2-Phenylpyrazolo[4,3-d]pyrimidin-7-one as a new scaffold to obtain potent and selective human a3 adenosine receptor antagonists: new insights into the receptor-antagonist recognition

Article abstract of DOI:10.1021/jm900718w

A molecular simplification approach of previously reported 2-arylpyrazolo[3,4-c]quinolin-4-ones was applied to design 2-arylpyrazolo[4,3-d] pyrimidin-7-one derivatives as new human A₃ adenosine receptor antagonists. Substituents with different

Full text of DOI:10.1021/jm900718w

7640 J. Med. Chem. 2009, 52, 7640–7652
DOI: 10.1021/jm900718w
2-Phenylpyrazolo[4,3-d]pyrimidin-7-one as a New Scaffold To Obtain Potent and Selective Human A3
Adenosine Receptor Antagonists: New Insights into the Receptor-Antagonist Recognition†
Ombretta Lenzi,‡ Vittoria Colotta,*,‡ Daniela Catarzi,‡ Flavia Varano,‡ Daniela Poli,‡ Guido Filacchioni,‡ Katia Varani,§
Fabrizio Vincenzi,§ Pier Andrea Borea,§ Silvia Paoletta,^ Erika Morizzo,^ and Stefano Moro*,^
‡Dipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente Attivi, Universitaꢀdi Firenze,
Polo Scientifico, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy, §Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia,
Universitaꢀ di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy, and ^Molecular Modeling Section (MMS), Dipartimento di Scienze
Farmaceutiche, Universitaꢀ di Padova, Via Marzolo 5, 35131 Padova, Italy
Received May 27, 2009
A molecular simplification approach of previously reported 2-arylpyrazolo[3,4-c]quinolin-4-ones
was applied to design 2-arylpyrazolo[4,3-d]pyrimidin-7-one derivatives as new human A3 adenosine
receptor antagonists. Substituents with different lipophilicity and steric hindrance were introduced at
the 5-position of the bicyclic scaffold (R5=H, Me, Et, Ph, CH2Ph) and on the 2-phenyl ring (OMe, Me).
Most of the synthesized derivatives were highly potent hA3 adenosine receptor antagonists, the best
being the 2-(4-methoxyphenyl)pyrazolo[4,3-d]pyrimidin-7-one (Ki=1.2 nM). The new compounds were
also highly selective, being completely devoid of affinity toward hA1, hA2A, and hA2B adenosine
receptors. On the basis of the recently published human A2A receptor crystallographic information, we
propose a novel receptor-driven hypothesis to explain both A3 AR affinity and A3 versus A2A selectivity
profiles of these new antagonists.
Introduction
death, depending on the degree of receptor activation and/or
the cell type or the toxic insult.7-9 As a consequence of this dual
effect, both A3 receptor agonists and antagonists might be
effective therapeutics in cancer.9-11 In the brain and in other
tissues, such as kidney, lung, and eye, activation of the A3 AR
may induce both pro- and antisurvival effects, thus affording
either protection or damage, depending on the situation.9 In the
immune system, the A3-mediated effects can be either proin-
flammatory or antiinflammatory,9 depending on the investi-
gated models. Thus, even though it is clear that the A3 AR is
involved in many disease pathways, much remains to be
clarified about its role. Therefore, the search for new selective
A3 AR ligands, either agonists or antagonists, continues to be
attractive.
In our laboratory, we have directed much effort toward the
study of human (h) A3 AR antagonists belonging to different
classes of heteroaromatic systems.12-20 Of these, we have
identified a class of tricyclic hA3 antagonists, the pyrazolo-
[3,4-c]quinolin-4-one (PQ) derivatives (Chart 1).13,17 The PQ
derivatives show high affinities for the hA3 receptor (Ki =
3-30 nM) and also high hA3 versus hA2A selectivity, since they
do not bind the hA2A receptor. They possess quite good
affinities for the hA1 AR and scarce hA3 versus hA1 selectivity.
Thus, to develop a new class of compounds targeting the A3
receptor, but with a better selectivity profile, we performed a
molecular simplification of the PQ structure to yield the
2-arylpyrazolo[4,3-d]pyrimidin-7-one derivatives 1-12 (Chart 1).
Substituents with different lipophilicity and steric hindrance were
introduced at the 5-position of the 2-phenylpyrazolopyri-
midin-7-one scaffold (R5 = H, Me, Et, Ph, CH2Ph) to give
compounds 1-5. Certain substituents that were profitable for
hA3 affinity and selectivity in the PQ lead series were introduced
on the 2-phenyl ring. In particular, a 4-methoxy group was
The neuromodulator adenosine affects a wide variety
of physiopathological processes through activation of four
G-protein-coupled receptors (GPCRsa), classified as A1,
A2A, A2B, and A3 subtypes.1,2 Activation of adenosine recep-
tors (ARs) can induce inhibition (A1 and A3) or activation (A2A
and A2B) of the adenylate cyclase.1 Moreover, other second
messenger signaling pathways can be associated with stimula-
tion of ARs. The A3 AR positively modulates phospolipase C3
and D,4 KATP channel,5 inositol triphosphate, and intracellular
calcium.5,6 Activation of this receptor subtype leads to modu-
lation of mitogen-activated protein kinases (MAPK), such as
the extracellular signal-regulated kinase (ERK) 1/2 and the
stress-activated protein kinase p38.7 What is known about
A3-signaling in several processes is still controversial. The A3
regulation of the cell cycle may induce cell protection or cell
†The authors are deeply honored to contribute to this special Cen-
tennial issue, and they are also extremely grateful to the Division of
Medicinal Chemistry of the American Chemical Society for its out-
standing support toward the advancement of medicinal chemistry.
*To whom correspondence should be addressed. For V.C.: phone,
þ39 055 4573731; fax, þ39 055 4573780; e-mail, vittoria.colotta@unifi.
it. For S.M.: phone, þ39 049 8275704; fax, þ39 049 8275366; e-mail,
stefano.moro@unipd.it.
a Abbreviations: GPCRs, G-protein-coupled receptors; AR, adenosine
receptor; MAPK, mitogen-activated protein kinase; ERK, extracel-
lular signal-regulated kinase; h, human; NECA, 50-(N-ethylcarboxami-
do)adenosine; cAMP, cyclic adenosine monophosphate; Cl-IB-MECA,
2-chloro-N6-(3-iodobenzyl)-50-(N-methylcarbamoyl)adenosine; DPCPX,
8-cyclopentyl-1,3-dipropylxanthine; ZM-241385, 4-(2-[7-amino-2-(2-furyl)-
[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol; I-AB-MECA,
N6-(4-amino-3-iodobenzyl)-50-(N-methylcarbamoyl)adenosine; SAR,
structure-affinity relationship; TM, transmembrane; rmsd, root-
mean-square deviation; EL2, second extracellular loop; MOE, Mole-
cular Operating Environment software.
r
pubs.acs.org/jmc
Published on Web 09/10/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7641
Scheme 2a
Chart 1. Molecular Simplification Approach from the Pyrazolo-
[3,4-c]quinolin-4-one to the Pyrazolo[4,3-d]pyrimidin-7-one Scaf-
fold
Scheme 1a
a (a) NEt3, CHCl3; (b) H2, Pd/C; (c) R5-C(OEt)3, NH4OAc, micro-
wave irradiation; (d) R5-CH2-CN, HClg, anhydrous dioxane; (e) neat,
T = 230 ꢀC.
into the 4-amino-1-arylpyrazole-3-carboxylic acids 2022-22.
These latter derivatives were cyclized with suitable triethyl
orthoesters and ammonium acetate via one-pot three-compo-
nent reaction and under solvent-free conditions. The reaction,
performed under microwave irradiation, afforded the desired
5-substituted 1-arylpyrazolo[4,3-d]pyrimidin-7(6H)-ones 1-4,
7-9, and 11. Since the yields in this last step were quite low
(from 18% to 40%), a new synthetic pathway was followed to
obtain the target bicyclic derivatives 5, 6, 10, and 12
(Scheme 2). Compounds 1 and 2 were also resynthesized to
evaluate whether the second synthetic method was better than
the previous one. Allowing N,N-dimethyl-2-nitroethenea-
mine25 to react with suitable ethyl N1-arylhydrazono-N2-
chloroacetates,26,27 in refluxing chloroform and in the presence
of triethylamine, the ethyl 1-aryl-4-nitropyrazole-3-carboxy-
lates 2328-25 were obtained. The 4-nitro-substituted com-
pounds 23-25 were reduced to the corresponding 4-amino
derivatives 26-28 which were cyclized with triethyl orthoesters
and ammonium acetate, under microwave irradiation, to give
the 1-arylpyrazolo[4,3-d]pyrimidines 1, 2, 6, 12. Interestingly,
compounds 1 and 2 were obtained with significantly higher
yields (about 70%) than those obtained by the above-de-
scribed cyclization of derivative 20. Intermediates 26 and 27
were also reacted with benzyl cyanide, in anhydrous dioxane
and hydrogen chloride, to give the ethyl 4-(2-phenylacet-
amidoimido)-1-arylpyrazole-3-carboxylate hydrochlorides 29
and 30 which were heated at 250 ꢀC to provide the corres-
ponding 1-aryl-5-benzylpyrazolo[4,3-d]pyrimidin-7(6H)-ones 5
and 10. Finally, Scheme 3 describes the synthesis of the
2-phenylpyrazolo[4,3-d]pyrimidin-5,7-dione 13. Reaction of
a (a) 20% KOH/EtOH; (b) 85% H3PO4; (c) R5-C(OEt)3, NH4OAc,
microwave irradiation.
appended on the 2-phenyl ring of derivatives 1-5 to afford
compounds 6-10. 3-Methyl or 4-methyl groups were also inserted
on the 2-phenyl ring of the 5-methyl derivative 2 (compounds 11
and 12, respectively). Finally, an oxo function was introduced at the
5-position to give the 2-phenylpyrazolo[4,3-d]pyrimidin-5,7-dione
13. We propose a novel receptor-driven hypothesis to explain both
the hA3 affinity and hA3 versus A2A receptor selectivity profiles of
these
new
anta-
gonists. This is based on the recently published structure of
the hA2A AR, in conjunction with the high affinity hA2A
AR antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a]-
[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385).21
Chemistry
The 5-substituted 2-arylpyrazolo[4,3-d]pyrimidin-7-one de-
rivatives 1-12 were prepared following two synthetic path-
ways, as depicted in Schemes 1 and 2. Scheme 1 shows the
synthesis of compounds 1-4, 7-9, and 11 starting from ethyl
4-amino-1-arylpyrazole-3,5-dicarboxylates 14-16 which
were prepared as previously described.22-24 Compounds
14-16 were hydrolyzed to the corresponding 4-amino-
1-arylpyrazole-3,5-dicarboxylic acids 1722-19 which were
heated at 100 ꢀC in 85% phosphoric acid to be transformed

7642 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lenzi et al.
ethyl 4-nitro-1-phenylpyrazole-3-carboxylate 23 with saturated
aqueous solution of ammonia yielded the 4-nitro-
1-phenyl-3-carboxamide 31, which was catalytically reduced to
give the corresponding 4-amino derivative 32.22 The bicyclic
derivative 13 was obtained by allowing compound 32 to react
with triphosgene.
cAMP production in CHO cells, stably expressing hA3 ARs.
All pharmacological data are collected in Table 1, together
with those of ZM-241385, taken as a reference compound.
Results and Discussion
The binding results indicate that the herein reported mole-
cular simplification of the PQ structure was successful, since it
not only maintained high affinity for the hA3 AR but also
increased the hA3 selectivity. Indeed, most of the newly
synthesized compounds displayed hA3 AR affinities in the
low nanomolar range (Ki = 1.2-72 nM) and are totally
inactive at the other three investigated ARs. Structure-affi-
nity relationship (SAR) analysis shows that both R2 and R5
substituents play a key role in anchoring to the hA3 receptor
site. Both the lipophilicity and steric hindrance of the R5
substituent are critical for hA3 affinity. Compound 1, bearing
a hydrogen atom at the 5-position, shows a good hA3 affi-
nity (Ki=185 nM) which was significantly enhanced when the
5-hydrogen atom was replaced by a 5-ethyl moiety (com-
pound 3) or, even better, by a 5-methyl group (compound 2).
The presence at the 5-position of the bulkier phenyl ring was
detrimental, with derivative 4 showing a null hA3 binding
activity. Replacement of the 5-phenyl group with a 5-benzyl
moiety restored the hA3 AR affinity (compound 5). However,
it was significantly lower than those of the less hindered
5-substituted derivatives 1-3. These results indicate that
the presence of a quite small lipophilic substituent at the
5-position of the pyrazolo[4,3-d]pyrimidin-7-one scaffold is
important. This is probably because this group engages
hydrophobic bonds with a lipophilic receptor pocket of
limited size. The presence of a substituent on the 2-phenyl
ring (R2) was also a crucial feature of these new hA3 AR
Pharmacological Assays
The synthesized derivatives 1-13 were tested to evaluate
their affinity at hA1, hA2A, and hA3 ARs. Compounds were
also tested at the hA2B AR subtype by measuring their
inhibitory effects on NECA-stimulated cAMP levels in
CHO cells stably transfected with the hA2B AR. Finally, the
antagonistic potency of all the pyrazolopirimidin-7-one deri-
vatives able to bind at the hA3 AR (1-3, 5-8, 10-12) was
assessed by evaluating their effect on Cl-IB-MECA-inhibited
Scheme 3a
a (a) 33% aqueous NH3; (b) H2, 10% Pd/C, EtOH; (c) triphosgene,
NEt3, anhydrous tetrahydrofuran.
Table 1. Binding Affinity (Ki) at hA1, hA2A, and hA3 ARs and Potency (IC50) at hA2B and hA3 ARs
binding experiments,
Ki (nM) or I (%)
cAMP assays,
IC50 (nM) or I (%)
a
b
c
d
e
R5
R2
hA3
hA1
1%
hA2A
1%
hA2B
3%
hA3
1
H
Me
H
H
H
H
H
185 ( 19
16 ( 2
52 ( 5
10%
725 ( 64
87 ( 9
245 ( 23
2
9%
1%
6%
10%
1%
1%
1%
1%
1%
1%
1%
1%
5%
2%
1%
4%
4%
3%
2%
5%
5%
4%
4%
2%
2%
42 ( 5
3
Et
Ph
11%
22%
11%
2%
4
5
Ph-CH2
H
Me
900 ( 95
54 ( 6
1.2 ( 0.1
14 ( 2
16%
6%
6
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-Me
270 ( 26
5.2 ( 0.5
63 ( 7
7
5%
1%
8
Et
Ph
9
10
1%
Ph-CH2
Me
Me
250 ( 27
10 ( 1
72 ( 8
12%
10%
1%
850 ( 76
46 ( 5
354 ( 33
11
12
3-Me
4%
4%
13
ZM-241385
5%
180 ( 16
1.2 ( 0.2
2%
a Displacement of specific [125I]AB-MECA binding to hA3CHO cells, where Ki values are mean values ( SEM of four separate assays each performed
in duplicate. Percentage of inhibition in [125I]AB-MECA competition binding assays to hA3CHO cells at 1 μM tested compounds. b Percentage of
inhibition in [3H]DPCPX competition binding assays to hA1CHO cells at 1 μM tested compounds. c Percentage of inhibition in [3H]ZM241385
competition binding assays to hA2ACHO cells at 1 μM tested compounds. d Percentage of inhibition on cAMP experiments in hA2BCHO cells,
stimulated by 200 nM NECA, at 1 μM examined compounds. e IC50 values are expressed as mean values ( SEM of four separate cAMP experiments in
hA3CHO cells, inhibited by 100 nM Cl-IB-MECA.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7643
Figure 1. Topology of the hA3 AR built using bovine rhodopsin (A) and human A2A AR (B) as templates. The molecular surfaces and the
calculated volumes refer to the ligand binding pockets.
Table 2. Root Mean Square Deviation (rmsd) of the Backbone of the Aligned Models of the hA3AR Compared with the Available Crystallo-
graphic Templatesa
all TMs
all loops
TM1
TM2
TM3
TM4
TM5
TM6
TM7
2.04
HX8
1.64
EL2
˚
rmsd (A) with Respect to the hA3AR Model from Bovine Rhodopsin (Backbone)
A3_A2A
2.43
10.06
˚
2.55
2.40
2.78
2.45
2.85
2.02
14.30
rmsd (A) with Respect to the hA3AR Model from hβ2-Adrenergic Receptor (Backbone)
A3_rho
A3_A2A
2.29
2.57
10.86
7.46
2.82
3.84
2.12
1.89
1.98
2.02
2.01
1.73
2.07
2.09
2.19
2.71
1.85
2.23
3.73
3.66
11.44
6.18
a The main difference among the models is due to the loops, which represent the most variable region of the templates and consequently of the models.
Particular attention has to be given to EL2 because it is part of the binding pocket and it can directly interact with ligands.
antagonists. When a 4-methoxy group was introduced on
the 2-phenyl ring of compounds 1-5, a significant enhance-
ment of the hA3 affinity was obtained (compounds 6, 8-10).
Among the 2-(4-methoxyphenyl) derivatives 6-10, the
5-methylsubstituted compound 7 emerged as the most active
(Ki =1.2 nM), confirming that the best substituent for the
5-position was the methyl group. Thus, taking 2 as the lead
derivative, we tested two further substituents on the 2-phenyl
ring because in the triciclic PQ series they afforded the high-
est hA3 AR affinities, i.e., the 4-Me and 3-Me groups (com-
pounds 11 and 12). Both compounds 11 and 12 show high
hA3 affinity, in particular the 2-(4-methylphenyl)-substi-
tuted compound 11 (Ki =10 nM), but the 4-methoxy sub-
stituted derivative 7 remains the best in terms of hA3 affinity.
Finally, introduction of an oxo function at the 5-position of
the pyrazolo[4,3-d]pyrimidin-7-one nucleus exerted a dele-
terious effect, with compound 13 displaying null affinity for
the hA3 receptor. All the new derivatives 1-3, 5-8, 10-12
were antagonists at the hA3 AR. They show inhibitory
effects on Cl-IB-MECA-inhibited cAMP production, and
their potencies are coherent with their hA3 affinity values.
Molecular Modeling Studies
The recently published structure of the hA2A AR, in con-
junction with the high affinity antagonist ZM-241385, pro-
vides a new template for GPCR modeling, particularly for
ARs.21 Therefore, we built a new homology model of the
hA3 AR using the crystal structure of the hA2A receptor (PDB
code 3EML) as a template (methodological details are sum-
marized in the Experimental Section).
The helical arrangement is similar to the previously pub-
lished rhodopsin-based homology models as shown in
Figure 1.14-18,20 However, the transmembrane (TM) helices
are slightly shifted, and the differences among their relative
positions result in a root-mean-square deviation (rmsd)
˚
around 2.50 A for the backbone atoms. A detailed compar-
ison of the superimposed models is presented in Table 2, in
which we report the values of rsmd for each TM helix. As seen

7644 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lenzi et al.
Figure 2. (A) Crystallographic binding mode of ZM-241385 inside the hA2A receptor (PDB code 3EML) and hypothetical binding mode of
compound 7 obtained after docking simulations (B) inside the hA2A AR binding site and (C) inside the hA3 AR binding site. Side chains of some
amino acids important for ligand recognition and H-bonding interactions are highlighted. Hydrogen atoms are not displayed. Beside each
pose, the graph displays the electrostatic interaction energy (in kcal/mol) between the ligand and each single amino acid involved in ligand
recognition.
with the templates, the main difference between the two
models of the hA3 ARis in the loop region. Theligand binding
pocket of the crystal structure of the hA2A AR is shifted closer
toTM6and TM7, andthe position of the hA2A ARantagonist
is closer to these helices (Figure 2, panel A on the left).21
Important interactions are also established with the second

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7645
Table 3. Comparison of Different Molecular Docking Protocols in the Reproduction of the Binding Mode of ZM-241385 at the hA2A ARa
˚
best rmsd (A)
˚
best ranked pose rmsd (A)
˚
mean poses rmsd (A)
˚
number of poses with rmsd < 2.5 A
docking protocol
MOE tabu search
MOE simulated annealing
MOE genetic algorithm
GOLD (GoldScore)
GOLD (ChemScore)
GOLD (asp)
1.61
2.17
2.25
0.63
1.31
0.61
0.79
0.93
0.84
1.97
3.35
4.36
9.06
1.95
3.90
4.96
2.71
1.98
1.93
11.80
5.65
6.47
6.66
1.20
3.13
1.50
6.82
6.96
6.70
8.22
4/25
1/25
2/25
25/25
11/25
23/25
7/25
GLIDE
PLANTS (chemplp)
PLANTS (plp)
PLANTS (plp95)
3/25
6/25
4/25
a Different scoring functions for the same protocol are reported in parentheses if available.
extracellular loop (EL2), in particular with Glu169. Indeed, the
position of ZM-241385 is significantly different from that of
11-cis-retinal into the bovine rhodopsin29 or of carazolol into
the human β2-adrenergic receptor.30 Even though GPCRs share
a common topology, ligands may bind in a different fashion and
interact with different positions of the receptor. The model built
starting from the hA2A AR template is different from previous
models of the hA3 AR; the binding pocket is closer to TM6 and
TM7 and open to the extracellular side. Because of the open
conformation to the extracellular side of the EL2, the volume
region of the hA3 AR. Ligand recognition occurs in the upper
region of the TM bundle, and the pyrazolo[4,3-d]pyrimidin-
7-one scaffold is surrounded by TMs 3, 5, 6, 7 with the 2-phenyl
ring pointing toward EL2 and the substituent at the 5-position
(R5) oriented toward the intracellular environment.
Figure 2 (panel C on the left) shows the hypothetical
binding mode of compound 7, which possesses the highest
hA3 affinity among all the newly synthesized derivatives (hA3
AR Ki = 1.2 nM). This compound is anchored, inside the
binding cleft, by two stabilizing hydrogen-bonding inter-
actions with the amide moiety of Asn250 (6.55) side chain.
The two hydrogen bonds involve the carbonyl group at the
7-position and one of the N atoms of the pyrazole ring,
respectively. The asparagine residue 6.55, conserved among
all AR subtypes, was already found to be important for ligand
binding at both the hA3 and hA2A ARs.32,33 Compound 7
also forms hydrophobic interactions with many residues
of the binding cleft including Leu90 (3.32), Leu91 (3.33),
Phe168 (EL2), Val169 (EL2), Met177 (5.38), Trp243 (6.48),
Leu246 (6.51), Leu264 (7.35), Tyr265 (7.36), Ile268 (7.39).
In particular, the 2-phenyl ring forms an aromatic
π-π stacking interaction with Phe168 (EL2), while the
pyrazolo[4,3-d]pyrimidin-7-one core interacts with the highly
conserved Trp243 (6.48), an important residue in receptor
activation and in antagonist binding.33
The 2-arylpyrazolo[4,3-d]pyrimidin-7-one derivatives bear-
ing nonbulky moieties at the 5-position, such as 5-hydrogen
atom (compounds 1 and 6), 5-methyl group (compounds 2,
11, and 12), and 5-ethyl group (compounds 3 and 8), show a
similarbinding mode compared tocompound7. In fact, for all
these derivatives, the pyrazolo[4,3-d]pyrimidin-7-one core is
perfectly aligned inside the TM region of the hA3 receptor. In
particular, the two hydrogen bonds with Asn250 (6.55), the
aromatic stacking interaction with Phe168 (EL2), and the
hydrophobic interaction with Trp243 (6.48) are conserved.
In contrast, binding poses of compounds bearing bulkier
substituents, including 5-phenyl and 5-benzyl groups (com-
pounds 4, 5, 9, and 10), are quite different. The 2-phenyl ring
of these compounds is directed toward the intracellular
environment rather than toward EL2, while the substituent
at the 5-position (R5) points toward TM2. Because of this
different orientation of the molecule inside the binding cleft,
these derivatives lose one of the two H-bonding interactions
with Asn250 (6.55) and the π-π stacking interaction between
Phe168 (EL2) and the 2-phenyl ring (data not shown). The
lack of these important interactions seems to be why deriva-
tives with bulky R5 groups show low or null hA3 AR affinities.
Compound 13 (bearing an oxo function at the 5-position)
shows a completely different docking pose inside the hA3
binding pocket, compared to all the other derivatives, and its
3
˚
of the binding site, estimated as ∼1930 A , was larger than
the volume of the binding site of the rhodopsin-based model
˚
3
(∼660 A ), as summarized in Figure 1.
Although the hA3 AR possesses a higher percentage of
identity with the A2A subtype than with the previously
reported rhodopsin and β2-adrenergic structures, the confor-
mations of the EL2 and, consequently, of the binding pocket
of the hA3 AR might be different from those of the hA2A AR.
The peculiarity of the hA2A AR is the presence of three
disulfide bridges on EL2, which are not conserved among
ARs. Moreover, one additional disulfide bridge is present in
the crystal structure of the hA2A AR between Cys259 and
Cys262 on EL3. Human A3 and A2B ARs do not present
cysteine residues in the same positions, and the link cannot be
formed in these two receptor subtypes. According to the
alignment of the sequences, the hA1AR presents two cysteines
(Cys260 and Cys263) in the corresponding positions of the
cysteines of the hA2AAR. The formation of the disulfide bond
is possible, but mutagenesis analysis showed that mutation of
cysteine residues to alanine or serine does not change the
affinity for agonists or antagonists.31 An in-depth investiga-
tion using lipid membrane molecular dynamics simulations is
underway in our laboratory to verify the influence of EL2
conformation in the antagonist recognition process.
Starting from this novel hA3AR model, a molecular model-
ing study was performed on the new 2-arylpyrazolo[4,3-d]-
pyrimidin-7-one derivatives 1-13 to identify the hypothetical
binding mode at the hA3 adenosine receptor and to rationalize
the observed structure-activity relationships. In particular,
molecular docking studies were carried out on both hA3 and
hA2A adenosine receptors. As reported in the Experimental
Section, four different programs were used to calibrate our
docking protocol using the crystallographic pose of ZM-241385
into the hA2A receptor as a reference (Table 3). On the basis of
the lowest average ligand rmsd value obtained from the different
docking algorithms, we decided to used Gold as a docking pro-
gram for the pose inspection of the novel 2-arylpyrazolo[4,3-d]-
pyrimidin-7-one derivatives in both hA2A and hA3 receptors.
From the docking simulation analysis, almost all the deri-
vatives were seen to share a similar binding pose in the TM

7646 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lenzi et al.
Figure 3. Access to the binding pocket of hA2A (A) and hA3 (B) ARs. Receptors are viewed from the extracellular side. Gaussian surface
of some important amino acids is displayed. Surface color shows a screened electrostatic potential (where scale parameters are as follows: red,
-35 kcal/mol; white, 0 kcal/mol; blue, þ35 kcal/mol).
orientation is almost parallel to the membrane plane. Even
though this molecule forms two H-bonding interactions with
Asn250 (6.55), it is too far to interact with Trp243 (6.48), and
so it loses the interaction with this important residue (data not
shown).
With regard to the presence of a substituent on the 2-phenyl
ring (R2), it emerged that small groups (methyl and methoxy)
enhance the hA3 affinity even though they do not seem to be
involved in particular interactions with residues of the binding
pocket. Nevertheless, these substituents, because of their
electron-donating properties, could reinforce the π-π stack-
ing interaction of the 2-phenyl ring with the receptor. In
addition, the methyl and methoxy groups increase the topo-
logical complementarity of the compounds with the TM
binding cavity.
As shown in Figure 2 (panel B on the left), the hypothetical
binding pose of compound 7, obtained after molecular dock-
ing to the crystal structure of the hA2A receptor, is quite
different compared to the pose of the same compound on the
hA3 subtype (Figure 2, panel C on the left), although ligand
recognition occurs in the same region of the TM bundle. In
particular, the 2-phenyl ring points toward the intracellular
environment and the substituent at the 5-position (R5) is
oriented toward EL2. The molecule forms two H-bonds, with
Asn253 (6.55) and Glu169 (EL2), and an aromatic interaction
with Phe168 (EL2).
All the newly synthesized derivatives 1-13 show this same
binding mode inside the hA2A receptor pocket. Although the
predicted binding affinities are lower with respect to those
estimated for the corresponding binding to the hA3 receptor
(around 3-9 kcal/mol), this only partially justifies the absence
of A2A binding observed experimentally. To analyze the
possible ligand-receptor recognition mechanism in a more
quantitative way, we calculated the individual electrostatic
contribution (ΔEelint) to the interaction energy (ΔEint) of each
receptor residue (see Experimental Section for more details).
From this study, one of the most critical residues affecting the
affinity at ARs seems to be the asparagine 6.55 (Asn253 in
hA2A and Asn250inhA3). Inparticular, Asn253 is responsible
for two stabilizing interactions with ZM-241385 into human
A2A AR. This is confirmed by the electrostatic contribution of
around -10 kcal/mol to the whole interaction energy. Con-
sidering the binding poses of compound 7, the electrostatic
contribution of this asparagine residue to the interaction
energy is completely different between hA2A and hA3 recep-
tors (see Figure 2, panels B and C on the right, respectively).
Asn250 (6.55) strongly stabilizes the ligand-hA3 receptor
complex (negative electrostatic interaction energy) because of
the two hydrogen bonding interactions, while Asn253 (6.55)
destabilizes the ligand-hA2A receptor complex (positive elec-
trostatic interaction energy). This detrimental contribution to
the stability of the complex is due to the electrostatic repulsion
between the oxo moiety of the pyrazolo[4,3-d]pyrimidin-7-one
nucleus and the carbonyl group of Asn253 side chain. This
could considerably reduce the permanence of these 2-aryl-
pyrazolo[4,3-d]pyrimidin-7-one analogues in the TM binding
pocket and explain the null affinity of these new derivatives for
the hA2A AR (I=1-10%).
Moreover, comparing the docking pose of compound 7 on
the hA2A receptor with the crystallographic pose of the
antagonist ZM-241385, we note that the bicyclic core of the
pyrazolo[4,3-d]pyrimidin-7-one derivative is almost aligned
with the bicyclic region of ZM-241385. Nevertheless, the
exocyclic amino group (H-bond donor) of ZM-241385 is
replaced with a carbonyl group (H-bond acceptor) in com-
pound 7, and this could lead to the substantially different
affinities at the hA2A receptor (see Table 1). The values of the
electrostatic contributions to the binding energies are also
coherent with this recognition scenario. Therefore, in the
7-hA2A receptor complex, Glu169 (EL2) is the only amino
acid that has a notable negative electrostatic interaction
energy. Site-directed mutagenesis studies have shown that

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7647
this glutamate residue is important for ligand binding at the
hA2A AR.34
19: yield 52%; mp 243-245 ꢀC (acetonitrile/MeOH); 1H
NMR 2.37 (s, 3H, Me), 7.25 (d, 2H, ar, J = 8.3 Hz), 7.30
(d, 2H, ar, J=8.3 Hz). Anal. (C12H11N3O4) C, H, N.
We note that Glu169 (EL2) of the hA2A receptor subtype is
not present in the corresponding position of the hA3 receptor,
where the residue is replaced by a valine (Val169). This
difference could influence not only the binding mode but also
the entrance of ligands to the TM region of the receptors. As
shown in Figure 3, the electrostatic potentials of amino acids
present at the binding pocket entrance from the extracellular
site are very different in the two receptors. In the hA3 AR, the
binding pocket gate is surrounded essentially by side chains of
hydrophobic residues including Phe168 (EL2), Val169 (EL2),
Ile253 (6.58), Val259 (EL3), Leu264 (7.35) (Figure 3B). In the
hA2A AR, there are ionic residues, such as Glu169 (EL2) and
His264 (EL3) among the amino acids delimiting the binding
site access (Figure 3A). We speculate that the presence of this
charged gate may affect both the ligand orientation, while
approaching the binding pocket, and its accommodation into
the final TM binding cleft. Several studies are in progress in
our laboratories to experimentally support our hypothesis.
General Procedure for the Synthesis of 4-Amino-1-arylpyra-
zole-3-carboxylic Acids 20-22. A suspension of 4-amino-1-
arylpyrazole-3,5-dicarboxylic acids 17-19 in 85% H3PO4 was
heated at 100 ꢀC for 45 min. After cooling at room temperature,
the solution was alkalinized to pH 4-5 with 40% NaOH
solution. The solid that precipitated was collected by filtration,
washed with a few drops of water, and recrystallized
20: yield 60%; mp 225-226 ꢀC (EtOH) (lit. mp 223-225 ꢀC).22
1
21: yield 58%; mp 208-210 ꢀC (EtOAc/EtOH); H NMR
3.80 (s, 3H, OMe), 7.04 (d, 2H, ar, J=8.9 Hz), 7.69-7.71 (m, 3H,
2ar þ H5). Anal. (C11H11N3O3) C, H, N.
22: yield 55%; mp 213-214 ꢀC (EtOAc/EtOH); 1H NMR
2.33 (s, 3H, Me), 7.29 (d, 2H, ar, J=8.3 Hz), 7.66 (d, 2H, ar, J=
8.3 Hz), 7.76 (s, 1H, H5). Anal. (C11H11N3O2) C, H, N.
General Procedure for the Synthesis of 5-Substituted 2-Aryl-
pyrazolo[4,3-d]pyrimidin-7(6H)-ones 1-4, 7-9, 11. A mixture of
4-amino-1-arylpyrazole-3-carboxylic acids 20-22 (2.5 mmol),
ammonium acetate (3.2 mmol), and the suitable orthoesters
(3.2 mmol) was microwave-irradiated at 150 ꢀC for 15 min. The
suspension was cooled at room temperature, the solid was
collected by filtration, washed with water and diethyl ether,
and recrystallized. The crude compound 11 was purified by
column chromatography (eluting system CH2Cl2/MeOH, 9:1)
and then recrystallized.
Conclusion
The present study identifies a new class of highly potent
and selective hA3 AR antagonists, the 2-arylpyrazolo[4,3-d]-
pyridine-7-one derivatives, which were designed as simplified
analogues of our previously reported hA3 antagonists. To
depict the binding mode of these new hA3 antagonists, we
built a novel model of the hA3 receptor, based on the recently
published structure of the hA2A receptor. This new A3 recep-
tor model can correctly interpret the SAR observed for the
new antagonists in terms of both hA3 affinity and hA3 versus
hA2A selectivity. Moreover, we propose a very preliminary
hypothesis concerning the specific roles of a few crucial amino
acids in affecting the molecular mechanism of both the ligand-
entering process and the TM-recognition process.
1
1: yield 25%; mp 299-300 ꢀC (acetonitrile); H NMR 7.47
(t, 1H, ar, J=7.3 Hz), 7.60 (t, 2H, ar, J=7.3 Hz), 7.86 (s, 1H,
H5), 8.03 (d, 2H, ar, J=7.8 Hz), 9.14 (s, 1H, H3), 12.00 (br s, 1H,
NH); IR 1673, 1724, 3170. Anal. (C11H8N4O) C, H, N.
1
2: yield 18%; mp >300 ꢀC (2-methoxyethanol); H NMR
2.31 (s, 3H, Me), 7.46 (t, 1H, ar, J=7.4 Hz), 7.59 (t, 2H, ar, J=
7.4 Hz), 8.01 (d, 2H, ar, J=8.0 Hz), 8.98 (s, 1H, H3), 11.92
(s, 1H, NH); IR 1683. Anal. (C12H10N4O) C, H, N.
3: yield 30%; mp 270-272 ꢀC (2-methoxyethanol); 1H NMR
1.22 (t, 3H, Me, J=7.5 Hz), 2.61 (q, 2H, CH2, J=7.5 Hz), 7.46
(t, 1H, ar, J=7.3 Hz), 7.57-7.61 (m, 2H, ar), 8.01 (d, 2H, ar, J=
8.1 Hz), 9.02 (s, 1H, H3), 11.89 (br s, 1H, NH); IR 1688, 3150.
Anal. (C13H12N4O) C, H, N.
1
4: yield 30%; mp >300 ꢀC (2-methoxyethanol); H NMR
Experimental Section
7.47-7.65 (m, 6H, ar), 8.05 (d, 2H, ar, J=7.7 Hz), 8.10 (d, 2H,
ar, J=7.8 Hz), 9.20 (s, 1H, H3), 12.26 (br s, 1H, NH); IR 1684,
3155. Anal. (C17H12N4O) C, H, N.
(A) Chemistry. The microwave-assisted syntheses were per-
formed using an Initiator EXP Biotage microwave instrument
(frequency of irradiation: 2.45 GHz). Silica gel plates (Merck
F254) and silica gel 60 (Merck, 70-230 mesh) were used for
analytical and column chromatography, respectively. All melt-
ing points were determined 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-Elmer Spectrum RX I
spectrometer in Nujol mulls and are expressed in cm-1. The
1H NMR spectra were obtained with a Bruker Avance 400 MHz
instrument. The chemical shifts are reported in δ (ppm) and are
relative to the central peak of the solvent which was always
DMSO-d6. The following abbreviations are used: s=singlet, d=
doublet, t=triplet, m=multiplet, br=broad, and ar=aromatic
protons.
1
7: yield 25%; mp >300 ꢀC (2-methoxyethanol); H NMR
2.30(s, 3H, Me), 3.80(s, 3H, OMe), 7.11(d, 2H, ar,J=9.0 Hz), 7.92
(d, 2H, ar, J=9.0 Hz), 8.87 (s, 1H, H3), 11.67 (br s, 1H, NH); IR
1678. Anal. (C13H12N4O2) C, H, N.
8: yield 25%; mp 278-280 ꢀC (EtOH); 1H NMR 1.22 (t, 3H,
Me, J=7.5 Hz), 2.59 (q, 2H, CH2, J=7.5 Hz), 3.84 (s, 3H, OMe),
7.13 (d, 2H, ar, J=8.9 Hz), 7.91 (d, 2H, ar, J=8.9 Hz), 8.91 (s, 1H,
H3), 11.83 (br s, 1H, NH); IR 1702. Anal. (C14H14N4O2) C, H, N.
9: yield 18%; mp 291-293 ꢀC (2-methoxyethanol); 1H NMR
3.85 (s, 3H, OMe), 7.16 (d, 2H, ar, J=7.0 Hz), 7.51-7.57 (m, 3H,
ar), 7.97 (d, 2H, ar, J=7.0 Hz), 8.11 (d, 2H, ar, J=7.7 Hz),
9.09 (s, 1H, H3), 12.19 (br s, 1H, NH); IR 1681, 3180. Anal.
(C18H14N4O2) C, H, N.
1
11: yield 23%; mp >300 ꢀC (MeOH/EtOH) H NMR 2.31
(s, 3H, Me), 2.38 (s, 3H, Me), 7.39 (d, 2H, ar, J =8.3 Hz), 7.89
(d, 2H, ar, J=8.3 Hz), 8.91 (s, 1H, H3), 11.86 (br s, 1H, NH); IR
1685. Anal. (C13H12N4O) C, H, N.
General Procedure for the Synthesis of 4-Amino-1-arylpyra-
zole-3,5-dicarboxylic Acids 17-19. A suspension of ethyl 4-
amino-1-arylpyrazole-3,5-dicarboxylates 14-1622-24 (1.64
mmol) in 20% KOH ethanolic solution (15 mL) was heated at
80 ꢀC for 15 min. The mixture was cooled at room temperature
and acidified at pH 2 with 12 N HCl. The solid was collected by
filtration, washed with water, and recrystallized.
General Procedure for the Synthesis of Ethyl 1-Aryl-5-nitro-
pyrazole-3-carboxylates 23-25. A solution of N,N-dimethyl-
2-nitroetheneamine25 (5.5 mmol), suitable ethyl N1-arylhydra-
zono-N2-chloroacetates26,27 (11 mmol), and triethylamine (11 mmol)
in chloroform (30 mL) was refluxed for 48 h. After the mixture was
cooled at room temperature, the solvent was evaporated at reduced
pressure to give a residue which was taken up with ethanol (about
20 mL) to give crude compounds 23 and 24, which were collected by
filtration. To obtain derivative 25, the residue was treated with
17: yield 50%; mp 198-200 ꢀC (EtOH) (lit. mp 197-199 ꢀC).22
18: yield 55%; mp 180-182 ꢀC (acetonitrile/MeOH); 1H
NMR 3.82 (s, 3H, OMe), 6.99 (d, 2H, ar, J=8.5 Hz), 7.35 (d,
2H, ar, J=8.5 Hz). Anal. (C12H11N3O5) C, H, N.

7648 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lenzi et al.
1
cyclohexane/EtOAc, the solid was filtered off, and the solvents were
evaporated at reduced pressure. The solid obtained was purified by
column chromatography (eluting system, EtOAc).
30: yield 80%; H NMR 1.18 (t, 3H, Me, J=7.2 Hz), 3.83
(s, 3H, OMe), 4.02 (s, 2H, benzyl CH2), 4.18 (q, 2H, CH2,
J =7.2 Hz), 7.12 (d, 2H, ar, J=7.0 Hz), 7.37-7.43 (m, 3H, ar),
7.60 (d, 2H, ar, J=6.9 Hz), 7.83 (d, 2H, ar, J=7.0 Hz), 8.87
(s, 1H, H5), 8.91 (br s, 1H, NH), 10.00 (br s, 1H, NH2þ proton),
11.62 (br s, 1H, NH2þ proton).
23: yield 45%; mp 149-150 ꢀC (EtOH) (lit. 150-151 ꢀC).28
24: yield 25%; mp 91-92 ꢀC (diethyl ether/petroleum ether 40-
60 ꢀC); 1H NMR 1.33 (t, 3H, Me, J=7.1Hz), 3.83(s,3H, MeO), 4.41
(q, 2H, CH2, J=7.1 Hz), 7.13 (d, 2H, ar, J=6.9 Hz), 7.85 (d, 2H, ar,
J=6.9 Hz), 9.61 (s, 1H, H5). Anal. (C13H13N3O5) C, H, N.
25: yield 40%; mp 118-120 ꢀC (diethyl ether); 1H NMR 1.33
(t, 3H, Me, J=7.1 Hz), 2.41 (s, 3H, Me), 4.43 (q, 2H, CH2, J=
7.1 Hz), 7.31 (d, 1H, ar, J=7.6 Hz), 7.47 (t, 1H, ar, J=7.8 Hz),
7.74 (d, 1H, ar, J=8.1 Hz), 7.81 (s, 1H, ar), 9.70 (s, 1H, H5).
Anal. (C13H13N3O4) C, H, N.
General Procedure for the Synthesis of 2-Aryl-5-benzyl-
pyrazolo[4,3-d]pyrimidin-7(6H)-ones 5 and 10. Intermediates 29
and 30 were heated at 230 ꢀC under nitrogen atmosphere for
about 1-2 h. Compound 5 was purified by recrystallization
from ethanol, while derivative 10 was chromatographed on
silica gel column (eluent, cyclohexane/EtOAc, 2:8) and then
recrystallized from 2-methoxyethanol.
General Procedure for the Synthesis of Ethyl 4-Amino-
1-arylpyrazole-3-carboxylates 26-28. The nitro derivatives
23-25 (3 mmol) were dissolved in a suitable solvent (23 in
boiling ethanol, 100 mL; 24 and 25 in EtOAc, 30 mL), and 10%
Pd/C (10% p/p) was added to the solution. The mixture was
hydrogenated in a Parr apparatus at 35 psi for 4 h and then the
catalyst was filtered off and the solvent evaporated to dryness
under reduced pressure to give a solid which was recrystallized.
26: yield 90%; mp 129-130 ꢀC (cycloexane/EtOAc); 1H
NMR 1.32 (t, 3H, Me, J = 7.0 Hz), 4.32 (q, 2H, CH2, J =
7.0 Hz), 4.90 (s, 2H, NH2), 7.35 (t, 1H, ar, J=7.4 Hz), 7.50 (t, 2H,
ar, J=7.4 Hz), 7.79 (d, 2H, ar, J=8.2 Hz), 7.85 (s, 1H, H5). Anal.
(C12H13N3O2) C, H, N.
5: yield 75%; mp >300 ꢀC; 1H NMR 3.92 (s, 2H, CH2),
7.23-7.38 (m, 5H, ar), 7.44 (t, 1H, ar, J=7.6 Hz), 7.59 (t, 2H, ar,
J=7.7 Hz), 7.91 (d, 2H, ar, J=8.2 Hz), 9.03 (s, 1H, H3), 12.13
(br s, 1H, NH); IR 1702. Anal. (C18H14N4O) C, H, N.
10: yield 55%; mp 293 ꢀC dec; 1H NMR 3.83 (s, 3H, OMe),
3.91 (s, 2H, CH2), 7.13 (d, 2H, ar, J=8.8 Hz), 7.23-7.37 (m, 5H,
ar), 7.89 (d, 2H, ar, J=8.9 Hz), 8.91 (s, 1H, H3), 12.09 (br s, 1H,
NH); IR 1673. Anal. (C19H16N4O2) C, H, N.
Synthesis of 4-Nitro-2-phenylpyrazole-4-carboxamide 31. A
stream of ammonia was bubbled through a suspension of the
ester 23 (3.0 mmol) in 33% aqueous ammonia solution (40 mL)
for about 2 h. Then the suspension was stirred at room tem-
perature for an additional 6 h. The solid was collected by
filtration, washed with water, and recrystallized from 2-ethoxy-
ethanol. Yield 85%; mp 230-232 ꢀC; 1H NMR 7.24 (t, 1H, ar,
J=7.3 Hz), 7.59 (t, 2H, ar, J=6.9 Hz), 7.90 (br s, 1H, NH2 amide
proton), 7.96 (d, 2H, ar, J =7.6 Hz), 8.18 (br s, 1H, NH2 amide
proton), 9.64 (s, 1H, H5). Anal. (C10H8N4O3) C, H, N.
Synthesis of 4-Amino-2-phenylpyrazole-4-carboxamide 32.22
The nitro derivative 31 (2.0 mmol) was dissolved in boiling
ethanol (about 150 mL), and 10% Pd/C (0.05 g) was added to
the solution. The mixture was hydrogenated in a Parr apparatus
at 35 psi for 4 h. The catalyst was filtered off and the solvent
evaporated at reduced pressure to give an oil that spontaneously
solidified. Yield 60%; mp 196-198 ꢀC (MeOH), (lit. 189-
191 ꢀC);22 1H NMR 4.86 (s, 2H, NH2), 7.24 (br s, 1H, NH2
proton), 7.29 (t, 1H, ar, J=7.4 Hz), 7.46-7.50 (m, 3H, 2ar þ
NH2 proton), 7.79 (s, 1H, H5), 7.83 (d, 2H, ar, J=8.7 Hz). Anal.
(C10H10N4O) C, H, N.
Synthesis of 2-Phenylpyrazolo[4,3-d]pyrimidin-5,7-(4H,6H)-
dione 13. A solution of triethylamine (2.16 mmol) in anhydrous
tetrahydrofuran (3 mL) was added dropwise to a mixture of
compound 32 (0.92 mmol) and triphosgene (0.36 mmol) in
anhydrous tetrahydrofuran (20 mL). The mixture was refluxed
for 8 h, then cooled at room temperature and diluted with
ice-water. The solid was collected by filtration, washed with
water, and recrystallized from dimethylformamide. Yield 55%;
mp >300 ꢀC; 1H NMR 7.41 (t, 1H, ar, J=7.4 Hz), 7.55 (t, 2H,
ar, J=7.6 Hz), 7.95 (d, 2H, ar, J=7.8 Hz), 11.03 (br s, 1H, NH),
11.09 (br s, 1H, NH); IR 1715, 1748, 3059, 3150. Anal.
(C11H8N4O2) C, H, N.
(B) Computational Methodologies. All modeling studies were
carried out on a 20 CPU (Intel Core2 Quad CPU 2.40 GHz) Linux
cluster. Homology modeling, energy calculation, and analyses of
docking poses were performed using the Molecular Operating
Environment (MOE, version 2008.10) suite.35 The software pack-
age MOPAC (version 7),36 implemented in the MOE suite, was
utilized for all quantum mechanical calculations. Docking simula-
tion were performed using the GOLD suite.37
Homology Model of the hA3 AR. On the basis of the assump-
tion that GPCRs share similar TM boundaries and overall
topology, a homology model of the hA3 adenosine receptor
was constructed using as template the recently published crystal
structure of the hA2A receptor.21 In Figure 4, we show the
alignment of the aminoacidic sequences of the two human
adenosine receptors considered in our study (hA3/hA2A seq-
uence identity of ∼42%).
27: yield 78%; mp 122-124 ꢀC (cycloexane/EtOAc); 1H
NMR 1.31 (t, 3H, Me, J = 7.1 Hz), 3.80 (s, 3H, MeO), 4.30
(q, 2H, CH2, J=7.1 Hz), 4.86 (s, 2H, NH2), 7.04 (d, 2H, ar, J=
9.0 Hz), 7.70 (d, 2H, ar, J=9.0 Hz), 7.75 (s, 1H, H5). Anal.
(C13H15N3O3) C, H, N.
28: yield 80%; mp 87-88 ꢀC (petroleum ether 40-60 ꢀC/diethyl
ether); 1H NMR 1.32 (t, 3H, Me, J=7.1 Hz), 2.38 (s, 3H, Me), 4.31
(q, 2H, CH2, J=7.1 Hz), 4.89(s, 2H, NH2), 7.15 (d, 1H, ar, J=
7.5 Hz), 7.36 (t, 1H, ar, J=7.8 Hz), 7.57 (d, 1H, J=8.1 Hz), 7.63
(s, 1H, ar), 7.82 (s, 1H, H5). Anal. (C13H15N3O2) C, H, N.
General Procedure for the Synthesis of 5-Substituted 2-Aryl-
pyrazolo[4,3-d]pyrimidin-7(6H)-ones 1, 2, 6, 12. The title com-
pounds were prepared from ethyl 4-amino-1-arylpyrazole-
3-carboxylates 26-28 (2.5 mmol), ammonium acetate (3.2 mmol),
and the suitable orthoesters (3.2 mmol) under microwave irradia-
tion at 150 ꢀC for 15 min. The suspension was cooled at room
temperature, and the solid was collected by filtration, washed with
water and diethyl ether, and recrystallized. This procedure provided
compounds 1 and 2 with higher yields (70% and 58%, respectively)
than those obtained by the cyclization of the 4-amino-1-phenyl-
pyrazole-3-carboxylic acid 20, reported above.
1
6: yield 80%; mp 277-278 ꢀC (acetonitrile); H NMR 3.84
(s, 3H, Me), 7.12 (d, 2H, ar, J=6.9 Hz), 7.84 (s, 1H, H5), 7.93
(d, 2H, ar, J=6.9 Hz), 9.03 (s, 1H, H3), 11.96 (br s, 1H, NH); IR
1694. Anal. (C12H10N4O2) C, H, N.
12: yield 75%; mp 299-300 ꢀC (2-methoxyethanol); 1H NMR
2.31 (s, 3H, Me), 2.42 (s, 3H, Me), 7.27 (d, 1H, ar, J=7.5 Hz),
7.46 (t, 1H, ar, J=7.8 Hz), 7.79 (d, 1H, ar, J=8.1 Hz), 7.85
(s, 1H, ar), 8.94 (s, 1H, H3), 11.90 (br s, 1H, NH); IR 1692. Anal.
(C13H12N4O) C, H, N.
General Procedure for the Synthesis of Ethyl 4-(2-Phenyl-
acetamidoimido)-1-arylpyrazole-3-carboxylate Hydrochlorides
29 and 30. Dry hydrogen chloride was bubbled for about 30 min
through a mixture of phenylacetonitrile (4.4 mmol) and ethyl
4-amino-1-arylpyrazole-3-carboxylate 26 or 27 (2.2 mmol) in an-
hydrous dioxane (20 mL). The mixture was stirred at room
temperature for a further 3-4 h. Then the solid was collected by
filtration and washed with diethyl ether. Crude compounds 29 and
30 were not recrystallized but were directly used for the next step.
1
29: yield 60%; H NMR 1.18 (t, 3H, Me, J=7.1 Hz), 4.08
(s, 2H, benzyl CH2), 4.12 (q, 2H, CH2), 7.33-7.46 (m, 4H, ar),
7.58 (t, 2H, ar, J=7.4 Hz), 7.71 (d, 2H, ar, J=7.6 Hz), 7.91
(d, 2H, ar, J=7.4 Hz), 8.91 (br s, 1H, NH), 8.98 (s, 1H, H5),
10.23 (s, 1H, NH2þ proton), 12.00 (s, 1H, NH2þ proton).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7649
Figure 4. Sequence alignment of hA3 and hA2A ARs. Conserved residues are identified by asterisks.
The numbering of the amino acids in parentheses follows
the arbitrary scheme by Ballesteros and Weinstein. Accord-
ing to this scheme, each amino acid identifier starts with the
helix number, followed by the position relative to a ref-
erence residue among the most conserved amino acid in that
helix. The number 50 is arbitrarily assigned to the reference
residue.38
bridge between cysteines in TM3 and EL2. Since this covalent
link is conserved in all receptors considered in the current study,
the EL2 loop was modeled using a constrained geometry around
the EL2-TM3 disulfide bridge. The constraints were applied
before the construction of the homology model, in particular
during the sequences alignment. The cysteine residues, involved
in the disulfide bridge in the hA2A receptor, were selected to be
constrained with the corresponding cysteines in the hA3 receptor
sequence. In particular, Cys166 (EL2) and Cys77 (3.25) of the
hA2A receptor were constrained, respectively, with Cys166
(EL2) and Cys83 (3.25) of the hA3 receptor. During the align-
ment, MOE-Align attempts to minimize the number of con-
straint violations. Then, after running the homology modeling,
the presence of the conserved disulfide bridge in the model was
manually checked. After the heavy atoms were modeled, all
hydrogen atoms were added and were then minimized with
MOE using the AMBER99 force field.39 The minimizations
were carried out by the 1000 steps of steepest descent followed
First, the amino acid sequences of TM helices of the hA3
receptor were aligned with those of the template, guided by the
highly conserved amino acid residues, including the DRY motif
(Asp3.49, Arg3.50, and Tyr3.51) and three proline residues
(Pro4.60, Pro6.50, and Pro7.50) in the TM segments of GPCRs.
The same boundaries were applied for the TM helices of the hA3
receptor as they were identified from the 3D structure for the
corresponding sequences of the template, the coordinates of
which were used to construct the seven TM helices for the hA3
receptor. The loop domains were constructed by the loop search
method implemented in MOE on the basis of the structure of
compatible fragments found in the Protein Data Bank. In
particular, loops are modeled first in random order. For each
loop, a contact energy function analyzes the list of candidates
collected in the segment searching stage, taking into account all
atoms already modeled and any atoms specified by the user as
belonging to the model environment. These energies are then
used to make a Boltzmann-weighted choice from the candidates,
the coordinates of which are then copied to the model. Side
chains were modeled using a library of rotamers generated by
systematic clustering of the Protein Data Bank data, using the
same procedure. Side chains belonging to residues, whose back-
bone coordinates were copied from a template, are modeled
first, followed by side chains of modeled loops. Outgaps and
their side chains are modeled last. Special caution has to be given
to EL2 because amino acids of this loop could be involved in
direct interactions with the ligands. A driving force to the
peculiar fold of the EL2 loop might be the presence of a disulfide
by conjugate gradient minimization until the rms gradient of
˚
the potential energy was less than 0.1 kcal mol-1
A
-1. We used
the Protonate 3D methodology, part of the MOE suite, for
protonation state assignment by selecting a protonation state
for each chemical group that minimizes the total free energy of
the system (taking titration into account).40
Protein stereochemistry evaluation was then performed by
several tools (Ramachandran and χ plots measure j/ψ and
χ1/χ2 angles, clash contacts reports) implemented in the MOE
suite.35
Molecular Docking of hA3 AR Antagonists. Ligand structures
were built using the MOE-builder tool, part of the MOE suite,35
and were subjected to MMFF94x energy minimization until the
-1. Charges
˚
A
rms of conjugate gradient was 0.05 kcal mol-1
were calculated using PM3/ESP methodology.
Four different programs were used to calibrate our docking
protocols: MOE-Dock,35 GOLD,37 Glide,41 and PLANTS.42

7650 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lenzi et al.
ZM-241385 was redocked to the crystal structure of the hA2A
AR (PDB code 3EML) with different docking algorithms and
scoring functions (see Table 3). Each docking was performed
automatically to the binding site of the hA2A AR without any
constraints and without the presence of water molecules. For all
the different docking simulations, the center of the docking box
or of the docking sphere was set in the same point (obtained
from the experimental pose of ZM-241385 inside the crystal
structure of hA2A AR), and the number of independent docking
runs was set to 25. Then rmsd values between predicted and
crystallographic positions of ZM-241385 were calculated.
As shown in Table 2, for each docking result there is at least
one pose in good agreement with the experimental binding mode
radioactivity was counted by a Packard Tri Carb 2500 TR scintilla-
tion counter with an efficiency of 58%.
Measurement of Cyclic AMP Levels in CHO Cells Transfected
with hA2B or hA3 AR. CHO cells transfected with hAR subtypes
were washed with phosphate-buffered saline and diluted tripsine
and centrifuged for 10 min at 200g. The pellet containing the
CHO cells (1 ꢀ 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 phospho-
diesterase inhibitor and preincubated for 10 min in a shaking
bath at 37 ꢀC. The potency of antagonists to the A2B AR was
determined by antagonism of NECA (200 nM) induced stimula-
tion of cyclic AMP levels. In addition, the potency of anta-
gonists to the A3 receptor was determined in the presence of
1 μM forskolin and 100 nM Cl-IB-MECA that mediated
inhibition of cyclic AMP levels. The reaction was terminated
by the addition of cold 6% trichloroacetic acid (TCA). The TCA
suspension was centrifuged at 2000g for 10 min at 4 ꢀC, and the
supernatant was extracted 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 pmol) were added to each test tube
containing [3H]cAMP and the incubation buffer (0.1 M trizma
base, 8.0 mM aminophylline, 6.0 mM 2-mercaptoethanol, 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, was centrifuged at 2000g for
10 min. The clear supernatant was counted in a Packard Tri
Carb 2500 TR scintillation counter with an efficiency of 58%.46
Data Analysis. The protein concentration was determined
according to a Bio-Rad method47 with bovine albumin as a
standard reference. Inhibitory binding constant (Ki) values were
calculated from IC50 according to the Cheng and Prusoff
equation Ki=IC50/(1þ[C*]/KD*), where [C*] is the concentra-
tion of the radioligand and KD* its dissociation constant.48
A weighted nonlinear least-squares curve-fitting program LI-
GAND49 was used for computer analysis of inhibition experi-
ments. EC50 and IC50 values obtained in a cyclic AMP assay
were calculated by nonlinear regression analysis using the
equation for a sigmoid concentration-response curve (Graph-
PAD Prism, San Diego, CA).
˚
(rmsd value of <2.5 A). These poses with the lowest rmsd value
differ from the crystallographic pose of ZM-241385 mainly in
the position of the phenylethylamine chain, while the bicyclic
triazolotriazine core is almost aligned. However, the mean rmsd
value is quite high for most of the docking protocols tested
except for Gold. Docking performed with Gold gives the lowest
rmsd value, the lowest mean rmsd value, and the highest number
˚
of poses with rmsd value of <2.5 A.
On the basis of the best docking performance, all antagonist
structures were docked into the hypothetical TM binding site of
the hA3 AR model and of the crystal structure of the hA2A AR,
by using the dock tool part of the GOLD suite.37 Searching is
conducted within a user-specified docking sphere, using the
Genetic Algorithm protocol and the GoldScore scoring func-
tion. GOLD performs a user-specified number of independent
docking runs (25 in our specific case) and writes the resulting
conformations and their energies in a molecular database file.
The resulting docked complexes were subjected to MMFF94x
energy minimization until the rms of the conjugate gradient was
-1. Charges for the ligands were imported
˚
A
<0.1 kcal mol-1
from the MOPAC output files using PM3/ESP methodology.
Prediction of the antagonist-receptor complex stability (in
terms of corresponding pKi value) and the quantitative analysis
for nonbonded intermolecular interactions (H-bonds, transition
metal, water bridges, hydrophobic, electrostatic) were calcu-
lated and visualized using several tools implemented in the
MOE suite.35
Electrostatic contributions to the binding energy of indivi-
dual amino acids have been calculated as implemented in the
MOE suite.35 To estimate the electrostatic contributions, atomic
charges for the ligands were calculated using PM3/ESP metho-
dology. Partial charges for protein amino acids were calculated
on the basis of the AMBER99 force field.
(C) Pharmacological Assays. Human Cloned A1, A2A, and A3
AR Binding Assay. All synthesized compounds were tested to
evaluate their affinity at human A1, A2A, and A3 adenosine
receptors. Displacement experiments of [3H]DPCPX (1 nM) to
hA1 CHO membranes (50 μg 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.43
Nonspecific binding was determined in the presence of 10 μM
CHA (e10% of the total binding). Binding of [3H]ZM-241385
(1 nM) to hA2A CHO membranes (50 μg of protein/assay) was
performed by using 50 mM Tris-HCl buffer and 10 mM MgCl2,
pH 7.4, and at least six to eight different concentrations of
antagonists were studied for an incubation time of 60 min at
4 ꢀC.44 Nonspecific binding was determined in the presence of
1 μM ZM-241385 and was about 20% of total binding. Compe-
tition binding experiments to hA3 CHO membranes (50 μg of
protein/assay) 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 different concentrations of examined
ligands were studied for 120 min at 4 ꢀC.45 Nonspecific binding
was defined as binding in the presence of 1 μM 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 by using a Brandel cell harvester. The filter bound
Acknowledgment. The synthetic work was supported by a
grant of the Italian Ministry for University and Research
(MIUR, FIRB RBNE03YA3L Project). The molecular mod-
eling work coordinated by S.M. was carried out with financial
support from the University of Padova, Italy, and the Italian
Ministry for University and Research (MIUR), Rome, Italy.
S.M. is also very grateful to Chemical Computing Group for
the scientific and technical partnership.
Supporting Information Available: Combustion analysis data
of the synthesized compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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