1,2,4-Triazolo[4,3-a]quinoxalin-1-one: A versatile tool for the synthesis of potent and selective adenosine receptor antagonists

Article abstract of DOI:10.1021/jm991096e

4-Amino-6-benzylamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3- a]quinoxalin-1-one (1) has been found to be an A(2A) versus A₁ selective antagonist (Colotta et al. Arch. Pharm. Pharm. Med. Chem. 1999, 332, 39-41). In this paper some novel triazoloquinoxalin-1-ones 4-25 bearing different substituents on the 2-phenyl and/or 4-amino moiety of the parent 4-amino-1,2- dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (3) have been synthesized and tested in radioligand binding assays at bovine A₁ and A(2A) and cloned human A₃ adenosine receptors (AR). Moreover, the binding activities at the above-mentioned AR subtypes of the 1,4-dione parent compounds 26-31 and their 5-N-alkyl derivatives 33-37 were also evaluated. The substituent on the 2-phenyl ring exerted a different effect on AR subtypes, while replacement of a hydrogen atom of the 4-amino group with suitable substituents yielded selective A₁ or A₃ antagonists. Replacement of a hydrogen atom of the 4-NH₂ with an acyl group, or replacement of the whole 4-NH₂ with a 4-oxo moiety, shifted the binding activity toward the A₃ AR. The binding results allowed elucidation of the structural requirements for the binding of these novel tricyclic derivatives at each receptor subtype. In particular, A₁ and A₂A binding required the presence of a proton donor group at position-4, while for A₃ affinity the presence of a proton acceptor in this same region was of paramount importance.

Full text of DOI:10.1021/jm991096e

1158
J . Med. Chem. 2000, 43, 1158-1164
1,2,4-Tr ia zolo[4,3-a ]qu in oxa lin -1-on e: A Ver sa tile Tool for th e Syn th esis of
P oten t a n d Selective Ad en osin e Recep tor An ta gon ists
Vittoria Colotta,† Daniela Catarzi,† Flavia Varano,† Lucia Cecchi,*,† Guido Filacchioni,† Claudia Martini,‡
Letizia Trincavelli,‡ and Antonio Lucacchini‡
Dipartimento di Scienze Farmaceutiche, Universita’ di Firenze, Via G. Capponi, 9, 50121 Firenze, Italy, and Dipartimento di
Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita’ di Pisa, Via Bonanno, 6, 50126 Pisa, Italy
Received J une 11, 1999
4-Amino-6-benzylamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (1) has been
found to be an A2A versus A1 selective antagonist (Colotta et al. Arch. Pharm. Pharm. Med.
Chem. 1999, 332, 39-41). In this paper some novel triazoloquinoxalin-1-ones 4-25 bearing
different substituents on the 2-phenyl and/or 4-amino moiety of the parent 4-amino-1,2-dihydro-
2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (3) have been synthesized and tested in radio-
ligand binding assays at bovine A1 and A2A and cloned human A3 adenosine receptors (AR).
Moreover, the binding activities at the above-mentioned AR subtypes of the 1,4-dione parent
compounds 26-31 and their 5-N-alkyl derivatives 33-37 were also evaluated. The substituent
on the 2-phenyl ring exerted a different effect on AR subtypes, while replacement of a hydrogen
atom of the 4-amino group with suitable substituents yielded selective A1 or A3 antagonists.
Replacement of a hydrogen atom of the 4-NH2 with an acyl group, or replacement of the whole
4-NH2 with a 4-oxo moiety, shifted the binding activity toward the A3 AR. The binding results
allowed elucidation of the structural requirements for the binding of these novel tricyclic
derivatives at each receptor subtype. In particular, A1 and A2A binding required the presence
of a proton donor group at position-4, while for A3 affinity the presence of a proton acceptor in
this same region was of paramount importance.
Ch a r t 1
In tr od u ction
Adenosine is a ubiquitous neuromodulator in both the
periphery and the central nervous system. The effects
elicited by adenosine are mediated by its interactions
with four receptor subtypes, termed A1, A2A, A2B, and
A3, belonging to the G-protein-coupled receptor family.1,2
All four adenosine receptor (AR) subtypes have been
identified on a pharmacological level as well as on a
molecular level.3 ARs from different species show amino
acid sequence homology (82-93%) with the only excep-
tion being the A3 subtype which only exhibits 74%
primary sequence homology between rat and human or
sheep.4-6
In the last two decades, many efforts have been in-
vested in the synthesis of selective AR ligands for their
potential therapeutic use. This research has resulted
in the synthesis of a number of AR agonists and antag-
onists.7-9 Particularly, selective AR subtype antagonists
are sought as renal protective,10,11 anti-Parkinson,12
antiinflammatory, antiasthmatic, and antiischemic
agents.13-16
In recent years some studies in our laboratory have
been directed toward the synthesis and structure-
activity relationship (SAR) studies of AR antagonists.17-21
A recent paper21 reported the synthesis of 4-amino-6-
benzylamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]-
quinoxalin-1-one (1) and its 6-N-desbenzyl derivative
2 (Chart 1). In preliminary binding screenings at the
A1 and A2A ARs, compound 1 was a potent and selective
A2A versus A1 antagonist, while 2 was 2-fold more
selective for the A1 versus A2A subtype. To investigate
the SAR on the new triazoloquinoxalin-1-one system,
we here describe the synthesis and the A1, A2A, and A3
binding activities of some novel triazoloquinoxalin-1-
ones 3-25 bearing different substituents on the 2-phen-
yl and/or 4-amino moiety. Moreover, the binding activi-
ties at the above-mentioned AR subtypes of the 1,4-
dione parent compounds 26-31, of their 5-N-alkyl
derivatives 33-37, and of the 1,2,4,5-tetrahydro-2-(4-
methylphenyl)-1,2,4-triazolo[4,3-a]quinoxalin-4-one (32)
are also evaluated.
* To whom correspondence should be addressed. Tel: +39 55
2757282. Fax: +39 55 240776. E-mail: cecchi@farmfi.scifarm.unifi.it.
† Universita’ di Firenze.
‡ Universita’ di Pisa.
10.1021/jm991096e CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/03/2000

1,2,4-Triazolo[4,3-a]quinoxalin-1-one
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1159
Sch em e 1a
2-(4-chlorophenyl)-1,2,4-triazolo[4,3-a]quinoxaline-1,4-
dione (31) was obtained from the corresponding 3-aryl-
hydrazonoquinoxalin-2-one 43 as described for the
preparation of 26-30.23 The 1,2,4,5-tetrahydro-1-(4-
methylphenyl)-1,2,4-triazolo[4,3-a ]quinoxalin-4-one
(32) was obtained by cyclizing the corresponding hy-
drazonoquinoxaline 4023 with formaldehyde. The 5-N-
alkyl-2-aryl-1,2,4,5-tetrahydro-1,2,4-triazolo[4,3-a]qui-
noxaline-1,4-diones 33-37 were prepared by reacting
the key intermediates 26, 28, and 31 with alkyl halides
(Scheme 1).
By reacting the key intermediates 26-31 with phos-
phorus pentachloride and phosphorus oxychloride, the
unstable 2-aryl-4-chloro-1,2-dihydro-1,2,4-triazolo[4,3-
a]quinoxalin-1-ones 44-49 were isolated (Scheme 2).
Reaction of 44-49 with ammonia or amines yielded the
final 4-amino-substituted derivatives 3-19. Allowing
the 4-amino-2-phenyl-1,2-dihydro-1,2,4-triazolo[4,3-a]-
quinoxalin-1-one (3) to react with either acyl chlorides
or aryl isocyanates, the 4-amido 20-23 or 4-ureido
derivatives 24-25 were obtained, respectively.
a
Bioch em istr y
(a) (Cl3CO)2CO, THF; (b) 40% HCHO, ethylene glycol; (c) R1-
halide, NaH, DMF.
Compounds 3-37 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]-5′-(N-ethylcarbam-
oyl)adenosine ([3H]CGS 21680) from A2A AR in bovine
striatal membranes, and [125I]N6-(4-amino-3-iodoben-
zyl)-5′-N-methylcarbamoyladenosine ([125I]AB-MECA)
from cloned human A3 AR stably expressed in HEK-
293 cells. In fact, due to the species differences in A3
primary amino acid sequence, new A3 AR ligands had
to be tested on cloned human A3 ARs.4-6 On the
contrary, for A1 and A2A AR subtypes there is a good
amino acid sequence homology,9 since standard antago-
nists, such as theophilline and 1,3-dipropyl-8-cyclopen-
tylxanthine (DPCPX), showed an affinity at bovine A1
and A2A ARs comparable to those reported at the cloned
human ones.24-26
Sch em e 2a
The binding results of 3-37 are shown in Table 1. In
this table the binding activity at bovine A1 and A2A and
human cloned A3 ARs of the previously synthesized21
compounds 1 and 2 are also reported together with those
of theophilline and DPCPX, included as antagonist
reference compounds.
a
Resu lts a n d Con clu sion s
R and R1 are defined in Tables 1 and 2. (a) PCl5/POCl3,
pyridine; (b) NH3(g), EtOH; (c) R1NH2, NEt3, EtOH; (d) R2COCl,
pyridine, CH2Cl2, or R2NCO, THF.
The results on the binding activities of compounds
1-37 displayed in Table 1 show that we have produced
some potent and selective AR subtype antagonists. It
is worth noting that a more careful screening of the A1
affinity of 1 revealed a higher affinity (Ki ) 730 nM)
than that reported (Ki ) 17 500 nM).21 Nevertheless,
due to its inactivity at the A3 subtype (I% ) 30),
compound 1 is still a potent and selective A2A antago-
nist. Compound 2, on the contrary, is a nonselective AR
antagonist displaying nanomolar affinity at all three
receptor subtypes.
With the aim of defining the SAR in the 1,2,4-
triazoloquinoxalin-1-one system, we synthesized the
4-amino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one
(3) which is the parent compound of the whole series.
Compound 3 was equipotent to 2 at the A1, less potent
Ch em istr y
The synthesis of the novel triazoloquinoxalin-1-one
derivatives 3-37 is illustrated in Schemes 1 and 2.
Scheme 1 shows the preparation of the 1,4-dione parent
compounds 26-31, their 5-N-alkyl derivatives 33-37,
and the 1,2,4,5-tetrahydro-1-(4-methylphenyl)-1,2,4-
triazolo[4,3-a]quinoxalin-4-one (32), while in Scheme 2
the synthesis of the 4-amino-1-ones 3-8 and the 4-amino-
substituted-1-ones 9-25 is described.
Briefly, compound 43 was prepared by reacting the
commercially available o-phenylenediamine with N1-(4-
chlorophenyl)hydrazono-N2-chloroacetate22 following the
procedure described to prepare compounds 38-42.23 The

1160 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Ta ble 1. Binding Activity at Bovine A1 and A2A and Human A3 ARs
Colotta et al.
a
b
The Ki values are means ( SEM of four separate assays, each performed in triplicate. Displacement of specific [3H]CHA binding in
bovine brain membranes or percentage of inhibition (I%) of specific binding at 20 µM concentration. c Displacement of specific [3H]CGS
d
21680 binding in 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 ARs expressed in HEK-293 cells or percentage of inhibition (I%) of specific binding at 1
µM concentration.
at the A2A, and inactive at the A3 ARs. The first
variation on the structure of the parent compound 3 was
the introduction of simple substituents at the 3- or
4-position of the 2-phenyl ring (compounds 4-8). In fact,
nothing was known about the influence of a substituent
on the 2-phenyl ring toward the AR affinity. None of
these 2-phenyl-substituted derivatives 4-8 exceeded the
affinity of 3 at the A1 and A2A ARs, with the exception
of 2-(3-methylphenyl) derivative 4 which was about
3-fold more potent than 3 at the A2A subtype. The
generally negative effect of the substituent on the
2-phenyl ring toward A1 and A2A affinity is stressed in
the 2-(4-methoxyphenyl) (7) and 2-(4-chlorophenyl) (8)
derivatives which showed dramatically reduced A1 and
A2A binding activities. On the contrary, the presence of
the substituent on the 2-phenyl ring has a favorable
effect for A3 receptor-ligand interaction. In fact, com-
pounds 4-8 displayed higher A3 receptor affinity than
that of the parent compound 3. Among these 2-phenyl-
substituted 4-amino derivatives 4-8 the 2-(3-meth-
ylphenyl) compound (4) showed the highest A3 receptor
affinity (Ki ) 28.5 nM), while the best A3 receptor
selectivity was achieved with the 2-(4-methoxyphenyl)
substituent (7). In fact compound 7 was about 6- and
8-fold more potent on A3 than on A1 and A2A AR
subtypes, respectively. These data suggest that in A1
and A2A ARs the lipophilic region that accommodates
the 2-aryl moiety has different structural requirements
with respect to those of the A3 area.
The second modification we performed on the parent
structure 3 concerned the replacement of a hydrogen
atom of the 4-amino group with suitable substituents,
such as cycloalkyl, aralkyl, and acyl, to obtain A1 or A3
subtype selective antagonists. The 4-cycloalkylamino
derivatives 9-15 were prepared as potential A1 selective
antagonists since the cycloalkyl substituent in several
tricyclic systems of similar size and shape yielded A1
selective ligands.8,27,28 As expected, the 4-aminocy-
cloalkyl derivatives 9-15 displayed nanomolar A1 af-
finity. However, compounds 9-15 were also active at
the A3 ARs, although the A1 affinities were on the whole
higher than the A3 ones. The A2A affinities of 9-15 were
low or null, with the exception of the 2-(3-fluorophenyl)
derivatives 11 and 15 which displayed an A2A affinity

1,2,4-Triazolo[4,3-a]quinoxalin-1-one
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1161
in the nanomolar range (Ki values of 66 and 148 nM,
respectively). The negative effect of the substituent on
the 2-phenyl ring toward the A1 binding activity is
present in this series also, as shown by the decreased
binding activity at this receptor of the 4-N-cyclohexyl
(10-12) and 4-N-cyclopentyl (14, 15) derivatives, as
compared to those of their corresponding 2-phenyl
derivatives 9 and 13, respectively. The generally favor-
able effect of the presence of a substituent on the
2-phenyl moiety toward A3 affinity is also confirmed in
these 4-N-cycloalkyl derivatives 9-15. Indeed, the
2-aryl compounds 11, 12, and 14 are more potent than
the corresponding 2-phenyl derivatives 9 and 13, re-
spectively.
respectively). The similar A1 affinity of 16, 22 and 17,
23 suggests that high affinity at this receptor subtype
depends on the number of carbon atoms of the spacer
between the phenyl moiety and the NH group.
Finally, the synthesis of the 4-N-carbamoyl deriva-
tives 24 and 25 was pursued due to the A3 affinity in
the low nanomolar range of some adenosine agonists.32
Nevertheless, in the present series a ureido group at
position-4 did not offer any advantage in receptor-
ligand interaction. In fact, compounds 24 and 25 were
much less active at all three receptor subtypes than the
corresponding amides 20-23.
Evaluation of the importance of the 4-amino proton
donor group was the rationale for testing the intermedi-
ate 1,4-diones 26-31. As Table 1 shows, these xanthine-
like compounds are completely inactive at the A2A AR
and less active at the A1 AR than the corresponding
4-amino derivatives 3-8 confirming the importance of
the 4-amino donor group in A1 and A2A receptor recogni-
tion.19 This is not the case for receptor-ligand interac-
tion at the A3 subtype since the 1,4-diones 26-31
display at this subtype a higher affinity than the
corresponding 4-amino derivatives 3-8, with the only
exception being 27 which was less active than its
corresponding 4-amino derivative 4. Moreover, compari-
son of the A1 and A3 affinity of the 2-phenyl-unsubsti-
tuted 26 with those of the 2-phenyl-substituted 27-31
indicated that the substituent on the 2-phenyl ring
increased both A1 and A3 binding activities, with the
exception of the 2-(4-methoxyphenyl) (30) and 2-(4-
chlorophenyl) (31) derivatives which, in agreement with
the results mentioned above, showed a decreased A1
affinity. The 4-methoxy and 4-chloro groups have how-
ever contrasting effects on the A3 affinity. In fact, while
the 2-(4-methoxyphenyl) 30 was a potent and selective
A3 ligand, the 2-(4-chlorophenyl) 31 showed the lowest
A3 binding activity among the 1,4-diones 26-31. The
A1 and A3 affinities of 30 confirmed the different
structural requirements of the A1 and A3 subtypes in
the region that binds the 2-aryl moiety of the triazolo-
quinoxaline system.
Evaluation of the effect of the 5-N-alkylation on the
1,4-diones 26, 28, and 31 was the rationale for the
synthesis of the 5-N-alkylated-1,4-diones 33-37. The
5-N-alkylation offered no advantage on A2A affinity since
compounds 33-37 are devoid of affinity at this receptor
subtype, while it is advantageous for A1 and A3 affinities
only in the case of the 5-N-methyl derivative 33. In fact,
compound 33 was more active at the A1 (Ki value of 309
nM) and A3 (Ki value of 36.6 nM) than its 5-N-desmethyl
analogue 26. Elongation of the 5-N-alkyl chain (36) or
the presence of a triple bond (37) decreased A1 and A3
potency. It has to be noted that in these 5-N-alkyl
derivatives the negative effect of the substituent on the
2-phenyl ring (compounds 34, 35) appears not only for
A1 affinities but also for A3 affinities.
Replacement of a hydrogen atom of the 4-amino group
of the parent structure 3 with an aralkyl substituent
(16-19) had contrasting effects depending on AR sub-
type. The 4-N-aralkylamino-2-phenyl derivatives 16-
19 were all inactive at the A2A AR. The N-benzyl 16 was
less potent at the A1 (Ki ) 55.0 nM) and more potent at
the A3 (Ki ) 1700 nM) than 3. Homologation of the
N-alkyl chain (compound 17) produced a strong incre-
ment in A1 (Ki ) 4.8 nM) and A3 (Ki ) 201 nM) affinities.
Indeed, the N-phenylethyl derivative 17 is a potent and
selective A1 antagonist (A1/A3 ) 41). Further homolo-
gation of the N-alkyl chain (18) reduced, by about 4-fold,
the A1 affinity (Ki ) 17.9 nM) while it increased, by the
same order, the A3 affinity (Ki ) 40.9 nM). The affinity
at the A1 and A3 ARs dropped significantly when a
second phenyl ring (19) was present in the ethylene
spacer chain of 17.
Replacement of a hydrogen atom of the 4-amino group
of 3 with an acyl moiety (20-23) yielded, in agreement
with the literature data,29-31 a strong increment in A3
potency. Compounds 20-23 were all inactive at the A2A
AR. It has to be noted that the aliphatic 4-acetylamide
20 and 4-propionylamide 21 were potent (Ki values 2.0
and 15.8 nM, respectively) but not A3 selective since
they displayed Ki values of 4.3 and 9.3 nM on A1,
respectively. On the contrary, the aromatic 4-benzoyl-
amide 22 was 60-fold more potent at human A3 (Ki )
1.4 nM) than at bovine A1 subtype. Homologation of 22
afforded the 4-phenylacetylamide 23 which, like 20 and
21, was an A1 and A3 potent nonselective antagonist.
In our series of triazoloquinoxalin-1-ones the importance
of the presence of the CdO amide group at position-4
in A3 receptor-ligand interaction was stressed by the
comparison of the A3 affinity of the 4-N-benzoylamido
22 (Ki ) 1.47 nM) versus 4-N-benzylamino 16 (Ki ) 1700
nM) and the 4-N-phenylacetylamido 23 (Ki ) 3.75 nM)
versus 4-N-phenethylamino 17 (Ki ) 201 nM). Since we
presume that the exocyclic N-4 region of the triazolo-
quinoxalin-1-ones corresponds to that of the N-6 of the
adenosine, the improvement in A3 potency of 20-23
could be due (i) to the enhanced acidity of the NH proton
donor because of the presence of the electron-withdraw-
ing CdO group and/or (ii) to the presence in the A3
subtype of a proton donor site which binds to the CdO
acceptor. In contrast, the 4-CdO amide group it is not
necessary for A1 receptor-ligand interaction since the
4-N-benzylamino 16 and 4-N-benzoylamido 22 showed
the same order of A1 affinity (Ki values of 55.0 and 89.6
nM, respectively) as the 4-N-phenethylamino 17 and
4-N-phenylacetylamido 23 (Ki values of 4.8 and 6.3 nM,
The 1-descarbonyl derivative 32 was devoid of A1 and
A2A binding activity but mantained some A3 affinity (Ki
) 197 nM). These data showed that the presence of the
CdO proton acceptor at position-119,21 is essential for
A1 and A2A affinity but is not necessary for A3 receptor-
ligand recognition.
In conclusion, the synthesis of these novel triazolo-
quinoxalin-1-ones has allowed us to elucidate the struc-

1162 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Colotta et al.
Ta ble 2. Physical Data of the Newly Synthesized Compounds
tural requirements for the binding of this new tricyclic
system at each AR subtype. The AR affinities of
compounds 9-18 showed that the presence of a 4-N-
cycloalkyl or 4-N-aralkyl group gives rise to A1 potent
and selective antagonists. The introduction in the
triazoloquinoxaline moiety of a 4-N-amido (compounds
20-23) or 4-oxo (compounds 26-31, 33-37) function
affords selective and/or potent A3 receptor antagonists.
These findings indicate that a CdO group, either
extranuclear (as in the 4-amido 20-23) or nuclear (as
in the 1,4-diones 26-31, 33-37) is necessary for A3
affinity. This suggests the importance for A3 receptor-
ligand interaction of (i) a strong acidic NH proton donor
and/or (ii) a CdO proton acceptor able to engage a
hydrogen bond with a proton donor present on the A3
recognition site. Examination of the AR affinity of 26-
31 and 33-37 suggests that the 4-NH2 proton donor
group is essential for A1 and A2A receptor-ligand
interaction while it is not necessary for A3 receptor
recognition. Finally, the binding results of the 2-aryl
derivatives, in both the 4-amino (3-15) and 4-oxo (26-
31) series, indicate that the presence and the nature of
the substituent on the 2-phenyl moiety affect the A1 and
A3 receptor affinities differently. Thus, the introduction
of suitable groups on the 2-phenyl ring can be used to
shift the selectivity toward A1 or A3 ARs.
In conclusion, the triazoloquinoxalin-1-one core seems
to be a versatile tool to obtain potent and selective AR
antagonists.
Exp er im en ta l Section
(A) Ch em istr y. Silica gel plates (Merck F254) were used for
analytical chromatography. All melting points were deter-
mined on a Gallenkamp melting point apparatus. Microanaly-
ses were performed with
a Perkin-Elmer 260 elemental
analyzer for C, H, N, and the results were within (0.4% of
the theoretical values. The IR spectra were recorded with a
Perkin-Elmer 1420 spectrometer in Nujol mulls and are
1
expressed in cm-1. The H 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. The following abbreviations are used: s
) singlet, d ) doublet, dd ) double doublet, t ) triplet, m )
multiplet, br ) broad, and ar ) aromatic protons. Physical
data of the newly synthesized compounds are listed in Table
2.
3-(4-Ch lor op h en yl)h yd r a zon o-1,2,3,4-t et r a h yd r oq u i-
n oxa lin -2-on e (43). The title compound was obtained from
ethyl N1-(4-chlorophenyl)hydrazono-N2-chloroacetate22 (9 mmol),
o-phenylenediamine (9 mmol) and triethylamine (10.8 mmol)
as described in ref 23 to prepare 38-42. Compound 43 may
exist, like 38-42,23 in either one of the two tautomeric forms
A and B:
a
Recrystallization solvents: A ) ethanol, B ) dioxane, C )
methanol, D ) ethyl acetate, E ) cyclohexane, F ) acetonitrile,
G ) glacial acetic acid, H ) ethylene glycol.
8.89 (s, NH of tautomer A), 9.45 (d, NH of tautomer B, J )
1.4 Hz), 9.63 (s, NH of tautomer A), 11.15 (s, lactam NH of
tautomer A), 12.30 (s, lactam NH of tautomer B).
2-(4-Ch lor op h en yl)-1,2,4,5-t et r a h yd r o-1,2,4-t r ia zolo-
[4,3-a ]qu in oxa lin e-1,4-d ion e (31). The title compound was
obtained from 43 (4 mmol) and triphosgene (4 mmol) as
described in ref 23 to prepare 26-30: 1H NMR (DMSO-d6)
7.28-7.40 (m, 3H, ar), 7.65 (d, 2H, ar, J ) 8.9 Hz), 8.06 (d,
2H, ar, J ) 8.9 Hz), 8.60 (d, 1H, ar, J ) 7.7 Hz), 12.01 (br s,
1H, NH).
2-(4-Met h ylp h en yl)-1,2,4,5-t et r a h yd r o-1,2,4-t r ia zolo-
[4,3-a ]qu in oxa lin -4-on e (32). A mixture of 4023 (0.89 mmol)
in ethylene glycol (3 mL) and aqueous formaldehyde (40%, 0.4
mL) was heated at reflux for 2-3 min. Dilution with water
(10 mL) yielded a yellow solid which was collected, washed
with water and crystallized: 1H NMR (DMSO-d6) 2.25 (s, 3H,
CH3), 5.72 (s, 2H, CH2), 6.84-7.18 (m, 8H, ar), 11.48 (br s,
1H, NH); IR 1670, 3160.
Tautomer A was easily distinguished from tautomer B since
in the former each exchangeable proton was present as singlet,
while in the latter the two hydrazine protons appeared as
1
doublets. The H NMR spectrum of compound 43 revealed the
existence of both tautomers A and B (ratio 1:2) since there
are six protons which exchange with D2O: 1H NMR (DMSO-
d6) 6.73-7.34 (m, ar), 8.05 (d, NH of tautomer B, J ) 1.4 Hz),

1,2,4-Triazolo[4,3-a]quinoxalin-1-one
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1163
Gen er a l P r oced u r e To P r ep a r e 2-Ar yl-4-ch lor o-1,2-
d ih yd r o-1,2,4-tr ia zolo[4,3-a ]qu in oxa lin -1-on es 44-49. A
mixture of 26-3123 (2 mmol) and phosphorus pentachloride
(1 mmol) in phosphorus oxychloride (30 mL) and anhydrous
pyridine (0.2 mL) was heated at reflux until the disappearance
(TLC monitoring) of the starting material (2-8 h). Evaporation
at reduced pressure of the excess of phosphorus oxychloride
yielded a residue which was treated with water (50 mL),
collected and washed with cyclohexane. These 4-chloro deriva-
tives were very unstable; however they were pure enough to
be characterized and used without further purification. Com-
pound 44 displayed the following: 1H NMR (DMSO-d6) 7.41
(t, 1H, ar, J ) 7.2 Hz), 7.55-7.65 (m, 3H, ar), 7.77 (t, 1H, ar,
J ) 6.6 Hz), 7.90 (dd, 1H, ar, J ) 8.0, 1.3 Hz), 8.06 (dd, 2H,
ar, J ) 7.4, 1.3 Hz), 8.77 (dd, 1H, ar, J ) 8.0, 1.3 Hz).
Gen er a l P r oced u r e To P r ep a r e 4-Am in o-2-a r yl-1,2-
dih ydr o-1,2,4-tr iazolo[4,3-a ]qu in oxalin -1-on es 3-8. A mix-
ture of 44-49 (2 mmol) in absolute ethanol (30 mL) saturated
with ammonia was heated overnight at 120 °C in a sealed tube.
Upon cooling, a solid precipitated which was collected, washed
with water and crystallized. Compound 3 displayed the fol-
lowing spectral data: 1H NMR (DMSO-d6) 7.25-7.50 (m, 3H,
ar), 7.51-7.63 (m, 5H, ar + NH2), 8.09 (d, 2H, ar, J ) 8.2 Hz),
8.64 (d, 1H, ar, J ) 8.1 Hz); IR 1660, 1735, 3020-3220, 3320,
3460.
Gen er a l P r oced u r e To P r ep a r e 4-Cycloh exyla m in o-
2-a r yl-1,2-d ih yd r o-1,2,4-t r ia zolo[4,3-a ]q u in oxa lin -1-
on es 9-12. A mixture of 44, 45, 47, 49 (1 mmol), cyclohexyl-
amine (1,2 mmol) and triethylamine (2 mmol) in absolute
ethanol (5 mL) was heated overnight at 120 °C in a sealed
tube. Upon cooling, a solid was obtained which was collected,
washed with water and crystallized. Compound 9 displayed
the following spectral data: 1H NMR (DMSO-d6) 1.10-2.05
(m, 10H, aliphatic protons), 4.13-4.18 (m, 1H, aliphatic
proton), 7.22-7.67 (m, 7H, 6 ar + NH), 8.10 (d, 2H, ar, J )
8.5 Hz), 8.63 (d, 1H, ar, J ) 7.8 Hz); IR 1730, 3420.
Gen er a l P r oced u r e To P r ep a r e 4-Cyclop en tyla m in o-
2-a r yl-1,2-d ih yd r o-1,2,4-t r ia zolo[4,3-a ]q u in oxa lin -1-
on es 13-15. The title compounds were prepared from 44, 45,
47 (1 mmol) and cyclopentylamine (1.2 mmol) following the
experimental conditions described above to obtain 9-12.
Compound 13 displayed the following spectral data: 1H NMR
(DMSO-d6) 1.29-1.78 (m, 8H, aliphatic protons), 4.20-4.35
(m, 1H, aliphatic proton), 6.94-7.33 (m, 6H, 5 ar + NH), 7.54
(d, 1H, ar, J ) 7.3 Hz), 7.82 (d, 2H, ar, J ) 8.3 Hz), 8.34 (d,
1H, ar, J ) 7.9 Hz).
The resulting solid was collected and crystallized. Compound
24 displayed the following: 1H NMR (DMSO-d6) 7.10-7.94 (m,
11H, ar), 8.20 (d, 2H, ar, J ) 8.0 Hz), 8.68-8.74 (m, 1H, ar),
10.24 (s, 1H, NH), 11.68 (s, 1H, NH).
Gen er al P r ocedu r e To P r epar e 5-N-Alkyl-2-ar yl-1,2,4,5-
tetr ah ydr o-1,2,4-tr iazolo[4,3-a ]qu in oxalin e-1,4-dion es 33-
37. The suitable alkyl halide (1.65 mmol of methyl iodide or
propargyl bromide, 4 mmol of n-propyl bromide) and sodium
hydride (80% dispersion in mineral oil, 2.42 mmol) were added
to a suspension of 26, 28, 31 (1.1 mmol) in anhydrous
dimethylformamide (DMF) (3 mL). The mixture was stirred
at room temperature for 90 min in the case of methyl iodide
and propargyl bromide or for 36 h in the case of the less
reactive n-propyl bromide. Addition of water (40 mL) to the
mixture afforded a solid which was collected and crystallized.
Compound 33 displayed the following spectral data: 1H NMR
(DMSO-d6) 3.61 (s, 3H, CH3), 7.31-7.65 (m, 6H, ar), 8.03 (d,
2H, ar, J ) 7.5 Hz), 8.75 (d, 1H, ar, J ) 7.8 Hz); IR 1690,
1720.
(B) Bioch em istr y. A1 a n d A2A r ecep tor bin d in g: Dis-
placement of [3H]CHA from A1 AR in bovine cortical mem-
branes and [3H]CGS 21680 from A2A AR in bovine striatal
membranes was performed as described.33
A3 r ecep tor bin d in g: The displacement of [125I]AB-MECA
in membranes prepared from HEK-293 cells (Sigma-Aldrich,
Milano) stably expressing the human A3 AR was performed
as described.34 The assay medium consisted of a buffer
containing 50 mM Tris-HCl, 10 mM MgCl2, and 1 mM EDTA
at pH 8.12. The glass incubation tubes, containing 20 µL of
the membrane suspension (0.2 mg of protein/mL, stored at -80
°C in the same buffer), 20 µL of [125I]AB-MECA (final concen-
tration 0.2 nM), and 10 µL of the tested ligand, were incubated
for 60 min at 25 °C in a total volume of 100 µL. After
incubation the samples were filtered on Whatman GF/C filters
presoaked for 1 h in 0.5% poly(ethylenimine) followed by three
washes with 5 mL of ice-cold incubation buffer. Nonspecific
binding was determined in the presence of 200 µM NECA.
Specific binding was obtained by subtracting nonspecific
binding from 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 binding.
Protein estimation was based on a reported method,35 after
solubilization with 0.75 N sodium hydroxide, using bovine
serum albumin as standard.
The concentration of the tested compound that produced
50% inhibition of specific [3H]CHA, [3H]CGS 21680, or [125I]-
AB-MECA binding (IC50) was calculated using a nonlinear
regression method implemented in the InPlot program (Graph-
Pad, San Diego, CA) with five concentrations of displacer, each
performed in triplicate. Inhibition constants (Ki) were calcu-
lated according to the Cheng-Prusoff equation.36 The dis-
sociation constants (Kd) of [3H]CHA, [3H]CGS 21680, and
Gen er a l P r oced u r e To P r ep a r e 4-Ar a lk yla m in o-2-
p h e n yl-1,2-d ih yd r o-1,2,4-t r ia zolo[4,3-a ]q u in oxa lin -1-
on es 16-19. The title compounds were prepare from 44 and
aralkylamine following the experimental conditions described
above to obtain 9-12. Compound 16 displayed the following
spectral data: 1H NMR (DMSO-d6) 4.75 (d, 2H, CH2, J ) 5.8
Hz), 7.23-7.61 (m, 11H, ar), 8.08 (d, 2H, ar, J ) 8.5 Hz), 8.51-
8.65 (m, 2H, 1H ar + NH).
[
125I]AB-MECA were 1.2, 14, and 0.86 nM,37 respectively.
Gen er a l P r oced u r e To P r ep a r e 4-Am id o-1,2-d ih yd r o-
2-p h en yl-1,2,4-tr ia zolo[4,3-a ]qu in oxa lin -1-on es 20-23. A
solution of acyl chloride (2 mmol) in anhydrous dichlo-
romethane (2 mL) was slowly added at 0 °C to a suspension
of 3 (1.1 mmol) in anhydrous dichloromethane (6 mL) and
anydrous pyridine (0.4 mL). During the addition the temper-
ature of the mixture was kept at 0 °C. The mixture was stirred
at room-temperature overnight. Evaporation at reduced pres-
sure of the solvent yielded a residue which was treated with
ethanol (10 mL), collected and crystallized. Compound 20
displayed the following spectral data: 1H NMR (DMSO-d6)
2.37 (s, 3H, CH3), 7.39 (t, 1H, ar, J ) 7.0 Hz), 7.52-7.64 (m,
4H, ar), 7.53 (dd, 1H, ar, J ) 7.3, 1.1 Hz), 8.13 (d, 2H, ar, J )
8.6 Hz), 8.73 (d, 1H, ar, J ) 7.9 Hz), 10.57 (br s, 1H, NH); IR
1700, 1750, 3220.
Refer en ces
(1) J acobson, K. A.; van Galen, P. J . M.; Williams, M. Adenosine
receptors - pharmacology, structure-activity relationships, and
therapeutic potential. J . Med. Chem. 1992, 35, 407-422.
(2) Von Lubitz, D. K. J . E.; J acobson, K. A. Behavioral effects of
adenosine receptor stimulation. In Adenosine and Adenine
Nucleotides: From Molecular Biology to Integrative Physiology;
Bellardinelli, L., Pelleg, A., Eds.; Kluwer: Norwell, 1995; pp
489-498.
(3) Collis, M. G.; Hourani, S. M. O. Adenosine receptor subtypes.
Trends Pharmacol. Sci. 1993, 14, 360-366.
(4) Salvatore, C. A.; J acobson, M. A.; Taylor, H. E.; Linden, J .;
J ohnson, R. G. Molecular cloning and characterization of the
human A3 adenosine receptor. Proc. Natl. Acad. Sci. U.S.A. 1993,
90, 10365-10369.
(5) Linden, J .; Taylor, H. E.; Robeva, A. S.; Tucker, A. L.; Stehle, J .
H.; Rivkees, S. A.; Fink, J . S.; Reppert, S. M. Molecular-cloning
and functional expression of sheep A3 adenosine receptor with
widespread tissue distribution. Mol. Pharmacol. 1993, 44, 524-
532.
(6) J i, X.-D.; von Lubitz, D.; Olah, M. E.; Stiles, G. L.; J acobson, K.
A. Species differences in ligand affinity at central A3-adenosine
receptors. Drug Dev. Res. 1994, 33, 51-59.
Gen er a l P r oced u r e To P r ep a r e 4-Ar ylu r eid o-1,2-d i-
h ydr o-2-ph en yl-1,2,4-tr iazolo[4,3-a ]qu in oxalin -1-on es 24-
25. Aryl isocyanate (1.65 mmol) was added to a suspension of
3 (1.1 mmol) in anydrous tetrahydrofuran (50 mL). The
mixture was refluxed for 30 min under nitrogen atmosphere.

1164 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Colotta et al.
(7) Mu¨ller, C. E.; Stein, B. Adenosine receptor antagonists: struc-
tures and potential therapeutic applications. Curr. Pharm. Des.
1996, 2, 501-530.
(23) Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filacchioni, G.;
Galli, A.; Costagli, C. Tricyclic heteroaromatic systems. 1,2,4-
Triazolo[4,3-a]quinoxalines and 1,2,4-triazino[4,3-a]quinoxalines:
synthesis and central benzodiazepine receptor activity. Arch.
Pharm. Pharm. Med. Chem. 1997, 330, 387-391.
(8) Mu¨ller, C. E. A1-adenosine receptor antagonists. Exp. Opin. Ther.
Patents 1997, 7, 419-440.
(9) Poulsen, S.-A.; Quinn, R. J . Adenosine receptors for future drugs.
Bioorg. Med. Chem. 1998, 6, 619-641.
(24) Nakata, H. Biochemical and immunological characterization of
A1
adenosine receptors purified from human brain membranes.
(10) Akahane, A.; Katayama, H.; Takafumi, M.; Kato, T.; Kinoshita,
T.; Kita, Y.; Kusunoki, T.; Terai, T.; Yoshida, K.; Shiokawa, Y.
Discovery of 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-
pyridazinebutanoic acid (FK 838): a novel non-xanthine ad-
enosine A1 receptor antagonist with potent diuretic activity. J .
Med. Chem. 1999, 42, 779-783.
Eur. J . Biochem. 1992, 206, 171-177.
(25) Klotz, K. N.; Hessling, J .; Hegler, J .; Owman, C.; Kull, B.;
Fredholm, B. B.; Lohes, M. J . Comparative pharmacology of
human adenosine receptor subtypes-characterization of stably
transfected receptors in CHO cells. Naunyn-Schiedeberg’s Arch.
Pharmacol. 1998, 357, 1-9.
(11) Suzuki, F.; Shimada, J .; Mizumoto, H.; Karasawa, A.; Kubo, K.;
Nonaka, H.; Ishii, A.; Kawakita, T. Adenosine-A1 antagonists.
2. Structure-activity relationships on diuretic activities and
protective effects against acute renal failure. J . Med. Chem.
1992, 35, 3066-3075.
(12) Richardson, P. J .; Kase, H.; J enner, P. G. Adenosine A2A receptor
antagonists as new agents for the treatment of Parkinson’s
disease. Trends Pharmacol. Sci. 1997, 18, 338-344.
(13) Beaven, M. A.; Ramkumar, V.; Ali, H. Adenosine-A(3) receptors
in mast-cells. Trends Pharmacol. Sci. 1994, 15, 13-14.
(14) Knight, D.; Zheng, X.; Rocchini, C.; J acobson, M. A.; Bai, T.;
Walker, B. Adenosine A3 stimulation inhibits migration of
human eosinophils. J . Leukoc. Biol. 1997, 62, 465-468.
(15) von Lubitz, D. K. J . E.; Lin, R. C. S.; Popik, P.; Carter, M. F.;
J acobson, K. A. Adenosine A3 receptor stimulation and cerebral
ischemia. Eur. J . Pharmacol. 1994, 263, 59-67.
(16) von Lubitz, D. K. J . E.; Lin, R. C. S.; J acobson, K. A. Adenosine
A3 receptor antagonists and protection cerebral ischemic damage
in gerbils. Soc. Neurosci. 1997, 23, Abstr. 745.16, 1924.
(17) Colotta, V.; Cecchi, L.; Catarzi, D.; Melani, F.; Filacchioni, G.;
Martini, C.; Tacchi, P.; Lucacchini, A. 1-(3-Aminophenyl)-3-
methyl[1]benzopyrano[2,3-c]pyrazol-4-one: a new selective A2
adenosine receptor antagonist. Pharm. Pharmacol. Lett. 1992,
2, 74-76.
(18) Colotta, V.; Cecchi, L.; Catarzi, D.; Melani, F.; Filacchioni, G.;
Martini, C.; Tacchi, P.; Lucacchini, A. Novel adenosine receptor
ligands: 1,3-disubstituted[1]benzopyrano[2,3-c]pyrazol-4-ones.
Synthesis and structure-activity relationships. Recept. Channels
1993, 1, 111-119.
(19) Colotta, V.; Cecchi, L.; Catarzi, D.; Filacchioni, G.; Martini, C.;
Tacchi, P.; Lucacchini, A. Synthesis of some tricyclic heteroaro-
matic systems and their A1 and A2a adenosine binding activity.
Eur. J . Med. Chem. 1995, 30, 133-139.
(20) Catarzi, D.; Cecchi, L.; Colotta, V.; Filacchioni, G.; Martini, C.;
Tacchi, P.; Lucacchini, A. Tricyclic heteroaromatic systems.
Synthesis and A1 and A2a adenosine binding activities of some
1-aryl-1,4-dihydro-3-methyl[1]benzopyrano[2,3-c]pyrazol-4-
ones, 1-aryl-4,9-dihydro-3-methyl-1H-pyrazolo[3,4-b]quinolin-4-
ones, and 1-aryl-1H-imidazo[4,5-b]quinoxalines. J . Med. Chem.
1995, 38, 1330-1336.
(21) Colotta, V.; Catarzi, D.; Varano, F.; Cecchi, L.; Filacchioni, G.;
Martini, C.; Trincavelli, L.; Lucacchini, A. 4-Amino-6-benzy-
lamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-
(26) Camaioni, E.; Costanzi, S.; Vittori, S.; Volpini, R.; Klotz, K. N.;
Cristalli, G. New substituted 9-alkylpurines as adenosine recep-
tor ligands. Bioorg. Med. Chem. 1998, 6, 523-553.
(27) Ijzerman, A. P.; Van Galen, P. J . M.; Van Vlijmen H. W. Th.;
Soudijin, W.; Nissen, P.; Van Wijngaarden, I. Molecular mod-
elling of the antagonist binding site on the adenosine A1 receptor.
Nucleosides Nucleotides 1991, 10, 1039-1047.
(28) Sarges, R.; Howard, H. R.; Browne, R. G.; Lebel, L. A.; Seymour,
P. A.; Koe, B. K. 4-Amino[1,2,4]triazolo[4,3-a]quinoxalines. A
novel class of potent adenosine receptor antagonists and poten-
tial rapid-onset antidepressants. J . Med. Chem. 1990, 33, 2240-
2254.
(29) Kim, Y.-C.; de Zwart, M.; Chang, L.; Moro, S.; von Frijtag Drabbe
Ku¨nzel, J . K.; Melman, N.; Ijzerman, A. P.; J acobson, K. A.
Derivatives of the triazoloquinazoline adenosine antagonist (CGS
15943) having high potency at human A2B and A3 receptor
subtypes. J . Med. Chem. 1998, 41, 2835-2845.
(30) Kim, Y.-C.; J i, X.-D.; J acobson, K. A. Derivatives of the triazo-
loquinazoline adenosine antagonist (CGS 15943) are selective
for human A3 receptor subtype. J . Med. Chem. 1996, 39, 4142-
4148.
(31) van Tilburg, E. W.; Muijlwijk-Koezen, J . E.; Ijzerman, A. P.
Medicinal chemistry of the human A3 receptor. Drug. Dev. Res.
1998, 45, 182-189.
(32) Baraldi, P. G.; Cacciari, B.; Spalluto, G.; J i, X.-d.; Olah, M. E.;
Stiles, G.; Dionisotti, D.; Zocchi, C.; Ongini, E.; J acobson, A. K.
Novel N6-(substituted-phenylcarbamoyl)adenosine-5′-uronamides
as potent agonists for A3 adenosine receptors. J . Med. Chem.
1996, 39, 802-806.
(33) Colotta, V.; Catarzi, D.; Varano, F.; Melani, F.; Filacchioni, G.;
Cecchi, L.; Trincavelli, L.; Martini, C.; Lucacchini, A. Synthesis
and A1 and A2A adenosine binding activity of some pyrano[2,3-
c]pyrazol-4-ones. Farmaco 1998, 53, 189-196.
(34) Olah, M. E.; Gallo-Rodriguez, C.; J acobson, K. A. [125I]AB-MECA,
a high affinity radioligand for the rat A3 adenosine receptors.
Mol. Pharmacol. 1994, 45, 978-982.
(35) Lowry, O. H.; Rosenbrough, N. J .; Farr, A. L.; Randall, R. J .
Protein measurement with Folin phenol reagent. J . Biol. Chem.
1951, 193, 265-275.
(36) Cheng, Y. C.; Prusoff, W. H. Relation between the inhibition
constant Ki and the concentration of inhibitor which causes fifty
percent inhibition (IC50) of an enzyme reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
(37) Varani, K.; Cacciari, B.; Baraldi, P. G.; Dionisotti, S.; Ongini
E.; Borea, P. A. Binding affinity of adenosine receptor agonist
at human cloned A3 adenosine receptors. Life Sci. 1998, 63,
81-87.
one:
a new A2A adenosine receptor antagonist with high
selectivity versus A1 receptors. Arch. Pharm. Pharm. Med. Chem.
1999, 332, 39-41.
(22) Shawali, A. S.; Albar, H. A. Kinetics and mechanism of dehy-
drochlorination of N-aryl-C-ethoxycarbonylformohydrazidoyl chlo-
rides. Can. J . Chem. 1986, 64, 871-875.
J M991096E