Syntheses of optically pure β-hydroxyaspartate derivatives as glutamate transporter blockers

Article abstract of DOI:10.1016/S0960-894X(00)00487-X

DL-threo-β-Benzyloxyaspartate (DL-TBOA) is a non-transportable blocker of the glutamate transporters that serves as an indispensable tool for the investigation of the physiological roles of the transporters. To examine the precise interaction between a blocker and the transporters, we synthesized the optically pure isomers (L- and D-TBOA) and its erythro-isomers. L-TBOA is the most potent blocker for the human excitatory amino acid transporters (EAAT1-3), while D-TBOA revealed a difference in the pharmacophores between EAAT1 and EAAT3. We also synthesized the substituent variants (methyl or naphthylmethyl derivatives) of L-TBOA. The results obtained here suggest that bulky substituents are crucial for non-transportable blockers. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00487-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2407±2410
Syntheses of Optically Pure ꢀ-Hydroxyaspartate Derivatives as
Glutamate Transporter Blockers
Keiko Shimamoto,a,* Yasushi Shigeri,b Yoshimi Yasuda-Kamatani,a
Bruno Lebrun,a,y Noboru Yumotob and Terumi Nakajimaa
aSuntory Institute for Bioorganic Research, Wakayamadai, Shimamoto, Mishima, Osaka, 618-8503, Japan
bOsaka National Research Institute (AIST, MITI), Midorigaoka, Ikeda, Osaka, 563-8577, Japan
Received 29 June 2000; accepted 12 August 2000
AbstractÐdl-threo-b-Benzyloxyaspartate (dl-TBOA) is a non-transportable blocker of the glutamate transporters that serves as an
indispensable tool for the investigation of the physiological roles of the transporters. To examine the precise interaction between a
blocker and the transporters, we synthesized the optically pure isomers (l- and d-TBOA) and its erythro-isomers. l-TBOA is the
most potent blocker for the human excitatory amino acid transporters (EAAT1±3), while d-TBOA revealed a di€erence in the
pharmacophores between EAAT1 and EAAT3. We also synthesized the substituent variants (methyl or naphthylmethyl derivatives)
of l-TBOA. The results obtained here suggest that bulky substituents are crucial for non-transportable blockers. # 2000 Elsevier
Science Ltd. All rights reserved.
l-Glutamate acts as the principal excitatory neuro-
transmitter in the mammalian central nervous systems
(CNS). The extracellular glutamate concentrations are
tightly controlled by a transport system that limits the
activation of receptors during signalling and maintains
below excitotoxic level.1 It has been reported that gluta-
mate transporters transport Na+ ions and one proton
with glutamate while one K+ ion is counter-transported.2
Therefore, net inward movement of positive charges into
the cell is observed as a transport current. A number of
pharmacological agents have been shown to inhibit glu-
tamate transport but most of them indeed act as com-
petitive substrates, which was demonstrated by their
ability to induce transport currents and heteroexchange
with intracellular glutamate.3 Therefore, non-transport-
able blockers are indispensable tools for the investiga-
tion of the physiological roles of glutamate transporters.
racemic mixture of TBOA. Optically pure compounds
are needed for the precise discussion of structure±activity
relationships. l-Glutamate is a high anity substrate,
whereas d-glutamate is poorly transported. On the other
hand, both l- and d-aspartate are excellent substrates. It
would be interesting to know whether TBOA shows such
stereoselectivity. We describe here the syntheses and
characterization of the optically pure TBOA and its
erythro-derivatives and some new substituent variants.
Chemistry
We synthesized optically pure hydroxyaspartate deriva-
tives by the route shown in Scheme 1. In accordance
with the literature precedence,6 addition of vinyl mag-
nesiumbromide to (R)-Garner aldehyde (1)7 gave erythro
(anti)-2 with a moderate selectivity (threo:erythro=1:3)
whereas reaction with vinylzinc chloride showed the
opposite diastereoselectivity (threo:erythro=6:1). A dia-
stereomixture of methyl ester 4 was prepared by the fol-
lowing steps; (1) benzylation of 2, (2) oxidative cleavage
of the vinyl group of 3, (3) further oxidation to car-
boxylic acid, and (4) esteri®cation. Separation of dia-
stereomers by a ¯ash column chromatography followed by
removal of the acetonide group provided pure threo-5 and
erythro-5. Jones oxidation of the hydroxyl group of threo-5
gave monomethyl ester 6. Deprotection of threo-6 was
accomplished without epimerization to give l-
Recently, we reported that some derivatives of dl-threo-
b-hydroxyaspartate (dl-THA) acted as blockers.4,5
Among them, dl-threo-b-benzyloxyaspartate (dl-
TBOA) was the most potent blocker for the human
excitatory amino acid transporters, EAAT1 and EAAT2.5
In the previous paper, however, we have characterized a
*Corresponding author: Tel.: +81-75-962-3742; fax: +81-75-962-2115;
e-mail: shimamot@sunbor.or.jp
yPresent address: CNRS ESA 6034, INRA UA 1033, Faculte des Sci-
ences et Techniques de Saint Jerome, Cases 351-352, 13397 Marseille
Cedex 20, France.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00487-X

2408
K. Shimamoto et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2407±2410
Scheme 1.
TBOA.8 10 l-erythro-b-Benzyloxyaspartate (l-EBOA)
was prepared from erythro-5 in a similar manner. d-
TBOA and d-EBOA were synthesized from (S)-1.
Introduction of other substituents was performed from
the lactone 10 because it would be a useful common
intermediate for various analogues. The benzyl group of
threo-9 was removed by hydrogenation and the hydroxyl
group of threo-10 was substituted by methyl, 1-, or 2-
naphthylmethyl group (11a±c). Since trans-substituted
lactone was thermodynamically more stable than cis-
substituted lactone, etheri®cation of erythro-10 also gave
trans-products (11a±c) predominantly (3:1±6:1). Ring
opening of lactones followed by oxidation and depro-
tection provided the substituent variants of l-TBOA.
EAAT-transfected cells accumulated ®ve to ninefold
more [14C] glutamate than vector-transfected cells. All
four isomers of benzyloxyaspartate markedly inhibited
[14C]glutamate uptake in COS-1 cells (Fig. 1). Among
them, l-TBOA showed the most potent inhibitory action
for each EAAT subtype. threo-Isomers were more potent
than erythro-isomers and l-isomers were more potent
than d-isomers. The IC50 values are summarized in
Table 1. Concerning the substituent variants, l-TMOA,
l-TNOA1, and l-TNOA2 showed inhibitory activities
almost comparable to that of l-TBOA (Table 1).
Discrimination Between Blockers and Substrates
We performed electrophysiological analyses on Xenopus
oocytes expressing EAAT1±3 to determine whether each
compound acts as a blocker or a substrate.4,5 None of
the four benzyl derivatives elicited a detectable current
in voltage-clamped oocytes but all of them did reduce
Inhibition of Glutamate Uptake
Inhibition of radiolabeled glutamate uptake was mea-
sured in COS-1 cells transiently expressing EAAT1±3.5
Figure 1. All isomers of benzyloxyaspartate inhibited uptake of [14C]glutamate in COS-1 cells expressing EAAT1±3. l-TBOA is the most potent for
all EAAT subtypes. Values are presented as mean Æ S.E.M. of at least three determinations.

K. Shimamoto et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2407±2410
2409
Table 1. IC50 values of THA derivatives for blocking [14C]glutamate
uptake in COS-1 cells expressing EAAT1±3
Discussion
l-threo-b-Hydroxyaspartate derivatives possessing bulky
substituents, l-TBOA, l-TNOA1 and l-TNOA2, are
found to be potent blockers for all transporter subtypes.
On the other hand, l-TMOA acts as a substrate for
EAAT1 and EAAT3, but a blocker for EAAT2, like
(2S,4R)-4-methylglutamate (SYM 2081)12 and l-anti-
endo-3,4-methanopyrrolidine dicarboxylate (MPDC).13,14
According to our [14C]Glu uptake assays, l-TMOA
seems to have almost the same anity for the binding
site of transporters as that of l-TBOA. Based on our
results, it is likely that the bulky substituent in l-TBOA
inhibits the translocation step of the transporters,
whereas a smaller substituent would be sucient for
such an inhibition on EAAT2.
EAAT1
(IC50 mM)
EAAT2
(IC50 mM)
EAAT3
(IC50 mM)
dl-TBOA
l-TBOA
d-TBOA
l-EBOA
d-EBOA
l-TMOA
l-TNOA1
l-TNOA2
48Æ4.1
23Æ4.4
637Æ175
817Æ455
>1000
7.0Æ1.1
3.8Æ1.0
64Æ14
8.0Æ1.9
7.0Æ1.5
21Æ2.3
164Æ42
221Æ39
2.0Æ0.34
1.3Æ0.44
2.2Æ0.45
134Æ14
161Æ41
8.4Æ1.9
4.8Æ0.79
6.5Æ1.6
69Æ13
15Æ2.4
16Æ3.4
the glutamate (100 mM)-induced inward currents, con-
®rming they act as blockers for EAAT1±3. Among the
substituent variants, l-TMOA proved to be a substrate
for EAAT1 and EAAT3, while being a blocker for
EAAT2. The other substituent variants are blockers for
EAAT1±3. Relative transport currents obtained for
each compound (100 mM) in the absence or presence of
100 mM glutamate are shown in Table 2.
The synthesis of optically pure l-TBOA isomers
allowed us to investigate the importance of the bulky
substituent's spatial orientation. It has been proposed
that the active conformation of glutamate to transport-
ers is the folded form and that of aspartate is the
extended form.5,15 Recently Bridges et al. proposed a
pharmacophore model of blockers using the structure of
MPDC as a template.13 l-TBOA appears to ®t with this
pharmacophore model, in which the aspartate backbone
of l-TBOA takes an extended form. Inspection of
molecular models suggested that the bulky substituents
of l-TBOA and dihydrokainate (DHKA), a selective
EAAT2 blocker, have large overlaps (Fig. 2).16 When
E€ects on Glutamate Receptors
Next, we analyzed the activities of our compounds on
the ionotropic glutamate receptors by binding assay using
rat brain synaptic membranes. N-Methyl-d-aspartate
(NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazolepro-
pionic acid (AMPA), and kainate (KA) receptors were
labeled with [3H]CGP 39653 [dl-(E)-2-amino-4-propyl-
5-phosphono-3-pentenoic acid], [3H]AMPA, and
[3H]KA, respectively.5 l-TBOA showed a low anity
for AMPA and KA-type receptors (IC50>1 mM).
However, its anity for NMDA receptors was relatively
high (IC50=65Æ5.4 mM).11 In order to examine whether
l-TBOA activates ionotropic receptors, we measured the
inward currents in oocytes injected with rat whole brain
mRNA. At 100 mM, none of the compounds elicited
inward currents nor did they antagonize NMDA-evoked
inward currents. In addition, none of the compounds at
1 mM showed agonist or antagonist activity on the
metabotropic glutamate receptors (mGluRs) stably
expressed in CHO cells.5
Figure 2. Molecular models of MPDC, DHKA, and l-TBOA.16
Table 2. Potency of the hydroxyaspartate derivatives to induce a transport current in the absence or presence of L-Glu (100 mM) in oocytes
expressing EAAT1-3. The results are expressed relative to the current obtained with 100 mM L-Glu in the same cells (mean Æ S.D., n=3)
EAAT1
I/Icont (%)
EAAT2
I/Icont (%)
EAAT3
I/Icont (%)
Compound
(100 mM)
+Glu
(100 mM)
Compound
(100 mM)
+Glu
(100 mM)
Compound
(100 mM)
+Glu
(100 mM)
dl-TBOA
l-TBOA
d-TBOA
l-EBOA
d-EBOA
l-TMOA
l-TNOA1
l-TNOA2
0
0
0
0
30.1Æ3.9
14.5Æ0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
12.7Æ1.5
8.0Æ2.7
84.7Æ0.7
77.0Æ4.0
99.6Æ5.1
33.3Æ4.2
10.0Æ2.0
18.1Æ3.6
27.6Æ1.5
64.4Æ2.7
83.1Æ4.2
25.4Æ2.0
13.0Æ0.1
29.8Æ4.1
0
60.7Æ3.6
0
4.7Æ1.3
0
0
0
5.9Æ0.1
0
0
0
0

2410
K. Shimamoto et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2407±2410
the functional groups of the other stereoisomers (d- or
erythro-isomers) ®t with the pharmacophore, their sub-
stituents can not occupy the same space as that of the
bulky substituent of DHKA or l-TBOA.
6. Coleman, R. S.; Carpenter, A. J. Tetrahedron Lett. 1992,
33, 1697.
7. Garner, P.; Park, J. M. Org. Synth. 1991, 70, 18.
8. Purity of each isomer of 6 was con®rmed by conversion to
dimethyl ester 7. No peaks corresponding to methyl esters of
the other isomer were detected by 400 MHz NMR (3.61 and
3.77 ppm for threo-7; 3.73 and 3.79 ppm for erythro-7). How-
ever, deprotection of the dimethyl ester threo-7 was accom-
panied with the epimerization (ꢀ30%).
So far, it is well known that the pharmacological pro-
®les of EAAT1 and EAAT3 are very similar while
EAAT2 shows di€erent selectivity from these subtypes.
Interestingly, d-TBOA shows a major loss of activity on
EAAT1 (30-fold less potent than l-TBOA), while it
remains substantially active on EAAT3 (only threefold
less potent than l-TBOA). EAAT3 is more tolerant to
the d-isomers.
9. Enantiomeric purity (>95%) of each compound was con-
®rmed by chiral HPLC [column; CROWNPAK CR(+) or
CR( ), Daicel Chem. Ind. Ltd (Japan), eluent; 1% aqueous
HClO4].
10. Analytical data for l-TBOA: mp 172±175 ꢁC (dec.); [a]D
473.6ꢁ (c 0.68, 1 N HCl); 1H NMR (1 N DCl/D2O,
400 MHz): d 4.57 (d, 1H, J=2.5 Hz), 4.62 (d, 1H, J=11.5 Hz),
4.71 (d, 1H, J=2.5 Hz), 4.87 (d, 1H, J=11.5 Hz), 7.45 (m,
5H); 13C NMR (1 N DCl/D2O, 100 MHz): d 55.4, 74.5, 75.1,
129.6, 129.7, 129.8, 136.8, 169.4, 172.5; HRMS (FAB) m/z
calcd for C11H14O5N (M+H)+ 240.0871, found 240.0863.
11. The IC50 values of both l- and d-TBOA were lower than the
reported value for dl-TBOA (ref 5), since [3H]CGP 39653
(2nM) was used as a radioligand instead of [3H]CGS 19755. IC50
values for [3H]CGP 39653 binding were as follows (mM); d-
TBOA 181Æ23, l-EBOA 680Æ58, d-EBOA 26Æ1.3, l-TMOA
511Æ17, l-TNOA1 151Æ20, l-TNOA2 194Æ7.0. l-TMOA
inhibited [3H]KA binding (48Æ5.6 mM) and [3H]AMPA binding
(222Æ42 mM). The other derivatives showed very low anity
(>500 mM) for KA and AMPA receptors.
In summary, the optically pure isomers provided precise
information on the structure±activity relationship of
glutamate transporter blockers. These compounds can
be used as lead compounds to design new blockers with
greater subtype selectivity.
Acknowledgements
We wish to thank Prof. S. Nakanishi at Kyoto Uni-
versity (Japan) for providing the CHO cells expressing
mGluRs and Dr. R. P. Seal at Oregon Health Sciences
University for critical reading of this manuscript. This
work was supported by a Grant-in-Aid from the Ministry
of Education, Science, Sports, and Culture, Japan.
12. Vandenberg, R. J.; Mitrovic, A. D.; Chebib, M.; Balcar,
V. W.; Johnston, G. A. R. Mol. Pharmacol. 1997, 51, 809.
13. (a) Bridges, R. J.; Lovering F. E.; Koch, H.; Cotman, C.
W.; Chamberlin, A. R. Neurosci. Lett. 1994, 174, 193. (b)
Esslinger, C. S.; Koch, H. P.; Kavanaugh, M. P.; Philips, D.
P.; Chamberlin, A. R.; Thompson, C. M.; Bridges, R. J.
Bioorg. Med. Chem. Lett. 1998, 8, 3101.
References and Notes
14. In our study, MPDC (100 mM) also evoked the transport
currents in EAAT1 (16% to control) and EAAT3 (8%)
although it blocked EAAT2 as shown in ref 13b.
1. (a) Danbolt, N. C.; Chaudhry, F. A.; Dehnes, Y.; Lehre, K.
P.; Levy, L. M.; Ullensvang, K.; Storm-Mathisen, J. Prog.
Brain Res. 1998, 116, 23. (b) Billups, B.; Rossi, D.; Oshima, T.;
Warr, O.; Takahashi, M.; Sarantis, M.; Szatkowski, M.; Att-
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S. G. Annu. Rev. Pharmacol. Toxicol. 1999, 39, 431.
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macol. 1998, 53, 195.
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man, C. W.; Chamberlin, A. R. J. Med. Chem. 1991, 34, 717. (b)
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16. Molecular modelings were performed by using
QUANTA/CHARMm system (Molecular Simulations Inc.).
The CHARMm energy minimization with distance-dependent
dielectric term (e=80) was applied. See Shimamoto, K.;
Ohfune, Y. J. Med. Chem. 1996, 39, 407. In this reference we
have also reported the preparation of MPDC as CMP-III.