Synthesis and pharmacology of 3-isoxazolol amino acids as selective antagonists at group I metabotropic glutamic acid receptors

Article abstract of DOI:10.1021/jm000441t

Using ibotenic acid (2) as a lead, two series of 3-isoxazolol amino acid ligands for (S)-glutamic acid (Glu, 1) receptors have been developed. Whereas analogues of (RS)-2-amino-3-(3-hydroxy- 5-methyl-4-isoxazolyl)propionic acid [AMPA, (RS)-3] interact sel

Full text of DOI:10.1021/jm000441t

J . Med. Chem. 2001, 44, 1051-1059
1051
Syn th esis a n d P h a r m a cology of 3-Isoxa zolol Am in o Acid s a s Selective
An ta gon ists a t Gr ou p I Meta botr op ic Glu ta m ic Acid Recep tor s
Ulf Madsen,* Hans Bra¨uner-Osborne, Karla Frydenvang, Lise Hvene, Tommy N. J ohansen, Birgitte Nielsen,
Connie Sa´nchez,† Tine B. Stensbøl, Francois Bischoff, and Povl Krogsgaard-Larsen
Centre for Drug Design and Transport, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy,
Universitetsparken 2, DK-2100 Copenhagen, Denmark, and Department of Pharmacology, H. Lundbeck A/ S, Ottiliavej 9,
DK-2500 Valby, Denmark
Received October 10, 2000
Using ibotenic acid (2) as a lead, two series of 3-isoxazolol amino acid ligands for (S)-glutamic
acid (Glu, 1) receptors have been developed. Whereas analogues of (RS)-2-amino-3-(3-hydroxy-
5-methyl-4-isoxazolyl)propionic acid [AMPA, (RS)-3] interact selectively with ionotropic Glu
receptors (iGluRs), the few analogues of (RS)-2-amino-3-(3-hydroxy-5-isoxazolyl)propionic acid
[HIBO, (RS)-4] so far known typically interact with iGluRs as well as metabotropic Glu receptors
(mGluRs). We here report the synthesis and pharmacology of a series of 4-substituted analogues
of HIBO. The hexyl analogue 9 was shown to be an antagonist at group I mGluRs. The effects
of 9 were shown to reside exclusively in (S)-9 (Kb ) 30 µM at mGlu1 and Kb ) 61 µM at mGlu5).
The lower homologue of 9, compound 8, showed comparable effects at mGluRs, but 8 also was
a weak agonist at the AMPA subtype of iGluRs. Like 9, the higher homologue, compound 10,
did not interact with iGluRs, but 10 selectively antagonized mGlu1 (Kb ) 160 µM) showing
very weak antagonist effect at mGlu5 (Kb ) 990 µM). The phenyl analogue 11 turned out to be
an AMPA agonist and an antagonist at mGlu1 and mGlu5, and these effects were shown to
originate in (S)-11 (EC50 ) 395 µM, Kb ) 86 and 90 µM, respectively). Compound 9, administered
icv, but not sc, was shown to protect mice against convulsions induced by N-methyl-D-aspartic
acid (NMDA). Compounds 9 and 11 were resolved using chiral HPLC, and the configurational
assignments of the enantiomers were based on X-ray crystallographic analyses.
In tr od u ction
The main excitatory amino acid neurotransmitter in
the central nervous system (CNS), (S)-glutamic acid
(Glu, 1), operates through two main groups of receptors:
the ionotropic Glu receptors (iGluRs) and the metabo-
tropic Glu receptors (mGluRs).1-3 Each of these groups
of receptors is divided into three subgroups: the iGluRs
comprise the N-methyl-D-aspartic acid (NMDA), 2-
amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid
(AMPA), and kainic acid (KA) receptors, and the mGluR
subgroups are named I, II, and III. The iGluR subgroups
each consists of a number of subtypes forming homo-
or heteromeric receptors in tetra- or pentameric con-
figurations.4,5 The mGluR groups consist of the following
example, led to (S)-AMPA (3) and (S)-homoibotenic acid
[(S)-HIBO, 4].12-14 (S)-HIBO and the 4-alkyl-substituted
analogues 5 and 6 have previously been shown to be
agonists at AMPA receptors and antagonists at group I
mGluRs.15-17 On the other hand, no activity was ob-
served for the 4-octyl-substituted analogue of HIBO (7)
at iGluRs18 or mGluRs (results of this work).
individual subtypes: group I, mGlu1,5; group II, mGlu2,3
;
6,7
and group III, mGlu4,6-8
.
It is generally agreed that
iGluRs as well as mGluRs are important in the healthy
as well as the diseased CNS, and all subtypes of these
receptors are thus potential targets for therapeutic
intervention in a number of neurologic and psychiatric
diseases.8-10
These observations prompted us to synthesize com-
pounds 8-11 in order to study in detail the relationship
between the structure of the 4-substituents and the
pharmacology of these compounds. Two of these ana-
logues, compounds 9 and 11, were resolved by chiral
HPLC, and the absolute configuration was assigned on
the basis of X-ray crystallographic analyses. All com-
pounds synthesized have been tested pharmacologically
in receptor binding assays for iGluR activity, in an in
vitro electrophysiological model, and in second-mes-
A number of selective ligands have been developed
for iGluRs, notably for NMDA and AMPA receptors,
whereas relatively few selective ligands have been
reported for mGluRs.9,11 Ibotenic acid (2) has been used
as a lead in the search for selective agents at different
subtypes of GluRs, and these lines of research have, for
* To whom correspondence should be addressed. Phone: (+45) 35
30 62 43. Fax: (+45) 35 30 60 40. E-mail um@dfh.dk.
† H. Lundbeck A/S.
10.1021/jm000441t CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/13/2001

1052 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 7
Madsen et al.
Sch em e 2a
senger assays for mGlu1, mGlu2, mGlu4, and mGlu5
receptor activity. In addition compound 9 has been
tested for activity against NMDA-induced convulsions
in mice.
Resu lts
Syn th esis. The synthesis of all of the 4-alkyl-HIBO
analogues 8-10 started from methyl acetoacetate (12)
(Scheme 1). The alkyl substituents were introduced
using sodium methoxide and alkylation with the ap-
propriate alkyl bromide. Cyclization with hydroxyl-
amine afforded the 4-substituted 3-isoxazolols 14a -c,
which were subsequently converted into the O-ethyl-
protected analogues 15a -c. Deprotections were ac-
complished using concentrated hydrobromic acid. The
final products 8-10 all crystallized as zwitterions
directly after evaporation of the hydrobromic acid solu-
tions and subsequent re-evaporation with water. Com-
pounds 8-10 were all highly insoluble in water.
Compound 11 was synthesized from 5-methyl-4-
phenyl-3-isoxazolol (18)19 (Scheme 2), which in analogy
to the syntheses described above was O-ethyl protected
and then converted into the target compound 11 via
intermediates 20 and 21. However, the bromination of
19 was carried out with NBS, and the final product was
obtained as a zwitterion after adjustment of a solution
of the hydrobromide salt to pH 3-4 using triethylamine.
Ch r om a togr a p h ic Resolu tion . Compounds 9 and
11 were resolved by chiral HPLC using the Chirobiotic
T column,20 which shows good enantioseparation for
a
Reagents: (i) K2CO3, ethyl bromide; (ii) NBS; (iii) NaH/
(H5C2OOC)2CHNHCOCH3; (iv) 48% HBr.
naturally occurring amino acids as well as 3-isoxazolol
acidic amino acids.21,22 In both chromatographic resolu-
tions, volatile mobile phases could be used, thus facili-
tating the workup procedure of the pure enantiomers.
Upon crystallization, all four enantiomers were obtained
with high enantiomeric excess (ee g 98.7%). Determi-
nation of the enantiomeric excess of the late eluting (-)-
enantiomers on the Chirobiotic T column, (-)-9 and (-)-
11, was performed on an analytical column. To precisely
determine the enantiomeric excess of the two (+)-
enantiomers, (+)-9 and (+)-11, an (S)-pipecolic acid
ligand-exchange column and a Crownpak CR(+) col-
umn, respectively, were used. In general, both of these
columns show opposite elution order as compared to that
of the Chirobiotic T column.23,24
X-r ay Cr ystallogr aph ic An alyses. Perspective draw-
ings25 of the molecular structures of zwitterionic mono-
hydrates of (R)-(-)-9 and (R)-(-)-11 with atomic labeling
are depicted in Figure 1. The molecular geometry of the
two compounds is comparable, and only minor differ-
ences are observed in the molecules due to the different
substituents on carbon atom C4. The orientation of the
amino acid side chain is slightly different [(-)-9: O1-
C5-C6-C7 ) 67.0(2)°; (-)-11: O1-C5-C6-C7 )
47.8(2)°], the hexyl moiety of (-)-9 is observed in an
extended conformation, and the interplanar angle be-
tween the phenyl ring and the isoxazole ring of (-)-11
is 38.94(7)°.
Sch em e 1a
The unit cell and the crystal packing characteristics
of the two compounds are comparable. The crystal
packings of the two compounds are observed with
alternating hydrophobic and hydrophilic layers. The
hexyl and the phenyl moieties of 9 and 11, respectively,
are packed in the hydrophobic layers of the crystal
packing. Due to the difference in the size of these
substituents, the unit cell of (-)-9 is elongated along
the c-axis [(-)-9: c ) 35.331(5) Å; (-)-11: c ) 28.603(4)
Å]. In the hydrophilic layers the amino acid moieties,
the 3-isoxazolol rings, and the water molecules are
found.
Con figu r a tion a l Assign m en t. For (-)-9 the Flack
absolute structure26,27 parameter for the R-configuration
was calculated to x ) 0.00(18) and for the S-configura-
tion to x ) 0.96(16). For (-)-11 the Flack absolute
structure parameter for the R-configuration was calcu-
a
Reagents: (i) CH3ONa/CH3OH, alkyl bromide; (ii) NH2OH;
(iii) NaH, ethyl bromide; (iv) Br2/AcOH; (v) NaH/(R1OOC)2-
CHNHCOCH3; (vi) 48% HBr.

Synthesis/ Pharmacology of 3-Isoxazolol Amino Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 7 1053
Ta ble 1. Receptor Binding Affinities and Electrophysiological
Data from the Cortical Slice Model
IC50 (µM)
EC50 (µM)
compd
[3H]AMPA
0.8 ( 0.4 >100
[3H]CPP [3H]KA electrophysiology
(S)-HIBO (4)a
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
330 ( 45
18
17 ( 1
>500*
630 ( 80
>1000
>1000
>1000
>500*
590 ( 75
395 ( 45
>1000
5b
0.32
0.48
>100
11 ( 4
>100
>100
>100
>100
39 ( 3
23 ( 5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6c
7d
8
9
(S)-9
(R)-9
10
11
(S)-11
(R)-11
31 ( 4 >100
a
b
d
Ref 14. Ref 15. c Ref 17. Ref 18. Data represent the mean
( SEM of at least three independent results. *Estimated value.
Due to very low solubility compounds 7 and 10 were only tested
at 200 µM, at which concentration no significant responses were
observed.
types of chiral columns,17,21-24,28,29 the observed elution
orders for the two pairs of enantiomers under investiga-
tion are in accordance with the assignments based on
the crystallographic analyses.
Additional information on the configuration of the
isolated isomers was obtained from the circular dichro-
ism (CD) spectra. In analogy to what has been shown
for a number of structurally similar compounds, namely
(S)-(+)-HIBO (4), (S)-(+)-Br-HIBO, (S)-(+)-Bu-HIBO (6),
and (S)-(+)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)-
propionic acid [(S)-(+)-APPA] (compounds with known
absolute configuration from heavy atom method X-ray
crystallographic analyses),14,17,28,30 compounds (+)-9 and
(+)-11 show a positive Cotton effect at 215-220 nm in
acidic solution, strongly supporting the assignment of
both these compounds as having the S-configuration.
Recep tor Bin d in g Affin ities. The affinities for
iGluRs were determined using [3H]AMPA,31 [3H]-3-(2-
carboxy-4-piperazinyl)propyl-1-phosphonic acid32 ([3H]-
CPP), and [3H]KA33 as radioligands for AMPA, NMDA,
and KA receptors, respectively. (S)-HIBO (4), 5, and 6
have previously been shown to possess moderate affinity
for the [3H]AMPA binding site (Table 1). Compounds 8
and 11 showed weak affinity for the [3H]AMPA binding
site, the latter with the S-form as the active one. None
of the other compounds synthesized showed significant
affinity for the [3H]AMPA binding site. Only (R)-11
showed detectable affinity for the [3H]CPP binding site,
whereas all other compounds showed no affinity (IC50
> 100 µM) for this binding site or for the [3H]KA binding
site.
In Vitr o Electr op h ysiology. The excitatory activity
of all compounds were investigated in the rat cortical
slice model34 (Table 1). Compounds 515 and 617 have
previously been shown to express potent agonist activity
at AMPA receptors, whereas (S)-HIBO (4) is a weak
agonist14 and 7 is inactive.18 However, due to very low
solubility in the buffer solution, 7 could only be tested
up to 200 µM, at which concentration no significant
activity was observed. Compounds 8, 11, and (S)-11
were found to be weak agonists at AMPA receptors,
whereas 9, (S)-9, (R)-9, 10, and (R)-11 were found to be
inactive (EC50 > 1 mM). In analogy with 7, compound
10 was, due to low solubility, tested at concentrations
up to 200 µM. All compounds showing no agonist
F igu r e 1. Perspective drawings25 of (R)-9 (top) and (R)-11
(bottom). Displacement ellipsoids enclose 50% probability.
Hydrogen atoms are represented by spheres of arbitrary size.
The hydrogen bonds observed between the compounds and the
water molecules are indicated by thin lines.
lated to x ) 0.00(17) and for the S-configuration to x )
0.97(16). These results strongly suggest that (-)-9 and
(-)-11 both possess the R-configuration. However, a
distinct statement is not possible due to the high
standard deviation of the Flack parameter.
To support the configurational assignment based on
the X-ray analyses, the elution orders of the isomers
were studied on three analytical HPLC columns relying
on three different modes of chiral recognition. On the
Chirobiotic T and Crownpak CR(-) columns the two (+)-
enantiomers, compounds (+)-9 and (+)-11, elute before
the corresponding (-)-enantiomers, whereas opposite
elution orders were seen on the (S)-pipecolic acid ligand-
exchange column. On the basis of the elution orders for
a large number of natural R-amino acids and 3-isox-
azolol-containing R-amino acids studied on these three

1054 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 7
Madsen et al.
Ta ble 2. Pharmacological Effects at Cloned mGluRs Expressed
in Chinese Hamster Ovary Cells
Kb (µM)
compd
mGlu1R
mGlu5a
mGlu2 mGlu4a
(S)-HIBO (4)a
250 ( 27
190 ( 18
110 ( 3
>100
490 ( 8
180 ( 37
97 ( 9
>1000 >1000
>1000 >1000
>1000 >1000
5a
6b
7
8
9
(S)-9
(R)-9
10
11
(S)-11
(R)-11
nd
>100
>100
140 ( 17
140 ( 40
30 ( 5
190 ( 51
110 ( 16
61 ( 11
>1000 >1000
>1000 >1000
>1000 >1000
>1000 >1000
>1000
>1000
160 ( 19
160 ( 10
86 ( 2
990 ( 290* >1000 >1000
nd
90 ( 15
>1000
>1000 >1000
>1000 >1000
>1000 >1000
>1000
a
b
Ref 16. Ref 17. nd, not determined. Data represent the mean
( SEM of at least two independent results. *Estimated from
partial concentration-response curves.
activity were also tested for antagonist activity (at 1
mM; compounds 7 and 10 at 100 µM) toward AMPA (5
µM)-, NMDA (10 µM)-, and KA (5 µM)-induced re-
sponses, and no significant antagonist effects were
observed for any of these compounds.
F igu r e 2. Histograms illustrating the differences in affinity
toward [3H]AMPA (black bars) binding and activities at mGlu1
(gray bars) and mGlu5 (white bars) for (RS)-6, 8, 9, and 10.
The respective affinity and activities of (RS)-6 are estimated
to be twice the values given in Tables 1 and 2 for the S-form
(6), and these values are set to 1.0; the values for 8-10 are
normalized relative to this. # < 0.05.
Effects a t m Glu Rs. The interaction of the com-
pounds with cloned mGluRs was measured using second-
messenger assays.16 The activities were determined at
group I mGluRs (mGlu1R and mGlu5a) and at mGlu2 and
mGlu4a as representatives of groups II and III, respec-
tively. (S)-HIBO (4), 5, and 6 have previously been
shown to exhibit antagonist effects at mGlu1 and
mGlu516,17 (Table 2). Similar antagonist effects at group
I mGluRs were found for 8, 9, (S)-9, 11, and (S)-11, the
most potent antagonist being (S)-9 (Kb ) 30 and 61 µM
at mGlu1 and mGlu5, respectively). In contrast, com-
pound 10 was found to be a selective antagonist at
mGlu1, showing a very weak effect at mGlu5. None of
the compounds tested showed any agonist or antagonist
activity at mGlu2 or mGlu4a. The R-forms of 9 and 11
were inactive at all receptor subtypes as both agonists
and antagonists (Table 2).
An ticon vu lsa n t Activity. Compound 9 was tested
for anticonvulsant activity against NMDA-induced con-
vulsions in mice. NMDA was given iv 20 mg/kg, and
compound 9, administered 15 min before NMDA, an-
tagonized the convulsions completely at 12.5 and 6.3 µg/
mouse when given icv. Compound 9 did not induce any
behavioral changes at these doses. When administered
sc (up to 50 mg/kg) 30 min before NMDA, no anticon-
vulsant activity of 9 was observed toward the NMDA-
induced convulsions.
mGluRs (Tables 1 and 2), whereas no activity at iGluRs
was observed for the octyl analogue 7. This indicates
that the tolerance for the size of substituents in the
4-position of HIBO analogues is limited. In the search
for selective antagonists at group I mGluRs, a new
series of HIBO analogues has now been synthesized,
namely the pentyl-, hexyl-, heptyl-, and phenyl-substi-
tuted analogues, compounds 8-11, respectively. For
compounds 8 and 11 a dramatic loss in activity at
AMPA receptors compared to 617 was observed, and 9
and 10 did not show detectable AMPA receptor activity.
In contrast, these analogues showed antagonist activity
at group I mGluRs. Compounds 8, 9, and 11 showed
approximately equipotent effects at mGlu1 and mGlu5
receptors.
Resolution of 9 and 11 by chiral HPLC afforded the
enantiomers (ee g 98.7%) of the two compounds. The
absolute configurations were established by X-ray chrys-
tallographic analyses, elution orders in three chiral
HPLC systems, and CD spectra. The antagonist effects
were shown to reside in the S-forms, and (S)-9 was
found to be the most potent mGlu1 and mGlu5 antago-
nist of the compounds tested. Compound 10 showed a
different and quite interesting pharmacological profile,
being an antagonist at mGlu1, equipotent with 8 and 9,
but with very low effect at mGlu5. Thus, 10 is a rather
selective antagonist at mGlu1, showing weak or no effect
at other mGluRs or iGluRs.
Discu ssion
There is accumulating evidence to suggest that mGluR
ligands may have therapeutic potential in certain neu-
rologic and psychiatric diseases.8,9 Notably, antagonists
at group I mGluRs have shown anticonvulsant activity,
neuroprotective properties, or analgesic effects in animal
models.35-41 Thus, selective antagonists for group I
mGluRs have considerable pharmacological interest.
HIBO [(RS)-4] and a few 4-alkyl-substituted HIBO
analogues have previously been synthesized and shown
to be agonists at AMPA receptors and antagonists at
group I mGluRs. Compounds 5 and 6 were both potent
AMPA receptor agonists and antagonists at group I
A comparison of the affinity for AMPA receptors and
the activity at mGlu1 and mGlu5 receptors of (RS)-6, 8,
9, and 10 is illustrated in Figure 2. The affinities for
AMPA receptors disappear with increasing length of the
alkyl side chains. The effects at mGlu1 are of the same
order of magnitude for the four compounds, whereas at
mGlu5 the activity peaks for 9 and is almost lost for 10.
Compound 10 showed limited solubility in water and
buffer solutions in agreement with what was previously
observed for compound 7.18 However, the solubility of 9

Synthesis/ Pharmacology of 3-Isoxazolol Amino Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 7 1055
added a solution of NaOH (0.84 g, 21 mmol) in water (0.5 mL)
and MeOH (4 mL). This mixture was cooled to -50 °C, stirred
for 10 min, and added to the above NH2OH solution. The
reaction mixture was stirred at -50 °C for 2.5 h and acetone
(2.1 g) was added. The reaction mixture was then quickly
added to 4 M HCl (24 mL) heated to 85 °C and stirred for 45
min at 85 °C. The MeOH was evaporated and CC (toluene-
AcOEt-AcOH 20:2:1) gave 14a (1.2 g, 65%) as an oil: 1H NMR
(CDCl3) δ 2.26 (t, J ) 7.1 Hz, 2H), 2.24 (s, 3H), 1.52 (quintet,
J ) 7.1 Hz, 2H), 1.30 (m, 4H), 0.89 (t, J ) 7.1 Hz, 3H).
4-Hexyl-5-m eth yl-3-isoxa zolol (14b). Prepared according
to the procedure described for compound 14a . 14b (3.7 g, 58%)
was obtained as an oil after CC: 1H NMR (CDCl3) δ 2.27 (t, J
) 7.1 Hz, 2H), 2.24 (s, 3H), 1.52 (quintet, J ) 7.1 Hz, 2H),
1.29 (m, 6H), 0.88 (t, J ) 7.1 Hz, 3H).
4-Hep tyl-5-m eth yl-3-isoxa zolol (14c). Prepared according
to the procedure described for compound 14a . 14c (3.1 g, 75%)
was obtained as an oil after CC: 1H NMR (CDCl3) δ 2.27 (t, J
) 7.1 Hz, 2H), 2.24 (s, 3H), 1.52 (quintet, J ) 7.1 Hz, 2H),
1.30 (m, 8H), 0.89 (t, J ) 7.1 Hz, 3H).
3-Eth oxy-5-m eth yl-4-p en tylisoxa zole (15a ). NaH (620
mg, 60%, 15.4 mmol) was added to a solution of 14a (2.18 g,
12.9 mmol) in dry DMF (50 mL) and the mixture stirred for
45 min. Ethyl bromide (1.6 mL, 20.9 mmol) was added over a
period of 10 min and the reaction mixture stirred at room
temperature overnight. After evaporation and addition of
water (100 mL) the mixture was extracted with AcOEt (3 ×
120 mL). The organic phases were dried (MgSO4), evaporated
and CC (toluene-AcOEt 5:1) of the residue gave 15a (1.2 g,
47%) as an oil: 1H NMR (CDCl3) δ 4.25 (q, J ) 7 Hz, 2H), 2.22
(s, 3H), 2.20 (t, J ) 7 Hz, 2H), 1.47 (m, 2H), 1.40 (t, J ) 7 Hz,
3H), 1.27 (m, 4H), 0.87 (t, J ) 7 Hz, 3H).
3-Eth oxy-4-h exyl-5-m eth ylisoxa zole (15b). Prepared ac-
cording to the procedure described for compound 15a . 15b (1.6
g, 45%) was obtained as an oil after CC: 1H NMR (CDCl3) δ
4.25 (q, J ) 7 Hz, 2H), 2.22 (s, 3H), 2.20 (t, J ) 7 Hz, 2H),
1.45 (m, 2H), 1.40 (t, J ) 7 Hz, 3H), 1.24 (m, 6H), 0.87 (t, J )
7 Hz, 3H).
3-Eth oxy-4-h ep tyl-5-m eth ylisoxa zole (15c). Prepared
according to the procedure described for compound 15a . 15c
(2.1 g, 59%) was obtained as an oil after CC: 1H NMR (CDCl3)
δ 4.27 (q, J ) 7 Hz, 2H), 2.22 (s, 3H), 2.20 (t, J ) 7 Hz, 2H),
1.48 (m, 2H), 1.37 (t, J ) 7 Hz, 3H), 1.27 (m, 8H), 0.87 (t, J )
7 Hz, 3H).
5-Br om om eth yl-3-eth oxy-4-p en tylisoxa zole (16a ). To a
solution of 15a (1.20 g, 6.1 mmol) in CCl4 (5 mL) and AcOH (2
mL) was added bromine (2 g, 13 mmol). The reaction mixture
was left stirring at room temperature for 6 days. Water (150
mL) was added and NaHSO3 until no bromine color was left.
The solution was extracted with CH2Cl2 (3 × 150 mL), dried
and evaporated. CC (toluene-AcOEt 20:1) gave 16a (440 mg,
26%) as an oil: 1H NMR (CDCl3) δ 4.33 (s, 2H), 4.32 (q, J ) 7
Hz, 2H), 2.22 (t, J ) 7 Hz, 2H), 1.48 (quintet, J ) 7 Hz, 2H),
1.40 (t, J ) 7 Hz, 3H), 1.28 (m, 4H), 0.89 (t, J ) 7 Hz, 3H).
5-Br om om eth yl-3-eth oxy-4-h exylisoxa zole (16b). Pre-
pared according to the procedure described for compound 16a .
16b (1.5 g, 73%) was obtained after CC as an oil: 1H NMR
(CDCl3) δ 4.33 (s, 2H), 4.32 (q, J ) 7 Hz, 2H), 2.29 (t, J ) 7
Hz, 2H), 1.54 (m, 2H), 1.41 (t, J ) 7 Hz, 3H), 1.30 (m, 6H),
0.89 (t, J ) 7 Hz, 3H).
was high enough to enable testing at concentrations up
to 1 mM, and it was decided to study 9 for anticonvul-
sant activity. Compound 9 antagonized NMDA-induced
convulsions when given icv, whereas no activity was
observed when 9 was given sc. This indicates that 9 is
not capable of penetrating the blood-brain barrier and/
or that 9 is metabolized in vivo to an extent that
prevents sufficient concentrations to be reached in the
CNS.
In conclusion, new selective group I mGluR antago-
nists have been synthesized and characterized. (S)-9 is
the most potent antagonist in this series at mGlu1 and
mGlu5, whereas 10 was selective at mGlu1, showing
very low effect at mGlu5 and no activity at other GluRs.
The results of the present structure-activity studies
seem to suggest that the group I mGlu1 and mGlu5
receptor recognition sites contain lipophilic pockets of
slightly different sizes, which may be exploited in the
attempts to design mGlu1- and mGlu5-selective antago-
nists.
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Compounds were vi-
sualized on TLC plates (Merck silica gel 60 F254) using UV light
or a KMnO4 spraying reagent. Column chromatography (CC)
was performed using silica gel C60-H (230-400 mesh, Rhoˆne-
Poulenc). Compounds containing amino groups were visualized
using a ninhydrin spraying reagent. Melting points were
1
determined in capillary tubes and are uncorrected. H NMR
spectra were recorded on a Bruker AC-200F (200 MHz) or
Varian Gemini-2000 BB spectrometer (300 MHz). J values are
given in hertz (Hz). Chemical shifts (δ) are given in ppm
relative to TMS for compounds dissolved in CDCl3. Elemental
analyses were performed at Analytical Research Department,
H. Lundbeck A/S, Denmark, or at Microanalytical Laboratory
at the Department of Physical Chemistry, University of
Vienna, Austria, and were within (0.4% of the theoretical
values unless otherwise stated. IR spectra were recorded from
KBr disks on a Perkin-Elmer 781 grating infrared spectro-
photometer. Optical rotations were measured in thermostated
cuvettes on a Perkin-Elmer 241 polarimeter. Circular dichro-
ism (CD) spectra were recorded in 0.1 M HCl in 1.0-cm
cuvettes at room temperature on a J asco J -720 spectropola-
rimeter.
Meth yl (RS)-2-Acetylh ep ta n oa te (13a ). Sodium (4.3 g,
187 mmol) was dissolved in MeOH (100 mL) and methyl
acetoacetate (40 mL, 373 mmol) added over 15 min. The
solution was cooled to 0 °C and n-pentyl bromide (23.5 mL,
186 mmol) added over 10 min at 0 °C. The reaction mixture
was stirred overnight at 50 °C and then evaporated. Water
(100 mL) was added and extracted with CH2Cl2 (4 × 100 mL).
The organic phases were dried (MgSO4) and evaporated, and
the residue was distilled (0.3 mmHg, 78-92 °C) using a
vigreaux column, which gave 13a (20.0 g, 58%) as an oil: 1H
NMR (CDCl3) δ 3.7 (s, 3H), 3.4 (t, J ) 7 Hz, 1H), 2.2 (s, 3H),
1.8 (m, 2H), 1.3 (m, 6H), 0.9 (t, J ) 7 Hz, 3H).
Meth yl (RS)-2-Acetylocta n oa te (13b). Prepared accord-
ing to the procedure described for compound 13a . 13b (12.0 g,
32%) was obtained after distillation (0.2 mmHg, 62-74 °C):
1H NMR (CDCl3) δ 3.7 (s, 3H), 3.4 (t, J ) 7.3 Hz, 1H), 2.2 (s,
3H), 1.85 (m, 2H), 1.3 (m, 8H), 0.9 (t, J ) 7.1 Hz, 3H).
Meth yl (RS)-2-Acetyln on a n oa te (13c). Prepared accord-
ing to the procedure described for compound 13a . 13c (22.3 g,
56%) was obtained after distillation (0.3 mmHg, 94-95 °C):
1H NMR (CDCl3) δ 3.7 (s, 3H), 3.4 (t, J ) 7 Hz, 1H), 2.2 (s,
3H), 1.8 (m, 2H), 1.3 (m, 10H), 0.9 (t, J ) 7 Hz, 3H).
5-Meth yl-4-p en tyl-3-isoxa zolol (14a ). To a solution of
NH2OH‚HCl (2.9 g, 42 mmol) in MeOH (10 mL) heated to 60
°C was added a solution of NaOH (1.7 g, 42 mmol) in water (1
mL) and MeOH (10 mL). The mixture was cooled to -50 °C.
To a solution of 13a (3.72 g, 20 mmol) in MeOH (4 mL) was
5-Br om om eth yl-3-eth oxy-4-h ep tylisoxa zole (16c). Pre-
pared according to the procedure described for compound 16a .
1
16c (300 mg, 69%) was obtained after CC as an oil: H NMR
(CDCl3) δ 4.33 (s, 2H), 4.32 (q, J ) 7 Hz, 2H), 2.30 (t, J ) 7
Hz, 2H), 1.53 (quintet, J ) 7 Hz, 2H), 1.40 (t, J ) 7 Hz, 3H),
1.28 (m, 8H), 0.89 (t, J ) 7 Hz, 3H).
Meth yl 2-Acetam ido-3-(3-eth oxy-4-pen tyl-5-isoxazolyl)-
2-(m eth oxyca r bon yl)p r op ion a te (17a ). To a solution of
dimethyl acetamidomalonate (650 mg, 1.8 mmol) in dry DMF
(5 mL) was added NaH (75 mg, 60%, 1.8 mmol). After stirring
for 30 min a solution of 16a (440 mg, 1.6 mmol) in dry DMF
(5 mL) was added. The reaction mixture was stirred at room
temperature overnight, evaporated and the residue added
water (25 mL) and extracted with AcOEt (3 × 25 mL). The

1056 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 7
Madsen et al.
organic phases were extracted with 1 M NaOH (25 mL), water
(2 × 25 mL), dried (MgSO4) and evaporated. CC (toluene-
AcOEt 6:1) gave after recrystallization (toluene-light petro-
leum) 17a (180 mg, 29%) as colorless crystals: mp 105.5-106.5
°C; 1H NMR (CDCl3) δ 6.73 (s, 1H), 4.25 (q, J ) 7 Hz, 2H),
3.82 (s, 6H), 3.70 (s, 2H), 2.13 (t, J ) 7 Hz, 2H), 2.00 (s, 3H),
1.42 (m, 2H), 1.40 (t, J ) 7 Hz, 3H), 1.28 (m, 4H), 0.89 (t, J )
7 Hz, 3H). Anal. (C18H28N2O7) C, H, N.
E t h yl 2-Acet a m id o-2-et h oxyca r b on yl-3-(3-et h oxy-4-
p h en yl-5-isoxa zolyl)p r op ion a te (21). To a stirred suspen-
sion of NaH (57 mg, 80%, 1.98 mmol) in dry DMF (6 mL), was
added, at room temperature, diethyl acetamidomalonate (430
mg, 1.98 mmol), and this solution was stirred for 15 min. 20
(560 mg, 1.98 mmol) dissolved in dry DMF (2 mL) was
dropwise added and the mixture was stirred at room temper-
ature for 24 h. The resulting solution was evaporated, diluted
with water and extracted with AcOEt. The organic layer was
dried (MgSO4), evaporated and CC (toluene-AcOEt 4:1 and
1% AcOH) gave 21 (500 mg, 60%) after recrystallization
(AcOEt-light petroleum): mp 129-130 °C; 1H NMR (CDCl3)
δ 7.2-7.5 (m, 5H), 6.62 (br s, 1H), 4.33 (q, J ) 7.1 Hz, 2H),
4.0-4.2 (m, 4H), 3.96 (s, 2H), 1.65 (s, 3H), 1.40 (t, J ) 7.1 Hz,
3H), 1.19 (t, J ) 7.1 Hz, 6H). Anal. (C21H26N2O7) C, H, N.
(RS)-2-Am in o-3-(3-h yd r oxy-4-p h en yl-5-isoxa zolyl)p r o-
p ion ic Acid (11). 21 (450 mg, 1.07 mmol) was dissolved in
48% hydrobromic acid (15 mL) and refluxed at 140 °C for 30
min. The solution was rapidly cooled and evaporated, then
twice dissolved in water and re-evaporated. After drying in
vacuo (over KOH) the crude hydrobromide salt was dissolved
in water (500 mL) and EtOH (300 mL) and pH adjusted to
3-4 with a solution of triethylamine in EtOH. The precipitate
was recrystallized from water to give zwitterionic 11 (226 mg,
85%): mp 220 °C dec; 1H NMR (D2O) δ 7.1-7.5 (m, 5H), 3.40
(dd, J ) 5.6 and 8.0 Hz, 1H), 2.90 (dd, J ) 5.6 and 14.7 Hz,
1H), 2.73 (dd, J ) 8.0 and 14.7 Hz, 1H). Anal. (C12H12N2O4‚
0.25H2O) C, H, N.
E t h yl 2-Acet a m id o-2-et h oxyca r b on yl-3-(3-et h oxy-4-
h exyl-5-isoxa zolyl)p r op ion a te (17b). Prepared according to
the procedure described for compound 17a using diethyl
acetamidomalonate. 17b (600 mg, 45%) was obtained after CC
and recrystallization as colorless crystals: mp 54.0-56.5 °C;
1H NMR (CDCl3) δ 6.75 (s, 1H), 4.29 (q, J ) 7 Hz, 4H), 4.25
(q, J ) 7 Hz, 2H), 3.71 (s, 2H), 2.15 (t, J ) 7 Hz, 2H), 1.99 (s,
3H), 1.40 (m, 2H), 1.36 (t, J ) 7 Hz, 3H), 1.29 (t, J ) 7 Hz,
6H), 1.26 (m, 6H), 0.87 (t, J ) 7 Hz, 3H). Anal. (C21H34N2O7)
C, H, N.
Meth yl 2-Acetam ido-3-(3-eth oxy-4-h eptyl-5-isoxazolyl)-
2-(m eth oxyca r bon yl)p r op ion a te (17c). Prepared according
to the procedure described for compound 17a . 17c (310 mg,
52%) was obtained after CC and recrystallization as colorless
1
crystals: mp 84.0-86.0 °C; H NMR (CDCl3) δ 6.75 (s, 1H),
4.26 (q, J ) 7 Hz, 2H), 3.83 (s, 6H), 3.71 (s, 2H), 2.13 (t, J )
7 Hz, 2H), 2.00 (s, 3H), 1.41 (m, 2H), 1.39 (t, J ) 7 Hz, 3H),
1.25 (m, 8H), 0.87 (t, J ) 7 Hz, 3H). Anal. (C20H32N2O7) C, H,
N.
(RS)-2-Am in o-3-(3-h yd r oxy-4-p en tyl-5-isoxa zolyl)p r o-
p ion ic Acid (8). A mixture of 17a (180 mg, 0.47 mmol) in
concentrated hydrobromic acid (48%, 10 mL) was refluxed for
1 h. The reaction mixture was evaporated to dryness and
reevaporated after addition of water (10 mL). The crystalline
residue was washed with hot AcOEt and subsequently with
hot water to give 8 (76 mg, 67%) as colorless crystals: mp 221-
226 °C dec; 1H NMR (NaOD, D2O) δ 3.48 (dd, J ) 5.1 and 7.8
Hz, 1H), 2.88 (dd, J ) 5.1 and 14.7 Hz, 1H), 2.76 (dd, J ) 7.8
and 14.7 Hz, 1H), 2.15 (t, J ) 7.3 Hz, 2H), 1.40 (quintet, J )
7.3 Hz, 2H), 1.23 (m, 4H), 0.82 (t, J ) 6.9 Hz, 3H). Anal.
(C11H18N2O4) C, H, N.
Ch ir a l Liq u id Ch r om a t ogr a p h y. Analytical ligand-
exchange HPLC was performed on a column (120 × 4.6 mm)
containing (S)-pipecolic acid chemically bound following a
procedure used for attachment of (S)-proline to a silica-based
packing material.42 The column was connected to an HPLC
instrumentation consisting of a Waters M510 pump, a Waters
U6K injector, and a Waters 991 photodiode array detector with
a detection wavelength set at 220 nm. The column was
thermostated at 50 °C using a LKB 2155 HPLC column oven
and eluted at 1.0 mL/min with aqueous KH2PO4 (50 mM, pH
4.5) containing CuSO4 (0.12 mM). Analytical HPLC using the
Chirobiotic T column (150 × 4.6 mm) was performed at room
temperature. The column was connected to the same HPLC
instrumentation as described above for the ligand-exchange
column and was eluted at 0.5 mL/min. For (R)-9 the mobile
phase consisted of an aqueous NH4OAc/AcOH buffer (15 mM,
pH 4.0) and EtOH (70:30), whereas for (R)-11 aqueous AcOH
(pH 4.0) and EtOH (60:40) was used. For the determination
of the elution order of the enantiomers of 9 and 11 on the
Crownpak CR(-) column (150 × 4 mm) the column was eluted
at 0.4 mL/min with aqueous HClO4 (pH 3.9) at 50 °C or water
at 45 °C, respectively. Analytical HPLC using the Crownpak
CR(+) column (150 × 10 mm) was performed at 45 °C eluting
with aqueous trifluoroacetic acid (pH 3) at 2 mL/min. The
HPLC instrumentation used for both Crownpak CR columns
consisted of a J asco 880-PU pump, a Rheodyne injector model
7125 equipped with a 5.0-mL loop, and a Waters model 480
detector set at 210 nm. Preparative chiral chromatographic
separations were performed at room temperature on a Chiro-
biotic T column (500 × 10 mm) equipped with a guard column
(50 × 4.6 mm). The column was connected to the same HPLC
instrumentation as described above for the Crownpak CR
columns.
(RS)-2-Am in o-3-(4-h exyl-3-h yd r oxy-5-isoxa zolyl)p r op i-
on ic Acid (9). Prepared according to the procedure described
for compound 8. 9 (290 mg, 80%) was obtained as colorles
1
crystals: mp 207-219 °C dec; H NMR (NaOD, D2O) δ 3.55
(dd, J ) 4.8 and 8.4 Hz, 1H), 2.97 (dd, J ) 4.8 and 14.7 Hz,
1H), 2.81 (dd, J ) 8.4 and 14.7 Hz, 1H), 2.24 (t, J ) 7.3 Hz,
2H), 1.48 (m, 2H), 1.32 (m, 6H), 0.90 (t, J ) 6.8 Hz, 3H). Anal.
(C12H20N2O4) C, H, N.
(RS)-2-Am in o-3-(4-h ep tyl-3-h yd r oxy-5-isoxa zolyl)p r o-
p ion ic Acid (10). Prepared according to the procedure
described for compound 8. 10 (52 mg, 70%) was obtained as
colorles crystals: mp 221-227 °C dec; 1H NMR (NaOD, D2O)
δ 3.48 (dd, J ) 5.4 and 7.8 Hz, 1H), 2.88 (dd, J ) 5.4 and 14.7
Hz, 1H), 2.76 (dd, J ) 7.8 and 14.7 Hz, 1H), 2.15 (t, J ) 7.3
Hz, 2H), 1.40 (m, 2H), 1.23 (m, 8H), 0.81 (t, J ) 6.8 Hz, 3H).
Anal. (C13H22N2O4‚0.5H2O) C, H, N.
3-Eth oxy-5-m eth yl-4-p h en ylisoxa zole (19). To a solution
of 5-methyl-4-phenyl-3-isoxazolol19 (18) (1.8 g, 10.27 mmol) in
acetone (60 mL) was added potassium carbonate (2.8 g, 20.54
mmol) and the mixture was stirred at 60 °C for 30 min. Ethyl
bromide (1.68 g, 15.4 mmol) was added in quarter portions at
15-min intervals. The reaction mixture was refluxed for 4 h
and then cooled, filtered and evaporated. CC (toluene-AcOEt
19:1) gave 19 (1.4 g, 67%) as an oil: 1H NMR (CDCl3) δ 7.7-
7.5 (m, 5H), 4.37 (q, J ) 7.1 Hz, 2H), 2.44 (s, 3H), 1.43 (t, J )
7.1 Hz, 3H).
(S)-(+)-2-Am in o-3-(4-h exyl-3-h yd r oxy-5-isoxa zolyl)p r o-
p ion ic Acid [(S)-9] a n d (R)-(-)-2-Am in o-3-(4-h exyl-3-h y-
d r oxy-5-isoxa zolyl)p r op ion ic Acid [(R)-9]. Compound 9
(140 mg, 0.55 mmol) was dissolved in a mixture of an aqueous
NH4OAc/AcOH buffer (300 mM, pH 6.0) and EtOH (30:70) (110
mL), passed through a Millex HV filter (45 µm, Millipore), and
resolved on the Chirobiotic T column (500 × 10 mm) in
approximately 6 mg injections eluting the column with a
mixture of an aqueous NH4OAc/AcOH buffer (100 mM, pH 4.0)
and EtOH (80:20) at 1.1 mL/min with a detection wavelength
at 210 nm. Each run was divided into two main fractions. The
combined fractions of each of the resolved enantiomers were
evaporated, reevaporated three times from water and finally
5-Br om om eth yl-3-eth oxy-4-p h en ylisoxa zole (20). To a
solution 19 (1.1 g, 5.41 mmol) in CCl4 (25 mL) were added, in
quarter portions at 90-min intervals, dibenzoyl peroxide (100
mg) and NBS (1.06 g, 5.95 mmol). The mixture was refluxed
for 15 h and cooled. Light petroleum (25 mL) was added and
the solution was filtered and evaporated. CC (toluene) gave
20 (1.1 g, 72%) as an oil: 1H NMR (CDCl3) δ 7.3-7.6 (m, 5H),
4.45 (s, 2H), 4.40 (q, J ) 7.1 Hz, 2H), 1.44 (t, J ) 7.1 Hz, 3H).

Synthesis/ Pharmacology of 3-Isoxazolol Amino Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 7 1057
dried in vacuo. Recrystallization (water) of the first eluting
solution in water. Crystal data: C12H12N2O4‚H2O, Mr ) 266.25,
orthorhombic, space group P212121 (No. 19), a ) 5.4161(7) Å,
b ) 8.0495(9) Å, c ) 28.603(4) Å, V ) 1247.0(3) Å3, Z ) 4, Dc
) 1.418 Mg m-3, F(000) ) 560, µ(Cu KR) ) 0.948 mm-1, T )
122.0(5) K, crystal dimensions ) 0.28 × 0.10 × 0.04 mm.
Da ta Collection a n d P r ocessin g. Diffraction data were
collected on an Enraf-Nonius CAD-4 diffractometer using
graphite monochromated Cu KR radiation (λ ) 1.54184 Å).43
Intensities were collected using the ω/2θ scan mode. Unit cell
dimensions were determined by least-squares refinement of
20 reflections (θ range 38.81-40.26°).43 The reflections were
measured in the range -6 < h < 6, -10 < k < 10, -35 < l <
35 (3.09° < θ < 74.86°). Data were reduced using the programs
of Blessing (DREADD).44,45 The intensities of five standard
reflections were monitored every 104 s (decay 4.1%, corrected).
Absorption correction was applied using the program ABSORB
(Tmin ) 0.860; Tmax ) 0.965).46 A total of 6715 reflections were
averaged according to the point group symmetry 222 resulting
in 2560 unique reflections (Rint ) 0.0158 on Fo2).
(+)-enantiomer afforded compound (S)-9 (42.2 mg, 57%): mp
212-220 °C dec; ee ) 99.7%; [R]25 ) +18.9 (c ) 0.173, 1.0
589
M HCl); ∆ꢀ (220 nm) ) +0.20 m2/mol. Anal. (C12H20N2O4‚
0.75H2O) C, N; H: calcd, 8.03; found, 7.57. Recrystallization
(water) of the second eluting (-)-enantiomer gave compound
(R)-9 (36.5 mg, 48%): mp 212-220 °C dec; ee ) 98.7%; [R]25
589
) -16.5 (c ) 0.194, 1.0 M HCl); ∆ꢀ (220 nm) ) -0.20 m2/mol.
Anal. (C12H20N2O4‚H2O) C, N; H: calcd, 8.08; found, 7.59.
(S)-(+)-2-Am in o-3-(3-h yd r oxy-4-p h en yl-5-isoxa zolyl)-
pr opion ic Acid [(S)-11] an d (R)-(-)-2-Am in o-3-(3-h ydr oxy-
4-p h en yl-5-isoxa zolyl)p r op ion ic Acid [(R)-11]. Compound
11 (124 mg, 0.489 mmol) was dissolved in aqueous ammonia
(10 mM) in a concentration of 2 mg/mL, filtered through a
Millex HV filter (0.45 mm, Millipore) and resolved in 4 mg
injections on the Chirobiotic T column. The column was eluted
with a mixture of aqueous AcOH (pH 4) and EtOH (60:40) at
1.5 mL/min with a detection wavelength at 254 nm. The
collected fractions of the first peak were pooled, evaporated,
and reevaporated twice from water. After drying in vacuo, the
crystalline residue was recrystallized (water) to give (S)-11
Str u ctu r e Solu tion a n d Refin em en t. The structure was
solved by the direct method using the program SHELXS9747,48
and refined using the program SHELXL97.27 Full matrix least-
(51.9 mg, 80%): mp 223-224 °C dec; ee ) 99.9%; [R]25
)
589
squares refinement on F2 was performed, minimizing Σw(Fo
2
+28.2° (c ) 0.39, 0.1 M HCl); ∆ꢀ (214 nm) ) +0.23 m2/mol; IR
3450 (m), 3220 (m), 3100-2600 (multiple, m), 1600 (s), 1520
(s) cm-1. Anal. (C12H12N2O4‚H2O) C, H, N. The collected
fractions of the second peak were treated as described for the
first eluting enantiomer to give (R)-11 (50.1 mg, 77%): mp
226-227 °C dec; ee ) 99.7%; [R]25589 ) -29.3° (c ) 0.39, 0.1 M
HCl); ∆ꢀ (214 nm) ) -0.25 m2/mol; IR spectrum of (R)-11 was
identical with that of (S)-11.
- Fc2)2, with anisotropic displacement parameters for the non-
hydrogen atoms. The positions of the hydrogen atoms were
located on intermediate difference electron density maps and
refined with fixed isotropic displacement parameters. Correc-
tion for extinction was applied (coefficient: 0.0041(4)). The
refinement (216 parameters, 2560 reflections) with the mol-
ecule having the R-configuration converged at RF ) 0.0270,
wRF2 ) 0.0633 for 2383 reflections with Fo > 4σ(Fo); w ) 1/[σ2-
X-r a y Cr ysta llogr a p h ic An a lysis of (R)-(-)-9 Mon oh y-
d r a te. Colorless single crystals were obtained from a solution
in water. Crystal data: C12H20N2O4‚H2O, Mr ) 274.32, orthor-
hombic, space group P212121 (No. 19), a ) 5.3916(9) Å, b )
7.8160(10) Å, c ) 35.331(5) Å, V ) 1488.9(4) Å3, Z ) 4, Dc )
1.224 Mg m-3, F(000) ) 592, µ(Cu KR) ) 0.795 mm-1, T )
122.0(5) K, crystal dimensions ) 0.16 × 0.09 × 0.08 mm.
(Fo2) + (0.0330P)2 + 0.0745 P], where P ) (Fo + 2Fc2)/3; S )
2
1.064. In the final difference Fourier map maximum and
minimum electron densities were 0.221 and -0.157 e Å-3
,
respectively. Refinement of the Flack absolute structure factor
x in the final refinement gave x ) 0.00(17).26,27 The large
standard deviation of x is due to the atom types present in
the compound (C, H, N, and O), which produce minor anoma-
lous scattering. Complex atomic scattering factors for neutral
atoms were as incorporated in SHELXL97.27,49
Da ta Collection a n d P r ocessin g. Diffraction data were
collected on an Enraf-Nonius CAD-4 diffractometer using
graphite monochromated Cu KR radiation (λ ) 1.54184 Å).43
Intensities were collected using the ω/2θ scan mode. Unit cell
dimensions were determined by least-squares refinement of
24 reflections (θ range 31.65-37.18°).43 The reflections were
measured in the range -6 < h < 4, -9 < k < 9, -43 < l < 44
(2.50° < θ < 74.85°). Data were reduced using the programs
of Blessing (DREADD).44,45 The intensities of five standard
reflections were monitored every 104 s (decay 3.1%, corrected).
Absorption correction was applied using the program ABSORB
(Tmin ) 0.908; Tmax ) 0.945).46 A total of 11327 reflections were
averaged according to the point group symmetry 222 resulting
in 3067 unique reflections (Rint ) 0.0353 on Fo2).
R ecep t or Bin d in g. Affinity for AMPA, KA, and NMDA
receptors was determined using the ligands [3H]AMPA, [3H]-
KA, and [3H]CPP, respectively,31-33 with the modifications
previously described.50 The membrane preparations used in
all the receptor binding experiments were prepared according
to the method of Ransom and Stec.51
In Vitr o Electr op h ysiology. The rat cortical slice prepa-
ration for determination of excitatory amino acid-evoked
depolarizations described by Harrison and Simmonds34 was
used in a modified version.52 Application of agonists were made
for 90 s at each concentration tested. The receptor selectivity
of agonists was tested using the AMPA receptor antagonist
NBQX or the NMDA receptor antagonist CPP. In antagonist
experiments, the antagonists were applied alone for 90 s
followed by co-application with agonists for another 90 s.
Str u ctu r e Solu tion a n d Refin em en t. The structure was
solved by the direct method using the program SHELXS9747,48
and refined using the program SHELXL97.27 Full matrix least-
squares refinement on F2 was performed, minimizing Σw(Fo
2
Secon d -Messen ger Assa ys. The mGluR subtypes mGlu1R
,
- Fc2)2, with anisotropic displacement parameters for the non-
hydrogen atoms. The positions of the hydrogen atoms were
located on intermediate difference electron density maps and
refined with fixed isotropic displacement parameters. Correc-
tion for extinction was applied (coefficient: 0.0022(2)). The
refinement (240 parameters, 3067 reflections) with the mol-
ecule having the R-configuration converged at RF ) 0.0279,
wRF2 ) 0.0656 for 2772 reflections with Fo > 4σ(Fo); w ) 1/[σ2-
(Fo2) + (0.0289P)2], where P ) (Fo2 + 2Fc2)/3; S ) 1.053. In the
final difference Fourier map maximum and minimum electron
densities were 0.167 and -0.144 e Å-3, respectively. Refine-
ment of the Flack absolute structure factor x in the final
refinement gave x ) 0.00(18).26,27 The large standard deviation
of x is due to the atom types present in the compound (C, H,
N, and O), which produce minor anomalous scattering. Com-
plex atomic scattering factors for neutral atoms were as
incorporated in SHELXL97.27,49
mGlu2, mGlu4a, and mGlu5a were expressed in Chinese ham-
ster ovary cell lines. All compounds were tested for agonist
and antagonist activity at 1 mM concentrations, unless
otherwise stated, by the method previously described.16
An ticon vu lsa n t Activity. Male mice (NMRI/BOM, SPF,
Bomholtgård, Denmark) were used for the studies of anticon-
vulsant effects. The mice were kept in groups of 10 in plastic
cages (35 × 30 × 12 cm) under a 12-h day/night cycle (lights
on 6 a.m.). They had free access to food and water. Each dose
group consisted of 5-10 mice. Compound 9 was administered
sc (10 mL/kg) 30 min or icv (5 µL/mouse) 15 min before NMDA
(140 µmol/kg ) 20 mg/kg) was given iv (10 mL/kg). The icv
administration was given into the third ventricle by free hand
injection as previously described.53 The mice were observed
for behavioral changes before NMDA and subsequently for up
to 1 h for presence of clonic/tonic convulsions.
Ack n ow led gm en t. The work was supported by
grants form the Lundbeck Foundation, H. Lundbeck
X-r a y Cr ysta llogr a p h ic An a lysis of (R)-(-)-11 Mon o-
h yd r a t e. Colorless single crystals were obtained from a

1058 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 7
Madsen et al.
(20) Armstrong, D. W.; Liu, Y.; Ekborgott, K. H. A covalently bonded
teicoplanin chiral stationary phase for HPLC enantiosepara-
tions. Chirality 1995, 7, 474-497.
(21) Berthod, A.; Liu, Y.; Bagwill, C.; Armstrong, D. W. Facile liquid
chromatographic enantioresolution of native amino acids and
peptides using a teicoplanin chiral stationary phase. J . Chro-
matogr. 1996, A731, 123-137.
(22) J ohansen, T. N.; Ebert, B.; Falch, E.; Krogsgaard-Larsen, P.
AMPA receptor agonists: Resolution, configurational assign-
ment, and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-
hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA).
Chirality 1997, 9, 274-280.
(23) Gu¨bitz, G. Direct separation of enantiomers by high performance
ligand exchange chromatography on chemically bonded chiral
phases. J . Liq. Chromatogr. 1986, 9, 519-535.
(24) Shinbo, T.; Yamagushi, T.; Nishimura, K.; Sugiura, M. Chro-
matographic separation of racemic amino acids by use of chiral
crown ether-coated reversed-phase packings. J . Chromatogr.
1987, 405, 145-153.
(25) J ohnson, C. K., Ed. ORTEP II Report ORNL-5138. A Fortran
Thermal-Ellipsoid Plot Program for Crystal Structure Illustra-
tions; Oak Ridge National Laboratory: Oak Ridge, TN, 1976.
(26) Flack, H. D. On enantiomorph-polarity estimation. Acta Crys-
tallogr. 1983, A39, 876-881.
(27) Sheldrick, G. M., Eds. SHELXL-97. Program for crystal structure
refinement; University of Go¨ttingen: Go¨ttingen, Germany, 1997.
(28) Ebert, B.; Lenz, S. M.; Brehm, L.; Bregnedal, P.; Hansen, J . J .;
Frederiksen, K.; Bøgesø, K. P.; Krogsgaard-Larsen, P. Resolu-
tion, absolute stereochemistry, and pharmacology of the S-(+)-
and R-(-)-isomers of the apparent partial AMPA receptor
agonist (R,S)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propi-
onic acid [(R,S)-APPA]. J . Med. Chem. 1994, 37, 878-884.
(29) Falch, E.; Brehm, L.; Mikkelsen, I.; J ohansen, T. N.; Skjærbæk,
N.; Nielsen, B.; Stensbøl, T. B.; Ebert, B.; Krogsgaard-Larsen,
P. Heteroaryl analogues of AMPA. 2. Synthesis, absolute ster-
eochemistry, photochemistry, and structure-activity relation-
ships. J . Med. Chem. 1998, 41, 2513-2523.
A/S, the Alfred Benzon Foundation, and the Danish
Medical Research Council. The secretarial assistance of
Ms. Anne Nordly and the technical assistance of Mr.
Flemming Hansen, Ms. Karen J ørgensen, Ms. Anette
L. Eriksen, Ms. Lisbeth Eriksen, Ms. Annette Kris-
tensen, and Ms. Heidi Petersen are gratefully acknowl-
edged.
Su p p or tin g In for m a tion Ava ila ble: Tables for com-
pounds (R)-9 and (R)-11, listing final atomic coordinates,
equivalent isotropic displacement parameters, anisotropic
displacement parameters for non-hydrogen atoms, and a full
list of bond lengths, bond angles, torsion angles, hydrogen bond
dimensions; an illustration of the crystal packings; lists of
structure factors. This material is available free of charge via
the Internet at http://pubs.acs.org.
Refer en ces
(1) Hollmann, M.; Heinemann, S. Cloned glutamate receptors.
Annu. Rev. Neurosci. 1994, 17, 31-108.
(2) Nakanishi, S.; Masu, M. Molecular diversity and functions of
glutamate receptors. Annu. Rev. Biophys. Biomol. Struct. 1994,
23, 319-348.
(3) Wheal, H. V., Thomson, A. M., Eds. Excitatory Amino Acids and
Synaptic Transmission; Academic Press: London, 1995.
(4) Monaghan, D. T., Wenthold, R. J ., Eds. The Ionotropic Glutamate
Receptors; Humana Press: Totowa, NJ , 1997.
(5) Dingledine, R.; Borges, K.; Bowie, D.; Traynelis, S. F. The
glutamate receptor ion channels. Pharmacol. Rev. 1999, 51,
7-61.
(6) Conn, P. J ., Patel, J ., Eds. The Metabotropic Glutamate Recep-
tors; Humana Press: Totowa, NJ , 1994.
(7) Moroni, F., Nicoletti, F., Pellegrini-Giampietro, D. E., Eds.
Metabotropic Glutamate Receptors and Brain Function; Portland
Press: London, 1998.
(8) Parsons, C. G.; Danysz, W.; Quack, G. Glutamate in CNS
disorders as a target for drug development: an update. Drug
News Perspect. 1998, 11, 523-569.
(9) Bra¨uner-Osborne, H.; Egebjerg, J .; Nielsen, E. Ø.; Madsen, U.;
Krogsgaard-Larsen, P. Ligands for glutamate receptors: design
and therapeutic prospects. J . Med. Chem. 2000, 43, 2609-2645
(10) Lees, G. J . Pharmacology of AMPA/kainate receptor ligands and
their therapeutic potential in neurological and psychiatric
disorders. Drugs 2000, 59, 33-78.
(11) Schoepp, D. D.; J ane, D. E.; Monn, J . A. Pharmacological agents
acting at subtypes of metabotropic glutamate receptors. Neu-
ropharmacology 1999, 38, 1431-1476.
(12) Krogsgaard-Larsen, P.; Honore´, T.; Hansen, J . J .; Curtis, D. R.;
Lodge, D. New class of glutamate agonist structurally related
to ibotenic acid. Nature 1980, 284, 64-66.
(13) Hansen, J . J .; Lauridsen, J .; Nielsen, E.; Krogsgaard-Larsen,
P. Enzymic resolution and binding to rat brain membranes of
the glutamic acid agonist, R-amino-3-hydroxy-5-methyl-4-isox-
azolepropionic acid. J . Med. Chem. 1983, 26, 901-903.
(14) Bischoff, F.; J ohansen, T. N.; Ebert, B.; Krogsgaard-Larsen, P.;
Madsen, U. Excitatory amino acid receptor ligands: Asymmetric
synthesis, absolute stereochemistry and pharmacology of (R)-
and (S)-homoibotenic acid. Bioorg. Med. Chem. 1995, 3, 553-
558.
(15) Hansen, J . J .; J ørgensen, F. S.; Lund, T. M.; Nielsen, B.;
Reinhardt, A.; Breum, I.; Brehm, L.; Krogsgaard-Larsen, P.
AMPA receptor agonists: structural, conformational and ster-
eochemical aspects. In Excitatory amino acid receptors. Design
of agonists and antagonists; Krogsgaard-Larsen, P., Hansen, J .
J ., Eds.; Ellis Horwood: Chichester, 1992; pp 216-245.
(16) Bra¨uner-Osborne, H.; Nielsen, B.; Krogsgaard-Larsen, P. Mo-
lecular pharmacology of homologues of ibotenic acid at cloned
metabotropic glutamic acid receptors. Eur. J . Pharmacol. 1998,
350, 311-316.
(30) Hansen, J . J .; Nielsen, B.; Krogsgaard-Larsen, P.; Brehm, L.;
Nielsen, E. Ø.; Curtis, D. R. Excitatory amino acid agonists.
Enzymic resolution, X-ray structure and enantioselective activi-
ties of (R)- and (S)-bromohomoibotenic acid. J . Med. Chem. 1989,
32, 2252-2260.
(31) Honore´, T.; Nielsen, M. Complex structure of quisqualate-
sensitive glutamate receptors in rat cortex. Neurosci. Lett. 1985,
54, 27-32.
(32) Murphy, D. E.; Schneider, J .; Boehm, C.; Lehmann, J .; Williams,
K. Binding of [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic
acid to rat membranes. A selective high affinity ligand for
N-methyl-D-aspartate receptors. J . Pharmacol. Exp. Ther. 1987,
240, 778-783.
(33) Braitman, D. J .; Coyle, J . T. Inhibition of [3H]kainic acid receptor
binding by divalent cations correlates with ion affinity for the
calcium channel. Neuropharmacology 1987, 26, 1247-1251.
(34) Harrison, N. L.; Simmonds, M. A. Quantitative studies on some
antagonists of N-methyl-D-aspartate in slices of rat cerebral
cortex. Br. J . Pharmacol. 1985, 84, 381-391.
(35) Kno¨pfel, T.; Kuhn, R.; Allgeier, H. Metabotropic glutamate
receptors: novel targets for drug development. J . Med. Chem.
1995, 38, 1417-1426.
(36) Dalby, N. O.; Thomsen, C. Modulation of seizure activity in mice
by metabotropic glutamate receptor ligands. J . Pharmacol. Exp.
Ther. 1996, 276, 516-522.
(37) Thomsen, C.; Dalby, N. O. Roles of metabotropic glutamate
receptor subtypes in modulation of pentylenetetrazole-induced
seizure activity in mice. Neuropharmacology 1998, 37, 1465-
1473.
(38) Chapman, A. G.; Yip, P. K.; Yap, J . S.; Quinn, L. P.; Tang, E.;
Harris, J . R.; Meldrum, B. S. Anticonvulsant actions of LY
367385 ((+)-2-methyl-4-carboxyphenylglycine) and AIDA ((R,S)-
1-aminoindan-1,5-dicarboxylic acid). Eur. J . Pharmacol. 1999,
368, 17-24.
(39) Pellegrini-Giampietro, D. E.; Peruginelli, F.; Meli, E.; Cozzi, A.;
Albani-Torregrossa, S.; Pellicciari, R.; Moroni, F. Protection with
metabotropic glutamate 1 receptor antagonists in models of
ischemic neuronal death: time course and mechanisms. Neu-
ropharmacology 1999, 38, 1607-1619.
(40) Chapman, A. G. Glutamate and epilepsy. J . Nutr. 2000, 130,
1043S-1045S.
(41) Chen, Y.; Bacon, G.; Sher, E.; Clark, B. P.; Kallman, M. J .;
Wright, R. A.; J ohnson, B. G.; Schoepp, D. D.; Kingston, A. E.
Evaluation of the activity of a novel metabotropic glutamate
receptor antagonist (()-2-amino-2-(3-cis and trans)-carboxycy-
clobutyl-3-(9-thioxanthyl acid) in the in vitro neonatal spinal cord
and in an in vivo pain model. Neuroscience 2000, 95, 787-793.
(42) Gu¨bitz, G.; J ellenz, W.; Santi, W. Resolution of the optical
isomers of underivatisized amino acids on chemically bonded
chiral phases by ligand exchange chromatography. J . Liq.
Chromatogr. 1981, 4, 701-712.
(17) J ohansen, T. N.; Ebert, B.; Bra¨uner-Osborne, H.; Didriksen, M.;
Christensen, I. T.; Søby, K. K.; Madsen, U.; Krogsgaard-Larsen,
P.; Brehm, L. Excitatory amino acid receptor ligands: resolution,
absolute stereochemistry, and enantiopharmacology of 2-amino-
3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid. J . Med. Chem.
1998, 41, 930-939.
(18) Christensen, I. T.; Ebert, B.; Madsen, U.; Nielsen, B.; Brehm,
L.; Krogsgaard-Larsen, P. Excitatory amino acid receptor ligands.
Synthesis and biological activity of 3-isoxazolol amino acids
structurally related to homoibotenic acid. J . Med. Chem. 1992,
35, 3512-3519.
(19) Nakamura, N.; Tajima, Y.; Sakai, K. Direct phenylation of
isoxazoles using palladium catalysts synthesis of 4-phenylmus-
cimol. Heterocycles 1982, 17, 235-245.

Synthesis/ Pharmacology of 3-Isoxazolol Amino Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 7 1059
(43) CAD-4 Software, version 5.0; Enraf-Nonius: Delft, The Neth-
erlands, 1989.
(50) Stensbøl, T. B.; Borre, L.; J ohansen, T. N.; Egebjerg, J .; Madsen,
U.; Ebert, B.; Krogsgaard-Larsen, P. Resolution, absolute ster-
eochemistry and molecular pharmacology of the enantiomers of
ATPA. Eur. J . Pharmacol. 1999, 380, 153-162.
(44) Blessing, R. H. Data reduction and error analysis for accurate
single-crystal diffraction intensities. Crystallogr. Rev. 1987, 1,
3-58.
(51) Ransom, R. W.; Stec, N. L. Cooperative modulation of [3H]MK-
801 binding to the N-methyl-D-aspartate receptor ion channel
complex by L-glutamate, glycine and polyamines. J . Neurochem.
1988, 51, 830-836.
(45) Blessing, R. H. DREADD - data reduction and error analysis
for single-crystal diffractometer data. J . Appl. Crystallogr. 1989,
22, 396-397.
(46) DeTitta, G. T. ABSORB: an absorption correction programme
for crystals enclosed in capillaries with trapped mother liquor.
J . Appl. Crystallogr. 1985, 18, 75-79.
(52) Madsen, U.; Frølund, B.; Lund, T. M.; Ebert, B.; Krogsgaard-
Larsen, P. Design, synthesis and pharmacology of model com-
pounds for indirect elucidation of the topography of AMPA
receptor sites. Eur. J . Med. Chem. 1993, 28, 791-800.
(53) Sa´nchez, C.; Meier, E. Central and peripheral mediation of
hypothermia, tremor and salivation induced by muscarinic
agonists in mice. Pharmacol. Toxicol. 1993, 72, 262-267.
(47) Sheldrick, G. M., Ed. SHELXS-97. Program for the solution of
crystal structures; University of Go¨ttingen: Go¨ttingen, Germany,
1997.
(48) Sheldrick, G. M. Phase annealing in SHELX-90: Direct methods
for large structures. Acta Crystallogr. 1990, A46, 467-473.
(49) Wilson, A. J . C., Ed. International Tables for Crystallography,
Vol. C. Tables 6.1.1.4 and 4.2.6.8; Kluwer Academic Publish-
ers: Dordrecht, The Netherlands, 1995.
J M000441T