Design and synthesis of enantiomers of 3,5-dinitro-o-tyrosine: α- amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptor antagonists

Article abstract of DOI:10.1021/jm970276q

The R- and S-isomers of 3,5-dinitro-o-tyrosine (6a,b) have been synthesized though the use of chemoenzymatic synthesis and shown to bind differentially with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA, 3) receptors. The phenolic functional group of these o-tyrosine analogues was designed to act as a bioisostere of the γ-carboxyl group of glutamate. The S-isomer of 3,5-dinitro-o-tyrosine (6b) was 6.5 times more potent than the R-isomer (6a) in inhibiting [³H]AMPA binding with IC₅₀ values of 13 ± 7 and 84 ± 26 μM, respectively. The phenolic group was important for binding affinity since the methoxy compound 7 was less potent than the phenolic compound 6 in inhibiting the binding of AMPA. The free amino group was also shown to be important since the N-acetyl analogue 15 and the N-t-BOC compounds 16 and 17 exhibited very low affinity for the AMPA receptors. AMPA receptor functional tests showed that the o-tyrosine analogues are antagonists and that S-isomer 6b (IC₅₀ = 630 ± 140 μM) was more potent than the racemate 6 (IC₅₀ = 730 ± 88 μM) while the R-isomer 6a was inactive up to 1 mM concentration, which is consistent with the S- isomer having higher binding affinity than the R-isomer.

Full text of DOI:10.1021/jm970276q

1034
J . Med. Chem. 1998, 41, 1034-1041
Design a n d Syn th esis of En a n tiom er s of 3,5-Din itr o-o-tyr osin e:
r-Am in o-3-h yd r oxy-5-m eth yl-4-isoxa zolep r op a n oic Acid (AMP A) Recep tor
An ta gon ists
Guoping Sun,† Meri Slavica,‡ Norman J . Uretsky,‡ Lane J . Wallace,‡ Gamal Shams,‡ David M. Weinstein,‡
J ohn C. Miller,† and Duane D. Miller*,†
Department of Pharmaceutical Sciences, University of Tennessee, Memphis, Tennessee 38163, and College of Pharmacy,
The Ohio State University, Columbus, Ohio 43210
Received April 28, 1997
The R- and S-isomers of 3,5-dinitro-o-tyrosine (6a ,b) have been synthesized through the use
of chemoenzymatic synthesis and shown to bind differentially with the R-amino-3-hydroxy-5-
methyl-4-isoxazolepropanoic acid (AMPA, 3) receptors. The phenolic functional group of these
o-tyrosine analogues was designed to act as a bioisostere of the γ-carboxyl group of glutamate.
The S-isomer of 3,5-dinitro-o-tyrosine (6b) was 6.5 times more potent than the R-isomer (6a )
in inhibiting [3H]AMPA binding with IC50 values of 13 ( 7 and 84 ( 26 µM, respectively. The
phenolic group was important for binding affinity since the methoxy compound 7 was less potent
than the phenolic compound 6 in inhibiting the binding of AMPA. The free amino group was
also shown to be important since the N-acetyl analogue 15 and the N-t-BOC compounds 16
and 17 exhibited very low affinity for the AMPA receptors. AMPA receptor functional tests
showed that the o-tyrosine analogues are antagonists and that the S-isomer 6b (IC50 ) 630 (
140 µM) was more potent than the racemate 6 (IC50 ) 730 ( 88 µM) while the R-isomer 6a
was inactive up to 1 mM concentration, which is consistent with the S-isomer having higher
binding affinity than the R-isomer.
In tr od u ction
lation through activation of dopaminergic neurotrans-
mission in the NAC.6 Our biological studies have shown
that in addition to the well-documented dopamine
component, AMPA receptors (named after AMPA, 3, a
very selective and potent agonist) in the NAC and
ventral pallidum (VP) of rats are important in the
psychostimulant actions of amphetamine and cocaine.
We have shown that AMPA antagonists, such as (γ-
glutamylamino)methanesulfonic acid (GAMS, 4) and
6,7-dinitroquinoxaline-2,3-dione (DNQX, 5), block the
locomotor activity elicited by systemic administration
of amphetamine and/or cocaine.7 This experiment
strongly suggests that activation of AMPA receptors in
the NAC is important for the effects of amphetamine.
Glutamate (1) (Chart 1) and to a lesser extent
aspartate (2) have been established as excitatory neu-
rotransmitters in the mammalian central nervous sys-
tem (CNS), exerting their action on most neurons via
excitatory amino acid (EAA) receptors.1 In analogy to
other neurotransmitter receptors, heterogeneity among
EAA receptors has been observed. The EAA receptors
are classified based on electrophysiological, radioligand
binding, and biochemical studies into five subtypes:
N-methyl-D-aspartic acid (NMDA), (R,S)-R-amino-3-
hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA, 3),
kainate, (S)-2-amino-4-phosphonobutanoic acid (AP4),
and metabotropic receptors.2 EAA receptors appear to
be involved in a number of neuronal growth and
maturation processes. However, several lines of evi-
dence indicate that hyperactivation of EAA receptors
may be involved in the pathophysiology of a number of
neurological diseases such as ischemia, stroke, epilepsy,
dementia of the Alzheimer type, and Parkinsonism.3,4
Furthermore, it has been shown that endogenous
glutamate activity within the nucleus accumbens (NAC)
modulates the psychomotor activation induced by co-
caine and heroin.5 Thus, the development of EAA
antagonists might be useful as therapeutic intervention
in neurodegenerative diseases as well as providing a
new pharmacological strategy for treating the problems
associated with drug addiction.
To extend the structure-activity relationship studies
for AMPA receptor ligands, 3,5-dinitro-o-tyrosine (6) was
synthesized.8 The rationale behind this design was that
the phenolate anion (pKa ) 3.4)9 in 6 might serve as a
bioisostere for the γ-carboxyl anion of glutamate (1), as
the 3-hydroxyisoxazole anion of AMPA (3) presumably
does. However, compound 6 was found to be an
antagonist at AMPA receptors, inhibiting the locomotor
stimulation produced by amphetamine. Although 6 is
less potent than AMPA (3) in inhibiting specific [3H]-
AMPA binding in rat brain homogenate, it shows much
higher selectivity than other antagonists for AMPA vs
kainate receptors (approximately 20-fold more selective
for AMPA than for kainate receptors).8 In addition, in
an attempt to determine the importance of the pheno-
late in 6 in AMPA receptor binding, compound 7 was
designed and synthesized in which the phenolate was
masked with a methoxy group.
Psychostimulant drugs such as amphetamine and
cocaine are believed to produce their behavioral stimu-
* To whom correspondence should be addressed.
† University of Tennessee.
‡ The Ohio State University.
S0022-2623(97)00276-8 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/03/1998

AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1035
Ch a r t 1
Sch em e 1a
a
Reagents: (a) (1) n-BuLi, THF, -78 °C, (2) N-(phenylsulfonyl)-L-serine; (b) Et3SiH, CF3COOH; (c) CrO3/H2SO4, acetone, room
temperature; (d) 48% HBr, phenol; (e) NO2BF4, CH3CN, 0-5 °C; (f) L-amino acid oxidase, O2, catalase, tris-maleate buffer.
The AMPA receptor shows a pronounced stereoselec-
tivity toward the L-enantiomers of agonists. For ex-
ample, L-AMPA shows 4000 times higher affinity for
AMPA receptors than D-AMPA.10 The neuroexcitatory
effects of two other AMPA agonists, 4-bromohomoi-
botenic acid and 3-hydroxy-4,5,6,7-tetrahydroisoxazolo-
[5,4-c]pyridine-5-carboxylate also reside only with the
S-enantiomers. Therefore, one of our objectives was to
synthesize the optical isomers of 3,5-dinitro-o-tyrosine,
6a ,b, and to determine the enantioselectivity of the
AMPA receptors for binding and functional activity for
these AMPA ligands.
Homologation is a classical approach used to convert
agonists to antagonists. It was therefore our objective
to use o-tyrosine (o-tyrosine itself is unable to inhibit
specific [3H]AMPA binding in rat forebrain homoge-
nates) as a flexible template for designing AMPA
antagonists, and â-[2-(carboxymethoxy)phenyl]-R-ala-
nine (8) was designed to probe the distance between the
two carboxylic groups (six atoms apart in comparison
with a three-atom separation in Glu, 1, or AMPA, 3).
Rapoport et al.11 as outlined in Scheme 1, in which
L-serine is utilized as the chiral source. The S-isomer
of 6a can also be synthesized by using D-serine as the
starting material. N-(Phenylsulfonyl)-L-serine was syn-
thesized according to the procedure of Rapoport et al.11
Treatment of o-bromoanisole (9) with n-BuLi and the
lithium salt of N-(phenylsulfonyl)-L-serine at -78 °C
gave (S)-2-methoxy-R-[(phenylsulfonyl)amino]-â-hydrox-
ypropiophenone (10) in 35-50% yield. The carbonyl
group in 10 was reduced with Et3SiH in CF3COOH,11
resulting in the formation of (R)-2-[(phenylsulfonyl)-
amino]-3-(o-methoxyphenyl)propan-1-ol (11) with a 59%
yield. Inverse addition of an acetone solution of 11 to
J ones reagent at 0 °C gave (R)-2-[(phenylsulfonyl)-
amino]-3-(o-methoxyphenyl)propionic acid (59%) which
was refluxed with 48% HBr in phenol11 followed by
nitration with NO2BF412 at 0-5 °C to give 6a . However,
the product had an optical purity of only 50% ee,
possibly due to partial racemization in the oxidation
(step c) and/or deprotection (step d) under strong acidic
conditions. The nitration reaction should not cause
racemization since under the same conditions an opti-
cally pure 6a was obtained when using 13a as the
precursor, as demonstrated in Scheme 2.
Ch em istr y
Our initial attempt to synthesize R-enantiomeric
isomer 6a was based on the methodology developed by

1036 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Sun et al.
Sch em e 2a
a
Reagents: (a) L-amino acid oxidase, O2, 38 °C, 72 h, tris-maleate buffer; (b) NO2BF4, CH3CN, 0-5 °C; (c) same as b; (d) (1) (CH3CO)2O,
NaOH, (2) 2 N NaOH, room temperature, 30 min; (e) Aspergillus acylase I, pH 7.5-8.0, 40 °C; (f) [(CH3)3COCO]2O, NaOH, THF; (g) CH3I,
K2CO3, DMSO; (h) CF3CO2H, 45 min, room temperature.
Since de novo synthesis of 6a as well as 6b gave only
50% ee, we decided to take a different approach,
biological resolution of racemic amino acids with ap-
propriate enzymes, to obtain our target compound(s)
with higher optical purity. Hooker and Schellman13
obtained (R)-o-tyrosine by using the L-amino acid oxi-
dase (L-AAOx) method of Parikh et al.14 for the resolu-
tion of the racemic mixture of amino acids. L-AAOx is
a flavoenzyme that catalyzes the oxidation of L-amino
acids utilizing FAD as its redox coenzyme. The result-
ing FADH2 is reoxidized by O2. Oxidation of 1 mol of
amino acid results in 1 mol of keto acid, NH3, and H2O2,
with consumption of 1 mol of O2.15 Phenylalanine is an
excellent substrate for this enzyme, and examples from
the literature show that even substituted phenylala-
nines (-NO2, -F, -OCH3, -CH3, -CF3) still undergo oxida-
tion with L-AAOx.16
Kin etic Resolu tion of (R)-3,5-Din itr o-o-tyr osin e
(6a ) a n d (R)-o-Tyr osin e by L-AAOx. Encouraged by
the literature reports,13 we decided to resolve 6a from
the combined 6a and 6b product obtained by the
procedure described in Scheme 1 or from racemate 6,
using L-AAOx as a chemoenzymatic agent. However,
we were unable to obtain optically pure R-isomer 6a by
this method. We do not know why the enzyme L-AAOx
does not oxidize 6b into the R-keto acid 12. The two
nitro groups on the benzene ring somehow block the
oxidation (by steric bulk and/or electronic effect?). Since
this attempt failed, we decided to resolve (R)-o-tyrosine
(13a ) from the racemic mixture of commercially avail-
able o-tyrosine (13) and nitrate it to obtain 6a , as
outlined in Scheme 2. In this experiment, the S-
enantiomer of 13 was readily oxidized to R-keto acid 14
and removed, and 6a was obtained in 31% yield with
high optical purity (>99% ee).
Pirrung et al. have suggested that D-amino acid
oxidase (D-AAOx) could be used to obtain L-isomers of
aromatic amino acids.17 However Parikh et al. have
shown that attempts to prepare the L-isomers of phe-
nylalanine, tryptophan, tyrosine, and hydroxyproline by
using D-AAOx led to products largely contaminated with
the respective D-isomers.14 Hellermann et al.18 have
shown that D-AAOx is considerably inhibited by many
aromatic compounds such as benzoic acid, phenylpyru-
vic acid, and indole-2-carboxylic acid. Therefore, we
predicted that the oxidation of (R)-o-tyrosine with the
D-AAOx would be effectively hindered by the products
of the reaction.
Kin etic Resolu tion of (S)-3,5-Din itr o-o-tyr osin e
(6b) by Asper gillu s Acyla se I. Chemoenzymatic
synthesis of enantioisomer 6b is outlined in Scheme 2.
Racemic o-tyrosine was nitrated with NO2BF4 in CH3-
CN to give 6, and acylation of 6 under Schotten-
Baumann conditions gave N-acetyl-3,5-dinitro-o-ty-
rosine (15)19,20 as a racemic mixture. The method of
Whitesides21 was employed to obtain 6b in 41% yield
with optical purity of >99% ee. Progress of the reaction
of 15 with Aspergillus acylase I (Sigma) was monitored
by quantitative HPLC analysis of the amount of pro-
duced 6b (tR ) 4.32 min), using 13 as internal standard

AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1037
Ta ble 1. Percent Displacement of [3H]AMPA Binding to Rat
Brain Membranes by a 100 µM Concentration of
Dinitro-o-tyrosine and o-Tyrosine Analogues
Sch em e 3a
compd
config
substitutions
[3H]AMPA
6
6a
6b
7
8
15
16
17
DNQX
racemate
R
S
racemate
racemate
racemate
racemate
racemate
R ) OH
R ) OH
R ) OH
R ) OCH3
76 ( 3
58 ( 8
78 ( 5
66 ( 4
20 ( 11
14 ( 7
17 ( 7
26 ( 5
99 ( 0.4
a
Reagents: (a) [(CH3)3COCO]2O, NaOH, H2O, THF; (b) (1)
BrCH2CO2Et, K2CO3, acetone, (2) 2 N HCl.
(tR ) 2.22 min) [HPLC conditions: column, ODS 4.8-
mm i.d. × 15-mm L; eluent, 8% CH3CN in acetate buffer
(0.02 M, pH ) 4.0); flow rate, 2 mL/min; UV detector, λ
) 280 mm; sample injected, 10 µL]. The completion of
the conversion took approximately 30 h. The S-amino
acid 6b was separated from nonhydrolyzed N-acetyl
amino acid by extracting the latter with EtOAc. An
attempt to obtain 6a was also made by hydrolyzing (R)-
N-acetyl-3,5-dinitro-o-tyrosine (15a ). However, hydrol-
ysis in 2 N HCl for 70 min gave 6a in only 80% ee. Basic
hydrolysis (2.5 N NaOH) destroyed the product com-
pletely.
The synthesis of â-3,5-dinitro-2-(methoxyphenyl)-R-
alanine (7) is also outlined in Scheme 2. Treatment of
6 with di-tert-butyl dicarbonate under Schotten-Bou-
mann conditions gave (R,S)-N-(tert-butoxycarbonyl)-3,5-
dinitro-o-tyrosine (16).22,23 Methylation of 16 with CH3I
in the presence of K2CO3 in DMSO gave N-(tert-
butoxycarbonyl)-â-(3,5-dinitro-2-methoxyphenyl)-R-ala-
nine (17). However, when a stronger base (e.g., NaOH,
KOH) was used, methylation of the protected amino
group took place. The deprotection of the amino group
of compound 17 was carried out in CF3COOH, and
purification by ion-exchange column afforded 7.
Scheme 3 shows the synthesis of â-[(2-carboxymethox-
y)phenyl]-R-alanine (8). Reaction of 13 with di-tert-
butyl dicarbonate gave (R,S)-N-(tert-butoxycarbonyl)-o-
tyrosine (18).22,23 Treatment of 18 with ethyl bromo-
acetate and K2CO3 in acetone followed by reflux in 2 N
HCl gave the product 8.
F igu r e 1. Effects of DNQX, (R)- and (S)-dinitro-o-tyrosine
(6a ,b), and their racemate (6) on specific [3H]AMPA binding
(IC50 values of DNQX, 6, 6a , and 6b are 0.93 ( 0.42, 29 ( 6,
84 ( 26, and 13 ( 7 µM, respectively).
3,5-dinitro-o-tyrosine (15), (R,S)-N-(tert-butoxycarbo-
nyl)-3,5-dinitro-o-tyrosine (16), and N-(tert-butoxycar-
bonyl)-â-(3,5-dinitro-2-methoxyphenyl)-R-alanine (17)]
were examined for binding to AMPA receptors. The
reason for testing of the latter compounds was to
examine the necessity of the free amino group in the
binding to the AMPA receptors. Table 1 summarizes
the effect of a 100 µM concentration of different 3,5-
dinitro-o-tyrosine and o-tyrosine analogues on [3H]-
AMPA binding to brain homogenates. The results
clearly demonstrate that an amino acid function on the
side chain and two nitro groups on the ring are
important for optimum binding activity. Concentra-
tion-response experiments (Figure 1) yielded IC50
values for 6b,a of 13 ( 7 and 84 ( 26 µM, respectively.
Thus the S-isomer is 6.5-fold more potent than the
R-isomer. The reference compound DNQX had an IC50
value of 0.93 ( 0.42 µM. The racemic mixture of â-(3,5-
dinitro-2-methoxyphenyl)-R-alanine (7), which lacks
phenolate anion, was less potent than 6. It was
surprising that this compound still significantly inhib-
ited [3H]AMPA binding (66 ( 4% inhibition). At this
time we do not have an explanation for the activity
observed with 7. â-[2-(Carboxymethoxy)phenyl]-R-ala-
nine (8) did not show significant inhibition of specific
[3H]AMPA binding. A possibility for the low activity of
8 is that the two carboxy groups may be separated by
too large of a distance. Intermediates 15-17, all of
which have protected amino groups, did not inhibit [3H]-
AMPA binding at 10-4 M. Thus the free amino group
appears necessary in order for these compounds to bind
to AMPA receptors.
Deter m in a tion of Op tica l P u r ity. The optical
purity of 6a ,b was determined by diastereoisomer
formation with Marfey’s reagent (1-fluoro-2,4-dinitro-
phenyl-5-L-alanine amide) and analyzed by HPLC.24,25
Parallel reaction with the racemic 3,5-dinitro-o-tyrosine
allowed us to develop the HPLC method for separation
of diastereoisomers. The blank probe (containing only
Marfey’s reagent) was used to determine the retention
time for the hydrolyzed reagent (1-hydroxy-2,4-dinitro-
phenyl-5-L-alanine amide).
Biologica l Resu lts
Racemic 3,5-dinitro-o-tyrosine (6), â-(3,5-dinitro-2-
methoxyphenyl)-R-alanine (7), â-[2-(carboxymethoxy)-
phenyl]-R-alanine (8), and optical isomers 6a ,b were
examined for the inhibition of [3H]AMPA binding to rat
brain membranes. In addition to these analogues,
intermediates with protected amino groups [N-acetyl-
The two enantiomers 6a ,b and the racemic compound
6 were examined for functional activity in an assay in

1038 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Sun et al.
which AMPA agonists promote release of [3H]norepi-
nephrine from hippocampal slices and AMPA antago-
nists inhibit the AMPA-stimulated release. These
compounds proved to be antagonists, and the S-isomer
6b (IC50 ) 630 ( 140 µM) was more potent than the
racemate (6) (IC50 ) 730 ( 88 µM) while the R-isomer
6a was inactive up to 1 mM which is consistent with
the S-isomer having higher binding affinity than the
R-isomer (the reference compound DNQX had an IC50
value of 41 ( 6 µM). This relative order of binding
potency for the isomers follows the same trend as found
for the AMPA receptor agonists, AMPA and glutamate.
This could imply that the tyrosine antagonists bind in
a manner similar to agonists but do not produce any
intrinsic activity. In conclusion we are reporting that
the S-isomer 6b is an AMPA receptor antagonist and
represents a lead for a new structural class of AMPA
antagonists.26
The cooling bath was removed after completion of addition and
the mixture was allowed to warm to room temperature. The
mixture was then poured into ice-cold 1.2 N HCl (500 mL) and
extracted with Et2O (3 × 300 mL). The combined organic
extracts were washed with saturated NaHCO3 (300 mL) and
brine (300 mL) and dried over Na2SO4. After filtration, the
solution was concentrated under reduced pressure, and the
product was crystallized from EtOAc/Hex to give slightly
yellow crystals of 10: yield 3.39 g (51%); mp 109-110.5 °C;
1H NMR (CDCl3, TMS, 250 MHz) δ 7.88-7.84 (dd, 2H, J )
1.3, 8.1 Hz, ArH), 7.58-7.55 (dd, 1H, J ) 1.8, 7.75 Hz, ArH),
7.54-7.40 (m, 4H, ArH), 7.00-6.90 (m, 2H, ArH), 6.17-6.14
(d, 1H, J ) 7.0 Hz, NH), 5.13-5.07 (m, 1H, CH), 3.99-3.94
(dd, 1H, J vic ) 3.3 Hz, J gem ) 11.6 Hz, CH2), 3.84 (s, 3H, OCH3),
3.74-3.67 (dd, 1H, J vic ) 4.3 Hz, CH2), 2.32-2.27 (t, 1H, J )
6.8 Hz, OH); IR (KBr, cm-1) 3540 (OH stretching), 3270 (NH
stretching), 1693 (CdO stretching); MS (EI) m/z 336 (M+), 200
(M+ - Ph(OCH3)CO), 135 (base); [R]25 ) +94.2° (c 1.0,
D
acetone). Anal. (C16H17NO5S) C, H, N.
Meth od B: N-(Phenylsulfonyl)-L-serine (2.45 g, 10 mmol)
in freshly distilled THF (100 mL) was cooled to -78 °C in a
dry ice-acetone bath and stirred under nitrogen atmosphere.
n-BuLi (8 mL, 20 mmol) was added dropwise via a syringe,
and the mixture was stirred for an additional 30 min at -78
°C. The mixture was then treated with 2-methoxyphenylmag-
nesium bromide in THF. Stirring was continued at room
temperature for another 40 h. It was then poured into 1 N
HCl (150 mL) which was cooled in an ice-bath and extracted
with ether (3 × 100 mL). The combined extracts were washed
with saturated NaHCO3 (250 mL) and brine (250 mL), dried
over Na2SO4, filtered, and concentrated under reduced pres-
sure on a rotavapor. Crystallization of the residue from
EtOAc/Hex gave 10 as a white crystalline solid: yield 1.76 g
(53%); mp 109-111 °C.
Exp er im en ta l Section
Gen er a l. Melting points were determined on a Thomas-
Hoover capillary melting point apparatus and are uncorrected.
Infrared data were collected on an Analect RFX-40 FTIR
spectrophotometer (The Ohio State University, College of
Pharmacy) or on a Perkin-Elmer 2000 FT-IR spectrophotom-
eter (University of Tennessee, College of Pharmacy). The
NMR spectra were obtained either on an IBM AF-250 FTNMR
spectrometer (250 MHz) at The Ohio State University, College
of Pharmacy, or on an Bruker ARX 300 FT NMR spectrometer
(300 MHz) at the University of Tennessee, College of Phar-
macy. 1H NMR chemical shifts are reported in ppm relative
to CDCl3 (δ 7.24, or TMS), acetone-d6 (δ 2.04), and DMSO-d6
(δ 2.49). 13C chemical shifts are reported in ppm relative to
CDCl3 (δ 77.00, or TMS), acetone-d6 (δ 29.8), and DMSO-d6 (δ
39.5). Mass spectra were obtained at the College of Pharmacy
by use of a Kratos MS25RFA mass spectrophotometer or at
The Ohio State University Campus Chemical Instrumentation
Center by use of a VG 70-250S or Kratos MS-30 mass
spectrometer. Elemental analyses were performed by Gal-
braith Laboratories, Inc., Knoxville, TN, or by Atlantic Micro-
lab, Inc., Norcross, GA, and were within (0.4% of the
theoretical values for the elements indicated. The solvents
used in these experiments were purchased and were not
further purified unless otherwise stated. Tetrahydrofuran was
dried by refluxing with and distillation from sliced sodium and
distilled under Ar, using benzophenone as an indicator for
dryness. Ion-exchange resin Dowex 50 × 8 200, as well as
Dowex 1 × 8 200, was purified prior to use. Enzymes acylase
I (EC 3.5.1.14; 0.47 units/mg of solid), L-amino acid oxidase
(EC 1.4.3.2; 0.53 units/mg of solid) and catalase (EC 1.11.1.6;
1600 units/mg of solid) were purchased from Sigma Chemical
Co. Derivatizing reagent N-R-(2,4-dinitro-5-fluorophenyl)-L-
alanine amide was purchased from TCI America, Inc. The pH
values were determined with Fisher Scientific Accumet pH
meter 15. Optical rotations were recorded on an Autopol III
at λ ) 589 nm at room temperature. High-performance liquid
chromatography was performed on a Waters HPLC system
equipped with a model Waters 510 Waters 501 pump, a U6K
injector, and a model Waters 486 detector. All solvents for
HPLC operation were HPLC grade and were filtered prior to
use. The chromatograph was operated isocratically.
(R)-2-[(P h en ylsu lfon yl)a m in o]-3-(o-m et h oxyp h en yl)-
p r op a n -1-ol (11). To a solution of 10 (5 g, 15 mmol) in CF3-
COOH (34 g, 298 mmol) was added triethylsilane (12.4 g, 66
mmol) via a dropping funnel while stirring and cooling in an
ice bath. The mixture was then stirred and heated on an oil
bath at 50-55 °C for 2 h. After the mixture to cooled room
temperature, a mixture of H2O (20 mL) and MeOH (20 mL)
was added and stirring was continued overnight. The mixture
was neutralized with NaHCO3 and extracted with EtOAc (3
× 100 mL). Combined organic extracts were washed with
brine, dried over Na2SO4, and concentrated under reduced
pressure to give a yellow oil. This residue was chromato-
graphed on a silica gel column (mobile phase Hex/EtOAc, 8/2
and then 1/1). The product 11 was crystallized from EtOAc/
Hex in the form of white crystals: yield 2.84 g (59%); mp 77-
1
79 °C; H NMR (CDCl3, 250 MHz) δ 7.63-7.59 (dd, 2H, J )
1.3, 7.5 Hz, ArH), 7.49-7.44 (m, 1H, ArH), 7.36-7.30 (t, 2H,
J ) 7.9, 8.2 Hz, ArH), 7.19-7.12 (td, 1H, J ) 1.7, 7.7 Hz, ArH),
6.95-6.91 (dd, 1H, J ) 1.7, 7.4 Hz, ArH), 6.82-6.72 (q, 2H, J
) 7.4, 8.7 Hz, ArH), 5.36-5.33 (d, 1H, J ) 7 Hz, NH), 3.75 (s,
3H, OCH3), 3.55 (s, 2H, CH2), 3.49-3.39 (m, 1H, CH), 2.80-
2.72 (t, 2H, J ) 5.8, 7.9, 6.6 Hz, CH2OH), 2.67 (br s, 1H, OH);
IR (KBr, cm-1) 3509.8 (OH stretching), 3299.6 (NH stretching);
MS(EI) m/z 321 (M+), 290 (M+ - OCH3), 200 (M+ - C6H5-
(OCH3)CH2), 77 (base) (C6H5); [R]25D ) +66.5° (c 1.0, acetone).
Anal. (C16H19NO4S) C, H, N.
(R)-3,5-Din itr o-o-tyr osin e (6a ). Meth od A: To a solution
of J ones reagent (0.118 g of CrO3/mL in 1.5 M H2SO4) (12 mL)
was added a solution of 11 (1.0 g, 3.11 mmol) in acetone (30
mL) via a dropping funnel over a period of 30 min at 5-10 °C.
The mixture continued to stir at room temperature for 24 h,
was poured into Et2O (70 mL), and was extracted with brine
(3 × 100 mL). The Et2O layer was then washed with 1 N
NaOH, and the combined basic extract was acidified with 10
M H2SO4 and extracted with Et2O (3 × 100 mL). The Et2O
extracts were combined, washed with H2O (2 × 100 mL) and
brine (150 mL), dried over Na2SO4, and concentrated under
reduced pressure on a rotavapor to give a brown oil. The
product was crystallized from EtOAc/Hex in the form of
slightly yellow crystals which were identified as (R)-2-[(phe-
(S)-2-Meth oxy-r-[(ph en ylsu lfon yl)am in o]-â-h ydr oxypr o-
p iop h en on e (10). Meth od A: 2-Bromoanisole (9) (22.4 g, 120
mmol) was dissolved in dry THF (100 mL) with stirring under
argon atmosphere, and the flask was then cooled to -78 °C
with a dry ice-acetone bath. n-BuLi (53 mL, 133 mmol) was
added via a syringe over a period of 5 min, and stirring was
continued for an additional 1 h at -78 °C. The solution of
N-(phenylsulfonyl)-L-serine (4.9 g, 20 mmol) in dry THF (400
mL) was added via a dropping funnel over 50 min at -78 °C.

AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1039
nylsulfonyl)amino]-3-(o-methoxyphenyl)propionic acid: yield
0.62 g (59%); mp 146-147 °C; H NMR (CDCl3, 250 MHz) δ
(R, S)-N-Acetyl-3,5-d in itr o-o-tyr osin e (15). A racemic
mixture of 3,5-dinitro-o-tyrosine (6) (3.3 g, 9.8 mmol), obtained
from the nitration of 13 by NO2BF4, was dissolved in H2O (20
mL) and 2 N NaOH (5 mL) and cooled in an ice-acetone bath
at 0 °C. Redistilled acetic anhydride (2 mL, 22 mmol) and 2
N NaOH (24 mL) were added in small equal portions. After
completion of addition, stirring was continued at room tem-
perature for additional 40 min. The mixture was acidified with
10 N H2SO4 and extracted with EtOAc (3 × 70 mL). Organic
extracts were combined, washed with brine (100 mL), dried
over Na2SO4, filtered, and concentrated under reduced pres-
sure on a rotavapor. Crystallization of the residual material
from acetone gave 15 as a yellow powder: yield 3.0 g (98%);
mp 174-175 °C; 1H NMR (acetone-d6, 300 MHz) δ 8.85-8.84
(d, 1H, J ) 3.0 Hz, ArH), 8.46-8.45 (d, 1H, ArH), 7.51-7.49
(br d, 1H, J ) 8.0 Hz, NH), 4.93-4.85 (m, 1H, CH), 3.57-3.51
(dd, 1H, J vic ) 5.0 Hz, J gem ) 14.0 Hz, CH2), 3.17-3.09 (dd,
1H, J vic ) 9.0 Hz, CH2), 1.84 (s, 3H, CH3); MS(EI) m/z 313
(M+), 268 (M+ - COOH), 74 (base). Anal. (C11H11N3O7) C, H,
N.
1
7.59-7.56 (dd, 2H, J ) 1.4, 8.1 Hz, ArH), 7.50-7.44 (m, 1H,
ArH), 7.44-7.30 (m, 2H, ArH), 7.22-7.15 (td, 1H, J ) 1.6, 8.0
Hz, ArH), 6.99-6.95 (dd, 1H, J ) 1.7, 7.5 Hz, ArH), 6.83-
6.71 (m, 2H, ArH), 5.39-5.35 (d, 1H, J ) 8.4 Hz, NH), 4.2-
4.11 (m, 1H, CH), 3.69 (s, 3H, OCH3), 3.02-2.99 (d, 2H, J )
6.8 Hz, CH2); IR (KBr, cm-1) 3334 (OH stretching), 3004 (br
COOH stretching), 1727.9 (CdO stretching); MS(EI) m/z 335
(M+), 290 (M+ - COOH), 121 (base) (C6H5(OCH3)CH2); [R]25
) +38.9° (c 1.0, acetone). Anal. (C16H17NO5S) C, H, N.
D
A mixture of (R)-2-[(phenylsulfonyl)amino]-3-(o-methox-
yphenyl)propionic acid (1.7 g, 4.7 mmol), phenol (1.8 g, 19
mmol), and freshly distilled 48% HBr (20.9 g, 258 mmol) was
refluxed for 2.5 h. Progress of the reaction was monitored by
assaying aliquots (0.5 mL) on HPLC (ODS column, mobile
phase of 15% MeOH in H2O, UV detector λ ) 254 nm) in 30-
min intervals. The reaction mixture was allowed to cool to
room temperature, diluted with H2O (50 mL), and extracted
with EtOAc (50 mL). The water layer was purified over cation-
exchange column Dowex 50 × 8 (H+). The product was eluted
from the column with 0.3 M NH4OH. Solvent was removed
under reduced pressure to give (R)-o-tyrosine as a white
powder: yield 0.53 g (62%); mp 248-250 °C (lit. mp 249-250
°C11); 1H NMR (D2O, DSS, 300 MHz) δ 7.28-7.20 (m, 2H, ArH),
6.97-6.92 (t, 2H, ArH), 4.07-4.03 (m, 1H, CH), 3.38-3.32 (dd,
1H, J vic ) 5.0 Hz, J gem ) 14.0 Hz, CH2), 3.08-3.00 (q, 1H, CH2);
[R]25 ) +17.46° (c 1.0, 1 M HCl) [lit. [R]25 ) +25.0° (c 0.84,
(S)-3,5-Din itr o-o-tyr osin e (6b). Racemic N-acetyl-3,5-
dinitro-o-tyrosine (15) (3.0 g, 9.6 mmol) was suspended in
distilled H2O (50 mL). 2 N KOH (10 mL) was added to make
the final solution pH 7.5-8.0, and the solution was diluted
with distilled H2O to 100 mL total volume. Aspergillus acylase
I (Sigma; 0.47 units/mg, 40 mg) and CoCl2 (15 mg) were added
to the solution. The mixture was stirred in a water bath at
40 °C. Progress of the reaction was monitored by assaying
aliquots (0.5 or 0.25 mL) for free amino acid on HPLC.
Resolution proceeded for 24 h, but reaction was stopped after
27 h. The pH of the reaction mixture was adjusted to ∼5 with
1 N HCl. Norite (100 mg) was added, and the mixture was
stirred at 40 °C for 30 min, filtered over Celite, cooled to room
temperature, and acidified to pH ∼1.5-2.0 with 1 N HCl. The
filtrate was then extracted with EtOAc (3 × 80 mL) to remove
unhydrolyzed starting material ((R)-N-acetyl-3,5-dinitro-o-
tyrosine). The aqueous layer was purified by cation-exchange
column Dowex 50 × 8 (H+). After the sample was loaded, the
column was washed with H2O (2 L) and EtOH (500 mL), and
the product was eluted with 0.3 M NH4OH. The aqueous layer
was concentrated under reduced pressure to 5 mL, pH was
adjusted to ∼3.5-4.2, and the product 6b crystallized in the
form of yellow crystals: yield 0.54 g (41%); mp 235 °C
D
D
1 M HCl)11]. The nitration reaction of the obtained (R)-o-
tyrosine with NO2BF4 in CH3CN gave 6a in 50% ee (detailed
procedure is the same as and described in method B).
Meth od B: A racemic mixture of 13 (2.72 g, 15 mmol) was
suspended in tris-maleate buffer (250 mL, 0.05 M, pH 7.8).
To this mixture was added KCl (1.86 g, 25 mmol) followed by
40 mg of L-amino acid oxidase (Sigma; type I, activity 0.53
units/mg) and 10 mg of catalase. The reaction mixture was
vigorously stirred in a water bath at 35-40 °C for 48 h. The
mixture was then acidified to pH 6-7 with 1 N HCl, heated
with Norite on a steam bath, and filtered over Celite. The
filtrate was alkalized to pH 10-11 and purified over anion-
exchange column Dowex 1 × 8 (OH-), eluting the amino acid
from the column with 1 M CH3COOH followed by purification
over cation-exchange column Dowex 50 × 8 (H+). The column
was washed with H2O and EtOH, and the amino acid was
eluted with 0.3 M NH4OH. The solvent was removed under
reduced pressure, and the product was crystallized from water
to give (R)-o-tyrosine (13a ) as white crystals: yield 1.2 g (88%);
1
(decomposition starts), 243-245 °C (decomposed rapidly); H
NMR, FT-IR, and elemental analysis data same as for 6a ;
[R]25 ) +4.44° (c 1.92, 0.05 M HCl).
D
(R,S)-N-(ter t-Bu t oxyca r b on yl)-3,5-d in it r o-o-t yr osin e
(16). To a solution of 6 (1.4 g, 4.2 mmol) in H2O (100 mL)
were added 2 N NaOH (30 mL) and THF (150 mL). Di-tert-
butyl dicarbonate (1.1 g, 4.6 mmol) was added to the vigorously
stirred solution, and stirring was continued at room temper-
ature for 2 h. Silica gel TLC of the reaction mixture [Rf )
0.05 (starting material), Rf ) 0.57 (product); eluting solvent,
1% MeOH in CH2Cl2 + 2 drops of AcOH] showed that the
reaction was incomplete. Additional di-tert-butyl dicarbonate
(2.0 g, 9 mmol) was added, and the solution was stirred at
room temperature overnight. The mixture was then poured
into H2O (200 mL) and extracted with n-hexanes to remove
excess of di-tert-butyl dicarbonate. Water layer was cooled in
an ice-acetone bath (∼0-5 °C), acidified with 4 N HCl to pH
∼2, and extracted with EtOAc (3 × 100 mL). Organic extracts
were combined, dried over Na2SO4, and filtered. Removal of
solvent under reduced pressure on a rotavapor gave a yellow
powder. Recrystallization of the powder from EtOAc/Hex gave
beige crystals of 16: yield 1.5 g (96%); mp 187-189 °C; 1H
NMR (acetone-d6, 300 MHz) δ 8.88-8.87 (d, 1H, J ) 2.6 Hz,
ArH), 8.49-8.48 (d, 1H, ArH), 6.36-6.33 (br d, J ) 8.5 Hz,
1H, NH), 4.67-4.59 (m, 1H, CH), 3.62-3.56 (dd, J ) 4.0, 14.0
Hz, CH2), 3.16-3.08 (m, 1H, CH2), 1.27 (s, 9H, 3 × CH3). Anal.
(C14H17N3O9) C, H, N.
1
mp 249-251 °C; H NMR was the same as for (R)-o-tyrosine
obtained by method A; [R]25 ) +31.5° (c 0.84, 1 M HCl).
D
A suspension of 13a (0.41 g, 2.3 mmol) in CH3CN (10 mL)
was cooled in an ice-acetone bath at ∼0-5 °C, and NO2BF4
(0.8 g, 5.8 mmol) was added in small portions over a period of
20 min while stirring. Progress of the reaction was monitored
by assaying aliquots with HPLC (ODS column, mobile phase
of 8% CH3CN in 0.1% CF3COOH in H2O, flow rate 2 mL/min,
UV detector λ ) 254 nm). The reaction was completed after 8
h. The mixture was diluted with water (120 mL), stirred
overnight, and filtered over Celite. The filtrate was purified
by cation-exchange chromatography on Dowex 8 × 50 (H+).
After the column was loaded and washed it with H2O (2 L)
and EtOH (500 mL), the amino acid was eluted with 0.3 M
NH4OH. The eluent was concentrated under reduced pressure
to 5 mL, pH was adjusted to ∼3.5 with 1 N HCl, and 6a was
crystallized as yellow crystals which had optical purity of
>99% ee: yield 0.19 g (31%); mp 235-240 °C; 1H NMR
(DMSO-d6, 300 MHz) δ 8.54-8.53 (d, 1H, J ) 3.1 Hz, ArH),
7.89-7.88 (d, 1H, ArH), 4.14-4.11 (m, 1H, CH), 3.12-3.06 (dd,
1H, J vic ) 3.8 Hz, J gem ) 14.0 Hz, CH2), 2.89-2.82 (dd, 1H,
J vic ) 7.4 Hz, CH2); 13C NMR (DMSO-d6, 75 MHz) δ 170.37
(COOH), 162.33 (ArC), 136.02 (ArC), 134.61 (ArC), 127.90
(ArC), 127.76 (ArC), 123.29 (ArC), 54.61 (CHNH2), 35.78 (CH2);
IR (KBr, cm-1) 3500-2500 (COOH), 3278 (OH), 3083 (NH),
N-(ter t-Bu toxyca r bon yl)-â-(3,5-d in itr o-2-m eth oxyp h e-
n yl)-r-a la n in e (17). To a suspension of K2CO3 (0.4 g, 2.7
mmol) in DMSO (5 mL) was added 16 (0.4 g, 1.1 mmol)
followed immediately by the addition of methyl iodide (0.42 g,
1637 (CdO); MS (FAB+) m/z 272 (M+ + H); [R]25 ) -5.66° (c
D
2.0, 0.05 M HCl). Anal. (C9H9N3O7) C, H, N.

1040 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Sun et al.
2.2 mmol). The mixture was vigorously stirred overnight at
room temperature. Completion of the reaction was confirmed
by silica gel TLC [Rf ) 0.5 (starting material), Rf ) 0.64
(product); eluting solvent, 1% MeOH in CH2Cl2 + 2 drops of
AcOH]. The mixture was poured into water (50 mL), acidified
with 4 N HCl while cooling in an ice-acetone bath (0-5 °C),
and extracted with EtOAc (3 × 50 mL). Organic extracts were
combined, dried over Na2SO4, and concentrated to dryness
under reduced pressure on a rotavapor. Recrystallization of
the material from acetone/hexane gave 17 as yellow crystals:
yield 0.30 g (72%); mp 153-154 °C; 1H NMR (acetone-d6, 300
MHz) δ 8.88-8.87 (d, J ) 2.8 Hz, 1H, ArH), 8.47-8.46 (d, J )
2.8 Hz, 1H, ArH), 6.44-6.41 (br d, J ) 8.8 Hz, 1H, NH), 4.66-
4.58 (m, 1H, CH), 3.72 (s, 3H, OCH3), 3.55-3.49 (dd, J ) 4.8
Hz, 1H, CH2), 3.16-3.80 (m, 1H, CH2), 1.27 (s, 9H, 3 × CH3).
Anal. (C15H19N3O9) C, H, N.
NMR (DMSO-d6, 300 MHz) δ 8.37 (br s, 3H, NH3+), 7.26-7.17
(m, 2H, ArH), 6.93-6.85 (m, 2H, ArH), 4.72 (s, 2H, OCH2),
4.19-4.15 (t, 1H, J ) 6.5 Hz, CH), 3.24-3.17 (dd, J vic ) 6.3
Hz, J gem ) 14.0 Hz, 1H, CH2), 3.09-3.05 (m, 1H, CH2); 13C
(DMSO-d6, 75 MHz) δ 170.8 (COOH), 170.6 (COOH), 156.2
(ArC), 131.6 (ArC), 129.0 (ArC), 123.5 (ArC), 121.2 (ArC), 111.9
(ArC), 64.9 (OCH2), 52.3 (CHNH2), 31.6 (CH2); MS (EI) m/z
221 (M+ - H2O), 107 (base). Anal. (C11H13NO5‚0.9HCl) C, H,
N.
Der iva tiza tion a n d Deter m in a tion of En a n tiom er ic
Excess of (S)- a n d (R)-3,5-Din itr o-o-tyr osin e. (S)-3,5-
Dinitro-o-tyrosine (6b) (100 µL, 5 µmol) was dissolved in 100
µL of 0.5 M NaHCO3. To the solution was added 200 µL of a
1% solution of Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-
L-alanine amide) in acetone. The mixture was incubated at
40 °C for 90 min and allowed to cool to room temperature prior
to the addition of 50 µL of 2 N HCl. The sample was allowed
to degas and was diluted with EtOH (1.2 mL); 10-µL aliquots
of the sample were used for HPLC injection. The R-enanti-
omer 6a and racemic mixture 6 were derivatized in the same
fashion, and 6 was used as a standard. The diastereoisomers
were separated by reverse-phase HPLC on an ODS column.
The enantiomeric excess of 6a ,b was calculated based on the
peak integration.
â-(3,5-Din itr o-2-m eth oxyp h en yl)-r-a la n in e (7). Com-
pound 17 (0.6 g, 1.6 mmol) was dissolved in CF3COOH and
stirred at room temperature for 45 min. At this point TLC of
the reaction mixture showed there was no starting material
left [Rf ) 0.79 (starting material), Rf ) 0.40 (product) on silica
gel TLC; eluting solvent, 5% MeOH in CH2Cl2 + 2 drops of
AcOH]. The mixture was poured into H2O (100 mL) and
extracted with EtOAc (3 × 50 mL). Organic extracts were
combined, dried over Na2SO4, filtered, and concentrated in
vacuo to ∼5 mL. Triethylamine was added to adjust the pH
to ∼5, and addition of water afforded 7 purified by ion-
exchange column to give orange crystals:27 yield 0.248 g (56%);
mp 227-228 °C dec; 1H NMR (DMSO-d6, 300 MHz) δ 8.59 (br
s, 3H, NH3+), 8.54-8.53 (d, J ) 3.0 Hz, 1H, ArH), 7.87-7.86
(d, J ) 3.0 Hz, 1H, ArH), 4.27 (s, 1H, CH), 3.66 (s, 3H, OCH3),
3.11-3.05 (dd, J vic ) 4.2 Hz, J gem ) 14.0 Hz, 1H, CH2), 2.93-
2.86 (m, 1H, CH2); 13C NMR (DMSO-d6, 75 MHz) δ 169.76
(COOH), 169.72 (ArC), 136.58 (ArC), 131.01 (ArC), 128.78
(ArC), 127.94 (ArC), 124.06 (ArC), 52.91 (OCH3), 52.19 (CHNH2),
33.47 (CH2); MS(EI) m/z 285 (M+), 88 (base). Anal.
(C10H11N3O7) C, H, N.
(R,S)-N-(ter t-Bu toxyca r bon yl)-o-tyr osin e (18). o-Ty-
rosine (13) (1.0 g, 5.5 mmol) was dissolved in a mixture of H2O
(30 mL), 2 N NaOH (10 mL), and THF (30 mL). Di-tert-butyl
dicarbonate (1.32 g, 6.1 mmol) was added to the vigorously
stirred mixture, and stirring was continued for 5 h at room
temperature. The mixture was poured into water (100 mL)
and extracted with n-hexanes to remove unreacted di-tert-butyl
dicarbonate. Water layer was then acidified with 4 N HCl
while cooling in an ice-acetone bath (0-5 °C) and extracted
with EtOAc (3 × 50 mL). Organic extracts were combined,
dried over Na2SO4, filtered, and concentrated on a rotavapor
to give a white solid. Recrystallization of the solid from
acetone/hexane gave 18 as white crystals: yield 1.41 g (91%);
1H NMR (acetone-d6, 300 MHz) δ 7.18-7.15 (dd, J ) 1.6, J )
7.5 Hz, 1H, ArH), 7.08-7.03 (t, J ) 7.0 Hz, 1H, ArH), 6.92-
6.70 (m, 2H, ArH), 6.12-6.09 (br d, J ) 7.0 Hz, 1H, NH), 4.48-
4.41 (m, 1H, CH), 3.24-3.17 (dd, J ) 4.7, 13.5 Hz, 1H, CH2),
2.98-2.90 (m, 1H, CH2), 1.33 (s, 9H, 3 × CH3).
Biologica l Meth od s. 1. Bin d in g Stu d ies. The interac-
tion of compounds with AMPA receptors was assessed by
inhibition of specific [3H]AMPA binding in washed membrane
preparations of rat brain following the procedure originally
published by Honore et al.28 Washed membranes were incu-
bated with [3H]AMPA (1.4 nM final concentration) and solu-
tions of test compounds at appropriate concentrations in 50
mM Tris-HCl buffer containing 2.5 mM CaCl2 and 100 mM
KSCN, pH 7.2. The mixture was incubated on ice for 60 min
with periodic shaking, after which bound and free ligands were
separated by rapid filtration through Whatman GF/C glass
fiber filters. The resulting filter disks, containing membrane-
bound radioligand, were dissolved in Scintiverse-E cocktail for
scintillation counting. Nonspecific binding was determined in
the presence of 1 mM glutamate. For determination of
potency, a range of test compound concentrations (0.01-100
µM) was evaluated. IC50 values were calculated using non-
linear regression analysis.
2. Neu r otr a n sm itter Relea se Assa y. This assay is based
on the fact that AMPA receptor activation induces norepi-
nephrine release from hippocampal nerve endings.29 The
procedure used was adapted from the method of Desai et al.30
Briefly, mouse hippocampi were dissected out and chopped into
0.3-mm × 0.3-mm slices. Slices were incubated with 0.2 µM
[3H]norepinephrine in Krebs buffer for 30 min and then
transferred to superfusion chambers and washed for 60 min
with warmed, oxygenated Krebs buffer at 0.3 mL/min. Fol-
lowing this, 10 5-min fractions were collected. After the third
collection (i.e., fraction 3), buffer containing 50 µM cyclothiaz-
ide was introduced. The presence of cyclothiazide diminishes
AMPA receptor desensitization, thus allowing for a more
readily quantifiable AMPA response. After the collection of
fraction 4, buffer containing drug, cyclothiazide, and 100 µM
AMPA was introduced for 5 min followed by normal buffer
until the end. For initial screening purposes, tissue slices were
exposed to 1 mM test compound alone (test for possible agonist
activity) run in parallel with tissue slices exposed to test
compound plus 100 µM AMPA (test for antagonist). Each
condition was run in triplicate. At least two experiments were
conducted to generate an n ) 6 for each treatment. For dose-
response data, a minimum of four concentrations of each test
compound were run against 100 µM AMPA (for antagonists)
or alone (for agonists). Determination of IC50 values was
accomplished utilizing median effect plot analysis.
â-[2-(Ca r boxym eth oxy)p h en yl]-r-a la n in e (8). Finely
powdered K2CO3 (0.98 g, 7.1 mmol) and compound 18 (0.8 g,
2.8 mmol) were stirred in acetone at 50 °C in an oil bath for
1.5 h. Ethyl bromoacetate (0.52 g, 3.1 mmol) was added to
the mixture, and stirred at 50 °C for 3 h. Silica gel TLC of
the reaction mixture at this point showed the reaction was
incomplete [Rf ) 0.17 (compound 18); eluting solvent, hexanes/
EtOH (8/2) + 2 drops of AcOH]. Additional ethyl bromoacetate
(0.94 g, 5.6 mmol) was added and the mixture stirred at 50 °C
for another 4 h when all starting material disappeared. The
mixture was allowed to cool to room temperature, poured into
50 mL of water, acidified with 4 N HCl (pH ∼2), and extracted
with EtOAc (3 × 50 mL). Organic extracts were combined and
concentrated under reduced pressure on a rotavapor to give a
slightly yellow oil. This residue was suspended in 2 N HCl
(50 mL), refluxed for 24 h, and cooled to room temperature.
Removal of solvent under reduced pressure gave a white solid.
Recrystallization of the solid from acetone/water/Et2O afforded
Ack n ow led gm en t. We thank the National Institute
of Drug Abuse in the form of Grant DA06776 for partial
support of this research.
1
8 as white crystals: yield 0.203 g (26%); mp 203-204 °C; H

AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1041
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Amino Acid Oxidase. J . Org. Chem. 1993, 58, 957-958.
(18) Frisell, W. R.; Lowe, H. J .; Hellerman, L. Flavoenzyme Catalysis.
Substrate-Competitive Inhibition of D-Amino Acid Oxidase. J .
Biol. Chem. 1956, 223 (1), 75-83.
(19) DuVigneaud, V.; Meyer, C. E. The Racemization of Amino Acids
in Aqueous Solution by Acetic Anhydride. J . Biol. Chem. 1932,
98, 295-308.
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