Synthesis and pharmacological characterization of a new series of 5,7-disubstituted-[1,2,4]triazolo[1,5-a][1,3,5]triazine derivatives as adenosine receptor antagonists: A preliminary inspection of ligand-receptor recognition process

Article abstract of DOI:10.1016/j.bmc.2010.02.039

A new series of triazolotriazines variously substituted at the C5 and N7 (5-25) positions was synthesized and fully characterized at the four adenosine receptor (AR) subtypes. In particular, arylacetyl or arylcarbamoyl moieties were introduced at the N7 position, which enhanced affinity at the hA_2B and hA₃ ARs, respectively, when utilized on the pyrazolo-triazolopyrimidine nucleus as we reported in the past. In general, compounds with a free amino group at the 7 position (5, 6), showed good affinity at the rat (r) A_2A AR (range 18.3-96.5 nM), while the introduction of a phenylcarbamoyl moiety at the N7 position (12, 19, 24) slightly increased the affinity at the hA₃ AR (range 311-633 nM) with respect to the unsubstituted derivatives. The binding profiles of the synthesized analogues seemed to correlate with the substitutions at the C5 and N7 positions. At the hA_2B AR, derivative 5, which contained a free amino group at the 7 position, was the most potent (EC₅₀ 3.42 μM) and could represent a starting point for searching new non-xanthine hA_2B AR antagonists. Molecular models of the rA_2A and hA₃ ARs were constructed by homology to the recently reported crystallographic structure of the hA_2A AR. A preliminary receptor-driven structure-activity relationship (SAR) based on the analysis of antagonist docking has been provided.

Full text of DOI:10.1016/j.bmc.2010.02.039

Bioorganic & Medicinal Chemistry 18 (2010) 2524–2536
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological characterization of a new series
of 5,7-disubstituted-[1,2,4]triazolo[1,5-a][1,3,5]triazine derivatives
as adenosine receptor antagonists: A preliminary inspection of ligand–receptor
recognition process
Giorgia Pastorin a, Stephanie Federico b, Silvia Paoletta c, Marta Corradino c, Francesca Cateni b,
Barbara Cacciari d, Karl-Norbert Klotz e, Zhan-Guo Gao f, Kenneth A. Jacobson f, Giampiero Spalluto b,
,
*
Stefano Moro c,
*
a Department of Pharmacy, National University of Singapore, Block S4, 18 Science Drive 4, Singapore
b Dipartimento di Scienze Farmaceutiche, Università degli Studi di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy
c Molecular Modeling Section (MMS), Dipartimento di Scienze Farmaceutiche, Università di Padova, Via Marzolo 5, I-35131 Padova, Italy
d Dipartimento di Scienze Farmaceutiche, Università degli Studi di Ferrara, Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy
e Institut für Pharmakologie und Toxikologie, Universität Würzburg, Versbacher Str. 9, D-97078 Würzburg, Germany
f Molecular Recognition Section (MRS), Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of triazolotriazines variously substituted at the C5 and N7 (5–25) positions was synthesized
and fully characterized at the four adenosine receptor (AR) subtypes. In particular, arylacetyl or arylcar-
bamoyl moieties were introduced at the N7 position, which enhanced affinity at the hA2B and hA3 ARs,
respectively, when utilized on the pyrazolo-triazolopyrimidine nucleus as we reported in the past. In gen-
eral, compounds with a free amino group at the 7 position (5, 6), showed good affinity at the rat (r) A2A AR
(range 18.3–96.5 nM), while the introduction of a phenylcarbamoyl moiety at the N7 position (12, 19, 24)
slightly increased the affinity at the hA3 AR (range 311–633 nM) with respect to the unsubstituted deriv-
atives. The binding profiles of the synthesized analogues seemed to correlate with the substitutions at the
C5 and N7 positions. At the hA2B AR, derivative 5, which contained a free amino group at the 7 position,
Received 4 December 2009
Revised 17 February 2010
Accepted 21 February 2010
Available online 25 February 2010
Keywords:
G Protein-coupled receptors
Adenosine receptor antagonists
Triazolo-triazine
was the most potent (EC50 3.42 lM) and could represent a starting point for searching new non-xanthine
Ligand–receptor modeling studies
hA2B AR antagonists. Molecular models of the rA2A and hA3 ARs were constructed by homology to the
recently reported crystallographic structure of the hA2A AR. A preliminary receptor-driven structure–
activity relationship (SAR) based on the analysis of antagonist docking has been provided.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Activation or blockade of adenosine receptors (ARs) is responsi-
ble for a broad range of effects in various organ systems suggesting
that regulation of ARs has substantial therapeutic potential. Recent
Abbreviations: GPCRs, G protein-coupled receptors; AR, adenosine receptor;
ADA, adenosine deaminase; cAMP, cyclic adenosine monophosphate; h, human;
SAR, structure–affinity relationship; r, rat; DPCPX, 8-cyclopentyl-1,3-dipropyl-
xanthine; CGS 21680, 2-[4-(2-carboxyethyl)phenethyl]amino-50-(N-ethylcarba-
the focus has been increased on the cardioprotective1,2 and neuro-
protective3,4 effects associated with AR activation during periods of
cardiac and cerebral ischemia, respectively. It has also been pro-
posed that antagonists of distinct AR subtypes may be used in
the treatment of asthma5,6 or certain neurological diseases such
as Parkinson’s disease.6,7 A complete summary of the physiological
roles of ARs and their potential as clinical targets in a variety of dis-
ease states have been recently reported.6–11
moyl)adenosine;
I-AB-MECA,
N6-(4-amino-3-iodobenzyl)-50-(N-methylcarba-
moyl)adenosine; NECA, 50-(N-ethyl-carboxamido)adenosine; Cl-IB-MECA, 2-
chloro-N6-(3-iodobenzyl)-50-(N-methylcarbamoyl)adenosine; TM, transmembrane;
RMSD, root mean square deviation; EL2, second extracellular loop; MOE, molecular
operating environment; ZM241385, 4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-
a}{1,3,5,}triazin-5-yl amino]ethyl)phenol.
* Corresponding authors. Tel.: +39 040 6762525; fax: +39 040 52572 (G.S.); tel.:
+39 049 8275704; fax: +39 049 8275366 (S.M.).
E-mail addresses: spalluto@univ.trieste.it (G. Spalluto), stefano.moro@unipd.it
(S. Moro).
All ARs are members of the superfamily of G protein-coupled
receptors (GPCRs), with four subtypes currently recognized, the
A1AR, A2AAR, A2BAR, and A3AR,12 which exert their physiological
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.039

G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
2525
Taking into account these experimental observations, we
decided to better investigate the potential of modifying this nu-
cleus at the C5 and N7 positions (5–25). In particular, arylacetyl
or arylcarbamoyl moieties at the N7 position on the pyrazolo-triaz-
olopyrimidine nucleus enhanced affinity at the A2B and A3 ARs,
respectively (Chart 3).17,21
All the compounds have been fully characterized at the four ARs
with the aim of better understanding the structure–activity rela-
tionship (SAR) profile of this class of compounds and optimizing
the substitution in order to modulate both AR affinity and selectiv-
ity. Moreover, a preliminary receptor-driven SAR based on the re-
cently published crystallographic structure of the hA2A AR has been
provided.
O
N
NH2
N
HN
NH
N
N
N
N
N
7
O
N
N
O
N
N
N8
2
SCH 58261, 1
rA1 = 128 nM
hA1 = 594 nM
hA2A = 381 nM
hA2B = 222 nM
hA3 = 0.16 nM
rA2A = 2.3 nM
hA2B = > 10000 nM
hA3 = > 10,000 nM
Chart 1. Pyrazolo-triazolopyrimidine derivatives as A2A and A3 AR antagonists.
2. Results and discussion
2.1. Chemistry
role by activation or inhibition of several second messenger sys-
tems. In particular, the modulation of adenylate cyclase activity
could be considered to be the principal signal mediated by these
receptors.1,2
The compounds with the free amino group bearing a phenoxy
(5) or methylthio (6) residue at the C5 position were prepared as
previously described by Caulkett et al.,32 while introduction of a
dimethylamino group at the C5 position (7) was performed by
heating compound 5 at 120 °C in a sealed tube with an ethanolic
solution (33%) of dimethylamine.
Derivatives substituted at the N7 position (8–25) were prepared
by treating compounds 5–7 with the appropriate acylchloride (in
presence of triethylamine) or arylisocyanate in dioxane at reflux
(Scheme 1).
In the last 20 years intense medicinal chemical efforts led to the
synthesis of a variety of AR agonists and antagonists for the phar-
macological characterization of this family of G protein-coupled
receptors.13 With respect to antagonists, several classes of hetero-
cyclic derivatives have been reported as AR antagonists with high
levels of both affinity and selectivity. In particular, in recent years
we investigated in depth the nucleus of triazolo-pyrazolopyrimi-
dine as a basis for designing ARs antagonists. Modulating the sub-
stitution at the C5, N7 and N8 positions (Chart 1) led to potent and
selective A2A and A3 AR antagonists (compounds 1 and 2).14–21
Nevertheless, this class of compounds, like other tricyclic struc-
tures, was subject to poor water solubility and more importantly
complicated synthetic routes. In consideration of these problems,
medicinal chemists recently focused their attention on the synthe-
sis of more simplified heterocyclic derivatives, in particular bicyclic
systems such as adenine,22 triazolopyrazine23–25 and triazolotri-
azine.26–28 One of the most appealing bicyclic cores is represented
by the triazolotriazine nucleus, which led in the past to the discov-
ery of ZM241385 (3) that could be considered as one of the most
potent A2A AR antagonists yet reported.29,30 In addition, 3 also
binds with high affinity at the human (h) A2B AR (28 nM), and its
tritiated form is suitable for use as a radioligand in binding studies
at this receptor subtype.31 Very recently a large number of triazol-
otriazine derivatives bearing different substitution at the 5-posi-
tion have been reported,26 and compound 4 proved to be potent
and selective for the A2A in comparison to the A1 AR. Nevertheless,
the lack of binding data at the A2B and A3 ARs prevents a compar-
ison with other fully characterized derivatives (Chart 2).26
2.2. Biological activity
The receptor binding affinities of the synthesized compounds (5–
25) are reported in Table 1. Binding was conducted at the following
ARs: rat A1 (from rat cerebral cortex membranes),33 rat A2A (from rat
striatal membranes),34 and hA3 (from HEK-293 cells expressing
the hA3 AR).35 [3H]N6-Phenylisopropyladenosine ([3H]R-PIA),
(A1)33 [3H]2-[4-[(2-carboxyethyl)-phenyl]ethylamino]-50-N-ethylc-
arbamoyladenosine, ([3H]CGS21680) (A2A 24 and [125I]N6-(4-amino-
)
3-iodobenzyl)-50-N-methylcarbamoyladenosine ([125I]I-AB-MECA)
(A3)26 have been used as radioligands in binding assays. Instead,
for evaluating potency at the hA2B AR, due to the lack of a readily
available radioligand, the activity of antagonists was determined
in adenylyl cyclase experiments in CHO (Chinese hamster ovary)
cells expressing the hA2B AR.37,38
All of the synthesized compounds showed affinities at the four
ARs in the high nanomolar or micromolar range without significant
levels of selectivity. The analysis of the data reported in Table 1,
clearly indicates that in general, with the exception of compound
NH2
N
N
N
NH2
O
N
N
N
HO
N
N
N
N
N
O
NH
N
O
4
ZM241385, 3
rA1 = 2,040 nM
rA1 = 3,300 nM
F
rA2A = 0.2 nM
rA2A = 0.3 nM
gpA2B = 87 nM
rA3 = > 10,000 nM
Chart 2. Triazolotriazine derivatives as AR antagonists.

2526
G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
COCHPh2, COCH2Ph, COCH2Ph-4-OCH3
CO(CH2)4CH3, CONHPh, CONHPh-4-CH3
R
N
R
HN
HN
N
N
N
N
N
N
O
HN
O
N
R1
N
N
H
R1
5-25
R1 = OPh R = H, 5
R1 = SCH3 R = H, 6
R1 = N(CH3)2 R = H, 7
Chart 3. Molecular simplification approach applied for the design of the novel triazolotriazine analogs.
(A)
NH2
NH2
i
N
N
N
N
N
N
N
O
N
N
O
N
PhO
N
5
7
(B)
CO NHR
N
HN
N
N
ii
O
N
R1
N
12,13,18,19,24,25
5-7
COR
HN
iii
N
N
N
O
N
R1
N
8-11,14-17.20-23
Scheme 1. Reagents: (i) EtOH, dimethylamine, 120 °C sealed tube, 4 h; (ii) R–N@C@O, dioxane, Et3N, reflux, 12 h; (iii) R–COCl, dioxane, reflux, 12 h.
7, compounds with a free amino group at the 7 position (5 and 6)
showed good affinity at the A2A AR (range 18.3–96.5 nM) with
quite significant levels of selectivity versus the other AR subtypes.
A significantly different selectivity profile could be noted when
theaminogroupatthe7 positionwassubstitutedwithanacylgroup,
and most importantly the affinities at the four AR subtypes seemed
to be very sensitive to the substitution at both the 5 and 7 positions.
In particular, when a phenylacetyl moiety was introduced at the
N7 position (9, 15, and 21) the binding profiles of the derivatives
were quite different with respect to the N7 unsubstituted deriva-
tives (5–7), and it was also significantly modified by the substitu-
tion at the 5 position. In fact, the presence of a methylthio (9) or
phenoxy (15) group at the 5 position enhanced affinity at the A2A
AR (range 136–429 nM), while the potency at the other receptor
or dimethylamino (20) group at the 5 position, favored A1 and A3
ARs affinities (range 170–950 nM). In contrast, the presence of a
phenoxy group at the 5 position reduced affinity at all the ARs.
Different binding affinities were observed when an acyl chain
was introduced at the N7 position. In fact, a combination of an acyl
chain at the N7 position with a methylthio (11) or a phenoxy (17)
groups at the 5 position afforded good affinity at the A2A AR (range
180–400 nM), while the affinities at the other AR subtypes was
poor. However, the presence of a dimethylamino group at the 5 po-
sition (23) was detrimental to affinity at all the AR subtypes.
Incorporation of an arylcarbamoyl moiety at the N5 position of
the pyrazolo-triazolopyrimidine nucleus (e.g., compound 2) en-
hanced hA3 AR affinity. In general, the affinity at the A1 AR was
poor (range from 3 lM to >10 lM) independent of the substitution
subtypes was poor (range 1–7
dimethylamino group at the 5 position (21) reduced affinity (1–
M) at all the ARs. When the phenylacetyl moiety at the N7
l
M). In contrast, the presence of a
at the 5 position (12, 13, 18, 19, 24, 25). However, phenoxy (18, 19)
or dimethylamino (24) groups at the 5 position enhanced the affin-
ity at the A2A subtype (range 39–580 nM).
7
l
was substituted at the para position with a methoxy group (10,
16, and 22), a general decrease of affinity versus all the ARs were
observed in comparison to the unsubstituted derivatives (9, 15,
and 21). If a bulky arylacetyl moiety (such as diphenylacetyl-8,
14, and 20) was introduced at the N7 position, a completely differ-
ent biological profile was evident. In fact, the presence of a bulky
substituent at the N7, position combined with a methylthio (8)
Thus, most of these derivatives were nearly inactive at the hA3
AR, with the exception of compounds 12, 18, and 24, which showed
hA3 affinity in the high nanomolar range (range 311–633 nM)
and were characterized by the presence of an unsubstituted
phenylcarbamoyl moiety at the N7 position, independently of
the substitution at the N5 position. The low affinity at the hA3
AR, was quite surprising considering that triazolotriazine

G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
2527
Table 1
Structures and binding profile of synthesized compounds 5–25
R
N
HN
N
N
N
O
R
1
N
5-25
#
R1
R
rA1
rA2A
(Ki, nM)b
hA2B
EC50 (nM)c
hA3 (Ki, nM)
or % displ. at
rA1/rA2A
rA1/hA3
rA2A/hA3
(Ki, nM)
% displ. at 10
l
Ma
10l
Md
5
6
7
8
OPh
H
H
H
2,720 680
1730 360
31,400 8000
557 205
1420 260
5000 1170
1520 170
10,000 2900
3440 690
18.3 3.4
96.5 36.1
3800 1230
> 10,000
429 38
1570 660
427 96
1660 1120
742 194
3,420 200
14,900 1,700
33,700 1,800
> 100,000
> 100,000
42,400 9,900
> 100,000
17,100 2,700
16,800 3,100
489 63
2580 780
7270 2230
170 45
4200 950
2100 400
24 3%
148
18
0.04
0.7
4.3
3.2
0.33
2.4
< 0.04
24
1.5
5.5
0.04
0.5
59
0.1
0.7
< 0.15
4
0.3
SCH3
N(CH3)2
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
8.3
< 0.05
3.3
3.2
3.5
6
COCHPh2
COCH2Ph
COCH2-Ph-4OCH3
CO(CH2)4CH3
CONHPh
9
10
11
12
13
414 29
2200 54
CONH-Ph-4CH3
4.6
Compd
R1
R
rA1
(Ki, nM)
<10% inhib
rA2A
(Ki, nM)
1880 310
hA2B
hA3 (Ki, nM)
or% displ. at 10
4400 1150
1020 173
5200 1300
2200 470
633 37
750 130
473 85
1050 466
2700 345
2200 370
311 122
rA1/rA2A
rA1/hA3
rA2A/hA3
EC50 (nM)
> 100,000
> 100,000
> 100,000
> 100,000
8,870 1,680
20,000 3,600
> 100,000
> 100,000
> 100,000
> 100,000
> 100,000
> 100,000
lM
14
15
16
17
18
19
20
21
22
23
24
25
OPh
OPh
OPh
OPh
OPh
OPh
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
COCHPh2
COCH2Ph
> 5.3
53
> 11
15
> 257
> 46
0.2
0.9
0.8
< 0.16
5.4
1
> 2.2
7
0.4
0.13
0.17
0.08
0.06
0.3
8.6
6.4
3.8
> 4.5
1.8
7260 2820
136 48
892 252
189 26
38.9 3.5
214 72
COCH2-Ph-4-OCH3
CO(CH2)4CH3
CONHPh
CONH-Ph-4CH3
COCHPh2
12 2% inhib
2900 710
17 5% inhib
35 5% inhib
951 88
6080 1040
8740 590
1570 380
3150 550
2720 830
> 2
1.3
> 16
> 13
2
5.8
3.2
0.7
10
4090 1360
6700 3200
10,300 1,300
> 10,000
580 100
2720 2090
COCH2Ph
COCH2-Ph-4-OCH3
CO(CH2)4CH3
CONHPh
CONH-Ph-4CH3
9600 1200
0.3
0.3
a
b
c
Displacement of specific [3H]R-PIA binding (A1) in rat brain membranes.
Displacement of specific [3H]CGS 21680 binding (A2A) in rat striatal membranes.
Measurement of adenylyl cyclase activity in CHO cells stably transfected with human recombinant A2B AR, expressed as EC50
(lM).
d
Displacement of specific [125I]-I-AB-MECA binding at hA3 receptors expressed in CHO cells. Data are expressed as Ki SEM in nM or as % of displacement at 10
l
M(n = 3-6).
derivatives were simplified analogs of the pyrazolo-triazolopyr-
imidine antagonists of the hA3 AR. Nevertheless, a careful struc-
tural analysis of these two classes of compounds, by comparing
the derivatives 24 and 2, clearly indicated that the dimethyl-
amino group on the triazolotriazine nucleus is not favored. In
fact, the dimethylamino group seemed not to correspond to the
N8-methyl group of derivative 2, but was similar to its N7 pat-
tern of substitution, which was extensively demonstrated to be
inactive at the hA3 AR.13–15
Regarding the activity of this series at the hA2B AR, most of com-
pounds were almost inactive at this receptor subtype. Only two
compounds, bearing a phenoxy group at the 5 position (5, 18),
showed promising activity in the adenylyl cyclase assays at the
formed in parallel at the A2A and A3 ARs. Therefore, we built models
of the rA2A and hA3 receptors by homology modeling, using as tem-
plate the crystal structure of hA2A receptor (PDB code: 3EML);39
methodological details are summarized in Section 4. Figure 1 shows
the alignment of the three amino acid sequences (hA2A/rA2A
sequence identity 82%; hA2A/hA3 sequence identity 41%).
Then, we performed docking studies to recognize the hypothet-
ical binding motif of the newly synthesized 5,7-disubstituted-
[1,2,4]triazolo[1,5-a][1,3,5]triazine derivatives. We compared our
docking simulation results with the docking poses of ZM241385
at both receptors. As reported in Section 4, four different programs
have been used to calibrate our docking protocol using the crystal-
lographic pose of ZM241385 into human A2A as reference. Based on
the lowest average ligand RMSD value obtained from the different
docking algorithms, we decided to use GOLD as docking program for
the pose inspection of the novel 5,7-disubstituted-[1,2,4]triazol-
o[1,5-a][1,3,5]triazine derivatives.
As shown in Figure 2, the binding motif of ZM241385 at the rA2A
receptor was similar to the crystallographic pose on the hA2A
receptor; this was consistent with the binding affinity of ZM-
241385 at the two receptors (Ki = 0.21–0.50 nM and Ki = 0.7–
1.0 nM, respectively).40,41 Consistently, all the residues belonging
to the binding pocket are conserved in both receptors. The num-
bering of the amino acids in parenthesis follows the numbering
convention of Ballesteros and Weinstein: according to this scheme,
each amino acid identifier starts with the helix number, followed
by the position relative to a reference residue among the most con-
served amino acid in that helix, with the reference residue arbi-
trarily assigned the number 50.42
A2B AR, with an EC50 ranging from 3.4 to 8.8 lM. In particular,
derivative 5, which contained a free amino group at the 7 position,
was the most potent at the hA2B AR, and could represent a starting
point for new non-xanthine hA2B AR antagonists. Nevertheless, the
high potency of this compound at the A2A AR (18.3 nM) clearly
indicated that further investigation would be needed in order to
delineate the activities at these two ARs. A summary of the most
relevant structure–activity features of the novel triazolotriazine
analogs has been reported in Chart 4.
2.3. Molecular modeling
It was clear from the data analysis that it was very difficult to de-
fine a robust SAR profile of this new class of compounds. For this rea-
son and with the aim to better understand these pharmacological
results, molecular modeling and docking studies have been per-

2528
G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
R = COR is detrimental for affinity
at the four adenosine receptors
R = H seems essential for
A2A and A2B affinity
R = CONHR increases affinity at
the hA3 adenosine receptor, without
significant selectivity
R
HN
N
N
N
N
O
R1
N
R1 plays a role only with the free amino group at the 7 position.
Dimethylamino results detrimental in terms of affinity
at the four adenosine receptors
Chart 4. Summary of the most relevant SAR features of the novel triazolotriazine analogs.
Figure 1. Multiple sequence alignment of hA2A receptor, rat A2A receptor and hA3 receptor. Conserved residues in all the sequences are identified by stars. Differences in
sequences of rA2A and hA3 compared to hA2A are shown in boldface type.
From the analysis of docking of ZM241385 in the rA2A AR, it ap-
peared that the bicyclic triazolotriazine core was anchored through
an aromatic stacking interaction with Phe163 (EL2), an aliphatic
hydrophobic interaction with Ile269 (7.39), and a hydrogen bond-
ing interaction with Asn248 (6.55). The exocyclic amino group,
linked to the bicyclic core of ZM241385, interacts with two polar

G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
2529
Figure 2. Structure superimposition of crystallographic pose (in magenta) and docking pose (in green) of ZM241385 inside the rA2A AR binding site. Side chains of some
amino acids important for ligand recognition and H-bonding interactions are highlighted. Hydrogen atoms are not displayed.
residues, Asn248 (6.55) and Glu164 (EL2). The phenyl ring forms
hydrophobic interactions with Pro262 (7.32) and Met265 (7.35).
The phenylethylamine substituent was directed towards the more
solvent exposed extracellular region (EL2 and EL3) rather than to-
wards the transmembrane domains of the receptor. The furan ring
was located deep in the ligand binding cavity and directed towards
TM5 and TM7; it forms hydrophobic interactions with the highly
conserved Trp241 (6.48), an important residue in receptor activa-
tion,43 His245 (6.52) and Leu244 (6.51).
The triazolotriazine derivatives with a free amino group at the 7
position (5–7) show a similar binding motif to ZM-241385 inside
the transmembrane region of the rA2A receptor, as reported in Fig-
ure 3. In particular, the three H-bonding interactions with Asn248
(6.55) and Glu164 (EL2) are conserved.
Among these, compound 5 (Ki rA2AAR = 18.3 3.4) was the most
potent because the phenoxy group forms strong hydrophobic
interactions with side chains of the following residues: Leu162
(EL2), Phe163 (EL2), Pro262 (7.32), Met265 (7.35), Tyr266 (7.36)
and Ile269 (7.39). Calculated log P values of compounds 6 and 7
(1.01 and ꢀ0.24, respectively) could explain the different affinities
at the receptor, even though the two R1 substituents were not in-
volved in particular interactions with residues of the binding pock-
et. The hydrogen bonding network with Asn248 (6.55) and Glu164
(EL2) seems to be critical both for the recognition of these antago-
nist structures and for receptor selectivity versus hA3. In particular,
Glu164 (EL2) of rA2A receptor subtype (Glu169 in hA2A) was not
present in the corresponding position of hA3 receptor, where this
amino acid was replaced by valine (Val169); in fact molecular
docking results on hA3 suggest that compounds 5, 6 and 7 (Ki
hA3AR = 489 63, 2580 780, 7270 2230, respectively) form
only two (with Asn250) of the three hydrogen bonding interactions
(data not shown).
binding pose of compound 18 results to be quite different com-
pared with the unsubstituted derivatives (ZM241385, 5–7). In fact,
the R substituent was directed towards TM2 and TM3 rather than
towards TM5 and TM6 and was located in a hydrophobic pocket
delimited by Ala56 (2.57), Ile57 (2.58), Phe59 (2.60), Ala60
(2.61), Ile63 (2.64), Phe77 (3.28), Ala78 (3.29), Phe80 (3.31) and
Val81 (3.32). Nevertheless, compound 18 formed also the same
hydrophobic interactions as compound 5 and ZM241385, but only
one hydrogen bonding interaction with Asn248 (6.55). Moreover
the bicyclic triazolotriazine core of the disubstituted ligand was
aligned with the same region of the other two compounds.
In the hA3 receptor, the presence of the less bulky side chain of
Val169 (EL2) allows the phenylcarbamoyl moiety of compound 18
to direct towards TM5 and TM6 and so it looses the interactions
with the residues of the hydrophobic pocket. Moreover, all the
5,7-disubstituted-[1,2,4]triazolo[1,5-a][1,3,5]triazine derivatives
were located deeper in the ligand binding cavity of hA3; conse-
quently they lose the p–p stacking interaction with Phe168 (EL2).
With respect to the R1 moieties, the SAR at rA2A was similar for
compounds 8–25 and compounds 5–7. It seems that the presence
of too bulky substituents at the N7 position was not well tolerated
because of unfavourable steric interactions.
To analyze in a more quantitative way the possible ligand–
receptor recognition mechanism, the individual electrostatic
el
int
(DE
) and hydrophobic (
D
Ehinytd) contributions to the interaction en-
ergy (DEint) of each receptor residue has been calculated (see Sec-
tion 4 for more details). Analyzing the results of this study
(collected in Figs. 4 and 5) it is clear that, from the electrostatic
point of view, two of the most critical residues affecting the affinity
at ARs seem to be the asparagine 6.55 (Asn253 in hA2A, Asn248 in
rA2A and Asn250 in hA3) and the glutamic acid located within EL2
of both human and rat A2A (Glu169 and Glu164, respectively) but
mutated in valine in hA3 (Val169). In particular, Asn 6.55 is respon-
sible of two stabilizing interactions with ZM241385 in both human
and rat A2A and this is supported by the electrostatic contribution
Among other compounds (8–25) the most potent at the rA2A
receptor was 18 (Ki rA2AAR = 38.9 3.5). As shown in Figure 3, be-
cause of the presence of the phenylcarbamoyl moiety at N7, the

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G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
Figure 3. Structure superimposition of docked conformations of compound 5 (in red, Ki rA2AAR = 18.3 3.4) and compound 18 (in magenta, Ki rA2AAR = 38.9 3.5) inside the
rA2A AR binding pocket. Side chains of some amino acids important for ligand recognition and H-bonding interactions are highlighted. Hydrogen atoms are not displayed.
of around ꢀ13 kcal/mol to the whole interaction energy, in partic-
ular ꢀ9.5 kcal/mol in hA2A and ꢀ17.9 kcal/mol in rA2A (Fig. 4). In
addition, the glutamic acid on EL2 can strongly interact through
an additional hydrogen-bond with the exocyclic amino group of
ZM-241385 as supported by the stabilizing electrostatic contribu-
tion of around ꢀ13 kcal/mol to the whole interaction energy, in
particular ꢀ9.0 kcal/mol in hA2A and ꢀ17.6 kcal/mol in rA2A
(Fig. 4). Consistently, this specific interaction is missing in hA3.
Interestingly, compound 5 presents a very similar electrostatic
energy contributions to ZM241385 supporting the hypothesis of
a common TM binding motif. Conversely, compound 18 completely
abolishes the capability to interact with both asparagine 6.55 and
glutamic acid on EL2 due to the presence of phenylcarbamoyl moi-
ety that forces the triazolotriazine moiety to flip 180° (around its
parallel TM axis) inside the TM binding cleft. However, the lack
of these two stabilizing interactions seems to be balanced by the
presence of several additional hydrophobic interactions as mapped
in Figure 5.
nists, a novel model of the rA2A and hA3 receptor, based on the re-
cently published structure of the human A2A receptor, was built.
These new AR models provide a self-consistent framework that
rationalizes the available SAR data. Moreover, a very preliminary
hypothesis, concerning the specific roles of a few crucial amino
acids in affecting the molecular mechanism of both ligand-entering
process and TM-recognition process, has been proposed.
4. Experimental section
4.1. Chemistry
4.1.1. General
Reactions were routinely monitored by thin-layer chromatogra-
phy (TLC) on silica gel (precoated F254 Merck plates). Infrared spec-
tra (IR) were measured on a Jasco FT-IR instrument. 1H NMR were
determined in CDCl3 or DMSO-d6 solutions with a Varian Gemini
200 spectrometer, peaks positions are given in parts per million
(d) downfield from tetramethylsilane as internal standard, and J
values are given in hertz. Light petroleum ether refers to the frac-
tions boiling at 40–60 °C. Melting points were determined on a Bu-
chi-Tottoli instrument and are uncorrected. Flash chromatography
was performed using Merck 60–200 mesh silica gel. Elemental
analyses were performed by the microanalytical laboratory of
Dipartimento di Chimica, University of Trieste, and were within
0.4% of the theoretical values for C, H and N.
In fact, besides the three hydrophobic contributions mediated
by the conserved phenylalanine on EL2 (Phe168 in hA2A, Phe163
in rA2A and Phe168 in hA3), the leucine 6.51 (Leu249 in hA2A
,
Leu244 in rA2A and Leu246 in hA3) and the tryptophan 6.48
(Trp246 in hA2A, Trp241 in rA2A and Trp243 in hA3), the phenylcar-
bamoyl moiety at N7 is surround by several hydrophobic side
chains such as, for example for the rA2A, Ala56 (2.57), Ile57
(2.58), Phe59 (2.60), Ala60 (2.61), Ile63 (2.64), Phe77 (3.28),
Ala78 (3.29), Phe80 (3.31) and Val81 (3.32).
4.1.1.1. 7-Amino-5-dimethylammino-2-(2-furyl)-[1,2,4]triazol-
o[1,5-a]-[1,3,5] triazine (7). To a solution of phenoxy derivative
5 (0.3 g, 1.01 mmol) in ethanol (5 mL) was added a large excess
(5 equiv) of an ethanolic solution (33%) of dimethylamine
(0.7 mL). The resulting solution was transferred to a sealed tube
and heated at 120 °C for 4 h. Then the solvent was removed at re-
duced pressure and the crude residue purified by flash chromatog-
raphy (EtOAc–light petroleum 1:1) to afford the desired product
3. Conclusion
The present study has led to the identification of a new class of
promising AR antagonists, the 5,7-disubstituted-[1,2,4]triazol-
o[1,5-a][1,3,5]triazine derivatives, which were designed as simpli-
fied analogues of our previously reported hA2A and hA3
antagonists. To depict the binding motif of these new AR antago-

G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
2531
Figure 4. Electrostatic interaction energy (in kcal/mol) between the ligand and each single amino acid involved in ligand recognition calculated from: (A) crystallographic
binding mode of ZM241385 inside the hA2A receptor (PDB code: 3EML); (B) hypothetical binding mode of ZM241385 inside the rA2A receptor obtained after docking
simulations; (C) hypothetical binding mode of compound 5 inside the rA2A receptor obtained after docking simulations; and (D) hypothetical binding mode of compound 18
inside the rA2A receptor obtained after docking simulations.

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G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
Figure 5. Hydrophobic interaction energy (in arbitrary hydrophobic unit) between the ligand and each single amino acid involved in ligand recognition calculated from: (A)
crystallographic binding mode of ZM241385 inside the hA2A receptor (PDB code: 3EML); (B) hypothetical binding mode of ZM241385 inside the rA2A receptor obtained after
docking simulations; (C) hypothetical binding mode of compound 5 inside the rA2A receptor obtained after docking simulations; and (D) hypothetical binding mode of
compound 18 inside the rA2A receptor obtained after docking simulations.
(7) as a pale yellow solid in a good yield (80%). Mp 260–262 °C
(EtOAc–light petroleum); IR (KBr): 3300, 1620, 1510 cmꢀ1 1H
NMR (DMSO-d6) d: 3.1 (s, 6H); 6.7 (dd, 1H, J = 2, J = 4), 7.1 (d, 1H,
J = 2); 7.9 (d, 1H, J = 4); 8.2 (br s, 2H). MW 245.24. Anal. Calcd for
C10H11N7O: C, 48.98; H, 4.52; N, 39.98. Found: C, 49.15; H, 4.50;
N, 39.77.
4.1.2. General procedure for the preparation of the 7-(aralkyl-
carbonyl)amino-2-(2-furyl)-5-substituted[1,2,4]triazolo[1,5-
a][1,3,5]triazine derivatives (8–11, 14–17, 20–23)
A solution of amino derivative (5–7) (1 mmol), appropriate acyl
chloride (1.2 equiv) and triethylamine (1.2 equiv) in dry dioxane
(10 mL) was poured at reflux under argon atmosphere for 12 h.
;

G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
2533
The solvent was removed in vacuo, and the crude residue purified
by flash chromatography (EtOAc–light petroleum 3:7) to afford the
desired compound.
1450 cmꢀ1 1H NMR (DMSO-d6) d: 0.93 (t, 3H, J = 7); 1.05–1.19
;
(m, 4H); 1.43–1.65 (m, 2H); 3.35 (t, 2H, J = 7); 6.68 (dd, 1H, J = 2,
J = 4), 7.21 (d, 1H, J = 2); 7.35–7.63 (m, 5H); 7.94 (d, 1H, J = 4);
10.58 (br s, 1H). MW 392.41. Anal. Calcd for C20H20N6O3: C,
61.21; H, 5.14; N, 21.42. Found: C, 61.53; H, 5.23; N, 21.58.
4.1.2.1. 7-Diphenyl-acetylamino-2-(2-furyl)-5-methylthio[1,2,4]-
triazolo[1,5-a]-[1,3,5]triazine (8). Yield 65%; pale yellow solid;
mp 172–173 °C (EtOAc–light petroleum); IR (KBr): 1680, 1520,
4.1.2.9. 5-Dimethylamino-7-diphenylacetylamino-2-(2-furyl)-
[1,2,4]triazolo[1,5-a]-[1,3,5]triazine (20). Yield 70%; yellow so-
lid; mp 105–107 °C (EtOAc–light petroleum); IR (KBr): 1680,
1420 cmꢀ1 1H NMR (DMSO-d6) d: 2.53 (s, 3H); 5.1 (s, 1H); 6.7
;
(dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2);7.2–7.6 (m, 10H); 7.9 (d, 1H,
J = 4); 9.87 (br s, 1H). MW 442.49. Anal. Calcd for C23H18N6O2S: C,
62.43; H, 4.10; N, 18.99. Found: C, 62.66; H, 4.18; N, 19.13.
1520, 1425 cmꢀ1 1H NMR (DMSO-d6) d: 3.13 (s, 6H); 4.32 (s,
;
1H); 6.68 (dd, 1H, J = 2, J=4), 7.21 (d, 1H, J = 2); 7.32–7.65 (s,
10H); 7.88 (d, 1H, J = 4); 10.12 (br s, 1H). MW 439.47. Anal. Calcd
for C24H21N7O2: C, 65.59; H, 4.82; N, 22.31. Found: C, 65.77; H,
4.82; N, 22.43.
4.1.2.2. 2-(2-Furyl)-5-methylthio-7-phenylacetylamino-[1,2,4]-
triazolo[1,5-a]-[1,3,5]triazine (9). Yield 75%; brown solid; mp
70–72 °C (EtOAc–light petroleum); IR (KBr): 1680, 1510,
1450 cmꢀ1 1H NMR (DMSO-d6) d: 2.51 (s, 3H); 4.0 (s, 2H); 6.7
;
4.1.2.10. 5-Dimethylamino-2-(2-furyl)-7-phenylacetylamino-
[1,2,4]triazolo[1,5-a]-[1,3,5]triazine (21). Yield 75%; yellow
solid; mp 226–228 °C (EtOAc–light petroleum); IR (KBr): 1680,
(dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.2–7.6 (m, 5H); 7.9 (d, 1H,
J = 4); 11.8 (br s, 1H). MW 366.40. Anal. Calcd for C17H14N6O2S: C,
55.73; H, 3.85; N, 22.94. Found: C, 55.63; H, 3.79; N, 22.90.
1525, 1455 cmꢀ1 1H NMR (DMSO-d6) d: 3.15 (s, 6H); 4.28 (s,
;
2H); 6.69 (dd, 1H, J = 2, J = 4), 7.08 (d, 1H, J = 2); 7.4–7.55 (m,
5H); 7.85 (d, 1H, J = 4); 11.02 (br s, 1H). MW 363.37. Anal. Calcd
for C18H17N7O2: C, 59.50; H, 4.72; N, 26.98. Found: C, 59.68; H,
4.71; N, 26.75.
4.1.2.3. 2-(2-Furyl)-7-[4-(methoxyphenyl)acetyl]amino-5-
methylthio [1,2,4]triazolo[1,5-a][1,3,5]triazine (10). Yield 78%;
pale yellow; mp 138–141 °C (EtOAc–light petroleum); IR (KBr):
1680, 1525, 1455 cmꢀ1 1H NMR (DMSO-d6) d: 2.5 (s, 3H); 3.8 (s,
;
3H); 4.3 (s, 3H); 4.5 (s,2H); 6.7 (dd, 1H, J = 2, J = 4); 6.9 (d, 2H,
J = 9); 7.1 (d, 1H, J = 2); 7.2 (d, 2H, J = 9); 7.9 (d, 1H, J = 4); 11.8
(br s, 1H). MW 396.42. Anal. Calcd for C18H16N6O3S: C, 54.54; H,
4.07; N, 21.20. Found: C, 54.70; H, 4.11; N, 21.08.
4.1.2.11. 5-Dimethylamino-2-(2-furyl)-7-[4-(methoxyphenyl)-
acetyl]amino-[1,2,4]triazolo[1,5-a]-[1,3,5]triazine (22). Yield
73%; pale yellow solid; mp 168–171 °C (EtOAc–light petroleum);
IR (KBr): 1680, 1530, 1460 cmꢀ1 1H NMR (DMSO-d6) d: 3.09 (s,
;
6H); 3.88 (s, 3H); 4.45 (s, 2H); 6.73 (dd, 1H, J = 2, J = 4), 7.12 (d,
1H, J = 2); 7.18 (d, 2H, J = 9); 7.47 (d, 2H, J = 9); 7.85 (d, 1H,
J = 4); 10.43 (br s, 1H). MW 393.41. Anal. Calcd for C19H19N7O3:
C, 58.01; H, 4.87; N, 24.92. Found: C, 57.78; H, 4.74; N, 24.83.
4.1.2.4. 2-(2-Furyl)-5-methylthio-7-n-pentylcarbonylamino- [1,2,4]-
triazolo[1,5-a]-[1,3,5]triazine (11). Yield 70%; white solid; mp
125–127 °C (EtOAc–light petroleum); IR (KBr): 1675, 1520,
1450 cmꢀ1 1H NMR (DMSO-d6) d: 0.91 (t, 3H, J = 7); 1.01–1.2 (m,
;
4H); 1.4–1.61 (m, 2H); 2.5 (s, 3H); 3.32 (t, 2H, J = 7); 6.7 (dd, 1H,
J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.9 (d, 1H, J = 4); 10.93 (br s, 1H).
MW 346.41. Anal. Calcd for C15H18N6O2S: C, 52.01; H, 5.24; N,
24.26. Found: C, 52.15; H, 5.20; N, 24.07.
4.1.2.12. 5-Dimethylamino-2-(2-furyl)-7-n-pentylcarbonylami-
no-[1,2,4]triazolo[1,5-a]-[1,3,5]triazine (23). Yield 68%; white
solid; mp 147–148 °C (EtOAc–light petroleum); IR (KBr): 1685,
1515, 1450 cmꢀ1 1H NMR (DMSO-d6) d: 0.89 (t,3H, J = 7); 0.99–
;
1.05 (m, 4H); 1.32–1.55 (m,2H); 3.14 (s, 6H); 3.29 (t, 2H, J = 7);
6.72 (dd, 1H, J = 2, J = 4); 7.15 (d, 1H, J = 2); 7.86 (d, 1H, J = 4);
10.04 (br s, 1H). MW 343.38. Anal. Calcd for C16H21N7O2: C,
55.96; H, 6.16; N, 28.55. Found: C, 56.13; H, 6.23; N, 28.73.
4.1.2.5.
7-Diphenylacetylamino-2-(2-furyl)-5-phenoxy[1,2,4]-
triazolo[1,5-a]-[1,3,5]triazine (14). Yield 67%; pale yellow solid;
mp 136–139 °C (EtOAc–light petroleum); IR (KBr): 1680, 1520,
1425 cmꢀ1 1H NMR (DMSO-d6) d: 5.12 (s, 1H); 6.73 (dd, 1H,
;
J = 2, J = 4), 7.1 (d, 1H, J = 2); 7.2–7.6 (m, 15H); 7.9 (d, 1H, J = 4);
10.15 (br s, 1H). MW 488.50. Anal. Calcd for C28H20N6O3: C,
68.84; H, 4.13; N, 17.20. Found: C, 68.91; H, 4.21; N, 17.08.
4.1.3. General procedure for the preparation of the 7-(arylcar-
bamoyl)amino-2-(2-furyl)-5-substituted)[1,2,4]triazolo[1,5-a]-
[1,3,5]triazine derivatives (12, 13, 18, 19, 24, 25)
A solution of amino compound (1 mmol) (5–7), appropriate iso-
cyanate (1.2 equiv) in dry dioxane was refluxed under argon for
18 h. The solvent was removed in vacuo, and the crude product
was purified by flash chromatography (EtOAc–light petroleum
4:6) to afford the final compound (12, 13, 18, 19, 24, 25).
4.1.2.6. 2-(2-Furyl)-5-phenoxy 7-phenylacetylamino- [1,2,4]tria-
zolo[1,5-a]-[1,3,5]triazine (15). Yield 65%; white solid; mp 208–
210 °C (EtOAc–light petroleum); IR (KBr): 1680, 1520, 1425 cmꢀ1
;
1H NMR (DMSO-d6) d: 4.2 (s, 2H); 6.7 (dd, 1H, J = 2, J = 4), 7.1
(d, 1H, J = 2,); 7.2–7.6 (m, 10H); 7.9 (d, 1H, J = 4); 11.01 (br s,
1H). MW 412.40. Anal. Calcd for C22H16N6O3: C, 64.07; H, 3.91;
N, 20.38. Found: C, 64.23; H, 3.88; N, 20.28.
4.1.3.1. 2-(2-Furyl)-5-methylthio-7-(phenylcarbamoyl)amino-
[1,2,4]triazolo[1,5-a]-[1,3,5]triazine (12). Yield 75%; yellow so-
lid; mp 165–167 °C (EtOAc–light petroleum); IR (KBr): 1660,
4.1.2.7. 2-(2-Furyl)-7-[4-(methoxyphenyl)acetyl]amino-5-phen-
oxy[1,2,4]triazolo[1,5-a]-[1,3,5]triazine (16). Yield 70%; white
solid; mp 178–181 °C (EtOAc–light petroleum); IR (KBr): 1680,
1510, 1450 cmꢀ1 1H NMR (DMSO-d6) d: 2.54 (s, 3H); 6.65 (dd,
;
1H, J = 2, J = 4); 7.13 (d, 1H, J = 2); 7.25–7.54 (m, 5H); 7.92 (d,
1H, J = 4); 8.76 (br s, 1H); 11.55 (br s, 1H). MW 367.39. Anal. Calcd
for C16H13N7O2S: C, 52.31; H, 3.57; N, 26.29. Found: C, 52.45; H,
3.63; N, 26.61.
1525, 1455 cmꢀ1 1H NMR (DMSO-d6) d: 3.8 (s, 3H); 4.2 (s, 2H);
;
6.73 (dd, 1H, J = 2, J = 4), 7.12 (d, 1H, J = 2); 7.2–7.45 (m, 7H);
7.62 (d, 2H, J = 9); 7.9 (d, 1H, J = 4); 10.77 (br s, 1H). MW 442.43.
Anal. Calcd for C23H18N6O4: C, 62.44; H, 4.10; N, 19.00. Found: C,
62.28; H, 4.15; N, 18.89.
4.1.3.2. 2-(2-Furyl)-7-[4-methyl(phenylcarbamoyl)]amino-5-
methylthio [1,2,4]triazolo[1,5-a]-[1,3,5]triazine (13). Yield
69%; yellow solid; mp 169–171 °C (EtOAc–light petroleum); IR
4.1.2.8. 2-(2-Furyl)-7-n-pentylcarbonylamino-5-phenoxy[1,2,4]-
triazolo[1,5-a]-[1,3,5]triazine (17). Yield 71%; white solid; mp
157–159 °C (EtOAc–light petroleum); IR (KBr): 1685, 1515,
(KBr): 1665, 1525, 1450 cmꢀ1 1H NMR (DMSO-d6) d: 2.28 (s,
;
3H); 2.55 (s, 3H); 6.66 (dd, 1H, J = 2, J = 4); 7.07 (d, 1H, J = 2);
7.23 (d, 2H, J = 9); 7.65 (d, 2H, J = 9); 7.83 (d, 1H, J = 4); 8.32 (br

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G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
s, 1H); 11.03 (br s, 1H). MW 381.41. Anal. Calcd for C17H15N7O2S: C,
53.53; H, 3.96; N, 25.71. Found: C, 53.57; H, 3.90; N, 25.59.
trations of competitor, spanning 3 orders of magnitude adjusted
appropriately for the IC50 of each compound, were used. IC50 val-
ues, calculated with the nonlinear regression method implemented
in Graph-Pad (Prism, San Diego, CA), were converted to dissocia-
tion constants (Ki) as described.47 Hill coefficients of the tested
compounds were in the range of 0.8–1.1.
4.1.3.3. 2-(2-Furyl)-5-phenoxy-7-(phenylcarbamoyl)amino[1,2,4]-
triazolo[1,5-a]-[1,3,5]triazine (18). Yield 65%; pale yellow solid;
mp 196–198 °C (EtOAc–light petroleum); IR (KBr): 1658, 1509,
1455 cmꢀ1 1H NMR (DMSO-d6) d: 6.62 (dd, 1H, J = 2, J = 4), 7.03
;
(d, 1H, J = 2); 7.2–7.6 (m, 10H); 7.85 (d, 1H, J = 4); 8.77 (br s, 1H);
10.78 (br s, 1H). MW 414.39. Anal. Calcd for C21H15N7O3: C,
61.01; H, 3.66; N, 23.72. Found: C, 61.25; H, 3.72; N, 23.85.
4.2.3. Adenylyl cyclase activity
Due to the lack of a suitable radioligand the affinity of antago-
nists and the relative potency of agonists at A2B. ARs was deter-
mined in adenylyl cyclase experiments. The procedure was
carried out as described previously37,38 with minor modifications.
4.1.3.4.
2-(2-Furyl)-7-[4-methyl(phenylcarbamoyl)]amino-5-
phenoxy[1,2,4]triazolo[1,5-a]-[1,3,5]triazine (19). Yield 73%;
pale yellow solid; mp 203–205 °C (EtOAc–light petroleum); IR
Membranes were incubated with about 150,000 cpm of [a-
32P]ATP
for 20 min in the incubation mixture as described37,38 without
EGTA and NaCl. For agonists the EC50-values for the stimulation
of adenylyl cyclase were calculated with the Hill equation. Hill
coefficients in all experiments were near unity. IC50 values for con-
centration-dependent inhibition of NECA-stimulated adenylyl cy-
clase caused by antagonists were calculated accordingly. Ki
values for antagonists were then calculated with the Cheng and
Prusoff equation.48
(KBr): 1668, 1530, 1465 cmꢀ1 1H NMR (DMSO-d6) d: 2.31 (s,
;
3H); 6.71 (dd, 1H, J = 2, J = 4), 7.12 (d, 1H, J = 2); 7.23–7.45 (m,
7H); 7.57 (d, 2H, J = 9); 7.86 (d, 1H, J = 4); 8.45 (br s, 1H); 10.08
(br s, 1H). MW 427.42. Anal. Calcd for C22H17N7O3: C, 61.82; H,
4.01; N, 22.94. Found: C, 62.01; H, 4.13; N, 23.07.
4.1.3.5. 5-Dimethylamino-2-(2-furyl)-7-(phenylcarbamoyl)amino-
[1,2,4]triazolo[1,5-a]-[1,3,5]triazine (24). Yield 80%; white solid;
mp 270–274 °C (EtOAc–light petroleum); IR (KBr): 1665, 1515,
4.3. Computational methodologies
1460 cmꢀ1 1H NMR (DMSO-d6) d: 3.14 (s, 6H); 6.70 (dd, 1H,
;
J = 2, J = 4), 7.09 (d, 1H, J = 2); 7.34–7.58 (m, 5H); 7.89 (d, 1H,
J = 4); 8.32 (br s, 1H); 10.65 (br s, 1H). MW 364.40. Anal. Calcd
for C17H16N8O2: C, 56.04; H, 4.43; N, 30.75. Found: C, 56.21; H,
4.38; N, 30.58.
All modeling studies were carried out on a 20 CPU (Intel
CoreTM2 Quad CPU 2.40 GHz) linux cluster. Homology modeling,
energy calculation, and analyses of docking poses were performed
using the Molecular Operating Environment (MOE, version
2008.10) suite.49 The software package MOPAC (version 7),50
implemented in MOE suite, was utilized for all quantum mechan-
ical calculations. Docking simulations were performed using GOLD
suite.51 Log P values were calculated using ACDLABS 10.0.52
4.1.3.6. 5-Dimethylamino-2-(2-furyl)-7-[4-methyl(phenylcar-
bamoyl)]amino-[1,2,4]triazolo[1,5-a]-[1,3,5]triazine (25). Yield
83%; white solid; mp 263–266 °C (EtOAc–light petroleum); IR
(KBr): 1665, 1520, 1455 cmꢀ1 1H NMR (DMSO-d6) d: 2.24 (s,
;
3H); 3.16 (s, 6H); 6.69 (dd, 1H, J = 2, J = 4), 7.11 (d, 1H, J = 2);
7.24 (d, 2H, J = 9); 7.58 (d, 2H, J = 9); 7.86 (d, 1H, J = 4); 8.76 (br
s, 1H); 11.03 (br s, 1H). MW 378.39. Anal. Calcd for C18H18N8O2:
C, 57.74; H, 4.79; N, 29.61. Found: C, 57.35; H, 4.77; N, 29.78.
4.3.1. Homology model of rA2A AR and hA3 AR
Based on the assumption that GPCRs share similar TM bound-
aries and overall topology, homology models of the ARs were con-
structed. First, the amino acid sequences of TM helices of rA2A and
hA3 receptor were aligned with those of hA2A AR,39 guided by the
highly conserved amino acid residues, including the DRY motif
(Asp3.49, Arg3.50, and Tyr3.51) and three proline residues
(Pro4.60, Pro6.50, and Pro7.50) in the TM segments of GPCRs.
The same boundaries were applied for the TM helices of rA2A and
hA3 ARs as they were identified from the X-ray crystal structure
for the corresponding sequences of hA2A AR,39 the CR coordinates
of which were used to construct the seven TM helices for the
rA2A and hA3 ARs. The loop domains were constructed by the loop
search method implemented in MOE on the basis of the structure
of compatible fragments found in the Protein Data Bank. In partic-
ular, loops were modeled first in random order. For each loop, a
contact energy function analyzes the list of candidates collected
in the segment searching stage, taking into account all atoms al-
ready modeled and any atoms specified by the user as belonging
to the model environment. These energies were then used to make
a Boltzmann-weighted choice from the candidates, the coordinates
of which were then copied to the model. Any missing side chain
atoms were modeled using the same procedure. Side chains are
modeled using a library of rotamers generated by systematic clus-
tering of the Protein Data Bank data, using the same procedure.
Side chains belonging to residues whose backbone coordinates
were copied from a template were modeled first, followed by side
chains of modeled loops. Outgaps and their side chains were mod-
eled last. Special caution has to be given to the second extracellular
loop (EL2) because amino acids of this loop could be involved in di-
rect interactions with the ligands. A driving force to this peculiar
fold of the EL2 loop might be the presence of a disulfide bridge be-
tween cysteines in TM3 and EL2. The constraints were applied
4.2. Biological activity
4.2.1. Radioligand binding to rA1 and rA2A ARs
Procedures for preparation of rat brain membranes were re-
ported previously.44,45 For binding experiments, membranes were
frozen and stored at ꢀ20 °C for 62 months. Adenosine deaminase
(ADA) was from Boehringer Mannheim (Indianapolis, IN). [3H]R-
PIA was from Amersham (Arlington Heights, IL), and [3H]CGS
21680 was from DuPont NEN (Boston, MA).
Binding of [3H]R-PIA to the A1 AR from rat cortical membranes
and of [3H]CGS 21680 to the A2A AR from rat striatal membranes
was performed as described previously.46 Adenosine deaminase
(ADA, 2 units/mL) was present during the preparation of brain
membranes. Additional ADA was not added during incubation with
the radioligand.
4.2.2. Radioligand binding to hA3 ARs
[
125I]-AB-MECA was utilized in radioligand binding assays to
membranes prepared from CHO cells expressing recombinant
hA3 ARs, as previously described.36 ADA (3 units/mL) was present
during the preparation of the membranes, in a preincubation of
30 min at 30 °C, and during the incubation with the radioligands.
All nonradioactive compounds were initially dissolved in DMSO
and diluted with buffer to the final concentration, where the
amount of DMSO never exceeded 2%. Incubations were terminated
by rapid filtration over Whatman GF/B filters, using a Brandell cell
harvester (Brandell, Gaithersburg, MD). The tubes were rinsed
three times with 3 mL of buffer each. At least six different concen-

G. Pastorin et al. / Bioorg. Med. Chem. 18 (2010) 2524–2536
2535
Table 2
Comparison of different molecular docking protocols
Docking protocol
Best RMSD (Å)
Best ranked
pose RMSD (Å)
Mean poses
RMSD (Å)
Number of poses
with RMSD <2.5 Å
MOE tabù search
MOE simulated annealing
MOE genetic algorithm
1.61
3.35
4.36
9.06
1.95
5.65
6.47
6.66
1.20
4/25
1/25
2/25
25/25
2.17
2.25
0.63
GOLD (goldscore)
GOLD (chemscore)
1.31
0.61
3.90
4.96
3.13
1.50
11/25
23/25
GOLD (asp)
GLIDE
0.79
0.93
2.71
1.98
6.82
6.96
7/25
3/25
PLANTS (chemplp)
PLANTS (plp)
0.84
1.97
1.93
6.70
8.22
6/25
4/25
11.80
PLANTS (plp95)
before the construction of the homology model, in particular dur-
ing the sequences alignment, selecting the cysteine residues in-
volved in the disulfide bridge in hA2A to be constrained with the
corresponding cysteines in hA3 sequence. In particular, Cys166
(EL2) in hA2A was constrained with Cys166 (EL2) in hA3 and
Cys77 (3.25) in hA2A with Cys83 (3.25) in hA3. When performing
the alignment, MOE-Align attempts to minimize the number of
constraint violations. Then, after running the homology modeling,
the presence of the conserved disulfide bridge in the model was
manually checked. Since this covalent link was conserved in all
ARs modeled in the current study, the EL2 loop was modeled using
a hA2A-like constrained geometry around the EL2-TM3 disulfide
bridge. In rA2A receptor all the three disulfide bridge that charac-
terize the template were conserved; for this reason the loop was
modeled constraining all its residues. After the heavy atoms were
modeled, all hydrogen atoms were added and were then mini-
mized with MOE using the AMBER9953 force field. The minimiza-
tions were carried out by the 1000 steps of steepest descent
followed by conjugate gradient minimization until the rms gradi-
ent of the potential energy was less than 0.1 kcal molꢀ1 Åꢀ1. We
used Protonate 3D methodology, part of the MOE suite, for proton-
ation state assignment by selecting a protonation state for each
chemical group that minimizes the total free energy of the system
(taking titration into account).54
from the crystallographic pose of ZM241385 mainly for the posi-
tion of the phenylethylamine chain, while the bicyclic triazolotri-
azine core is almost aligned. However, the mean RMSD value is
quite high for the most part of the docking protocols tested except
for GOLD. Docking performed with GOLD gives the lowest RMSD value,
the lowest mean RMSD value and the highest number of poses
with RMSD value <2.5 Å.
Based on the best docking performance, all antagonist struc-
tures were docked into the hypothetical TM binding site of the
hA3 AR and the rA2A AR models, by using the dock tool part of
the GOLD suite. Searching is conducted within a user-specified dock-
ing sphere, using the Genetic Algorithm protocol and the GoldScore
scoring function. GOLD performs a user-specified number of inde-
pendent docking runs (25 in our specific case) and writes the
resulting conformations and their energies in a molecular database
file. The resulting docked complexes were subjected to MMFF94x
energy minimization until the rms of conjugate gradient was
<0.1 kcal molꢀ1 Åꢀ1. Charges for the ligands were imported from
the MOPAC output files using PM3/ESP methodology.
Prediction of antagonist–receptor complex stability (in terms of
corresponding pKi value) and the quantitative analysis for non-
bonded intermolecular interactions (H-bonds, transition metal,
water bridges, hydrophobic, electrostatic) were calculated and
visualized using several tools implemented in MOE suite.39
Electrostatic and hydrophobic contributions to the binding en-
ergy of individual amino acids have been calculated based on the
AMBER99 force field as implemented in MOE suite.39 To estimate
the electrostatic contributions, atomic charges for the ligands were
calculated using PM3/ESP methodology; instead partial charges for
protein amino acids were calculated based on the AMBER99 force
field.
Protein stereochemical evaluation was then performed using
several tools (Ramachandran and Chi plots measure phi/psi and
chi1/chi2 angles, clash contacts reports) implemented in MOE
suite.49
4.3.2. Molecular docking of rA2A AR and hA3 AR antagonists
Ligand structures were built using MOE-builder tool, part of the
MOE suite,49 and were subjected to MMFF94x55 energy minimiza-
tion until the rms of conjugate gradient was 0.05 kcal molꢀ1 Åꢀ1
Charges were calculated using ESP methodology.
.
Acknowledgements
Four different programs have been used to calibrate our docking
The molecular modeling work coordinated by S.M. was carried
out with financial support from the University of Padova, Italy, and
the Italian Ministry for University and Research (MIUR), Rome,
Italy. S.M. is also very grateful to Chemical Computing Group for
the scientific and technical partnership. This research was sup-
ported in part by the Intramural Research Program of the NIH, Na-
tional Institute of Diabetes and Digestive and Kidney Diseases.
49
51
56
57
protocols: MOE-DOCK
,
GOLD
,
GLIDE
,
and PLANTS.
Therefore ZM241385 was re-docked to the crystal structure of
the hA2A AR (PDB code: 3EML) with different docking algorithms
and scoring functions (see Table 2).
Each docking was performed automatically to the binding site
of the hA2A AR without any constraints and without the presence
of water molecules. For all the different docking simulations, the
center of the docking box or of the docking sphere was set in the
same point (got from the experimental pose of ZM241385 inside
the crystal structure of hA2A AR) and the number of independent
docking runs was set to 25. Then RMSD values between predicted
and crystallographic positions of ZM241385 were calculated.
As shown in Table 2, for each docking result there is at least one
pose in good agreement with the experimental binding mode
(RMSD value <2.5 Å). These poses with lowest RMSD value differ
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