Heteroaryl analogues of AMPA. 2. Synthesis, absolute stereochemistry, photochemistry, and structure-activity relationships

Article abstract of DOI:10.1021/jm9801206

We have previously shown that (S)-2-amino-3-(3-hydroxy-5-phenyl-4- isoxazolyl)propionic acid [(S)-APPA, 2] is a weak agonist at (RS)-2-amino-3- (3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, specifically activated by (S)-AMPA (1), whereas (S)-2-amino-3-[3-hydroxy-5- (2-pyridyl)-4-isoxazolyl]propionic acid [(S)-2-Py-AMPA, 5] and (RS)-2-amino- 3-[3-hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (4) are potent AMPA agonists. On the other hand, (R)-APPA (3) and (R)-2-Py-AMPA (6) have been shown to be weak AMPA antagonists. We now report the synthesis of 2-Py-AMPA (7a) and the isomeric compounds 3-Py-AMPA (7b) and 4-Py-AMPA (7c) as well as the 7a analogues, (RS)-2-amino-3-[3-hydroxy-5-(6-methyl-2-pyridyl)-4- isoxazolyl]propionic acid (7d) and (RS)-2-amino-3-[3-hydroxy-5-(2- quinolinyl)-4-isoxazolyl]propionic acid (7e). Furthermore, (RS)-2-amino-3- [3-hydroxy-5-(2-furyl)-4-isoxazolyl]-propionic acid (2-Fu-AMPA, 7f) and its 5-bromo-2-furyl derivative (7g) were synthesized, and (S)-2-Fu-AMPA (8) and (R)-2-Fu-AMPA (9) were prepared by semipreparative chiral HPLC resolution of 7f. HPLC analyses and circular dichroism spectroscopy indicated the absolute stereochemistry of 8 and 9 to be S and R, respectively. This was confirmed by an X-ray crystallographic analysis of 9·HCl. In receptor binding (IC₅₀ values) and rat cortical wedge electrophysiological (EC₅₀ values) studies, 7c (IC₅₀ = 5.5 ± 0.6 μM; EC₅₀ = 96 ± 5 μM) was shown to be markedly weaker than 7a (IC₅₀ = 0.57 ± 0.16 μM; EC₅₀ = 7.4 ± 0.2 μM) as an AMPA agonist, whereas 7b,d,e were inactive. The very potent AMPA agonist effect of 7f (IC₅₀ = 0.15 ± 0.03 μM; EC₅₀ = 1.7 ± 0.2 μM) was shown to reside exclusively in 8 (IC₅₀ = 0.11 ± 0.01 μM; EC₅₀ = 0.71 ± 0.11 μM), whereas 9 did not interact significantly with AMPA receptors, either as an agonist or as an antagonist. 8 was shown to be photochemically active and is a potential photoaffinity label for the recognition site of the AMPA receptors. Compound 7g turned out to be a very weak AMPA receptor agonist (IC₅₀ = 12 ± 0.7 μM; EC₅₀ = 160 ± 15 μM). None of these new compounds showed detectable effects at N-methyl-D-aspartic acid (NMDA) or kainic acid receptors in vitro. The present studies have emphasized that the presence of a heteroatom in the 2-position of the heteroaryl 5-substituent greatly facilitates AMPA receptor agonist activity.

Full text of DOI:10.1021/jm9801206

J . Med. Chem. 1998, 41, 2513-2523
2513
Heter oa r yl An a logu es of AMP A. 2. Syn th esis, Absolu te Ster eoch em istr y,
P h otoch em istr y, a n d Str u ctu r e-Activity Rela tion sh ip s
Erik Falch, Lotte Brehm, Ivan Mikkelsen, Tommy N. J ohansen, Niels Skjærbæk, Birgitte Nielsen,
Tine B. Stensbøl, Bjarke Ebert, and Povl Krogsgaard-Larsen*
PharmaBiotec Research Center, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy,
2 Universitetsparken, DK-2100 Copenhagen, Denmark
Received February 23, 1998
We have previously shown that (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid
[(S)-APPA, 2] is a weak agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic
acid (AMPA) receptors, specifically activated by (S)-AMPA (1), whereas (S)-2-amino-3-[3-
hydroxy-5-(2-pyridyl)-4-isoxazolyl]propionic acid [(S)-2-Py-AMPA, 5] and (RS)-2-amino-3-[3-
hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (4) are potent AMPA agonists. On the other
hand, (R)-APPA (3) and (R)-2-Py-AMPA (6) have been shown to be weak AMPA antagonists.
We now report the synthesis of 2-Py-AMPA (7a ) and the isomeric compounds 3-Py-AMPA (7b)
and 4-Py-AMPA (7c) as well as the 7a analogues, (RS)-2-amino-3-[3-hydroxy-5-(6-methyl-2-
pyridyl)-4-isoxazolyl]propionic acid (7d ) and (RS)-2-amino-3-[3-hydroxy-5-(2-quinolinyl)-4-
isoxazolyl]propionic acid (7e). Furthermore, (RS)-2-amino-3-[3-hydroxy-5-(2-furyl)-4-isoxazolyl]-
propionic acid (2-Fu-AMPA, 7f) and its 5-bromo-2-furyl derivative (7g) were synthesized, and
(S)-2-Fu-AMPA (8) and (R)-2-Fu-AMPA (9) were prepared by semipreparative chiral HPLC
resolution of 7f. HPLC analyses and circular dichroism spectroscopy indicated the absolute
stereochemistry of 8 and 9 to be S and R, respectively. This was confirmed by an X-ray
crystallographic analysis of 9‚HCl. In receptor binding (IC50 values) and rat cortical wedge
electrophysiological (EC50 values) studies, 7c (IC50 ) 5.5 ( 0.6 µM; EC50 ) 96 ( 5 µM) was
shown to be markedly weaker than 7a (IC50 ) 0.57 ( 0.16 µM; EC50 ) 7.4 ( 0.2 µM) as an
AMPA agonist, whereas 7b,d ,e were inactive. The very potent AMPA agonist effect of 7f (IC50
) 0.15 ( 0.03 µM; EC50 ) 1.7 ( 0.2 µM) was shown to reside exclusively in 8 (IC50 ) 0.11 (
0.01 µM; EC50 ) 0.71 ( 0.11 µM), whereas 9 did not interact significantly with AMPA receptors,
either as an agonist or as an antagonist. 8 was shown to be photochemically active and is a
potential photoaffinity label for the recognition site of the AMPA receptors. Compound 7g
turned out to be a very weak AMPA receptor agonist (IC50 ) 12 ( 0.7 µM; EC50 ) 160 ( 15
µM). None of these new compounds showed detectable effects at N-methyl-D-aspartic acid
(NMDA) or kainic acid receptors in vitro. The present studies have emphasized that the
presence of a heteroatom in the 2-position of the heteroaryl 5-substituent greatly facilitates
AMPA receptor agonist activity.
In tr od u ction
administered together, 2 and 3 produced functional
partial agonism.10-12 Like (RS)-2-amino-3-[3-hydroxy-
5-(2-thiazolyl)-4-isoxazolyl]propionic acid (4),13,14 (S)-2-
amino-3-[3-hydroxy-5-(2-pyridyl)-4-isoxazolyl]propion-
ic acid [(S)-2-Py-AMPA, 5] (Figure 1) is markedly more
potent than 2 as an AMPA agonist, whereas (R)-2-Py-
AMPA (6) and 3 are equipotent as AMPA antagonists.15
The enantiomers of 2-Py-AMPA15 as well as the S- and
R-forms of the 4-fluorophenyl analogue of APPA16 also
produce functional partial agonism after coadministra-
tion.
The central excitatory amino acid (EAA) neurotrans-
mitter (S)-glutamic acid [(S)-Glu] operates through
three heterogeneous classes of ionotropic receptors
named N-methyl-D-aspartic acid (NMDA), (RS)-2-amino-
3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic
acid
(AMPA), and kainic acid receptors1-4 and a number of
subtypes of metabotropic receptors.5 These or perhaps
distinct subtypes of these receptors have been associated
with certain central nervous system (CNS) diseases and
are potential therapeutic targets in such diseases.6,7
Our projects on the design of therapeutically useful
EAA receptor ligands have recently been focused on aryl
and heteroaryl analogues of AMPA. This line of re-
search was initiated by the synthesis of (RS)-2-amino-
3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid (AP-
PA)8 and the observation that (S)-APPA (2) (Figure 1),
like (S)-AMPA (1),9 shows AMPA receptor agonism,
whereas (R)-APPA (3) is an AMPA antagonist.10 When
On the basis of these observations and structure-
activity studies on these heteroaryl analogues of AMPA
containing five-membered heterocyclic rings, we con-
cluded that the presence of a 2-positioned heteroatom
of the ring substituent at the 5-position greatly en-
hances AMPA receptor binding affinity and agonist
potency.14
To further substantiate this correlation between
structural, stereochemical, and electrostatic character-
istics of AMPA analogues and their AMPA agonist
potencies, and as an attempt to develop analogous
* Address correspondence to Professor Povl Krogsgaard-Larsen:
phone, (+45) 35 37 08 50, ext. 511; fax, (+45) 35 37 22 09.
S0022-2623(98)00120-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/16/1998

2514 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Falch et al.
of O-methylated (13a -e) and N-methylated (14a -e)
products. Reaction of the sodium salts of 12a -e with
dimethyl sulfate in dry DMF was shown to give the best
obtainable yields and the highest ratios between 13a -e
and the byproducts 14a -e.
Deprotonation of the isoxazole rings of 13a -e using
butyllithium and subsequent treatment of the lithium
salts formed by excess of paraformaldehyde gave the
hydroxymethyl analogues, 16a -e, in varying yields. The
4-pyridyl analogue, 16c, was obtained in the highest
yield, whereas 13b, containing a 3-pyridyl group, under
the same reaction conditions gave a complex reaction
mixture, from which 16b could not be isolated. The
brominated derivative of 13b, compound 15b, was,
however, readily lithiated and subsequently converted
into 16b by treatment with paraformaldehyde (Scheme
1).
Compounds 16a -e were transformed into the corre-
sponding dimethyl acetamidomalonates 18a-e via 17a-
e, and deprotection of 18a -e by treatment with hydro-
bromic acid gave the target compounds, 7a -e, isolated
in the zwitterionic forms.
The synthesis of 7f is outlined in Scheme 2. The key
intermediate in this reaction sequence, 5-(2-furyl)-4-
methyl-3-isoxazolol (21), was synthesized from the â-oxo
ester 19 and hydroxylamine. According to the litera-
ture,19,20 this synthesis can be accomplished with con-
comitant formation of only minor amounts of the
isomeric 3-isoxazolin-5-one 20. Using the procedure
described by Sato et al.,19 where the sodium salt of 19
was treated with hydroxylamine at -35 °C, compound
21 was formed selectively and could be isolated in 68%
yield. Treatment of 19 with hydroxylamine under the
conditions described by J acobsen et al.20 gave the isomer
20 as the only product.
The hydroxy group of 21 was protected as the (piv-
aloyloxy)methyl ether, a protecting group which can be
removed under mild acidic or basic conditions. Bromi-
nation of this protected intermediate 22 with NBS in
the absence of benzoyl peroxide catalyst gave 24, which
was converted into the Boc-protected diethyl aminoma-
lonate 25. Compound 25 was stepwise deprotected
using aqueous base and subsequently aqueous acid to
give 7f.
As an attempt to synthesize the 3-isoxazolin-5-one
analogue of 7f, compound 30, intermediate 20 was
N-benzoylated to give 27 and substantial amounts of
the isomeric compound 26. NBS bromination of 27 gave
28, which was converted into the Boc-protected diethyl
aminomalonate 29 with concomitant loss of the benzoyl
group. Attempts to deprotect 29 to the target compound
30 were, however, unsuccessful.
Bromination of compound 22 in the presence of
benzoyl peroxide gave a mixture of 24 and a compound
shown by NMR spectroscopy to be the 5-bromofuryl
analogue of 24. Separation of this 5-bromofuryl com-
pound, a potential intermediate for the synthesis of 7g,
from 24 failed, and an alternative route for the synthesis
of 7g was developed (Scheme 3). Treatment of 21 with
pivaloyl chloride gave 31 in high yield, and in the
absence of benzoyl peroxide catalyst, NBS bromination
of 31 produced 32. In the presence of benzoyl peroxide
and by using an excess of NBS, an inseparable 1:2
mixture of 32 and 33 was formed. Treatment of this
F igu r e 1. Structures of (S)-AMPA (1), (S)-APPA (2), (R)-
APPA (3), and a number of AMPA analogues containing
heteroaromatic 5-substituents.
correlations for structurally related AMPA antagonists,
we have now synthesized and pharmacologically char-
acterized the Py-AMPA analogues 7a -e, (RS)-2-amino-
3-[3-hydroxy-5-(2-furyl)-4-isoxazolyl]propionic acid (2-
Fu-AMPA, 7f), and its bromo-substituted analogue 7g
and optical isomers, (S)-2-Fu-AMPA (8) and (R)-2-Fu-
AMPA (9) (Figure 1). A further aim of this work was
to develop a potent and photochemically active AMPA
agonist for future radio- and photolabeling of AMPA
receptor recognition site(s).
Resu lts
Ch em istr y. It has been reported17 that addition of
bromine to 3-pyridylpropenoates is accompanied by
precipitation of perbromide adducts, and a similar
observation was made in connection with the conversion
of 10a -e into 11a -e (Scheme 1). Compounds 11a -e
were, however, obtained as hydrobromides in high yields
by addition of bromine to solutions of the hydrobromides
of 10a -e in glacial acetic acid, followed by heating (60
°C) of the suspensions of precipitated products. Treat-
ment of some 2,3-dihalogenopropionates with hydroxy-
lamine has previously been reported18 to give 3-isox-
azololes. Following this procedure, compounds 11a -e
were converted into the 3-isoxazolols 12a -e in good
yields. Methylation of compounds 12a -e gave mixtures

Heteroaryl Analogues of AMPA
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2515
Sch em e 1a
a
(i) Br2; (ii) NH2OH, NaOH; (iii) Me2SO4, NaOH; (iv) Br2; (v) n-BuLi, (CH2O)n; (vi) SOCl2; (vii) AcNHCH (COOMe)2, NaH; (viii) 48%
aq HBr.
Sch em e 2a
a
(i) NH2OH, NaOH, pH 10; (ii) NH2OH, NaOH, -35 °C; (iii) (CH3)3CCOOCH2I, K2CO3; (iv) NBS; (v) BocNHCH(COOEt)2, NaH; (vi)
NaOH, EtOH, 4 M HCl; (vii) PhCOCl, TEA; (viii) NBS; (ix) BocNHCH(COOEt)2, NaH; (x) NaOH, EtOH, 4 M HCl.
mixture with Boc-protected diethyl aminomalonate gave
a mixture of 34 and its nonbrominated analogue, from
which 34 was isolated chromatographically and depro-
tected to 7g under conditions similar to those used for
the conversion of 25 into 7f (Scheme 2).
A HPLC analysis of 7g did, however, disclose the
presence of approximately 0.25% of 7f. Prior to receptor
binding and electrophysiological studies on 7g, a sample
of this compound was further purified by preparative
HPLC, resulting in a sample of 7g containing less than
0.05% of 7f as determined by analytical reverse-phase
HPLC.
as chiral selector.21 In analogy with previous reports
on resolution of R-amino acids,15,22 the S-enantiomer,
compound 8, was less retained than the R-enantiomer,
compound 9, on this chiral HPLC column. Compounds
8 and 9 were isolated having enantiomeric excess (ee)
> 99.8%. Due to unsatisfactory separation of the
enantiomers on the Chirobiotic T column, only the
enantiomeric excess of the late-eluting enantiomer, 9,
could be precisely determined using this column. For
the analysis of 8, a ligand-exchange (S)-pipecolic acid
column, which showed the reverse elution order for the
enantiomers, was chosen.
Ch ir a l HP LC Resolu tion of 7f. Compound 7f was
resolved by chiral HPLC using a Chirobiotic T column,
which contains the macrocyclic glycopeptide Teicoplanin
The UV spectrum of 8, which was identical to that of
9, only showed one broad absorption band (250-300 nm)
with a maximum at 278 nm. This relatively strong UV

2516 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Falch et al.
Sch em e 3a
F igu r e 3. Time course of the photochemical degradation of
7f (b) and AMPA (9), both dissolved in water, using a 125-W
UV lamp. Each data point represents the relative amount of
compound left in the solution determined by HPLC analysis
and originates from single injections.
a
(i) (CH3)3CCOCl, TEA; (ii) NBS, (PhCO)2O2; (iii) BocNHCH-
(COOEt)2, NaH; (iv) NaOH, EtOH, 4 M HCl.
F igu r e 2. Circular dichroism spectra of 8 and 9.
F igu r e 4. Hydrogen-bonding pattern in crystals of 9‚HCl
(Table 2). Two cations and four chloride anions are shown in
perspective drawing.25 Displacement ellipsoids of the non-
hydrogen atoms are shown at the 50% probability level.
Hydrogen atoms are represented by spheres of arbitrary size.
Hydrogen bonds are indicated by thin lines.
absorption is most likely due to the conjugated aromatic
bicyclic ring system of 8 and 9. In the circular dichroism
(CD) spectra of 8 and 9 (Figure 2) only minor absorption
bands were found in the same region, indicating that
the contribution of the bicyclic ring system to the CD
spectra is small. The CD spectrum of 8 showed a
positive Cotton effect (∆ꢀ ) +0.18 m2/mol) at 210-220
nm, which is in agreement with what is generally
observed for R-amino acids having S-configuration.15,23
P h otoch em ica l Sta bility of 7f. The photochemical
stability of compound 7f in aqueous solution was
investigated using a 125-W UV lamp and was compared
to that of AMPA. The time course of the degradation
process is shown in Figure 3. Under the conditions
used, 7f was relatively quickly decomposed, t1/2 being
less than 30 min, whereas no detectable decomposition
was observed for AMPA even after UV light exposure
for 2 h. The UV light treatment of 7f resulted in
formation of a number of products, all of which were
more polar than the starting material. However, no
attempts were made to isolate or characterize any of
the degradation products formed.
X-r a y Cr ysta llogr a p h ic An a lysis of Com p ou n d
9‚HCl. A perspective drawing of the molecular struc-
ture of 9‚HCl with atomic labeling is depicted in Figure
4. The absolute configuration was established to be R
according to the procedure of Flack.26,27 The bond
lengths and angles are consistent with the general
pattern of related compounds.10,28-30 The isoxazole and
furan rings are both planar within the limits of experi-
mental errors, the interplanar angle being 10.4(1)°. The

Heteroaryl Analogues of AMPA
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2517
Ta ble 1. Selected Torsion Angles (deg) for
(R)-2-Amino-3-[5-(2-furyl)-3-hydroxy-4-isoxazolyl]propionic Acid
(9) Hydrochloridea
has been proposed for the AMPA receptor recognition
site containing a lipophilic cavity of limited size in
addition to binding sites recognizing stereospecifically
the charged groups of AMPA analogues.35-38 Enantio-
pharmacological studies on APPA (Figure 1) accordingly
disclosed weak AMPA agonist effects of 2, whereas 3
showed competitive AMPA receptor antagonism of
comparable potency.10 An analysis of these effects led
to the development of the concept of functional partial
agonism stating that partial agonism at any desired
level of relative efficacy can be established by coadmin-
istration of an agonist of a competitive antagonist at
appropriate concentrations.10-12
torsion angles
O3-C9-C5-C4
O1-C5-C9-C10
C3-C4-C6-C7
C5-C4-C6-C7
C4-C6-C7-N1
10.0(2)
9.6(2)
-89.7(2)
95.4(2)
62.0(1)
C4-C6-C7-C8
C6-C7-C8-O4
N1-C7-C8-O5
O4-C8-O5-H5
N2-C3-O2-H2
-176.6(1)
16.8(2)
-42.8(1)
6(2)
-3(1)
a
Estimated standard deviations are given in parentheses.
Ta ble 2. Hydrogen Bonds and Close Contacts24 Geometries
(Å, deg) in Crystals of (R)-2-Amino-3-[5-(2-furyl)-3-hydroxy-
4-isoxazolyl]propionic Acid (9) Hydrochloridea,b
D-H‚‚‚A
D-H
H‚‚‚A
D‚‚‚A
< DHA
Replacement of the phenyl group of APPA by the
isosteric 2-thienyl group quite surprisingly provided an
AMPA receptor agonist much more potent than 2 and
approximately equipotent with AMPA.13 This observa-
tion was suggested to reflect an electrostatic interaction
with the receptor protein and/or a stabilization of a
receptor-active conformation of the agonist by the het-
eroatom in the 2-position of the 5-substituent of the
isoxazole ring and prompted us to synthesize and
pharmacologically characterize a series of structurally
related compounds.14 Some of these analogues, includ-
ing 4 (Figure 1), all of which contained sulfur and/or
nitrogen in the 2-position of the ring, also contained ring
methyl substituents. A few of these compounds, notably
4, showed very potent AMPA receptor agonism.14 The
structure-activity analysis revealed highly regioselec-
tive steric effects of the methyl groups and, furthermore,
vanishingly low AMPA agonist effects of analogues
containing heterocyclic substituents carrying partial
positive or negative charges.14
Intramolecular
0.92(2)
N1-H1C‚‚‚O5i
2.478(2)
2.709(2)
94(1)
Intermolecular
0.93(3)
O5-H5‚‚‚N2ii
O2-H2‚‚‚Cliii
N1-H1A‚‚‚Cli
N1-H1B‚‚‚Cliv
N1-H1C‚‚‚Clv
C7-H7‚‚‚O4vi
C12-H12‚‚‚O4vii
C6-H6B‚‚‚O1iv
1.80(3)
2.10(2)
2.21(2)
2.23(2)
2.33(2)
2.46(2)
2.55(2)
2.60(1)
2.718(2)
2.961(1)
3.160(1)
3.148(1)
3.238(1)
3.346(2)
3.254(2)
3.287(2)
170(3)
173(2)
164(2)
163(2)
169(2)
150(1)
138(2)
127(1)
0.86(2)
0.98(2)
0.94(2)
0.92(2)
0.98(2)
0.87(2)
0.98(2)
a
Symmetry code: (i) x, y, z; (ii) -1 + x, -1 + y, z; (iii) 1 - x, 1/2
+ y, 1/2 - z; (iv) -1 + x, y, z; (v) 1 - x, -1/2 + y, 1/2 - z; (vi) 1 +
x, y, z; (vii) 1/2 + x, 3/2 - y, -z. Estimated standard deviations
are given in parentheses.
b
conformation of the cation is described by the selected
torsion angles given in Table 1. A close intramolecular
contact is observed between the ammonium group and
the hydroxyl oxygen atom of the carboxyl group (Table
2). This contact may be classified as the minor compo-
nent of a three-center hydrogen bond.31 All of the five
hydrogen atoms bonded to oxygen and nitrogen atoms
of the cation are utilized in the formation of hydrogen
bonds (Figure 4, Table 2).
In Vitr o P h a r m a cology. All compounds were char-
acterized in receptor binding studies, using rat brain
membranes, and electrophysiologically based on the rat
cortical wedge preparation for testing depolarizing
activity of EAA receptor ligands. Whereas none of the
new compounds showed significant affinity for [3H]-
kainic acid binding sites32 or NMDA receptor sites,
labeled by tritiated (RS)-3-(2-carboxypiperazin-4-yl)-
propyl-1-phosphonic acid ([3H]CPP),33 compounds 5,
7a ,c,f, and 8 were shown to be potent inhibitors of [3H]-
AMPA receptor binding34 (Table 3). Compounds 5,
7a ,c,f, and 8 turned out to be AMPA receptor agonists
showing full agonism (data not shown) and relative
potencies in agreement with their relative receptor
affinities (Figure 5). Thus, the AMPA agonist activities
of 7a ,f have been shown to reside exclusively in the
S-enantiomers, compounds 515 and 8, respectively.
Whereas the R-enantiomer of 7a , compound 6, previ-
ously has been shown to be a weak AMPA receptor
antagonist;15 the R-form of 7f, compound 9, was inactive
as an agonist as well as an antagonist at AMPA
receptors.
To further elucidate the structural factors determin-
ing the pharmacology of this class of AMPA analogues,
we have now synthesized (Scheme 1) the 2-, 3-, and
4-pyridyl analogues of APPA, compounds 7a -c, and
7d ,e which, like 7a , contain a nitrogen atom in the
2-position of the 5-substituent (Figure 1). Within this
group of compounds, only 7a showed potent AMPA
receptor agonism (Table 3). Whereas 7c was a weak
AMPA agonist, 7b,d ,e were inactive, emphasizing the
critical importance of a 2-positioned heteroatom for
effective interaction with the AMPA receptor and the
sensitivity of this interaction to steric effects.
Pharmacological studies on the enantiomers of 2-Py-
AMPA, obtained using chiral HPLC techniques, have
identified 5 as a potent AMPA agonist and 6 as a weak
AMPA antagonist equipotent with 3.15 In light of these
observations, we have now synthesized the 2-furyl
analogue of 7a , compound 7f (Scheme 2). In agreement
with previous structure-activity studies on heteroaryl
analogues of AMPA,13,14,16 7f turned out to be a very
potent AMPA agonist (Table 3). This, in turn, prompted
us to subject 7f to a chiral HPLC resolution and to
establish the absolute stereochemistry of the enanti-
omers by an X-ray crystallographic analysis on 9‚HCl
(Figure 4). Circular dichroism (CD) spectra of the
enantiomers of Fu-AMPA (Figure 2) were in agreement
with the stereochemical assignment.
Discu ssion
As predicted, the AMPA agonist effect of 7f was
shown to reside specifically in the S-enantiomer (8), but
whereas 310 as well as 615 showed competitive AMPA
receptor antagonism, 9 turned out to be pharmacologi-
On the basis of quite extensive structure-activity
studies on analogues of AMPA containing different alkyl
groups in the 5-position of the isoxazole ring, a model

2518 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Falch et al.
Ta ble 3. Receptor Binding and Electrophysiological Data (Values ( SEM, n ) 3-6)
receptor binding IC50 (µM)
electropharmacology
compound
[3H]AMPA
[3H]kainic acid
[3H]CPP
agonism EC50 (µM)
antagonism Ki (µM)
(S)-AMPA (1)
2
3
4
5
6
7a
7b
7c
7d
7e
7f
7g
8
9
0.040 ( 0.014
5.5 ( 2.2b
>100
>100
>100b
>100b
>100a
>100c
>100c
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.5 ( 0.2
230 ( 12b
>100b
>100b
>100b
286 ( 24b
270 ( 50c
0.094a
4.9a
2.3a
0.19 ( 0.06c
>100c
>100c
>100
>100
>100
>100
>100
>100
>100
>100
>100
4.5 ( 0.3c
>100c
0.57 ( 0.16
>100
5.5 ( 0.6
>100
>100
7.4 ( 0.2
>1000
96 ( 5
>1000
>1000
0.15 ( 0.03
1.7 ( 0.2
160 ( 15
0.71 ( 0.11
>1000
12 ( 1
0.11 ( 0.01
100
kainic acid
4.0 ( 1.2
0.007 ( 0.002
Nd
a
b
Reference 14. Reference 10. c Reference 15. Nd, not determined.
Exp er im en ta l Section
Ch em istr y. Melting points were determined in capillary
tubes and are uncorrected. Elemental analyses were per-
formed by Mr. G. Cornali, Microanalytical Laboratory, LEO
Pharmaceutical Products, Denmark, and are within (0.4% of
the calculated values. Flash chromatography (FC) was per-
formed on Merck silica gel 60 H (5-40 µm) and column
chromatography (CC) on Merck silica gel 60 (70-230 mesh,
ASTM). Analytical thin-layer chromatography (TLC) was
carried out using Merck silica gel F254 plates. Compounds
containing the 3-isoxazolol moiety were visualized using a
FeCl3 spraying reagent, and compounds containing amino
groups were visualized using a ninhydrin spraying reagent.
All compounds under study were also detected on TLC plates
using UV light and a KMnO4 spraying reagent. 1H NMR
spectra were recorded on a Bruker AC 200F (200-MHz) or a
Varian 360L (60-MHz) spectrometer. DMF was dried over
CaH2 and distilled. IR spectra were recorded from KBr disks
on a Perkin-Elmer 781 grating infrared spectrophotometer. CD
and UV spectra of 8 and 9 (0.06 mM, 0.1 M HCl) were recorded
at ambient temperature in 1.0-cm cuvettes on a J asco J -720
spectropolarimeter and on a Perkin-Elmer Lambda 2 spectro-
photometer, respectively. Optical rotations were measured in
thermostated cuvettes on a Perkin-Elmer 241 polarimeter.
High -P er for m an ce Liqu id Ch r om atogr aph y. The HPLC
instrumentation used for the Chirobiotic T column (10 × 500
mm, ASTEC) consisted of a J asco 880PU pump, a Rheodyne
7125 injector equipped with a 5.0-mL loop, and a Waters M480
detector set at 254 nm connected to a Merck-Hitachi Chro-
mato-Integrator D-2000. The column was eluted at 1.2 mL/
min with 10 mM aqueous NH4OAc/EtOH (20:80). Chiral
HPLC with the analytical Chirobiotic T column (4.6 × 150 mm,
ASTEC) was performed using a Waters M510 pump connected
to a Waters U6K injector and a Waters 991 photodiode array
detector. The column was eluted at ambient temperature with
0.5 mL/min 10 mM NH4OAc (pH 4.0)/EtOH (20:80). Chiral
ligand-exchange HPLC was performed on a column (4.6 × 120
mm) containing (S)-pipecolic acid chemically bound to a silica-
based packing material following a procedure used for attach-
ment of (S)-proline.39 The column (50 °C) was eluted with 1.0
mL/min 50 mM KH2PO4 (pH 4.6) containing 0.1 mM CuSO4
and was connected to the same HPLC instrumentation used
for the analytical Chirobiotic T column. Reverse-phase HPLC
with a Knauer RP18 (20 × 250 mm) column was performed
using a Shimadzu LC-6A pump, a Rheodyne injector equipped
with a 5.0-mL loop, and a Shimadzu SPD-6A detector set at
254 nm. The column was eluted at 5.0 mL/min with 100 mM
aqueous HOAc/MeOH (60:40). Analytical reverse-phase HPLC
was performed on a Supelco LC-18-DB column (4.6 × 150 mm).
The column was eluted at ambient temperature with 1.0 mL/
min 15 mM aqueous HOAc/MeOH (70:30) and was connected
to the HPLC instrumentation used for the analytical Chiro-
F igu r e 5. Comparison of [3H]AMPA receptor binding with
electrophysiological data for compounds 8, 7f, 5, 7a , 7c, and
7g showing a decreasing order of affinity and potency. Data
from Table 3.
cally inactive (Table 3). Thus, within this class of
compounds, the AMPA receptors impose different and
strict stereochemical and structural demands on ago-
nists and antagonists. This conclusion is supported by
the observation that the bromo analogue of 7f, com-
pound 7g, is some 2 orders of magnitude weaker than
the parent compound as an AMPA agonist (Table 3).
The synthesis of 7f (Scheme 2) was seriously impeded
by the sensitivity of the furan ring to acidic reaction
conditions. Furthermore, 7f turned out to be highly
sensitive to light. This photosensitivity was confirmed
in a series of photochemical decomposition studies as
illustrated in Figure 3. Considering the high AMPA
receptor affinity and agonist potency of 8, this photo-
chemical sensitivity is interesting, and we have initiated
the characterization of 8 as a potentially useful photo-
label for native and recombinant AMPA receptors. The
results on these studies are in progress and will be
reported separately.

Heteroaryl Analogues of AMPA
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2519
biotic T column. The ee values for 8 and 9 and the chemical
purity of compound 7g were based on peak areas at 280 nm.
Syn th eses of 10a -e, 11c, a n d 12c. The methyl 2-prope-
noates (10a -e) were prepared in analogy with the preparation
of ethyl 3-(3-pyridyl)propenoate40 from the acids corresponding
to 10a ,41 10b,40 10d ,42 and 10e.43 The syntheses of compounds
11c and 12c have previously been reported.44
Gen er a l P r oced u r e for th e P r ep a r a tion of (2RS,3SR)-
Meth yl 2,3-Dibr om opr opion ate Hydr obr om ides 11a,b,d,e.
To a solution of 10a ,b,d , or e (25 mmol) in HOAc (35 mL) was
added HBr in HOAc (33%, 25 mmol) followed by a solution of
Br2 (25 mmol) in HOAc (35 mL). The mixture was stirred at
60 °C for 4 h and left at room temperature overnight. The
precipitate was collected, washed with HOAc and Et2O, and
recrystallized from MeOH-Et2O.
7.35 (1H, dd, J ) 5 and 7 Hz), 6.55 (1H, s), 4.05 (3H, s). Anal.
(C9H8N2O2) C, H, N.
14a : yield 14%; mp 137-139 °C (EtOAc-light petroleum);
1H NMR (60 MHz, CDCl3) δ 8.75 (1H, dt, J ) 2 and 6 Hz),
8.0-7.6 (2H, m), 7.50-7.30 (1H, m), 6.35 (1H, s), 3.65 (3H, s).
Anal. (C9H8N2O2) C, H, N.
13b: yield 47%; mp 80-81 °C (heptane); 1H NMR (60 MHz,
CDCl3) δ 9.05 (1H, d, J ) 3 Hz), 8.70 (1H, dd, J ) 2 and 5
Hz), 8.10 (1H, dt, J ) 2 and 9 Hz), 7.40 (1H, dd, J ) 5 and 9
Hz), 6.25 (1H, s), 4.05 (3H, s). Anal. (C9H8N2O2) C, H, N.
14b: yield 10%; mp 98-99.5 °C (EtOAc-light petroleum);
1H NMR (60 MHz, CDCl3) δ 9.05 (1H, d, J ) 3 Hz), 8.85 (1H,
dd, J ) 2 and 5 Hz), 8.05 (1H, dt, J ) 2 and 9 Hz), 7.55 (1H,
dd, J ) 5 and 9 Hz), 6.25 (1H, s), 3.65 (3H, s). Anal.
(C9H8N2O2) C, H, N.
13c: yield 60%; mp 126-128 °C (heptane); 1H NMR (60
MHz, CDCl3) δ 8.55 (2H, d, J ) 5 Hz), 7.40 (2H, dd, J ) 1 and
5 Hz), 6.10 (1H, s), 3.90 (3H, s). Anal. (C9H8N2O2) C, H, N.
14c: yield 19%; mp 134-137 °C (EtOAc-light petroleum);
1H NMR (60 MHz, CDCl3) δ 8.65 (2H, dd, J ) 1 and 5 Hz),
7.45 (2H, dd, J ) 1 and 5 Hz), 6.15 (1H, s), 3.55 (3H, s). Anal.
(C9H8N2O2) C, H, N.
11a ‚HBr : yield 91%; mp 164-167 °C; 1H NMR (60 MHz,
DMSO-d6) δ 8.75 (1H, dd, J ) 2 and 6 Hz), 8.1-7.85 (2H, m),
7.7-7.4 (1H, m), 5.85 (1H, d, J ) 11 Hz), 5.40 (1H, d, J ) 11
Hz), 3.85 (3H, s). Anal. (C9H9Br2NO2‚HBr) C, H, Br, N.
11b‚HBr : yield 87%; mp 182-184 °C; 1H NMR (60 MHz,
DMSO-d6) δ 9.05 (1H, br s), 8.9-8.6 (2H, m), 8.00 (1H, dd, J
) 1.5 and 8 Hz), 5.75 (1H, d, J ) 11 Hz), 5.40 (1H, d, J ) 11
Hz), 3.80 (3H, s). Anal. (C9H9Br2NO2‚HBr) C, H, Br, N.
13d : yield 51%; mp 58-60 °C (heptane); 1H NMR (60 MHz,
CDCl3) δ 8.85-7.75 (2H, m), 7.35 (1H, dd, J ) 5 and 10 Hz),
6.60 (1H, s), 4.05 (3H, s), 2.55 (3H, s). Anal. (C10H10N2O2) C,
H, N.
1
11d ‚HBr : yield 94%; mp 189-190 °C; H NMR (60 MHz,
DMSO-d6) δ 8.05 (1H, d, J ) 9 Hz), 7.85 (1H, dd, J ) 2 and 9
Hz), 7.55 (1H, dd, J ) 2 and 8 Hz), 5.80 (1H, d, J ) 11 Hz),
5.45 (1H, d, J ) 11 Hz), 3.85 (3H, s), 2.60 (3H, s). Anal.
(C10H11Br2NO2‚HBr) C, H, N.
14d : yield 17%; mp 77-79 °C (EtOAc-light petroleum); 1H
NMR (60 MHz, CDCl3) δ 8.0-7.25 (3H, m), 6.45 (1H, s), 3.70
(3H, s), 2.65 (3H, s). Anal. (C10H10N2O2) C, H, N.
13e: yield 59%; mp 93-94 °C (heptane); 1H NMR (60 MHz,
CDCl3) δ 8.4-7.5 (6H, m), 6.70 (1H, s), 4.10 (3H, s). Anal.
(C13H10N2O2) C, H, N.
11e‚HBr : yield 94%; mp 171-174 °C; 1H NMR (60 MHz,
DMSO-d6) δ 8.55 (1H, d, J ) 9 Hz), 8.2-7.65 (5H, m), 5.80
(1H, d, J ) 11 Hz), 5.55 (1H, d, J ) 11 Hz), 3.85 (3H, s). Anal.
(C13H11Br2NO2‚HBr) C, H, Br, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Su bsti-
tu ted 3-Isoxa zolols 12a ,b,d ,e. To an ice-cooled solution of
NaOH (140 mmol) in MeOH (100 mL) was added hydroxy-
lamine hydrochloride (50 mmol). The mixture was stirred for
10 min, and 11a ,b,d , or e (20 mmol) was added in portions
during a period of 1 h. After 1 h at 0 °C, the mixture was
refluxed for 2 h and evaporated. H2O (50 mL) was added to
the residue, and the pH of the solution was adjusted to 4 with
concentrated HCl. The precipitate was collected and washed
with H2O.
14e: yield 17%; 1H NMR (60 MHz, CDCl3) δ 8.45-7.40 (6H,
m), 6.60 (1H, s), 3.60 (3H, s).
3-Met h oxy-4-b r om o-5-(3-p yr id yl)isoxa zole (15b ). Br2
(0.76 mL, 15 mmol) in HOAc (13 mL) was added to a solution
of 13b (2.36 g, 13.6 mmol) in HOAc (13 mL). The mixture
was stirred at 60 °C for 2 days and cooled. The precipitate
was collected and added to NaHCO3 (5%, 15 mL). The mixture
was extracted with CH2Cl2 (2 × 35 mL), and the combined
extracts were washed with NaHSO3 (10%, 20 mL), dried, and
evaporated. FC of the residue [tol-EtOAc (0-30%)] gave 15b
1
12a : yield 63%; mp > 225 °C (MeOH-H2O); H NMR [60
1
(1.67 g, 49%): mp 111-113 °C (heptane); H NMR (60 MHz,
MHz, CDCl3-DMSO-d6 (1:1)] δ 8.70 (1H, dt, J ) 1.5 and 6
Hz), 8.0-7.75 (2H, m), 7.55-7.25 (1H, m), 6.55 (1H, s). Anal.
(C8H6N2O2) C, H, N.
CDCl3) δ 9.30 (1H, d, J ) 3 Hz), 8.75 (1H, dd, J ) 2 and 5
Hz), 8.25 (1H, dt, J ) 2 and 8 Hz), 7.45 (1H, dd, J ) 5 and 8
Hz), 4.10 (3H, s). Anal. (C9H7BrN2O2) C, H, Br, N.
12b: yield 56%; mp 211-215 °C dec (EtOH-H2O); 1H NMR
(60 MHz, DMSO-d6) δ 9.10 (1H, d, J ) 3 Hz), 8.75 (1H, dd, J
) 2 and 5 Hz), 8.25 (1H, dt, J ) 2 and 9 Hz), 7.60 (1H, dd, J
) 5 and 9 Hz), 6.75 (1H, s). Anal. (C8H6N2O2) C, H, N.
12d : yield 58%; mp 206-208 °C (EtOH-H2O); 1H NMR (60
MHz, CDCl3-DMSO-d6 [9:1]) δ 8.0-7.6 (2H, m), 7.25 (1H, dd,
J ) 2 and 7 Hz), 6.55 (1H, s), 2.55 (3H, s). Anal. (C9H8N2O2)
C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Su bsti-
tu ted 3-Meth oxy-4-(h yd r oxym eth yl)isoxa zoles 16. A so-
lution of 13a ,c-e or 15b (5 mmol) in dry THF (35 mL) was
cooled to -78 °C, and a solution of n-butyllithium in hexane
(1.6 M, 7.5 mmol) was added during 10 min. Paraformalde-
hyde (45 mmol) was added, and the mixture was stirred at
-78 °C for 30 min and then at room temperature for 2 h. The
reaction mixture was evaporated, and H2O (20 mL) and CH2-
Cl2 (30 mL) were added to the residue. The pH was adjusted
to 6 with 4 M HCl, and the phases were separated. The
aqueous phase was extracted with CH2Cl2 (2 × 30 mL), and
the combined organic phases were dried and evaporated.
Compounds 16 were isolated by FC [tol-EtOAc (10-50%)] and
crystallized from EtOAc-light petroleum.
12e: The crude product was extracted with boiling EtOAc,
and the extracts were submitted to FC [tol-EtOAc (0-25%)];
yield 43%; mp 207-209 °C (EtOAc); 1H NMR (60 MHz, DMSO-
d6) δ 8.45 (1H, d, J ) 9 Hz), 8.25-7.6 (5H, m), 6.75 (1H, s).
Anal. (C12H8N2O2) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Su bsti-
tu ted 3-Meth oxyisoxa zoles 13 a n d 5-Su bstitu ted 2-Meth -
ylisoxa zolin -3-on es 14. A solution of 12 (10 mmol) and
NaOH (10 mmol) in H2O (15 mL) and EtOH (15 mL) was
evaporated to dryness and further dried for 2 h at 2 Pa. The
residue was suspended in dry DMF (15 mL) and cooled to -10
°C. Me2SO4 (11 mmol) was added dropwise, and the mixture
was stirred at -10 °C for 1 h and then at room temperature
for 15 h. The resulting solution was evaporated, and H2O (25
mL) was added to the residue. Extraction with CH2Cl2 (3 ×
50 mL), drying, and evaporation gave a mixture of 13 and 14,
which was submitted to FC [tol-EtOAc (1:1)]. The first
fractions contained 13 and the later fractions 14.
1
16a : yield 62%; mp 90-92 °C; H NMR (60 MHz, CDCl3) δ
8.70 (1H, dt, J ) 1 and 5 Hz), 7.90 (2H, m), 7.35 (1H, m), 4.60
(2H, s), 4.05 (3H, s). Anal. (C10H10N2O3) C, H, N.
16b : from 15b; yield 58%; mp 136-139 °C; 1H NMR (60
MHz, CDCl3) δ 9.10 (1H, d, J ) 3 Hz), 8.70 (1H, dd, J ) 2 and
5 Hz), 8.20 (1H, dt, J ) 2 and 8 Hz), 7.45 (1H, dd, J ) 5 and
8 Hz), 4.65 (2H, s), 4.10 (3H, s). Anal. (C10H10N2O3) C, H, N.
16c: yield 76%; mp 135-137 °C; 1H NMR (60 MHz, CDCl3)
δ 8.70 (2H, m), 7.75 (2H, dd, J ) 2 and 6 Hz), 4.60 (2H, s),
4.05 (3H, s). Anal. (C10H10N2O3) C, H, N.
16d : yield 51%; mp 119-121 °C; 1H NMR (60 MHz, CDCl3)
δ 7.90 (2H, d, J ) 5 Hz), 7.35 (1H, m), 4.65 (2H, s), 4.05 (3H,
s), 2.65 (3H, s). Anal. (C11H12N2O3) C, H, N.
13a : yield 59%; mp 45-47 °C (heptane); 1H NMR (60 MHz,
CDCl3) δ 8.70 (1H, dt, J ) 2 and 7 Hz), 7.95-7.75 (2H, m),

2520 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Falch et al.
16e: yield 52%; mp 143-145 °C; 1H NMR (200 MHz, CDCl3)
δ 8.42 (1H, d, J ) 9 Hz), 8.2-7.4 (5H, m), 4.76 (2H, s), 4.12
(3H, s). Anal. (C14H12N2O3) C, H, N.
7b: yield 69%; mp > 225 °C; 1H NMR (200 MHz, DMSO-d6)
δ 8.84 (1H, d, J ) 1.4 Hz), 8.67 (1H, dd, J ) 1.4 and 4 Hz),
8.08 (1H, d, J ) 8 Hz), 7.55 (1H, dd, J ) 4 and 8 Hz), 3.68
(1H, m), 2.93 (1H, dd, J ) 7 and 15 Hz), 2.76 (1H, dd, J ) 4
and 15 Hz). Anal. (C11H11N3O4‚0.33H2O) C, H, N.
7c: yield 76%; mp > 225 °C; 1H NMR (200 MHz, DMSO-d6)
δ 8.72 (2H, d, J ) 6 Hz), 7.63 (2H, d, J ) 6 Hz), 3.73 (1H, dd,
J ) 3.6 and 6 Hz), 3.07-2.81 (2H, m). Anal. (C11H11N3O4‚
H2O) C, H, N.
7d : yield 61%; mp 215-217 °C; 1H NMR (200 MHz, DMSO-
d6) δ 7.87 (1H, t, J ) 8 Hz), 7.62 (1H, d, J ) 7 Hz), 7.36 (1H,
d, J ) 8 Hz), 3.75 (1H, br s), 3.45 (1H, dd, J ) 4.5 and 15 Hz),
3.11 (1H, dd, J ) 7 and 15 Hz), 2.55 (3H, s). Anal. (C12H13N3O4‚
H2O) C, H, N.
7e: yield 32%; mp > 225 °C; 1H NMR (200 MHz, DMSO-d6)
δ 8.55 (1H, d, J ) 8 Hz), 8.19 (1H, d, J ) 8 Hz), 8.07-7.68
(4H, m), 3.84 (1H, br s), 3.63 (1H, d, J ) 14 Hz), 3.34 (1H, dd,
J ) 3 and 14 Hz). Anal. (C15H13N3O4‚H2O) C, H, N.
3-(2-F u r yl)-4-m eth ylisoxa zolin -5-on e (20). A solution of
hydroxylamine hydrochloride (1.0 g, 14 mmol) in NaOH (0.5
M, 20 mL) at 2 °C was adjusted to pH 10 with 0.5 M NaOH.
The pH of the reaction mixture was kept at 10.0 ( 0.2 by using
a pH-state (TTT80 combined with an ABU80 autoburet; both
from Radiometer, Copenhagen) while 1945 (2.0 g, 11 mmol) was
added dropwise over a period of 30 min. The mixture was
stirred at room temperature for 2 h, and concentrated HCl
(10 mL) was added in one portion. The mixture was left at 5
°C for 20 h, and the precipitate (1.42 g, 71%) was collected. A
sample was recrystallized (EtOAc-light petroleum) to give
20: mp 134-136 °C; 1H NMR (200 MHz, DMSO-d6) δ 7.48
(1H, dd, J ) 0.6 and 1.8 Hz), 6.73 (1H, dd, J ) 0.6 and 3.5
Hz), 6.43 (1H, dd, J ) 1.8 and 3.5 Hz), 1.88 (3H, s). Anal.
(C8H7NO3) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Su bsti-
tu ted 3-Meth oxy-4-(ch lor om eth yl)isoxa zoles 17. A mix-
ture of 16 (2 mmol) and thionyl chloride (10 mL) was refluxed
for 2 h and evaporated. Crystallization of the respective
residues gave 17b‚HCl and 17c‚HCl. Otherwise, NaHCO3
(5%, 15 mL) was added to the residue, and the mixture was
extracted with CH2Cl2 (2 × 25 mL). The combined extracts
were dried, evaporated, and crystallized.
1
17a : yield 83%; mp 88-89 °C (Et2O-light petroleum); H
NMR (60 MHz, CDCl3) δ 8.75 (1H, dt, J ) 1.5 and 5 Hz), 7.85
(2H, m), 7.30 (1H, m), 5.05 (2H, s), 4.15 (3H, s). Anal. (C10H9-
ClN2O2) C, H, Cl, N.
17b‚HCl: yield 84%; mp 212-215 °C (MeOH-Et2O); 1H
NMR (200 MHz, DMSO-d6) δ 8.96 (1H, s), 8.72 (1H, d, J ) 5
Hz), 8.48 (1H, d, J ) 8 Hz), 7.88 (1H, dd, J ) 5 and 8 Hz),
4.39 (2H, s), 3.85 (3H, s). Anal. (C10H9ClN2O2‚HCl) C, H, Cl,
N.
17c‚HCl: yield 63%; mp > 225 °C (MeOH-Et2O); 1H NMR
(60 MHz, D2O) δ 9.30 (2H, dd, J ) 2 and 7 Hz), 8.55 (2H, dd,
J ) 2 and 7 Hz), 4.90 (2H, s), 4.20 (3H, s). Anal. (C10H9-
ClN2O2‚HCl) C, H, Cl, N.
17d : yield 87%; mp 102-104 °C (Et2O-light petroleum); 1H
NMR (60 MHz, CDCl3) δ 7.90-7.75 (2H, m), 7.45-7.25 (1H,
m), 5.15 (2H, s), 4.20 (3H, s), 3.65 (3H, s). Anal. (C11H11
ClN2O2) C, H, N.
-
17e: yield 64%; mp 123-126 °C (EtOAc-Et2O-light petro-
leum); 1H NMR (60 MHz, CDCl3) δ 8.4-7.55 (6H, m), 5.15 (2H,
s), 4.15 (3H, s). Anal. (C14H11ClN2O2) C, H, Cl, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Meth yl
2-Acet a m id o-2-(m et h oxyca r b on yl)-3-(3-m et h oxy-5-su b -
stitu ted-4-isoxazolyl)pr opion ates 18. Sodium hydride (60%
in mineral oil, 2.5 mmol) was added in small portions to a
solution of dimethyl acetaminomalonate (2.2 mmol) in dry
DMF (5 mL). The mixture was stirred at room temperature
for 1 h. A solution of 17 (2.0 mmol) in dry DMF (5 mL) was
added, and stirring was continued for 6-18 h. The mixture
was evaporated, and H2O (20 mL) was added to the residue.
Extraction with CH2Cl2 (3 × 25 mL), drying, and FC of the
residue [tol-EtOAc (10-80%)] gave compounds 18.
5-(2-F u r yl)-4-m eth ylisoxa zol-3-ol (21). Hydroxylamine
hydrochloride (2.45 g, 35 mmol) and NaOH (1.44 g, 36 mmol)
were suspended in MeOH (25 mL) and water (2.5 mL), and
the mixture was cooled in an ice bath. After stirring for 10
min, the mixture was filtered. The filtrate was dropwise added
to a solution of 1945 (3.20 g, 17.6 mmol) and NaOH (0.70 g,
17.6 mmol) in MeOH (25 mL) and H2O (2.5 mL) at -35 °C.
The mixture was stirred at -35 °C for 3 h and then at 5 °C
for 30 min. Concentrated HCl (35 mL) was added in one
portion, and the mixture was evaporated. H2O (35 mL) was
added to the residue, and the mixture was left at 5 °C for 20
h. The precipitate (1.99 g, 68%) (mp 160-163 °C) was almost
pure 21 contaminated with 20. FC [tol-EtOAc (10:1)] followed
by recrystallization (EtOAc-light petroleum) gave pure 21:
1
18a : yield 76%; oil; H NMR (60 MHz, CDCl3) δ 8.70 (1H,
dt, J ) 2 and 5 Hz), 7.95-7.80 (2H, m), 7.35 (1H, m), 4.05
(3H, s), 3.85 (2H, s), 3.70 (6H, s), 1.70 (3H, s).
18b: yield 70%; oil; 1H NMR (200 MHz, CDCl3) δ 8.93 (1H,
d, J ) 2 Hz), 8.69 (1H, d, J ) 5 Hz), 7.95 (1H, dt, J ) 2 and
8 Hz), 7.43 (1H, dd, J ) 5 and 8 Hz), 4.01 (3H, s), 3.69 (2H, s),
3.60 (6H, s), 1.63 (3H, s).
1
mp 164-166 °C; H NMR (200 MHz, DMSO-d6) δ 7.45 (1H,
dd, J ) 0.6 and 1.8 Hz), 6.64 (1H, d, J ) 3.4 Hz), 6.42 (1H, dd,
J ) 1.8 and 3.4 Hz), 2.01 (3H, s). Anal. (C8H7NO3) C, H, N.
18c: yield 58%; mp 174-175 °C (EtOAc-light petroleum);
1H NMR (60 MHz, CDCl3) δ 8.80 (2H, dd, J ) 2 and 5 Hz),
7.65 (2H, dd, J ) 2 and 5 Hz), 4.05 (3H, s), 3.75 (2H, s), 3.65
(6H, s), 1.60 (3H, s). Anal. (C17H19N3O7) C, H, N.
18d : yield 75%; mp 118-120 °C (light petroleum); 1H NMR
(60 MHz, CDCl3) δ 8.75-8.60 (1H, m), 7.4-7.1 (2H, m), 3.95
(3H, s), 3.75 (2H, s), 3.65 (6H, s), 2.55 (3H, s), 1.55 (3H, s).
Anal. (C18H21N3O7) C, H, N.
18e: yield 36%; mp 167-168 °C (EtOAc-light petroleum);
1H NMR (60 MHz, CDCl3) δ 8.5-7.4 (6H, m), 4.05 (3H, s), 3.80
(2H, s), 3.55 (6H, s), 1.55 (3H, s). Anal. (C21H21N3O7) C, H,
N.
Gen er a l P r oced u r e for th e P r ep a r a tion of (RS)-2-
Am in o-3-(3-h yd r oxy-5-su b st it u t ed -4-isoxa zolyl)p r op i-
on ic Acid s 7a -e. A solution of 18 in aqueous HBr (48%, 20
mL) was refluxed for 1 h. The reaction mixture was evapo-
rated, and the residue was dissolved in H2O (10 mL). The
solution was treated with charcoal, and 7 was precipitated by
addition of 1 M Na2CO3 to pH 3.
3-[(P iva loyloxy)m et h yloxy]-4-m et h yl-5-(2-fu r yl)isox-
a zole (22) a n d 2-[(P iva loyloxy)m et h yl]-4-m et h yl-5-(2-
fu r yl)isoxa zolin -3-on e (23). Chloromethyl pivalate (5.80
mL, 40 mmol) was added to a solution of NaI (6.0 g, 40 mmol)
in acetone (50 mL), and the mixture was stirred at room
temperature for 2 h. After filtration, the filtrate was evapo-
rated and the iodomethyl pivalate was dissolved in DMF (20
mL). A mixture of 21 (2.23 g, 13.5 mmol) and K2CO3 (3.73 g,
27 mmol) in DMF (60 mL) was stirred at 40 °C for 1 h, and
the solution of iodomethyl pivalate was added. Stirring was
continued for 20 h at 40 °C, and the mixture was evaporated.
Water (50 mL) was added, and the mixture was extracted with
EtOAc (3 × 75 mL). The combined organic extracts were dried
and evaporated to give a mixture of 22 and 23. The com-
pounds were separated by FC (tol). The first fractions
contained 22 (oil, 2.55 g, 68%): 1H NMR (60 MHz, CDCl3) δ
7.90 (1H, d, J ) 2 Hz), 7.10 (1H, d, J ) 3.5 Hz), 6.85 (1H, dd,
J ) 2 and 3.5 Hz), 6.25 (2H, s), 2.25 (3H, s), 1.25 (9H, s). The
latter fractions contained 23 (0.80 g, 21%): mp 91-92 °C
(Et2O-light petroleum); 1H NMR (60 MHz, CDCl3) δ 7.85 (1H,
d, J ) 2 Hz), 7.10 (1H, d, J ) 3.5 Hz), 6.75 (1H, dd, J ) 2 and
7a : yield 76%; mp > 225 °C; 1H NMR (200 MHz, DMSO-d6)
δ 8.68 (1H, d, J ) 5 Hz), 7.98 (1H, t, J ) 7 Hz), 7.85 (1H, d, J
) 7 Hz), 7.47 (1H, t, J ) 5 Hz), 3.74 (1H, m), 3.41 (1H, dd, J
) 3 and 12 Hz), 3.15 (1H, dd, J ) 3 and 12 Hz). Anal.
(C11H11N3O4‚0.25H2O) C, H, N.
3.5 Hz), 6.00 (2H, s), 2.20 (3H, s), 1.20 (9H, s). Anal. (C14H17
NO5) C, H, N.
-

Heteroaryl Analogues of AMPA
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2521
3-[(P ivaloyloxy)m eth yloxy]-4-(br om om eth yl)-5-(2-fu r yl)-
isoxa zole (24). A mixture of 22 (4.62 g, 16.5 mmol) and
N-bromosuccinimide (NBS) (3.50 g, 19.8 mmol) in CCl4 (200
mL) was refluxed for 20 h. The reaction mixture was filtered,
and the filtrate was evaporated. FC (tol) of the residue gave
24 (3.70 g, 63%) as an oil: 1H NMR (60 MHz, CDCl3) δ 7.85
(1H, d, J ) 2 Hz), 7.20 (1H, d, J ) 3.5 Hz), 6.80 (1H, dd, J )
2 and 3.5 Hz), 6.15 (2H, s), 4.50 (2H, s), 1.25 (9H, s).
3-(P ivaloyloxy)-4-m eth yl-5-(2-fu r yl)isoxazole (31). Com-
pound 31 was prepared from 21 by the method described for
the synthesis of 27. FC (tol) and recrystallization (EtOAc-
1
light petroleum) gave 31 (87%): mp 73-74 °C; H NMR (60
MHz, CDCl3) δ 7.65 (1H, d, J ) 1.5 Hz), 6.85 (1H, d, J ) 3.5
Hz), 6.55 (1H, dd, J ) 1.5 and 3.5 Hz), 2.05 (3H, s), 1.40 (9H,
s). Anal. (C13H15NO4) C, H, N.
3-(P iva loyloxy)-4-(br om om et h yl)-5-(2-fu r yl)isoxa zole
(32). Compound 32 was prepared from 31 by the method
described for the synthesis of 24. FC (tol) and recrystallization
Eth yl 2-[N-(ter t-Bu tyloxyca r bon yl)a m in o]-2-(eth oxy-
car bon yl)-3-[3-[(pivaloyloxy)m eth yloxy]-5-(2-fu r yl)-4-isox-
a zolyl]p r op ion a te (25). Sodium hydride (60% in mineral oil,
0.64 g, 16 mmol) was added to a solution of diethyl N-(tert-
butyloxycarbonyl)aminomalonate46 (3.32 g, 12 mmol) in DMF
(20 mL). The mixture was stirred at room temperature for 1
h. A solution of 24 (3.58 g, 10 mmol) in DMF (20 mL) was
added, and stirring was continued for 20 h. After evaporation,
H2O (40 mL) was added and the mixture was extracted with
EtOAc (3 × 50 mL). The extracts were dried and evaporated.
FC [tol-EtOAc (0-10%)] of the residue afforded 3.70 g (67%)
of almost pure 25 as an oil. CC (tol-EtOAc) of the oil followed
by recrystallization (Et2O-light petroleum) gave pure 25: mp
1
(light petroleum) gave 32 (76%): mp 66-67 °C; H NMR (60
MHz, CDCl3) δ 7.60 (1H, d, J ) 1.5 Hz), 7.00 (1H, d, J ) 3.5
Hz), 6.55 (1H, dd, J ) 1.5 and 3.5 Hz), 4.40 (2H, s), 1.40 (9H,
s). Anal. (C13H14BrNO4) C, H, N.
(RS)-2-Am in o-3-[3-h yd r oxy-5-(5-br om o-2-fu r yl)-4-isox-
a zolyl]p r op ion ic Acid (7g). A mixture of 31 (2.04 g, 8.18
mmol), NBS (1.74 g, 9.77 mmol), and benzoyl peroxide (50 mg,
0.2 mmol) in CCl4 (100 mL) was refluxed for 20 h. Another
portion of NBS (1.74 g) and benzoyl peroxide (50 mg) were
added, and the mixture was refluxed for an additional 20 h.
Filtration and evaporation followed by FC (tol) gave 2.44 g of
a 1:2 mixture of 32 and 33, respectively. This mixture was
reacted with diethyl N-(tert-butyloxycarbonyl)aminomalonate
by the method described for the synthesis of 25. FC (tol) gave
34 (oil, 2.1 g): 1H NMR (60 MHz, CDCl3) δ 6.85 (1H, d, J )
3.5 Hz), 6.40 (1H, d, J ) 3.5 Hz), 4.4-3.9 (4H, m), 3.45 (2H,
br s), 1.45 (9H, s), 1.40 (9H, s), 1.6-1.1 (6H, m).
Compound 34 was deprotected by the method described for
the synthesis of 7f. Crude 7g was dissolved in dilute HCl,
treated with charcoal, and reprecipitated by addition of Na2-
CO3 (1 M) to give 7g (0.48 g, 46%): mp > 230 °C; 1H NMR
(200 MHz, DMSO-d6) δ 7.02 (1H, d, J ) 3.5 Hz), 6.84 (1H, d,
J ) 3.5 Hz), 3.68 (1H, t, J ) 5.1 Hz), 2.91 (2H, d, J ) 5.1 Hz).
Anal. (C10H9BrN2O5) C, H, N.
1
96-98 °C; H NMR (200 MHz, CDCl3) δ 7.54 (1H, d, J ) 1.8
Hz), 6.92 (1H, d, J ) 3.4 Hz), 6.47 (1H, dd, J ) 1.8 and 3.4
Hz), 5.88 (2H, s), 4.4-4.2 (2H, m), 4.2-4.0 (2H, m), 3.66 (2H,
br s), 1.34 (9H, s), 1.23 (9H, s), 1.5-1.2 (6H, m). Anal.
(C26H36N2O11) C, H, N.
(RS)-2-Am in o-3-[3-h ydr oxy-5-(2-fu r yl)-4-isoxazolyl]pr o-
p ion ic Acid (7f). A mixture of 25 (1.16 g, 2.10 mmol), NaOH
(2 M, 10 mL), and EtOH (4 mL) was refluxed for 1.5 h and
evaporated. The residue was dissolved in H2O (20 mL), and
the aqueous solution was washed with EtOAc (2 × 20 mL).
To the aqueous solution was added HCl (4 M, 8 mL), and the
mixture was refluxed for 1 h, cooled, and washed with EtOAc
(2 × 25 mL). The aqueous solution was treated with charcoal
and evaporated. The residue was dissolved in H2O (6 mL) at
80 °C, and 1 M Na2CO3 was added to pH 3. After 20 h at 5
°C, the precipitate (435 mg, 81%) was collected. Recrystalli-
zation (EtOH-H2O) afforded 7f: mp > 240 °C; 1H NMR (200
MHz, DMSO-d6) δ 7.90 (1H, d, J ) 1.1 Hz), 7.00 (1H, d, J )
3.4 Hz), 6.70 (1H, dd, J ) 1.1 and 3.4 Hz), 3.67 (1H, m), 2.98
(2H, m). Anal. (C10H10N2O5‚H2O) C, H, N.
3-(2-F u r yl)-4-m eth yl-5-(ben zoyloxy)isoxa zole (26) a n d
2-Ben zoyl-3-(2-fu r yl)-4-m eth ylisoxa zolin -5-on e (27). Tri-
ethylamine (4.67 mL, 33.5 mmol) was added to a mixture of
20 (4.61 g, 27.9 mmol) and benzoyl chloride (3.40 mL, 29.3
mmol) in tol (110 mL). After stirring for 20 h at room
temperature, the reaction mixture was washed with NaHCO3
(5%, 100 mL) and H2O (100 mL), dried, and evaporated. FC
(tol) of the residue first eluted 26 (1.68 g, 22%) and subse-
quently 27 (3.91 g, 52%). Both compounds were recrystallized
from EtOAc-light petroleum.
The isolated 7g was shown to contain 0.25% of 7f as
determined on the Supelco-LC-18-DB column. Therefore, 7g
was submitted to preparative reverse-phase HPLC using the
Knauer RP18 column (20 × 250 mm). Appropriate fractions
containing 7g were combined and evaporated. Recrystalliza-
tion of the crystalline residue (H2O) gave 7g with a content of
7f < 0.05% as determined by analytical reverse-phase HPLC.
(S)-2-Am in o-3-[3-h yd r oxy-5-(2-fu r yl)-4-isoxa zolyl]p r o-
p ion ic Acid (8) a n d (R)-2-Am in o-3-[3-h yd r oxy-5-(2-fu r yl)-
4-isoxa zolyl]p r op ion ic Acid (9). Compound 7f (250 mg,
0.98 mmol) was dissolved in H2O (30 mL) and resolved on the
Chirobiotic T column (10 × 500 mm) in 12 injections each of
2.5 mL. Fractions containing the first enantiomer, compound
8, were pooled and evaporated, reevaporated from H2O, and
finally lyophilized from H2O. Recrystallization of the crude
1
product (H2O) gave 8 (64 mg, 55%): mp > 240 °C; H NMR
(200 MHz, DMSO-d6 containing several drops of D2O) δ 7.72
(1H, d, J ) 1.5 Hz), 6.94 (1H, d, J ) 3.6 Hz), 6.61 (1H, dd, J
) 1.5 and 3.6 Hz), 3.69 (1H, d, J ) 1.5 Hz), 2.98 (2H, m); ee )
26: mp 88-90 °C; 1H NMR (60 MHz, CDCl3) δ 8.35 (2H, dd,
J ) 1.5 and 8 Hz), 7.85-7.60 (4H, m), 7.10 (1H, d, J ) 3.5
Hz), 6.70 (1H, dd, J ) 1.5 and 3.5 Hz), 2.10 (3H, s). Anal.
(C15H11NO4) C, H, N.
99.9%; [R]22.9 ) +22.5 (c ) 0.36, 1 M HCl); λmax (log ꢀ) ) 278
D
nm (4.3); ∆ꢀ(210 nm) ) +0.18 m2/mol. Anal. (C10H10N2O5) C,
H, N. The combined fractions containing the second enanti-
omer, compound 9, were processed as described above for 8
1
27: mp 149-150 °C; H NMR (60 MHz, CDCl3) δ 8.15 (2H,
affording 9 (65 mg, 56%): mp > 240 °C; ee > 99.8%; [R]22.9
)
m), 7.85-7.60 (4H, m), 7.10 (1H, d, J ) 3.5 Hz), 6.75 (1H, dd,
J ) 1.5 and 3.5 Hz), 2.20 (3H, s). Anal. (C15H11NO4) C, H, N.
D
-22.4 (c ) 0.39, 1 M HCl); λmax (log ꢀ) ) 278 nm (4.3); ∆ꢀ(210
1
nm) ) -0.20 m2/mol; H NMR and IR spectra were identical
2-Be n zoyl-3-(2-fu r yl)-4-(b r om om e t h yl)isoxa zolin -5-
on e (28). Compound 28 was prepared from 27 by the method
described for the synthesis of 24. 28: yield 79%; mp 131-
133 °C (EtOAc-light petroleum); 1H NMR (60 MHz, CDCl3) δ
8.40-7.65 (6H, m), 7.45 (1H, d, J ) 3.5 Hz), 6.85 (1H, dd, J )
1.5 and 3.5 Hz), 4.60 (2H, s). Anal. (C15H10BrNO4) C, H, N.
with those of compound 8.
X-r a y Cr ysta llogr a p h ic An a lysis of (R)-2-Am in o-3-[3-
h yd r oxy-5-(2-fu r yl)-4-isoxa zolyl]p r op ion ic Acid , Hyd r o-
ch lor id e (9‚HCl). Single crystals of 9‚HCl suitable for X-ray
crystallographic analysis were obtained from a solution of
compound 9 (4.5 mg, 19 µmol) in aqueous 4 M HCl (30 µL)
Eth yl 2-[N-(ter t-Bu tyloxyca r bon yl)a m in o]-2-(eth oxy-
c a r b o n y l)-3-[3-(2-fu r y l)-5-o x o -4-is o x a zo lin y l]p r o p i-
on a te (29). Compound 29 was prepared from 28 by the
method described for the synthesis of 25. FC (tol) and
recrystallization (EtOAc) gave 29 (67%): mp 155-157 °C; 1H
NMR (60 MHz, CDCl3) δ 7.75 (1H, br s), 7.35 (1H, d, J ) 3.5
Hz), 6.70 (1H, dd, J ) 1.5 and 3.5 Hz), 4.40 (4H, q, J ) 8 Hz),
3.65 (2H, s), 1.40 (9H, s), 1.30 (6H, t, J ) 8 Hz). Anal.
(C20H26N2O9) C, H, N.
and HOAc (200 µL). Crystal data: C10H10N2O5‚HCl, Mr
)
274.66, colorless elongated prisms, orthorhombic, space group
P212121 (No. 19), a ) 5.2616(9) Å, b ) 7.7536(11) Å, c )
28.326(4) Å, V ) 1155.6(3) Å3, Z ) 4, Dc ) 1.579 Mg m-3, F(000)
) 568, µ(Cu KR) ) 3.12 mm-1, T ) 122 ( 0.5 K, crystal
dimensions ) 0.14 × 0.23 × 0.42 mm.
Da ta Collection a n d P r ocessin g. Diffraction data were
collected on an Enraf-Nonius CAD-4 diffractometer,47 using

2522 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Falch et al.
graphite monochromated Cu KR radiation (λ ) 1.54184 Å) and
the ω - 2θ scan mode (θ ) 3-75°, (h, (k, (l). Unit cell
dimensions were determined by least-squares refinement of
20 reflections (θ range 41.31-42.61°). Data were reduced
using the programs of Blessing (DREADD).48,49 Five standard
reflections were monitored every 104 s, and appropiate scaling
was performed using the program SCALE3.48,49 Absorption
chloride, tables listing final atomic coordinates, equivalent
isotropic (non-hydrogen atoms) or isotropic (hydrogen atoms)
displacement parameters, anisotropic displacement param-
eters of the non-hydrogen atoms, and selected bond lengths
and angles (3 pages). Ordering information is given on any
current masthead page.
correction was applied using the program ABSORB (Tmin
)
Refer en ces
0.442, Tmax ) 0.683).48-50 A total of 9565 reflections were
averaged (Rint ) 0.028 on Fo2) according to the point group
symmetry 222 resulting in 2381 unique reflections.
(1) Krogsgaard-Larsen, P., Hansen, J . J ., Eds. Excitatory Amino
Acid Receptors: Design of Agonists and Antagonists; Ellis
Horwood: Chichester, 1992.
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Oxford University Press: Oxford, 1994.
(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: New J ersey, 1997.
(5) Conn, P. J ., Patel, J ., Eds. The Metabotropic Glutamate Recep-
tors; Humana Press: New J ersey, 1994.
(6) Danysz, W.; Parsons, C. G.; Bresink, I.; Quack, G. Glutamate
in CNS disorders. Drug News Perspect. 1995, 8, 261-277.
(7) Kno¨pfel, T.; Kuhn, R.; Allgeier, H. Metabotropic glutamate recep-
tors: Novel targets for drug development. J . Med. Chem. 1995,
38, 1417-1426.
(8) Christensen, I. T.; Reinhardt, A.; Nielsen, B.; Ebert, B.; Madsen,
U.; Nielsen, E. Ø.; Brehm, L.; Krogsgaard-Larsen, P. Excitatory
amino acid agonists and partial agonists. Drug Des. Del. 1989,
5, 57-71.
(9) Hansen, J . J .; Lauridsen, J .; Nielsen, E. Ø.; 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.
(10) 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.
(11) Ebert, B.; Madsen, U.; Lund, T. M.; Lenz, S. M.; Krogsgaard-
Larsen, P. Molecular pharmacology of the AMPA agonist, (S)-
2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-
APPA] and the AMPA antagonist, (R)-APPA. Neurochem. Int.
1994, 24, 507-515.
(12) Ebert, B.; Madsen, U.; Søby, K. K.; Krogsgaard-Larsen, P.
Functional partial agonism at ionotropic excitatory amino acid
receptors. Neurochem. Int. 1996, 29, 309-316.
(13) Moltzen, S. L.; Falch, E.; Bøgesø, K. P.; Krogsgaard-Larsen, P.
5-Arylisoxazolyl-4-substituted 2-amino carboxylic acid com-
pounds. PCT Int. Appl. No. 9512, 587; Chem. Abstr. 1995, 123,
112056n.
(14) Bang-Andersen, B.; Lenz, S. M.; Skjærbæk, N.; Søby, K. K.;
Hansen, H. O.; Ebert, B.; Bøgesø, K. P.; Krogsgaard-Larsen, P.
Heteroaryl analogues of AMPA. Synthesis and quantitative
structure-activity relationships. J . Med. Chem. 1997, 40, 2831-
2842.
(15) 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.
(16) Skjærbæk, N.; Brehm, L.; J ohansen, T. N.; Hansen, L. M.;
Nielsen, B.; Ebert, B.; Søby, K. K.; Stensbøl, T. B.; Falch, E.;
Krogsgaard-Larsen, P. Aryl and cycloalkyl analogues of AMPA.
Synthetic, pharmacological and stereochemical aspects. Bioorg.
Med. Chem. 1998, 6, 119-131.
(17) Alberts, A. A.; Bachman, G. B. Dehalogenation of R,â-dibromo
acids. III. Acids of the pyridine and quinoline series. J . Am.
Chem. Soc. 1935, 57, 1284-1287.
(18) Tomita, K. Hymexazol, a new plant protecting agent. Ann.
Sankyo Res. Lab. 1973, 25, 1-51.
Str u ctu r e Solu tion a n d Refin em en t. The structure was
solved by direct methods using the program SHELXS-97.51
Full-matrix least-squares refinement (SHELXL-97)26 was
performed on F2 for all unique reflections, minimizing ∑w(Fo
2
- Fc2), 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 isotropic displacement parameters. Extinction
correction was applied, extinction coefficient: 0.0074(5).26 The
refinement (208 parameters, 2381 reflections) with the mol-
ecule having the R configuration converged at RF ) 0.0221,
RwF ) 0.0603 for 2367 reflections with Fo > 4σ(Fo); w-1
)
2
σ(Fo2) + (0.0294P)2 + 0.2351P, where P ) (Fo + 2Fc2)/3; S )
1.093. The Flack absolute structure factor: 0.00(1).26,27 Maxi-
mum and minimum electron density in the final difference
Fourier map was 0.29 and -0.27 e Å-3, respectively (both near
the Cl- atom). Complex atomic scattering factors for neutral
atoms were as incorporated in SHELXL-97.26,52
2
Recep tor Bin d in g Assa ys. Affinity for AMPA receptors
was determined using the ligand [3H]AMPA,34 and for deter-
mination of NMDA and kainic acid receptor affinities, [3H]-
CPP33 and [3H]kainic acid,32 respectively, were used. The
membrane preparations used in all of the receptor binding
experiments were prepared according to the method of Ransom
and Stec.53
In Vitr o Electr op h ysiology. A rat cortical wedge prepa-
ration for determination of EAA-evoked depolarizations de-
scribed by Harrison and Simmonds54 was used in a slightly
modified version. Wedges (500 µm thick) of rat brain, contain-
ing cerebral cortex and corpus callosum, were placed through
a grease barrier for electrical isolation with each part in
contact with a Ag/AgCl pellet electrode. The cortex was
superfused with Mg2+-free Krebs buffer containing 25 mM
CaCl2, whereas the corpus callosum part was superfused with
Mg2+- and Ca2+-free Krebs buffer at 25 °C. The test com-
pounds were added to the cortex superfusion medium contain-
ing 2.5 mM CaCl2, and the induced potential difference
between the electrodes was recorded on a chart recorder.
Agonists were applied for 90 s at each concentration tested.
The sensitivity of agonist effects to the AMPA receptor
antagonist NBQX (5 µM) was tested at agonist concentrations
producing at least 50% of maximal responses. Under these
conditions, all of the recorded agonist responses were revers-
ibly reduced by at least 70%. In experiments designed to
detect antagonist effects of AMPA analogues at 1 mM concen-
trations, the compounds were applied alone for 90 s followed
by coapplication of agonist (AMPA, 5 µM) and potential
antagonist for another 90 s.
Ack n ow led gm en t. This work was supported by
grants from the Lundbeck Foundation, The Danish
Natural Science Research Council, and The Danish
State Biotechnology Program (1991-1995). The techni-
cal assistance of Karen J ørgensen and Flemming Hans-
en, Department of Chemistry, University of Copen-
hagen, on the recording of CD and UV spectra and with
the collection of X-ray data, respectively, is gratefully
acknowledged. We wish to express our gratitude to Tina
Lindgreen and Anne Nordly for technical and secretarial
assistance, respectively.
(19) Sato, K.; Sugai, S.; Tomita, K. Synthesis of 3-hydroxyisoxazoles
from â-ketoesters and hydroxylamine. Agric. Biol. Chem. 1986,
50, 1831-1837.
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3-isoxazolols revisited. Diketene and â-ketoesters as starting
materials. Can. J . Chem. 1984, 62, 1940-1944.
(21) Armstrong, D. W.; Liu, Y.; Ekborgott, K. H. A covalently bonded
teicoplanin chiral stationary phase for HPLC enantiosepara-
tions. Chirality 1995, 7, 474-497.
(22) 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.
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