Pyrazolo[1,5-c]quinazoline derivatives and their simplified analogues as adenosine receptor antagonists: Synthesis, structure-affinity relationships and molecular modeling studies

Article abstract of DOI:10.1016/j.bmc.2012.10.031

A number of 5-oxo-pyrazolo[1,5-c]quinazolines (series B-1), bearing at position-2 the claimed (hetero)aryl moiety (compounds 1-8) but also a carboxylate group (9-14), were designed as hA₃ AR antagonists. This study produced some interesting com

Full text of DOI:10.1016/j.bmc.2012.10.031

Bioorganic & Medicinal Chemistry 21 (2013) 283–294
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Bioorganic & Medicinal Chemistry
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Pyrazolo[1,5-c]quinazoline derivatives and their simplified analogues as
adenosine receptor antagonists: Synthesis, structure–affinity relationships
and molecular modeling studies
Daniela Catarzi a, , Vittoria Colotta a, Flavia Varano a, Daniela Poli a, Lucia Squarcialupi a,
⇑
Guido Filacchioni a, Katia Varani b, Fabrizio Vincenzi b, Pier Andrea Borea b, Diego Dal Ben c,
Catia Lambertucci c, Gloria Cristalli c
a Dipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente Attivi, Universita’ di Firenze, Polo Scientifico, Via Ugo Schiff 6,
50019 Sesto Fiorentino, FI, Italy
b Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Università di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy
c School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of 5-oxo-pyrazolo[1,5-c]quinazolines (series B-1), bearing at position-2 the claimed (het-
ero)aryl moiety (compounds 1–8) but also a carboxylate group (9–14), were designed as hA3 AR antag-
onists. This study produced some interesting compounds endowed with good hA3 receptor affinity and
high selectivity, being totally inactive at all the other AR subtypes. In contrast, the corresponding 5-
ammino derivatives (series B-2) do not bind or bind with very low affinity at the hA3 AR, the only excep-
Received 5 September 2012
Revised 3 October 2012
Accepted 14 October 2012
Available online 29 October 2012
tion being the 5-N-benzoyl compound 19 that shows a hA3 Ki value in the high l-molar range. Evaluation
Keywords:
of the synthetic intermediates led to the identification of some 5(3)-(2-aminophenyl)-3(5)-(hetero)aryl-
pyrazoles 20–24 with modest affinity but high selectivity toward the hA3 AR subtype. Molecular docking
of the herein reported tricyclic and simplified derivatives was carried out to depict their hypothetical
binding mode to our model of hA3 receptor.
G protein-coupled receptors
Adenosine receptor antagonists
Pyrazoloquinazolines
Tricyclic heteroaromatic systems
Ligand–receptor modeling studies
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
of A3 AR subtype has recently emerged as particularly interesting
for the selective control of acute and chronic disorders such as
Adenosine is an endogenous nucleoside that plays a key role in
different pathophysiological conditions by interacting with specific
cell-surface G protein-coupled receptors (GPCRs) which are termed
A1, A2A, A2B and A3 adenosine receptors (ARs).1 Each receptor
subtype mediates distinct physiological actions by coupling with
different secondary messenger systems, such as adenylate cyclase
(AC), calcium or potassium channels, phospholipase C and D. How-
ever, AC is considered the principal effector system which can be
either inhibited (A1 and A3) or stimulated (A2A and A2B) thus
decreasing or increasing, respectively, the cAMP production.1–3
Due to the widespread distribution of ARs in peripheral organs
and in the central nervous system (CNS),1,3 and to the ubiquity of
the natural ligand adenosine, selective modulation of each receptor
subtype could be exploited for some therapeutic advantages. ARs
have been recently recognized as novel and potential drug targets
for therapeutic intervention in many pathological conditions such
as cardiac and cerebral ischemia, asthma, renal failure, CNS disor-
ders, inflammatory and neurodegenerative diseases.4 Modulation
cerebral and cardiac ischemia,5,6 inflammation, and normal and
tumor cell regulation.7,8 Consequently, highly selective A3 AR
antagonists have been proposed for the acute treatment of stroke
and for the development of cerebroprotective and anti-inflamma-
tory drugs. The potential use of A3 AR antagonists as therapeutics
in cancers is still under debate.7
The putative therapeutic applications of A3 AR antagonists as
well as the growing need for pharmacological tools to deeply
investigate A3 AR roles, has made the identification of potent and
selective antagonists at this receptor subtype, endowed with
adequate pharmacokinetic properties, an emerging topic.
Our research group has developed several classes of tricyclic AR
antagonists6,9–16 including the 2-arylpyrazolo[3,4-c]quinoline
series (PQ)6,12–14 (Fig. 1). By modulating substituents at positions
2 and 4 of this scaffold, we have obtained highly potent and
selective hA3 AR antagonists.6,12,13 Structure–activity relationships
(SAR) of these derivatives showed that the 2-aryl-4-oxo-substi-
tuted compounds (Series A-1) have, on the whole, a higher affinity
for the hA3 AR6,12 than the corresponding 4-amino derivatives
(Series A-2).12Interestingly, the hA3 receptor affinities of the
2-arylpyrazolo[3,4-c]quinolin-4-amines were enhanced when acyl
⇑
Corresponding author. Tel.: +39 055 4573722; fax: +39 055 4573780.
E-mail address: daniela.catarzi@unifi.it (D. Catarzi).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.031

284
D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
R4
N
N
OH HN
HN
X
O
HN
R2
4
4
N
N
N
HN
2
N
2
N
N
I
R
R
X = NH2
R2 = aryl,
Serie A-1
20-24
25
Serie A-2
heteroaryl,
COOEt
R4 = H, COAlk, COAr
R = H, OCH3, CH3, CF3, Cl, F, NO2
X = OH
R2 = C6H5
Figure 1. Previously reported pyrazolo[3,4-c]quinoline (PQ) derivatives as hA3
adenosine receptor antagonists.
Figure 3. Simplified adenosine receptor antagonists: 3,5-Diarylpyrazole derivatives.
residues were appended on the 4-amino group.6,12,13 Moreover, in
both the 4-oxo- (A-1) and 4-amino (A-2) series the presence of a
methoxy substituent at the para-position of the 2-phenyl ring
ameliorated the hA3 AR binding affinity.6,12 The SAR on these
pyrazoloquinolines resemble those of other classes of tricyclic AR
antagonists which were extensively studied in our labora-
tory.9,10,15,16 On this basis, we have designed a new series of pyraz-
olo[1,5-c]quinazoline derivatives differently substituted at
position-2 and 5 (Series B-1 and B-2, Fig. 2). The present study
was performed to evaluate the versatility of the pirazoloquinazo-
line scaffold to obtain new potent and selective AR antagonists.
Together with the classical 5-oxo- and 5-amino- patterns,
different aryl, heteroaryl and carboxyalkyl substituents were intro-
duced at position-2 of the tricyclic ring system. These modifica-
tions were performed on the basis of the SARs on structurally
correlated AR antagonists synthesized in our laboratory.6,12,13,15,16
Moreover, our attention was also addressed to the development
of more simplified heterocyclic compounds.17–19 In fact, structural
simplification can represent a drug design strategy to shorten the
synthetic route and to improve physicochemical properties of the
original candidate. Thus, looking at the intermediates in the syn-
thetic pathway which leads to the pyrazolo[1,5-c]quinazoline
derivatives, we have identified the 3(5)-(2-aminophenyl)-5(3)-
(hetero)aryl-pyrazoles 20–2420,21 (Fig. 3) as possible candidates
for our pharmacological assays. In fact, these derivatives seem to
have, on the whole, the minimal structural requirements (i.e., the
primary amino group and the correctly oriented aryl moieties) to
profitably interact with the AR binding pocket. The presence of
the primary amino group is also important for solubility purposes,
as is the polar pyrazole NH function. Moreover, other evidence
prompted us to investigate in this direction. In fact, five-member
monocyclic cores have been evaluated as AR antagonists22 in
recent years. Among them, our attention was caught by the
5-(2-hydroxyphenyl)-3-phenyl-1,2,4-triazole I (Fig. 3) which was
reported as a weak hA2A AR antagonist (Ki = 0.7 lM) together
with other triazole derivatives.23 In this regard, we also considered
the corresponding 5-(2-hydroxyphenyl)-3-phenylpyrazole 2520
(Fig. 3) as a link between the 3(5)-substituted 5(3)-(2-aminophe-
nyl)-pyrazole series 20–24 and the lead triazole I. In fact, the close
structural similarities between these two different monocyclic
scaffolds seem intriguing.
2. Chemistry
The 5,6-dihydro-2-(hetero)arylpyrazolo[1,5-c]quinazolin-5-ones
1, 3–5, 8, the alkyl 5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazolin-
2-carboxylates 9–11, together with the synthetic intermediates
20 and 24, and the diethyl 5,6-dihydro-5-oxo-pyrazolo[1,5-c]qui-
nazolin-1,2-dicarboxylate 14, were synthesized as reported in
previously published papers.20,21,25 The synthesis of the 2-arylpy-
razolo[1,5-c]quinazoline derivatives 2, 6–7, 15–19 and that of the
2-carboxy-substituted analogues 12–13 is depicted in Schemes 1
and 2, respectively. Preparation of compounds 6–7 was performed
following different procedures with respect to those previously
reported.21
By reacting commercially available 2-nitrobenzaldehyde with
the suitable (hetero)aryl-methylketone in the presence of boron tri-
fluoride acetic acid complex, in glacial acetic acid, the 1,3-diaryl-2-
propenones 26–2826–28 were obtained with high yields (Scheme 1).
The latters were then reacted with hydrazine hydrate yielding the
4,5-dihydro-3,5-diaryl-1H-pyrazoles 29–3121 which were dehy-
drogenated to the 3(5)-(2-nitrophenyl)-5(3)-(hetero)aryl-pyra-
zoles 32–34 by reaction with lead tetracetate followed by
treatment with concentrated hydrochloric acid in boiling ethanol.
Catalytic reduction of the nitro group of 32–34 afforded the corre-
sponding amino derivatives 21–2321 which were cyclized to the tri-
cyclic compounds 5-oxo- (2, 6–7) and 5-amino- (16–18)
substituted by reacting, respectively, with triphosgene and cyana-
mide. The tricyclic 2-phenyl-5-amino substituted derivative 15, ob-
tained by treating the 5-(2-aminophenyl)-3-phenyl-1,2,4-triazole
2020 with cyanamide, was reacted with benzoyl chloride in the
presence of anhydrous pyridine to give the corresponding 5-benz-
amido compound 19. Hydrolysis of the ethyl 5,6-dihydro-5-oxo-
pyrazolo[1,5-c]quinazolin-2-carboxylate 1020 (Scheme 2) yielded
the corresponding 2-carboxylic acid 3521 that was transformed into
the 2-carbonyl chloride by reacting with an excess of thionyl chlo-
ride. The crude product was heated at reflux with benzyl alcohol to
give the corresponding benzyl 2-carboxylate 12. By reacting 35
with benzylamine in the presence of N-(3-dimethylaminopropyl)-
N0-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole
hydrate, the 2-benzylcarboxamido derivative 13 was obtained.
R5
HN
O
5
N
N
N
N
HN
N
5
R2
2
R2
2
R1
Serie B-2
15-19
R5 = H, COC6H5
Serie B-1
1-13 R1 = H
14 R1 = COOEt
R2
=
aryl,
heteroaryl,
COR
C6H5
2-furyl
OEt
4CH3OC6H4 2-thienyl
OCH2C6H5
NHCH2C6H5
4CH3C6H4
4ClC6H4
2FC6H4
2-pyridil
Figure 2. Currently reported pyrazolo[1,5-c]quinazoline derivatives.

D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
285
NO2
O
N
NO2
NO2HN
O
R2
b
a
c
CHO
R2
H3C
R2
26-28
29-31
O
N
NO
2HN
N
NH2HN
N
HN
N
R2
e
d
R2
R2
32-34
20-23
2, 6-7
f
O
C6H5
N
HN
NH2
N
N
g
N
N
N
R2
15-18
19
R2
C6H5
n
15, 19, 20
2, 16, 26, 29, 32, 21
6, 17, 27, 30, 33, 22
7, 18, 28, 31, 34, 23
4-CH3OC6H4
2-furyl
2-thienyl
Scheme 1. Synthesis of 2-arylpyrazolo[1,5-c]quinazoline derivatives. Reagents and conditions: (a) BF3ꢀ(CH3COOH)2, acetic acid, room temperature; (b) NH2NH2ꢀH2O, EtOH,
reflux; (c) (i) Pb(OAc)4, anhydrous CH2Cl2, room temperature; (ii) conc. HCl, EtOH, reflux; (d) H2, 10% Pd/C, EtOAc; (e) (CCl3O)2CO, anhydrous THF, room temperature; (f)
NH2CN, pTsOH, NMP, reflux; (g) C6H5COCl, anhydrous pyridine, anhydrous CH2Cl2, room temperature.
O
O
O
N
N
HN
N
N
HN
N
HN
N
COOEt
b, c or d
COOH
a
COR
10
12 R = OCH2C6H5
13 R = NHCH2C6H5
35
Scheme 2. Synthesis of 2-carboxy-substituted pyrazolo[1,5-c]quinazoline derivatives. Reagents and conditions. (a) 6N HCl, glacial acetic acid, reflux; (b) SOCl2, reflux; (c)
C6H5CH2OH, reflux; (d) C6H5CH2NH2, N-(3-dimethylamino-propyl)-N0-ethylcarbodiimideꢀHCl, 1-hydroxybenzotriazole hydrate.
the pirazolo[1,5-c]quinazoline series B-1 and B-2. In particular,
while the 5-oxo-substituted derivatives (series B-1) are endowed
with good hA3 receptor affinity and selectivity, the corresponding
5-ammino compounds (series B-2) do not bind or bind with very
low affinity at the same receptor subtype, the only exception being
the 5-benzamido substituted derivative 19. In contrast, the 4-ami-
no compound 17, bearing a 2-furyl moiety at position-2, is the only
compound among series B-1 and B-2 which binds at all the hARs
with the exception of the hA3 subtype. Moreover, evaluation of
the synthetic intermediates 20–24 for the binding at the hARs gave
good results thus leading to some modest but selective hA3 AR
antagonists despite their highly simplified structure.
It has to be noted that both the 5-oxo and 5-amino derivatives 1
and 15, respectively, bearing a phenyl substituent at position-2, are
devoid of significant affinity toward all the human AR subtypes.
This result was almost unexpected considering the close structural
similarities between the pyrazolo[1,5-c]quinazoline scaffold and
the pyrazolo[3,4-c]quinoline (PQ) core of the lead compounds
(Fig. 1). In fact, all the structural features which are considered
as essential for a profitable receptor–ligand interaction are present
in both the 2-phenylpyrazolo[1,5-c]quinazoline either 5-oxo (1) or
5-amino (15) substituted. Replacement of the 2-phenyl group of 1
3. Pharmacology
All the reported compounds 1–25, were tested for their ability
to displace specific [3H]DPCPX, [3H]ZM241385 and [125I]AB-MECA
binding from cloned hA1, hA2A and hA3 receptors, respectively, sta-
bly expressed in CHO cells. Compounds 1–25 were also tested at
the hA2B subtype by measuring their inhibitory effects on NECA-
stimulated cAMP levels in CHO cells stably transfected with the
hA2B receptor. The results of binding experiments and cAMP assays
in hA2B CHO cells are reported in Tables 1 and 2, where hA1, hA2A
and hA3 affinities of I are also included as reference data. Moreover,
to evaluate their hA3 AR antagonistic effect, compounds 2–4, 6 and
7 were tested for their ability to counteract the Cl-IB-MECA-med-
iated inhibition of cAMP production in CHO cells stably expressing
hA3 AR (Table 3).
4. Results and discussion
4.1. Structure–affinity relationships
The binding results reported in Tables 1 and 2 indicate that we
have produced some new selective hA3AR antagonists belonging to

286
D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
Table 1
Binding affinity (Ki) at hA1, hA2A, and hA3 ARs and potencies (IC50) at hA2B
R5
O
HN
N
N
5
HN
N
N
N
R2
R2
R1
Series B-1
(1-14)
Series B-2
(15-19)
Series
n°
R1
R2
R5
Binding experiments Kia (nM) or I%
cAMP assay I%
b
c
d
e
hA3
hA1
hA2A
hA2B
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
H
H
H
H
H
H
H
H
H
H
H
H
C6H5
—
—
—
—
—
—
—
—
28%
100 12
25
23
140 16
12%
4%
1%
6%
1%
5%
7%
4%
1%
3%
1%
3%
2%
12%
1%
7%
35
1%
1%
12%
1%
11%
1%
20%
1%
3%
5%
1%
4%
5%
1%
5%
15%
80
20%
1%
4%
2%
2%
3%
1%
3%
6%
2%
6%
3%
4%
2%
1%
5%
3%
3%
4-OCH3C6H4
4-CH3C6H4
4-ClC6H4
2-FC6H4
2
2
2-Furyl
80
40
7
3
B-1
2-Thienyl
2-Pyridil
COOMe
252 13
440 44
315 20
450 42
1%
25%
101 12
15%
45%
35%
18%
520 60
COOEt
—
COOCH(CH3)2
COOCH2C6H5
CONHCH2C6H5
COOEt
C6H5
4-OCH3C6H4
2-Furyl
—
—
—
NH2
NH2
NH2
NH2
H
COOEt
—
—
—
—
—
B-2
2
7
120 13
3%
4%
2-Thienyl
C6H5
44%
13%
NHCOC6H5
a
Ki values are means SEM of four separate assays each performed in duplicate. Percentage of inhibition (I%) are determined at 1
compounds.
lM concentration of the tested
b
Displacement of specific [125I]AB-MECA competition binding to hA3CHO cells.
c
d
e
Displacement of specific [3H]DPCPX competition binding to hA1CHO cells.
Displacement of specific [3H]ZM241385 competition binding to hA2ACHO cells.
Percentage of inhibition on cAMP experiments in hA2BCHO cells, stimulated by 200 nM NECA, at 1
l
M concentration of the examined compounds.
Table 2
Binding affinity (Ki) at hA1, hA2A, and hA3 ARs and potencies (IC50) at hA2B AR
N
N
N
HN
HN
OH
X
R2
20-25
I
R2
X
Binding experiments Kia (nM) or I%
cAMP assay I%
b
c
d
e
hA3
hA1 (%)
hA2A (%)
hA2B (%)
20
21
22
23
24
25
If
C6H5
NH2
NH2
NH2
NH2
NH2
OH
680 62
480 53
1%
600 57
23%
9
2
4
2
3
1
9
6
1
1
1
2
2
5
1
3
2
4
8
4-CH3OC6H4
2-Furyl
2-Thienyl
COOEt
C6H5
7%
39%
13
47
—
—
a
Ki values are means SEM of four separate assays each performed in duplicate. Percentage of inhibition (I%) are determined at 1
compounds.
l
M concentration of the tested
b
Displacement of specific [125I]AB-MECA competition binding to hA3CHO cells.
c
d
e
f
Displacement of specific [3H]DPCPX competition binding to hA1CHO cells.
Displacement of specific [3H]ZM241385 competition binding to hA2ACHO cells.
Percentage of inhibition on cAMP experiments in hA2BCHO cells, stimulated by 200 nM NECA, at 1
Ref.23,24
l
M concentration of the examined compounds.
with suitable moieties significantly reinforces the receptor–ligand
interaction, thus leading to compounds 2–8 that possess enhanced
hA3 binding affinities. In particular, the ameliorated binding of the
2-(4-methoxyphenyl)-substituted compound 2, with respect to
the parent 1, was expected on the basis of the lead SARs.6,12 In fact,
the 4-methoxy group could improve the receptor–ligand interac-
tion by forming a weak hydrogen bond with a suitable receptor
site6 or by reinforcing the
p–p stacking interaction of the 2-phenyl
ring with the receptor, due to its electron-donating properties.
The lipophilic contribution of the methoxy group to the ligand

D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
287
Table 3
Potencies (IC50
derivatives at hA3AR
ascribed also to an additional hydrogen bond interactions of the
5-amide function with specific receptor sites.
) of some selected pyrazoloquinoline
Furthermore, the effect of compounds 2–4, 6 and 7 in limiting
the Cl-IB-MECA-inhibited cAMP accumulation in hA3 CHO cells
was determined. In accordance with their hA3 affinity, all the se-
lected pyrazoloquinoline derivatives proved to be very potent in
this test, showing an antagonistic behavior (Table 3).
The results obtained up to now on these new tricyclic AR antag-
onists are, in our opinion, not so intriguing to encourage further
investigation in such direction. However, we considered it very
interesting to biologically evaluate the synthetic pyrazole interme-
diates 20–24. These 3(5)-(2-aminophenyl)-5(3)-aryl-pyrazoles are
closely correlate with the hA2A AR antagonist 5-(2-hydroxy-
phenyl)-3-phenyl-1,2,4-triazole I23,24 which was re-tested in our
binding assays together with the pyrazole analogue 2-hydroxy-
phenyl substituted 25.20 Compound I turned out to be totally inac-
tive at the hA2A and hA2B AR subtypes, while it shows I% values of
cAMP assays hA3 IC50 (nM)a
324 32
2
3
4
6
7
88
82
9
8
138 13
263 25
a
IC50 values represent the mean SEM of four sep-
arate cAMP experiments in hA3 CHO cells, inhibited by
100 nM Cl-IB-MECA.
interaction is negligible. In contrast, introduction of either the 4-
methyl or the 4-chloro substituent (compound 3 and 4, respec-
tively) strongly affected the hA3 binding affinity probably by
increasing the hydrophobic interactions at the level of the 2-phe-
nyl substituent. Moreover, it is evident that introduction at posi-
tion-2 of the pyrazoloquinazoline scaffold of a 2-fluorophenyl
(compound 5), 2-furyl (6), 2-thienyl (7) or 2-pyridil (8) moiety im-
proves the hA3 receptor–ligand interactions with respect to the
parent 1. This could be due to the presence, in the 2-substituent,
of an heteroatom able to reinforce the binding with the receptor
through a profitable hydrogen bond. The improved hA3 binding
affinity of the 5-oxo-substituted compounds 5–8 compared to
the corresponding 2-phenyl derivative 1, suggested introduction
at position-2 of an alkyl carboxylate group (compounds 9–11), that
could ameliorate interaction with the receptor as hypothesized
above for the 2-substituent of compounds 5–8. As expected, com-
pounds 9–11 are endowed with hA3 binding affinity higher than
that of the reference 1, and comparable to that of the 2-pyridil
compound 8. The positive effect exerted by the alkyl 2-carboxylate
groups on this scaffold prompted us to introduce at position-2 a
benzyl-carboxylate ester and amide (compounds 12 and 13,
respectively) in order to increase hydrophobic interactions. Unfor-
tunately, this modification does not positively affect the hA3 affin-
ity as observed in other series of tricyclic AR antagonists.16 It is
clear that the A3 binding pocket which accommodates the 2-sub-
stituent has precise steric requirements, being compounds 12
and 13 completely inactive. In contrast, introduction of a further
ethyl carboxylate function at position-1 of 10 yielded compound
14 which shows a three-fold enhanced hA3 binding affinity with
respect to that of the parent mono-substituted 10. This result can
be interpreted considering the presence at the receptor level of a
cavity large enough to well accommodate this group.
39 and 47, respectively, at the hA3 and hA1 subtypes, at 1 lM con-
centration. It has to be noted that our biological result on the hA2A
AR (I = 2%) is in total disagreement with that reported in the liter-
ature (Ki = 0.7 lM), but can be considered in accordance with the
data obtained on the pyrazole analogue 25.
Interestingly, despite the extreme structural simplification of
these monocyclic compounds 20–24 with respect to the tricyclic
derivatives 15–18, some of them are endowed with hA3 binding
affinity in the l-molar range and, more importantly, they have to-
tal selectivity, being completely inactive at all the other AR sub-
types. However, the SAR obtained for these compounds do not
resemble those of the pyrazoloquinolines herein reported. In fact,
while the 3(5)-phenylpyrazole 20 is endowed with a hA3 affinity
in the high
l-molar range, the corresponding furyl substituted
derivative 22 is totally inactive at the same receptor subtype. An
opposite trend can be observed in the tricyclic series: the 2-phenyl
substituted compound 1 is totally inactive at the hA3 AR, while the
2-furyl substituted 6 shows high affinity and selectivity for this
receptor subtype.
4.2. Molecular modeling studies
To define the structural features at the base of the different
binding affinities of the new derivatives, a molecular docking anal-
ysis was performed on homology models of hA3AR developed by
using three recently published crystal structures of the A2AAR in
complex with the ZM241385 antagonist as templates (PDB code:
3EML; 2.6-Å resolution;29 PDB code: 3PWH; 3.3-Å resolution;30
PDB code: 3VG9; 2.7-Å resolution31). The A2AAR crystal structure
allows improvement of the accuracy of AR homology models, due
to the high residue conservation in the primary sequences of the
AR subtypes, which share a sequence identity of ꢁ57% within the
transmembrane (TM) domains.32 The residues located within the
seven TM domains in the upper part of ARs, corresponding to the
ligand binding site, are conserved with an average identity of
71%.33 Furthermore, the A2AAR crystal structure is solved in com-
plex with the high affinity antagonist ZM241385, hence presenting
a cavity suitable as binding site for docking analysis. The obtained
hA3AR homology models were checked by using the Protein Geom-
etry Monitor application within MOE,34 which provides a variety of
stereochemical measurements for inspection of the structural
quality in a given protein, such as backbone bond lengths, angles
However, although these new 5-oxo-pyrazoloquinazoline
derivatives 1–14 do not possess very high hA3 receptor affinity,
they are, without any doubt, highly selective due to their total
inactivity at all the other three AR subtypes.
The lower binding affinities of the 5-amino substituted deriva-
tives 15–18 with respect to those of the corresponding 5-oxo com-
pounds 1–2, 6–7 was expected12 even if not to such extent.
However, the dramatic increase of the hA2A, A2B and A1 binding
affinities of the 5-amino-2-furyl derivative 17 with respect to those
of the corresponding 2-phenyl one 15 confirms the profitable effect
exerted by the presence of the 2-furyl ring. It is, however, difficult
to explain not only the low or null hA3 affinity of both the 2-(2-fur-
yl) compound 17 and the corresponding 2-(2-thienyl) derivative
18, but also the total discrepancy of binding results between 18
and 17 at the other AR subtypes. In fact, these data are not in accor-
dance with those obtained on the 5-oxo-pyrazoloquinazoline 6, 7
and on other tricyclic hA3 AR antagonists.16 To increase the hydro-
phobic interactions of these 5-amino derivatives with the receptor
site, the 5-benzoylamino substituted derivative 19 was synthe-
sized. An ameliorated hA3 affinity was observed, which could be
and dihedrals, Ramachandran
u–w dihedral plots, and sidechain
rotamer and nonbonded contact quality.
Structures of hA3AR were then used as target for the docking
analysis of synthesized derivatives. All ligand structures were opti-
mized using RHF/AM1 semi-empirical calculations and the soft-
ware package MOPAC implemented in MOE was utilized for
these calculations.35 The compounds were then docked into the

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D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
binding site of the hA3AR models by using the MOE Dock tool. Top-
score docking poses of each compound were subjected to energy
minimization and then rescored using three available methods
implemented in MOE: the London dG scoring function that esti-
mates the free energy of binding of the ligand from a given pose;
the Affinity dG scoring tool that estimates the enthalpic contribu-
tion to the free energy of binding; the dock-pKi predictor that uses
the MOE scoring.svl script to estimate for each ligand a pKi value,
which is described by the H-bonds, transition metal interactions,
and hydrophobic interactions energy. For each compound, the
three top-score docking poses, according to at least two out of
three scoring functions, were selected for final ligand–target inter-
action analysis.
The docking methodology was first tested by comparing the pre-
dicted binding mode of ZM241385 within the A2AAR pocket to the
X-ray data. The docking method showed good ability to reproduce
the ZM241385 binding mode observed in the experimental data. In
particular, the RMSD from the comparison of the crystal structure
and the docking conformation was calculated as 1.4547, 1.3302,
and 2.7320 Å (for the 3EML, 3PWH, and 3VG9 crystal structure,
respectively), with the best matching being given by the 2-(furan-
2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-amine moiety and the
highest deviation being given by a slightly different orientation of
the 4-(2-aminoethyl)phenol group.
The three hA3AR models developed present highly similar bind-
ing sites by considering both pocket volumes and receptor residue
orientation. In the binding pocket only subtle rearrangements of
some flexible residues are observed, while higher conformational
variability is observed at EL domains in peripheral regions of the
binding site. As consequence, it is not a surprise that the docking
analysis of the synthesized compounds at the three receptor models
led to analogous results. Considering the general binding mode of
the 5-oxo-substituted pyrazoloquinazoline derivatives in the
hA3AR, two main sets of docking conformations were observed with
all three hA3AR models. The first set of conformations (from this
point on called ‘family 1’) are depicted in Figure 4 where compound
4 is shown as template. Family 1 conformations present the pyrazo-
loquinazoline moiety located approximately in correspondence
Figure 4. Panel A: Family 1 docking conformations, binding mode of compound 4
(yellow) at 3EML-based hA3AR model is shown as example; key receptor residues
are indicated. Panel B: Superimposition of the three hA3AR models and docking
conformations of compound 4; 3EML-, 3PWH-, and 3VG9-based hA3AR models are
colored in orange, magenta, and cyan, respectively; docking conformations of
compound 4 at the three receptor models are colored accordingly.
Figure 5. Panel A: Family 2 docking conformations, binding mode of compound 4
(yellow) at 3EML-based hA3AR model is shown as example; key receptor residues
are indicated. Panel B: Superimposition of the three hA3AR models and docking
conformations of compound 4; 3EML-, 3PWH-, and 3VG9-based hA3AR models are
colored in orange, magenta, and cyan, respectively; docking conformations of
compound 4 at the three receptor models are colored accordingly.

D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
289
with the [1,2,4]triazolo[1,5-a][1,3,5]triazine scaffold of the co-crys-
tallized ZM241385 antagonist. The 2-substituent is pointed towards
the central transmembrane core and located in a mainly hydropho-
bic sub-pocket in proximity of Leu90, Leu91, Met177, Trp243, and
Leu246. Superimposition of the family 1 docking conformations of
each compound at the three hA3AR models shows that the binding
modes and the interactions are almost identical at the three binding
sites (Fig. 4B). This data confirms the high conservation of residue
orientation within the core region of the binding site of the three
models.
The second set of conformations (‘family 2’ conformations,
Fig. 5) presents the pyrazoloquinazoline moiety located approxi-
mately in the same position as family 1 conformations but with
opposite orientation, hence presenting the 2-substituent pointed
towards the extracellular region. Superimposition of the family 2
docking conformations of each compound at the three A3AR mod-
els shows that there are some differences in compound orientation
(Fig. 5B). In particular the 2-substituent results differently posi-
tioned at the three A3AR models and this is due to only partial con-
servation of residue orientation at the extracellular region of the
three models.
Asn250, hence suggesting the presence of an aromatic group in
that compound position as structural requirement. In the case of
family 2 conformations, a H-bond interaction is still observed be-
tween pyrazoloquinazoline derivatives and Asn250, and it is given
by the 5-carbonyl group and the NH2 segment of the asparagine
amide moiety.
It must be underlined that the receptor affinity seems directly
correlated to compound hydrophobicity. As a consequence, given
the general pyrazoloquinazoline structure as constant, the different
compound hydrophobicity is provided by the chemical–physical
profile of 2-substituent. Considering its position within the binding
pocket, it can be observed that family 1 conformations present this
group inserted within TM3, TM5, and TM6 residues (Leu91, Thr94,
Met177, Ser181, Ile186, Trp243, Ser247, and Asn250) in a sub-pock-
et characterized mainly by hydrophobic properties. The different
chemical–physical profile of the 2-substituent could hence enhance
or reduce the interaction with this sub-pocket. In the case of family
2 conformations, the 2-substituent is partially exposed to the extra-
cellular environment. In this case the role of this substituent could
be mainly related to its impact in compound solvation/desolvation,
with the less water soluble compounds having higher affinity with
respect to the more hydrophilic ones. These results are somehow
confirmed by comparing the docking scores with the pKi data. In
fact, best matching between docking scores and binding affinity
trends is given by two scoring functions of MOE, dock-pKi and Affin-
ity dG (Fig. 6). The first tool is based on an empirical scoring function
consisting of a directional hydrogen-bonding term, a directional
hydrophobic interaction term, and an entropic term (ligand rotat-
able bonds immobilized in binding). In particular the trend of the
hydrophobic interaction term values for the top scoring pose of
each compound present high correspondence with pKi binding data
tendency. The second scoring function shows significant correspon-
dence with logS and logP data, hence highlighting the importance
of compound solubility for receptor activity. It is important to point
out that, in any case, if on one hand higher hydrophobicity is often
correlated with higher binding activity, on the other hand hydro-
phobicity that is too high makes compounds difficult to be handle
in experimental (and therapeutic) conditions. LogP analysis of all
synthesized compounds shows that they all present predicted logP
values <4, hence still in an acceptable range.
Considering both families of docking conformations, the 5-oxo-
pyrazoloquinazoline derivatives herein reported form hydrophobic
interactions with the hA3 AR binding cleft, such as the p-stacking
interaction between the compound scaffold and the Phe168 resi-
due within the EL2 segment. In contrast, the double polar interac-
tion of asparagine 6.55 amide group (Asn250) with both the NH2
group and the N1 atom of ZM241385 (particularly evident in the
3EML crystal structure) is not observed for the 5-oxo pyrazoloqui-
nazolines. It has to be noted that the asparagine residue 6.55, con-
served among all ARs subtypes, was found to be important for
ligand binding at both hA3 and hA2A ARs. Considering the family
1 conformations, the possible interactions of Asn250 with both
the 5-oxo substituent and the N3 atom of the ligand is reduced
to a single interaction. In fact, only a single H-bond link between
the 5-oxo group or the 3-nitrogen atom and the Asn250 amine
function is allowed, due also to the steric hindrance of the 2-aro-
matic ring that obstructs second possible interaction.
Replacement of the 2-aromatic substituent with an alkyl car-
boxylate group does not significantly improve the interaction with
Figure 6. Comparison of docking scores with experimental binding data. Plots of dock-pKi scores versus experimental pKi data and Affinity dG scores versus experimental pKi
data from docking analysis at 3EML-based hA3 AR model are shown.

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D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
A docking analysis was performed also to simulate the possible
exchangeable protons were confirmed by addition of D2O. The fol-
lowing abbreviations are used: s = singlet, d = doublet, dd = double
doublet, t = triplet, m = multiplet, br = broad, ar = aromatic protons.
binding modes of simplified pyrazole derivatives 20–24 at hA3AR
binding site. The same docking and post-docking protocols were
employed.
Due to their greater flexibility, these compounds present several
combinations of conformation and orientation within the receptor
binding site. Among them, two binding modes are particularly
interesting due to their similarity with the above described family
1 and 2 docking conformations of pyrazoloquinazoline derivatives.
The similarity is given in particular by a matching of the pyrazole
ring of simplified and tricyclic compounds and a matching of the
aromatic ring of the 2-aminophenyl group of pyrazole derivatives
with the phenyl ring of the pyrazoloquinazoline scaffold, with
the nitrogen atom of the free amino group of pyrazole derivatives
being almost superimposed to the nitrogen atom at 6 position of
the pyrazoloquinazoline scaffold (Fig. 7). Compounds 20 and 4
are shown as template for the pyrazole and tricyclic derivatives,
respectively. The planarity of the simplified pyrazole derivatives
is given by an internal H-bond interaction between the free amino
group and one of the nitrogen atoms of the pyrazole scaffold. These
compounds do not present the carbonyl group of the tricyclic
derivatives hence the H-bond acceptor function interacting with
Asn250 is missing. Therefore, while the planarity typical of AR
antagonists is conserved, on the other hand the ability of the com-
pound to give hydrophilic interactions with binding site residues is
partially reduced with respect to the original tricyclic derivatives.
Further optimization of these simplified derivatives could possibly
restore the H-bonding ability.
6.1.1. General method for the synthesis of 1-(hetero)aryl-3-(2-
nitrophenyl)-2-propen-1-ones (26–28)
A solution of equimolar amounts (16 mmol) of 2-nitrobenzalde-
hyde and the suitable (etero)aryl methyl ketone in glacial acetic
acid (8 mL) was treated with boron trifluoride acetic acid complex
(48 mL). The reaction mixture was stirred at room temperature un-
til the disappearance of the starting material (TLC monitoring). Sat-
urated solution of sodium acetate (8 mL) and 20% NaOH solution
were added to the resulting solution until pH 7 was reached. The
solid which precipitates, was then collected by filtration and
washed with water.
6.1.1.1. 1-(4-Methoxyphenyl)-3-(2-nitrophenyl)-2-propen-1-one
(26)26
. Yield: 78%, mp 112–114 °C (cyclohexane/EtOAc) (Lit. mp
93–94 °C from EtOH); 1H NMR(CDCl3) d 3.92 (s, 3H, OCH3), 7.01
(d, 2H, ar, J = 8.95 Hz), 7.35 (d, 1H, H-2, J = 15.64 Hz), 7.58 (t, 1H,
ar, J = 7.42 Hz), 7.70 (t, 1H ar, J = 7.42 Hz), 7.76 (d, 1H, ar,
J = 7.42 Hz), 8.05 (d, 2H ar, J = 8.95 Hz), 8.09 (d, 1H, ar,
J = 7.42 Hz), 8.15 (d, 1H, H-3, J = 15.64 Hz), IR: 1653, 1571, 1353.
Anal. Calcd for C16H13NO4: C, 67.84; H, 4.63; N, 4.94. Found C,
68.02; H, 4.39; N, 5.13.
6.1.1.2. 1-(2-Furyl)-3-(2-nitrophenyl)-2-propen-1-one (27)27
. Col-
umn chromatography, eluting system cyclohexane/EtOAc 1:1;
yield: 41%; mp 132–135 °C (EtOH); 1H NMR (DMSO-d6) d 6.82–
6.84 (m, 1H, ar), 7.69–7.74 (m, 2H, ar+H-2), 7.85 (t, 1H, ar,
J = 7.60 Hz), 7.89 (d, 1H, ar, J = 3.56 Hz), 7.99 (d, 1H, H-3,
J = 15.61 Hz), 8.10–8.16 (m, 3H, ar). IR: 1690, 1550, 1330. Anal.
Calcd for C13H9NO4: C, 64.20; H, 3.73; N, 5.76. Found C, 64.03; H,
3.98; N, 5.82.
5. Conclusion
The present study has led to the identification of some pyrazol-
o[1,5-c]quinazoline derivatives as hA3 AR antagonists. In particular,
the 5-oxo-substituted derivatives (series B-1) are endowed with
good hA3 receptor affinity and high selectivity, being totally inac-
tive at all the other AR subtypes. In contrast, the corresponding
5-amino compounds (series B-2) do not bind or bind with very
low affinity at the hA3 AR the only exception being the 5-N-benzoyl
6.1.1.3. 1-(2-Thienyl)-3-(2-nitrophenyl)-2-propen-1-one (28)28
Yield: 50%; mp 133–134 °C (EtOH) (Lit. mp 136 °C); 1H NMR
(DMSO-d6) d: 7.35 (m, 1H, ar), 7.72 (t, 1H, ar, J = 8.36 Hz), 7.84–7.90
(m, ar+H-2), 7.99 (d, 1H, H-3, J = 15.49 Hz), 8.10–8.14 (m, 2H, ar),
8.20 (d, 1H, ar, J = 7.00 Hz), 8.38 (m, 1H, ar). IR: 1690, 1550, 1330.
Anal. Calcd for C13H9NO3S: C, 60.22; H, 3.50; N, 5.40. Found C,
59.99; H, 3.29; N, 5.31.
.
derivative 19 that shows a hA3 Ki value in the high l-molar range.
Moreover, the synthetic intermediates 20–24, despite their simpli-
fied structure, possess the minimal molecular requirements for
binding at the hA3 AR, showing modest binding affinity for this
receptor subtype but total selectivity versus all the other ARs.
Molecular docking of tricyclic and simplified derivatives identified
their hypothetical binding mode to our model of hA3 receptor. As a
new finding, the pyrazole derivatives emerged as lead candidates
for the development of new monocyclic AR antagonists with im-
proved physicochemical properties and, as a consequence, better
bioavailability with respect to tricyclic compounds.
6.1.2. General procedure for the synthesis of 4,5-dihydro-3-
(hetero)aryl-5-(2-nitrophenyl)-1H-pyrazoles (29–31)
A solution of the suitable 1-(hetero)aryl-3-(2-nitrofenil)-2-pro-
pen-1-one 26–28 (6.7 mmol) and hydrazine hydrate (65%,
18.1 mmol) in EtOH (50 mL) was heated at reflux until the disap-
pearance of the starting material (TLC monitoring). After cooling,
a solid precipitated (compounds 29, 31) which was collected by fil-
tration and washed with diethyl ether. A second crop of the desired
compound was obtained after evaporation under reduced pressure
of the mother ethanolic liquor and work-up with diethyl ether. The
resulting crude product was purified by crystallization. In the case
of compound 30, the resulting solid was removed by filtration and
the solvent was evaporated under reduced pressure. The residue
was purified by column chromatography, eluting system methy-
lene chloride/EtOAc 9:1, to yield a dark red oil which was the pure
product.
6. Experimental section
6.1. Chemistry
Silica gel plates (Merck F254) and silica gel 60 (Merck; 70–230
mesh) were used for analytical and column chromatography,
respectively. All melting points were determined on a Gallenkamp
melting point apparatus. Microanalyses were performed with a
Flash E1112 Thermofinnigan elemental analyzer for C, H, N, and
the results were within 0.4% of the theoretical values except
where stated otherwise. The IR spectra were recorded with a Per-
kin–Elmer Spectrum RX I spectrometer in Nujol mulls and are ex-
pressed in cmꢂ1. The 1H NMR spectra were obtained with a Bruker
Avance 400 MHz instrument. The chemical shifts are reported in d
(ppm) and are relative to the central peak of the solvent. All the
6.1.2.1. 4,5-Dihydro-3-(4-methoxyphenyl)-5-(2-nitrophenyl)-1H-
pyrazole (29). Yield: 88%; mp 72–73 °C (cyclohexane); 1H NMR
(DMSO-d6) d: 1H NMR (CDCl3) d: 2.94 (dd, 1H, H-4, J = 16.6,
9.92 Hz), 3.73 (dd, 1H, H-4, J = 16.56, 10.80 Hz), 3.81 (s, 3H,
OCH3), 5.34 (t, 1H, H-5, J = 10.28 Hz), 6.14 (br s, 1H, NH), 6.88 (d,
2H, ar, J = 8.76 Hz), 7.41 (t, 1H, ar, J = 8.00 Hz), 7.58–7.62 (m, 3H,

D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
291
1251. Anal. Calcd for C13H11N3O2S: C, 57.13; H, 4.06; N,15.37.
Found C, 57.08; H, 4.27; N, 15.09.
6.1.3. General procedure for the synthesis of 3(5)-(hetero)aryl-
5(3)-(2-nitrophenyl)pyrazoles (32–34)
A solution of lead tetraacetate (3.74 mmol) in anhydrous
dichloromethane (25 mL) was added dropwise to a solution of
equimolar amount of 29–31 in anhydrous dichloromethane
(25 mL). The addition of the oxidant agent was performed in about
20 min. The reaction mixture was stirred at room temperature for
15 min, and then, after addition of water (70 mL), a solid precipi-
tated, which was removed by filtration. After separation of the
resulting two layers, the organic solution was washed with water
(50 mL ꢃ 3), anhydrified (Na2SO4) and evaporated under reduced
pressure. The dark red oily residue was dissolved in a mixture of
concentrated HCl (0.15 mL) and EtOH (20 mL) and heated at reflux
for 30 min. The resulting solution was diluted with 2.5% NaHCO3
and extracted with dichloromethane (50 mL). The organic layer
was washed with water (50 mL ꢃ 3), anhydrified (Na2SO4) and
evaporated under reduced pressure. The resulting crude product
was purified by column chromatography, eluting system dichloro-
methane/EtOAc 9:1.
6.1.3.1. 3-(4-Methoxyphenyl)-5-(2-nitrophenyl)pyrazole (32).
Yield: 60%; mp 148–149 °C (cyclohexane/EtOAc); 1H NMR
(DMSO-d6) d: 3.81 (s, 3H, OCH3), 6.94 (s, 1H, pyrazole), 7.06 (d,
2H, ar, J = 8.56 Hz), 7.56 (t, 1H, ar, J = 7.56 Hz), 7.70–7.73 (m, 3H,
ar), 7.81 (d, 1H, ar, J = 7.96 Hz), 7.85 (d, 1H, ar, J = 7.68 Hz), 13.48 (s,
1H, NH); IR: 3215, 1511, 1462. Anal. Calcd for C16H13N3O3: C,
65.08; H, 4.44; N, 14.23. Found C, 65.01; H, 4.57; N, 14.01.
6.1.3.2. 3-(2-Furyl)-5-(2-nitrophenyl)pyrazole (33)21
44%; mp 121–123 °C (Lit. mp 121 °C dec) Anal. Calcd for
13H9N3O3 C, 61.18; H, 3.55; N, 16.46. Found C, 59.92; H, 3.29; N,
. Yield:
C
16.70.
6.1.3.3. 5-(2-Nitrophenyl)-3-(2-thienyl)pyrazole (34)21
. Yield:
52%; mp 104–106 °C (Lit. mp 105–106 °C) C; Anal. Calcd for
13H9N3O2S: C, 57.55; H, 3.34; N, 15.49. Found C, 57.48; H, 3.08;
C
N, 15.27.
6.1.4. General procedure for the synthesis of 3(5)-(hetero)aryl-
5(3)-(2-aminophenyl)pyrazoles (21–23)
Figure 7. Docking conformations of simplified pyrazoles at 3EML-based hA3AR
model and comparison with family 1 (Panel A) and 2 (Panel B) docking conforma-
tions of pyrazoloquinazoline derivatives. Compounds 20 (ball and sticks, cyan) and
4 (line, yellow) are shown. Key receptor residues are indicated.
A mixture of 32–34 (1.7 mmol) and 30% (w/w) Pd/C (10%) in
EtOAc (100 mL) was hydrogenated in a Parr apparatus at 30 psi un-
til the disappearance of the starting material. Elimination of the
catalyst and evaporation of the solvent at reduced pressure gave
a solid which was purified by crystallization.
ar); 7.93 (m, 2H, ar); IR: 3353, 1515, 1463, 1341, 1250. Anal. Calcd
for C16H15N3O3: C,64.64; H,5.09; N,14.13. Found C, 64.48; H, 4.89;
N, 13.96.
6.1.4.1. 5-(2-Aminophenyl)-3-(4-methoxyphenyl)pyrazole
(21). Yield: 90%; mp 172–173 °C (toluene); 1H NMR (DMSO-d6) d
3.81 (s, 3H, OCH3), 6.35 (s, 2H, NH2), 6.59 (t, 1H, ar, J = 7.32 Hz),
6.74 (d, 1H, ar, J = 8.00 Hz), 7.00 (t, 1H, ar, J = 7.68 Hz), 7.05–7.07
(m, 2H, ar+pyrazole), 7.57 (d, 1H, ar, J = 7.60 Hz), 7.76 (d, 2H, ar,
J = 8.52 Hz), 13.15 (br s, 1H, NH); IR: 3363, 3291, 1610, 1459. Anal.
Calcd for C16H15N3O: C, 72.43; H, 5.70; N, 15.84. Found C, 72.31; H,
5.98; N, 16.02.
6.1.2.2. 4,5-Dihydro-3-(2-furyl)-5-(2-nitrophenyl)-1H-pyrazole
(30). Yield: 84%; (oil) 1H NMR (CDCl3) d 2.99 (dd, 1H, H-4,
J = 16.64, 9.8 Hz) 3.57 (dd, 1H, H-4, J = 16.52, 10.92 Hz), 5.42 (t,
1H, H-5, J = 10.36 Hz), 6.04 (s, 1H, NH), 6.48 (dd, 1H, ar, J = 3.40,
1.84 Hz), 6.58 (d, 1H, ar, J = 3.36 Hz), 7.47 (t, 1H, ar, J = 8.44 Hz),
7.51 (d, 1H, ar, J = 1.84 Hz), 7.67 (t, 1H, ar, J = 8.44 Hz), 7.95 (d,
1H, ar, J = 7.92 Hz), 8.01 (d, 1H, ar, J = 8.17 Hz); IR: 3300, 1650,
1550, 1340, 1250. Anal. Calcd for C13H11N3O3: C, 60.70; H, 4.31;
N, 16.33. Found C, 60.58; H, 4.23; N, 16.17.
6.1.4.2. 5-(2-Aminophenyl)-3-(2-furyl)pyrazole (22)21
90%; mp 127–128 °C (Lit. mp 127–128 °C). Anal. Calcd for
13H11N3O: C, 69.32; H, 4.92; N, 18.66. Found C, 69.54; H, 5.11;
. Yield:
C
6.1.2.3. 4,5-Dihydro-5-(2-nitrophenyl)-3-(2-thienyl)-1H-pyra-
zole (31). Yield: 66%; mp 79–81 °C (MeOH); 1H NMR (DMSO-d6) d
2.97 (dd, 1H, H-4, J = 16.52, 9.72), 3.62 (dd, 1H, H-4, 16.56, 11.08),
5.18 (td, 1H, H-5, J = 10.44, 3.56), 7.06 (dd, 1H, ar, J = 5.0, 3.64 Hz),
7.17 (d, 1H, ar, J = 4.28), 7.51–7.58 (m, 3H, ar+NH), 7.73–7.81 (m,
2H, ar), 7.99 (d, 1H, ar, J = 8.96 Hz); IR: 3340, 1654, 1540, 1338,
N, 18.84.
6.1.4.3. 5-(2-aminophenyl)-3-(2-thienyl)pyrazole (23)21
. Yield:
70%; mp 174–177 °C (Lit. mp 176–178 °C) Anal. Calcd for
13H11N3S: C, 64.70; H, 4.59; N, 17.41. Found C, 64.53; H, 4.72; N,
17.19.
C

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D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
6.1.5. General procedure for the synthesis of 5,6-dihydro-2-
(hetero)aryl-pyrazolo[1,5-c]quinazolin-5-ones (2, 6–7)
Triethylamine (3.36 mmol) and triphosgene (0.56 mmol) were
successively added to a solution of 21–23 (1.4 mmol) in anhydrous
tetrahydrofuran (10 mL). The reaction mixture was stirred at room
temperature until the disappearance of the starting material and
then diluted with water (30 mL). The resulting solid was collected
by filtration and washed with water.
6.1.6.4. 2-(2-Thienyl)-pyrazolo[1,5-c]quinazolin-5-amine
(18). Yield: 71%; mp 170–171 °C (2-methoxyethanol); 1H NMR
(DMSO-d6) d: 7.22–7.24 (m, 1H, ar), 7.32 (t, 1H, ar, J = 7.40 Hz),
7.48–7.61 (m, 4H, ar+NH2), 7.62 (s, 1H, pyrazole), 6.69 (d, 1H, ar,
J = 4.88 Hz), 7.74 (d, 1H, ar, J = 3.48 Hz), 8.07 (d, 1H, ar
J = 7.68 Hz); IR: 3441, 1462. Anal. Calcd for C14H10N4S: C, 63.14;
H, 3.78; N, 21.04. Found C, 62.99; H, 4.01; N, 20.88.
6.1.7. 2-Phenylpyrazolo[1,5-c]quinazolin-5-benzamide (19)
A solution of benzoyl chloride (3 mmol) in anhydrous dichloro-
methane (5 mL) was dropwise added to a cold (0 °C) suspension of
15 (1 mmol) in anhydrous dichloromethane (30 mL) and pyridine
(10 mmol). The reaction mixture was stirred at room temperature
for 12 h. The resulting solution was washed with water
(30 mL ꢃ 3), anhydrified (Na2SO4) and evaporated under reduced
pressure to yield a red oily residue which was purified by silica
gel column chromatography, eluting system cyclohexane/EtOAc
6:4. Yield: 33%; mp 178–180 °C (EtOH); 1H NMR (DMSO-d6) d:
7.46 (t, 1H, ar, J = 7.28 Hz), 7.53 (t, 2H, ar, J = 7.38 Hz), 7.60–7.79
(m, 5H, ar), 7.86 (d, 1H, ar, J = 7.88 Hz), 7.96 (s, 1H, pyrazole),
8.06 (d, 2H, ar, J = 7.12 Hz), 8.13–8.16 (m, 2H, ar), 8.30 (d, 2H, ar,
J = 7.81 Hz), 11.29 (s, 1H, NH); IR: 3350, 1632. Anal. Calcd for
6.1.5.1. 5,6-Dihydro-2-(4-methoxyphenyl)pyrazolo[1,5-c]qui-
nazolin-4-one (2). Yield: 88%; mp >300 °C (acetic acid); 1H
NMR (DMSO-d6) d: 3.84 (s, 3H, OCH3), 7.09 (d, 2H, ar,
J = 8.76 Hz), 7.31–7.36 (m, 2H, ar), 7.53 (t, 1H, ar, J = 7.07 Hz),
7.67 (s, 1H, pyrazole), 7.96 (d, 2H, ar, J = 8.72 Hz), 8.05 (d, 1H, ar,
J = 7.84 Hz), 11.82 (s, 1H, NH); IR: 3197, 1725, 1682, 1453, 1376,
1254. Anal. Calcd for C17H13N3O2: C, 70.09; H, 4.50; N, 14.42. Found
C, 69.81; H, 4.70; N, 14.01.
6.1.5.2. 5,6-Dihydro-2-(2-furyl)pyrazolo[1,5-c]quinazolin-4-one
(6)21
. Yield: 90%; mp 303–304 °C (acetic acid) (Lit. mp 304–
305 °C); Anal. Calcd for C14H9N3O2: C, 66.93; H, 3.61; N, 16.73.
Found C, 67.01; H, 3.41; N, 16.58.
C23H16N4O: C, 75.81; H, 4.43; N, 15.38. Found C, 76.00; H, 4.21;
N, 15.60.
6.1.5.3. 5,6-Dihydro-2-(2-thienyl)pyrazolo[1,5-c]quinazolin-4-
. Yield: 75%; mp 297–299 °C (acetic acid) (Lit. mp
299–300 °C); Anal. Calcd for C14H9N3OS: C, 62.91; H, 3.39; N,
one (7)21
6.1.8. 5,6-Dihydro-5-oxo-pyrazolo[1,5-c]quinazolin-2-
carboxylic acid (35)21
A suspension of 1020 (6.64 mmol) in a mixture of glacial acetic
acid (26 mL) and 6 N HCl (5 mL) was heated at reflux for 30 h. After
cooling a solid precipitated which was collected by filtration and
washed with water. Yield: 91%; mp 283 °C dec (acetic acid/H2O)
(Lit. mp 282 °C dec). Anal. Calcd for C11H7N3O3: C, 57.65; H, 3.08;
N, 18.33. Found C, 57.80; H, 3.00; N, 18.25.
15.72. Found C, 63.12; H, 3.18; N, 15.98.
6.1.6. General procedure for the synthesis of 2-(hetero)aryl-
pyrazolo[1,5-c]quinazolin-5-amines (15–18)
A solution of 20–23 (1.06 mmol), para-toluensulfonic acid
(1.06 mmol) and an excess of cyanamide (6.12 mmol) in N-
methyl-2-pyrrolidone (5 mL) was heated at reflux until the disap-
pearance of the starting material (TLC monitoring). The reaction
mixture was diluted with EtOAc (100 mL) and the resulting solid
was eliminated by filtration. After concentration up to about
30 mL, the organic solution was washed with water (20 mL ꢃ 3)
and anhydrified (Na2SO4). Evaporation of the solvent under re-
duced pressure gave a residue which was purified by silica gel col-
umn chromatography, eluting system cyclohexane/EtOAc 5:5
(compounds 15–16) or CHCl3/MeOH 9.5:0.5 (compounds 17–18).
6.1.9. Benzyl 5,6-dihydro-5-oxo-pyrazolo[1,5-c]quinazolin-2-
carboxylate (12)
A suspension of 35 (1.74 mmol) in thionyl chloride (20 mL) was
heated at reflux for 4 h. After evaporation at reduced pressure of
the excess of thionyl chloride, the last traces of the chlorinating
agent were removed by repeated treatment of the resulting residue
with cyclohexane (30 mL ꢃ 3) and subsequent distillation of the
solvent. A suspension of the crude 5,6-dihydro-5-oxo-pyrazol-
o[1,5-c]quinazolin-2-carbonyl
chloride
in
tetrahydrofuran
(50 mL) and benzyl alcohol (23 mL) was heated at reflux for 7 h.
Elimination of the solvent by evaporation at reduced pressure gave
a solid which was dissolved in EtOAc (70 mL). The organic layer
was washed with water (40 mL ꢃ 4) and anhydrified (Na2SO4).
Evaporation of the solvent under reduced pressure gave an oily res-
idue which solidified after treatment with EtOH. Yield: 43%; mp
270–272 °C (2-methoxyethanol); 1H NMR (DMSO-d6) d: 5.43 (s,
2H, CH2), 7.29–7.46 (m, 5H, ar), 7.51–7.57 (m, 3H, ar), 7.75 (s,
1H, pyrazole), 8.17 (d, 1H, ar, J = 7.84 Hz), 12.12 (s, 1H, NH); IR:
3232, 1733, 1710. Anal. Calcd for C18H13N3O3: C, 67.71; H, 4.10;
N, 13.16. Found C, 67.49; H, 4.29; N, 13.00.
6.1.6.1. 2-Phenylpyrazolo[1,5-c]quinazolin-5-amine (15). Yield:
40%; mp 233–235 °C (EtOAc); 1H NMR (DMSO-d6) d: 7.32 (t, 1H,
ar, J = 7.35 Hz), 7.45–7.57 (m, 5H, ar), 7.60 (s, 2H, NH2), 7.73 (s,
1H, pyrazole), 8.08–8.13 (m, 3H, ar); IR: 3450, 3405. Anal. Calcd
for C16H12N4: C, 73.83; H, 4.65; N, 21.52. Found C, 74.06; H, 4.81;
N, 21.76.
6.1.6.2. 2-(4-Methoxyphenyl)-pyrazolo[1,5-c]quinazolin-5-
amine (16). Yield: 87%; mp 243–244 °C (toluene); 1H NMR
(DMSO-d6) d: 3.84 (s, 3H, OCH3), 7.09 (d, 2H, ar, J = 7.12 Hz), 7.30
(t, 1H, ar, J = 7.24 Hz), 7.47–7.55 (m, 4H, ar+NH2), 7.62 (s, 1H, pyr-
azole), 8.03–8.07 (m, 3H, ar); IR: 3447, 1610, 1461. Anal. Calcd for
6.1.10. 5,6-Dihydro-5-oxo-pyrazolo[1,5-c]quinazolin-2-
benzylcarboxamide (13)
C17H14N4O: C, 70.33; H, 4.86; N, 19.30. Found C, 70.51; H, 5.01; N,
19.12.
A
solution of 35 (1.74 mmol), benzylamine (2.60 mmol),
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
(1,74 mmol) and 1-hydroxybenzotriazole hydrate (2 mmol) in
anhydrous dimethylformamide (33 mL) was heated at reflux for
5 h. After cooling the resulting solution was diluted with water
(70 mL) and the resulting solid was collected by filtration and
washed with water. Yield: 52%; mp >300 °C (2-methoxyethanol);
1H NMR (DMSO-d6) d: 4.49 (d, 2H, CH2, J = 6.28 Hz), 7.23–7.26
(m, 1H, ar), 7.30–7.38 (m, 6H, ar), 7.55 (t, 1H, ar, J = 7.24 Hz),
6.1.6.3. 2-(2-Furyl)-pyrazolo[1,5-c]quinazolin-5-amine (17). Yield:
37%; mp 253–255 °C (EtOAc); 1H NMR (DMSO-d6) d: 6.70–7.73 (m,
1H, ar), 7.08 (d, 1H, ar, J = 3.21 Hz), 7.33 (t, 1H, ar, J = 7.28 Hz),
7.49–7.58 (m, 3H, ar), 7.70 (s br, 2H, NH2), 7.90 (s, 1H, pyrazole),
8.10 (d, 1H, ar, J = 7.56 Hz); IR: 3445, 3405, 1676. Anal. Calcd for
C14H10N4O: C, 67.19; H, 4.03; N, 22.39. Found C, 69.95; H, 3.86;
N, 22.01.

D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
293
7.61 (s, 1H, pyrazole), 8.14 (d, 1H, ar, J = 7.16 Hz), 9.24 (t, 1H, NH,
J = 6.20 Hz), 12.01 (s br, 1H, NH); IR: 339, 3111, 1732. Anal. Calcd
for C18H14N4O2: C, 67.91; H, 4.43; N, 17.60. Found C, 68.08; H,
4.15; N, 17.90.
coordinates were kept fixed. AMBER99 partial charges of receptor
and MOPAC output partial charges of ligands were utilized. Once
the compound-binding site energy minimization was completed,
receptor coordinates were fixed and a second energy minimization
stage was performed leaving free to move only compound atoms.
MMFF9437–43force field was applied. For each compound, the min-
imized docking poses were then rescored using London dG and
Affinity dG scoring functions and the dock-pKi predictor. The latter
tool allows estimation of the pKi for each ligand using the ‘scor-
ing.svl’ script retrievable at the SVL exchange service (Chemical
Computing Group, Inc. SVL exchange: http://svl.chemcomp.com).
The algorithm is based on an empirical scoring function consisting
of a directional hydrogen-bonding term, a directional hydrophobic
interaction term, and an entropic term (ligand rotatable bonds
immobilized in binding). For each compound, the three top-score
docking poses according to at least two out of three scoring func-
tions were selected for final ligand–target interaction analysis.
6.2. Computational methodologies
All molecular modeling studies were performed on a 2 CPU (PIV
2.0-3.0 GHZ) Linux PC. Homology modeling, energy minimization,
and docking studies were carried out using Molecular Operating
Environment (MOE, version 2010.10) suite. All ligand structures
were optimized using RHF/AM1 semi-empirical calculations and
the software package MOPAC implemented in MOE was utilized
for these calculations.
6.2.1. Homology modeling of the human A3AR
Homology models of the hA3AR were built using recently solved
X-ray structures of the hA2AAR in complex with ZM241385 as tem-
plates (pdb code: 3EML; 2.6-Å resolution;29 PDB code: 3PWH; 3.3-
Å resolution;30 PDB code: 3VG9; 2.7-Å resolution31). A multiple
alignment of the AR primary sequences was built within MOE as
preliminary step. For all hA3AR models, the boundaries identified
from the utilized X-ray crystal structure of hA2AAR were then ap-
plied for the corresponding sequences of the TM helices of the
hA3AR. The missing loop domains were built by the loop search
method implemented in MOE. Once the heavy atoms were mod-
eled, all hydrogen atoms were added, and the protein coordinates
were then minimized with MOE using the AMBER99 force field.36
The minimizations were performed by 1000 steps of steepest des-
cent followed by conjugate gradient minimization until the RMS
6.3. Pharmacology
6.3.1. Human cloned A1, A2A and A3 AR binding assay
All synthesized compounds were tested to evaluate their affin-
ity at hA1, hA2A and hA3 adenosine receptors. Displacement exper-
iments of [3H]DPCPX (1 nM) to hA1 CHO membranes (50
lg of
protein/assay) and at least six to eight different concentrations of
antagonists for 120 min at 25 °C in 50 mM Tris HCl buffer pH 7.4
were performed.44 Non specific binding was determined in the
presence of 1
l
M of DPCPX (610% of the total binding). Binding
g of pro-
of [3H]ZM-241385 (1 nM) to hA2ACHO membranes (50
l
tein/assay) was performed using 50 mM Tris HCl buffer, 10 mM
MgCl2 pH 7.4 and at least six to eight different concentrations of
antagonists studied for an incubation time of 60 min at 4 °C.45
gradient of the potential energy was less than 0.05 kJ molꢂ1 Åꢂ1
.
Reliability and quality of these models were checked using the Pro-
tein Geometry Monitor application within MOE, which provides a
variety of stereochemical measurements for inspection of the
structural quality in a given protein, like backbone bond lengths,
Non specific binding was determined in the presence of 1
lM
ZM241385 and was about 20% of total binding. Competition bind-
ing experiments to hA3 CHO membranes (50 lg of protein/assay)
angles and dihedrals, Ramachandran
u
–w
dihedral plots, and side-
and 0.5 nM [125I]AB-MECA, 50 mM Tris HCl buffer, 10 mM MgCl2,
1 mM EDTA, pH 7.4, were conducted, and at least six to eight dif-
ferent concentrations of examined ligands for 120 min at 4 °C.46
Non specific binding was defined as binding in the presence of
chain rotamer and nonbonded contact quality.
6.2.2. Molecular docking analysis
All compound structures were docked into the binding site of
the three hA3AR models using the MOE Dock tool. This method is
divided into a number of stages. Conformational analysis of ligands:
The algorithm generated conformations from a single 3D confor-
mation by conducting a systematic search. In this way, all combi-
nations of angles were created for each ligand. Placement: A
collection of poses was generated from the pool of ligand confor-
mations using Triangle Matcher placement method. Poses were
generated by superposition of ligand atom triplets and triplet
points in the receptor binding site. The receptor site points are al-
pha sphere centers which represent locations of tight packing. At
each iteration a random conformation was selected, a random trip-
let of ligand atoms and a random triplet of alpha sphere centers
were used to determine the pose. Scoring: Poses generated by the
placement methodology were scored using two available methods
implemented in MOE, the London dG scoring function which esti-
mates the free energy of binding of the ligand from a given pose,
and Affinity dG scoring which estimates the enthalpic contribution
to the free energy of binding. The top 30 poses for each ligand were
output in a MOE database.
1 lM AB-MECA and was about 20% of total binding. Bound and free
radioactivity were separated by filtering the assay mixture through
Whatman GF/B glass fiber filters using a Brandel cell harvester. The
filter bound radioactivity was counted by Scintillation Counter
Packard Tri Carb 2810 TR with an efficiency of 62%.
6.3.2. Measurement of cyclic AMP levels in CHO cells transfected
with hA2B or hA3 ARs
CHO cells transfected with hAR subtypes were washed with
phosphate-buffered saline, diluted tripsine and centrifuged for
10 min at 200 g. The pellet containing the CHO cells (1 ꢃ 106
cells/assay) was suspended in 0.5 ml of incubation mixture
(mM): NaCl 15, KCl 0.27, NaH2PO4 0.037, MgSO4 0.1, CaCl2 0.1,
Hepes 0.01, MgCl2 1, glucose 0.5, pH 7.4 at 37 °C, 2 IU/ml adenosine
deaminase and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone
(Ro 20-1724) as phosphodiesterase inhibitor and preincubated
for 10 min in a shaking bath at 37 °C. The potency of antagonists
to A2B ARs was determined by antagonism of NECA (200 nM)-in-
duced stimulation of cyclic AMP levels. In addition, the potency
of antagonists to A3 receptors was determined in the presence of
forskolin 1 lM and Cl-IB-MECA (100 nM) that mediated inhibition
6.2.3. Post docking analysis
of cyclic AMP levels. The reaction was terminated by the addition
of cold 6% trichloroacetic acid (TCA). The TCA suspension was cen-
trifuged at 2000 g for 10 min at 4 °C and the supernatant was ex-
tracted four times with water saturated diethyl ether. The final
aqueous solution was tested for cyclic AMP levels by a competition
protein binding assay. Samples of cyclic AMP standard (0–10
The five top-score docking poses of each compound were then
subjected to AMBER99 force field energy minimization until the
RMS gradient of the potential energy was less than
0.05 kJ molꢂ1 Åꢂ1. Receptor residues within 6 Å distance from the
ligand were left free to move, while the remaining receptor

294
D. Catarzi et al. / Bioorg. Med. Chem. 21 (2013) 283–294
pmoles) were added to each test tube containing [3H] cyclic AMP
and the incubation buffer (trizma base 0.1 M, aminophylline
8.0 mM, 2-mercaptoethanol 6.0 mM, pH 7.4). The binding protein
prepared from beef adrenals, was added to the samples previously
incubated at 4 °C for 150 min, and after the addition of charcoal
were centrifuged at 2000 g for 10 min. The clear supernatant was
counted in a Scintillation Counter Packard Tri Carb 2500 TR with
an efficiency of 58%.47
15. Catarzi, D.; Colotta, V.; Varano, F.; Calabri, F. R.; Lenzi, O.; Filacchioni, G.;
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6.3.3. Data analysis
The protein concentration was determined according to a Bio-
Rad method48 with bovine albumin as a standard reference. Inhib-
itory binding constant (Ki) values were calculated from those of
IC50 according to Cheng & Prusoff equation Ki = IC50/(1 + [C⁄]/KD⁄),
where [C⁄] is the concentration of the radioligand and KD⁄ its disso-
ciation constant.49 A weighted non linear least-squares curve fit-
ting program LIGAND50 was used for computer analysis of
inhibition experiments. EC50 and IC50 values obtained in cyclic
AMP assay were calculated by non linear regression analysis using
the equation for a sigmoid concentration-response curve (Graph-
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Acknowledgment
28. Kushawaha, S. J. Indian Chem. Soc. 1968, 45, 752.
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This work was supported by a Grant from the Italian Ministry
for University and research (MIUR, FIRB RBNE03YA3L project).
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