Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes

Article abstract of DOI:10.1021/acschemneuro.5b00311

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins, so-called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O-Benzylated l-threo-β-hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy.

Full text of DOI:10.1021/acschemneuro.5b00311

Letter
pubs.acs.org/chemneuro
Concise Asymmetric Synthesis and Pharmacological Characterization
of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA
and Synthesis of EAAT Photoaffinity Probes
Michele Leuenberger,, Andreas Ritler, Alexandre Simonin, Matthias A. Hediger,‡,§
†
,§
†
‡,§
†,§
and Martin Lochner*
†
Department of Chemistry and Biochemistry, University of Bern, 3012 Bern, Switzerland
‡
Institute of Biochemistry and Molecular Medicine, University of Bern, 3012 Bern, Switzerland
§
Swiss National Centre of Competence in Research, NCCR TransCure, 3008 Bern, Switzerland
*
S Supporting Information
ABSTRACT: Glutamate is the major excitatory neuro-
transmitter in the mammalian brain. Its rapid clearance after
the release into the synaptic cleft is vital in order to avoid toxic
effects and is ensured by several transmembrane transport
proteins, so-called excitatory amino acid transporters
(
EAATs). Impairment of glutamate removal has been linked
to several neurodegenerative diseases and EAATs have
therefore received increased attention as therapeutic targets.
O-Benzylated L-threo-β-hydroxyaspartate derivatives have been
developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)-
benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four
stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory
activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters
EAAT1−3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy.
KEYWORDS: Glutamate transporters, asymmetric synthesis, inhibitors, aspartate derivatives, isoform selectivity, photoaffinity probes
he involvement of glutamate in electrophysiological
In fact, several studies have linked the reduced function or
expression of glutamate transporters to neurodegenerative
diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer,
T
processes in the brain was first proposed and later
1
confirmed in the 1950s. It has been established since that the
glutamatergic system regulates most of the excitatory activities in
the central nervous system and it is estimated that 80−90% of the
neurons use glutamate as neurotransmitter. A crucial step is the
rapid termination of glutamate excitation since a high
concentration of glutamate in the synaptic cleft, as upon release
from presynaptic vesicles, is leading to excitotoxicity over time,
2
,3
Parkinson, and Huntington disease. Thus, enhancing gluta-
mate transport is a viable therapeutic strategy for neuro-
protection, and corresponding studies both in vitro and in vivo
in a mouse model of ALS, for instance, have provided some
4
encouraging results. Addressing glutamate transporters with
modulators does not only pose a challenge in terms of isoform
selectivity and dynamic expression levels; it has very recently
been shown for GLT-1 at least, that they are highly mobile at the
surface of astrocytes, which in turn shapes synaptic trans-
2
which in turn can result in neuronal damage.
Rapid removal of extracellular glutamate is achieved by five
selective transmembrane transport proteins belonging to the
solute carrier (SLC) 1 family: EAAT1 (human gene SLC1A3,
termed GLAST in rodents), EAAT2 (SLC1A2, GLT-1), EAAT3
5,6
mission.
Several research groups have investigated the activity of small
molecules, in particular L-glutamate (1) and L-aspartate (2)
derivatives (Chart 1), to inhibit specific glutamate transporter
(
SLC1A1, EAAC1), EAAT4 (SLC1A6), and EAAT5 (SLC1A7).
The first three are generally expressed in the brain and play a
fundamental role in the termination of glutamate-mediated
excitation at synapses. The latter two are very tissue specific and
only found in Purkinje cells in the cerebellum (EAAT4) and in
retina (EAAT5), respectively. EAAT3 is mainly located on the
surface of postsynaptic neurons, whereas EAAT1 and EAAT2 are
expressed in astrocytes. EAAT2 alone is responsible for the
transport of >90% of extracellular glutamate into astrocytes. Not
surprisingly, impairment of the glial glutamate transporters in
particular, is leading to excitotoxic effects and death of neurons.
7
isoforms.
Early on, threo-L-β-hydroxyaspartate ((2S,3S)-3) was discov-
ered to inhibit glutamate transport in synaptosomes and later
8
identified as EAAC1 blocker. Shimamoto and co-workers
Received: November 30, 2015
Accepted: February 26, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/acschemneuro.5b00311
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Letter
Chart 1. EAAT Transporter Substrates L-Glu and L-Asp and Inhibitors 3−5 Derived Thereof
Scheme 1. Retrosynthetic Analysis and Synthetic Strategy to Access All Four Isomers of 5
developed a series of threo-L-β-hydroxyaspartate derivatives that
are the most potent inhibitors of EAAT1−3 to date. From their
first two studies, L-TBOA ((2S,3S)-4, L-threo-β-benzyloxyaspar-
tate) emerged as the most active compound (IC values in the
RESULTS AND DISCUSSION
■
Chemistry. In our retrosynthetic analysis, the benzyl ether in
was disconnected which led to bromide 6 and the key
intermediate β-hydroxyaspartate 3 (Scheme 1). After some
literature research, we envisaged to use asymmetric amino-
hydroxlyation developed by Sharpless and co-workers
install the two stereogenic centers in one reaction with high syn-
selectivity to access the threo (2,3-syn) isomers (TBOA, Scheme
, retrosynthetic path a). For the projected erythro (2,3-anti)
isomers (EBOA, Scheme 1, retrosynthetic path b), we chose to
utilize the Davis α-hydroxylation methodology to diaster-
5
5
0
9
−11
low micromolar range).
L-TBOA was used in numerous
neurobiological studies and, among other things, cocrystallized
with the homologous prokaryotic aspartate transporter GltPh
20−22
to
12
from Pyroccocus hirokoshii (PDB ID: 2NWW). Notably,
extending the structure at the phenyl ring, as exemplified by
the best compound TFB-TBOA ((2S,3S)-5), yielded EAAT2
and EAAT3 inhibitors with IC values in the low nanomolar
1
23
5
0
1
3
range.
Using the information from GltPh crystal structures,
eoselectively install the stereogenic center at C-3. Thus, the
projected synthetic plan would allow us to access all four
stereoisomers of 5 in a very short four-step sequence starting
either from fumarate 7 or protected aspartate 8.
1
2,14
other
groups have used a structure-based screening approach to
identify conformationally constrained analogues of aspartate and
15
glutamate in a virtual compound library. This led to the
discovery of a norbornane-type aspartate derivative which
exhibited an IC50 of 1.4 μM for EAAT2 and showed no
inhibition of EAAT3. Moving away from amino acid scaffolds,
Bunch and co-workers synthesized a library of substituted 5-oxo-
First, dimethyl fumarate (7) was converted to the tert-butyl
diester 9 in order to streamline deprotection conditions at the
end of the synthesis (Scheme 2). Following a recently published
24
modification of the Sharpless aminohydroxylation protocol
yielded protected hydroxyaspartate 10 with complete diaster-
eoselectivity (only syn isomers formed) in 65%. The major
improvement of this protocol is the use of 4-chlorobenzoylox-
ycarbamate (11) instead of the commonly used N-chlorocarba-
5,6,7,8-tetrahydro-4H-chromenes and identified congeners that
were potent inhibitors of EAAT1 (IC50 0.43 μM for best
compound) but displayed no noticeable inhibitory activity at
16−18
25
EAAT2 and EAAT3.
mate that has to be formed in situ from tert-butyl carbamate
The published asymmetric syntheses of L-TBOA ((2S,3S)-4)
and TFB-TBOA ((2S,3S)-5) are rather elaborate (10 and 21
and freshly prepared tert-butyl hypochlorite under aqueous basic
conditions. In the presence of chiral ligand (DHQD) PHAL the
aminohydroxylation led to (2S,3S)-10 as major enantiomer,
2
11,19
synthetic steps, respectively).
Our goal was to establish a
general and concise synthetic procedure to prepare derivatives of
TFB-TBOA that we envision to develop into molecular probes
for studying glutamate transporters in cells. Here, we report the
synthesis of all four stereoisomers of TFB-TBOA (5) in only four
synthetic steps and their inhibitory activity at human isoforms
EAAT1−3. Furthermore, we used our novel synthetic strategy to
generate two potent photoaffinity probes based on the TFB-
TBOA scaffold.
whereas using the pseudoenantiomer (DHQ) PHAL as ligand
2
gave mainly (2R,3R)-10. In both cases, the enantiomeric ratio
(er) was determined to be 7:3 by chiral HPLC. It is important to
run the aminohydroxylation reaction at −15 °C in order to
suppress formation of 1,2-diol side products resulting from
asymmetric dihydroxylation that is also operational under the
reaction conditions. The free hydroxyl group of 10 was O-
26
alkylated with bromide 6 (for synthesis details, see the
B
DOI: 10.1021/acschemneuro.5b00311
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Letter
a
27
Scheme 2. Synthesis of 2,3-syn Isomers (threo) of 5
recently published protocol utilizing diisopropylcarbodiimide
DIPC), t-BuOH, and CuCl as reagents ultimately worked best
(
for us. The α-hydroxylation reaction developed by Davis and co-
workers is a powerful methodology to stereoselectively install
28
hydroxyl groups next to carbonyl functions. In our particular
case, treatment of fully protected aspartate 13 with NaHMDS at
low temperature generated the corresponding ester enolate
which was then almost exclusively attacked by the oxaziridine 14
from the opposite face of the N-Boc group, yielding the 2,3-anti
hydroxylated product with high diastereoselectivity (dr 92:8−
9
3:7). The isolated yield of the α-hydroxylated products was low
and a large amount of unreacted starting material was usually
recovered. The O-alkylation of the 2,3-anti (erythro) alcohols 10
proved much more troublesome as we observed epimerization
and elimination at reaction temperatures above 5 °C. Monitoring
1
epimerization (by H NMR) is advisible since the separation of
diastereomers 2,3-anti-12 and 2,3-syn-12 is extremely difficult.
Interestingly, once purified and isolated, the 2,3-anti isomers of
a
1
2 appear to be configurationally stable even at elevated
Reagents and conditions: (a) t-BuOH, n-BuLi, THF, 0°C; then 7,
°C to rt, 71%. (b) for (2S,3S)-10 (shown): OsO , (DHQD) PHAL,
0
1
7
temperature (60 °C for 24 h in DMSO-d ). Global deprotection
6
4
2
1, H O/MeCN, −15°C; then 9, H O/MeCN, −15° to rt, 65%, er
to yield the final compounds (2S,3R)-5 and (2R,3S)-5 was
achieved by treatment with TFA in chloroform. Notably, the 2,3-
anti isomers did not epimerize under the strong acidic
conditions. For the biological assessment we also synthesized
the racemic mixture of the 2,3-anti isomers of 5 by starting from
racemic N-Boc aspartate and following the synthetic route
depicted in Scheme 3.
The synthetic route depicted in Scheme 2 is amenable to
rapidly access TFB-TBOA analogues. For instance, by alkylating
alcohol (2S,3S)-10 with the appropriate building blocks we
generated diazirine- and benzophenone-TFB-TBOA photo-
affinity probes 15 and 16 (Chart 2), respectively (for details of
2
2
0:30. For (2R,3R)-10 (not shown): OsO , (DHQ) PHAL, 11, H O/
4
2
2
MeCN, −15°C; then 9, H O/MeCN, −15° to rt, 63%, er 69:31. (c)
2
NaH, DMF, −15°C; then 6, −15°C to rt, 99% for (2S,3S)-12, 98% for
(
(
2R,3R)-12. (d) TFA, CHCl , rt, 84% for (2S,3S)-5, quant. for
2R,3R)-5.
3
Supporting Information). Global deprotection using TFA in
chloroform led to enantioenriched isomers (2S,3S)-5 or
(2R,3R)-5. By following the same synthetic route as shown in
Scheme 2 but omitting the chiral phthalazine ligand in the
asymmetric aminohydroxylation step, we also synthesized the
racemic mixture of the 2,3-syn (threo) isomers of 5 for biological
assessment.
Chart 2. Structures of EAAT Photoaffinity Probes 15 and 16
The synthesis of the two 2,3-anti (erythro) enantiomers started
from enantiomerically pure N-Boc protected L- or D-aspartate
that was first transformed into the corresponding di-tert-butyl
ester 13 (Scheme 3) in order to simplify deprotection at the end.
Unexpectedly, this protection step failed or resulted in very
low yields when employing commonly used conditions. A
a
Scheme 3. Synthesis of 2,3-anti Isomers (erythro) of 5
their synthesis, see the Supporting Information). Diazirine and
benzophenone moieties appended to pharmacophores are
commonly used for photo-cross-linking studies with proteins
29,30
and can help to map the binding site of the pharmacophore.
Biological Activity. With all four stereoisomers of 5 in hand,
we next examined their ability to inhibit glutamate uptake into
cells. To this end, HEK293 cell lines stably expressing either
3
human EAAT1, 2, or 3 were generated and transport of [ H]-L-
glutamate into these cells in the presence of 5 was determined
using a similar assay set up as described previously (using 10 μM
31
L-glutamate in the transport assay). For all four stereoisomers
3
of 5, we observed dose-dependent inhibition of [ H]-L-glutamate
transport (Figure 1).
a
Overall, the 2,3-syn (threo, TFB-TBOA) isomers of 5 are more
potent inhibitors of glutamate uptake facilitated by EAAT1, 2, or
Reagents and conditions: (a) CuCl, DIPC, t-BuOH; then 8, CH Cl ,
2
2
reflux, 79% for (S)-13, 97% for (R)-13. (b) NaHMDS, THF, −78°C;
3
than the 2,3-anti (erythro, TFB-EBOA) isomers by 1−2 orders
then 14, THF, −78°C, 22% and dr 93:7 for (2S,3R)-10, 26% and dr
of magnitude (Table 1). This is in line with what was observed
for the stereoisomers of β-benzyloxyaspartate, where for instance
L-TBOA ((2S,3S)-4) was a more active inhibitor than L-EBOA
9
2:8 for (2R,3S)-10. (c) NaH, 6, DMF, −15°C to 0°C, 19% for
(
2S,3R)-12, 21% for (2R,3S)-12. (d) TFA, CHCl , rt, 82% for
3
(
2S,3R)-5, 96% for (2R,3S)-5.
C
DOI: 10.1021/acschemneuro.5b00311
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Letter
3
Figure 1. Dose-dependent inhibition of [ H]-L-glutamate transport mediated by EAAT1 (▲), EAAT2 (■), or EAAT3 (●), by stereoisomers of 5. Top
panels: 2,3-syn isomers (threo, TFB-TBOA). Bottom panels: 2,3-anti isomers (erythro, TFB-EBOA). Dose−inhibition curves shown are representative
experiments. L-Glutamate concentration used in the transport assay was 10 μM. All data are means of three measurements ± SD.
3
Table 1. Inhibiton of Human Glutamate Transporter-Mediated [ H]-L-Glutamate Uptake by TFB-TBOA and TFB-EBOA
a
Stereoisomers
IC50 (nM)
compd
EAAT1
EAAT2
EAAT3
2
,3-syn, threo TFB-TBOA
,3-anti, erythro TFB-EBOA
commercial (S,S)-TFB-TBOA
8.2 ± 3.6
20.7 ± 3.9
19.2 ± 4.2
10.1 ± 1.5
1051 ± 97
335 ± 96
619 ± 73
9.3 ± 1.0
9.9 ± 1.3
12.2 ± 4.1
6.4 ± 1.8
175 ± 65
181 ± 68
179 ± 88
248 ± 76
284 ± 34
412 ± 86
b
synthetic TFB-TBOA, (2S,3S)-5
b
(2R,3R)-5
rac-(2RS,3RS)-5
(2S,3R)-5
228 ± 37
2
13824 ± 5007
2399 ± 1690
5208 ± 2830
(2R,3S)-5
rac-(2RS,3SR)-5
a
L-Glutamate concentration used in the transport assay was 10 μM. All data are means of at least three independent experiments performed in
b
triplicate ± SD. Enantioenriched, er 7:3.
1
1
(
(2S,3R)-4). It is interesting to note, however, that the
inhibitor of EAAT3 as (2R,3R)-5, but has very similar inhibitory
activity at EAAT1 and EAAT2 compared to its enantiomer.
Interestingly, the racemic mixture (50:50) of both 2,3-syn (threo)
enantiomers (rac-(2RS,3RS)-5) gave very similar IC50 values
compared to commercially available (S,S)-TFB-TBOA. This lack
of stereoselectivity in inhibitor binding by EAAT1 and EAAT2
might seem unusual but is not unprecedented for transporters.
Poly specific binding of enantiomeric cyclic peptide inhibitors
was also observed in the case of the ABC transporter P-
glycoprotein (P-gp) due to mainly hydrophobic and aromatic
difference in inhibitory potency of the β-benzyloxyaspartate
stereoisomers 4 is more or less pronounced depending on the
glutamate transporter isoform studied; an observation that we
have also made with our synthesized stereoisomers of 5.
In terms of isoform selectivity, EAAT2 was inhibited most
strongly by all four stereoisomers of 5, followed by EAAT1 and
then EAAT3, for the latter of which we obtained IC values in
5
0
the high nanomolar to low micromolar range.
The IC50 values for our synthetic TFB-TBOA ((2S,3S)-5),
even though only enantioenriched, were almost identical for
EAAT2 and EAAT3 and very similar for EAAT1 compared to the
values we obtained with commercially available (S,S)-TFB-
TBOA. Furthermore, the IC values obtained with our assay are
32
interactions between the inhibitor and the transporter.
The stereoselectivity in terms of inhibitor activity is more
pronounced for the 2,3-anti (erythro, TFB-EBOA) isomers of 5,
in particular in the case of EAAT3; the (2R,3S)-isomer of 5 was
about five times more active than its enantiomer (2S,3R)-5.
Furthermore, IC values for the racemic mixture of both TFB-
5
0
very comparable to literature values for (S,S)-TFB-TBOA, where
1
4
[
C]-L-glutamate uptake into COS-1 cells transiently expressing
50
EAAT1, 2, or 3 gave IC values of 22 ± 0.4 nM, 17 ± 1 nM and
EBOA enantiomers lay between values of the two individual
enantiomers. As for the 2,3-syn (threo) isomers above, the
stereoselection of EAAT1 and EAAT2 for the 2,3-syn (erythro)
isomers was weakly manifested.
5
0
13
3
00 ± 45 nM, respectively. When comparing the enantiomers
2S,3S)-5 and (2R,3R)-5 (both enantioenriched to the same
degree, er 7:3) it appears that (2S,3S)-5 is only twice as potent an
(
D
DOI: 10.1021/acschemneuro.5b00311
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Letter
Both photoaffinity probes 15 and 16 inhibited transport of
syn-β-hydroxyaspartate as single diastereomer. For spectral data of
[3
(
1
H]-L-glutamate via EAAT1−3 in a dose-dependent manner
Figure S1). Inhibition of EAAT1 was significantly stronger by
5 than 16 (IC50 0.11 ± 0.02 μM and 1.23 ± 0.29 μM,
respectively). Similarly, diazirine 15 was the slightly more active
inhibitor than 16 at EAAT2 (IC50 0.40 ± 0.03 μM and 0.82 ±
.08 μM, respectively), however, for EAAT3 this was the
(
2S,3S)-10 and (2R,3R)-10, see the Supporting Information.
General Procedure for the Synthesis of 2,3-anti-β-Hydrox-
yaspartates (2S,3R)-10 and (2R,3S)-10. A solution of N-Boc
protected (S)- or (R)-aspartate diester 13 (0.87 mmol) in dry THF
(
30 mL) was cooled to −78 °C and a solution of NaHMDS in THF (1M,
2.17 mL, 2.17 mmol) was added dropwise. After stirring for 2 h at −78
°C a solution of oxazirine 14 (1.04 mmol; for synthesis details, see the
Supporting Information) in dry THF (6 mL) was added at −78 °C to
the reaction mixture. The progress of the reaction was monitored by
TLC (silica gel, EtOAc/hexane 2:8) and was complete after 30−45 min.
The reaction was quenched at −78 °C by addition of a solution of
camphorsulfonic acid (3.6 mmol) in THF (3.6 mL) and further diluted
with EtOAc (100 mL). The organic phase was washed with aqueous
0
opposite (15: IC 4.03 ± 0.80 μM; 16: IC 2.11 ± 0.58 μM).
50
50
Overall, both photoaffinity probes are more potent inhibitors
than the standardly used DL-TBOA (IC values 48.0 ± 4.1, 7.0 ±
5
0
1
.1 and 8.0 ± 1.9 μM for EAAT1, EAAT2 and EAAT3,
11
respectively).
HCl (10%, 100 mL), sat. aqueous NaHCO solution (100 mL) and
3
CONCLUSIONS
brine (100 mL), dried over Na SO , filtered, and concentrated under
2
4
■
reduced pressure. The diastereomeric purity of the crude products
We have developed a short and concise synthesis for the
preparation of all four stereoisomers of TFB-TBOA, a known
and potent inhibitor of glutamate uptake by excitatory amino
acid transporters EAAT1−3. Our only four-step synthetic route
relies on the Sharpless asymmetric aminohydroxylation to access
the 2,3-syn (threo) isomers, while we have employed the Davis
asymmetric α-hydroxylation to prepare the 2,3-anti (erythro)
isomers of TFB-TBOA. For the first time, all four stereoisomers
have been tested on human glutamate transporters EAAT1−3
and our data show that the correct relative stereochemistry is
crucial for high inhibitory activity, with the 2,3-syn (threo)
isomers being clearly more active than the 2,3-anti (erythro)
isomers. On the other hand, the absolute stereochemistry plays a
minor role and enantioselectivity was effectively only observed
for EAAT3. This observation implies that it is probably not
necessary to use enantiomerically pure (S,S)-TFB-TBOA for
biological studies but the racemic mixture, that is very easily
synthesized following our developed protocol, is equally potent.
Following our synthetic strategy, two TFB-TBOA-derived
photoaffinity probes have been synthesized which exhibited
submicromolar to micromolar inhibitory activity at EAAT1−3
and therefore could be useful molecular tools for photo-cross-
linking studies. Efforts to develop further EAAT molecular
probes with improved activities are underway in our laboratory
and will be reported in due course.
1
(
determined by H NMR) was dr 92:8−94:6. The crude was purified
twice by flash column chromatography (silica gel, first column: CH Cl /
2
2
MeOH 99:1, second column EtOAc/hexane 1:9) to afford anti-β-
hydroxyaspartate as single diastereomer. For spectral data of (2S,3R)-10
and (2R,3S)-10, see the Supporting Information.
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschemneur-
o.5b00311.
Synthesis procedures and spectral data for synthetic
intermediates and final compounds, purity assessment of
final compounds and details of biological procedures and
pharmacological evaluation (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: +41 31 631 3311. Fax +41 31 631 4272. E-mail martin.
lochner@dcb.unibe.ch.
■
Author Contributions
M.L.: developed novel synthetic route, synthesized and
characterized intermediate and target compounds, and con-
tributed to writing of manuscript. A.R.: synthesized and
characterized intermediate and target compounds. A.S.:
generated stable cell lines, performed pharmacological assays,
data analysis, and contributed to writing of manuscript. M.H.:
supervised the pharmacological analysis. M.L.: supervised the
chemistry, designed experiments, and wrote the manuscript.
METHODS
■
General Procedure for the Synthesis of 2,3-syn-β-Hydrox-
yaspartates (2S,3S)-10 and (2R,3R)-10. OsO (5 μmol) was added
as an aqueous stock solution (61 μL of 2% OsO in H O) to a stirred
solution of either (DHQD) PHAL (14.5 μmol, for obtaining the S,S-
4
4
2
2
Funding
isomer) or (DHQ)2PHAL (for obtaining the R,R-isomer) and 4-
chlorobenzoyloxycarbamate 11 (0.438 mmol; for synthesis details, see
the Supporting Information) in MeCN (2.5 mL). After stirring at room
temperature for 12−25 min, the yellow-brown reaction mixture was
cooled to −15 °C and a solution of fumarate 9 (0.438 mmol; for
This study was supported by the Swiss National Science
Foundation (SNSF professorship PP00P2_123536 and
PP00P2_146321 to M.L., and through the National Center of
Competence in Research (NCCR) TransCure) and the
University of Bern.
synthesis details, see the Supporting Information) in MeCN/H O 4:1
2
(
2.5 mL) was added slowly over a period of 15−45 min at −15 °C. Upon
Notes
addition, the color of the reaction mixture changed to dark green. After
completed addition, it was left to warm up to room temperature,
whereupon the color changed again to pale yellow. Reaction control
revealed no more product formation after 4 h. The reaction was
quenched by addition of sat. aqueous K S O soln. (1 mL) which
resulted in the precipitation of a fine brown powder. After addition of
H O (20 mL) and EtOAc (20 mL), the phases were separated and the
aqueous layer was extracted with EtOAc (3 × 20 mL). The combined
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the analytical services from the Department of
Chemistry and Biochemistry, University of Bern, for measuring
■
2
2
5
1
13
2
H NMR, C NMR, and MS spectra.
organic layers were washed with sat. aqueous NaHCO solution (2 × 15
3
ABBREVIATIONS
mL) and brine (15 mL), dried over Na SO , filtered, and concentrated
■
2
4
under reduced pressure to afford the crude product. This was purified by
flash column chromatography (silica gel, EtOAc/hexane 1:9) and gave
ALS, amyotrophic lateral sclerosis; DIPC, N,N′-diisopropylcar-
bodiimide; DMAP, 4-dimethylaminopyridine; EAAT, excitatory
E
DOI: 10.1021/acschemneuro.5b00311
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Letter
amino acid transporter; EBOA, erythro-β-benzyloxyaspartate,
Frydenvang, K., Jensen, A. A., and Bunch, L. (2010) Structure−activity
relationship study of first selective inhibitor of excitatory amino acid
transporter subtype 1:2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-
yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-
(
2,3-anti)-2-amino-3-(benzyloxy)succinic acid; HMDS, hexam-
ethyldisilazane; SLC, solute carrier (group of membrane
transporter proteins); TBOA, threo-β-benzyloxyaspartate, (2,3-
syn)-2-amino-3-(benzyloxy)succinic acid; TFA, trifluoroacetic
acid; TFB-TBOA, (2, 3-syn)-2-amino-3-((3-(4-
1
(
01). J. Med. Chem. 53, 7180−7191.
18) Huynh, T. H. V., Shim, I., Bohr, H., Abrahamsen, B., Nielsen, B.,
Jensen, A. A., and Bunch, L. (2012) Structure−activity relationship
study of selective excitatory amino acid transporter subtype 1 (EAAT1)
inhibitor 2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-
(trifluoromethyl)benzamido)benzyl)oxy)succinic acid
5
,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (UCPH-101) and abso-
REFERENCES
■
lute configurational assignment using infrared and vibrational circular
dichroism spectroscopy in combination with ab initio Hartree−Fock
calculations. J. Med. Chem. 55, 5403−5412.
(
1) Watkins, J. C., and Jane, D. E. (2006) The glutamate story. Br. J.
Pharmacol. 147, S100−S108.
2) Benarroch, E. E. (2010) Glutamate transporters: diversity,
function, and involvement in neurological disease. Neurology 74, 259−
64.
3) Bunch, L., Erichsen, M. N., and Jensen, A. A. (2009) Excitatory
amino acid transporters as potential drug targets. Expert Opin. Ther.
Targets 13, 719−731.
4) Rothstein, J. D., Patel, S., Regan, M. R., Haenggeli, C., Huang, Y. H.,
(
(
19) Shimamoto, K. (2003) β-Benzyloxyaspartate derivatives with
amino group on benzene ring. Patent WO2003/000698 A1.
20) Li, G., Chang, H.-T., and Sharpless, K. B. (1996) Catalytic
2
(
(
asymmetric aminohydroxylation (AA) of olefins. Angew. Chem., Int. Ed.
Engl. 35, 451−454.
(
21) Bodkin, J. A., and McLeod, M. D. (2002) The Sharpless
(
asymmetric aminohydroxylation. J. Chem. Soc., Perkin Trans. 1, 2733−
746.
22) Herranz, E., Biller, S. A., and Sharpless, K. B. (1978) Osmium-
Bergles, D. E., Jin, L., Dykes Hoberg, M., Vidensky, S., Chung, D. S.,
Toan, S. V., Bruijn, L. I., Su, Z.-z., Gupta, P., and Fisher, P. B. (2005) β-
Lactam antibiotics offer neuroprotection by increasing glutamate
transporter expression. Nature 433, 73−77.
2
(
catalyzed vicinal oxyamination of olefins by N-chloro-N-argentocarba-
mates. J. Am. Chem. Soc. 100, 3596−3598.
(
(
5) Murphy-Royal, C., Dupuis, J. P., Varela, J. A., Panatier, A., Pinson,
23) Davis, F. A., Chattopadhyay, S., Towson, J. C., Lal, S., and Reddy,
B., Baufreton, J., Groc, L., and Oliet, S. H. R. (2015) Surface diffusion of
astrocytic glutamate transporters shapes synaptic transmission. Nat.
Neurosci. 18, 219−226.
T. (1988) Chemistry of oxaziridines. 9. Synthesis of 2-sulfonyl- and 2-
sulfamyloxaziridines using potassium peroxymonosulfate (oxone). J.
Org. Chem. 53, 2087−2089.
(
6) Edwards, R. H. (2015) Mobile binding sites regulate glutamate
clearance. Nat. Neurosci. 18, 166−168.
7) Kanai, Y., Clemencon, B., Simonin, A., Leuenberger, M., Lochner,
(
24) Harris, L., Mee, S. P. H., Furneaux, R. H., Gainsford, G. J., and
Luxenburger, A. (2011) Alkyl 4-chlorobenzoyloxycarbamates as highly
effective nitrogen source reagents for the base-free, intermolecular
aminohydroxylation reaction. J. Org. Chem. 76, 358−372.
(
́
̧
M., Weisstanner, M., and Hediger, M. A. (2013) The SLC1 high-affinity
glutamate and neutral amino acid transporter family. Mol. Aspects Med.
(
25) Li, G., Angert, H. H., and Sharpless, K. B. (1996) N-
3
(
4, 108−120.
Halocarbamate salts lead to more efficient catalytic asymmetric
aminohydroxylation. Angew. Chem., Int. Ed. Engl. 35, 2813−2817.
(
8) Kanai, Y., and Hediger, M. A. (1992) Primary structure and
functional characterization of a high-affinity glutamate transporter.
Nature 360, 467−471.
9) Lebrun, B., Sakaitani, M., Shimamoto, K., Yasuda-Kamatani, Y., and
26) Pignataro, L., Bovio, C., Civera, M., Piarulli, U., and Gennari, C.
(2012) A library approach to the development of BenzaPhos: Highly
(
efficient chiral supramolecular ligands for asymmetric hydrogenation.
Chem. - Eur. J. 18, 10368−10381.
(
N- Boc L-glutathione dimethyl and di-tert-butyl esters: Versatile
synthetic building blocks. Bioorg. Med. Chem. 15, 1062−1066.
(
Nakajima, T. (1997) New β-hydroxyaspartate derivatives are com-
petitive blockers for the bovine glutamate/aspartate transporter. J. Biol.
Chem. 272, 20336−20339.
27) Falck, J. R., Sangras, B., and Capdevila, J. H. (2007) Preparation of
t
(
10) Shimamoto, K., Lebrun, B., Yasuda-Kamatani, Y., Sakaitani, M.,
Shigeri, Y., Yumoto, N., and Nakajima, T. (1998) DL-threo-β-
Benzyloxyaspartate, a potent blocker of excitatory amino acid
transporters. Mol. Pharmacol. 53, 195−201.
28) Hanessian, S., and Vanasse, B. (1993) Novel access to (3R)- and
(
3S)-3-hydroxy-L-aspartic acids, (4S)-4-hydroxy-L-glutamic acid, and
related amino acids. Can. J. Chem. 71, 1401−1406.
29) Dubinsky, L., Krom, B. P., and Meijler, M. M. (2012) Diazirine
based photoaffinity labeling. Bioorg. Med. Chem. 20, 554−570.
30) Tanaka, Y., Bond, M. R., and Kohler, J. J. (2008) Photo-
crosslinkers illuminate interactions in living cells. Mol. BioSyst. 4, 473−
80.
́
31) Simonin, A., Montalbetti, N., Gyimesi, G., Pujol-Gimenez, J., and
(
11) Shimamoto, K., Shigeri, Y., Yasuda-Kamatani, Y., Lebrun, B.,
(
Yumoto, N., and Nakajima, T. (2000) Syntheses of optically pure β-
hydroxyaspartate derivatives as glutamate transporter blockers. Bioorg.
Med. Chem. Lett. 10, 2407−2410.
(
(
12) Boudker, O., Ryan, R. M., Yernool, D., Shimamoto, K., and
4
(
Gouaux, E. (2007) Coupling substrate and ion binding to extracellular
gate of a sodium-dependent aspartate transporter. Nature 445, 387−393.
13) Shimamoto, K., Sakai, R., Takaoka, K., Yumoto, N., Nakajima, T.,
Hediger, M. A. (2015) The hydroxyl side chain of a highly conserved
serine residue is required for cation selectivity and substrate transport in
the glial glutamate transporter GLT-1/SLC1A2. J. Biol. Chem. 290,
(
Amara, S. G., and Shigeri, Y. (2004) Characterization of novel L-threo-β-
benzyloxyaspartate derivatives, potent blockers of the glutamate
transporters. Mol. Pharmacol. 65, 1008−1015.
14) Yernool, D., Boudker, O., Jin, Y., and Gouaux, E. (2004) Structure
of a glutamate transporter homologue from Pyrococcus horikoshii. Nature
31, 811−818.
15) Luethi, E., Nguyen, K. T., Bu
Hediger, M., and Reymond, J.-L. (2010) Identification of selective
norbornane-type aspartate analogue inhibitors of the glutamate
transporter 1 (GLT-1) from the chemical universe generated database
3
(
0464−30474.
32) Aller, S. G., Yu, J., Ward, A., Weng, Y., Chittaboina, S., Zhuo, R.,
Harrell, P. M., Trinh, Y. T., Zhang, Q., Urbatsch, I. L., and Chang, G.
2009) Structure of P-glycoprotein reveals a molecular basis for poly-
specific drug binding. Science 323, 1718−1722.
(
(
4
(
̈
rzle, M., Blum, L. C., Suzuki, Y.,
(
GDB). J. Med. Chem. 53, 7236−7250.
(
16) Jensen, A. A., Erichsen, M. N., Nielsen, C. W., Stensbøl, T. B.,
Kehler, J., and Bunch, L. (2009) Discovery of the first selective inhibitor
of excitatory amino acid transporter subtype 1. J. Med. Chem. 52, 912−
9
15.
(
17) Erichsen, M. N., Huynh, T. H. V., Abrahamsen, B., Bastlund, J. F.,
Bundgaard, C., Monrad, O., Bekker-Jensen, A., Nielsen, C. W.,
F
DOI: 10.1021/acschemneuro.5b00311
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX