Tables 1 and 2). However, some of
the structural alterations did change the affinity for
certain NR2 subunits and also changed the pattern of
NR2 subunit selectivity observed with 16b. The most
profound effect was observed upon substituting the
biphenyl ring of 16b with a phenanthrene ring to form
16e. This has the effect of locking the first and last
aromatic rings of the biphenyl substituent into a
coplanar arrangement, which is different than that
observed with the biphenyl ring system, where the
benzene rings are twisted out of the plane. The effect
on the affinity across all four NR2 subunits was
dramatic as 16e had 94- and 64-fold higher affinity for
NR2C and NR2D, respectively, while increases in af-
finity on NR2A and NR2B were more modest. The
pattern of selectivity was NR2C > NR2D > NR2B >
NR2A and 16e was almost 6-fold selective for NR2C vs
NR2A.
Figure 3. Schild analysis of the antagonism of NMDA-
induced depolarizations of neonatal rat motoneurones by five
different concentrations of 16e. Each point represents the
mean of three independent determinations of the dose ratio
(DR).
Activity at NMDA Receptors on Neonatal Rat Spinal
Motoneurones. The most potent antagonist identified in
this study 16e was tested as an antagonist of NMDA-
induced depolarizations of neonatal rat motoneurones.
To calculate the pA2 value for the antagonism of NMDA-
induced depolarizations by 16e, noncumulative concen-
tration-response curves for NMDA were constructed
both in the absence and presence of 0.5, 0.7, 1, 2, and 3
µM 16e. From a Schild plot of the data (Figure 3) a pA2
value of 6.45 and a slope of 0.96 ( 0.07 (n ) 3 for each
data point) was calculated suggestive of a competitive
mode of antagonism.
Discussion
Most of the competitive NMDA receptor antagonists
reported to date are conformationally restricted ana-
logues based on the structure of 2 or 3.23,24 These
antagonists tend to display a typical pattern of selectiv-
ity on recombinant NR2 subunits with the rank order
of affinity being NR2A > NR2B > NR2C > NR2D.8,14,15
We have previously evaluated the four reference sub-
stances in Table 1 including the well-established an-
tagonists 2, 3, and 412,13 and the recently reported
NR2A-selective antagonist 516 as antagonists of recom-
binant NMDA receptors. Three of the reference sub-
stances (2-4) used in this study displayed the “typical”
competitive antagonist profile noted for antagonists
based on the AP5 or AP7 structure. Indeed, 4 displayed
the greatest selectivity for NR2A vs NR2D, with ap-
proximately 50-fold higher affinity for NR2A than
NR2D. However, 5 displayed a higher affinity for NR2A-
and NR2C-containing receptors than for NR2B- and
NR2D-containing receptors, but in our hands it did not
display the >100-fold difference in affinity between
NR2A and NR2B reported by Auberson et al.16 This may
be due to a species difference, as rat rather than human
recombinant NMDA receptors were used in the present
study.
We have previously reported that a few compet-
itive NMDA receptor antagonists display an altered
pattern of NR2 subunit selectivity. The conforma-
tionally restricted AP7 analogue (3S,4aR,6S,8aR)-6-
(1H-tetrazol-5-ylmethyl)decahydroisoquinoline-3-car-
boxylic acid (LY233536) and the AP5 analogue with a
bulky biphenyl-containing substituent (S)-2-amino-3-(5-
phosphonomethylbiphenyl-3-yl)propionic acid (EAB-
It has been previously reported that the N1-4-bromo-
benzoyl and N1-4-chlorobenzoyl analogues of cis-piper-
azine-2,3-dicarboxylic acid are selective for NMDA vs
non-NMDA receptors in the hippocampus, but the
selectivity is reversed when these compounds were
tested on neonatal rat spinal cord motoneurones.27,28

2632 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Morley et al.
The activity of these compounds on non-NMDA recep-
tors was fairly weak (KD values > 500 µM). Preliminary
data from binding studies suggests that 16e has about
100-fold higher affinity at NMDA vs AMPA receptor
binding sites (D.T.M., unpublished observations).
A positional isomer of 16e with the carbonyl linker
attached to the 3-position of the phenanthrene ring (16f)
had 40- and 20-fold lower affinity for NR2C and NR2D,
respectively; however, this compound had a similar
pattern of selectivity to 16e. Indeed 16f was more
selective than 16e, being 5- and 7-fold selective for
NR2D vs NR2A and NR2B, respectively making it the
most selective NR2D antagonist reported to date. Thus,
the position of substitution of the carbonyl linker on the
aromatic moiety has a profound effect on the affinity
for each of the NR2 subunits and also influences NR2D
selectivity.
Substituting the phenanthrene ring of 16e for a 9,10-
dihydrophenanthrene ring to form 16h resulted in a
3-5-fold reduction of affinity for NR2C and NR2D and
also lowered NR2C selectivity, suggesting that a co-
planar arrangement of the aromatic rings is favored for
binding to NR2C and NR2D. An additional consider-
ation is that the π electrons in the middle ring may be
contributing to the interaction between the ligand and
the receptor. If either a methylene (16j) or keto (16k)
group was used to link the two phenyl rings of 16b, then
there was a decrease in affinity across all four NR2
subunits as well as a decrease in NR2C selectivity,
suggesting that the middle ring needs to be a six-
membered aromatic ring as found in 16e for optimal
NR2C binding.
To gain insight into the optimal number of phenyl
rings required for high NR2 subunit affinity compounds
16a,j,m,n were synthesized. When one phenyl ring was
removed from 16b to form 16a, there was a total loss of
affinity across all four NR2 subunits. Interestingly, if
the first aromatic ring of 16b was replaced with an (E)-
ethenyl spacer, then AP5-like subunit selectivity was
observed as 16l displayed the following rank order of
affinity: NR2A > NR2B > NR2C > NR2D. A generally
similar rank order of affinity was observed for compound
16n, where the first phenyl ring of 16e was replaced
with an (E)-ethenyl substituent. Thus it would appear
that the first phenyl ring is essential for NR2C/NR2D
vs NR2A/NR2B selectivity. Removal of the last aromatic
ring of the phenanthrene ring of 16e led to compound
16m, which had a reduced affinity across all four NR2
subunits compared to 16e but an unusual pattern of
selectivity: NR2C > NR2A > NR2B > NR2D. Thus it
would appear that all three rings in 16e contribute to
the observed high affinity and NR2C/NR2D selectivity.
tion on NR2 subunit affinity and pattern of selectivity
vary depending on the nature of the aromatic substitu-
ent and the position of halo substitution on the aromatic
ring. This pharmacological heterogeneity may be im-
portant in the design of subunit selective antagonists
in the future.
Changing the group linking the aromatic substituent
to the piperazine ring of 16b from a carbonyl group to
a sulfonyl group (17) reduced affinity at NR2B and
NR2D, thereby removing the overall NR2B/NR2D se-
lectivity of 16b. Replacement of the carbonyl linker of
16e with a methylene group (23) led to an ∼30-fold drop
in affinity for NR2C and NR2D; however, the pattern
of NR2 subunit selectivity was similar to that of 16e.
Thus, a carbonyl linker is preferred over a methylene
or sulfonyl group for enhancing potency at NR2D,
though for binding to NR2C a methylene group is
detrimental but a sulfonyl group is an acceptable linker.
The profound difference in affinity observed on replace-
ment of the carbonyl group linker is likely due to the
tight restrictions imposed by the hydrophobic binding
site in the NR2 subunit on the orientation of the bulky
aromatic ring.
The configuration of the carboxylic acid groups on the
piperazine ring has a profound influence on subunit
selectivity as a cis arrangement in 16e leads to high
affinity across all NR2 subunits and a NR2C/NR2D
selectivity while the trans-isomer (13) has an ∼60-fold
lower affinity for NR2C and NR2D and very little
selectivity for NR2C/NR2D vs NR2A/NR2B.
As 16e was the most potent antagonist identified in
this study it was characterized as an antagonist of
native NMDA receptors expressed on neonatal rat
spinal motoneurones. 16e was found to be a potent
antagonist, and Schild analysis revealed a pA2 value of
6.45. The Schild plot was a straight line with a slope
close to 1 suggestive of a competitive mode of antago-
nism for 16e.
We have developed homology models of the S1/S2
domain of NR2 subunits based upon the X-ray crystal
structure of the glutamate binding core of the structur-
ally related GluR2 receptor29 and more recently based
on the X-ray crystal structure of NR1.30 Molecular
modeling studies in our laboratory using these homology
models suggest that the hydrophobic pocket for bulky
aromatic substituents represents the amino acids lining
the cleft between the S1 and S2 domains (Kinarsky, L.;
et al., submitted). In the case of 16e, the hydrophobic
pocket may represent the groove found in S2 at the base
of the ‘H’ helix29 that leads directly away from the
glutamate binding site, the S1/S2 hinge region, and out
toward the surface of the receptor. Most of the NR2
subunit-specific amino acid residues in the area around
the glutamate binding pocket are found near the surface
of the S1/S2 domain. Thus, only compounds with large
aromatic residues can come within reach of the subunit-
specific amino acid residues, and this is probably the
main reason only antagonists such as 16b, 16e, and 5
show altered subunit selectivity compared to more
typical AP5-like antagonists. Though the subunit se-
lectivity of 16b, 16e, and 16f is only modest, it might
be possible to improve NR2 subunit selectivity by adding
substituents to the distal benzene ring of these antago-
nists to target NR2 subunit specific amino acid residues
Halogen substitution of the biphenyl ring of 16b
resulted in differential effects on NMDA receptor sub-
unit selectivity depending on the position of ring sub-
stitution. Thus, 2′-bromo substitution of the biphenyl
ring decreased affinity at all NR2 subunits except
NR2C, while 4′-fluoro substitution enhanced affinity for
NR2A but decreased affinity at NR2B and NR2D.
However, 7-bromo substitution (16g) had marginal
effects on both affinity and pattern of selectivity across
all NR2 subunits. In contrast, 7-bromo substitution of
compound 16h led to an enhancement of selectivity for
NR2C vs NR2A. Thus, the effects of halogen substitu-

N1-Substituted Piperazine-2,3-dicarboxylic Acids
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2633
solvents were obtained from the Aldrich Chemical Co., unless
otherwise stated. The carboxylic acids (14a-n) were either
commercially available or were synthesized by standard
methods.
around the S1-S2 cleft. Further structure activity
relationship studies around the structure of 16e and 16f
are underway to investigate this theory.
(2R*,3S*)-Piperazine-2,3-dicarboxylic Acid (7). Pyr-
azine-2,3-dicarboxylic acid (6) (100 g, 0.595 mol) and potassium
hydroxide (66.8 g, 1.19 mol) were dissolved in water (1500 mL)
and hydrogenated under 3 atm of hydrogen in the presence of
platinum(IV) oxide (1 g) for 3 days. The reaction mixture was
filtered through Celite filter agent and concentrated under
reduced pressure to a volume of 200 mL. The solution was
acidified to pH 3.8 with concentrated nitric acid, yielding a
fine white precipitate that was filtered and air-dried to give 7
(57.8 g, 51%) as a white solid. The filtrate was concentrated
under reduced pressure and applied to a column of Dowex
50WX8-400 resin H+ form (100 mL). The column was eluted
with water until the eluent had a pH of 5 and then the prod-
uct was eluted with 10% aqueous ammonia solution. The
ninhydrin-positive fractions were combined and evaporated to
dryness under reduced pressure. The solid was purified by
crystallization from boiling water to give a further 48.4 g (42%)
Conclusions
We have developed a synthesis for a series of piper-
azine-2,3-dicarboxylic acid analogues based on the lead
structure 16b. One compound, 16e, proved to have
much higher affinity for NR2C and NR2D than 16b and
also showed an ∼6-fold selectivity for NR2C vs NR2A.
A positional isomer of 16e, 16f, though less potent than
16e, had a 7-fold selectivity for NR2D vs NR2B and is
therefore a useful lead for the design of NR2D selective
antagonists. The compounds synthesized in this study
extend previous work showing that NMDA receptor
antagonists bearing bulky hydrophobic residues are
capable of probing a different part of the antagonist
binding pocket, leading to a different subunit selectivity
compared to “typical” competitive antagonists such as
2. It would appear from our studies that for optimal
binding to NR2C or NR2D subunits that the bulky
hydrophobic residue is preferentially a planar aromatic
residue such as a phenanthrene ring.
Though the NR2C vs NR2A selectivity of 16e is only
∼6-fold when this compound is used in combination with
NR2A vs NR2D selective antagonists such as 4, the
involvement of individual subunits in physiological
processes can begin to be elucidated.31,32
Given the NR2C/NR2D subunit selectivity of 16e and
16f, they represent leads for the discovery of more
potent and NR2 subunit selective antagonists in the
future. These will not only be important in elucidating
the involvement of NR2C and NR2D subunits in physi-
ological processes in the CNS but may also have
therapeutic importance given that at present only
NR2A/NR2B or NR2B selective antagonists have been
investigated for therapeutic application.
1
of 7: mp 295-298 °C (dec); H NMR (400 MHz, D2O/NaOD,
pH 11) δ 2.67 (m, 2H, HNCH2), 2.78 (m, 2H, HNCH2), 3.53 (s,
2H, HNCHCO2H); 13C NMR (100 MHz, D2O/NaOD, pH 11) δ
45.34, 62.23, 181.34; MS (ES-) m/z ) 173.1 (M - H). Anal.
(C6H10N2O4‚H2O) C, H, N.
(2R*,3S*)-1,4-Dibenzylpiperazine-2,3-dicarboxylic Acid
Diethyl Ester (10). A solution of N,N-dibenzylethylene-1,2-
diamine (9) (48.3 g, 0.2 mol) and triethylamine (40.5 g, 0.4
mol) in anhydrous benzene (50 mL) was added dropwise to a
mechanically stirred solution of 2,3-dibromosuccinic acid di-
ethyl ester (8)20 (66.4 g, 0.2 mol) in anhydrous benzene (160
mL) at 40 °C under a dry argon atmosphere. The reaction
mixture was heated to 80 °C and stirred for a further 3 h. Upon
cooling the reaction mixture was filtered to remove triethyl-
amine hydrobromide. The filtrate was washed with water,
dried (MgSO4), and concentrated under reduced pressure. The
crude product was preabsorbed onto silca gel and flash
chromatographed over silica gel. Elution with petroleum ether/
diethyl ether (9:1) yielded an off white crystalline solid.
Crystallization from absolute ethanol gave 10 (26.1 g, 32%)
as a white crystalline solid: mp 91-92 °C (dec); 1H NMR (400
MHz, CDCl3) δ 1.23 (t, J ) 6.9 Hz, 6H, CH3), 2.53 (d, JA,B
)
7.3 Hz, 2H, BnNCH2), 3.29 (d, JBA ) 7.3 Hz, 2H, BnNCH2),
3.89 (s, 2H, NCHCO2Et), 3.93 (s, 4H, ArCH2N), 4.18 (m, 4H,
CO2CH2CH3), 7.23 (m, 10H, Ar); 13C NMR (100 MHz, CDCl3)
δ 14.10, 46.51, 59.21, 60.09, 62.63, 126.82, 127.99, 128.49,
138.89, 170.72; MS (EI) m/z ) 410 (M+), 337, 264, 173, 91.
Anal. (C24H30N2O4) C, H, N.
Experimental Section
Chemistry. General Procedures. Proton NMR spectra
were measured on a JEOL NMR spectrometer at either 399.78
or 270.18 MHz. Carbon NMR spectra were run on a 399.78
MHz JEOL NMR spectrometer at 100.54 MHz or a 270.18
MHz JEOL spectrometer at 67.94 MHz. 3-(Trimethylsilyl)-
propionic-2,2,3,3-d4 acid, sodium salt in D2O, or tetramethyl-
silane in CDCl3 and DMSO-d6 were used as internal standards.
Chemical shifts (δ) are expressed in ppm and coupling
constants (J) in Hertz; bs ) broad singlet and usp ) under
solvent peak (chemical shifts correlated via 2D COSY spectra).
Elemental analyses were performed in the microanalytical
laboratory in the Department of Chemistry, University of
Bristol, Bristol, UK. Mass spectrometry was performed in the
School of Chemistry at the University of Southampton,
Southampton, UK utilizing Electron Ionization (EI) and posi-
tive (ES+) and negative (ES-) electrospray ionization tech-
niques. Melting points were determined in capillary tubes on
Electrothermal IA9100 electronic melting point equipment and
are uncorrected. Thin-layer chromatography was performed
on Merck silica gel 60 F254 plastic sheets. Silica gel for flash
chromatography was silica gel 60 (220-440 mesh) from Fluka.
The eluents for thin-layer chromatography of amino acids
included [2 (pyridine:acetic acid:water (3:8:11)):3 (n-butanol)]
and [propan-2-ol:35% aqueous ammonia (70:30)]. Amino acids
were detected by spraying plates with a 2% solution of
ninhydrin in 70% ethanol. Ion-exchange resin chromatography
was carried out using Dowex 50WX8-400 acid form resin
obtained from Aldrich Chemical Co., UK. All reagents and dry
(2R*,3R*)-Piperazine-2,3-dicarboxylic Acid Diethyl Es-
ter (11). (2R*,3R*)-1,4-Dibenzylpiperazine-2,3-dicarboxylic
acid diethyl ester (10) (6.5 g, 16 mmol) was dissolved in
anhydrous ethanol (100 mL) and stirred under an atmosphere
of hydrogen in the presence of 10 wt % palladium on activated
carbon (0.5 g). The reaction mixture was filtered through Celite
filter agent, and solvents were removed under reduced pres-
sure. The crude product was flash chromatographed over silica
gel; elution first with diethyl ether removed a trace impurity
and then the product was eluted with ethyl acetate/methanol
(9:1). The eluent was evaporated to dryness under reduced
pressure, and the product was redissolved in diethyl ether,
filtered to remove traces of silica gel, and then evaporated to
dryness under reduced pressure to yield 11 (3.0 g, 82%) as an
amber colored oil: 1H NMR (270 MHz, CDCl3) δ 1.30 (t, J )
7.3 Hz, 6H, CH3), 2.02 (bs, 2H, NH), 2.74 (d, J ) 8.6 Hz, 2H,
HNCH2), 3.00 (d, J ) 8.6 Hz, 2H, HNCH2), 3.81 (s, 2H,
HNCHCO2Et), 4.24 (m, 4H, CO2CH2CH3); 13C NMR (100 MHz,
CDCl3) δ 13.55, 43.47, 58.57, 60.51, 170.35; MS (ES+) m/z )
231.2 (MH+). Anal. (C10H18N2O4) C, H, N.
(2R*,3R*)-Piperazine-2,3-dicarboxylic Acid (12). (2R*,
3R*)-Piperazine-2,3-dicarboxylic acid diethyl ester (11) (3 g,
13 mmol) and sodium hydroxide (2 g, 50 mmol) were stirred
in absolute ethanol for 12 h. Solvents were removed under
reduced pressure. The residue was redissolved in 20 mL of

2634 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Morley et al.
water and the pH of the solution was adjusted to 3.8 with
concentrated nitric acid. Dowex 50WX8-400 resin H+ form (10
mL) was added and the mixture was stirred for 30 min. The
slurry was then applied to a column of Dowex 50WX8-400 resin
H+ form (10 mL) and the column was eluted with water until
the eluent had a pH of 5. The product was eluted with 10%
aqueous ammonia solution, and the ninhydrin-positive frac-
tions were combined and evaporated to dryness under reduced
pressure. The solid was purified by crystallization from boiling
water to give 12 (1.8 g, 78%) as a white solid: mp 283-285
(2R*,3S*)-1-(2′-Bromobiphenylyl-4-carbonyl)piperazine-
2,3-dicarboxylic Acid (16c). Acid chloride 15c (5.17 g, 17.5
mmol) in dioxane (50 mL) and (2R*,3S*)-piperazine-2,3-
dicarboxylic acid (7) (3.05 g, 15.9 mmol) and sodium hydroxide
(1.91 g, 47.7 mmol) in water/dioxane (1:1) (50 mL) gave 16c
(1.16 g, 15%) as a white solid: mp 238-240 °C (dec); 1H NMR
(270 MHz, D2O/NaOD, pH 11) δ 2.63-2.84 (m, 1H, HNCH2),
2.91-3.07 (m, 1H, HNCH2), 3.12-3.28 (m, 1H, CONCH2), 3.35
(d, J ) 3.6 Hz, 0.5H, HNCHCO2H), 3.39 (d, J ) 3.6 Hz, 0.5H,
HNCHCO2H), 3.62 (d, J ) 13.2 Hz, 0.5H, CONCH2), 4.36 (d,
J ) 13.2 Hz, 0.5H, CONCH2), 4.82 (usp, 0.5H, CONCHCO2H),
5.54 (d, J ) 3.6 Hz, 0.5H, CONCHCO2H), 7.31-7.38 (m, 1H,
Ar), 7.41-7.53 (m, 2H, Ar), 7.54-7.64 (m, 4H, Ar), 7.78 (d, J
) 7.9 Hz, 1H, Ar); MS (ES+) m/z ) 434.2, 435.2 (M + H),
455.2. 457.2 (M + H + Na). Anal. (C19H17N2O5Br) C, H, N.
1
°C (dec); H NMR (400 MHz, D2O/NaOD, pH 11) δ 2.69 (m,
2H, NHCH2), 2.90 (m, 2H, NHCH2), 3.25 (s, 2H, NHCHCO2H);
13C NMR (100 MHz, D2O/NaOD, pH 11) δ 46.23, 65.79, 181.07;
MS (ES-) m/z ) 173.1 (M - H). Anal. (C6H10N2O4‚H2O) C, H,
N.
General Procedure for Preparing Acid Chlorides
(15a-n). The carboxylic acid (14a-n) (10 mmol) was refluxed
with excess thionyl chloride (5 mL) in anhydrous benzene (50
mL) under a dry argon atmosphere for up to 12 h. Once all
the carboxylic acid had dissolved, the solution was allowed to
cool and was evaporated to dryness under reduced pressure.
The product was redissolved in a second aliquot of anhydrous
benzene (50 mL) and this was again evaporated to dryness
under reduced pressure, ensuring complete removal of any
excess thionyl chloride. The acid chlorides (15a-n) were used
without any further purification.
General Procedure for Coupling of Acid Chloride
(15a-n) and Piperazine-2,3-dicarboxylic Acid (7 or 12).
A solution of the acid chloride (15a-n) (1 equiv) in dioxane
(20 mL) was added dropwise to a rapidly stirring solution of
the piperazine-2,3-dicarboxylic acid (7 or 12) (1 equiv) and
sodium hydroxide (3 equiv) dissolved in water/dioxane (1:1)
at 0 °C. The reaction mixture was stirred at 0 °C for 2 h and
then allowed to warm to room temperature and stirred for a
further 3 h. The pH of the reaction mixture was adjusted to 7,
diluted to twice the volume with water, and reduced to half
volume under reduced pressure. The reaction mixture was
acidified to pH 3 with 2 M aqueous hydrochloric acid, and the
precipitate formed was filtered and washed with water and
then three times with hot dioxane. The solid was redissolved
in a minimum volume of aqueous sodium hydroxide, filtered,
and then precipitated with 2 M aqueous hydrochloric acid at
a pH of 3. The precipitate was washed with water and air-
dried.
(2R*,3S*)-1-(4′-Fluorobiphenylyl-4-carbonyl)piperazine-
2,3-dicarboxylic Acid (16d). Acid chloride 15d (9.32 g, 39.7
mmol) in dioxane (100 mL) and (2R*,3S*)-piperazine-2,3-
dicarboxylic acid (7) (7.62 g, 39.7 mmol) and sodium hydroxide
(4.76 g, 0.12 mol) in water/dioxane (1:1) (100 mL) gave the
monosodium salt of 16d (3.58 g, 20%) as a white solid: mp
258-263 °C (dec); δ 2.63-2.83 (m, 1H, HNCH2), 2.90-3.05
(m, 1H, HNCH2), 3.11-3.27 (m, 1H, CONCH2), 3.34 (d, J )
3.6 Hz, 0.5H, HNCHCO2H), 3.39 (d, J ) 3.6 Hz, 0.5H,
HNCHCO2H), 3.57 (d, J ) 13.2 Hz, 0.5H, CONCH2), 4.35 (d,
J ) 13.2 Hz, 0.5H, CONCH2), 4.77 (d, J ) 3.6 Hz, 0.5H,
CONCHCO2H), 5.53 (d, J ) 3.6 Hz, 0.5H, CONCHCO2H),
7.21-7.30 (m, 2H, Ar), 7.55-7.63 (m, 2H, Ar), 7.67-7.78 (m,
4H, Ar); MS (ES+) m/z ) 373.2 (M + H), 395.2 (M + H + Na).
Anal. (C19H16N2O5FNa‚2.75H2O) C, H, N.
(2R*,3S*)-1-(Phenanthrenyl-2-carbonyl)piperazine-2,3-
dicarboxylic Acid (16e). Acid chloride 15e (10.80 g, 45.0
mmol) in dioxane (100 mL) and (2R*,3S*)-piperazine-2,3-
dicarboxylic acid (7) (8.64 g, 45.0 mmol) and sodium hydroxide
(5.40 g, 0.13 mol) in water/dioxane (1:1) (100 mL) gave 16e
(5.84 g, 30%) as a white solid; mp 223-226 °C (dec);1H NMR
(270 MHz, D2O/NaOD, pH 11) δ 2.62-3.29 (m, 3H, HNCH2,
CONCH2), 3.38 (d, J ) 3.6 Hz, 0.5H, HNCHCO2H), 3.43 (d, J
) 3.6 Hz, 0.5H, HNCHCO2H), 3.54 (d, J ) 13.9 Hz, 0.5H,
CONCH2), 4.43 (d, J ) 13.9 Hz, 0.5H, CONCH2), 4.79 (usp,
0.5H, CONCHCO2H), 5.60 (d, J ) 3.6 Hz, 0.5H, CONCHCO2H),
7.63-8.12 (m, 7H, Ar), 8.58-8.79 (m, 2H, Ar); MS (ES-) m/z
) 377.2 (M - H), 399.2 (M - H + Na). Anal. (C21H18N2O5‚
3.0H2O) C, H, N.
(2R*,3S*)-1-Benzoylpiperazine-2,3-dicarboxylic Acid
(16a). Acid chloride 15a (1.46 g, 10.4 mmol) in dioxane (40
mL) and (2R*,3S*)-piperazine-2,3-dicarboxylic acid (7) (2.00
g, 10.4 mmol) and sodium hydroxide (0.83 g, 31.2 mmol) in
water/dioxane (1:1) (40 mL) gave a solid that was washed with
ethanol followed by diethyl ether to yield the monosodium salt
of 16a (2.0 g, 52%) as white fluffy crystals: mp 255-258 °C
(dec); 1H NMR (270 MHz, D2O/NaOD, pH 11) δ 2.60-2.85 (m,
1H, HNCH2), 2.88-3.05 (m, 1H, HNCH2), 3.10-3.23 (m, 1H,
CONCH2), 3.31 (d, J ) 3.6 Hz, 0.5H, HNCHCO2H), 3.37 (d, J
) 3.6 Hz, 0.5H, HNCHCO2H), 3.55 (d, J ) 13.5 Hz, 0.5H,
CONCH2), 4.33 (d, J ) 13.5 Hz, 0.5H, CONCH2), 4.69 (d, J )
3.6 Hz, 0.5H, CONCHCO2H), 5.53 (d, J ) 3.6 Hz, 0.5H,
CONCHCO2H), 7.54 (m, 5H, Ar); MS (ES+) m/z ) 279.2 (M +
H), 301.2 (M + H + Na), 323.2 (M + H + 2Na). Anal.
(C13H13N2O5Na‚4H2O) C, H, N.
(2R*,3S*)-1-(Phenanthrenyl-3-carbonyl)piperazine-2,3-
dicarboxylic Acid (16f). Acid chloride 15f (2.16 g, 9.0 mmol)
in dioxane (40 mL) and (2R*,3S*)-piperazine-2,3-dicarboxylic
acid (7) (1.73 g, 9.0 mmol) and sodium hydroxide (1.08 g, 27.0
mmol) in water/dioxane (1:1) (40 mL) gave 16f (410 mg, 12%)
1
as a white solid: mp 205-208 °C (dec); H NMR (270 MHz,
D2O/NaOD, pH 11) δ 2.45-2.59 (m, 0.5H, HNCH2), 2.68-2.92
(m, 1H, HNCH2), 3.03-3.26 (m, 1.5H, CONCH2, HNCH2),
3.30-3.42 (m, 1.5H, CONCH2, HNCHCOOH), 4.45 (d, J ) 12.5
Hz, 0.5H, CONCH2), 4.80 (usp, 0.5H, CONCHCOOH), 5.59 (d,
J ) 3.6 Hz, 0.5H, CONCHCOOH), 7.42-7.85 (m, 6.5H, Ar),
7.98 (d, J ) 8.6 Hz, 0.5H, Ar), 8.51 (d, J ) 8.2 Hz, 0.5H, Ar),
8.61 (d, J ) 8.2 Hz, 0.5H, Ar), 8.65 (s, 0.5H, Ar), 8.87 (s, 0.5H,
Ar); MS (ES+) m/z ) 379.2 (M + H), 401.2 (M + Na), 423.2
(M + 2Na). Anal. (C21H18N2O5‚H2O) C, H, N.
(2R*,3S*)-1-(7-Bromophenanthrenyl-2-carbonyl)piper-
azine-2,3-dicarboxylic Acid (16g). Acid chloride 15g (1.06
g, 3.3 mmol) in dioxane (40 mL) and (2R*,3S*)-piperazine-2,3-
dicarboxylic acid (7) (0.64 g, 3.3 mmol) and sodium hydroxide
(0.40 g, 9.9 mmol) in water/dioxane (1:1) (40 mL) gave 16g
(163 mg, 11%) as a brown solid: mp 225-228 °C (dec); 1H NMR
(270 MHz, D2O/NaOD, pH 11) δ 2.58-2.72 (m, 0.5H, HNCH2),
2.80-2.97 (m, 1H, CONCH2, HNCH2), 3.00-3.12 (m, 0.5H,
HNCH2), 3.18-3.31 (m, 1H, CONCH2), 3.40-3.51 (m, 1.5H,
CONCH2, HNCHCO2H), 4.43 (d, J ) 12.5 Hz, 0.5H, CONCH2),
4.89 (d, J ) 3.6 Hz, 0.5H, CONCHCO2H), 5.60 (d, J ) 3.6 Hz,
0.5H, CONCHCO2H), 7.06 (d, J ) 8.6 Hz, 0.5H, Ar), 7.21 (d,
J ) 8.6 Hz, 0.5H, Ar), 7.27 (d, J ) 8.9 Hz, 0.5H, Ar), 7.34-
7.85 (m, 4.5H, Ar), 7.96-8.11 (m, 1.5H, Ar), 8.33 (d, J ) 7.9
(2R*,3S*)-(Biphenylyl-4-carbonyl)piperazine-2,3-di-
carboxylic Acid (16b). Acid chloride 15b (2.00 g, 9.2 mmol)
in dioxane (40 mL) and (2R*,3S*)-piperazine-2,3-dicarboxylic
acid (7) (1.77 g, 9.2 mmol) and sodium hydroxide (1.11 g, 27.7
mmol) in water/dioxane (1:1) (40 mL) gave 16b (1.21 g, 31%)
1
as a white solid: mp 204-206 °C (dec); H NMR (270 MHz,
D2O/NaOD, pH 11) δ 2.54-3.06 (m, 2H, HNCH2), 3.10-3.24
(m, 1H, CONCH2), 3.33 (d, J ) 3.6 Hz, 0.5H, HNCHCO2H),
3.35 (d, J ) 3.6 Hz, 0.5H, HNCHCO2H), 3.57 (d, J ) 13.9 Hz,
0.5H, CONCH2), 4.35 (d, J ) 13.9 Hz, 0.5H, CONCH2), 4.75
(d, J ) 3.6 Hz, 0.5H, CONCHCO2H), 5.58 (d, J ) 3.6 Hz, 0.5H,
CONCHCO2H), 7.40-7.79 (m, 8H, Ar); MS (ES+) m/z ) 355.3
(M + H), 377.2 (M + H + Na), 399.2 (M + H + 2Na). Anal.
(C19H18N2O5‚2H2O) C, H, N.

N1-Substituted Piperazine-2,3-dicarboxylic Acids
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2635
Hz, 0.5H, Ar); MS (ES+) m/z ) 457.2, 459.1 (M + H), 479.2,
481.1 (M + H + Na). Anal. (C21H17N2O5Br‚2.0H2O) C, H, N.
(d, J ) 13.5 Hz, 0.5H, CONCH2), 4.31 (d, J ) 13.5 Hz, 0.5H,
CONCH2), 4.77 (d, J ) 3.6 Hz, 0.5H, CONCHCO2H), 5.53 (d,
J ) 3.6 Hz, 0.5H, CONCHCO2H), 7.12 (d, 0.5H, J ) 15.8 Hz,
(2R*,3S*)-(9,10-Dihydrophenanthrenyl-2-carbonyl)-
piperazine-2,3-dicarboxylic Acid (16h). Acid chloride 15h
(2.15 g, 8.9 mmol) in dioxane (40 mL) and (2R*,3S*)-piper-
azine-2,3-dicarboxylic acid (7) (1.71 g, 8.9 mmol) and sodium
hydroxide (1.07 g, 26.7 mmol) in water/dioxane (1:1) (40 mL)
gave 16h (252 mg, 7%) as a white solid: mp 205-208 °C (dec);
1H NMR (270 MHz, D2O/NaOD, pH 11) δ 2.61-2.83 (m, 1H,
HNCH2), 2.87 (s, 2H, ArCH2CH2Ar), 2.89 (s, 2H, ArCH2CH2Ar),
2.94-3.07 (m, 1H, HNCH2), 3.11-3.26 (m, 1H, CONCH2), 3.32
(d, J ) 3.6 Hz, 0.5H, HNCHCO2H), 3.37 (d, J ) 3.6 Hz, 0.5H,
HNCHCO2H), 3.59 (d, J ) 13.9 Hz, 0.5H, CONCH2), 4.34 (d,
J ) 13.9 Hz, 0.5H, CONCH2), 4.84 (usp, 0.5H, CONCHCO2H),
5.53 (d, J ) 3.6 Hz, 0.5H, CONCHCO2H), 7.33-7.49 (m, 5H,
Ar), 7.85-7.94 (m, 1H, Ar); MS (ES+) m/z ) 381.3 (M + H),
403.3 (M + H + Na), 425.3 (M + H + 2Na). Anal. (C21H20N2O5‚
H2O) C, H, N.
(2R*,3S*)-1-(7-Bromo-9,10-dihydrophenanthrenyl-2-
carbonyl)piperazine-2,3-dicarboxylic Acid (16i). Acid
chloride 15i (1.06 g, 3.3 mmol) in dioxane (40 mL) and
(2R*,3S*)-piperazine-2,3-dicarboxylic acid (7) (0.63 g, 3.3
mmol) and sodium hydroxide (0.40 g, 9.9 mmol) in water/
dioxane (1:1) (40 mL) gave 16i (190 mg, 11%) as a white solid:
mp 209-211 °C (dec); 1H NMR (270 MHz, D2O/NaOD, pH 11)
δ 2.63-2.88 (m, 5H, ArCH2CH2Ar, HNCH2), 2.91-3.06 (m, 1H,
HNCH2), 3.10-3.28 (m, 1H, CONCH2), 3.32 (d, J ) 3.6 Hz,
0.5H, HNCHCO2H), 3.38 (d, J ) 3.6 Hz, 0.5H, HNCHCO2H),
3.59 (d, J ) 13.5 Hz, 0.5H, CONCH2), 4.34 (d, J ) 13.5 Hz,
0.5H, CONCH2), 4.75 (d, J ) 3.6 Hz, 0.5H, CONCHCO2H),
5.52 (d, J ) 3.6 Hz, 0.5H, CONCHCO2H), 7.37-7.53 (m, 4H,
Ar), 7.66 (d, J ) 8.9 Hz, 0.5H, Ar), 7.72 (d, J ) 8.9 Hz, 0.5H,
Ar), 7.80 (d, J ) 7.9 Hz, 0.5H, Ar), 7.86 (d, J ) 7.9 Hz, 0.5H,
Ar); MS (ES+) m/z ) 459.2 (M + H), 482.2 (M + H + Na),
505.2 (M + H + 2Na). Anal. (C21H19N2O5Br‚3.0H2O) C, H, N.
(2R*,3S*)-1-(9H-Fluorenyl-2-carbonyl)piperazine-2,3-
dicarboxylic Acid (16j). Acid chloride 15j (2.17 g, 9.5 mmol)
in dioxane (40 mL) and (2R*,3S*)-piperazine-2,3-dicarboxylic
acid (7) (1.83 g, 9.5 mmol) and sodium hydroxide (1.14 g, 28.6
mmol) in water/dioxane (1:1) (40 mL) gave 16j (440 mg, 12%)
as a pale yellow solid: mp 230-233 °C (dec); 1H NMR (270
MHz, D2O/NaOD, pH 11) δ 2.66-2.84 (m, 1H, HNCH2), 2.89-
3.06 (m, 1H, HNCH2), 3.11-3.28 (m, 1H, CONCH2), 3.34 (d, J
) 3.6 Hz, 0.5H, HNCHCO2H), 3.39 (d, J ) 3.6 Hz, 0.5H,
HNCHCO2H), 3.60 (d, J ) 13.2 Hz, 0.5H, CONCH2), 3.91 (m,
2H, ArCH2Ar), 4.36 (d, J ) 13.2 Hz, 0.5H, CONCH2), 4.81 (usp,
0.5H, CONCHCO2H), 5.54 (d, J ) 3.3 Hz, 0.5H, CONCHCO2H),
7.33-7.72 (m, 5.5H, Ar), 7.79-7.95 (m, 1.5H, Ar); MS (ES+)
m/z ) 367.3 (M + H), 389.3 (M + H + Na), 411.2 (M + H +
2Na). Anal. (C20H18N2O5‚0.5H2O) C, H, N.
Hvic), 7.24 (d, 0.5H, J ) 15.8 Hz, Hvic), 7.45-7.51 (m, 2.5H,
Ar), 7.58 (d, 0.5H, J ) 15.8 Hz, Hvic), 7.60 (d, 0.5H, J ) 15.8
Hz, Hvic), 7.65-7.74 (m, 2.5H, Ar); MS (ES+) m/z ) 305.3 (M
+ H), 327.2 (M + H + Na), 349.2 (M + H + 2Na). Anal.
(C15H16N2O5‚H2O) C, H, N.
(2R*,3S*)-1-(Naphthalenyl-2-carbonyl)piperazine-2,3-
dicarboxylic Acid (16m). Acid chloride (15m) (2.0 g, 10.5
mmol) in dioxane (40 mL) and (2R*,3S*)-piperazine-2,3-
dicarboxylic acid (7) (2.01 g, 10.5 mmol) and sodium hydroxide
(1.26 g, 31.5 mmol) in water/dioxane (1:1) (40 mL) gave the
monosodium salt of 16m (1.41 g, 37%) as a white solid: mp
1
250-253 °C (dec); H NMR (270 MHz, D2O/NaOD, pH 11) δ
2.66-2.87 (m, 1H, HNCH2), 2.89-3.09 (m, 1H, HNCH2), 3.13-
3.28 (m, 1H, CONCH2), 3.35 (d, J ) 3.6 Hz, 0.5H, HNCHCO2H),
3.43 (d, J ) 3.6 Hz, 0.5H, HNCHCO2H), 3.59 (d, J ) 13.9 Hz,
0.5H, CONCH2), 4.40 (d, J ) 13.9 Hz, 0.5H, CONCH2), 4.78
(usp, 0.5H, CONCHCO2H), 5.58 (d, J ) 3.6 Hz, 0.5H,
CONCHCO2H), 7.58-7.70 (m, 3H, Ar), 8.00-8.11 (m, 4H, Ar);
MS (ES+) m/z ) 329.2 (M + H), 351.2 (M + H + Na), 373.2
(M + H + 2Na). Anal. (C17H15N2O5‚H2O) C, H, N.
(2R*,3S*,E)-1-(3-Naphthalen-2-ylacryloyl)piperazine-
2,3-dicarboxylic Acid (16n). Acid chloride 15n (1.13 g, 5.2
mmol) in dioxane (40 mL) and (2R*,3S*)-piperazine-2,3-
dicarboxylic acid (7) (1.00 g, 5.2 mmol) and sodium hydroxide
(0.63 g, 15.6 mmol) in water/dioxane (1:1) (40 mL) gave 16n
(530 mg, 31%) as a white solid: mp 285-287 °C (dec); 1H NMR
(270 MHz, D2O/NaOD, pH 11) δ 2.59-2.84 (m, 1.5H, HNCH2),
3.20-3.30 (m, 1.5H, HNCHCO2H, CONCH2), 3.98 (d, J ) 13.4
Hz, 0.5H, CONCH2), 4.36 (d, J ) 13.4 Hz, 0.5H, CONCH2),
5.17 (d, J ) 3.6 Hz, 0.5H, CONCHCO2H), 5.52 (d, J ) 3.6 Hz,
0.5H, CONCHCO2H), 7.06 (d, J ) 15.1 Hz, 0.5H, Hvic), 7.23
(d, J ) 15.1 Hz, 0.5H, Hvic), 7.53-8.04 (m, 8H, Ar, Hvic); MS
(ES+) m/z ) 355.2 (M + H), 377.2 (M + H + Na), 399.2 (M +
H + 2Na). Anal. (C19H18N2O5‚1.5H2O) C, H, N.
(2R*,3S*)-1-(Biphenylyl-4-sulfonyl)piperazine-2,3-di-
carboxylic Acid (17). Biphenyl-4-sulfonyl chloride (1.32 g,
5.2 mmol) in dioxane (40 mL) and (2R*,3S*)-piperazine-2,3-
dicarboxylic acid (7) (1.00 g, 5.2 mmol) and sodium hydroxide
(0.63 g, 15.6 mmol) in water/dioxane (1:1) (40 mL) gave 17
(1.61 g, 37%) as a white solid: mp 198-200 °C (dec); 1H NMR
(270 MHz, D2O/NaOD, pH 11) 2.22-2.39 (m, 1H, HNCH2), 2.84
(d, J ) 13.9 Hz, 1H, OSONCH2), 3.12 (d, J ) 3.0 Hz, 1H,
HNCHCO2H), 3.18-3.32 (m, 1H, HNCH2), 4.42 (d, J ) 13.9
Hz, 1H, OSONCH2), 4.80 (d, J ) 3.0 Hz, 1H, OSONCHCO2H),
7.22-7.37 (m, 5H, Ar), 7.45 (d, J ) 8.4 Hz, 2H, Ar), 7.76 (d, J
) 8.4 Hz, 2H, Ar); MS (ES+) m/z ) 391.2 (M + H), 413.1 (M
+ H + Na). Anal. (C18H18N2O6S‚1.5H2O) C, H, N.
(2R*,3S*)-1-(9-Oxo-9H-fluorenyl-2-carbonyl)piperazine-
2,3-dicarboxylic Acid (16k). Acid chloride 15k (0.84 g, 3.5
mmol) in dioxane (20 mL) and (2R*,3S*)-piperazine-2,3-
dicarboxylic acid (7) (0.5 g, 2.6 mmol) and sodium hydroxide
(0.31 g, 7.8 mmol) in water/dioxane (1:1) (20 mL) gave 16k
(2R*,3R*)-1-(Phenanthrenyl-2-carbonyl)piperazine-
2,3-dicarboxylic Acid (13). Acid chloride 15e (1.08 g, 4.5
mmol) in dioxane (40 mL) and (2R*,3R*)-piperazine-2,3-
dicarboxylic acid (12) (0.78 g, 4.5 mmol) and sodium hydroxide
(0.54 g, 13.5 mmol) in water/dioxane (1:1) (40 mL) gave 13
(295 mg, 17%) as a white solid: mp 250-252 °C (dec); 1H NMR
(270 MHz, D2O/NaOD pH 11) δ 2.63-3.11 (m, 2.0H, HNCH2),
3.26-3.60 (m, 1.5H, CONCH2), 3.93 (d, J ) 1.3 Hz, 0.5H,
HNCHCO2H), 4.17 (d, J ) 1.3 Hz, 0.5H, HNCHCO2H), 4.41
(d, J ) 13.5 Hz, 0.5H, CONCH2), 4.86 (d, J ) 1.3 Hz, 0.5H,
CONCHCO2H), 5.65 (d, J ) 1.3 Hz, 0.5H, CONCHCO2H),
7.69-7.80 (m, 3H, Ar), 7.87-7.91 (m, 2H, Ar), 7.99-8.08 (m,
2H, Ar), 8.76-8.88 (m, 2H, Ar); MS (ES+) m/z ) 379.3 (M +
H), 401.2 (M + H + Na), 423.2 (M + H + 2Na). Anal.
(C21H18N2O5‚3.0H2O) C, H, N.
1
(89 mg, 7%) as a pale yellow solid: mp 225-228 °C (dec); H
NMR (270 MHz, D2O/NaOD, pH 11) δ 2.71-2.85 (m, 1H,
HNCH2), 2.91-3.36 (m, 2H, CONCH2, HNCH2), 3.38 (d, J )
3.6 Hz, 0.5H, HNCHCO2H), 3.41 (d, J ) 3.6 Hz, 0.5H,
HNCHCO2H), 3.60 (d, J ) 13.5 Hz, 0.5H, CONCH2), 4.34 (d,
J ) 13.5 Hz, 0.5H, CONCH2), 4.78 (d, J ) 3.6 Hz, 0.5H,
CONCHCO2H), 5.53 (d, J ) 3.6 Hz, 0.5H, CONCHCO2H),
7.16-7.28 (m, 2H, Ar), 7.31-7.46 (m, 3H, Ar), 7.52-7.65 (m,
2H, Ar); MS (ES+) m/z ) 381.2 (M + H), 403.2 (M + H + Na),
425.2 (M + H + 2Na). Anal. (C20H16N2O6‚0.7H2O) C, H, N.
(2R*,3S*,E)-(3-Phenylacryloyl)piperazine-2,3-dicar-
boxylic Acid (16l). Acid chloride 15l (0.57 g, 3.5 mmol) in
dioxane (20 mL) and (2R*,3S*)-piperazine-2,3-dicarboxylic acid
(7) (0.50 g, 2.6 mmol) and sodium hydroxide (0.31 g, 7.8 mmol)
in water/dioxane (1:1) (20 mL) gave 16l (288 mg, 26%) as a
2-Iodomethylphenanthrene (19). To a stirring solution
of 2-hydroxymethylphenanthrene (18)21 (4.2 g, 20 mmol) in
anhydrous dioxane (100 mL) at room temperature under an
argon atmosphere were added potassium iodide (3.35 g, 20
mmol) and boron trifluoride diethyl etherate (2.86 g, 20 mmol).
After stirring for 12 h the solution was diluted with 200 mL
of diethyl ether and washed with a 10% sodium metabisulfite
solution (100 mL) followed by water (2 × 100 mL). The organics
were dried over magnesium sulfate and concentrated to yield
1
white solid: mp 251-253 °C (dec); H NMR (270 MHz, D2O/
NaOD, pH 11) δ 2.61-2.87 (m, 1.5H, HNCH2), 3.05-3.31
(m, 1.5H, CONCH2, HNCH2), 3.27 (d, J ) 3.6 Hz, 0.5H,
HNCHCO2H), 3.34 (d, J ) 3.6 Hz, 0.5H, HNCHCO2H), 4.04

2636 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Morley et al.
19 (6.0 g, 94%) as an off-white powder: mp 81-83 °C (dec);
1H NMR (270 MHz, CDCl3) δ 4.65 (s, 2H, ICH2Ar), 7.55-7.88
(m, 7H, Ar), 8.60 (m, 2H, Ar); MS (EI) m/z ) 318 (M+), 191
(M+ - I). Anal. (C15H11I) C, H, N.
scribed in vitro with T3 (NR2A, NR2C), SP6 (NR2B), or T7
(NR1a, NR2D) RNA polymerase using the mMessage
mMachine Transcription Kits (Ambion, Austin, TX).
Oocytes were removed from mature female Xenopus laevis
(Xenopus One, Ann Arbor, MI) as previously described.8
NMDA receptor subunit RNAs were dissolved in sterile
distilled H2O. NR1a and NR2 RNAs were mixed in a molar
ratio of 1:3. In all, 50 nL of the final RNA mixture was
microinjected (15-30 ng total) into the oocyte cytoplasm.
Oocytes were incubated in ND-96 solution at 17 °C prior to
electrophysiological assay (1-5 days).
(2R*,3S*)-Piperazine-2,3-dicarboxylic Acid Dimethyl
Ester (21). Pyrazine-2,3-dicarboxylic acid dimethyl ester (20)
(2.0 g, 10 mmol) was dissolved in anhydrous ethanol (30 mL)
and stirred under an atmosphere of hydrogen in the presence
of 10 wt % palladium on activated carbon for 24 h. The reaction
mixture was filtered through Celite filter agent and concen-
trated under reduced pressure. The crude product was flash
chromatographed over silica gel with ethyl acetate as the
eluent, which removed impurities, followed by methanol, which
eluted the product. The eluent was evaporated to dryness
under reduced pressure, and the product was redissolved in
diethyl ether, filtered to remove traces of silica gel, and then
evaporated to dryness under reduced pressure to yield 21 (1.6
g, 78%) as a viscous amber oil: 1H NMR (270 MHz, CDCl3) δ
2.54 (2H, NH, bs), 2.75-2.96 (4H, CH2, m), 3.76 (6H, CH3, s),
3.90 (2H, CH, s); 13C NMR (270 MHz, CDCl3) δ 44.00, 51.85,
58.48, 171.37; MS (ES+) m/z ) 203.1 (MH+). Anal. (C8H14N2O4)
C, H, N.
(2R*,3S*)-1-Phenanthren-2-ylmethylpiperazine-2,3-di-
carboxylic Acid Dimethyl Ester (22). A solution of 2-iodo-
methylphenanthrene (19) (0.79 g, 2.5 mmol) in tetrahydro-
furan (10 mL) was added dropwise to a stirred solution of
(2R*,3S*)-piperazine-2,3-dicarboxylic acid dimethyl ester (21)
(1.0 g, 5.0 mmol) and triethylamine (0.25 g, 2.5 mmol) in
tetrahydrofuran (30 mL) at 0 °C for 2 h. The solution was
allowed to warm to room temperature and continued to stir
for 12 h. Solvents were removed under reduced pressure, and
the residue was redissolved in ethyl acetate (50 mL). The
organics were washed with water (2 × 50 mL), dried over
magnesium sulfate, and concentrated. The crude product was
flash chromatographed over silica gel with ethyl acetate as
the eluent. The eluted product was evaporated to dryness
under reduced pressure, and the product was redissolved in
diethyl ether, filtered to remove traces of silica gel, and then
evaporated to dryness under reduced pressure to yield 22 (0.5
g, 51%) as an amber solid: mp 116-117 °C; 1H NMR (270
MHz, CDCl3) δ 2.11 (bs, 1H, NH), 2.54 (m, 1H, HNCH2), 2.90
(m, 2H, HNCH2, HNCH2CH2), 3.06 (m, 1H, HNCH2CH2), 3.70
(s, 1H, CH3), 3.73 (s, 1H, CH3), 3.86 (d, J ) 3.6 Hz, 1H,
HNCHCO2CH3), 3.93 (d, J ) 3.6 Hz, 1H, NCHCO2CH3), 3.97
(d, JA,B ) 13.9 Hz, 1H, NCH2Ar), 4.08 (d, JB,A ) 13.9 Hz, 1H,
NCH2Ar), 7.53-7.75 (m, 5H, Ar), 7.79 (s, 1H, Ar), 7.87 (d, J )
7.6 Hz, 1H, Ar), 8.65 (m, 2H, Ar); MS (ES+) m/z ) 393 (MH+).
Anal. (C23H24N2O4) C, H, N.
Electrophysiological Characterization of Recombi-
nant NMDA Receptors. Electrophysiological responses were
measured using a standard two-microelectrode voltage clamp
as previously described.8 The voltage clamp used was a Warner
Instruments (Hamden, CT) model OC-725B Oocyte Clamp,
designed to provide fast clamp of large cells. The recording
buffer contained 116 mM NaCl, 2 mM KCl, 2 mM BaCl2, and
5 mM HEPES, pH 7.4. Response magnitude was determined
by the steady plateau response elicited by bath application of
10 µM l-glutamate plus 10 µM glycine at a holding potential
of 60 mV. Five oocytes were tested against 100 µM NMDA/10
µM glycine-evoked responses. Response amplitudes for the four
heteromers were generally between 30 and 100 nA. Attempts
were made to keep response magnitudes within this range to
minimize activation of the endogenous Cl- current. The
presence of a plateau response was taken as an indication of
the lack of significant activation of the endogenous Cl- current
by Ba2+ in these cells. Antagonist inhibition curves were fit
(GraphPad Prism, ISI Software, San Diego, CA) according to
the equation I ) Imax - Imax/[1 + (IC50/A)n], where Imax is the
current response in the absence of antagonist, A is the
antagonist concentration, and IC50 is the antagonist concentra-
tion producing half-maximal inhibition. Apparent Ki values
were determined by correcting for agonist affinity according
to the equation IC50 ) IC50(obs)/1 + ([agonist]/EC50) as
described.33
Electrophysiological Characterization of 16e on Neo-
natal Rat Motoneurones. All experiments were carried out
in 1-4-day-old Wistar rats of either sex as described previ-
ously.12 To examine the effects of 16e on direct depolarizations
of motoneurones induced by NMDA (50 µM), experiments were
conducted in standard medium containing TTX (10 µM for 2
min then 0.1 µM continuously). NMDA was applied for 1 min
and the degree of depolarization of the motoneurone was
measured by peak amplitude. The ability of 16e to selectively
reduce the peak amplitude of the NMDA-induced responses
was then measured. NMDA-induced responses recovered after
a 30 min washout of the antagonist. To calculate the pA2 value
for the antagonism of NMDA-induced depolarizations by 16e,
noncumulative concentration-response curves were con-
structed both in the absence and presence of 0.5, 0.7, 1, 2, and
3 µM 16e and dose ratios (DR) estimated from the equation
DR ) EC50 of NMDA in the presence of 16e/EC50 of NMDA in
the absence of 16e. A graph (Schild plot) of log(DR - 1) vs log
[PPDA] M was then plotted using GraphPad Prism version
2.0.
(2R*,3S*)-1-(Phenanthren-2-ylmethyl)piperazine-2,3-
dicarboxylic Acid (23). (2R*,3S*)-1-Phenanthren-2-ylmethyl-
piperazine-2,3-dicarboxylic acid dimethyl ester (22) (0.40 g, 1.0
mmol) was stirred for 24 h at room temperature in tetra-
hydrofuran/water (60 mL, 2:1) with excess lithium hydroxide
(0.42 g, 10.0 mmol). The solution was then acidified to pH 3
with 2 M HCl (aq), evaporated to half volume under reduced
pressure, diluted with water (30 mL), and again evaporated
to half volume under reduced pressure. The solid was collected
by filtration, washed with water and diethyl ether, and allowed
to air-dry, giving 23 (320 mg, 81%) as a white solid: mp 190-
193 °C (dec); 1H NMR (270 MHz, D2O/NaOD, pH 11) δ 2.32-
2.66 (m, 2H, HNCH2), 2.89-3.06 (m, 2H, HNCH2CH2), 3.49
(d, J ) 4.0 Hz, 1H, NCHCO2H), 3.63 (d, JA,B ) 12.9 Hz, 1H,
Acknowledgment. We thank Drs Shigetada Na-
kanishi, David Lynch, and Peter Seeburg, for provid-
ing NMDA receptor cDNA constructs, and Dr Yves
Auberson at Novartis Pharma AG, Basel, Switzerland,
for providing PEAQX. This work was supported by NIH
Grant MH60252.
NCH2Ar), 3.71 (d, J ) 4.0 Hz, 1H, NCHCO2H), 3.99 (d, JB,A
)
12.9 Hz, 1H, NCH2Ar), 7.53-7.71 (m, 5H, Ar), 7.76-7.90 (m,
2H, Ar), 8.45-8.61 (m, 2H, Ar); MS (ES+) m/z ) 391.2 (M +
H), 413.1 (M + H + Na). Anal. (C21H20N2O4‚1.5H2O) C, H, N.
Supporting Information Available: Six tables contain-
ing the X-ray crystallographic crystal data of derivative 10.
This material is available free of charge via the Internet at
http://pubs.acs.org.
NR Subunit Expression in Xenopus oocytes. cDNA
encoding the NMDAR1a subunit was a generous gift of Dr
Shigetada Nakanishi (Kyoto, Japan). cDNAs encoding NR2A,
NR2C, and NR2D were kindly provided by Dr. Peter Seeburg
(Heidelburg, Germany) and the NR2B [5′UTR]-cDNA was a
generous gift of Drs. Dolan Pritchett and David Lynch
(Philadelphia, PA). Plasmids were linearized with NotI (NR1a),
EcoRI (NR2A, NR2C and NR2D), or SalI (NR2B), and tran-
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