Synthesis of a set of ethyl 1-carbamoyl-3-oxoquinoxaline-2-carboxylates and of their constrained analogue imidazo[1,5-a]quinoxaline-1,3,4-triones as glycine/NMDA receptor antagonists

Article abstract of DOI:10.1016/S0223-5234(00)01203-4

The synthesis and glycine/NMDA and AMPA receptor affinities of a set of ethyl (±) 1-N-carbamoyl-1,2,3,4-tetrahydro-3-oxoquinoxaline-2-carboxylates 1-11 and those of their constrained analogue (±) 1,2,3,3a,4,5-hexahydroimidazo[1,5-a]quinoxaline-1,3,4-triones 12-24 are reported. Compounds 1-11 bear a side-chain at position 1 which has been spatially constrained in compounds 12-24. Most of the reported tricyclic derivatives 12-24 showed glycine/NMDA binding activity comparable to that of their corresponding bicyclic analogues 1-11 providing further evidence that the spatial orientation of the side-chain is an important structural requirement for glycine/NMDA receptor antagonists.

Full text of DOI:10.1016/S0223-5234(00)01203-4

Eur. J. Med. Chem. 36 (2001) 203–209
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(00)01203-4/FLA
Short communication
Synthesis of a set of ethyl 1-carbamoyl-3-oxoquinoxaline-2-carboxylates and of
their constrained analogue imidazo[1,5-a]quinoxaline-1,3,4-triones as
glycine/NMDA receptor antagonists
Flavia Varanoa, Daniela Catarzia, Vittoria Colottaa, Lucia Cecchia*, Guido Filacchionia,
Alessandro Gallib, Chiara Costaglib
aDipartimento di Scienze Farmaceutiche, Universita’ di Firenze, Via G. Capponi, 9, 50121 Firenze, Italy
bDipartimento di Farmacologia Preclinica e Clinica, Universita’ di Firenze, Viale G. Pieraccini, 6, 50134 Firenze, Italy
Received 19 July 2000; revised 7 November 2000; accepted 23 November 2000
Abstract – The synthesis and glycine/NMDA and AMPA receptor affinities of a set of ethyl ( ) 1-N-carbamoyl-1,2,3,4-tetrahydro-
3-oxoquinoxaline-2-carboxylates 1–11 and those of their constrained analogue ( ) 1,2,3,3a,4,5-hexahydroimidazo[1,5-a]quinoxaline-
1,3,4-triones 12–24 are reported. Compounds 1–11 bear a side-chain at position 1 which has been spatially constrained in
compounds 12–24. Most of the reported tricyclic derivatives 12–24 showed glycine/NMDA binding activity comparable to that of
their corresponding bicyclic analogues 1–11 providing further evidence that the spatial orientation of the side-chain is an important
´
structural requirement for glycine/NMDA receptor antagonists. © 2001 Editions scientifiques et me´dicales Elsevier SAS
quinoxaline derivatives / glycine/NMDA receptor / AMPA receptor / antagonists
1. Introduction
(glycine/NMDA), is particularly important as a co-
agonist of glutamate for NMDA receptor activation
[3].
Ionotropic glutamate receptors (iGluRs), namely N
- methyl -
D
- aspartate (NMDA), (RS) - 2 - amino-
Structure–activity relationship studies on quinoxa-
line-2,3-diones, which have been reported to have
glycine/NMDA and AMPA receptor antagonist ac-
tivities [4–7], have revealed that there are remarkable
structural similarities in the binding sites of both
receptor types [8, 9]. As shown in figure 1, both
glycine/NMDA and AMPA antagonists possess a
NH proton donor which binds to a proton acceptor
of the receptors, as well as negatively-charged het-
eroatoms able to form a Coulombic interaction with a
positive site of the receptors. Both glycine/NMDA
and AMPA receptors can tolerate a polar side-chain
called X (figure 1), and both prefer to accommodate
electron-withdrawing group(s) at R1 and/or R2, al-
though some quinoxaline-2,3-diones with R1=R2 =
Me have been reported to bind at both receptors with
low micromolar affinity [10]. However, glycine/
NMDA or AMPA selectivity can be modulated by
3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid
(AMPA) and kainic acid (KA), play key roles in
neuronal transmission in the mammalian central ner-
vous system, but are also likely to be involved in
numerous pathological and excitotoxic processes [1,
2]. Selective iGluR agonists and antagonists have
provided the tools for understanding the physiologi-
cal and pathological roles of each receptor type [2].
Like other ligand-gated ion channels, the NMDA
receptor is modulated by a number of different
molecules and among them the amino acid glycine,
binding at the strychnine-insensitive binding site
Abbreviations: AMPA, (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isox-
azolyl) propionic acid; DMSO, dimethylsulfoxide; glycine/NMDA,
strychnine-insensitive binding site; iGluRs, ionotropic glutamate re-
ceptors; NMDA, N-methyl-D-aspartate.
* Correspondence and reprints.
E-mail address: cecchi@farmfi.scifarm.unifi.it (L. Cecchi).

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F. Varano et al. / European Journal of Medicinal Chemistry 36 (2001) 203–209
varying the nature of R1, R2 and the spatial orienta-
tion of the X polar side-chain. Generally, bulky elec-
tron-withdrawing NO2, CF3 or Br substituents and a
nitrogen-containing heterocycle are preferred by the
AMPA receptor in R1 and R2, respectively, while the
glycine/NMDA site preferably accommodates chlo-
rine atoms in R1 and R2. Moreover, the AMPA
receptor prefers polar side-chains oriented towards
the benzo-fused moiety (i.e. north-western orienta-
tion), while the glycine/NMDA one accommodates
polar side-chains on the opposite side (i.e. north-east-
ern orientation) [6].
N-[(2,4-dinitrophenyl)amino]malonate 28 [13] yielded
the ethyl 6-amino-3,4-dihydro-3-oxoquinoxaline-2-
On this basis, the present paper reports the synthe-
sis and glycine/NMDA and AMPA receptor-binding
activity of a set of ethyl ( ) 1-N-carbamoyl-1,2,3,4-te-
trahydro-3-oxoquinoxaline-2-carboxylates 1–11 and
of their constrained analogue ( ) 1,2,3,3a,4,5-hexahy-
Figure 1. Common structural requirements for glycine/
NMDA and AMPA receptor antagonists.
droimidazo[1,5-a]quinoxaline-1,3,4-triones
12–24
(figure 2).
The
tetrahydro-3-oxoquinoxaline-2-carboxylates
1–11 can be regarded as analogues of the 3-nitro-3,4-
dihydro-2(1H)-quinolones [11] which are selective
glycine/NMDA receptor antagonists. In fact, the elec-
tron-withdrawing 2-carboxylate group of compounds
1–11, like the 3-nitro one of the quinolone analogues,
confers acidity to the 2-hydrogen atom, increasing the
charge density on the lactam oxygen at position-3.
Moreover, compounds 1–11 can be easily trans-
formed by cyclization into compounds 12–24, where
the possibility of delocalization of the negative charge
increases the charge density, not only on the 4-lactam
oxygen, but also on the 3-oxo function. Finally, the
tricyclic derivatives 12–24 offer a further advantage
over the corresponding bicyclic analogues 1–11 by
bearing a side-chain constrained in the north-eastern
region which could improve the glycine/NMDA re-
ceptor affinity.
Figure 2. The newly synthesized compounds.
2. Chemistry
The target compounds 1–24 were obtained starting
from the 3-oxoquinoxaline-2-carboxylates 25–27. The
unsubstituted-3-oxoquinoxaline-2-carboxylate 25 and
its 6,7-dimethyl-substituted analogue 27 were pre-
pared following a reported method [12]. The synthesis
of the unknown ethyl ( ) 6-chloro-1,2,3,4-tetrahydro-
3-oxoquinoxaline-2-carboxylate 26 is illustrated in
figure 3. Briefly, catalytic reduction of the diethyl
Figure 3. Synthesis of ethyl ( ) 6-chloro-1,2,3,4-tetrahydro-3-
oxoquinoxaline-2-carboxylate 26. (a) H2/Pd/C; (b) CuCl2, t-
BuONO; (c) Na2S2O4.

F. Varano et al. / European Journal of Medicinal Chemistry 36 (2001) 203–209
205
pounds, namely 2, 11 and 24, displayed micromolar
AMPA affinity.
carboxylate 29 which was transformed into its cor-
responding 6-chloro-analogue 30 with CuCl2 and t-
butyl nitrite. Reduction of 30 with sodium
hydrosulfite yielded the tetrahydro-derivative 26.
Allowing 25–27 to react with isocyanates, the
ethyl ( ) 1-N-carbamoyl-3-oxoquinoxaline-2-car-
boxylates 1–11 were prepared (figure 4). By heating
1–11 with diluted HCl the tricyclic 2-aryl- 12–18,
2-benzyl- 19, 2-ethyl- 20 and 2-acetic acid deriva-
tives 23–24 were obtained. The 2-acetic acid deriva-
tives 23–24 resulted from cyclization and hydrolysis
of the corresponding bicyclic esters 10–11. The ace-
tic esters 21 and 22 were prepared by heating their
corresponding bicyclic analogues 10 and 11 over
their melting points.
The 1-N-phenylcarbamoyl derivative 1 and its 2-
phenyl constrained analogue 12 displayed compara-
ble glycine/NMDA receptor affinity (Ki=57.3 and
48.5 mM, respectively), indicating that a polar side-
chain bearing a lipophilic phenyl moiety in the
north-eastern region of the molecules is recognized
by the glycine/NMDA receptor. Introduction of an
electron-withdrawing chlorine atom at position 6 of
1 and at position 7 of 12 yielded compounds 2 and
13, respectively, which showed a 17-fold enhanced
glycine/NMDA affinity with respect to their corre-
sponding unsubstituted derivatives (1 and 12). In
agreement with the literature data [11], the presence
of two electron-donating methyl groups in R1 and
R2 are tolerated by the glycine/NMDA receptor (see
3 and 14 vs. 1 and 12, respectively). However, com-
pounds 3 and 14 were 26- and 10-fold less active
than their corresponding chloro-substituted deriva-
tives 2 and 13, respectively.
The introduction of a para-substituent on the
phenyl ring of the parent compounds 1–2 yielded
the bicyclic derivatives 4–7 which were all, except
for the 6-chloro-1-N-(4-methylphenyl)- derivative 5
(Ki =5.2 mM), inactive at the glycine/NMDA recep-
tor. On the contrary, all the para-substituted tri-
cyclic derivatives 15–18 bind to the glycine/NMDA
receptor with comparable or higher affinities than
their 2-phenyl-unsubstituted derivatives 12–13.
Introduction of a methylene spacer between the
carbamoyl or the tricyclic core and the phenyl moi-
ety (as in the benzyl derivatives 8 and 19) into com-
pounds 1 and 12, and the replacement of their
lipophilic phenyl ring with the less lipophilic ethyl
group (as in the ethyl derivatives 9 and 20) dramati-
cally affected glycine/NMDA receptor affinity.
Finally, the lipophilic phenyl moiety of 1–2 and
12–13 was replaced with an acetate or an acetic
acid group yielding compounds 10–11 and 21–24,
respectively. It was, in fact, demonstrated that in
bicyclic and tricyclic derivatives of similar size and
shape a carbethoxy or a free carboxylic group
yielded potent glycine/NMDA receptor antagonists
[14–17]. In our case, compounds bearing an acetate
or an acetic acid group showed an affinity compara-
ble to that of the corresponding phenyl-substituted
derivatives only when a chloro substituent on the
3. Results and discussion
Compounds 1–24 were tested for their ability to
displace both tritiated glycine and AMPA from
their specific binding in rat cortical membranes.The
binding data, shown in table I, indicated that com-
pounds 1–24 bind to the glycine/NMDA receptor
with low micromolar affinity while only three com-
Figure 4. Synthesis of ethyl ( ) 1-N-carbamoyl-1,2,3,4-te-
trahydro-3-oxoquinoxaline-2-carboxylates 1–11 and
( )
1,2,3,3a,4,5-hexahydroimidazo[1,5-a]quinoxaline-1,3,4-triones
12–24. (a) R(CH2)nNCO; (b) 2 N HCl or heating above
melting point.

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F. Varano et al. / European Journal of Medicinal Chemistry 36 (2001) 203–209
Table I. Glycine/NMDA and AMPA binding activity.
Ki (mM) or % inhibitiona
Compd.
R
n
R1
R2
[3H]Glycine
[3H]AMPA
1
C6H5
C6H5
C6H5
4-Me-C6H4
4-Me-C6H4
4-OMe-C6H4
4-Cl-C6H4
C6H5
0
0
0
0
0
0
0
1
0
1
1
0
0
0
0
0
0
0
1
0
1
1
1
1
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
57.399.0
3.490.8
88.3915.0
34%
5.290.8
26%
35%
0%
23%
97.098.1
3.190.6
48.597.9
2.890.5
20.291.9
10.992.5
5.091.1
45.597.5
15.091.8
0%
10%
30%
4.190.7
71.2912.1
2.290.3
21%
61.3918.1
20%
30%
30%
15%
5%
16%
25%
17%
72.1912
28%
42%
45%
21%
24%
0%
15%
2
Cl
Me
H
Cl
H
H
H
H
H
Cl
H
Cl
Me
H
Cl
H
H
H
H
H
Cl
H
3
4
5
6
7
8
9
C2H5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
COOC2H5
COOC2H5
C6H5
C6H5
C6H5
4-Me-C6H4
4-Me-C6H4
4-OMe-C6H4
4-Cl-C6H4
C6H5
24%
17%
0%
40%
26%
51.194
C2H5
COOC2H5
COOC2H5
COOH
COOH
Cl
a
Ki values are means9S.E.M. of 3–4 separate determinations in triplicate or percentage of inhibition (I%) of specific binding at
100 mM concentration.
ments to obtain glycine/NMDA receptor antago-
nists.
benzo-fused moiety was present (compare com-
pounds 11 and 22, 24 with 2 and 13, respectively).
Some selected compounds of our two series were
shown to inhibit the binding of the NMDA receptor
channel-blocking agent [3H]-(+)-MK-801 to rat cor-
tical membranes incubated with 10 mM glutamate
and 0.1 mM glycine (table II). The IC50 values of
these compounds for glutamate-stimulated [3H]-(+)-
MK-801 binding showed a trend similar to that of
their Ki values for [3H]-glycine binding, thus indicat-
ing that these new ligands behave like weak glycine/
NMDA receptor antagonists [18–20].
In conclusion, the synthesis and glycine/NMDA
receptor affinity of the ethyl ( ) 1-N-carbamoyl-
1,2,3,4-tetrahydro-3-oxoquinoxaline-2-carboxylates
1–11 and of their constrained tricyclic analogues
12–24 provide further evidence that the spatial ori-
entation of the polar side-chain in the north-eastern
region of the molecule combined with the presence
of a chlorine atom in R1 are two important require-
Table II. Inhibition of stimulated [H3]-(+)-MK-801 binding.
Compd.
[3H]-(+)-MK-801 IC50 (mM)a
2
14.692.1
23.991.2
32.095.0
45.093.0
18.091.1
21.093.0
36.894.2
61.097.0
19.391.5
20.591.6
5
11
12
13
15
16
17
22
24
a
Concentrations giving 50% inhibition of stimulated [3H]-(+
)-MK-801 binding. All assays were carried out in the presence
of 10 mM glutamate and 0.1 mM glycine. The results were
calculated from 3–4 separate determinations in triplicate.

F. Varano et al. / European Journal of Medicinal Chemistry 36 (2001) 203–209
207
4. Experimental protocols
mixture was cooled and the resulting solid was collected
and washed with water. The title compound could not
be recrystallized because it easily dehydrogenated to
lead compound 30. Nevertheless, compound 26 was
pure enough to be characterized and used in the next
preparation without further purification.
4.1. Chemistry
Silica gel plates (Merck F254) were used for analytical
chromatography. All melting points were determined on
a Gallenkamp melting point apparatus. Microanalyses
were performed with a Perkin-Elmer 260 elemental ana-
lyzer for C, H, N, and the results were within 0.4% of
4.1.4. General procedure for the preparation of ethyl
( ) 1-N-carbamoyl-1,2,3,4-tetrahydro-3-
oxoquinoxaline-2-carboxylates 1–9 and ( ) N-(2-car-
boxyethyl-1,2,3,4-tetrahydro-3-oxoquinoxalinyl-1-N-carb
onyl)glycine ethyl esters 10–11
A solution of 25–27 [12] (3.63 mmol) and the suitable
isocyanate (18 mmol) in anhydrous benzene (40 mL)
was heated at reflux under nitrogen atmosphere until the
disappearance of the starting material (TLC monitor-
ing). The cooled mixture yielded a solid which was
collected, thoroughly washed with diethyl ether and
recrystallized.
1
the theoretical values. The H-NMR spectra were ob-
tained with a Varian Gemini 200 instrument at 200
MHz. The chemical shifts are reported in l (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;
ar, aromatic protons and br, broad. Physical and analyt-
ical data of the newly synthesized compounds are listed
in table III; the legend also reports the spectral data of
some significant compounds.
4.1.1. Ethyl
4.1.5. General procedure for the preparation of ( )
1,2,3,3a,4,5-hexahydroimidazo[1,5-a]quinoxaline-
1,3,4-triones 12–20 and ( ) 1,2,3,3a,4,5-hexahydro-
1,3,4-trioxoimidazo[1,5-a]quinoxalin-2-acetic acids
23–24
A mixture of 1–11 (1.47 mmol) in HCl (2 N, 10 mL)
was heated at 110 °C for 2 h. The cooled mixture
yielded a solid which was collected, washed with water
and then recrystallized.
6-amino-3,4-dihydro-3-oxoquinoxaline-2-carboxylate 29
A
mixture of diethyl N-[(2,4-dinitrophenyl)-
amino]malonate 28 [13] (1.47 mmol) and Pd/C (10%,
0.06 g) in absolute ethanol (100 mL) was hydrogenated
in a Parr apparatus at 30 psi for 12 h. Elimination of the
catalyst yielded a solution which was further stirred at
room temperature for another 12 h yielding a green
suspension. Evaporation of the solvent at reduced pres-
sure yielded a green solid which was washed with diethyl
ether and recrystallized.
4.1.6. General procedure for the preparation of ethyl
( ) 1,2,3,3a,4,5-hexahydro-1,3,4-
4.1.2. Ethyl
trioxoimidazo[1,5-a]quinoxaline-2-acetates 21–22
Compound 10 or 11 (0.7 mmol) was heated over its
melting point for 30 min. The cooled mass was treated
with diethyl ether, collected and recrystallized.
6-chloro-3,4-dihydro-3-oxoquinoxaline-2-carboxylate 30
Compound 29 (1.07 mmol) was slowly (1 h) added to
a cooled (0–5 °C) mixture of CuCl2 (1.34 mmol) and
tert-butyl nitrite (1.60 mmol) in acetonitrile (20 mL).
The mixture was allowed to stand for 90 min. Then HCl
(6 N, 20 mL) was added to the mixture which was
extracted with ethyl acetate (20 mL×3). The combined
organic extracts were washed with water (20 mL×3) and
dried (Na2SO4). Evaporation at reduced pressure of the
solvent yielded a solid which was treated with diethyl
ether, collected and recrystallized.
4.2. Biochemistry
Rat cortical synaptic membrane preparation,
[3H]glycine and [3H]AMPA binding experiments were
performed according to previously described procedures
[21, 22].
4.2.1. [3H]-(+)-MK-801 binding
4.1.3. Ethyl ( ) 6-chloro-1,2,3,4-tetrahydro-3-
oxoquinoxaline-2-carboxylate 26
An excess of Na2S2O4 (9.3 mmol) was added to a
suspension of 30 (3.72 mmol) in ethanol/water (1:1, 20
mL). The mixture was heated at 80 °C for 90 min. The
[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 Ogita
[23] with slight modifications. Preparations of frozen rat
cortical membranes were resuspended (0.5 mg protein

208
F. Varano et al. / European Journal of Medicinal Chemistry 36 (2001) 203–209
Table III. Physical and analytical data of newly synthesized compounds.
Compd.
R
n
R1
R2
m.p., °C
Cryst. solv.a
% Yield
Analysis (C, H, N)
1
C6H5
C6H5
C6H5
4-Me-C6H4
4-Me-C6H4
4-OMe-C6H4
4-Cl-C6H4
C6H5
0
0
0
0
0
0
0
1
0
1
1
0
0
0
0
0
0
0
1
0
1
1
1
1
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
196–198
210–212
200–204
198–199
206–208
204–207
206–208
186–188
169–171
169–172
172–174
256–259
269–272
\300
A
A
A
A
B
A
A
A
C
A
A
A
A
D
E
D
A
A
D
A
A
B
80
60
70
85
35
90
90
85
60
80
60
85
70
80
80
80
80
90
90
50
50
40
40
60
50
80
50
C18H17N3O4
C18H16ClN3O4
C20H21N3O4
C19H19N3O4
C19H18ClN3O4
C19H19N3O5
C18H16ClN3O4
C19H19N3O4
C14H17N3O4
C16H19N3O6
C16H18ClN3O6
C16H11N3O3
C16H10ClN3O3
C18H15N3O3
C17H13N3O3
C17H12ClN3O3
C17H13N3O4
C16H10ClN3O3
C17H13N3O3
C12H11N3O3
C14H13N3O5
C14H12ClN3O5
C12H9N3O5
2b
3
Cl
Me
H
Cl
H
H
H
H
H
Cl
H
Cl
Me
H
Cl
H
H
H
H
H
Cl
H
Cl
Cl
NH2
Cl
4
5
6
7
8
9c
C2H5
10
11d
12
13e
14
15
16
17
18
19
20f
21
22g
23
24h
26i
29j
30k
COOC2H5
COOC2H5
C6H5
C6H5
C6H5
4-Me-C6H4
4-Me-C6H4
4-OMe-C6H4
4-Cl-C6H4
C6H5
278–279
\300
263–265
294–297
277–280
272–274
220–222
236–238
\300
263–266 dec
196–201
260–264 dec
235–237
C2H5
COOC2H5
COOC2H5
COOH
F
A
COOH
C12H8ClN3O5
A
B
C11H11N3O3
C11H9ClN2O3
a
Recrystallization solvents: A, ethanol; B, ethyl acetate; C, cyclohexane/ethyl acetate; D, nitromethane; E, glacial acetic acid; F,
dimethylformamide/water.
b
1H-NMR (DMSO-d6): 1.10 (t; 3H, CH3, J=7.0 Hz); 4.06–4.15 (m; 2H, CH2); 5.49 (s; 1H, H-2); 7.03–7.14 (m; 3H, ar); 7.27–7.50
(m; 5H, ar); 9.37 (s; 1H, carbamoyl NH); 11.11 (s; 1H, NH at position-4).
c
1H-NMR (DMSO-d6): 1.02–1.12 (m; 6H, 2CH3); 3.08–3.17 (m; 2H, CH2); 4.04–4.08 (m; 2H, CH2); 5.52 (s; 1H, H-2); 6.97–7.11
(m; 4H, 3 ar+carbamoyl NH); 7.45 (d; 1H, ar, J=6.0 Hz); 10.90 (s; 1H, NH at position-4).
d
1H-NMR (DMSO-d6): 1.05 (t; 3H, CH3, J=6.2 Hz); 1.19 (t; 3H, CH3, J=7.0 Hz); 3.78–4.11 (m; 6H, 3CH2); 5.46 (s; 1H, H-2);
6.98 (s; 1H, ar); 7.11 (d; 1H, ar, J=8.7 Hz); 7.15–7.61 (m; 2H, 1 ar+carbamoyl NH); 11.05 (s; 1H, NH at position-4).
e
1H-NMR (DMSO-d6): 5.39 (s; 1H, H-3a, it exchanges with D2O); 7.02 (d; 1H, ar, J=2.2 Hz); 7.15 (dd; 1H, ar, J=8.7, 2.2 Hz);
7.39–7.52 (m; 5H, ar); 7.65 (d; 1H, ar, J=8.7 Hz); 10.98 (s; 1H, NH).
f
1H-NMR (DMSO-d6): 1.13 (t; 3H, CH3, J=7.1 Hz); 3.45 (q; 2H, CH2, J=7.1 Hz); 5.23 (s; 1H, H-3a, it exchanges with D2O);
6.95–7.15 (m; 3H, ar); 7.61 (d; 1H, ar, J=7.3 Hz); 10.83 (s; 1H, NH).
g
1H-NMR (DMSO-d6): 1.20 (t; 3H, CH3, J=7.0 Hz); 4.10–4.27 (m; 4H, 2CH2); 5.49 (s; 1H, H-3a, it exchanges with D2O); 7.01
(d; 1H, ar, J=2.2 Hz); 7.15 (dd; 1H, ar, J=8.7, 2.2 Hz); 7.59 (d; 1H, ar, J=8.7 Hz); 11.05 (s; 1H, NH).
h
1H-NMR (DMSO-d6): 4.15 (s; 2H, CH2); 5.47 (s; 1H, H-3a, it exchanges with D2O); 7.01 (d; 1H, ar, J=2.2 Hz); 7.15 (dd; 1H,
ar, J=8.5, 2.2 Hz); 7.60 (d; 1H, ar, J=8.5 Hz); 11.05 (s; 1H, NH).
i
This compound easily dehydrogenated, thus it could not be recrystallized. Nevertheless, it was pure enough to be characterized.
1H-NMR (DMSO-d6): 1.16 (t; 3H, CH3, J=7.1 Hz); 4.11 (q; 2H, CH2, J=7.1 Hz); 4.60 (d; 1H, H-2, J=2.1 Hz); 6.74–6.87 (m;
4H, 3 ar+NH at position-1); 10.68 (s; 1H, NH at position-4).
j
1H-NMR (DMSO-d6): 1.29 (t; 3H, CH3, J=7.1 Hz); 4.28 (q; 2H, CH2, J=7.1 Hz); 6.32 (d; 1H, ar, J=2.2 Hz); 6.47 (s; 2H, NH2);
6.63 (dd; 1H, ar, J=8.8, 2.2 Hz); 7.44 (d; 1H, ar, J=8.8 Hz); 12.28 (s; 1H, NH).
k
1H-NMR (DMSO-d6): 1.34 (t; 3H, CH3, J=7.1 Hz); 4.39 (q; 2H, CH2, J=7.1 Hz); 7.35–7.45 (m; 2H, ar); 7.87 (d; 1H, ar, J=8.7
Hz); 12.93 (s; 1H, NH).
mL–1) in ice-cold 5 mM Tris-HCl buffer, pH 7.4, con-
taining 0.08% v/v Triton X-100 and stirred for 10 min at
0–2 °C. They were then collected by centrifugation
(48 000×g for 10 min) and submitted to four additional
resuspension and centrifugation cycles before finally be-
ing resuspended in the appropriate volume of buffer

F. Varano et al. / European Journal of Medicinal Chemistry 36 (2001) 203–209
209
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(0.2–0.3 mg 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–1), 10 mM glutamic acid and 0.1 mM glycine 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%
polyethylenimmine and washed with ice-cold buffer (3×
5 mL). Non-specific binding was determined in the
presence of 100 mM phencyclidine hydrochloride.
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4.2.2. Sample preparation and result calculation
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A 1 mM stock solution of the test compound was
prepared in 50% dimethylsulfoxide (DMSO). Subse-
quent dilutions were accomplished in buffer. The IC50
values were calculated from 3–4 displacement curves
based on 4–6 scalar concentrations of the test com-
pound in triplicate using the ALLFIT computer pro-
gram [24] and, in the case of tritiated glycine and
AMPA binding, converted to Ki values by application of
the Cheng-Prusoff equation [25]. In our experimental
conditions the dissociation constants (KD) for
[3H]glycine (10 nM) and [3H]AMPA (8 nM) were 75 6
and 28 3 nM, respectively.
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Valuable assistance in animal handling was provided
by M.G. Giovannini.
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