Synthesis, biological and modeling studies of 1,3-di-n-propyl-2,4-dioxo-6- methyl-8-(substituted) 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-purines as adenosine receptor antagonists

Article abstract of DOI:10.1016/j.farmac.2005.04.012

A new series of potential adenosine receptor antagonists with a [1,2,4]-triazolo-[3,4-f]-purine structure bearing at the 1 and 3 position n-propyl groups have been synthesized, and their affinities at the four human adenosine receptor subtypes (A₁

Full text of DOI:10.1016/j.farmac.2005.04.012

Il Farmaco 60 (2005) 643–651
http://france.elsevier.com/direct/FARMAC/
Original article
Synthesis, biological and modeling studies
of 1,3-di-n-propyl-2,4-dioxo-6-methyl-8-(substituted) 1,2,3,4-tetrahydro
[1,2,4]-triazolo [3,4-f]-purines as adenosine receptor antagonists
G. Pastorin a, C. Bolcato a, B. Cacciari b, S. Kachler c, K.-N. Klotz c, C. Montopoli d,
S. Moro d,*, G. Spalluto a,*
a Dipartimento di Scienze Farmaceutiche, Università di Trieste, Piazzale Europa 1, 34127 Trieste, Italy
b Dipartimento di Scienze Farmaceutiche, Università degli Studi di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy
c Institut für Pharmakologie und Toxikologie, Universität Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany
d Molecular Modeling Section, Dipartimento di Scienze Farmaceutiche, Università degli Studi di Padova, via Marzolo 5, 35131 Padova, Italy
Received 28 February 2005; received in revised form 27 April 2005; accepted 28 April 2005
Available online 14 June 2005
Abstract
A new series of potential adenosine receptor antagonists with a [1,2,4]-triazolo-[3,4-f]-purine structure bearing at the 1 and 3 position
n-propyl groups have been synthesized, and their affinities at the four human adenosine receptor subtypes (A1, A2A, A2B and A3) have been
evaluated. In this case the presence of n-propyl groups seems to induce potency at the A2A and A3 adenosine receptor subtypes as opposed to
our previously reported series bearing methyl substituents at the 1 and 3 positions. In particular the non-acylated derivative 17 showed affinity
at these two receptor subtypes in the micromolar range. Indeed, preliminary molecular modeling investigations according to the experimental
binding data indicate a modest steric and electrostatic antagonist-receptor complementarity.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Adenosine receptors; Antagonists; Xanthine; G-protein-coupled receptors; Cycloaddition
1. Introduction
the last decades. Such compounds could be used as pharma-
cological tools for receptor characterization as well as poten-
tial drug candidates [7,8].
In the field of antagonists, naturally occurring xanthines
like caffeine or theophylline are adenosine receptor ligands
with micromolar affinity. These compounds represent an ideal
starting point for the development of potent and selective
adenosine receptor antagonists.
Several substitutions at the 1-, 3-, and 8-positions led to
the discovery of potent and selective antagonists for the dif-
ferent adenosine receptor subtypes. (Fig. 1) In particular, sub-
stitution at the 8-position with a cyclopentyl group led to
DPCPX 1 [9] which is one of the most potent and selective
A1 adenosine receptor antagonists. The introduction at the
same position of styryl (2) [10] or bulky aryl substituents (3)
[11,12] permitted to obtain A2A and A2B adenosine receptor
antagonists (2, 3). Recently it has been demonstrated that ana-
logs constrained between 7- and 8- (4) [13] or 3- and 4- posi-
tions (5) [14] possess A3 affinity and selectivity (Fig. 1).
Adenosine is a widely distributed modulator, which regu-
lates many physiological functions [1,2]. The action of
adenosine occurs through the stimulation of P1 purinergic
receptors, which are subdivided into A1, A2A, A2B and A3
adenosine receptors subtypes [1,2]. All these receptors are
coupled to G-proteins and whileA1 andA3 stimulation induces
adenylyl cyclase inhibition and consequently reduced cAMP
levels, the activation of A2A and A2B produces the opposite
effect [3–6]. All these receptors are widely distributed and
they are strongly involved in several patho-physiological func-
tions and/or disorders as extensively reported [1].
For this reason great efforts have been dedicated to the
search for potent and selective agonists and antagonists over
* Corresponding authors. Tel.: +39 040 558 3726; fax: +39 040 52 572.
E-mail address: spalluto@units.it (G. Spalluto).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.04.012

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G. Pastorin et al. / Il Farmaco 60 (2005) 643–651
absence of n-propyl or bulkier groups at the 1 and 3 positions
(see reference compounds 1–5), which on these bases could
be considered an ideal structural requirement for having bind-
ing affinity at the adenosine receptor subtypes.
For this reasons we designed a new series of derivatives in
which the methyl groups at the 1 and 3 positions were replaced
by n-propyl groups that are characteristics of adenosine recep-
tor antagonists with the xanthine nucleus (Fig. 2).
2. Chemistry
The designed compounds (7a–j) have been prepared by
one-pot cycloaddition reaction between the 8-substituted xan-
thine with an appropriate hydrazonoyl halide [16,17] as pre-
viously reported. The appropriate 8-substituted dipropyl xan-
thine was prepared following a standard procedure and briefly
reported in Scheme 1.
Reacting N-N′-dipropyl urea 8 with cyanacetic acid 9 in
presence of acetic anhydride afforded the 5-aminouracil 10,
which by treatment with sodium nitrite in acidic conditions
gave the nitroso derivative 11. The latter was then reduced
with hydrazine and C–Pd 10% to afford the diaminouracil 12
[18] which was then cyclized to dipropylxanthine 13 by treat-
ment with formic acid and then with NaOH [19,20]. Reac-
tion of 13 with HNO3 70% yield to the desired 8-nitro-
dipropyl xanthine 14 [21–23]. Finally, reacting at reflux (10 h)
in presence of triethylamine, compound 14 with the N-(2-
bromo-4-nitrophenyl) acetohydrazonoyl bromide (15) [24] the
corresponding cycloadduct 16 was obtained in a good yield
Fig. 1. Structures of xanthine derivatives as potent and selective adenosine
receptor antagonists.
Taking into account these experimental observations we
have recently reported a new series of 1,3-dimethyl-2,4-dioxo-
6-methyl-8-(substituted) 1,2,3,4-tetrahydro [1,2,4]-triazolo
[3,4-f]-purines of general formula 6 as potential adenosine
receptor antagonists. (Fig. 2) Unfortunately, all the synthe-
sized compounds resulted to be totally inactive at all four
adenosine receptor subtypes [15].
A molecular modeling study clearly indicated that a poor
steric and electrostatic complementarity for the hA3 binding
site. In particular, this inactivity could be attributed to the
Fig. 2
.
Rational design of newly synthesized derivatives.
Scheme 1.

G. Pastorin et al. / Il Farmaco 60 (2005) 643–651
645
Table 1
Binding affinities at rat A1 and rat A2A adenosine receptors of synthesized
compounds
Compound R
rA1 a % displ
(10 µM) (%)
< 10
rA2A b % displ
(10 µM) (%)
< 10
16
17
7a
7b
7c
7d
7e
7f
NO2
NH2
< 10
47
4
NHCOCH2Ph
NHCOCHPh2
NHCOCH3
< 10
< 10
< 10
24
< 10
< 10
4
NHCOPh
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
NHCO(CH2)2CH3
NHCO(CH2)4CH3
NHCOPh-4-Ph
NHCO-Adamantyl
NHCO-Thienyl
NHCO-4-OCH3-Ph
< 10
< 10
7 g
7 h
7i
< 10
< 10
< 10
7j
< 10
a Displacement of specific [3H]R-PIA binding (A1) in rat brain membra-
nes.
b Displacement of specific [3H]CGS 21680 binding (A2A) in rat striatal
membranes.
Table 2
Binding affinities (A1, A2A, A3) and potency (A2B) of selected compounds at
four human adenosine receptor subtypes
a
c
Compound hA1
hA2A Ki nM
or b % displ
(10 µM)
2,050 520
25%
hA2B
EC50
(µM)
> 100
> 30
hA3 Ki nM
or d % displ
(10 µM)
1,330 470
26%
% displ
(10 µM)
28
17
7a
7c
7f
24
24
64%
> 30
48%
Scheme 2
.
18
42%
> 30
56%
7h
7j
16
23%
> 30
15%
[25]. The latter was then reduced with hydrazine in the pres-
ence of C/Pd 10% [18] to afford the corresponding amino
derivative 17, which was reacted in dioxane at room tempera-
ture in the presence of an equivalent amount of trietylamine
with an appropriate acyl chloride (18a–j) to yield the final
compounds 7a–j (Scheme 2).
24
24%
> 100
5%
a Displacement of specific [3H]DPCPX binding at human A1 receptors
expressed in HEK-293 cells.
b Displacement of specific [125I] -ZM241385 binding at humanA2A recep-
tors expressed. In HEK-293 cells. Data are expressed as Ki S.E.M. in nM
(n = 3–6).
c Measurement of adenylyl cyclase activity in CHO cells stably transfec-
ted with human recombinant A2B adenosine receptor, expressed as EC50
(µM).
d Displacement of specific [125I]-I-AB-MECA binding at humanA3 recep-
tors expressed in CHO cells. Data are expressed as Ki S.E.M. in µM (n = 3–
6).
3. Results and discussion
In Tables 1 and 2 the receptor binding affinities of the syn-
thesized compounds (16, 17, 7a–j) are reported. They were
determined at the rat A1, (from rat cerebral cortex mem-
branes) with [3H]-N6-phenylisopropyladenosine ([3H]R-
PIA), [26] ratA2A (from rat striatal membranes), with [3H]-2-
[4-[(2-carboxyethyl)-phenyl]ethylamino]-5′-N-
antagonists was determined in adenylyl cyclase experiments
in Chinese hamster ovary (CHO) cells expressing humanA2B
adenosine receptors [31,32].
In a preliminary screening at the rA1 and rA2A adenosine
receptors, all the synthesized compounds resulted to be totally
inactive (Table 1).
Nevertheless, considering the differences between species
for the adenosine receptors [31] we tested some selected com-
pounds (17, 7a, c, f, h, j) at the four cloned human adenosine
receptor subtypes (Table 2).
As clearly indicated while the derivatives acylated at the
amino function (7a, c, f, h, j) resulted to be inactive at all four
adenosine receptor subtypes the unsubstituted derivative 17
displayed affinity in the micromolar range at the human A2A
andA3 receptors. This activity could be most probably attrib-
uted at the structural similarity between derivative 17 and ref-
ethylcarbamoyladenosine, ([3H]-CGS21680) (A2A) [27].
Binding affinities at human A1, A2A, and A3 receptors
expressed in CHO cells were determined using [3H]-1,3-
dipropyl-8-cyclopentyl xanthine ([3H]DPCPX), (A1) [28,58],
[
125I]- (4-(2-[7-amino-2-(2-furyl) [1,2,4]triazolo[2,3-a]
[1,3,5]triazin-5-yl-amino]ethyl)-4-iodo-phenol, ([125I]-
ZM241385) (A2A) [29] and [125I]-N6-(4-amino-3-iodo-
benzyl)-5′-N-methylcarbamoyladenosine ([125I]-AB-MECA)
(A3) [30]as radioligands.
Instead of evaluating binding affinity at humanA2B recep-
tors due to the lack of a suitable radioligand, the potency of

646
G. Pastorin et al. / Il Farmaco 60 (2005) 643–651
erence compounds 2 and 4. In order to analyze these results
in more detail we performed a molecular modeling investi-
gation, at the human A3 adenosine receptor.
other compounds or their intensities are questionable, in par-
ticular with the key residues His95 (TM3), Ser247 (TM6),
Asn250 (TM6), and Lys152 (EL2). An intense modeling
evaluation is running to improve receptor complementarity
using this interesting triazolo-purine scaffold. In fact, identi-
fication of microscopic complementarity between residues of
the putative receptor binding site and the docked high-
affinity ligands, i.e. energetically stabilizing elements in ligand
recognition, has an useful tool in the design process of new
potent and selective antagonists.
In fact, we have recently revisited the rhodopsin-based
model of the humanA3 receptor in its resting state (antagonist-
like state), which can be considered a further refinement in
building the hypothetical binding site of the A3 receptor
antagonists already proposed [33–37]. Special care had to be
given to the second extracellular (EL2) loop, which has been
described in bovine rhodopsin to fold back over transmem-
brane helices, and, therefore, controls the size of the recog-
nition cavity. Similar to rhodopsin, the A3 receptor model
reveals a seven-helix bundle with a central cavity surrounded
by helices 1–7 and 5–6. TM4 is not part of the cavity wall
and makes contacts only with TM3. Moreover, hairpin (EL2)
between TM4 and TM5 controls access of ligands from the
periplasmic environment. This hairpin lies between the heli-
ces, roughly parallel to the membrane surface. It has contacts
with side chains of most of the helices. The most prominent
contact is a disulfide bridge to helix 3 (TM3). The ligand rec-
ognition seems to occur in the upper region of the TM helical
bundle. TM 3, 5, 6 and 7 seem to be crucial for the recogni-
tion of both agonists and antagonists. To test the quality of
our theoretical model, we decided to mutate several residues
of the hA3 receptor within TMs 3 and 6 and EL2, which have
been predicted by previous molecular modeling to be involved
in the ligand recognition, including His95, Trp243, Leu244,
Ser247,Asn250, and Lys152 The N250A mutant receptor lost
the ability to bind both radiolabeled agonist and antagonist
[38–40]. We carried out molecular docking simulations to
evaluate the complementarity between the TM cleft and the
new series of the hA3 receptor antagonists. According to the
experimental binding data a modest steric and electrostatic
complementarity has been observed for all the new synthe-
sized triazolo-purines. One of the best docked conformation
of derivative 17 is shown in Fig. 3.
4. Experimental section
4.1. Chemistry
General Remarks: Reactions were routinely monitored by
thin-layer chromatography (TLC) on silica gel (precoated F254
Merck plates) and products visualized with iodine or aque-
ous potassium permanganate. Infrared spectra (IR) were mea-
sured on a Jasco FT-IR instrument using KBr powder (DRIFT
system). 1H NMR spectra were determined in DMSO-d6 solu-
tions, unless otherwise noted, at 200 MHz, with a Varian
Gemini 200 spectrometer, peaks positions are given in parts
per million (d) downfield, and J values are given in Hz. Melt-
ing points were determined on a Buchi-Tottoli instrument and
are uncorrected. Chromatographies were performed with
Merck 60–200 mesh silica gel. All products reported showed
IR and 1H NMR spectra in agreement with the assigned struc-
tures. Organic solutions were dried over anhydrous magne-
sium sulfate. Elemental analyses (C, H, N) were performed
by the microanalytical laboratory of Dipartimento di Chimica,
University of Trieste, and were within 0.4% of the theoreti-
cal values.
4.1.1. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-(4-nitrophe-
nyl) 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-purine (16)
To a mixture of hydrazonoyl bromide 15 (0.46 g, 1.8 mmol)
and 8-nitrodipropylxanthine 14 (0.5 g, 1.8 mmol) in dry diox-
ane (10 ml), was added triethyamine (0.26 ml, 1.8 mmol).
The mixture was then refluxed for 10 h. The excess of sol-
vent was removed under reduced pressure, the crude solid
residue was then filtered, washed with ethanol and crystal-
lized from ethyl acetate-light petroleum to give final product
16 as a brown solid in a good yield (0.68 g, 92%). MW 411.42;
m.p.: 246 °C (ethyl acetate-light petroleum); IR-DRIFT
(KBr): cm–1 1684, 1673, 1610, 1545, 1350; 1H NMR
(DMSO): d: 1.01 (t, 6H, J = 6.7), 1.71–1.78 (m, 4H), 2.95 (s,
3H), 3.96 (t, 2H, J = 6.7), 4,12 (t, 2H, J = 6.7), 8.26 (d, 2H,
J = 9), 8.33 (d, 2H, J = 9); Anal. (C19H21N7O4): Calc.: C
55.47, H 5.14, N 23.83; Found: C 55.08; H 5.09, N 23.57.
Indeed, the most crucial interactions detected between the
receptor cavity and the antagonist structures are missing for
Fig. 3. Triazolo-purine derivatives binding site in the human A3 receptor.
4.1.2. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-(4-aminophe-
nyl) 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-purine (17)
To a solution of nitroderivative 16 (1 g, 2.4 mmol) in diox-
ane (20 ml), hydrazine hydrate (0.52 ml, 16.37 mmol) and
Derivative 17 docked into the ligand binding crevice of the human A3 recep-
tor viewed from the membrane side facing TM helices 5 and 6. TM helical
6 is not shown to facilitate the inspection of the receptor cavity. Hydrogen
atoms are not displayed.

G. Pastorin et al. / Il Farmaco 60 (2005) 643–651
647
C–Pd 10% (0.08 mg) were added. The mixture was then
stirred at reflux for 20 min. The misxture was then filter
through a celite pan and the solvent removed under reduced
pressure. The crude solid was crystallized from ethyl acetate-
light petroleum to give the amino derivative 17 as a white
solid in excellent yield (0.90 g, 99%). MW 381.44; m.p.:
225 °C (ethyl acetate-light petroleum); IR-DRIFT (KBr):
cm–1 3353, 1685, 1675, 1611; 1H NMR (DMSO): d:1.02 (t,
6H, J = 6.7), 1.73–1.81 (m, 4H), 2.90 (s, 3H), 3.85 (bs, 2H),
3.95 (t, 2H, J = 6.7), 4,14 (t, 2H, J = 6.7), 6.80 (d, 2H, J = 9),
7.81 (d, 2H, J = 9); Anal. (C19H23N7O2): Calc.: C 59.83, H
6.08, N 25.70; Found: C 56.03; H 6.25, N 25.43.
(d, 2H, J = 9), 10.10 (bs, 1H); Anal. (C21H25N7O3): Calc.: C
59.56, H 5.95, N 23.15; Found: C 59.81; H 6.01, N 23.01.
4.1.7. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-[4-(benzoy-
lamino)phenyl] 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-
purine (7d)
Yield: 77%; white solid; MW 473.53; m.p.: 215–218 °C
(ethyl acetate-light petroleum); IR-DRIFT (KBr): cm–1 3325,
1690, 1672, 1665, 1605; 1H NMR (DMSO): d: 1.04 (t, 6H,
J = 6.7), 1.73–1.80 (m, 4H), 2.77 (s, 3H). 3.90 (t, 2H, J = 6.7),
4,21 (t, 2H, J = 6.7), 7.45–7.57 (m, 5H); 7.88 (s, 4H), 10.31
(bs, 1H); Anal. (C25H27N7O3): Calc.: C 63.41, H 5.75, N
20.71; Found: C 63.18; H 5.83, N 20.48.
4.1.3. General procedures for the preparation of 1,3-Di-n-
propyl-2,4-dioxo-6-methyl-8-(substituted) 1,2,3,4-
tetrahydro [1,2,4]-triazolo [3,4-f]-purine. (7a–j)
4.1.8. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-[4-((4-methox-
yphenylcarbonyl) amino)phenyl] 1,2,3,4-tetrahydro
[1,2,4]-triazolo [3,4-f]-purine (7e)
To a solution of amino compounds 17 (106 mg, 0.28 mmol)
in dry dioxane (10 ml) triethylamine (48.5 µl, 0.34 mmol)
and appropriate acyl chloride 18a–j (1.2 eq) were added. Then
the mixture was stirred at room temperature for 6 h. The tri-
ethylamine hydrochloride war removed by filtration and the
solvent concentrated under reduced pressure. The crude resi-
due was crystallized from an appropriate mixture of solvents
to afford final compounds 7a–j in a good yield as solids.
Yield: 93%; pale yellow solid; MW 515.57; m.p.: 265 °C
(ethyl acetate-light petroleum); IR-DRIFT (KBr): cm–1 3323,
1688, 1675, 1667, 1605; 1H NMR (DMSO): d: 1.02 (t, 6H,
J = 6.7), 1.73–1.81 (m, 4H), 2.77 (s, 3H), 3.30 (s, 3H), 3.88
(t, 2H, J = 6.7), 4,20 (t, 2H, J = 6.7), 7.02 (d, 2H, J = 9); 7.89
(s, 4H), 7.98 (d, 2H, J = 9), 10.23 (bs, 1H); Anal.
(C27H29N7O4): Calc.: C 62.90, H 5.67, N 19.02; Found: C
63.08; H 5.70, N 18.89.
4.1.4. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-[4-((phenyl-
acetyl)amino)phenyl] 1,2,3,4-tetrahydro [1,2,4]-triazolo
[3,4-f]-purine (7a)
4.1.9. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-[4-((butanoy-
l)amino)phenyl] 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-
purine (7f)
yield: 80%; white solid; MW 499.57; m.p.: 202 °C (ethyl
acetate-light petroleum); IR-DRIFT (KBr): cm–1 3345, 1698,
1683, 1675, 1600; 1H NMR (DMSO): d: 1.04 (t, 6H, J = 6.7),
1.70−1.80 (m, 4H), 2.91 (s, 3H), 3.97 (t, 2H, J = 6.7), 4,15 (t,
2H, J = 6.7), 5.10 (s, 2H), 7.28–7.45 (m, 5H). 7.62 (d, 2H,
J = 9), 7.88 (d, 2H, J = 9), 9.20 (bs, 1H);Anal. (C27H29N7O3):
Calc.: C 64.92, H 5.85, N 19.63; Found: C 65.12; H 5.99, N
19.78.
Yield: 77%; white solid; MW 451.53; m.p.: 205 °C (ethyl
acetate-light petroleum); IR-DRIFT (KBr): cm–1 3325, 1694,
1673, 1665, 1610; 1H NMR (DMSO): d: 0.97 (t, 3H, J 0 6.7),
1.02 (t, 6H, J = 6.7), 1.22–1.27 (m, 2H), 1.73–1.81 (m, 4H),
2.15 (t, 2H, J = 6.7), 2.78 (s, 3H), 3.85 (t, 2H, J = 6.7), 4,11
(t, 2H, J = 6.7), 7.70 (d, 2H, J = 9); 7.91 (d, 2H, J = 9), 10.13
(bs, 1H); Anal. (C23H29N7O3): Calc.: C 61.18, H 6.47, N
21.71; Found: C 60.93; H 6.53, N 21.52.
4.1.5. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-[4-((diphenyl–
acetyl)amino)phenyl] 1,2,3,4-tetrahydro [1,2,4]-triazolo
[3,4-f]-purine (7b)
Yield: 80%; white solid; MW 575.67; m.p.: 230 °C (ethyl
acetate-light petroleum); IR-DRIFT (KBr): cm–1 3320, 1690,
1670, 1665, 1610; 1H NMR (DMSO): d: 1.04 (t, 6H, J = 1.70–
1.80 (m, 4H), 2.85 (s, 3H), 3.96 (t, 2H, J = 6.7), 4,20 (t, 2H,
J = 6.7), 4.87 (s, 1H), 7.17–7.35 (m, 10H). 7.75 (d, 2H, J = 9),
7.83 (d, 2H, J = 9), 10.35 (bs, 1H);Anal. (C33H33N7O3): Calc.:
C 68.85, H 5.78, N 17.03; Found: C 68.60; H 5.71, N 16.91.
4.1.10. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-[4-((esanoy-
l)amino)phenyl] 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-
purine (7g)
Yield: 76%; white solid; MW 479.58; m.p.: 195 °C (ethyl
acetate-light petroleum); IR-DRIFT (KBr): cm–1 3323, 1692,
1673, 1663, 1610; 1H NMR (DMSO): d: 0.91 (t, 3H, J = 6.7),
1.02 (t, 6H, J = 6.7), 1.21–1.30 (m, 4H), 1.47–1.59 (m, 2H),
1.47–1.59 (m, 2H), 1.73–1.81 (m, 4H), 2.29 (t, 2H, J = 6.7),
2.81 (s, 3H), 3.89 (t, 2H, J = 6.7), 4,17 (t, 2H, J = 6.7), 7.72
(d, 2H, J = 9); 7.97 (d, 2H, J = 9), 10.01 (bs, 1H); Anal.
(C25H33N7O3): Calc.: C 62.61, H 6.94, N 20.44; Found: C
62.33; H 7.00, N 20.63.
4.1.6. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-[4-(acetylami-
no)phenyl] 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-
purine (7c)
4.1.11. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-[4-((4-biphe-
nylyl)amino)phenyl] 1,2,3,4-tetrahydro [1,2,4]-triazolo
[3,4-f]-purine (7h)
Yield: 95%; white solid; MW 423.47; m.p.: 263–265 °C
(ethyl acetate-light petroleum); IR-DRIFT (KBr): cm–1 3335,
1690, 1675, 1668, 1605; 1H NMR (DMSO): d: 1.01 (t, 6H,
J = 6.7), 1.71–1.76 (m, 4H), 2.05 (s, 3H); 2.77 (s, 3H), 3.92
(t, 2H, J = 6.7), 4,23 (t, 2H, J = 6.7), 7.65 (d, 2H, J = 9), 7.84
Yield: 92%; whitesolid; MW 561.64; m.p.: 229 °C (ethyl
acetate-light petroleum); IR-DRIFT (KBr): cm–1 3330, 1693,

648
G. Pastorin et al. / Il Farmaco 60 (2005) 643–651
1677, 1665, 1605; 1H NMR (DMSO): d: 1.03 (t, 6H, J = 6.7),
1.70–1.83 (m, 4H), 2.75 (s, 3H), 3.90 (t, 2H, J = 6.7), 4,22 (t,
2H, J = 6.7), 7.20–7.35 (m, 4H), 7.47–7.90 (m, 9H); 10.32
(bs, 1H); Anal. (C32H31N7O3): Calc.: C 68.43, H 5.53, N
17.46; Found: C 68.17; H 5.60, N 17.21.
tion of the membranes, in a preincubation of 30 min at 30 °C,
and during the incubation with the radioligands. All nonra-
dioactive compounds were initially dissolved in DMSO and
diluted with buffer to the final concentration, where the
amount of DMSO never exceeded 2%. Incubations were ter-
minated by rapid filtration over Whatman GF/B filters, using
a Brandell cell harvester (Brandell, Gaithersburg, MD). The
tubes were rinsed three times with 3 ml of buffer each. At
least six different concentrations of competitor, spanning three
orders of magnitude adjusted appropriately for the IC50 of
each compound, were used. IC50 values, calculated with the
nonlinear regression method implemented in Graph-Pad
(Prism, San Diego, CA), were converted to Ki values as
described [44]. Hill coefficients of the tested compounds were
in the range of 0.8–1.1.
4.1.12. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-[4-((adaman-
tyl-carbonyl)amino)phenyl] 1,2,3,4-tetrahydro [1,2,4]-
triazolo [3,4-f]-purine (7i)
Yield: 80%; pale yellow solid; MW 544.68; m.p.: 164 °C
(ethyl acetate/light petroleum); IR-DRIFT (KBr): cm–1 3335,
1
1693, 1675, 1662, 1600; H NMR (DMSO): d: 1.03 (t, 6H
J = 6.7), 1.53–2.03 (m, 10H), , 2.06–2.18 (m, 6H), 2.57–2.88
(m, 3H), 2.82 (s, 3H), 3.04–3.15 (m, 1H), 3.93 (t, 2H, J = 6.7),
4,20 (t, 2H, J = 6.7), 7.90 (s, 4H), 9.45 (bs, 1H); Anal.
(C30H38N7O3): Calc.: C 66.15, H 7.03, N 18.00; Found:
C 65.84; H 6.97, N 17.82.
4.2.3. Adenylyl cyclase activity
Due to the lack of a suitable radioligand the affinity of
antagonists and the relative potency of agonists atA2B adenos-
ine receptors was determined in adenylyl cyclase experi-
ments. The procedure was carried out as described previ-
ously [31,32] with minor modifications. Membranes were
incubated with about 150,000 cpm of [a-32 P]ATP for 20 min
in the incubation mixture as described [31,32] without EGTA
and NaCl. For agonists the EC50-values for the stimulation of
adenylyl cyclase were calculated with the Hill equation. Hill
coefficients in all experiments were near unity. IC50-values
for concentration-dependent inhibition of NECA-stimulated
adenylyl cyclase caused by antagonists was calculated accord-
ingly. Dissociation constants (Ki) for antagonist were then
calculated with the Cheng and Prusoff equation [45].
4.1.13. 1,3-Di-n-propyl-2,4-dioxo-6-methyl-8-[4-((2-thie-
nyl-carbonyl)amino)phenyl] 1,2,3,4-tetrahydro [1,2,4]-
triazolo [3,4-f]-purine (7j)
Yield: 73%; pale yellow solid; MW 491.57; m.p.: 250–
253 °C (dec.) (ethyl acetate-light petroleum); IR-DRIFT
(KBr): cm–1 3335, 1693, 1670, 1661, 1620, 1600; 1H NMR
(DMSO): d: 1.03 (t, 6H, J = 6.7), 1.70–1.83 (m, 4H), 2.78 (s,
3H), 3.92 (t, 2H, J = 6.7), 4,18 (t, 2H, J = 6.7), 7.20–7.25 (m,
1H), 7.73–7.81 (m, 1H), 7.87 (s, 4H), 7.91–8.02 (m, 1H),
10.33 (bs, 1H);Anal. (C24H25N7O3S): Calc.: C 58.64, H 5.13,
N 19.95, S 6.52; Found: C 58.38; H 5.04, N 20.04, S 6.47.
4.2. Biology
4.2.4. Computational methodologies
All molecular modeling studies were carried out on a six
CPU (PIV 2.0–3.0 GHZ) linux cluster running under open-
Mosix architecture [46].
Homology modeling, energy calculation, and docking stud-
ies have been done using Molecular Operating Environment
(MOE, version 2003.03) suite [47].
The ground state geometry of all, charged and uncharged,
docked structures were fully optimized without geometry con-
straints using RHF/3-21G(*) ab initio calculations. Vibra-
tional frequency analysis was used to characterize the minima
stationary points (zero imaginary frequencies). The software
package Spartan O2 was utilized for all quantum mechanical
calculations [48].
4.2.1. Binding at rA1 and rA2A adenosine receptors assays
Procedures for preparation of rat brain membranes were
reported previously [41,42]. For binding experiments, mem-
brane membranes were frozen and stored at –20 °C
for ≤ 2 months. Adenosine deaminase (ADA) was from Boe-
hringer Mannheim (Indianapolis, IN). [3H]R-PIA was from
Amersham (Arlington Heights, IL), and [3H]CGS 21680 was
from DuPont NEN (Boston, MA).
Binding of [3H]R-PIA to A1 receptors from rat cortical
membranes and of [3H]CGS 21680 to A2A receptors for rat
striatal membranes was performed as described previously
[43]. ADA (2 units ml–1) was present during the preparation
of brain membranes. Additional deaminase was not added
during incubation with the radioligand.
4.2.4.1. Homology model of the hA3 AR. Based on the assump-
tion that GPCRs share similar TM boundaries and overall
topology, a homology model of the hA3 receptor was con-
structed. First, the amino acid sequences of TM helices of the
A3 receptor were aligned with those of bovine rhodopsin,
guided by the highly conserved amino acid residues, includ-
ing the DRY motif (D3.49, R3.50, and Y3.51) and three Pro
residues (P4.60, P6.50, and P7.50) in the TM segments of
GPCRs. The same boundaries were applied for the TM heli-
4.2.2. Radioligand binding to human A1, A2A and A3
adenosine receptors
[3H]DPCPX,30 125I] -ZM241385, and ([125I]-AB-MECA)
[
were utilized in radioligand binding assays to membranes pre-
pared from CHO cells expressing recombinant human A1,
A
2A, and A3 ARs, respectively, as previously described
[28,30]. ADA (3 units ml–1) was present during the prepara-

G. Pastorin et al. / Il Farmaco 60 (2005) 643–651
649
ces of the A3 receptor as were identified from the X-ray crys-
Acknowledgments
tal structure for the corresponding sequences of bovine
rhodopsin, the Ca coordinates of which were used to con-
struct the seven TM helices for the human A3 receptor. The
loop domains of the hA3 receptor were constructed by the
loop search method implemented in MOE. In particular, loops
are modeled first, in random order. For each loop, a contact
energy function analyzes the list of candidates collected in
the segment searching stage, taking into account all atoms
already modeled and any atoms specified by the user as
belonging to the model environment. These energies are then
used to make a Boltzmann-weighted choice from the candi-
dates, the coordinates of which are then copied to the model.
Any missing sidechain atoms are modeled using the same
procedure. Sidechains belonging to residues whose back-
bone coordinates were copied from a template is modeled
first, followed by side chains of modeled loops. Outgaps and
their sidechains are modeled last. Special caution had to be
given to the second extracellular (E2) loop, which has been
described in bovine rhodopsin to fold back over transmem-
brane helices, and, therefore, limits the size of the active site.
Hence, amino acids of this loop could be involved in direct
interactions with the ligands. A driving force to this peculiar
fold of the E2 loop might be the presence of a disulfide bridge
between cysteines in TM3 and E2. Since this covalent link is
conserved in all receptors modeled in the current study, the
E2 loop was modeled using a rhodopsin-like constrained
geometry around the E2-TM3 disulfide bridge.After the heavy
atoms were modeled, all hydrogen atoms were added, and
the protein coordinates were then minimized with MOE using
the AMBER94 force field [49]. The minimizations were car-
ried out by 1000 steps of steepest descent followed by con-
jugate gradient minimization until the rms gradient of the
potential energy was less than 0.1 kcal mol–1 Å.
We wish to thank the Regione FriuliVenezia Giulia (Fondo
2000) and the University of Trieste (fondo 60%) for financial
support. The molecular modeling work coordinated by S.
Moro has been carried out with financial support from Asso-
ciazione Italiana per la Ricerca sul Cancro (AIRC), Milan,
and the Italian Ministry for University and Research (MIUR),
Rome, Italy. S. Moro is also grateful to Chemical Computing
Group for their scientific and technical partnership. We wish
also to thank Dr. Ken A. Jacobson and Dr. Zhan-Guo Gao
(Bethesda) for binding data and helpful discussion.
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