Structural determinants of AMPA agonist activity in analogues of 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid: Synthesis and pharmacology

Article abstract of DOI:10.1021/jm0003586

We have previously shown that the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA, 2), binds to AMPA receptors in a manner different from that of AMPA (1) i

Full text of DOI:10.1021/jm0003586

4910
J . Med. Chem. 2000, 43, 4910-4918
Str u ctu r a l Deter m in a n ts of AMP A Agon ist Activity in An a logu es of
2-Am in o-3-(3-ca r boxy-5-m eth yl-4-isoxa zolyl)p r op ion ic Acid : Syn th esis a n d
P h a r m a cology
Benny Bang-Andersen,† Haleh Ahmadian,‡ Sibylle M. Lenz,† Tine B. Stensbøl,‡ Ulf Madsen,‡
Klaus P. Bøgesø,† and Povl Krogsgaard-Larsen*,‡
Centre for Drug Design and Transport, Medicinal Chemistry Research, H. Lundbeck A/ S, 9 Ottiliavej, DK-2500 Valby,
Denmark, and Department of Medicinal Chemistry, The Royal Danish School of Pharmacy, 2 Universitetsparken,
DK-2100 Copenhagen, Denmark
Received August 17, 2000
We have previously shown that the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid
(AMPA) receptor agonist, 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA, 2),
binds to AMPA receptors in a manner different from that of AMPA (1) itself and that 2, in
contrast to 1, also binds to kainic acid receptor sites. To elucidate the structural requirements
for selective activation of the site/conformation of AMPA receptors recognized by 2, a number
of isosteric analogues of 2 have now been synthesized and pharmacologically characterized.
The compound 2-amino-3-(5-carboxy-3-methoxy-4-isoxazolyl)propionic acid (3a ) (IC50 ) 0.11
µM; EC50 ) 1.2 µM), which is a regioisostere of 2 with a methoxy group substituted for the
methyl group, was approximately equipotent with 2 (IC50 ) 0.020 µM; EC50 ) 1.0 µM) as an
inhibitor of [3H]AMPA binding and as an AMPA agonist, respectively, whereas the correspond-
ing 3-ethoxy analogue 3b (IC50 ) 1.0 µM; EC50 ) 4.8 µM) was slightly weaker. The analogues
3c-e, containing C3 alkoxy groups, were an order of magnitude weaker than 3b, whereas the
additional steric bulk of the alkoxy groups of 3f-i or the presence of an acidic hydroxyl group
at the 3-position of the isoxazole ring of 3j prevented interaction with AMPA receptor sites.
The 2-amino-3-(2-alkyl-5-carboxy-3-oxo-4-isoxazolyl)propionic acids 4a ,b ,i, which are regio-
isosteric analogues of 3a ,b,i, showed negligible interaction with AMPA recognition sites.
Similarly, replacement of the carboxyl group of 3b by isosteric tetrazolyl or 1,2,4-triazolyl groups
to give 5 and 6, respectively, or conversion of 3b into analogue 7, in which the diaminosquaric
acid group has been bioisosterically substituted for the R-aminocarboxylic acid unit, provided
compounds completely devoid of effect at AMPA receptors. In contrast to the parent compound
ACPA (2) (IC50 ) 6.3 µM), none of the analogues described showed detectable inhibitory effect
on [3H]kainic acid receptor binding.
In tr od u ction
Recently, the recombinant S1-S2 binding domain of
an AMPA receptor subtype, cocrystallized with the
atypical AMPA agonist kainic acid, was subjected to an
X-ray crystallographic structure analysis,12 and subse-
quently, a number of AMPA receptor ligands, including
AMPA (1), have been cocrystallized with the S1-S2
binding domain.13 Different structural classes of recep-
tor ligand may interact with the AMPA recognition
site(s) in a different manner, and an important step
toward design of AMPA receptor ligands on a rational
basis is mapping of the structural determinants of
receptor-ligand interactions.
Extensive structure-activity studies (SARs) on ana-
logues of AMPA (1) (Figure 1), in which a variety of
alkyl,14 aryl,15 or heteroaryl groups16-18 have been
substituted for the methyl group, have shed some light
on the structural requirements for activation of AMPA
receptors by this class of ligand. These studies have
supported the view that, in addition to binding sites for
the three charged groups of AMPA agonists, this recep-
tor contains a pocket capable of accommodating lipo-
philic groups of limited size.19
The central excitatory neurotransmitter effects of (S)-
glutamic acid [(S)-Glu] are mediated by three hetero-
geneous classes of ionotropic receptors named N-methyl-
D-aspartic acid (NMDA), 2-amino-3-(3-hydroxy-5-methyl-
4-isoxazolyl)propionic acid (AMPA), and kainic acid
receptors1-3 and a number of subtypes of metabotropic
receptors.3,4 These or perhaps distinct subtypes of these
receptors have been associated with certain neurologic
and psychiatric diseases and are potential therapeutic
targets in such diseases.3-6
In recent years, much interest has been directed
toward the role of AMPA receptors in the mechanisms
associated with cognitive functions,7-9 and enhancement
of AMPA receptor functions has been shown to facilitate
learning and memory.3,10,11 Although AMPA receptor
agonists may not be used therapeutically due to poten-
tial neurotoxicity, these observations have focused
interest on the molecular mechanisms of receptor
activation and, thus, on the structural basis of AMPA
receptor-agonist interactions.
* To whom correspondence should be addressed. Phone: (+45) 35
30 65 11. Fax: (+45) 35 30 60 40. E-mail: ano@dfh.dk.
† H. Lundbeck A/S.
We have recently shown that AMPA (1) and the
AMPA receptor agonist 2-amino-3-(3-carboxy-5-methyl-
‡ The Royal Danish School of Pharmacy.
10.1021/jm0003586 CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/10/2000

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4911
Sch em e 1a
F igu r e 1. Structures of (S)-glutamic acid [(S)-Glu], the AMPA
receptor agonists 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-
propionic acid (AMPA, 1) and 2-amino-3-(3-carboxy-5-methyl-
4-isoxazolyl)propionic acid (ACPA, 2), and the new ACPA
analogues and bioisosteres 3a -j, 4a ,b,i, and 5-7.
4-isoxazolyl)propionic acid (ACPA, 2)20 bind to AMPA
receptor sites in a dissimilar manner,21 suggesting that
the structural determinants of AMPA receptor activa-
tion by 1 and 2 are different. These aspects prompted
us to synthesize and pharmacologically characterize a
series of compounds (3a -j, 4a ,b,i, and 5-7) in which
different parts of the molecule of ACPA (2) have been
isosterically modified (Figure 1).
Resu lts
Ch em istr y. The analogues 3a -j, 4a ,b,i, and 7 con-
taining a carboxyl group in the 5-position of the isox-
azole ring (Figure 1) were synthesized as shown in
Schemes 1 and 2, and the analogues 5 and 6 containing
heterocyclic carboxyl group bioisosteres were synthe-
sized as outlined in Scheme 2. Two strategies were
followed for the synthesis of the former group of
analogues, involving introduction of the different O- or
N-alkyl groups at an early stage in the synthetic
sequence or as a later synthetic step (Scheme 1).
Following the first strategy, the ethoxy group of 816 was
hydrolyzed using 47% aqueous HBr to give 9. The ethyl
ester of 9, compound 10, was treated with appropriate
a
Reagents and conditions: (a) 47% HBr (aq), reflux; (b)
C2H5OH, HCl, reflux; (c) alkyl halide, K2CO3, (DMF, acetone, or
EtOH), heat; (d) NBS, dibenzoyl peroxide, CCl4, reflux; (e)
CH3CONHCH(COOC2H5)2, (CH3)3COK, NMP, rt; (f) 0.5 M HCl
(aq), reflux; (g) 1 M HCl (aq), reflux; (h) 47% HBr (aq), reflux; (i)
(Boc)2O, TEA, THF, H2O, rt; (j) 1 M NaOH (aq), EtOH, 90 °C; (k)
1 M HCl (aq), 40 °C.

4912 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
Sch em e 2a
the respective target compounds 3c,f,h and 4i, whereas
20e,g,i, which contain unsaturated alkyl substituents
or an O-benzyl substituent, were deprotected under
basic conditions. The deprotections of 20e,g were ac-
companied by the formation of several byproducts, and
purification of 3e,g required ion-exchange chromato-
graphic procedures.
The target compounds 5 and 6 were synthesized from
the earlier described intermediates 22 and 23,16 respec-
tively (Scheme 2). The squaric acid analogue 7 was
synthesized from intermediate 13b, which via the
phthalimide derivative 24 was converted into the amino
acid 25. Under basic conditions, 25 was reacted with
3-amino-4-ethoxy-3-cyclobutene-1,2-dione to give 7.
In Vitr o P h a r m a cology. The 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 excitatory amino acid receptor ligands. With
the exception of ACPA (2) itself, none of the compounds
showed significant affinity for [3H]kainic acid binding
sites22 (Table 1). Compounds 3a ,b containing a 3-meth-
oxy and a 3-ethoxy group, respectively, were potent
inhibitors of [3H]AMPA receptor binding,23 and these
compounds showed agonist potencies comparable with
that of 2. The larger alkoxy groups of 3c-e resulted in
reduced affinity for and proportionally reduced agonist
activity at AMPA receptors, whereas 3f-i were es-
sentially inactive. Similarly, compound 3j containing an
acidic hydroxy group in the 3-position of the isoxazole
ring did not interact with AMPA receptors. The N-
alkylated isomers of 3a ,b, compounds 4a ,b, were totally
inactive at AMPA receptors, whereas the N-benzyl
analogue 4i was a very weak inhibitor of NMDA-
induced depolarization (Table 1). Among the compounds
tested, only 7 showed weak affinity for NMDA receptor
sites labeled by tritiated 3-(2-carboxypiperazin-4-yl)-
propyl-1-phosphonic acid ([3H]CPP),24 and this affinity
turned out to reflect a marginally weak NMDA antago-
nist effect.
a
Reagents and conditions: (a) 1
M HCl (aq), reflux; (b)
potassium phthalimide, DMF, 90 °C; (c) 1 M NaOH (aq), reflux;
(d) 3-amino-4-ethoxy-3-cyclobutene-1,2-dione, NaOH, rt.
alkyl halides to give the O-alkyl derivatives 11a ,b,d and
the corresponding N-alkylated products 12a ,b,d in
different ratios depending on the structure of the alkyl
halides. For each alkyl halide, this ratio was strongly
dependent on the reaction conditions, in particular the
nature of the solvent used. Thus, for example, alkylation
of 10 with methyl iodide, using potassium carbonate as
base, gave 11a and 12a at ratios of 15:1, 2:1, and 1:1
using DMF, acetone, or ethanol, respectively, as solvents
(experimental details not given for acetone). The ethoxy
derivative 11b was most conveniently synthesized by
esterification of 8,16 whereas the N-ethyl isomer 12b was
isolated from a 2:1 mixture of 11b and 12b, prepared
by treatment of 9 with ethyl iodide and potassium
carbonate in ethanol.
The intermediates 15a ,b,d and 16a ,b were synthe-
sized by NBS bromination of 11a ,b,d and 12a ,b, re-
spectively, and subsequent treatment with diethyl
acetamidomalonate under basic conditions, where 16a
was extensively hydrolyzed to give 17a . Acid deprotec-
tion of these intermediates under different conditions
gave the respective target compounds 3a ,b,d ,j and 4a ,b,
which were isolated in the zwitterionic forms.
The second strategy for the synthesis of target com-
pounds containing carboxyl groups in the 5-position of
the isoxazole ring was based on 3j as the starting
material (Scheme 1). Compound 3j, synthesized from 8
via 11b, 13b, and 15b, was converted into the N-Boc-
protected diester 19, which was treated with appropri-
ate alkyl halides. Using DMF as solvent, the O-alkyl
derivatives 20c,e-h were obtained in excellent yield
(>80%) with formation of the corresponding N-alkyl
derivatives in trace amounts. Compounds 20i and 21i
were isolated from a mixture of 20i and 21i prepared
by treatment of 19 with benzyl bromide using acetone
as solvent. Acid deprotection of 20c,f,h , and 21i gave
Discu ssion
Like AMPA (1), ACPA (2) contains an isoxazole ring,
but whereas 1 is a 3-isoxazolol bioisostere of Glu,
compound 2 is an aminodicarboxylic acid homologue of
Glu (Figure 1). Despite these quite profound dissimilari-
ties, both 1 and 2 are potent AMPA receptor agonists,
but in contrast to 1, compound 2 also displays significant
affinity for kainic acid receptor sites (Table 1).
In a number of cases, homologation of heterocyclic Glu
analogues showing AMPA agonist activity has been
shown to provide metabotropic Glu receptor ligands.3
Although ACPA (2) is a homologue of Glu, neither
enantiomer of 2 shows detectable affinity for metabo-
tropic Glu receptor subtypes (H. Bra¨uner-Osborne and
T. N. J ohansen, unpublished results). Consequently,
none of the ACPA analogues 3-7 described here were
tested for effect at metabotropic Glu receptors.
On the basis of quite extensive SAR studies on 1 and
structurally related compounds, the methyl group of 1
appears to fit into a cavity at the AMPA recognition site
capable of accommodating lipophilic groups of limited
size.14-19 The aim of the present project was to elucidate
the structural determinants of AMPA agonist activity

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4913
Ta ble 1. Receptor Binding Data and AMPA Agonist or NMDA Antagonist Effects (values ( SEM, n ) 3-7)
in analogues of 2 and, furthermore, to develop analogues
of 2 devoid of the kainic acid receptor affinity. All of
the analogues of 2 synthesized contain an alanine side
chain with a 3-oxygenated isoxazole ring structure
(Figure 1).
In continuation of the successful X-ray crystallo-
graphic analysis of the recombinant S1-S2 binding
domain of the AMPA receptor cocrystallized with the
atypical AMPA agonist, kainic acid,12 and the successful
crystallization of this binding domain bound to AMPA
(1) and structurally related potent AMPA agonists,3,13
attempts will be made to subject ACPA (2) and, for
example, 3a to similar biostructural studies. Studies
along these lines may pave the way for future design of
AMPA receptor ligands on a rational basis.
Interestingly, none of the compounds synthesized
showed detectable affinity for kainic acid receptor sites
(Table 1). In analogy with the observations for analogues
of 1 containing different alkyl substituents, the AMPA
recognition site was shown to impose constraints on the
size of the alkoxy substituents of the analogues of 2.
Whereas 3a ,b containing a 3-methoxy and a 3-ethoxy
group, respectively, are potent AMPA receptor agonists,
3c-e containing C3 alkoxy groups are markedly weaker,
and incorporation of one or more additional C atoms into
the alkoxy groups essentially abolished activity. The
inactivity of the N-alkyl isomers of 3a ,b, compounds
4a ,b, seems to indicate that only substituents of limited
size and proper orientation can be accommodated by the
proposed lipophilic pocket, which does not recognize the
3-hydroxy group of 3j, which is deprotonated at pH 7.
Exp er im en ta l Section
Ch em istr y. All reactions were performed under nitrogen.
Reagents were purchased commercially and used without
purification. Melting points were determined in capillary tubes
(Bu¨chi 535 apparatus) and are uncorrected. All evaporations
of solvents were performed in vacuo at 15 mmHg. 1H NMR
was recorded on a Bruker AC 250 MHz spectrometer. 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, h ) heptet, dd ) double
doublet, ddd ) double doublet of doublets, dt ) doublet of
triplet, dq ) doublet of quartet, tdd ) triplets of double
doublets, m ) multiplet. NMR signals corresponding to acidic
protons are omitted. 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) or in a mixture of acetonitrile/water/aqueous ammonia
(25%) (25:25:1) 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 inter-
mediates 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 an FeCl3 spraying reagent (yellow color).
Compounds containing amino groups were visualized using a
ninhydrin spraying reagent. Microanalyses were performed by
A similar degree of structural specificity was exhibited
by the site of the AMPA receptor which binds the
heteroaromatic carboxyl groups of 2 and analogues such
as 3a ,b. Thus, compounds 5 and 6, in which a tetrazolyl
and a 1,2,4-triazolyl group, respectively, have been
substituted for the carboxyl group of 3b (Figure 1), were
totally inactive. Since the tetrazolyl group of 5 is
markedly more acidic than the 1,2,4-triazolyl group of
6,16 steric rather than, or in addition to, pKa effects of
these azole rings seem to prevent interaction with the
AMPA recognition site. Compound 7 is not recognized
by AMPA or kainic acid receptor sites. The very weak
NMDA antagonist effect of 7 is, however, consistent
with the reported NMDA antagonist effect of certain
amino acids, in which the R-amino acid functionality has
been bioisosterically replaced by a 3,4-diamino-3-cy-
clobutene-1,2-dione unit.25

4914 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
the Lundbeck Analytical Department, and results obtained
were within (0.4% of the theoretical values if not otherwise
stated.
was subjected to flash chromatography [silica gel, eluent:
heptane/EtOAc (2:1 then 1:2)] to give compound 11b as a
colorless solid (2.6 g, 47%; for analytical data see above) and
crude 12b which was crystallized (heptane) to give 12b as
yellow crystals (0.64 g, 12%): mp 65-67 °C; 1H NMR (CDCl3)
δ 1.36 (t, 3H), 1.41 (t, 3H), 2.15 (s, 3H), 4.15 (q, 2H), 4.42 (q,
2H). Anal. (C9H13NO4) C, H, N.
3-Hyd r oxy-4-m eth ylisoxa zole-5-ca r boxylic Acid (9). A
mixture of 816 (15 g, 88 mmol) and 47% aqueous HBr (150 mL)
was heated under reflux for 6 h. The solution was cooled, and
crystalline 9 was collected by filtration (8.7 g, 69%): mp 257-
259 °C. Water (100 mL) was added to the residual solution,
which was left after filtration, and the aqueous phase was
extracted with Et2O (6 × 400 mL). The combined organic phase
was washed with brine (100 mL), dried (MgSO4), and concen-
trated to give crude 9 (3.0 g, 24%): 1H NMR (DMSO-d6) δ 2.05
(s, 3H); MS ((M - H)-) m/z 142. Anal. (C5H5NO4) C, H, N. A
mixture of the two crops was used in the next step.
Eth yl 3-Hyd r oxy-4-m eth ylisoxa zole-5-ca r boxyla te (10).
A mixture of 9 (6.0 g, 42 mmol) and a saturated solution of
HCl in EtOH (110 mL) was heated under reflux for 4 h. The
resulting solution was concentrated, and the residue was
dissolved in EtOAc. The organic phase was dried (MgSO4) and
concentrated to give crude 10 (7.2 g, 100%). A small sample
was recrystallized (EtOAc/heptane) to give colorless crystals:
mp 133-134 °C; 1H NMR (CDCl3) δ 1.43 (t, 3H), 2.20 (s, 3H),
4.44 (q, 2H). Anal. (C7H9NO4) C, H, N. The crude product was
used in the next step without further purification.
E t h yl 3-Isop r op oxy-4-m et h ylisoxa zole-5-ca r b oxyla t e
(11d ) a n d E t h yl 2,3-Dih yd r o-2-isop r op yl-4-m et h yl-3-
oxoisoxa zole-5-ca r boxyla te (12d ). A mixture of 10 (3.4 g,
20 mmol), K2CO3 (5.5 g, 40 mmol), and acetone (75 mL) was
heated under reflux for a total of 40 h. 2-Bromopropane (3.7
mL, 40 mL) was added after 1, 4.5, 20, and 26 h, and additional
K2CO3 (2.8 g, 20 mmol) was added after 26 h. The mixture
was cooled, filtered, and concentrated. Flash chromatography
[silica gel, eluent: EtOAc/heptane (1:2)] gave crude 11d as a
yellow oil (3.2 g, 76%): 1H NMR (CDCl3) δ 1.40 (d, 6H), 1.40
(t, 3H), 2.13 (s, 3H), 4.40 (q, 2H), 4.99 (h, 1H). The crude
product was used in the next step without further purification.
Further elution furnished compound 12d as a brown oil (0.25
g, 6%). A small sample was crystallized (heptane) to give
1
colorless crystals: mp 67-68 °C; H NMR (CDCl3) δ 1.40 (d,
6H), 1.41 (t, 3H), 2.13 (s, 3H), 4.41 (q, 2H), 4.73 (h, 1H). Anal.
(C10H15NO4) C, H, N.
Eth yl 4-(Br om om eth yl)-3-m eth oxyisoxa zole-5-ca r box-
yla te (13a ). A mixture of 11a (1.3 g, 7.0 mmol), NBS (1.4 g,
7.9 mmol), dibenzoyl peroxide (catalytic amount), and CCl4 (40
mL) was heated under reflux for 10 h. The mixture was cooled
to room temperature, filtered, and concentrated to give crude
13a as a yellow oil (1.8 g, 97%): 1H NMR (CDCl3) δ 1.44 (t,
3H), 4.10 (s, 3H), 4.46 (q, 2H), 4.48 (s, 2H). The crude product
was used in the next step without further purification.
Compounds 13b,d and 14a were prepared in a similar
manner as described for 13a .
Eth yl 3-Meth oxy-4-m eth ylisoxazole-5-car boxylate (11a).
A mixture of 10 (3.0 g, 17.5 mmol), methyl iodide (1.1 mL, 17.5
mmol), K2CO3 (4.8 g, 35.1 mmol), and DMF (40 mL) was heated
at 40 °C for 1 h. The mixture was poured onto an ice/water
mixture (100 mL), and the aqueous phase was extracted with
Et2O (3 × 100 mL). The combined organic phase was washed
with water (2 × 50 mL) and brine (50 mL), dried (MgSO4),
and concentrated (2.4 g, 74%; according to 1H NMR, a 15:1
mixture of 11a and 12a was obtained). The residue was
subjected to flash chromatography [silica gel, eluent: CH2Cl2/
Et2O (9:1)] affording crude 11a as a yellow oil (1.4 g, 43%):
1H NMR (CDCl3) δ 1.41 (t, 3H), 2.15 (s, 3H), 4.05 (s, 3H), 4.41
(q, 2H). No attempt was made to isolate compound 12a from
this mixture. The crude product was used in the next step
without further purification.
Eth yl 4-(Br om om eth yl)-3-eth oxyisoxa zole-5-ca r boxyl-
a te (13b). Yield: 5.3 g, 100%; 1H NMR (CDCl3) δ 1.44 (t, 3H),
1.47 (t, 3H), 4.44 (q, 2H), 4.46 (q, 2H), 4.50 (s, 2H). The crude
product was used in the next step without further purification.
Eth yl 4-(Br om om eth yl)-3-isop r op oxyisoxa zole-5-ca r -
1
boxyla te (13d ). Yield: 4.0 g, 100%; H NMR (CDCl3) δ 1.43
Eth yl 3-Meth oxy-4-m eth ylisoxazole-5-car boxylate (11a)
a n d E t h yl 2,3-Dih yd r o-2,4-d im et h yl-3-oxoisoxa zole-5-
ca r boxyla t e (12a ). A mixture of 10 (2.0 g, 11.6 mmol),
K2CO3 (4.0 g, 29 mmol), and EtOH (50 mL) was heated at 40
°C for 1 h followed by the addition of methyl iodide (0.8 mL,
13 mmol). The resulting mixture was heated at 40 °C for 25
h, and during this time additional methyl iodide (0.8 mL, 13
mmol) was added three times. The mixture was filtered and
concentrated (according to 1H NMR, a 1:1 mixture of 11a and
12a was obtained). Flash chromatography [silica gel, eluent:
CH2Cl2/Et2O (9:1 then 1:1)] gave compound 11a as a yellow
oil (0.40 g, 18%; for analytical data see above) and compound
12a (0.45 g, 21%). A small sample of 12a was recrystallized
(t, 3H), 1.44 (d, 6H), 4.46 (q, 2H), 4.47 (s, 2H), 5.04 (h, 1H).
The crude product was used in the next step without further
purification.
Eth yl 4-(Br om om eth yl)-2,3-dih ydr o-2-m eth yl-3-oxoisox-
a zole-5-ca r boxyla te (14a ). Yield: 1.0 g, 100%; 1H NMR
(CDCl3) δ 1.45 (t, 3H), 3.64 (s, 3H), 4.46 (s, 2H), 4.48 (q, 2H).
The crude product was used in the next step without further
purification.
E t h yl 2-Acet a m id o-2-(et h oxyca r b on yl)-3-[5-(et h oxy-
ca r bon yl)-3-m et h oxy-4-isoxa zolyl]p r op ion a t e (15a ). A
mixture of diethyl acetamidomalonate (1.6 g, 7.4 mmol) and
potassium tert-butoxide (0.9 g, 8.0 mmol) in N-methylpyrro-
lidin-2-one (NMP) (30 mL) was stirred at room temperature
for 30 min followed by the addition of 13a (1.8 g, 6.8 mmol) in
NMP (10 mL) (temperature 22-28 °C). The resulting mixture
was stirred at room temperature for 1.5 h, and the mixture
was poured onto an ice/water mixture (100 mL). The aqueous
phase was extracted with EtOAc (3 × 150 mL), and the
combined organic phase was washed with an aqueous solution
of potassium tert-butoxide in water, water (100 mL), and brine
(100 mL), dried (MgSO4), and concentrated. Flash chromatog-
raphy [silica gel, eluent: EtOAc/heptane (1:1)] afforded crude
15a (1.8 g, 66%). A small sample was recrystallized (EtOAc/
heptane) to give colorless crystals: mp 78-80 °C; 1H NMR
(CDCl3) δ 1.28 (t, 6H), 1.39 (t, 3H), 1.95 (s, 3H), 3.68 (s, 2H),
4.00 (s, 3H), 4.10-4.34 (m, 4H), 4.37 (q, 2H), 6.50 (br s, 1H).
Anal. (C17H24N2O9) C, H, N. The crude product was used in
the next step without further purification.
1
(EtOAc/heptane) to give colorless crystals: mp 64-65 °C; H
NMR (CDCl3) δ 1.41 (t, 3H), 2.15 (s, 3H), 3.60 (s, 3H), 4.42 (q,
2H). Anal. (C8H11NO4) C, H, N. Crude 12a was used in the
next step without further purification.
Eth yl 3-Eth oxy-4-m eth ylisoxa zole-5-ca r boxyla te (11b).
A mixture of 816 (3.6 g, 21 mmol) and a saturated solution of
HCl in EtOH (100 mL) was heated under reflux for 2 h. The
mixture was concentrated, and the residue was dissolved in
CH2Cl2 (200 mL), dried (MgSO4), and concentrated. Flash
chromatography [silica gel, eluent: heptane/EtOAc (4:1)] gave
compound 11b as a colorless solid (4.0 g, 95%): mp 34-36 °C;
1H NMR (CDCl3) δ 1.40 (t, 3H), 1.45 (t, 3H), 2.15 (s, 3H), 4.36
(q, 2H), 4.40 (q, 2H). Anal. (C9H13NO4) C, H, N.
Eth yl 3-Eth oxy-4-m eth ylisoxa zole-5-ca r boxyla te (11b)
a n d Eth yl 2,3-Dih yd r o-2-eth yl-4-m eth yl-3-oxoisoxa zole-
5-ca r boxyla te (12b). A mixture of 9 (3.7 g, 26 mmol), K2CO3
(5.8 g, 42 mmol), ethyl iodide (2.8 mL, 35 mmol), and EtOH
(70 mL) was heated under reflux for a total of 68 h. Additional
ethyl iodide (2.8 mL, 35 mmol) was added after 16, 24, and 44
h, and additional K2CO3 (5.8 g, 42 mmol) was added after 44
h. The mixture was filtered and concentrated (according to 1H
NMR, a 2:1 mixture of 11b and 12b was obtained). The residue
Compounds 15b,d , 16a , and 17 were prepared in a similar
manner as described for 15a .
Eth yl 2-Aceta m id o-2-(eth oxyca r bon yl)-3-[3-eth oxy-5-
(eth oxyca r bon yl)-4-isoxa zolyl]p r op ion a te (15b). Flash
chromatography [silica gel, eluent: EtOAc/heptane (1:2 then
2:1)] afforded crude 15b as a yellow oil (3.0 g, 67%). A small

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4915
sample was crystallized (2-propanol) to give colorless crys-
tals: mp 94-95 °C; H NMR (CDCl3) δ 1.28 (t, 6H), 1.39 (t,
3H), 1.41 (t, 3H), 1.95 (s, 3H), 3.68 (s, 2H), 4.08-4.33 (m, 4H),
4.33 (q, 2H), 4.36 (q, 2H), 6.49 (br s, 1H). Anal. (C18H26N2O9)
C, H, N. The crude product was used in the next step without
further purification.
and concentrated. The residue was triturated with Et2O
followed by the addition of water (10 mL). Compound 3b was
obtained as colorless crystals (1.0 g, 68%): mp 238-240 °C
dec; 1H NMR (DMSO-d6) δ 1.34 (t, 3H), 2.90 (dd, 1H), 3.03
(dd, 1H), 3.96 (dd, 1H), 4.23 (q, 2H); MS ((M + H)+) m/z 245.
Anal. (C9H12N2O6) C, H, N.
1
(RS)-2-Am in o-3-(5-ca r boxy-3-isop r op oxy-4-isoxa zolyl)-
p r op ion ic Acid (3d ). A suspension of 15d (1.15 g, 2.7 mmol)
in 1 M HCl (100 mL) was heated under reflux for 24 h. The
mixture was cooled to room temperature and washed with
EtOAc (3 × 100 mL). The aqueous phase was filtered and
concentrated. The residue was dissolved in water (10 mL), and
the precipitate that formed was stirred at room temperature
E t h yl 2-Acet a m id o-2-(et h oxyca r b on yl)-3-[5-(et h oxy-
ca r b on yl)-3-isop r op oxy-4-isoxa zolyl]p r op ion a t e (15d ).
Flash chromatography [silica gel, eluent: EtOAc/heptane (1:2
then 1:1)] gave crude 15d (1.6 g, 73%). A small sample was
recrystallized (2-propanol) to give colorless crystals: mp 73-
1
74 °C; H NMR (CDCl3) δ 1.27 (t, 6H), 1.38 (d, 6H), 1.39 (t,
3H), 1.96 (s, 3H), 3.66 (s, 2H), 4.07-4.37 (m, 4H), 4.36 (q, 2H),
4.95 (h, 1H), 6.50 (br s, 1H). Anal. (C19H28N2O9) C, H, N. The
crude product was used in the next step without further
purification.
1
for 16 h affording 3d (0.46 g, 66%): mp 242-243 °C dec; H
NMR (DMSO-d6) δ 1.32 (dd, 6H), 2.88 (dd, 1H), 3.01 (dd, 1H),
3.96 (dd, 1H), 4.79 (h, 1H); MS ((M + H)+) m/z 259. Anal.
(C10H14N2O6) C, H, N.
E t h yl 2-Acet a m id o-2-(et h oxyca r b on yl)-3-[5-(et h oxy-
ca r bon yl)-2,3-d ih yd r o-2-m eth yl-3-oxo-4-isoxa zolyl]p r op i-
on a te (16a ) a n d Eth yl 2-Aceta m id o-2-(eth oxyca r bon yl)-
3-(5-carboxy-2,3-dihydro-2-methyl-3-oxo-4-isoxazolyl)propio-
n a te (17a ). Flash chromatography [silica gel, eluent: EtOAc/
heptane (3:1 then 5:1)] followed by crystallization (EtOAc/
heptane) gave compound 16a as colorless crystals (0.10 g,
6%): mp 121-123 °C; 1H NMR (CDCl3) δ 1.28 (t, 6H), 1.39 (t,
3H), 1.95 (s, 3H), 3.57 (s, 3H), 3.65 (s, 2H), 4.18-4.33 (m, 4H),
4.39 (q, 2H), 6.84 (br s, 1H). Anal. (C17H24N2O9) C, H, N. The
potassium tert-butoxide aqueous solution, after extracted by
EtOAc to give 16a , was acidified with concentrated aqueous
HCl and extracted with EtOAc (3 × 150 mL). The combined
organic phase was washed with brine, dried (MgSO4), and
concentrated. The residue was crystallized (EtOH/Et2O) to give
compound 17a as colorless crystals (0.3 g, 20%): mp 203-205
°C; 1H NMR (CDCl3, DMSO-d6) δ 1.27 (t, 3H), 1.97 (s, 3H),
3.56 (s, 3H), 3.60 (s, 2H), 4.15-4.31 (m, 4H), 7.09 (br s, 1H).
Anal. (C15H20N2O9) C, H, N.
E t h yl 2-Acet a m id o-2-(et h oxyca r b on yl)-3-[5-(et h oxy-
ca r bon yl)-2-et h yl-2,3-d ih yd r o-3-oxo-4-isoxa zolyl]p r op i-
on a te (16b). A mixture of 12b (0.56 g, 2.8 mmol), NBS (0.55
g, 3.1 mmol), dibenzoyl peroxide (catalytic amount), and CCl4
(40 mL) was heated under reflux for 4 h. The mixture was
cooled to room temperature, filtered, and concentrated to give
crude 14b as a yellow oil (0.8 g, 100%). The crude product was
dissolved in NMP (10 mL) and subsequently added to a
mixture of diethyl acetamidomalonate (0.67 g, 3.1 mmol) and
potassium tert-butoxide (0.38 g, 3.4 mmol) in NMP (20 mL)
(temperature 22-28 °C). The reaction mixture was stirred at
room temperature for 1 h and poured onto an ice/water mixture
(100 mL). The aqueous phase was extracted with EtOAc (4 ×
75 mL), and the combined organic phase was washed with
brine, dried (MgSO4), and concentrated. Flash chromatography
[silica gel, eluent: EtOAc/heptane (3:1)] followed by crystal-
lization (EtOAc/heptane) gave compound 16b as colorless
crystals (88 mg, 8%): mp 113-114 °C; 1H NMR (CDCl3) δ 1.27
(t, 6H), 1.34 (t, 3H), 1.40 (t, 3H), 1.96 (s, 3H), 3.64 (s, 3H),
3.99 (q, 2H), 4.16-4.36 (m, 4H), 4.39 (q, 2H), 6.86 (br s, 1H).
Anal. (C18H26N2O9) C, H, N.
(RS)-2-Am in o-3-(5-car boxy-3-h ydr oxy-4-isoxazolyl)pr o-
p ion ic Acid Hyd r a te (3j). A suspension of 15b (1.1 g, 2.7
mmol) in 47% HBr (aq) (5 mL) was heated under reflux for 1
h. The solution was cooled to room temperature and washed
with CH2Cl2 (3 × 30 mL), concentrated and triturated with
Et2O. The residue was dissolved in water, and pH of the
solution was adjusted to about 3.5 by addition of 0.1 M NaOH.
Compound 3j was obtained as colorless crystals (0.34 g, 57%):
1
mp 175-177 °C; H NMR (DMSO-d6) δ 3.00 (d, 2H), 3.88 (t,
1H); MS ((M + H)+) m/z 217. Anal. (C7H8N2O6‚0.25H2O) C, H,
N.
(RS)-2-Am in o-3-(5-car boxy-2,3-dih ydr o-2-m eth yl-3-oxo-
4-isoxa zolyl)p r op ion ic Acid Hyd r a te (4a ). A suspension
of 16a (0.05 g, 0.1 mmol), 17a (0.30 g, 0.8 mmol), and 1.0 M
HCl (55 mL) was heated under reflux for 16 h. The mixture
was cooled to room temperature, washed with EtOAc (3 × 50
mL), and concentrated. The residue was dissolved in water (4
drops) and EtOH (5 mL), and pH of the solution was adjusted
to about 3 by addition of TEA. The precipitate that formed
was collected by filtration to give 4a as colorless crystals (0.07
g, 72%): mp 211-212 °C dec; 1H NMR (DMSO-d6) δ 2.87 (dd,
1H), 2.97 (dd, 1H), 3.43 (s, 3H), 3.92 (dd, 1H); MS ((M + H)+)
m/z 231. Anal. (C8H10N2O6‚0.25H2O) C, H, N.
(RS)-2-Am in o-3-(5-ca r boxy-2-eth yl-2,3-d ih yd r o-3-oxo-
4-isoxa zolyl)p r op ion ic Acid Mon oh yd r a te (4b). A suspen-
sion of 16b (60 mg, 0.1 mmol) and 1.0 M HCl (30 mL) was
boiled under reflux for 18 h. The mixture was cooled to room
temperature, washed with EtOAc (3 × 30 mL), and concen-
trated. The residue was dissolved in water (2 drops) and EtOH
(1 mL), and pH of the solution was adjusted to about 3 by
addition of TEA. The resulting mixture was concentrated, and
the residue recrystallized twice (EtOH) to yield 4b as colorless
crystals (5 mg, 13%): 1H NMR (D2O, 1,4-dioxane δ 3.70) δ 1.28
(t, 3H), 3.19 (d, 2H), 4.01 (q, 2H), 4.18 (t, 1H); MS ((M + H)+)
m/z 245. Anal. (C9H12N2O6‚H2O) C, N; H: calcd, 5.38; found,
4.74.
Eth yl (RS)-2-Am in o-3-[5-(eth oxyca r bon yl)-3-h yd r oxy-
4-isoxa zolyl]p r op ion a te (18). A mixture of 3j (3.5 g, 11.8
mmol) and a saturated solution of HCl in EtOH (50 mL) was
heated under reflux for 2.5 h and evaporated to dryness to
give crude 18 (4.15 g, 100%): 1H NMR (CDCl3) δ 1.16 (t, 3H),
1.34 (t, 3H), 3.20 (dd, 1H), 3.32 (dd, 1H), 4.18 (q, 2H), 4.36-
4.42 (m, 3H). The crude product was used in the next step
without further purification.
(RS)-2-Am in o-3-(5-ca r b oxy-3-m et h oxy-4-isoxa zolyl)-
p r op ion ic Acid Hyd r a te (3a ). A suspension of 15a (1.2 g,
3.0 mmol) in 0.5 M HCl (100 mL) was heated under reflux for
48 h. The mixture was cooled to room temperature, washed
with CH2Cl2 (100 mL) and Et2O (2 × 100 mL), filtered, and
concentrated. The residue was dissolved in water (5 mL), and
pH of the solution was adjusted to about 3 by addition of NaOH
(0.1 and 1 M). The aqueous phase was reduced to about 2 mL,
and the precipitate that formed was collected by filtration. The
crude product was stirred in water (2 mL) at room temperature
E t h yl
(RS)-2-[(ter t-Bu t yloxyca r b on yl)a m in o]-3-[5-
(eth oxycar bon yl)-3-h ydr oxy-4-isoxazolyl]pr opion ate (19).
To a solution of 18 (19.0 g, 70.0 mmol) in THF (175 mL) and
water (175 mL) was added a solution of (Boc)2O (19.5 g, 89.2
mmol) and TEA (34 mL, 243 mmol) in THF (50 mL). The
resulting solution was stirred at room temperature for 3 days
and then concentrated. To the residue was added water (250
mL), and pH of the aqueous phase was adjusted to 3 with 1 M
HCl. The aqueous phase was extracted with EtOAc (3 × 400
mL), and the combined organic phase was dried (MgSO4) and
evaporated to dryness. The residue was subjected to flash
chromatography [silica gel, eluent: EtOAc/heptane/HOAc (1:
5:1%)] to give crude 19 (20.7 g, 79%). A sample was recrystal-
1
for 24 h affording 3a (70 mg, 10%): mp 222-225 °C dec; H
NMR (DMSO-d6) δ 2.88 (dd, 1H), 3.01 (dd, 1H), 3.85-3.96 (m,
1H), 3.90 (s, 3H); MS ((M + H)+) m/z 231. Anal. (C8H10N2O6‚
0.25H2O) C, H, N.
(RS)-2-Am in o-3-(5-ca r boxy-3-eth oxy-4-isoxa zolyl)p r o-
p ion ic Acid (3b). A suspension of 15b (2.5 g, 6 mmol) in 1 M
HCl (200 mL) was heated under reflux for 20 h. The reaction
mixture was cooled to room temperature, washed with Et2O

4916 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25
lized (heptane/EtOAc): mp 91-93 °C; 1H NMR (CDCl3) δ 1.27
(t, 3H), 1.40 (s, 9H), 1.42 (t, 3H), 3.02 (dd, 1H), 3.25 (dd, 1H),
4.14-4.25 (m, 2H), 4.43 (q, 2H), 4.40-4.50 (m, 1H), 5.40 (br s,
1H). Anal. (C16H24N2O8) C, H, N. The crude product was used
in the next step without further purification.
(q, 2H), 4.40 (q, 2H), 4.46-4.60 (m, 1H), 5.13 (d, 2H), 5.72 (br
d, 1H), 7.35 (s, 5H).
(RS)-2-Am in o-3-(5-car boxy-3-pr opoxy-4-isoxazolyl)pr o-
p ion ic Acid (3c). A mixture of 20c (1.0 g, 2.4 mmol) and 1 M
HCl (100 mL) was heated under reflux for 5 h and then heated
at 70 °C for 16 h. The mixture was cooled to room temperature,
washed with EtOAc (3 × 100 mL), and concentrated. The
residue was recrystallized (water) to give 3c (0.32 g, 52%): mp
250-251 °C dec; 1H NMR (D2O, NaOD) δ 0.95 (t, 3H), 1.76
(sextet, 2H), 2.78 (dd, 1H), 2.90 (dd, 1H), 3.42 (dd, 1H), 4.17
(t, 2H); MS ((M + H)+) m/z 259. Anal. (C10H14N2O6) C, H, N.
Compounds 3f,h were prepared using methods analogous
to that described for 3c.
(RS)-2-Am in o-3-(3-bu toxy-5-ca r boxy-4-isoxa zolyl)p r o-
p ion ic Acid (3f). Yield: 0.33 g, 58%; mp 238-240 °C dec; 1H
NMR (D2O, NaOD) δ 0.95 (t, 3H), 1.43 (sextet, 2H), 1.76
(quintet, 2H), 2.80 (dd, 1H), 2.91 (dd, 1H), 3.44 (dd, 1H), 4.25
(t, 2H); MS ((M + H)+) m/z 273. Anal. (C11H16N2O6) C, H, N.
(RS)-2-Am in o-3-(5-ca r boxy-3-p en tyloxy-4-isoxa zolyl)-
p r op ion ic Acid (3h ). Yield: 0.55 g, 70%; mp 244-245 °C dec;
1H NMR (DMSO-d6) δ 0.88 (t, 3H), 1.32-1.40 (m, 4H), 1.73
(quintet, 2H), 2.90 (dd, 1H), 3.02 (dd, 1H), 3.95 (dd, 1H), 4.17
(t, 2H); MS ((M + H)+) m/z 287. Anal. (C12H18N2O6) C, H, N.
(RS)-2-Am in o-3-(3-a llyloxy-5-ca r boxy-4-isoxa zolyl)p r o-
p ion ic Acid Hem ih yd r a te Hyd r och lor id e (3e). A mixture
of 20e (0.7 g, 1.69 mmol), 1 M NaOH (60 mL), and EtOH (30
mL) was heated at 90 °C for 16 h. The mixture was cooled to
room temperature, acidified with 4 M HCl, heated at 40 °C
for 4 h, and then concentrated. The residue was suspended in
water (10 mL) and filtered, and the filtrate was subjected to
ion-exchange chromatography (IRA-400, eluent: 1 M HOAc).
Collection and evaporation of the ninhydrin-reactive fractions
followed by recrystallization (water) gave 3e as the hydrochlo-
ride salt (0.12 g, 28%): mp 243-244 °C dec; 1H NMR (DMSO-
d6) δ 2.93 (dd, 1H), 3.05 (dd, 1H), 3.99 (dd, 1H), 4.73 (d, 2H),
5.29 (dd, 1H), 5.44 (dd, 1H), 6.05 (tdd, 1H). Anal. (C10H12N2O6‚
HCl‚0.5H2O) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Eth yl (RS)-
2-[(ter t-Bu tyloxyca r bon yl)a m in o]-3-[3-a lk oxy-5-(eth oxy-
ca r bon yl)-4-isoxa zolyl]p r op ion a tes 20c,e-h . A solution of
19 and K2CO3 in DMF (40 mL) was stirred at 40 °C for 1 h
followed by addition of alkyl bromide, and the resulting
mixture was stirred at 40 °C for an additional 4 h. The mixture
was then poured onto an ice/water mixture (100 mL), and the
aqueous phase was extracted with Et2O (3 × 100 mL). The
combined organic phase was washed with water (2 × 50 mL)
and brine (50 mL), dried (MgSO4), and concentrated. Flash
chromatography [silica gel, eluent: EtOAc/heptane (1:7)] gave
compounds 20c,e-h as oils, which were used in the next step
without further purification.
E t h yl
(RS)-2-[(ter t-Bu t yloxyca r b on yl)a m in o]-3-[5-
(eth oxycar bon yl)-3-pr opoxy-4-isoxazolyl]pr opion ate (20c).
Compound 19 (1.4 g, 3.8 mmol), K2CO3 (1.0 g, 7.5 mmol), and
propyl bromide (0.69 g, 5.6 mmol) were used to prepare 20c
(1.4 g, 92%): 1H NMR (CDCl3) δ 1.04 (t, 3H), 1.26 (t, 3H), 1.38
(s, 9H), 1.42 (t, 3H), 1.85 (sextet, 2H), 2.95 (dd, 1H), 3.13 (dd,
1H), 4.17 (q, 2H), 4.29 (t, 2H), 4.43 (q, 2H), 4.40-4.50 (m, 1H),
5.25 (br d, 1H).
Eth yl (RS)-2-[(ter t-Bu tyloxyca r bon yl)a m in o]-3-[3-a l-
lyloxy-5-(eth oxyca r bon yl)-4-isoxa zolyl]p r op ion a te (20e).
Compound 19 (1.0 g, 2.7 mmol), K2CO3 (0.75 g, 5.4 mmol), and
allyl bromide (0.49 g, 4.0 mmol) were used to prepare 20e (1.0
g, 90%): 1H NMR (CDCl3) δ 1.26 (t, 3H), 1.38 (s, 9H), 1.42 (t,
3H), 2.98 (dd, 1H), 3.15 (dd, 1H), 4.18 (q, 2H), 4.43 (q, 2H),
4.40-4.50 (m, 1H), 4.82 (dt, 2H), 5.22 (br d, 1H), 5.32 (ddd,
1H), 5.48 (ddd, 1H), 6.09 (tdd, 1H).
Eth yl (RS)-2-[(ter t-Bu tyloxyca r bon yl)a m in o]-3-[3-bu -
toxy-5-(eth oxyca r bon yl)-4-isoxa zolyl]p r op ion a te (20f).
Compound 19 (1.0 g, 2.8 mmol), K2CO3 (0.74 g, 5.4 mmol), and
butyl bromide (0.55 g, 4.0 mmol) were used to prepare 20f (1.1
g, 95%): 1H NMR (CDCl3) δ 0.95 (t, 3H), 1.27 (t, 3H), 1.35 (s,
9H), 1.42 (t, 3H), 1.50 (sextet, 2H), 1.82 (quintet, 2H), 2.98
(dd, 1H), 3.12 (dd, 1H), 4.18 (q, 2H), 4.35 (t, 2H), 4.43 (q, 2H),
4.45-4.51 (m, 1H), 5.22 (br d, 1H).
Compounds 3g,i were prepared using methods analogous
to that described for 3e.
(RS)-2-Am in o-3-[5-ca r b oxy-3-(tr a n s-2-b u t en yloxy)-4-
isoxa zolyl]p r op ion ic Acid (3g). Ion-exchange chromatog-
raphy (IRA-400, eluent: 1 M HOAc) gave crude 3g that was
subjected to ion-exchange chromatography (Dowex-50, elu-
ent: 1 M NH3). Concentration of the ninhydrin-reactive
fractions followed by recrystallization of the residue gave 3g
(40 mg, 13%): mp 203-204 °C dec; 1H NMR (DMSO-d6) δ 1.71
(dd, 3H), 2.75 (dd, 1H), 2.90 (dd, 1H), 3.10 (dd, 1H), 4.65 (d,
2H), 5.73 (dt, 1H), 5.87 (dq, 1H); MS ((M + H)+) m/z 271. Anal.
(C11H14N2O6) C, H, N.
E t h yl
(RS)-2-[(ter t-Bu t yloxyca r b on yl)a m in o]-3-[5-
(et h oxyca r b on yl)-3-(tr a n s-2-b u t en yloxy)-4-isoxa zolyl]-
p r op ion a te (20g). Compound 19 (1.4 g, 3.8 mmol), K2CO3 (1.0
g, 7.5 mmol), and crotyl bromide (0.90 g, 5.6 mmol) were used
to prepare 20g (0.80 g, 50%): 1H NMR (CDCl3) δ 1.26 (t, 3H),
1.38 (s, 9H), 1.42 (t, 3H), 1.76 (d, 3H), 2.97 (dd, 1H), 3.14 (dd,
1H), 4.18 (q, 2H), 4.43 (q, 2H), 4.40-4.50 (m, 1H), 4.75 (d, 2H),
5.22 (br d, 1H), 5.76 (dt, 1H), 5.89 (dq, 1H).
(RS)-2-Am in o-3-(3-ben zyloxy-5-ca r boxy-4-isoxa zolyl)-
p r op ion ic Acid (3i). A mixture of 20i (0.65 g, 1.4 mmol) and
1 M NaOH (50 mL) was heated under reflux for 16 h. The
mixture was cooled (5 °C), acidified with 4 M HCl, and
concentrated. The residue was recrystallized from water to give
E t h yl
(RS)-2-[(ter t-Bu t yloxyca r b on yl)a m in o]-3-[5-
(et h oxyca r b on yl)-3-p en t yloxy-4-isoxa zolyl]p r op ion a t e
(20h ). 19 (1.7 g, 4.6 mmol), K2CO3 (1.3 g, 9.3 mmol), and pentyl
bromide (1.1 g, 7.0 mmol) were used to prepared 20h (1.8 g,
76%): 1H NMR (CDCl3) δ 0.94 (t, 3H), 1.26 (t, 3H), 1.38 (s,
9H), 1.39-1.45 (m, 7H), 1.84 (quintet, 2H), 2.97 (dd, 1H), 3.11
(dd, 1H), 4.18 (q, 2H), 4.33 (t, 2H), 4.43 (q, 2H), 4.40-4.50 (m,
1H), 5.22 (br d, 1H).
1
3i (0.1 g, 23%): mp 209-211 °C dec; H NMR (DMSO-d6) δ
2.95 (dd, 1H), 3.05 (dd, 1H), 3.99 (t, 1H), 5.26 (s, 2H), 7.31-
7.52 (m, 5H); MS ((M + H)+) m/z 307. Anal. (C14H14N2O6) H,
N; C: calcd, 54.90; found, 54.31.
(RS)-2-Am in o-3-(2-ben zyl-5-ca r boxy-2,3-d ih yd r o-3-oxo-
4-isoxa zolyl)p r op ion ic Acid Mon oh yd r a te (4i). A mixture
of 21i (0.9 g, 1.9 mmol) and 1 M HCl was heated under reflux
for 5 h. The mixture was concentrated to give 4i (0.56 g,
80%): mp 146-148 °C dec; 1H NMR (DMSO-d6) δ 3.08 (dd,
1H), 3.19 (dd, 1H), 4.17 (br s, 1H), 5.16 (s, 2H), 7.24-7.45 (m,
5H); MS ((M + H)+) m/z 307. Anal. (C14H14N2O6‚H2O) C, H,
N.
(RS)-2-Am in o-3-[3-et h oxy-5-(1H -t et r a zol-5-yl)-4-isox-
a zolyl]p r op ion ic Acid (5). A suspension of 2216 (2.0 g, 4.9
mmol) in 1 M HCl (150 mL) was heated under reflux for 24 h.
The mixture was concentrated, and the residue was dissolved
in water. The pH of the solution was adjusted to about 3.5 by
addition of NaOH (0.1 and 1 M), and 5 was collected by
filtration (1.0 g, 76%): mp 273-275 °C dec; 1H NMR (DMSO-
d6) δ 1.38 (t, 3H), 3.08 (dd, 1H), 3.19 (dd, 1H), 4.23-4.36 (m,
Eth yl (RS)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-[3-ben -
zyloxy-5-(eth oxyca r bon yl)-4-isoxa zolyl]p r op ion a te (20i)
a n d Eth yl (RS)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-(2-ben -
zyl-5-eth oxyca r bon yl-2,3-d ih yd r o-3-oxo-4-isoxa zolyl)p r o-
p ion a te (21i). A mixture of 19 (3.2 g, 8.6 mmol), K2CO3 (2.4
g, 17.2 mmol), and acetone (40 mL) was heated to reflux
temperature followed by addition of benzyl bromide (2.2 g, 12.9
mmol). The resulting mixture was heated under reflux for 1.5
h
and concentrated. The residue was subjected to flash
chromatography [silica gel, eluent: EtOAc/heptane (1:2)] to
give 20i (1.64 g, 41%): 1H NMR (CDCl3) δ 1.20 (t, 3H), 1.39
(s, 9H), 1.43 (t, 3H), 2.96 (dd, 1H), 3.19 (dd, 1H), 4.12 (q, 2H),
4.35-4.60 (m, 1H), 4.45 (q, 2H), 5.20 (br d, 1H), 5.35 (d, 2H),
7.32-7.55 (m, 5H). And 21i (0.7 g, 18%): 1H NMR (CDCl3) δ
1.25 (t, 3H), 1.40 (s, 9H), 1.41 (t, 3H), 2.99-3.18 (m, 2H), 4.18

J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 25 4917
3H); MS ((M + H)+) m/z 269. Anal. (C9H12N6O4) C, H; N: calcd,
ibly reduced by at least 70%. In experiments designed to detect
antagonist effects of ACPA analogues at 1 mM concentrations,
the compounds were applied alone for 90 s followed by
coapplication of the appropriate agonist (5 µM AMPA, 10 µM
NMDA, or 5 µM kainic acid) and potential antagonist for
another 90 s.
31.33; found, 30.72.
(RS)-2-Am in o-3-[3-eth oxy-5-(1H-1,2,4-tr iazol-3-yl)-4-isox-
a zolyl]p r op ion ic Acid Hyd r a te (6). A suspension of 2316 (1.5
g, 2.3 mmol) in 1 M HCl (aq) (150 mL) was heated under reflux
for 24 h. The solution was cooled to room temperature, washed
with Et2O (2 × 150 mL) and CH2Cl2 (150 mL), filtered, and
concentrated. The residue was dissolved in water (5 mL), and
pH of the solution was adjusted to about 3.5 by addition of
NaOH (0.1 and 1 M) affording 6 (0.35 g, 56%): mp 225-227
°C dec; 1H NMR (DMSO-d6) δ 1.38 (t, 3H), 2.94 (dd, 1H), 3.18
(dd, 1H), 3.58 (dd, 1H), 4.30 (q, 2H), 8.64 (s, 1H); MS ((M +
H)+) m/z 268. Anal. (C10H13N5O4‚0.25H2O) C, H, N.
Ack n ow led gm en t. This work was supported by
grants from the Lundbeck Foundation. We also express
our gratitude to Heidi Petersen and Annette Kristensen
for technical assistance and Anne Nordly for secretarial
assistance.
Eth yl 3-Eth oxy-4-(p h th a lim id om eth yl)isoxa zole-5-ca r -
boxyla te (24). A suspension of potassium phthalimide (3.6
g, 19.7 mmol) in DMF (125 mL) was heated to 90 °C followed
by the addition of a solution of 13b (5.0 g, 17.9 mmol) in DMF
(85 mL). The mixture was stirred at 90 °C for 45 min, cooled,
and concentrated. To the residue was added water (250 mL),
and the aqueous phase was extracted with Et2O (2 × 200 mL).
The combined organic phase was dried (MgSO4) and concen-
trated. The residue was recrystallized (EtOH) to give 24 (3.7
Su p p or tin g In for m a tion Ava ila ble: Elemental analyses
for 3a -j, 4a ,b,i, 5-7, 9, 10, 11b, 12a ,b,d , 13b, 15a ,b,d , 16a ,b,
17a , 19, 24, and 25. This material is available free of charge
via the Internet at http://pubs.acs.org.
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