(S)-Homo-AMPA, a specific agonist at the mGlu6 subtype of metabotropic glutamic acid receptors

Article abstract of DOI:10.1021/jm9703597

Our previous publication (J. Med. Chem. 1996, 39, 3188-3194) described (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (Homo-AMPA) as a highly selective agonist at the mGlu₆ subtype of metabotropic excitatory amino acid (EAA) recep

Full text of DOI:10.1021/jm9703597

3700
J . Med. Chem. 1997, 40, 3700-3705
Notes
(S)-Hom o-AMP A, a Sp ecific Agon ist a t th e m Glu 6 Su btyp e of Meta botr op ic
Glu ta m ic Acid Recep tor s
Haleh Ahmadian,† Birgitte Nielsen,† Hans Bra¨uner-Osborne,† Tommy N. J ohansen,† Tine B. Stensbøl,†
Frank A. Sløk,† Naohiro Sekiyama,‡,§ Shigetada Nakanishi,‡ Povl Krogsgaard-Larsen,*,† and Ulf Madsen†
PharmaBiotec Research Center, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy,
2 Universitetsparken, DK-2100 Copenhagen, Denmark, and Department of Biological Sciences, Kyoto University, Faculty of
Medicine, Yosida, Sakyo-ku, Kyoto 606, J apan
Received May 29, 1997X
Our previous publication (J . Med. Chem. 1996, 39, 3188-3194) described (RS)-2-amino-4-(3-
hydroxy-5-methylisoxazol-4-yl)butyric acid (Homo-AMPA) as a highly selective agonist at the
mGlu6 subtype of metabotropic excitatory amino acid (EAA) receptors. Homo-AMPA has
already become a standard agonist for the pharmacological characterization of mGlu6 (Trends
Pharmacol. Sci. Suppl. 1997, 37-39), and we here report the resolution, configurational
assignment, and pharmacology of (S)- (6) and (R)- (7) Homo-AMPA. Using the “Ugi
four-component condensation”, 3-(3-ethoxy-5-methylisoxazol-4-yl)propanal (10) was converted
into the separable diastereomeric derivatives of 6 and 7, compounds 12 and 11, respectively.
Deprotection of 12, in one or two steps, gave extensively racemized 6, which was converted in
low yield into 6 (99.0% ee) through several crystallizations. 6 (99.7% ee) and 7 (99.9% ee)
were finally obtained by preparative chiral HPLC. The configurational assignments of 6 and
1
7 were based on H NMR spectroscopic studies on 12 and 11, respectively, and circular dichroism
studies on 6 and 7. Values of optical rotations using different solvents and the chiral HPLC
elution order of 6 and 7 supported the results of the spectroscopic configurational assignments.
The activities of 6 and 7 at ionotropic EAA (iGlu) receptors and at mGlu1-7 were studied. (S)-
Homo-AMPA (6) was shown to be a specific agonist at mGlu6 (EC50 ) 58 ( 11 µM) comparable
in potency with the endogenous mGlu agonist (S)-glutamic acid (EC50 ) 20 ( 3 µM). Although
Homo-AMPA did not show significant effects at iGlu receptors, (R)-Homo-AMPA (7), which
was inactive at mGlu1-7, turned out to be a weak N-methyl-D-aspartic acid (NMDA) receptor
antagonist (IC50 ) 131 ( 18 µM).
In tr od u ction
the S-form of the homologous 3-isoxazolol amino acid,
(S)-AMPA (3), is a specific AMPA receptor agonist11
(Chart 1). The higher homologue of AMPA, (RS)-2-
amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid
(Homo-AMPA), on the other hand, does not interact
significantly with iGlu receptors,12 but Homo-AMPA
was shown to be a highly selective mGlu6 receptor
agonist.13 Homo-AMPA has already become a standard
ligand for the pharmacological characterization of mGlu6
receptors,14 and we here report the preparation, con-
figurational assignment, and pharmacology of (S)-
Homo-AMPA (6) and (R)-Homo-AMPA (7). The phar-
macological effects of 6 and 7 are compared with those
of the S- and R-forms of the glutamic acid homologue
2-aminoadipic acid, compounds 4 and 5, respectively
(Chart 1).
Based on pharmacological, electrophysiological, and
molecular cloning studies, the excitatory amino acid
(EAA) receptors, operated by (S)-glutamic acid [(S)-Glu,
1], have been classified into two major classes: the
ionotropic EAA (iGlu) receptors and the metabotropic
EAA (mGlu) receptors coupled to G-proteins. The
former class of EAA receptors comprises the N-methyl-
D-aspartic acid (NMDA), (RS)-2-amino-3-(3-hydroxy-5-
methylisoxazol-4-yl)propionic acid (AMPA), and kainic
acid subgroups of receptors, all of which are hetero-
geneous.1-4 So far, eight different subtypes of mGlu
receptors (mGlu1-8) have been cloned.5-8
A prerequisite for the determination of the physi-
ological role and pharmacological importance of the
subgroups of iGlu receptors and the subtypes of mGlu
receptors is the availability of highly selective agonists
and antagonists.6,9 We have previously shown that (R)-
2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid
[(R)-AMAA, 2] is a selective NMDA agonist,10 whereas
Resu lts
Ch em istr y a n d Ster eoch em istr y. Under “Ugi
four-component condensation” reaction conditions,15 us-
ing (S)-1-phenylethylamine [(S)-PEA] as a chiral aux-
iliary, 3-(3-ethoxy-5-methylisoxazol-4-yl)propanal (10)
was converted into (2R)-N-tert-butyl-4-(3-ethoxy-5-me-
thylisoxazol-4-yl)-2-[N-[(S)-1-phenylethyl]benzamido]bu-
tyramide (11) and the 2S-diastereomeric compound 12
(Scheme 1). Compounds 11 and 12 were readily sepa-
* Address correspondence to this author. Phone: (+45) 35 37 08
50, ext. 511. Fax: (+45) 35 37 22 09.
† The Royal Danish School of Pharmacy.
‡ Kyoto University.
§ Present address: Department of Molecular Biology, Biomolecular
Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565,
J apan.
X Abstract published in Advance ACS Abstracts, October 1, 1997.
S0022-2623(97)00359-2 CCC: $14.00 © 1997 American Chemical Society

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3701
Sch em e 1a
(1.13 ppm) were assigned the 2R- and 2S-configurations,
respectively.
Compound 12 was subjected to one- and two-step
deprotection reactions to give (S)-Homo-AMPA (6) under
previously reported conditions.15,17 In the two-step
procedure, 12 was debenzylated using formic acid to give
13, which was subsequently converted into 6‚HCl by
treatment with 6 M hydrochloric acid at 105-110 °C
for 16 h. Chiral HPLC analysis of the fully deprotected
product before recrystallization disclosed pronounced
racemization, the crude 6‚HCl showing an enantiomeric
excess (ee) of 62%. 1H NMR analyses of the starting
material 12, to which different amounts of 11 were
added, indicated a diastereomeric excess (de) of 12
higher than 90%. The stereochemical purity of 13 was
analyzed by chiral HPLC. To be able to identify the
enantiomeric impurity in the HPLC chromatogram of
compound 13, the enantiomer 14 was synthesized as a
reference compound. Compound 14 was synthesized in
analogy with 13 by treatment of compound 11 with
formic acid (Scheme 1). The HPLC analysis showed
both compounds 13 and 14 to have ee g 96%, indicating
that no significant racemization takes place during the
formic acid treatment.
a
Reagents: (i) EtBr, K2CO3; (ii) diisobutylaluminum hydride;
In the one-step deprotection reaction, a solution of 12
in 48% hydrobromic acid was heated at 100 °C for 1 h
providing extensively racemized crude 6‚HBr (45% ee).
Additional heating of this solution at 100 °C for more
than 3 h did not result in further racemization, indicat-
ing stereochemical stability of 6 under strongly acidic
conditions. After four crystallizations, crude 6‚HCl (62%
ee) was converted into 6‚HCl of relatively high optical
purity (99% ee) but at a low yield (20%). The results
prompted us to develop a chromatographic procedure
for the preparation of 6 and 7.
(iii) (S)-PEA, benzoic acid, tert-butylisonitrile; (iv) formic acid; (v)
6 M HCl; (vi) 48% aqueous HBr.
Ch a r t 1. Structures of (S)-Glutamic Acid [(S)-Glu, 1]
and a Number of Analogues and Homologues
The preparative resolution of Homo-AMPA was car-
ried out on a preparative Crownpak CR(+) column (150
× 10 mm) containing 18-crown-6-type crown ether as
the chiral selector. The column was eluted at 0-1 °C
with aqueous acetic acid. Under these conditions, 10
mg samples of racemic compound dissolved in the
mobile phase could be separated with baseline resolu-
tion in 30 min. Collection and evaporation of appropri-
ate fractions gave after recrystallization 6 (99.7% ee)
and 7 (99.9% ee) in 73% and 80% yields, respectively
(Figure 1).
It has been shown that R-forms of R-amino acids elute
faster than the corresponding S-forms on Crownpak CR-
(+) columns,18 and accordingly, compound 6, which was
1
assigned the S-configuration on the basis of H NMR
studies, was eluted more slowly than 7, assigned the
R-configuration, using the Crownpak CR(+) column.
The elution sequence of 6 and 7 was reversed, when the
enantiomeric Crownpak CR(-) column was used (Figure
1). Furthermore, the elution order of 6 and 7 was
studied using a chiral ligand exchange HPLC column
containing (S)-pipecolic acid bound to silica gel and
rable by silica gel column chromatography (CC). The
1H NMR chemical shifts of the tert-butyl groups of this
type of diastereomeric products, containing a (S)-PEA
unit, have empirically been used to assign their relative,
and consequently their absolute, stereochemistry.15-17
Thus, a resonance signal for this group in the region of
1.3-1.4 ppm is consistent with R,S-configuration,
whereas a signal near 1.1 ppm indicates S,S-configu-
ration. On the basis of an 1H NMR spectroscopic
analysis, compound 11 (1.37 ppm) and compound 12
chelated with Cu2+
. In agreement with the general
observations that (R)-R-amino acids elute before the
corresponding S-forms using a similar (S)-proline-based
column,19,20 7 eluted before 6 on the (S)-pipecolic acid-
based column.
CD spectra of 6 and 7, recorded in 0.1 M hydrochloric
acid, clearly are mirror images (Figure 2), demonstrat-
ing the enantiomeric relationship. The positive Cotton

3702 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Notes
agonist or antagonist effects at these receptors using
the rat cortical wedge preparation.26 Compound 6 was
inactive when tested as an agonist (EC50 > 1000 µM)
or an antagonist (IC50 > 1000 µM) at iGlu receptors.
Compound 7 marginally (IC50 > 500 µM) reduced
depolarizations induced by AMPA (5 µM) or kainic acid
(10 µM) and turned out to be a weak antagonist at
NMDA receptors (IC50 ) 131 ( 18 µM).
Discu ssion
The preparation, configurational assignment, and in
vitro pharmacology of (S)-Homo-AMPA (6) and (R)-
Homo-AMPA (7) are described. The “Ugi four-compo-
nent condensation”15 reaction was used to synthesize
the diastereomeric condensation products 11 and 12,
which were separated chromatographically. Deprotec-
tion of 12 to give 6 using one- or two-step procedures,
both involving hydrolysis under strongly acidic condi-
tions (Scheme 1), provided extensively racemized 6. This
amino acid was shown to be stereochemically stable
under these reaction conditions indicating that an as
yet unidentified intermediate in the transformation of
12 into 6 must be susceptible to acid-catalyzed racem-
ization. In any case, the “Ugi four-component conden-
sation” reaction does not seem to be a versatile proce-
dure for the synthesis of optically pure R-amino acids.
Compounds 6 and 7 of high enantiomeric purities and
in good yields were finally obtained by preparative
chiral HPLC separation of Homo-AMPA12 (Figure 1).
The configurational assignments of 6 and 7 were
F igu r e 1. Chromatographic separation of 10 mg of Homo-
AMPA on a 150 × 10 mm Crownpak CR(+) column eluted at
1.5 mL/min with 15 mM aqueous AcOH at 0-1 °C (A). Chiral
HPLC analyses of the chromatographically resolved (S)-(+)-
Homo-AMPA (6) (B) and (R)-(-)-Homo-AMPA (7) (C) on a 150
× 4 mm Crownpak CR(-) column eluted at 0.4 mL/min with
aqueous HClO4, pH 2, at room temperature. Spike experiments
using Homo-AMPA confirmed the identity of the enantiomeric
impurities indicated in panels B and C.
effect of 6 at 210 nm (∆ꢀ ) +0.14 m2/mol) is in
agreement with published data describing positive
Cotton effects of (S)-R-amino acids in acidic solu-
tions.21,22 Under similar conditions, (S)-AMPA (3) also
showed a positive Cotton effect (∆ꢀ ) +0.19 m2/mol)
(Figure 2), supporting the configurational assignment
of 6.
The chiral chromatographic behavior, IR spectrum,
optical rotations in hydrochloric acid, and CD spectrum
of 6‚HCl, prepared from 6 obtained by preparative chiral
HPLC, were identical with those of 6‚HCl obtained by
deprotection of compound 12 (Scheme 1) and crystallized
to an ee of 99%.
In Vitr o P h a r m a cology. So far, eight different
mGlu receptors (mGlu1-8) have been cloned.5-8 In this
study we tested the effects of (S)-Homo-AMPA (6) and
(R)-Homo-AMPA (7) at mGlu1-7 essentially following
published procedures,23-25 whereas mGlu8 was not
available to us. Initially, we tested 6 and 7 at 1 mM
concentrations (10 mM in the case of mGlu7) alone and
in the presence of 30 µM (S)-Glu (1) [in the case of
mGlu7, 1 mM of (S)-2-amino-4-phosphonobutyric acid
[(S)-AP4] was used due to the low potency of (S)-Glu
(1)]. Compound 6 showed agonist potency at mGlu6 but
did not show significant activities at the remaining
mGlu receptors, neither as an agonist nor as an an-
tagonist (Table 1). As shown in Figure 3 and Table 1,
6 had an EC50 value of 58 ( 11 µM at mGlu6, being
almost equipotent with (S)-Glu (1) at this subtype of
mGlu receptors. Whereas (S)-2-aminoadipic acid (4)
previously has been shown to activate mGlu2 as well
as mGlu6, (R)-2-aminoadipic acid (5) is inactive at these
receptors,13 and in the present studies, compound 7
was inactive at all seven mGlu receptors, when tested
for both agonist and antagonist effects (Table 1 and
Figure 3).
1
based on H NMR chemical shift values for the tert-butyl
groups of 12 and 11. These values have been shown to
be predictive of the absolute configuration of the R-ami-
no acid units of such condensation products.15-17 The
results of this empirical determination of the absolute
stereochemistry of 6 and 7 were corroborated by the
results of comparative CD spectral analyses of 6, 7, and
(S)-AMPA (3) (Figure 2) and by elution orders of 6 and
7 under different chiral HPLC conditions (Figure 1).
Finally, the signs of optical rotations of 6 and 7, recorded
in water and hydrochloric acid, were in accordance with
the empirical Clough-Lutz-J irgenson’s rule.27 These
results together are consistent with (+)-Homo-AMPA
(6) having S-configuration and (-)-Homo-AMPA (7)
R-configuration.
(S)-Glu (1) is capable of activating all iGlu and mGlu
receptors, and (S)-Glu (1) probably is the endogenous
ligand for all of these receptors.1-7 In light of this total
lack of selectivity of (S)-Glu (1), it is remarkable that
(S)-AMPA (3), which is the 3-isoxazolol bioisostere of
(S)-Glu (1) (Chart 1), is a specific agonist at AMPA
receptors.4 The higher homologue of (S)-Glu (1), (S)-2-
aminoadipic acid (4), interacts with a number of
glutamatergic synaptic mechanisms,13 including activa-
tion of mGlu2 and mGlu6.13 We have now shown that
the 3-isoxazolol bioisostere of 4, (S)-Homo-AMPA (6)
(Chart 1), is a specific agonist at mGlu6, approximately
equipotent with (S)-Glu (1) (Table 1).
(R)-Homo-AMPA (7) turned out to be a weak NMDA
antagonist reflecting the structural relationship of 7
with (R)-2-aminoadipic acid (5) (Chart 1), which has
previously been shown to be an NMDA antagonist.2
Homo-AMPA has previously been shown to be inac-
tive when tested as an agonist (at 1000 µM) and as an
antagonist (at 100 µM) at iGlu receptors.12 Neverthe-
less, (S)- (6) and (R)- (7) Homo-AMPA were tested for
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were determined in
capillary tubes and are uncorrected. CC was performed on
Merck silica gel 60 (0.063-0.200 mm). Compounds were

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3703
Ta ble 1. Pharmacological Profiles of (S)- (6) and (R)- (7) Homo-AMPA and Some Structurally Related Amino Acids at the Cloned
mGlu Receptors
EC50 (µM)a
b
c
c
d
b
d
d
compound
mGlu1R
mGlu2
mGlu3
mGlu4a
mGlu5
mGlu6
20 ( 3
mGlu7
(S)-Glu (1)
10 ( 2
>1000
>1000
>1000
>1000
>1000
>1000
4.0 ( 0.5
35 ( 1
>1000
>1000
>1000
>1000
>1000
9.0 ( 4.4
nd
12 ( 2
>3000
nd
4.4 ( 0.8
nd
5400 ( 2800
nd
(S)-2-aminoadipic acid (4)e
(R)-2-aminoadipic acid (5)e
(S)-AP4
140 ( 35
>1000
0.57 ( 0.23
82 ( 15
58 ( 11
>1000
nd
nd
nd
>1000
>1000
nd
>1000
nd
>1000
>1000
nd
0.91 ( 0.50
252 ( 23
Homo-AMPAe
>3000
nd
(S)-Homo-AMPA (6)
(R)-Homo-AMPA (7)
>1000
>5000
>5000
>1000
a
b
EC50 values ( SEM of at least two independent experiments. mGlu1R and mGlu5 (group I) are positively coupled to the hydrolysis
of phosphatidylinositol (PI), and agonism at these receptors was tested as the fold increase in PI level as compared to control. c mGlu2
and mGlu3 (group II) are negatively coupled to the formation of cyclic AMP, and agonism at these receptors was tested as percent decrease
d
of cyclic AMP level, in the presence of forskolin, as compared to control. mGlu4a, mGlu6, and mGlu7 (group III) are also negatively
coupled to the formation of cyclic AMP, and agonism at these receptors was tested as indicated above for group II mGlu receptors (for
details, see ref 13). e Data from ref 13.
are within (0.4% of the calculated values unless otherwise
stated. The HPLC system for the preparative resolution and
the analytical determinations of the enantiomeric purities of
6 and 7 consisted of a J asco 880 pump, a Rheodyne 7125
injector, and a Waters 480 UV detector (220 nm), connected
to a Hitachi D-2000 Chromato-Integrator. A Waters HPLC
system consisting of a M510 pump, a U6K injector, and a M991
photodiode array detector was used for the analytical separa-
tion of 13 and 14. The ee values were calculated from peak
areas. The preparative chromatographic resolution of 6 and
7 was performed on a 150 × 10 mm Crownpak CR(+) column
equipped with a 10 × 4.0 mm Crownpak CR(+) guard column,
both obtained from Daicel and eluted with 1.5 mL/min aqueous
F igu r e 2. CD spectra of the chromatographically resolved (S)-
AcOH (15 mM) at 0-1 °C. Chiral HPLC analyses of 13 and
Homo-AMPA (6) and (R)-Homo-AMPA (7) (A) and (S)-AMPA
14 were performed on a 100 × 4.0 mm CHIRAL-AGP column
(3) (B) in 0.1 M HCl.
(ChromTech, Sweden) eluted at ambient temperature with 0.5
mL/min of 10% 2-PrOH in aqueous potassium phosphate (50
mM), pH 7.0, and the HPLC analyses of 6 and 7 were
performed on a 150 × 4.0 mm Crownpak CR(-) column
(Daicel) eluted with 0.4 mL/min aqueous HClO4, pH 2, at
ambient temperature.
Eth yl 3-(3-Eth oxy-5-m eth ylisoxazol-4-yl)pr opion ate (9).
A mixture of ethyl 3-(3-hydroxy-5-methylisoxazol-4-yl)propi-
onate (8)28 (15.0 g, 75.4 mmol) and K2CO3 (20.8 g, 150.8 mmol)
in acetone (600 mL) was heated at 60 °C for 30 min. After
the mixture cooled to 21 °C, ethyl bromide (12.3 g, 113 mmol)
was added dropwise, and the resulting suspension was heated
at 60 °C for 24 h. The reaction mixture was cooled to 21 °C,
filtered, and concentrated in vacuo. CC [toluene (tol)-EtOAc
(20:1)] of the residue gave 9 (8.0 g, 35.2 mmol) as a slightly
yellow oil in 48% yield. A small sample was distilled in a
Kugelrohr apparatus (200 °C, 0.1 mmHg): 1H NMR (CDCl3)
δ 1.23 (3H, t, J ) 7.2 Hz), 1.40 (3H, t, J ) 7.1 Hz), 2.26 (3H,
s), 2.51-2.56 (4H, m), 4.10 (2H, q, J ) 7.2 Hz), 4.28 (2H, q, J
) 7.1 Hz); 13C NMR (CDCl3) δ 11.2, 13.4, 14.5, 16.5, 32.8, 60.2,
65.2, 103.0, 165.8, 170.5, 172.5. Anal. (C11H17NO4) C, H, N.
3-(3-Eth oxy-5-m eth ylisoxa zol-4-yl)p r op a n a l (10). Com-
pound 9 (5.5 g, 24.0 mmol) was dissolved in dry tol (120 mL)
under a N2 atmosphere, and the solution was cooled to -78
°C. Diisobutylaluminum hydride (60 mL of a 1 M solution in
hexane, 60.0 mmol) was added, and after 6 min the reaction
was quenched with CH3OH (6 mL) followed by addition of a
Rochelle salt solution (45 mL of a saturated aqueous solution
of sodium potassium tartrate). The mixture was allowed to
warm to 25 °C, and the product was extracted with 3 × 150
mL of Et2O. The combined organic phases were dried (MgSO4),
filtered, and concentrated in vacuo to give 10 (3.0 g, 68%) as
a yellow oil: 1H NMR (CDCl3) δ 1.40 (3H, t, J ) 7.1 Hz), 2.27
(3H, s), 2.56 (2H, m), 2.68 (2H, m), 4.28 (2H, q, J ) 7.1 Hz),
9.77 (1H, t, J ) 1.0 Hz). Crude 10 was, without further
purification, converted into a mixture of 11 and 12.
F igu r e 3. Dose-response curves of (S)-Glu (1) (b), (S)-Homo-
AMPA (6) (9), and (R)-Homo-AMPA (7) (2) at CHO cells
expressing mGlu6 receptors. Cells were incubated with the
agonists in the presence of 10 µM forskolin for 10 min, and
cyclic AMP levels were determined by a RIA assay (Amer-
sham). Data are the mean ((SD) of a representative experi-
ment performed in triplicate.
visualized on TLC plates (Merck silica gel 60 F254) using UV
light or a KMnO4 spraying reagent. Compounds containing
an amino group or 3-isoxazolol moiety were visualized using
a ninhydrin or FeCl3 spraying reagent, respectively; 4 Å
molecular sieves were used to dry solvents at least 2 days prior
to use. 1H and 13C NMR spectra were recorded on a Bruker
AC 200F (200 MHz) spectrometer using TMS or 1,4-dioxane
as internal standards for spectra recorded in CDCl3 or D2O,
respectively. IR spectra were recorded from KBr disks on a
Perkin-Elmer 781 grating infrared spectrophotometer. Optical
rotations were determined on a Perkin-Elmer 241 polarimeter.
CD spectra were recorded at room temperature in 1.0 cm
cuvettes on a J asco J -720 spectropolarimeter. Elemental
analyses were performed at Analytical Research Department,
H. Lundbeck A/S, Denmark, or by Mrs. K. Linthoe, Depart-
ment of Chemistry, University of Copenhagen, Denmark, and
(2R)- a n d (2S)-N-ter t-Bu tyl-4-(3-eth oxy-5-m eth ylisox-
a zol-4-yl)-2-[N -[(S )-1-p h e n yle t h yl]b e n za m id o]b u t yr a -
m id e (11 a n d 12). A mixture of 10 (3.0 g, 16.4 mmol) and
(S)-PEA (1.98 mL, 16.4 mmol) in CH3OH (28 mL) was refluxed
at 100 °C for 30 min. After the mixture cooled to 25 °C, benzoic
acid (2.0 g, 16.4 mmol) and tert-butylisonitrile (1.36 g, 16.4

3704 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Notes
mmol) were added, and the resulting solution was stirred at
25 °C for 24 h. Evaporation of the solvent followed by CC [tol-
EtOAc (9:1)] of the residue produced 11 (3.0 g, 38%) as a yellow
oil: [R]23D ) -120°, [R]23365 ) -490° (c ) 0.52, EtOH); 1H NMR
(CDCl3) δ 1.37 (12H, s and t, J ) 7.1 Hz), 1.62 (3H, d, J ) 7.0
Hz), 1.70 (1H, m), 1.90 (2H, m), 2.08 (3H, s), 2.65 (1H, m),
3.35 (1H, m), 4.19 (2H, q, J ) 7.1 Hz), 5.05 (1H, q, J ) 7.0
Hz), 7.29 (5H, m), 7.46 (5H, bs), 8.0 (1H, bs); 13C NMR (CDCl3)
δ 11.1, 14.5, 17.3, 17.9, 28.4, 30.3, 50.6, 58.6, 60.2, 64.9, 103.3,
125.4, 127.0, 128.0, 128.3, 128.8, 129.5, 137.0, 138.5, 165.4,
170.3, 171.3, 173.0. Anal. (C29H37N3O4) H, N; C: calcd, 70.85;
found, 69.44.
and dried chiral HPLC fractions of the first peak (tR ) 10 min,
81.6 mg, corresponding to 94% yield) with an enantiomeric
purity of 99.5% were dissolved in H2O, filtered, evaporated,
and recrystallized in H2O/EtOH to give 69.3 mg of 7 (80%)
with an ee of 99.9%: mp 211-212 °C dec; [R]25 ) -17.4°,
D
[R]25
) -61.1° (c ) 0.44, H2O); IR 3280 (m), 3100-2800
365
(multiple, m), 1585 (s), 1510 (s), 1460 (s), 1400 (s), 1290 (s)
cm-1. Anal. (C8H12N2O4) H, N; C: calcd, 48.00; found, 48.61.
Recrystallization (H2O/EtOH) of the mother liquor furnished
an additional 7.8 mg of 7 (total yield: 89%) with an ee of
99.4%: mp 210-211 °C dec; IR spectrum identical with that
of the first crop of crystals.
Further elution gave after recrystallization (Et2O-light
(S)-2-Am in o-4-(3-h yd r oxy-5-m et h yl-4-isoxa zolyl)b u -
tyr ic Acid [(S)-(+)-Hom o-AMP A, 6]. The pooled and dried
chiral HPLC fractions of the second peak from the separation
procedure described above for 7 (tR ) 16 min, 81.5 mg,
corresponding to 94% yield) with an enantiomeric purity of
99.5% were dissolved in H2O, filtered, and evaporated. Re-
crystallization (H2O/EtOH) gave 63.1 mg of 6 (73%) with an
petroleum ether) 12 (2.0 g, 25%): mp 108-110 °C; [R]23
)
D
-34.8°, [R]23 ) -142° (c ) 0.51, EtOH); 1H NMR (CDCl3) δ
365
1.13 (9H, s), 1.40 (3H, t, J ) 7.1 Hz), 1.46 (3H, d, J ) 7.0 Hz),
2.0 (1H, m), 2.20 (3H, s), 2.45 (2H, t, J ) 8.7 Hz), 2.72 (1H,
m), 3.45 (1H, m), 4.30 (2H, q, J ) 7.1 Hz), 5.10 (1H, q, J ) 7.0
Hz), 7.14-7.40 (5H, m), 7.47 (5H, bs); 13C NMR (CDCl3) δ 11.3,
14.7, 16.9, 18.8, 28.4, 29.9, 50.3, 57.7, 59.3, 65.2, 103.8, 125.7,
127.3, 128.3, 128.8, 128.9, 129.6, 137.0, 166.3, 169.5, 171.0,
172.0. Anal. (C29H37N3O4) C, H, N.
ee of 99.7%: mp 211-212 °C dec; [R]25 ) +17.4°, [R]25
)
D
365
+61.0° (c ) 0.41, H2O), [R]23 ) +29.1°, [R]23 ) +103° (c )
D
365
1
0.37, 0.1 M HCl); H NMR (D2O) δ 2.0 (2H, m), 2.22 (3H, s),
2.36 (2H, m), 3.67 (1H, t, J ) 6.4 Hz); IR spectrum identical
with that of 7.
(S)-N-ter t-Bu tyl-2-ben zam ido-4-(3-eth oxy-5-m eth ylisox-
a zol-4-yl)bu tyr a m id e (13). A solution of 12 (0.40 g, 0.81
mmol) in 98% formic acid (20 mL) was stirred at 21 °C for 30
min and then at 60 °C for 2.5 h. After the mixture cooled to
21 °C and was concentrated in vacuo, the residue was purified
by CC [tol-EtOAc (3:1)] to give 13 (0.20 g, 64%): mp 142-
143 °C; [R]23D ) -3.9°, [R]23365 ) -17.9° (c ) 0.38, EtOH); 97%
The mother liquor furnished after recrystallization (H2O/
EtOH) an additional 9.2 mg of 6 (total yield: 84%) with an ee
of 99.0%: mp 208-210 °C dec; IR spectrum identical with that
of the first crop of crystals of 6 and with that of 7. The solution
used for optical rotation in 0.1 M HCl was evaporated once
and recrystallized from AcOH to give 2.9 mg of 6‚HCl. The
IR spectrum was identical with that of 6‚HCl obtained after
deprotection of compound 13.
Cell Cu ltu r e. The Chinese hamster ovary (CHO) cell lines
expressing mGlu1R, mGlu2, mGlu3, mGlu4a, mGlu5, mGlu6, and
mGlu7 were maintained as described previously.29-34 The cell
lines were grown in a humidified 5% CO2/95% air atmosphere
at 37 °C in DMEM containing a reduced concentration of (S)-
glutamine (2 mM), 1% proline, penicillin (100 U/mL), strep-
tomycin (100 mg/mL), and 10% dialyzed fetal calf serum (all
GIBCO, Paisley, Scotland). Two days before assay 1.8 × 106
cells were divided into the wells of 24-well plates.
1
ee; H NMR (CDCl3) δ 1.35 (12H, s and t, J ) 7.1 Hz), 2.05
(2H, m), 2.20 (3H, s), 2.36 (2H, m), 4.24 (2H, q, J ) 7.1 Hz),
4.60 (1H, dd, J ) 12.7, 6.0 Hz), 6.40 (1H, s), 7.45 (5H, m); 13
C
NMR (CDCl3) δ 11.4, 14.6, 17.1, 28.6, 31.6, 51.6, 53.2, 65.4,
103.4, 127.0, 128.3, 128.5, 130.0, 131.8, 133.7, 165.8, 167.2,
170.4, 170.6. Anal. (C21H29N3O4‚0.25H2O) C, H, N.
(R)-N-ter t-Bu tyl-2-ben zam ido-4-(3-eth oxy-5-m eth ylisox-
a zol-4-yl)bu tyr a m id e (14). Compound 14 (0.11 g, 63%) with
ee ) 96% was synthesized from 11 (0.23 g, 0.45 mmol) by a
method similar to that used for compound 13. Compound 14:
mp 141-143 °C; [R]23 ) +3.3°, [R]23
) +16.0° (c ) 0.36,
D
365
EtOH); 96% ee; 1H and 13C NMR spectra of 14 were identical
to those obtained for compound 13.
Mea su r em en t of P I Hyd r olysis a n d Cyclic AMP F or -
m a tion . PI hydrolysis was essentially measured as described
previously.23-25 Briefly, the cells were labeled with [3H]inositol
(2 µCi/mL) 24 h prior to the assay. For agonist assay, the cells
were incubated with ligand dissolved in PBS-LiCl for 20 min,
(S)-2-Am in o-4-(3-h yd r oxy-5-m et h ylisoxa zol-4-yl)b u -
tyr ic Acid [(S)-Hom o-AMP A, 6], Hyd r och lor id e. A solu-
tion of 13 (0.20 g, 0.52 mmol) in 6 M HCl (15 mL) was heated
at 105-110 °C for 16 h, then cooled to 21 °C, washed with 3
× 15 mL of CH2Cl2, and concentrated in vacuo to give 100 mg
of crude product with an ee of 62%. Recrystallization from
AcOH gave 50 mg of colorless crystals with an ee of 88%. This
procedure was repeated three times to afford 20 mg of 6‚HCl
3
and agonist activity was determined by measurement of H-
labeled mono-, bis-, and tris-inositol phosphates by ion ex-
change chromatography. For antagonist assay, the cells were
preincubated with the ligand dissolved in PBS-LiCl for 20
min prior to incubation with ligand and 30 µM (S)-Glu (1).
The antagonist activity was then determined as the inhibitory
effect of the (S)-Glu (1)-mediated response. The assay of cyclic
AMP formation was performed as described previously.23-25
Briefly, the cells were incubated for 10 min in PBS containing
the ligand, 10 µM forskolin, and 1 mM 3-isobutyl-1-methyl-
xanthine (IBMX) (Sigma Chemicals, St. Louis, MO). The
agonist activity was then determined as the inhibitory effect
of the forskolin-induced cyclic AMP formation. For antagonist
assay, the cells were preincubated with ligand dissolved in PBS
containing 1 mM IBMX for 20 min prior to a 10 min incubation
in PBS containing the ligand, 30 µM (S)-Glu (1) [in the case
of mGlu7, 1 mM (S)-AP4 was used], 10 µM forskolin, and 1
mM IBMX. Cyclic AMP levels were determined by use of a
RIA assay (Amersham). All experiments were performed at
least twice in triplicate.
in 20% yield with an ee of 99%: mp 164-165 °C dec; [R]23
)
D
+27.9°, [R]23 ) +103° [c ) 0.408 (concentration of zwitte-
365
rion), 0.1 M HCl]; 1H NMR (D2O) δ 2.12 (2H, m), 2.22 (3H, s),
2.43 (2H, t, J ) 7.4 Hz), 4.00 (1H, t, J ) 6.4 Hz); 13C NMR
(D2O) δ 11.7, 17.0, 29.7, 53.4, 105.4, 169.9, 171.3, 173.2; IR
3450 (m), 3200-2900 (s), 1720 (m), 1620 (s), 1520 (s) cm-1
.
Cr u d e (S)-2-Am in o-4-(3-h yd r oxy-5-m eth ylisoxa zol-4-
yl)bu tyr ic Acid [(S)-Hom o-AMP A, 6], Hyd r obr om id e. A
solution of 12 (5 mg, 0.40 mmol) in 48% aqueous HBr (0.5 mL)
was heated at 115 °C for 1.5 h, then cooled to 20 °C, and
concentrated in vacuo to give 4 mg of crude 6‚HBr with an ee
of 45%. This crude product was redissolved in 48% aqueous
HBr, and after heating at 115 °C for 3 h, this solution was
evaporated in vacuo to give crude 6‚HBr with an ee of 45%.
This sample of crude 6‚HBr, which cochromatographed [Crown-
pak CR(-)] with 6‚HCl prepared from compound 13, was not
worked up.
(R)-2-Am in o-4-(3-h yd r oxy-5-m et h yl-4-isoxa zolyl)b u -
tyr ic Acid [(R)-(-)-Hom o-AMP A, 7]. Homo-AMPA12 (173
mg) was dissolved in 15 mM aqueous AcOH (34.5 mL) by
ultrasonication and heating and filtered through a 0.45 µm
membrane filter (Millex-HV, Millipore). This amount of Homo-
AMPA was resolved in 20 injections, each of 1.0-2.0 mL of
solution. The collected fractions from each of the two peaks
were pooled and evaporated. These two residues were re-
evaporated twice from water and dried in vacuo. The pooled
Electr op h ysiology in Vitr o. A rat cortical wedge prepa-
ration26 was used for the determination of the depolarizing
effects of the EAA amino acid analogues under study, as
described previously.12
Ack n ow led gm en t. This work was supported by a
postdoctoral grant to H.B.-O. from H. Lundbeck A/S, by
the Lundbeck Foundation, and by the Danish State
Biotechnology Programme (1991-1995). The collabora-

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3705
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