New strategies for the synthesis of A3 adenosine receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(03)00484-X

New A₃ adenosine receptor antagonists were synthesized and tested at human adenosine receptor subtypes. An advanced synthetic strategy permitted us to obtain a large amount of the key intermediate 5 that was then submitted to alkylation procedures in order to obtain the derivatives 6-8. These compounds were then functionalised into ureas at the 5-position (compounds 9-11, 18 and 19) to evaluate their affinity and selectivity versus hA₃ adenosine receptor subtype; in particular, compounds 18 and 19 displayed a value of affinity of 4.9 and 1.3 nM, respectively. Starting from 5, the synthetic methodologies employed permitted us to perform a rapid and a convenient divergent synthesis. A further improvement allowed the regioselective preparation of the N⁸-substituted compound 7. This method could be used as an helpful general procedure for the design of novel A₃ adenosine receptor antagonists without the difficulty of separating the N⁸-substituted pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines from the corresponding N ⁷-isomers.

Full text of DOI:10.1016/S0968-0896(03)00484-X

Bioorganic & Medicinal Chemistry 11 (2003) 4161–4169
New Strategies for the Synthesis of A3 Adenosine Receptor
Antagonists
Pier Giovanni Baraldi,a,* Andrea Bovero,a Francesca Fruttarolo,a Romeo Romagnoli,a
Mojgan Aghazadeh Tabrizi,a Delia Preti,a Katia Varani,b Pier Andrea Boreab
and Allan R. Moormanc
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, Via Fossato di Mortara, 17/19 44100 Ferrara, Italy
b
`
Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, Universita di Ferrara, Via Fossato di Mortara,
17/19 44100 Ferrara, Italy
cKing Pharmaceuticals Research and Development, 4000 CentreGreen Way, Suite 300, Cary, NC 27513, USA
Received 4 March 2003; accepted 18 July 2003
Abstract—New A3 adenosine receptor antagonists were synthesized and tested at human adenosine receptor subtypes. An advanced
synthetic strategy permitted us to obtain a large amount of the key intermediate 5 that was then submitted to alkylation procedures
in order to obtain the derivatives 6–8. These compounds were then functionalised into ureas at the 5-position (compounds 9–11, 18
and 19) to evaluate their affinity and selectivity versus hA3 adenosine receptor subtype; in particular, compounds 18 and 19 dis-
played a value of affinity of 4.9 and 1.3 nM, respectively. Starting from 5, the synthetic methodologies employed permitted us to
perform a rapid and a convenient divergent synthesis. A further improvement allowed the regioselective preparation of the N8-
substituted compound 7. This method could be used as an helpful general procedure for the design of novel A3 adenosine receptor
antagonists without the difficulty of separating the N8-substituted pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines from the
corresponding N7-isomers.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
treatment of inflammation and in regulation of cell
growth.7,8
Adenosine, an endogenous modulator of a wide range
of biological functions in the nervous, cardiovascular,1
renal, and immune systems, interacts with at least four
cell surface receptor subtypes classified as A1, A2A, A2B
and A3. These receptor subtypes belong to the super-
family of G protein-coupled receptors and have been
cloned from several animal species.2 In particular, the
A3 adenosine receptor subtype, which is distributed in
different organs (lung, liver, heart, kidney, and, in low
density, in the brain)3 exerts its action through the
modulation of two second messengers systems: stimula-
tion of phospholipases C4 and D5 and inhibition of
adenylate cyclase.6 The potential therapeutic applica-
tions of activating or antagonizing this receptor subtype
have been investigated in recent years and in particular,
antagonists for A3 receptor promise to be useful for the
Recently, our research group reported a large series of
pyrazolo-triazolo-pyrimidines bearing substituted phe-
nylcarbamoyl residues at the amino group at the 5-
position (Chart 1, compound a) as highly potent and
selective antagonists of the human A3 adenosine recep-
tor.9,10 Among these, Compound b (5-[(phenyl)amino]-
carbonyl]amino-8-methyl-2-(2-furyl)-pyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c] pyrimidine (Chart 1) showed highly
favorable binding affinity and selectivity for the human
A3 adenosine receptor.11
Unfortunately, the major problem within this class of
compounds is the typical low water solubility that has
limited the in vivo pharmacological screening. Starting
from these experimental observations, we decided to
introduce oxygenated functions like b-hydroxyethyl,
acetic, and diethyloxyethyl groups at the 8-position of
the pyrazole nitrogen (Fig. 1) with the aim of increasing
the water solubility of the final compounds obtained
*Corresponding author. Tel.: +39-0532-291293; fax: +39-0532-
291296; e-mail: pgb@dns.unife.it
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00484-X

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P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 4161–4169
(10, 11 and 19). As in the typical A3-antagonist struc-
tures previously synthesized by our group, the 5-posi-
tion of the new N8-substituted-pyrazolo[4,3-e]1,2,4-
triazolo[1,5-c]pyrimidine derivatives was functionalized
as 3-chlorophenyl or simply phenyl ureas in order to
evaluate their affinity and selectivity versus the human
A3 adenosine receptor subtype.
procedures. However, the alkylation of compound 5
furnished a mixture of the N7- and N8-alkylated regio-
isomers, not easily separated by chromatography. In
this paper, we also described a multistep regioselective
strategy, reported in the chemistry section, to prepare
the N8-substituted derivative 7 in good yield, devoid of
the corresponding N7-isomer. This synthetic route pro-
vides only substituted 3-amino-4-cyano-pyrazoles that
are then utilized to obtain tricyclic pyrazolo-triazolo-
pyrimidine derivatives functionalized at the 8-position
of the pyrazole nitrogen.
These new derivatives were obtained by direct alkyl-
ation of the 2-furan-2-yl-7H-pyrazolo[4,3-e]-1,2,4-tria-
zolo[1,5-c]pyrimidin-5-yl amine (5) with the appropriate
alkyl halides. The tricyclic starting compound 5 was
synthesized using an expedited synthetic methodology12
that permitted us to obtain this important key inter-
mediate in a very good yield. In the past the same
intermediate was obtained by a tedious de-tert-butyla-
tion procedure, performed by the treatment of 2-furan-
2-yl-7-tert-butyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]-
pyrimidin-5-yl-amine with 99% formic acid for 48 h.10
Chemistry
Synthesis of 5 has been performed using a new synthetic
strategy described in the patent literature and depicted
in Scheme 1.12 Starting from diethyl malonate and gua-
nidine hydrochloride, both commercially available,
condensation in basic conditions gave the intermediate
2-amino-4,6-dihydroxypyrimidine 1.13 The correspond-
ing transformation into 2 was achieved in good yield by
treatment with POCl3 and DMF (Vilsmeier’s reaction)14
at refluxing temperature. Compound 2 was then reacted
with 2-furoic acid hydrazide. Compound 3 was then
cyclized by treatment with hydrazine in ethanol to fur-
nish 4. Treatment of 4 with DMF, HMDS and BSA at
220 ꢀC (Dimroth-type rearrangement) induced the
formation of compound 5 in good yield which was sub-
sequently utilized for alkylation reactions.
The new synthetic steps employed allowed us to acquire
a large amount of 5 in a very short time and the inter-
mediates obtained did not require tedious purifications
The intermediate 5 was functionalised as reported in
Scheme 2. Alkylation of 5 with bromoacetaldeyde di-
ethylacetal, 2-iodoethanol and 2-bromoacetic acid tert-
butyl ester in DMF and 60% NaH gave compounds 6–8
which are separated from the N7-isomers by flash chro-
matography.
The free amino group at the 5-position of compounds
6 and 8 was converted into the corresponding ureas
(9–10) by treatment with phenylisocyanate or 3-chloro-
phenylisocyanate and catalytic amount of TEA (Scheme
3). The tert-butyl ester at the 7-position of 9 was
Figure 1. New A3 adenosine receptor antagonists synthesized.
Chart 1. Structure of the pyrazolo-triazolo-pyrimidines a and b as hA3 adenosine receptor antagonists.

P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 4161–4169
4163
removed by treatment with TFA at reflux temperature.
The free acid function was conceived to increase the
water solubility of the final compound 11.
concentrated HCl provided only 13 in good yield. The
free hydroxyl group was then protected by treatment
with benzylbromide and the free amino group of the
compound 14 was transformed into the imidate 15 by
refluxing in HC(OEt)3. The pyrazolo[4,3-e]1,2,4-tri-
azolo[1,5-c]pyrimidine 17 was obtained using the syn-
thetic steps already employed for the synthesis of the
tricyclic compounds and reported in our previous
papers.9,10 The imidate 15 was reacted with 2-furoic acid
hydrazide in refluxing 2-methoxyethanol to provide the
pyrazolo[4,3-e]pyrimidine intermediate, following the
For the synthesis of compound 7, was utilized a regio-
selective synthetic procedure that permitted the pre-
paration only of the N8-isomer as depicted in Scheme 4.
The reaction between benzaldehyde and 2-hydroxy-
ethylhydrazine, commercially available, gave the corre-
sponding hydrazone 12. Subsequent reaction with
ethoxymethylenemalononitrile and hydrolysis with
.
Scheme 1. Reagents: (i) EtONa, EtOH abs; (ii) POCl3, DMF, Rfx; (iii) 2-furoic acid hydrazide, THF; (iv) hydrazine H2O, 2-methoxyethanol;
(v) HMDS, BSA, DMF.
Scheme 2. Reagents: (i) bromacetaldehyde diethylacetal, NaH, 80 ꢀC; (ii) iodoethanol, 60% NaH, rt; (iii) 2-bromoacetic acid t-butylester, 60%
NaH, 80 ꢀC.

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P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 4161–4169
method of Gatta et al.15 The latter compound was con-
verted through a thermally-induced cyclization in
diphenylether to the tricyclic derivative 16 in good yield.
Treatment of 16 with dilute hydrochloric acid induced
pyrimidine ring opening. The intermediate was then
converted into 17 by treatment with an excess of cyan-
amide in 1-methyl-2-pyrrolidone at 160 ꢀC. The urea
derivative 19 was obtained by treatment of 17 with
phenyl isocyanate to afford the O-protected 18. The
final deprotection of the hydroxylic group provided 19
in a good yield. The O-debenzylation at the 8-position,
necessary to reduce the reactivity of the hydroxylic
group in the side chain, was achieved by treatment with
HCO2NH4 and 10% Pd/C to afford 7.
of the pyrimidine ring, show poor affinity and low
selectivity only versus the A2A adenosine receptor sub-
type. The transformation of the amino function into
urea leads to a significant increase of the A3 interaction
and a drastic decrease in A2A interaction (compounds
9–11). This behavior confirms that functionalization of
the free amino group in addition to an alkyl or arylalkyl
substituent at the 8-position of the pyrazole nitrogen is
necessary for strong interaction with the A3 adenosine
receptor.
The best results in terms of affinity and selectivity versus
the human A3 adenosine receptor subtype are shown by
compound 19, that display an hydroxyethyl group at
the 8-position of the pyrazole nitrogen and a phenylur-
eido function at the 5-position. The corresponding O-
protected compound 18 also has good affinity versus the
hA3 receptor, but less than the deprotected analogue,
probably due to the steric hindrance introduced by the
benzyl group; however, it’s important to highlight that
the presence of this protecting group is favorable for the
interaction with the A1 adenosine receptor subtype
(compounds 17 and 18, Ki A1=15 and 32 nM, respec-
tively).
Results and Discussion
The biological results of the synthesized compounds at
human A1, A2A, A2B and A3 adenosine receptors,
expressed as affinity values (Ki), are shown in Table 1.
Alkylation of the key intemediate 5 furnished com-
pounds 6–8 that were also tested in binding experi-
ments, with particular regard to the affinity value versus
the A3 adenosine subtype. We can note that these com-
pounds, displaying a free amino group at the 5-position
The free carboxylic acid function of 11 was designed to
improve the water solubility of the compound by the
transformation of the carboxylic group into a salt; the
selectivity of this molecule is very good but the affinity
versus the A3 receptor subtype is quite low and less than
the tert-butyl derivative 9. The diethyloxyethyl group of
10 also represents a versatile functionality, with the
possibility of deprotecting the aldehydic group and
coupling it with amines or other nucleophiles to obtain
compounds with enhanced water-solubility.
In order to evaluate the increase of the hydrophilic
profile of the new derivatives obtained, the hydrophilic/
lipophilic balance was determined by the calculation of
the ClogP values derived from ChemDraw Ultra ana-
lyses, version 6.0.1, Cambridge Software. The ClogP
values calculated for 11 and 19 are 3.23 and 2.93,
respectively, versus compound b that displayed a lipo-
philic value of 3.63.
The methodology applied to obtain 5 was demonstrated
to be a convenient synthetic route: the intermediates are
obtained in good yield, minor synthetic steps are
employed, and easy purifications are the principals
advantages in contrast to the de-tert-butylation path-
way used in the past by our research group.10
Starting from 5, the synthetic pathway used for the
synthesis of the compounds described in this work per-
mitted us to prepare several molecules endowed with
adenosine antagonist activity, performing a rapid and a
divergent synthesis. Further, the regioselective route to
achieve the N8-substituted tricyclic derivatives, per-
formed for the synthesis of 7, is an useful and convenient
methodology that permits the direct preparation of N8-
functionalized compounds with high affinity and selec-
tivity versus the A3 adenosine receptor subtype, after the
transformation of the free amino group into an urea.
Scheme 3. Reagents: (i) phenylisocyanate, TEA, dry CH3CN; (ii) 3-
chlorophenylisocyanate, TEA, dry CH3CN; (iii) TFA, rt.

P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 4161–4169
4165
Scheme 4. Reagents: (i) 2-hydroxyethylhydrazine, abs EtOH; (ii) ethoxymethylene-malono nitrile, benzene; (iii) concd HCl; (iv) benzylbromide, 60%
NaH; (v) HC(OEt)3, rfx; (vi) 2-furoic acid hydrazide, 2-methoxyethanol; (vii) Ph2O, 260 ꢀC; (viii) aq HCl 10%; (ix) NH2CN, pTsOH; (x) HCO2NH4,
Pd/C 10%; (xi) phenylisocyanate, TEA.
Conclusion
Experimental
In summary, we have reported new synthetic meth-
odologies to obtain the key intermediate 5 in large
amount and an extendible regioselective route to obtain
selectively N8-substituted pyrazolo[4,3-e]-1,2,4-triazolo-
[1,5-c]pyrimidine-N5-ureas. The compounds described
in this work have an high affinity and selectivity toward
the hA3 adenosine receptor subtype displaying hydro-
philic functionalities, an important feature to improved
the pharmacological profile and to make in vivo phar-
macological screening easier.
Reactions were routinely monitored by thin-layer chro-
matography (TLC) on silica gel (precoated F245 Merck
plates) and products visualized with iodine or potassium
1
permanganate solution. H NMR were determined in
CDCl3 or DMSO-d6 solutions with a Bruker AC 200
spectrometer. Peak positions are given in parts per mil-
lion (d) downfield from tetramethylsilane as internal
standard, and J values are given in Hz. Light petroleum
ether refers to the fractions boiling at 40–60 ꢀC. Melting
points were determined on a Buchi-Tottoli instrument

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P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 4161–4169
and are uncorrected. Chromatographies were performed
using Merck 60–200 mesh silica gel. All products
reported showed H NMR spectra in agreement with
the assigned structures. Organic solutions were dried
over anhydrous magnesium sulphate. Elemental ana-
lyses were performed by the micro-analytical laboratory
of Dipartimento di Chimica, University of Ferrara, and
were within ꢁ0.4% of the theoretical values for C, H,
and N. Elemental analyses are reported in Table 2.
(0.5 g, 1.7 mmol) in 2-methoxyethanol (25 mL) was
added hydrazine monohydrate (0.16 mL, 3.4 mmol) and
the mixture was stirred at room temperature for 1.5 h.
Then the solvent was removed at reduced pressure and
the residue was crystallized from a mixture of methanol
and ethyl ether (8:1). The solid formed was filtered off
and dried to furnish 4 as a yellow solid (1.4 mmol,
83%). Mp 272 ꢀC; 1H NMR (DMSO-d6) d: 6.72 (s, 1H),
7.52 (s, 1H), 7.96 (bs, 2H), 8.02 (s, 1H), 8.54 (s, 1H),
10.97 (bs, 1H), 11.38 (bs, 1H), 13.35 (bs, 1H).
1
Furan-2-carboxylic acid N-(2-amino-6-chloro-5-formyl-
pyrimidin-4-yl)-hydrazide (3)
2-Furan-2-yl-7H-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyri-
midin-5-yl amine (5). To a suspension of 4 (0.5 g,
1.9 mmol) in DMF (12 mL) was added HMDS (4 mL,
19 mmol) and BSA (4.6 mL, 19 mmol). The mixture was
heated at 230 ꢀC for 30 min, then the solvent was
removed under vacuum. The residue obtained was
crystallized from methanol to furnish 5 as yellow solid
(1.2 mmol, 67%). Mp >300 C; H NMR (DMSO-d6)
d: 6.72 (t, 1H, J=1.4), 7.20 (d, 1H, J=1.4), 7.90 (d, 1H,
J=1.5), 7.92 (bs, 2H), 8.18 (s, 1H), 13.30 (bs, 1H).
To a suspension of 2 (1 g, 5.2 mmol) in dry THF
(40 mL) was added 2-furoic acid hydrazide (730 mg,
5.72 mmol) and TEA (5.2 mmol). The mixture was
heated at reflux for 2 h, then the solvent was removed at
reduced pressure. The residue was crystallized from a
mixture of methanol and ethyl ether (8:1). The solid
formed was filtered off and dried to furnish 3 as a yellow
ꢀ
1
solid (4.2 mmol, 82%). Mp 130 ꢀC; H NMR (DMSO-
1
d6) d: 6.67 (t, 1H, J=2), 7.25 (d, 1H, J=2.1), 7.88 (bs,
2H), 7.91 (d, 1H, J=2), 9.95 (s, 1H), 10.36 (bs, 1H),
10.69 (bs, 1H).
General procedure for the preparation of compounds 6–8
To a stirred suspension of 60% NaH (1.2 mmol) in dry
DMF (15 mL), chilled in an ice-bath, was added 5
(1.2 mmol) in small portions and the mixture was stirred
at room temperature for several minutes. Then was
added the appropriate alkyl halide (1.2 mmol) in dry
DMF (2 mL) and the reaction was heated at 80 ꢀC until
the TLC analyses indicated the disappearance of the
starting material (14–16 h, 1:1 EtOAc/light petroleum).
The solvent was removed at reduced pressure and the
residue was purified by flash chromatography (1:1
EtOAc/light petroleum) to afford 6–8 as solids, free of
N7-isomers.
Furan-2-carboxylic acid N-(6-amino-1H-pyrazolo[3,4-
d]pyrimidin-4-yl)-hydrazide (4). To a solution of 3
Table 1. Biological data of synthesized compounds 6–19
Compd
A1
Ki (nM)a
A2A
Ki (nM)b
A2B
Ki (nM)c
A3
Ki (nM)d
6
7
8
130 (106–158) 100 (82–120) 376 (332–426) >1000 (84%)
136 (110–167) 88 (75–104) 71 (54–94) 900 (829–976)
>1000 (72%) 250 (202–301) >1000 (80%) >1000 (77%)
>1000 (69%) >1000 (68%) >1000 (81%) 39 (20–80)
250 (208–299) 796 (717–883) 876 (831–924) 5.5 (3.2–9.4)
>1000 (76%) >1000 (75%) >1000 (80%) 62 (50–77)
35 (27–45) 609 (547–678)
103 (79–134) 202 (152–267) 4.9 (3.4–7.2)
9
10
11
17
18
19
6: 8-(2,2-Diethoxy-ethyl)-2-furan-2-yl-8H-pyrazolo[4,3-
e]-1,2,4-triazolo[1,5-c] pyrimidin-5-yl amine. White solid,
yield 60%, 0.72 mmol; mp 213 ꢀC; 1H NMR (DMSO-d6)
d: 1.04 (t, 6H, J=7.6), 3.42 (m, 2H), 3.65 (m, 2H), 4.35
(d, 2H, J=6), 4.92 (t, 1H, J=7.4), 6.73 (m, 1H), 7.21 (d,
1H, J=2), 7.65 (bs, 2H), 7.93 (s, 1H), 8.56 (bs, 1H).
15 (12–19)
32 (22–46)
14 (7–27)
776 (731–824) 816 (754–884) >1000 (83%) 1.3 (1.0–1.6)
aDisplacement of [3H]DPCPX binding at human A1 adenosine recep-
tors expressed in CHO cells.
bDisplacement of [3H]SCH58261 binding at human A2A adenosine
receptors expressed in CHO cells.
7: 8-(2-Hydroxy-ethyl)-2-furan-2-yl-8H-pyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c] pyrimidin-5-yl amine. White solid,
yield 51%, 0.62 mmol; mp 269 ꢀC; 1H NMR (DMSO-d6)
d: 3.81 (m, 2H), 4.30 (t, 2H, J=7.6), 4.90 (bs, 1H), 6.73
cDisplacement of [3H]DPCPX binding at human A2B adenosine
receptors expressed in HEK-293 cells.
dDisplacement of [3H]MRE 3008F20 binding at human A3 adenosine
receptors expressed in CHO cells.
Table 2. Elemental analyses of the final compounds described 5–19
Compd
Formula
Mr
Calculated
% H
Found
% C
% N
% C
% H
% N
5
6
7
8
C10H7N7O
C16H19N7O3
C12H11N7O2
C16H17N7O3
C23H22N8O4
C23H23ClN8O4
C19H14N8O4
C19H17N7O2
C26H22N8O3
C19H16N8O3
241.21
357.37
285.26
355.35
474.47
510.93
418.37
357.38
494.51
404.38
49.79
53.77
52.35
54.08
58.22
54.07
54.55
60.79
63.15
56.43
2.93
5.36
4.06
4.82
4.67
4.54
3.37
4.56
4.48
3.99
40.65
27.44
32.87
27.59
23.62
21.93
26.78
26.18
22.66
27.71
49.77
53.76
52.33
54.06
58.21
54.05
54.54
60.78
63.13
56.42
2.92
5.34
4.05
4.80
4.65
4.53
3.35
4.55
4.46
3.98
40.64
27.44
32.86
27.57
23.61
21.91
26.76
26.16
22.65
27.69
9
10
11
17
18
19

P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 4161–4169
4167
(t, 1H, J=7.80), 7.20 (d, 1H, J=1.6), 7.63 (bs, 2H), 7.94
(s, 1H), 8.55 (s, 1H).
benzene (20 mL). The mixture was heated at reflux for
1 h, then the solvent was removed at reduce pressure to
furnish 12a as yellow solid (12.5 mmol, 41.6%),
1
8: (5-Amino-2-furan-2-yl-pyrazolo4,3-e]-1,2,4-triazolo[1,5-
c]pyrimidin-8-yl)-acetic acid tert-butyl ester. White solid,
yield 52%, 1.04 mmol, mp 194–197 ꢀC; 1H NMR
(DMSO-d6) d: 1.44 (s, 9H), 5.13 (s, 2H), 6.73 (s, 1H),
7.22 (s, 1H), 7.70 (bs, 2H), 7.94 (s, 1H), 8.59 (s, 1H).
directly utilized for the next synthetic step. H NMR
(DMSO-d6) d: 3.62 (t, 2H, J=6.3), 4.11 (t, 2H,
J=6.9), 5.12 (t, 1H, J=6.2), 7.48 (m, 3H), 7.91 (m,
3H), 8.38 (s, 1H).
3-Amino-1-(2-hydroxyethyl)-1H-pyrazole-4-carbonitrile
hydrochloride (13). To a solution of 12a (12.5 mmol) in
abs EtOH (20 mL) was added 36% HCl (1.41 mL). The
reaction was heated at reflux for 30 min, then the sol-
vent was removed at reduced pressure and the residue
was recrystallized from boiling Et2O to afford 13 as
yellow solid (10.5 mmol, 84%), mp 232 ꢀC, IR (KBr):
[2-Furan-2-yl-5(3-phenylureido)-pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c] pyrimidin-8-yl] acetic acid tert-butyl ester
(9). To a suspension of 8 (0.56 mmol) in dry CH3CN
(10 mL) was added a catalytic amount of TEA and
phenylisocyanate (0.89 mmol) and the resultant mixture
was heated at 70 ꢀC for 6–8 h. Then the solvent was
removed at reduce pressure and the residue was purified
by flash chromatography (EtOAc/light petroleum 1:1)
to afford 9 as white solid (0.27 mmol, 51%), mp 194–
197 C; H NMR (DMSO-d6) d: 1.46 (s, 9H), 5.27 (s,
2H), 6.76 (s, 1H), 7.13 (s, 1H), 7.33 (m, 3H), 7.59 (m,
2H), 7.99 (s, 1H), 8.80 (s, 1H), 9.76 (bs, 1H), 10.69 (bs,
1H).
1516, 1569, 1653, 2222, 3207, 3439 cmꢂ1 1H NMR
;
(DMSO-d6) d: 3.64 (t, 2H, J=6.4), 3.88 (t, 2H, J=6.2),
4.89 (bs, 1H), 5.53 (bs, 2H), 7.35 (bs, 1H), 8.02 (s, 1H).
ꢀ
1
3-Amino-1-(2-benzyloxy-ethyl)-1H-pyrazole-4-carboni-
trile (14). To a suspension of 60% NaH (6.7 mmol) in
dry DMF (25 mL) at 0 ꢀC was added 13 (3.9 g,
25.4 mmol) in small portions. The mixture was stirred at
room temperature for several min and then benzyl-
bromide (26.9 mmol) in dry DMF (8 mL) was added.
The resulting mixture was stirred at room temperature
for 1 h then the solvent was removed at reduced pres-
sure. To the residue was added water (40 mL) and the
aqueous layer was extracted with EtOAc (5ꢃ25 mL).
The organic layers were dried (Na2SO4) and evaporated
under vacuum to afford a solid purified by flash chro-
matography (EtOAc/light petroleum 4:1) to furnish 14
as a pale yellow solid (12.4 mmol, 49.5%), mp 210 ꢀC,
1H NMR (DMSO-d6) d: 3.70 (t, 2H, J=6), 4.04 (t, 2H,
J=6.1), 4.44 (s, 2H), 5.57 (bs, 2H), 7.3 (m, 5H), 8.08 (s,
1H).
1-(3-Chlorophenyl)-3-[8-(2,2-diethoxyethyl)-2-furan-2-yl-
8H-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidin-5-yl]urea
(10). To a suspension of 6 (0.20 mmol) in dry CH3CN
(15 mL) was added a catalytic amount of TEA and 3-
chlorophenylisocyanate (1.1 mmol) and the resultant
mixture was heated at 90 ꢀC for 12 h. Then the solvent
was removed at reduce pressure and the residue was
purified by flash chromatography (EtOAc/light petro-
leum 4:6) to afford 10 as white solid (0.13 mmol, 65%),
mp 187–190 ꢀC; H NMR (DMSO-d6) d: 1.06 (t, 6H,
1
J=6), 3.43 (m, 2H), 3.65 (m, 2H), 4.48 (d, 2H, J=8.1),
4.97 (t, 1H, J=6.1), 6.76 (m, 1H), 7.30 (m, 4H), 7.80 (s,
1H), 8.00 (s, 1H), 8.79 (s, 1H), 9.90 (bs, 1H), 10.67 (bs,
1H).
N-[1-(2-Benzyloxy-ethyl)-4-cyano-1H-pyrazol-3-yl]-formi-
midic acid ethyl ester (15). A solution of 14 (3 g,
12.4 mmol) in HC(OEt)3 (25 mL) was heated at reflux
for 16 h. Then the solvent was removed at reduced
pressure and the residue was recrystallized from a mix-
ture of light petroleum/Et2O (1:1) to afford 15 as white
solid (9.9 mmol, 79.8%), mp 198 ꢀC, 1H NMR (DMSO-
d6) d: 1.30 (t, 3H, J=6.2), 3.76 (t, 2H, J=6.6), 4.26 (m,
4H), 4.46 (s, 2H), 7.26 (m, 5H), 8.27 (s, 1H), 8.42 (s,
1H).
[2-Furan-2-yl-5-(3-phenylureido)-pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidin-8-yl] acetic acid (11). A solution
of 9 (70 mg, 0.15 mmol) in TFA (1.5 mL) was stirred at
room temperature for 15 min. The solid formed was fil-
tered off, washed with cold water, and recrystallized
from a mixture of DMF/water (1:1) to afford 11 as
white solid (0.09 mmol, 60%), mp 290 ꢀC, 1H NMR
(DMSO-d6) d: 5.27 (s, 2H), 6.76 (s, 1H), 7.30 (s, 1H),
7.33 (m, 3H), 7.55 (m, 2H), 7.99 (s, 1H), 8.80 (s, 1H),
9.73 (bs, 1H), 10.70 (bs, 1H), 13.09 (bs, 1H).
8-(2-Benzyloxy-ethyl)-2-(furan-2-yl)-8H-pyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c]pyrimidine (16). To a solution of 15
(2.95 g, 9.9 mmol) in 2-methoxyethanol (35 mL) was
added 2-furoic acid hydrazide (9.9 mmol) and the mix-
ture was heated at reflux for 12 h. After cooling, the
solvent was removed under reduced pressure and the
oily residue was cyclized without other purification in
diphenyl ether (50 mL) at 260 ꢀC for 1.5 h. Then the
mixture was poured into light petroleum (300 mL) and
cooled. The precipitate was filtered off and purified by
recrystallization from EtOAc to afford 16 as a white
2-(N0-Benzylidene-hydrazino) ethanol (12). To a solution
of benzaldehyde (10 mL, 98 mmol) in abs. EtOH
(50 mL) was added 2-hydroxyethylhydrazine (6.67 mL,
98 mmol) in abs. EtOH (10 mL). The solution was
heated at reflux for 1 h, then the solvent was removed at
reduced pressure to furnish 12 as yellow oil (97.5 mmol,
99%), 1H NMR (CDCl3) d: 3.37 (t, 2H, J=6.2), 3.83 (t,
1H, J=7), 3.85 (t, 2H, J=6.1), 7.33 (m, 4H), 7.54 (d,
2H, J=3.9), 7.65 (s, 1H).
2-[N0-Benzylidene-N-(2-hydroxyethyl)-hydrazinomethyl-
ene]-malononitrile (12a). To a solution of 12 (5 g,
30 mmol) in benzene (20 mL) was added ethoxy-
methylenemalononitrile (3.72 g, 30 mmol) dissolved in
solid (8.3 mmol, 84%), mp 232 ꢀC, H NMR (DMSO-
1
d6) d: 3.95 (t, 2H, J=6.3), 4.49 (s, 2H), 4.67 (t, 2H,
J=6.2), 6.74 (m, 1H), 7.24 (m, 7H), 7.96 (bs, 1H), 8.93
(s, 1H).

4168
P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 4161–4169
8-(2-Benzyloxy ethyl)-2-(furan-2-yl)-8H-pyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c]pyrimidin-5-yl amine (17). A solution
of 16 (3 g, 8.3 mmol) in aqueous 10% HCl (30 mL) and
dioxan (15mL) was refluxed for 1 h. Then the solution was
cooled, basified with 10% NaOH at 0 ꢀC, and extracted
with EtOAc (3ꢃ50 mL). The organic layers were dried
over anhyd. Na2SO4 and evaporated under vacuum. The
residue obtained was recrystallized from EtOAc to afford
the hydrolyzed compound as a solid utilized without other
purification for the next step of reaction.
acetone (20 mL) was added HCO2NH4 (2.13 mmol) and
10% Pd/C (100 mg) and the resulting mixture was
heated at reflux for 8 h. The solution was cooled and the
catalyst was removed by filtration. The solvent was
evaporated at reduced pressure and the residue was
washed with water (100 mL). The aqueous layer was
extracted with EtOAc (5ꢃ25 mL) and the recombined
organic phases were dried over anhyd. Na2SO4 and
evaporated under reduced pressure to afford 7 as a
white solid (0.19 mmol, 75%). Physical properties
reported above.
To a solution of the hydrolyzed intermediate (1 g,
2.8 mmol) in N-methylpyrrolidone (8 mL) were added
cyanamide (16.8 mmol) and p-toluenesulphonic acid
(4.2 mmol), and the mixture was heated at 160 ꢀC for
4 h. Then cyanamide (16.8 mmol) was added again and
the solution was heated overnight. Then the solution
was diluted with EtOAc (100 mL) and the precipitate
(excess of cyanamide) was filtered off; the filtrate was
concentrated under reduced pressure, and washed with
water (3ꢃ50 mL). The organic layer was dried (Na2SO4)
and evaporated under vacuum. The residue was purified
by chromatography (EtOAc/light petroleum 1:1) to
afford 17 as a solid (2.9 mmol, 35% from 16), mp 181–
Pharmacology
All synthesized compounds have been tested for their affi-
nity to human A1, A2A, A2B and A3 adenosine receptors.
The affinity values were determined by receptor binding
assays at human A1, A2A, A2B and A3 adenosine recep-
tor subtypes cloned in CHO and HEK-293 cells using
[3H]DPCPX, [3H]SCH 58261, [3H]DPCPX and
[3H]MRE 3008F20, respectively.
Human cloned adenosine receptor binding assay
183 ꢀC, H NMR (DMSO-d6) d: 3.88 (t, 2H, J=6.3),
1
4.48 (s, 4H), 6.58 (bs, 2H), 6.72 (m, 1H), 7.25 (m, 5H),
7.55 (s, 1H), 7.93 (d, 1H, J=1.4), 8.61 (s, 1H).
The cells were grown adherently and maintained in
Dulbecco’s modified Eagle’s medium with nutrient
mixture F12 without nucleosides at 37 ꢀC in 5% CO2/
95% air. The cells were washed with phosphate-buffered
saline and scrapped off flasks in ice cold hypotonic buf-
fer (5 mM Tris–HCl, 2 mM EDTA, pH 7.4). The cell
suspension was homogenized with a Polytron and the
homogenate was centrifuged for 30 min at 48,000g. The
membrane pellet was resuspended in 50 mM Tris–HCl
buffer at pH 7.4 for A1 adenosine receptors, in 50 mM
Tris–HCl, 10 mM MgCl2 at pH 7.4 for A2A adenosine
receptors, in 50 mM Tris–HCl, 10 mM MgCl2, 1 mM
EDTA at pH 7.4 for A3 adenosine receptors and were
utilized for binding assays. HEK 293 cells transfected
with the human recombinant A2B adenosine receptor
were obtained from Receptor Biology, Inc. (Beltsville,
MD, USA).
1-[8-(2-Benzyloxy-ethyl)-2-furan-2-yl-8H-pyrazolo[4,3-
e]-1,2,4-triazolo[1,5-c]pyrimidin-5-yl]-3-phenyl urea (18).
To a solution of 17 (260 mg, 0.69 mmol) in dry CH3CN
(10 mL) was added phenylisocyanate (2.6 mmol) and a
catalytic amount of TEA. The solution was heated at
80 ꢀC for 24 h, then the solvent was removed at reduced
pressure and the oily residue was purified by flash
chromatography (EtOAc/light petroleum 1:1) to afford
18 as a white solid (0.30 mmol, 55%), mp 185–187 ꢀC,
1H NMR (DMSO-d6) d: 3.94 (t, 2H, J=6.2), 4.50 (s,
2H), 4.60 (t, 2H, J=6.5), 6.76 (m, 1H), 7.10 (t, 1H,
J=2.1), 7.25 (m, 8H), 7.42 (m, 2H), 7.98 (s, 1H), 8.83 (s,
1H), 9.71 (bs, 1H), 10.67 (bs, 1H).
1-[8-(2-Hydroxy-ethyl)-2-furan-2-yl-8H-pyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c]pyrimidin-5-yl]-3-phenyl urea (19).
To a solution of 18 (100 mg, 0.2 mmol) in dry acetone
(15 mL) was added HCO2NH4 (1.6 mmol) and 10% Pd/
C (100 mg).The resulting mixture was heated at reflux
for 8 h.The solution was cooled, the catalyst was
removed by filtration, and the solvent was removed at
reduced pressure. The residue was washed with water
(100 mL), the aqueous layer was extracted with EtOAc
(3ꢃ30 mL) and the recombined organic phases were
dried over anhyd. Na2SO4 and evaporated under
reduced pressure to afford 19 as a white solid
Binding of [3H]-DPCPX to CHO cells transfected with
the human recombinant A1 adenosine receptor was
performed according to the method previously descri-
bed by Varani et al.16 Displacement experiments were
performed for 120 min at 25 ꢀC in 200 mL of buffer con-
taining 1 nM [3H]-DPCPX, 20 mL of diluted membranes
(50 mg of protein/assay) and at least 6–8 different con-
centrations of examined compounds. Non-specific
binding was determined in the presence of 10 mM of
CHA and this is always ꢄ10% of the total binding.
(0.17 mmol, 85%), mp 192–194 ꢀC, H NMR (DMSO-
Binding of [3H]-SCH58261 to CHO cells transfected
with the human recombinant A2A adenosine receptors
(50 mg of protein/assay) was performed according to
Varani et al.16
1
d6) d: 4.01 (t, 2H, J=6.2), 4.41 (t, 2H, J=6.3), 5.15 (t,
1H, J=6.8), 6.75 (bs, 1H), 7.27 (t, 1H, J=1.5), 7.38 (m,
3H), 7.55 (d, 2H, J=1.4), 9.97 (s, 1H), 8.71 (d, 1H,
J=1.8), 9.73 (bs, 1H); 10.75 (bs, 1H).
In competition studies, at least 6–8 different concentra-
tions of compounds were used and non-specific binding
was determined in the presence of 1 mM SCH58261 for an
incubation time of 60 min at the temperature of 25 ꢀC.
8-(2-Hydroxy-ethyl)-2-furan-2-yl-8H-pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidin-5-yl amine (7) (O-debenzyla-
tion). To a solution of 17 (100 mg, 0.26 mmol) in dry

P. G. Baraldi et al. / Bioorg. Med. Chem. 11 (2003) 4161–4169
4169
Binding of [3H]-DPCPX to HEK 293 cells transfected
with the human recombinant A2B adenosine receptors
were performed essentially to the method described by
Varani et al.16
William, J. Greenlee for the helpful discussion on the
Dimroth-type rearrangement. We also thank the King
Pharmaceuticals Research and Development for finan-
cial support and Dr. Eddie Leung for useful discussions.
In particular, assays were carried out for 60 min at 25 ꢀC
in 100 mL of 50 mM Tris–HCl buffer, 10 mM MgCl2,
1 mM EDTA, 0.1 mM benzamidine pH 7.4, 2 IU/mL
adenosine deaminase containing 40 nM [3H]-DPCPX,
diluted membranes (20 mg of protein/assay) and at least
6–8 different concentration of tested compounds. Non
specific binding was determined in the presence of 100 mM
of NECA and was always ꢄ30% of the total binding.
Binding of [3H]MRE3008 F20 to CHO cells transfected
with the human recombinant A3 adenosine receptors
was performed according to Varani et al.16 Competition
experiments were carried out in duplicate in a finale
volume of 250 mL in test tubes containing 1 nM
[3H]MRE3008 F20, 50 mM Tris–HCl buffer, 10 mM
MgCl2, pH 7.4 and 100 mL of diluted membranes (50 mg
protein/assay) and at least 6–8 different concentrations
of examined ligands for 120 min at 4 ꢀC. Non-specific
binding was defined as binding in the presence of 1 mM
of MRE3008 F20 and was about 25% of total binding.
References and Notes
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Data analysis
The protein concentration was determined according to
a Bio-Rad method17 with bovine albumin as a standard
reference. Inhibitory binding constant, Ki, values were
calculated from those of IC50 according to Cheng and
Prusoff equation.18 A weighted non linear least-squares
curve fitting program LIGAND19 was used for compu-
ter analysis of saturation and inhibition experiments.
12. Schering Corporation, NJ 07033-0530 (US), International
Publication Number: WO01/92264 A1, 2001.
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Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, G.;
Borea, P. A. Mol. Pharmacol. 2000, 57, 968.
Data are expressed as geometric mean, with 95 or 99%
confidence limits in parentheses.
17. Bradfor, M. M. Anal. Biochem. 1976, 72, 248.
18. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
Acknowledgements
We wish to express gratitude to the Schering Plough
Corporation, NJ 07033-0530 (US) and in particular Dr.
19. Munson, P. J.; Rodbard, D. Anal. Biochem. 1980, 107,
220.