2-Arylpyrazolo[4,3-d]pyrimidin-7-amino derivatives as new potent and selective human A3 adenosine receptor antagonists. Molecular modeling studies and pharmacological evaluation

Article abstract of DOI:10.1021/jm400068e

On the basis of our previously reported 2-arylpyrazolo[4,3-d]pyrimidin-7- ones, a set of 2-arylpyrazolo[4,3-d]pyrimidin-7-amines were designed as new human (h) A₃ adenosine receptor (AR) antagonists. Lipophilic groups with different steric bulk were introduced at the 5-position of the bicyclic scaffold (R₅ = Me, Ph, CH₂Ph), and different acyl and carbamoyl moieties (R₇) were appended on the 7-amino group, as well as a para-methoxy group inserted on the 2-phenyl ring. The presence of acyl groups turned out to be of paramount importance for an efficient and selective binding at the hA₃ AR. In fact, most of the 7-acylamino derivatives showed low nanomolar affinity (K_i = 2.5-45 nM) and high selectivity toward this receptor. A few selected pyrazolo[4,3-d]pyrimidin-7-amides were effective in counteracting oxaliplatin-induced apoptosis in rat astrocyte cell cultures, an in vitro model of neurotoxicity. Through an in silico receptor-driven approach the obtained binding data were rationalized and the molecular bases of the observed hA₃ AR affinity and hA₃ versus hA_2A AR selectivity were explained.

Full text of DOI:10.1021/jm400068e

Article
pubs.acs.org/jmc
2‑Arylpyrazolo[4,3‑d]pyrimidin-7-amino Derivatives As New Potent
and Selective Human A3 Adenosine Receptor Antagonists. Molecular
Modeling Studies and Pharmacological Evaluation
Lucia Squarcialupi,† Vittoria Colotta,*,† Daniela Catarzi,† Flavia Varano,† Guido Filacchioni,†
Katia Varani,‡ Carmen Corciulo,‡ Fabrizio Vincenzi,‡ Pier Andrea Borea,‡ Carla Ghelardini,§
Lorenzo Di Cesare Mannelli,§ Antonella Ciancetta,# and Stefano Moro*,#
†Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e Nutraceutica,
Universita
̀
di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy
‡Dipartimento di Scienze Mediche, Istituto di Farmacologia, Universita
̀
di Ferrara,Via Fossato di Mortara 17-19, 44121 Ferrara, Italy
§Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmacologia e Tossicologia, Viale
Pieraccini, 6, 50139 Firenze, Italy
#Molecular Modeling Section (MMS), Dipartimento di Scienze del Farmaco, Universita
̀
di Padova, via Marzolo 5, 35131 Padova, Italy
S
* Supporting Information
ABSTRACT: On the basis of our previously reported 2-
arylpyrazolo[4,3-d]pyrimidin-7-ones, a set of 2-arylpyrazolo[4,3-
d]pyrimidin-7-amines were designed as new human (h) A3
adenosine receptor (AR) antagonists. Lipophilic groups with
different steric bulk were introduced at the 5-position of the
bicyclic scaffold (R5 = Me, Ph, CH2Ph), and different acyl and
carbamoyl moieties (R7) were appended on the 7-amino group, as
well as a para-methoxy group inserted on the 2-phenyl ring. The
presence of acyl groups turned out to be of paramount importance
for an efficient and selective binding at the hA3 AR. In fact, most of the 7-acylamino derivatives showed low nanomolar affinity
(Ki = 2.5−45 nM) and high selectivity toward this receptor. A few selected pyrazolo[4,3-d]pyrimidin-7-amides were effective in
counteracting oxaliplatin-induced apoptosis in rat astrocyte cell cultures, an in vitro model of neurotoxicity. Through an in silico
receptor-driven approach the obtained binding data were rationalized and the molecular bases of the observed hA3 AR affinity
and hA3 versus hA2A AR selectivity were explained.
overexpressed in different tumor cell types.7 The A3 AR
INTRODUCTION
■
regulation of the cell cycle may induce both cell protection or
cell death, depending on the degree of receptor activation and/
or the cell type or toxic insult.6−9 Thus, both A3 receptor
agonists and antagonists might be effective agents in cancer
therapy.7−9 In the central nervous system (CNS), the A3 AR is
expressed both in neurons and glial cells, such as microglia and
astrocytes, which are recognized both as structural support for
neurons and as active participants in various pathological
conditions, as neurodegenerative diseases, trauma, and neuro-
pathic pain.10−12 Also in the CNS, activation of the A3 AR may
afford both pro- and antisurvival effects, thus inducing either
protection or damage, depending on the situation.6,8,13 Hence,
even though it is has been clearly demonstrated that the A3 AR
is involved in many disease pathways, much research is needed
to understand in depth the roles played by this receptor.
Therefore, the search for new selective A3 AR ligands, either
agonists or antagonists, still remains an attractive objective.14
Adenosine is a ubiquitous purine nucleoside that acts as a
modulator of many cell functions, both in normal and
pathological conditions. The wide variety of effects exerted by
adenosine is due to activation of four G-protein-coupled
receptors (GPCRs), classified as A1, A2A, A2B, and A3
subtypes.1−3 Adenosine receptors (ARs) are coupled to
adenylate cyclase that can be both inhibited (A1 and A3) or
activated (A2A and A2B).1 Moreover, ARs are also associated to
other second messenger signaling pathways. In particular, the
A3 AR activates phospolipase C,4 causing an increase of
intracellular calcium levels, and modulates protein kinase C
activity4 as well as KATP channel.5 The A3 AR also controls the
activity of mitogen-activated protein kinases (MAPK), such as
the extracellular signal-regulated kinase (ERK) 1/2 and the
stress-activated protein kinase p38.6 These latter effects account
for the role of adenosine in the regulation of cell proliferation
and differentiation, although the understanding of the A3-
signaling in these processes is far from being completely
clarified. In humans, the A3 AR was found in many peripheral
tissues such as lung, liver, and immune cells, and it is
Received: September 24, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
In our laboratory we have devoted much research to the
study of AR antagonists belonging to different heterocyclic
classes. Within the investigated series, many potent and
selective antagonists for the A3 receptor subtype were
identified.15−26 Most of them are tricyclic derivatives, thus
highly lipophilic and with low water solubility, and this often
makes it difficult to test them in pharmacological assays. To
overcome this drawback, our recent studies have been directed
to the identification of new bicyclic scaffolds21,25,26 to obtain
more soluble AR antagonists. This strategy afforded diverse
classes of compounds, such as the recently investigated
pyrazolo[4,3-d]pyrimidin-7-one derivatives25 (PP-7-oxo series),
designed as analogues of previously reported pyrazolo[3,4-
c]quinolin-4-ones (PQ-1 series, Chart 1). Most of the PP-7-oxo
the 2-phenyl ring, and acyl residues (R7= COMe, COAr,
COCH2Ph, COHeteroaryl) or carbamoyl moieties (R7
=
CONHPh, CONHCH2Ph) were placed on the 7-amino
group. The choice of these R7 and R2 substituents was made
since they increased hA3AR affinity and selectivity both in our
previously described derivatives15−24 and in many other classes
of AR antagonists of similar size and shape.14 Molecular
modeling studies were carried out to explain both hA3 affinity
and selectivity profiles of the new antagonists. Since A3AR
modulation was described as relevant in chemotherapy-induced
neuropathy,12 some selected pyrazolo[4,3-d]pyrimidines (com-
pounds 8, 17, 26, 27), showing high hA3 AR affinity and
selectivity, were evaluated in a rat cellular model of chemo-
therapy-induced neurotoxicity.
CHEMISTRY
Chart 1. Structural Modification Approach from the
Pyrazolo[3,4-c]quinoline Series to the Pyrazolo[4,3-
d]pyrimidine Derivatives
■
The synthesis of the pyrazolo[4,3-d]pyrimidin-7-amines 1−6
(Scheme 1) started from the ethyl 1-aryl-4-nitropyrazole-3-
a
Scheme 1
compounds were highly potent and selective hA3 antagonists,
thus indicating that the pyrazolo[4,3-d]pyrimidine ring system
can be considered as a good scaffold for the development of
hA3 AR antagonists. Hence, we decided to replace the 7-oxo
function of the PP series with an amino group (PP-7-amino
series, Chart 1), as it was performed on the previously reported
PQ-1 series to give the corresponding 4-amines (PQ-2 series,
Chart 1) which turned out potent AR antagonists.16,20,23,27 The
herein reported 2-arylpyrazolo[4,3-d]pyrimidin-7-amino deriv-
atives 1−34 (Chart 2) bear substituents with different
lipophilicity and steric bulk at the 5-position (R5 = Me, Ph,
CH2Ph). Moreover, a 4-methoxy group (R2) was inserted on
a
Reagents and conditions: (a) 33% aqueous NH3; (b) POCl3, mw,
120 or 160 °C; (c) cyclohexene, Pd/C, mw, 150 °C; (d) CH3−
C(OEt)3 or Ph−C(OEt)3 or PhCH2−C(NH)OEt hydrochloride,
NH4OAc, mw, 110 °C.
carboxylates 35 and 36,25 which were treated with 33% aqueous
solution of ammonia to yield the 4-nitro-1-aryl-3-carboxamides
37 and 38.25 These compounds were reacted with phosphorus
oxychloride under microwave irradiation to give the 4-nitro-1-
aryl-3-carbonitriles 39 and 40. Permitting compounds 39 and
40 to react with cyclohexene and Pd/C, under microwave
irradiation, the 4-amino derivatives 41 and 42 were obtained.
These compounds were transformed into the 5-substituted
pyrazolopyrimidin-7-amines 1−6 by a one-pot, three-compo-
nent, solvent-free reaction with the commercially available
triethyl orthoacetate (compounds 1 and 4) or triethyl
orthobenzoate (compounds 2 and 5), and ammonium acetate
under microwave irradiation. Since triethyl orthophenylacetate
was not commercial, the ethyl phenyliminoacetate28 was
employed to introduce the benzyl group at the 5-position
(compounds 3 and 6). The 7-acylamines 7−27 were prepared
Chart 2. Herein Reported 2-Arylpyrazolo[4,3-d]pyrimidin-7-
amino Derivatives 1−34
B
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
by using two different reaction conditions (Scheme 2).
Derivatives 7, 9, 11, 14, 16−18, 21, and 24−27 were obtained
Scheme 4
a
Scheme 2
a
Reagents and conditions: (a) R-NCO, anhydrous tetrahydrofuran,
reflux.
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 some
selected pyrazolopyrimidin-7-amino derivatives (8, 14, 15, 17,
22, 26−28) was assessed by evaluating their effect on Cl-IB-
MECA-inhibited cAMP production in CHO cells, stably
expressing hA3 ARs. All pharmacological data are collected in
Table 1. The selected pyrazolopyrimidine derivatives 8, 17,
26−27 were tested to evaluate their effect in counteracting
oxaliplatin-induced apoptosis in rat astrocyte cell cultures, an in
vitro model of neurotoxicity. As a consequence of this study,
these compounds were evaluated for their affinity at the rat A3
AR, stably expressed in HEK cells. The rat A3 binding data are
included in Table 1.
a
Reagent and conditions: (a) RCOCl, anhydrous pyridine, anhydrous
methylene chloride, room temperature or reflux; (b) suitable
carboxylic acid, 1-hydroxybenzotriazole, NEt3, 4-(dimethylamino)-
pyridine, 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydro-
chloride, DMF, room temperature.
by reacting the pyrazolopyrimidin-7-amines 1, 2, 4, and 5 with
the suitable acyl chloride in anhydrous methylene chloride and
pyridine. Instead, compounds 8, 10, 12, 13, 15, 19, 20, 22, and
23 were synthesized by reacting the 7-amines 1, 2, and 4 with
the suitable carboxylic acid in dimethylformamide and in the
presence of 1-(3-(dimethylamino)propyl))-3-ethyl-carbodii-
mide hydrochloride, 1-hydroxybenzotriazole, triethylamine,
and 4-(dimethylamino)pyridine. When compounds 1 and 2
were treated with an excess of benzoyl- and 2-furoyl-chloride, in
anhydrous methylene chloride and pyridine, the corresponding
7-diacylamino-substituted derivatives 28−30 were obtained
(Scheme 3). Finally, the synthesis of the 7-ureido derivatives
31−34 was achieved by refluxing compounds 1, 4, and the
suitable isocyanate in anhydrous tetrahydrofuran (Scheme 4).
MOLECULAR MODELING
■
To rationalize the structure-affinity relationships (SARs) and
the selectivity profiles of the new pyrazolopyrimidin-7-amino
derivatives, a receptor-driven molecular modeling investigation
was carried out. With the aim to identify the hypothetical
binding modes of the new compounds, docking simulations
inside the binding cavity of the hA3 AR receptor subtype were
performed. As, to date, no crystallographic information about
the A3 AR is available, we used a previously reported hA3 AR
homology model,25 which was built by using the crystal
structure of the hA2A AR29 as a template. Moreover, docking
simulations at the hA2A AR binding site were also carried out to
explain the observed hA2A/hA3 AR selectivity profiles.
PHARMACOLOGICAL ASSAYS
■
The synthesized derivatives 1−34 were tested to evaluate their
affinity at hA1, hA2A, and hA3 ARs. Compounds were also tested
a
Scheme 3
Furthermore, to analyze the ligand−receptor recognition
mechanism in a more quantitative fashion, the individual
electrostatic and hydrophobic contributions to the interaction
energy of each receptor residue involved in the binding with the
ligands were also calculated for selected binding poses. The
analysis of these contributions afforded “interaction energy
fingerprints” (IEFs), i.e., interaction energy patterns, graphically
displayed as histograms, reporting the key residues involved in
the binding with the considered ligands along with a
quantitative estimate of the occurring interactions. This analysis
has provided clues on the underlying recognition mechanism
that might occur between the new derivatives and the
considered receptor subtypes. Moreover, the semiquantitative
estimate of the interactions has allowed a direct comparison of
different ligands with respect to the quality of ligand−receptor
contacts.
a
Reagents and conditions: (a) RCOCl, anhydrous pyridine, methylene
chloride, room temperature or reflux.
C
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. Binding Affinity (Ki) at hA1, hA2A, and hA3 ARs and Potencies (IC50) at hA2B and hA3 ARs
a
binding experiments Ki (nM) or I%
cAMP assays IC50 (nM) or I%
b
c
d
e
f
g
R5
R2
R7
hA1
1%
hA2A
1%
hA3
rA3
hA2B
2%
hA3
A
Me
Ph
H
H
H
H
H
H
16
2
B
22%
11%
70
10%
1%
10%
4%
4%
C
CH2Ph
Me
Ph
900 95
40%
1
H
6
7
246 23
325 34
110 10
1%
320 35
440 43
420 38
2%
2
H
75
48%
3
Ph−CH2
Me
Ph
H
150 12
30%
12%
40%
25%
30%
4%
39%
4
4-OMe
4-OMe
4-OMe
H
H
38%
5
H
35%
37%
4%
75
8
3%
6
Ph−CH2
Me
Me
Me
Me
Me
Me
Me
Ph
H
45%
33%
7
COMe
COPh
300 26
5.6 0.5
2.4 0.2
120 11
5%
8
H
1%
6%
2%
19.7 1.8
9
H
CO-C4H4-4-OMe
COCH2Ph
CO-2-furyl
CO-3-pyridyl
CO-4-pyridyl
COMe
1%
1%
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
H
510 47
20%
12%
12%
5%
13%
1%
1%
H
20
2
1%
H
1%
5.3 0.5
5.2 0.5
1%
H
1%
H
5%
32
20
3
2
2%
2%
1%
2%
1%
1%
1%
30%
1%
11%
1%
1%
2%
2%
5%
1%
1%
1%
10%
13%
1%
103 10
Ph
H
COPh
5%
5%
65
6
Ph
H
CO-C4H4-4-OMe
COCH2Ph
CO-2-furyl
CO-3-pyridyl
CO-4-pyridyl
COMe
3%
1%
7.3 0.7
18
Ph
H
5%
5%
2
2%
61
5
Ph
H
11%
1%
1%
24 2.3
3.2 0.3
Ph
H
1%
Ph
H
1%
1%
25
100 11
35
520 52
45
98 10
2
Me
Me
Me
Me
Ph
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
30%
1%
30%
7%
COPh
4
128 11
COCH2Ph
CO-2-furyl
COMe
9%
11%
6%
6%
4
1%
11%
1%
Ph
COPh
3%
18
2
3
4
3
6
12%
3%
63
80
5
9
Ph
COCH2Ph
COPh
29%
6%
18%
1%
24
Me
Me
Ph
33
115 12
CO-2-furyl
CO-2-furyl
CONHPh
CONHCH2Ph
CONHPh
CONHCH2Ph
4%
1%
33
3%
1%
75
Me
Me
Me
Me
H
1%
1%
30%
1%
1%
6%
H
26%
5%
7%
4-OMe
4-OMe
13%
7%
8%
a
Ki values are means SEM of four separate assays each performed in duplicate. Percentage of inhibition (I%) is determined at 1 μM concentration
b
c
of the tested compounds. Displacement of specific [3H]DPCPX competition binding assays to hA1CHO cells. Displacement of specific
d
[3H]ZM241385 competition binding to hA2ACHO cells. Displacement of specific [125I]AB-MECA competition binding to hA3CHO cells.
e
f
Percentage of inhibition (I%) in [125I]AB-MECA competition binding assays to rA3HEK cells. cAMP experiments in hA2BCHO cells, stimulated by
200 nM NECA. IC50 values are expressed as means SEM of four separate cAMP experiments. Percentage of inhibition (I%) is determined at 1 μM
g
concentration of the tested compounds. IC50 values are expressed as means SEM of four separate cAMP experiments in hA3CHO cells, in the
presence of 100 nM Cl-IB-MECA.
A more detailed description of all the methods used to
perform the receptor-driven molecular modeling investigation
is reported in the Experimental Section.
C, included as reference compounds. The obtained results
indicate that we achieved our goal because most of the 7-
acylamino-substituted derivatives 7−30, designed to target the
hA3 receptor, are endowed with nanomolar affinity and high
selectivity toward this receptor subtype, the best being the 7-(4-
methoxybenzoylamino)-5-methyl-2-phenyl-substituted deriva-
tive 9 (Ki = 2.4 nM).
RESULTS AND DISCUSSION
■
Structure-Affinity Relationship Studies. The affinity
data of the newly synthesized pyrazolo[4,3-d]pyrimidin-7-
amino derivatives 1−34 at ARs are reported in Table 1,
together with those of the pyrazolo[4,3-d]pyrimidin-7-ones A−
Analyzing the binding results, we can observe that
replacement of the 7-oxo group (derivatives A−C) with the
D
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. (A) Superimposition of the most energetically favored docking poses of compound 1, 2 and 3 inside the hA2A AR binding site. The poses
are viewed from the membrane side facing TM6, TM7, and TM1. To aid visualization, the view of TM7 is partially omitted and hydrogen atoms are
not displayed. Side chains of some amino acids important for ligand recognition and hydrogen bond interactions are highlighted. (B) Interaction
energy fingerprints (IEFs) computed for the most energetically favored docking poses of compounds 1, 2, and 3: electrostatic interaction energy
values and hydrophobic interaction scores are expressed in kcal/mol and arbitrary hydrophobic units, respectively.
7-amino (compounds 1−3) significantly modified AR affinity
and selectivity. Indeed, while the 7-oxo substituted compounds
A, C are hA3 AR selective antagonists, and B is devoid of
affinity for all the ARs, the corresponding 7-amino derivatives
1−3 show good affinity for hA1, hA2A, and hA2B ARs, and low
binding activity at the hA3 receptor (I% = 39−48 at 1 μM). To
increase affinity for this last subtype a para methoxy group was
introduced on the 2-phenyl ring of derivatives 1−3 to give
compounds 4−6. This modification was undertaken because in
several AR antagonists belonging to our series it was one of the
most advantageous for improving hA3 AR affinity and
selectivity.15−24 Contrary to our expectations, it did not always
induce a profitable effect in the herein reported compounds.
Indeed, among 2-(4-methoxyphenyl)-substituted derivatives 4−
6, only compound 5 (R5 = Ph) possesses nanomolar hA3 AR
affinity and high selectivity, while both derivatives 4 (R5 = Me)
and 6 (R5 = CH2Ph) maintain the low hA3 affinity of the parent
2-phenyl-substituted 1 and 3, and they are also scarcely active at
the other ARs. Notable results were obtained when acyl groups
were appended on the 7-amino function of the 2-phenyl
derivatives 1 (R5 = Me) and 2 (R5 = Ph), to give compounds
7−13 and 14−20, respectively. This structural modification
completely shifted affinity toward the hA3 receptor. In fact, all
the 7-amido-substituted pyrazolopyrimidines showed nano-
molar affinities for the hA3 AR and almost null activities at the
other AR subtypes, the only exception being the 5-methyl-7-
phenylacetamido derivative 10 which was endowed with a
certain hA1 AR binding affinity (Ki = 510 nM). The
acetylamino-substituted derivatives 7 and 14 demonstrated,
respectively, good (Ki = 300 nM) and high (Ki = 32 nM)
affinity for the hA3 receptor. Replacement of the small acetyl
group with the bulkier and more lipophilic benzoyl moiety
(derivatives 8 and 15) increased the capability to bind the hA3
receptor, in particular for compound 8, which is about 50-fold
more active than the corresponding 7-acetamido derivative 7.
When the benzoyl group was replaced with the phenylacetyl
moiety, the A3 AR affinity was 20-fold reduced (compound 10)
or unchanged (compound 17).
Because the 7-benzoylamino-substituted derivatives 8 and 15
showed the higher hA3 affinity and selectivity they were taken
as leads to be optimized. Thus, a para-methoxy substituent was
introduced on the benzoyl group to afford compounds 9 and
16, respectively. This modification caused a further enhance-
ment of the hA3 receptor binding and led to the best antagonist
E
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. Most energetically favored docking poses obtained for compound 9 (A) and 19 (B) inside the hA3 AR binding site. The poses are viewed
from the membrane side facing TM6, TM7, and TM1. To aid visualization, the view of TM7 is partially omitted and hydrogen atoms are not
displayed. Side chains of some amino acids important for ligand recognition and hydrogen bond interactions are highlighted. (C) Interaction energy
fingerprints (IEFs) computed for the most energetically favored docking poses of compounds 9 and 19: electrostatic interaction energy values and
hydrophobic interaction scores are expressed in kcal/mol and arbitrary hydrophobic units, respectively.
among the herein reported compounds, i.e., derivative 9 (Ki =
2.4 nM).
Cl-IB-MECA-inhibited cAMP accumulation in CHO cells
expressing the hA3 receptor. In accordance with the hA3 AR
affinity values, the IC50 results (Table 1) showed that the tested
compounds are hA3 AR antagonists endowed with significant
potencies.
Subsequently, the benzoyl moiety of derivatives 8 and 15 was
replaced with the 2-furoyl (compounds 11 and 18) and the 3-
or 4-pyridoyl residues (derivatives 12, 19 and 13, 20,
respectively). These isosteric modifications were performed
since in other classes of our previously reported AR
antagonists22,23 maintained the capability of the molecules to
properly interact with the hA3 recognition site. The new
derivatives 11−13 and 18 and 20 showed both nanomolar hA3
affinity and high selectivity, similar to those of the parent
compounds 8 and 15 and, more interestingly, the 7-(3-
pyridylamido)-substituted derivative 19 had 6-fold higher hA3
affinity than the parent 15.
Also the presence of two acyl groups on the 7-amino
substituent preserved high hA3 AR affinity (compounds 28−
30), thus indicating the existence of a roomy receptor pocket
able to hold the two bulky moieties. Introduction of the
methoxy group as the R2 substituent of the 7-acylamino
derivatives 7−8, 10, 11 and 14, 15, 17 to give compounds 21−
24 and 25−27, respectively, did not produce a profitable effect.
In fact, in only one case the hA3 affinity was 3-fold enhanced
(compound 21), while in the others it was decreased
(compounds 22, 23, 24, 25) or unchanged (compounds 26
and 27). In contrast to the advantageous role of the acyl
groups, a phenyl- or benzyl-carbamoyl moiety (compounds 31
and 32, respectively) exerted a deleterious effect for the binding
at the hA3 receptor, as well as for the other receptor subtypes.
Insertion of a para methoxy group on the 2-phenyl ring of the
7-ureido derivatives 31 and 32 did not enhance the AR affinity,
being derivatives 33 and 34 as inactive as the parent derivatives
31 and 32. The selected compounds 8, 14, 15, 17, 22, 26−28
were tested to evaluate their antagonistic potencies to modulate
Molecular Modeling Studies. With the aim of ration-
alizing the observed binding data, all the newly synthesized
analogues were subjected to a molecular modeling inves-
tigation. We performed docking simulations at both the hA3 AR
model25 and the crystallographic structure of hA2A AR,29 and
we computed the per-residue electrostatic and hydrophobic
contributions to the interaction energy for selected docking
poses. The analysis of these contributions afforded IEFs: these
interaction energy patterns highlight the key residues involved
in the binding with the considered ligands, by providing a
semiquantitative estimate of the occurring interactions and
allowing a direct comparison of different ligands with respect to
the quality of the ligand−receptor contacts.
The data reported in Table 1 reveal that the introduction of
an amino moiety (1−3) in place of an oxo group (A−C) at the
7-position of the pyrazolo[4,3-d]pyrimidine scaffold causes a
change of the affinity and selectivity profiles toward the
different AR subtypes, by conferring to the new derivatives a
good affinity for the A2A AR. The most favorable docking poses
obtained for compounds 1−3 at the A2A AR help to explain the
observed behavior. In Figure 1A, the corresponding hypo-
thetical binding modes at the A2A AR are depicted: the ligands
(1−3) reside in the upper region of the transmembrane (TM)
bundle and are anchored inside the binding cleft by a tight
hydrogen bond network with the side chains of the highly
conserved Asn253 (6.55)30,31 and Glu169 (EL2). The
pyrazolo[4,3-d]pyrimidine (PP) core establishes an aromatic
π−π stacking interaction with Phe168 (EL2) and hydrophobic
F
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
contacts with Trp246 (6.48) and Leu249 (6.51). The IEFs
analysis (Figure 1B) identifies Asn253 (6.55) and Phe168
(EL2) as the residues mainly contributing to the interaction
energy, with corresponding electrostatic energies and hydro-
phobic scores in the range 5−15 kcal/mol and 40−60 arbitrary
units, respectively.
showed by the 7-ureido-substituted derivatives to the adoption
of such a conformation, that might remarkably change the
orientation of the PP scaffold with respect to the binding
modes predicted for the other derivatives (Figure 2A,B).
Finally, some of the most relevant ADME and physicochem-
ical properties of all the new pyrazolopyrimidine derivatives
were calculated (see Supporting Information, Table S1). The
predicted data indicated that some properties, such as water
solubility and blood−brain barrier penetration, could be
ameliorated to improve the druggability profile of these
derivatives.
Pharmacological Studies. Very recently it has been
reported that A3 AR modulation is relevant in chemotherapy-
induced neuropathy.12 On this basis, we tested some of the
newly synthesized derivatives (8, 17, 26, 27) by evaluating their
effect on oxaliplatin-induced apoptosis in rat astrocyte cell
cultures. It is well-known that astroglia cells play a key role in
the central homeostasis, and therefore they would be expected
to form an integral part of a response to CNS injury. Astrocytes
activate several mechanisms that tend to decrease neuronal
injury. In fact, they produce trophic factors, regulate transmitter
and ion concentrations, thus having a direct influence on
neuronal survival, synaptic transmission, and neural repair.33
Hence, astrocytic functional impairments have the potential to
induce neuronal dysfunction.33 Moreover, astroglial cells have a
pivotal role in neuropathy development.34,35
The loss of A2A AR activity due to the introduction of an acyl
moiety on the 7-amino group, to yield the corresponding 7-
amido derivatives 7−30, can be ascribed to the loss of those key
interactions. It is conceivable that the presence of a bulky
substituent at this position forces the scaffold to adopt a
different orientation into the binding cleft, by hampering the
ligand to properly approach Asn253 (6.55) and Glu169 (EL2)
and establish the above-described hydrogen bond network. On
the contrary, the presence of sterically hindered substituents is
well tolerated at the hA3 AR. In this receptor subtype, the
Glu169 residue is mutated to Val169: as a consequence, its
binding pocket enables accommodation of bulky groups better
than the other AR subtypes. Indeed, as indicated by the data in
Table 1, the introduction of lipophilic and bulky substituents at
the R7 position (7−30) confers to the pyrazolo[4,3-d]-
pyrimidine scaffold a high degree of selectivity toward the
hA3 AR subtype.
To shed light on the molecular bases underlying this
behavior, we report here the most favorable docking poses of
two selected compounds, 9 and 19, which represent two among
the most active derivatives of the methyl- (R5= CH3) and
phenyl- (R5 = Ph) substituted pyrazolopyrimidin-7-amino
series, respectively.
The antineoplastic agent oxaliplatin is the standard treatment
for advanced colorectal cancer; its limiting side effect is
neurotoxicity that results in a neuropathic syndrome.36,37 In a
primary cell culture of rat astrocytes, oxaliplatin was able to
reduce cell viability (MTT assay, see Experimental Section) in a
concentration-dependent manner showing LC50% > 100 μM
In Figure 2A,B the hypothetical binding modes of 9 (Ki = 2.4
nM) and 19 (Ki = 3.2 nM), respectively, are depicted. The
compounds share a common hypothetical binding mode: the
ligand recognition occurs in the upper region of the TM
bundle, the PP scaffold is surrounded by TMs 3, 5, 6, and 7
with the 2-phenyl ring and the R5 groups pointing toward TM2
and TM6, respectively. The ligands are anchored inside the
binding cleft by two stabilizing hydrogen bonds with the side
chain of Asn250 (6.55) and an aromatic π−π stacking
interaction with the side chain of Phe168 (EL2). As a result
of this orientation, the R7 substituent is directed outward from
the binding cleft. The IEFs analysis for the two selected
compounds (Figure 2C) reveals that further contributions to
the whole interaction energy are afforded by hydrophobic
contacts with other residues, namely, Val72 (2.64), Val169
(EL2), Leu246 (6.51), Ile253 (6.58), Leu264 (7.35), and
Ile268 (7.39). At variance with derivative 9, bearing a small
alkyl group at the R5 position, compound 19 also establishes a
hydrophobic contact with Trp243 (6.48), an important residue
in receptor activation and antagonist recognition.31 The
placement of the ligands into the binding cleft also highlight
that there is enough space to accommodate a para-methoxy
moiety on the 2-phenyl ring.
From the docking analysis it is not easy to rationalize the
observed low affinities of derivatives 31−34 bearing an urea
moiety at the R7 position. As it is generally recognized that the
activity of disubstituted ureas strongly depends on their
conformational state,32 we performed an ab initio study to
identify the most stable conformer of the 7-ureido-pyrazolopyr-
imidine derivatives (see Supporting Information). From this
analysis, it resulted that the most energetically favored species is
the E,Zb conformer (see Figure S1B) structure in which an
intramolecular hydrogen bond leads to the formation of a six-
termed pseudo cycle. We therefore ascribed the low affinity
after 24 h incubation, and 16.0
0.1 μM after 48 h. These
values were in accordance with previous results indicating the
strong resistance of astrocytes to noxious stimuli.38,39 Aimed at
evaluating an early apoptotic effect we used 100 μM oxaliplatin
for 4 h incubation. This concentration was comparable to that
used in an animal model of oxaliplatin-induced neuropathy (2.4
mg kg−1, ∼600 μM)37 and to that clinically used.40
After 4 h incubation, 100 μM oxaliplatin caused programmed
cell death increasing caspase 3 activity up to 150%, with respect
to the control condition fixed at 100% (Figure 3). All the tested
pyrazolopyrimidines 8, 17, 26, 27 were able to significantly
prevent the oxaliplatin-dependent apoptosis when coincubated
at micromolar concentration (10−0.1 μM). Figure 3 shows the
effect of compounds (1 μM) on caspase 3 activity. Among
them, compound 27 was the most effective in reducing the
enzymatic activity. Subsequently, derivative 27 was tested on a
human colon adenocarcinoma cell-line (HT-29) to assess
whether it interferes with the oxaliplatin antineoplastic in vitro
mechanism. The viability of HT-29 was measured in the
presence of increasing concentration (0.1−100 μM) of
oxaliplatin, and its concentration-dependent lethal effect after
48 h incubation is described in Table 2. In the presence of 27
(10 μM), oxaliplatin-dependent decrease of cell viability
remained unaffected, suggesting a lack of interference with
the anticancer activity. This evidence highlights a difference in
oxaliplatin toxicity mechanism in normal nerve cells versus
tumoral cells. Compound 27 selectively intervenes in neuro-
toxicity exerting protective effects on astrocytes.
In light of these interesting results, derivatives 8, 17, 26, and
27 were tested for their ability to bind the rat A3 AR. All the
tested compounds displayed null rat A3 receptor affinity,
G
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
d]pirimidines as protective agents against oxaliplatin-induced
neurotoxicity.
EXPERIMENTAL SECTION
■
A. Chemistry. The microwave-assisted syntheses were performed
using an Initiator EXP Microwave Biotage 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 melting points were determined
on a Gallenkamp melting point apparatus. Elemental analyses were
performed with a Flash E1112 Thermofinnigan elemental analyzer for
C, H, N, and the results were within 0.4% of the theoretical values.
All final compounds revealed a purity not less than 95%. 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. The following abbreviations
are used: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad,
and ar = aromatic protons.
General Procedure for the Synthesis of 1-Aryl-4-nitropyrazole-3-
carboxamides 3725 and 38. Compound 38 was prepared from the
ester 36 in the same conditions previously described to prepare
derivative 37 from 35.25 Briefly, a stream of ammonia was bubbled
through a suspension of the ethyl pyrazole-3-carboxylates 35−3625
(3.0 mmol) in 33% aqueous ammonia solution (40 mL) for about 30
min. Then the suspension was stirred at room temperature for about
48 h (for 37) or 24 h (for 38). The solid was collected by filtration,
washed with water, and recrystallized from 2-ethoxyethanol.
Figure 3. Antiapoptotic profile of derivatives 8, 17, 26, 27. Rat primary
cortical astrocytes were exposed to 100 μM oxaliplatin for 4 h in the
absence and in the presence of 1 μM of the tested compounds.
Caspase 3 activity was evaluated by a fluorimetric assay. Values are
expressed in percentage as mean SEM. Control condition is fixed at
100%. ∧∧P < 0.01 with respect to control; *P < 0.05 and **P < 0.01
with respect to oxaliplatin.
notwithstanding their nanomolar affinity for the hA3 receptor
(Table 1). This result is in line with data obtained on most of
the reported hA3 antagonists, and it is due to the well-known
species differences between human and rodent A3 receptor.1,2
The null affinities for the rat A3 AR of the tested compounds
indicate that their protective effect on astrocytes should be
ascribed to an A3 receptor-independent mechanism.
In any case, these pyrazolopyrimidine derivatives still
maintain high interest for their protective effect against
oxaliplatin-induced toxicity in rat astrocytes.
1-(4-Methoxyphenyl)-4-nitro-2-phenylpyrazole-3-carboxamide
1
38. Yield 75% ; mp 232−233 °C; H NMR 3.83 (s, 3H, OMe), 7.13
(d, 2H, ar, J = 9.4 Hz), 7.85−7.88 (m, 3H, 2 ar +amide proton), 8.16
(br s, 1H, amide proton), 9.52 (s, 1H, H-5); Anal. Calc. for
C11H10N4O4.
General Procedure for the Synthesis of 1-Aryl-4-nitropyrazole-3-
carbonitriles 39−40. A suspension of 37 or 38 (2 mmol) in
phosphorus oxychloride (5 mL) was microwave irradiated, respec-
tively, at 160 °C for 30 min or 120 °C for 3 min. The excess of
phosphorus oxychloride was distilled off and the residue was treated
with water (about 5−10 mL). The obtained solid was collected by
filtration and recrystallized.
CONCLUSION
■
The study reported here has led to the identification of a new
series of potent and selective hA3 AR antagonists, namely, the
2-arylpyrazolo[4,3-d]pyrimidin-7-amino derivatives. The pres-
ence of acyl moiety on the 7-amino group was found to be of
paramount importance for both hA3 AR affinity and selectivity.
Indeed most of the 7-acylamino derivatives were potent and
selective hA3 AR antagonists. Molecular docking investigations
performed at the hA3 AR model permitted identification of the
hypothetical binding mode of these new antagonists and to
rationalize the observed SARs.
4-Nitro-1-phenylpyrazole-3-carbonitrile 39. Yield 82%; mp 143−
1
145 °C (cyclohexane/EtOH). H NMR (DMSO-d6) 7.54−7.63 (m,
3H, ar), 7.74−7.76 (m, 2H, ar), 8.72 (s, 1H, H-5); IR 2256. Anal. Calc.
for C10H6N4O2.
1-(4-Methoxyphenyl)-4-nitropyrazole-3-carbonitrile 40. Yield
1
76%; mp 152−154 °C (EtOH). H NMR (DMSO-d6) 3.85 (s, 3H,
OMe), 7.15 (d, 2H, ar, J = 9.1 Hz) 7.89 (d, 2H, ar, J= 9.1 Hz), 9.81 (s,
1H, H-5). IR 2251, 1538, 1342. Anal. Calc. for C11H8N4O3.
General Procedure for the Synthesis of 4-Amino-1-arylpyrazole-
3-carbonitriles 41−42. A mixture of 4-nitropyrazoles 39, 40 (2
mmol), cyclohexene (8 mmol), and 10% Pd/C (15% w/w with respect
to the nitropyrazole) in EtOH (10 mL) was microwave irradiated at
150 °C for 15 min (compound 41) or 10 min (compounds 42). After
being cooled at room temperature, the catalyst was filtered off and the
solution was evaporated at reduced pressure to give a solid which was
recrystallized from suitable solvent.
Some selected pyrazolo[4,3-d]pyrimidin-7-amides (8, 17, 26,
27) proved to be effective in counteracting oxaliplatin-induced
apoptosis, in rat astrocyte cell cultures, with a non-A3AR-
dependent mechanism. Very interestingly, the most active
compound 27 did not interfere with the antitumor activity of
oxaliplatin on colon cancer HT-29 cell lines. Due to this
intriguing pharmacological behavior, further investigations are
in progress in our laboratory to develop new pyrazolo[4,3-
4-Amino-1-phenylpyrazole-3-carbonitrile 41. Yield 80%; mp
1
108−109 °C (H2O). H NMR (DMSO-d6) 5.06 (br s, 2H, NH2),
Table 2. Compound 27 Effect on Oxaliplatin-Affected HT-29 Cell Viability
a
HT-29 cell viability
oxaliplatin (μM)
oxaliplatin + 27 (10 μM)
0
0.3
1
3
10
30
100
%
100.0
97.6 0.9
95.1 2.4
94.9 1.3
90.2 2.3
92.3 1.0
86.6 1.2
87.2 0.9
82.2 1.7
81.9 0.9
78.8 1.7
52.2 0.5
54.8 0.6
a
HT-29 cell viability was evaluated by MTT assay. HT-29 cells were treated with increasing oxaliplatin concentration (0.3−100 μM) in the presence
or in the absence of 27 (10 μM) for 48 h. Values are expressed in percentage as mean SEM. Control condition is fixed at 100%.
H
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7.38 (t, 1H, ar, J = 7.5 Hz), 7.52 (t, 2H, ar, J = 7.5 Hz), 7.78 (s, 2H, ar,
J = 8.4 Hz), 7.91 (s, 1H, H-5); IR 3377, 3340, 2331. Anal. Calc. for
C10H8N4.
heated at reflux for 5 h (compounds 11 and 26), 24 h (compound 25),
36 h (compounds 18 and 27), 72 h (compounds 16 and 24) or 6 days
(compound 9). The suspension was diluted with water (15 mL) and
methylene chloride (15 mL). The organic phase was washed with
water (15 mL × 2) and anhydrified (Na2SO4). Evaporation of the
solvent at reduced pressure afforded an oil which solidified upon
treatment with diethyl ether (2−3 mL). All the crude derivatives were
purified by recrystallization, except compounds 9, 24, and 26 which
were chromatographed on silica gel column (eluent CH2Cl2/EtOAc/
MeOH 7:3:1, EtOAc/cyclohexane/MeOH 8:3:1, and CH2Cl2/cyclo-
hexane/CH3CN 4.5:4.5:1, respectively) and then recrystallized.
7-Acetylamino-5-methyl-2-phenyl-2H-pyrazolo[4,3-d]pyrimidine
4-Amino-1-(4-methoxyphenyl)pyrazole-3-carbonitrile 42. Yield
1
55%; mp 146−147 °C (H2O/EtOH). H NMR (DMSO-d6) 3.81 (s,
3H, OMe), 5.02 (br s, 2H, NH2), 7.05 (d, 2H, ar, J = 6.3 Hz), 7.70 (d,
2H, ar, J = 6.3 Hz), 7.81 (s, 1H, H-5); IR 3343, 3338, 3330, 2218.
Anal. Calc. for C11H10N4O.
General Procedure for the Synthesis of 2-Arylpyrazolo[4,3-
d]pyrimidin-7-amines 1−2, 4−5. A mixture of the 4-amino-
pyrazole-3-carbonitrile derivatives 41, 42 (1 mmol), anhydrous
ammonium acetate (2 mmol), and the suitable orthoester (1.5
mmol) was heated under microwave irradiation at 110 °C for 20 min
(compound 1), 130 °C for 30 min (compound 2) or 10 min
(compound 4), and at 150 °C for 25 min (compound 5). The mixture
was cooled at room temperature and taken up with cyclohexane (3−5
mL). The resulting solid was collected, then suspended in 0.15 M
NaHCO3 solution (5−6 mL), and kept under stirring for 1−2 min.
The crude product was collected by filtration, washed with water, and
recrystallized.
1
7. Yield 46%; mp 234−235 °C (EtOAc). H NMR (DMSO-d6) 2.37
(s, 3H, Me), 2.59 (s, 3H, Me), 7.53 (t, 1H, ar, J = 7.5 Hz), 7.64 (t, 2H,
ar, 7.5 Hz), 8.13 (d, 2H, ar, J = 7.9 Hz), 9.25 (s, 1H, H-3), 10.73 (s,
1H, NH). IR 3221, 3197, 1760. Anal. Calc. for C14H13N5O.
5-Methyl-7-(4-methoxybenzoylamino)-2-phenyl-2H-pyrazolo-
[4,3-d]pyrimidine 9. Yield 25%; mp 227−228 °C (toluene). 1H NMR
(CDCl3) 2.62 (s, 3H, Me), 3.92 (s, 3H, OMe), 6.99 (d, 2H, ar, J = 8.3
Hz), 7.49 (t, 1H, ar, J = 7.7 Hz), 7.59 (t, 2H, ar, J = 7.5 Hz), 7.97 (d,
2H, ar, J = 7.9 Hz), 8.38 (s, 1H, H-3), 8.45 (d, 2H, ar, J = 8.5 Hz). IR
3399, 1643. Anal. Calc. for C20H17N5O2.
5-Methyl-2-phenyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine 1. Yield
1
80%; mp 295−297 °C (EtOAc). H NMR (DMSO-d6) 3.38 (s, 3H,
7-(2-Furoylamino)-5-methyl-2-phenyl-2H-pyrazolo[4,3-d]-
pyrimidine 11. Yield 35%; mp 229−231 °C (EtOH). 1H NMR
(CDCl3) 2.62 (s, 3H, Me), 6.60 (s, 1H, furane proton), 7.50 (t, 1H, ar,
J = 7.4 Hz), 7.57−7.61 (m, 3H, 2 ar + furane proton), 7.67 (s, 1H,
furane proton), 7.97 (d, 2H, ar, J = 7.9 Hz), 8.41 (br s, 1H, H-3), 14.22
(br s, 1H, exchangeable with D2O). Anal. Calc. for C17H13N5O2.
7-Acetylamino-2,5-diphenyl-2H-pyrazolo[4,3-d]pyrimidine 14.
CH3), 7.47 (t, 1H, ar, J = 6.7 Hz), 7.58−7.62 (m, 4H, 2ar + NH2),
8.05 (d, 2H, ar, J = 7.6 Hz), 8.91 (s, 1H, H-3); IR 3471, 3445. Anal.
Calc. for C12H11N5.
2,5-Diphenyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine 2. Yield 62%;
1
mp 196−198 °C (cyclohexane/EtOAc). H NMR (DMSO-d6) 7.42−
7.52 (m, 4H, ar), 7.62−7.66 (m, 2H, ar), 7.81 (br s, 2H, NH2), 8.10
(d, 2H, ar, J = 7.7 Hz), 8.41 (d, 2H, ar, J = 8.0 Hz), 9.13 (s, 1H, H-3);
IR 3456, 3280, 1623. Anal. calc. for C17H13N5.
1
Yield 62%; mp 228−230 °C (toluene). H NMR (DMSO-d6) 2.51
(s, 3H, Me), 7.53 (m, 4H, ar), 7.67 (t, 2H, ar, J = 7.8 Hz), 8.18 (d, 2H,
ar, J = 7.8 Hz), 8.45 (d, 2H, ar, J = 8.0 Hz), 9.46 (s, 1H, H-3), 10.88 (s,
1H, NH). IR 3246, 1676. Anal. Calc. for C19H15N5O.
2-(4-Methoxyphenyl)-5-methyl-2H-pyrazolo[4,3-d]pyrimidin-7-
1
amine 4. Yield 72%; mp 269−270 °C (H2O). H NMR (DMSO-d6)
2.37 (s, 3H, Me), 3.84 (s, 3H, OMe), 7.14 (d, 2H ar, J = 9.1 Hz), 7.61
(br s, 2H, NH2), 7.95 (d, 2H, ar, J = 9.1 Hz), 8.80 (s, 1H, H-3). IR
3461, 3296, 1650. Anal. Calc. for C13H13N5O.
7-(4-Methoxybenzoylamino)-2,5-diphenyl-2H-pyrazolo[4,3-d]-
pyrimidine 16. Yield 78%; mp 233−235 °C (CH3CN). 1H NMR
(DMSO-d6) 3.89 (s, 3H, OMe), 7.13 (d, 2H, ar, J = 8.6 Hz), 7.52−
7.56 (m, 4H, ar), 7.65 (t, 2H, ar, J = 7.8 Hz), 8.09 (d, 2H, ar, J = 8.6
Hz), 8.14 (d, 2H, ar, J = 8.2 Hz), 8.43 (d, 2H, ar, J = 5.8 Hz), 9.50 (s,
1H, H-3), 11.23 (s, 1H, NH). Anal. Calc. for C25H19N5O2.
2,5-Diphenyl-7-phenylacetylamino-2H-pyrazolo[4,3-d]-
pyrimidine 17. Yield 75%; mp 205−206 °C (toluene). 1H NMR
(DMSO-d6) 4.16 (s, 2H, CH2), 7.28 (t, 1H, ar, J = 7.1 Hz), 7.38 (m,
4H, ar) 7.52 (m, 4H, ar), 7.68 (t, 2H, ar, J = 7.6 Hz), 8.18 (d, 2H, ar, J
= 8.4 Hz), 8.45 (d, 2H, ar, J = 7.9 Hz), 9.47 (s, 1H, H-3), 11.18, (s,
1H, NH). IR 3485, 3385, 1709. Anal. Calc. for C25H19N5O.
2,5-Diphenyl-7-(2-furoylamino)-2H-pyrazolo[4,3-d]pyrimidine
18. Yield 75%; mp 193−195 °C (EtOAc/cyclohexane). 1H NMR
(DMSO-d6) 6.79−6.81(m, 1H, furane proton), 7.53−7.58 (m, 4H, ar),
7.64−7.69 (m, 3H, ar), 8.06 (s, 1H, furane proton), 8.16 (d, 2H, ar, J =
7.7 Hz), 8.47 (d, 2H, ar, J = 8.1 Hz), 9.52 (s, 1H, H-3), 11.14 (s, 1H,
NH). Anal. Calc. for C22H15N5O2.
2-(4-Methoxyphenyl)-5-phenyl-2H-pyrazolo[4,3-d]pyrimidin-7-
amine 5. Yield 66%, mp 206−208 °C (EtOH). 1H NMR (DMSO-d6)
3.86 (s, 3H, OMe), 7.17 (d, 2H, ar, J = 9.0 Hz), 7.43−7.46 (m, 3H,
ar), 7.74 (br s, 2H, NH2), 8.00 (d, 2H, ar, J = 9.0 Hz), 8.38 (d, 2H, ar, J
= 7.5 Hz), 9.01 (s, 1H, H-3). IR 3465, 3296. Anal. Calc. for
C18H15N5O.
General Procedure for the Synthesis of 2-Aryl-5-benzylpyrazolo-
[4,3-d]pyrimidin-7-amines 3, 6. A mixture of the 4-aminopyrazole-3-
carbonitrile derivatives 41, 42 (1 mmol), anhydrous ammonium
acetate (1.8 mmol), and ethyl phenyliminoacetate hydrochloride28
(1.3 mmol) was heated under microwave irradiation at 150 °C for 15
min (compound 3) or 10 min (compound 6). The mixture, cooled at
room temperature, was taken up with EtOAc (2−3 mL), and the solid
which precipitated was filtered, washed with water (5−10 mL), and
recrystallized.
5-Benzyl-2-phenyl-2H-pyrazolo[4,3-d]pyrimidin-7-amine 3. Yield
7-Acetylamino-2-(4-methoxyphenyl)-5-methyl-2H-pyrazolo[4,3-
1
1
35%; mp 263−264 °C (EtOH). H NMR (DMSO-d6) 3.96 (s, 2H,
d]pyrimidine 21. Yield 46%; mp 189−190 °C (EtOAc). H NMR
CH2), 7.17 (s, 1H, ar, J = 7.1 Hz), 7.19−7.33 (m, 4H, ar), 7.48 (t, 1H,
ar, J = 7.1 Hz), 7.59−7.64 (m, 4H, 2 ar + NH2), 8.04 (d, 2H, ar, J = 7.6
Hz), 8.96 (s, 1H, H-3); IR 3438, 3315, 1661. Anal. Calc. for C18H15N5.
5-Benzyl-2-(4-methoxyphenyl)-2H-pyrazolo[4,3-d]pyrimidin-7-
(DMSO-d6) 2.36 (s, 3H, Me), 2.68 (s, 3H, Me), 3.68 (s, 3H, OMe),
7.17 (d, 2H, ar, J = 9.0 Hz), 8.04 (d, 2H, ar, J = 9.0 Hz), 9.14 (s, 1H,
H-3), 10.69 (s, 1H, NH). IR 3336, 1709. Anal. Calc. for C15H15N5O2.
7-(2-Furoylamino)-5-methyl-2-(4-methoxyphenyl)-2H-pyrazolo-
[4,3-d]pyrimidine 24. Yield 25%; mp 221−223 °C (EtOH). 1H NMR
(CDCl3) 2.63 (br s, 3H, Me), 3.91 (s, 3H, OMe), 6.59 (br s, 1H,
furane proton), 7.07 (d, 2H, ar, J = 8.8 Hz), 7.54 (br s, 1H, furane
proton), 7.67 (s, 1H, furane proton), 7.87 (d, 2H, ar, J = 8.8 Hz), 8.31
(br s, 1H, H-3), 14.31 (br s, 1H, exchangeable with D2O). Anal. Calc.
for C18H15N5O3.
1
amine 6. Yield 30%; mp 251−252 °C (2-ethoxyethanol). H NMR
(DMSO-d6) 3.84 (s, 3H, OMe), 3.95 (s, 2H, CH2), 7.13−7.19 (m, 3H,
ar), 7.25−7.31 (m, 4H, ar), 7.61 (br s, 2H, NH2), 7.93 (d, 2H, ar, J =
6.90 Hz), 8.85 (s, 1H, H-3). IR: 3441, 3315. Anal. Calc. for
C19H17N5O.
General Procedure for the Synthesis of 7-Amido-substituted
Pyrazolo[4,3-d]pyrimidine Derivatives 7, 9, 11, 14, 16−18, 21, 24−
27. The title compounds were prepared by reacting the pyrazolopyr-
imidin-7-amino derivatives 1, 2, 4, and 5 (1.3 mmol) with an excess of
acetyl chloride (2.6 mmol), 4-methoxybenzoyl chloride (2 mmol),
benzoyl chloride (2 mmol), 2-furoyl chloride (2.6 mmol), and
phenylacetyl chloride (2.6 mmol) in anhydrous CH2Cl2 (15 mL) and
pyridine (13 mmol). The mixture was stirred at room temperature for
2 h (compounds 14 and 17) or 24 h (compounds 7 and 21), or it was
7-Acetylamino-2-(4-methoxyphenyl)-5-phenyl-2H-pyrazolo[4,3-
d]pyrimidine 25. Yield 47%; mp 213-215 °C (cyclohexane/EtOAc).
1H NMR (DMSO-d6) 2.50 (s, 3H, Me), 3.87 (s, 3H, OMe), 7.21 (d,
2H, ar, J = 9.0 Hz), 7.51−7.53 (m, 3H, ar), 8.09 (d, 2H, ar, J = 9.0 Hz),
8.44 (d, 2H, ar, J = 7.4 Hz), 9.34 (s, 1H, H-3), 10.82 (s, 1H, NH). IR
3224, 1700. Anal. Calc. for C20H17N5O2.
7-Benzoylamino-2-(4-methoxyphenyl)-5-phenyl-2H-pyrazolo-
[4,3-d]pyrimidine 26. Yield 40%; mp 205−207 °C (EtOH). 1H NMR
I
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(DMSO-d6) 3.86 (s, 3H, Me), 7.19 (d, 2H, ar, J = 8.7 Hz), 7.48−7.51
(m, 3H, ar), 7.59 (t, 2H, ar, J = 7.3 Hz), 7.67 (t, 1H, ar, J = 7.0 Hz),
8.06−8.07 (m, 4H, ar), 8.37−8.39 (m, 2H, ar), 9.41 (s, 1H, H-3),
11.38 (s, 1H, NH). IR 3259, 1662. Anal. Calc. for C25H19N5O2.
2-(4-Methoxyphenyl)-5-phenyl-7-phenylacetylamino-2H-
pyrazolo[4,3-d]pyrimidine 27. Yield 25%; mp 168−170 °C
7-Benzoylamino-2-(4-Methoxyphenyl)-5-Methyl-2H-pyrazolo-
[4,3-d]pyrimidine 22. Yield 68%; mp 216−217 °C (EtOAc). 1H NMR
(DMSO-d6) 2.50 (s, 3H, ar), 3.84 (s, 3H, OMe), 7.14 (d, 2H, ar, J =
9.0 Hz), 7.52−7.62 (m, 3H, ar), 7.93 (br s, 2H, ar), 8.14 (br s, 2H, ar),
9.01 (br s, 1H, H-3). Anal. Calc. for C20H17N5O2.
2-(4-Methoxyphenyl)-5-methyl-7-phenylacetylamino-2H-
pyrazolo[4,3-d]pyrimidine 23. Yield 52%; mp 206−208 °C (EtOH).
1H NMR (DMSO-d6) 2.58 (s, 3H, Me), 3.86 (s, 3H, OMe), 4.02 (s,
2H, CH2), 7.18 (d, 2H, ar, J = 9.0 Hz), 7.26 (t, 1H, ar, J = 7.1 Hz),
7.33−7.40 (m, 4H, ar), 8.04 (d, 2H, ar, J = 8.9 Hz), 9.17 (s, 1H, H-3),
10.99 (s, 1H, NH). IR 3204, 1686. Anal. Calc. for C21H19N5O2.
General Procedure for the Synthesis of 7-Diacylamino-
substituted Pyrazolo[4,3-d]pyrimidine Derivatives 28−30. The title
compounds were obtained by refluxing the 7-amino derivatives 1 or 2
(2 mmol) with an excess of benzoyl chloride (10 mmol) or 2-furoyl
chloride (15 mmol) in anhydrous methylene chloride (5 mL) and
pyridine (20 mmol). The suspension was stirred at room temperature
for 4 h (compound 28) or refluxed for 96 h (compounds 29 and 30),
then diluted with water (10 mL), and the resulting mixture was
extracted with methylene chloride (15 mL × 3). The organic phase
was anhydrified (Na2SO4) and evaporated at reduced pressure to yield
a solid which was purified by column chromatography (compound 28,
eluent cyclohexane/EtOAc 7:3) or by recrystallization (compounds 29
and 30).
7-Dibenzoylamino-2-phenyl-2H-pyrazolo[4,3-d]pyrimidine 28.
Yield 20%; mp 249−250 °C. 1H NMR (DMSO-d6) 2.57 (s, 3H,
Me), 7.37−7.41 (m, 4H, ar), 7.44−7.54 (m, 5H, ar), 7.85 (d, 2H, ar, J
= 7.9 Hz), 7.90 (d, 4H, ar, J = 7.3 Hz), 8.58 (s, 1H, H-3). Anal. Calc.
for C26H19N5O2.
5-Methyl-7-(difuro-2-ylamino)-2-phenyl-2H-pyrazolo[4,3-d]-
pyrimidine 29. Yield 35%; mp 231−233 °C (EtOH). 1H NMR
(DMSO-d6) 2.55 (s, 3H, Me), 6.73−6.86 (m, 2H, furane protons),
7.45−7.46 (m, 2H, furane protons), 7.54 (t, 1H, ar, J = 7.0 Hz), 7.62
(t, 2H, ar, J = 7.4 Hz), 7.93 (s, 2H, furane protons), 7.98 (d, 2H, ar),
9.46 (s, 1H, H-3). IR 1702. Anal. Calc. for C22H15N5O4.
2,5-Diphenyl-7-(difuro-2-ylamino)-2H-pyrazolo[4,3-d]pyrimidine
30. Yield 15%; mp 269−271 °C (EtOAc/cyclohexane). 1H NMR
(CDCl3) 6.53−6.55 (m, 2H, furane protons), 7.40−7.57 (m, 10H, 8ar
+ 2 furane protons), 7.89 (d, 2H, ar, J = 7.8 Hz), 8.22−8.25 (m, 2H,
furane protons), 8.72 (s, 1H, H-3). Anal. Calc. for C27H17N5O4.
General Procedure for the Synthesis of 7-Ureido-Substituted
Pyrazolo[4,3-d]pyrimidine Derivatives 31−34. The suitable isocya-
nate (1.5 mmol) was added to a suspension of the 7-amino derivatives
1 or 4 (1 mmol) in anhydrous tetrahydrofuran (20 mL). The mixture
was refluxed under nitrogen atmosphere until the disappearance of the
starting material (about 1−2 h). The suspension was diluted with
water (5−10 mL), and the resulting solid was collected, washed with
diethyl ether and recrystallized. Compound 31 was purified by column
chromatography (eluent EtOAc).
1
(CH3CN). H NMR (DMSO-d6) 3.87 (s, 3H, OMe), 4,15 (s, 2H,
CH2), 7.22 (d, 2H, ar, J = 9.1 Hz), 7.28 (t, 1H, ar, J = 7.1 Hz), 7.34−
7.42 (m, 4H, ar), 7.49−7.51 (m, 3H, ar), 8.10 (d, 2H, ar, J = 9.0 Hz),
8.44 (d, 2H, ar, J = 9.1 Hz), 9.36 (s, 1H, H-3), 11.CH3CN12 (s, 1H,
NH). IR 3320, 1710. Anal. Calc. for C26H21N5O2.
General Procedure for the Synthesis of 7-Amido-substituted
Pyrazolo[4,3-d]pyrimidine Derivatives 8, 10, 12, 13, 15, 19, 20, 22,
23. A mixture of the pyrazolopyrimidin-7-amino derivatives 1, 2, 4 (1
mmol), the suitable carboxylic acid (6 mmol), 1-(3-(dimethylamino)-
propyl))-3-ethyl-carbodiimide hydrochloride (6 mmol), 1-hydroxy-
benzotriazole hydrochloride (6 mmol), triethylamine (15 mmol), and
4-(dimethylamino)pyridine (0.1 mmol) in anhydrous dimethylforma-
mide (2−3 mL) was stirred at room temperature for about 4 h
(compound 22), 7 h (compound 8), 72 h (compounds 12, 15, 19 and
20), and one week (compounds 10 and 23). The suspension was
diluted with water (about 15−20 mL), and the resulting solid was
collected by filtration (compounds 8, 15, 19, and 20) or extracted with
CH2Cl2 (15 mL × 3) (compounds 10, 12, 13, 22, and 23). The
combined organic extracts were anhydrified (Na2SO4), and the solvent
was evaporated at reduced pressure. Derivatives 12, 13, 15, 19, 20, 22,
and 23 were recrystallized, while compounds 8 and 10 were purified
by column chromatography (eluent cyclohexane/EtOAc/MeOH 2:3:1
and EtOAc, respectively) and then recrystallized.
7-Benzoylamino-5-methyl-2-phenyl-2H-pyrazolo[4,3-d]-
pyrimidine 8. Yield 35%; mp 222−223 °C (EtOAc). 1H NMR
(CDCl3) 2.72 (s, 3H, Me), 7.46−7.59 (m, 6H, ar), 7.95 (d, 2H, ar, J =
7.7 Hz), 8.38−8.39 (m, 2H, ar), 8.44 (s, 1H, H-3). IR 3156, 1621.
Anal. Calc. for C19H15N5O.
5-Methyl-2-phenyl-7-phenylacetylamino-2H-pyrazolo[4,3-d]-
pyrimidine 10. Yield 20%; mp 193−194 °C (MeOH). 1H NMR
(DMSO-d6) 2.59 (s, 3H, Me), 4.04 (s, 2H, CH2), 7.27 (t, 1H, ar, J =
7.2 Hz), 7.35−7.40 (m, 4H, ar), 7.53 (t, 1H, ar, J = 7.4 Hz), 7.64 (t,
2H, ar, J = 7.7 Hz), 8.12 (d, 2H, ar, J = 7.8 Hz), 9.26 (s, 1H, H-3),
10.98 (br s, 1H, NH). Anal. Calc. for C20H17N5O.
5-Methyl-2-phenyl-7-(3-pyridoylamino)-2H-pyrazolo[4,3-d]-
pyrimidine 12. Yield 65%; mp 217−219 °C (EtOAc). 1H NMR
(CDCl3) 2.67 (s, 3H, Me), 7.44−7.53 (m, 2H, 1 ar + pyridine proton),
7.60 (t, 2H, ar, J = 7.4 Hz), 7.98 (d, 2H, ar, J = 8.5 Hz), 8.44 (s, 1H,
pyridine H-2 proton), 8.72−8.79 (m, 2H, pyridine protons), 9.64 (s,
1H, H-3), 14.57 (br s, 1H, exchangeable with D2O). IR 3368, 1622.
Anal. Calc. for C18H14N6O.
5-Methyl-2-phenyl-7-(4-pyridoylamino)-2H-pyrazolo[4,3-d]-
pyrimidine 13. Yield 55%; mp 200−201 °C (EtOH). 1H NMR
(DMSO-d6) 2.56 (s, 3H, Me), 7.49 (t, 1H, ar, J = 7.3 Hz), 7.60 (t, 2H,
ar, J = 7.6 Hz), 7.99−8.03 (m, 4H, 2 ar +2 pyridine protons), 8.80 (d,
2H, pyridine protons, J = 5.8 Hz), 9.18 (br s, 1H, H-3). Anal. Calc. for
C18H14N6O.
7-Benzoylamino-2,5-diphenyl-2H-pyrazolo[4,3-d]pyrimidine 15.
Yield 40%; mp 248−249 °C (tetrahydrofurane/H2O). 1H NMR
(DMSO-d6) 7.51−7.71 (m, 9 H, ar), 8.08 (d, 2H, ar, J = 7.5 Hz), 8.15
(d, 2H, ar, J = 7.6 Hz), 8.40−8.41 (m, 2H, ar), 9.42 (s, 1H, H-3), 11.45
(br s, 1H, NH). IR 3243, 1676. Anal. Calc. for C24H17N5O.
2,5-Diphenyl-7-(3-pyridoylamino)-2H-pyrazolo[4,3-d]pyrimidine
19. Yield 75%; mp 227−229 °C (EtOH). 1H NMR (DMSO-d6) 7.51-
7.68 (m, 7H, 6 ar + pyridine proton), 8.16 (d, 2H, ar, J = 7.9 Hz),
8.35−8.37 (m, 3H, 2 ar + pyridine proton), 8.88 (d, 1H, pyridine
proton, J = 4.6 Hz), 9.18 (br s, 1H, pyridine proton), 9.52 (s, 1H, H-
3), 11.70 (s, 1H, NH). Anal. Calc. for C23H16N6O.
2,5-Diphenyl-7-(4-pyridoylamino)-2H-pyrazolo[4,3-d]pyrimidine
20. Yield 68%; mp 262−264 °C (EtOAc). 1H NMR (DMSO-d6)
7.50−7.66 (m, 6H, ar), 7.91−7.94 (d, 2H, pyridine protons, J = 4.4
Hz), 8.16 (d, 2H, ar, J = 7.7 Hz), 8.31−8.30 (m, 2H, ar), 8.83 (d, 2H,
pyridine protons, J = 4.4 Hz), 9.54 (s, 1H, H-3), 11.77 (s, 1H, NH).
Anal. Calc. for C23H16N6O.
5-Methyl-2-phenyl-7-(3-phenylureido)-2H-pyrazolo[4,3-d]-
1
pyrimidine 31. Yield 30%; mp 240−242 °C. H NMR (CDCl3) 2.80
(s, 3H, Me), 7.17 (t, 1H, ar, J = 7.3 Hz), 7.41 (t, 2H, ar, J = 7.7 Hz),
7.50−7.54 (m, 1H, ar), 7.61 (t, 2H, ar, J = 7.6 Hz), 7.67 (d, 2H, ar, J =
7.9 Hz), 7.90 (d, 2H, ar, J = 7.9 Hz), 8.15 (br s, 1H, NH), 8.48 (s, 1H,
H-3), 12.10 (br s, 1H, NH). Anal. Calc. for C19H16N6O2.
7-(3-Benzylureido)-5-methyl-2-phenyl-2H-pyrazolo[4,3-d]-
pyrimidine 32. Yield 70%; mp 255−257 °C (toluene). 1H NMR
(CDCl3) 2.67 (s, 3H, Me), 4.71 (d, 2H, CH2, J = 5.1 Hz), 7.34−7.41
(m, 4H, ar), 7.51−7.58 (m, 4H, ar), 7.89 (d, 2H, ar, J = 7.5 Hz), 8.19
(br s, 1H, NH), 8.51 (s, 1H, H-3), 10.00 (br s, 1H, NH). Anal. Calc.
for C20H18N6O.
2-(4-Methoxyphenyl)-5-methyl-7-(3-phenylureido)-2H-pyrazolo-
[4,3-d]pyrimidine 33. Yield 65%; mp 241−243 °C (nitromethane).
1H NMR (CDCl3) 2.83 (s, 3H, Me), 3.93 (s, 3H, OMe), 7.09 (d, 2H,
ar, J = 8.0 Hz), 7.17 (t, 1H, ar, J = 7.2 Hz), 7.41 (t, 2H, ar, J = 7.3 Hz),
7.66 (d, 2H, ar, J = 8.0 Hz), 7.82 (d, 2H, ar, J = 7.5 Hz), 8.24 (br s, 1H,
NH), 8.45 (s, 1H, H-3), 12.00 (br s, 1H, NH). IR 3466, 1692. Anal.
Calc. for C20H18N6O2.
7-(3-Benzylureido)-2-(4-methoxyphenyl)-5-methyl-2H-pyrazolo-
[4,3-d]pyrimidine 34. Yield 68%; mp 243−244 °C (EtOH/EtOAc).
1H NMR (CDCl3) 2.64 (s, 3H, Me), 3.93 (s, 3H, OMe), 4.70 (d, 2H,
J
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
CH2, J = 5.1 Hz), 7.08 (d, 2H, ar, J = 9.0 Hz), 7.28−7.46 (m, 5H, ar),
7.78 (d, 2H, ar, J = 8.56 Hz), 8.20 (br s, 1H, NH), 8.43 (s, 1H, H-3),
10.02 (s, 1H, NH). IR 3376, 1694. Anal. Calc. for C21H20N6O2.
B. Computational Studies. All modeling studies were carried out
on a 20 CPU (Intel Core2 Quad CPU 2.40 GHz) Linux cluster.
Energy calculations and analyses of docking poses were performed
with the Molecular Operating Environment (MOE, version 2010.10)
suite.41
Three-Dimensional Structures of hA2A AR and hA3 AR. The
crystallographic structure of hA2A AR, in complex with the high affinity
antagonist ZM241385 (PDB access code: 3EML),29 and a previously
proposed homology model of the hA3AR25 were used to perform
molecular docking studies at these receptor subtypes.
The numbering of the amino acids follows the arbitrary scheme by
Ballesteros and Weinstein: each amino acid identifier starts with the
helix number, followed by the position relative to a reference residue
among the most conserved amino acids in that helix, to which the
number 50 is arbitrarily assigned.42
Molecular Docking of hA3 AR Antagonists. Ligand structures were
built using the MOE-builder tool, as part of the MOE suite,41 and were
subjected to a MMFF94x energy minimization until the rms conjugate
gradient was <0.05 kcal mol−1 Å−1.
All antagonists were docked into the hypothetical TM binding site
of the hA3 AR model and the orthosteric binding site of the hA2A AR
crystal structure, by employing the docking tool of the GOLD suite.43
For each compound, 25 independent docking runs were performed
and searching was conducted within a user-specified docking sphere
with the Genetic Algorithm protocol and the GoldScore scoring
function.
Prediction of antagonist-receptor complex stability (in terms of
corresponding pKi value) and quantitative analysis for nonbonded
intermolecular interactions (H-bonds, transition metal, water bridges,
hydrophobic, electrostatic) were calculated and visualized using several
tools implemented in the MOE suite.41 Electrostatic and hydrophobic
contributions to the interaction energy of individual residues were
calculated using MOE.41 To estimate the electrostatic contributions,
atomic charges for the ligands were calculated with MOPAC44 and the
PM3/ESP methodology, whereas partial charges for the protein amino
acids were computed with the AMBER99 force field.
Interaction Energy Fingerprints (IEFs). To analyze the ligand−
receptor recognition mechanism in a more quantitative manner, we
calculated the individual electrostatic and hydrophobic contributions
to the interaction energy of each receptor residue involved in the
binding with the ligand. In particular, the electrostatic contribution has
been computed on the basis of the nonbonded electrostatic interaction
energy term of the force field, whereas the hydrophobic contributions
has been calculated by using the directional hydrophobic interaction
term based on contact surfaces as implemented in the MOE scoring
function.41 As a consequence, an energy (expressed in kcal/mol) is
associated with the electrostatic contribution, whereas a score (the
higher the better) is related to the hydrophobic contribution.
The analysis of these contributions have been reported as
“interaction energy fingerprints” (IEFs), e.g., interaction energy
patterns (graphically displayed as histograms) reporting the key
residues involved in the binding with the considered ligands along with
a quantitative estimate of the occurring interactions.
of 60 min at 4 °C.47 Nonspecific binding was determined in the
presence of 1 μM ZM-241385 and was about 20% of total binding.
Competition binding experiments to hA3 CHO membranes (50 μg of
protein/assay) were performed incubating 0.5 nM [125I]AB-MECA, 50
mM Tris HCl buffer, 10 mM MgCl2, 1 mM EDTA, pH 7.4, and at
least 6−8 different concentrations of examined ligands for 60 min at
37 °C.48 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 radioactivity was counted by Scintillation Counter
Packard Tri Carb 2810 TR with an efficiency of 58%.
Rat A3 Receptor Binding Assay. Selected compounds were tested
for evaluating their affinity at rat A3 adenosine receptors expressed in
HEK293 cells (Perkin-Elmer, Boston, USA). Competition binding
experiments to rA3 HEK membranes (10 μg of protein/assay) were
performed incubating 0.5 nM [125I]AB-MECA, 50 mM Tris−HCl
buffer, 10 mM MgCl2, 1 mM EDTA, pH 7.4, and at least 6−8 different
concentrations of examined ligands for 60 min at 37 °C.49 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
radioactivity was counted by Scintillation Counter Packard Tri Carb
2810 TR with an efficiency of 58%.
Measurement of Cyclic AMP Levels in CHO Cells Transfected with
hA2B or hA3 ARs. CHO cells transfected with hAR subtypes were
washed with phosphate-buffered saline, diluted tripsine and centri-
fuged for 10 min at 200g. The cells (1 × 106 cells/assay) were
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 phosphodies-
terase inhibitor and preincubated for 10 min in a shaking bath at 37
°C. The potency of antagonists to the A2B AR was determined by the
inhibition of NECA (200 nM)-induced cyclic AMP production. In
addition, the potency of antagonists to the A3 ARs was determined in
the presence of forskolin 1 μM and Cl-IB-MECA (100 nM) 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] cyclic
AMP and incubation buffer (trizma base 0.1 M, aminophylline 8.0
mM, 2-mercaptoethanol 6.0 mM, pH 7.4). The binding protein
prepared from beef adrenals was added to the samples previously
incubated at 4 °C for 150 min, and, after the addition of charcoal, was
centrifuged at 2000g for 10 min. The clear supernatant was counted in
a Scintillation Counter Packard Tri Carb 2810 TR with an efficiency of
58%.49
Data Analysis. The protein concentration was determined
according to a Bio-Rad method50 with bovine albumin as a standard
reference. Inhibitory binding constant (Ki) values were calculated from
those of IC50 according to Cheng & Prusoff equation Ki = IC50/(1 +
[C*]/KD*), where [C*] is the concentration of the radioligand and
KD* its dissociation constant.51 A weighted nonlinear least-squares
curve fitting program LIGAND52 was used for computer analysis of
inhibition experiments. IC50 values obtained in cyclic AMP assay were
calculated by nonlinear regression analysis using the equation for a
sigmoid concentration−response curve (Graph-PAD Prism, San
Diego, CA, U.S.A).
ADME and Physicochemical Properties. The predicted ADME and
physicochemical properties have been calculated using StarDrop
program.45
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 ARs. Displacement experiments
of [3H]DPCPX (1 nM) to hA1 CHO membranes (50 μg of protein/
assay) and at least 6−8 different concentrations of antagonists for 120
min at 25 °C in 50 mM Tris HCl buffer pH 7.4 were performed.46
Nonspecific binding was determined in the presence 1 μM of DPCPX
(≤10% of the total binding). Binding of [3H]ZM-241385 (1 nM) to
hA2ACHO membranes (50 μg of protein/assay) was performed by
using 50 mM Tris HCl buffer, 10 mM MgCl2 pH 7.4 and at least 6−8
different concentrations of antagonists studied for an incubation time
Cell Cultures. Human colon cancer cell line HT-29 was obtained
from American Type Culture Collection (Rockville, MD). HT-29
were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) high
glucose with 20% fetal bovine serum (FBS). Media contained 2 mM L-
glutamine, 1% essential aminoacid mix, 100 IU mL−1 penicillin and
100 μg mL−1 streptomycin (Sigma, Germany) in 5% CO2 atmosphere
at 37 °C.
K
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Primary cultures of astrocytes were obtained according to the
method described by McCarthy and De Vellis.53 Briefly, the cerebral
cortex of newborn (P1−P3) Sprague−Dawley rats (Harlan, Italy) were
dissociated in Hank’s balanced salt solution containing 0.5% trypsin/
EDTA and 1% DNase (Sigma, Germany) for 30 min at 37 °C.
Suspension was mechanically homogenized and filtered. Cells were
plated in DMEM high glucose with 20% FBS. At confluency, primary
glial cultures were used to isolate astrocytes removing microglia and
oligodendrocytes by shaking. The purity of the astrocyte culture was
determined by immunocytochemically staining for glial fibrillary acidic
protein (GFAP) (Dako, Denmark). The cells were fixed in 4%
paraformaldehyde, then incubated with the antibody (1:200) and
visualized using Alexafluor conjugated secondary antibody. Of the cells
in astrocyte cultures, 98% were positive for GFAP. Experiments were
performed 21 days after cell isolation. Formal approval to conduct the
experiments described was obtained from the Animal Subjects Review
Board of the University of Florence. The ethical policy of the
University of Florence complies with the Guide for the Care and Use
of Laboratory Animals of the US National Institutes of Health (NIH
Publication No. 85-23, revised 1996; University of Florence assurance
number: A5278-01).
ACKNOWLEDGMENTS
■
The synthetic work was supported by a grant of the Italian
Ministry for University and Research (MIUR, FIRB
RBNE03YA3L project). The molecular modeling 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.
ABBREVIATIONS USED
■
AR, adenosine receptor; NECA, 5′-(N-ethyl-carboxamido)-
adenosine; cAMP, cyclic adenosine monophosphate; Cl-IB-
M E C A , 2 - c h l o r o - N 6 - ( 3 - i o d o b e n z y l ) 5 ′ - ( N -
methylcarboxamido)adenosine; DPCPX, 8-cyclopentyl-1,3-di-
propyl-xanthine; 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-
M E C A , N 6 - ( 4 - a m i n o - 3 - i o d o b e n z y l ) - 5 ′ - ( N -
methylcarboxamido)adenosine; CHO, chinese hamster ovary;
IEFs, interaction energy fingerprints; TM, transmembrane;
EL2, second extracellular loop; PP, pyrazolo[4,3-d]pyrimidine;
RMSD, root-mean-square deviation; MOE, molecular operat-
ing environment; PES, potential energy surface; DMEM,
Dulbecco’s modified Eagle’s medium; FBS, fetal bovine
serum; GFAP, glial fibrillary acidic protein; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
HT-29 and astrocytes were starved in serum-free DMEM overnight
before all treatments.
Cells were incubated with 0.1−100 μM oxaliplatin as described for
each measurement (see below). The effects of tested compounds were
evaluated allowing a concomitant incubation with oxaliplatin.
The duration time and concentration used in each set of
experiments were chosen with respect to the method sensibility and
specificity.
Caspase 3 Activity. Astrocytes were plated in 6-well plates (5 ×
105/well) and grown in until 90% to 100% confluence. Cells were
incubated with 100 μM oxaliplatin for 4 h. After treatment cells were
scraped in 100 μM lysis buffer (200 mM Tris-HCl buffer, pH 7.5,
containing 2 M NaCl, 20 mM EDTA, and 0.2% Triton X-100). Fifty
microliters of the supernatants were incubated with 0.025 mM
fluorogenic peptide caspase substrate rhodamine 110 bis-(N-CBZ-L-
aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide) (Z-DEVD-R110;
Molecular Probes) at 25 °C for 30 min. The amount of cleaved
substrate in each sample was measured in a 96-well plate fluorescent
spectrometer (Perkin-Elmer; excitation at 496 nm and emission at 520
nm).
Cell Viability Assay. Cell viability was evaluated by the reduction of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
as an index of mitochondrial compartment functionality. Cells were
plated onto 96-well plates for cell culture (1 × 104 for astrocytes and 5
× 103 for HT-29), grown until confluence, and then treated for 24 or
48 h with different concentrations of oxaliplatin in DMEM. After
extensive washing, 1 mg/mL MTT was added into each well and
incubated for 2 h at 37 °C. After washing, the formazan crystals were
dissolved in 100 μL dimethyl sulfoxide. The absorbance was measured
at 580 nm. Experiments were performed in quadruplicate on at least
three different cell batches.
REFERENCES
■
(1) Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz, K. N.;
Linden, J. International union of Pharmacology XXV. Nomenclature
and classification of adenosine receptors. Pharmacol. Rev. 2001, 53,
527−552.
(2) Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Linden, J.;
Muller, C. E. International union of Pharmacology LXXXI.
Nomenclature and classification of adenosine receptors. An up date.
Pharmacol. Rev. 2011, 63, 1−34.
(3) Jacobson, K. A.; Knutsen, L. J. S. P1 and P2 purine and
pyrimidine receptor ligands. In Purinergic and Pyrimidinergic Signalling;
Abbracchio, M. P., Williams, M., Eds; Springer: Berlin, 2001;
Handbook of Experimental Pharmacology, Vol. 151/1, pp 129−175.
(4) Borea, P. A.; Gessi, S.; Bar-Yehuda, S.; Fishman, P. A3 adenosine
receptor: pharmacology and role in disease. Handb. Exp. Pharmacol.
2009, 193, 297−327.
(5) Shneyvays, V.; Leshem, D.; Zinman, T.; Mamedova, L. K.;
Jacobson, K. A.; Shainberg, A. Role of adenosine A1 and A3 receptors
in regulation of cardiomyocyte homeostasis after mitochondrial
respiratory chain injury. Am. J. Physiol. Heart Circ. Physiol. 2005,
288, H279−H2801.
(6) Schulte, G.; Fredholm, B. B. Signalling from adenosine receptors
to mitogen-activated protein kinases. Cell. Signal. 2003, 15, 813−827.
(7) Gessi, S.; Merighi, S.; Sacchetto, V.; Simioni, C.; Borea, P. A.
Adenosine receptors and cancer. Biochim. Biophys. Acta 2011, 1808,
1400−1412.
(8) 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 affect cell cycle
progression and cell growth. Naunyn-Schmiedeberg’s Arch. Pharmacol.
2000, 361, 225−234.
(9) Gessi, S.; Merighi, S.; Varani, K.; Leung, E.; Mac Lennan, S.;
Borea, P. A. The A3 adenosine receptor: an enigmatic player in cell
biology. Pharmacol. Ther. 2008, 117, 123−140.
ASSOCIATED CONTENT
* Supporting Information
Conformational analysis data of the 7-ureido-pyrazolo[4,3-
d]pyrimidine derivatives 31−34, ADME properties and
combustion analysis data of the newly synthesized pyrazolopyr-
imidine derivatives. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel +39 055 4573731 (V.C.); +39 049 8275704 (S.M.); fax
+39 055 4573780 (V.C.), +39 049 8275366 (S.M.); e-mail:
vittoria.colotta@unifi.it (V.C.); stefano.moro@unipd.it (S.M.).
■
(10) Boison, D.; Chen, J.-F.; Fredholm, B. B. Adenosine signaling
and function in glial cells. Cell Death Differ. 2010, 17, 1071−1082.
(11) Liu, W.; Tang, Y.; Feung, J. Cross talk between activation of
microglia and astrocytes in pathological conditions in the central
nervous system. Life Sci. 2011, 89, 141−146.
Notes
The authors declare no competing financial interest.
L
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(12) Chen, Z.; Janes, K.; Chen, C.; Doyle, T.; Bryant, L.; Tosh, D. K.;
Jacobson, K. A.; Salvemini, D. Controlling murine and rat chronic pain
through A3 adenosine receptor activation. FASEB J. 2012, 26, 1855−
1865.
(13) Pedata, F.; Pugliese, A. M.; Coppi, E.; Popoli, P.; Morelli, M.;
Schwarzschild, M. A.; Melani, A. Adenosine in the central nervous
system: effects on neurotransmission and neuroprotection. Immun.,
Endoc. Metab. Agents Med. Chem. 2007, 7, 304−321.
(14) Taliani, S.; Bellandi, M.; La Motta, C.; Da Settimo, F. A3
receptor ligands: past, present and future trends. Curr. Top. Med.
Chem. 2010, 10, 942−975.
(15) Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filacchioni, G.;
Martini, C.; Trincavelli, L.; Lucacchini, A. 1,2,4-Triazolo[4,3-a]-
quinoxalin-1-one: a versatile tool for the synthesis of potent and
selective adenosine receptor antagonists. J. Med. Chem. 2000, 43,
1158−1164.
(16) Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filacchioni, G.;
Martini, C.; Trincavelli, L.; Lucacchini, A. Synthesis and structure-
activity relationships of a new sets of 2-arylpyrazolo[3,4-c]quinoline
derivatives as adenosine receptor antagonists. J. Med. Chem. 2000, 43,
3118−3124.
(17) Colotta, V.; Catarzi, D.; Varano, F.; Calabri, F. R.; Lenzi, O.;
Filacchioni, G.; Trincavelli, L.; Martini, C.; Deflorian, F.; Moro, S.
1,2,4-Triazolo[4,3-a]quinoxalin-1-one moiety as an attractive scaffold
to develop new potent and selective human A3 adenosine receptor
antagonists: synthesis, pharmacological and ligand-receptor modeling
studies. J. Med. Chem. 2004, 47, 3580−3590.
(18) Catarzi, D.; Colotta, V.; Varano, F.; Lenzi, O.; Filacchioni, G.;
Trincavelli, L.; Martini, C.; Montopoli, C.; Moro, S. 1,2,4-Triazolo[1,5-
a]quinoxaline as a versatile tool for the design of selective human A3
adenosine receptor antagonists: synthesis, biological evaluation and
molecular modeling studies of 2-(hetero)aryl- and 2-carboxy-
substituted derivatives. J. Med. Chem. 2005, 48, 7932−7945.
(19) Lenzi, O.; Colotta, V.; Catarzi, D.; Varano, F.; Filacchioni, G.;
Martini, C.; Trincavelli, L.; Ciampi, O.; Marighetti, F.; Morizzo, E.;
Moro, S. 4-Amido-2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-ones as new
potent and selective human A3 adenosine receptor antagonists.
Synthesis, pharmacological evaluation and and ligand-receptor
modeling studies. J. Med. Chem. 2006, 49, 3916−3925.
(20) Colotta, V.; Catarzi, D.; Varano, F.; Capelli, F.; Lenzi, O.;
Filacchioni, G.; Martini, C.; Trincavelli, L.; Ciampi, O.; Pugliese, A.
M.; Pedata, F.; Schiesaro, A.; Morizzo, E.; Moro, S. New 2-
arylpyrazolo[3,4-c]quinoline derivatives as potent and selective
human A3 adenosine receptor antagonists: synthesis, pharmacological
evaluation, and ligand-receptor modeling studies. J. Med. Chem. 2007,
50, 4061−4074.
(21) Morizzo, E.; Capelli, F.; Lenzi, O.; Catarzi, D.; Varano, F.;
Filacchioni, G.; Vincenzi, F.; Varani, K.; Borea, P. A.; Colotta, V.;
Moro, S. Scouting human A3 adenosine receptor antagonist binding
mode using a molecular simplification approach: from triazoloquinoxa-
line to a pyrimidine skeleton as a key study. J. Med. Chem. 2007, 50,
6596−6606.
(22) Colotta, V.; Catarzi, D.; Varano, F.; Lenzi, O.; Filacchioni, G.;
Martini, C.; Trincavelli, L.; Ciampi, O.; Traini, C.; Pugliese, A. M.;
Pedata, F.; Morizzo, E.; Moro, S. Synthesis, ligand-receptor modeling
studies and pharmacological evaluation of novel 4-modified-2-aryl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives as potent and selec-
tive human A3 Adenosine Receptor Antagonists. Bioorg. Med. Chem.
2008, 16, 6086−6102.
(23) Colotta, V.; Capelli, F.; Lenzi, O.; Catarzi, D.; Varano, F.; Poli,
D.; Vincenzi, F.; Varani, K.; Borea, P. A.; Dal Ben, D.; Volpini, R.;
Cristalli, G.; Filacchioni, G. Novel potent and highly selective human
A3 adenosine receptor antagonists belonging to the 4-amido-2-
arylpyrazolo[3,4-c]quinoline series: molecular docking analysis and
pharmacological studies. Bioorg. Med. Chem. 2009, 17, 401−410.
(24) Colotta, V.; Lenzi, O.; Catarzi, D.; Varano, F.; Filacchioni, G.;
Martini, C.; Trincavelli, L.; Ciampi, O.; Pugliese, A. M.; Traini, C.;
Pedata, F.; Schiesaro, A.; Morizzo, E.; Moro, S. Pyrido[2,3-e]-1,2,4-
triazolo[4,3-a]pyrazin-1-one as a new scaffold to develop potent and
selective human A3 adenosine receptor antagonists. Pharmacological
evaluation, and ligand-receptor modeling studies. J. Med. Chem. 2009,
52, 2407−2419.
(25) 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 Scaffold to obtain Potent and Selective Human A3 Adenosine
Receptor Antagonists: New Insights into the Receptor-Antagonist
Recognition. J. Med. Chem. 2009, 52, 7640−7652.
(26) Poli, D.; Catarzi, D.; Colotta, V.; Varano, F.; Filacchioni, G.;
Daniele, S.; Trincavelli, L.; Martini, C.; Paoletta, S.; Moro, S.. The
Identification of the 2-Phenylphthalazin-1(2H)-one Scaffold as a New
Decorable Core Skeleton for the Design of Potent and Selective
Human A3 Adenosine Receptor Antagonists. J. Med. Chem. 2011, 54,
2102−2113.
(27) Lenzi, O.; Colotta, V.; Catarzi, D.; Varano, F.; Squarcialupi, L.;
Filacchioni, G.; Varani, K.; Vincenzi, F.; Borea, P. A.; Dal Ben, D.;
Lambertucci, C.; Cristalli, G. Synthesis, structure-affinity relationships
and molecular modeling studies of novel pyrazolo[3,4-c]quinoline
derivatives as adenosine receptor antagonists. Bioorg. Med. Chem.
2011, 19, 3757−3768.
(28) McElvain, S. M.; Stevens, C. L. Ketene Acetals. XVI.
Phenylketene Diethyl- and Dimethylacetals from the Pyrolysis of the
Corresponding Orthoesters. J. Am. Chem. Soc. 1946, 68, 1917−1921.
(29) 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.
(30) 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.
(31) 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.
(32) Galabov, A.; Galabov, B.; Neykova, N. Structure-Activity
Relationship of Diphenylthiourea Antivirals. J. Med. Chem. 1980, 23,
1048.
(33) Aschner, M.; Sonnewald, U.; Tan, K. H. Astrocyte modulation
of neurotoxic injury. Brain Pathol. 2002, 12, 475−481.
(34) Marchand, F.; Perretti, M.; McMahon, S. B. Role of the immune
system in chronic pain. Nat. Rev. Neurosci. 2005, 6, 521−532.
(35) Scholz, J.; Woolf, C. J. The neuropathic pain triad: neurons,
immune cells and glia. Nat. Neurosci. 2007, 10, 1361−1368.
(36) Gamelin, E.; Gamelin, L.; Bossi, L.; Quasthoff, S. Clinical
aspects and molecular basis of oxaliplatin neurotoxicity: current
management and development of preventive measures. Semin. Oncol.
2002, 29, 21−33.
(37) Di Cesare Mannelli, L.; Zanardelli, M.; Failli, P.; Ghelardini, C.
Oxaliplatin-induced neuropathy: oxidative stress as pathological
mechanism. Protective effect of silibinin. J. Pain 2012, 13, 276−284.
(38) Elekes, O.; Venema, K.; Postema, F.; Dringen, R.; Hamprecht,
B.; Korf, J. Evidence that stress activates glial lactate formation in vivo
assessed with rat hippocampus lactography. Neurosci. Lett. 1996, 208,
69−72.
(39) Milligan, E. D.; Watkins, L. R. Pathological and protective roles
of glia in chronic pain. Nat. Rev. Neurosci. 2009, 10, 23−36.
̀
(40) Cavaletti, G.; Tredici, G.; Petruccioli, M. G.; Donde, E.; Tredici,
P.; Marmiroli, P.; Minoia, C.; Ronchi, A.; Bayssas, M.; Erienne, G. G.
Effects of different schedules of oxaliplatin treatment on the peripheral
nervous system of the rat. Eur. J. Cancer 2001, 37, 2457−2463.
(41) MOE (Molecular Operating Environment), version 2010.10;
Chemical Computing Group Inc.: Montreal, Quebec, Canada, 2010.
(42) Ballesteros, J. A.; Weinstein, H. Integrated methods for the
construction of three dimensional models and computational probing
of structure-function relationships in G-protein coupled receptors.
Methods Neurosci. 1995, 25, 366−428.
M
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(43) GOLD suite, version 5.1; Cambridge Crystallographic Data
Centre: Cambridge.
(44) Stewart, J. J. P. MOPAC 7; Fujitsu Limited: Tokyo, Japan, 1993.
(45) StarDrop, version 5.3; Optibrium Ltd: Cambridge, UK.
(46) Borea, P. A.; Dalpiaz, A.; Varani, K.; Gessi, S.; Gilli, G. Binding
thermodynamics at A1 and A2A adenosine receptors. Life Sci. 1996, 59,
1373−88.
(47) Varani, K.; Rigamonti, D.; Sipione, S.; Camurri, A.; Borea, P. A.;
Cattabeni, F.; Abbracchio, M. P.; Cattaneo, E. Aberrant amplification
of A2A receptor signalling in striatal cells expressing mutant huntigtin.
FASEB J. 2001, 5, 1245−1247.
(48) Varani, K.; Cacciari, B.; Baraldi, P. G.; Dionisotti, S.; Ongini, E.;
Borea, P. A. Binding affinity of adenosine receptor agonists and
antagonists at human cloned A3 adenosine receptors. Life Sci. 1998, 63,
81−87.
(49) Varani, K.; Gessi, S.; Merighi, S.; Vincenzi, F.; Cattabriga, E.;
Benini, A.; Klotz, K.-N.; Baraldi, P. G.; Tabrizi, M.-A.; Mac Lennan, S.;
Leung, E.; Borea, P. A. Pharmacological characterization of novel
adenosine ligands in recombinant and native human A2B receptors.
Biochem. Pharmacol. 2005, 70, 1601−1612.
(50) Bradford, M. M. A rapid and sensitive method for the
quantification of microgram quantities of protein utilizing the principle
of protein dye-binding. Anal. Biochem. 1976, 72, 248−254.
(51) Cheng, Y. C.; Prusoff, W. H. Relationships between the
inhibition constant (Ki) and the concentration of inhibitor which
causes 50% inhibition (IC50) of an enzymatic reaction. Biochem.
Pharmacol. 1973, 22, 3099−3108.
(52) Munson, P. J.; Rodbard, D. Ligand: a versatile computerized
approach for the characterization of ligand binding systems. Anal.
Biochem. 1980, 107, 220−239.
(53) McCarthy, K. D. de Vellis, J. Preparation of separate astroglial
and oligodendroglial cell cultures from rat cerebral tissue. J. Cell. Biol.
1980, 85, 890−902.
N
dx.doi.org/10.1021/jm400068e | J. Med. Chem. XXXX, XXX, XXX−XXX