A new highly selective metabotropic excitatory amino acid agonist: 2- Amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid

Article abstract of DOI:10.1021/jm9602569

The homologous series of acidic amino acids, ranging from aspartic acid (1) to 2-aminosuberic acid (5), and the corresponding series of 3-isoxazolol bioisosteres of these amino acids, ranging from (RS)-2-amino-2-(3-hydroxy-5- methylisoxazol-4-yl)acetic acid (AMAA, 6) to (RS)-2-amino-6-(3-hydroxy-5- methylisoxazol-4-yl)hexanoic acid (10), were tested as ligands for metabotropic excitatory amino acid receptors (mGlu(1α), mGlu₂, mGlu(4a), and mGlu₆). Whereas AMAA (6) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol- 4-yl)propionic acid (AMPA, 7) are potent and highly selective agonists at N- methyl-D-aspartic acid (NMDA) and AMPA receptors, respectively, the higher homologue of AMPA (7), (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4- yl)butyric acid (homo-AMPA, 8), is inactive at ionotropic excitatory amino acid receptors. Homo-AMPA (8), which is a 3-isoxazolol bioisostere of 2- aminoadipic acid (3), was, however, shown to be a specific and rather potent agonist at mGlu₆, approximately 4 times weaker than the nonselective excitatory amino acid receptor agonist (S)-glutamic acid. 2-Aminoadipic acid (3), which shows a complex excitatory amino acid synaptic pharmacology, was an agonist at mGlu₆ as well as mGlu₂. AMPA (7) and the higher homologue of homo-AMPA (8), (RS)-2-amino-5-(3-hydroxy-5-methylisoxazol-4-yl)pentanoic acid (9), showed relatively weak agonist effects at mGlu₆. It is concluded that homo-AMPA (8) is likely to be a useful tool for studies of the pharmacology and physiological role of mGlu₆. We describe a new versatile synthesis of this homologue of AMPA and the synthesis of compound 10.

Full text of DOI:10.1021/jm9602569

3188
J . Med. Chem. 1996, 39, 3188-3194
A New High ly Selective Meta botr op ic Excita tor y Am in o Acid Agon ist:
2-Am in o-4-(3-h yd r oxy-5-m eth ylisoxa zol-4-yl)bu tyr ic Acid
Hans Bra¨uner-Osborne,† Frank A. Sløk,† Niels Skjærbæk,† Bjarke Ebert,† Naohiro Sekiyama,‡
Shigetada Nakanishi,‡ and Povl Krogsgaard-Larsen*,†
PharmaBiotec Research Center, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy,
DK-2100 Copenhagen, Denmark, and Department of Biological Sciences, Kyoto University, Faculty of Medicine,
Yosida, Sakyo-ku, Kyoto 606, J apan
Received April 2, 1996X
The homologous series of acidic amino acids, ranging from aspartic acid (1) to 2-aminosuberic
acid (5), and the corresponding series of 3-isoxazolol bioisosteres of these amino acids, ranging
from (RS)-2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid (AMAA, 6) to (RS)-2-amino-
6-(3-hydroxy-5-methylisoxazol-4-yl)hexanoic acid (10), were tested as ligands for metabotropic
excitatory amino acid receptors (mGlu1R, mGlu2, mGlu4a, and mGlu6). Whereas AMAA (6) and
(RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA, 7) are potent and highly
selective agonists at N-methyl-D-aspartic acid (NMDA) and AMPA receptors, respectively, the
higher homologue of AMPA (7), (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid
(homo-AMPA, 8), is inactive at ionotropic excitatory amino acid receptors. Homo-AMPA (8),
which is a 3-isoxazolol bioisostere of 2-aminoadipic acid (3), was, however, shown to be a specific
and rather potent agonist at mGlu6, approximately 4 times weaker than the nonselective
excitatory amino acid receptor agonist (S)-glutamic acid. 2-Aminoadipic acid (3), which shows
a complex excitatory amino acid synaptic pharmacology, was an agonist at mGlu6 as well as
mGlu2. AMPA (7) and the higher homologue of homo-AMPA (8), (RS)-2-amino-5-(3-hydroxy-
5-methylisoxazol-4-yl)pentanoic acid (9), showed relatively weak agonist effects at mGlu6. It
is concluded that homo-AMPA (8) is likely to be a useful tool for studies of the pharmacology
and physiological role of mGlu6. We describe a new versatile synthesis of this homologue of
AMPA and the synthesis of compound 10.
In tr od u ction
acid [(1S,3R)-ACPD] and (2S,1′R,2′R,3′R)-2-(2,3-dicar-
boxycyclopropyl)glycine (DCG-IV).8,12,13 Group III com-
prises mGlu4, mGlu6, mGlu7, and mGlu8, which also are
coupled to inhibition of cyclic AMP formation and are
selectively activated by (S)-2-amino-4-phosphonobutyric
acid (L-AP4).9-11,13
(S)-Glutamic acid [(S)-Glu, (S)-2] and perhaps also (S)-
aspartic acid [(S)-1] are the major excitatory neu-
rotransmitters in the central nervous system and play
an important role in many natural processes such as
neural plasticity, memory, and neurotoxicity.1-4 On the
basis of pharmacology, electrophysiology, and molecular
cloning, the excitatory amino acid (EAA) receptors
operated by (S)-Glu have been classified into two major
classes: the ionotropic glutamic acid (iGlu) receptors
and the metabotropic glutamic acid (mGlu) receptors
belonging to the superfamily of G protein-coupled recep-
tors.4 Whereas 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 heterogeneous, eight different sub-
types of mGlu receptors have, so far, been cloned.5-11
On the basis of pharmacology, signal transduction
pathways, and sequence homology, the mGlu receptors
have been subdivided into three groups (I-III). Group
I consists of mGlu1 and mGlu5, which are coupled to the
hydrolysis of phosphatidylinositol (PI) and are selec-
tively activated by quisqualic acid.5-7 mGlu2 and
mGlu3, which constitute group II, are coupled to inhibi-
tion of cyclic AMP formation and are selectively acti-
vated by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic
A prerequisite for the determination of the physi-
ological role and pharmacological importance of the
mGlu receptors is the availability of highly selective
agonists and antagonists for each subtype of this
heterogeneous family of EAA receptors.14,15 Recently,
2-amino-3-(4-bromo-3-hydroxyisoxazol-5-yl)propionic acid
(Br-HIBO), which is a potent AMPA receptor agonist,16
has been shown to be a mGlu receptor antagonist.17 Like
1-aminoindan-1,5-dicarboxylic acid,18 Br-HIBO reduces
the function of mGlu1 without affecting significantly
other mGlu receptors.17 However, for the other sub-
types of mGlu receptors, no potent and selective an-
tagonists have yet been reported.
We have previously shown that 2-amino-2-(3-hydroxy-
5-methylisoxazol-4-yl)acetic acid (AMAA, 6), a 3-isox-
azolol bioisostere of aspartic acid (1), is a specific agonist
at NMDA receptors,19 whereas AMPA (7), bioisosteri-
cally derived from glutamic acid (2) (Figure 1), is a
highly selective AMPA receptor agonist.16 Along this
line of EAA receptor ligand design, we have synthesized
and pharmacologically characterized the two higher
homologues of AMPA (7), i.e., (RS)-2-amino-4-(3-hy-
droxy-5-methylisoxazol-4-yl)butyric acid (homo-AMPA,
8)20 and (RS)-2-amino-5-(3-hydroxy-5-methylisoxazol-4-
yl)pentanoic acid (9).21 Whereas homo-AMPA (8) is
completely devoid of effects at the iGlu receptors,20
* Corresponding author. Phone: (+45) 35 37 67 77, ext. 247. Fax:
(+45) 35 37 22 09.
† The Royal Danish School of Pharmacy.
‡ Kyoto University.
X Abstract published in Advance ACS Abstracts, J uly 1, 1996.
S0022-2623(96)00256-7 CCC: $12.00 © 1996 American Chemical Society

Homo-AMPA: A Highly Selective mGlu Receptor Agonist
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3189
Sch em e 1a
F igu r e 1. Structures of the acidic amino acids and corre-
sponding 3-isoxazolol bioisosteres tested in this study.
compound 9 was shown to be a very weak AMPA
receptor agonist.21
We here report the results of a comparative study of
the effects of the homologous series of amino acids
ranging from aspartic acid (1) to 2-aminosuberic acid
(5) and the corresponding series of 3-isoxazolol amino
acids ranging from AMAA (6) to (RS)-2-amino-6-(3-
hydroxy-5-methylisoxazol-4-yl)hexanoic acid (10) (Fig-
ure 1) on membrane-bound iGlu receptors and repre-
sentative subtypes of recombinant mGlu receptors. As
a notable result of this study, homo-AMPA (8) was
shown to be a highly selective agonist at mGlu6, 2 times
as potent as the S-form of its amino acid analogue,
2-aminoadipic acid (3) (Figure 1), which was shown also
to be an agonist at mGlu2. As part of this study we
report the synthesis of compound 10, which was phar-
macologically inactive, and an improved synthesis of
homo-AMPA (8).
a
Reagents: (a) 1,3,5-trioxane, HBr (62%), then MeOH; (b)
AcNHCH(COOCH3)2, NaH, DMF; (c) CF3COOH (1 M), IRA-400;
(d) NaOEt, NaI; (e) NH2OH‚HCl, NaOH, then HCl; (f) NaOH,
DMF, then EtI; (g) AlH[CH2CH(CH3)2]2 (1 M), toluene; (h) KCN,
(NH4)2CO3, MeOH/H2O; (i) HCl (6 M).
Ch em istr y
The potential utility of homo-AMPA (8) for pharma-
cological studies of mGlu6 prompted us to develop a new
and effective synthesis of this compound (Scheme 1, top)
to replace the earlier described low-yield procedure.20
The key steps in this new reaction sequence are the
transformations of 3-hydroxy-4-(2-hydroxyethyl)-5-
methylisoxazole (11)22 into compound 12 and subse-
quently into the precursor for 8, compound 13. This
reaction was optimized to provide 13 in 62% yield,
though with the formation of compound 14 (14%) as an
unvoidable side reaction. Compound 13 was easily sep-
arated from 14 and transformed into homo-AMPA (8).
The synthesis of the new compound, 10, is also shown
in Scheme 1 (bottom). The key steps in this reaction
sequence are the regioselective conversion of 16 into the
3-isoxazolol 17 by treatment with the binucleophile hy-
droxylamine and the reduction of the ester group of 18
into the aldehyde group in 19. These reactions were
optimized to provide respectively 17 in 63% yield and
19 in a yield of 94%. The decomposition of the hydan-
toin nucleus of 20 to an R-amino acid function and the
deprotection of the 3-isoxazolol unit were accomplished
in one reaction step to provide 10 in 34% yield.

3190 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Bra¨uner-Osborne et al.
Ta ble 1. Agonist Activities of the 3-Isoxazolol Amino Acid
Bioisosteres at Ionotropic Excitatory Amino Acid Receptors
IC50 (µM)a
EC50 (µM)a
[3H]kainic cortical
compd [3H]CPP [3H]AMPA [3H]MK-801
acid
wedge
6d
4.5 >100
0.02
76
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
12b
(S)-7e >100
3.5c
(R)-7e >100
580c
>1000
540c
8f
>100
>100
>100
>100
9g
10
49
>100
>1000
a
b
Mean of three to four experiments. NMDA agonist. c AMPA
agonist. From ref 19. e From ref 23. f From ref 20. From ref 21.
d
g
In Vitr o P h a r m a cology
Activity a t iGlu Recep tor s. In Table 1 the effects
of the homologous series of 3-isoxazolol amino acids,
AMAA (6)-10, at membrane-bound NMDA, AMPA, and
kainic acid receptors are summarized. The earlier
reported specific NMDA agonist effect of AMAA (6)19
and the highly stereoselective AMPA agonist action of
the S-form of AMPA (7)23 are emphasized. With the
exception of the very weak AMPA agonist effect of 9,21
the three higher homologues of AMPA tested did not
show significant effect at iGlu receptors.
Activity a t m Glu Recep tor s. The eight subtypes
of mGlu receptors can be divided in three groups based
on pharmacology, signal transduction pathway, and
sequence homology: mGlu1/mGlu5, mGlu2/mGlu3, and
mGlu4/mGlu6/mGlu7/mGlu8. In this study, we have
chosen mGlu1R, mGlu2, and mGlu4a/mGlu6 as represen-
tatives for these three groups. Initially, we tested the
compounds depicted in Figure 1 as racemates or enan-
tiomers, when available, at 1 mM concentration in the
three cell lines. In mGlu1R-expressing cells, (S)-Glu
increased the total level of inositol phosphates about
6-fold (Figure 2a) with an EC50 value of 19 ( 3 µM.
However, none of the remaining compounds showed any
significant agonist activity at mGlu1R. At the mGlu2-
expressing cell line, (S)-Glu inhibited the forskolin-
stimulated cyclic AMP level to 10-20% of control levels
(Figure 2b). Of all the tested compounds, only (S)-2-
aminoadipic acid [(S)-3] displayed any significant ago-
nist activity and was able to inhibit the forskolin-
stimulated cyclic AMP level to the same extent as (S)-
2, indicating full agonism (Figure 2b). When examined
in greater detail (S)-3 had an EC50 value of 35 ( 1 µM,
some 3-4-fold higher than that of (S)-Glu (see Figure
3a and Table 2). This is in agreement with previously
published results using mGlu2-expressing baby hamster
kidney cell. However, in that study, (S)-3 was about
80 times weaker than (S)-Glu.24 Finally, we tested the
compounds at mGlu6-expressing cells. (S)-Glu inhibited
the forskolin-stimulated cyclic AMP level to 40-50% of
control levels (Figure 2c). Of the tested compounds, (S)-
3, (S)-7, and homo-AMPA (8), as well as compound 9,
were able to inhibit the forskolin-stimulated cyclic AMP
formation to approximately the same extent as (S)-Glu
(Figure 2c). Dose-response curves of these compounds
revealed a rank order of potency of (S)-Glu > 8 g (S)-3
g (S)-7 g 9 (Figure 3b and Table 2). The effect of (S)-7
is in agreement with a previous report in which (RS)-7
at 1 mM could inhibit 50% of the response.9 On our
cell line, (S)-3 was some 7-fold weaker than (S)-Glu.
(S)-3 has previously been tested in mGlu4-expressing
BHK cells and found to be inactive even in 1 mM
F igu r e 2. Agonist activities of the acidic amino acids and
corresponding 3-isoxazolol bioisosteres at CHO cells expressing
mGlu1R, mGlu2, and mGlu6. (a) mGlu1R-expressing cells were
incubated with ligands at a concentration of 1 mM for 20 min.
Total IP formation was determined by ion-exchange assay and
the fold increase in IP level calculated compared to control
cells (incubated in buffer only). (b and c) mGlu2- and mGlu6-
expressing cells were incubated with ligands (1 mM) for 10
min in the presence of 10 µM forskolin, and cyclic AMP levels
were measured by a RIA assay (Amersham). Cyclic AMP
levels in control cells (incubated in buffer only) were set to
100%. Data are the means ((SD) of representative experi-
ments performed in triplicate.
concentration.24 Thus, there appears to be a disagree-
ment between these two results, since both mGlu4 and
mGlu6 belong to group III and previously have been
shown to exhibit very similar pharmacology.
In order to investigate this apparent disagreement,
we tested all compounds that had shown mGlu6 activity
in mGlu4a-expressing cells. As seen in Figure 4, (S)-
Glu inhibited the forskolin-stimulated cyclic AMP for-
mation to 50-60% of control levels, whereas none of the
other compounds tested, even at a concentration of 1
mM, had any measurable effect. Thus, the discrepancy
appears to be the result of receptor subtype selectivity
rather than a result of using different cell lines.
We also tested the compounds showing no significant
agonist effect as antagonists at the mGlu receptors.

Homo-AMPA: A Highly Selective mGlu Receptor Agonist
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3191
F igu r e 3. Dose-response curves for agonists at CHO cells expressing mGlu2 (a) or mGlu6 (b). Cells were incubated with glutamic
acid (b), (S)-2-aminoadipic acid [(S)-3] (9), (S)-AMPA [(S)-7] (2), homo-AMPA (8) (O), and compound 9 (0). For further details,
see Figure 2.
Ta ble 2. Agonist Activities of the Tested Ligands at
Metabotropic Excitatory Amino Acid Receptors Expressed in
CHO Cells
EC50 (µM)a
compd
mGlu1R
>1000
>1000
19 ( 3
mGlu2
>1000
>1000
mGlu4a
mGlu6
>1000
>1000
20 ( 4
>1000
140 ( 35
>1000
>1000
>1000
>1000
220 ( 83
>1000
82 ( 15
410 ( 110
>1000
(S)-1
(R)-1
(S)-Glu
(R)-Glu >1000
(S)-3
(R)-3
4
5
6
(S)-7
(R)-7
8
9
10
nd
nd
8.5 ( 0.1 15 ( 6
>1000
35 ( 1
>1000
nd
>3000
nd
nd
nd
nd
>3000
nd
>3000
>3000
nd
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
F igu r e 4. Agonist and antagonist activities at CHO cells
expressing mGlu4a of ligands with significant agonist activities
at mGlu6-expressing cells. In agonist assays, cells were
incubated with glutamic acid (1 mM) or test ligands (1 mM)
for 10 min in the presence of 10 µM forskolin. In antagonist
assays, cells were preincubated with ligand (1 mM) for 20 min
and then incubated with ligand (1 mM) for 10 min in the
presence of 50 µM (S)-Glu and 10 µM forskolin. For further
details, see Figure 2.
a
Mean ( standard error of mean of at least two independent
experiments.
However, as exemplified in Figure 4, none of these
compounds had any significant antagonist effect.
Discu ssion
effects at mGlu receptors was (S)-2-aminoadipic acid
[(S)-3], the higher homologue of (S)-Glu. (S)-3 was
shown to be an agonist at mGlu2 and mGlu6 but was
inactive at mGlu1R and mGlu4a, indicating that this
amino acid is capable of discriminating between the
mGlu receptors of group III.
We have previously reported the design and pharma-
cology of specific agonists for iGlu receptors using the
nonselective 3-isoxazolol amino acid ibotenic acid as a
lead structure. Thus, the 3-isoxazolol bioisostere of
aspartic acid (1), AMAA (6), is a specific NMDA ago-
nist,19 whereas the corresponding glutamic acid (2)
analogue, AMPA (7), is a highly selective AMPA recep-
tor agonist16 (Figure 1). Within the framework of this
EAA ligand design project, the higher homologue of
AMPA (7), homo-AMPA (8), was shown to be completely
inactive at iGlu receptors,20 whereas the next homo-
logue, compound 9, is a very weak agonist at AMPA
receptors21 (Table 1).
These observations represent a new facet of the
complex pharmacology of 3, the R-form of which shows
NMDA antagonist effect,2,25 whereas the S-form is an
inhibitor of EAA uptake mechanism(s).26,27 Further-
more, 3 has previously been shown to protect against
kainic acid-induced neurotoxicity in the striatum28 and
the retina,29 regions which express mGlu230 and mGlu6,9
respectively. Finally, (S)-3 has been shown31 to antago-
nize the priming effect of quisqualic acid on depolar-
izations by (R)-Br-HIBO32 or L-AP4.33 The mecha-
nism(s) underlying these neuroprotective and antiprim-
ing effects of 3 are not very well understood29,31 and may
involve activation of mGlu receptors. A prerequisite for
testing this possible mechanism is the availability of
selective agonists for the subtypes of mGlu receptors.
In this context, the testing of 3-isoxazolol amino acids
on subtypes of mGlu receptors provided interesting
In light of these observations, we decided to perform
a comparative study of the homologous series of acidic
amino acids ranging from aspartic acid (1) to 2-amino-
suberic acid (5) and the respective 3-isoxazolol amino
acids ranging from AMAA (6) to compound 10 (Figure
1). On all subtypes of mGlu receptors tested (mGlu1R
,
mGlu2, mGlu4a, and mGlu6), (S)-Glu was the most
potent agonist (Figures 2-4). In addition to (S)-Glu,
the only member of this series of amino acids showing

3192 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Bra¨uner-Osborne et al.
Meth yl 2-Aceta m id o-2-(m eth oxyca r bon yl)-4-[2-(m eth -
oxym eth yl)-5-m eth yl-3-oxoisoxa zolin -4-yl]bu tyr a te (13)
a n d 4-Eth en yl-2-(m eth oxym eth yl)-5-m eth ylisoxa zolin -3-
on e (14). Dimethyl acetamidomalonate (5.20 g, 27.5 mmol)
was dissolved in dry DMF (180 mL), and a suspension (60%)
of sodium hydride (1.10 g, 27.5 mmol) in mineral oil was added.
The reaction mixture was stirred for 1 h, and a solution of 12
(6.30 g, 25.2 mmol) in dry DMF (20 mL) was added. This
reaction mixture was left at 22 °C for 14 days and then at 40
°C for 2 days. CH3COOH (5 mL) was added, and the reaction
mixture was evaporated. Water (200 mL) was added and the
mixture extracted with CH2Cl2 (4 × 150 mL). The organic
phase was dried (MgSO4) and evaporated, and the product was
subjected to CC [eluent: ethyl acetate-light petroleum (1:1)]
to provide 13 as a crystalline product (5.55 g, 62%), mp 79-
80 °C, and 14 as an oil (0.59 g, 14%). Data for 13: 1H NMR
(CDCl3, TMS): δ 2.09 (3H, s), 2.18 (2H, m), 2.21 (3H, s), 2.55
(2H, m), 3.40 (3H, s), 3.77 (6H, s), 5.11 (2H, s), 7.12 (1H, s).
13C NMR (CDCl3): δ 11.5, 15.4, 22.6, 30.9, 53.2, 56.8, 65.5,
75.0, 107.2, 166.0, 167.6, 168.0, 169.2. Anal. (C15H22N2O8) C,
H, N. Data for 14: 1H NMR (CDCl3, TMS): δ 2.31 (3H, s),
results. Thus, whereas (S)-AMPA [(S)-7] and homo-
AMPA (8), as well as compound 9, were inactive at
mGlu1R, mGlu2, and mGlu4a, all of these 3-isoxazolol
amino acids showed agonist effects at mGlu6. Within
this series of homologous amino acids, homo-AMPA (8)
was the most potent compound, showing activity some
4 times weaker than (S)-Glu (Table 2). Since homo-
AMPA (8) is the 3-isoxazolol analogue of 3 (Figure 1),
this structure-activity relationship indicates that mGlu6,
in contrast to mGlu2, is capable of accommodating the
3-isoxazolol unit as a bioisostere of the carboxyl group.
It may be postulated that the slightly reduced confor-
mational flexibility of homo-AMPA (8), as compared
with 3, can explain this selectivity.
In conclusion, homo-AMPA (8) has been shown to be
a very selective and rather potent agonist at mGlu6,
being inactive at iGlu receptors and at mGlu1R, mGlu2,
and mGlu4a. This observation is interesting in light of
the pharmacology of structurally related 3-isoxazolol
amino acids. Thus, the lower homologues AMAA (6)
and AMPA (7) are highly selective agonists at NMDA
and AMPA receptors, respectively, whereas the higher
homologue 9 is a weak AMPA agonist, and the higher
homologue 10 is inactive. Although homo-AMPA (8) is
not as potent as L-AP4 (EC50 ) 0.9 µM at mGlu6),9 we
envisage that 8 will be a useful tool for studies of the
pharmacology and physiology of mGlu6 in the retina,
since 8, in contrast to L-AP4, shows selectivity within
group III mGlu receptors.
3.41 (3H, s), 5.15 (2H, s), 5.34 (1H, dd, J gem ) 3.1 Hz, J cis
)
10.3 Hz), 6.17 (1H, dd, J gem ) 3.1 Hz, J trans ) 17.6 Hz), 6.31
(1H, dd, J cis ) 10.3 Hz, J trans ) 17.6 Hz). 13C NMR (CDCl3):
δ 11.7, 56.9, 74.9, 107.2, 116.7, 122.4, 165.7, 166.1. Anal.
(C8H11NO3) C, H, N.
2-Am in o-4-(3-h yd r oxy-5-m et h ylisoxa zol-4-yl)b u t yr ic
Acid (Hom o-AMP A, 8). Compound 13 (1.85 g, 5.2 mmol) was
refluxed in 1 M aqueous CF3COOH (100 mL, 100 mmol) for
18 h. To the evaporated reaction product was added water
(25 mL), and the mixture was evaporated. This procedure was
repeated twice. A solution of the evaporated product in water
(15 mL) was passed through a column containing Amberlite
IRA-400 (OH- form, 60 mL). The column was washed with
water until the eluent was neutral. The product was eluted
with 1 M aqueous CH3COOH. The fractions containing
product were collected and evaporated. The crude product was
dissolved in water (50 mL) and the solution treated with
activated carbon, filtered, and left at 5 °C for crystallization.
The yield of homo-AMPA (8) was 451 mg (43%), mp 230 °C
dec. 1H NMR (D2O, dioxane, 1 M NaOD): δ 1.68 (2H, m), 2.11
(3H, s), 2.17 (2H, t, J ) 8.0 Hz), 3.22 (1H, t, J ) 6.5 Hz). 13C
NMR (D2O, dioxane, 1 M NaOD): δ 11.9, 18.2, 34.9, 56.5,
107.5, 166.6, 178.5, 183.1. Anal. (C8H12N2O4‚0.25H2O) C, H,
N.
Dieth yl 1-Acetylp en ta n e-1,5-d ica r boxyla te (16). Ethyl
acetoacetate (5.0 g, 38.4 mmol) was added to a solution of
sodium ethoxide prepared from sodium (883 mg, 38.4 mmol)
and ethanol (100 mL), and the solution was heated to reflux.
Ethyl 5-bromovalerate (8.82 g, 42.2 mmol) was slowly added
together with sodium iodide (6.63 g, 42.2 mmol), and the
mixture was refluxed for 18 h. The mixture was evaporated
to dryness followed by distillation (0.1 mmHg, 200 °C) in a
Kugelrohr apparatus to produce 16 (8.53 g, 86.0%). 1H NMR
(CDCl3, TMS): δ 1.28 (6H, dt, J ) 7.2, 1.8 Hz), 1.48-1.71 (4H,
m), 1.86 (2H, quintet, J ) 7.2 Hz), 2.23 (3H, s), 2.30 (2H, t, J
) 7.2 Hz), 3.42 (1H, t, J ) 7.2 Hz), 4.12 (4H, dq, J ) 7.2, 1.8
Hz). 13C NMR (CDCl3): δ 13.9, 14.0, 24.4, 26.6, 27.5, 28.6,
33.7, 59.3, 60.0, 61.1, 169.5, 173.1, 203.1. Anal. (C13H22O5)
C, H.
Eth yl 5-(3-Hyd r oxy-5-m eth ylisoxa zol-4-yl)p en ta n oa te
(17). A solution of hydroxylammonium chloride (3.50 g, 50.3
mmol) in ethanol (20 mL) and water (1 mL) was added to a
solution of sodium hydroxide (1.06 g, 26.4 mmol) in ethanol
(20 mL) and water (1 mL) at 60 °C. This reaction mixture
was filtered and, after cooling to -60 °C, added to a solution
of 16 (6.50 g, 25.2 mmol) in ethanol (5 mL). Stirring was
continued at -60 °C for 2 h. Acetone (1.85 mL, 25.2 mmol)
was added to the reaction mixture followed by addition of a
80 °C solution of concentrated HCl (10 mL) and water (20 mL),
additional stirring at 80 °C for 30 min, evaporation to dryness,
extraction with CH2Cl2 (3 × 50 mL), addition of acetyl chloride
(10 mL) in ethanol (90 mL), refluxing for 3 h, and evaporation
to dryness followed by reevaporation three times with toluene.
The residue was subjected to CC [eluent: toluene-ethyl
acetate (1:1)] which produced 17 as a crystalline product (3.6
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. The structures of all
new compounds were established by infrared (IR), 1H and 13C
NMR spectroscopy, and elemental analyses. IR spectra were
recorded on a Perkin-Elmer 781 grating infrared spectropho-
tometer. 1H and 13C NMR spectra were recorded at 200 and
50.32 MHz, respectively, on a Bruker AC-200 instrument, and
chemical shift (δ) values are in parts per million with respect
to an internal standard. Column chromatography (CC) was
performed using silica gel 60, 0.063-0.200 mm (Merck).
Analytical and preparative thin layer chromatography (TLC)
was performed using silica gel 60 PF254. Compounds contain-
ing the 3-isoxazolol unit were visualized on TLC plates using
ultraviolet light and a FeCl3 spraying reagent, and amino acids
were visualized using a ninhydrin spraying reagent. Solvents
were removed in vacuo by rotary evaporation at 15 mmHg.
Melting points were determined in capillary tubes and are
uncorrected. Elemental analyses were performed by Mr. G.
Cornali, Microanalytical Laboratory, Leo Pharmaceutical Prod-
ucts, Denmark, and were within (0.4% of the calculated
values.
4-(2-Br om oeth yl)-2-(m eth oxym eth yl)-5-m eth ylisoxa zo-
lin -3-on e (12). A solution of compound 1122 (4.29 g, 30 mmol)
in aqueous HBr (80 mL, 62%) was heated to 60 °C for 18 h in
a sealed flask. The reaction mixture was cooled to 22 °C,
and 1,3,5-trioxane (4.05 g, 45 mmol) was added. The flask
was resealed and heated to 60 °C for 18 h. The reaction
mixture was extracted with CH2Cl2 (4 × 100 mL), CH3OH
(200 mL) was added to the organic phase, and this reaction
mixture was left at 22 °C for 30 min, after which CH2Cl2 (300
mL) and water (500 mL) were added. After extraction the
organic phase was separated, and the aqueous phase was
extracted with CH2Cl2 (100 mL). The combined CH2Cl2 phases
were washed with water (2 × 250 mL), dried (MgSO4), and
evaporated to give crude 12 (8.4 g), which was subjected to
CC [eluent: ethyl acetate-light petroleum (1:1)] to give 12
(6.8 g, 91%) as a colorless oil. 1H NMR (CDCl3, TMS):
δ
2.29 (3H, s), 2.83 (2H, t, J ) 6.5 Hz), 3.39 (3H, s), 3.61
(2H, t, J ) 6.5 Hz), 5.14 (2H, s). 13C NMR (CDCl3): δ 11.6,
25.0, 30.9, 56.6, 74.7, 105.8, 166.9, 167.1. Anal. (C8H12NO3-
Br) C, H, N, Br.

Homo-AMPA: A Highly Selective mGlu Receptor Agonist
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3193
g, 63%), mp 57-58 °C. 1H NMR (CDCl3, TMS): δ 1.25 (3H, t,
J ) 6.8 Hz), 1.50-1.75 (4H, m), 2.25 (3H, s), 2.26-2.36 (4H,
m), 4.12 (2H, q, J ) 6.8 Hz), 11.71 (1H, s). 13C NMR (CDCl3):
δ 11.2, 14.0, 20.4, 24.2, 28.0, 33.7, 60.0, 105.0, 165.3, 170.2,
173.3. Anal. (C11H17NO4) H, N; C: calcd, 58.14; found, 58.89.
E t h yl 5-(3-E t h oxy-5-m et h ylisoxa zol-4-yl)p en t a n oa t e
(18). Compound 17 (2.97 g, 13.9 mmol) was dissolved in a
solution of sodium hydroxide (0.56 g, 13.9 mmol) in water (10
mL) followed by evaporation to dryness using an oil pump (0.05
mmHg). The residue was dissolved in dry DMF (10 mL) and
cooled to -40 °C. Ethyl iodide (1.69 mL, 20.9 mmol) was
added, and stirring was continued for 15 min at -40 °C.
Further stirring at 22 °C for 30 min and evaporation to dryness
followed by preparative TLC [eluent: toluene-ethyl acetate
(1:1)] produced 18 (2.3 g, 65%) as an oil. 1H NMR (CDCl3,
TMS): δ 1.25 (3H, t, J ) 7.2 Hz), 1.40 (3H, t, J ) 7.1 Hz),
1.45-1.72 (4H, m), 2.24 (3H, s), 2.25-2.36 (4H, m), 4.13 (2H,
q, J ) 7.1 Hz), 4.28 (2H, q, J ) 7.2 Hz). 13C NMR (CDCl3): δ
11.3, 14.1, 14.5, 20.5, 24.2, 28.2, 33.8, 60.1, 65.1, 104.3, 165.2,
170.8, 173.4. Anal. (C13H21NO4) C, H, N.
5-(3-Eth oxy-5-m eth ylisoxa zol-4-yl)p en ta n a l (19). Un-
der a nitrogen atmosphere, a solution of diisobutylaluminum
hydride (1 M, 3.92 mL, 3.92 mmol) was added to a solution of
18 (400 mg, 1.56 mmol) in dry toluene (6.7 mL). Stirring was
continued for 6 min, after which a saturated aqueous solution
of sodium potassium tartrate (4.15 mL) was added to quench
the reaction. Extraction with ether (4 × 25 mL) and evapora-
tion to dryness. CC of the residue [eluent: toluene-ethyl
acetate (3:1)] produced 19 (311 mg, 94%) as an oil. 1H NMR
(CDCl3, TMS): δ 1.40 (3H, t, J ) 7.1 Hz), 1.47-1.68 (4H, m),
2.24 (3H, s), 2.23-2.30 (2H, m), 2.47 (2H, dt, J ) 1.6, 7.1 Hz),
4.28 (2H, q, J ) 7.1 Hz), 8.76 (1H, t, J ) 1.6 Hz). 13C NMR
(CDCl3): δ 11.4, 14.6, 20.6, 21.3, 28.2, 43.4, 65.2, 104.2, 165.3,
170.8, 202.3. Anal. (C11H17NO3) C, H, N.
5-[4-(3-E t h oxy-5-m et h ylisoxa zol-4-yl)b u t yl]h yd a n t o-
in (20). Compound 19 (200 mg, 0.95 mmol), potassium
cyanide (616 mg, 9.46 mmol), ammonium carbonate (473 mg,
4.73 mmol), and CH3OH (50%, 3.5 mL) were refluxed for 16
h. Evaporation to dryness, extraction with ethyl acetate (3 ×
10 mL), and reevaporation produced a crude product, which
was purified by preparative TLC [eluent: toluene-ethyl
acetate (1:4)] to give 20 (105 mg, 39%) as colorless crystals,
mp 222.5-223.0 °C. 1H NMR (CDCl3, TMS): δ 1.39 (3H, t, J
) 7.1 Hz), 1.43-1.98 (6H, m), 2.23 (3H, s), 2.27 (2H, t, J ) 6.8
Hz), 4.09 (1H, dt, J ) 2.9, 4.5 Hz), 4.29 (2H, q, J ) 7.1 Hz),
6.38 (1H, s), 8.52 (1H, s). 13C NMR (CDCl3): δ 11.4, 14.6, 20.6,
24.2, 28.3, 31.3, 58.6, 65.3, 104.3, 157.3, 165.4, 171.0, 174.4.
Anal. (C13H19N3O4) C, H, N.
2-Am in o -6-(3-h y d r o x y -5-m e t h y lis o x a zo l-4-y l)h e x -
a n oic Acid (10). A solution of 20 (55 mg, 0.20 mmol) in HCl
(6 M, 4 mL) was heated to 100 °C for 24 h in a sealed ampule.
Evaporation of the solvent produced a crude product, which
was subjected to preparative TLC [eluent: acetonitrile-acetic
acid-water (8:1:1)] to give 10 (15 mg, 34%) after recrystalli-
zation from water, mp 198-199 °C dec. 1H NMR (D2O,
dioxane, 1 M NaOD): δ 1.28-1.60 (4H, m), 1.82-1.99 (2H,
m), 2.20 (3H, s), 2.23 (3H, s), 2.23 (2H, t, J ) 7.0 Hz), 4.02
(1H, t, J ) 6.3 Hz). 13C NMR (D2O, dioxane, 1 M NaOD): δ
11.7, 20.4, 24.3, 28.1, 30.3, 53.6, 67.4, 107.3, 169.4, 172.9.
Anal. (C10H16N2O4) C, H, N.
of 5 nM was used instead of 2 nM. [3H]Kainic acid binding
was performed as described by Braitman and Coyle38 with the
following modifications: The concentration of [3H]kainic acid
was 5 nM rather than 1 nM, and the reaction was terminated
by filtration through Whatman GF/B filters followed by
washing with ice cold 50 mM Tris-HCl buffer (2 × 5 mL, pH
7.1).
Electr op h ysiology in Vitr o. A rat cortical slice prepara-
tion for testing EAAs described by Harrison and Simmonds39
was used as modified by Wheatley.40 Wedges (500 µm thick)
of rat brain containing cerebral cortex and corpus callosum
were placed with the corpus callosum on a wick of an Ag/AgCl
electrode electrically insulated from the cortex part, which was
placed between two layers of nappy liner and constantly
perfused with a Mg2+-free oxygenated Krebs solution (at room
temperature). A reference electrode was placed in contact with
the nappy liner, and the potential difference between the
electrodes was recorded on an ABB SE120 chart recorder.
Standard and test compounds were dissolved in the superfu-
sion medium.
Cell Cu ltu r e. The Chinese hamster ovary (CHO) cell lines
expressing mGlu1R, mGlu2, mGlu4a, and mGlu6 have been
described previously.8,9,13,41 They were maintained at 37 °C
in a humidified 5% CO2 incubator in DMEM which contained
a reduced concentration of (S)-glutamine (2 mM) and was
supplemented with 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 12-well plates (PI assay)
or 24-well plates (cyclic AMP assay).
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 measured as described previ-
ously.42,43 Briefly, the cells were labeled with [3H]inositol (1
µCi/mL) 24 h prior to the assay. For agonist assay, the cells
were incubated with ligand dissolved in PBS-LiCl for 20 min,
and agonist activity was determined by measurement of the
level of 3H-labeled mono-, bis-, and tris-inositol phosphates by
ion-exchange chromatography. For antagonist assay, the cells
were preincubated with the ligand dissolved in PBS-LiCl for
20 min prior to incubation with ligand and 10 µM (S)-Glu for
20 min. The antagonist activity was then determined as the
inhibitory effect of the (S)-Glu-mediated response. The assay
of cyclic AMP formation was performed as described previ-
ously.42,43 Briefly, the cells were incubated for 10 min in PBS
containing the ligand, 10 µM forskolin, and 1 mM 3-isobutyl-
1-methylxanthine (IBMX) (both 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, (S)-Glu, 10
µM forskolin, and 1 mM IBMX. All experiments were per-
formed at least twice in triplicate.
Ack n ow led gm en t. This work was supported by the
Lundbeck Foundation and the Danish State Biotech-
nology Programme (1991-1995). The secretarial as-
sistance of Mrs. Anne Nordly is gratefully acknowl-
edged.
Recep tor Bin d in g Assa ys. The membrane preparation
used in the [3H]-4-(3-phosphonoprop-1-yl)piperazine-2-car-
boxylic acid ([3H]CPP) and [3H]-5-methyl-10,11-dihydro-5H-
dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) (two NMDA
receptor antagonist radioligands), [3H]AMPA, and [3H]kainic
acid binding assays was prepared according to the method of
Ransom and Stec.34 [3H]CPP binding was studied following a
published procedure,35 where termination of the assays was
accomplished using filtration through Whatman GF/B filters
[presoaked in 0.1% poly(ethylenimine)] using a Brandell
M-48R cell harvester rather than by centrifugation. [3H]-
AMPA binding was performed following a published procedure
by Honore´ and Nielsen.36 [3H]MK-801 binding to fully stimu-
lated membranes was performed essentially as described
earlier,37 although the incubation time was increased from 1
to 4 h, and, furthermore, a concentration of radioactive ligand
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