Chemo-enzymatic synthesis of (2S,4R)-2-amino-4-(3-(2,2-diphenylethylamino)- 3-oxopropyl)pentanedioic acid: A novel selective inhibitor of human excitatory amino acid transporter subtype 2

Article abstract of DOI:10.1021/jm800091e

In the mammalian central nervous system (CNS), the action of sodium dependent excitatory amino acid transporters (EAATs) is responsible for termination of glutamatergic neurotransmission by reuptake of (S)-glutamate (Glu) from the synaptic cleft. Five EAAT subtypes have been identified, of which EAAT1-4 are present in the CNS, while EAAT5 is localized exclusively in the retina. In this study, we have used an enantioselective chemo-enzymatic strategy to synthesize 10 new Glu analogues 2a-k (2d is exempt) with different functionalities in the 4R-position and characterized their pharmacological properties at the human EAAT1-3. In particular, one compound, 2k, displayed a significant preference as inhibitor of the EAAT2 subtype over EAAT1,3. The compound also displayed very low affinities toward ionotropic and metabotropic Glu receptors, making it the most selective EAAT2 inhibitor described so far.

Full text of DOI:10.1021/jm800091e

J. Med. Chem. 2008, 51, 4085–4092
4085
Chemo-Enzymatic Synthesis of (2S,4R)-2-Amino-4-(3-(2,2-diphenylethylamino)-3-
oxopropyl)pentanedioic Acid: A Novel Selective Inhibitor of Human Excitatory Amino Acid
Transporter Subtype 2
Emanuelle Sagot,‡ Anders A. Jensen,† Darryl S. Pickering,§ Xiaosui Pu,| Michelle Umberti,| Tine B. Stensbøl,
Birgitte Nielsen,† Zeinab Assaf,‡ Be´tina Aboab,‡ Jean Bolte,‡ Thierry Gefflaut,*,‡ and Lennart Bunch*,†
Department of Medicinal Chemistry, The Faculty of Pharmaceutical Sciences, UniVersity of Copenhagen, UniVersitetsparken 2, DK-2100
Copenhagen, Denmark, De´partement de Chimie, UniVersite´ Blaise Pascal, 63177 Aubie`re Cedex, France, Department of Pharmacology and
Pharmacotherapy, The Faculty of Pharmaceutical Sciences, UniVersity of Copenhagen, UniVersitetsparken 2, DK-2100 Copenhagen, Denmark,
Biological Research, H. Lundbeck A/S, 215 College Road, Paramus, New Jersey 07652, Department of Neurobiology, H. Lundbeck A/S,
OttiliaVej 9, DK-2500 Valby, Denmark
ReceiVed January 30, 2008
In the mammalian central nervous system (CNS), the action of sodium dependent excitatory amino acid
transporters (EAATs) is responsible for termination of glutamatergic neurotransmission by reuptake of (S)-
glutamate (Glu) from the synaptic cleft. Five EAAT subtypes have been identified, of which EAAT1-4 are
present in the CNS, while EAAT5 is localized exclusively in the retina. In this study, we have used an
enantioselective chemo-enzymatic strategy to synthesize 10 new Glu analogues 2a-k (2d is exempt) with
different functionalities in the 4R-position and characterized their pharmacological properties at the human
EAAT1-3. In particular, one compound, 2k, displayed a significant preference as inhibitor of the EAAT2
subtype over EAAT1,3. The compound also displayed very low affinities toward ionotropic and metabotropic
Glu receptors, making it the most selective EAAT2 inhibitor described so far.
Introduction
the metabotropic Glu receptors (mGluR), or the Glu reuptake
system (EAATs).3,4
In the mammalian central nervous system (CNS) glutamater-
gic neurotransmission is terminated by reuptake of (S)-glutamate
(Glu) from the synaptic cleft by the action of a family of sodium
dependent excitatory amino acid transporters (EAATs).1,2 To
date, five transporter subtypes have been identified, of which
four, EAAT1-4, are present in the mammalian CNS while
EAAT5 is localized exclusively in the retina.a EAAT1-3 are
localized on neurons and/or in astroglial cells and exhibit high
capacity for transporting Glu across the membrane, whereas
EAAT4 and EAAT5 predominately function as Glu-gated
chloride channels.1 In the healthy CNS, activation of Glu
receptors is involved in important neurophysiological processes
underlying memory and learning, motor functions, and neural
plasticity and development.3 However, under conditions of
metabolic stress and oxygen deprivation, Glu is a neurotoxic
agent. Thus, it is believed that neurodegenerative diseases such
as Alzheimer’s disease, dementia, Huntington’s disease, amyo-
trophic lateral sclerosis, epilepsy, and cerebral stroke may be
related to disordered glutamatergic neurotransmission originating
from dysfunction of either the ionotropic Glu receptors (iGluR),
Inseveralrecentstudies,wehaveexploredthestructure-activity
relationship (SAR) of ligands targeting human EAATs, with
the ultimate goal of identifying new EAAT subtype selective
inhibitors and substrates.5–8 In this paper, we present the
pharmacological evaluation of Glu analogues 2a-k (2b is
exempt) at human EAAT1-3 and the chemo-enzymatic syn-
thetic strategy developed for the preparation of amide analogues
2 g-k. Furthermore, a modeling study addressing the observed
structure-activity relations is presented.
Chemistry
The enzyme aspartate aminotransferase (AAT), isolated from
E. coli, may catalyze the stereoselective conversion of substi-
tuted R-ketoglutarates (KG) into their corresponding L-Glu
analogues. Using this approach, we have previously prepared a
series of L-2,4-syn-4-alkyl Glu analogues starting from their
corresponding racemic KGs,6 thus demonstrating the KG
substrate tolerance and that AAT is highly enantioselective. In
this paper, we show that this methodology can be extended to
also cover a range of functionalized side chains in the 4-position
of KG (Scheme 1).
Whereas the enantioselective preparation of Glu analogues
2a-f, including their corresponding KG precursors, is reported
elsewhere by us,9 the synthesis of 4-amide functionalized KGs
1 g-k is based on an original approach starting from the
cyclohexanone 3 (Scheme 2). The well-known disubstituted
cyclohexanone 3 was readily prepared from methyl malonate
and methyl acrylate following a described four-step procedure
involving a double Michael condensation and a Dieckmann
reaction.10 This cyclic ꢀ-keto ester was then converted in high
yield to the enamines 4g-k by treatment with ammonia or
several primary amines. Oxidative cleavage by ozone of the
various enamines 4g-k afforded simultaneously the R-keto ester
* To whom correspondence should be addressed. For L.B.: Phone, +45
35336244; fax, +45 35336040; e-mail, lebu@farma.ku.dk. For T.G. (for
correspondence regarding the synthetic work): Phone, +33 473407866, fax,
+33 473407717; e-mail, thierry.gefflaut@univ-bpclermont.fr.
†
Department of Medicinal Chemistry, The Faculty of Pharmaceutical
Sciences, University of Copenhagen.
‡
De´partement de Chimie, Universite´ Blaise Pascal.
Department of Pharmacology and Pharmacotherapy, The Faculty of
§
Pharmaceutical Sciences, University of Copenhagen.
|
Biological Research, H. Lundbeck A/S.
Department of Neurobiology, H. Lundbeck A/S.
a Abbreviations: iGluR, ionotropic Glu receptors; mGluR, metabotropic
Glu receptors; EAAT, excitatory amino acid transporter; KG, R-ketoglut-
arates; AAT, aspartate aminotransferase; DHK, dihydrokainic acid; TBOA,
threo-benzyloxyaspartate; AMPA, 2-amino-3-(3-hydroxy-5-methyl-4-isox-
azolyl)propionic acid; KA, kainic acid; NMDA, N-methyl-D-aspartic acid;
FMP, FLIPR membrane potential.
10.1021/jm800091e CCC: $40.75
2008 American Chemical Society
Published on Web 06/25/2008

4086 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Sagot et al.
Table 1. Kinetic Parameters of AAT Catalyzed Transaminations of KG
Analogues 1g-ka
Scheme 1. AAT Catalyzed Synthesis of 4-Substituted Glu
Analogues 2a-k
Scheme 2. Synthesis of 4-Amide Functionalized KGs 1g-k
a Values and standard errors were calculated from the Hanes-Woolf
plot according to the least-squares method and Gauss’s error propagation
law. b The absolute kcat value measured in our experimental conditions was
39.6 ( 1.2 s-1
.
however, the kcat measured with 1g-k are all close to 10% of
that of KG thus offering the opportunity of synthetic applica-
tions. Preparative scale transaminations were carried out as
previously described, by an equilibrium shifted reaction using
cysteine sulfinic acid (CSA) as the amino donor substrate: during
the reaction, CSA is converted into the very unstable pyruvyl
sulfinic acid that decomposes into sulfur dioxide and pyruvic
acid, which is not a substrate for AAT. The transamination
reaction was monitored by enzymatic titration of pyruvic acid
using lactate dehydrogenase and NADH. When a conversion
rate around 40% was reached, the reaction was stopped to
achieve the kinetic resolution of the keto acid substrates. Glu
analogues were selectively adsorbed on a short column of
sulfonic resin (H+ form) and then eluted with aqueous ammonia.
Finally, ion exchange chromatography on cationic dowex 1 resin
(AcO- form) afforded 2g-k, each compound being isolated as
a single stereomer in 35-46% yield. AAT was previously shown
to display a very high stereoselectivity in favor of the L-2,4-
syn-4-alkyl Glu derivatives.6,11 The L-2,4-syn configuration was
thus expected for the new Glu analogues 2g-k. The configu-
ration was demonstrated, as an example, in the case of the amide
2k: an aqueous solution of this compound was heated to form
a lactame which was then converted into the ester 7 in the
presence of methanol and thionyl chloride. NMR analysis of
this pyro-Glu derivative confirmed the expected trans config-
uration (Scheme 4): the measured coupling constants are in
agreement with the expected preferred conformation of (3S,5S)-
7. On the contrary, these data are inconsistent with the (3R,5S)-
cis configuration for which H2 (and H3) would present close
values of coupling constants with H1 and H4. These experiments
confirmed previous findings with the 4-alkyl-Glu derivatives
and were considered to be demonstrative of the general
Scheme 3. Oxidation of Enamines 4g-k
moiety and the amide functionality. The ketoglutarates 5g-k
were thus isolated with overall yields of 25-55% from 3. This
modest yield resulted from a competitive oxidation reaction,
giving, in every case, the 2-hydroxy cyclohexanone 6, presum-
ably via an elimination of O2 from the primary ozonides formed
from 4g-k (Scheme 3). Despite this side reaction, the procedure
described in Scheme 2 offers an easy and short route to a variety
of amide-substituted KGs. Ester hydrolysis was finally done with
a stoichiometric amount of LiOH and gave the desired KGs
1g-k in quantitative yields.
The substituted KGs were evaluated as substrate for E. coli
AAT on the basis of the Michaelis-Menten model. Table 1
summarizes the kinetic constants measured for the transamina-
tion reaction of 1g-k in the presence of aspartic acid used as
the amino donor in quasisaturating concentration (40 mM).
The Km values measured with 1g-k are in the mmol range,
1 to 2 orders of magnitude higher than that of the natural
substrate (KG). This result contrasts with the observations made
previously with the 4-alkyl derivatives, which showed very good
affinities for the enzyme active site.6 More interestingly,

Synthesis of a Pentanedioic Acid
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4087
Scheme 4. Synthesis and NMR Analysis of Lactam 7
a Coupling constants were estimated from the preferred calculated conformations I-IV.
stereoselectivity of AAT in favor of the L-2,4-syn configuration
of Glu analogues substituted at position 4.
subtypes iGluR6 and iGluR7. We also characterized 2k in a
functional assay at the metabotropic Glu receptors (mGluRs)
subtypes mGluR1 and mGluR5 (group I), mGluR2 (group II),
and mGluR4 (group III).9 In these assays, 2k proved unable to
agonize, antagonize, or modulate any of aforementioned mGluR
subtypes. Furthermore, in binding assays at cloned human
mGluR2 and mGluR3 subtypes, 2k proved without affinity (both
IC50 >1000 µM).
The EAAT2-Inhibitor Pharmacophore. On the basis of
several SAR studies, we5–8 and others14–16 have proposed that
EAAT substrates and inhibitors, which contain a Glu skeleton,
bind in two distinct ways: while substrates bind to the transporter
in an extended Glu conformation, the binding conformation of
inhibitors is assumed to be equivalent to the folded Glu
conformation previously identified as the agonist binding
conformation at the iGluRs by X-ray crystallographic studies17
and rational ligand design studies.18
Pharmacology
The Glu analogues 2a-k (2d is exempt) were characterized
pharmacologically at human EAAT1, EAAT2, and EAAT3
stably expressed in HEK293 cells using the FLIPR membrane
potential (FMP) assay (see Experimental Section for details).
The assay was performed essentially as described previously,12
and results are summarized in Table 2. All analogues were either
inactive or displayed very weak activities as nonsubstrate
inhibitors at EAAT1 and EAAT3. At EAAT2, compounds
2a,b,e,g,h,j,k displayed weak-to-moderate inhibitory activities,
whereas 2k, which holds a bulky substituent on the amide
nitrogen (N-2,2-diphenylethyl), displayed a significant preference
for inhibition of EAAT2 over EAAT1 and EAAT3, with a
midrange micromolar potency (IC50 ) 75 µM).
To address the origin of the inhibitory preference for EAAT2
by 2k, we submitted the compounds 2j, 2k, and DHK to a
stochastic conformational search (see Experimental Section for
details). The observed low-energy folded Glu conformations (up
to +1 kcal/mol) of 2j, 2k, and DHK were superimposed by
fitting the ammonium and the two carboxylate groups (Figure
1). Interestingly, the isopropyl group of DHK, which is believed
to be the molecular feature determining the EAAT2 subtype
selectivity of the compound,5 does not occupy the same area in
space as the bulky substituent on the amide nitrogen (N-2,2-
diphenylethyl) of 2k. This suggests that the EAAT2 preference
displayed by 2k arises from a different molecular mechanism
than that of DHK. The lack of exact three-dimensional structural
information of EAAT2 makes it impossible for us to point out
the specific structural features of neither the EAAT2 protein
nor the 2k ligand, which underlie the EAAT2 selectivity profile
of this novel compound.
The Glu analogues 2a-k were also characterized at the
EAAT-HEK293 cell lines in a conventional [3H]-D-Asp assay
(see Experimental Section for details). The inhibition profiles
exhibited by the compounds in this assay correlated well with
the results from the FMP assay (Table 3). For example, 2k
displayed a 10-30 fold higher inhibitory potency at EAAT2
compared with EAAT1 and EAAT3.
In other studies, dihydrokainic acid (DHK, Table 2) has been
shown to be a selective inhibitor of the EAAT2 transporter
subtype in medium-range micromolar potency.12,13 However,
DHK also displays moderate affinity (IC50 ) 6 µM) for native
kainic acid receptors as well as the cloned homomeric subtypes
iGluR5-7 (Table 4).13 On the other hand, the well-described
EAAT inhibitor, L-threo-benzyloxyaspartate13 (TBOA) displays
very low affinity for the iGluRs (Table 4) but also no selectivity
among the EAAT subtypes (Table 2).
These facts prompted us to investigate 2k at native 2-amino-
3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA)
receptors, kainic acid (KA, Table 2) receptors, and the N-methyl-
D-aspartic acid (NMDA) receptors in a binding assay using rat
brain homogenate. As summarized in Table 4, 2k displayed low
affinities (Ki > 100 µM) for all three groups of native iGluRs.
Given the fact that affinities for KA receptor subunits iGluR5-7
are poorly estimated in the [3H]KA binding assay, we further
investigated 2k, and DHK, in binding assays at cloned rat
iGluR5-7 subtypes. Whereas DHK displayed low micromolar
affinity for iGluR5-7, compound 2k showed only midrange
micromolar affinity for iGluR5 and no affinity for receptor
Conclusion
In conclusion, we have described a chemo-enzymatic enan-
tioselective synthetic route toward compounds 2g-k and
presented the pharmacological characteristics of Glu analogues
2a-k at human EAAT1-3. Notably, 2k was found to be a novel
inhibitor with significant preference for EAAT2 over EAAT1
and EAAT3. In comparison with the often used EAAT2-
selective inhibitor, DHK, compound 2k displays a significantly
higher degree of selectivity toward EAAT2 over iGluRs and
mGluRs. Thus 2k may be a more useful pharmacological tool

4088 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Sagot et al.
Table 2. Inhibition of Glu-Induced Depolarization in EAAT1-, EAAT2-, and EAAT3-HEK293 Cells in the FMP Assaya
a The IC50 values of the nonsubstrate inhibitors were obtained using EC80-EC90 concentrations of Glu at the three EAAT subtypes. The Km values for
substrates (bold) and IC50 values for inhibitors, are given in µM (with pKm ( SEM and pIC50 ( SEM values in brackets, respectively). b Values taken from
ref 12.
procedures. Cysteine sulfinic acid was prepared from cystine
following a described procedure.19 Bovine heart malic dehydro-
genase and rabbit muscle lactic dehydrogenase were purchased from
Sigma. Escherichia. coli AAT was produced and purified following
described procedures from overexpressing E. coli strains JM103
transformed with pUC119-aspC (AAT).20 Enzyme kinetic measure-
ments were performed at 25 °C in 0.1 M potassium phosphate
buffer, pH 7.6, Asp (40 mM), NADH (0.2 mM), ketoacid substrate
(0.1-10 mM), AAT (0.05 UI), and malic dehydrogenase (2 UI) in
a total volume of 1 mL. Rates were calculated from the OD linear
for the investigation of the physiological roles governed by the
EAAT2. The design and synthesis of analogues of 2k, which
may improve potency, is currently in progress in our laboratories.
Experimental Section
Chemistry. Melting points were determined on a Reichert hot-
stage apparatus and are uncorrected. IR spectra were recorded on
1
a Perkin-Elmer 801 spectrophotometer. H and 13C NMR spectra
were recorded on a Bruker Avance 400 MHz spectrometer.
Chemical shifts are reported in ppm (δ) relative to TMS as internal
standard. HRMS were recorded on a q-tof Micromass spectrometer.
Optical rotations were determined with a JASCO DIP 370 pola-
rimeter and are reported at the sodium D line (589 nm). Elemental
analyses were performed at the Service Central d’Analyze du
CNRS, Solaize, France. Silicagel 60 (Merck, 40-63 µm) and
precoated F254 plates were used for column and TLC chromatog-
raphy. All solvents were purified by distillation following usual
decay at 340 nm using ꢀNADH ) 6220 cm-1 ·M-1
.
Dimethyl 4-Aminocyclohex-3-ene-1-3-dicarboxylate 4g. To a
solution of 310 (1 g, 4.6 mmol) in MeOH (5 mL), was added a
20% aqueous solution of NH3 (1.3 mL, 13.8 mmol). The reaction
mixture was stirred at room temperature for 48 h. After addition
of brine (10 mL), the aqueous solution was extracted with AcOEt
(3 × 10 mL). The combined organic layers were dried over MgSO4

Synthesis of a Pentanedioic Acid
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4089
Table 3. Inhibition of [3H]-D-Asp Uptake in EAAT1-, EAAT2-, and
solution). After 30 min, the excess ozone was eliminated by oxygen
bubbling. Triphenylphosphine (1.2 g, 4.5 mmol) was added, and
the reaction mixture was allowed to warm to room temperature.
The solution was washed with water (20 mL), brine (20 mL), dried
over MgSO4, and concentrated under reduced pressure. Flash
chromatography (eluent, cyclohexane-AcOEt, 7:3 to AcOEt-MeOH,
9:1, v/v) afforded 5g-k as colorless liquids. 6 was also isolated as
a 1:2 mixture of isomers.
EAAT3-HEK293 Cellsa
EAAT1
EAAT2
EAAT3
Glu
2a
2b
2c
35 [4.5 ( 0.04]
62 [4.2 ( 0.03] 51 [4.3 ( 0.02]
140 [3.9 ( 0.05] ∼3000 [∼2.5]
290 [3.5 ( 0.03]b 1000-3000 [2.5-3]
∼3000 [∼2.5]
450 [3.3 ( 0.05]b
>3000 [<2.5]
>3000 [<2.5]
>3000 [<2.5]
>3000 [<2.5]
>3000 [<2.5]
>3000 [<2.5]
2f
2e 1000-3000 [2.5-3]
180 [3.8 ( 0.04] ∼3000 [∼2.5]
290 [3.5 ( 0.04]b >3000 [<2.5]
680 [3.2 ( 0.05]b >3000 [<2.5]
Dimethyl 2-(3-Amino-3-oxopropyl)-4-oxoglutarate 5g. Yield 55%
2g
2h
2i
>3000 [<2.5]
>3000 [<2.5]
>3000 [<2.5]
∼1000 [∼3]
from 3. IR (neat film) 3453, 3363, 3206, 1730, 1667 cm-1 1H
.
NMR (400 MHz, CDCl3) δ 5.73 (2H, s, broad), 3.86 (3H, s), 3.67
(3H,s), 3.31 (1H, dd, J ) 10.1 and 20.0 Hz) 2.96 (1H, dd, J ) 4.9
and 20.0 Hz), 2.97 (1H, m), 2.29 (2H, m), 1.95 (2H, m). 13C NMR
(100 MHz, CDCl3) δ 191.9, 174.3, 174.2, 160.8, 53.1, 52.2, 40.9,
39.2, 32.8, 26.9. HRMS (ES+) m/z 268.0798 ([M + Na]+,
C10H15NaNO6 requires 268.0797). Anal. (C10H15NO6): C, H, N.
Dimethyl 2-(3-Methylamino-3-oxopropyl)-4-oxoglutarate 5h.
Yield 35% from 3. IR (neat film) 3393, 3313, 3103, 1735, 1652
1000-3000 [2.5-3]
>3000 [<2.5]
410 [3.4 ( 0.05]b ∼3000 [∼2.5]
95 [4.0 ( 0.04] ∼3000 [∼2.5]
2j
2k 1000-3000 [2.5-3]
a The Ki values are given in µM with pKi ( SEM values in brackets.
b The specific [3H]-D-Asp uptake at the EAAT was not completely inhibited
by the maximal concentration of the compound (3 mM), and thus the Ki
value is determined from the fit of the concentration-inhibition curve for
the compound.
1
cm-1. H NMR (400 MHz, CDCl3) δ 6.03 (1H, s, broad), 3.88
(3H, s), 3.67 (3H, s), 3.32 (1H, dd, J ) 10.2 and 19.8 Hz) 2.96
(1H, dd, J ) 4.6 and 19.8 Hz), 2.96 (1H, m), 2.81 (3H, d, J ) 4.8
Hz), 2.23 (2H, m), 1.97 (2H, m). 13C NMR (100 MHz, CDCl3) δ
191.9, 174.4, 172.4, 160.8, 53.1, 52.1, 40.9, 39.2, 33.6, 27.3, 26.3.
HRMS (ES+) m/z 282.0968 ([M + Na]+, C11H17NaNO6 requires
282.0954). Anal. (C11H17NO6): C, H, N.
and concentrated under reduced pressure. 4g was isolated as a white
solid (0.98 g) and used in the next reaction without further
purification; mp 56 °C (lit.21 mp 58-60.5 °C). 1H NMR (400 MHz,
D2O) δ 7.5-4.5 (2H, s, broad), 3.69 (3H, s), 3.68 (3H, s), 2.66
(1H, dd, J ) 5.1 and 15.6 Hz), 2.52 (1H, tdd, J ) 2.9, 5.3 and
10.3 Hz) 2.40-2.30 (2H, m), 2.25 (1H, ddd, J ) 3.6, 5.7 and 16.8
Hz), 2.00 (1H, m), 1.74 (1H, tdd, J ) 5.8, 10.9 and 13.0 Hz). 13
C
Dimethyl 2-(3-Propylamino-3-oxopropyl)-4-oxoglutarate 5i.
Yield 35% from 3. IR (neat film) 3389, 3306, 3100, 1732, 1648
NMR (100 MHz, D2O) δ 175.8, 170.2, 155.9, 89.9, 51.7, 50.4,
39.4, 29.4, 25.9, 24.2.
1
cm-1. H NMR (400 MHz, CDCl3) δ 5.64 (1H, s, broad), 3.85
(3H, s), 3.66 (3H, s), 3.30 (1H, dd, J ) 10.1 and 19.7 Hz), 3.18
(2H, q, J ) 6.8 Hz), 2.94 (1H, dd, J ) 4.6 and 19.8 Hz), 2.92 (1H,
m), 2.20 (2H, m), 1.93 (2H, m), 1.49 (2H, hex, J ) 7.2 Hz), 0.89
(3H, t, J ) 7.4 Hz). 13C NMR (100 MHz, CDCl3) δ 191.9, 174.4,
171.5, 160.8, 53.1, 52.1, 41.3, 40.9, 39.2, 33.4, 27.4, 22.8, 11.3.
HRMS (ES+) m/z 310.1274 ([M + Na]+, C13H21NaNO6 requires
310.1267). Anal. (C11H17NO6): C, H, N.
General Procedure for the Synthesis of 4h-k. To a solution of
3 (1 g, 4.6 mmol) in anhydrous toluene (4 mL) was added 4 Å
molecular sieves (1 g) and the amine (6.9 mmol). For 4h synthesis,
a 2 M solution of MeNH2 in THF was used. In the case of 4k
synthesis, 4.6 mmol of Ph2CHCH2NH2 and 40 mL of toluene were
used. The reaction mixture was stirred at room temperature for 48 h.
After filtration, the solution was concentrated under reduced
pressure. Compounds 4h-k were isolated as white solids (4h and
4k) or as colorless liquids (4i and 4j) and were used in the next
reaction without further purification. In every case, 1H NMR
analysis indicated a yield over 90%.
Dimethyl 2-(3-Benzylamino-3-oxopropyl)-4-oxoglutarate 5j.
Yield 43% from 3. IR (neat film) 3384, 3303, 1734, 1654 cm-1
.
1H NMR (400 MHz, CDCl3) δ 7.28 (5H, m), 6.19 (1H, s, broad),
4.39 (2H, d, J ) 5.7 Hz), 3.85 (3H, s), 3.64 (3H, s), 3.29 (1H, dd,
J ) 10.2 and 19.8 Hz), 2.94 (1H, m), 2.93 (1H, dd, J ) 4.4 and
19.8 Hz), 2.25 (2H, m), 1.96 (2H, m). 13C NMR (100 MHz, CDCl3)
δ 191.9, 174.4, 171.5, 160.8, 138.2, 128.7, 127.8, 127.5, 53.1, 52.1,
43.6, 40.9, 39.2, 33.6, 27.3. HRMS (ES+) m/z 358.1267 ([M +
Na]+, C17H21NaNO6 requires 358.1267). Anal. (C11H17NO6): C, H,
N.
Dimethyl 4-Methylaminocyclohex-3-ene-1-3-dicarboxylate 4h.
1
Melting point 68-70 °C. H NMR (400 MHz, D2O) δ 8.76 (1H,
s, broad), 3.61 (3H, s), 3.57 (3H, s), 2.79 (3H, d, J ) 5.2 Hz), 2.66
(1H, dd, J ) 4.5 and 15.0 Hz), 2.50-2.15 (4H, m), 1.98 (1H, m),
1.65 (1H, m). 13C NMR (100 MHz, D2O) δ 175.6, 170.4, 159.1,
87.4, 51.6, 50.1, 39.2, 29.0, 29.4, 26.4, 25.2, 24.2.
Dimethyl 4-Propylaminocyclohex-3-ene-1-3-dicarboxylate 4i. 1H
NMR (400 MHz, D2O) δ 8.93 (1H, s, broad), 3.69 (3H, s), 3.66
(3H, s), 3.11 (2H, m), 2.68 (1H, dd, J ) 4.8 and 15.0 Hz),
2.56-2.24 (4H, m), 2.05 (1H, m), 1.70 (1H, tdd, J ) 5.8, 11.0 and
13.1 Hz), 1.58 (2H, hex, J ) 7.2 Hz), 0.97 (3H, t, J ) 7.3 Hz).
13C NMR (100 MHz, D2O) δ 175.7, 170.8, 158.7, 87.1, 51.7, 50.3,
44.1, 39.3, 26.4, 25.6, 24.3, 23.5, 11.5.
Dimethyl 2-(2,2-Diphenylethylamino-3-oxopropyl)-4-oxoglut-
arate 5k. Yield 25% from 3. IR (CCl4) 3450, 1735, 1668 cm-1
.
1H NMR (400 MHz, CDCl3) δ 7.25 (10H, m), 5.50 (1H, s, broad),
4.18 (1H, t, J ) 7.8 Hz), 3.90 (2H, m), 3.87 (3H, s), 3.62 (3H, s),
3.26 (1H, dd, J ) 8.8 and 18.6 Hz), 2.89 (1H, dd, J ) 4.3 and
18.5 Hz), 2.83 (1H, m), 2.11 (2H, m), 1.85 (2H, m). 13C NMR
(100 MHz, CDCl3) δ 191.8, 174.3, 171.6, 160.8, 141.8, 128.7,
128.0, 126.8, 53.1, 52.0, 50.6, 43.8, 40.9, 39.1, 33.6, 27.2. HRMS
(ES+) m/z 426.1933 ([M + H]+, C24H28NO6 requires 426.1917).
Anal. (C11H17NO6, 0.25H2O): C, H, N.
Dimethyl 4-Benzylaminocyclohex-3-ene-1-3-dicarboxylate 4j. 1H
NMR (400 MHz, D2O) δ 9.31 (1H, s, broad), 7.35-7.10 (5H, m),
4.37 (2H, m), 3.66 (6H, s), 2.68 (1H, dd, J ) 4.4 and 14.8 Hz),
2.55-2.35 (3H, m), 2.24 (1H, ddd, J ) 6.3, 10.8 and 17.5 Hz),
1.98 (1H, m), 1.66 (1H, tdd, J ) 5.8, 10.9 and 13.1 Hz). 13C NMR
(100 MHz, D2O) δ 175.6, 170.7, 158.4, 139.1, 128.8, 127.2, 126.7,
88.7, 51.7, 50.5, 46.2, 39.3, 26.4, 25.4, 24.3.
Dimethyl 3-Hydroxy-4-oxocyclohexanedicarboxylate 6. Yield
20-50%. IR (neat) 3456, 1728 cm-1. 1H NMR (400 MHz, CDCl3)
δ 4.39 (1H, s), 4.00 (1H, s), 3.83 (3H, s), 3.78 (3H, s), 3.70 (2 ×
3H, s), 3.08 (2 × 1H, m), 2.89 (2 × 1H, m), 2.67 (1H, m), 2.50 (3
× 1H, m), 2.38-2.23 (3 × 1H, m), 2.03-1.81 (3 × 1H, m). 13C
NMR (100 MHz, CDCl3) δ 205.7, 205.2, 174.2, 173.4, 171.9, 169.8,
79.7, 78.9, 53.3, 53.2, 52.2, 52.0, 39.5, 39.1, 37.7, 37.1, 37.0, 36.2,
29.1, 28.4. HRMS (ES+) m/z 253.0674 ([M + Na]+, C10H14NaO6
requires 253.0688).
General Procedure for the Synthesis of Dilithium Oxoglut-
arate 1g-k. To a solution of 5g-k (2 mmol) in MeOH (10 mL)
was added dropwise a 0.4 M solution of LiOH (10.5 mL, 4.2 mmol).
The mixture was stirred at room temperature for 5 to 15 h, until
completion. After evaporation of MeOH, the pH of the aqueous
solution was adjusted to 7.6 by addition of dowex 50WX8 resin
(H+ form). The resin was removed by filtration before evaporation
Dimethyl 4-(2,2-Diphenylethylamino)cyclohex-3-ene-1-3-dicar-
1
boxylate 4k. Melting point 145 °C. H NMR (400 MHz, D2O) δ
8.83 (1H, t, J ) 5.0 Hz), 7.25-7.10 (10H, m), 4.06 (1H, t, J ) 7.2
Hz), 3.71 (2H, m), 3.60 (3H, s), 3.51 (3H, s), 2.56 (1H, dd, J )
4.2 and 14.7 Hz), 2.40-2.15 (3H, m), 2.08 (1H, m), 1.90 (1H, m),
1.55 (1H, m). 13C NMR (100 MHz, D2O) δ 175.7, 170.4, 157.7,
141.9, 128.7, 128.1, 126.8, 88.2, 52.1, 51.7, 50.4, 47.4, 39.2, 26.5,
25.7, 24.3.
General ozonolysis procedure for the synthesis of 5g-k. A
solution of enamine 4g-k (approximately 4 mmol) in anhydrous
CH2Cl2 (40 mL) was treated at -70 °C with a mixture of O2 and
O3 at a rate of 10 L/h until saturation (blue coloration of the

4090 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Sagot et al.
Table 4. Binding Affinities of 2k, DHK, and TBOA at Native AMPA-, KA-, NMDA Receptors (Rat Synaptosomes) as well as at Rat Homomeric
iGluR5-7
compound
AMPA IC50 (µM)
KA IC50 (µM)
NMDA IC50 (µM)
iGluR5 Ki (µM)
iGluR6 Ki (µM)
iGluR7 Ki(µM)
2k
DHK
TBOA
>100
1100a
>100
6a
>100
350a
470a
37.2 [4.44 ( 0.05]
8.18 [5.09 ( 0.02]
nd
>100
>100
12.3 [4.92 ( 0.05]
0.376 [6.44 ( 0.07]
>1000a
550a
nd
nd
a Data are taken from ref 13. Mean pKi (M) ( SEM is given in square brackets (3-5 experiments conducted in triplicate). Hill values were unity for DHK
and 2k at iGluR5-7. nd: no data available.
M-1 ·cm-1. When a conversion rate of 40% was reached, the
reaction mixture was rapidly passed through a column of dowex
50WX8 resin (H+ form, 25 mL). The column was then washed
with water (100 mL) until complete elution of CSA and then eluted
with 1 M NH4OH. The ninhydrin positive fractions were combined
and concentrated to dryness under reduced pressure. The residue
was diluted in water (5 mL) and, if necessary, the pH adjusted to
7.0 with 1 M NaOH before adsorption of the product on a column
of dowex 1 × 8 resin (200-400 mesh, AcO- form, 1.5 cm × 12
cm). The column was washed with water (50 mL) and then eluted
with an AcOH gradient (0.1-1M) and, in the case of 2k, with 1 M
AcOH in H2O-MeOH (1:1, v/v). The ninhydrin positive fractions
Figure 1. Superimposition of calculated low-energy conformations
were combined and concentrated under reduced pressure. The oily
residue was dissolved in water (4 mL), and the solution was
lyophilized to afford 2g-k as hygroscopic white solids.
(2S,4R)-4-(3-Amino-3-oxopropyl)glutamic Acid 2g. Yield 35 mg,
35%; mp 120 °C; [R]25D ) +34.2 (c 1.0, 6 N HCl). 1H NMR (400
MHz, D2O) δ 3.78 (1H, dd, J ) 5.4 and 8.3 Hz), 2.62 (1H, m),
2.34 (2H, m), 2.29 (1H, ddd, J ) 5.5, 10.0 and 15.0 Hz), 1.92
(3H, m). 13C NMR (100 MHz, D2O) δ 178.7, 178.5, 173.6, 53.0,
41.6, 32.3, 27.8. HRMS (ES-) m/z 217.0838 ([M - H]-,
C8H13N2O5 requires 217.0824). Anal. (C8H14N2O5 ·0.75H2O) C, H,
N.
(up to +1 kcal/mol) of 2k (type code), 2j (yellow), and DHK (purple),
by fitting ammonium groups and two carboxylate groups.
of the water under reduced pressure. 2g-k were isolated in
quantitative yield as white solids.
Dilithium 2-(3-Amino-3-oxopropyl)-4-oxoglutarate 1g. 1H NMR
(400 MHz, D2O) δ 3.02 (1H, dd, J ) 9.0 and 19.5 Hz), 2.84 (1H,
dd, J ) 6.7 and 19.2 Hz), 2.61 (1H, m), 2.24 (2H, m), 1.74 (2H,m).
13C NMR (100 MHz, D2O) δ 204.3, 182.6, 179.2, 167.9, 42.6,
42.0, 33.0, 28.2. HRMS (ES-) m/z 216.0509 ([M - 2Li + H]-,
C8H10NO6 requires 216.0508).
Dilithium 2-(3-Methylamino-3-oxopropyl)-4-oxoglutarate 1h. 1H
NMR (400 MHz, D2O) δ 3.02 (1H, dd, J ) 8.8 and 18.4 Hz), 2.83
(1H, dd, J ) 5.4 and 18.6 Hz), 2.68 (3H, s), 2.59 (1H, m), 2.21
(2H, m), 1.73 (2H,m). 13C NMR (100 MHz, D2O) δ 204.4, 182.9,
176.5, 169.7, 42.6, 42.0, 33.7, 28.4, 25.8. HRMS (ES-) m/z
236.0761 ([M - 2Li + H]-, C9H12NO6 requires 236.0746).
Dilithium 2-(3-Propylamino-3-oxopropyl)-4-oxoglutarate 1i. 1H
NMR (400 MHz, D2O) δ 3.12 (3H, t, J ) 6.8 Hz), 3.05 (1H, dd,
J ) 8.4 and 18.4 Hz), 2.86 (1H, dd, J ) 5.2 and 18.4 Hz), 2.62
(1H, m), 2.24 (2H, m), 1.75 (2H,m), 1.50 (2H, hex, J ) 7.4 Hz),
0.87 (3H, t, J ) 7.4 Hz). 13C NMR (100 MHz, D2O) δ 204.4,
181.6, 175.7, 170.8, 41.5, 41.2, 33.7, 27.9, 21.7, 10.6. HRMS (ES-)
m/z 258.0995 ([M - 2Li + H]-, C11H16NO6 requires 258.0978).
Dilithium 2-(3-Benzylamino-3-oxopropyl)-4-oxoglutarate 1j. 1H
NMR (400 MHz, D2O) δ 7.35 (5H, m), 4.36 (2H, s), 3.03 (1H, dd,
J ) 9.1 and 17.6 Hz), 2.84 (1H, dd, J ) 5.5 and 17.8 Hz), 2.63
(1H, m), 2.29 (2H, m), 1.78 (2H,m). 13C NMR (100 MHz, D2O) δ
204.3, 182.3, 175.8, 170.0, 137.9, 128.7, 127.4, 127.2, 43.0, 42.3,
41.7, 33.8, 28.2. HRMS (ES-) m/z 306.0991 ([M - 2Li + H]-,
C15H16NO6 requires 306.0978).
(2S,4R)-4-(3-Methylamino-3-oxopropyl)glutamic Acid 2h. Yield
1
74 mg, 46%; mp 124 °C; [R]25 ) +30.8 (c 1.0, 6 N HCl). H
D
NMR (400 MHz, D2O) δ 3.75 (1H, dd, J ) 5.0 and 8.5 Hz), 2.71
(3H, s), 2.54 (1H, m), 2.29 (2H, m), 2.25 (1H, ddd, J ) 5.2, 9.7
and 14.8 Hz), 1.89 (3H, m). 13C NMR (100 MHz, D2O) δ 179.4,
176.0, 173.9, 53.1, 42.2, 33.1, 32.5, 28.2, 25.8. HRMS (ES+) m/z
255.0961 ([M + Na]+, C9H16NaN2O5 requires 255.0957). Anal.
(C9H16N2O5) C, H, N.
(2S,4R)-4-(3-Propylamino-3-oxopropyl)glutamic acid 2i. Yield
1
94 mg, 38%; mp 145 °C; [R]25 ) +27.6 (c 1.0, 6 N HCl). H
D
NMR (400 MHz, D2O) δ 3.76 (1H, dd, J ) 5.3 and 8.4 Hz), 3.11
(2H, t, J ) 6.9 Hz), 2.57 (1H, m), 2.28 (2H, m), 2.27 (1H, ddd, J
) 5.2, 9.6 and 15.0 Hz), 1.90 (3H, m), 1.48 (hex, J ) 7.3 Hz),
0.87 (3H, t, J ) 7.4 Hz). 13C NMR (100 MHz, D2O) δ 178.9,
175.3, 173.7, 53.1, 41.8, 41.2, 33.2, 32.4, 28.2, 21.7, 10.6. HRMS
(ES+) m/z 283.1277 ([M + Na]+, C11H20NaN2O5 requires 283.1270).
Anal. (C11H20N2O5) C, H, N.
(2S,4R)-4-(3-Benzylamino-3-oxopropyl)glutamic acid 2j. Yield
1
70 mg, 39%; mp 110 °C; [R]25 ) +22.0 (c 1.1, 6 N HCl). H
D
Dilithium 2-(2,2-Diphenylethylamino-3-oxopropyl)-4-oxoglut-
arate 1k. 1H NMR (400 MHz, D2O) δ 7.30 (10H, m), 4.28 (1H, t,
J ) 8.2 Hz), 3.86 (2H, m), 2.93 (1H, dd, J ) 8.6 and 18.7 Hz),
2.71 (1H, dd, J ) 5.5 and 18.7 Hz), 2.44 (1H, m), 2.09 (2H, t, J )
8.0 Hz), 1.54 (1H,m), 1.50 (1H, m). 13C NMR (100 MHz, D2O) δ
205.6, 184.3, 177.2, 171.2, 143.6, 130.3, 129.6, 128.4, 51.6, 45.2,
44.4, 43.6, 35.5, 30.4. HRMS (ES-) m/z 396.1451 ([M - 2Li +
H]-, C22H22NO6 requires 396.1447).
General Procedure for the Synthesis of Substituted Glutamic
Acids 2g-k. To a solution of racemic 1g-k (0.5 mmol) in water
(25 mL) was added cysteine sulfinic acid (0.5 mmol). The pH of
the solution was adjusted to 7.6 with 1 M NaOH before the addition
of E. coli AAT (20-100 Units). The reaction was stirred slowly at
room temperature and monitored by titration of pyruvate: 5 µL
aliquots of the reaction mixture were added to 995 µL of 0.1 M
potassium phosphate buffer, pH 7.6 containing NADH (0.2 mM),
and lactate dehydrogenase (1 unit). Pyruvate concentration was
calculated from the ∆OD measured at 340 nm using ꢀNADH ) 6220
NMR (400 MHz, D2O) δ 7.33 (5H, m), 4.33 (2H, s), 4.03 (1H, t,
J ) 7.2 Hz), 2.68 (1H, m), 2.34 (3H, m), 1.96 (1H, ddd, J ) 4.4,
7.2 and 14.4 Hz), 1.92 (2H, m). 13C NMR (100 MHz, D2O) δ 177.8,
175.1, 171.4, 137.9, 128.8, 127.5, 127.3, 51.4, 43.0, 40.9, 32.8,
31.6, 27.9. HRMS (ES+) m/z 331.1268 ([M
+
Na]+,
C15H20NaN2O5 requires 331.1270); Anal. (C15H20N2O5 ·0.75H2O)
C, H, N.
(2S,4R)-4-(2,2-Diphenylethylamino-3-oxopropyl)glutamic acid
2k. Yield 108 mg, 37%; mp 135 °C; [R]25 ) +16.9 (c 0.9, 6 N
D
1
HCl). H NMR (400 MHz, D2O) δ 7.33 (8H, m), 7.25 (2H, m),
4.24 (1H, t, J ) 8.3 Hz), 3.94 (1H, t, J ) 7.1 Hz), 3.82 (2H, d, J
) 8.3 Hz), 2.50 (1H, m), 2.25 (1H, ddd, J ) 7.0, 9.3 and 14.8 Hz),
2.12 (2H, m), 1.87 (1H, ddd, J ) 4.4, 7.3 and 14.7 Hz), 1.68 (2H,
m). 13C NMR (100 MHz, D2O) δ 177.8, 175.0, 171.4, 142.1, 128.9,
127.9, 127.0, 51.3, 49.9, 43.3, 40.7, 32.8, 31.3, 27.9. HRMS (ES+)
m/z 421.1742 ([M + Na]+, C22H26NaN2O5 requires 421.1739). Anal.
(C22H26N2O5 · 2.75 H2O) C, H, N.

Synthesis of a Pentanedioic Acid
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4091
cally in a [3H]-D-Asp assay essentially as previously described.12
Briefly, cells were split into PDL-coated white 96-well plates
(Perkin-Elmer, Boston, MA) in culture medium. At 16-24 h
later, the medium was aspirated, and cells were washed 3 times
with 100 µL of assay buffer (Hanks Buffered Saline Solution
supplemented with 1 mM CaCl2 and 1 mM MgCl2). Then 50
µL of assay buffer supplemented with 30 nM [3H]-D-Asp and
various concentrations of different ligands was added to each
well, and the plate was incubated at 37 °C for 7 min. The wells
were then washed with 3 × 100 µL of ice-cold assay buffer,
and 150 µL of Microscint20 scintillation fluid (Perkin-Elmer)
was added to each well. The plate was shaken for 1 h and
counted in a TopCounter (Perkin-Elmer). Nonspecific [3H]-D-
Asp transport in the cell lines was determined using 3 mM Glu.
Methyl (2S,4R)-4-(2,2-Diphenylethylamino-3-oxopropyl)pyro-
glutamate 7. A solution of 2k (20 mg, 0.05 mmol) in water (5
mL) was heated under reflux for 48 h until complete disappearance
of 2k. After evaporation of water under reduced pressure, the solid
residue was dried by addition and evaporation of MeOH (2 × 5
mL). It was then dissolved in MeOH (2 mL) and SOCl2 (25 µL,
0.35 mmol) was added. The mixture was stirred at room temperature
for 18 h before concentration under reduced pressure. Flash
chromatography (eluent, AcOEt/acetone, 8:2, v/v) afforded 7 (11
mg, 56%) isolated as a colorless viscous liquid: [R]25D ) +20.8 (c
0.6, CHCl3). 1H NMR (400 MHz, C6D6) δ 7.14 (8H, m), 7.02 (2H,
m), 5.98 (1H, s, broad), 5.65 (1H, t, J ) 5.5 Hz); 4.18 (1H, t, J )
8.0 Hz), 3.93 (1H, ddd, J ) 6.3, 7.9 and 13.8 Hz), 3.77 (1H, ddd,
J ) 5.6, 7.8 and 13.5 Hz), 3.39 (1H, dd, J ) 3.5 and 9.0 Hz), 3.22
(3H, s), 2.09-1.93 (3H, m); 1.85 (1H, ddd, J ) 3.5, 8.8 and 12.8
Hz), 1.69 (2H, m), 1.43 (1H, td, J ) 8.7 and 13.0 Hz). 13C NMR
(100 MHz, D2O) δ 178.9, 172.5, 171.7, 142.8, 128.9, 128.6, 126.8,
53.4, 51.7, 51.2, 44.2, 38.1, 33.8, 31.7, 27.7. HRMS (ES+) m/z
395.1976 ([M + H]+, C23H27N2O4 requires 395.1971).
AMPA, KA and NMDA Binding Assays. Glu analogues
were evaluated for AMPA, KA, NMDA (CGP 39653) binding
affinity in native rat synaptosomes, in accordance with previ-
ously described experimental procedure.22
Modeling
iGluR5-7 Binding Assay. Rat iGluR5(Q)1b, iGluR6-
(V,C,R)a, and iGluR7a were inserted into recombinant bacu-
loviruses, receptors expressed by infection of Sf9 insect cells
and infected Sf9 cell membranes utilized for radioligand binding
assays. Cells were maintained in BaculoGold Max-XP serum-
free medium (BD Biosciences-Pharmingen, San Diego, CA)
according to standard protocols in “Guide to Baculovirus
Expression Vector Systems and Insect Cell Culture Techniques”
(Life Technologies, Paisley, UK) and “Baculovirus Expression
Vector System: Procedures and Methods Manual”, 2nd ed.,
(Pharmingen). [3H]-SYM2081 ([3H]-(2S,4R)-4-methylglutamic
acid) (47.9 Ci/mmol; ARC Inc., St. Louis, MO) microtiter plate
binding assays were performed in 250 µL of assay buffer (50
mM Tris-HCl, pH 7.1 at 4 °C) at a protein concentration from
10 to 50 µg/mL, incubating for 1-2 h at 4 °C. Bound and free
radiolabel were separated by cold filtration through GF/B glass
fiber filters (UniFilter-96, Perkin-Elmer) on a Perkin-Elmer
FilterMate manifold using two washes with cold assay buffer.
Nonspecific binding was determined in the presence of 1 mM
Glu. Competition studies were performed using 1-5 nM
radiolabel in the presence of 10 nM to 1 mM cold ligand. Filters
were dried 1 h at 50 °C and then 50 µL/well of Microscint 20
was added. Radioactivity was detected as DPM using a
TopCounter. The affinity of the radiolabel for the kainate
receptors was determined from saturation binding experiments,
Kd (mean ( SEM): iGluR5(Q)1b, 0.663 ( 0.035 nM;
iGluR6(V,C,R)a, 17.0 ( 3.0 nM; iGluR7a, 5.69 ( 1.12 nM.
Competition data were analyzed using Grafit v3.00 (Erithacus
Software Ltd., Horley, UK) and fit to equations as previously
described for the determination of Ki.23
In Silico Study of 4-Substituted Glu Analogues and
Selected Ligands. The modeling study was performed using
the software package MOE (Molecular Operating Environment,
v2006.08, Chemical Computing Group, 2006) using the built-
in mmff94x forcefield and the GB/SA continuum solvent model.
General procedure for compounds 2g-k: The γ-carboxylate
group was protonated and the compound submitted to a
stochastic conformational search (standard setup). Conforma-
tions which hold intramolecular hydrogen bond(s) or π-cation
interactions were discarded. Superimpositions of selected con-
formations were carried out using the built-in function in MOE
by fitting the ammonium group and the two carboxylate groups.
Pharmacology
FLIPR Membrane Potential (FMP) Assay. The construc-
tion of Human Embryonic Kidney 293 (HEK293) cell lines
stably expressing human EAAT1, EAAT2, and EAAT3 have
been reported previously, and the pharmacological characteriza-
tion of 2a-k (2d is exempt) at the cell lines in the FMP assay
was performed essentially as described.12 Briefly, cells were
split into poly-D-lysine (PDL)-coated black 96-well plates with
a clear bottom (BD Biosciences, Palo Alto, CA) in Dulbecco’s
modified Eagle medium supplemented with penicillin (100
U/mL), streptomycin (100 µg/mL), 10% dialyzed fetal bovine
serum, and 1 mg/mL G-418 (culture medium). At 16-24 h later,
the medium was aspirated and the cells were washed with 100
µL Krebs buffer [140 mM NaCl/4.7 mM KCl/2.5 mM CaCl2/
1.2 mM MgCl2/11 mM HEPES/10 mM D-glucose, pH 7.4].
Then 50 µL of Krebs buffer was added to each well (in the
characterization of nonsubstrate inhibitors, the inhibitors were
added to this buffer). Then 50 µL of Krebs buffer supplemented
with FMP assay dye was then added to each well (in the
experiments with the nonsubstrate inhibitors, the compounds
were added to this solution), and the plate was incubated at 37
°C for 30 min. The plate was assayed at 30 °C in a NOVOstar
plate reader measuring emission at 560 nm caused by excitation
at 530 nm before and up to 1 min after addition of 33 µL of
substrate solution. The experiments were performed in duplicate
at least three times for each compound. For the characterization
of nonsubstrate inhibitors, 30 µM Glu was used as substrate
concentration at the EAAT1- and EAAT3-cell lines, whereas
50 µM Glu was used as substrate concentration at the EAAT2-
cell line.
mGluR Assays. Binding and functional assays were carried
out as described in ref 9.
Acknowledgment. We thank the The Carlsberg Foundation,
Lundbeck Foundation, The Danish Medical Research Council,
the French National Center for Scientific Research (CNRS), and
the Novo Nordisk Foundation for funding. We are grateful to
Prof. Kagamiyama’s group (Osaka Medical College) for provid-
ing us with the AAT overexpressing E. coli strains. Dr. Susan
Amara is thanked for providing us with the EAAT cDNAs.
Supporting Information Available: Combustion analysis data
1
of compounds 2g-k and 5g-k, and H and 13C NMR spectra of
[3H]-D-Asp Uptake Assay. The EΑΑΤ1-, EAAT2-, and
EAAT3-HEK293 cell lines were characterized pharmacologi-
compounds 2 g-k. HPLC analyses of compound 2k. This material
is available free of charge via the Internet at http://pubs.acs.org.

4092 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Sagot et al.
(13) Shimamoto, K.; LeBrun, B.; Yasuda-Kamatani, Y.; Sakaitani, M.;
Shigeri, Y.; Yumoto, N.; Nakajima, T. DL-threo-beta-benzyloxyas-
partate, a potent blocker of excitatory amino acid transporters. Mol.
Pharmacol. 1998, 53, 195–201.
References
(1) Campiani, G.; Fattorusso, C.; De Angelis, M.; Catalanotti, B.; Butini,
S.; Fattorusso, R.; Fiorini, I.; Nacci, V.; Novellino, E. Neuronal high-
affinity sodium-dependent glutamate transporters (EAATs): targets for
the development of novel therapeutics against neurodegenerative
diseases. Curr. Pharm. Des. 2003, 9, 599–625.
(2) Seal, R. P.; Amara, S. G. Excitatory amino acid transporters: a family
in flux. Annu. ReV. Pharmacol. Toxicol. 1999, 39, 431–456.
(3) Bra¨uner-Osborne, H.; Egebjerg, J.; Nielsen, E. O.; Madsen, U.;
Krogsgaard-Larsen, P. Ligands for glutamate receptors: design and
therapeutic prospects. J. Med. Chem. 2000, 43, 2609–2645.
(4) Meldrum, B. S. Glutamate as a neurotransmitter in the brain: review
of physiology and pathology. J. Nutr. 2000, 130, 1007S–1015S.
(5) Bunch, L.; Nielsen, B.; Jensen, A. A.; Brauner-Osborne, H. Rational
design and enantioselective synthesis of (1R,4S,5R,6S)-3-
azabicyclo[3.3.0]octane-4,6-dicarboxylic acid: a novel inhibitor at
human glutamate transporter subtypes 1, 2, and 3. J. Med. Chem. 2006,
49, 172–178.
(6) Alaux, S.; Kusk, M.; Sagot, E.; Bolte, J.; Jensen, A. A.; Brauner-
Osborne, H.; Gefflaut, T.; Bunch, L. Chemoenzymatic synthesis of a
series of 4-substituted glutamate analogues and pharmacological
characterization at human glutamate transporters subtypes 1-3. J. Med.
Chem. 2005, 48, 7980–7992.
(7) Bra¨uner-Osborne, H.; Bunch, L.; Chopin, N.; Couty, F.; Evano, G.;
Jensen, A. A.; Kusk, M.; Nielsen, B.; Rabasso, N. Azetidinic Amino
Acids: Stereocontrolled Synthesis and Pharmacological Characteriza-
tion as Ligands for Glutamate Receptors and Transporters. Org.
Biomol. Chem. 2005, 3, 3926–3936.
(8) Faure, S.; Jensen, A. A.; Maurat, V.; Gu, X.; Sagot, E.; Aitken, D. J.;
Bolte, J.; Gefflaut, T.; Bunch, L. Stereoselective chemoenzymatic
synthesis of the four stereoisomers of L-2-(2-carboxycyclobutyl)glycine
and pharmacological characterization at human excitatory amino acid
transporter subtypes 1, 2, and 3. J. Med. Chem. 2006, 49, 6532–6538.
(9) Sagot, E.; Pickering, D. S.; Pu, X.; Umberti, M.; Stensbøl, T. B.;
Nielsen, B.; Chapelet, M.; Bolte, J.; Gefflaut, T.; Bunch, L. Chemo-
Enzymatic Synthesis of a Series of 2,4-Syn-Functionalized (S)-
Glutamate Analogues: New Insight into the Structure-Activity
Relation of Ionotropic Glutamate Receptor Subtypes 5, 6 and 7. J. Med.
Chem. 2008, 51, 4093–4103.
(10) Chakrabortty, P. N. D., R.; Dasgupta, S. K.; Ghosh, S. R.; Ghatak,
U. R. New synthetic routes to tetracyclic bridged-bicyclo[3.2.1]octane
intermediates by intramolecular alkylation reactions through [alpha]-
diazomethyl ketones of hydroaromatic [gamma],[delta]-unsaturated
acids. Tetrahedron 1972, 28, 4653–4665.
(11) Echalier, F.; Constant, O.; Bolte, J. Enzymatic-Synthesis of 4-Methyl-
L-Glutamic and 4-Ethyl-L-Glutamic Acids. J. Org. Chem. 1993, 58,
2747–2750.
(12) Jensen, A. A.; Bra¨uner-Osborne, H. Pharmacological characterization
of human excitatory amino acid transporters EAAT1, EAAT2, and
EAAT3 in a fluorescence-based membrane potential assay. Biochem.
Pharmacol. 2004, 67, 2115–2127.
(14) Campiani, G.; De Angelis, M.; Armaroli, S.; Fattorusso, C.; Catalanotti,
B.; Ramunno, A.; Nacci, V.; Novellino, E.; Grewer, C.; Ionescu, D.;
Rauen, T.; Griffiths, R.; Sinclair, C.; Fumagalli, E.; Mennini, T. A
rational approach to the design of selective substrates and potent
nontransportable inhibitors of the excitatory amino acid transporter
EAAC1 (EAAT3). New glutamate and aspartate analogues as potential
neuroprotective agents. J. Med. Chem. 2001, 44, 2507–2510.
(15) Mennini, T.; Fumagalli, E.; Gobbi, M.; Fattorusso, C.; Catalanotti,
B.; Campiani, G. Substrate inhibitors and blockers of excitatory amino
acid transporters in the treatment of neurodegeneration: critical
considerations. Eur. J. Pharmacol. 2003, 479, 291–296.
(16) Koch, H. P.; Kavanaugh, M. P.; Esslinger, C. S.; Zerangue, N.;
Humphrey, J. M.; Amara, S. G.; Chamberlin, A. R.; Bridges, R. J.
Differentiation of substrate and nonsubstrate inhibitors of the high-
affinity, sodium-dependent glutamate transporters. Mol. Pharmacol.
1999, 56, 1095–1104.
(17) Hogner, A.; Kastrup, J. S.; Jin, R.; Liljefors, T.; Mayer, M. L.;
Egebjerg, J.; Larsen, I. K.; Gouaux, E. Structural basis for AMPA
receptor activation and ligand selectivity: Crystal structures of five
agonist complexes with the GluR2 ligand-binding core. J. Mol. Biol.
2002, 322, 93–109.
(18) Bunch, L.; Liljefors, T.; Greenwood, J. R.; Frydenvang, K.; Brauner-
Osborne, H.; Krogsgaard-Larsen, P.; Madsen, U. Rational design,
synthesis, and pharmacological evaluation of 2-azanorbornane-3-exo,5-
endo-dicarboxylic acid: A novel conformationally restricted glutamic
acid analogue. J. Org. Chem. 2002, 68, 1489–1495.
(19) Emiliozzi, R.; Pichat, L. A simple method for the preparation of
cysteinesulfinic acid. Bull. Soc. Chim. Fr. 1959, 1887–1888.
(20) Kamitori, S.; Hirotsu, K.; Higuchi, T.; Kondo, K.; Inoue, K.;
Kuramitsu, S.; Kagamiyama, H.; Higuchi, Y.; Yasuoka, N.; Kusunoki,
M.; Matsuura, Y. Overproduction and Preliminary X-Ray Character-
ization of Aspartate-Aminotransferase from Escherichia coli. J. Bio-
chem. 1987, 101, 813–816.
(21) Augustine, R. L.; Bellina, R. F.; Gustavsen, A. J. Hydrogenation of
beta-Amino-alpha,beta-Unsaturated Esters. J. Org. Chem. 1968, 33,
1287–1288.
(22) Hermit, M. B.; Greenwood, J. R.; Nielsen, B.; Bunch, L.; Jorgensen,
C. G.; Vestergaard, H. T.; Stensbol, T. B.; Sanchez, C.; Krogsgaard-
Larsen, P.; Madsen, U.; Bra¨uner-Osborne, H. Ibotenic acid and
thioibotenic acid: a remarkable difference in activity at group III
metabotropic glutamate receptors. Eur. J. Pharmacol. 2004, 486, 241–
250.
(23) Nielsen, B. S.; Banke, T. G.; Schousboe, A.; Pickering, D. S.
Pharmacological properties of homomeric and heteromeric GluR1(o)
and GluR3(o) receptors. Eur. J. Pharmacol. 1998, 360, 227–238.
JM800091E