Synthesis and Structure-Activity Relationships of 4-Cycloalkylamino-1,2,4-triazolo[4,3-a]quinoxalin-1-one Derivatives as A 1 and A3 Adenosine Receptor Antagonists

Article abstract of DOI:10.1002/ardp.200300816

In a previous paper (Colotta V. et al., J. Med. Chem. 2000, 43, 1158-1164) we reported the synthesis and binding activity of 4-cycloalkylamino-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives, differently substituted on the appended 2-phenyl ring, some o

Full text of DOI:10.1002/ardp.200300816

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 35–41
Synthesis of A1 Adenosine Receptor Antagonists 35
Vittoria Colottaa,
Synthesis and Structure-Activity Relationships of
4-Cycloalkylamino-1,2,4-triazolo[4,3-a]quinoxalin-
1-one Derivatives as A1 and A3 Adenosine Receptor
Antagonists
Daniela Catarzia,
Flavia Varanoa,
Guido Filacchionia,
Claudia Martinib,
Letizia Trincavellib,
Antonio Lucacchinib
In a previous paper (ColottaV.et al., J.Med.Chem.2000, 43, 1158–1164) we report-
ed the synthesis and binding activity of 4-cycloalkylamino-1,2,4-triazolo[4,3-a]quin-
oxalin-1-one derivatives, differently substituted on the appended 2-phenyl ring,
some of which were potent and selective A1 adenosine receptor (AR) antagonists.In
the present paper several 4-cycloalkylamino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxa-
lin-1-one derivatives (1–11), bearing simple substituents on the benzofused moiety,
are reported.The binding data of bovine A1 and A2A and human A3 AR show that we
have obtained highly potent A1 AR antagonists.In particular, the 4-cyclohexylamino
derivatives 1–5 show higher A1 vs A2A selectivity than the parent compound A, which
lacks substituents on the benzofused moiety.Moreover, compounds 1–11 display, in
general, good A3 AR affinity. Finally, SAR studies provide some new insights about
the steric requirements of the A3 receptor pocket, which accommodates the benzo-
fused moiety of our 4-amino-triazoloquinoxalin-1-one derivatives.
a
Dipartimento di Scienze
Farmaceutiche,
Universita’ di Firenze,
Firenze, Italy
b
Dipartimento di Psichiatria,
Neurobiologia,
Farmacologia e
Biotecnologie,
Università di Pisa,
Pisa, Italy
Keywords:G Protein-coupled receptors;Adenosine receptor antagonists;Triazolo-
quinoxalines; Tricyclic heteroaromatic systems
Received: May 14, 2003; Accepted: September 1, 2003 [FP816]
DOI 10.1002/ardp.200300816
human (h) cloned A3 ARs of 2-aryl-4-amino-1,2,4-
triazolo[4,3-a]quinoxalin-1-one derivatives bearing
Introduction
Adenosine is a neuromodulator, which produces many
important biological functions by activation of G protein-
coupled receptors classified into A1, A2A, A2B and A3 sub-
types [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 hu-
man [3–5].In the last few years, much effort has been di-
rected towards the synthesis of selective AR antagonists
since they are attractive tools for pharmacological inter-
vention in many pathophysiological conditions [6].In par-
ticular, A1 AR selective antagonists are developed as an-
tihypertensives and potassium-saving diuretics [7], cog-
nition enhancers [6] and useful therapeutics in allevia-
tion of the symptoms of Alzheimer’s disease [6, 7], while
A3 AR antagonists are considered potential anti-inflam-
matory and anti-asthmatic agents [8].
some substituents on the 2-phenyl ring and the 4-amino
group (Figure 1) [16]. Structure-Activity Relationship
(SAR) studies indicated that introduction of a substituent
on the 2-phenyl moiety did not increase A1 AR affinity
and selectivity while the presence of a cycloalkyl ring on
the 4-amino group was an important feature to obtain
highly potent A1 and A1 vs A2A selective antagonists.The
4-cycloalkylamino derivatives also displayed A3 AR af-
finity in the nanomolar range [16].Continuing our studies
of this class of AR antagonists, in the present paper we
have focused our attention on A1 selective ligands.
Therefore, taking the potent and selective A1 antagonists
2-phenyl-4-cycloalkylamino derivatives A and B [16] as
lead compounds (Figure 1), new 4-cycloalkylamino-2-
phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives
(1–11) were prepared and tested at bA1, bA2A and hA3
ARs. Compounds 1–11 were designed to evaluate the
influence on A1 affinity and selectivity of some simple
substituents (chloro, nitro or amino groups) at different
positions of the benzofused moiety. Moreover, we were
interested in investigating the effect of all these substitu-
ents on hA3 AR affinity.
As a part of our research aimed at finding new AR selec-
tive antagonists [9–15] we recently reported the synthe-
sis and binding activity of bovine (b) A1 and A2A and
Correspondence: Prof. Vittoria Colotta, Dipartimento di
Scienze Farmaceutiche, Università di Firenze, Polo Scientifico,
Via Ugo Schiff, 6, 50016 Sesto Fiorentino (FI), Italy.
Phone: +39 55 4573731, Fax: +39 55 4573671; e-mail
vittoria.colotta@unifi.it
Introduction of lipophilic chlorine atoms at the 7,8-posi-
tion of our triazoloquinoxalines was pursued since this
substitution was profitable for A1 and/or A2A AR affinity of
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

36 Colotta et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 35–41
some tricyclic AR ligands of similar size and shape [17,
18]. In addition, we also decided to evaluate the effect of
chlorine atoms at the 6,8-positions and of substituents
(the nitro or amino group) at the 6- or 8-position, able to
engage hydrogen bonds with the binding site.It has to be
noted that, to our knowledge, the effects of these substit-
uents on AR affinity have never been investigated in tri-
cyclic AR antagonists.
Chemistry
The novel 4-cycloalkylamino-triazoloquinoxalin-1-one
derivatives 1–11 were prepared as illustrated in Scheme
1. Briefly, the key intermediates 4-chloro-triazoloqui-
noxalin-1-ones 12–16 were obtained by treatment of the
corresponding 1,4-dione derivatives 17–21 [13, 15] with
Figure 1. Previously reported 4-amino-2-phenyl-1,2,4-
triazolo[4,3-a]quinoxalin-1-ones A and B.
Scheme 1.Reagents:(a) PCl5/POCl3, pyridine;(b) Cyclohexylamine, Et3N, absolute EtOH;(c) Cyclopentylamine, Et3N,
absolute EtOH; (d) H2, Pd/C, AcOH or THF.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 35–41
Synthesis of A1 Adenosine Receptor Antagonists 37
phosphorous pentachloride and phosphorous oxychlo-
ride. Reaction of the 4-chloro derivatives 12–15 with
cyclohexylamine yielded compounds 1–4, while deriva-
tives 6–9 were obtained by reacting 12, 14–16 with
cyclopentylamine. Finally, catalytic reduction (Pd/C) of
the nitro derivatives 4, 8 and 9 afforded the correspond-
ing amino-compounds 5, 10 and 11.
stituents on the benzofused moiety of compounds A and
B affects A3 AR affinity differently.In fact, compounds 2, 3
are significantly less active at this subtype than A and the
same can be said for 7 with respect to B.On the contrary,
compounds 10–11 display higher A3 affinity than B. In
particular, the 4-cyclopentylamino derivative 11, bearing
an 8-amino group, possesses the highest A3 AR affinity
(Ki = 2.7 nM) among the herein reported ligands.
The presence of two chlorine atoms, at the 7,8- position
(compounds 1, 6), did not elicit the beneficial effects for
A1 and A2A affinity exerted in other tricyclic derivatives
[17, 18] and was not profitable for A3 AR affinity either.
The same applies to the 6,8-dichloro (compound 2) and
to the 6-nitro-8-chloro (compounds 3 and 7) substitu-
tions, with particular regard to A3 AR affinity. The nega-
tive effects of the presence of two substituents (chloro
and nitro) on the benzofused moiety of compounds 1–3,
6–7 could be attributed to their steric bulk, which may
hinder the correct anchoring to the AR recognition site.
This hypothesis can be confirmed by comparison of the
A1 and A3 AR affinities of the 8-chloro-6-nitro derivatives
3 and 7 with those of compounds 4 and 8, lacking the
bulky chlorine atom at the 8-position. In fact, 4 and 8 are
significantly more active than 3 and 7.On the other hand,
the importance of steric requirements for interaction with
the A3 AR binding pocket has already been shown in pre-
vious studies about this class of AR antagonists [14] and
other tricyclic antagonists of similar size and shape
[21].
Biochemistry
Compounds 1–11 were tested for their ability to displace
[3H]N6-cyclohexyladenosine ([3H]CHA) from A1 AR in
bovine cerebral cortical membranes, [3H]-2-[[4-(2-
carboxyethyl)phenethyl]amino]-5Ј-(N-ethyl-carbamoyl)-
adenosine ([3H]CGS 21680) from A2A AR in bovine stri-
atal membranes, and [125I]N6-(4-amino-3-iodobenzyl)-
5Ј-N-methylcarbamoyladenosine ([125I]AB-MECA) from
human cloned A3 receptor stably expressed in CHO
cells.In fact, due to the high species differences in the A3
primary amino acid sequence [3, 19, 20], we tested our
A3 AR ligands on cloned human A3 receptors.
Results and discussion
The binding results of 1–11 are shown in Table 1. In this
table the binding affinities of compounds A and B [16]
are also reported, together with those of theophylline
and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), in-
cluded as antagonist reference compounds.
Comparison of the A3 AR affinity of compound 4 with that
of compound A confirms that the presence of a 6-nitro
group is profitable for anchoring of 4-amino-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives to the
A3 AR recognition site [15]. The advantageous effect of
this group has been previously rationalized [15] on the
basis of a recent rhodopsin-based model of human A3
AR [22] which has been docked with the 9-chloro-2-(2-
furyl)-1,2,4-triazolo[1,5-c]quinazolin-5-amine (CGS
15943) [23] as reference ligand. Due to their similar size
and shape, we have hypothesized [15] a similar binding
mode of our 4-amino-triazolo[4,3-a]quinoxalin-1-one
derivatives and CGS 15943 [14].This model assumes a
hydrogen bond interaction between the N-6 atom of
CGS 15943, corresponding to the N-5 of our ligands,
and Ser247 (TM6). Moreover, the 5-NH2 group of CGS
15943 also seems to give a hydrogen bond being sur-
rounded by Ser 242(TM6), Ser271 (TM7) and Ser275
(TM7).Thus, although the 6-nitro group of 4 reduces 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-ni-
tro group of 4, due to its electron-withdrawing properties,
increases acidity of the 4-amino protons, thus enhancing
the strenght of interaction of this group with the receptor
Due to the presence of the 4-cycloalkyl moiety, which is
well-known to yield A1 vs A2A selective ligand [7, 11, 14,
16–18], compounds 1–11 possess nanomolar or subna-
nomolar A1 affinity, while they are on the whole scarcely
active or inactive at the A2A receptor subtype. Further-
more, in accordance with our previous data [16], the
4-cyclohexylamino derivatives 3–5 show higher A1 vs
A2A selectivity than the corresponding 4-cyclopentylami-
no derivatives 7, 8, 10. Compounds 1–11 are also active
at the A3 AR since they display, generally, A3 AR affinities
in the nanomolar range. Moreover, confirming our previ-
ous findings [16], most of the 4-cyclohexylamino deriva-
tives show lower A3 AR affinity than the corresponding
4-cyclopentylamino compounds (compare 1, 3, 4 and 5
with 6, 7, 8 and 10, respectively).
Introduction of substituents on the benzofused moiety of
the 4-cyclohexylamino derivative A generally reduces
the A1 and, especially, the A2A AR affinity, thus improving
the A1 vs A2A selectivity.In fact, compounds 1–5 are more
A1 vs A2A-selective than compound A. Among the 4-cy-
clopentylamino derivatives, compounds 6–8 and 9–11
show, respectively, lower and similar A1 affinity than their
parent compound B, while compounds 6 and 9 display
higher A1 vs A2A selectivity than B.The presence of sub-
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

38 Colotta et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 35–41
Table 1. Binding activity at bovine A1 and A2A and human A3 ARs.
Ki (nM)† or %-inhibition
#
$
‡
Compound
R6
R7
R8
R
A1
A2A
A3
A͵
1
H
H
H
Cl
H
H
H
H
H
H
Cl
Cl
Cl
H
1.43 0.1
90 7.2
1370 118
15 %
506 43
1360 120
16 %
2
Cl
48 3.8
0 %
3
NO2
NO2
NH2
H
17 0.9
54 %
3 %
4
1.98 0.15
0.39 0.02
0.42 0.03
32 %
281 24
316 29
55.4 4.2
5
H
2000 170
986 82
B͵
H
6
H
NO2
NO2
H
Cl
H
H
H
H
H
Cl
Cl
1.07 0.1
11.9 0.9
2.36 0.11
0.35 0.02
0.55 0.04
0.83 0.05
3800 340
0.5 0.03
55 %
8800 410
5000 290
25 %
144 11
53 %
7
8
H
116 24
212 18
18 1.5
9
NO2
H
10
11
NH2
H
310 28
NH2
1142 109
21000 1800
337 28
2.7 0.15
86000 7800
1300 125
Theophylline
DPCPX
†
The Ki values are means SEM of 4 separate assays, each performed in triplicate.
#
$
‡
͵
Displacement of specific [3H]CHA binding in bovine brain membranes or percentage of inhibition (I%) of specific bind-
ing at 20 µM concentration.
Displacement of specific [3H]CGS 21680 binding from bovine striatal membranes or percentage of inhibition (I%) of
specific binding at 20 µM concentration.
Displacement of specific [125I]AB-MECA binding at human A3 receptors expressed in CHO cells or percentage of in-
hibition (I%) of specific binding at 1 µM concentration.
Reference [16].
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 35–41
Synthesis of A1 Adenosine Receptor Antagonists 39
proton acceptor site. Nevertheless, we must admit that
this hypothesis is in contrast with the 2-fold lower A3 affin-
ity of the 6-nitro derivative 8 with respect to compound B.
Experimental
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 Gal-
lenkamp melting point apparatus. Microanalyses were per-
formed with a Perkin-Elmer 260 elemental analyzer for C, H, N,
and the results were within 0.4 % of the theoretical. The IR
spectra were recorded with a Perkin-Elmer 1420 spectrometer
in Nujol mulls and are expressed in cm–1.The 1H-NMR spectra
were obtained with a Varian Gemini 200 instrument at
200 MHz. The chemical shifts are reported in δ (ppm) and are
relative to the central peak of the solvent that is always DMSO-
d6. The following abbreviations are used: s = singlet, d = dou-
blet, dd = double doublet, t = triplet, m = multiplet, br = broad,
and ar = aromatic protons.
The A3 receptor model, cited above, could also justify the
beneficial effect of the 6-amino group for A3 affinity of
compounds 5, 10 and of some previuosly reported
triazolo[4,3-a]quinoxalin-1-one derivatives [15]. In fact,
the increased A3 receptor affinity of 5 and 10 with respect
to A and B, could be due to the capability of the 6-amino
group to form a hydrogen bond with Ser247, thus rein-
forcing the interaction of this serine residue with the N-5
area of 5 and 10.
In contrast, the influence of the hydrophilic 6-amino
group on A1 and A2A affinity is difficult to rationalize since
this substituent elicits opposite effects on the 4-cy-
clohexylamino derivative A and on the 4-cyclopentylami-
no derivative B. In fact, while compound 5 possesses an
increased A1 affinity and A1 vs A2A selectivity, with re-
spect to A, compound 10 shows similar A1 affinity and
reduced A1 vs A2A selectivity compared to B.
General Procedure for the Synthesis of 4-Cycloalkylamino-1,2-
dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-ones 1–4,
6–9
The title compounds were obtained starting from the triazolo-
quinoxaline-1,4-dione derivatives 17–21 [13, 15] (2 mmol)
which were reacted with phosphorus pentachloride (4 mmol)
by refluxing phosphorus oxychloride (40 mL) and anhydrous
pyridine (0.2 mL) until the disappearance of the starting materi-
al (12–24 h) could be observed byTLC monitoring.Evaporation
at reduced pressure of the excess phosphorus oxychloride af-
forded a solid, which was treated with ice water (50 mL), col-
lected and washed with cyclohexane. The 4-chloro derivatives
12–16 [16], obtained in high overall yields (85–90 %), were un-
stable, nevertheless they were pure enough to be used without
further purification.
Also the beneficial effect of the 8-amino group on com-
pound B (compound 11) could be rationalized on the ba-
sis of the previously cited A3 receptor model which hy-
pothesizes that the benzofused moiety of CGS 15943 re-
sides in a hydrophobic pocket in which polar amino ac-
ids, such as Thr94 (TM3) and Ser97 (TM3), are present.
Thus, the significantly increased affinity of 11, compared
to B, could be explained by hypothesizing a hydrogen
bond interaction betweenThr94 or Ser97 and the 8-ami-
no group.
The 4-cyclohexylamino derivatives 1–4 were prepared from the
4-chloro derivatives 12–15 (1 mmol) which were reacted over-
night at 120 °C, in a sealed tube with cyclohexylamine
(1.2 mmol) in absolute ethanol (5 mL) and triethylamine
(2 mmol). Upon cooling, a solid was obtained which was col-
lected and washed with water.
In conclusion, introduction of simple substituents on the
benzofused moiety of the previously reported triazolo-
quinoxalin-1-one derivatives A and B was advantageous
since it afforded some highly potent A1 and A1 vs A2A se-
lective antagonists.In particular, all the 4-cyclohexylami-
no derivatives (1–5) showed higher A1 vs A2A selectivity
than the parent compound A. Furthermore, on the basis
of the SAR studies, we confirmed the important role
played by the steric factors for the A3 receptor-ligand in-
teraction. In addition, we highlighted the importance of
the presence on the triazoloquinoxalin-1-one framework
of a group (6-nitro, 6- or 8-amino) able to form hydrogen
bonds with a proton donor or acceptor site of the A3 re-
ceptor-binding pocket.
7,8-Dichloro-4-cyclohexylamino-1,2-dihydro-2-phenyl-1,2,4-
triazolo[4,3-a]quinoxalin-1-one (1)
1
Yield: 95 %; mp 223–225 °C (AcOH). H-NMR 1.06–2.07 (m,
10 H, aliphatic protons), 4.02–4.29 (m, 1 H, aliphatic proton),
7.42 (t, 1 H, ar, J = 7.0 Hz), 7.58 (t, 2 H, ar, J = 7.4 Hz), 7.67 (s,
1 H, H-6), 8.05–8.15 (m, 3 H, 2ar + NH), 8.71 (s, 1 H, H-9).Anal.
(C21H19Cl2N5O) C, H, N.
6,8-Dichloro-4-cyclohexylamino-1,2-dihydro-2-phenyl-1,2,4-
triazolo[4,3-a]quinoxalin-1-one (2)
1
Yield: 98 %; mp 222–224 °C (AcOH). H-NMR 1.09–2.10 (m,
10 H, aliphatic protons), 4.08–4.30 (m, 1 H, aliphatic proton),
7.37 (t, 1 H, ar, J = 7.3 Hz), 7.50–7.62 (m, 3 H, ar), 8.06 (d, 2 H,
ar, J = 7.7 Hz), 8.22 (d, 1 H, NH, J = 7.9 Hz), 8.56 (d, 1 H, H-9, J
= 2.4 Hz). Anal. (C21H19Cl2N5O) C, H, N.
8-Chloro-4-cyclohexylamino-1,2-dihydro-6-nitro-2-phenyl-1,2,4-
triazolo[4,3-a]quinoxalin-1-one (3)
Yield: 88 %; mp 214–215 °C (Cyclohexane/EtOAc). 1H-NMR
1.04–2.08 (m, 10 H, aliphatic protons), 3.79–4.09 (m, 1 H,
aliphatic proton), 7.39 (t, 1 H, ar, J = 7.6 Hz), 7.56 (t, 2 H, ar, J =
7.6 Hz), 7.99–8.06 (m, 3 H, ar), 8.55 (d, 1 H, NH, J = 7.7 Hz),
8.70 (d, 1 H, H-9, J = 2.2 Hz). Anal. (C21H19ClN6O3) C, H, N.
Acknowledgment
We thank Dr. Karl-Norbert Klotz of the University of
Würzburg, Germany, for providing cloned human A3 re-
ceptors expressed in CHO cells.
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

40 Colotta et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 35–41
10: Yield: 75 %; mp 212–213 °C (Cyclohexane/EtOAc). 1H-
NMR 1.50–2.10 (m, 8 H, aliphatic protons), 4.35–4.52 (m, 1 H,
aliphatic proton), 5.50 (br s, 2 H, NH2), 6.67 (d, 1 H, ar, J = 7.8
Hz), 7.10–7.47 (m, 3 H, 2 ar + NH), 7.51 (t, 2 H, ar, J = 7.7 Hz),
7.95 (d, 1 H, H-9, J = 3.1 Hz), 8.08 (d, 2 H, ar, J = 8.1 Hz); IR
3480, 3400–3200, 1700. Anal. (C20H20N6O) C, H, N.
4-Cyclohexylamino-1,2-dihydro-6-nitro-2-phenyl-1,2,4-tri-
azolo[4,3-a]quinoxalin-1-one (4)
1
Yield: 90 %; mp 184–185 °C (EtOH). H-NMR 1.05–2.11 (m,
10 H, aliphatic protons), 3.87–4.13 (m, 1 H, aliphatic proton),
7.33–7.42 (m, 2 H, ar), 7.59 (t, 2 H, ar, J = 7.6 Hz), 7.80 (d, 1 H, J
= 7.9 Hz), 8.09 (d, 2 H, ar, J = 7.8 Hz), 8.60 (d, 1 H, NH, J = 7.3
Hz), 8.79 (d, 1 H, H-9, J = 7.0 Hz). Anal. (C21H20N6O3) C, H,
N.
11: Yield: 80 %; mp 178–180 °C (Cyclohexane/EtOAc). 1H-
NMR 1.49–2.23 (m, 8 H, aliphatic protons), 4.50–4.60 (m, 1 H,
aliphatic proton), 5.46 (br s, 2 H, NH2), 6.69 (d, 1 H, J = 7.7 Hz),
6.99 (t, 1 H, ar, J = 8.3 Hz), 7.36 (t, 1 H, ar, J = 7.2 Hz), 7.50–
7.60 (m, 3 H, 2 ar + NH), 7.86 (d, 1 H, ar, J = 7.9 Hz), 8.10 (d,
2 H, ar, J = 7.8 Hz). Anal. (C20H20N6O) C, H, N.
The 4-cyclopentylamino derivatives 6–9 were obtained from
12, 14–16 (1 mmol) and cyclopentylamine (1.2 mmol) following
the experimental conditions described above to obtain 1–4.
7,8-Dichloro-4-cyclopentylamino-1,2-dihydro-2-phenyl-1,2,4-tri-
azolo[4,3-a]quinoxalin-1-one (6)
Biochemistry
Bovine A1 and A2A Receptor binding: Displacement of [3H]CHA
from A1 AR in bovine cerebral cortical membranes and
[3H]CGS 21680 from A2A AR in bovine striatal membranes was
performed as described in [24].
Yield: 88 %; mp 220–222 °C (EtOAc). 1H-NMR 1.52–2.15 (m,
8 H, aliphatic protons), 4.42–4.63 (m, 1 H, aliphatic proton),
7.40 (t, 1 H, ar, J = 7.6 Hz), 7.54–7.66 (m, 3 H, ar), 8.07 (d, 2 H,
ar, J = 7.3 Hz), 8.22 (d, 1 H, NH, J = 7.3 Hz), 8.71 (s, 1 H, H-9).
Anal. (C20H17Cl2N5O) C, H, N.
Human A3 Receptor binding: Binding experiments at human A3
adenosine receptors were performed on crude membranes ob-
tained from CHO cells [25], using [125I]AB-MECA according to a
procedure previously described [16].
8-Chloro-4-cyclopentylamino-1,2-dihydro-6-nitro-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (7)
Yield: 86 %; mp 212–214 °C (Cyclohexane/EtOAc). 1H-NMR
1.43–2.12 (m, 8 H, aliphatic protons), 4.21–4.40 (m, 1 H,
aliphatic proton), 7.40 (t, 1 H, ar, J = 7.6 Hz), 7.56 (t, 2 H, ar, J =
7.6 Hz), 7.99–8.06 (m, 3 H, ar), 8.60–8.69 (m, 2 H, H-9 + NH).
Anal. (C20H17ClN6O3) C, H, N.
The concentration of the tested compounds that produced
50 % inhibition of specific [3H]CHA, [3H]CGS 21680 or [125I]AB-
MECA binding (IC50) was calculated using a non-linear regres-
sion method implemented in the InPlot program (Graph-Pad,
San Diego, CA) with 5 concentrations of the displacer, each
performed in triplicate.Inhibition constants (Ki) were calculated
according to the Cheng-Prusoff equation [26].The dissociation
constant (Kd) of [3H]CHA and [3H]CGS 21680 in cortical and
striatal bovine brain membranes were 1.2 nM and 14 nM, re-
spectively. The Kd value of [125I]AB-MECA in human A3 AR in
CHO cell membranes was 1.4 nM.
4-Cyclopentylamino-1,2-dihydro-6-nitro-2-phenyl-1,2,4-
triazolo[4,3-a]quinoxalin-1-one (8)
Yield: 70 %; mp 216–217 °C (EtOAc). 1H-NMR 1.45–2.16 (m,
8 H, aliphatic protons), 4.42–4.50 (m, 1 H, aliphatic proton),
7.38 (t, 2 H, ar, J = 8.1 Hz), 7.59 (t, 2 H, ar, J = 7.7 Hz), 7.77 (d,
1 H, ar, J = 8.2 Hz), 8.09 (d, 2 H, ar, J = 7.7 Hz), 8.52 (d, 1 H, NH,
J = 6.9 Hz), 8.78 (d, 1 H, H-9, J = 8.2 Hz). Anal. (C20H18N6O3) C,
H, N.
References
4-Cyclopentylamino-1,2-dihydro-8-nitro-2-phenyl-1,2,4-
triazolo[4,3-a]quinoxalin-1-one (9)
[1] K. A. Jacobson, P. J. M. van Galen, M. Williams, J. Med.
Chem. 1992, 35, 407–422.
1
Yield: 80 %; mp 276–277 °C (EtOAc). H-NMR 1.58–1.9 (m,
6 H, aliphatic protons), 2.04–2.18 (m, 2 H, aliphatic proton),
4.52–4.62 (m, 1 H, aliphatic proton), 7.42 (t, 1 H, ar, J = 7.2 Hz),
7.51–7.64 (m, 3 H, ar), 8.03–8.23 (m, 3 H, ar), 8.72 (d, 1 H, NH,
J = 7.3 Hz), 9.38 (d, 1 H, H-9, J = 2,7 Hz); IR 3380, 1740. Anal.
(C20H18N6O3) C, H, N.
[2] S.-A. Poulsen, R. J Quinn, Bioorg. Med. Chem. 1998, 6,
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General Procedure for the Synthesis of 6-Amino-4-cyclohexyl-
amino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-
one (5), 6-Amino-4-cyclopentylamino-1,2-dihydro-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (10) and 8-Amino-4-
cyclopentylamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]-
quinoxalin-1-one (11)
[4] J.Linden, H.E.Taylor, A.S.Robeva, A.L.Tucker, J.H.Steh-
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Compounds 4, 8 or 9 (0.8 mmol) were dissolved in hot glacial
acetic acid (200 mL).Pd/C 10 % (0.05 g) was added to the solu-
tion and the mixture was hydrogenated overnight in a Parr ap-
paratus at 30 psi.The suspension was heated and the catalyst
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filtered.
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[9] V. Colotta, L. Cecchi, D. Catarzi, F. Melani, G. Filacchioni,
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5:Yield: 82 %; mp 191–192 °C (EtOAc). 1H-NMR 1.04–2.08 (m,
10 H, aliphatic protons), 4.07–4.33 (m, 1 H, aliphatic proton),
5.44 (br s, 2 H, NH2), 6.69 (d, 1 H, ar, J = 7.9 Hz), 6.99 (t, 1 H, ar,
J = 7.8 Hz), 7.30–7.61 (m, 4 H, 3 ar + NH), 7.86 (d, 1 H, ar, J =
8.3 Hz), 8.10 (d, 2 H, ar, J = 8.5 Hz). Anal. (C21H22N6O) C, H, N.
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© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim