Discovery of the first selective inhibitor of excitatory amino acid transporter subtype 1

Article abstract of DOI:10.1021/jm8013458

The discovery of the first class of subtype-selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rat orthologue GLAST is reported. An opening structure-activity relationship of 25 analogues is presented that addresses the influence of substitutions at the 4- and 7-positions of the parental skeleton 2-amino-5-oxo-5,6,7,8-tetrahydro-4H- chromene-3-carbonitrile. The most potent analogue 1o displays high nanomolar inhibitory activity at EAAT1 and a >400-fold selectivity over EAAT2 and EAAT3, making it a highly valuable pharmacological tool.

Full text of DOI:10.1021/jm8013458

912
J. Med. Chem. 2009, 52, 912–915
Discovery of the First Selective Inhibitor of
Excitatory Amino Acid Transporter Subtype 1
Anders A. Jensen,*,† Mette N. Erichsen,†
Christina W. Nielsen,† Tine B. Stensbøl,‡ Jan Kehler,‡ and
Lennart Bunch*,†
Department of Medicinal Chemistry, Faculty of Pharmaceutical
Sciences, UniVersity of Copenhagen, UniVersitetsparken 2, DK-2100
Copenhagen, Denmark, and H. Lundbeck A/S, OttiliaVej 9,
DK-2500 Valby, Denmark
Figure 1. EAAT ligands L-serine-O-sulfate (L-SOS), (RS)-2-amino-
3-(1-hydroxy-1,2,3-triazol-5-yl)propionate, and (4R)-4-methylglutamate
(4-Me-Glu).
ReceiVed October 24, 2008
Abstract: The discovery of the first class of subtype-selective inhibitors
of the human excitatory amino acid transporter subtype 1 (EAAT1)
and its rat orthologue GLAST is reported. An opening structure-activity
relationship of 25 analogues is presented that addresses the influence
of substitutions at the 4- and 7-positions of the parental skeleton
2-amino-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile. The most
potent analogue 1o displays high nanomolar inhibitory activity at
EAAT1 and a >400-fold selectivity over EAAT2 and EAAT3, making
it a highly valuable pharmacological tool.
specific transporter subtype, as the absence of physiologic and
phenotypic effects in the knockout mice may also be ascribed
to compensatory mechanisms. Hence, we believe that valuable
information about the physiological functions and thus thera-
peutic potentials of a specific EAAT subtype may be addressed
in a more subtle way by studying the actions of a selective
ligand.4
Over the past 4 decades, the interest in glutamatergic
neurotransmission as a therapeutic target has prompted the
synthesis of an astonishing number of ligands targeting iono-
tropic and metabotropic Glu receptors.11-13 In contrast, me-
dicinal chemistry efforts in the EAAT field have been far more
limited.14,15 The vast majority of EAAT ligands published to
date are analogues of endogenous substrates Glu and Asp and
display nonselective activities as substrates or inhibitors at the
EAAT1, EAAT2, and EAAT3 subtypes.14,15 However, the
EAAT2 subtype appears to be more susceptible to inhibition
by such amino acid analogues compared with EAAT1 and
EAAT3, given the number of ligands found to display preference
or selectivity for this subtype.14,15 In contrast, no truly EAAT1-
selective ligands have been reported. Nevertheless, the interest
in such compounds is reflected in the notable attention paid to
the few compounds that display a bare minimal of preference
for the subtype.3,15 Best two examples are (RS)-2-amino-3-(1-
hydroxy-1,2,3-triazol-5-yl)propionate and L-serine-O-sulfate (L-
SOS) (Figure 1). The former displays very weak activity at the
EAAT1 subtype (IC50 ≈ 100 µM) and only 3- and >10-fold
lower activities at EAAT2 and EAAT3, respectively.16 It has
not been established whether it is a substrate or an inhibitor of
the transporters. L-SOS displays weak substrate activity at
EAAT1 and EAAT3 (IC50 ≈ 100 µM) and only a 10-fold lower
activity at EAAT2.17 Although the Glu analogue (4R)-4-
methylglutamate (4-Me-Glu) is a completely nonselective EAAT
ligand, it is actually the most EAAT1-discriminating ligand
described to date, being a substrate at EAAT1 and an inhibitor
of EAAT2,3.18,19 However, since the compound will inhibit Glu
uptake through EAAT1-3 with similar potencies and further-
more is a very potent iGluR5 agonist, it is not well-suited as a
pharmacological tool for studies of EAATs in native tissues.13
In this paper, we present the discovery of the first subtype-
selectiveinhibitorofEAAT1andtheopeningofthestructure-activity-
relation (SAR) which investigates the influence of the substi-
tutions at the 4- and 7-positions of the parental skeleton 2-amino-
5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile for the
subtype-selective EAAT1-activity.
The excitatory amino acid transporters (EAATs) belong to
the solute carrier family 1 (SLC1), and carry out the concentra-
tive uptake of (S)-glutamate (Glu) [and aspartate (Asp)] from
the glutamatergic synaptic cleft.1-3 Five EAAT subtypes have
been identified to date, in humans termed EAAT1-EAAT5.
For historical reasons the nomenclature differs in rodents: the
orthologues of human EAAT1, EAAT2 and EAAT3 subtypes
are termed GLAST, GLT-1 and EAAC-1, respectively, whereas
the nomenclature for EAAT4,5 is conserved across the species.
The EAATs display differential localization and overall
distribution patterns in the adult rodent brain. Whereas GLAST
(EAAT1) and GLT-1 (EAAT2) are astroglial transporters,
EAAC-1 (EAAT3) and EAAT4 are predominantly found in
neurons.4 GLT-1 (EAAT2) is the predominantly expressed
subtype, found throughout the brain and spinal cord, and has
been shown to be responsible for >90% of the Glu uptake in
the adult brain.4-6 GLAST (EAAT1) is found at the highest
levels in the cerebellum with lower levels in the cortex and
spinal cord,7 and EAAC-1 (EAAT3) is expressed in high
densities at postsynaptic terminals in the whole brain, particu-
larly in the hippocampus, cerebellum, and basal ganglia.4
EAAT4 is highly enriched in the Purkinje cells of the cerebel-
lum, but the transporter has also been detected in the rat fore-
and midbrain albeit in dramatically lower levels.4,5,8,9 Finally,
EAAT5 is found exclusively in the retina.4,5,10 In agreement
with the distribution and densities of the CNS subtypes, EAAT2
knockout mice have displayed pronounced loss of hippocampal
neurons, seizures, and 50% mortality at 6 weeks of age. In
contrast, knockout of GLAST (EAAT1), EAAC-1 (EAAT3),
or EAAT4 in mice have been reported not to cause significant
abnormalities in anatomy or in CNS-related phenotypes.4,6
However, such studies may not be fully conclusive when it
comes to elucidation of the physiological importance of the
* To whom correspondence should be addressed. For A.A.J.: phone, +45
35336491; fax, +45 35336040; e-mail, aaj@farma.ku.dk. For L.B.: phone,
+45 35336244; fax, +45 35336040; e-mail, lebu@farma.ku.dk.
†
A small commercially available compound library consisting
of 3040 compounds (obtained from Chembridge Corporation)
University of Copenhagen.
H. Lundbeck A/S.
‡
10.1021/jm8013458 CCC: $40.75
2009 American Chemical Society
Published on Web 01/22/2009

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 913
Table 1. Inhibition of [3H]-D-Asp Uptake in HEK293 Cells Stably Expressing Human EAAT1, EAAT2, and EAAT3a
a The IC50 for analogues 1a-y are given in µM with pIC50 ( SEM in brackets (n ) 3-7).
was screened for inhibitory activity at EAAT1-, EAAT2-, and
EAAT3-expressing HEK293 cell lines in a [3H]-D-Asp uptake
assay. Interestingly, we found 1a to inhibit the [3H]-D-Asp
uptake through EAAT1 selectively, showing no inhibitory
activity at EAAT2 or EAAT3. A subsequent verification of this
preliminary screening result gave IC50 values for 1a of 4.7 µM,
>100 µM, and >100 µM at EAAT1, -2, and -3, respectively
(Table 1). In comparison, the standard EAAT inhibitor DL-threo-
ꢀ-benzyloxyaspartate (TBOA) inhibited [3H]-D-Asp transport
through all three EAAT subtypes with IC50 values in the low
micromolar range (data not shown).20,21
The discovery of 1a prompted us to commence the building
of a SAR for this class of EAAT1 inhibitors by exploring the
4- and/or 7-position. The parental skeleton 2-amino-5-oxo-
5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile comprising sub-
stituents in the 4- and/or 7-positions (1a-y) is readily available
as a mixture of the four stereoisomers from the multicomponent
reaction (MCR) of 1,3-cyclohexandinone (which delivers the
R1 substituent), an aldehyde (which allows the incorporation
of the R2 substituent), and malononitrile (Scheme 1). The MCR
has been described under a large variety of reaction conditions
such as in EtOH/amine base,22 H2O/catalytic HTMAB,23 H2O/
DAHP,24 and solvent-free/NaBr under microware conditions.25
In all, 25 compounds were synthesized by us or obtained from
commercial suppliers and their pharmacological activities
determined at the EAAT1-, EAAT2-, and EAAT3-expressing
HEK293 cell lines in the [3H]-D-Asp uptake assay. The IC50
values of the analogues are summarized in Table 1, and
concentration-inhibition curves for selected analogues are
shown in Figure 2. The inhibitory activities displayed by the
analogues at EAAT1 were found to be dependent on the nature
of the R1 and R2 substituents (Table 1). None of the analogues
1a-y showed any significant inhibitory activity at the EAAT2
and EAAT3 subtypes at high-micromolar concentrations (all
IC50 values >100 µM, Table 1).
Building the SAR, analogues 1a-t which bear one aromatic
substituent in both the 4- and 7-positions show differential
inhibitory potencies at EAAT1 with 1o being the most potent
inhibitor (IC50 ) 0.66 µM). In detail, inhibitory activity is
maintained while the R1 substituent spans a phenyl (non-, mono-,
or disubstituted), a naphthyl, or a five-membered hetereoaromatic
moiety (furan and thiophene). For the R2 substituent a mono or
disubstituted phenyl ring is allowed; however, larger o-substituents
may result in decreased activity (o-fluoro in 1a vs the o-methoxy
group in 1f). The volume size of the pocket occluding the R2-
substituent is probed successfully with a naphthyl group (1i,j), a
biphenyl group (1k), and an additional trisubstituted phenyl ring
attached via a short linker (1l). But limitations to the bulk of the
R2-substituent are also apparent given that 1q shows a marked
decrease in inhibitory activity (IC50 ≈ 100 µM at EAAT1). As for
analogues in which R2 is a heteroaromatic group, the marked
decrease in inhibitory potency displayed by 1s,t points to disfavored

914 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Letters
Figure 2. Concentration-inhibition curves for selected analogues at
the EAAT1-HEK293 cell line in the [3H]-D-Asp uptake assay using
30 nM [3H]-D-Asp as tracer concentration. (A) Influence of inhibitory
activity of the chemical nature of R1 by comparing analogues
1a,m-p,r,x,y which bear comparable R2 substituents ((un)substituted
phenyl groups). (B) Influence of inhibitory activity of the chemical
nature of R2 by comparing analogues with conserved R1 substituents
(phenyl). The figure depicts representative experiments. For experi-
mental details, see Supporting Information.
Figure 3. Pharmacological characterization of 1o at human and
rat EAATs in the FLIPR Membrane Potential Blue assay: (A)
concentration-inhibition curves for 1o at the EAAT1-, EAAT2-, and
EAAT3-HEK293 cell lines using 50 µM Glu as substrate concentration;
(B) concentration-inhibition curves for 1o at the rat EAAT subtypes
GLAST (EAAT1), GLT-1 (EAAT2), and EAAC-1 (EAAT3) transiently
expressed in tsA-201 cells using 50 µM Glu as substrate concentration.
The figure depicts representative experiments. For further details, see
Supporting Information.
Scheme 1. Multicomponent Reaction (MCR) Methodology
Applied for the Synthesis of 1a-ya
4) at EAAT1, EAAT2, and EAAT3, respectively. Application of
compound 1o at concentrations up to 100 µM onto the three cell
lines did not give rise to fluorescent responses in this assay, which
demonstrates that 1o is not a substrate at the EAATs (data not
shown). However, compound 1o was found to be a potent inhibitor
of EAAT1, inhibiting the fluorescent response induced by 50 µM
Glu in EAAT1-HEK293 cells in a concentration-dependent manner
with an IC50 of 120 nM (pIC50 ( SEM, 6.91 ( 0.03, n ) 4),
whereas the compound did not display any significant inhibition
of the Glu-induced responses in EAAT2- or EAAT3-cells at
concentrations up to 100 µM (Figure 3A). In contrast to the
EAAT1-selectivity displayed by 1o and in agreement with the
findings of a previous study,20 the competitive EAAT inhibitor
TBOA displayed low-micromolar Ki values at all the three subtypes
(data not shown).
The pharmacological profile of 1o was also determined at
the rat EAAT subtypes GLAST (EAAT1), GLT-1 (EAAT2),
and EAAC-1 (EAAT3) transiently expressed in tsA-201 cells
using the FMP Blue assay. In this assay, Glu displayed Km
values of 37 µM (pKm ( SEM, 4.43 ( 0.04, n ) 3) at GLAST
(EAAT1), 45 µM (pKm ( SEM, 4.35 ( 0.05, n ) 3) at GLT-1
(EAAT2), and 35 µM (pKm ( SEM, 4.45 ( 0.04, n ) 3) at
EAAC-1 (EAAT3). In concordance with its EAAT1 selectivity
at the human transporters, 1o was shown to be a selective
inhibitor of GLAST (EAAT1), displaying an IC50 values of 970
nM (pIC50 ( SEM, 6.02 ( 0.05, n ) 4) at this subtype. No
significant inhibition of the Glu-induced responses in GLT-1
(EAAT2)- or EAAC-1 (EAAT3)-expressing cells was observed
for 1o at concentrations up to 100 µM (Figure 3B).
a For details, see Supporting Information.
electrostatic interactions with the transporter protein. The suitable
physical chemical nature of the R2 substituent is investigated further
in the three analogues 1u-w: a benzyl group, 1u, results in a 10-
fold decrease in potency, whereas a methyl group, 1v, is fully
acceptable. In contrast, no substituent (1w; R2 ) H) results in the
complete loss of activity. This finding is quite intriguing, and its
origin will be addressed in future studies. Analogously, a distinct
change in pharmacology is also observed when R1 is a methyl
group, 1x, or a hydrogen, 1y (Table 1). Again, all inhibitory activity
is lost, which points to the loss of an essential π-cation or π-π
stacking interaction.
The EAAT1-selective inhibitory activity displayed by this class
of compounds in the [3H]-D-Asp uptake assay was verified for the
most prominent compound in the series, 1o, in a different functional
assay. In a previous study, we have found the pharmacological
properties displayed by a series of standard EAAT substrates and
nonsubstrate inhibitors at the EAAT1-, EAAT2-, and EAAT3-
HEK293 cell lines in a fluorescence-based high throughput assay,
the FLIPR Membrane Potential Blue (FMP Blue) assay, to be in
good agreement with those observed for the compounds in other
assays.20 In the FMP Blue assay, Glu displayed Km values of 16
µM (pKm ( SEM, 4.79 ( 0.04, n ) 4), 27 µM (pKm ( SEM,
4.57 ( 0.05, n ) 3), and 21 µM (pKm ( SEM, 4.68 ( 0.04, n )
The compounds in this study are structurally distinct from
the endogenous EAAT substrates Glu and Asp. Thus, it is not
surprising that the active analogues in the 1a-y series are not
substrates but rather inhibitors of the EAAT1. Whether the
compounds target the orthosteric site in EAAT1, i.e., the binding

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 915
Okuyama, S.; Kawashima, N.; Hori, S.; Takimoto, M.; Wada, K.
Epilepsy and exacerbation of brain injury in mice lacking the glutamate
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site for Glu and Asp, or are negative allosteric (noncompetitive)
modulators inhibiting the uptake through EAAT1 via binding
to other regions in the transporter is currently under investigation.
It is important to stress that the 25 analogues only have been
characterized pharmacologically at three of the five EAAT
subtypes. Thus, we cannot exclude the possibility that the
compounds in addition to their EAAT1 activity could target
EAAT4 and/or EAAT5. However, the EAAT1-3 and EAAT4,5
subtypes make up two distinct subgroups within the EAAT
family in terms of transporter function and ion conductance,
despite the fact that a 50-60% sequence homology exists
between all of the subtypes.3,9,10 We are currently investigating
whether 1o and other compounds in the series target EAAT4,
the fourth CNS EAAT subtype. Nevertheless, we find it very
unlikely that a compound exhibiting >400-fold selectivity for
one subtype within the EAAT1-3 subgroup would possess any
significant activity at EAAT4,5.
In conclusion, we have presented the first class of compounds
that show fully selective inhibition of the human EAAT1
subtype over EAAT2 and EAAT3. A SAR was built from the
pharmacological investigation of 25 analogues, varying the
substituents in the 4- and 7-positions of the parental skeleton:
2-amino-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carboni-
trile. From this study, we have shown that the presence of an
aromatic ring in the 7-position (R1-substituent) is crucial for
the inhibitory activity at EAAT1. On the other hand, the
4-position (R2-substituent) may accommodate small and larger
groups, not being restricted to aromatics only, although a
substituent in this position is mandatory. The most potent
analogue in the series, 1o, displayed high nanomolar inhibitory
activity (IC50 ) 0.66 µM) at EAAT1, with more than 400-fold
selectivity compared to EAAT2 and EAAT3. This selectivity
profile was also observed at the rat orthologues, which in all
makes 1o a highly attractive pharmacological tool for future
explorations of the physiological role of the EAAT1 subtype.
Expansion of the SAR, studies addressing the bioavailability
and pharmacokinetic properties of 1o, and detailed investigations
of its binding mode at EAAT1 are all ongoing projects in our
laboratories.
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Acknowledgment. We thank the Lundbeck Foundation, the
Carlsberg Foundation, and the Danish Research Council for
financial support. Drs. S. G. Amara and T. Rauen are thanked
for their generous gifts of cDNAs for the human and rat EAATs,
respectively. Shahrokh Padrah is thanked for technical assistance
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Supporting Information Available: Experimental procedures
for the synthesis of analogues 1c,n,o,w and for the pharmacological
assays. This material is available free of charge via the Internet at
http://pubs.acs.org.
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