Novel N 2-Substituted pyrazolo[3,4- d ]pyrimidine adenosine A3 receptor antagonists: Inhibition of A3-Mediated human glioblastoma cell proliferation

Article abstract of DOI:10.1021/jm901785w

Adenosine induces glioma cell proliferation by means of an antiapoptotic effect, which is blocked by cotreatment with selective A₃ AR antagonists. In this study, a novel series of N²-substituted pyrazolo[3,4-d]pyrimidines 2a-u was de

Full text of DOI:10.1021/jm901785w

3954 J. Med. Chem. 2010, 53, 3954–3963
DOI: 10.1021/jm901785w
Novel N2-Substituted Pyrazolo[3,4-d]pyrimidine Adenosine A3 Receptor Antagonists:
Inhibition of A3-Mediated Human Glioblastoma Cell Proliferation†
Sabrina Taliani,‡ Concettina La Motta,*,‡ Laura Mugnaini,‡ Francesca Simorini,‡ Silvia Salerno,‡ Anna Maria Marini,‡
Federico Da Settimo,‡ Sandro Cosconati,§ Barbara Cosimelli,§ Giovanni Greco,§ Vittorio Limongelli,§ Luciana Marinelli,§
Ettore Novellino,§ Osele Ciampi, Simona Daniele, Maria Letizia Trincavelli, and Claudia Martini
‡
§
Dipartimento di Scienze Farmaceutiche, Universita di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Chimica Farmaceutica e
ꢀ
Tossicologica, Universita di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy, and Dipartimento di Psichiatria,
ꢀ
Neurobiologia, Farmacologia e Biotecnologie, Universita di Pisa, Via Bonanno 6, 56126 Pisa, Italy
ꢀ
Received December 2, 2009
Adenosine induces glioma cell proliferation by means of an antiapoptotic effect, which is blocked by
cotreatment with selective A3 AR antagonists. In this study, a novel series of N2-substituted pyrazolo-
[3,4-d ]pyrimidines 2a-u was developed as highly potent and selective A3 AR antagonists. The most
performing compounds were derivatives 2a (R1 = CH3 and R2 = COC6H5; Ki 334, 728, and 0.60 nM at
the human A1, A2A, and A3 ARs, respectively) and 2b (R1 = CH3 and R2 = COC6H4-4-OCH3; Ki 1037,
3179, and 0.18 nM at the human A1, A2A, and A3 ARs, respectively), which counteracted the effect of the
A3 AR agonists Cl-IB-MECA and IB-MECA on human glioma U87MG cell proliferation. This effect
was concentration-dependent, with IC50 values comparable to A3 AR binding affinity values of 2a and
2b, thereby suggesting that their effects were receptor-mediated. Furthermore, the antiproliferative
activity of the new compounds was demonstrated to be mediated by the block of A3 AR agonist
activation of intracellular kinases ERK 1/2.
Introduction
subject of medicinal chemistry research for more than three
decades.5,6
Adenosine is an endogenous purine nucleoside that modu-
lates a wide variety of physiological actions by triggering specific
cell membrane G-protein-coupled receptors (GPCRsa). Adeno-
sine receptors (ARs) are widely distributed in mammalian
tissues and have been classified into the four following sub-
classes: A1, A2A, A2B, and A3.1-3
The adenosine A1 and A3 ARs are coupled to the Gi
protein, which inhibits adenylate cyclase. In contrast, the
A2A and A2B ARs stimulate adenylatecyclase via a Gs protein.
Additional couplings to other second messenger systems have
been described, including the stimulation of phospholipase C
(A1, A2B, and A3) as well as the activation and inhibition of
potassium and calcium channels, (A1) respectively.4
ARs are currently of great interest as targets for therapeutic
intervention in many pathological conditions such as renal
failure, cardiac and cerebral ischemia, central nervous system
(CNS) disorders, neurodegenerative diseases, and inflamma-
tory pathologies such as asthma.5,6 Indeed, the development
of potent and selective synthetic AR antagonists has been the
The A3 AR is widely distributed in peripheral organs and is
found in high levels in the testis and in lower levels in the lung,
kidney, and heart aswell asinregionsof the CNS.7 The A3 AR
is involved in a variety of important physiological processes,
including modulation of cerebral and cardiac ischemic
damage,8-10 inflammation,11 modulation of intraocular pres-
sure,12 regulation of normal and tumor cell growth,13-15 and
immunosuppression.16 Consequently, A3 AR selective anta-
gonists have been proposed as novel anti-inflammatory drugs,
as cerebroprotective agents for preventing ischemic brain
damage, and as new agents for the treatment of glaucoma.16
Furthermore, the significant overexpression of A3 AR in
several types of tumor cells,17 together with the pro-survival
and antiapoptotic effects of A3 AR stimulation, has recently
led Baraldi et al. to propose that antagonists at this receptor
might sensitize tumor cells to chemotherapeutic drugs.14,18 In
this regard, recent studies have shown that the specific A3 AR
antagonist MRS1220 blocked adenosine-mediated glioma cell
proliferation, thereby suggesting that A3 AR antagonists are
possible adjuvant agents in glioma chemotherapy.19 Recently,
it has been demonstrated that extracellular regulated kinases
(ERK 1/2), as well as p38 and Akt kinases, are intracellular
phosphorylative pathways involved in A3 AR-mediated pro-
liferative effects in glioblastoma cell lines.20
†This paper is dedicated to Prof. Fulvio Gualtieri on the occasion of
his retirement.
*To whom correspondence should be addressed. Phone: þ390502219593.
Fax: þ390502219605. E-mail: lamotta@farm.unipi.it.
a Abbreviations: AB-MECA, 4-aminobenzyl-50-N-methyl-carboxa-
midoadenosine; ADA, adenosine deaminase; AR, adenosine receptor;
Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)adenosine-50-N-methylcar-
boxamide; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; EHNA, ery-
thro-9-(2-hydroxy-3-nonyl)adenine; ERK 1/2, extracellular regulated
kinase 1 and 2; GPCRs, G-protein-coupled receptors; IB-MECA,
N6-(3-iodobenzyl)-adenosine-50-N-methylcarboxamide; NECA, adeno-
sine-50-(N-ethylcarboxamide); SEM, standard error of mean.
Our research group has developed several classes of A1 and A3
AR antagonists,21-26 including the imidazo[1,2-a][1,3,5]triazines
1 (Chart 1).22,23 Among this series, derivative 1a (Chart 1, R1 =
CH3 and R2 = COPh) exhibited the highest potency at the A3
AR and demonstrated fair selectivity for A3 over the A1 and A2A
r
pubs.acs.org/jmc
Published on Web 04/21/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3955
ARs (Ki values measured on human A3, A1, and A2A ARs were
3 nM, 350 nM, and >10 μM, respectively).22,23
the key role of NH-acyl side chains in ligand recognition by
the A3 AR.32-36
Furthermore, we have recently reported a series of
pyrazolo[3,4-d ]pyrimidines 2 (Chart 1) that act as adenosine
deaminase (ADA) inhibitors,27,28 which are isosterically re-
lated to imidazo[1,2-a][1,3,5]triazines 1. The most active
compound of series 2 was found to be effective at reducing
systemic and intestinal inflammatory alterations in an experi-
mental model of colitis.28
The present paper describes the synthesis, the biological
evaluation, andthe molecularmodeling studies of anumberof
pyrazolo[3,4-d ]pyrimidines 2a-u as A3 AR antagonists. Two
of the best performing new compounds, 2a and 2b, were
evaluated for their inhibitory activity on the A3 AR-mediated
glioma cell proliferation.
As a logical extension of these projects, we designed novel
N2-substituted pyrazolo[3,4-d]pyrimidine derivatives of series 2
as A3 AR antagonists. Indeed, a large number of AR antagonists
featuring the pyrazolo[3,4-d]pyrimidine scaffold have been re-
ported, but all of them have been N1-substituted and have shown
a high affinity and selectivity for the A1 or A2A ARs.29-31
In almost all the new compounds 2, the 6 position of the
bicyclic scaffold possesses a phenyl ring in consideration of its
pharmacophoric role as evidenced in the AR antagonist series
Chemistry
The synthesis of the target compounds 2a-u was performed
as outlined in Scheme 1. The 3-amino-N1-substituted-4-pyr-
azolecarbonitriles 3-5 were cyclized to the corresponding
6-phenylpyrazole[3,4-d]pyrimidines 6-8 through a reaction
with benzonitrile in the presence of potassium tert-butoxide
and under microwave irradiation according to our previously
published procedure.27 Cyclization of 4with boiling formamide
provided the 6-unsubstituted pyrazolo[3,4-d]pyrimidine 9.27
The key intermediates 6-9 yielded the N4-aroyl com-
pounds 2a-c, 2g-i, 2m-o, and 2s-u by reaction with the
suitable aroylchloride in the presence of triethylamine. These
same intermediates also yielded the N4-carbamoyl derivatives
2d-f, 2j-l, and 2p-r by treatment with the appropriate
isocyanate. Both procedures were carried out under micro-
wave irradiation (Scheme 1).
1
22 and of its unfavorable effect on ADA inhibitory activity.27
Substituents at the 2 position were represented by a methyl
group, in analogy to the lead compound 1a, or by a phenyl-
alkyl group known to be detrimental to ADA inhibitory
activity. Finally, the R2 substituents attached to the exocyclic
N4 were selected by taking into account literature data about
Chart 1. Structures of Known (1) and New (2) A3 AR Antagonists
Biology
The affinities of the new compounds toward human A1,
A2A, and A3 ARs were evaluated by competition experi-
ments by assessing their respective abilities to displace
[3H]DPCPX, [3H]NECA, or [125I]AB-MECA binding from
Scheme 1. Synthesis of Target Compounds 2a-ua
a Reaction conditions: (a) benzonitrile, t-BuOK, MW, or boiling formamide, 12 h;26 (b) substituted-benzoylchloride, NEt3, MW; (c) substituted-
phenylisocyanate, MW.

3956 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Taliani et al.
Table 1. Binding Affinity at Human A1, A2A, and A3 ARs of 2-Substituted-pyrazolo[3,4-d]pyrimidine derivatives 6-8 and 2a-u
Ki (nM)a
b
c
d
no.
R1
R2
hA1
hA2A
hA3
6
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
COC6H5
584.7 ( 57.0
334.0 ( 32.0
1037.0 ( 105.0
1081.6 ( 110.0
>10000
>10000
922.0 ( 19.0
0.60 ( 0.10
0.18 ( 0.02
5.9 ( 0.9
20.9 ( 1.2
357.9 ( 35.8
29.1 ( 2.4
93.0 ( 5.0
3.0 ( 0.1
22.1 ( 2.4
3.7 ( 0.3
2.9 ( 0.5
2.9 ( 0.1
120.8 ( 11.3
503 ( 17
43.7 ( 2.4
45.2 ( 5.3
22.3 ( 1.3
36.3 ( 2.1
>1000
2a
2b
2c
2d
2e
2f
7
728.1 ( 70.3
3179.0 ( 315.0
886.0 ( 42.0
>10000
COC6H4-4-OCH3
COC6H4-3-Cl
CONHC6H5
CONHC6H4-4-OCH3
CONHC6H4-3-Cl
H
>10000
>10000
>10000
>10000
CH2-C6H5
CH2-C6H5
312.0 ( 11.0
53.0 ( 1.1
>10000
121.1 ( 1.0
180.6 ( 1.7
>10000
2g
2h
2i
COC6H5
CH2-C6H5
CH2-C6H5
COC6H4-4-OCH3
COC6H4-3-Cl
CONHC6H5
>10000
>10000
>10000
2j
CH2-C6H5
CH2-C6H5
CH2-C6H5
290.0 ( 11.0
>10000
>10000
2k
2l
CONHC6H4-4-OCH3
CONHC6H4-3-Cl
H
>10000
>10000
8
CH2CH2-C6H5
CH2CH2-C6H5
CH2CH2-C6H5
CH2CH2-C6H5
CH2CH2-C6H5
CH2CH2-C6H5
CH2CH2-C6H5
CH2-C6H5
2500.0 ( 170.0
27.0 ( 6.1
2400.0 ( 110.0
1050.0 ( 99.0
1700.0 ( 123.0
>10000
1000.0 ( 200.0
227.0 ( 12.0
1136.0 ( 95.0
314.0 ( 22.0
96.0 ( 6.1
>10000
2m
2n
2o
2p
2q
2r
2s
2t
2u
COC6H5
COC6H4-4-OCH3
COC6H4-3-Cl
CONHC6H5
CONHC6H4-4-OCH3
CONHC6H4-3-Cl
COC6H5
>10000
>10000
>10000
>10000
>10000
>1000
>1000
CH2-C6H5
CH2-C6H5
COC6H4-4-OCH3
COC6H4-3-Cl
>10000
>10000
>1000
>1000
4900.0 ( 360.0
130
20
DPCPX
NECA
C1-IBMECA
3.9
14.0
4000
6.2
120
2100
11
a The Ki values are means ( SEM derived from an iterative curve-fitting procedure (Prism program, GraphPad, San Diego, CA). b Displacement of
specific [3H]DPCPX binding in membranes obtained from hA1 AR stably expressed in CHO cells. c Displacement of specific [3H]NECA binding in
membranes obtained from hA2A AR stably expressed in CHO cells. d Displacement of specific [125I]AB-MECA binding in membranes obtained from
hA3 AR stably expressed in CHO cells.
transfected CHO cells. Experiments were performed as des-
cribed previously.23 Compounds 2a, 2b, 2g, and 2i-k, which
showed a high A3 AR affinity, were tested in functional assays at
human A2B and A3 ARs by measuring their effects on NECA-
mediated cAMP modulation in transfected CHO cells.37
Compounds 6-8, 2a-g, and 2i-k were also evaluated
for their inhibitory activity against bovine spleen ADA using
(þ)-EHNA as the reference standard, as previously reported.27
Compounds 2a and 2b were tested for their effect on the
proliferation of U87MG human glioma cells. U87MG cells,
which were blocked in G1 phase, were treated with the A3 AR
agonists Cl-IB-MECA or IB-MECA in the absence or in the
presence of compounds 2a or 2b for 48 h. At the end of this
period, the number of living cells was determined by the
Trypan blue exclusion test. In this assay, cells with a damaged
cell membrane stain blue, while cells that maintain plasma
membrane integrity resist dye entry and remain unstained
(apoptotic and healthy cells, respectively). Finally, the effects
of the new compounds on A3 AR-mediated activation of
intracellular ERK 1/2 were evaluated by incubating U87MG
cells with Cl-IB-MECA in the presence of different 2a or 2b
compound concentrations. Following 30 min cell incubation,
the phosphorylated ERK 1/2 levels were quantified using an
ELISA kit.
Results and Discussion
Table1 lists the binding affinities atthe humanA1, A2A, and
A3 ARs of the newly synthesized 2-substituted pyrazolo[3,4-d]-
pyrimidines 2a-u expressed as Ki values. The intermediate
products 6-8, which were also assayed, exhibited moderate
potency and selectivity for the A3 AR, and no ADA inhibitory
activity at 10 μM concentration (data not shown).
Within the set of 2-methyl derivatives (R1 = CH3), the
introduction of a benzoyl group attached to the N4 of 6 yielded
2a (R2 =COC6H5), 2b (R2 =COC6H4-4-OCH3),and2c (R2 =
COC6H4-3-Cl) and dramatically increased the potency and
selectivity at the A3 AR. Among the above NH-acyl derivatives,
2b stood out as a subnanomolar A3 AR antagonist with micro-
molar affinity at the A1 and A2A ARs.
Replacement of the benzoylamino moiety of 2a-c with a
phenylcarbamoyl moiety to give 2d-f lowered the affinity to
all the ARs.
None of the 2-benzyl derivatives (R1 = CH2-C6H5) was
more potent at the A3 AR than the 2-methyl derivative 2b.
Generally, the structure-affinity relationships (SARs) within
the set of 2-benzyl derivatives 7, 2g-l did not exactly parallel
those of the 2-methyl derivatives 6, 2a-f. As an example, the
CONHC6H4-4-OCH3 chain attached to N4 gave the best and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3957
Table 2. Potency of Compounds 2a, 2b, 2g, and 2i-k versus hA2B and hA3 Adenosine Receptor Subtypes
IC50 (nM) in cAMP assaysa
no.
R1
CH3
CH3
R2
hA2B CHO cells
hA3 CHO cells
2a
2b
2g
2i
COC6H5
COC6H4-4-OCH3
COC6H5
49.8 ( 2.8
53.9 ( 5.9
272.0 ( 25.0
1327.0 ( 137.0
1324.0 ( 130.0
1467.0 ( 130.0
1.9 ( 0.1
2.4 ( 0.2
53.0 ( 4.3
7.6 ( 0.7
5.7 ( 0.6
8.2 ( 0.8
CH2-C6H5
CH2-C6H5
CH2-C6H5
CH2-C6H5
COC6H4-3-Cl
CONHC6H5
CONHC6H4-4-OCH3
2j
2k
a The data are expressed as the mean ( SEM of four independent experiments performed in triplicate.
Figure 1. Top (a) and front (b) view of the 2b/hA3 AR complex.
the worst results in the series of 2-benzyl (2k) and 2-methyl
(2e) derivatives, respectively.
by forskolin, thereby indicating neutral antagonism (data not
shown).
Finally, compounds 2a-g and 2i-k were also evaluated
for their inhibitory activity against bovine spleen ADA using
(þ)-EHNA as the reference standard, as previously reported.27
Notably, all of the compounds tested displayed no appreciable
inhibitory activity at 10 μM concentration (data not shown).
Molecular modeling studies were undertaken to rationalize
the SARs regarding the binding of compounds 2a-u to the A3
AR.
Replacement of the 2-benzyl group of 7, 2g-l, with a
2-phenylethyl moiety to give the corresponding homologues
8, 2m-r produced a general decrease in A3 AR affinity,
probably due to unfavorable steric effects, and different
effects on the binding for the A1 and A2A ARs.
Finally, the poor affinity of compounds 2s-u for all the
ARs suggests that the 6-phenyl substituent is a key pharma-
cophoric element for the recognition of our pyrazolo[3,4-d ]-
pyrimidines with each of these receptors.
Compounds 2a, 2b, 2g, and 2i-k were also tested at the A2B
AR by evaluating their inhibitory effects on NECA-mediated
cAMP accumulation in CHO cells stably expressing this
receptor subtype (Table 2). Interestingly, the N2-methyl deri-
vatives 2a and 2b displayed a nanomolar activity in this assay
even though maintaining a good selectivity for the A3 AR.
These products may represent promising lead compounds for
the development of potent and selective A2B AR antagonists
featuring the 2-substituted-pyrazolo[3,4-d ]pyrimidine scaf-
fold. Conversely, the other selected derivatives 2g and 2i-k
showed submicromolar/micromolar IC50 values, therebyindi-
cating a poor to moderate affinity toward the A2B AR.
Given the recent discoveries regarding non-nucleoside AR
agonists,38,39 the efficacy profiles of 2a, 2b, 2g, and 2i-k were
evaluated in cAMP functional assays at the A3 AR (Table 2).
As expected, all of these compounds displayed full antagon-
ism with potencies similar to their binding affinity. Further-
more, when tested in the absence of NECA, they did not show
any significant effect on the cAMP level even after stimulation
A homology model of the human A3 AR was constructed
using as a template the recently published crystal structure of
the human A2A AR (PDB code 3EML).40
The sequence alignment between the two receptors and the
positions of the disulfide bridges were attained consistently
with what was already suggested by Moro and co-workers.41
The structure of the A3 AR was then used to perform docking
calculations on 2b, 2i, 2o, and 2t employing the software
AudoDock4 (AD4).42 The obtained docking poses were
evaluated for their consistency with the available SARs as
well as for the calculated binding free energy (ΔGAD4).
The docking results achieved for 2b revealed that this
ligand binds to the outer portion of the A3 AR being sur-
rounded by TMs III, V, VI,VII helices and ELII (Figure 1a,b).
More precisely, its methyl group (R1) points to the outer part
of the receptor taking contact with the side chains of F168 and
L246 belonging to the ELII, its aroyl chain (R2) is embedded
in the fissure between TMII and TMIII, and its pendant
6-phenyl ring is oriented toward the inner portions of TMV
and TMVI.

3958 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Taliani et al.
As depicted in Figure 2, this binding pose allows the
pyrazolopyrimidine scaffold of 2b to engage a π-stacking
interaction with the F168 side chain and two H-bonds bet-
ween its N1 and N7 nitrogens and the N250 side chain.
Interestingly, the latter residue was found to be critical for
ligand binding at both the A3 and A2A ARs.43,44 Moreover,
the 6-phenyl ring of the ligand is embedded in a hydrophobic
cleft formed by I186, L91, W243, L246, and S247. Among
these residues, W243 was considered to play a crucial role in
receptor activation and antagonist binding.44 Additionally,
the aroyl moiety is located in a lipophilic pocket, at the
interface between TMII and TMIII, made up by L68, A69,
V72, T87, and L89. The tight interaction of this group with the
receptor might explain why the nature of the R2 substituent
affects so strongly the potency at the A3 AR (see Ki values of 6
and 2a-f).
According to our docking calculations, 2i is oriented diffe-
rently from 2b at the A3 AR. In particular, its aroyl chain (R2)
points to the extracellular portion of the receptor taking
favorable contacts with the EL2 region, while its benzyl group
(R1) is located within the lipophilic cleft at the crevice bet-
ween TMII and TMIII (Figure 3a). The above differences
might explain why for 7 and 2g-l the bulkiness of the R2
substituent (which now points to the roomier extracellular
region) influences affinity to the A3 AR to a lower extent
compared with what happens for 6 and 2a-f. The orientation
of the pyrazolopyrimidine system of 2i within the A3 AR still
allows a double H-bond with N250 and a tight interaction of
the pendant 6-phenyl ring with W243.
The docking results achieved for 2o indicate that the
binding pose of this ligand (Figure 3b) is roughly similar to
that of 2i (Figure 3a). However, for steric reasons, the
phenylethyl moiety of 2o is oriented outward the lipophilic
cleft hosting the benzylchain of2i. Thisdifference isconsistent
with the drop of affinity to the A3 AR associated with the
replacement of the benzyl with the phenylethyl substituent.
Docking calculations on 2u were performed to rationalize
the lack of affinity to the A3 AR characterizing all the
compounds devoid of the 6-phenyl ring (2s-u). The binding
pose of 2u (Figure 4) resembles that of 2b, as both molecules
orient the R1 and the R2 substituents in the same receptor
regions. As expected, the absence of the 6-phenyl ring in the
structure of 2u implies the loss of crucial interactions with the
W243 side chain.
To estimate the drugability of compounds 2a and 2b,
provided with the best affinity and selectivity at the A3 AR,
we calculated some of their physicochemical properties,
namely log P, ASA, PSA, the number of H-bond donors
and acceptors, the number of atoms and bonds, the number of
rotatable bonds, and the molecular weight (Table 3).45 These
data suggest that 2a and 2b display a drug-like character and
that basically fulfill the physicochemical requirements for an
adequate distribution into the CNS.
Prompted by these results, we investigated the effects of 2a
and 2b on proliferation of the U87MG human glioblastoma
cell line.
In U87MG cells, the A3 agonists Cl-IB-MECA (100 nM)
and IB-MECA (12 nM) were able to stimulate cell prolifera-
tion. Compounds 2a and 2b counteracted the effects of both
agonists, Cl-IB-MECA and IB-MECA, with IC50 values in
the subnanomolar range, comparable to the affinity of these
compounds toward A3 AR (Figure 5). These results suggest
that the effects of 2a and 2b on cell proliferation may be
ascribed to their selective interaction with A3 AR subtype.
Furthermore, the compounds alone did not modulate glioma
cell proliferation at the tested concentrations (data not
shown), thereby demonstrating that they behave as A3 AR
antagonists.
Figure 2. Calculated binding pose for 2b in the hA3 AR model.
Figure 3. Calculated binding pose for 2i (a) and 2o (b) in the hA3 AR model.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3959
Figure 6. Kinetic of A3 AR-mediated ERK 1/2 phosphorylation.
U87MG cells were treated for different times (5-120 min) with 100
nM Cl-IB-MECA and then ERK 1/2 phosphorylation levels were
quantified by Fast Activated Cell-Based ELISA kits. Data (mean (
SEM) were expressed as percentage of phosphorylated ERK 1/2
with respect to control cells (set to 100%). *P < 0.05 vs control;
**P < 0.01 vs control, Student t test.
Figure 4. Calculated binding pose for 2u in the hA3 AR model.
Figure 7. Effect of 2a and 2b compounds on Cl-IB-MECA induced
ERK 1/2 phosphorylation in U87MG cells. U87MG cells were
treated for 30 min with 100 nM Cl-IB-MECA in the absence or in
the presence of compound 2a (0.25-10 nM) and 2b (0.05-1 nM).
ERK 1/2 phosphorylation levels were quantified by Fast Activated
Cell-Based ELISA kits. Data (mean ( SEM) were expressed as
percentage of phosphorylated ERK 1/2 with respect to control cells
(set to 100%).
Figure 5. Effect of 2a and 2b compounds on Cl-IB-MECA or IB-
MECA-induced U87MG cell proliferation. Cells were treated for
48 h with Cl-IB-MECA (100 nM) or IB-MECA (12 nM) in the
absence or in the presence of compound 2a (0.1-10 nM) and 2b
(0.01-5 nM). Live cell counting was determined by using the trypan
blue exclusion test. Data (mean ( SEM) were expressed as percent-
age of living cells with respect to control cells (set to 100%).
constant values for A3 AR (Figure 7). Furthermore, the
compounds alone did not modulate ERK 1/2 activation state
at the tested concentrations (data not shown), thereby demons-
trating that they behave as A3 AR antagonists. These results
demonstrated that A3 AR induced U87MG cell proliferation
occurred through the recruitment of ERK as intracellular
phosphorylative pathway.
Table 3. Physicochemical Predicted Properties of Compounds 2a, 2b45
2a
2b
log Pa
ASA (A )
˚
TPSA (A )
donor sites
3.8
675.4
69.0
1
4.0
609.0
59.8
1
2 b
˚
2 c
acceptor sites
atom count
bond count
4
45
3
41
Conclusions
A number of pyrazolo[3,4-d]pyrimidines with the general
formula 2 were developed as A3 AR antagonists with high
potency and selectivity. These compounds were designed by
taking the imidazo[1,2-a][1,3,5]triazine 1a (Chart 1, R1 = CH3
and R2 = COPh) as a reference structure22,23 and utilizing our
experience in the preparation of pyrazolo[3,4-d]pyrimidines as
ADA inhibitors.27,28 The main SARs were substantiated by
docking studies performed by means of a hA3 AR model
constructed using as a template the recently published crystal
structure of the hA2A AR. The highest affinity and selectivity
toward the A3 AR were exhibited by the N2-methyl derivatives
2a (R1 = CH3 and R2 = COC6H5; Ki 334, 728, and 0.60 nM at
the human A1, A2A, and A3 ARs, respectively) and 2b (R1 =
CH3 and R2 = COC6H4-4-OCH3; Ki 1037, 3179, and 0.18 nM
at the human A1, A2A, andA3 ARs, respectively), which showed
48
4
359.38
44
3
329.36
rotatable bond count
molecular weight
a Calculated n-octanol/water partition coefficient. b Calculated acces-
sible solvent area. c Topological polar surface area.
To explore the intracellular phosphorylative pathways
involved in A3-mediated proliferative effects, the ability of
the agonist Cl-IB-MECA (100 nM) to stimulate ERK 1/2
phosphorylation was investigated. The results, depicted in
Figure 6, demonstrated that Cl-IB-MECA induced a time-
dependent stimulation of ERK 1/2 phosphorylation with a
maximum after 30 min of cell incubation. The compounds 2a
and 2b counteracted the Cl-IB-MECA-mediated activation of
ERK 1/2 with a potency corresponding to their affinity

3960 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Taliani et al.
no appreciable ADA inhibitory activity. Both compounds,
when tested on human glioma U87MG cells, counteracted the
A3 AR agonists Cl-IB-MECA- and IB-MECA-mediated pro-
liferation through the inhibition of A3 AR agonist-mediated
ERK 1/2 activation. Furthermore, the IC50 values obtained in
functional assays were comparable to their A3 AR binding
affinity constants, thereby suggesting a receptor-mediated
effect. In the light of these results and thanks to their drug-like
physicochemical properties, 2a and 2b may represent promising
lead compounds for the development of adjuvant agents in
glioma chemotherapy.
suitable aroyl chloride (11.0 mmol), and triethylamine (1.53 mL,
11.0 mmol) was mixed thoroughly and irradiated with microwaves
at 110 °C for 5 min. The cooled residue was then diluted with
ice-water, and the solid separated was filtered and purified by
recrystallization with the appropriate solvent to give target com-
pounds 2s-u (see Supporting Information).
Biological Methods. Materials. [3H]DPCPX, [3H]NECA, and
[
125I]AB-MECA were obtained from DuPont-NEN (Boston,
MA). Adenosine deaminase (ADA) type IX from bovine spleen
(150-200 U/mg) and adenosine were from Sigma Chemical Co.
(St. Louis, MO). All other reagents were from standard com-
mercial sources and of the highest commercially available grade.
CHO cells stably expressing human A1, A2A, A2B, and A3
receptors were kindly supplied by Prof. K.-N. Klotz, Wurzburg
University, Germany.47
Experimental Section
Chemistry. 1H NMR spectra were recorded on a Varian
Gemini 200 spectrometer using DMSO-d6 as the solvent. IR
spectra were recorded with a Pye Unicam Infracord model
PU956 in Nujol mulls. Melting points were determined using a
Adenosine Receptor Binding Assay. Human A1 Adenosine
Receptors. Aliquots of membranes (30 μg proteins) obtained
from A1 CHO cells were incubated at 25 °C for 180 min in 500 μL
of T1 buffer (50 mM Tris-HCl, 2 mM MgCl2, and 2 units/mL
ADA, pH 7.4) containing [3H]DPCPX (3 nM) and six different
concentrations of the newly synthesized compounds. Non-
specific binding was determined in the presence of 50 μM
RPIA.23 The dissociation constant (Kd) of [3H]DPCPX in A1
CHO cell membranes was 3 nM.
Human A2A Adenosine Receptors. Aliquots of cell membranes
(30 μg) were incubated at 25 °C for 90 min in 500 μL of T2 buffer
(50 mM Tris-HCl, 2 mM MgCl2, and 2 units/mL ADA, pH 7.4)
in the presence of 30 nM of [3H]NECA and six different
concentrations of the newly synthesized compounds. Non-
specific binding was determined in the presence of 100 μM
NECA.23 The dissociation constant (Kd) of [3H]NECA in A2A
CHO cell membranes was 30 nM.
€
Reichert-Kofler hot-stage apparatus and were uncorrected.
Thin-layer chromatography (TLC) was performed by using
aluminum sheets precoated with silica gel (60 F-254, Merck
0.2 mm), followed by visualization via irradiation with a UV
lamp. Flash chromatography was performed using the Flash
system (Biotage Corporation). Combustion analyses on target
compounds were performed by our Analytical Laboratory in
Pisa. All compounds showed g95% purity.
The microwave-assisted reactions were performed using a
dedicated microwave CEM Discover (CEM Corporation). The
wattage was automatically adjusted so as to maintain the desired
temperature.
All reagents used were obtained from commercial sources
(Sigma-Aldrich). All solvents were of an analytical grade.
The 3-amino-1-methyl-4-pyrazolecarbonitrile 3,46 3-amino-1-
benzyl-4-pyrazolecarbonitrile 4,27 the 3-amino-1-phenylethyl-4-pyr-
azolecarbonitrile 5,27 and 4-amino-2-benzylpyrazolo[3,4-d ]pyrimi-
dine 927 were prepared essentially as previously described.
General Procedure for the Synthesis of 4-Amino-2-substituted-
6-phenylpyrazolo[3,4-d ]pyrimidine Derivatives 6-8. A mixture
of 3-amino-1-substituted-4-pyrazolecarbonitrile 3-5(10.0 mmol),
benzonitrile (1.24 mL, 12.0 mmol), and potassium tert-butoxide
(0.561 g, 5.00 mmol) was mixed thoroughly and irradiated with
microwaves at 150 °C for 3 min. The cooled residue was then
diluted with ice-water, and the solid separated was filtered,
washed with water, and purified by recrystallization with the
appropriate solvent to give the target compounds 6-8 (see
Supporting Information).
General Procedure for the Synthesis of 6-Phenyl-2-substituted-
4-(substituted-aroylamino)pyrazolo[3,4-d ]pyrimidine Derivatives
2a-c, 2g-i, and 2m-o. A mixture of 4-amino-2-substituted-
6-phenylpyrazolo[3,4-d ]pyrimidine 6-8 (10.0 mmol), the suita-
ble aroyl chloride (11.0 mmol), and triethylamine (1.53 mL,
11.0 mmol) was mixed thoroughly and irradiated with micro-
waves at 110 °C for 5 min. The cooled residue was then diluted
with ice-water, and the solid separated was filtered and purified by
recrystallization with the appropriate solvent to give target com-
pounds 2a-c, 2g-i, and 2m-o (see Supporting Information).
General Procedure for the Synthesis of 6-Phenyl-2-substituted-
4-(substituted-phenylalkylaminocarbamoyl)pyrazolo[3,4-d ]pyrimi-
dine Derivatives 2d-f, 2j-l, and 2p-r. A mixture of 4-amino-6-
phenyl-2-substituted-pyrazolo[3,4-d ]pyrimidine6-8 (10.0 mmol)
and the suitable isocyanate (11.0 mmol) was mixed thoroughly
and irradiated with microwaves at 150 °C for 3 min. The cooled
residue was then diluted with ice-water, and the solid separated
was filtered and purified by recrystallization with the appropriate
solvent to give target compounds, 2d-f, 2j-l, and 2p-r (see
Supporting Information).
Human A3 Adenosine Receptors. Aliquots of cell membranes
(40 μg) were incubated at 25 °C for 90 min in 100 μL of T3 buffer
(50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, and 2 units/mL
ADA, pH 7.4) in the presence of 1.4 nM [125I]ABMECA and six
different concentrations of the newly synthesized compounds.
Non-specific binding was determined in the presence of 50 μM
RPIA.23 The dissociation constant (Kd) of [125I]AB-MECA in
A3 CHO cell membranes was 1.4 nM.
Measurement of Cyclic AMP Levels on hA2B CHO and hA3
CHO Cells. Intracellular cyclic AMP (cAMP) levels were mea-
sured using a competitive protein binding method.37 CHO cells,
expressing recombinant hA2B ARs or hA3 ARs, were harvested
by trypsinization. After centrifugation and resuspension in
medium, cells (∼48000) were plated in 24-well plates in 0.5 mL
of medium. After 48 h, the medium was removed, and the cells
were incubated at 37 °C for 15 min with 0.5 mL of DMEM in the
presence of adenosine deaminase (1 U/mL) and the phospho-
diesterase inhibitor Ro 20-1724 (20 μM). The antagonism
profile of the new compounds toward A2B AR was evaluated
by assessing their ability to inhibit 100 nM NECA-mediated
accumulation of cAMP. The antagonism profile of the new
compounds toward A3 AR was evaluated by assessing their
ability to counteract 100 nM NECA-mediated inhibition of
cAMP stimulated by 1 μM forskolin. Cells were incubated in
the reaction medium (15 min at 37 °C) with different concen-
trations of the target compound (1 nM to 10 μM) and then
were treated with NECA. In parallel, aliquots of cells were
treated with the compound alone (10 μM) in the absence or in
the presence of forskolin. The reaction was terminated by the
removal of the medium and the addition of 0.4 N HCl. After
30 min, lysates were neutralized with 4 N KOH, and the
suspension was centrifuged at 800g for 5 min. For the deter-
mination of cAMP production, bovine adrenal cAMP bind-
ing protein was incubated with [3H]cAMP (2 nM) and 50 μL of
cell lysate or cAMP standard (0-16 pmol) at 0 °C for 150 min
in a total volume of 300 μL. Bound radioactivity was sepa-
rated by rapid filtration through GF/C glass fiber filters
and washed twice with 4 mL 50 mM Tris/HCl, pH 7.4. The
General Procedure for the Synthesis of 2-Benzyl-4-(substituted-
aroylamino)pyrazolo[3,4-d ]pyrimidine derivatives 2s-u. A mixture
of 4-amino-2-benzylpyrazolo[3,4-d]pyrimidine 927 (10.0 mmol), the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3961
radioactivity was measured by liquid scintillation spectro-
metry.
were calculated by subtracting the mean background from the
values obtained from each test condition and were expressed as
the percentage of the control (untreated cells).
To evaluate the antagonist profile of the new synthetized
compounds on A3 AR agonist-mediated ERK 1/2 activation,
the cells were treated for 30 min with 100 nM Cl-IBMECA in the
presence of different 2a or 2b compound concentrations. The
levels of ERK 1/2 phosphorylation were quantified as above-
described.
All compounds were routinely dissolved in DMSO and
diluted with assay buffer to the final concentration with the
final amount of DMSO never exceeding 2%. Percent inhibi-
tion values for specific radiolabeled ligand binding results at
1-10 μM concentration are means ( SEM of at least three
determinations. At least six different concentrations (spanning
3 orders of magnitude) were adjusted appropriately for the
IC50 of each compound examined. IC50 values were computer-
generated using a nonlinear regression formula (Graph-Pad,
San Diego, CA) and were converted to Ki values using the
known Kd values of the radioligands in the different tissues and
using the Cheng-Prusoff equation.48 Ki values were means (
SEM of at least three determinations.
Enzymatic Assay. The activity of the test enzyme was deter-
mined spectrophotometrically by monitoring the change in
absorbance at 262 nm for 2 min, which was due to the deamina-
tion of adenosine catalyzed by ADA. The change in adenosine
concentration/min was determined using a Beckman DU-64
kinetics software program (Solf Pack TM module). ADA
activity was assayed at 30 °C in a reaction mixture containing
50 μM of adenosine, 50 mM of potassium phosphate buffer,
pH = 7.2, and 0.3 nM of enzyme solution in a total volume of
500 μL. The inhibitory activity of the newly synthesized com-
pounds was assayed by adding 100 μL of the inhibitor solution
to the reaction mixture described above. All the inhibitors were
dissolved in water, and the solubility was facilitated with
DMSO, whose concentration never exceeded 4% in the final
reaction mixture. To correct for the nonenzymatic changes in
adenosine concentration and the absorption values of the test
compounds, a reference blank containing all the above assay
components except the substrate was prepared. The inhibitory
effect of the new derivatives was routinely estimated at a
concentration of 10-5 M.
Modeling Studies. Homology Modeling. The homology model
50
€
of the hA3 AR receptor was built using Schrodinger Prime
software accessible through the Maestro interface.51 Unless
otherwise stated, default parameters were used throughout.
The sequence of the hA3 AR (P33765) was aligned against the
sequence of hA2A AR (P29274). Alignment of anchor residues
within each TM domain was attained according to the sequence
alignment suggested by Moro and co-workers.41 During the
homology model building, Prime keeps the backbone rigid for
the cases in which the backbone does not need to be recon-
structed due to gaps in the alignment. The loop refinements were
carried out using Prime with default parameter settings, if not
mentioned otherwise. Prior to refinement, the protein structures
were subjected to a protein preparation step to reorientate side
chain hydroxyl groups and alleviate potential steric clashes. The
implemented loop modeling protocol consists of several steps.52
First, large numbers of loops are created by a conformational
search in dihedral angle space. Clustering of loop conformations
and side chain optimization is performed to select representative
solutions. On the basis of the user parameters, a limited number
of structures is then processed using complete energy minimiza-
tion. The top ranked solution in terms of Prime energy is
considered best. The short loops (ELI and ELIII) were refined
using default sampling rates in the initial step, while the extend-
ed highest sampling rate was chosen for the larger amino acid
˚
loops (ELII). Side chains were unfrozen within 7.5 A of the
Cell Culture and Cell Counting. Cells were grown in culture
flasks in RPMI 1640 supplemented with 10% fetal bovine serum
(FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and
2 mM L-glutamine, which were seeded in 24-well plates at
densities of 5 ꢀ 103 to 1 ꢀ 104 cells/well in 500 μL of medium
per well. Cells were grown for 24 h in 10% FBS and then were
blocked in G1 by reducing the concentration of FBS to 0.5% for
48 h. The medium was changed 2 h prior to treatment, after
which cells were treated with the A3 AR agonist Cl-IB-MECA
(100 nM) or IB-MECA (12 nM) in the absence or in the presence
of different antagonist concentrations (2a: 0.1-10 nM; 2b:
0.01-5 nM) for 48 h.
At the end of incubation time, living cell counts were deter-
mined using the Trypan blue exclusion test.19,49 Briefly, cells
were harvested with trypsin and pooled together with cells
floating in the culture medium. An aliquot of this cell suspension
was mixed with 0.4% trypan blue in phosphate buffered saline
(PBS). Cells were scored on a phase contrast microscope using a
Burcke improved counting chamber. Cells with a damaged cell
membrane stained blue, while cells that maintained plasma
membrane integrity resisted dye entry and remained unstained
(apoptotic and healthy cells, respectively).
ERK 1/2 Phosphorylation Assays. A3 AR-mediated activation
of ERK 1/2 phosphorylation in U87MG cells was evaluated by
kinetic studies incubating cells with 100 nM Cl-IBMECA for
different times (5-120 min). The ERK 1/2 activation was
assessed by Fast Activated Cell-Based ELISA kits following
the manufacturer’s instruction. Following stimulation the cells
were rapidly fixed to preserve activation of specific protein
modification. Each well was then incubated with primary anti-
body that recognized phosphorylated ERK 1/2. Subsequent
incubation with secondary HRP-conjugated antibody and devel-
oping solution allowed a colorimetric quantification of phos-
phorylated ERK levels. The relative number of cells in each well
was then determined using crystal violet solution. The results
corresponding loop, and the energy cutoff was set to 10 kcal.
AD4 Docking Calculations. The new version of the docking
program AutoDock (version 4, AD4),53 as implemented
through the graphical user interface called AutoDockTools
(ADT), was used to dock 2b, 2i, 2o, and 2u. Ligand structures
were built using the builder in the Maestro package of Schroe-
dinger Suite 2007 and optimized using a version of MacroModel
also included. The constructed compounds and the receptor
structure were converted to AD4 format files using ADT
generating automatically all other atom values. The docking
area was centered around the putative binding site. A set of grids
˚
˚
˚
˚
of 60 A ꢀ 60 A ꢀ 60 A with 0.375 A spacing was calculated
around the docking area for the ligand atom types using
AutoGrid4. For each ligand, 100 separate docking calculations
were performed. Each docking calculation consisted of 10
million energy evaluations using the Lamarckian genetic algo-
rithm local search (GALS) method. The GALS method evalu-
ates a population of possible docking solutions and propagates
the most successful individuals from each generation into the
subsequent generation of possible solutions. A low-frequency
local search according to the method of Solis and Wets is applied
to docking trials to ensure that the final solution represents a
local minimum. All dockings described in this paper were
performed with a population size of 250, and 300 rounds of
Solis and Wets local search were applied with a probability of
0.06. A mutation rate of 0.02 and a crossover rate of 0.8 were
used to generate new docking trials for subsequent generations,
and the best individual from each generation was propagated
over the next generation. The docking results from each of the
100 calculations were clustered on the basis of root-mean square
˚
deviation (rmsd) (solutions differing by less than 2.0 A) between
the Cartesian coordinates of the atoms and were ranked on the
basis of free energy of binding (ΔGAD4). The top-ranked com-
pounds were visually inspected for good chemical geometry.
Because AD4 does not perform any structural optimization and

3962 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Taliani et al.
energy minimization of the complexes found, a molecular
mechanics/energy minimization (MM/EM) approach was app-
lied to refine the AD4 output. The computational protocol
applied consisted of the application of 100000 steps of the
(17) Gessi, G.; Merighi, S.; Varani, K.; Cattabriga, E.; Benini, A.;
Mirandola, P.; Leung, E.; Mac Lennan, S.; Feo, C.; Baraldi, S.;
Borea, P. A. Adenosine Receptors in Colon Carcinoma Tissues and
Colon Tumoral Cell Lines: Focus on the A3 Adenosine Subtype.
J. Cell. Physiol. 2007, 211, 826–836.
(18) Merighi, S.; Mirandola, P.; Varani, K.; Gessi, S.; Capitani, S.;
Leung, E.; Baraldi, P. G.; Tabrizi, M. A.; Borea, P. A. Pyrazolo-
triazolopyrimidine derivatives sensitive melanoma cells to the
chemiotherapic drugs: taxol and vindesine. Biochem. Pharmacol.
2003, 66, 739–748.
ꢁ
Polak-Ribiere conjugate gradients (PRCG) or until the deri-
vative convergence was 0.05 kJ/mol. All complexes pictures
were rendered employing the UCSF Chimera software.54
Acknowledgment. This work was financially supported by
MIUR (ex 40%). We thank Prof. Dr. Karl-Norbert Klotz for
his generous gift of transfected CHO cells expressing human
A1, A2A, A2B, and A3 adenosine receptors.
(19) Morrone, F. B.; Jacques-Silva, M. C.; Horn, A. P.; Bernardi, F.;
Schwartsmann, G.; Rodnight, R.; Lenz, G. Extracellular nucleo-
tides and nucleosides induce proliferation and increase nucleoside
transport in human glioma cell lines. J. Neuro-Oncol. 2003, 64, 211–
218.
(20) Merighi, S.; Benini, A.; Mirandola, P.; Gessi, S.; Varani, K.; Leung,
E.; Maclennan, S.; Borea, P. A. Adenosine modulates vascular
endothelial growth factor expression via hypoxia-inducible factor-
1 in human glioblastoma cells. Biochem. Pharmacol. 2006, 72,
19–31.
(21) Da Settimo, F.; Primofiore, G.; Taliani, S.; Marini, A. M.;
La Motta, C.; Novellino, E.; Greco, G.; Lavecchia, A; Trincavelli,
M. L.; Martini, C. 3-Aryl[1,2,4]triazino[4,3-a]benzimidazol-
4(10H)-ones: a new class of selective A1 adenosine receptor
antagonists. J. Med. Chem. 2001, 44, 316–327.
(22) Novellino, E.; Abignente, E.; Cosimelli, B.; Greco, G.; Iadanza, M.;
Laneri, S.; Lavecchia, A.; Rimoli, M. G.; Da Settimo, F.; Primofiore,
G.; Tuscano, D.; Trincavelli, L.; Martini, C. Design, synthesis and
biological evaluation of novel N-alkyl- and N-acyl-(7-substituted-2-
phenylimidazo[1,2-a][1,3,5]triazin-4-yl]amines (ITAs) as novel
A1 adenosine receptor antagonists. J. Med. Chem. 2002, 45,
5030–5036.
(23) Da Settimo, F.; Primofiore, G.; Taliani, S.; La Motta, C.; Novellino,
E.; Greco, G.; Lavecchia, A.; Cosimelli, B.; Iadanza, M.; Klotz,
K.-N.; Tuscano, D.; Trincavelli, M. L.; Martini, C. A1 Adenosine
receptor antagonists, 3-aryl[1,2,4]triazinobenzimidazol-4-(10H)-
ones (ATBIs) and N-alkyl and N-acyl-(7-substituted-2-phenyl-
imidazo[1,2-a][1,3,5]triazin-4-yl)amines (ITAs): different recogni-
tion of bovine and human binding sites. Drug Dev. Res. 2004, 63,
1–7.
Supporting Information Available: Physical properties and
spectral data of compounds 6-8 and 2a-u, and analytical data
of compounds 6-8 and 2a-u. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) Poulsen, S.-A.; Quinn, R. J. Adenosine receptors: new opportu-
nities for future drugs. Bioorg. Med. Chem. 1998, 6, 619–641.
(2) Fredholm, B. B.; Arslan, G.; Halldner, L.; Kull, B.; Schulte, G.;
Wasserman, W. Structure and function of adenosine receptors and
their genes. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2000, 362,
364–374.
(3) Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz, K.-N.;
Linden, J. International Union of Pharmacology. XXV. Nomen-
clature and classification of adenosine receptors. Pharmacol. Rev.
2001, 53, 527–552.
(4) Schulte, G.; Fredholm, B. B. Signalling from adenosine receptors
to mitogen-activated protein kinases. Cell. Signalling 2003, 15,
813–827.
(5) Jacobson, K. A.; Gao, Z.-G. Adenosine receptors as therapeutic
targets. Nat. Rev. Drug Discovery 2006, 5, 247–264.
(6) Moro, S.; Gao, Z.-G.; Jacobson, K. A.; Spalluto, G. Progress in
the pursuit of therapeutic adenosine receptor antagonists. Med.
Res. Rev. 2006, 26, 131–159.
(24) Novellino, E.; Cosimelli, B.; Ehlardo, M.; Greco, G.; Iadanza, M.;
Lavecchia, A.; Rimoli, M. G.; Sala, A.; Da Settimo, A.; Primofiore,
G.; Da Settimo, F.; Taliani, S.; La Motta, C.; Klotz, K.-N.;
Tuscano, D.; Trincavelli, M. L.; Martini, C. 2-(Benzimidazol-2-
yl)quinoxalines: A Novel Class of Selective Antagonists at Human
A1 and A3 Adenosine Receptors Designed by 3D Database Search-
ing. J. Med. Chem. 2005, 48, 8253–8260.
(7) Linden, J.; Taylor, H. E.; Robeva, A. S.; Tucker, A. L.; Stehle,
J. H.; Rivkees, S. A.; Fink, J. S.; Reppert, S. M. Molecular Cloning
and Functional Expression of a Sheep A3 Adenosine Receptor with
Widespread Tissue Distribution. Mol. Pharmacol. 1993, 44, 524–
532.
(25) Da Settimo, F.; Primofiore, G.; Taliani, S.; Marini, A. M.;
La Motta, C.; Simorini, F.; Salerno, S.; Sergianni, V.; Tuccinardi,
T.; Martinelli, A.; Cosimelli, B.; Greco, G.; Novellino, E.; Ciampi,
O.; Trincavelli, M. L.; Martini, C. 5-Amino-2-phenyl[1,2,3]triazolo-
[1,2-a][1,2,4]benzotriazin-1-one: A Versatile Scaffold To Obtain
Potent and Selective A3 Adenosine Receptor Antagonists. J. Med.
Chem. 2007, 50, 5676–5684.
(8) Linden, J. Cloned adenosine A3 receptors: pharmacological prop-
erties, species differences and receptor functions. Trends Pharma-
col. Sci. 1994, 15, 298–306.
(9) Liang, B. T.; Jacobson, K. A. A physiological role of the adenosine
A3 receptor: sustained cardioprotection. Proc. Natl. Acad. Sci. U.S.
A. 1998, 95, 6995–6999.
(10) Headrick, J. P.; Peart, J. A3 adenosine receptor-mediated protec-
(26) Cosimelli, B.; Greco, G.; Ehlardo, M.; Novellino, E.; Da Settimo,
F.; Taliani, S.; La Motta, C.; Bellandi, M.; Tuccinardi, T.; Marti-
nelli, A.; Ciampi, O.; Trincavelli, M. L.; Martini, C. Derivatives of
4-Amino-6-hydroxy-2-mercaptopyrimidine as Novel, Potent, and
Selective A3 Adenosine Receptor Antagonists. J. Med. Chem. 2008,
51, 1764–1770.
(27) Da Settimo, F.; Primofiore, G.; La Motta, C.; Taliani, S.; Simorini,
F.; Marini, A. M.; Mugnaini, L.; Lavecchia, A.; Novellino, E.;
Tuscano, D.; Martini, C. Novel, Highly Potent Adenosine Deami-
nase Inhibitors Containing the Pyrazolo[3,4-d ]pyrimidine Ring
System. Synthesis, Structure-Activity Relationships, and Mole-
cola Modeling Studies. J. Med. Chem. 2005, 48, 5162–5174.
(28) Antonioli, L.; Fornai, M.; Colucci, R.; Ghisu, N.; Da Settimo, F.;
Natale, G.; Kastsiuchenka, O.; Duranti, E.; Virdis, A.; Vassalle, C.;
La Motta, C.; Mugnaini, L.; Breschi, M. C.; Blandizzi, C.; Del
Tacca, M. Inhibition of Adenosine Deaminase Attenuates Inflam-
mation in Experimental Colitis. J. Pharmacol. Exp. Ther. 2007,
322, 435–442.
(29) Chebib, M.; Quinn, R. J. Pyrazolo[3,4-d ]pyrimidines; Adenosine
Receptor Selectivity. Bioorg. Med. Chem. Lett. 1995, 5, 2409–2412.
(30) Chebib, M.; Quinn, R. J. 1-Phenylpyrazolo[3,4-d ]pyrimidines as
Adenosine Antagonists: The Effects of Substituents at C4 and C6.
Bioorg. Med. Chem. 1997, 5, 311–322.
(31) Briel, D.; Aurich, R.; Egerland, U.; Unverferth, K. Synthesis of
substituted 6-phenylpyrazolo[3,4-d ]pyrimidines with potential
adenosin-A2A antagonistic activity. Pharmazie 2005, 60, 732–735.
(32) Kim, Y.-C.; Ji, X.-D.; Jacobson, K. A. Derivatives of the Triazo-
loquinazoline Adenosine Antagonist (CGS 15943) Are Selective
tion of the ischemic heart. Vasc. Pharmacol. 2005, 42, 271–279.
(11) Akkari, R.; Burbiel, J. C.; Hockemeyer, J.; Muller, C. E. Recent
€
Progress in the Development of Adenosine Receptor Ligands as
Antiinflammatory Drugs. Curr. Top. Med. Chem. 2006, 6, 1375–
1399.
(12) Mitchell, C. H.; Peterson-Yantorno, K.; Carre, D. A.; McGlinn,
A. M.; Coca-Prados, M.; Stone, R. A.; Civan, M. M. A3 adenosine
receptors regulate Cl- channels of nonpigmented ciliary epithelial
cells. Am. J. Physiol. 1999, 276, 659–666.
(13) Brambilla, R.; Cattabeni, F.; Ceruti, S.; Barbieri, D.; Franceschi,
C.; Kim, Y.-C.; Jacobson, K. A.; Klotz, K.-N.; Lohse, M. J.;
Abbracchio, M. P. Activation of the A3 adenosine receptor affects
cell cycle progression and cell growth. Naunyn-Schmiedeberg’s
Arch. Pharmacol. 2000, 361, 225–234.
(14) Baraldi, P. G.; Tabrizi, M. A.; Romagnoli, R.; Fruttarolo, F.;
Merighi, S.; Varani, K.; Gessi, S.; Borea, P. A. Pyrazolo[4,3-
e]1,2,4-triazolo[1,5-c]pyrimidine Ligands, New Tools to Charac-
terize A3 Adenosine Receptors in Human Tumor Cell Lines. Curr.
Med. Chem. 2005, 12, 1319–1329.
ꢀ
(15) Gessi, S.; Cattabriga, E.; Avitabile, A.; Gafa, R.; Lanza, G.;
Cavazzini, L.; Bianchi, N.; Gambari, R.; Feo, C.; Liboni, A.;
Gullini, S.; Leung, E.; Mac-Lennan, S.; Borea, P. A. Elevated
Expression of A3 Adenosine Receptors in Human Colorectal
Cancer is Reflected in Peripheral Blood Cells. Clin. Cancer Res.
2004, 10, 5895–5901.
(16) Fishman, P.; Bar-Yehuda, S. Pharmacology and Therapeutic
Applications of A3 Receptor Subtype. Curr. Top. Med. Chem.
2003, 3, 463–469.

Article
for the Human A3 Receptor Subtypes. J. Med. Chem. 1996, 39,
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 3963
Antagonists: New Insights into the Receptor-Antagonist Recogni-
tion. J. Med. Chem. 2009, 52, 7640–7652.
4142–4148.
(33) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Merighi, S.; Varani,
K.; Borea, P. A.; Spalluto, G. A3 Adenosine Receptor Ligands:
History and Perspectives. Med. Res. Rev. 2000, 20, 103–128.
(34) Baraldi, P. G.; Cacciari, B.; Borea, P. A.; Varani, K.; Pastorin, G.;
Da Ros, T.; Tabrizi, M. A.; Fruttarolo, F.; Spalluto, G. Pyrazolo-
Triazolo-Pyrimidine Derivatives as Adenosine Receptor Antago-
nists: A Possible Template for Adenosine Receptor Subtypes?
Curr. Pharm. Des. 2002, 8, 2299–2332.
(35) Van Muijlwijk-Koezen, J. E.; Timmerman, H.; Link, R.; van der
Goot, H.; IJzerman, A. P. A Novel Class of Adenosine A3 Recep-
tor Ligands. 2. Structure-Affinity Profile of a Series of Isoquino-
line and Quinazoline Compounds. J. Med. Chem. 1998, 41, 3994–
4000.
(36) Lenzi, O.; Colotta, V.; Catarzi, D.; Varano, F.; Filacchioni, G.;
Martini, C.; Trincavelli, L.; Ciampi, O.; Varani, K.; Marighetti, F.;
Morizzo, E.; Moro, S. 4-Amido-2-aryl-1,2,4-triazolo[4,3-a]quino-
xalin-1-ones as new potent and selective human A3 adenosine
receptor antagonists. Synthesis, pharmacological evaluation, and
ligand-receptor modeling studies. J. Med. Chem. 2006, 49, 3916–
3925.
(37) Nordstedt, C.; Fredholm, B. B. A modification of a protein-
binding method for rapid quantification of cAMP in cell-culture
supernatants and body fluid. Anal. Biochem. 1990, 189, 231–234.
(38) Beukers, M. W.; Chang, L. C. W.; von Frijtag Drabbe Kunzel, J. K;
Mulder-Krieger, T.; Spanjersberg, R. F.; Brussee, J.; IJzerman,
A. P. New, non-adenosine, high-potency agonists for the human
adenosine A2B receptor with an improved selectivity profile com-
pared to the reference agonist N-ethylcarboxamidoadenosine.
J. Med. Chem. 2004, 47, 3707–3709.
(42) Huey, R.; Morris, G. M.; Olson, A. J.; Goodsell, D. S. A Semi-
empirical Free Energy Force Field with Charge-Based Desolva-
tion. J. Comput. Chem. 2007, 28, 1145–1152.
(43) Kim, J.; Wess, J.; van Rhee, M.; Schoneberg, T.; Jacobson, K. A.
Site-directed mutagenesis identifies residues involved in ligand
recognition in the human A2A adenosine receptor. J. Biol. Chem.
1995, 270, 13987–13997.
(44) Gao, Z.-G.; Chen, A.; Barak, D.; Kim, S.-K.; Muller, C. E.;
Jacobson, K. A. Identification by site-directed mutagenesis of
residues involved in ligand recognition and activation of the human
A3 adenosine receptor. J. Biol. Chem. 2002, 277, 19056–19063.
(45) The CLogP, ASA, and TPSA calculations were performed with the
Marvin Sketch and Calculator Plugins on the website http://www.
chemaxon.com/demosite/marvin/index.html.
(46) Baraldi, P. G.; Borea, P. A. Preparation of pyrazolo[4,3-e]1,2,4-
triazolo[1,5-c]pyrimidines and analogs as adenosine A3 receptor
modulators for therapeutic and diagnostic use. U.S. Patent Appl.
US6407236 B1 20020618, 2002.
(47) Klotz, K.-N.; Hessling, J.; Hegler, J.; Owman, C.; Kull, B.;
Fredholm, B. B.; Lohse, M. J. Comparative pharmacology of
human adenosine receptor subtypes characterization of stably
transfected receptors in CHO cells. Naunyn-Schmiedebergs Arch.
Pharmacol. 1998, 357, 1–9.
(48) Cheng, Y. C.; Prusoff, W. H. Relation between the inhibition
constant Ki and the concentration of inhibitor which causes fifty
percent inhibition (IC50) of an enzymatic reaction. Biochem. Phar-
macol. 1973, 22, 3099–3108.
ꢀ
(49) Bjorklund, O.; Shang, M.; Tonazzini, I.; Dare, E.; Fredholm, B. B.
Adenosine A1 and A3 receptors protect astrocytes from hypoxic
damage. Eur. J. Pharmacol. 2008, 596, 6–13.
(39) Chang, L. C. W.; von Frijtag Drabbe Kunzel, J. K.; Mulder-
Krieger, T.; Spanjersberg, R. F.; Roerink, S. F.; van den Hout,
G.; Beukers, M. W.; Brussee, J.; IJzerman, A. P. A series of ligands
displaying a remarkable agonistic-antagonistic profile at the
adenosine A1 receptor. J. Med. Chem. 2005, 48, 2045–2053.
(40) Jaakola, V. P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.;
Chien, E. Y. T.; Lane, J. R.; IJzerman, A. P.; Stevens, R. C. The 2.6
angstrom crystal structure of a human A2A adenosine receptor
bound to an antagonist. Science 2008, 322, 1211–1217.
€
(50) Prime, version 2.0; Schrodinger, LLC, New York, 2005.
€
(51) Maestro, version 8.5; Schrodinger, LLC, New York, 2006.
(52) Jacobson, M. P.; Pincus, D. L.; Rapp, C. S.; Day, T. J.; Honig, B.;
Shaw, D. E.; Friesner, R. A. A hierarchical approach to all-atom
protein loop prediction. Proteins 2004, 55, 351–367.
(53) Huey, R.; Morris, G. M.; Olson, A. J.; Goodsell, D. S. A Semi-
empirical Free Energy Force Field with Charge-Based Desolva-
tion. J. Comput. Chem. 2007, 28, 1145–1152.
(41) Lenzi, O.; Colotta, V.; Catarzi, D.; Varano, F.; Poli, D.; Filacchioni,
G.; Varani, K.; Vincenzi, F.; Borea, P. A.; Paoletta, S.; Morizzo, E.;
Moro, S. 2-Phenylpyrazolo[4,3-d ]pyrimidin-7-one as a New Scaf-
fold To Obtain Potent and Selective Human A3 Adenosine Receptor
(54) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera;A
Visualization System for Exploratory Research and Analysis.
J. Comput. Chem. 2004, 25, 1605–1612.