Synthesis and pharmacological studies at the Gly/NMDA, AMPA and Kainate receptors of new oxazolo[4,5-c]quinolin-4-one derivatives bearing different substituents at position-2 and on the fused benzo ring

Article abstract of DOI:10.1016/j.ejmech.2005.03.017

The synthesis and biological evaluation at the Gly/NMDA, AMPA and Kainate receptors of new oxazolo[4,5-c]quinolin-4-one derivatives are reported. Different substituents were introduced at the 2-position (mercapto, carbonyl and methyl groups) and on the fu

Full text of DOI:10.1016/j.ejmech.2005.03.017

European Journal of Medicinal Chemistry 40 (2005) 897–907
www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological studies at the Gly/NMDA, AMPA
and Kainate receptors of new oxazolo[4,5-c]quinolin-4-one derivatives
bearing different substituents at position-2 and on the fused benzo ring
Francesca Romana Calabri a, Vittoria Colotta a,*, Daniela Catarzi a, Flavia Varano a,
Ombretta Lenzi a, Guido Filacchioni a, Chiara Costagli b, Alessandro Galli b
a Dipartimento di Scienze Farmaceutiche, Universita’ degli Studi di Firenze, Polo Scientifico, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, FI, Italy
b Dipartimento di Farmacologia Preclinica e Clinica, Universita’ degli Studi di Firenze, Viale Pieraccini, 6, 50134 Firenze, Italy
Received 30 September 2004; received in revised form 21 March 2005; accepted 23 March 2005
Available online 24 May 2005
Abstract
The synthesis and biological evaluation at the Gly/NMDA, AMPA and Kainate receptors of new oxazolo[4,5-c]quinolin-4-one derivatives
are reported. Different substituents were introduced at the 2-position (mercapto, carbonyl and methyl groups) and on the fused benzo ring
(chlorine atom(s) and trifluoromethyl group). Among the herein reported compounds, the 2-mercapto-derivatives 1–4 showed the highest
Gly/NMDA affinities, comparable to that of 5,7-dichlorokynurenic acid. The most active compound was the 7-chloro-substituted derivative 1
(Ki = 0.082 µM) which possesses a Gly/NMDA selectivity of 50- and 500-fold with respect to AMPA and KA receptors, respectively. Func-
tional antagonism studies performed on some selected 2-mercapto compounds, at bothAMPA and NMDA receptor-ion channels, assessed the
antagonistic properties of these derivatives. SAR studies pointed out the importance of the concurrent presence of electron-rich moieties at
both the 2- and 3-positions of the oxazolo[4,5-c]quinolin-4-one framework. In fact, the 3-sp2-nitrogen atom plays a significant role in rein-
forcing the hydrogen bond that the 4-carbonyl oxygen probably forms with the arginine residue (R523) of the Gly/NMDA receptor site. The
presence of 2-substituent able to form a hydrogen bonding interaction was also proved to be important for a good Gly/NMDA receptor affinity.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Oxazoloquinoline derivatives; Ionotropic glutamate receptor antagonists; Glycine/NMDA receptor antagonists; AMPA receptor antagonists
1. Introduction
NMDA receptor itself [3]. In the last few years it has been
well established that overstimulation of iGluRs and the sub-
sequent excess of intracellular Ca2+ are involved in many
pathological conditions such as Parkinson’s,Alzheimer’s and
Huntington’s diseases, stroke, cerebral ischemia and pain
[1,4–8].As a consequence, iGluRs antagonists have been con-
sidered attractive targets for their potential therapeutic use in
the above cited pathologies. While competitive NMDA recep-
tor antagonists have shown many side effects, such as ataxia,
neurotoxicity, memory deficits, psichotomimetic effects, the
Gly/NMDA receptor antagonists have demonstrated to pos-
sess fewer adverse reactions [9–11]. Thus, in the last few years
much effort has been directed towards the study of
Gly/NMDA receptor antagonists belonging to various chemi-
cal classes. Among them, different series of tricyclic deriva-
tives have been developed [1,9–12]. As a part of a program
aimed at finding novel iGluR receptor antagonists [13–17],
The excitatory neurotransmitter glutamate (Glu) plays an
essential role in many physiological functions of the mam-
malian central nervous system where it activates two recep-
tor classes: the metabotropic and ionotropic receptors. The
ionotropic receptors (iGluRs) are ligand-gated ion channels,
permeable to Na+, K+ and Ca2+ ions, and are presently clas-
sified into three major subclasses: N-methyl-D-aspartate
(NMDA) (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-
propionic acid (AMPA) and kainate (KA) receptors [1,2]. On
the NMDA receptor complex different binding sites are
present including the glycine (Gly/NMDA) receptor. Bind-
ing of glycine to this site is necessary for activation of the
* Corresponding author. Tel.: +39 055 457 3731; fax: +39 055 457 3780.
E-mail address: vittoria.colotta@unifi.it (V. Colotta).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.03.017

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F.R. Calabri et al. / European Journal of Medicinal Chemistry 40 (2005) 897–907
we have published some papers on 4-oxo-1,2,4-triazolo[1,5-
a]quinoxaline-2-carboxylate derivatives (TQX series) [13] and
5-oxo-pyrazolo[1,5-c]quinazoline-2-carboxylate compounds
(PQZ series) [17] (Fig. 1) some of which showed significant
Gly/NMDA affinity. Indeed, derivatives of both series pos-
sess some important structural requirements of Gly/NMDA
receptor antagonists [11,18]: (a) a flat hydrophobic area rep-
resented by the fused benzo ring; (b) a NH hydrogen bond
donor that binds a proton acceptor of the receptor; and (c) a
d-negatively charged moiety, represented by both the carbo-
nyl group, adjacent to the NH proton donor, and the 3-nitrogen
atom, which could form a hydrogen bond with a cationic
hydrogen bond donor–receptor site. Structure–activity rela-
tionship (SAR) studies in the TQX and PQZ series have
pointed out the importance of the ethyl 2-carboxylate func-
tion or, even better, of the acidic 2-carboxyl group for anchor-
ing to the Gly/NMDA binding site. The positive effect of both
these substituents has been attributed to their capability of
interacting with a putative hydrogen bond donor–receptor site
[13,17]. Electron-withdrawing substituents, such as chlorine
atom(s) and a trifluoromethyl group, on precise position(s)
of the fused benzo ring have also been profitable for
Gly/NMDA affinity of TQX and PQZ derivatives. On this
basis and with the purpose of extending our investigation to
other tricyclic iGluR antagonists strictly correlated to the TQX
and PQZ series, we designed the synthesis of new com-
pounds, namely the oxazolo[4,5-c]quinolin-4-one deriva-
tives 1–12 (Fig. 2). The 2-mercapto-compounds 1–4 still
maintain the important pharmacophoric descriptors described
above: (i) the NH proton donor, (ii) the d-negatively charged
N-3 and 4-carbonyl oxygen atom, (iii) the 2-substituent which
can engage a hydrogen bond, acting as a proton acceptor or,
more likely, as proton donor, and (iv) the chlorine atom(s) or
trifluoromethyl substituent on suitable position(s) of the fused
benzo ring. The choice of introducing the 2-mercapto group
in compounds 1–4 was made since to our knowledge this sub-
stituent has never been investigated in analogous tricyclic
Gly/NMDA receptor antagonists. Subsequently, the
2-mercapto group was replaced by the 2-oxo function (deriva-
tives 5–8): this latter group is able to form a hydrogen bond
acting as a proton acceptor group. Moreover, introduction of
the 2-C=O group implied replacement of the 3-nitrogen atom
with a NH moiety which was likely to interact with different
strength with the cationic hydrogen bond donor–receptor
site. However, since some 2,3,4,5-tetrahydro-oxazolo[4,5-
c]quinoline-2,4-diones, bearing an 8-heteroaryl substituent,
were already described as AMPA receptor antagonists [19],
it was predictable that compounds 5–8 would possess
Gly/NMDA affinity, being similar the structural require-
ments of these two receptors [18].
2. Chemistry
Compounds 1–12 (Fig. 2) were prepared using as key inter-
mediates the 4-hydroxy-3-nitroquinolin-2(1H)ones 19–21 and
27 which were synthesized as described in Figs. 3 and 4,
respectively. The 7-chloro-4-hydroxy-3-nitroquinolin-2(1H)-
one 19 was obtained, differently from the reported procedure
[20], by heating at 50 °C in anhydrous tetrahydrofuran the
7-chloro-1,2-dihydro-3,1-benzoxazine-2,4-dione 13 [21] and
ethyl nitroacetate. The synthesis of the 5,7-dichloro-4-
hydroxy-3-nitroquinolin-2(1H)-one 20 was performed as
described in ref. 20, i.e. by allowing the 5,7-dichloro-4-
hydroxyquinolin-2(1H)-one 17 [22] to react with 70% HNO3
in glacial acetic acid at room temperature. The 6,7-dichloro-
4-hydroxy-3-nitroquinolin-2(1H)-one 21 was obtained dif-
ferently from the previously described method [20], which in
our hands was inconvenient. Derivative 21 was synthesized
starting from the 6,7-dichloro-1,2-dihydro-3,1-benzoxazine-
2,4-dione 14, which ensued from treatment of 13 with sulfu-
ryl chloride, as previously reported [23]. By heating deriva-
tive 14 and NaOH in absolute methanol at 80 °C, the methyl
Fig. 1. Previously reported TQX and PQZ series.
Fig. 3. Synthesis of 4-hydroxy-3-nitroquinolin-2(1H)-ones 19–21. (a)
NO2CH2COOEt, THF, NEt3; (b) 70% HNO3, glacial AcOH; (c) SO2Cl2,
glacial AcOH, I2; (d) anhydrous MeOH, NaOH; (e) Ac2O, dioxane; (f) (i)
[(CH3)3Si]2NK, anhydrous THF; (ii) 6 N HCl.
Fig. 2. Currently reported oxazolo[4,5-c]quinolin-4-one derivatives.

F.R. Calabri et al. / European Journal of Medicinal Chemistry 40 (2005) 897–907
899
Fig. 6. Tautomers A and B of derivatives 1–8.
3-amino-4-hydroxyquinolin-2(1H)-ones, which were iso-
lated as hydrochlorides 28–31 (Fig. 5). These compounds are
very unstable for their photosensibility and transform into co-
loured compounds. Thus, they cannot be recrystallized and
must be handled and stored sheltered from light. However,
the crude 28–31 were pure enough to be used without further
purifications. Reflux of a hydroalcoholic solution of 28–31
and carbon disulfide, in the presence of KOH, gave the desid-
ered 4,5-dihydro-2-mercapto-oxazolo[4,5-c]quinolin-4-one
derivatives 1–4. Oxidation of compounds 1–4 with hydrogen
peroxide in aqueous NaOH solution furnished the 2,3,4,5-
tetrahydro-oxazolo[4,5-c]quinoline-2,4-diones 5–8.
Fig. 4. Synthesis of 7-trifluoromethyl-4-hydroxy-3-nitroquinolin-2(1H)-
ones 27. (a) CrO3, glacial AcOH, Ac2O; (b) anhydrous MeOH, NaOH; (c)
Ac2O, dioxane; (d) (i) [(CH3)3Si]2NK, anhydrous THF; (ii) 6 N HCl; (e)
70% HNO3, glacial AcOH.
2-amino-4,5-dichlorobenzoate 15 was obtained which was
acetylated to yield the 2-acetamido derivative 16. This last
was cyclized with potassium bis(trimethylsilyl)amide in anhy-
drous tetrahydrofuran to afford the 6,7-dichloro-4-
hydroxyquinolin-2(1H)-one 18 [22], which was nitrated to
yield the 3-nitro derivatives 21 [20]. The 7-trifluoromethyl-
4-hydroxy-3-nitroquinolin-2(1H)-one 27 (Fig. 4) was ob-
tained following the pathway above described for the prepa-
ration of 21. Oxidation with cromic anhydride of the
6-trifluoromethylisatin 22 [24] yielded the 6-trifluoromethyl-
1,2-dihydro-3,1-benzoxazine-2,4-dione 23 which was trans-
formed into the methyl 2-amino-4-trifluoromethylbenzoate
24 [25]. Acetylation of 24 afforded the 2-acetamido deriva-
tive 25, which was cyclized to the 7-trifluoromethyl-4-
hydroxyquinolin-2(1H)-one 26. Treatment of 26 with nitric
acid gave the corresponding 3-nitro derivative 27. The
4-hydroxy-3-nitroquinolin-(1H)-one derivatives 19–21, 27
were reduced with sodium dithionite to the corresponding
Reaction of 28–30 with acetyl chloride afforded the
3-acetamido derivatives 32–34 which were cyclized in reflux-
ing acetic anhydride to the 4,5-dihydro-2-methyl-oxazolo[4,5-
c]quinolin-4-one derivatives 9–11. Treatment of the
7-trifluoromethyl derivative 31 with acetyl chloride gave a
complex mixture made up by of desidered 4-acetamido com-
1
pound 35 and poly-acetylated derivatives (from H-NMR
analysis of the crude product). By refluxing the crude mix-
ture with acetic anhydride, the 2-methyl-7-trifluoromethyl-
oxazolo[4,5-c]quinolin-4-one 12 was obtained with satisfac-
tory yield. Derivative 35 was easily isolated from the crude
product due to its insolubility in boiling ethanol.
It has to be noted that derivatives 1–4 and 5–8 may exist in
either one of the two tautomeric forms A and B (Fig. 6). As
Fig. 5
. Synthesis of oxazolo[4,5-c]quinolin-4-ones 1–12. (a) (i) Na2S2O4, NaOH; (ii) 6 N HCl; (b) (i) CS2, EtOH/H2O, KOH; (ii) 6 N HCl; (c) 35% H2O2,
NaOH; (d) CH3COCl, NEt3, THF; (e) Ac2O.

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F.R. Calabri et al. / European Journal of Medicinal Chemistry 40 (2005) 897–907
regards compounds 1–4, neither the 1H-NMR nor IR spectral
data indicated which of the two forms exists, respectively, in
DMSO solution or in the solid state. Similarly, no informa-
A are more stable than the 2-thioxo forms B, having 26–
30 kcal mol–1 lower energy than the latter. Conversely, for
compounds 5–8, tautomers B, i.e. the 2-oxo forms, are sig-
nificantly more stable than tautomers A, the difference in
energy being 63–66 kcal mol–1. These latter results are also
in accordance with the IR data of 5–8.
1
tion can be obtained from the H-NMR data of derivatives
5–8. In contrast, their IR spectra showed two strong bands
between 1700 and 1780 cm–1 thus indicating that derivatives
5–8 exist in the solid state in the 2,4-dione form (tautomer
B). To know which were the most stable tautomers, ab initio
quantum chemical computational calculation has been per-
formed on derivatives 1–8. By using the program GAMESS
[26] the energy in vacuo of both forms A and B have been
calculated. The results of these studies, reported in Table 1,
clearly indicate that for compounds 1–4, the 2-mercapto forms
3. Results and discussion
As stated above, computational studies indicated that the
most stable tautomers for compounds 1–4 and 5–8 are, respec-
tively, the 2-mercapto- and the 2-oxo-forms. However, it is
well-known that the most stable structure of a molecule drawn
from spectroscopical or theoretical studies may not be the
most stable in the biological environment. In addition, the
receptor–ligand interaction itself can stabilize a molecule in
a high energy state. Nevertheless, as a great difference in
energy was found between the two tautomeric forms of deriva-
tives 1–8, we can assume that the most stable tautomers are
also the pharmacologically active ones.
Compounds 1–12 were tested for their ability to displace
[3H]glycine, [3H]AMPA and [3H]kainate from their specific
sites in rat cerebral cortex membranes. The binding data are
shown in Table 2 together with those of the 2-carboxylic acids
7-Cl-TQX and 8-Cl-PQZ and of DCKA (5,7-dichloro-
Table 1
Energies of tautomers A and B of 1–8 and differences in energy between A
and B (DE A – B)
Compound number Ea A
Ea B
DEb A – B
–26.3
–28.5
–26.4
–29.8
66.3
1
2
3
4
5
6
7
8
–2977.029105
–2976.987183
–3890.013292
–3890.022856
–2730.426285
–2334.957335
–3247.980077
–3247.990539
–2088.401441
–3890.058786
–3890.065003
–2730.473749
–2334.851725
–3247.878306
–3247.886252
–2088.297168
63.9
65.4
65.4
a Energies values are expressed in Hartree (Hartree × 627.5 = kcal mol–1).
b DE values are expressed in kcal mol–1
.
Table 2
[3H]Glycine, [3H]AMPA and [3H]KA binding assays
Ki (µM)a or I%b
AMPA
IC50 (µM)c or I%b
KA
R2
R7
Cl
R8
H
R9
H
Gly
1
2
3
4
5
6
7
8
9
10
11
12
SH
SH
SH
SH
0.083 0.006
0.2 0.07
0.10 0.02
0.28 0.04
3.4 0.5
51 7.0
4.2 1.4
1.7 0.1
4.2 0.4
2.7 0.4
27%
42 17
Cl
H
Cl
H
7.4 1.4
27 1.1
Cl
Cl
H
CF3
Cl
H
32
0%
4
H
H
Cl
H
Cl
H
25
4
0%
20%
9%
Cl
Cl
H
1.2 0.2
45%
10%
52
CF3
Cl
H
63
8
CH3
CH3
CH3
CH3
H
H
46%
25%
25%
48%
8
10%
0%
Cl
H
Cl
H
35%
25%
Cl
Cl
H
0%
CF3
H
87 10
0.78 0.15
2.3 0.4
5%
0%
7-Cl-TQXd
8-Cl-PQZf
DCKA
0.17 0.02
0.48 0.04
0.09 0.02
NTe
NTe
8%
a Ki values are mean S.E.M. of three or four separate determinations in triplicate.
b Percentage of inhibition (I%) of specific binding at 100 µM concentration.
c IC50 values are mean S.E.M. of three or four separate determinations in triplicate.
d Ref. [13].
e Not Tested.
f Ref. [17].

F.R. Calabri et al. / European Journal of Medicinal Chemistry 40 (2005) 897–907
901
Table 3
than the corresponding compounds 1–4. It has to be noted
that, differently from the 2-mercapto substituent, the 2-oxo
group of 5–8 is only able to act as a hydrogen bond acceptor.
Furthermore, the significantly different Gly/NMDA receptor
affinity of compounds 1–4, with respect to derivatives 5–8,
may also be associated with the replacement of the 3-nitrogen
atom with the 3-NH substituent and, consequently, with the
different strength of interaction of these two diverse moieties
with the cationic hydrogen bond donor–receptor site. This
site is reported to be a protonated arginine residue (R523),
which provides an essential contribution to the binding of
both agonists and antagonists of the Gly/NMDA receptor [27].
Recently, docking studies of TQX and PQZ series to a
homology-based model of the Gly/NMDA receptor [28] have
pointed out a bidentate interaction of R523 with both the
N-3 and the adjacent carbonyl group of TQX and PQZ deriva-
tives. Consequently, we also presumed a similar interaction
for the herein reported oxazoloquinolin-4-one derivatives.
Therefore, in order to explain the different Gly/NMDA affini-
ties of the 2-mercapto derivatives 1–4 with respect to the 2-oxo
compounds 5–8, we performed an ab initio computational
study to calculate the different interaction strength of the
3-nitrogen-4-C=O moiety of 1–8 with the N-methyl-
guanidinium ion (probe I) which was used to model the pro-
tonated side-chain of the arginine residue. In addition, since
we hypothesized that the 2-substituents of 1–4 and 5–8, SH
and C=O, respectively, were directly involved in the anchor-
ing to the receptor site by forming a hydrogen bond, we also
evaluated the different interaction strengths of these groups
with MeOH (probe II), chosen because it can act as either
hydrogen bond donor or acceptor. The computational calcu-
lations were performed on the 7-chloro-2-mercapto-
oxazoloquinolin-4-one 1 and on the corresponding 2-oxo
compound 5. To evaluate the contribution energy of each
probe to the total interaction energy, we considered one probe
at a time. Thus, probes I and II were placed, respectively, in
the neighborhood of the 3,4- and 1,2-positions of compounds
1 and 5. The four complexes were minimized with GAMESS
and their structures reported in Figs. 7 and 8. The interaction
energies of derivatives 1 and 5 with each probe are shown in
Table 4. As can be seen from the energy values, the strength
of interaction of the 2-mercapto derivative 1 with probe I
[3H]Glycine, [3H]AMPA and [3H]KA binding assays
Ki (µM)a or I%b
I%b
KA
0%
X
H
Cl
H
H
Y
H
H
Cl
H
Z
Gly
63
AMPA
8%
32
33
34
35
Cl
Cl
Cl
CF3
9
8
90
17%
18%
6%
0%
12.5 1.9
40%
20%
0%
a Ki values are mean S.E.M. of three or four separate determinations in
triplicate.
b Percentage of inhibition (I%) of specific binding at 100 µM concentra-
tion.
kynurenic acid), included as reference compounds. The inter-
mediates 32–35 were also tested in the above cited assays
(Table 3) since they belong to the class of the 4-hydroxy-
quinolin-2(1H)-ones thus they were likely to possess some
Gly/NMDA receptor affinity.
The binding results indicate that the 2-mercapto-
oxazoloquinolin-4-one derivatives 1–4 are endowed with good
Gly/NMDA affinity, comparable to that of DCKA. More-
over, compounds 1–4 display similar Gly/NMDA affinities
with respect to the corresponding TQX and PQZ derivatives
[13,17], but are more selective towards this receptor. The most
active and selective oxazoloquinoline derivative is the
7-chloro-substituted compound 1 (Ki = 0.083 µM) which is
about 50- and 500-fold less active at theAMPA and KA recep-
tors, respectively, than at the Gly/NMDA one. The 7,9-
dichloro- derivative 2 and the 7,8-dichloro-derivative 3 also
display similar good Gly/NMDA affinities. Nevertheless,
while 3 is quite selective (42- and 270-fold towards AMPA
and KA receptor, respectively), compound 2 is only 8.5-fold
less active at the AMPA receptor than at the Gly/NMDA site,
while displaying a 37-fold selectivity with respect to the KA
receptor. Replacement of the 7-chloro substituent of com-
pound 1 with a trifluoromethyl group afforded derivative 4
which shows, compared to 1, a decreased Gly/NMDA selec-
tivity vs. either AMPA or KA receptors. The comparable
Gly/NMDA receptor affinities of the 2-mercapto-oxazolo-
quinoline derivatives 1–4 and their analogue TQX and PQZ
2-carboxylic acids [13,17], together with the close structural
similarities of the three series, would suggest a similar bind-
ing mode of compounds 1–4 and of TQX or PQZ derivatives
to the glycine receptor site. Therefore, the newly probed
2-mercapto substituent seems to replace well the previously
investigated 2-carboxyl group in a hypothetical hydrogen
bond with a receptor subsite.
(DE
= –37.94 kcal mol–1) is higher than that of the 2-oxo
probe I
compound 5 (DEprobe I = –25.20 kcal mol–1), in accordance
with the higher affinity of 1 with respect to 5. The energy
difference resulted from the different binding modes of this
probe with the two compounds (Fig. 7). Indeed, the
N-methylguanidinium ion forms a bidentate interaction with
both the 3-nitrogen and the 4-carbonyl oxygen of the
2-mercapto derivative 1, while its interaction with compound
5 involves only the 4-carbonyl oxygen, due to the presence of
the NH group at the 3-position.
The important role of the 2-mercapto substituent emerged
when it was replaced with the 2-oxo group. This structural
modification drastically decreased the Gly/NMDA affinity.
In fact, compounds 5–8 are from 12- to 255-fold less active
Interaction of MeOH (probe II) with the 2-substituent of
compounds 1 and 5 occurs in two different modes (Fig. 8), as
the 2-mercapto group and the 2-oxo are, respectively, a hydro-
gen bond donor and acceptor. A difference of 2.37 kcal mol–1

902
F.R. Calabri et al. / European Journal of Medicinal Chemistry 40 (2005) 897–907
Fig. 7
.
Optimized geometries of the complexes between N-methylguanidinium ion (probe I) and compounds 1 (on the left) and 5 (on the right).
cal calculations carried out on the 2-methyl-7-chloro-
derivative 9 show that interaction energy with probe I
(DEprobeI = –38.53 kcal mol–1) is as high as that of the corre-
sponding 2-mercapto derivative 1. Thus, the complete lack of
affinity of 9–12 has to be attributed to the incapability of the
2-methyl group to give a hydrogen bond interaction with the
receptor site. These data point out that in this class of com-
pounds the presence of a 3-sp2-nitrogen atom is an important
but not the only requirement since a 2-substituent able to form
a hydrogen bond, with a putative hydrogen bond donor or
Fig. 8. Optimized geometries of the complexes between MeOH (probe II)
and compounds 1 (on the left) and 5 (on the right).
acceptor, is also necessary to permit an efficacious binding to
the Gly/NMDA receptor site.
Both these structural features were also essential forAMPA
and KA receptor recognition. Indeed, in contrast to com-
pounds 1–4, which bind to these receptors with micromolar
affinities, derivatives 5–8 and 9–12 are inactive both atAMPA
and KA receptors, with the exceptions of compounds 6, 9
and 12, which show AMPA affinities in the high micromolar
range. However, as previously stated, some 8-heteroaryl-
oxazolo[4,5-c]quinoline-2,4-dione derivatives have already
been described as AMPA antagonists [18]. Thus, at first
glance, the very low AMPA affinity of our oxazoloquinoline-
2,4-dione derivatives 5–8 could be quite unexpected. Actu-
ally, inactivity of 5–8 has to be ascribed to the lack of the
8-heteroaryl moiety. In fact, such a substituent is known to
enhance AMPA affinity of different classes of antagonists
[14–17,19] and, accordingly, it was revealed to be of para-
mount importance in the oxazolo[4,5-c]quinoline-2,4-dione
series.
As expected, the 3-acetamido-4-hydroxyquinolin-2-(1H)-
ones 32–34, bearing chloro-substituent(s) on the benzofused
ring, display micromolar Gly/NMDA affinities while the
7-trifluoromethyl- derivative 35 is much less active. Com-
pounds 32–35 are completely devoid of both AMPA and KA
receptor affinities.
The selected compounds 1, 3–4 were tested to assess their
antagonistic activity at the Gly/NMDA receptor by evaluat-
ing their ability to inhibit the stimulated [3H]-(+)-MK-801
((+)-5-methyl-10,11-dihydro-5H-benzo[a,d]cyclohepten-
5,10-imine) binding [29–31]. The binding results, reported
in Table 5, indicate that derivatives 1, 3–4 are antagonists
with potencies similar to that of DCKA, included as refer-
ence antagonist. Compounds 1, 3–4 and DCKA were also
evaluated for their ability to inhibit depolarization induced
by AMPA or NMDA in mouse cortical wedge preparations
Table 4
Interaction energies (DE) of compounds 1, 5 and 9 with N-methylguanidinium
ion (probe I) and MeOH (probe II)
a
a
DEprobe I
–37.94
–25.20
–38.53
DEprobe II
–9.91
–7.54
ncb
1
5
9
a The interaction energies values, expressed in kcal mol–1, were calcula-
ted as follows: DE = Ecomplex – (Ecompound + Eprobe).
b Not calculated due to the incapability of the methyl group to form a hydro-
gen bond.
was found, as the hydrogen bond engaged with compound 1
(DEprobe II = –9.91 kcal mol–1) is slightly stronger. There-
fore, on the basis of these quantum chemical calculations, we
can assume that the significantly higher affinities of the
2-mercapto derivatives 1–4, with respect to the correspond-
ing 2-oxo derivatives 5–8, are above all due to the stronger
hydrogen bond with the arginine receptor site. This stronger
interaction, in its turn, is due to the presence of the 3-sp2-
nitrogen atom, which can reinforce the hydrogen bond that
the 4-carbonyl oxygen forms with the arginine residue.
Obviously, the calculated interaction energy values are not
binding energy, therefore, they can only be used to compare
the relative stabilities of the complexes. Thus, although the
interaction energies of derivatives 1 and 5 with probe I are
significantly higher than those resulting from interaction with
probe II, the 3-nitrogen-4-CO moiety may not be the only
one to play a pivotal role in the anchoring to the receptor site.
On the contrary, the binding data of the 2-methyl-derivatives
9–12 point out the importance of a suitable substituent at the
2-position. In fact, although these derivatives might be hypoth-
esized to bind efficaciously to the arginine site, they are com-
pletely inactive at the Gly/NMDA receptor. Quantum chemi-

F.R. Calabri et al. / European Journal of Medicinal Chemistry 40 (2005) 897–907
903
Table 5
C, H, N (and S for derivatives 1–4) and the results were
within 0.4% of the theoretical, unless otherwise stated. The
IR spectra were recorded with a Perkin-Elmer Spectrum RX
I spectrometer in Nujol mulls and are expressed in cm–1. The
1H-NMR spectra were obtained with a Varian Gemini 200.
The chemical shifts are reported in d (ppm) and are relative
to the central peak of the solvent that is always DMSO-d6.
Inhibition of stimulated [3H] (+)MK-801 binding and functional antago-
nism at NMDA and AMPA sites of some selected derivatives
IC50 (µM)a
[3H] (+)MK-801
1.5 0.2
IC50 (µM)b
AMPA
4.2 0.5
13
NMDA
1
3
4
0.84 0.1
1.7 0.2
0.3 0.05
4.7 0.9
0.9 0.1
2
1.1 0.15
1.5 0.2
52 11
DCKA
0.74 0.09
a Concentration giving 50% inhibition of stimulated [3H] (+)MK-
801 binding. All assays were carried out in the presence of 10 µM Glu and
0.1 µM glycine. IC50 values are mean S.E.M. of three or four separate deter-
minations in triplicate.
5.1.1. 7-Chloro-4-hydroxy-3-nitroquinolin-2(1H)-one (19)
[20]
The 7-chloro-1,2-dihydro-3,1-benzoxazine-2,4-dione (13)
[21] (5 g, 25.3 mmol) was added to a solution of ethyl nitro-
acetate (4.2 ml, 37.9 mmol) and anhydrous NEt3 (5.3 ml,
37.9 mmol) in anhydrous THF (100 ml). The mixture was
heated at 55 °C for 27 h. Evaporation of the solvent at reduced
pressure yielded a turry residue, which was treated with Et2O
(50 ml). The ethereal phase was decanted and this treatment
was repeated twice. Finally, the crude residue was taken up
with EtOAc (5 ml) and scratched to give a suspension, which
was kept overnight in a refrigerator. The solid was filtered,
washed with Et2O and dissolved in H2O (50 ml). Acidifica-
tion of the solution with 6 N HCl afforded a solid, which was
collected by filtration and washed with water. Yield: 35%;
m.p. 221–223 °C dec (EtOH) (lit. 220–222 °C [20]); 1H-NMR
7.23–7.29 (m, 2H, H5 + H8), 7.97 (dd, 1H, H6, J = 8.8 Hz,
4.4 Hz), 11.88 (s, 1H, exchangeable with D2O); IR 1368,
1523, 1676, 3190.
b Concentration necessary for 50% inhibition (IC50) of depolarization indu-
ced by S-AMPA or NMDA in mouse cortical wedge preparation. IC50 values
are mean S.E.M. of three separate determinations.
(Table 5). All the tested compounds inhibited AMPA and
NMDA responses in a reversible manner. The potencies of
derivatives 1, 3–4 are in agreement with their binding affini-
ties since they show higher inhibitory potencies on NMDA-
evoked response than on AMPA-induced depolarization.
Moreover, 1, 3–4 showed, on the whole, higher inhibitory
potencies than DCKA at the NMDA receptor.
4. Conclusion
The present study has produced new oxazolo[4,5-
c]quinolin-4-one derivatives, which are endowed with a good
affinity and selectivity toward the Gly/NMDA receptor (com-
pounds 1–4). SAR studies have pointed out the necessary
structural requirements of these tricyclic compounds to
achieve a good receptor–ligand interaction. In particular, as a
new result, the profitable effect of the 2-mercapto group has
been determined. The significantly higher affinity of the
2-mercapto derivatives 1–4 with respect to the 2-oxo deriva-
tives 5–8 has been explained on the basis of ab initio quan-
tum chemical calculations which showed the importance of a
3-sp2-nitrogen atom which, together with the 4-carbonyl oxy-
gen, is likely to be involved in a hydrogen bond with the
hydrogen- bond donor arginine site of the Gly/NMDA recep-
tor. Nevertheless, the complete inactivity of the 2-methyl
derivatives 9–12 indicates that the presence of the 3-sp2-
nitrogen atom is not the only requirement for a good affinity,
but a 2-substituent able to form a hydrogen bond is also nec-
essary.
5.1.2. 5,7-Dichloro-4-hydroxy-3-nitroquinolin-2(1H)-one
(20) [20]
Seventy percent HNO3 (5.2 ml) was added to a suspension
of 5,7-dichloro-4-hydroxyquinolin-2(1H)-one (17) [22] (4 g,
17.4 mmol) in glacialAcOH (24 ml). The mixture was heated
at 90 °C for 2 h, then cooled at room temperature. The solid
was collected by filtration, abundantly washed with water and
then with Et2O. Yield: 82%; m.p. 198–200 °C dec (EtOH)
(lit. 170–171 °C [20]); 1H-NMR 7.18–7.24 (m, 2H, ar), 11.48
(s, 1H, exchangeable with D2O); IR 1353, 1525, 1670.
5.1.3. 6,7-Dichloro-1,2-dihydro-3,1-benzoxazine-2,4-dione
(14) [23]
A suspension of derivative 13 (5 g, 25.3 mmol) in SO2Cl2
(165 ml) and glacial AcOH (45 ml), in the presence of cata-
lytic amount of I2, was heated at 80 °C for 65 h. Evaporation
of the solvent at reduced pressure afforded a solid which was
suspended in H2O (30 ml) and filtered.Yield: 80%; m.p. 295–
1
297 °C (EtOH); H-NMR 7.29 (s, 1H, ar), 8.08 (s, 1H, ar),
5. Experimental protocols
11.95 (s, 1H, NH).
5.1. Chemistry
5.1.4. Methyl 2-amino-4,5-dichlorobenzoate (15)
Compound 14 (12.7 mmol) was suspended in an anhy-
drous methanolic 0.1 M NaOH solution (60 ml). The mixture
was heated at 80 °C for a few minutes, until it became a
brownish solution.After cooling at room temperature and dilu-
tion with H2O, a solid precipitated which was collected and
washed with H2O. Yield: 85%; m.p. 119–121 °C (MeOH);
Silica gel plates (Merck F254) and silica gel 60 (Merck,
70–230 mesh) were used for analytical and column chroma-
tography, respectively. All melting points were determined
on a Gallenkamp melting point apparatus. Microanalyses were
performed with a Perkin–Elmer 260 elemental analyzer for

904
F.R. Calabri et al. / European Journal of Medicinal Chemistry 40 (2005) 897–907
1H-NMR 3.79 (s, 3H, CH3), 6.89 (s, 2H, NH2), 7.04 (s, 1H,
ar), 7.78 (s, 1H, ar); IR 1703, 3375, 3491. ‘Anal. C8H7Cl2NO2
(C, H, N)’.
(H2O/EtOH) (lit. 60–62 °C [25]); 1H-NMR 3.80 (s, 3H, CH3),
6.76 (d, 1H, ar, J = 8.4 Hz), 6.96 (s, 2H, NH2), 7.12 (s, 1H,
H-3), 7.85 (d, 1H, J = 8.4 Hz); IR 1687, 3368, 3398, 3480.
5.1.5. Methyl 2-acetamido-4,5-dichlorobenzoate (16)
A solution of 15 (4.5 mmol) in Ac2O (5 ml) and anhy-
drous dioxane (8 ml) was heated at 50 °C, under nitrogen
atmosphere, for 13 h. The mixture was diluted with H2O
(2.5 ml) and stirred for about 15 min, then the solvent was
evaporated at reduced pressure to give an oil, which slowly
solidified. Yield: 75%; m.p. 122–124 °C (MeOH); 1H-NMR
2.13 (s, 3H, CH3 amide), 3.85 (s, 3H, CH3), 8.00 (s, 1H, ar),
8.49 (s, 1H, ar), 10.54 (s, 1H, NH); IR 1698, 1716, 3305.
‘Anal.C10H9ClNO3 (C, H, N)’.
5.1.10. Methyl 2-acetamido-4-trifluoromethylbenzoate (25)
The title compound was obtained from 24 as described
above for the preparation of 16. Yield: 94%; m.p. 88–90 °C
(petroleum ether 30–60°); 1H-NMR 2.12 (s, 3H, CH3 amide),
3.86 (s, 3H, CH3), 7.51 (d, 1H, ar, J = 8.2 Hz), 8.04 (d, 1H,
ar, J = 8.2 Hz), 8.50 (s, 1H, H-3), 10.58 (s, 1H, NH); IR 1590,
1701, 3263. ‘Anal.C11H10F3NO3 (C, H, N)’.
5.1.11. 7-Trifluoromethyl-4-hydroxyquinolin-2(1H)-one
(26)
The title compound was prepared from 25 as described
above for the synthesis of 18. Yield: 60%; m.p. > 300 °C
(2-Methoxyethanol); 1H-NMR 5.82 (s, 1H, H-3), 7.42 (d, 1H,
ar, J = 8.4 Hz), 7.55 (s, 1H, ar), 7.95 (d, 1H, ar, J = 8.4 Hz),
11.45 (s, 1H, exchangeable with D2O), 11.67 (br s, 1H,
exchangeable with D2O); IR 1586, 1610, 3400. ‘Anal.
C10H6F3NO2 (C, H, N)’.
5.1.6. 6,7-Dichloro-4-hydroxy-quinolin-2(1H)-ones (18)
[22]
A solution of compounds 16 (7.6 mmol) in anhydrous THF
(30 ml) was added dropwise to a 0.5 M solution of potassium
bis(trimethylsilyl)amide (72.5 ml, 36.3 mmol) in toluene at
–78 °C under nitrogen atmosphere. After the addition was
completed, the mixture was kept at –78 °C for about 1 h, then
it was left to return to room temperature. The mixture was
quenched with ice H2O (150 ml) and stirred for 15 min. The
aqueous layer was washed with EtOAc (15 ml × 3) and then
acidified with 6 N HCl. The solid was collected, abundantly
washed with H2O and dried. Yield: 68%; m.p. > 300 °C
(DMF) (lit. 335 °C [22]); 1H-NMR 5.73 (s, 1H, H-3), 7.42 (s,
1H, ar), 7.85 (s, 1H, ar), 11.37 (s, 1H, exchangeable with
D2O), 11.65 (s, 1H, exchangeable with D2O); IR 1689, 3621.
5.1.12. 7-Trifluoromethyl-4-hydroxy-3-nitroquinolin-
2(1H)-one (27)
The title compounds was prepared from 26 as described
above for the synthesis of 21. Yield: 82%; m.p. 182–183 °C
(EtOAc); 1H-NMR 7.53 (d, 1H, ar, J = 7.8 Hz), 7.60 (s, 1H,
H-8), 8.20 (d, 1H, ar, J = 7.8 Hz), 11.92 (s, 1H, exchangeable
with D2O); IR 1334, 1558, 1668, 1684, 3167. ‘Anal.
C10H5F3N2O4 (C, H, N)’.
5.1.13. General procedure for the synthesis of 3-amino-4-
hydroxyquinolin-2(1H)-one hydrochlorides 28–31
Na2S2O4 (2.1 g, 11.9 mmol) was added to a 1 M NaOH
aqueous solution (20 ml) of the 3-nitro derivatives 19–21, 27
(2.1 mmol). The mixture was sheltered from light and stirred
for 30 min. After cooling at 0 °C, the solution was acidified
with 6 N HCl and the solid which precipitated was quickly
collected by filtration, washed with a few drops of cold water
and subsequently with petroleum ether and then dried in
vacuo. The crude compounds 28–31, obtained in almost quan-
titative yields, were photosensitive, thus they could not be
recrystallized and had to be handled and stored sheltered from
light. Nevertheless, they were pure enough to be used in the
next step.
5.1.7. 6,7-Dichloro-4-hydroxy-3-nitroquinolin-2(1H)-ones
(21) [20]
Compound 18 was nitrated following the experimental con-
ditions described above to prepare 20 from 17, with the only
difference being the reaction time that, for 18, was shorter
(about 10 min). Yield: 74%; m.p. 170–171 °C (EtOH) (lit.
235–236 °C [20]); 1H-NMR 7.40 (s, 1H, ar), 8.08 (s, 1H, ar),
11.49 (s, 1H, exchangeable with D2O); IR 1377, 1528, 1664,
3162, 3400. ‘Anal.C9H4Cl2N2O4 (C, H, N)’.
5.1.8. 7-Trifluoromethyl-1,2-dihydro-3,1-benzoxazine-2,4-
dione (23)
CrO3 (3.6 g, 36.7 mmol) was added portionwise to a hot
(90 °C) suspension of the 6-trifluoromethylisatin (22) [24]
(5 g, 21.6 mmol) in glacial AcOH (20 ml) and Ac2O (20 ml).
The mixture was heated at 90 °C for 2 h. After cooling at
room temperature, the suspension was diluted with water
(50 ml) and the solid collected and abundantly washed with
H2O. Yield: 86%; m.p. 240–242 °C (EtOAc); 1H-NMR 7.37
(s, 1H, ar), 7.54 (d, 1H, ar, J = 8.4 Hz), 8.10 (d, 1H, ar, J
= 8.4 Hz), 11.95 (s, 1H, NH). ‘Anal.C9H4F3NO3 (C, H, N)’.
5.1.13.1. 3-Amino-7-chloro-4-hydroxyquinolin-2(1H)-one
+
hydrochloride (28). 1H-NMR 5.00 (br s, 3H, NH3 ), 7.11(d
1H, ar J = 8.4 Hz), 7.24 (s, 1H, ar), 7.67 (dd, 1H, ar,
J = 8.4 Hz, J = 2.2 Hz), 11.62 (br s 1H, exchangeable with
D2O).
5.1.13.2. 3-Amino-5,7-dichloro-4-hydroxyquinolin-2(1H)-
+
5.1.9. Methyl 2-amino-4-trifluoromethylbenzoate (24) [25]
The title compound was obtained from 23 as described
above for the preparation of 15. Yield: 80%; m.p. 65–68 °C
one hydrochloride (29). 1H-NMR 6.20 (br s, 3H, NH3 ), 7.16
(s, 1H, ar), 7.22 (s, 1H, ar), 11.80 (br s, 1H, exchangeable
with D2O).

F.R. Calabri et al. / European Journal of Medicinal Chemistry 40 (2005) 897–907
905
5.1.13.3. 3-Amino-6,7-dichloro-4-hydroxyquinolin-2(1H)-
(d, 1H, J = 8.6 Hz), 12.18 (s, 1H, exchangeable with D2O),
12.32 (s, 1H, exchangeable with D2O); IR 1697, 1789, 3220.
‘Anal. C10H5ClN2O3 (C, H, N)’.
+
one hydrochloride (30). 1H-NMR 5.80 (br s, 3H, NH3 ), 7.37
(s, 1H, ar), 7.78 (s, 1H, ar), 11.73 (br s 1H, exchangeable
with D2O).
5.1.15.2. 7,9-Dichloro-2,3,4,5-tetrahydro-oxazolo[4,5-
c]quinoline-2,4-dione (6). Yield: 68%; m.p. > 300 °C (DMF);
1H-NMR 7.40 (s, 1H, ar), 7.49 (s, 1H, ar), 12.39 (s, 1H,
exchangeable with D2O), 12.49 (s, 1H, exchangeable with
D2O); IR 1701, 1750, 3194. ‘Anal. C10H4Cl2N2O3 (C, H, N)’.
5.1.13.4. 3-Amino-7-trifluoromethyl-4-hydroxyquinolin-
1
2(1H)-one hydrochloride (31). H-NMR 6.30 (br s, 3H,
+
NH3 ), 7.41 (d, 1H, ar, J = 8.2 Hz), 7.54 (s, 1H, H-8), 7.87
(d, 1H, ar, J = 8.2 Hz), 11.84 (s, 1H, exchangeable with D2O).
5.1.14. General procedure for the synthesis of 4,5-dihydro-
2-mercapto-oxazolo[4,5-c]quinolin-4-ones 1–4
5.1.15.3. 7,8-Dichloro-2,3,4,5-tetrahydro-oxazolo[4,5-
c]quinoline-2,4-dione (7). Yield: 60%; m.p. > 300 °C (EtOH);
1H-NMR 7.59 (s, 1H, ar), 7.93 (s, 1H, ar), 12.25 (s, 1H,
exchangeable with D2O), 12.44 (s, 1H, exchangeable with
D2O); IR 1700, 1780, 3120. ‘Anal. C10H4Cl2N2O3 (C, H, N)’.
To a suspension of crude 28–31 (3.6 mmol) in aqueous
EtOH (12 ml of EtOH and 2 ml of H2O), KOH (0.45 g,
8.0 mmol) and CS2 (2.2 ml, 36.4 mmol) were added. After
refluxing for 5 h, the suspension was cooled at room tempera-
ture, diluted with H2O (40 ml) and acidified with 6 N HCl.
The precipitate was collected, washed with H2O and dried.
5.1.15.4. 2,3,4,5-Tetrahydro-7-trifluoromethyl-oxazolo[4,5-
c]quinolin-2,4-dione (8). Yield: 55%; m.p. > 300 °C (DMF);
1H-NMR 7.60 (d, 1H, ar, J = 8.2 Hz), 7,76 (s, 1H, H-6), 7.92
(d, 1H, ar, J = 8.2 Hz), 12.37 (s, 1H, exchangeable with D2O),
12.50 (s, 1H, exchangeable with D2O); IR 1700, 1780, 3230.
‘Anal. C11H5F3N2O3 (C, H, N)’.
5.1.14.1. 7-Chloro-4,5-dihydro-2-mercapto-oxazolo[4,5-
c]quinolin-4-one (1). Yield: 68%; m.p. > 300 °C (EtOH);
1H-NMR 7.35 (d, 1H, ar, J = 8.8 Hz), 7.46 (s, 1H, ar), 7.84
(d, 1H, ar, J = 8.8 Hz), 12.3 (br s, 1H, exchangeable with
D2O), 14.10 (br s, 1H, exchangeable with D2O); IR 1674,
3400–3650. ‘Anal. C10H5ClN2O2S (C, H, N, S)’.
5.1.16. General procedure for the synthesis of 3-aceta-
mido-4-hydroxyquinolin-2(1H)ones 32–34
5.1.14.2. 7,9-Dichloro-4,5-dihydro-2-mercapto-oxazolo[4,5-
c]quinolin-4-one (2). Yield: 56%; m.p. > 300 °C (2-
Methoxyethanol); 1H-NMR 7.42 (s, 1H, ar), 7.56 (s, 1H, ar),
12.51 (s, 1H, exchangeable with D2O), 14.50 (br s,1H,
exchangeable with D2O); IR 1697. ‘Anal. C10H4Cl2N2O2S
(C, H, N, S)’.
Anhydrous NEt3 (0.59 ml, 4.25 mmol) and CH3COCl
(0.18 ml, 2.55 mmol) were dropwise added to a suspension
of derivatives 28–30 (1.70 mmol) in anhydrous THF (10 ml).
The mixture, sheltered from light, was refluxed for 5 h, cooled
at room temperature, diluted with H2O (20 ml) and acidified
with 6 N HCl. The solid was collected and washed with water.
5.1.14.3. 7,8-Dichloro-4,5-dihydro-2-mercapto-oxazolo[4,5-
c]quinolin-4-one (3). Yield: 90%; m.p. > 300 °C (EtOH);
1H-NMR 7.61 (s, 1H, ar), 8.10 (s, 1H, ar), 12.38 (br s, 1H,
exchangeable with D2O), 14.20 (br s, 1H, exchangeable with
D2O); IR 1700, 3383. ‘Anal. C10H4Cl2N2O2S (C, H, N, S)’.
5.1.16.1. 3-Acetamido-7-chloro-4-hydroxyquinolin-2(1H)one
(32). Yield: 98%; m.p. > 300 °C (2-Methoxyethanol);
1H-NMR 2.20 (s, 3H, CH3), 7.22 (d, 1H, ar, J = 8.8 Hz), 7.28
(s, 1H, H-8), 7.83 (d, 1H, ar, J = 8.8 Hz), 9.73(s, 1H, NHAc),
11.88 (s,1H, exchangeable with D2O), 11.99 (br s, 1H,
exchangeable with D2O); IR 1606, 1633, 1658, 3326. ‘Anal.
C11H9ClN2O3 (C, H, N)’.
5.1.14.4. 4,5-Dihydro-2-mercapto-7-trifluoromethyl-oxazolo-
[4,5-c]quinolin-4-one (4). Yield: 53%; m. p. > 300 °C
1
(EtOH); H-NMR 7.63 (d, 1H, ar, J = 8.4 Hz), 7.78 (s, 1H,
H-6), 8.05 (d, 1H, ar, J = 8.4 Hz), 12.50 (s, 1H, exchangeable
with D2O), 14.75 (br s, 1H, exchangeable with D2O); IR 1687,
1710. ‘Anal. C11H5F3N2O2S (C, H, N, S)’.
5.1.16.2. 3-Acetamido-5,7-dichloro-4-hydroxyquinolin-
2(1H)one (33). Yield: 54%; m.p. 298 °C dec (2-
Methoxyethanol); 1H-NMR 2.18 (s, 3H, CH3), 7.28 (d, 1H,
ar, J = 1.8 Hz), 7.33 (d, 1H, ar, J = 1.8 Hz), 9.70 (s, 1H,
NHAc), 11.68 (s, 1H, exchangeable with D2O), 12.03 (s, 1H,
exchangeable with D2O); IR 1620, 1661, 3313. ‘Anal.
C11H8Cl2N2O3 (C, H, N)’.
5.1.15. General procedure for the synthesis of 2,3,4,5-
tetrahydro-oxazolo[4,5-c]quinoline-2,4-diones 5–8
Thirty-five percent H2O2 (5 ml) was dropwise added to a
10% NaOH solution (22 ml) of compounds 1–4 (1.71 mmol)
while maintaining the temperature at 15 °C. After the addi-
tion, the mixture was stirred at 15 °C for 30 min, then it was
left to return to room temperature. The suspension was acidi-
fied to pH 2 with 6 N HCl and the solid collected and washed
with H2O.
5.1.16.3. 3-Acetamido-6,7-dichloro-4-hydroxyquinolin-
2(1H)one (34). Yield: 68%; m.p. > 300 °C (2-
Methoxyethanol); 1H-NMR 2.20 (s, 3H, CH3), 7.43 (s, 1H,
ar), 7.93 (s, 1H, ar), 9.78 (s, 1H, NHAc), 11.97 (s, 1H,
exchangeable with D2O), 12.11 (s, 1H, exchangeable with
D2O); IR 1603, 1629, 1661, 3329. ‘Anal. C11H8Cl2N2O3 (C,
H, N)’.
5.1.15.1. 7-Chloro-2,3,4,5-tetrahydro-oxazolo[4,5-c]quino-
line-2,4-dione (5). Yield: 84%; m.p. > 300 °C (DMF);
1H-NMR 7.33 (d, 1H, ar, J = 8.6 Hz), 7.45 (s. 1H, H-6), 7.72

906
F.R. Calabri et al. / European Journal of Medicinal Chemistry 40 (2005) 897–907
5.1.17. 3-Acetamido-4-hydroxy-7-trifluoromethylquinolin-
2(1H)one (35)
performed following the procedure reported in Refs. [32,33]
and [29], respectively. The high affinity [3H]kainate binding
assays were performed on rat cortical membrane, according
to a previously described method [17].
Anhydrous NEt3 (0.76 ml, 5.48 mmol) and CH3COCl
(0.23 ml, 3.29 mmol) were dropwise added to a suspension
of derivative 31 (2.19 mmol) in anhydrous THF (15 ml). The
mixture, sheltered from light, was refluxed for 1 h 45 min,
cooled at room temperature, diluted with H2O (50 ml) and
acidified with 6 N HCl. The crude solid, made up of a mix-
ture of 35 and poly-acetylated derivatives (1H-NMR analy-
sis), was collected, washed with H2O and used without fur-
ther purification for the synthesis of derivative 12. Pure
derivative 35 was obtained by treating the crude product with
boiling EtOH. The hot suspension was filtered and the solid
collected. Yield: 20%; m.p. > 300 °C (2-Methoxyethanol)
1H-NMR 2.25 (s, 3H, CH3), 7.52 (d, 1H, H-6, J = 8.2 Hz),
7.60 (s, 1H, H-8), 8.06 (d, 1H, H-5, J = 8.2 Hz), 9.84 (s, 1H,
exchangeable with D2O), 12.10 (s, 2H, exchangeable with
D2O); IR 1660, 1623, 3328. ‘Anal. C12H9F3N2O3 (C, H, N)’.
5.2.2. Electrophysiological assays
The mouse cortical wedge preparations described by Man-
naioni et al. [34] was used while the electrophysiological
assays were performed following the procedures described
in Ref. [17].
5.2.3. Sample preparation and result calculation
A stock 1 mM solution of the tested compound was pre-
pared in 50% DMSO and the subsequent dilutions were
accomplished in buffer. The IC50 values were calculated from
three or four displacement curves based on four to six scalar
concentrations of the tested compound in triplicate using the
ALLFIT computer program [35]. Ki values were calculated
according to the Cheng-Prusoff equation [36]. KL for [3H]
glycine and [3H]-DL-AMPA were 75 6 and 28 3 nM,
respectively.
5.1.18. General procedure for the synthesis of 4,5-dihydro-
2-methyl-oxazolo[4,5-c]quinolin-4-ones 9–12
Derivatives 32–34 (2.02 mmol) or the crude mixture
(0.31 g) described above in the synthesis of 35, were refluxed
in Ac2O (4 ml) until the disappearance of the starting mate-
rial (16–25 h). After cooling at room temperature, the solid
was collected by filtration and washed with H2O.
5.3. Quantum chemical computational calculations
Ab initio quantum chemical calculations were performed
using the program package GAMESS [26]. Geometries of
molecules and molecule-probe complexes were optimized in
vacuo at the restricted Hartree Fock (RHF) level with the N21-
3 basis set.
5.1.18.1. 7-Chloro-4,5-dihydro-2-methyl-oxazolo[4,5-
c]quinolin-4-one (9). Yield: 80%; m.p. > 300 °C (DMF);
1H-NMR 2.63 (s, 3H, CH3), 7.31 (d, 1H, ar, J = 8.4 Hz), 7.47
(s, 1H, H-8), 7.83 (d, 1H, J = 8.4 Hz), 11.98 (s, 1H, NH); IR
1693, 3228. ‘Anal. C11H7ClN2O2 (C, H, N)’.
Acknowledgments
5.1.18.2. 7,9-Dichloro-4,5-dihydro-2-methyl-oxazolo[4,5-
c]quinolin-4-one (10). Yield: 90%; m.p. > 300 °C (DMF);
1H-NMR 2.65 (s, 3H, CH3), 7.44 (s, 1H, ar), 7.53 (s, 1H, ar),
12.22 (s, 1H, NH); IR 1698. ‘Anal. C11H6Cl2N2O2 (C, H,
N)’.
We thank Professor Fabrizio Melani of the University of
Florence, Italy, for his involvement in the molecular model-
ing study.
5.1.18.3. 7,8-Dichloro-4,5-dihydro-2-methyl-oxazolo[4,5-
c]quinolin-4-one (11). Yield: 85%; m.p. > 300 °C (DMF)
1H-NMR 2.65 (s, 3H, CH3), 7.64 (s, 1H, ar), 8.13 (s, 1H, ar),
12.10 (s, 1H, NH); IR 1689. ‘Anal. C11H6Cl2N2O2 (C, H,
N)’.
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