Structure-activity studies for α-amino-3-hydroxy-5-methyl-4- isoxazolepropanoic acid receptors: Acidic hydroxyphenylalanines

Article abstract of DOI:10.1021/jm950028z

Antagonists of α-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptors may have therapeutic potential as psychotropic agents. A series of mononitro- and dinitro-2- and 3-hydroxyphenylalanines was prepared, and their activity compared with wi

Full text of DOI:10.1021/jm950028z

3182
J . Med. Chem. 1997, 40, 3182-3191
Str u ctu r e-Activity Stu d ies for r-Am in o-3-h yd r oxy-5-m eth yl-
4-isoxa zolep r op a n oic Acid Recep tor s: Acid ic Hyd r oxyp h en yla la n in es
Ronald A. Hill,†,§ Lane J . Wallace,‡ Duane D. Miller,*,† David M. Weinstein,‡ Gamal Shams,‡ Henry Tai,‡
Richard T. Layer,‡ David Willins,‡ Norman J . Uretsky,‡ and Satyavijayan Narasimhan Danthi§
College of Pharmacy, The Ohio State University, Columbus, Ohio 43210-1291
Received J anuary 17, 1995X
Antagonists of R-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptors may
have therapeutic potential as psychotropic agents. A series of mononitro- and dinitro-2- and
3-hydroxyphenylalanines was prepared, and their activity compared with willardiine, 5-ni-
trowillardiine, AMPA, and 2,4,5-trihydroxyphenylalanine (6-hydroxydopa) as inhibitors of
specific [3H]AMPA and [3H]kainate binding in rat brain homogenates. The most active
compounds were highly acidic (pKa 3-4), namely, 2-hydroxy-3,5-dinitro-DL-phenylalanine (13;
[3H]AMPA IC50 ≈ 25 µM) and 3-hydroxy-2,4-dinitro-DL-phenylalanine (19; [3H]AMPA IC50 ≈ 5
µM). Two other dinitro-3-hydroxyphenylalanines, and 3,5-dinitro-DL-tyrosine, were considerably
less active. Various mononitrohydroxyphenylalanines, which are less acidic, were also less
active or inactive, and 2- and 3-hydroxyphenylalanine (o- and m-tyrosine) were inactive.
Compounds 13 and 19, DL-willardiine (pKa 9.3, [3H]AMPA IC50 ) 2 µM), and 5-nitro-DL-
willardiine (pKa 6.4, [3H]AMPA IC50 ) 0.2 µM) displayed AMPA . kainate selectivity in binding
studies. Compound 19 was an AMPA-like agonist, but 13 was an antagonist in an AMPA-
evoked norepinephrine release assay in rat hippocampal nerve endings. Also, compound 13
injected into the rat ventral pallidum antagonized the locomotor activity elicited by systemic
amphetamine.
In tr od u ction
Excitatory amino acid neurotransmission is ubiqui-
tous in the mammalian central nervous system (CNS).1
Glutamate and other excitatory amino acid neurotrans-
mitters evoke fast excitatory postsynaptic potentials
through activation of ionotropic receptors, one family
of which includes those at which depolarizations are
elicited by exogenously applied R-amino-3-hydroxy-5-
methyl-4-isoxazolepropanoic acid (AMPA, 1; Figure 1)
or kainate (2).2 Receptors of the AMPA/kainate family
are clearly quite diverse and are composed of several
subunit proteins;3 diversity of function is also evident
by marked differences in responses to various agonists
in various physiological test systems. The quest to
understand the workings of the brain, as well as possible
therapeutic applications, makes the discovery of com-
pounds that can selectively interact with receptor
subtypes an important goal.
Our initial objective was to add to the understanding
of structural requirements for AMPA receptor agonists
in order to discover new lead compounds that might be
converted to antagonists through structural modifica-
tion.4 The most potent such agonists identified to date
have five-membered heterocylic rings in place of the
γ-carboxylate group of glutamate, as in AMPA (1) and
quisqualic acid (3) (Figure 1). The 2,4-imidazolidine-
dione (hydantoin) 4 was also recently shown to be a
potent, AMPA-like agonist in the mammalian hippo-
F igu r e 1. Selected glutamate analogs that are AMPA and
kainate receptor agonists or partial agonists.
campus.5 Substitution with various alkyl and aryl sub-
stituents at the 5-position of 1 has given agonists,6 while
elaboration to the amino acids 5a -d and 6 (Figure 2)
provided modestly potent AMPA receptor antagonists.7
In this work we addressed six-membered ring sys-
tems. Willardiine (7; Figure 1) is a â-(1-uracil)-
substituted alanine that is approximately equipotent
* To whom correspondence should be addressed. Current address:
College of Pharmacy, The University of Tennessee, Memphis, TN
38163.
† Division of Medicinal Chemistry and Pharmacognosy.
‡ Division of Pharmacology.
§ Current address: School of Pharmacy, Northeast Louisiana Uni-
versity, Monroe, LA 71209-0470.
X Abstract published in Advance ACS Abstracts, September 1, 1997.
S0022-2623(95)00028-8 CCC: $14.00 © 1997 American Chemical Society

Acidic Hydroxyphenylalanines
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3183
Sch em e 1a
F igu r e 2. Amino acid glutamate antagonists.
with glutamate in competing for [3H]AMPA binding and
in physiological studies.8 Recently, a number of 5-sub-
stituted L-willardiine analogs were shown to be potent
AMPA agonists.9-11 It was also reported that 2,4,5-
trihydroxyphenylalanine (6-hydroxydopa, 8) potently
depolarized frog spinal neurons12 and more recently that
8 inhibited [3H]AMPA binding;13 it is now clear, how-
ever, that the apparent activity of 8 is actually due to
the rapid, spontaneous oxidation to the quinone 9 in
aqueous solution.14 We postulated that a phenolate
anion with enhanced acidity corresponding to the o- or
m-hydroxyl groups in 8 might serve as a bioisostere for
the γ-carboxylate group of glutamate; such an isostere
would potentially provide a more flexible template on
which to build AMPA antagonists. As a first approach
to testing this hypothesis, nitrated 2- and 3-hydroxy-
phenylalanines were synthesized.
a
(a) 23% HNO3, 2 h/0 °C; (b) cation exchange chromatography;
(c) BOC-ON, Et3N, H2O/dioxane; (d) 30% CF3CO2H, CH2Cl2; (e)
Et3N.
Sch em e 2a
Resu lts
Syn th esis of 2-Hyd r oxyp h en yla la n in e An a logs.
Syntheses of 10 from the corresponding benzyl halides
have previously been reported,15,16 but we wished to
obtain both the 3- and 5-nitro cogeners (11 and 10,
respectively) for biological evaluation. The Dall’Asta
and Farrario17 synthesis of 3-nitrotyrosine was adapted
for the nitration of commercially available 2-hydroxy-
DL-phenylalanine (12, Scheme 1). The nitrated product
mixture, which contained a small amount of the dinitro
product 13, was treated with BOC-ON; the tert-butyl-
oxycarbonyl-protected derivatives 14 and 15 were iso-
lated by preparative chromatography on silica gel,
crystallized from ethyl acetate/hexane, and deprotected
with 30% trifluoroacetic acid in methylene chloride to
give 10 and 11, respectively. The small amount of BOC-
protected dinitro derivative could not be purified. Un-
der one set of conditions intended to increase the
amount of 13 (Scheme 2), no significant quantities of
any of the three amino acids were detected by HPLC,
and compound 16 was isolated in 32% yield. Initially,
it was unclear whether 16 was the R-hydroxy or â-hy-
a
(a) 60% aq HNO3, 2 h/0 °C, then 5 h/25 °C; (b) adsorb on
MgSO4 and dry, incubate over fuming HNO3; (c) NO2BF4/CH3CN,
2 h/0 °C; (d) cation exchange chromatography.
achieved with nitronium tetrafluoroborate in acetoni-
trile; the product was isolated as the hydrated am-
monium phenolate salt, and this form was found to be
stable for at least 1 year. Two separately prepared lots
of material assumed the same hydration state (‚5/2H2O).
Syn th esis of 3-Hyd r oxyp h en yla la n in e An a logs
a n d 3,5-Din itr o-DL-tyr osin e. Nitration of DL-3-hy-
droxyphenylalanine (17; Scheme 3) with nitronium
tetrafluoroborate gave a mixture of three products (18-
20); this observation is consistent with the work of
J ackson,18 who nitrated with nitric acid/sulfuric acid
and separated the three isomers through extensive
fractional crystallization of barium salts. J ackson later
confirmed the structures of two of the isomers (4,6- and
1
droxy acid, because the H- and 13C-NMR spectra were
consistent with either isomer. A peak corresponding to
the loss of HO2CCHO was observed in the high-
resolution EI mass spectrum, however, and the carbon-
carbon connectivity was ultimately verified using a 13C
INADEQUATE experiment.
The dinitro compound 13 was produced in 44% yield
and free of mononitrated products by adsorbing 12 on
dry magnesium sulfate and incubating in a chamber
containing fuming nitric acid (Scheme 2). This proce-
dure was costly and difficult to scale, and a more
convenient, higher-yielding preparation of 13 was

3184 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Hill et al.
Sch em e 3a
Sch em e 5a
a
(a-f) as in Scheme 4; (g) recrystallization of 20.
thesized (unambiguously) by further nitrating the two
mononitro compounds 21 and 22 to give mixtures of 18
with each of the other two dinitrated products (as in
Schemes 4 and 5). The synthesis of 21 by the Scheme
5 route was previously reported in the literature.20
Compound 22 was synthesized by the same route
(Scheme 4), through the known intermediate 23,21 and
was nitrated to the expected mixture of 18 and 19.
These compounds were directly separated, without
resorting to chromatography, by fractional crystalliza-
tion alone. In this nitration, the hydrobromide salt of
22 could not serve as starting material because ad-
ditional (unidentified) products were produced in sub-
stantial amounts. Also, excess nitronium tetrafluoro-
borate was needed to ensure complete reaction; otherwise
the starting material (22) was difficult to remove from
the products. The known compound 21 was similarly
nitrated to give 18 and 20, Scheme 5.
a
(a) NO2BF4/CH3CN, 27 h/0 °C; (b) cation exchange chroma-
tography; (c) separation of 18 by fractional crystallization; (d)
preparative C18 chromatographic separation of 19 and 20.
Sch em e 4a
Finally, 3,5-dinitro-DL-tyrosine (24) was smoothly
synthesized from DL-tyrosine (25), again with nitronium
tetrafluoroborate in acetonitrile. This compound was
also readily isolated as the hydrated ammonium salt,
apparently the first reported instance of this.
p Ka Stu d ies. The protolysis of various compounds
was studied by UV spectrophotometric methods22 at a
constant ionic strength of 0.1 M. Unexpectedly, the
measured ionization constants for 10, 11, and 13 (Table
1) were nearly 1 log unit lower than the literature
values23 for 2,4-dinitro-o-cresol (26; see structure ac-
companying Table 1, pKa 4.35), o- and p-nitrophenols
(pKa 7.23 and 7.15, respectively), and 4-nitro-o-cresol
(27; pKa 7.85 by titration in 50% methanol).24 We
determined the protolysis constants for the appropriate
o-cresols (26-28) under the same conditions as for the
amino acids (Table 1) and found them to be in excellent
agreement with the literature values.25 Clearly, the
amino acid side chain exerts a substantial electron-
withdrawing effect on the aromatic ring in these com-
pounds, and most of this effect must result from the
positively charged R-ammonium group because the pKa
of the R-hydroxy acid 16 is similar to that of the o-cresol
26 and considerably higher than that of the R-amino
acid 13.
a
(a) NBS, cat. PhCO2OCOPh/CCl4, 18 h reflux; (b) AcNHCH-
(CO2Et)2, NaOEt/EtOH, 10 min/rt; (c) 48% HBr, 5 h reflux; (d)
NO2BF4/CH3CN, 1 h/0 °C; (e) cation exchange chromatography;
(f) separation of 18 by fractional crystallization; (g) recrystalliza-
tion of 19.
2,6-dinitro) via unambiguous synthesis of the corre-
sponding 3-methoxybenzyl amino acids from the requi-
site dinitromethoxybenzaldehydes.19 In the present
work, the ammonium salt 18 was found to have a much
lower aqueous solubility than 19 or 20, and 18 could be
crystallized from mixed solutions almost quantitatively.
Fractional crystallization failed to separate the remain-
ing two compounds, but the mixture could be separated
on a medium-pressure liquid chromatography column
packed with C18-derivatized silica gel. Either 18 or 19
was the predominant product, apparently depending on
the rate at which the nitronium tetrafluoroborate was
added (this dependence was not fully characterized).
The product ratios were, however, generally similar to
the ratio reported by J ackson.18
Ra d ioliga n d Bin d in g Stu d ies. None of the unsub-
stituted hydroxyphenylalanines, namely, 2-hydroxy-DL-
phenylalanine (12), 3-hydroxy-DL-phenylalanine (17), or
DL-tyrosine (25), competed significantly for specific
[3H]AMPA binding in rat cortex homogenates (Table 1);
It was impossible to differentiate 19 and 20 using 1H-
NMR, and large-scale preparative separation was not
practical. These two compounds were, however, syn-

Acidic Hydroxyphenylalanines
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3185
Ta ble 1. pKa Values and Inhibition of [3H]AMPA Specific Binding at 10-4 M Concentrations of Various Hydroxyphenylalanines and
Related Compounds
analytical
wavelength (nm)
[3H]AMPA
inhibtnb
a
compd
configuration
R2
R3
R4
R5
R6
X
Y
pKa
+
+
+
+
+
+
+
8
7
DL
DL
DL
DL
DL
DL
DL
R,S
DL
DL
DL
DL
DL
DL
DL
DL
L
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
NO2
NO2
H
H
H
H
H
H
H
OH
OH
OH
H
H
H
H
H
H
H
NO2
NO2
NO2
H
OH
OH
OH
OH
OH
H
NO2
H
NO2
H
NO2
NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
H
NO2
H
NO2
H
H
H
H
C
N
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
NH3
NH3
NH3
NH3
NH3
NH3
NH3
OH
NH3
NH3
NH3
NH3
NH3
NH3
NH3
NH3
NH3
NH3
ND
9.3
6.4
∼50
258
326
100
31
12
10
11
13
16
17
21
22
18
19
20
25
24
29
30
100
8.67c
0, 0, 4
20, 57
37, 35
>90
0
0, 0, 0
ND
0
H
413
430
380
371
6.4 (27, 7.2)d
NO2
NO2
NO2
OH
OH
OH
OH
OH
OH
H
6.6 (28, 7.7)d
6.4 (26, 4.4)d
4.2
ND
6.8
6.6
3.8
3.2
3.6
ND
ND
6.8
3.2
+
+
+
+
+
+
+
+
+
+
398
417
360
410
381
∼50
100
H
H
NO2
H
NO2
∼50
0, 0, 0
33
0, 30
0,0
NO2
NO2
NO2
429
444
L
H
a
b
Protolysis constant at an ionic strength of 0.1 M. Percent inhibition of specific [3H]AMPA binding by 100 µM test compound; replicate
results represent determinations in different tissue preparations. c Protolysis constant at ionic strength ) 0.2 (KCl).31 pKa of the
corresponding o-cresol; see accompanying structure and text for discussion. ND, not determined.
d
Ta ble 2. Inhibition of Specific [3H]AMPA and [3H]Kainate Binding, and Functional Activity in Hippocampal Nerve Ending
Norepinephrine Release Assay
IC50 (µM)a
NE release
compound
[3H]AMPA
[3H]kainate
EC50 (µM)b
IC50 (µM)c
L-glutamate
AMPA (1)
kainate (2)
DL-willardiine (7)
31
1.2 ( 0.7
0.013 ( 0.003
ND
1.8 ( 0.1
0.12 ( 0.025
4.9 ( 0.9
25 ( 2.5
0.93 ( 0.42
0.54 ( 0.14
0.7 ( 0.3
ND
92 ( 8
6.7 ( 2.4
3.7 ( 1.2
ND
15 ( 1.6
74 ( 5.1
0.0054 ( 0.0013
97 ( 1.7
3.7 ( 0.9
34 ( 6
>100
2.2 ( 0.4
2.4 ( 0.4
19
13
730 ( 88
41 ( 6
ND
DNQX (32)
CNQX (39)
a
b
50% inhibition of specific radioligand binding, mean ( standard error of the mean of at least three independent determinations. In
vitro stimulation of norepinephrine release from hippocampal nerve endings; see text for experimental details. c Inhibition of the agonist
activity elicited by the simultaneous presence of 100 µM AMPA.
however, the more acidic mononitro compounds 10 and
11, and the dinitro compound 13, did. For the series
12, 11, 10, and 13, there is a suggestive correlation
between extent of ionization at physiological pH and
affinity for binding sites labeled by [3H]AMPA. The
R-hydroxy acid 16 was inactive, reiterating the require-
ment for the R-amino group. Of the 3-hydroxypheny-
lalanine analogs, 19 exhibited substantial activity,
whereas the other two dinitro analogs (18 and 20) were
considerably less active. 3-Nitro-L-tyrosine (29) and 3,5-
dinitro-L-tyrosine (30) were inactive; however, 3,5-
dinitro-DL-tyrosine (24) gave some displacement of
[3H]AMPA in the 100 µM screening assay, comparable
to that observed with 18 and 20.
Compounds effecting greater than 50% inhibition of
specific [3H]AMPA binding at 100 µM concentration
were further evaluated for potency as inhibitors of both
[3H]AMPA and [3H]kainate specific binding (Table 2).
The 2- and 3-hydroxyphenylalanines 13 and 19 inhib-
ited [3H]AMPA binding with low-micromolar potency.
These two compounds were somewhat less active as
inhibitors of [3H]kainate specific binding, and the ratio

3186 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Hill et al.
of IC50 values provides one index of relative selectivity.
The 2-hydroxyphenylalanine 13 was approximately
5-fold selective for AMPA over kainate binding inhibi-
tion and the 3-hydroxyphenylalanine 19 about 10-fold
selective.
DL-Willardiine (7) is surprisingly potent considering
that the 1-substituted uracil ring has a pKa of about 9.3.
We had predicted that the 5-nitro group of DL-5-
nitrowillardiine (31) would, via increased acidity, confer
as much as 2 orders of magnitude increase in potency;
at pH 7.4, willardiine (pKa 9.3) is about 1% ionized,
compared with 90% for 5-nitrowillardiine (pKa 6.4).
(This work was carried out in 1989-1990, prior to the
reports of Patneau et al.,9 Wong et al.,11 and Hawkins
et al.,10 and we first reported the pKa and preliminary
binding data for 31 in early 1991.4) Compound 31 was,
in fact, about 10-fold more potent than willardiine as
an inhibitor of specific [3H]AMPA binding (Table 2).
Compounds 7 and 31 were also quite selective for AMPA
vs kainate receptors in these binding studies.
F u n ction a l P h a r m a cologica l Stu d ies. The most
potent compounds were further studied in functional
assays. One assay is based on the fact that AMPA/
kainate receptor activation induces norepinephrine
release from hippocampal nerve endings.26,27 Com-
pound 19 was an agonist, albeit less potent than AMPA
and 5-nitrowillardiine (31) (Table 2). Compound 13,
however, was an antagonist that was somewhat less
potent than 1,4-dihydro-6,7-dinitro-2,3-quinoxalinedione
(32, DNQX; see structure in Table 2), in agreement with
the results of the equilibrium binding studies.
Another test derives from the observation that some
glutamatergic neurons become activated in parallel with
certain behavioral responses to amphetamine. One
example is that the AMPA/kainate antagonist DNQX,
when injected into the rat ventral pallidum, attenuates
the locomotor response elicited by systemic amphet-
amine (Figure 3; cf. Willins et al.28). The 2-hydroxy-
phenylalanine 13 had a similar effect, while 6-amino-
7-fluoro-1,4-dihydro-2,3-quinoxalinedione (33), which
has very low potency in competing for [3H]AMPA
binding sites, was inactive. The correlation between
potency in the binding assay and functional ability to
antagonize effects of amphetamine, coupled with the
antagonist action of 13 in the neurotransmitter release
assay, strongly suggests that the locomotor antagonism
of 13 is brought about by AMPA or kainate receptor
blockade. In another functional study, compound 13
was unable to stimulate acetylcholine release in rat
striatal slices, a model in which NMDA produces a
marked effect (data not shown), or to block the effect of
NMDA in this test system. Compound 13 also did not
antagonize the specific binding of [3H]NMDA, [3H]-
MK801, or [3H]PCP to rat brain preparations.29
F igu r e 3. Effect of AMPA receptor antagonists injected into
the ventral pallidum on hypermotility elicited by systemic
injection of stimulants. Microinjections of 1 µg of DNQX (32),
1 µg of compound 33, 1 µg of compound 13, or vehicle were
made into the ventral pallidum of anesthetized rats. Im-
mediately following this injection, the rats were given 0.5 mg/
kg amphetamine. Ambulatory locomotor activity was moni-
tored during a 1 h period immediately following recovery from
anesthesia. Control hypermotility (systemic stimulant; vehicle
in ventral pallidum) was 2000-3000 locomotor counts with
the doses of stimulants used, and baseline motility in the
absence of any drugs was approximately 500 locomotor counts.
Data are expressed as mean ( SEM for groups of 6-8 animals.
See main text for further details.
of AMPA (pKa 4.8)32 or quisqualate (pKa 4.5).33 Nitro-
tyrosines 10 and 21 were previously studied for the pH
dependence of their uptake in Ehrlich ascites tumor
cells via the glutamic acid uptake system.34 The rate
of uptake was well correlated with the degree of proto-
nation of these two compounds in pH-dependence stud-
ies, as it was for glutamate. Exploiting this same
concept, a 4-hydroxy-3-cyclobutene-1,2-dione (squaric
acid) analog was recently reported to be a potent AMPA-
like agonist.35 Even more recently, Matzen et al.6
reported that the less acidic isothiazole analog of AMPA
(pKa 7.00 for the 3-isothiazolol group) was about 50
times less potent than AMPA as an inhibitor of [3H]-
AMPA specific binding.
Despite the compelling correlation between acidity of
the γ-carboxyl group surrogate and activity, willardiine
(pKa >9) retains good biological activity. The consider-
able potency of willardiine might be accounted for if the
acidity of willardiine is enhanced by appropriate recep-
tor interactions, such as a hydrogen bond to the 4-oxo
group. Nonetheless, 5-nitrowillardiine (31) was about
10-fold more potent than willardiine in our binding
studies, and electron-withdrawing groups have been
found by others9-11 to enhance the potency of willardi-
ines at AMPA-preferring receptors.
Discu ssion
The notion that a phenolate anion might be bioisos-
teric for the γ-carboxyl group in glutamate is certainly
not new; in fact, Curtis and Watkins tested the activity
of 2-hydroxyphenylalanine (12) in their early structure-
activity studies (ca. 1960).30 They found, however, that
12 could not depolarize cat dorsal horn interneurons,
Renshaw cells, or motoneurons. The lack of activity is
not surprising, since the pKa of the phenolic hydroxyl
group in 12 is about 8.7,31 in contrast to the γ-carboxyl
group of glutamate (pKa 4.3)23 or the heterocyclic rings
Regarding the antagonist action of 13, the o-nitro-
phenolate moiety would be expected to engage the
receptor in a different manner than the γ-carboxylate
group of glutamate or the carboxylate isosteres of
agonists such as AMPA or willardiine; all of the three

Acidic Hydroxyphenylalanines
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3187
activity. We have thus shown that the phenolate anion
can serve as a bioisosteric replacement for the γ-car-
boxylate of glutamate in AMPA receptor ligands. In one
case (13), antagonist properties were conferred, and a
suitably positioned o-nitrohydroxylate moiety is there-
fore an important structural lead.
Exp er im en ta l Section
Gen er a l P r oced u r es. Melting points were determined on
a Thomas-Hoover melting point apparatus; emergent stem
corrections were not applied. Unless otherwise noted, 1H-NMR
spectra were acquired using an IBM AF/250 spectrometer (250
MHz). Some 13C-NMR and other special experiments were
conducted with IBM AF/270 (270 MHz) or Bru¨ker AMX-500
(500 MHz) spectrometers. Infrared spectra were obtained
using a Laser Precision Analytical RFX-40 FTIR. High-
resolution electron impact (EI) mass spectra were obtained at
The Ohio State University Chemical Instrumentation Center
using a Kratos MS-30 spectrometer, while fast atom bombard-
ment (FAB) mass spectra were acquired using either a VG 70-
250S or Finnigan MAT-90 spectrometer on “magic bullet” or
3-nitrobenzyl alcohol, using DMSO as an additional solvent
when necessary. High-pressure liquid chromatography (HPLC)
was carried out on a Beckman 421 system, equipped with
Model 112 pumps and a Model 153 fixed-wavelength detector,
or a Beckman System Gold equipped with a Model 167
scanning UV-visible detector, Model 406 analog interface, and
Model 110B pumps. Unless otherwise noted, chromatograms
were obtained on an IBM 5 µm EC-C8 column, 4.6 mm i.d. ×
15 cm length, using a mobile phase flow rate of 2.0 mL/min
and detection by ultraviolet absorption at 254 nm. Ultraviolet/
visible spectra were acquired with a Kontron UVIKON 860,
IBM Model 9420, or Gilford REPONSE spectrometer. El-
emental analyses were performed by Galbraith Laboratories,
Inc., Knoxville, TN, Oneida Research Services, Inc., Whites-
boro, NY, or Atlantic Microlab, Inc., Norcross, GA.
F igu r e 4. Structural comparison of the AMPA antagonist 13
with AMPA (1), 5-nitrowillardiine (31), and 3-carboxyphenyl-
alanine (34).
Miscella n eou s Com p ou n d s. DL-Willardiine (7) was syn-
thesized by the method of Martinez and Lee.42 DL-5-Nitro-
willardiine (31) was reported as a synthetic intermediate by
Martinez, Lee, and Goodman;43 we readily repeated their
procedure for the nitration of willardiine. 3-Nitro-L-tyrosine
(29) and 3,5-dinitro-L-tyrosine (30) were obtained from Sigma.
p Ka Deter m in a tion s. An adaptation of published spec-
trophotometric methods22 served to determine the proton
dissociation constants for various compounds. Compound (1-2
mg) was dissolved in a solution that contained 0.01 N NaOH
and 0.09 M KCl (solution A) to make the ionic strength equal
to 0.1; the pH of the resulting solutions was ca. 11.8, and the
volume was sufficient to give an optical density of ca. 1 AU at
a suitable wavelength (Table 1). The solution was titrated
with 0.1 N HCl, graphical estimates of the pKa and the acid-
and base-form absorbance values were obtained, and the pKa
was then calculated by fitting the data to the Henderson-
Hasselbach equation (recast in terms of UV absorbances) using
the program NONLIN84.44
N-[[(1,1-Dim eth yleth yl)oxy]ca r bon yl]-2-h yd r oxy-5-n i-
t r o-DL-p h en yla la n in e (14) a n d N-[[(1,1-d im et h ylet h yl)-
oxy]ca r bon yl]-2-h yd r oxy-3-n itr o-DL-p h en yla la n in e (15),
Meth od A. Compound 12 (2-hydroxy-DL-phenylalanine; 5.0
g, 27.6 mmol) was suspended in 20.7 mL of H2O, and 5.2 mL
of 50% HNO3 was added during 30 min at room temperature
while stirring. The solution was cooled in an ice bath, and an
additional 11.0 mL of 50% HNO3 was added dropwise during
2 h. The solution was allowed to warm to room temperature
during 5 h and then placed in a refrigerator overnight. H2O
(50 mL) was added, and the mixture was extracted with
benzene (2 × 100 mL). The aqueous fraction was neutralized
with Na2CO3, again extracted with benzene (2 × 100 mL), and
evaporated to dryness in vacuo using i-PrOH for azeotropic
removal of the last portion of H2O. The solid residue was taken
up in 100 mL of 1.2 N HCl (a black solid remained undissolved)
and eluted through a short 1 in. i.d. column containing 5 g of
Bio-beads (SM-16, 20-50 mesh; Biorad, Inc.). The solution
was diluted 10-fold with H2O and eluted through a cation
exchange column (2.5 cm i.d. × 9 cm length, Biorad AG50W-
F igu r e 5. Structural comparison of the agonist 19 to selected
glutamate homologs.
latter compounds have a pair of heteroatoms positioned
to interact with the receptor in a similar fashion, and
different than for the antagonist 13 (see Figure 4).
Furthermore, it is noteworthy that certain invertebrate
neurons are depolarized by 3-carboxyphenylalanine (34;
Figure 4).36 In 13, either of the two nitro groups could
occupy the same region of receptor space as the 3-car-
boxy group in 34. The 3-hydroxyphenylalanine 19, on
the other hand, elicited the anticipated agonist activity;
the glutamate homologs â-oxalylaminoalanine (35),37
4-bromo- and 4-methylhomoibotenic acids (36 and 37),38
and compound 3839 (Figure 5) are also all potent AMPA
receptor agonists.
It is now known40 that the enantioselectivity for
compound 13 is the same as for AMPA (L >> D).41 The
screening data for 3,5-dinitro-L-tyrosine (30) and 3,5-
dinitro-DL-tyrosine (24) suggest the opposite enantiose-
lectivity (Table 1). The enantiospecific synthesis of L-13
was sufficiently challenging that it will be the subject
of a subsequent publication.
Con clu sion s
Of the nitrated hydroxyphenylalanines tested for
inhibition of specific [3H]AMPA binding in rat brain
homogenates, acidic hydroxy groups at the 2- and
3-positions of the phenyl ring conferred substantial

3188 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Hill et al.
X12, sulfonic acid type, hydrogen form, 200-400 mesh). The
column was washed with H2O (ca. 1500 mL) and then eluted
with 0.75 N NH4OH. Evaporation to dryness in vacuo gave
2.57 g of a solid that was estimated by H-NMR to contain 10
and 11 in about a 7:3 (para:ortho) ratio, along with <10 mol
% of 13.
zwitterionic form: 230 mg of the trifluoroacetate salt was
dissolved in 90 mL of 0.1 N HCl, and this solution was eluted
through a 2.5 cm i.d. × 3.5 cm length cation exchange column
(see above). The column was washed with 600 mL of H2O,
and the product was eluted with 550 mL of 0.75 N NH4OH.
The volume was reduced in vacuo to ca. 15 mL, i-PrOH (50
mL) was added, and the volume was again reduced to ca. 20
mL. The product was collected and washed successively with
i-PrOH and Et2O. Recovery from the column was ca. 94%:
dec 246-256 °C (lit.15 dec 252-257 °C); 1H-NMR (DCl/D2O,
DSS) δ 8.15 (s, 1H, ArH, overlapped with δ 8.13), 8.13 (dd
overlapped with δ 8.15, 1H, J ≈ 2.8 Hz, ArH), 7.04 (dd, 1H, J
) 7.8, 1.6 Hz, ArH), 4.48 (dd, 1H, J ) 5.8, 7.3 Hz, HR), 3.44
(dd, 1H, J ) 5.8, 14.5 Hz, CH2), 3.27 (dd, 1H, J ) 7.3, 14.5
Hz, CH2); MS (FAB) m/e 227 (M + 1). Anal. (C9H10N2O5) C,
H, N.
2-Hyd r oxy-3-n itr o-DL-p h en yla la n in e (11). Compound
15 (0.20 g, 0.61 mmol) was suspended in 7 mL of CH2Cl2 and
treated with 3 mL of CF3CO2H while stirring. After 7 h at
room temperature, the reaction mixture was neutralized with
ca. 30 mL of 10% Et3N in CH2Cl2 to give 0.12 g (83%) of 11 in
two crops as a yellow powder: dec 236-237 °C; 1H-NMR (DCl/
D2O, DSS) δ 8.07 (dd, 1H, J ) 8.5, 1.3 Hz, ArH), 7.67 (dd, 1H,
J ) 7.4, 1.3 Hz, ArH), 7.10 (asym t, 1H, ArH), 4.24 (asym t,
1H, HR), 3.37 (dd, 1H, J ) 6.9, 14.0 Hz, CH2), 3.24 (dd, 1H, J
) 7.3, 14.1 Hz, CH2); MS (FAB) m/e 227 (M + 1); reversed-
phase HPLC showed the product to contain <0.5% of 10. Anal.
(C9H10N2O5) C, H, N.
2-Hyd r oxy-3,5-d in itr o-DL-p h en yla la n in e, Am m on iu m
Sa lt, 5/2 Hyd r a te (13). A suspension of 12 (0.50 g, 2.76 mmol)
in reagent-grade CH3CN (8 mL) was cooled to 0-5 °C, and
1.0 g (6.4 mmol) of 85% NO2BF4 was added in small portions
during 10 min. Cooling and stirring were continued for 8 h,
H2O (120 mL) was added, and the mixture was filtered after
standing overnight to remove a dark-brown solid. The filtrate
was eluted through a 2.5 cm i.d. × 6 cm length cation exchange
column (Biorad AG50W-X12, see above), and the column was
washed with H2O (2500 mL). The product was eluted with
0.4 N NH4OH, the volume was reduced to ca. 10 mL in vacuo,
i-PrOH (100 mL) was added, and the suspension was heated
briefly. After cooling, Et2O (500 mL) was slowly added and
the flask placed in a refrigerator overnight. The product was
collected, washed with small amounts of i-PrOH and Et2O, and
dried in air to give 0.625 g (68%) of a fluffy, yellow-orange
powder: melting behaviorsheating at about 5 °C/min, the
material visibly changed form at ca. 160 °C, discolored at >180
°C, and rapidly decomposed at >190 °C; 1H-NMR (D2O, DSS)
δ 8.78 (d, 1H, J ) 3.1 Hz, ArH), 8.05 (d, 1H, J ) 3.1 Hz, ArH),
4.02 (dd, 1H, J ) 3.6, 7.7 Hz, HR), 3.27 (dd, 1H, J ) 3.6, 14.6
Hz, CH2), 3.00 (dd, 1H, J ) 7.7, 14.6 Hz, CH2); in DMSO-d6
there was a broad peak centered at ∼7.5 ppm (7H, NH3+ and
NH4+); MS (FAB) m/e 272 (M + 1), 226 (-NO2). Anal.
(C9H9N3O7‚NH3‚5/2H2O) C, H, N.
2-H yd r oxy-3-(2′-h yd r oxy-3′,5′-d in it r op h e n yl)p r op a -
n oic Acid (16). A solution of concentrated HNO3 (5 mL) in
H2O (6 mL) was cooled in an ice bath, and 12 (1.00 g, 5.52
mmol) was added in small portions during 10 min while
stirring. After 2.5 h of continued stirring at 0-5 °C, fuming
HNO3 (11 mL) was added during 15 min. After 1 h, the ice
bath was removed and the solution stirred 7 h longer. Water
(400 mL) was added, and the solution was extracted twice with
400-mL portions of 1:9 CH3CN/EtOAc. The extracts were
shaken with 200 mL of brine, dried over MgSO4, filtered, and
concentrated in vacuo. Hexane was added portionwise during
2 days while crystallization proceeded, yielding 0.48 g (32%)
of pale-yellow crystals: mp 140-141 °C after an additional
recrystallization from EtOAc/hexane: 1H-NMR (acetone-d6,
TMS) δ 8.85 (d, 1H, J ) 2.8 Hz, ArH), 8.53 (d, 1H, J ) 2.8 Hz,
ArH), 4.57 (dd, 1H, J ) 4.2, 8.9 Hz, HR), 3.46 (dd, 1H, J ) 4.2,
14.2 Hz, CH2), 3.11 (dd, 1H, J ) 8.9, 14.2 Hz, CH2); 13C-NMR
(acetone-d6, TMS) δ 175 (s, C1), 158.1 (s, C2′), 140.1 (s, C5′),
134.2 (s, C3′), 133.0 (d, C6′), 131.9 (s, C1′), 120.7 (d, C4′), 69.7
(d, C2), 35.2 (t, C3); these assignments were confirmed by 1H-
13C heteronuclear correlation and by a 13C INADEQUATE
experiment; MS (EI) m/e 272.025 (calcd 272.028, M+), 254
(-H2O), 227 (-CO2H), 198 (-HO2CCHO), 76 (base). Anal.
(C9H8N2O8‚1/2H2O) C, H, N.
1
A 1.5 g portion (ca. 6.6 mmol) of the above mixture was
dissolved in 100 mL of 1:1 dioxane-H2O, and 4.0 mL (30 mmol)
of Et3N and 3.59 g (14.6 mmol) of 2-[[(tert-butyloxycarbonyl)-
oxy]imino]-2-phenylacetonitrile ("BOC-ON") was added. After
stirring for 16 h at room temperature, 200 mL of H2O was
added and the solution was extracted with EtOAc (2 × 200
mL). The aqueous phase was acidified with 5% citric acid (75
mL) and 1.2 N HCl (20 mL) and extracted with EtOAc (2 ×
150 mL). The extracts were dried (MgSO4) and concentrated
in vacuo. This solution was divided into two portions, and each
was chromatographed on fresh silica gel (40-63 µm, E. Merck,
dry-packed in a 51 mm i.d. × 43 cm length Michel-Miller
column (Ace Glass, Inc.) and pre-equilibrated with CHCl3),
eluting with 8% i-PrOH/2% MeOH/0.7% CH3CO2H in CHCl3
at 20 mL/min. Appropriate fractions were combined, concen-
trated in vacuo, and coevaporated with toluene to dryness. The
residues were taken up in EtOAc and triturated with hexane
to yield 0.88 g of 14 (pale-yellow crystals, 16.8% from 12) and
0.35 g of 15 (yellow crystals, 6.6% from 12).
Analytical data for 14: dec 205 °C; 1H-NMR (acetone-d6,
TMS) δ 8.13 (d, 1H, J ) 2.7 Hz, ArH), 8.03 (dd, 1H, J ) 8.9,
2.7 Hz, ArH), 7.03 (d, 1H, J ) 8.9 Hz, ArH), 6.26 (bd, J ) 7.2
Hz, NH), 4.54-4.62 (bm, 1H, HR), 3.41 (dd, 1H, J ) 4.6, 13.6
Hz, CH2), 3.00 (dd, 1H, J ) 9.8, 13.6 Hz, CH2), 1.31 (s, 9H,
CH3); HPLC (5 µm IBM RP-phenyl, 4.6 mm i.d. × 15 cm
length, 3 min at 10% MeOH/0.1% CF3CO2H/0.2% CH3CO2H/
H2O, then a linear gradient during 15 min to 21% MeOH; see
also General Procedures) indicated <1% contamination of the
product with 15. Anal. (C14H18N2O7) C, H, N.
Analytical data for 15: dec 139-140 °C; 1H-NMR (acetone-
d6, TMS) δ 8.04 (d, 1H, J ) 8.1 Hz, peak-broadening due to
meta-coupling to δ 7.67 proton, ArH), 7.67 (d, 1H, J ) 6.8, ∼1
Hz, ArH), 7.03 (asym t, 1H, ArH), 6.18 (bd, J ) 7.9 Hz, NH),
4.59 (bdd, 1H, J ) 4.7, 9.8 Hz, HR), 3.44 (dd, 1H, J ) 4.7, 13.6
Hz, CH2), 3.03 (dd, 1H, J ) 9.8, 13.6 Hz, CH2), 1.31 (s, 9H,
CH3); HPLC (same conditions as for 14) showed the product
to contain ∼2% of 14, and there were no other significant
peaks. Anal. (C14H18N2O7) C, H, N.
Meth od B. A suspension of 12 (2.94 g, 16.2 mmol) in 50
mL of reagent-grade CH3CN under dry argon was cooled to
-18 °C, and 85% NO2BF4 (2.53 g, 16.2 mmol) was added in
small portions during 30 min. After another 30 min of cooling,
the reaction was quenched by pouring the mixture into 470
mL of H2O and adding 28 mL of 1.2 N HCl. The solution was
allowed to stand overnight, filtered to remove a brown
precipitate, and eluted through a cation exchange column as
in method A. The ammoniacal eluates were evaporated to
dryness in vacuo, the residue was suspended in 10 mL of H2O,
and the grayish solid was collected, washed with i-PrOH, and
dried to a constant weight of 2.26 g. 1H-NMR (DCl/DMSO)
showed the mixture to contain 10, 11, and 12, ca. 33:22:45
(molar basis), corresponding to 22% and 14% yields of 10 and
11, respectively, and 30% recovery of unreacted 12.
A portion of this mixture (2.03 g, ca. 9.0 mmol) was treated
with BOC-ON as in method A. The EtOAc extracts were
concentrated in vacuo to a volume of ca. 5 mL and diluted to
40 mL with CHCl3; this solution was chromatographed as in
method A, but in a single portion. The two mononitro products
were crystallized as before to give 0.73 g of 14 (15% from 12)
and 0.58 g of 15 (12% from 12).
2-Hyd r oxy-5-n itr o-DL-p h en yla la n in e (10). Compound
14 (0.50 g, 1.5 mmol) was suspended in CH2Cl2 (14 mL) and
treated with CF3CO2H (6 mL) while stirring. The starting
material dissolved, and stirring was continued for 3 h at room
temperature. The resulting white precipitate was collected
and air-dried to yield 0.42 g (80%) of the trifluoroacetate salt,
dec 208-209 °C. Reversed-phase HPLC (see General Proce-
dures for column and detection; eluting 8 min with 8% CH3-
CN/0.1% CF3CO2H/H2O, then a linear gradient to 70% CH3CN
during 10 min) showed the material to be free of detectable
(<0.01%) quantitities of 11. A portion was converted to the

Acidic Hydroxyphenylalanines
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3189
5-Hyd r oxy-2,4-d in itr o-DL-p h en yla la n in e, Am m on iu m
Sa lt (18) a n d 3-Hyd r oxy-2,6-d in itr o-DL-p h en yla la n in e,
Am m on iu m Sa lt, /2 Hyd r a te (20). Commercially obtained
mmol) of 4-(bromomethyl)-2-methoxy-1-nitrobenzene21 with
strong stirring. The reaction was rapid; after 4-5 min, strong
precipitation occurred and the suspension quickly became an
unstirrable cake, whereupon heating was discontinued. After
another 15 min, the reaction flask was placed in a refrigerator.
After 3 days, 50 mL of EtOH was added and the cake was
broken up, collected by filtration, resuspended in 400 mL of
EtOH, and stirred vigorously for 1 h. The solid was again
collected, washed (EtOH, 2 × 25 mL), and dried in air to give
23, 18.98 g (70.9%): mp 165.0-165.5 °C (lit.21 163-164 °C);
1H-NMR (CDCl3, TMS) δ 7.76 (d, 1H, J ) 8.3 Hz, ArH), 6.73
(d, 1H, J ) 1.3 Hz, ArH), 6.66 (dd, 1H, J ) 8.3, 1.3 Hz, ArH),
6.59 (s, 1H, NH), 4.20-4.36 (m, 4H, CH2), 3.90 (s, 3H,
ArOCH3), 3.73 (s, 2H, ArCH2), 2.04 (s, 3H, COCH3), 1.31 (t,
6H, J ) 7.1 Hz, CH3); MS (EI) m/e 382.136 (calcd 382.138,
M+), 323 (-CH3CONH2), 276, 43 (base, CH3CO+). Anal.
(C17H22N2O8) C, H, N.
7
3-hydroxy-DL-phenylalanine (17; 1.00 g, 5.52 mmol) was
suspended in 15 mL of reagent-grade CH3CN under dry argon.
This suspension was cooled in an ice bath and treated with
solid 85% NO2BF4 (2.07 g, 13.2 mmol) in small portions during
10 min with constant stirring and cooling. The resulting deep-
red solution was stirred for 2 h more in the ice bath and then
allowed to come to room temperature during another 5.5 h.
The reaction vessel was placed in a freezer overnight, warmed
to room temperature during 2 h, and diluted with H2O to 500
mL. After cation exchange chromatography and concentration
of the ammoniacal eluates, 18 crystallized almost quantita-
tively. After several days of refrigeration, the nearly pure
crystals of 18 were collected (the mother liquor was set aside),
washed twice with H2O (1-2 mL), and dried in air overnight
and in a dessicator for 3 days, yielding 0.43 g (27%) of 18 as
the anhydrous ammonium salt. The product was recrystal-
lized from H2O: melting behaviorsheating at about 5 °C/min,
the yellow-orange salt slowly discolored at temperatures above
3-Hyd r oxy-4-n itr o-DL-p h en yla la n in e (22) a n d 3-Hy-
d r oxy-4-n itr o-DL-p h en yla la n in e Hyd r obr om id e. A sus-
pension of 9.56 g (25.0 mmol) of 23 in 95 mL of 48% HBr was
brought to gentle reflux. Heating was discontinued after 20
h, and the HBr salt slowly crystallized from solution. After 3
days in a freezer, the salt was collected, and the damp cake
was dissolved in H2O (50 mL) and titrated to pH 5 with
concentrated NH4OH. After overnight refrigeration another
50 mL of H2O was added; the product was collected, washed
with H2O, and dried in air and then under high vacuum for
18 h to yield 4.31 g (76.3%) of 22 as light-yellow crystals: dec
1
200 °C and rapidly turned dark-brown at ∼245 °C; H-NMR
(DCl/D2O, DSS) δ 9.02 (s, 1H, ArH), 7.33 (s, 1H, ArH), 4.52
(asym t, 1H, HR), 3.77 (dd, 1H, J ) 7.6, 13.8 Hz, CH2), 3.58
(dd, 1H, J ) 7.5, 13.8 Hz, CH2); MS (FAB) m/e 289 (M‚NH4+),
272 (M + 1). The amounts of the other two isomers in this
material were quantitated by HPLC (mobile phase 8% CH3-
CN/0.1% CF3CO2H/H2O), using authentic standards due to the
considerable differences in response factors: 19 (<0.5%) and
20 (<0.5%). Anal. (C9H9N3O7‚NH3) C, H, N.
1
229 °C (at 2 °C/min from 226 °C); H-NMR (DCl/D2O, DSS) δ
8.13 (d, 1H, J ) 8.7 Hz, ArH), 7.15 (d, 1H, J ) 1.7 Hz, ArH),
7.04 (dd, 1H, J ) 8.7, 1.7 Hz, ArH), 4.48 (dd, 1H, J ) 6.1, 7.5
Hz, HR), 3.47 (dd, 1H, J ) 6.1, 14.5 Hz, CH2), 3.26 (dd, 1H, J
) 7.5, 14.5 Hz, CH2); MS (FAB) m/e 227 (M + 1), 181 (-NO2).
Anal. Calcd for C9H10N2O5: C, 47.79; H, 4.46; N, 12.39.
Found: C, 47.12; H, 4.38; N, 12.23.
The mother liquors from above, containing 19 and 20, were
slowly treated with i-PrOH to a volume of ca. 400 mL; Et2O
(150 mL) was then added. The suspension was concentrated
in vacuo to ca. 50 mL, i-PrOH (150 mL) and Et2O (150 mL)
were added, and crystallization was allowed to proceed over-
night at room temperature. The solid was collected and air-
dried to yield 0.63 g of material that contained 19 and 20 in a
2:1 molar ratio (1H-NMR). These compounds were separated
by preparative reversed-phase (C18) chromatography as fol-
lows: 10 60-mg portions were each dissolved in 1 mL of 1.2 N
HCl and injected onto a 47 mm i.d. × 38 cm length Michel-
Miller column packed with 40-63 µm silica gel (E. Merck) that
had been derivatized with octadecyltrichlorosilane. The com-
pounds were eluted with 15% CH3CN/1% CF3CO2H/H2O at 20
mL/min, monitoring by UV at 280 nm. The fractions were
analyzed by HPLC (4% CH3CN/0.1% CF3CO2H/H2O, 2.0 mL/
min); those containing 20 were combined, and HCl was added
to make the solution 0.1 N. Cation exchange chromatography
and evaporation of the ammoniacal eluates gave a damp cake.
Water (10 mL) was added, and the solution was treated with
i-PrOH to the point of initial cloudiness. Crystallization was
continued for 1 week in a freezer, and the product was
collected, washed with i-PrOH, and dried in air to give 0.13 g
(6.7%) of 20: dec 170-180 °C; 1H-NMR (D2O, DSS) δ 8.07 (d,
1H, J ) 9.6 Hz, ArH), 6.58 (d, 1H, J ) 9.6 Hz, ArH), 3.92 (dd,
1H, J ) 6.9, 8.7 Hz, HR), 3.50 (dd, 1H, J ) 6.9, 14.3 Hz, CH2),
3.04 (dd, 1H, J ) 8.7, 14.3 Hz, CH2); MS (FAB) m/e 272 (M +
1). The amounts of the other two isomers in this product,
quantitated by HPLC vs authentic standards, were ∼0.35%
of 19 and ∼0.04% of 18. Anal. (C9H9N3O7‚NH3‚7/2H2O) C, H,
N.
The fractions containing mostly 19 were combined, and HCl
was added to make the solution 0.1 N. Cation exchange
chromatography was carried out, the eluates were concen-
trated to ca. 5 mL, i-PrOH was added to the point of cloudiness,
and crystallization was allowed to proceed slowly in the
freezer. The crystals were collected, washed with i-PrOH, and
dried in air to yield compound 19 (0.31 g, ca. 19%), which was
contaminated with 20 (3 mol %) and 18 (1 mol %). This
material was not purified further because an alternate syn-
thesis was in progress (see below). The total yield for the three
isolated compounds (18-20) was 53%.
Dieth yl 1-Aceta m id o-2-(3′-m eth oxy-4′-n itr op h en yl)-1,1-
eth a n ed ica r boxyla te (23). Sodium metal (1.77 g, 77.0
mmol) was dissolved in 375 mL of EtOH, and acetamidoma-
lonic acid diethyl ester (15.2 g, 70.0 mmol) was added. The
solution was heated to 60 °C and treated with 17.22 g (70.0
In a separate trial (2/5 scale), the hydrolysis took only 5 h
(monitored by HPLC), and the HBr salt was isolated by
washing the filter cake thrice with i-PrOH. Drying in air and
then under high vacuum for 3.5 h gave 22‚HBr: mp 238-239
°C (at 2 °C/min from 235 °C). Anal. (C9H11BrN2O5) C, H, N.
5-Hyd r oxy-2,4-d in itr o-DL-p h en yla la n in e, Am m on iu m
Sa lt (18) a n d 3-Hyd r oxy-2,4-d in itr o-DL-p h en yla la n in e,
Am m on iu m Sa lt, Dih yd r a te (19). Compound 22 (free
zwitterion, 2.26 g, 10.0 mmol) was suspended in CH3
CN (10
mL) under dry argon gas and cooled to 0-4 °C. NO2BF4 (85%,
3.9 g, 25 mmol) was added during 5 min with continued
cooling. After 2.7 h, the contents were poured into 0.1 N HCl
(1000 mL), and the solution was eluted through a cation
exchange column. The column was washed (H2O, 600 mL) and
eluted (0.3 N NH4OH, 1000 mL), and the eluates were
evaporated in vacuo to a damp residue. H2O (10 mL) was
added, and after 5 days of refrigeration the crystals were
collected (setting the mother liquor aside), washed twice with
H2
O, and dried in air to a constant weight (ca. 4 days),
yielding 0.75 g (26%) of 18.
The mother liquor was slowly treated with i-PrOH just to
the point of persistent cloudiness; after 3 weeks in a freezer,
the product was collected, washed (i-PrOH), and dried in air
to give 0.27 g of 19 as deep-orange crystals of the ammonium
salt dihydrate: slow dec >170 °C, rapid dec >185 °C. This
crop contained 0.9% 18 (HPLC, 8% CH3CN/0.1% CF3CO2H/
H2O at 2.0 mL/min). A second crop was obtained (0.04 g, total
11%): 1H-NMR (D2O, DSS) δ 7.96 (d, 1H, J ) 8.9 Hz, ArH),
6.38 (d, 1H, J ) 8.9 Hz, ArH), 3.98 (dd, 1H, J ) 5.2, 9.0 Hz,
HR), 3.20 (dd, 1H, J ) 5.2, 14.8 Hz, CH2), 2.92 (dd, 1H, J )
9.0, 14.8 Hz, CH2); the spectrum in DMSO-d6 gave a broad
+
peak centered near 7.5 ppm (NH4 and RNH3+); MS (FAB)
m/e 272 (M + 1). Anal. Calcd for C9H9N3O7‚NH3‚2H2O: C,
33.34; H, 4.97; N, 17.28. Found: C, 33.76, 33.81; H, 4.69, 4.73;
N, 17.03, 17.33.
5-Hyd r oxy-2,4-d in itr o-DL-p h en yla la n in e, Am m on iu m
Sa lt (18) a n d 3-Hyd r oxy-2,6-d in itr o-DL-p h en yla la n in e,
Am m on iu m Sa lt (20). Compound 2120 (1.70 g, 7.52 mmol)
was nitrated under conditions similar to those for compound
22, except the NO2BF4 was added in portions during 12 min.
The products (18 and 20) were isolated by ion exchange
chromatography and fractional crystallization in similar fash-

3190 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Hill et al.
ion (the volume of the mother liquor from the first crystal-
lization of 18 was 8 mL). The yield of 18 was 1.10 g (50.7%),
and the yield of 20 was 0.35 g (13%).
diminishes AMPA receptor desensitization, thus allowing for
a more readily quantifiable AMPA response.) After fraction
4, buffer containing test compound, 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 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 EC50 or IC50 values
was accomplished utilizing median effect plot analysis.46
4-Hyd r oxy-3,5-d in itr o-DL-p h en yla la n in e, Am m on iu m
Sa lt, Mon oh yd r a te (24). DL-Tyrosine (25; 1.0 g, 5.5 mmol)
was suspended in 16 mL of CH3CN, and NO2BF4 (95%, 1.8 g,
13 mmol) was added portionwise during a period of 20 min
while the temperature was maintained at 0-5 °C. Stirring
was continued until no starting material or 3-nitrotyrosine was
in evidence by HPLC (ca. 1 h); the mixture was then poured
onto 50 g of ice and refrigerated for ca. 12 h. The solution
was filtered and diluted with H2O (1000 mL). Cation exchange
chromatography, concentration of the eluates in vacuo to ca.
10 mL, addition of i-PrOH (100 mL) followed by Et2O (500 mL),
and refrigeration for ca. 12 h gave orange crystals. These were
collected, washed with Et2O (10 mL), and air-dried to yield
0.96 g (57%) of 24: melting behaviorsdarkened at 190 °C, dec
Locom otor Stu d ies. Microinjections of 1 µg of DNQX (32),
1 µg of 33, 1 µg of 13, or vehicle were made into the ventral
pallidum of anesthetized rats using a stereotaxic apparatus
(David Kopf Instruments, Tujunga, CA). Immediately follow-
ing this injection, the rats were given 0.5 mg/kg amphetamine
ip. Ambulatory locomotor activity was monitored during a 1-h
period immediately following recovery from anesthesia using
activity cages equipped with infrared beams and photocells
(Columbus Instruments, Columbus, OH). Control hypermo-
tility (systemic stimulant; vehicle in ventral pallidum) was
2000-3000 locomotor counts with the doses of stumulants
used, and baseline motility in the absence of any drugs was
approximately 500 locomotor counts. Study groups contained
6-8 animals.
1
>200 °C; H-NMR (90 MHz, D2O, DSS) δ 7.97 (s, 2H, ArH),
3.92 (dd, 1H, J ) 4.9, 8.3 Hz, HR), 3.18 (m, 1H, CHaHb), 2.91
(m, 1H, CHaHb); 13C-NMR (90 MHz, D2O, DSS) δ 176.6 (CdO),
161.7 (C4), 145.0 (C3 and C5), 136.0 (C2 and C6), 118.8 (C1),
58.6 (CH), 37.8 (CH2). Anal. (C9H9N3O7‚NH3‚H2O) C, H, N.
P h a r m a cology. Bin d in g Stu d ies. The interaction of
compounds with AMPA receptors was assessed by inhibition
of specific [3H]AMPA binding in washed membrane prepara-
tions of rat brain following the procedure originally published
by Honore et al.45 After the freeze/thaw cycle and two
additional washings, the preparation was incubated with
0.04% Triton X-100 at 37 °C for 30 min to remove an inhibitor
protein. After another wash, binding was performed by one
of two procedures.
Ack n ow led gm en t. This work was financially sup-
ported in part by an enrichment grant from The Ohio
State University College of Pharmacy and by National
Institute of Drug Abuse Grant DA06776. R.A.H. was
supported in part by a fellowship from The American
Foundation for Pharmaceutical Education.
P r oced u r e A: Membranes were incubated with [3H]AMPA
(1.0 nM final concentration) and solutions of test compounds
at appropriate concentrations in 30 mM Tris-acetate contain-
ing 2.5 mM CaCl2 and 100 mM KSCN, pH 7.4. The mixture
was incubated on ice for 30 min, and bound ligand was then
separated from free ligand by centrifugation. The pellet was
surface-washed and then dissolved in scintillation fluid for
counting.
Refer en ces
P r oced u r e B: Membranes were incubated with [3H]AMPA
(1.4 nM final concentration) and solutions 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,
following which bound ligand was separated from free ligand
by rapid filtration through Whatman GF/C filter paper using
a Brandel M-12R cell harvester (Gaithersburg, MD). The
resulting filter disks, containing membrane-bound radioligand,
were dissolved in Scintiverse-E cocktail for scintillation count-
ing. For both procedures, 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 tested. IC50 values were calculated using nonlinear
regression analysis (InPlot software, GraphPad, Inc.).
Interaction of compounds with kainate receptors was as-
sessed by inhibition of specific [3H]kainate binding sites.45
Tissue preparation was exactly the same as for the AMPA
studies to the point of the freeze/thaw cycle. After thawing,
the membranes were washed twice with 50 mM Tris-HCl
buffer containing 2.5 mM CaCl2, pH 7.1. Binding was then
performed by incubating membranes with [3H]kainate (1 nM
final concentration) and solutions of test compounds at ap-
propriate concentrations, in a 50 mM Tris-citrate buffer, pH
7.4. The mixture was incubated for 60 min on ice before
separating bound and free ligand by one of the two methods
described above.
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,26 and is
adapted from Desai et al.27 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, then transferred to superfusion chambers,
and washed for 60 min with warmed, oxygenated Krebs buffer
at 0.3 mL/min. Following this, 10 5-min fractions were
collected. After the third fraction was collected, buffer con-
taining 50 µM cyclothiazide was introduced. (Cyclothiazide
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