Synthesis and Biological Evaluation of Analogues of 7-Chloro-4,5-dihydro-4-oxo-8-(1,2,4-triazol-4-yl)-1,2,4-triazolo[1,5-a] quinoxaline-2-carboxylic Acid (TQX-173) as Novel Selective AMPA Receptor Antagonists

Article abstract of DOI:10.1021/jm030906q

In recent papers (Catarzi, D.; et al. J. Med. Chem. 2000, 43, 3824-3826; 2001, 44, 3157-3165) we reported chemical and biological studies on 4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a]-quinoxaline-2-carboxylates (TQXs) bearing different nitrogen-containing he

Full text of DOI:10.1021/jm030906q

262
J . Med. Chem. 2004, 47, 262-272
Syn th esis a n d Biologica l Eva lu a tion of An a logu es of 7-Ch lor o-4,5-d ih yd r o-4-
oxo-8-(1,2,4-tr ia zol-4-yl)-1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid
(TQX-173) a s Novel Selective AMP A Recep tor An ta gon ists
Daniela Catarzi,*,† Vittoria Colotta,† Flavia Varano,† Francesca Romana Calabri,† Guido Filacchioni,†
Alessandro Galli,‡ Chiara Costagli,‡ and Vincenzo Carla`‡
Dipartimento di Scienze Farmaceutiche, Polo Scientifico, Universita’degli Studi di Firenze, Via Ugo Schiff, 6,
Sesto Fiorentino (FI), Italy, and Dipartimento di Farmacologia Preclinica e Clinica, Universita’ degli Studi di Firenze,
Viale G. Pieraccini, 6, 50134 Firenze, Italy
Received May 23, 2003
In recent papers (Catarzi, D.; et al. J . Med. Chem. 2000, 43, 3824-3826; 2001, 44, 3157-
3165) we reported chemical and biological studies on 4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a]-
quinoxaline-2-carboxylates (TQXs) bearing different nitrogen-containing heterocycles at
position-8. In particular, from these studies it emerged that both the 7-chloro-4,5-dihydro-4-
oxo-8-(1,2,4-triazol-4-yl)-1,2,4-triazolo[1,5-a] quinoxaline-2-carboxylic acid TQX-173 (compound
B) and its corresponding ethyl ester (compound A) were the most active and selective compounds
of this series. In pursuing our investigation on the structure-activity relationships of these
TQX derivatives, different electron-withdrawing groups (CF3, NO2) were introduced at position
7 on the TQX ring system, replacing the 7-chloro substituent of B and of other selected
8-heteroaryltriazoloquinoxaline-2-carboxylates previously described. All the newly synthesized
compounds were biologically evaluated for their binding at the Gly/NMDA, AMPA, and KA
high-affinity receptors. Gly/NMDA binding assays were performed to assess the selectivity of
the reported compounds toward the AMPA receptor. Compounds endowed with micromolar
binding affinity for the KA high-affinity binding site were also evaluated for their binding at
the KA low-affinity receptor. Some selected compounds were also tested for their functional
antagonist activity at the AMPA and NMDA receptor-ion channel complex. The results obtained
in this study have pointed out that 4,5-dihydro-7-nitro-4-oxo-8-(3-carboxypyrrol-1-yl)-1,2,4-
triazolo[1,5-a]quinoxaline-2-carboxylic acid (9b) and its corresponding ethyl ester (9a ) are the
most potent and selective AMPA receptor antagonists reported to date among the TQX series.
In tr od u ction
trary, the physiological function of the KA receptor
family, comprising the low-affinity binding site (GluR5-7
subunits) and the high-affinity one (KA1-2 subunits),
is still an open question.1,3-5
Glutamate (Glu) is the major excitatory neurotrans-
mitter in the central nervous system (CNS), where it is
involved in the physiological regulation of processes
such as learning, memory, and synaptic plasticity. On
the other hand, glutamatergic hyperactivity may lead
to neurotoxicity. In fact, excessive endogenous Glu is
implicated in a number of acute and chronic neurode-
generative pathologies such as cerebral ischaemia,
epilepsy, amiotrophic lateral sclerosis, and Parkinson’s
diseases.1
Glu activates specific receptors that belong to the
classes of metabotropic receptors (mGluRs, coupled to
G-protein) and ionotropic receptors (iGluRs, ligand-
gated ion channel), the latter consisting of two primary
families: N-methyl-D-aspartic acid (NMDA) receptor
family and non-NMDA receptor family. The non-NMDA
family is composed of the kainic acid (KA) receptor and
the (R,S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)-
propionic acid (AMPA) receptor according to their
preferential synthetic agonists.1,2 The physiological and
pathological roles of both AMPA and NMDA receptor-
ion channel complex are well understood; on the con-
The well-known physiological, but also pathological,
role of Glu has resulted in a great interest for the
development of Glu receptor (GluR) ligands as research
tools and potential therapeutic agents.1
Several AMPA receptor antagonists have been re-
ported in the literature and show promise in terms of
their therapeutic potential as neuroprotective agents for
the treatment and prevention of acute and chronic
neurological disorders.1,3-4,6-11
However, only a limited number of KA receptor
antagonists have been developed, which, in general,
have shown mixed AMPA/KA inhibitor activity.1,3-4,6
This is due to the fact that structural similarities and
differences between these two diverse receptors are still
unknown. For this reason, there are some difficulties
in pharmacologically differentiating AMPA and KA
receptors. To our knowledge, only the 5-nitro-6,7,8,9-
tetrahydro-1H-benz[g]indole-2,3-dione 3-oxime NS-102
shows a high affinity and selectivity for the low-affinity
KA receptor binding site.1,12 It has been reported that
compounds showing high affinity for both AMPA and
KA low-affinity binding sites are more potent anticon-
vulsants than AMPA receptor selective drugs.13 Thus,
* To whom correspondence should be addressed: Tel: +39 55
4573722. Fax: +39 55 4573667. E-mail: daniela.catarzi@unifi.it.
† Dipartimento di Scienze Farmaceutiche.
‡ Dipartimento di Farmacologia Preclinica e Clinica.
10.1021/jm030906q CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/26/2003

Novel Selective AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 263
Ch a r t 1
Sch em e 1a
KA and AMPA receptors can be considered as potential
targets for therapeutic prevention of epileptic sei-
zures.11,13
Moreover, an alternative approach to antagonize the
overstimulation of postsynaptic iGluRs by excessive
endogenous Glu is represented by the use of non-
competitive NMDA antagonists acting at the strychnine-
insensitive glycine site (Gly/NMDA) on the NMDA
receptor. In fact, glycine (Gly) is a necessary coagonist
of Glu and exerts a primary role in the activation of the
NMDA receptor by interacting with its allosteric modu-
latory site.14,15 Thus, blockade of the Gly/NMDA recep-
tor by antagonists results in a complete inhibition of
NMDA-mediated neurodegenerative effects.1,15-19
a
Reagents: (a) 90% HNO3; (b) iron, glacial AcOH; (c) (OHC-
NH)2, anhydrous pyridine, Me3SiCl, Et3N; (d) 2,5-dimethoxy-3-
tetrahydrofurancarboxyaldehyde, glacial AcOH; (e) 2,5-diethoxy-
tetrahydrofuran, glacial AcOH.
receptor,7-9 different electron-withdrawing groups (CF3,
NO2) were introduced at position-7 on the herein
reported TQX compounds, replacing the 7-chloro sub-
stituent of B and of other selected 8-heteroaryltriazolo-
quinoxaline-2-carboxylates previously described.
All of the newly synthesized compounds were biologi-
cally evaluated for their binding at the Gly/NMDA,
AMPA, and KA high-affinity receptors. Gly/NMDA and
KA binding assays were performed to assess the selec-
tivity of the reported compounds toward the AMPA
receptor. Compounds endowed with micromolar binding
affinity for the KA high-affinity binding site were also
evaluated for their binding at the KA low-affinity
receptor.
Accordingly, there has been a growing interest in
competitive and non-competitive iGluR antagonists due
to their promising therapeutic potential.
A non-selective blockade of both Gly/NMDA and
AMPA/KA receptors was shown by quinoxalinedione
and heterocyclic-fused quinoxalinone antagonists.15,20-24
Extensive structure-activity relationship (SAR) studies
on these derivatives have pointed out not only some
important common structural requirements for anchor-
ing at both receptor sites7,9,21-22 but also some different
features that are important in distinguishing the AMPA/
KA receptors from the Gly/NMDA one.6-9,21-22,25-28 In
particular, introduction of suitable substituents on
precise positions on the benzofused moiety of these
antagonists yielded selective and competitive blockade
of the AMPA/KA receptor.6-7,9,21-22,26-28
Some selected compounds were also tested for their
functional antagonist activity at the AMPA and NMDA
receptor-ion channel complex.
Ch em istr y
The ethyl 8-substituted-4,5-dihydro-4-oxo-1,2,4-tria-
zolo[1,5-a]quinoxaline-2-carboxylates 1a -13a were pre-
pared following two different synthetic strategies illus-
trated in Schemes 1-4.
In the course of our efforts to find novel competitive
and non-competitive iGluR antagonists,29-37 we have
published works that report the synthesis and biological
studies on 4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a]quinox-
aline-2-carboxylates (TQXs) bearing different nitrogen-
containing heterocycles at position-8.34,36 These previous
studies have well-established that the presence of a N3-
nitrogen-containing heterocycle at position-8 of the TQX
framework is an essential feature for potent and selec-
tive AMPA receptor antagonists. In particular, it has
emerged that both the 7-chloro-4,5-dihydro-4-oxo-8-
(1,2,4-triazol-4-yl)-1,2,4-triazolo[1,5-a]quinoxaline-2-car-
boxylic acid TQX-173 (compound B) and its correspond-
ing ethyl ester (compound A) (Chart 1) are the most
active and selective compounds among this series.34,36
The previously reported ethyl 4,5-dihydro-4-oxo-7-
trifluoromethyl-1,2,4-triazolo[1,5-a]quinoxaline-2-car-
boxylate33 was regioselectively nitrated at position-8 to
yield 10a , which was reduced to the corresponding
8-amino derivative 11a (Scheme 1). By reacting 11a
with diformylhydrazine, the 8-(1,2,4-triazol-4-yl) ester
1a was prepared. The 8-(3-formylpyrrol-1-yl) ester 2a
and the 8-(pyrrol-1-yl) ester 3a were obtained by react-
ing 11a with either 2,5-dimethoxy-3-tetrahydrofuran-
carboxaldehyde or 2,5-diethoxytetrahydrofuran, respec-
tively.
Oxidation of the 8-(3-formylpyrrol-1-yl) derivative 2a
with potassium permanganate yielded a mixture of the
8-(3-carboxypyrrol-1-yl) ester 4a and the 8-(3-formyl-
2,5-dioxopyrrol-1-yl) 5a (Scheme 2), which were sepa-
rated by using silica gel column chromatography.
By reacting the commercially available 2,4-dichloro-
5-nitrobenzotrifluoride with ammonia, the 5-chloro-2-
nitro-4-trifluoromethylaniline 1438 was isolated (Scheme
In pursuing our studies on the SAR of these TQX
derivatives and in order to develop new AMPA receptor
antagonists with increased potency and selectivity, we
have introduced some modifications on the tricyclic
system, in particular at position-7. In fact, on the basis
of a reported pharmacophore model of the AMPA

264 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Catarzi et al.
Sch em e 2a
Sch em e 4a
a
Reagents: (a) KMnO4, acetone/water 1:1.
Sch em e 3a
a
Reagents: (a) Ac2O, glacial AcOH; (b) 90% HNO3; (c) 6 N HCl,
EtOH; (d) 2,5-dimethoxy-3-tetrahydrofurancarboxyaldehyde, gla-
cial AcOH; (e) 2,5-diethoxytetrahydrofuran, glacial AcOH; (f)
KMnO4, acetone/water.
Oxidation of compound 7a with potassium permanga-
nate afforded the 8-(3-carboxypyrrol-1-yl) derivative 9a .
Finally, the hydrolysis of the esters 1a -4a and 6a -
13a to yield the corresponding 2-carboxylic acids 1b-
4b and 6b-13b is depicted in Scheme 5.
Resu lts a n d Discu ssion
a
Reagents: (a) NH3(gas), 2-methoxyethanol; (b) imidazole,
The triazoloquinoxalines 1a ,b-4a ,b, 5a , and 6a ,b-
13a ,b together with NBQX (2,3-dihydroxy-6-nitro-7-
sulfamoylbenzo[f]quinoxaline) and DCKA (5,7-dichlo-
rokinurenic acid) as standard compounds, were tested
for their ability to displace tritiated AMPA and Gly from
their specific binding in rat cortical membranes. High-
affinity KA binding assays were also performed. The
binding results are shown in Table 1 together with those
of previously reported 2-carboxylic acid derivative B and
its corresponding ester A included as reference com-
pounds.34,36 Some selected compounds (1b, 2b, 4b, 7b,
8b) were also evaluated for their binding at the KA low-
affinity receptor, and the results are shown in Table 2.
Since most of the newly synthesized compounds were
AMPA vs KA receptor non-selective, with the exception
of 1a ,b, 2a , 3a , 4b, 6a ,b, 9a ,b, and 10a together with
the reference B (Ki KA vs AMPA g 80), only AMPA vs
Gly/NMDA receptor selectivity of these TQX derivatives
has been considered in the discussion of the herein
reported results.
In general, the affinities of the TQX derivatives 1a ,b-
4a ,b, 5a , and 6a ,b-13a ,b for the Gly/NMDA receptor
are lower than those for the AMPA receptor, confirming
previously reported data. In fact, the binding data
shown in Table 1 indicate that replacement of the
7-chloro substituent of some selected previously reported
8-heteroaryl TQX derivatives,34,36 with a trifluoromethyl
or a nitro group, has produced new highly potent and
selective AMPA receptor antagonists.
KOH, anhydrous DMF; (c) NaNO2/conc H2SO4, H2O; (d) CH3CO-
CHClCOOEt, MeOH; (e) NH3 (gas), anhydrous dioxane; (f) ClCO-
COOEt, anhydrous toluene; (g) iron powder, glacial AcOH.
3). Reaction of 14 with imidazole in alkaline conditions
yielded the corresponding 5-(imidazol-1-yl)-2-nitro-4-
trifluoromethylaniline 15,39 which was transformed into
its diazonium sulfate by diazotization with NaNO2 in
H2SO4. The diazonium salt of 15 was reacted with ethyl
2-chloro-3-oxobutanoate to yield the N2-chloroacetate 16,
which was transformed with ammonia into its corre-
sponding N2-oxamidrazonate 17. By reacting 17 with
ethyloxalyl chloride, the diethyl 1-[5-(imidazol-1-yl)-2-
nitro-4-trifluoromethylphenyl]-1,2,4-triazole-3,5-dicar-
boxylate 18 was directly obtained. Reduction of the nitro
group of 18 and contemporary cyclization afforded the
tricyclic ester 6a .
Reaction of the ethyl 8-amino-4,5-dihydro-4-oxo-1,2,4-
triazolo[1,5-a]quinoxaline-2-carboxylate36 (Scheme 4)
with acetic anhydride yielded the corresponding 8-acetyl-
amino derivative 19, which was nitrated at position-7
with 90% HNO3 to afford 12a . Acidic hydrolysis of the
acetyl group of 12a yielded the 8-amino-7-nitro deriva-
tive 13a .
By reacting 13a with either 2,5-dimethoxy-3-tetrahy-
drofurancarboxaldehyde or 2,5-diethoxytetrahydrofuran
in glacial AcOH, the 8-(3-formylpyrrol-1-yl) 7a and the
8-(pyrrol-1-yl) 8a esters were obtained, respectively.

Novel Selective AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 265
Sch em e 5a
Ta ble 1. Binding Affinity at AMPA, Gly/NMDA, and KA
High-Affinity Receptors of TQX Derivativesa
a
Reagents: (a) 0.8 N NaOH/EtOH, 6 N HCl or glacial AcOH;
(b) HCl conc.
In particular, the binding data indicate that all the
2-carboxylic acids 1b-4b and 6b-9b are, in general,
more active than their corresponding ethyl esters 1a -
4a and 6a -9a at the AMPA receptor. In accordance
with our previously reported results,33-34,36 the differ-
ence between the binding affinity of the esters with
respect to the corresponding acids is generally depend-
ent on the nature of the 8-heteroaryl substituent. In fact,
the 2-carboxylic acid derivatives bearing at position-8
a N3-nitrogen-containing heterocycle (1b and 6b), a
3-formyl-substituted heterocycle (2b and 7b), or a
3-carboxy-substituted heterocycle (9b) are only 2-3-fold
more active at the AMPA receptor than their corre-
sponding esters (1a , 6a , 2a , 7a , and 9a ). On the
contrary, the 2-carboxylic acid 3b and 8b, bearing a
8-heterocyclic ring lacking the 3-formyl group, are about
10-15-fold more active than their corresponding esters
3a and 8a .
Thus, these results confirm that the presence of a free
carboxylic group at position-2 is not an essential feature
for the anchoring to the AMPA receptor when the
8-heteroaryl substituent bears at position-3 a proton-
acceptor atom/group.34 Indeed, the N3-nitrogen atom,
the 3-formyl, or the 3-carboxy group could act as proton
acceptors in a hydrogen bond with a specific receptor
site, thus reinforcing the receptor-ligand interaction.
a
The tested compounds were dissolved in DMSO and then
b
diluted with the appropriate buffer. Inhibition constant (Ki)
values were means ( SEM of three or four separate determina-
tions in triplicate. c Percentage of inhibition (I%) of specific binding
d
at 100 µM concentration. Concentration necessary for 50%
inhibition (IC50). The IC50 values were means ( SEM of three or
four separate determinations in triplicate.34
It has to be noted that the 2-carboxylic acid 4b,
bearing at position-8 a 3-carboxylic acid substituted
heterocycle, is 10-fold more active than its corresponding
ester 4a . This is an unexpected result on the basis of
the above-reported data, because the carboxylic acid
function on the 8-heterocycle substituent is a powerful
proton acceptor group.
Replacement of the 7-chloro substituent of B and its
corresponding 2-carboxylic ester A with a trifluoro-

266 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Catarzi et al.
Ta ble 2. Binding Affinity at KA Low-Affinity Binding Site of
On the contrary, oxidation of both 2- and 5-positions
of the 8-(3-formylpyrrol-1-yl) moiety of 2a gives com-
pound 5a , which is about 30-fold less active at the
AMPA receptor than 2a , indicating that an effect of
steric hindrance may be operative at the 8-position. In
fact, due to the steric hindrance exerted by the 2,5-dioxo
function, the 8-substituent of 5a is probably twisted out
of the plane of the TQX framework, owing to an
unfavorable steric interaction at this level. These data
could suggest that in the AMPA receptor both sites that
accommodate, respectively, the 8-heteroaryl substituent
and the tricyclic ring system of AMPA antagonists such
as TQXs lie in the same plane.
Some Selected TQX Derivativesa
IC50 (µM)b
IC50
IC50 (µM)b
IC50
compd
[3H]KA
ratioc
compd
[3H]KA
ratioc
1b
2b
4b
1.8 ( 0.2
1.0 ( 0.2
2.6 ( 0.3
0.19
0.18
0.48
7b
8b
NBQX
1.2 ( 0.09
1.7 ( 0.4
1.5 ( 0.07
0.15
0.33
0.21
a
The tested compounds were dissolved in DMSO and then
b
diluted with the appropriate buffer. Concentration necessary for
50% inhibition (IC50). The IC50 values were means ( SEM of three
or four separate determinations in triplicate. c IC50 at KA low-
affinity receptor site/IC50 at KA high-affinity receptor site (see
Table 1).
methyl moiety produces compounds 1b and 1a , which
show comparable and 3.5-fold increased AMPA binding
affinities compared to those of the reference compounds,
respectively. While compound 1a is one of the most
AMPA-selective compounds among the TQX series, its
corresponding acid 1b is much less selective than B, due
to its 10-fold increased binding affinity at the Gly/
NMDA receptor. The high AMPA selectivity of com-
pound 1a can be considered an exception. In fact, as
discussed below, the contemporary presence of the
2-carboxylic acid function together with the 7-trifluo-
romethyl substituent on the TQX framework produces
an ameliorated interaction with both the Gly/NMDA
and AMPA receptor sites, thus positively affecting
binding affinities to a small degree, but not selectivity.
No data about the replacement of the 7-chloro sub-
stituent of B with a nitro group are available. In fact,
the synthesis of the 7-nitro analogue of A, following the
same procedures used to prepare 1a , was not achieved,
probably because of the very low reactivity of the
8-amino group of the starting compound 13a . Other
synthetic strategies (data not published) have not given
the desired compound either.
Replacement of the 7-trifluoromethyl group of 2a ,b
with a nitro group gives compounds 7a ,b, which are
about equiactive at the AMPA receptor as their parents
2a ,b. Both the 2-carboxylic ester 7a and its correspond-
ing 2-carboxylic acid 7b, due to the inactivity at the Gly/
NMDA receptor, were much more selective toward the
AMPA receptor than 2a and 2b, respectively. Increased
AMPA activity and selectivity was observed when the
nitro group replaced the trifluoromethyl substituent of
the 2-carboxylic acid 3b to give 8b. On the contrary, the
same modification on the 2-carboxylic ester 3a unex-
pectedly produced only a 1.5-2-fold increase of AMPA
binding affinity and selectivity (compare 8a to 3a ).
These data indicate that the presence of the meso-
meric electron-withdrawing nitro group at the 7-position
of our TQX derivatives is an important feature for
potent and selective AMPA receptor antagonists, inde-
pendent from both the nature of the 8-heteroaryl
substituent and the presence of the 2-carboxylic acid
function. In fact, the 8-(pyrrol-1-yl)-substituted deriva-
tive 8b , although bearing both a N3-nitrogen-lacking
heterocycle at position-8 and a carboxylic acid function
at position-2, is one of the most AMPA receptor selective
compounds among this series.
Maintaining the 7-trifluoromethyl substitution, the
8-(1,2,4-triazol-4-yl) moiety was replaced by the 8-(3-
formylpyrrol-1-yl) or 8-(imidazol-1-yl) moiety, giving
compounds 2a ,b and 6a ,b, respectively, which have
AMPA binding affinity values comparable to those of
1a ,b. It has to be noted that, while compounds 1b and
2b, as discussed above, show a low AMPA selectivity,
6b is one of the most AMPA-selective 2-carboxylic acid
derivatives among this series. The high AMPA selectiv-
ity of 6b can be explained by hypothesizing that the
8-(imidazol-1-yl) moiety is not well-tolerated by the Gly/
NMDA receptor site.
Elimination of the 3-formyl group from the 8-sub-
stituent of compounds 2a ,b gives the corresponding
8-(pyrrol-1-yl) derivatives 3a ,b, which are 6- and 2,3-
fold less active at the AMPA receptor than their parents
2a ,b.
These data confirm that the N3-nitrogen atom or the
3-formyl group can act as a proton acceptor in a
hydrogen bond with a receptor site, thus reinforcing the
receptor-ligand interaction (compare 1a ,b, 2a ,b, and
6a ,b to 3a ,b, respectively).
Taking into consideration the positive effect exerted
on AMPA binding affinity and selectivity by both the
7-nitro substituent and the 8-(3-carboxypyrrol-1-yl)
moiety, compounds 9a ,b were rationally designed. The
binding results of these compounds showed that the
contemporary presence on the TQX framework of these
two profitable groups is optimal to achieve significant
AMPA binding affinity and selectivity. In fact, com-
pounds 9a ,b are not only highly selective for AMPA vs
Gly/NMDA (selectivity ratio > 1500) but also AMPA vs
KA (selectivity ratio > 900). Thus, 9a and 9b are the
most potent and selective AMPA receptor antagonists
among the TQX series.
In summary, replacement of the 7-chlorine atom of
the analogues of compound B with a powerful electron-
withdrawing substituent such as trifluoromethyl or
nitro group yields, in general, AMPA receptor antago-
nists more potent than the 7-chloro substituted ones.
Moreover, the presence of the strong electron-withdraw-
Replacement of the 3-formyl group on the 8-substitu-
ent of 2a ,b with a free carboxylic group gives compounds
4a ,b, which are equiactive and about 3-fold more active
at the AMPA receptor than 2a ,b, respectively. More
importantly, an increased selectivity toward the AMPA
receptor was observed. In fact, the 2-carboxylic acid 4b
is one of the most potent and selective antagonists
among the TQX series. This result can be explained
hypothesizing that a 3-anionic carboxylate residue on
the 8-substituent is able to engage a strong interaction
with a cationic proton donor site of the AMPA receptor,
but it is not well-tolerated by the Gly/NMDA receptor
site. This hypothesis is supported by the high AMPA
vs Gly/NMDA selectivity of the 2-ethyl carboxylate 4a .

Novel Selective AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 267
Ta ble 3. Functional Antagonism (IC50, µM) at AMPA and
ing nitro group also enhances the AMPA selectivity of
these derivatives. In fact, in accordance with the
reported AMPA pharmacophore model,6-9 a nitro sub-
stitution pattern at this level is, in general, unfavorable
at the Gly/NMDA site but preferable for the AMPA
receptor.
Replacement of the 8-heteroaryl moiety of the 7-tri-
fluoromethyl-substituted derivatives 1a ,b-4a ,b, 5a ,
and 6a ,b with a nitro or amino group gives compounds
10a ,b and 11a ,b, respectively, which show in general a
decreased AMPA binding affinity.
A similar effect exerted by both the 8-nitro and the
8-amino groups on the AMPA binding affinity was also
previously observed.36 The 8-nitro substituent, due to
its electron-withdrawing effect, could increase the NH
lactam acidity, thus reinforcing the essential hydrogen-
bond receptor-ligand interaction at this level. On the
other hand, the 8-amino group could act as a proton
acceptor in a hydrogen-bond interaction with a receptor
site. The positive effect of the 8-amino group is also
exerted on the Gly/NMDA binding affinity. In fact, the
8-amino derivative 11a is 11-fold more active than the
corresponding 8-nitro compound 10a . The profitable
effect of the 8-amino group is confirmed by the binding
activity of compound 13b that, although bearing a
7-nitro substituent that is not well-tolerated by the Gly/
NMDA receptor site, has a comparable binding affinity
to that of the 7-trifluoromethyl-substituted derivatives
11b.
NMDA Receptors of Some Selected TQX Derivatives
[3H]NE
compd
AMPAa
NMDAa
releaseb
[3H]-(+)-MK-801c
1a
1b
2a
2b
3b
4b
7b
8b
Be
12 ( 2
>150
NT
5.8 ( 1
NT
8.1 ( 0.5
11.0 ( 1.8
7.5 ( 0.6
12 ( 2.5
19.0 ( 3.0
28.0 ( 3.1
4.0 ( 0.7
NT
29 ( 3
NT
11 ( 0.8
12.7 ( 0.7
NT
NT
NT
0.8 ( 0. 1
7.8 ( 0.9
60 ( 5
d
1.0 ( 0.15 50 ( 4
1.5 ( 0.2
1.3 ( 0.2
3.1 ( 0.4
1.0 ( 0.2
2.3 ( 0.4
80 ( 9
60 ( 15
52 ( 5
50 ( 8
46 ( 4
68 ( 4
NT
NT
d
NBQX 0.2 ( 0.02
DCKA 52 ( 11
4.7 ( 0.9 NT
a
Concentration necessary for 50% inhibition (IC50) of depolar-
ization induced by 5 µM S-AMPA or NMDA in mouse cortical
wedge preparation. The IC50 values were mens ( SEM of three
b
separate determinations. Concentration that inhibits 50% S-
AMPA-stimulated (50 µM) [3H]noradrenaline release (IC50) from
rat hippocampal synaptosomes. S-AMPA stimulatory action was
assessed in the presence of 50 µM cyclothiazide; data represent
the means ( SEM of three separate determinations in duplicate.
c Concentration giving 50% inhibition of stimulated [3H]-(+)-MK-
801 binding: all assays were carried out in the presence of 10 µM
Glu and 0.1 µM Gly. The results were calculated from three or
d
four separate determinations in triplicate. At 10 µM concentra-
tion, the inhibition was not significant. e Reference 34.
rations41 (compounds 1a ,b, 2a ,b, 3b, 4b, 7b, and 8b)
and [3H]norepinephrine release from rat hippocampal
synaptosomes (compounds 1b, 2b, 3b, 4b, 7b, and 8b).
In fact, since it has been reported that AMPA receptor
activation induces norepinephrine release from hippo-
campal nerve endings,42 the same compounds were
assayed for their ability to antagonize this action in rat
[3H]norepinephrine-preloaded hippocampal synapto-
somes. Moreover, functional antagonism at the NMDA
receptor-ion channel complex was also evaluated by the
ability of some selected TQX derivatives to inhibit both
the NMDA-induced depolarization in mouse cortical
wedge preparations (compounds 1a ,b, 2a ,b, 3b, 4b, 7b,
and 8b) and the binding of the channel-blocking agent
[3H]-(+)-MK-801((+)-5-methyl-10,11-dihydro-5H-benzo-
[a,d]cyclohepten-5,10-imine)25,43-44 in rat cortical mem-
branes incubated with 10 µM glutamate and 0.1 µM
glycine. The results obtained in the functional studies
on the newly synthesized derivatives, together with
those of compound B, NBQX, and DCKA as reference
compounds, are listed in Table 3. All the tested com-
pounds show an antagonistic profile and inhibit AMPA
and NMDA responses in a reversible manner. The
results obtained in the electrophysiological assays in-
dicate that the inhibitory actions of 1a ,b, 2a ,b, 3b, 4b,
7b, and 8b on depolarization induced by 5 µM AMPA
are much higher than those on NMDA-evoked response,
confirming that these derivatives are more potent
antagonists at the AMPA than the Gly/NMDA receptor.
Moreover, compounds 1b-4b and 8b are more potent
AMPA receptor antagonists than the reference B.
However, when the 7-trifluoromethyl group of 11a
was replaced by a nitro group, compound 13a was
obtained, showing about a 3-4-fold increased AMPA
binding affinity and selectivity.
Compound 12a , which is the 8-N-acetyl derivative of
13a , is about 6-fold less active at the AMPA receptor
than 13a . On the contrary, its corresponding 2-carboxy-
lic acid 12b is equiactive to 13b, but more AMPA
selective, due to its low Gly/NMDA receptor affinity.
Finally, the triazoloquinoxalines 1a,b-4a,b, 5a, 6a,b-
13a ,b are, in general, active in the high micromolar
range at the high-affinity KA receptor, with the excep-
tions being the 2-carboxylic acids 1b, 2b, 4b, 7b, and
8b, which display IC50 values in the range 5-10 µM.
These data confirm that the presence of a free carboxylic
group at position-2 seems to be of paramount impor-
tance for KA receptor-ligand interaction.
All the most active TQX derivatives at the KA high-
affinity binding site (1b, 2b, 4b, 7b, and 8b) together
with NBQX as reference compound were also evaluated
for their binding at the KA low-affinity binding site.39
While the well-known quinoxalinedione-like antagonist
NBQX shows only a slight preference for the KA-low
affinity site with respect to the KA high-affinity one
(low/high-affinity IC50 ratio ) 0.21), so far, only the
isatin-oxime NS-102 is reported to selectively interact
with the KA low-affinity site (IC50 ratio < 0.06).12,40 In
our experiments (Table 2) the IC50 ratio of the tested
compounds 1b, 2b, 4b, 7b, and 8b (0.14-0.48) are in
the range of the quinoxalinedione-like antagonist NBQX.
To correlate the binding data with functional AMPA
receptor activity, some of the herein reported com-
pounds, endowed with high affinity for AMPA receptor,
were tested for their ability to inhibit both AMPA-
induced depolarization in mouse cortical wedge prepa-
The IC50 values to inhibit AMPA-stimulated [3H]-
norepinephrine release were considerably higher than
those effective on cortical depolarization. This is prob-
ably due to the fact that a high concentration of S-AMPA
(50µM) is necessary to obtain a measurable [3H]cate-
cholamine efflux from synaptosomes.
Moreover, 1b, 2b, and 3b, being the most potent
compounds at the Gly/NMDA receptor, show IC50 values

268 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Catarzi et al.
for glutamate stimulated [3H]-(+)-MK-801 binding that
correlate with their Ki values on [3H]glycine binding
assays.
Ta ble 4. Physical and Analytical Data of the Newly
Synthesized Compounds
compd
mp (°C)
solventa
yield (%)
C, H, N
In conclusion, this study has confirmed that replace-
ment of the 7-chloro atom of compound B and of other
8-heterocyclic-substituted TQX derivatives previously
reported with electron-withdrawing substituents more
powerful than a chlorine atom has produced some potent
and selective AMPA receptor antagonists. Moreover,
this study has pointed out that the presence of a free
carboxylate function on the 8-heteroaryl substituent
could be of paramount importance to increase AMPA
receptor affinity and selectivity. In fact, it has emerged
that 4,5-dihydro-7-nitro-4-oxo-8-(3-carboxypyrrol-1-yl)-
1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylic acid (9b)
and its corresponding ester (9a ) are the most potent and
selective AMPA receptor antagonists among the TQX
series.
1a
1b
2a
2b
3a
3b
4a
4b
5a
6a
6b
7a
7b
8a
8b
9a
>300
A
B
C
B
C
B
D
E
64
95
67
72
78
81
8
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
15H10F3N7O3
13H6F3N7O3
18H12F3N5O4
16H8F3N5O4
17H12F3N5O3
15H8F3N5O3
18H12F3N5O5
16H8F3N5O5
18H10F3N5O6
16H11F3N6O3
14H7F3N6O3
17H12N6O6
15H8N6O6
16H12N6O5
14H8N6O5
17H12N6O7
15H8N6O7
13H8F3N5O5
11H4F3N5O5
13H10F3N5O3
11H6F3N5O3
14H12N6O6
12H8N6O6
>300
289 dec
>300
272-273
>300
262 dec
250 dec
>300
96
35
60
80
88
95
70
90
46
90
92
87
70
44
75
92
56
78
98
60
40
92
35
89
D
A
F
266 dec
>300
>300
>300
C
C
C
B
D
B
A
A
A
F
C
A
A
G
H
H
H
E
276 dec
>300
237 dec
270 dec
292-293
245 dec
>300
9b
10a
10b
11a
11b
12a
12b
13a
13b
14b
15b
16
This result will be taken into consideration for the
future design of novel potent and selective AMPA
receptor antagonists.
>300
>300
>300
Exp er im en ta l Section
297 dec
>300
12H10N6O5
10H6N6O5
Ch em istr y. Silica gel plates (Merck F254) and silica gel
60 (Merck; 70-230 mesh) were used for analytical and column
chromatography, respectively. All melting points were deter-
mined on a Gallenkamp melting point apparatus. Microanaly-
113-114
223-224
145-147
224 dec
172-174
>300
C7H4ClF3N2O2
C
C
C
C
C
10H7F3N4O2
14H11ClF3N5O4
14H13F3N6O4
18H15F3N6O6
14H13N5O4
17
18
19
ses were performed with
a Perkin-Elmer 260 elemental
I
A
analyzer for C, H, N, and the results were within (0.4% of
the theoretical values, except where stated otherwise (Table
4). The IR spectra were recorded with a Perkin-Elmer 1420
spectrometer in Nujol mulls and are expressed in cm-1. The
1H NMR spectra were obtained with a Varian Gemini 200
instrument at 200 MHz. The chemical shifts are reported in δ
(ppm) and are relative to the central peak of the solvent. All
the exchangeable protons were confirmed by addition of D2O.
The physical and analytical data of the newly synthesized
compounds are shown in Table 4.
Eth yl 4,5-Dih ydr o-8-n itr o-4-oxo-7-tr iflu or om eth yl-1,2,4-
tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxyla te (10a ). A solution
of ethyl 4,5-dihydro-7-trifluoromethyl-4-oxo-1,2,4-triazolo[1,5-
a]quinoxaline-2-carboxylate33 (6.13 mmol) in HNO3 (90%, 20
mL) was heated at 40 °C for 1 h. The reaction mixture was
poured onto ice (40 g) and the resulting solid was collected
and washed with water: 1H NMR (DMSO-d6) 1.37 (t, 3H, CH3,
J ) 6.96 Hz), 4.45 (q, 2H, CH2, J ) 6.96 Hz), 7.94 (s, 1H, ar),
8.87 (s, 1H, ar), 13.0 (br s, 1H, NH); IR 3190, 3085, 1720.
E t h yl 8-Am in o-4,5-d ih yd r o-4-oxo-7-t r iflu or om et h yl-
1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxyla te (11a ). Iron
powder (3.0 g) was added to a solution of 10a (3.0 mmol) in
glacial acetic acid (18 mL). The mixture was heated at reflux
for 1 h. Evaporation at reduced pressure of the solvent yielded
a residue which was dried and extracted in Soxhlet with
acetone (250 mL). Most of the solvent was evaporated to yield
a solid which was collected by filtration: 1H NMR (DMSO-d6)
1.37 (t, 3H, CH3, J ) 6.96 Hz), 4.42 (q, 2H, CH2, J ) 6.96 Hz),
5.5 (br s, 2H, NH2), 7.47 (s, 1H, ar), 7.64 (s, 1H, ar), 12.2 (br
s 1H, NH); IR 3385, 1740, 1685.
Eth yl 4,5-Dih yd r o-4-oxo-8-(1,2,4-tr ia zol-4-yl)-7-tr iflu o-
r om e t h yl-1,2,4-t r ia zolo[1,5-a ]q u in oxa lin e -2-ca r b oxy-
la te (1a ). Diformylhydrazine (4.41 mmol) and then, drop by
drop, trimethylsilyl chloride (22.6 mmol) and triethylamine
(10.3 mmol) were added to a suspension of 11a (1.47 mmol) in
anhydrous pyridine (7 mL). The mixture was heated at 100
°C for an overall time of 72 h. The reaction was monitored by
TLC [eluting system CHCl3/MeOH (90:10)]. After 46 h, di-
formylhydrazine (1.24 mmol), trimethylsilyl chloride (6.1
mmol), and triethylamine (2.73 mmol) were added. Another
addition of trimethylsilyl chloride (11.03 mmol) and triethyl-
amine (5.02 mmol) was done after 56 h. Evaporation at reduced
pressure of the solvent yielded a solid which was treated with
a
Recrystallization solvents: A ) ethanol; B ) the title com-
pound was dissolved in the minimal amount of 1 N NaOH, the
insoluble material was filtered off, and the resulting clear solution
acidified with 1 N HCl. The resulting solid was collected, treated
with boiling ethanol, collected again, and washed with fresh
ethanol; C ) glacial acetic acid; D ) purification of the title
compound was performed by silica gel column cromatography as
reported in the experimental; E ) ethanol/diethyl ether; F ) the
title compound was treated as in B, but after dissolution in 1 N
NaOH, the acid was precipitated by addition of glacial acetic acid;
G ) ethanol/water; H ) ethyl acetate; I ) ethyl acetate/diethyl
b
ether. Reference 38.
water (50 mL), collected, and purified by silica gel column
chromatography [eluting system CHCl3/MeOH (90:10)]: 1H
NMR (DMSO-d6) 1.35 (t, 3H, CH3, J ) 6.96 Hz), 4.43 (q, 2H,
CH2, J ) 6.96 Hz), 7.93 (s, 1H, ar), 8.54 (s, 1H, ar), 8.83 (s,
2H, triazole H-2 and H-5),12.8 (br s, 1H, NH); IR 3140, 1735,
1715.
Eth yl 4,5-Dih yd r o-4-oxo-8-(3-for m ylp yr r ol-1-yl)-7-tr i-
flu or om eth yl-1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxy-
la t e (2a ). A solution of 2,5-dimethoxytetrahydrofuran-3-
carboxaldehyde (3.08 mmol) in glacial acetic acid (10 mL) was
dropwise added to a suspension of 11a (2.05 mmol) in glacial
acetic acid (23 mL). The reaction mixture was heated at 90 °C
for 10 min. After cooling, the mixture was diluted with water
(200 mL) to yield a solid which was collected and washed with
water. A second crop of 2a was obtained by extracting the
mother liquor with ethyl acetate (50 mL × 3). Evaporation of
the dried (Na2SO4) organic layers afforded a solid which was
purified by crystallization: 1H NMR (DMSO-d6) 1.35 (t, 3H,
CH3, J ) 6.96 Hz), 4.43 (q, 2H, CH2, J ) 6.96 Hz), 6.68 (d, 1H,
pyrrole H-4, J ) 1,47 Hz), 7.17 (d, 1H, pyrrole H-5, J ) 1.47
Hz), 7.93 (s, 2H, ar + pyrrole H-2), 8.26 (s, 1H, ar), 9.79 (s,
1H, CHO), 12.8 (br s, 1H, NH); IR 3100, 1755, 1720, 1640.
E t h yl
4,5-Dih yd r o-4-oxo-8-(p yr r ol-1-yl)-7-t r iflu or o-
m eth yl-1,2,4-tr iazolo[1,5-a ]qu in oxalin e-2-car boxylate (3a ).
A solution of 2,5-diethoxytetrahydrofuran (4.38 mmol) in
glacial acetic acid (9 mL) was added dropwise to a hot (90 °C)
suspension of 11a (1.46 mmol) in glacial acetic acid (18 mL).
The reaction mixture was kept at 90 °C for 5 min. Dilution
with water (40 mL) afforded a solid which was collected and

Novel Selective AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 269
washed with water: 1H NMR (DMSO-d6) 1.35 (t, 3H, CH3, J
) 6.96 Hz), 4.43 (q, 2H, CH2, J ) 6.96 Hz), 6.29 (t, 2H, pyrrole
H-3 and H-4, J ) 2.2 Hz), 7.01 (t, 2H, pyrrole H-2 and H-5, J
) 2.2 Hz), 7.90 (s, 1H, ar), 8.02 (s, 1H, ar), 12.79 (br s, 1H,
NH); IR 3270, 3070, 1740, 1710.
anhydrous dioxane (8 mL). The mixture was stirred at room
temperature for 1 h and then was diluted with water (30 mL)
to yield an orange solid, which was collected and washed with
water. 1H NMR (DMSO-d6) 1.22 (t, 3H, CH3, J ) 6.8 Hz), 4.24
(q, 2H, CH2, J ) 6.8 Hz), 7.11-7.13 (m, 3H, NH2 + imidazole
H-4), 7.47 (d, 1H, imidazole H-5, J ) 1.22 Hz), 7.62 (s, 1H,
ar), 7.92 (s, 1H, imidazole H-2), 8.46 (s, 1H, ar), 10.26 (br s,
1H, NH); IR 3420, 3300, 1710.
Diet h yl 1-[5-(Im id a zol-1-yl)-2-n it r o-4-t r iflu or om et h -
ylp h en yl]-1,2,4-t r ia zolo]-3,5-d ica r b oxyla t e (18). Ethyl-
oxalyl chloride (1.71 mmol) was added to a suspension of the
amidrazone 17 (0.85 mmol) in anhydrous toluene (10 mL). The
reaction mixture was heated at reflux for 3 h. Upon cooling, a
solid precipitates which was collected and washed with a little
diethyl ether: 1HNMR (DMSO-d6) 1.20 (t, 3H, CH3, J ) 7.0
Hz), 1.32 (t, 3H, CH3, J ) 7.0 Hz), 4.29 (q, 2H, CH2, J ) 7.0
Hz), 4.39 (q, 2H, CH2, J ) 7.0 Hz) 7.16 (s, 1H, imidazole H-4),
7.56 (s, 1H, imidazole H-5), 8.0 (s, 1H, imidazole H-2), 8.39 (s,
1H, ar), 8.84 (s, 1H, ar); IR 1740.
Eth yl 4,5-Dih yd r o-8-(im id a zol-1-yl)-4-oxo-7-tr iflu or o-
m eth yl-1,2,4-tr iazolo[1,5-a ]qu in oxalin e-2-car boxylate (6a ).
Iron powder (0.75 g) was added to a solution of 18 (0.75 mmol)
in glacial acetic acid (6 mL). The mixture was heated at 90°C
for 10 min. Evaporation at reduced pressure of the solvent
yielded a residue which was treated with water (25 mL). The
solid was collected by filtration and extracted in Soxlhet with
acetone (250 mL). Evaporation of the solvent yielded a solid
which was worked up with diethyl ether (10 mL) and collected.
1H NMR (DMSO-d6) 1.35 (t, 3H, CH3, J ) 6.96 Hz), 4.43 (q,
2H, CH2, J ) 6.96 Hz), 7.12 (s, 1H, imidazole H-4), 7.46 (s,
1H, imidazole H-5), 7.89 (s, 1H, imidazole H-2), 7.92 (s, 1H,
ar), 8.22 (s, 1H, ar), 12.80 (br s, 1H, NH); IR 1750, 1710.
Eth yl 8-Acetyla m in o-4,5-d ih yd r o-4-oxo-1,2,4-tr ia zolo-
[1,5-a ]qu in oxa lin e-2-ca r boxyla te (19). Acetic anhydride
(3.07 mmol) was added to a suspension of the ethyl 8-amino-
4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxy-
late36 (2.56 mmol) in glacial acetic acid (18 mL). The reaction
mixture was heated at reflux for 20 min. After cooling, the
resulting solution was diluted with water (170 mL), affording
a suspension which was stirred at room temperature for 2 h.
The yellow solid was collected and washed with water: 1H
NMR (DMSO-d6) 1.38 (t, 3H, CH3, J ) 6.96 Hz), 2.09 (s, 3H,
CH3), 4.44 (q, 2H, CH2, J ) 6.96 Hz), 7.44 (d, 1H, ar, J ) 8.8
Hz), 7.58 (dd, 1H, ar, J ) 8.8, 2.2 Hz), 8.64 (d, 1H, ar, J ) 2.2
Hz), 10.3 (br s 1H, NH), 12.4 (br s 1H, NH); IR 3370, 1730,
1670.
E t h yl 8-Acet yla m in o-4,5-d ih yd r o-7-n it r o-4-oxo-1,2,4-
t r ia zolo[1,5-a ]q u in oxa lin e-2-ca r b oxyla t e (12a ). Com-
pound 19 (2.22 mmol) was added portionwise to cold (-15 °C)
HNO3 (90%, 15 mL). The reaction mixture was stirred at -15
°C for 6 h and then was quenched with ice (70 g). The
suspension was kept at room temperature for one night and
the resulting solid was collected and washed with water: 1H
NMR (DMSO-d6) 1.37 (t, 3H, CH3, J ) 6.96 Hz), 2.11 (s, 3H,
CH3), 4.44 (q, 2H, CH2, J ) 6.96 Hz), 7.80 (s, 1H, ar), 8.42 (s,
1H, ar), 10.4 (br s 1H, NH), 12.6 (br s 1H, NH); IR 3410, 1740,
1700, 1615.
Eth yl 8-Am in o-4,5-d ih yd r o-7-n itr o-4-oxo-1,2,4-tr ia zolo-
[1,5-a ]qu in oxa lin e-2-ca r boxyla te (13a ). HCl (6 N, 5 mL)
was added to a hot (90 °C) solution of 12a (0.69 mmol) in EtOH
(5 mL). The reaction mixture was heated at reflux for 1 h and
then diluted with water (20 mL). The resulting solid was
filtered, resuspended in water (10 mL), and basified with
saturated NaHCO3 solution. The suspension was kept at room
temperature with stirring for 1 h. The orange solid was
collected and washed with water 1H NMR (DMSO-d6) 1.36 (t,
3H, CH3, J ) 6.96 Hz), 4.43 (q, 2H, CH2, J ) 6.96 Hz), 7.5 (br
s, 2H, NH2), 7.79 (s, 1H, ar), 8.06 (s, 1H, ar), 12.2 (br s 1H,
NH); IR 3470, 3210, 1755, 1725.
Eth yl 4,5-Dih yd r o-4-oxo-8-(3-ca r boxyp yr r ol-1-yl)-7-tr i-
flu or om eth yl-1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxy-
la te (4a ) a n d Eth yl 4,5-Dih yd r o-4-oxo-8-(3-for m yl-2,5-
d ioxop yr r ol-1-yl)-7-t r iflu or om et h yl-1,2,4-t r ia zolo[1,5-
a ]q u in oxa lin e-2-ca r b oxyla t e (5a ). An excess of solid
potassium permanganate (3.0 mmol) was added portionwise
to a cooled (0 °C) suspension of 2a (1.19 mmol) in a 1:1 acetone/
water mixture (10 mL). The reaction mixture was stirred at 0
°C for 4 h and then was diluted with ice/water (30 g); the excess
of potassium permanganate was quenched with a 40% solution
of sodium bisulfite and the resulting white solid was collected,
washed with water, and purified by a silica gel column
chromatography (eluting system CHCl3/MeOH/AcOH 9:0.5:
0.5). Evaporation of the first eluates gives 5a (35% yield), while
the second crops of eluates afforded the desired compound 4a
(7.2% yield). Compound 4a displays the following spectral data:
1HNMR (DMSO-d6) 1.33 (t, 3H, CH3, J ) 6.96 Hz), 4.41 (q,
2H, CH2, J ) 6.96 Hz), 6.58 (d, 1H, pyrrole H-4, J ) 1.47 Hz),
7.04 (d, 1H, pyrrole H-5, J ) 1.47 Hz), 7.56 (s, 1H, pyrrole
H-2), 7.88 (s, 1H, ar), 8.17 (s, 1H, ar), 12.1 (br s, 1H, COOH),
12.8 (br s, 1H, NH); IR 3560, 3415, 1735, 1695, 1670.
Compound 5a shows the following spectral data: 1H NMR
(DMSO-d6) 1.36 (t, 3H, CH3, J ) 6.96 Hz), 4.43 (q, 2H, CH2, J
) 6.96 Hz), 7.76 (s, 1H, ar), 8.40 (s, 1H, ar), 8.71 (s, 1H, pyrrole
H-4), 10.06 (s, 1H, CHO), 12.6 (br s, 1H, NH); IR 3240, 1740,
1720, 1655.
5-Ch lor o-2-n itr o-4-tr iflu or om eth yla n ilin e (14).38 The
title compound was synthesized following the procedure
reported in ref 38 with some modification. A mixture of the
commercially available 2,4-dichloro-5-nitrobenzotrifluoride (7.7
mmol) in 2-methoxyethanol (6 mL) saturated with ammonia
was heated in a sealed tube at 100 °C for 16 h. The reaction
mixture was diluted with water (100 mL) and a solid precipi-
tated which was collected and washed with water. 1HNMR
(DMSO-d6) 7.25 (s, 1H, ar), 8.05 (s, 2H, NH2), 8.27 (s, 1H, ar);
IR 3510, 3400, 1640.
5-(Im idazol-1-yl)-2-n itr o-4-tr iflu or om eth ylan ilin e (15).38
5-Chloro-2-nitro-4-trifluoromethylaniline 14 (4.16 mmol) was
added to a solution of imidazole (16.0 mmol) and KOH (6.23
mmol) in anhydrous DMF (10 mL). The reaction mixture was
heated at 100°C for 1.5 h. The resulting dark red solution was
diluted with water (100 mL) and the solid which precipitated
was extracted with ethyl acetate (50 mL × 3). The dried (Na2-
SO4) organic layers were decolored with animal carbon and
evaporated to a small volume. The solid which crystallized was
collected and washed with a little of diethyl ether. 1H NMR
(DMSO-d6) 7.07-7.09 (m, 2H, ar + imidazole H-4), 7.41 (d,
1H, imidazole H-5, J ) 1.22 Hz), 7.86 (s, 1H, imidazole H-2),
8.12 (s, 2H, NH2), 8.37 (s, 1H, ar); IR 3460, 3280, 1650.
E t h yl N1-[5-(Im id a zol-1-yl)-2-n it r o-4-t r iflu or om et h -
ylp h en yl]h yd r a zon o-N2-ch lor oa ceta te (16). Concentrated
H2SO4 (5.9 mL) was added to a suspension of 15 (3.67 mmol)
in water (6 mL). A solution of NaNO2 (5%, 5.6 mL) was added
dropwise to the cooled (-5 °C) mixture, during an overall time
of 45 min. Cold (0 °C) methanol (6 mL) and an equimolar
amount of ethyl 2-chloro-3-oxobutanoate were added to the red
solution, which was kept at 0 °C for 10 min and then at room
temperature for 4 h. The yellow solution was diluted with
water (80 mL) and extracted with chloroform (60 mL × 4).
Evaporation of the dried (Na2SO4) organic layers yielded a solid
which was treated with ethyl acetate and collected: 1H NMR
(DMSO-d6) 1.26 (t, 3H, CH3, J ) 7.0 Hz), 4.33 (q, 2H, CH2, J
) 7.0 Hz), 7.18 (d, 1H, imidazole H-4, J ) 1.22 Hz), 7.55 (d,
1H, imidazole H-5, J ) 1.22 Hz), 7.70 (s, 1H, ar), 8.01 (s, 1H,
imidazole H-2), 8.59 (s, 1H, ar), 11,20 (br s, 1H, NH); IR 3265,
1730, 1630.
E t h yl 4,5-Dih yd r o-7-n it r o-4-oxo-8-(3-for m ylp yr r ol-1-
yl)-1,2,4-tr iazolo[1,5-a ]qu in oxalin e-2-car boxylate (7a ). The
title compound was prepared by reacting 13a (2.05 mmol) with
2,5-dimethoxy-3-tetrahydrofurancarboxaldehyde (3.08 mmol)
as described above for the preparation of 2a : 1H NMR (DMSO-
E t h yl N1-[5-(Im id a zol-1-yl)-2-n it r o-4-t r iflu or om et h -
ylp h en yl]-N2-oxa m id r a zon a te (17). Ammonia was bubbled
until saturation into a stirred solution of 16 (0.98 mmol) in

270 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Catarzi et al.
d6) 1.35 (t, 3H, CH3, J ) 6.96 Hz), 4.43 (q, 2H, CH2, J ) 6.96
Hz), 6.67 (d, 1H, pyrrole, H-4, J ) 1.47 Hz), 7.24 (d, 1H, pyrrole
H-5, J ) 1.47 Hz), 8.04 (s, 1H, pyrrole H-2), 8.18 (s, 1H, ar),
8.32 (s, 1H, ar), 9,77 (s, 1H, CHO), 12.9 (br s, 1H, NH); IR
3140, 1745, 1715, 1655.
E t h yl 4,5-Dih yd r o-7-n it r o-4-oxo-8-(p yr r ol-1-yl)-1,2,4-
tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxyla te (8a ). The title
compound was prepared by reacting 13a (1.25 mmol) with 2,5-
diethoxytetrahydrofuran (3.76 mmol) as described above for
the preparation of 3a : 1H NMR (DMSO-d6) 1.35 (t, 3H, CH3,
J ) 6.98 Hz), 4.43 (q, 2H, CH2, J ) 6.98 Hz), 6.29 (t, 2H,
pyrrole H-3 and H-4, J ) 2.19 Hz), 7.06 (t, 2H, pyrrole H-2
and H-5, J ) 2.19 Hz), 8.06 (s, 1H, ar), 8.10 (s, 1H, ar), 12.8
(br s, 1H, NH); IR 3160, 3070, 1715.
× 2). Evaporation of the dried (Na2SO4) organic layers yielded
a solid which was worked up with a little diethyl ether and
collected by filtration: 1H NMR (DMSO-d6) 6.59 (d, 1H, pyrrole
H-4, J ) 1.47 Hz), 7.07 (d, 1H, pyrrole H-5, J ) 1.47 Hz), 7.57
(s, 1H, pyrrole H-2), 7.90 (s, 1H, ar), 8.16 (s, 1H, ar), 12.8 (br
s, 1H, NH); IR 3600-3000, 1700, 1670.
4,5-Dih yd r o-8-(im id a zol-1-yl)-4-oxo-7-tr iflu or om eth yl-
1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid (6b). A
solution of NaOH (1 N, 2 mL) was added to a suspension of
6a (0.1 mmol) in EtOH (2.5 mL). The reaction mixture was
stirred at room temperature for 1 h and then was acidified
with glacial acetic acid. The resulting solid was collected and
washed with water: 1H NMR (DMSO-d6) 7.11 (s, 1H, imidazole
H-4), 7.45 (s, 1H, imidazole H-5), 7.88 (s, 1H, imidazole H-2),
7.92 (s, 1H, ar), 8.17 (s, 1H, ar), 12.8 (br s, 1H, NH); IR 3600-
3000, 1695, 1605.
4,5-Dih yd r o-7-n itr o-4-oxo-8-(3-for m ylp yr r ol-1-yl)-1,2,4-
tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid (7b). A solu-
tion of NaOH (3%, 12 mL) was added to a suspention of 7a
(0.5 mmol) in EtOH (13 mL). The reaction mixture was stirred
at room temperature for 1 h, diluted with water (10 mL), and
acidified with 6 N HCl. The resulting solid was collected by
filtration and washed with water. A second crop of 7b was
obtained by extracting the acidic mother liquor with ethyl
acetate (30 mL × 3); evaporation of the dried (Na2SO4) organic
layers yielded a solid which was worked up with a small
amount of diethyl ether and filtered. 1H NMR (DMSO-d6) 6.67
(d, 1H, pyrrole, H-4, J ) 1.47 Hz), 7.25 (d, 1H, pyrrole H-5, J
) 1.47 Hz), 8.04 (s, 1H, pyrrole H-2), 8,18 (s, 1H, ar), 8.29 (s,
1H, ar), 9,77 (s, 1H, CHO), 12.9 (br s, 1H, NH); IR 3140, 1735,
1675.
4,5-Dih yd r o-7-n itr o-4-oxo-8-(p yr r ol-1-yl)-1,2,4-tr ia zolo-
[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid (8b). A solution of
NaOH (3%, 10 mL) was added to a suspension of 8a (0.41
mmol) in EtOH (10 mL). The reaction mixture was stirred at
room temperature for 5 min and then was acidified with 6 N
HCl. After stirring at room temperature for 3 h, the suspension
was filtered under vacuum and the resulting solid was washed
with water: 1H NMR (DMSO-d6) 6.29 (t, 2H, pyrrole H-3 and
H-4, J ) 2.2 Hz), 7.05 (t, 2H, pyrrole H-2 and H-5, J ) 2.2
Hz), 8.05 (s, 1H, ar), 8.09 (s, 1H, ar), 12.8 (br s, 1H, NH); IR
3130, 1730.
Eth yl 4,5-Dih yd r o-7-n itr o-4-oxo-8-(3-ca r boxyp yr r ol-1-
yl)-1,2,4-tr iazolo[1,5-a ]qu in oxalin e-2-car boxylate (9a ). An
excess of solid potassium permanganate (1.2 mmol) was
portionwise added to a cooled (0-5 °C) suspension of 7a (0.48
mmol) in a 1:1 acetone/water mixture (10 mL). Small additions
were done in sequence at the disappearance of the character-
istic violet color of the oxidizing agent, in a total time of 10 h.
After stirring at room temperature for one night, the reaction
mixture was then diluted with ice-water (20 g) and the excess
of potassium permanganate was quenched with a 40% solution
of sodium bisulfite. The resulting solution was extracted with
ethyl acetate (50 mL × 3), and then evaporation of the dried
(Na2SO4) organic layers afforded a solid which was collected
and purified by silica gel column chromatography [eluting
1
system CHCl3/EtOH (9:1)]: H NMR (DMSO-d6) 1.34 (t, 3H,
CH3, J ) 6.96 Hz), 4.41 (q, 2H, CH2, J ) 6.96 Hz), 6.58 (d, 1H,
pyrrole H-4, J ) 1.47 Hz), 7.12 (d, 1H, pyrrole H-5, J ) 1.47
Hz), 7.68 (s, 1H, pyrrole H-2), 8.13 (s, 1H, ar), 8.24 (s, 1H, ar),
12.1 (br s, 1H, COOH), 12.9 (br s, 1H, NH); IR 1740, 1695.
4,5-Dih ydr o-4-oxo-8-(1,2,4-tr iazol-4-yl)-7-tr iflu or om eth yl-
1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid (1b). A
solution of NaOH (3%, 11 mL) was added to a suspension of
1a (0.46 mmol) in EtOH (11 mL). The mixture was stirred at
room temperature for 1.5 h. The solid was collected by
filtration and dissolved in the minimal amount of water;
acidification of the cold (5 °C) and clear solution by 6 N HCl
yielded a solid that, after standing 3 h in a ice bath, was
collected and washed with water: 1H NMR (DMSO-d6) 7.92
(s, 1H, ar), 8.47 (s, 1H, ar), 8.83 (s, 2H, triazole H-2 and H-5),
12.8 (br s, 1H, NH); IR 3520, 3360, 3140, 1725, 1685.
4,5-Dih yd r o-4-oxo-8-(3-for m ylp yr r ol-1-yl)-7-t r iflu or o-
m eth yl-1,2,4-tr iazolo[1,5-a ]qu in oxalin e-2-car boxylic Acid
(2b). A solution of NaOH (3%, 8 mL) was added to a
suspension of 2a (0.29 mmol) in EtOH (9 mL). The mixture
was stirred at room temperature for 15 min and then diluted
with water (20 mL). After cooling (0-5°C), the resulting
suspension was acidified with 6 N HCl and kept in an ice bath
for one night. The solid was collected by filtration and washed
with water: 1H NMR (DMSO-d6) 6.67 (d, 1H, pyrrole H-4, J
) 1,47 Hz), 7.16 (d, 1H, pyrrole H-5, J ) 1.47 Hz), 7.93 (s, 2H,
ar + pyrrole H-2), 8.22 (s, 1H, ar), 9.78 (s, 1H, CHO), 12.82 (s,
1H, NH); IR 1720, 1675, 1650.
4,5-Dih ydr o-7-n itr o-4-oxo-8-(3-car boxypyr r ol-1-yl)-1,2,4-
tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid (9b). A solu-
tion of NaOH (0.8 N, 2.5 mL) was added to a suspension of 9b
(0.097 mmol) in EtOH (2.5 mL). The reaction mixture was
stirred at room temperature for 1 h and then was diluted with
water (10 mL). The resulting solution was decolored with
activated carbon, acidified with 6 N HCl, and then extracted
with ethyl acetate (15 mL × 3). Evaporation of the dried (Na2-
SO4) organic layers yielded a yellow solid which was worked
up with a little diethyl ether and collected by filtration:
1HNMR (DMSO-d6) 6.61 (d, 1H, pyrrole H-4, J ) 1.47 Hz),
7.14 (d, 1H, pyrrole H-5, J ) 1.47 Hz), 7.71 (s, 1H, pyrrole
H-2), 8.16 (s, 1H, ar), 8.25 (s, 1H, ar), 12.2 (br s, 1H, COOH),
12.88 (s, 1H, NH); IR 1730.
4,5-Dih yd r o-4-oxo-8-(p yr r ol-1-yl)-7-t r iflu or om et h yl-
1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid (3b). A
solution of NaOH (3%, 13.5 mL) was added to a suspension of
3a (0.51 mmol) in EtOH (14 mL). The reaction mixture was
stirred at room temperature for 5 min and then, after cooling
(0-5 °C), was acidified with 6 N HCl. The resulting suspension
was stirred in an ice bath for 2 h and then the solid was
collected and washed with water: 1H NMR (DMSO-d6) 6.29
(t, 2H, pyrrole H-3 and H-4, J ) 2.2 Hz), 7.00 (t, 2H, pyrrole
H-2 and H-5, J ) 2.2 Hz), 7.92 (s, 1H, ar), 7.99 (s, 1H, ar),
12.81 (s, 1H, NH); IR 3195, 1730, 1715.
4,5-Dih yd r o-4-oxo-8-(3-ca r b oxyp yr r ol-1-yl)-7-t r iflu o-
r om et h yl-1,2,4-t r ia zolo[1,5-a ]q u in oxa lin e-2-ca r b oxylic
Acid (4b). A solution of NaOH (0.8 N, 5.8 mL) was added to
a suspension of 4a (0.23 mmol) in EtOH (5 mL). The reaction
mixture was stirred at room temperature for 1.5 h and then,
after cooling (0-5 °C), was acidified with 6 N HCl. The
resulting suspension was extracted with ethyl acetate (15 mL
4,5-Dih yd r o-8-n itr o-4-oxo-7-tr iflu or om eth yl-1,2,4-tr ia -
zolo[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid (10b). A solution
of NaOH (3%, 10 mL) was added to a suspension of 10a (0.4
mmol) in EtOH (11 mL). The reaction mixture was stirred at
room temperature for 3 h. The solid was collected and dissolved
in the minimum amount of water. The resulting solution was
acidified with 6 N HCl and then kept at room temperature
for 30 min. The suspension was filtered under vacuum and
the solid was washed with water: 1H NMR(DMSO-d6) 7.93
(s, 1H, ar), 8.84 (s, 1H, ar), 13.0 (br s, 1H, NH); IR 1735.
8-Am in o-4,5-d ih yd r o-4-oxo-7-tr iflu or om eth yl-1,2,4-tr i-
a zolo[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid (11b). A solu-
tion of NaOH (1 N, 4.5 mL) was added to a suspension of 11a
(0.73 mmol) in EtOH (3.7 mL). The reaction mixture was
heated at 90°C for 1 h. After cooling, a solid precipitated which
was collected and dissolved in a small amount of water (3 mL).
Acidification with glacial acetic acid of the resulting solution
yielded a solid which was collected and washed with water. A

Novel Selective AMPA Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 271
second crop of 10b was obtained after evaporation, at small
volume, of the hydroalcolic alkaline mother liquor and acidi-
fication with glacial acetic acid. The resulting solid was
collected and washed with water: 1H NMR (DMSO-d6) 5.87
(s, 2H, NH2), 7.47 (s, 1H, ar), 7.60 (s, 1H, ar), 12.12 (s 1H,
NH); IR 3600, 3380, 3260, 1685.
8-Acetyla m in o-4,5-d ih yd r o-7-n itr o-4-oxo-1,2,4-tr ia zolo-
[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid (12b). A solution of
NaOH (3%, 15 mL) was added to a suspension of 12a (0.55
mmol) in EtOH (15 mL). The reaction mixture was stirred at
room temperature for 30 min and then was acidified with
glacial acetic acid. The resulting suspension was kept at room
temperature for 30 min and then the solid was collected and
washed with water: 1H NMR (DMSO-d6) 2.11 (s, 3H, CH3),
7.91 (s, 1H, ar), 8.31 (s, 1H, ar), 10.36 (s, 1H, NH), 12.3 (br s
1H, NH); IR 3230, 1725, 1680.
8-Am in o-4,5-d ih yd r o-7-n it r o-4-oxo-1,2,4-t r ia zolo[1,5-
a ]qu in oxa lin e-2-ca r boxylic Acid (13b). A solution of 13a
(0.22 mmol) in concentrated HCl (2 mL) was heated at reflux
for 1 h. The resulting solid was collected and washed with
water: 1H NMR (DMSO-d6) 7.4 (br s, 2H, NH2), 7.77 (s, 1H,
ar), 8.07 (s, 1H, ar), 12.21 (s, 1H, NH); IR 3480, 3370, 3280,
1700, 1650.
P h a r m a cology. Bin d in g Assa y. Rat cortical synaptic
membrane preparation, [3H]glycine, [3H]AMPA, and [3H]-(+)-
MK-801 binding experiments were performed following the
procedures described in refs 30, 45, and 35, respectively.
[3H]Ka in a te Bin d in g. High-affinity and low-affinity [3H]-
kainate binding assays were performed on rat cortical mem-
branes according to the method of J ohansen et al.12 The
standard high-affinity assay was performed at 2 nM [3H]-
kainate (NEN Life Science Products; Boston, MA; specific
activity 58 Ci/mmol) in Tris-HCl buffer (50 mM, pH 7.4),
whereas the low-affinity binding assay was performed at 20
nM [3H]kainate in Tris-HCl buffer containing 20 mM calcium
chloride. Binding was terminated by rapid filtration over glass
fiber filters (Millipore APFC02500). Non-specific binding was
determined in the presence of 1 mM glutamate.
ratio obtained under control conditions (buffer plus 50 µM
cyclothiazide only).
IC50 values for antagonists were determined from inhibition
curves, based on four different drug concentrations, using a
function fitting program (ALLFIT).
Sa m p le P r ep a r a tion a n d Resu lt Ca lcu la tion . A stock
1 mM solution of the test compound was prepared in 50%
DMSO. Subsequent dilutions were accomplished in buffer. The
IC50 values were calculated from three or four displacement
curves based on four to six scalar concentrations of the test
compound in triplicate using the ALLFIT computer program49
and, in the case of tritiated glycine and AMPA binding,
converted to Ki values by application of the Cheng-Prusoff
equation.50 Under our experimental conditions the dissociation
constants (KD) for [3H]glycine (10 nM) and [3H]-DL-AMPA (8
nM) were 75 ( 6 and 28 ( 3 nM, respectively.
Refer en ces
(1) Bra¨uner-Osborne, H.; Egebjerg, J .; Nielsen, E. Ø.; Madsen, U.;
Krogsgaard-Larsen, P. Ligands for glutamate receptor: Design
and therapeutic prospects. J . Med. Chem. 2000, 43, 2609-2645.
(2) Dingledine, R.; Borges, K.; Bowie, D.; Traynelis, S. F. The
glutamate receptor ion channels. Pharmacol. Rev. 1999, 51,
7-61.
(3) Lees, G. J . Pharmacology of AMPA/Kainate receptor ligands and
their therapeutic potential in neurological and psychiatric
disorders. Drugs 2000, 59, 33-78.
(4) Madsen, U.; Stensbøl, T. B.; Krogsgaard-Larsen, P. Inhibitors
of AMPA and kainate receptors. Curr. Med. Chem. 2001, 8,
1291-1301.
(5) Ruscheweyh, R.; Sandku¨hler. Role of Kainate receptors in
nociception. J . Brain. Res. Rev. 2002, 40, 215-222.
(6) Bigge, C. F.; Boxer, P. A.; Ortwine, D. F. AMPA/Kainate
Receptors. Curr. Pharm. Des. 1996, 2, 397-412.
(7) Bigge, C. F.; Nikam, S. S. AMPA receptor agonists, antagonists
and modulators: Their potential for clinical utility. Exp. Opin.
Ther. Patents 1997, 7, 1099-1114.
(8) Chimirri, A.; Gitto, R.; Zappala`, M. AMPA receptor antagonists.
Exp. Opin. Ther. Patents 1999, 9, 557-570.
(9) Nikam, S. S.; Kornberg, B. E. AMPA receptor antagonists. Curr.
Med. Chem. 2001, 8, 155-170.
Electr op h ysiologica l Assa y. The cortical wedge prepara-
tion described by Mannaioni et al.46 was used while the
electrophysiological assays were performed following the
procedures described in ref 37.
(10) Stensbøl, T. B.; Madsen, U.; Krogsgaard-Larsen, P. The AMPA
receptor binding site: Focus on agonists and competitive an-
tagonists. Curr. Pharm. Des. 2002, 8, 857-872.
(11) Auberson, Y. P. Competitive AMPA antagonism:
A novel
mechanism for antiepileptic drugs? Drugs Future 2001, 26, 463-
471.
[3H]NE Relea se fr om Syn a p tosom es. The following
procedure was adapted from the method of Pittaluga et al.;47
cyclothiazide was added to potentiate AMPA-induced [3H]-
norepinephrine release.48 Briefly, crude synaptosomes pre-
pared from rat hippocampi were resuspended in an oxygenated
(95% O2 and 5% CO2) physiological standard medium having
the following composition (mM): NaCl 125, KCl 3, MgSO4 1.2,
CaCl2 1.2, NaH2PO4 1.0, NaHCO3 22, glucose 10; pH 7.2-7.4.
Synaptosomes were then incubated at 37 °C for 15 min with
50 nM levo-[ring-2,5,6-3H]norepinephrine (NEN Life Science
Products; specific activity 56.4 Ci/mmol) in the presence of 1
µM 6-nitroquipazine (Sigma-RBI) to prevent [3H]catecholamine
uptake by serotoninergic nerve terminals. Identical aliquots
of the labeled synaptosomal suspension were then introduced
in parallel superfusion chambers maintained at 37 °C. Super-
fusion was carried out at a rate of 0.6 mL/min, using media
aerated with 95% O2-5% CO2, for a total period of 58 min. After
46 min of superfusion to equilibrate the system, four consecu-
tive 3 min fractions were collected. Cyclothiazide (Sigma-RBI)
(50 µM) was added 6 min prior to collecting the first fraction.
S-AMPA (Tocris) (50 µM) was introduced 1 min before com-
pleting the first fraction collection, while antagonists were
added 8 min before AMPA, that is, together with cyclothiazide.
The fractions were collected and the superfused synaptosomes
counted for radioactivity.
(12) J ohansen, T. H.; Drejer, J .; Watjen, F.; Nielsen, E. O. A novel
non-NMDA receptor antagonist shows selective displacement of
low-affinity [3H]kainate binding. Eur. J . Pharmacol. 1993, 246,
195-204.
(13) Loscher, W.; Lehmann, H.; Behl, B.; Seemann, D.Teschendorf,
H. J . Hofmann, H. P.; Lubisch, W.; Hoger, T.; Lemaire, H. G.;
Gross, G. A new pyrrolyl-quinoxalinedione series of non-NMDA
glutamate receptor antagonists: Pharmacological characteriza-
tion and comparison with NBQX and valproate in the kindling
model of epilepsy. Eur. J . Neurosci. 1999, 11, 250-262.
(14) J ohnson, J . W.; Ascher, P. Glycine potentiates the NMDA
response in cultured mouse brain neurons. Nature 1987, 325,
529-531.
(15) Danysz, W.; Parsons, C. G. Glycine and N-Methyl-D-Aspartate
receptors: Physiological significance and possible therapeutic
applications. Pharmacol. Rev. 1998, 50, 597-664.
(16) Donati, D.; Micheli, F. Recent development in glycine antago-
nists. Exp. Opin. Ther. Patents. 2000, 10, 667-673.
(17) Wolfe, J . F.; Greenwood, T. D.; Mulheron, J . M. Recent trends
in the development of new anti-epileptic drugs. Exp. Opin. Ther.
Patents 1998, 8, 361-381.
(18) Kulagowski, J . J . Glycine-site NMDA receptor antagonists: An
update. Exp. Opin. Ther. Patents 1996, 6, 1069-1079.
(19) Kulagowski, J . J .; Leeson, P. D. Glycine-site NMDA receptor
antagonists. Exp. Opin. Ther. Patents 1995, 5, 1061-1075.
(20) Keana, J . F. W.; Kher, S. M.; Cai, S. X.; Dinsmore, C. M.; Glenn,
A. G.; Guastella, J .; Huang, J . C.; Ilyn, V.; Lu¨, Y.; Mouser, P.
L.; Woodward, R. M.; Weber, E. Synthesis and structure-activity
relationships of substituted 1,4-dihydroquinoxaline-2,3-diones:
Antagonists of N-methyl-D-aspartate(NMDA) receptor glycine
sites and non-NMDA glutamate receptors. J . Med. Chem. 1995,
38, 4367-4379.
The amount of radioactivity released into each superfusate
fraction was expressed as a percentage of the total synapto-
somal radioactivity at the start of the collected fraction. Drug
effects were evaluated from the ratio between the [3H]-
norepinephrine release in the fraction corresponding to the
maximal effect (third fraction) and that in the first fraction
(basal efflux). This ratio was compared with the corresponding
(21) Bigge, C. F.; Malone, T. C.; Boxer, P. A.; Nelson, C. B.; Ortwine,
T. F.; Schelkun, R. M.; Retz, D. M.; Lescosky, L. J .; Borosky, S.
A.; Vartanian, M. G.; Schwarz, R. D.; Campbell, G. W.; Ro-
bichaud, L. J .; Wa¨tjen, F. Synthesis of 1,4,7,8,9,10-hexahydro-

272 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Catarzi et al.
9-methyl-6-nitropyrido[3,4-f]-quinoxaline-2,3-dione and related
quinoxalinediones: Characterization of R-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid (and N-methyl-D-aspartate)
receptor and anticonvulsant activity. J . Med. Chem. 1995, 38,
3720-3740.
(35) Varano, F.; Catarzi, D.; Colotta, V.; Cecchi, L.; Filacchioni G.;
Galli, A.; Costagli, C. Synthesis of a set of ethyl 1-carbamoyl-
3-oxoquinoxaline-2-carboxylates and of their constrained ana-
logue imidazo[1,5-a]quinoxaline-1,3,4-triones as glycine/NMDA
receptor antagonists. Eur. J . Med. Chem. 2001, 36, 203-209.
(36) Catarzi, D.; Colotta; V.; Varano; F.; Filacchioni, G.; Galli, A.;
Costagli, C.; Carla`, V. Synthesis, ionotropic glutamate receptor
binding affinity and structure-activity relationships of a new
set of 4,5-dihydro-8-heteroaryl-4-oxo-1,2,4-triazolo[1,5-a]quinox-
aline-2-carboxylates analogues of TQX-173. J . Med. Chem. 2001,
44, 3157-3165.
(37) Varano, F.; Catarzi, D.; Colotta, V.; Filacchioni, G.; Galli, A.;
Costagli, C.; Carla`, V. Synthesis and biological evaluation of a
new set of pyrazolo[1,5-c]quinazoline-2-carboxylates as novel
excitatory amino acid antagonists. J . Med. Chem. 2002, 45,
1035-1044.
(38) Gu¨ngo¨r, T.; Fouquet, A.; Teulon, J . M.; Provost, D.; Cazes, M.;
Cloarec, A. Cardiotonic agents. Synthesis and cardiovascular
properties of novel 2-arylbenzimidazole and azabenzimidazole.
J . Med. Chem. 1992, 35, 4455-4463.
(39) Honore´, T.; Drejer, J .; Nielsen, M. Calcium discriminates two
[3H]kainate binding sites with different target sizes in rat cortex.
Neurosci. Lett. 1986, 65, 47-52.
(22) Nikam, S. S.; Cordon, J . J .; Ortwine, D. F.; Heimbach, T. H.;
Blackburn, A. C.; Vartanian, M. G.; Nelson, C. B.; Schwarz, R.
D.; Boxer, P. A.; Rafferty, M. F. Design and synthesis of novel
quinoxaline-2,3-dione AMPA/GlyN receptor antagonists: Amino
acid derivatives. J . Med. Chem. 1999, 42, 2266-2271.
(23) Cai, S. X.; Huang, J . C.; Espitia, S. A.; Tran, M.; Ilyn, V. I.;
Hawkinson, J . E.; Woodward, R. M.; Weber, E.; Keana, J . F. W.
5-(N-oxyaza)-7-substituted-1,4-dihydroquinoxaline-2,3-diones: Novel,
systematically active and broad spectrum antagonists for NMDA/
glycine, AMPA, and kainate receptors. J . Med. Chem. 1997, 40,
3679-3686.
(24) Auberson, Y. P.; Acklin, P.; Bishoff, S.; Moretti, R.; Ofner, S.
N-Phosphonoalkyl-5-aminomethylquinoxaline-2,3-diones: In vivo
active AMPA and NMDA (Glycine) antagonists. Bioorg. Med.
Chem. Lett. 1999, 9, 249-254.
(25) Leeson, P. D.; Iversen, L. L. The glycine site on the NMDA
receptor: Structure-activity relationships and therapeutic po-
tential. J . Med. Chem. 1994, 37, 4053-4067.
(26) Homori, J .; Shimizu-Sasamata, M.; Okada, M.; Sakamoto, S.
8-(1H-imidazol-1-yl)-7-nitro-4-(5H)-imidazo[1,2-a]quinoxalin-
one and related compounds: Synthesis and structure activity
relationships for the AMPA-type non-NMDA receptor. J . Med.
Chem. 1997, 40, 2053-2063.
(27) Armstrong, N.; Gouaux, E. Mechanism for activation and
antagonism of an AMPA-sensitive glutamate receptor: Crystal
strucure of the GluR2 ligand binding core. Neuron 2000, 28,
165-181.
(40) Verdoorn, T. A.; J ohansen, T. H.; Drejer, J .; Nielsen, E. O.
Selective block of recombinant GluR6 receptors by NS-102, a
novel non-NMDA receptor antagonist. Eur. J . Pharmacol. 1994,
269, 43-49.
(41) Burton, N. R.; Smith, D. A. S.; Stone, T. W. A quantitative
pharmacological analysis of some excitatory amino acid receptors
in the mouse neocortex in vitro. Br. J . Pharmacol. 1988, 93,
693-701.
(28) Hogner, A.; Greenwood, J . R. Liljefors, T.; Lunn, M. L.; Egebjerg,
J .; Larsen, I. K.; Gouaux, E.; Kastrup, J . S. Competitive
antagonism of AMPA receptors by ligands of different classes:
Crystal structure of ATPO bound to the GluR2 ligand-binding
core, in comparison with DNQX. J . Med. Chem. 2003, 46, 214-
221.
(29) Varano, F.; Catarzi, D.; Colotta, V.; Cecchi, L.; Filacchioni G.;
Galli, A.; Costagli, C. Tricyclic heteroaromatic systems. 3-sub-
stituted 1,2,3,5,6,7-hexahydro-2,5,6-trioxopyrazino[1,2,3-d,e]-qui-
noxalines as novel antagonists at the Glycine-NMDA site.
Farmaco 1997, 52, 179-182.
(30) Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filacchioni, G.;
Galli, A.; Costagli, C. Synthesis and biological evaluation of a
series of quinazoline-2-carboxylic acids and quinazoline-2,4-
diones as glycine-NMDA antagonists: A pharmacophore model
based approach. Arch. Pharm. Pharm. Med. Chem. 1997, 330,
129-134.
(31) Varano, F.; Catarzi, D.; Colotta, V.; Filacchioni G.; Cecchi, L.;
Galli, A.; Costagli, C. Synthesis of 2-substituted-6,8-dichloro-
3,4-dihydro-3-oxo-2H-1,4-benzothiazine-1,1-dioxides and -1-
oxides as Glycine-NMDA receptor antagonists. Farmaco 1998,
53, 752-757.
(32) Varano, F.; Catarzi, D.; Colotta, V.; Cecchi, L.; Filacchioni G.;
Galli, A.; Costagli, C. Synthesis, Glycine/NMDA and AMPA
binding activity of some new 2,5,6-trioxopyrazino[1,2,3-de]qui-
noxalines and of their restricted analogues 2,5-dioxo- and 4,5-
dioxoimidazo[1,5,4-de]quinoxalines. Arch. Pharm. Pharm. Med.
Chem. 1999, 332, 201-207.
(33) Catarzi, D.; Colotta, V.; Varano, F.; Cecchi, L.; Filacchioni, G.;
Galli, A.; Costagli, C. 4,5-Dihydro-1,2,4-triazolo[1,5-a]quinoxalin-
4-ones: Excitatory amino acid antagonists with combined gly-
cine/NMDA and AMPA receptor affinity. J . Med. Chem. 1999,
42, 2478-2484.
(34) Catarzi, D.; Colotta, V.; Varano, F.; Cecchi, L.; Filacchioni, G.;
Galli, A.; Costagli, C.; Carla`, V: 7-Chloro-4,5-dihydro-8-(1,2,4-
triazol-4-yl)-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxy-
lates as novel highly selective AMPA receptor antagonists. J .
Med. Chem. 2000, 43, 3824-3826.
(42) Pittaluga, A.; Raiteri, M. N-methyl-D-aspartic acid (NMDA) and
non-NMDA receptors regulating hippocampal norepinephrine
release. I. Location on axon terminals and pharmacological
characterization. J . Pharmacol. Exp. Ther. 1992, 260, 232-237.
(43) Wong, E. H. F.; Kemp, J . A.; Priestley, T.; Knight, A. R.;
Woodruff, G. N.; Iverson, L. L. The anticonvulsant MK-801 is a
potent N-methyl-D-aspartate antagonist. Proc. Natl. Acad. Sci.
U.S.A. 1986, 83, 7104-7108.
(44) Wong, E. H. F.; Knight, A. R.; Ranson, R. Glycine modulates
[3H]MK-801 binding to the NMDA receptor in rat brain. Eur. J .
Pharmacol. 1987, 142, 487-488.
(45) Nielsen, E. O.; Madsen, U.; Schaumburg, K.; Brehm, L.; Krogs-
gaard-Larsen, P. Studies on receptor-active conformations of
excitatory amino acid agonists and antagonists. Eur. J . Chem.-
Chim. Ther. 1986, 21, 433-437.
(46) Mannaioni, G.; Carla`, V.; Moroni, F. Pharmacological charac-
terization of metabotropic glutamate receptors potentiating
NMDA responses in mouse cortical wedge preparations. Br. J .
Pharmacol. 1996, 118, 1530-1536.
(47) Pittaluga, A.; Thellung, S.; Maura, G.; Raiteri, M. Characteriza-
tion of two central AMPA-preferring receptors having distinct
location, function and pharmacology. Naunyn-Schmiedeberg’s
Arch. Pharmacol. 1994, 349, 555-558.
(48) Desai, M. A.; Burnett, J . P.; Schoepp, D. D. Cyclothiazide
selectively potentiates AMPA- and kainate-induced [3H]norepi-
nephrine release from rat hippocampal slices. J . Neurochem.
1994, 63, 231-237.
(49) De Lean, A.; Munson, P. J .; Rodbard, D. Simultaneous analysis
of families of sigmoidal curves: Application to bioassay, radio-
ligand assay and physiological dose-response curves. Am. J .
Physiol. 1978, 235, E97-102.
(50) Cheng, Y.-C.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes
50% inhibition (IC50) of an enzymatic reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
J M030906Q