Synthesis and structure-activity relationships of a new set of 1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives as adenosine receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(03)00338-9

In a previous paper (Colotta V. et al., J. Med. Chem. 2000, 43, 1158), we reported the synthesis and the binding activity of some 4-oxo (A) and 4-amino (B) substituted 1,2,4-triazolo[4,3-a]quinoxalin-1-ones, bearing different substituents on the appended

Full text of DOI:10.1016/S0968-0896(03)00338-9

Bioorganic & Medicinal Chemistry 11 (2003) 3541–3550
Synthesis and Structure–Activity Relationships of a New Set of
1,2,4-Triazolo[4,3-a]quinoxalin-1-one Derivatives as Adenosine
Receptor Antagonists
Vittoria Colotta,a,* Daniela Catarzi,a Flavia Varano,a Guido Filacchioni,a
Claudia Martini,b Letizia Trincavellib and Antonio Lucacchinib
aDipartimento di Scienze Farmaceutiche, Universita’ di Firenze, Via G. Capponi, 9, 50121 Florence, Italy
bDipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Via Bonanno, 6, 50126 Pisa, Italy
Received 23 January 2003; accepted 16 May 2003
Abstract—In a previous paper (Colotta V. et al., J. Med. Chem. 2000, 43, 1158), we reported the synthesis and the binding activity
of some 4-oxo (A) and 4-amino (B) substituted 1,2,4-triazolo[4,3-a]quinoxalin-1-ones, bearing different substituents on the appen-
ded 2-phenyl ring (region 1), some of which were potent and selective A1 or A3 antagonists. To further investigate the SAR in this
class of antagonists, in the present paper some 2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives of both series A and B,
bearing simple substituents on the benzofused moiety (region 2), are reported. The binding data at bovine A1 (bA1) and A2A(bA2A
)
and at human A3 (hA3) adenosine receptors (ARs) show that in series A (compounds 1, 4–11) the presence of substituents on the
benzofused moiety is, in general, not advantageous for anchoring at all three AR subtypes, while within series B (compounds
12–21) it exerts a beneficial effect for both bA1 and hA3 AR affinities which span the low nanomolar range. In particular, among the
4-amino derivatives 12–21, the 8-chloro-6-nitro (compound 17) and the 6-nitro (compound 18) substitutions afford, respectively, the
highest bA1 and hA3 AR affinity. Moreover, compound 18, additionally investigated in binding assays at human A1 (hA1) recep-
tors, shows a 183-fold selectivity for hA3 versus hA1 receptors. Finally, the SAR studies provide some new insights about the steric
and lipophilic requirements of the hA3 receptor binding pocket which accommodates the benzofused moiety of our 4-amino-tria-
zoloquinoxalin-1-one derivatives.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
synthesis of selective AR ligands in order to shed light on
the different structural requirements of each AR subtype.
Adenosine is a neuromodulator which produces many
important biological functions by activation of G pro-
As a part of our research aimed at finding new AR
selective antagonists,15ꢀ20 we recently reported the
synthesis and the binding activity at bovine A1 (bA1)
and A2A (bA2A) and at human cloned A3 (hA3) ARs of
2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-ones, that is the
4-oxo (series A) and the 4-amino substituted (series B)
derivatives (Chart 1), bearing diverse substituents on the
appended 2-phenyl ring (region 1).21 The binding results
showed that some of these compounds were potent and
selective A1 or A3 antagonists. In particular, the
1,4-dione derivatives (series A) were, on the whole, more
active at the A3 AR than the corresponding 4-amino-1-
one compounds (series B). On the contrary, compounds
of series B possessed significantly higher A1 AR affinity
than those of derivatives of series A. Thus, these results
indicated that the presence of the 4-amino proton donor
group was important for high bA1 receptor affinity,
tein-coupled receptors that are classified into A1, A2A
,
A2B and A3 subtypes.1,2 Adenosine receptors (ARs)
from different species show 82–93% amino acid
sequence homology, the only exception being the A3
subtype which exibits 74% primary sequence homology
between rat and human.3ꢀ5
Recent studies indicate that ARs are attractive targets for
pharmacological intervention in many pathophysiologi-
cal conditions2 such as renal failure,6 cardiac7 and cere-
bral8,9 ischemia, Parkinson’s disease,10 schizophrenia11
and chronic inflammatory diseases.12ꢀ14 Thus, in the last
few years, much effort has been directed towards the
*Corresponding author. Tel.: +39-055-275-7282; fax: +39-055-
240776; e-mail: vittoria.colotta@unifi.it
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00338-9

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V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 3541–3550
Chart 1. Previously reported 1,2,4-triazolo[4,3-a]quinoxaline derivatives.
while it was not necessary for hA3 receptor recognition.
To further investigate the SAR in this class of antago-
nists, in the present paper we report the synthesis and
bA1, bA2A and hA3 AR binding activities of some
2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1,4-diones (ser-
ies A, 1, 4–11) and 4-amino-1-ones (series B, 12–21)
(Chart 2), bearing simple substituents (chloro, nitro or
amino groups) at different positions of the benzofused
moiety (region 2). Taking the previously reported tri-
azoloquinoxalin-1,4-dione 1A and 4-amino-1-one 1B21
as lead compounds (Chart 1), derivatives 1–11 and 12–
21 were designed, respectively.
Scheme 1. (a) NEt3, EtOH; (b) (Cl3CO)2CO, THF; (c) H2, Pd/C, THF
.
or AcOH; (d) SnCl2 2H2O, EtOH/DMSO; (e) ref 19.
of
the
2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1,4-
diones 1–11. Compounds 1–6 were prepared following
the reported procedure described to obtain derivative 7.19
Reaction of the 4,5-dichloro-1,2-phenylenediamine (22)
with ethyl N1-phenylhydrazono-N2-chloroacetate affor-
ded the 3-(6,7-dichlorophenyl)hydrazono-1,2,3,4-tetra-
hydroquinoxalin-2-one (28). Starting from the
1,2-phenylenediamines 23 and 24,25 two couples of
regioisomers, that is compounds 29, 30 and 31, 32 were
obtained, respectively. On the contrary, from the di-
amines 25, 26,26 27 only one compound was obtained,
that is the corresponding 3-phenylhydrazono-1,2,3,4-
tetrahydroquinoxalin-2-ones 33, 34, 35.19 The two iso-
mers 31 and 32 were easily separated by their different
solubility in the reaction medium, while the two isomers
29, 30 were used as inseparable mixture for the next
step, due to their instability on silica gel column. The
structure of derivatives 31, 33 and 34 was assigned by
means of 1H/1H NOE experiments on compounds
36–38 obtained by reacting 31, 33 and 34 with formal-
dehyde (Scheme 2). Pre-irradiation of the 1-methylene
protons of 36–38 caused a clear enhancement of the
signals of the two ortho protons of the 2-phenyl ring and
of the diagnostic proton at position 9. Thus, the bicyclic
compounds 36 and 38 were 6,8-dichloro and 6-chloro-8-
nitro-substituted derivatives, respectively. The 6-nitro-
structure of compound 33 was assigned on the basis of
the multiplicity and the coupling constant of the H-9
signal of 37, which appears as a doublet (J=1.8 Hz),
Chart 2. Currently reported 1,2,4-triazolo[4,3-a]quinoxaline 1-one
derivatives.
Introduction of a lipophilic chlorine atom(s) at the 7-
and/or 8-position of our triazoloquinoxalines was pur-
sued due to the increased A1 and/or A2A AR affinity,
observed in different tricyclic AR ligands of similar size
and shape.17,22ꢀ24 In addition, we decided also to eval-
uate the effect of chlorine atoms at the 6,8-positions and
that of a polar or hydrophilic group (the nitro or amino
group, respectively) at the 6- or 8-position, since, to our
knowledge, these effects on AR affinity have never been
investigated in tricyclic AR antagonists. Moreover, little
was known about the influence of all these substituents
on A3 AR affinity.20
Chemistry
The triazoloquinoxalines 1–21 were prepared as illu-
strated in Schemes 1 and 3. Scheme 1 shows the synthesis

V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 3541–3550
3543
Scheme 2. (a) 40% HCHO, ethylene glycol.
due to the coupling with the H-7. Finally, on the basis
of the structure of 31, the 5,7-dichloro-structure of its
regioisomer 32 was assigned.
The intermediates 28, 31, 33, 34, 3519 were cyclized with
triphosgene to yield the 1,2,4-triazolo[4,3-a]quinoxaline-
1,4-diones 1, 4–6, 719 (Scheme 1). The 3-phenylhy-
drazono-1,2,3,4-tetrahydro-5,7-dichloroquinoxalin-2-one
(32), obtained in small amounts, was not cyclized to the
corresponding triazoloquinoxalin-1,4-dione. By reacting
the mixture of the regioisomers 29 and 30 with tri-
phosgene a mixture of the tricyclic derivatives 2 and 3
was obtained. We were unable to separate the isomers 2
and 3 with satisfactory yields, thus their inseparable
mixture was used for the next step. Nevertheless, by
column chromatography a few milligrams of pure 2 and
3, only sufficient to characterize them, were obtained.
The structures of 2 and 3 were assigned on the basis of
Scheme 3. (a) PCl5/POCl3, pyridine; (b) NH3(g), absolute EtOH;
(c) H2, Pd/C, AcOH or THF.
1
basis of their H NMR spectra, using as a key tool the
coupling constant of the H-9 signal, as above illustrated
for the structural attribution of their corresponding
1,4-dione derivatives 2 and 3. In the 1H NMR spectra of
13 and 14 the H-9 signal appears as a doublet at 8.59 ppm
with a coupling constant of 8.4 and 2.1 Hz, respectively.
Indeed, compounds 13 and 14 were 7-chloro and
8-chloro substituted derivatives, respectively.
The nitro derivatives 16–18 were catalytically reduced to
afford the corresponding amino compounds 19, 20, 21.19
1
their H NMR spectra, using as key tool the coupling
constant value of the H-9 signal which is easily identi-
fied in this class of tricyclic derivatives. In fact, the H-9
is in general the most deshielded aromatic proton
(about 8.6–9.4 ppm) due to the paramagnetic effect of
Biochemistry
1
the 1-carbonyl group.19,21,27 In both the H NMR spec-
Compounds 1, 4–21 were tested for their ability to dis-
place [3H]N6-cyclohexyladenosine ([3H]CHA) from A1
ARs in bovine cerebral cortical membranes, [3H]-2-{4-
(2-carboxyethyl)phenethyl]amino}-50 -(N-ethyl-carba-
moyl)adenosine ([3H]CGS 21680) from A2A ARs in
bovine striatal membranes, and [125I]N6-(4-amino-3-
iodobenzyl)-50-N-methylcarbamoyladenosine ([125I]AB-
MECA) from human cloned A3 receptors stably
expressed in CHO cells. In fact, due to the high species
differences in the A3 primary amino acid sequence,28ꢀ30
we tested our A3 AR ligands on cloned human A3
receptors.
tra of the isomers 2 and 3, the H-9 signal appeared as a
doublet at 8.59 ppm, but with different coupling con-
stants: while in the spectrum of 2 the H-9 coupling
constant value was 8.4 Hz, in that of 3 it was 2.3 Hz.
Thus 2 and 3 were 7-chloro- and 8-chloro-substituted
derivatives, respectively.
Compounds 5 and 719 were catalytically reduced (Pd/C)
to the corresponding amino-substituted derivatives 8
and 10,19 while the 6-amino-8-chloro derivative 9 was
obtained by reacting 6 with tin(II) chloride (Scheme 1).
The 6-benzylamino-1,4-dione derivative 11 was pre-
pared as described in ref 19, that is by reacting 10 with
benzaldehyde and then reducing the corresponding
Schiff base with sodium borohydride.
The binding results of 1, 4–11 and 12–21 are shown in
Table 1 together with those of their parent compounds
1A and 1B (described in ref 21). Moreover, the binding
data of theophylline and 1,3 - dipropyl - 8 - cyclo-
pentylxanthine (DPCPX), included as antagonist refer-
ence compounds, are also reported.
By reacting the 1,4-dione derivatives 1, 4–7 and the
mixture of 2 and 3 with phosphorus pentachloride and
phosphorus oxychloride the unstable 4-chloro deriva-
tives 39, 42–45 and the mixture of 40 and 41 were
obtained (Scheme 3). The final 4-amino-triazoloquinox-
alin-1-ones 12–17, 1819 ensued from the reaction of 39–
45 with ammonia. The mixture of the 7- and 8-chloro
isomers 13 and 14 was separated by column chromato-
graphy. The structure of 13 and 14 was assigned on the
In addition, compound 18, which displayed the highest
hA3 affinity, was tested for its ability to displace
[3H]CHA from cloned human A1 AR (hA1), in order to
establish its A3 versus A1 selectivity within the same
species. The hA1 binding result of 18 together with those
of theophylline and DPCPX are reported in Table 2.

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V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 3541–3550
Table 1. Binding activity at bovine A1 and A2A and human A3 ARs
On the contrary, compounds 1, 4–11 show A3 AR affi-
nities which are, on the whole, significantly lower than
that of compound 1A (Ki=80nM) thus indicating that,
in series A, the presence of substituents on the benzo-
fused moiety is not well tolerated for anchoring to the
A3 AR recognition site. In fact, it should be noted that
the previously reported 1,4-dione derivatives,21 lacking
substituent(s) on the benzofused moiety, showed higher
A3 affinity than those of the herein reported series A.
Compd
R6
R7 R8
Ki (nM)a or I %
Introduction of a polar or hydrophilic substituent at the
8-position of 1A is very advantageous for A1 AR
recognition. In fact, both the 8-nitro compound 5 and
the 8-amino derivative 8 possess high A1 AR affinity.
The nanomolar A1 affinities of the 1,4-dione derivatives
5 and 8 are noteworthy since they represent a new find-
ing with respect to our previous results21 which indi-
cated that the presence of a 4-amino group on the
triazoloquinoxaline framework was an important fea-
ture for obtaining high bA1 affinity.
b
c
d
A1
A2A
A3
1Ae
1
4
5
6
7
8
9
10
11
1Be
12
13
14
15
16
17
18
19
20
21f
H
H
Cl
H
NO2
NO2
H
NH2
NH2
H
H
515Æ43
48%
36%
64%
5%
7%
28%
80.0Æ6.3
936Æ81
1360Æ118
241Æ21
Æ80
Cl Cl
Cl
H
H NO2 99Æ8.2
H
H
Cl 3540Æ2905%
529Æ47
856
H
0%
279Æ23
34%
H NH2 45.1Æ3.6 478.1Æ42
H
H
Cl 382Æ31
24%
42%
16%
152Æ11
418Æ39
399Æ29
490Æ41
91.5Æ8.3
Æ0.9
H
H
H
195Æ14
NHCH2Ph H
33%
11.0Æ0.9
Displacement of the nitro and amino group of com-
pounds 5 and 8, respectively, from the 8- to the 6-posi-
tion reduces the A1 affinity about 5-fold (compare
compounds 5 and 8 to 7 and 10, respectively).
H
H
H
H
Cl
H
NO2
NO2
H
NH2
NH2
H
49Æ3.7
712Æ63
113Æ.21010
10 Æ9.3 2
150Æ13
49.2Æ4.3
256Æ21
75.8Æ6.9
152Æ11
56.1Æ4.8
18.7Æ1.4
Cl Cl 212Æ32
Cl
H
H
H
47.8Æ3.8
Cl 17.1Æ1
Cl 42.7Æ1
11Æ1
1140Æ101
40Æ3.2
112Æ9.3
4.75Æ0.3
34Æ2.1
23.3Æ1.9
54Æ4.2
H NO2 8.25Æ0.6
H
H
Cl
H
0
Æ.20.01
Introduction of a lipophilic chlorine atom at the 8-
position of the 6-nitro compound 7 and of the 6-amino
derivative 10 yields compounds 6 and 9, respectively.
These latter compounds show 7- and 2-fold reduced A1
AR affinity, with respect to their 8-deschloro derivatives
7 and 10. The negative effect of the 8-chloro substituent
on A1 affinity could be attributed to the steric bulk of
the chlorine atom which may hinder the correct
anchoring to the receptor recognition site. On this basis,
it is possible to hypothesize that the benzofused moiety
(region 2) of the 1,2,4-triazolo[4,3-a]quinoxalin-1,4-
diones (series A) interacts with a hydrophobic pocket of
the bA1 receptor that possesses strict steric require-
ments. The importance of the steric factors for inter-
action of these derivatives with the bA1 AR is also
supported by the null A1 affinity of either the dichloro-
substituted derivatives 1 and 4 or the 6-N-benzylamino
compound 11. Nevertheless, the significantly different
A1 AR affinity of compounds 4 and 6, bearing at the
6-position a chlorine atom and a nitro group, respec-
tively, indicates that not only the steric factors but also
the electronic properties play an important role in the
bA1 receptor–ligand interaction. In fact, since these two
substituents have comparable steric hindrance, the
higher A1 AR affinity of 6 can be explained by assuming
that the negative steric effect of the 6-nitro group is
compensated by its electronic properties.
82Æ7.4
H NH2 7.33Æ0.4
H
H
Cl 28.6Æ1.4
9.2Æ0.8
H
Theophylline
DPCPX
3800Æ340 21,000Æ1800 86,000Æ7800
0.5Æ0.03
337Æ28
1300Æ125
aThe Ki values are meansÆSEM of four separate assays, each per-
formed in triplicate.
bDisplacement of specific [3H]CHA binding in bovine brain mem-
branes or percentage of inhibition (I%) of specific binding at 20 mM
concentration.
cDisplacement of specific [3H]CGS 21680binding from bovine striatal
membranes or percentage of inhibition (I%) of specific binding at
20 mM concentration.
dDisplacement of specific [125I]AB-MECA binding at human A3
receptors expressed in CHO cells or percentage of inhibition (I%) of
specific binding at 1 mM concentration.
eRef 21. It is worth noting that a more careful screening of the A3
affinity of 1B revealed a higher affinity (Ki=490nM) than that repor-
ted in ref 21.
fThe A1 and A2A binding data are reported in ref 19.
Results and Discussion
The binding results reported in Table 1 show that we
have produced some potent bA1 and hA3 AR antago-
nists, while only a few compounds (16, 18, 20 and 21)
display good bA2A receptor affinity.
In the 1,4-dione derivatives 1, 4–11 (series A) the pre-
sence of substituent(s) on the benzofused moiety, on the
whole, negatively affects A2A AR affinity while it exerts
different effects on A1 binding activity. In fact, while
derivatives 7, 9 and 5, 8, 10 are, respectively, equi-active
and 3- to 10-fold more active than 1A at this receptor
subtype, compounds 1, 4, 11 and 6 show, respectively,
null and 7-fold lower A1 affinity than that of the lead
compound 1A.
The 4-amino-1-one derivatives 12–21 (series B) display, on
the whole, nanomolar affinity for all three AR subtypes.
Moreover, we should point out the significantly higher A3
AR affinities of compounds 12, 16–21 with respect to those
of the corresponding 1,4-dione derivatives 1, 5–10 (series
A), since this is a new finding with respect to our previous
results21 which showed that the 1,4-dione derivatives were,
on the whole, more active at the A3 AR subtype than the
corresponding 4-amino-1-one derivatives.

V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 3541–3550
3545
The SAR of the 4-amino-1-one derivatives 12–21 are
generally different from those of the 1,4-diones (series
A) and only a few similarities have been found.
such as the 8-amino substituent (compound 19).
The presence of an 8-nitro or 8-amino group is favor-
able also for A3 AR affinity, being 16 and 19, respec-
tively, 12- and 14-fold more active than 1B at this AR
subtype.
The presence of substituent(s) on the benzofused moiety
of 12–21 affects the A1 affinity differently while it gen-
erally reduces the A2A AR affinity, with respect to those
of the lead compound 1B. In particular, the 8-chloro-6-
nitro derivative 17 is the most potent bA1 AR antagonist
(Ki=0.2 nM) among the herein reported compounds.
The beneficial effect of either a lipophilic (chloro or
nitro) or a hydrophilic substituent (amino) at the 7- or
8-position on the 4-amino-triazoloquinoxalin-1-ones
could be rationalized on the basis of a recent rhodopsin-
based model of human A3 AR receptor,32 which has
been docked with the 9-chloro-2-(2-furyl)-1,2,4-tri-
azolo[1,5-c]quinazolin-5-amine (CGS 15943)33 as refer-
ence ligand. This model hypothesizes that the
benzofused moiety of CGS 15943 resides in a hydro-
phobic pocket delimited by apolar amino acids, such as
Leu90(TM3), Phe182 (TM5) and Ile185 (TM5), which
are considered important for the binding of antagonists.
Also in this pocket polar amino acids, such as Thr94
(TM3) and Ser97 (TM3) are present, but are instead
considered not important for the binding of antagonists.
Due to their similar size and shape, it is possible to
hypothesize a similar binding mode of our 4-amino-1-
one derivatives (series B) and of CGS 15943. Thus, the
significantly increased affinity of the 7- or of the
8-chloro derivatives 13 and 14, with respect to that of
1B, could be explained by the improvement of the
hydrophobic interaction of the benzofused moiety of
compounds 13 and 14 with the three above cited apolar
amino acids. On the other hand, it is also possible to
rationalize the increased A3 affinity of both the 8-nitro
derivative 16 and the 8-amino 19 with respect to 1B, by
hypothesizing a hydrogen bond interaction between
Thr94 or Ser97 and the 8-nitro or the 8-amino group.
Nevertheless, comparison of the A3 binding data of the
8-chloro derivative 14 (Ki=11 nM) with those of 16 and
19, could confirm the major role that the hydrophobic
interactions play in the anchoring of antagonists to this
receptor pocket.
In contrast, the A3 AR affinities of compounds 12–21
are significantly enhanced, thus indicating that in series
B the presence of substituents on the benzofused moiety
is advantageous for anchoring to the hA3 AR, in parti-
cular the 6-nitro group (compound 18) confers the
highest A3 AR affinity (Ki=4.75 nM).
Introduction of a chloro substituent either at the 7- or at
the 8-position of 1B (compounds 13 and 14, respec-
tively) does not elicit the beneficial effect on A1 and A2A
AR affinity, which was observed in other series of tri-
cyclic antagonists of similar size and shape.17,22ꢀ24 The
same applies to the 7,8- or 6,8-dichloro derivatives 12
and 15, respectively.
On the contrary, the presence of a 7- or 8-chloro sub-
stituent is favorable for the binding to the A3 AR sub-
type. In fact, compounds 13 and 14 show similar
nanomolar A3 affinity, which is significantly higher than
that of 1B. These results indicate that the lipophilic
pocket of the A3 AR subtype, which accommodates the
benzofused moiety (region 2) of the 4-amino-triazolo-
quinoxalines 12–21, well tolerates a small hydrophobic
substituent, such as a chlorine atom, at the 7- or
8-position of the triazoloquinoxaline framework.
The importance of lipophilic requirements for anchor-
ing to the A3 AR recognition site is confirmed by the
binding data of the 7,8-dichloro derivative 12 which is
5-fold more active than the parent compound 1B.
Nevertheless, compound 12 is less active at this subtype
than the mono-chloro derivatives 13 and 14. These
results indicate that not only lipophilic factors, but also
the steric ones are important for the correct anchoring
of the 4-amino-1-one derivatives to the A3 receptor
recognition site, in accordance with recent literature
data regarding tricyclic AR antagonists of similar size
and shape.31 Moreover, the important role of the steric
factors is confirmed by the A3 binding data of the
6,8-dichloro derivative 15 which exhibits the lowest A3
AR affinity within the 4-amino derivatives 12–21.
The human A3 receptor model, cited above, made it
possible to explain also the A3 binding data of the
6-nitro derivative 18. In fact, in this model, a hydrogen
bond interaction between the N-6 atom of CGS 15943,
corresponding to the N-5 of our triazoloquinoxalin-4-
amines, and Ser247 (TM6) has been hypothesized.
Moreover, the 5-NH2 group of CGS 15943 also seems
to be involved in a hydrogen bond, being surrounded by
three polar amino acids: Ser242 (TM6), Ser271 (TM7)
and Ser275 (TM7). Thus, although the 6-nitro group of
18 reduces the nucleophilicity and, consequently, the
capability of the N-5 to act as hydrogen bond acceptor,
it could reinforce the hydrogen bond with Ser247, being
able itself to interact with this residue. Moreover, the
6-nitro group of 18, due to its electron-withdrawing
properties, increases acidity of the 4-amino protons,
thus enhancing the strength of interaction of this group
with the receptor proton acceptor site. Similarly to the
6-nitro group of 18, also the 8-chloro substituent of 14
could affect the N-5 nucleophilicity and the 4-amino
proton acidity, even if in minor extent, due to its lower
electron-withdrawing properties. The advantageous
Introduction of a nitro or amino group at the 8-position
of 1B affords compounds 16 and 19, respectively, which
both display similar A1 affinity to that of 1B. The high
A1 affinity (Ki=7.33 nM) of the 8-amino derivative 19
should be highlighted. This result indicates that the bA1
AR lipophilic pocket that accomodates the benzofused
moiety of the 4-amino-1-one derivatives (series B) well
tolerates not only hydrophobic substituents, such as the
7- or 8-chloro (compounds 13 and 14, respectively) and
the 8-nitro (compound 16), but also hydrophilic groups,

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V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 3541–3550
effect of the 8-chloro substituent (compound 14) and that
of the 6-nitro group (compound 18) for A3 AR affinity
was the rationale for the synthesis of compound 17
which, bearing both these substituents on the benzofused
moiety, was expected to be more active than compounds
14 and 18. Instead, the 8-chloro-6-nitro derivative 17 is a
potent bA1 AR antagonist. While it is difficult to ration-
alize the unexpected high A1 AR affinity of 17, its lower
A3 AR affinity, compared to those of 14 and 18, could be
attributed to the steric hindrance of the two substituents,
confirming the above discussed importance of the steric
factors for the A3 receptor–ligand interaction.
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 Æ0.4% of the theoretical (Table 3). The IR
spectra were recorded with a Perkin-Elmer 1420spec-
trometer in Nujol mulls and are expressed in cmꢀ1. The
1H NMR spectra were obtained with a Varian Gemini
200 instrument at 200 MHz. The chemical shifts are
reported in d (ppm) and are relative to the central peak
of the solvent that is always DMSO-d6. The following
abbreviations are used: s=singlet, d=doublet,
dd=double
br=broad, and ar=aromatic protons.
doublet,
t=triplet,
m=multiplet,
It has to be noted that compound 18 possesses not only
the highest A3 AR affinity but also the highest hA3 ver-
sus bA1 selectivity ðA1=A3Þ ¼ 17, among the herein
reported compounds. Thus, in order to rigorously
establish the true selectivity of this compound we tested
it on human A1 (hA1) ARs. The hA1 binding result,
reported in Table 2, indicates that 18 is about 10-fold
less active at hA1 than at bA1 AR and consequently it
possesses a 183-fold hA1 versus hA3 selectivity.
General procedure for the synthesis of 1,2,3,4-tetra-
hydro-3-phenylhydrazono-quinoxalin-2-ones 28–34, 3519
Compounds 28–34 were synthesized following the
method described to prepare compound 35.19 Briefly,
ethyl N1-phenylhydrazono-N2-chloroacetate (9 mmol),
suitable ortho-phenylendiamines 22, 23, 24,25 25, 26,26
27 (9 mmol) and triethylamine (10.8 mmol) were reacted
in refluxing ethanol (80mL) for 3 h. The suspension was
cooled and the solid collected and washed with water
(30–40 mL). The crude solid was made up of com-
pounds 28, 31, 33–35 and of the mixture of regioisomers
29, 30. Compounds 29, 30 (overall yield 95%), due to
their instability on silica gel column, were used as inse-
parable mixture for the next step. Compound 32, which
is the regioisomer of 31, was obtained as follows: the
mother liquor of 31 was reduced to dryness by evap-
oration of the solvent at reduced pressure. The residue
was treated with acetone (50mL) and the solid filtered
off. Evaporation of the solvent gave a residue which was
chromatographed on a silica gel column, eluting system
chloroform/ethyl acetate 9:1. From the central eluates
compound 32 was obtained. The intermediates 28, 31,
33, 34, as the previously described 35,19 may exist in
either one of the two tautomeric forms a and b. In fact,
Table 2. Binding activity at human A1 ARs
Compd
Ki (nM)a,b
18
Theophylline
DPCPX
870Æ22
3.2Æ0.2
6200Æ530
aThe Ki values are meansÆSEM of four separate assays, each per-
formed in triplicate.
bDisplacement of specific [3H]CHA binding at hA1 receptors expressed
in CHO cells.
In conclusion, the synthesis of the herein reported tri-
azoloquinoxalin-1-one derivatives has allowed us to fur-
ther investigate the SAR of the two series, A and B. The
results of this study show that the presence of sub-
stituent(s) on the benzofused moiety (region 2) of the
1,4-dione derivatives 1, 4–11 (series A) lowers the hA3
affinity and, generally, it is also unprofitable for the bA1
receptor binding. Nevertheless, the 8-amino substituent
has been found to confer high bA1 affinity (derivative 8).
1
their H NMR spectra revealed the existence of both
tautomers a and b because there are more than three
signals relative to protons that exchange with D2O. On
1
the contrary, the H NMR spectrum of compound 32
showed the existence of only one tautomer.
Introduction of substituent(s) on the benzofused moiety
of the 4-amino-1-one derivatives 12–21 (series B) is
advantageous for A1 and for A3 AR affinity to a greater
extent than in series A. In particular, the 8-chloro-6-nitro
substitution (compound 17) achieves the highest bA1 AR
affinity while the 6-nitro group (compound 18) produces
not only the highest hA3 affinity but also high hA3 versus
hA1 selectivity. Taking this new and promising finding
into account, together with our previous ones,21 further
modification of the 1,2,4-triazolo[4,3-a]quinoxalin-1-one
derivatives are in progress to improve both the A3
receptor affinity and hA3 versus hA1 selectivity.
6,7-Dichloro-1,2,3,4-tetrahydro-3-phenylhydrazono-qui-
noxalin-2-one (28). Yield: 60%; mp 273–275 ꢁC dec
1
(AcOH). H NMR 6.64–7.49 (m, ar), 7.86 (br s, NH),
8.77 (br s, NH), 9.72 (br s, NH), 11.25 (br s, NH), 12.40
(br s, NH). Anal. (C14H10Cl2N4O) C, H, N.
Experimental
Chemistry
6,8-Dichloro-1,2,3,4-tetrahydro-3-phenylhydrazono-qui-
noxalin-2-one (31). Yield: 45%; mp 248–249 ꢁC dec
(AcOH). H NMR 6.73–7.42 (m, ar), 7.92 (br s, NH),
8.85 (br s, NH), 9.80 ( br s, NH), 10.65 (br s, NH), 12.00
(br s, NH). Anal. (C14H10Cl2N4O) C, H, N.
1
Silica gel plates (Merck F254) and silica gel 60(Merck,
70–230 mesh) were used for analytical and column

V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 3541–3550
3547
5,7-Dichloro-1,2,3,4-tetrahydro-3-phenylhydrazono-qui-
noxalin-2-one (32). Yield: 2%; mp 236–238 ꢁC dec
(EtOAc). 1H NMR 6.75–6.90(m, 3H, ar), 7.10–7.30(m,
4H, ar), 7.74 (br s, 1H, NH), 9.87 (br s, 1H, NH), 12.50
(br s, 1H, NH). Anal. (C14H10Cl2N4O) C, H, N.
(4 mmol) were reacted with triphosgene (4 mmol) in
refluxing anhydrous tetrahydrofuran (40mL) for 2–3 h.
The suspension was diluted with water (40mL) and the
solid collected by filtration. The mixture of isomers 2 and
3 (overall yield 94%) was chromatographed on silica gel
column, eluting system cyclohexane/ethyl acetate 6:4.
Evaporation of the solvent of the first and the last eluates
gave few milligrams of pure 2 and 3, respectively. From
the central eluates the inseparable mixture of 2 and 3 was
obtained which was used for the next step.
1,2,3,4-Tetrahydro-6-nitro-3-phenylhydrazono-quinoxa-
lin-2-one (33). Yield: 55%; mp 190–192 ꢁC dec (AcOH).
1H NMR 6.62–6.85 (m, ar), 7.05–7.38 (m, ar), 7.70–8.05
(m, ar+NH), 8.82 (br s, NH), 9.86 (br s, NH), 10.09 (br
s, NH), 11.62 (br s, NH), 12.78 (br s, NH). Anal.
(C14H11N5O3) C, H, N.
7,8 - Dichloro - 1,2,4,5 - tetrahydro - 2 - phenyl - 1,2,4 - tria-
zolo[4,3-a]quinoxalin-1,4-dione (1). Yield: 98%; mp
>300 ꢁC (AcOH). 1H NMR 7.35–7.46 (m, 2H, ar), 7.57
(t, 2H, ar, J=8.1 Hz), 8.01 (d, 2H, ar J=8.1 Hz), 8.73 (s,
1H, H-9), 12.05 (br s, 1H, NH); IR 3200, 1740, 1700.
Anal. (C15H8Cl2N4O2) C, H, N.
6-Chloro-1,2,3,4-tetrahydro-8-nitro-3-phenylhydrazono-
quinoxalin-2-one (34). Yield: 50%; mp 172–173 ꢁC
(AcOH).1H NMR 6.65–7.37 (m, ar), 7.62–7.70(m, ar),
7.92–8.04 (m, ar+NH), 9.07 (br s, NH), 10.11 (br s,
NH), 10.46 (br s, NH), 11.60 (br s, NH). Anal.
(C14H10ClN5O3) C, H, N.
7-Chloro-1,2,4,5-tetrahydro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1,4-dione (2). Mp >300 ꢁC (AcOH). 1H
NMR 7.24–7.43 (m, 3H, ar), 7.57 (t, 2H, ar, J=8.2 Hz),
7.03 ( d, 2H, ar, J=7.5 Hz), 8.61 (d, 1H, H-9, J=8.4 Hz),
12.09 (br s, 1H, NH). Anal.(C15H9ClN4O2) C, H, N.
1,2,3,4-Tetrahydro-8-nitro-3-phenylhydrazono-quinoxa-
lin-2-one (35). See ref 19.
General procedure for the synthesis of 1,2,4,5-tetra-
hydro-2-phenyl-1,2,4-triazolo[4,3- a]quinoxalin-4-ones
36–38
8-Chloro-1,2,4,5-tetrahydro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1,4-dione (3). Mp >300 ꢁC (AcOH). 1H
NMR 7.28–7.63 (m, 5H, ar), 8.01 (d, 2H, ar, J=7.8 Hz),
8.60(d, 1H, H-9, J=2.3 Hz), 12.11 (br s, 1H, NH).
Anal. (C15H9ClN4O2) C, H, N.
A mixture of 31 or 33 or 34 (0.9 mmol) and aqueous
formaldehyde (40%, 0.4 mL) in ethylene glycol (3 mL)
was heated at reflux for 2–3 min. Dilution with water
(10mL) yielded a yellow solid which was collected and
washed with water.
6,8 - Dichloro - 1,2,4,5 - tetrahydro - 2 - phenyl - 1,2,4 - tri-
azolo[4,3-a]quinoxalin-1,4-dione (4). Yield: 90%; mp
296–298 ꢁC (AcOH). 1H NMR 7.40(t, 1H, ar,
J=7.7Hz), 7.58 (t, 2H, ar, J=7.7Hz), 7.75 (d, 1H, H-7,
J=2.3Hz), 7.99 (d, 2H, ar, J=7.7 Hz), 8.63 (d, 1H, H-9,
J=2.3 Hz), 11.61 (br s, 1H, NH). Anal. (C15H8Cl2N4O2)
C, H, N.
6,8 - Dichloro - 1,2,4,5 - tetrahydro - 2 - phenyl - 1,2,4 - tria-
zolo[4,3-a]quinoxalin-4-one (36). Yield: 85%; mp
ꢁ
1
289–291 C dec (ethylene glycol). H NMR 5.70(s, 2H,
CH2), 6.84–6.97 (m, 4H, 3ar+H-9), 7.19 (s, 1H, H-7),
7.29–7.37 (m, 2H, ar), 11.04 (br s, 1H, NH). Anal.
(C15H10Cl2N4O) C, H, N.
1,2,4,5-Tetrahydro-8-nitro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1,4-dione (5). Yield: 93%; mp 277–278 ꢁC
1
1,2,4,5-Tetrahydro-8-nitro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-4-one (37). Yield: 80%; mp 294–296 ꢁC dec
(EtOAc). H NMR 7.35–7.52 (m, 2H, ar), 7.60(t, 2H,
J=7.6 Hz), 8.02 (d, 2H, J=8.1 Hz), 8.29 (dd, 1H, H-7,
J=8.9, 2.6 Hz), 9.40(d, 1H, H-9, J=2.6 Hz). Anal.
(C15H9N5O4) C, H, N.
1
(ethylene glycol). H NMR 5.81 (s, 2H, CH2), 6.91 (t,
1H, ar, J=7.6 Hz), 7.04 (dd, 2H, J=8.4, 1.5 Hz), 7.16
(dd, 1H, H-6, J=8.7, 1.8 Hz), 7.35 (t, 2H, ar,
J=7.6 Hz), 7.64 (d, 1H, H-9, J=1.8 Hz), 7.90(dd, 1H,
H-7, J=8.7, 1.8 Hz), 11.98 (br s, 1H, NH). Anal.
(C15H11N5O3) C, H, N.
8-Chloro-1,2,4,5-tetrahydro-6-nitro-2-phenyl-1,2,4-tria-
zolo[4,3-a]quinoxalin-1,4-dione (6). Yield: 98%; mp
>300 ꢁC (MeOH/EtOAc). 1H NMR 7.42 (t, 1H, ar,
J=7.2 Hz), 7.61 (t, 2H, ar, J=7.2 Hz), 8.01 (d, 2H, ar,
J=8.3 Hz), 8.25 (d, 1H, H-7, J=2.4 Hz), 8.98 (d, 1H,
H-9, J=2.4 Hz), 11.46 (br s, 1H, NH). IR 3330, 3100,
1740, 1710. Anal. (C15H8ClN5O4) C, H, N.
8-Chloro-1,2,4,5-tetrahydro-6-nitro-2-phenyl-1,2,4-tria-
zolo[4,3-a]quinoxalin-4-one (38). Yield: 97%; mp
ꢁ
1
244–246 C dec (ethylene glycol). H NMR 5.80(s, 2H,
CH2), 6.60–6.99 (m, 3H, ar), 7.30 (d, 1H, H-9, J=2.4 Hz),
7.39 (t, 2H, ar, J=7.6 Hz), 7.77 (d, 1H, H-7, J=2.4 Hz),
10.56 (br s, 1H, NH). Anal.(C15H10ClN5O3) C, H, N.
1,2,4,5-Tetrahydro-6-nitro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1,4-dione (7). See ref 19.
General procedure for the synthesis of 8-amino-1,2,4,5-
tetrahydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1,4-
dione (8) and 6-amino-1,2,4,5-tetrahydro-2-phenyl-1,2,4-
triazolo[4,3-a]quinoxalin-1,4-dione (10)19
General procedure for the synthesis of 1,2,4,5-tetra-
hydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1,4-diones
1–6, 719
Compounds 1–6 were synthesized following the reported
procedure to prepare 7 from 35.19 Briefly, compounds
28, 31, 33, 34 and the mixture of regioisomers 29, 30
Pd/C 10% (0.05 g) was added to a hot solution of 5 or 719
(0.8 mmol) in tetrahydrofuran (200 mL). The mixture

3548
V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 3541–3550
was hydrogenated overnight in a Parr apparatus at
30psi. The suspension was heated and the catalyst fil-
tered off. Evaporation of the solvent at reduced pressure
gave a residue which was suspended in diethyl ether
(5–10mL) and filtered.
further purification. The isomers 40 and 41 were used as
a mixture for the next step.
4,7,8-Trichloro-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1-one (39). 1H NMR 7.48 (t, 1H, ar,
J=7.4 Hz), 7.62 (t, 2H, ar, J=7.6 Hz), 8.04 (d, 2H, ar,
J=7.6 Hz), 8.29 (s, 1H, H-6), 8.85 (s, 1H, H-9).
8. Yield: 98%; mp >300 ꢁC (EtOH/AcOH). H NMR
1
5.37 (br s, 2H, NH2), 6.55 (dd, 1H, H-7, J=2.1, 8.4 Hz),
6.96 (d, 1H, H-6, J=8.4 Hz), 7.36 (t, 1H, ar, J=7.3 Hz),
7.54 (t, 2H, ar, J=7.7 Hz), 7.91 (d, 1H, H-9, J=2.2 Hz),
7.98 (d, 2H, ar, J=8.1 Hz), 11.6 (br s, 1H, NH). Anal.
(C15H11N5O2) C, H, N.
4,6,8-Trichloro-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1-one (42). 1H NMR 7.46 (t, 1H, ar,
J=7.3 Hz), 7.62 (t, 2H, ar, J=7.3 Hz), 7.98–8.08 (m,
3H, 2ar+H-7), 8.71 (d, 1H, H-9, J=1.6 Hz).
10. See ref 19.
4-Chloro-1,2-dihydro-8-nitro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1-one (43). 1H NMR 7.45 (t, 1H, ar,
J=7.4 Hz), 7.63 (t, 2H, ar, J=7.4 Hz), 7.98–8.18 (m,
3H, ar), 8.42 (dd, 1H, H-7, J=8.1, 1.4 Hz), 9.46 (d, 1H,
H-9, J=1.4 Hz).
Synthesis
phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1,4-dione (9).
of
6-amino-8-chloro-1,2,4,5-tetrahydro-2-
A
suspension of compound 6 (0.6 mmol) and tin(II) chlor-
ide dihydrate (2.8 mmol) in ethanol (10mL) and dime-
thylsulfoxide (0.3 mL) was refluxed for 90 min and then
cooled at room temperature. The solid was filtered,
resuspended in water (100 mL) and stirred for 15 min at
room temperature. The resulting solid was collected by
filtration and washed with water (20–30 mL). Yield:
45%; mp >300 ꢁC (AcOH).1H NMR 5.93 (br s, 2H,
NH2), 6.75 (d, 1H, H-7, J=2.3 Hz), 7.39 (t, 1H, ar,
J=7.4 Hz), 7.57 (t, 2H, ar, J=7.3 Hz), 7.94–8.02 (m,
3H, ar). Anal. (C15H10ClN5O2) C, H, N.
4,8-Dichloro-1,2-dihydro-6-nitro-2-phenyl-1,2,4-tria-
1
zolo[4,3-a]quinoxalin-1-one (44). H NMR 7.46 (t, 1H,
ar, J=7.4 Hz), 7.65 (t, 2H, ar, J=7.4 Hz), 8.05 (d, 2H,
ar, J=7.4 Hz), 8.42 (d, 1H, H-7, J=1.7 Hz), 8.88 (d,
1H, H-9, J=1.7 Hz).
4-Chloro-1,2-dihydro-6-nitro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1-one (45). 1H NMR 7.42 (t, 1H, ar,
J=7.3 Hz), 7.60(t, 2H, ar, J=7.3 Hz), 7.85–8.16 (m,
4H, ar), 8.92 (d, 1H, H-9, J=7.0Hz).
Synthesis of 6-benzylamino-1,2,4,5-tetrahydro-2-phenyl-
1,2,4-triazolo[4,3-a] quinoxalin-1,4-dione (11).19 A mix-
ture of 1019 (3.4 mmol), benzaldehyde (4.1 mmol) and
anhydrous zinc chloride (1.7 mmol) in anhydrous tetra-
hydrofuran (50mL) was refluxed under nitrogen atmos-
phere for 5 h. The solid, made up of the corresponding
Schiff base, that is the 4-amino-6-(N-benzyliden)amino-
1,2,4,5-tetrahydro-2-phenyl-1,2,4-triazolo[4,3-a]quinox-
alin-1,4-dione,19 was filtered off, washed with water and
dried. Sodium borohydride (4.2 mmol) was added por-
tionwise in 30min to a suspension of the Schiff base
(2.1 mmol), in refluxing anhydrous methanol (40mL).
The suspension was refluxed for 3 h, then it was cooled
at room temperature and diluted with water (20mL).
The solid was filtered off and washed with water. Yield:
General procedure for the synthesis of 4-amino-1,2-dihydro-
2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-ones 12–17, 1819
A suspension of 39, 42–44 and of the mixture of isomers
40 and 41 (2 mmol) in absolute ethanol (30mL) satu-
rated with ammonia was heated overnight at 120 ꢁC in a
sealed tube. After cooling, the solid was collected and
washed with water. The two isomers 13 and 14 were
separated by column chromatography, eluting system
cyclohexane/ethyl acetate 1:1. Evaporation of the sol-
vent of the first and the second eluates gave compounds
13 and 14, respectively.
4-Amino-7,8-dichloro-1,2-dihydro-2-phenyl-1,2,4-tria-
zolo[4,3-a]quinoxalin-1-one (12). Yield: 90%; mp
257–258 ꢁC (AcOH).1H NMR 7.38 (t, 1H, ar,
J=7.5 Hz), 7.55–7.63 (m, 3H, 2ar+H-6), 7.88 (br s, 2H,
NH2), 8.04 (d, 2H, ar, J=8.4 Hz), 8.72 (s, 1H, H-9).
Anal. (C15H9Cl2N5O) C, H, N.
78%; mp 283–285 ꢁC (AcOH). H NMR 4.43 (d, 2H,
1
CH2, J=4.4 Hz), 6.40(t, 1H, NH, J=4.4 Hz), 6.63 (d,
1H, ar, J=8.3 Hz), 7.08 (t, 1H, ar, J=8.1 Hz), 7.27–7.62
(m, 7H, ar), 8.00–8.10 (m, 3H, ar), 11.21 (br s, 1H, NH).
General procedure for the synthesis of 4-chloro-1,2-dihy-
dro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-ones 39–45
4 - Amino - 7 - chloro - 1,2 - dihydro - 2 - phenyl - 1,2,4 - tria-
zolo[4,3-a]quinoxalin-1-one (13). Yield: 40%; mp
>300 ꢁC (AcOH). 1H NMR 7.24–7.40(m, 3H, ar), 7.56
(t, 2H, ar, J=7.8 Hz), 7.76 (br s, 2H, NH2), 8.05 (d, 2H,
ar, J=8.1 Hz), 8.59 (d, 1H, H-9, J=8.4 Hz); IR 3480,
3310, 3260–3020, 1740. Anal. (C15H10ClN5O) C, H, N.
Compounds 1, 4–6, 719 and the mixture of 2 and 3
(2 mmol) were reacted with phosphorus pentachloride
(4 mmol) in refluxing phosphorus oxychloride (40mL)
and anhydrous pyridine (0.2 mL) until the dis-
appearance (TLC monitoring) of the starting material
(12–24 h). Evaporation at reduced pressure of the excess
phosphorus oxychloride afforded a solid which was
treated with iced water (50mL), collected and washed
with cyclohexane. The 4-chloro derivatives 39–45,
obtained in high overall yields (85–90%), were instable,
nevertheless they were pure enough to be used without
4 - Amino - 8 - chloro - 1,2 - dihydro - 2 - phenyl - 1,2,4 - tria-
zolo[4,3-a]quinoxalin-1-one (14). Yield: 40%; mp
275–277 ꢁC (AcOH). H NMR 7.36–7.41 (m, 3H, ar),
1
7.53–7.66 (m, 4H, 2ar+NH2 ), 8.04 (d, 2H, ar,
J=8.4 Hz), 8.59 (d, 1H, H-9, J=2.1 Hz). Anal.
(C15H10ClN5O) C, H, N.

V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 3541–3550
3549
4-Amino-6,8-dichloro-1,2-dihydro-2-phenyl-1,2,4-tria-
zolo[4,3-a]quinoxalin-1-one (15). Yield: 70%; mp
>300 ꢁC (AcOH).1H NMR 7.42 (t, 1H, ar, J=7.32 Hz),
7.55–7.65 (m, 3H, 2ar+H-7), 7.98–8.07 (m, 4H,
2ar+NH2), 8.61 (d, 1H, H-9, J=2.4 Hz). Anal.
(C15H9Cl2N5O) C, H, N.
A1 adenosine receptor binding assay was performed
using [3H]CHA as radioligand. The assay medium con-
sisted of a buffer containing 50mM Tris/HCl at pH 7.4.
The glass incubation tubes, containing 20 mg of mem-
brane proteins, 0.2 U/mL adenosine deaminase, 1 nM
[3H]CHA and 10 mL of the tested ligand, were incubated
for 2 h at 25 ꢁC. After incubation, samples were filtered
under vacuum on Whatman GF/C filters and washed
three times with 5 mL of ice-cold assay buffer. Non-
specific binding was determined in the presence of
50 mM NECA. Specific binding, obtained by subtract-
ing nonspecific binding from total binding, corre-
sponded to 90% of the total binding. Compounds were
dissolved in DMSO (buffer/concentration of 2%) and
added to the assay mixture. Blank experiments were
carried out to determine the effect of solvent on
[3H]CHA binding.
4-Amino-1,2-dihydro-8-nitro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1-one (16). Yield: 78%; mp >300 ꢁC
1
(AcOH). H NMR 7.35–7.62 (m, 4H, ar), 8.05 (d, 2H,
ar, J=7.8 Hz), 8.15–8.42 (m, 3H, 1ar+NH2), 9.36 (d,
1H, H-9, J=2.5 Hz). Anal. (C15H10N6O3) C, H, N.
4-Amino-8-chloro-1,2-dihydro-6-nitro-2-phenyl-1,2,4-tria-
zolo[4,3-a]quinoxalin-1-one (17). Yield: 85%; mp
ꢁ
1
>300 C (AcOH). H NMR 7.43 (t, 1H, ar, J=7.5 Hz),
7.60(t, 2H, ar, J=7.5 Hz), 8.03–8.08 (m, 3H, ar),
8.15–8.40(br s, 2H, NH 2), 8.73 (d, 1H, H-9, J=2.3 Hz);
IR 3480, 3340, 3280–3080, 1730, 1460, 1360. Anal.
(C15H9ClN6O3) C, H, N.
Displacement of [125I]AB-MECA from hA3 ARs stably
expressed in CHO cells was performed as previously
described.20
4-Amino-1,2-dihydro-6-nitro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1-one (18). See ref 19.
The concentration of the tested compounds that pro-
duced 50% inhibition of specific [3H]CHA, [3H]CGS
21680or [ 125I]AB-MECA binding (IC50) was calculated
using a non-linear regression method implemented in
the InPlot program (Graph-Pad, San Diego, CA, USA)
with five concentrations of displacer, each performed in
triplicate. Inhibition constants (Ki) were calculated
according to the Cheng–Prusoff equation.36 The dis-
sociation constant (Kd) of [3H]CHA and [3H]CGS 21680
in cortical and striatal bovine brain membranes were 1.2
and 14 nM, respectively. The Kd values of [3H]CHA and
General procedure for the synthesis of 4,8-diamino-1,2-di-
hydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (19),
4,6-diamino-8-chloro-1,2-dihydro-2-phenyl-1,2,4-triazolo
[4,3-a]quinoxalin-1-one (20) and 4,6-diamino-1,2-dihydro-
2-phenyl-1,2,4-triazolo[4,3-a] quinoxalin-1-one (21)19
Compounds 19 and 2119 were prepared by hydrogenat-
ing 16 and 1819 (0.8 mmol), dissolved in hot glacial ace-
tic acid (200 mL), following the experimental conditions
described above to obtain compounds 8 and 10. Simi-
larly, compound 20 was prepared from 17 (0.8 mmol)
which was dissolved in hot tetrahydrofuran (200 mL)
and hydrogenated for 3 days.
[
membranes were 1.9 and 1.4 nM, respectively.
125I]AB-MECA in hA1 and hA3 ARs in CHO cell
19. Yield: 94%; mp >300 ꢁC (EtOAc/AcOH).1H NMR
5.40(br s, 2H, NH at the 8-position), 6.64 (dd, 1H, ar,
Table 3. Analytical data of the newly synthesized compounds
2
Compd
Formula
C
H
N
J=2.4, 8.6 Hz), 6.83 (s, 2H, NH2 at 4-position), 7.16 (d,
1H, ar, J=8.6 Hz), 7.36 (t, 1H, ar, J=7.3 Hz), 7.57 (t,
2H, ar, J=7.7 Hz), 7.95 (d, 1H, H-9, J=2.4 Hz), 8.08
(d, 2H, ar, J=7.7 Hz). Anal. (C15H12N6O) C, H, N.
calcd–
found
calcd–
found
calcd–
found
1
2
3
4
5
6
8
9
12
13
14
15
16
17
19
20
28
31
32
33
34
36
37
38
C15H8Cl2N4O2
C15H9ClN4O2
C15H9ClN4O2
C15H8Cl2N4O2
C15H9N5O4
51.89–51.71 2.33–2.32
57.61–57.802.91–2.9017.92–17.87
16.14–16.10
57.61–57.45
51.89–51.75
55.72–55.88
50.36–50.18
61.42–61.61
54.97–55.09
52.04–52.27
57.79–57.61
57.79–57.95
52.04–52.21
2.91–2.92
2.33–2.34
2.81–2.8021.67–22.75
2.26–2.27
3.79–3.8023.88–23.98
3.08–3.07
2.63–2.64
3.24–3.25
3.24–3.23
2.63–2.61
17.92–17.96
16.14–16.18
20. Yield: 92%; mp 237–238 ꢁC (MeOH).1H NMR 5.61
(br s, 2H, NH2 at the 6-position), 6.69 (d, 1H, H-7,
J=2.5 Hz), 7.30–7.37 (m, 3H, 1ar+NH2 at the 4-position),
7.56 (t, 2H, ar, J=7.7 Hz), 7.85 (d, 1H, H-9, J=1.8 Hz),
8.04 (d, 2H, ar, J=8.4 Hz). Anal. (C15H11ClN6O) C, H, N.
C15H8ClN5O4
C15H11N5O2
19.58–19.50
C15H10ClN5O2
C15H9Cl2N5O
C15H10ClN5O
C15H10ClN5O
C15H9Cl2N5O
C15H10N6O3
21.37–21.29
20.23–20.29
22.47–22.55
22.47–22.56
20.63–20.56
21. See ref 19.
55.89–55.703.13–3.14
286–.206.16
C15H9ClN6O3
C15H12N6O
50.50–50.34
61.63–61.39
55.13–55.31
52.35–52.59
52.35–52.16
52.35–52.47
56.56–56.74
50.68–50.48
54.07–54.35
58.24–58.02
52.41–52.28
2.55–2.53
23.56–23.65
28.76–28.67
25.72–25.62
17.45–17.59
17.45–17.39
17.45–17.58
23.56–23.64
21.12–21.20
16.82–16.65
22.65–22.72
20.38–20.30
Biochemistry
4.15–4.16
3.40–3.39
3.14–3.28
3.14–3.15
3.14–3.25
3.74–3.75
3.04–3.03
3.03–3.11
3.59–3.60
2.94–2.93
C15H11ClN6O
C14H10Cl2N4O
C14H10Cl2N4O
C14H10Cl2N4O
C14H11N5O3
C14H10ClN5O3
C15H10Cl2N4O
C15H11N5O3
Bovine A1 and A2A receptor binding. Displacement of
[3H]CHA from A1 ARs in bovine cerebral cortical mem-
branes and [3H]CGS 21680from A
striatal membranes was performed as described in ref 34.
ARs in bovine
2A
Human A1 and A3 receptor binding. Binding experiments
at hA1 and hA3 adenosine receptors were performed on
crude membranes obtained from CHO cells.35
C15H10ClN5O3

3550
V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 3541–3550
19. Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filac-
Acknowledgements
chioni, G.; Martini, C.; Trincavelli, L.; Lucacchini, A. Arch.
Pharm. Pharm. Med. Chem. 1999, 332, 39.
20. Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filac-
chioni, G.; Martini, C.; Trincavelli, L.; Lucacchini, A. J. Med.
Chem. 2000, 43, 3118.
We thank Dr. Karl-Norbert Klotz of the University of
Wuzburg, Germany, for providing cloned hA1 and hA3
receptors expressed in CHO cells.
21. Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filac-
chioni, G.; Martini, C.; Trincavelli, L.; Lucacchini, A. J. Med.
Chem. 2000, 43, 1158.
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