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

Article abstract of DOI:10.1021/jm981102r

A series of 4,5-dihydro-1,2,4-triazolo[1,5-a]quinoxalin-4-ones bearing different substituents on the benzo-fused ring and at position 2 were synthesized and biologically evaluated for their binding at glycine/NMDA and AMPA receptors. Most of the reported compounds show combined glycine/NMDA and AMPA receptor binding activity providing further evidences of the structural similarities of the binding pockets of both receptor recognition sites. Moreover, this study has pointed out some differences for the binding at each receptor type. In particular, for the glycine/NMDA receptor-ligand interaction, the presence of a free acidic function at position 2 and an electron-withdrawing substituent(s) nonbulkier than chlorine atom(s) on the benzo-fused moiety is required. Functional antagonism at the NMDA receptor- ion channel complex was also performed on some selected compounds.

Full text of DOI:10.1021/jm981102r

2478
J . Med. Chem. 1999, 42, 2478-2484
Brief Articles
4,5-Dih yd r o-1,2,4-tr ia zolo[1,5-a ]qu in oxa lin -4-on es: Excita tor y Am in o Acid
An ta gon ists w ith Com bin ed Glycin e/NMDA a n d AMP A Recep tor Affin ity
Daniela Catarzi,† Vittoria Colotta,† Flavia Varano,† Lucia Cecchi,*,† Guido Filacchioni,† Alessandro Galli,‡ and
Chiara Costagli‡
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 Pieraccini, 6, 50134 Firenze, Italy
Received February 12, 1999
A series of 4,5-dihydro-1,2,4-triazolo[1,5-a]quinoxalin-4-ones bearing different substituents on
the benzo-fused ring and at position 2 were synthesized and biologically evaluated for their
binding at glycine/NMDA and AMPA receptors. Most of the reported compounds show combined
glycine/NMDA and AMPA receptor binding activity providing further evidences of the structural
similarities of the binding pockets of both receptor recognition sites. Moreover, this study has
pointed out some differences for the binding at each receptor type. In particular, for the glycine/
NMDA receptor-ligand interaction, the presence of a free acidic function at position 2 and an
electron-withdrawing substituent(s) nonbulkier than chlorine atom(s) on the benzo-fused moiety
is required. Functional antagonism at the NMDA receptor-ion channel complex was also
performed on some selected compounds.
In tr od u ction
oxaline-2,3-dione derivatives, which have been reported
to have glycine/NMDA and AMPA receptor affinity,
have led to the identification of the common structural
requirements for their anchoring to the recognition site
of both receptors.9,12-17
Thus, the purpose of this study was to prepare some
1,2,4-triazolo[1,5-a]quinoxalin-4-ones with combined gly-
cine/NMDA and AMPA receptor antagonist activities.
In these new tricyclic quinoxalin-4-ones the nitrogen
atom at position 3 is replacing the 3-carbonyl oxygen of
the bicyclic quinoxalinediones. Different substituents
have been placed on the benzo-fused ring and at position
2 of the tricyclic system to assess the similarities and/
or differences for the binding at each receptor in these
new kinds of ligands.
Glutamate receptors (GluRs) mediate most of the
excitatory neurotransmission in the mammalian central
nervous system. GluRs have been classified into two
general classes of receptors: those that form ion chan-
nels or “ionotropic”, and those that are linked to G-
protein or “metabotropic”.1,2 The ionotropic GluRs are
further subdivided according to their preferential ago-
nists as NMDA (N-methyl-D-aspartate), AMPA (R-
amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid),
and KA (kainic acid).3 Like other ligand-gated ion
channels, the NMDA receptor is modulated by a number
of different molecules acting at different sites, and
among them the amino acid glycine, binding at the
allosteric strycnine-insensitive glycine site (glycine/
NMDA), is particularly important for its synergic action
with glutamate for the NMDA receptor activation.4
Ch em istr y
The synthesis of the 4,5-dihydro-1,2,4-triazolo[1,5-a]-
quinoxaline derivatives 1a -i, 2a -g,i-l, and 3-21 is
illustrated in Schemes 1 and 2. The triazoloquinoxaline-
2-carboxylic esters 1a -f,h were prepared through the
intermediates 22-25a -g, following reported proce-
dures18,19 (Scheme 1). The tricyclic 7-nitro and 7-bromo
esters 1g,i were prepared from the diazonium salt of
the 7-amino ester 1h with sodium nitrite or copper(II)
bromide, respectively. The hydrolysis of the esters 1a -
g,i gave the corresponding acids 2a -g,i, while the nitro
acids 2j-l ensued by nitration of the acids 2a -c.
By reacting the tricyclic 7-chloro ester 1b with ethyl
iodide (Scheme 2), a mixture of 4-ethoxy ester 3 and 5-N-
ethyl ester 4 was obtained. After chromatographic
separation, the main product of the alkylation reaction,
i.e., the N-ethyl ester 4, was hydrolyzed to the corre-
sponding acid 5. In addition, reduction of the tricyclic
Overstimulation of both NMDA and AMPA receptors
by excessive endogenous glutamate, which occurs in
several pathological conditions, can cause excess excita-
tion that initiates neuronal cell death. Thus, antagonists
at both NMDA and AMPA receptors are attractive
therapeutic targets in many neurological diseases, such
as focal and global ischemia5,6 and seizure disorders,7-9
and in the protection of neuronal degeneration in the
retina.10 It has been established that there is a consid-
erable similarity in structural requirements for binding
at the glycine/NMDA and AMPA recognition sites.9,11-12
In fact, structure-activity relationships (SAR) on quin-
* 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/jm981102r CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/16/1999

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2479
Sch em e 1a
Sch em e 2a
k
a
(a) EtI; (b) NaOH, HCl; (c) LiBH4; (d) PBr3; (e) NaCN; (f) concd
a
(a) Diazotization; (b) CH3COCHClCOOEt; (c) NH3(g); (d)
H2SO4/EtOH; (g) NaOH, CH3COOH; (h) NH2OH‚HCl/NaOH; (i)
N2H4‚H2O; (j) SOCl2, NH4OH; (k) RNH2.
ClCOCOOEt; (e) heating above mp or concd H2SO4; (f) Fe/
CH3COOH; (g) diazotization, NaNO2 or CuBr2; (h) NaOH, HCl;
(i) 90% HNO3.
ylate residue at position 2 and a cationic proton donor
site of the receptor.20
7-chloro ester 1b yielded the alcohol 6, which was
reacted with phosphorus tribromide to give the bro-
momethyl derivative 7. By reacting 7 with sodium
cyanide the acetonitrile 8 was obtained. The latter was
transformed into the ethyl acetate 9, which was hydro-
lyzed to give the corresponding 2-acetic acid 10. Finally,
by reacting 1b with hydroxylamine hydrochloride or
hydrazine hydrate, the 2-hydroxamic acid 11 and 2-hy-
drazide 12 were obtained, respectively.
The preparation of the 2-carboxamide derivatives 13-
20 from the 7-chloro acid 2b is also illustrated in
Scheme 2. The glycine methyl ester 20 was hydrolyzed
to the corresponding acid 21.
The importance of the presence of the NH proton
donor and of electron-withdrawing substituent(s) on the
benzo-fused moiety is illustrated by the inactivity of the
5-N-ethyl acid 5 and 7-amino ester 1h , respectively,
which are inactive at both receptors. However, the
position and the nature of the substituent on the benzo-
fused moiety are very important. A chlorine atom at
position 7 enhances receptor affinity (see 2b vs 2a ),
while the same halogen at position 8 decreases it (see
2c vs 2b) at both receptors. The same applies to the
7-nitro acid 2g and 8-nitro acid 2j. However, the
replacement of the 7-chlorine atom of 2b with a bulkier
substituent such as a trifluoromethyl or nitro group or
a bromine atom (compounds 2f,g,i, respectively) de-
creases glycine/NMDA affinity, while it has little or no
effect on AMPA binding activity, with the exception of
the 7-bromo derivative 2i which, in agreement with the
literature data,9,16 is 3-fold less active than 2b. It follows
that in the glycine/NMDA receptor there is a size-
limited region which cannot well-accommodate a 7- or
8-substituent bulkier than a chlorine atom.
Resu lts a n d Discu ssion
The triazoloquinoxalines 1a -f,h -i, 2a -g,i-l, 5,6,
and 8-21 were tested for their ability to displace both
tritiated glycine and AMPA from their specific binding
in rat cortical membranes. The binding data are shown
in Tables 1 and 2. The results listed in Table 1 show
that all the acids 2 are more active than the correspond-
ing esters 1 especially at the level of the glycine/NMDA
receptor. The enhanced glycine/NMDA affinity of the
acids 2 with respect to the corresponding esters 1 arises
from a strong interaction between the anionic carbox-
The disubstitution of the benzo-fused moiety has
different effects on the binding activity depending on
the receptor type. In fact, the 7,9-dichloro acid 2d and
7,8-dichloro acid 2e are respectively equally active or

2480 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Brief Articles
Ta ble 1. Binding Activities at Glycine/NMDA and AMPA
Receptors of Tricyclic Esters and Acids
Ta ble 3. Functional Antagonism at Glycine/NMDA Site of
Some Selected Triazoloquinoxalin-4-ones
compd
[3H]MK-801a EC50 (µM)
1a
1b
1e
1h
2a
2b
2c
2d
2e
2f
2g
2i
2j
2l
5
69.0 ( 12.0
22.1 ( 1.5
1.5 ( 0.28
>100
4.9 ( 0.9
2.9 ( 0.3
32.5 ( 1.9
1.35 ( 1.2
0.30 ( 0.05
3.18 ( 0.21
7.8 ( 0.7
3.7 ( 0.24
>100
Kia (µM)
compd
R
R6
R7
R8 R9
[3H]glycine
[3H]AMPA
1a
1b
1c
1d
1e
1f
1h
1i
2a
2b
2c
2d
2e
2f
2g
2i
Et
Et
Et
Et
Et
Et
Et
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
Cl
H
H
Cl
H
Cl
H
H
H
H
H
Cl
H
Cl
H
H
H
NO2
NO2
Cl
H
H
H
16.7 ( 1.2
3.0 ( 0.4
28.5 ( 1.2
2.6 ( 0.5
72.1 ( 19.5
4.9 ( 1.4
33.9 ( 5.9
3.6 ( 0.4
>100
3.3 ( 0.7
13.6 ( 0.8
0.78 ( 0.15
18.4 ( 3.4
1.53 ( 0.2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
>100
>100
0.33 ( 0.05
88.2 ( 15.0
Cl 4.8 ( 0.5
5,7-dichlorokynurenic acid
Cl
H
H
H
H
H
H
H
0.71 ( 0.08
1.8 ( 0.3
>100
2.6 ( 0.2
1.1 ( 0.02
0.17 ( 0.02
3.5 ( 0.8
a
Concentrations giving 50% inhibition of stimulated [3H]-(+)-
CF3
NH2
Br
H
Cl
H
Cl
Cl
CF3
NO2
Br
H
MK-801 binding. All assays were carried out in the presence of
10 µM glutamate: 100% binding ) glutamate + buffer; 0% binding
) glutamate + phencyclidine hydrochloride (100 µM). The results
were calculated from 3-4 separate determinations in triplicate.
Cl 0.14 ( 0.02
7,8-dichloro acid 2e have similar affinities at the AMPA
receptor, demonstrating that in this receptor there is a
larger lipophilic pocket able to accommodate bulky
substituent(s). As expected, the 6-nitro-8-chloro acid 2l
displays low affinity at both receptors.
H
H
H
H
H
H
H
0.074 ( 0.003 1.3 ( 0.2
0.57 ( 0.08
0.71 ( 0.04
0.39 ( 0.09
79.4 ( 8.8
2.2 ( 0.3
4.5 ( 0.5
88.0 ( 11.5
0.90 ( 0.09
0.60 ( 0.08
4.4 ( 0.7
10.7 ( 0.7
1.2 ( 0.08
4.0 ( 0.5
>100
2j
2k
2l
Cl
H
The importance in the triazoloquinoxalines of a free
acidic group at position 2 is further confirmed by the
introduction of different substituents in this position of
the parent acid 2b. The binding results shown in Table
2 indicate that, in general, the replacement of the
2-carboxylic group of 2b results in a reduction in binding
activity at both receptors. Moreover, the decreased
binding activities of the 2-acetic acid 10 and hydroxamic
acid 11 suggest that the spatial position of the free acidic
function is critical for receptor-ligand interaction at
both receptors. Moving the acidic function in the side
chain at position 2 further, as in compounds 18 and 21,
produces a progressive reduction in binding activity at
glycine/NMDA receptor. The amides 14-17, prepared
to investigate the influence of a lipophilic moiety on the
side chain at position 2, unfortunately could not be
tested because of their insolubility in the medium of the
binding assay.
Functional antagonism at the NMDA receptor-ion
channel complex was demonstrated by the ability of
some triazoloquinoxalines to inhibit the binding of the
channel-blocking agent [3H]-(+)-MK-80112,21-22 in mem-
branes incubated with 10 µM glutamate and in the
nominal absence of glycine (Table 3). The results shown
in Table 3 indicate that, as in the case of [3H]glycine
displacement assays, compound 2e is the most potent
compound tested with an EC50 of 0.30 ( 0.05 µM. This
value is very close to that obtained in our experiments
with the glycine/NMDA antagonist 5,7-dichlorokynurenic
acid (0.33 ( 0.05 µM). The EC50 values of these
compounds for glutamate-stimulated [3H]-(+)-MK-801
binding closely correlated with their Ki values on [3H]-
glycine binding. Figure 1 shows that the inhibition of
[3H]-(+)-MK-801 binding to the channel of activated
NMDA receptor by 2e can be completely reversed by
increasing glycine concentrations, indicating competi-
tion between the two compounds for the regulatory site
on the receptor.
5
a
Ki values are means ( SEM of 3-4 separate determinations
in triplicate. The tested compounds were dissolved in DMSO.
Ta ble 2. Binding Activity at Glycine/NMDA and AMPA
Receptors of Compounds 6 and 8-21
Kia (µM)
[3H]glycine
compd
R
[3H]AMPA
6
8
9
CH2OH
CH2CN
CH2COOEt
CH2COOH
CONHOH
CONHNH2
CONH2
CONHC6H5
CONHCH2C6H5
CONH-3-OCH3C6H4
CONH-3-OHC6H4
CONH-5-tetrazolyl
CONHCH2CH2OH
CONHCH2COOCH3
CONHCH2COOH
7.5 ( 0.9
9.7 ( 1.8
6.5 ( 1.8
1.1 ( 0.04
1.1 ( 0.09
9.6 ( 0.5
7.2 ( 1.4
NTb
4.4 ( 0.8
2.0 ( 0.2
2.1 ( 0.4
3.7 ( 0.5
3.0 ( 0.4
5.0 ( 0.5
11.7 ( 1.7
NTb
10
11
12
13
14
15
16
17
18
19
20
21
NTb
NTb
NTb
NTb
NTb
NTb
5.1 ( 0.3
27.7 ( 5.9
5.4 ( 0.7
8.8 ( 0.9
2.4 ( 0.2
7.7 ( 1.9
4.1 ( 0.4
3.2 ( 0.4
a
b
See Table 1. Due to the insolubility of the compound in the
medium binding assay, testing was not possible.
more active at the glycine/NMDA receptor and less
active at the AMPA receptor than the 7-monochloro acid
2b. In the case of glycine/NMDA receptor, the size-
limited tolerance observed in the monosubstitution of
the benzo-fused moiety is confirmed by the binding
results of the 7,8-disubstituted derivatives (see 2k vs
2e). On the contrary, the 7-chloro-8-nitro acid 2k and

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2481
Ta ble 4. Physical Data of the Final Tricyclic Compounds
compd
mp (°C)
cryst solv
% yield
1b
1d
1e
1f
1g
1h
1i
2b
2c
2d
2e
2f
2g
2i
2j
2k
2l
3
4
5
6
7
8
280-282
263-264
298-300
>300
acetone
acetone
EtOH
78
70
82
86
45
92
18
93
94
83
84
98
82
90
67
68
63
10
40
95
98
65
30
49
88
87
98
90
90
90
71
89
57
62
85
81
EtOH
280-281
>300
acetone
acetone
acetonitrile
H2O
EtOH/H2O
EtOH
EtOH
EtOH
EtOH
EtOH
284-285
>300
264-266 dec
>300
>300
>300
>300
>300
237-240
>300
EtOH
EtOH
EtOH
F igu r e 1. Effect of increasing glycine concentrations on the
antagonist action of 2e on NMDA receptor-mediated [3H]-(+)-
MK-801 binding to rat cortical membranes. Ordinate values
represent percentages of specific [3H]-(+)-MK-801 binding in
the nominal absence of glycine and glutamate. In the absence
of glycine, the specific [3H]-(+)-MK-801 binding was equal to
568% or 41% of controls in the presence of 10 µM glutamate
plus buffer or 10 µM glutamate plus 10 µM 2e, respectively.
Points on the graph represent the means ( SEM from three
experiments in triplicate.
>300
>300a
>300a
>300
EtOH
EtOH/DMF
EtOH
acetone
EtOH
EtOH
265-267
>300
279-280
200-202
254-255
>300
9
10
11
12
13
14
15
16
17
18
19
20
21
EtOH
>300
DMF/H2O
DMF/H2O
DMF/H2O
DMF/H2O
DMF/H2O
DMF/H2O
DMF
DMF/H2O
DMF
DMF/H2O
In conclusion, the synthesis of these triazoloquinoxa-
lines has produced some nonselective antagonists of the
glycine/NMDA and AMPA receptors. This result pro-
vides further evidence of the structural similarities of
the binding pockets of both receptor recognition sites
and confirms our original hypothesis that the 3-carbonyl
oxygen of the bicyclic quinoxalinediones can be replaced
by the nitrogen atom at position 3 of the triazoloquin-
oxalines. Moreover, the SAR on these new kinds of
glycine/NMDA and AMPA receptor ligands have pointed
out some differences for the binding at each receptor
type. In particular, for the glycine/NMDA receptor-
ligand interaction, the presence of a free acidic function
at position 2 and substituent(s) on the benzo-fused
moiety nonbulkier than chlorine atom(s) is required.
It follows that the triazoloquinoxalin-4-one tricyclic
system may represent a structure which, suitably
modified, could lead to selective glycine/NMDA or
AMPA receptor ligands. Further modifications of these
molecules to improve biological activity and/or selectiv-
ity are in progress.
>300
>300
>300
>300
>300
>300
298-300 dec
>300
>300
a
Compound purified by column chromatography, eluting system
ethyl acetate/cyclohexane (1:1).
Ta ble 5. Physical Data of the Intermediate Compounds
compd
mp (°C)
cryst solv
% yield
22d
22e
22f
22g
23d
23e
23f
23g
24b
24d
24e
24f
24g
25b
25d
25e
25f
25g
84-85
EtOH
EtOH
EtOH
EtOH
Et2O/petroleum ether
EtOH
Et2O
EtOH
MeOH
MeOH
EtOH
Et2O
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
20
57
68
76
98
86
87
76
74
54
86
55
77
63
95
87
41
69
125-126
95-96
120-121
97-98
179-180
108-109
108-109
158-159
126-127
167-168
167-169
152-155
165-166
149-150
163-165
141-142
164-165
Exp er im en ta l Section
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-
ses were performed with
a Perkin-Elmer 260 elemental
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
2-nitroaryldiazonium chloride.18,23 Compounds 22d -g were
obtained by reacting at room temperature equimolar amount
(40 mmol) of the suitable 2-nitroaryldiazonium tetrafluoborate
and ethyl 2-chloro-3-oxobutanoate in a small amount of MeOH.
The synthesis of 4,6-dichloro-2-nitrophenyl- and 2,4-dinitro-
phenyldiazonium tetrafluoborate is reported in refs 24 and 25,
respectively. The unknown 4,5-dichloro-2-nitrophenyl- and
4-(trifluoromethyl)-2-nitrophenyldiazonium tetrafluoborates
were obtained following the method described in ref 24.
Compound 22b displays the following spectral data: 1H NMR
(CDCl3) 1.44 (t, 3H, CH3), 4.43 (q, 2H, CH2), 7.60 (dd, 1H, ar,
J ) 9.1, 2.3 Hz), 7.94 (d, 1H, ar, J ) 9.1 Hz), 8.24 (d, 1H, ar,
J ) 2.3 Hz), 11.33 (s, 1H, NH); IR 1750, 3100, 3120, 3300.
1
expressed in cm-1. The H 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.
Physical data of the newly synthesized compounds are listed
in Tables 4 and 5.
E t h yl N1-(2-Nit r oa r yl)h yd r a zon o-N2-ch lor oa cet a t es
22a -g. All the title compounds were prepared by reacting the
diazonium salt of the suitable 2-nitroaniline with ethyl 2-chloro-
3-oxobutanoate. Compounds 22a -c were prepared from the

2482 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Brief Articles
Eth yl N1-(2-Nitr oa r yl)-N2-oxa m id r a zon a tes 23a -g. The
title compounds were obtained from 22a -g (49 mmol) as des-
cribed for the synthesis of 23a -b.18,23 Compound 23b displays
the following spectral data: 1H NMR (CDCl3) 1.43 (t, 3H, CH3),
4.42 (q, 2H, CH2), 4.97 (br s, 2H, NH2), 7.51 (dd, 1H, ar, J )
9.2, 2.4 Hz), 7.87 (d, 1H, ar, J ) 9.2 Hz), 8.17 (d, 1H, ar, J )
2.4 Hz), 10.04 (br s, 1H, NH); IR 1730, 3320, 3340, 3430.
Eth yl N1-(2-Nitr oa r yl)-N3-eth oxa lyl-N2-oxa m id r a zon -
a tes 24a -g. The title compounds were obtained from 23a -g
(2.18 mmol) and ethyloxalyl chloride (4.4 mmol) as described
for the synthesis of 24a ,c.18,19 Compound 24b displays the
following spectral data: 1H NMR (CDCl3) 1.44 (t, 3H, CH3),
1.49 (t, 3H, CH3), 4.43 (q, 2H, CH2), 4.54 (q, 2H, CH2), 7.53
(dd, 1H, ar, J ) 9.2, 2.3 Hz), 8.76 (d, 1H, ar, J ) 9.2 Hz), 8.20
(d, 1H, ar, J ) 2.3 Hz), 9.30 (br s, 1H, NH), 13.27 (br s, 1H,
NH); IR 1720, 1730, 1775, 3100, 3280, 3400, 3420.
7.62 ((m, 2H, ar), 8.09 (d, 1H, ar, J ) 8.7 Hz), 12.56 (br s, 1H,
NH); IR 1690, 1740.
4,5-Dih yd r o-4-oxo-1,2,4-t r ia zolo[1,5-a ]q u in oxa lin e-2-
ca r boxylic Acid s 2g,i. The title compounds were obtained
from the esters 1g,i (2.4 mmol) as described for the synthesis
of 2a .19 Compound 2g displays the following spectral data: 1H
NMR (DMSO-d6) 8.23-8.35 (m, 3H, ar), 12.77 (br s, 1H, NH);
IR 1720.
Gen er a l P r oced u r e to P r ep a r e Tr icyclic Nitr oa r yl
Acid s 2j-l. Compounds 2a -c (0.6 mmol) were carefully added
to HNO3 (90%, 2 mL). The solution was stirred at 0-5 °C until
the disappearance of the starting material (TLC monitoring).
The mixture was then poured onto crushed ice (20 g). The
resulting solid was collected and washed with H2O. Compound
2k displays the following spectral data: 1H NMR (DMSO-d6)
7.65 (s, 1H, ar), 8.79 (s, 1H, ar), 12.9 (br s, 1H, NH); IR 1730.
Eth yl 7-Ch lor o-4-eth oxy-1,2,4-tr ia zolo[1,5-a ]qu in oxa -
lin e-2-ca r boxyla te (3) a n d Eth yl 7-Ch lor o-5-N-eth yl-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 te (4). Ethyl iodide (4.29 mmol) and NaH (8.3 mmol) were
successively added to a stirred solution of 1b (3.1 mmol) in
anhydrous dimethylformamide (DMF) (35 mL). The mixture
was stirred at room temperature for 6 h and then poured onto
crushed ice (40 g) to give a mixture of compounds 3 and 4.
The mixture was collected, washed with H2O, and separated
by column chromatography, eluting system cyclohexane/ethyl
acetate (1:1). Compound 3 was obtained by evaporation at
reduced pressure of the first running eluates, while compound
4 was obtained from evaporation at reduced pressure of the
second running eluates. Compounds 3 and 4 display the
following spectral data. 3: 1H NMR (DMSO-d6) 1.49 (t, 3H,
CH3), 1.51 (t, 3H, CH3), 4.48 (q, 2H, CH2), 4.69 (q, 2H, CH2),
7.75 (dd, 1H, ar, J ) 8.8, 2.3 Hz), 8.02 (d, 1H, ar, J ) 2.3 Hz),
8.36 (d, 1H, ar, J ) 8.8 Hz); IR 1740. 4: 1H NMR (DMSO-d6)
1.28 (t, 3H, CH3), 1.39 (t, 3H, CH3), 4.35-4.51 (m, 4H, 2CH2),
7.54 (dd, 1H, ar, J ) 8.7, 1.9 Hz), 7.94 (d, 1H, ar, J ) 1.9 Hz),
8.24 (d, 1H, ar, J ) 8.7 Hz); IR 1690, 1735.
7-Ch lor o-5-N-eth yl-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 (5). The title acid was
obtained from 4 (0.46 mmol) as described for the synthesis of
2a :19 1H NMR (DMSO-d6) 1.27 (t, 3H, CH3), 4.38 (q, 2H, CH2),
7.55 (dd, 1H, ar, J ) 8.8, 1.8 Hz), 7.93 (d, 1H, ar, J ) 1.8 Hz),
8.23 (d, 1H, ar, J ) 8.8 Hz); IR 1730, 3100, 3420, 3540.
7-Ch lor o-4,5-d ih yd r o-2-(h yd r oxym eth yl)-1,2,4-tr ia zolo-
[1,5-a ]qu in oxa lin -4-on e (6). LiBH4 (3.6 mmol) was carefully
added to a stirred solution of 1b (2.4 mmol) in anhydrous
tetrahydrofuran (THF) (12 mL). The mixture was stirred at
room temperature until the disappearance of the starting
material (TLC monitoring) and by adding some more LiBH4
(1.8 mmol each time) every 8 h. Evaporation of almost all the
solvent gave a white suspension which was treated with HCl
(2 N) until the end of the evolution of gas. After dilution with
iced H2O (5-6 mL) the solid was collected: 1H NMR (DMSO-
d6) 4.69 (d, 2H, CH2, J ) 3.5 Hz), 5.62-5.69 (m, 1H, OH), 7.39-
7.46 (m, 2H, ar), 8.06 (d, 1H, ar, J ) 8.6 Hz), 12.43 (br s, 1H,
NH); IR 1700, 3500.
Diet h yl 1-(2-Nit r oa r yl)-1,2,4-t r ia zole-3,5-d ica r b oxyl-
a tes 25a -g. Meth od A. Compounds 25a -c,e-g were ob-
tained from 24a -c,e-g (5.2 mmol) as described for the
synthesis of 25a ,c.18-19
Meth od B. Concentrated H2SO4 (12 mL) was added drop-
wise and under stirring to finely crushed 24d (2 mmol). The
solution was stirred at room temperature for 1 h and then
poured onto crushed ice (80 g). The resulting suspension was
extracted with ethyl acetate (50 mL × 3). The organic layers
were washed with a diluted solution of NaHCO3 (1%, 80 mL
× 3) and H2O (100 mL × 2) and then dried (Na2SO4).
Evaporation at reduced pressure of the solvent yielded solid
crude 25d . Compound 25b displays the following spectral
data: 1H NMR (CDCl3) 1.36 (t, 3H, CH3), 1.46 (t, 3H, CH3),
4.37 (q, 2H, CH2), 4.54 (q, 2H, CH2), 7.41 (d, 1H, ar, J ) 8.4
Hz), 7.80 (dd, 1H, ar, J ) 8.4, 1.6 Hz), 8.30 (d, 1H, ar, J ) 1.6
Hz); IR 1735, 1755.
Eth yl 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 boxyla tes 1a -f,h . The title compounds were ob-
tained from 25a -g (5.2 mmol) as described for the synthesis
of 1a ,c.18,19 Compound 1b displays the following spectral
data: 1H NMR (DMSO-d6) 1.39 (t, 3H, CH3), 4.45 (q, 2H, CH2),
7.42-7.46 (m, 2H, ar), 8.13 (d, 1H, ar, J ) 8.4 Hz), 12.54 (br
s, 1H, NH); IR 1705, 1750.
4,5-Dih yd r o-4-oxo-1,2,4-t r ia zolo[1,5-a ]q u in oxa lin e-2-
ca r boxylic Acid s 2a -f. The title compounds were obtained
by hydrolysis of 1a -f (2.4 mmol) as described for the synthesis
of 2a .19 Compound 2b displays the following spectral data: 1H
NMR (DMSO-d6) 7.48-7.42 (m, 2H, ar), 8.13 (d, 1H, ar, J )
8.4 Hz), 12.54 (s, 1H, NH); IR 1695, 1725.
E t h yl 4,5-Dih yd r o-7-n it r o-4-oxo-1,2,4-t r ia zolo[1,5-a ]-
qu in oxa lin e-2-ca r boxyla te (1g). Compound 1h (1.1 mmol)
was carefully added to a frozen (-5 °C) solution of nitrosyl-
sulfuric acid (1.1 mmol) in concentrated H2SO4 (1 mL). The
mixture was stirred at 0 °C for 2 h and then at 10 °C for 1 h
and finally poured onto crushed ice (15 g). The solid was
quickly filtered off, and the solution was cooled (0 °C) and
treated dropwise with a concentrated solution of NaBF4 (5.9
mmol in 2 mL of H2O). The mixture was stirred at 0-5 °C for
1.5 h. The resulting solid was collected and washed with a
diluted solution (25%) of NaBF4 and then with cold MeOH.
This 7-aryldiazonium tetrafluoborate was carefully added to
a stirred and warm (30 °C) solution of NaNO2 (10%, 55 mL).
The mixture was stirred at room temperature overnight and
then extracted with ethyl acetate (50 mL × 3). Evaporation of
the dried (Na2SO4) organic solvent yielded the crude title
compound: 1H NMR (DMSO-d6) 1.40 (t, 3H, CH3), 4.47 (q, 2H,
CH2), 8.18-8.39 (m, 3H, ar), 12.78 (br s, 1H, NH); IR 1695,
1735.
Eth yl 7-Br om o-4,5-d ih yd r o-4-oxo-1,2,4-tr ia zolo[1,5-a ]-
qu in oxa lin e-2-ca r boxyla te (1i). Compound 1h (1.83 mmol)
was carefully added to a cooled (0-5 °C) mixture of CuBr2 (2.29
mmol) and tert-butyl nitrite (2.79 mmol). The mixture was
stirred at room temperature overnight and then poured onto
crushed ice (20 g) and bleached with HCl (6 N). The resulting
solid was collected, washed with H2O, and purified by column
chromatography, eluting system CHCl3/acetonitrile (8.5:1.5):
1H NMR (DMSO-d6) 1.38 (t, 3H, CH3), 4.45 (q, 2H, CH2), 7.57-
2-(Br om om et h yl)-7-ch lor o-4,5-d ih yd r o-1,2,4-t r ia zolo-
[1,5-a ]qu in oxa lin -4-on e (7). PBr3 (28 mmol) was added
dropwise to a solution of 6 (2.8 mmol) in anhydrous THF (120
mL). The mixture was stirred at room temperature for 24 h
and then diluted with iced H2O (100 mL). The mixture was
treated with a solution of NaOH (10%) until pH ) 8-9 and
extracted with ethyl acetate (100 mL × 3). The dried (Na2-
SO4) organic extracts were evaporated at reduced pressure to
give a solid which was treated with Et2O and collected: 1H
NMR (DMSO-d6) 4.89 (s, 2H, CH2), 7.40-7.46 (m, 2H, ar), 8.06
(d, 1H, ar, J ) 8.4 Hz), 12.50 (s, 1H, NH); IR 1700.
7-Ch lor o-4,5-d ih yd r o-4-oxo-1,2,4-t r ia zolo[1,5-a ]q u in -
oxa lin e-2-a ceton itr ile (8). Compound 7 (1.5 mmol) was
added portionwise to a solution of NaCN (1.7 mmol) in DMSO
(10 mL). The yellow mixture was stirred at room temperature
for 4 days and then diluted with iced H2O (100 mL) and basi-
fied with a solution of NaOH (10%). The resulting solid was
collected, washed with H2O, and purified by column chroma-

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2483
tography, eluting system CHCl3/MeOH (9:1). The central
eluates were evaporated at reduced pressure to yield crude
compound 8. A second crop of 8 was obtained by extracting
with ethyl acetate (50 mL × 3) the alkaline mother solution
and evaporating at reduced pressure the organic solvent: 1H
NMR (DMSO-d6) 4.52 (s, 2H, CH2), 7.41-7.48 (m, 2H, ar), 8.01
(d, 1H, ar, J ) 8.7 Hz), 12.50 (br s, 1H, NH); IR 1700, 2260.
Eth yl 7-Ch lor 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-a ceta te (9). Concentrated H2SO4 (0.5 mL) was
added dropwise to a solution of 8 (0.46 mmol) in EtOH (1 mL).
The mixture was refluxed for 2.5 h, then cooled, and diluted
with H2O (10 mL). Neutralization of the solution with a
saturated solution of NaHCO3 yielded a solid which was
collected and washed with H2O: 1H NMR (DMSO-d6) 1.23 (t,
3H, CH3), 4.06 (s, 2H, CH2), 4.16 (q, 2H, CH2), 7.41-7.46 (m,
2H, ar), 8.06 (d, 1H, ar, J ) 8.5 Hz), 12.48 (br s, 1H, NH); IR
1700, 1735.
Hz), 9.61 (t, 1H, NH, J ) 6. 0 Hz), 12.52 (br s, 1H, NH); IR
1680, 1705, 3400.
N-(7-Ch lor o-4,5-d ih yd r o-4-oxo-1,2,4-tr ia zolo[1,5-a ]qu in -
oxa lin yl-2-ca r bon yl)glycin e Meth yl Ester (20). Triethyl-
amine (1.08 mmol) was added to a solution of glycine methyl
ester hydrochloride (1.08 mmol) in anhydrous DMF (20 mL).
The mixture was stirred at room temperature for 10 min.
Compound 2b (0.9 mmol), N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (1.8 mmol), and 1-hydroxy-
benzotriazole (1.8 mmol) were successively added to the
mixture, which was then stirred at room temperature for 15
h. Dilution with iced H2O (80 mL) yielded a solid which was
collected and washed with H2O: 1H NMR (DMSO-d6) 3.69 (s,
3H, CH3), 4.08 (d, 2H, CH2, J ) 5.6 Hz), 7.45-7.50 (m, 2H,
ar), 8.14 (d, 1H, ar, J ) 8.9 Hz), 9.37 (t, 1H, NH, J ) 5.6 Hz),
12.50 (br s, 1H, NH); IR 1695, 1740, 3370.
N-(7-Ch lor o-4,5-d ih yd r o-4-oxo-1,2,4-tr ia zolo[1,5-a ]qu in -
oxa lin yl-2-ca r bon yl)glycin e (21). A solution of NaOH (1 M,
1.8 mL) was added to a suspension of 20 (0.53 mmol) in THF/
methanol (1:1, 10 mL). The mixture was stirred at room
temperature overnight. The resulting solid was collected and
dissolved in H2O (10 mL). The solution was acidified with HCl
(6 N) to afford a solid which was collected and washed with
H2O: 1H NMR (DMSO-d6) 3.97 (d, 2H, CH2, J ) 6.0 Hz), 7.45-
7.50 (m, 2H, ar), 8.14 (d, 1H, ar, J ) 8.4 Hz), 9.17 (t, 1H, NH,
J ) 6.0 Hz), 12.50 (br s, 1H, NH); IR 1690, 1740, 3240, 3360.
Bioch em istr y. Synaptic membrane preparation and [3H]-
glycine and [3H]-DL-AMPA binding experiments were per-
formed following described procedures.26,27
7-Ch lor o-4,5-d ih yd r o-4-oxo-1,2,4-t r ia zolo[1,5-a ]q u in -
oxa lin e-2-a cetic Acid (10). A solution of NaOH (1 M, 0.5 mL)
was added to a solution of 9 (0.16 mmol) in THF (5 mL). The
mixture was stirred at room temperature for 24 h, then diluted
with iced H2O (15 mL), and acidified with glacial acetic acid.
The mixture was extracted with ethyl acetate (25 mL × 3).
The organic extracts were washed with H2O (25 mL × 3), dried
(Na2SO4), and evaporated at reduced pressure to produce a
solid: 1H NMR (DMSO-d6) 3.95 (s, 2H, CH2), 7.40-7.46 (m,
2H, ar), 8.06 (d, 1H, ar, J ) 8.7 Hz), 12.47 (br s, 1H, NH),
12.73 (br s, 1H, COOH); IR 1605, 1700, 3400.
[3H]-(+)-MK-801 Bin d in g. [3H]-(+)-MK-801 ((+)-5-methyl-
10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate)
binding assays were carried out according to Yoneda and
Ogita28 with slight modifications. Membranes were resus-
pended (0.5 mg of 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 centrifuga-
tion (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 temperature for 120 min with 2.5
nM [3H]-(+)-MK-801 (22.5 Ci/mmol) and 10 µM glutamic acid
in the presence and absence of the test compound in a total
volume of 0.5 mL. Bound radioactivity was separated by
filtration through GF/C filters presoaked in 0.05% poly-
(ethylenimine) and washed with ice-cold buffer (3 × 5 mL).
Nonspecific binding was determined in the presence of 100 µM
phencyclidine hydrochloride.
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 3-4 displacement curves
based on 4-6 scalar concentrations of the test compound in
triplicate using the ALLFIT computer program29 and, in the
case of tritiated glycine and AMPA binding, converted to Ki
values by application of the Cheng-Prusoff equation.30 In 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.
7-Ch lor o-4,5-d ih yd r o-4-oxo-1,2,4-t r ia zolo[1,5-a ]q u in -
oxa lin e-2-ca r boh yd r oxa m ic Acid (11). Hydroxylamine hy-
drochloride (10.2 mmol) was added to a solution of NaOH (10.2
mmol) in absolute EtOH (50 mL). The mixture was stirred at
room temperature overnight. The solid was filtered off. Com-
pound 1b (1.02 mmol) and NaOH (10.2 mmol) were added to
the solution which was then stirred at room temperature until
disappearance of the staring material (TLC monitoring).
Acidification with HCl (6 N) gave a solid which was filtered
off. The solution was concentrated at reduced pressure to yield
a solid which was collected: 1H NMR (DMSO-d6) 7.46 (dd, 1H,
ar, J ) 8.8, 2.2 Hz), 7.60 (d, 1H, ar, J ) 2.2 Hz), 8.12 (d, 1H,
ar, J ) 8.8 Hz), 9.43 (br s, 1H, NH/OH), 11.84 (br s, 1H, OH/
NH); IR 1720, 3100, 3200, 3440.
7-Ch lor o-4,5-d ih yd r o-4-oxo-1,2,4-t r ia zolo[1,5-a ]q u in -
oxa lin e-2-ca r boh yd r a zid e (12). Hydrazine monohydrate (32
mmol) was added dropwise to a suspension of 1b (1.7 mmol)
in EtOH (20 mL). The resulting solid was collected and washed
with H2O: 1H NMR (DMSO-d6) 3.37 (br s, 2H, NH2), 5.30 (br
s, 1H, NH), 7.42-7.49 (m, 2H, ar), 8.13 (d, 1H, ar, J ) 9.2
Hz), 12.50 (br s, 1H, NH); IR 1695, 1730, 3300.
7-Ch lor o-4,5-d ih yd r o-4-oxo-1,2,4-t r ia zolo[1,5-a ]q u in -
oxa lin e-2-ca r boxa m id e (13). A suspension of 2b (2.6 mmol)
in thionyl chloride (10 mL) was refluxed for 4 h. Evaporation
at reduced pressure of the solvent yielded a residue which was
washed twice with cyclohexane (30 mL) and immediately
reacted with NH4OH (30%, 50 mL) under stirring and cooling
(ice bath). The mixture was then stirred at room temperature
overnight. Dilution with H2O (20 mL) yielded a solid which
was collected and washed with H2O: 1H NMR (DMSO-d6)
7.45-7.48 (m, 2H, ar), 7.92 (br s, 1H, NH), 8.13 (d, 1H, ar, J
) 9.5 Hz), 8.33 (br s, 1H, NH), 12.50 (br s, 1H, NH); IR 1700,
3460.
Ack n ow led gm en t. Valuable assistance in animal
handling was provided by M. G. Giovannini.
Refer en ces
Gen er a l P r oced u r e for th e P r ep a r a tion of Ca r boxa m -
id es 14-19. N-3-(Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (2.26 mmol), 1-hydroxybenzotriazole (2.26 mmol),
and the suitable amine (1.69 mmol) were successively added
to a solution of 2b (1.13 mmol) in anhydrous DMF (20 mL).
The mixture was stirred at room temperature for 24 h.
Compounds 14-17 and 19 were precipitated by diluting the
solution with a small amount of H2O, while compound 18
precipitated without dilution. Compounds 14-19 were col-
lected and washed with H2O. Compound 15 displays the
following spectral data: 1H NMR (DMSO-d6) 4.51 (d, 2H, CH2,
J ) 6.0 Hz), 7.25-7.49 (m, 7H, ar), 8.14 (d, 1H, ar, J ) 9.1
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