2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual A1/A3 adenosine receptor antagonists: Synthesis and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2019.06.034

From a collection containing more than 1500 academic compounds, in silico screening identified a hit for the human A₁ adenosine receptor containing a new purine scaffold. To study the structure activity relationships of this new chemical series

Full text of DOI:10.1016/j.bmc.2019.06.034

Accepted Manuscript
2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual A1/A3 ade-
nosine receptor antagonists: synthesis and structure-activity relationships
Filipe Areias, Carla Correia, Ashly Rocha, José Brea, Marián Castro, Maria I.
Loza, M. Fernanda Proença, M. Alice Carvalho
PII:
S0968-0896(19)30393-1
https://doi.org/10.1016/j.bmc.2019.06.034
BMC 14971
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 March 2019
18 June 2019
19 June 2019
Please cite this article as: Areias, F., Correia, C., Rocha, A., Brea, J., Castro, M., Loza, M.I., Fernanda Proença, M.,
Alice Carvalho, M., 2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual A1/A3 adenosine receptor
antagonists: synthesis and structure-activity relationships, Bioorganic & Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.bmc.2019.06.034
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Graphical Abstract
2-Aryladenine derivatives as a potent
scaffold for A1, A3 and dual A1/A3
adenosine receptor antagonists:
synthesis and structure-activity
relationships
Leave this area blank for abstract info.
Filipe Areiasa,b,c,#, Carla Correiaa,#, Ashly Rochaa, José Breab, Marian Castrob, Maria I. Lozab, M.
Fernanda Proençaa,#, M. Alice Carvalhoa,#
a Center of Chemistry, Campus de Gualtar, Universidade do Minho, 4710-057 Braga, Portugal
b Department of Pharmacology, Universidade de Santiago de Compostela, Edificio CIMUS, Avda de
Barcelona, 15782 Santiago de Compostela, Spain
c
School of Chemical Sciences & Engineering, Yachay Tech University, Yachay City of Knowledge,
100119 Urcuquí, Ecuador
# First author equal contribution
* Corresponding author equal contribution
Crucial for
Selectivity for
high potency
adenosine receptors
Y
4
5
6
H
3
2
A1
A3
A1/A3
3-Cl
N
N
N
2-HO
Y= 3-HO
4-HO
2,5-Cl2
3,4-Cl2
4-Cl
N
selectivity
Control
N
X
X=CH2, NMe

2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual
A1/A3 adenosine receptor antagonists: synthesis and structure-activity
relationships
Filipe Areiasa,b,c,#, Carla Correiaa,#, Ashly Rochaa, José Breab, Marián Castrob, Maria I. Lozab,
M. Fernanda Proençaa,*, M. Alice Carvalhoa,*
a Center of Chemistry, Campus de Gualtar, Universidade do Minho, 4710-057 Braga,
Portugal
b Department of Pharmacology, Universidade de Santiago de Compostela, Edificio
CIMUS, Avda de Barcelona, 15782 Santiago de Compostela, Spain
c
School of Chemical Sciences & Engineering, Yachay Tech University, Yachay City of
Knowledge, 100119 Urcuquí, Ecuador
# First author equal contribution
* Corresponding author equal contribution
Keywords: Adenine derivatives
adenosine receptor antagonists
2-arylpurine derivatives
Abstract
From a collection containing more than 1500 academic compounds, in silico
screening identified a hit for the human A1 adenosine receptor containing a new purine
scaffold. To study the structure activity relationships of this new chemical series for
adenosine receptors, a library of 24 purines was synthesized and tested in radioligand
binding assays at human A1, A2A, A2B and A3 adenosine receptor subtypes. Fourteen
molecules showed potent antagonism at A1, A3 or dual A1/A3 adenosine receptors. This
purine scaffold is an important source for novel biochemical tools and/or therapeutic
drugs.
1. Introduction
Adenosine is an endogenous purinergic nucleoside, occurring in all body cells
and modulates many physiological and pathological conditions related to
cardiovascular, immune, metabolic and neurological functions [1,2]. Cellular signalling
by adenosine occurs through four adenosine receptor subtypes (A1, A2A, A2B, and A3),
belonging to the G protein-coupled receptor (GPCR) superfamily [3]. The A1 and A3

receptors principally couple to Gi/o proteins, inducing an inhibitory effect on adenylyl
cyclase activity, while the A2A and A2B receptors couple to Gs proteins, stimulating
cyclic AMP production [4-5]. Therefore, it is of therapeutic interest to develop new
drugs active on adenosine receptors, as evidenced by the continuous patent claims on
new compounds modulating adenosine receptors or new uses for selective ligands [6-8].
Intensive efforts in medicinal chemistry in the adenosine receptor field yielded selective
agonists and antagonists for the four receptor subtypes as well as allosteric modulators
[3,9], some of them advancing to clinical trials or to FDA approval for diagnostic or
therapeutic uses [2,3,5,8-9]. For example, A1 ligands are under development for
cardiovascular diseases, pain indications, glaucoma and PET imaging agents [3,5,8-10].
Also, A2B antagonists and dual A2B/A3 antagonists are being investigated for their use in
asthma, diabetes, and cancer. In addition, A2A agonists are in clinical trials for cardiac
imaging diagnostic and wound healing and some have already been approved for
cardiac perfusion imaging [3,5,8-9], while A2A antagonists are under development for
the treatment of cancer [11]. Finally, A3 agonists have been linked to inflammatory
diseases, such as rheumatoid arthritis and psoriasis, liver cancer, hepatitis, and liver
regeneration, and showed efficacy in clinical trials for dry eye syndrome [3,5,8-9,12].
Typically, selective adenosine ligands were developed as heterocyclic-based
scaffolds, comprising one, two or three fused rings having nitrogen and sometimes
sulphur as heteroatoms [5, 13-14]. In particular, adenosine receptor ligands based on
purine scaffold were reported as adenine derivatives and aza-and deazapurines.
Furthermore, the reported adenine derivatives have a hydrogen atom, an alkyl, an aryl, a
sugar or a sugar-like unit on N9, an amino group, primary or secundary amine on C6 and
an aryl, an alkyl or alkynyl, a proton or a chlorine atom on C2. As far as we know
adenine derivatives having a proton or a methyl group on N9, combined with a
secondary amine on C6 and different aryl units on C2 were never reported as adenosine
receptor ligands.
The current work is part of an ongoing interdisciplinary project integrating
organic and medicinal chemistry with in silico and in vitro pharmacology. In a previous
study, we showed that in silico target profiling was able to identify the targets at which
a chemical library of biologically-orphan molecules should be tested, leading to the
identification of novel antagonists for all members of the adenosine receptor family
[15]. Subsequently in silico target profiling of the library of 1584 compounds led to the

identification of compound 3a as a new hit for the A1 receptor subtype containing a
purine scaffold (Fig. 1).
In this work we introduce the new family of compounds based on the general
structure of the purine that was present in the original hit (3a). This purine series
showed a rich variety of affinity profiles for adenosine receptors, some with marked
selectivity for the A1, A3 and dual A1/A3 receptor subtypes.
Me
N
N
N
OH
N
N
N
Figure 1. Purine hit (3a) for A1 receptor subtype identified by in silico target
profiling.
2. Results and discussion
2.1. In silico target profiling
A collection of 1584 biologically-orphan molecules was profiled against ligand-
based models available for 4643 proteins [15]. This in silico target profiling identified
one molecule (3a) containing a novel purine scaffold for which affinity for adenosine
receptor subtypes was predicted. The compound was then synthesized and tested in
vitro on all human adenosine receptors (A1, A2A, A2B and A3) and presented
submicromolar affinity for the A1 adenosine receptor (Table 1). These results set the
basis for initiating a medicinal chemistry program around a novel purine series.
The new purine series 3a-x was built by combining different groups at N9, C6
and C2 of the purine nucleus. A hydrogen or a methyl group were incorporated at N9, a
piperidinyl or a 4-methylpiperazinyl group were introduced in C6 and different
substituent groups were incorporated in the aryl substituent present in C2 of the purine
ring.
2.2. Chemistry

The synthetic approach followed to prepare the target molecules 3a-x is described in
Scheme 1. The synthesis of starting materials 1a-b, was described in previous works
[16-17] and compound 2a was also described [18]. Compounds 3c, d, g and j were
previously reported [19] however their synthesis is described here for the first time. The
new derivatives 2b-d were obtained following an experimental protocol similar to that
used for the synthesis of 2a. The imidazoles 1, in acetonitrile at room temperature, were
reacted with an excess of the corresponding secondary amine. The pure products
precipitated from solution and were isolated, in very good to excellent yield, by simple
filtration.
In order to obtain compounds 3a-x, imidazoles 2a-d were reacted with different
aldehydes (Scheme 1) using ethanol as solvent and triethylamine as base. The reactions
started at room temperature until the reagents evolved to a yellow solution. The
o
temperature was then raised to 40 C and the reactions continued at that temperature to
complete consumption. These reactions led to black-greyish solids that required
purification. This was achieved by filtration of a dichloromethane solution of the solids
through a silica gel column.

R
R
R1
N
N
N
NH2
NH
R1CHO
+
N
N
N
N
X
4
N
a R1=2-HOC6H4
b R1=3-HOC6H4
c R1=4-HOC6H4
d R1=2,3-(HO)2C6H3
e R1=2-ClC6H4
f R1=3-ClC6H4
g R1=4-ClC6H4
h R1=3,4-Cl2C6H3
i R1=2,5-Cl2C6H3
j R1=H
2
3
X
a R=CH3, X=NCH3, R1=2-HOC6H4
b R=CH3, X=NCH3, R1=3-HOC6H4
c R=CH3, X=NCH3, R1=3-ClC6H4
d R=CH3, X=NCH3, R1=3,4-Cl2C6H3
e R=CH3, X=CH2, R1=2-HOC6H4
f R=CH3, X=CH2, R1=2,3-(HO)2C6H3
g R=CH3, X=CH2, R1=2-ClC6H4
h R=CH3, X=CH2, R1=2,5-Cl2C6H3
i R=CH3, X=CH2, R1=3-HOC6H4
j R=CH3, X=CH2, R1=3-ClC6H4
k R=H, X=NCH3, R1=H
l R=H, X=NCH3, R1=2,5-Cl2C6H3
m R=H, X=NCH3, R1=3-HOC6H4
n R=H, X=NCH3, R1=3-ClC6H4
o R=H, X=NCH3, R1=3,4-Cl2C6H3
p R=H, X=NCH3, R1=4-Cl-3-CF3C6H3
q R=H, X=NCH3, R1=4-HOC6H4
r R=H, X=NCH3, R1=4-ClC6H4
s R=H, X=CH2, R1=2,5-Cl2C6H3
t R=H, X=CH2, R1=3-HOC6H4
u R=H, X=CH2, R1=3-ClC6H4
a R=H, X=CH2
b R=CH3, X=CH2
c R=H, X=NCH3
d R=CH3, X=NCH3
X
k R1=4-Cl-3-CF3C6H3
N
H
R
N
NH2
NH
CN
N
1
a R=H
b R=CH3
v R=H, X=CH2, R1=2,4-Cl2C6H3
w R=H, X=CH2, R1=4-HOC6H4
x R=H, X=CH2, R1=4-ClC6H4
Scheme 1 Synthesis of 2-aryl-adenine derivatives 3a-x
When the same methodology was applied to the reaction of compounds 2b and 2d with
phenolic aldehydes, a black solution resulted shortly after mixing the reagents that
evolved to a black oil. In order to obtain the phenolic derivatives the reactions were
performed in acidic medium until complete consumption of reagents (evidenced by
o
TLC) and then continued in basic medium, at 40 C, according to the previously
described procedure [20]. The pure products were obtained following the purification
approach described above for the other derivatives.
2.3. Pharmacology
All synthesized compounds (3a-x) were screened by radioligand binding
experiments at all human adenosine receptor subtypes (A1, A2A, A2B and A3) expressed

in mammalian cell lines. The percentage of inhibition of radioligand binding (%inhib
)
was obtained for all the tested compounds at a concentration of 10 µM. Those
compounds showing a %inhib higher than 80 % were assayed at different concentrations
for the calculation of their affinity (measured as pKi) at the studied receptor (Table 1).
Binding tests confirmed the virtual study results and the 9-methyl-6-(4-
methypiperazinyl)purine 3a presented a pKi value of 6.2 ± 0.3 for A1 receptor with a
good selectivity for this receptor (see Table 1). When the affinity of 3a for adenosine
receptor subtypes was compared with that of the other 9-methyl-6-(4-
methypiperazinyl)purines (3b, 3c and 3d), we observed that 3a was the most potent
compound of this group. We observed also that 3c and 3d had higher affinity for A1
receptor than 3b, while 3b had greater affinity for A3 receptor.
Some 9-methyl-6-piperidinylpurines (3e-j) were also synthesized and tested. The
purines 3e and 3i had higher affinity for A2A and A1 receptors, respectively, with
inhibition percentages of 74.8 ± 4.6 % and 78.5 ± 1.3 % at 10 µM, respectively. The
other purine derivatives of this group (3f, 3g, 3h and 3j) presented weak affinities for all
adenosine receptor subtypes. When the results of 9-methyl-6-piperidinylpurines 3e, 3i
and 3j were compared to the respective 6-(4-methypiperazinyl) derivatives (3a, 3b and
3c), we observed only small to moderate changes in the affinity of these compounds for
adenosine receptor subtypes. These results indicated that piperidine versus N-
methypiperazine subunit in C6 of the purine nucleus, having a methyl group in N9, had
only limited importance for the affinity at adenosine receptors. Indeed, all purines
belonging to these two groups had weak to moderate affinities for the adenosine
receptors except 3a.
Table 1
Binding affinities of the new compounds 3 at all human adenosine receptors (%inhib at
10 µM or pKi, mean ± SEM).
R
R1
N
N
N
N
N
X
Human adenosine receptors
R
X
R1
hA1
hA2A
hA2B
hA3

3a
3b
Me NMe
Me NMe
6.2 ± 0.3
59.6 ± 1.0 % 36.0 ± 3.2 % 15.3 ± 1.1 %
HO
OH
Cl
27.1 ± 3.0 %
4.2 ± 0.3 %
47.8 ± 2.4 % 63.8 ± 1.4 %
3c
Me NMe
Me NMe
45.7 ± 0.9 % 21.0 ± 1.8 % 19.2 ± 1.1 % 38.3 ± 3.9 %
65.2 ± 1.9 % 25.0 ± 2.1 % 28.3 ± 1.7 % 35.3 ± 3.0 %
Cl
Cl
3d
3e
3f
Me CH2
Me CH2
50.5 ± 3.0 % 74.8 ± 4.6 % 30.2 ± 2.5 % 20.0 ± 2.1 %
17.2 ± 0.4 % 24.1 ± 1.9 % 26.0 ± 1.5 % 33.7 ± 2.0 %
HO
HO
OH
Cl
3g
3h
Me CH2
Me CH2
35.2 ± 2.9 % 36.4 ± 3.5 % 32.7 ± 0.7 %
2.1 ± 0.5 %
5.4 ± 0.6 %
Cl
Cl
13.2 ± 1.0 %
2.4 ± 0.5 %
31.0 ± 2.7 %
OH
Cl
3i
3j
Me CH2
Me CH2
78.5 ± 1.3 % 54.8 ± 2.0 % 29.3 ± 0.5 % 49.1 ± 5.0 %
22.4 ± 0.5 % 17.0 ± 0.3 % 24.2 ± 1.9 %
6.1 ± 0.7 %
3k
3l
H
H
NMe
NMe
H
9.2 ± 0.7 %
19.1 ± 2.0 % 14.0 ± 0.9 % 26.9 ± 4.6 %
Cl
5.6 ± 0.3
9.1 ± 0.2 % 13.0 ± 0.47 %
6.6 ± 0.2
Cl
OH
Cl
3m
H
NMe
6.8 ± 0.1
7.1 ± 0.2
6.2 ± 0.3
1.0 ± 1.0 %
27.0 ± 2.0 %
6.3 ± 0.5
7.0 ± 0.1
7.4 ± 0.2
3n
3o
H
H
NMe
NMe
52.7 ± 3.0 % 29.9 ± 2.6 %
12.0 ± 0.6 % 24.1 ± 2.3 %
Cl
Cl
CF3
Cl
3p
H
NMe
52.4 ± 2.1 % 32.0 ± 3.3 % 25.1 ± 2.7 %
6.1 ± 0.4
3q
3r
H
H
NMe
NMe
OH
6.4 ± 0.2
6.4 ± 0.3
37.0 ± 3.0 % 53.0 ± 2.0 %
15.9 ± 1.7 % 6.2 ± 0.5 %
48 ± 1 %
7.4 ± 0.1
Cl

Cl
3s
3t
H
H
CH2
CH2
1.4 ± 0.1 %
21.0 ± 0.5 % 12.1 ± 1.0 %
6.7 ± 0.4
Cl
OH
Cl
8.0 ± 0.1
7.3 ± 0.1
6.5 ± 0.4
43.5 ± 1.3 %
7.0 ± 0.6 %
6.8 ± 0.1
6.0 ± 0.4
7.2 ± 0.2
7.4 ± 0.3
7.1 ± 0.1
3u
3v
H
H
CH2
CH2
Cl
Cl
24.1 ± 4.8 %
0.7 ± 0.1 %
3w
3x
H
H
CH2
CH2
OH
Cl
8.0 ± 0.2
6.9 ± 0.4
7.0 ± 0.5
5.5 ± 0.3
7.1 ± 0.2
7.4 ± 0.3
8.1 ± 0.1
1.4 ± 0.7 %
In order to evaluate the importance of the group present in N9 of the purine
nucleus (hydrogen atom versus methyl group) in the affinity of these compounds for
adenosine receptors the new derivatives 9-H-6-(4-methypiperazinyl)purine (3l-r) were
synthesized, tested and the results are presented in Table 1. The first observation that
should be highlighted is that by comparing the affinities of 9-H-6-(4-
methypiperazinyl)purines (3m, 3n and 3o) with the respective 9-methyl-6-(4-
methypiperazinyl) derivatives (3b, 3c and 3d) for adenosine receptors, we observed a
great increase in the affinity values for A1 and A3 receptors. These results showed the
importance of the group present in N9 (hydrogen atom over methyl group) for activity.
Furthermore, the purine 3q (pKi = 6.4 ± 0.2) was selective for A1 receptors. The
compounds 3m and 3n had similar affinity for both A1 (pKi = 6.8 ± 0.1; pKi = 7.1 ± 0.2)
and A3 (pKi = 6.3 ± 0.5; pKi = 7.0 ± 0.1) receptors, respectively. On the other side, the
purines 3o and 3r were the most potent compounds for A3 receptors in this chemical
series (pKi = 7.4 ± 0.2 and 7.4 ± 0.1, respectively), being 3o slightly more selective than
3r. In addition, compounds 3l (pKi = 6.6 ± 0.2) and 3p (pKi = 6.1 ± 0.4) were also
selective for A3 receptors but they were less potent than 3o and 3r.
The series 9-H-6-piperidinylpurines (3s-x) was synthesized to assess the
importance of the group present in C6 of the purine nucleus in the affinity of these
compounds for adenosine receptors. The results, presented in Table 1, showed that the

presence of a piridinyl group in C6 of the purine nucleus instead of a 4-
methylpiperidinyl group led to compounds with higher potency (3m vs 3t, 3n vs 3u, 3r
vs 3x) or to compounds with similar potency but having higher selectivity (3l vs 3s, 3o
vs 3v). Compounds 3t and 3w, with the same affinity for A1 receptors (pKi = 8.0), were
the most potent and 3t was more selective for this receptor. Figure 2(a) shows a
representative competition binding curve for compound 3t at A1 receptors. Compound
3x was the most potent and, at the same time, the more selective compound for A3
receptors (pKi = 8.1 ± 0.1). The purine 3u had almost the same affinity for A1 (pKi = 7.3
± 0.1) and A3 (pKi = 7.4 ± 0.3) receptors. Compounds 3s and 3v were selective for A3
receptors with pKi values of 6.7 ± 0.4 and 7.1 ± 0.1, respectively. Additionally, we
observed a high increase in the affinities of 9-H-6-piperidinylpurines (3s, 3t and 3u)
over their respective 9-methyl-6-piperidinylpurines (3h, 3i and 3j) for A1 and A3
adenosine receptors, confirming the importance of the hydrogen atom over methyl
group in N9 position of purine as observed previously in this work.
In order to complete our structure-activity relationship (SAR) study, compound
3k was also synthesized and tested. The results (Table 1) showed that compound 3k
had little affinity for all adenosine receptor subtypes, showing that the aromatic ring
present in position C2 of the purine ring is crucial for the affinity of these purines for
adenosine receptors. For purines with submicromolar affinity for adenosine receptors,
the aromatic substituents (R1) present in C2 of the purine ring are also important to
obtain potent and selective ligands. When R1 = 2-HOC6H4, 3-HOC6H4, 4-HOC6H4,
selective compounds for A1 receptors were obtained (3a, 3m, 3q, 3t and 3w). Selective
ligands for A3 receptors were achieved when R3 = 2,5-Cl2C6H3, 3,4-Cl2C6H3, 4-ClC6H4
(3l, 3s, 3o, 3v, 3r, 3x). Dual ligands for A1/A3 receptors were generated when R3 = 3-
ClC6H4 (purines 3n and 3u). This dual affinity at A1/A3 receptors can be of therapeutic
interest because A1 and A3 receptors principally couple to Gi/o proteins and induce a
complementary inhibition on adenylyl cyclase activity [4-6]. Therefore, the compounds
3n and 3u are dual ligands of A1/A3 receptors and they can antagonize simultaneously
the inhibitor effect on adenylyl cyclase activity by A1 and A3 receptors.
From the most potent and selective compounds for A1 and A3 receptors, two
representative compounds were selected and tested in cyclic AMP assays to study their
functional activity. The functional behaviour of compounds 3t and 3x at A1 and A3
receptors, respectively, was examined by measuring their effect on the modulation of
intracellular cAMP levels by the non-selective adenosine receptor agonist NECA.

Figure 2(b) shows the results of a representative experiment for the antagonist potency
of compound 3x at A3 receptors. The results confirmed that the compounds 3t and 3x
were antagonists at A1 and A3 receptors, respectively. The pKB values express the
antagonist potency of these compounds (Table 2), which was in good agreement with
their affinity values (pKi) at the corresponding receptor. We extrapolated that all
compounds of our series are antagonists of adenosine receptors based on their structural
similarity.
Table 2
Antagonistic potency (pKB) of selected compounds at human hA1 and hA3 adenosine
receptors in cAMP assays.
Compound
Human adenosine receptors
hA1
hA3
ND
3t
7.9 ± 0.2
3x
ND
8.2 ± 0.1
ND – Not determined.
(a)
(b)
Figure 2. (a) Competition binding curve for compound 3t at A1 receptors. (b)
Concentration-response curve of 3x in the presence of 0.1 µM NECA at human A3
receptors expressed in CHO cells. Points represent the mean ± standard deviation
(vertical bars) of duplicate measurements.
3. Conclusion
In summary, several purine-based compounds having different substituents in
C2, C6 and N9 of the purine nucleus were designed, synthesized and pharmacologically
tested for adenosine receptors. From the synthesized compounds, five showed high
potency and selectivity for A1 receptors and seven showed high potency and selectivity
for A3 receptors. Two of the compounds showed high potency for both receptors A1/A3

and represent compounds with dual affinity for those receptors. From SAR analysis, we
observed that the substituent groups present in C2, C6 and N9 of the purine ring affect
the potency and selectivity of the compounds for adenosine receptors. In order to
generate highly potent and selective ligands for A1, A3 and dual A1/A3 receptors a
hydrogen atom must be present in N9 and an aryl group in C2. The group in C6
(piperidinyl vs 4-methylpirazinyl) combined with a specific aryl group in C2 seems to
govern the selectivity however a complex relationship between this substituents is
observed. Functional studies indicated that these structurally related ligands have
antagonist activity at A1 and A3 receptors. In conclusion, a number of purine-based
compounds are described in this study, several of them showed selective antagonism for
A1, A3 and dual A1/A3 receptors at submicromolar concentration.
4. Experimental protocols
4.1. Chemistry
The 5-amino-4-(cyanoformimidoyl)imidazoles 1 used in this work were synthesized
according to previously described procedures [16-18] and compound 2a was
synthesized according to procedure described in [18]. Solvents and other chemicals
commercially available were used as shipped. The melting points were determined with
a Gallenkamp melting point apparatus and are uncorrected. The reactions were
monitored by thin layer chromatography (TLC) using Silica Gel 60 F254 (Merck) with
detection by UV light. The NMR spectra were recorded on a Varian Unity Plus (1H: 300
MHz, 13C: 75 MHz) or on a Bruker Avance III NMR spectrometer (1H: 400 MHz, 13C:
1
100 MHz) including the H and 13C correlation spectra (HMQC and HMBC), for
solutions in [D6]-DMSO [residual [D6]-DMSO (δH = 2.49 ppm) or [D6]-DMSO (δC =
39.5 ppm) as internal standard] at 298 K. Chemical shifts () were reported in parts per
million (ppm) and the coupling constants, J, were reported in hertz (Hz). The purities of
all tested compounds were higher than 95 % by elemental analysis, which were reported
to be within 0.4 % of calculated values. IR spectra were recorded with a FT-IR Bomem
MB 104 using nujol mulls and NaCl cells. Elemental analyses were performed with a
LECO CHNS-932 instrument.
4.1. General procedure for the synthesis of 2b-d

The secondary amine (5.0 molar equivalent) was added to a suspension of 5-amino-4-
cyanoformimidoylimidazole 1a-b in acetonitrile. The mixture was stirred at room
temperature until all the starting material was consumed (evidence by TLC. The off-
white solid that precipitated from the reaction mixture was filtered and washed with
acetonitrile and diethyl ether.
4.1.1. 4-(Imino(piperidin-1-yl)methyl)-1-methyl-1H-imidazol-5-amine (2b)
o
Off-white solid; Yield: 0.60 g, 72 %; m.p. 133 – 135 C (Found: C, 57.80; H, 8.30; N,
33.90. C H N requires C, 57.97; H, 8.21; N, 33.82); IR (Nujol mull) 
: 3332,
max
10 17
5
3109, 1578, 1522 cm-1;  (300 MHz, DMSO-d )  7.24 (s, 1H, H-2), 4.80 – 6.80 (br
6
13
s, 3H, NH), 3.41 (m, 7H), 1.57 (s, 6H); C (75 MHz, DMSO-d )  160.33, 141.67,
6
132.32, 110.25, 48.41, 30.00, 25.56, 23.92.
4.1.2. 4-(Imino(4-methylpiperazin-1-yl)methyl)-1H-imidazol-5-amine (2c)
Off-white solid; Yield: 0.65 g, 94 %; m.p. = 182 – 184 oC (Found: C, 55.34; H, 6.48; N,
38.42. C H N requires C, 55.05; H, 6.42; N, 38.53); IR (Nujol mull) 
: 3328,
max
10 14
6
3212, 1498 cm-1;  (300 MHz, DMSO-d )  10.00 – 7.20 (br s, 1H, NH), 6.99 (s, 1H,
6
H-2), 4.81 (br s, 2H, NH), 3.68 (m, 4H), 2.37 (t, J = 4.8 Hz, 4H), 2.18 (s, 3H); 13C (75
MHz, DMSO-d )  159.57, 147.63, 135.19, 107.10, 54.52, 46.56, 45.76.
6
4.1.3. 4-(Imino(4-methylpiperazin-1-yl)methyl)-1-methyl-1H-imidazol-5-amine (2d)
Off-white solid; Yield: 0.16 g, 74 %; m.p. = 131 – 133 oC (Found: C, 50.06; H, 8.23; N,
34.78. C H N .H O requires C, 50.00; H, 8.33; N, 35.00); IR (Nujol mull) 
:
max
10 18
6
2
3375, 3257, 3148, 1596, 1557, 1533 cm-1;  (300 MHz, DMSO-d )  7.12 (s, 1H, H-
6
2), 6.80 – 4.80 (br s, 3H, NH), 3.38 (s, 3H), 3.28 (t, J = 4.8 Hz, 4H), 2.34 (t, J = 4.8 Hz,
13
4H), 2.17 (s, 3H); C (75 MHz, DMSO-d )  163.18, 139.85, 130.68, 113.80, 54.56,
6
46.51, 45.77, 29.71.
4.2. General procedure for the synthesis of 3a-x
Method A: The aldehyde (1.1 molar equivalent) and trifluoroacetic acid (1.3 molar
equivalent) were added to a suspension of 2, in ethanol, under efficient magnetic

stirring, at room temperature. A yellow solution resulted and was maintained under the
previous reaction conditions until TLC showed absence of starting material and the
presence of a new spot. The solution was concentrated to dryness in the rotary
evaporator. Ethanol (1 mL) and triethylamine (10 molar equivalent) were added to the
solid residue and the reaction was maintained at 40 oC until TLC showed only one spot
for the product. The dark solution was concentrated and water was added. A dark solid
precipitated, was filtered, washed with water followed by diethyl ether and then
dissolved in DCM. The dark solution obtained was filtered through a column of silica
gel (1 cm height). The solution was concentrated, diethyl ether was added and an off
white solid precipitated. The solid was filtered and washed with diethyl ether and
identified as 3.
Method B: The aldehyde (1.1 molar equivalent) and triethylamine (10 molar equivalent)
were added to a suspension of 2, in ethanol, under efficient magnetic stirring, at room
o
temperature. A yellow solution resulted and the reaction was mantained at 40 C until
TLC showed the absence of starting material. The solution was concentrated to dryness
in the rotary evaporator. The solid residue was dissolved in DCM and filtered through a
column of silica gel (1 cm height). The solution was concentrated, diethyl ether was
added and an off white solid precipitated. The solid was filtered and washed with
diethyl ether and identified as 3.
4.2.1. 2-(9-Methyl-6-(4-methylpiperazin-1-yl)-9H-purin-2-yl)phenol 3a
Method A: Off-white solid; Yield: 0.08 g, 67 %; m.p. = 193 – 195 oC (Found: C, 63.33;
H, 5.85; N, 25.73. C17H20N6O requires C, 62.96; H, 6.17; N, 25.92); IR (Nujol mull)


: 3013, 1599, 1581, 1513 cm-1;  (300 MHz, DMSO-d )  13.56 (s, 1H, OH),
max
6
8.36 (dd, J = 7.6 Hz, J = 1.2 Hz, 1H), 8.16 (s, 1H), 7.31 (dt, J = 7.6 Hz, J = 1.2 Hz, 1H),
6.90 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 4.22 (br s, 4H), 3.76 (s, 3H), 2.43 –
2.50 (m, 4H), 2.22 (s, 3H); 13C (75 MHz, DMSO-d )  157.41, 152.18, 149.68, 141.11,
6
131.82, 119.31, 117.66, 117.12, 54.41, 45.66, 44.75, 29.56.
4.2.2. 3-(9-Methyl-6-(4-methylpiperazin-1-yl)-9H-purin-2-yl)phenol 3b

Method A: Off-white solid; Yield: 0.10 g, 55 %; m.p. = 234 – 235 oC (Found: C, 62.61;
H, 6.33; N, 25.73. C17H20N6O requires C, 62.96; H, 6.17; N, 25.93); IR (Nujol mull)


: 3108, 3084, 1599, 1569 cm-1;  (300 MHz, DMSO-d ) 9.51 (s, 1H), 8.14 (s,
max
6
1H), 7.80 – 7.85 (m, 2H), 7.24 (t, J = 8.4 Hz, 1H), 6.81 (dt, J = 8.1 Hz, J = 1.8 Hz, 1H),
13
4.28 (br s, 4H), 3.78 (s, 3H), 2.45 – 2.48 (m, 4H), 2.22 (s, 3H); C (75 MHz, DMSO-
d )  157.33, 156.85, 152.84, 151.98, 140.92, 139.74, 129.07, 118.53, 118.05, 116.66,
6
114.48, 54.60, 45.79, 44.43, 29.38.
4.2.3. 2-(3-Chlorophenyl)-9-methyl-6-(4-methylpiperazin-1-yl)-9H-purine 3c
Method B: Off-white solid; Yield: 0.12 g, 72 %; m.p. = 132 – 134 oC (Found: C, 59.25;
H, 5.66; N, 24.28. C17H19N6Cl requires C, 59.56; H, 5.55; N, 24.50); IR (Nujol mull)


: 1670, 1592, 1570 cm-1;  (300 MHz, DMSO-d )  8.20-8.40 (m, 2H), 8.17 (s,
max
6
1H), 7.48 – 7.51 (m, 2H), 4.28 (br s, 4H), 3.79 (s, 3H), 2.45 (t, J = 5.1 Hz, 4H), 2.22 (s,
3H); 13C (75 MHz, DMSO-d )  155.26, 152.90, 151.81, 141.27, 140.49, 133.14,
6
130.22, 129.38, 127.02, 126.19, 118.33, 54.58, 45.76, 44.49, 29.45.
4.2.4. 2-(3,4-Dichlorophenyl)-9-methyl-6-(4-methylpiperazin-1-yl)-9H-purine 3d
Method B: Off-white solid; Yield: 0.12 g, 60 %; m.p. = 136 – 138 oC (Found: C, 54.04;
H, 4.67; N, 22.16. C17H18N6Cl2 requires C, 54.11; H, 4.47; N, 22.28); IR (Nujol mull)


: 1727, 1591, 1560 cm-1;  (300 MHz, DMSO-d )  8.47 (d, J = 1.8 Hz, 1H),
max
6
8.31 (dd, J = 8.5 Hz, J = 2.4 Hz, 1H), 8.17 (s, 1H), 7.71 (d, J = 8.4 Hz, 1H), 4.27 (br s,
13
4H), 3.78 (s, 3H), 2.44 (m, 4H), 2.22 (s, 3H); C (75 MHz, DMSO-d )  154.41,
6
152.86, 151.72, 141.38, 138.96, 132.21, 131.13, 131.59, 128.95, 127.60, 118.37, 54.60,
45.79, 44.54, 29.49 .
4.2.5. 2-(9-Methyl-6-(piperidin-1-yl)-9H-purin-2-yl)phenol 3e
Method A: Off-white solid; Yield: 0.13 g, 70 %; m.p. = 184 – 186 oC (Found: C, 65.63;
H, 6.17; N, 22.62. C17H19N5O.0.1H2O requires C, 65.64; H, 6.18; N, 22.52); IR (Nujol

mull) 
: 3013, 1560, 1576 cm-1;  (300 MHz, DMSO-d 
max
6
9.83 (br s, 1H), 8.50
J =
J =
J =
(s, 1H), 8.17 (d,
8.7 Hz, 2H), 7.79 (d,
8.4 Hz, 2H), 7.42 (d,
8.4 Hz, 2H), 6.82
J =
; 13C (75 MHz, DMSO-d )
(d,
8.7 Hz, 2H), 4.30 (br s, 4H), 3.77 (s, 4H), 2.40 (s, 3H)
6

 159.35, 157.47, 152.03, 149.72, 140.85, 131.77, 128.85, 119.40, 118.49, 117.54,
117.12, 46.08, 29.55, 25.57, 24.06.
4.2.6. 3-(9-Methyl-6-(piperidin-1-yl)-9H-purin-2-yl)benzene-1,2-diol 3f
Method A: Off-white solid; Yield: 0.12 g, 41 %; m.p. = 222 – 224 oC (Found: C, 62.90;
H, 5.55; N, 21.78. C17H19N5O2 requires C, 62.77; H, 5.85; N, 21.54); IR (Nujol mull)


: 3462, 1611, 1580 cm-1;  (300 MHz, DMSO-d )  13.95 (s, 1H), 8.80 (br s,
max
6
1H), 8.17 (s, 1H), 7.83 (dd, J = 8.0 Hz, J = 1.5 Hz, 1H), 6.84 (dd, J = 7.8 Hz, J = 1.5
Hz, 1H), 6.70 (t, J = 7.8 Hz, 1H), 4.22 (s, 4H), 3.78 (s, 3H), 1.66 (s, 6H); 13C (75 MHz,
DMSO-d )  157.95, 151.97, 149.86, 148.34, 146.07, 140.89, 119.56, 118.96, 117.82,
6
117.42, 117.20, 46.11, 29.55, 25.59, 24.07.
4.2.7. 2-(2-Chlorophenyl)-9-methyl-6-(piperidin-1-yl)-9H-purine 3g
Method B: Off-white solid; Yield: 0.14 g, 40 %; m.p. = 145 – 147 oC (Found: C, 62.47;
H, 5.63; N, 21.65. C17H18N5Cl requires C, 62.29; H, 5.50; N, 21.37); IR (Nujol mull)

: 1582 cm-1;  (300 MHz, DMSO-d )  8.16 (s, 1H), 7.71 – 7.74 (m, 1H), 7.49 –
max
6
7.53 (m, 1H), 7.39 – 7.44 (m, 2H), 4.21 (br s, 4H), 3.74 (s, 3H), 1.66 (d, J = 4.2 Hz,
13
2H), 1.57 (d, J = 3.9 Hz, 4H); C (75 MHz, DMSO-d )  157.71, 152.62, 151.47,
6
140.78, 138.92, 131.62, 131.48, 130.02, 129.75, 126.81, 117.55, 45.59, 29.50, 25.71,
24.27.
4.2.8. 2-(2,5-Dichlorophenyl)-9-methyl-6-(piperidin-1-yl)-9H-purine 3h
Method B: Off-white solid; Yield: 0.15 g, 45 %; m.p. = 116 – 118 oC (Found: C, 56.15;
H, 4.70; N, 18.93. C H N
5Cl2
requires C, 56.35; H, 4.69; N, 19.34); IR (Nujol mull)
17 17


: 3052, 1692, 1592 cm-1;  (300 MHz, DMSO-d )  8.18 (s, 1H), 7.77 (d, J =
max
6
2.4 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.51 (dd, J = 8.7 Hz, J = 2.4 Hz, 1H), 4.22 (br s,
13
4H), 3.75 (s, 3H), 1.67 (d, J = 4.8 Hz, 2H), 1.59 (d, J = 4.2 Hz, 4H); C (75 MHz,
DMSO-d )  156.31, 152.60, 151.36, 140.98, 140.28, 131.89, 131.37, 130.86, 130.43,
6
129.56, 117.70, 45.71, 29.53, 25.68, 24.22.

4.2.9. 3-(9-Methyl-6-(piperidin-1-yl)-9H-purin-2-yl)phenol 3i
Method A: Off-white solid; Yield: 0.15 g, 67 %; m.p. = 210 – 212 oC (Found: C, 64.78;
H, 5.93; N, 21.97. C17H19N5O.0.3H2O requires C, 64.88; H, 6.23; N, 22.26); IR (Nujol
mull) 
max
: 3165, 1569 cm-1;  (300 MHz, DMSO-d )  13.45 (s, 1H), 8.56 (s, 1H),
6
8.82 (br s, 1H), 7.79 (dd, J = 8.2 Hz, J = 1.8 Hz, 1H), 7.67 (d, J = 8.1 Hz, 2H), 7.46 (d,
J = 8.1 Hz, 2H), 6.83 (dd, J = 8.0 Hz, J = 1.5 Hz, 1H), 6.69 (t, J = 7.8 Hz, 1H), 4.30 (br
13
s, 4H), 3.81 (s, 4H), 2.42 (s, 3H); C (75 MHz, DMSO-d )  157.24, 156.85, 152.79,
6
151.89, 140.56, 139.90, 129.05, 118.53, 117.93, 116.54, 114.44, 45.42, 29.36, 25.66,
24.33.
4.2.10. 2-(3-Chlorophenyl)-9-methyl-6-(piperidin-1-yl)-9H-purine 3j
Method B: Off-white solid; Yield: 0.10 g, 35 %; m.p. = 139 – 141 oC (Found: C, 61.03;
H, 5.40; N, 20.66. C17H18N5Cl.0.4H2O requires C, 60.95; H, 5.62; N, 20.91); IR (Nujol

mull) 
: 1699, 1595, 1585, 1569 cm-1;  (300 MHz, DMSO-d )  8.14 (s, 1H),
max
6
8.30 – 8.35 (m, 2H), 7.40 – 7.52 (m, 2H), 4.27 (br s, 4H), 3.79 (s, 3H), 1.68 (t, J = 4.0
Hz, 2H), 1.57 (d, J = 4.4 Hz, 4H); 13C (75 MHz, DMSO-d )  155.26, 152.83, 151.73,
6
140.90, 140.65, 133.10, 130.19, 129.28, 126.99, 126.14, 118.21, 45.59, 29.42, 25.71,
24.27.
4.2.11. 6-(4-Methylpiperazin-1-yl)-9H-purine 3k
Method B: Off-white solid; Yield: 0.03 g, 72 %; m.p. = 274 – 276 oC (Found: C, 55.34;
H, 6.48; N, 38.42. C10H14N6 requires C, 55.05; H, 6.42; N, 38.53); IR (Nujol mull)


: 3067, 1582, 1517 cm-1;  (300 MHz, DMSO-d )  12.99 8br s, 1H), 8.19 (s,
max
6
13
1H), 8.10 (s, 1H), 4.20 (br s, 4H), 3.38 (s, 3H), 2.40 – 2.50 (m, 4H), 2.20 (s, 3H); C
(75 MHz, DMSO-d )  153.12, 151.73, 151.44, 138.14, 118.73, 54.59, 45.77, 44.45.
6
4.2.12. 2-(2,5-Dichlorophenyl)-6-(4-methylpiperazin-1-yl)-9H-purine 3l
o
Method B: Off-white solid; Yield: 0.090 g, 53 %; m.p. = 271 – 273 C (Found: C,
52.77; H, 4.35; N, 22.95. C16H16N6Cl2 requires C, 52.91; H, 4.44; N, 23.14); IR (Nujol

mull) 
: 3257, 3148, 1596, 1557 cm-1;  (300 MHz, DMSO-d )  8.08 (s, 1H),
max
6

7.74 (d, J = 2.7 Hz, 1H), 7.46 (dd, J = 8.7 Hz, J = 2.7 Hz, 1H), 4.80 (br s, 4H), 2.41 (m,
4H), 2.19 (s, 3H); 13C (75 MHz, DMSO-d )  155.34, 154.11, 152.54, 141.61, 140.77,
6
131.85, 131.33, 130.97, 130.39, 129.28, 118.31, 54.69, 45.83, 44.58.
4.2.13. 3-(6-(4-Methylpiperazin-1-yl)-9H-purin-2-yl)phenol 3m
Method B: Off-white solid; Yield: 0.23 g, 68 %; m.p. = 266 – 268 oC (Found: C, 57.93;
H, 5.10; N, 24.26. C16H18N6O requires C, 58.01; H, 5.07; N, 24.09.); IR (Nujol mull)

: 3125, 1596, 1568, 1516 cm-1;  (400 MHz, DMSO-d )  13.03 (br s, 1H), 9.44
max
6
(br s, 1H), 8.11 (s, 1H), 7.77-7.78 (m, 2H), 7.23 (t, J = 8.0 Hz, 1H), 6.81 (dd, J = 2.4
Hz, J = 8.0 Hz, 1H), 4.28 (br s, 4H), 2.45 (s, 4H), 2.22 (s, 3H); 13C (100 MHz, DMSO-
d )  157.28, 156.93, 152.86, 152.54, 139.97, 138.57, 129.13, 118.45, 117.91, 116.54,
6
114.43, 54.65, 45.83, 44.46.
4.2.14. 2-(3-Chlorophenyl)-6-(4-methylpiperazin-1-yl)-9H-purine 3n
Method B: Off-white solid; Yield: 0.11 g, 43 %; m.p. = 239 – 241 oC (Found: C, 58.26;
H, 5,11; N, 25.38. C16H17N6Cl requires C, 58.45; H, 5.18; N, 25.57); IR (Nujol mull)


: 3110, 1588, 1573 cm-1;  (300 MHz, DMSO-d )  13.13 (br s, 1H), 8.25 –
max
6
8.31 (m, 2H), 8.16 (s, 1H), 7.49 – 7.51 (m, 2H), 4.29 (br s, 4H), 4.20 – 4.50 (m, 4H),
2.23 (s, 3H); 13C (75 MHz, DMSO-d )  155.34, 152.89, 152.40, 140.70, 138.92,
6
133.12, 130.21, 129.33, 126.98, 126.04, 118.14, 55.60, 45.77, 44.42.
4.2.15. 2-(3,4-Dichlorophenyl)-6-(4-methylpiperazin-1-yl)-9H-purine 3o
Method B: Off-white solid; Yield: 0.13 g, 48 %; m.p. = 257 – 259 oC (Found: C, 52.88;
H, 4.64; N, 22.98. C16H16N6Cl2 requires C, 52.89; H, 4.41; N, 23.14); IR (Nujol mull)

: 3093, 1696, 1584, 1559 cm-1;  (300 MHz, DMSO-d )  13.13 (br s, 1H), 8.43
max
6
(d, J = 1.8 Hz, 1H), 8.27 (dd, J = 8.7 Hz, J = 2.1 Hz, 1H), 8.16 (s, 1H), 7.69 (d, J = 8.7
13
Hz, 1H), 4.28 (br s, 4H), 2.40 – 2.50 (m, 4H), 2.22 (s, 3H); C (75 MHz, DMSO-d )
6
 154.49, 152.85, 152.34, 139.18, 139.08, 132.07, 131.10, 130.59, 128.90, 127.47,
118.18, 54.60, 45.78, 44.47.

4.2.16. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-6-(4-methylpiperazin-1-yl)-9H-purine
3p
Method B: Off-white solid; Yield: 0.12 g, 48 %; m.p. = 232 – 234 oC (Found: C, 51.38;
H, 4.00; N, 21.33. C17H16N6ClF3 requires C, 51.45; H, 4.03; N, 21.19); IR (Nujol mull)

: 3093, 1600, 1586, 1575 cm-1;  (300 MHz, DMSO-d )  13.13 (br s, 1H), 8.72
max
6
(d, J = 2.1 Hz, 1H), 8.60 (dd, J = 8.1 Hz, J = 1.8 Hz, 1H), 8.18 (s, 1H), 7.83 (d, J = 8.4
13
Hz, 1H), 4.30 (br s, 4H), 2.43 – 2.50 (m, 4H), 2.23 (s, 3H); C (75 MHz, DMSO-d )
6
 154.32, 152.87, 152.33, 139.14, 137.95, 132.63, 131.84, 131.58 (d, J = 1Hz), 126.55
(q, J = 31Hz), 126.04 (q, J = 5 Hz), 122.89 (q, J = 272 Hz), 118.25, 54.57, 45.74, 44.54.
4.2.17. 4-(6-(4-Methylpiperazin-1-yl)-9H-purin-2-yl)phenol 3q
o
Method B: Dark orange solid; Yield: 0. 23 g, 76 %; m.p. = 253 – 255 C (Found: C,
58.12; H, 4.99; N, 24.23. C16H18N6O requires C, 58.01; H, 5.07; N, 24.09.); IR (Nujol
mull) 
: 3188, 1660, 1572 cm-1;  (400 MHz, DMSO-d )  12.96 (br s, 1H), 9.77
max
6
(br s, 1H), 8.17 (d, J = 8.8 Hz, 2H), 8.05 (s, 1H), 6.82 (d, J = 8.8 Hz, 2H), 4.27 (br s,
13
4H), 2.50-2.49 (m, 4H), 2.22 (s, 3H); C (100 MHz, DMSO-d )  158.96, 157.19,
6
152.88, 152.68, 138.08, 129.56, 129.14, 117.40, 114.95, 54.67, 45.83, 44.40.
4.2.18. 2-(4-Chlorophenyl)-6-(4-methylpiperazin-1-yl)-9H-purine 3r
Method B: Off-white solid; Yield: 0.12 g, 42 %; m.p. = 133 – 134 oC (Found: C, 58.35;
H, 5.33; N, 25.87. C16H17N6Cl requires C, 58.44; H, 5.22; N, 25.56); IR (Nujol mull)


: 3191, 3087, 1583 cm-1;  (300 MHz, DMSO-d )  13.08 (s, 1H), 8.34 (d, J =
max
6
8.4 Hz, 2H), 8.14 (s, 1H), 7.50 (d, J = 8.4 Hz, 2H), 4.29 (br s, 4H), 2.45 (m, 4H), 2.22
13
(s, 3H); C (75 MHz, DMSO-d )  155.80, 152.90, 152.49, 138.82, 137.41, 134.30,
6
129.23, 128.31, 117.99, 54.63, 45.80, 44.46.
4.2.19. 2-(2,5-Dichlorophenyl)-6-(piperidin-1-yl)-9H-purine 3s
Method B: Off-white solid; Yield: 0.05 g, 45 %; m.p. = 288 – 290 oC (Found: C, 54.98;
H, 4.44; N, 20.35. C16H15N5Cl2 requires C, 55.17; H, 4.31; N, 20.11); IR (Nujol mull)

: 3049, 1594, 1573, 1557 cm-1;  (300 MHz, DMSO-d )  13.11 (br s, 1H), 8.16
max
6

(s, 1H), 7.74 (d, J = 2.4 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.49 (dd, J = 8.4 Hz, J = 2.8
13
Hz, 1H), 4.22 (br s, 4H), 1.67 (d, J = 4.8 Hz, 2H), 1.59 (d, J = 4.0 Hz, 4H); C (75
MHz, DMSO-d )  156.33, 152.61, 151.80, 140.44, 138.60, 131.87, 131.35, 130.89,
6
130.35, 129.46, 117.52, 45.55, 25.68, 24.25.
4.2.20. 3-(6-(Piperidin-1-yl)-9H-purin-2-yl)phenol 3t
o
Method B: Light yellow solid; Yield: 0.17 g, 63 %; m.p. = 291 – 293 C (Found: C,
64.93; H, 5.74; N, 23.93. C16H17N5O requires C, 65.07; H, 5.80; N, 23.71); IR (Nujol
mull) 
: 3165, 3109, 1569, 1510 cm-1;  (400 MHz, DMSO-d )  12.97(br s, 1H),
max
6
9.43 (br s, 1H), 8.08 (s, 1H), 7.76-7.78 (m, 2H), 7.23 (t, J = 8.0 Hz, 1H), 6.80 (m, 1H),
13
4.28 (br s, 4H), 1.70 – 1.62 (m, 6H); C (100 MHz, DMSO-d )  157.25, 156.94,
6
152.80, 152.44, 140.13, 138.17, 129.08, 118.40, 117.77, 116.43, 114.42, 45.48, 25.70,
24.37.
4.2.21. 2-(3-Chlorophenyl)-6-(piperidin-1-yl)-9H-purine 3u
Method B: Off-white solid; Yield: 0.05 g, 20 %; m.p. = 245 – 247 oC (Found: C, 60.97;
H, 5.10; N, 21.85. C16H16N5Cl.0.1H2O requires C, 60.89; H, 5.14; N, 22.20); IR (Nujol

mull) 
max
: 3055, 1600, 1581, 1567 cm-1;  (300 MHz, DMSO-d )  13.05 (br s,
6
1H), 8.26 – 8.30 (m, 2H), 8.13 (s, 1H), 7.48 – 7.51(m, 2H), 4.28 (br s, 4H), 1.60 – 1.70
13
(m, 6H); C (75 MHz, DMSO-d )  155.36, 152.83, 152.35, 140.87, 138.61, 133.11,
6
130.24, 129.18, 126.97, 126.02, 118.02, 45.62, 25.68, 24.32.
4.2.22. 2-(3,4-Dichlorophenyl)-6-(piperidin-1-yl)-9H-purine 3v
Method B: Off-white solid; Yield: 0.11 g, 33 %; m.p. = 142 – 143 oC (Found: C, 55.45;
H, 4.45; N, 20.49. C16H15N5Cl2 requires C, 55.18; H, 4.35; N, 20.11); IR (Nujol mull)


: 3202, 3111, 1599, 1576, 1556 cm-1;  (300 MHz, DMSO-d )  13.08 (br s,
max
6
1H), 8.42 (d, J = 2.1 Hz, 1H), 8.27 (dd, J = 8.4 Hz, J = 2.1 Hz, 1H), 8.14 (s, 1H), 7.70
(d, J = 8.4 Hz, 1H), 4.27 (br s, 4H), 1.63 (m, 6H); 13C (75 MHz, DMSO-d )  154.54,
6
152.81, 152.18, 139.33, 138.67, 132.01, 131.09, 130.61, 128.88, 127.45, 118.11, 45.61,
25.68, 24.29.

4.2.23. 4-(6-(Piperidin-1-yl)-9H-purin-2-yl)phenol 3w
Method B: Light yellow solid; Yield: 0. 19 g, 72 %; m.p. > 300 oC (Found: C, 58.34; H,
6.65; N, 20.88. C16H17N5O. 1.90H2O requires C, 58.31; H, 6.36; N, 21.25); IR (Nujol
mull) 
max
: 3208, 3125, 1608, 1569, 1518 cm-1;  (400 MHz, DMSO-d )  12.86 (br
6
s, 1H), 9.69 (br s, 1H), 8.17 (d, J = 8.8 Hz, 2H), 8.02 (s, 1H), 6.81 (d, J = 8.8 Hz, 2H),
13
4.25 (br s, 4H), 1.69 – 1.60 (m, 6H); C (100 MHz, DMSO-d )  158.87, 157.21,
6
152.83, 152.60, 140.13, 137.71, 129.10, 117.28, 114.93, 45.49, 25.71, 24.40.
4.2.24. 2-(4-Chlorophenyl)-6-(piperidin-1-yl)-9H-purine 3x
Method B: Off-white solid; Yield: 0.06 g, 18 %; m.p. = 141 – 143 oC (Found: C, 61.06;
H, 5.17; N, 22.46. C16H16N5Cl requires C, 61.23; H, 5.15; N, 22.32); IR (Nujol mull)


: 3250, 1622, 1581 cm-1;  (300 MHz, DMSO-d )  8.32 (d, J = 6.9 Hz, 2H),
max
6
13
8.18 (s, 1H), 7.52 (d, J = 6.9 Hz, 2H), 4.26 (br s, 4H), 1.63 (m, 6H); C (75 MHz,
DMSO-d )  155.69, 152.69, 152.01, 138.71, 137.13, 134.46, 129.29, 128.38, 117.28,
6
45.71, 25.71, 24.28.
4.2. Radioligand binding assays
The percentage of inhibition of specific radioligand binding at the different
receptors by the compounds was assayed at the compound concentration of 10 µM at all
adenosine receptors following the conditions stated below. Competition binding curves
at all receptors were carried out by assaying 6 different concentrations (range from 10
nM to 100 M) for all the compounds showing an inhibition percentage above 80%.
The -log of the inhibition constant (pKi) of each compound was calculated by the
Cheng-Prusoff equation, Ki = IC50/(1 + [L]/Kd), where IC50 is the concentration of
compound that displaces the binding of the radioligand by 50 %, [L] is the free
radioligand concentration and Kd is the dissociation constant of each radioligand. IC50
values were obtained by non-linear regression fitting the data, using Prism 2.1 software
(GraphPad, San Diego, CA).

4.2.1. Human A1 receptors
Adenosine A1 receptor competition binding experiments were carried out in
membranes from CHO-A1 cells (Euroscreen, Gosselies, Belgium). On the day of assay,
membranes were defrosted and re-suspended in incubation buffer 20 mM Hepes, 100
mM NaCl, 10 mM MgCl2, 2 UI/ml adenosine deaminase (pH = 7.4). Each reaction well
of a GF/C multiscreen plate (Millipore, Madrid, Spain), prepared in duplicate, contained
15 g of protein, 2 nM [3H]DPCPX and test compound. Non-specific binding was
determined in the presence of 10 M (R)-PIA. The reaction mixture was incubated at 25
°C for 60 min, after which samples were filtered and measured in a microplate beta
scintillation counter (Microbeta Trilux, Perkin Elmer, Madrid, Spain).
4.2.2. Human A2A receptors
Adenosine A2A receptor competition binding experiments were carried out in
membranes from HeLa-A2A cells. On the day of assay, membranes were defrosted and
re-suspended in incubation buffer 50 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl2 and 2
UI/mL adenosine deaminase (pH = 7.4). Each reaction well of a GF/C multiscreen plate
(Millipore, Madrid, Spain), prepared in duplicate, contained 10 g of protein, 3 nM
[3H]ZM241385 and test compound. Nonspecific binding was determined in the
presence of 50 M NECA. The reaction mixture was incubated at 25 °C for 30 min,
after which samples were filtered and measured in a microplate beta scintillation
counter (Microbeta Trilux, Perkin Elmer, Madrid, Spain).
4.2.3. Human A2B receptors
Adenosine A2B receptor competition binding experiments were carried out in
membranes from HEK-293-A2B cells (Euroscreen, Gosselies, Belgium) prepared
following the provider’s protocol. On the day of assay, membranes were defrosted and
re-suspended in incubation buffer 50 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl2, 0.1
mM benzamidine, 10 g/mL bacitracine and 2 UI/mL adenosine deaminase (pH = 6.5).
Each reaction well prepared in duplicate, contained 18 g of protein, 35 nM

[3H]DPCPX and test compound. Non-specific binding was determined in the presence
of 400 M NECA. The reaction mixture was incubated at 25 °C for 30 min, after which
samples were filtered through a multiscreen GF/C microplate and measured in a
microplate beta scintillation counter (Microbeta Trilux, Perkin Elmer, Madrid, Spain).
4.2.4. Human A3 receptors
Adenosine A3 receptor competition binding experiments were carried out in
membranes from HeLa-A3 cells. On the day of assay, membranes were defrosted and
re-suspended in incubation buffer 50 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl2 and 2
UI/mL adenosine deaminase (pH = 7.4). Each reaction well of a GF/B multiscreen plate
(Millipore, Madrid, Spain), prepared in triplicate, contained 90 g of protein, 30 nM
[3H]NECA and test compound. Non-specific binding was determined in the presence of
100 M (R)-PIA. The reaction mixture was incubated at 25 °C for 180 min, after which
samples were filtered and measured in a microplate beta scintillation counter (Microbeta
Trilux, Perkin Elmer, Madrid, Spain).
4.3 Functional studies
4.3.1. Human A1 receptors
The antagonist behaviour of test compounds at A1 receptors was evaluated in
CHO-A1 cells stably expressing the receptors, by measuring NECA-mediated inhibition
of forkolin-stimulated cAMP accumulation, in the absence or presence of test
compounds. Briefly, cells grown in 96 well plates with growth medium containing
dialyzed fetal bovine serum, were washed twice with F-12 nutrient mixture medium
containing 25 mM HEPES pH 7.4 and 20 µM of the phosphodiesterase inhibitor
rolipram (incubation buffer). Then the test compounds were preincubated for 15 min in
assay medium and after this time, different concentrations (0.1 nM - 1 mM) of the
agonist NECA and 3 µM of forskolin were added to each well. The incubation was
continued for 15 min and reaction was stopped with a lysis buffer included in a cAMP

enzyme immunoassay kit (GE Healthcare). Cell lysates were transferred to a plate
coated with anti-rabbit IgG and rabbit-anticAMP in the presence of cAMP conjugated
with peroxidase for 60 min. After this time wells were washed four times with the wash
buffer provided in the kit and TMB was added to each well and incubated for 60 min.
Peroxidase reaction was stopped with 1 M sulphuric acid and the cAMP quantity was
calculate from the optical density at 450 nm read on a Tecan M100 reader.
4.3.2. Human A3 receptors
The antagonist behaviour of test compounds at A3 receptors was evaluated in
CHO- A3 cells stably expressing the receptors, by measuring NECA-mediated inhibition
of forskolin-stimulated cAMP accumulation, in the absence or presence of test
compounds. Briefly, cells grown in 96 well plates with growth medium containing
dialyzed fetal bovine serum, were washed twice with DMEM F-12 nutrient mixture
medium containing 25 mM HEPES pH 7.4 and 30 µM of the phosphodiesterase
inhibitor rolipram (incubation buffer). Then the test compounds were preincubated for
15 min in assay medium and after this time, different concentrations (0.1 nM - 1 mM)
of the agonist NECA and 10 µM of forskolin were added to each well. The incubation
was continued for 15 min and cAMP was quantified by an enzyme immunoassay
(Perkin Elmer).
NECA concentration-response curves in cAMP assays were fitted to the
following equations with Prism 2.1 (Graph Pad, San Diego, CA) and Kaleidagraph
software (Synergy Software, Reading, PA), respectively:
E = Emax x [A]s/(ECs50 + [A]s)
where Emax, [A], and s represent the maximum response, agonist concentration, and
curve slope, respectively. EC50 is the concentration of agonist that produces 50 % of the
maximal response. The antagonist potency was expressed as pKB (-log of the
dissociation constant, KB), calculated from a concentration-response curve of the
antagonist over the effect elicited by NECA following the equation:
KB = IC50/(2+(A/A50)n)1/n-1

where IC50 is concentration of antagonist that inhibits the agonist effect by 50 %, A is
the concentration of agonist, A50 is the concentration of agonist that exerts 50 % of the
maximal effect and n is the Hill-slope of the inhibition curve.
Acknowledgments
This research was funded by the Spanish Ministerio de Ciencia e Innovación (grants
HF2007-0055 and BIO2008-02329), the Portuguese Fundação para a Ciência e
Tecnologia (PPCDT/QUI/ 59356/2004), the Xunta de Galicia (07CSA003203PR and
08CSA020203PR), and the Instituto de Salud Carlos III. Funds provided by FCT
through the Chemistry Research Centre of the University of Minho (Ref.
UID/QUI/00686/2013 and UID/QUI/0686/2016) are also gratefully acknowledged.
Filipe Areias gratefully acknowledge Post-PhD grants from the Portuguese FCT
(SFRH/BPD/26106/2005). Carla Correia and Ashly Rocha also acknowledge to FCT
the PhD grants (SFRH/BD/22270/2005; SFRH/BD/85937/2012). The NMR
spectrometer (Bruker 400 Avance III) is part of the National NMR Network supported
by funds from FCT.
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