Design, synthesis, and biological evaluation of C9- and C2-substituted pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as new A2A and A3 adenosine receptors antagonists

Article abstract of DOI:10.1021/jm021023m

In the past few years, our group has been involved in the development of A_2A and A₃ adenosine receptor antagonists which led to the synthesis of SCH58261 (5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine, 61), potent and very selective at the A_2A receptor subtype, and N⁸-substituted-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidines-N⁵-urea or amide (MRE series, b), very selective at the human A₃ adenosine receptor subtype. We now describe a large series of C⁹- and C²-substituted pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines to represent an extension of structure-activity relationship work on this class of tricyclic compounds. The introduction of a substituent at 9 position of the tricyclic antagonistic structure led to retention of receptor affinity but a loss of selectivity in respect to the lead compounds b, N⁸-substituted-pirazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidines-N⁵-urea or -amide. The substitution of the furanyl moiety of compound 61, necessary for receptor binding, with a phenyl or a substituted aromatic ring (compounds 5a-d, 6-8), caused a complete loss of the affinity at all the adenosine receptor subtypes, demonstrating that the furanyl ring is a necessary structural element to guarantee interaction with the adenosine receptor surface. The introduction of an ethoxy group at the ortho position of the aromatic ring to mimic the oxygen of the furan (compound 5c, 5-amino-7-(2-phenylethyl)-2-(2-ethoxyphenyl)pyrazolo[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine) did not enhance affinity. The introduction of the cycloaminomethyl function by Mannich reaction at the 5′ position of the furanyl ring of 61 and the C⁹-substituted compound 41 (5-amino-8-methyl-9-methylsulfanyl-2-(2-furyl)-pyrazolo[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine) resulted in complete water solubility but a loss of receptor affinity. We can conclude that modifications or substitutions at the furanyl ring are not allowed and the introduction of a substituent at the 9-position of the core pyrazolo-triazolo-pyrimidine structure caused a severe loss of selectivity, probably due to an increased steric hindrance of the radical introduced.

Full text of DOI:10.1021/jm021023m

J . Med. Chem. 2003, 46, 1229-1241
1229
Design , Syn th esis, a n d Biologica l Eva lu a tion of C9- a n d C2-Su bstitu ted
P yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in es a s New A2A a n d A3 Ad en osin e
Recep tor s An ta gon ists
Pier Giovanni Baraldi,*,† Francesca Fruttarolo,† Mojgan Aghazadeh Tabrizi,† Delia Preti,† Romeo Romagnoli,†
Hussein El-Kashef,§ Allan Moorman,| Katia Varani,‡ Stefania Gessi,‡ Stefania Merighi,‡ and Pier Andrea Borea‡
Dipartimento di Scienze Farmaceutiche and Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia,
Universita` di Ferrara, 44100 Ferrara, Italy, Department of Chemistry, Faculty of Science, University of Assiut, Egypt, and
King Pharmaceutical, 7001 Weston Parkway Suit 300, Cary, North Carolina 27513
Received August 8, 2002
In the past few years, our group has been involved in the development of A2A and A3 adenosine
receptor antagonists which led to the synthesis of SCH58261 (5-amino-7-(2-phenylethyl)-2-(2-
furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, 61), potent and very selective at the A2A
receptor subtype, and N8-substituted-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines-N5-urea
or amide (MRE series, b), very selective at the human A3 adenosine receptor subtype. We now
describe a large series of C9- and C2-substituted pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines
to represent an extension of structure-activity relationship work on this class of tricyclic
compounds. The introduction of a substituent at 9 position of the tricyclic antagonistic structure
led to retention of receptor affinity but a loss of selectivity in respect to the lead compounds b,
N8-substituted-pirazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines-N5-urea or -amide. The substitu-
tion of the furanyl moiety of compound 61, necessary for receptor binding, with a phenyl or a
substituted aromatic ring (compounds 5a -d , 6-8), caused a complete loss of the affinity at all
the adenosine receptor subtypes, demonstrating that the furanyl ring is a necessary structural
element to guarantee interaction with the adenosine receptor surface. The introduction of an
ethoxy group at the ortho position of the aromatic ring to mimic the oxygen of the furan
(compound 5c, 5-amino-7-(2-phenylethyl)-2-(2-ethoxyphenyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-
c]pyrimidine) did not enhance affinity. The introduction of the cycloaminomethyl function by
Mannich reaction at the 5′ position of the furanyl ring of 61 and the C9-substituted compound
41 (5-amino-8-methyl-9-methylsulfanyl-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimi-
dine) resulted in complete water solubility but a loss of receptor affinity. We can conclude that
modifications or substitutions at the furanyl ring are not allowed and the introduction of a
substituent at the 9-position of the core pyrazolo-triazolo-pyrimidine structure caused a severe
loss of selectivity, probably due to an increased steric hindrance of the radical introduced.
In tr od u ction
633902 (5-amino-7-(3-phenylpropyl)-2-(2-furyl)pyrazolo-
[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine), and related com-
pounds which possess hydrophilic groups at the para
and ortho positions of the aromatic ring have been found
to be potent and selective adenosine A2A antagonists,3
and SCH 58261 is widely used as a tool for character-
izing the adenosine A2A receptor subtype.4,5 At the same
time different classes of compounds have been reported
to be selective A3 receptor antagonists (eight classes
with nonxanthine structure, including dihydropyridine
and pyridine analogues, flavonoid, isoquinoline and
triazoloquinazoline derivatives, triazolonaphthiridine
and thiazolopyrimidine analogues).6-13 The best results
in terms of A3-antagonism were obtained with the
synthesis of 5-N-(substituted phenylcarbamoyl)amino-
8-substituted-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo-
[1,5-c]pyrimidines, where the best substitution on the
phenyl ring of the phenyl carbamoyl moiety was a
methoxy at the para position or a chlorine atom at the
meta position (MRE series).14-17
Adenosine, an endogenous modulator of a wide range
of biological functions in the nervous, cardiovascular,1a
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 (Fredholm et al.,
2000).1b
In the past 10 years, great efforts by medicinal
chemists and pharmacologists have been devoted to the
design of potent and selective antagonists for A2A and
A3 receptors. Thus, the pyrazolotriazolopyrimidines
SCH 58261 (5-amino-7-(2-phenylethyl)-2-(2-furyl)pyra-
zolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine, 61), SCH
* To whom correspondence should be addressed: Prof. Pier Giovanni
Baraldi, Dipartimento di Scienze Farmaceutiche, Universita` di Fer-
rara, Via Fossato di Mortara 17-19, 44100 Ferrara Italy, Tel: +39-
0532-291293, Fax: +39-0532-291296, E-mail: pgb@dns.unife.it.
† Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
‡ Dipartimento di Medicina Clinica
Farmacologia, Universita` di Ferrara.
§ University of Assiut.
e Sperimentale-Sezione di
Starting from these observations, and on the basis of
data derived from an enlarged series of SCH 58261 (61)
analogues and MRE series analogues previously re-
| King Pharmaceutical.
10.1021/jm021023m CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/28/2003

1230 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Baraldi et al.
Ta ble 1. C9-Substituted Compounds Synthesized
ported (compounds of general formula b), we synthe-
sized new molecules structurally modified in different
positions with respect to the lead compounds 61 and b
structure in order to evaluate the change in affinity and
selectivity of the new compounds obtained and provide
a better understanding of the important features about
the associated structure-activity relationships (SAR).
In particular, we investigated two positions of the
tricyclic pyrazolotriazolopyrimidine structure: the 2-
and the 9-position. For all the A2A adenosine receptor
antagonists, the furanyl group at the 2-position was
shown to be important for the binding activity of the
molecule. Substitution of this heterocycle with other
heterocyclic rings, e.g., thiophene or tetrahydrofuranyl18
led to a severe loss of affinity of the compound for this
relevant adenosine receptor. We tried to introduce in
the same position a phenyl or an aromatic ring substi-
tuted in the para position with suitable substitution
groups with electron negative centers to interact with
the adenosine receptors (e.g., halogens, free hydroxyl
group, amide, and free carboxylic acid functions; com-
pounds of general formula a , see below). The ortho
position of the aromatic ring was also functionalized
with an ethoxy group in an attempt to mimic the oxygen
in the furanyl ring. Structure-activity relationship
study was evaluated based on the lead compound 61 to
appreciate the change in receptor affinity and selectiv-
ity.
compd
R
R1
R2
38
39
40
41
42
43
44
45
46
47
48
49
50
52
53
54
CH3
NHCH2CH3
NH-Ph-4-OMe
N-Me-piperazine
SCH3
H
H
H
H
H
H
CH3
CH3
CH3
CH3
S(CH2)2CH3
Ph(CH2)3 SCH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
NHCH2CH3
N-Me-piperazine
SCH3
SCH3
SCH3
CONHPh-4-OMe
CONHPh-4-OMe
CONHPh-4-OMe
COCH2Ph-4-OMe
COCH2Ph-4-isobutyl
COCH2Ph-3,4-Medioxy
COCH2Ph-3,4-Medioxy
H
SCH3
NHCH2CH3
NH-Ph-4-OH
NHCH2CH3‚HCl
N-Me-piperazine ‚
2HCl
H
H
55
CH3
NHCH2CH3 ‚HCl
CONHPh-4-OMe
tion of the free amino group into an amide or urea
function for A3-antagonists, were maintained.
All new compounds synthesized are pyrazolotria-
zolopyrimidines, N8-substituted principally with small
alkyl groups. Compounds with substituents introduced
at the 9 position were examined for steric hindrance and
hydrophilic/lipophilic balance: they are cycloalkyl, alkyl,
or amine functions bound to the tricyclic core by
thioether or amine functions (Table 1). The principal
aim of these innovative modifications was to evaluate
the influence of these variations on receptor interaction.
All compounds synthesized were evaluated in binding
assays on all four adenosine receptor subtypes, paying
particular attention to the results obtained from the
interaction with A2A and A3 receptor subtypes.
The principal difficulty in evaluating the adenosine
tricyclic antagonists previously reported by our group
were their very lipophilic nature. Starting from these
observations, we introduced, by Mannich reaction,
another structural modification at the 5′-position of the
furanyl ring of the lead compound 61 and C9-substituted
compound 41, which showed the best results in terms
of receptor affinity. The aim of this modification was to
improve the water solubility of the new compounds
We have also investigated the effects of various
substituents at the 9-position of the antagonist pyrazolo-
triazolo-pyrimidine structure. To evaluate the change
in affinity and selectivity versus A2A and A3 adenosine
receptor subtypes, all the other structural requirements
necessary for antagonism and present in the lead
compounds 61 and b, such as planar structure, alkyl or
arylalkyl substituents on the pyrazole ring, free exocy-
clic amino group for A2A-antagonists, and transforma-

New A2A and A3 Adenosine Receptors Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1231
Sch em e 1a
Sch em e 2a
a
Reagents: (i) 2-chloroacetyl chloride, 2-chloro-N-(4-iodophen-
yl)acetamide, K2CO3, DMF, rt; (ii) 10% HCl, dioxane, 60 °C.
converted into the final compounds 5a -d by reaction
with an excess of cyanamide in 1-methyl-2-pyrrolidone
at 140 °C.
The tricyclic compound 5a was functionalized at the
phenolic hydroxyl group by treatment with 2-chloro-
acetyl chloride or 2-chloro-N-(4-iodophenyl)acetamide22
in DMF to obtain the derivatives 6 and 8, respectively.
Finally, derivative 6 was converted into 7 by treatment
with aqueous HCl in dioxane (Scheme 2).
a
Reagents: (i) triethyl orthoformate, reflux; (ii) (substituted)-
benzoic acid hydrazides, 2-methoxyethanol; (iii) Ph2O, 260 °C; (iv)
The synthesis of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]-
pyrimidine derivatives substituted at the 9-position is
depicted in Schemes 3-6. The reaction between mal-
ononitrile, carbon disulfide, and methyl iodide in the
presence of K2CO3 and DMF, gave the 2-(bis(methyl-
sulfanyl)methylene)malononitrile 9.23 Reaction with
methylhydrazine or 3-phenylpropylhydrazine in ethanol
gave the amino cyanopyrazoles 14 and 15 (Scheme 3).
It’s interesting to note that the only isomer obtained
is the 3-amino-4-cyanopyrazole, confirmed by NOESY
analyses.23
Transformation of pyrazoles 14 and 15 to the corre-
sponding imidates 23 and 24 was achieved by refluxing
in triethyl orthoformate according to the procedure of
Gatta et al.19 The imidates were reacted with 2-furoic
acid hydrazide in refluxing 2-methoxyethanol to provide
the pyrazolo[4,3-e]pyrimidine intermediates. The latter
compounds were converted to the derivatives 29 and 30,
in good overall yield, through a thermal cyclization in
diphenyl ether. Treatment of 29 and 30 with dilute
hydrochloric acid at reflux temperature induced pyri-
midine ring opening to furnish the 5-amino-4-(1H-1,2,4-
triazol-5-yl)pyrazoles 35 and 37 in a very good yield.
These derivatives were converted into the final com-
pounds 41 and 43 by reaction with an excess of
cyanamide in 1-methyl-2-pyrrolidone at 140 °C. The
reaction between malononitrile, carbon disulfide, and
1-bromopropane gave the unsaturated intermediate 10
which was submitted to the same synthetic steps to
obtain the final compound 42.
10% HCl; (v) cyanamide, 1-methyl-2-pyrrolidone, p-TsOH, 140 °C.
obtained and to further evaluate the change in terms
of affinity and selectivity of the molecules (compounds
of general formula c). The amines used for this type of
reaction were morpholine and N-methylpiperazine.
Treatment with hydrochloric acid solution to form the
corresponding salt was effected to increase the water
solubility and to make pharmacological testing easier.
The purposes of this research effort are to better
understand what structural modifications introduced on
the tricyclic antagonist core pyrazolo-triazolo-pyrimidine
play an important role on ligand-receptor interaction.
In this way, we can determine what position of the
heterocyclic structure should not be modified and, on
the contrary, what position is susceptible to modifica-
tions or functionalizations, to develop new drugs which
target A2A and A3 adenosine receptor subtypes.
Ch em istr y
The general synthesis of 2-aryl-pyrazolo-triazolo-
pyrimidines is depicted in Scheme 1. The synthetic steps
for the synthesis of this new class of compounds are the
same utilized for the synthesis of 61 and analogues
according to Gatta et al.,19 except for the (substituted)-
hydrazide utilized. The 4-cyano-5-amino-1-(2-phenyl-
ethyl)pyrazole20 1 was transformed into the correspond-
ing imidate 2 by refluxing in triethyl orthoformate. The
imidate was then reacted with benzoic acid hydrazide,
4-hydroxybenzoic acid hydrazide, 4-chlorobenzoic acid
hydrazide (commercially available), or 2-ethoxybenzoic
acid hydrazide21 in refluxing 2-methoxyethanol to pro-
vide the pyrazolo[4,3-e]pyrimidine intermediates. The
latter compounds were converted through a thermally
induced cyclization in diphenyl ether to the tricyclic
derivatives 3a -d in good yield.
Compound 9, by reaction with amines IV, V, and VI
in ethanol, furnished the intermediates 11, 12, and 13,
as depicted in Scheme 4. Compounds 11-13 are then
converted into aminocyanopyrazoles by cyclization with
methylhydrazine in ethanol at reflux. The synthesis of
the final compounds 38-40 was performed using the
same synthetic methodology employed for compounds
41 and 43. The pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyri-
midine derivatives 38, 40, and 41 were converted into
the corresponding ureas (44, 45, and 46) by reaction
Treatment of 3a -d with dilute hydrochloric acid
induced pyrimidine ring opening to furnish the amines
4a -d in quantitative yield. These derivatives were

1232 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Baraldi et al.
Sch em e 3a
a
Reagents: (i) CS2, CH3I; (ii) (aryl)alkylhydrazine, reflux; (iii) HC(OEt)3, reflux; (iv) 2-furoic acid hydrazide, MeO(CH2)2OH; (v) Ph2O,
260 °C; (vi) 10% HCl; (vii) NH2CN, p-TsOH, 1-methyl-2-pyrrolidone, 140 °C; (viii) 4-methoxyphenyl isocyanate, TEA; (ix) acyl chlorides.
with 4-methoxyphenyl isocyanate and a catalytic amount
of TEA (Schemes 3 and 4). The tricyclic compounds 38
and 41 were converted into compounds 47-50 (Schemes
3 and 4) by reaction with acyl chlorides in benzene and
DMF as solvents. This permitted the introduction of an
amide function in the 5-position to evaluate the changes
in affinity and selectivity at the A3 adenosine receptor
subtype.
Derivative 39 was converted into 52 by treatment
with acetic acid and iodic acid (Scheme 5). The free
hydroxyl group was introduced to increase the water
solubility of the final compound.
of 61 and 41, we performed the Mannich reaction, as
depicted in Scheme 6. The appropriate tricyclic deriva-
tives were reacted with N-methylpiperazine or morpho-
line and 36% aqueous formaldehyde in glacial acetic acid
to give the target compounds 51 and 57-58 in 20-30%
yield.
The treatment of compounds 57, 58, and 61 with a
saturated solution of hydrochloric acid in methanol gave
the salts 56, 59, and 60 which displayed significant
water solubility.
Resu lts a n d Discu ssion
The final compounds which own a free amino function
were transformed into a salt by treatment with a
saturated methanolic solution of hydrochloric acid. For
the modifications at the 5′ position of the furanyl ring
Tables 2, 3, and 5 show the receptor affinity profiles
of structurally modified compounds described in this
work. The affinity values were determined by receptor
binding assays at human A1, A2A, A2B, and A3 adenosine

New A2A and A3 Adenosine Receptors Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1233
Sch em e 4a
a
IV: N-methylpiperazine; V: 4-methoxyaniline; VI: ethylamine. Reagents: (i) CS2, CH3I; (ii) (aryl)alkylhydrazine, reflux; (iii) HC(OEt)3,
reflux; (iv) 2-furoic acid hydrazide, MeO(CH2)2OH; (v) Ph2O, 260 °C; (vi) 10% HCl; (vii) NH2CN, p-TsOH, 1-methyl-2-pyrrolidone, 140 °C;
(viii) 4-methoxyphenyl isocyanate, TEA; (ix) acyl chlorides.
Sch em e 5a
electronic condition for the interaction with the adenos-
ine receptor.
The introduction of an ethoxy group at the 2′-position
of the aromatic ring, to imitate the oxygen of the furan,
proved unsuccessful for A2A interaction. The introduc-
tion of an ester, acid, or amide function in the para-
position, introduced to promote the formation of a
hydrogen bond between the molecule and the adenosine
receptor surface, also proved fruitless. Only compound
5c shows a poor affinity for A3 adenosine receptor
subtype, but with complete selectivity. The biological
results of the compounds modified at 9-position are
showed in Table 3.
a
Reagents: (i) acetic acid, iodic acid, reflux.
receptor subtypes cloned in CHO and HEK-293 cells
using [3H]DPCPX, [3H]SCH 58261, [3H]DPCPX, and
[3H]MRE 3008F20, respectively.
As showed in the table, the introduction of a sub-
stituent at the 9-position instead of a hydrogen leads
to a loss of selectivity that is present in the lead
compound 61 and in compounds b, but the receptor
affinity is maintained. In general the methylthio group
at the 9-position is the best tolerated. A methyl group
at 8-position is better tolerated than a phenylpropyl
substituent by A2A adenosine receptor subtype. Com-
pound 41, which has a free amino group at the 5-posi-
tion, shows good affinity for the A2A adenosine receptor
but, unfortunately, low selectivity. Transformation of
the amino group into urea (compound 46) or amide
The biological results of the new series of compounds
5a -8 modified in the 2-position are shown in Table 2.
In general we observe that substitution of the furanyl
moiety with a (substituted)aromatic function or a phenyl
ring causes a complete loss of affinity at the A2A
adenosine receptor subtype with respect to the lead
compound 61. This provides supportive evidence that
the furanyl ring at the 2-position of the tricyclic struc-
ture is a necessary element to guarantee the activity of
the molecule, probably because in this heterocycle is
present an oxygen atom that produces a favorable

1234 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Baraldi et al.
Sch em e 6a
a
Reagents: (i) 36% aqueous formaldehyde, glacial acetic acid, (ii) N-methylpiperazine or morpholine.
Ta ble 2. Biological Results of Compounds 5a -8
compd
X
A1 Ki nM
121
A2A Ki nM
A2B Ki nM
A3 Ki nM
61a
5a b
5bb
5cb
5d b
6b
-
2.3
>1000
>1000-
>1000
>1000
>1000
>1000
>1000
>1000
>1000
4-OH
4-Cl
2-OEt
H
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
348 (267-453)
>1000
235 (181-305)
>1000
4-OCH2CO2Et
4-OCH2CO2H
4-OCH2CONHPh-4′-I
>1000
7b
>1000
>1000
>1000
8b
>1000
a
Displacement of [3H]CHA binding (A1) at rat cortical membrane, displacement of [3H]CGS 21680 binding (A2A) at rat striatal membranes,
and displacement of [125I]AB MECA binding at human A3 adenosine receptors expressed in HEK-293 cells. Displacement of [3H]DPCPX
binding (A1, A2B) at human A1 and A2B adenosine receptors expressed in CHO and HEK-293 cells, displacement of [3H]SCH58261 binding
(A2A) at human A2Aadenosine receptors expressed in CHO cells, and displacement of [3H]MRE 3008F20 binding at human A3 adenosine
receptors expressed in CHO cells.
b
functions (compounds 47, 48, and 49) preserves A2A
affinity, but interaction with the A3 adenosine receptor
subtype decreases. In this case we can say that the
presence of a substituent at the 9-position does not
permit the A3-interaction like that obtained by similar
functionalization of the free amino group at the 5-posi-
tion of the simpler pyrazolotriazolopyrimidine struc-
ture.17
The substitution of the methylthio group by a thio-
propyl group at the 9-position maintains the A2A affinity
and increases the selectivity (compound 42). Introduc-
tion of a phenylpropyl group at N8-position, instead of
a methyl group (compound 43), leads to a decrease of
A2A interaction. In contrast, compound 38, which pos-
sess an ethylamino group at the 9-position, shows good
affinity for the A2A adenosine receptor subtype. Func-
tionalization of the amino group into a p-methoxy-
phenylurea causes a decrease of A2A affinity, but a good
increase in A3 affinity (compound 44). The interaction
with A3 receptor is also increased by the salification of
this molecule (compound 55), probably due to increased
water solubility. However, selectivity of this compound
versus the other adenosine receptor subtypes is very
low.
40, and 52), was ineffectual for A2A-interaction. Func-
tionalization of the amino group of compound 40 into
urea was also negative for A3 interaction, possibly due
to an important steric interaction of the radicals intro-
duced (compound 45).
The synthesized compounds were also tested to verify
the antagonist behavior. In particular, the most inter-
esting of the examined compounds (41, 42, 46, 49, 53,
and 55) were evaluated for blocking cAMP generation
after typical agonist-modulation of A2A and A3 receptors
(Table 4).
The compounds 41 and 42 that show an high affinity
versus A2A adenosine receptors are also the most potent
antagonists revealing IC50 values in the nanomolar
range (6.1-14.8 nM). The compound 55 with high
affinity versus A3 adenosine receptors is also the most
potent A3 antagonist. All these data suggest that the
potency of the antagonists in the cAMP assays strictly
correlated with the affinity values observed in binding
assays.
We can conclude that modifications at the 9-position
maintain antagonistic activity, particularly with small
groups introduced, like methylthio or ethylamino radi-
cals, but lead to a significant loss of selectivity.
The compound 61 and compound 41, which displayed
a better antagonistic activity on A2A adenosine receptor
subtype, were modified at 5′-position of the furanyl ring
The introduction of hindered amino functions at
9-position, such as p-methoxyphenylamino, N-meth-
ylpiperazine, or p-hydroxyphenylamino (compounds 39,

New A2A and A3 Adenosine Receptors Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1235
Ta ble 3. Biological Results of Compounds 38-55
a
b
c
d
compd
R
R1
NHCH2CH3
NH-Ph-p-OMe
N-Me-piperazine
SCH3
R2
A1 Ki nM
A
2A Ki nM
A
2B Ki nM
A3 Ki nM
38
39
40
41
42
43
44
45
46
47
48
49
50
52
53
54
55
CH3
H
H
H
H
H
H
50 (41-61)
10 (7-14)
81 (70-94)
>1000
225 (177 (288)
>1000
CH3
CH3
CH3
CH3
260 (201-332) >1000
30 (22-41)
156 (123-198) 35 (27-45)
>1000
8.4 (4.9-14.5) 1.2 (0.8-1.8)
10.3 (7.9-13.4) 35 (27-45)
S(CH2)2CH3
9 (7-11)
2.1 (1.5-3.0)
69 (55-87)
31 (25-38)
37 (27-42)
26 (17-38)
24 (17-35)
45 (34-56)
224 (186-269)
>1000
Ph(CH2)3 SCH3
175 (134-229) 22 (12-43)
150 (132-169) 21 (16-27)
316 (268-372) >1000
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
NHCH2CH3
N-Me-piperazine
SCH3
SCH3
SCH3
CONHPh-4-OMe
CONHPh-4-OMe
CONHPh-4-OMe
COCH2Ph-4-OMe
COCH2Ph-4-isobutyl
14 (10-21)
>1000
70 (53-90)
3.1 (1.3-5.2)
15 (10-22)
212 (162-278)
>1000
80 (63-100)
780 (700-820) 50 (41-60)
180 (140-233) >1000
SCH3
COCH2Ph-3,4-Medioxy 70 (61-80)
4.1 (1.9-7.0)
30 (22-41)
65 (56-75)
>1000
36 (25-52)
12 (8-18)
23 (18-29)
110 (59-165)
NHCH2CH3
NH-Ph-p-OH
NHCH2CH3‚HCl
N-Me-piperazine‚2HCl
NHCH2CH3‚HCl
COCH2Ph-3,4-Medioxy 136 (110-168) 61 (50-74)
183 (157-213)
308 (250-380)
25 (17-35)
>1000
H
H
H
666 (593-748) >1000
41 (30-54)
48 (36-65)
100 (83-120)
10 (7-14)
135 (97-188)
16 (13-20)
CONHPh-4-OMe
9 (6-12)
a
b
Displacement of [3H]DPCPX binding at human A1 adenosine receptors expressed in CHO cells. Displacement of [3H]SCH58261
binding at human A2A adenosine receptors expressed in CHO cells. c Displacement of [3H]DPCPX binding at human A2B adenosine receptors
expressed in HEK-293 cells. Displacement of [3H]MRE 3008F20 binding at human A3 adenosine receptors expressed in CHO cells.
d
Ta ble 4. Functional Assay. Effect of Selected Antagonists on
Ta ble 5. Biological Results of 5′-Substituted Compounds
Agonist-Mediated CAMP Productionc
compound hA2A receptora IC50 (nM) hA3 receptorb IC50 (nM)
41
42
46
49
53
55
6.1 (4.2-8.8)
14.8 (10.2-21.5)
17.5 (13.4-22.9)
21.5 (15.8-29.2)
48 (40-59)
188 (166-213)
852 (764-951)
813 (740-892)
466 (414-525)
103 (79-134)
45 (30-66)
A1
A2A
A2B
A3
61 (48-77)
compd
R
Ki nMa Ki nMb Ki nMc Ki nMd
a
hA2A adenosine receptors were stimulated with 100 nM NECA
b
51
57
58
N-methylpiperazine >1000 >1000 >1000 >1000
N-methylpiperazine >1000 >1000 >1000 >1000
hA3 adenosine receptors were inhibited with 100 nM Cl-IB-
MECA c Values are the means of at least three experiments and
in parentheses the 95% confidence limits are shown.
morpholine
>1000 >1000 >1000 >1000
Displacement of [3H]DPCPX binding at human A1 adenosine
receptors expressed in CHO cells. b Displacement of [3H]SCH58261
binding at human A2A adenosine receptors expressed in CHO cells.
c Displacement of [3H]DPCPX binding at human A2B adenosine
a
by Mannich reaction, introducing cycloaminomethyl
functions. These functions, easily salified, increased
water solubility, but a complete loss of affinity at all
the adenosine receptors subtypes (see Ta ble 5) was
observed. Also for compounds 5a -8 we can confirm that
the furanyl moiety is a necessary element for receptor
interaction and that no modifications are allowed.
d
receptors expressed in HEK-293 cells. Displacement of [3H]MRE
3008F20 binding at human A3 adenosine receptors expressed in
CHO cells.
selectivity was more or less complete. When a small
substitution group is at N8 and 9 positions, compound
such as 41, showed the highest A2A binding affinity.
Whereas, compounds with small alkyl amino substi-
tution group at the 9 position together with an ureidic
function at 5 position displayed the highest A3 subtype
selective binding (compound 44).
These structural modifications introduced to the
tricyclic antagonistic structure allowed us to obtain
important information about the SAR of pyrazolo-
triazolo-pyrimidines: (1) the furanyl ring is an indis-
pensable structural element for adenosine receptor
binding, and (2) increased steric hindrance leads to a
decrease in receptor affinity.
Con clu sion
In the present study we have described the affinity
at adenosine receptor subtypes of a new series of
pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine compounds,
structurally modified with respect to lead compounds
previously synthesized in our laboratory. These lead
molecules are very potent and selective antagonists on
A2A and A3 adenosine receptor subtypes. Modifications
at the 2-position and at the 5′-position (Mannich reac-
tion) of 61 have decreased affinity at the A2A receptor
subtype, probably due to an increased steric hindrance
by the (substituted)aromatic rings introduced. Modifica-
tions at 9-position of the tricyclic structure of the
adenosine antagonists allowed the affinity at adenosine
receptor subtypes to be maintained, but the loss of
Exp er im en ta l Section
Gen er a l. Reactions were routinely monitored by thin-layer
chromatography (TLC) on silica gel (precoated F245 Merck

1236 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Baraldi et al.
plates) and products visualized with iodine or potassium
permanganate solution. 1H NMR were determined in CDCl3
or DMSO-d6 solutions with a Bruker AC 200 spectrometer,
peaks positions are given in parts per million (δ) 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 and are uncorrected. Chromatogra-
phy was performed using Merk 60-200 mesh silica gel. All
p-toluenesulfonic acid (1.5 molar equiv), and the mixture was
heated at 160 °C for 4 h. Then cyanamide (6 molar equiv) 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 concen-
trated under reduced pressure and washed with water (3 ×
50 mL). The organic layer was dried (Na2SO4) and evaporated
under vacuum. The residue was purified by chromatography
(EtOAc/light petroleum 1:1) to afford the final products 5a -d
as solids.
1
products reported showed H NMR spectra in agreement with
the assigned structures. Organic solutions were dried over
anhydrous magnesium sulfate. Elemental analyses were per-
formed 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.
Gen er a l P r oced u r e for th e P r ep a r a tion of 7-(2-P h en -
yleth yl)-2-(su bstitu ted p h en yl)p yr a zolo[4,3-e]-1,2,4-tr ia -
zolo[1,5-c]p yr im id in es 3a -d . The imino ether 2 (3 g, 15
mmol) dissolved in 2-methoxyethanol (35 mL) was added to
the selected hydrazide (15 mmol), and the mixture was heated
at reflux for 12 h. After being cooled, 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 crystallization from EtOAc to afford the compounds
as solids.
7-(2-P h en ylet h yl)-2-(4-h yd r oxid p h en yl)p yr a zolo[4,3-
e]-1,2,4-tr ia zolo[1,5-c]p yr im id in e (3a ). Yield 50%; yellow
solid; mp 246-247 °C; 1H NMR (DMSO-d6) δ 3.32 (t, 2H, J )
8), 4.73 (t, 2H, J ) 8), 6.94 (d, 2H, J ) 8), 7.13 (m, 5H), 8.07
(d, 2H, J ) 8), 8.51 (s, 1H), 9.52 (s, 1H), 10.22 (bs, 1H).
7-(2-P h en ylet h yl)-2-(4-ch lor op h en yl)p yr a zolo[4,3-e]-
1,2,4-tr ia zolo[1,5-c]p yr im id in e (3b). Yield 76%; yellow solid;
mp 237-239 °C; 1H NMR (DMSO-d6) δ 3.25 (t, 2H, J ) 8),
4.76 (t, 2H, J ) 8), 7.17 (m, 5H), 7.67 (d, 2H, J ) 8), 7.96 (m,
1H), 8.25 (d, 1H, J ) 8), 8.56 (s, 1H), 9.60 (s, 1H).
7-(2-P h en ylet h yl)-2-(2-et h oxyp h en yl)p yr a zolo[4,3-e]-
1,2,4-tr ia zolo[1,5-c]p yr im id in e (3c). Yield 80%, yellow oil
used for the next step of reaction without other purifications.
7-(2-P h en yleth yl)-2-ph en ylpyr azolo[4,3-e]-1,2,4-tr iazolo-
[1,5-c]p yr im id in e (3d ). Yield 75%, orange oil used for the
next step of reaction without purifications.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Am in o-
1-(2-ph en yleth yl)-4-[3-(su bstitu ted-ph en yl)-1,2,4-tr ia zolo-
5-yl]p yr a zoles 4a -d . A solution of the mixture of 3a -d (10
mmol) in aqueous 10% HCl (20 mL) and dioxane (25 mL) was
refluxed for 45 min. Then the solution was cooled and basified
with 10% NaOH at 0 °C. The compounds were extracted with
EtOAc (3 × 50 mL); the organic layers were dried over Na2-
SO4 and evaporated under vacuum. The residues obtained
were recrystallized from EtOAc to afford the desired com-
pounds as solids.
5-Am in o-1-(2-ph en yleth yl)-4-[3-(4-h ydr oxyph en yl)-1,2,4-
tr ia zolo-5-yl]p yr a zole (4a ). Yield 86%; yellow solid; mp 168-
170 °C; 1H NMR (DMSO-d6) δ 3.03 (t, 2H, J ) 9), 4.26 (t, 2H,
J ) 8), 5.48 (bs, 2H), 6.98 (d, 2H, J ) 8), 7.24 (m, 5H), 8.11 (d,
2H, J ) 8), 8.21 (s, 1H).
5-Am in o-7-(2-p h en ylet h yl)-2-(4-h yd r oxyp h en yl)p yr a -
zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in e (5a ). Yield 32%;
pale yellow solid; mp 296-297 °C; 1H NMR (DMSO-d6) δ 3.18
(t, 2H, J ) 8), 4.49 (t, 2H, J ) 8), 6.90 (d, 2H, J ) 8), 7.16 (m,
5H), 8.02 (bs, 2H), 8.08 (d, 2H, J ) 8), 8.16 (s, 1H), 9.97 (s,
1H). Anal. (C20H17N7O) C, H, N.
5-Am in o-7-(2-ph en yleth yl)-2-(4-ch lor oph en yl)pyr azolo-
[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in e (5b). Yield 48%; pale
1
yellow solid; mp 272-273 °C; H NMR (DMSO-d6) δ 3.19 (t,
2H, J ) 8), 4.49 (t, 2H, J ) 8), 7.21 (m, 5H), 7.65 (d, 2H, J )
8), 8.09 (bs, 2H), 8.20 (d, 2H, J ) 8), 8.24 (s, 1H). Anal. (C20H16
ClN7) C, H, N.
-
5-Am in o-7-(2-ph en yleth yl)-2-(2-eth oxyph en yl)pyr azolo-
[4,3-e]-1,2,4-tr iazolo[1,5-c]pyr im idin e (5c). Yield 38%; white
solid; mp 159-160 °C; 1H NMR (DMSO-d6) δ 1.33 (t, 3H, J )
8), 3.19 (t, 2H, J ) 8), 4.11 (q, 2H, J ) 8), 4.54 (t, 2H, J ) 8),
7.10 (m, 2H), 7.19 (m, 5H); 7.44 (m, 1H), 7.84 (d, 1H, J ) 8),
7.92 (bs, 2H), 8.16 (s, 1H). Anal. (C22H21N7O) C, H, N.
5-Am in o-7-(2-p h en ylet h yl)-2-p h en yl-p yr a zolo[4,3-e]-
1,2,4-tr ia zolo[1,5-c]p yr im id in e (5d ). Yield 42%; pale yellow
solid; mp 240-242 °C; NMR (DMSO-d6) δ 3.21 (t, 2H, J ) 8),
4.52 (t, 2H, J ) 8), 5.90 (bs, 2H), 7.22 (m, 6H), 7.60 (m, 2H),
8.27 (m, 3H). Anal. (C20H17N7) C, H, N.
P r oced u r e for th e P r ep a r a tion of 2-[4-(5-Am in o-7-(2-
p h en yleth yl)-7H-p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr i-
m id in -2-yl)p h en oxy]a cetic Acid Eth yl Ester 6. To a solu-
tion of compound 5a (100 mg, 0.27 mmol) in dry DMF (10 mL)
was added anhyd K2CO3 (44.7 mg, 0.32 mmol), and the
resulting mixture was stirred at room temperature for 10 min.
Then was added 2-chloroethyl acetate (34 µL, 0.32 mmol),
and the solution was stirred at the same conditions for 12 h.
The solvent was removed under reduced pressure, and to the
residue was added water (30 mL).The aqueous phase was
extracted with EtOAc (3 × 30 mL), and the organic phases
were dried (Na2SO4) and evaporated to dryness under vacuum.
The oily residue was recrystallized from EtOAc to furnish the
desired compound as solid.
1
Yield 63%; white solid; mp 205-206 °C; H NMR (DMSO-
d6) δ 1.22 (t, 3H, J ) 8), 3.20 (t, 2H, J ) 8), 4.21 (q, 2H, J )
6), 4.49 (t, 2H, J ) 8), 4.88 (s, 2H), 7.08 (bs, 2H), 7.16 (m, 9H),
8.16 (s, 1H). Anal. (C24H23N7O3) C, H, N.
P r oced u r e for th e P r ep a r a tion of 2-[4-(5-Am in o-7-(2-
p h en yleth yl)-7H-p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr i-
m id in -2-yl)p h en oxy]a cetic Acid 7. A solution of the com-
pound 6 (50 mg, 0.11 mmol) in aqueous 10% HCl (3 mL) and
dioxane (5 mL) was heated at 60 °C for 3 h. Then the solution
was cooled and basified with 10% NaOH at 0 °C. The solid
formed was filtered off and washed with cold water to afford
the desired compound.
5-Am in o-1-(2-p h en yleth yl)-4-[3-(4-ch lor op h en yl)-1,2,4-
tr ia zolo-5-yl]p yr a zole (4b). Yield 90%; yellow solid; mp 202-
204 °C; 1H NMR (DMSO-d6) δ 3.04 (t, 2H, J ) 8), 4.19 (t, 2H,
J ) 8), 6.27 (bs, 2H), 7.27 (m, 5H), 7.23 (d, 2H, J ) 8), 7.69 (s,
1H), 8.11 (d, 2H, J ) 8), 13.81 (s, 1H).
1
Yield 94%; white solid; mp 250-251 °C; H NMR (DMSO-
d6) δ 3.18 (t, 2H, J ) 8), 4.49 8t, 2H, J ) 8), 4.75 (s, 2H), 7.18
(m, 9H), 8.02 (bs, 2H), 8.17 (s, 1H), 11.03 (bs, 1H). Anal.
(C22H19N7O3) C, H, N.
5-Am in o-1-(2-p h en yleth yl)-4-[3-(2-eth oxyp h en yl)-1,2,4-
tr ia zolo-5-yl]p yr a zole (4c). Yield 83%, orange solid, used for
the next step of reaction without purifications.
5-Am in o-1-(2-ph en yleth yl)-4-[3-(2-ph en yl)-1,2,4-tr iazolo-
5-yl]p yr a zole (4d ). Yield 72%, yellow solid, used for the next
step of reaction without purifications.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Am in o-
7-(2-p h en yleth yl)-2-(su bstitu ted p h en yl)p yr a zolo[4,3-e]-
1,2,4-tr ia zolo[1,5-c]p yr im id in es 5a -d . To a solution of
tricycles derivatives 4a -d (1.5 mmol) in N-methylpyrrolidone
(5 mL) were added cyanamide (380 mg, 6 molar equiv) and
P r oced u r e for th e P r ep a r a tion of 2-[4-(5-Am in o-7-(2-
p h en yleth yl)-7H-p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr i-
m id in -2-yl)p h en oxy]-N-(4-iod op h en yl)a ceta m id e 8. To a
solution of compound 5a (100 mg, 0.27 mmol) in DMF (10 mL)
was added K2CO3 (44.7 mg, 0.32 mmol), and the mixture was
stirred at room temperature for 10 min. Then was added
2-chloro-N-(4-iodophenyl)acetamide (95.5 mg, 0.32 mmol) dis-
solved in DMF (3 mL), and the resulting mixture was heated
at 50 °C for 5 h. The solvent was removed under reduced
pressure, and the residue obtained was washed with water
(30 mL) and extracted with EtOAc (3 × 35 mL). The organic

New A2A and A3 Adenosine Receptors Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1237
layer was dried (Na2SO4) and evaporated under vacuum to
afford a residue that was crystallized from a mixture of EtOAc/
CH3OH to furnish the desired compound.
Yield 71%; white solid; mp >300 °C; 1H NMR (DMSO-d6) δ
3.14 (t, 2H, J ) 8), 4.49 (t, 2H, J ) 8), 4.80 (s, 2H), 7.19 (m,
7H), 7.52 (d, 2H, J ) 8), 7.69 (d, 2H, J ) 8), 8.15 (bs, 2H), 8.17
(m, 3H), 10.25 (bs, 1H). Anal. (C28H23IN8O2) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 1H-1-Meth -
yl-3-a m in o-4-cya n o-5-su bstitu ted P yr a zoles 17-19. To a
solution of compounds 11-13 (35 mmol) in absol EtOH (50
mL) was added methylhydrazine (1.5 molar equiv), and the
mixture was heated at reflux 18 h. The solvent was evaporated
under reduced pressure, and the residue was recrystallized
from absol EtOH.
1H-1-Meth yl-3-a m in o-4-cya n o-5-(4-m eth ylp ip er a zin -1-
yl)p yr a zole (17). Yield 78%; yellow solid; mp 202-203 °C;
1H NMR (CDCl3) δ 2.33 (s, 3H), 2.52 (t, 4H, J ) 5), 3.2 (t, 4H,
J ) 5), 3.48 (s, 3H), 3.97 (bs, 2H).
1H-1-Meth yl-3-a m in o-4-cya n o-5-(4-m eth oxyp h en yla m i-
n o)p yr a zole (18). Yield 44%; pale yellow solid; mp 144°C; 1H
NMR (DMSO-d6) δ 3.4 (s, 3H), 3.67 (s, 3H), 6.45 (s, 2H), 6.76
(d, 2H, J ) 9), 7.42 (d, 2H, J ) 9), 8.15 (s, 1H).
1H -1-Met h yl-3-a m in o-4-cya n o-5-et h yla m in op yr a zole
(19). Yield 79%; yellow solid, mp 120 °C; 1H NMR (DMSO-d6)
δ 1.15 (t, 3H, J ) 8), 3.3 (m, 2H, J ) 8), 3.35 (s, 3H), 5.03 (bs,
2H), 6.3 (bs, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of 1H-1-Meth -
yl-3-[(et h oxym et h ylen e)a m in o]-4-cya n o-5-su b st it u t ed
P yr a zoles 20-22. The 3-amino-4-cyanopyrazoles 17-19 (29
mmol) were dissolved in triethyl orthoformate (80 mL), and
the solution was refluxed under nitrogen for 12 h. Then the
solvent was removed under reduced pressure, and the oily
residue was recrystallized from absol EtOH to afford the
corresponding imino ethers 20-22.
Gen er a l P r oced u r e for th e P r ep a r a tion of 2-(Bis-
(m eth ylsu lfa n yl)m eth ylen e)m a lon on itr ile 9 a n d 2-(Bis-
(p r op ylsu lfa n yl)m eth ylen e)m a lon on itr ile 10. To a solu-
tion of malononitrile (8 g, 0.12 mol) in DMF (40 mL) was added
an excess of anhydrous K2CO3 (12 g) and, after 10 min, CS2 in
small amount (7.2 mL, 0.12 mol). After 10 min was added a
catalytic amount of tetrabutylammonium bromide and methyl
iodide or propyl bromide (0.12 mol) in small portions, and the
mixture was stirred at room temperature for 30 min, heated
at 50 °C for 2 h, and finally stirred at room temperature
overnight. The residue was washed with water (100 mL), and
the solid formed was collected by filtration and washed with
cold water.
2-(Bis(m et h ylsu lfa n yl)m et h ylen e)m a lon on it r ile (9).
Yield 83%; yellow solid; mp 79-81 °C; IR (KBr): 2190, 2210
1
cm-1; H NMR (CDCl3) δ 2.7 (s, 6H).
2-(Bis(p r op ylsu lfa n yl)m eth ylen e)m a lon on itr ile (10).
Yield 70%; yellow solid; mp 85-86; IR(KBr) 1456, 2218, 2967
1
cm-1; H NMR (CDCl3) δ 1.05 (t, 6H, J ) 7.3), 1.75 (m, 4H, J
) 7.3), 3.2 (t, 4H, J ) 7.3).
Gen er a l P r oced u r e for th e P r ep a r a tion of 1H-1-Su b-
stitu ted -3-a m in o-4-cya n o-5-m eth ylsu lfa n ylp yr a zoles 14
a n d 15. To a solution of compound 9 (5 g, 29 mmol) in absol
EtOH (50 mL) was added the appropriate hydrazine (29
mmol), and then the mixture was refluxed for 6 h. The solvent
was removed at reduced pressure, and the residue was washed
with water (50 mL) and extracted with EtOAc (3 × 35 mL).
The organic phases were dried (Na2SO4) and evaporated, and
the residue obtained was purified by chromatography (EtOAc/
light petroleum, 7:3) to afford the products as solids in good
yield.
1H -1-Met h yl-3-a m in o-4-cya n o-5-m et h ylsu lfa n ylp yr a -
zole (14). Yield 95%; yellow solid; mp 120 °C; 1H NMR (CDCl3)
δ 2.54 (s, 3H), 3.70 (s, 3H), 4.17 (bs, 2H).
1H -1-(3-P h en ylp r op yl)-3-a m in o-4-cya n o-5-m et h ylsu l-
fa n ylp yr a zole (15). Yield 60%; yellow solid; mp 110-113 °C;
1H NMR (CDCl3) δ 2.11 (m, 2H), 2.53 (s, 3H), 2.63 (t, 2H, J )
7.3), 4.05 (t, 2H, J ) 7.3), 4.1 (bs, 2H), 7.2 (m, 5H).
P r ep a r a tion of 1H-1-Meth yl-3-a m in o-4-cya n o-5-p r op -
ylsu lfa n ylp yr a zole 16. To a solution of 26 (6 g, 26 mmol) in
absol EtOH (70 mL) was added methylhydrazine (26 mmol),
and then the mixture was refluxed for 4 h before evaporating
the solvent. The residue was crystallized from a mixture of
Et2O/light petroleum (1:1).
Yield 83%; pale yellow solid; mp 56-58 °C; 1H NMR (CDCl3)
δ 1.02 (t, 3H, J ) 7.3), 1.63 (m, 2H), 2.92 (t, 2H, J ) 7.3), 3.74
(s, 3H), 4.11 (bs, 2H).
1H-Meth yl-3-[(eth oxym eth ylen e)a m in o]-4-cya n o-5-(4-
m et h ylp ip er a zin -1-yl)p yr a zole (20). Yield 70%; yellow
crystals, mp 108-110 °C; 1H NMR (DMSO-d6) δ 1.28 (t, 3H, J
) 7), 2.22 (s, 3H), 2.45 (t, 4H, J ) 5), 3.14 (t, 4H, J ) 5), 3.55
(s, 3H), 4.25 (q, 2H, J ) 7), 8.23 (s, 1H).
1H-Meth yl-3-[(eth oxym eth ylen e)a m in o]-4-cya n o-5-(4-
m eth oxyp h en yla m in o)p yr a zole (21). Yield 60%; yellow
solid; mp 125-128 °C; 1H NMR (DMSO-d6) δ 1.34 (t, 3H, J )
7), 3.54 (s, 3H); 3.69 (s, 3H), 4.30 (q, 2H, J ) 7), 6.84 (m, 3H),
7.43 (d, 2H, J ) 9), 8.46 (s, 1H).
1H-Meth yl-3-[(eth oxym eth ylen e)a m in o]-4-cya n o-5-eth -
yla m in op yr a zole (22). Yield 76%; yellow solid; mp 110-111
1
°C; H NMR (DMSO-d6) δ 1.18 (t, 3H, J ) 6.5), 1.28 (t, 3H, J
) 7), 3.34 (m, 2H), 3.42 (s, 3H), 4.22 (q, 2H, J ) 7), 6.62 (bs,
1H), 8.17 (s, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of 1H-1-m eth -
yl/(3-ph en ylpr opyl)-3-[(eth oxym eth ylen e)am in o]-4-cyan o-
5-su bstitu ted P yr a zoles 23-25. The 3-amino-4-cyanopyra-
zoles 14-16 (23 mmol) were dissolved in triethyl orthoformate
(80 mL), and the solution was refluxed under nitrogen for 12
h. Then the solvent was removed under reduced pressure, and
the oily residue was recrystallized from absol EtOH to afford
the corresponding imino ethers 23-25.
1H -1-Met h yl-3-[(et h oxym et h ylen e)a m in o]-4-cya n o-5-
m eth ylsu lfa n ylp yr a zole (23). Yield 94%; yellow crystals; mp
1
35-36°C; H NMR (DMSO-d6) δ 1.29 (t, 3H, J ) 7), 2.54 (s,
3H); 3.81 (s, 3H), 4.26 (q, 2H, J ) 7), 8.30 (s, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of 2-Su bsti-
tu ted -m eth ylsu lfa n ylm eth ylen em a lon on itr ile 11-13. To
a solution of compound 9 (7 g, 41 mmol) in absol EtOH (80
mL) was added the appropriate amine (41 mmol), and the
mixture was heated at reflux for 4-6 h. Then the solvent was
evaporated under reduced pressure, and the residue was
crystallized from EtOH to afford the product as a solid.
2-[(4-Meth yl-1-p yp er a zin yl)m eth ylsu lfa n ylm eth ylen e]-
m a lon on itr ile (11). Yield 80%; yellow solid, mp 115-116 °C;
1H NMR (DMSO-d6) δ 2.21 (s, 3H), 2.44 (t, 4H, J ) 5), 2.52 (s,
3H), 3.72 (t, 4H, J ) 5).
2-[(4-Meth oxyph en yla m in o)m eth ylsu lfa n ylm eth ylen e]-
m a lon on itr ile (12). Yield 70%; green solid; mp 150-152 °C;
1H NMR (DMSO-d6) δ 2.53 (s, 3H), 3.76 (s, 3H), 6.9 (d, 2H, J
) 6), 7.2 (d, 2H, J ) 6), 10.37 (bs, 1H).
2-[(e t h yla m in o)m e t h ylsu lfa n ylm e t h yle n e ]m a lon o-
n itr ile (13). Yield 61%; yellow solid; mp 74°C; 1H NMR
(DMSO-d6) δ 1.18 (t, 3H, J ) 7), 2.55 (s, 3H), 3.5 (m, 2H, J )
7), 8.68 (bs, 1H).
1H-1-(3-P h en ylp r op yl)-3-[(eth oxym eth ylen e)a m in o]-4-
cya n o-5-m eth ylsu lfa n ylp yr a zole (24). Yield 74%; orange
oil; 1H NMR (CDCl3) δ 1.35 (t, 3H, J ) 7), 2.15 (m, 2H, J ) 7),
2.55 (s, 3H), 2.61 (t, 2H, J ) 7.5), 4.00 (t, 2H, J ) 7.5), 4.22 (q,
2H, J ) 7), 7.20 (m, 5H), 8.35 (s, 1H).
1H -1-Met h yl-3-[(et h oxym et h ylen e)a m in o]-4-cya n o-5-
p r op ylsu lfa n ylp yr a zole (25). Yield 94%; orange oil; 1H NMR
(DMSO-d6) δ 1.02 (t, 3H, J ) 7), 1.37 (t, 3H, J ) 7), 1.63 (m,
2H, J ) 7), 2.94 (t, 2H, J ) 7), 3.84 (s, 3H), 4.37 (q, 2H, J )
7), 8.20 (s, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of 8-Meth yl-
2-(2-fu r yl)-9-su b st it u t ed -p yr a zolo[4,3-e]-1,2,4-t r ia zolo-
[1,5-c]p yr im id in es 26-28. Imino ethers 20-22 (18 mmol)
were dissolved in 2-methoxyethanol (70 mL), and 2-furoic acid
hydrazide (18 mmol) was added. The mixture was refluxed for
12 h, then, 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)

1238 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Baraldi et al.
and cooled. The precipitate was filtered off and purified by
crystallization from a mixture of CH2Cl2/light petroleum.
3-Am in o-1H -1-m e t h yl-5-p r op ylsu lfa n yl-4-[3-(2-fu r -
yl)1,2,4-tr ia zol-5-yl]p yr a zole (36). Yield 67%; yellow solid;
mp 154-155 °C; 1H NMR (DMSO-d6) δ 0.88 (t, 3H, J ) 7),
1.42 (m, 2H, J ) 7), 2.81 (t, 2H, J ) 7), 3.75 (s, 3H), 5.56 (bs,
2H), 6.66 (s, 1H), 7.00 (s, 1H), 7.85 (s, 1H), 13.60 (bs, 1H).
3-Am in o-1H-1-(3-p h en ylp r op yl)-5-m eth ylsu lfa n yl-4-[3-
(2-fu r yl)1,2,4-tr ia zol-5-yl]p yr a zole (37). Yield 80%; yellow
solid; mp 205-207°C; 1H NMR (DMSO-d6) δ 2.20 (m, 2H, J )
7), 2.67 (s, 3H), 4.21 (t, 2H, J ) 7), 4.41 (t, 2H, J ) 7), 5.80
(bs, 2H), 6.56 (s, 1H), 7.25 (m, 5H), 7.36 (s, 1H), 7.58 (s, 1H),
13.50 (bs, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Am in o-
8-a lk yl/a r yla lk yl-9-su bstitu ted -2-(2-fu r yl)p yr a zolo[4,3-e]-
1,2,4-tr ia zolo[1,5-c]p yr im id in es 38-43. To a solution of
pyrazole derivatives 32-37 (3 mmol) in N-methylpyrrolidone
(20 mL) were added cyanamide (18 mmol) and p-toluene-
sulfonic acid (6 mmol), and the mixture was heated at 160 °C
for 4 h. Then cyanamide (18 mmol) was added again, and the
solution was heated at 160 °C for additional 18 h. Then the
solution was diluted with EtOAc (100 mL), and the precipitate
(excess of cyanamide) was filtered off; the filtrate was concen-
trated under reduced pressure and washed with water (3 ×
50 mL). The organic layer was dried (Na2SO4) and evaporated
under vacuum. The residue was purified by chromatography
(EtOAc/light petroleum 1:1) to afford the final products 38-
43 as solids.
8-Meth yl-2-(2-fu r yl)-9-(4-m eth ylpiper azin -1-yl)-pyr azolo-
[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in e (26). Yield 50%; yel-
low solid; mp 223-225 °C; 1H NMR (DMSO-d6) δ 2.30 (s, 3H),
2.56 (m, 4H), 3.37 (m, 4H), 3.97 (s, 3H), 6.70 (m, 1H), 7.20 (m,
1H), 7.95 (m, 1H), 9.32 (s, 1H).
8-Meth yl-2-(2-fu r yl)-9-(4-m eth oxyp h en yla m in o)-p yr a -
zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in e (27). Yield 35%;
1
yellow solid; mp 211-213 °C; H NMR (DMSO-d6) δ 3.72 (s,
3H), 3.96 (s, 3H), 6.74 (m, 1H), 6.85 (d, 2H, J ) 8.6), 6.96 (d,
1H, J ) 3.5), 7.60 (d, 2H, J ) 8.6), 7.97 (d, 1H, J ) 1), 8.40 (s,
1H), 9.52 (s, 1H).
8-Meth yl-2-(2-fu r yl)-9-(eth ylam in o)-pyr azolo[4,3-e]-1,2,4-
tr ia zolo[1,5-c]p yr im id in e (28). Yield 67%; yellow solid; mp
1
277-280 °C; H NMR (DMSO-d6) δ 1.22 (t, 3H, J ) 7), 3.78
(s, 3H), 4.00 (q, 2H), 6.70 (m, 2H), 7.13 (d, 1H, J ) 3), 7.92 (s,
1H), 9.14 (s, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of 8-Su bsti-
tu ted -2-(2-fu r yl)-9-su bstitu ted -p yr a zolo[4,3-e]1,2,4-tr ia -
zolo[1,5-c]p yr im id in es 29-31. Imino ethers 23-25 (18
mmol) were dissolved in 2-methoxyethanol (70 mL), and
2-furoic acid hydrazide (18 mmol) was added. The mixture was
refluxed for 12 h, then, 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 crystallization from EtOAc.
8-Me t h yl-2-(2-fu r yl)-9-m e t h ylsu lfa n ylp yr a zolo[4,3-
e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (29). Yield 30%; white
solid; mp 140-142 °C; 1H NMR (DMSO-d6) δ 2.82 (s, 3H), 4.16
(s, 3H), 6.74 (d, 1H, J ) 3.5), 7.25 (d, 1H, J ) 3.4), 7.97 (s,
1H), 9.42 (s, 1H).
8-(3-P h en ylp r op yl)-2-(2-fu r yl)-9-m et h ylsu lfa n ylp yr a -
zolo[4,3-e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (30). Yield 22%;
yellow solid; mp 154-157 °C; 1H NMR (CDCl3) δ 2.30 (m, 2H),
2.67 (t, 2H, J ) 7), 2.80 (s, 3H), 4.53 (t, 2H, J ) 7), 6.62 (d,
1H, J ) 2), 7.20 (m, 5H), 7.35 (d, 1H, J ) 2), 7.65 (s, 1H), 9.07
(s, 1H).
8-Me t h yl-2-(2-fu r yl)-9-p r op ylsu lfa n yl-p yr a zolo[4,3-
e]1,2,4-tr ia zolo[1,5-c]p yr im id in e (31). Yield 35%; yellow
solid; mp 130-132 °C; 1H NMR (DMSO-d6) δ 0.93 (t, 3H, J )
7), 1.45 (m, 2H, J ) 7), 3.31 (t, 2H, J ) 7), 4.10 (s, 3H), 6.75
(d, 1H, J ) 2), 7.24 (d, 1H, J ) 3), 7.98 (s, 1H), 9.43 (s, 1H).
5-Am in o-8-m eth yl-9-(eth yla m in o)-2-(2-fu r yl)p yr a zolo-
[4,3-e]-1,2,4-tr iazolo[1,5-c]pyr im idin e (38). Yield 42%; white
1
crystals; mp >300 °C; H NMR (DMSO-d6) δ 1.19 (t, 3H, J )
7), 3.64 (s, 3H), 3.99 (q, 2H, J ) 7), 6.39 (bs, 2H), 6.70 (s, 1H),
7.08 (s, 1H), 7.35 (bs, 2H), 7.91 (s, 1H). Anal. (C13H14N8O) C,
H, N.
5-Am in o-8-m e t h yl-9-(4-m e t h oxyp h e n yla m in o)-2-(2-
fu r yl)p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c] p yr im id in e (39).
1
Yield 35%; white crystals; mp 230 °C; H NMR (DMSO-d6) δ
3.71 (s, 3H), 3.76 (s, 3H), 6.73 (m, 1H), 6.85 (d, 2H, J ) 9),
7.22 (d, 1H, J ) 3), 7.58 (d, 2H, J ) 9), 7.94 (bs, 2H), 8.02 (bs,
2H). Anal. (C18H16N8O2) C, H, N.
5-Am in o-8-m eth yl-9-(4-m eth ylpiper azin -1-yl)-2-(2-fu r yl)-
p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c] p yr im id in e (40). Yield
1
33%; white crystals; mp 276-278 °C; H NMR (DMSO-d6) δ
2.27 (s, 3H), 2.50 (m, 4H), 3.31 (m, 4H), 3.79 (s, 3H), 6.70 (m,
1H), 7.12 (d, 1H, J ) 4), 7.54 (bs, 2H), 7.92 (s, 1H). Anal.
(C16H19N9O) C, H, N.
5-Am in o-8-m eth yl-9-m eth ylsu lfan yl-2-(2-fu r yl)-pyr azolo-
[4,3-e]-1,2,4-tr iazolo[1,5-c]pyr im idin e (41). Yield 83%; white
1
Gen er a l P r oced u r e for th e P r ep a r a tion of 3-Am in o-
1H-1-su bstitu ted -5-su bstitu ted -4-[3-(2-fu r yl)-1,2,4-tr ia zol-
5-yl]p yr a zoles 32-37. A solution of the mixture of 26-31 (7
mmol) in aqueous 10% HCl (20 mL) and dioxane (15 mL) was
refluxed for 1 h. Then the solution was cooled and basified
with 10% NaOH at 0 °C. The compounds were extracted with
EtOAc (3 × 50 mL); the organic layers were dried over Na2-
SO4 and evaporated under vacuum. The residues obtained
were recrystallized from EtOAc to afford the desired com-
pounds as solids.
crystals; mp 298 °C; H NMR (DMSO-d6) δ 2.77 (s, 3H), 4.01
(s, 3H), 6.76 (s, 1H), 7.22 (d, 1H, J ) 3), 7.70 (bs, 2H), 7.95 (s,
1H). Anal. (C12H11N7OS) C, H, N.
5-Am in o-8-m eth yl-9-pr opylsu lfan yl-2-(2-fu r yl)-pyr azolo-
[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in e (42). Yield 31%; yel-
low solid; mp 298-300 °C; 1H NMR (DMSO-d6) δ 0.92 (t, 3H,
J ) 7), 1.47 (m, 2H, J ) 7), 3.32 (t, 2H, J ) 7), 4.02 (s, 3H),
6.72 (s, 1H), 7.20 (s, 1H), 7.90 (s, 1H), 7.91 (bs, 1H). Anal.
(C14H15N7OS) C, H, N.
5-Am in o-8-(3-p h e n ylp r op yl)-9-m e t h ylsu lfa n yl-2-(2-
fu r yl)-p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in e (43).
Yield 23%; white crystals; mp 190-192 °C; 1H NMR (DMSO-
d6) δ 2.27 (m, 2H), 2.69 (t, 2H, J ) 7), 2.76 (s, 3H), 4.33 (t, 2H,
J ) 7), 5.95 (bs, 2H), 6.60 (s, 1H), 7.24 (m, 5H), 7.32 (s, 1H),
7.63 (s, 1H). Anal. (C20H19N7OS) C, H, N.
3-Am in o-1H-1-m eth yl-5-(eth ylam in o)-4-[3-(2-fu r yl)1,2,4-
tr ia zol-5-yl]p yr a zole (32). Yield 94%; yellow solid; mp 230
1
°C; H NMR (DMSO-d6) δ 1.05 (t, 3H, J ) 7), 3.07 (m, 2H),
3.43 (s, 3H), 5.13 (bs, 2H), 6.57 (s, 1H), 6.87 (s, 1H), 7.87 (s,
1H), 13.30 (bs, 1H).
3-Am in o-1H-1-m eth yl-5-(4-m eth oxyp h en yla m in o)-4-[3-
(2-fu r yl)1,2,4-tr ia zol-5-yl]p yr a zole (33). Yield 90%; yellow
oil; used without further purifications.
3-Am in o-1H-1-m eth yl-5-[4-(m eth ylp ip er a zin -1-yl)]-4-[3-
(2-fu r yl)1,2,4-tr ia zol-5-yl]p yr a zole (34). Yield 62%; yellow
solid; mp 207-209 °C; 1H NMR (DMSO-d6) δ 2.21 (s, 3H), 2.41
(m, 4H), 3.11 (m, 4H), 3.48 (s, 3H), 5.10 (bs, 2H), 6.65 (m, 1H),
7.87 (d, 1H, J ) 3), 7.85 (s, 1H), 13.50 (bs, 1H).
Gen er a l P r oced u r e for th e P r ep a r a tion of 1-[2-(2-
F u r yl)-8-m et h yl-9-su b st it u t ed -8H -p yr a zolo[4,3-e]-1,2,4-
tr ia zolo[1,5-c] p yr im id in -5-yl]-3-(4-m eth oxyp h en yl)u r ea
44-46. To a solution of compounds 38, 40, and 41 (1.7 mmol)
in dioxane (20 mL) were added 4-methoxyphenyl isocyanate
(7.47 mmol, 4.5 molar equiv) and a catalytic amount of TEA,
and the solution was heated at reflux for 18 h. The solvent
was removed under reduced pressure, and the residue was
purified by chromatography (EtOAc/light petroleum 1:1) to
afford the final products 44-46 as solids.
3-Am in o-1H -1-m e t h yl-5-m e t h ylsu lfa n yl-4-[3-(2-fu r -
yl)1,2,4-tr ia zol-5-yl]p yr a zole (35). Yield 84%; yellow solid;
1
mp 220-222 °C; H NMR (DMSO-d6) δ 2.37 (s, 3H), 3.86 (s,
1-[2-(2-F u r yl)-8-m et h yl-9-(et h yla m in o)-8H -p yr a zolo-
[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in -5-yl]-3-(4-m eth oxy-
p h en yl)u r ea (44). Yield 60%; white solid; mp >300 °C; 1H
3H), 5.15 (bs, 2H), 6.50 (m, 1H), 7.00 (d, 1H, J ) 3), 7.53 (s,
1H), 12.00 (bs, 1H).

New A2A and A3 Adenosine Receptors Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7 1239
NMR (DMSO-d6) δ 1.22 (t, 3H, J ) 6), 3.74 (d, 6H), 4.00 (m,
2H), 6.66 (t, 1H; J ) 4), 6.71 (m, 1H), 6.93 (d, 2H, J ) 8), 7.18
(d, 1H, J ) 2), 7.45 (d, 2H, J ) 8), 7.95 (m, 1H), 9.23 (bs, 1H),
10.73 (bs, 1H). Anal. (C21H21N9O3) C, H, N.
1-[2-(2-F u r yl)-8-m et h yl-9-(4-m et h yl-p yp er a zin -1-yl)-
8H-p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c] p yr im id in -5-yl]-3-
(4-m eth oxyp h en yl)u r ea (45). Yield 50%; white solid; mp
199-200 °C; 1H NMR (DMSO-d6) δ 2.42 (s, 3H), 2.76 (m, 4H),
3.42 (m, 4H), 3.75 (s, 3H), 3.91 (s, 3H), 6.76 (s, 1H), 6.95 (d,
2H, J ) 9), 7.28 (s, 1H), 7.45 (d, 2H, J ) 9), 7.98 (s, 1H), 9.55
(bs, 1H), 10.59 (s, 1H). Anal. (C24H26N10O3) C, H, N.
1-[2-(2-F u r yl)-8-m eth yl-9-m eth ylsu lfa n yl-8H-p yr a zolo-
[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in -5-yl]-3-(4-m eth oxy-
p h en yl)u r ea (46). Yield 30%; white solid; mp 195-197 °C;
1H NMR (DMSO-d6) δ 2.81 (s, 3H), 3.75 (s, 3H), 4.10 (s, 3H),
6.75 (m, 1H), 6.95 (d, 2H, J ) 10), 7.30 (d, 1H, J ) 2), 7.46 (d,
2H, J ) 10), 7.99 (bs, 1H), 9.60 (bs, 1H), 10.50 (bs, 1H). Anal.
(C20H18N8O3S) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of N-[2-(2-
F u r yl)-8-m eth yl-9-m eth ylsu lfa n yl/eth yla m in o-8H-p yr a -
zolo[4,3-e]-1,2,4-t r ia zolo[1,5-c]p yr im id in -5-yl]-2-su b st i-
tu ted p h en yl)a ceta m id e 47-50. To a solution of compounds
38 and 41 (100 mg, 0.335 mmol) in dioxane (15 mL) were added
the appropriate freshly prepared acyl chloride (0.4 mmol) and
pyridine (0.335 mmol), and the mixture was refluxed under
argon for 8 h. Then the solvent was removed under reduced
pressure, and the residue was purified by chromatography
(EtOAc/light petroleum 1:1) to afford the final products 47-
50.
Gen er a l P r oced u r e for th e P r ep a r a tion of Hyd r och lo-
r ic Sa lts 53-55. The compounds 38, 40, and 44 (0.28 mmol)
were dissolved in a methanolic satured solution of hydrochloric
acid (0.57 mmol), and the solution was stirred at 0 °C for 10
min and then at room temperature for 10 min. The solvent
was removed at reduced pressure, and the residue was
coevaporated with dry Et2O and purified by crystallization
from absol EtOH to afford the desired products 53-55.
5-Am in o-8-m eth yl-9-(eth yla m in o)-2-(2-fu r yl)p yr a zolo-
[4,3-e]-1,2,4-tr iazolo[1,5-c]pyr im idin e Hydr och lor ide (53).
Yield 95%; white solid; mp >300 °C. 1H NMR (DMSO-d6) δ
1.23 (t, 3H, J ) 7), 3.72 (s, 3H), 4.00 (q, 2H, J ) 7), 6.10 (bs,
3H), 6.72 (m, 1H), 7.16 (d, 1H, J ) 4), 7.96 (s, 1H), 8.49 (bs,
2H). Anal. (C13H15ClN8O) C, H, N.
5-Am in o-8-m eth yl-9-(4-m eth ylpiper azin -1-yl)-2-(2-fu r yl)-
p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in e Dih yd r o-
1
ch lor id e (54). Yield 90%; white solid; mp >300 °C. H NMR
(DMSO-d6) δ 2.45 (s, 3H), 2.50 (m, 4H), 3.62 (m, 4H), 3.85 (s,
3H), 6.70 (s, 1H), 7.35 (s, 1H), 7.59 (bs, 2H), 7.94 (s, 1H), 9.00
(bs, 1H), 11.4 (bs, 1H). Anal. (C16H21Cl2N9O) C, H, N.
1-(2-(2-F u r yl)-8-m et h yl-9-(et h yla m in o)-8H -p yr a zolo-
[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in -5-yl)-3-(4-m eth oxy-
p h en yl)u r ea Hyd r och lor id e (55). Yield 93%; white solid;
1
mp >300 °C. H NMR (DMSO-d6) δ 1.22 (t, 3H, J ) 6), 3.73
(d, 6H), 4.00 (m, 2H, J ) 6), 4.47 (bs, 2H), 6.72(m, 1H), 6.95-
(d, 2H, J ) 8), 7.19 (m, 1H), 7.45 (d, 2H, J ) 8), 7.95 (s, 1H),
10.68 (bs, 1H). Anal. (C21H22ClN9O3) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Am in o-
8-m eth yl-9-m eth ylsu lfa n yl-2-[5-(4-m eth ylp ip er a zin -1-yl-
m et h ylen e)-2-fu r yl]p yr a zolo[4,3-e]-1,2,4-t r ia zolo[1,5-c]-
p yr im id in e 51 a n d 5-Am in o-7-(2-p h en ylet h yl)-2-[5-(4-
m eth ylpiper azin /m or ph olin -1-yl-m eth ylen e)-2-fu r yl]pyr a-
zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in es 57, 58. To a
solution of compounds 41 and 61 (180 mg, 0.6 mmol) in acetic
acid (20 mL) were added N-methylpiperazine or morpholine
(1.62 mmol) and 36% formaldehyde (0.96 mmol), and the
mixture was heated at reflux until the disappearance of
starting material. The solvent was removed under reduced
pressure, and the residue was purified by chromatography
(CH2Cl2/MeOH 9:1) to afford the desired compounds as solid.
8-Meth yl-2-[5-(4-m eth ylp ip er a zin -1-yl-m eth yl)fu r a n -2-
yl]-9-m eth ylsu lfa n yl-8H-p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-
c]p yr im id in -5-yla m in e (51). Yield 20%; white solid; mp 258-
260 °C; 1H NMR (DMSO-d6) δ 2.15 (s, 3H), 2.50 (bs, 4H), 2.76
(s, 3H), 3.34 (bs, 4H), 3.57 (s, 2H), 4.01 (s, 3H), 6.54 (d, 1H, J
) 3), 7.15 (d, 1H, J ) 3), 7.72 (s, 2H). Anal. (C18H23N9OS) C,
H, N.
2-[5-(4-Meth yl-piper azin -1-ylm eth yl)fu r an -2-yl]-7-ph en -
eth yl-7H-p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in -5-
yla m in e (57). Yield 22%; pale yellow solid; mp 206-207 °C;
1H NMR (DMSO-d6) δ 2.13 (s, 3H), 2.42 (bs, 4H), 3.21 (t, 2H,
J ) 8), 3.34 (bs, 4H), 3.55 (s, 2H), 4.48 (t, 2H, J ) 8), 6.55 (d,
1H, J ) 4), 7.18 (bs, 5H), 7.24 (d, 1H, J ) 4), 8.08 (bs, 2H),
8.16 (s, 1H). Anal. (C24H27N9O) C, H, N.
2-(5-Mor p h olin -4-yl-m et h yl-fu r a n -2-yl)-7-p h en et h yl-
7H -p y r a zo lo [4,3-e]-1,2,4-t r ia zo lo [1,5-c]p y r im id in -5-
yla m in e (58). Yield 20%; pale yellow solid; mp 200-202 °C;
1H NMR (DMSO-d6) δ 2.42 (bs, 4H), 3.17 (t, 2H, J ) 8), 3.34
(s, 2H), 3.57 (bs, 4H), 4.48 (t, 2H, J ) 8), 6.57 (d, 1H, J ) 4),
7.21 (m, 5H), 7.23 (d, 1H, J ) 4), 8.09 (bs, 2H), 8.18 (s, 1H).
Anal. (C23H24N8O2) C, H, N.
N-[2-(2-F u r yl)-8-m eth yl-9-m eth ylsu lfa n yl-8H-p yr a zolo-
[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in -5-yl]-2-(4-m eth oxy-
p h en yl)a ceta m id e (47). Yield 40%; white solid; mp 199-200
1
°C; H NMR (DMSO-d6) δ 2.81 (s, 3H), 3.74 (s, 3H), 3.93 (s,
2H), 4.12 (s, 3H), 6.76 (m, 1H), 6.90 (d, 2H, J ) 8), 7.26 (m,
1H), 7.30 (d, 2H, J ) 8), 8.00 (m, 1H), 10.37 (bs, 1H). Anal.
(C21H19N7O3S) C, H, N.
N-[2-(2-F u r yl)-8-m eth yl-9-m eth ylsu lfa n yl-8H-p yr a zolo-
[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in -5-yl]-2-(4-isobu tyl)-
1
a ceta m id e (48). Yield 15%; white solid; mp 106-107 °C; H
NMR (DMSO-d6) δ 0.85 (m, 6H), 1.79 (m, 1H), 2.41 (d, 2H, J
) 8), 2.81 (s, 3H), 3.16 (d, 2H, J ) 3), 4.12 (s, 3H), 6.76 (m,
1H), 7.13 (d, 2H, J ) 8), 7.22 (m, 1H), 7.37 (d, 2H, J ) 8), 7.98
(m, 1H), 10.98 (bs, 1H). Anal. (C24H25N7O2S) C, H, N.
N-[2-(2-F u r yl)-8-m eth yl-9-m eth ylsu lfa n yl-8H-p yr a zolo-
[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in -5-yl]-2-(3,4-m eth yl-
en d ioxyp h en yl)a ceta m id e (49). Yield 43%; white solid; mp
1
209-210 °C; H NMR (DMSO-d6) δ 2.81 (s, 3H), 3.90 (s, 2H),
4.12 (s, 3H), 6.00 (s, 2H), 6.76 (m, 1H), 6.87 (m, 2H), 6.99 (s,
1H), 7.27 (d, 1H, J ) 2), 7.99 (bs, 1H), 11.03 (bs, 1H). Anal.
(C21H17N7O4S) C, H, N.
N-[2-(2-F u r yl)-8-m et h yl-9-(et h yla m in o)-8H -p yr a zolo-
[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in -5-yl]-2-(3,4-m eth yl-
en d ioxy p h en yl)a ceta m id e (50). Yield 13%; white solid; mp
1
>300 °C; H NMR (DMSO-d6) δ 1.07 (t, 3H, J ) 7), 3.50 (s,
3H), 3.67 (s, 2H), 4.00 (m, 2H), 5.87 (s, 1H), 6.30 (d, 2H, J )
8), 6.47 (s, 1H), 6.60 (d, 2H, J ) 8), 6.70 (m, 1H, J ) 2), 7.14
(d, 1H, J ) 2), 7.94 (s, 1H), 10.50 (bs, 1H). Anal. (C22H20N8O4)
C, H, N.
P r oced u r e for th e P r ep a r a tion of 5-Am in o-8-m eth yl-
9-(4-h ydr oxyph en ylam in o)-2-(2-fu r yl)pyr azolo[4,3-e]-1,2,4-
tr ia zolo[1,5-c]p yr im id in e 52. A solution of compound 39 (60
mg, 0.16 mmol) in acetic acid (0.6 mL) and HI (0.4 mL) was
refluxed for 5 h. Then acetic acid (0.6 mL) and HI (0.4 mL)
were added again, and the solution was heated at reflux for
additional 5 h. The mixture was diluted with water (8 mL),
and the aqueous layer was extracted with EtOAc (3 × 50 mL).
The organic phases were dried (Na2SO4) and evaporated to
obtain a residue crystallized from absol EtOH to afford the
product 52.
Gen er a l P r oced u r e for th e P r ep a r a tion of Hyd r och lo-
r ic Sa lts 56, 59, 60. The compounds 51, 57, 58 (0.24 mmol)
were dissolved in a methanolic satured solution of hydrochloric
acid (0.48 mmol), and the solution was stirred at 0 °C for
several minutes and then at room temperature for 10 min.
The solvent was removed at reduced pressure, and the residue
was coevaporated with dry Et2O and purified by crystallization
from EtOH to afford the desired products as hydrochloric salts.
5-Am in o -8-m e t h y l-9-m e t h y ls u lfa n y l-2-[5-(4-m e t h -
ylp yp er a zin -1-ylm eth ylen e)-2-fu r yl]p yr a zolo[4,3-e]-1,2,4-
tr ia zolo[1,5-c]p yr im id in e Hyd r och lor id e (56). Yield 76%;
white solid; mp 255-256 °C; 1H NMR (DMSO-d6) δ 2.50 (s,
3H), 2.75 (s, 3H), 2.79 (s, 3H), 3.30 (m, 4H), 3.66 (m, 4H), 4.01
Yield 70%; pale yellow solid; mp>300 °C; 1H NMR (DMSO-
d6) δ 3.74 (s, 3H), 6.72 (m, 3H), 7.20 (d, 1H, J ) 3), 7.45 (d,
2H, J ) 8), 7.70 (s, 1H), 7.94 (s, 1H), 8.04 (bs, 2H), 8.86 (s,
1H). Anal. (C17H14N8O2) C, H, N.

1240 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 7
Baraldi et al.
(s, 2H), 6.91 (d, 1H, J ) 4), 7.26 (d, 1H, J ) 4), 7.72 (bs, 2H),
11.71 (bs, 1H). Anal. (C18H24ClN9OS) C, H, N.
Nonspecific binding was defined as binding in the presence of
1 µM of MRE3008 F20 and was about 25% of total binding.
Bound and free radioactivity were separated by rapid
filtration through Whatman GF/B glass-fiber filters which
were washed three times with ice cold buffer. The filter bound
radioactivity was counted in a Beckman LS-1800 spectrometer
(efficiency 55%).
Da ta An a lysis. The protein concentration was determined
according to a Bio-Rad method26 with bovine albumin as a
standard reference. Inhibitory binding constant, Ki, values
were calculated from those of IC50 according to Cheng &
Prusoff equation27 (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 LIGAND28 was used for computer analysis of satura-
tion and inhibition experiments.
5-Am in o-7-(2-p h en yleth yl)-2-[5-(4-m eth ylp ip er a zin -1-
ylm eth ylen e)-2-fu r yl]p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]-
p yr im id in e Hyd r och lor id e (59). Yield 81%; white solid; mp
210-212 °C; 1H NMR (DMSO-d6) δ 2.24 (s, 3H), 2.51 (bs, 4H),
3.26 (t, 2H, J ) 4), 3.41 (bs, 4H), 3.62 (s, 2H), 4.52 (t, 2H, J )
8), 6.62 (d, 1H, J ) 4), 7.28 (bs, 5H), 7.31 (d, 1H, J ) 4), 8.15
(bs, 2H), 8.22 (s, 1H), 10.81 (bs, 1H). Anal. (C24H28ClN9O) C,
H, N.
5-Am in o-7-(2-p h en yleth yl)-2-[5-(4-m or p h olin -1-yl-m e-
th ylen e)-2-fu r yl]p yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr i-
m id in e Hyd r och lor id e (60). Yield 78%; pale yellow solid;
1
mp 215-216 °C; H NMR (DMSO-d6) δ 2.53 (bs, 4H), 3.23 (t,
2H, J ) 8), 3.61 (s, 2H), 3.65 (bs, 4H), 4.53 (t, 2H, J ) 8), 7.01
(d, 1H, J ) 4), 7.23 (m, 5H), 7.37 (d, 1H, J ) 4), 8.24 (bs, 2H),
8.31 (s, 1H), 10.22 (bs, 1H). Anal. (C23H25ClN8O2) C, H, N.
Biology Exp er im en ts. All synthesized compounds have
been tested for their affinity to human A1, A2A, A2B, and A3
adenosine receptors.
Data are expressed as geometric mean, with 95% or 99%
confidence limits in parentheses.
Mea su r em en t of Cyclic AMP Levels in CHO Cells
Tr a n sfected w ith Hu m a n A2A or A3 Ad en osin e Recep tor s.
CHO cells transfected with human A2A or A3 adenosine
receptors were washed with phosphate-buffered saline, diluted
with trypsin, and centrifuged for 10 min at 200g. The pellet
containing CHO cells (1 × 106 cells/assay) was suspended in
0.5 mL of incubation mixture (mM): NaCl 15, KCl 0.27, NaH2-
PO4 0.037, MgSO4 0.1, CaCl2 0.1, Hepes 0.01, MgCl2 1, glucose
0.5, pH 7.4 at 37 °C, 2 IU/mL adenosine deaminase and 4-(3-
butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) as
phosphodiesterase inhibitor and preincubated for 10 min in a
shaking bath at 37 °C. The potency of antagonists (IC50, nM)
on A2A adenosine receptors were determined by antagonism
of NECA (100 nM)-induced stimulation of cyclic AMP levels.
The potency of antagonists (IC50, nM) on A3 adenosine recep-
tors was determined by antagonism of Cl-IB-MECA (100 nM)-
induced inhibition of cyclic AMP levels. The reaction was
terminated by the addition of cold 6% trichloroacetic acid
(TCA). The TCA suspension was centrifuged at 2000g for 10
min at 4 °C, and the supernatant was extracted four times
with water saturated diethyl ether. The final aqueous solution
was tested for cyclic AMP levels by a competition protein
binding assay. Samples of cyclic AMP standard (0-10 pmol)
were added to each test tube containing the incubation buffer
(trizma base 0.1 M, aminophyllin 8.0 mM, 2 mercaptoethanol
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
adrenals, 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 Beckman scintillation counter.
Hu m a n Clon ed Ad en osin e Recep tor Bin d in g Assa y.
The expression of the human A1, A2A, and A3 receptors in CHO
cells has been previously described.25 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
buffer (5 mM Tris HCl, 2 mM EDTA, pH 7.4). The cell
suspension was homogenized with a Polytron, and the homo-
genate 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, 1mM 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).
Bin d in g of [3H]-DP CP X to CHO cells tr a n sfected w ith
th e h u m a n r ecom bin a n t A1 a d en osin e r ecep tor was
performed according to the method previously described by
Varani et al.16 Displacement experiments were performed for
120 min at 25 °C in 200 µL of buffer containing 1 nM [3H]-
DPCPX, 20 µL of diluted membranes (50 µg of protein/assay)
and at least 6-8 different concentrations of examined com-
pounds. Nonspecific binding was determined in the presence
of 10 µM of CHA and this is always e10% of the total binding.
Bin d in g of [3H]-SCH58261 to CHO cells tr a n sfected
w ith th e h u m a n r ecom bin a n t A2A a d en osin e r ecep tor s
(50 µg of protein/assay) was performed according to Varani et
al.16
Ack n ow led gm en t. We thank King Pharmaceutical
R & D, 7001 Weston Parkway Suit 300, Cary, NC 27513
for financial support, in particular, Dr. Eddie J ung for
useful discussion. We also thank Prof. Karl-Norbert
Klotz for cDNA encoding the human adenosine recep-
tors.
In competition studies, at least 6-8 different concentrations
of compounds were used, and nonspecific binding was deter-
mined in the presence of 1 µM SCH58261 for an incubation
time of 60 min at the temperature of 25 °C.
Bin d in g of [3H]-DP CP X to HEK 293 cells tr a n sfected
w ith th e h u m a n r ecom bin a n t A2B a d en osin e r ecep tor s
were performed essentially to the method described by Varani
et al.16
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