Synthesis of new β- and γ-benzyloxy-S-glutamic acid derivatives and evaluation of their activity as inhibitors of excitatory amino acid transporters

Article abstract of DOI:10.1016/j.tet.2009.05.054

An efficient stereoselective preparation of the two enantiomerically pure diasteroisomers of γ-benzyloxy-S-glutamic acid was performed using trans-4-hydroxy-l-proline as a source of chirality, while the erythro and threo isomers of β-benzyloxy-S-glutamic

Full text of DOI:10.1016/j.tet.2009.05.054

Tetrahedron 65 (2009) 6083–6089
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of new
b- and g-benzyloxy-S-glutamic acid derivatives and evaluation
of their activity as inhibitors of excitatory amino acid transporters
,
a
b
b
a
Lucia Tamborini a, Paola Conti a , Andrea Pinto , Simona Colleoni , Marco Gobbi , Carlo De Micheli
*
a
`
Dipartimento di Scienze Farmaceutiche ‘Pietro Pratesi’, Universita degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy
b Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via La Masa 19, 20156 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient stereoselective preparation of the two enantiomerically pure diasteroisomers of
g
-benzyl-
Received 5 February 2009
Received in revised form 6 May 2009
Accepted 21 May 2009
oxy-S-glutamic acid was performed using trans-4-hydroxy-
L-proline as a source of chirality, while the
erythro and threo isomers of -benzyloxy-S-glutamic acid were prepared starting from (R)-Garner’s al-
b
dehyde. All new derivatives were tested for their inhibitory activity against excitatory amino acid
transporters in a rat synaptosomal preparation and their IC50 values were compared to that of TBOA,
a one carbon lower homologue commonly used as the reference blocker of glutamate transporters.
Ó 2009 Elsevier Ltd. All rights reserved.
Available online 27 May 2009
1. Introduction
synaptic communication, and (iii) the recycling of the transmitter
via the glutamine cycle.
L
-Glutamate (Glu) is an important nutrient involved in several
In pathological conditions in which energy levels fall and the
transmembrane gradient of Naþ collapses (e.g., ischaemia, neuro-
trauma), EAATs release additional Glu through the reversed mode
of operation, thus contributing to neuronal cell death.5,6 This sce-
nario supports the idea that the blockade of EAATs by non-trans-
biochemical pathways such as gluconeogenesis and ammonia de-
toxification.1 In the mammalian central nervous system (CNS) Glu is
the most abundant excitatory neurotransmitter; it is involved in
physiological functions of utmost importance such as neuronal
development and synaptic plasticity, learning and memory, cog-
nition, and pain perception (nociception).2 An abnormal increase in
the extracellular concentration of Glu induces toxicity (excitotox-
icity) leading to neuronal cell death.
portable inhibitors (blockers) could be
a useful therapeutic
approach to prevent glutamate release and neuronal death after
cerebral ischaemia.7–9 Unfortunately, EAAT blockers are likely to be
neurotoxic under physiological conditions.10,11
Under physiological conditions, the concentration of Glu in the
synaptic cleft is kept at low level by a number of plasma membrane
proteins acting as excitatory amino acid transporters (EAATs).3 So
far, five different human EAATs (EAAT1–5) have been cloned. EAATs
are mainly localised at the glial level; once taken up in the glial
cells, the neurotransmitter is then recycled through intracellular
metabolism and returned to the nerve endings in the form of glu-
tamine (Gln), its precursor.4 In contrast, the fraction of Glu removed
by the EAATs located in the presynaptic nerve endings is directly
stored in presynaptic vescicles, ready to be released in the extra-
cellular space.
Selective and potent inhibitors are needed to identify the
physiological roles of transporters in the regulation of synaptic
transmission or in the pathogenesis of neurological diseases. An
interesting compound is
blocker, which contains the aspartate backbone functionalised with
a benzyloxy moiety at the position (Fig. 1). A survey of the liter-
L-TBOA, a non-selective but potent EAAT
b
ature shows that the same type of functionalisation applied to
glutamic acid has never been investigated, even though it is known
that the insertion of a substituent such a methyl in the
Glu gives rise to a selective EAAT2 blocker.12 Substitution at the
position of Glu was recently reported:13 a series of 4-alkyl-
b position of
g
The rapid removal of glutamate from the synaptic cleft by
high-affinity uptake is thought to influence the kinetics of Glu
receptor activation, and to contribute to (i) the termination of the
excitatory signal, (ii) the maintenance of extracellular glutamate
levels below excitotoxic concentrations, though maintaining
substituted glutamic acid derivatives proved to act as EAAT in-
hibitors, in particular when a bulky substituent was introduced.
O
NH2
OH
HO
OBn O
L-TBOA
Figure 1. Model compound.
* Corresponding author. Tel.: þ39 02 50319329; fax: þ39 02 50319326.
E-mail address: paola.conti@unimi.it (P. Conti).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.05.054

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L. Tamborini et al. / Tetrahedron 65 (2009) 6083–6089
Again, the impact of the benzyloxy group on the biological activity
was never investigated. Therefore, it appeared highly relevant to
(2S,4R)-6
(2S,4S)-7
a
a
89%
92%
synthesise both
b and g benzyloxy glutamic acid derivatives and to
test their activity as EAAT inhibitors.
MeOCO
MeOCO
O
Since a common feature of EAAT inhibitors is the S configuration
at the
a
-amino acidic centre, we planned the synthesis of the
a
S
COOMe
O
COOMe
N
N
diastereomers, and we varied the sole configuration at the 3- or 4-
position, thus obtaining the four possible isomers (2S,4R)-1,
(2S,4S)-2, (2S,3R)-3 and (2S,3S)-4 (Fig. 2).
Boc
(2S,4S)-10
Boc
(2S,4R)-8
b, c
b, c
70%
75%
OBn NH2
OBn NH2
OBn NHBoc
OBn NHBoc
HO
OH
HO
OH
MeO
HO
OMe
MeO
OMe
O
O
O
O
O
O
O
O
O
O
(2S,4S)-2
(2S,4R)-1
(2S,4S)-11
(2S,4R)-9
NH2
NH2
d,e
d,e
58%
57%
HO
OH
HO
OH
OBn O
OBn O
(2S,3R)-3
OBn NH2
OBn NH2
(2S,3S)-4
OH
HO
OH
O
O
O
O
Figure 2. Target compounds.
(2S,4S)-2
(2S,4R)-1
2. Results
Scheme 2. (a) RuO2xH2O, NaIO4, H2O, AcOEt; (b) PS-CO3, MeOH; (c) Ag2O, PhCH2Br,
Et2O; (d) NaOH, MeOH; (e) 30% CF3COOH/CH2Cl2.
The stereoselective synthesis of enantiomerically pure amino
acids (2S,4R)-1 and (2S,4S)-2 was achieved using as a source of
chirality trans-4-hydroxy-L-proline, which is commercially
available.
In a first attempt, the preparation of amino acids (2S,3R)-3 and
(2S,3S)-4 was tackled by using trans-3-hydroxy-
material following the synthetic strategy described above.
Unfortunately, we have been unable to prepare the -benzyloxy-
L-proline as starting
trans-4-Hydroxy-L-proline was protected at the carboxylic acid
b
and amino functions, following the procedure reported in the lit-
erature14 to afford compound (2S,4R)-5 (Scheme 1). This in-
termediate was in part acetylated using acetic anhydride and
dimethylamino pyridine (DMAP) to give compound 6, having
a 2S,4R configuration at the stereogenic centres, thus being a suit-
able precursor of the final amino acid (2S,4R)-1.
Alternatively, intermediate (2S,4R)-5 was submitted to a Mitsu-
nobu reaction with acetic acid, triphenylphosphine, and dieth-
ylazodicarboxylate (DEAD), to induce a complete inversion of
configuration at the stereogenic centre in position 4, leading to
compound (2S,4S)-7, the suitable precursor of amino acid (2S,4S)-2
(Scheme 1).
glutamatederivativesincethesubstrateprovedtobeunreactivewhen
treated with the mixture of benzyl bromide and silver oxide, whereas,
under conventional reaction conditions, i.e., benzyl bromide/sodium
hydride, it gave the corresponding unsaturated derivative.
Consequently, we moved on to another route starting from (R)-
Garner’s aldehyde, obtained following a literature procedure.17 The
aldehyde was submitted to a Grignard’s reaction, using allyl mag-
nesium chloride in anhydrous tetrahydrofuran, to obtain the mix-
ture of the two diasteroisomers 12a,b. At this stage the two isomers
could not be separated and were used as such in the next step. The
mixture of 12a,b was alkylated by using benzyl bromide/sodium
hydride in anhydrous tetrahydrofuran to yield the mixture of the
corresponding benzyl derivatives 13a,b (Scheme 3). Also at this
stage the two isomers could not be separated by column chromato-
graphy; nevertheless we could determine their 1:3 relative ratio by
HRGC analysis.
MeOCO
a
COOMe
N
HO
97%
Boc
(2S,4R)-6
COOMe
N
Boc
MeOCO
OBn
Boc
O
OH
b
H
95%
(2S,4R)-5
COOMe
a
88%
b
63%
N
O
N
O
N
O
N
Boc
Boc
Boc
(2S,4S)-7
12a:
12b:
13a:
13b:
(R)-Garner's aldehyde
Scheme 1. Reagents and conditions: (a) Ac2O, DMAP, MeCN; (b) HAc, Ph3P, DEAD, THF.
13a : 13b = 1 : 3
Intermediates (2S,4R)-6 and (2S,4S)-7 were oxidised with a cat-
alytic amount of hydrated ruthenium(IV) oxide and a 10% aqueous
solution of sodium periodate in a biphasic system water/ethyl ac-
etate.15 Treatment of (2S,4R)-8 and (2S,4S)-10 with polymer sup-
ported sodium carbonate (PS-CO3) in methanol allowed the direct
deprotection of the hydroxy group in position 4 and the opening of
the lactam ring, to give the corresponding methyl esters, which
were directly benzylated using freshly prepared silver oxide and
benzyl bromide in diethyl ether16 to yield intermediates (2S,4R)-9
and (2S,4S)-11. The latters were then hydrolysed and deprotected at
the amine function to produce the desired enantiopure amino acids
(2S,4R)-1 and (2S,4S)-2 (Scheme 2).
Scheme 3. (a) CH2]CHCH2MgCl, ZnCl2, THF; (b) BnBr, NaH 60%, THF.
The mixture of intermediates 13a,b was then oxidised to the
corresponding diols by using 4-methylmorpholine-N-oxide and
a 4% aqueous solution of OsO4. The crude reaction mixture was
treated with sodium periodate, to yield the aldehyde, followed by
treatment with sodium chlorite and sulfamic acid to produce the
mixture of carboxylic acids 14a,b. After the usual reaction work-up,
the mixture of 14a,b was converted into the corresponding methyl
esters 15a,b by treating with iodomethane and potassium car-
bonate. At this stage, the two diasteroisomers 15a and 15b were
separated by flash chromatography (Scheme 4).

L. Tamborini et al. / Tetrahedron 65 (2009) 6083–6089
6085
O
O
O
OBn
OBn
Boc
OBn
Boc
NH2
NH2
HO
OH
OH
HO
HO
OH
OH
a
OMe
OH
OMe
a
b
O
N
O
N
O
N
13
O
OBn O
(2S,3R)-3
O
O
OH O
Boc
67%
(2S,3R)-22
(2 steps)
15a
15b
14a:
14b:
NH2
NH2
HO
a
Scheme 4. (a) (i) 4-Methylmorpholine-N-oxide, 4% OsO4 in H2O, 1,4-dioxane, H2O; (ii)
NaIO4; (iii) NaOCl2, NH2SO3H; (b) CH3I, K2CO3, acetone.
O
OBn O
OH O
(2S,3R)-23
(2S,3S)-4
Compounds 15a and 15b were treated with a weakly acidic so-
lution of acetic acid and water (5:1),18 in order to remove the ace-
tonide protective group. Compound 15a gave almost exclusively the
expected intermediate 16 with trace amounts of lactone 17, derived
from the intramolecular cyclisation of the alcohol. On the contrary,
the same transformation carried out on derivative 15b yielded
lactone 19 as the major product and alcohol 18 as the byproduct
(ratio 19/18¼86:14) (Scheme 5). This different outcome could be
due to the different spatial orientation of the benzyloxy group.
Scheme 7. (a) H2, Pd/C 5%, MeOH/H2O.
Amino acids 1–4 were tested as inhibitors of [3H]
L-glutamate
uptake in a crude synaptosomal fraction (P2) of the rat brain cortex
and their IC50 values are reported in Table 1, compared to the ac-
tivity of the reference compound (ꢀ)-TBOA.
Table 1
Inhibition of the EAATs-mediated uptake
Compounds
[3H]
L-Glutamate uptake
in synaptosomes IC50 (mM)
O
OBn
OBn
NHBoc
OMe
(2S,4R)-1
(2S,4S)-2
(2S,3R)-3
(2S,3S)-4
(ꢀ)-TBOA
>1000
213.5 (103–442)a
187.7 (163–216)a
>300
a
HO
NHBoc
15a
+
O
O
64%
(2R,3R)-16
(3R,4R)-17
5.4 (4.3–6.7)a
16 : 17 = 99.4 : 0.6
a
95% Confidence intervals (C.I.).
O
OBn
OBn
O
NHBoc
As shown in Table 1, all new derivatives are significantly less
active than TBOA as inhibitors of the Glu reuptake, demonstrating
that homologation of the amino acidic chain worsens the in-
teraction with the EAA transporter proteins. Among the four tested
compounds, the most active stereoisomers (lowest IC50 values) are
(2S,4S)-2 and (2S,3R)-3. Noteworthy, compound (2S,3R)-3 shares
with TBOA the threo arrangement of the amino and the benzyloxy
groups while the erythro counterpart (2S,3S)-4 is basically inactive.
This outcome confirms the high stereoselectivity of the interaction
between the transporters and their inhibitors.
OMe
a
HO
NHBoc
15b
+
O
85%
(2R,3S)-18
18 : 19 = 14 : 86
Scheme 5. (a) H2O/AcOH (1:5).
(3R,4S)-19
The two major fractions, i.e., (2R,3R)-16 and (3R,4S)-19, were
then hydrolysed to carboxylic acids (3R,4R)-20 and (3S,4R)-21, re-
spectively. The u-alcohol function of 20 and 21 was oxidised to the
corresponding carboxylate group by using pyridinium dichromate
in N,N-dimethylformamide and, subsequently, the amine was
deprotected by treatment with a 30% solution of trifluoroacetic acid
in dichloromethane, to give final amino acids (2S,3R)-3 and (2S,3S)-
4 (Scheme 6).
3. Conclusion
The present work describes a very efficient stereoselective
preparation of the two enantiomerically pure diastereoisomers of
g
-benzyloxy glutamic acid, using as a source of chirality trans-4-
hydroxy- -proline. Moreover, starting from (R)-Garner’s aldehyde,
the erythro and threo isomers of -benzyloxy glutamic acid, in an
L
NHBoc
OH
NH2
b
HO
HO
OH
c, d
39%
a
(2R,3R)-16
enantiomeric pure form, were prepared. The four amino acids are
one carbon higher homologues of TBOA, which is widely consid-
ered the reference compound in the field of glutamate transporter
inhibitors due to its high potency as an EAAT blocker, even if it is
unable to discriminate the different EAAT subtypes. Unfortunately,
our new derivatives showed a markedly decreased activity as in-
hibitors of glutamate reuptake. Nevertheless, the four stereoiso-
mers confirm the importance of the stereochemistry in the
interaction of the inhibitors with the active site of the excitatory
amino acid transporters.
O
OBn
O
OBn O
(2S,3R)-3
(3 steps)
(3R,4R)-20
NHBoc
OH
NH2
HO
HO
OH
c,d
34%
b
(3R,4S)-19
O
OBn
O
OBn O
(3 steps)
(3S,4R)-21
(2S,3S)-4
Scheme 6. (a) NaOH 1 N, MeOH; (b) NaOH 1 N, 1,4-dioxane; (c) PDC, DMF; (d) 30%
CF3COOH/CH2Cl2.
In order to assign the relative stereochemistry to stereoisomeric
amino acids (2S,3R)-3 and (2S,3S)-4 as well as to their precursors,
they were submitted to hydrogenation with a catalytic amount of
4. Experimental
4.1. Material and methods
5% palladium on charcoal to produce the corresponding b-hydroxy
amino acids (2S,3R)-22 and (2S,3S)-23 (Scheme 7), whose structure
was previously assigned, and their 1H NMR spectra were compared
to those reported in the literature.19
L
-Glutamic acid was obtained from Sigma (St Louis, MO).
[3H]
-Glutamate was purchased from GE Healthcare (Chalfont St.
Giles, UK). TBOA was obtained from Tocris (Ellisville, MO).
L

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L. Tamborini et al. / Tetrahedron 65 (2009) 6083–6089
IR spectra were registered with
spectrometer.
1H NMR and 13C NMR spectra were recorded with a Varian
Mercury 300 (300 MHz) spectrometer. Chemical shifts ( ) are
a
Perkin–Elmer FT-IR
(s, 3H), 4.70 (dd, J¼1.9, 9.9, 1H), 5.35 (dd, J¼8.8, 9.9, 1H); 13C NMR
(DMSO-d6, T¼100 ꢁC): 20.7, 28.2, 53.0, 55.8, 56.0, 69.8, 83.9, 149.1,
168.7, 169.7, 171.7. Anal. Calcd for C13H19NO7 (301.29): C, 51.82; H,
6.36; N, 4.65. Found: C, 52.02; H, 6.53; N, 4.54.
d
expressed in parts per million and coupling constants (J) in hertz.
Rotary power determinations were carried out with a Jasco J-
810 spectropolarimeter coupled with a Haake N3-B thermostat. TLC
analyses were performed on commercial silica gel 60 F254 alu-
minium sheets; spots were further evidenced by spraying with
a dilute alkaline potassium permanganate solution or with ninhy-
drin. Melting points were determined on a model B 540 Bu¨chi
apparatus and are uncorrected. Microanalyses (C, H, N) of new
compounds agreed with the theoretical value within ꢀ0.4%.
The HRGC-FID analysis was performed using a Trace GC (Thermo
Electron) equipped with a split–splitless injector and flame ion-
isation detector (FID). The column used was a Varian VF-5ms (5%
phenyl 95% dimethylpolysiloxane) fused-silica capillary column
4.4. Synthesis of tert-butyl (1S,3R)-1,3-di(methoxycarbonyl)-
3-(benzyloxy)propylcarbamate (2S,4R)-9
(a) To a solution of compound (2S,4R)-8 (260 mg, 0.86 mmol) in
1.5 mL of methanol was added PS-CO3 (245 mg, 0.86 mmol,
3.5 mmol/g). The reaction mixture was stirred for 1 h at room
temperature. PS-CO3 was filtered off and washed with MeOH. The
solvent was evaporated to give
a pale yellow oil (227 mg,
0.78 mmol), which was directly submitted to the next step.
(b) The crude derivative obtained from the previous step
(227 mg, 0.78 mmol) was dissolved in 3 mL of anhydrous Et2O and
benzyl bromide (0.093 mL, 0.78 mmol) followed by freshly pre-
pared Ag2O (180 mg, 0.78 mmol) were added. The reaction mixture
was stirred for 48 h at room temperature. After disappearance of
the starting material, Ag2O was filtered off and washed with Et2O.
The solvent was evaporated and the crude material was purified by
column chromatography (eluent: petroleum ether/AcOEt 9:1) to
give 230 mg of compound (2S,4R)-9 (70% overall yield).
(30 m, 0.25 mm i.d., 0.25 mm film thickness). He was used as the
carrier gas at a flow rate of 1.0 mL/min. The temperature was pro-
grammed as follows: from 70 ꢁC to 180 ꢁC at 40 ꢁC/min, from 180 ꢁC
to 300 ꢁC at 10 ꢁC/min, then at 300 ꢁC for 7.25 min. The injector and
detector temperatures were 250 and 280 ꢁC, respectively. The in-
jector was operated in split mode with a split rate of 30:1.
4.2. Synthesis of (2S,4R)-1-tert-butyl 2-methyl 4-
acetoxypyrrolidine-1,2-dicarboxylate (2S,4R)-6
4.4.1. Compound (2S,4R)-9
Crystallised from diisopropyl ether as white prisms; mp>234 ꢁC
20
dec; Rf: 0.42 (petroleum ether/AcOEt 8:2); [
a
]
þ82.3 (c 1.00,
D
To a solution of compound (2S,4R)-5 (245 mg, 1.00 mmol) in
MeCN (2.5 mL) were added DMAP (61 mg, 0.50 mmol) and Ac2O
(0.38 mL, 4.00 mmol). The reaction mixture was stirred for 2 h at
room temperature. The solvent was evaporated and the crude
material was purified by column chromatography (eluent: petro-
leum ether/AcOEt 8:2), obtaining 278 mg of compound (2S,4R)-6
(0.97 mmol, 97% yield).
CHCl3); IR (neat, cmꢂ1
)
nmax: 3368, 2977, 1750, 1718, 1522, 1368,
1
1224, 1162; H NMR (DMSO-d6, T¼100 ꢁC): 1.38 (s, 9H), 2.05 (ddd,
J¼7.0, 7.6, 14.3, 1H), 2.18 (ddd, J¼5.2, 6.4, 14.3, 1H), 3.56 (s, 3H), 3.67
(s, 3H), 4.14 (dd, J¼5.2, 7.0, 1H), 4.18 (dd, J¼6.4, 7.6, 1H), 4.44 (d,
J¼11.1,1H), 4.58 (d, J¼11.1, 1H), 6.72 (br s,1H), 7.25–7.35 (m, 5H); 13C
NMR (DMSO-d6, T¼100 ꢁC): 28.8, 34.9, 50.5, 52.4, 52.5, 72.2, 75.2,
79.0, 128.3, 128.5, 128.8, 138.2, 155.8, 172.5, 172.9. Anal. Calcd for
C19H27NO7 (381.42): C, 59.83; H, 7.14; N, 3.67. Found: C, 59.83; H,
7.20; N, 3.64.
4.2.1. Compound (2S,4R)-6
20
Pale yellow oil; Rf 0.37 (petroleum ether/AcOEt 8:2); [
a
]
ꢂ42.3
D
(c 1.00, CHCl3); IR (neat, cmꢂ1
)
nmax: 3339, 2853, 1734, 1654, 1426,
1232, 1161; 1H NMR (DMSO-d6, T¼100 ꢁC): 1.38 (s, 9H), 2.00 (s, 3H),
2.18 (m, 1H), 2.31 (m, 1H), 3.44 (d, J¼12.0, 1H), 3.60 (dd, J¼4.9, 12.0,
1H), 3.68 (s, 3H), 4.28 (t, J¼7.6, 1H), 5.20 (ddd, J¼2.9, 4.9, 7.9, 1H);
13C NMR (DMSO-d6, T¼100 ꢁC): 21.01, 28.8, 39.2, 52.3, 55.1, 58.4,
68.9, 79.4, 154.3, 172.0, 174.5. Anal. Calcd for C13H21NO6 (287.21): C,
54.35; H, 7.37; N, 4.88. Found: C, 54.49; H, 7.52; N, 4.75.
4.5. Synthesis of (2S,4R)-2-amino-4-(benzyloxy)pentanedioic
acid (2S,4R)-1
(a) Compound (2S,4R)-9 (230 mg, 0.60 mmol) was dissolved in
MeOH (2 mL) and 1 N NaOH (0.60 mL) was added. The mixture was
stirred overnight at room temperature. The solvent was evapo-
rated, water (6 mL) was added and the aqueous phase was
extracted with Et2O (1ꢃ4 mL). The aqueous layer was made acidic
with 2 N HCl and newly extracted with AcOEt (4ꢃ3 mL). The or-
ganic extracts were pooled and dried over anhydrous sodium sul-
fate. The solvent was removed under vacuum to give 212 mg (100%
yield) of the carboxylic acid intermediate, which was directly
submitted to next step.
(b) The crude derivative obtained from the previous step
(212 mg, 0.60 mmol) was treated with a 30% dichloromethane so-
lution of trifluoroacetic acid (1.60 mL) at 0 ꢁC. The reaction mixture
was stirred at room temperature until disappearance of the starting
material (3 h). The volatiles were removed under vacuum and the
residue was taken up with acetone and filtered under vacuum to
give 89 mg (58% yield) of (2S,4R)-1 as white foam.
4.3. Synthesis of (2S,4R)-1-tert-butyl 2-methyl 4-acetoxy-5-
oxopyrrolidine-1,2-dicarboxylate (2S,4R)-8
To a magnetically stirred 10% aqueous solution of NaIO4 (10 mL),
hydrated RuO2 (13 mg, 0.10 mmol) was added. Such a mixture was
immediately poured into a well stirred solution of (2S,4R)-6
(278 mg, 0.97 mmol) in AcOEt (2.5 mL) in an Erlenmeyer flask. The
resulting biphasic mixture was stirred for 48 h at room tempera-
ture. Isopropanol was added until the solution became black. The
mixture was filtered through a Celite pad and washed with AcOEt.
The aqueous layer was extracted with AcOEt (3ꢃ15 mL). The or-
ganic layer was dried over anhydrous Na2SO4 and the solvent was
evaporated under vacuum. The crude material was purified by flash
chromatography (eluent: petroleum ether/AcOEt 7:3) to give
260 mg of compound (2S,4R)-8 (89% yield).
4.5.1. Compound (2S,4R)-1
White foam (hygroscopic); Rf: 0.46 (BuOH/H2O/AcOH 4:2:1);
20
4.3.1. Compound (2S,4R)-8
[a
]
þ53.5 (c 0.55, H2O); IR (neat, cmꢂ1
) nmax: 3246, 2361, 2343,
D
20
Colourless oil; Rf: 0.31 (petroleum ether/AcOEt 6:4); [
a]
þ20.8
1718, 1429, 1267, 1214, 1118; 1H NMR (DMSO-d6): 2.02 (ddd, J¼4.6,
10.2, 14.6, 1H), 2.16 (ddd, J¼3.2, 8.5, 14.6, 1H), 3.82 (dd, J¼4.6, 8.5,
1H), 4.23 (dd, J¼3.2, 10.2, 1H), 4.42 (d, J¼11.1, 1H), 4.58 (d, J¼11.1,
1H), 7.22–7.40 (m, 5H); 13C NMR (DMSO-d6): 34.2, 50.5, 72.2, 75.5,
D
(c 1.00, CHCl3); IR (neat, cmꢂ1
) nmax: 2980, 1800, 1750, 1371, 1288,
1228, 1151; 1H NMR (DMSO-d6, T¼100 ꢁC): 1.40 (s, 9H), 2.05 (s, 3H),
2.36 (ddd, J¼9.9, 9.9, 13.4, 1H), 2.49 (ddd, J¼1.9, 8.8, 13.4, 1H), 3.75

L. Tamborini et al. / Tetrahedron 65 (2009) 6083–6089
6087
128.1, 128.3, 128.8, 138.5, 171.2, 173.6. Anal. Calcd for C12H15NO5
(253.25): C, 56.91; H, 5.97; N, 5.53. Found: C, 56.72; H, 6.03; N, 5.48.
4.9. Synthesis of (2S,4S)-2-amino-4-(benzyloxy)pentanedioic
acid (2S,4S)-2
The same procedure used for the synthesis of (2S,4R)-2-amino-
4-(benzyloxy)pentanedioic acid (2S,4R)-1 was applied to com-
pound (2S,4S)-11 (249 mg, 0.65 mmol) to give compound (2S,4S)-2
(93 mg, 57% yield).
4.6. Synthesis of (2S,4S)-1-tert-butyl 2-methyl 4-
acetoxypyrrolidine-1,2-dicarboxylate (2S,4S)-7
To a solution of compound (2S,4R)-5 (245 mg, 1.00 mmol) in
anhydrous THF (15 mL) was added triphenylphosphine (1.0 g,
4.00 mmol) followed by glacial acetic acid (0.114 mL, 2.00 mmol).
4.9.1. Compound (2S,4S)-2
20
Whitefoam (hygroscopic);Rf:0.50(BuOH/H2O/AcOH4:2:1);[a]
D
The mixture was cooled to 0 ꢁC and
a solution of dieth-
ꢂ40.3 (c 0.255, H2O); IR (neat, cmꢂ1
) nmax: 3247, 2359, 2340, 1718,
ylazadicarboxylate (solution 40% in toluene, 1.7 mL, 4.00 mmol) in
4 mL of anhydrous THF was added dropwise over 10 min. The re-
action mixture was allowed to warm at room temperature and
stirred for 6 h. The solvent was evaporated and the crude material
was purified by flash chromatography (eluent: petroleum ether/
AcOEt 8:2) to give 273 mg of compound (2S,4S)-7 (95% yield).
1425, 1259, 1216, 1128; 1H NMR (DMSO-d6): 2.33 (m, 2H), 3.92 (dd,
J¼6.9, 6.9, 1H), 4.26 (dd, J¼5.5, 7.9, 1H), 4.51 (d, J¼11.2, 1H), 4.60 (d,
J¼11.2, 1H), 7.22–7.42 (m, 5H); 13C NMR (DMSO-d6): 34.0, 49.9, 71.8,
74.4, 128.2, 128.4, 128.8, 138.5, 171.1, 173.3. Anal. Calcd for C12H15NO5
(253.25): C, 56.91; H, 5.97; N, 5.53. Found: C, 56.74; H, 6.03; N, 5.48.
4.10. Synthesis of (R)-tert-butyl 4-((R)-1-hydroxybut-3-enyl)-
2,2-dimethyloxazolidine-3-carboxylate 12a and (R)-tert-butyl
4-((S)-1-hydroxybut-3-enyl)-2,2-dimethyloxazolidine-3-
carboxylate 12b
4.6.1. Compound (2S,4S)-7
20
Pale yellow oil; Rf: 0.20 (petroleum ether/AcOEt 8:2); [a]
D
ꢂ34.0 (c 1.00, CHCl3); IR (neat, cmꢂ1
) nmax: 3341, 2852, 1736, 1651,
1429,1230,1158; 1H NMR (DMSO-d6, T¼100 ꢁC): 1.38 (s, 9H),1.85 (s,
3H), 2.05 (m, 1H), 2.54 (ddd, J¼5.5, 9.3, 14.0, 1H), 3.32 (dd, J¼2.5,
12.0, 1H), 3.66 (s, 3H), 3.68 (dd, J¼5.8, 12.0, 1H), 4.34 (dd, J¼2.9, 9.3,
1H), 5.15 (ddd, J¼2.5, 5.8, 8.2, 1H); 13C NMR (DMSO-d6, T¼100 ꢁC):
21.1, 28.4, 35.9, 52.1, 52.3, 57.9, 72.6, 79.9, 153.7, 170.2, 172.0. Anal.
Calcd for C13H21NO6 (287.21): C, 54.35; H, 7.37; N, 4.88. Found: C,
54.47; H, 7.50; N, 4.76.
To a stirred mixture of (R)-Garner aldehyde (3.5 g, 15.3 mmol)
and ZnCl2 (3.2 g, 22.9 mmol) in anhydrous THF (32 mL) at ꢂ78 ꢁC,
was added dropwise a solution of vinyl magnesium bromide 2 M in
THF (11.4 mL, 22.9 mmol). The suspension was allowed to warm at
room temperature and then stirred for additional 2 h. The reaction
was quenched with a saturated solution of NH4Cl (20 mL) and the
aqueous layer was extracted with Et2O (3ꢃ30 mL). The organic layer
was dried over anhydrous Na2SO4 and the solvent was evaporated
under vacuum. The crude material was purified by flash chroma-
tography (eluent: petroleum ether/AcOEt 85:15) to give compounds
12a and 12b as an unsplittable mixture (3.6 g, 88% yield).
Compounds 12a,b: pale yellow oil; Rf: 0.20 (petroleum ether/
AcOEt 85:15).
4.7. Synthesis of (2S,4S)-1-tert-butyl 2-methyl 4-acetoxy-5-
oxopyrrolidine-1,2-dicarboxylate (2S,4S)-10
The same procedure used for the synthesis of (2S,4R)-1-tert-
butyl
2-methyl 4-acetoxy-5-oxopyrrolidine-1,2-dicarboxylate
(2S,4R)-8 was applied to compound (2S,4S)-7 (273 mg, 0.95 mmol)
to give compound (2S,4S)-10 (263 mg, 92% yield).
4.11. Synthesis of (R)-tert-butyl 4-((R)-1-(benzyloxy)but-3-
enyl)-2,2-dimethyloxazolidine-3-carboxylate 13a and (R)-tert-
butyl 4-((S)-1-(benzyloxy)but-3-enyl)-2,2-
4.7.1. Compound (2S,4S)-10
Crystallised from diisopropyl ether as white prisms, mp 68–
20
71 ꢁC; Rf: 0.26 (petroleum ether/AcOEt 8:2); [
a
]
ꢂ33.8 (c 1.07,
D
dimethyloxazolidine-3-carboxylate 13b
CHCl3); IR (neat, cmꢂ1
) nmax: 2981, 1797, 1750, 1370, 1291, 1220,
1153; 1H NMR (CDCl3): 1.50 (s, 9H), 1.96 (ddd, J¼8.4, 8.4, 13.5, 1H),
2.14 (s, 3H), 2.82 (ddd, J¼8.4, 8.4, 13.5, 1H), 3.80 (s, 3H), 4.50 (dd,
J¼8.4, 8.4, 1H), 5.38 (dd, J¼8.4, 8.4, 1H); 13C NMR (CDCl3): 20.7, 28.5,
52.8, 55.6, 55.7, 69.7, 84.6, 149.0, 168.4, 169.9, 170.9. Anal. Calcd for
C13H19NO7 (301.29): C, 51.82; H, 6.36; N, 4.65. Found: C, 51.98; H,
6.49; N, 4.56.
To a solution of 12a,b (3.6 g, 13.4 mmol) in anhydrous THF
(26 mL) was added NaH (60% in mineral oil, 804 mg, 20.1 mmol) at
0 ꢁC and the mixture was stirred for 10 min under nitrogen atmo-
sphere. Then, benzyl bromide (2.4 mL, 20.1 mmol) was added
dropwise. The reaction mixture was stirred at room temperature
for 12 h and then was quenched with water (20 mL). The aqueous
layer was extracted with Et2O (3ꢃ20 mL). The organic layer was
dried over anhydrous Na2SO4 and the solvent was evaporated un-
der vacuum. The crude material was purified by flash chromato-
graphy (eluent: petroleum ether/AcOEt 98:2) to give an unsplittable
mixture of compounds 13a and 13b (3.0 g, 63% yield).
4.8. Synthesis of tert-butyl (1S,3S)-1,3-di(methoxycarbonyl)-
3-(benzyloxy)propylcarbamate (2S,4S)-11
The same procedure used for the synthesis of tert-butyl (1S,3R)-
1,3-di(methoxycarbonyl)-3-(benzyloxy)propylcarbamate (2S,4R)-9
was applied to compound (2S,4S)-10 (263 mg, 0.87 mmol) to give
compound (2S,4S)-11 (249 mg, 75% yield).
Compounds 13a,b: yellowoil; Rf: 0.19 (petroleum ether/AcOEt99:1).
4.12. Synthesis of (R)-tert-butyl 4-((R)-2-(methoxycarbonyl)-1-
(benzyloxy)ethyl)-2,2-dimethyloxazolidine-3-carboxylate 15a
and (R)-tert-butyl 4-((S)-2-(methoxycarbonyl)-1-(benzyloxy)-
ethyl)-2,2-dimethyloxazolidine-3-carboxylate 15b
4.8.1. Compound (2S,4S)-11
20
Colourless oil; Rf: 0.31 (petroleum ether/AcOEt 8:2); [
a
]
D
ꢂ35.5
(c 1.00, CHCl3); IR (neat, cmꢂ1
)
nmax: 3369, 2976, 1744, 1718, 1498,
1
1366, 1210, 1163; H NMR (DMSO-d6, T¼100 ꢁC): 1.40 (s, 9H), 2.08
(m, 2H), 3.60 (s, 3H), 3.70 (s, 3H), 4.06 (dd, J¼6.0, 8.2, 1H), 4.30 (m,
1H), 4.42 (d, J¼11.2, 1H), 4.62 (d, J¼11.2, 1H), 6.80 (br s, 1H), 7.20–
(a) To a stirred solution of 13a,b (3.0 g, 8.4 mmol) in dioxane
(12 mL) and H2O (12 mL) were added 4-methyl-morpholine N-ox-
ide (1.5 g, 12.6 mmol) and osmium tetraoxide (4% in H2O, 1.1 mL).
After stirring at room temperature for 1 h, NaIO4 (2.7 g, 12.6 mmol)
was added, and the suspension was stirred at room temperature for
additional 1.5 h. The reaction mixture was cooled to 0 ꢁC and so-
dium chlorite (3.0 g, 33.6 mmol) and sulfamic acid (3.3 g,
13
7.35 (m, 5H); C NMR (DMSO-d6, T¼100 ꢁC): 28.7, 34.8, 51.1, 52.1,
52.3, 72.8, 75.6, 79.1, 128.3, 128.8, 129.2, 138.3, 155.9, 172.5, 173.0.
Anal. Calcd for C19H27NO7 (381.42): C, 59.83; H, 7.14; N, 3.67. Found:
C, 59.83; H, 7.23; N, 3.62.

6088
L. Tamborini et al. / Tetrahedron 65 (2009) 6083–6089
33.6 mmol) were added, and the resulting mixture was removed
from the cold bath and stirred for 2 h. To the mixture was added 5%
aqueous HCl (10 mL), and the resulting solution was extracted with
CH2Cl2 (3ꢃ20 mL). The combined organic extracts were washed
with 5% aqueous HCl, dried over Na2SO4 and the solvent was
evaporated under vacuum to give the mixture of the acids as
a yellow oil (2.7 g, 7.1 mmol).
(b) The crude material was treated with iodomethane (0.88 mL,
14.2 mmol) and K2CO3 (1.9 g, 14.2 mmol) in refluxing acetone
(20 mL) for 1 h. The solvent was evaporated under reduced pressure
and the residue was dissolved in H2O (20 mL); the aqueous layer
was extracted with Et2O (3ꢃ15 mL) and the organic layer was dried
over anhydrous Na2SO4 and the solvent was evaporated under
vacuum. The crude material was purified by flash chromatography
(eluent: petroleum ether/AcOEt 95:5) to give compounds 15a
(0.55 g, 1.40 mmol) and 15b (1.65 g, 4.20 mmol). Overall yield: 67%.
stirred at room temperature for 3 h. The solvent was evaporated,
water (6 mL) was added and the aqueous phase was extracted with
Et2O (1ꢃ4 mL). The aqueous layer was made acidic with 2 N HCl
and newly extracted with AcOEt (4ꢃ3 mL). The organic extracts
were pooled and dried over anhydrous sodium sulfate. The solvent
was removed under vacuum to give 298 mg (98% yield) of the
carboxylic acid intermediate, which was directly submitted to next
step.
(b) To a solution of the crude derivative obtained from the
previous step (298 mg, 0.88 mmol) in DMF (4.4 mL) pyridinium
dichromate (PDC) (4.4 g, 13.2 mmol) was added and the mixture
was stirred at room temperature for 4 h. The progress of the
reaction was monitored by TLC (CHCl3/MeOH 9:1þ1% acetic
acid). Water was added (6 mL) and the mixture was extracted
with AcOEt (3ꢃ5 mL). The organic layer was extracted with
NaHCO3 (3ꢃ5 mL) and the aqueous phase was made acidic with
2 N HCl and extracted with AcOEt (3ꢃ5 mL). The organic extracts
were washed with brine, dried over anhydrous Na2SO4, and the
solvent evaporated to give the carboxylic acid (282 mg, 90%
yield).
4.12.1. Compound 15a
Crystallised from hexane as white prisms; mp 57–58 ꢁC; Rf: 0.33
20
(petroleum ether/AcOEt 9:1); [
a
]
D
þ7.3 (c 0.98, CHCl3); IR (neat,
cmꢂ1
)
nmax: 2978, 1739, 1697, 1454, 1366, 1254, 1172, 1096, 1072; 1H
(c) The intermediate from the previous step (282 mg,
0.81 mmol) was treated with a 30% CH2Cl2 solution (0.15 mL) of
trifluoroacetic acid (0.048 mL, 7.8 mmol) at 0 ꢁC. The solution was
stirred at room temperature for 4 h until disappearance of the
starting material. The volatiles were removed under vacuum and
the residue was taken up with MeOH, filtered, washed with MeOH
and Et2O, and dried under vacuum to give amino acid (2S,3R)-3
(79 mg, 39% yield).
NMR (DMSO-d6, 100 ꢁC): 1.40 (s, 3H), 1.42 (s, 9H), 1.50 (s, 3H), 2.44
(m, 1H), 2.57 (dd, J¼3.8, 15.9, 1H), 3.58 (s, 3H), 3.88–4.02 (m, 2H),
4.14 (m, 1H), 4.30 (m, 1H), 4.50 (d, J¼11.8, 1H), 4.58 (d, J¼11.8, 1H),
7.20–7.38 (m, 5H); 13C NMR (DMSO-d6): 22.8, 26.5, 28.6, 35.7, 52.0,
55.5, 57.1, 63.5, 72.1, 75.8, 80.1, 94.3, 128.0, 128.1, 128.8, 138.9, 152.1,
172.4. Anal. Calcd for C21H31NO6 (393.47): C, 64.10; H, 7.94; N, 3.56.
Found: C, 64.49; H, 8.14; N, 3.49.
4.12.2. Compound 15b
4.14.1. Compound (2S,3R)-3
20
Colourless oil; Rf: 0.25 (petroleum ether/AcOEt 9:1); [
a]
þ30.1
White powder; mp>155 ꢁC dec; Rf: 0.35 (BuOH/H2O/AcOH
D
20
(c 1.02, CHCl3); IR (neat, cmꢂ1
)
nmax: 2980, 1738, 1697, 1450, 1368,
3:2:1); [
a
]
þ3.3 (c 0.28, H2O); IR (neat, cmꢂ1
) nmax: 2945, 1699,
D
1
1251, 1166, 1091, 1067; H NMR (DMSO-d6, 100 ꢁC): 1.42 (s, 15H),
2.54 (m, 2H), 3.59 (s, 3H), 3.88–3.98 (m, 3H), 4.26 (m, 1H), 4.54 (s,
2H), 7.20–7.35 (m, 5H); 13C NMR (DMSO-d6): 23.7, 26.9, 28.7, 37.7,
52.1, 60.3, 64.0, 73.0, 75.6, 80.1, 94.2, 128.2, 128.5, 128.8, 138.9, 152.0,
172.0. Anal. Calcd for C21H31NO6 (393.47): C, 64.10; H, 7.94; N, 3.56.
Found: C, 64.44; H, 8.11; N, 3.50.
1547, 1420, 1215, 1181, 1063; 1H NMR (D2O): 2.68 (d, J¼6.4, 2H), 3.83
(d, J¼3.2, 1H), 4.44 (ddd, J¼3.2, 6.4, 6.4, 1H), 4.52 (s, 2H), 7.23–7.38
(m, 5H); 13C NMR (D2O): 37.5, 57.1, 73.1, 73.8, 128.7, 128.8, 128.9,
137.0, 171.7, 174.5. Anal. Calcd for C12H15NO5 (253.25): C, 56.91; H,
5.97; N, 5.53. Found: C, 56.61; H, 6.07; N, 5.44.
4.15. Synthesis of tert-butyl (3R,4S)-4-(benzyloxy)-
tetrahydro-6-oxo-2H-pyran-3-ylcarbamate (3R,4S)-19
4.13. Synthesis of tert-butyl (2R,3R)-4-(methoxycarbonyl)-3-
(benzyloxy)-1-hydroxybutan-2-yl carbamate (2R,3R)-16
The same procedure used for the synthesis of (2R,3R)-16 was
applied to compound 15b (1.6 g, 4.2 mmol) to give compound
(3R,4S)-19 (984 mg, 73% yield) and compound (2R,3S)-18 (12%
yield) as the minor product.
Compound 15a (550 mg, 1.40 mmol) was dissolved in a 1:5
mixture of H2O and acetic acid (14 mL). The solution was stirred at
room temperature for 24 h. After disappearance of the starting
material, the solvent was evaporated under reduced pressure and
the crude material was purified by flash chromatography (eluent:
petroleum ether/AcOEt 7:3), obtaining the amino alcohol (2R,3R)-
16, as the major product (317 mg, 64% yield) and lactone (3R,4R)-17
as the minor product (0.4% yield).
4.15.1. Compound (3R,4S)-19
Crystallised from diisopropyl ether as white prisms, mp
20
85–86 ꢁC; Rf: 0.16 (petroleum ether/AcOEt 7:3); [
a
]
þ49.4 (c
D
1.00, CHCl3); IR (neat, cmꢂ1
) nmax: 3361, 2360, 2325, 1756,
1682, 1527, 1364, 1222, 1177, 1072; 1H NMR (CDCl3): 1.42 (s,
9H), 2.70 (dd, J¼4.1, 18.1, 1H), 2.92 (dd, J¼3.3, 18.1, 1H), 3.92
(m, 1H), 4.14 (m, 1H), 4.22–4.42 (m, 2H), 4.50 (d, J¼11.6, 1H),
4.70 (d, J¼11.6, 1H), 4.84 (br s, 1H), 7.23–7.42 (m, 5H); 13C
NMR (CDCl3): 28.5, 34.3, 46.6, 67.1, 71.3, 71.9, 80.6, 128.0,
128.5, 128.9, 137.1, 155.3, 168.3. Anal. Calcd for C17H23NO5
(321.27): C, 63.54; H, 7.21; N, 4.36. Found: C, 63.44; H, 7.38; N,
4.25.
4.13.1. Compound (2R,3R)-16
Colourless oil; Rf: 0.21 (petroleum ether/AcOEt 7:3); [a D20
]
þ4.60 (c
0.99, CHCl3); IR (neat, cmꢂ1
) nmax: 3338, 2923, 2852, 1718, 1499, 1458,
1366,1248,1165,1061; 1HNMR(CDCl3):1.42(s, 9H), 2.59(dd, J¼6.0,16.2,
1H), 2.69 (dd, J¼6.8, 16.2, 1H), 3.60 (m, 1H), 3.65 (s, 3H), 3.70–3.83 (m,
2H), 4.23(m,1H), 4.51 (d, J¼11.0,1H), 4.63(d, J¼11.0,1H), 5.04(brd, J¼8.8,
1H), 7.25–7.40 (m, 5H); 13C NMR (CDCl3): 28.5, 37.0, 52.0, 55.1, 63.3, 73.3,
75.3, 80.0,128.1,128.2,128.7,137.9,156.6,172.1. Anal. Calcd for C18H27NO6
(353.41): C, 61.17; H, 7.70; N, 3.96. Found: C, 61.14; H, 7.86; N, 3.85.
4.16. Synthesis of (2S,3S)-2-amino-3-(benzyloxy)pentanedioic
acid (2S,3S)-4
4.14. Synthesis of (2S,3R)-2-amino-3-(benzyloxy)pentanedioic
acid (2S,3R)-3
The same procedure used for the synthesis of amino acid
(2S,3R)-3 was applied to compound (3R,4S)-19 (984 mg, 3.0 mmol)
to give compound (2S,3S)-4 (258 mg, 34% yield).
(a) Compound (2S,3R)-16 (317 mg, 0.90 mmol) was dissolved in
MeOH (2 mL) and 1 N NaOH (0.9 mL) was added. The mixture was

L. Tamborini et al. / Tetrahedron 65 (2009) 6083–6089
6089
4.16.1. Compound (2S,3S)-4
about 3 mg of tissue/mL, wet weight, in 10 mM Tris-acetate, pH 7.4,
containing 128 mM NaCl, 10 mM -glucose, 5 mM KCl, 1.5 mM
NaH2PO4, 1 mM MgSO4 and 1 mM CaCl2. Samples of 0.5 mL were
preincubated for 7 min at 35 ꢁC in a water bath with or without the
compounds to be tested. Nonspecific uptake was determined in the
White powder; mp>140 ꢁC dec; Rf: 0.37 (BuOH/H2O/AcOH
D
20
3:2:1); [
a
]
ꢂ15.0 (c 0.25, H2O); IR (neat, cmꢂ1
)
nmax: 2943, 1700,
D
1551, 1419, 1227, 1186, 1069; 1H NMR (D2O): 2.50 (m, 1H), 2.53 (m,
1H), 4.08 (d, J¼3.8, 1H), 4.34 (ddd, J¼3.8, 5.5, 7.6, 1H), 4.52 (d, J¼11.7,
1H), 4.62 (d, J¼11.7, 1H), 7.25–7.38 (m, 5H); 13C NMR (D2O): 38.3,
58.4, 74.4, 74.8, 74.5, 131.3, 131.3, 131.5, 139.5, 173.5, 177.2. Anal.
Calcd for C12H15NO5 (253.25): C, 56.91; H, 5.97; N, 5.53. Found: C,
56.64; H, 6.06; N, 5.45.
presence of 300
mM L-glutamate. Uptake was started by adding
10 -glutamate (initial specific activity, 49 Ci/mmol; specific
m
M [3H]
L
activity after isotopic dilution with unlabelled Glu, 0.049 Ci/mmol)
and was stopped 4 min later by adding 2 ml of ice-chilled assay
buffer. Samples were immediately filtered through 0.65 mm cellu-
4.17. Synthesis of (2S,3R)-2-amino-3-hydroxypentanedioic
acid (2S,3R)-22
lose mixed ester filters (Millipore Corporation, Cork, Ireland), which
were washed with 2 mL of assay buffer and counted for radioac-
tivity in 4 mL of Ultima Gold MV (PerkinElmer Life and Analytical
Sciences, Waltham, MA), in a Tri-Carb 2800TR rack-beta liquid
scintillation counter (PerkinElmer Life and Analytical Sciences) with
a counting efficiency of about 60%.
For each drug, the percentages inhibition of specific uptake were
determined for three to six concentrations in three different ex-
periments and the inhibition curves were fitted using the ‘one-site
competition’ equation built into Prism version 5.0 for Windows
(GraphPad Software, San Diego, CA). This analysis gives the IC50
(i.e., the drug concentration inhibiting specific binding by 50%),
with its 95% confidence intervals (C.I.).
Amino acid (2S,3R)-3 (30 mg, 0.12 mmol) was dissolved in
methanol (1.0 mL) and a catalytic amount of 5% Pd/C was added.
The mixture was hydrogenated for 10 h. After disappearance of the
starting material, the catalyst was filtered off, and the solvent was
evaporated under vacuum to give 19.4 mg of the corresponding
hydroxy amino acid (0.12 mmol, 100% yield) as a colourless foam.
4.17.1. Compound (2S,3R)-22
20
Mp 180–182 ꢁC; [
a
]
D
þ21.0 (c 0.25, H2O); 1H NMR (D2O): 2.55
(dd, J¼8.80, 16.22, 1H), 2.72 (dd, J¼4.13, 16.22, 1H), 3.66 (d, J¼5.22,
1H), 4.39 (ddd, J¼4.13, 5.22, 8.80, 1H); 13C NMR (D2O): 39.3, 58.8,
66.4, 172.1, 175.0. Anal. Calcd for C5H9NO5 (163.13): C, 36.81; H,
5.56; N, 8.59. Found: C, 36.64; H, 5.59; N, 8.55.
Acknowledgements
`
We are grateful to MIUR (PRIN 2007, Rome) and Universita degli
4.18. Synthesis of (2S,3S)-2-amino-3-hydroxypentanedioic
acid (2S,3S)-23
Studi di Milano (PUR) for their financial support. The technical
assistance of Mr. G.L. Visconti is gratefully acknowledged.
The same procedure used for the synthesis of (2S,3R)-22 was
applied to amino acid (2S,3S)-4 (30 mg, 0.12 mmol) to give com-
pound (2S,3S)-23 (19.4 mg, 100% yield).
References and notes
1. Headley, P. M.; Grillner, S. Trends Pharmacol. Sci. 1990, 11, 205.
2. (a) Bra¨uner-Osborne, H.; Egebjerg, J.; Nielsen, E. Ø.; Madsen, U.; Krogsgaard-
Larsen, P. J. Med. Chem. 2000, 43, 2609; (b) Kew, J. N. C.; Kemp, J. A. Psycho-
pharmacology 2005, 179, 4.
3. Beart, P. M.; O’Shea, R. D. Br. J. Pharmacol. 2007, 150, 5.
4. Broer, S.; Brooks, N. J. Neurochem. 2001, 77, 705.
4.18.1. Compound (2S,3S)-23
20
Mp 172–174 ꢁC dec; [
a]
þ19.8 (c 0.25, H2O); 1H NMR (D2O):
D
2.58 (d, J¼6.74, 2H); 3.81 (d, J¼3.52, 1H); 4.41 (ddd, J¼3.52, 6.74,
6.74, 1H); 13C NMR (D2O): 37.7, 58.4, 66.3, 171.0, 175.2. Anal. Calcd
for C5H9NO5 (163.13): C, 36.81; H, 5.56; N, 8.59. Found: C, 36.59; H,
5.60; N, 8.53.
5. Levi, G.; Raiteri, M. Trends Neurosci. 1993, 16, 415.
6. McMahon, H. T.; Nicholls, D. G. Biochim. Biophys. Acta 1991, 1059, 243.
7. Mennini, T.; Fumagalli, E.; Gobbi, M.; Fattorusso, C.; Catalanotti, B.; Campiani, G.
Eur. J. Pharmacol. 2003, 479, 291.
8. Campiani, G.; Fattorusso, C.; De Angelis, M.; Catalanotti, B.; Butini, S.; Fattor-
usso, S.; Fiorini, I.; Nacci, V.; Novellino, E. Curr. Pharm. Des. 2003, 9, 599.
9. O’Shea, R. D.; Fodera, M. V.; Aprico, K.; Dehnes, Y.; Danbolt, N. C.; Crawford, D.;
Beart, P. M. Neurochem. Res. 2002, 27, 5.
10. Kanai, Y.; Bhide, P. G.; DiFiglia, M.; Hediger, M. A. Neuroreport 1995, 6, 2357.
11. Fairman, W. A.; Vandenberg, R. J.; Arriza, J. L.; Kavanaugh, M. P.; Amara, S. G.
Nature 1995, 375, 599.
12. Wehbe, J.; Rolland, V.; Roumestant, M. L.; Martinez, J. Tetrahedron: Asymmetry
2003, 14, 1123.
13. Alaux, S.; Kusk, M.; Sagot, E.; Bolte, J.; Jensen, A. A.; Bra¨uner-Osborne, H.;
Gefflaut, T.; Bunch, L. J. Med. Chem. 2005, 48, 7980.
14. Ru¨eger, H.; Benn, M. H. Can. J. Chem. 1982, 60, 2918.
15. Zhang, X.; Schmitt, A. C.; Jiang, W. Tetrahedron Lett. 2001, 42, 5335.
16. De Amici, M.; Dellanoce, C.; De Micheli, C.; Grana, E.; Barbieri, A.; Ladinsky, H.;
Schiavi, G. B.; Zonta, F. J. Med. Chem. 1992, 35, 1915.
17. Garner, P.; Park, J. M. J. Org. Chem. 1987, 52, 2361.
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2003, 68, 6172.
4.19. Biological assays
[3H]Glutamate uptake was measured as previously described20
in a crude synaptosomal fraction (P2) obtained from rat brain
cortex. Adult male CRL:CD(SD)BR rats (Charles River, Calco, Italy)
were used. Procedures involving animals and their care were con-
ducted in conformity with the institutional guidelines that are in
compliance with national (D.L. n.116G.U., suppl. 40, 1992 Feb 18)
and international laws and policies (EEC Council Directive 86/609,
OJ L 358, 1, 1987 Dec 12; Guide for the Care and Use of Laboratory
Animals, US National Research Council, 1996). Rats were killed by
decapitation and their brain cortices homogenised in 40 volumes of
ice-chilled 0.32 M sucrose, pH 7.4, in a glass homogeniser with
a Teflon pestle. The homogenates were centrifuged at 1000ꢃg for
5 min and the supernatants centrifuged again at 12,000ꢃg for
20 min at 4 ꢁC. The pellets (P2) were diluted to a concentration of
19. Vidal-Cros, A.; Gaudry, M.; Marquet, A. J. Org. Chem. 1985, 50, 3163.
20. Funicello, M.; Conti, P.; De Amici, M.; De Micheli, C.; Mennini, T.; Gobbi, M. Mol.
Pharmacol. 2004, 66, 522.