Scouting human A3 adenosine receptor antagonist binding mode using a molecular simplification approach: From triazoloquinoxaline to a pyrimidine skeleton as a key study

Article abstract of DOI:10.1021/jm070852a

The concept of molecular simplification as a drug design strategy to shorten synthetic routes, while keeping or enhancing the biological activity of the lead drug, has been applied to design new classes of human A₃ adenosine receptor (AR) antag

Full text of DOI:10.1021/jm070852a

6596
J. Med. Chem. 2007, 50, 6596–6606
Scouting Human A3 Adenosine Receptor Antagonist Binding Mode Using a Molecular
Simplification Approach: From Triazoloquinoxaline to a Pyrimidine Skeleton as a Key Study
Erika Morizzo,† Francesca Capelli,‡ Ombretta Lenzi,‡ Daniela Catarzi,‡ Flavia Varano,‡ Guido Filacchioni,‡ Fabrizio Vincenzi,§
Katia Varani,§ Pier Andrea Borea,§ Vittoria Colotta,*,‡ and Stefano Moro*,†
Molecular Modeling Section, Dipartimento di Scienze Farmaceutiche, UniVersità di PadoVa, Via Marzolo 5, PadoVa, Italy, Dipartimento di
Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente AttiVi, UniVersità di Firenze, Polo
Scientifico, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy, and Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia,
UniVersità di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara
ReceiVed July 13, 2007
The concept of molecular simplification as a drug design strategy to shorten synthetic routes, while keeping
or enhancing the biological activity of the lead drug, has been applied to design new classes of human A3
adenosine receptor (AR) antagonists. Over the past decade, we have focused a part of our research on the
study of AR antagonists belonging to strictly correlated classes of tricyclic compounds. One of these classes
is represented by the 2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives, either 4-amino or 4-oxo-
substituted, which were intensively investigated by evaluating the effect of different substituents on the
2-phenyl ring and on the 4-amino group. Using an in silico molecular simplification approach, a new series
of easily synthesizable 2-amino/2-oxoquinazoline-4-carboxamido derivatives have been discovered, presenting
high affinity and selectivity against human A3 AR.
Chart 1. Previously Reported
2-Aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-ones (TQX Series)
Introduction
The A3 adenosine receptor (ARa) is one of the four G-protein-
coupled receptors (GPCRs) activated by adenosine;1 it is
expressed in a broad spectrum of tissues and couples to Gi/o
proteins.1,2 The ligand-stimulated A3 AR leads to the elevation
of intracellular Ca2+ concentration ([Ca2+]i) and the activation
of phosphoinositide 3-kinase (PI3K)γ.3,4 The activated PI3Kγ
leads to the phosphorylation of protein kinase B (PKB) and
members of the mitogen-activated protein kinase (MAPK)
family, including extracellular signal-regulated kinase (ERK)
1/2 and p38.3–5 This A3 AR-stimulated PI3Kγ-dependent
signaling pathway is essential for the potentiation of IgE/antigen-
dependent mast cell degranulation6 and the A3 AR internaliza-
tion.7
treatment of stroke and glaucoma and also as antiasthmatic and
antiallergic drugs.2,9,10
In particular, the development of antagonists for the A3 ARs
has so far been directed by traditional medicinal chemistry.11
However, in the past few years we optimized a multidisciplinary
integrated approach to speed up the discovery and the structural
refinement of new potent and selective A3 AR antagonists.
Adenosine receptor antagonists, including A3 AR-selective
compounds, have been extensively reviewed in previous
articles.12–17
Over the past decade, we have focused a part of our research
on the study of AR antagonists belonging to strictly correlated
classes of tricyclic compounds.18–24 One of these classes is
represented by the 2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one
derivatives (TQX series), either 4-amino or 4-oxo-substituted,
which were intensively investigated by evaluating the effect of
different substituents on the 2-phenyl ring and on the 4-amino
group (Chart 1).18–21,24 These studies led to the identification
of some groups which, introduced one by one in a suitable
position of the parent compounds 4-amino-2-phenyl-1,2,4-
triazolo[4,5-a]quinoxalin-1-one A and 2-phenyl-1,2,4-triazolo[4,5-
a]quinoxalin-1,4-dione B, afforded high human (h) A3 AR
affinity and good selectivity.
The A3 AR is implicated in a variety of important physi-
ological processes.2 Activation of A3 ARs increases the release
of inflammatory mediators, such as histamine, from rodent mast
cells8 and inhibits the production of tumor necrosis factor-R.9
The activation of the A3 ARs is also suggested to be involved
in immunosuppression and in the protection from ischemia of
the brain and heart.10 It is becoming increasingly apparent that
agonists of A3 ARs have potential as therapeutic agents for the
treatment of rheumatoid arthritis and colorectal cancer, whereas
antagonists might be therapeutically useful for the acute
* To whom correspondence should be addressed. Tel.: +39 049
8275704(S.M.); +39 55 4573731(V.C.). Fax: +39 049 827 5366 (S.M.);
+39554573780(V.C.).E-mail:stefano.moro@unipd.it(S.M.);vittoria.colotta@unifi.it
(V.C.).
†
Università di Padova.
Università di Firenze.
Università di Ferrara.
‡
§
aAbbreviations: AR, adenosine receptor; h, human; TM, transmembrane;
EL2, second extracellular loop; SAR, structure-affinity relationship;
DPCPX, 8-cyclopentyl-1,3-dipropyl-xanthine; I-AB-MECA, N6-(4-amino-
3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine; NECA, 5′-(N-ethyl-car-
boxamido)adenosine; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)-5′-(N-
methylcarbamoyl)adenosine; GPCR, G-protein-coupled receptor; PI3Kγ,
phosphoinositide 3-kinase γ; MAPK, mitogen-activated protein kinase; PKB,
protein kinase B; ERK, extracellular signal-regulated kinase.
These groups are the para-methoxy substituent on the
2-phenyl ring (compounds C and D)18,24 and acyl residues, such
as the acetyl or benzoyl groups, on the 4-amino group
(compounds E and F).18,24 However, besides potency and
10.1021/jm070852a CCC: $37.00
2007 American Chemical Society
Published on Web 11/30/2007

Human A3 Adenosine Receptor Antagonist Binding
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6597
Chart 2. Hereby Reported 1,2,4-Triazoloquinoxalin-1-one
Simplified Analogues
structures. In fact, a major structural difference between the
hypothetical binding sites in these receptor subtypes is that the
hA3 receptor does not contain the histidine residue in TM6
(6.52), common to all A1 (His251 in hA1) and A2 (His250 in
hA2A and His251 in hA2B) 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).1,2
Starting from these binding requirements, we decided to
perform an in silico molecular simplification approach to identify
a suitable fragmentation route of the 4-amino-triazoloquinoxalin-
1-one scaffold and explore which of the structural features were
essential to guarantee an efficient ligand–receptor recognition.
A schematic representation of our molecular simplification is
shown in Figure 2.
selectivity, the straightforward synthesis and pharmacokinetic
profile represent crucial requirements in developing new possible
therapeutic agents. Structural simplification represents a drug
design strategy to shorten synthetic routes while keeping or
enhancing the biological activity of the original candidate.
Following this strategy, we have carried out an in silico
molecular simplification approach to identify a suitable frag-
mentation route and explore which of the structural features
are essential to guarantee an efficient ligand–receptor recogni-
tion. In this context, three series of triazoloquinoxalin-1-one
analogues were prepared (Chart 2) and, among them, the easily
synthesizable 2-amino/2-oxoquinazoline-4-carboxamido deriva-
tives 1–11 (QZ series) proved to be highly potent and selective
against the hA3 AR.
The first step was to verify the effects of the 4-amino-
triazoloquinoxalin-1-one (TQX series) replacement with the
2-amino-quinazoline scaffold bearing a CO-NH-C6H4-R1 moiety
at the 4-position (QZ series). Interestingly, the formation of an
intramolecular H-bond between the nitrogen at the 3-position
of the quinazoline system and the NH of the amide moiety at
the 4-position simulates the presence of a planar tricycle with
similar steric properties with respect to the original triazolo-
quinoxalinone analog. Quantum chemistry calculations support
the crucial role of the intramolecular H-bond in stabilizing the
cyclic conformer (see Supporting Information for more details).
Indeed, it is worth noting that a different entropy contribution,
between the TQX and the QZ series, could differently affect
the total free energy of binding. Unfortunately, in our docking
simulations, the entropy effect could not accurately be taken
into account. In particular, using the 4-amino-2-(4-methoxyphe-
nyl)-triazoloquinoxalin-1-one derivative C (Chart 1) as primary
reference compound, the corresponding 2-aminoquinazoline-4-
carboxyamide derivative 1 (Chart 2, R ) C6H4-p-OMe) was
investigated. As shown in Figure 3, molecular docking simula-
tions confirm that the new compound 1 is efficiently accom-
modated in the TM binding cavity, maintaining all crucial
interactions above-mentioned (π-π stacking interactions at least
with both side chains of Phe168 (EL2) and Phe182 (5.43), two
H-bonds with Gln167 (EL2) and Asn250 (6.55), and a H-bond
interaction with His95 (3.37). In particular, His95 (3.37) is
involved in a H-bond interaction with the amino group at the
2-position of the quinazoline-4-carboxyamide moiety. Analo-
gously, we decided to extend our investigation, also considering
the corresponding 2-oxo analogue of 1, that is, the 2-oxo-
quinazoline-4-carboxyamide derivative 6 (Chart 2, R ) C6H4-
p-OMe), which can also be considered the simplified analogue
of the triazoloquinoxalin-1,4-dione derivative D (Chart 1). As
is clearly shown in Figure 3, the 2-oxo derivative 6 assumes a
binding conformation very similar to that of the 2-amino-
quinazoline derivative 1. In compound 6, the 2-oxo group
interacts through a H-bond interaction with His95 (3.37).
Results and Discussion
In Silico Design of New 2-Aryl-1,2,4-triazolo[4,3-a]quinoxa-
lin-1-one Simplified Analogues. As previously reported, 4-amino-
2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives nicely
bind to hA3 AR.18–24 We recognized the hypothetical binding
site of the triazolo-quinoxalinone moiety surrounded by trans-
membrane (TM) regions 3, 5, 6, and 7, with the carbonyl group
at the 1-position pointing toward the second extracellular loop
(EL2) and the amide moiety in the 4-position oriented toward
the intracellular environment.21,24 The phenyl ring at the
2-position is positioned close to TM3, TM6, and TM7. The
asymmetric topology of the binding cavity is characterized by
a major axis (measured from TM1 toward TM5) of about 17 Å
and by a minor axis (measured from TM3 toward TM6) of about
6 Å, as shown in Figure 1. The peculiar geometric properties
of the hA3 AR binding pocket effortlessly rationalize the
experimental evidence that planar polyaromatic systems are
usually suitable scaffolds to design potent and selective hA3
AR antagonists. Moreover, planar polyaromatic systems seem
to interact through π-π stacking interactions at least with one
of the two side chains of Phe168 (EL2) and Phe182 (5.43), as
shown in Figure 1. This interaction has already been described
as a crucial pharmacophoric feature in the hA3 AR recognition.
All triazolo-quinoxalinone derivatives also share at least two
stabilizing hydrogen-bonding interactions inside the binding cleft
(Figure 1). The first hydrogen bonding is between the carbonyl
group at the 1-position, which points toward the EL2, and the
NH of the Gln167. This hydrogen-bonding distance is calculated
around 2.7 Å for all docked compounds. Moreover, the
1-carbonyl group is also at the hydrogen-bonding distance with
the amide moiety of Asn250 (6.55) side chain. This asparagine
residue, conserved among all adenosine receptor subtypes, was
found to be important for ligand binding. Second, the NH2 or
NHR moiety at the 4-position is surrounded by three polar amino
acids: Thr94 (3.36), His95 (3.37), and Ser247 (6.52). This region
seems to be very critical for the recognition of all antagonist
Subsequently, docking studies were also carried out to
evaluate whether the presence of acyl residues on the 2-amino
group of the new quinazoline-4-carboxamido series (Figure 2,
QZ series, R2 ) acyl) was tolerated. The docking simulations,
performed on the 2-acetylaminoquinazoline-4-carboxyanilide 10
(Chart 2, R ) Ph) showed that the acetyl substituent is not only
well tolerated, but it might reinforce the binding to the hA3 AR
(Figure 3). Indeed, consistently with that observed in the
triazoloquinoxaline series,24 an additional H-bond interaction
takes place between the carbonyl moiety of the 2-acylamino
group and the side chain of Ser247 (6.52).

6598 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Morizzo et al.
Figure 1. Hypothetical TM binding cleft topology of the human A3 receptor model obtained by the conventional rhodopsin-based homology
modeling approach (on the left); and hypothetical binding motif of the reference derivative C (on the right). The most energetically favorable
docked conformation is viewed from the membrane side facing TM helices 5, 6, and 7. To clarify the TM cavity, the view of TM6 from Pro245
to Cys251, was voluntarily omitted. Side chains of some amino acids, important for ligand recognition, are highlighted. Hydrogen atoms are not
displayed.
tions (in particular, the two H-bonds with Gln167 and
Asn250)anddrasticallyreducedtheircavityshapecomplementarity.
Finally, to explore how reducible was the extension of the
planar aromatic ring, starting from a 2-aminoquinazoline scaf-
fold, we designed the corresponding 2-aminopyrimidines bearing
a CO-NHC6H4-R1 moiety at the 4-position (Figure 2, PYRM
series). As above-described for the quinazoline derivatives, also
in this series, we can observe the formation of the intramolecular
H-bond between the 3-nitrogen atom of the pyrimidine system
and the NH of the 4-amide moiety, which allows a simulation
of the presence of a planar bicycle with a missing benzene ring,
with respect to the original triazolo-quinoxalinone analogs.
As shown in Figure 5 for the 2-aminopyrimidine-4-carboxy-
(4-methoxyphenyl)amide 16 (Chart 2), molecular docking
simulations indicate that 2-amino-pyrimidine skeleton main-
tains the stabilizing π-π stacking interactions with both
Phe168 and Phe182. However, the shift of the ligand position
into the binding cleft abolishes the possibility an interaction
through the H-bond with His95, Gln167, and Asn250,
reducing the stability of the corresponding antagonist/receptor
Figure 2. Flowchart of our in silico simplification approach.
complex.
From these theoretical hypotheses, we synthesized and
To demonstrate the important role of the intramolecular
H-bond interaction in maintaining the coplanarity of both
2-amino- and 2-oxo-quinazoline scaffolds and the CO-NHC6H4-
R1 moiety at the 4-position, we decided to design a new class
of analogs: the 2-aminoquinoline-4-carboxamides (Figure 2, QN
series) and the corresponding 2-oxo derivatives. In fact, in these
quinoline derivatives, the formation of the intramolecular
H-bond is not allowed and, consequently, the CO-NHC6H4-R1
is twisted with respect to the quinoline ring of about 135°, as
suggested by the systematic conformational analysis of the
corresponding dihedral angle (data not shown). The impossibility
of both 2-amino- and 2-oxo-quinoline systems to adopt a planar
conformation is also confirmed by the docking simulations. In
fact, as shown in Figure 4 for the 2-aminoquinoline-4-carboxya-
mide derivative 12 (Chart 2, R ) C6H4-p-OMe) and its 2-oxo
analogue 14 (Chart 2, R ) C6H4-p-OMe), the corresponding
energetically more stable docking pose is still twisted (of about
121°) and, in this conformation, the 2-amino-quinoline deriva-
tives completely missed some of the most important interac-
pharmacologically characterized some derivatives belonging to
the three designed classes of triazolo-quinoxalinone simplified
analogs (see Chart 2, Tables 1 and 2), that is, the 2-amino/2-
oxoquinazoline-4-carboxamides 1–11 (QZ series), the 2-amino/
2-oxoquinoline-4-carboxamides 12–15 (QN series), and the
2-aminopyrimidine-4-carboxyamides 16–18 (PYRM series).
Among these compounds, there are the above cited and
theoretically investigated quinazolines 1, 6, and 10, quinolines
12 and 14, and pyrimidines 16, all except one (10) bearing the
4-carboxy-(4-methoxyphenyl)amide function. To perform a
preliminary structure-affinity relationship (SAR) study, in the
first two series, we synthesized derivatives lacking the methoxy
group on the 4-carboxyamide moiety, that is, the 4-carboxya-
nilide compounds 2, 7, 13, and 15. In the quinazoline series,
the methoxy group was also replaced by lipophilic substituents,
such as methyl (compounds 3 and 8) or bromine (compounds
4 and 9). In addition, to evaluate the importance of the aromatic

Human A3 Adenosine Receptor Antagonist Binding
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6599
Figure 3. Hypothetical binding motif of the newly synthesized A3 antagonists: 1 (top on the left), 6 (top on the right), and 10 (bottom). The most
energetically favorable docked conformations are viewed from the membrane side facing TM helices 5, 6, and 7. To clarify the TM cavity, the view
of TM6 from Pro245 to Cys251 was voluntarily omitted. Side chains of some amino acids, important for ligand recognition, are highlighted.
Hydrogen atoms are not displayed.
Figure 4. Hypothetical binding motif of the newly synthesized analogs 12 (on the left) and 14 (on the right). The most energetically favorable
docked conformations are viewed from the membrane side facing TM helices 5, 6, and 7. To clarify the TM cavity, the view of TM6 from Pro245
to Cys251 was voluntarily omitted. Side chains of some amino acids important for ligand recognition are highlighted. Hydrogen atoms are not
displayed.
phenyl ring on the carboxyamide function, the 2-aminoquinazo-
line-4-carboxy-cyclohexylamide 5 was synthesized. The effect
of a benzoyl residue on the 2-amino function was evaluated
both in the quinazoline (compound 11) and in the pyrimidine
(compound 17) series, and in the latter, the 2-dibenzoylamino
derivative 18 was also prepared.
synthesis of the 2-amino-quinazoline-4-carboxamido derivatives
1–5 and the 2-oxo-quinazoline-4-carboxamido derivatives 6–9,
which were prepared by coupling the corresponding 2-amino-
and 2-oxo-quinazoline-4-carboxylic acids25 with the suitable
amines 19–23 (Scheme 1). By reaction of the 2-amino-
quinazoline-4-carboxyanilide 2 with acetyl and benzoyl chloride,
the 2-amido-quinazoline-4-carboxyamides 10 and 11 were
obtained, respectively. The 2-aminoquinoline-4-carboxamides
12 and 13 (Scheme 2) were synthesized starting from the
2-chloroquinoline-4-carboxamides 24 and 25,26,27 which were
reacted with hydrazine monohydrate under microwave irradia-
tion to yield the corresponding 2-hydrazinoquinoline-4-car-
Chemistry
The quinazoline-4-carboxamido derivatives 1–11, their 3-dea-
za analogues quinoline-4-carboxamides 12–15, and the pyri-
midine-4-carboxamides 16–18 were obtained as depicted in
Schemes 1 and 2. Scheme 1 showed the straightforward

6600 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Morizzo et al.
Figure 5. Hypothetical binding motif of the newly synthesized analog 16. The most energetically favorable docked conformation is viewed from
the membrane side facing TM helices 5, 6, and 7. To clarify the TM cavity, the view of TM6 from Pro245 to Cys251 was voluntarily omitted. Side
chains of some amino acids important for ligand recognition are highlighted. Hydrogen atoms are not displayed.
Scheme 1a
Scheme 2a
a Reagents: (a) R-NH2, 1-hydroxybenzotriazole, NEt3, 4-(dimethyl-
amino)pyridine, 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydro-
chloride, DMF; (b) R-COCl, anhydrous pyridine, methylene chloride.
a Reagents: (a) hydrazine monohydrate, EtOH, microwave irradiation;
(b) H2, Ni/Raney, EtOH; (c) thionyl chloride; (d) aniline derivative, Et3N,
anhydrous methylene chloride; (e) 4-methoxyaniline, 1-hydroxybenzotria-
zole, NEt3, 4-(dimethylamino)pyridine, 1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride, DMF; (f) PhCOCl, anhydrous pyridine,
methylene chloride.
boxyamides 26 and 27.26,27 Hydrazinolysis of compounds 26
and 27 afforded the desired 2-aminoquinoline derivatives 12
and 13. Synthesis of 2-oxo-1,2-dihydroquinoline-4-carboxamides
14 and 15 was achieved by reacting suitable aniline derivatives
with the 2-oxo-1,2-dihydroquinoline-4-carbonyl chloride 28,
obtained from the corresponding 2-oxo-1,2-dihydroquinoline-
4-carboxylic acid28 in boiling thionyl chloride. The 2-amino-
pyrimidine-4-carboxamido compounds 16–18 were prepared
starting from the 2-aminopyrimidine-4-carboxylic acid,29 which
was reacted with 4-methoxyaniline to give the 2-aminopyrimi-
dine-4-carboxamide 16. Reaction of 16 with benzoyl chloride
gave a mixture of the 2-benzoylamino- and 2-dibenzoylamino-

Human A3 Adenosine Receptor Antagonist Binding
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6601
Table 1. Binding Affinity (Ki) at hA1, hA2A, and hA3 ARs and Potency
(IC50) at hA2B and A3 ARs
on our in silico simplification approach. In fact, the new
quinazoline derivatives 1–11 are potent antagonists at the hA3
AR and are also highly selective, being completely inactive at
the other AR subtypes (Table 1). The antagonist properties at
the hA3 AR of this series of simplified triazoloquinoxalinone
analogues were assessed by testing three selected derivatives
(6, 7, and 10) in the AMPc assays. Compounds 6, 7, and 10
antagonized the Cl-IB-MECA-inhibited cAMP production with
potencies in accordance with the hA3 AR affinities.
Ki (nM) or I%
IC50 (nM) or I% cAMP
a
b
c
d
e
R
hA3
hA1 hA2A
8% 6%
40% 17%
hA2B
hA3
In contrast to the high hA3 AR affinity of the quinazoline
derivatives 1–11, but consistent with the predictions, the
quinoline derivatives 12–15, as well as the pyrimidine com-
pounds 16–18, completely lacked affinities at the hA3 AR (Table
2). In fact, at 1 µM concentration, they inhibited the radioligand
binding at this subtype, as well as at hA1 and hA2A ARs, for
only 1–38%. Compounds 12–18 were also inactive in inhibiting
the NECA-stimulated cAMP accumulation in hA2B CHO cells
(I% ) 2–5%).
1
2
3
4
5
6
7
8
C6H4-4-OMe 87.5 ( 6.6
23%
5%
4%
2%
1%
9%
4%
2%
2%
5%
3%
C6H5
350 ( 40
98.3 ( 7.3
550 ( 47
21%
C6H4-4-Me
C6H4-4-Br
C6H11
3%
1%
2%
4%
22%
5%
1%
3%
1%
1%
2%
1%
7%
C6H4-4-OMe 19.5 ( 2.2
125 ( 10
238 ( 21
C6H5
50 ( 4
C6H4-4-Me
C6H4-4-Br
26.7 ( 3.3 21%
27.2 ( 3.1 3%
25.3 ( 2.8 25%
182 ( 10
9
10
11
140 ( 13
7% 10%
The structure-affinity relationships (SAR) in the quinazoline
series resemble those previously observed in the leads.18,24 It
is confirmed that the advantageous role of the p-methoxy
substituent, indeed its removing from the 4-carboxamide phenyl
ring, which mimics the 2-phenyl ring of the triazoloquinoxaline
derivatives, caused a reduction of the hA3 AR affinity (compare
the 4-carboxy-(p-methoxyphenyl)amides 1 and 6 with the
corresponding 4-carboxyanilides 2 and 7). Replacement of
the para-methoxy residue (compounds 1 and 6) with lipophilic
substituents, such as the methyl (derivatives 3 and 8) or bromine
(compounds 4 and 9), maintained the hA3 affinity in the
nanomolar range, the only exception being compound 4, which
possesses a 5-fold reduced binding activity, compared to
derivative 1. The profitable effect of the above cited groups is
coherent with the observation that these residues are positioned
in a small hydrophobic pocket in between TM3 and TM7.
As observed in the triazoloquinoxalin-1-one series,18,24
replacement of the 2-amino group with the 2-oxo residue is
advantageous for the anchoring to the A3 AR binding site
(compare 6–9 with 1–4), in particular, due to a probably stronger
H-bond interaction with His95 (3.37).
a Displacement of specific [125I]AB-MECA binding to hA3 CHO cells.
Ki values are means ( SEM of four separate assays, each performed in
duplicate. b Percentage of inhibition in [3H]DPCPX competition binding
assays to hA1 CHO cells at 1 µM concentration of the tested compounds.
c Percentage of inhibition in [3H]ZM241385 competition binding assays to
hA2A CHO cells at 1 µM concentration of the tested compounds.
d Percentage of inhibition on cAMP experiments in hA2B CHO cells,
stimulated by 200 nM NECA, at 1 µM concentration of the examined
compounds. e IC50 values are expressed as means ( SEM of four separate
cAMP experiments in hA3 CHO cells, inhibited by 100 nM Cl-IB-MECA.
derivatives 17 and 18, which were separated by column
chromatography.
Pharmacological Assays
All the synthesized derivatives 1–18 were tested to evaluate
their affinity at hA1, hA2A, and hA3 ARs. Compounds 1–18 were
also tested at the hA2B subtype by measuring their inhibitory
effects on NECA-stimulated cAMP levels in CHO cells stably
transfected with the hA2B AR. Finally, the antagonistic potencies
of the three selected quinazoline derivatives 6, 7, and 10 were
assessed by evaluating their inhibition on Cl-IB-MECA-inhibited
cAMP production in CHO cells, stably expressing hA3 ARs.
All pharmacological data are collected in Tables 1 and 2.
SAR Commentary. Impressively, the pharmacological data,
reported in Tables 12, are coherent with the predictions based
In agreement with our previous findings in the triazoloqui-
noxalin-1-one derivatives18,24 and with the docking predictions,
introduction of acyl substituents on the 2-amino group enhances
the hA3 AR affinity (compare compounds 10 and 11 with
Table 2. Inhibition of Specific Binding at hA1, hA2A, and hA3 ARs and of cAMP Production at hA2B AR
binding experiments
cAMP assays
a
b
c
d
R1
R2
hA3
hA1
hA2A
hA2B
13
12
14
15
16
17
18
H
6%
1%
1%
2%
4%
1%
3%
6%
5%
2%
3%
1%
1%
6%
6%
1%
5%
3%
3%
2%
3%
2%
3%
OMe
OMe
H
26%
38%
15%
22%
14%
H
COPh
a Percentage of inhibition in [125I]AB-MECA competition binding assays to hA3CHO cells at 1 µM concentration of the tested compounds. b Percentage
of inhibition in [3H]-DPCPX competition binding assays to hA1CHO cells at 1 µM concentration of the tested compounds. c Percentage of inhibition in
[3H]-ZM 241385 competition binding assays to hA2ACHO cells at 1 µM concentration of the tested compounds. d Percentage of inhibition on cAMP experiments
in hA2B CHO cells, stimulated by 200 nM NECA, at 1 µM concentration of the tested compounds.

6602 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Morizzo et al.
Table 3. Comparison between the hA3 AR Affinities of the
Quinazoline-4-carboxamides and the Corresponding
Triazoloquinoxalin-1-ones
Experimental Section
(A) Computational Methodologies. All modeling studies were
carried out on a 10 CPU (PIV-3.0GHZ and AMD64) linux cluster.
Homology modeling, energy calculation, and docking studies
were performed using the Molecular Operating Environment (MOE,
version 2006.08) suite.37
All docked structures were fully optimized without geometry
constraints using RHF/AM1 semiempirical calculations. Vibrational
frequency analysis was used to characterize the minima stationary
points (zero imaginary frequencies). The software package MOPAC
(ver. 7),38 implemented in MOE suite, was utilized for all quantum
mechanical calculations.
Ki (nM) hA3
R
R2
quinazolinesa
triazoloquinoxalinesb
C6H4-p-OMe
C6H5
C6H4-p-Me
C6H4-p-OMe
C6H5
H
H
H
1
2
3
6
7
8
10
11
87.5 ( 6.6
350 ( 40
98.3 ( 7.3
19.5 ( 2.2
50 ( 4
26.7 ( 3.3
25.3 ( 2.8
182 ( 10
C
45.3 ( 3.8
490 ( 41
48.3 ( 3.6
16 ( 1.2
A
G
D
B
H
E
F
Homology Model of the hA3 AR. Based on the assumption that
GPCRs share similar TM boundaries and overall topology, a
homology model of the hA3 receptor was constructed. 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, including the DRY motif (D3.49, R3.50, and
Y3.51) and three proline 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 they were identified from the
X-ray crystal structure for the corresponding sequences of bovine
rhodopsin,39 the CR coordinates of which were used to construct
the seven TM helices for the hA3 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
candidates, 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 has to be given to the EL2, which
has been described in bovine rhodopsin as folding back over
transmembrane helices and, therefore, limiting the size of the active
site.39 Hence, amino acids of this loop could be involved in direct
interactions with the ligands. A driving force to this peculiar fold
of the EL2 loop might be the presence of a disulfide bridge between
cysteines in TM3 and EL2. Because this covalent link is conserved
in all receptors modeled in the current study, the EL2 loop was
modeled using a rhodopsin-like constrained geometry around the
EL2-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.40 The
minimizations were carried out by the 1000 steps of steepest descent
followed by conjugate gradient minimization until the rms gradient
of the potential energy was less than 0.1 kcal mol-1 Å-1. Protein
stereochemistry evaluation was performed by several tools (Ram-
achandran and Chi plots measure phi/psi and chi1/chi2 angles, clash
contacts reports) implemented in the MOE suite.37
80 ( 4
C6H4-p-Me
C6H5
25.0 ( 1.6
2.0 ( 0.13
1.47 ( 0.1
COMe
COPh
C6H5
a Data from Table 1. b Data from refs 18, 19, and 21.
derivative 2), probably because of the hydrogen-bonding
interaction that the acyl oxygen engages with the Ser247 side
chain.
Replacement of the 4-carboxyanilide moiety of compound 2
with a 4-carboxycyclohexylamide group (compound 5) dramati-
cally affected the hA3 affinity. As already described, the
asymmetric topology of the TM binding cavity, characterized
by a major axis of about 17 Å and by a minor axis of about 6
Å, does not allow to accommodate the bulky cyclohexyl moiety,
causing an unfavorable van der Waals repulsive interaction (data
not shown).
Finally, it has to be pointed out that, on the whole, the hA3
AR affinities of the quinazoline-4-carboxamido derivatives are
comparable to those of the corresponding triazoloquinoxalin-
1-one leads,18,19,21 as it appears by comparing the hA3 AR
binding data of some derivatives of the two series (Table 3).
There are two exceptions: the 2-acylamino derivatives 10 and
11 that possess, respectively, a 10- and 120-fold reduced affinity,
in comparison to that of corresponding parent compounds E
and F. Moreover, another relevant finding was the higher
solubility of the new quinazoline derivatives. Indeed, experi-
mentally, the quinazolines derivatives showed a significantly
enhanced solubility in the most common organic solvents with
respect to that of the tryciclic parent derivatives.
Conclusions
As already reported, rhodopsin-based homology modeling
represents a widely used and well-consolidated approach to
visualize GPCR three-dimensional models. Modeling ligan-
d–receptor complexes, as provided by the crystallographic
structure of rhodopsin, enables the study of specific interactions
at higher resolution. In this paper we have once again
demonstrated, by using our in silico simplification approach,
how crucial computational models are to generate hypotheses
and how the experimental findings are, in turn, used to refine
the model. The success of this approach is due to the synergistic
interaction between theory and experiment. Indeed, within the
new easily synthesizable quinazoline-4-carboxyamide series, we
have identified some highly potent and selective hA3 AR
antagonists and we have clarified which chemical features are
crucial to efficiently recognize the TM binding cleft.
Considering the acceptable topological complementarity of all
A3 antagonists described in the present work, our recently proposed
ligand-based homology modeling approach has been not carried
out.41
Molecular Docking of the hA3 AR Antagonists. All antagonist
structures were docked into the hypothetical TM binding site by
using the MOE-dock tool, part of the MOE suite. Searching is
conducted within a user-specified 3D docking box (the standard
protocol selects all atoms inside 12 Å from the center of mass of
the binding cavity), using the Tabu Search42 protocol (standard
parameters are: 1000 steps/run, 10 attempts/step, and 10 Tabu list
length), and the MMFF94 force field.43 MOE-dock performs a user-
specified number of independent docking runs (50 in our specific
case) and writes 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

Human A3 Adenosine Receptor Antagonist Binding
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6603
Table 4. Predicted Binding Affinity (pKi) of the Most Relevant New
Simplified Triazoloquinoxaline Analogs
2-Aminoquinazoline-4-carboxy-(4-methylphenyl)amide (3),
2-Aminoquinazoline-4-carboxy-(4-bromophenyl)amide (4). The
title compounds were obtained from the reaction mixture, as
described above for compound 1. Compound 3: yield, 78%; mp
293–295 °C (EtOH); 1H NMR 2.30 (s, 3H, Me), 7.11 (s, 2H, NH2),
7.19–7.26 (m, 3H, ar), 7.51 (d, 1H, ar, J ) 8.4 Hz), 7.66–7.74 (m,
3H, ar), 8.06 (d, 1H, ar, J ) 8.1 Hz), 10.74 (s, 1H, NH); IR 1664,
1688, 3321, 3385; Anal. (C16H14N4O) C, H, N. Compound 4: yield,
#
pKia
C
1
6
10
12
14
16
6.6
6.6
7.3
7.2
4.8
5.1
4.6
1
76%; mp 244–247 °C (EtOH); H NMR 7.11 (s, 2H, NH2), 7.27
(t, 1H, ar, J ) 7.9 Hz), 7.51–7.61 (m, 3H, ar), 7.73–7.76 (m, 3H,
ar), 8.07 (d, 1H, ar, J ) 8.3 Hz), 10.96 (s, 1H, NH); IR 1630,
1674, 3288, 3347, 3478; Anal. (C15H11BrN4O) C, H, N.
a pKi values were calculated and visualized using the MOE-scoring tool
implemented into the MOE suite.37
2-Aminoquinazoline-4-carboxycyclohexylamide (5). The reac-
tion mixture was a solution that was cooled at 0 °C and diluted
with water (5–10 mL) to yield a solid, which was collected.
Extraction of the mother liquor with chloroform (30 mL × 3) and
evaporation of the anhydrified (Na2SO4) organic phases under
reduced pressure gave a solid that, collected to the first, was
recrystallized. Yield, 22%; mp 176–177 °C (EtOH); 1H NMR
1.07–1.38 (m, 5H, cyclohexyl), 1.60-1.91 (m, 5H, cyclohexyl),
3.81–3.90 (m, 1H, cyclohexyl), 7.03 (s, 2H, NH2), 7.24 (t, 1H, ar,
J )7.6 Hz), 7.45 (d, 1H, ar, J ) 8.4 Hz), 7.71 (t, 1H, ar, J ) 7.6
Hz), 8.01 (d, 1H, ar, J ) 8.32), 8.66 (d, 1H, NH, J ) 7.7 Hz); IR
1637, 3297, 3464; Anal. (C15H18N4O) C, H, N.
conjugate gradient was <0.1 kcal mol-1 Å-1. Charges for the
ligands were imported from the MOPAC output files. To better
refine all antagonist-receptor complexes, a rotamer exploration of
all side chains involved in the antagonist-binding was carried out.
Rotamer exploration methodology was implemented in the MOE
suite.37 Prediction of antagonist-receptor complex stability (in
terms of corresponding pKi value) and the quantitative analysis for
nonbonded intermolecular interactions (H-bonds, transition metal,
water bridges, hydrophobic) were calculated and visualized using
the MOE-scoring tool implemented into the MOE suite.37
A
summary of the most relevant pKi value predictions are collected
General Procedure for the Synthesis of 2-Oxoquinazoline-4-
carboxamido Derivatives 6–9. An excess of 2-oxoquinazoline-4-
carboxylic acid25(2.64 mmol) was reacted with the amines 19, 21,
22 (0.88 mmol), and 20 (0.59 mmol) in the presence of 1-(3-
(dimethylamino)propyl))-3-ethyl-carbodiimide hydrochloride (2.64
mmol), 1-hydroxybenzotriazole (2.64 mmol), triethylamine (4.4
mmol), and 4-(dimethylamino)pyridine (0.08 mmol) in anhydrous
dioxane (5 mL, derivative 7) or anhydrous dimethylformamide (2–3
mL, compounds 6, 8, 9). The mixture was stirred at room
temperature for 4 h (compound 6), 24 h (compound 8), 72 h
(compound 9), or 168 h (compound 7) The title compounds were
obtained by different workup of the reaction mixtures.
in Table 4.
(B) Chemistry. The microwave-assisted syntheses were per-
formed using an Initiator EXP Microwave Biotage instrument
(frequency of irradiation: 2.45 GHz). Silica gel plates (Merck F254
)
and silica gel 60 (Merck, 70–230 mesh) were used for analytical
and column chromatography, respectively. All melting points were
determined on a Gallenkamp melting point apparatus. Microanalyses
were performed with a Perkin-Elmer 260 elemental analyzer for
C, H, N, and the results were within (0.4% of the theoretical values,
unless otherwise stated. The IR spectra were recorded with a Perkin-
Elmer Spectrum RX I spectrometer in Nujol mulls and are expressed
in cm-1. The 1H NMR spectra were obtained with a Brucker
Avance 400 MHz instrument. The chemical shifts are reported in
δ (ppm) and are relative to the central peak of the solvent, which
was always DMSO-d6. The following abbreviations are used: s )
singlet, d ) doublet, t ) triplet, m ) multiplet, br ) broad, and ar
) aromatic protons.
General Procedure for the Synthesis of 2-Aminoquinazoline-4-
carboxamido Derivatives 1–5. An excess of 2-aminoquinazoline-
4-carboxylic acid25 (2.64 mmol) was reacted with the amines 19,
21–23 (0.88 mmol), and 20 (1.32 mmol), in the presence of 1-(3-
(dimethylamino)propyl))-3-ethyl-carbodiimide hydrochloride (2.64
mmol), 1-hydroxybenzotriazole (2.64 mmol), triethylamine (4.4
mmol), and 4-(dimethylamino)pyridine (0.08 mmol) in anhydrous
dimethylformamide (2–3 mL). The mixture was stirred at room
temperature for 26 h (compound 3), 96 h (compounds 1, 4, and 5),
and 168 h (compound 2). The title compounds were obtained by a
different workup of the reaction mixtures.
1,2-Dihydro-2-oxoquinazoline-4-carboxy-(4-methoxyphenyl)
amide (6). The solid was filtered off and the dimethylformamide
was distilled at reduced pressure. By treating the residue with an
EtOH/EtOAc mixture, a solid was obtained that was collected and
1
recrystallized. Yield, 60%; mp > 300 °C (EtOH); H NMR 3.77
(s, 3H, OMe), 6.97 (d, 2H, ar, J ) 8.8 Hz), 7.29 (t, 1H, ar, J ) 7.7
Hz), 7.38 (d, 1H, ar, J ) 8.6 Hz), 7.71 (d, 2H, ar, J ) 8.8 Hz),
7.79 (t, 1H, ar, J ) 7.7 Hz), 8.07 (d, 1H, ar, J ) 7.8 Hz), 10.79 (s,
1H, NH), 12.20 (s, 1H, NH); IR 1669, 3326; Anal. (C16H13N3O3)
C, H, N.
1,2-Dihydro-2-oxoquinazoline-4-carboxyanilide (7). The solid
was filtered off and the clear dioxane solution was diluted with
water (15 mL) and extracted with chloroform (15 mL × 3). The
organic phases were anhydrified, and the solvent was evaporated
at reduced pressure to yield a solid, which was recrystallized. Yield,
48%; mp > 300 °C (2-ethoxyethanol); 1H NMR 7.18 (t, 1H, ar, J
) 7.3 Hz), 7.30 (t, 1H, ar, J ) 7.7 Hz), 7.38–7.43 (m, 3H, ar),
7.78–7.83 (m, 3H, ar), 8.05 (d, 1H, ar, J ) 8.2 Hz), 10.92 (s, 1H,
NH), 12.02 (s, 1H, NH); IR 1666, 3319; Anal. (C15H112N3O2) C,
H, N.
1,2-Dihydro-2-oxoquinazoline-4-carboxy-(4-methylphenyl)a-
mide (8). The reaction mixture was diluted with water (15–20 mL)
and extracted with chloroform (15 mL × 3). The anydrified
(Na2SO4) organic phases were evaporated at reduced pressure to
give an oil that solidified upon treatment with a few drops of EtOH
and petroleum ether. Yield, 65%; mp 193–195 °C (EtOH); 1H NMR
2.30 (s, 3H, Me), 7.21 (d, 2H, ar, J ) 8.2 Hz), 7.29 (t, 1H, ar, J )
7.7 Hz), 7.38 (d, 1H, ar, J ) 8.3 Hz), 7.67 (d, 2H, ar, J ) 7.7 Hz),
7.80 (t, 1H, ar, J ) 7.7 Hz), 8.02 (d, 1H, ar, J ) 8.1 Hz), 10.82 (s,
1H, NH), 12.21 (s, 1H, NH); IR 1668, 3228; Anal. (C16H13N3O2)
C, H, N.
2-Aminoquinazoline-4-carboxy-(4-methoxyphenyl)amide (1).
The insoluble solid was filtered off from the reaction mixture and
water was added to the cooled (0 °C) dimethylformamide solution
until a solid was obtained, which was collected and recrystallized.
Yield, 63%; mp 217–220 °C (EtOH); 1H NMR 3.77 (s, 3H, OMe),
6.97 (d, 2H, ar, J ) 8.3 Hz), 7.08 (s, 2H, NH2), 7.26 (t, 1H, ar, J
) 7.3 Hz), 7.51 (d, 1H, ar, J ) 8.2 Hz), 7.70–7.76 (m, 3H, ar),
8.10 (d, 1H, ar, J ) 8.4 Hz), 10.66 (s, 1H, NH); IR 1664, 3303,
3437; Anal. (C16H14N4O2) C, H, N.
2-Aminoquinazoline-4-carboxyanilide (2). The insolubile solid
was collected by filtration and washed with water. A second crop
of solid was obtained by dilution with water of the cooled (0 °C)
mother liquor. The two solids were put together and recrystallized.
Yield, 54%; mp 191–193 °C (EtOH); 1H NMR 7.12 (s, 2H, NH2),
7.17 (t, 1H, ar, J ) 7.4 Hz), 7.27 (t, 1H, ar, J ) 7.6 Hz), 7.41 (t,
2H, ar, J ) 7.8 Hz), 7.52 (d, 1H, ar, J ) 8.4 Hz), 7.73–7.80 (m,
3H, ar), 8.07 (d, 1H, ar, J ) 8.4 Hz), 10.82 (s, 1H, NH); IR 1664,
3303, 3462; Anal. (C15H12N4O) C, H, N.
1,2-Dihydro-2-oxoquinazoline-4-carboxy-(4-bromophenyl)
amide (9). The cooled (0 °C) reaction mixture was diluted with
water (15–20 mL), and the solid was collected by filtration and
1
recrystallized. Yield, 67%; mp > 300 °C (EtOH); H NMR 7.30

6604 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Morizzo et al.
(t, 1H, ar, J ) 7.8 Hz), 7.39 (d, 1H, ar, J ) 8.4 Hz), 7.61 (d, 2H,
ar, J ) 8.6 Hz), 7.77–7.83 (m, 3H, ar), 8.07 (d, 1H, ar, J ) 8.1
Hz), 11.08 (s, 1H, NH), 12.25 (s, 1H, NH); IR 1667, 3335; Anal.
(C15H10BrN3O2) C, H, N.
oxoquinoline-4-carboxyanilide (15). 2-Oxo-1,2-dihydroquinoline-
4-carboxylic acid28 (2.6 mmol) was refluxed in thionyl chloride
(10 mL) for 2 h. Excess of thionyl chloride was removed at reduced
pressure, and anhydrous cyclohexane (10 mL) was added to the
residue and then evaporated at reduced pressure. The residue, that
is, 2-oxo-quinoline-4-carbonyl chloride (28), was dissolved in
anhydrous methylene chloride (10 mL), and the suitable aniline
derivative (2.17 mmol) and triethylamine (2.17 mmol) were added
to the solution. The mixture was refluxed for 1–3 h and, after
cooling at room temperature, the solid was collected by filtration,
washed with diethyl ether, and recrystallized. Compound 14: yield,
90%; mp > 300 °C (2-methoxyethanol); 1H NMR 3.76 (s, 3H,
OMe), 6.69 (s, 1H, H-3), 6.96 (d, 2H, ar, J ) 8.9 Hz), 7.22 (t, 1H,
ar, J ) 7.6 Hz), 7.39 (d, 1H, ar, J ) 8.2 Hz), 7.57 (t, 1H, ar, J )
7.6 Hz), 7.67 (d, 2H, ar, J ) 8.9 Hz), 7.74 (d, 1H, ar, J ) 8.0 Hz),
10.59 (s, 1H, NH), 11.99 (s, 1H, NH); IR 1649, 1667, 3271; Anal.
(C17H14N2O3) C, H, N. Compound 15: yield, 87%; mp > 300 °C
(2-methoxyethanol); 1H NMR 6.71 (s, 1H, H-3), 7.16 (t, 1H, ar, J
) 7.3 Hz), 7.23 (t, 1H, ar, J ) 7.6), 7.37–7.39 (m, 3H, ar), 7.58 (t,
1H, ar, J ) 7.7 Hz), 7.72–7.78 (m, 3H, ar), 10.74 (s, 1H, NH),
12.01 (s, 1H, NH); IR 1650, 1669, 3276; Anal. (C16H12N2O2) C,
H, N.
Synthesis of 2-Acetylaminoquinazoline-4-carboxyanilide
(10). Acetyl chloride (1.51 mmol) was added to a suspension of
2-aminoquinazoline derivative 2 (1.26 mmol) in anhydrous meth-
ylene chloride (20 mL) and pyridine (0.2 mL). The mixture was
stirred at room temperature for 5 h. The solid was collected by
filtration and washed with water. The methylene chloride solution
was diluted with diethyl ether (10–15 mL) to yield a second crop
of solid that was collected by filtration and, together with the first
solid, recrystallized. Yield, 79%; mp 232–233 °C (EtOH); 1H NMR
2.31 (s, 3H, Me), 7.19 (t, 1H, ar, J ) 7.3 Hz), 7.42 (t, 2H, ar, J )
7.4 Hz), 7.61 (t, 1H, ar, J ) 7.1 Hz), 7.81 (d, 2H, ar, J ) 7.4 Hz),
7.91 (d 1H, ar, J ) 8.6 Hz), 8.01 (t, 1H, ar, J ) 8.5 Hz), 8.41 (d,
1H, ar, J ) 8.5 Hz), 10.92 (s, 2H, 2NH); IR 1680, 3322; Anal.
(C17H14N4O2) C, H, N.
Synthesis of 2-Benzoylaminoquinazoline-4-carboxyanilide
(11). Benzoyl chloride (1.26 mmol) was added to a suspension of
2-aminoquinazoline derivative 2 (1.26 mmol) in anhydrous meth-
ylene chloride (20 mL) and pyridine (0.2 mL). The reaction mixture
was refluxed for 30 h, and after cooling, the solid was collected by
filtration and washed with water. The clear solution was evaporated
under reduced pressure to afford a solid that, collected with the
first solid, was chromatographed on a silica gel column (eluting
system CHCl3/cyclohehane/EtOAc, 6:2:2). Yield, 80%; mp 192–195
Synthesis of the 2-Aminopyrimidine-4-carboxy-(4-methoxyphe-
nyl)amide (16). 2-Aminopyrimidine-4-carboxylic acid29 (5.64 mmol),
4-methoxyaniline (1.88 mmol), 1-(3-(dimethylamino)propyl))-3-
ethyl-carbodiimide hydrochloride (5.64 mmol), 1-hydroxybenzot-
riazole (2.64 mmol), triethylamine (9.4 mmol), and 4-(dimethy-
lamino)pyridine (0.12 mmol) in anhydrous dimethylformamide (3–4
mL) were stirred at room temperature for 44 h. The mixture was
diluted with water and cooled at 0 °C. The solid, which precipitated,
was collected by filtration and recrystallized. Yield, 50%; mp
1
°C (acetonitrile); H NMR 7.21 (t, 1H, ar, J ) 7.3 Hz), 7.45 (t,
2H, ar, J ) 7.9 Hz), 7.57 (t, 2H, ar, J ) 7.5 Hz), 7.63–7.62 (m,
2H, ar), 7.83 (d, 2H, ar, J ) 7.6 Hz), 7.99 (d, 1H, ar, J ) 8.2 Hz),
8.05–8.06 (m, 3H, ar), 8.54 (d, 1H, ar, J ) 8.2 Hz), 10.96 (s, 1H,
NH), 11.38 (s, 1H, NH); IR 1688; Anal. (C22H16N4O2) C, H, N.
1
215–216 °C (EtOH); H NMR 3.75 (s, 3H, OMe), 6.94–6.97 (m,
4H, 2ar + NH2), 7.14 (d, 1H, H-5, J ) 4.9 Hz), 7.71 (d, 2H, ar, J
) 6.9 Hz), 8.51 (d, 1H, ar, J ) 4.9 Hz), 10.14 (s, 1H, NH); IR
1655, 3190, 3334; Anal. (C12H12N4O2) C, H, N.
General Procedure for the Synthesis of Hydrazinoquinoline-4-
carboxy-(4-methoxyphenyl)amide (26) and 2-Hydrazinoquinoline-
4-carboxanilide (27). A mixture of 2-chloroquinoline-4-carboxa-
mides 24 and 2526,27 (3.5 mmol) and hydrazine monohydrate (4.16
mmol) in ethanol (2 mL) and water (0.5 mL) was microwave
irradiated at 150 °C for 8 min. The mixture was cooled and diluted
with water, and the solid was collected by filtration, washed with
water, and recrystallized. Compound 26: yield, 75%; mp 230–231
Synthesis of the 2-Benzoylaminopyrimidine-4-carboxy-(4-meth-
oxyphenyl)amide (17) and 2-Dibenzoylaminopyrimidine-4-car-
boxy-(4-methoxyphenyl)amide (18). A mixture of compound 16
(1.31 mmol), benzoyl chloride (2.32 mmol), and anhydrous pyridine
(4.96 mmol) in anhydrous methylene chloride (20 mL) was refluxed
for 48 h. The solid was filtered off and the methylene chloride
solution was washed with water (2 × 10 mL). The anhydrified
(Na2SO4) organic phase was evaporated at reduced pressure. The
solid residue was a mixture of compounds 17 and 18, which were
separated by column chromatography (SiO2, eluting system EtOAc/
CH2Cl2, 1:9). Compound 17: yield, 15%; mp 197–199 °C (EtOH);
1H NMR 3.76 (s, 3H, OMe), 6.99 (d, 2H, ar, J ) 9.0 Hz), 7.57 (t,
2H, ar, J ) 7.8 Hz), 7.64 (t, 1H, ar, J ) 7.8 Hz), 7.71 (d, 2H, ar,
J ) 9.0 Hz), 7.80 (d, 1H, H-5, J ) 4.9 Hz), 8.01 (d, 2H, ar, J )
7.5 Hz), 9.02 (d, 1H, H-6, J ) 4.9 Hz), 10.26 (s, 1H, NH), 11.23
(s, 1H, NH); IR 1660, 1681, 3225, 3305; Anal. (C19H16N4O3) C,
H, N. Compound 18: yield, 45%; mp 230–232 °C (EtOH); 1H
NMR 3.75 (s, 3H, OMe), 6.95 (d, 2H, ar, J) 9.1 Hz), 7.47–7.65
(m, 8H, ar), 7.84 (d, 4H, ar, J ) 7.7 Hz), 7.91 (d, 1H, H-5), 9.01
(d, 1H, H-6, J ) 5.0 Hz), 10.09 (s, 1H, NH); IR 1681, 1695, 3351;
Anal. (C26H20N4O4) C, H, N.
1
°C (lit.27 229–231 °C, EtOH); H NMR 3.73 (s, 3H, OMe), 4.48
(s, 2H, NH2), 6.96–7.01 (m, 3H, ar), 7.23 (t, 1H, ar, J ) 7.3 Hz),
7.54–7.64 (m, 2H, ar), 7.69 (d, 2H, ar, J ) 8.3 Hz), 7.81 (d, 1H,
ar, J ) 8.0 Hz), 8.28 (s, 1H, NH), 10.56 (s, 1H, NHCO); Anal.
(C17H16N4O2) C, H, N. Compound 27: yield, 68%; mp 226–228
1
°C (lit.26 225–227 °C, acetonitrile); H NMR 4.42 (s, 2H, NH2),
7.02 (s, 1H, H-3), 7.15 (t, 1H, ar, J ) 7.4 Hz), 7.23 (t, 1H, ar, J )
7.0 Hz), 7.39 (t, 2H, ar, J ) 7.7 Hz), 7.56 (t, 1H, ar, J ) 7.1 Hz),
7.63 (d, 1H, ar, J ) 8.2 Hz), 7.77–7.81 (m, 3H, ar), 8.23 (s, 1H,
NH), 10.70 (s, 1H, NHCO); Anal. (C16H14N4O) C, H, N.
General Procedure for the Synthesis of 2-Aminoquinoline-4-
carboxy-(4-methoxyphenyl)amide (12) and 2-Aminoquinoline-4-
carboxanilide (13). Compounds 26 and 27 (1.66 mmol) were
dissolved in boiling EtOH (100 mL), then an excess of Ni/Raney
(50% slurry in water, 4 g) was added. The suspension was
hydrogenated in a Parr apparatus (50 psi) for 14 h, Ni/Raney was
filtered off, and the solvent was evaporated at reduced pressure.
The residue was treated with diethyl ether (4–6 mL), collected by
filtration, and recrystallized. Compound 12: yield, 70%; mp
(C) Pharmacological Assays. Human Cloned A1, A2A, and
A3 Adenosine Receptor Binding Assay. All synthesized com-
pounds were tested for evaluating their affinity at human A1, A2A
,
and A3 adenosine receptors. Displacement experiments of [3H]-
DPCPX (1 nM) to hA1 CHO membranes (50 µg of protein/assay)
and at least 6–8 different concentrations of antagonists for 120 min
at 25 °C in 50 mM Tris HCl buffer, pH 7.4, were performed.30
Nonspecific binding was determined in the presence of 10 µM of
CHA (e10% of the total binding). Binding of [3H]ZM-241385 (1
nM) to hA2A CHO membranes (50 µg of protein/assay) was
performed by using 50 mM Tris HCl buffer, 10 mM MgCl2, pH
7.4, and at least 6–8 different concentrations of antagonists studied
for an incubation time of 60 min at 4 °C.31 Nonspecific binding
was determined in the presence of 1 µM ZM-241385 and was about
20% of total binding. Competition binding experiments to hA3 CHO
1
257–258 °C (acetonitrile); H NMR 3.77 (s, 3H, OMe), 6.63 (s,
2H, NH2), 6.89 (s, 1H, H-3), 6.96 (d, 2H, ar, J ) 8.96 Hz),
7.18–7.22 (m, 1H, ar), 7.52–7.53 (m, 2H, ar), 7.69 (d, 2H, ar, J )
8.9 Hz), 7.78–7.80 (m, 3H, ar), 10.53 (s, 1H, NH); IR 1658, 3187,
3303, 3469; Anal. (C17H15N3O2) C, H, N. Compound 13: yield,
1
68%; mp 288 °C dec (2-methoxyethanol); H NMR 6.66 (s, 2H,
NH2), 6.91 (s, 1H, H-3), 7.13–7.23 (m, 2H, ar), 7.37–7.54 (m, 4H,
ar), 7.78–7.80 (m, 3H, ar), 10.69 (s, 1H, NH); IR 1628, 1662, 3192,
3298, 3467. Anal. (C16H13N3O) C, H, N.
General Procedure for the Synthesis of 1,2-Dihydro-2-oxoquino-
line-4-carboxy-(4-methoxyphenyl)amide (14) and 1,2-Dihydro-2-

Human A3 Adenosine Receptor Antagonist Binding
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6605
membranes (50 µg of protein/assay) and 0.5 nM [125I]AB-MECA,
50 mM Tris HCl buffer, 10 mM MgCl2, 1 mM EDTA, pH 7.4,
and at least 6–8 different concentrations of examined ligands for
120 min at 4 °C.32 Nonspecific binding was defined as binding in
the presence of 1 µM AB-MECA and was about 20% of total
binding. Bound and free radioactivity were separated by filtering
the assay mixture through Whatman GF/B glass fiber filters by using
a Brandel cell harvester. The filter bound radioactivity was counted
by Scintillation Counter Packard Tri Carb 2500 TR with an
efficiency of 58%.
Measurement of Cyclic AMP Levels in CHO Cells
Transfected with hA2B or hA3 Adenosine Receptors. CHO cells
transfected with hAR subtypes were washed with phosphate-
buffered saline and diluted tripsine and centrifuged for 10 min at
200 g. 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 antagonists to
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A2B receptors was determined by antagonism of NECA (200 nM)-
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induced stimulation of cyclic AMP levels. In addition, the potency
of antagonists to A3 receptors was determined in the presence of
forskolin 1 µM and Cl-IB-MECA (100 nM) that mediated inhibition
of cyclic AMP levels. The reaction was terminated by the addition
of cold 6% trichloroacetic acid (TCA). The TCA suspension was
centrifuged at 2000 g 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
pmoles) were added to each test tube containing [3H] cyclic AMP
and the incubation buffer (trizma base, 0.1 M; aminophylline, 8.0
mM; 2-mercaptoethanol, 6.0 mM; pH 7.4). The binding protein
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 2000 g for 10 min. The clear supernatant was
counted in a Scintillation Counter Packard Tri Carb 2500 TR with
an efficiency of 58%.33
513, 249–280.
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a novel 3D-pharmacophore model of human A3 adenosine receptor
antagonists: Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives
as a key study. J. Med. Chem. 2005, 48, 152–162.
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molecular electrostatic potential surface properties combined with
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Data Analysis
The protein concentration was determined according to a Bio-
Rad method,34 with bovine albumin as a standard reference.
Inhibitory binding constant, Ki, values were calculated from
those of IC50 according to Cheng-Prusoff equation: Ki ) IC50/
(1 + [C*]/KD*), where [C*] is the concentration of the
radioligand and KD* is the dissociation constant.35 A weighted
nonlinear least-squares curve fitting program LIGAND36 was
used for computer analysis of inhibition experiments. EC50 and
IC50 values obtained in the cyclic AMP assay were calculated
by nonlinear regression analysis using the equation for a sigmoid
concentration–response curve (Graph-PAD Prism, San Diego,
CA, U.S.A).
(20) Colotta, V.; Catarzi, D.; Varano, F.; Filacchioni, G.; Martini, C.;
Trincavelli, L.; Lucacchini, A. Synthesis of 4-amino-6-(hetero)aryla-
lkylamino-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives as potent
A2A adenosine receptor antagonists. Bioorg. Med. Chem. 2003, 11,
5509–5518.
(21) Colotta, V.; Catarzi, D.; Varano, F.; Calabri, F. R.; Lenzi, O.;
Filacchioni, G.; Trincavelli, L.; Martini, C.; Deflorian, F.; Moro, S.
1,2,4-Triazolo[4,3-a]quinoxalin-1-one moiety as an attractive scaffold
to develop new potent and selective human A3 adenosine receptor
antagonists: Synthesis, pharmacological, and ligand-receptor modeling
studies. J. Med. Chem. 2004, 47, 3580–3590.
Acknowledgment. The molecular modeling work coordi-
nated by S.M. has been carried out with financial support from
the University of Padova, Italy, and the Italian Ministry for
University and Research (MIUR), Rome, Italy. S.M. is also very
grateful to Chemical Computing Group for the scientific and
technical partnership. The synthetic work was supported by a
grant of the Italian Ministry for University and Research (MIUR,
FIRB RBNE03YA3L project).
(22) Catarzi, D.; Colotta, V.; Varano, F.; Calabri, F. R.; Lenzi, O.;
Filacchioni, G.; Trincavelli, L.; Martini, C.; Tralli, A.; Montopoli, C.;
Moro, S. 2-Aryl-8-chloro-1,2,4-triazolo[1,5-a]quinoxalin-4-amines as
highly potent A1 and A3 adenosine receptor antagonists. Bioorg. Med.
Chem. 2005, 13, 705–715.
(23) Catarzi, D.; Colotta, V.; Varano, F.; Lenzi, O.; Filacchioni, G.;
Trincavelli, L.; Martini, C.; Montopoli, C.; Moro, S. 1,2,4-Triazolo[1,5-
a]quinoxaline as a versatile tool for the design of selective human A3
Supporting Information Available: Combustion analysis data
of the synthesized compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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