Heteroaryl analogues of AMPA. Synthesis and quantitative structure- activity relationships

Article abstract of DOI:10.1021/jm970253b

A number of 3-isoxazolol bioisosteres, 7a-i, of (S)-glutamic acid (Glu), in which the methyl group of (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4- yl)propionic acid (AMPA, 1) was replaced by different 5-membered heterocyclic rings, were synthesized. Comp

Full text of DOI:10.1021/jm970253b

J . Med. Chem. 1997, 40, 2831-2842
2831
Heter oa r yl An a logu es of AMP A. Syn th esis a n d Qu a n tita tive Str u ctu r e-Activity
Rela tion sh ip s
Benny Bang-Andersen, Sibylle M. Lenz, Niels Skjærbæk,† Karina K. Søby, Hans O. Hansen, Bjarke Ebert,†
Klaus P. Bøgesø, and Povl Krogsgaard-Larsen*,†
Research Departments, H. Lundbeck A/ S, 9 Ottiliavej, DK-2500 Valby, Copenhagen, Denmark, and Department of Medicinal
Chemistry, The Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark
Received April 16, 1997X
A number of 3-isoxazolol bioisosteres, 7a -i, of (S)-glutamic acid (Glu), in which the methyl
group of (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA, 1) was replaced
by different 5-membered heterocyclic rings, were synthesized. Comparative in vitro pharma-
cological studies on this series of AMPA analogues were performed using receptor binding assays
(IC50 values) and the electrophysiological rat cortical slice model (EC50 values). None of these
compounds showed detectable affinity for the N-methyl-D-aspartic acid subtype of Glu receptors.
Some of the compounds were weak inhibitors of [3H]kainic acid binding. The inhibitory effects
on [3H]AMPA binding and agonist potencies at AMPA receptors of 7a -i were strictly dependent
on the structure, electrostatic potential, and methyl substitution of the heterocyclic 5-sub-
stituent. Thus, while 7a (IC50 ) 0.094 µM; EC50 ) 2.3 µM) was approximately equipotent
with AMPA (IC50 ) 0.023 µM; EC50 ) 5.4 µM), (RS)-2-amino-3-[3-hydroxy-5-(1H-imidazol-2-
yl)isoxazol-4-yl]propionic acid (7b) (IC50 ) 48 µM; EC50 ) 550 µM) was some 2 orders of
magnitude weaker than AMPA, and (RS)-2-amino-3-[3-hydroxy-5-(1-methyl-1H-imidazol-2-yl)-
isoxazol-4-yl]propionic acid (7c) (IC50 > 100 µM; EC50 > 1000 µM) was inactive. Furthermore,
(RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (7i) (IC50
) 0.030 µM; EC50 ) 0.92 µM) was more potent than AMPA, whereas its N-1 methyl isomer,
(RS)-2-amino-3-[3-hydroxy-5-(1-methyl-1H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (7h ) (IC50
) 54 µM; EC50 > 1000 µM) was inactive as an AMPA agonist. A quantitative structure-
activity relationship (QSAR) analysis revealed a positive correlation between receptor affinity,
electrostatic potential near the nitrogen atom at the “ortho” position of the heterocyclic
5-substituent, and the rotational energy barrier around the bond connecting the two rings.
We envisage that a hydrogen bond between the protonated amino group and an ortho-positioned
heteroatom of the ring substituent at the 5-position stabilize receptor-active conformations of
these AMPA analogues.
In tr od u ction
schizophrenia.12,13 As in Alzheimer’s disease (AD), the
role of these receptors in the etiology and the clinical
manifestations of schizophrenia is still very incom-
pletely understood, but there is evidence to suggest that
hypoactivity at EAA receptors also is a factor of impor-
tance in the latter CNS disorder.13-15 Thus, in AD as
well as schizophrenia compounds capable of activating
EAA receptors may have therapeutic interest.15
(S)-Glutamic acid (Glu, Figure 1), which is the main
excitatory neurotransmitter in the central nervous
system (CNS), and other excitatory amino acids (EAAs)
operate through four different classes of receptors. In
addition to the three heterogeneous classes of ionotropic
EAA receptors (iGluRs), named N-methyl-D-aspartic
acid (NMDA), (RS)-2-amino-3-(3-hydroxy-5-methylisox-
azol-4-yl)propionic acid (AMPA), and kainic acid (KAIN)
receptors,1-4 a heterogeneous class of metabotropic
receptors (mGluRs) has been shown to have important
functions in neurotransmission processes in the CNS.5
It is now generally agreed that all subtypes of these
receptors are potential targets for therapeutic interven-
tion in a number of CNS diseases.6,7
EAA receptors are involved in the mechanisms of
long-term potentiation, which is believed to play an
important role in learning and memory functions, and
the deficits of these functions in Alzheimer patients
may, to some extent, be caused by hypoactivity at
iGluRs and/or mGluRs in the brain.8-11 There also is
growing evidence of an implication of EAA receptors in
During the past years we have developed a number
of agonist and antagonist ligands for pharmacological
characterization of subtypes of EAA receptors, notably
the AMPA receptors.16,17 Whereas AMPA (1) is a high-
affinity and very potent AMPA receptor agonist,18,19
(RS)-2-amino-(3-hydroxyisoxazol-4-yl)propionic acid (dem-
ethyl-AMPA, 2) (Figure 1) is a high-affinity but very
low-potency AMPA agonist.20 (RS)-2-Amino-3-(3-hy-
droxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA, 3),
on the other hand, is relatively weak both as an AMPA
agonist and as an inhibitor of [3H]AMPA binding.20,21
Replacement of the bulky tert-butyl group of ATPA (3)
by a hydroxymethyl group, to give (RS)-2-amino-3-[3-
hydroxy-5-(hydroxymethyl)isoxazol-4-yl)propionic acid
(4),22 or by a phenyl group, to give (RS)-2-amino-3-(3-
hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA,
5),23,24 further reduced AMPA receptor agonist activity.
These structure-activity relationships and the recent
observation that substitution of a 2-thienyl group for
the phenyl group of APPA (5) provided the high-affinity
* Address correspondence to: Professor Povl Krogsgaard-Larsen,
Department of Medicinal Chemistry, The Royal Danish School of
Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark.
Phone: (+45) 35370850, ext. 511. Fax: (+45) 35372209.
† The Royal Danish School of Pharmacy.
X Abstract published in Advance ACS Abstracts, August 1, 1997.
S0022-2623(97)00253-7 CCC: $14.00 © 1997 American Chemical Society

2832 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Bang-Andersen et al.
Sch em e 1a
a
Reagents and conditions: (i) Br2, CCl4, room temperature; (ii)
H2O/NMP (2:1), reflux; (iii) CrO3, H2SO4, CH3COOH, room tem-
perature.
derivative 9. The crude reaction mixture was subse-
quently treated with a mixture of water and N-meth-
ylpyrrolidone (2:1) to furnish a mixture of starting
material 8 and alcohol 10. Alcohol 10 was cleanly
oxidized to carboxylic acid 11 by addition of an excess
of chromium trioxide in a mixture of acetic acid and
sulfuric acid.28 The synthetic sequences to the AMPA
analogues 7a ,c-f,h ,i were as follows: (1) construction
of the heterocyclic 5-substituent from the carboxy group
of carboxylic acid 11 (compounds 13a ,c-i; Schemes 2
and 3); (2) regioselective bromination of the methyl
group of 5-heteroaryl-4-methylisoxazoles with N-bro-
mosuccinimide (compounds 21a ,c-f,h ,i; Scheme 4); (3)
introduction of the acetamidomalonate group by a
Sorensen synthesis (compounds 22a ,c-f,h ,i; Scheme 4);
and (4) complete deprotection in 47% aqueous HBr or 6
M HCl (Scheme 4).
Construction of the heterocyclic 5-substituent, cf. (1)
above, is a key transformation and will be discussed in
more detail. Unsubstituted tetrazole 13g and the two
methyltetrazoles 13h ,i were synthesized as shown in
Scheme 2. The amide 14 was obtained from carboxylic
acid 11 by addition of the corresponding freshly pre-
pared acyl chloride to 25% aqueous ammonia. Amide
14 was dehydrated with neat phosphoryl chloride to give
nitrile 15, which was then converted into the unsubsti-
tuted tetrazole 13g with triethylammonium azide29,30
in 1,2-dimethoxyethane. The methyltetrazoles 13h ,i
were prepared by methylation of the unsubstituted
tetrazole 13g with methyl iodide under basic condition.
Thereby, a 1:2 mixture of 1-methyltetrazole 13h , which
has the methyl group “ortho” to the isoxazole ring, and
its 2-methyl isomer 13i was obtained. However, the two
isomers were not easily separated by either flash
chromatography or crystallization, but pure 1-meth-
yltetrazole 13h was finally obtained after several crys-
tallizations. The 2-methyltetrazole 13i could only be
enriched in the mother liquor to a degree of 80% and
was used in the next step without further purification.
It may be noted that the bromomethyl derivative of
2-methyltetrazole 13i, compound 21i (Scheme 4), could
be obtained in pure form after flash chromatography
and crystallization. The ratio between the methyltet-
razoles 13h ,i was not affected by the reaction temper-
ature (methyl iodide in acetone at reflux temperature,
room temperature, or -10 °C), the methyl halide (meth-
yl iodide or methyl bromide in acetone at room temper-
ature), or the solvent (methyl bromide in acetone or a
1:1 mixture of water and ethanol at room temperature).
1-Methyltetrazole 13h was prepared by a regioselective
F igu r e 1. Structures of Glu, AMPA (1), demethyl-AMPA (2),
and a number of 5-substituted AMPA analogues (3-6). The
compounds synthesized (7a -i) are depicted in the box.
and very potent AMPA agonist (RS)-2-amino-3-[3-hy-
droxy-5-(2-thienyl)isoxazol-4-yl)propionic acid (6)25 em-
phasized that the pharmacology of AMPA analogues is
strongly affected by the structure of the 5-substituent
of these compounds.
Mapping of the structural parameters of importance
for AMPA agonist-receptor interactions may open up
the prospects of designing on a rational basis AMPA
receptor ligands showing desired pharmacological pro-
files. These prospects prompted us to synthesize and
pharmacologically characterize the heterocyclic AMPA
analogues 7a-i and to perform a quantitative structure-
activity relationship (QSAR) analysis.
Resu lts
Ch em istr y. The bicyclic analogues of AMPA were
synthesized as shown in Schemes 1-5. It was envis-
aged that the different heterocyclic rings at the 5-posi-
tion of the isoxazole ring could be constructed from a
carboxylic acid functionality. Hence, synthesis of the
common intermediate 11 was vital to the strategy.
Carboxylic acid 11 was synthesized as shown in Scheme
1. In analogy with a published procedure,26 treatment
of dimethyl derivative 827 with bromine gave a 1:1
mixture of starting material 8 and 5-bromomethyl

Heteroaryl Analogues of AMPA
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2833
Sch em e 2a
a
Reagents and conditions: (i) SOCl2, reflux; NH2CH3, 5 °C; (ii) POCl3, reflux; (iii) NaN3, DMF, room temperature; (iv) SOCl2, reflux;
NH3/H2O (25%), 5 °C; (v) NaN3, TEA‚HCl, DME, reflux; (vi) CH3I, K2CO3, acetone, reflux; (vii) CH(OCH3)2N(CH3)2, 120 °C; (viii) NH2NHCH3,
CH3COOH, 90 °C; (ix) NH2NH2, CH3COOH, 90 °C; (x) Ph3CCl, TEA, DMF, room temperature.
Sch em e 3a
a
Reagents and conditions: (i) CH2(SH)CH(OCH3)2, TEA, -10 °C; (ii) TsOH, toluene, reflux; (iii) NaOCH3/CH3OH, room temperature;
NH(CH3)CH2CH(OCH3)2, CH3COOH, reflux; (iv) 1 M HCl, reflux; (v) NH2NHCH3, C2H5OH, 5 °C; (vi) HCOOH, reflux.
synthesis in order to prove the structure of the two
methyltetrazoles. N-Methylamide 12 was prepared
from carboxylic acid 11 by addition of the corresponding
freshly prepared acyl chloride to 40% aqueous methy-
lamine. N-Methylamide 12 was subsequently treated
with phosphoryl chloride to give the imidoyl chloride,
which was then added to sodium azide in N,N-dimeth-
ylformamide31 to furnish 1-methyltetrazole 13h (Scheme
2). The two methyltetrazoles 13h ,i were easily distin-
of acylamidine 16 with either hydrazine or methylhy-
drazine in acetic acid32 afforded unsubstituted triazole
17 and 2-methyltriazole 13e, respectively. The struc-
tural proof of 2-methyltriazole 13e is based on the
unambiguous synthesis of its N-1 methyl isomer 13f
(Scheme 3). The unsubstituted triazole 17 was pro-
tected with a trityl group to avoid complications with
the acidic hydrogen of the triazole ring in the following
synthesis steps. Treatment of triazole 17 with trityl
chloride gave a 7:1 mixture of two isomers, termed
trityltriazole 13d , which were tentatively assigned to
1-trityltriazole (major isomer) and the 2-trityltriazole
(minor isomer) in agreement with general observations
for alkylation of 3-substituted 1,2,4-triazoles.33 The
mixture of the two isomers was used in the next step
without further purification.
1
guished by H NMR spectroscopy.
Unsubstituted 1,2,4-triazole 17 (1,2,4-triazole is ab-
briviated triazole in the following) and 2-methyltriazole
13e, which has the methyl group ortho to the isoxazole
ring, were synthesized as shown in Scheme 2. Acyla-
midine 16 was prepared by treatment of amide 14 with
N,N-dimethylformamide dimethyl acetal.32 Treatment

2834 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Bang-Andersen et al.
Sch em e 4a
Sch em e 5a
a
Reagents and conditions: (i) NBS, CCl4, reflux; (ii) CH3CO-
NHCH(COOC2H5)2, (CH3)3COK, NMP, room temperature; (iii) 47%
HBr(aq), reflux; (iv) 6 M HCl, reflux. Substituents, R, are found
in Table 1.
Thiazole 13a and methylimidazole 13c were synthe-
sized as shown in Scheme 3. Appropriate nucleophiles
were added to nitrile 15, and the addition products 18,
19 cyclized in acidic media to give the corresponding
cyclization products 13a ,c. The 1-methyltriazole 13f
was synthesized as shown in Scheme 3. Addition of
methylhydrazine to nitrile 15 in ethanol34 gave amidra-
zone 20. Methylhydrazine has two nucleophilic centers,
and addition to the nitrile could theoretically give two
a
Reagents and conditions: (i) NBS, dibenzoyl peroxide, CCl4,
reflux; (ii) CH3CONHCH(COOC2H5)2, (CH3)3COK, NMP, room
temperature; (iii) NaN3, TEA‚HCl, DME, reflux; (iv) NaOCH3/
CH3OH, room temperature; NH2CH2CH(OCH3)2, CH3COOH, room
temperature; 1 M HCl, reflux; (v) C2H5OH/HCl, reflux; (vi) 47%
HBr(aq), reflux.
1
isomeric amidrazones. On the basis of H NMR spec-
troscopy it was clear that a single compound was
obtained, which was assigned structure 20. The single
proton on the nitrogen atom (CH3NHN′) (δ 5.04) dis-
played a coupling constant of 4.6 Hz to the protons of
the N-methyl group (CH3NHN′) (δ 2.74), thus ruling out
the structure where the methyl group is attached to
nitrogen atom N′. Condensation of 20 with formic acid
afforded 1-methyltriazole 13f, which was easily distin-
1
guished from 2-methyltriazole 13e on the basis of H
NMR spectroscopy. A difference-NOE experiment was
performed on 13f to further support the regioisomeric
proof. Thus, by saturation of the N-1 methyl group of
the triazole ring, NOE was observed at the hydrogen
atom at the 5-position.
The imidazole and tetrazole analogues 7b,g were
synthesized following a slightly different scheme, where
the imidazole and tetrazole rings were constructed at a
later stage in the syntheses (Scheme 5). Acetamidoma-
lonate derivative 23, which was derived from nitrile 15
(Scheme 2), was treated with triethylammonium azide29,30
in 1,2-dimethoxyethane to give tetrazole derivative 24.
Analogously, the nitrile group of acetamidomalonate
derivative 23 was converted into the imidazole ring by
addition of aminoacetaldehyde dimethyl acetal and
subsequent cyclization in 1 M HCl. Thereby, a decar-
boxylated and partly deprotected imidazole intermediate
was obtained, which was then treated with a solution
of HCl in ethanol to give ester 25. Imidazole and
tetrazole analogues 7b,g were obtained after complete
deprotection in 47% aqueous HBr.
F igu r e 2. Dose-response curves as determined in the rat
cortical slice model for compounds 7e,f,i compared to AMPA
(1). The data are the mean of at least three determinations
(see Table 1).
compounds were also studied in the electrophysiological
rat cortical slice model38 (Table 1).
None of the compounds displayed significant affinity
for NMDA receptors (IC50 > 100 µM), whereas the
affinities for AMPA receptors ranged from insignificant
affinity to very high affinities comparable with that of
AMPA (1) (Table 1). The compounds with high affinity
for AMPA receptors, except for 7f, also displayed some
affinity for KAIN receptors, but mostly, the compounds
showed selectivity for AMPA receptors. Furthermore,
it was shown in the rat cortical slice model that the
compounds were potent neuronal excitants. Concentra-
tion-response curves were constructed for every com-
pound, and it was shown that the compounds were full
agonists, as exemplified in Figure 2. The depolarizing
In Vitr o P h a r m a cology. The bicyclic analogues of
AMPA were studied in different receptor binding assays
in order to determine their affinity for various iGluRs.
The ligands [3H]CPG 39653,35 [3H]AMPA,36 and [3H]-
KAIN37 were used to determine affinity for NMDA,
AMPA, and KAIN receptors, respectively (Table 1). The

Heteroaryl Analogues of AMPA
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2835
Ta ble 1. Receptor Binding, Electrophysiological Data and pKa Valuesa
a
Binding data are expressed as IC50 values in µM and they are the antilog to the log mean of at least two determinations. Two full
concentration-inhibition curves were measured by using five concentrations of test drug in triplicate (covering three decades). Standard
deviation ratios (SDR) of each of the assay methods were obtained by calculating the variance of repeated measures of ratios between the
first and the second determination for a series of 30 ([3H]AMPA- and [3H]KAIN-binding) and 16 ([3H]CPG39653-binding) drugs. If the
ratio was greater than two times the SDR (95% confidence interval), extra determinations were performed and outliers were discarded.
The following SDR’s were calculated: [3H]AMPA-binding, 1.6; [3H]KAIN-binding, 1.7; [3H]CPG39653-binding, 1.3. Electrophysiological
data are expressed as EC50 values in µM and are the antilog to the log mean of at least three determinations with the minimum and
b
d
maximum values in brackets. AMPA agonists. c See Experimental Section for details. No antagonism at 300 µM toward NMDA (10
µM) and AMPA (5 µM).
effects of these compounds could be antagonized by the
competitive non-NMDA receptor antagonist 6-nitro-7-
sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) (5 µM),
but not by the competitive NMDA receptor antagonist
(RS)-[3-(2-carboxypiperazin-4-yl)propyl]phosphonic acid
(CPP) (5 µM), indicating that the compounds are non-
NMDA receptor agonists. Thus, while replacement of
the methyl group of AMPA with a thiazole ring, to give
compound 7a , resulted in a compound equipotent with
AMPA, the imidazole analogue 7b was some 2 orders
of magnitude weaker than AMPA. The methylimidazole
analogue 7c was inactive. Replacement of the methyl
group of AMPA with a 1,2,4-triazole ring or a tetrazole
ring containing methyl substituents ortho or meta to the
isoxazole ring, resulted in comparable structure-activ-
ity relationships. Thus, whereas the meta methyl
isomers 7f,i were as potent as AMPA itself, the corre-
sponding ortho methyl isomers 7e,h were nearly inac-
tive. The unsubstituted 1,2,4-triazole and tetrazole
analogues 7d ,g were weak or inactive as AMPA ago-
nists, respectively. The compounds showing no excita-
tory activity in the slice model were tested for NMDA
and non-NMDA receptor antagonism. None of these
compounds showed significant antagonism at 300 µM

2836 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Bang-Andersen et al.
Ta ble 2. Calculated Parameter Values for the 5-Substituted
4-Methyl-3-isoxazolol Moieties (13a ,b,d -f,h ,i) of the Test
Compounds (7a ,b,d -f,h ,i)Used in the QSAR Analysis
compd
V(r) (kcal/mol)
E(ω) (kcal/mol)
log IC50 (µM)
7a
7b
7d
7e
7f
-106.3
-121.8
-106.8
-103.6
-111.5
-94.7
2.14
4.52
4.59
6.73
2.17
6.94
2.63
-1.03
1.68
0.18
0.76
-0.15
1.73
7h
7i
-97.0
-1.52
toward excitation induced by either NMDA (10 µM) or
AMPA (5 µM).
QSAR An a lysis. QSAR analysis was performed on
seven of the AMPA analogues (compounds 7a ,b,d -
f,h ,i). The properties of the 5-substituents were de-
scribed by two parameters: (1) the electrostatic poten-
tial (V(r)) at a position 2.8 Å in the lonepair direction of
the ortho-positioned nitrogen atom, and in the plane of
the heterocyclic ring; and (2) the energy barrier (E(ω))
for the rotation of the substituted heterocyclic ring by
360°. These torsional drives were performed on the
5-substituted 4-methyl-3-isoxazolol moieties (Table 2).
The calculated log IC50 values were plotted against the
experimental log IC50 values (Figure 3) by performing
a multiple linear regression, and a r2 value of 0.91 was
obtained.
F igu r e 3. Plot of calculated versus experimental [3H]AMPA
binding data (IC50 value in µM). The calculated data are
derived from eq 1.
AMPA analogues were shown to be full agonists, we
hypothesized that occupancy of the proposed cavity by
an appropriately sized alkyl group contributes to the
stabilization of the agonist conformation of the AMPA
receptor.20 We have recently shown that substitution
of a 2-thienyl group for the phenyl group of the weak
AMPA agonist, APPA (5)23,24 provided the AMPA ago-
nist 6, approximately equipotent with AMPA.25 On the
other hand, the hydroxymethyl analogue of AMPA,
compound 4, which contains a potential hydrogen bond
donor group turned out to be a low-affinity, low-potency,
but full AMPA agonist.22 These observations suggested
that AMPA analogues containing appropriately sized
5-substituents producing oriented electrostatic poten-
tials and capable of acting as hydrogen bond acceptors
might show potent agonist effects.
This proposal led to the synthesis and pharmacologi-
cal characterization of the heterocyclic AMPA analogues
7a -i, all of which contain at least one unsubstituted
ortho-positioned nitrogen atom in the heterocyclic ring
substituent (Figure 1). In preparation for a QSAR
analysis, we introduced methyl substituents into se-
lected positions of some of these new compounds.
In the QSAR analysis, the electronic and steric effects
of the heterocyclic rings located at C-5 of the 3-isoxazolol
unit were described by the electrostatic potential near
the ortho-positioned nitrogen atom and the rotational
barrier around the C-C bond connecting the two rings.
This analysis revealed a linear relationship between
experimental and calculated receptor binding data (IC50
values), using multiple linear regression methods (Fig-
ure 3).
log IC50 ) 0.56E(ω) + 0.06V(r) - 8.00
n ) 7, r2 ) 0.91, s ) 0.46, F ) 20.11
(1)
The binding affinity of the seven compounds can partly
be explained by these two parameters in the QSAR
model and by the equation described above. A favorable
correlation was obtained with E(ω), a positive V(r) value,
and a negative constant. A large bulky substituent at
the 5-position of the isoxazole ring will therefore in-
crease the IC50 value, and a more negative electrostatic
potential at the ortho position of the heterocyclic ring
gives a lower IC50 value.
Discu ssion
The 3-isoxazolol amino acid, AMPA (1) is a high-
affinity, potent, and very selective AMPA receptor
agonist,18,19 and it is generally agreed that the Glu
structural element of AMPA (1) (Figure 1) is recognized
and bound by the AMPA receptor recognition site. On
the basis of structure-activity studies on AMPA and a
series of AMPA analogues containing alkyl substituents
at the 5-position of the ring, we have earlier proposed
that the alkyl group of these AMPA agonists may be
accommodated by a lipophilic cavity of limited size
located at or near the AMPA recognition site.39,40 Thus,
whereas substitution of ethyl40 or trifluoromethyl41
groups for the methyl group of AMPA led to AMPA
agonists slightly more potent than AMPA, the tert-butyl
analogue of AMPA, ATPA (3) was markedly weaker
than AMPA,20,21 and incorporation of even more bulky
groups like 4-heptyl or 2,2-dimethylpropyl groups pro-
vided totally inactive compounds.20 Demethyl-AMPA
(2), on the other hand, turned out to be a potent
inhibitor of [3H]AMPA binding, but showing remarkably
low AMPA agonist activity.20
On the basis of this analysis, we postulate that the
receptor-active conformations of this group of com-
pounds are largely determined by intramolecular hy-
drogen bonds between the protonated amino groups and
the ortho-positioned nitrogen atoms of the heterocyclic
ring substituents. We envisage that the geometry of the
hydrogen-bonded structures are influenced by the loca-
tion of the methyl substituents in these rings (com-
pounds 7c,e,f,h ,i). Thus, due to steric interactions
between the methyl groups at the second ortho position
of the rings of 7c,e,h and the oxygen atom of the
On the basis of these structure-activity relationships
and the observation that all active alkyl-substituted

Heteroaryl Analogues of AMPA
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2837
nitrogen atoms of 7f and 7i does not significantly
influence the rotational energy barriers calculated for
these compounds (Table 2). The high AMPA receptor
affinities and agonist activities of 7f,i, in particular of
7i, are consistent with the results of these calculations
and seem to indicate that these substituted rings are
easily accommodated by the proposed AMPA receptor
cavity.
In agreement with earlier findings for alkyl-substi-
tuted AMPA analogues,20,40,41 the compounds under
study were either full agonists (Figure 2) or inactive.
In no case were partial agonist or antagonist effects
observed. The results of this structure-activity analy-
sis may, however, be further developed into principles
for the design of partial AMPA receptor agonists, which
may have particular therapeutic interest in certain CNS
disorders.17
Exp er im en ta l Section
Ch em istr y. All reactions were performed under an inert
atmosphere (nitrogen). Reagents were purchased commer-
cially and used without purification. Melting points were
determined in capillary tubes (Bu¨chi 535 apparatus) and are
uncorrected. 1H NMR was recorded on a Bruker AC 250
spectrometer at 250 MHz. Unless otherwise stated, chemical
shift values (δ) are expressed in ppm relative to TMS. The
following abbreviations are used for multiplicity of NMR
signals: br ) broad, s ) singlet, d ) doublet, t ) triplet, q )
quartet, dd ) double doublet, m ) multiplet. NMR signals
corresponding to acidic protons are omitted. Difference-NOE
spectra were obtained by use of standard Bruker software.
Mass spectra were obtained on a Quattro MS-MS system from
VG Biotech, Fisons Instruments. The MS-MS system was
connected to an HP 1050 modular HPLC system. A volume
of 20-50 µL of the sample (10 µg/mL) dissolved in a mixture
of acetonitrile/water/acetic acid ) 250:250:1 (v/v/v) or in a
mixture of acetonitril/water/aqueous ammonia (25%) ) 25:25:1
(v/v/v) was introduced via the autosampler at a flow of 30 µL/
min into the electrospray source. Spectra were recorded for
all target compounds and for selected key intermediates at
standard operating conditions to obtain molecular weight
information ((M + H)+) or ((M - H)-). The background was
subtracted. Compounds containing the 3-isoxazolol moiety
were visualized on TLC plates (Merck silica gel 60 F254) using
UV light and a FeCl3 spraying reagent (yellow colors). Com-
pounds containing amino groups were visualized using a
ninhydrin spraying reagent. Microanalyses were performed
by Lundbeck Analytical Department, and results obtained
were within (0.4% of the theoretical values if not otherwise
stated. It shall be mentioned that the relatively high nitrogen
content of the target compounds in several cases caused
instrumental problems, and higher deviations from the theo-
retical values than (0.4% were observed.
(3-Eth oxy-4-m eth ylisoxa zol-5-yl)m eth a n ol (10). A mix-
ture of 3-ethoxy-4,5-dimethylisoxazole (8) (114 g, 0.8 mol),
bromine (83 mL, 1.6 mol), and CCl4 (150 mL) was stirred in
the dark at room temperature for 6 days. Water was added,
and NaHSO3 was added until the phases were colorless. The
phases were separated, and the aqueous phase was extracted
with CH2Cl2. The organic extracts were washed with water
and brine and dried (MgSO4), and the solvents were evapo-
rated in vacuo to give crude 5-(bromomethyl)-3-ethoxy-4-
methylisoxazole (9) (according to 1H NMR, a 1:1 mixture of 8
and 9 was obtained). The crude 9 was dissolved in a water/
N-methylpyrrolidone mixture (2:1, 750 mL) and stirred at 105
°C for 2 days. The mixture was cooled and extracted with
ether. The organic extracts were washed with brine and dried
(MgSO4), and the solvent was evaporated in vacuo. The
residue was subjected to flash chromatography (silica gel,
eluent: heptane/EtOAc ) 3:1 then 1:2) to give crude starting
material 8 (45.6 g, 40%) and pure 10 as a colorless solid (50 g,
39%): mp 40-41 °C; 1H NMR (CDCl3) δ 1.43 (t, 3 H), 1.90 (s,
3 H), 4.30 (q, 2 H), 4.59 (s, 2 H). Anal. (C7H11NO3) C, H, N.
F igu r e 4. Energy minimized structures of (S)-7b in stereo-
views. Two conformations are represented with plausible
hydrogen bonds between the protonated amino group and the
imidazole ring.
F igu r e 5. Possible “charge-transfer” hydrogen bonds for
compounds 7b and 7g.
isoxazole ring the ring substituents may be twisted out
of the rather planar receptor-active conformations (Fig-
ure 4). These interactions are reflected by the relatively
high rotational energy barriers calculated for 7e,h
(Table 2).
Although 7b contains an unsubstituted imidazole
ring, a rather high IC50 value for inhibition of [3H]AMPA
binding (Table 1) and a high rotational energy barrier
(Table 2) was measured and calculated, respectively, for
this compound. The latter value is assumed to reflect
the formation of a stable “charge-transfer” hydrogen
bond between the protonated amino group and the
weakly basic imidazole ring (pKa ) 3.4) (Figure 5). The
unexpectedly low affinity of 7b for the AMPA receptor
site (IC50 ) 48 µM) may reflect that the imidazole ring
carrying a partial positive charge is not optimally
accommodated by the proposed lipophilic cavity at this
receptor site. Similar effects in addition to infavorable
steric effects of the ortho-positioned methyl group may
explain the inactivity of 7c.
Compound 7g containing an unsubstituted tetrazole
ring substituent (pKa < 3) also showed unexpectedly
weak AMPA receptor affinity (IC50 ) 72 µM) and no
detectable AMPA receptor agonist effect (Table 1).
Under the test conditions, this negatively charged
tetrazole ring may form a “charge-transfer” hydrogen
bond, as depicted in Figure 5, and may not interact
optimally with the lipophilic AMPA receptor cavity in
analogy with the partially charged imidazole ring of 7b.
The presence of a methyl group at the meta-positioned

2838 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Bang-Andersen et al.
3-Eth oxy-4-m eth ylisoxa zole-5-ca r boxylic Acid (11). To
a solution of 10 (50 g, 0.31 mol) in H2SO4/water/AcOH (1:2:7,
400 mL) was added a mixture of CrO3/H2O/AcOH (1:1:2, 400
mL), and the resulting mixture was stirred at room temper-
ature for 2 h. Water was added (500 mL), and the aqueous
phase was extracted with ether (3 × 1 L). The organic extracts
were washed with water and brine and dried (MgSO4), and
the solvent was evaporated in vacuo to give 11 as a grayish
and the pH adjusted to ca. 1 by addition of 6 M HCl. The
aqueous phase was extracted with EtOAc (3 × 75 mL), the
organic extracts were washed with water and brine and dried
(MgSO4), and the solvent was removed in vacuo to give crude
13g (1.6 g, 83%). A small sample was recrystallized (EtOH)
to give colorless crystals: mp 147-149 °C; 1H NMR (CDCl3) δ
1.52 (t, 3 H), 2.34 (s, 3 H), 4.48 (q, 2 H); MS ((M + H)+) m/z
196. Anal. (C7H9N5O2) C, H, N. The crude product was used
in the next step without further purification.
1
crystalline compound (38 g, 70%): mp 120-122 °C; H NMR
(CDCl3) δ 1.45 (t, 3 H), 2.18 (s, 3 H), 4.39 (q, 2 H); MS ((M -
5-(3-Eth oxy-4-m eth ylisoxa zol-5-yl)-2-m eth yl-2H-tetr a -
zole (13i). A mixture of 13g (0.64 g, 3.2 mmol) and K2CO3
(0.9 g, 6.5 mmol) in acetone was stirred at room temperature
for 5 min, and a solution of methyl iodide (0.3 mL, 4.8 mmol)
in acetone (5 mL) was added. The resulting mixture was
stirred at room temperature for 16 h, and the solvent was
removed in vacuo (according to 1H NMR, a 2:1 mixture of 13i
and 13h was obtained). Crude product equivalent to 1.8 g (9.1
mmol) of 13g was subjected to flash chromatography (silica
gel, eluent: EtOAc/heptane ) 1:2). The residue was crystal-
lized (EtOAc) to give pure 13h (for analytical data see
elsewhere), and crude 13i was enriched in the mother liquor
H)-) m/z 170. Anal. (C7H9NO4) C, H, N.
N -Me t h y l-3-e t h o x y -4-m e t h y lis o x a zo le -5-c a r b o x a -
m id e (12). A mixture of 11 (5.0 g, 29.2 mmol), thionyl chloride
(10 mL), and DMF (1 mL) in CH2Cl2 (50 mL) was boiled under
reflux for 3.5 h. The solvents were evaporated to dryness in
vacuo, and the residue dissolved in THF (50 mL) was poured
onto an aqueous solution of methylamine (40%) at 0-5 °C. The
mixture was allowed to warm to room temperature and stirred
for another 30 min. The aqueous phase was extracted with
EtOAc (3 × 100 mL), and the organic extracts were washed
with brine and dried (MgSO4). The solvent was removed in
vacuo, and the residue was crystallized (EtOAc) to give 12 as
colorless crystals (3.3 g, 61%): mp 113-115 °C; 1H NMR
(CDCl3) δ 1.44 (t, 3 H), 2.18 (s, 3 H), 2.98 (d, 3 H), 4.34 (q, 2
H), 6.49 (br s, 1 H). Anal. (C8H12N2O3) C, H, N.
1
(1.0 g, 53%, according to H NMR, a 4:1 mixture of 13i and
13h was obtained): 1H NMR (CDCl3) δ 1.47 (t, 3 H), 2.26 (s,
3 H), 4.40 (q, 2 H), 4.45 (s, 3 H). Crude 13i was used in the
next step without further purification.
5-(3-Eth oxy-4-m eth ylisoxa zol-5-yl)-1-m eth yl-1H-tetr a -
zole (13h ). A mixture of 12 (3.8 g, 20.6 mmol) and phosphoryl
chloride (40 mL) was boiled under reflux for 3 h. The solvent
was removed in vacuo, and the residue dissolved in DMF was
added to a suspension of NaN3 (2.7 g, 41.5 mmol) in DMF (18
mL) at 25-35 °C. The resulting suspension was stirred at
room temperature for 1 h and then poured onto water (150
mL). The aqueous phase was extracted with EtOAc (3 × 150
mL), the organic extracts were washed with water and brine
and dried (MgSO4), and the solvent was removed in vacuo.
Flash chromatography (silica gel, eluent: EtOAc/heptane )
1:2) gave crude 13h (0.6 g, 14%). A small sample was
recrystallized (EtOAc) to give colorless crystals: mp 136-138
°C; 1H NMR (CDCl3) δ 1.49 (t, 3 H), 2.31 (s, 3 H), 4.38 (s, 3 H),
4.40 (q, 2 H); MS ((M + H)+) m/z 210. Anal. (C8H11N5O2) C,
H, N. The crude product was used in the next step without
further purification.
N-[(Dim eth yla m in o)m eth ylid en e]-3-eth oxy-4-m eth yl-
isoxa zole-5-ca r boxa m id e (16). A mixture of 14 (3.5 g, 21
mmol) and DMF dimethyl acetal (15 mL) was stirred at 120
°C for 15 min. After being cooled, pure 16 was collected by
filtration as colorless crystals (4.2 g, 91%): mp 115-117 °C;
1H NMR (CDCl3) δ 1.44 (t, 3 H), 2.21 (s, 3 H), 3.20 (s, 3 H),
3.22 (s, 3 H), 4.36 (q, 2 H), 8.64 (s, 1 H). Anal. (C10H15N3O3)
C, H, N.
3-(3-Eth oxy-4-m eth ylisoxa zol-5-yl)-2-m eth yl-2H-1,2,4-
tr ia zole (13e). To a solution of methylhydrazine (0.6 mL, 11
mmol) in AcOH (15 mL) was added 16 (2.2 g, 10 mmol). The
resulting reaction mixture was stirred at 90 °C for 1 h, and
the solvents were evaporated in vacuo. Flash chromatography
(silica gel, eluent: EtOAc/heptane ) 1:2) gave compound 13e
as colorless crystals (1.0 g, 49%): mp 86-87 °C; 1H NMR
(CDCl3) δ 1.48 (t, 3 H), 2.23 (s, 3 H), 4.20 (s, 3 H), 4.39 (q, 2
H), 7.98 (s, 1 H); MS ((M + H)+) m/z 209. Anal. (C9H12N4O2)
C, H, N.
3-(3-Eth oxy-4-m eth ylisoxazol-5-yl)-1H-1,2,4-tr iazole (17).
To a solution of hydrazine hydrate (0.6 mL, 12.4 mmol) in
AcOH (15 mL) was added 16 (1.8 g, 8.0 mmol). The resulting
reaction mixture was stirred at 90 °C for 15 min and then
allowed to cool to room temperature. Pure 17 was collected
by filtration (1.2 g, 77%): mp 194-196 °C. Water (40 mL)
was added to the filtrate, and the aqueous phase was extracted
with EtOAc (3 × 30 mL). The organic extracts were washed
with brine and dried (MgSO4), and the solvent was removed
in vacuo to give crude 17 (0.3 g, 20%). Overall yield of 17:
97%. 1H NMR (DMSO-d6) δ 1.40 (t, 3 H), 2.15 (s, 3 H), 4.31
(q, 2 H), 8.74 (s, 1 H); MS ((M + H)+) m/z 195. Anal.
(C8H10N4O2) C, H, N. A mixture of the two crops were used
in the next step.
3-Eth oxy-4-m eth ylisoxazole-5-car boxam ide (14). A mix-
ture of 11 (5.0 g, 29 mmol), thionyl chloride (11 mL, 150 mmol),
and DMF (0.5 mL) in CH2Cl2 (75 mL) was boiled under reflux
for 2 h. The solvents were evaporated in vacuo, and the
residue dissolved in THF (30 mL) was poured onto an aqueous
solution of ammonia (25%) at 0-5 °C. The mixture was
allowed to warm to room temperature and then stirred for
another 1 h. The aqueous phase was extracted with ether (3
× 150 mL), the organic extracts were washed with water and
brine and dried (MgSO4), and the solvent was removed in
vacuo to give crude 14 (4.5 g, 91%). A small sample was
recrystallized (EtOH) to give colorless crystals: mp 170-172
1
°C; H NMR (DMSO-d6) δ 1.37 (t, 3 H), 2.05 (s, 3 H), 4.29 (q,
2 H), 7.84 (br s, 1 H), 8.09 (br s, 1 H). Anal. (C7H10N2O3) C,
H, N. The crude product was used in the next step without
further purification.
3-(3-Eth oxy-4-m eth ylisoxa zol-5-yl)-1-tr ityl-1H-1,2,4-tr i-
a zole (13d ). To a mixture of 17 (1.1 g, 5.7 mmol), TEA (2.5
mL, 18 mmol), and DMF (20 mL) was added trityl chloride
(1.6 g, 5.7 mmol) in DMF (5 mL). The mixture was stirred at
room temperature for 5 h and then poured onto an ice/water
mixture (200 mL). The aqueous phase was extracted with
ether (3 × 200 mL), and the organic extracts were washed with
an aqueous solution of Na2CO3 (10%) and brine. The solution
was dried (Na2SO4) and concentrated in vacuo to give crude
3-Eth oxy-4-m eth ylisoxa zole-5-ca r bon itr ile (15). A mix-
ture of 14 (10 g, 59 mmol) and phosphoryl chloride (120 mL)
was boiled under reflux for 45 min. The solvent was removed
in vacuo, and the residue dissolved in CH2Cl2 (200 mL) was
poured onto an ice/water mixture (200 mL). The two layers
were separated, and the aqueous phase was extracted with
CH2Cl2 (2 × 200 mL). The organic extracts were washed with
water and brine and dried (MgSO4), and the solvent was
removed in vacuo to give crude 15 as a brownish oil (7.9 g,
88%): 1H NMR (CDCl3) δ 1.45 (t, 3 H), 2.08 (s, 3 H), 4.37 (q,
2 H). The crude product was used in the next step without
further purification.
5-(3-Eth oxy-4-m eth ylisoxa zol-5-yl)-1H-tetr a zole (13g).
A suspension of 15 (1.5 g, 9.9 mmol), NaN3 (0.77 g, 11.8 mmol),
and TEA hydrochloride (1.63 g, 11.8 mmol) in 1,2-dimethoxy-
ethane (50 mL) was boiled under reflux for 48 h. The solvent
was evaporated in vacuo, and the residue was dissolved in
water (50 mL). The aqueous phase was washed with EtOAc
1
13d (2.5 g, 100%, according to H NMR, a 7:1 mixture of two
isomers was obtained). A small sample was crystallized
(EtOAc) to give a single isomer as colorless crystals: mp 181-
183 °C; 1H NMR (CDCl3) δ 1.44 (t, 3 H), 2.11 (s, 3 H), 4.37 (q,
2 H), 7.08-7.22 (m, 6 H), 7.22-7.42 (m, 9 H), 8.03 (s, 1 H).
Anal. (C27H24N4O2) C, H, N. The crude product was used in
the next step without further purification.
S-(2,2-Dim eth oxyeth yl) 3-Eth oxy-4-m eth ylisoxa zole-5-
th ioca r boxim id a te (18). A mixture of 15 (2.6 g, 17 mmol),
sulfanylacetaldehyde dimethyl acetal42 (3.6 g, 34 mmol), and

Heteroaryl Analogues of AMPA
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2839
TEA (4.8 mL, 34 mmol) was stirred at (-10 °C) for 48 h. Flash
chromatography (silica gel, eluent: EtOAc/heptane ) 1:3)
afforded crude 18 as a yellow oil (3.6 g, 77%): 1H NMR (CDCl3)
δ 1.44 (t, 3 H), 2.14 (s, 3 H), 3.40 (t, 2 H), 3.43 (s, 6 H), 4.34 (q,
2 H), 4.58 (br t, 1 H). The crude product was used in the next
step without further purification.
diethyl acetamidomalonate (0.62 g, 2.9 mmol) and potassium
tert-butoxide (0.35 g, 3.1 mmol) in N-methylpyrrolidone (15
mL) was stirred at room temperature for 30 min. Compound
21a (0.75 g, 2.6 mmol) in N-methylpyrrolidone (5 mL) was
added, and the resulting mixture was stirred at room tem-
perature for 2 h. The mixture was poured onto an ice/water
mixture (100 mL), and the aqueous phase was extracted with
EtOAc (3 × 100 mL). The organic extracts were washed with
an aqueous solution of base and brine and dried (MgSO4), and
the solvent was removed in vacuo. Flash chromatography
(silica gel, eluent: heptane/EtOAc ) 1:1 then 1:2) followed by
crystallization (2-propanol) gave 22a as slightly yellow crystals
3-Eth oxy-4-m eth yl-5-(th ia zol-2-yl)isoxa zole (13a ).
A
mixture of 18 (2.5 g, 9 mmol) and p-toluenesulfonic acid
(catalytic amount) in toluene (75 mL) was boiled under reflux
for 3 days. The mixture was concentrated in vacuo and
subjected to flash chromatography (silica gel, eluent: EtOAc/
heptane ) 1:5), affording crude 13a (0.85 g, 45%). A small
sample was recrystallized (heptane) to give a yellow crystalline
compound: mp 84-86 °C; 1H NMR (CDCl3) δ 1.46 (t, 3 H),
2.26 (s, 3 H), 4.39 (q, 2 H), 7.47 (d, 1 H), 7.99 (d, 1 H); MS ((M
+ H)+) m/z 211. Anal. (C9H10N2O2S) C, H, N. The crude
product was used in the next step without further purification.
3-Eth oxy-4-m eth yl-5-(1-m eth yl-1H-im id a zol-2-yl)isox-
a zole (13c). A mixture of 15 (3.0 g, 19.7 mmol), 5.4 M NaOMe
(0.7 mL, 3.8 mmol), and MeOH (60 mL) was stirred at room
temperature for 45 min. The mixture was cooled (0-5 °C),
and AcOH (2.6 mL, 45.4 mmol) was added. N-(Methylamino)-
acetaldehyde dimethyl acetal (2.8 mL, 21.8 mmol) was added,
and the resulting mixture was boiled under reflux for 6 h. The
reaction mixture was reduced in vacuo, added a boiling
solution of 1 M HCl (150 mL), and boiled under reflux for 4 h.
The reaction mixture was cooled, washed with ether, and
basified by addition of 24% NaOH. The aqueous phase was
extracted with EtOAc (3 × 150 mL), the organic extracts were
washed with brine and dried (MgSO4), and the solvent was
evaporated in vacuo. Flash chromatography (silica gel, elu-
ent: EtOAc/heptane ) 4:1) followed by treatment with char-
coal, filtration, and concentration in vacuo gave pure 13c as
slightly brown crystals (2.5 g, 61%): mp 103-104 °C; 1H NMR
(CDCl3) δ 1.46 (t, 3 H), 2.20 (s, 3 H), 3.93 (s, 3 H), 4.37 (q, 2
H), 6.94 (d, 1 H), 7.19 (d, 1 H); MS ((M + H)+) m/z 208. Anal.
(C10H13N3O2) C, H, N.
1
(0.63 g, 57%): mp 136-137 °C; H NMR (CDCl3) δ 1.25 (t, 6
H), 1.45 (t, 3 H), 1.80 (s, 3 H), 3.82 (s, 2 H), 4.00-4.29 (m, 4
H), 4.36 (q, 2 H), 6.80 (br s, 1 H), 7.50 (d, 1 H), 7.96 (d, 1 H).
Anal. (C18H23N3O7S) C, H, N.
(RS)-2-Am in o-3-[3-h yd r oxy-5-(t h ia zol-2-yl)isoxa zol-4-
yl]p r op ion ic Acid Hem ih yd r a te (7a ). A suspension of 22a
(0.6 g, 1.4 mmol) in 6 M HCl (150 mL) was boiled under reflux
for 8 days. The reaction mixture was concentrated in vacuo,
and the residue was dissolved in water (40 mL). The aqueous
phase was washed with EtOAc, treated with charcoal, filtered,
and concentrated in vacuo. The residue was dissolved in
water, and the pH was adjusted to ca. 3 by addition of 1 M
NaOH. The resulting precipitate was stirred at room tem-
perature for 16 h, and 7a was collected by filtration as colorless
crystals (0.26 g, 70%): mp 218-219 °C dec; 1H NMR (DMSO-
d6) δ 3.08 (dd, 1 H), 3.43 (dd, 1 H), 3.76 (dd, 1 H), 7.98 (d, 1
H), 8.08 (d, 1 H); MS ((M - H)-) m/z 254. Anal. (C9H9N3O4S‚
0.5H2O) C, H, N.
4-(Br om om eth yl)-3-eth oxy-5-(1-m eth yl-1H-im id a zol-2-
yl)isoxa zole (21c). Compound 21c was prepared from 13c
(1.0 g, 4.8 mmol) by the method described for compound 21a .
Crude product (1.4 g, 100%): 1H NMR (CDCl3) δ 1.49 (t, 3 H),
3.98 (s, 3 H), 4.42 (q, 2 H), 4.78 (s, 2 H), 6.98 (d, 1 H), 7.24 (d,
1 H). The crude product was used in the next step without
further purification.
N-Meth yl-3-eth oxy-4-m eth ylisoxazole-5-car boxam idr a-
zon e (20). Compound 15 (2.2 g, 14.5 mmol) in EtOH (5 mL)
at 5 °C was added methylhydrazine (7 mL, 130 mmol), and
the reaction mixture was stirred at room temperature for 45
min. Water (25 mL) was added, and crude 20 was collected
by filtration (1.9 g, 66%). A small sample was recrystallized
(heptane) to give colorless crystals: mp 117-118 °C; 1H NMR
(DMSO-d6) δ 1.35 (t, 3 H), 1.96 (s, 3 H), 2.74 (d, 3 H, J ) 4.6
Hz), 4.24 (q, 2 H), 5.04 (q, 1 H, J ) 4.6 Hz), 5.61 (br s, 2 H).
Anal. (C8H14N4O2) C, H, N. The crude product was used in
the next step without further purification.
Eth yl 2-Aceta m id o-3-[3-eth oxy-5-(1-m eth yl-1H-im id a -
zol-2-yl)isoxazol-4-yl]-2-(eth oxycar bon yl)pr opion ate (22c).
Compound 22c was prepared from 21c (1.5 g, 5.2 mmol) by
the method described for compound 22a . Flash chromatog-
raphy (silica gel, eluent: EtOAc/heptane ) 4:1 then EtOAc)
followed by crystallization (2-propanol) gave 22c as colorless
crystals (1.2 g, 54%): mp 112-114 °C; 1H NMR (CDCl3) δ 1.23
(t, 6 H), 1.44 (t, 3 H), 1.89 (s, 3 H), 3.58 (s, 2 H), 3.91 (s, 3 H),
3.99-4.26 (m, 4 H), 4.34 (q, 2 H), 6.98 (d, 1 H), 7.14 (d, 1 H),
8.23 (br s, 1 H). Anal. (C19H26N4O7) C, H, N.
(RS)-2-Am in o-3-[3-h yd r oxy-5-(1-m eth yl-1H-im id a zol-2-
3-(3-Eth oxy-4-m eth ylisoxa zol-5-yl)-1-m eth yl-1H-1,2,4-
tr ia zole (13f). A mixture of formic acid (10 mL) and 20 (1.9
g, 9.6 mmol) was boiled under reflux for 1 h. The reaction
mixture was concentrated in vacuo, and the residue was
dissolved in EtOAc (100 mL) and poured onto brine (100 mL)
and K2CO3 (5 g). The two layers were separated, and the
aqueous phase was extracted with EtOAc (2 × 100 mL). The
organic extracts were washed with brine and dried (MgSO4),
and the solvent was evaporated in vacuo to give crude 13f as
colorless crystals (2.0 g, 100%). A small sample was recrystal-
yl)isoxa zol-4-yl]p r op ion ic Acid Mon oh yd r a te (7c). A
suspension of 21c (1.0 g, 2.4 mmol) in 47% HBr(aq) (40 mL)
was boiled under reflux for 1.5 h. The solvent was removed
in vacuo, and the residue was dissolved in water (50 mL). The
aqueous phase was washed with EtOAc and reduced in vacuo
(5 mL). A pH of ca. 3 was obtained by addition of 1 M NaOH,
and 7c was collected by filtration as colorless crystals (0.44 g,
1
69%): mp 234-237 °C dec; H NMR (DMSO-d6) δ 2.96 (dd, 1
H), 3.18 (dd, 1 H), 3.72 (dd, 1 H), 3.85 (s, 3 H), 7.11 (d, 1 H),
7.41 (d, 1 H); MS ((M + H)+) m/z 253. Anal. (C10H12N4O4‚H2O)
C, H, N.
1
lized (EtOAc) to give colorless crystals: mp 129-131 °C; H
NMR (CDCl3) δ 1.45 (t, 3 H), 2.21 (s, 3 H), 4.01 (s, 3 H), 4.38
(q, 2 H), 8.14 (s, 1 H); MS ((M + H)+) m/z 209. Anal.
(C9H12N4O2) C, H, N. The crude product was used in the next
step without further purification.
3-[4-(Br om om eth yl)-3-eth oxyisoxa zol-5-yl]-1-tr ityl-1H-
1,2,4-tr ia zole (21d ). Compound 21d was prepared from 13d
(2.4 g, 5.5 mmol) by the method described for compound 21a .
Crude product (2.8 g, 100%, according to 1H NMR, a 15:1
mixture of two isomers was obtained): 1H NMR (CDCl3) δ 1.47
(t, 3 H), 4.42 (q, 2 H), 4.57 (s, 2 H), 7.11-7.23 (m, 6 H), 7.23-
7.44 (m, 9 H), 8.09 (s, 1 H). The crude product was used in
the next step without further purification.
Eth yl 2-Aceta m id o-3-[3-eth oxy-5-(1-tr ityl-1H-1,2,4-tr ia -
zol-3-yl)isoxazol-4-yl]-2-(eth oxycar bon yl)pr opion ate (22d).
Compound 22d was prepared from 21d (2.8 g, 5.4 mmol) by
the method described for compound 22a . Flash chromatog-
raphy (silica gel, eluent: EtOAc/heptane/TEA ) 50:50:2) gave
compound 22d (2.2 g, 62%, according to 1H NMR, a single
isomer was obtained): mp 145-149 °C; 1H NMR (CDCl3) δ
1.16 (t, 6 H), 1.43 (t, 3 H), 1.80 (s, 3 H), 3.77 (s, 2 H), 3.93-
4.10 (m, 2 H), 4.10-4.28 (m, 2 H), 4.35 (q, 2 H), 6.82 (br s, 1
4-(Br om om e t h yl)-3-e t h oxy-5-(t h ia zol-2-yl)isoxa zole
(21a ). A mixture of 13a (0.85 g, 4.0 mmol) and NBS (0.8 g,
4.5 mmol) in CCl4 (70 mL) was boiled under reflux for a total
of 64 h. Additional NBS was added after 16 h (0.8 g, 4.5 mmol)
and again after 40 h (0.4 g, 2.2 mmol). The reaction mixture
was cooled and filtered, and the solvent was evaporated in
vacuo. Flash chromatography (silica gel, eluent: EtOAc/
heptane ) 1:6) gave crude 21a as a yellow oil (0.75 g, 64%):
1H NMR (CDCl3) δ 1.51 (t, 3 H), 4.44 (q, 2 H), 4.77 (s, 2 H),
7.57 (d, 1 H), 8.08 (d, 1 H). The crude product was used in
the next step without further purification.
Eth yl 2-Aceta m id o-3-[3-eth oxy-5-(th ia zol-2-yl)isoxa zol-
4-yl]-2-(eth oxyca r bon yl)p r op ion a te (22a ). A mixture of

2840 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Bang-Andersen et al.
H), 7.10-7.22 (m, 6 H), 7.29-7.42 (m, 9 H), 8.02 (s, 1 H). Anal.
(C36H37N5O7) C, H, N.
3 H), 4.18 (br s, 1 H), 8.71 (s, 1 H); MS ((M + H)+) m/z 254.
Anal. (C9H11N5O4‚0.25H2O) C, H, N.
(RS)-2-Am in o-3-[3-h ydr oxy-5-(1H-1,2,4-tr iazol-3-yl)isox-
a zol-4-yl]p r op ion ic Acid Mon oh yd r a te (7d ). A suspension
of 22d (0.7 g, 1.1 mmol) in 47% HBr(aq) (60 mL) was boiled
under reflux for 1.5 h. Water (100 mL) was added, and the
aqueous phase was washed with ether and CH2Cl2 and
concentrated in vacuo. Water (1 mL) was added, and the pH
was adjusted to ca. 3 by addition of 1 M NaOH. The
precipitate which formed was collected by filtration, resus-
pended in water (10 mL), and stirred for another 24 h at room
temperature. Compound 7d was collected by filtration as
colorless crystals (0.16 g, 58%): mp 193-196 °C dec; 1H NMR
(DMSO-d6) δ 3.01 (dd, 1 H), 3.35 (dd, 1 H), 3.69 (dd, 1 H), 8.68
(s, 1 H); MS ((M + H)+) m/z 240. Anal. (C8H9N5O4‚H2O) C,
H; N: calcd, 27.23; found, 26.67.
3-[4-(Br om om et h yl)-3-et h oxyisoxa zol-5-yl]-2-m et h yl-
2H-1,2,4-tr ia zole (21e). Compound 21e was prepared from
13e (1.0 g, 4.8 mmol) by the method described for compound
21a . Crude product (1.34 g, 97%): 1H NMR (CDCl3) δ 1.52 (t,
3 H), 4.25 (s, 3 H), 4.45 (q, 2 H), 4.73 (s, 2 H), 8.03 (s, 1 H).
The crude product was used in the next step without further
purification.
5-[4-(Br om om et h yl)-3-et h oxyisoxa zol-5-yl]-1-m et h yl-
1H-tetr a zole (21h ). Compound 21h was prepared from 13h
(0.6 g, 2.9 mmol) by the method described for compound 21a .
Crude product (0.8 g, 97%): 1H NMR (CDCl3) δ 1.52 (t, 3 H),
4.41 (s, 3 H), 4.47 (q, 2 H), 4.72 (s, 2 H). The crude product
was used in the next step without further purification.
Eth yl 2-Aceta m id o-3-[3-eth oxy-5-(1-m eth yl-1H-tetr a -
zol-5-yl)isoxazol-4-yl]-2-(eth oxycar bon yl)pr opion ate (22h ).
Compound 22h was prepared from 21h (0.8 g, 2.8 mmol) by
the method described for compound 22a . Flash chromatog-
raphy (silica gel, eluent: EtOAc/heptane ) 2:1) followed by
crystallization (2-propanol) gave 22h as colorless crystals (0.73
g, 62%): mp 139-141 °C; 1H NMR (CDCl3) δ 1.25 (t, 6 H),
1.47 (t, 3 H), 1.94 (s, 3 H), 3.80 (s, 2 H), 3.97-4.25 (m, 4 H),
4.33 (s, 3 H), 4.37 (q, 2 H), 6.53 (br s, 1 H). Anal. (C17H24N6O7)
C, H, N.
(RS)-2-Am in o-3-[3-h yd r oxy-5-(1-m eth yl-1H-tetr a zol-5-
yl)isoxa zol-4-yl]p r op ion ic Acid Dih yd r a te (7h ). A sus-
pension of 22h (0.65 g, 1.5 mmol) in 47% HBr(aq) (40 mL) was
boiled under reflux for 2.5 h. The reaction mixture was
concentrated in vacuo and the residue dissolved in water (50
mL). The aqueous phase was washed with EtOAc, filtered,
and reduced in vacuo (3 mL). A pH of ca. 3 was obtained by
addition of 1 M NaOH, affording 7h as colorless crystals (0.36
g, 81%): mp 255-257 °C dec; 1H NMR (DMSO-d6) δ 3.10 (dd,
1 H), 3.31 (dd, 1 H), 3.78 (dd, 1 H), 4.27 (s, 3 H); MS ((M +
H)+) m/z 255. Anal. (C8H10N6O4‚2H2O) C, H, N.
5-[4-(Br om om et h yl)-3-et h oxyisoxa zol-5-yl]-2-m et h yl-
2H-tetr a zole (21i). Compound 21i was prepared from 13i
(1.0 g, 4.8 mmol) by the method described for compound 21a .
Flash chromatography (silica gel, toluene/EtOAc ) 15:1) gave
crude 21i (0.34 g, 25%). A small sample was recrystallized
(EtOAc) to give colorless crystals: mp 97-99 °C; 1H NMR
(CDCl3) δ 1.50 (t, 3 H), 4.46 (q, 2 H), 4.49 (s, 3 H), 4.69 (s, 2
H). Anal. (C8H10BrN5O2) C, H, N.
Eth yl 2-Aceta m id o-3-[3-eth oxy-5-(2-m eth yl-2H-tetr a -
zol-5-yl)isoxazol-4-yl]-2-(eth oxycar bon yl)pr opion ate (22i).
Compound 22i was prepared from 21i (0.32 g, 1.1 mmol) by
the method described for compound 22a . Flash chromatog-
raphy (silica gel, eluent: EtOAc/heptane ) 2:1) followed by
crystallization (2-propanol) gave 22i as colorless crystals (0.33
g, 70%): mp 144-145 °C; 1H NMR (CDCl3) δ 1.25 (t, 6 H),
1.45 (t, 3 H), 1.78 (s, 3 H), 3.78 (s, 2 H), 4.04-4.33 (m, 4 H),
4.37 (q, 2 H), 4.44 (s, 3 H), 6.52 (br s, 1 H). Anal. (C17H24N6O7)
C, H, N.
E t h yl 2-Acet a m id o-3-[3-et h oxy-5-(2-m et h yl-2H -1,2,4-
t r ia zo l-3-y l)is o x a zo l-4-y l]-2-(e t h o x y c a r b o n y l)p r o p i-
on a te (22e). Compound 22e was prepared from 21e (1.34 g,
4.7 mmol) by the method described for compound 22a . Flash
chromatography (silica gel, eluent: EtOAc/heptane ) 2:1 then
4:1) followed by crystallization (2-propanol) gave compound
1
22e (1.35 g, 68%): mp 130-131 °C; H NMR (CDCl3) δ 1.23
(t, 6 H), 1.45 (t, 3 H), 1.88 (s, 3 H), 3.73 (s, 2 H), 3.94-4.00 (m,
2 H), 4.00-4.27 (m, 2 H), 4.15 (s, 3 H), 4.37 (q, 2 H), 6.91 (br
s, 1 H), 7.95 (s, 1 H). Anal. (C18H25N5O7) C, H, N.
(RS)-2-Am in o-3-[3-h ydr oxy-5-(2-m eth yl-2H-1,2,4-tr iazol-
3-yl)isoxa zol-4-yl]p r op ion ic Acid Mon oh yd r a te (7e).
A
suspension of 22e (1.1 g, 2.6 mmol) in 47% HBr(aq) (60 mL)
was boiled under reflux for 1.5 h. The reaction mixture was
treated with charcoal and filtered. Water (90 mL) was added,
and the aqueous phase was washed with CH2Cl2 and concen-
trated in vacuo. The residue was dissolved in water (5 mL),
and pH of the solution was adjusted to ca. 3 by addition of 1
M NaOH. The precipitate which formed was collected by
filtration, resuspended in water, and stirred at room temper-
ature for another 16 h. Compound 7e was collected by
filtration (0.52 g, 74%): mp 222-223 °C dec; 1H NMR (DMSO-
d6) δ 3.04 (dd, 1 H), 3.24 (dd, 1 H), 3.72 (dd, 1 H), 4.06 (s, 3 H),
8.15 (s, 1 H); MS ((M + H)+) m/z 254. Anal. (C9H11N5O4‚H2O)
C, H, N.
(RS)-2-Am in o-3-[3-h yd r oxy-5-(2-m eth yl-2H-tetr a zol-5-
3-[4-(Br om om et h yl)-3-et h oxyisoxa zol-5-yl]-1-m et h yl-
1H-1,2,4-tr ia zole (21f). Compound 21f was prepared from
13f (2.0 g, 9.6 mmol) by the method described for compound
21a . Crude product (2.6 g, 94%): 1H NMR (CDCl3) δ 1.49 (t,
3 H), 4.05 (s, 3 H), 4.44 (q, 2 H), 4.72 (s, 2 H), 8.17 (s, 1 H).
The crude product was used in the next step without further
purification.
E t h yl 2-Acet a m id o-3-[3-et h oxy-5-(1-m et h yl-1H -1,2,4-
t r ia zo l-3-y l)is o x a zo l-4-y l]-2-(e t h o x y c a r b o n y l)p r o p i-
on a te (22f). Compound 22f was prepared from 21f (2.6 g,
9.1 mmol) by the method described for compound 22a . Flash
chromatography (silica gel, eluent: EtOAc/heptane ) 5:1 then
EtOAc/MeOH ) 95:5) followed by crystallization (2-propanol)
gave compound 22f as colorless crystals (2.45 g, 64%): mp
122-123 °C; 1H NMR (CDCl3) δ 1.24 (t, 6 H), 1.42 (t, 3 H),
1.74 (s, 3 H), 3.77 (s, 2 H), 4.00 (s, 3 H), 4.05-4.33 (m, 4 H),
4.35 (q, 2 H), 6.70 (br s, 1 H), 8.11 (s, 1 H). Anal. (C18H25N5O7)
C, H, N.
yl)isoxa zol-4-yl]p r op ion ic Acid Hem ih yd r a t e (7i).
A
suspension of 22i (0.3 g, 0.7 mmol) in 47% HBr(aq) (40 mL)
was boiled under reflux for 2.5 h. The reaction mixture was
concentrated in vacuo, and the residue was dissolved in water
(40 mL). The aqueous phase was washed with EtOAc, filtered,
and reduced in vacuo. The residue was dissolved in water,
and pH of the solution was adjusted to ca. 3 by addition of 1
M NaOH. The resulting precipitate was recrystallized (water),
affording 7i as colorless crystals (0.12 g, 64%): mp 236-238
1
°C dec; H NMR (DMSO-d6) δ 3.06 (dd, 1 H), 3.29 (dd, 1 H),
3.73 (dd, 1 H), 4.47 (s, 3 H); MS ((M + H)+) m/z 255. Anal.
(C8H10N6O4‚0.5H2O) C, H; N: calcd, 31.93; found, 30.89.
Eth yl 2-Aceta m id o-3-(5-cya n o-3-eth oxyisoxa zol-4-yl)-
2-(eth oxyca r bon yl)p r op ion a te (23). A mixture of 15 (2.0
g, 13.1 mmol), NBS (2.6 g, 14.5 mmol), and dibenzoyl peroxide
(catalytic amount) in CCl4 (100 mL) was boiled under reflux
for 3 days. The reaction mixture was filtered and evaporated
to dryness in vacuo (3.0 g, 100%, according to 1H NMR, a 4:2:1
mixture of 4-(bromomethyl)-3-ethoxyisoxazole-5-carbonitrile,
15, and 4-(dibromomethyl)-3-ethoxyisoxazole-5-carbonitrile
was obtained). Compound 23 was prepared from the residue
by the method described for compound 22a . Flash chroma-
tography (silica gel, eluent: EtOAc/heptane ) 1:3 then 1:2)
followed by crystallization (EtOAc/heptane) gave compound 23
(RS)-2-Am in o-3-[3-h ydr oxy-5-(1-m eth yl-1H-1,2,4-tr iazol-
3-yl)isoxa zol-4-yl]p r op ion ic Acid Hyd r a te (7f). A suspen-
sion of 22f (2.0 g, 4.7 mmol) in 47% HBr(aq) (70 mL) was boiled
under reflux for 2 h. The reaction mixture was concentrated
in vacuo, and the residue was dissolved in water (70 mL). The
aqueous phase was washed with EtOAc, treated with charcoal,
filtered, and reduced in vacuo (20 mL). A pH of ca. 3 was
obtained by addition of 1 M NaOH, affording compound 7f as
1
as colorless crystals (1.5 g, 55%): mp 103-104 °C; H NMR
(CDCl3) δ 1.28 (t, 6 H), 1.42 (t, 3 H), 2.05 (s, 3 H), 3.58 (s, 2
H), 4.15-4.41 (m, 6 H), 6.61 (br s, 1 H). Anal. (C16H21N3O7)
C, H, N.
1
colorless crystals (1.0 g, 82%): mp 250-252 °C dec; H NMR
(DMSO-d6, CF3COOH) δ 3.11 (dd, 1 H), 3.32 (dd, 1 H), 3.98 (s,

Heteroaryl Analogues of AMPA
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18 2841
E t h yl 2-Acet a m id o-3-[3-et h oxy-5-(1H -t et r a zol-5-yl)-
isoxa zol-4-yl]-2-(eth oxyca r bon yl)p r op ion a te (24). A sus-
pension of 23 (9.8 g, 26.7 mmol), NaN3 (3.5 g, 53.4 mmol), and
TEA hydrochloride (7.4 g, 53.4 mmol) in 1,2-dimethoxyethane
(250 mL) was boiled under reflux for 17 h. The mixture was
cooled and poured onto water (200 mL). pH of the aqueous
phase was adjusted to ca. 1 by addition of 37% HCl(aq), and
the aquoeus phase was extracted with ether. The organic
extracts were dried (Na2SO4), and the solvent was removed
in vacuo. Flash chromatography (silica gel, eluent: 1,2-
dimethoxyethane/1% AcOH) afforded a residue, which was was
stirred with acetone. Crude 24 was collected by filtration (6.3
g, 58%). A small sample was recrystallized (2-propanol) to give
colorless crystals: mp 193-195 °C; 1H NMR (DMSO-d6) δ 1.11
(t, 6 H), 1.37 (t, 3 H), 1.61 (s, 3 H), 3.58 (s, 2 H), 3.85-4.14 (m,
4 H), 4.30 (q, 2 H), 8.19 (br s, 1 H). Anal. (C16H22N6O7) C, H,
N. The crude product was used in the next step without
further purification.
(RS)-2-Am in o-3-[3-h ydr oxy-5-(1H-tetr azol-5-yl)isoxazol-
4-yl]p r op ion ic Acid Hyd r a te (7g). A suspension of 24 (1.0
g, 2.5 mmol) in 47% HBr(aq) (15 mL) was boiled under reflux
for 3 h. The reaction mixture was concentrated in vacuo, and
the residue dissolved in water (12 mL). The precipitate which
formed was collected by filtration to give 7g as colorless
crystals (0.52 g, 83%): mp 227-230 °C dec; 1H NMR (DMSO-
d6) δ 3.00-3.23 (m, 2 H), 4.23 (t, 1 H); MS ((M + H)+) m/z
241. Anal. (C7H8N6O4‚H2O) C, H; N: calcd, 32.56; found,
31.49.
with an Ag/AgCl pellet electrode. The cortex and corpus
callosum parts were constantly superfused with a Mg2+-free
oxygenated Krebs buffer at room temperature. The test
compounds were added to the cortex superfusion medium, and
the potential difference between the electrodes was recorded
on a chart recorder. Application of agonists were done for 90
s, and for antagonist experiments, the antagonists were
applied alone for 90 s followed by coapplication of the agonist
and antagonist for another 90 s.
P oten tiom etr ic Titr a tion s. The method by Clarke and
Cahoon44 was used in a slightly modified version. A solution
of the compound to be titrated was prepared in dilute HCl
containing 150 mM NaCl. The molarity and volume of the
solution were determined by the solubility of the compound.
All compounds were of relative low solubility in water and
titrated in volumes up to 100 mL of dilute HCl of molarity
down to 5 mM. As titrant was used 0.5 M NaOH. During
the titration procedure the sample solution was bubled with
nitrogen. Due to the low solubility of the compounds, the
titration curve of solvent alone was subtracted from that of
the solution. The resulting difference curve was then analyzed
by a curve-fitting algorithm developed in Lotus 1-2-3 program
(version 5.0 for Windows). The spreadsheet provided a fitted
curve to the actual measured points based on the dependent
variables. These are the pKa values, the equivalence volume,
and the initial titrant volume. The measured pH during the
titration is treated as an independent variable. The method
allowed pKa determination in the pH range from 3 to 10.
E t h yl (RS)-2-Am in o-3-[3-et h oxy-5-(1H-im id a zol-2-yl)-
isoxa zol-4-yl]p r op ion a te (25). A mixture of 23 (1.1 g, 3.0
mmol), 5.4 M NaOMe (0.1 mL, 0.5 mmol), and MeOH (5 mL)
was stirred at room temperature for 30 min. The mixture was
cooled (0-5 °C) and AcOH added (0.6 mL, 10.5 mmol).
Aminoacetaldehyde dimethyl acetal (0.4 mL, 3.7 mmol) was
added, and the resulting mixture was stirred at room tem-
perature for 24 h. The solvents were removed in vacuo, and
the residue was added a boiling solution of 1 M HCl (30 mL)
and boiled under reflux for 48 h. The solution was cooled,
filtered, and evaporated to dryness in vacuo. The residue was
dissolved in a saturated solution of HCl in EtOH (110 mL)
and boiled under reflux for 4 h. The solvent was evaporated
in vacuo, and the residue was dissolved in an ice/water
mixture, which was basified by careful addition of an aquoeus
solution of Na2CO3 (5%). The aquoeus phase was extracted
with EtOAc (3 × 50 mL), and the organic extracts were washed
with brine, dried (MgSO4), and concentrated in vacuo. The
resulting residue was crystallized (EtOH) to give crude 25 (0.45
g, 51%). A small sample was recrystallized (EtOH) to give
Com p u t a t ion a l Met h od s. The construction of input
structures for the illustrations were done by means of the
molecular modeling program MacMimic.45 The statistical
analysis (Multiple Linear Regression) was performed by the
use of the Microsoft Excel program (version 5.0) for Macintosh
computers. Semiemperical calculations were performed in
SPARTAN 4.1.1.46
Ack n ow led gm en t. We acknowledge Dr. Henrik
Pedersen for the difference-NOE experiment and Mrs.
Anne Nordly for the secretarial assistance.
Refer en ces
(1) Krogsgaard-Larsen, P., Hansen, J . J ., Eds. Excitatory Amino
Acid Receptors: Design of Agonists and Antagonists; Ellis
Horwood: Chichester, 1992.
(2) Collingridge, G. L., Watkins, J . C., Eds. The NMDA Receptor;
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.
1
colorless crystals: mp 122-123 °C; H NMR (CDCl3) δ 1.32
(t, 3 H), 1.43 (t, 3 H), 2.89 (dd, 1 H), 3.04 (dd, 1 H) 3.85 (dd, 1
H), 4.15-4.31 (m, 2 H), 4.37 (q, 2 H), 7.20 (s, 2 H); MS ((M +
H)+) m/z 295. Anal. (C13H18N4O4) C, H, N. The crude product
was used in the next step without further purification.
(RS)-2-Am in o-3-[3-h ydr oxy-5-(1H-im idazol-2-yl)isoxazol-
4-yl]p r op ion ic Acid Hem ih yd r a te (7b). A suspension of
25 (0.44 g, 1.5 mmol) in 47% HBr(aq) (30 mL) was boiled under
reflux for 2.5 h. The reaction mixture was concentrated in
vacuo, and the residue dissolved in water (40 mL). The
aqueous phase was washed with EtOAc, filtered and reduced
in vacuo (1 mL). A pH of ca. 3 was obtained by addition of 1
M NaOH, and compound 7b was collected as colorless crystals
(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
receptors: Novel targets for drug development. J . Med. Chem.
1995, 38, 1417-1426.
(8) Deutsch, S. I.; Morihisa, J . M. Glutamatergic abnormalities in
Alzheimer’s disease and
a rationale for clinical trials with
L-glutamate. Clin. Neuropharmacol. 1988, 11, 18-35.
(9) Greenamyre, J . T.; Young, A. B. Excitatory amino acids and
Alzheimer’s disease. Neurobiol. Ag. 1989, 10, 593-602.
(10) Bowen, D. M. Treatment of Alzheimer’s disease. Molecular
pathology versus neurotransmitter-based therapy. Br. J . Psy-
chiat. 1990, 157, 327-330.
(11) Madsen, U.; Ebert, B.; Krogsgaard-Larsen, P. Modulation of
AMPA receptor function in relation to glutamatergic abnormali-
ties in Alzheimer’s disease. Biomed. Pharmacother. 1994, 48,
305-311.
(12) Carlsson, M.; Carlsson, A. Interactions between glutamatergic
and monoaminergic systems within the basal ganglia - implica-
tions for schizophrenia and Parkinson’s disease. Trends Neuro-
sci. 1990, 13, 272-276.
(13) Ulas, J .; Cotman, C. W. Excitatory amino acid receptors in
schizophrenia. Schizophrenia Bull. 1993, 19, 105-117.
(14) Deutsch, S. I.; Mastropaolo, J .; Schwartz, B. L.; Rosse, R. B.;
Morihisa, J . M. A “glutamatergic hypothesis” of schizophrenia.
Rationale for pharmacotherapy with glycine. Clin. Neurophar-
macol. 1989, 12, 1-13.
1
(0.24 g, 65%): mp 266-269 °C dec; H NMR (DMSO-d6, CF3-
COOH) δ 3.21 (d, 2 H), 4.24 (br s, 1 H), 7.77 (s, 2 H); MS ((M
+ H)+) m/z 239. Anal. (C9H10N4O4‚0.5H2O) C, H, N.
Ra d ioliga n d Bin d in g Assa ys. Affinities for NMDA,
AMPA, and KAIN receptors were determined using the ligands
[3H]CGP 39653,35 [3H]AMPA,36 and [3H]KAIN,37 respectively.
The membrane preparations used in all the receptor binding
experiments were prepared according to the method of Ransom
and Stec.43
In Vitr o Electr op h ysiology. A rat cortical slice model for
determination of EAA-evoked depolarizations described by
Harrison and Simmonds38 was used in a slighly modified
version. Wedges (500 µm thick) of rat brain, containing
cerebral cortex and corpus callosum, were placed through a
grease barrier for electrical isolation with each part in contact

2842 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 18
Bang-Andersen et al.
(15) Ebert, B.; Søby, K. K.; Madsen, U.; Krogsgaard-Larsen, P.
Glutamic acid receptors in schizophrenia and Alzheimer’s
disease: Functional partial agonism and receptor modulation
as potential therapeutic approaches. In Schizophrenia - An
Integrated View; Fog, R., Gerlach, J ., Hemmingsen, R., Eds.;
Munksgaard: Copenhagen, 1995; pp 379-398.
(29) Finnegan, W. G.; Henry, R. A.; Lofquist, R. An improved
synthesis of 5-substituted tetrazoles. J . Am. Chem. Soc. 1958,
80, 3908-3911.
(30) Reese, C. B.; Zhang, P. Z. Phosphotriester approach to the
synthesis of oligonucleotides: A reappraisal. J . Chem. Soc.,
Perkin Trans. 1 1993, 2291-2301.
(31) Kadaba, P. K. Protic-dipolar aprotic solvents in 1,3-cycloaddition
reactions. Role of dipolar aprotic solvents in cycloadditions
involving anionic 1,3-dipoles and an improved synthesis of 1,5-
substituted tetrazoles. Synth. Commun. 1971, 1, 1-5.
(32) Lin, Y. I.; Lang, S. A., J r.; Lovell, M. F.; Perkinson, N. A. New
synthesis of 1,2,4-triazoles and 1,2,4-oxadiazoles. J . Org. Chem.
1979, 44, 4160-4164.
(33) Atkinson, M. R.; Polya, J . B. Triazoles. Part III. Mono- and di-
methyl(phenyl)-1,2,4-triazoles. J . Chem. Soc., Perkin Trans. 1
1954, 3319-3324.
(16) Hansen, J . J .; Krogsgaard-Larsen, P. Structural, conformational,
and stereochemical requirements of central excitatory amino acid
receptors. Med. Res. Rev. 1990, 10, 55-94.
(17) Krogsgaard-Larsen, P.; Ebert, B.; Lund, T. M.; Bra¨uner-Osborne,
H.; Sløk, F. A.; J ohansen, T. N.; Brehm, L.; Madsen, U. Design
of excitatory amino acid receptor agonists, partial agonists and
antagonists: ibotenic acid as a key lead structure. Eur. J . Med.
Chem. 1996, 31, 515-537.
(18) Krogsgaard-Larsen, P.; Honore´, T.; Hansen, J . J .; Curtis, D. R.;
Lodge, D. New class of glutamate agonist structurally related
to ibotenic acid. Nature (London) 1980, 284, 64-66.
(19) Krogsgaard-Larsen, P.; Hansen, J . J .; Lauridsen, J .; Peet, M.
J .; Leah, J . D.; Curtis, D. R. Glutamic acid agonists, stereo-
chemical and conformational studies of DL-R-amino-3-hydroxy-
5-methyl-4-isoxazolepropionic acid (AMPA) and related com-
pounds. Neurosci. Lett. 1982, 31, 313-317.
(20) Sløk, F. A.; Ebert, B.; Lang, Y.; Krogsgaard-Larsen, P.; Lenz, S.
M.; Madsen, U. Excitatory amino-acid receptor agonists. Syn-
thesis and pharmacology of analogues of 2-amino-3-(3-hydroxy-
5-methylisoxazol-4-yl)propionic acid. Eur. J . Med. Chem. 1997,
32, 329-338.
(21) Lauridsen, J .; Honore´, T.; Krogsgaard-Larsen, P. Ibotenic acid
analogues. Synthesis, molecular flexibility, and in vitro activity
of agonists and antagonists at central glutamic acid receptors.
J . Med. Chem. 1985, 28, 668-672.
(22) J ohansen, T. N.; Frydenvang, K.; Ebert, B.; Krogsgaard-Larsen,
P.; Madsen, U. Synthesis and structure-activity studies on acidic
amino acids and related diacids as NMDA receptor ligands. J .
Med. Chem. 1994, 37, 3252-3262.
(23) 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)propionic acid
[(R,S)APPA]. J . Med. Chem. 1994, 37, 878-884.
(24) 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.
(25) Moltzen, S. L.; Falch, E.; Bøgesø, K. P.; Krogsgaard-Larsen, P.
5-Arylisoxazolyl-4-yl-substituted 2-amino carboxylic acid com-
pounds. PCT Int. Appl. No. 9512, 587; Chem. Abstr. 1995, 123,
112056n.
(34) Case, F. H. The preparation of hydrazidines and as-triazines
related to substituted 2-cyanopyridines. J . Org. Chem. 1965, 30,
931-933.
(35) Sills, M. A.; Fagg, G.; Pozza, M.; Angst, C.; Brundish, D. E.; Hurt,
3H]CGP 39653: A new
S. D.; Wilusz, E. J .; Williams, M. [
N-methyl-D-aspartate antagonist radioligand with low nano-
molar affinity in rat brain. Eur. J . Pharmacol. 1991, 192, 19-
24.
(36) Honore´, T.; Nielsen, M. Complex structure of quisqualate-
sensitive glutamate receptors in rat cortex. Neurosci. Lett. 1985,
54, 27-32.
(37) 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.
(38) 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.
(39) Brehm, L.; J ørgensen, F. S.; Hansen, J . J .; Krogsgaard-Larsen,
P. Agonists and antagonists for central glutamic acid receptors.
Drug News Perspect. 1988, 1, 138-144.
(40) 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.
(41) Madsen, U.; Ebert, B.; Krogsgaard-Larsen, P.; Wong, E. H. F.
Synthesis and pharmacology of (RS)-2-amino-3-(3-hydroxy-5-
trifluoromethyl-4-isoxazolyl)propionic acid, a potent AMPA re-
ceptor agonist. Eur. J . Med. Chem. 1992, 27, 479-484.
(42) Parham, W. E.; Wynberg, H.; Ramp, F. L. Heterocyclic Vinyl
Ethers. III. The Synthesis of 1,4-Dithiadiene and 1,4-Dithiadiene
Disulfone. J . Am. Chem. Soc. 1953, 75, 2065-2069.
(43) 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 . Neuro-
chem. 1988, 51, 830-836.
(44) Clarke, F. H.; Cahoon, N. M. Ionization constants by curve
fitting: Determination of partition and distribution coefficients
of acids and bases and their ions. J . Pharm. Sci. 1987, 76, 611-
620.
(45) MacMimic, 2.0. The program MacMimic and a Macintosh version
of the MM2(91) program is available from InStar Software AB,
Ideon Research Park, S-223 70 Lund, Sweden.
(26) 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.
(27) Madsen, U.; Nielsen, E. Ø.; Curtis, D. R.; Beattie, D. T.;
Krogsgaard-Larsen, P. Synthesis and single cell pharmacology
of potential heterocyclic bioisosteres of the excitatory amino acid
antagonist glutamic acid diethyl ester. Acta Chem. Scand. 1990,
44, 96-102.
(28) Newman, M. S.; Arkell, A.; Fukunaga, T. The synthesis and some
reactions of di-t-butylacetic acid and di-t-butylketene. J . Am.
Chem. Soc. 1960, 82, 2498-2501.
(46) SPARTAN Wavefunction, Inc., 18401 Von Karman Ave., Suite
370, Irvine, CA 92715.
J M970253B