Conformationally-restricted amino acid analogues bearing a distal sulfonic acid show selective inhibition of system xc- over the vesicular glutamate transporter

Article abstract of DOI:10.1016/j.bmcl.2009.10.020

A panel of amino acid analogs and conformationally-restricted amino acids bearing a sulfonic acid were synthesized and tested for their ability to preferentially inhibit the obligate cysteine-glutamate transporter system x_c^- versus t

Full text of DOI:10.1016/j.bmcl.2009.10.020

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2680–2683
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Conformationally-restricted amino acid analogues bearing a distal sulfonic
acid show selective inhibition of system xꢀc over the vesicular glutamate
transporter
Jean-Louis G. Etoga a, S. Kaleem Ahmed b, Sarjubhai Patel b, Richard J. Bridges b, Charles M. Thompson b,c,
*
a Department of Chemistry and Biochemistry, The University of Montana, Missoula, MT 59812, USA
b COBRE Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
c ATERIS Technologies LLC, 901 N Orange Street, Missoula, MT 59802, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A panel of amino acid analogs and conformationally-restricted amino acids bearing a sulfonic acid were
synthesized and tested for their ability to preferentially inhibit the obligate cysteine–glutamate trans-
porter system xꢀc versus the vesicular glutamate transporter (VGLUT). Several promising candidate
molecules were identified: R/S-4-[40-carboxyphenyl]-phenylglycine, a biphenyl substituted analog of
4-carboxyphenylglycine and 2-thiopheneglycine-5-sulfonic acid both of which reduced glutamate uptake
at system xꢀc by 70–75% while having modest to no effect on glutamate uptake at VGLUT.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 1 September 2009
Revised 5 October 2009
Accepted 5 October 2009
Available online 12 October 2009
Keywords:
Glutamate
Amino acid analog
Inhibitor
VGLUT
System xꢀc
Sulfonic acid
L
-Glutamate (1) is a key neurotransmitter responsible for the
System xꢀc and VGLUT are structurally and functionally distinct
from the EAATs and also from each other. Although system xꢀc and
VGLUT differ pharmacologically,5,8,15,16 both transporters remove
vast majority of the fast excitatory synaptic communication in
the mammalian CNS. L-Glutamate acts at ionotropic glutamate
receptors to mediate ligand gated ion channels and at metabotro-
pic glutamate receptors to couple intracellular second messenger
L
-glutamate from the cytosol. In principle, therefore, intracellular
-glutamate levels could be regulated by modulating one or both
L
systems via G-proteins.1–5 The importance of
L-glutamate as a
of these transporters.16 As such, the development of inhibitors that
selectively block system xꢀc and/or VGLUT represents an interesting
pharmacologic challenge. Some inhibitors9,16,17 have been reported
for system xcꢀ and for VGLUT6,15,18–26 (Fig. 1). Two interesting fea-
tures common to several system xꢀc and VGLUT inhibitors are the
use of aromatic rings to conformationally lock27 the acid groups
(e.g., CPG) and sulfonic acid isosteres24,25,28 in place of a carboxylic
acid. Seeing these as opportunities to explore similarities and dif-
ferences in the specificities of system xꢀc and VGLUT, we prepared
a number of conformationally-restricted glutamate analogs bear-
contributor to higher order processing required in development,
plasticity, learning, and memory is well established.4,6,7 However,
glutamatergic excitotoxicity can result when an excess of
mate occurs and continually activates glutamate receptors.2,5,8 To
maintain the proper titer of -glutamate there is a network of stra-
tegically positioned transporters that shuttle -glutamate in and
L-gluta-
L
L
out of cells and organelles. Most notable among these transporters
are the excitatory amino acid transporters (EAATs) that facilitate
the uptake of L
-glutamate into neurons.8
In addition to EAATs, other transporters maintain intra- and
extra-cellular levels of glutamate including; system xꢀc , a chlo-
ride-dependent, sodium-independent obligate exchanger that cou-
ing a sulfonic acid group in place of the distal (
of glutamate.
c) carboxylic acid
The target compounds were synthesized as shown in Schemes 1
and 2. Simple amino acid analogs (3a–e) of phenylglycine were
synthesized via hydrolysis of the corresponding hydantoin inter-
mediates (2a–e).29–32 The preparation of sulfonic acid analogs
5a–i was carried out by reaction of commercially available amino
acids with fuming sulfuric acid to afford monosulfonic acid
analogs.33 To explore the relative contribution of the amino acid
center to inhibition two additional targets, compounds 7a–b, were
ples the export of intracellular
L-glutamate with the import of
extra-cellular
L
-cystine9–12 and the vesicular glutamate transporter
(VGLUT) that mediates the uptake of intracellular glutamate into
synaptic vesicles.6,13,14
* Corresponding author. Tel.: +1 406 243 4643.
E-mail address: charles.thompson@umontana.edu (C.M. Thompson).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.020

Jean-Louis G. Etoga et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2680–2683
2681
uptake of 3H-
L
-gluamate by either system xꢀc or VGLUT. System
-glutamate (100 M) was measured in
-O2C
CO2
-
xꢀc mediated uptake of
L
l
+
H
NH3
SNB19 glioma cells under Na-free conditions, corrected for non-
specific uptake, and normalized to protein content.9 VGLUT medi-
1: L-Glutamate
ated uptake of L-glutamate (250 lM) was measured in synaptic
HO3S
N
vesicles isolated from rat brain, corrected for non-specific uptake,
and normalized to protein content 20.The rationale for testing com-
pounds 3a–f was based on the fact that a thienylglycine heterocy-
cle contains an embedded cysteine. The imidazole structure was
prepared as a control analog. Structure 3e was built as a chain ex-
tended homologue of 4-CPG, which was found to be a good inhib-
itor of system xꢀc but a poor inhibitor of VGLUT. The activity of 3e
also suggests the likelihood that the compounds are interacting
with lipophilic domains associated with the transporter, as has also
been shown to occur with EAAT inhibitors.12 Interestingly, all the
thiophene-containing structures showed inhibition of system xcꢀ
and VGLUT with the 5-bromo thienylglycine 3d and benzothienyl-
glycine 3f blocking about 60% and 70% of VGLUT uptake, respec-
tively (Table 1). The imidazo analog 3c was completely inactive
indicating the importance of the thiophene ring and/or possibly
contribution by the sulfur atom. Most surprising in this initial
screen was the finding that the biphenyl analog of CPG 3e blocked
73% of glutamate uptake at system xꢀc but was a poor inhibitor of
VGLUT.
NH2
H3C
OH
HO3S
N
N
SO3H
N
HO
CH3
H2N
SO3H
O
Trypan Blue
VGLUT ~ 50 nM
-O2C
-
CO2
NH
-O2C
N
H
+
+ O
NH3
O
H
NH3
Quisqualate
system xc- ~ 5 µM
S-4-Carboxyphenylglycine
system xc- ~ 5 µM
-
-
-O2C
SO3
-O2C
SO3
O
+
+
H
NH3
H
NH3
Sulfonic acid analogs of the amino acids phenylglycine, phenyl-
alanine and thienylglycine 5a–i were prepared to determine the
role of stereochemistry, isostere contribution and limitations of
L-Homocysteate
system xc- ~ 35 µM
L-Serine-o-sulfate
system xc- ~ 26 µM
Figure 1. Structures of glutamate, VGLUT and system xꢀc inhibitors and their
corresponding IC50 values.
the
are inhibitors of system xꢀc and therefore, CPG analogs bearing a
-sulfonic acid would be more potent, and potentially highly
selective inhibitors when compared as inhibitors of VGLUT. Neither
- or -4-sulfophenylalanine 5ab nor -methyl 4-sulfophenylala-
c-carboxylic acid group. We rationalized that CPG and cysteate
c
O
D
L
a
O
NH2
R1
CO2H
HN
R1
i
ii
nine 5d blocked glutamate uptake at either transporter (Table 1).
Sulfonation of 4-bromophenylalanine was conducted to afford
the phenylalanine-2-sulfonic acid analog 5c to reduce the distance
between the two acid groups, and render it a conformationally-re-
stricted analog of homocysteate.
However, compound 5c was a poor inhibitor of both transport-
ers. (R)-4-Sulfo-phenylglycine 5e did not block glutamate uptake
at either transporter, yet 5f was a selective inhibitor of system
xꢀc . We attribute this selectivity to the fact that system xꢀc generally
requires an S-configured amino acid center for inhibitors whereas
VGLUT shows no need for this center and, in fact, does not require
a basic amine.
NH
R1
1a-e
1a: R1 =
H
O
H
2a-e
3a-e
1d: R1 =
1e: R1 =
Br
S
S
S
H3C
O
1b: R1 =
1c: R1 =
HO
H3C
S
N
1f: R1 =
HN
Using (S)-4-sulfophenylglycine as a new lead, we prepared the
thiophene analogs that position the sulfonic acid and amino acid
groups at a distance midway between 4-sulfophenylglycine and
homocysteic acid. Both (R)-5h and (S)-4-sulfothienylglycine 5g
blocked uptake of glutamate at system xꢀc , 45% and 70%, respec-
tively (Table 1). The latter compound proved as potent as the
S
Scheme 1. Synthesis of amino acid analogs 3a–f. Reagents and conditions: (i)
(NH4)2CO3, KCN, 1:1 MeOH, H2O, 50–60 °C, 3 h; (ii) Ba(CO3)2, H2O, 100 °C, 72 h.
Y
X
Y
X
endogenous substrate
potent at VGLUT.
L
-cystine. Unlike system xꢀc both were less
2N NaOH
rt, 3h
OCH3
OH
Z
Z
S
S
The last set of analogs we prepared to test specificity differences
between system xꢀc and VGLUT were aminothiophenecarboxylic
acids 7ab. Since the thiophene scaffold showed promise in system
xꢀc inhibitors, we next queried whether or not replacement of the -
amino acid group with an aniline-type amine and carboxylic acid
would preferentially block VGLUT. In both instances, glutamate up-
take was blocked at system xꢀc and not VGLUT indicating that the
O
O
6a/6b
7a/7b
a: X = NH2, Y = CH3, Z = H;
b: X = CH3, Y = CO2H, Z = NH2
Scheme 2. Preparation of thiophene analogs 7a/7b.
presence of an
xꢀc inhibitor structure. This is also consistent with the activity of
sulfazaline, an inhibitor of system xꢀc , which lacks the free
-amino
acid head group that typifies the majority of known inhibitors. Sul-
fasalazine is of particular interest because it suggests that system
xꢀc may represent a viable point of therapeutic intervention in
the treatment of glial brain tumors.35
a-amino acid group is not a requirement for system
synthesized by hydrolysis of the commercially available structures
6a–b using 2 N NaOH. Each synthesized compound was character-
ized by 1H NMR, IR, and mass spectral analysis34 prior to testing at
the two transporter systems (Table 1). Activity was assessed by
quantifying the ability of the compounds to inhibit the specific
a

2682
Jean-Louis G. Etoga et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2680–2683
Table 1
Percent of L-glutamate uptake in system xcꢀ and VGLUT for compounds 3a–f, 5a–i and 7ab. System xꢀc assay: 100
glutamate and 5 mM of inhibitor
l
M L-glutamate and 500
9
lM of inhibitor. VGLUT assay: 250 lM L-
Entry
Structure or name
System xꢀc (% of uptake)
VGLUT22 (% of uptake)
53
57 10
3a
3b
3c
3d
3e
3f
R/S-5-(2-Thienyl)-2-thienylglycine
R/S-4,5-Dimethyl-2-thienylglycine
R/S-5-Imidazoylglycine
69
73
96
1
3
6
4
95
37
75
31
4
6
7
4
R/S-5-Bromo-2-thienylglycine
R/S-4-[40-Carboxyphenyl]-phenylglycine
R/S-Benzothiophene-3-glycine
83 17
27
53
2
2
SO3H
NH2
5a
5b
90
3
131 31
102 10
HO2C
SO3H
NH2
101
6
HO2C
Br
NH2
5c
83
2
77
9
HO2C
SO3H
SO3H
NH2
H3C
5d
5e
5f
106 13
98 13
HO2C
HO2C
SO3H
H2N
98
55
32
9
4
1
108
9
HO2C
SO3
H
105 10
H2N
CO2H
NH2
HO3S
HO3S
HO3S
5g
92
4
S
S
S
CO2H
NH2
5h
5i
55
26
3
3
104
81
9
CO2
NH2
H
7
7a
7b
3-Amino, 4-methylthiophene-2-carboxylic acid
3-Methyl, 5-aminothiophene-2,4-dicarboxylic acid
83
57
2
4
91
44
6
3
Cntrls
22
3
n/a
31
L
-Cystine
Congo red
n/a
2
Overall, the only sulfonic acid analog of phenylglycine or phen-
ylalanine that showed activity was 5f that selectively blocked 45%
transport at system xꢀc . Substituted thienyl- and benzthienylgly-
cines blocked VGLUT with marginal selectivity over system xꢀc ,
however, the presence of a sulfonic acid group on the thiophene
(5fgh) afforded selective system xꢀc transport inhibitors. We are
currently developing a system xꢀc pharmacophore model using
the thiophene template to produce better inhibitors. One inhibitor
that effectively reduced the uptake of glutamate at both transport-
ers was R/S-benzothiophene-3-glycine. In summary, we have
identified new system xꢀc inhibitors that we envision will become
important pharmacologic tools, but additional work is needed to
identify more effective dual inhibitors.
Acknowledgements
The authors thank J. Gerdes and N. Natale for advice. This re-
search was supported in part by NIH NS38248 (C.M.T.), SBIR Grants
to ATERIS Technologies LLC (R43 ES016392 U44 NS058229),
NS30570 (R.J.B.) and NS42077 (R.J.B.), as well as by the Core Labo-

Jean-Louis G. Etoga et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2680–2683
2683
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mp >300 °C; 1H NMR (400 MHz, D2O): d 7.32 (d, J = 7.35 Hz, 1H), 7.11 (d,
J = 7.35 Hz, 1H), 5.07 (s, 1H); 13C: d 173.5, 148.3, 141.2, 131.8, 131.5, 55.7;
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