Synthesis and biological studies of a new series of 5- heteroarylcarbamoylaminopyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines as human A3 adenosine receptor antagonists. Influence of the heteroaryl substituent on binding affinity and molecular modeling investigations

Article abstract of DOI:10.1021/jm051147+

Some pyrazolotriazolopyrimidines bearing different heteroarylcarbamoylamino moieties at the N5-position are described. We previously reported the synthesis of a water soluble compound with high potency and selectivity versus the human A₃ adenosine receptor as antagonist, and herein we present an enlarged series of compounds related to the previously mentioned one. These compounds showed A₃ adenosine receptor affinity in the nanomolar range and different levels of selectivity evaluated in radioligand binding assays at human A₁, A_2A, A_2B, and A₃ adenosine receptors. In particular, the effect of the heteroaryl substituents at the N5 position has been analyzed. This study allows us to recognize that the presence of a pyridinium moiety in this position not only increases water solubility but also improves or retains potency and selectivity at the human A₃ adenosine receptors. In contrast, replacement of pyridine with different heterocycles produces loss of affinity and selectivity at the human A₃ adenosine receptors. A molecular modeling study has been carried out with the aim to explain these various binding profiles.

Full text of DOI:10.1021/jm051147+

1720
J. Med. Chem. 2006, 49, 1720-1729
Synthesis and Biological Studies of a New Series of
5-Heteroarylcarbamoylaminopyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidines as Human A3
Adenosine Receptor Antagonists. Influence of the Heteroaryl Substituent on Binding Affinity
and Molecular Modeling Investigations
Giorgia Pastorin,† Tatiana Da Ros,† Chiara Bolcato,† Christian Montopoli,‡ Stefano Moro,*,‡ Barbara Cacciari,§
Pier Giovanni Baraldi,§ Katia Varani, Pier Andrea Borea, and Giampiero Spalluto*,†
Dipartimento di Scienze Farmaceutiche, UniVersita` degli Studi di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy, Dipartimento di Scienze
Farmaceutiche, UniVersita` degli Studi di Ferrara, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy, Molecular Modeling Section,
Dipartimento di Scienze Farmaceutiche, UniVersita` di PadoVa, Via Marzolo 5, I-35131 PadoVa, Italy, and Dipartimento di Medicina Clinica e
Sperimentale - Sezione di Farmacologia, UniVersita` degli Studi di Ferrara, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy
ReceiVed NoVember 15, 2005
Some pyrazolotriazolopyrimidines bearing different heteroarylcarbamoylamino moieties at the N5-position
are described. We previously reported the synthesis of a water soluble compound with high potency and
selectivity versus the human A3 adenosine receptor as antagonist, and herein we present an enlarged series
of compounds related to the previously mentioned one. These compounds showed A3 adenosine receptor
affinity in the nanomolar range and different levels of selectivity evaluated in radioligand binding assays at
human A1, A2A, A2B, and A3 adenosine receptors. In particular, the effect of the heteroaryl substituents at
the N5 position has been analyzed. This study allows us to recognize that the presence of a pyridinium
moiety in this position not only increases water solubility but also improves or retains potency and selectivity
at the human A3 adenosine receptors. In contrast, replacement of pyridine with different heterocycles produces
loss of affinity and selectivity at the human A3 adenosine receptors. A molecular modeling study has been
carried out with the aim to explain these various binding profiles.
Introduction
selective human A3 adenosine receptors ever reported (e.g.
compounds 1 and 2).20 (Chart 1) Unfortunately, a major problem
of these compounds is the typical low water solubility, which
has limited their use as pharmacological and diagnostic tools.
This aspect has been avoided with the synthesis, previously
reported in a preliminary form, of derivative 3 (5-[[(4-pyridyl)-
amino]carbonyl]amino-8-methyl-2-(2-furyl)-pyrazolo[4,3-e]-
1,2,4-triazolo[1,5-c]pyrimidine hydrochloride);22 this derivative
was highly water soluble (15 mM) and also displayed a
significantly increased potency and selectivity at the A3 ad-
enosine receptor with respect to the reference compounds 1 and
2 (Chart 1).
This exceptional potency has preliminarily been rationalized
by a molecular modeling investigation.22 Our pilot docking
studies have highlighted a strong electrostatic interaction among
the pyridinium moiety of 3 and two carbonyl backbone moieties
of the TM region of human A3 adenosine receptor.22 These
electrostatic interactions might be responsible for the increase
of the affinity in the protonated form, i.e., the hydrochloride
derivative 3.
To strengthen our preliminary modeling studies, we synthe-
sized an enlarged series of N8-methyl pyrazolo-triazolo-pyrim-
idines bearing different heteroarylcarbamoyl moieties (4-20)
at the 5-position. In particular: (i) the position of the nitrogen
on pyridine nucleus was modified, (ii) the basicity of pyridine
nitrogen was deleted by formation of N-oxide or N-methyl
derivatives, (iii) the pyridine moiety was replaced with different
heteroaryl groups (Chart 2).
Chemistry. The desired compounds (4, 5, 7, 15-20) were
prepared by reaction of the appropriate acyl azides 21-29 (2
equiv), obtained from commercially available acid and diphen-
ylphosphoryl azide (DPPA),23 and the well-known methyl
derivative 3019 in dry dioxane at reflux (18 h). Under these
Interaction of adenosine with its receptors, classified as A1,
A2A, A2B, and A3, could modulate several physiological func-
tions. These receptor subtypes belong to the family of the G
protein coupled receptors and exert their physiological role by
activation or inhibition of different second messenger systems.1,2
In recent years, intense efforts by medicinal chemists have
led to the discovery of several potent and selective adenosine
receptor agonists and antagonists, which permitted the phar-
macological characterization of this family of G-protein coupled
receptors. In particular A1, A2A, and A3 subtypes have been well
characterized through the use of potent and selective ligands.1,3
The A3 adenosine receptor subtype has been cloned from
different species (e.g. rat, human, dog, sheep),4-9 and it is found
to modulate two second messenger systems: inhibition of
adenylyl cyclase3 and stimulation of phospholipase C10 and D.11
The potential therapeutic applications of activating or antagoniz-
ing this receptor subtype have been investigated in recent years.
In particular, antagonists for the A3 receptor promise to be useful
for the treatment of inflammation12 and in the regulation of the
cell growth.13,14
In this field, many different classes of compounds have been
proposed, with both good affinity (nM range) and selectivity.15-17
In particular our group proposed a series of pyrazolo-triazolo-
pyrimidines18-22 which proved to be the most potent and
* To whom correspondence should be addressed. (G.P.) Phone: +39
040 5583726, fax: +39 040 52572, e-mail: spalluto@univ.trieste.it.
† Universita` degli Studi di Trieste.
§ Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di
Ferrara.
‡ Universita` di Padova.
Dipartimento di Medicina Clinica e Sperimentale - Sezione di Farma-
cologia, Universita` degli Studi di Ferrara.
10.1021/jm051147+ CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/04/2006

Adenosine Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1721
Chart 1. Structures and Binding Affinities at the Four Adenosine Receptor Subtypes of Reference Compounds
Chart 2. General Structures of Designed and Synthesized
Compounds
the synthesized compounds showed affinity in the low nano-
molar range for the human A3 adenosine receptors with different
degrees of selectivity versus the other receptor subtypes.
Pyridine derivatives 3-8 showed the best profile in terms of
both affinity and selectivity at the hA3 adenosine receptors, and
no significant differences emerged comparing the free bases (4,
5, 7) and the hydrochloride forms (3, 6, 8); nevertheless, the
salts present an exceptional water solubility (15 mM), which
renders these derivatives as ideal candidates for pharmacological
studies. On the other hand, it should be noted that the nitrogen
position on the pyridine nucleus significantly influences affinity
at the human A3 adenosine receptors with a consequent reduction
of selectivity versus the other receptor subtypes. In fact the best
affinity at the A3 receptors was obtained by using the 4-pyridyl
moiety (0.014-0.04 nM), while a significant reduction of
affinity (10-30-fold) was observed when nitrogen was at the
3- or 2-position. These results strongly suggested that the
electrostatic interactions hypothesized for derivative 322 were
not possible with the other isomers (5-8), and these compounds
showed an affinity versus the human A3 adenosine receptors
very similar to reference compound 1 (e.g. compound 5: Ki
hA3 0.44 nM vs compound 1: Ki hA3 0.16 nM).
condition, the acyl azide led to appropriate isocyanate by Curtius
rearrangement,24 which reacted with the amino function to give
the final compounds (Scheme 1).
The pyridine derivatives as hydrochlorides (3, 6, 8) were
prepared by treating the free base (4, 5, 7) for 30 min at 0 °C
with methanol saturated with HCl gas. The corresponding
N-methylpyridinium salt (9-11) and the N-oxide derivatives
(12-14) were obtained by treating the pyridine analogues (4,
5, 7) with methyl iodide or 3-chloroperbenzoic acid, respec-
tively, under standard conditions (Scheme 2).
A completely different biological profile was observed when
pyridine nitrogen was alkylated or oxidized. In the N-meth-
ylpyridinium series (9-11) a significant reduction of binding
affinity at the human A3 adenosine receptors (ranging from 1.3
to 28 nM) with respect to the pyridine analogues 3-8 was
obtained (100-2000-fold). Moreover the position of the N-
methylpyridinium moiety demonstrated a strong effect on
binding affinity, which seems to contradict the experimental
observations made for the pyridine analogues 3-8. In fact
derivative 9 was the least potent of this series (Ki hA3 28 nM)
while compound 11 showed a recovered potency at the hA3
adenosine receptors (Ki hA3 1.3 nM). These results suggested
that the presence of the methylpyridinium moiety had a negative
effect on binding affinity at the hA3 adenosine receptors.
Comparing binding affinities of reference compound 2 (Chart
1) to its bioisostere 9, a reduction of almost 100 fold in terms
of affinity at the hA3 adenosine receptors was observed
(compound 2: Ki hA3 0.31 nM vs compound 9: Ki hA3 28
nM). As we clarify later in the modeling discussion, we found
a reasonable interpretation of these data based on the ligand-
receptor complementarity analysis. Interestingly, it should be
also noted that an N-methylpyridinium salt at the 2- or
3-positions induces good potency on 100 nM NECA-stimulated
cAMP accumulation in CHO cells expressing hA2B receptors
(244-280 nM), while compounds 3-8 and the 4-N-methylpy-
ridinium derivative 9 did not bind this receptor subtype at
concentrations up to 1 µM.
The regioselective preparation of N8 methyl derivative 30
was novel. Up to now we reported the synthesis of N8-
substituted pyrazolo-triazolo-pyrimidines by standard procedure,
described by Gatta and co-workers,25 starting from the appropri-
ate alkylated 5-amino-4-cyanopyrazole.19 The alkylation per-
formed under typical conditions (NaH, in dry DMF) using a
small alkylating agent led to a 1:1 mixture of two regioisomers
with a consequent reduction of final yield. To overcome this
problem, we regioselectively prepared N2-methyl-4-cyano-5-
aminopyrazole 31 following a well-known procedure.26,27
Results and Discussion
In Table 1 the receptor binding affinities of the synthesized
compounds (3-20) are reported. They were determined at the
human A1, A2A, and A3 receptors expressed in CHO (A1, A2A
,
A3) cells; [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX)
(A1),28,29 [3H]-4-[2-[[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a]-
[1,3,5]triazin-5-yl]amino]ethyl]phenol ([3H]-ZM241385) (A2A),30
and [3H]-5-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-
furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([3H]-
MRE3008-F20) (A3)29 have been used as radioligands in binding
assays.
To evaluate the potency at human A2B receptors, due to the
lack of a suitable radioligand, the activity of antagonists was
determined in a functional assay based on the antagonism of
NECA (100 nM)-induced stimulation of cyclic AMP levels. All

1722 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Scheme 1. Synthesis of 4, 5, 7, 15-20a
Pastorin et al.
a Reagents and conditions: i: DMF, DPPA, Et3N, ii: dioxane reflux, 18 h.
Scheme 2. Synthesis of 3, 6, 8-14a
a Reagents and conditions: i: MeOH, HCl gas, 0 °C, 30 min; ii: MeCN, MeI, 40 °C, 5 h; iii: THF, mCPBA, rt, 12 h.
All these observations are strongly supported by the analysis
of binding data for the pyridinium N-oxide series (12-14),
which are exactly the same. In fact derivative 12 showed an
affinity for the hA3 adenosine receptors comparable with the
4-N-methylpyridinium derivative 9 (compound 12: Ki hA3 21
nM vs compound 9: Ki hA3 28 nM). Similarly, as mentioned
above for derivatives 9-11, the 2- and 3-pyridinium N-oxide
derivatives (13, 14) possess a better affinity for the hA3
adenosine receptor subtype with respect to the 4-pyridinium
derivative (13: Ki hA3 10 nM vs 10: Ki hA3 5.3 nM; 14: Ki
hA3 3.5 nM vs 11: Ki hA3 1.3 nM). Also in this series,
derivatives 13 and 14 proved to be quite potent versus the hA2B
adenosine receptor subtype.
induces a significant decrease of affinity of more than 100 fold
(e.g. compound 3: Ki hA3 0.046 nM vs compound 15: Ki hA3
16 nM). Most surprisingly, the replacement with thiophene (19,
20)31 produced a significant reduction of affinity not only with
respect to the pyridine derivative 3, but also with respect to
reference compound 2 (e.g. compound 2: Ki hA3 0.16 nM vs
compound 20: Ki hA3 3.5 nM).
If more complex heteroaryl systems such as benzofuran (17)
or quinoline (18) were introduced at the 5-position, a significant
decrease of affinity at the hA3 adenosine receptors was generally
observed, but while the introduction of a benzofuryl moiety (17)
retains a quite good affinity at the hA3 receptors (Ki hA3 12
nM) and also at the A2A (Ki hA2a 60 nM) and A2B (IC50 hA2B
250 nM) subtypes, the quinoline nucleus (18) induces a
significant decrease of affinity at all four adenosine receptor
subtypes.
A quite different biological profile was found when the
pyridine moiety was replaced with other heterocycles. Substitu-
tion with a furan ring, linked at the 2- (15) or 3-position (16),

Adenosine Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1723
Table 1. Structures and Binding Affinity at hA1, hA2A, hA3 and Potency at hA2B Adenosine Receptors of Synthesized Compounds (3-20)
a Displacement of specific [3H]-DPCPX binding at human A1 receptors expressed in CHO cells (n ) 3-6). b Displacement of specific [3H]-ZM241385
binding at human A2A receptors expressed in HEK-293 cells. c IC50 values of the inhibition of the tested compounds on 100 nM NECA-stimulated cAMP
accumulation in CHO cells expressing hA2B receptors. d Displacement of specific [3H]MRE3008-F20 binding at human A3 receptors expressed in HEK-293
cells. Data are expressed as means, with ( SEM. Data in parentheses are expressed as percentage of inhibition of specific binding at a concentration of 1
µM of the tested compounds. e Data taken from ref 22.
A new series of docking simulations have been carried out
to better rationalize the experimental binding affinities of all
newly synthesized N8-methylpyrazolo-triazolo-pyrimidines bear-
ing different heteroarylcarbomoyl moieties (4-20) at the
5-position.
Following our recently reported modeling approaches, we
used an improved model of the human A3 receptor, obtained
by a rhodopsin-based homology modeling approach, to recog-
nize the hypothetical binding motif of these new water soluble
human A3 antagonists.16,32,33 Considering docking simulations,
all new pyrazolo-triazolo-pyrimidine derivatives share the
common binding motif inside the TM region of human A3
receptor, as previously described.16,32,33 Before starting the
molecular modeling description of this new series of pyrazolo-
triazolo-pyrimidine derivatives, it is very interesting to remember
that derivative 3 undergoes to the acid-base equilibrium,
forming both protonated and unprotonated forms as a function
of the environment pH value. Of course, under buffer conditions

1724 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Pastorin et al.
Figure 1. The general topology of the human A3 receptor model. Reference compounds 3 (on the left) and 9 (on the right) are docked inside the
transmembrane recognition site (see Experimental Section for details) viewed from the membrane side facing TM helices 5 and 6. To clarify the
TM cavity viewing, TM6 has been voluntarily omitted. Side chains of some amino acids important for ligand recognition are highlighted.
both free base and hydrochloride give rise to the same
equilibrium. On the contrary, the experimental data seem to
indicate that the hydrochloride derivative presents a higher
affinity compared with the free base.16 We have already
speculated that the kinetic of the solubilization process might
play a critical role in determining the final concentration of both
compounds in solution. In this sense, hydrochloride solubilizes
faster than the corresponding free base and, consequently, can
guarantee a higher concentration of antagonist in solution.
Moreover, we have also found that the best docking energy for
the free base is around 20 kcal/mol less stabilizing compared
to the corresponding protonated form of derivative 3. We have
explained this result as a sort of receptor-driven selection of
the protonated versus the unprotonated form of the antagonist.
Indeed, other positively charged antagonists have been already
reported to be nicely accommodated into human A3 binding
region such as the water-soluble 3,5-diacyl-1,2,4-trialkylpyri-
dinium salts already reported by K. A. Jacobson et al. (J. Med.
Chem. 1999, 42, 4232). As we describe in detail later, from a
molecular point of view, we have found that the TM5 region
closed to Ile186 could mediate this stabilizing interaction.
As shown in Figure 1, we identified the hypothetical binding
site of the pyrazolo-triazolo-pyrimidine moiety surrounded by
TMs 3, 5, 6, and 7 with furan ring and the N8-substituents
pointing toward the EL2, and the carbamoyl moiety in the
5-position oriented toward the intracellular environment. The
furan ring is positioned between TM5 and TM3 whereas the
N8-substituents are surrounded by TM2 and TM7. Interestingly,
these pharmacophore feature models are nicely coherent with
our recently proposed receptor-based pharmacophore model.
Analyzing in detail our model, all pyrazolo-triazolo-pyrimidine
derivatives present the carbamoyl moiety in the 5-position
surrounded by two polar amino acids: His95 (TM3) and Ser247
(TM6). This region seems to be very critical for the recognition
of the antagonist structures. In fact, a major structural difference
between the hypothetical binding sites in these receptor subtypes
is that the A3 receptor does not contain the histidine residue in
TM6 common to all A1 (His251 in hA1) and A2 (His250 in
hA2A) receptors. This histidine has been shown to participate
in both agonist and antagonist binding to A2A receptors. In the
A3 receptor this histidine in TM6 is replaced by a serine residue
(Ser247 in hA3).34 The stabilizing interactions among the
carbamoyl moiety and these polar amino acids orient the
carbamoyl phenyl ring in the middle of the TM bundle. In
particular, the N-H of His95 (TM3) and the oxygen atom of
the carbamoyl group are separated by 2.6 Å and appropriately
oriented to form an H-bonding interaction. The side chain of
Ser247 (TM6) is within hydrogen-bonding distance of NH of
the carbamoyl group at 2.9 Å. According to the recently
published mutagenesis results, His95 is crucial for ligand
recognition, whereas Ser247 slightly affects the binding of both
agonists and antagonists.34 The receptor region around the aryl
or heteroaryl ring of the carbamoyl moiety is mostly hydro-
phobic and characterized by three nonpolar amino acids: Ile98
(TM3), Ile186 (TM5), Leu244 (TM6). However, a very strong
charge-dipole interaction between the carbonyl backbone moiety
of Ile186 (TM5) and the 4-pyridinium system of 3 seems to
strongly influence the binding affinity versus the human A3
receptor. The strength of this important charge-dipole interaction
is greatly reduced in intensity for both 3-pyridinium (6) and
2-pyridinium (8) analogues as a consequence of the increased
distance between the positive pyridinium charge of derivatives
3, 6, 8 and the carbonyl moiety of Ile186 (TM5), as shown in
Figures2-4. The evaluation of the ligand binding pocket of this
specific region of the A3 receptor reveals that a very limited
empty space is present between TM5 and TM6, and conse-
quently a severe steric control seems to take place around the
4-position of the aryl ring. As already demonstrated, substituents
at the 4-position of the N5-phenyl ring of pyrazolo-triazolo-
pyrimidine scaffold induced a decrease of affinity at the human
A3 adenosine receptors of about 2-5-fold with respect to the
nonsubstituted derivatives.20,22 Using this argument, we can
explain the drop of the activities of both p-N-methyl (9) and
p-N-oxide (12) derivatives compared with the analogue 3.
Moreover, also the nature of electrostatic interactions among
the carbonyl backbone moiety of Ile186 (TM5) and the
positively charged N-methyl (9) or the zwitterionic N-oxide (12)
groups are less effective than the positively charged 4-pyri-

Adenosine Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1725
Figure 2. Molecular docking of ortho (8), meta (6) and para (3) pyridinium compounds. Side chains of some amino acids important for ligand
recognition are highlighted.
Figure 3. Molecular docking of ortho (11), meta (10) and para (9) methylpyridinium compounds. Side chains of some amino acids important for
ligand recognition are highlighted.
dinium system of 3. In fact from a chemical point of view, the
carbonyl backbone moiety of Ile186 (TM5) interacts differently
with the positively charged N-H of derivative 3 in which a
hydrogen-mediated interaction is still possible with respect to
the positively charged N-Me of derivative 9 that cannot
establish a hydrogen-mediated interaction with the carbonyl
backbone moiety of Ile186, and finally, with the formally neutral
N+-O- moiety of derivative 12. Accordingly, bulkier but planar
substituents, such as benzofuran (17) or quinoline (18), also
slightly reduce the receptor complementarity. Moreover, smaller
heteroaromatic substituents such as methylthiophene (19 and
20) or furan (15 and 16) can be also accommodated into the
recognition pocket, partially loosening the receptor shape
complementarity (data not shown). However, it is useful to note
that all the above-mentioned derivatives (15-20) are still active
at a low nanomolar range as human A3 antagonists. Following
our previously published results, substituents at the ortho
position of the N5-phenyl ring of the pyrazolo-triazolo-pyrimi-
dine scaffold seem to occupy an empty region of the binding
receptor cavity.20
As already described, in the chlorine-substituted series the
change of the position (2- or 4-phenyl) does not seem to
influence affinity at the human A1, human A2A, and human A2B
receptors compared with the 3-chloro-substituted compounds,
maintaining it in the high nanomolar range (100-500 nM),
while significant differences appeared in binding affinity to the
human A3 adenosine receptors.20 Also with these newly
synthesized N-methyl and N-oxide derivatives, we observed a
similar chemical behavior. Indeed, both o-N-methyl (11) and
N-oxide (14) derivatives are more active than the corresponding

1726 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Pastorin et al.
Figure 4. Molecular docking of ortho (14), meta (13) and para (12) NO compounds. Side chains of some amino acids important for ligand
recognition are highlighted.
methanol. Flash chromatography was performed using Merck 60-
200 mesh silica gel. Elemental analyses were performed by the
microanalytical laboratory of Dipartimento di Chimica, University
of Trieste, and were within (0.4% of the theoretical values for C,
H, and N.
General Procedures for the Preparation of 5-Heteroarylcar-
bamoylamino-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines (4,
5, 7, 15-20). Amino compound 30 (0.5 g, 1.95 mmol) was
dissolved in freshly distilled dioxane (15 mL) and the freshly
prepared arylacyl azide (21-29) (5 eq) was added. The mixture
was refluxed under argon for 18 h. Then the solvent was removed
under reduced pressure and the residue was purified by flash
chromatography (EtOAc-Methanol 9:1 gradient EtOAc-Methanol
8:2) on silica gel pretreated with ammonia gas, to afford the desired
final compounds (4, 5, 7, 15-20) as a solid 365 in a quite good
yield.
p-N-methyl (9) and N-oxide (12) isomers (see Table 1 and
Figures 2-4).
Finally, a similar steric control takes place when substituents
larger than hydrogen are also present at the 3-position of the
aryl or heteroaryl ring. In this case, a steric repulsion among
substituents, at the 4-position, and amino acid side chains of
TM6 and TM7 could significantly reduce the affinity at the
human A3 receptor. Consistently, both m-N-methyl (10) and
N-oxide (13) derivatives are less active than the corresponding
o-N-methyl (11) and N-oxide (14) isomers (see Table 1 and
Figures 2-4).
Conclusions
The present study provides important information concerning
the structural requirements necessary for the recognition of the
human A3 adenosine receptor. In particular the effect of a
heteroaryl carbamoyl moiety at the N5 position has been strongly
investigated. It has been confirmed that a pyridinium moiety
increases water solubility and increases or retains affinity and
selectivity at the human A3 adenosine receptor subtype. In
particular the position of the nitrogen and its nature significantly
influence both affinity and selectivity. In contrast, when the
pyridinium moiety was replaced with other heterocycles, such
as furan or thiophene, a significant reduction of both affinity
and selectivity vs the hA3 adenosine receptors was observed.
All these experimental observations have been studied with
molecular modeling simulation, permitting validatation of the
recently proposed receptor-based pharmacophore model.16,32
5-[[(3-Pyridyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-
pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (5): yield 55%;
pale yellow solid; mp 183 °C (methanol-diethyl ether); IR (KBr):
1
3245-2965, 1666, 1620, 1512, 1453 cm-1; H NMR (DMSO-d6)
δ: 4.09 (s, 3H); 6.74 (dd, 1H, J ) 2, J ) 4); 7.23 (d, 1H, J ) 4);
7.36-7.41 (m, 1H); 7.91 (s, 1H); 7.94-8.01 (m, 1H); 8.37 (d, 1H,
J ) 2); 8.65-8.71 (m, 1H); 8.82 (s, 1H); 9.95 (bs, 1H); 10.60 (bs,
1H). ES-MS: (MH+) 376.5. Anal. (C17H13N9O2) C, H, N.
5-[[(2-Pyridyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-
pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (7): yield 64%;
light brown solid; mp 175 °C (methanol-diethyl ether); IR (KBr):
1
3245-2968, 1665, 1615, 1510, 1450 cm-1; H NMR (DMSO-d6)
δ: 4.10 (s, 3H); 6.73 (dd, 1H, J ) 2, J ) 4); 7.02-7.19 (m, 1H);
7.22 (d, 1H, J ) 4); 7.43-7.61 (m, 1H); 7.79-7.85 (m, 1H); 7.90-
8.01 (m, 1H); 8.24 (d, 1H, J ) 2); 8.72 (s, 1H); 8.95 (bs, 1H);
10.92 (bs, 1H). ES-MS: (MH+) 376.5. Anal. (C17H13N9O2) C, H,
N.
Experimental Section
5-[[(2-Furyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-pyra-
zolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (15): yield 45%; brown
solid; mp 182 °C (methanol-diethyl ether); IR (KBr): 3245-2970,
Chemistry. General. Reactions were routinely monitored by
thin-layer chromatography (TLC) on silica gel (precoated F254
Merck plates). Infrared spectra (IR) were measured on a Jasco FT-
1
1660, 1610, 1510, 1445 cm-1; H NMR (DMSO-d6) δ: 4.15 (s,
1
IT instrument. H NMR were determined in CDCl3 or DMSO-d6
3H); 6.73-6.85 (m, 2H); 7.02-7.19 (m, 2H); 7.79-7.85 (m, 1H);
7.90-8.01 (m, 1H); 8.24 (d, 1H, J ) 2); 8.74 (s, 1H); 8.97 (bs,
1H); 11.02 (bs, 1H). ES-MS: (MH+) 365.3. Anal. (C16H12N8O3)
C, H, N.
5-[[(3-Furyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-pyra-
zolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (16): yield 63%; brown
solid; mp 193 °C (methanol-diethyl ether); IR (KBr): 3240-2985,
solutions with a Varian Gemini 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.
Electrospray mass spectra were recorded on a Perkin-Elmer PE
SCIEX API 1 spectrometer, and compounds were dissolved in

Adenosine Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1727
1
1665, 1610, 1520, 1450 cm-1; H NMR (DMSO-d6) δ: 4.08 (s,
3H); 6.62-6.73 (m, 2H); 7.22-7.35 (m, 2H); 7.59-7.65 (m, 1H);
7.95 (s, 1H); 8.11 (s, 1H); 8.63 (bs, 1H); 10.89 (bs, 1H). ES-MS:
(MH+) 365.3. Anal. (C16H12N8O3) C, H, N.
(KBr): 3340-2750, 1665, 1615, 1610, 1505, 1450 cm-1; 1H NMR
(DMSO-d6) δ: 3.41 (s, 3H); 4.07 (s, 3H); 6.78 (dd, 1H, J ) 2, J
) 4); 7.21 (d, 1H, J ) 4); 7.99 (d, 2H, J ) 9); 8.01 (d, 1H, J )
2); 8.65 (d, 2H, J ) 9); 8.79 (s, 1H); 9.05 (bs, 1H); 11.18 (bs, 1H).
ES-MS: (MH+) 391.4. Anal. (C18H16N9O2I) C, H, N.
5-[[(Benzofuran-2-yl)amino]carbonyl]amino-8-methyl-2-(2-fu-
ryl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (17): yield
58%; yellow solid; mp 192 °C (methanol-diethyl ether); IR
3-[3-(2-Furan-2-yl-8-methy-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)-ureido]-N-methylpyridinium iodide (10):
yellow solid; mp 224 °C (methanol-diethyl ether); IR (KBr):
(KBr): 3242-2975, 1668, 1610, 1515, 1460 cm-1 1H NMR
;
(DMSO-d6) δ: 4.09 (s, 3H); 6.79 (dd, 1H, J ) 2, J ) 4); 7.05-
7.19 (m, 2H); 7.24 (d, 1H, J ) 4); 7.42-7.59 (m, 2H); 8.01 (d,
1H, J ) 2); 8.79 (s, 1H); 10.22 (bs, 1H); 11.59 (bs, 1H). ES-MS:
(MH+) 415.4. Anal. (C20H14N8O3) C, H, N.
3335-2760, 1668, 1615, 1605, 1515, 1465 cm-1 1H NMR
;
(DMSO-d6) δ: 3.43 (s, 3H); 4.05 (s, 3H); 6.68 (dd, 1H, J ) 2, J
) 4); 7.21 (d, 1H, J ) 4); 7.35-7.42 (m, 1H); 7.95 (s, 1H); 7.97-
8.03 (m, 1H); 8.21 (d, 1H, J ) 4); 8.61-8.75 (m, 1H); 8.79 (s,
1H). 9.99 (bs, 1H); 10.61 (bs, 1H). ES-MS: (MH+) 391.4. Anal.
(C18H16N9O2I) C, H, N.
5-[[(Quinolin-2-yl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-
pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (18): yield 60%;
brown solid; mp 190 °C (methanol-diethyl ether); IR (KBr):
2-[3-(2-Furan-2-yl-8-methy-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)-ureido]-N-methylpyridinium iodide (11):
yellow solid; mp 194 °C (methanol-diethyl ether); IR (KBr):
3240-2985, 1665, 1600, 1515, 1450 cm-1; H NMR (DMSO-d6)
1
δ: 4.07 (s, 3H); 6.78 (dd, 1H, J ) 2, J ) 4); 7.22 (d, 1H, J ) 4);
7.40-7.57 (m, 2H); 7.63-7.85 (m, 3H); 8.03 (d, 1H, J ) 2); 8.23-
8.31 (m, 1H); 8.67 (s, 1H); 10.21 (bs, 1H); 11.13 (bs, 1H). ES-
MS: (MH+) 426.4. Anal. (C21H15N9O2) C, H, N.
3350-2770, 1670, 1625, 1610, 1510, 1455 cm-1 1H NMR
;
(DMSO-d6) δ: 3.18 (s, 1H); 4.06 (s, 3H); 6.79 (dd, 1H, J ) 2, J
) 4); 7.05-7.19 (m, 1H); 7.25 (d, 1H, J ) 4); 7.59-7.91 (m, 2H);
8.04 (d, 1H, J ) 2); 8.21-8.25 (d, 1H, J ) 2); 8.79 (s, 1H); 8.90
(bs, 1H); 10.87 (bs, 1H). ES-MS: (MH+) 391.4. Anal. (C18H16N9O2I)
C, H, N.
5-[[(2-Methyl-thiophen-3-yl)amino]carbonyl]amino-8-methyl-
2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (19):
yield 70%; brown solid; mp 195 °C (methanol-diethyl ether); IR
(KBr): 3240-2975, 1670, 1605, 1510, 1455 cm-1 1H NMR
;
General Procedures for the Preparation of [3-(2-Furan-2-yl-
8-methy-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl)-
ureido]-pyridinium N-Oxide Derivatives (12-14). Pyrido de-
rivative (4, 5, 7) (50 mg, 0.13 mmol) was dissolved in dry THF
(10 mL), and an excess of mCPBA (3 equivalents) was added. The
reaction was stirred at room temperature for 12 h. The final products
precipitated from reaction mixture that was filtered off as a solid
in high yield.
4-[3-(2-Furan-2-yl-8-methy-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)-ureido]pyridinium N-oxide (12): yield
75%; pale yellow solid; mp 198 °C (methanol-diethyl ether); IR
(KBr): 3345-2745, 1668, 1615, 1610, 1505, 1450 cm-1; 1H NMR
(DMSO-d6) δ: 4.05 (s, 3H); 6.77 (dd, 1H, J ) 2, J ) 4); 7.20 (d,
1H, J ) 4); 7.59 (d, 2H, J ) 9); 7.99 (d, 1H, J ) 2); 8.21 (d, 2H,
J ) 9); 8.65 (s, 1H); 9.15 (bs, 1H); 10.98 (bs, 1H). ES-MS: (MH+)
392.3. Anal. (C17H13N9O3) C, H, N.
(DMSO-d6) δ: 2.27 (s, 3H); 4.05 (s, 3H); 6.34 (d, 1H, J ) 4);
6.62 (dd, 1H, J ) 2, J ) 4); 6.98 (d, 1H, J ) 4); 7.21 (d, 1H, J )
4); 7.78 (d, 1H, J ) 2); 8.21 (s, 1H); 9.65 (bs, 1H); 11.38 (bs, 1H).
ES-MS: (MH+) 395.4 Anal. (C17H14N9O2S) C, H, N.
5-[[(3-Methyl-thiophen-2-yl)amino]carbonyl]amino-8-methyl-
2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (20):
yield 55%; brown solid; mp 170 °C (methanol-diethyl ether); IR
(KBr): 3245-2965, 1667, 1615, 1513, 1445 cm-1 1H NMR
;
(DMSO-d6) δ: 2.39 (s, 3H); 4.05 (s, 3H); 6.62 (dd, 1H, J ) 2, J
) 4); 6.79 (d, 1H, J ) 4); 6.97 (d, 1H, J ) 4); 7.21 (d, 1H, J )
4); 7.82 (d, 1H, J ) 2); 8.19 (s, 1H); 8.65 (bs, 1H); 10.01 (bs, 1H).
ES-MS: (MH+) 395.4 Anal. (C17H14N9O2S) C, H, N.
General Procedure for the Preparation of 5-[[(Pyridyl)amino]-
carbonyl]amino-8-methyl-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-tria-
zolo[1,5-c]pyrimidine Hydrochloride (3, 6, 8). Pyridyl derivative
(4, 5, 7) (50 mg, 0.13 mmol) was dissolved in methanol (1 mL),
and a saturated solution of methanol with HCl gas (2 mL) was
added at 0 °C. The reaction was stirred at the same temperature
for 30 min, and then the solvent was removed under reduced
pressure to afford the correspondent salt as a solid in quantitative
yield.
3-[3-(2-Furan-2-yl-8-methy-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)-ureido]pyridinium N-oxide (13): yield
80%; pale yellow solid; mp 185 °C (methanol-diethyl ether); IR
(KBr): 3245-2975, 1665, 1620, 1520, 1450 cm-1 1H NMR
;
(DMSO-d6) δ: 4.04 (s, 3H); 6.78 (dd, 1H, J ) 2, J ) 4); 7.21 (d,
1H, J ) 4); 7.38-7.44 (m, 1H); 7.96 (s, 1H); 7.99-8.07 (m, 1H);
8.47 (d, 1H, J ) 2); 8.75-8.91 (m, 1H); 8.95 (s, 1H); 9.65 (bs,
1H); 10.73 (bs, 1H). ES-MS: (MH+) 392.3. Anal. (C17H13N9O3)
C, H, N.
5-[[(3-Pyridyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-
pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine hydrochloride
(6): white solid, mp 223-225 °C (dec) (methanol-diethyl ether);
IR (KBr): 3425-2900, 1665, 1625, 1610, 1520, 1455 cm-1; H
1
2-[3-(2-Furan-2-yl-8-methy-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)-ureido]pyridinium N-oxide (14): yield
78%; pale yellow solid; mp 192 °C (methanol-diethyl ether); IR
NMR (D2O) δ: 3.60 (s, 3H); 6.18 (dd, 1H, J ) 2, J ) 4); 6.57 (d,
1H, J ) 4); 7.20 (d, 2H, J ) 9); 7.61-7.78 (m, 1H); 7.80 (s, 1H);
7.95-8.01 (m, 1H); 8.21 (d, 1H, J ) 4); 8.62 (s, 1H). Anal.
(C17H14N9O2Cl) C, H, N.
(KBr): 3255-2975, 1668, 1610, 1511, 1465 cm-1 1H NMR
;
(DMSO-d6) δ: 4.06 (s, 3H); 6.68 (dd, 1H, J ) 2, J ) 4); 7.00-
7.15 (m, 1H); 7.20 (d, 1H, J ) 4); 7.36-7.41 (m, 1H); 7.75-7.82
(m, 1H); 7.97-8.04 (m, 1H); 8.27 (d, 1H, J ) 2); 8.79 (s, 1H);
10.95 (bs, 1H); 12.01 (bs, 1H). ES-MS: (MH+
5-[[(2-Pyridyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-
pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine hydrochloride
(8): white solid, mp 223-225 °C (dec) (methanol-diethyl ether);
) 392.3. Anal.
IR (KBr): 3425-2900, 1665, 1625, 1610, 1520, 1455 cm-1; H
(C17H13N9O3) C, H, N.
1
NMR (D2O) δ: 3.62 (s, 3H); 6.21 (dd, 1H, J ) 2, J ) 4); 6.55 (d,
1H, J ) 4); 7.18 (d, 2H, J ) 9); 7.31-7.45 (m, 1H); 7.58 (s, 1H);
7.75-7.93 (m, 1H); 8.23 (s, 1H). Anal. (C17H14N9O2Cl) C, H, N.
General Procedures for the Preparation of [3-(2-Furan-2-yl-
8-methy-8H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-yl)-
ureido]-N-methylpyridinium Iodide Derivatives (9-11). Pyrido
derivative (4, 5, 7) (50 mg, 0.13 mmol) was dissolved in acetonitrile
(10 mL), and a slight excess of methyl iodide (2 equivalents) was
added. The reaction was stirred at 40 °C for 5 h. Then the solvent
was removed under reduced pressure to afford the corresponding
salt as a solid in quantitative yield.
Biology. CHO Membrane Preparation. The expression of the
human A1, A2A, and A3 receptors in CHO cells has been previously
described.35 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. Cells were split
two or three times weekly, and then the culture medium was
removed for membrane preparations. The cells were washed with
phosphate-buffered saline solution and scraped 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
homogenate was centrifuged for 30 min at 48 000g. The membrane
pellet was resuspended in 50 mM Tris HCl buffer at pH 7.4 for A1
adenosine receptors, in 50 mM Tris HCl, 10 mM MgCl2 at pH 7.4
for A2A adenosine receptors, in 50 mM Tris HCl, 10 mM MgCl2,
4-[3-(2-Furan-2-yl-8-methy-8H-pyrazolo[4,3-e][1,2,4]triazolo-
[1,5-c]pyrimidin-5-yl)-ureido]-N-methylpyridinium iodide (9):
yellow solid; mp 221 °C (dec) (methanol-diethyl ether); IR

1728 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Pastorin et al.
1 mM EDTA at pH 7.4 for A3 adenosine receptors, and were utilized
for the binding assay.
TM helices of the resting state A3 receptor were aligned with those
of bovine rhodopsin, guided by the highly conserved amino acid
residues, including 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 helices
of the A3 receptor as were identified from the X-ray crystal structure
for the corresponding sequences of bovine rhodopsin,40 the CR
coordinates which were used to construct the seven TM helices
for the human A3 receptor. The loop domains of the human A3
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 atom 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 side chain atoms are modeled using the same procedure.
Side chains belonging to residues, whose backbone coordinates were
copied from a template, are modeled first, followed by side chains
of modeled loops. Outgaps and their side chains 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 transmembrane helices,40 and, therefore, it limits the size of
the active site. Hence, amino acids of this loop could be involved
in direct interactions with the ligands. The presence of a disulfide
bridge between cysteines in TM3 and E2 might be the driving force
to this peculiar fold of the E2 loop. 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 coordi-
nates were minimized with MOE using the AMBER94 force field.41
The minimizations were carried out by 1000 steps of steepest
descent followed by conjugate gradient minimization until the rms
Human Cloned A1, A2A, and A3 Adenosine Receptor Binding
Assay. Binding of [3H]-DPCPX to CHO cells transfected with the
human recombinant A1 adenosine receptor was performed as
previously described.28 Displacement experiments were performed
for 120 min at 25 °C in 0.20 mL of buffer containing 1 nM [3H]-
DPCPX, 20 mL of diluted membranes (50 mg of protein/assay)
and at least 6-8 different concentrations of examined compounds.
Nonspecific binding was determined in the presence of 10 mM of
CHA, and this is always e10% of the total binding. Binding of
[3H]-ZM241385 to CHO cells transfected with the human recom-
binant A2A adenosine receptors (50 mg of protein/assay) was
performed according to Ongini et al.30 In competition studies, at
least 6-8 different concentrations of compounds were used and
nonspecific binding was determined in the presence of 50 mM
NECA for an incubation time of 60 min at 25 °C.
Binding of [3H]MRE3008-F20 to CHO cells transfected with
the human recombinant A3 adenosine receptors was performed
according to Varani et al.29 Competition experiments were carried
out in duplicate in a final volume of 250 mL in test tubes containing
1 nM [3H] MRE3008-F20, 50 mM Tris HCl buffer, 10 mM MgCl2,
pH 7.4, and 100 mL of diluted membranes (50 mg protein/assay),
and at least 6-8 different concentrations of examined ligands.
Incubation time was 120 min at 4 °C, according to the results of
previous time-course experiments.29 Nonspecific binding was
defined as binding in the presence of 1 mM of MRE3008-F20 and
was about 25% of total binding. Bound and free radioactivities were
separated by filtering the assay mixture through Whatman GF/B
glass-fiber filters using a Micro-Mate 196 cell harvester (Packard
Instrument Company). The filter bound radioactivity was counted
on Top Count (efficiency 57%) with Micro-Scint 20. The protein
concentration was determined according to the Bio-Rad method36
with bovine albumin as reference standard.
Measurement of Cyclic AMP Levels in CHO Cells Trans-
fected with Human A2B Adenosine Receptors. CHO cells trans-
fected with human A2B adenosine receptors were washed with
phosphate-buffered saline and diluted trypsin and centrifuged for
10 min at 200g. The pellet containing the CHO cells (1 × 106 cells/
assay) was suspended in 0.5 mL of incubation mixture (mM): NaCl
15, KCl 0.27, NaH2PO4 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 studied
antagonists was determined by antagonism of NECA (100 nM)-
induced stimulation 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, aminophylline 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.
gradient of the potential energy was less than 0.1 kcal mol-1 Å-1
.
Molecular Docking of the Human A3 Receptor Antagonists.
All antagonist structures were docked into the hypothetical TM
binding site by using the MULTIDOCK docking program, which
is part of the MOE suite. Conformational samplings were conducted
within a user-specified 3D docking box, using the Tabu` Search
protocol42 and MMFF94 force field.44 MOE-Dock performs a user-
specified number of independent docking runs (50 in our specific
case) and wrote the resulting conformations and their energies in a
molecular database file. The resulting docked complexes were
subjected to MMFF94 energy minimization until the rms of the
conjugate gradient was <0.1 kcal mol-1 × Å -1. Charges for the
ligands were imported directly from the MMFF94 force field.
The interaction energy values were calculated as follows:
∆Ebinding ) Ecomplex - (Eligand + Ereceptor). These energies are not
rigorous thermodynamic quantities, but can only be used to compare
the relative stabilities of the complexes. Therefore, these interaction
energy values cannot be used to calculate binding affinities since
changes in entropy and solvation effects are not taken into account.
Acknowledgment. We thank the Regione Friuli Venezia
Giulia (Fondo 2000) for financial support. The molecular
modeling work coordinated by S. Moro has been carried out
with financial support of the University of Padova, Italy, and
the Italian Ministry for University and Research (MIUR), Rome,
Italy. S. Moro is also very grateful to Chemical Computing
Group for the scientific and technical partnership.
Computational Methodologies. All molecular modeling studies
were carried out on a 10 CPU (AMD 64Athon and PIV 2.0-30
GHz) Linux cluster running under openMosix architecture.37
Homology modeling, energy calculation, and docking studies have
been done using Molecular Operating Environment (MOE, version
2004.03) suite.38 Quantum calculations used throughout this study
were performed using MOPAC (version 7.0).39
Homology Model of the Resting State of Human A3 Receptor.
On the basis of the assumption that GPCRs share similar TM
boundaries and overall topology, a homology model of the human
A3 receptor was constructed. First, the amino acid sequences of
Supporting Information Available: Elemental analysis table;
figure showing the best docked conformation of the thiophene
derivative. This material is available free of charge via the Internet
at http://pubs.acs.org
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