Novel AMPA and kainate receptor antagonists containing the pyrazolo[1,5-c]quinazoline ring system: Synthesis and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2007.11.046

This paper reports the synthesis and AMPA, Gly/NMDA, and KA receptor binding affinities of a new set of 1,9-disubstituted-8-chloro-pyrazolo[1,5-c]quinazoline-2-carboxylates 2-34. Binding data show that, in general, compounds 2-34 bind to the AMPA receptor with good affinity and selectivity. In particular, the obtained results indicate that the contemporary presence of a 1,2-dicarboxylic acid moiety and suitable benzo-substituents on the PQZ system is important to gain selective AMPA receptor antagonists. Moreover, this study shows that the presence of a 2-carboxybenzoylamino substituent at position-9 (compounds 33-34) is important for obtaining selective KA receptor antagonists. Some selected compounds were also tested for their functional antagonistic activity at both AMPA and NMDA receptor-ion channels.

Full text of DOI:10.1016/j.bmc.2007.11.046

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2617–2626
Novel AMPA and kainate receptor antagonists containing
the pyrazolo[1,5-c]quinazoline ring system: Synthesis
and structure–activity relationships
Flavia Varano,a,* Daniela Catarzi,a Vittoria Colotta,a Ombretta Lenzi,a
Guido Filacchioni,a Alessandro Gallib and Chiara Costaglib
aDipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente Attivi,
`
Universita degli Studi di Firenze, Polo Scientifico, via Ugo Schiff, 6, 50019 Sesto Fiorentino (FI), Italy
`
Dipartimento di Farmacologia Preclinica e Clinica, Universita degli Studi di Firenze, viale G. Pieraccini, 6, 50134 Firenze, Italy
b
Received 15 May 2007; revised 12 November 2007; accepted 16 November 2007
Available online 22 November 2007
Abstract—This paper reports the synthesis and AMPA, Gly/NMDA, and KA receptor binding affinities of a new set of 1,9-disub-
stituted-8-chloro-pyrazolo[1,5-c]quinazoline-2-carboxylates 2–34. Binding data show that, in general, compounds 2–34 bind to the
AMPA receptor with good affinity and selectivity. In particular, the obtained results indicate that the contemporary presence of a
1,2-dicarboxylic acid moiety and suitable benzo-substituents on the PQZ system is important to gain selective AMPA receptor
antagonists. Moreover, this study shows that the presence of a 2-carboxybenzoylamino substituent at position-9 (compounds
33–34) is important for obtaining selective KA receptor antagonists. Some selected compounds were also tested for their functional
antagonistic activity at both AMPA and NMDA receptor-ion channels.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
such as hypoxia/ischemia, brain damage,4 epilepsy,1
Parkinson’s5 and Alzheimer’s diseases,6 and multiple
sclerosis.7 Accordingly, there is great interest for com-
petitive iGluR antagonists as well as for non-competi-
tive antagonists acting at the Gly/NMDA receptor for
their potential therapeutic use in the above mentioned
pathologies.1,8,9
Glutamic acid (Glu) is the major excitatory neurotrans-
mitter in the central nervous system (CNS). The effects
of Glu are mediated by ionotropic (iGluRs) and metab-
otropic receptors (mGluRs).1
The iGluRs have been divided into three main groups:
NMDA (N-methyl-D-aspartic acid), AMPA (2-amino-3-
(3-hydroxy-5-methylisoxazol-4-yl)-propionic acid), and
KA (kainic acid) receptors, based on their pharmacology,
and more recently on their amino acid sequence homol-
ogy.1,2 The NMDA receptor complex possesses different
binding sites including the glycine coagonist receptor
(Gly/NMDA).3
Availability of selective Gly/NMDA and AMPA recep-
tor agonists and antagonists has made it possible to
clearly establish both the physiological and pathophysi-
ological role of Gly/NMDA and AMPA receptors.1,8,9
In contrast, knowledge about the role of KA receptors
is still in its early stages since only recently were some
selective KA receptor antagonists reported.10–15
It is generally agreed that iGluRs are implicated in a
variety of CNS functions including learning and mem-
ory. However, over-stimulation of postsynaptic iGluRs
is known to worsen neuro-degeneration in conditions
In the last few years in our laboratory we prepared two
series of 5,6-dihydro-pyrazolo[1,5-c]quinazoline-2-car-
boxylate (PQZ), 1A and 1B (Chart 1), as Gly/NMDA
and/or AMPA receptor antagonists.16,17
Structure–activity relationship (SAR) studies on series
1A have highlighted that the contemporary presence of
a chlorine atom and a nitrogen-containing heterocycle
at position-8 and -9, respectively, shifted affinity and
Keywords: Ionotropic glutamate receptors; AMPA receptor antago-
nists; KA receptor antagonists; Pyrazolo[1,5-c]quinazolines.
*
Corresponding author. Tel.: +39 55 4573721; fax: +39 55 4573780;
e-mail: flavia.varano@unifi.it
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.046

2618
F. Varano et al. / Bioorg. Med. Chem. 16 (2008) 2617–2626
COOR2
ring of our 7-chloro-3-hydroxy-quinazoline-2,4-dione
scaffold afforded a selective KA receptor antagonist.15
COOR2
N
R1
1
2
10
7
N
9
R9
Cl
R9
Cl
N
N
5
8
N
O
N
O
2. Chemistry
H
H
1A
1B
Compounds 2–34 were synthesized as depicted in
Schemes 1–4. Briefly, the 1-chloro-9-nitro-2-ethyl ester
221 (Scheme 1) was obtained by chlorination of the cor-
responding 1-unsubstituted-9-nitro ester 35,16 while the
1,9-dinitro-2-ethyl ester 3 was prepared by nitration of
the known ethyl 8-chloro-5,6-dihydro-5-oxo-pyrazol-
o[1,5-c]quinazoline-2-carboxylate 36.16
R2 = H, Et
R9 = H, Cl, NO2
heterocycles
R1 = COOEt, COOH,
Cl, Br, CH3
R9 = H, Cl
Chart 1. Previously reported PQZ derivatives.
selectivity of PQZ derivatives toward the AMPA recep-
tor.16 Furthermore, even if PQZ derivatives show lower
affinities for the KA receptor than for the AMPA one,
the SARs of these two receptors are comparable, thus
confirming the high structural similarity of their binding
pockets.16
COOEt
N
R1
COOEt
N
O2N
Cl
R9
Cl
N
N
O
N
O
N
H
H
a
b
2 R1 = Cl
3 R1 = NO2
35 R9 = NO2
36 R9 = H
Further studies on PQZ derivatives, also supported by
molecular modeling investigations, pointed out that
the contemporary presence of substituents on the fused
benzo ring and at position-1 seems to affect both po-
tency and receptor selectivity (series 1B).17
Scheme 1. Reagents and conditions: (a) SO2Cl2, glacial AcOH, I2,
50 °C; (b) 90% HNO3, 0 °C to room temperature.
As a continuation of our research, we report in the pres-
ent paper the synthesis of two new sets of 1,9-disubsti-
tuted-8-chloro-pyrazolo[1,5-c]quinazoline-2-carboxylates
2–13 and 14–34 (Chart 2) designed as AMPA and/or
KA receptor antagonists.
COOEt
EtOOC
COOEt
EtOOC
a
O2N
Cl
N
N
b
N
N
O
N
O
N
Cl
H
H
37
4
Compounds 2–13 were synthesized in order to identify
the best R1 group to enhance affinity and selectivity to-
ward the AMPA receptor. In fact, derivatives 2–13 bear
diverse substituents at position-1 (Cl, NO2, COOEt,
COOH) and at position-9 display substituents (NO2,
heterocycles) which are well known to enhance AMPA
receptor affinity and selectivity in many classes of
AMPA receptor antagonists.8,16,18–20 Compounds 14–
32 were designed holding the 1-carboxylate function
constant and by introducing at position-9 some ureido
moieties which we did not investigate in our previously
reported PQZ derivatives. Finally, the synthesis of the
9-(2-carboxybenzoylamino) derivatives 33 and 34,
thought to be selective KA receptor antagonists, was
performed. In fact, the 2-carboxybenzoylamino substi-
tuent introduced in a similar position on the fused benzo
COOEt
N
EtOOC
COOEt
EtOOC
R9
Cl
H2N
Cl
N
N
c/d/e/f
N
O
N
O
N
H
H
38
5-7, 14-23
n°
5
n°
R9
R9
N
NHCONHC6H5
18
N
N
N
N
NHCONHCH2C6H5
6
7
19
20
NHCONHCH2CH2C6H5
CHO
COOR2
R1
COOR2
N
NHCONHCH2COOEt
NHCONH(3-COOEtC6H4)
NHCONH(4-COOEtC6H4)
NHCONHCH(COOMe)CH2C6H5
R1OOC
14
15
16
17
21
22
23
N
O
NHCONHCH2CH2COOEt
NHCONHCH2CH2CH2COOEt
NHCONHCH2CH3
R9
Cl
R9
Cl
N
N
N
N
O
H
R2 = Et, H
H
R1 = Et, H
R9 = NHCONHR
R1 = Cl, NO2, COOEt
COOH
R9 = NO2, heterocycles
14-32
33-34
Scheme 2. Reagents and conditions: (a) 90% HNO3, 0 °C; (b) iron
powder, glacial AcOH, reflux; (c) diformylhydrazine, Me3SiCl, Et3N,
pyridine, reflux; (d) 2,5-diethoxytetrahydrofuran, glacial AcOH, 90 °C;
(e) 2,5-dimethoxy-3-tetrahydrofurancarboxyaldehyde, glacial AcOH,
90 °C; (f) RNCO, tetrahydrofuran, 70 °C.
2-13
R1 = Et, H
R9 = NHCO-(2-COOHC6H5)
Chart 2. Currently reported PQZ derivatives.

F. Varano et al. / Bioorg. Med. Chem. 16 (2008) 2617–2626
2619
obtained. Reaction of 38 with an excess of 2,5-dime-
thoxy-3-tetrahydrofurancarboxaldehyde yielded the 9-
(3-formylpyrrol-1-yl) derivative 7. Finally, reaction of
the 9-amino 38 with an excess of suitable isocyanates
afforded the corresponding 9-ureido derivatives 14–23.
COOH
N
R1 COOEt
R1
R9
Cl
N
R9
Cl
a
N
N
O
O
N
N
H
H
2-6
8-13
Alkaline hydrolysis of the esters 2–6, 14–15, and 17–23
carried out at room temperature or at reflux, yielded
acids 8–1321 and 24–32 (Scheme 3).
entry
R9
NO2
NO2
NO2
NO2
R1
Cl
NO2
COOEt
COOEt
COOEt
method
n˚
8
9
10
11
12
R1
Cl
NO2
COOEt
COOH
COOH
2
3
4
4
5
A
A
B
C
C
Finally, reaction of the 9-amino derivative 38 with
phthalic anhydride afforded the 9-(2-carboxybenzoyla-
mino)-1,2-diethyl ester 33 that yielded the correspond-
ing 1,2-dicarboxylic acid 34 by alkaline hydrolysis
(Scheme 4).
N
N
N
N
6
COOEt
D
13
COOH
COOH
N
COOEt
HOOC
EtOOC
H
N
H
N
H
H
N
3. Results and discussion
N
O
N
R9
R9
N
N
b
O
O
The pyrazoloquinazolines 2–34 were evaluated for their
binding at AMPA, Gly/NMDA, and KA receptors and
the results are listed in Tables 1–3. In Table 1 the bind-
ing data of the previously reported 1-unsubstituted
derivative 1a are also included as references.
O
Cl
Cl
N
H
24-32
N
H
14-15, 17-23
entry
14
15
17
18
19
20
21
22
R9
n˚
R9
CH2COOH
CH2CH2COOH
CH2CH3
CH2COOEt
CH2CH2COOEt
CH2CH3
24
25
26
27
28
29
30
31
Compounds 2–13 (Table 1) were synthesized in order to
identify the best R1 substituent to enhance affinity and/
or selectivity toward the AMPA receptor.
C6H5
CH2C6H5
C6H5
CH2C6H5
CH2CH2C6H5
3-COOEtC6H4
4-COOEtHC6H4
CH2CH2C6H5
3-COOHC6H4
4-COOHC6H4
To reach our goal, we first synthesized the 9-nitro-2-
ethyl esters 2–4 and their corresponding 2-carboxylic
acids 8–11 bearing diverse substituents (Cl, NO2, COO-
Et, COOH) at position-1. The binding data show that
acids 8–11 are more active toward the AMPA and
Gly/NMDA receptors than their corresponding esters
2–4. These results are in accordance with our previously
reported molecular docking studies which showed
that the presence of a carboxylic acid group at
position-2 of the PQZ system is important for the bind-
ing to AMPA and Gly/NMDA receptors due to an
electrostatic interaction between the negatively
charged carboxylate group and a cationic site on the
receptors.17
23
CH(COOMe)CH2C6H5 32 CH(COOH)CH2C6H5
Scheme 3. Reagents and conditions: (a) Method A: i—10% KOH,
MeOH, reflux; ii—6 N HCl; Method B: i—10% KOH, MeOH, room
temperature; ii—12 N HCl; Method C: i—10% KOH, EtOH, reflux;
ii—12 N HCl; Method D: i—1 N NaOH, room temperature; ii—6 N
HCl; (b) Method E: i—10% NaOH, EtOH, reflux; ii—6 N HCl.
COOR2
N
R1OOC
COOH
HN
a
N
38
O
Cl
N
O
H
Among the 2 carboxylic acids 8–11, the highest AMPA
receptor affinity appears when a carboxylic acid group is
present at position-1 (see compound 11). This could be
rationalized on the basis of our previous docking inves-
tigations17 which clearly indicated that the AMPA site
environment around the R1 position is a polar region
which better accommodates hydrophilic groups like car-
boxylic acids rather than lipophilic moieties like a chlo-
rine atom, a nitro group, or an ethyl carboxylate group
(compare 11 with 8–10).
33 R1=R2=Et
34 R1=R2=H
b
Scheme 4. Reagents and conditions: (a) phthalic anhydride, NaOAc,
glacial AcOH, 60 °C; (b) i—1 N NaOH, room temperature; ii—6 N
HCl.
In Scheme 2, the synthesis of the ethyl 1,2-dicarboxyl-
ates 4–7 and 14–23 is reported.
Replacement of the 9-nitro group of 1,2-diethyl ester 4
and of its corresponding acid 11 with heterocycle moie-
ties yields esters 5–7 and acids 12–13. The positive effect
exerted by the presence of a 9-heterocycle group is
clearly highlighted by the ameliorated AMPA receptor
binding affinity of esters 5–7 with respect to their parent
compound 4. In particular, the 9-(1,2,4-triazol-4-yl)
derivative 5 shows a micromolar AMPA receptor affin-
Allowing the known ethyl 8-chloro-1,2-dicarboxylate
3717 to react with 90% nitric acid, the corresponding
9-nitro derivative 4 was obtained, which was reduced
to afford the corresponding 9-amino derivative 38. By
reacting the latter either with 1,2-diformylhydrazine or
2,5-diethoxytetrahydrofuran, the 9-(1,2,4-triazol-4-yl)
ester 5 and the 9-(pyrrol-1-yl) ester 6, respectively, were

2620
F. Varano et al. / Bioorg. Med. Chem. 16 (2008) 2617–2626
Table 1. Binding affinity at AMPA, Gly/NMDA, and KA receptors
R2
N
R1
R9
Cl
N
O
N
H
1a, 2-13
Kia (lM) or I%b
AMPA versus Gly/NMDA
selectivity ratio
IC50 (lM) or I%b
c
Compound
R1
R2
R9
[3H]AMPA
[3H]glycine
[3H]KA
N
N
1ad
H
COOH
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOH
COOH
COOH
COOH
COOH
COOH
N
0.14 0.02
8.3 2.0
59.3
>1.5
0.4
5.1 1.4
2
Cl
NO2
NO2
63
7
45%
25%
16%
0%
3
NO2
45%
40%
36.2 3.1
30%
4
COOEt
COOEt
COOEt
COOEt
Cl
NO2
N
N
5
N
1.2 0.2
18%
>80
>2
82
9
6
48
6
13%
12%
17%
94
N
OHC
7
15.7 2.1
1.3 0.1
20%
>6
N
8
NO2
NO2
NO2
2.5 0.1
20.5 6.0
4.7 0.4
1.25 0.25
43%
1.9
1.7
5
7
5
9
NO2
11.9 3.5
0.94 0.07
0.59 0.11
0.1 0.02
0.13 0.03
29%
60
10
11
12
13
COOEt
COOH
COOH
COOH
NO2
2
11.7 2.1
2.4 0.7
4.5 0.3
N
N
>1000
153.8
N
20
3
N
a Ki values are means SEM of three or four separate determinations in triplicate.
b Percentage of inhibition (I%) of specific binding at 100 lM concentration.
c IC50 values are means SEM of three or four separate determinations in triplicate.
d Ref. 16.
ity and also a notable AMPA versus Gly/NMDA recep-
tor selectivity.
4-triazol-4-yl) derivative 12 and the 9-(pyrrol-1-yl)
derivative 13 are equipotent to the reference compound
1a16 at the AMPA receptor, but they are much more
AMPA versus Gly/NMDA selective. Indeed, com-
pound 12 (Ki = 0.1 lM; selectivity ratio > 1000) shows
the highest affinity and AMPA versus Gly/NMDA
receptor selectivity among PQZ derivatives reported
till now.
The binding data of acids 12–13 are even more interest-
ing. They show somewhat improved AMPA receptor
affinities with respect to the parent acid 11 and a con-
siderable increase in AMPA versus Gly/NMDA recep-
tor selectivity. It is worth noting that the 9-(1,2,

F. Varano et al. / Bioorg. Med. Chem. 16 (2008) 2617–2626
2621
Table 2. Functional antagonism at AMPA and NMDA sites
tor-ligand interaction. In fact, while the 2-ethyl esters 2–
7 are inactive or scarcely active, the corresponding acids
8–13 display some KA receptor affinity, the only excep-
tion being compound 9.
a
Compound
IC50 (lM)
AMPA
NMDA
5
5.8 0.7
2.5 0.3
0.5 0.07
>>20
>10
20 2.0
12
1ab
Moreover, the presence of a carboxylic acid group at po-
sition-1 is advantageous for the binding to KA receptor.
Indeed, among the 2 carboxylic acids 8–13, the 1,2-
dicarboxylic acids 11–13 are endowed with the highest
KA receptor affinity.
a Concentration necessary for 50% inhibition (IC50) of depolarization
induced by S-AMPA or NMDA in mouse cortical wedge prepara-
tion. IC50 values are means SEM of three separate determinations.
b Ref. 16.
Thus, keeping the 1,2-dicarboxylate substitution pattern
constant, we synthesized a new set of PQZ derivatives
14–32 (Table 3) bearing at position-9 diverse ureido
moieties which were not investigated in our previously
reported AMPA/KA receptor antagonists of similar size
and shape.16–20
Compounds 5 and 12 were tested to evaluate their
antagonistic activity by assessing their ability to inhibit
depolarization induced by 5 lM AMPA and NMDA
in cortical wedge preparations.19 The results obtained
from these functional antagonism studies are reported
in Table 2. Compounds 5 and 12 inhibit AMPA and
NMDA response in a reversible manner and their inhib-
itory activities are in agreement with their binding
affinities.
Interestingly, the results indicate that 14–32 are in gen-
eral more active toward the AMPA receptor than to-
ward the Gly/NMDA one. Indeed, 1,2-dicarboxylic
acids 24–32 show submicromolar AMPA receptor bind-
ing activities and noteworthy AMPA versus Gly/
NMDA receptor selectivities, compound 25 being the
most active and selective (Ki = 0.16 lM; selectivity
ratio = 425).
Regarding KA receptor binding activities of 2–13, the
data reported in Table 1 show that the presence of a car-
boxylic acid group at position-2 is important for recep-
Table 3. Binding affinity at AMPA, Gly/NMDA, and KA receptors
R2
N
R2
N
R1
R1
H
N
H
N
H
N
R9
R9
N
N
O
O
Cl
N
H
O
Cl
N
O
H
33-34
14-32
c
Compound R1
R2
R9
Kia (lM) or I%b
[3H]AMPA [3H]glycine
AMPA versus Gly/NMDA IC50 (lM) or I%b
selectivity ratio
[3H]KA
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
COOEt COOEt CH2COOEt
8.9 2.1
3.8 0.5
3.5 0.4
43%
32%
>11
>26
28
19%
32%
COOEt COOEt CH2CH2COOEt
COOEt COOEt CH2CH2CH2COOEt
COOEt COOEt CH2CH3
99 9.0
44%
11%
24%
13%
27
4
>3.7
—
COOEt COOEt C6H5
COOEt COOEt CH2C6H5
20%
44%
44%
18%
5%
0%
0%
19%
49
—
COOEt COOEt CH2CH2C6H5
COOEt COOEt 3-COOEtC6H4
COOEt COOEt 4-COOEtC6H4
4
0.49
—
9%
0%
0%
5%
16%
54
—
—
15%
16%
COOEt COOEt CH(COOMe)CH2C6H5 46%
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
CH2COOH
0.48 0.02
0.16 0.04
0.56 0.03
0.14 0.02
0.37 0.04
0.48 0.06
0.42 0.04
1.1 0.2
6
112.5
425
73.3
83.5
44
4.4 0.08
4.6 0.2
9.8 1.1
CH2CH2COOH
CH2CH3
C6H5
68 7.0
41
4
11.7 2.5
16.3 2.7
11
11
17
3
1
2
CH2C6H5
CH2CH2C6H5
3-COOHC6H4
4-COOHC6H4
CH(COOH)CH2C6H5
31
35
49
61
36
27
4
3
6
6
4
3
31
83
2
0.2
44
13 2.0
40
0.47 0.06
2.8 0.4
1.3 0.1
129
12.8
20.7
5
COOEt COOEt 2-COOHC6H4
COOH COOH 2-COOHC6H4
0.11 0.02
0.52 0.09
a Ki values are means SEM of three or four separate determinations in triplicate.
b Percentage of inhibition (I%) of specific binding at 100 lM concentration.
c IC50 values are means SEM of three or four separate determinations in triplicate.

2622
F. Varano et al. / Bioorg. Med. Chem. 16 (2008) 2617–2626
In particular, acids 24–32 are more active than their cor-
responding esters 14–23, indicating that the 1,2-dicar-
boxylic acid moiety is of paramount importance for
anchoring to the AMPA receptor.
in the submicromolar range (33: IC50 = 0.11 lM; 34:
IC50 = 0.52 lM), they are the most active compounds
among the PQZs reported till now. Moreover, it is
worth noting that compound 33 shows KA receptor
selectivity both versus Gly/NMDA and, most impor-
tantly, versus AMPA receptors.
Since acids 24–32 show comparable and good AMPA
receptor binding activities, we can deduce that R9 sub-
stituents with different electronic and steric properties
are similarly well tolerated by the receptor site. In fact,
AMPA receptor affinities of 24 and 25, which bear a
hydrophilic R9 carboxyalkyl chain on the 9-ureido,
match those of 28, 29, and 27, respectively, which bear
lipophilic and bulky R9 ureido substituents.
4. Conclusions
The present study has produced new 1,9-disubstituted-8-
chloro-pyrazolo[1,5-c]quinazolines which are endowed
with good affinity and improved selectivity toward the
AMPA receptor. As a new result, SAR studies have
pointed out that the contemporary presence of 1,2-
dicarboxylic acid moiety and suitable benzo substituents
is important to enhance AMPA receptor selectivity. In
particular, among PQZ derivatives reported till now,
the 9-(1,2,4-triazol-4-yl)-1,2-dicarboxylic acid derivative
12 shows the highest affinity (Ki = 0.1 lM) and AMPA
versus Gly/NMDA receptor selectivity (selectivity ra-
tio > 1000). Moreover, the new 9-ureido-1,2-dicarbox-
ylic acid derivatives 24–32 display good AMPA
receptor affinities (0.14 lM 6 Ki 6 1.1 lM) and note-
worthy AMPA versus Gly/NMDA receptor selectivities.
Indeed, the 9-(carboxypropionic acid)-ureido derivative
25 shows high AMPA receptor affinity (Ki = 0.14 lM)
and improved AMPA versus Gly/NMDA receptor
selectivity (selectivity ratio = 425) when compared with
previously reported PQZ series.
Introduction of a carboxylic acid group in meta or para
on the 9-phenylureido moiety of 27 yields derivatives 30
and 31, respectively, which show comparable or slightly
reduced AMPA receptor affinities with respect to 27.
Moreover, introduction of a carboxylic acid group on
the ethyl chain of the 9-(phenylethyl)ureido derivative
29 afforded compound 32 which results equipotent to
29.
Concerning AMPA versus Gly/NMDA receptor selec-
tivity of 24–32, the results are difficult to rationalize
since all derivatives show comparable and good AMPA
receptor selectivity apart from the nature of the R9 sub-
stituent on the 9-ureido group. However, compounds
with AMPA versus Gly/NMDA receptor selectivity ra-
tio > 100 bear a R9 carboxy alkyl chain (see compounds
24, 25, and 32). These data lead us to hypothesize that a
R9 hydrophilic substituent, such as the carboxyalkyl
one, can shift selectivity toward the AMPA receptor. In-
deed, previous molecular modeling studies pointed out
the hydrophilic nature of the AMPA receptor binding
pocket which accommodates the R9 substituent.17
However, the most significant result is the finding that
the 2-carboxybenzoylamino substituent at the 9-position
of the tricyclic PQZ scaffold is able to shift affinity
and selectivity toward the KA receptor. Indeed, both
the 9-(2-carboxybenzoylamino) derivatives 33 (IC50
=
0.11 lM) and 34 (IC50 = 0.52 lM) display good KA
receptor affinity, and compound 33 also possesses KA
receptor selectivity both versus the Gly/NMDA and,
even more importantly, versus the AMPA receptors.
Analysing KA receptor binding activities of 9-ureides
14–32, the data reported in Table 3 show that the 1,2-
dicarboxylic acids 24–32 display affinity for the KA
receptor in the micromolar range with the only excep-
tion being compound 32. In contrast, their correspond-
ing esters 14–23 are inactive, suggesting the importance
of the 1,2-dicarboxylic acid residue for anchoring to the
KA receptor, and also indicating that ureido moieties,
with different steric and electronic properties, are simi-
larly tolerated by the receptor. As in the set of com-
pounds 2–13, the SARs at the KA receptor are similar
to those at the AMPA one, and none of the acids 24–
32 show KA versus AMPA receptor selectivity.
5. Experimental
5.1. Chemistry
Silica gel plates (Merck F254) and silica gel 60 (Merck;
70–230 mesh) were used for analytical and column chro-
matography, respectively. All melting points were deter-
mined on a Gallenkamp melting point apparatus.
Microanalyses were performed with a Perkin-Elmer
260 elemental analyzer for C, H, N, and the results were
within 0.4% of the theoretical values unless otherwise
stated. The IR spectra were recorded with a Perkin-El-
mer Spectrum RX I spectrometer in Nujol mulls and
However, while we were obtaining the pyrazoloquinazo-
lines 2–32, the 6-(2-carboxybenzoylamino)-3-hydroxy-
1H-quinazolin-2,4-dione was synthesized in our labora-
tory. This compound possesses good affinity for KA
receptor and also good selectivity not only versus the
Gly/NMDA receptor but also, and most importantly,
versus the AMPA one.15 On this basis, we synthesized
the 1,2-dicarboxylate derivatives 33 and 34 bearing the
2-carboxybenzoylamino group at position-9, which
resembles the 6 position of our quinazoline-dione lead.
As expected, 33 and 34 show very interesting phar-
macological profiles. In fact, with KA receptor affinities
1
are expressed in cmÀ1. The H NMR spectra were ob-
tained with a Varian Gemini 200. The chemical shifts
are reported in d (ppm) and are relative to the central
peak of the solvent, which is always DMSO-d6 unless
otherwise stated. All the exchangeable protons were
confirmed by addition of D2O. The following abbrevia-
tions are used: s, singlet; d ,doublet; dd, double doublet;
t, triplet; m, multiplet; br, broad; ar, aromatic protons;

F. Varano et al. / Bioorg. Med. Chem. 16 (2008) 2617–2626
2623
al, aliphatic protons. The physical and analytical data
of the newly synthesized compounds are shown in
Table 4.
5.1.2. Synthesis of ethyl 8-chloro-5,6-dihydro-1,9-dinitro-
5-oxo-pyrazolo[1,5-c]quinazoline-2-carboxylate (3). Ethyl
8-chloro-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-
2-carboxylate 3616 (1.03 mmol) was portionwise added
to cooled (0–5 °C) HNO3 (90%, 4 mL). The reaction
mixture was stirred at room temperature for 48 h. The
solution was poured onto ice and the resulting solid
5.1.1. Synthesis of ethyl 1,8-dichloro-5,6-dihydro-9-nitro-
5-oxo-pyrazolo[1,5-c]quinazoline-2-carboxylate (2).21
To a suspension of ethyl 8-chloro-5,6-dihydro-9-nitro
1
-5-oxo-pyrazolo[1,5-c]quinazoline-2-carboxylate
3516
was collected and washed with water. H NMR 1.34
(1.2 mmol) in glacial acetic acid (10 mL), sulfuryl chlo-
ride (2.4 mmol) and a crystal of iodine as catalyst were
added. The reaction mixture was heated at 60 °C for
3 h, and then cooled at room temperature. The solid
(t, 3H, al, J = 7.2 Hz), 4.46 (q, 2H, al, J = 7.2 Hz),
7.61 (s, 1H, ar), 9.32 (s, 1H, ar), 13.21 (br s, 1H, NH).
IR 1740, 3200, 3420.
1
was collected by filtration and washed with water. H
5.1.3. Synthesis of diethyl 8-chloro-5,6-dihydro-9-nitro-
5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylate (4).
Diethyl 8-chloro-5,6-dihydro-5-oxo-pyrazolo[1,5-c]qui-
nazoline-1,2-dicarboxylate 3717 (2.75 mmol) was por-
tionwise added to cooled (0–5 °C) HNO3 (90%,
10 mL). The reaction mixture was stirred at 0–5 °C for
20 min and then poured onto ice. The solid was collected
and washed with water. 1H NMR 1.30–1.36 (m, 6H, al),
4.36–4.44 (m, 4H, al), 7.58 (s, 1H, ar), 9.64 (s, 1H, ar),
12.92 (br s, 1H, NH).
NMR 1.36 (t, 3H, al, J = 7.3 Hz), 4.40 (q, 2H, al,
J = 7.3 Hz), 7.54 (s, 1H, ar), 9.07 (s, 1H, ar), 12.77 (br
s, 1H, NH).
Table 4. Physical and analytical data of compounds 2–34 and 38
Compound
Mp (°C)
Solv.a
Yield (%)
C, H, N
2
292–295
191–194
294–296
>300
A
B
70
75
90
80
80
50
85
70
60
80
80
55
65
50
55
60
75
50
50
70
78
60
60
70
70
65
50
45
90
65
55
70
60
70
C13H8Cl2N4O5
3
C13H8ClN5O7
C16H13ClN4O7
C18H15ClN6O5
C20H17ClN4O5
C21H17ClN4O6
C11H4Cl2N4O5
C11H4ClN5O7
C14H9ClN4O7
C12H5ClN4O7
C14H7ClN6O5
C16H9ClN4O5
C21H22ClN5O8
C22H24ClN5O8
C23H26ClN5O8
C19H20ClN5O6
C23H20ClN5O6
C24H22ClN5O6
C25H24ClN5O6
C26H24ClN5O8
C26H24ClN5O8
C27H26ClN5O8
C15H10ClN5O8
C16H12ClN5O8
C15H12ClN5O6
C19H12ClN5O6
C20H14ClN5O6
C21H16ClN5O6
C20H12ClN5O8
C20H12ClN5O8
C22H16ClN5O8
C24H19ClN4O8
C20H11ClN4O8
C16H15ClN4O5
4
C
D
C
A
E
5
5.1.4. Synthesis of diethyl 9-amino-8-chloro-5,6-dihydro-
5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylate (38).
To a hot (90 °C) solution of 4 (2.4 mmol) in glacial ace-
tic acid (20 mL), iron powder (2.4 g) was added. The
mixture was refluxed for 30 min and then evaporation
at reduced pressure of the solvent afforded a solid resi-
due that was dried and extracted in Soxhlet for one
day with acetone (500 mL). The resulting solution was
filtered through SiO2. Evaporation at reduced pressure
of the solvent yielded a solid that was treated with
diethyl ether and collected. 1H NMR 1.31–1.35 (m,
6H, al), 4.37–4.42 (m, 4H, al), 5.63 (s, 2H, NH2), 7.28
(s, 1H, ar), 7.70 (s, 1H, ar), 12.03 (br s, 1H, NH). IR
1711, 1752, 3368, 3465.
6
>300
>300
>300
7
8
9
286–288
248–250
>300
E
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
38
C
F
>300
>300
A
G
C
C
C
C
H
C
C
C
C
C
G
G
G
G
G
G
G
G
G
I
>300
>300
238–240
>300
>300
>300
>300
>300
>300
5.1.5. Synthesis of diethyl 8-chloro-5,6-dihydro-5-oxo-9-
(1,2,4-triazol-4-yl)-pyrazolo[1,5-c]quinazoline-1,2-dicarbox-
ylate (5). To a suspension of 38 (1.1 mmol) in anhydrous
pyridine (6 mL), diformylhydrazine (3.3 mmol) and, drop
by drop, trimethylsilylchloride (16.5 mmol) and triethyl-
amine (7.7 mmol) were successively added. The resulting
solution was refluxed for 4 h, then cooled at room temper-
ature, and diluted with water (30 mL). The resulting solid
>300
>300
>300
>300
>300
>300
>300
>300
1
>300
>300
was collected and washed with water. H NMR 1.21 (t,
3H, al, J = 7.2 Hz), 1.32 (t, 3H, al, J = 7.3 Hz), 4.28–
4.39 (m, 4H, al), 7.61 (s, 1H, ar), 8.90–8.93 (m, 3H, ar),
12.70 (br s, 1H, NH). IR 1722, 1738, 3105.
>300
>300
>300
G
C
a Recrystallization solvents: A, glacial acetic acid; B, toluene; C, eth-
anol; D, dimethylformamide; E, dimethylformamide/water; F, gla-
cial acetic acid/dimethylformamide; G, the title compound was
dissolved in the minimal amount of 1 N NaOH, the insoluble
material was filtered off, and the resulting clear solution acidified
with 1 N HCl; the resulting solid was collected, treated with boiling
ethanol, collected again, and washed with fresh ethanol; H, acetone;
I, the title compound material was dissolved in the minimal amount
of NaHCO3 saturated solution, the insoluble was filtered off, and the
resulting clear solution acidified with 6 N HCl; the resulting solid
was collected, treated with boiling ethanol, collected again, and
washed with fresh ethanol.
5.1.6. Synthesis of diethyl 8-chloro-5,6-dihydro-5-oxo-9-
(pyrrol-1-yl)-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylate
(6). To a hot (90 °C) suspension of 38 (0.8 mmol) in gla-
cial acetic acid (10 mL),
a solution of 2,5-diet-
hoxytetrahydrofuran (1.18 mmol) in glacial acetic acid
(5 mL) was added dropwise.
The mixture was heated at 90 °C for 15 min and then
cooled and diluted with water (20 mL). The solid was
collected and washed with water. 1H NMR 1.24 (t,
3H, al, J = 7.2 Hz), 1.34 (t, 3H, al, J = 7.2 Hz), 4.31

2624
F. Varano et al. / Bioorg. Med. Chem. 16 (2008) 2617–2626
(q, 2H, al, J = 7.2 Hz), 4.40 (q, 2H, al, J = 7.2 Hz), 6.30–
6.31 (m, 2H, ar), 7.02–7.03 (m, 2H, ar), 7.58 (s, 1H, ar),
8.86 (s, 1H, ar), 12.61 (br s, 1H, NH). IR 1727, 1747,
3564.
1H NMR 7.52 (s, 1H, ar), 9.86 (s, 1H, ar), 12.79 (br s,
1H, exchangeable proton). IR 1753, 3171, 3431, 3494.
5.1.12. Synthesis of 8-chloro-5,6-dihydro-5-oxo-9-(1,2,4-
triazol-4-yl)-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylic
acid (12). The title compound was obtained from 5 fol-
lowing Section 5 described above for the preparation
5.1.7. Synthesis of diethyl 8-chloro-5,6-dihydro-9-(3-
formylpyrrol-1-yl)-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-
dicarboxylate (7). To a hot (90 °C) suspension of 38
( 0.58 mmol) in glacial acetic acid (4 mL), a solution of
2,5-dimethoxy-3-tetrahydrofurancarboxaldehyde (0.87
mmol) in glacial acetic acid (3 mL) was added dropwise.
The mixture was heated at 90 °C for 20 min and then
evaporation at reduced pressure of the solvent yielded
a residue that was collected and purified by column
chromatography (eluting system acetone/chloroform/
cyclohexane 6/4/0.2). Evaporation of the first eluates
1
of 11. H NMR 7.59 (s, 1H, ar), 8.93 (s, 2H ar), 9.22
(s, 1H, ar), 12.63 (br s, 1H, exchangeable proton). IR
1712, 1737, 3257, 3444.
5.1.13. Synthesis of 8-chloro-5,6-dihydro-5-oxo-9-(pyrrol-
1-yl)-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylic acid
(13). The ester 6 (8.4 mmol) was dissolved at room tem-
perature in 15 mL of 1 M NaOH. The mixture was stir-
red at room temperature for 12 h, and then diluted with
water (20 mL). Acidification in an ice bath with 6 N HCl
afforded an unfilterable suspension that after stirring at
room temperature for 12 h yielded a solid which was col-
1
afforded the desired compound. H NMR 1.23 (t, 3H,
al, J = 7.1 Hz), 1.34 (t, 3H, al, J = 7.1 Hz), 4.31 (q,
2H, al, J = 7.1 Hz), 4.40 (q, 2H, al, J = 7.1 Hz), 6.72
(s, 1H, ar), 7.21 (s, 1H, ar), 7.61 (s, 1H, ar), 7.97 (s,
1H, ar), 8.97 (s, 1H, ar), 9.81 (s, 1H, CHO), 12.70 (br
s, 1H, NH). IR 1731, 1751, 3113.
1
lected and washed with water. H NMR 6.29 (m, 2H,
pyrrole protons), 7.01 (m, 2H, pyrrole protons), 7.56
(s, 1H, ar), 9.05 (s, 1H, ar), 12.47 (br s 1H, exchangeable
proton). IR 1720, 1741, 3248, 3456.
5.1.8. Synthesis of 1,8-dichloro-5,6-dihydro-9-nitro-5-
oxo-pyrazolo[1,5-c]quinazoline-2-carboxylic acid (8)21.
To a suspension of the ester 2 (0.54 mmol) in methanol
(4 mL), an aqueous solution of KOH (10% w/w, 3.7 mL)
was added and then the mixture was refluxed for 10 min.
After cooling at room temperature, addition of water
(20 mL) and acidification with 6 N HCl afforded a solid
residue that was collected by filtration and washed with
5.1.14. General procedure to prepare diethyl 8-chloro-9-
[3-substituted-ureido]-5,6-dihydro-5-oxo-pyrazolo[1,5-c]qui-
nazoline-1,2-dicarboxylates (14–23). To a hot (70 °C)
solution of 38 (0.80 mmol) in anhydrous tetrahydrofuran
(25 mL), an excess of the appropriate isocyanate
(1.1 mmol) was added. The reaction mixture was stirred
at 70 °C and under nitrogen atmosphere for 3 days. Once
a day, an equimolar amount of the suitable isocyanate
was added. When the reaction was complete (TLC moni-
toring: eluting system CHCl3/MeOH 9:1), the mixture
was cooled at room temperature. Compounds 14, 17–22
precipitated and were then collected by filtration and
recrystallized. Compounds 15–16 and 23 were soluble
and thus evaporation at reduced pressure of the solvent
yielded a residue which was purified by column chroma-
tography (compound 15 eluting system chloroform/etha-
nol 9.5:0.5) or by crystallization (compounds 16 and 23).
1
water. H NMR 7.51 (s, 1H, ar), 9.07 (s, 1H, ar), 13.20
(br s, 1H, exchangeable proton).
5.1.9. Synthesis of 8-chloro-5,6-dihydro-1,9-dinitro-5-
oxo-pyrazolo[1,5-c]quinazoline-2-carboxylic acid (9).21
The title compound was prepared from 3 (0.40 mmol)
following the experimental procedure described above
for the preparation of 8. 1H NMR 7.58 (s, 1H, ar),
9.24 (s, 1H, ar). IR 1750, 1950, 3200, 3450.
5.1.10. Synthesis of 8-chloro-1-ethoxycarbonyl-5,6-dihy-
dro-9-nitro-5-oxo-pyrazolo[1,5-c]quinazoline-2-carboxylic
acid (10).21 To a suspension of 4 (0.74 mmol) in metha-
nol (6 mL), an aqueous solution of KOH (10% w/w,
6 mL) was added. The mixture was left at room temper-
ature under stirring for 1 h. Addition of water (15 mL)
and acidification with 12 N HCl afforded a suspension
that was kept at room temperature for 1 h. The solid
5.1.14.1. Diethyl 8-chloro-9-[3-(ethoxycarbonylmeth-
yl)ureido]-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-
dicarboxylate (14). 1H NMR 1.20–1.35 (m, 9H, al),
3.92–3.93 (m, 2H, al), 4.13 (q, 2H, al, J = 7.0 Hz),
4.37–4.43 (m, 4H, al), 7.29–7.32 (m, 1H, NH), 7.42 (s,
1H, ar), 8.48 (s, 1H, NH), 8.99 (s, 1H, ar), 12.26 (br s,
1H, NH). IR 1693, 1727, 1740, 3352.
1
was collected and washed with water. H NMR 1.30
(t, 3H, al, J = 6.9 Hz), 4.35 (q, 2H, al, J = 6.9 Hz),
7.52 (s, 1H, ar), 9.51 (s, 1H, ar), 12.84 (br s, 1H,
exchangeable proton). IR 1697, 1743, 3206, 3390, 3497.
5.1.14.2. Diethyl 8-chloro-9-[3-(2-ethoxycarbonyleth-
yl)ureido]-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-
1
dicarboxylate (15). H NMR (pyridine-d5) 1.05 (t, 3H,
al, J = 7.1 Hz), 1.23 (t, 3H, al, J = 7.0 Hz), 1.47 (t, 3H,
al, J = 6.9 Hz), 2.69–2.74 (m, 2H, al), 3.79–3.81 (m,
2H, al), 3.99–4.03 (m, 2H, al), 4.40–4.45 (m, 2H, al),
4.79–4.83 (m, 2H, al,), 7.43 (s, 1H, ar), 7.84–7.89 (m,
1H, NH), 8.54 (s, 1H, NH), 9.77 (s, 1H, ar).
5.1.11. Synthesis of 8-chloro-5,6-dihydro-9-nitro-5-oxo-
pyrazolo[1,5-c]quinazoline-1,2-dicarboxylic acid (11).21
A solution of KOH (10% w/w, 7 mL) was added to a
suspension of 4 (0.6 mmol) in ethanol (7 mL). The mix-
ture was heated at 100 °C for 1 h and then diluted with
water (15 mL). Acidification with 12 N HCl yielded a
suspension that, after heating at 100 °C for 30 min and
stirring at room temperature for 12 h, afforded a solid
which was collected by filtration and washed with water.
5.1.14.3. Diethyl 8-chloro-9-[3-(3-ethoxycarbonylpro-
pyl)ureido]-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-
1,2-dicarboxylate (16). 1H NMR 1.18 (t, 3H, al,
J = 7.1 Hz), 1.30–1.35 (m, 6H, al), 1.69–1.74 (m, 2H,

F. Varano et al. / Bioorg. Med. Chem. 16 (2008) 2617–2626
2625
al), 2.35 (t, 2H, al, J = 7.5 Hz), 3.11–3.16 (m, 2H, al),
4.06 (q, 2H, al, J = 7.1 Hz), 4.37 (q, 2H, al,
J = 7.1 Hz), 4.43 (q, 2H, al, J = 7.1 Hz), 6.99–7.02 (m,
1H, NH), 7.40 (s, 1H, ar), 8.14 (s, 1H, NH), 9.01 (s,
1H, ar), 12.26 (br s, 1H, NH). IR 1731, 3319.
5.1.15. General procedure to prepare 8-chloro-9-[3-substi-
tuted-ureido]-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,
2-dicarboxylic acids (24–32). To a suspension of the es-
ters 14–15, 17–23 (0.84 mmol) in a mixture of water
and ethanol (1:1; 10 mL), a 10% w/w NaOH solution
(5 mL) was added. The reaction mixture was refluxed
until disappearance of the starting material (TLC mon-
itoring: eluting system CHCl3/MeOH/AcOH 7:1:2). The
solution was diluted with water (15 mL) and then acid-
ified with 6 N HCl. The suspension was refluxed for
10 min and then cooled to room temperature. The solid
was collected and washed with water.
5.1.14.4. Diethyl 8-chloro-9-(3-ethylureido)-5,6-dihy-
dro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylate (17).
1H NMR 1.07 (t, 3H, al, J = 5.5 Hz), 1.31–1.36 (m,
6H, al), 3.13–3.16 (m, 2H, al), 4.38 (q, 2H, al,
J = 7.0 Hz), 4.45 (q, 2H, al, J = 7.0 Hz), 6.92–6.94
(m, 1H, NH), 7.41 (s, 1H, ar), 8.12 (s, 1H, NH),
9.02 (s, 1H, ar), 12.23 (br s, 1H, NH). IR 1731, 3127,
3328.
5.1.15.1. 8-Chloro-9-[3-(carboxymethyl)ureido]-5,
6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarbox-
ylic acid (24). 1H NMR 3.85 (d, 2H, al, J = 5.0 Hz), 7.20
(br s, 1H, NH), 7.41 (s, 1H, ar), 8.42 (s, 1H, NH), 9.25
(s, 1H, ar), 12.19 (s, 1H, exchangeable proton).
5.1.14.5. Diethyl 8-chloro-9-(3-phenylureido)-5,6-dihy-
dro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylate (18).
1H NMR 1.32–1.37 (m, 6H, al), 4.39 (q, 2H, al,
J = 6.9 Hz), 4.47 (q, 2H, al, J = 6.9 Hz), 6.99–7.03 (m,
1H, ar), 7.30–7.34 (m, 2H, ar), 7.46–7.50 (m, 3H, ar),
8.47 (s, 1H, NH), 9.08 (s, 1H, ar), 9.39 (s, 1H, NH),
12.31 (s, 1H, NH).
5.1.15.2. 8-Chloro-9-[3-(2-carboxyethyl)ureido]-5,6-
dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylic
acid (25). 1H NMR 7.02 (br s, 1H, NH), 7.39 (s, 1H, ar),
8.25 (s, 1H, NH), 9.27 (s, 1H, ar), 12.17 (s, 1H,
exchangeable proton). 1H NMR (CD3OD-d4) 2.59–
2.60 (m, 2H, al), 3.51–3.52 (m, 2H, al), 7.43 (s, 1H,
ar), 9.39 (s, 1H, ar). IR 1722, 3360.
5.1.14.6. Diethyl 8-chloro-9-(3-benzylureido)-5,6-dihy-
dro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylate (19).
1H NMR 1.29–1.35 (m, 6H, al), 4.33–4.44 (m, 6H, al),
7.27–7.42 (m, 7H, 6ar + NH), 8.27 (s, 1H, NH), 9.04
(s, 1H, ar), 12.25 (br s, 1H, NH).
5.1.15.3.
8-Chloro-9-(3-ethylureido)-5,6-dihydro-5-
oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylic acid (26).
1H NMR 1.07 (t, 3H, al, J = 7.2 Hz), 3.10–3.16 (m,
2H, al), 6.82 (br s, 1H, NH), 7.38 (s, 1H, ar), 8.08 (s,
1H, NH), 9.28 (s, 1H, ar), 12.18 (s, 1H, exchangeable
proton). IR 1710, 1743, 3328.
5.1.14.7. Diethyl 8-chloro-9-[3-(2-ethylphenyl)ureido]-
5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarbox-
ylate (20). 1H NMR 1.32–1.36 (m, 6H, al), 2.77–2.80 (m,
2H, al), 3.36–3.39 (m, 2H, al), 4.38 (q, 2H, al,
J = 6.9 Hz), 4.46 (q, 2H, al, J = 7.0 Hz), 6.95–6.98 (m,
1H, NH), 7.21–7.35 (m, 5H, ar), 7.44 (s, 1H, ar), 8.18
(s, 1H, NH), 9.02 (s, 1H, ar), 12.21 (br s, 1H, NH).
5.1.15.4. 8-Chloro-9-(3-phenylureido)-5,6-dihydro-5-
oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylic acid (27).
1H NMR 6.98–7.02 (m, 1H, ar), 7.29–7.33 (m, 2H, ar),
7.45–7.49 (m, 3H, ar), 8.39 (s, 1H, NH), 9.26 (s, 1H, ar),
9.48 (s, 1H, NH), 12.21 (s, 1H, exchangeable proton).
5.1.14.8. Diethyl 8-chloro-9-[3-(3-ethoxycarbonylphe-
nyl)ureido]-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-
1
1,2-dicarboxylate (21). H NMR 1.34–1.39 (m, 9H, al),
5.1.15.5. 8-Chloro-9-(3-benzylureido)-5,6-dihydro-5-
oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylic acid (28).
1H NMR 4.35 (d, 2H, al, J = 5.5 Hz), 7.27–7.41 (m, 7H,
6ar + NH), 8.22 (s, 1H, NH), 9.33 (s, 1H, ar), 12.17 (s,
1H, exchangeable proton).
4.33 (q, 2H, al, J = 7.3 Hz), 4.36 (q, 2H, al,
J = 7.3 Hz), 4.50 (q, 2H, al, J = 7.0 Hz), 7.46–7.49 (m,
2H, ar), 7.60–7.62 (m, 2H, ar), 8.25 (s, 1H, ar), 8.50 (s,
1H, NH), 9.09 (s, 1H, ar), 9.66 (s, 1H, NH), 12.33 (br
s, 1H, NH).
5.1.15.6. 8-Chloro-9-[3-(2-phenylethyl)ureido]-5,6-dihy-
dro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylic acid
(29). 1H NMR 2.76–2.79 (m, 2H, al), 3.36–3.39 (m,
2H, al), 6.86–6.87 (m, 1H, NH), 7.22–7.33 (m, 5H, ar),
7.44 (s, 1H, ar), 8.16 (s, 1H, NH), 9.23 (s, 1H, ar).
5.1.14.9. Diethyl 8-chloro-9-[3-(4-ethoxycarbonylphe-
nyl)ureido]-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-
1
1,2-dicarboxylate (22). H NMR 1.29–1.37 (m, 9H, al),
4.28 (q, 2H, al, J = 7.1 Hz), 4.38 (q, 2H, al, J = 7.1
Hz), 4.46 (q, 2H, al, J = 7.1 Hz), 7.46 (s, 1H, ar), 7.62
(d, 2H, ar, J = 8.6 Hz), 7.93 (d, 2H, ar, J = 8.6 Hz),
8.60 (s, 1H, NH), 9.07 (s, 1H, ar), 9.78 (s, 1H, NH),
12.34 (br s, 1H, NH).
5.1.15.7. 8-Chloro-9-[3-(3-carboxyphenyl)ureido]-5,6-
dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylic
1
acid (30). H NMR 7.40–7.43 (m, 2H, ar), 7.56 (d, 1H,
ar, J = 7.6 Hz), 7.67 (d, 1H, ar, J = 7.8 Hz), 8.14 (s,
1H, ar), 8.40 (s, 1H, NH), 9.38 (s, 1H, ar), 9.89 (br s,
1H, NH), 12.21 (s, 1H, exchangeable proton) 12.96 (br
s, 1H, exchangeable proton). IR 1693, 1746, 3284.
5.1.14.10. Diethyl ( )8-chloro-9-[3-(1-methoxycar-
bonyl-1-benzyl-methyl)ureido]-5,6-dihydro-5-oxo-pyrazolo-
[1,5-c]quinazoline-1,2-dicarboxylate (23). H NMR 1.28–
1
1.34 (m, 6H, al), 2.96–3.01 (m, 1H, al), 3.06–3.11 (m, 1H,
al), 3.65 (s, 3H, al), 4.35–4.43 (m, 4H, al), 4.52–4.57 (m,
1H, al), 7.21–7.41 (m, 7H, 6ar + NH), 8.44 (s, 1H, NH),
8.96 (s, 1H ar), 12.25 (br s, 1H, NH). IR 1732, 3287, 3368.
5.1.15.8. 8-Chloro-9-[3-(4-carboxyphenyl)ureido]-5,6-
dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-dicarboxylic
1
acid (31). H NMR 7.46 (s, 1H, ar), 7.59 (d, 2H, ar,

2626
F. Varano et al. / Bioorg. Med. Chem. 16 (2008) 2617–2626
J = 8.7 Hz), 7.90 (d, 2H, ar, J = 8.7 Hz), 8.55 (s, 1H,
NH), 9.37 (s, 1H, ar), 9.68 (s, 1H, NH), 12.27 (br s,
1H, exchangeable proton). IR 1723, 3359, 3611.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2007.
11.046.
5.1.15.9. ( ) 8-Chloro-9-[3-(1-benzyl-1-carboxymethyl)
ureido]-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-
1
dicarboxylic acid (32). H NMR 2.93–2.98 (m, 1H, al),
3.09–3.14 (m, 1H, al), 4.47–4.48 (m, 1H, al), 7.20–7.34
(m, 6H, 5ar + NH), 7.40 (s, 1H, ar), 8.39 (s, 1H, NH),
9.28 (s, 1H ar), 12.18 (br s, 1H, exchangeable proton).
References and notes
1. Bra¨uner-Osborne, H.; Egebjerg, J.; Nielsen, E. Ø.; Madsen,
U.; Krogsgaard-Larsen, P. J. Med. Chem. 2000, 43, 2609.
2. Dingledine, R.; Borges, K.; Bowie, D.; Traynelis, S. F.
Pharmacol. Rev. 1999, 51, 7.
3. Johnson, J. W.; Ascher, P. Nature 1987, 325, 529.
4. Wahlgren, N. G. Int. Rev. Neurobiol. 1997, 40, 337.
5. Hallett, P. J.; Standaert, D. G. Pharmacol. Ther. 2004,
102, 155.
6. Hynd, M. R.; Scott, H. L.; Dodd, P. R. Neurochem. Int.
2004, 45, 583.
7. Pitt, D.; Werner, P.; Raine, C. S. Nature 2000, 6, 67.
8. Catarzi, D.; Colotta, V.; Varano, F. Med. Res. Rev. 2007,
27, 239.
5.1.16. Synthesis of diethyl 8-chloro-9-[(2-carboxyben-
zoyl)amino]-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,
2-dicarboxylate (33). To a hot (60 °C) suspension of 38
(0.90 mmol) in glacial acetic acid (5 mL), sodium acetate
(0.90 mmol) and phthalic anhydride (1.18 mmol) were
added. The reaction mixture was heated at 60 °C for
10 min, then addition of water afforded a solid which
1
was collected and washed with water. H NMR 1.31–
1.36 (m, 6H, al), 4.37–4.45 (m, 4H, al), 7.50 (s, 1H,
ar), 7.59–7.61 (m, 2H, ar), 7.67–7.70 (m, 1H, ar), 7.88–
7.90 (m, 1H, ar), 8.77 (s, 1H, ar), 10.23 (br s, 1H,
exchangeable proton), 12.43 (s, 1H, exchangeable pro-
ton). IR 1715, 1756, 3160, 3461.
9. Catarzi, D.; Colotta, V.; Varano, F. Curr. Top. Med.
Chem. 2006, 6, 809.
10. Bleakman, D.;Lodge, D. Neuropharmacology1998, 37, 1187.
11. Lerma, J.; Paternain, A. V.; Rodriguez-Moreno, A.;
`
`
Lopez-Garcıa, J. C. Physiol. Rev. 2001, 81, 971.
12. Bleakman, D.; Gates, M. R.; Ogden, A. M.; Mackowiak,
M. Curr. Pharm. Des. 2002, 8, 873.
5.1.17. Synthesis of 8-chloro-9-[(2-carboxybenzoyl)
amino]-5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazoline-1,2-
dicarboxylic acid (34). The title compound was obtained
by alkaline hydrolysis of ester 33 (0.46 mmol) following
13. Shou, X.; Chamberlin, A. R. Exp. Opin. Ther. Patents
2004, 14, 471.
1
Section 5 described above for the synthesis of 13. H
14. Dolman, N. P.; Troop, H. M.; More, J. C. A.; Alt, A.;
Knaus, J. L.; Nistico, R.; Jack, S.; Morley, R. M.;
Bortolotto, Z. A.; Roberts, P. J.; Bleackman, D.; Colling-
ridge, G. L.; Jane, D. E. J. Med. Chem. 2005, 48, 7867.
15. Colotta, V.; Catarzi, D.; Varano, F.; Lenzi, O.; Filacchi-
oni, G.; Costagli, C.; Galli, A.; Ghelardini, C.; Galeotti,
N.; Gratteri, P.; Sgrignani, J.; Deflorian, F.; Moro, S.
J. Med. Chem. 2006, 49, 6015.
NMR 7.50 (s, 1H, ar), 7.60–7.68 (m, 3H, ar), 7.85–
7.87 (m, 1H, ar), 9.12 (s, 1H, ar), 10.22 (br s, 1H,
NH), 12.33 (br s, 1H, exchangeable proton).
6. Biochemistry
16. Varano, F.; Catarzi, D.; Colotta, V.; Filacchioni, G.; Galli,
`
6.1. Binding assay
A.; Costagli, C.; Carla, V. J. Med. Chem. 2002, 45, 1035.
17. Varano, F.; Catarzi, D.; Colotta, V.; Calabri, F. R.; Lenzi,
O.; Filacchioni, G.; Galli, A.; Costagli, C.; Deflorian, F.;
Moro, S. Bioorg. Med. Chem. 2005, 13, 5536.
18. Catarzi, D.; Colotta, V.; Varano, F.; Cecchi, L.; Filacchi-
`
oni, G.; Galli, A.; Costagli, C.; Carla, V. J. Med. Chem.
Rat cortical synaptic membrane preparation and
[3H]glycine, [3H]AMPA, and high-affinity [3H]kainate
binding experiments were performed following the pro-
cedures described in Refs. 16, 22, and 23, respectively.
2000, 43, 3824.
19. Catarzi, D.; Colotta, V.; Varano, F.; Filacchioni, G.;
`
Galli, A.; Costagli, C.; Carla, V. J. Med. Chem. 2001, 44,
6.2. Electrophysiological assay
3157.
20. Catarzi, D.; Colotta, V.; Varano, F.; Calabri, F. R.;
The cortical wedge preparation described by Mannaioni
et al.24 was used while the electrophysiological assays were
performed following the procedures described in Ref. 16.
`
Filacchioni, G.; Galli, A.; Costagli, C.; Carla, V. J. Med.
Chem. 2004, 47, 262.
21. Moro, S.; Varano, F.; Cozza, G.; Pagano, M.; Fagotto, G.;
Chilin, A.; Guiotto, A.; Catarzi, D.; Colotta, V.; Pinna, L.
A.; Meggio, F. Lett. Drug Des. Discov. 2006, 3, 281.
22. Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filacchi-
oni, G.; Galli, A.; Costagli, C. Arch. Pharm. Pharm. Med.
Chem. 1997, 330, 129.
23. Nielsen, E. O.; Madsen, U.; Schaumburg, K.; Brehm, L.;
Krogsgaard-Larsen, P. Eur. J. Chem. Chim. Ther. 1986,
21, 433.
`
24. Mannaioni, G.; Carla, V.; Moroni, F. Br. J. Pharmacol.
1996, 118, 1530.
25. De Lean, A.; Munson, P. J.; Rodbard, D. Am. J. Physiol.
1978, 235, E97.
26. Cheng, Y.-C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
6.3. Sample preparation and results calculation
A stock 1 mM solution of the tested compound was pre-
pared in 50% DMSO. Subsequent dilutions were accom-
plished in buffer. The IC50 values were calculated from
three or four displacement curves based on four to six
scalar concentrations of the test compound using the
ALLFIT computer program25 and, in the case of triti-
ated glycine and AMPA binding, converted to Ki values
by application of the Cheng–Prusoff equation.26 Under
our experimental conditions, the dissociation constants
(KD) for [3H]glycine (10 nM) and [3H]-DL-AMPA
(8 nM) were 75 6 and 28 3 nM, respectively.