Exploring the 2- and 5-positions of the pyrazolo[4,3-d]pyrimidin-7-amino scaffold to target human A1 and A2A adenosine receptors

Article abstract of DOI:10.1016/j.bmc.2016.04.048

A new series of 7-aminopyrazolo[4,3-d]pyrimidine derivatives (1-31) were synthesized to evaluate some structural modifications at the 2- and 5-positions aimed at shifting affinity towards the human (h) A_2A adenosine receptor (AR) or both hA_2A and hA₁ ARs. The most active compounds were those featured by a 2-furyl or 5-methylfuran-2-yl moiety at position 5, combined with a benzyl or a substituted-benzyl group at position 2. Several of these derivatives (22-31) displayed nanomolar affinity for the hA_2A AR (K_i = 3.62-57 nM) and slightly lower for the hA₁ ARs, thus showing different degrees (3-22 fold) of hA_2A versus hA₁ selectivity. In particular, the 2-(2-methoxybenzyl)-5-(5-methylfuran-2-yl) derivative 25 possessed the highest hA_2A and hA₁ AR affinities (K_i = 3.62 nM and 18 nM, respectively) and behaved as potent antagonist at both these receptors (cAMP assays). Its 2-(2-hydroxybenzyl) analog 26 also showed a high affinity for the hA_2A AR (K_i = 5.26 nM) and was 22-fold selective versus the hA₁ subtype. Molecular docking investigations performed at the hA_2A AR crystal structure and at a homology model of the hA₁ AR allowed us to represent the hypothetical binding mode of our derivatives and to rationalize the observed SARs.

Full text of DOI:10.1016/j.bmc.2016.04.048

Accepted Manuscript
Exploring the 2- and 5-positions of the pyrazolo[4,3-d]pyrimidin-7-amino scaf-
fold to target human A and A adenosine receptors
1 2A
Lucia Squarcialupi, Matteo Falsini, Daniela Catarzi, Flavia Varano, Marco
Betti, Katia Varani, Fabrizio Vincenzi, Diego Dal Ben, Catia Lambertucci,
Rosaria Volpini, Vittoria Colotta
PII:
S0968-0896(16)30291-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.048
BMC 12963
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
14 January 2016
20 April 2016
23 April 2016
Please cite this article as: Squarcialupi, L., Falsini, M., Catarzi, D., Varano, F., Betti, M., Varani, K., Vincenzi, F.,
Ben, D.D., Lambertucci, C., Volpini, R., Colotta, V., Exploring the 2- and 5-positions of the pyrazolo[4,3-
d]pyrimidin-7-amino scaffold to target human A and A adenosine receptors, Bioorganic & Medicinal
1
2A
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.04.048
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Exploring the 2- and 5-positions of the pyrazolo[4,3-
d]pyrimidin-7-amino scaffold to target human A and A
1
2A
adenosine receptors
a
a
a
a
a
b
Lucia Squarcialupi , Matteo Falsini , Daniela Catarzi , Flavia Varano , Marco Betti , Katia Varani ,
b
c
c
c
a*
Fabrizio Vincenzi , Diego Dal Ben , Catia Lambertucci , Rosaria Volpini , Vittoria Colotta
a
Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e
Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy.
b
Dipartimento di Scienze Mediche, Sezione di Farmacologia, Università degli Studi di Ferrara, Via Fossato di Mortara
1
7-19, 44121 Ferrara, Italy.
c
Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università degli Studi di Camerino, via S.Agostino 1, 62032
Camerino (MC), Italy.
Key words: G protein-coupled receptors, A and A2A adenosine receptor antagonists, pyrazolo[4,3-
1
d]pyrimidines, ligand-adenosine receptor modeling studies.
Corresponding authors. e-mail: vittoria.colotta@unifi.it.
1
Abbreviations: AR, adenosine receptor; PD, Parkinson’s disease; CCPA, 2-chloro-N(6)-
cyclopentyladenosine; NECA, 5’-(N-ethyl-carboxamido)adenosine; cAMP, cyclic adenosine
monophosphate; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)5’-(N-methylcarbamoyl)adenosine
DPCPX, 8-cyclopentyl-1,3-dipropyl-xanthine; ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]-
6
triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol; I-AB-MECA, N -(4-amino-3-iodobenzyl)-5’-
(
N-methylcarbamoyl)adenosine; EL2, second extracellular loop; MOE, molecular operating
environment
1

Abstract
A new series of 7-aminopyrazolo[4,3-d]pyrimidine derivatives (1-31) were synthesized to evaluate
some structural modifications at the 2- and 5- positions aimed at shifting affinity towards the human
(
h) A2A adenosine receptor (AR) or both hA2A and hA ARs. The most active compounds were
1
those featured by a 2-furyl or 5-methylfuran-2-yl moiety at position 5, combined with a benzyl or a
substituted-benzyl group at position 2. Several of these derivatives (22-31) displayed nanomolar
affinity for the hA2A AR (K
different degrees (3-22 fold) of hA2A versus hA
-(5-methylfuran-2-yl) derivative 25 possessed the highest hA2A and hA
nM and 18 nM, respectively) and behaved as potent antagonist at both these receptors (cAMP
assays). Its 2-(2-hydroxybenzyl) analog 26 also showed a high affinity for the hA2A AR (K = 5.26
nM) and was 22-fold selective versus the hA subtype. Molecular docking investigations performed
at the hA2A AR crystal structure and at a homology model of the hA AR allowed us to represent
the hypothetical binding mode of our derivatives and to rationalize the observed SARs.
i
= 3.62-57 nM) and slightly lower for the hA
selectivity. In particular, the 2-(2-methoxybenzyl)-
AR affinities (K = 3.62
1
ARs, thus showing
1
5
1
i
i
1
1
2

1
. Introduction
The neuromodulator adenosine elicits its biological effects through the activation of G-protein-
1
,2
coupled receptors, classified as A
typically coupled to adenylyl cyclase which can be inhibited (A
2B), but other intracellular pathways can be modulated, depending on the cell type and on the
contingent situation.
and A2A ARs are coupled to mitogen-activated protein kinases (MAPK), K-ATP channel and
phospholipase C. A receptor is the most widely distributed subtype with the highest level in the
brain, in particular in the hippocampus and prefrontal cortex, which are areas implicated in the
1
, A2A, A2B and A
3
subtypes. Adenosine receptors (ARs) are
1
and A ) or activated (A2A and
3
A
A
1
1
3
-4
control of emotions and cognition functions.
A
1
AR is expressed with intermediate density in
AR antagonists are sought as
1
,2
peripheral organs such as heart and kidney. Coherently, A
1
3
-5
therapeutic agents for mental dysfunction, such as dementia and anxiety and they have also been
5
-6
shown to be protectant in models of renal ischemia-reperfusion injury and vasoconstriction
although cardiac disorders have been reported as their common adverse events. A2A AR subtype is
extensively distributed in different organs, including heart, liver and lung, as well as in the brain
where the higher density is in the striatum, nucleus acumens, cortex and hippocampus. A2A AR
blockade in the brain elicits protective effect in both chronic and acute neurodegenerative diseases,
7
,8
9-11
such as cerebral ischemia and Parkinson’s disease (PD)
respectively. Related to PD, several
clinical trials have been conducted with A2A AR antagonists which proved to be effective in
counteracting extrapyramidal symptoms, and, recently, istradefylline has been approved for
1
2
marketing in Japan. Preclinical studies have also shown that PD can benefit from the use of dual
/A2A antagonists because they reduce both motor (A2A) and cognitive (A ) deficits associated to
A
1
1
5
,13-15
the disease.
Very recent research has demonstrated the effect of the A2A AR blockade on enhancing
immunologic response, highlighting that A2A receptor antagonists have the potential to markedly
improve anti-tumor immunity in mouse models and to promote tumor regression. Accordingly, A2A
3

AR antagonists have been shown to enhance the effect of tumor vaccines during T cell activation,
hence they might work in concert with other immune checkpoint inhibitors in cancer
1
6-17
immunotherapy.
In our laboratory, much research has been addressed to the study of AR antagonists belonging to
1
8-27
25-27
different classes
including the 2-arylpyrazolo[4,3-d]pyrimidine-7-amino series
(Figure 1).
Figure 1. Previously reported pyrazolo[4,3-d]pyrimidine derivatives A-D.
A recent structure-activity relationship (SAR) study on this series has highlighted that the presence
of a methyl and a phenyl group at the 5-position (R ) of the pyrazolo[4,3-d]pyrimidine (PP) scaffold
Figure 1, compounds A and B respectively) affords good affinity for hA
whereas a benzyl and, even better, a 3-phenylpropyl chain (Figure 1, derivatives C and D,
5
2
5
(
1
, hA2A and hA2B ARs,
2
6
respectively) elicit an enhancement of the A2A AR affinity and selectivity. Moreover, introduction
of a methoxy/hydroxy group at the ortho, meta or para positions of the appended 3-phenylpropyl
2
6
moiety of D completely reversed selectivity, affording potent and selective A
Hence, we decided to further investigate this series with the aim of obtaining more potent
antagonists for the hA2A AR or balanced antagonists for hA and hA2A ARs. Thus, we performed
1
AR antagonists.
1
some modifications on the previously reported compounds A-D (compounds 1-31, Figure 2) by
replacing the 2-phenyl residue with aryl groups or other substituents with different lipophilicity and
steric hindrance (methyl, benzyl, arylmethyl, phenethyl). Moreover, the effect of some heteroaryl
moieties at the 5-position of the bicyclic scaffold was evaluated.
4

Figure 2. Herein reported pyrazolo[4,3-d]pyrimidine derivatives 1-31.
2
Chemistry
The herein described pyrazolo[4,3-d]pyrimidin-7-amines 1-31 were prepared as depicted in
Schemes 1-3. The pyrazolopyrimidines 1-14, bearing an aryl moiety at the 2-position were obtained
as shown in Scheme 1.
2
8
Allowing N,N-dimethyl-2-nitroetheneamine to react with the suitable N
1
-arylhydrazono-N -
2
2
9-30
27
chloroacetates 32-33
and 34 in chloroform, the ethyl 4-nitropyrazole-3-carboxylates 35, 36
2
7
and 37 were obtained. These compounds were transformed into the corresponding amides 38, 39
and 40 by reaction with 33% aqueous ammonia solution. Treatment of the 3-carboxamides 38-40
2
7
with phosphorous oxychloride under microwave irradiation gave the 4-nitro derivatives 41, 42 and
3 which were reduced with cyclohexene or hydrogen, in the presence of Pd/C, to provide the
4
2
7
corresponding 4-amino derivatives 44, 45 and 46. These compounds were cyclized by treatment
with ammonium acetate and triethyl orthoacetate or the suitably synthetized ethyl iminoesters
hydrochlorides [31-34] to yield the pyrazolo[4,3-d]pyrimidin-7-amine derivatives 1-3, 5, 7, 8, 10,
1
1 and 13. The 2-(2-methoxyphenyl)-derivatives 3, 5, 8, 11 and 13 were demethylated with boron
tribromide to obtain the corresponding 2-(2-hydroxyphenyl)-derivatives 4, 6, 9, 12, 14.
5

R
3
3
3
2,35,38,41,44 H
3,36,39,42,45 2-F
4,37,40,43,46 2-OMe
R
Me
(CH
(CH
(CH
CH
CH
5
R
F
F
R
5
R
1
2
3
4
5
6
7
8
9
2-furyl
2-furyl
2-(5-methylfuryl)
2-(5-methylfuryl)
2-(5-methylfuryl)
2-thienyl
OMe
OH
H
OMe
OH
OMe
OH
2
2
2
)
)
)
Ph
Ph
3
3
3
Ph
Ph
Ph
OMe 10
OH
11
OMe 12
OH
H
2
2
13
14
2-furyl
2-thienyl
.
Scheme 1. (a) NEt
3
, CHCl3, mw
,
140 °C; (b) 33% aqueous NH
or R -C(OEt)NH hydrochloride, NH
, CH Cl , reflux.
3
, r.t.; (c) POCl
3
, mw, 150 °C; (d)
cyclohexene, Pd/C, mw, 110-130 °C; (e) Me-C(OEt)
sealed tube or mw, 90-150 °C; (f) R= OMe, BBr
3
5
4
OAc,
3
2
2
The pyrazolopyrimidines 15-23, bearing a methyl or an arylalkyl moiety at the 2-position, were
obtained as depicted in Scheme 2.
The synthetic pathway started from the 4-nitropyrazole-3-carbonitrile 47 which was regioselectively
alkylated with methyl iodide and benzylbromide in the presence of sodium hydride, in anhydrous
tetrahydrofuran, to give the previously reported 4-nitropyrazole-3-carbonitrile derivatives 48 and
2
7
4
9. Alkylation of 47 with phenethylbromide, in the same conditions, afforded the 1-phenethyl-4-
6

nitropyrazole-3-carbonitrile 50 as the major isomer, along with the 1-phenethyl-3-nitropyrazole-5-
1
carbonitrile 50a (the molar ratio between the two isomers 50 and 50a was about 6:1 from the H-
NMR spectrum of the crude material). The structure of compound 50 was determined by means of
NOESY experiments, which showed a spatial closeness of the pyrazole hydrogen atom to
methylene protons.
R
5
R
2
1
1
1
1
1
2
2
2
2
5
6
7
8
9
0
1
2
3
CH
(CH
Me
2
Ph
Ph
Me
Me
CH
2
)
3
2
Ph
Me
(CH
2
)
2
Ph
CH
2
Ph
CH
CH
2
Ph
Ph
2-furyl
2-furyl
2-furyl
2-furyl
2
(CH
2
)
C
C
2
6
6
Ph
CH
CH
2
H
H
4
-2-OMe
-2-OH
2
4
Scheme 2. (a) CH
3
I or Ph-(CH
2
)
n
Br, NaH, THF, room temperature (48, 49) or reflux (50); (b) H
or R -C(OEt)NH
OAc, mw at 130 °C or, for 22, sealed tube at 120°C; (e) 22, BBr , CH Cl
2
,
Pd/C, Parr apparatus, 30 psi; (c) cyclohexene, Pd/C, mw, 150 °C; (d) Me-C(OEt)
hydrochloride, NH
reflux.
3
5
4
3
2
2
,
7

The 4-nitropyrazole derivatives 48-50 were transformed into the corresponding 4-aminopyrazoles
2
7
27
by reduction with hydrogen (51) or cyclohexene (52 and 53), in the presence of Pd/C. Treatment
3
1-33
of 51-53 with triethyl orthoacetate or the suitable imminoester hydrochlorides
and ammonium
acetate yielded the pyrazolopyrimidines 15-22. Demethylation of the 2-(2-methoxybenzyl)-
derivative 22 with boron tribromide afforded the respective 2-(2-hydroxybenzyl) derivative 23. To
prepare the set of pyrazolopyrimidine 24-31, bearing the (5-methyl-furan-2-yl)- moiety at the 5-
position, a new synthetic pathway was developed (Scheme 3).
R
2
2
2
2
2
2
3
3
4
5
6
7
8
9
0
1
H
2-OMe
3-OMe
2-F
3-F
2-Cl
2-OH
3-OH
Scheme 3. (a) H
2
, Pd/C, Parr apparatus, 35 psi; (b) NH
4
OAc, sealed tube, 120 °C; (c) Ar-CH
Cl , r.t. or reflux.
2
-Cl,
K
2
CO , CH CN/DMF, r.t. or 60 °C; (d) 25 or 26, BBr
3
3
3
, CH
2
2
3
5
The 4-aminopyrazole-3-carbonitrile 54, obtained from the corresponding 4-nitro derivative 47,
was reacted with ammonium acetate and ethyl 5-methylfuran-2-carboximidate hydrochloride 55 to
provide the 7-amino-5-(5-methyl-furan-2-yl)-2H-pyrazolo[4,3-d]pyrimidine 56. This compound
was transformed into the pyrazolopyrimidines 24-29 by regioselective alkylation with suitable
benzyl chlorides, all commercially available, except the 2-methoxybenzyl chloride which was
36
prepared following a reported procedure. Alkylation was carried out in a mixture of
8

dimethylformamide/acetonitrile, in the presence of potassium carbonate. The 2-substituted structure
of 24-29 was determined by means of NOESY experiments showing a spatial proximity between
the pyrazole hydrogen atom and the methylene protons.
Finally, the methoxy-derivatives 25 and 26 were demethylated with boron tribromide to give the
respective hydroxy-substituted compounds 30 and 31.
3
3
. Results and Discussion
.1. Structure-affinity relationship studies
The synthesized compounds 1-31 were tested in binding assays to evaluate their affinity at cloned
hA , hA2A and hA ARs, stably expressed in CHO cells. The new compounds were also tested at the
1
3
hA2B receptor by measuring their inhibitory effects on NECA-stimulated cAMP levels in CHO
cells. The results of binding experiments and cAMP assays are reported in Table 1, where the data
of compounds A-D are also included as references.
The reported results indicate that our aim of obtaining pyrazolopyrimidine derivatives endowed
with high affinity for the hA2A receptor or both hA and hA2A receptors has been achieved. The
1
most interesting findings were found within the set of compounds 20-31 in which a benzyl or a
substituted benzyl group at the 2-position was combined with a 2-furyl or a 2-(5-methylfuryl)
moiety at position 5. Indeed, most of these derivatives (22-31) bind to the hA2A AR with a
nanomolar affinity and different degrees of selectivity (3-22 fold) versus the hA
1
ARs. Moreover,
they showed good to moderate affinities for A and hA2B subtypes.
3
The first set of synthesized derivatives (1-6) were designed as analogues of compounds A, C-D
which were modified by introduction of hydrogen bond acceptor groups (F, OMe, OH) on the ortho
position of the 2-phenyl ring. This choice was based on the results of docking studies at the hA2A
crystal structure previously performed on the pyrazolopyrimidine series, including derivatives A-D
2
5,26
(
Figure 1).
In the best docking poses, these derivatives were anchored inside the binding cleft by
a tight hydrogen bond network with the highly conserved residues Asn253 (TM6) and E172/169
9

(
EL2). In particular, a hydrogen bond between the NH amide moiety of Asn253 (TM6) and the N1-
2
pyrazole atom has been evidenced. Hence, the hypothesis that a hydrogen bond acceptor group at
the ortho position of the 2-phenyl ring might reinforce the anchoring of the molecule at the level of
Asn253 (TM6) residue prompted us to synthesize compounds 1-6. Actually, these modifications
caused, on the whole, a reduction of the affinity for the hA2A receptor (compare 1 to A; 2 and 3 to
C; 5 and 6 to D) and for the other hAR subtypes.
Table 1. Binding Affinity (K
i
) at hA
1
, hA2A and hA
3
ARs and potencies (IC50) at hA2B ARs.
a
cAMP assays
Binding experiments
IC50(nM) or
I%
K
i
(nM) or I%
b
c
d
e
R
5
R
2
hA
1
hA2A
hA
3
hA2B
f
f
f
A
B
C
Me
Ph
PhCH
Ph(CH
Me
PhCH
PhCH
Ph-(CH
Ph-(CH
Ph-(CH
2-furyl
2-furyl
2-furyl
Ph
Ph
Ph
Ph
70 ±6
75 ± 7
150 ±12
5.3±04
410 ±42
25%
246 ± 23
325 ± 34
110 ±10
55±5
23%
43%
647 ±62
240±22
34%
40%
48%
39%
12%
8%
163±15
6%
5%
405 ±38
19%
320±35
440 ±43
420 ±38
42%
8%
1%
21%
5%
1%
1%
1%
12%
42%
32%
1%
1%
1%
2
g
D
2
)
3
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
C
6
H
4
-2-F
2
2
2
2
2
C
6
H
4
-2-OMe
C
6
H
4
-2-OH
-2-F
-2-OMe
-2-OH
49±4
)
)
)
3
3
3
C
6
H
4
17 ± 2
103 ± 9
52 ± 4
206 ±17
31%
372 ± 33
42 ± 3
1%
1%
24%
159 ± 16
9%
C
6
H
4
C
6
H
4
21%
Ph
-2-OMe
195±14
362 ± 34
89 ± 8
99 ± 8
518 ± 47
147 ± 13
27%
39%
37%
36%
C
6
H
4
C
6
H
4
-2-OH
Ph
1
2-(5-methylfuryl)
2-(5-methylfuryl)
2-(5-methylfuryl)
2-thienyl
147±12
297 ± 26
375 ± 32
150±13
209±18
1%
1
1
1
1
1
1
1
C
6
H
4
-2-OMe
C
6
H
4
-2-OH
C
6
H
4
-2-OMe
2-thienyl
PhCH
Ph(CH
Me
C
6
H
4
-2-OH
Me
Me
CH Ph
412±39
1%
28%
20%
2%
2
2
)
3
36%
1%
1%
1%
30%
1%
2
1%
10

18
19
20
21
22
23
24
25
26
27
28
29
30
31
Me
(CH
CH
CH
2
)
2
Ph
10%
1%
32%
12%
98±8
242±21
136±12
18±2
120±11
309±8
252±21
60±5
12%
1%
320 ± 28
33%
5.37±0.39
17±2
9.23 ±0.85 269 ± 25
3.62±0.34
5.26±0.47
57 ± 6
51 ± 5
6.21±0.58
38 ±4
3%
15%
2%
4%
196±17
905±88
3%
1%
37%
CH
2
Ph
2
2
Ph
Ph
2-furyl
2-furyl
2-furyl
2-furyl
(CH
-C
-C
CH
-C
-C
-C
-C
CH -C H -2-F
2
)
2
Ph
3%
CH
CH
2
6
H
4
-2-OMe
512±49
112±11
20%
2
6
H
4
-2-OH
Ph
-2-OMe
-2-OH
-3-OMe
-3-OH
2-(5-methylfuryl)
2-(5-methylfuryl)
2-(5-methylfuryl)
2-(5-methylfuryl)
2-(5-methylfuryl)
2-(5-methylfuryl)
2-(5-methylfuryl)
2-(5-methylfuryl)
2
CH
CH
CH
CH
2
6
H
4
82 ± 7
88±6
498 ± 47
427 ± 41
71±6
30%
2
6
H
4
293±26
354 ± 32
421 ± 39
152±14
395 ± 38
633±52
2
6
H
4
2
6
H
4
2
6
4
CH -C H -3-F
2
6
4
111±10
65±5
122 ± 11
225±21
CH -C H -2-Cl
2
6
4
14±2
a
K values are means ± SEM of four separate assays each performed in duplicate. Percentage of
i
b
inhibition (I%) are determined at 1µM concentration of the tested compounds. Displacement of
specific [ H]DPCPX competition binding assays to hA
[
3
c
1
CHO cells. Displacement of specific
3
d
125
H]ZM241385 competition binding to hA2ACHO cells. Displacement of specific [ I]AB-MECA
e
competition binding to hA
3
CHO cells. cAMP experiments in hA2BCHO cells, stimulated by 200
nM NECA. Percentage of inhibition (I%) are determined at 1µM concentration of the tested
f
g
compounds. Ref. 25. Ref. 26.
Replacement of the 5-phenyl ring of B with a 2-furyl (compound 7) was more profitable because it
achieved a shift of affinity, albeit small, towards the A2A subtype. In fact, with respect to B,
compound 7 bind slightly better the hA2A receptor (K
of the 2-(5-methyl)furyl group at the 5-position further enhanced the hA2A affinity (K
not the selectivity, compound 10 showing high and good affinity, respectively, for the hA
nM) and hA (Ki= 147 nM) subtypes. In light of these findings, compounds 7 and 10 were modified
by introduction of a 2-methoxy or 2-hydroxy group on the 2-phenyl ring. All the resulting
compounds (8, 9 and 11, 12) showed decreased affinity for the hA2A AR and even more for the hA
subtype. There was only one exception: the 2-(2-hydroxyphenyl) derivative 9 (R = 2-furyl) which
was two-fold more active than 7 at the hA2A AR subtype (K = 89 nM). A selective hA AR ligand,
instead, ensued from the combination of the 2-methoxyphenyl group at position 5 with a 2-thienyl
i
= 195 nM) and worse the hA
1
one. Insertion
= 99 nM) but
(K = 42
i
1
i
3
1
5
i
3
11

residue at the 2-position (derivative 13), thus confirming that the 2-thienyl group is very profitable
2
7
for the anchoring to the hA AR, unlike the 2-furyl that shifts affinity towards the hA2A AR.
3
A dramatic reduction of affinity was obtained when the 2-phenyl ring of compounds C and D was
replaced with the smaller and less lipophilic methyl group (derivatives 15 and 16) or when the 2-
phenyl ring of compound A was replaced with a benzyl (17) or phenethyl moiety (18). In fact,
compounds 15-18 are completely inactive at both the target hA2A and hA receptors and also at the
1
other hAR subtypes. The same applies to derivative 19, characterized by the presence of a benzyl
chain at both the 2- and 5-positions.
An interesting finding was achieved when the benzyl group at the 2-position was combined with the
2
-furyl ring at the 5-position of the pyrazolopyrimidine scaffold to provide derivative 20. With
respect to the corresponding 2-phenyl derivative 7, compound 20 showed higher hA2A AR
selectivity, even if its hA2A AR affinity is slightly lower (K = 320 nM). Elongation of the 2-benzyl
chain of 20 to 2-phenethyl dropped hA2A AR affinity (compound 21), while replacement of the 5-
2-furyl) ring of 20 with the 5-(5-methylfuran-2-yl)- substituent (compound 24) significantly
improved it. Derivative 24, in fact, binds the hA2A receptor with high affinity (K = 9.23 nM) and
some selectivity versus both hA (15-fold) and hA (29-fold) subtypes, thus confirming the
i
(
i
1
3
favorable role of the 5-(5-methylfuran-2-yl)- substituent for the hA2A AR-ligand interaction (see
above, 10 versus 7).
Based on these results, both compounds 20 and 24 were modified by introduction of a methoxy or
hydroxy residue at the ortho position on the benzyl group. All the resulting pyrazolopyrimidines 22,
2
3 (R
nM < K
Moreover, the 5-(methylfuran-2-yl)- derivative 26 exhibited the highest hA2A versus hA
5
= 2-furyl) and 25, 26 (R
< 17 nM), being improved with respect to those of the corresponding 2-benzyl derivatives.
AR
5
= 5-methylfuran-2-yl) possessed very high hA2A AR affinities (3.6
i
1
selectivity. Hence, we sought to synthesize new compounds featured by the 5-(5-methylfuryl)
residue combined with other substituted benzyl groups at position 2 (27-31). Insertion of a methoxy
and hydroxy substituent at the meta position of the benzyl moiety afforded derivatives 27 and 28,
12

whose hA2A affinities were still in the low nanomolar range (K = 57 and 51 nM, respectively),
i
although decreased with respect to those of the ortho substituted derivatives 25 and 26. A similar
trend of hA2A affinity was found for compounds 29 and 30, bearing the fluorine atom as hydrogen
bond acceptor, respectively, at the ortho and meta position of the benzyl group. In fact, the ortho-
fluorobenzyl derivative 29 displayed a higher hA2A affinity (K
= 38 nM). Finally, insertion of a chlorine atom at the ortho position on the benzyl moiety also
afforded high hA2A affinity (K = 14 nM).
In summary, the structural investigation carried out at the 5- and 2- position of pyrazolopyrimidine
scaffold highlighted that high affinity for the hA2A AR or for both hA2A and hA ARs can be
i
= 6.21 nM) than the meta-isomer 30
(
K
i
i
1
achieved through the 5-(5-methylfuran-2-yl)- and 5-(2-furyl) substitution in combination with a 2-
benzyl or 2-benzyl-substituted moiety.
Some selected derivatives (22, 24-26) were tested to assess their antagonistic potencies at hA
1
and
6
hA2A ARs. Thus, their capability to reduce the inhibitory effect of 2-chloro-N -
cyclopentyladenosine (CCPA) in cAMP production on hA
1
CHO cells and to inhibit NECA-
stimulated cAMP production on hA2A CHO cells, were evaluated (Table 2).
Table 2. Antagonistic potencies of selected compounds at hA
1
and hA2A ARs.
a
cAMP assay IC50 (nM)
hA
1
hA2A
2
2
2
2
2
4
5
6
106 ± 10
152 ± 14
21 ± 2
6.21 ± 0.57
10.91 ± 1.07
4.35 ± 0.42
6.42 ± 0.61
134 ± 12
a
cAMP experiments: the compounds were tested in hA CHO or hA2ACHO cells in the presence of
1
CCPA (1 nM) or NECA (10 nM), respectively. IC50 values are expressed as means ± SEM of four
separate experiments.
13

The tested compounds behaved as antagonists at both hA
correlated to their affinities. Among them, compounds 25 proved to be the most potent dual A2A and
AR antagonist, although it was able to bind also to the hA AR.
1
and hA2A ARs with potencies well-
A
1
3
3
.2 Molecular modeling studies
A molecular modeling study was performed to simulate and analyse the interaction of the
synthesised compounds with the binding site of the human A2A AR. For this analysis, the crystal
structure of the same protein solved in complex with the well-known antagonist ZM241385 (Protein
3
7,38
Data Bank -pdb- code: 3EML; 2.6-Å resolution
) was employed. The crystal structure was re-
modelled by firstly removing the external T4L segment and secondly by performing a building of
missing receptor regions (the missing section of the extracellular 2 -EL2- or intracellular 3 -IL3-
domains). The obtained A2A AR model was checked by inspection of backbone bond lengths,
angles and dihedrals, Ramachandran φ-ψ dihedral plots, and sidechain rotamer and nonbonded
contact quality. These steps were performed within Molecular Operating Environment (MOE,
3
9
version 2010.10) suite. The A2A AR structure was then used as target for the docking analysis of
4
0-42
the synthesised derivatives by using Autodock 4.2.6 software and PyRx interface.
results were then imported into MOE for post-docking energy minimization and analysis. A set of
derivatives was also subjected to molecular docking analysis at a homology model of the human A
Docking
1
AR built by using the above cited A2A AR crystal structure as a template.
The pyrazolopyrimidine scaffold shows structural similarity to the triazolotriazine one of the co-
crystallized reference compound ZM241385. The docking results for the synthesized molecules
showed two possible binding modes, one of which (binding mode 1, Figure 3A) being comparable
to the one observed for ZM241385 at the A2A AR crystal structure (pdb code: 3EML) and similar
2
6
also to the binding mode of other pyrazolopyrimidine derivatives previously reported. In details,
the bicyclic scaffold is vertically oriented and positioned between residues of the EL2 and
6
.51
43
transmembrane (TM) 6 domains (Phe168 and Leu249 , respectively ), with the 7-amino group
14

6
.55
and the N1 nitrogen atom providing a double H-bond interaction with the Asn253 residue. The 2-
substituent is oriented towards the receptor core and is located between residues of TM3, TM5, and
3
.33
3.36
5.38
5.42
6.48
6.51
6.52
TM6 segments (Leu85 , Thr88 , Met177 , Asn181 , Trp246 , Leu249 , and His250 ),
while the 5-substituent points toward the extracellular space and is located between TM1, TM2,
1
.35
2.61
2.64
7.35
7.36
EL2, EL3, and TM7 residues (Tyr9 , Ala63 , Ile66 , Leu167, Leu267, Met270 , Tyr271
,
7
.39
and Ile274 ).
Figure 3. Docking conformations at the A2A AR. Panel A. Binding mode 1 conformation (see text
for details) of compound C. Key receptor residues and domains are indicated. Panel B. Binding
mode 2 conformation of compound 3.
In the second binding mode (binding mode 2, Figure 3B) the derivatives were located in an
analogue position but were oppositely oriented considering the 2- and 5- substituents. The double
1
5

6
.55
H-bond interaction with the Asn253
residue is given by the 7-amino group and the N6 nitrogen
atom of the pyrazolopyrimidine moiety. In general, the derivatives bearing an arylalkyl substituent
at the 2-position and an aryl group at the 5-position, or vice versa, are positioned with the more
flexible arylalkyl substituent oriented towards the extracellular space. In this way, the docking
6
.55
poses allow the above cited interaction with the Asn253
residue, while those with the arylalkyl
substituent in the depth of the binding pocket partially lose such interaction with the receptor and
are associated to a significantly lower docking score. Exceptions are compounds 2, 3, 5, 6, bearing a
hydroxy- or methoxy-substituted phenyl ring as 2-substituent. For these compounds, docking results
showed the 2-substituent as externally oriented and the phenylalkyl group at the 5-position as
located in the depth of the cavity. This binding mode still allows the 5-benzyl substituted
6
.55
derivatives 2 and 3 to partially interact with Asn253
and Glu169 (Figure 3B), providing a fair
affinity at least for compound 3. In the case of compounds 5 and 6 (inactive at the A2A AR), the
longer 5-substituent prevents the scaffold from properly interacting with the two above cited
receptor residues.
The conformations predicted for compounds 20-31, bearing a 2-furyl or a 2-(5-methyl)furyl group
at the 5-position and an arylalkyl substituent at the 2-position, are generally belonging to the
“binding mode 2” arrangement (see details of docking results for compound 25, Figure 4).
16

Figure 4. Docking conformation of compound 25 at the A2A AR binding site. Panel A. Global view
of the ligand-receptor interaction, with indication of key receptor residues. Panels B-C. Detail of the
interaction between the receptor and the 2- and 5-substituents of 25, respectively.
Interestingly, the superimposition of the top-score docking conformations of this set of molecules
presents the bicyclic scaffold as occupying an almost identical position, hence not being influenced
by the replacement of the 2-furyl ring with a 2-(5-methyl)furyl group. We performed a post-docking
analysis of the interactions between the compounds and the receptor binding site by using the IF-E
4
4
6
.0 tool that is retrievable at the SVL exchange service (Chemical Computing Group, Inc. SVL
exchange: http://svl.chemcomp.com). This tool was previously employed for other analyses at
2
4,45,46
ARs.
The script calculates and displays atomic and residue interaction forces as 3D vectors
and calculates the per-residue interaction energies (values in kcal/mol), where negative and positive
energy values are associated to favorable and unfavorable interactions, respectively. In this study,
the analysis was focused on the interaction between the 5-substituent (2-furyl or 2-(5-methyl)furyl
group) and the residues located in its proximity. For this task we employed four derivatives bearing
17

the same substituted benzyl group at the 2-position and a 2-furyl (22, 23) or 2-(5-methyl)furyl group
25, 26) at the 5-position. The results of this analysis are reported in Table 3.
(
Table 3. Interaction energies (values in kcal/mol) between compounds 22, 23, 25, 26 and 8, 9, 11,
2 and the binding site residues located in proximity of the 5-position of the analyzed compounds.
1
See text for details.
res
cpd
2
2
25
23
26
8
11
9
12
Leu85
Thr88
-0.51
0.07
-0.46
-0.51
-2.28
-1.05
-1.59
-2.37
-1.47
-5.88
-15.61
0.16
-0,36
-0,71
-2,27
-0,89
-1,73
-2,01
-0,80
-5,21
-13,98
-0,71
-1,03
-1,19
-0,97
-0,01
-1,05
-0,81
-5,17
-10,92
-0,44
-0,50
-2,45
-0,64
-1,85
-2,72
-1,48
-5,42
-15,49
-0,78
-0,71
-1,97
-0,90
0,31
-0.35
-1.91
-1.29
-1.73
-1.19
-1.52
-6.39
-14.88
-1.02
-2.13
-1.09
-1.44
-1.63
-1.79
-6.49
-15.51
-1.10
-2.54
-0.97
-2.04
-2.15
-1.64
-6.24
-16.52
Met177
Asn181
Trp246
Leu249
His250
Asn253
tot
-3,30
-0,73
-6,04
-14,12
The two derivatives bearing at the 5-position a 2-(5-methyl)furyl group present a slightly better
interaction with the binding site residues (located in proximity of the 5-position) respect to the
corresponding 2-furyl substituted compounds. This is probably due to the mainly hydrophobic
properties of the receptor residues involved in the interaction with this part of the molecules. In this
sense, the presence of an additional methyl group could lead to a more favorable interaction with
respect to the unsubstituted furan ring. It should be taken into account also that the presence of the
additional methyl group within the 5-substituent may lead to a higher occupancy of the TM3-TM5-
TM6 sub pocket and hence to a higher topological complementarity between the compounds and
binding site (Figure 5A). These factors could help to depict the slightly higher affinity of the
analyzed 2-benzyl-5-(5-methyl-2-furyl) substituted compounds with respect to the corresponding 5-
(
2-furyl) substituted analogues.
18

Figure 5. Comparison of docking conformations. Panel A. Superimposition of docking
conformations of compounds 22 (light) and 25 (dark). Panel B. Superimposition of docking
conformations of compounds 8 (light) and 11 (dark).
Derivatives bearing aryl substituents at both the 2- and 5- positions (7-14) show both the binding
modes, as similarly scored by the docking tool. For all these compounds the top score conformation
always presents the 2-furyl or the 2-(5-methyl)furyl oriented toward the receptor core and the 2-
phenyl ring as externally located. Moreover, comparison of the binding modes of derivatives
bearing the same 2-substituent and a 2-furyl or a 2-(5-methyl)furyl group at the 5-position suggests
that the presence of a methyl group on the furan ring causes a slight rearrangement of the compound
conformation. This can be observed in Figure 5B where superimposition of the docking
conformations of compounds 8 and 11 is reported.
19

Differently from compounds 20-31, in which the flexible 2-substituent allows the compounds to
adapt their conformation to the binding pocket, in compounds 7-14 the 2-aryl substituents, being
directly bound to the scaffold, does not permit this fitting. Hence, the presence of a methyl group on
the 5-(2-furyl) residue can be a further hinder to the receptor-ligand accommodation, thus resulting
generally detrimental for the compound affinity. Also in this case we performed the analysis of
ligand-receptor interaction by using the above cited IF-E 6.0 tool and by considering for
comparison the compounds 8, 9, 11, and 12. The results are reported in Table 3. Both derivatives 11
and 12, substituted at the 5-position with a 2-(5-methyl)furyl group, give weaker interactions with
the binding site residues, located in proximity of the 5-position, with respect to the corresponding 2-
furyl substituted compounds 8 and 9. In addition, rearrangement of the conformation (Figure 5B)
could lead to an analogue occupancy of the TM3-TM5-TM6 binding sub-pocket but also to a partial
6
.55
loss of the interaction with Asn253 . These results seem partially in accordance with the binding
data of this set of derivatives, with the exception of compound 10 where the effect on the 5-
substituent appears to be different when compared with 7. Considering the 2-substituent, binding
data highlighted the higher affinity of the benzyl-substituted compounds (20, 22-31) with respect to
the phenyl-substituted analogues (1-14). As previously discussed, the more flexible benzyl
substituents allow the molecules to get more deeply inserted inside the binding pocket. Regarding
the substituent on the 2-benzyl or 2-phenyl moieties, we observed that the hydroxyl group at the 2-
position of the aromatic ring generally engages an internal H-bond interaction with the N1 atom,
while the methoxy group in the same position probably gives hydrophobic interaction with residues
1
.35
2.61
2.64
located at the entrance of the binding cavity (i.e. Tyr9 , Ala63 , Ile66 , Leu167, Leu267,
7
.35
7.36
7.39
Met270 , Tyr271 , and Ile274 , see Figure 4B). Docking results do not provide a clear
explanation about the ten-fold decreased affinity of compounds bearing hydroxyl or methoxy group
at the meta-position of the benzyl moiety with respect to the corresponding ortho-substituted
derivatives (compare 27 and 28 with 25 and 26, see Table 1). At the basis of the diverse biological
behavior there could be a possible different polar interaction between the ortho- and the meta-
20

substituted compounds with the backbone atoms of Leu167-Phe169 residues and also a different
solvation/desolvation effect of these groups that are exposed toward the external environment.
A set of derivatives (20-31) was also subjected to molecular docking analysis at a homology model
of the hA
homology modeling and docking protocols were the same as the ones used for the refinement of the
EML hA2A AR crystal structure and the docking at the same receptor (see above). Docking results
at the hA AR present some similarities to those obtained at the hA2A AR. The arrangement of the
1
AR built by using the above cited 3EML hA2A AR crystal structure as a template. The
3
1
derivatives bearing a 2-furyl or a 2-(5-methyl)furyl group at the 5-position and a benzyl or
substituted-benzyl group at position 2 is very similar at the two receptors (Figure 4 and 6) and also
the role of the methyl group on the furyl moiety appears analogue. Hence, it is no particularly
surprising that these compounds show affinity also for the hA AR as the depth of the two binding
1
cavities presents a high degree of conservation. Though, a series of differences among the two
receptors may be observed at the entrance of the binding cavities where the position of some
7
.35
hydrophobic residues of the hA2A AR (Leu167 in EL2, Leu267 in EL3, and Met270 ) is occupied
7
.35
47
by polar amino acids in the hA
As this sub-region is occupied by the 2-arylmethyl moiety of the docked compounds, the different
chemical-physical profile of the residues in proximity (between hA2A AR and hA AR) may help to
explain the generally higher affinity of this set of ligands for the hA2A AR with respect to the hA
1
AR (Glu170 in EL2, Ser267 in EL3, and Thr270 , respectively).
1
1
AR. Furthermore, as observed at the hA2A AR, the different affinity of compounds bearing hydroxyl
or methoxyl group at the ortho- or meta-position of the 2-benzyl moiety could be related to the
diverse interaction with receptor residues. Docking results, in fact, suggest an interaction between
the ortho-substituted derivatives and the charged amino group of Lys265 (EL3) (Figure 6). Affinity
data show that the ortho-methoxy substituted derivatives (such as 25) are more active at the hA AR
1
than their corresponding hydroxy-substituted analogues (such as 26). This could be due to some
factors. Firstly, the presence of an H-bond acceptor feature at the ortho-position, such as the
methoxy (derivative 25) appears more suitable for the interaction with Lys265 than an hydroxy H-
21

bond donor group (derivative 26). Moreover, this latter substituent appears involved in an internal
H-bond with the N1 atom of the scaffold, hence not presenting an optimal orientation for a strong
interaction with Lys265. Instead, the methoxy group of 25, not establishing this intramolecular
bond, may assume the correct position to engage the H-bond with Lys265. Finally, the easier
desolvation of the methoxy-substituted derivatives with respect to the hydroxy-substituted ones
could be taken into account to explain the different receptor affinities.
Figure 6. Docking conformation of compounds 25 (A) and 27 (B) at the hA
1
AR binding site. Key
receptor residues and domains are indicated.
22

4
. Conclusion
In the study reported here we evaluated different substituents at the 5- and 2- position of the
pyrazolo[4,3-d]pyrimidine scaffold to shift affinity towards the hA2A AR or both hA2A and hA
1
ARs. Among the synthesized compounds, those featured by the 2-furyl or 5-methylfuran-2-yl
moiety at position 5, combined with a benzyl or benzyl-substituted group at position 2, resulted in
the most active. Several of these derivatives (22-31) displayed hA2A AR affinities in the low
nanomolar range (K
showing different degrees of A2A versus A
methoxybenzyl)-5-(5-methylfuran-2-yl) derivative 22 resulted in the most potent A2A and A
antagonist herein reported, both in binding and in functional assays. Its 2-(2-hydroxybenzyl) analog
6 also showed a high affinity for the A2A AR (K = 5.26 nM) and was 22-fold selective versus the
subtype. Molecular docking investigations performed at the hA2A AR crystal structure and at a
homology model of the A AR permitted us to represent the hypothetical binding mode of our
derivatives as well as to rationalize the observed SARs.
i
= 3.62-57 nM) and a little lower for the hA
selectivity (3-22 fold). In particular, the 2-(2-
AR
1
ARs (K
i
= 18-234 nM), thus
1
1
2
i
A
1
1
5
5
. Experimental section
.1. Chemistry
The microwave-assisted syntheses were performed using an Initiator EXP Microwave
Biotage instrument (frequency of irradiation: 2.45 GHz). Analytical silica gel plates (Merck F254),
preparative silica gel plates (Merck F254, 2 mm) and silica gel 60 (Merck, 70-230 mesh) were used
for analytical and preparative TLC, and for column chromatography, respectively. All melting
points were determined on a Gallenkamp melting point apparatus and are uncorrected. 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 purity not
less than 95%. The IR spectra were recorded with a Perkin-Elmer Spectrum RX I spectrometer in
23

-1
Nujol mulls and are expressed in cm . NMR spectra were recorded on a Bruker Avance 400
1
13
spectrometer (400 MHz for H NMR, 100 MHz for C NMR). The chemical shifts are reported in δ
ppm) and are relative to the central peak of the solvent which was CDCl or DMSOd . The
(
3
6
following abbreviations are used: s= singlet, d= doublet, t= triplet, q= quartet, m= multiplet, br=
broad and ar= aromatic protons.
1
2
5
.1.1. Synthesis of Ethyl N -2-methoxyphenylhydrazono-N -chloroacetate 34.
The title compound was synthesized following the procedure previously reported to prepare
2
8,29
compounds 32 and 33
methoxyaniline (53 mmol) in 6 N HCl (32 mL) was cooled at -5 – 0 °C and 1 M NaNO
53 mL) was added dropwise, keeping the temperature below 0 °C. The solution of the diazonium
from the corresponding arylamines. Briefly, a mixture of 2-
2
solution
(
salt was poured into a cold (0 °C) solution of ethyl-2-chloroacetoacetate (53 mmol) and sodium
acetate (54.2 mmol) in MeOH (200 mL), while stirring and keeping the temperature below 0 °C.
The mixture was then left in a refrigerator for 16 h. The solid was collected by filtration, washed
with water (200 mL) and dried.
1
Yield 62%; m.p.73-74 °C (cyclohexane); H NMR (CDCl
3
) 1.42 (t, 3H, CH , J = 7.1 Hz), 3.96 (s,
3
3
H, OCH
m, 1H, ar), 8.83 (s, 1H, NH). Anal. Calcd. for C11
C, 51,28; H 5.36; N, 11.22.
3
), 4.41 (q, 2H, CH
2
, J = 7.1 Hz), 6.90-6.92 (m, 1H, ar), 6.99-7.01 (m, 2H, ar), 7.55-7.58
(
H13ClN
2
O
3
: C, 51,47; H 5.10; N, 10.91. Found:
5
3
.1.2. General procedure for the synthesis of ethyl 1-aryl-4-nitro-1H-pyrazole-3-carboxylates
6 and 37.
2
1
Derivative 36 and 37 were synthesized following the procedure previously described for 35.
Briefly, a mixture of N,N-dimethyl-2-nitroethenamine (11 mmol), triethylamine (22 mmol) and the
suitable idrazone 33 or 34 in CHCl (5 mL) was reacted under microwave irradiation for 30 min at
40 °C. The solvent was eliminated at reduced pressure to give a black slurry. Derivative 36
3
1
24

solidified upon treatment with isopropyl alcohol (2 mL). The solid obtained was collected by
filtration, washed with water and recrystallized. To isolate compound 37, the black residue was first
purified by chromatography (eluent: cyclohexane/EtOAc, 3:1) and then the compound was
recrystallized.
Ethyl 1-(2-fluorophenyl)-4-nitro-1H-pyrazole-3-carboxylate 36. Yield 23%; m.p. 112-114 °C
1
(
(
EtOH); H NMR (CDCl
3
) 1.45 (t, 3H, CH
3
, J = 7.1 Hz), 4.52 (q, 2H, CH J = 7.1 Hz), 7.31-7.37
2
m, 2H, ar), 7.44-7.50 (m, 1H, ar), 7.95 (t, 1H, ar, J = 8.4 Hz), 8.72 (s, 1H, H-5). Anal. Calcd. for
: C, 51.62; H, 3.61; N, 15.05. Found: C, 51.44; H, 3.85; N, 15.27.
C
12
H
10FN
3
O
4
Ethyl 1-(2-methoxyphenyl)-4-nitro-1H-pyrazole-3-carboxylate 37. Yield 36%; m.p. 68-69°C
1
(
cyclohexane); H NMR (CDCl
3
) 1.45 (t, 3H, CH
3
, J = 7.1 Hz), 3.97 (s, 3H, OCH ), 4.52 (q, 2H,
3
CH J = 7.1 Hz), 7.10-7.14 (m, 2H, ar), 7.44 (t, 1H, ar, J = 7.9 Hz), 7.82 (d, 1H, ar, J = 7.9), 8.79 (s,
2
1
1
H, H-3). Anal. Calcd. for C13
4.60.
H
13
N
3
5
O : C, 53.61; H, 4.50; N, 14.43. Found: C, 53.46; H, 4.36; N,
5
.1.3. General procedure for the synthesis of 1-aryl-4-nitro-1H-pyrazole-3-carboxamides 39
and 40
25
Derivatives 39 and 40 were prepared following the same procedure employed to prepare 38.
Briefly, a suspension of ethyl pyrazole-3-carboxylates 36 or 37 (3.0 mmol) in 33% aqueous
ammonia solution (40 mL) was stirred at room temperature for about 48 h. The solid was collected
by filtration, washed with water and recrystallized.
5
.1.3.1. 1-(2-Fluorophenyl)-4-nitro-1H-pyrazole-3-carboxamide 39. Yield 72%; m.p. 212-213
1
°
C (EtOH); H NMR (DMSO-d
6
) 7.41-7.48 (m, 1H, ar), 7.54-7.64 (m, 2H, ar), 7.79-7.84 (m, 1H,
FN : C,
ar), 7.94 (br s, 1H, NH), 8.21 (br s, 1H, NH), 9.34 (s, 1H, H-5). Anal. Calcd. for C10
H
7
4
O
4
4
8.01; H, 2.82; N, 22.39. Found: C, 48.18; H, 2.64; N, 22.45.
5
.1.3.2. 1-(2-Methoxyphenyl)-4-nitro-1H-pyrazole-3-carboxamide 40. Yield 88%; m .p. 200-202
1
°
C (EtOAc/MeOH); H NMR (DMSO-d
6
) 3.90 (s, 3H, OCH ), 7.15 (t, 1H, ar J = 7.8 Hz), 7.33 (d,
3
25

1
H, ar, J = 8.4 Hz), 7.51 (t, 1H, ar J = 7.5 Hz), 7.64 (d, 1H, ar, J = 8.0 Hz), 7.85 (br s, 1H, NH), 8.14
(
br s, 1H, NH), 9.16 (s, 1H, H-5). Anal. Calcd. for C11 : C, 50.38; H, 3.84; N, 21.37. Found:
H
10
N
4
O
4
C, 50.64; H, 3.75; N, 21.25.
5
4
.1.4. General procedure for the synthesis of 1-aryl-4-nitro-pyrazole-3-carbonitrile derivatives
2-43
2
5
Compounds 42 and 43 were synthesized as previously described to prepare 41 from 38. Briefly, a
suspension of the suitable amide 39 or 40 (2 mmol) in phosphorus oxychloride (5 mL) was
microwave irradiated at 150 °C for 10-20 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.
5
.1.4.1. 1-(2-Fluorophenyl)-4-nitro-1H-pyrazole-3-carbonitrile 42. Yield 96%; mp 108−110 °C
1
(
H
2
O/EtOH); H NMR (CDCl
3
) 7.36-7.43 (m, 2H, ar), 7.49-7.58 (m, 1H, ar), 7.93 (t, 1H, ar, J = 7.9
Hz), 8.81 (s, 1H, H-5). Anal. Calcd. for C10
H, 2.09; N, 24.32.
H
5
FN
4
2
O : C, 51.73; H, 2.17; N, 24.13. Found C, 51.58;
5
.1.4.2. 1-(2-Methoxyphenyl)-4-nitro-1H-pyrazole-3-carbonitrile 43. Yield 89%; mp 134−136
1
°
C (cyclohexane/EtOAc); H NMR (CDCl
H, ar, J = 7.8 Hz), 7.79 (d, 1H, ar, J = 7.9 Hz), 8.89 (s, 1H, H-5). IR 2251, 1538, 1342. Anal.
Calcd. for C11 : C, 54.10; H, 3.30; N, 22.94. Found C, 54.21; H, 3.18; N, 22.78.
3
) 4.00 (s, 3H, OMe), 7.17 (t, 2H, ar, J = 8.4 Hz), 7.51 (t,
1
H
8
N
4
O
3
5
.1.5. General procedure for the synthesis of 4-amino-1-aryl-1H-pyrazole-3-carbonitriles 45
and 46.
2
5
Compounds 45 and 46 were prepared as previously described for derivative 44. Briefly, a mixture
of 4-nitropyrazoles 42 or 43 (2 mmol), cyclohexene (8 mmol) and 10% Pd/C (15% w/w with
respect to the nitropyrazole) in EtOH (10 mL) was microwave irradiated, respectively, at 110 °C for
1
0 min and 130°C for 30 min. After cooling at room temperature, the catalyst was filtered off and
26

the solvent was evaporated at reduced pressure to give a solid which was recrystallized from
suitable solvent.
5
.1.5.1. 4-Amino-1-(2-fluorophenyl)-1H-pyrazole-3-carbonitrile 45. Yield 55%; m.p. 106−107
1
°
C (H
2
O/EtOH). H NMR (DMSO-d
6
) 5.05 (br s, 2H, NH
2
), 7.35-7.39 (m, 1H, ar), 7.48-7.51 (m,
FN : C, 59.40; H, 3.49;
2
H, ar), 7.67 (s, 1H, H-5), 7.77 (t, 1H, ar, J = 8.3 Hz). Anal. Calcd. for C10
H
7
4
N, 27.71. Found C, 59.58; H, 3.63; N, 27.61.
5
.1.5.2. 4-Amino-1-(2-methoxyphenyl)pyrazole-3-carbonitrile 46. Yield 80%; m.p. 108−109 °C
1
(
(
Light petroleum ether/Et
2
O). H NMR (CDCl
3
) 3.49 (br s, 2H, NH
2
), 3.91 (s, 3H, OMe), 7.06-7.10
O:
m, 2H, ar), 7.38 (t, 1H, ar, J = 7.5 Hz), 7.66-7.68 (m, 2H, 1 ar + H-5). Anal. Calcd. for C11
H
10
N
4
C, 61.67; H, 4.71; N, 26.15. Found C, 61.48; H, 4.55; N, 26.30.
5
.1.6. General procedure for the synthesis of 2-arylpyrazolo[4,3-d]pyrimidin-7-amine
derivatives 1-3, 5, 7, 8, 10, 11, 13.
A mixture of the 4-aminopyrazole-3-carbonitrile derivatives 44-46 (0.84 mmol), anhydrous
ammonium acetate (1.64 mmol), and triethyl orthoacetate (1.5 mmol) or ethyl imidates
3
1
32
33
34
hydrochlorides (1.64 mmol) (R
5
= CH
2
Ph, (CH
2
) Ph, 2-furyl, 2-thienyl, 5-methyl-furan-2-yl
3
5
5 (see 5.1.15) was heated in a sealed tube or under microwave irradiation in the conditions
described below for each compound. The obtained mixture was cooled at room temperature and
treated with EtOH (0.5 mL) and diethyl ether (1-3 mL). The resulting solid was collected by
filtration and washed, in sequence, with water (10-15 mL), NaHCO saturated solution (1-2 mL)
3
and water (5-10 mL), then dried and recrystallized. The crude compounds 5 and 7 were purified by
chromatography (see below for details), then the latter was also recrystallized.
5
.1.6.1. 7-amino-2-(2-fluorophenyl)-5-methyl-2H-pyrazolo[4,3-d]pyrimidine 1. The title
compound was obtained by heating the reaction mixture under microwave irradiation at 130 °C for
1
1
7
5 min. Yield 85%; m.p. 212-214 °C (cyclohexane/EtOAc); H NMR (DMSO-d
6
) 2.37 (s, 3H, Me),
.45 (t, 1H ar, J = 6.8 Hz), 7.54-7.62 (m, 2H, ar), 7.68 (br s, 2H, NH
2
), 7.89 (t, 1H ar, J = 7.8 Hz),
27

8
4
5
.61 (s, 1H, H-3). Anal. Calcd. for C12
.27; N, 28.58.
H
10FN : C, 59.25; H, 4.14; N, 28.79. Found C, 59.09; H,
5
.1.6.2. 7-Amino-2-(2-fluorophenyl)-5-(3-phenylpropyl)-2H-pyrazolo[4,3-d]pyrimidine 2. The
title compound was obtained by heating the reaction mixture under microwave irradiation at 150 °C
1
for 10-15 min. Yield 63%; m.p. 188-189 °C (cyclohexane/EtOAc); H NMR (DMSO-d
6
) 2.01-2.06
(
(
m, 2H, CH
m, 2H, ar), 7.67 (br s, 2H, NH
: C, 69.15; H, 5.22; N, 20.16. Found C, 69.01; H, 5.08; N, 20.30.
.1.6.3. 7-Amino-2-(2-methoxyphenyl)-5-(3-phenylpropyl)-2H-pyrazolo[4,3-d]pyrimidine 3.
2
), 2.63-2.66 (m, 4H, CH
2
), 7.16-7.31 (m, 5H, ar), 7.45 (t, 1H, ar, J = 6.9 Hz), 7.54-7.59
2
), 7.89 (t, 1H, ar, J = 7.7 Hz), 8.65 (s, 1H, H-3). Anal. Calcd. for
C
20
H18FN
5
5
The title compound was obtained by heating the reaction mixture in sealed tube at 120 °C for 2 h.
1
Yield 40%; m.p. 199-200 °C (cyclohexane/EtOAc); H NMR (DMSO-d
6
) 2.01-2.05 (m, 2H, CH ),
2
2
7
1
1
.63-2.65 (m, 4H, 2CH
2
), 3.87 (s, 3H, OMe), 7.20-7.33 (m, 7H, ar), 7.50-7.54 (m, 3H, 1 ar + NH
2
),
1
3
.68 (d, 1H, ar, J= 7.8 Hz), 8.50 (s, 1H, H-3). C NMR (DMSO-d
6
) 30.36, 35.38, 38.87, 56.53,
13.44, 121.16, 125.38, 126.16, 126.93, 128.69, 128.86, 129.71, 130.71, 131.33, 139.81, 142.70,
52.65, 156.26, 164.60. Anal. Calcd. for C21 O: C, 70.17; H, 5.89; N, 19.48. Found C, 70.32;
H
21
N
5
H, 5.65; N, 19.35.
.1.6.4. 7-Amino-2-(2-methoxyphenyl)-5-benzyl-2H-pyrazolo[4,3-d]pyrimidine 5. The title
5
compound was obtained by heating the reaction mixture in sealed tube at 120 °C for 5h. The crude
compound was purified by preparative TLC (eluent cyclohexane/EtOAc/MeOH, 6:4:1). Yield 65%;
1
m.p. 141-142 °C; H NMR (DMSO-d
6
) 3.86 (s, 3H, OMe), 3.96 (s, 2H, CH
2
), 7.13-7.18 (m, 2H, ar),
), 8.54 (s, 1H, H-3).
O: C, 68.87; H, 5.17; N, 21.13. Found C, 68.59; H, 4.95; N, 21.02.
.1.6.5. 7-Amino-5-(2-furyl)-2-phenyl-2H-pyrazolo[4,3-d]pyrimidine 7. The title compound was
7
.20-7.30 (m, 5H, ar), 7.52 (t, 1H, J= 7.4 Hz), 7.66-7.68 (m, 3H, 1ar + NH
Anal. Calcd. for C19
2
H
17
N
5
5
obtained by heating the reaction mixture under microwave irradiation at 130 °C for 10 min. The
crude derivative was purified by column chromatography (eluent cyclohexane/EtOAc 6:4). Yield
1
4
8%; m.p. 208-210 °C (EtOAc); H NMR (DMSO-d
6
) 6.62-6.63 (m, 1H, furan proton), 7.07-7.08
28

(
m, 1H, furan proton), 7.50 (t, 1H, ar, J = 9.0 Hz), 7.63 (t, 2H, ar, J= 9.0 Hz), 7.80 (s, 1H, furan
proton), 7.84 (br s, 2H, NH ), 8.08 (d, 2H, ar, J = 9.0 Hz), 9.06 (s, 1H, H-3). IR 3335, 3150, 1591.
Anal. Calcd. for C15 O: C, 64.97; H, 4.00; N, 25.26. Found C, 64.75; H, 3.88; N, 25.01.
.1.6.6. 7-Amino-5-(2-furyl)-2-(2-methoxyphenyl)-2H-pyrazolo[4,3-d]pyrimidine 8. The title
2
H
11
N
5
5
compound was obtained by heating the reaction mixture at 130 °C for 15 min in a sealed tube. Yield
1
3
8%; m.p. 295-296 °C (MeNO
2
). H NMR (DMSO-d
6
) 3.89 (s, 3H, OMe), 6.61-6.62 (m, 1H, furan
proton), 7.06-7.07 (m, 1H, H furan proton), 7.17 (t, 1H, ar, J = 7.7 Hz), 7.33 (d, 1H, ar, J = 7.5 Hz),
7
8
4
5
.53 (t, 1H, ar, J = 8.3 Hz), 7.72 (d, 1H, ar, J = 7.8 Hz), 7.77-7.79 (m, 3H, NH
2
+ 1 H furan proton),
.64 (s, 1H, H-3). Anal. Calcd. for C16
.45; N, 22.65.
H
13
N
5
O
2
: C, 62.53; H, 4.26; N, 22.79. Found C, 62.42; H,
.1.6.7. 7-Amino-5-(5-methylfuran-2-yl)-2-phenyl-2H-pyrazolo[4,3-d]pyrimidine 10. The title
compound was obtained by heating the reaction mixture at 110 °C for 1 h in a sealed tube. Yield
1
3
8%; m.p. 257-258 °C (H
2
O/EtOH); H NMR (DMSO-d
6
) 2.37 (s, 3H, Me), 6.23-6.24 (m, 1H,
furan proton), 6.97-6.98 (m, 1H, furan proton), 7.49 (t, 1H, ar, J = 7.4 Hz), 7.62 (t, 1H, ar, J = 7.7
Hz), 7.79 (br s, 2H, NH ), 8.06 (d, 2H, ar, J = 7.8 Hz), 9.00 (s, 1H, H-3). Anal. Calcd. for
O; C, 65.97; H, 4.50; N, 24.04. Found C, 65.75; H, 4.72; N, 24.21.
2
C
16
H
13
N
5
5
1
.1.6.8. 7-Amino-5-(5-methylfuran-2-yl)-2-(2-methoxyphenyl)-2H-pyrazolo[4,3-d]pyrimidine
1. The title compound was obtained by heating the reaction mixture at 120 °C for 2 h in a sealed
1
tube. Yield 58%; m.p. 216-217 °C (cyclohexane/EtOAc); H NMR (DMSO-d
6
) 2.36 (s, 3H, Me),
3
.88 (s, 3H, OMe), 6.22-6.23 (m, 1H, furan proton), 6.95-6.96 (m, 1H, furan proton), 7.16 (t, 1H, ar,
J= 7.7 Hz), 7.33 (d, 1H, ar, J= 8.4 Hz), 7.53 (t, 1H, ar, J= 7.6 Hz), 7.71-7.73 (m, 3H, 1 ar + NH ),
: C, 63.54; H, 4.71; N, 21.79. Found C, 63.70; H,
2
8
4
5
.61 (s, 1H, H-3). Anal. Calcd. for C17
.65; N, 21.68.
H
15
N
15
O
2
.1.6.9. 7-Amino-2-(2-methoxyphenyl)-5-(2-thienyl)-2H-pyrazolo[4,3-d]pyrimidine 13. The title
compound was obtained by heating the reaction mixture at 110 °C for 2 h in a sealed tube. Yield
1
7
0%; m.p. 244-245 °C (MeNO
2
); H NMR (DMSO-d
6
) 3.89 (s, 3H, OMe), 7.13-7.19 (m, 2H, 1ar +
29