Synthesis and pharmacological characterization at glutamate receptors of the four enantiopure isomers of tricholomic acid

Article abstract of DOI:10.1021/jm701394a

The two enantiomeric pairs of erythro- and threo-amino-(3′-hydroxy- 4′,5′-dihydro-isoxazol-5′-yl)-acetic acids were synthesized via the 1,3-dipolar cycloaddition of bromonitrile oxide to (R)- or (S)-3-(tert-butoxycarbonyl)-2,2-dimethyl-4-vinyloxazolidine.

Full text of DOI:10.1021/jm701394a

J. Med. Chem. 2008, 51, 2311–2315
2311
Synthesis and Pharmacological Characterization at Glutamate Receptors of the Four
Enantiopure Isomers of Tricholomic Acid
Andrea Pinto,† Paola Conti,*,† Marco De Amici,† Lucia Tamborini,† Ulf Madsen,‡ Birgitte Nielsen,‡ Thomas Christesen,‡
Hans Bräuner-Osborne,‡ and Carlo De Micheli†
Istituto di Chimica Farmaceutica e Tossicologica “P. Pratesi”, UniVersità degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy, and
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, UniVersity of Copenhagen, UniVersitetsparken 2,
DK-2100 Copenhagen, Denmark
ReceiVed NoVember 7, 2007
The two enantiomeric pairs of erythro- and threo-amino-(3′-hydroxy-4′,5′-dihydro-isoxazol-5′-yl)-acetic acids
were synthesized via the 1,3-dipolar cycloaddition of bromonitrile oxide to (R)- or (S)-3-(tert-butoxycarbonyl)-
2,2-dimethyl-4-vinyloxazolidine. The pharmacological profiles of the studied amino acids reflect the
relationship between the activity/selectivity and the stereochemistry of the two stereogenic centers: while
the (2S,5′S) stereoisomer is an agonist at the AMPA and KA receptors, its (2R,5′R) enantiomer interacts
selectively with the NMDA receptors; the (2S,5′R) stereoisomer is the only one capable to activate the
mGluRs.
Introduction
The acidic amino acid neurotransmitter L-glutamate (Glua)
plays a pivotal role in the excitatory pathways of the mammalian
central nervous system (CNS).1,2 Once released from the
presynaptic neurons into the glutamatergic synaptic cleft, Glu
activates two main classes of receptors: G-protein-coupled
metabotropic Glu receptors (mGluRs) and ligand-gated iono-
tropic Glu receptors (iGluRs). The mGluRs exert modulatory
effects on neuronal excitability and synaptic transmission. So
far, eight mGluR subtypes (mGluR1–8) have been cloned from
mammalian brain.3 On the other hand, the iGluRs are the major
Figure 1. Structures of reference [(()-1a, (()-1b, (()-2a, and (()-
2b] and target compounds [(-)-2a, (+)-2a, (-)-2b, and (+)-2b].
players in the fast neuronal signaling and represent a potential
therapeutic target for the treatment of a number of neurological
and psychiatric disorders, for example, chronic pain, stroke,
low-affinity, open-channel blocker that has been approved by
epilepsy, drug addiction, schizophrenia, and Parkinson’s disease.4,5
the EMEA and the FDA for the treatment of Alzheimer’s
On the basis of agonist selectivity, iGluRs have been subclas-
disease.6
In a recent paper7 we reported the synthesis and the
sified into N-methyl-D-aspartate (NMDA) receptors, 2-amino-
3-(3-hydroxy-5-methyl-4-isoxazolyl)propionate (AMPA) recep-
pharmacological characterization at Glu receptors of the racemic
tors, and kainate (KA) receptors.1,2 The functional ion channel
form of the two acidic amino acids, 5-(2-amino-2-carboxyethyl)-
4,5-dihydroisoxazole-3-carboxylic acids (()-1a and (()-1b
is composed of four subunits, which can assemble either
homomerically or heteromerically. A total of seven NMDA
subunits (NR1, NR2A-D, NR3A,B), four AMPA subunits
(GluR1–4), and five KA subunits (GluR5–7 and KA1,2) have
been cloned and characterized.
(Figure 1). Later on, we prepared and tested their lower
homologues (()-2a and (()-2b,8 which are Glu analogues in a
partially locked conformation. While both (()-1a and (()-1b
were endowed with a remarkable NMDA antagonist activity
and were devoid of any activity at mGluRs, amino acids (()-
2a and (()-2b activated all three iGluRs and, marginally, some
mGluR subtypes. Therefore, they were classified as nonselective
agonists of the Glu receptors. Furthermore, the erythro isomer
(()-2a was roughly 10 times more active at iGluRs than its
threo counterpart (()-2b, whereas the opposite held true
concerning mGluRs activity. It is worth pointing out that the
(2S,5′S)-diastereomer [(+)-2a] is a naturally occurring amino
acid, termed tricholomic acid. It is a flycidal substance isolated
from different species of mushrooms such as Tricholoma
muscarium,9 Amanita strobiliformis,10 and Ustilago maydis.11
Its biological activity, evaluated on rat cortical neurones12 and
on giant neurons of an African giant snail (Achatina fulica,
Férrusac),13,14 turned out to be similar to that displayed by Glu.
Because at that time the classification of the different Glu
Although an extensive preclinical literature suggests a
therapeutic potential for a number of NMDA, AMPA, KA, and
metabotropic receptor–ligands, only NMDA antagonists have
been clinically investigated in detail. At present, Memantine is
the only drug acting at NMDA receptors as an uncompetitive,
* To whom correspondence should be addressed. Phone: +39 02
50319329. Fax: +39 02 50319326. E-mail: paola.conti@unimi.it.
†
Università degli Studi di Milano.
University of Copenhagen.
‡
a Abbreviations: Glu, L-glutamate; mGluRs, metabotropic glutamate
receptors; iGluRs, ionotropic glutamate receptors; NMDA, N-methyl-D-
aspartate; AMPA, (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) pro-
pionic acid; KA, kainate; EMEA, European Agency for the Evaluation of
Medicinal Products; FDA, Food and Drug Administration; HEK, Human
Embryonic Kidney; CHO, Chinese Hamster Ovary.
10.1021/jm701394a CCC: $40.75
2008 American Chemical Society
Published on Web 03/14/2008

2312 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Brief Articles
Scheme 1. Cycloaddition of Bromonitrile Oxide to (R)- or (S)-3-(tert-Butoxycarbonyl)-2,2-dimethyl-4-vinyloxazolidine
Scheme 2. Synthesis of the Target Amino Acids
receptors was unknown, the pharmacological profile of tricho-
lomic acid was not ascertained and it is, at present, undefined.
To investigate in depth their pharmacological profile, we now
report a simplified and versatile synthesis15,16 of the enantiomers
of erythro-[(-)-2a, -(+)-2a] and threo-[(-)-2b, -(+)-2b] tri-
cholomic acid along with their characterization at iGluRs
performed by in vitro binding to rat cortical membranes and
calcium imaging assays on HEK cells expressing the different
iGluR1–6 cloned subtypes. The activity of the test compounds
at representative mGluRs was ascertained by second messenger
assays at cloned receptors, expressed in Chinese hamster ovary
(CHO) cells.
formation, the desired isomeric amino alcohols (-)-5a/(+)-5a
and (+)-5b/(-)-5b were obtained in acceptable average yield
(71%) only by treatment of the intermediates with a 5:1 acetic
acid/water mixture.20 Derivatives 5 were then transformed into
final amino acids erythro-(-)-2a/(+)-2a and threo-(-)-2b/(+)-
2b via the sequential oxidation of the primary alcohols to the
corresponding carboxylic acids 6 followed by hydrogenolysis
of the benzyl group and treatment with a dichloromethane
solution of trifluoroacetic acid.
Because the relative stereochemistry of the corresponding
racemates (()-2a and (()-2b had been previously assigned by
1H NMR spectroscopic data,8 the absolute configuration at the
stereogenic centers of the pairs of amino acids (-)-2a/(+)-2b
and (+)-2a/(-)-2b was attributed by chemical correlation with
the enantiopure dipolarophiles (R)-3 and (S)-3, respectively.
Chiral HPLC analyses, carried out on final amino acids (+)-2b
and (-)-2b, gave a value of enantiomeric excess higher than
99%. Conversely, chromatographic resolution of (-)-2a/(+)-
2a was not achieved.
The two pairs of enantiomeric acidic amino acids (-)-2a/
(+)-2a and (-)-2b/(+)-2b were tested in vitro by means of
receptor binding techniques, calcium imaging assays, and second
messenger assays. The receptor affinities for NMDA, AMPA,
and KA receptors were determined by use of the radioligands
[3H]CGP39653, [3H]AMPA, and [3H]KA, respectively,21–23
while the activity at cloned rat iGluR1–6 subtypes, expressed
in HEK cells, was evaluated with a calcium imaging assay.24
The mGluRs activities of the new compounds were measured
at rat mGluR1a (representative of group I), mGluR2 (represen-
tative of group II), and mGluR4 (representative of group III)
expressed in CHO cells.25
Results and Discussion
The key step in the synthesis of target amino acids (-)-2a,
(+)-2a, (-)-2b, and (+)-2b is the 1,3-dipolar cycloaddition of
bromonitrile oxide to either (R)- or (S)-3-(tert-butoxycarbonyl)-
2,2-dimethyl-4-vinyloxazolidine [(R)-3 or (S)-3; Scheme 1].
Dipolarophiles (R)-3 and (S)-3 were prepared from (S)- and (R)-
Garner’s aldehyde,17 respectively, following the procedure
reported in the literature.18 The two pairs of diastereomeric
cycloadducts erythro-(-)-4a/threo-(+)-4b and erythro-(+)-4a/
threo-(-)-4b, obtained in a 65:35 ratio19 (Scheme 1), were
separated by silica gel column chromatography and then
separately submitted to the reaction sequences depicted in
Scheme 2. Nucleophilic displacement of the 3-bromo moiety
by the benzyloxy anion gave the expected benzyloxy-substituted
intermediates in high yield (93%), whereas the subsequent
opening of the oxazolidine ring to yield amino alcohols 5 turned
out to be rather problematic. Indeed, among the different
methodologies reported in the literature to achieve this trans-

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2313
Table 1. Affinity for iGluRs Using Rat Cortical Membranesa,b
cmpd
(()-2ac
(-)-(2R,5′R)-2a
(+)-(2S,5′S)-2a
(()-2bc
(+)-(2R,5′S)-2b
(-)-(2S,5′R)-2b
ibotenic acidd
[3H]AMPA IC50 (µM)
[3H]KA IC50 (µM)
[3H]CGP Ki (µM)
1.4 [5.86 ( 0.04]
>100
0.76 [6.14 ( 0.08]
>100
0.29 [6.55 ( 0.06]
6.0 [5.22 ( 0.03]
11 [4.95 ( 0.04]
36 [4.45 ( 0.04 ]
22 [4.66 ( 0.07]
1.5 [5.82 ( 0.05]
0.67 [6.19 ( 0.05]
41 [4.40 ( 0.07]
73 [4.14 ( 0.06]
75 [4.13 ( 0.03]
95 [4.03 ( 0.05]
5.3 [5.28 ( 0.04]
0.95 [6.02 ( 0.01]
19 [4.73 ( 0.04]
12 [4.92 ( 0.03]
>100
>100
a Data are given as mean [mean pIC50 ( SEM or mean pKi ( SEM] of at least three individual experiments. b The membrane preparations used in all
the receptor-binding experiments were prepared according to the method of Ransom and Stec (ref 26). c Data from ref 8. d Data from ref 27.
Table 2. Calcium Imaging Assay (Fluo-4) Using HEK-Cells for Expression of Cloned Rat iGluR1–6 Subtypesa
iGluR1i
EC50(µM)
iGluR2(Q)i
EC50(µM)
iGluR3i
EC50(µM)
iGluR4i
EC50(µM)
iGluR5(Q)
EC50(µM)
iGluR6(Q)
EC50(µM)
cmpd
L-Glu
%
%
%
%
%
%
35 [4.49 (
0.12]
100
140 [3.87 (
0.05]
100
100 [3.99 (
0.05]
100
17 [4.78 (
0.03]
100
130 [3.93 (
0.13]
100
66 [4.23 (
0.15]
100
ibotenic acid
(-)-(2R,5′R)-2a
(+)-(2S,5′S)-2a
>1000
6
15
90
>1000
2
13
74
>1000
1
10
88
>1000
>1000
30 [4.53 (
0.06]
32
9
91
>1000
8
10
91
>1000
>1000
82 [4.09 (
0.04]
96
11
91
>1000
>1000
>1000
>1000
120 [3.94 (
0.02]
480 [3.35 (
0.11]
170 [3.81 (
0.15]
280 [3.60 (
0.13]
(+)-(2R,5′S)-2b
(-)- (2S,5′R)-2b
>1000
>1000
13
35
>1000
>1000
14
20
>1000
>1000
10
23
>1000
>1000
7
90
>1000
>1000
8
12
>1000
>1000
15
11
a Numbers in square brackets are pEC50 ( SEM. Numbers in the right column are maximal response as % of the Glu response. Testing performed as
duplicates and repeated three times, except for ibotenic acid (n ) 2 due to limited amount of compound).
Table 3. Activities at Cloned Rat mGlu Receptors Expressed in CHO Cellsa
cmpd
(()-2ab
(-)-(2R,5′R)-2a
(+)-(2S,5′S)-2a
(()-2bb
(+)-(2R,5′S)-2b
(-)-(2S,5′R)-2b
ibotenic acidc
mGluR1a EC50(µM)
mGluR2 EC50(µM)
mGluR4 EC50(µM)
790 [3.1 ( 0.1]
>1000
610 [3.24 ( 0.11]
110 [4.1 ( 0.3]
>1000
82 [4.10 ( 0.08]
43 [4.37 ( 0.01]
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
100 [4.0 ( 0.1]
>1000
84 [4.08 ( 0.07]
110 [3.97 ( 0.04]
a Data are given as mean [mean pEC50 ( SEM] of at least three independent experiments. b Data from ref 8. c Data from ref 27.
The racemic form of tricholomic acid (()-2a binds efficiently
to all three iGluRs with similar IC50 values (Table 1). When
comparing the data obtained for the two enantiomers (-)-2a
and (+)-2a, we observe that while the (-)-form is a ligand
highly selective for the NMDA receptors, the (+)-form pos-
sesses affinity for the AMPA and KA receptors. Thus, the lack
of selectivity for iGluRs, previously determined for the racemate
(()-2a, is due to the overlap of selective activities of the
individual enantiomers. The picture is less clear for the
corresponding stereoisomers (+)-2b and (-)-2b, generally
showing somewhat lower affinities, (+)-2b with some selectivity
for the AMPA and KA receptors and (-)-2b with a slight
preference for the KA receptors. The results of these binding
studies are confirmed by the data obtained on cloned rat AMPA
(iGluR1–4) and KA (iGluR5,6) receptor subtypes (Table 2).
Amino acid (-)-2a does not activate any of the six subtypes,
whereas its enantiomer (+)-2a is an agonist at all subtypes,
slightly weaker than Glu but with a similar profile. On the other
hand, both amino acids (+)-2b and (-)-2b are inactive at all
the tested subtypes.
The data reported in Table 3 reveal that the activity at
mGluRs, previously observed for the two racemates (()-2a and
(()-2b, is due in both cases to a single enantiomer, (+)-2a and
(-)-2b, respectively. It is worth noting that the potency of (-)-
2b is close to that of ibotenic acid27 and that (-)-2b has an
overall pharmacology similar to that of ibotenic acid.
In summary, we herein report the synthesis of the four
enantiopure isomers of tricholomic acid and the evaluation of
their activity at iGluRs and mGluRs. Particularly interesting are
the pharmacological profiles of enantiomers (-)-2a and (+)-
2a at iGluRs. While the first one interacts exclusively with the
NMDA receptors, its mirror image is an agonist of the AMPA
and KA receptors. As a consequence, it can be deduced that
the natural amino acid (+)-2a, which is found in a number of
mushrooms, is most likely contributing to the poisonous activity
by activation of the AMPA and KA receptors present in the
mammalian nervous system.
Experimental Section
Material and Methods. All reagents were purchased from
Sigma. Dibromoformaldoxime28 was prepared according to a
literature procedure. Dipolarophiles (R)-3-(tert-butoxycarbonyl)-
2,2-dimethyl-4-vinyloxazolidine (R)-3 and (S)-3-(tert-butoxycar-
bonyl)-2,2-dimethyl-4-vinyloxazolidine (S)-3 were prepared from
(S)- and (R)-Garner’s aldehyde,17 respectively, following the
procedure reported in the literature.18 1H NMR (300 MHz) and
13C NMR (75 MHz) spectra were recorded with a Varian Mercury
300 spectrometer in DMSO-d6 or D2O. Analysis of the crude
mixture of cycloadducts 4a/4b was carried out using an Agilent
1100 HPLC with diode array detector coupled with an ion trap
Bruker Esquire 3000+ with ESI+ interface. HPLC analyses of final
amino acids were carried out using a Merck Hitachi L7100 HPLC
with a HP 1050 DAD. Specific optical rotations were determined
with a Jasco J-810 polarimeter coupled with a Haake N3-B
thermostat. TLC analyses were performed on commercial silica gel
60 F254 aluminum sheets; spots were visualized by spraying with a
dilute alkaline potassium permanganate solution or with ninhydrin.
Melting points were determined on a model B 540 Büchi apparatus
and are uncorrected. Microanalyses (C, H, N) of new compounds
agreed with the theoretical value within (0.4%.

2314 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Brief Articles
(4S,5′R)-4-(3′-Bromo-4′,5′-dihydro-isoxazol-5′-yl)-2,2-dimeth-
yl-N-Boc-oxazolidine [(-)-4a] and (4S,5′S)-4-(3′-Bromo-4′,5′-
dihydro-isoxazol-5′-yl)-2,2-dimethyl-N-Boc-oxazolidine [(+)-4b].
To a solution of (R)-3 (5.0 g, 22 mmol) in AcOEt (100 mL) was
added dibromoformaldoxime (6.75 g, 33 mmol) and NaHCO3 (9.2
g). The mixture was vigorously stirred for 24 h at room temperature;
the progress of the reaction was monitored by TLC (petroleum ether/
AcOEt 95:5). Water was added and the organic layer was separated
and dried over anhydrous Na2SO4. The crude material, obtained
after evaporation of the solvent, was chromatographed on silica
gel (petroleum ether/AcOEt 95:5) to give (-)-4a (4.37 g) and (+)-
4b (2.35 g). Overall yield: 88%. Compound (-)-4a: crystallized
from hexane as white needles; mp 82–84 °C; Rf 0.4 (petroleum
extracted with AcOEt (3 × 100 mL). The organic extracts were
washed with brine, dried over anhydrous Na2SO4, and the solvent
evaporated to give (-)-6a (2.15 g, yield 75%). Compound (-)-6a:
colorless prisms; mp 143–146 °C; [R]20 ) -126.12 (c ) 1.0,
D
CHCl3); Anal. (C17H22N2O6) C, H, N.
The above-described methodology was also applied to (+)-5a,
(+)-5b, and (-)-5b to give derivatives (+)-6a, (+)-6b, and (-)-
6b, respectively, in comparable yield. Compound (+)-6a: colorless
prisms; mp 143–145 °C; [R]20D ) +125.23 (c ) 1.0, CHCl3); Anal.
(C17H22N2O6) C, H, N. Compound (+)-6b: colorless oil; [R]20
)
D
+62.04 (c ) 1.004, CHCl3); Anal. (C17H22N2O6) C, H, N.
Compound (-)-6b: colorless oil; [R]20 ) -63.13 (c ) 1.006,
D
CHCl3); Anal. (C17H22N2O6) C, H, N.
ether/AcOEt 9:1); [R]20 ) -405.3 (c ) 1.104, CHCl3); HPLC
(2R,5′R)-Amino-(3′-hydroxy-4′,5′-dihydro-isoxazol-5′-yl)-ace-
tic Acid [(-)-2a]. To a solution of (-)-6a (2.15 g, 6.1 mmol) in
MeOH (100 mL), 5% palladium on carbon powder (Engelhard
cod.5011) was added and the mixture was stirred in a hydrogen
atmosphere at room temperature for 15 min. The progress of the
reaction was monitored by TLC (CHCl3/MeOH 9:1 + 1%
CH3COOH). The mixture was filtered, and the solvent was
evaporated to give 1.4 g of a white solid, which was directly treated
with 15 mL of a 30% CH2Cl2 solution of trifluoroacetic acid at 0
°C. The solution was stirred at room temperature for 5 h. 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 (-)-2a (570 mg, yield 58%).
Compound (-)-2a: crystallized from water/ethanol as white prisms;
mp 188–190 °C dec.; [R]20 ) -105.0 (c ) 0.204, H2O); [R]20
365
retention time, 6.21 min; Anal. (C13H21BrN2O4) C, H, N. Compound
(+)-4b: crystallized from diisopropyl ether as white needles; mp
131–132 °C; Rf 0.23 (petroleum ether/AcOEt 9:1); [R]20365 ) +35.1
(c ) 1.112, CHCl3); HPLC retention time, 5.92 min; Anal.
(C13H21BrN2O4) C, H, N.
(4R,5′S)-4-(3′-Bromo-4′,5′-dihydro-isoxazol-5′-yl)-2,2-dimeth-
yl-N-Boc-oxazolidine [(+)-4a] and (4R,5′R)-4-(3′-Bromo-4′,5′-
dihydro-isoxazol-5′-yl)-2,2-dimethyl-N-Boc-oxazolidine [(-)-4b].
The procedure described above was applied to dipolarophile (S)-3
to yield cycloadducts (+)-4a and (-)-4b in identical ratio.
Compound (+)-4a: crystallized from hexane as white needles; mp
83–85 °C; Rf 0.4 (petroleum ether/AcOEt 9:1); [R]20365 ) +403.0
(c ) 1.12, CHCl3); Anal. (C13H21BrN2O4) C, H, N. Compound (-)-
4b: crystallized from diisopropyl ether as white needles; mp
130–131 °C; Rf 0.23 (petroleum ether/AcOEt 9:1); [R]20365 ) -35.8
(c ) 1.0, CHCl3); Anal. (C13H21BrN2O4) C, H, N.
D
D
lit.15 ) -101 (c ) 0.2, H2O); HPLC retention time, 5.6 min; Anal.
(C5H8N2O4) C, H, N.
(2S,5′R)-N-Boc-2-amino-2-(3′-benzyloxy-4′,5′-dihydro-isoxazol-
5′-yl)ethanol [(-)-5a]. To a solution of benzyl alcohol (7.75 mL,
75 mmol) in dry THF (50 mL) was added in small portions NaH
(0.9 g, 37.5 mmol), and the mixture was stirred at room temperature
under a nitrogen atmosphere for 30 min. A solution of (-)-4a (4.37
g, 12.5 mmol) in dry THF (20 mL) was then added with a syringe,
and the mixture was refluxed for 2 h. HCl (2 N) was added to the
reaction mixture, and after evaporation of the solvent, the aqueous
layer was extracted with Et2O. The organic phase was dried over
anhydrous Na2SO4 and the solvent was evaporated. Excess benzyl
alcohol was removed by Kugelrohr distillation under reduced
pressure. The residue was then purified by column chromatography
(petroleum ether/AcOEt 9:1) to give 4.4 g of a yellow oil which
was directly treated with 100 mL of a mixture of water and acetic
acid (1:5 v/v). After stirring for 48 h, the solution was evaporated
under reduced pressure and the residue was purified by column
chromatography (petroleum ether/AcOEt 7:3) to give (-)-5a (2.77
g, overall yield 66%). Compound (-)-5a: white prisms from
diisopropyl ether; mp 75–76 °C; Rf 0.43 (petroleum ether/AcOEt
The above-described methodology was also applied to (+)-6a,
(+)-6b, and (-)-6b to give derivatives (+)-2a, (+)-2b, and (-)-
2b, respectively, in comparable yield. Compound (+)-2a: crystal-
lized from water/ethanol as white prisms; mp 187–189 °C dec.;
[R]20D ) +100.6 (c ) 0.202, H2O); [R]20D lit.15 ) +103 (c ) 0.2,
H2O); HPLC retention time, 5.6 min; Anal. (C5H8N2O4) C, H, N.
Compound (+)-2b: crystallized from methanol/diethyl ether as
white prisms; mp > 180 °C dec.; [R]20D ) +52.5 (c ) 0.2, H2O);
[R]20D lit.15 ) +63 (c ) 0.2, H2O); HPLC retention time, 7.1 min.;
ee > 99%; Anal. (C5H8N2O4) C, H, N. Compound (-)-2b:
crystallized from methanol/diethyl ether as white prisms; mp >
180 °C dec.; [R]20 ) -54.5 (c ) 0.202, H2O); [R]20 lit.15
)
D
D
-65 (c ) 0.2, H2O); HPLC retention time, 4.6 min.; ee > 99%;
Anal. (C5H8N2O4) C, H, N.
Acknowledgment. Professor Shigetada Nakanishi and Dr.
Jan Egebjerg are gratefully acknowledged for their kind gifts
of the mGluR expressing CHO cell lines and iGluR1-6 cDNAs,
respectively. This work was financially supported by MIUR
(COFIN 2005, Rome) and Università degli Studi di Milano
(FIRST), the Danish Medical Research Council (HBO), the
Direktør Ib Henriksen Foundation (HBO), and the Augustinus
Foundation (HBO).
1:1); [R]20 ) -54.02 (c ) 1.004, CHCl3); Anal. (C17H24N2O5)
D
C, H, N.
The above-described methodology was applied to (+)-4a, (+)-
4b, and (-)-4b to give derivatives (+)-5a, (+)-5b, and (-)-5b,
respectively, in comparable yield. Compound (+)-5a: white prisms
from diisopropyl ether; mp 75–76 °C; Rf 0.43 (petroleum ether/
1
Supporting Information Available: Elemental Analyses; H-
AcOEt 1:1); [R]20 ) +53.83 (c ) 1.002, CHCl3); Anal.
and 13C NMR data; HPLC and LC-MS experimental conditions;
information on the binding and in vitro functional assays at cloned
iGlu and mGlu receptor subtypes. This material is available free
of charge via the Internet at http://pubs.asc.org.
D
(C17H24N2O5) C, H, N. Compound (+)-5b: white prisms from
diisopropyl ether; mp 96–98 °C; Rf 0.33 (petroleum ether/AcOEt
1:1); [R]20 ) +53.55 (c ) 1.004, CHCl3); Anal. (C17H24N2O5)
D
C, H, N. Compound (-)-5b: white prisms from diisopropyl ether;
mp 94–95 °C; [R]20 ) -56.57 (c ) 1.0, CHCl3); Anal.
D
References
(C17H24N2O5) C, H, N.
(2R,5′R)-N-Boc-2-amino-2-(3′-benzyloxy-4′,5′-dihydro-isoxazol-
5′-yl) Acetic Acid [(-)-6a]. To a solution of (-)-5a (2.77 g, 8.2
mmol) in DMF (45 mL), pyridinium dichromate (45.7 g, 123 mmol)
was added, and the mixture was stirred at room temperature for
6 h. The progress of the reaction was monitored by TLC (CHCl3/
MeOH 9:1 + 1% acetic acid). Water (150 mL) was added, and the
mixture was extracted with AcOEt (3 × 100 mL). The pooled
organic layers were then extracted with a 1 N NaOH solution (4 ×
80 mL), the aqueous phase was made acidic with 2 N HCl, and
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