Bioisosteric modifications of 2-arylureidobenzoic acids: Selective noncompetitive antagonists for the homomeric kainate receptor subtype GluR5

Article abstract of DOI:10.1021/jm030638w

2-Arylureidobenzoic acids (AUBAs) have recently been presented as the first series of selective noncompetitive GluR5 antagonists. In this paper we have modified the acidic moiety of the AUBAs by introducing different acidic and neutral groups, and similarly, we have replaced the urea linker of the AUBAs with other structurally related linkers. Replacing the acid with neutral substituents led to inactive compounds in all instances, showing that an acidic moiety is necessary for activity. Replacing the carboxylic moiety in 2a with a sulfonic acid (5c) or a tetrazole ring (5d) improved the potency at GluR5 receptors (compounds 5c and 5d showed IC₅₀ values of 1.5 and 2.0 μM, respectively, compared to compound 2a with IC₅₀ = 4.8 μM). Compound 5c did not show improved in vivo activity in the ATPA rigidity test compared to 2a, whereas compound 5d was 4 times more potent than 2a. All compounds wherein the urea linker had been replaced showed lower or no activity. The results described extend the knowledge of structure-activity relationships for the AUBAs, and compound 5d may prove to be a good candidate for studying GluR5 receptors in vitro and in vivo.

Full text of DOI:10.1021/jm030638w

6948
J. Med. Chem. 2004, 47, 6948-6957
Bioisosteric Modifications of 2-Arylureidobenzoic Acids: Selective
Noncompetitive Antagonists for the Homomeric Kainate Receptor Subtype
GluR5
Jon Valgeirsson,†,# Elsebet Ø. Nielsen,‡ Dan Peters,‡ Claus Mathiesen,‡ Anders S. Kristensen,§ and
Ulf Madsen*,†
Department of Medicinal Chemistry and Department of Pharmacology, The Danish University of Pharmaceutical Sciences,
2 Universitetsparken, DK-2100 Copenhagen, Denmark, and NeuroSearch A/S, 93 Pederstrupvej, DK-2750 Ballerup, Denmark
Received December 23, 2003
2-Arylureidobenzoic acids (AUBAs) have recently been presented as the first series of selective
noncompetitive GluR5 antagonists. In this paper we have modified the acidic moiety of the
AUBAs by introducing different acidic and neutral groups, and similarly, we have replaced
the urea linker of the AUBAs with other structurally related linkers. Replacing the acid with
neutral substituents led to inactive compounds in all instances, showing that an acidic moiety
is necessary for activity. Replacing the carboxylic moiety in 2a with a sulfonic acid (5c) or a
tetrazole ring (5d) improved the potency at GluR5 receptors (compounds 5c and 5d showed
IC50 values of 1.5 and 2.0 µM, respectively, compared to compound 2a with IC50 ) 4.8 µM).
Compound 5c did not show improved in vivo activity in the ATPA rigidity test compared to
2a, whereas compound 5d was 4 times more potent than 2a. All compounds wherein the urea
linker had been replaced showed lower or no activity. The results described extend the
knowledge of structure-activity relationships for the AUBAs, and compound 5d may prove to
be a good candidate for studying GluR5 receptors in vitro and in vivo.
Introduction
(S)-Glutamic acid (Glu) is the main excitatory neuro-
transmitter in the central nervous system (CNS). The
effects of Glu and other excitatory amino acids (EAAs)
are mediated by three heterogeneous classes of iono-
tropic receptors (iGluRs) (N-methyl-D-aspartic acid
(NMDA), 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-
propionic acid (AMPA), and kainic acid (KA) receptors)
and by three heterogeneous classes of metabotropic
receptors (mGluRs) (namely, groups I, II, and III).1 The
iGluRs are composed of different subunits that assemble
into tetrameric structures that form functional cation
channels. Seven NMDA subunits (NR1, NR2A-D, and
NR3A, NR3B), four AMPA subunits (GluR1-4), and five
KA subunits (GluR5-7, KA1, and KA2) have been
cloned and characterized.2
All three classes of iGluRs have been implicated in
different diseases, and although NMDA and AMPA
selective ligands have held much of the research focus,
KA selective ligands and in particular ligands for the
GluR5 subunit have received increasing attention fol-
lowing the cloning and ongoing physiological charac-
terization of GluR5 and other KA receptors.1,3,4 GluR5
selective antagonists have been proposed to be poten-
tially useful in the treatment of various diseases such
as ischemic conditions,5,6 pain,7 epilepsy,8 and mi-
Figure 1. Structures of the noncompetitive GluR5 selective
antagonists 1, 2a, and 2b and generalized structures of the
modified AUBAs presented in this paper. See Tables 1 and 2
for lists of substituents.
graine.9 A particular advantage of GluR5 selective
antagonists is that blocking this subunit has so far not
been associated with serious side effects, whereas non-
subunit selective AMPA and NMDA antagonists have
been found to induce, for example, ataxia and psychoto-
mimetic effects, respectively.1
We have previously published a series of 2-aryl-
ureidobenzoic acids (AUBAs) that showed GluR5 an-
tagonistic activity in vitro and in vivo.10 Therein, we
investigated the structure-activity relationship (SAR)
around the two aromatic rings in the AUBAs and
* To whom correspondence should be addressed. Phone: (+45)
35306243. Fax: (+45) 35306040. E-mail: um@dfuni.dk.
† Department of Medicinal Chemistry, The Danish University of
Pharmaceutical Sciences.
# Present address: Actavis, Reykjavikurvegi 76-78, 220 Hafnarfirdi,
Iceland.
‡ Department of Pharmacology, The Danish University of Pharma-
ceutical Sciences.
§ NeuroSearch A/S.
10.1021/jm030638w CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/02/2004

Modifications of 2-Arylureidobenzoic Acids
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6949
Scheme 1a
a Reagents and conditions: (a) NIS, AcOH, room temp; (b) NH2OH‚HCl, NaHCO3, EtOH, reflux; (c) NaOH, H2O, reflux; (d) methyl
acrylate, Pd/C, K2CO3, DME, 100 °C; (e) H2SO4 (conc), heat; (f) NaN3, Et3N‚HCl, toluene; (g) NaOEt, (EtO)2CO, EtOH, reflux; (h)
3-bromophenyl isocyanate or 3-trifluoromethylphenyl isocyanate, THF, room temp; (i) H2, Raney Ni, EtOH; (j) H2, Pd/C, EtOH; (k) NaOH,
EtOH, room temp. See Table 1 for a list of substituents.
optimized these for in vitro GluR5 potency. However,
the compound that was most potent in vitro (1, see
Figure 1) turned out to be inactive in vivo (ED50 > 30
mg/kg in the ATPA-induced rigidity test), whereas
compound 2a, which was 5 times less potent in vitro
compared to 1, was active in vivo (ED50 ) 4 and 24 mg/
kg after 5 and 30 min, respectively). This exemplifies
the difficulties in correlating in vitro with in vivo
activities and calls for further optimization and atten-
tion to the pharmacokinetic properties of the com-
pounds. Here, we present a new series of AUBA
derivatives where the acid moiety on ring A (see Figure
1) has been replaced by other acidic and neutral groups,
thus exploring the pharmacological effects of using
moieties other than benzoic acid, which is prone to
conjugation and other metabolic processes. Compounds
5a, 5c-l, and 5n are analogues of the two equipotent
analogues from the first series of AUBAs, 2a or 2b.
Furthermore, we have exchanged the urea moiety,
which links the two aromatic ring systems of the
AUBAs, for other linkers of similar size and structure
to yield compounds 7, 8, 10, 13, and 15. These modifica-
tions of the acid and urea moieties, combined with the
observations around the two aryl rings from our previ-
ous paper,10 constitute a more comprehensive SAR for
the AUBAs as GluR5 antagonists.
Results
Chemistry. The compounds with modified acid moi-
eties (5a-k) were prepared by condensing the ap-
propriately substituted anilines (4a-j) with 3-bromo-
phenyl isocyanate or 3-trifluoromethylphenyl isocyanate
(see Scheme 1). Attempts to hydrogenate 5k using Pd/C
as catalyst gave a complex mixture of compounds, but
exchanging Pd/C with Raney Ni yielded 1-(2-amino-5-

6950 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Valgeirsson et al.
Scheme 2a
a Reagents and conditions: (a) 3-bromophenyl isothiocyanate, Et3N, THF; (b) 3-bromobenzoyl chloride, pyridine, room temp; (c)
3-bromoaniline, THF, room temp; (d) 3-bromoaniline, EtOH, room temp; (e) 2-amino-4-chlorobenzoic acid, Et3N, MeCN, reflux; (f) (1)
NaNO2, HCl, H2O, 0 °C; (2) NaSO3, H2O, room temp; (3) HCl, room temp; (g) 3-bromophenyl isocyanate, pyridine, room temp.
chlorophenyl)-3-(3-trifluoromethylphenyl)urea (5l) as
the only product in a good yield. Phenylpropionic acid
5n was prepared from the acrylic acid ester 5b in two
steps. Catalytic hydrogenation of 5b using Pd/C yielded
5m, which was then hydrolyzed with NaOH to yield 5n.
The anilines 4f-j were commercially available, and
anilines 4a-e were prepared as follows. (2-Amino-4-
chlorophenyl)acetic acid (4a) was prepared from 6-
chlorooxindole (3a) by reflux in aqueous NaOH and
precipitation of 4a as a zwitterion. 3-(2-Amino-4-
chlorophenyl)acrylic acid methyl ester (4b) was pre-
pared in two steps. 5-Chloroaniline (3c) was treated
with N-iodosuccinimide (NIS) to yield 5-chloro-2-iodo-
aniline (3b), which was subsequently cross-coupled with
acrylic acid methyl ester to yield 4b, using Pd/C as a
catalyst.11 Attempts to couple 3b with propynoic acid
methyl ester under modified12 Sonogashira conditions
failed. 2-Amino-4-chlorobenzenesulfonic acid (4c) was
prepared from 3-bromoaniline (3c) by modification of
the sulfonation procedure of Allen et al.13 Tetrazole-
substituted aniline 4d was prepared from 2-amino-4-
chlorobenzonitrile (3d) as described by Koguro et al.14
Oxadiazolone 4e was prepared in two steps. Nucleo-
philic addition of hydroxylamine to 2-amino-4-chloro-
benzonitrile (3d) gave 2-amino-4-chloro-N-hydroxy-
benzamidine (3e),15 which was treated with diethyl
carbonate under basic conditions to form the 4H-[1,2,4]-
oxadiazol-5-one ring of 4e.16
because the reaction conditions that were necessary to
make the salicylic acid react (1 equiv of Et3N in THF
at 50 °C) caused immediate and quantitative cycliza-
tion of the desired product into 3-(3-bromophenyl)-7-
chlorobenzo[e][1,3]oxazine-2,4-dione. This is analogous
to the results obtained previously when applying these
conditions to similar compounds.10 However, 10 could
be prepared from 4-chlorosalicylic acid in two steps
through 7-chlorobenzo[1,3]dioxine-2,4-dione (9), which
reacts with 3-bromoaniline at the carbamate carbonyl
as reported in the literature for similar compounds.17
Compound 13 was prepared by reacting 3,4-diethoxy-
cyclobut-3-ene-1,2-dione (11) with 3-bromoaniline and
aniline 6 successively. The ring-fused analogue 15 was
synthesized by condensing 3-bromophenyl isocyanate
with 6-chloro-1H-indazol-3-ol 14, which was synthesized
by reacting anthranilic acid 6 with NaNO2.
In Vitro Pharmacology. The antagonistic effects of
the compounds on different recombinant homomeric rat
AMPA (GluR1-4) and human KA receptors (GluR5-
6), stably expressed in HEK293 cells, were evaluated
by functional assays using the FLIPR (fluorescent
imaging plate reader) technology. The antagonistic
activity of the test compounds was quantified as the
inhibition of the increase in intracellular Ca2+ concen-
tration, measured by light emission from a Ca2+ sensi-
tive fluorophore, following the addition of a known
agonist (Glu for GluR1-4 and domoic acid for GluR5,6)
to cells expressing the appropriate receptor.
Thiourea 7 and amide 8 were prepared from aniline
6 by reaction with 3-bromophenyl isothiocyanate and
3-bromobenzoyl chloride, respectively (Scheme 2). At-
tempts to synthesize carbamate 10 from 4-chlorosalicylic
acid and 3-bromophenyl isocyanate were unsuccessful
All the compounds were screened for activity at GluR5
(see IC50 values in Tables 1 and 2). None of the neutral
compounds 5f-l, bearing CO2Me, OH, CH2OH, CN,
NO2, or NH2 instead of the carboxylic acid group of 2a

Modifications of 2-Arylureidobenzoic Acids
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6951
Table 1. Antagonist Effects of Compounds with Different
Acidic or Neutral Substitutions for the Carboxylic Acid Group
of Compounds 2a and 2ba
Table 2. GluR5 Antagonist Effects, Measured by FLIPR Assay
on HEK293 Cells, of Compounds with Different Linker
Moietiesa
a IC50 values were determined with a FLIPR assay using
HEK293 cells expressing GluR5. Results are the mean ( SEM
for at least three separate experiments.
terized in functional assays (FLIPR) using GluR1-4 and
GluR6 and in binding studies using recombinant re-
ceptors and the GluR5-selective agonist [3H]-(RS)-2-
amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid
([3H]ATPA) (see Table 3). None of the compounds
showed any GluR1-4 antagonistic activity at 30 µM and
only weak antagonism (less than 50%) at 100 µM. The
potency at GluR6 could only be determined for 5c, for
which the GluR6 potency was about 25 times lower than
at the GluR5. The other compounds were not sufficiently
soluble to enable accurate determination of their IC50
for GluR6 (see Table 3). No significant inhibition of
[3H]ATPA binding to GluR5 receptors was observed at
the concentrations tested (3-30 µM).
In Vivo Pharmacology. The in vivo activities of
compounds 5a and 5c-e were evaluated in the ATPA-
induced rigidity test in mice. The test compounds were
administered iv 30 min prior to the administration of
the GluR5 agonist ATPA, and the degree of rigidity
induced by ATPA was observed. The benzoic acid 2a has
previously been reported to have an ED50 of 24 mg/kg
in this model.10 The sulfonic acid 5c had a potency
similar to 2a (ED50 ) 29 mg/kg), whereas the tetrazole
5d was notably more potent, having an ED50 of 5.5 mg/
kg. Phenylacetic acid 5a and oxadiazolone 5e were
inactive at the doses tested.
a IC50 values were determined with a FLIPR assay using
HEK293 cells expressing GluR5. Results are the mean ( SEM
for at least three separate experiments.
and 2b, showed any activity at the concentrations
tested. The concentrations tested of the neutral com-
pounds were limited by their low aqueous solubility
because the lack of an acidic moiety lowered the solubil-
ity markedly. All but one of the acidic compounds were
active at the GluR5. The tetrazole 5d was approximately
twice as potent as 2a, and the oxadiazolone 5e was
equipotent with 2a. The phenylacetic acid 5a was also
equipotent with 2a, but the phenylpropanoic acid ana-
logue 5n was inactive. The sulfonic acid analogue 5c
was about 3 times more potent than 2a.
Of the compounds with modified urea moieties, only
the thiourea 7 and amide 8 were active, although 2 and
4 times less potent than 2a, respectively. The carbamate
10, the dione analogue 13, and the ring fused analogue
15 were all inactive.
Discussion
Acid and urea modified AUBA analogues were de-
signed and synthesized in a concise manner. A series
of 12 compounds (5a, 5c-l, and 5n), wherein the acid
moiety of 2a or 2b is replaced by other acidic or neutral
groups, have been synthesized. Care was taken to
introduce diverse acidic functionalities, e.g., homologues
The compounds that were equally or more potent than
2a in the GluR5 assay (5a, 5c-e) were further charac-

6952 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Valgeirsson et al.
Table 3. Potencies of Selected Compounds in Functional Assays for GluR1-6 (FLIPR Assays and Electrophysiology on GluR5),a
[3H]ATPA Binding (All as IC50 in µM),a and ATPA Rigidity (as ED50 in mg/kg)
a Results are the mean ( SEM for at least three separate experiments.
(5a and 5n), heterocycles (5d and 5e), and a sulfonic
acid (5c). Furthermore, mesoionic (5j and 5k) and other
neutral compounds (5f-i and 5l) were prepared. An
additional five compounds where the urea moiety of 2a
was modified were synthesized.
it is a hydrogen bond acceptor but does not have a net
charge. However, the nitro compounds 5j and 5k were
not active at the concentrations tested (3 µM). It also
became apparent that the presence of acidic function-
alities was necessary for adequate aqueous solubility;
compounds 5f-l were only soluble in low concentrations
in the assay buffers.
Most of the compounds in which the urea moiety was
modified were inactive. However, limited activity was
retained for thiourea 7 and amide 8, which were about
2 and 3 times less potent than 2a, respectively. These
results indicate that although urea is the best linker
tested, it is possible to modify the linker without causing
complete loss of activity. The ring-fused analogue 15 was
designed on the basis of the observation that an intra-
molecular hydrogen bond between the proximal -NH
of the urea and the carboxylate anion seems very
favorable, and therefore, the carboxylic acid and the
nitrogen can be envisioned to form a six-membered ring.
Compound 15 forms an analogous five-membered 3-hy-
droxypyrazole ring, which restricts the conformational
freedom of the acidic functionality. Unfortunately 15
was completely inactive at GluR5, indicating that this
particular restriction is not suitable.
The noncompetitive activity observed for the previous
series of AUBAs10 was confirmed for the present com-
pounds by lack of inhibition of [3H]ATPA binding to
GluR5 (Table 3). The in vivo effects of the acidic bio-
isosteres 5a,c-e were measured using ATPA-induced
rigidity. Sulfonic acid 5c was about equipotent with the
carboxylic acid 2a. However, the tetrazole 5d gave 4-fold
higher in vivo activity compared to 2a. Part of this can
be attributed to the higher in vitro activity of 5d (2.0
versus 4.8 µM, respectively), but it should also be
considered that tetrazoles are known to be more meta-
bolically stable than carboxylic acids and therefore more
likely to be active in vivo.18 Furthermore, the more
lipophilic tetrazole may improve penetration of the
blood-brain barrier compared to the carboxylic acid
group. Surprisingly, the phenylacetic acid 5a and the
oxadiazolone 5e were devoid of in vivo activity (tested
As demonstrated by the activities of compounds 2a
and 2b (Table 1), it is not crucial for GluR5 activity
whether ring B of the AUBAs bears a 3-bromo or
3-trifluoromethyl substitutent (IC50 values of 4.8 and
5.0, respectively). We have included seven compounds
bearing neutral moieties instead of the carboxylic acid
of 2a and 2b, and it can be concluded that an acidic
functionality is needed for GluR5 activity (Table 1).
All the acidic analogues, except the two-carbon ho-
mologue 5n, showed GluR5 activity comparable to that
of 2a. The one-carbon homologue 5a was about equi-
potent with 2a (3.6 versus 4.8 µM, respectively) and
more selective than 2a for GluR5 over GluR6. The two-
carbon homologue 5n was found to be inactive at the
concentration tested (30 µM), suggesting some specific
limitations with regard to the binding site of the acidic
moiety. The sulfonic acid 5c was approximately 3 times
more potent than 2a, further establishing that ionic
interactions are needed for GluR5 activity. The oxa-
diazolone 5e was about equipotent with 2a, but the
tetrazole 5d was more than twice as potent as 2a. The
higher affinity of the tetrazole might be attributed to
the increased lipophilicity of the tetrazolyl anion com-
pared to the carboxylate anion because of the delocal-
ization opportunities in the former and because of the
possibilities of making multiple hydrogen bonds to the
receptor through the ring nitrogens.18
Thus, the receptor can accommodate acidic groups
that are considerably larger than the carboxylate.
Because the presence of ionized groups enhances the
solvation of ligands in the aqueous environment outside
the receptor (makes the free energy of solvation more
negative) and thereby inherently decreases the affinity
of the compounds, we investigated nonionized alterna-
tives to replace the carboxylate. The mesoionic nitro
group was our prime candidate in this regard because

Modifications of 2-Arylureidobenzoic Acids
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6953
in THF and DMSO but 24 h when run in toluene). If both
starting materials were detected after 24 h, then the reaction
mixture was refluxed until the reaction was complete. The
solution was cooled, and the product precipitated with heptane
when necessary. The product was then filtered off, washed,
and recrystallized as described below for each compound.
5-Chloro-2-iodoaniline (3b). 3-Chloroaniline (3c) (30.0 g,
236 mmol) and N-iodosuccinimide (NIS) (55.8 g, 248 mmol)
were dissolved in glacial acetic acid (250 mL) and stirred for
12 h. The mixture was evaporated down to 50 mL, and succinic
anhydride was precipitated from the solution by addition of
EtOAc and heptane and filtered off. The mother liquor was
washed with 1 M NaOH, dried with Na2SO4, and evaporated.
The product was recrystallized from Et2O/heptane to yield
faintly yellow crystalline 3b (42 g, 70%). Mp: 64-66 °C (lit.
40-42 and 75-77 °C).20,21 The 1H NMR spectrum was in
agreement with the literature.21 Anal. (C6H5ClIN) C, H, N.
2-Amino-4-chloro-N-hydroxybenzamidine (3e). 2-Amino-
4-chlorobenzonitrile (3d) (5.3 g, 35 mmol) was dissolved in
EtOH (180 mL) by heating. An aqueous solution of hydroxyl-
amine hydrochloride (4.9 g, 70 mmol) and sodium bicarbonate
(11.1 g, 105 mmol) was added, and the mixture was refluxed
for 8 h. The solvent was evaporated and the product was
crystallized by suspension in water (100 mL) to yield colorless
crystalline 3e (5.6 g, 86%). Mp: 131-133 °C (lit. 127-128 °C).22
1H NMR (DMSO-d6) δ 9.66 (1H, s), 7.36 (1H, d, J ) 8.5 Hz),
6.70 (1H, s), 6.55 (1H, br s), 6.52 (1H, d, J ) 8.5 Hz), 5.78 (2H,
br s). Anal. (C7H8ClN3O) C, H, N.
3-(2-Amino-4-chlorophenyl)acrylic Acid Methyl Ester
(4b). Iodoaniline 3b (1.25 g, 5.0 mmol), methyl acrylate (750
mg, 7.50 mmol), K2CO3 (1.05 g, 7.50 mmol), and Pd/C (630 mg,
0.30 mmol) were dissolved in DME (20 mL) and stirred at 100
°C for 48 h. The mixture was diluted with EtOAc and filtered
through Celite. The solvent was evaporated and the product
was recrystallized from EtOAc/heptane to yield colorless
crystalline 4b (0.95 g, 90%). Mp: 115-121 °C (lit. 124-125
°C).23 The 1H NMR spectrum was in agreement with the
literature.23 Anal. (C10H10ClNO2) C, H. N: calcd, 6.62; found,
6.01.
2-Amino-4-chlorobenzenesulfonic Acid (4c). Concen-
trated sulfuric acid (specific gravity 1.84) (13.9 mL, 250 mmol)
was diluted with 100 mL of water, and 3-chloro-aniline (3c)
(31.9 g, 250 mmol) was added over 30 min while the temper-
ature was kept at 85 °C. During the addition some precipita-
tion occurred. The suspension was allowed to cool to room
temperature, whereupon heavy precipitation occurred. The
suspension was filtered to yield a colorless crystalline solid
that was heated slowly to 200 °C under vacuum (water
aspirator) and heated for 7 h. Water was lost from the
crystalline material. The solid was allowed to cool and then
dissolved in dilute NaOH. The product was precipitated by
addition of 4 M HCl, filtered off, and dried to yield colorless
crystalline 4c (25 g, 48%). Mp: >250 °C (lit. >310 °C).24 1H
NMR (DMSO-d6) δ 7.48 (1H, d, J ) 8.5 Hz), 6.83 (1H, s), 6.71
(1H, d, J ) 8.5 Hz). Anal. (C6H6ClNO3S) C, H, N.
Figure 2. Comparison of the dose-response curves, obtained
in the in vivo ATPA-induced rigidity test, for carboxylic acid
2a (ED50 ) 24 mg/kg) and tetrazole 5d (ED50 ) 5.5 mg/kg).
up to 30 and 100 mg/kg, respectively), although they
were equipotent with 2a in vitro. This was especially
disappointing in the case of the oxadiazolone 5e because
this heterocycle has previously been used as a lipophilic
carboxylic bioisostere with good results, for example, in
ACE inhibitors.19
Conclusion
The acid and urea moieties of AUBAs, which are
GluR5 selective antagonists, have been modified in
order to elucidate the SAR of these compounds and as
an attempt to improve their in vitro activity. The
importance of the acidic moiety has been established,
as all the neutral compounds were inactive, and the
structural requirements of the receptor with respect to
the acidic group do not seem to be very stringent, as all
but one of the acidic compounds retained or were
increased in activity compared to the carboxylic acid 2a.
Similarly, the urea linker can be modified without
abolishing all activity, but the urea moiety still yields
the most active compounds. When considered together
with our previous publication,10 SAR information has
been gathered for all the components of the AUBAs, that
is, both aromatic rings A and B, the urea linker, and
the acid moiety.
The tetrazolyl group (analogue 5d) improved the in
vivo potency significantly compared to the previous
carboxylic acid derivatives (Table 1 and Figure 2). This
makes 5d a more suitable candidate for in vivo char-
acterization of GluR5 pharmacology than 2a, 2b, or 1.
3-(2-Amino-4-chlorophenyl)-4H-[1,2,4]oxadiazol-5-
one (4e). Sodium metal (0.92 g, 40 mmol) was dissolved in 70
mL of EtOH, and 3e (3.71 g, 20 mmol) was added, followed by
diethyl carbonate (9.44 g, 80 mmol). The mixture was refluxed
for 18 h and then reduced to dryness under vacuum. The
residue was triturated with 0.5 M HCl, and the product was
filtered off. The product was then triturated with toluene and
filtered off to yield red-brown crystalline 4a (3.4 g, 80%). Mp:
Experimental Section
Chemistry. All reagents are commercially available unless
stated otherwise. All reactions were carried out under nitrogen
atmosphere. Melting points were measured on a capillary
melting point apparatus and are uncorrected. 1H NMR spectra
were recorded on a Varian 300 MHz spectrometer. Residual
solvent peaks were used as an internal reference in the NMR
spectra. Elemental analyses were within (0.4% of the theo-
retical value unless stated otherwise.
General Procedure A: Synthesis of AUBAs 5a-k. The
appropriate isocyanate (2.1 mmol) and the appropriate aniline
(2.0 mmol) were dissolved/suspended in dry THF (10 mL),
toluene (100 mL), or DMSO (10 mL), and the mixture was
stirred at room temperature until one or both starting materi-
als could not be detected by TLC (typically 2-3 h when run
1
159-161 °C. H NMR (DMSO-d6) δ 7.52 (1H, d, J ) 8.5 Hz),
6.94 (1H, s), 6.68 (1H, d, J ) 8.5 Hz). Anal. (C7H8ClN3O‚H2O)
C, H, N.
{2-[3-(3-Bromophenyl)ureido]-4-chlorophenyl}acetic
Acid (5a). 6-Chlorooxindole (3a) (2.50 g, 14.9 mmol) was
suspended in 4 M aqueous NaOH (30 mL), the mixture was
refluxed for 6 h and allowed to cool, and the product was
filtered off as the sodium salt. The solid was dissolved in water
(100 mL) and precipitated as an HCl salt by addition of 4 M
HCl at 0 °C. Crude 4a was filtered off and dried under vacuum
1
to yield colorless crystals (2.0 g). Mp: 251-254 °C (dec). H

6954 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Valgeirsson et al.
NMR (D2O) δ 10.00 (1H, s), 8.89 (1H, s), 8.36 (1H, d, J ) 8.5
Hz), 8.29 (1H, t, J ) 8.0 Hz), 8.21 (1H, d, J ) 8.0 Hz), 3.55
(2H, s). Crude 4a (700 mg, 3.8 mmol) was dissolved in pyridine
(25 mL), 1-bromophenyl 3-isocyanate (811 mg, 4.1 mmol) was
added dropwise over 10 min, and the mixture was stirred for
90 min. The mixture was diluted with 150 mL of EtOAc and
washed twice with 1 M HCl. The volume of the organic phase
was reduced under vacuum and the product was recrystallized
from EtOAc/heptane to yield colorless crystalline 5a (680 mg,
and the suspension was stirred at 100 °C for 1 h. When the
mixture was cooled, the product precipitated and was filtered
off, dried, and recrystallized (acetone/water) to yield yellow
crystalline 5i (795 mg, 76%). Mp: 221-222 °C. 1H NMR
(DMSO-d6) δ 9.72 (1H, s), 8.97 (1H, s), 8.26 (1H, s), 7.89 (1H,
s), 7.84 (1H, d, J ) 8.5 Hz), 7.30 (3H, m), 7.24 (1H, t, J ) 7.5
Hz). Anal. (C14H9BrClN3O) C, H, N.
1-(3-Bromophenyl)-3-(5-chloro-2-nitrophenyl)urea (5j).
5-Chloro-2-nitroaniline (4j) (518 mg, 3.00 mmol), 3-bromo-
phenyl isocyanate (693 mg, 3.00 mmol), and p-toluenesulfonic
acid (52 mg, 0.30 mmol) were suspended in toluene, and the
suspension was stirred at 100 °C for 1 h. When the mixture
was cooled, the product precipitated and was filtered off, dried,
and recrystallized (acetone/water) to yield yellow crystalline
5j (830 mg, 79%). Mp: 199-200 °C. 1H NMR (DMSO-d6) δ
10.23 (1H, s), 9.81 (1H, s), 8.50 (1H, s), 8.18 (1H, d, J ) 9.0
Hz), 7.88 (1H, s), 7.36 (1H, d, J ) 8.0 Hz), 7.30 (2H, m), 7.24
(1H, d, J ) 8.0 Hz). Anal. (C13H9BrClN3O3) C, H, N.
1-(5-Chloro-2-nitrophenyl)-3-(3-trifluoromethylphenyl)-
urea (5k). 5-Chloro-2-nitroaniline (4j) (1036 mg, 6.00 mmol),
3-trifluoromethylphenyl isocyanate (1.12 g, 6.00 mmol), and
p-toluenesulfonic acid (52 mg, 0.30 mmol) were suspended in
toluene, and the suspension was stirred at 100 °C for 1 h.
When the mixture was cooled, the product precipitated and
was filtered off, dried, and recrystallized (acetone/water) to
yield yellow crystalline 5k (2.16 g, 94%). Mp: 196-197 °C.
1H NMR (DMSO-d6) δ 10.39 (1H, s), 9.83 (1H, s), 8.50 (1H, s),
8.17 (1H, d, J ) 8.5 Hz), 8.00 (1H, s), 7.64 (1H, d, J ) 7.5 Hz),
7.56 (1H, t, J ) 7.5 Hz), 7.39 (1H, d, J ) 7.5 Hz), 7.29 (1H, d,
J ) 8.5 Hz). Anal. (C14H9ClF3N3O3) C, H, N.
1-(2-Amino-5-chlorophenyl)-3-(3-trifluoromethyl-
phenyl)urea (5l). 5k (1.00 g, 2.78 mmol) was dissolved in
EtOH (50 mL) and treated with Raney Ni and H2 (1 atm)
under vigorous stirring for 24 h. The mixture was filtered
through Celite and concentrated under vacuum. The crude
product was recrystallized from EtOAc/heptane to yield red
crystalline 5l (0.8 g, 87%). Mp 181-182 °C. 1H NMR (DMSO-
d6) δ 9.27 (1H, s), 8.01 (1H, s), 7.97 (1H, s), 7.52 (3H, m), 7.30
(1H, d, J ) 7.5 Hz), 6.87 (1H, d, J ) 8.5 Hz), 6.74 (1H, d, J )
8.5 Hz), 4.97 (2H, br s). Anal. (C14H11ClF3N3O) C, H, N.
1
47%). Mp: 195-196 °C. H NMR (DMSO-d6) δ 12.57 (1H, s),
9.41 (1H, s), 8.20 (1H, s), 7.93 (1H, d, J ) 2 Hz), 7.89 (1H, t,
J ) 2 Hz), 7.26 (1H, m), 7.16 (1H, dt, J ) 7.5 Hz, 2 Hz), 7.08
(1H, dd, J ) 8.5 Hz, 2 Hz), 3.64 (2H, s). Anal. (C15H12BrClN2O3‚
1/3H2O) C, H, N.
3-{2-[3-(3-Bromophenyl)ureido]-4-chlorophenyl}-
acrylic Acid Methyl Ester (5b). 5b was prepared according
to general procedure A to yield a colorless crystalline solid.
Yield: 93%. Mp: 186-190 °C. 1H NMR (DMSO-d6) δ 9.23 (1H,
s), 9.06 (1H, s), 7.84 (4H, m), 7.33 (2H, m), 7.23 (1H, t, J ) 8.0
Hz), 7.15 (1H, d, J ) 8.0 Hz), 6.58 (1H, d, J ) 15.5 Hz), 3.72
(3H, s). Anal. (C17H14BrClN2O3) C, H, N. C: calcd, 49.84; found,
50.35. N: calcd, 6.84; found, 6.27.
2-[3-(3-Bromophenyl)ureido]-4-chlorobenzenesulfon-
ic Acid, Triethylammonium Salt (5c). 5c was prepared
according to a modified version of general procedure A (1 equiv
of Et3N was added to the reaction mixture along with the
starting materials) to yield a colorless crystalline solid. Yield:
1
70%. Mp: 178-179 °C. H NMR (DMSO-d6) δ 10.00 (1H, s),
9.49 (1H, s), 8.85 (1H, br s), 8.13 (1H, s), 7.91 (1H, s), 7.66
(1H, d, J ) 8.5 Hz), 7.47 (1H, d, J ) 8.0 Hz), 7.23 (1H, t, J )
8.0 Hz), 7.14 (1H, d, J ) 8.0 Hz), 7.02 (1H, d, J ) 8.5 Hz),
3.09 (6H, q, J ) 7.5 Hz), 1.16 (9H, t, J ) 7.5 Hz). Anal. (C13H10-
BrClN2O4S‚C6H15N) C, H, N.
1-(3-Bromophenyl)-3-[5-chloro-2-(1H-tetrazol-5-yl-phen-
yl]urea (5d). 5d was prepared from 5-chloro-2-(1H-tetrazol-
5-yl)phenylamine14 (4d) and 3-bromophenyl isocyanate by
general procedure A to yield a colorless crystalline solid.
Yield: 78%. Mp: 197-199 °C. 1H NMR (DMSO-d6) δ 10.08
(1H, s), 9.97 (1H, s), 8.42 (1H, s), 7.91 (2H, m), 7.42 (1H, d, J
) 8.0 Hz), 7.33 (1H, d, J ) 8.0 Hz), 7.26 (1H, t, J ) 8.0 Hz),
7.19 (1H, d, J ) 8.0 Hz). Anal. (C14H10BrClN6O) C, H, N.
1-[5-Chloro-2-(5-oxo-4,5-dihydro-[1,2,4]oxadiazol-3-yl)-
phenyl]-3-(3-bromophenyl)urea (5e). 5e was prepared from
4e according to general procedure A to yield a colorless
crystalline solid. Yield: 63%. Mp: 246-248 °C (decp). 1H NMR
(DMSO-d6) δ 13.07 (1H, s), 9.97 (1H, s), 9.08 (1H, s), 8.32 (1H,
s), 7.67 (1H, d, J ) 8.5 Hz), 7.37 (1H, d, J ) 8.0 Hz), 7.32 (1H,
d, J ) 8.5 Hz), 7.27 (1H, t, J ) 8.0 Hz), 7.20 (1H, d, J ) 8.0
Hz). Anal. (C15H10BrClN4O3‚11/4H2O) C, H, N.
3-{2-[3-(3-Bromophenyl)ureido]-4-chlorophenyl}-
propionic Acid Methyl Ester (5m). 5b (600 mg, 1.46 mmol)
and Pd/C (60 mg) were dissolved in EtOH (30 mL) and
subjected to H2 (3 atm) with vigorous shaking for 24 h. The
solution was filtered through Celite, and the product was
precipitated by addition of water, filtered off, dried, and
recrystallized from EtOAc/heptane to yield colorless crystalline
1
5m (435 mg, 73%). Mp: 189-190 °C. H NMR (DMSO-d6) δ
8.80 (1H, s), 8.73 (1H, s), 7.70 (1H, s), 7.44 (1H, d, J ) 8.5
Hz), 7.25 (4H, m), 6.97 (1H, t, J ) 7.5 Hz), 3.60 (3H, s), 2.89
(2H, t, J ) 7.5 Hz), 2.60 (2H, t, J ) 7.5 Hz). Anal. (C17H16-
BrClN2O3) C, H. N: calcd, 6.80; found, 6.34.
2-[3-(3-Bromophenyl)ureido]-4-chlorobenzoic Acid
Methyl Ester (5f). 5f was prepared according to general
procedure A to yield a colorless crystalline solid. Yield: 88%.
1
Mp: 164-166 °C. H NMR (DMSO-d6) δ 10.22 (1H, s), 10.17
3-{2-[3-(3-Bromophenyl)ureido]-4-chlorophenyl}-
propionic Acid (5n). 5m (300 mg, 0.73 mmol) was dissolved
in 40 mL of EtOH, NaOH (4 mL, 12.5 M, 50 mmol) was added
dropwise, and the mixture was stirred under N2 for 6 h. The
mixture was neutralized with 4 M HCl, and the volume was
reduced to 50 mL. The product was precipitated by addition
of water and recrystallized from EtOAc/heptane to yield
(1H, s), 8.50 (1H, s), 7.92 (1H, d, J ) 8.5 Hz), 7.88 (1H, s),
7.40 (1H, d, J ) 8.5 Hz), 7.25 (1H, t, J ) 8.0 Hz), 7.17 (1H, d,
J ) 8.0 Hz), 7.11 (1H, d, J ) 8.5 Hz), 3.88 (3H, s). Anal. (C15H12-
BrClN2O3) C, H, N.
1-(5-Chloro-2-hydroxyphenyl)-3-(3-trifluoromethyl-
phenyl)urea (5g). 5g was prepared according to general
procedure A to yield a colorless crystalline solid. Yield: 96%.
1
1
colorless crystalline 5n (220 mg, 76%). Mp: 193-196 °C. H
Mp: 177-178 °C. H NMR (DMSO-d6) δ 10.36 (1H, s), 9.75
NMR (DMSO-d6) δ 12.23 (1H, s), 8.79 (1H, s), 8.71 (1H, s),
7.69 (1H, s), 7.45 (1H, s), 7.43 (1H, s), 7.25 (3H, m), 6.97 (t, J
) 7.5 Hz), 2.85 (2H, t, J ) 8.0 Hz), 2.55 (2H, t. J ) 8.0 Hz).
Anal. (C16H14BrClN2O3) C, H, N.
(1H, s), 8.39 (1H, s), 8.16 (1H, s), 8.03 (1H, s), 7.51 (2H, s),
7.32 (1H, s), 6.85 (2H, s). Anal. (C14H10ClF3N2O2) C, H, N.
1-(5-Chloro-2-hydroxymethylphenyl)-3-(3-trifluoro-
methylphenyl)urea (5h). 5h was prepared according to
general procedure A to yield a colorless crystalline solid.
Yield: 74%. Mp: 217-218 °C. 1H NMR (DMSO-d6) δ 9.71 (1H,
s), 8.30 (1H, s), 8.01 (1H, s), 7.88 (1H, d, J ) 8.5 Hz), 7.59
(1H, d, J ) 8.5 Hz), 7.51 (1H, t, J ) 8.5 Hz), 7.38 (1H, s), 7.28
(2H, m), 4.52 (2H, s). Anal. (C15H12ClF3N2O2) C, H, N.
1-(3-Bromophenyl)-3-(5-chloro-2-cyanophenyl)urea (5i).
2-Amino-4-chlorobenzonitrile (4i) (450 mg, 2.95 mmol), 3-bro-
mophenyl isocyanate (594 mg, 3.00 mmol), and p-toluene-
sulfonic acid (52 mg, 0.30 mmol) were suspended in toluene,
2-[3-(3-Bromophenyl)thioureido]-4-chlorobenzoic Acid
(7). 2-Amino-4-chlorobenzoic acid (6) (600 mg, 3.48 mmol),
3-bromophenyl isothiocyanate (800 mg, 3.73 mmol), and Et3N
(380 mg, 3.73 mmol) were dissolved in THF (10 mL), and the
solution was stirred at room temperature for 6 h. The product
was precipitated by adding heptane, filtered off, and recrystal-
lized (acetone/water) to yield colorless crystalline 7 (1150 mg,
1
86%). Mp: 164-167 °C. H NMR (DMSO-d6) δ 13.70 (1H, s),
11.00 (1H, s), 10.77 (1H, s), 8.61 (1H, s), 7.91 (1H, d, J ) 8.5

Modifications of 2-Arylureidobenzoic Acids
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6955
Hz), 7.79 (1H, s), 7.39 (3H, m), 7.24 (1H, d, J ) 8.5 Hz). Anal.
6-Chloro-3-hydroxyindazole-1-carboxylic Acid (3-Bro-
mophenyl)amide (15). Indazolol 14 (700 mg, 4.15 mmol) was
dissolved in 25 mL of pyridine, together with 3-bromophenyl
isocyanate (990 mg, 5 mmol). The mixture was stirred at room
temperature for 4 h, then diluted with EtOAc (100 mL) and
washed twice with 1 M HCl. The organic phase was dried with
Na2SO4 and evaporated under vacuum. The residue was
dissolved in 1 M NaOH and washed with EtOAc, and the
product was precipitated with HCl. The precipitate was
extracted into EtOAc, and the solvent was removed under
vacuum. The crude product was recrystallized twice from
EtOAc/heptane to yield colorless crystalline 15 (770 mg 55%).
(C14H10BrClN2O2S) C, H, N.
2-(3-Bromobenzoylamino)-4-chlorobenzoic Acid (8).
2-Amino-4-chlorobenzoic acid (6) (1026 mg, 6.00 mmol) and
3-bromobenzoyl chloride (1.66 g, 7.20 mmol) were dissolved
in dry pyridine (20 mL) at 0 °C, and the solution was stirred
for 18 h. The mixture was diluted with EtOAc and washed
with 1 M HCl. The organic phase was extracted with 1 M
NaOH, the pH was adjusted to 2 with HCl, and the product
was extracted into EtOAc. The extracts were dried with
Na2SO4 and evaporated. The product was recrystallized from
EtOAc/heptane to yield colorless crystalline 8 (830 mg, 39%).
1
1
Mp: 223-225 °C. H NMR (DMSO-d6) δ 12.20 (1H, s), 10.01
Mp: 242-244 °C (dec). H NMR (DMSO-d6) δ 12.28 (1H, s),
(1H, s), 8.23 (1H, s), 8.06 (1H, s), 7.95 (1H, d, J ) 8.5 Hz),
7.72 (1H, d, J ) 7.5 Hz), 7.37 (1H, d, J ) 8.5 Hz), 7.30 (2H,
m). Anal. (C14H9BrClN3O2) C, H, N.
8.73 (1H, s), 8.09 (1H, s), 8.05 (1H, d, J ) 8.5 Hz), 7.93 (1H, d,
J ) 8.0 Hz), 7.87 (1H, d, J ) 8.0 Hz), 7.57 (1H, t, J ) 8.0 Hz),
7.30 (1H, d, J ) 8.5 Hz). Anal. (C14H9BrClNO3) C, H, N.
Stable Cell Lines. HEK293 cell lines stably expressing
homomeric human GluR5-1a and human GluR6 were estab-
lished as described previously.27 HEK293 cell lines stably
expressing homomeric rat GluR1-4 were established using a
modified version of the bicistronic expression vector pIRES
(ClonTech, Palo Alto, CA), pIRES-BLAS-AN,28 using blastici-
din selection for incorporation of the flip isoforms of rat
GluR1-4. Cell lines were grown in Dulbecco’s modified Eagle’s
medium supplemented with 10% (v/v) fetal calf serum in
polystyrene culture flasks (175 cm2) in a humidified atmo-
sphere of 5% CO2 and 95% O2 at 37 °C. Growth media for
GluR1-4 cell lines were furthermore supplemented with 100
µg/mL SPD 502, an AMPA receptor antagonist.27,29 Cells were
cultured to 80-90% confluency before plating. The cells were
rinsed with 10 mL of phosphate buffered saline (PBS), 1.5 mL
of trypsin-EDTA (0.1% (w/v) trypsin) was added, and the cells
were left in the incubator for 5 min. After addition of 10 mL
of growth media, cells were resuspended by trituration with a
10 mL pipet. The cells were seeded at a density of (0.5-5) ×
106 cells/mL (100 µL/well) in black-walled, clear-bottom 96-
well plates pretreated with 0.001% (w/v) polycation poly-
ethyleneimine (PEI) solution (75 µL/well for g30 min). Plated
cells were allowed to proliferate for 24 h before loading with
dye.
2-[(3-Bromophenyl)carbamoyloxy]-4-chlorobenzoicAcid
(10). 4-Chlorosalicylic acid (2.07 g, 12 mmol) dissolved in dry
Et2O (10 mL) was added dropwise to a solution of phosgene
(7.5 mL, 20% in heptane, 15 mmol) in Et2O (30 mL) at -20
°C. After this mixture was stirred for 30 min at -20 °C, Et3N
(2.73 g, 27.0 mmol) diluted with Et2O (20 mL) was added
dropwise over 3 h, keeping the temperature between -10 and
-20 °C. The mixture was allowed to reach room temperature,
stirred for 18 h, and filtered. The mother liquor was evaporated
and 7-chlorobenzo[1,3]dioxine-2,4-dione (9) was recrystallized
from Et2O to yield a colorless crystalline solid (1450 mg, 61%).
1H NMR (DMSO-d6) δ 7.48 (1H, d, J ) 8.5 Hz), 7.31 (1H, d, J
) 8.5 Hz), 7.18 (1H, s). Compound 9 (700 mg, 3.53 mmol) was
dissolved in THF, and 3-bromoaniline (2.00 g, 11.6 mmol) was
added. The mixture was stirred for 48 h at room temperature,
then diluted with EtOAc and washed with dilute HCl. The
organic phase was extracted with dilute NaOH and with water.
The product was precipitated from the combined aqueous
phases by addition of HCl, filtered off, dried, and recrystallized
from EtOAc/heptane to yield colorless crystalline 10 (840 mg,
1
64%). Mp: 212-215 °C. H NMR (DMSO-d6) δ 11.85 (1H, s),
10.43 (1H, s), 8.05 (1H, s), 7.86 (1H, d, J ) 8.5 Hz), 7.63 (1H,
m), 7.31 (2H, m), 7.03 (2H, m). Anal. (C14H9BrClNO4) C, H,
N.
Fluorescence Measurements. On the day of experiment
the medium was removed from the wells, and 50 µL of the
Fluo-4-AM (cell-permeant acetoxymethyl ester of the Ca2+
indicator Fluo-4; Molecular Probes) loading solution (2 µM in
medium) was added to each well. The plates were sealed and
incubated at room temperature for 60 min (GluR5,6) or 30 min
(GluR1-4). After the loading period, the loading media was
aspirated and the cells were washed twice with 100 µL of Na+-
free Ringer (10 mM HEPES, 140 mM choline chloride, 5 mM
KCl, 1 mM MgCl2, 10 mM CaCl2; pH 7.4) to remove extra-
cellular dye. Na+-free Ringer (100 µL) was added to each well,
and the fluorescence was measured at room temperature
(excitation 488 nm, emission 510-570 nm band-pass interfer-
ence filter) in the FLIPR (Molecular Devices, Sunnyvale, CA).
Cells were preincubated for 1.5 min with test compound (50
µL) before addition of agonist (50 µL) to a final concentration
of 2 µM domoic acid (for GluR5), 0.2 µM domoic acid (for
GluR6), or 25 µM Glu (for GluR1-4). For GluR1-4, all
incubation solutions contained 100 µM cyclothiazide to inhibit
receptor desensitization. Stock solutions of test substances
were prepared in EtOH or DMSO, with the final concentration
of these solvents never exceeding 1.0% in the prepared test
solutions.
Ligand Binding Studies. GluR5-expressing cells were
harvested, washed once with 50 mM Tris-HCl (pH 7.1), and
stored at -80 °C until the day of experiment. The thawed
membrane pellets were resuspended in >100 volumes of ice-
cold Tris-HCl buffer and centrifuged at 27000g for 10 min. The
final pellet was resuspended in Tris-HCl buffer and used for
binding experiments. All procedures were performed at 0-4
°C.
Binding conditions for GluR5 were as described previously.27
Briefly, binding to GluR5 receptors was performed using 3 nM
[3H]ATPA and 46-84 µg of protein/assay. The samples were
2-[2-(3-Bromophenylamino)-3,4-dioxocyclobut-1-enyl-
amino]-4-chlorobenzoic Acid (13). 3-Bromoaniline (0.86 g,
5.0 mmol) and 3,4-diethoxycyclobut-3-ene-1,2-dione (0.85 g, 5.0
mmol) were dissolved in EtOH (10 mL) and stirred at room
temperature for 48 h. The product was precipitated by adding
toluene, filtered off, and dried to yield crude 3-(3-bromo-
phenylamino)-4-ethoxycyclobut-3-ene-1,2-dione (12) as a yellow
crystalline solid. Crude 12 (0.30 g, 1.0 mmol) was dissolved in
20 mL of acetonitrile together with 2-amino-4-chlorobenzoic
acid (6) (0.18 g, 1.0 mmol) and Et3N (0.10 g, 1.0 mmol). The
mixture was refluxed for 24 h and then concentrated to a
volume of about 4 mL, and the product was precipitated by
adding water (20 mL) and 4 M HCl (the pH was adjusted to
1). The product was recrystallized (EtOH/0.5 M HCl) to give
colorless crystalline 13 (0.30 g, 70%). Mp: 280-290 °C (dec).
1H NMR (DMSO-d6) δ 10.91 (1H, s), 10.54 (1H, s), 7.94 (1H,
d, J ) 8.5 Hz), 7.79 (1H, s), 7.66 (1H, s), 7.33 (3H, m), 7.21
(1H, d, J ) 8.5 Hz). Anal. (C17H10BrClN2O4‚H2O) C, H, N.
6-Chloro-1H-indazol-3-ol (14). 2-Amino-4-chlorobenzoic
acid (6) (17.16 g, 100 mmol) was suspended in 90 mL of water
and 20 mL of concentrated HCl. NaNO2 (6.9 g, 100 mmol) was
dissolved in water (15 mL) and added slowly at 0 °C. The
mixture was stirred at 0 °C for 30 min, and Na2SO3 (34.0 g,
270 mmol) was added, dissolved in 150 mL of water. The
mixture was then stirred for 2 h at room temperature.
Concentrated HCl (30 mL) was added, and the mixture was
stirred overnight at room temperature and then at 80 °C for
2 h. The pH of the mixture was adjusted to 5.5 with 1 M
NaOH. The precipitate was filtered off and triturated with
EtOH to yield crystalline 14 (13.8 g, 82%). Mp: 292-296 °C
(lit. 298-302 and 275-276 °C).25,26 1H NMR (DMSO-d6) δ 11.64
(1H, s), 10.58 (1H, s), 7.62 (1H, d, J ) 8.5 Hz), 7.35 (1H, s),
6.97 (1H, d). Anal. (C7H5ClN2O) C, H. N: calcd, 16.62; found,
15.89.

6956 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27
Valgeirsson et al.
(11) Merlic, C. A.; Semmelhack, M. F. An interesting chloride ion
effect in the Heck reaction. J. Organomet. Chem. 1990, 391,
C23-C27.
(12) Anastasia, L.; Negishi, E. Highly satisfactory procedures for the
Pd-catalyzed cross coupling of aryl electrophiles with in situ
generated alkynylzinc derivatives. Org. Lett. 2001, 3, 3111-
3114.
(13) Allen van, A. o-Toluidinsulfonic acid. Org. Synth. 1927, 27, 88.
(14) Koguro, K.; Oga, T.; Mitsui, S.; Orita, R. Novel synthesis of
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914.
(15) Reichert, A.; Froehlich, R.; Ferguson, R.; Kraft, A. Binding
interactions between 3-aryl-1,2,4-oxadiazol-5-ones and a trisimid-
azoline base. J. Chem. Soc., Perkin Trans. 1 2001, 1321-1328.
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1995, 328, 397-402.
incubated in a final volume of 550 µL for 60 min at 2 °C.
Nonspecific binding was determined in the presence of 0.6 mM
Glu, and binding was terminated by rapid filtration. Radio-
activity was determined by conventional liquid scintillation
counting.
ATPA-Induced Rigidity. Rigidity in mice was induced by
intravenous administration of ATPA. ATPA is systemically
active30 and has agonistic activity primarily at the GluR5
subtype.31,32 Intravenous administration of ATPA in 20-25 g
mice (female NMRI, Taconic M&B, Denmark) produces a
characteristic muscle rigidity that can be blocked by KA/AMPA
receptor antagonists.33,34 ATPA was dissolved in water by
addition of one drop of 1 M NaOH, addition of saline, and
titration back to pH 7 (3 mg/mL). Test substances were
administrated intravenously (n ) 8 per dose) 5 or 30 min prior
to the ATPA (30 mg/kg) administration. Rigidity was scored
by the number of mice showing loss of righting and lack of
movement on 45° grid grib 30 min after ATPA administration.
The data are presented as the percent of mice for each
experimental group showing rigidity.
(17) Dean, F. M.; Small, S.; Hindley, K. B. Synthesis of dis-disalicylide
and of flavones containing a chromeno[4,3-B]chromen nucleus.
J. Chem. Soc., Perkin Trans. 1 1972, 16, 2007-2010.
(18) Herr, R. J. 5-Substituted-1H-tetrazoles as carboxylic acid iso-
steres: medicinal chemistry and synthetic methods. Bioorg. Med.
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Acknowledgment. The technical assistance of Mrs.
Anne B. Fischer, Kirsten V. Haugegaard and Christine
Klæsøe is gratefully acknowledged.
Supporting Information Available: Results from ele-
mental analysis. This material is available free of charge via
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Note Added after ASAP Publication. This manu-
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the artwork and footnotes of Scheme 2 and in the listing
of the starting material for the preparation of 10 (in
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