Pyrrolo- and pyrazolo-[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines as adenosine receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2011.11.037

The discovery and development of adenosine receptor antagonists have represented for years an attractive field of research from the perspective of identifying new drugs for the treatment of widespread disorders such as inflammation, asthma and Parkinson's

Full text of DOI:10.1016/j.bmc.2011.11.037

Bioorganic & Medicinal Chemistry 20 (2012) 1046–1059
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Bioorganic & Medicinal Chemistry
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Pyrrolo- and pyrazolo-[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines as adenosine
receptor antagonists
Pier Giovanni Baraldi a, , Giulia Saponaro a, Mojgan Aghazadeh Tabrizi a, Stefania Baraldi a,
⇑
Romeo Romagnoli a, Allan R. Moorman c, Katia Varani b, Pier Andrea Borea b, Delia Preti a
a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, 44100 Ferrara, Italy
b Dipartimento di Medicina, Clinica e Sperimentale-Sezione di Farmacologia, Università di Ferrara, 44100 Ferrara, Italy
c King Pharmaceuticals, Inc., Research & Development, 4000 CentreGreen Way, Suite 300, Cary, NC 27513, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and development of adenosine receptor antagonists have represented for years an attrac-
tive field of research from the perspective of identifying new drugs for the treatment of widespread dis-
orders such as inflammation, asthma and Parkinson’s disease. The present work can be considered as an
extension of our structure-activity relationship studies on the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrim-
idine (PTP) nucleus, extensively investigated by us as a useful template, in particular, for the identifica-
tion of A2A and A3 adenosine receptor antagonists. In order to explore the role of the nitrogen at the
7-position, we performed a new synthetic strategy for the preparation of pyrrolo[3,4-e][1,2,4]triazol-
o[1,5-c]pyrimidine derivatives which can be considered as 7-deaza analogues of the parent PTPs. We also
synthesised a novel series of pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines as junction isomers of the
reference compounds. In both cases we obtained some examples of potent antagonists (Ki in the low
nanomolar range) with variable selectivity profiles in relation to the nature of substituents introduced
at the C5-, N8- and/or N9-positions. The pyrrolo-triazolo-pyrimidine derivative 9b appeared to be a potent
A3 adenosine receptor antagonist (Ki = 10 nM) with good selectivity over hA1 (74-fold) and hA2A (20-fold)
adenosine receptors combined with low activity at the hA2B subtype (IC50 = 906 nM). Moreover, some
examples of high-affinity A1/A2A dual antagonists have been identified in both series. This work consti-
tutes a new and important contribution for the comprehension of the interaction between PTPs and
adenosine receptors.
Received 12 October 2011
Revised 17 November 2011
Accepted 18 November 2011
Available online 1 December 2011
Keywords:
Adenosine
Pyrazolo-triazolo-pyrimidines
Pyrrolo-triazolo-pyrimidines
Adenosine antagonists
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
asthma.4 The selective blockade of A2A AR proved to be effective
in clinical trials for the treatment of extrapyramidal neurodegener-
Adenosine is an ubiquitous nucleoside essential for the proper
functioning of every cell in mammalian species. It is directly linked
to energy metabolism through ATP (adenosine triphosphate), ADP
(adenosine diphosphate) and AMP (adenosine monophosphate),
while at the extracellular level it regulates a wide range of biolog-
ical functions through activation of specific receptors (adenosine
receptors, ARs)1–3 that belong to the superfamily of G-protein cou-
pled receptors (GPCRs) and are classified as A1, A2A, A2B and A3.
The discovery and development of AR antagonists have repre-
sented for years an attractive field of research from the perspective
of identifying new drugs for the treatment of widespread disor-
ders. A1 AR antagonists have been studied for the treatment of
heart failure with related renal impairment, cystic-fibrosis and
ative disorders such as Parkinson’s disease.5,6 A growing number of
A2B AR antagonists are under clinical evaluation in the therapeutic
area of respiratory disorders (asthma, COPD).7 Although very po-
tent and selective A3 AR antagonists have been reported in the last
decade, none of these molecules has yet entered human trials.
Nonetheless, it is becoming increasingly apparent that A3 AR
antagonists might be therapeutically useful for the acute treatment
of stroke and glaucoma,8 inflammation,9–11 and in the develop-
ment of cerebroprotective,12,13 antiasthmatic and antiallergic
drugs.14,15 Furthermore, the recent evidence16–20 of high levels of
expression of A3 ARs in several cell lines has suggested potential
applications for selective antagonists in cancer chemotherapy.
The pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP) nu-
cleus has distinguished itself as an attractive scaffold for obtaining
adenosine receptor antagonists based upon its strong structural
correlation with the non-selective AR antagonist CGS15943 (9-
chloro-2-(furan-2-yl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine). A
wide number of compounds originated from our structure-activity
⇑
Corresponding author. Address: Department of Pharmaceutical Sciences, Uni-
versity of Ferrara, Via Fossato di Mortara 17-19, 44121, Ferrara Italy. Tel./fax: +39
0532 455921.
E-mail addresses: baraldi@dns.unife.it, baraldi@unife.it (P.G. Baraldi).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.037

P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
1047
optimization work based on the systematic substitution of the C5-,
N7-, N8-, C2- or C9-positions of PTPs,21 which allowed us to delin-
eate a structure–activity relationship (SAR) profile mainly concern-
ing the effect of substitutions on subtype selectivity. The 2-furyl
ring at the 2-position of the nucleus appeared important for the
affinity toward all four AR subtypes. The combined presence of a
free amino group at the 5-position and an arylalkyl chain at the
N7-position of PTPs was found essential for both affinity and selec-
tivity at the A2A AR subtype. Two compounds of this family named
SCH-58261 (Fig. 1) and SCH-63390 (Fig. 1), bearing N7-arylalkyl
functions, proved to be potent and selective A2A AR antagonists
both in rat and human models.7,22 On the other hand, the concur-
rent presence of a 4-methoxy-phenylcarbamoyl moiety and small
alkyl chains (methyl or propyl) at the 5- and 8- positions, respec-
tively, led to the identification of highly potent and selective hu-
man A3 AR antagonists. Of this class, MRE-3008-F20 (Fig. 1),
displays a Ki value of 0.29 nM in binding assays to human A3 recep-
tors expressed in CHO cells with high selectivity over hA1 and hA2A
ARs (Ki = 1100 and 140 nM, respectively).23 The isosteric replace-
ment of the phenylcarbamoyl moiety with a salifiable 4-pyridylc-
arbamoyl moiety improved water solubility and led to the most
potent hA3 antagonist known to date (MRE-3005-F20, Fig. 1).7 Fi-
nally, the introduction of substituents at the C9-position led to
the identification of AR antagonists with low selectivity but high
potency.24
synthesised a
novel series of N8/N9-substituted-pyrazolo[3,4-
e][1,2,4]triazolo[1,5-c]pyrimidines as junction isomers of the refer-
ence compounds (Fig. 2, B and C).
Starting from the data obtained with the previous series of
PTPs,7 we introduced at the N8 or N9 positions small alkyl chains,
such as a methyl- or a propyl-, or arylalkyl chains, such as a
phenylethyl- or a phenylpropyl-function. These modifications al-
lowed us to further explore the contribution of this side of the
molecule to the interaction with adenosine receptors. In both
series we also studied the 5-position of the tricyclic structure,
introducing an amino group, a 4-methoxy-phenylcarbamoyl-, a
4-pyridylcarbamoyl moiety, a morpholine or an N-substituted
piperazine.
We obtained some examples of potent ARs antagonists (Ki val-
ues from binding assays in the low nanomolar range) with variable
selectivity profiles in relation to the nature of the substituents
introduced at the C5-, N8- and N9-positions.
2. Results and discussion
2.1. Chemistry
2.1.1. Pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines
As depicted in Scheme 1, 1,3-dibenzyl-1H-pyrrolo[3,4-d]pyrim-
idine-2,4(3H,6H)-dione 226 was alkylated with the appropriate al-
kyl halide to furnish 6-alkyl-derivatives 3a–d. Debenzylation at
the 1- and 3-positions with AlCl3 in anhydrous toluene provided
derivatives 4a–d. The 2,4-dichloro-6-alkyl-6H-pyrrolo[3,4-
d]pyrimidines 5a–d were obtained by treatment of 4a–d with
POCl3 and DBU. Selective substitution of the chlorine atom at the
4-position with furoic acid hydrazide followed by a Dimroth
rearrangement led to the desired pyrrolo[3,4-e][1,2,4]triazol-
o[1,5-c]pyrimidine nucleus (7a–d). Compounds 8a–d were ob-
tained by treating derivatives 7a–d with a saturated solution of
ammonia in ethanol. These were converted into the corresponding
The present work can be considered an extension of our SAR
studies on the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine nu-
cleus (general structure 1, Fig. 2), in which we focused our atten-
tion on the isosteric replacement of the pyrazole ring.
Specifically, in order to explore the role of the nitrogen at the 7-po-
sition, we performed a new synthetic strategy for the preparation
of pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives which
can be considered as 7-deaza-analogues of the classical PTPs
(Fig. 2A). A series of 8-deaza-PTPs has been claimed recently for
their potent and selective activity as A2A AR antagonists.25 We also
O
O
N
N
N
N
N
N
N
N
N
N
H2N
N
H2N
N
SCH-582617
SCH-633907
Ki (hA1) = 549 nM
Ki (hA2A) = 1.1 nM
Ki (hA2B) > 10000 nM
Ki (hA3) > 10000 nM
Ki (hA1) = 350 nM
Ki (hA2A) = 1.2 nM
Ki (hA2B) > 10000 nM
Ki (hA3) > 10000 nM
O
O
N
N
N
N
O
O
N
N
O
N
N
N
N
N
N
H
N
H
N
N
H
N
H
N
MRE-3008-F2023
MRE-3005-F2027
Ki (hA1) = 250 nM
Ki (hA2A) = 60 nM
Ki (hA2B) = 200 nM
Ki (hA3) = 0.04 nM
Ki (hA1) = 1100 nM
Ki (hA2A) = 140 nM
Ki (hA2B) = 2100 nM
Ki (hA3) = 0.29 nM
Figure 1. Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines as A2A and A3 adenosine receptor antagonists.

1048
P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
R
R'
O
R
H
CH3
N
H CO
3
O
O
N
CH2CH2CH3
CH2CH2Ph
N
N
H
N
R'
N
N
N
H
N
1
CH2CH2CH2Ph
N
H
A
B
C
O
R
O
O
N
N
N
R'
N
N
N
N
N
N
N
N
N R'
N
N R'
R
R
N
H
N
N
H
N
N
H
N
8 a-d, 9 a-d, 10
40-47 a
40-47 b
Figure 2. General structures of the newly designed molecules.
O
O
O
Cl
Bn
Bn
HN
N
O
ii
N
N
iii
i
N R
NH
N R
N R
O
N
H
4a-d
O
N
Bn
O
N
Bn
Cl
N
2
3a-d
5a-d
O
O
O
HN
N
NH
N
N
N
iv
v
vi
N
N
N
N R
N R
N R
H2N
N
8a-d
Cl
N
Cl
N
6a-d
7a-d
O
O
N
a = CH3
b = CH2CH2CH3
c = CH2CH2Ph
N
MeO
N
vii
N R
d = CH2CH2CH2Ph
N
N
H
N
H
9a-d
Scheme 1. Synthesis of pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines. Reagents and conditions: (i) K2CO3, RX, DMF, 40 °C, 4 h; (ii) AlCl3, toluene, rt, 2 h; (iii) POCl3, DBU,
50 °C, 8 h; (iv) 2-furoic acid hydrazide, TEA, 1,4-dioxane, 80–90 °C, 5 h; (v) HMDS, BSA, 120 °C, 18 h; (vi) EtOH satd ammonia soln, 60 °C, 18 h; (vii) 4-OCH3-phenyl isocyanate,
THF, 50 °C, 18 h.
4-methoxy-phenyl urea derivatives 9a–d by reaction with 4-meth-
oxy-phenylisocyanate.
O
O
Compound 10 was prepared according to a similar efficient
strategy previously reported27 and depicted in Scheme 2. Com-
pound 8a was heated for 5 h in dry toluene with 4-pyridyl isocya-
nate to give the desired urea derivative 10 (Scheme 2).
N
N
i
N
N
N
8a
N
H
N
H
N
2.1.2. Pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines
10
For the synthesis of these compounds the synthetic strategy de-
picted in Scheme 3 has been followed. 4-Nitro-1H-pyrazole-3-car-
boxylic acid amide 1128 was alkylated with the appropriate alkyl
halide in the presence of K2CO3 in DMF to give an approximately
Scheme 2. Synthesis of 5-{[(4-pyridinyl)-carbamoyl]amino}-2-(furan-2-yl) -8-
methyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine 10. Reagents and condi-
tions: (1) 4-pyridine isocyanate, toluene, 5 h, 100 °C.27

P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
1049
O
Cl
N
CONH2 O2N
CONH2
ii
O2N
H2N
CONH2
iii
R
N
R
N
N
HN
N
O
iv
Cl
i
N
N
N
N
N
N
H
N
O
N
H
R
R
11
12-15a,b
16-19a,b
20-23a,b
24-27a,b
v
O
O
O
HN
N
N
NH
N
N
N
vii
vi
R
N
R
R
N
N
N
N
N
N
N
H2N
N
Cl
N
Cl
N
40-43a,b
28-31a,b
32-35a,b
viii
ix
R
N
N
O
N
O
N
N R
N
N
a
b
N
MeO
R
N
N
N
O
N
N
N
N
H
N
H
N
R'
N
44-47a,b
36-39
Scheme 3. Synthesis of pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines. Reagents and conditions: (i) alkyl halide, K2CO3, DMF, rt, 6 h; (ii) H2, 10% Pd/C, EtOH; (iii) urea, neat,
250 °C; (iv) POCl3, DBU, 80 °C, 8 h; (v) 2-furoic acid hydrazide, TEA, 1,4-dioxane, 80–90 °C, 5 h; (vi) HMDS, BSA, 120 °C, 18 h; (vii) EtOH satd ammonia soln, 60 °C, 18 h; (viii) 4-
OCH3-phenylisocyanate, THF, 50 °C, 18 h; (ix) amines, 2-methoxyethanol, 100 °C, 3 h. R = CH3 (12, 16, 20, 24, 28, 32, 40, 44); R = (CH2)2CH3 (13, 17, 21, 25, 29, 33, 41, 45);
R = (CH2)2Ph (14, 18, 22, 26, 30, 34, 42, 46); R = (CH2)3Ph (15, 19, 23, 27, 31, 35, 43, 47).
1:1 mixture of the two isomers (a and b) which were efficiently
separated via column chromatography. The nitro group was then
reduced by hydrogenation in the presence of a catalytic amount
of 10% Pd/C and intermediates 16–19a,b were converted into the
2.2.1. Pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines
The pyrrolo-triazolo-pyrimidine derivatives 8a-d (Table 1)
bearing a free amino group at the 5- position were substituted at
the N8-position with a methyl-, propyl-, phenylethyl- and phenyl-
propyl- group, respectively, in accordance with the reference com-
pounds depicted in Figure 1. Both N8-methyl (8a) and N8-propyl
(8b) derivatives showed good Ki values for A2A AR (Ki = 20 and
30 nM, respectively) but they also bind to hA1/hA3 and block
hA2B subtype in the nanomolar range. The N8-phenylethyl 8c dis-
played high and comparable affinity toward the A1 and A2A recep-
tors (Ki = 4.3 and 3.9 nM, respectively) while a lower affinity/
efficacy has been detected at the remaining subtypes (IC50
hA2B = 46 and Ki hA3 = 124 nM). The elongation of the spacer be-
tween N8 and the phenyl ring (8d) appeared somewhat detrimen-
tal for A1/A2A affinity. These molecules therefore can be considered
quite potent (low nanomolar range) but low selectivity A1/A2A dual
ligands. In this subset, the affinity at the hA3 receptor decreased
with the increase of the steric hindrance around the N8-position,
with the phenylpropyl-derivative 8d being fivefold less potent
than the N8-methyl analogue 8a.
corresponding
1/2-methyl-1,4-dihydro-pyrazolo[4,3-d]pyrimi-
dine-5,7-dione 20–23a,b by heating with an excess of urea.
The 5,7-dichloro-1/2-methyl-pyrazolo[4,3-d]pyrimidines 24–
27a,b were obtained by treatment of 20–23a,b with POCl3 and
DBU. Selective substitution of the chlorine atom at the 7-position
with furoic acid hydrazide followed by a Dimroth rearrangement
led to the desired pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine
nucleus (32–35a,b). Compounds 40–43a,b were obtained by
treating derivatives 32–35a,b with
a saturated solution of
ammonia in ethanol. These were converted into the corresponding
4-methoxy-phenyl urea derivatives 44–47a,b by reaction with
4-methoxy-phenylisocyanate. The intermediate 5-chloro-2-(2-fur-
yl)-9-methyl-9H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine
32a was also reacted with different primary and secondary amines
to give the final derivatives 36–39.
2.2. Biological activity
As previously noted with the pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidine series,7 when the amino group at the 5-position was
replaced by a phenylcarbamoyl moiety (derivatives 9a–d), an in-
crease of A3 affinity was observed resulting in different degrees
of selectivity over the other AR subtypes. Both the N8-methyl
(9a) and the N8-propyl (9b) derivatives showed high affinity at
the human A3 AR (Ki values of 15 and 10 nM, respectively) with
similar patterns of selectivity. Short alkyl chains at the 8-position
of the pyrrolo-triazolo-pyrimidine urea derivatives are preferred to
N8-arylalkyl chains (see 9a,b vs 9c,d) for promoting hA3 selectivity
All the synthesized compounds were evaluated in radioligand
binding assays to determine their affinities at the human A1, A2A
and A3 adenosine receptors. Potency of the compounds at the
hA2B AR was studied evaluating their capability to inhibit NECA
(100 nM) stimulated cAMP production. Affinity data for A1, A2A
and A3 receptors, expressed as Ki values, and IC50 values derived
from the cAMP assay carried out for hA2B subtype, are listed in Ta-
bles 1 and 2.

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P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
Table 1
Binding (hA1, hA2A and hA3) and functional (hA2B) parameters of the synthesized pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines towards adenosine receptors
O
O
O
O
O
N
N
N
N
N
N
O
N
N
N
N
N R
N CH3
N R
N
H
N
H
N
N
H
N
H
N
H2N
8a-d
N
H
9a-d
10
a
b
c
d
Compd
R
hA1 Ki (nM)
hA2A Ki (nM)
hA2B IC50 (nM)
hA3 Ki (nM)
8a
8b
8c
8d
9a
9b
9c
9d
10
CH3
100 (83–120)
35 (27–45)
4.3 (3.1–6.0)
18 (13–23)
800 (701–913)
743 (671–821)
178 (148–213)
210 (162–258)
355 (289–437)
20 (12–31)
30 (23–38)
3.9 (2.5–6.3)
50 (41–60)
500 (420–595)
200 (166–240)
148 (126–173)
130 (92–163)
>1000 (13%)
42 (31–57)
90 (81–99)
46 (37–56)
251 (205–306)
838 (713–984)
906 (852–964)
740 (722–759)
>1000 (12%)
>1000 (8%)
50 (41–60)
55 (46–65)
124 (96–161)
241 (176–330)
15 (10–21)
10 (6–17)
12 (10–16)
13 (11–19)
111 (74–167)
(CH2)2CH3
(CH2)2Ph
(CH2)3Ph
CH3
(CH2)2CH3
(CH2)2Ph
(CH2)3Ph
—
a
b
c
Displacement of specific [3H]-DPCPX binding to human A1 receptors expressed in CHO cells (Ki nM).
Displacement of specific [3H]-ZM 241385 binding to human A2A receptors expressed in CHO cells (Ki nM).
cAMP assay in CHO cells expressing hA2B receptors (IC50 nM).
d
Displacement of specific [3H]-MRE3008F20 binding to human A3 receptors expressed in CHO cells (Ki nM).
Table 2
Binding (hA1, hA2A and hA3) and functional (hA2B) parameters of the synthesized pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines towards adenosine receptors
O
O
N
N
R'
N
N
N
N
N
N
N
N R'
R
N
R
N
40-47a, 36-39
40-47b
a
b
c
d
Compd
R
R’
CH3
(CH2)2CH3
(CH2)2Ph
(CH2)3Ph
CH3
(CH2)2CH3
(CH2)2Ph
(CH2)3Ph
CH3
(CH2)2CH3
(CH2)2Ph
(CH2)3Ph
CH3
hA1 Ki (nM)
hA2A Ki (nM)
hA2B IC50 (nM)
hA3 Ki (nM)
40a
41a
42a
43a
44a
45a
46a
47a
40b
41b
42b
43b
44b
45b
46b
47b
36
NH2
NH2
NH2
NH2
10 (8–13)
14 (11–20)
3.6 (2.5–5.3)
12 (8–18)
42 (37–47)
122 (86–172)
68 (55–84)
20 (14–29)
626 (546–717)
>1000 (32%)
8.1 (6.9–9.7)
17 (12–24)
9.2 (7.9–10.6)
11 (9–13)
222 (181–273)
208 (177–245)
672 (621–729)
>1000 (31%)
119 (87–162)
>1000 (4%)
31 (25–38)
40 (35–46)
749 (662–847)
83 (70–99)
603 (542–670)
802 (719–895)
61 (42–88)
33 (28–38)
85 (68–105)
129 (97–172)
61 (44–85)
524 (421–652)
>1000 (38%)
30 (24–38)
22 (18–27)
4.9 (3.4–7.2)
7.1 (5.7–8.9)
923 (878–970)
240 (222–259)
753 (712–798)
>1000 (35%)
12 (8–18)
319 (275–371)
595 (502–706)
122 (84–177)
32 (26–45)
772 (660–902)
>1000 (32%)
33 (28–37)
45 (40–51)
27 (23–32)
32 (28–37)
580 (453–742)
863 (801–930)
548 (456–634)
>1000 (31%)
915 (786–1064)
>1000 (4%)
(4-OCH3-Ph)NHCONH
(4-OCH3-Ph)NHCONH
(4-OCH3-Ph)NHCONH
(4-OCH3-Ph)NHCONH
NH2
NH2
NH2
NH2
(4-OCH3-Ph)NHCONH
(4-OCH3-Ph)NHCONH
(4-OCH3-Ph)NHCONH
(4-OCH3-Ph)NHCONH
Cyclohexyl-NH
Morpholine
161 (132–196)
208 (153–283)
>1000 (25%)
125 (85–182)
432 (363–514)
315 (255–390)
526 (482–575)
110 (93–130)
50 (45–56)
314 (269–358)
815 (722–908)
507 (443–582)
>1000 (1%)
(CH2)2CH3
(CH2)2Ph
(CH2)3Ph
CH3
37
CH3
>1000 (6%)
38
39
4-Me-piperazine
4-Ph-piperazine
CH3
CH3
>1000 (5%)
>1000 (8%)
>1000 (2%)
>1000 (9%)
>1000 (2%)
>1000 (5%)
>1000 (1%)
>1000 (1%)
a
b
c
Displacement of specific [3H]DPCPX binding at human A1 receptors expressed in CHO cells (Ki nM, n = 3–6).
Displacement of specific [3H]ZM241385 binding at human A2A receptors expressed in CHO cells (Ki nM, n = 3–6).
Potency (IC50, nM) of examined compounds to inhibit 100 nM NECA stimulation cAMP levels in hA2B CHO cells.
d
Displacement of specific [3H]MRE3008F20 binding at human A3 receptors expressed in CHO cells (Ki nM, n = 3–6). For the compounds 47a, 36, 37, 38 and 39 are reported
in parentheses the % of inhibition at the 1
lM concentration versus hA1, A2A, A2B and A3 adenosine receptors, respectively. The data are expressed as geometric means with
95% or 99% confidence limits in parentheses.
vs hA1 and hA2A subtypes. The introduction of an N8-arylalkyl moi-
ety seems indeed related to an increase of A1/A2A affinity with a
resulting decrease of A3 selectivity. These data are mostly in agree-
ment with previously reported studies on N8-substituted pyrazol-
o[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines.29,30
The pyridyl-urea derivative 10 was synthesised as this
structural modification proved to improve hA3 affinity and
water-solubility when introduced at the same position of the pyr-
azolo-triazolo-pyrimidine tricycle27 (see MRE3005-F20, Fig. 1).
Quite surprisingly, compound 10 (Ki hA3 = 111 nM) was found to

P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
1051
be more than 7-fold less potent than the corresponding 4-OCH3–
phenylurea derivative 9a (Ki hA3 = 15 nM) in binding the hA3
receptor.
9b IC50= 50 (41-63) nM
9a IC50= 75 (66-84) nM
45b IC50= 250 (208-300) nM
41a IC50= 500 (463-540) nM
100
80
60
40
20
0
From the pair-wise comparison between binding affinities of
compounds 8, 9a–d and 10 with the analogously substituted and
previously reported PTPs,29 some general observation can be de-
rived. The removal of the nitrogen at the 7-position did not signif-
icantly affect hA1 and hA2B affinity/efficacy both for the 5-NH2
derivatives 8a-d and the 5-urea derivatives 9a–d. This would sug-
gest that N7 is not involved in crucial interaction with these AR
subtypes. Conversely, the 5-amino-7-deaza derivatives appeared
to be significantly less potent (up to 300-fold29 in the case of 8d)
than the corresponding N8-substituted pyrazole-containing tricy-
cles toward the A2A receptor. With respect to the A3 subtype, the
5-amino-7-deaza derivatives appeared from 6- to 22-fold more po-
tent than the corresponding PTP, while the 5-urea derivatives dis-
played a lower affinity (see compound 9b and 10 vs MRE3008-F20
and MRE3005-F20, respectively, Fig. 1). The overall results suggest
that the removal of the nitrogen at the 7-position was detrimental
to the selectivity of the pyrrole derivatives when compared to the
corresponding 7-aza analogues,7 probably due to a significant
alteration of the receptor binding interactions, mainly with the
A2A and/or A3 receptors.
-10
-9
-8
-7
-6
-5
-4
log drug concentration (M)
Figure 3. Inhibition curves of cAMP accumulation in hA3 CHO cells by adenosine
antagonists blocking the effect of 100 nM Cl-IB-MECA.
tolerated. These data confirm our previous results on C9-substi-
tuted-PTPs,24 and are in agreement with recent QSAR analyses per-
formed at the A3 AR.31 The amino derivatives (40–43a) exhibit
good affinity toward A2A AR (Ki from 3.6 to 122 nM). Unfortunately,
the ligands bind to the A1 and inhibit the A2B subtypes in the same
range of concentration. The conversion of the amino group into the
corresponding 4-OCH3–phenylurea moiety (44–47a) did not pro-
vide the desired improvement of affinity/selectivity at the A3 AR
in the N9-substituted series (40–43a vs 44–47a).
2.2.2. Pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines
Among compounds 36–39, in which the chlorine atom at the 5-
position of intermediate 32a (see Scheme 3) was substituted with
cyclohexylamine, morpholine, and 4-methyl/phenyl-piperazine,
only the cyclohexyl-amino derivative 36 showed some affinity ver-
sus ARs, especially for the A1 receptor (Ki = 12 nM). This would con-
firm previously reported studies indicating that the amino group at
the 5-position is involved in a crucial interaction with ARs as a
hydrogen bond donor. Bisubstitution of this nitrogen, by incorpo-
rating it in a ring, completely abolished the affinity. Interestingly,
compound 36 is characterized by the presence at the 5-position
of a cycloalkyl group, which is the typical N6-substitution of NECA
(50-N-ethylcarboxamidoadenosine)-related A1 AR agonists.32 This
findings would be in agreement with the previously observed par-
allelism between the C5-position of PTPs and the C6-position of
NECA-related agonists.33
With the aim to better investigate the role of the pyrazole ring
on the affinity toward ARs we also synthesised a novel series of 8-
or 9-substituted pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines
(see Table 2) as junction isomers of the reference compounds re-
ported in Figure 1. These series allowed us to evaluate the effect
of the shift of a pyrazole nitrogen from the 7- to the 9-position
of the tricycle.
The 5-amino-N8-alkyl derivatives (40–43b) showed comparable
or slightly increased affinity/potency at the A1, A2A and A2B sub-
types in comparison with the corresponding 5-amino-N8-alkyl pyr-
rolo-triazolo-pyrimidines (8a–d) while the affinity at the A3
receptor was found to be from 2- to 8-fold lower. Introduction of
a 4-methoxy-phenylurea moiety at the 5-position yielded com-
pounds 44–47b, exerting generally lower affinities if compared to
40–43b at the A1 (from 11- to 153-fold) and A2A (from 12- to 90-
fold) subtypes. Lower potency at the A2B (from 17- to 37-fold) sub-
type was also seen. The 4-methoxy-phenylurea moiety produced a
significant increase of A3 affinity (almost ninefold) only in the case
of compound 45b compared to 41b. The N8-propyl-substituted 45b
(Ki = 50 nM) exerted twofold higher affinity at A3 AR compared to
the N8-methyl- derivative 44b (Ki = 110 nM) while the introduction
of N8-arylalkyl chains (46–47b) was somewhat detrimental in
terms of A3 affinity. Comparing the 5-urea-N8-alkyl-pyrazolo-triaz-
olo-pyrimidine derivatives (44–47b) to the corresponding 5-urea-
N8-alkyl-pyrrolo-triazolo-pyrimidines (9a–d), the main effect of
the introduction of nitrogen at the 9-position was a significantly
reduced hA3 affinity, especially in the case of N8-arylalkyl substitu-
tion (see compounds 46–47b vs 9c,d). The comparison between
40–47b and their isomeric N8-substituted pyrazolo[4,3-e]triazol-
o-pyrimidines29 showed how the shift of the pyrazole nitrogen
from the 7- to the 9-position of the tricycle exerted a remarkable
decrease of hA3 affinity and selectivity of the 5-urea-derivatives
(see Fig. 1 compound MRE3008-F20 vs 45b).
3. Conclusions
Herein, we described a novel synthetic strategy for the prepara-
tion of previously unreported pyrrolo- and pyrazolo-[3,
4-e][1,2,4]triazolo[1,5-c]pyrimidines that can be, respectively,
considered as 7-deaza analogues and junction isomers of A2A/A3
AR antagonists based on the pyrazolo[4,3-e][1,2,4]triazolo[1,5-
c]pyrimidine nucleus.7 In both series, we identified examples of
potent (nanomolar range) AR antagonists characterized by broad
selectivity profiles.
The removal of the nitrogen at the 7-position was found to sig-
nificantly affect both A2A and A3 affinities, reflected in a substantial
loss of potency/selectivity of the newly examined molecules. The
introduction of a 5-phenylcarbamoyl moiety, to promote A3 affin-
ity and selectivity, appeared effective only in the pyrrole series of
which the urea derivatives 9a and 9b distinguished as high affinity
(Ki = 15 and 10 nM, respectively) and potent (IC50 = 75 and 50 nM,
respectively, see Fig. 3) hA3 antagonists.
The synthesis of compounds 40–47a allowed us to evaluate the
effect of N9- vs N8-substitution. When small alkyl chains were
shifted from N8- to N9-position, the general effect (excepting 40a
vs 40b at hA3) was a random increase of affinity/potency at the
four AR subtypes, especially for the 5-urea derivatives (see
44–45a vs 44–45b). An opposite trend was instead observed when
arylalkyl chains were shifted (see 42–43a vs 42–43b). Thus, the
increase of the steric hindrance around N9-position seems poorly
The comparison between the new N8-substituted pyrazolo[3,4-
e]triazolo-pyrimidines (40–47b) and their structural isomers N8-
substituted pyrazolo[4,3-e]triazolo-pyrimidines29 showed how
the shift of the pyrazole nitrogen from the 7- to the 9-position ex-
erted, as a main effect, a remarkable decrease of hA3 affinity and
selectivity of the 5-urea-derivatives (see Fig.
1 compound
MRE3008-F20 vs 45b).

1052
P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
When small alkyl chains were shifted from the N8- to N9-posi-
d6) d 7.46 (d, J = 2 Hz, 1H), 7.29–7.13 (m, 15H), 6.83 (d, J = 2 Hz, 1H),
5.04 (s, 2H), 4.93 (s, 2H), 4.18 (t, J = 7.6 Hz, 2H), 3.02 (t, J = 7.2 Hz,
2H). MS (ESI): [MH]+ = 436.8.
tion, the general effect was a random increase of affinity/potency
at the four AR subtypes especially for the 5-urea derivatives (see
44–45a vs 44–45b). In contrast, N9-arylalkyl substitution appeared
generally detrimental. Thus, steric hindrance around this position
seems poorly tolerated, in accordance with previous findings.24,31
The present work can be considered as an innovative and
important contribution to the understanding of the SAR of PTPs
based AR antagonists.
4.1.2.4. 1,3-Dibenzyl-6-(3-phenyl-propyl)-1,6-dihydro-pyrrolo[3,4-
d]pyrimidine-2,4-dione (3d). Column chromatography eluting
with a mixture EtOAc/petroleum ether 1:4. White solid; 82% yield;
mp 129–130 °C; 1H NMR (400 MHz, DMSO-d6) d 7.57 (d, J = 2 Hz,
1H), 7.33–7.12 (m, 15H), 6.86 (d, J = 2 Hz, 1H), 5.08 (s, 2H), 4.99 (s,
2H), 3.97 (t, J = 7.2 Hz, 2H), 2.51–2.44 (m, 2H), 2.03 (m, 2H). MS
(ESI): [MH]+ = 450.8.
4. Experimental section
4.1. Chemistry
4.1.3. General procedure for preparation of 6-alkyl-1,6-dihydro-
pyrrolo[3,4-d]pyrimidine-2,4-diones (4a–d)
To a solution of the appropriate dibenzyl derivative 3a–d
(4 mmol) in anhydrous toluene (50 mL) was added AlCl3
(28 mmol) and the resulting suspension was stirred at room tem-
perature for 2 h. The solvent was concentrated in vacuo to obtain
a red residue that was treated with crushed ice and stirred at
0 °C till the disappearance of the red colour. The aqueous phase
was then extracted with EtOAc (3 ꢀ 50 mL), the combined organic
layers were dried over Na2SO4, filtered, and the solvent was evap-
orated to give a solid residue that was crystallized from EtOAc/
Et2O.
4.1.1. Materials and methods
Reaction progress and product mixtures were monitored by
thin-layer chromatography (TLC) on silica gel (precoated F254
Merck plates) and visualized with aqueous potassium permanga-
nate. 1H NMR data were determined in CDCl3 or DMSO-d6 solutions
with a Varian VXR 200 spectrometer or a Varian Mercury Plus 400
spectrometer. Peak positions are given in parts per million (d)
downfield from tetramethylsilane as internal standard, and J values
are given in Hertz. All products reported showed 1H NMR spectra
in agreement with the assigned structures. Light petroleum refers
to the fractions boiling at 40–60 °C. Melting points were deter-
mined on a Buchi–Tottoli instrument and are uncorrected. Chro-
matography was performed on Merck 230–400 mesh silica gel.
Organic solutions were dried over anhydrous sodium sulfate. Ele-
mental analyses were performed by the microanalytical laboratory
of Dipartimento di Chimica, University of Ferrara, and were within
0.4% of the theoretical values for C, H, and N. All final compounds
exhibited a purity of not less than 95%. The mass spectra were ob-
tained on an ESI Micromass ZMD 2000 mass spectrometer in posi-
tive scan mode using direct injection of the purified compound
solution (MH+).
4.1.3.1. 6-Methyl-1,6-dihydro-pyrrolo[3,4-d]pyrimidine-2,4-dione
(4a). White solid; 82% yield; mp >300 °C; 1H NMR (200 MHz,
DMSO-d6) d 10.36 (br s, 1H), 10.28 (br s, 1H), 7.29 (s, 1H), 6.44
(s, 1H), 3.67 (s, 3H). MS (ESI): [MH]+ = 166.1.
4.1.3.2. 6-Propyl-1,6-dihydro-pyrrolo[3,4-d]pyrimidine-2,4-dione
(4b). Pale yellow solid; 87% yield; mp 270 °C dec.; 1H NMR
(200 MHz, DMSO-d6) d 10.37 (br s, 1H), 10.27 (br s, 1H), 7.35 (d,
J = 2.2 Hz, 1H), 6.47 (d, J = 2.2 Hz, 1H), 3.89 (t, J = 7 Hz, 2H), 1.77–
1.66 (m, 2H), 0.79 (t, J = 7.4 Hz, 3H). MS (ESI): [MH]+ = 194.1.
4.1.3.3. 6-Phenethyl-1,6-dihydro-pyrrolo[3,4-d]pyrimidine-2,4-
dione (4c). White solid; 81% yield; mp 263 °C dec.; 1H NMR
(400 MHz, DMSO-d6) d 10.38 (br s, 1H), 10.28 (br s, 1H), 7.30–
7.10 (m, 6H), 6.49 (d, J = 2.4 Hz, 1H), 4.19 (t, J = 7.6 Hz, 2H), 3.03
(t, J = 7.2 Hz, 2H). MS (ESI): [MH]+ = 256.3.
4.1.2. General procedure for preparation of 1,3-dibenzyl-6-
alkyl-1,6-dihydro-pyrrolo[3,4-d]pyrimidine-2,4-diones (3a–d)
To a solution of 1,3-dibenzyl-1,6-dihydro-pyrrolo[3,4-d]pyrimi-
dine-2,4-dione (2,26 1.5 mmol) in anhydrous DMF (5 mL), K2CO3
was added (4.5 mmol) and the resulting mixture stirred at 40 °C
for 100. After cooling at room temperature, the appropriate alkyl
halide (4.5 mmol) was added and the reaction heated at 40 °C for
further 4 h. The solvents were removed in vacuo and the residue
was suspended in water and extracted with EtOAc. The organic
phase was dried over anhydrous Na2SO4 and the solvents removed
under reduced pressure to obtain a crude solid which was purified
by crystallization or flash chromatography.
4.1.3.4. 6-(3-Phenyl-propyl)-1,6-dihydro-pyrrolo[3,4-d]pyrimi-
dine-2,4-dione (4d). White solid; 80% yield; mp 225 °C dec.;
1H NMR (400 MHz, DMSO-d6) d 10.40 (br s, 1H), 10.30 (br s, 1H),
7.38 (d, J = 2 Hz, 1H), 7.30–7.17 (m, 5H), 6.51 (d, J = 2 Hz, 1H),
3.96 (t, J = 6.8 Hz, 2H), 2.52–2.48 (m, 2H), 2.051–2.00 (m, 2H). MS
(ESI): [MH]+ = 270.3.
4.1.4. General procedure for preparation of 1-alkyl-4-nitro-1H-
pyrazole-5-carboxamide and 1-alkyl-4-nitro-1H-pyrazole-3-car-
boxamide (12–15a,b)
4.1.2.1. 1,3-Dibenzyl-6-methyl-1,6-dihydro-pyrrolo[3,4-d]pyrimi-
dine-2,4-dione (3a). Crystallization from Et2O/petroleum ether.
White solid; 97% yield; mp 164–166 °C; 1H NMR (200 MHz, CDCl3)
d 7.52–7.22 (m, 10H), 7.18 (d, J = 2.2 Hz, 1H), 6.13 (d, J = 2.4 Hz,
1H), 5.24 (s, 2H), 5.01 (s, 2H), 3.66 (s, 3H). MS (ESI): [MH]+ = 346.4.
To a solution of the nitroamide 1128 (1.2 mmol) in anhydrous
DMF (36 mL) was added K2CO3 (2.3 mmol) and the resulting mix-
ture was stirred for 100. Then, the appropriate alkyl halide
(1.4 mmol) was added and the mixture was stirred for 6 h. The sol-
vent was removed in vacuo and the residue was suspended in
water and extracted with EtOAc. The organic phase was dried over
anhydrous Na2SO4, filtered and the solvents removed under re-
duced pressure to obtain a crude solid that was purified via column
chromatography (gradient from EtOAc/petroleum ether 1:1 to
EtOAc) to obtain the desired products.
4.1.2.2. 1,3-Dibenzyl-6-propyl-1,6-dihydro-pyrrolo[3,4-d]pyrimi-
dine-2,4-dione (3b). Crystallization from Et2O/petroleum ether.
White solid; 89% yield; mp 122 °C; 1H NMR (200 MHz, CDCl3) d
7.51–7.48 (m, 2H), 7.29–7.22 (m, 9H), 6.15 (d, J = 2.4 Hz, 1H), 5.24
(s, 2H), 5.01 (s, 2H), 3.80 (t, J = 7.2 Hz, 2H), 1.77–1.73 (m,
J = 7.2 Hz, 2H), 0.86 (t, J = 7.4 Hz, 3H) MS (ESI): [MH]+ = 374.4.
4.1.2.3. 1,3-Dibenzyl-6-phenethyl-1,6-dihydro-pyrrolo[3,4-d]pyrim-
idine-2,4-dione (3c). Crystallization from Et2O/petroleum ether.
White solid; 76% yield; mp 159–160 °C; 1H NMR (400 MHz, DMSO-
4.1.4.1. 1-Methyl-4-nitro-1H-pyrazole-5-carboxamide (12a).White
solid; 40% yield; mp 167 °C; 1H NMR (400 MHz, DMSO-d6) d 8.49
(br s, 1H), 8.34 (br s, 1H), 8.27 (s, 1H), 3.86 (s, 3H).

P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
1053
4.1.4.2. 1-Methyl-4-nitro-1H-pyrazole-3-carboxamide (12b).
White solid; 38% yield; mp 166 °C; 1H NMR (400 MHz, DMSO-
d6) d 8.83 (s, 1H), 8.03 (br s, 1H), 7.79 (br s, 1H), 3.90 (s, 3H).
4.1.5.5. 4-Amino-1-(2-phenylethyl)-1H-pyrazole-5-carboxamide
(18a). White solid; 68% yield; mp 87–88 °C; 1H NMR (200 MHz,
DMSO-d6) d 7.40 (br s, 2H), 7.27–7.15 (m, 5H), 7.05 (s, 1H), 4.53
(t, J = 7.6 Hz, 2H), 4.37 (br s, 2H), 2.94 (t, J = 7.8 Hz, 2H).
4.1.4.3. 1-Propyl-4-nitro-1H-pyrazole-5-carboxamide (13a). White
1
solid; 48% yield; mp 98–100 °C; H NMR (400 MHz, DMSO-d6) d 8.48
4.1.5.6. 4-Amino-1-(2-phenylethyl)-1H-pyrazole-3-carboxamide
(18b). White solid; 87% yield; mp 82–83 °C; 1H NMR (200 MHz,
DMSO-d6) d 7.29–7.17 (m, 5H), 7.11 (br s, 1H), 7.04 (s, 1H), 6.97
(br s, 1H), 4.61 (br s, 2H), 4.22 (t, J = 7 Hz, 2H), 3.07 (t, J = 7.6
Hz, 2H).
(br s, 1H), 8.28–8.27 (m, 2H), 4.08 (t, J = 6.8 Hz, 2H), 1.81–1.76 (m,
2H), 0.813 (t, J = 7.6 Hz, 3H).
4.1.4.4. 1-Propyl-4-nitro-1H-pyrazole-3-carboxamide (13b). White
solid; 43% yield; mp 115–116 °C; 1H NMR (400 MHz, DMSO-d6) d 8.87
(s, 1H), 7.98 (br s, 1H), 7.73 (br s, 1H), 4.10 (t, J = 6.8 Hz, 2H), 1.83–1.78
(m, 2H), 0.83 (t, J = 7.2 Hz, 3H).
4.1.5.7. 4-Amino-1-(3-phenylpropyl)-1H-pyrazole-5-carboxam-
ide (19a). White solid; 87% yield; mp 78 °C; 1H NMR (200 MHz,
DMSO-d6) d 7.42 (br s, 2H), 7.26–7.15 (m, 5H), 7.07 (s, 1H), 4.38–
4.31 (m, 4H), 2.51–2.43 (m, 2H), 1.97–1.93 (m, 2H).
4.1.4.5. 4-Nitro-1-(2-phenylethyl)-1H-pyrazole-5-carboxamide (14a).
White solid; 25% yield; mp 133 °C; 1H NMR (400 MHz, DMSO-d6) d
8.45 (br s, 1H), 8.31 (br s, 1H), 8.25 (s, 1H), 7.29–7.16 (m, 5H), 4.38–
4.35 (m, 2H), 3.09 (t, J = 7.6 Hz, 2H).
4.1.5.8. 4-Amino-1-(3-phenylpropyl)-1H-pyrazole-3-carboxamide
(19b). White solid; 90% yield; mp 111–112 °C; 1H NMR (200 MHz,
DMSO-d6) d 7.32–7.11 (m, 7H), 6.97 (br s, 1H), 4.65 (br s, 2H), 3.98 (t,
J = 6.8 Hz, 2H), 2.58–2.49 (m, 2H), 2.08–2.00 (m, 2H).
4.1.4.6. 4-Nitro-1-(2-phenylethyl)-1H-pyrazole-3-carboxamide
(14b). White solid; 50% yield; mp 158–160 °C; 1H NMR
(400 MHz, DMSO-d6) d 8.74 (s, 1H), 8.00 (br s, 1H), 7.76 (br s,
1H), 7.30–7.18 (m,5H), 4.40 (t, J = 7.2 Hz, 2H), 3.13 (t, J = 7.6 Hz,
2H).
4.1.6. General procedure for preparation of 1/2-alkyl-1/2H-
pyrazolo[4,3-d]pyrimidine-5,7-(4H,6H)-diones (20–23a,b)
The appropriate aminoamide 16–19a,b (7.1 mmol) and urea
(33.3 mmol) were finely mixed without solvent in a round bottom
flask which was then heated at 200 °C for 2 h. During the reaction
course, complete fusion followed by resolidification was observed.
The crude product was purified by crystallization from 10% NaOH/
acetic acid.
4.1.4.7. 4-Nitro-1-(3-phenylpropyl)-1H-pyrazole-5-carboxamide
(15a). White solid; 34% yield; mp 93–95 °C; 1H NMR (400 MHz,
DMSO-d6) d 8.53 (br s, 1H), 8.33–8.32 (m, 2H), 7.30–7.19 (m, 5H),
4.16 (t, J = 6.8 Hz, 2H), 2.57 (t, J = 7.2 Hz, 2H), 2.11–2.08 (m, 2H).
4.1.4.8. 4-Nitro-1-(3-phenylpropyl)-1H-pyrazole-3-carboxamide
(15b). White solid; 57% yield; mp 111–113 °C; 1H NMR
(400 MHz, DMSO-d6) d 8.90 (s, 1H), 8.00 (br s, 1H), 7.77 (br s,
1H), 7.31–7.19 (m, 5H), 4.18 (t, J = 7.2 Hz, 2H), 2.59 (t, J = 7.2 Hz,
2H), 2.15–2.12 (m, 2H).
4.1.6.1. 1-Methyl-1H-pyrazolo[4,3-d]pyrimidine-5,7-(4H,6H)-dione
(20a). White solid; quantitative yield; mp >300 °C; 1H NMR
(200 MHz, DMSO-d6) d 11.15 (br s, 1H), 10.96 (br s, 1H), 8.16 (s,
1H), 4.04 (s, 3H). MS (ESI): [MH]+ = 167.1.
4.1.6.2. 2-Methyl-2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H,6H)-dione
(20b). White solid; 81% yield; mp >300 °C; 1H NMR (400 MHz,
DMSO-d6) d 10.87 (br s, 1H), 10.71 (br s, 1H), 7.64 (s, 1H), 3.94
(s, 3H). MS (ESI): [MH]+ = 167.1.
4.1.5. General procedure for preparation of 4-amino-1-alkyl-
1H-pyrazole-5-carboxamide and 4-amino-1-alkyl-1H-pyrazole-
3-carboxamide (16–19a,b)
The appropriate nitroamide 12–15a,b (4 mmol) was dissolved
in EtOH (200 mL), then Pd on activated charcoal (10%, 150 mg)
was added and the mixture was hydrogenated at 50 psi in a Parr
apparatus for 5 h at room temperature. The mixture was filtered
on Celite and then the solvent was removed under reduced pres-
sure to obtain the crude product which was purified by crystalliza-
tion from EtOAc/Et2O.
4.1.6.3. 1-Propyl-1H-pyrazolo[4,3-d]pyrimidine-5,7-(4H,6H)-dione
(21a). White solid; quantitative yield; mp >300 °C; 1H NMR
(200 MHz, DMSO-d6) d 9.34 (br s, 2H), 7.37 (s, 1H), 4.35 (t,
J = 6.8 Hz, 2H), 1.81–1.70 (m, 2H), 0.78 (t, J = 7.2 Hz, 3H). MS
(ESI): [MH]+ = 195.1.
4.1.6.4. 2-Propyl-2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H,6H)-dione
(21b). White solid; 74% yield; mp >300 °C; 1H NMR (200 MHz,
DMSO-d6) d 10.48 (br s, 2H), 7.68 (s, 1H), 4.14 (t, J = 6.8 Hz, 2H),
1.85–1.74 (m, 2H), 0.80 (t, J = 7.4 Hz, 3H). MS (ESI): [MH]+ = 195.1.
4.1.5.1. 4-Amino-1-methyl-1H-pyrazole-5-carboxamide (16a).
White solid; 60% yield; mp 174–175 °C; 1H NMR (200 MHz,
DMSO-d6) d 7.37 (br s, 2H), 7.01 (s, 1H); 4,39 (br s, 2H), 3.89 (s,
3H).
4.1.6.5.
1-(2-Phenylethyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-
4.1.5.2. 4-Amino-1-methyl-1H-pyrazole-3-carboxamide (16b). White
solid; 60% yield; mp 171–172 °C; 1H NMR (200 MHz, DMSO-d6) d 7.10 (br
s, 1H), 7.06 (s, 1H), 6.95 (br s, 1H), 4.63 (br s, 2H), 3.72 (s, 3H).
(4H,6H)-dione (22a). White solid; quantitative yield; mp
>300 °C; 1H NMR (200 MHz, DMSO-d6) d 11.08 (br s, 2H), 7.34 (s,
1H), 7.27–7.10 (m, 5H), 4.62 (t, J = 7.2 Hz, 2H), 3.07 (t, J = 7.4 Hz,
2H). MS (ESI): [MH]+ = 257.1.
4.1.5.3. 4-Amino-1-propyl-1H-pyrazole-5-carboxamide (17a) ..
White solid; 65% yield; mp 91–92 °C; 1H NMR (200 MHz, DMSO-
d6) d 7.40 (br s, 2H), 7.04 (s, 1H), 4.33–4.24 (br m, 4H), 1.69 (m,
2H), 0.75 (t, J = 7.6 Hz, 3H).
4.1.6.6.
2-(2-Phenylethyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7-
(4H,6H)-dione (22b). White solid; 70% yield; mp >300 °C; 1H
NMR (200 MHz, DMSO-d6) d 10.91 (br s, 1H), 10.38 (br s, 1H),
7.58 (s, 1H), 7.32–7.17 (m, 5H), 4.45 (t, J = 7.4 Hz, 2H), 3.14 (t
J = 7.2 Hz, 2H). MS (ESI): [MH]+ = 257.1.
4.1.5.4. 4-Amino-1-propyl-1H-pyrazole-3-carboxamide (17b).
White solid; 91% yield; mp 115 °C; 1H NMR (200 MHz, DMSO-
d6) d 7.10 (s, 1H), 7.07 (br s, 1H), 6.95 (br s, 1H), 4.62 (br s,
2H), 3.92 (t, J = 6.8 Hz, 2H), 1.79–1.69 (m, 2H), 0.82 (t,
J = 7.2 Hz, 3H).
4.1.6.7. 1-(3-Phenylpropyl)-1H-pyrazolo[4,3-d]pyrimidine-5,7-
(4H,6H)-dione (23a). White solid; 63% yield; mp 280 °C; 1H
NMR (200 MHz, DMSO-d6) d 11.19 (br s, 1H), 10.02 (br s, 1H),

1054
P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
7.38 (s, 1H), 7.26–7.154 (m, 5H), 4.43 (t, J = 6.8 Hz, 2H), 2.57–2.50
(200 MHz, CDCl3) d 8.22 (s, 1H), 7.26–7.23 (m, 3H), 7.10–7.07 (m,
2H), 4.94 (t, J = 7.4 Hz, 2H), 3.23 (t, J = 7.6 Hz, 2H).
(m, 2H), 2.11–2.04 (m, 2H). MS (ESI): [MH]+ = 271.1.
4.1.6.8. 2-(3-Phenylpropyl)-2H-pyrazolo[4,3-d]pyrimidine-5,7-
(4H,6H)-dione (23b). White solid; 55% yield; mp >300 °C; 1H
NMR (200 MHz, DMSO-d6) d 10.51 (br s, 2H), 7.71 (s, 1H), 7.28–
7.17 (m, 5H), 4.22 (t, J = 6.8 Hz, 2H), 2.58–2.48 (m, 2H), 2.15–2.11
(m, 2H). MS (ESI): [MH]+ = 271.1.
4.1.7.10. 5,7-Dichloro-2-phenethyl-2H-pyrazolo[4,3-d]pyrimidine
(26b). Pale yellow solid; 50% yield; crude product; 1H NMR
(200 MHz, CDCl3) d 7.83 (s, 1H), 7.29–7.26 (m, 3H), 7.08–7.04 (m,
2H), 4.75 (t, J = 7.2 Hz, 2H), 3.34 (t, J = 7 Hz, 2H). MS (ESI):
[MH]+ = 292.1.
4.1.7. General procedure for preparation of 2,4-dichloro-6-
alkyl-6H-pyrrolo[3,4-d]pyrimidines (5a–d) and 5,7-dichloro-1/
2-alkyl-1/2H-pyrazolo[4,3-d]pyrimidines (24–27a,b)
4.1.7.11. 5,7-Dichloro-1-(3-phenyl-propyl)-1H-pyrazolo[4,3-d]
pyrimidine (27a). Pale yellow solid; 58% yield; crude product;
1H NMR (200 MHz, CDCl3) d 8.19 (s, 1H), 7.27–7.14 (m, 5H), 4.73
(t, J = 7 Hz, 2H), 2.71 (t, J = 7.4 Hz, 2H), 2.33–2.62 (m, 2H).
A mixture of 4a-d or 20–23a,b (6.1 mmol) and phosphorous
oxychloride (60 mmol) was heated at 50 °C under an argon atmo-
sphere and DBU (36.6 mmol) was added dropwise under vigorous
stirring. The reaction was then heated for further 8 h at 80 °C. After
cooling to room temperature, the reaction mixture was slowly
poured into cold water and treated with a 50% aqueous solution
of NaOH to pH 7. The solution was extracted with Et2O, the organic
phase was dried over anhydrous Na2SO4, filtered and the solvent
was removed under reduced pressure to obtain the desired inter-
mediate. Due to instability, these compounds were used for the
next reaction without further purification. The NMR analyses were
performed on the crude product.
4.1.7.12. 5,7-Dichloro-2-(3-phenyl-propyl)-2H-pyrazolo[4,3-d]
pyrimidine (27b). Pale yellow solid; 62% yield; crude product;
1H NMR (200 MHz, CDCl3) d 7.77 (s, 1H), 7.30–7.14 (m, 5H),
4.55–4.35 (m, 2H), 2.65 (t, J = 7.4 Hz, 2H), 2.45–2.30 (m, 2H).
4.1.8. General procedure for preparation of furan-2-carboxylic
acid N0-(2-chloro-6-alkyl-6H-pyrrolo[3,4-d]pyrimidin-4-yl)-
hydrazides (6a–d) and furan-2-carboxylic acid N0-(5-chloro-1/2-
alkyl-1/2H-pyrazolo[4,3-d]pyrimidin-7-yl)-hydrazides (28–
31a,b)
To
a solution of dichloro derivatives 5a-d or 24–27a,b
4.1.7.1. 2,4-Dichloro-6-methyl-6H-pyrrolo[3,4-d]pyrimidine (5a).
Pale yellow solid; 75% yield; crude product; 1H NMR (400 MHz,
CDCl3) d 7.32 (s, 1H), 7.24 (s, 1H), 4.07 (s, 3H). MS (ESI):
[MH]+ = 202.1.
(0.5 mmol) in anhydrous 1,4-dioxane (4 mL) was added TEA
(0.5 mmol) and furan-2-carboxylic acid hydrazide (0.5 mmol).
The reaction was heated at 80–90 °C for 5 h. The solvent was re-
moved under reduced pressure to obtain a crude solid that was
purified via column chromatography eluting with a mixture of
CH2Cl2/CH3OH 9:1.
4.1.7.2. 2,4-Dichloro-6-propyl-6H-pyrrolo[3,4-d]pyrimidine (5b).
Pale yellow solid; 63% yield; crude product; 1H NMR (200 MHz,
CDCl3) d 7.34 (d, J = 2.2 Hz, 1H), 7.25 (s, 1H), 4.20 (t, J = 7 Hz, 2H),
2.02–1.92 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). MS (ESI): [MH]+ = 230.1.
4.1.8.1. Furan-2-carboxylic acid N0-(2-chloro-6-methyl-6H-pyr-
rolo[3,4-d]pyrimidin-4-yl)-hydrazide (6a). Pale yellow solid;
65% yield; mp 250–251 °C dec.; 1H NMR (400 MHz, DMSO-d6) d
11.45 (br s, 1H), 10.40 (br s, 1H), 7.89 (s, 1H), 7.77 (t, J = 4.8 Hz,
1H), 7.40 (br s, 1H), 7.23 (d, J = 2.8 Hz, 1H), 6.66–6.65 (m, 1H),
3.45 (s, 3H). MS (ESI): [MH]+ = 292.2.
4.1.7.3. 2,4-Dichloro-6-phenethyl-6H-pyrrolo[3,4-d]pyrimidine
(5c). Pale yellow solid; 73% yield; crude product; 1H NMR
(400 MHz, DMSO-d6) d 7.88 (d, J = 2 Hz, 1H), 7.72 (d, J = 2 Hz, 1H),
7.29–7.19 (m, 5H), 4.59 (t, J = 7.2 Hz, 2H), 3.21 (t, J = 7.6 Hz, 2H).
MS (ESI): [MH]+ = 293.1.
4.1.8.2. Furan-2-carboxylic acid N’-(2-chloro-6-propyl-6H-pyr-
rolo[3,4-d]pyrimidin-4-yl)-hydrazide (6b). Pale yellow solid;
72% yield; mp 202–203 °C; 1H NMR (200 MHz, DMSO-d6) d 10.60
(br s, 1H), 9.91 (br s, 1H), 7.96 (s, 1H), 7.52 (s, 1H), 7.31–7.28 (m,
1H), 7.18 (br s, 1H), 6.72–6.71 (m, 1H), 4.17 (m, 2H), 1.81 (m,
2H), 0.88–0.80 (m, 3H). MS (ESI): [MH]+ = 320.2.
4.1.7.4. 2,4-Dichloro-6-(3-phenyl-propyl)-6H-pyrrolo[3,4-d]pyrimi-
dine (5d). Pale yellow oil; 81% yield; crude product; 1H NMR
(400 MHz, CDCl3) d 7.33–7.14 (m, 7H), 4.23 (t, J = 7.2 Hz, 2H), 2.65 (t,
J = 6.8 Hz, 2H), 2.31–2.28 (m, 2H). MS (ESI): [MH]+ = 307.1.
4.1.7.5. 5,7-Dichloro-1-methyl-1H-pyrazolo[4,3-d]pyrimidine (24a).
Pale yellow solid; 63% yield; crude product; 1H NMR (400 MHz, CDCl3)
d 8.17 (s, 1H), 4.41 (s, 3H).
4.1.8.3. Furan-2-carboxylic acid N0-(2-chloro-6-phenethyl-6H-
pyrrolo[3,4-d]pyrimidin-4-yl)-hydrazide (6c). Pale yellow so-
lid; 48% yield; mp 209–210 °C; 1H NMR (200 MHz, DMSO-d6) d
10.58 (br s, 1H), 10.17 (br s, 1H), 7.95 (s, 1H), 7.45 (s, 1H), 7.27–
7.12 (m, 8H), 6.71 (s, 1H), 4.47–4.41 (m, 2H), 3.15–3.12 (m, 1H).
MS (ESI): [MH]+ = 382.3.
4.1.7.6.
5,7-Dichloro-2-methyl-2H-pyrazolo[4,3-d]pyrimidine
(24b). Pale yellow solid; 63% yield; crude product; 1H NMR
(400 MHz, CDCl3) d 8.17 (s, 1H), 4.41 (s, 3H).
4.1.8.4. Furan-2-carboxylic acid N0-[2-chloro-6-(3-phenyl-pro-
4.1.7.7. 5,7-Dichloro-1-propyl-1H-pyrazolo[4,3-d]pyrimidine (25a).
Pale yellow oil; 57% yield; crude product; 1H NMR (200 MHz, CDCl3) d
8.20 (s, 1H), 4.69 (t, J = 7 Hz, 2H), 2.04–1.92 (m, 2H), 0.96 (t, J = 7.2 Hz,
3H).
pyl)-6H-pyrrolo[3,4-d]pyrimidin-4-yl]-hydrazide
(6d). Pale
yellow solid; 56% yield; mp 204–205 °C dec.; 1H NMR (400 MHz,
DMSO-d6) d 10.63 (br s, 1H), 9.97 (br s, 1H), 7.96 (s, 1H), 7.55 (s,
1H), 7.32–6.99 (m, 7H), 6.71 (s, 1H), 4.22 (t, J = 6.8 Hz, 2H), 2.56
(t, J = 7.2 Hz, 2H), 2.16- 2.12 (m, 2H). MS (ESI): [MH]+ = 396.3.
4.1.7.8. 5,7-Dichloro-2-propyl-2H-pyrazolo[4,3-d]pyrimidine (25b).
Pale yellow solid; 55% yield; crude product; 1H NMR (200 MHz, CDCl3)
d 8.16 (s, 1H), 4.49 (t, J = 7.2 Hz, 2H), 2.11–2.04 (m, 2H), 0.99 (t,
J = 7.6 Hz, 3H).
4.1.8.5. Furan-2-carboxylic acid N0-(5-chloro-1-methyl-1H-pyr-
azolo[4,3-d]pyrimidin-7-yl)-hydrazide (28a). Pale yellow solid;
82% yield; mp 205 °C dec.; 1H NMR (400 MHz, DMSO-d6) d 10.76
(br s, 1H), 9.98 (br s, 1H), 8.07 (s, 1H), 7.98 (d, J = 1.2 Hz, 1H),
7.33 (d, J = 3.2 Hz, 1H), 6.73–6.72 (m, 1H), 4.29 (s, 3H). MS (ESI):
[MH]+ = 293.3.
4.1.7.9. 5,7-Dichloro-1-phenethyl-1H-pyrazolo[4,3-d]pyrimidine
(26a). Pale yellow solid; 58% yield; crude product; 1H NMR

P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
1055
4.1.8.6. Furan-2-carboxylic acid N0-(5-chloro-2-methyl-2H-pyr-
azolo[4,3-d]pyrimidin-7-yl)-hydrazide (28b). White solid; 40%
yield; mp 158 °C dec.; 1H NMR (400 MHz, DMSO-d6) d 10.67 (br
s, 1H), 10.61 (br s, 1H), 8.42 (s, 1H), 7.96 (d, J = 1.2 Hz, 1H), 7.29
(d, J = 3.2 Hz, 1H), 6.72–6.70 (m, 1H), 4.19 (s, 3H). MS (ESI):
[MH]+ = 293.3.
(d, J = 2 Hz, 1H), 7.62 (d, J = 2 Hz, 1H), 7.04–7.03 (m, 1H), 6.76–
6.74 (m, 1H), 3.96 (s, 3H). MS (ESI): [MH]+ = 274.2.
4.1.9.2. 5-Chloro-(2-furan-2-yl)-8-propyl-8H-pyrrolo[3,4-e][1,2,4]
triazolo[1,5-c]pyrimidine (7b). White solid; 63% yield; mp 81–
82 °C; 1H NMR (200 MHz, DMSO-d6) d 8.02 (d, J = 1 Hz, 1H), 7.93
(d, J = 2 Hz, 1H), 7.68 (d, J = 2 Hz, 1H), 7.02–7.01 (m, 1H), 6.76–
6.75 (m, 1H), 4.18 (t, J = 7.4 Hz, 2H), 1.91–1.79 (m, 2H), 0.85 (t,
J = 7.4 Hz, 3H). MS (ESI): [MH]+ = 302.1.
4.1.8.7. Furan-2-carboxylic acid N’-(5-chloro-1-propyl-1H-pyr-
azolo[4,3-d]pyrimidin-7-yl)-hydrazide (29a). White solid; 70%
yield; mp 217 °C dec.; 1H NMR (200 MHz, DMSO-d6) d 10.77 (br
s, 1H), 9.86 (br s, 1H), 8.11 (s, 1H), 7.98–7.97 (m, 1H), 7.32 (d,
J = 3.4 Hz, 1H), 6.74–6.71 (m, 1H), 4.61 (t, J = 7 Hz, 2H), 1.85–1.75
(m, 2H), 0.80 (t, J = 7.4 Hz, 3H). MS (ESI): [MH]+ = 321.0.
4.1.9.3. 5-Chloro-(2-furan-2-yl)-8-phenethyl-8H-pyrrolo[3,4-e][1,2,4]
triazolo[1,5-c]pyrimidine (7c). White solid; 60% yield; mp
182–183 °C; 1H NMR (400 MHz, DMSO-d6) d 8.02–8.01 (m, 1H),
7.87 (d, J = 2 Hz, 1H), 7.63 (d, J = 1.6 Hz, 1H), 7.29–7.20 (m, 5H),
7.02–7.01 (m, 1H), 6.75–6.74 (m, 1H), 4.49 (t, J = 7.2 Hz, 2H), 3.19
(t, J = 7.2 Hz, 2H). MS (ESI): [MH]+ = 364.0.
4.1.8.8. Furan-2-carboxylic acid N0-(5-chloro-2-propyl-2H-pyr-
azolo[4,3-d]pyrimidin-7-yl)-hydrazide (29b). White solid; 50%
yield; mp 164–165 °C; 1H NMR (200 MHz, DMSO-d6) d 10.66 (br
s, 2H), 8.48 (s, 1H), 7.96 (s, 1H), 7.29 (d, J = 3.6 Hz, 1H), 6.72–6.70
(m, 1H), 4.41 (t, J = 7.2 Hz, 2H), 1.97–1.90 (m, 2H), 0.87 (t,
J = 7.4 Hz, 3H). MS (ESI): [MH]+ = 321.0.
4.1.9.4. 5-Chloro-(2-furan-2-yl)-8-(3-phenyl-propyl)-8H-pyrrol-
o[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (7d). White solid; 65%
yield; mp 138 °C; 1H NMR (400 MHz, DMSO-d6)
d 8.02 (t,
J = 0.8 Hz, 1H), 7.97 (d, J = 2 Hz, 1H), 7.72 (d, J = 2 Hz, 1H), 7.29–
7.19 (m, 5H), 7.03 (d, J = 3.2 Hz, 1H), 6.76–6.74 (m, 1H), 4.26 (t,
J = 6.8 Hz, 2H), 2.55 (t, J = 8.4 Hz, 2H), 2.18 (m, 2H). MS (ESI):
[MH]+ = 379.2.
4.1.8.9. Furan-2-carboxylic acid N’-(5-chloro-1-phenethyl-1H-
pyrazolo[4,3-d]pyrimidin-7-yl)-hydrazide (30a). White solid;
60% yield; mp 197–198 °C dec.; 1H NMR (200 MHz, DMSO-d6) d
10.78 (br s, 1H), 10.04 (br s, 1H), 8.03 (s, 1H), 7.98 (s, 1H), 7.33
(d, J = 3.4 Hz, 1H), 7.24–7.20 (m, 5H), 6.74–6.72 (m, 1H), 4.94–
4.87 (m, 2H), 3.16–3.09 (m, 2H). MS (ESI): [MH]+ = 383.0.
4.1.9.5. 5-Chloro-(2-furan-2-yl)-9-methyl-9H-pyrazolo[3,4-e][1,2,4]
triazolo[1,5-c]pyrimidine (32a). White solid; 43% yield; mp
221–222 °C; 1H NMR (400 MHz, DMSO-d6) d 8.31 (s, 1H), 8.08–
8.07 (m, 1H), 7.10–7.09 (m, 1H), 6.80–6.79 (m, 1H), 4.42 (s, 3H).
MS (ESI): [MH]+ = 275.2.
4.1.8.10. Furan-2-carboxylic acid N0-(5-chloro-2-phenethyl-2H-
pyrazolo[4,3-d]pyrimidin-7-yl)-hydrazide (30b). White solid;
42% yield; mp 137–138 °C dec.; 1H NMR (200 MHz, DMSO-d6) d
10.66 (br s, 2H), 8.37 (s, 1H),7.96 (s, 1H), 7.32–7.17 (m, 6H),
6.72–6.70 (m, 1H), 4.71 (t, J = 6.8 Hz, 2H), 3.36–3.24 (m, 2H). MS
(ESI): [MH]+ = 383.0.
4.1.9.6. 5-Chloro-(2-furan-2-yl)-8-methyl-8H-pyrazolo[3,4-e][1,2,4]
triazolo[1,5-c]pyrimidine (32b). White solid; 52% yield; mp
238 °C; 1H NMR (400 MHz, DMSO-d6) d 8.70 (s, 1H), 8.06–8.05
(m, 1H), 7.07–7.06 (m, 1H), 6.78–6.77 (m, 1H), 4.20 (s, 3H). MS
(ESI): [MH]+ = 275.2.
4.1.8.11. Furan-2-carboxylic acid N0-[5-chloro-1-(3-phenyl-pro-
pyl)-1H-pyrazolo[4,3-d]pyrimidin-7-yl]-hydrazide (31a). Pale
yellow solid; 50% yield; mp 115–116 °C dec.; 1H NMR (200 MHz,
DMSO-d6) d 10.79 (br s, 1H), 9.98 (br s, 1H), 8.10 (s, 1H), 7.99–
7.98 (m, 1H), 7.34–7.16 (m, 6H), 6.74–6.71 (m, 1H), 4.70 (t,
J = 7 Hz, 2H), 2.59–2.49 (m, 2H), 2.13–2.05 (m, 2H). MS (ESI):
[MH]+ = 397.0.
4.1.9.7. 5-Chloro-(2-furan-2-yl)-9-propyl-9H-pyrazolo[3,4-e][1,2,4]
triazolo[1,5-c]pyrimidine (33a). White solid; 76% yield; mp
104–105 °C; 1H NMR (200 MHz, DMSO-d6) d 8.32 (s, 1H), 8.08–
8.06 (m, 1H), 7.10–7.07 (m, 1H), 6.80–6.78 (m, 1H), 4.73 (t,
J = 7 Hz, 2H), 2.02–1.99 (m, 2H), 0.88 (t, J = 7.4 Hz, 3H). MS (ESI):
[MH]+ = 303.0.
4.1.8.12. Furan-2-carboxylic acid N0-[5-chloro-2-(3-phenyl-pro-
pyl)-2H-pyrazolo[4,3-d]pyrimidin-7-yl]-hydrazide (31b). Pale
yellow solid; 56% yield; mp 169–170 °C dec.; 1H NMR (200 MHz,
DMSO-d6) d 10.67 (br s, 2H), 8.51 (s, 1H), 7.97–7.96 (m, 1H),
7.30–7.19 (m, 6H), 6.72–6.70 (m, 1H), 4.49–4.43 (m, 2H), 2.62–
2.49 (m, 2H), 2.29–2.21 (m, 2H). MS (ESI): [MH]+ = 397.0.
4.1.9.8. 5-Chloro-(2-furan-2-yl)-8-propyl-8H-pyrazolo[3,4-e][1,2,4]
triazolo[1,5-c]pyrimidine (33b). White solid; 46% yield; mp
155 °C; 1H NMR (200 MHz, CDCl3) d 8.04 (s, 1H), 7.71–7.70 (m,
1H), 7.00–6.98 (m, 1H), 6.64–6.62 (m, 1H), 4.41 (t, J = 7.2 Hz, 2H),
2.10–2.06 (m, 2H), 0.97 (t, J = 7.6 Hz, 3H). MS (ESI): [MH]+ = 303.0.
4.1.9.9. 5-Chloro-(2-furan-2-yl)-9-(2-phenylethyl)-9H-pyrazol-
o[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (34a). White solid;
89% yield; mp 178 °C; 1H NMR (200 MHz, DMSO-d6) d 8.30 (s,
1H), 8.08–8.07 (m, 1H), 7.26–7.21 (m, 5H), 7.11–7.09 (m, 1H),
6.81–6.78 (m, 1H), 4.99 (t, J = 7.2 Hz, 2H), 3.33 (t, J = 7 Hz, 2H).
MS (ESI): [MH]+ = 365.0.
4.1.9. General procedure for preparation of 5-chloro-2-(furan-2-
yl)-8-alkyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines
(7a–d) and 5-chloro-(2-furan-2-yl)-8/9-alkyl-8/9H-pyrazolo[3,4-
e][1,2,4]triazolo[1,5-c]pyrimidines (32–35a,b)
The pyrrolo- or pyrazolopyrimidines 6a–d or 28–31a,b
(0.2 mmol) were suspended in a mixture of hexamethyldisilazane
(0.5 mL) and bis(trimethylsilyl)acetamide (0.5 mL) and the reac-
tion was heated at 120 °C for 18 h. The excess of reagents was re-
moved under reduced pressure and the residue was purified via
column chromatography eluting with a mixture CH2Cl2/CH3OH
9.5:0.5.
4.1.9.10. 5-Chloro-(2-furan-2-yl)-8-(2-phenylethyl)-8H-pyrazolo
[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (34b). White solid; 57%
yield; mp 186 °C; 1H NMR (200 MHz, CDCl3) d 7.71–7.70 (m, 2H),
7.25–7.24 (m, 3H), 7.11–7.02 (m, 2H), 6.99 (m, 1H), 6.61 (m, 1H),
4.66 (t, J = 7.2 Hz, 2H), 3.35 (t, J = 7 Hz, 2H). MS (ESI):
[MH]+ = 365.0.
4.1.9.1. 5-Chloro-(2-furan-2-yl)-8-methyl-8H-pyrrolo[3,4-e][1,2,4]
triazolo[1,5-c]pyrimidine (7a). White solid; 75% yield; mp
275 °C; 1H NMR (400 MHz, DMSO-d6) d 8.02–8.01 (m, 1H), 7.86
4.1.9.11. 5-Chloro-(2-furan-2-yl)-9-(3-phenylpropyl)-9H-pyraz-
olo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (35a). White solid;

1056
P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
76% yield; mp 155–156 °C; 1H NMR (400 MHz, DMSO-d6) d 8.34 (s,
1H), 8.08–8.07 (m, 1H), 7.25–7.15 (m, 5H), 7.10–7.09 (m, 1H),
6.80–6.79 (m, 1H), 4.81 (t, J = 6.8 Hz, 2H), 2.63 (t, J = 7.2 Hz, 2H),
2.33–2.29 (m, 2H). MS (ESI): [MH]+ = 378.9.
4.1.10.7. 5-Amino-(2-furan-2-yl)-9-propyl-9H-pyrazolo[3,4-e]
[1,2,4]triazolo[1,5-c]pyrimidine (41a). White solid; 82% yield;
mp 229 °C; 1H NMR (200 MHz, DMSO-d6) d 7.98 (s, 1H), 7.88 (s,
1H), 7.47 (br s, 2H), 7.26 (d, J = 3.2 Hz, 1H), 6.77–6.74 (m, 1H),
4.58 (t, J = 7 Hz, 2H), 1.99–1.95 (m, 2H), 0.85 (t, J = 7.6 Hz, 3H).
MS (ESI): [MH]+ = 284.2. Anal. (C13H13N7O) C, H, N.
4.1.9.12. 5-Chloro-(2-furan-2-yl)-8-(3-phenylpropyl)-8H-pyraz-
olo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (35b). White solid;
62% yield; mp 173 °C; 1H NMR (400 MHz, DMSO-d6) d 8.09 (s,
1H), 7.89–7.81 (m, 1H), 7.35–7.22 (m, 5H), 7.01–6.99 (m, 1H),
6.75–6.67 (m, 1H), 4.32 (t, J = 6.8 Hz, 2H), 2.54 (t, J = 7.2 Hz, 2H),
2.25–2.19 (m, 2H). MS (ESI): [MH]+ = 378.9.
4.1.10.8. 5-Amino-(2-furan-2-yl)-8-propyl-8H-pyrazolo[3,4-e]
[1,2,4]triazolo[1,5-c]pyrimidine (41b). White solid; 78% yield;
mp 198 °C; 1H NMR (200 MHz, DMSO-d6) d 8.18 (s, 1H), 7.96–
7.95 (m, 1H), 7.36 (br s, 2H), 7.25–7.23 (m, 1H), 6.76–6.73 (m,
1H), 4.32 (t, J = 7 Hz, 2H), 1.94–1.90 (m, 2H), 0.86 (t, J = 7.4 Hz,
3H). MS (ESI): [MH]+ = 284.2. Anal. (C13H13N7O) C, H, N.
4.1.10. General procedure for preparation of 5-amino-(2-furan-
2-yl)-8-alkyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidines
(8a–d) and 5-amino-(2-furan-2-yl)-8/9-alkyl-8/9H-pyrazolo[3,4-
e][1,2,4]triazolo[1,5-c]pyrimidines (40–43a,b)
The appropriate pyrrolo- or pyrazolo-triazolo-pyrimidine 7a–d
or 32–35a,b (0.2 mmol) was dissolved in 20 mL of EtOH previously
saturated at 0 °C with ammonia. The mixture was heated in a steel
bomb at 60 °C for 18 h. The solvent was removed under reduced
pressure and the residue was purified via column chromatography
eluting with a mixture CH2Cl2/CH3OH 9.5:0.5.
4.1.10.9. 5-Amino-(2-furan-2-yl)-9-(2-phenylethyl)-9H-pyrazol-
o[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (42a). White solid; 85%
yield; mp 239 °C; 1H NMR (200 MHz, DMSO-d6)
d 7.99 (d,
J = 1.2 Hz, 1H), 7.85 (s, 1H), 7.47 (br s, 2H), 7.28–7.14 (m, 6H),
6.78–6.76 (m, 1H), 4.84 (t, J = 6.8 Hz, 2H), 3.33–3.24 (m, 2H). MS
(ESI): [MH]+ = 346.4. Anal. (C18H15N7O) C, H, N.
4.1.10.10. 5-Amine-(2-furan-2-yl)-8-(2-phenylethyl)-8H-pyraz-
olo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (42b). White solid;
82% yield; mp 218 °C; 1H NMR (200 MHz, DMSO-d6) d 8.07 (s,
1H), 7.96 (d, J = 1 Hz, 1H), 7.37 (br s, 2H), 7.27–7.18 (m, 6H),
6.76–6.74 (m, 1H), 4.61 (t, J = 7 Hz, 2H), 3.35–3.25 (m, 2H). MS
(ESI): [MH]+ = 346.4. Anal. (C18H15N7O) C, H, N.
4.1.10.1. 5-Amino-(2-furan-2-yl)-8-methyl-8H-pyrrolo[3,4-e][1,2,4]
triazolo[1,5-c]pyrimidine (8a). White solid; 80% yield; mp
273 °C; 1H NMR (200 MHz, DMSO-d6) d 7.92 (d, J = 1 Hz, 1H), 7.55
(d, J = 2.2 Hz, 1H), 7.16 (t, J = 3.4 Hz, 1H), 7.01–7.00 (m, 3H), 6.72
(m, 1H), 3.87 (s, 3H). MS (ESI): [MH]+ = 255.2. Anal. (C12H10N6O)
C, H, N.
4.1.10.11. 5-Amino-(2-furan-2-yl)-9-(3-phenylpropyl)-9H-pyr-
azolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (43a). White solid;
78% yield; mp 208 °C; 1H NMR (200 MHz, DMSO-d6) d 7.99–7.98
(m, 1H), 7.89 (s, 1H), 7.48 (br s, 2H), 7.25–7.15 (m, 6H), 6.78–
6.75 (m, 1H), 4.63 (t, J = 7 Hz, 2H), 2.61 (t, J = 7.2 Hz, 2H), 2.32–
2.24 (m, 2H). MS (ESI): [MH]+ = 360.4. Anal. (C19H17N7O) C, H, N.
4.1.10.2. 5-Amino-(2-furan-2-yl)-8-propyl-8H-pyrrolo[3,4-e][1,2,4]
triazolo[1,5-c]pyrimidine (8b). White solid; 75% yield; mp
176 °C; 1H NMR (200 MHz, DMSO-d6) d 7.92–7.90 (m, 1H), 7.61
(d, J = 2.2 Hz, 1H), 7.31 (m, 1H), 7.17–7.15 (m, 1H), 7.06 (d,
J = 2.2 Hz, 1H), 6.98 (s, 1H), 6.72–6.70 (m, 1H), 4.08 (t, J = 6.8 Hz,
2H), 1.84–1.77 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H). MS (ESI):
[MH]+ = 283.1. Anal. (C14H14N6O) C, H, N.
4.1.10.12. 5-Amino-(2-furan-2-yl)-8-(3-phenylpropyl)-8H-pyr-
azolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (43b). White solid;
84% yield; mp 162 °C; 1H NMR (200 MHz, CDCl3) d 7.78 (s, 1H),
7.65–7.63 (m, 1H), 7.37–7.16 (m, 6H), 6.62–6.60 (m, 1H), 6.21 (br
s, 2H), 4.37 (t, J = 7.2 Hz, 2H), 2.66 (t, J = 7.8 Hz, 2H), 2.41–2.37
(m, 2H). MS (ESI): [MH]+ = 360.4. Anal. (C19H17N7O) C, H, N.
4.1.10.3. 5-Amino-(2-furan-2-yl)-8-phenethyl-8H-pyrrolo[3,4-e]
[1,2,4]triazolo[1,5-c]pyrimidine (8c). White solid; 77% yield;
mp 149 °C; 1H NMR (400 MHz, DMSO-d6) d 7.91–7.90 (m, 1H),
7.53 (d, J = 2 Hz, 1H), 7.29–7.20 (m, 5H), 7.16–7.15 (m, 1H), 7.05
(d, J = 2 Hz, 1H), 6.99 (br s, 2H), 6.71–6.70 (m, 1H), 4.38 (t,
J = 7.2 Hz, 2H), 3.15 (t, J = 7.2 Hz, 2H). MS (ESI): [MH]+ = 345.4.
Anal. (C19H16N6O) C, H, N.
4.1.11. General procedure for preparation of 5-alkyl-(2-furan-2-
yl)-9-methyl-9H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine
(36–39)
The intermediate 32a (0.5 mmol) was dissolved in 2 mL of 2-
methoxyethanol and 1 mL of the appropriate amine was added to
the solution. The mixture was heated in a steel bomb at 100 °C
for 3 h. The solvent was removed under reduced pressure and
the residue was purified via column chromatography eluting with
a mixture petroleum ether/EtOAc 1:4.
4.1.10.4. 5-Amino-(2-furan-2-yl)-8-(3-phenyl-propyl)-8H-pyr-
rolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (8d). White solid;
77% yield; mp 156 °C; 1H NMR (400 MHz, CDCl3) d 7.62 (m, 1H),
7.44 (m, 1H), 7.30–7.15 (m, 6H), 6.97 (s, 1H), 6.59 (m, 1H), 6.19
(br s, 2H), 4.08 (m, 2H), 2.61 (m, 2H), 2.23 (m, 2H). MS (ESI):
[MH]+ = 359.2. Anal. (C20H18N6O) C, H, N.
4.1.11.1.
N-Cyclohexyl-(2-furan-2-yl)-9-methyl-9H-pyrazol-
4.1.10.5. 5-Amino-(2-furan-2-yl)-9-methyl-9H-pyrazolo[3,4-e]
[1,2,4]triazolo[1,5-c]pyrimidine (40a). White solid; 72% yield;
mp 270 °C; 1H NMR (400 MHz, DMSO-d6) d 7.98–7.97 (m, 1H),
7.85 (s, 1H), 7.47 (br s, 2H), 7.27–7.26 (m, 1H), 6.76–6.75 (m,
1H), 4.28 (s, 3H). MS (ESI): [MH]+ = 256.2. Anal. (C11H9N7O) C, H, N.
o[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (36). White solid; 76%
yield; mp 175 °C; 1H NMR (400 MHz, DMSO-d6) d 7.98 (m, 1H),
7.94 (s, 1H), 7.37 (d, J = 8 Hz, 1H), 7.29–7.28 (m, 1H), 6.76–6.75
(m, 1H), 4.28 (s, 3H), 4.12–3.82 (m, 1H), 1.95–1.13 (m, 10H). MS
(ESI): [MH]+ = 338.4. Anal. (C17H19N7O) C, H, N.
4.1.10.6. 5-Amino-(2-furan-2-yl)-8-methyl-8H-pyrazolo[3,4-e]
[1,2,4]triazolo[1,5-c]pyrimidine (40b). White solid; 77% yield;
mp 295 °C; 1H NMR (400 MHz, DMSO-d6) d 8.14 (s, 1H), 7.96 (d,
J = 1.2 Hz, 1H), 7.37 (br s, 2H), 7.24 (d, J = 3.6 Hz, 1H), 6.75–6.74
(m, 1H), 4.11 (s, 3H). MS (ESI): [MH]+ = 256.2. Anal. (C11H9N7O) C,
H, N.
4.1.11.2. (2-Furan-2-yl)-9-methyl-5-morpholin-4-yl-9H-pyraz-
olo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (37). White solid;
68% yield; mp 193 °C; 1H NMR (400 MHz, DMSO-d6) d 8.15 (s,
1H), 8.08 (m, 1H), 7.14–7.13 (m, 1H), 6.83–6.82 (m, 1H), 4.38 (s,
3H), 3.33 (m, 4H), 2.98 (m, 4H). MS (ESI): [MH]+ = 326.3. Anal.
(C15H15N7O2) C, H, N.

P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
1057
4.1.11.3. (2-Furan-2-yl)-9-methyl-5-(4-methylpiperazin-1-yl)-
9H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (38). White
solid; 73% yield; mp 159 °C; 1H NMR (400 MHz, DMSO-d6) d 8.13
(s, 1H), 8.05–8.04 (m, 1H), 7.11–7.10 (m, 1H), 6.81–6.80 (m, 1H),
4.37 (s, 3H), 3.33 (m, 4H), 3.01 (m, 4H), 2.10 (s, 3H). MS (ESI):
[MH]+ = 339.4. Anal. (C16H18N8O) C, H, N.
DMSO-d6) d 10.35 (br s, 1H), 9.62 (br s, 1H), 8.24 (s, 1H), 8.03–
8.02 (m, 1H), 7.51 (dd, J = 8.8 Hz, 2H), 7.37 (d, J = 3.2 Hz, 1H),
6.95 (dd, J = 8.8 Hz, 2H), 6.78–6.78 (m, 1H), 4.36 (s, 3H), 3.75 (s,
3H). MS (ESI): [MH]+ = 405.0. Anal. (C19H16N8O3) C, H, N.
4.1.12.6. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-2-
yl)-8-methyl-8H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine
(44b). White solid; 61% yield; mp 254 °C; 1H NMR (400 MHz,
DMSO-d6) d 10.38 (br s, 1H), 9.50 (br s, 1H), 8.56 (s, 1H), 8.01–
8.00 (m, 1H), 7.50 (dd, J = 9.2 Hz, 2H), 7.34 (d, J = 2.8 Hz, 1H),
6.95 (dd, J = 9.2 Hz, 2H), 6.78–6.77 (m, 1H), 4.20 (s, 3H), 3.75 (s,
3H). MS (ESI): [MH]+ = 405.0. Anal. (C19H16N8O3) C, H, N.
4.1.11.4. (2-Furan-2-yl)-9-methyl-5-(4-phenylpiperazin-1-yl)-
9H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (39). White
solid; 67% yield; mp 105 °C; 1H NMR (400 MHz, DMSO-d6) d 8.16
(s, 1H),8.03–8.02 (m, 1H), 7.23–7.19 (m, 2H), 7.15–7.14 (m, 1H),
6.91–6.89 (m, 2H), 6.80–6.77 (m, 2H), 4.39 (s, 3H), 3.58 (m, 4H),
3.15 (m, 4H). MS (ESI): [MH]+ = 401.4. Anal. (C21H20N8O) C, H, N.
4.1.12.7. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-2-
yl)-9-propyl-9H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine
(45a). White solid; 50% yield; mp 202 °C; 1H NMR (200 MHz,
DMSO-d6) d 10.37 (br s, 1H), 9.61 (br s, 1H), 8.26 (s, 1H), 8.02 (d,
J =1 Hz, 1H), 7.52 (dd, J = 8.8 Hz, 2H), 7.36 (d, J = 2.6 Hz, 1H), 6.95
(dd, J = 9 Hz, 2H), 6.78–6.77 (m, 1H), 4.66 (t, J = 6.6 Hz, 2H), 3.75
(s, 3H), 2.08–1.99 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H). MS (ESI):
[MH]+ = 433.0. Anal. (C21H20N8O3) C, H, N.
4.1.12. General procedure for preparation of 5-{[(4-methoxy-
phenyl)carbamoyl]amino}-8-alkyl-(2-furan-2-yl)-8H-pyrrolo[3,
4-e][1,2,4]triazolo[1,5-c]pyrimidine (9a-d) and 5-{[(4-methoxy-
phenyl)carbamoyl]amino}-8/9-alkyl-(2-furan-2-yl)-8/9H-pyrazolo
[3,4 e][1,2,4]triazolo[1,5-c]pyrimidine (44–47a,b)
To a solution of the amino derivatives 8a-d or 40–43a,b
(0.27 mmol) in anhydrous THF (5 mL) was added 4-methoxy-
phenyl-isocyanate (0.54 mmol). The mixture was heated at 50 °C
for 18 h. The solvent was removed under reduced pressure and
the residue was purified via column chromatography eluting with
EtOAc. The resulting solid was further purified by crystallization
from CH3OH.
4.1.12.8. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-2-
yl)-8-propyl-8H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine
(45b). White solid; 40% yield; mp 238 °C; 1H NMR (200 MHz,
DMSO-d6) d 10.43 (br s, 1H), 9.58 (br s, 1H), 8.60 (s, 1H), 8.01
(s, 1H), 7.51 (dd, J = 8.8 Hz, 2H), 7.33 (d, J = 3.4 Hz, 1H), 6.95 (dd,
J = 9 Hz, 2H), 6.79–6.76 (m, 1H), 4.41 (t, J = 6.8 Hz, 2H), 3.75 (s,
3H), 1.98–1.94 (m, 2H), 0.88 (t, J = 7.4 Hz, 3H). MS (ESI):
[MH]+ = 433.1. Anal. (C21H20N8O3) C, H, N.
4.1.12.1. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-2-
yl)-8-methyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine
(9a). White solid; 37% yield; mp 215 °C; 1H NMR (200 MHz,
DMSO-d6) d 10.68 (br s, 1H), 9.18 (br s, 1H), 7.96–7.95 (m, 1H),
7.74 (d, J = 2 Hz, 1H), 7.52 (dd, J = 9.2 Hz, 2H), 7.46 (s, 1H), 7.26
(d, J = 3.2 Hz, 1H), 6.94 (dd, J = 9 Hz, 2H), 6.75–6.73 (m, 1H), 3.95
(s, 3H), 3.76 (s, 3H). MS (ESI): [MH]+ = 404.3. Anal. (C19H17N7O3)
C, H, N.
4.1.12.9. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-2-
yl)-9-(2-phenylethyl)-9H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-
c]pyrimidine (46a). White solid; 31% yield; mp 199 °C; 1H NMR
(400 MHz, DMSO-d6) d 10.39 (br s, 1H), 9.55 (br s, 1H), 8.23 (s, 1H),
8.04 (s, 1H), 7.51 (dd, J = 8.8 Hz, 2H), 7.37 (d, J = 3.6 Hz, 1H), 7.24–
7.23 (m, 5H), 6.95 (dd, J = 8.8 Hz, 2H), 6.80 (d, J = 1.2 Hz, 1H), 4.92
(t, J = 7 Hz, 2H), 3.75 (s, 3H), 3.34–3.27 (m, 2H). MS (ESI):
[MH]+ = 495.5. Anal. (C26H22N8O3) C, H, N.
4.1.12.2. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-2-
yl)-8-propyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine
(9b). White solid; 40% yield; mp 198 °C; 1H NMR (200 MHz,
DMSO-d6) d 10.72 (br s, 1H), 9.16 (br s, 1H), 7.97–7.96 (m, 1H),
7.82 (m, 1H), 7.55–7.50 (m, 3H), 7.26 (d, J = 3.4 Hz, 1H), 6.95
(d, J = 9.2 Hz, 2H), 6.76–6.73 (m, 1H), 4.18 (t, J = 7.4 Hz, 2H), 3.75
(s, 3H), 1.89–1.85 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H). MS (ESI):
[MH]+ = 432.1. Anal. (C22H21N7O3) C, H, N.
4.1.12.10. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-
2-yl)-8-(2-phenylethyl)-8H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]
pyrimidine (46b). White solid; 30% yield; mp 227 °C; 1H NMR
(400 MHz, DMSO-d6) d 10.42 (br s, 1H), 9.51 (br s, 1H), 8.41 (s,
1H), 8.03 (s, 1H), 7.51 (dd, J = 8.8 Hz, 2H), 7.34 (d, J = 3.6 Hz, 1H),
7.22–7.18 (m, 5H), 6.95 (dd, J = 8.8 Hz, 2H), 6.78 (d, J = 1.2 Hz,
1H), 4.73 (t, J = 7 Hz, 2H), 3.75 (s, 3H), 3.33–3.28 (m, 2H). MS
(ESI): [MH]+ = 495.5. Anal. (C26H22N8O3) C, H, N.
4.1.12.3. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-2-
yl)-8-phenylethyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimi-
dine (9c). White solid; 35% yield; mp 182 °C; 1H NMR (200 MHz,
DMSO-d6) d 10.66 (br s, 1H), 9.14 (br s, 1H), 7.96 (s, 1H), 7.74 (d,
J = 2 Hz, 1H), 7.54 (d, J = 2 Hz, 1H), 7.51 (dd, J = 9 Hz, 2H), 7.28–
7.21 (m, 6H), 6.95 (dd, J = 9 Hz, 2H), 6.76–6.73 (m, 1H), 4.49 (t,
J = 7.2 Hz, 2H), 3.76 (s, 3H), 3.20 (t, J = 7.2 Hz, 2H). MS (ESI):
[MH]+ = 494.2. Anal. (C27H23N7O3) C, H, N.
4.1.12.11. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-
2-yl)-9-(3-phenylpropyl)-9H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]
pyrimidine (47a). White solid; 32% yield; mp 206 °C; 1H NMR
(200 MHz, DMSO-d6) d 10.39 (br s, 1H), 9.68 (br s, 1H), 8.26 (s,
1H), 8.03–8.02 (m, 1H), 7.52 (dd, J =9 Hz, 2H), 7.33–7.31 (m, 1H),
7.22–7.19 (m, 5H), 6.95 (dd, J = 9 Hz, 2H), 6.81–6.78 (m, 1H), 4.72
(t, J = 7 Hz, 2H), 3.75 (s, 3H), 2.62 (t, J = 7.2 Hz, 2H), 2.39–2.21 (m,
2H). MS (ESI): [MH]+ = 509.6. Anal. (C27H24N8O3) C, H, N.
4.1.12.4. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-2-
yl)-8-phenylpropyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimi-
dine (9d). White solid; 38% yield; mp 176 °C; 1H NMR (200 MHz,
DMSO-d6) d 10.39 (br s, 1H), 9.68 (br s, 1H), 8.26 (s, 1H), 8.03–8.02
(m, 1H), 7.52 (dd, J =9 Hz, 2H), 7.33–7.31 (m, 1H), 7.22–7.19 (m, 6H),
6.95 (dd, J = 9 Hz, 2H), 6.81–6.78 (m, 1H), 4.72 (t, J = 7 Hz, 2H), 3.75
(s, 3H), 2.62 (t, J = 7.2 Hz, 2H), 2.39–2.21 (m, 2H). MS (ESI):
[MH]+ = 508.3. Anal. (C28H25N7O3) C, H, N.
4.1.12.12. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-
2-yl)-8-(3-phenylpropyl)-8H-pyrazolo[3,4-e][1,2,4]triazolo[1,5-c]
pyrimidine (47b). White solid; 35% yield; mp 236 °C; 1H NMR
(200 MHz, DMSO-d6) d 10.41 (br s, 1H), 9.63 (br s, 1H), 8.52 (s,
1H), 8.01(m, 1H), 7.52 (dd, J = 9 Hz, 2H), 7.30–7.29 (m, 1H), 7.22–
7.18 (m, 5H), 6.95 (dd, J = 9 Hz, 2H), 6.80 (m, 1H), 4.71 (t, J = 7 Hz,
2H), 3.75 (s, 3H), 2.62 (t, J = 7.2 Hz, 2H), 2.37–2.20 (m, 2H). MS
(ESI): [MH]+ = 509.6. Anal. (C27H24N8O3) C, H, N.
4.1.12.5. 5-{[(4-Methoxy-phenyl)carbamoyl]amino}-(2-furan-2-
yl)-9-methyl-9H-pyrazolo[3,4-e][1,2,4]triazolo[1,5c]pyrimidine
(44a). White solid; 55% yield; mp 223 °C; 1H NMR (400 MHz,

1058
P. G. Baraldi et al. / Bioorg. Med. Chem. 20 (2012) 1046–1059
4.1.13. 5-{[(4-Pyridinyl)-carbamoyl]amino}-2-(2-furyl)-8-
methyl-8H-pyrrolo[3,4-e][1,2,4]triazolo[1,5-c]pyrimidine (10)
The compound was prepared as previously reported.27 White
solid; 45% yield; mp 206 °C; 1H NMR (200 MHz, DMSO-d6) d 10.8
(br s, 1H), 9.69 (br s, 1H),8.45–8.44 (m, 2H), 7.96–7.95 (m, 1H),
7.75 (m, 1H), 7.60–7.58 (m, 2H), 7.48 (m, 1H), 7.25–7.24 (m, 1H),
6.75–6.73 (m, 1H), 3.96 (s, 3H). MS (ESI): [MH]+ = 375.4. Anal.
(C18H14N8O2) C, H, N.
0.1, Hepes 0.01, MgCl2 1, glucose 0.5, pH 7.4 at 37 °C, 2 IU/mL aden-
osine deaminase and 4-(3-butoxy-4-methoxybenzyl)-2-imidazo-
lidinone (Ro 20-1724) as phosphodiesterase inhibitor and
preincubated for 10 min in a shaking bath at 37 °C. The potencies
of novel compounds at hA2B adenosine receptors were determined
by antagonism of NECA (100 nM)-induced stimulation of cyclic
AMP levels. The potencies of the examined ligands to hA3 adeno-
sine receptors were determined in the presence of Forskolin
(1 lM) by antagonism of the Cl-IB-MECA (100 nM)-induced inhibi-
4.2. Biology experiments
tion of cyclic AMP levels.37 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 compe-
tition protein binding assay. Samples of cyclic AMP standard (0–
10 pmol) were added to each test tube containing the incubation
buffer (trizma base 0.1 M, aminophylline 8.0 mM, 2 mercap-
toethanol 6.0 mM, pH 7.4) and [3H] cyclic AMP in a total volume
of 0.5 mL. The binding protein previously prepared from beef adre-
nals, was added to the samples previously incubated at 4 °C for
150 min, and after the addition of charcoal were centrifuged at
2000g for 10 min. The clear supernatant was counted in a 2500-
TR Packard scintillation counter.
4.2.1. CHO membrane preparation
The human A1, A2A, A2B and A3 adenosine receptors have been
transfected in CHO cells according to the method previously de-
scribed.34 The cells were grown adherently and maintained in Dul-
becco’s modified Eagles medium with nutrient mixture F12
(DMEM/F12) without nucleosides, containing 10% fetal calf serum,
penicillin (100 U/mL), streptomycin (100
l
g/mL),
L-glutamine
(2 mM) and Geneticin (G418, 0.2 mg/mL) at 37 °C in 5% CO2/95%
air. For membrane preparations, the culture medium was removed
and the cells were washed with PBS and scraped off T75 flasks in
ice-cold hypotonic buffer (5 mM Tris HCl, 2 mM EDTA, pH 7.4).
The cell suspension was homogenized with a Polytron and the
homogenate was spun for 10 min at 1000g. The supernatant was
then centrifuged for 30 min at 100,000g. The membrane pellet
was suspended in: (a) 50 mM Tris HCl buffer pH 7.4 for A1 adeno-
sine receptors; (b) 50 mM Tris HCl, 10 mM MgCl2 buffer pH 7.4 for
A2A adenosine receptors; (c) 50 mM Tris HCl, 10 mM MgCl2, 1 mM
EDTA buffer pH 7.4 for A3 adenosine receptors. The cell suspen-
sions were incubated with 2 IU/mL of adenosine deaminase for
30 min at 37 °C. The membrane preparation was used to perform
competition binding experiments.
4.2.4. Data analysis
The protein concentration was determined according to a Bio-
Rad method38 with bovine albumin as a standard reference. Inhib-
itory binding constants, Ki values, were calculated from those of
IC50 according to the Cheng and Prusoff equation39 Ki = IC50
/
(1 + [C⁄]/KD⁄), where [C⁄] is the concentration of the radioligand
⁄
and KD its dissociation constant. A weighted non-linear least-
squares curve fitting program LIGAND40 was also used for
computer analysis of inhibition experiments. Potency values
(IC50) obtained in cyclic AMP assays were calculated by non-linear
regression analysis using the equation for a sigmoid concentration-
response curve (Graph PAD Prism, San Diego, CA, USA). Affinity or
potency values are expressed as geometric mean, with 95% or 99%
confidence limits.
4.2.2. Human cloned A1, A2A and A3 adenosine receptor binding
assay
All synthesized compounds have been tested for their affinity at
human A1, A2A and A3 adenosine receptors. Displacement binding
experiments of [3H]-DPCPX (1 nM) to hA1CHO membranes (50
lg
of protein/assay) and at least 6–8 different concentrations of the
examined compounds were performed for 120 min at 25 °C. Non-
specific binding was determined in the presence of 1 M of DPCPX
and this was always 610% of the total binding.35
Acknowledgments
We thank King Pharmaceuticals, Inc., Research & Development,
4000 Centre Green Way, Suite 300, Cary, NC 27513, USA, for finan-
cial support. We also thank Professor Karl-Norbert Klotz for cDNA
encoding the human adenosine receptors.
Displacement binding experiments of [3H]-ZM 241385 (2 nM)
to hA2ACHO membranes (50 lg of protein/assay) and at least 6–8
different concentrations of the tested ligands were performed for
60 min at 4 °C. Non-specific binding was determined in the pres-
ence of 1
Displacement binding experiments of [3H]-MRE-3008-F20
(1 nM) to hA3CHO membranes (50 g of protein/assay) and at least
l
M ZM 241385 and was about 20% of total binding.36
References and notes
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