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]quinoxaline-2- carboxylates analogues of TQX-173

Article abstract of DOI:10.1021/jm010862q

A series of 4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a] quinoxaline-2-carboxylates analogues of TQX-173 (1b), bearing different nitrogen-containing heterocycles at position-8, were synthesized as AMPA receptor antagonists. All the reported compounds were also

Full text of DOI:10.1021/jm010862q

J . Med. Chem. 2001, 44, 3157-3165
3157
Syn th esis, Ion otr op ic Glu ta m a te Recep tor Bin d in g Affin ity, a n d
Str u ctu r e-Activity Rela tion sh ip s of a New Set of
4,5-Dih yd r o-8-h eter oa r yl-4-oxo-1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxyla tes
An a logu es of TQX-173
Daniela Catarzi,*,† Vittoria Colotta,† Flavia Varano,† Guido Filacchioni,† Alessandro Galli,‡ Chiara Costagli,‡ and
Vincenzo Carla`‡
Dipartimento di Scienze Farmaceutiche, Universita’ di Firenze, Via G. Capponi, 9, 50121 Firenze, Italy, and Dipartimento di
Farmacologia Preclinica e Clinica, Universita’ di Firenze, Viale G. Pieraccini, 6, 50134 Firenze, Italy
Received March 2, 2001
A series of 4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylates analogues of TQX-
173 (1b), bearing different nitrogen-containing heterocycles at position-8, were synthesized as
AMPA receptor antagonists. All the reported compounds were also biologically evaluated for
their binding at glycine/NMDA and KA receptors to better assess their selectivity toward the
AMPA receptor. Structure-activity relationships (SAR) on these TQX derivatives have
evidenced that the precise positioning of the nitrogen atoms and the specific electronic
topography of the 8-heteroaromatic ring are both important for the anchoring to the AMPA
receptor. In fact, it has been 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 selective
AMPA receptor antagonists. Functional antagonism at both AMPA receptor and NMDA
receptor-ion channel complex was evaluated by assessing the ability of some selected compounds
to inhibit depolarization induced by 5 µM AMPA or NMDA in mouse cortical wedge
preparations.
In tr od u ction
complete inhibition of NMDA-mediated neurodegenera-
tive effects.
It has been well-established that many neurological
disorders are caused by excessive release of glutamate
(Glu) from presynaptic terminals which overstimulate
postsynaptic glutamate receptors (GluRs). GluRs medi-
ate neurotransmission at the majority of fast excitatory
synapses in the mammalian central nervous system.
The postsynaptic activity of Glu is mediated by both
ionotropic receptors (iGluRs), ligand-gated ion channels,
and metabotropic receptors (mGluRs), coupled to G-
proteins. Historically the iGluRs were broadly subdi-
vided into N-methyl-D-aspartate (NMDA), (R,S)-2-amino-
3-(3-hydroxy-5-methyl-4-isoxazolyl)propionicacid (AMPA),
and kainate (KA) receptors on the basis of their respec-
tive affinities for these exogenous amino acids.1
There has been considerable interest in mixed AMPA
and Gly/NMDA antagonists as well as selective AMPA
antagonists that have demonstrated significant neuro-
protective action in several models of cerebral ischemia
and neuronal injury and have provided the basis for
extensive research in this area.2-4,9-10
As part of a program aimed at finding novel competi-
tive and noncompetitive iGluR antagonists11-17, we
previously studied a set of 4,5-dihydro-4-oxo-1,2,4-
triazolo[1,5-a]quinoxaline-2-carboxylates (TQXs) that
possessed combined Gly/NMDA and AMPA receptor
affinity,15 thus providing further evidence of the struc-
tural similarities and differences of the binding pockets
of both receptor recognition sites.2,18 On this basis we
hypothesized that the TQX framework, suitably modi-
fied, could lead to selective AMPA receptor antagonists.
Thus, we have recently reported, in preliminary
form,16 the AMPA receptor selective antagonists 7-chloro-
4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-car-
boxylates 1a ,b and 2a ,b, each bearing at position-8 a
(1,2,4-triazol-4-yl) and a (imidazol-1-yl) moiety (Chart
1). Compounds 2a ,b were rationally designed as AMPA
receptor antagonists.3,19 The emerging novelty of the
work16 is the fact that introduction of the 8-(1,2,4-
triazol-4-yl) substituent has led to a more potent and
selective AMPA receptor antagonist (TQX-173, i.e.,
compound 1b) than that bearing the claimed 8-(imida-
zol-1-yl) moiety (compound 2b).
Competitive iGluR antagonists have been proposed
as potentially useful neuroprotective agents for the
prevention and treatment of several acute and chronic
neurological disorders.1-4 Moreover, an alternative ap-
proach to antagonize the overstimulation of postsynaptic
GluRs by excessive endogenous Glu was represented by
the use of noncompetitive NMDA antagonists acting at
the strychnine-insensitive glycine site (Gly/NMDA) on
the NMDA receptor.5-7 In fact, a unique feature of the
NMDA receptor is the presence of an allosteric modula-
tory site at which the amino acid glycine acts as a
necessary coagonist in receptor activation.8 Thus, block-
age of the Gly/NMDA site by antagonists results in a
* To whom correspondence should be addressed: Tel: +39 55
2757294. Fax: +39 55 240776. E-mail: daniela.catarzi@unifi.i.
† Dipartimento di Scienze Farmaceutiche.
To shed more light on the SAR of these triazoloqui-
noxaline derivatives, we report, in this fully detailed
version, the synthesis and binding affinity at AMPA,
‡ Dipartimento di Farmacologia Preclinica e Clinica.
10.1021/jm010862q CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/11/2001

3158 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Catarzi et al.
Ch a r t 1
Sch em e 2a
Sch em e 1a
a
Reagents and conditions: (a) Het-H, KOH, DMF; (b) NaNO2/
conc H2SO4, NaBF4; (c) CH3COCHClCOOEt; (d) NH3(g), dioxane.;
(e) ClCOCOOEt, toluene; (f) conc H2SO4; (g) iron, AcOH.
its 7-unsubstituted analog15 were regioselectively ni-
trated at position-8 to yield 9a 15 and 10a , respectively,
which were reduced to their corresponding 8-amino
derivatives 11a 16 and 12a (Scheme 1). By reacting 11a
and 12a with diformylhydrazine, the 8-(1,2,4-triazol-4-
yl) esters 1a 16 and 3a were prepared. The 8-(pyrrol-1-
yl) ester 4a and the 8-(3-formylpyrrol-1-yl) ester 5a were
obtained by reacting 11a either with 2,5-diethoxytet-
rahydrofuran or 2,5-dimethoxy-3-tetrahydrofurancar-
boxaldehyde, respectively. The 7-(1,2,4-triazol-4-yl) ester
6a was obtained from the reaction of ethyl 7-amino-4,5-
dihydro-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carbox-
ylate15 with diformylhydrazine (Scheme 1).
By reacting the commercially available 4,5-dichloro-
2-nitroaniline with the suitable heterocycle, the corre-
sponding 4-chloro-5-heteroaryl-2-nitroanilines 1316,20
-
15 were isolated (Scheme 2). The diazonium tetrafluobor-
ates of 13-15 were reacted with ethyl 2-chloro-3-
oxobutanoate to yield the N2-chloroacetates 1616-18,
which were transformed with ammonia into their cor-
responding N2-oxamidrazonates 1916-21. Reaction of 20
and 21 with ethyloxalyl chloride yielded the N3-ethox-
alyl derivatives 22 and 23, respectively. By reacting the
N2-oxamidrazonate 19 with ethyloxalyl chloride the
diethyl 1-[4-chloro-5-(imidazol-1-yl)-2-nitrophenyl]-1,2,4-
triazole-3,5-dicarboxylate 2416 was directly obtained.
The diethyl triazole-3,5-dicaboxylates 25 and 26 were
obtained by cyclization with concentrated H2SO4 of the
N3-ethoxalyl derivatives 22 and 23, respectively. Reduc-
tion of the nitro group of 24-26 afforded the tricyclic
esters 2a 16,7a , and 8a .
a
Reagents and conditions: (a) 90% HNO3; (b) iron, AcOH; (c)
(OHCNH)2, pyridine, Me3SiCl, Et3N; (d) 2,5-diethoxytetrahydro-
furan, AcOH; (e) 2,5-dimethoxy-3-tetrahydrofurancarboxyalde-
hyde, AcOH.
Gly/NMDA, and KA receptors of all the triazoloquinoxa-
line-2-carboxylates bearing different substituents at
position-8.
Ch em istr y
Finally, the hydrolysis of the esters 1a -12a to their
corresponding acids 1b-12b is depicted in Scheme 3.
The ethyl esters of the 8-heteroaryl-4,5-dihydro-4-oxo-
1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylates 1a -8a
were prepared following the two different synthetic
strategies illustrated in Schemes 1 and 2.
Resu lts a n d Con clu sion s
The previously reported ethyl 7-chloro-4,5-dihydro-
The triazoloquinoxalines 1a ,b-12a ,b, together with
NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]qui-
4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylate15 and

A New Set of Analogues of TQX-173
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3159
Sch em e 3a
of the N3-nitrogen from the imidazole ring at position-8
of compounds 2a ,b to give the 8-(pyrrol-1-yl) derivatives
4a ,b causes an 8-fold reduction in potency in the
2-carboxylic ester derivative (compare 4a to 2a ) but does
not affect the binding affinity of the 2-carboxylic acid
derivative (compare 4b to 2b). The same applies to the
8-(pyrazol-1-yl) derivatives 7a ,b with respect to those
bearing the 8-(1,2,4-triazol-1-yl) moiety 8a ,b. Thus,
these results indicate that the presence of a free
carboxylic group at position-2 is not an essential feature
when a N3-nitrogen-containing heterocycle is present at
position-8.
These data led to the hypothesis that the N3-nitrogen
atom could act as a proton acceptor in a hydrogen bond
with a receptor site, thus reinforcing the receptor-
ligand interaction.10,18 This hypothesis is confirmed by
the binding affinity of compound 5a , which is formally
obtained by the introduction of a 3-formyl group on the
8-(pyrrol-1-yl) moiety of 4a . In fact, 5a shows a 10-fold
increased AMPA binding affinity with respect to 4a but
is comparable to that of the 8-(imidazol-1-yl) derivative
2a . Furthermore, the 2-carboxylic acid 5b is only 3-fold
more active at the AMPA receptor than the correspond-
ing ester 5a . These data indicate that the 3-formyl group
could replace the N3-nitrogen atom of the 8-heteroaryl
ring acting as a proton acceptor group in a hypothetical
hydrogen bond receptor-ligand interaction.
Furthermore, the AMPA binding results of the 7-chlo-
ro-8-heteroaryl-2-carboxylic acid derivatives 1b, 2b, 4b,
5b, 7b, and 8b indicate that the precise positioning of
the nitrogen atoms and the specific electronic topogra-
phy of the 8-heterocyclic rings are both important for
potent but also selective AMPA receptor antagonists
belonging to the TQX series. In fact, compound 4b,
which bears the pyrrole ring at position-8, is equipotent
at both AMPA and Gly/NMDA receptors. Introduction
of a N3-nitrogen atom on the 8-(pyrrol-1-yl) moiety of
4b gives the potent and AMPA selective 8-(imidazol-1-
yl) derivative 2b. On the contrary, introduction of a N2-
nitrogen atom on the pyrrole ring of 4b yields the
8-(pyrazol-1-yl) derivative 7b, which is as nonselective
as the parent compound 4b. These results are confirmed
by the binding data of the compound 8b, which bears
both the N2- and N3 (i.e., the N4 of the 8-(1,2,4-triazol-
1-yl) moiety) nitrogen atoms on the heterocyclic ring.
In fact, because of the beneficial effect of the N3-nitrogen
atom, compound 8b, though bearing the N2-nitrogen,
is as selective as 2b.
a
Reagents and conditions: (a) 0.8 N NaOH, EtOH, 6 N HCl or
AcOH.
noxaline) and DCKA (5,7-dichlorokynurenic acid) as
standard compounds, were tested for their ability to
displace tritiated AMPA, glycine, and KA from their
specific binding sites in rat cortical membranes. The
binding results, shown in Table 1, indicate that intro-
duction of different heteroaromatic rings at position-8,
holding the 7-chloro substitution pattern constant, leads
to some AMPA receptor antagonists belonging to the
TQX series. In particular, the previously reported
7-chloro-4,5-dihydro-8-(1,2,4-triazol-4-yl)-4-oxo-1,2,4-
triazolo[1,5-a]quinoxaline-2-carboxylates 1a ,b16 are the
most potent and selective AMPA receptor antagonists
among the herein reported compounds. These data point
out that the (1,2,4-triazol-4-yl) moiety at position-8 of
these tricyclic derivatives could be an adequate sub-
stituent in distinguishing the AMPA receptor from the
glycine/NMDA site.
Replacement of the 8-nitro group of 9b15,16 with an
amino one gives compound 11b, which has an AMPA
binding affinity comparable to that of 9b. The same
applies to the corresponding 2-carboxylic ester 11a with
respect to 9a . The similar effect exerted by both the
nitro and the amino groups on the AMPA binding
affinity might be explained as follows. The 8-nitro
substituent, due to its electron-withdrawing effect, could
increase the NH lactame acidity, thus reinforcing the
essential hydrogen-bond receptor-ligand interaction at
this level.2,10,18,19 On the other hand, the 8-amino group
could act as a proton acceptor in a hydrogen-bond
interaction with a receptor site.2,10,18 The unquestion-
ably different nature of the nitro and amino group might
explain the reduced difference between the Ki values of
11a ,b relative to that of 9a ,b. In fact, while the 8-amino-
We previously reported that the presence of a free
carboxylic group at position-2 on the TQX framework
was not an essential feature for the anchoring to the
AMPA receptor.15 On the basis of the present study, this
statement can be generally accepted, but it is necessary
to go into greater depth. In fact, as with most of the
previously reported combined glycine/NMDA and AMPA
antagonists belonging to the TQX series,15 the carboxylic
acid derivatives 1b, 2b,16 and 8b, bearing a N3-nitrogen-
containing heterocycle at position-8, are only 2-5-fold
more active at the AMPA receptor than their corre-
sponding esters 1a , 2a ,16 and 8a , respectively. On the
contrary, the 2-carboxylic acid derivatives 4b and 7b,
bearing a 8-heterocyclic ring lacking the N3- nitrogen
atom, are about 20-fold more active than their corre-
sponding ester derivatives 4a and 7a . In fact, removal

3160 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Ta ble 1. Binding Activity at iGluRs of TQX Derivativesa
Catarzi et al.
a
b
The tested compounds were dissolved in DMSO and then diluted with the appropriate buffer. Inhibition constant (Ki) values were
means ( SEM of three or four separate determinations in triplicate. c Percentage of inhibition (I%) of specific binding at 100 µM
d
concentration. Concentrations necessary for 50% inhibition (IC50). The IC50 values were means ( SEM of three or four separate
determinations in triplicate. e Reference 16. f Reference 15. Not tested.
g
2-carboxylic acid 11b is about only 4-fold more active
than its corresponding ester 11a , the 8-nitro-2-carboxyl-
ic acid 9b shows about a 40-fold increased binding
activity with respect to its corresponding ester 9a .
Removal of the chlorine atom at position-7 of 1a ,b to
give compounds 3a ,b causes a dramatic decrease in
AMPA binding affinity, confirming the importance of
the presence of electron-withdrawing groups at this
position to obtain potent AMPA receptor antagonists.
The same applies to compounds 10a ,b and 12a ,b15
compared to 9a ,b and 11a ,b, respectively, although not
to the same extent. In accordance with the reported

A New Set of Analogues of TQX-173
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3161
Ta ble 2. Functional Antagonism at NMDA and AMPA Sites
AMPA receptor pharmacophore model,2,19 the crucial
effect of the 7-chlorine atom is probably due to the
increased acidity of the NH lactam proton.
mouse cortical wedge preparation:
IC50 (µM) vs agonist-induced depolarizationsa
It has to be noted that the presence of a proton-
acceptor substituent at position-7 of the TQX frame-
work, capable of engaging a hydrogen-bond receptor-
ligand interaction, is well tolerated by the AMPA
receptor.2,18 In fact, the 7-(1,2,4-triazol-4-yl) derivatives
6a ,b, though lacking a substituent at position-8, show
AMPA binding affinity comparable to that of some
7-chloro-8-heteroaryl derivatives. Moreover, the 7-(1,2,4-
triazol-4-yl) derivatives 6a ,b show more than 4-fold
increased binding affinity with respect to compounds
3a ,b bearing the heteroaromatic ring at the crucial
position-8.
compd
AMPA
NMDA
1b (TQX-173)b
2.3 ( 0.4
3.0 ( 0.3
1.2 ( 0.2
3.0 ( 0.4
12 ( 2
0.20 ( 0.02
52 ( 11
46 ( 4
52 ( 6.0
23 ( 3
150 ( 12
350 ( 30
c
4b
5b
7b
8b
NBQX
DCKA
4.7 ( 0.9
a
Concentration that inhibits by 50% depolarizations (IC50
)
induced by 5 µM AMPA or NMDA. The IC50 values were means (
SEM of four separate determinations. Reference 16. c At 10 µM
b
concentration the inhibition was not significant.
Ta ble 3. Inhibition of Stimulated [3H]-(+)-MK-801 Binding
Affinities of the triazoloquinoxalines 1a ,b-5a ,b, 7a ,b,
and 8a ,b for the glycine/NMDA receptor are lower than
those for the AMPA receptor. In fact, replacement of
the 8-nitro group of the mixed Gly/NMDA and AMPA
antagonists 9a ,b with a 8-heterocyclic substituent leads
to a decrease in glycine/NMDA binding affinity, with
the exceptions of the 8-(pyrrol-1-yl) 4b and the 8-(pyra-
zol-1-yl) 7a ,b derivatives, which show affinities compa-
rable to those of compounds 9a ,b. Thus, the presence
of an 8-nitrogen-containing heterocycle, lacking the N3-
nitrogen atom, seems to be well-tolerated by the glycine/
NMDA receptor. These data indicate that the pyrrole
and pyrazole rings could be considered bioisosters of the
nitro group for the anchoring of these derivatives at the
glycine/NMDA receptor site. The same applies also to
the amino group. In fact, the 8-amino substituted
derivatives 11a ,b display comparable glycine/NMDA
binding affinity with respect to the corresponding 8-ni-
tro compounds 9a ,b.
Introduction of the 8-heterocyclic substituent leads,
in general, to an increase in KA binding activity of the
2-carboxylic acid derivatives 1b-5b,7b, and 8b with
respect to the inactive 8-nitro compound 9b. However,
all the reported 8-heteroaryl compounds show affinity
in the micromolar range, 1b and 5b being the most
active compounds toward the KA receptor. On the
contrary, all the 8-heteroaryl-2-carboxyethyl derivatives
1a -5a ,7a , and 8a are inactive at the KA receptor, with
the only exception being 5a . Replacement of the 8-nitro
group of 9a ,b with an amino one gives compounds
11a ,b, which show an increased KA receptor affinity
comparable to that of most of the 8-heteroaryl-2-
carboxylic acid derivatives.
Removal of the chlorine atom at position-7 of the TQX
framework causes a drastic reduction in glycine/NMDA
binding affinity, thus confirming previously reported
data.15 In fact, compounds 10a ,b and 12a ,b are more
than 10-fold less active than their corresponding 7-chlo-
ro derivatives 9a ,b and 11a ,b. To a minor extent, the
same applies to KA receptor binding affinity (compare
compounds 3b and 12b to 1b and 11b, respectively).
It has to be noted that the 2-carboxylic ester deriva-
tives are in general less active than the corresponding
acids toward the glycine/NMDA receptor, independent
of the benzo-fused substitutions. These results confirm
previously reported data on glycine/NMDA receptor
antagonists.15
[3H]-(+)-MK-801
[3H]-(+)-MK-801
compd
IC50a or I%b
compd
IC50a or I%b
1b
68 ( 4 µM
7b
16.8 ( 1.4 µM
(TQX-173)
2b
3b
4b
5b
45%
20%
11.7 ( 0.7 µM
43 ( 2.0 µM
8b
9b
11b
30%
17.8 ( 4.0 µM
5.6 ( 0.4 µM
Concentration giving 50% inhibition of stimulated [3H]-(+)-
MK-801 binding: all assays were carried out in the presence of
10 µM glutamate and 0.1 µM glycine. The results were calculated
from three or four separate determinations in triplicate. Per-
centage of inhibition (I%) of specific binding at 100 µM concentra-
a
b
tion.
and DCKA were evaluated for functional antagonist
activity by assessing their ability to inhibit depolariza-
tion induced by 5 µM AMPA or NMDA in mouse cortical
wedge preparations. All the tested compounds inhibited
AMPA and NMDA responses in a reversible manner
(Table 2). The electrophysiological potencies of com-
pounds 1b, 5b, and 8b closely correlate with the results
obtained in the AMPA and Gly/NMDA binding assays,
thus confirming that these derivatives are selective
AMPA antagonists. In fact, in agreement with [3H]-
AMPA and [3H]glycine binding results, the inhibitory
action of 1b, 5b, and 8b on depolarization induced by 5
µM AMPA was higher than that on NMDA-evoked
responses. On the contrary, the functional antagonist
activity vs NMDA of the nonselective AMPA and Gly/
NMDA antagonists 4b and 7b are not in agreement
with the [3H]glycine binding results. On the basis of [3H]-
AMPA and [3H]glycine binding data, the respective
values of the inhibitory action of 4b on depolarization
induced by 5 µM AMPA and NMDA were expected to
be similar. Instead, the inhibitory action of this deriva-
tive on the depolarization induced by 5 µM AMPA was
much higher than that on NMDA-evoked responses.
Compound 7b follows the same pattern.
To shed light on this contradictory data, the functional
antagonism at the NMDA receptor-ion channel complex
was demonstrated by the ability of compounds 4b and
7b, together with some other selected compounds (1b-
3b, 5b, 8b, 9b, and 11b), to inhibit the binding of the
channel blocking agent [3H]-(+)-MK-801 ((+)-5-methyl-
10,11-dihydro-5H-benzo[a,d]cyclohepten-5,10-imine mal-
eate)21-23 in rat cortical membranes incubated with
10µM glutamate and 0.1 µM glycine (Table 3). In
general, the IC50 values of these compounds for gluta-
mate-stimulated [3H]-(+)-MK-801 binding closely cor-
related with their Ki values on [3H]glycine binding. In
Some selected triazoloquinoxaline derivatives (1b,4b,
5b, 7b, and 8b) together with the well-known NBQX

3162 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Catarzi et al.
Ta ble 4. Physical and Analytical Data of the Synthesized
particular, the IC50 values of compounds 4b and 7b are
in agreement with the results obtained in the [3H]-
glycine displacement assays.
Compounds
compd
mp, °C
cryst solv
EtOH
EtOH
EtOH/AcOH
DMF
EtOH/DMF
DMF
AcOH
AcOH
EtOH
% yield
C, H, N
1a a
1ba
2a a
2ba
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
>300
>300
275 dec
>300
>300
>300
>300
250 dec
230 dec
228 dec
>300
>300
>300
73
82
85
92
39
97
80
88
80
68
34
80
96
61
73
28
80
68
70
67
44
67
63
41
58
86
89
59
63
41
86
92
77
91
82
51
50
50
C14H10ClN7O3
C12H6ClN7O3
C15H11ClN6O3
C13H7ClN6O3
C14H11N7O3
In conclusion, this study has shown that the introduc-
tion of different heteroaromatic rings at position-8 of
the TQX framework leads to other selective AMPA
receptor antagonists. In fact, the herein reported data
suggest that the presence of a N3-nitrogen containing
heterocycle at position-8 of the TQX framework is an
essential feature for highly selective AMPA receptor
antagonists.
We believe that these results could contribute to a
greater understanding about the structural require-
ments of a ligand for the anchoring to the AMPA
receptor in order to design novel potent and selective
AMPA receptor antagonists.
C12H7N7O3
C16H12ClN5O3
C14H8ClN5O3
C16H12ClN5O4
C14H8ClN5O4
C14H11N7O3
AcOH
AcOH/H2O
AcOH/H2O
AcOH
C12H7N7O3
C15H11ClN6O3
C13H7ClN6O3
C14H10ClN7O3
C12H6ClN7O3
C12H8ClN5O5
C10H4ClN5O5
C12H9N5O5
263-265 EtOH/DMF
279 dec
>300
EtOH
EtOH/DMF
8b
9a a
283-285 EtOH
On the basis of these new findings, further modifica-
tions of these TQX derivatives to improve biological
activity and selectivity are in progress.
9bb
>300
EtOH
288-300 EtOH
237-240 EtOH
10a
10bb
11a a
11b
12a
12b
13a
14
C10H5N5O5
>300
AcOH
C12H10ClN5O3
C10H6ClN5O3
C12H11N5O3
Exp er im en ta l Section
>300
>300
>300
EtOH/H2O
EtOH/DMF
EtOH/H2O
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-
C10H7N5O3
208-210 EtOH
C9H7ClN4O2
C9H7ClN4O2
C8H6ClN5O2
C13H11Cl2N5O4
C13H11Cl2N5O4
C12H10Cl2N6O4
C13H13ClN6O4
C13H13ClN6O4
C12H12ClN7O4
C17H17ClN6O7
C16H16ClN7O7
C17H15ClN6O6
C17H15ClN6O6
C16H14ClN7O6
170-172 EtOH/H2O
262-264 EtOH/DMF
172-174 EtOH
15
ses were performed with
a Perkin-Elmer 260 elemental
16a
17
analyzer for C, H, N, and the results were within (0.4% of
the theoretical values. The IR spectra were recorded with a
Perkin-Elmer 1420 spectrometer in Nujol mulls and are
159-161 EtOH
18
182-184 EtOH/DMF
235-237 EtOH
19a
20
1
expressed in cm-1. The H NMR spectra were obtained with a
180-182 EtOH
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. The following abbreviations are used: s
) singlet, d ) doublet, dd ) double doublet, t ) triplet, q )
quartet, m ) multiplet, br ) broad, and ar ) aromatic protons.
Physical and analytical data of the synthesized compounds are
listed in Table 4.
21
22
206-208 dioxane/H2O
160-162 EtOH
23
165-167 EtOH
24a
25
177-179 EtOH
136-138 EtOH
26
175-177 EtOH
a
b
Reference 16. Reference 15.
Gen er a l P r oced u r e To P r ep a r e Eth yl 4,5-Dih yd r o-8-
n itr o-4-oxo-1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxyl-
a tes 9a 16 a n d 10a . Ethyl 7-chloro-4,5-dihydro-4-oxo-1,2,4-
triazolo[1,5-a]quinoxaline-2-carboxylate15 or its 7-unsubstituted
analogue15 (0.65 mmol) was portionwise added to HNO3 (90%,
2 mL). The solution was stirred at 0-5 °C until the disap-
pearance of the starting material (TLC monitoring). The
mixture was then poured onto ice (20 g) and the resulting solid
was collected and washed with H2O. Compound 9a displayed
the following spectral data: 1H NMR (DMSO-d6) 1.40 (t, 3H,
CH3), 4.47 (q, 2H, CH2), 7.65 (s, 1H, ar), 8.81 (s, 1H, ar), 12.9
(br s, 1H, NH); IR 3170, 3060, 1715.
Gen er a l P r oced u r e To P r ep a r e Eth yl 8-Am 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 box-
yla tes 11a 16 a n d 12a . Iron powder (5.2 g) was added to a
solution of 9a or 10a (5.2 mmol) in glacial acetic acid (25 mL).
The mixture was heated at 90 °C for 30 min. Evaporation at
reduced pressure of the solvent yielded a residue which was
dried and exhaustively Soxhlet extracted with acetone (500
mL). Most of the solvent was evaporated to yield a solid, which
was collected. Compound 11a displayed the following spectral
data: 1H NMR (DMSO-d6) 1.38 (t, 3H, CH3), 4.44 (q, 2H, CH2),
5.8 (br s, 2H NH2), 7.34 (s, 1H, ar), 7.60 (s, 1H, ar), 12.2 (br s,
1H, NH); IR 3400, 3260, 1700, 1640.
Gen er a l P r oced u r e To P r ep a r e Eth yl 4,5-Dih 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 boxyla tes 1a 16 a n d 3a . Diformylhydrazine (4.0 mmol)
and then, drop by drop, trimethylsilyl chloride (20 mmol) and
triethylamine (9.3 mmol) were added to a suspension of 11a
or 12a (1.33 mmol) in anhydrous pyridine (6 mL). The mixture
was heated at 100 °C until the disappearance of the starting
material (TLC monitoring). Evaporation at reduced pressure
of the solvent yielded a solid that was treated with H2O (6
mL), collected, and washed with H2O. Compound 1a was
directly recrystallized, while compound 3a , before recrystal-
lization, was purified by column chromatography [eluting
system CHCl3/MeOH/AcOH (80:15:5)]. Compound 1a displayed
the following spectral data: 1H NMR (DMSO-d6) 1.37 (t, 3H,
CH3), 4.45 (q, 2H, CH2), 7.70 (s, 1H, ar), 8.49 (s, 1H, ar), 8.95
(s, 2H, triazole H-3 and H-5), 12.7 (br s, 1H, NH); IR 3620,
3520, 3140, 3120, 1730, 1715.
Eth yl 7-Ch lor o-4,5-d ih yd 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 4a . A solution of
2,5-diethoxytetrahydrofuran (2.43 mmol) in AcOH (5 mL) was
dropwise added to a hot (90 °C) suspension of 11a (0.81 mmol)
in AcOH (10 mL). The mixture was kept at 90 °C for 5 min.
Upon cooling a solid was obtained, which was collected and
washed with EtOH: 1H NMR (DMSO-d6) 1.37 (t, 3H, CH3),
4.44 (q, 2H, CH2), 6.32 (t, 2H, pyrrole H-3 and H-4, J ) 2.12
Hz), 7.12 (t, 2H, pyrrole H-2 and H-5, J ) 2.12 Hz), 7.65 (s,
1H, ar), 8.00 (s, 1H, ar), 12.6 (br s, 1H, NH); IR 3300, 3115,
1750, 1710.
Eth yl 7-Ch lor o-4,5-d ih yd r o-4-oxo-8-(3-for m ylp yr r ol-1-
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 . A
solution of 2,5-dimethoxy-3-tetrahydrofurancarboxaldehyde
(2.91 mmol) in AcOH (10 mL) was dropwise added to a
suspension of 11a (1.94 mmol) in AcOH (20 mL). The mixture
was heated at 90 °C for 10 min. The mixture was cooled and
diluted with H2O (200 mL) to yield a solid, which was collected
and washed with H2O. A second crop of 5a was obtained by
extracting the mother liquor with ethyl acetate (3 × 50 mL).
Evaporation of the dried (Na2SO4) organic layers afforded a
solid, which was purified by column chromatography [eluting
system CHCl3/MeOH (90:10)]: 1H NMR (DMSO-d6) 1.35 (t, 3H,
CH3), 4.45 (q, 2H, CH2), 6.71 (t, 1H, pyrrole H-4, J ) 1.47 Hz),
7.27 (d, 1H, pyrrole H-5, J ) 2.28 Hz), 7.69 (s, 1H, ar), 8.04 (s,

A New Set of Analogues of TQX-173
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3163
1H, pyrrole H-2), 8.24 (s, 1H, ar), 9.82 (s, 1H, CHO), 12.7 (br
s, 1H, NH); IR 3300, 1750, 1670.
a tes 22 a n d 23. Compound 20 or 21 (1.13 mmol) was dropwise
added to a refluxing solution of ethyloxalyl chloride (2.26
mmol) in anhydrous toluene (70 mL). The resulting yellow
solution was refluxed until the disappearance of the starting
material (TLC monitoring). Upon cooling compound 23 pre-
cipitated as a yellow solid and was collected and washed with
diethyl ether. Compound 22, which does not precipitate, was
isolated after evaporation of the solvent, treatment with
diethyl ether, and filtration. Compound 22 displayed the
following spectral data: 1H NMR (DMSO-d6) 1.23 (t, 3H, CH3),
1.32 (t, 3H, CH3), 4.25 (q, 2H, CH2), 4.36 (q, 2H, CH2), 6.64
(dd, 1H, pyrazole H-4, J ) 1.47, 2.56 Hz), 7.90 (d, 1H, pyrazole
H-3, J ) 1.47 Hz), 7.98 (s, 1H, ar), 8.43 (d, 1H, pyrazole H-5,
J ) 2.56 Hz), 8.44 (s, 1H, ar), 10.9 (br s, 1H, NH), 13.0 (br s,
1H, NH); IR 3300, 1730, 1715.
Dieth yl 1-[4-Ch lor o-5-(im id a zol-1-yl)-2-n itr op h en yl]-
1,2,4-t r ia zolo-3,5-d ica r boxyla t e 24.16 Compound 19 (1.13
mmol) was portionwise added to a boiling solution of ethylox-
alyl chloride (2.26 mmol) in anhydrous toluene (50 mL). The
resulting yellow solution was heated at reflux for 5 h. Elimina-
tion at reduced pressure of the solvent yielded a solid, which
was dissolved in ethyl acetate (100 mL). The organic solution
was washed with a solution of NaOH (0.5%, 40 mL × 2) and
then with H2O (40 mL). The dried (Na2SO4) organic layer was
evaporated to afford a solid, which was treated with diethyl
ether and collected: 1H NMR (DMSO-d6) 1.20 (t, 3H, CH3),
1.33 (t, 3H, CH3), 4.28 (q, 2H, CH2), 4.40 (q, 2H, CH2), 7.18 (d,
1H, imidazole H-4, J ) 1.22 Hz), 7.64 (d, 1H, imidazole H-5,
J ) 1,22 Hz), 8.13 (s, 1H, imidazole H-2), 8.30 (s, 1H, ar), 8.80
(s, 1H, ar); IR 1750.
Gen er a l P r oced u r e To P r ep a r e Dieth yl 1-[4-Ch lor o-
5-h eter oa r yl-2-n itr op h en yl]-1,2,4-tr ia zolo-3,5-d ica r boxyl-
a tes 25 a n d 26. Concentrated H2SO4 (4 mL) was dropwise
added to finely powdered 22 and 23 (0.88 mmol) under stirring.
The solution was stirred at room temperature for 12 h, poured
onto ice (200 g), and finally extracted with ethyl acetate (2 ×
100 mL). The organic layers were washed with a solution of
NaHCO3 (1%, 2 × 100 mL) and then with H2O (100 mL).
Evaporation of the dried (Na2SO4) solvent yielded a solid.
Compound 25 displayed the following spectral data: 1H NMR
(DMSO-d6) 1.19 (t, 3H, CH3), 1.33 (t, 3H, CH3), 4.28 (q, 2H,
CH2), 4.45 (q, 2H, CH2), 6.67 (dd, 1H, pyrazole H-4, J ) 1.47,
2.56 Hz), 7.92 (d, 1H, pyrazole H-3, J ) 1.47 Hz), 8.33 (s, 1H,
ar), 8.49 (d, 1H, pyrazole H-5, J ) 2.56 Hz), 8.77 (s, 1H, ar);
IR 1750, 1715.
Gen er a l P r oced u r e To P r ep a r e Eth yl 7-Ch lor o-4,5-
d ih yd r o-8-h eter oa r yl-4-oxo-1,2,4-tr ia zolo[1,5-a ]qu in oxa -
lin e-2-ca r boxyla tes 2a 16, 7a , a n d 8a . The title compounds
were obtained by reducing 24-26 (0.69 mmol) with iron
powder (0.69 g) and AcOH (6 mL) following the procedure
described above for the synthesis of 11a and 12a . Compound
2a displayed the following spectral data: 1H NMR (DMSO-
d6) 1.35 (t, 3H, CH3), 4.44 (q, 2H, CH2), 7.15 (d, 1H, imidazole
H-4, J ) 1.22 Hz), 7.54 (d, 1H, imidazole H-5, J ) 1.22 Hz),
7.67 (s, 1H, ar), 8.0 (s, 1H, imidazole H-2), 8.20 (s, 1H, ar),
12.7 (br s, 1H, NH); IR 1750, 1715.
Eth yl 4,5-Dih yd r o-4-oxo-7-(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 boxyla tes 6a . The title com-
pound was prepared by reacting ethyl 7-amino-4,5-dihydro-4-
oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylate 15 (0.91 mmol)
with diformylhydrazine (2.74 mmol), trimethylsilyl chloride
(13.6 mmol), and triethylamine (6.2 mmol), as described above
for the preparation of 1a and 3a :1H NMR (DMSO-d6) 1.37 (t,
3H, CH3), 4.45 (q, 2H, CH2), 7.60 (d, 1H, ar, J ) 2.48 Hz),
7.71 (dd, 1H, ar, J ) 8.8, 2.48 Hz), 8.30 (d, 1H, ar, J ) 8.8
Hz), 9.18 (s, 2H, triazole protons), 12.7 (br s, 1H, NH); IR 3500,
3300, 1750, 1730.
4-Ch lor o-5-(im id a zol-1-yl)-2-n itr oa n ilin e 13.16,20 An ex-
cess of imidazole (38.4 mmol) and KOH (14.4 mmol) were
added to a solution of 4,5-dichloro-2-nitroaniline (9.6 mmol)
in anhydrous dimethylformamide (DMF) (20 mL). The mixture
was heated at 80 °C for 1 h and then poured onto ice/water
(50 mL). Extraction with ethyl acetate (200 mL × 2), anhy-
drification of the organic layers, and evaporation at reduced
pressure of the solvent yielded a solid, which was treated with
diethyl ether and collected: 1H NMR (DMSO-d6) 7.14 (d, 2H,
ar + imidazole H-2), 7.49 (d, 1H, imidazole H-4, J ) 1.22 Hz),
7.7 (br s, 2H, NH2), 7.98 (d, 1H, imidazole H-5, J ) 1.22 Hz),
8.22 (s, 1H, ar); IR 3400, 3300, 3150.
Gen er a l P r oced u r e To P r ep a r e 4-Ch lor o-5-h eter oa r yl-
2-n itr oa n ilin es 14 a n d 15. An excess of the suitable hetero-
cycle (38.4 mmol) and KOH (14.4 mmol) were added to a
solution of 4,5-dichloro-2-nitroaniline (9.6 mmol) in anhydrous
DMF (20 mL). The mixture was heated at 100 °C for 6 h and
then set aside at room temperature for 12 h. The resulting
solid was collected and washed with a few drops of DMF.
Compound 14 displayed the following spectral data: 1H NMR
(DMSO-d6) 6.58 (dd, 1H, pyrazole H-4, J ) 1.46, 2.56 Hz), 7.31
(s, 1H, ar), 7.7 (br s, 2H, NH2), 7.82 (d, 1H, pyrazole H-3, J )
1.46 Hz), 8.19 (s, 1H, ar), 8.25 (d, 1H, pyrazole H-5, J ) 2.56
Hz); IR 3450, 3350.
Gen er a l P r oced u r e To P r ep a r e Eth yl N1-[4-ch lor o-5-
h et er oa r yl-2-n it r op h en yl]id r a zon o-N2-ch lor oa cet a t es
1616-18. Concentrated H2SO4 (7 mL) was added dropwise to
a suspension of 13-15 (4.19 mmol) in H2O (10 mL). A solution
of NaNO2 (5%, 6 mL) was added to the cooled (0 °C) mixture,
which was then stirred for 15 min to yield a solid that was
quickly filtered off. A solution of NaBF4 (17%, 4 mL) was added
to the cold (0-5 °C) solution, which was allowed to stand in
ice/water for 30 min. Methanol (25 mL) and ethyl 2-chloro-3-
oxobutanoate (4.19 mmol) was added to the mixture, which
was then stirred at room temperature for 3 h. Compounds 17
and 18 were obtained as solid and were collected and washed
with H2O. The reaction mixture containing compound 16 was
instead diluted with H2O (70 mL) and extracted with chloro-
form (60 mL × 3). Evaporation at reduced pressure of the dried
(Na2SO4) organic solvent yielded a solid, which was treated
with diethyl ether and collected. Compound 16 displayed the
following spectral data: 1H NMR (DMSO-d6) 1.28 (t, 3H, CH3),
4.33 (q, 2H, CH2), 7.19 (d, 1H, imidazole H-4, J ) 1.22 Hz),
7.62 (d, 1H, imidazole H-5, J ) 1.22 Hz), 7.71 (s, 1H, ar), 8.10
(s, 1H, imidazole H-2), 8.53 (s, 1H, ar), 11.01 (s, 1H, NH); IR
3300, 1700.
Gen er a l P r oced u r e To P r ep a r e 4,5-Dih yd r o-4-oxo-
1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid s 1b16
,
3b-8b, 9b, a n d 10b15. A solution of NaOH (0.8 N, 25 mL)
was added to a suspension of 1a , 3a -10a (0.97 mmol) in EtOH
(25 mL). The mixture was stirred at room temperature for 3
h. Acidification of the clear solution with HCl (6 N) yielded a
solid, which was collected and washed with H2O. Compound
1b displayed the following spectral data: 1H NMR (DMSO-
d6) 7.73 (s, 1H, ar), 8.44 (s, 1H, ar), 8.95 (s, 2H, triazole H-3
and H-5), 12.8 (br s, 1H, NH), 13.9 (br s, 1H, COOH); IR 3140,
3060, 1715.
Gen er a l P r oced u r e To P r ep a r e Eth yl N1-[4-ch lor o-5-
h eter oa r yl-2-n itr op h en yl]-N2-oxa m id r a zon a tes 1916-21.
Ammonia was bubbled until saturation into a stirred solution
of 16-18 (0.81 mmol) in anhydrous dioxane (12 mL). The
mixture was stirred at room temperature until the disappear-
ance of the starting material (TLC monitoring). The mixture
was then diluted with H2O (50 mL) to yield a red solid, which
was collected and washed with H2O. Compound 19 displayed
the following spectral data: 1H NMR (DMSO-d6) 1.24 (t, 3H,
CH3), 4.24 (q, 2H, CH2), 6.9 (br s, 2H, NH2), 7.15 (d, 1H,
imidazole H-4, J ) 1.22 Hz), 7.55 (d, 1H, imidazole H-5, J )
1.22 Hz), 7.69 (s, 1H, ar), 8.03 (s, 1H, imidazole H-2), 8.35 (s,
1H, ar), 10.04 (s, 1H, NH); IR 3480, 3360, 3300, 1760.
7-Ch lor o-4,5-d ih yd r o-8-(im id a zol-1-yl)-4-oxo-1,2,4-tr ia -
zolo[1,5-a ]qu in oxa lin e-2-ca r boxylic Acid 2b.16 A solution
of NaOH (0.8 N, 15 mL) was added to a suspension of 2a (0.59
mmol) in EtOH (15 mL). The mixture was stirred at room
temperature for 1 h. The resulting solid was collected and
dissolved in a small volume of H2O. The solution was acidified
with AcOH to afford a suspension that was stirred for 30 min.
Gen er a l P r oced u r e To P r ep a r e Eth yl N1-(4-ch lor o-5-
h eter oa r yl-2-n itr op h en yl)-N3-eth oxa lyl-N2-oxa m id r a zon -

3164 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Catarzi et al.
The solid was collected and washed with H2O: 1H NMR
(DMSO-d6) 7.14 (d, 1H, imidazole H-4, J ) 1.22 Hz), 7.53 (d,
1H, imidazole H-5, J ) 1.22 Hz), 7.65 (s, 1H, ar), 7.99 (s, 1H,
imidazole H-2), 8.14 (s, 1H, ar), 12.6 (br s, 1H, NH); IR 3500,
3300, 1720, 1620.
of 0.5 mL. Bound radioactivity was separated by filtration
through GF/C filters presoaked in 0.05% polyethylenimmine
and washed with ice-cold buffer (3 × 5 mL). Nonspecific
binding was determined in the presence of 100 µM phencyc-
lidine hydrochloride.
Gen er a l P r oced u r e To P r ep a r e 8-Am 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 boxylic Acid s
11b a n d 12b. A solution of NaOH (1N, 4 mL) was added to a
suspension of 11a and 12a (0.65 mmol) in EtOH (3 mL). The
mixture was heated at 90 °C for 2 h. Upon cooling the resulting
jelly mixture was acidified with AcOH, stirred several hours,
and then set aside overnight. The solid was collected and
washed with H2O. Compound 11b displayed the following
spectral data: 1H NMR (DMSO-d6) 5.65 (s, 2H, NH2), 7.32 (s,
1H, ar), 7.53 (s, 1H, ar), 12.0 (s, 1H, NH); IR 3440, 3330, 3220,
3060, 1700, 1640.
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 on the basis of four to six scalar concentrations of the
test compound in triplicate using the ALLFIT computer
program27 and, in the case of tritiated glycine and AMPA
binding, converted to Ki values by application of the Cheng-
Prusoff equation.28 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.
P h a r m a cology. Bin d in g Assa y. Rat cortical synaptic
membrane preparation and [3H]glycine and [3H]AMPA binding
experiments were performed following described procedures.12,24
Refer en ces
High -Affin ity [3H]Ka in a te Bin d in g. Frozen membrane
aliquots were resuspended (0.5 mg protein/mL) in 0.05 M Tris-
citrate buffer, pH 7.4, and incubated at 37 °C for 30 min. The
membranes were then washed with fresh ice-cold buffer by
three centrifugation and resuspension cycles as described for
[3H]glycine and [3H]AMPA binding assays. The final mem-
brane pellets were then resuspended in buffer to give 0.2-0.3
mg of protein/400 µL. Binding assays were carried out in ice
for 60 min in the presence of 5 nM [3H]kainate (NEN Life
Science Products, Boston, MA; specific activity 58 Ci/mmol)
and tested compounds in a total volume of 0.5 mL. Nonspecific
binding was assessed in the presence of 1 mM glutamate.
Bound radioactivity was separated by rapid filtration through
glass fiber paper (GF/C) in a Brandel harvester and washed
with 3 × 5 mL ice-cold buffer.
(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) 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.
(3) Chimirri, A.; Gitto, R.; Zappala`, M. AMPA receptor antagonists.
Exp. Opin. Ther. Patents 1999, 9, 557-570.
(4) Lees, G. J . Pharmacology of AMPA/Kainate Receptor Ligands
and their Therapeutic Potential in Neurological and Psychiatric
Disorders. Drugs 2000, 59, 33-78.
(5) Kulagowski, J . J .; Leeson, P. D. Glycine-site NMDA receptor
antagonists. Exp. Opin. Ther. Patents 1995, 5, 1061-1075.
(6) Kulagowski, J . J . Glycine-site NMDA receptor antagonists: an
update. Exp. Opin. Ther. Patents 1996, 6, 1069-1079.
(7) Danysz, W.; Parsons, C. G. Glycine and N-Methyl-D-Aspartate
Receptors: Physiological Significance and Possible Therapeutic
Applications. Pharmacol. Rev. 1998, 50, 597-664.
(8) J ohnson, J , W.; Ascher, P. Glycine potentiates the NMDA
response in cultured mouse brain neurons. Nature 1987, 325,
529-531.
(9) Arias, R. L.; Tasse, J . R. P.; Bowlby, M. R. Neuroprotective
interaction effects of NMDA and AMPA receptor antagonists in
an in vivo model of cerebral ischemia. Brain Res. 1999, 816,
299-308.
(10) Bigge, C. F.; Boxer, P. A.; Ortwine, D. F. AMPA/Kainate
Receptors. Curr. Pharm. Design. 1996, 2, 397-412.
(11) 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]-
quinoxalines as Novel Antagonists at the Glycine-NMDA Site.
Il Farmaco 1997, 52, 179-182.
(12) 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.
(13) 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. Il Farmaco 1998,
53, 752-757.
(14) 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]-
quinoxalines 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.
Electr op h ysiologica l Assa y. The cortical wedge prepara-
tion described by Mannaioni et al.25 was used. Briefly, wedges
obtained from white Swiss mice (male 15-25 g) were placed
in a two-compartment bath so that most of the cortical tissue
was contained in one chamber and the callosal tissue in the
other. Silicone grease had been previously placed between the
two portions of the incubation bath. The wedges were incu-
bated at room temperature and perfused with Krebs solution
(mM: NaCl, 135; CaCl2, 2.4; KH2PO4, 1.3; MgCl2, 1.2; NaH-
CO3, 16.2; and glucose, 7.7), gassed with 95% O2 and 5% CO2
at a flow rate of 2 mL min-1. After stabilization, the gray
matter was perfused with a Mg2+-free medium. AMPA (5 µM)
and NMDA (5 µM) were repeatedly applied for 2 min, every
15 min, until response stabilization (control peaks). Starting
from this moment, the wedges were continuously superfused
with the antagonists, while AMPA and NMDA were applied
every 15 min as described above. At the end of the assays, the
wedges were washed out with antagonists-free Krebs solution
and AMPA and NMDA applied again at 15-min intervals for
30-60 min to verify tissue response recovery. The variations
of the dc potentials between the two compartments were
monitored via Ag/AgCl electrodes and displayed on a chart
recorder. The inhibitory potencies of the tested compounds
were calculated by comparing the depolarization peaks ob-
tained in the presence of both agonist and antagonist with
control peaks.
[3H]-(+)-MK-801 Bin d in g. [3H]-(+)-MK-801 binding assays
were carried out according to the method of Yoneda and Ogita26
with slight modification.
Rat cortical membranes were resuspended (0.5 mg protein/
mL) in ice-cold 5 mM Tris-HCl buffer, pH 7.4, containing 0.08%
v/v Triton X-100 and stirred for 10 min at 0-2 °C. They were
then collected by centrifugation (48000g for 10 min) and
submitted to four additional resuspension and centrifugation
cycles before being finally resuspended in the appropriate
volume of buffer (0.2-0.3 mg of protein/tube) for the binding
assay. The assay incubations were carried out at room tem-
perature for 120 min with 2.5 nM [3H]-(+)-MK-801 (22.5 Ci/
mmol), 10 µM glutamic acid, and 0.1 µM glycine in the
presence and absence of the tested compound in a total volume
(15) 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 glycine/
NMDA and AMPA receptor affinity. J . Med. Chem. 1999, 42,
2478-2484.
(16) 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-carboxyl-
ates as Novel Highly Selective AMPA Receptor Antagonists. J .
Med. Chem. 2000, 43, 3824-3826.
(17) Varano, F.; Catarzi, D.; Colotta, V.; Cecchi, L.; Filacchioni G.;
Galli, A.; Costagli, C. Synthesis of a set of ethyl 1-carbamoyl-

A New Set of Analogues of TQX-173
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3165
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.
(18) Armstrong, N.; Gouaux, E. Mechanisms for activation and
antagonism of an AMPA-sensitive glutamate receptor: crystal
structure of the GluR2 ligand binding core. Neuron. 2000, 28,
165-181.
(19) Ohmori, J .; Shimizu-Sasamata, M.; Okada, M.; Sakamoto, S.
8-(1H-imidazol-1-yl)-7-nitro-4-(5H)-imidazo[1,2-a]quinoxalino-
ne and related compounds: synthesis and structure-activity
relationships for the AMPA-type non-NMDA receptor. J . Med.
Chem. 1997, 40, 2053-2063.
(20) Gu¨ngo¨r, T.; Fouquet, A.; Teulon, J .-M.; Provost, D.; Cazes, M.;
Cloarec, A. Cardiotonic agents. Synthesis and cardiovascular
properties of novel 2-arylbenzimidazoles and azabenzimidazoles.
J . Med. Chem. 1992, 35, 4455-4463.
(21) 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.
(23) 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.
(24) 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.
(25) 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.
(26) Yoneda, Y.; Ogita, K.; Suzuku, T. Interaction of strychnine-
insensitive glycine binding with MK-801 in brain synaptic
membranes. J . Neurochem. 1990, 55, 237-244.
(27) De Lean, A.; Munson, P. J .; Rodbard, D. Am. J . Physiol. 1978,
235, E97-102.
(28) Cheng, Y.-C.; Prusoff, W. H.; Biochem. Pharmacol. 1973, 22,
3099-3108.
(22) 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.
USA 1986, 83, 7104-7108.
J M010862Q