Design, synthesis, and biological evaluation of a second generation of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as potent and selective A(2A) adenosine receptor antagonists

Article abstract of DOI:10.1021/jm9708689

New A(2A) adenosine receptor antagonists in the series of pyrazolo[4,3- e]-1,2,4-triazolo[1,5-c]pyrimidines, bearing oxygenated substituents on the phenylalkyl chains on the 7-position, have been synthesized. The compounds were tested in binding and funct

Full text of DOI:10.1021/jm9708689

2126
J . Med. Chem. 1998, 41, 2126-2133
Design , Syn th esis, a n d Biologica l Eva lu a tion of a Secon d Gen er a tion of
P yr a zolo[4,3-e]-1,2,4-tr ia zolo[1,5-c]p yr im id in es a s P oten t a n d Selective A2A
Ad en osin e Recep tor An ta gon ists
Pier Giovanni Baraldi,*,† Barbara Cacciari,† Giampiero Spalluto,† Manuela Bergonzoni,† Silvio Dionisotti,‡
Ennio Ongini,‡ Katia Varani,§ and P. A. Borea§
Dipartimento di Scienze Farmaceutiche and Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia,
Universita` di Ferrara, Via Fossato di Mortara 17/ 19, 44100 Ferrara, Italy, and Centro Ricerche Schering-Plough,
Parco Scientifico San Raffaele, Via Olgettina 58, 20132 Milano, Italy
Received December 29, 1997
New A2A adenosine receptor antagonists in the series of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]-
pyrimidines, bearing oxygenated substituents on the phenylalkyl chains on the 7-position, have
been synthesized. The compounds were tested in binding and functional assays to evaluate
affinity, potency, and selectivity for rat A2A compared to rat A1 and human A3 receptor subtypes.
The most interesting compounds (5d ,e,h ) were tested also in binding to human A1 and A2A
adenosine receptors. They showed very good affinity (Ki ) 0.94 nM for compound 5h ) and
interesting selectivity with respect to both rA1 and hA3 (compound 5h : rA1/rA2A ) 787, hA3/
rA2A > 10 000). These important findings make this new series of compounds the first really
selective for A2A adenosine receptors. Thermodynamic parameters were evaluated; all the tested
compounds displayed an enthalpy-driven binding as expected for antagonists. Moreover,
compound 5h showed a negative entropy value. The highly negative enthalpic and entropic
contributions could mean that 5h fits very well in the binding site where, probably, an
electrostatic interaction is present associated to a scarce solvent reorganization around the
receptor binding site. These compounds deserve to be further developed to assess their potential
for treatment of neurodegenerative disorders such as Parkinson’s disease.
Ch a r t 1
In tr od u ction
Adenosine modulates physiological functions acting
via specific cell surface receptors which have been
identified as A1, A2A, A2B, and A3.1,2 The A2A receptor
subtype which is coupled to stimulation of adenylyl
cyclase activity, is a high-affinity receptor found in large
amounts in the brain striatum.3 Intensive efforts have
been made over the past few years with the aim to
synthesize novel compounds, either xanthine or non-
xanthine, interacting selectively with the A2A adenosine
receptor type. 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, 1) and SCH
63390 (5-amino-7-(3-phenylpropyl)-2-(2-furyl)pyrazolo-
[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, 2) have been found
to be potent and selective adenosine A2A antagonists,
and SCH 58261 is widely used as a tool for character-
izing the adenosine A2A receptor subtype (Chart 1).4,5
Moreover, the labeled form of SCH 58261 has been
synthesized and well characterized as described in a
variety of tissues and cell types.6,7 Another compound
of interest is ZM 241385 (4-[2-[[7-amino-2-(2-furyl)[1,2,4]-
triazolo[2,3-a][1,3,5]triazin-5-yl]amino]ethyl]phenol, 3)8
which, like SCH 58261, has been derived from the
prototype CGS 15943 (5-amino-9-chloro-2-(2-furyl)-1,2,4-
triazolo[1,5-c]quinazoline, 4) (Chart 1).9 ZM 241385 is
an A2A adenosine antagonist endowed with good affinity
and selectivity for A2A vs A1, and it is quite hydrophilic,
an interesting characteristic because, usually, the A2A
adenosine antagonists are lipophilic compounds.
Starting from these observations and on the basis of
data derived from an enlarged series of SCH 58261
analogues previously reported,4 we have investigated
the effects of variously substituted hydroxylic functions
on the phenyl ring at the side chain in the 7-position in
analogy with ZM 241385, to improve the hydrophilic
character, which was the main problem of our previous
series (Chart 2).
* To whom correspondence should be addressed. Phone: +39-(0)-
532-291293. Fax: +39-(0)532-291296. E-mail: pgb@ifeuniv.unife.it.
† Dipartimento di Scienze Farmaceutiche.
Particular attention was given to the cloned human
A3 receptor due to recent data showing that CGS 15943,
the original precursor of the currently examined pyra-
zolotriazolopyrimidine series, has been reported to be
‡ Centro Ricerche Schering-Plough.
§ Dipartimento di Medicina Clinica
Farmacologia.
e Sperimentale-Sezione di
S0022-2623(97)00868-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/13/1998

Second-Generation Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2127
Sch em e 1a
a
Reagents: (i) (ethoxymethylene)malonitrile; (ii) HC(OEt)3, reflux; (iii) furoic hydrazide, MeO(CH2)2OH; (iv) Ph2O, 260 °C; (v) 10%
HCl; reflux; (vi) NH2CN, 1-methyl-2-pyrrolidone, pTsOH, 140 °C; (vii) 10% C-Pd, H2 (70 psi), dioxane, 3 h; (viii) BCl3.
corresponding halides.13 Pyrazoles 7a -d were prepared
Ch a r t 2
by reacting (ethoxymethylene)malononitrile with the
appropriate hydrazines 6a -d .14,15 This strategy af-
forded only the N1 isomer and allowed us to avoid
tedious purification procedures. Unfortunately, the
preparation of the hydrazine 6e failed, so we got the
pyrazole 7e by alkylating 2-amino-3-cyanopyrazole with
3-(3,4-dimethoxyphenyl)propyl bromide; the two isomers
were separated by chromatography, and their structure
was assigned unequivocately by NOE experiment. The
designed compounds 5a -i were synthesized according
to Gatta et al.16 for the synthesis of pyrazolo[4,3-e]-1,2,4-
inactive at rat A3 receptors,10 but its affinity was found
triazolo[1,5-c]pyrimidines which involved transforma-
tion of pyrazoles 7a -e to corresponding imidates 8a -e
to be high at the human A3 receptor (Ki ) 14 nM).11
Moreover, we have undertaken systematic investiga-
tion on the thermodynamic aspects of the binding
equilibrium of this new series of compounds to adenos-
ine A2A receptors aimed at having a direct knowledge
of the enthalpic (∆H°) and entropic (∆S°) contributions
to the equilibrium standard free energy (∆G° ) ∆H°
-T∆S° ) -RT ln KA; KA ) association constant).
Thermodynamic parameters of the binding equilibrium
of all antagonists tested were determined by means of
affinity measurements, carried out on rat striatal
membranes at six different temperatures (0, 10, 20, 25,
30, 35 °C), and van’t Hoff plots.12 Affinity constants (KA)
were obtained from inhibition experiments utilizing as
radioligand a selective A2A agonist, [3H]-2-[[p-(2-car-
boxyethyl)phenethyl]amino]-5′-(N-ethylcarboxamido)ad-
enosine ([3H]CGS 21680). In the adenylyl cyclase assay
the compounds examined exhibited a rank order of
potency very close to that observed in binding experi-
ments.
by refluxing in triethyl orthoformate. The imidates
were reacted with 2-furoic acid hydrazide in refluxing
2-methoxyethanol to provide the pyrazolo[4,3-e]pyrimi-
dine intermediates. The latter compounds were con-
verted through a thermally induced cyclization in
diphenyl ether to the derivatives 9a -e in good overall
yield.
Treatment of 9a -e with dilute hydrochloric acid at
reflux temperature induced pyrimidine ring opening to
furnish the 5-amino-4-(1H-1,2,4-triazol-5-yl)pyrazoles
10a -e in excellent yield. These derivatives were con-
verted into the final compounds 5a -e by reaction with
an excess of cyanamide in 1-methyl-2-pyrrolidone at 140
°C. Compounds 5f-h were obtained by catalytic hy-
drogenation of the corresponding O-benzyl-protected
compounds 5a -c. The compound 5i was derived from
the deprotection of 5d by reaction with boron trichloride
at 5 °C.17
Resu lts a n d Discu ssion
Ch em istr y
Table 1 gives the receptor affinity profile of com-
pounds 5a -i, determined by receptor binding assay at
rat A1 and A2A receptors using [3H]-N6-cyclohexylad-
enosine ([3H] CHA) and [3H]-2-[[4-(2-carboxyethyl)-
phenethyl]amino]-5′-(N-ethylcarboxamido)adenosine
([3H]CGS 21680), respectively.18,19Species differences
are well-known among adenosine receptor subtypes; in
particular, this evidence is really important for the A3
The preparation of the compounds 5a -i was per-
formed following the general synthetic pathway depicted
in Scheme 1.
To obtain only the N1-substituted isomer of the
pyrazoles 7a -e, we synthesized the appropriate (phe-
nylalkyl)hydrazines bearing different substituents, fol-
lowing a well-known procedure, starting from the

2128 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Baraldi et al.
Ta ble 1. Biological Activity of a Series of Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines 5a -i
Ki (nM)
rA2
selectivity
a
b
c
d
compound
rA1
121
hA3
rA1/rA2A
hA3/rA2A
cyclic AMP assay, IC50 (nM)
2.3
12
1, SCH 58261
(103-123)
(2.0-2.7)
>10000
53
>4347
>4166
14.5
(9-17)
504
2.4
18
2, SCH 63390
(329-773)
6.4
(6.2-6.6)
9450
(1.9-2.9)
0.95
(0.8-1.1)
246
>10000
14
210
(15-21)
12
(10-14)
500
4, CGS 15943
(9.9-18)11
6.7
5a
5b
5c
5d
5e
5f
(7650-11700)
(109-554)
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
38
>169
32
>40
(484-516)
90
(86-94)
300
(282-321)
8
(7-10)
20
(19-22)
149
(138-162)
14
(11-17)
10
(8-12)
60
59
>10000
(44-78)
45
(25-28)
3.8
(2.1-6.9)
5.3
(4.1-8.3)
53
(47-70)
1.7
(1.3-2.1)
0.94
(0.58-1.7)
49
(23-104)
>169
1460
(1400-1660)
1841
(1100-3060)
2825
(2000-3990)
13400
(11100-16100)
444
(338-582)
741
(545-902)
2250
(895-5640)
>222
484
487
252
261
787
46
>2631
>1886
>188
5g
5h
>5882
>10638
>204
5i
(56-65)
Data are expressed as geometric means, with 95% confidence limits. Displacement of [3H]CHA binding (A1) at rat cortical membranes.
Displacement of [3H]CGS 21680 binding (A2A) at rat striatal membranes. c Displacement of [125]AB-MECA binding at human A3 adenosine
a
b
receptors expressed in HEK-293 cells. Ki values, calculated from the Cheng-Prusoff equation32 using 1.0, 18.5, and 0.9 nM as Kd values
d
in A1, A2A, and A3 binding assays, respectively. IC50 values of the test compounds for inhibition of NECA (1 µM)-stimulated cyclic AMP
levels in human platelets.
Ta ble 2. Affinity and Selectivity of Selected A2A Receptor
Antagonists at Human A1, A2A, and A3 Adenosine Receptor
Subtypes
receptor subtype: CGS 15943 did not show affinity for
the rat A3 receptor subtype but displayed good affinity
for the human A3 receptor. For this reason the affinity
selectivity
of 5a -i for cloned human A3 adenosine receptors
expressed in HEK-293 cells, using [125I]-N6-(4-amino-
Ki nM
hA1/
hA2A hA2A
hA3/
a
b
c
3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine ([125I]-
compound
hA1
549
hA2A
hA3
AB-MECA) as radioligand, was examined.20,21
1.1
SCH 58261
(322-987)
(0.75-1.6) >10000 499 >9090
The results show that all the tested compounds 5a -i
display high affinity at A2A receptors: high rA2A vs rA1
and, most importantly, rA2A vs hA3 selectivity. Despite
the structural similarity with CGS 15943, our series of
compounds does not show affinity for the human A3
receptor subtype; this important relevance makes 5a -i
the first compounds really selective versus A2A adenos-
ine receptors. Binding data indicate that the presence
of the hydroxylic function at the para position on a
phenyl ring increases the affinity and in particular A2A
receptor selectivity. In fact, comparing the derivatives
5g,h with the corresponding unsubstituted analogues
1 and 2, the affinity is slightly increased, while instead
the A2A receptor selectivity is highly enhanced. Fur-
thermore, we confirmed the previous observation4 that
the best length for the side chain is three methylene
groups: compound 5h shows the highest affinity and
selectivity in binding assays (Ki(A2A) ) 0.94 nM, A1/A2A
) 787) and potency in the A2A receptor adenylyl cyclase
assay (IC50 ) 10 nM). This statement was confirmed
also for compounds 5d ,e, bearing the same side chain,
which are endowed with good activity and very high
selectivity. Compound 5c was less active probably due
to the presence of the bulky group at the para position
of the phenyl ring.
2170
3.3
5d
5e
(1260-3740) (2.9-3.8)
1650 2.7
(1530-1780) (1.8-4.0)
253
>10000 658 >3030
>10000 611 >3700
>10000 169 >6670
1.5
(1.2-1.9)
5h
(110-585)
a
Displacement of [3H]CHA binding at human A1 adenosine
receptors expressed in CHO cells. Displacement of [3H]SCH
b
58261 binding at human A2A adenosine receptors expressed in
CHO cells. c Displacement of [125I]AB-MECA binding at human
A3 adenosine receptors expressed in HEK-293 cells.
compounds were unable to displace [125I]AB-MECA
binding at human A3 adenosine receptors up to the
highest concentration examined (Ki > 10 µM), thus
indicating the absence of interaction with this receptor
subtype.
We thought that it could be interesting to test some
compounds of this new series also at human A1 and A2A
adenosine receptors. The results are shown in Table
2; surprisingly, the best compound so far evaluated, 5h ,
at human receptors displays lower selectivity for A2A
vs A1, whereas the former SCH 58261 and 5d ,e show
very high selectivity.
The A2A vs A2B selectivity of these compounds was not
evaluated. However, in CHO cells transfected with the
human A2B receptor, SCH 58261 at micromolar concen-
trations was unable to antagonize NECA-induced cAMP
accumulation.24 Moreover, SCH 58261 and its prototype
8FB-PTP (5-amino-8-(4-fluorobenzyl)-2-(2-furyl)pyrazolo-
As already mentioned, marked species differences
have been found in the affinity of antagonists at rat A3
receptors;22,23 to this purpose, we have tested the
compounds 5a -i on human A3 receptors. All the tested

Second-Generation Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2129
Ta ble 3. Rm Values of Compounds 5a -i Measured at pH 7.0
by TLC in Methanol-Water System
compound
Rm(0)a
1, SCH 58261
2, SCH 63390
3.5 ( 0.2
3.6 ( 0.2
3.7 ( 0.2
3.6 ( 0.2
3.4 ( 0.2
3.6 ( 0.2
3.5 ( 0.2
2.8 ( 0.1
2.5 ( 0.1
2.9 ( 0.1
3.2 ( 0.2
5a
5b
5c
5d
5e
5f
5g
5h
5i
a
The Rm values of 1, 2, and 5a -i were measured with a mobile
phase of different concentrations of CH3OH/H2O. Rm values are
reported as theoretical at 0% organic solvent in the mobile phase
(Rm(0)).
F igu r e 1. Representative van’t Hoff plots showing the effect
of temperature on the equilibrium association constants, KA,
for tested compounds 5a -i. All plots are essentially linear (r
) 0.95) in the temperature range from 0 to 30 °C.
[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine)16 were found to
have little or no activity in CHO cells transfected with
human A2B receptors25 and in the guinea pig aorta,5
which is a functional model specific for the evaluation
of A2B-mediated effects.
The functional A2A receptor-mediated antagonist prop-
erties of compounds 5a -i were tested on NECA (1 µM)-
induced cAMP stimulation in human platelets (Table
1), which are known to represent a reliable assay to
examine potency of adenosine antagonists.26,27 Most of
the tested compounds showed activity in the low-
nanomolar range, with 5d being the most potent com-
pound.
Ta ble 4. Thermodynamic Parameters (Given at 298.15 K) for
the Binding Equilibrium of Antagonists to Adenosine A2A
Receptors
ligand
∆G° (kJ /mol) ∆H° (kJ /mol) ∆S° (J /mol/deg)
1, SCH 58261
2, SCH 63390
-49.07
-46.52
-37.79
-42.11
-39.42
-50.15
-46.11
-41.17
-47.23
-49.99
-43.38
-44.9
-32.0
-24.8
-39.1
-33.0
-47.9
-25.8
-43.4
-38.8
-59.5
-40.3
14
49
44
10
21
7
68
-2
28
5a
5b
5c
5d
5e
5f
5g
5h
5i
Furthermore, the new series did not show, as we
hoped, a remarkable increase in water solubility, mea-
sured by evaluation of Rm values (Rm ) log(1/Rf - 1))
through reversed-phase TLC experiments (Table 3).28
-32
10
If we compared compounds SCH 58261 and SCH
63390 with the corresponding compounds bearing the
hydroxy moiety at the para position on the phenyl ring
in the side chain (5g,h ), it is possible to notice an
appreciable improvement in hydrophilic properties,
unfortunately not enough to make our compounds
water-soluble. However, these data prove that our
hypothesis to introduce oxygenated functions was cor-
rect.
mostly enthalpy-driven (-59.5 ) ∆H° ) -24.8 kJ /mol;
-32 ) ∆S° ) 68 J /mol/K).
From the equilibrium thermodynamic parameters,
binding of all compounds resulted to be mostly enthalpy-
driven as expected for adenosine receptor antagonists;12
moreover, the enthalpic contribution was greater for 5h
with respect to the corresponding derivative lacking the
hydroxylic group (2), while the entropic contribution was
practically negligible.
In thermodynamic studies, inhibitory binding con-
stants (Ki) were measured at the six chosen tempera-
tures for the selected 11 ligands by displacement of
[3H]CGS 21680 from rat striatum adenosine A2A recep-
tors. All adenosine receptor antagonists studied display
Ki values that depend on the temperature showing a
lower affinity at room temperature. This behavior is
in perfect agreement with that already reported in rat
striatum for xanthine antagonists.12 The temperature
dependence of the affinity constants, KA ) 1/Ki, is
exemplified by the van’t Hoff plots, ln KA versus 1/T, of
Figure 1 which report typical results for all antagonists
studied.
The van’t Hoff plots are linear in the range 0-30 °C,
and the slopes are consistently positive with affinities
decreasing with the increase in temperature. Final
thermodynamic parameters calculated for the binding
equilibria of the different compounds investigated are
reported in Table 4. ∆G° values range from -50.2 to
-37.8 kJ /mol; equilibrium standard enthalpy ∆H° and
entropy ∆S° values show that binding of antagonists is
It is remarkable that 5h has a higher binding equi-
librium enthalpy value than all the other compounds
and, in particular, of compound 2 which is similar but
lacks the hydroxyl group. This could be indicative of
the presence of an electrostatic interaction (most prob-
ably a hydrogen bond) formed by the hydroxyl moiety
with the recognition of the binding site. In light of this
observation, it is possible to understand the affinity and
selectivity of 5d ,e. Moreover, the entropic contribution
to the binding of compound 5h is low with respect to
that of other compounds (1,2,5a -g), which could be
explained by hypothesizing either scarce or null solvent
reorganization around the receptor binding site. This,
speculatively, might be indicative of the presence of a
pocket where the aromatic ring of 5h fits well via
electrostatic forces with hydrogen bond acceptors or
donors present on the receptor binding site.
Con clu sion s
In the present study we have described the affinity
and selectivity at adenosine receptors of a series of SCH

2130 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Baraldi et al.
(EtOAc-Et2O); IR (KBr) 3350-3160, 2200, 1655, 1460 cm-1
;
58261 derivatives, in which different substituents, such
as hydroxy, methoxy, or methylenedioxy groups, have
been introduced on the phenyl ring. Considerable
progress has been made with the introduction of the
hydroxylic function at the para position, together with
the presence of three methylene groups at the side
chain. In fact, all the tested compounds 5a -i showed
high A2A affinity and, in particular, the very high
selectivity for both human and rat A2A vs A1 and A2A vs
A3 adenosine receptors. This is particularly important
because of the affinity of CGS 15943, structurally
correlated to our compounds, for the human A3 adenos-
ine receptor. These new compounds 5a -i are the first
A2A adenosine antagonists really selective versus the
human A3 adenosine receptor subtype. Since it is now
clear that the stimulation of A2A adenosine receptors
decreases the affinity of D2 dopamine receptor agonists
in striatopallidal neurons, potent and selective A2A
receptor antagonists may be a novel therapeutic ap-
proach to Parkinson’s disease.29,30
1H NMR (CDCl3) δ 2.04-2.15 (m, 2H), 2.57 (t, 2H, J ) 8), 3.85
(t, 2H, J ) 8), 4.23 (bs, 2H), 5.93 (s, 2H), 6.63-6.76 (m, 3H),
7.5 (s, 1H). Anal. (C14H14N4O2) C, H, N.
P r ep a r a tion of 5-Am in o-4-cya n o-1-[3-(3,4-d im eth oxy-
p h en yl)p r op yl]p yr a zole (7e). Potassium carbonate (1.4 g,
1.1 equiv) and 3-amino-4-cyanopyrazole (1 g, 9.25 mmol) were
suspended in dry DMF (20 mL), and 3-(3,4-dimethoxyphenyl)-
propyl bromide (2.63 g, 1.1 equiv) was added. The reaction
was stirred at room temperature for 12 h; then the solvent
was removed and the mixture of the two isomers purified by
flash chromatography (EtOAc/light petroleum, 1:4) to afford
7e as a yellow solid (yield 68%): mp 153-155 °C (EtOAc-
1
Et2O); IR (KBr) 3355-3050, 2220, 1665, 1440 cm-1; H NMR
(CDCl3) δ 2.04-2.17 (m, 2H), 2.62 (t, 2H, J ) 7), 3.67 (t, 2H,
J ) 7), 3.85 (s, 3H), 3.86 (s,3H), 4.38 (bs, 2H), 6.66-6.82 (m,
3H), 7.5 (s, 1H), the 2D 1H-1H NOE NMR (CDCl3) did not
display a cross-peak for C1′-H and C3-H. Anal. (C15H18N4O2)
C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 1-Su bsti-
tu ted-4-cyan o-5-[(eth oxym eth ylen e)am in o]pyr azoles 8a-
e. The 4-cyano-5-aminopyrazoles 7a -e (20 mmol) were dis-
solved in triethyl orthoformate (40 mL), and the solution was
refluxed under nitrogen for 8 h. Then the solvent was removed
under reduced pressure, and the oily residue was dissolved in
ether and roughly purified on silica gel (EtOAc-light petro-
leum, 1:9) to afford the corresponding imino ethers. The
following spectral data are reported as examples.
1-[4-(Be n zyloxy)b e n zyl]-4-cya n o-5-[(e t h oxym e t h yl-
en e)a m in o]p yr a zole (8a ): yield 85%, pale-yellow oil; IR
(neat) 2230, 1650 cm-1; 1H NMR (CDCl3) δ 1.43 (t, 3H, J ) 7),
4.43 (q, 2H, J ) 7), 5.05 (s, 2H), 5.2 (s, 2H), 6.94 (d, 2H, J )
8), 7.2 (d, 2H, J ) 8), 7.32-7.44 (m, 5H), 7.66 (s, 1H), 8.4 (s,
1H). Anal. (C21H20N4O2) C, H, N.
Exp er im en ta l Section
Ch em istr y. Reaction courses and product mixtures were
routinely monitored by thin-layer chromatography (TLC) on
silica gel (precoated F254 Merck plates) and visualized with
iodine or aqueous potassium permanganate. Infrared spectra
(IR) were measured on Perkin-Elmer 257 instruments. 1H
NMR were determined in CDCl3 or DMSO-d6 solutions with
a Bruker AC 200 spectrometer, peak positions are given in
parts per million (δ) downfield from tetramethylsilane as
internal standard, and J values are given in Hz. Light
petroleum refers to the fractions boiling at 40-60 °C. Melting
points were determined on a Buchi-Tottoli instrument and are
uncorrected. Chromatography was performed with Merck 60-
1-[2-[4-(Ben zyloxy)p h en yl]et h yl]-4-cya n o-5-[(et h oxy-
m eth ylen e)a m in o]p yr a zole (8b): yield 87%, pale-yellow oil;
1
IR (neat) 2215, 1640, 1420 cm-1; H NMR (CDCl3) δ 1.33 (t,
1
3H, J ) 7), 2.97 (t, 2H, J ) 7), 4.18-4.26 (m, 4H), 5.01 (s,
2H), 6.82 (s, 4H), 7.33-7.41 (m, 5H), 7.64 (s, 1H), 7.77 (s, 1H).
Anal. (C22H22N4O2) C, H, N.
200 mesh silica gel. All products reported showed IR and H
NMR spectra in agreement with the assigned structures.
Organic solutions were dried over anhydrous magnesium
sulfate. Elemental analyses were performed by the microana-
lytical 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 N-Su bsti-
tu ted -5-a m in o-4-cya n op yr a zoles 7a -e. The appropriate
hydrazine (65 mmol) was dissolved in EtOH (150 mL), and
(ethoxymethylene)malononitrile (8 g, 65 mmol) was added in
small portions. Then the mixture was heated at 70 °C for 18
h before evaporating the solvent. The solid residue was
purified by chromatography (EtOAc/light petroleum, 1:3) to
afford the product as a solid in good yield. The following
spectral data are reported as examples.
1 -[ 3 -[ 4 -( B e n z y l o x y ) p h e n y l ] p r o p y l ] -4 -c y a n o -5 -
[(eth oxym eth ylen e)a m in o]p yr a zole (8c): yield 92%, yel-
1
low oil; IR (neat) 2210, 1650, 1520 cm-1; H NMR (CDCl3) δ
1.36 (t, 3H, J ) 7), 2.03-2.2 (m, 2H), 2.54 (t, 2H, J ) 7), 4.06
(t, 2H, J ) 7), 4.28 (q, 2H, J ) 7), 5.02 (s, 2H), 6.88 (d, 2H, J
) 8), 7.07 (d, 2H, J ) 8), 7.33-7.40 (m, 5H), 7.64 (s, 1H), 8.38
(s, 1H). Anal. (C23H24N4O2) C, H, N.
4-Cya n o-5-[(eth oxym eth ylen e)a m in o]-1-[3-[3,4-(m eth -
ylen ed ioxy)p h en yl]p r op yl]p yr a zole (8d ): yield 93%, yel-
1
low oil; IR (neat) 2225, 1630, 1510 cm-1; H NMR (CDCl3) δ
1.39 (t, 3H, J ) 7), 2.01-2.19 (m, 2H), 2.52 (t, 2H, J ) 7), 4.05
(t, 2H, J ) 7), 4.25 (q, 2H, J ) 7), 5.91 (s, 2H), 6.62-6.73 (m,
3H), 7.65 (s, 1H), 8.39 (s, 1H). Anal. (C17H18N4O3) C, H, N.
4-C y a n o -1-[3-[3,4-(d i m e t h o x y )p h e n y l]p r o p y l]-5-
[(eth oxym eth ylen e)a m in o]p yr a zole (8e): yield 89%, pale-
yellow oil; IR (neat) 2210, 1640 cm-1; 1H NMR (CDCl3) δ 1.37
(t, 3H, J ) 7), 2.02-2.23 (m, 2H), 2.57 (t, 2H, J ) 7), 3.85 (s,
3H), 3.86 (s, 3H), 4.08 (t, 2H, J ) 7), 4.35 (q, 2H, J ) 7), 6.66-
6.78 (m, 5H), 7.66 (s, 1H), 8.23 (s, 1H). Anal. (C18H22N4O3)
C, H, N.
5-Am in o-4-cya n o-1-[4-(ben zyloxy)ben zyl]p yr a zole (7a ):
yield 72%, yellow solid; mp 205-207 °C (EtOAc-light petro-
leum); IR (KBr) 3450-2950, 2200, 1670, 1580, 1420 cm-1; 1H
NMR (DMSO-d6) δ 5.07 (s, 4H), 6.73 (bs, 2H), 6.96 (d, 2H, J )
8), 7.14 (d, 2H, J ) 8),7.3-7.44 (m, 5H), 7.56 (s, 1H). Anal.
(C18H16N4O) C, H, N.
5-Am in o-1-[2-[4-(b e n zyloxy)p h e n yl]e t h yl]-4-cya n o-
p yr a zole (7b): yield 82%, yellow solid; mp 189-190 °C
(EtOAc-light petroleum); IR (KBr) 3350-3150, 2210, 1670,
Gen er a l P r oced u r es for th e P r ep a r a tion of 7-Su bsti-
tu ted -2-(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 es 9a -e. Imino ethers 8a -e (20 mmol) were dissolved
in 2-methoxyethanol (50 mL), and 2-furoic acid hydrazide (2.5
g, 22 mmol) was added. The mixture was refluxed for 5-10
h, then, after cooling, the solvent was removed under reduced
pressure, and the dark oily residue was cyclized without any
other purification in diphenyl ether (50 mL) at 260 °C using a
Dean-Stark trap for the azeotropic elimination of water
produced in the reaction. After 1.5 h, the mixture was poured
onto hexane (200 mL) and cooled. The precipitate was filtered
off and purified by chromatography (EtOAc/ hexane, 1:1). In
this way, different compounds were obtained. The following
spectral data are reported as examples.
1
1470 cm-1; H NMR (DMSO-d6) δ 2.97 (t, 2H, J ) 7), 4.09 (t,
2H, J ) 7), 5.03 (s, 2H), 5.7 (bs, 2H), 6.88 (d, 2H, J ) 8), 7.09
(d, 2H, J ) 8), 7.3-7.41 (m, 6H). Anal. (C19H18N4O) C, H, N.
5-Am in o-4-cya n o-1-[3-[4-(b en zyloxy)p h en yl]p r op yl]-
p yr a zole (7c): yield 79%, pale-yellow solid; mp 160-162 °C
(EtOAc-Et2O); IR (KBr) 3350-3170, 2215, 1650, 1440 cm-1
;
1H NMR (DMSO-d6) δ 1.9-2.1 (m, 2H), 2.56 (t, 2H, J ) 7),
3.91 (t, 2H, J ) 7), 5.02 (s, 2H), 5.93 (bs, 2H), 6.87 (d, 2H, J )
8), 7.09 (d, 2H, J ) 8), 7.33-7.39 (m, 6H). Anal. (C20H20N4O)
C, H, N.
5-Am in o-4-cyan o-1-[3-[3,4-(m eth ylen edioxy)ph en yl]pr o-
p yl]p yr a zole (7d ): yield 83%, yellow solid; mp 156 °C

Second-Generation Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2131
7-[4-(Ben zyloxy)ben zyl]-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 (9a ): yield 65%, white solid; mp
5-Am in o-1-[3-(3,4-d im e t h oxyp h e n yl)p r op yl]-4-[3-(2-
fu r yl)-1,2,4-tr ia zol-5-yl]p yr a zole (10e): yield 69%, yellow
solid; mp 73 °C (EtOAc); IR (KBr) 3295-3155, 1640, 1480
168-170 °C (EtOAc); IR (KBr) 1530, 1420 cm-1 1H NMR
;
1
(DMSO-d6) δ 5.06 (s, 2H), 5.65 (s, 2H), 6.71-6.74 (m, 1H),
6.93-7.01 (m, 3H), 7.1 (d, 2H, J ) 8), 7.3-7.4 (m, 6H), 7.55
(s, 1H), 8.37 (s, 1H). Anal. (C24H18N6O2) C, H, N.
cm-1; H NMR (CDCl3) δ 2.1-2.22 (m, 2H), 2.59 (t, 2H, J )
7), 3.82 (s, 3H), 3.83 (s, 3H), 3.93 (t, 2H, J ) 7), 5.08 (bs, 2H),
6.48-6.51 (m, 1H), 6.67-6.74 (m, 3H), 6.97 (d, 1H, J ) 4),
7.48 (d, 1H, J ) 2), 7.74 (s, 1H), 13.51 (bs, 1H). Anal.
(C20H22N6O3) C, H, N.
7-[2-[4-(Ben zyloxy)p h en yl]et h yl]-2-(2-fu r yl)p yr a zolo-
[4,3-e]-1,2,4-tr iazolo[1,5-c]pyr im idin e (9b): yield 72%, white
solid; mp 193-194 °C (EtOAc-light petroleum); IR (KBr) 1620,
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-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 5a -e. To a solution of pyrazole derivatives
10a -e (10 mmol) in N-methylpyrrolidone (40 mL) were added
cyanamide (0.42 g, 60 mmol) and p-toluenesulfonic acid (2.85
g, 15 mmol), and the mixture was heated at 160 °C for 4 h.
Then cyanamide (0.42 g, 60 mmol) was added again and the
solution was heated overnight. Then the solution was diluted
with EtOAc (80 mL) and the precipitate (excess of cyanamide)
was filtered off; the filtrate was concentrated under reduced
pressure and washed with water (3 × 30 mL). The organic
layer was dried (Na2SO4) and evaporated under vacuum. The
residue was purified by chromatography (EtOAc/light petro-
leum, 4:1) to afford the final products 5a -e as solids.
5-Am in o-7-[4-(b en zyloxy)b en zyl]-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 (5a ): white solid; mp
188-190 °C; IR (KBr) 3490-3050, 1660, 1640, 1610, 1555,
1440 cm-1; 1H NMR (DMSO-d6) δ 5.06 (s, 2H), 5.4 (s, 2H), 6.7-
6.74 (m, 1H), 6.95 (d, 2H, J ) 8), 7.1-7.2 (m, 3H), 7.32-7.42
(m, 5H), 7.94 (s, 1H), 8.13 (bs, 2H), 8.17 (s, 1H). Anal.
(C24H19N7O2) C, H, N.
1
1510, 1440 cm-1; H NMR (CDCl3) δ 3.21 (t, 2H, J ) 8), 4.72
(t, 2H, J ) 8), 4.97 (s, 2H), 6.58 (dd, 1H, J ) 2, 4), 6.81 (d, 2H,
J ) 8), 6.87 (d, 1H, J ) 2), 7.03 (d, 2H, J ) 8), 7.28-7.36 (m,
5H), 7.62 (s, 1H), 8.37 (s, 1H), 9.0 (s, 1H). Anal. (C25H20N6O2)
C, H, N.
7-[3-[4-(Ben zyloxy)p h en yl]p r op 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 (9c): yield 74%, mp
183-186 °C (EtOH); IR (KBr) 1650, 1510, 1430 cm-1; 1H NMR
(CDCl3) δ 2.23-2.36 (m, 2H), 2.62 (t, 2H, J ) 7), 4.57 (t, 2H,
J ) 7), 5.01 (s, 2H), 6.6-6.63 (m, 1H), 6.87 (d, 2H, J ) 8),
7.05 (d, 2H, J ) 8), 7.3-7.46 (m, 6H), 7.66 (s, 1H), 8.38 (s,
1H), 9.1 (s, 1H). Anal. (C26H22N6O2) C, H, N.
7-[3-[3,4-(Meth ylen ed ioxy)p h en yl]p r op 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 (9d ): yield
82%, pale-yellow solid; mp 145 °C (EtOAc); IR (KBr) 1640,
1
1510, 1445 cm-1; H NMR (CDCl3) δ 2.05-2.2 (m, 2H), 2.57
(t, 2H, J ) 8), 4.56 (t, 2H, J ) 8), 5.92 (s, 2H), 6.58-6.76 (m,
4H), 7.29 (d, 1H, J ) 4), 7.66 (d, 1H, J ) 2), 8.39 (s, 1H), 9.1
(s, 1H). Anal. (C20H16N6O3) C, H, N.
7-[3-[3,4-(Dim eth oxy)ph en yl]pr opyl]-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 (9e): yield 78%, pale-
5-Am in o-7-[2-[4-(b en zyloxy)p h en yl]et h 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 (5b): white
solid; mp 225-228 °C; IR (KBr) 3400-2955, 1680, 1650, 1620,
1570, 1440 cm-1; 1H NMR (DMSO-d6) δ 3.1 (t, 2H, J ) 7), 4.44
(t, 2H, J ) 7), 1.80 (m, 2H), 5.02 (s, 2H), 6.71-6.72 (m, 1H),
6.87 (d, 2H, J ) 8), 7.07 (d, 2H, J ) 8), 7.22 (d, 1H, J ) 4),
7.32-7.39 (m, 5H), 7.94 (s, 1H), 8.08 (bs, 2H), 8.16 (s, 1H).
Anal. (C25H21N7O2) C, H, N.
yellow solid; mp 181 °C (EtOAc); IR (KBr) 1610, 1520 cm-1
;
1H NMR (CDCl3) δ 2.2-2.4 (m, 2H), 2.63 (t, 2H, J ) 8), 3.84
(s, 3H), 3.86 (s, 3H), 4.58 (t, 2H, J ) 8), 6.6-6.63 (m, 1H),
6.71-6.76 (m, 3H), 7.28 (d, 1H, J ) 2), 7.66 (s, 1H), 8.39 (s,
1H), 9.1 (s, 1H). Anal. (C21H20N6O3) 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-Su bsti-
t u t ed -4-[3-(2-fu r yl)-1,2,4-t r ia zol-5-yl]-5-a m in op yr a zoles
10a -e. A solution of the mixture of 9a -e (10 mmol) in
aqueous 10% HCl (20 mL) and dioxane (30 mL) was refluxed
for 3 h. Then the solution was cooled and basified with
concentrated ammonium hydroxide at 0 °C. The compounds
were extracted with EtOAc (3 × 20 mL); the organic layers
were dried with Na2SO4 and evaporated under vacuum. The
residue was purified by chromatography (EtOAc/light petro-
leum, 2:1) to afford the desired compound as a solid. The
following spectral data are reported as examples.
5-Am in o-7-[3-[4-(ben zyloxy)p h en yl]p r op 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 (5c): white
solid; mp 187 °C; IR (KBr) 3520-2960, 1660, 1645, 1610, 1560,
1430 cm-1 1H NMR (DMSO-d6) δ 2.08-2.16 (m, 2H), 2.5-
;
2.57 (m, 2H), 4.25 (t, 2H, J ) 7), 5.03 (s, 2H), 6.72-6.74 (m,
1H), 6.9 (d, 2H, J ) 8), 7.13 (d, 2H, J ) 8), 7.21 (d, 1H, J ) 4),
7.3-7.43 (m, 5H), 7.94 (s, 1H), 8.09 (bs, 2H), 8.16 (s, 1H). Anal.
(C26H23N7O2) C, H, N.
5-Am in o-7-[3-[3,4-(m eth ylen ed ioxy)p h en yl]p r op yl]-2-
(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
(5d ): white solid; mp 210-211 °C; IR (KBr) 3520-2950, 1670,
1630, 1620, 1545, 1450 cm-1; 1H NMR (DMSO-d6) δ 2.01-2.12
(m, 2H), 2.49-2.51 (m, 2H), 4.24 (t, 2H, J ) 7), 5.94 (s, 2H),
6.68-6.82 (m, 4H), 7.23 (d, 1H, J ) 4), 7.94 (s, 1H), 8.09 (bs,
2H), 8.17 (s, 1H). Anal. (C20H17N7O3) C, H, N.
5-Am in o-1-[4-(b en zyloxy)b en zyl]-4-[3-(2-fu r yl)-1,2,4-
tr ia zol-5-yl]p yr a zole (10a ): yield 75%, yellow oil; IR (neat)
3340-3150, 1610 cm-1; 1H NMR (CDCl3) δ 5.01 (bs, 4H), 5.15
(s, 2H), 5.46-5.49 (m, 1H), 6.87-6.95 (m, 3H), 7.12 (d, 2H, J
) 8), 7.32-7.41 (m, 5H), 7.46 (s, 1H), 7.77 (s, 1H), 14.51 (bs,
1H). Anal. (C23H20N6O2) C, H, N.
5-Am in o-1-[2-[4-(ben zyloxy)ph en yl]eth yl]-4-[3-(2-fu r yl)-
1,2,4-tr ia zol-5-yl]p yr a zole (10b): yield 79%, yellow oil; IR
(neat) 3345-3140, 1630 cm-1; 1H NMR (CDCl3) δ 2.95 (t, 2H,
J ) 7), 4.08 (t, 2H, J ) 7), 4.86 (bs, 2H), 4.91 (s, 2H), 6.38-
6.40 (m, 1H), 6.77 (d, 2H, J ) 8), 6.90-6.94 (m, 3H), 7.24-
7.37 (m, 6H), 7.79 (s, 1H), 14.41 (bs, 1H). Anal. (C24H22N6O2)
C, H, N.
5-Am in o-1-[3-[4-(Be n zyloxy)p h e n yl]p r op yl]-4-[3-(2-
fu r yl)-1,2,4-tr ia zol-5-yl]p yr a zole (10c): yield 83%, yellow
oil; IR (neat) 3320-3160, 1635 cm-1; 1H NMR (CDCl3) δ 2.1-
2.25 (m, 2H), 2.55 (t, 2H, J ) 7), 3.92 (t, 2H, J ) 7), 5.01 (s,
2H), 5.2 (bs, 2H), 6.48-6.55 (m, 1H), 6.88 (d, 2H, J ) 8), 6.99
(d, 1H, J ) 4), 7.08 (d, 2H, J ) 8), 7.31-7.4 (m, 5H), 7.48 (d,
1H, J ) 2), 7.8 (s, 1H), 13.81 (bs, 1H). Anal. (C25H24N6O2) C,
H, N.
5-Am in o-7-[3-(3,4-dim eth oxyph en yl)pr opyl]-2-(2-fu r yl)-
pyr azolo[4,3-e]-1,2,4-tr iazolo[1,5-c] pyr im idin e (5e): white
solid; mp 148 °C; IR (KBr) 3500-2950, 1655, 1640, 1630, 1550,
1440 cm-1 1H NMR (DMSO-d6) δ 2.03-2.2 (m, 2H), 2.48-
;
2.54 (m, 2H), 3.69 (s, 3H), 3.71 (s, 3H), 4.26 (t, 2H, J ) 7),
6.72-6.85 (m, 4H), 7.23 (d, 1H, J ) 4), 7.95 (s, 1H), 8.08 (bs,
2H), 8.17 (s, 1H). Anal. (C21H21N7O3) C, H, N.
Gen er a l P r oced u r e for O-Ben zyl Dep r otection (5f-h ).
A solution of the benzyl-protected compounds (0.25 mmol, 5a -
c) in a mixture of CH3OH/dioxane (1:3) (40 mL) was hydro-
genated over 10% palladium on charcoal (100 mg) at room
temperature (50 psi, 4 h). Then the catalyst was filtered off
on Celite and the filtrate concentrated under reduced pressure
to afford compounds 5f-h in very good yield.
5-Am in o-1-[3-[3,4-(m eth ylen ed ioxy)p h en yl]p r op yl]-4-
[3-(2-fu r yl)-1,2,4-tr ia zol-5-yl]p yr a zole (10d ): yield 82%,
5-Am in o-7-(4-h yd r oxyben zyl)-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 (5f): off-white solid; mp
>280 °C (CH3OH); IR (KBr) 3580-2950, 1665, 1620, 1610,
yellow oil; IR (neat) 3460-3120, 1650, 1460 cm-1 1H NMR
;
(CDCl3) δ 1.99-2.09 (m, 2H), 2.49 (t, 2H, J ) 7), 3.87 (t, 2H,
J ) 7), 5.22 (bs, 2H), 5.85 (s, 2H), 6.43-6.45 (m, 1H), 6.5-
6.66 (m, 3H), 6.93 (d, 1H, J ) 4), 7.42 (d, 1H, J ) 2), 7.73 (s,
1H), 13.6 (bs, 1H). Anal. (C19H18N6O3) C, H, N.
1545, 1450 cm-1 1H NMR (DMSO-d6) δ 5.35 (s, 2H), 6.66-
;
6.72 (m, 3H), 7.08 (d, 2H, J ) 8), 7.22 (d, 1H, J ) 4), 7.95 (s,
1H), 8.16 (bs, 2H), 8.24 (s, 1H), 9.4 (s, 1H). Anal. (C17H13N7O2)
C, H, N.

2132 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Baraldi et al.
5-Am in o-7-[2-(4-h yd r oxyp h en yl)eth yl]-2-(2-fu r yl)p yr a -
zolo[4,3-e]-1,2,4-tr iazolo[1,5-c]pyr im idin e (5 g): white solid;
mp 265 °C with dec (EtOH); IR (KBr) 3570-2910, 1660, 1620,
1605, 1540, 1460 cm-1; 1H NMR (DMSO-d6) δ 3.04 (t, 2H, J )
7), 4.41 (t, 2H, J ) 7), 6.62 (d, 2H, J ) 8), 6.72-6.75 (m, 1H),
6.95 (d, 2H, J ) 8), 7.22 (d, 1H, J ) 4), 7.94 (s, 1H), 8.07 (bs,
2H), 8.16 (s, 1H), 9.22 (s, 1H). Anal. (C18H15N7O2) C, H, N.
5-Am in o-7-[3-(4-h ydr oxyph en yl)pr opyl]-2-(2-fu r yl)pyr a-
zolo[4,3-e]-1,2,4-tr iazolo[1,5-c]pyr im idin e (5h ): white solid;
mp 189-191 °C (EtOH); IR (KBr) 3550-2930, 1670, 1640,
were calculated from the Cheng-Prusoff equation.32 All
binding data were analyzed using the nonlinear regression
curve-fitting computer program LIGAND.33
2. Hu m a n Clon ed A1 a n d A2A Ad en osin e Recep tor
Bin d in g Assa ys. Chinese hamster ovary cells (CHO cells)
stably expressing the human A1 adenosine receptor were used
for binding experiments. Thawed membranes were resus-
pended in the same buffer to a final protein concentration of
2.5 mg/mL. Adenosine deaminase (2 IU/mL) was added for
30 min at 37 °C, before radioligand binding assays, to remove
endogenous adenosine. Binding of [3H]CHA to human A1
adenosine receptor was performed as described by Bruns et
al.18 Assays were carried out in duplicate and in a final volume
of 0.5 mL, containing 1.0 nM [3H]CHA, 50 mM Tris HCl buffer,
pH 7.4, and CHO membrane suspension (0.25 mg of protein/
assay). In competition studies, at least seven different con-
centrations of the tested compounds were used. Nonspecific
binding was determined in the presence of 10 µM CHA.
CHO cells stably expressing the human A2A adenosine
receptor were used for binding experiments. Binding of [3H]-
SCH 58261 to human A2A adenosine receptors was performed
as described in rat striatal membranes.7 Assays were carried
out in duplicate and in a final volume of 0.5 mL, containing
0.5 nM [3H]SCH 58261, 50 mM Tris HCl buffer, pH 7.4, and
CHO membrane suspension (0.1 mg of protein/assay). Non-
specific binding was determined in the presence of 10 µM
NECA.
1
1625, 1535, 1440 cm-1; H NMR (DMSO-d6) δ 1.99-2.11 (m,
2H), 2.45-2.50 (m, 2H), 4.24 (t, 2H, J ) 7), 6.66 (d, 2H, J )
8), 6.72-6.74 (m, 1H), 7.0 (d, 2H, J ) 8), 7.22 (d, 1H, J ) 4),
7.94 (s, 1H), 8.09 (bs, 2H), 8.17 (s, 1H), 9.17 (s, 1H). Anal.
(C19H17N7O2) C, H, N.
P r ep a r a tion of 5-Am in o-7-[3-(3,4-d ih yd r oxyp h en yl)-
p r op yl]-2-(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 (5i): To a solution of 5d (0.12 mmol) in methylene
chloride (5 mL) at 0 °C was added a solution of 1 M boron
trichloride in CH2Cl2 (0.25 mL), and the mixture was stirred
at 5 °C for 5 h. Then methanol was added (1 mL), and the
solvent was evaporated under reduced pressure to afford 5i
as a white solid (92%): mp 272 °C with dec (CH3OH); IR (KBr)
3570-2920, 1650, 1635, 1620, 1535, 1440 cm-1 1H NMR
;
(DMSO-d6) δ 2.01-2.15 (m, 2H), 2.43 (t, 2H, J ) 7), 4.23 (t,
2H, J ) 7), 6.43 (d, 1H, J ) 3), 6.47 (d, 1H, J ) 3), 6.57-6.6
(m, 3H), 6.71-6.74 (m, 1H), 7.22 (d, 1H, J ) 4), 7.94 (s, 1H),
8.08 (bs, 2H), 8.16 (s, 1H). Anal. (C19H17N7O3) C, H, N.
Deter m in a tion of Rm Va lu es by C18 RP -HP TLC. The
HPTLC determinations were carried out on Whatman KC18F
plates as previously described.28 Solvent mixtures of methanol-
water buffer at pH 7.0 were used as mobile phase. The
methanol concentration ranged from 60% to 90%. The solutes
were detected under UV 254-nm light.
After 30-min incubation at 25 °C, samples were filtered
through Whatman GF/B filters using a Brandel cell harvester
(Gaithersburg, MD). Radioactivity was determined in a LS-
6000IC Beckman liquid scintillation counter (Beckman Instru-
ments Inc., Fullerton, CA), at an efficiency of 50-60%. Protein
concentration was determined by the method of Lowry et al.34
with bovine serum albumin used as standard.
Biologica l Stu d ies. 1. Ra t A1 a n d A2A Ad en osin e
Recep tor Bin d in g Assa ys. Male Wistar rats (200-250 g)
were decapitated and the whole brain and striatum dissected
on ice. The tissues were disrupted in a Polytron homogenizer
at a setting of 5 for 30 s in 25 volumes of 50 mM Tris HCl, pH
7.4, containing 10 mM MgCl2. The homogenate was centri-
fuged at 48000g for 10 min, and the pellet was resuspended
in the same buffer containing 2 IU/mL adenosine deaminase.
After 30-min incubation at 37 °C, the membranes were
centrifuged and pellets were stored at -80 °C. Prior to
freezing, an aliquot of homogenate was removed for protein
assay according to a Bio-Rad method31 with bovine albumin
as reference standard.
Binding assays3,19 were performed on rat brain and striatum
membranes, respectively, in the presence of 10 mM MgCl2 at
0, 10, 15, 20, 25, and 30 °C. All buffer solutions were adjusted
to maintain a constant pH of 7.4 at the desired temperature.
Displacement experiments were performed in 500 µL of Tris
HCl buffer containing 1 nM of the selective adenosine A1
receptor ligand [3H]CHA (N6-cyclohexyladenosine) and mem-
branes of rat brain (150-200 µg of protein/assay).
Displacement experiments were performed in 500 µL of Tris
HCl buffer containing 10 mM MgCl2, 5 nM of the selective
adenosine A2A receptor ligand [3H]CGS 21680 (2-[[4-(2-car-
boxyethyl)phenethyl]amino]-5′-(N-ethylcarbamoyl)adeno-
sine) and membranes of rat striatum (80-100 µg of protein/
assay). To determine IC50 values (where IC50 is the inhibitor
concentration displacing 50% of labeled ligand) the test
compounds were added in triplicate to binding assay samples
at a minimum of six different concentrations. Separation of
bound from free radioligand was performed by rapid filtration
through Whatman GF/B filters which were washed three times
with ice-cold buffer. Filter-bound radioactivity was measured
by scintillation spectrometry after addition of 5 mL of Aquas-
sure. Nonspecific binding was defined as binding in the
presence of 10 µM R-PIA (N6-(phenylisopropyl)adenosine) and
10 µM NECA (5′-(N-ethylcarboxamido)adenosine), respectively,
and was always 10% of the total binding. Incubation time
ranged from 150 min at 0 °C to 75 min at 30 °C according to
the results of previous time-course experiments. Ki values
Hu m a n Clon ed A3 Ad en osin e Recep tor Bin d in g As-
sa ys. An aliquot of membranes (8 mg of protein/mL) from
HEK-293 cells transfected with the human recombinant A3
adenosine receptor was used for binding assays.35 Inhibition
experiments were carried out in duplicate in a final volume of
100 µL in test tubes containing 0.3 nM [125I]AB-MECA (N6-
(4-amino-3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine), 50
mM Tris HCl buffer, 10 mM MgCl2, pH 7.4, 20 µL of diluted
membranes (12.4 mg of protein/mL), and at least 6-8 different
concentrations of typical adenosine receptor antagonists.
Nonspecific binding was defined in the presence of 50 µM
R-PIA and was about 30% of total binding. Incubation time
was 60 min at 37 °C, according to the results of previous time-
course experiments. Bound and free radioactivity was sepa-
rated by filtering the assay mixture through Whatman GF/B
glass-fiber filters using a Brandel cell harvester.
4. Th er m od yn a m ic P a r a m eter Deter m in a tion . The
standard free energy was calculated as ∆G° ) -RT ln KA
(where KA is the association constant of the equilibrium drug
+ receptor h drug-receptor) at 298.15 K. The standard
enthalpy, ∆H°, was obtained from the van’t Hoff plot of ln KA
versus 1/T (whose slope is -∆H°/R), and the standard entropy
was ∆S° ) (∆H° - ∆G°)/T, with T ) 298.15 K and R ) 8.314
J /mol/K.36
5. Cyclic AMP Assa y. Washed human platelets from
peripheral blood of healthy volunteers were prepared as
previously described by Korth et al.37 The final suspending
medium was a Tyrode’s buffer, pH 7.4, of the following
composition (mM): NaCl, 137; KCl, 2.68; NaHCO3, 11.9;
MgCl2, 1.0; NaHPO4, 0.4; glucose, 5.5. Platelets (6-8 × 104
cells) were suspended in 0.5 mL of incubation mixture (Ty-
rode’s buffer containing 0.25% BSA, 1 IU/mL adenosine
deaminase, and 0.5 mM Ro 20-1724 as a phosphodiesterase
inhibitor) and preincubated for 10 min in a shaking bath at
37 °C. Then, the synthesized compounds, NECA (1 µM), and
forskolin (1 µM) were added, and the incubation was continued
for a further 5 min. The reaction was terminated by the
addition of cold 6% trichloroacetic acid (TCA). TCA suspension
was centrifuged at 2000g for 10 min at 4 °C, and the
supernatant was extracted four times with water-saturated

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