Pyrido[2,3-e]-1,2,4-triazolo[4,3-α]pyrazin-1-one as a new scaffold to develop potent and selective human A3 adenosine receptor antagonists. Synthesis, pharmacological evaluation, and ligand-receptor modeling studies

Article abstract of DOI:10.1021/jm8014876

The paper describes a new class of human (h) A₃ adenosine receptor antagonists, the 2-arylpyrido[2,3-e]- l,2,4-triazolo[4,3-α] pyrazin-1-one derivatives (PTP), either 4-oxo (1-6, series A) or 4-amino-substituted (7-20, series B). In both series

Full text of DOI:10.1021/jm8014876

J. Med. Chem. 2009, 52, 2407–2419
2407
Pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one as a New Scaffold To Develop Potent and
Selective Human A3 Adenosine Receptor Antagonists. Synthesis, Pharmacological Evaluation,
and Ligand-Receptor Modeling Studies
Vittoria Colotta,*,† Ombretta Lenzi,† Daniela Catarzi,† Flavia Varano,† Guido Filacchioni,† Claudia Martini,‡
Letizia Trincavelli,‡ Osele Ciampi,‡ Anna Maria Pugliese,§ Chiara Traini,§ Felicita Pedata,§ Erika Morizzo,| and Stefano Moro|
Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente AttiVi, Dipartimento di Scienze Farmaceutiche, UniVersita` di
Firenze, Polo Scientifico, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy, Dipartimento di Psichiatria, Neurobiologia, Farmacologia e
Biotecnologie, UniVersita` di Pisa, Via Bonanno, 6, 56126 Pisa, Italy, Dipartimento di Farmacologia Preclinica e Clinica, UniVersita` di
Firenze, Viale Pieraccini 6, 50139 Firenze, Italy, and Molecular Modeling Section (MMS), Dipartimento di Scienze Farmaceutiche, UniVersita`
di PadoVa, Via Marzolo 5, 35131 PadoVa, Italy
ReceiVed NoVember 26, 2008
The paper describes a new class of human (h) A3 adenosine receptor antagonists, the 2-arylpyrido[2,3-e]-
1,2,4-triazolo[4,3-a]pyrazin-1-one derivatives (PTP), either 4-oxo (1-6, series A) or 4-amino-substituted
(7-20, series B). In both series A and B, substituents able to act as hydrogen bond acceptors (OMe, OH,
F, COOEt) were inserted on the 2-phenyl ring. In series B, cycloalkyl and acyl residues were introduced on
the 4-amino group. Some of the new derivatives showed high hA3 AR affinities (Ki < 50 nM) and selectivities
vs both hA1 and hA2A receptors. The selected 4-benzoylamino-2-(4-methoxyphenyl)pyrido[2,3-e]-1,2,4-
triazolo[4,3-a]pyrazin-1-one (18), tested in an in vitro rat model of cerebral ischemia, proved to be effective
in preventing the failure of synaptic activity induced by oxygen and glucose deprivation in the hippocampus.
Molecular docking of this new class of hA3 AR antagonists was carried out to depict their hypothetical
binding mode to our refined model of hA3 receptor.
Introduction
of glioblastoma multiforme,9 and as antiglaucoma agents.10
Recently, A3 AR antagonists have also been described as
potential neuroprotective agents in ischemia- and aging-associ-
ated brain damages.7,11-14 Nevertheless, studies regarding the
role of the A3 AR receptor in the pathophysiology of cerebral
ischemia have provided contrasting results. In fact, it has been
proven that A3 receptor activation can induce both cell protection
and cell damage, depending on the degree of receptor activation
and/or the cell type.6,7,14
In the past years much of our research has been directed
toward the discovery of potent and selective hA3 AR antagonists
belonging to different heterocyclic classes,13,15-24 and among
them the 2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives
(TQX), either 4-oxo- or 4-amino-substituted, have been deeply
investigated (Chart 1).15-19,22-24 On the basis of the observed
structure-affinity relationships (SAR) in the TQX compounds,
a new class of AR antagonists was designed, synthesized, and
pharmacologically tested: the 2-aryl-pyrido[2,3-e]-1,2,4-tria-
zolo[4,3-a]pyrazin-1-one derivatives (PTP), 6-aza analogues of
TQX (Chart 1).
Adenosine is well recognized as an important neuromodulator
that affects a large variety of physiopathological functions
through activation of at least four specific cell surface receptors,
termed A1, A2A, A2B, A3, which belong to the G-protein-coupled
receptor family.1,2 Stimulation of adenosine receptors (ARsa)
inhibits (A1 and A3) or activates (A2A and A2B) adenylyl cyclase,
thus decreasing or increasing, respectively, cAMP production.2
Other second messenger signaling pathways have been de-
scribed. A positive modulation of phospholipase C3 and D4 and
KATP channels5 has been reported for the A3 AR that also couples
to members of the mitogen-activated protein kinase (MAPK)
family, such as the extracellular signal-regulated kinase (ERK)
1/2 and p38.6 Modulation of MAPK is thought to have a role
in the effects that the A3 AR elicits on cell growth, survival,
and death.6,7 Growing evidence indicates that A3 AR antagonists
could have therapeutic potential in renal injury,8 in the treatment
* To whom correspondence should be addressed. Phone: +39 55
4573731. Fax: +39 55 4573780. E-mail: vittoria.colotta@unifi.it.
†
Dipartimento di Scienze Farmaceutiche, Universita` di Firenze.
Universita` di Pisa.
Dipartimento di Farmacologia Preclinica e Clinica, Universita` di
‡
Rational Design of the
Pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one
Derivatives
§
Firenze.
|
Universita` di Padova.
a Abbreviations: GPCRs, G-protein-coupled receptors; AR, adenosine
receptor; cAMP, cyclic adenosine monophosphate; MAPK, mitogen-activated
protein kinase; ERK, extracellular signal-regulated kinase; h, human; SAR,
structure-affinity relationship; r, rat; DPCPX, 8-cyclopentyl-1,3-dipropylxan-
thine; CGS 21680, 2-[4-(2-carboxyethyl)phenethyl]amino-5′-(N-ethylcarbam-
oyl)adenosine; I-AB-MECA, N6-(4-amino-3-iodobenzyl)-5′-(N-methylcarbam-
oyl)adenosine;NECA,5′-(N-ethylcarboxamido)adenosine;C1-IB-MECA,2-chloro-
N6-(3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine; TM, transmembrane;
RBHM, rhodopsin-based homology modeling; LBHM, ligand-based homology
modeling; EL2, second extracellular loop; MOE, molecular operating environ-
ment; OGD, oxygen and glucose deprivation; AD, anoxic depolarization; fEPSP,
field excitatory postsinaptic potential; dc, direct current; aCSF, artificial cerebral
spinal fluid.
An intensive study of the SAR in the 2-aryltriazoloquinoxalin-
1-one derivatives TQX led to the identification of some
substituents (R, R1, R6) which, introduced one by one in a
suitable position, generally afforded high hA3 affinity and
selectivity15,16,22 (Chart 1). These substituents are the p-methoxy
(R) on the 2-phenyl ring, the nitro group (R6) at the 6-position
(4-oxo and 4-amino series), and acyl residues (R1) on the
4-amino group (4-amino series). Even best results, in terms of
hA3 affinity and especially of selectivity, ensued from the
combination of R ) OMe with R6 ) NO2 or with R1 ) acyl.19,22
10.1021/jm8014876 CCC: $40.75
2009 American Chemical Society
Published on Web 03/20/2009

2408 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Colotta et al.
Chart 1. Previously Reported
Scheme 1a
2-Aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one Derivatives (TQX)
and Newly Synthesized
2-Arylpyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one
Bioisosters (PTP)
a (a) Et3N, EtOH; (b) (Cl3CO)2CO, THF; (c) 48% HBr, AcOH; (d) (i)
0.8 N NaOH/EtOH; (ii) 6 N HCl.
Instead, when R6 ) NO2 was combined with R1 ) acyl, not
always a good hA3 affinity was obtained.22,24 The same applies
when the three substituents R, R1, and R6 were simultaneously
introduced in the key positions.22 Molecular modeling studies
permitted depiction of the putative binding motif of these
derivatives on a refined model of the hA3 AR and to rationalize
the observed SAR.19,22,24 At least two hydrogen-bonding
interactions stabilize the anchoring of the tricyclic scaffold inside
the binding cleft: one involving the 1-carbonyl group and the
other the 4-oxo/4-amino function. Additional hydrogen bonds
can be engaged by the nitro group in R6, the methoxy in R, and
the acyl carbonyl group in R1, thus explaining the positive effects
of these substituents for the hA3 AR affinity. Both the 2-aryl
and the fused benzo rings are positioned in two size-limited
hydrophobic pockets, and as a consequence, the volume of the
whole molecule is critical in the fitting with the receptor. For
the 4-amino series it has been found that the bulkiness of the
R1 acyl residue plays a key role in the fine control of the
orientation of the molecule in the binding site and that it
influences the positioning of the 6-nitro substituent that does
not always exert a profitable effect.22,24 The unfavorable steric
interactions of the nitro group with the receptor site have been
thought to be responsible for the reduction of affinity of some
6-nitro-4-amido-substituted derivatives. On this basis, we
designed a new series of AR antagonists deriving from the
replacement of the 6-nitro group of the 1,2,4-triazolo[4,3-
a]quinoxalin-1-one derivatives with an endocyclic nitrogen atom
that could act as hydrogen-bond acceptor without hindering
anchoring to the receptor site. Thus, applying the isosteric
replacement of the nitrobenzene moiety with a pyridine ring,
the 6-aza analogues of the TQX derivatives, namely, the 2-aryl-
pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one derivatives (PTP,
Chart 1), both 4-oxo (1-6, series A) and 4-amino-substituted
(7-20, series B), were synthesized. In both series A and B,
besides introducing the p-methoxy group on the 2-phenyl ring,
other substituents able to act as hydrogen bond acceptors (OH,
F, COOEt) were inserted. In series B, cycloalkyl and acyl
residues were introduced on the 4-amino group. The new
derivatives 1-20 were tested to evaluate their affinities at ARs,
and one selected compound (18) was also tested in an in vitro
Figure 1. Two tautomeric forms of intermediates 25-28.
TXQ derivative proved to be effective in preventing the failure
of synaptic activity induced by oxygen and glucose deprivation
in the hippocampus.
Molecular docking of this new class of hA3 AR antagonists
was carried out to depict their hypothetical binding mode to
our refined model of hA3 receptor.
Chemistry
The target pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one
derivatives 1-20 were prepared as outlined in Schemes 1-3.
Scheme 1 shows the synthesis of the 2-arylpyrido[2,3-e]-1,2,4-
triazolo[4,3-a]pyrazin-1,4-diones 1-6 which were synthesized
by reacting the commercially available 2,3-diaminopyridine with
a suitable ethyl N1-arylhydrazono-N2-chloroacetate 21-24.25-27
From the reaction, the 2-arylhydrazono-1,2-dihydropyrido[2,3-
b]pirazin-3(4H)-ones 25-28 were isolated in high yields. These
derivatives resulted from the displacement of the chlorine atom
of compounds 21-24 by the more nucleophilic amino group
of the 2,3-diaminopyridine and subsequent cyclization at ester
function level. The intermediates 25-28 may exist in two
1
tautomeric forms (Figure 1). Indeed, their H NMR spectra
reveals the existence of both tautomers because there are more
than three protons that exchange with D2O. In compounds 26
(R ) OMe) and 28 (R ) COOEt) the existence of both species
a and b is also revealed by the presence of doubled signals for
the methoxy and carbethoxy substituents. The 2-arylhydrazono
structure of derivatives 25-28 was assigned on the basis of
1
the H NMR spectra of the corresponding tricyclic derivatives
1-4, obtained from 25-28 and triphosgene (Scheme 1). In the
1H NMR spectra of 1-4 the signal of the two most deshielded
protons appears as a doublet at 8.83-8.84 ppm and at 8.33-8.35
ppm with, respectively, a coupling constant of about 8.1 and
rat model of cerebral ischemia, since in a previous study24
a

Pyrazin-1-one Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2409
Scheme 2a
was prepared by demethylation of the 2-(4-methoxyphenyl)
derivative 8. The 4-cycloalkylamino derivatives 12 and 13 were
prepared by reacting the 4-chloro derivative 7 with, respectively,
cyclohexylamine and cyclopentylamine. The 4-amido derivatives
14-20 were obtained as illustrated in Scheme 3. Reaction of
the 4-amino derivatives 7 and 8 with the suitable acyl chlorides
gave the corresponding 4-amido-substituted compounds 14-18,
while from treatment of 7 and 8 with an excess of benzoyl
chloride the 4-dibenzoylamino derivatives 19 and 20 were
obtained, respectively.
Pharmacology
Compounds 1-20, rationally designed to obtain hA3 receptor
antagonists, were tested for their ability to displace [125I]N6-(4-
amino-3-iodobenzyl)-5′-(N-methylcarbamoyl)adenosine ([125I]AB-
MECA) from cloned hA3 receptor stably expressed in CHO
cells. Subsequently, the selected compounds 1, 2, 8, 12-18,
and 20, which showed the highest A3 AR affinities (Ki e 250
nM), were tested for their ability to displace [3H]DPCPX from
cloned hA1 ARs and [3H]5′-(N-ethylcarboxamido)adenosine
([3H]NECA) from cloned hA2A ARs, to establish their hA3
selectivities both vs hA1 and hA2A ARs.
a (a) PCl5/POCl3, pyridine; (b) NH3(g), absolute EtOH; (c) 48% HBr,
AcOH; (d) NH-R1, absolute EtOH.
Compounds 1-20 were also tested at the bovine (b) A1 AR,
from cerebral cortical membranes, and at the bA2A AR, from
striatal membranes, using respectively [3H]8-cyclopentyl-1,3-
dipropylxanthine ([3H]DPCPX) and [3H]2-[4-(2-carboxyeth-
yl)phenethyl]amino-5′-(N-ethylcarbamoyl)adenosine ([3H]CGS
21680) as radioligands.
Scheme 3a
The binding results of 1-20, together with those of theo-
phylline and DPCPX included as antagonist reference com-
pounds, are reported in Table 1.
Compound 18, which showed the highest hA3 affinity and
selectivity among the herein reported compounds, both vs hA1
and hA2A ARs, was selected for further investigations. It was
tested at the hA2B receptor by evaluating its inhibitory effect
on NECA-stimulated cAMP levels in hA2B CHO cells. The same
functional assay was performed on hA3 CHO cells in order to
assess the antagonism of compound 18 on 2-chloro-N6-(3-
iodobenzyl)-5′-(N-methylcarbamoyl)adenosine (Cl-IB-MECA)-
inhibited cAMP production. Moreover, rat (r) A1, A2A, and A3
AR affinities were evaluated using [3H]DPCPX (rat cortex),
[3H]CGS 21680 (rat striatum), and [125I]AB-MECA (HEK cells
stably transfected with the rat A3 receptor).
Compound 18 was selected to evaluate its effect in an in vitro
rat model of cerebral ischemia obtained by oxygen and glucose
deprivation (OGD).
a (a) R4COCl, anhydrous pyridine; (b) excess of PhCOCl, anhydrous
pyridine.
4.8 Hz, this latter value being typical of a 2-pyridine proton.
Thus, considering both the chemical shifts and the J values,
the second signal was assigned to the H-7 proton and the first
to the H-9, this latter proton being more deshielded because of
the paramagnetic effect of the 1-carbonyl group. Indeed,
compounds 1-4, and consequently 25-28, were pyrido[2,3-
b]pirazino derivatives.
Demethylation of the 2-(4-methoxyphenyl) derivative 2
afforded the 2-(4-hydroxyphenyl) compound 5. From alkaline
hydrolysis of the ethyl carboxylic ester 4, the corresponding
carboxylic acid 6 was obtained. The 4-amino-2-arylpyrido[2,3-
e]-1,2,4-triazolo[4,3-a]pyrazin-1-ones 7-11 and the 4-cy-
cloalkyamino derivatives 12, 13 were synthesized as depicted
in Scheme 2. The 1,4-dione derivatives 1-4 were reacted with
phosphorus pentachloride and phosphorus oxychloride to give
the corresponding 4-chloro derivatives 29-32 which were
transformed into the desired 4-amino-substituted compounds
7-10 with ammonia. The 2-(4-hydroxyphenyl) derivative 11
Results and Discussion
(A) Structure-Affinity Relationship Studies. The hA3 AR
binding data, reported in Table 1, show that the pyrido[2,3-e]-
1,2,4-triazolo[4,3-a]pyrazin-1-one tricyclic system is a new
scaffold to obtain potent and selective hA3 AR antagonists. In
fact, some of the novel derivatives showed high hA3 AR
affinities (Ki < 50 nM) and selectivities vs hA1 (compounds 2,
12, 13, 16-18, 20) and vs hA2A (2, 17, 18, 20) receptors. Good
affinity for the hA2A receptor was observed for compounds 12,
13, and 16.
This new class of AR antagonists and their triazoloquinoxa-
line leads share common SAR at the hA3 receptor. First of all
is the profitable effect of the p-methoxy substituent on the
2-phenyl ring. In fact, this substituent increases the hA3 affinity,
both in the 4-oxo series (compare compound 2 to 1) and in the
4-amino series (compare compound 8 to 7), as previously
described for the leads.15,19 The 2-(4-methoxyphenyl)-4-one

2410 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Colotta et al.
Table 1. Binding Affinity at Human A1, A2A, A3 and Bovine A1 and A2A ARs
b
a The Ki values are mean values ( SEM of four separate assays, each performed in triplicate. Displacement of specific [125I]AB-MECA binding at
human A3 receptors expressed in CHO cells or percentage of inhibition (I%) of specific binding at 1 µM concentration. c Displacement of specific [3H]DPCPX
and [3H]NECA binding at, respectively, hA1 and hA2A receptors expressed in CHO cells or percentage of inhibition (I%) of specific binding at 10 µM
d
concentration. Displacement of specific [3H]DPCPX binding in bovine brain membranes or percentage of inhibition (I%) of specific binding at 20 µM
concentration. e Displacement of specific [3H]CGS 21680 binding from bovine striatal membranes or percentage of inhibition (I%) of specific binding at 20
µM concentration.
derivative 2 also showed good hA3 selectivity vs both hA1 and,
especially, hA2A receptors. Replacement of the methoxy group
of derivatives 2 and 8 with a hydroxy group (compounds 5 and
11), a fluorine atom (compounds 3 and 9), and carboxy acid/
ester groups (compounds 4, 6, 10) caused a significant drop of
the hA3 affinity. Introduction of a cyclohexyl or a cyclopentyl
moiety on the 4-amino group of derivative 7 significantly
increases the hA3 affinity (compounds 12 and 13, respectively),
as in the triazoloquinoxaline-1-one class,15 and also affords high
selectivity vs the hA1 receptor. Similarly, when acyl groups were
appended on the 4-amino substituent of 7 (derivatives 14-16,
19), a significant gain in the hA3 affinity, linked to the steric
hindrance of the acyl moiety, was obtained. In particular, the
benzoyl (compound 15) and phenylacetyl groups (compound
16) resulted in being the most advantageous and conferred high
hA3 vs hA1 selectivity as well. Also in the 4-amido-substituted
derivatives, introduction of the p-methoxy group on the 2-phenyl
ring ameliorated both the affinity and selectivity for the hA3
receptor, as can be observed by comparing the binding data of
compounds 17, 18, and 20 to those of derivatives 14, 15, and
19, respectively.
data of some new derivatives to those of the corresponding
triazoloquinoxalines (Table 2), the PTP compounds showed
similar (1, 2, 5, 15) or even higher (11-14, 17-20) hA3
affinities with respect to those of the corresponding 6-NO2 TQX
derivatives.
Among the new PTP compounds, the 4-benzoylamino-2-(4-
methoxyphenyl)pyridotriazolopyrazin-1-one derivative 18 pos-
sesses the highest hA3 affinity (Ki ) 4.54 nM) and selectivity
vs both hA1 and hA2A receptors. To determine also its hA3 vs
hA2B selectivity we tested compound 18 in cAMP assays which
evidenced a lack of hA2B affinity, being that 18 is ineffective
in inhibiting NECA-stimulated cAMP levels in hA2B CHO cells
(IC50 > 10 000). Furthermore, the effect of compound 18 in
limiting the NECA-inhibited cAMP accumulation in hA3 CHO
cells was determined. Coherently with its high hA3 affinity,
derivative 18 proved to be very potent in this test, showing an
antagonistic behavior (Figure 2, EC50 ) 36.3 ( 3.1 nM). Since
compound 18 was tested in an in vitro rat model of cerebral
ischemia, its binding affinities for the rA1, A2A, and A3 receptors
were measured. The rA1 affinity value (Ki ) 587 ( 58 nM)
was similar to the bA1 one, while the affinities for the rA2A and
rA3 receptors were, respectively, very low (I% at 10 µM ) 34)
and null (I% at 1 µM ) 0).
Finally, it is pointed out that the nitrobenzene moiety of the
TQX derivatives can be conveniently replaced by the pyridine
ring to afford the PTP derivatives as a new class of hA3 AR
antagonists. In fact, as appears evident comparing the binding
Before discussion of the binding data at the bA1 and bA2A
receptors, it is pointed out that the choice of testing the PTP

Pyrazin-1-one Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2411
Table 2. Comparison between the hA3 AR Affinities of the
Pyridotriazolopyrazin-1-ones (X ) N) and the Corresponding
6-Nitrotriazoloquinoxalin-1-ones (X ) C-NO2)
observed in the TQX series, insertion of the cyclohexyl and
cyclopentyl rings on the 4-amino group of 7 (derivatives 12
and 13, respectively) ameliorated the bA1 affinity, which falls
in the subnanomolar range, while it worsened the bA2A binding
activity. The presence of an acetyl (compound 14) or a
phenylacetyl (compound 16) residue in R1 maintained a high
affinity for the bA1 receptor while introduction of a benzoyl
group decreased it (derivative 15). When a second benzoyl
moiety was inserted on the 4-amino group of compound 15,
the affinity for the bA1 receptor was enhanced 2-fold (derivative
19). As expected, all the acyl groups caused a drop in the bA2A
affinity (compare compounds 14-16, 19 to derivative 7), and
introduction of the p-methoxy group on the 2-phenyl ring of
the 4-amido derivatives 14 and 15 reduced both the bA1 and
bA2A binding activity (compounds 17 and 18).
Finally, it is pointed out that the replacement of the nitroben-
zene moiety of the TQX series with the pyridine ring, although
performed to obtain hA3 AR antagonists, resulted in being
advantageous also for the binding at the bA1 and A2A receptors.
In fact, in general the PTP compounds 1, 2, 5-8, 11-15, 17-20
showed higher affinities at both receptors than those of the
corresponding 6-NO2 TQX derivatives16,18,19,22 (see Table 2 in
Supporting Information).
Ki (nM)
Ki (nM)
hA3 or I%
(1 µM)
hA3 or I%
(1 µM)
b
R
R4
X ) Na
X ) C-NO2
H
1
2
5
7
8
11
12
13
14
15
17
18
19
20
251 ( 16
3.3 ( 0.2
32%
33
34
35
36
37
38
39
40
41
42
43
44
45
46
279 ( 16
4.7 ( 0.52
21%
4.75 ( 0.3
47 ( 1.2
45%
281 ( 24
116 ( 24
18%
22 ( 2.60
36%
217 ( 20
27%
342 ( 21
OMe
OH
H
OMe
OH
H
H
H
H
H
H
H
656 ( 41
158 ( 9.8
1335 ( 112
15.5 ( 1.2
8.4 ( 0.9
138 ( 12
70.3 ( 6
41 ( 3.6
4.54 ( 0.2
335 ( 22
7.75 ( 0.8
NHC6H11
NHC5H9
NHCOMe
NHCOPh
OMe NHCOMe
OMe NHCOPh
H
(B) Molecular Modeling Studies. Following our recently
reported modeling investigations, we used our improved model
of the hA3 receptor, obtained by a rhodopsin-based homology
modeling (RBHM) approach,28-31 to recognize the hypothetical
binding motif of the newly synthesized PTP derivatives.
Rhodopsin-based homology modeling had been used for many
years to obtain three-dimensional models of the A3 AR, and
different A3 AR models have been published describing the
hypothetical interactions with known A3 AR ligands having
different chemical scaffolds. The recently published structures
of human ꢀ2 and turkey ꢀ1 adrenergic receptors32,33 and, in
particular, of human A2A AR34 very recently deposited provide
alternative templates for GPCR modeling. However, even if the
A2AAR as a template for modeling other AR subtypes is under
intense investigation in our laboratory, we believe that the A2A
AR-driven models is not sufficiently validated to replace
toutcour the previous one. Consequently, for the appropriate
comparison of our previously reported modeling studies, in
particular those concerning TQX analogues, we decided to
utilized the rhodopsin-based homology model in tandem with
the ligand-based homology modeling (LBHM) approach used
to simulate the conformational changes induced by ligand
binding.35
N(COPh)2
OMe N(COPh)2
a Data from Table 1. b Data from refs 16, 18, 19, and 22.
Figure 2. Effect of compound 18 on NECA-mediated cAMP ac-
cumulation (vs forskolin, set to 100%) in CHO cells stably expressing
hA3 AR. Data represent the mean ( SEM from three separate
experiments. The EC50 value for 18 was 36.3 ( 3.1 nM.
derivatives 1-20 at these receptors was made to compare the
A1 and A2A affinities of the new compounds with those of the
corresponding 6-NO2 TQX derivatives, which were tested at
the bovine species receptors.16,18,19,22
It is noted that some PTP compounds show high (derivatives
7, 12-14, 16) to good bA1 AR affinities (compounds 9, 11,
15) while they all possess low (8, 9, 11-13, 15, 16) to null
(10, 14, 17, 20) affinities for the bA2A subtype, the only
exception being the 4-amino-2-phenyl derivative 7 (Ki ) 92.6
nM).
The 4-oxo derivatives 1-6 possess low or null bA1 and bA2A
receptor affinity. In contrast, in the 4-amino series (derivatives
7-20), several compounds displayed high bA1 affinity and some
of them also showed some affinity for the bA2A receptor. The
4-amino-2-phenylpyridotriazolopyrazin-1-one derivative 7 re-
sulted in being one of the most active at either the bA1 (Ki )
3.1 nM) or the bA2A receptor (Ki ) 92.6 nM). All the
substituents introduced on the 2-phenyl moiety of 7 significantly
weakened the anchoring to both these receptor subtypes
(compounds 8-11). As expected on the basis of the SAR
The topological properties of the ligands change depending
on the bulkiness of the R1 substituents. According to the
volumes, shapes, and chemical complementarities of the ana-
lyzed compounds, we obtained three different conformational
models of the hA3 receptor using the LBHM approach.35 The
volume of the transmembrane (TM) binding cavity changes from
660 Å3 of the standard RBHM-driven model to 850 and 1000
Å3 of the LBHM-driven models. The conventional rhodopsin-
based model was used to detect the specific interaction at atomic
level of this class of compounds. This model is suitable to
rationalize the structure-activity relationships of compounds
1-11, 14, and 17. The first ligand-based homology model was
built by using compound 15 as reference, and the binding pocket
of this model has a volume of 850 Å3. The model was used to
describe the receptor-ligand interactions of compounds 12, 13,
15, 16, and 18. For compounds 19 and 20, the volume of the
cavity was expanded to 1000 Å3. The most important receptor
modeling perturbation, obtained by the application of the LBHM

2412 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Colotta et al.
and hA2A receptor subtypes. On the contrary, the hydroxyl group
of Ser247 (6.52) of hA3 receptor is appropriately positioned to
form a hydrogen bonding interaction with the carbonyl oxygen
of the 4-amido group of compounds 14-20. These observations
support the importance of a N-acyl group in modulating receptor
selectivity. Specifically with reference to 4-N-acylated deriva-
tives, the hA3 receptor affinity increases with the bulkiness of
the R1 substituent (compare the 4-amino compounds 7 and 8 to
the 4-acetylamino derivatives 14 and 17 and to the 4-benzoy-
lamino compounds 15 and 18).
The hydrophobic environment of the five nonpolar amino
acids, Ile98 (3.40), Ile186 (5.47), Leu190 (5.51), Phe239 (6.43),
and Leu244 (6.49), can justify this trend of affinity. To support
this theory, hydrophobic substituents were introduced on
4-amino derivatives: cyclohexyl (compound 12) and cyclopentyl
(compound 13) interact with this hydrophobic pocket increasing
the hA3 receptor affinity (compare compounds 12 and 13 to the
unsubstituted 4-amino derivatives 7).
Finally, as shown in Figure 3 by the comparison of the best
docking poses of both the pyrido[2,3-e]-1,2,4-triazolo[4,3-
a]pyrazin-1-one (in green, compound 20, Ki hA3 ) 7.75 ( 0.8
nM) and the 4-amido-6-nitro-2-phenyl-1,2,4-triazolo[4,3-a]qui-
noxalin-1-one antagonists (in magenta, compound 44, Ki hA3
) 342 ( 21 nM), the described hydrogen bond interaction
between the 6-nitro group of 44 with the side chain of Ser181
(5.42) is now replaced by the interaction with the same amino
acid side chain and the endocyclic nitrogen atom of 20.
Figure 3. Left: Structure superimposition hypothetical binding motif
of a representative newly synthesized pyrido[2,3-e]-1,2,4-triazolo[4,3-
a]pyrazin-1-one antagonists (in green, compound 20, Ki hA3AR ) 7.75
( 0.8) and a representative compound of 4-amido-2-aryl-1,2,4-
triazolo[4,3-a]quinoxalin-1-ones antagonists (in magenta, compound 44,
Ki hA3AR ) 342 ( 21). The most energetically favorable docked
conformations of derivatives 20 and 44 are viewed from the membrane
side facing TM helices 3 and 4. To clarify the TM cavity, the view of
TM4 from Ser138 to Thr144 has been omitted. The surface shows the
shape of the binding pocket that corresponds to residues of TMs 5, 6,
and 7. Right: Hypothetical binding motif of compound 20. Side chains
of some amino acids important for ligand recognition are highlighted.
Hydrogen atoms are not displayed.
technique, is the modification of both shape and volume of the
hA3 TM binding cavity, without altering the conventional
rhodopsin-like receptor topology.
The binding cavity reorganization induced by ligand binding
is due to the conformational change in several amino acid side
chains, such as Leu90 (3.32), Leu91 (3.33), Thr94 (3.36), His95
(3.37), Ile98 (3.40), Gln167 (EL2), Phe168 (EL2), Phe182
(5.43), Ile186 (5.47), Leu190 (5.51), Phe239 (6.44), Trp243
(6.48), Leu244 (6.49), Leu264 (7.35), Ile268 (7.39). The
numbering of the amino acids in parentheses follows the
arbitrary scheme by Ballesteros and Weinstein:36 according to
this scheme, each amino acid identifier starts with the helix
number, followed by the position relative to a reference residue
among the most conserved amino acid in that helix. The
reference residue is arbitrarily assigned the number 50.36
From the docking simulation analysis it resulted that all the
PTP derivatives share a similar binding pose in the TM region
of the hA3 adenosine receptor. As shown in Figure 3, ligand
recognition occurs in the upper region of the TM bundle and
the PTP scaffold is surrounded by the TMs 3, 5, 6, 7 with the
1-carbonyl group pointing toward the EL2 and the substituent
in the 4-position oriented toward the intracellular environment.
The phenyl ring at the 2-position is close to TMs 3, 6, and 7.
As observed for the TQX derivatives, the PTP antagonists
present a π-π stacking interaction with both side chains of
Phe168 (EL2) and Phe182 (5.43) and a hydrogen bonding
network in the most energetically stable docked conformations.
The first hydrogen bond is between the 1-carbonyl group and
the NH of the Glu167 (EL2) and Phe168 (EL2) amidic bond.
A second important hydrogen bond involves the side chains of
Thr94 (3.36), His95 (3.37), and Ser247 (6.52) that interact with
the 4-carbonyl oxygen of compounds 1-6, the 4-amino group
of compounds 7-13, or the 4-acylamino group of compounds
14-20. This region seems to be critical both for the recognition
of all antagonist structures and for receptor selectivity. In
particular, Ser247 (6.52) of hA3 receptor subtype is not present
in the corresponding position of A1 and A2 receptors, where
this amino acid is replaced by histidine (His251 in hA1, His250
in hA2A, and His251 in hA2B). The histidine side chain is bulkier
than serine, and probably for this reason large substituents at
the 4-position of PTP framework are not well tolerated by hA1
Considering the substituent on the 2-phenyl ring, the methoxy
group turned out advantageous in all the PTP derivatives, either
4-oxo, 4-amino, or 4-amido substituted. The 2-(4-methoxyphe-
nyl) derivatives 2, 8, 17, 18, and 20 possess higher hA3 AR
affinities than the corresponding 2-phenyl derivatives 1, 7, 14,
15, and 19 because the methoxy substituent can favorably
interact with Ser165 (EL2). The hydroxyl group of Ser165 is
separated by 2 Å from the p-methoxy group and correctly
oriented to form a weak hydrogen bond. The side chains of
Leu90 (3.32) and Ile268 (7.39) delimit a small hydrophobic
pocket that can accommodate the methoxy substituent but create
unfavorable steric and dipolar interaction with the other groups
(OH, F, COOH/COOEt) introduced on the 2-phenyl ring
(derivatives 3-6, 9-11). Compounds 5 and 11 present a
hydroxyl group that loses the hydrophobic interactions with
Leu90 (3.32) and Ile268 (7.39) and decreases the hA3AR
affinity. The bulkiness of the carboxy acid/ester groups of
compounds 4, 6, 10 determines the lack of affinity of these
derivatives. The fluorine atom seems to have no effect because
the 2-(4-fluorophenyl) derivatives 3 and 9 display comparable
affinities to those of the 2-phenyl compounds 1 and 7. The
fluorine atom could interact as hydrogen bond acceptor, but in
the most energetically stable conformations of compounds 3
and 9, the distance between the fluorine and the hydroxy group
of Ser165 (EL2) is more than 3 Å.
In summary, as stated above, the nitrobenzene moiety of the
triazoloquinoxaline-1-one derivatives can be conveniently re-
placed by the pyridine ring to afford a new class of AR
antagonists, the pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one
derivatives. The steric clashes created by the nitrobenzene with
the backbone of TM5, and in particular with the peptide bond
of Ser181 (5.42) and Phe182 (5.43), have been overcome, while
the electrostatic effect is conserved. Moreover, the endocyclic
nitrogen atom can favorably interact with the side chain of
Ser181 (5.42) through a hydrogen-bond interaction. This
structural modification turned out particularly beneficial in the
4-amino series B when the volume of the molecule is increased

Pyrazin-1-one Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2413
by the presence of cycloalkyl and acyl substituents on the
4-amino group. Indeed, the hA3 affinities of the PTP derivatives
12-15, 17-20 are significantly higher than those of the
corresponding TQX 39-46,16,18,19,22 with the only exception
being the 4-benzoylamino derivative 15 that shows a 3-fold
reduced A3 receptor affinity compared to the triazoloquinoxaline
analogue 42 (Table 2).
(C) Electrophysiological Studies. Compound 18 was inves-
tigated for its effect on synaptic transmission during severe
oxygen glucose deprivation (OGD) in the CA1 region of rat
hippocampalslices.Inagreementwithourpreviousresults,11,13,12,24
a direct current (dc) shift, that is, an electrophysiological
signature of anoxic depolarization (AD), was always present
when we applied an OGD episode of 7 min (n ) 8, Figure 4).
AD consists in a drop of neuron and glia membrane potential
and represents an unequivocal sign of neuronal injury during
ischemia both in vivo and in vitro.37 The dc shift presented a
mean latency of 6.6 ( 0.1 min (calculated from the beginning
of OGD) and a mean peak amplitude of 7.9 ( 0.5 mV (n ) 8)
(Figure 4A). In addition, 7-min OGD completely abolished the
field excitatory postsinaptic potentials (fEPSPs) which did not
recover their amplitude after return to oxygenated solution
(Figure 4C). Compound 18 (10 nM, n ) 8) was checked for its
effects on 7 min OGD. Figure 4B,C shows that derivative 18
prevented AD in seven out of the eight slices and delayed it in
the remaining slice (Figure 4B). Moreover, it induced a
significant fEPSP recovery of 77.6 ( 10.4%, (n ) 8, p < 0.001,
one-way ANOVA, Newman-Keuls multiple-comparison post
hoc test) when compared to that found in untreated OGD slices
(2.3 ( 2.0%, n ) 8).
It is mentioned that fEPSP reduction in the CA1 hippocampus
during OGD is most likely due to a reduction of glutamate
release due to activation of presynaptic A1 receptors induced
by extracellular adenosine. This effect is detectable by the first
2 min of ischemia.38 At the concentration used in our experi-
ments, the A1 antagonistic effect of compound 18 did not appear,
since the compound did not modify the depression of fEPSP
induced by OGD (Figure 4C).
In order to better characterize the effects of the A3 antagonist
on AD development, we prolonged the duration of ischemic
insult from 7 to 30 min, an OGD time duration invariably
harmful for the tissue.11,39 As illustrated in Figure 4D, 30 min
of OGD elicited the appearance of AD in all experimental
groups. When OGD was applied in the presence of different
concentrations of compound 18, the latency of AD was always
delayed (Figure 4B). A statistically significant difference from
control was reached at the concentration of 10 nM (from 6.8 (
0.1 min, n ) 15, in the absence to 8.9 ( 0.8, n ) 5, P < 0.01,
in the presence of 10 nM compound 18).
The effects of derivative 18, on both fEPSPs and the AD
delay, are similar to those previously obtained11-13,24 with other
structurally diverse compounds which proved to be effective at
nanomolar concentrations in the same rat model. It is pointed
out that all the previously tested derivatives11-13,24 possessed
nanomolar affinities for the hA3 receptor but most of them
showed a very low affinity for the rA3 receptor. The discrepancy
between the potency demonstrated in the rat hippocampus
experiments and the rA3 binding affinity is much more striking
for compound 18, since it shows no affinity for the cloned rA3
receptor. We might hypothesize that the specific conformation
assumed by A3 receptors in CA1 hippocampal slices during
ischemia may differ from that assessed in receptor binding
experiments on disrupted HEK cell membranes. Alternatively,
there could be another mechanism responsible for the electro-
Figure 4. Compound 18 blocks or delays the appearance of AD
induced by OGD and prevents the irreversible loss of neurotransmission
induced by 7 min OGD in CA1 rat hippocampus. AD was recorded as
the negative dc shift in response to 7 min OGD (solid bar) in the absence
(A) (n ) 8) and in the presence (B) (n ) 8) of 10 nM compound 18
(open bar). (C) Graph shows the time course of 7 min OGD effects on
fepsp amplitude in untreated (black circles, mean ( SEM, n ) 8) OGD
slices and in 10 nM compound 18 (open circles, mean ( SEM, n ) 8)
treated OGD slices. fEPSP amplitude is expressed as percent of baseline.
Note that after reperfusion in oxygenated solution, a significant recovery
of fEPSP was found only in treated OGD slices. (D) Each column
represents the mean ( SEM of AD latency recorded in hippocampal
slices during 30 min of OGD and measured from the beginning of
ischemic insult. Note also that compound 18 significantly postpones
AD development when used at a concentration of 10 nM: (*) p < 0.01,
unpaired two-tailed Student’s t test in comparison to untreated OGD
slices. The number (n) of slices tested is reported inside columns.
physiological effects of 18. Since it is a very lipophilic
compound, it might affect AD development and recovery of
fEPSP by acting at an intracellular molecular target.
In any case, compound 18 delays the occurrence of AD,
brought about by a severe OGD in the CA1 region of rat
hippocampal slices, and induces a significant recovery of an
otherwise disrupted neurotransmission. Since AD is an un-
equivocal sign of brain damage during ischemia,38 compounds
that delay the AD initiation or propagation may be developed
as neuroprotective agents in brain ischemia. The availability of
drugs able to counteract stroke-induced neurodegeneration is
an as yet unmet need.

2414 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Colotta et al.
suspension, cooled at room temperature, was diluted with water
(about 50 mL) and the solid was collected by filtration.
1: yield 80%; mp >300 °C (AcOH); H NMR 7.33-7.40 (m,
(D) Conclusion. The present study has led to the identification
of the pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one tricyclic
system as a new scaffold to obtain potent and selective hA3
AR antagonists. In fact, some of the newly synthesized
derivatives showed high hA3 AR affinities (Ki < 50 nM) and
selectivities vs both hA1 and hA2A receptors. Our recently
described ligand-based homology model of the hA3 receptor
has permitted depiction of the hypothetical binding mode of
thenewderivativesandtorationalizetheobservedstructure-affinity
relationships. The selected 4-benzoylamino-2-(4-methoxyphe-
nyl)pyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1-one (18), tested
in an in vitro rat model of cerebral ischemia, proved to be
effective in preventing the failure of synaptic activity induced
by 7 min of OGD and delayed the occurrence of AD caused by
a prolonged (30 min) OGD.
1
2H, 1 ar + H-8), 7.56-7.60 (t, 2H, ar), 8.01 (d, 2H, ar, J ) 8.6
Hz), 8.34 (d, 1H, H-7, J ) 4.0 Hz), 8.84 (d, 1H, H-9, J ) 8.1 Hz),
12.45 (br s, 1H, NH). Anal. (C14H9N5O2) C, H, N.
2: yield 65%; mp >300 °C (DMF); 1H NMR 3.82 (s, 3H, CH3),
8.13 (d, 2H, ar, J ) 9.1 Hz), 7.34 (dd, 1H, H-8, J ) 8.1 and 4.8
Hz), 7.87 (d, 2H, ar, J ) 9.1 Hz), 8.33 (d, 1H, H-7, J ) 4.8 Hz),
8.83 (d, 1H, H-9, J ) 8.1 Hz), 12.41 (s, 1H, NH). Anal.
(C15H11N5O3) C, H, N.
3: yield 40%; mp >300 °C (AcOH); 1H NMR 7.35 (dd, 1H, H-8,
J ) 8.0 and 4.8 Hz), 7.41-7.45 (m, 2H, ar), 8.00-8.04 (m, 2H,
ar), 8.34 (d, 1H, H-7, J ) 4.8 Hz), 8.83 (d, 1H, H-9, J ) 8.0 Hz),
12.47 (br s, 1H, NH); IR 3414, 1728, 1705. Anal. (C14H8FN5O2)
C, H, N.
1
4: yield 45%; mp >300 °C (DMF); H NMR 1.35 (t, 3H, CH3,
J ) 7.1 Hz), 4.35 (q, 2H, CH2, J ) 7.1 Hz), 7.36 (dd, 1H, H-8, J
) 8.1 and 4.8 Hz), 8.16 (d, 2H, ar, J ) 8.8 Hz), 8.21 (d, 2H, ar,
J ) 8.8 Hz), 8.35 (d, 1H, H-7, J ) 4.8 Hz), 8.83 (d, 1H, H-9, J)
8.1 Hz), 12.52 (br s, 1H, NH); IR 3402, 1722, 1708, 1693. Anal.
(C17H13N5O4) C, H, N.
Experimental Section
(A) Chemistry. 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 DMSO-
d6 or CDCl3. The following abbreviations are used: s ) singlet, d
) doublet, dd ) double doublet, t ) triplet, m ) multiplet, br )
broad, ar ) aromatic protons, al ) aliphatic protons.
General Procedure for the Synthesis of 2-Arylhydrazono-1,2-
dihydropyrido[2,3-b]pyrazin-3(4H)-ones 25-28. Ethyl N1-arylhy-
drazono-N2-chloroacetates 21-2425-27 (11 mmol) were reacted
with 1,2-diamminopyridine (9.2 mmol) in refluxing ethanol (50 mL)
and triethylamine (1.5 mL) for 3-4 h. The suspension was cooled
at room temperature, and the solid was collected by filtration and
washed with water and then with diethyl ether.
2-(4-Hydroxyphenyl)-1,2-dihydropyrido[2,3-e]-1,2,4-triazolo[4,3-
a]pyrazin-1,4(2H,5H)-dione 5. A BBr3 methylene chloride solution
(1 M, 5.8 mL) was dropwise added at 0 °C to a suspension of
compound 2 (0.97 mmol) in anhydrous methylene chloride (15 mL)
under nitrogen atmosphere. The suspension was stirred at room
temperature for 5 days, then diluted with water (4 mL) and the
mixture neutralized with 1 M NaOH solution. The solid was
collected by filtration and washed with water. Yield 90%; mp >300
1
°C (DMF); H NMR 6.92 (d, 2H, ar, J ) 8.9 Hz), 7.33 (dd, 1H,
H-8, J ) 8.1 and 4.8 Hz), 7.72 (d, 2H, ar, J ) 8.9 Hz), 8.32 (d,
1H, H-7, J ) 4.8 Hz), 8.83 (d, 1H, H-9, J ) 8.1 Hz), 9.76 (s, 1H,
OH), 12.40 (s, 1H, NH); IR 3420, 3370, 1712, 1688. Anal.
(C14H9N5O3) C, H, N.
2-(4-Carboxyphenyl)-1,2-dihydropyrido[2,3-e]-1,2,4-triazolo[4,3-
a]pyrazin-1,4(2H,5H)-dione 6. A suspension of the ester 4 (0.85
mmol) in ethanol (20 mL) and 0.8 M NaOH aqueous solution (20
mL) was stirred at room temperature for 24 h, then acidified with
6 N HCl. The solid was collected by filtration and washed with
1
water. Yield 75%; mp >300 °C (DMF); H NMR 7.36 (dd, 1H,
1
25: yield 80%; mp >300 °C (DMF); H NMR 6.65-6.78 (m,
H-8, J ) 8.1 and 4.8 Hz), 8.13 (d, 2H, ar, J ) 8.8 Hz), 8.18 (d,
2H, ar, J ) 8.8 Hz), 8.35 (d, 1H, H-7, J ) 4.8 Hz), 8.83 (d, 1H,
H-9, J) 8.1 Hz), 12.50 (br s, 1H, NH), 13.07 (br s, 1H, OH); IR
3395, 1723, 1695. Anal. (C15H9N5O4) C, H, N.
ar), 6.91-7.06 (m, ar), 7.21-7.29 (m, ar), 7.55 (d, ar, J ) 8.4
Hz), 7.65 (d, ar, J ) 7.8 Hz), 7.78 (d, H-7, J ) 4.8 Hz), 7.89 (s,
NH), 8.12 (d, H-9, J ) 3.9 Hz), 9.65 (s, NH), 9.74 (br s, NH),
10.97 (s, NH), 11.38 (s, NH), 12.6 (s, NH). Anal. (C13H11N5O) C,
H, N.
General Procedure for the Synthesis of 2-Aryl-4-chloro-1,2-
dihydropyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1(2H)-ones 29-32.
A mixture of compounds 1-4 (4 mmol), phosphorus penthachloride
(4 mmol), phosphorus oxychloride (40 mL), and anhydrous pyridine
(0.3 mL) was refluxed until the disappearance (TLC monitoring)
of the starting material (2-18 h). Evaporation of the excess of
phosphorus oxychloride at reduced pressure gave a residue that was
treated with ice-water (50 mL). The solid was filtered, washed
with water, and dried. The 4-chloro derivatives, obtained in high
overall yields (75-90%) were unstable upon recrystallization;
however, they were pure enough to be used without further
purification.
26: yield 68%; mp 273-275 °C (DMF); 1H NMR 3.65 (s, CH3),
3.69 (s, CH3), 6.81 (d, ar, J ) 8.8 Hz), 6.84-6.97 (m, ar), 7.10 (d,
ar, J ) 8.8 Hz), 7.51-7.53 (m, ar + NH), 7.63 (d, ar, J ) 6.7 Hz),
7.75 (d, H-7, J ) 3.5 Hz), 8.13 (d, H-7, J ) 3.5 Hz), 9.61 (s, NH),
9.84 (s, NH), 11.16 (s, NH), 11.29 (s, NH), 12.66 (s, NH). Anal.
(C14H13N5O2) C, H, N.
1
27: yield 96%; mp 288-289 °C (DMF); H NMR 6.77-6.79
(m, ar), 6.99-7.17 (m, ar), 7.51 (d, ar, J ) 7.8 Hz), 7.65 (d, ar, J
) 7.7 Hz), 7.79 (d, H-7, J ) 4.3 Hz), 7.85 (s, NH), 8.15 (d, H-7,
J ) 3.9 Hz), 9.59 (s, NH), 9.94-9.96 (m, NH), 10.96-11.16 (m,
NH), 11.35 (br s, NH), 12.67 (br s NH). Anal. (C13H10FN5O) C,
H, N.
1
29: H NMR 7.40 (t, 1H, ar, J) 6.7 Hz), 7.52-7.59 (m, 2H,
ar), 7.76 (dd, 1H, H-8, J ) 8.1 and 4.4 Hz), 8.01 (d, 2H, ar, J )
8.0 Hz), 8.73 (d, 1H, H-7, J ) 4.4 Hz), 9.05 (s, 1H, H-9, J ) 8.1
Hz).
1
28: yield 85%; mp 273-275 °C (2-methoxyethanol); H NMR
1.20-1.34 (m, CH3), 4.20-4.38 (m, CH2), 6.72-6.85 (m, ar),
6.99-7.18 (m, ar), 7.23 (d, ar, J ) 8.6 Hz), 7.56-7.82 (m, ar),
7.83 (d, ar, J ) 9.4 Hz), 8.16 (d, H-7, J ) 4.7 Hz), 8.67 (s, NH),
9.79 (s, NH), 10.33 (s, NH), 10.71 (s, NH), 11.46 (s, NH), 11.53
(s, NH), 12.74 (s, NH). Anal. (C16H15N5O3) C, H, N.
1
30: H NMR 3.81 (s, 3H, Me), 7.14 (d, 2H, ar, J ) 9.1 Hz),
7.77 (dd, 1H, H-8, J ) 8.1 and 4.4 Hz), 7.89 (d, 2H, ar, J ) 9.1
Hz), 8.73 (d, 1H, H-7, J ) 4.4 Hz), 9.05 (d, 1H, H-9, J ) 8.1 Hz).
31: 1H NMR 7.44-7.48 (m, 2H, ar), 7.79 (dd, 1H, H-8, J ) 8.0
and 4.8 Hz), 8.04-8.08 (m, 2H, ar), 8.76 (d, 1H, H-7, J ) 4.8
Hz), 9.06 (d, 1H, H-9, J ) 8.0 Hz).
General Procedure for the Synthesis of 2-Aryl-1,2-dihydropy-
rido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1,4(2H,5H)-diones 1-4. De-
rivatives 25-28 (4 mmol) were reacted with triphosgene (4 mmol,
with 25 and 26; 12 and 8 mmol with 27 and 28, respectively) in
refluxing anhydrous tetrahydrofuran (50 mL) until the disappearance
(TLC monitoring) of the starting hydrazones (8-20 h). The
1
32: H NMR 1.36 (t, 3H, CH3, J ) 7.1 Hz), 4.36 (q, 2H, CH2,
J ) 7.1 Hz),7.81 (dd, 1H, H-8, J ) 8.2 and 4.7 Hz), 8.12-8.15
(m, 4H, ar), 8.76 (d, 1H, H-7, J ) 4.7 Hz), 9.05 (d, 1H, H-9, J )
8.2 Hz).

Pyrazin-1-one Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2415
General Procedure for the Synthesis of 4-Amino-2-aryl-1,2-
dihydropyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1(2H)-ones 7-10.
A suspension of the 4-chloro derivatives 29-32 (2 mmol), in
absolute ethanol (30 mL) saturated with ammonia, was heated
overnight at 120 °C in a sealed tube. After the mixture was cooled
at room temperature, the solid was collected by filtration and washed
with water.
14: yield 70%; mp >300 °C (DMF); 1H NMR 2.46 (s, 3H, CH3),
7.40 (t, 1H, ar, J ) 7.3 Hz), 7.57-7.61 (m, 3H, 2ar + H-7), 8.11
(d, 2H, ar, J ) 8.0 Hz), 8.63 (d, 1H, H-7, J ) 4.8 Hz), 8.99 (d,
1H, H-9, J ) 8.1 Hz), 10.72 (br s, 1H, NH); IR 1715, 1695. Anal.
(C16H12N6O2) C, H, N.
1
15: yield 84%; mp 244-246 °C (2-methoxyethanol); H NMR
7.38 (t, 1H, ar, J ) 7.4 Hz), 7.55-7.69 (m, 6H, ar), 7.99 (d, 2H,
ar, J ) 7.6 Hz), 8.03-8.13 (m, 2H, ar), 8.57 (br s, 1H, ar), 8.98
(br s, 1H, H-9), 11.45 (br s, 1H, NH); IR 1719, 1654. Anal.
(C21H14N6O2) C, H, N.
1
7: yield 75%; mp >300 °C (AcOH); H NMR 7.25-7.40 (m,
4H, ar), 7.56 (t, 2H, ar, J ) 7.7 Hz), 7.90-8.06 (m, 4H, 2ar +
NH2), 8.42 (d, 1H, H-7, J ) 4.8 Hz), 8.85 (d, 1H, H-9, J ) 8.1
Hz); IR 3420, 3240, 1700. Anal. (C14H10N6O) C, H, N.
8: yield 70%; mp >300 °C (AcOH); 1H NMR 3.82 (s, 3H, CH3),
7.13 (d, 2H, ar, J ) 9.1 Hz), 7.27 (dd, 1H, H-8, J ) 8.0 and 4.8
Hz), 7.89-8.05 (m, 4H, 2ar + NH2), 8.43 (d, 1H, H-7, J ) 4.8
Hz), 8.85 (d, 1H, H-9, J ) 8.0 Hz); IR 3428, 3230, 1710. Anal.
(C15H12N6O2) C, H, N.
16: yield 35%; mp 240-241 °C (acetonitrile); 1H NMR 4.11 (s,
2H, CH2), 7.28-7.43 (m, 6H, ar), 7.58-7.62 (m, 3H, ar), 8.09 (d,
2H, ar, J ) 7.7 Hz), 8.64 (d, 1H, H-7, J ) 4.7 Hz), 8.99 (d, 1H,
H-9, J ) 8.1 Hz), 10.95 (s, 1H, NH); IR 3340-3430, 1727, 1688.
Anal. (C22H16N6O2) C, H, N.
1
17: yield 55%; mp 278-280 °C (2-ethoxyethanol); H NMR
1
2.42 (s, 3H, COCH3), 3.83 (s, 3H, OCH3), 7.14 (d, 2H, ar, J ) 9.0
Hz), 7.57 (dd, 1H, H-8, J ) 7.9 and 4.5 Hz), 7.96 (d, 2H, ar, J )
9.0 Hz), 8.63 (d, 1H, H-7, J ) 4.5 Hz), 8.98 (d, 1H, H-9, J ) 7.9
Hz), 10.73 (br s, 1H, NH); IR 3485-3358 1716, 1691. Anal.
(C17H14N6O3) C, H, N.
9: yield 95%; mp >300 °C (2-methoxyethanol); H NMR 7.28
(dd, 1H, H-8, J ) 8.0 and 4.8 Hz), 7.44 (t, 2H, J ) 8.8 Hz),
7.80-8.08 (m, 4H, 2ar + NH2), 8.44 (d, 1H, H-7, J ) 4.8 Hz),
8.85 (d, 1H, H-9, J ) 8.0 Hz); IR 3482, 1715. Anal. (C14H9FN6O)
C, H, N.
10: yield 64%; mp >300 °C (DMF); 1H NMR 1.36 (t, 3H, CH3,
J ) 7.1 Hz), 4.36 (q, 2H, CH2, J ) 7.1 Hz), 7.29 (dd, 1H, H-8, J
) 8.0 and 4.8 Hz), 8.01 (br s, 2H, NH2), 8.16 (d, 2H, ar, J ) 8.7
Hz), 8.26 (d, 2H, ar, J ) 8.7 Hz), 8.45 (d, 1H, H-7, J ) 4.8 Hz),
8.85 (d, 1H, H-9, J ) 8.0 Hz); IR 3380, 1727, 1715. Anal.
(C17H14N6O3) C, H, N.
1
18: yield 88%; mp 250-252 °C (2-ethoxyethanol); H NMR
3.81 (s, 3H, CH3), 7.12 (d, 2H, ar, J ) 9.0 Hz), 7.55-7.69 (m, 4H,
ar), 7.85 (d, 2H, ar, J ) 8.3 Hz), 8.11-8.13 (m, 2H, ar), 8.52-8.56
(m, 1H, ar), 9.02 (br s, 1H, H-9), 11.42 (br s, 1H, NH); IR 1717.
Anal. (C22H16N6O3) C, H, N.
4-Dibenzoylamino-2-phenyl-1,2-dihydropyrido[2,3-e]-1,2,4-tria-
zolo[4,3-a]pyrazin-1(2H)-ones 19. A mixture of the 4-amino deriva-
tive 7 (1.8 mmol) and benzoyl chloride (16 mmol) in anhydrous
pyridine was refluxed for 28 h. After cooling at room temperature,
the suspension was diluted with water and the solid collected by
filtration and recrystallized. Yield 60%; mp 276-278 °C (DMF);
1H NMR 7.36 (t, 1H, ar, J ) 7.4 Hz), 7.51-7.55 (m, 6H, ar),
7.65-7.68 (m, 2H, ar), 7.73 (dd, 1H, H-8, J ) 8.2 and 4.5 Hz),
7.87 (d, 2H, ar, J ) 8.0 Hz), 7.93 (d, 4H, ar, J ) 7.4 Hz), 8.63 (d,
1H, H-7, J ) 4.5 Hz), 9.07 (d, 1H, H-9, J ) 8.2 Hz); IR 1725,
1710. Anal. (C28H18N6O3) C, H, N.
4-Amino-2-(4-hydroxyphenyl)-1,2-dihydropyrido[2,3-e]-1,2,4-
triazolo[4,3-a]pyrazin-1(2H)-one 11. A BBr3 methylene chloride
solution (1M, 5.8 mL) was dropwise added at 0 °C to a suspension
of compound 8 (0.97 mmol) in anhydrous methylene chloride (15
mL) under nitrogen atmosphere. The suspension was stirred at room
temperature for 4 days, then diluted with water (4 mL) and the
mixture neutralized with 1 M NaOH solution. The solid was
collected by filtration and washed with water. Yield 95%; mp >300
1
°C (DMF); H NMR 6.92 (d, 2H, ar, J ) 8.9 Hz), 7.26 (dd, 1H,
H-8, J ) 8.0 and 4.7 Hz), 7.75 (d, 2H, ar, J ) 8.9 Hz), 7.80 (br s,
2H, NH2), 8.43 (d, 1H, H-7, J ) 4.7 Hz), 8.85 (d, 1H, H-9, J )
8.0 Hz), 9.77 (s, 1H, OH); IR 3440, 3315, 1720. Anal. (C14H10N6O2)
C, H, N.
4-Dibenzoylamino-2-(4-methoxyphenyl)-1,2-dihydropyrido[2,3-
e]-1,2,4-triazolo[4,3-a]pyrazin-1(2H)-ones 20. A mixture of the
4-amino derivative 8 (1.3 mmol) and benzoyl chloride (3.9 mmol)
in anhydrous pyridine was refluxed for 30 h. After cooling at room
temperature, the suspension was diluted with water and the solid
was collected by filtration. The crude solid was a mixture of
4-Cyclohexylamino-2-phenyl-1,2-dihydropyrido[2,3-e]-1,2,4-tria-
zolo[4,3-a]pyrazin-1(2H)-one 12. A mixture of the 4-chloro deriva-
tive 29 (1 mmol) and cyclohexylamine (1.2 mmol) in absolute
ethanol (5 mL) was heated overnight at 120 °C in a sealed tube.
After the mixture was cooled at room temperature, the solid was
collected by filtration and washed with water. Yield 78%; mp
199-200 °C (cyclohexane); 1H NMR 1.18-2.0 (m, 10H, al),
4.10-4.25 (m, 1H, al), 7.20-7.39 (m, 2H, ar + H-8), 7.57 (t, 2H,
ar, J ) 7.2 Hz), 8.06 (d, 2H, ar, J ) 7.2 Hz), 8.19 (d, 1H, NH, J
) 8.7 Hz), 8.41 (d, 1H, H-7, J ) 3.7 Hz), 8.85(d, 1H, H-9, J )
6.6 Hz); IR 3500, 3370, 1700. Anal. (C20H20N6O) C, H, N.
1
compounds 20 and 15 (about 1:1 ratio, from H NMR spectrum)
which where separated by column chromatography (eluent CH2Cl2/
EtOAc, 7:3). Evaporation of the first eluates afforded the desired
compound 20; from the second eluates the 4-benzoylamino deriva-
tive 15 and also the 1,4-dione 2 was obtained. Yield 8%; mp
287-289 °C (2-methoxyethanol); 1H NMR 3.79 (s, 3H, CH3), 7.10
(d, 2H, ar, J ) 9.1 Hz), 7.53 (t, 4H, ar, J ) 7.7 Hz), 7.67 (t, 2H,
ar, J) 7.7 Hz), 7.72-7.77 (m, 3H, 2ar + H-8), 7.93 (d, 4H, ar, J
) 7.7 Hz), 8.63 (d, 1H, H-7, J ) 4.7 Hz), 9.07 (d, 1H, H-9, J )
8.2 Hz); IR 1715, 1704. Anal. (C29H20N6O4) C, H, N.
(B) Computational Methodologies. All modeling studies were
carried out on a 16 CPU (Intel Core 2 Quad CPU 2.40 GHz) Linux
cluster. Homology modeling, energy calculation, and docking
studies were performed using the Molecular Operating Environment
(MOE, version 2007.09) suite.40 All docked structures were fully
optimized without geometry constraints using RHF/AM1 semiem-
pirical calculations. Vibrational frequency analysis was used to
characterize the minima stationary points (zero imaginary frequen-
cies). The software package MOPAC (version 7),41 implemented
in MOE suite, was utilized for all quantum mechanical calculations.
Homology Model of the hA3 AR. On the basis of 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,42 guided by the highly conserved
amino acid residues, including the DRY motif (Asp3.49, Arg3.50,
and Tyr3.51) and three proline residues (Pro4.60, Pro6.50, and
Pro7.50) in the TM segments of GPCRs. The same boundaries were
4-Cyclopentylamino-2-phenyl-1,2-dihydropyrido[2,3-e]-1,2,4-
triazolo[4,3-a]pyrazin-1(2H)-one 13. The title compound was
prepared by reacting derivative 29 (1.0 mmol) and cyclopentylamine
(1.2 mmol) in the conditions described above to prepare compound
1
12. Yield 75%; mp 190-191 °C (cyclohexane/EtOAc); H NMR
1.5-2.5 (m, 8H, al), 4.42-4.65 (m, 1H, al), 7.21-7.39 (m, 2H, ar
+ H-8), 7.57 (t, 2H, ar, J ) 7.3 Hz), 8.06 (d, 2H, ar, J ) 7.3 Hz),
8.29 (d, 1H, NH, J ) 7.7 Hz), 8.41 (d, 1H, H-7, J ) 4.7 Hz), 8.84
(d, 1H, H-9, J ) 8.0 Hz); IR 3290, 1730. Anal. (C19H18N6O) C, H,
N.
General Procedure for the Synthesis of 4-Amido-2-aryl-1,2-
dihydropyrido[2,3-e]-1,2,4-triazolo[4,3-a]pyrazin-1(2H)-ones 14-18.
A mixture of the 4-amino derivative 7 or 8 (1.1 mmol) and acetyl
chloride (1.2 mmol), benzoyl chloride (1.7 mmol), or phenylacetyl
chloride (3.2 mmol) in anhydrous pyridine (10 mL) was refluxed
until the disappearance (TLC monitoring) of the starting 4-amino
derivative (2-5 h). After cooling at room temperature, the
suspension was diluted with water (30 mL) and the solid was
collected and washed with water.

2416 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Colotta et al.
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,42 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 second
extracellular loop (EL2), which has been described in bovine
rhodopsin as folding back over transmembrane helices42 and,
therefore, limiting the size of the active site. Hence, amino acids
of this loop could be involved in direct interactions with the ligands.
A driving force to this peculiar fold of the EL2 loop might be the
presence of a disulfide bridge between cysteines in TM3 and EL2.
Since 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.43 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 (Ramachandran and ꢁ plots measure
ꢂ/ψ and ꢁ1/ꢁ2 angles, clash contacts reports) implemented in MOE
suite.40
Search protocol45 and the MMFF94 force field.46 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 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 explora-
tion of all side chains involved in the antagonist-binding was carried
out. Rotamer exploration methodology was implemented in MOE
suite.40
(C) Biochemistry. Bovine A1 and A2A Receptor Binding.
Displacement of [3H]DPCPX from A1 ARs in bovine cortical
membranes and [3H]CGS 21680 from A2A ARs in bovine striatal
membranes was performed as described in refs 47 and 48,
respectively.
Human A1, A2A, and A3 Receptor Binding. Binding experiments
at hA1 and hA2A ARs, stably expressed in CHO cells, were
performed as previously described,49 using [3H]DPCPX and [3H]N-
ECA, respectively, as radioligands. Displacement of [125I]AB-
MECA from hA3 AR, stably expressed in CHO cells, was
performed as reported in ref 16.
Rat A1 and A2A Receptor Binding. Rat brain cortex was dissected
from male Wistar rats. Membrane preparation was carried out as
previously reported for the bovine membrane preparation.48 The
A1 binding assays were performed in triplicate by incubating
aliquots of brain cortex membranes (40-50 µg of protein) at 25
°C for 180 min in 0.5 mL of binding buffer (50 mM Tris-HCl, 2
mM MgCl2 pH 7.7) containing 0.2-0.5 nM [3H]DPCPX. Nonspe-
cific binding was defined in the presence of 20 µM R-PIA.
Incubation was terminated by rapid filtration through Whatman
GF/C glass microfiber filters and washing twice with 4 mL of ice-
cold buffer. The dissociation constant (Kd) of [3H]DPCPX on brain
cortex was 0.51 nM.50 Binding assays at the rA2A were performed
as previously described.48
Ligand-Based Homology Modeling. We have recently revisited
the rhodopsin-based model of the human A3 receptor in its resting
state (antagonist-like state), taking into account a novel strategy to
simulate the possible receptor reorganization induce by the antagonist-
binding.35 We called this new strategy ligand-based homology
modeling. Briefly, the ligand-based homology modeling technique
is an evolution of a conventional homology modeling algorithm
based on a Boltzmann-weighted randomized modeling procedure
adapted from Levitt,44 combined with specialized logic for the
proper handling of insertions and deletions any selected atoms. The
ligand-based option is very useful when one wishes to build a
homology model in the presence of a ligand docked to the primary
template, or other proteins known to be complexed with the
sequence to be modeled.40 In this specific case both model building
and refinement take into account the presence of the ligand in terms
of specific steric and chemical features. In order to generate an
initial ensemble of ligand poses, a conventional docking procedure
(see next paragraphs for details) with reduced van der Waals radii
(equal to 75%) and an increased Coulomb-vdW cutoff (cutoff on
10 Å, cutoff on 12 Å) has been performed. For each pose, a
homology model is then generated to accommodate the ligand by
reorienting nearby side chains. These residues and the ligand are
then locally minimized. Finally, each ligand is redocked into its
corresponding low energy protein structures and the resulting
complexes are ranked according to MOEScore.40
Different quantitative measures of molecular volume of the
receptor binding cavities have been carried out using MOE suite.40
Prediction of antagonist-receptor complex stability (in terms of
corresponding pKi value) and the quantitative analysis for non-
bonded intermolecular interactions (H-bonds, transition metal, water
bridges, hydrophobic) were calculated and visualized using several
tools implemented into MOE suite.40
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.40 Searching is
conducted within a user-specified 3D docking box, using the Tabu`
Rat A3 Receptor Binding. Membrane preparation of rat A3
adenosine receptor expressed in HEK-293/EBNA cells was pur-
chased from PerkinElmer Life Sciences (Boston, MA), and the
radioligand binding assay was performed following manufacturer’s
instructions. Briefly, aliquots of cell membranes (3.9 µg) were
incubated at 25 °C for 120 min in 100 µL of buffer (50 mM Tris-
HCl, 10 mM MgCl2, 1 mM EDTA, 1.5 units/mL ADA, pH 7.6) in
the presence of 2.1 nM [125I]ABMECA. Nonspecific binding was
determined in the presence of 100 µM R-PIA. The Kd value of
[
125I]AB-MECA in rat A3 HEK-293/EBNA cell membranes was
1.97 nM. Bound radioactivity was separated by rapid filtration
through GF/C glass fiber filters presoaked in 0.5% polyethylenimine
and washed nine times with 0.5 mL of ice-cold 50 mM Tris-HCl,
10 mM MgCl2, 1 mM EDTA, pH 7.6. The radioactivity was
measured by γ scintillation spectrometry.
Measurement of cAMP Levels on CHO Cells Transfected
with Human A2B and A3 ARs. Intracellular cAMP levels were
measured using a competitive protein binding method.51 CHO cells
(∼60 000), stably expressing hA2B or hA3 ARs, were plated in 24-
well plates. After 48 h, the medium was removed, and the cells
were incubated at 37 °C for 15 min with 0.5 mL of di DMEM in
the presence of Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)imida-
zolidin-2-one) (20 µM) and adenosine deaminase (1 U/mL). A stock
1 mM solution of the tested compound was prepared in DMSO,
and subsequent dilutions were accomplished in distilled water. The
antagonistic profile of the new compound toward hA2B AR was
evaluated assessing its ability to inhibit 100 nM NECA-mediated
accumulation of cAMP. The antagonistic profile of the new
compound toward hA3 AR was evaluated by assessing its ability
to counteract 100 nM NECA-mediated inhibition of cAMP ac-
cumulation stimulated by 1 µM forskolin. Cells were incubated in
the reaction medium (15 min at 37 °C) with different compound
concentrations (1 nM to 10 µM) and then treated with NECA.
The reaction was terminated by removing the medium and adding
0.4 N HCl. After 30 min the lysate was neutralized with 4 N KOH
and the suspension was centrifuged at 800g for 5 min. To determine

Pyrazin-1-one Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2417
cyclic AMP production, the binding protein, prepared from beef
adrenal glands, was incubated with [3H]cAMP (2 nM) in distilled
water, 50 µL of cell lysate, or standard cAMP (0-16 pmol) at 4
°C for 150 min in a total volume of 300 µL. Bound radioactivity
was separated by rapid filtration through GF/C glass fiber filters
and washed twice with 4 mL of 50 mM Tris-HCl, pH 7.4. The
radioactivity was measured by liquid scintillation spectrometry.
Data Analysis. The concentration of the tested compounds that
produced 50% inhibition of specific [3H]DPCPX, [3H]NECA,
[3H]CGS 21680, and [125I]AB-MECA binding (IC50) was calculated
using a nonlinear regression method implemented by the InPlot
program (Graph-Pad, San Diego, CA) with five concentrations of
displacer, each performed in triplicate. Inhibition constants (Ki) were
calculated according to the Cheng--Prusoff equation.52 The
dissociation constant (Kd) values of [3H]DPCPX and [3H]CGS
21680 in cortical and striatal bovine brain membranes were 0.3
and 14 nM, respectively.The Kd values of [3H]DPCPX, [3H]NECA,
and [125I]AB-MECA in hA1, hA2A, and hA3 ARs in CHO cell
membranes were 3, 30, and 1.4 nM, respectively. EC50 values
obtained in cAMP assays were calculated by nonlinear regression
analysis using the equation for a sigmoid concentration-response
curve (Graph-Pad, San Diego, CA).
(D) Electrophysiological Assays. Slice Preparation. All animal
procedures were conducted according to the European Community
Guidelines for Animal Care, DL 116/92, application of the European
Communities Council Directive (86/609/EEC). Experiments were
carried out on acute hippocampal slices,11 prepared from male
Wistar rats (Harlan Italy; Udine Italy, 150-200 g body weight).
Animals were killed with a guillotine under anesthesia with ether,
and their hippocampi were rapidly removed and placed in ice-cold
oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid
(aCSF) of the following composition (mM): NaCl 125, KCl 3,
NaH2PO4 1.25, MgSO4 1, CaCl2 2, NaHCO3 25, and D-glucose 10.
Slices (400 µm thick) were cut using a McIlwain tissue chopper
(The Mickle Laboratory, Engineering, Co. Ltd., Gomshall, U.K.)
and kept in oxygenated aCSF for at least 1 h at room temperature.
A single slice was then placed on a nylon mesh, completely
submerged in a small chamber (0.8 mL), and superfused with
oxygenated aCSF (31-32 °C) at a constant flow rate of 1.5-1.8
mL min-1. The treated solutions reached the preparation in 60 s,
and this delay was taken into account in our calculations.
Extracellular Recording. Test pulses (80 µs, 0.066 Hz) were
delivered through a bipolar nichrome electrode positioned in the
stratum radiatum. Evoked extracellular potentials were recorded
with glass microelectrodes (2-10 MΩ, Harvard Apparatus LTD,
Edenbridge, U.K.) filled with 150 mM NaCl and placed in the CA1
region of the stratum radiatum. Responses were amplified (BM 622,
Mangoni, Pisa, Italy), digitized (sample rate, 33.33 kHz), and stored
for later analysis with LTP (version 2.30D) program.53 Stimulus-
response curves were obtained by gradual increases in stimulus
strength at the beginning of each experiment, when a stable baseline
of evoked response was reached. The test stimulus pulse was then
adjusted to produce a field excitatory postsynaptic potential (fepsp)
whose slope and amplitude was 40-50% of the maximum and was
kept constant throughout the experiment. The fepsp amplitude was
routinely measured and expressed as the percentage of the average
amplitude of the potentials measured during the 5 min preceding
exposure of the hippocampal slices to OGD. In some experiments,
both the amplitude and initial fepsp slope were quantified, but
because no appreciable differences between these two parameters
were observed in drug effect and OGD, only the amplitude
measurement is expressed in the figures. Simultaneously with fepsp
amplitude, AD was recorded as negative extracellular direct current
(dc) shifts induced by 7 or 30 min of OGD. The dc potential is an
extracellular recording considered to provide an index of the
polarization of cells surrounding the tip of the glass electrode.54
Application of OGD and Drugs. OGD was obtained by
perfusing the slice with aCSF without glucose and gassed with
nitrogen (95% N2 and 5% CO2).55 This caused a drop in pO2 in
the recording chamber from ∼500 mmHg (normoxia) to a range
of 35-75 mmHg (after 7 min of OGD).56 At the end of the ischemic
period, the slice was again superfused with normal, glucose-
containing, oxygenated aCSF. When slices were subjected to 7 min
of OGD, if the recovery of fEPSP amplitude after 15 min of
reperfusion with glucose-containing and normally oxygenated aCSF
was e15% of the preischemic value, a second slice from the same
rat was submitted to a 7 min OGD insult in the presence of
compound 18. To confirm the result obtained in the treated group,
a third slice was taken from the same rat and another 7 min OGD
was performed in control conditions to verify that no difference
between slices was caused by the time gap between the experiments.
The selective A3 adenosine receptor antagonist was applied 15 min
before, during, and 5 min after OGD. The compound was made up
to a 100 µM stock solution in dimethyl sulfoxide (DMSO). These
stock solutions were aliquoted, stored at -20 °C, and diluted in
aCSF before use. The final concentration of DMSO (0.05% and
0.01% in aCSF) used in our experiments did not affect either fepsp
amplitude or the depression of synaptic potentials induced by OGD
(not shown).
Statistics. Data were analyzed using Prism 3.02 software
(Graphpad Software, San Diego, CA). All numerical data are
expressed as the mean ( SEM. Data were tested for statistical
significance with the paired two-tailed Student’s t test or by analysis
of variance (one-way ANOVA), as appropriate. When significant
differences were observed, the Newman-Keuls multiple compari-
son test was done. A value of p < 0.05 was considered significant.
Acknowledgment. This work was supported by a grant of
the Italian Ministry for University and Research (MIUR, FIRB
RBNE03YA3L Project). The molecular modeling work coor-
dinated by S.M. was carried out with financial support from
the University of Padova, Italy, and the Italian Ministry for
University and Research (MIUR), Rome, Italy. The electro-
physiological work was supported by a grant from the University
of Florence and by Ente Cassa di Risparmio di Firenze. We
thank Dr. Karl-Norbert Klotz of the University of Wu¨zburg,
Germany, for providing cloned hA1, hA2A, and hA3 receptors
expressed in CHO cells. S.M. is also very grateful to Chemical
Computing Group for the scientific and technical partnership.
Supporting Information Available: Combustion analysis data
of the newly synthesized compounds; binding affinities of some
PTP derivatives and the corresponding 6-nitro-TQX compounds
at bovine A1 and A2A ARs. This material is available free of charge
via the Internet at http://pubs.acs.org.
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