3-Hydroxy-quinazoline-2,4-dione as a useful scaffold to obtain selective Gly/NMDA and AMPA receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.01.109

The synthesis and Gly/NMDA, AMPA and KA receptor binding activities of some 3-hydroxy-quinazoline-2,4-dione derivatives are reported. The binding data, together with functional antagonism studies, showed that the 3-hydroxy-quinazoline-2,4-dione moiety can be considered a useful scaffold to obtain selective Gly/NMDA and AMPA receptor antagonists. In fact, introduction of chlorine atom(s) on precise position(s) of the benzofused moiety yielded Gly/NMDA selective antagonists, while the presence of the 6-(1,2,4-triazol-4-yl) group shifted the affinity and selectivity towards the AMPA receptor.

Full text of DOI:10.1016/j.bmcl.2004.01.109

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2345–2349
3-Hydroxy-quinazoline-2,4-dione as a useful scaffold to obtain
selective Gly/NMDAand AMPAreceptor antagonists
Vittoria Colotta,a,* Daniela Catarzi,a Flavia Varano,a Francesca Romana Calabri,a
Guido Filacchioni,a Chiara Costaglib and Alessandro Gallib
aDipartimento di Scienze Farmaceutiche, Universita’ degli Studi di Firenze, Polo Scientifico, Via Ugo Schiff, 6,
50019 Sesto Fiorentino, (FI), Italy
bDipartimento di Farmacologia Preclinica e Clinica, Universita’ degli Studi di Firenze, Viale Pieraccini, 6, 50134 Firenze, Italy
Received 3 December 2003; revised 21January 2004; accepted 27 January 2004
Abstract—The synthesis and Gly/NMDA, AMPA and KA receptor binding activities of some 3-hydroxy-quinazoline-2,4-dione
derivatives are reported. The binding data, together with functional antagonism studies, showed that the 3-hydroxy-quinazoline-2,4-
dione moiety can be considered a useful scaffold to obtain selective Gly/NMDA and AMPA receptor antagonists. In fact, intro-
duction of chlorine atom(s) on precise position(s) of the benzofused moiety yielded Gly/NMDA selective antagonists, while the
presence of the 6-(1,2,4-triazol-4-yl) group shifted the affinity and selectivity towards the AMPA receptor.
Ó 2004 Elsevier Ltd. All rights reserved.
Glutamate (Glu), the major excitatory neurotransmitter
in the mammalian central nervous system, exerts its
physiological roles via metabotropic (mGluRs) and
ionotropic receptors (iGluRs). The iGluRs, ligand-gated
ion channels, are subdivided into three major subclasses,
receptor types.7–13 In particular, we prepared a series of
3-aryl-quinazoline-2,4-dione derivatives7 (Chart 1) some
of which showed a binding affinity at the Gly/NMDA
receptors in the micromolar range. In fact, these deriv-
atives possess all the pharmacophoric descriptors of the
Gly/NMDA receptor antagonists proposed by Leeson
and Iversen:14 (a) a flat hydrophobic area, profitably
substituted with chlorine atoms, represented by the
fused benzo ring; (b) a NH hydrogen-bond donor that
binds a proton acceptor of the receptor; (c) a d-nega-
tively charged carbonyl oxygen atom that forms a cou-
lombic interaction with a positive receptor site; (d) a
hydrogen-bond acceptor, represented by the 4-carbonyl
group, able to interact with a receptor hydrogen-bond
donor. Moreover, an attractive interaction between the
3-aryl ring and the positively charged receptor site could
be hypothesized.
namely N-methyl-D-aspartate (NMDA), (S)-2-amino-3-
(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA)
and kainate (KA) receptors.1;2 The NMDA receptor
complex possesses different binding sites including the
glycine co-transmitter receptor (Gly/NMDA).3 It is well
known that overstimulation of iGluRs is involved in
acute and chronic degenerative disorders such as stroke,
ischaemia, Parkinsonꢀs and Alzheimerꢀs diseases, epi-
lepsy and pain.4–6 Accordingly, iGluR antagonists, as
well as the Gly/NMDA receptor antagonists are under
investigation for their potential therapeutic use in the
above mentioned pathologies.
In recent years, in our laboratory much effort has been
directed towards the study of novel competitive and
uncompetitive iGluR antagonists in order to shed light
on the different structural requirements of the diverse
Thus, to continue the study of this class of derivatives
and to ameliorate their affinity, we replaced the 3-aryl
ring with a 3-hydroxy substituent (compounds 1–6,
Chart 1). In fact, it seemed obvious that the acidic
3-hydroxy group could engage, with the positively
charged receptor site, a stronger interaction, compared
to that formed by the 3-aryl ring. Moreover, the less
hindered 3-hydroxy group could make the approach to
the positively charged receptor subsite easier. Finally,
this substitution could improve the water solubility of
these compounds.
Keywords: Ionotropic glutamate receptors; Glycine/NMDA receptor
antagonists; AMPA receptor antagonists; Quinazoline-2,4-dione deriv-
atives.
* Corresponding author. Tel.: +39-055-4573731; fax: +39-055-4573-
780; e-mail: vittoria.colotta@unifi.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.109

2346
V. Colotta et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2345–2349
D
H
R5
O
Cl
O
R
R6
Cl
OH
O
N
N
N
H
1-6
Cl
N
H
O
R5= H, Cl
A
N
N
R6= H, Cl, NO2, NH2,
N
Chart 1. Previously and currently reported quinazoline-2,4-dione derivatives.
Therefore, compounds 1–6 were prepared and tested at
the Gly/NMDA site. In particular, in this preliminary
work, we introduced chlorine atom(s) on precise posi-
tion(s) of the fused benzene ring (compounds 1–3) since
it is well known that their presence increases the affinity
of several classes of Gly/NMDA receptor antagonists.
Compounds 1–6 were also tested at the AMPA receptor.
In fact, it has to be noted that 1–6 also possess the
structural requirements described by Bigge and Nikam
in the AMPA receptor pharmacophore model proposed
for the quinoxaline-2,3-dione derivatives.4 Therefore, we
rationally designed the 7-chloro-6-(1,2,4-triazol-4-yl)
derivative 6, since, from our experience with 1,2,4-
triazolo[1,5-a]quinoxalin-4-one derivatives,9;11;13 the
presence of this heterocycle at such a position could be
particularly profitable for AMPA receptor affinity and
selectivity. Finally, we also evaluated the binding affinity
of derivatives 1–6 at KA receptor to obtain information
about the structural requirements of this still little
known iGluR subtype.
described.7;16 The 6,7-dichloro-derivative 9 was prepared
by chlorination of 7 with SO2Cl2 in glacial acetic acid
and catalytic amount of iodine, differently from the
previously reported method.17 The 2-amino-N-benzyl-
oxybenzamides 10–12 were prepared by allowing 7–9 to
react with O-benzylhydroxylamine in refluxing ethanol.
Cyclization of 10–12 with triphosgene afforded the
3-benzyloxy-quinazoline-2,4-dione derivatives 13–15,
which were debenzylated with 48% HBr in glacial acetic
acid to give the desired 3-hydroxy-quinazoline-2,4-di-
ones 1–3. The 3-hydroxy derivative 1 was transformed
into the corresponding 3-acetate 16 by refluxing in acetic
anhydride (Scheme 2). Derivative 16 was regioselectively
nitrated to afford the 6-nitro compound 17. Desacetyl-
ation of 17 with an aqueous NaOH solution followed
by acidification yielded the 7-chloro-3-hydroxy-6-nitro-
quinazoline-2,4-dione 4. Catalytical reduction of 17 gave
the 6-amino derivative 18. This compound was hydro-
lyzed to the final 6-amino-7-chloro-3-hydroxy-quinazo-
line-2,4-dione 5. Reaction of derivative 18 with
diformylhydrazine afforded compound 19, which was
desacetylated to give the desired 7-chloro-3-hydroxy-6-
(1,2,4-triazol-4-yl)-quinazoline-2,4-dione 6.
Compounds 1–615 were prepared as depicted in Schemes
1 and 2. The 1,2-dihydro-3,1-benzoxazine-2,4-diones 7
and 8, bearing the 7-chloro and 5,7-dichloro substitu-
ents, respectively, were prepared as previously
The binding affinities of compounds 1–6 at the Gly/
NMDA and AMPA receptors were evaluated by mea-
suring their ability to displace [3H]glycine and
[3H]AMPA, respectively, from rat cerebral cortex syn-
aptic membranes, following the procedure described in
Ref. 7 and 18. The high affinity [3H] kainate binding
assays were performed on rat cortical membrane,
according to a previously described method.12
R5
O
R5
O
R6
Cl
R6
Cl
O
b
O
N
H
N
H
O
NH2
7, 8
9
10-12
a
The Gly/NMDA binding data of 1–6, reported in Table 1
together with that of the previously described 3-phenyl-
quinazolin-2,4-dione A7, confirm our working hypoth-
esis. Replacement of the 3-aryl moiety of the previously
reported quinazoline-2,4-dione derivatives with the
3-hydroxy group resulted in a significant enhancement
of the Gly/NMDA receptor affinity (compare 2 with A).
Indeed, compounds 1–3, designed as Gly/NMDA
ligands, show an affinity towards this receptor in the low
micromolar range. Moreover, 1–3 also possess a good
selectivity for the Gly/NMDA site towards both AMPA
receptor (from 40- to 50-fold) and KA receptor (more
than 220-fold).
c
R5
O
R5
O
R6
Cl
OH
R6
Cl
O
d
N
N
N
H
O
N
H
O
1-3
13-15
R5
H
Cl
H
R6
H
H
1, 7, 10, 13
2, 8, 11, 14
3, 9, 12, 15
Cl
Scheme 1. Reagents and conditions: (a) SO2Cl2, glacial AcOH, I2,
50 °C; (b) PhCH2ONH2 Æ HCl, NEt3, EtOH, reflux; (c) Triphosgene,
THF, NEt3, rt; (d) 48% HBr, AcOH, reflux.
Derivative 6, designed as an AMPA selective antagonist,
is very interesting. It shows not only a good AMPA

V. Colotta et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2345–2349
2347
O
O
CH3
CH3
O2N
Cl
O
O
O
O
b
N
a
N
1
O
O
N
H
17
Cl
N
H
16
O
O
CH3
H2N
O
O
O2N
Cl
OH
O
N
N
O
Cl
N
N
H
H
4
18
e
c
N
O
O
N
H2N
Cl
OH
N
OR
N
N
N
H
O
Cl
N
H
O
5
19 R= COCH3
6 R= H
c
Scheme 2. Reagents and conditions: (a) Refluxing Ac2O; (b) 90% HNO3, )10 °C; (c) (i) 2.5% NaOH, 40 °C; (ii) 6 N HCl; (d) 10% Pd/C, EtOAc,
40 psi; (e) Diformylhydrazine, Me3SiCl, pyridine, NEt3, 1 00°C.
receptor affinity, but also a high selectivity, displaying
more than 400-fold higher affinity for the AMPA than
for the Gly/NMDA receptor. These data, firstly, confirm
the advantageous effect of the 6-(1,2,4-triazol-4-yl)
moiety for AMPA receptor affinity and selectivity,
which as previously hypothesized,9;11 could be due to a
hydrogen bond that the N3 atom of the triazole ring
form with a putative hydrogen-bond donor of the
receptor. Secondly, the binding data of 6 indicate that
the fused benzo ring of these 3-hydroxy-quinazoline-2,4-
dione derivatives interacts with a large lipophilic pocket
of the AMPA receptor, which well tolerates a bulky
group, such as the 6-(1,2,4-triazol-4-yl) ring. On the
contrary, the corresponding lipophilic area of the Gly/
NMDA receptor is a size-limited binding pocket which
does not permit the anchoring of a bulky 6-substituent.
All these findings agree well with our previous results
obtained for the 1,2,4-triazolo[1,5-a]quinoxalin-4-one
derivatives8;9;11–13 and confirm the structural require-
ments proposed in the AMPA and Gly/NMDA receptor
antagonist pharmacophore models.4;14
Introduction of a nitro group at the 6-position of the
7-chloro derivative 1 reduces the Gly/NMDA affinity
Table 1. Binding activity at Gly/NMDA, AMPA and KA receptors
Cl
O
R5
O
R6
Cl
OH
O
N
N
Cl
N
H
O
N
H
A
1-6
Compound
R5
R6
Ki (lM)a
IC50 (lM)b
[3H]KA
[3H]glycine
[3H]AMPA
1
2
3
4
5
H
Cl
H
H
H
H
0.24 0.02
0.20 0.03
0.3 0.03
1.1 0.1
11.6 1.9
9.8 1.4
12.5 2.6
1.3 0.1
9.7 1.5
140 15
H
Cl
52
68
4
7
NO2
NH2
19.2
70
1
10.6 1.2
6
N
N
6
H
>100
0.25 0.04
NTd
7.0 0.5
NTd
N
Ac
23 6
a Ki values were means SEM of three or four separate determinations in triplicate.
b IC50 values were means SEM of three or four separate determinations in triplicate.
c Ref. 6.
d Not tested.

2348
V. Colotta et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2345–2349
Table 3. Functional antagonism at NMDA and AMPA sites of some
selected quinazoline-2,4-diones
while it increases that towards the AMPA receptor, thus
leading to a nonselective Gly/NMDA versus AMPA
antagonist (compound 4). The lower Gly/NMDA
affinity of 4, with respect to 1, confirms the negative
effect of a 6-substituent bulkier than a chlorine atom
for anchoring to the Gly/NMDA receptor. The 10-fold
increased AMPA affinity of 4, compared to that of 1, is
consistent with the hypothesis of a hydrogen bonding
interaction at the level of the 6-substituent. Neverthe-
less, the profitable effect of the 6-nitro group probably
also arises from its electron-withdrawing properties that
increases the NH acidity, thus reinforcing the hydrogen-
bonding interaction with the receptor proton donor
site.4;11 Reduction of the 6-nitro group of 4 gave the
6-amino derivative 5, which compared to the former,
displays a reduced affinity for both the Gly/NMDA and
AMPA receptor. As regards the binding data of 1–6, at
the high affinity KA receptor, resemble those at the
AMPA receptor, even if their values are from 5- to 30-
fold lower. In particular, compound 6, the most active at
the AMPA receptor, also shows the highest KA affinity,
among the herein reported ligands.
Compound
IC50 (lM)a
NMDA
AMPA
2
42
36
5
4
33
29
4
2
4
6
84 10
4.7 0.9
(*)b
4.6 0.5
52 11
0.20 0.02
DCKA
NBQX
a Concentration necessary for 50% inhibition (IC50) of depolarization
induced by 5 lM S-AMPA or NMDA in mouse cortical wedge
preparation. IC50 values were means SEM of three separate deter-
minations.
b At 10 lM concentration the inhibition was not significant.
NMDA responses in a reversible manner. The electro-
physiological potencies of compounds 4 and 6 closely
correlate with their binding affinities at the Gly/NMDA
and AMPA receptors. In fact, 4 shows similar potencies
in the Gly/NMDA and AMPA functional assays while
the inhibitory action of 6 on depolarization induced by
AMPA was higher than that on NMDA-evoked
response. On the contrary, the functional antagonist
activity of compound 2 versus NMDA receptor is not in
agreement with the [3H]glycine binding result since 2
shows about 50-fold higher affinity on the Gly/NMDA
site than on AMPA receptor, while, it appears equipo-
tent in inhibiting NMDA- and AMPA-evoked
responses.
In order to assess the functional antagonism at the
NMDA receptor–ion channel complex, we tested
derivatives 1–6 for their ability to inhibit the binding
of the channel blocking agent [3H]-(þ)-MK-801((þ)-
5-methyl-10,11-dihydro-5H-benzo[a,d]cyclohepten-5,10-
imine)10;19;20 in rat cortical membranes incubated with
10 lM glutamate and 0.1 lM glycine. The results ob-
tained are listed in Table 2. In general, the IC50 values of
these derivatives closely correlate with their Ki values on
[3H]glycine binding, the only exception being compound
1, which, in this assay, results about 6-fold less active
than derivatives 2 and 3, which both show binding
activity similar to 1.
In conclusion, the herein reported preliminary study has
identified the 3-hydroxy-quinazoline-2,4-dione as a new
scaffold to obtain selective Gly/NMDA and AMPA
receptor antagonists. In fact, introduction of suitable
substituents on the benzofused moiety have led to either
Gly/NMDA or AMPA selective antagonists. Moreover,
this work has confirmed some different structural
requirements of the Gly/NMDA and AMPA receptors.
Taking into account these findings, further modifica-
tions of these derivatives, to improve both affinity and
selectivity, are in progress.
The selected 3-hydroxy-quinazoline-2,4-dione deriva-
tives 2, 4, 6, together with NBQX (2,3-dihydroxy-6-
nitro-7-sulphamoylbenzo[f]quinoxaline) and DCKA
(5,7-dichlorokynurenic acid), as reference compounds,
were evaluated for functional antagonist activity by
assessing their ability to inhibit depolarization induced
by 5 lM AMPA or NMDA in mouse cortical wedge
preparations21 (Table 3). The electrophysiological assays
were performed following the procedures described in
Ref. 12. All the tested compounds inhibited AMPA and
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