1,2,4-Triazolo[1,5-a]quinoxaline derivatives and their simplified analogues as adenosine A3 receptor antagonists. Synthesis, structure-affinity relationships and molecular modeling studies

Article abstract of DOI:10.1016/j.bmc.2014.11.033

The 1,2,4-triazolo[1,5-a]quinoxaline (TQX) scaffold was extensively investigated in our previously reported studies and recently, our attention was focused at position 5 of the tricyclic nucleus where different acyl and carboxylate moieties were introduced (compounds 2-15). This study produced some interesting compounds endowed with good hA₃ receptor affinity and selectivity. In addition, to find new insights about the structural requirements for hA₃ receptor-ligand interaction, the tricyclic TQX ring was destroyed yielding some 1,2,4-triazole derivatives (compounds 16-23). These simplified compounds, though maintaining the crucial structural requirements for adenosine receptor-ligand interaction, have a very low hA₃ adenosine receptor affinity, the only exception being compound 23 (1-[3-(4-methoxyphenyl)-1-phenyl-1H-1,2,4-triazol-5-yl]-3-phenylurea) endowed with a K_i value in the micro-molar range and high hA₃ selectivity versus both hA₁ and hA_2A AR subtypes. Evaluation of the side products obtained in the herein reported synthetic pathways led to the identification of some new triazolo[1,5-a]quinoxalines as hA₃AR antagonists (compounds 24-27). These derivatives, though lacking the classical structural requirements for the anchoring at the hA₃ receptor site, show high hA₃ affinity and in some case selectivity versus hA₁ and hA_2A subtypes. Molecular docking of the herein reported tricyclic and simplified derivatives was carried out to depict their hypothetical binding mode to our model of hA₃ receptor.

Full text of DOI:10.1016/j.bmc.2014.11.033

Bioorganic & Medicinal Chemistry 23 (2015) 9–21
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,2,4-Triazolo[1,5-a]quinoxaline derivatives and their simplified
analogues as adenosine A3 receptor antagonists. Synthesis,
structure–affinity relationships and molecular modeling studies
a
a
a
a
b
Daniela Catarzi a, , Flavia Varano , Daniela Poli , Lucia Squarcialupi , Marco Betti , Letizia Trincavelli ,
⇑
Claudia Martini b, Diego Dal Ben c, Ajiroghene Thomas c, Rosaria Volpini c, Vittoria Colotta a
a Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e Nutraceutica, Università di Firenze, Polo Scientifico, Via Ugo Schiff
6, 50019 Sesto Fiorentino, FI, Italy
b Dipartimento di Farmacia, Università di Pisa, Via Bonanno Pisano 6, 56126 Pisa, Italy
c School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 July 2014
Revised 30 October 2014
Accepted 21 November 2014
Available online 27 November 2014
The 1,2,4-triazolo[1,5-a]quinoxaline (TQX) scaffold was extensively investigated in our previously
reported studies and recently, our attention was focused at position 5 of the tricyclic nucleus where dif-
ferent acyl and carboxylate moieties were introduced (compounds 2–15). This study produced some
interesting compounds endowed with good hA3 receptor affinity and selectivity. In addition, to find
new insights about the structural requirements for hA3 receptor–ligand interaction, the tricyclic TQX ring
was destroyed yielding some 1,2,4-triazole derivatives (compounds 16–23). These simplified compounds,
though maintaining the crucial structural requirements for adenosine receptor–ligand interaction, have a
very low hA3 adenosine receptor affinity, the only exception being compound 23 (1-[3-(4-methoxy-
phenyl)-1-phenyl-1H-1,2,4-triazol-5-yl]-3-phenylurea) endowed with a Ki value in the micro-molar
range and high hA3 selectivity versus both hA1 and hA2A AR subtypes. Evaluation of the side products
obtained in the herein reported synthetic pathways led to the identification of some new triazolo[1,5-
a]quinoxalines as hA3AR antagonists (compounds 24–27). These derivatives, though lacking the classical
structural requirements for the anchoring at the hA3 receptor site, show high hA3 affinity and in some
case selectivity versus hA1 and hA2A subtypes. Molecular docking of the herein reported tricyclic and sim-
plified derivatives was carried out to depict their hypothetical binding mode to our model of hA3
receptor.
Keywords:
G protein-coupled receptors
Adenosine receptor antagonists
Triazoloquinoxalines
Tricyclic heteroaromatic systems
Ligand–receptor modeling studies
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
distributed in the human body, both in peripheral organs and in dis-
tinct regions of the central nervous system (CNS), though in low
Adenosine is a ubiquitous nucleoside that regulates a large
number of physiological and patho-physiological processes by trig-
gering specific adenosine receptors (ARs) at the extracellular level.
The ARs are four different subtypes of G protein-coupled receptors
(GPCRs) classified as A1, A2A, A2B and A3 on the basis of their tissue
localization, respective coupling to adenylate cyclase (AC) and spe-
cific pharmacological criteria.1
The adenosine A3 receptor (A3AR), the most recently character-
ized, was originally isolated from rat testis2 and subsequently
cloned from a variety of species.3 For the A3AR, significant differ-
ences (72%) in sequence similarity and tissue distribution have
been observed between species.4,5 However, this receptor is widely
levels. The A3AR is reported to be related to various second messen-
ger systems. Its activation leads to inhibition of AC and stimulation
of phospholipase C and D6,7 through Gi and Gq proteins, respec-
tively. Moreover, additional intracellular pathways have been
described to be important for intracellular signal transduction in
the adenosine biochemical system.6,8 The improved understanding
of the physiological effects mediated by the A3AR and of its biology
has provided substantial evidence that this AR subtype is an inter-
esting target for different therapeutic interventions. In particular,
A3AR antagonists are being investigated for the treatment of glau-
coma, asthma, inflammation9 and cerebral ischemia.10,11 A3AR
antagonists have also been reported to have potential efficacy in
both glioblastoma multiforme and colon cancer therapy.12,13
However, the role of A3AR antagonists as potential therapeutics
in many pathological diseases such as inflammation, cerebral
⇑
Corresponding author. Tel.: +39 055 4573722; fax: +39 055 4573780.
E-mail address: daniela.catarzi@unifi.it (D. Catarzi).
http://dx.doi.org/10.1016/j.bmc.2014.11.033
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

10
D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
ischemia and cancer is still ambiguous and widely debated.14 Thus,
the search for potent and selective human (h) A3AR antagonists has
become an attractive goal for many scientists. In the last few years,
much effort has been directed toward design and development of
potent and selective AR antagonists belonging to diverse classes
of heterocyclic derivatives with different structures.9
O
O
N
N
N
N
R5
N
R5
NH
R1
R1
N
N
Series C (2-15)
R8
R8 Series D1 (16-19)
R
1 = H, OCH3
R1 = OCH3
The 1,2,4-triazolo[1,5-a]quinoxaline (TQX) ring system is a
recurrent structural core which has been used to obtain tricyclic
AR antagonists.15–18 The first AR antagonists belonging to this series
were designed as structural analogues of CGS15943 (9-chloro-2-(2-
furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine)19 in the mid-
nineties15 and, since then, many other TQX derivatives of interest
have been developed by our research group. In particular, the 8-
R5 = C2H5, OC2H5, OCH3,
OC3H7, OCH2C CH,
CH2C6H5, OCH2C6H5,
R8 = H, Cl, CH3
R5 = OC2H5, OCH2C CH,
CH2C6H5, OCH2C6H5
R8 = CH3
R5
R
O
N
N
N
R1
N
N
N
R1
N
chloro-2-phenyl-1,2,4-triazolo[1,5-a]quinoxalin-4-amine17
(1A,
N
Fig. 1) was modified by introducing suitable substituents either on
the 4-amino group or the 2-phenyl ring (Series A,17 Fig. 1). The pres-
ence of an acyl moiety on the 4-amino group, as in compound 2A,
led to potent and selective hA3AR antagonists. The improvement
of hA3 affinity and selectivity was hypothesized to be due to the
amide carbonyl at position-4 that can act as a proton acceptor in a
hydrogen bonding interaction with a proton donor binding site. A
further increase in affinity was observed when a methoxy group
which can engage an additional hydrogen bond with the receptor
site was introduced in the para position of the 2-phenyl
substituent.17
These promising results indicated that the TQX ring system is a
versatile scaffold which can be further modified to develop new AR
antagonists. Thus, we studied a series of 2-(hetero)aryl-1,2,4-triaz-
olo[1,5-a]quinoxaline derivatives bearing a 4-oxo function replac-
ing the 4-amino group of the previously reported Series A
(Series B,18 Fig. 1). Some interesting hA3 AR antagonists were pro-
duced starting from the 8-chloro-2-phenyl-4,5-dihydro-1,2,4-
triazolo[1,5-a]quinoxalin-4-one 1B, selected as lead compound.18
Profitable modifications were made either by introducing different
aryl and heteroaryl groups at position 2, or by replacing the 8-
chloro substituent with a methyl group or a hydrogen atom. In
order to further investigate the potentiality of the TQX ring system,
we decided to move the 4-carbonyl function of Series B into an
exonuclear position by introducing different acyl or carboxyalkyl
substituents at N-5 (Series C, compounds 2–15, Fig. 2). We also
introduced the above cited suitable substituents, that is, the 8-
chloro or 8-methyl on the fused benzo moiety and the crucial 4-
methoxy on the 2-phenyl ring, onto the TQX scaffold.
R8
Series E (24-27)
Series D2 (20-23)
R8
R1 = H, OCH3
R8 = H, Cl, CH3
R1 = OCH3
R5 = CH3, C6H5
R = H, COCH3, COC6H5
R8 = CH3
Figure 2. Currently reported 1,2,4-triazolo[1,5-a]quinoxaline (TQX) derivatives and
their simplified 1,3-diaryl-1,2,4-triazole analogues.
the new 1,3-diaryl-1,2,4-triazole monocyclic core (Series D1, com-
pounds 16–19, Fig. 2) which can be considered a simplified struc-
ture of Series C. Contemporarily, analogues of Series D1, called
Series D2 (compounds 20–23), were designed by moving the
substituted NH group from the ortho-position of the 1-aryl moiety
to the 5-position on the 1,2,4-triazole core. Hence, both Series D1
and D2 maintain the substituted NH group and the two aryl moie-
ties which could be important requirements for a profitable inter-
action with the AR binding pockets.9 It has to be noted that only a
few other monocyclic cores have been evaluated as possible candi-
dates for developing AR antagonists.20–25
In addition, looking at the side products in the synthetic path-
way which leads to the targeted TQX compounds, we have identi-
fied the 4,5-dehydro-derivatives (Series E, compounds 24–27,
Figure 2) as possible candidates for our pharmacological studies.
Unlike the other series herein reported, these compounds lack all
the classical structural requirements considered important for
anchoring at the receptor binding sites, while maintaining only
the nude tricyclic TQX scaffold.
To find new insights into the structural requirements for hA3
receptor–ligand interaction, the tricyclic TQX ring was destroyed
by eliminating the 4-methylene bridge of Series C, generating
2. Chemistry
The synthetic pathways which yielded compounds 2–15, 16–
19, 20–23 and 24–2726 are illustrated in Schemes 1–3. Compounds
2–15 (Series C) were obtained starting from the 2-aryl-4,5-dihy-
dro-1,2,4-triazolo[1,5-a]quinoxalin-4-ones 28–31,18,26 as reported
in Scheme 1. Reduction of the 4-oxo function of 28–31 with LiAlH4
led to a mixture of the key intermediates 32–3526 and small
amounts of the 4,5-dehydro-derivatives 24–27,26 which were suc-
cessively evaluated as AR antagonists. An increased quantity of 25
and 27 was obtained by treating 33 and 35, respectively, with gla-
cial acetic acid at reflux. Derivatives 32–35 were reacted with the
suitable acyl chloride or chloroformates in the presence of pyridine
to yield the final N-5-substituted compounds 2–15. In order to find
an alternative synthetic pathway to achieve the target compounds
32–35 with higher yields, we synthesized 35 starting from the 1,3-
diaryl-5-chloromethyl-1,2,4-triazole 37 which was obtained by
treating the amidrazone 3618 with chloroacetyl chloride. The inter-
mediate 37 was transformed into the tricyclic derivative 35 with
SnCl2 dihydrate, and only traces of compound 27 were obtained
(1H NMR determination). In this way the synthetic procedure for
preparing 35 has been shortened and the total yield improved.
R4
NH
O
4
N
N
N
N
2
N
HN
R2
R2
N
N
1A: R2 = C6H5
1B : R2 = C6H5
R4 = H
R8 = Cl
R8
R8
R8
= Cl
Series A
Series B
R2 = C6H5, 4CH3OC6H4
R4 = H, COR
R8 = Cl, H
R2 = C6H5, 4CH3OC6H4,
Heteroaryl
R8 = Cl, H, CH3
2A: R2 = C6H5
R4 = COC2H5
R8
= Cl
Figure 1. Previously reported 4-amino- and 4-oxo-substituted 2-(hetero)aryl-
1,2,4-triazolo[1,5-a]quinoxaline (TQX) derivatives as human A3 adenosine receptor
antagonists.

D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
11
R5
O
R1
R8
N
N
R5
2, 4, 7, 11
CH2CH3
OCH2CH3
OCH3
N
R1
28, 32, 24, 2, 3
29, 33, 25, 4-6
30, 34, 26, 7-10
H
H
H
Cl
H
CH3
3, 6, 9, 12
N
5, 8
10
13
14
15
OCH2CH2CH3
OCH2C CH
CH2C6H5
31, 35, 27, 11-15 OCH3 CH3
2-15
R8
OCH2C6H5
b
c
O
N
N
N
HN
HN
N
R1
R1
R1
a
N
N
N
N
N
N
+
28-31
32-35
24-27
R8
R8
R8
e (to 35, 27)
Cl
N
N
NH2
NO2
NO2
H
N
N
d
N
OCH3
OCH3
37
36
CH3
CH3
Scheme 1. Reagents and conditions. (a) LiAlH4, anhydrous tetrahydrofuran, nitrogen atmosphere, reflux; (b) glacial acetic acid, reflux; (c) R5COCl, pyridine, anhydrous
dichloromethane, nitrogen atmosphere; (d) ClCOCH2Cl, anhydrous toluene, 80 °C; (e) SnCl2 dihydrate, ethanol, reflux.
which was reacted with the suitable acyl chloride or chlorofor-
mates to provide compounds 16–19 with high yields.
N
NH2
NO2
NO2
H
N
OCH3
N
b
a
N
N
The synthesis of compounds 20–23 (Series D2) was performed
as reported in Scheme 3. By reacting the hydrazide 4027,28 with a
mixture of POCl3 and PCl5 at reflux, the unstable chloroimine
4129 was obtained which was immediately reacted with cyana-
mide at 100 °C in solvent-free conditions to give the intermediate
42. Then, the latter was transformed into the corresponding acyl-
and carbamoyl-derivatives by reacting with acetic anhydride
(compound 20), benzoyl chloride (21, 22) or phenyl isocyanate
(23).
OCH3
38
36
CH3
NH2
CH3
O
N
N
R5
N
NH
OCH3
OCH3
N
N
c
N
39
16-19
CH3
CH3
R5
16
17
18
19
OCH2CH3
OCH2C CH
CH2C6H5
3. Pharmacology
OCH2C6H5
The newly synthesized derivatives 2–15 (Table 1), 16–23
(Table 2), and 24–27 (Table 3) were tested for their ability to dis-
Scheme 2. Reagents and condition: (a) CH(OEt)3, p-toluensulfonic acid, 100 °C; (b)
10% Pd/C, H2, ethyl acetate; (c) R5COCl, pyridine, anhydrous dichloromethane, 0 °C.
place
[
125I]N6-(4-amino-3-iodobenzyl)-50-(N-methylcarba-
moyl)adenosine ([125I]AB-MECA) from
a
cloned hA3 receptor
stably expressed in CHO cells. Subsequently, all compounds except
3 and 25, were evaluated for their ability to displace [3H]8-cyclo-
pentyl-1,3-dipropylxantine ([3H]DPCPX) from cloned hA1 ARs,
and [3H]50-(N-ethylcarboxamido)adenosine ([3H]NECA) from
cloned hA2A ARs, to establish their A3 versus A1 and versus A2A
selectivity. In Table 1, the binding results of the reference com-
pound 1B18 (Fig. 1) at hA3 AR is reported.
To determine hA3 versus hA2B selectivity, some selected com-
pounds (2, 7–9, 11, 13–14, 27) were tested at the hA2B subtype
by measuring their effects on cyclic adenosine monophosphate
(cAMP) accumulation in CHO cells stably transfected with the
hA2B AR (Table 4).
O
Cl
H
N
H
N
b
a
N
H
N
OCH3
OCH3
40
41
R5
R
O
H2N
N
N
N
N
OCH3
OCH3
N
N
c
N
42
20-23
R
R5
20
21
COCH3
H
CH3
C6H5
C6H5
22 COC6H5
4. Results and discussion
23
H
NHC6H5
Scheme 3. Reagents and condition. (a) POCl3, PCl5, reflux; (b) NH2CN, 100 °C; (c)
acetic anhydride, pyridine, reflux; or C6H5COCl, pyridine, anhydrous tetrahydrofu-
ran, reflux; or C6H5NCO, anhydrous dichloromethane, 5 °C/room temperature.
4.1. Structure–affinity relationships
The binding results reported in Tables 1–3 indicate that we
have produced some new potent and selective hA3 AR antagonists
belonging to the 1,2,4-triazolo[1,5-a]quinoxaline series (Series C
and E, Table 1 and 3, respectively). Some of the novel derivatives
show high hA3 AR affinity (Ki <100 nM) and selectivity versus the
hA2A receptor (compounds 2, 7–9 and 11–14) and, in some cases,
also good selectivity versus the hA1 subtype. The choice to test
The simplified 1,2,4-triazole derivatives of Series D1 (16–19)
were obtained as reported in Scheme 2. Reaction of 36 with triethyl
orthoformate in the presence of para-toluenesulfonic acid, led to
1,2,4-triazole cyclization (compound 38). By reduction of the nitro
group of 38, the corresponding amino-derivative 39 was obtained

12
D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
Table 1
Table 2
Binding affinity (Ki) at hA3, hA1 and hA2A ARs of the 5-substituted 2-aryl-4,5-dihydro-
1,2,4-triazolo[1,5-a]quinoxaline derivatives (Series C)
Binding affinity (Ki) at hA3, hA1 and hA2A ARs of the simplified 1,3-diaryl-1,2,4-
triazole analogues (Series D1 and D2)
O
O
O
O
R5
N
N
NH
R5
C
HN
N
N
N
R5
N
N
R
N
R1
N
OCH3
N
N
N
N
OCH3
N
1B
2-15
16-19
R8
20-23
Cl
R8
R8
Series D1
Series D2
R1
R5
R8
Kia (nM) or I%
R
R5
R8
Kia (nM) or I%
b
c
d
hA3
hA1
hA2A
b
c
d
hA3
hA1
hA2A
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1Be
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
—
C2H5
OC2H5
C2H5
OCH3
OC2H5
C2H5
OCH3
OC2H5
OC3H7
C2H5
Cl
Cl
H
H
72 6.8
166 15
55%
245 24
22%
39%
n.d.
1360 50
34%
4600 118
134.3
1481 145
556 55
565 72
1543 61
24%
n.d.
52%
50%
33%
33%
32%
9%
56%
6%
2%
13%
0%
4%
16
17
18
19
20
21
22
23
—
—
—
—
OC2H5
OCH2C„CH
CH2C6H5
OCH2C6H5
CH3
C6H5
C6H5
NHC6H5
CH3
CH3
CH3
CH3
H
H
H
H
34%
35%
45%
47%
19%
50%
44%
6%
13%
16%
1%
7%
4%
43%
13%
7%
n.d.
44%
2%
26%
25%
55%
H
COCH3
H
COC6H5
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
—
6.5 0.5
7
31.9
3
12.5 1.1
171 16
545 48
1%
a
25.1
39.4
2
3
Ki values are means SEM of four separate assays, each performed in triplicate.
Displacement of specific [125I]AB-MECA binding at hA3 receptors expressed in
b
OC2H5
86
8
OCH2CꢀCH
CH2C6H5
OCH2C6H5
—
23.3 1.8
54.8
49%
39%
45%
n.d.
CHO cells or percentage of inhibition (I%) of specific binding at 1
l
M.
Percentage of inhibition (I%) of specific [3H]DPCPX binding at hA1 receptors
expressed in CHO cells, at 10 M concentration.
Percentage of inhibition (I%) of specific [3H]NECA binding at hA2A receptors
c
5
231.4 18
163 13
l
d
n.d.
expressed in CHO cells, at 10 lM concentration.
a
Ki values are means SEM of four separate assays, each performed in triplicate.
b
Displacement of specific [125I]AB-MECA binding at hA3 receptors expressed in
CHO cells or percentage of inhibition (I%) of specific binding at 1
l
M.
Displacement of specific [3H]DPCPX binding at hA1 receptors expressed in CHO
cells or percentage of inhibition (I%) of specific binding at 10 M concentration.
Percentage of inhibition (I%) of specific [3H]NECA binding at hA2A receptors
c
Table 3
l
Binding affinity (Ki) at hA3, hA1 and hA2A ARs of the 1,2,4-triazolo[1,5-a]quinoxaline
derivatives (Series E)
d
expressed in CHO cells, at 10
lM concentration.
N
e
N
Ref. 18.
R1
N
N
24-27
R8
the side products 24–27 (Series E, Table 3) which turned out to be
potent hA3AR antagonists and, in the case of 26 and 27 also selec-
tive versus both the hA1 and hA2A subtypes in the binding assays
was fortunate. In contrast, the simplified 1,2,4-triazole derivatives
(Series D1 and D2, Table 2) were inactive or had very low activity
at all the AR subtypes, the only exception being compound 23
endowed with a Ki value at the hA3AR in the micromolar range
and high selectivity versus both hA1 and hA2A subtypes.
Focusing on the results reported in Table 1, we can observe that
elimination of the 8-chloro substituent is detrimental for hA3
receptor–ligand interaction (compare 2 and 3 to 4 and 6, respec-
tively), while its replacement with a methyl group leads to a strong
increase in hA3 affinity (compare compounds 7 and 9 to 2 and 3,
respectively) and maintains good hA3 selectivity versus both hA1
and hA2A receptors. High hA3 AR binding activity is also observed
for compounds 11–14 which hold the methyl group at position 8
but, unlike derivatives 7–10, are decorated with a para-methoxy
group on the 2-phenyl ring. These modifications maintain very
high selectivity versus both the hA2A and the hA1 ARs, the only
exception being compound 12 which shows, compared to the par-
ent 9, a dramatic increase in hA1 AR affinity and a total loss of hA3
versus hA1 selectivity. Nevertheless, the introduction of the para-
methoxy group does not exert the positive effect on hA3 affinity
observed in the previously reported Series A and B17,18 (Fig. 1).
In fact, it has to be noted that compounds 11 and 12 possess a 3-
fold reduced hA3 affinity compared to 7 and 9.
R1
R8
Kia (nM) or I%
b
c
d
hA3
hA1
hA2A
24
25
26
27
H
H
H
OCH3
Cl
H
CH3
CH3
15.2 1.6
91.5 8.9
23.9 2.19
127.4 13
n.d.
1322 135
2016 100
19%
n.d.
2401 246
30%
26.6
1
a
Ki values are means SEM of four separate assays each performed in triplicate.
Displacement of specific [125I]AB-MECA binding at hA3 receptors expressed in
b
CHO cells.
Displacement of specific [3H]DPCPX binding at hA1 receptors expressed in CHO
c
cells.
Displacement of specific [3H]NECA binding at hA2A receptors expressed in CHO
cells or percentage of inhibition (I%) of specific binding at 10 M concentration.
d
l
the 4-carbonyl function of Series B with exonuclear acyl or carbo-
xyalkyl groups is well tolerated by the hA3 receptor.
The effect on hA3 AR affinity and selectivity exerted by the sub-
stituents inserted at position 5 of the TQX scaffold is very difficult
to explain. By restricting our attention to the 8-methyl substituted
compounds 7–15, we can observe that the binding affinity is main-
tained in the nanomolar range apart from the nature of the substi-
tuent at position-5. Hence, the steric hindrance at this position
does not seem to be critical for receptor–ligand interaction. It is
worth noting that the 5-N-propionyl substituted derivative 7 is
the most active compound at the hA3 AR with a Ki value of
6.5 nM (Table 1). Furthermore, compound 13, bearing the propar-
gyl carboxylate group at the same position, is about 3.5-fold less
active than 7, but much more selective versus the hA1 subtype.
The opening of the tricyclic TQX scaffold of Series C to produce
the 1,3-diaryl-1,2,4-triazole system (Series D1, Table 2) is
A comparison of the binding data of the previously reported 1B
(Fig. 1) with those of the 5-substituted herein reported (com-
pounds 2–15) highlights that the hA3 binding affinities of 2–15
are similar or higher than that of 1B, with the only exceptions
being the 8-unsubstituted derivatives 4, 5 and 6 and compound
15 which are less active. These data suggest that replacement of

D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
13
Table 4
using four X-ray structures of the antagonist-bound hA2A AR as
templates (pdb code: 3EML; 2.6-Å resolution;30 pdb code: 3PWH;
3.3-Å resolution;31 pdb code: 3REY; 3.3-Å resolution;31 pdb code:
3UZA; 3.3-Å resolution32). The A2AAR crystal structure provides
improved accuracy of AR homology models, due to high residue
conservation in the primary sequences of the AR subtypes, which
share a sequence identity of ꢁ57% within the transmembrane
(TM) domains.33 The residues located within the seven TM
domains in the upper part of ARs, corresponding to the ligand
binding site, are conserved with an average identity of 71%.34 Fur-
thermore, the above cited A2AAR crystal structures have been
solved in complex with high affinity antagonists (ZM241385,
XAC, and the 6-(2,6-dimethylpyridin-4-yl)-5-phenyl-1,2,4-triazin-
3-amine, see cited articles for details), hence presenting a cavity
suitable as a binding site for docking analysis. Each obtained hA3AR
homology model was checked by using the Protein Geometry Mon-
itor application within MOE35 and then employed for a preliminary
docking analysis performed by manually docking the high affinity
antagonist MRS 1220 (N-[9-chloro-2-(2-furyl)[1,2,4]-triazolo[1,5-
c]quinazolin-5-yl]benzene acetamide, Ki hA3AR = 0.65 nM36) struc-
ture within the respective binding site. The obtained hA3AR–MRS
1220 complexes were then subjected to energy minimization and
to Monte Carlo analysis to explore the favorable binding conforma-
tions. During this analysis, the ligand was left free to be continu-
ously re-oriented and re-positioned within the binding site and
the conformation of both ligand and nearby residues could be
explored and reciprocally relaxed. The remaining receptor atoms
were kept fixed. This stage was crucial to provide A3AR binding
sites with conformations able to accommodate the analyzed antag-
onists. For each A3AR model, the best receptor–MRS 1220 complex
was saved and energetically minimized.
Once the MRS 1220 compound was removed, each hA3AR
model was then used as target for the docking analysis of the syn-
thesized derivatives. All ligand structures were optimized using
RHF/AM1 semi-empirical calculations (with the aid of the software
package MOPAC37 implemented in MOE) and then docked into the
binding site of the hA3AR models by using the MOE Dock tool. Top-
score docking poses of each compound were subjected to energy
minimization and then rescored using three available methods
implemented in MOE: the London dG scoring function, the Affinity
dG scoring tool, and the dock-pKi predictor. For each compound,
the four top-score docking poses, according to at least two out of
three scoring functions, were selected for final ligand-target inter-
action analysis.
Effect of some selected compounds on cAMP production in CHO cells expressing hA2B
ARa
% of cAMP productionb
2
7
8
63.7 4.7
68.6 4.6
74.9 6.3
103.8 5.1
102.4 6.7
108.9 10.2
89.5 8.3
42.6 1.9
9
11
13
14
27
a
The effect of each compound at 10
l
M concentration was evaluated. Each
compound was tested in the presence of an EC50 concentration of agonist NECA
(100 nM, determined on the same day as each assay).
b
Data are expressed as percentage of cAMP production versus agonist set to
100%. All data represent the mean SEM of two different experiments each per-
formed in duplicate.
detrimental for hA3 AR affinity, although the resulting compounds
(16–19) maintain some ability to bind the hA3 subtype
(35 < I% < 50). Similar results were obtained when the substituted
amino group was moved from the 1-aryl-moiety of Series D1 to
the 5-position of the 1,2,4-triazole core (compounds 20–23, Series
D2, Table 2). However, in contrast with the low hA3 AR affinity
(19 < I% < 50) of the 5-amido-derivatives 20–22, there is the micro-
molar hA3 Ki value and high selectivity versus both hA1 and hA2A
subtypes of compound 23. Thus, this compound could represent
a suitable lead for the development of hA3 AR antagonists endowed
with a small heterocyclic core.
In contrast to compounds 16–23 (Series D1 and D2), derivatives
24–27 (Series E, Table 3) hold the tricyclic ring system constant
but lack both the claimed NH function and the carbonyl group
which are considered important requirements for AR-ligand inter-
action.9 Although these derivatives have only the endonuclear
nitrogen atoms able to give hydrogen bonding interactions, they
show high hA3 affinity and in some cases selectivity versus hA1
and hA2A subtypes.
This series also confirms the profitable effect of the presence of
a small substituent (chloro or methyl) on the fused benzo moiety
for hA3 receptor–ligand interaction. In fact, the 8-chloro- and 8-
methyl-substituted compounds 24 and 26, respectively, are equi-
potent at the hA3AR with a Ki value in the nanomolar range and
are 4-6-fold more active than the unsubstituted derivative 25.
These data suggest that these small lipophilic groups could posi-
tively interact with a hydrophobic receptor pocket. The presence
of a para-methoxy substituent on the 2-phenyl ring (compound
27) is not cooperative as in Series A and B, leaving unchanged
the ability to bind the hA3AR compared to compound 26.
To evaluate the hA3 versus hA2B selectivity, the affinities of
some selected derivatives (2, 7–9, 11, 13–14, 27) at the hA2B AR
were evaluated by cAMP functional assay using A2B transfected
cells. In general, all the compounds tested alone are not effective
in stimulating cAMP accumulation (data not shown). In addition,
they showed low or null ability to inhibit cAMP accumulation
evoked by the agonist NECA (Table 4). Thus, this study demon-
strates that the tested derivatives 2, 7–9, 11, 13–14, 27 have not
agonist/antagonist activity toward A2B AR subtype.
The four developed hA3AR models present highly similar bind-
ing sites by considering both pocket volumes and receptor residues
orientation. In particular, the binding pockets present only subtle
rearrangements of some flexible residues, while the EL domains
in peripheral regions of binding site contain higher conformational
variability. For example, the side chain of hA2A AR Asn250 (a criti-
cal residue for ligand interaction due to its position in the core of
the binding pocket) is observed as having different conformations
by comparing the hA2A AR crystal structures used as templates and
analogue variability is obtained within the developed hA3AR mod-
els for the same amino acid (Asn250). Furthermore, the different
interaction and distance between EL2 and EL3 domains (even
due to a different orientation of Glu169) are observed in the hA2A
AR X-ray structures and also in the developed hA3AR models. Con-
sequently, it is not surprising that the docking analysis of the syn-
thesized compounds at the four receptor models led to analogue
results.
All together these data confirm that this work produced some
compounds endowed with good hA3 affinity and also selectivity
versus all the other ARs.
4.2. Molecular modeling studies
Considering the general binding mode of TQX derivatives
belonging to Series C (compounds 2–15) in the hA3AR, two main
sets of docking conformations were observed in all four hA3AR
models. Figures 3 and 4 show the binding modes at the hA3 AR
model of compounds 7 as representative of Series C compounds.
To define the structural features at the basis of the different
binding affinities of the new derivatives, a molecular docking anal-
ysis was performed on homology models of hA3AR developed by

14
D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
Figure 3. Panel A. Family 1 docking conformations. The binding mode of compound 7 at 3EML-based hA3AR model is shown as example. Panels B and C. Detailed view of
ligand-target interaction considering the 8- and the 5-substituent, respectively.
Figure 4. Panel A. Family 2 docking conformations. The binding mode of compound 7 at 3EML-based hA3AR model is shown as example. Panels B and C. Detailed view of
ligand-target interaction considering the 5- and the 8-substituent, respectively.
The first set of conformations (from now on called ‘family 1’ con-
formations, Fig. 3, Panel A) presents the TQX moiety located in
the center of the binding site with the quinoxaline ring being posi-
tioned between Phe168 (EL2) and Ile268 (TM7) side chains. The 2-
substituent points towards the central transmembrane core and is
located in a mainly hydrophobic subpocket in proximity of Leu90
and Leu91 (TM3), Met177 (TM5), Trp243 and Leu246 (TM6), while
the fused phenyl ring is internally oriented and located in a region
given by Ala69 and Val72 (TM2), Leu90 (TM3), Phe168 (EL2), and
Ile268 (TM7) residues.
Superimposition of the family 1 docking conformations of each
compound in the four hA3AR models shows that the binding modes
and the interactions are almost identical at the four binding sites.
These conformations are only marginally influenced by the slight
rearrangement of external binding site residues as shown by the
comparison of the four models. The interaction with the binding
site is mainly hydrophobic, the unique exception being given by
a possible H-bond between a nitrogen atom of the triazole core
and the polar hydrogen of the amide function of Asn250 (TM6) res-
idue. The presence of small substituents on the 2-phenyl ring mod-
ulates the interaction with TM5-6 residues, while small groups
inserted at the 8-position provide an additional hydrophobic inter-
action with TM2-3 amino acids. A detailed view of the interaction
of compound 7 (taken as template of Series C) with the hA3 AR
model is depicted in Figure 3, Panels B and C. The presence of sub-
stituents at the 5-position (hence linked to the nitrogen atom of
the quinoxaline ring) seems important but not critical for com-
pound activity. The presence of small hydrophobic groups at this
position improves the affinity for the receptor; this is not surpris-
ing and has already been shown even in the case of hA3AR ago-
nists.38–41 Binding data indicate that, on the whole, the propionyl
group (COEt) (compounds 2, 7, 11) or the ethyl (COOEt, compounds
3, 9, 12) and methyl (COOMe, 5 and 8) carboxylate chain at the
5-position have a good effect on compound affinity. This is

D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
15
particularly true for derivatives bearing an 8-substituent, and can
be considered true on the basis of the docking scores of the respec-
tive compounds. In the family 1 docking conformations, the phenyl
group on the 5-substituent of compounds 14 and 15 is inserted
between the hydrophobic side chains of Val169 (EL2) and Ile264
(TM7).
The second set of conformations (‘family 2’, Fig. 4, Panel A) is a
mirror version of family 1, with analogue location of both the TQX
scaffold and the 2-substituent, but with the fused phenyl ring
pointing externally and located between Met174, Phe168, and
Val169 (EL2), Ile249 and Ile253 (TM6), and Leu264 (TM7).
Just as for the family 1 conformations, the interaction with the
binding site is mainly hydrophobic, the unique exception being a
possible H-bond between a nitrogen atom of the triazole core
and the polar hydrogen of the amide function of Asn250 (TM6) res-
idue. The position and role of eventual small substituents on the 2-
phenyl ring is analogous to family 1 conformations. Interestingly,
the small groups, inserted at the 8-position and externally ori-
ented, are located in analogous positions of the (small) 5-substitu-
ents in family 1 conformations. Conversely, the 5-substituents are
positioned in an analogous position of 8-substituents in the case of
family 1 conformations. Superimposition of the family 2 docking
conformations of each compound on the four A3AR models shows
that there are also some differences in compound orientation. The
resulting family 2 conformations for compounds 14 and 15 present
the tricyclic scaffold more externally oriented with the loss of H-
bond interaction with Asn250. Moreover, the phenyl group on
the 5-substituent of 14 and 15 could make the family 2 conforma-
tions for these two compounds difficult as there is not enough
space to accommodate the 5-substituents in the subcavity
between TM2 and TM3. This difficulty can be particularly evi-
denced for compound 15 that is endowed with reduced hA3 bind-
ing activity compared to most of the other N-5 substituted TQX
derivatives. Thus, the lower affinity of this compound could be
ascribed to its lower ability to assume both binding modes.
All together, the modeling results suggest that the com-
pounds interact with the binding site through generally hydro-
phobic contact without the presence of strong H-bond or
electrostatic interactions. On the other hand, the ability to pres-
ent two reasonable ways of binding could be the key factor that
leads to a higher affinity for the receptor. This result is particu-
larly true for the compounds bearing the 8-substituent and small
groups at N-5, for which the two docking conformations are
almost equivalent from both the energy and score points of
view. Compounds 24–27 seem able to assume both conforma-
tions as well, and the presence of the 8-substituent provides
higher affinity (compare 24 and 26 to 25).
A docking analysis was performed also to simulate the possible
binding modes of the simplified triazole derivatives 16–23 at the
hA3AR binding site. The same docking and post-docking protocols
were employed. Among these derivatives, only compound 23
showed nanomolar affinity at the hA3AR. The highest docking score
conformation of this compound shows some similarities with fam-
ily 1 conformations of tricyclic compounds described above. In par-
ticular, the triazole ring and the 3-aryl substituent of 23 are located
in an analogous position of the triazole ring and the 2-substituent
of the tricyclic derivatives 2–15, respectively. The 1-aryl ring mim-
ics the role of the fused phenyl ring of the above described com-
pounds, while the phenyl-urea function of 23 is externally
oriented, with the phenyl ring inserted between Val169 (EL2)
and Leu264 (TM7). An H-bond interaction occurs between the 4-
nitrogen of triazole and a polar hydrogen atom of Asn250 (TM6)
and we cannot exclude a possible second interaction involving a
polar hydrogen atom of the compound urea function and the
Asn250 carbonyl group. Among the monocyclic derivatives
described in this work, compound 23 seems the only derivative
Figure 5. Panel A. Superimposition of family
1
(light) and
2
(dark) docking
conformations. The binding mode of compound 7 at 3EML-based hA3AR model is
shown as example. The superimpositions of 5- and 8-substituents (I and II) and 2-
substituents (III) are highlighted. Panel B. Docking conformations of simplified
triazoles (compound 23) at 3EML-based hA3AR model.
able to fit the three subpockets of the hA3AR binding site indicated
as I-III in Figure 5, Panel A, while the other simplified derivatives
16–19, lacking the side chain at the 5-position, do not seem able
to properly interact with the binding site.
5. Conclusion
The present study has led to the identification of some 1,2,4-
triazolo[1,5-a]quinoxalines as new hA3AR antagonists. In particu-
lar, the 5-substituted-4,5-dihydro derivatives 2–15 show, on the
whole, good hA3 receptor affinity and in some cases selectivity ver-
sus all the other AR subtypes. Surprisingly, similar results are
obtained with some TQX compounds (24–27), obtained as side
products, which lack all the classical structural requirements for
anchoring at the hA3 receptor site, and maintain only the nude tri-
cyclic scaffold. In contrast, the 1,2,4-triazole derivatives 16–23,
designed as simplified structures from TQX compounds and pre-
serving both the crucial NH and carbonyl groups and the two aryl
moieties, turn out to be inactive or have very little activity at all the
AR subtypes. The only exception is compound 23 which is
endowed with micromolar hA3AR affinity and high selectivity ver-
sus both hA1 and hA2A subtypes. As a new finding, this triazole
derivative emerges as lead candidate for the development of new
monocyclic AR antagonists. On the whole, our results lead to
new interesting insights about the structural requirements for
hA3 receptor–ligand interaction. Molecular docking of tricyclic
and simplified derivatives identify their hypothetical binding mode
to our hA3 receptor model.

16
D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
ar, J = 8.57). Anal. Calcd for (C17H16N4O): C, 69.85; H, 5.52; N
19.17; Found: C, 70.12; H, 5.75; N, 19.32.
6. Experimental section
6.1. Chemistry
6.1.1.5.
(24).
8-Chloro-2-phenyl-1,2,4-triazolo[1,5-a]quinoxaline
Yield: 24%; mp 217–219 °C (ethyl acetate). 1H NMR
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
Flash E1112 Thermofinnigan elemental analyzer for C, H, N, and
the results were within 0.4% of the theoretical values except
where stated otherwise. All final compounds revealed a purity
not less than 95%. The IR spectra were recorded with a Perkin–
(DMSO-d6) d: 7.58–7.61 (m, 3H, ar), 7.86 (dd, 1H, ar, J = 8.79, 2.2),
8.25–8.35 (m, 3H, ar), 8.52 (d, 1H, ar, J = 2.2), 9.51 (s, 1H, ar). IR:
1090, 820. Anal. Calcd for (C15H9ClN4): C, 64.18; H, 3.23; N
19.96; Found: C, 64.86; H, 3.74; N, 20.13.
6.1.1.6. 2-Phenyl-1,2,4-triazolo[1,5-a]quinoxaline (25).
5%; mp 174–176 °C (ethanol) (lit. mp 181–183 °C).26
Yield:
Elmer Spectrum RX
I spectrometer in Nujol mulls and are
expressed in cmꢂ1. The 1H NMR spectra were obtained with a Bru-
ker Avance 400 MHz instrument. The chemical shifts are reported
in d (ppm) and are relative to the central peak of the solvent. The
coupling constant (J) are expressed in Hz. All the exchangeable pro-
tons were confirmed by addition of D2O. The following abbrevia-
tions are used: s = singlet, d = doublet, dd = double doublet,
t = triplet, m = multiplet, br = broad, ar = aromatic protons.
6.1.1.7.
(26).
8-Methyl-2-phenyl-1,2,4-triazolo[1,5-a]quinoxaline
Yield: 10%; mp 162–164 °C (ethanol). 1H NMR (DMSO-
d6) d: 2.64 (s, 3H, CH3), 7.58–7.66 (m, 4H, ar), 8.13 (d, 1H, ar,
J = 8.06), 8.29–8.32 (m, 3H, ar), 9.40 (s, 1H, ar). Anal. Calcd for
(C16H12N4): C, 73.83; H, 4.65; N 21.52; Found: C, 74.23; H, 4.05;
N, 20.97.
6.1.1.8.
a]quinoxaline (27).
2-(4-Methoxyphenyl)-8-methyl-1,2,4-triazolo[1,5-
Yield: 30%; mp 180–182 °C (cyclohex-
6.1.1. General procedure for the synthesis of 8-substituted 2-
aryl-4,5-dihydro-1,2,4-triazolo[1,5-a]quinoxalines (32–35)26
and the corresponding 4,5-dehydro-derivatives (24–27)26
To a solution of the previously reported 2-aryl-4,5-dihydro-
1,2,4-triazolo[1,5-a]quinoxalin-4-ones 28–3118,26 (2.7 mmol) in
anhydrous tetrahydrofuran (200 mL), heated at reflux under nitro-
gen atmosphere, an excess (21.5 mmol) of LiAlH4 was added por-
tion by portion. At the end of the addition, the reaction mixture
was maintained at reflux for 30 min. Then ice (200 g) was carefully
added and the mixture was kept under stirring until gas evolution
ended. The aqueous phase was extracted with ethyl acetate
(100 mL ꢃ 2), and the separated organic layers were washed with
water (60 mL ꢃ 2), anhydrified (Na2SO4) and evaporated under
reduced pressure. The crude mixture, composed by the 4,5-dihy-
dro-derivatives 32–35 and the corresponding dehydro-compounds
24–27, was separated by silica gel column chromatography, elut-
ing system chloroform/methanol 9.5:0.5 (24 and 32), chloroform/
methanol 9:1 (25 and 33), chloroform/acetone 8:2 (26 and 34),
dichloromethane/cyclohexane/ethyl acetate 9:0.5:0.5 (27 and 35).
ane). 1H NMR (DMSO-d6) d: 2.65 (s, 3H, CH3), 3.87 (s, 3H, OCH3),
7.16 (d, 2H, ar, J = 8.59), 7.64 (d, 1H, ar, J = 8.16), 8.13 (d, 1H, ar,
J = 8.24), 8.25 (d, 2H, ar, J = 8.56), 8.32 (s, 1H, ar), 9.38 (s, 1H, CH).
Anal. Calcd for (C17H14N4O): C, 70.33; H, 4.86; N 19.30; Found: C,
69.76; H, 5.01; N, 19.12.
6.1.2. Synthesis of 2-Phenyl-1,2,4-triazolo[1,5-a]quinoxalines
(25, 27)
A mixture of compounds 25, 33 or 27, 35 (0.2 g) in glacial acetic
acid (5 mL) was heated at reflux for 5 h. Evaporation of the solvent
at reduced pressure to small volume produced separation of a solid
which was collected and washed with diethyl ether.
2-Phenyl-1,2,4-triazolo[1,5-a]quinoxaline (25).26 Yield: 26%.
2-(4-Methoxyphenyl)-8-methyl-1,2,4-triazolo[1,5-a]quinoxa-
line (27). Yield: 30%.
6.1.3. General procedure for the synthesis of N-5-substituted 2-
aryl-4,5-dihydro-1,2,4-triazolo[1,5-a]quinoxaline derivatives
(2–15)
To a suspension of compounds 32–3526 (1.2 mmol) and small
amounts of the corresponding 4,5-dehydro-derivatives 24–27 in
anhydrous dichloromethane (40 mL) and anhydrous pyridine
(0.1 mL) kept at 0 °C and under nitrogen atmosphere, a solution
of the suitable acyl chloride or chloroformates (3.6 mmol) in
anhydrous dichloromethane (4.0 mL) was drop by drop added.
The reaction was stirred at 0 °C for 30 min (compounds 11–
15), at room temperature for 1 h (4–5, 7, 9–10), 4 h (compound
8), otherwise at reflux for 3 h (2–3, 6). After evaporation of the
solvent at reduced pressure, the crude mixture was purified by
silica gel column chromatography by using the suitable eluting
system.
6.1.1.1.
a]quinoxaline (32).
8-Chloro-2-phenyl-4,5-dihydro-1,2,4-triazolo[1,5-
Yield: 24%; mp 176–178 °C dec (ethanol).
1H NMR (DMSO-d6) d: 4.78 (s, 2H, CH2), 6.74 (s, 1H, NH), 6.85 (d,
1H, ar J = 8.79), 7.12 (d, 1H, ar, J = 8.06), 7.49–7.58 (m, 4H, ar),
8.08–8.10 (m, 2H, ar). IR: 3295. Anal. Calcd for (C15H11ClN4): C,
63.72; H, 3.92; N 19.82; Found: C, 62.99; H, 3.41; N, 19.98.
6.1.1.2. 2-Phenyl-4,5-dihydro-1,2,4-triazolo[1,5-a]quinoxaline
(33).
Yield: 30%; mp 126–128 °C (ethyl acetate) (lit. mp
126–128 °C).26
6.1.1.3.
a]quinoxaline (34).
8-Methyl-2-phenyl-4,5-dihydro-1,2,4-triazolo[1,5-
Yield: 37%; mp 177–179 °C (ethanol).
6.1.3.1. 1-(8-Chloro-2-phenyl-1,2,4-triazolo[1,5-a]quinoxalin-
1H NMR (DMSO-d6) d: 2.26 (s, 3H, CH3), 4.68 (s, 2H, CH2), 6.36 (s,
1H, NH), 6.75 (d, 1H, ar, J = 8,06), 6.88 (d, 1H, ar, J = 8.06), 7.44–
7.54 (m, 4H, ar), 8.05–8.10 (m, 2H, ar). IR 3300. Anal. Calcd for
(C16H14N4): C, 73.26; H, 5.38; N 21.36; Found: C, 73.58; H, 4.71;
N, 20.87.
5(4H)-yl)-propan-1-one (2).
Eluting system: chloroform/
methanol 9.9:0.1. Yield: 25%; mp 142–144 °C (cyclohexane). 1H
NMR (DMSO-d6) d: 0.99 (t, 3H, CH3, J = 7.33), 2.58 (q, 2H, CH2,
J = 7.33), 5.24 (s, 2H, CH2), 7.43–7.53 (m, 4H, ar), 7.79–7.83 (m,
2H, ar), 8.09–8.13 (m, 2H, ar). IR: 1680. Anal. Calcd for (C18H15ClN4-
O) C, 63.81; H, 4.46; N 16.54; Found: C, 63.24; H, 4.15; N, 17.02.
6.1.1.4. 2-(4-Methoxyphenyl)-8-methyl-4,5-dihydro-1,2,4-triaz-
olo[1,5-a]quinoxaline (35).
Yield: 35% (impure); 1H NMR
6.1.3.2. Ethyl 8-chloro-2-phenyl-1,2,4-triazolo[1,5-a]quinoxa-
(DMSO-d6) d: 2.27 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 4.67 (s, 2H,
CH2), 6.32 (s, 1H, NH), 6.75 (d, 1H, ar, J = 8.19), 6.89 (d, 1H, ar,
J = 8.17 Hz), 7.06 (d, 2H, ar, J = 8.53), 7.43 (s, 1H, ar), 8.01 (d, 2H,
lin-5(4H)-carboxylate (3).
Eluting system: chloroform/meth-
anol 9.9:0.1. Yield: 48%; mp 177–179 °C (ethanol). 1H NMR (DMSO-
d6) d: 1.26 (t, 3H, CH3, J = 6.96), 4.22 (q, 2H, CH2, J = 6.96), 5.21 (s,

D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
17
2H, CH2), 7.42–7.58 (m, 4H, ar), 7.80–7.84 (m, 2H, ar), 8.07–8.13
(m, 2H, ar). IR: 1715. Anal. Calcd for (C18H15ClN4O2): C, 60.94; H,
4.26; N 15.79; Found: C, 61.78; H, 4.18; N, 16.06.
6.1.3.10. 1-[2-(4-Methoxyphenyl)-8-methyl-1,2,4-triazolo[1,5-
a]quinoxalin-5(4H)-yl]-propan-1-one (11). Eluting system:
cyclohexane/ethyl acetate/methanol 9:2:1. Yield: 21%; mp 132–
133 °C (cyclohexane); 1H NMR (DMSO-d6) d: 1.00 (t, 3H, CH3,
J = 6.64), 2.44–2.53 (m, 5H, CH2+CH3), 3.83 (s, 3H, OCH3), 5.20 (s,
2H, CH2N), 7.08 (d, 2H, ar, J = 8.52), 7.20 (d, 1H, ar, J = 8.32), 7.63
(d, 1H, ar, J = 7.60), 7.68 (s, 1H, ar), 8.03 (d, 2H, ar, J = 8.52). Anal.
Calcd for (C20H20N4O2): C, 68.95; H, 5.79; N 16.08; Found: C,
68.13; H, 5.99; N, 15.83.
6.1.3.3. 1-(2-Phenyl-1,2,4-triazolo[1,5-a]quinoxalin-5(4H)-yl)-
propan-1-one (4).
Eluting system: cyclohexane/ethyl ace-
tate/methanol 6:3.5:0.5. Yield: 28%; mp 126–128 °C (ethanol). 1H
NMR (DMSO-d6) d: 1.06 (t, 3H, CH3, J = 7.29), 2.58 (q, 2H, CH2,
J = 7.29), 5.25 (s, 2H, CH2), 7.40–7.53 (m, 5H, ar), 7.77 (d, 1H, ar,
J = 6.73), 7.87 (d, 1H, ar, J = 7.40), 8.11 (d, 2H, ar, J = 6.73). IR:
1680. Anal. Calcd for (C18H16N4O): C, 71.04; H, 5.30; N 18.41;
Found: C, 71.37; H, 4.83; N, 18.07.
6.1.3.11. Ethyl 2-(4-methoxyphenyl)-8-methyl-1,2,4-triazol-
o[1,5-a]quinoxalin-5(4H)-carboxylate (12).
Eluting system:
dichloromethane/cyclohexane/ethylacetate 9:0.5:0.5. Yield: 21%;
mp 141–142 °C (cyclohexane); 1H NMR (DMSO-d6) d: 1.26 (t, 3H,
CH3, J = 7.08), 2.42 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 4.19 (q, 2H,
CH2, J = 7.08), 5.17 (s, 2H, CH2), 7.08 (d, 2H, ar, J = 8.76), 7.19 (d,
1H, ar, J = 8.40), 7.65–7.67 (m, 2H, ar), 8.03 (d, 2H, ar, J = 8.76).
IR: 1708. Anal. Calcd for (C20H20N4O3): C, 65.92; H, 5.53; N
15.38; Found: C, 66.66; H, 5.78; N, 14.87.
6.1.3.4. Methyl 2-phenyl-1,2,4-triazolo[1,5-a]quinoxalin-5(4H)-
carboxylate (5).
Eluting system: cyclohexane/ethyl acetate/
methanol 8:3:0.15. Yield: 21%; mp 149–151 °C (methanol). 1H
NMR (DMSO-d6) d: 3.77 (s, 3H, OCH3), 5.22 (s, 2H, CH2), 7.40–
7.47 (m, 2H, ar), 7.49–7.54 (m, 3H, ar), 7.79 (d, 1H, ar, J = 8.51),
7.86 (d, 1H, ar, J = 6.73), 8.11 (d, 2H, ar, J = 6.73). IR: 1700. Anal.
Calcd for (C17H14N4O2): C, 66.66; H, 4.61; N 18.29; Found: C,
65.98; H, 4.33; N, 18.71.
6.1.3.12. Propyn-2-yl 2-(4-methoxyphenyl)-8-methyl-1,2,4-
triazolo[1,5-a]quinoxalin-5(4H)-carboxylate (13).
Eluting
6.1.3.5. Ethyl 2-phenyl-1,2,4-triazolo[1,5-a]quinoxalin-5(4H)-
system: cyclohexane/ethyl acetate 6:4. Yield 21%. mp 134–135 °C
(cyclohexane); 1H NMR (DMSO-d6) d: 2.42 (s, 3H, CH3), 3.63 (s,
1H, CCH), 3.84 (s, 3H, OCH3), 4.84 (s, 2H, OCH2), 5.18 (s, 2H,
NCH2), 7.09 (d, 2H, ar, J = 8.40), 7.21 (d, 1H, ar, J = 7.68), 7.62 (d,
1H, ar, J = 7.68), 7.68 (s, 1H, ar), 8.04 (d, 2H, ar, J = 8.48). IR: 3260,
1721. Anal. Calcd for (C21H18N4O3): C, 67.37; H, 4.85; N 14.96;
Found: C, 67.97; H, 4.05; N, 15.13.
carboxylate (6).
Eluting system: dichloromethane/methanol
9.8:0.2. Yield: 38%; mp 116–118 °C (methanol). 1H NMR (DMSO-
d6) d: 1.27 (t, 3H, CH3, J = 6.96), 4.23 (q, 2H, CH2, J = 6.96), 5.22 (s,
2H, CH2), 7.40–7.47 (m, 2H, ar), 7.49–7.54 (m, 3H, ar), 7.80 (d,
1H, ar, J = 6.73), 7.85 (d, 1H, ar, J = 4.94), 8.11 (d, 2H, ar, J = 6.73).
IR 1700. Anal. Calcd for (C18H16N4O2): C, 67.49; H, 5.03; N 17.49;
Found: C, 68.93; H, 4.71; N, 17.94.
6.1.3.13.
2-Phenyl-1-[2-(4-methoxyphenyl)-8-methyl-1,2,4-
Eluting
6.1.3.6. 1-(8-Methyl-2-phenyl-1,2,4-triazolo[1,5-a]quinoxalin-
triazolo[1,5-a]quinoxalin-5(4H)-yl)-ethanone (14).
5(4H)-yl)-propan-1-one (7).
Eluting system: chloroform/ace-
tone 9:1. Yield 31%; mp 150–152 °C (cyclohexane). 1H NMR
(DMSO-d6) d: 0.98 (t, 3H, CH3, J = 6.96), 2.43–2.57 (m, 5H, CH2+-
CH3), 5.21 (s, 2H, CH2), 7.20 (d, 1H, ar, J = 8.06), 7.49–7.52 (m, 3H,
ar), 7.61–7.690(m, 2H, ar), 8.08–8.12 (m, 2H, ar). IR: 1670. Anal.
Calcd for (C19H18N4O): C, 71.68; H, 5.70; N 17.60; Found: C,
70.85; H, 5.51; N, 18.10.
system: dichloromethane/acetone 9:1. Yield: 22%; mp 188–
190 °C (cyclohexane); 1H NMR (DMSO-d6) d: 2.44 (s, 3H, CH3),
3.84 (s, 3H, OCH3), 3.96 (s, 2H, COCH2), 5.22 (s, 2H, CH2N),
7.08 (d, 2H, ar, J = 7.12), 7.10–7.23 (m, 6H, ar), 7.66–7.70 (m,
2H, ar), 8.03 (d, 2H, ar, J = 7.12). IR: 1662. Anal. Calcd for
(C25H22N4O2): C, 73.15; H, 5.40; N 13.65; Found: C, 72.54; H,
5.63; N, 14.15.
6.1.3.7. Methyl 8-methyl-2-phenyl-1,2,4-triazolo[1,5-a]quinox-
6.1.3.14. Benzyl 2-(4-methoxyphenyl)-8-methyl-1,2,4-triazol-
alin-5(4H)-carboxylate (8).
Eluting system: dichlorometh-
ane/methanol 9.8:0.2. Yield: 12%; mp 132–134 °C (methanol). 1H
NMR (DMSO-d6) d: 2.40 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 5.17 (s,
2H, CH2), 7.18 (d, 1H, ar, J = 8.06), 7.49–7.52 (m, 3H, ar), 7.61–7.66
(m, 2H, ar), 8.07–8.11 (m, 2H, ar). IR: 1720. Anal. Calcd for
(C18H16N4O2): C, 67.49; H, 5.03; N 17.49; Found: C, 68.15; H, 4.67;
N, 17.95.
o[1,5-a]quinoxalin-5(4H)-carboxylate (15).
Eluting system:
cyclohexane/ethyl acetate 6:4. Yield: 31%, mp 119–120 °C (cyclo-
hexane); 1H NMR (DMSO-d6) d: 2.40 (s, 3H, CH3), 3.83 (s, 3H,
OCH3), 5.19 (s, 2H, OCH2), 5.23 (s, 2H, NCH2), 7.10 (d, 2H, ar,
J = 8.61), 7.17 (d, 1H, ar, J = 8.24), 7.35–7.45 (m, 5H, ar), 7.66 (m,
2H, ar), 8.03 (d, 2H, ar, J = 8.60). IR: 1722. Anal. Calcd for
(C25H22N4O3): C, 70.41; H, 5.20; N 13.14; Found: C, 70.59; H,
4.66; N, 13.91.
6.1.3.8. Ethyl 8-methyl-2-phenyl-1,2,4-triazolo[1,5-a]quinoxa-
lin-5(4H)-carboxylate (9).
Eluting system: dichlorometh-
6.1.4. 3-(4-Methoxyphenyl)-1-(5-methyl-2-nitrophenyl)-1,2,4-
triazole-5-chloromethyl (37)
ane/acetone/cyclohexane 8.8:0.7:0.5. Yield: 56%; mp 176–178 °C
(ethyl acetate). 1H NMR (DMSO-d6) d: 1.25 (t, 3H, CH3, J = 6.96),
2.41 (s, 3H, CH3), 4.19 (q, 2H, CH2, J = 6.96), 5.18 (s, 2H, CH2N),
7.19 (d, 1H, ar, J = 8.79), 7.50–7.53 (m, 3H, ar), 7.63–7.68 (m, 2H,
ar), 8.08–8.12 (m, 2H, ar). IR: 1715. Anal. Calcd for (C19H18N4O2):
C, 68.25; H, 5.43; N 16.76; Found: C, 68.77; H, 4.66; N, 16.12.
To a solution of chloroacetyl chloride (3.4 mmol) in anhy-
drous toluene (15 mL) at 80 °C, the amidrazone 3618 (1.7 mmol)
was added portion by portion. The reaction mixture was heated
at reflux for 3 h. Then, the solvent was removed under reduced
pressure and the oily residue was worked up with a mixture
of ethyl acetate/petroleum ether 1:1. The solid which separated
was collected by filtration and washed with petroleum ether.
Yield: 72%; mp 127–128 °C (ethanol); 1H NMR (DMSO-d6) d:
2.51 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 4.95 (s, 2H, CH2), 7.07 (d,
2H, ar, J = 8.88), 7.72 (d, 1H, ar, J = 8.32), 7.81(s, 1H, ar), 7.93
(d, 2H, ar, J = 8.88), 8.23 (d, 1H, ar, J = 8.40). Anal. Calcd for
(C17H15Cl N4O3): C, 56.91; H, 4.21; N 15.66; Found: C, 57.27;
H, 4.10; N, 16.09.
6.1.3.9. n-Propyl 8-methyl-2-phenyl-1,2,4-triazolo[1,5-a]qui-
noxalin-5(4H)-carboxylate (10).
Eluting system: chloroform.
Yield: 70%; mp 117–119 °C (ethanol). 1H NMR (DMSO-d6) d: 0.90 (t,
3H, CH3, J = 7.41), 1.58–1.69 (m, 2H, CH2), 2.40 (s, 3H, CH3), 4.10 (t,
2H, CH2O, J = 6.23), 5.17 (s, 2H, CH2N), 7.18 (d, 1H, ar, J = 8.42),
7.49–7.58 (m, 3H, ar), 7.62–7.66 (m, 2H, ar), 8.08–8.10 (m, 2H, ar).
IR: 3070, 1725. Anal. Calcd for (C20H20N4O2): C, 68.95; H, 5.79; N
16.08; Found: C, 69.56; H, 5.47; N, 16.27.

18
D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
6.1.5. 2-(4-Methoxyphenyl)-8-methyl-4,5-dihydro-1,2,4-
triazolo[1,5-a]quinoxaline (35)
9.13 (s, 1H, NH). IR: 1726. Anal. Calcd for. (C19H20N4O3): C, 64.76;
H, 5.72; N 15.90; Found: C, 64.66; H, 5.01; N, 16.18.
To a solution of the 5-chloromethyl-1,2,4-triazole derivative 37
(1.59 mmol) in ethanol (75 mL), an excess of SnCl2 dihydrate
(4.77 mmol) was added under nitrogen atmosphere. Then, the
reaction mixture was heated at reflux, under nitrogen atmosphere,
for 40 h. After evaporation of the solvent at reduced pressure, the
resulting solid was worked up with diethyl ether, collected by fil-
tration and washed with a large amount of water. The crude prod-
uct, containing a small amount of the dehydro-derivative 27 (27/
35, ratio 1:10, 1H NMR evaluation) was used as it is for the next
step. Yield: 83%.
6.1.8.2. Propyn-2-yl 2-[3-(4-methoxyphenyl)-1H-1,2,4-triazol-1-
yl]-4-methylphenylcarbamate (17).
Yield: 54%; mp 132–
133 °C (cyclohexane); 1H NMR (DMSO-d6) d: 2.37 (s, 3H, CH3),
3.53 (s, 1H, CH), 3.83 (s, 3H, OCH3), 4.68 (s, 2H, CH2), 7.07 (d, 2H,
ar, J = 8.88), 7.32 (d, 1H, ar, J = 8.28), 7.46 (s, 1H, ar), 7.59 (d, 1H,
ar, J = 8.04), 8.01 (d, 2H, ar, J = 8.90), 8.84 (s, 1H, CH), 9.37 (s, 1H,
NH). IR: 1720. Anal. Calcd for (C20H18N4O3): C, 66.29; H, 5.01; N
15.46; Found: C, 66.76; H, 4.55; N, 14.93.
6.1.8.3.
N-[2-(3-(4-methoxyphenyl)-1H-1,2,4-triazol-1-yl)-4-
Yield: 76%; mp
6.1.6. 3-(4-Methoxyphenyl)-1-(5-methyl-2-nitrophenyl)-1,2,4-
triazole (38)
methylphenyl]-2-phenylacetamide (18).
138–139 °C (ethanol); 1H NMR (DMSO-d6) d: 2.36 (s, 3H, CH3),
3.59 (s, 2H, CH2), 3.84 (s, 3H, OCH3), 7.09 (d, 2H, ar, J = 8.80), 7.19
(s, 5H, ar), 7.30 (d, 1H, ar, J = 8.44), 7.43 (s, 1H, ar), 7.71 (d, 1H,
ar, J = 8.32), 8.01 (d, 2H, ar, J = 8.80), 8.50 (s, 1H, CH), 9.72 (s, 1H,
NH). IR: 3247, 3096, 1688. Anal. Calcd for (C24H22N4O2): C, 72.34;
H, 5.57; N 14.06; Found: C, 73.05; H, 5.74; N, 14.54.
To a suspension of the amidrazone 3618 (1.0 mmol) in ethyl
orthoformate (1.25 mL), p-toluenesulfonic acid (10 mg) was added.
The reaction mixture was heated at 100 °C for 30 min. Upon cool-
ing, a orange solid precipitated which was collected by filtration
and washed with diethyl ether. Yield 54%; mp 138–139 °C (etha-
nol); 1H NMR (DMSO-d6) d: 2.50 (s, 3H, CH3), 3.82 (s, 3H, OCH3),
7.06 (d, 2H, ar, J = 8.76), 7.60 (d, 1H, ar, J = 8.36), 7.80 (s, 1H, ar),
7.94 (d, 2H, ar, J = 8.70), 8.09 (d, 1H, ar, J = 8.36), 9.09 (s, 1H, CH).
Anal. Calcd for (C16H14N4O3): C, 61.93; H, 4.55; N 18.06; Found:
C, 61.13; H, 4.89; N, 18.72.
6.1.8.4. Benzyl 2-[3-(4-methoxyphenyl)-1H-1,2,4-triazol-1-yl]-4-
methylphenylcarbamate (19).
Yield: 34%; mp 149–150 °C
(ethanol); 1H NMR (DMSO-d6) d: 2.37 (s, 3H, CH3), 3.83 (s, 3H,
OCH3), 5.06 (s, 2H, CH2), 7.06 (d, 2H, ar, J = 8.68), 7.31 (s, 6H, ar),
7.46 (s, 1H, ar), 7.61 (d, 1H, ar, J = 8.08), 8.00 (d, 2H, ar, J = 8.61),
8.85 (s, 1H, CH), 9.34 (s, 1H, NH). IR: 3227, 3109, 1728. Anal. Calcd
for (C24H22N4O3): C, 69.55; H, 5.35; N 13.52; Found: C, 68.88; H,
4.95; N, 14.18.
6.1.7. 2-[3-(4-Methoxyphenyl)-1,2,4-triazol-1-yl]-4-methyl-
phenylamine (39)
To a solution of the 2-nitrophenyl-1,2,4-triazole derivative 38
(2.0 mmol) in ethyl acetate (50 mL), the catalyst (10% Pd/C,
50 mg) was added. Hydrogenation of the resulting mixture was
performed at 30 Psi until disappearance of the starting material
(TLC monitoring, eluting system cyclohexane/ethyl acetate 6:4).
The catalyst was removed by filtration and the solvent was dis-
tilled under reduced pressure to yield a solid. Yield 69%; mp
131–132 °C (ethanol); 1H NMR (DMSO-d6) d: 2.22 (s, 3H, CH3),
3.82 (s, 3H, OCH3), 5.31 (s, 2H, NH2), 6.84 (d, 1H, ar, J = 8.24),
7.02 (d, 1H, ar, J = 8.20), 7.06 (d, 2H, ar, J = 8.80), 7.14 (s, 1H, ar),
8.02 (d, 2H, ar, J = 8.76), 8.83 (s, 1H, CH). IR: 3448, 3348 Anal. Calcd
for (C16H16N4O): C, 68.55; H, 5.75; N 19.99; Found: C, 67.71; H,
5.05; N, 20.17.
6.1.9. N1-Phenyl-N2-(4-methoxybenzoyl)-hydrazide (40)27,28
To a mixture of phenylhydrazine (9.25 mmol) in anhydrous
pyridine (10 mL) a solution of equimolar amount of p-anisoyl
chloride in anhydrous pyridine (5 mL) was drop by drop added.
The reaction mixture was heated at reflux for 5 h. The excess
of pyridine was removed by distillation under reduced pressure.
The residue was treated with HCl 2 M solution (30 mL) and the
resulting solid was collected by filtration and well washed with
water. Yield: 75%; mp 165–168 °C (ethanol) (lit. Mp 177–
178 °C).28
6.1.10. N1-Phenyl-N2-[
hydrazine (41)29
a-chloro-(4-methoxybenzyliden)]
6.1.8. General procedure for the synthesis of 2-[3-(4-
methoxyphenyl)-1,2,4-triazol-1-yl]-4-methyl-
A
suspension of equimolar amount of hydrazide 4027,28
phenylcarbamates (16–19)
(4.13 mmol) and PCl5 in POCl3 (7 mL) was heated at reflux for
4 h. Then, another portion (4.13 mmol) of PCl5 was added and
the heating continued for 2 h. Evaporation at reduced pressure of
the excess of POCl3 gave an oily residue which was treated with
cold water (50 mL) and quickly collected. The compound was used
as it is without further purification. Yield: 90%; mp 117–121 °C (lit.
mp 119–120 °C). 1H NMR (CDCl3) d: 3.89 (s, 3H, OCH3), 6.98 (d, 2H,
ar, J = 8.60), 7.33–7.35 (m, 1H, ar), 7.38–7.46 (m, 4H, ar), 8.06 (d,
2H, ar, J = 8.61).
A solution of the suitable chloroformates (compounds 16–17,
19) (3.2 mmol) or phenacetyl chloride (18) (3.2 mmol) in anhy-
drous dichloromethane (3.2 mL) was drop by drop added to a solu-
tion of the triazole derivative 39 (1.07 mmol) in anhydrous
dichloromethane (32 mL) and anhydrous pyridine (0.05 mL) at
0 °C. The reaction mixture was kept at 0 °C for 2 h. Then, the sol-
vent was removed under reduced pressure and the solid was
worked up with water (20 mL), collected and washed with water
(compounds 16–17, 19). Otherwise (compound 18), the solid was
worked up with 10% aqueous solution of NaHCO3 (20 mL), and
the resulting mixture extracted with ethyl acetate (15 mL ꢃ 3).
The organic layers were washed with water (20 mL), anhydrified
(Na2SO4) and evaporated under reduced pressure to give a yellow
solid.
6.1.11. 3-(4-Methoxyphenyl)-1-phenyl-1H-1,2,4-triazol-5-amine
(42)
A mixture of the chloro-derivative 4129 (4.12 mmol) and an
excess of cyanamide (20.6 mmol) was heated at 100 °C for
20 min. The crude mass was worked up with ethyl acetate
(20 mL) and the resulting solid was filtered off and well washed
with ethyl acetate. The mother liquors were evaporated under
reduced pressure to yield a solid which was purified by silica gel
column chromatography, eluting system: dichloromethane/meth-
anol 9:1, and then dichloromethane/ethyl acetate 8:2. Yield: 45%;
mp 152–155 °C (toluene). 1H NMR (DMSO-d6) d: 3.80 (s, 3H,
OCH3), 6.51 (br s, 2H, NH2), 7.01 (d, 2H, ar, J = 8.68), 7.39 (t, 1H,
6.1.8.1. Ethyl 2-[3-(4-methoxyphenyl)-1H-1,2,4-triazol-1-yl]-4-
methylphenylcarbamate (16).
Yield: 85%; mp 117–118 °C
(cyclohexane); 1H NMR (DMSO-d6) d: 1.12 (t, 3H, CH3, J = 7.08),
2.37 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 4.03 (q, 2H, CH2, J = 7.08),
7.07 (d, 2H, ar, J = 8.88), 7.30 (d, 1H, ar, J = 8.41), 7.45 (s, 1H, ar),
7.58 (d, 1H, ar, J = 8.16), 8.00 (d, 2H, ar, J = 8.84), 8.84 (s, 1H, CH),

D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
19
ar, J = 7.28), 7.54 (t, 2H, ar, J = 7.64), 7.62 (d, 2H, ar, J = 7.88), 7.88 (d,
6.2. Computational methodologies
2H, ar, J = 8.60). IR: 3303. Anal. Calcd for (C15H14N4O): C, 67.65; H,
5.30; N 21.04; Found: C, 67.93; H, 4.87; N, 21.27.
All molecular modeling studies were performed on a 2 CPU (PIV
2.0–3.0 GHZ) Linux PC. Homology modeling, energy minimization,
and docking studies were carried out using Molecular Operating
Environment (MOE, version 2010.10) suite.35 Manual docking
and Monte Carlo studies of the MRS 1220 binding mode were done
using MOE and Schrodinger Macromodel (ver. 8.0)42 with Schro-
dinger Maestro interface. Compounds docking analyses were then
performed with MOE. All ligand structures were optimized using
RHF/AM1 semiempirical calculations and the software package
MOPAC implemented in MOE was utilized for these calculations.37
6.1.12. N-Acetyl-N-[3-(4-methoxyphenyl)-1-phenyl-1H-1,2,4-
triazol-5-yl]acetamide (20)
A mixture of the triazole 42 (0.75 mmol) and acetic anhydride
(2.25 mmol) in anhydrous pyridine (3 mL) was heated at reflux
for 8 h. The crude mass was worked up with water and the result-
ing solid collected by filtration ad purified by silica gel column
chromatography, eluting system dichloromethane/ethyl acetate
8:2. Yield: 38%; mp 130–132 °C (cyclohexane/ethyl acetate 8:2);
1H NMR (DMSO-d6) d: 2.29 (2, 6H, 2CH3), 3.83 (s, 3H, OCH3), 7.09
(d, 2H, ar, J = 8.64), 7.54–7.60 (m, 5H, ar), 8.01 (d, 2H, ar,
J = 8.64). IR: 1749, 1715. Anal. Calcd for (C19H18N4O3): C, 65.13;
H, 5.18; N 15.99; Found: C, 64.66; H, 5.45; N, 16.35.
6.2.1. Homology modeling of the human A3AR
Homology models of the hA3AR were built using recently solved
X-ray structures of the antagonist-bound hA2A AR as templates
(pdb code: 3EML; 2.6-Å resolution;30 pdb code: 3PWH; 3.3-Å res-
olution;31 pdb code: 3REY; 3.3-Å resolution;31 pdb code: 3UZA;
3.3-Å resolution32). A multiple alignment of the AR primary
sequences was built within MOE as preliminary step. For all hA3AR
models, the boundaries identified from the used X-ray crystal
structure of hA2A AR were then applied for the corresponding
sequences of the TM helices of the hA3AR. The missing loop
domains were built by the loop search method implemented in
MOE. Once the heavy atoms were modeled, all hydrogen atoms
were added, and the protein coordinates were then minimized
with MOE using the AMBER99 force field.43 The minimizations
were performed by 1000 steps of steepest descent followed by con-
jugate gradient minimization until the RMS gradient of the poten-
tial energy was less than 0.05 kJ molꢂ1 Åꢂ1. The reliability and
quality of these models were checked using the Protein Geometry
Monitor application within MOE, which provides a variety of ste-
reochemical measurements for inspection of the structural quality
in a given protein, like backbone bond lengths, angles and dihe-
6.1.13. General procedure for the synthesis of N-[3-(4-
methoxyphenyl)1-phenyl-1H-1,2,4-triazol-5-yl]benzamide (21)
and N-benzoyl-N-[3-(4-methoxyphenyl)-1-phenyl-1H-1,2,4-
triazol-5-yl]benzamide (22)
A solution of a little excess of benzoyl chloride (0.54 mmol) in
anhydrous tetrahydrofuran (2 mL) was drop by drop added to a
solution of the 1,2,4-triazol-5-amino derivative 42 (0.45 mmol) in
anhydrous tetrahydrofuran (8 mL) and anhydrous pyridine
(3 mL). The reaction mixture was heated at reflux for 25 h. After
10 h, another portion (0.5 mmol) of benzoyl chloride was added.
After cooling, water (30 mL) and ice (20 g) were added and the
resulting solution was extracted with ethyl acetate (30 mL ꢃ 4).
The organic layers were washed with water (30 mL ꢃ 4), with a
saturated solution of NaHCO3 (30 mL), and again with water
(40 mL), then anhydrified (Na2SO4) and evaporated under reduced
pressure. The resulting oily residue was purified by silica gel col-
umn chromatography, eluting system dichloromethane/ethyl ace-
tate 1:1.
drals, Ramachandran
u–w dihedral plots, and sidechain rotamer
and non-bonded contact quality.
6.1.13.1. Compound (21).
Yield: 20%; mp 156–158 °C
(diethyl ether/petroleum ether); 1H NMR (DMSO-d6) d: 3.84 (s,
3H, OCH3), 7.08 (d, 2H, ar, J = 8.44), 7.42 (t, 1H, ar, J = 6.88), 7.50–
7.56 (m, 4H, ar), 7.65 (d, 3H, ar, J = 7.16), 7.91 (d, 2H, ar, J = 7.40),
8.01 (d, 2H, ar, J = 8.36), 11.17 (s, br, 1H, NH). IR: 3194, 1667. Anal.
Calcd for (C22H18N4O2): C, 71.34; H, 4.90; N 15.13; Found: C, 70.67;
H, 5.30; N, 14.59.
6.2.2. Preliminary docking analysis with MRS 1220
A preliminary docking analysis was performed by manually
docking MRS 1220 structure within each hA3AR model binding
site. The obtained hA3AR–MRS 1220 complexes were then sub-
jected to energy minimization refinement and to Monte Carlo anal-
ysis to explore the favorable binding conformations. This analysis
was conducted by Monte Carlo Conformational Search protocol
implemented in Schrodinger Macromodel. The input structure con-
sisted of the ligand and a shell of receptor amino acids within the
specified distance (6 Å) from the ligand. A second external shell of
all the residues within a distance of 8 Å from the first shell was
kept fixed. During the Monte Carlo conformational searching, the
input structure was modified by random changes in user-specified
torsion angles (for all input structure residues), and molecular
position (for the ligand). Hence, the ligand was left free to be con-
tinuously re-oriented within the binding site and the conformation
of both ligand and internal shell residues could be explored and
reciprocally relaxed. The method consisted of 10,000 Conforma-
tional Search steps with MMFF94s force field.44–50 For each
6.1.13.2. Compound (22).
Yield: 60%; mp 154–156 °C (etha-
nol); 1H NMR (DMSO-d6) d: 3.82 (s, 3H, OCH3), 7.05 (d, 2H, ar,
J = 6.88), 7.41–7.45 (m, 6H, ar), 7.50–7.55 (m, 3H, ar), 7.59 (t, 2H,
ar, J = 7.48), 7.63 (d, 4H, ar, J = 7.12), 7.93 (d, 2H, ar, J = 4.88). IR:
1708. Anal. Calcd for. (C29H22N4O3): C, 73.40; H, 4.67; N 11.81;
Found: C, 73.01; H, 5.10; N, 11.12.
6.1.14. 1-[3-(4-Methoxyphenyl)-1-phenyl-1H-1,2,4-triazol-5-yl]-
3-phenylurea (23)
A
solution of phenylisocyanate (0.26 mmol) in anhydrous
dichloromethane (4 mL) was drop by drop added to an equimolar
amount of the 5-aminotriazole 42 in anhydrous dichloromethane
(4 mL) maintained at 5 °C. The reaction mixture was then kept at
room temperature for 20 days. The solvent was removed until
small volume and the resulting solid was collected. Yield 42%;
mp 202–204 °C (2-methoxyethanol); 1H NMR (DMSO-d6) d: 3.83
(s, 3H, OCH3), 7.02 (t, 1H, ar, J = 7.20), 7.07 (d, 2H, ar, J = 8.44),
7.30 (t, 2H, ar, J = 7.25), 7.44–7.49 (m, 3H, ar), 7.57 (t, 2H, ar,
J = 7.44), 7.66 (d, 2H, ar, J = 7.84), 7.99 (d, 2H, ar, J = 8.32), 9.43 (s,
1H, NH), 9.75 (s, 1H, NH). IR: 3182, 3145, 1685. Anal. Calcd for
(C22H19N5O2): C, 68.56; H, 4.97; N 18.17; Found: C, 69.07; H,
5.31; N, 18.44.
A2AAR-based model, the best hA3AR–MRS 1220 complex was
saved. The four final complexes served as input in MOE and were
subjected to energy minimization with the same protocol as above.
This protocol was recently used to prepare hA3AR models for dock-
ing and dynamics studies of nucleoside agonists at the same
receptor.41,51
6.2.3. Molecular docking analysis
All compound structures were docked into the binding site of
the four hA3AR models using the MOE Dock tool. This method is

20
D. Catarzi et al. / Bioorg. Med. Chem. 23 (2015) 9–21
divided into a number of stages: Conformational Analysis of ligands.
The algorithm generated conformations from a single 3D confor-
mation by conducting a systematic search. In this way, all combi-
nations of angles were created for each ligand. Placement. A
collection of poses was generated from the pool of ligand confor-
mations using Triangle Matcher placement method. Poses were
generated by superposition of ligand atom triplets and triplet
points in the receptor binding site. The receptor site points are
alpha sphere centers which represent locations of tight packing.
At each iteration a random conformation was selected, a random
triplet of ligand atoms and a random triplet of alpha sphere centers
were used to determine the pose. Scoring. Poses generated by the
placement methodology were scored using two available methods
implemented in MOE, the London dG scoring function which esti-
mates the free energy of binding of the ligand from a given pose,
and Affinity dG scoring which estimates the enthalpic contribution
to the free energy of binding. The top 30 poses for each ligand were
output in a MOE database.
reaction medium (15 min at 37 °C) with the target compounds
(10 M) and then were treated with the agonist.
l
Following incubation, the reaction was terminated by the
removal of the medium and the addition of 0.4 N HCl. After
30 min, lysates were neutralized with 4 N KOH, and the suspension
was centrifuged at 800 g for 5 min. For the determination of cAMP
production, bovine adrenal cAMP binding protein was incubated
with [3H]cAMP (2 nM) and 50
lL of cell lysate or cAMP standard
(0–160 pmol) at 0 °C for 150 min in a total volume of 300 L.
l
Bound radioactivity was separated by rapid filtration through GF/
C glass fiber filters and washed twice with 4 mL 50 mM Tris–HCl,
pH 7.4. The radioactivity was measured by liquid scintillation
spectrometry.
6.3.3. Data analysis
The concentration of the tested compounds that produced 50%
inhibition of specific [3H]DPCPX, [3H]NECA,
[
125I]AB-MECA,
[3H]CHA and [3H]CGS 21680 binding (IC50) was calculated using
a non-linear regression method implemented by the InPlot pro-
gram (Graph-Pad, San Diego, CA, U.S.A.) with five concentrations
of displacer, each performed in triplicate. Inhibition constants
(Ki) were calculated according to the Cheng–Prusoff equation.55
The Kd values of [3H]DPCPX, [3H]NECA and [125I]AB-MECA in hA1,
hA2A and hA3 ARs in CHO cell membranes were 3 nM, 30 nM and
1.4 nM, respectively. The dissociation constant (Kd) of [3H]CHA
and [3H]CGS 21680 in cortical and striatal bovine brain membranes
were 1.2 and 14 nM, respectively.
6.2.4. Post docking analysis
The five top-score docking poses of each compound were then
subjected to AMBER99 force field energy minimization until the
RMS gradient of the potential energy was less than 0.05 kJ molꢂ1 Å-
ꢂ1. Receptor residues within 6 Å distance from the ligand were left
free to move, while the remaining receptor coordinates were kept
fixed. AMBER99 partial charges of receptor and MOPAC output par-
tial charges of ligands were utilized. Once the compound-binding
site energy minimization was completed, receptor coordinates
were fixed and a second energy minimization stage was performed
leaving only compound atoms free to move. MMFF94 force field
was applied. For each compound, the minimized docking poses
were then rescored using London dG and Affinity dG scoring func-
tions and the dock-pKi predictor. The latter tool estimates the pKi
for each ligand using the ‘scoring.svl’ script retrievable at the SVL
exchange service (Chemical Computing Group, Inc. SVL exchange:
http://svl.chemcomp.com). The algorithm is based on an empirical
scoring function consisting of a directional hydrogen-bonding
term, a directional hydrophobic interaction term, and an entropic
term (ligand rotatable bonds immobilized in binding). The four
top-score docking poses according to at least two out of three scor-
ing functions were selected for final ligand-target interaction anal-
ysis for each compound.
Acknowledgment
This work was supported by a Grant from the Italian Ministry
for University and Research (MIUR, FIRB RBNE03YA3L project).
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