Synthesis of 4-amino-6-(hetero)arylalkylamino-1,2,4-triazolo[4,3-a] quinoxalin-1-one derivatives as potent A2A adenosine receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2003.09.019

In previous papers (Colotta, V. et al. Arch. Pharm. Pharm. Med. Chem. 1999, 332, 39. Colotta, V. et al. J. Med. Chem. 2000, 43, 1158) we reported the synthesis and binding affinity at bovine (b) A₁ and A_2A and human (h) A₃ adenosine receptors (ARs) of the 4-amino-6-benzylamino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (compound A) which resulted in a potent and selective A_2A AR antagonist. Compound A provided the lead compound of a series of 6- or 8-(hetero) arylalkylamino-4-amino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives (compounds 1-20) which are the object of this paper. Most of the newly synthesized compounds are inactive at hA₃ ARs while they possess both nanomolar bA_2A affinities and different degrees of bA_2A versus bA₁ selectivity. The binding data show that hydrophilic substituents on the benzyl moiety are the most profitable for bA_2A receptor affinity. Furthermore, their steric hindrance seems to play an important role for the bA_2A AR interaction, thus suggesting that the 6-aralkylamino moiety of these ligands interacts with a size-limited binding pocket of this AR subtype. Thus, the SAR studies provided us some new insights about the structural requirements of the bA_2A AR recognition site.

Full text of DOI:10.1016/j.bmc.2003.09.019

Bioorganic & Medicinal Chemistry 11 (2003) 5509–5518
Synthesis of 4-Amino-6-(hetero)arylalkylamino-
1,2,4-triazolo[4,3-a]quinoxalin-1-one Derivatives as
Potent A2A Adenosine Receptor Antagonists
Vittoria Colotta,a,* Daniela Catarzi,a Flavia Varano,a Guido Filacchioni,a
Claudia Martini,b Letizia Trincavellib and Antonio Lucacchinib
a
´
Dipartimento di Scienze Farmaceutiche, Polo Scientifico, Universita di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino (FJ), Italy
bDipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita’ di Pisa, Via Bonanno, 6, 50126 Pisa, Italy
Received 10 June 2003; accepted 12 September 2003
Abstract—In previous papers (Colotta, V. et al. Arch. Pharm. Pharm. Med. Chem. 1999, 332, 39. Colotta, V. et al. J. Med. Chem.
2000, 43, 1158) we reported the synthesis and binding affinity at bovine (b) A1 and A2A and human (h) A3 adenosine receptors
(ARs) of the 4-amino-6-benzylamino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (compound A) which resulted in a potent and
selective A2A AR antagonist. Compound A provided the lead compound of a series of 6- or 8-(hetero)arylalkylamino-4-amino-2-
phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives (compounds 1–20) which are the object of this paper. Most of the newly
synthesized compounds are inactive at hA3 ARs while they possess both nanomolar bA2A affinities and different degrees of bA2A
versus bA1 selectivity. The binding data show that hydrophilic substituents on the benzyl moiety are the most profitable for bA2A
receptor affinity. Furthermore, their steric hindrance seems to play an important role for the bA2A AR interaction, thus suggesting
that the 6-aralkylamino moiety of these ligands interacts with a size-limited binding pocket of this AR subtype. Thus, the SAR
studies provided us some new insights about the structural requirements of the bA2A AR recognition site.
# 2003Elsevier Ltd. All rights reserved.
Introduction
such effects has yet to be elucidated, A2A ARs seem to
be involved in the regulation of glutamatergic transmis-
sion.6,7 Furthermore, recent data support the hypothesis
that blockade of A2A ARs produces anti-nociceptive8
and antidepressant9 effects.
Adenosine receptors (ARs) belong to the G-protein
coupled receptor family and presently are classified into
A1, A2A, A2B and A3 subtypes.1 In recent years, much
effort has been directed towards the study of potent and
selective AR antagonists which are needed to define the
specific requirements of each AR subtype. Moreover,
selective AR antagonists have attracted attention for
their potential therapeutic use.2,3 In particular, over the
last few years there is a growing interest toward the
development of A2A selective antagonists since they are
sought as novel therapeutics for the treatment of Par-
kinson’s disease, both for their capability to alleviate
parkinsonian symptoms in animal models and for their
chronic neuroprotective actions.4,5 In fact, A2A AR
antagonists show neuroprotective effects in disease
models, such as cerebral ischemia, caused by excitotoxic
mechanism.6,7 Although the mechanism responsible for
In our laboratory much effort has been directed towards
the study of AR antagonists.10ꢀ14 As a part of this pro-
gram, we reported the synthesis and binding affinity at
bovine (b) A1 and A2A and human (h) A3 ARs of a ser-
ies of 4-amino - 1,2,4 - triazolo[4,3- a]quinoxalin - 1 - one
derivatives among which the 4-amino-6-benzylamino-2-
phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one A15,16 was
a potent and selective A2A antagonist (Chart 1). SAR
studies on this class of derivatives indicated that the
presence of the 6-benzylamino moiety was an important
requisite for obtaining high A2A affinity and selectivity,
in accordance with reported data on different classes of
potent and selective A2A AR antagonists, such as the 2-
(2-furyl)-1,2,4-triazolo[2,3-a]-1,3,5-triazino derivative ZM
24138518 and the 2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-tria-
zolo[1,5-c]pyrimidine derivatives of the SCH series19,20
(Chart 1). In fact, due to the similar size and shape of
*Corresponding author. Tel.: +39-55-4573731; fax: +39-55-4573667;
e-mail: vittoria.colotta@unifi.it
0968-0896/$ - see front matter # 2003Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.09.019

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V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 5509–5518
these latter derivatives and compound A, we hypothe-
sized that the appended aralkyl chain of the SCH series
derivatives could interact with the A2A receptor subsite
which binds the benzyl moiety of A. Thus, taking A as
lead compound, we prepared and tested at ARs a set of
6- or 8-(hetero)arylalkylamino-4-amino-2-phenyl-1,2,4-
triazolo[4,3-a]quinoxalin-1-one derivatives (compounds
1–20, Chart 2) which are the object of this paper. We
performed various structural modifications on the ben-
zyl group of A. The first was introduction of different
substituents at different positions on the phenyl moiety.
Most of the substituents (F, OMe, OH, COOR) were
chosen on the basis of their capability to engage hydro-
gen bonds since it was reported that in the SCH series
the presence on the appended aryl moiety of a group
possessing this feature often increased A2A AR affinity
and selectivity.19,20 In addition, some of these groups
(OMe, OH, COOH), due to their hydrophilic proper-
ties, may be able to improve the water solubility of
compounds and to make the binding assays easier. The
second structural modification was replacement of the
phenyl ring of the benzyl chain with an heteroaryl ring
such as furyl, thienyl or pyridil. This change was made
because these heterocyclic substituents, isosters of the
benzene ring, are able to act as hydrogen bond accep-
tors. Thus, they were expected to reinforce the anchor-
ing to the A2A receptor binding site. Moreover, the
pyridine ring, possessing a hydrophilic character, could
increase the water solubility of compounds. The third
modification performed on the benzyl moiety of A was
homologation of the alkyl spacer. This change was
made in order to evaluate the importance of the dis-
tance between the 6-amino-triazoloquinoxalin-1-one
framework and the lipophilic area constituted by the
benzene ring. Finally, the benzylamino substituent of A
was moved from the 6- to the 8-position.
Chemistry
The target compounds 1–20 were prepared as depicted
in Schemes 1–3. Scheme 1 shows the synthesis of the 6-
[(hetero)arylmethyl]amino-substituted derivatives 1–15.
Reaction of the 4,6-diamino-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1-one 2116,17 with suitable (hetero)aryl
aldehydes produced the Schiff’s bases 22–33. The 6-
[(hetero)arylmethylene]amino-structure of 22–33 was
1
assigned by comparing the H NMR spectra of 22–33
with that of the 4,6-diamino derivative 21. The 1H
NMR spectrum of 21 shows two signals at 5.36 and
7.28 ppm, assigned to the 6-amino and the 4-amino
group, respectively, this last substituent being more
deshielded due to the electron-withdrawing effect of the
N-5 atom. In the 1H NMR spectra of compounds 22–33
the signal of the amino group appears at 7.2–7.6 ppm,
thus this substituent is at the 4-position and, conse-
quently, the [(hetero)arylmethylene]amino group is at
the 6-position. The structure of 22–33 was also con-
firmed by the downfield shift of their H-9 signal (about
8.5 ppm), with respect to that of the 6-amino derivative
21 (7.88 ppm). This difference of chemical shift clearly
indicates the transformation of the electron-donating 6-
amino group of 21 into the electron-withdrawing 6-
azomethine function of 22–33. In fact, it is well estab-
lished that the H-9 signal of the 1,2,4-triazolo[4,3-a]
quinoxalin-1-one derivatives is the most deshielded aro-
matic proton (over 8.50 ppm) with the exceptions being
those compounds containing the electron-donating 6- or
8-amino group (under 8.0 ppm).15ꢀ17
Reduction of 22–33 with sodium borohydride yielded
the desired 6-[(hetero)arylmethyl]amino derivatives 1–3,
5, 7, 9–15. The H NMR spectra of these compounds
1
shows the H-9 signal under 8.0 ppm. The upfield shift of
these signals, with respect to the H-9 signals of the cor-
responding Schiff’s bases 22–33, was due to the change
of the electronic properties of the 6-substituent and it
further confirmed the 6-position of the appended sub-
stituents. Moreover, the 6-substitued structure of 1–3, 5,
7, 9–15 was consistent with the chemical shifts of the
two amino group proton(s) which are easily identified
for their different multiplicity and integral value. In fact,
the NH signals appear as triplets (J=5.5–6.2 Hz) at 5.8–
6.2 ppm; those of NH2 as broad singlets at about 7.3–
7.4 ppm. Reaction of compound 21 with either the 2-
formylbenzoic acid or the 3-formylbenzoic acid, per-
formed in the experimental conditions followed to pre-
pare 22–33, did not afford the corresponding Schiff’s
bases. In fact, in the first case, the 6-(1,3-dihydro-3-oxo-
isobenzofuran-1-yl)amino-substituted derivative 16 was
obtained, while in the latter, the 3-formylbenzoate of
the amine 21, was isolated. The synthesis of the desired
6-(3-carboxybenzyl)amino derivative 6 was achieved by
reductive amination of the 3-formylbenzoic acid with
compound 21, in the presence of sodium triacetoxyboro-
hydride. The 6-arylmethyl-substituted structure of 6
was assigned on the basis of the chemical shifts of the
two amino group proton(s). In fact, the NH signal
appears as a triplet (J=5.9 Hz) at 6.12 ppm while the
NH2 signal as a broad singlet at about 7.3ppm. These
significantly different chemical shifts, in accordance with
Chart 1. Previously reported A2A AR selective antagonists.
Chart 2. Presently reported 1,2,4-triazolo[4,3-a]quinoxalin-1-one
derivatives.

V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 5509–5518
5511
Scheme 1. (a) RCHO, ZnCl2, THF; (b) 2-formylbenzoic acid, ZnCl2, THF; (c) NaBH4, MeOH; (d) 3-formylbenzoic acid, ZnCl2, NaB(O2CCH3)3,
THF; (e) BBr3, CH2Cl2; (f) NaOH, H2O/MeOH.
Scheme 2. (a) K2CO3, DMF; (b) H2O, HCl.
the spectral data of compound 21 and of the 6-[(het-
ero)arylmethyl]amino- derivatives 1–3, 5, 7, 9–15 (see
above), indicate that the NH and NH2 groups are,
respectively, at the 6- and the 4-positions. The 6-(4-
methoxybenzyl)amino-triazoloquinoxalin-1-one deriva-
tive 3 was demethylated to give the corresponding 6-(4-
hydroxybenzyl)amino compound 4. Alkaline hydrolysis
of the methyl ester 7 gave the corresponding carboxylic
acid 8. The 6-(2-phenylethyl)amino derivative 17 and
the 6-[2-(4-methoxyphenyl)ethyl]amino compound 18
were prepared as described in Scheme 2, that is by
reacting the 4,6-diamino compound 21 with, respec-
tively, 2-phenylethyl bromide 34 and 2-(4-methoxy-
phenyl)ethyl bromide 3521 in DMF at 70ꢁ C in the
presence of potassium carbonate. These conditions
afforded a mixture of 17 or 18 and the corresponding 4-
(N,N-dimethylaminomethylene)amino derivatives 36 or
37. These latter compounds were neither isolated nor
1
characterized but were identified in the H NMR spec-
tra of the mixtures. By treatment of the crude mixture of
17 and 36 or 18 and 37 with aqueous hydrochloric acid
the transformation of 36 and 37 into 17 and 18, respec-
tively, was achieved. The structure of both compounds
17 and 18 was assigned on the basis of the chemical
shifts of the two amino group proton(s), similarly to
compound 6 structure attribution (see the discussion
reported above). In fact, the NH signal of 17 and 18
appears as a triplet (J=7.0 Hz) at 5.6 ppm while that of

5512
V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 5509–5518
(6.87 ppm) protons were too close to be unambiguously
assigned to 4-amino and 8-amino groups, respectively.
Thus, the structure of 19 was attributed by means of
1H/1H nuclear Overhauser enhancement (NOE) experi-
ment. Preirradiation of the methylene protons
(4.31 ppm) caused a significant enhancement of the H-9
proton signal (7.97 ppm) together with the two ortho
benzyl (about 7.4 ppm) and NH (6.64 ppm) proton sig-
nals. The 8-dibenzylamino structure of compound 20
was assigned on the basis of both the structure of 19 and
the chemical shift of the NH2 signal (7.00 ppm) of 20.
Scheme 3. (a) Benzyl bromide, K2CO3, DMF.
NH2 as a broad singlet at about 7.3ppm. These sig-
nificantly different chemical shifts indicate that the NH
and NH2 groups are, respectively, at the 6- and the 4-
positions. Finally, when the 4,8-diamino-triazoloqui-
noxalin-1-one derivative 3817 was reacted with benzyl
bromide, both the 8-benzylamino derivative 19 and the
8-dibenzylamino compound 20 were obtained. It was
not possible to assign the 8-benzylamino-substituted
structure of 19 merely on the basis of the different che-
mical shifts of its two amino groups. In fact, the chemi-
cal shifts of the NH (about 6.6 ppm) and NH2
Biochemistry
Compounds 1–20 were tested for their ability to displace
[3H]N6-cyclohexyladenosine ([3H]CHA) from A1 ARs in
bovine cerebral cortical membranes, [3H]2-{[4-(2-carboxy-
ethyl)phenethyl]amino}-50-(N-ethyl-carbamoyl)adenosine
([3H]CGS 21680) from A2A ARs in bovine striatal
Table 1. Binding activity at bovine A1 and A2A and human A3 ARs
Compd
R6
R8
Ki (nM)a or I%
b
c
d
A1
A2A
A3
Ae
1
2
3
4
5
6
7
NHCH2–C6H5
NHCH2–C6H4–2F
NHCH2–C6H4–4F
NHCH2–C6H4–4OMe
NHCH2–C6H4–4OH
NHCH2–C6H4–4Cl
NHCH2–C6H4–3COOH
NHCH2–C6H4–4COOMe
NHCH2–C6H4–4COOH
NHCH2-2-furyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
73 Æ75.1
93.9Æ8.8
37Æ3.9
0 6.5Æ0.7
152Æ16.4
74Æ6.9
30%
31%
750Æ76.8
830Æ81.1
23.9%
44%
365Æ39.2
650Æ55.4
74%
42.5Æ3.9
49.7Æ5.2
229Æ23.5
15.2Æ1.6
74Æ6.6
92Æ7.8
817Æ79.6
627Æ59.4
94Æ8.322
189.4Æ22.4
281Æ27.2
90Æ8.5
52%
41%
8
9
Æ1.9
8.66Æ0.9
22Æ2.4
6%
50%
10
11
12
13
14
15
NHCH2-2-(5-methylfuryl)
NHCH2-3-furyl
NHCH2-2-thienyl
15Æ1.4
327Æ29.4
171Æ15.9
259Æ16.2
124Æ10.5
3260Æ343
18.6Æ1.7
10Æ1.9
37%
NHCH2-3-thienyl
NHCH2-3-pyridil
NHCH2-4-pyridil
31.5%
40.8%
38.7%
26Æ1.9
65.2Æ7.2
16
H
16Æ1.5
31.5Æ2.9
25Æ2.2
17
18
19
20
NH(CH2)2–C6H5
NH(CH2)2–C6H4–4OCH3
H
H
116Æ10.5
204Æ19.6
9.4Æ0.8
151Æ13.9
42.3Æ5.1
36%
0%
H
H
NHCH2–C6H5
N(CH2–C6H5)2
549Æ48.6
2086Æ199
21,000Æ1800
337Æ28
62Æ5.9
56Æ4.8
120Æ11.4
86,000Æ7800
1300Æ125
Theophylline
DPCPX
3800Æ340
0.5Æ0.03
aThe Ki values are meansÆSEM of four separate assays, each performed in triplicate.
bDisplacement of specific [3H]CHA binding in bovine brain membranes or percentage of inhibition (I%) of specific binding at 20 mM concentration.
cDisplacement of specific [3H]CGS 21680 binding 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.
eRefs 15 and 16.

V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 5509–5518
5513
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,22ꢀ24 we tested our A3 AR
ligands on cloned human A3 receptors.
possessing a hydrogen bond acceptor, that is the car-
bonyl oxygen of the lattone moiety. These results could
suggest that a hydrogen bonding interaction does not
play an important role for anchoring of the 6-aralkyla-
mino moiety of our triazoloquinoxaline derivatives to
the A2A receptor, differently from what reported for the
SCH series.19,20 Alternatively, we can suppose that the
6-appended moiety of these ligands interacts with a size-
limited binding pocket and hence only the 6-benzyla-
mino group would possess the right steric bulk to best
fit the A2A receptor subsite. The importance of the steric
hindrance of the 6-substituent seems to be confirmed by
the 3-fold reduced A2A affinity of compound 7, ensuing
by replacement of the para-carboxylic function of 8 with
the bulkier para-carboxymethyl group. Nevertheless, the
difference in A2A affinity of 8 and 7 could also be ascri-
bed to the reduced hydrophilicity of the ester group
with respect to the carboxylic group. The negative
influence of steric hindrance and/or lipophilic properties
of the substituent seems to be confirmed by the binding
datum of compound 5, bearing a para-chloro sub-
stituent, which possesses a 35-fold reduced A2A binding
activity, compared to A. The same applies to the para-
fluoro substituted compound 2, even though to a minor
extent and probably due to either lower lipophilicity or
steric hindrance of fluorine with respect to chlorine
atom. The ortho-fluoro-substituted derivative 1 also
exibits a significantly reduced A2A affinity (23-fold),
compared to A. This result could be explained by
assuming that the ortho-fluorine atom stabilizes a bio-
inactive conformation of the molecule as it may engage
an intramolecular hydrogen bond with the 6-amino
group.
The binding results of 1–20 are shown in Table 1 toge-
ther with those of the lead A. The binding data of
theophylline and 1,3-dipropyl-8-cyclopentylxanthine
(DPCPX), included as antagonist reference compounds,
are also reported.
Results and Discussion
The binding results reported in Table 1 show that we
have produced some potent bA2A antagonists endowed
with an A2A affinity in the low nanomolar range.
Nevertheless, none of the newly synthesized compounds
1–20 exceeded the A2A affinity and A2A versus A1 selec-
tivity of the parent compound A (Ki ratio A1/
A2A=112). Instead, most of compounds 1–20 are inac-
tive or scarcely active at the hA3 AR, similar to A.
There are only three exceptions: the 6-(1,3-dihydro-3-
oxo-isobenzofuran-1-yl)amino-substituted derivative 16,
the 8-benzylamino- derivative 19 and the 8-dibenzyl-
amino- compound 20 which showed good A3 AR affi-
nity. All these data indicate that the presence of a
6-(hetero)aralkylamino moiety on the 4-amino-1,2,4-
triazoloquinoxalin-1-one core is well tolerated by the
bA1 and bA2A ARs while, in general, it is detrimental
for anchoring to the hA3 receptor. On the contrary, the
presence of the 8-benzylamino- group (compound 19)
and the 8-dibenzylamino-substituent (compound 20)
significantly reduced the A2A affinity while it is profit-
able for anchoring to the A1 AR.
Better results in terms of A2A affinity were obtained
among the 6-(heteroarylmethyl)amino derivatives 9–15.
In fact, the 6-[(2-furylmethyl)]amino derivative 9, the 6-
[(3-furylmethyl)]amino derivative 11 and their corre-
sponding thienyl derivatives 12 and 13 show compar-
able or only slightly reduced (Ki=9–19 nM) A2A affinity
with respect to that of A. Compounds 9 and 13 also
display some A2A versus A1 selectivity (21- and 25-fold,
respectively). The above cited influence of the steric
hindrance of the 6-appended moiety on the A2A affinity
was also observed among these heterocycle-substituted
derivatives. In fact, the 6-[2-(5-methylfuryl)methyl]-
The first modification performed on A was introduction
of various substituents on the phenyl ring of its 6-ben-
zylamino group (compounds 1–8). Some substituents,
such as the para-hydroxy (compound 4) and the para-
carboxymethyl (compound 7) afforded comparable A1
affinity, with respect to A. All the others increased from
2- to 8-fold the A1 affinity of A, while only the para-
chloro substituent (compound 5) significantly reduced
the A1 binding activity of the lead A. The A2A affinities
of compounds 1–8 were comparable or lower than that
of A. Consequently, these derivatives were scarcely A2A
versus A1 selective, suggesting that the A1 and A2A
lipophilic areas which accommodate the 6-(hetero)-
aralkylamino group possess similar requirements. The
best substituents for A2A receptor–ligand interaction
were the hydrophilic ones, such as methoxy, hydroxy or
carboxy group (compounds 3, 4, 6, 8). In particular, a
free carboxylic group, at either the meta- or para- posi-
tion (compounds 6 and 8, respectively), produced high
A2A affinities, only slightly decreased with respect to
that of the lead compound A. Nevertheless, although
these groups are able to engage hydrogen bonds, their
presence did not increase the A2A affinity of A. A similar
consideration applies to compound 16 which shows a 5-
fold reduced A2A affinity, with respect to A, although
amino-substituted derivative 10 exibits
a 2.5-fold
reduction of A2A affinity with respect to the less bulky 6-
[(2-furylmethyl)]amino- derivative 9. The 6-(pyr-
idylmethyl)amino-substituted derivatives 14 and 15
show, among the heterocycle-substituted derivatives 9–
15, the lowest A2A AR affinities. Nevertheless, the 6-(4-
pyridylmethyl)amino- derivative 15, due to its very low
A1 affinity, possesses the highest A2A versus A1 selec-
tivity (selectivity ratio=50) among the herein reported
antagonists.
Homologation of the alkyl chain of the lead compound
A and derivative 3 (compounds 17 and 18, respectively)
elicited contrasting effects on A2A affinity and selectiv-
ity. In fact, while the 6-phenethylamino- derivative 17
shows a reduced (23-fold) A2A affinity and a loss of A2A
versus A1 selectivity, compared to A, compound 18

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V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 5509–5518
Table 2. Analytical data of the newly synthesized compounds
In conclusion, some of our modifications on the 6-ben-
zylamino moiety of the lead compound A have main-
tained high nanomolar A2A affinity, although reducing
the A2A versus A1 selectivity. Moreover, the SAR study
of compounds 1–20 gave us some useful insights about
the structural requirements of the bA2 AR subtype
which may be taken into consideration in the design of
new A2A AR antagonists.
Compd
Formula
C
Calcd–found Calcd–found Calcd–found
H
N
1
2
3
4
5
6
7
8
C22H17FN6O 65.98–65.67
C22H17FN6O 65.98–65.75
C23H20N6O2 66.97–66.78
C22H18N6O2 66.31–66.52
C22H17ClN6O 63.38–63.59
4.29–4.48
4.29–4.37
4.90–4.85
4.56–4.35
4.12–4.34
4.26–4.12
4.59–4.41
20.99–21.15
20.99–21.23
20.38–20.25
21.10–20.99
20.16–20.01
19.71–19.89
19.08–19.22
C23H18N6O3
C24H20N6O3
C23H18N6O3
C20H16N6O2
C21H18N6O2
C20H16N6O2
C20H16N6OS
C20H16N6OS
C21H17N7O
C21H17N7O
C23H16N6O3
C23H20N6O
C24H22N6O2
C22H18N6O
C29H24N6O
64.77–64.61
65.43–65.32
64.77–64.59
64.50–64.69
65.26–65.01
64.50–64.41
61.83–61.69
61.83–61.63
65.77–65.60
4.26–4.0319.71–19.99
9
4.34–4.52
4.70–4.96
4.34–4.20
4.16–4.34
4.16–4.01
4.48–4.67
22.57–22.34
Experimental
Chemistry
10
11
12
13
14
15
16
17
18
19
20
21.75–21.58
22.57–22.89
21.64–21.81
21.64–21.49
25.58–25.35
Silica gel plates (Merck F254) and silica gel 60 (Merck,
70–230 mesh) were used for analytical and column
chromatography, respectively. All melting points were
determined on a Gallenkamp melting point apparatus.
Microanalyses were performed with a Perkin-Elmer 260
elemental analyzer for C, H, N, and the results were
within Æ0.4% of the theoretical (Table 2). The IR
spectra were recorded with a Perkin-Elmer 1420 spec-
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,
65.77–65.534.480–4.3 25.58–25.75
65.08–65.25
69.67–69.48
67.58–67.85
69.08–69.27
73.70–73.65
3.81–3.97
5.09–5.27
5.21–5.01
4.75–4.96
5.13–5.30
19.80–19.64
21.20–21.46
19.71–19.60
21.98–21.74
17.79–17.58
exibits similar A2A affinity and selectivity with respect to
3. These opposing data regarding A2A affinity do not
permit us to draw any conclusion about the influence of
the distance between the 6-amino-triazoloquinoxaline
framework and the lipophilic aryl ring for the anchoring
to this receptor subtype.
dd=double
br=broad, ar=aromatic protons.
doublet,
t=triplet,
m=multiplet,
Movement of the benzylamino chain of A from the 6- to
the 8-position yielded compound 19 which showed a
dropped A2A and a significantly increased A1 affinity.
Consequently, 19 showed a reversed selectivity with
respect to A. These data indicate that the presence of a
bulky substituent at the 8-position of the 1,2,4-triazolo-
quinoxaline framework, while being well tolerated by
the A1 subtype, it is deleterious for anchoring to the A2A
AR. The detrimental effect of the steric hindrance of the
8-substituent is confirmed by the very low A2A affinity
(Ki=2086 nM) of compound 20 bearing the bulky 8-
dibenzylamino moiety.
General procedure for the synthesis of 4-amino-6-[(het-
ero)arylmethylene]amino-1,2-dihydro-2-phenyl-1,2,4-tria-
zolo[4,3-a]quinoxalin-1-ones 22–33
A mixture of compound 21 (2.05 mmol),16,17 the suitable
aldehyde (2.46 mmol) and anhydrous zinc chloride
(4.10 mmol) was refluxed in anhydrous tetrahydrofuran
(30 mL), under nitrogen atmosphere, until the dis-
appearance (TLC monitoring) of the starting material
(4–8 h). The solid was filtered, washed with water and
dried. The Schiff’s bases 22–33, obtained in high overall
yields (70–95%), were instable upon recrystallization,
nevertheless they were pure enough to be used without
purification. Compounds 22–33 displayed the following
1H NMR data.
To summarize, the binding data show that hydrophilic
substituents on the benzyl moiety of the lead compound
A are the most profitable for the A2A AR affinity, and
the best is the carboxy group. Nevertheless, although
these substituents could engage hydrogen bonds, their
presence, as stated above, do not reinforce the binding
to the A2A receptor. These results suggest that: (i) a
hydrogen bonding interaction does not play a crucial
role for anchoring of the 6-aralkylamino moiety of our
triazoloquinoxaline derivatives to the bA2A receptor,
and (ii) the 6-appended moiety of these ligands may
interact with a size-limited binding pocket. In fact,
among the 6-aralkylamino-substituents (compounds A,
1–8), the 6-benzylamino group of A seems to possess the
best balance of hydrophobic and steric properties to
permit a good fit with the receptor pocket. The existence
of strict steric and lipophilic requirements can be con-
firmed by the high binding affinity of compounds 9, 11–
13 in which the phenyl ring of A was replaced by the less
hindered bioisoster furyl or thienyl rings.
4-Amino-1,2-dihydro-6-[(2-fluorophenyl)methylene]amino
-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (22). 7.08
(d, 1H, ar, J=6.4 Hz), 7.28–7.47 (m, 4H, 2 ar+NH2),
7.50–7.63(m, 5H, ar), 8.16–8.20 (m, 3H, ar), 8.58 (d,
1H, H-9, J=6.7 Hz), 8.76 (s, 1H, ¼CH).
4-Amino-1,2-dihydro-6-[(4-fluorophenyl)methylene]amino
-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (23). 7.00
(d, 1H, ar, J=7.1 Hz), 7.20–7.59 (m, 8H, 6 ar+NH2),
7.98–8.08 (m, 4H, ar), 8.51–8.57 (m, 2H, H-9+
¼CH).
4-Amino-1,2-dihydro - 6 - [(4 - methoxyphenyl)methylene]
amino-2-phenyl-1,2,4-triazolo[4,3 -a]quinoxalin-1-one
(24). 3.85 (s, 3H, OMe), 6.98 (d, 1H, ar, J=7.7 Hz),
7.09 (d, 2H, ar, J=8.1 Hz), 7.22–7.39 (m, 2H, ar),

V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 5509–5518
5515
7.52–7.60 (m, 4H, 2 ar+NH2), 7.91 (d, 2H, ar,
J=8.4 Hz), 8.07 (d, 2H, ar, J=8.4 Hz), 8.48 (s, 1H,
¼CH), 8.52 (d, 1H, H-9, J=8.4 Hz).
General procedure for the synthesis of 4-amino-6-[(hetero)
arylmethyl]amino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1-ones 1–3, 5, 7, 9–16
4-Amino - 1,2 - dihydro - 6 - [(4 - chlorophenyl)methylene]
amino-2-phenyl-1,2,4-triazolo[4,3-a] quinoxalin-1-one (25).
7.04 (d, 1H, ar, J=7.7 Hz), 7.24–7.39 (m, 2H, ar), 7.53–
7.65 (m, 6H, 4 ar+NH2), 7.99 (d, 2H, ar, J=8.4 Hz),
8.08 (d, 2H, ar, J=8.4 Hz), 8.55 (d, 1H, H-9,
J=8.1 Hz), 8.61 (s, 1H, ¼CH).
Sodium borohydride (4.6 mmol) was added portionwise,
over 10 min, to a boiling suspension of compounds 22–
33 (2.3mmol) in anhydrous methanol (20 mL), under
nitrogen atmosphere. The mixture was refluxed until the
disappearance (TLC monitoring) of the starting mate-
rial (2–3h), cooled at room temperature and then
quenched with ice water (15 mL). The solid was col-
lected, washed with water and recrystallized.
Methyl 4-[(4-amino-1,2-dihydro-1-oxo-2-phenyl-1,2,4-tria-
zolo[4,3-a]quinoxalin-6-yl)amino] methylene benzoate (26).
3.89 (s, 3H, CH3), 7.05 (d, 1H, ar, J=7.9 Hz), 7.24–7.38
(m, 2H, ar), 7.51–7.59 (m, 4H, 2 ar+NH2), 8.04–8.10 (m,
6H, ar), 8.55 (d, 1H, H-9, J=8.1 Hz), 8.59 (s, 1H, ¼CH).
4-Amino-1,2-dihydro-6-[(2-fluorophenyl)methyl]amino-2-
phenyl-1,2,4-triazolꢁo[4,3-a]quinoxalin-1-one (1). Yield
1
70%; mp 228–229 C (CH3CN); H NMR 4.52 (d, 2H,
CH2, J=6.2 Hz), 6.02 (t, 1H, NH, J=6.2 Hz), 6.58 (d,
1H, J=8.0 Hz), 7.02–7.48 (m, 8H, 6 ar+NH2), 7.57 (t,
2H, ar, J=7.4 Hz), 7.93(d, 1H, ar, J=8.2 Hz), 8.08 (d,
2H, ar, J=8.5 Hz); IR 1710, 3315, 3405, 3495. Anal.
(C22H17FN6O) C, H, N.
4-Amino-1,2-dihydro-6-(2-furylmethylene)amino-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (27). 6.79–6.80 (m,
1H, ar), 7.01–7.12 (m, 1H, ar), 7.20–7.49 (m, 4H, 2
ar+NH2), 7.52–7.63 (m, 3H, ar), 8.02–8.12 (m, 3H, ar),
8.40–8.55 (m, 1H, ¼CH), 8.55 (d, 1H, H-9, J=8.2 Hz).
4-Amino-1,2-dihydro-6-[(4-fluorophenyl)methyl]amino-2-
phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (2). Yield
4-Amino-1,2-dihydro-6-[2-(5-methylfuryl)methylene]ami-
no-2- phenyl- 1,2,4- triazolo[4,3-a]quinoxalin-1-one (28).
2.42 (s, 3H, CH3), 6.39 (br s, 1H, furane proton), 6.92–
7.18 (m, 2H, ar), 7.20–7.40 (m, 2H, ar), 7.42–7.60 (m,
4H, 2 ar+NH2), 8.05 (d, 2H, ar, J=8.4 Hz), 8.25 (br s,
1H, ¼CH), 8.51 (d, 1H, H-9, J=6.3Hz).
74%; mp 204–205 ꢁC (DMF); H NMR 4.42 (d, 2H,
1
CH2, J=5.9 Hz), 6.12 (t, 1H, NH, J=5.8 Hz), 6.56 (d,
1H, ar, J=7.3 Hz), 6.98–7.19 (m, 3H, ar), 7.23–7.59 (m,
7H, 5 ar+NH2), 7.89 (d, 1H, ar, J=8.8 Hz), 8.1 (d, 2H,
ar, J=8.1 Hz); IR 1709, 3338, 3360, 3417, 3463. Anal.
(C22H17FN6O) C, H, N.
4-Amino-1,2-dihydro-6-(3-furylmethylene)amino-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (29). 6.96–7.01 (m,
2H, ar), 7.20–7.40 (m, 2H, ar), 7.48–7.60 (m, 4H, 2
ar+NH2), 7.83(br s, 1H, furane proton), 8.06 (d, 2H,
ar, J=8.4 Hz), 8.31 (br s, 1H, furane proton), 8.48–8.56
(m, 2H, H-9+ ¼CH).
4-Amino-1,2-dihydro-6-[(4-methoxyphenyl)methyl]amino-
2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (3). Yield
85%; mp 210–212 ꢁC (EtOH); H NMR 3.74 (s, 3H,
1
OCH3), 4.35 (d, 2H, CH2, J=5.9 Hz), 5.89 (t, 1H, NH,
J=5.9 Hz), 6.57 (d, 1H, ar, J=8.1 Hz), 6.92 (d, 2H, ar,
J=8.7 Hz), 7.05 (t, 1H, ar, J=8.4 Hz), 7.29–7.36 (m,
5H, 3ar+NH 2), 7.56 (t, 2H, ar, J=7.3Hz), 7.90 (d, 1H,
H-9, J=7.3Hz), 8.06 (d, 2H, ar, J=8.1 Hz); IR 1670,
3290, 3400. Anal. (C23H20N6O2) C, H, N.
4-Amino - 1,2 - dihydro - 6 - (2 - thienylmethylene)amino-2-
phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (30). 7.06
(d, 1H, ar, J=7.0 Hz), 7.22–7.43(m, 4H, 2 ar+NH ),
2
7.50–7.91 (m, 5H, ar), 8.09 (d, 2H, ar, J=8.2 Hz), 8.55
(d, 1H, H-9, J=8.1 Hz), 8.78 (s, 1H, ¼CH).
4-Amino-1,2-dihydro-6-[(4-chlorophenyl)methyl]amino-2-
phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (5). Yield
80%; mp 235–237 ꢁC (EtOAc); H NMR 4.46 (d, 2H,
1
4-Amino - 1,2 - dihydro - 6 - (3 - thienylmethylene)amino-2-
phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (31). 6.98
(d, 1H, ar, J=7.2 Hz), 7.20–7.40 (m, 2H, ar), 7.42–7.68
(m, 6H, 4 ar+NH2), 8.05 (d, 2H, ar, J=8.1 Hz), 8.18–
8.20 (m, 1H, ar), 8.48–8.58 (m, 2H, H-9+ ¼CH).
CH2, J=6.2 Hz), 6.09 (t, 1H, NH, J=6.2 Hz), 6.48 (d,
1H, ar, J=8.1 Hz), 7.02 (d, 1H, ar, J=8.4 Hz), 7.31–
7.40 (m, 7H, 5 ar +NH2), 7.56 (t, 2H, ar, J=7.7 Hz),
7.90 (d, 1H, H-9, J=8.1 Hz), 8.07 (d, 2H, ar,
J=8.4 Hz); IR 1715, 3330, 3410, 3460. Anal.
(C22H17ClN6O) C, H, N.
4-Amino-1,2-dihydro-6-(3-pyridylmethylene)amino-2-
phenyl - 1,2,4 - triazolo[4,3-a]quinoxalin-1-one (32). 7.07
(d, 1H, ar, J=6.6 Hz), 7.24–7.38 (m, 2H, ar), 7.45–7.62
(m, 5H, 3ar+NH 2), 8.05 (d, 2H, ar, J=8.4 Hz), 8.35 (d,
1H, ar, J=6.6 Hz), 8.55 (d, 1H, H-9, J=8.4 Hz), 8.67–
8.76 (m, 2H, ar+ ¼CH), 9.07 (s, 1H, pyridine proton).
Methyl 4-[(4-amino-1,2-dihydro-1-oxo-2-phenyl-1,2,4-tri-
azolo[4,3-a]quinoxalin-6-yl)amino]methyl benzoate (7).
Yield 65%; mp 208–210 ꢁC (CH3CN); H NMR 3 .81 (s,
1
3H, CH3), 4.53(d, 2H, CH 2, J=6.2Hz), 6.18 (t, 1H, NH,
J=6.2 Hz), 6.41 (d, 1H, ar, J=8.1 Hz), 6.98 (t, 1H, ar,
8.7 Hz), 7.36–7.57 (m, 7H, 5 ar+NH2), 7.82–7.88 (m, 3H,
ar), 8.05 (d, 2H, ar, J=7.7 Hz); IR 1627, 1714, 3300, 3348,
3443. Anal. (C24H20N6O3) C, H, N.
4-Amino - 1,2 - dihydro - 6 - (4 - pyridylmethylene)amino-2-
phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (33). 7.08
(d, 1H, ar, J=7.1 Hz), 7.22–7.28 (m, 2H, ar), 7.51–7.55
(m, 4H, 2 ar+NH2), 7.89 (d, 2H, pyridine proton,
J=5.9 Hz), 8.05 (d, 2H, ar, J=8.1 Hz), 8.55 (d, 1H, H-
9, J=8.4 Hz), 8.67 (s, 1H, ¼CH), 8.76 (d, 2H, pyridine
proton, J=5.9 Hz).
4-Amino-1,2-dihydro-6-(2-furylmethyl)amino-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (9). Yield 80%; mp
209–211 ꢁC (CH3CN); 1H NMR 4.46 (d, 2H, CH2,

5516
V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 5509–5518
J=5.9 Hz), 5.90 (t, 1H, NH, J=5.9 Hz), 6.31–6.42 (m,
2H, ar), 6.73(d, 1H, ar, J=8.1 Hz), 7.11 (t, 1H, ar,
J=8.2 Hz), 7.30–7.50 (m, 3H, 1 ar+NH2), 7.51–7.78
(m, 3H, ar,), 7.95 (d, 1H, ar, J=8.2 Hz), 8.09 (d, 2H, ar,
J=8.1 Hz); IR 1705, 3320, 3400, 3500. Anal.
(C20H16N6O2) C, H, N.
ar, J=8.4 Hz), 8.05 (d, 2H, ar, J=8.4 Hz), 8.42 (d, 2H,
pyridine protons); IR 1711, 3320, 3390, 3457. Anal.
(C21H17N7O) C, H, N.
Synthesis of 3-[(4-amino - 1,2 - dihydro - 1 - oxo-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-6-yl)amino]methylbenzoic
acid (6). A mixture of compound 21 (0.68 mmol), 3-
formylbenzoic acid (0.82 mmol), anhydrous zinc
chloride (1.36 mmol) and sodiumtriacetoxyborohydride
(3.06 mmol) in anhydrous tetrahydrofuran (20 mL) and
glacial acetic acid (0.05 mL) was refluxed under nitrogen
atmosphere for 6 h. After cooling at room temperature,
the suspension was diluted with water and acidified with
glacial acetic acid. The solid was collected by filtration,
washed with water and then ethanol. Yield 95%; mp
253–255 ꢁC (EtOH/AcOH); 1H NMR 4.49 (d, 2H, CH2,
J=5.9 Hz), 6.12 (t, 1H, NH, J=5.9 Hz), 6.50 (d, 1H, ar,
J=9.1 Hz), 7.00 (t, 1H, ar, J=7.7 Hz), 7.23–7.57 (m,
7H, 5 ar+NH2), 7.74–8.06 (m, 5H, ar), 12.50 (br s, 1H,
OH); IR 1690, 1727, 3396, 3506. Anal. (C23H18N6O3) C,
H, N.
4-Amino-1,2-dihydro-6-[2-(5-methylfuryl)methyl]amino-
2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (10). Yield
76%; mp 190–192 ꢁC (Isopropyl alcohol); 1H NMR 2.22
(s, 3H, CH3), 4.35 (d, 2H, CH2, J=5.9 Hz), 5.80 (t, 1H,
NH, J=5.9 Hz), 5.90–6.01 (m, 1H, furane proton),
6.19–6.20 (m, 1H, furane proton), 6.67 (d, 1H, ar,
J=7.3Hz), 7.07 (t, 1H, ar, J=8.4 Hz), 7.33–7.58 (m,
5H, 3ar+NH ), 7.90 (d, 1H, ar, J=7.3Hz), 8.04 (d,
2
2H, ar, J=7.7 Hz); IR 1718, 3300, 3436, 3475. Anal.
(C21H18N6O2) C, H, N.
4-Amino-1,2-dihydro-6-(3-furylmethyl)amino-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (11). Yield 78%;
mp 208–210 ꢁC (DMF); 1H NMR 4.22 (d, 2H, CH2,
J=5.5 Hz), 5.49 (t, 1H, NH, J=5.5 Hz), 6.51 (s, 1H,
furane proton), 6.65 (d, 1H, ar, J=7.7 Hz), 7.06 (t, 1H,
ar, J=7.7 Hz), 7.25–7.36 (m, 3H, 2 ar+NH2), 7.53(t,
2H, ar, J=8.1 Hz), 7.64 (d, 2H, ar, J=8.8 Hz), 7.89 (d,
1H, ar, J=7.7 Hz), 8.05 (d, 2H, ar, J=7.7 Hz); IR 1705,
3186, 3321, 3393, 3446. Anal. (C20H16N6O2) C, H, N.
Synthesis of 4-amino-1,2-dihydro-6-(1,3-dihydro-3-oxo-
isobenzofuran-1-yl)amino - 2 - phenyl - 1,2,4 - triazolo[4,3-
a]quinoxalin-1-one (16). The title compound was
obtained by reacting compound 2116,17 (2.05 mmol) with
2-formyl benzoic acid (2.46 mmol), following the proce-
dure above described to prepare Schiff’s bases 22–33.
Yield 87%; mp 282–284 ꢁC (2-Methoxyethanol); 1H
NMR 6.59 (d, 1H, NH, J=11.3Hz), 7.19–7.58 (m, 8H,
5 ar+CH+NH2), 7.73–7.93 (m, 4H, ar), 8.04 (d, 2H,
ar, J=7.7 Hz), 8.16 (d, 1H, ar, J=7.5 Hz); IR 1736,
1754, 3367, 3489. Anal. (C23H16N6O3) C, H, N.
4-Amino-1,2-dihydro-6-(2-thienylmethyl)amino-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (12). Yield 72%;
mp 208–209 ꢁC (CH3CN); H NMR 4.61 (d, 2H, CH2,
1
J=6.2 Hz), 6.01 (t, 1H, NH, J=6.2 Hz), 6.66 (d, 1H, ar,
J=7.7 Hz), 6.90–7.10 (m, 3H, ar), 7.30–7.40 (m, 4H, 2
ar+NH2), 7.55 (t, 2H, ar, J=7.7 Hz), 7.92 (d, 1H, ar,
J=7.3Hz), 8.06 (d, 2H, ar, J=7.6 Hz); IR 1720, 3375,
3470. Anal. (C20H16N6SO) C, H, N.
Synthesis of 4-amino-1,2-dihydro-6-[(4-hydroxyphenyl)-
methyl]amino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-
one (4). 1 M solution of BBr3 in dichloromethane
(2.06 mL) was slowly added at 0 ꢁC, under nitrogen
4-Amino-1,2-dihydro-6-(3-thienylmethyl)amino-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (13). Yield 70%;
mp 211–212 ꢁC (DMF); 1H NMR 4.40 (d, 2H, CH2,
J=5.8 Hz), 5.85 (t, 1H, NH, J=5.8 Hz), 6.62 (d, 1H, ar,
J=8.4 Hz), 7.04–7.18 (m, 2H, ar), 7.29–7.41 (m, 4H, 2
ar+NH2), 7.49–7.58 (m, 3H, ar), 7.90 (d, 1H, ar,
J=8.4 Hz), 8.05 (d, 2H, ar, J=8.4 Hz); IR 1710, 3357,
3460. Anal. (C20H16N6SO) C, H, N.
atmosphere, to
a
suspension of compound
3
(1.02 mmol) in anhydrous dichloromethane (20 mL).
The mixture was stirred at 0 ꢁC for 2 h and then at room
temperature for 24 h. The mixture was diluted with
water (10 mL) and neutralized with a saturated solution
of sodium bicarbonate. The solid, which was filtered,
washed with water and dried, was a mixture of com-
1
pound 4 and 21 (ratio about 2:1 from H NMR spec-
4-Amino-1,2-dihydro-6-(3-pyridylmethyl)amino-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (14). Yield 75%;
trum). The crude solid was chromatographed on silica
gel column, eluting system cyclohexane/ethyl acetate
1:1. Evaporation of the first and the second eluates
afforded compound 4 and 21, respectively. 4: Yield
ꢁ
1
mp 259–260 C (2-Methoxyethanol); H NMR 4.49 (d,
2H, CH2, J=6.2 Hz), 6.12 (t, 1H, NH, J=6.2 Hz), 6.54
(d, 1H, ar, J=8.1 Hz), 7.02 (t, 1H, ar, J=8.1 Hz), 7.37–
7.66 (m, 4H, 2 ar+NH2), 7.53(t, 2H, ar, J=7.3Hz),
7.75 (d, 1H, ar, J=7.7 Hz), 7.89 (d, 1H, ar, J=8.1 Hz),
8.05 (d, 2H, ar, J=8.4 Hz), 8.45 (d, 1H, pyridine pro-
ton, J=4.7 Hz), 8.59 (s, 1H, pyridine proton); IR 1702,
3381. Anal. (C21H17N7O) C, H, N.
50%; mp 141–143 ꢁC (EtOAc); H NMR 4.28 (d, 2H,
1
CH2, J=6.0 Hz), 5.78 (t, 1H, NH, J=6.0 Hz), 6.58 (d,
1H, ar, J=8.1 Hz), 6.74 (d, 2H, ar, J=8.4 Hz), 7.06 (t,
1H, ar, J=8.1 Hz), 7.20 (d, 2H, ar, J=8.4 Hz), 7.36–
7.40 (m, 3H, 2 ar+NH2), 7.56 (t, 2H, ar, J=8.1 Hz),
7.90 (d, 1H, ar, J=8.1 Hz), 8.06 (d, 2H, ar, J=7.7 Hz),
9.35 (s, 1H, OH); IR 1690, 3340, 3400, 3480. Anal.
(C22H18N6O2) C, H, N.
4-Amino-1,2-dihydro-6-(4-pyridylmethyl)amino-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (15). Yield 72%;
mp 279–280 ꢁC (DMF); 1H NMR 4.51 (d, 2H, CH2,
J=6.2 Hz), 6.24 (t, 1H, NH, J=6.2 Hz), 6.38 (d, 1H, ar,
J=8.1 Hz), 6.99 (t, 1H, ar, J=8.1 Hz), 7.31–7.37 (m,
5H, 3ar+NH 2), 7.54 (t, 2H, ar, J=7.3Hz), 7.88 (d, 1H,
Synthesis of 4-[(4-amino-1,2-dihydro-1-oxo-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-6-yl)amino]methylbenzoic
acid (8). A solution of NaOH (1.8 M, 5 mL) was added
to a suspension of the methyl ester 7 (0.76 mmol) in

V. Colotta et al. / Bioorg. Med. Chem. 11 (2003) 5509–5518
5517
methanol (10 mL). The mixture was refluxed for 3h.
After cooling at room temperature, the suspension was
acidified to pH=6 with HCl 6 N. The solid was filtered
and recrystallized. Yield 80%; mp 284–286 ꢁC (EtOH/
AcOH); 1H NMR 4.54 (d, 2H, CH2, J=6.1 Hz), 6.15 (t,
1H, NH, J=6.1 Hz), 6.44 (d, 1H, ar, J=8.4 Hz), 6.99 (t,
1H, ar, J=8.4 Hz), 7.35–7.57 (m, 7H, ar, 5 ar+NH2),
7.89–8.04 (m, 5H, ar), 12.8 (br s, 1H, OH); IR 1698,
1723, 3295, 3426. Anal. (C23H18N6O3) C, H, N.
Evaporation of the first and the second eluates afforded
compounds 19 and 38, respectively.
19. Yield 30%; mp 237–239 ꢁC (DMF/MeOH); 1H
NMR 4.31 (d, 2H, CH2, J=5.5 Hz), 6.60–6.65 (m, 2H, 1
ar+NH), 6.87 (br s, 2H, NH2), 7.14–7.41 (m, 7H, ar),
7.54 (t, 2H, ar, J=7.7 Hz), 7.97 (d, 1H, H-9, J=2.6 Hz),
8.04 (d, 2H, ar, J=7.7 Hz); IR 1730, 3300, 3490. Anal.
(C22H18N6O) C, H, N.
Synthesis of 4-amino-1,2-dihydro-6-(2-phenylethyl)amino-
2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (17) and 4
-amino-1,2-dihydro-6-[2-(4-methoxyphenyl)ethyl]amino-2
-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (18). 2-Phe-
nylethyl bromide or 2-(4-methoxyphenyl)ethyl bro-
mide21 (2.65 mmol) was added to a suspension of
compound 2116,17 (1.03mmol) and potassium carbonate
(2.06 mmol) in anhydrous DMF (6 mL). The mixture
was stirred at 70ꢁ C for 3h, then cooled at room tem-
perature, diluted with water (20 mL) and extracted with
ethyl acetate (15 mL ꢂ 3). The organic layers were eva-
porated and the residue, made up of a mixture of 17 and
36 or 18 and 37, was diluted with water (20 mL) and
HCl 6 N (3–4 mL) and refluxed for 3 h. After cooling at
room temperature, the solution was neutralized with
NaOH 10% and extracted with ethyl acetate (10 mL ꢂ
3). The organic layers were dried (Na2SO4), evaporated
and the residue recrystallized.
ꢁ
1
20. Yield 51%; mp 232–234 C (CH3NO2); H NMR
4.76 (s, 4H, 2 CH2), 6.70 (dd, 1H, ar, J=7.9, 2.9 Hz),
7.00 (s, 2H, NH2), 7.18–7.37 (m, 12H, ar), 7.54 (t, 2H,
ar, J=7.7 Hz), 8.01 (d, 2H, ar, J=7.7 Hz), 8.17 (d, 1H,
H-9, J=2.6 Hz). IR 1680, 3200, 3385. Anal.
(C29H24N6O) C, H, N.
Biochemistry
Bovine A1 and A2A receptor binding. Displacement of
[3H]CHA from A1 ARs in bovine cerebral cortical
membranes and [3H]CGS 21680 from A2A ARs in
bovine striatal membranes was performed as described
in ref 25.
Human A3 receptor binding. Displacement of [125I]AB-
MECA from hA3 ARs stably expressed in CHO cells
was performed as previously described.14
17. Yield 41%; mp 208–209 ꢁC (EtOAc); H NMR
1
2.88 (t, 2H, CH2, J=7.1 Hz), 3.25–3.41 (m, 2H, CH2),
5.63(t, 1H, NH, J=7.1 Hz), 6.61 (d, 1H, ar, J=7.7 Hz),
7.09–7.37 (m, 9H, 7 ar+NH2), 7.53(t, 2H, ar,
J=7.7 Hz), 7.85 (d, 1H, ar, J=7.6 Hz), 8.01 (d, 2H, ar,
J=7.7 Hz); IR 1720, 3400, 3500. Anal. (C23H20N6O) C,
H, N.
The concentration of the tested compounds that pro-
duced 50% inhibition of specific [3H]CHA, [3H]CGS
21680 or [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.26 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 value of [125I]AB-
MECA in hA3 ARs in CHO cell membranes was 1.4 nM.
18. Yield 46%; mp 207–208 ꢁC (EtOAc/cyclohexane);
1H NMR 2.87 (t, 2H, CH2, J=7.3 Hz), 3.25–3.38 (m,
2H, CH2), 3.72 (s, 3H, OCH3), 5.60 (t, 1H, NH,
J=7.3Hz), 6.61 (d, 1H, ar, J=8.1 Hz), 6.88 (d, 2H, ar,
J=8.4 Hz), 7.10–7.24 (m, 3H, ar), 7.26–7.38 (m, 3H, 1
ar+NH2), 7.55 (t, 2H, ar, J=7.7 Hz), 7.90 (d, 1H ar,
J=7.7 Hz), 8.06 (d, 2H, ar, J=7.7 Hz); IR 1730, 3360,
3430, 3480. Anal. (C24H22N6O2) C, H, N.
Acknowledgements
We thank Dr. Karl-Norbert Klotz of the University of
Wuzburg, Germany, for providing cloned hA3 receptors
expressed in CHO cells.
Synthesis of 4-amino-1,2-dihydro-8-benzylamino-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (19) and 4-amino-
1,2-dihydro-8-dibenzylamino-2-phenyl-1,2,4-triazolo[4,3-
a]quinoxalin-1-one (20). Benzyl bromide (1.6 mmol) was
added to a suspension of compound 3817 (1.6 mmol)
and potassium carbonate (2.4 mmol) in anhydrous
DMF (0.6 mL). The mixture was stirred at room tem-
perature for 6 days, then it was diluted with water
(10 mL). The solid, made up of the 8-dibenzylamino
derivative 20, was filtered. Further dilution with water
(10 mL) of the clear solution yielded a solid which was
filtered and washed with water and then ethanol. This
crude product was a mixture of compound 19 and the
starting product 38, which were separated on silica gel
column, eluting system cyclohexane/ethyl acetate, 3:7.
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