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

Article abstract of DOI:10.1021/jm060822s

The four stereoisomers of L-2-(2-carboxycyclobutyl)glycine, L-CBG-I, L-CBG-II, L-CBG-III, and L-CBG-IV, were synthesized in good yield and high enantiomeric excess, from the corresponding cis and trans-2- oxalylcyclobutanecarboxylic acids 5 and 6 using the enzymes aspartate aminotransferase (AAT) and branched chain aminotransferase (BCAT) from Escherichia coli. The four stereoisomeric compounds were evaluated as potential ligands for the human excitatory amino acid transporters, subtypes 1, 2, and 3 (EAAT1, EAAT2, and EAAT3) in the FLIPR membrane potential assay. While the one trans-stereoisomer, L-CBG-I, displayed weak substrate activity at all three transporters, EAAT1-3, we found a particular pharmacological profile for the other trans-stereoisomer, L-CBG-II, which displayed EAAT1 substrate activity and inhibitory activity at EAAT2 and EAAT3. Whereas L-CBG-III was found to be a weak inhibitor at all three EAAT subtypes, the other cis-stereoisomer L-CBG-IV was a moderately potent inhibitor with 20-30-fold preference for EAAT2/3 over EAAT1.

Full text of DOI:10.1021/jm060822s

6532
J. Med. Chem. 2006, 49, 6532-6538
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
Sophie Faure,† Anders A. Jensen,‡ Vincent Maurat,† Xin Gu,† Emmanuelle Sagot,† David J. Aitken,† Jean Bolte,†
Thierry Gefflaut,*,† and Lennart Bunch*,‡
Department of Medicinal Chemistry, The Danish UniVersity of Pharmaceutical Sciences, UniVersitetsparken 2,
DK-2100 Copenhagen, Denmark, and De´partement de Chimie, UniVersite´ Blaise Pascal, 63177 Aubie`re Cedex, France
ReceiVed July 13, 2006
The four stereoisomers of L-2-(2-carboxycyclobutyl)glycine, L-CBG-I, L-CBG-II, L-CBG-III, and L-CBG-
IV, were synthesized in good yield and high enantiomeric excess, from the corresponding cis and trans-
2-oxalylcyclobutanecarboxylic acids 5 and 6 using the enzymes aspartate aminotransferase (AAT) and
branched chain aminotransferase (BCAT) from Escherichia coli. The four stereoisomeric compounds were
evaluated as potential ligands for the human excitatory amino acid transporters, subtypes 1, 2, and 3 (EAAT1,
EAAT2, and EAAT3) in the FLIPR membrane potential assay. While the one trans-stereoisomer, L-CBG-I,
displayed weak substrate activity at all three transporters, EAAT1-3, we found a particular pharmacological
profile for the other trans-stereoisomer, L-CBG-II, which displayed EAAT1 substrate activity and inhibitory
activity at EAAT2 and EAAT3. Whereas L-CBG-III was found to be a weak inhibitor at all three EAAT
subtypes, the other cis-stereoisomer L-CBG-IV was a moderately potent inhibitor with 20-30-fold preference
for EAAT2/3 over EAAT1.
Introduction
done with the cyclobutane analogues L-CBG. Only three isomers
have been prepared to date: L-CBG-I, L-CBG-III,11 and,
recently, L-CBG-II.12 We present here the stereoselective
synthesis of all four stereoisomers of L-2-(2-carboxycyclobutyl)-
glycine (L-CBG-I-IV) (Figure 1) and the pharmacological char-
acterization of these four compounds at human EAAT1-3.
In the mammalian central nervous system (CNS) glutamater-
gic neurotransmission is terminated by reuptake of L-glutamate
(Glu) from the synaptic cleft, by the action 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. EAAT1-3 are localized
on neurons and/or astroglial cells and exhibit high capacity for
transporting Glu across the membrane, whereas EAAT4 and
EAAT5 predominately function as chloride channels.1 In the
healthy CNS, activation of Glu receptors is involved in important
neurophysiological processes, such as memory and learning,
motor functions, and neural plasticity and development.3
However, under conditions of metabolic stress and oxygen
depravation, Glu is a neurotoxic agent. Thus, it is believed that
neurodegenerative diseases such as Alzheimer’s disease, de-
mentia, Huntington’s disease, amyotrophic lateral sclerosis
(ALS), epilepsy, and cerebral stroke may be directly related to
disordered glutamatergic neurotransmission originating from
dysfunction of either the Glu receptors (iGluR or mGluR) or
the Glu reuptake system (EAATs).3,4 We have recently reported
a numbers of studies in which we have explored the structure-
activity relationship (SAR) of ligands at the human EAAT1-
3, with the ultimate goal of identifying new EAAT subtype
selective inhibitors and substrates.5-7 For this purpose, con-
strained cyclic Glu analogues are of particular interest and many
different compounds have been prepared and evaluated for their
activity at the glutamatergic neurotransmitter system. Although
L-2-(2-carboxycyclopropyl)glycine (L-CCG) isomers have been
the subject of several studies,8-10 very limited work has been
Chemistry
Enzymatic transamination is a valuable approach for the
stereoselective preparation of amino acids. Aminotransferases
are very common enzymes with broad substrate spectra, and a
variety of biologically active compounds have been prepared,
including unnatural L- and D-R-amino acids13-20 as well as
â-amino acids or simple amines.21-24 Most naturally occurring
L-R-aminotransferases accept L-Glu as a preferred substrate.
Among them, aspartate aminotransferase (AAT) has proven
useful for the stereoselective preparation of Glu analogues from
the corresponding 2-oxoglutaric acid (KG) derivatives.5,25-28
AAT offers the opportunity to shift the transamination equilib-
rium through the use of aspartic acid or cysteine sulfinic acid29
(CSA) as the amino donor substrate: an unstable keto acid is
produced in both cases, which is decomposed into pyruvate,
which is not a substrate for the enzyme. AAT is active toward
many 4-substituted KG analogues, and its enantiopreference
allows the stereoselective syntheses of a variety of Glu analogues
and especially L-2,4-syn-4-alkylglutamic acids (Scheme 1).5
Recently, we have shown in a preliminary communication
that the scope of AAT transamination can be extended to
constrained Glu analogues with the synthesis of L-CBG-II from
(()-trans-2-oxalylcyclobutanecarboxylic acid 5. Branched chain
aminotransferase (BCAT) from Escherichia coli allowed the
preparation of the L-CBG-I stereomer from the same keto acid.12
In the present study, we present the synthesis of the cis- and
trans-cyclobutane keto acids 5 and 6 and their stereoselective
conversion into the four stereomers L-CBG-I to IV using E.
coli AAT and BCAT (Scheme 2).
* To whom correspondence should be addressed. L.B.: phone, +45
35306244; fax, +45 35306040; e-mail lebu@dfuni.dk. T.G. (for cor-
respondence regarding the synthetic work): phone, 33 473407866; fax, 33
473407717; e-mail: Thierry.GEFFLAUT@univ-bpclermont.fr.
† Universite´ Blaise Pascal.
‡ The Danish University of Pharmaceutical Sciences.
10.1021/jm060822s CCC: $33.50 © 2006 American Chemical Society
Published on Web 10/04/2006

Stereoisomers of L-2-(2-Carboxycyclobutyl)glycine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6533
Scheme 3. Synthesis of cis- and trans-Cyclobutane
R-Ketoglutaric Acids 5 and 6a
Figure 1. L-CBG isomers.
Scheme 1. AAT-Catalyzed Synthesis of L-2,4-syn-4-Alkyl-Glu
from the Corresponding KG Analogues
a Reagents and conditions: (a) acetophenone, CH3CN, rt, hν, Pyrex, 5h,
75%; (b) MeOH, reflux, 16 h, 77%; (c) BnOH, cat. DMAP, rt, 20 h, 63%;
(d) (triphenylphosphoranylidene)acetonitrile, EDCI, DMAP, CH2Cl2, rt, 4
h, 91%; (e) O3, CH2Cl2/MeOH 3:1, -78 °C, 1 h, 81%; (f) O3, CH2Cl2,
-78 °C, 1 h then BnOH, 2 h, 62%; (g) LiOH, MeOH, rt, 2 h then Dowex
resin H+, 100%; (h) H2, Pd/C, AcOEt, rt, 3 h then LiOH, H2O, 100%.
Scheme 2. Synthesis of L-CBG Isomers by Enzymatic
Transamination
yield.32 The R-keto cyanophosphorane 3b, obtained by coupling,
was submitted to ozonolysis followed by addition of benzyl
alcohol to provide (()-cis-2-oxalylcyclobutanecarboxylic diben-
zyl ester 4b in 62% yield. Quantitative hydrogenolysis of the
benzyl group in the presence of Pd/C followed by generation
of the dilithium salt by careful addition of lithium hydroxide
gave an aqueous solution of the cis isomer of cyclobutane
R-ketoglutaric acid 6. To minimize the isomerization process
this solution was used directly in the transamination reaction.
1H NMR analysis after concentration first revealed a 80:20 cis/
trans mixture rapidly evolving in a few hours to the major trans
isomer. The transamination substrates 5 and 6 were thus obtained
in five steps with respective overall yields of 42% and 27%
from maleic anhydride.
Synthesis of Glutamic Acid Analogues. Since the cyclobu-
tane KG analogues 5 and 6 were obtained as an enriched mixture
and are prone to isomerization under neutral conditions, we
could not determine reliable kinetic parameters for the ami-
notransferase-catalyzed reactions. However, analytical measure-
ments indicated that both racemates are substrates for E. coli
AAT and BCAT with kcat of around 1% of that of the natural
substrate KG. Because of the high specific activity of these
aminotransferases, this modest activity proved to be sufficient
for synthetic purposes.
AAT-catalyzed reactions were performed according to the
method depicted in Scheme 1 and previously described:5,12 an
aqueous solution of (()-5 or (()-6 (approximately 2 mmol)
was reacted with a stoechiometric amount of CSA in the
presence of AAT at pH 7.6. The reaction was monitored by
enzymatic titration of pyruvic acid formed from CSA and was
stopped when a conversion rate of approximately 40% was
reached, to observe a possible kinetic resolution. Glu analogues
were selectively adsorbed on a short column of sulfonic resin
(H+ form) and recovered by elution with 1 M aqueous
ammonium hydroxide. They were finally purified by adsorption
on a strongly basic Dowex 2 resin (AcO- form) followed by
Results and Discussion
Synthesis of 2-Oxalylcyclobutanecarboxylic Acids 5 and
6. Both cis- and trans-isomers of cyclobutane R-ketoglutaric
acids 5 and 6 were synthesized from cis-1,2-cyclobutane
dicarboxylic anhydride 1 as described in Scheme 3. A modifica-
tion of the [2 + 2] photocycloaddition reaction described by
Bloomfield and Owsley30 afforded cis-1,2-cyclobutanedicar-
boxylic anhydride 1 in 75% yield. Methanolysis of the anhydride
moiety gave (()-cis-2-methoxycarbonylcyclobutane carboxylic
acid 2a in good yield. Homologation of acid 2a into R-keto
ester was performed using cyanoketophosphorane methodology
developed by Wasserman’s group:31 First, the carboxylic acid
2a was coupled with (cyanomethylene)triphenylphosphorane to
give the R-keto cyanophosphorane 3a in 91% yield. Ozonolysis
of compound 3a then generated a diketo nitrile, which was
trapped in situ by methanol to provide (()-cis-2-oxalylcyclo-
butane carboxylic dimethyl ester 4a in 81% yield. Final ester
hydrolysis was carried out in the presence of a stoichiometric
amount of lithium hydroxide in MeOH. Under these reaction
conditions, regiospecific epimerization R to the ketone func-
tionality was observed to furnish thermodynamically more stable
trans-cyclobutane. The dilithium salt of 5 was isolated as a
90:10 trans/cis mixture determined by 1H NMR. To obtain the
cis isomer of cyclobutane R-ketoglutaric acid (()-6, we decided
to modify the above sequence to accommodate a benzyl ester
moiety that should be cleaved under neutral conditions, thus
avoiding epimerization. Opening of the anhydride moiety of 1
with benzyl alcohol catalyzed by DMAP gave (()-cis-2-
benzyloxycarbonylcyclobutane carboxylic acid 2b in 63%

6534 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Scheme 4. BCAT-Catalyzed Synthesis of L-CBG
Faure et al.
Table 1. L-CBG Isomers Prepared by Transamination
product
substrate
with AAT
with BCAT
(()-5
(()-6
L-CBG-II
L-CBG-III
L-CBG-IV
L-CBG-I
-
Table 2. Pharmacological Characterization of L-CBG-I, L-CBG-II,
L-CBG-III, and L-CBG-IV at Human Excitatory Amino Acid
Transporters EAAT1, EAAT2, and EAAT3 in the FMP Assaya
EAAT1
EAAT2
EAAT3
Glu
7.9 (5.1 ( 0.03)
300-1000b
96 (4.0 ( 0.03)c
∼200
21 (4.7 ( 0.02)
300-1000b
22 (4.7 ( 0.01)
110 (3.9 ( 0.02)
6.6 (5.2 ( 0.04)
9.9 (5.0 ( 0.02)
300-1000b
49 (4.3 ( 0.03)
52 (4.3 ( 0.02)
10 (5.0 ( 0.03)
L-CBG-I
L-CBG-II
L-CBG-III
L-CBG-IV
elution with an AcOH gradient. Three L-CBG isomers were thus
isolated with a very good purity. L-CBG-II was isolated in 32%
yield from (()-5. This result indicates that AAT is enantiose-
lective toward 5, allowing the kinetic resolution of this racemic
substrate. Reaction of (()-6 produced L-CBG-III and -IV in
19% and 17% respective yields. The two cis isomers were
consecutively eluted from the basic resin and were thus easily
separated. A small amount of L-CBG-II (10% yield) was also
isolated in that reaction and easily separated from the cis
isomers. L-CBG-II was likely formed from the isomerized trans-
keto acid present in the reaction mixture. In the case of the cis
derivative 6, the enzyme enantioselectivity is very low, affording
access to both stereomers L-CBG-III and IV. These products
should be isolated in better yields after a complete conversion
of (()-6 substrate.
∼200
a The Km values for substrate (in bold) and the Ki values for inhib-
itors are given in µM (with pKm ( SEM and pKi ( SEM values in
parentheses, respectively). b The concentration-response curves for the
substrate L-CBG-I at the three transporters did not reach saturation in the
concentration ranges used. The highest concentration of L-CBG-I used
(1000 µM) elicited substrate responses of 72 ( 6%, 21 ( 3% and 48 (
4% of the Rmax values of Glu at EAAT1, EAAT2 and EAAT3, respectively.
c
L-CBG-II was a “full substrate” at EAAT1, eliciting a maximal response
of 94 ( 7% of that of Glu.
Pharmacology and Modeling. The four stereoisomeric Glu
analogues L-CBG-I-IV were characterized pharmacologically
at EAAT1-3, using a FLIPR membrane potential (FMP) assay
(see Experimental Section for details). The assay was performed
essentially as described previously,33 and results are summarized
in Table 2. The trans-stereoisomer L-CBG-I was found to be a
weak substrate at all three EAAT subtypes. In contrast, the
stereoisomeric trans substituted analogue, L-CBG-II, was found
to be a substrate at EAAT1 (Km ) 96 µM) and an inhibitor at
subtypes EAAT2 and EAAT3 (Ki ) 22 and 49 µM, respec-
tively). A similar pharmacological profile (EAAT1 substrate vs
EAAT2 and EAAT3 inhibitor) has previously been observed
by us and others for (R)-4-methyl Glu.5,34 The cis substituted
analogue L-CBG-III was a weak inhibitor at all three subtypes
EAAT1, -2, and -3 (Ki ) ∼200, 110, and 52 µM, respectively),
whereas the other cis stereoisomer, L-CBG-IV, was found to a
weak inhibitor at EAAT1 (Ki ∼ 200) but a moderately potent
inhibitor at EAAT2 and EAAT3 (Ki ) 6.6 and 10 µM,
respectively).
To explain and eventually expand the SAR for substrates and
inhibitors at EAAT1-3, we carried out a modeling study of
L-CBG-I-IV. We have previously argued for a differentiation
of substrate and inhibitor activity at EAAT1-3 on the basis of
conformations of a set of diverse ligands. An extended Glu
conformation appears to be essential to allow substrate transport,
whereas inhibitors bind to the transporters in a folded Glu
conformation.5 Thus, we submitted the four stereoisomeric
analogues, L-CBG-I-IV, to a stochastic conformational search
to identify relevant low-energy folded and extended Glu
conformations, and results are summarized in Table 3. The two
trans stereoisomers, L-CBG-I and L-CBG-II, can both attain low-
energy extended and folded Glu conformations. Furthermore,
L-CBG-II is found in a -3 kcal/mol lower energy pseudoex-
tended Glu conformation compared with the extended one. The
two cis stereoisomers, L-CBG-III and L-CBG-IV, are both found
to attain a folded Glu conformation, exclusively.
The transaminations with BCAT were performed on a smaller
scale (0.3 mmol) according to Scheme 4 as previously de-
scribed.12
As CSA is not a substrate for BCAT, Glu was used as
catalytic amino donor and was regenerated by reductive
amination of KG using glutamic dehydrogenase (GluDH),
+
NADH, and NH4 ions. NADH itself was used catalytically
and regenerated using the equilibrium-shifted oxidation of
formic acid catalyzed by formate dehydrogenase (FDH). The
reaction was followed by TLC and stopped after 2-5 days. Glu
analogues were isolated as previously described for AAT-
catalyzed reactions. Starting from 5 or 6, the same L-CBG-I
isomer was isolated in 26% and 16% respective yields. This
result indicates that BCAT exhibits an enantioselectivity toward
5 that is complementary to that of AAT. Unfortunately, in the
conditions used, isomerization of 6 into 5 was faster than its
transamination and did not allow an access to cis-L-CBG
derivatives.
Configurations were attributed by comparison to the physi-
cochemical properties and spectroscopic data of the already
described L-CBG-I and L-CBG-III isomers.11 As these known
derivatives are respectively a trans and a cis isomer, it was easy
to attribute configurations to our two new analogues, corre-
sponding to trans-L-CBG-II and cis-L-CBG-IV. The L-CBG-II
stereochemistry was further confirmed, as previously de-
scribed,12 by the synthesis of (1R,2R)-5 and confirmation of
the AAT enantiopreference for this enantiomer. Because E. coli
aminotransferases are highly specific for L-amino acids, all CBG
analogues prepared in the present study were isolated with very
high enantiopurity. Table 1 summarizes the results obtained for
AAT- and BCAT-catalyzed transamination of 5 and 6. Our
chemoenzymatic approach appeared shorter than the previous
described syntheses11 of L-CBG-I and -III isomers. Moreover,
it allowed the preparation of the two new stereomers L-CBG-II
and -IV.
By superimposition of the ammonium group and the two
carboxylate groups in the two trans stereoisomers, L-CBG-I and
L-CBG-II, it becomes evident that the two cyclobutane rings
occupy very different areas in space (Figure 2). We propose

Stereoisomers of L-2-(2-Carboxycyclobutyl)glycine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6535
Table 3. Calculated Low-Energy Conformationsa
folded
extended
L-CBG-I
L-CBG-II
-117.255
-118.028
-115.902
-114.908
(-117.957b)
cnfc
L-CBG-III
L-CBG-IV
-112.491
-115.599
cnf
a All calculated ∆G values in kcal/mol, using the mmff94x force field
and gb/sa solvation model. For further details, see the Experimental Section.
b Energy of a pseudoextended conformation that fits the substrate EAAT
pharmacophore poorly. c Conformation not found.
Figure 4. EAAT1-3 inhibitory pharmacophore model. Superimposi-
tion by fitting the ammonium group and the two carboxylate groups
of calculated low-energy folded Glu conformations of L-CBG-II (type
code), dihydrokainate (DHK) (orange), and (1R,4S,5R,6S)-3-azabicyclo-
[3.3.0]octane-4,6-dicarboxylic acid (ABOD) (green).
Figure 2. Superimposition by fitting the ammonium group and the
two carboxylate groups of calculated low-energy extended Glu
conformations of L-CBG-I (green) and L-CBG-II (type code).
Figure 5. EAAT1-3 inhibitory pharmacophore model. Superimposi-
tion by fitting the ammonium group and the two carboxylate groups
of calculated low-energy folded Glu conformations of L-CBG-II (type
code), L-CBG-III (yellow), and L-CBG-IV (green).
extended Glu conformations does (4R)-4-methyl-Glu adapt,5
when transported by EAAT1? First, a molecular modeling study
like the one presented here provides static representations of
the dynamic transport process of a ligand across a membrane.
Second, ligand-protein interactions are acutely sensitive to
disfavored van der Waals forces. Hence, the minor differences
in spatial orientation that we predict between the cyclobutane
ring of L-CBG-II and the methyl group of (4S)-4-methyl-Glu
may be of detrimental nature to the transport of the ligand (4S)-
4-methyl-Glu.
Since L-CBG-II is an inhibitor at EAAT2/3, the two subtypes
are clearly not capable of facilitating transport, in contrast to
the substrate properties observed for this ligand at EAAT1. To
address this observation, we bring to the readers attention that
L-CBG-II, in addition to an extended Glu conformation, can
also adapt a folded Glu conformation (Table 3). Comparing the
folded L-CBG-II conformation with our previously published
inhibitory pharmacophore models of EAAT2 and EAAT3, a
clear resemblance between folded L-CBG-II and conformation-
ally restricted EAAT inhibitors (1R,4S,5R,6S)-3-azabicyclo-
[3.3.0]octane-4,6-dicarboxylic acid (ABOD)7 and dihydrokainate
(DHK) can be observed (Figure 4).
Superimposition of the two cis-stereoisomers, L-CBG-III and
L-CBG-IV, in their folded Glu conformation (Figure 5), together
with folded L-CBG-II, provides a clear three-point overlap and
unambiguously explains the pharmacological profile of L-CBG-
III and L-CBG-IV as EAAT1-3 inhibitors. Although DHK has
been reported to be selective EAAT2 inhibitors, most published
EAAT inhibitors to date show no or little subtype selectivity.33
In this context, the pharmacological profile of L-CBG-IV, with
its 20-30-fold preference for EAAT2/3 over EAAT1, is
remarkable.
Figure 3. EAAT1 substrate pharmacophore model. Superimposition
by fitting the ammonium group and the two carboxylate groups of
calculated low-energy extended Glu conformations of L-CBG-II (type
code) and L-2,4-methanopyrrolidine-2,4-dicarboxylate (L-2,4-MPDC)
(green), (4R)-4-Me-Glu (two conformations) (purple), and (4S)-4-Me-
Glu (yellow).
this to be the origin of the different potencies observed for these
two substrates at EAAT1 (Table 2).
The distinct pharmacological profile of L-CBG-II, being an
EAAT1 substrate and an EAAT2/3 inhibitor, has also been
observed for (4R)-4-methyl-Glu.34 In contrast, (4S)-4-methyl-
Glu is neither a substrate nor an inhibitor at EAAT1-3.34 In
our previous study, we concluded that when (4S)-4-methyl Glu
adapts its only extended Glu conformation found, there is
inadequate space in the EAAT1-3 proteins, because the
4-methyl group is pointing down. As a consequence of this we
put forward the hypothesis that (4R)-4-methyl-Glu binds in its
extended Glu conformation with the 4-methyl group pointing
up, and not down, and thus becomes a substrate at EAAT1.5
On superimposition and comparison of the extended Glu
conformation of L-CBG-II with the inactive ligand (4S)-4-
methyl-Glu, it becomes evident that part of the cyclobutane ring
in L-CBG-II occupies approximately the same region in space
as the 4-methyl substituent of (4S)-4-methyl-Glu (Figure 3).
Furthermore, L-CBG-II does not reach into the region in space
defined by the 4-methyl group of (4R)-4-methyl-Glu. Conse-
quently, we have to revisit the premises of our previous
hypothesis to address the following: What is the molecular basis
for (4S)-4-methyl-Glu being inactive at EAAT134 while L-CBG-
II is a substrate? And which of the two previously proposed
Conclusion
Our chemoenzymatic approach allowed a convenient access
to the four stereomers of L-CBG, including the two new
derivatives L-CBG-II and IV. These Glu analogues were isolated

6536 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Faure et al.
2H), 2.38-2.50 (m, 2H), 3.43-3.49 (m, 2H), 5.06 (d, J ) 12,2
Hz, 1H), 5.15 (d, J ) 12.2 Hz, 1H), 7.26-7.36 (m, 5H), 10.10 (bs,
1H); 13C NMR (100 MHz, CDCl3) δ 22.1, 22.2, 40.4, 40.5, 66.6,
128.2, 128.3, 128.5, 135.7, 173.0, 179.4.
in good yields and very high purity. The four compounds were
characterized at human EAAT1, EAAT2, and EAAT3, in the
FMP assay. Notably, a distinct pharmacological profile was
observed for the one trans-stereoisomer, L-CBG-II, which
displayed EAAT1 substrate activity and EAAT2/3 inhibitory
activity. The SAR study of this ligand adds new insight to the
EAAT1 substrate pharmacophore. The cis-stereoisomer L-CBG-
IV was found to be a moderately potent inhibitor with 20-30-
fold preference for EAAT2/3 over EAAT1.
General Procedure for Cyanophosphorane Formation. To a
solution of acid 2 (5 mmol) in CH2Cl2 (40 mL) under argon at 0
°C were added DMAP (0.5 mmol) and EDCI (5.5 mmol) followed
by (triphenylphosphoranylidene)acetonitrile (7.5 mmol). The reac-
tion mixture was stirred for 20 min at 0 °C and then 5 h at room
temperature. It was added to CH2Cl2/H2O (1:1; 30 mL) and the
aqueous phase was extracted again with CH2Cl2 (20 mL). Combined
organic extracts were washed with brine, dried over MgSO4, and
concentrated under reduce pressure to afford 3, which was purified
by flash chromatography on silica gel (eluent cyclohexanes-ethyl
acetate 1:1).
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
(()-cis-2-(Cyanotriphenylphosphorane)ketomethyl-
cyclobutanecarboxylic Acid Methyl Ester 3a. Compound 3a was
isolated as a white foam from (()-cis-cyclobutane 1,2-dicarboxylic
acid monomethyl ester 2a (0.780 g, 4.93 mmol): yield 1.97 g, 91%;
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 for the sodium D line (589 nm). Elemental
analyses were performed at the Service Central d’Analyse du
CNRS, Solaize, France. Silica gel 60 (Merck, 40-63 µm) and
precoated F254 plates were used for flash column and TLC
chromatographies. All solvents were purified by distillation fol-
lowing usual procedures. Cysteine sulfinic acid was prepared from
cystine following a described procedure.35 Rabbit muscle lactic
dehydrogenase, glutamic dehydrogenase from bovine liver, and
formate dehydrogenase from Candida boidinii were purchased from
Sigma. E. coli AAT and BCAT were produced and purified
following described procedures from overexpressing E. coli strains
TY103 transformed with pUC19-aspC (AAT) and pUC19-ilVE
(BCAT).36-38 Activities expressed as IU refers to Vmax measured
with natural substrates (Asp and KG for AAT, Glu and 4-methyl-
2-oxopentanoic acid for BCAT) in 0.1 M phosphate buffer, pH
7.6, as previously described.5 Both E. coli aminotransferases are
commercially available from Biocatalytics.
IR (KBr pellet) 2180, 1725, 1580 cm-1 1H NMR (400 MHz,
;
CDCl3) δ 2.05-2.20 (m, 1H), 2.24-2.43 (m, 3H), 3.35-3.45 (m,
1H), 3.48 (s, 3H), 4.10-4.17 (m, 1H), 7.48-7.64 (m, 15H); 13C
NMR (100 MHz, CDCl3) δ 21.8, 23.3, 40.4, 45.0, 47.5 (d, J )
125 Hz), 51.3, 122.2 (d, J ) 17 Hz), 123.3 (d, J ) 93 Hz), 129.1,
129.2, 133.0, 133.5, 133.6, 174.6, 195.9 (d, J ) 3 Hz).
(()-cis-2-(Cyanotriphenylphosphorane)ketomethyl-
cyclobutanecarboxylic Acid Benzyl Ester 3b. Compound 3b was
isolated as a white foam from (()-cis-cyclobutane 1,2-dicarboxylic
acid monobenzyl ester 2b (0.896 g, 3.82 mmol): yield 1.802 g,
91%; mp 131-133 °C; IR (KBr pellet) 2175, 1739, 1581 cm-1
;
1H NMR (400 MHz, CDCl3) δ 2.14-2.44 (m, 4H), 3.45-3.50 (m,
1H), 4.11-4.18 (m, 1H), 5.85 (d, J ) 13 Hz, 1H), 5.10 (d, J ) 13
Hz, 1H), 7.24-7.32 (m, 5H), 7.44-7.65 (m, 15H); 13C NMR (100
MHz, CDCl3) δ 21.8, 23.3, 40.8, 45.4, 47.8 (d, J ) 125 Hz), 65.6,
122.2 (d, J ) 16 Hz), 123.3 (d, J ) 92 Hz), 127.6, 128.3, 129.0,
129.1, 133.0, 133.6, 133.7, 174.1, 195.9.
(()-cis-2-Oxalylcyclobutanecarboxylic Acid Dimethyl Ester
4a. The R-keto cyanophosphorane 3a (1.932 g, 4.38 mmol) in a
7:3 mixture of CH2Cl2/MeOH (50 mL) at -78 °C, was treated with
ozone at a rate of 10L/h during 25 min. The excess of ozone was
eliminated by oxygen bubbling and then dimethylsulfur was added
and the reaction mixture was allowed to warm to room temperature
during 2 h. After evaporation of the solvent, the residue was
dissolved in CH2Cl2 (20 mL) and was washed three times with
water. The organic layer was dried over MgSO4 and concentrated
under reduced pressure. A short flash chromatography (eluent,
cyclohexanes-EtOAc acetate 7:3) gave 4a isolated as a colorless
oil (0.707 g, 81%): IR (neat film) 1733, 1277, 1207 cm-1; 1H NMR
(400 MHz, CDCl3) δ 2.00-2.15 (m, 2H), 2.22-2.35 (m, 1H),
2.41-2.51 (m, 1H), 3.58 (s, 3H), 3.61-3.70 (m, 1H), 3.80 (s, 3H),
3.85-3.92 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 20.4, 22.7,
43.2, 43.3, 52.1, 52.8, 160.7, 173.9, 192.7.
cis-Cyclobutanecarboxylic Anhydride 1. Maleic anhydride
(0.981 g, 10 mmol) was introduced into a cylindrical reactor
containing acetonitrile (500 mL) with acetophenone (117 µL, 1
mmol). The mixture was stirred at room temperature, degassed with
argon for 30 min, and then saturated with ethylene for 30 min.
While ethylene bubbling continued, the mixture was irradiated
with a 400 W medium-pressure mercury lamp fitted with a Pyrex
filter for 5 h. The solvent was evaporated and the solid resi-
due was recrystallized from a cyclohexane/ether solution. The
cyclobutane adduct 1 was obtained as a white solid (0.763 g,
75%): mp 74-75 °C; IR (KBr pellet) 1854, 1782 cm-1; 1H NMR
(400 MHz, acetone-d6) δ 2.25-2.45 (m, 2H), 2.70-2.85 (m, 2H),
3.65-3.72 (m, 2H); 13C NMR (100 MHz, acetone-d6) δ 23.2, 40.0,
206.3.
(()-cis-2-Methoxycarbonylcyclobutanecarboxylic Acid 2a. A
solution of cis-cyclobutane carboxylic anhydride 1 (0.240 g, 2.35
mmol) in methanol (5 mL) was stirred under reflux for 18 h. The
solvent was evaporated under reduce pressure. Flash chromatog-
raphy on silica gel (eluent cyclohexanes-ethyl acetate 1:1) afforded
2a as a colorless liquid (0.287 g, 77%): IR (neat film) 3200-
2860, 1735, 1705 cm-1; 1H NMR (400 MHz, CDCl3) δ 2.18-2.30
(m, 2H), 2.36-2.46 (m, 2H), 3.41-3.48 (m, 2H), 3.69 (s, 3H),
10.9 (bs, 1H); 13C NMR (100 MHz, CDCl3) δ 21.9, 22.1, 40.4,
40.5, 51.8, 173.7, 179.3.
(()-cis-2-Oxalylcyclobutanecarboxylic Acid Dibenzyl Ester
4b. A solution of 3b (1.801 g, 3.48 mmol) in CH2Cl2 (40 mL) at
-78 °C was treated with ozone at a rate of 10L/h during 1 h. The
excess of ozone was eliminated by argon bubbling and benzyl
alcohol (720 µL, 6.96 mmol) was added under argon. The reaction
mixture was stirred at -78 °C for 2 h and then triphenylphosphine
(1.826 g, 6.96 mmol) was added and the solution was allowed to
warm to room temperature over a period of 2 h. After evaporation
of the solvent, the residue was dissolved in CH2Cl2 (20 mL). The
solution was washed three times with water (3 × 20 mL), dried
over MgSO4, and concentrated under reduced pressure. Purification
by flash chromatography on silica gel (eluent, cyclohexanes-
EtOAC 8:2) afforded 4a as a yellow semisolid (0.761 g, 62%): IR
(()-cis-2-Benzyloxycarbonylcyclobutanecarboxylic Acid 2b.
A mixture of anhydride 1 (0.600 g, 5.88 mmol), benzyl alcohol
(1.2 mL, 11.75 mmol), and 4-(dimethylamino)pyridine (0.043 g,
0.35 mmol) was stirred under argon at room temperature during
20 h. A saturated aqueous solution of NaHCO3 (10 mL) was added.
The aqueous layer was washed three times with ether and then
acidified to pH 3 with a 1 M HCl solution. After extraction with
EtOAc, organic layers were washed with brine, dried over MgSO4,
and concentrated under reduce pressure to afford 2b as a white
solid (0.872 g, 63%): mp 67-69 °C; IR (KBr pellet) 3500-2850,
1
(film) 1732, 1270 cm-1; H NMR (400 MHz, CDCl3) δ 2.14-
2.24 (m, 2H), 2.32-2.37 (m, 1H), 2.53-2.58 (m, 1H), 3.69-3.71
(m, 1H), 3.94-3.96 (m, 1H), 4.98 (d, J ) 12.4 Hz, 1H), 5.06 (d,
J ) 12,4 Hz, 1H), 5.18 (s, 2H), 7.28-7.38 (m, 10H); 13C NMR
(100 MHz, CDCl3) δ 20.6, 22.7, 43.5, 67.2, 67.7, 128.5, 128.2,
128.3, 128.4, 128.6, 128.7, 134.8, 135.5, 159.8, 173.4, 192.6.
1
1743, 1698 cm-1; H NMR (400 MHz, CDCl3) δ 2.17-2.32 (m,

Stereoisomers of L-2-(2-Carboxycyclobutyl)glycine
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6537
(()-trans-2-Oxalylcyclobutanecarboxylic Acid Dilithium Salt
5. To a solution of R-keto ester 4a (0.204 g, 1.02 mmol) in methanol
(5 mL) was added dropwise an aqueous solution of LiOH (0.4 M,
5.4 mL). The reaction mixture was stirred for 2 h and then MeOH
was evaporated under reduced pressure. The pH was adjusted to
7.6 by addition of H+ resin (Dowex 50Wx8-100). Filtration and
evaporation of water under reduced pressure furnished quantitatively
compound 5 (187 mg) as a white solid and as a 10:90 cis/trans
mixture: 1H NMR (400 MHz, D2O) δ 1.91-2.31 (m, 4H), 3.24
(q, J ) 8.8 Hz, 1H), 3.80 (q, J ) 8.8 Hz, 1H); 13C NMR (100
MHz, D2O) δ 21.4, 22.3, 41.3, 45.3, 170.4, 182.8, 206.1.
BCAT-Catalyzed Transaminations. To an aqueous solution of
racemic 5 or 6 (0.3 mmol) were added glutamic acid (9 mg, 0.06
mmol), NADH (10 mg, 0.014 mmol), and HCO2NH4 (38 mg, 0.6
mmol). The pH of the solution was adjusted to 7.6 with 1 M NaOH
and the volume adjusted to 15 mL with water before addition of
glutamate dehydrogenase (1 mg, 8 IU), formate dehydrogenase (15
mg, 15 IU), and BCAT (2 mg, 4 IU). The reaction was stirred
slowly at room temperature for 2-7 d and the reaction mixture
was then passed through a column of Dowex 50 × 8 resin (H+
form, 25 mL). L-CBG-I was isolated in both cases as previously
described for AAT transaminations.
(()-cis-2-Oxalylcyclobutanecarboxylic Acid Dilithium Salt 6.
A mixture of R-keto ester 4b (0.530 g, 1.5 mmol) with palladium
on activated carbon (10% Pd, 50 mg) in EtOAc (50 mL) was stirred
under pressure of hydrogen (40 psi) during 3 h. After filtration
and evaporation of the solvent, the residue was dissolved in water
and the pH adjusted to 7.6 by careful addition of lithium hydroxide
(0.4 M). This solution was directly used for the transamination
reaction. A sample was concentrated under reduced pressure and
rapidly analyzed. A 80:20 cis/trans mixture was thus characterized
by NMR analysis: 1H NMR (400 MHz, D2O) δ 1.92-2.37 (m,
4H), 3.43 (td, J ) 8.8 and 6.4 Hz, 1H), 3.74-3.89 (m, 1H); 13C
NMR (100 MHz, D2O) δ 20.9, 22.8, 44.8, 45.2, 168.2, 182.6, 205.6.
AAT-Catalyzed Transaminations. To an aqueous solution of
racemic 5 or 6 (1.9-2.8 mmol) were added stoechiometric amounts
of cysteine sulfinic acid and acetaldehyde. The pH of the solution
was adjusted to 7.6 with 1 M NaOH and the volume adjusted to
100 mL with water before addition of AAT (250 IU, approximately
5 mg). 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 rabbit muscle lactate
dehydrogenase (1 unit). Pyruvate concentration was calculated from
(2S,1′R,2′R)-2-(2′-Carboxycyclobutyl)glycine L-CBG-I: yield
14 mg, 26% from 5; mp >245 °C (dec) (lit.11 mp 258 °C (dec));
1
[R]20 +96.4° (c 0.7, H2O) (lit.11 [R]20 +97.0° (c 0.5, H2O)); H
D
D
NMR (400 MHz, D2O) δ 1.79-1.95 (2H, m), 1.96-2.11 (2H, m),
2.78 (1H, dq, J ) 8.8, 9.2 Hz), 3.15 (1H, q, J ) 9.2 Hz), 3.67 (1H,
d, J ) 8.8 Hz); 13C NMR (100 MHz, D2O) δ 178.5, 172.6, 57.8,
41.7, 38.9, 21.4, 20.9; HRMS (ES, negative ion) m/z 172.0617 ([M
.
- H]-, C7H10NO4 requires 172.0610). Anal. (C7H11NO4 H2O) C,
H, N: calcd, 43.98, 6.85, 7.33; found, 43.66, 6.10, 7.41.
Molecular Modeling. The modeling study was performed using
the software package MOE (Molecular Operating Environment,
v2005.06, Chemical Computing Group) using the build-in mmff94x
force field and the GB/SA continuum solvation model. Relevant
low-energy conformations were sought by first methylating the
γ-carboxylate group to avoid the return of a large number of
collapsed conformations (holding intramolecular hydrogen bonds).
Each compound was then submitted to a stochastic conformational
search, and conformations above +7 kcal/mol were discarded. The
γ-carboxylate group was demasked and the structure energy-
minimized and used in the following superimposition studies. The
superimposition of ligands was carried out using the built-in
function in MOE, by fitting the ammonium group and the two
carboxylate groups.
the ∆DO measured at 340 nm using ꢀNADH ) 6220 M-1 cm-1
.
When a conversion rate of 40-45% was reached within ap-
proximately 4 h, the reaction mixture was rapidly passed through
a column of Dowex 50 × 8 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 2 × 8 resin (200-400 mesh, AcO-
form, 1.5 × 12 cm). The column was washed with water (50 mL)
and then eluted with an AcOH gradient (0.05-1 M). The ninhydrin-
positive fractions were combined and dried under reduced pressure
to afford L-CBG derivatives: L-CBG-II was isolated from (()-5
while L-CBG-IV, L-CBG-II, and L-CBG-III were consecutively
eluted from the basic resin in the case of (()-6 reaction.
(2S,1′R,2′R)-2-(2′-Carboxycyclobutyl)glycine L-CBG-II: yield
157 mg, 32%; mp 164-6 °C; [R]20D -53.0° (c 0.7, H2O); 1H NMR
(400 MHz, D2O) δ 1.79-2.13 (4H, m), 2.86 (1H, dq, J ) 8.0, 9.2
Hz), 3.07 (1H, q, J ) 9.2 Hz), 3.68 (1H, d, J ) 7.6 Hz); 13C NMR
(100 MHz, D2O) δ 178.5, 172.8, 57.4, 41.3, 38.0, 21.6, 21.0; HRMS
(ES, negative ion) m/z 172.0600 ([M - H]-, C7H10NO4 requires
172.0610). Anal. (C7H11NO4) C, H, N.
Pharmacological Characterization at Human EAAT1-3. The
pharmacological properties of l-CBG-I-IV at human EAAT1,
EAAT2, and EAAT3 were determined in the FLIPR membrane
potential (FMP) assay. The construction of human embryonic
kidney 293 (HEK293) cell lines stably expressing human EAAT1,
EAAT2, and EAAT3 have been reported previously, and the
pharmacological characterization of L-CBG-I-IV was performed
essentially as described here.33 Briefly, cells were split into poly-
d-lysine-coated black-walled clear-bottom 96-well plates in Dul-
becco’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. Then 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]. Next 50 µL of Krebs buffer
was added to each well (in the characterization of nonsubstrate
inhibitors, the inhibitors were added to this buffer), 50 µL of Krebs
buffer supplemented with FMP assay dye was then added to each
well, 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 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. IC50 values were converted to Ki
values by the use of the Cheng-Prusoff equation.39
(2S,1′S,2′R)-2-(2′-Carboxycyclobutyl)glycine L-CBG-III: yield
63 mg, 19%; mp 175-6 °C (lit.11 mp 165-6 °C); [R]20 +3.7°
D
(c 1.0, H2O) (lit.11 [R]20 +3.7° (c 0.5, H2O); 1H NMR (400
D
MHz, D2O) δ 2.05-2.32 (4H, m), 2.95-3.05 (1H, m), 3.37-3.43
(1H, m), 3.90 (1H, d, J ) 11.0 Hz); 13C NMR (100 MHz, D2O) δ
178.7, 172.8, 55.8, 41.0, 37.4, 23.1, 21.4; HRMS (ES, negative
ion) m/z 172.0614 ([M - H]-, C7H10NO4 requires 172.0610). Anal.
(C7H11NO4‚0.2H2O ) C, H, N.
Acknowledgment. We would like to thank The Danish
Medical Research Council, the French National Center for
Scientific Research (CNRS), the Lundbeck Foundation, and the
Novo Nordisk Foundation for funding. We are grateful to Pr.
Kagamiyama’s group (Osaka Medical College) for providing
us with the AAT-overexpressing E. coli strains.
(2S,1′R,2′S)-2-(2′-Carboxycyclobutyl)glycine L-CBG-IV: yield
57 mg, 17%; mp 165-6 °C; [R]20D +42.9° (c 1.1, H2O); 1H NMR
(400 MHz, D2O) δ 2.02-2.30 (4H, m), 2.95-3.09 (1H, m), 3.30-
3.40 (1H, m), 4.06 (1H, d, J ) 11.0 Hz); 13C NMR (100 MHz,
D2O) δ 178.7, 173.0, 54.9, 42.0, 37.1, 23.6, 21.3; HRMS (ES,
negative ion) m/z 172.0613 ([M - H]-, C7H10NO4 requires
172.0610). Anal. (C7H11NO4‚0.5H2O) C, H, N.
Supporting Information Available: A zipped file containing
Figures 2-5 in manipulatable 3D coordinates (file formats MOE

6538 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Faure et al.
(18) Lo, H.-H.; Hsu, S.-K.; Lin, W.-D.; Chan, N.-L.; Hsu, W.-H.
Asymmetrical synthesis of L-homophenylalanine using engineered
escherichia coli aspartate aminotransferase. Biotechnol. Prog. 2005,
21, 411-415.
and PDB), a pdf file containing combustion analysis data of
compounds L-CBG-I-IV, and 1H and 13C NMR spectra of L-CBG-
I. This material is available free of charge via the Internet at
http://pubs.acs.org.
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