Synthesis and biological evaluation of a new set of pyrazolo[1,5-c]quinazoline-2-carboxylates as novel excitatory amino acid antagonists

Article abstract of DOI:10.1021/jm010995b

In recent papers (Catarzi, D.; et al. J. Med. Chem. 1999, 42, 2478-2484; 2000, 43, 3824-3826; 2001, 44, 3157-3165) we reported the synthesis of a set of 4,5-dihydro-4-oxo-1,2,4-triazolo-[1,5-α]quinoxaline-2-carboxylates (TQXs) that were active at the Gly/

Full text of DOI:10.1021/jm010995b

J . Med. Chem. 2002, 45, 1035-1044
1035
Syn th esis a n d Biologica l Eva lu a tion of a New Set of
P yr a zolo[1,5-c]qu in a zolin e-2-ca r boxyla tes a s Novel Excita tor y Am in o Acid
An ta gon ists
Flavia Varano,† Daniela Catarzi,*,† Vittoria Colotta,† 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 J uly 19, 2001
In recent papers (Catarzi, D.; et al. J . Med. Chem. 1999, 42, 2478-2484; 2000, 43, 3824-
3826; 2001, 44, 3157-3165) we reported the synthesis of a set of 4,5-dihydro-4-oxo-1,2,4-triazolo-
[1,5-a]quinoxaline-2-carboxylates (TQXs) that were active at the Gly/NMDA and/or AMPA
receptors. In the present work the synthesis and Gly/NMDA, AMPA, and KA receptor binding
affinities of a set of 5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-2-carboxylates 1a ,b-4a ,b, 5a ,
6a , and 7a ,b-9a ,b, (()-5,6-dihydro-pyrazolo[1,5-c]quinazoline-2,5-dicarboxylates 10a ,b and
11a ,b, and (()-1,5,6,10b-tetrahydro-5-oxo-pyrazolo[1,5-c]quinazoline-2-carboxylates 12a ,b-
14a ,b are reported. The binding results indicate that compounds 1a ,b-4a ,b, 5a , 6a , and 7a ,b-
9a ,b show good Gly/NMDA and/or AMPA receptor binding affinities, demonstrating that the
pyrazoloquinazoline tricyclic system is an adequate alternative to the triazoloquinoxaline
framework for anchoring at both receptor types. Moreover, the inactivity of the 2,5-dicarboxylate
derivatives 10a ,b and 11a ,b at the Gly/NMDA and AMPA receptors indicates that the presence
of a glycine moiety in the southern portion of the pyrazoloquinazoline framework is deleterious
for receptor-ligand interaction. Finally, the binding data of compounds 12a ,b-14a ,b indicate
that lack of planarity in the northeastern region of the molecules shifts selectivity toward the
Gly/NMDA receptor, depending on the benzofused substitutions. In general, the pyrazolo-
quinazoline derivatives herein reported were inactive at the KA receptor. A study of the
functional antagonism at both the AMPA receptor and the NMDA receptor-ion channel complex
was also performed on some selected compounds.
Ch a r t 1. Structural Similarities and Differences for the
Binding of TQX Derivatives to the Gly/NMDA and
AMPA Receptors
In tr od u ction
Glutamate (Glu), the major excitatory neurotrans-
mitter in the central nervous system, has the potential
to influence the function of most neuronal circuits by
interacting with disparate receptors that are classified
as ionotropic (ion-channel forming) or metabotropic
(second messenger coupled) receptors. The ionotropic
glutamate receptors (iGluRs) are in their turn subdi-
vided, according to their preferential synthetic agonists,
into N-methyl-D-aspartate (NMDA), (R,S)-2-amino-3-(3-
hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA),
and kainate (KA) receptors.1,2 The NMDA receptor
possesses numerous bindingsites,includingthe glutamate
coagonist glycine binding site (Gly/NMDA).3
Overstimulation of iGluRs induces excitotoxic mech-
anisms and cell death, and it is involved in several
neurodegenerative syndromes such as Parkinson’s,
Huntington’s, and Alzheimer’s disease, as well as in
brain ischemia and epilepsy. Accordingly, competitive
iGluR antagonists, as well as noncompetitive NMDA
antagonists acting at the Gly/NMDA site, are of great
interest for their therapeutic potential in the treatment
of the above-mentioned neurological disorders.1,4-9
In the course of our efforts to find novel competitive
and noncompetitive iGluR antagonists,10-17 we have
recently published works on a new set of 4,5-dihydro-
4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylates(TQXs)
that are active at the Gly/NMDA and/or AMPA recep-
tors.14,15,17 Extensive structure-activity relationship
(SAR) studies on the TQX series have provided further
evidence of the structural similarities of each receptor
recognition site.4 As shown in Chart 1, some important
common requirements for anchoring of these antago-
nists to the Gly/NMDA and AMPA receptor sites are (i)
a NH proton donor that binds to a proton acceptor of
* To whom correspondence should be addressed: tel +39 55
2757294. Fax +39 55 240776. E-mail daniela.catarzi@unifi.it
† Dipartimento di Scienze Farmaceutiche.
‡ Dipartimento di Farmacologia Preclinica e Clinica.
10.1021/jm010995b CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/05/2002

1036 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Ch a r t 2. Newly Synthesized Compounds
Varano et al.
Sch em e 1a
the receptors; (ii) the 3-nitrogen atom and the oxygen
atom of the 4-carbonyl group that are δ-negatively
charged heteroatoms able to form a Coulombic interac-
tion with a positive site of the receptors; (iii) a carboxy-
late function at position-2 able to engage a strong
hydrogen-bond interaction with a cationic proton donor
site of the receptors; and (iv) an electron-withdrawing
substituent, such as a chlorine atom, at position 7.14
Furthermore, the SAR studies have also pointed out
some different structural requirements for binding at
the Gly/NMDA and AMPA receptors.14,15,17 In particu-
lar, it has been clarified that diverse substituents at
position-8 of the triazoloquinoxaline framework are able
to distinguish the AMPA receptor from the Gly/NMDA
site. In fact, the presence of a nitrogen-containing
heterocycle at position-8, the best being the (1,2,4-
triazol-4-yl) moiety (i.e., TQX-173, Chart 1), is required
for potent and selective AMPA receptor antagonists.15,17
On the other hand, the presence of a chlorine atom at
the same position on the benzofused moiety is of
paramount importance for Gly/NMDA receptor affinity
and selectivity (i.e. 7,8-Cl2TQX, Chart 1).14
Encouraged by these results, we have now replaced
the triazoloquinoxaline framework of the TQX com-
pounds with a pyrazoloquinazoline ring system in order
to investigate the influence of this structural modifica-
tion on Gly/NMDA, AMPA, and KA receptor binding
affinities. Thus, in this paper, we report the synthesis
and binding affinities of some 5,6-dihydro-5-oxo-pyra-
zolo[1,5-c]quinazoline-2-carboxylates 1a ,b-4a ,b, 5a , 6a ,
and 7a ,b-9a ,b (Chart 2), which contain the important
general requirements described above for the binding
at each receptor type.
a
Reagents: (a) diethyloxalate, EtONa; (b) 55% N2H4‚xH2O, 70%
AcOH; (c) H2/Pd/C; (d) (CCl3O)2CO, Et3N; (e) CHOCOOH‚H2O.
and 11a ,b. In addition, compounds 12a ,b-14a ,b were
synthesized in order to investigate the effect of a lack
of planarity in the northeastern region of the pyrazolo-
quinazoline moiety.
Ch em istr y
The synthesis of the pyrazolo[1,5-c]quinazoline-2-
carboxylic esters 1a 18,19-14a and that of the 2-carboxy-
lic acids 1b19,20-4b and 7b-14b are illustrated in
Schemes 1-6.
Scheme 1 shows the synthetic pathway that yielded
the 5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-2-car-
boxylic esters 1a and 2a , and the (()-5,6-dihydro-2-
carboxyethyl-pyrazolo[1,5-c]quinazoline-5-carboxylic ac-
ids 10a and 11a . Briefly, by reacting the commercially
available 2-nitroacetophenone 15 and its 4-chloro ana-
logue 1621 with diethyloxalate, the ethyl 4-(2-nitroaryl)-
2,4-dioxobutanoates 1722 and 18 were prepared. The
latter were transformed into the ethyl 5(3)-(2-nitroaryl)-
pyrazole-3(5)-carboxylates 1918 and 20 by reaction with
hydrazine hydrate. Catalytic reduction (Pd/C) of the
2-nitroarylpyrazoles 19 and 20 afforded the correspond-
ing amino derivatives 2118 and 22, which were cyclized
either with triphosgene or with glyoxylic acid to the final
5-oxo derivatives 1a and 2a and to the 5-carboxylic
tricyclic analogues 10a and 11a , respectively.
Compounds 3a and 4a were prepared by reacting 1a
and 2a with nitric acid (90%) (Scheme 2) and then
transformed into the corresponding 9-amino derivatives
5a and 6a by reduction with iron in glacial acetic acid.
Reaction of the 9-amino-8-chloro derivative 6a with 1,2-
diformylhydrazine yielded the 8-chloro-9-(1,2,4-triazol-
4-yl) ester 8a , while treatment of 6a with 2,5-dimethoxy-
3-tetrahydrofurancarboxyaldehyde gave the 8-chloro-9-
(3-formylpyrrol-1-yl) ester 9a .
Moreover, with the aim of extending the knowledge
on the structure-activity relationships of these tricyclic
derivatives, further modifications on the pyrazolo-
quinazoline framework were performed. Thus, some (()-
5,6-dihydro-pyrazolo[1,5-c]quinazoline-2,5-dicarboxy-
lates 10a ,b, 11a ,b, and some (()-1,5,6,10b-tetrahydro-
5-oxo-pyrazolo[1,5-c]quinazoline-2-carboxylates 12a ,b-
14a ,b were prepared and biologically evaluated (Chart
2). The effect of the introduction of a glycine moiety in
the southern portion of the pyrazoloquinazoline frame-
work was evaluated by synthesizing compounds 10a ,b
The synthesis of the (()-1,5,6,10b-tetrahydro-5-oxo-
pyrazolo[1,5-c]quinazoline-2-carboxylic esters 12a -14a

Novel Excitatory Amino Acid Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1037
Sch em e 2a
Sch em e 4a
a
Reagents: (a) tetrachloro-1,2-benzoquinone.
Sch em e 5a
a
Reagents: (a) 90% HNO3; (b) iron, AcOH; (c) diformylhydra-
zine, Me3SiCl, Et3N, pyridine; (d) 2,5-dimethoxy-3-tetrahydro-
furancarboxyaldehyde, AcOH.
Sch em e 3a
a
Reagents: (a) AcOH, 6 N HCl; (b) 1.5 N KOH/AcOH or 6 N
HCl.
Sch em e 6a
a
Reagents: (a) CH3COCOOH, HCl(g); (b) MeOH, HCl(g); (c)
N2H4, EtOH; (d) H2/PtO2; (e) (CCl3O)2CO, Et3N.
employed a totally different strategy, as shown in
Scheme 3. Allowing the suitable o-nitrobenzaldehydes
23-2523,24 to react with pyruvic acid, using dry hydro-
gen chloride as catalyst, the corresponding 3-hydroxy-
5-(2-nitroaryl)-5H-furan-2-ones 2625-28 were obtained.
By refluxing compounds 26-28 in methanol, saturated
with dry hydrogen chloride, the corresponding methyl
4-(2-nitroaryl)-2-oxo-3-butenoates 2925-31 were ob-
tained, which with anhydrous hydrazine were converted
into the methyl 4,5-dihydro-5-(2-nitroaryl)pyrazole-3-
carboxylates 32-34. Catalytic reduction (PtO2) of 32-
34 yielded the corresponding amino derivatives 35-37,
which were transformed into the final tricyclic deriva-
tives 12a -14a with triphosgene. Treatment of 12a and
a
Reagents: (a) 3 N NaOH/AcOH; (b) CHOCOOH‚H2O.
13a with nitric acid (90%) to obtain the corresponding
9-nitro derivatives afforded, in both cases, a mixture
containing prevalently the corresponding 9-nitro dehy-
dro derivatives 3a and 4a , respectively (by 1H NMR
analysis).
Dehydrogenation of compound 14a with tetrachloro-
1,2-benzoquinone gave the methyl 8,9-dichloro-5,6-di-
hydro-5-oxo-pyrazolo[1,5-c]quinazoline-2-carboxylate 7a
(Scheme 4).

1038 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Varano et al.
Ta ble 1. Displacement of [3H]Glycine, [3H]AMPA, and [3H]KA Bindinga
Ki (µM)b or I%c
IC50 (µM)d or I%c
[3H]KA
12%
26%
15%
compd
R
R8
R9
[3H]glycine
[3H]AMPA
1a
1b
2a
2b
3a
3b
4a
4b
5a
6a
7a
7b
8a
Et
H
Et
H
Et
H
Et
H
Et
Et
Me
H
H
H
Cl
Cl
H
H
H
H
H
NO2
NO2
NO2
NO2
NH2
NH2
Cl
33.3 ( 7
1.41 ( 0.3
26.5 ( 4.4
0.48 ( 0.04
15%
42 ( 6.8
12.4 ( 2.5
72 ( 2.3
2.3 ( 0.4
27%
60 ( 3
4%
H
35%
26 ( 9
24%
23%
23 ( 2
30%
11%
10%
41 ( 3
38%
Cl
Cl
H
Cl
Cl
Cl
Cl
10.6 ( 1.8
1.1 ( 0.1
30%
8.2 ( 2
0.74 ( 0.04
27%
40%
33%
42%
2.4 ( 0.8
0.87 (0.18
1.3 ( 0.2
0.16 ( 0.04
18%
Cl
Et
8b
9a
9b
H
Cl
Cl
Cl
8.3 ( 2.0
57 ( 9
0.14 ( 0.02
1.4 ( 0.2
5.1 ( 1.4
29%
Et
H
8.2 ( 3
0.27 (0.02
5.5 ( 0.4
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
Et
H
Et
H
Me
H
Me
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
Cl
Cl
15%
35%
5%
25%
12%
0%
6%
7%
0%
11%
1%
13%
0%
19.5 ( 0.5
3%
20%
9%
40%
29%
H
2.0 ( 0.4
10.6 ( 3.9
0.24 ( 0.03
4.0 ( 1.0
0.19 (0.02
0.074 ( 0.003
33.5 ( 5.3
96 ( 8
17.3 ( 4.1
7.2 ( 0.6
8.5 ( 1.3
2.65 (0.48
1.3 ( 0.2
0.14 ( 0.02
Cl
Cl
Cl
Cl
Me
H
17%
7,8-Cl2-TQXe
TQX-173g
NTf
11.6 ( 1.3
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 14. f Not tested. Reference 15.
g
Finally, the 5,6-dihydro-pyrazoloquinazoline esters
1a -4a , 7a -9a , and the 1,5,6,10b-tetrahydro-pyrazolo-
quinazoline esters 12a -14a were hydrolyzed to their
corresponding acids 1b-4b, 7b-9b, and 12b-14b,
respectively (Scheme 5).
Hydrolysis of the 2-ethyl ester of (()-5,6-dihydro-
pyrazolo[1,5-c]quinazoline-2,5-dicarboxylic acids 10a
and 11a , either in acidic or alkaline media, afforded the
corresponding acids 10b and 11b with very low yield.
Thus, compounds 10b and 11b were obtained by fol-
lowing a different procedure (Scheme 6). Alkaline hy-
drolysis of the 5-(2-aminoaryl)pyrazole esters 21 and 22
gave the corresponding acids 38 and 39, which were
cyclized to the final derivatives 10b and 11b with
glyoxylic acid.
previously reported 7,8-Cl2-TQX14 and TQX-17315 in-
cluded as reference compounds.
In general, the results listed in Table 1 indicate that
the 5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-2-car-
boxylates 1a ,b-4a ,b, 5a , 6a , and 7a ,b-9a ,b show good
Gly/NMDA and/or AMPA receptor binding affinities.
In particular, the binding data indicate that all the
2-carboxylic acids 1b-4b and 7b-9b are more active
than their corresponding esters 1a -4a and 7a -9a at
the Gly/NMDA and AMPA receptors. These data con-
firm that the presence in tricyclic derivatives of a
2-anionic carboxylate residue, able to engage a strong
interaction with a cationic proton donor site of the
receptors, is important for anchoring at both receptor
sites.14,15,17,26
The unsubstituted ethyl ester 1a displays comparable
Gly/NMDA (Ki ) 33.3 µM) and AMPA receptor affinity
(Ki ) 42 µM), while its corresponding 2-carboxylic acid
1b is about 9-fold more active at the Gly/NMDA receptor
(Ki) 1.41 µM) than at the AMPA one (Ki ) 12.4 µM).
Introduction of different substituents on the benzo-
fused moiety of the parents 1a ,b variously affects the
Resu lts a n d Discu ssion
The pyrazoloquinazolines 1a ,b-4a ,b, 5a , 6a , and
7a ,b-14a ,b were tested for their ability to displace
tritiated glycine, AMPA, and KA from their specific
binding sites in rat cortical membranes. The binding
data are shown in Table 1 together with those of

Novel Excitatory Amino Acid Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1039
receptor-ligand interaction. In fact, the presence of a
chlorine atom at position-8 (compounds 2a ,b) has
diverse effects on binding activities. While the 2-car-
boxylic ester 2a shows a comparable binding affinity to
that of 1a at both receptors, the corresponding acid 2b
is 3- and 6-fold more active than 1b at the Gly/NMDA
and AMPA receptors, respectively. Instead, the presence
of a 9-nitro (3a ,b) and a 9-amino group (6a ) dramatically
decreases Gly/NMDA and AMPA receptor affinities with
respect to those of 1a ,b, with the only exception being
compound 3b, which is only 2-fold less active than
compound 1b at the AMPA receptor.
Compounds 10a ,b and 11a ,b were formally obtained
by substitution with a glycine moiety of the lactam
group at position 5, 6 of 1a ,b and 2a ,b, respectively. In
fact, a glycine moiety in the southern portion of the
molecules is considered an important requirement for
highly potent and selective bicyclic Gly/NMDA receptor
antagonists,27,28 and to our knowledge, it has never been
introduced into tricyclic heteroaromatic derivatives in
order to obtain Gly/NMDA, AMPA, or KA receptor
antagonists. The inactivity of compounds 10a ,b and
11a ,b at all the three receptors indicates that a glycine
moiety is deleterious for the anchoring of pyrazolo-
quinazolines to the receptor sites. In fact, the replace-
ment of the 5,6-lactam group with a glycine moiety could
change the binding mode of the molecules, making other
pharmacophore portions, such as the important 2-car-
boxylate function, not optimized.
In addition, the presence of different substituents at
position-9, holding constant the 8-chloro-substitution
pattern, produces different effects on Gly/NMDA or
AMPA binding activities, depending on the nature of
the 9-substituent. In fact, while introduction of a
9-amino group is deleterious for the anchoring at both
receptors (see 6a vs 2a ), the presence of a 9-nitro
substituent is well-tolerated, especially for the binding
to the AMPA receptor (see 4a ,b vs 2a ,b, respectively).
Introduction of a chlorine atom at position-9 of com-
pound 2b affords 7b, which is about 3-fold more selec-
tive than 2b for the Gly/NMDA receptor. In fact, 7b is
15-fold more active at the Gly/NMDA receptor than at
the AMPA one and is the most active Gly/NMDA
antagonist herein reported. Furthermore, the 8,9-
dichloro substituted methyl ester 7a is one of the most
selective Gly/NMDA receptor ligands herein reported.
Introduction of a heteroaryl substituent at position-9
of compounds 2a ,b yields compounds 8a ,b and 9a ,b,
which, as expected on the basis of previous findings on
the TQX series,15,17 are selective AMPA receptor ligands,
being 30-60-fold more active at the AMPA receptor
than at the Gly/NMDA one.
Compounds 12a ,b-14a ,b were synthesized in order
to evaluate the importance of the lack of planarity in
the northeastern region of the pyrazoloquinazoline
derivatives in receptor ligand interaction. In fact, the
northern region of tricyclic and bicyclic iGluR antago-
nists was targeted for increasing the selectivity for
either AMPA or Gly/NMDA receptors.4
The binding results indicate that compounds 12a ,b-
14a ,b, like the planar analogues (1a ,b-4a ,b, 5a , 6a ,
and 7a ,b-9a ,b), are inactive at the KA receptor, while
they show good Gly/NMDA and/or AMPA binding af-
finities. In particular, the 2-carboxylic acid derivatives
12b-14b show higher affinities at both receptors than
their corresponding esters 12a -14a and are about 15-
50-fold more active at the Gly/NMDA receptor than at
the AMPA one. Moreover, the presence of a chlorine
atom at position-8 of the benzofused moiety significantly
increases the binding affinity at both receptors (see
13a ,b vs 12a ,b, respectively). Introduction of a chlorine
atom at position-9 of the 8-chloro derivatives 13a ,b
exerts different effects on the binding activities. In fact,
the 8,9-dichloro-substituted methyl ester 14a is about
2-fold more active than its 8-chloro analogue 13a at both
receptors. On the contrary, the 8,9-dichloro-substituted
2-carboxylic acid 14b is about 3-fold more active than
13b at the AMPA receptor, while it shows comparable
Gly/NMDA receptor affinity.
Finally, the pyrazoloquinazolines 1a ,b-4a ,b, 5a , 6a ,
and 7a ,b-9a ,b are in general inactive or active in the
high micromolar range at the KA receptor with the only
exceptions being the 2-carboxylic acids 8b and 9b, which
display IC50 values of about 5 µM. These data confirm
that the presence of a free carboxylic group at position-2
together with an 8-chloro substituent and a 9-hetero-
aryl group seems to be of paramount importance for KA
receptor-ligand interaction.15
Comparison of the binding affinities of the (()-1,5,6,-
10b-tetrahydro derivatives 12b, 13b, and 14a ,b with
those of their planar analogues 1b, 2b, and 7a ,b,
respectively, indicates that the lack of planarity in the
northeastern region of the pyrazoloquinazoline tricyclic
system does not significantly influence Gly/NMDA bind-
ing affinity, while it affects the anchoring to the AMPA
receptor depending on the benzofused substitutions. In
fact, the unsubstituted 12b and its 8-chloro analogue
13b are less active at the AMPA receptor than 1b and
2b, respectively, while they show comparable Gly/
NMDA binding affinities. Thus 12b and 13b are about
6-fold more selective for the Gly/NMDA receptor than
1b and 2b, respectively. On the contrary, the 8,9-
dichloro substituted methyl ester 14a is significantly
more active than its planar analogue 7a at the AMPA
receptor, while it is only 3-fold more active at the Gly/
NMDA receptor. Thus, 14a is a nonselective Gly/NMDA
receptor antagonist. Finally, the 8,9-dichloro-substituted
acid 14b is as selective as 7b toward the Gly/NMDA
To summarize, these results are in accordance with
previous findings on the TQX series and indicate that
the pyrazoloquinazoline framework could replace the
triazoloquinoxaline one for anchoring to the Gly/NMDA
and AMPA receptors. In fact, the 8,9-dichloro acid 7b
shows a comparable affinity and the same degree of
selectivity for the Gly/NMDA receptor with respect to
the reference 7,8-Cl2TQX (Table 1). Moreover, com-
pound 8b, bearing the claimed (1,2,4-triazol-4-yl) group
on the benzofused moiety, is equiactive to the reference
TQX-173 at the AMPA receptor, although about 4-fold
less selective.
On this basis we performed some structural modifica-
tions on the pyrazoloquinazoline framework in order to
further investigate the SAR of these tricyclic derivatives.
Thus we synthesized the (()-5,6-dihydro-pyrazolo[1,5-
c]quinazoline-2,5-dicarboxylates 10a ,b, 11a ,b, and the
(()-1,5,6,10b-tetrahydro-5-oxo-pyrazolo[1,5-c]quinazoline-
2-carboxylates 12a ,b-14a ,b (Table 1).

1040 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Ta ble 2. Inhibition of Stimulated [3H]-(+)-MK-801 Binding
Varano et al.
Morever, compounds 8a ,b and 9a ,b are more potent
AMPA receptor antagonists than the referenceTQX-
173.15
[3H]-(+)-MK-801
[3H]-(+)-MK-801
compd
IC50 (µM)a or I%b
compd
IC50 (µM)a or I%b
In conclusion, this study has shown that the pyra-
zoloquinazoline tricyclic system is an adequate alterna-
tive to the TQX framework for anchoring to the Gly/
NMDA and AMPA receptors. Moreover, the present
work has evidenced that introduction of a glycine moiety
in the southern portion of the pyrazoloquinazoline
tricyclic system is deleterious for receptor-ligand in-
teraction and that the lack of planarity in the north-
eastern region of the molecules could shift selectivity
toward the Gly/NMDA receptor, depending on the
benzofused substitutions.
1a
1b
2a
2b
4a
4b
7a
7b
8a
46 ( 4
10.4 ( 1.4
92 ( 8
1.8 ( 0.2
16.2 ( 2.1
4.33 ( 0.4
2.4 ( 0.3
0.89 ( 0.04
91 ( 8
8b
9a
9b
13.4 ( 1.1
34%
17 ( 2.0
17.5 ( 1.4
16.1 ( 1.1
2.1 ( 0.09
6.0 ( 0.46
0.79 ( 0.05
0.30 ( 0.05
12b
13a
13b
14a
14b
7,8-Cl2-TQXc
a
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
b
from three or four separate determinations in triplicate. Per-
On the basis of these preliminary findings, further
modifications on the pyrazoloquinazoline tricyclic sys-
tem are in progress to improve biological activity and
selectivity.
centage of inhibition (I%) of specific binding at 100 µM concentra-
tion. c Reference 14.
Ta ble 3. Functional Antagonism at AMPA and NMDA Sites
mouse cortical wedge preparation:
IC50 (µM) vs agonist-induced depolarizationsa
Exp er im en ta l Section
compd
8a
8b
9a
AMPA
NMDA
>50
20 ( 2.0
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-
1.75 ( 0.25
0.5 ( 0.07
1.0 ( 0.2
0.5 ( 0.1
2.3 ( 0.4
>50
9b
25 ( 4
46 ( 4
ses were performed with
a Perkin-Elmer 260 elemental
TQX-173b
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. The
following abbreviations are used: s ) singlet, d ) doublet, dd
) double doublet, t ) triplet, q ) quartet, m ) multiplet, br
) broad, and ar ) aromatic protons. The physical and
analytical data of the newly synthesized compounds are shown
in Table 4.
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 15.
b
receptor, because of their similar binding affinities not
only at the Gly/NMDA but also at the AMPA receptor.
Functional antagonism at the NMDA receptor-ion
channel complex was demonstrated by the ability of
some pyrazoloquinazolines (1a ,b, 2a ,b, 4a ,b, 7a ,b-
9a ,b, 12b, 13a ,b, and 14a ,b) to inhibit binding of the
channel blocking agent [3H]-(+)-MK-801 [(+)-5-methyl-
10,11-dihydro-5H-benzo[a ,d]cyclohepten-5,10-imine
maleate]28-30 in rat cortical membranes incubated with
10 µM glutamate and 0.1 µM glycine. The results are
listed in Table 2. In general, the IC50 values of these
compounds for glutamate stimulated [3H]-(+)-MK-801
binding are closely correlated with their Ki values on
[3H]glycine binding. In particular, as in the case of [3H]-
glycine displacement assays, compounds 7b and 14b,
bearing chlorine atoms at positions 8 and 9, are the most
potent compounds tested, with an IC50 of 0.89 ( 0.04
and 0.79 ( 0.05 µM, respectively. These values are very
close to that obtained with the reference 7,8-Cl2-TQX.14
Ma ter ia ls. Commercially available starting materials were
utilized as such, while the following products were prepared
according to reported methods: 1a ,18 1b,20 16,21 17,22 19,18 21,18
24,23 and 25.24
Eth yl 4-(4-Ch lor o-2-n itr op h en yl)-2,4-d ioxobu ta n oa te
(18). Diethyl oxalate (20 mmol) and then 4-chloro-2-nitroaceto-
phenone (16)21 (20 mmol) were added portionwise into a cold
(-5 °C) solution of sodium ethoxide (0.46 g of Na in 7 mL of
absolute ethanol). The reaction mixture was allowed to stand
at room temperature for 12 h and was then diluted with cold
water (200 mL) and filtered. The clear mother liquor was
acidified with glacial acetic acid to afford a suspension that
was set aside at 4 °C for 6 h. The solid obtained was collected
and washed with water: 1H NMR (DMSO-d6) 1.26 (t, 3H, CH3,
J
) 7.0 Hz), 4.25 (q, 2H, CH2, J ) 7.0 Hz), 6.38 (s,1H,
exchangeble with D2O), 7.74 (d, 1H, ar, J ) 8.2 Hz), 7.94 (d,
1H, ar, J ) 8.2 Hz), 8.26 (s, 1H, ar); IR 1650, 1750.
Furthermore, compounds 8a ,b and 9a ,b were evalu-
ated for functional antagonist activity at both the AMPA
receptor and NMDA receptor-ion channel complex by
assessing their ability to inhibit depolarization induced
by 5 µM AMPA or 5 µM NMDA in mouse cortical wedge
preparations (Table 3). All the tested compounds inhib-
ited AMPA and NMDA responses in a reversible man-
ner. The results obtained in the electrophysiological
assay confirm that compounds 8a ,b and 9a ,b, bearing
a heteroaryl substituent at position-9 of the benzofused
moiety, are potent and selective AMPA receptor antago-
nists. In fact, in agreement with [3H]AMPA and [3H]-
glycine binding data, the inhibitory actions of 8a ,b and
9a ,b on depolarization induced by 5 µM AMPA are
much higher than those on NMDA-evoked response.
Eth yl 5(3)-(4-Ch lor o-2-n itr op h en yl)p yr a zole-3(5)-ca r -
boxyla te (20). Hydrazine hydrate (55%, 2.3 mmol) was added
to a suspension of 18 (2.3 mmol) in glacial acetic acid (70%,
10 mL). The mixture was heated at 85 °C for 1 h. Evaporation
at reduced pressure of the solvent afforded an oily residue that
became solid upon treatment with cyclohexane: 1H NMR
(DMSO-d6) 1.31 (t, 3H, CH3, J ) 7.0 Hz), 4.33 (q, 2H, CH2, J
) 7.0 Hz), 7.21 (s, 1H, pyrazole H-4), 7.80 (d, 1H, ar, J ) 8.6
Hz), 7.86 (d, 1H, ar, J ) 8.6 Hz), 8.09 (s, 1H, ar); IR 1725,
3240.
Eth yl 5(3)-(2-Am in o-4-ch lor op h en yl)p yr a zole-3(5)-ca r -
boxyla te (22). A mixture of 20 (1.3 mmol) and 30% (w/w) Pd/C
(10%) in ethyl acetate (50 mL) was hydrogenated in a Parr
apparatus at 30 psi for 12 h. Elimination of the catalyst and
evaporation at reduced pressure of the solvent yielded a solid
that was treated with diethyl ether and collected: 1H NMR
(DMSO-d6) 1.33 (t, 3H, CH3, J ) 7.0 Hz), 4.34 (q, 2H, CH2, J

Novel Excitatory Amino Acid Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1041
following spectral data: 1H NMR (DMSO-d6) 1.30 (t, 3H, CH3,
J ) 7.0 Hz), 4.29 (q, 2H, CH2, J ) 7.0 Hz), 6.26 (d, 1H, H-5, J
) 2.7 Hz), 6.83 (dd, 1H, ar, J ) 8.2, 2.2 Hz), 6.96 (d, 1H, ar, J
) 2.2 Hz), 7.25 (s, 1H, H-1), 7.66 (d, 1H, ar, J ) 8.2 Hz), 7.87
(d, 1H, NH, J ) 2.7 Hz); IR 1700, 1740, 3380.
Gen er a l P r oced u r e To P r ep a r e Eth yl 5,6-Dih yd r o-9-
n itr o-5-oxo-pyr azolo[1,5-c]qu in azolin e-2-car boxylates (3a
a n d 4a ). Compound 1a 18-2a (1.2 mmol) was added portion-
wise to cooled (0-5 °C) HNO3 (90%, 4 mL). The reaction
mixture was stirred at 0-5 °C until the disappearance of the
starting material (TLC monitoring, eluting system AcOEt/
cyclohexane 7:3). The solution was then poured onto ice (20 g)
and the resulting solid was collected and washed with water.
Compound 4a displayed the following spectral data: 1H NMR
(DMSO-d6) 1.36 (t, 3H, CH3, J ) 7.1 Hz), 4.40 (q, 2H, CH2, J
) 7.1 Hz), 7.51 (s, 1H, ar), 7.94 (s, 1H, H-1), 9.07 (s, 1H, ar),
12.58 (s, 1H, NH); IR 1680, 1780, 3240.
Ta ble 4. Physical and Analytical Data of the Newly
Synthesized Compounds
compd
mp (°C)
solventa
yield (%)
C, H, N
2a
2b
3a
3b
4a
4b
5a
6a
7a
7b
8a
8b
9a
306-309
>300
A
B
C
B
A
D
E
A
C
D
B
B
A
A
A
A
A
A
F
D
A
D
A
D
G
H
H
I
90
98
85
90
85
90
30
65
85
80
60
90
70
65
80
40
95
65
75
55
90
60
75
95
45
95
95
70
90
70
65
40
30
90
40
70
80
75
77
90
80
C
13H10ClN3O3
C11H6ClN3O3
C13H10N4O5
C11H6N4O5
C13H9ClN4O5
C11H5ClN4O5
C13H12N4O3
C13H11ClN4O3
C12H7Cl2N3O3
C11H5Cl2N3O3
C15H11ClN6O3
C13H7ClN6O3
C18H13ClN4O4
C16H9ClN4O4
C14H13N3O4
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
295-296
>300
9b
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
18b
20
206-208
240-242
242-244
>300 dec
281-284
252-255
276-278
262-264
273-275
269-271
91-95
Gen er a l P r oced u r e To P r ep a r e Eth yl 9-Am in o-5,6-
d ih yd r o-5-oxo-p yr a zolo[1,5-c]q u in a zolin e -2-ca r b oxy-
la tes (5a a n d 6a ). Iron powder (1.3 g) was added to a solution
of 3a -4a (1.3 mmol) in glacial acetic acid (15 mL). The mixture
was heated at 90 °C for 1 h and then evaporation at reduced
pressure of the solvent yielded a solid residue that was dried
and extracted in Soxhlet with acetone (500 mL). Evaporation
at reduced pressure of the solvent afforded a solid that was
purified by column chromatography [eluting system CHCl3/
MeOH (9.5:0.5)]. Compound 6a displayed the following spectral
data: 1H NMR (DMSO-d6) 1.34 (t, 3H, CH3, J ) 7.0 Hz), 4.37
(q, 2H, CH2, J ) 7.0 Hz), 5.43 (s, 2H, NH2), 7.23 (s, 1H, ar),
7.37 (s, 1H, ar), 7.39 (s, 1H, H-1), 11.80 (br s, 1H, NH); IR
1750, 3300, 3520.
C12H9N3O4
C14H12ClN3O4
C12H8ClN3O4
C12H11N3O3
C11H9N3O3
C12H10ClN3O3
C11H8ClN3O3
C12H9Cl2N3O3
C11H7Cl2N3O3
C12H10ClNO6
C12H10ClN3O4
C12H12ClN3O2
137-139
184-186
140-142d
158-160d
169-171d
90-92
22
26c
27
I
I
J
J
28
29e
30f
31g
32
C11H9NO5
Eth yl 8-Ch lor o-5,6-d ih yd r o-5-oxo-9-(1,2,4-tr ia zol-4-yl)-
p yr a zolo[1,5-c]qu in a zolin e-2-ca r boxyla te (8a ). Diformyl-
hydrazine (4.5 mmol) and, drop by drop, trimethylsilyl chloride
(22.5 mmol) and triethylamine (10.2 mmol) were successively
added to a suspension of 6a (1.5 mmol) in anhydrous pyridine
(6 mL). The mixture was heated at 100 °C for 90 min, then
evaporation at reduced pressure of the solvent yielded a solid
that was treated with water (20 mL), collected, and washed
with water: 1H NMR (DMSO-d6) 1.35 (t, 3H, CH3, J ) 7.0
Hz), 4.38 (q, 2H, CH2, J ) 7.0 Hz), 7.57 (s, 1H, ar), 7.75 (s,
1H, H-1), 8.61 (s, 1H, ar), 8.92 (s, 2H, triazole H-3 and H-5),
12.43 (br s, 1H, NH); IR 1750.
98-99
C11H8ClNO5
C11H7Cl2NO5
C11H11N3O4
C11H10ClN3O4
C11H9Cl2N3O4
116-118
121-122
138-140
151-153
111-115d
119-123d
139-143 decd
265-267
278-280
J
A
A
A
I
I
I
33
34
35
36
37
38
39
D
D
C
10H9N3O2
C10H8ClN3O2
a
Recrystallization solvents: A ) ethanol, B ) dimethylforma-
mide, C ) glacial acetic acid, D ) dimethylformamide/water, E )
nitromethane, F ) acetonitrile, G ) cyclohexane, H ) cyclohexane/
ethyl acetate, I ) instable upon recrystallization, J ) methanol.
E t h yl 8-Ch lor o-5,6-d ih yd r o-9-(3-for m ylp yr r ol-1-yl)-5-
oxo-p yr a zolo[1,5-c]qu in a zolin e-2-ca r boxyla te (9a ). A so-
lution of 2,5-dimethoxy-3-tetrahydrofurancarboxaldehyde (0.94
mmol) in glacial acetic acid (3 mL) was added dropwise to a
hot (90 °C) suspension of 6a (0.62 mmol) in glacial acetic acid
(10 mL). The mixture was heated at 90 °C for 10 min and then
cooled and diluted with water (70 mL).The obtained solid was
collected and purified by column chromatography [eluting
system CHCl3/MeOH (9:1)]: 1H NMR (DMSO-d6) 1.32 (t, 3H,
CH3, J ) 7.3 Hz), 4.37 (q, 2H, CH2, J ) 7.3 Hz), 6.68 (s, 1H,
ar), 7.19 (s, 1H, ar), 7.51 (s, 1H, ar), 7.80 (s, 1H, H-1), 7.94 (s,
1H, ar), 8.52 (s, 1H, ar), 9.79 (s, 1H, CHO), 12.34 (br s, 1H,
NH).
b
Calcd (found) C, 48.09 (47.75); H, 3.37 (3.38); N, 4.67 (4.72). c Lit.
mp 138-139 °C. Melting point of the crude product. e Calcd
d
(found) C, 56.17 (56.49); H, 3.86 (3.82); N, 5.96 (6.01). f Calcd
g
(found) C, 48.99 (48.71); H, 3.00 (3.09); N, 5.20 (5.16). Calcd
(found) C, 43.44 (43.21); H, 2.32 (2.35); N, 4.61 (4.56).
) 7.0 Hz), 6.55 (br s, 3H, 1 ar + NH2), 6.80 (s, 1H, ar), 7.30 (s,
1H, pyrazole H-4), 7.60 (d, 1H, ar, J ) 8.0 Hz); IR 1740, 3320.
Eth yl 8-Ch lor o-5,6-d ih yd r o-5-oxo-p yr a zolo[1,5-c]qu in -
a zolin e-2-ca r boxyla t e (2a ). Triphosgene (0.37 mmol) and
triethylamine (2.3 mmol) were successively added to a solution
of 22 (0.94 mmol) in anhydrous tetrahydrofuran (10 mL). The
mixture was stirred at room temperature for 30 min and then
diluted with water (30 mL) to yield a solid, which was
collected: 1H NMR (DMSO-d6) 1.33 (t, 3H, CH3, J ) 7.0 Hz),
4.36 (q, 2H, CH2, J ) 7.0 Hz), 7.33-7.38 (m, 2H, ar), 7.70 (s,
1H, H-1), 8.17 (d, 1H, ar, J ) 8.9 Hz); IR 1710, 1740, 3240.
Gen er a l P r oced u r e To P r ep a r e th e 2-Eth yl Ester of
(()-5,6-Dih yd r o-p yr a zolo[1,5-c]qu in a zolin e-2,5-d ica r box-
ylic Acid s (10a a n d 11a ). An excess of glyoxylic acid
monohydrate (1.3 mmol) was added to a solution of 2118-22
(0.86 mmol) in anhydrous tetrahydrofuran (20 mL). The
mixture was refluxed under nitrogen atmosphere. The reaction
was monitored by TLC (eluting system CHCl3/MeOH/AcOH
8:0.5:0.5) and the heating was continued until the starting
material disappeared. Evaporation at reduced pressure of the
solvent afforded an oily residue that became solid upon
treatment with cyclohexane. Compound 11a displayed the
Gen er a l P r oced u r e To P r ep a r e 3-Hyd r oxy-5-(2-n itr o-
a r yl)-5H-fu r a n -2-on es (2625-28). The title compounds were
obtained from the suitable o-nitrobenzaldehyde (23-25)23,24
and pyruvic acid, which serves also as a solvent. The synthesis
was performed following the procedure described in ref 25 to
prepare 26 with modifications. Dry hydrogen chloride was
bubbled until saturation into a warmed (25-30 °C) mixture
of the suitable o-nitrobenzaldehyde 23-25 (10 mmol) and
pyruvic acid (21 mmol for the preparation of 26 and 27, 50
mmol for the preparation of 28). The mixture was left at room
temperature until disappearance of the starting material [2-7
days, TLC monitoring, eluting system CHCl3/MeOH (9:1)].
After addition of a small amount of petroleum ether and
ethanol (1:1), the main product was collected. The residue was
unstable upon recrystallization. Nevertheless, the crude prod-
uct was pure enough and thus used without further purifica-
tion.

1042 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Varano et al.
26: 1H NMR (DMSO-d6) 6.46-6.50 (m, 2H, furan H-4 and
H-5), 7.46 (d, 1H, ar, J ) 8.1 Hz), 7.59-7.68 (m, 1H, ar), 7.75-
7.82 (m, 1H, ar), 8.12 (dd, 1H, ar, J ) 8.1, 1.1 Hz), 10.77 (br
s, 1H, exchangeble with D2O).
27: 1H NMR (DMSO-d6) 6.44-6.46 (m, 2H, furan H-4 and
H-5), 7.48 (d, 1H, ar, J ) 8.6 Hz), 7.85 (d, 1H, ar, J ) 8.6 Hz),
8.21 (s, 1H, ar), 10.81 (br s, 1H, exchangeble with D2O).
28: 1H NMR (DMSO-d6) 6.43-6.48 (m, 2H, furan H-4 and
H-5), 7.69 (s, 1H, ar), 8.46 (s, 1H, ar), 10.86 (br s, 1H,
exchangeble with D2O).
Gen er a l P r oced u r e To P r ep a r e Meth yl 4-(2-Nitr oa r yl)-
2-oxo-3-bu ten oa tes (2925-31). A suspension of furane 26-
28 (8 mmol) in 30 mL of methanol, saturated with dry
hydrogen chloride, was refluxed for 3-5 h [TLC monitoring,
eluting system CHCl3/MeOH (9:1)] and then set aside at room
temperature for 12 h. The resulting solid was collected and
washed with diethyl ether. Compound 31 displayed the fol-
lowing spectral data: 1H NMR (DMSO-d6) 3.85 (s, 3H, CH3),
7.44 (d, 1H, butene H-3 or H-4, J ) 16.1 Hz), 7.95 (d, 1H,
butene H-4 or H-3, J ) 16.1 Hz), 8.33 (s, 1H, ar), 8.46 (s, 1H,
ar); IR 1700, 1740.
Gen er a l P r oced u r e To P r ep a r e Meth yl (()-4,5-Dih y-
d r o-5-(2-n itr oa r yl)p yr a zole-3-ca r boxyla tes (32-34). An-
hydrous hydrazine (2.3 mmol) was added to a hot (90 °C)
suspension of 29-31 (2.1 mmol) in absolute ethanol (5 mL).
The resulting solution was refluxed for 30 min. The solid,
which precipitated upon cooling, was collected and washed
with diethyl ether. Compound 34 displayed the following
spectral data: 1H NMR (DMSO-d6) 2.75-2.88 (m, 1H, pyra-
zoline H-4), 3.32-3.48 (m, 1H, pyrazoline H-4), 3.68 (s, 3H,
CH3), 5.29-5.34 (m, 1H, pyrazoline H-5), 7.73 (s, 1H, ar), 8.37
(s, 1H, ar), 8.75 (br s, 1H, NH); IR 1730, 3400.
Gen er a l P r oced u r e To P r ep a r e Meth yl (()-4,5-Dih y-
dr o-5-(2-am in oar yl)pyr azole-3-car boxylates (35-37). PtO2
[10% (w/w)] was added to a solution of 32 (3.2 mmol) in ethyl
acetate (40 mL) or 33-34 (1.6 mmol) in ethanol (150 mL). The
resulting mixture was hydrogenated in a Parr apparatus at
30 psi for 12 h. Elimination of the catalyst and evaporation at
reduced pressure of the solvent afforded an oily residue that
became solid upon treatment with a mixture of petroleum
ether/diethyl ether (1:1). The residue was unstable upon
recrystallization. Nevertheless, the crude product was pure
enough and thus used without purification.
35: 1H NMR (DMSO-d6) 2.47-2.55 (m, 1H, pyrazoline H-4),
3.21-3.34 (m, 1H, pyrazoline H-4), 3.68 (s, 3H, CH3), 4.95-
4.99 (m, 3H, pyrazoline H-5 + NH2), 6.52-6.66 (m, 2H, ar),
6.92-6.99 (m, 2H, ar), 8.68 (s, 1H, NH); IR 1720, 3320, 3430.
36: 1H NMR (DMSO-d6) 2.36-2.49 (m, 1H, pyrazoline H-4),
3.23-3.35 (m, 1H, pyrazoline H-4), 3.67 (s, 3H, CH3), 4.89-
5.01 (m, 1H, pyrazoline H-5), 5.32 (br s, 2H, NH2), 6.53 (d,
1H, ar, J ) 8.2 Hz), 6.67 (s, 1H, ar), 6.97 (d, 1H, ar, J ) 8.2
Hz), 8.70 (s, 1H, NH).
Gen er a l P r oced u r e To P r ep a r e 5,6-Dih yd r o-5-oxo-
p yr a zolo[1,5-c]q u in a zolin e-2-ca r b oxylic Acid s (2b-4b ,
8b). A suspension of 2a -4a , 8a (1.5 mmol) in glacial acetic
acid (6 mL) and 6 N HCl (1.2 mL) was heated at 100 °C until
disappearance of the starting material [TLC monitoring,
eluting system CHCl3/MeOH/AcOH (8:1.5:0.5)]. The mixture
was cooled and then the solid was collected and washed with
water. Compound 2b and 8b displayed the following spectral
data.
2b: 1H NMR (DMSO-d6) 7.35-7.40 (m, 2H, ar), 7.66 (s, 1H,
H-1), 8.17 (d, 1H, ar, J ) 8.5 Hz), 12.14 (s, 1H, exchangeble
with D2O), 13.34 (s, 1H, exchangeble with D2O); IR 1680, 1760,
3400.
8b: 1H NMR (DMSO-d6) 7.56 (s, 1H, ar), 7.67 (s, 1H, H-1),
8.58 (s, 1H, ar), 8.91 (s, 2H, triazole H-3 and H-5) 12.4 (br s,
1H, exchangeble with D2O); IR 1680, 1740, 3450.
8,9-Dich lor o-5,6-Dih ydr o-5-oxo-pyr azolo[1,5-c]qu in azo-
lin e-2-ca r boxylic Acid (7b). A solution of KOH (1.5 N, 12
mL) was added to a suspension of 7a (1.5 mmol) in methanol
(12 mL). The reaction mixture was heated at 100 °C for 15
min and then cooled. The resulting solid residue was filtered
and dissolved in the minimum amount of water. The solution
was acidified with glacial acetic acid to afford a solid that was
collected and washed with water: 1H NMR (DMSO-d6) 7.49
(s, 1H, ar), 7.75 (s, 1H, H-1), 8.35 (s, 1H, ar), 12.21 (s, 1H,
exchangeble with D2O), 13.43 (br s, 1H, exchangeble with D2O).
8-Ch lor o-5,6-dih ydr o-9-(3-for m ylpyr r ol-1-yl)-5-oxo-pyr a-
zolo[1,5-c]qu in a zolin e-2-ca r boxylic Acid (9b). A solution
of KOH (1.5 N, 12 mL) was added to a suspension of 9a (1.5
mmol) in methanol (12 mL). The reaction mixture was heated
at 100 °C for 15 min and then cooled and acidified with 6 N
HCl to yield a solid that was collected and washed with
water: 1H NMR (DMSO-d6) 6.69 (s, 1H, ar), 7.20 (s, 1H, ar),
7.53 (s, 1H, ar), 7.82 (s, 1H, H-1), 7.95 (s, 1H, ar), 8.51 (s, 1H,
ar), 9.80 (s, 1H, CHO), 12.36 (br s, 1H, exchangeble with D2O).
Gen er a l P r oced u r e To P r ep a r e (()-1,5,6,10b-Tetr a h y-
d r o-5-oxo-p yr a zolo[1,5-c]qu in a zolin e-2-ca r boxylic Acid s
(12b -14b ). The title compounds were obtained from 12a -
14a (1.4 mmol), by following the procedure described above
for the preparation of 2b-4b and 8b. Compound 14b displayed
the following spectral data: 1H NMR (DMSO-d6) 3.11-3.27
(m, 1H, H-1), 3.58-3.67 (m, 1H, H-1), 5.20-5.32 (m, 1H,
H-10b), 7.11 (s, 1H, ar), 7.53 (s, 1H, ar), 10.10 (s, 1H,
exchangeble with D2O), 13.40 (br s, 1H, exchangeble with D2O);
IR 1680, 1740, 3200.
Gen er a l P r oced u r e To P r ep a r e 5-(2-Am in oa r yl)p yr a -
zole-3-ca r boxylic Acid s (38 a n d 39). A suspension of 21-
22 (0.90 mmol) in 3 N NaOH (3 mL) was heated at 100 °C for
30 min. Dilution with water (10 mL) and acidification of the
clear solution with glacial acetic acid afforded a solid that was
collected and washed with water. Compound 39 displayed the
following spectral data: 1H NMR (DMSO-d6) 6.53-6.78 (m,
4H, 2ar + NH2), 7.14 (s, 1H, ar), 7.53 (d, 1H, ar, J ) 8.4 Hz);
IR 1680, 3240, 3380, 3480.
Gen er a l P r oced u r e To P r ep a r e (()-5,6-Dih yd r o-p yr a -
zolo[1,5-c]q u in a zolin e-2,5-d ica r boxylic Acid s (10b a n d
11b). The title compounds were prepared by reacting 38-39
(1 mmol), with an excess of glyoxylic acid monohydrate (1.5
mmol), as described above for the preparation of 10a and 11a .
Compound 11b displayed the following spectral data: 1H NMR
(DMSO-d6) 5.95 (s, 1H, H-5), 6.77 (d, 1H, ar, J ) 8.2 Hz), 6.92
(s, 1H, ar), 7.12 (s, 1H, H-1), 7.58 (d, 1H, ar, J ) 8.2 Hz), 7.68
(s, 1H, NH); IR 1700, 3250, 3500.
37: 1H NMR (DMSO-d6) 2.36-2.50 (m, 1H, pyrazoline H-4),
3.12-3.26 (m, 1H, pyrazoline H-4), 3.67 (s, 3H, CH3), 4.89-
5.03 (m, 1H, pyrazoline H-5), 5.51 (s, 2H, NH2), 6.82 (s, 1H,
ar), 7.19 (s, 1H, ar), 8.77 (s, 1H, NH).
Gen er a l P r oced u r e To P r ep a r e Meth yl (()-1,5,6,10b-
Tetr a h yd r o-5-oxo-p yr a zolo[1,5-c]qu in a zolin e-2-ca r boxy-
la tes (12a -14a ). The title compounds were prepared by
reacting 35-37 (2.7 mmol) with triphosgene (1.1 mmol) and
triethylamine (6.6 mmol), as described above for the prepara-
tion of 2a . Compound 14a displayed the following spectral
data: 1H NMR (DMSO-d6) 3.13-3.29 (m, 1H, H-1), 3.57-3.72
(m, 1H, H-1), 3.80 (s, 3H, CH3), 5.23-5.36 (m, 1H, H-10b), 7.12
(s, 1H, ar), 7.56 (s, 1H, ar), 10.16 (s, 1H, NH); IR 1740.
Meth yl 8,9-Dich lor o-5,6-d ih yd r o-5-oxo-p yr a zolo[1,5-c]-
qu in a zolin e-2-ca r boxyla te (7a ). A solution of tetrachloro-
1,2-benzoquinone (0.54 mmol) in anhydrous toluene (4 mL)
was added dropwise to a hot (120 °C) suspension of 14a (0.54
mmol) in anhydrous toluene (10 mL). The mixture was heated
at 120 °C for 1 h and then cooled at room temperature. The
resulting solid was collected and washed with diethyl ether:
1H NMR (DMSO-d6) 3.91 (s, 3H, CH3), 7.50 (s, 1H, ar), 7.84
(s, 1H, H-1), 8.56 (s, 1H, ar).
P h a r m a cology
Bin d in g Assa y. Rat cortical synaptic membrane prepara-
tion, [3H]glycine, [3H]AMPA, and [3H]-(+)-MK-801 binding
experiments were performed following the procedures de-
scribed in refs 11, 31, and 16, respectively.
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

Novel Excitatory Amino Acid Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 1043
[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; specific activity, 58 Ci/mmol) and
tested compounds in a total 0.5 mL volume. 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 of ice-cold buffer.
(11) 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.
(12) 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.
(13) 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.
(14) 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.
(15) 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-
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Med. Chem. 2000, 43, 3824-3826.
(16) 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.
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Costagli, C.; Carla`, V. Synthesis, ionotropic glutamate receptor
binding affinity and structure-activity relationships of a new
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Electr op h ysiologica l Assa y. The cortical wedge prepara-
tion described by Mannaioni et al. 32 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
(component, mM: NaCl, 135; CaCl2, 2.4; KH2PO4, 1.3; MgCl2,
1.2; NaHCO3, 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
(5µM) and NMDA (5µM) 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 antagonist-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 compart-
ments 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 obtained in the presence of both agonist and antagonist
with control peaks.
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 to four displacement
curves on the basis of four to six scalar concentrations of the
test compound in triplicate using the ALLFIT computer
program33 and, in the case of tritiated glycine and AMPA
binding, converted to Ki values by application of the Cheng-
Prusoff equation.34 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.
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