Synthesis and biological evaluation of a new series of 1, 2, 4-triazolo[1, 5-α]-1, 3, 5-triazines as human a2a adenosine receptor antagonists with improved water solubility

Article abstract of DOI:10.1021/jm101349u

The structure-activity relationship (SARof 1, 2, 4-triazolo[1, 5-a]-1, 3, 5-triazine derivatives related to ZM241385 as antagonists of the A_2A adenosine receptor (ARwas explored through the synthesis of analogues substituted at the 5 position. The A_2A AR X-ray structure was used to propose a structural basis for the activity and selectivity of the analogues and to direct the synthetic design strategy to provide access to solvent-exposed regions. Thus, we have identified a point of substitution for the attachment of solubilizing groups to enhance both aqueous solubility and physicochemical properties, maintaining potent interactions with the A_2A AR and, in some cases, receptor subtype selectivity. Among the most potent and selective novel compounds were a long-chain ether-containing amine congener 20 (K _i 11.5 nMand its urethane-protected derivative 14 (K_i 17.8 nM). Compounds 20 and 31 (K_i 11.5 and 16.9 nM, respectivelywere readily water-soluble up to 10 mM. The analogues were docked in the crystallographic structure of the hA_2A AR andina homology model of the hA₃ AR, and the per residue electrostatic and hydrophobic contributions to the binding were assessed and stabilizing factors were proposed.

Full text of DOI:10.1021/jm101349u

J. Med. Chem. 2011, 54, 877–889 877
DOI: 10.1021/jm101349u
Synthesis and Biological Evaluation of a New Series of 1,2,4-Triazolo[1,5-a]-1,3,5-triazines
as Human A2A Adenosine Receptor Antagonists with Improved Water Solubility
Stephanie Federico,# Silvia Paoletta,† Siew Lee Cheong,§ Giorgia Pastorin,§ Barbara Cacciari,‡ Stefano Stragliotto,†
Karl Norbert Klotz, Jeffrey Siegel,^ Zhan-Guo Gao,^ Kenneth A. Jacobson,^ Stefano Moro,*,† and Giampiero Spalluto*,#
#Dipartimento di Scienze Farmaceutiche, Universitaꢀ di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy, †Molecular Modeling Section (MMS),
Dipartimento di Scienze Farmaceutiche, Universitaꢀ di Padova, via Marzolo 5, I-35131 Padova, Italy, §Department of Pharmacy, National
University of Singapore, 3 Science Drive 2, Singapore 117543, ‡Dipartimento di Scienze Farmaceutiche, Universitaꢀ degli Studi di Ferrara,
via Fossato di Mortara 17-19, I-44100 Ferrara, Italy, Institut fu€r Pharmakologie, Universita€t of Wu€rzburg, D-97078 Wu€rzburg, Germany,
and ^Molecular Recognition Section (MRS), Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States
Received October 18, 2010
The structure-activity relationship (SAR) of 1,2,4-triazolo[1,5-a]-1,3,5-triazine derivatives related to
ZM241385 as antagonists of the A2A adenosine receptor (AR) was explored through the synthesis of
analogues substituted at the 5 position. The A2A AR X-ray structure was used to propose a structural basis
for the activity and selectivity of the analogues and to direct the synthetic design strategy to provide access to
solvent-exposed regions. Thus, we have identified a point of substitution for the attachment of solubilizing
groups to enhance both aqueous solubility and physicochemical properties, maintaining potent interactions
with the A2A AR and, in some cases, receptor subtype selectivity. Among the most potent and selective novel
compounds were a long-chain ether-containing amine congener 20 (Ki 11.5 nM) and its urethane-protected
derivative 14 (Ki 17.8 nM). Compounds 20 and 31 (Ki 11.5 and 16.9 nM, respectively) were readily water-
soluble up to 10 mM. The analogues were docked in the crystallographic structure of the hA2A AR and in a
homology model of the hA3 AR, and the per residue electrostatic and hydrophobic contributions to the
binding were assessed and stabilizing factors were proposed.
Adenosine receptors (ARs)a are members of the family of G
protein-coupled receptors (GPCRs), and are classified as four
subtypes, the A1AR, A2AAR, A2BAR, and A3AR,1 which exert
their physiological functions through the activation or inhibi-
tion of various second messenger systems. In particular, the
modulation of adenylyl cyclase activity could be considered to
be the principal signal mediated by these receptor subtypes.2,3
Activation or blockade of ARs is responsible for a wide
range of effects in numerous organ systems raising the possibi-
lity that the regulation of ARs has potential therapeutic
applications. The cardioprotective2,3 and neuroprotective4,5
effects associated with AR activation have been clearly demon-
strated during periods of cardiac and cerebral ischemia, res-
pectively. In addition, the use of antagonists of distinct AR
subtypes could be useful in the treatment of asthma6,7 or cer-
tain neurological diseases such as Parkinson’s disease.7,8 The
pathophysiological roles of ARs and their clinical potential
have been recently reviewed exhaustively.7-12
In recent years, an intensive effort performed by several
groups led to the synthesis of a large variety of AR agonists
and antagonists for the pharmacological characterization of
this family of G protein-coupled receptors.13 Among classes
of AR antagonists, diverse heterocyclic derivatives have been
proposed and found to display a range of affinities and selecti-
vities. In particular, our groups have extensively investigated
the pyrazolotriazolopyrimidine nucleus for AR antagonists.
Optimization of the substituents at the N5, N7, N8, and C2
positions led to potent and selective A2A and A3 AR antago-
nists, for example, compounds 1 and 2 (Chart 1).14-22
Nevertheless, most of these heterocyclic derivatives,
including other tricyclic structures, suffered from limited
aqueous solubility and, most importantly, difficulties in
their synthetic preparation.
Taking into account these problems, in recent years the
synthesis of more simplified heterocyclic derivatives has been
explored. In particular, bicyclic systems such as adenine,23
triazolopyrazine,24-26 and triazolotriazine27-29 could be consid-
ered some of the most promising targets. The triazolotriazine
nucleus was one of the most appealing bicyclic cores, which led
previously to the discovery of 4-[2-[7-amino-2-(2-furyl)-1,2,4-
triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenol (ZM241385,
3), that is, one of the most potent and selective A2A AR anta-
gonists known.30,31 This compound also binds with good affinity
to the human (h) A2B AR (28 nM), and its tritiated form is a
*To whom correspondence should be addressed. Tel.: þ39 040
5583726 (G.S.), þ39 049 8275704 (S.M.). Fax: þ39 040 52572 (G.S.),
þ39 049 8275366 (S.M.). E-mail: spalluto@units.it (G.S.); stefano.
moro@unipd.it (S.M.).
a Abbreviations: AR, adenosine receptor; CHO, Chinese hamster ovary;
DMSO, dimethylsulfoxide; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine;
EDTA, ethylenediaminetetraacetic acid; GPCR, G protein-coupled recep-
tor; HEK, human embryonic kidney; I-AB-MECA, 1-[6-[[(4-amino-3-
iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-β-D-ribofur-
anuronamide; NECA, 50-N-ethylcarboxamidoadenosine; TLC, thin layer
chromatography; ZM241385, 4-[2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a]-
[1,3,5]triazin-5-yl-amino]ethylphenol, SAR, structure-activity relation-
ship; TM, transmembrane; rmsd, root-mean-square deviation; EL2, second
extracellular loop.
r
2011 American Chemical Society
Published on Web 01/07/2011
pubs.acs.org/jmc

878 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Federico et al.
Chart 1. Pyrazolotriazolopyrimidines as A2A and A3 AR
Antagonists
Chart 3. Examples of Triazolotriazines as Nonselective A2B
and A3 AR Antagonists
Chart 4. Designed and Synthesized Compounds 7-43
Chart 2. Triazolotriazines as A2A AR Antagonists
useful radioligand for this receptor subtype.32 Recently, an inten-
sive study of the structure activity relationship (SAR) of the
triazolotriazine nucleus was reported, and compound 4proved to
be one of the most potent and selective for the A2A AR as
compared with the A1 AR. Nevertheless, the lack of binding data
at the A2B and A3 ARs prevented a comparison with other fully
characterized derivatives (Chart 2).27
Very recently, our group performed a study on this nucleus
trying to optimize substitution at the C5 and N7 positions with
the aim of improving affinity and selectivity versus the hA2B and
hA3 AR subtypes. In particular, inclusion at the N7 position of
arylcarbamoyl (for A3) or arylacetyl (for A2B) moieties, which
gave good results in the pyrazolotriazolopyrimidine family, has
been investigated.33 Unfortunately, none of these substitutions
led to the desired selectivity; in fact introduction of bulky
substituents at the N7 position (e.g., compound 5) significantly
increased the potency at the hA3 AR with respect to reference
compound 3 leading to low selectivity versus the A1 AR. In
contrast, a free amino group at the 7 position (compound 6)
provided intermediate potency at the A2B AR, but the potency
at the A2A AR still predominated (Chart 3).33
Considering theseexperimental observations, we decided to
further investigate the potential of this nucleus, in particular, by
exploring the C5 position through the introduction of substi-
tuted amino or diamino functions (Chart 4), with the aim to
modulate the activity at the A2A and A2B ARs and importantly
to improve the water solubility, which would otherwise limit
their use as pharmacological tools. In contrast, this new class of
derivatives mainly contained a free amino group at the 7
position, but small substituents were introduced in a few
compounds with the aim of increasing the interaction in the
receptor binding pocket.
Chemistry
The designed compounds (7-43) were synthesized as sum-
marized in Schemes 1-3. Reacting the well-known key
intermediate 4434 with pyrrolidine (51) or the appropriate
mono-tert-butyloxy-protected diamine (45-50), prepared
as previously reported,35 in ethanol solution in a sealed
tube at 120 ꢀC, the desired 5-substituted triazolotriazine
derivatives (7, 10-14) were obtained. Treatment of these
compounds (7, 10-14) with 10% trifluoroacetic acid in
CH2Cl2 at room temperature led to the corresponding
amino derivatives (9, 20, 30-33) as trifluoroacetate salts
(Scheme 1). Alternately, by reacting 44 with an excess of
mono-Boc-ethylendiamine (47) in a sealed tube at 180 ꢀC
the 5,7-disubstituted derivative (37) was obtained. After
N-Boc deprotection in acidic conditions, the bis-trifluoro-
acetate salt 38 was obtained (Scheme 1).
By treatment of derivative 9 with the appropriate benzyl
halide or acyl chloride in dry dioxane in the presence of
Et3N at room temperature, the corresponding N-benzyl
(15-19) or N-acyl (21-29, 34-36) derivatives were obtained
(Scheme 2).
Disubstituted derivatives at the 5 and 7 positions (39-43)
were prepared starting from 44 as depicted in Scheme 3.
Treating 44 with ethanolic ammonia in a sealed tube at
120 ꢀC afforded derivative 41. Otherwise, acylation at the
7 position with benzoyl chloride in standard conditions
led to compound 39, which after treatment in a sealed tube
at 120 ꢀC with ethanolic ammonia gave compound 43
(Scheme 3).
Instead, treatment of 44 with 2 N KOH in ethanol at reflux
led to the 5-hydroxy derivative 40, which after treatment with
benzoyl chloride in the presence of Et3N afforded the desired
product 42.
The analogues synthesized have been evaluated for potency
at all four hARs, and the results obtained were interpreted
with the help of computational methodologies.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 879
Scheme 1a
a Reagents: i: EtOH, sealed tube, amine excess, 180 ꢀC; ii: EtOH, sealed tube 120 ꢀC; iii: CF3COOH, CH2Cl2, rt.
Scheme 2a
a Reagents: i: RCH2Br, Et3N, dioxane, rt; ii: RCOCl, Et3N, dioxane, rt.
Molecular Modeling
synthesized compounds were docked to the binding cavity of
the A2A AR crystal structure, in order to evaluate their
binding mode at this receptor subtype. Moreover, to explain
the A2A/A3 selectivity profile, docking simulations were also
performed at the hA3 AR binding site. Since no crystallo-
graphic information is available for the A3 subtype, a pre-
viously proposed homology model of the hA3 AR based on
the crystal structure of the hA2A AR was used to perform the
docking studies.37,38
To better define the SAR profile of the AR antagonists
reported herein, a molecular modeling study was performed.
The recently published X-ray crystallographic structure of the
hA2A AR, in complex with the high affinity antagonist
ZM241385 (PDB code: 3EML),36 provides useful three-
dimensional structural information for performing molecular
docking studies of A2A AR antagonists. Therefore, all the

880 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Federico et al.
Scheme 3a
adenylyl cyclase experiments in CHO cells expressing the
hA2B AR.43,44
As clearly indicated in Table 1, all the analogues were in
general nearly inactive at the hA3 AR independent of the
substitution at the 5 and 7 positions, with the exception of
compounds 10 and 29 having affinity in the range of 1 μM. A
similar behavior, in contrast to expectations, could be ob-
served at the hA2B AR; in fact all the analogues were inactive
or poorly active (e.g., compounds 11, 31, 41, 43) in the
functional assay. Nevertheless, the binding profile of this class
of compounds at the hA2A AR demonstrated affinity in the
nanomolar range and with different degrees of selectivity
versus the hA1 AR subtype.
Insertion at the C5 position of amino or mono-Boc-diamino
(compounds 7, 8, 10-14) functionality led to compounds with
good affinity at the hA2A AR (range 18-70 nM) with variable
selectivity versus the A1 AR. In particular, the presence of a
pyrrolidine (8) at the 5 position favored affinity at the A2A AR
(59.1 nM) but poor selectivity (33-fold) versus the A1 subtype,
while introduction of Boc-piperidine (7) or Boc-ethylendiamine
(10) seem to improve both affinity and selectivity at the A2A AR
(e.g compound 7, hA2A = 21 nM, A1/A2A = 257). Interest-
ingly, elongation of the diamino chain at the C5 position to 3 or
5 carbon atoms (compounds 12, 13) led to derivatives which still
retained good affinity at the hA2A AR but less selectivity versus
a Reagents: i: NH3, EtOH, sealed tube, 120 ꢀC; ii: PhCOCl, Et3N,
dioxane reflux; iii: KOH, EtOH reflux.
the A1 subtype (e.g., compound 13 hA2A =44.7nM, A1/A2A
=
21.5), while longer chains such as a Boc-triethylenoxydiamino
moiety (14) gave good results both in terms of affinity and
selectivity (hA2A = 17.8 nM, A1/A2A = 89).
In the initial process of selecting a reliable docking protocol
to be employed in docking studies of the new derivatives, we
have evaluated the ability of different docking software pro-
grams to reproduce the crystallographic pose of ZM241385
inside the binding cavity of hA2A AR. As reported in the
Experimental Section, among the four different programs
used to calibrate our docking protocol, the Gold program
was finally chosen because it showed the best performance
with respect to the calculated rmsd values relative to the
crystallographic pose of ZM241385.37,39
Consequently, onthe basisofthe selected dockingprotocol,
weperformed docking simulationstoidentifythe hypothetical
binding mode of the new 1,2,4-triazolo[1,5-a]-1,3,5-triazines
inside the crystallographic structure of hA2A AR and the hA3
ARmodel. All the newly synthesized compoundsweredocked
into the orthosteric transmembrane (TM) binding cavities of
both ARs. Additionally, in order to analyze the possible
ligand-receptor recognition mechanism in a more quantita-
tive manner, we calculated the individual electrostatic and
hydrophobic contributions to the interaction energy of each
receptor residue involved in the binding with ligands. Using
the calculated electrostatic and hydrophobic contributions
values, color maps of electrostatic and hydrophobic interac-
tions per residue were constructed.
Trifluoroacetate salt derivatives (9, 30-33) were in general
less potent than the corresponding Boc derivatives (e.g.,
compound 7, hA2A = 21 nM, A1/A2A= 257 versus com-
pound 9, hA2A = 153 nM, A1/A2A = 65). An exception
occurred when long or bulky chains were present at the C5
position. In fact, compounds 20 and 31 were more potent at
the hA2A AR than the corresponding Boc derivatives (e.g.,
compound31, hA2A = 16.9 nM, A1/A2A = 79; compound11:
hA2A = 69.7 nM, A1/A2A = 56), but most importantly both
compounds were readily water-soluble up to 10 mM. In
particular, compound 20, which displayed an affinity at the
hA2A AR of 11.5 nM and good selectivity versus A1, was the
most potent compound of this series. Despite this relevant
improvement of the water solubility other pharmacokinetics
properties, such as cell permeation or metabolic stability, are
collecting in our laboratories before setting up any further in
vivo testing.
Interestingly, double substitution with diamino functions at
the C5 and N7 positions led to completely inactive com-
pounds at all four AR subtypes, both as protected (37) or
deprotected (38) forms.
An altered binding profile was observed for the piperazine
derivatives alkylated or acylated at the piperazine secondary
amine. In particular, in the N-benzyl series (15-19) good
affinity at the hA2A AR was retained, but also an increased
affinity at the hA1 was observed with a consequent reduction
of selectivity, independently of the type of substitution on the
phenyl ring.
Results and Discussion
Table 1 reports the receptor binding affinities of the synthe-
sized compounds (7-43). The binding properties were deter-
mined at the hA1, hA2A, and hA3 ARs expressed in human
embryonic kidney (HEK)-293 cells using as radioligands: [3H]8-
cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, A1);40 [3H]-
(4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-
5-yl-amino]ethyl)-4-iodo-phenol, ([3H]ZM241385, A2A);41 or in
Chinese hamster ovary (CHO) cells using [125I]N6-(4-amino-
3-iodobenzyl)-50-N-methylcarbamoyladenosine ([125I]I-AB-
MECA, A3).42 Because of the lack of a suitable hA2B AR
radioligand, the activity of antagonists was determined in
A more complex profile was present when an acyl group
was present on the piperazine secondary amine. In particular,
when a benzoyl group was introduced on the piperazine
nitrogen (26, 27) a significant reduction of affinity at the
hA2A AR (range 212-348 nM) was observed with a subse-
quent reduction of selectivity (35-48 fold). If the phenyl ring
was replaced with a more bulky substituent such as a naphthyl

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 881
Table 1. Structures and Binding Profile of Synthesized Compounds 7-43
a Displacement of specific [3H]DPCPX binding at the hA1 AR expressed in HEK-293 cells. b Displacement of specific [3H]ZM241385 binding at hA2A
AR expressed in HEK-293 cells. Data are expressed as Ki ( SEM in nM (n = 3-6). c Measurement of adenylyl cyclase activity in CHO cells stably
transfected with recombinant hA2B AR, expressed as IC50 (nM). d Displacement of specific [125I]I-AB-MECA binding at hA3 receptors expressed in
CHO cells. Data are expressed as Ki ( SEM in nM (n = 3-6). e Selectivity not determined or estimated.

882 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Federico et al.
nucleus (compound 28), a complete loss of affinity at all four
hARs was observed.
If the aroyl group was replaced with an aryl acetyl moiety,
significant differences could be observed in the binding pro-
file. In particular, introduction of a phenylacetyl (23) or a
4-substituted-phenylacetyl group (21, 29) at the piperazine
secondary amine led to compounds that still retained good
affinity at the hA2A AR in the range of 40-95 nM with poor
selectivity versus A1 (12-37 fold). In contrast, introduction of a
bulky substituent such as a diphenylacetyl moiety (34) provided
a compound with a quite good affinity at the hA2A AR (hA2A
=
93 nM). Importantly, the selectivity versus the hA1 subtype (A1/
A2A = 80) was high, which was exactly the opposite observed for
the nearly inactive bulky aroyl derivative 28.
A quite different profile could be observed when the
piperazine secondary amine was acylated with alkyl groups.
In particular, introduction of a small group (25) or unbranched
chains (22, 24) led to compounds which proved to be quite
potent (range 66-280 nM) at the hA2A AR. Branched chains
such as tert-butylcarbonyl (35) and 3,3-dimethylbutanoyl (35)
increased the affinity at the hA2A AR and significantly
increased selectivity versus the hA1 subtype. In particular,
derivative 36 showed high affinity at the hA2A AR (11.1 nM)
and 900-fold selectivity versus the hA1 subtype.
The derivatives substituted both at the C5 and N7 positions
with small groups (39-43) showed in general weak affinity at
the hA2A AR and low levels of selectivity. Moreover, the
diamino compound 41 and the corresponding 5-hydroxy
derivative 40 showed hA2A affinity in the high nanomolar
range (160-200 nM), but the levels of selectivity versus the
hA1 AR were very low, ranging from 2 to 12. When a benzoyl
group was present at the N7 position, the presence at the C5
position of a hydroxy (42) or phenoxy (39) moiety greatly
reduced activity at the four ARs, with affinity at the hA2A
subtype in the micromolar range. In contrast, when an amino
group was present at the C5 position, affinity at the hA2A AR
(hA2A = 44.1 nM) was recovered with good levels of selec-
tivity versus other receptor subtypes.
In order to rationalize the observed binding data, a molec-
ular modeling investigation was performed for all the newly
synthesized analogues using both the crystallographic struc-
ture of hA2A AR and the hA3 AR model. The analysis was
extended to docking simulations and per residue electrostatic
and hydrophobic contributions maps.
The first important consideration is that almost all the
selected poses at the hA2A AR of these new analogues showed
some common features, as highlighted by the calculated
electrostatic and hydrophobic contributions to the interaction
energy collected in Figure 1. In particular, all ligands made
contacts mainly with residues belonging to TM2, TM3, TM6,
TM7, and EL2.
The per residue electrostatic interaction energy map
(Figure 1A) showed two bands with negative energy
(colored in green) corresponding to Glu169 in EL2 and
Asn253 in TM6, indicating that these two residues were
responsible for the main electrostatic interactions with all
the tested analogues, with a few exceptions. On the other
hand, the map of the per residue hydrophobic interaction
score (Figure 1B) highlighted several residues involved in
hydrophobic contacts with ligands, including Leu85 in
TM3, Phe168 in EL2, Trp246, Leu249, His250 in TM6 and
Tyr271, Ile274 in TM7. Therefore, the analysis of these maps
gave important preliminary data concerning similarity and
differences in the binding modes at the hA2A AR of these new
Figure 1. (A) Per residue electrostatic interaction energy map and
(B) per residue hydrophobic interaction score map. The maps are
calculated for a selected pose of each compound (7-43) inside the
hA2A AR binding site. Electrostatic energy values are expressed in
kcal/mol, while hydrophobic scores are expressed in arbitrary
hydrophobic units.
compounds; this information was then confirmed by a detailed
investigation of the docking poses as reported below.
Docking of 7-Amino Derivatives (Compounds 7-36, 40,
and 41). From the docking simulation analysis, all the new
derivatives with free amino group at the 7 position, with the
exception of compound 28, were seen to share a similar
binding pose in the TM region of the hA2A AR. For these
compounds, ligand-recognition occurred in the upper region
of the TM bundle, and the triazolotriazine nucleus was
surrounded by TMs 3, 5, 6, 7 with the 2-furyl ring located
deep in the binding cavity.
Considering Figure 2, it is evident that the binding poses of
these ligands were very similar to the crystallographic pose of
ZM241385 bound to the hA2A AR.36 In fact, the triazolo-
triazine cores were completely superposable, while the only
slight difference was in the orientation of the substituents at
the 5-position. Moreover, all the crucial interactions estab-
lished by ZM241385 with amino acid residues of the hA2A
AR binding site were also found for all these new 7-amino
derivatives.
The analysis in Figure 3 (panel A) showing the hypothe-
tical binding pose of compound 36 (Ki hA2A = 11.1 nM) at
the hA2A AR helps to clarify this point. It appeared that the
bicyclic triazolotriazine core was anchored within the bind-
ing cleft through an aromatic stacking interaction with
Phe168 (EL2) and a H-bonding interaction with Asn253
(6.55). Moreover, the exocyclic amino group at the 7-posi-
tion of the bicyclic core interacted with two polar residues,
Asn253 (6.55) and Glu169 (EL2), forming two H-bonds.
Interestingly, the important role in ligand binding of these

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 883
compared to the ones at the hA2A AR. Moreover, the residues
present at the binding pocket entrance in the two AR subtypes
possess very different features, which could affect both the
orientation of the ligand while approaching the binding pocket
and its accommodation into the final TM binding cleft, as
already proposed for other compounds.37 Therefore, both the
lack of very strong interactions with the residues of the hA3 AR
and the differences at the binding site entrance are consistent
with a lack of hA3 affinity observed for the 7-amino derivative.
Among the 7-amino derivatives, only compound 28 (Ki
hA2A = 7680 nM) showed a different docking pose at the
hA2A AR (see Supporting Information). This ligand, prob-
ably due to the hindrance of the bulky and rigid R-naphthyl
group, was not able to occupy the same position as the other
7-amino derivatives and consequently lost important
H-bonding interactions with two critical residues of the
hA2A AR binding site, such as Glu169 (EL2) and Asn253
(6.55). In fact, in this docking pose the ligand formed only
one H-bond with Tyr271 (7.36). This finding explained why
compound 28 showed lower hA2A AR affinity compared to
the other 7-amino derivatives.
Docking of 7-(Alkyl/acyl)amino Derivatives (Compounds
37-39, 42 and 43). With the exception of compound 43, all
the 7-(alkyl/acyl)amino derivatives showed low affinity for
the hA2A AR (micromolar range). Docking studies revealed
that compounds 37-39 and 42 possessed a different binding
mode at the hA2A AR compared to the 7-amino derivatives
(see Supporting Information). In fact, the presence of an
(alkyl/acyl)amino group at the 7 position prevented these
compounds from forming a H-bonding network with
Asn253 (6.55) and Glu169 (EL2), already seen to be critical
for the binding of ZM241385 at this receptor subtype. This
fact led these compounds to assume a different orientation
inside the binding cavity of the receptor, although ligand-
recognition occurred in the same upper region of the TM
bundle, and the triazolotriazine nucleus was surrounded by
TMs 3, 5, 6, 7 with the 2-furyl ring directed toward the inner
part of the binding cavity. Therefore, their binding mode
showed only a weak H-bond with Glu169 (EL2) and a
stacking interaction with Phe168 (EL2). These findings were
in agreement with the experimental data showing micromolar
Ki values for these compounds.
In contrast to the 5 position derivatives described above,
compound 43 (Ki hA2A = 44.1 nM), due to the presence of a
free amino group at C5, showed a characteristic mode of
binding at the hA2A AR (see Supporting Information). The
triazolotriazine nucleus was oriented parallel to the membrane
plane, and the 2-furyl ring was directed toward TM2, while the
substituent at the 7 position was located in the inner part of the
binding cavity. Compound 43 formed three H-bonding inter-
actions, two with Asn253 (6.55) and one with Glu169 (EL2),
and a π-π stacking interaction with Phe168 (EL2). Therefore,
the presence of a H-bonding network with the residues of the
hA2A AR, similar to the one seen for the 7-amino derivatives,
seemed to explain why compound 43, among the 7-(alkyl/
acyl)amino derivatives, is the only one that showed affinity at
the hA2A AR in the nanomolar range.
In conclusion, we have synthesized AR antagonists of the
triazolotriazine class and use molecular modeling studies to
explain the SAR. Among the most potent and selective novel
compounds were a long-chain ether-containing amine con-
gener 20 and its urethane-protected derivative 14. Com-
pound 20 and a 5-(aminomethyl)cyclohexylmethyl-amino
derivative 31 were readily water-soluble up to 10 mM, thus
Figure 2. Structure superimposition of the crystallographic pose of
ZM241385 (in magenta) and of the docking poses of all the 7-amino
derivatives (in white) inside the hA2A AR binding site. Side chains of
some amino acids important for ligand recognition and H-bonding
interactions are highlighted. Hydrogen atoms are not displayed.
two residues was previously revealed by site-directed muta-
genesis studies.45,46 The 4-(3,3-dimethylbutanoyl)-piperazinyl
chain of the ligand was directed toward the more solvent-
exposed extracellular region (EL2 and EL3) and interacted,
through a H-bond, with Tyr271 (7.36). However, the furan ring
was located deep within the ligand binding cavity and formed
hydrophobic interactions with the highly conserved Trp246
(6.48), an important residue in receptor activation. Finally,
compound 36 also formed hydrophobic interactions with many
residues of the binding site including Val84 (3.32), Leu85 (3.33),
Met177 (5.38), Leu249 (6.51), and Ile274 (7.39).
Analyzing the electrostatic contribution per residue to the
whole interaction energy for the compound 36-hA2A AR
complex (Figure 3C), the three main stabilizing factors were
found to be related to Glu169 (EL2), Asn253 (6.55), and Tyr271
(7.36), due to the H-bonding interactions with the ligand above-
described, whereas, as shown in Figure 3 (panel E), the hydro-
phobic interaction scores pattern showed two strong stabilizing
contributions corresponding to the interactions of the bicyclic
core with Phe168 (EL2) and Leu249 (6.51).
Therefore, as exemplified by the binding pose of compound
36, all the newly synthesized 7-amino derivatives strongly inter-
acted with the hA2A AR in a manner similar to the crystallo-
graphic pose of ZM241385.36 Moreover, these compounds,
thanks to their different substituents at the 5 position, could
variously interact with residues of the upper region of the
receptor binding cavity, particularly in EL2 and EL3. Consider-
ing that the substituents at the 5 position were exposed to the
highly plastic EL region and to solvent, it was difficult to define a
clear SAR at the 5 position for this series.
On the other hand, the docking pose of compound 36 at
the hA3 AR was located in the same region of the TM bundle
as at the hA2A AR, but the orientation of the ligand was
different (Figure 3B). In this case, the ligand formed only two
H-bonds with Asn250 (6.55) and lost the aromatic interaction
with Phe168 (EL2). The patterns of electrostatic and hydro-
phobic contribution to the energy of hA3 AR-ligand com-
plexes (Figure 3D,F) showed weaker per residue contributions

884 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Federico et al.
Figure 3. Hypothetical binding modes of compound 36 obtained after docking simulations: (A) inside the hA2A AR binding site; (B) inside the
hA3 AR binding site. Poses are viewed from the membrane side facing TM6, TM7, and TM1. The view of TM7 is partially omitted. Side chains
of some amino acids important for ligand recognition and H-bonding interactions are highlighted. Hydrogen atoms are not displayed.
Electrostatic interaction energy (in kcal/mol) between the ligand and each single amino acid involved in ligand recognition observed from the
hypothetical binding modes of compound 36 inside (C) hA2A AR and (D) hA3 AR binding sites. Hydrophobic interaction scores (in arbitrary
hydrophobic units) between the ligand and each single amino acid involved in ligand recognition observed from the hypothetical binding modes
of compound 36 inside (E) hA2A AR and (F) hA3 AR binding sites.
overcoming a common limitation of other bicyclic and
tricyclic AR antagonists. N-Alkylated and N-acylated piper-
idine derivatives also displayed high affinity at the human
A2A AR, including an N-benzyl antagonist 19 and an N-3,3-
dimethylbutanoyl derivative 36 that was roughly 900-fold
selective versus both human A1 and A3 ARs. The analogues
were docked in the crystallographic structure of hA2A AR
and in a homology model of the hA3 AR, and the per residue
electrostatic and hydrophobic contributions to the binding
were assessed. The presence of a free amino group that
seemed to be critical for the hA2A affinity by allowing the
ligands to participate in a H-bonding network with two
critical residues of the binding site, Asn253 (6.55) and
Glu169 (EL2). Another interaction found to be important
for the hA2A affinity of this series was the aromatic stacking
between the triazole ring and Phe168 (EL2).
Conclusion
The present study has led to the identification of a new class
of promising AR antagonists, the 1,2,4-triazolo[1,5-a]-1,3,5-
triazine derivatives related to ZM241385. In particular, com-
pound 20 (Ki 11.5 nM) and a 5-(aminomethyl)cyclo-
hexylmethyl-amino derivative 31 (Ki 16.9 nM) were readily
water-soluble up to 10 mM, thus overcoming a common
limitation of other bicyclic and tricyclic AR antagonists. Finally,
a receptor-based SAR has been proposed using a molecular
modeling approach. All analogues were docked in the recently
published crystallographic structure of hA2A AR and in a
homology model of the hA3 AR, and the per residue electrostatic
and hydrophobic contributions to the binding were assessed.
Molecular modeling results highlighted that all the newly
synthesized 1,2,4-triazolo[1,5-a]-1,3,5-triazine derivatives with
free amino group at the 7 position are characterized by a

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 885
common binding mode very similar to the crystallographic one
of ZM241385 bound to the hA2A AR. In fact, all the interac-
tions found to be crucial for the X-ray structure, in particular
the hydrogen bonding network with Asn253 (6.55), the Glu169
(EL2), and the aromatic stacking with Phe168 (EL2), were also
important for anchoring these new derivatives to the hA2A AR
binding site. On the contrary, substitution at the 7 position was
detrimental for the affinity at the hA2A AR, as confirmed also
by the orientation of the 7-(alkyl/acyl)amino derivatives inside
the binding cavity that led to the loss of the stabilizing hydrogen
bonding network.
1H NMR (CDCl3) δ: 1.2-2.0 (m, 6H); 1.4 (s, 9H); 2.9-3.2
(m, 2H); 3.3-3.5 (m, 2H); 4.6 (bs, 1H); 5.4 (bs, 1H); 6.7 (dd, 1H,
J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.3 (bs, 2H); 7.9 (bs, 2H). IR
(Nujol) cm-1: 3450-3100, 1735, 1620, 1470. Anal. (C21H28-
N8O3) C, H, N.
7-Amino-5-[(2-tert-butyloxycarbonylamino)ethyl]-amino-2(2-
furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (10). Yield 63%; white
solid (EtOAc-light petroleum) mp 168 ꢀC. 1H NMR (DMSO-d6)
δ: 1.4 (s, 9H); 3.1-3.4 (m, 4H); 6.7 (dd, 1H, J = 2, J = 4); 6.9 (bs,
1H); 7.1 (d, 1H, J = 2); 7.4 (bs, 1H); 7.9 (d, 1H, J = 4); 8.2 (bs,
2H). IR (Nujol) cm-1: 3450-3150, 1740, 1630, 1500. Anal.
(C15H20N8O3) C, H, N.
7-Amino-5-[(3-tert-butyloxycarbonylamino)propyl]-amino-2(2-
furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (12). Yield 76%; white
solid (EtOAc-light petroleum) mp 155 ꢀC. 1H NMR (CDCl3) δ:
1.5 (s, 9H); 1.80-1.85 (m, 2H); 3.20-3.25 (m, 2H); 3.5-3.6 (m,
2H); 4.9 (bs, 1H); 5.4 (bs, 1H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d,
Thus, we have probed points of substitution for attachment
of solubilizing groups to enhance the aqueous solubility of this
class of triazolotriazines, which are characterized by poor
physicochemical properties. At the same time, potent interac-
tions with the A2A AR and, in some cases, receptor subtype
selectivity have been maintained. We have used the A2A AR
X-ray structure to propose a structural basis for the activity and
selectivity of this class of analogues and to direct the synthetic
design strategy to provide access to solvent-exposed regions.
In general, the strategy of grafting a terminal polar tail on a
pharmacophore, which increases the polar surface area, can
have a detrimental effect on bioavailability and ion channel
activity. Therefore, it will be necessary to evaluate these mole-
cules in further pharmacological testing to see if they will be
useful for in vivo studies.
1H, J = 2); 7.9 (d, 1H, J = 4); 7.9 (bs, 2H). IR (Nujol) cm-1
3400-3100, 1740, 1620, 1510. Anal. (C16H22N8O3) C, H, N.
7-Amino-5-[(5-tert-butyloxycarbonylamino)-n-pentyl]-amino-
:
2(2-furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (13). Yield 71%;
white solid (EtOAc-light petroleum) mp 140 ꢀC. 1H NMR
(CDCl3) δ: 1.4 (s, 9H); 1.6-1.8 (m, 6H); 3.2-3.3 (m, 2H);
3.50-3.55 (m, 2H); 4.7 (bs, 1H); 5.5 (bs, 1H); 6.40-6.43 (m,
2H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.9 (d, 1H,
J = 4). IR (Nujol) cm-1: 3450-3150, 1735, 1625, 1475. Anal.
(C18H26N8O3) C, H, N.
7-Amino-5-[(8-tert-butyloxycarbonylamino)triethylenoxy]-
amino-2(2-furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (14). Yield
64%; brown solid (EtOAc-light petroleum) mp 130 ꢀC. 1H
NMR (CDCl3) δ: 1.5 (s, 9H); 3.4-3.8 (m, 12H); 5.5 (bs, 1H);
6.2 (bs, 1H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.9 (d,
1H, J = 4); 9.4 (bs, 2H). IR (Nujol) cm-1: 3400-3100, 1740,
1610, 1450. Anal. (C19H28N8O5) C, H, N.
Procedure for Preparation of 5,7-bis-[(2-tert-butyloxycar-
bonylamino) ethyl]-amino-2-(2-furyl)-1,2,4-triazolo [1,5-a] 1,3,
5-triazine (37). A mixture of compound 44 (0.29 g, 1 mmol)
and the mono Boc ethylendiamine (47) (0.8 g, 5 equiv) in
absolute ethanol (5 mL) was poured into a sealed tube and
heated at 180 ꢀC for 8 h. Then the solvent was removed under
reduced pressure and the residue was crystallized from EtOAc-
light petroleum to afford the desired compound (37) as a white
solid in a good yield (70%). mp 154 ꢀC. 1H NMR (DMSO-d6) δ:
1.4 (s, 18H); 3.0-3.4 (m, 8H); 6.7 (dd, 1H, J = 2, J = 4); 6.9 (bs,
1H); 7.1 (d, 1H, J = 2);7.6 (bs, 1H); 7.9 (d, 1H, J = 4); 8.5 (bs,
1H); 8.7 (bs, 1H). IR (Nujol) cm-1: 3450, 1730, 1610, 1450. Anal.
(C22H33N9O5) C, H, N.
Experimental Section
Chemical Synthesis. General. Reactions were routinely mon-
itored by thin-layer chromatography (TLC) on silica gel
(precoated F254 Merck plates). Infrared spectra (IR) were
measured on a Jasco FT-IT 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 (δ)
downfield from tetramethylsilane as internal standard, and J values
are given in Hz. The following abbreviations were used: s, singlet;
bs, broad singlet; d, doublet; dd, double doublet; t, triplet; m,
multiplet.
Light petroleum ether refers to the fractions boiling at 40-60 ꢀC.
Melting points were determined on a Buchi-Tottoli instrument and
are uncorrected. Flash chromatography was performed using
Merck 60-200 mesh silica gel. Purity of compounds was detected
by elemental analyses performed at the laboratory of microanalysis
of the Department of Chemistry, University of Trieste (Italy), and
all the compounds were confirmed to achieve 95% purity.
General Procedure for Nucleophilic Substitution with Amino
Compounds (7, 8, 10-14). A mixture of compound 44 (0.29 g,
1 mmol) and the appropriate amine (45-51) (1.5 equiv) in abso-
lute ethanol (5 mL) was poured into a sealed tube and heated at
120 ꢀC for 3 h. Then the solvent was removed under reduced
pressure and the residue crystallized from EtOAc-light petro-
leum to afford the desired compounds (7, 8, 10-14) as solids.
7-Amino-5-(N-pyrrolidinyl)-2(2-furyl) 1,2,4-triazolo [1,5-a]
1,3,5-triazine (8). Yield: 88%; brown solid (EtOAc-light pet-
General Procedure for N-Boc Deprotection (9, 20, 30-33, 38).
Ten millimoles of the appropriate N-Boc derivative (7, 8, 10-14,
37) was suspended in a 10% solution of CF3COOH in dry
CH2Cl2 and stirred at room temperature for 3 h. Then the
solvent was removed under reduced pressure and the residue was
crystallized from AcOEt-light petroleum to give the desired
trifluoroacetate salts (9, 20, 30-33, 38) as solids.
5,7-Bis-(amino)ethyl-amino-2(2-furyl) 1,2,4-triazolo [1,5-a] 1,
3,5-triazine bis-trifluoroacetate (38). Yield 63%; brown solid
1
(EtOAc-light petroleum) mp 203 ꢀC. H NMR (DMSO-d6) δ:
1
3.4-3.6 (m, 8H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2);
roleum) mp > 300 ꢀC (dec.) H NMR (DMSO-d6) δ: 1.9-2.1
7.9 (d, 1H, J = 4); 8.0 (bs, 1H); 8.0 (bs, 3H). IR (Nujol) cm-1
3500-3100, 1630, 1500. Anal. (C16H19N9O5 F6) C, H, N.
:
(m, 4H); 3.4-3.7 (m, 4H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H,
J = 2); 7.9 (d, 1H, J = 4); 8.4 (bs, 2H). IR (Nujol) cm-1
3350-3200, 1620, 1480. Anal. (C12H13N7O) C, H, N.
:
7-Amino-5-(1-piperazinyl)-2(2-furyl) 1,2,4-triazolo [1,5-a] 1,3,
5-triazine trifluoroacetate (9). Yield 58%; brown solid (EtOAc-
light petroleum) mp 243 ꢀC. 1H NMR (DMSO-d6) δ: 3.20-3.25
(m, 4H); 4.0-4.1 (m, 4H); 6.0 (bs, 2H); 6.7 (dd, 1H, J = 2, J = 4);
7.1 (d, 1H, J = 2); 7.9 (d, 1H, J = 4); 9.0 (bs, 2H). IR (Nujol)
cm-1: 3500-3000, 1630, 1510. Anal. (C14H15N8O3F3) C, H, N.
7-Amino-5-(aminomethyl)cyclohexylmethyl-amino-2(2-furyl) 1,
2,4-triazolo [1,5-a] 1,3,5-triazine trifluoroacetate (31). Yield 66%;
brown solid (EtOAc-light petroleum) mp 150 ꢀC. 1H NMR
(DMSO-d6) δ: 1.2-2.0 (m, 10H); 2.4-3.0 (m, 4H); 6.7 (dd, 1H,
7-Amino-5-[(4-tert-butyloxycarbonyl)-1-piperazinyl]-2(2-furyl) 1,
2,4-triazolo [1,5-a] 1,3,5-triazine (7). Yield 73%; white solid
(EtOAc-light petroleum) mp 250 ꢀC. 1H NMR (DMSO-d6) δ: 1.4
(s, 9H); 3.4 (s, 4H); 3.8 (s, 4H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d,
1H, J = 2); 7.9 (d, 1H, J = 4); 8.4 (bs, 2H). IR (Nujol) cm-1
:
3400-3150, 1735, 1630, 1510. Anal. (C17H22N8O3) C, H, N.
7-Amino-5-[(4-tert-butyloxycarbonylaminomethyl)cyclohexyl-
methyl]-amino-2(2-furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (11).
Yield 76%; white solid (EtOAc-light petroleum) mp 148 ꢀC.

886 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Federico et al.
J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.9 (d, 1H, J = 4); 8.0 (bs, 2H).
IR (Nujol) cm-1: 3500-3050, 1640, 1500. Anal. (C18H23N8O3F3)
C, H, N.
(10 mL) was stirred at room temperature for 2 h. The solvent was
removed in vacuo and the residue was dissolved in EtOAc (15 mL)
and washed with water (3 ꢀ 5 mL). The organic phase was dried
and solvent was removed under reduced pressure. The crude was
purified by flash chromatography (EtOAc) to give the final com-
pounds (21-29, 34-36) as solids.
7-Amino-5-(2-aminoethyl)-amino-2(2-furyl) 1,2,4-triazolo [1,5-a]
1,3,5-triazine trifluoroacetate (30). Yield 54%; brown solid (EtOAc-
light petroleum) mp 243 ꢀC. 1H NMR (DMSO-d6) δ: 3.4-3.6 (m,
4H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.5 (d, 1H, J =
7-Amino-5-[(4-(pentanoyl))-1-piperazinyl]-2(2-furyl) 1,2,4-triazolo
[1,5-a] 1,3,5-triazine (22). Yield 66%; yellow solid (EtOAc-light
petroleum) mp 167 ꢀC. 1H NMR (DMSO-d6) δ: 0.9 (t, 3H, J = 7);
3.0-3.2 (m, 10H); 3.5 (s, 2H); 3.8 (s, 2H); 6.7 (dd, 1H, J=2, J=4);
4); 7.8 (bs, 2H); 8.4 (bs, 2H); 8.0 (bs, 3H). IR (Nujol) cm-1
:
3450-3150, 1630, 1510. Anal. (C12H13N8O3F3) C, H, N.
7-Amino-5-(3-amino)propyl-amino-2(2-furyl) 1,2,4-triazolo [1,5-a]
1,3,5-triazine trifluoroacetate (32). Yield 69%; brown solid (EtOAc-
light petroleum) mp 235 ꢀC. 1H NMR (DMSO-d6) δ: 3.4-3.6 (m,
6H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.5 (d, 1H, J =
7.1 (d, 1H, J=2);7.9 (d, 1H, J= 4); 8.4 (bs, 2H). IR (Nujol) cm-1
:
3350-3100, 1685, 1620, 1500. Anal. (C17H22N8O2) C, H, N.
7-Amino-5-[(4-(benzoyl))-1-piperazinyl]-2(2-furyl) 1,2,4-triazolo
[1,5-a] 1,3,5-triazine (27). Yield 69%; white solid (EtOAc-light
petroleum) mp 290 ꢀC. 1H NMR (DMSO-d6) δ: 3.4-3.9 (m,
8H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.5 (m, 5H);
7.9 (d, 1H, J = 4); 8.4 (bs, 2H). IR (Nujol) cm-1: 3360-3120, 1670,
1620, 1510. Anal. (C19H18N8O2) C, H, N.
4); 7.8 (bs, 2H); 8.4 (bs, 2H); 8.0 (bs, 3H). IR (Nujol) cm-1
:
3470-3140, 1640, 1510. Anal. (C13H15N8O3F3) C, H, N.
7-Amino-5-(5-amino)pentyl-amino-2(2-furyl) 1,2,4-triazolo [1,5-a]
1,3,5-triazine trifluoroacetate (33). Yield 71%; brown solid (EtOAc-
light petroleum) mp 160 ꢀC. 1H NMR (DMSO-d6) δ: 1.6-1.8 (m,
6H);3.2(m, 2H);3.5(m, 2H);5.5(bs, 1H);6.4(bd, 2H);6.7(dd, 1H,
J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.9 (d, 1H, J = 4); 8.0 (bs, 3H).
IR (Nujol) cm-1: 3480-3120, 1640, 1500. Anal. (C15H19N8O3F3)
C, H, N.
7-Amino-5-[(4-(benzyl-carbonyl))-1-piperazinyl]-2(2-furyl) 1,2,4-
triazolo [1,5-a] 1,3,5-triazine (23). Yield 64%; pale brown solid
1
(EtOAc-light petroleum) mp 240 ꢀC. H NMR (DMSO-d6) δ:
3.5-3.7 (m, 8H); 3.8 (s, 2H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H,
J =2); 7.2-7.4 (m, 5H); 7.9 (d, 1H, J = 4); 8.4 (bs, 2H). IR (Nujol)
cm-1: 3320-3100, 1680, 1610, 1500. Anal. (C20H20N8O2) C, H, N.
7-Amino-5-[(4-(2-bromo-benzyl-carbonyl))-1-piperazinyl]-2(2-
furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (21). Yield 76%; white
solid (EtOAc-light petroleum) mp 223 ꢀC. 1H NMR (DMSO-d6)
δ: 3.5-3.7 (m, 8H); 3.8 (s, 2H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d,
1H, J = 2); 7.2-7.4 (m, 4H); 7.9 (d, 1H, J = 4); 8.4 (bs, 2H). IR
(Nujol) cm-1: 3315-3080, 1670, 1620, 1500. Anal. (C20H19N8-
O2Br) C, H, N.
7-Amino-5-[(8-amino)triethylenoxy]-amino-2(2-furyl) 1,2,4-tria-
zolo [1,5-a] 1,3,5-triazine trifluoroacetate (20). Yield 52%; brown
solid (EtOAc-light petroleum) mp 243 ꢀC. 1H NMR (DMSO-d6) δ:
3.4-3.8 (m, 12H); 5.5 (bs, 1H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d,
1H, J = 2); 7.9 (d, 1H, J = 4); 9.4 (bs, 2H); 8.0 (bs, 3H). IR (Nujol)
cm-1: 3510-3150, 1630, 1510. Anal. (C15H22N8O5F3) C, H, N.
General Procedure for N-Benzylation of a Piperazine Moiety
(15-19). A mixture of compound 9 (0.2 g, 0.05 mmol) the
appropriate benzyl halide (1.2 equiv), Et3N (7.2 μL, 0.06 mmol)
in dry dioxane (10 mL) was stirred at room temperature for 6 h.
The solvent was removed in vacuo and the residue was dissolved
in EtOAc (15 mL) and washed with water (3 ꢀ 5 mL). The
organic phase was dried and solvent was removed under reduced
pressure. The crude was purified by flash chromatography
(EtOAc) to give the final compounds (15-19) as solids.
7-Amino-5-[(4-benzyl)-1-piperazinyl]-2(2-furyl) 1,2,4-triazolo
[1,5-a] 1,3,5-triazine (15). Yield 63%; white solid (EtOAc-light
petroleum) mp 242 ꢀC. 1H NMR (DMSO-d6) δ: 2.5 (s, 4H); 3.6
(s, 2H); 3.9 (s, 4H); 6.2 (bs, 2H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d,
1H, J = 2); 7.6 (d, 1H, J = 4); 7.1-7.4 (m, 5H). IR (Nujol)
cm-1: 3350-3100, 1610, 1470. Anal. (C19H20N8O) C, H, N.
7-Amino-5-[(4-(3-chloro-benzyl)-1-piperazinyl]-2(2-furyl) 1,2,4-
triazolo[1,5-a] 1,3,5-triazine (16). Yield 70%; white solid (EtOAc-
light petroleum) mp 227 ꢀC. 1H NMR (DMSO d6) δ: 2.5 (s, 4H);
3.5 (s, 2H); 3.8 (s, 4H); 6.7 (dd, 1H, J=2, J= 4); 7.1 (d, 1H, J=2);
7-Amino-5-[(4-(methoxycarbonylethylcarbonyl))-1-piperazinyl]-
2(2-furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (24). Yield 74%;
white solid (EtOAc-light petroleum) mp 160 ꢀC. 1H NMR
(DMSO-d6) δ: 2.3-2.7 (m, 4H); 3.3 (s, 3H); 3.7-3.9 (m, 8H);
6.7 (dd, 1H, J= 2, J= 4); 7.1 (d, 1H, J= 2); 7.9 (d, 1H, J=4);8.4
(bs, 2H). IR (Nujol) cm-1: 3330-3110, 1665, 1615, 1510. Anal.
(C17H20N8O4) C, H, N.
7-Amino-5-[(4-(dichloromethylcarbonyl))-1-piperazinyl]-2(2-
furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (25). Yield 70%; white
solid (EtOAc-light petroleum) mp 261 ꢀC. 1H NMR (DMSO-d6) δ:
3.6 (s, 4H); 3.8 (s, 4H); 6.7 (dd, 1H, J = 2, J = 4); 7.0 (s, 1H); 7.1 (d,
1H, J = 2); 7.9 (d, 1H, J = 4); 8.4 (bs, 2H). IR (Nujol) cm-1
:
3320-3100, 1670, 1610, 1520. Anal. (C14H14N8O2Cl2) C, H, N.
7-Amino-5-[(4-(4-nitrobenzoyl))-1-piperazinyl]-2(2-furyl) 1,2,4-
triazolo [1,5-a] 1,3,5-triazine (26). Yield 69%; pale yellow solid
(EtOAc-light petroleum) mp > 300 ꢀC (dec) 1H NMR (DMSO-
d6) δ:3.7-3.9 (m, 8H); 6.7 (dd, 1H, J=2, J= 4); 7.1 (d, 1H, J=2);
7.7 (d, 2H); 7.9 (d, 1H, J = 4); 8.3 (d, 2H); 8.4 (bs, 2H). IR (Nujol)
cm-1: 3300-3110, 1665, 1615, 1510. Anal. (C19H17N9O4) C, H, N.
7-Amino-5-[(4-(4-chloro-benzyl-carbonyl))-1-piperazinyl]-2(2-
furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (29). Yield 74%; white
solid (EtOAc-light petroleum) mp 260 ꢀC. 1H NMR (DMSO-d6)
δ: 3.7-3.9 (m, 8H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2);
7.2 (d, 2H); 7.3 (d, 2H); 7.7 (d, 2H, J = 9); 7.9 (d, 1H, J = 4); 8.3
(d, 2H, J = 9); 8.4 (bs, 2H). IR (Nujol) cm-1: 3300-3100, 1675,
1625, 1500. Anal. (C20H17N8O2Cl) C, H, N.
7-Amino-5-[(4-(3,3-dimethylbutanoyl))-1-piperazinyl]-2(2-furyl)
1,2,4-triazolo [1,5-a] 1,3,5-triazine (36). Yield 77%; white solid
(EtOAc-light petroleum) mp 297 ꢀC. 1H NMR (DMSO-d6) δ: 1.0
(s,9H);2.3(s,2H);3.6(s,4H);3.8(s,4H);6.7(dd,1H,J=2,J=4);
7.1 (d, 1H, J = 2); 7.9 (d, 1H, J = 4); 8.4 (bs, 2H). IR (Nujol)
cm-1: 3290-3100, 1680, 1615, 1510. Anal. (C18H24N8O2) C, H, N.
7-Amino-5-[(4-(diphenylmethylcarbonyl))-1-piperazinyl]-2(2-
furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (34). Yield 63%; white
solid (EtOAc-light petroleum) mp 295 ꢀC. 1H NMR (DMSO-d6)
δ: 3.6 (s, 4H); 3.8 (s, 4H); 5.6 (s, 1H); 6.7 (dd, 1H, J = 2, J = 4);
7.1 (d, 1H, J = 2); 7.2-7.4 (m, 10H); 7.9 (d, 1H, J = 4); 8.4 (bs,
2H). IR (Nujol) cm-1: 3310-3110, 1675, 1620, 1510. Anal.
(C26H24N8O2) C, H, N.
7.3-7.5(m, 4H);7.9 (d, 1H, J = 4);8.4 (bs, 2H). IR(Nujol)cm-1
3340-3100, 1620, 1480 Anal. (C19H19N8OCl) C, H, N.
:
7-Amino-5-[(4-(4-fluoro-benzyl)-1-piperazinyl]-2(2-furyl) 1,2,4-
triazolo[1,5-a] 1,3,5-triazine (17). Yield 66%; white solid (EtOAc-
light petroleum) mp 260 ꢀC. 1H NMR (DMSO-d6) δ: 2.5 (s, 4H);
3.5 (s, 2H); 3.8 (s, 4H); 6.7 (dd, 1H, J=2, J= 4); 7.1 (d, 1H, J=2);
7.1-7.4(m, 4H);7.9 (d, 1H, J = 4);8.4 (bs, 2H). IR(Nujol)cm-1
3340-3110, 1630, 1480. Anal. (C19H19N8OF) C, H, N.
:
7-Amino-5-[(4-(4-chloro-benzyl)-1-piperazinyl]-2(2-furyl) 1,2,4-
triazolo[1,5-a] 1,3,5-triazine (18). Yield 73%; white solid (EtOAc-
light petroleum) mp 274 ꢀC. 1H NMR (DMSO-d6) δ: 2.5 (s, 4H);
3.5 (s, 2H); 3.8 (s, 4H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J =
2); 7.3-7.5 (m, 4H); 7.9 (d, 1H, J = 4); 8.4 (bs, 2H). IR (Nujol)
cm-1: 3350-3110, 1620, 1470. Anal. (C19H19N8OCl) C, H, N.
7-Amino-5-[(4-(4-bromo-benzyl)-1-piperazinyl]-2(2-furyl) 1,2,4-
triazolo [1,5-a] 1,3,5-triazine (19). Yield 68%; white solid (EtOAc-
light petroleum) mp 284 ꢀC. 1H NMR (DMSO-d6) δ:2.5(s,4H);3.5
(s, 2H);3.8(s, 4H);6.7(dd, 1H, J=2, J=4);7.1(d, 1H, J=2);7.3
(d, 2H); 7.5 (d, 2H); 7.9 (d, 1H, J = 4); 8.4 (bs, 2H). IR (Nujol)
cm-1: 3340-3110, 1630, 1480. Anal. (C19H19N8OBr) C, H, N.
General Procedure for N-Piperazine Acylation (21-29, 34-36).
A mixture of compound 9 (0.2 g, 0.05 mmol) the appropriate acyl
chloride (1.2 equiv), Et3N (7.2 μL, 0.06 mmol) in dry dioxane

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3 887
7-Amino-5-[(4-(r-naphthoyl))-1-piperazinyl]-2(2-furyl) 1,2,4-
triazolo [1,5-a] 1,3,5-triazine (28). Yield 69%; white solid
from HEK-293 cells expressing recombinant hA1, and hA3 ARs,
and from CHO cells expressing the recombinant hA2A AR, as
previously described.40-42 Adenosine deaminase (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 con-
centration, 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, Gaithers-
burg, MD). The tubes were rinsed three times with 3 mL of
buffer each. At least six different concentrations of competitor,
spanning 3 orders of magnitude adjusted appropriately for the
IC50 of each compound, were used. IC50 values, calculated with
the nonlinear regression method implemented in GraphPad
(Prism, San Diego, CA), were converted to Ki values as
described.47 Hill coefficients of the tested compounds were in
the range of 0.8-1.1.
Adenylyl Cyclase Activity. Because of the lack of a suitable
radioligand the affinity of antagonists and the relative potency
of agonists at the A2B AR was determined in adenylyl cyclase
experiments. The procedure was carried out as described
previously43,44 with minor modifications. Membranes were
incubated with about 150 000 cpm of [R-32P]ATP for 20 min in
the incubation mixture as described43,44 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
antagonist concentration-dependent inhibition of adenylyl cyclase
stimulated by 50-N-ethylcarboxamidoadenosine (NECA) were
calculated accordingly.
Molecular Modeling. All modeling studies were carried out on
a 20 CPU (Intel Core2 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 2009.10) suite.48 The software package MOPAC
(version 7),49 implemented in MOE suite, was utilized for all
quantum mechanical calculations. Docking simulations were
performed using GOLD suite (version 1.3.2).50
Three-Dimensional Structures of hA2A AR and hA3 AR. The
recently published crystallographic structure of hA2A AR, in
complex with the high affinity antagonist ZM241385 (PDB
code: 3EML),36 was used to perform the molecular docking
studies at this receptor subtype.
1
(EtOAc-light petroleum) mp 305 ꢀC. H NMR (DMSO-d6) δ:
3.6-4.0 (m, 8H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2);
7.5-8.1 (m, 7H); 7.9 (d, 1H, J = 4); 8.4 (bs, 2H). IR (Nujol) cm-1
:
3305-3100, 1670, 1610, 1500. Anal. (C23H20N8O2) C, H, N.
7-Amino-5-[(4-(tert-butylcarbonyl))-1-piperazinyl]-2(2-furyl) 1,2,
4-triazolo [1,5-a] 1,3,5-triazine (35). Yield 74%; white solid (EtOAc-
light petroleum) mp > 300 ꢀC. 1H NMR (DMSO-d6) δ:1.2 (s, 9H);
3.6-3.8 (m, 8H); 6.7 (dd, 1H, J = 2, J = 4); 7.1 (d, 1H, J = 2); 7.9
(d, 1H, J = 4); 8.4 (bs, 2H). IR (Nujol) cm-1: 3300-3110, 1675,
1610, 1500. Anal. (C17H22N8O2) C, H, N.
Procedure for the Preparation of 5,7-Diamino-2(2-furyl) 1,2,4-
triazolo [1,5-a] 1,3,5-triazine (41). A mixture of compound 44
(0.29 g, 1 mmol) in absolute ethanol saturated with gas ammonia
(2 mL) was poured into a sealed tube and heated at 120 ꢀC for
3 h. Then the solvent was removed under reduced pressure and
the residue was crystallized from EtOAc-light petroleum to
afford the desired compound (41) as a solid in a good yield
(88%). White solid (EtOAc-light petroleum) mp > 300 ꢀC. 1H
NMR (DMSO-d6) δ: 6.68 (dd, 1H, J = 2, J = 4); 6.98 (bs, 2H);
7.13 (d, 1H, J = 2); 7.81 (d, 1H, J = 4); 8.03 (bs, 2H). IR (Nujol)
cm-1: 3300-3110, 1640, 1600, 1525. Anal. (C8H7N7O) C, H, N.
Procedure for the Preparation of 7-Amino 5-hydroxy-2(2-
furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine (40). A mixture of
compound 44 (0.29 g, 1 mmol) in 2 M KOH in absolute ethanol
(2 mL) was poured into a sealed tube and heated at 120 ꢀC for
3 h. Then the solvent was removed under reduced pressure and
the residuewascrystallizedfromEtOAc-lightpetroleum toafford
the desired compound (40) as a solid in a good yield (76%).
White solid (EtOAc-light petroleum) mp 206 ꢀC. 1H NMR
(DMSO-d6) δ: 6.65 (dd, 1H, J = 2, J = 4); 7.18 (d, 1H, J = 2);
7.93 (d, 1H, J = 4); 8.81 (bs, 2H); 12.13 (bs, 1H). IR (Nujol)
cm-1: 3350-3145, 1655, 1610, 1530. Anal. (C8H6N6O2) C, H, N.
General Procedure for the N7 Benzoylation (39, 42). A mixture
of the appropriate amino compound compound (40, 44) (0.1
mmol) benzoyl chloride (14 μL, 0.12 mmol), Et3N (14 μL, 0.12
mmol) in dry dioxane (10 mL) was stirred at reflux for 12 h. The
solvent was removed in vacuo and the residue was dissolved in
EtOAc (15 mL) and washed with water (3 ꢀ 5 mL). The organic
phase was dried and solvent was removed under reduced
pressure. The crude was purified by flash chromatography
(EtOAc) to give the final compounds (39, 42) as solids.
5-Hydroxy-7-phenylcarbonylamino-2(2-furyl) 1,2,4-triazolo
[1,5-a] 1,3,5-triazine (42). Yield 75%; white solid (EtOAc-light
petroleum) mp 201 ꢀC. 1H NMR (DMSO-d6) δ: 6.66 (dd, 1H, J =
2, J= 4); 7.21 (d, 1H, J= 2); 7.45-7.98 (m, 7H); 10.52 (bs, 1H). IR
(Nujol) cm-1: 3340-3150, 1687, 1635, 1615, 1530. Anal. (C15H10-
N6O3) C, H, N.
Moreover, on the basis of the assumption that GPCRs share
similar TM boundaries and overall topology, a homology model
of the hA3 AR was constructed with the software MOE, as
previously reported,37,38 using as template the crystal structure
of hA2A AR.36
5-Phenoxy-7-phenylcarbonylamino-2(2-furyl) 1,2,4-triazolo
[1,5-a] 1,3,5-triazine (39). Yield 87%; pale yellow solid (EtOAc-
light petroleum) mp 229 ꢀC (dec). H NMR (DMSO-d6) δ: 6.60
(dd, 1H, J = 2, J = 4); 7.12 (d, 1H, J = 2); 7.25-7.65 (m, 7H);
7.80-7.99 (m, 4H). IR (Nujol) cm-1: 3325-3070, 1695, 1620, 1510.
Anal. (C21H14N6O3) C, H, N.
The numbering of the amino acids follows the arbitrary
scheme by 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
conserved amino acid in that helix. The number 50 is arbitrarily
assigned to the reference residue.51
1
Procedure for the Preparation of 5-amino-7-phenylcarbonyla-
mino-2(2-furyl) 1,2,4-triazolo [1,5-a] 1,3,5-triazine trifluoroace-
tate (43). A mixture of compound 39 (0.39 g, 1 mmol) in absolute
ethanol saturated with gas ammonia (3 mL) was poured into a
sealed tube and heated at 120 ꢀC for 3 h. Then the solvent was
removed under reduced pressure and the residue was crystallized
from EtOAc-light petroleum to afford the desired compound (43)
as a solid in a good yield (70%). Pale brown solid (EtOAc-light
Molecular Docking of hA2A AR Antagonists. Ligand struc-
tures were built using MOE-builder tool, part of the MOE
suite,48 and were subjected to MMFF94x energy minimization
-1
until the rms conjugate gradient was <0.05 kcal mol-1
A
.
˚
Partial charges for the ligands were calculated using PM3/ESP
methodology.
Four different programs have been used to calibrate our
docking protocols: MOE-Dock,48 GOLD,50 Glide,52 and
PLANTS.53 In particular, ZM241385 was redocked into the
crystal structure of the hA2A AR (PDB code: 3EML) with
different docking algorithms and scoring functions, as already
described.37,39 Then, rmsd values between predicted and crystal-
lographic positions of ZM241385 were calculated for each of the
docking algorithms. The results showed that docking simula-
tions performed with GOLD gave the lowest rmsd value, the
1
petroleum) mp 187 ꢀC. H NMR (DMSO-d6) δ: 6.62 (dd, 1H,
J = 2, J = 4); 7.18 (d, 1H, J = 2); 7.38-7.52 (m, 3H); 7.60-7.81
(m, 4H);7.99(d, 1H, J = 4);8.98(bs, 1H). IR (Nujol) cm-1: 3325-
3105, 1690, 1640, 1605, 1540. Anal. (C15H11N7O2) C, H, N.
Biological Testing. Radioligand Binding to hA1, A2A and A3
ARs. [3H]DPCPX, [3H]ZM241385, and [125I]I-AB-MECA were
utilized in radioligand binding assays to membranes prepared

888 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 3
Federico et al.
lowest mean rmsd value and the highest number of poses with
˚
rmsd value < 2.5 A.
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On the basis of the best docking performance, all antagonist
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and their scores in a molecular database file. The resulting docked
˚
complexes (ligand and side chains of residues at 4.5 A from the
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the rms conjugate gradient was <1 kcal mol-1 -1. Partial charges
A
˚
for the ligands were calculated using MOPAC and PM3/ESP
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K.-N.; Leung, E.; Varani, K.; Gessi, S.; Merighi, S.; Borea, P. A.
Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly
potent and selective human A3 adenosine receptor antagonists.
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Prediction of antagonist-receptor complex stability (in terms
of corresponding pKi value) and the quantitative analysis for
nonbonded intermolecular interactions (H-bonds, transition
metal, water bridges, hydrophobic, electrostatic) were calcu-
lated and visualized using several tools implemented in MOE
suite.48 Electrostatic and hydrophobic contributions to the
binding energy of individual amino acids have been calculated
using MOE.48 To estimate the electrostatic contributions, atomic
charges for the ligands were calculated using PM3/ESP metho-
dology. Partial charges for protein amino acids were calculated
on the basis of the AMBER99 force field.
(18) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, G.; Moro, S.;
Klotz, K. N.; Leung, E.; Varani, K.; Gessi, S.; Merighi, S.; Borea,
P. A. Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as
highly potent and selective human A3 adenosine receptor antago-
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2000, 43, 4768–4780.
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Da Ros, T.; Klotz, K.-N.; Varani, K.; Gessi, S.; Borea, P. A.
Synthesis, biological activity, and molecular modeling investiga-
tion of new pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine deriva-
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2002, 45, 770–780.
Acknowledgment. The 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. This research was also supported in part
by the Intramural Research Program of the NIH, National
Institute of Diabetes and Digestive and Kidney Diseases. S.M.
is very grateful to Chemical Computing Group Inc. (Montreal,
Quebec, Canada) for the scientific and technical partnership.
(20) Maconi, A.; Pastorin, G.; Da Ros, T.; Spalluto, G.; Gao, Z. G.;
Jacobson, K. A.; Baraldi, P. G.; Cacciari, B.; Varani, K.; Borea,
P. A. Synthesis, biological properties and molecular modeling
investigation of the first potent, selective and water soluble
human A3 adenosine receptor antagonist. J. Med. Chem. 2002,
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Cacciari, B.; Baraldi, P. G.; Gessi, S.; Varani, K.; Borea, P. A.
Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives as ade-
nosine receptor antagonists. Influence of the N5 substituent on
the affinity at the human A3 and A2B adenosine receptor sub-
types: a molecular modeling investigation. J. Med. Chem. 2003, 46,
4287–96.
(22) Cheong, S. L.; Dolzhenko, A.; Kachler, S.; Paoletta, S.; Federico,
S.; Cacciari, B.; Dolzhenko, A.; Klotz, K. N.; Moro, S.; Spalluto,
G.; Pastorin, G. The significance of 2-furyl substitutions with a
2-(para-substituted) aryl group in a new series of pyrazolo-triazolo-
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Supporting Information Available: Elemental analyses and
modeling figures. This material is available free of charge via the
Internet at http://pubs.acs.org.
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