Structural investigation of the 7-chloro-3-hydroxy-1H-quinazoline-2,4-dione scaffold to obtain AMPA and kainate receptor selective antagonists. Synthesis, pharmacological, and molecular modeling studies

Article abstract of DOI:10.1021/jm0604880

In this paper, the study of new 7-chloro-3-hydroxy-1H-quinazoline-2,4-dione derivatives, designed as AMPA and kainate (KA) receptor antagonists, is reported. Some derivatives bear different carboxy-containing alkyl chains on the 3-hydroxy group, while various heterocyclic rings or amide moieties are present at the 6-position of other compounds. Binding data at Gly/NMDA, AMPA, and high-affinity KA receptors showed that the presence of the free 3-hydroxy group is of paramount importance for a good affinity at all three investigated receptors, while introduction of some 6-heterocyclic moieties yielded AMPA-selective antagonists. The most significant result was the finding of the 6-(2-carboxybenzoylamino)-3-hydroxy-1H-quinazolin-2,4-dione 12, which possesses good affinity for high-affinity and low-affinity KA receptors (K_i = 0.62 μM and 1.6 μM, respectively), as well as good selectivity. To rationalize the trend of affinities of the reported derivatives, an intensive molecular modeling study was carried out by docking compounds to models of the Gly/NMDA, AMPA, and KA receptors.

Full text of DOI:10.1021/jm0604880

J. Med. Chem. 2006, 49, 6015-6026
6015
Structural Investigation of the 7-Chloro-3-hydroxy-1H-quinazoline-2,4-dione Scaffold to Obtain
AMPA and Kainate Receptor Selective Antagonists. Synthesis, Pharmacological, and Molecular
Modeling Studies
Vittoria Colotta,*,† Daniela Catarzi,† Flavia Varano,† Ombretta Lenzi,† Guido Filacchioni,† Chiara Costagli,‡ Alessandro Galli,‡
Carla Ghelardini,‡ Nicoletta Galeotti,‡ Paola Gratteri,§ Jacopo Sgrignani,§ Francesca Deflorian,| and Stefano Moro|
Dipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente AttiVi, Polo Scientifico,
UniVersita` degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, (FI), Italy, Dipartimento di Farmacologia Preclinica e Clinica,
UniVersita` degli Studi di Firenze, Viale Pieraccini, 6, 50134 Firenze, Italy, Laboratorio di Molecular Modeling, Cheminformatics, and QSAR,
Dipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente AttiVi, Polo Scientifico,
UniVersita` degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, (FI), Italy, and Molecular Modeling Section, Dipartimento di
Scienze Farmaceutiche, UniVersita` degli Studi di PadoVa, Via Marzolo 5, 35131 PadoVa, Italy
ReceiVed April 26, 2006
In this paper, the study of new 7-chloro-3-hydroxy-1H-quinazoline-2,4-dione derivatives, designed as AMPA
and kainate (KA) receptor antagonists, is reported. Some derivatives bear different carboxy-containing alkyl
chains on the 3-hydroxy group, while various heterocyclic rings or amide moieties are present at the 6-position
of other compounds. Binding data at Gly/NMDA, AMPA, and high-affinity KA receptors showed that the
presence of the free 3-hydroxy group is of paramount importance for a good affinity at all three investigated
receptors, while introduction of some 6-heterocyclic moieties yielded AMPA-selective antagonists. The
most significant result was the finding of the 6-(2-carboxybenzoylamino)-3-hydroxy-1H-quinazolin-2,4-
dione 12, which possesses good affinity for high-affinity and low-affinity KA receptors (Ki ) 0.62 µM and
1.6 µM, respectively), as well as good selectivity. To rationalize the trend of affinities of the reported
derivatives, an intensive molecular modeling study was carried out by docking compounds to models of the
Gly/NMDA, AMPA, and KA receptors.
Introduction
the dorsal horn of rat and human spinal cords has been well-
documented,18,19 and many studies have demonstrated the key
role of the iGluRs for transmission of pain.20-22 Indeed, it has
been well-established that NMDA receptor antagonists have
antinociceptive activity in different types of chronic pain, both
in human and animal models, but their clinical use is limited
due to unacceptable central side effects such as hallucination
and ataxia.23 Similarly, application of mixed AMPA/KA receptor
antagonists as analgesics is generally not possible due to
undesirable ataxia,21,24 while KA receptor selective antagonists
seem to have more specific antinociceptive effects, at least in
rats,24 thus making them more promising antinociceptive
therapeutic agents.
In the last few decades, the research focused on iGluRs has
acquired considerable insights on Gly/NMDA and AMPA
receptors as a result of the availability of selective agonists and
antagonists, which have permitted clarification of the physi-
ological role of the two receptor types.1,2,25-29 In contrast, the
role of the KA receptor is much less understood because for a
long time there was a lack of selective antagonists, and some
KA receptor selective antagonists have only recently been
reported.29-33
Recently, we have directed efforts toward the study of novel
competitive and noncompetitive iGluR antagonists, with the aim
of shedding light on the different structural requirements of the
various receptor subtypes.34-42 Recently, we published a paper
where a preliminary study on 3-hydroxy-1H-quinazoline-2,4-
dione derivatives was reported40 (Chart 1). These derivatives
possess all the structural requirements for Gly/NMDA and
AMPA receptor recognition:27,43 (a) a flat hydrophobic area
represented by the fused benzo ring; (b) a NH hydrogen-bond
donor that binds a proton acceptor of the receptor; and (c) a
δ-negatively charged moiety, represented by both the 2-carbonyl
Glutamate (Glu) is the primary excitatory neurotransmitter
in both the central and the peripheral nervous systems where it
plays a pivotal role in a host of physiological processes such as
neuronal plasticity, learning, and memory. Glu exerts its effects
by activation of metabotropic (mGluRs) and ionotropic receptors
(iGluRs).1,2 The iGluRs are classified as N-methyl-D-aspartate
(NMDA), (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)pro-
pionic acid (AMPA), and kainate (KA) receptors. The NMDA
receptor complex possesses different binding sites, including
the glutamate coagonist glycine binding site (Gly/NMDA).3 To
date, molecular cloning has identified six subunits for the
NMDA receptor (NR1, NR2A-D, and N3A), four subunits for
the AMPA receptor (GluR1-4), and five subunits (GluR5-7
and KA1-2) for the KA receptor.1 KA1 and KA2 subunits
showed high-affinity binding for [3H]kainate,4,5 while GluR5-7
are low-affinity subunits.6,7 Excessive glutamatergic transmission
is involved in the pathogenesis of several neurological disorders,
including hypoxia/ischemia brain damage,8 Parkinson’s9 and
Alzheimer’s10,11diseases, epilepsy,1 and multiple sclerosis.12-13
Accordingly, the blockade of iGluRs could be employed for
the treatment of the aforementioned pathologies.1,14-15 A large
body of data also indicates that glutamatergic neurotransmission
is involved in nociceptive reflexes at the spinal cord level.16,17
In fact, the presence of NMDA, AMPA, and KA receptors in
* Corresponding author. Tel.: +39 055 4573731. Fax: +39 055
4573780. E-mail: vittoria.colotta@unifi.it.
† Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di
Firenze.
‡ Dipartimento di Farmacologia Preclinica e Clinica, Universita` degli
Studi di Firenze.
§ Laboratorio di Molecular Modeling, Cheminformatics, and QSAR,
Universita` degli Studi di Firenze.
| Universita` degli Studi di Padova.
10.1021/jm0604880 CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/12/2006

6016 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Colotta et al.
Chart 1. Previously and Currently Reported 7-Chloro-
quinazoline-2,4-dione Derivatives
or 2,5-dimethoxytetrahydrofuran-3-carbaldehyde in glacial acetic
acid at 90 °C gave, respectively, the 6-(pyrrol-1-yl)- derivative
22 and the 6-(3-formylpyrrol-1-yl)- derivative 23. This latter
was oxidized with potassium permanganate in a water/acetone
mixture to the corresponding 6-(3-carboxypyrrol-1-yl)- deriva-
tive 24. The 3-acetoxy derivatives 22-24 were hydrolyzed to
the desired 3-hydroxy derivatives 7-9. When the 6-amino
compound 21 was reacted with 2,5-dimethoxy-2,5-dihydrofuran,
in glacial acetic acid at 90 °C, the 6-(1,5-dihydropyrrol-2-oxo-
1-yl) derivative 25 was obtained, which was deacetylated with
a few drops of piperidine in boiling ethanol to give the
3-hydroxy- derivative 10. The 6-(4-oxo-4H-pyridin-4-yl)- de-
rivative 11 was prepared by heating the 6-amino compound 21
and the 4-oxo-4H-pyran-2,6-dicarboxylic acid (chelidonic acid)
in dimethyl sulfoxide at 90 °C.
Compounds 12-14 were prepared as shown in Scheme 3.
Reaction of the 6-amino derivative 21 with phthalic anhydride
and succinic anhydride, in glacial acetic acid at 60 °C, gave,
respectively, the 6-phthalimido- derivative 26 and the 6-succi-
namido- compound 27. Alkaline hydrolysis of compounds 26
and 27, followed by acidification with 6 N HCl, yielded the
3-hydroxy-6-phthalamido- derivative 12 and the 3-hydroxy-6-
succinamido- derivative 13. When the 6-succinamido- derivative
27 was reacted with sodium acetate in boiling acetic anhydride,
the corresponding 6-succinimido- compound 28 was obtained,
which was deacetylated to the corresponding 3-hydroxy deriva-
tive 14 with piperidine in refluxing ethanol.
The 6-aroylamino-3-hydroxyquinazoline-2,4-diones 15-19
were synthesized as depicted in Scheme 4. Reaction of the
6-amino-quinazoline-2,4-dione derivative 2140 with benzoyl
chloride, 3-carbomethoxybenzoyl chloride,44 or 4-carbomethox-
ybenzoyl chloride,45 in anhydrous methylene chloride, gave rise
to the corresponding 6-aroylamino-3-acetoxy-derivatives 29, 30,
and 31. Alkaline hydrolysis of 29-31 yielded compounds 15-
17, while treatment of compounds 30 and 31 with piperidine in
boiling ethanol afforded compounds 18 and 19.
group and the 3-hydroxy substituent, which could form a
hydrogen bond with a cationic hydrogen-bond donor receptor
site. Among the previously reported compounds, the 7-chloro-
3-hydroxy-1H-quinazoline-2,4-dione 1 (QZ 314, Chart 1) was
found to be a Gly/NMDA receptor-selective antagonist. Intro-
duction of the 6-(1,2,4-triazol-4-yl) residue on derivative 1
shifted the affinity and selectivity toward the AMPA receptor
(compound 2, i.e., QZ 323, Chart 1). In general, the 3-hydrox-
yquinazoline-2,4-dione derivatives showed lower affinities for
the KA receptor than for the AMPA one.
Taking into account these findings, we decided to continue
the study of this class of compounds to further explore the
structure-affinity relationships (SAR) and gain greater insight
about the differences among the structural requirements of the
three receptor types, in particular those between AMPA and
KA receptors. In fact, while known pharmacophore models of
Gly/NMDA and AMPA receptor antagonists point out the
different features of the two classes of ligands,27,43 it has not
yet been clarified how to shift the affinity from the AMPA to
the KA receptor. Indeed, so far only a few KA- versus AMPA-
selective antagonists have been reported,31-33 despite being
needed as tools for pharmacological characterization of the KA
receptor. Thus, to address this issue, in this paper the study of
the 7-chloro-quinazoline-2,4-dione derivatives 3-20 (Chart 1),
designed as AMPA- and/or KA-receptor antagonists, is de-
scribed. Compounds 3-6 are 3-O-modified analogues of
derivative 1. On the fused benzo ring compounds 7-11 bear
some typical heterocyclic substituents of previously reported
bicyclic and tricyclic selective AMPA- and KA-receptor an-
tagonists.27,28,32,36,38,39 In contrast, compounds 12-20 have some
selected amide or ureide moieties at the 6-position, which we
did not investigate in depth in our previously reported AMPA-
and KA-receptor antagonists.
The 6-benzylureido-3-hydroxyquinazoline-2,4-dione 20 was
prepared as shown in Scheme 5. By reacting the 6-amino
derivative 21 with benzylisocyanate, the 3-acetoxy-6-benzy-
lureido- compound 32 was obtained, which in alkaline medium
yielded the desired 3-hydroxy derivative 20.
Results and Discussion
The herein described 3-hydroxyquinazoline-2,4-diones 3-20
were designed taking the previously reported 7-chloro-3-
hydroxyquinazoline-2,4-dione 140 and the corresponding 6-(1,2,4-
triazol-4-yl)- derivative 240 as lead compounds. Compounds
3-20 were evaluated for their binding at AMPA, Gly/NMDA,
and high-affinity KA receptors.
Compounds 3-6 are 3-O-modified analogues of derivative
1 (Table 1). This set of compounds was synthesized to evaluate
the effects elicited by functionalization of the 3-hydroxy group
with carboxy-containing alkyl chains. The idea of introducing
such substituents on the 3-hydroxy group sprang from the
binding data of compound 33 (SPD 502)46,47 (Figure 1, Table
1), which belongs to the class of the 3-isatineoximes, described
as AMPA/KA receptor antagonists.48 Differently from the other
3-isatinoxime analogues, which bear the unsubstituted oxime
group, derivative 33 presents an attached carboxylic side chain
on the oxime function. This modification, made to improve
water solubility of the molecule, afforded high AMPA affinity
and selectivity.46 Because the 3-isatinoxime moiety displays
some similarities with the 3-hydroxy-quinazoline-2,4-dione
scaffold, we presumed a similar binding mode for these two
Chemistry
Compounds 3-20 were synthesized as described in Schemes
1-5. Scheme 1 depicts the synthesis of the 3-O-functionalized
compounds 3-6. Reaction of the 7-chloro-3-hydroxyquinazo-
line-2,4-dione 140 with R-bromo-γ-butyrolactone in methyl ethyl
ketone yielded to the 3-O-lactone derivative 3, which was
hydrolyzed to give the corresponding acid derivative 4. The 3-O-
(carbethoxymethyl)-substituted compound 5 was prepared by
treating compound 1 with ethyl bromoacetate and triethylamine
in ethanol. Alkaline hydrolysis of the ethyl ester 5 afforded the
corresponding acid 6.
Compounds 7-11 were obtained starting from the previously
reported 6-amino-7-chloro-3-acetoxyquinazoline-2,4-dione 2140
(Scheme 2). Reaction of 21 with 2,5-diethoxytetrahydrofuran

7-Chloro-3-hydroxy-1H-quinazoline-2,4-dione
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6017
Scheme 1
(a) (i) R-Bromo-γ-butyrolactone, K2CO3, ethyl methyl ketone, 90 °C; (ii) 6 N HCl; (b) (i) 1% NaOH, rt; (ii) 6 N HCl; (c) ethyl bromoacetate, NEt3,
EtOH, rt; (d) (i) 2.5% NaOH, rt; (ii) 6 N HCl.
Scheme 2
(a) 2,5-Diethoxytetrahydrofuran, glacial AcOH, 90 °C; (b) 2,5-dimethoxytetrahydrofuran-3-carbaldehyde, glacial AcOH, 90 °C; (c) (i) KMnO4, H2O/
acetone (1:1), 0 °C; (ii) 38% sodium hydrogen sulfite; (iii) 6 N HCl; (d) (i) 2.5% NaOH, rt; (ii) 6 N HCl; (e) piperidine, EtOH, reflux; (f) 2,5-dimethoxy-
2,5-dihydrofuran, NaOAc, glacial AcOH, 90 °C; (g) 4-oxo-4H-pyran-2,6-dicarboxylic acid (chelidonic acid), DMSO, 90 °C.
Scheme 3
in this class of compounds the free 3-hydroxy group plays a
pivotal role for anchoring to AMPA, Gly/NMDA, and KA
receptors.
Table 1. Binding Affinity at AMPA, Gly/NMDA, and High-affinity KA
(KAh-a) Receptors
(a) (i) Phthalic anhydride, NaOAc, glacial AcOH, 60 °C; (b) (i) 1%
NaOH, rt; (ii) 6 N HCl; (c) (i) succinic anhydride, NaOAc, glacial AcOH,
60 °C; (ii) 6 N HCl; (d) NaOAc, Ac2O, reflux; (e) piperidine, EtOH, reflux.
classes of compounds. Thus, we decided to first introduce the
same 2-(4-hydroxybutanoic) acid chain of derivative 33 on the
3-hydroxy group of the quinazoline-2,4-dione 1 (derivative 4).
In contrast with the isatinoxime derivative 33, compound 4
completely lacked affinity at all three investigated receptors.
The same applies to the corresponding 3-O-lactone derivative
3, a conformationally constrained analogue of derivative 4.
When the acetic acid side chain was positioned on the 3-hydroxy
group of derivative 1 (compound 6) some Gly/NMDA and
AMPA affinities were restored, while esterification of the acetic
group of compound 6 resulted in a complete loss of affinity at
all three receptors (compound 5). In summary, the scarce/null
affinities of the 3-O-substituted derivatives 3-6 highlight that
a Ki values are means ( SEM of three or four separate determinations
in triplicate. b Percentage of inhibition (I%) of specific binding at 100 µM
concentration. c IC50 values are means ( SEM of three or four separate
determinations in triplicate. d Reference 40. e Reference 46.

6018 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Colotta et al.
Scheme 4
(a) Anhydrous CH2Cl2, pyridine, 0 °C; (b) (i) 2% NaOH, rt; (ii) 6 N HCl; (c) piperidine, EtOH, reflux.
Table 2. Binding Affinity at AMPA, Gly/NMDA, and High-affinity KA
(KAh-a) Receptors
Scheme 5
(a) Benzylisocyanate, anhydrous THF, reflux; (b) (i) 1% NaOH, rt; (ii)
6 N HCl.
Figure 1. 3-Isatinoxime derivative 33-based structure of compound
4.
The set of derivatives 7-11 was designed taking as lead
compound the previously reported AMPA selective antagonist
240 (Table 2). In fact, new compounds formally resulted from
the replacement of the 6-(1,2,4-triazol-4-yl) group of the lead
with other heterocyclic rings, some of which were profitable in
well-known classes of AMPA-receptor antagonists.27,28,38,41 The
binding data of compounds 7-11, reported in Table 2, show
that, as expected, the presence of a suitable 6-heterocyclic
residue shifted the affinity toward the AMPA receptor, the only
exception being the pyrrol-1-yl group (compound 7), which
afforded higher affinity for the Gly/NMDA site than for the
AMPA receptor. The 6-(4-oxo-pyridin-1-yl)-derivative 11 dis-
played the highest AMPA receptor affinity, comparable to that
of the lead compound 2. KA receptor affinities of derivatives
7-11 are similar (7, 8, and 10) or lower (9 and 11) than the
AMPA ones.
Compound 11, which showed the highest AMPA receptor
affinity, was tested to evaluate its antagonistic activity by
assessing its ability to inhibit depolarization induced by 5 µM
AMPA and NMDA in cortical wedge preparations.38 The results
obtained from these functional antagonism studies are reported
in Table 3, where the inhibitory potencies of 2 and NBQX (2,3-
dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline) have also
been included. Compound 11 inhibits AMPA and NMDA
responses in a reversible manner. Its inhibitory activities are
similar to those of the lead compound 2 and are in agreement
with its binding affinities. In fact, compound 11 shows greater
a Ki values are means ( SEM of three or four separate determinations
in triplicate. b Percentage of inhibition (I%) of specific binding at 100 µM
concentration. c IC50 values are means ( SEM of three or four separate
determinations in triplicate. d Reference 40.
Table 3. Functional Antagonism of Derivative 11 at NMDA and
AMPA Sites
IC50a (µM)
AMPA
NMDA
11
3.5 ( 0.4
4.6 ( 0.5
0.20 ( 0.02
>80
2b
84 ( 10
NBQX
(*)c
a Concentration necessary for 50% inhibition (IC50) of depolarization
induced by S-AMPA or NMDA in mouse cortical wedge preparation. IC50
values are means ( SEM of three separate determinations. b Reference 40.
c At 10 µM concentration, the inhibition was not significant.
potency in inhibiting AMPA-evoked response than NMDA-
induced depolarization.
To interpret the AMPA binding affinities of derivatives 7-11,
we performed a molecular modeling study by docking these
compounds, and the leads 1 and 2, in the domain-open state of
the GluR2 ligand-binding core (GluR2-S1S2J/(S)-ATPO crystal
structure, pdb entry 1n0t),49 ((S)-ATPO, namely, (S)-2-amino-

7-Chloro-3-hydroxy-1H-quinazoline-2,4-dione
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6019
Figure 2. Compound 2 (panel a) and compounds 9 and 11 (orange and blue, respectively; panel b) docked into the X-ray GluR2-S1S2J/(S)-ATPO
crystal structure (1n0t). Best docking conformation of compounds 1 (orange) and 7 (blue) in the Gly/NMDA receptor binding site (panel c). The
ligand is shown in ball-and-stick representation. Side chains of receptor residues involved in the interactions are in stick representation. Standard
atomic colors are used (carbon ) gray, oxygen ) red, nitrogen ) blue, sulfur ) yellow, chlorine ) green).
3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl] propionic
acid). The antagonist binding site of glutamate ionotropic
receptors (iGluRs) is located in a clam shell-like ligand binding
region composed of two domains, domain 1 and domain 2. The
interactions of compounds 1, 2, and 7 with the AMPA receptor
binding pocket involve the highly conserved residue triad among
iGluRs: Arg485, Thr480, and Pro478 (numbering as in ref 49;
Figure 2, panel a; for clarity only compound 2 is shown). In
particular, the NH group of these derivatives engages a hydrogen
bond with Pro478 (backbone CO), the 2-carbonyl residue
interacts with Arg485 and Thr480 (guanidine side chain and
backbone NH, respectively), and the 3-hydroxy group interacts
with the guanidine residue of Arg485. Moreover, the aromatic
portion of the quinazoline scaffold is involved in a π-π stacking
interaction with the aromatic moiety of Tyr450, located at the
top of the binding pocket. In addition, the 6-heterocyclic rings
of both 7 and 2 occupy the binding cleft formed by Leu650,
Leu704, Thr655, Thr686, and Met708. Interestingly, the electron
deficiency of the 6-(1,2,4-triazol-1-yl) moiety of derivative 2
determines an additional interaction50 with the hydroxy group
of the Thr686 side chain. This interaction is likely to reinforce
the anchoring of this compound to the above-mentioned pocket
and, at least partly, might account for the higher AMPA receptor
affinity of 2 with respect to that of the 6-pyrrol derivative 7.
To interpret the trend of Gly/NMDA receptor affinity of
compounds 7-11, we docked these derivatives, and the lead
compounds 1 and 2, to a recently described model of the Gly/
NMDA receptor.42 The peculiar interactions between these
compounds and the Gly/NMDA receptor binding pocket are very
similar to the interactions found for the GluR2 receptor on
account of the fact that the highly conserved triad is involved
in the most significant hydrogen bonds. Briefly, in the Gly/
NMDA receptor, the residues of the triad are Arg523, Thr518,
and Pro516 (numbering as in ref 51). Considering compound 1
the most active ligand at the Gly/NMDA receptor among this
series, the NH group acts as a proton donor in favor of Pro516
(backbone CO), while the 2-carbonyl group accepts a hydrogen
bond from Thr518 (backbone NH; Figure 2, panel c). Both the
2-carbonyl and the 3-hydroxy groups of this compound interact
with Arg523 (guanidine residue). Moreover, at the top of the
binding pocket, the aromatic ring of Phe484 makes π-π
stacking interaction with the aromatic quinazoline scaffold. The
6-pyrrole-substituted derivative 7 shows a quite similar binding
mode to that of compound 1 (Figure 2, panel c). Indeed, 7 can
still interact with Thr518 and Arg523 residues through the
2-carbonyl and 3-hydroxy groups. Moreover, the 6-pyrrole ring
is settled in a cavity formed by nonconserved residues such as
Val735, Asp732, and Trp731.42 This subsite is less roomy and
more hydrophobic than the corresponding pocket in the GluR2-
S1S2J receptor, especially due to the steric hindrance of the
Trp731 indole moiety. For this reason, only the pyrrole ring
seems to be small enough to find space in the opening between
lobe S1 and lobe S2, very close to Trp731, making a
hydrophobic interaction with the aromatic side chain of this
residue and avoiding the closure of the ligand binding cleft. In
contrast, the 6-substituents of compounds 2 and 8-11 cannot
fit in this subsite, forcing compounds to find an alternative and
less-effective conformation in the binding site.
Very unexpectedly, the anchoring of derivatives 9 and 11 to
the AMPA receptor binding site (Figure 2, panel b) was different
with respect to that of derivatives 7 and 2. Moreover, 9 and 11
also differ between them for their binding mode. In compound
11, the NH group acts as a H-bond donor in favor of Pro478
(backbone CO), and the 2-carbonyl oxygen forms a hydrogen
bond with the hydroxy group of Tyr405, though the distance is
somewhat greater (3.2 Å) than a typical H-bond interaction.
The 3-hydroxy substituent and the 4-carbonyl group interact,
respectively, with Glu402 and Thr686, thus preventing interac-
tion between these interdomain residues, an interaction that is
crucial to stabilize the closed agonist state conformation of
GluR2-S1S2J. The position and distance (about 3.7 Å) of the
aromatic quinazoline ring of 11 are optimal for a π-π stacking
interaction with the aromatic Tyr450 residue. In regard to
compound 9, the NH and 2-carbonyl groups seem to not make
significant interactions with the recognition site, while the
3-hydroxy and 4-carbonyl groups form hydrogen bonds with
Glu402 and Thr686, respectively, thus avoiding the domain
closure and holding the antagonist-binding core in its open-
cleft conformation. The 6-substituent of 9 interacts, through the
carboxy group, with both Ser654 and Thr655.
To evaluate the effect of nonheterocyclic substituents at the
6-position, we synthesized the set of derivatives 12-20 that
bear different 6-amide or 6-ureide moieties. The binding results
of derivatives 12-20 are reported in Table 4, where the binding
affinities of the willardiine analogue UBP 30252,53 (compound
35, Figure 3) are also reported. The binding data show that the
6-amide and 6-ureide substituents shifted affinities toward the
AMPA and KA receptors. As the most important result, we have
obtained a potent and selective KA receptor antagonist: the 6-(2-
carboxybenzoylamino)-3-hydroxy-1H-quinazolin-2,4-dione 12
(QZ 443). This compound possesses good affinity and selectiv-
ity for the KA high-affinity receptor. Compound 12 was
designed as an analogue of derivative 9 (Figure 3). In fact,

6020 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Colotta et al.
Table 4. Binding Affinity at AMPA, Gly/NMDA, High-Affinity KA
(KAh-a), and Low-Affinity KA (KAl-a) Receptors
12 bears the ortho-carboxyphenyl moiety, similarly to the
willardiine analogue (R,S)-3-(2-carboxybenzyl)-willardiine 34
(UBP 296, Figure 3), which was recently reported in the
literature52,53 as a selective antagonist of the KA receptor.
Compound 34 was found to be a potent and selective antagonist
of the native GluR5-containing KA receptor in the spinal cord,
with activity residing in the S-enantiomer 35. Because no affinity
data at native KA receptors were reported for either 35 or its
racemate 34,52,53 we tested the commercially available 35 in
our experiments to evaluate its binding at the high-affinity native
KA receptor, which is described to be represented by KA1 and
KA2 subunits.4,5,6,54 Compound 35 was also tested at AMPA
and Gly/NMDA receptors. The binding results, reported in Table
4, showed that 35 possesses, respectively, low and null affinity
for AMPA and Gly/NMDA receptors. This derivative also has
low affinity for the high-affinity KA receptor, about 75-fold
lower than that of compound 12. Compound 12 and the
willardiine derivative 35 were also tested at the low-affinity
KA binding site,54 which consists of a combination of GluR5-7
subunits.6,7 As the binding results indicate (Table 4), derivatives
12 and 35 show, respectively, a halved and 3-fold higher affinity
at the low-affinity KA receptor with respect to the high-affinity
one. Most importantly, compound 12 possesses higher affinity
than derivative 35, even at the low-affinity site. It also has to
be highlighted that derivative 12, compared to 35, possesses a
higher selectivity toward both high- and low-affinity KA sites
with respect to the AMPA receptor.
The presence and position of the distal acidic carboxy group
play a critical role for KA receptor affinity and selectivity of
derivative 12. In fact, removal of this substituent (compound
15) or its movement from the ortho to the meta (compound 16)
or to the para position (compound 17) significantly decreased
the binding at the high-affinity KA receptor. However, it has
to be noted that derivative 17 still maintains higher affinity and
selectivity for this receptor with respect to those of the
willardiine derivative 35. Both compounds 16 and 17 are,
instead, totally unable to bind to the low-affinity KA site.
a Ki values are means ( SEM of three or four separate determinations
in triplicate. b Percentage of inhibition (I%) of specific binding at 100 µM
concentration. c IC50 values are means ( SEM of three or four separate
determinations in triplicate.
The replacement of the lipophilic benzene linker, between
the 6-carbamoyl spacer and the acidic distal function of 12, with
the more flexible ethylene chain afforded a 10-fold reduced KA
receptor affinity (compound 13), probably due to the free
rotation of the ethylene chain. Indeed, the benzene ring of
derivative 12 might constrain the carboxy group in the right
position for a good hydrogen-bonding interaction. Cyclization
of the 6-succininamido chain of 13 to afford derivative 14
drastically reduced affinity toward the KA receptor and, to a
lesser extent, toward the AMPA one. Similarly, a complete loss
of KA affinity ensued from esterification of the carboxy group
of compounds 16 and 17 (see derivatives 18 and 19). Also
derivative 20, which bears the long 3-benzylureido side chain
at the 6-position, completely lacks AMPA and KA receptor
affinities.
Due to the interesting binding affinity and selectivity of
derivative 12 at KA receptors, both high- and low-affinity ones,
we decided to evaluate its antinociceptive effect; it is well-
known that KA receptor antagonists are effective as analgesics
in various animal models of pain.21,24 Thus, compound 12 was
tested in the acetic acid-induced writhing assay and the results
are reported in Table 5. The effect of diclofenac (2-[(2,6-
dichlorophenyl)amino]benzeneacetic acid) is also reported for
comparison. Compound 12 showed antinociceptive properties
because it increased the pain threshold in a dose-dependent
manner starting from the dose of 1.0 mg kg-1 po. The dose of
0.1 mg kg-1 was devoid of any effect. Compound 12, at doses
Figure 3. Compounds 9, 12, and (R,S)-3-(2-carboxybenzyl)-willardiine
34 and its (S)-enantiomer 35.
compounds 12 and 9 share a carboxy group in a position that
is similarly spaced from the 3-hydroxy-quinazoline-2,4-dione
scaffold. Differently from 9, the carboxy function of derivative
12 was inserted on a benzo ring that was moved away from the
3-hydroxyquinazolin-2,4-dione nucleus, by means of the car-
bamoyl spacer, with the purpose of enhancing the volume of
the molecule in the neighborhood of the 6-position. Interestingly,
this modification shifted affinity and selectivity from the AMPA
(compound 7) to the KA receptor (compound 12). Compound

7-Chloro-3-hydroxy-1H-quinazoline-2,4-dione
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6021
Table 5. Effect of Compound 12 In Comparison with Diclofenac in the
Mouse Abdominal Constriction Test
treatment
per osa
dose
no. mice
mg kg-1
no. writhes
CMCb
12
12
12
12
8
11
12
14
35.1 ( 3.7
34.7 ( 2.9
26.1 ( 3.3c
24.5 ( 3.1c
18.2 ( 3.6c
0.1
1.0
10
diclofenac
5.0
a Compound 12 was administered 30 min before test. b Carboxymeth-
ylcellulose. c P < 0.01 versus CMC-treated mice.
Table 6. Effect of Compound 12 on Mice Rota-Rod Test
after treatmenta
treatment
per os
dose
before
mg kg-1 treatmenta
30 min
45 min
60 min
b
5.6 ( 0.4
5.1 ( 0.3
4.8 ( 0.3
4.0 ( 0.3 3.7 ( 0.2 2.0 ( 0.2
3.3 ( 0.4 2.6 ( 0.2 1.7 ( 0.3
4.8 ( 0.2 3.7 ( 0.3 3.2 ( 0.3
CMC
12
12
20
50
a Number of falls from the rod in 30 s. Each value represents the mean
of eight mice. b Carboxymethylcellulose.
Figure 4. Compound 12 docked into a homology model of GluR5/
ATPO built from the X-ray structure of the GluR2/DNQX and GluR2/
ATPO complexes. The ligand and receptor residues are represented
with the same coloring scheme for the atoms as in Figure 2.
20-50 times higher than those effective in increasing the pain
threshold, was unable to modify mouse motor coordination,
evaluated by means of the rota-rod test (Table 6). These results
not only rule out the possibility that the antinociceptive effect
observed was related to an altered viability of treated animals,
but also evidence a good tolerability of compound 12 in
laboratory animals.
Conclusion
Synthesis of the herein reported compounds has allowed us
to further explore the SAR in this class of derivatives, and it
has also afforded some selective AMPA receptor antagonists
characterized by good affinities and selectivities. However, the
most significant result of this work is the finding of a new KA
receptor antagonist (compound 12) that possesses good affinity
for both high- and low-affinity KA receptors and also good
selectivity not only toward the Gly/NMDA receptor, but also
toward the AMPA one. The importance of this result resides in
the fact that, currently, only a few examples of KA-receptor-
selective antagonists are known. It should also be highlighted
that in our binding experiments at the native KA receptor,
derivative 12 shows higher affinity than the willardiine analogue
35 that was recently reported as a potent and selective antagonist
at the native GluR5-containing KA receptor in rat spinal cord.
Modeling studies have provided valuable evidence of the
putative binding modes of the synthesized antagonists at all three
investigated receptors, pointing out different sets of interactions
that involve different amino acidic residues and contribute to
the affinity of the compounds. These studies prove themselves
as useful tools for guiding the design and synthesis of new
AMPA and KA receptor antagonists.
In an attempt to depict the binding mode of compound 12 at
the low-affinity KA receptor, the building of a homology model
of the antagonized state of the receptor was undertaken. In fact,
only the X-ray structures for the closed agonized state of GluR5
and GluR6 binding domains were available.55,56 Thus, taking
advantage of the almost identical secondary structures and the
high similarity in amino acidic sequence (50%) of the AMPA
receptor versus the KA receptor, a GluR5 homology model was
derived from the open state of the GluR2 ligand-binding core.
Passing from the GluR2 ligand binding pocket to the GluR5
one there is the exchange of five residues, all belonging to
domain 2, namely, Leu650 to Val670, Thr686 to Ser706, Tyr702
to Leu720, Leu704 to Met722, and Met708 to Ser726. These
changes make the volume of the binding cavity of GluR5 40%
larger than the volume of GluR2, increasing the number of
trapped water molecules and making the chemical features of
some subpockets in the two receptor types different.56
Experimental Section
The results of docking experiments on compound 12 in the
homology-built model of GluR5 (hmGluR5; Figure 4) point out
the interaction of the 6-carbamoyl CO group with the guanidine
residue of Arg508. The ortho-carboxy moiety accommodates
in the protein region favorable for a negatively charged group,
thus establishing a direct hydrogen bond with Ser674. The
substitution of the Met708 and Thr686 residues in GluR2 by
Ser726 and Ser706 in GluR5 permits a deeper insertion of the
quinazoline ring of 12 into the GluR5 binding pocket than in
the GluR2 one (not shown in figures), thus allowing a direct
interaction of the 3-OH group with the side chain of Glu426.
The quinazoline ring of 12 lies about 3.7 Å below and parallel
to the aromatic moiety of Tyr474, thus forming a π-π stacking
interaction. Moreover, it is likely that the formation of an
intramolecular hydrogen bond between the carboxy group and
the carbamoyl NH further stabilizes the bound conformation
of the compound.
Chemistry. Silica gel plates (Merck F254) were used for
analytical chromatography. All melting points were determined on
a Gallenkamp melting point apparatus. Microanalyses were per-
formed with a Perkin-Elmer 260 elemental analyzer for C, H, N,
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
1
cm-1. The H NMR spectra were obtained with a Varian Gemini
200. The chemical shifts are reported in δ (ppm) and are relative
to the central peak of the solvent that is always DMSO-d6. The
following abbreviations are used: s ) singlet, d ) doublet, dd )
double doublet, t ) triplet, m ) multiplet, br ) broad, ar ) aromatic
protons, and al ) aliphatic protons.
(()- 7-Chloro-3-[(2-oxo-tetrahydrofuran-3-yl)oxy]-1H-quinazo-
line-2,4-dione (3). K2CO3 (1.88 mmol) and (()-R-bromo-γ-
butyrolactone (1.1 mL) were added to a hot (90 °C) suspension of
3-hydroxy-7-chloro-1H-quinazoline-2,4-dione 140 (1.88 mmol) in
ethyl methyl ketone (30 mL). The suspension was refluxed for 48

6022 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Colotta et al.
h. After cooling at room temperature, the suspension was diluted
with water and acidified with 6 N HCl. The mixture was extracted
with ethyl acetate (20 mL × 3), and the collected organic layers
were anhydrified (Na2SO4) and evaporated at reduced pressure to
give an oil. Upon treatment with a small amount of ethanol, a solid
precipitated that was collected and recrystallized. Yield: 50%; mp
was collected, washed with water, and recrystallized. Yield: 85%;
mp 242-243 °C (EtOH). 1H NMR: 6.23 (s, 2H, pyrrole protons),
6.98 (s, 2H, pyrrole protons), 7.38 (s, 1H, ar), 7.79 (s, 1H, ar),
10.85 (br s, 1H, OH), 11.80 (s, 1H, NH). IR: 1690, 1750, 3100-
3600. Anal. (C12H8ClN3O3) C, H, N.
3-Acetoxy-7-chloro-6-(3-formylpyrrol-1-yl)-1H-quinazoline-
2,4-dione (23). A solution of 2,5-dimethoxytetrahydrofuran-3-
carbaldehyde (2.7 mmol) in glacial acetic acid (7 mL) was dropwise
added to a hot (90 °C) suspension of the 6-amino derivative 2140
(1.8 mmol) in glacial acetic acid (8 mL). After the addition was
completed, the mixture was stirred at room temperature for 30 min.
Evaporation of the solvent at reduced pressure yielded a solid that
was suspended in water (10-20 mL), collected, and recrystallized.
Yield: 70%; mp 233-234 °C (EtOH). 1H NMR: 2.39 (s, 3H, CH3),
6.65 (s, 1H, pyrrole proton), 7.17 (s, 1H, pyrrole proton), 7.44 (s,
1H, ar), 7.93 (s, 1H, pyrrole proton), 8.00 (s, 1H, ar), 9.76 (s, 1H,
CHO), 12.30 (br s, 1H, NH). IR: 1670, 1710, 1740, 1820, 3120,
3250. Anal. (C15H10ClN3O5) C, H, N.
7-Chloro-3-hydroxy-6-(3-formylpyrrol-1-yl)-1H-quinazoline-
2,4-dione (8). The title compound was obtained from 23 (0.8 mmol)
following the experimental procedure described above to prepare
7 from 22. Yield: 80%; mp > 300 °C (EtOH). 1H NMR: 6.65 (s,
1H, pyrrole proton), 7.17 (s, 1H, pyrrole proton), 7.39 (s, 1H, ar),
7.93 (s, 1H, pyrrole proton), 7.96 (s, 1H, ar), 9.76 (s, 1H, CHO),
10.80 (br s, 1H, OH), 11.85 (br s, 1H, NH). IR: 1690, 1740, 3100-
3600. Anal. (C13H8ClN3O4) C, H, N.
3-Acetoxy-7-chloro-6-(3-carboxypyrrol-1-yl)-1H-quinazoline-
2,4-dione (24). Potassium permanganate (0.27 g) was portionwise
added to a cooled (0 °C) suspension of compound 23 (0.57 mmol)
in a 1:1 acetone/water mixture (10 mL). Each small addition was
made after the disappearance of the violet color of the oxidizing
agent. After the additions were completed, the mixture was stirred
at room temperature for 1 h, then the excess of potassium
permanganate was quenched with a 38% solution of sodium
hydrogen sulfite, and the solution was acidified with 6 N HCl. The
solid was collected, washed with water, and recrystallized. Yield:
48%; mp > 300 °C (EtOH). 1H NMR: 2.40 (s, 3H, CH3), 6.56 (s,
1H, pyrrole proton), 7.03 (s, 1H, pyrrole proton), 7.37 (s, 1H, ar),
7.56 (s, 1H, pyrrole proton), 7.89 (s, 1H, ar), 12.10 (br s, 2H, NH
+ COOH). Anal. (C15H10ClN3O6) C, H, N.
7-Chloro-3-hydroxy-6-(3-carboxypyrrol-1-yl)-1H-quinazoline-
2,4-dione (9). A solution of the 3-acetoxy derivative 24 (0.3 mmol)
in ethanol (2 mL), containing 2-3 drops of piperidine, was refluxed
for 1 h. After cooling at room temperature and dilution with water
(10 mL), a solid was obtained that was collected and recrystallized.
Yield: 64%; mp > 300 °C (EtOH). 1H NMR: 6.56 (s, 1H, pyrrole
proton), 7.03 (s, 1H, pyrrole proton), 7.37 (s, 1H, H-8), 7.56 (s,
1H, pyrrole proton), 7.89 (s, 1H, H-5), 10.82 (s, 1H, OH), 11.82
(s, 1H, NH), 12.03 (br s, 1H, COOH). IR: 1700, 1740, 1750, 2500-
3600. Anal. (C13H8ClN3O5) C, H, N.
1
244-245 °C (CH3CN). H NMR: 2.38-2.46 (m, 1H, al), 2.59-
2.68 (m, 1H, al), 4.32-4.36 (m, 1H, al), 4.45-4.49 (m, 1H, al),
5.07 (dd, 1H, al, J ) 7.3, 5.0 Hz), 7.21 (d, 1H, H-8, J ) 1.9 Hz),
7.30 (dd, 1H, H-6, J ) 8.5, 1.9 Hz), 7.95 (d, 1H, H-5, J ) 8.5 Hz),
11.85 (s, 1H, NH). IR 1694, 1733, 1773, 3206. Anal. (C12H9ClN2O5)
C, H, N.
(()-2-[(7-Chloro-2,4-dioxo-2,4-dihydro-1H-quinazolin-3-yl)-
oxy]-4-hydroxybutanoic Acid (4). Derivative 3 (1.7 mmol) was
dissolved in 1% aqueous NaOH (20 mL), and the solution stirred
at room temperature for 30 min. After acidification with 6 N HCl,
the mixture was extracted with ethyl acetate (20 mL × 3). The
collected organic layers were anhydrified (Na2SO4) and evaporated
at reduced pressure to yield a solid that was treated with cyclo-
hexane (2-3 mL) and a few drops of ethyl acetate and collected
by filtration. The crude product cannot be recrystallized since it
changes into derivative 3. Thus, derivative 4 was purified by acid-
base exchange as follows: it was dissolved in 1% NaOH (about
20 mL), and the solution was extracted with ethyl acetate (about
20 mL). The aqueous phase was acidified to pH 1 with 6 N HCl
and extracted with ethyl acetate (10 mL × 3). The organic layers
were anhydrified (Na2SO4), and the solvent was evaporated at
reduced pressure. The residue was treated with cyclohexane/ethyl
acetate to yield a solid that was collected and dried. Yield: 74%;
1
mp 241-245 °C, after decomposition at about 150 °C. H NMR:
1.91-2.09 (m, 2H, al), 3.50-3.56 (m, 1H, al), 3.62-3.68 (m, 1H,
al), 4.55 (br s, 1H, OH), 4.61 (t, 1H, al, J ) 6.4 Hz), 7.20 (d, 1H,
H-8, J ) 1.8 Hz), 7.29 (dd, 1H, H-6, J ) 8.5, 1.8 Hz), 7.94 (d, 1H,
H-5, J ) 8.5 Hz), 11.77 (s, 1H, NH), 12.89 (br s, 1H, COOH). IR:
1681, 1732, 1758, 3070-3420. Anal. Calcd for C12H11ClN2O6: C,
45.80; H, 3.53; N, 8.90. Found: C, 46.40; H, 3.90; N, 8.40.
Ethyl (7-Chloro-2,4-dioxo-2,4-dihydro-1H-quinazolin-3-yl)-
oxyacetate (5). Ethyl bromoacetate (4.7 mmol) and triethylamine
(4.7 mmol) were added to a suspension of compound 140 (4.7 mmol)
in ethanol (60 mL). The mixture was stirred at room temperature
for 5 h, then the solid was collected and recrystallized. Yield: 57%;
1
mp 183-185 °C (EtOH). H NMR: 1.23 (t, 3H, CH3, J ) 7.1
Hz), 4.19 (q, 2H, CH2, J ) 7.1 Hz), 4.72 (s, 2H, CH2), 7.20 (d,
1H, H-8, J ) 1.9 Hz), 7.29 (dd, 1H, H-6, J ) 8.5, 1.9 Hz), 7.94 (d,
1H, H-5, J ) 8.5 Hz), 11.78 (s, 1H, NH). IR: 1674, 1732, 1760,
3192. Anal. (C12H11ClN2O5) C, H, N.
(7-Chloro-2,4-dioxo-2,4-dihydro-1H-quinazolin-3-yl)oxyace-
tic Acid (6). A suspension of the ester 5 (1.0 mmol) in 2.5%
aqueous NaOH (20 mL) was stirred at room temperature for 1 h.
The cooled (0 °C) solution was acidified to pH 1 with 6 N HCl,
and the resulting solid was collected, abundantly washed with water,
3-Acetoxy-7-chloro-6-(2-oxo-2,5-dihydropyrrol-1-yl)-1H-
quinazoline-2,4-dione (25). 2,5-Dimethoxy-2,5-dihydrofuran (1.1
mmol) and sodium acetate (2 mmol) were added to a suspension
of derivative 2140 in glacial acetic acid (5 mL), and the mixture
was heated at 90 °C for 2 h. After cooling at room temperature,
the solution was diluted with water (30-40 mL), and the resulting
solid was collected, washed with water, and recrystallized. Yield:
1
and recrystallized. Yield: 96%; mp > 300 °C (EtOH). H NMR:
4.63 (s, 2H, CH2), 7.21 (d, 1H, H-8, J ) 1.9 Hz), 7.28 (dd, 1H,
H-6, J ) 8.5, 1.9 Hz), 7.95 (d, 1H, H-5, J ) 8.5 Hz), 11.79 (br s,
1H, NH). IR: 1685, 1720, 1775, 3050-3290. Anal. (C10H7ClN2O5)
C, H, N.
3-Acetoxy-7-chloro-6-(pyrrol-1-yl)-1H-quinazoline-2,4-di-
one (22). A solution of 2,5-diethoxytetrahydrofuran (2.2 mmol) in
glacial acetic acid (3 mL) was dropwise added to a hot (90 °C)
suspension of the 6-amino derivative 2140 (0.74 mmol) in glacial
acetic acid (8 mL). The solution was heated at 90° C for 20 min,
then most of the solvent was distilled off at reduced pressure.
Dilution with water of the residue solution yielded a solid that was
collected and recrystallized. Yield: 87%; mp 248-250 °C (EtOH).
1H NMR: 2.39 (s, 3H, CH3), 6.24 (s, 2H, pyrrole protons), 7.01
(s, 2H, pyrrole protons), 7.41 (s, 1H, ar), 7.83 (s, 1H, ar), 12.15
(br s, 1H, NH). Anal. (C14H10ClN3O4) C, H, N.
1
82%; mp 280 °C dec (EtOH). H NMR: 2.39 (s, 3H, CH3), 4.42
(s, 2H, CH2), 6.25 (d, 1H, pyrrole proton, J ) 5.9 Hz), 7.31 (s,
1H, H-8), 7.50 (d, 1H, pyrrole proton, J ) 5.9 Hz), 7.97 (s, 1H,
H-5), 12.15 (br s, 1H, NH). IR: 1670, 1710, 1750, 1815. Anal.
(C14H10ClN3O5) C, H, N.
7-Chloro-3-hydroxy-6-(2-oxo-2,5-dihydropyrrol-1-yl)-1H-
quinazoline-2,4-dione (10). The title compound was prepared from
the 3-acetoxy derivative 25 (0.3 mmol) in the experimental
conditions described above to obtain 9 from 24. Yield: 82%; mp
> 300 °C (EtOH). 1H NMR: 4.43 (s, 2H, CH2), 6.25 (d, 1H, pyrrole
proton, J ) 5.9 Hz), 7.31 (s, 1H, H-8), 7.50 (d, 1H, pyrrole proton,
J ) 5.9 Hz), 7.97 (s, 1H, H-5), 10.72 (br s, 1H, OH), 11.72 (br s,
1H, NH). IR: 1680, 1740, 3100-3600. Anal. (C12H8ClN3O4) C,
H, N.
7-Chloro-3-hydroxy-6-(pyrrol-1-yl)-1H-quinazoline-2,4-di-
one (7). A solution of the 3-acetoxy derivative 22 (0.8 mmol) in
aqueous 2.5% NaOH (20 mL) was stirred at room temperature for
1 h and then acidified to pH 1 with 6 N HCl. The resulting solid

7-Chloro-3-hydroxy-1H-quinazoline-2,4-dione
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6023
7-Chloro-3-hydroxy-6-(4-oxo-4H-pyridin-1-yl)-1H-quinazoline-
2,4-dione (11). A solution of the 6-amino derivative 2140 (1.1 mmol)
and 4-oxo-4H-pyran-2,6-dicarboxylic acid (chelidonic acid; 1.4
mmol) in dimethyl sulfoxide (2 mL) was stirred at 90 °C for 58 h.
After cooling at room temperature, the solution was diluted with
water (50 mL), and the solid was filtered off. The solvent of the
clear solution was distilled at reduced pressure, and the oily residue
was taken up with acetone (5 mL) and a few drops of water. The
solid was collected and recrystallized. Yield: 15%; mp > 300 °C
8.01 (s, 1H, H-5), 10.70 (br s, 1H, OH), 11.81 (br s, 1H, NH). IR:
1690, 1710, 1730, 1750, 3120-3520. Anal. (C12H8ClN3O5) C, H,
N.
3-Acetoxy-6-benzoylamino-7-chloro-1H-quinazoline-2,4-di-
one (29). A solution of benzoyl chloride (1.1 mmol) in anhydrous
methylene chloride (3 mL) was dropwise added to a cooled (0 °C)
suspension of the 6-amino derivative 2140 (1.1 mmol) in anhydrous
methylene chloride (10 mL) and pyridine (1 mL). When the addition
was completed, the mixture was stirred at room temperature for
24 h. Evaporation of the solvent at reduced pressure gave an oily
residue that was treated with water (3 mL) and ethanol (3 mL).
The resulting solid was collected and recrystallized. Yield: 60%;
1
(AcOH). H NMR: 6.20 (d, 2H, pyridine protons, J ) 7.6 Hz),
7.41 (s, 1H, H-8), 7.73 (d, 2H, pyridine protons, J ) 7.6 Hz), 8.12
(s, 1H, H-6), 10.82 (s, 1H, OH), 11.90 (s, 1H, NH). IR: 1683,
1732, 3054-3600. Anal. (C13H8ClN3O4) C, H, N.
1
mp 259-261 °C (EtOH). H NMR: 2.39 (s, 3H, CH3), 7.37 (s,
1H, ar), 7.50-7.62 (m, 3H, ar), 7.97 (d, 2H, ar, J ) 7.7 Hz), 8.09
(s, 1H, ar), 10.25 (s, 1H, NH), 12.10 (br s, 1H, NH). Anal. (C17H12-
ClN3O5) C, H, N.
3-Acetoxy-7-chloro-6-phthalimido-1H-quinazoline-2,4-di-
one (26). A mixture of the 6-amino derivative 21 (0.9 mmol),
phthalic anhydride (1.6 mmol), and sodium acetate (0.9 mmol) in
glacial acetic acid (5 mL) was heated at 60 °C for about 15 h.
After cooling at room temperature, the suspension was diluted with
water (30 mL) and acidified to pH 1 with 6 N HCl. The solid was
collected, washed with water, and recrystallized. Yield: 65%; mp
General Procedure to Prepare 3-Acetoxy-6-[3-(methoxycar-
bonyl)benzoylamino]-7-chloro-1H-quinazoline-2,4-dione (30) and
3-Acetoxy-6-[4-(methoxycarbonyl)benzoylamino]-7-chloro-1H-
quinazoline-2,4-dione (31). A solution of 3-methoxycarbonylben-
zoyl chloride44 or 4-methoxycarbonylbenzoyl chloride45 (4 mmol)
in anhydrous methylene chloride (3 mL) and, in succession, a
solution of anhydrous pyridine (4 mmol) in anhydrous methylene
chloride (3 mL) were dropwise added, over a period of about 15
min, to a cooled (0 °C) suspension of the 6-amino derivative 2140
(2.1 mmol) in anhydrous methylene chloride (20 mL). The
suspension was stirred at room temperature for about 10 h
(compound 30) or 7 h (compound 31). The crude 30 was collected
by filtration, resuspended in EtOAc (100 mL), and stirred for a
few minutes, then it was collected, washed with water, and
recrystallized. The crude 31 was collected, washed with water, and
recrystallized.
1
> 300 °C (CH3NO2). H NMR: 2.42 (s, 3H, CH3), 7.48 (s, 1H,
H-8), 7.95-8.02 (m, 4H, ar), 8.32 (s, 1H, ar), 12.28 (br s, 1H,
NH). IR: 1702, 1750, 1777, 1813, 3210. Anal. (C18H10ClN3O6) C,
H, N.
7-Chloro-6-(2-carboxybenzoylamino)-3-hydroxy-1H-quinazo-
line-2,4-dione (12). A suspension of derivatives 26 (0.9 mmol) in
1% aqueous NaOH (15 mL) was stirred at room temperature for
about 30 min. The solution was acidified to pH 1 with 6 N HCl,
and the resulting solid was collected, washed with water, and
recrystallized. Yield: 85%; mp > 300 °C (EtOH). 1H NMR: 7.31
(s, 1H, H-8), 7.57-7.70 (m, 3H, ar), 7.91 (d, 1H, ar, J ) 7.6 Hz),
8.23 (s, 1H, H-5), 10.12 (s, 1H, exchangeable with D2O), 10.68 (s,
1H, exchangeable with D2O), 11.65 (s, 1H, exchangeable with D2O),
13.18 (s, 1H, exchangeable with D2O). IR: 1619, 1688, 1733,
2400-3600. Anal. (C16H10ClN3O6) C, H, N.
1
Compound 30. Yield: 40%; mp > 300 °C (EtOH). H NMR:
2.41 (s, 3H, COCH3), 3.92 (s, 3H, COOCH3), 7.41 (s, 1H, H-8),
7.72 (t, 1H, ar, J ) 8.0 Hz), 8.11 (s, 1H, H-5), 8.19 (d, 1H, ar, J
) 8.0 Hz), 8.26 (d, 1H, ar, J ) 8.0 Hz), 8.58 (s, 1H, ar), 10.51 (s,
1H, NH), 12.11 (s, 1H, NH). Anal. (C19H14ClN3O7) C, H, N.
3-Acetoxy-6-(3-carboxypropanoylamino)-7-chloro-1H-quinazo-
line-2,4-dione (27). A mixture of the 6-amino derivative 21 (1.3
mmol), succinic anhydride (1.3 mmol), and sodium acetate (1.3
mmol) in glacial acetic acid (5 mL) was heated at 60° C for about
6 h. After cooling at room temperature, the suspension was diluted
with water (30 mL) and acidified to pH 1 with 6 N HCl. The solid
was collected, washed with water, and recrystallized. Yield: 50%;
1
Compound 31. Yield: 70%; mp > 300 °C (EtOH). H NMR:
2.41 (s, 3H, COCH3), 3.91 (s, 3H, COOCH3), 7.40 (s, 1H, H-8),
8.10-8.15 (m, 5H, 4 ar + H-5), 10.47 (s, 1H, NH), 12.12 (s, 1H,
NH). Anal. (C19H14ClN3O7) C, H, N.
General Procedure to Prepare 6-Benzoylamino-7-chloro-3-
hydroxy-1H-quinazoline-2,4-dione (15), 6-(3-Carboxybenzoy-
lamino)-7-chloro-3-hydroxy-1H-quinazoline-2,4-dione (16), and
6-(4-Carboxybenzoylamino)-7-chloro-3-hydroxy-1H-quinazoline-
2,4-dione (17). A solution of the 3-acetoxy derivative 29, 30, or
31 (0.8 mmol) in aqueous 2% NaOH (10 mL) was stirred at room
temperature for 45 min and then acidified to pH 1 with 6 N HCl.
The resulting solid was collected, washed with water, and recrystal-
lized. Compounds 15 and 17 were recrystallized, while compound
16 was purified by acid/base exchange as follows: the crude product
was dissolved in aqueous 0.4% NaOH (10 mL), and the solution
was acidified to pH 1 with 6 N HCl to yield a solid that was
collected by filtration.
1
mp 223-225 °C (EtOAc). H NMR: 2.38 (s, 3H, CH3), 2.48-
2.63 (m, 4H, 2CH2), 7.30 (s, 1H, H-8), 8.22 (s, 1H, H-5), 9.72 (s,
1H, COOH), 12.00 (s, 1H, NH), 12.15 (s, 1H, NH). Anal. (C14H12-
ClN3O7) C, H, N.
7-Chloro-6-(3-carboxypropanoylamino)-3-hydroxy-1H-quinazo-
line-2,4-dione (13). The title compound was synthesized from
derivative 27 (0.9 mmol), as described above to prepare 12 from
1
26. Yield: 80%; mp 286-287 °C (EtOH). H NMR: 2.47-2.61
(m, 4H, al), 7.24 (s, 1H, ar), 8.17 (s, 1H, ar), 9.67 (s, 1H, COOH),
10.65 (br s, 1H, OH), 11.80 (br s, 2H, 2NH). Anal. (C12H10ClN3O6)
C, H, N.
1
Compound 15. Yield: 60%; mp > 300 °C (EtOH). H NMR:
3-Acetoxy-7-chloro-6-(2,5-dioxo-pyrrolidin-1-yl)-1H-quinazo-
line-2,4-dione (28). Sodium acetate (1.2 mmol) was added to a
suspension of compound 27 (0.7 mmol) in acetic anhydride (8 mL).
The mixture was refluxed for 2 h, the excess of acetic anhydride
was evaporated at reduced pressure, and the residue was taken up
with water (20 mL). The suspension was acidified to pH 1 with a
few drops of 6 N HCl, and the solid was collected, washed with
7.31 (s, 1H, ar), 7.50-7.62 (m, 3H, ar), 7.98 (d, 2H, ar, J ) 7.0
Hz), 8.07 (s, 1H, ar), 10.21 (s, 1H, exchangeable with D2O), 10.72
(br s, 1H, exchangeable with D2O), 11.63 (br s, 1H, exchangeable
with D2O). IR: 1680, 1740, 3060-3600. Anal. (C15H10ClN3O4)
C, H, N.
Compound 16. Yield: 87%; mp > 300 °C. 1H NMR: 7.34 (s,
1H, H-8), 7.65-7.70 (m, 1H, ar), 8.07 (s, 1H, H-5), 8.17-8.21
(m, 2H, ar), 8.57 (s, 1H, ar), 10.42 (s, 1H, exchangeable with D2O),
10.69 (s, 1H, exchangeable with D2O), 11.68 (s, 1H, exchangeable
with D2O), 13.27 (br s, 1H, exchangeable with D2O). IR: 1624,
1685, 1730, 2400-3600. Anal. Calcd for C16H10ClN3O6: C, 51.14;
H, 2.69; N, 11.19. Found: C, 51.64; H, 2.24; N, 11.61.
1
water, and recrystallized. Yield: 82%; mp > 300 °C (EtOH). H
NMR: 2.39 (s, 3H, CH3), 2.82-2.84 (m, 4H, 2CH2), 7.40 (s, 1H,
H-8), 8.06 (s, 1H, H-5), 12.2 (br s, 1H, NH). Anal. (C14H10ClN3O6)
C, H, N.
7-Chloro-3-hydroxy-6-(2,5-dioxo-pyrrolidin-1-yl)-1H-quinazo-
line-2,4-dione (14). The title compound was prepared from the
corresponding 3-acetoxy derivative 28 (0.3 mmol) as described
above to prepare derivative 9 from 24. Yield: 70%; mp > 300 °C
1
Compound 17. Yield: 74%; mp > 300 °C (EtOH). H NMR:
7.34 (s, 1H, H-8), 8.01-8.08 (m, 5H, 4 ar + H-5), 10.37 (s, 1H,
exchangeable with D2O), 10.70 (s, 1H, exchangeable with D2O),
11.69 (s, 1H, exchangeable with D2O), 13.27 (br s, 1H, exchange-
1
(EtOH). H NMR: 2.78-2.84 (m, 4H, 2CH2), 7.34 (s, 1H, H-8),

6024 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Colotta et al.
able with D2O). IR: 1625, 1667,1748, 2400-3600. Anal. (C16H10-
ClN3O6) C, H, N.
Molecular Modeling. Compounds were modeled using the
LigPrep Schrodinger ligands preparation procedure (pH 7.4) and
minimized with Macromodel8.562
General Procedure to Prepare 7-Chloro-3-hydroxy-6-[(3-
methoxycarbonyl)benzoylamino]-1H-quinazoline-2,4-dione (18)
and 7-Chloro-3-hydroxy-6-[(4-methoxycarbonyl)benzoylamino]-
1H-quinazoline-2,4-dione (19). The title compounds were prepared
from the corresponding 3-acetoxy derivatives 30 and 31 (0.7 mmol),
as described above, to obtain compound 9 from 24.
The experimental crystal structure of GluR2S1S2J/ATPO com-
plex (PDB entry: 1n0t) and a homology-built model of GluR5/
ATPO were used in the docking computation. Docking calculations
were performed using the Glide program (FirstDiscovery v3.0,
Schro¨dinger)63 on an AMD Athlon 2800 processor running Linux.
The crystal structure was prepared according to the protein
preparation procedure recommended. Default input parameters were
used in all computations (no scaling factor for the vdW radii of
nonpolar protein atoms, 0.8 scaling factor for nonpolar ligand
atoms). Upon completion of each docking calculation, three poses
per ligand were saved. The best-docked structure was chosen using
a model energy score (Emodel) derived from a combination of the
glide score (Gscore, a modified and extended version of the
empirically based ChemScore function64), coulombic, and the van
der Waals energies and the internal strain energy of the ligands.63
Molecular docking of all the compounds on the Gly/NMDA
receptor was carried out according to guidelines outlined in ref 42.
A comparative model of GluR5/ATPO was built by means of
the Prime Protein Structure Prediction Suite (Schrodinger)65 that
consists of the prime-structure prediction and prime refinement
options. The GluR5/glutamate (1ycj, chain A) was used as query
sequence, and both GluR2/ATPO (1n0t, chain A) and GluR2/
DNQX (1ftl, chain A) were used as template sequences. This made
an alignment based both on sequence and on secondary structure
information possible (prime align tool). The GluR5 3D model
structure was obtained through the build structure model tool, and
structural analysis of the homology-built model was carried out
with both the prime refinement tool program ProSa200366 and
Whatcheck.67
1
Compound 18. Yield: 74%; mp > 300 °C (DMF). H NMR:
3.92 (s, 3H, CH3), 7.32 (s, 1H, H-8), 7.72 (t, 1H, ar, J ) 8.0 Hz),
8.04 (s, 1H, H-5), 8.20 (d, 1H, ar, J ) 8 Hz), 8.24 (d, 1H, ar, J )
8.0 Hz), 8.58 (s, 1H, ar), 10.31 (br s, 1H, exchangeable with D2O),
11.02 (br s, 2H, exchangeable with D2O). Anal. (C17H12ClN3O6)
C, H, N.
Compound 19. Yield: 58%; mp > 300 °C (2-methoxyethanol).
1H NMR: 3.91 (s, 3H, CH3), 7.32 (s, 1H, H-8), 8.05 (s, 1H, H-5),
8.11 (s, 4H, ar), 10.42 (br s, 1H, exchangeable with D2O), 11.00
(br s, 2H, exchangeable with D2O). Anal. (C17H12ClN3O6) C, H,
N.
3-Acetoxy-6-(3-benzylureido)-7-chloro-1H-quinazoline-2,4-di-
one (32). A mixture of the 6-amino derivative 2140 (1.1 mmol)
and benzylisocyanate (1.7 mmol) in anhydrous tetrahydrofuran (15
mL) was refluxed for 5 h under a nitrogen atmosphere. The
suspension was cooled at room temperature and diluted with water
(40 mL). The solid was collected, washed with water, and
recrystallized. Yield: 90%; mp > 300 °C (EtOH). 1H NMR: 2.40
(s, 3H, CH3), 4.33 (d, 2H, CH2), 7.25-7.48 (m, 7H, ar), 8.29 (s,
1H, NH), 8.72 (s, 1H, NH), 11.88 (s, 1H, NH). IR: 1680, 1748,
1814, 3270. Anal. (C18H15ClN4O5) C, H, N.
6-(3-Benzylureido)-7-chloro-3-hydroxy-1H-quinazoline-2,4-di-
one (20). The title compound was prepared from the corresponding
3-acetoxy derivative 32 (0.7 mmol) in the conditions described
above to obtain 12 from 26. Yield: 80%; mp > 300 °C (MeOH).
1H NMR: 4.33 (s, 2H, CH2), 7.33-7.42 (m, 7H, ar), 8.21 (s, 1H,
NH), 8.69 (s, 1H, NH), 10.59 (s, 1H, OH), 11.45 (s, 1H, NH). IR:
1677, 1746, 3100-3500. Anal. (C16H13ClN4O4) C, H, N.
Pharmacology. Binding Assays. Rat cortical synaptic membrane
preparation and [3H]glycine, [3H]AMPA binding experiments were
performed following the procedure reported in refs 34 and 57,
respectively. The high-affinity and low-affinity [3H]kainate binding
assays were performed on rat cortical membranes according to
previously described methods.41
Electrophysiological Assays. The mouse cortical wedge prepa-
ration described by Mannaioni et al.58 was used, while the
electrophysiological assays were performed following the proce-
dures described in ref 38.
Sample Preparation and Result Calculation. A stock 1 mM
solution of the tested compound was prepared in 50% DMSO, and
the subsequent dilution was 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.59
Pharmacological Assays. Abdominal Constriction Test. Mice
were injected ip with a 0.6% solution of acetic acid (10 mL kg-1),
according to Koster et al.60 The number of stretching movements
was counted for 10 min, starting 5 min after acetic acid injection.
Rota-Rod Test. The apparatus consisted of a base platform and
a rotating rod of 3-cm diameter, with a nonslippery surface. The
rod was placed at a height of 15 cm from the base. The rod, 30 cm
in length, was divided into five equal sections by six disks. Thus,
up to five mice were tested simultaneously on the apparatus, with
a rod-rotating speed of 16 rpm. The integrity of motor coordination
was assessed on the basis of the number of falls from the rod in 30
s, according to Vaught et al.61 The performance time was measured
before treatment and 30, 45, and 60 min after treatment.
Administration of Drug. Compound 12 was dispersed in sodium
carboxymethylcellulose (CMC) 1% immediately before use. Drug
concentrations were prepared in such a way that the necessary dose
could be administered in a volume of 10 mL kg-1 by per os (po)
route 30 min before tests.
Supporting Information Available: Combustion analysis data
of the newly synthesized compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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