7-chloro-4,5-dihydro-8-(1,2,4-triazol-4-yl)-4-oxo-1,2,4-triazolo[1,5-a]quinox aline-2-carboxylates as novel highly selective AMPA receptor antagonists [3]

Full text of DOI:10.1021/jm0009686

3824
J . Med. Chem. 2000, 43, 3824-3826
Ch a r t 1
7-Ch lor o-4,5-d ih yd r o-8-(1,2,4-tr ia zol-4-yl)-
4-oxo-1,2,4-tr ia zolo[1,5-a ]qu in oxa lin e-2-
ca r boxyla tes a s Novel High ly Selective
AMP A Recep tor An ta gon ists
Daniela Catarzi,† Vittoria Colotta,† Flavia Varano,†
Lucia Cecchi,*,† 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 April 25, 2000
In tr od u ction . Glutamate (Glu) is probably the major
excitatory transmitter in the CNS, but it is also likely
to be involved in many pathological processes. The
excitotoxicity of Glu is mainly mediated by the over-
stimulation of the ionotropic Glu receptors (iGluRs): i.e.,
N-methyl-D-aspartate (NMDA), R-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid (AMPA), and kainate
(KA) receptors.1,2 Accordingly, there has been a growing
interest in the therapeutic potential of antagonists
acting at these iGluRs.3-6
Sch em e 1a
As part of a program aimed at finding novel iGluR
antagonists, we have recently reported the synthesis of
a set of 4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a]quinoxa-
line-2-carboxylates (TQXs).7 The combined glycine/
NMDA and AMPA receptor affinity of TQXs7 suggested
that this tricyclic system may represent a structure
which, suitably modified, could lead to selective glycine/
NMDA or AMPA receptor antagonists. We have hypoth-
esized that combining the TQX framework with a
8-(imidazol-1-yl) substituent might produce selective
AMPA antagonists. In fact, a pharmacophore model6,8
of the AMPA receptor for the binding of quinoxalinedi-
one and heterocyclic-fused quinoxalinone antagonists
claimed that the imidazol-1-yl substituent was essential
for high AMPA receptor activity. To verify this hypoth-
esis, we have replaced the 8-nitro substituent of our
mixed glycine/NMDA and AMPA antagonist 7-chloro-
8-nitro-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxy-
lic acid (1)7 with the bioisoster imidazol-1-yl moiety
(compounds 2 and 3, Chart 1). Moreover, although
several nitrogen-containing heterocycles have been
introduced as substituents on the benzo-fused moiety
of AMPA antagonists,9,10 to our knowledge the 1,2,4-
triazol-4-yl substituent has never been considered in the
structure-activity relationship (SAR) studies on quin-
oxalinone9 and heterocyclic-fused quinoxalinone antago-
nists.8 Thus, we synthesized and tested at iGluRs the
7-chloro-4,5-dihydro-8-(1,2,4-triazol-4-yl)-4-oxo-1,2,4-
triazolo[1,5-a]quinoxaline-2-carboxylates 4 and 5 (Chart
1) to evaluate the influence of the 8-(1,2,4-triazol-4-yl)
substituent at this crucial position in AMPA receptor
recognition.
a
Reagents and conditions: (a) NaNO2/H2SO4 concd, NaBF4,
0-5 °C; (b) CH3COCHClCOOEt, rt; (c) NH3(g), anhydrous dioxane,
rt, 1 h; (d) ClCOCOOEt, anhydrous toluene, reflux, 5 h; (e) iron,
glacial acetic acid, 90 °C, 30 min; (f) 0.8 N NaOH, ethanol, rt, 1 h,
then glacial acetic acid.
Resu lts a n d Discu ssion . The 8-(imidazol-1-yl) de-
rivatives 2 and 3 and the corresponding 8-(1,2,4-triazol-
4-yl) analogues 4 and 5 were prepared following the two
different synthetic strategies illustrated in Schemes 1
and 2. Briefly, by reacting the diazonium tetrafluoborate
of 4-chloro-5-(imidazol-1-yl)-2-nitroaniline (6)11 with
ethyl 2-chloro-3-oxobutanoate the N2-chloroacetate de-
rivative 7 was prepared (59%) (Scheme 1). The latter
was transformed with ammonia into its corresponding
oxamidrazonate 8 (86%). Reaction of 8 with ethyloxalyl
chloride directly yielded the diethyl 1-[4-chloro-5-(imi-
dazol-1-yl)-2-nitrophenyl]-1,2,4-triazole-3,5-dicarboxy-
late (9) (51%). Reduction with iron and glacial acetic
acid of the nitro group of 9 afforded the tricyclic ester 2
(85%) which was hydrolyzed to yield the 2-carboxylic
acid 3 (92%).
In Scheme 2 the synthetic strategy for the preparation
of the 8-(1,2,4-triazol-4-yl) derivatives 4 and 5 is il-
lustrated. The starting material, ethyl 7-chloro-4,5-
dihydro-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carbox-
ylate (10),7 was regioselectively nitrated at position 8
with 90% HNO3 to yield 11 (80%), which was reduced
* To whom correspondence should be addressed. Tel: +39 55
2757282. Fax: +39 55 240776. E-mail: cecchi@farmfi.scifarm.unifi.it.
† Dipartimento di Scienze Farmaceutiche.
‡ Dipartimento di Farmacologia Preclinica e Clinica.
10.1021/jm0009686 CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/29/2000

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3825
Sch em e 2a
branes. The binding results shown in Table 1 confirm
our hypothesis that by combining the triazoloquinoxa-
line-2-carboxylate framework with the presence of a
nitrogen-containing heterocycle at position 8 selective
AMPA receptor antagonists could be obtained. In fact,
while compound 17 is a mixed glycine/NMDA and AMPA
antagonist (see Table 1), the herein reported 8-het-
eroaryl derivatives 2-5 are AMPA-selective antago-
nists. In accordance with previously reported results7
on the TQX series, the presence of a free carboxylic
group at position 2 is not an important feature for
anchoring to the AMPA receptor. In fact, the carboxylic
acids 3 and 5 are only 2-5-fold more active at the AMPA
receptor than their corresponding esters 2 and 4,
respectively. On the contrary, the binding data shown
in Table 1 suggest that the presence of both a free
2-carboxylic group and a 8-heteroaryl substituent seems
to be essential for KA receptor-ligand interaction. In
fact, while the 8-nitro acid 1 and the 8-heteroaryl esters
2 and 4 are inactive as KA ligands, the 8-heteroaryl
acids 3 and 5 display micromolar KA receptor affinities.
It must be noted that the 8-(imidazol-1-yl) derivatives
2 and 3 are less active at the AMPA receptor than their
corresponding 8-(1,2,4-triazol-4-yl) analogues 4 and 5.
These data show that the replacement of the imidazole
moiety with a triazole one in the crucial position 8 of
the TQX framework leads to an increase in AMPA
receptor affinity. Moreover, the 8-(1,2,4-triazol-4-yl)
derivatives 4 and 5 are not only more potent but also
more selective AMPA antagonists than their corre-
sponding 8-(imidazol-1-yl) derivatives 2 and 3. In fact,
the beneficial effect of the 8-(1,2,4-triazol-4-yl) moiety
toward AMPA affinity and selectivity is indicated by the
a
Reagents and conditions: (a) 90% HNO3, 0-5 °C, 5 h; (b) iron,
glacial acetic acid, 90 °C, 1 h; (c) (OHCNH)2, pyridine, Me3SiCl,
100 °C, 15 h; (d) 0.8 N NaOH, ethanol, rt, 3 h, then 6 N HCl.
with iron and glacial acetic acid to its corresponding
8-amino derivative 12 (44%). By reacting 12 with
diformylhydrazine, the ethyl 7-chloro-4,5-dihydro-8-
(1,2,4-triazol-4-yl)-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-
2-carboxylate (4) was isolated (73%). Alkaline hydrolysis
of 4, followed by acidification, yielded the target 2-car-
boxylic acid 5 (82%).
Compounds 2-5, together with the previously re-
ported 17 and NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-
benzo[f]quinoxaline) and DCKA (5,7-dichlorokynurenic
acid) included as standard compounds, were tested for
their ability to displace tritiated glycine,12 AMPA,13 and
KA from their specific binding in rat cortical mem-
Ta ble 1. Binding Activity at Glycine/NMDA, AMPA, and KA Receptors and Functional Antagonism at NMDA and AMPA Sitesa
a
b
1 mM solutions of the tested compounds were prepared in DMSO/water (50%, v/v). Inhibition constant (Ki) values and concentrations
necessary for 50% inhibition of binding (IC50) were means ( SEM of 3-4 separate determinations in triplicate. c Percentage of inhibition
d
(I%) of specific binding at 100 µM concentration was based on 3 separate assays in triplicate. Concentrations that inhibit by 50%
depolarizations (IC50) induced by 5 µM NMDA or AMPA were means ( SEM of 4 separate determinations. e Ref 7. f Due to the insolubility
g
of the compound in the medium for electrophysiological assays, testing was not possible. At 10 µM concentration the inhibition was not
significant.

3826 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Communications to the Editor
(2) Michaelis, E. K. Molecular biology of glutamate receptors in the
central nervous system and their role in the excitotoxicity,
oxidative stress and aging. Prog. Neurobiol. 1998, 54, 369-415.
(3) Kulagowski, J . J .; Leeson, P. D. Glycine-site NMDA receptor
antagonists. Exp. Opin. Ther. Patents 1995, 5, 1061-1075.
(4) Kulagowski, J . J . Glycine-site NMDA receptor antagonists: an
update. Exp. Opin. Ther. Patents 1996, 6, 1069-1079.
(5) 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.
(6) Chimirri, A.; Gitto, R.; Zappala`, M. AMPA receptor antagonists.
Exp. Opin. Ther. Patents 1999, 9, 557-570.
(7) 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.
(8) Ohmori, 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.
(9) Ohmori, J .; Sakamoto, S.; Kubota, H.; Shimizu-Sasamata, M.;
Okada, M.; Kawasaki, S.; Hikada, K.; Togami, J .; Furuya, T.;
Murase, K. 6-(1H-Imidazol-1-yl)-7-nitro-2,3-(1H,4H)-quinoxa-
linedione hydrochloride (YM90K) and related compounds: Struc-
ture-activity relationships for the AMPA-type non-NMDA
receptor. J . Med. Chem. 1994, 37, 467-475.
(10) Sakamoto, S.; Ohmori, J . Y.; Sasamata, M.; Okada, M.; Hidaka,
K. Preparation of imidazoquinoxalinones as glutamate receptor
antagonists. Pat. Appl. WO 93/20077, 1993.
(11) 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.
binding data of the triazole ester 4, which is not only
equi-active to the imidazole acid 3 but also more AMPA-
selective than 3.
Compounds 1-5 together with the well-known NBQX
and DCKA were evaluated for functional antagonist
activity by assessing their ability to inhibit depolariza-
tions induced by 5 µM AMPA or NMDA in mouse
cortical wedge preparations14 (Table 1). Parallel experi-
ments have demonstrated that the inhibitory actions of
the tested compounds on AMPA- and NMDA-induced
mouse cortical wedge depolarization are competitive
with AMPA and glycine, respectively, since the actions
are reversed by increasing concentrations of these
agonists (data not shown).
In general, the results obtained in the electrophysi-
ological assays closely correlate with the binding data
on AMPA and glycine/NMDA receptors. In particular,
the electrophysiological data confirm that the 7-chloro-
4,5-dihydro-8-(1,2,4-triazol-4-yl)-4-oxo-1,2,4-triazolo[1,5-
a]quinoxaline-2-carboxylic acid (5, TQX-173) is the most
potent and selective AMPA antagonist herein reported.
In fact, in agreement with [3H]AMPA and [3H]glycine
binding results, the inhibitory action of 5 on depolar-
ization induced by 5 µM AMPA (IC50 ) 2.3 ( 0.4 µM)
was much higher than that on NMDA-evoked responses
(IC50 ) 46 ( 4 µM).
In conclusion, the synthesis of these novel 8-het-
eroaryl TQXs 2-5, their iGluR binding data, and the
electrophysiological results have evidenced that the
presence of the 8-(1,2,4-triazol-4-yl) moiety leads to more
potent and selective AMPA antagonists than those
bearing the claimed 8-(imidazol-1-yl) one.
(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) 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.
(14) 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.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails, spectral data, and analytical data are available free of
charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Danysz, W.; Parson, C. G.; Bresink, I.; Quack, G. Glutamate in
CNS disorders: A revived target for drug development? Drug
News Perspect. 1995, 8, 261-277.
J M0009686