New A2A adenosine receptor antagonists: a structure-based upside-down interaction in the receptor cavity

Article abstract of DOI:10.1016/j.bioorg.2019.103183

Adenosine receptor antagonists are generally based on heterocyclic core structures presenting substituents of various volumes and chemical-physical profiles. Adenine and purine-based adenosine receptor antagonists have been reported in literature. In this

Full text of DOI:10.1016/j.bioorg.2019.103183

Journal Pre-Proof
New A2A adenosine receptor antagonists: A structure-based upside-down inter-
action in the receptor cavity
Catia Lambertucci, Andrea Spinaci, Michela Buccioni, Diego Dal Ben, Michael
Alliance Ngouadjeu Ngnintedem, Sonja Kachler, Gabriella Marucci, Karl-
Norbert Klotz, Rosaria Volpini
PII:
S0045-2068(19)30707-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.103183
YBIOO 103183
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 May 2019
16 July 2019
1 August 2019
Please cite this article as: C. Lambertucci, A. Spinaci, M. Buccioni, D. Dal Ben, M. Alliance Ngouadjeu Ngnintedem,
S. Kachler, G. Marucci, K-N. Klotz, R. Volpini, New A2A adenosine receptor antagonists: A structure-based upside-
down interaction in the receptor cavity, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103183
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JOURNAL PRE-PROOF
NEW A2A ADENOSINE RECEPTOR ANTAGONISTS: A STRUCTURE-
BASED UPSIDE-DOWN INTERACTION IN THE RECEPTOR CAVITY
Catia Lambertuccia,#, Andrea Spinacia,#, Michela Buccionia, Diego Dal Bena, Michael Alliance Ngouadjeu
Ngnintedema, Sonja Kachlerb, Gabriella Maruccia, Karl-Norbert Klotzb, and Rosaria Volpinia*
aSchool of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Camerino, Italy; bUniversität
Würzburg, Institut für Pharmakologie und Toxikologie, Versbacher Str. 9, D-97078, Würzburg, Germany.
*Address correspondence to this author at the School of Pharmacy, University of Camerino, via S. Agostino,
1; 62032, Camerino (MC), Italy;
E-mail: rosaria.volpini@unicam.it
# C. Lambertucci and A. Spinaci equally contributed to the work
Abstract: Adenosine receptor antagonists are generally based on heterocyclic core structures presenting
substituents of various volumes and chemical-physical profiles. Adenine and purine-based adenosine
receptor antagonists have been reported in literature. In this work we combined various substituents in the 2,
6, and 8-positions of 9-ethylpurine to depict a structure-affinity relationship analysis at the human adenosine
receptors. Compounds were rationally designed trough molecular modeling analysis and then synthesized
and evaluated at radioligand binding studies at human adenosine receptors. The new compounds showed
affinity for the human adenosine receptors, with some derivatives endowed with low nanomolar Ki data, in
particular at the A2AAR subtype. The purine core proves to be a versatile core structure for the development
of novel adenosine receptor antagonists with nanomolar affinity for these membrane proteins.
Keywords: adenosine receptors; purinergic receptors; adenosine receptor ligands; adenosine receptor
antagonists; rational design; medicinal chemistry, molecular modeling; radioligand binding studies.
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Highlights:




2,6 And 8-substituted 9-ethylpurines were designed as adenosine receptors ligands.
Compounds were synthesized and the tested at human adenosine receptors.
Some derivatives showed low nanomolar Ki data at adenosine receptors.
The purine core proves to be a useful core to obtain adenosine receptor ligands.
1. INTRODUCTION
Extracellular adenosine (Ado) plays a number of physiological roles through the stimulation of G Protein-
Coupled Receptors called adenosine receptors (ARs) and known as four subtypes: A1, A2A, A2B, and A3 ARs
[1, 2]. Each subtype has unique pharmacological profile due to endogenous ligand affinity, intracellular
effector coupling, and synthetic ligands specificity. In fact, A1 and A2A ARs are endowed with nanomolar
affinity for Ado, while A2B and A3 ARs need micromolar concentrations (i.e. due to tissue damage) of the
endogenous ligand to be activated. Furthermore, while the activation of A2A and A2B ARs takes to an
increase of intracellular levels of cAMP due to stimulation of adenylyl cyclase (AC) activity, the stimulation
of A1 and A3 ARs takes to reduction of intracellular cAMP and stimulation of phospholipase C (PLC)
activity [1, 2]. Each AR subtype presents different levels of expression in various tissues. Through the
modulation of the activity of the different ARs, extracellular Ado regulates vascular smooth muscle tone and
blood flow [3, 4], heart rate and contractility [5], inflammation [6-8], sleep [9, 10], and cognitive
mechanisms [11]. Within the CNS, Ado modulates also neuronal plasticity and development, neuronal
excitability and arousal, release of neurotransmitters, and neuroinflammation [12-16]. All together, these
factors make the ARs potential (if not promising) therapeutic targets for a number of conditions including
cardiovascular, inflammatory, and neurodegenerative diseases [17-21]. Besides Ado itself, therapeutically
used as injectable antiarrhythmic agent, natural or synthetic AR ligands are available on the market as
agonists (i.e. the coronary vasodilator Regadenoson [22]) and antagonists (the CNS stimulator caffeine [23],
the bronchodilator theophylline [24], the anti-Parkinson tool istradefylline [25]). The prototypical AR
antagonists are xanthines and methylxanthines, whose modifications led to derivatives endowed with
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potency and AR subtype selectivity [26]. Among these compounds are the above cited caffeine, theophylline,
and istradefylline. A number of classes of non-xanthine derivatives have also been developed as potent and
selective AR antagonists [27-30].
In the last years we developed 9-alkyladenine derivatives substituted in the 2, N6, and 8-positions. These
compounds presented different degrees of affinity and selectivity for the various AR subtypes [31-37]. More
in detail, the 8-bromo-9-ethyladenine showed good affinity and moderate selectivity for the A2AAR (I; Ki
A2A= 52 nM, Fig. 1); substitution of the bromine atom with a 2-furyl ring enhanced both the A2A affinity and
selectivity, especially versus the A3 receptor subtype (II; Ki A2A = 3.7 nM). On the other hand, even the
presence of a phenylethylamino or a phenylethyloxy chain in the 2-position of 9-ethyladenine favoured the
interaction with the A2AAR subtype leading to compound III and IV, which are endowed with a similar
selectivity profile (see details within the Biological activity section). The combination of a phenylethylamino
or a phenylethyloxy group in the 2-position and a bromine atom in the 8-position led to compounds endowed
with enhanced A2AAR affinity (V; Ki = 19 nM and VI; Ki = 1.7 nM, Fig. 1) [33, 38].
PLEASE INSERT FIGURE 1 HERE
Since the replacement of the 8-substituent of the 8-bromo-9-ethyladenine with a 2-furyl ring led to an
improvement of the A2AAR affinity [39], we hence synthesised (and here we present) 9-ethyladenine
derivatives bearing a 2-furyl ring in the 8-position and a phenylethylamino or a phenylethyloxy group in the
2-position (designed molecules of general formula A, Fig. 1). On the other hand, the displacement of the
phenylethylamino or the phenylethyloxy chain of V and VI from the 2- to the 6-position led to a significative
decrease of the A2AAR affinity (compounds VII: Ki = 280 nM and VIII: Ki = 19,300 nM, Fig. 2) [31]. For
compounds VII and VIII, modeling studies suggested a particular binding mode in which the 6-substituents
mimicked the role of the analogue 2-substituents of V and VI, while the 8- and 9-substituents were located
almost in the same position (Fig. 2). This resulted in a sort of upside-down binding mode respect to the
“canonical” binding orientation observed i.e. for the reference A2A receptor ligand ZM241385. In this
binding mode, however, the 6-substituted derivatives lacked the H-bond donor function corresponding to the
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free amine function of the 2-substituted derivatives [40]. On these bases, we designed and developed 9-
ethyladenines bearing a phenylethylamino or a phenylethyloxy chain in the 6-position, a bromine atom in the
8-position, and an amine function in the 2-position (designed molecules of general formula B, Fig. 2). The
presence of the amino group in the 2-position is aimed at restoring the H-bond donor function suggested by
molecular modeling studies previously reported [40] and according to the “pharmacophore” hypothesis
showed in Fig. 2. The new compound bearing the phenylethylamino chain in the 6-position and an 8-bromo
substituent was further modified by replacing the bromine atom with a 2-furyl ring.
PLEASE INSERT FIGURE 2 HERE
Finally, we also designed and synthesized 9-ethyladenines bearing various groups in the 8-position and the
phenylethylamino substituent in both the 2- and 6-positions to test the effect of the combined presence of the
two arylalkyl groups on the AR affinity (designed molecules of general formula C, Fig. 3). All the
compounds were tested by radioligand binding studies at the human recombinant A1, A2A and A3 ARs and the
results were analysed with molecular docking studies.
PLEASE INSERT FIGURE 3 HERE
2. RESULTS AND DISCUSSION
2.1 Chemistry
The synthesis of the 9-ethyl-8-(2-furyl)-2-phenylethylamine-adenine (1) and its 2-alkoxy analogue 2 was
carried out by Suzuki coupling reaction conditions on the 8-bromo-9-ethyl-2-substituted adenine derivatives
V and VI, previously prepared in our laboratory [33].
PLEASE INSERT SCHEME 1 HERE
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The two compounds were in turn treated with 2-(tributylstannyl)furan and bis triphenylphosphine palladium
dichloride in tetrahydrofuran as solvent at 60 °C for 5 h in anhydrous conditions (Scheme 1). The synthesis
of purine derivatives 7-10 was carried out starting from commercially available 2-amino-6-chloropurine (3;
Scheme 2). Compound 3 was reacted with ethyl iodide in dry dimethylformamide (DMF), using potassium
carbonate as base, under anhydrous conditions at room temperature (r.t.) as reported before [31].
PLEASE INSERT SCHEME 2 HERE
After chromatography of the reaction mixture, 4 and 4a were obtained as pure products with 66% and 21%
yield, respectively. These compounds were obtained previously [31] and the structures were confirmed by
UV spectra and NMR chemical shifts. In this work, for a deeper characterization, the alkylation site of the
1
two isomers was assigned by H-NOE difference spectroscopy in DMSO of the N6-methyl analogues 5 and
6, obtained by reacting 4 and 4a with methylamine (Scheme 2). The choice to use the N6-methyl derivatives
5 and 6 for the alkylation site assignment was due to the fact that in the case of the N7-isomer the NOE
effect easily takes place when in the N6-position there is a small group (i.e. a methyl group) that makes the
H-N(6) less exchangeable. In fact, irradiation of the CH2 protons of 5 (at 3.95 ppm) yielded a NOE at the H-
C(8) and H3-C(CH2) protons while there was no effect on the H-N(6) proton in the resulting spectra. On the
contrary, irradiation of CH2 protons of 6 (at 4.26 ppm) yielded a NOE at the H-C(8), H3-C(CH2) and also at
the H-N(6) protons in the resulting spectra. These data allowed an unambiguous identification of 5 as the 9-
isomer and 6 as the 7-isomer (Fig. 4).
PLEASE INSERT FIGURE 4 HERE
Hence, 4 was treated with phenylethylamine or phenylethyl alcohol in order to substitute the 6-chlorine atom
and to obtain compounds 7 and 8, respectively (Scheme 2). The reactions were performed using the reagents
as solvents at r.t. In the case of reaction with phenylethyl alcohol, sodium hydroxide was added to catalyse
the reaction.
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The 6-substituted 2-amino-9-ethylpurine derivatives 7 and 8 were obtained with a yield of 78% and 57%,
respectively. Reaction of these latter compounds with N-bromosuccinimide (NBS) in DMF, at r.t., furnished
the desired 8-bromo derivatives 9 and 10 with 80 and 35% yield, respectively (Scheme 2).
To obtain the 8-furyl derivative 13, a different synthetic pathway was set up, which allows to synthesise 2-
amino-9-ethyl-8-(2-furyl)purine derivatives substituted in the 6-position with various chains. In fact,
compound 4 was brominated to obtain the polyfunctionalized intermediate 11 (Scheme 3). The 8-bromo-6-
cloroadenine derivative 11 was then reacted with 2-(tributylstannyl)furan in the presence of
bis(triphenylphosphine)palladium (II) dichloride in DMF using Stille coupling reaction conditions.
PLEASE INSERT SCHEME 3 HERE
The selective substitution of the 8-bromine atom was obtained using 2-(tributylstannyl)furan in a 1:1
equivalent ratio with the purine derivative 11; the 8-furyl analogue 12 was obtained with a 54% yield.
Replacement of the 6-chlorine atom of 12 with phenylethylamine, at r.t., furnished the desired
tetrasubstituted adenine derivative 13 in 90% yield. This approach could be very useful to obtain in good
yield further 6-substituted derivatives of compound 12. Compounds 17-20 were obtained by 3 synthetic steps
starting from 2,6-dichloro-9-ethylpurine (14, Scheme 4) obtained with the earlier reported procedure [38]. 14
Was treated with 2-phenylethylamine at 120 °C for 16 h to get the desired 9-ethyl-2-phenylethylamine-6-
phenylethyladenine (15), with 90% yield, which was then treated with bromine and sodium acetate in acetic
acid and left to react at r.t. for 12 h.
PLEASE INSERT SCHEME 4 HERE
The 8-bromoderivative 16 was obtained with 85% yield as pure compound. The substitution of the 8-
bromine atom with a 2-furyl group was obtained as previously described for compounds 1 and 2 (Scheme 1),
to get the 9-ethyl-8-(2-furyl)-2-phenylethylamino-2-phenylethyladenine (17) with 29% yield. Finally,
reaction of 16 with methanol or ethanol, used as solvent, or phenethyl alcohol (using tetrahydrofurane as
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solvent) in the presence of sodium hydroxide, furnished the 8-alkyloxy purine derivatives 18-20 (Scheme 4).
These compounds were obtained by reaction at 70 °C or reflux for 17-72 h, with yield ranging from 37 to
80%.
2.2 Biological activity and molecular modeling studies
The novel adenine derivatives (1, 2, 7-10, 13, 15-20) were tested by evaluating their affinity at the human A1,
A2A and A3 ARs stably expressed in Chinese hamster ovary (CHO) cells. Previously published derivatives V-
VIII were reported as reference compounds. A set of derivatives was also selected and tested at the hA2BAR
subtype by measuring their inhibitory effects on NECA-stimulated cAMP levels in hA2BAR CHO cells. The
results are presented in Table 1. Molecular docking analyses were performed to simulate the binding mode of
the synthesized compounds at the hA2AAR cavity. As molecular target, we chose the high-resolution crystal
structure of the hA2AAR in complex with the antagonist ZM241385 (http://www.rcsb.org; pdb code: 5NM4;
1.7-Å resolution [41]). Gold software [42] was employed to perform docking analyses to depict the potential
binding mode of the compounds at the hA2AAR binding cavity. For a subset of compounds, the binding
modes at a homology model of the hA3AR [43] were also simulated with the same tools and protocols.
As already reported in the introduction part (Fig. 1), the presence of a bromine atom in the 8-position of 9-
ethyl-2-phenylethylamino or 9-ethyl-2-phenylethyloxyadenine enhanced both the A2AAR affinity and
selectivity (III; Ki A2AAR = 150 nM, select. A1/A2A = 2, A3/A2A = 21 vs V; Ki A2AAR = 19 nM, select.
A1/A2A = 8, A3/A2A = 163; and IV; Ki A2AAR = 120 nM, select. A1/A2A = 1, A3/A2A = 60 vs VI; Ki A2AAR =
1.7 nM, select. A1/A2A = 13, A3/A2A = 640) [33]. Replacement of the 8-bromine atom of V and VI with a 2-
furyl ring led to compounds with generally enhanced affinity at ARs, but decreased selectivity for the A2AAR
subtype. In particular, the 9-ethyl-8-furyl-2-phenylethylaminoadenine (1: Ki A2AAR = 3.8 nM, select. A1/A2A
= 2, A1/A3 = 5) displayed a five-fold increased affinity respect to its 8-bromo analogue V, while the 9-ethyl-
8-furyl-2-phenylethyloxyadenine (2: Ki A2AAR = 2.2 nM, select. A1/A2A = 3, A1/A3 = 7) maintained the same
A2AAR affinity of VI.
PLEASE INSERT TABLE 1 HERE
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The results of biological evaluation of compounds 7-10 (Table 1) showed that the combination of a
phenylalkylamino or phenylalkyloxy group in the 6-position with a 2-amine function generally takes to
compounds endowed with micromolar or high nanomolar affinity for the human ARs, with a low selectivity
in some cases for the A2AAR subtype.
As expected, the introduction of an 8-bromo substituent improves the affinity at all the AR subtypes with
respect to the corresponding 8-unsubstituted derivatives. Among these molecules, compound 9 showed the
best A2AAR affinity and selectivity (9, Table 1; Ki A2AAR = 266 nM; A1/A2A = 7; A3/A2A = 4). Based on the
observation that compound 1 and 2, bearing a 2-furyl group in the 8-position, showed an improved A2AAR
affinity with respect to the corresponding 8-bromo substituted analogues V and VI, we analogously modified
compound 9 to obtain the derivative 13. This compound showed a remarkable improvement of affinity for
the ARs with respect to the corresponding 8-bromo substituted analogue 9 and resulted the compound
endowed with the highest A2AAR affinity (13, Table 2; Ki A2AAR = 12 nM) among the newly 6-substituted
2-amino synthesised compounds. Among these derivatives, compounds 9 and 13 showed activity as A2BAR
inhibitors, even if only at micromolar level.
Results of docking studies performed at the human A2AAR crystal structure showed that the 2-amino
derivatives 7-10, 13 may interact with the receptor by adopting two kinds of conformations (Fig. 5). The first
one (binding mode 1, Fig. 5A) presents the purine core oriented similarly to the co-crystallized A2AAR
antagonist ZM241385 and similarly also to other adenine-based A2AAR antagonists previously developed
and reported. In detail, the bicyclic core is inserted between residues of transmembrane (TM) domains 3, 6,
and 7, making polar and hydrophobic interactions with the receptor residues.
PLEASE INSERT FIGURE 5 HERE
The 8- and 9-substituents point toward the depth of the cavity while the N6-substituent is oriented toward the
extracellular environment. The alternative docking conformation (binding mode 2, Fig. 5B), presents the
compound inserted in the cavity with an upside-down orientation with respect to the binding mode 1
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arrangement. The 8- and 9-substituents are again pointing toward the depth of the cavity, while the 2-amino
group is externally oriented. The comparison (Fig. 5A-B) and superimposition (Fig. 5C) of the two
arrangements demonstrate a good agreement with the rationale presented in Figure 2 (considering i.e.
compounds V and 9, or 1 and 13). In fact, the phenylalkylamine group in the 6-position of compound 13
(binding mode 2, Fig. 5B) is located in almost the same position of the analogue group in the 2-position of 1
(Fig. 5D); analogously, the exocyclic amine groups of the same two compounds are similarly located,
making analogue polar interaction with the receptor residue Asn253.
PLEASE INSERT FIGURE 6 HERE
We further developed this series by introducing the phenylalkylamino group in both the 2- and 6-position,
with additional 8-modifications. Results (Table 1) show that these compounds (15-20) are generally
endowed with nanomolar affinity for the ARs, but with some selectivity for the A3AR subtype. Compound
17 (Fig. 6), presenting a 2-furyl group in the 8-position, is endowed with a significantly improved A2AAR
affinity (17, Table 1; Ki A2AAR = 104 nM) with respect to the 8-unsubstituted derivative 15 (15, Table 2; Ki
A2AAR = 593 nM); on the other hand, compound 17 lacks selectivity for the A2AAR and it preferentially
binds the A3AR subtype (17, Table 1; Ki A3AR = 45 nM).
PLEASE INSERT FIGURE 7 HERE
Compound 15 showed a low nanomolar A3AR affinity (15, Table 1; Ki A3AR = 13 nM) with 35- and 47-fold
selectivity vs the A1 and A2A AR subtypes, resulting the most A3AR selective ligand of the whole group. An
improved affinity for this AR subtype was not totally unexpected, as several A3AR ligands reported in
literature bear arylalkyl groups in a position corresponding to the N6-substituent of the here reported
compound series (i.e. the reference A3AR agonists IBMECA or 2-Cl-IBMECA, or other Ado/NECA
derivatives [21, 44]). Docking studies at a homology model of A3AR showed that this molecule may adopt
both the above described binding modes (Fig. 7A-B). Considering the binding mode 1 arrangement, the N6-
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arylalkyl substituent is located in proximity of a hydrophobic valine residue (Val169) that is substituted by a
negatively charged glutamate in the other AR subtypes (i.e. Glu169 in the A2AAR [45]). This feature takes to
an improved interaction of the A3AR with the N6-arylalkyl substituent considering the binding mode 1
arrangement, but also with the 2-arylalkyl substituent considering the binding 2 docking conformations.
Docking results at the A3AR show that favourable docking scores are assigned to both binding modes and
hence both binding modes could easily take place within this receptor cavity. The ability of compounds
presenting some symmetry to adopt various binding modes within the A3AR cavity was already observed
and reported [46]. Introduction of substituents in the 8-position of 15 led to a significant decrease of A3AR
affinity and selectivity, while the A2AAR affinity resulted generally slightly improved. This may due to
different interaction of the compounds with the depth of the A2A and A3 ARs binding cavity, but also with the
effect of the 8-substituent to make the compound able (or not) to adopt various binding modes within the
cavities, with consequent higher or lower interaction with the receptor. Among the derivatives of the second
set, compounds 15, 17, and 19 were tested as A2BAR inhibitors but they did not show any activity at this AR
subtype.
Selected derivatives (V, VI, 1, 7-10, 13) were tested in CHO cells, stably expressing hA2AAR, in order to
assess their ability to inhibit or stimulate the receptor through the evaluation of cAMP production. The
results showed that the compounds were not able to stimulate cAMP production (data not shown) and
inhibited the NECA-induced increase of cAMP accumulation (see Table 2), behaving as antagonists of the
A2AAR. For three of them, 7, 8, 10, endowed with micromolar A2AAR affinity, it was not possible to obtain
an IC50 value below 50 µM concentration. For the other compounds, the obtained IC50 values present an
analogue trend of activity compared to the affinity data at the same receptor.
PLEASE INSERT TABLE 2 HERE
Furthermore, in order to evaluate the efficacy of some new compounds in native tissues, 1 and 13 were tested
at rat aorta rings to evaluate their ability to counteract relaxation induced by the selective A2AAR agonist
CGS 21680 [47]. The A2AAR antagonist ZM 241385 was also tested for comparison. CGS 21680 showed an
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EC50 = 280 nM whereas ZM 241385 exhibited an IC50 = 64 nM. Results confirmed the antagonistic
behaviour of the new compounds 1 and 13, which counteracted the relaxation induced by CGS 21680 with
an IC50 of 136 nM and 589 nM, respectively (Table 3). These data reveal that 1 and 13 have analogue effect
at rat and human A2AARs demonstrating their usefulness as tools to study pathologies involving the
adenosine A2A receptor.
PLEASE INSERT TABLE 3 HERE
3. CONCLUSION
Based on the observation that previously reported 9-ethyladenine derivatives bearing arylalkyl groups in the
2-position and an 8-bromo substituent are endowed with nanomolar affinity for the human A2AAR, in this
work we further modified these compounds by substituting the 8-bromo atom with a 2-furyl ring, very often
associated in literature with high A2AAR affinity. The obtained compounds showed low nanomolar A2AAR
Ki data, confirming the hypothesis. From the further observation that 9-ethyladenine derivatives bearing
arylalkyl groups in the 2-position or in the N6-position and an 8-bromo substituent present nanomolar affinity
as AR ligands, and following a pharmacophore-based rationale, we developed also a set of 9-ethylpurine
derivatives bearing a phenylethylamino- or phenylethyloxy-group in the 6-position and a 2-amine function,
to test the effect of these substituents on the affinity at human ARs. Compounds showed low nanomolar
affinity in particular for the A2AAR, with the presence of a further 8-substituent modulating the affinity at all
the ARs. Docking studies suggested that the substituents inserted in the purine core could make the
compounds adopt a sort of upside-down arrangement within the receptor cavity, as compared to the
“canonical” binding mode of the reference A2AAR ligand ZM241385. Following these results and
observations, we developed a second group of compounds presenting arylalkyl groups at both 2- and 6-
positions, to test the effect on AR affinity and subtype selectivity. The second group proved to be slightly
A3AR selective, and again the 8-substituent gave a significant modulation of the AR affinity, with different
impact at the various AR subtypes. Docking studies suggested more than one potential binding mode for
these compounds, similarly to the first group. These data again suggest the purine core as a versatile scaffold
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to develop AR ligands with nanomolar affinity and various degrees of selectivity for the different AR
subtypes.
4. MATERIALS AND METHODS
4.1 Chemistry
Melting points were determined with a Büchi apparatus and are uncorrected. 1H NMR spectra were obtained
with Varian Gemini 200 or VX 300 MHz spectrometers (1H NMR), (13C NMR);  values are in ppm, J
values are in Hz. All exchangeable protons were confirmed by the addition of D2O. Mass spectra were
recorded on an HP 1100-MSD series instrument. All measurements were performed using electrospray
ionization (ESI-MS) on a single quadrupole analyser. Thin-layer chromatography (TLC) was carried out on
pre-coated TLC plates with silica gel 60 F254 (Fluka). For column chromatography, silica gel 60 (Merck) was
used. For preparative TLC, silica gel 20x20 cm plates (UV 254 indicator UNIPLATE from Analtech were
used. Elemental analyses were determined on Fisons Instruments Model EA 1108 CHNS-O model analyser
and are within 0.4% of theoretical values. Purity of the compounds is ≥ 98% according to elemental analysis
data.
General procedure for the synthesis of 8-(furan-2-yl) purine derivatives 1, 2, 12, and 17. To the suitable
8-bromo derivative V, VI, [33] 11, or 16 (70 mg, 0.19 mmol), in anhydrous THF (10 mL), 2-
(tributylstannyl)furan (in the appropriate amount) and (PPh3)2PdCl2 (8 mg, 0.011 mmol) were added and the
reaction was left under stirring, in nitrogen atmosphere, at 60 °C, unless otherwise noted, in an oil bath, for
the time necessary to the reaction completion. The solvent was removed under vacuum and the crude
worked-up as described below.
9-Ethyl-8-(furan-2-yl)-N2-phenethyl-9H-purine-2,6-diamine (1): The title compound was obtained by
reaction of V with 5 equivalents of 2-(tributylstannyl)furan, in 5 h the reaction was complete. The crude
residue was purified by preparative thin layer chromatography (TLC) eluting with AcOEt-cC6H12-CH3OH
(60:37:3). Compound 1 was obtained pure, with 61% yield, after crystallization from CH3CN. M.p. = 154-
156 °C; 1H‐NMR (DMSO-d6, 200 MHz) δ = 1.33 (t, J = 6.7 Hz, 3H, CH3); 2.87 (t, J = 7.6 Hz, 2H, CH2-Ph);
3.49 (m, 2H, NH-CH2); 4.30 (m, 2H, N-CH2); 6.46 (m, 1H, NH-CH2); 6.72 (m, 1H, H-Furyl); 6.86 (br s, 2H,
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NH2); 6.99 (d, J = 3.6 Hz, 1H, H-Furyl); 7.29 (m, 5H, H-Ph); 7.91 (d, J = 1.4 Hz, 1H, H-Furyl). ESI-MS:
positive mode m/z 349.1 [M+H]+, 371.0 [M+Na]+. Elemental analysis calcd for C19H20N6O: C, 65.50; H,
5.79; N, 24.12; found: C, 65.65; H, 5.83; N, 23.91.
9-Ethyl-8-(furan-2-yl)-2-phenethoxy-9H-purin-6-amine (2): The title compound was obtained by reaction
of VI with 5 equivalents of 2-(tributylstannyl)furan. The reaction was complete within 3 h. The crude
residue was purified by preparative TLC eluting with AcOEt-CHCl3-CH3OH (65:30:5). Compound 2 was
1
obtained pure, with 39% yield, after crystallization from CH3OH. M.p. = 173-175 °C; H-NMR (DMSO-d6,
200 MHz) δ = 1.33 (t, J = 7.0 Hz, 3H, CH3); 3.03 (t, J = 7.0 Hz, 2H, CH2-Ph); 4.35 (q, J = 7.0 Hz, 2H, N-
CH2); 4.45 (t, J = 7.0 Hz, 2H, O-CH2); 6.74 (m, 1H, H-Furyl); 7.07 (d, J = 3.6 Hz, 1H, H-Furyl); 7.17-7.40
(m, 7H, H-Ph and NH2); 7.95 (d, 1H, H-Furyl). ESI-MS: positive mode m/z 350.0 [M+H]+, 372.0 [M+Na]+,
699.1 [2M+H]+, 721.1 [2M+Na]+. Elemental analysis calcd for C19H19N5O2: C, 65.32; H, 5.48; N, 20.04;
found: C, 65.39; H, 5.72; N, 19.86.
6-Chloro-9-ethyl-8-(furan-2-yl)-9H-purin-2-amine (12): The title compound was obtained by reaction of
11 with 2-(tributylstannyl)furan in a 1:1 equivalent ratio. The reaction was complete within 3 h. The crude
was purified by normal column chromatography using a dry silica slurry and eluting with CHCl3 – MeCN
(99:5). Compound 12 was obtained pure, with 54% yield, after crystallization from CHCl3 – cC6H12. M.p. =
220-222 °C; 1H-NMR = (DMSO-d6) δ = 1.31 (t, J = 7.2 Hz, 3H, CH3); 4.34 (m, 2H, N-CH2); 6.76 (m, 1H, H-
Furyl); 7.01 (s, 2H, NH2); 7.26 (d, J = 3.6 Hz, 1H, H-Furyl); 8.00 (s, 1H, H-Furyl). ESI-MS: positive mode
m/z 264.0 [M+H]+, 549.0 [2M+Na]+.
9-Ethyl-8-(furan-2-yl)-N2,N6-diphenethyl-9H-purine-2,6-diamine (17): The title compound was obtained
by reaction of 16 with 5 equivalents of 2-(tributylstannyl)furan in an oil bath under reflux for 2.5 h till the
reaction was complete. The crude residue was purified by flash column chromatography, dry silica slurry,
eluting with nC6H14 – AcOEt (90:10 to 85:15). Compound 17 was obtained as a white solid in 29% yield
1
after crystallization from AcOEt – Et2O to get. M.p. = 171-173 °C; H-NMR (DMSO-d6) δ = 1.32 (m, 3H,
NCH2CH3), 2.88 (t, J = 6.8 Hz, 2H, CH2Ph), 2.93 (t, J = 8.0 Hz, 2H, CH2Ph), 3.56 (m, 2H, N2CH2), 3.67 (m,
2H, N6CH2), 4.29 (m, 2H, NCH2CH3), 6.72 (m, 1H, H-Furyl), 7.03 (m, 1H, H-Furyl), 7.19-7.36 (m, 12H, H-
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Ph, N2H, and N6H), 7.92 (m, 1H, H-Furyl). ESI-MS: positive mode m/z 453.0 [M+H]+, 474.9 [M+Na]+.
Elemental analysis calcd for C27H28N6O: C, 71.66; H, 6.24; N, 18.57; found: C, 71.86; H, 6.46; N, 18.34.
Synthesis of 6-chloro-9-ethyl-9H-purin-2-amine (4) and 6-chloro-7-ethyl-9H-purin-2-amine (4a). To a
solution of 2-amino-6-chloropurine (3, 210 mg, 1.24 mmol) in 6.2 mL of dry DMF K2CO3 (215 mg, 1.56
mmol) and 1 equivalent of iodoethane (99 µl; 1.24 mmol) were added. The mixture was left under stirring
and under nitrogen atmosphere at r.t. After 5 h the reaction was complete. After evaporation of the solvent, a
dry silica slurry was prepared and flash chromatography, eluting with nC6H14 – AcOEt (60:40 to AcOEt
100%), was used to separate the reaction products. Compounds 4 and 4a were obtained after crystallization
from MeCN as pure product with the yield of 66 and 21%, respectively. 4: m.p. = 154-156 °C; 1H-NMR
(DMSO-d6, 400 MHz) δ = 1.35 (t, J = 7.2 Hz, 3H, CH3); 4.05 (m, 2H, N-CH2); 6.88 (bs, 2H, NH2); 8.13 (s,
1H, H-8). 4a: m.p. = 178 °C (dec.); 1H-NMR (DMSO-d6, 400 MHz) δ = 1.38 (t, J = 7.2 Hz, 3H, CH3); 4.30
(m, 2H, N-CH2); 6.62 (br s, 2H, NH2); 8.37 (s, 1H, H-8).
General procedure for the synthesis of 9-ethyl-N6-methyl-9H-purine-2,6-diamine (5) and 7-ethyl-N6-
methyl-7H-purine-2,6-diamine (6): To 2 ml of methylamine, compound 4 or 4a (100 mg; 0.51 mmol) were
in turn added in a steel vial at -80 °C. The vial, well closed, was then brought to r.t. and the reaction was left
for 16 h until completion. After evaporation of methylamine at r.t. and coevaporation with MeOH,
compound 5 was obtained after crystallization from MeOH in 96% yield; the crude mixture of the reaction of
4a was chromatographed on silica gel flash column, eluting with CHCl3 – MeOH (93:7 to 90:10) to get
compound 6 in 64% yield.
5: m.p. = 212 - 214 °C; 1H-NMR (DMSO-d6, 400 MHz) δ = 1.30 (t, J = 7.2 Hz, 3H, CH3); 2.86 (bs, 3H, N-
CH3); 3.95 (m, 2H, N-CH2); 5.81 (bs, 2H, NH2); 7.11 (bs, 1H, NH), 7.67 (s, 1H, H-8). Elemental analysis
calcd for C8H12N6: C, 49.99; H, 6.29; N, 43.72; found: C, 50.32; H, 6.32; N, 43.65.
6: m.p. = 223 °C; 1H-NMR (DMSO-d6, 400 MHz) δ = 1.27 (t, J = 7.2 Hz, 3H, CH3); 2.88 (d, J = 4.8 Hz, 3H,
N-CH3); 4.26 (q, J = 6.0 Hz, 2H, N-CH2); 5.55 (bs, 2H, NH2); 6.45 (m, 1H, NH), 7.84 (s, 1H, H-8).
Synthesis of 9-ethyl-N6-phenethyl-9H-purine-2,6-diamine (7): Phenethylamine (7 mL) was added to
compound 4 (200 mg, 1.01 mmol). The reaction was left under stirring at r.t. for 3.5 h. The phenethylamine
was removed under vacuum at high temperature. After preparation of a dry silica slurry, the crude reaction
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mixture was chromatographed on a flash column, eluting with AcOEt – cC6H12 – MeOH (60:37:3).
1
Compound 7 was obtained after crystallization from MeCN in 80% yield. M.p. = 106-108 °C; H-NMR
(DMSO-d6, 400 MHz) δ = 1.31 (t, J = 7.2 Hz, 3H, CH3); 2.87 (t, J = 7.6 Hz, 2H, CH2-Ph); 3.59 (br s, 2H,
NH-CH2); 3.96 (q, J = 7.2, 2H, N-CH2); 5.84 (s, 2H, NH2); 7.17 (m, 1H, H-Ph); 7.28 (m, 5H, H-Ph and H-8);
7.69 (s, 1H, NH). Elemental analysis calcd for C15H18N6: C, 63.81; H, 6.43; N, 29.77; found: C, 64.05; H,
6.69; N, 29.54.
Synthesis of 9-ethyl-6-phenetoxy-9H-purin-2-amine (8): Phenethyl alcohol (5 mL) was added to
compound 4 (200 mg; 1.01 mmol) and, to the solution, 5 equivalents of NaOH (200 mg; 5 mmol) were
added and the reaction was left under stirring at r.t. until it was complete after 3.5 h. The solvent was
removed by distillation under vacuum; after preparation of a dry slurry, the crude reaction mixture was
separated by column chromatography eluting with CHCl3, to elute the phenethyl alcohol in excess, and then
with CHCl3-MeOH (99:1) to elute 8. Compound 8 was obtained after crystallization from MeCN in 57%
yield. M.p. = 146-148 °C; 1H-NMR (DMSO-d6, 400 MHz) δ = 1.32 (t, J = 7.4 Hz, 3H, CH3); 3.06 (t, J = 7.4
Hz, 2H, CH2-Ph); 4.01 (q, J = 7.2 Hz, 2H, N-CH2); 4.58 (t, J = 7.2 Hz, 2H, O-CH2); 6.37 (s, 2H, NH2); 7.31
(m, 5H, H-Ph); 7.58 (s, 1H, H-8). Elemental analysis calcd for C15H17N5O: C, 63.59; H, 6.05; N, 24.72;
found: C, 63.86; H, 6.41; N, 24.41.
General procedure for the preparation of the 8-bromoderivatives 9-11: To a solution (1 mmol) of the
suitable purine derivative 7, 8, or 4 in dry DMF (4 mL) the NBS (202 mg; 1.1 mmoles) was added and the
reaction mixture was left under stirring and under nitrogen atmosphere at r.t. for the time necessary to the
reaction completion. The volatiles were evaporated and a dry silica slurry was prepared with the crude
residue and used for the flash chromatography eluted with the suitable solvent.
8-Bromo-9-ethyl-N6-phenethyl-9H-purine-2,6-diamine (9): Compound 9 was prepared from compound 7.
The reaction took 10 min for the completion. The crude residue was chromatographed eluting with CHCl3 –
MeCN (98:2). Compound 9 was obtained in 80% yield. M.p. = 114-116 °C; 1H-NMR (DMSO-d6, 400 MHz)
δ = 1.23 (t, J = 7.0 Hz, 3H, CH3); 2.85 (t, J = 7.8 Hz, 2H, CH2-Ph); 3.57 (br s, 2H, NH-CH2); 3.96 (q, J = 7.0
Hz, 2H, N-CH2); 6.01 (s, 2H, NH2); 7.18 (m, 6H, H-Ph and H-8); 7.44 (s, 1H, NH); ESI-MS: positive mode
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m/z 361.0 [M+H]+, 383.0 [M+Na]+. Elemental analysis calcd for C15H17BrN6: C, 49.87; H, 4.74; N, 23.26;
found: C, 50.12; H, 4.98; N, 23.01.
8-Bromo-9-ethyl-6-phenethoxy-9H-purin-2-amine (10): Compound 10 was prepared from compound 8.
The reaction took 70 min for the completion. The crude residue was chromatographed eluting with cC6H12 –
AcOEt (60:40) to cC6H12 – AcOEt – MeOH (55:40:5). A further purification by preparative TLC was
necessary, hence, the obtained impure product was separated with preparative TLC, eluted with AcOEt –
cC6H12 - MeOH (60:39:1). After crystallization from MeCN, the desired product 10 was obtained in 35%
yield. M.p. = 168-171 °C; 1H-NMR (DMSO-d6, 400 MHz) δ = 1.24 (t, J = 7.2 Hz, 3H, CH3); 3.05 (t, J = 7.2
Hz, 2H, CH2-Ph); 4.00 (q, J = 7.0 Hz, 2H, N-CH2); 4.56 (t, J = 7.0 Hz, 2H, O-CH2); 6.59 (s, 2H, NH2); 7.21
(m, 1H, H-Ph); 7.31 (m, 4H, H-Ph). ESI-MS: positive mode m/z 362.0 [M+H]+, 384.0 [M+Na]+. Elemental
analysis calcd for C15H16BrN5O: C, 49.74; H, 4.45; N, 19.33; found: C, 49.86; H, 4.67; N, 19.14.
8-Bromo-6-chloro-9-ethyl-9H-purin-2-amine (11): Compound 11 was prepared from compound 4. The
reaction was left for 8 h, then stopped due to the formation of side products. The DMF was removed under
vacuum and the crude mixture was partitioned between H2O and AcOEt. The organic layers were collected,
dried over Na2SO4, and concentrated to dryness under vacuum. The crude residue was chromatographed
eluting with nC6H14 – AcOEt (75:25). The collected product was crystallized from CH2Cl2 and compound 11
1
was obtained in 75% yield. M.p. = 182 °C (dec); H-NMR (DMSO-d6) δ = 1.27 (t, J = 7.4 Hz, 3H, CH3);
4.04 (q, J = 7.2 Hz, 2H, N-CH2); 7.06 (s, 2H, NH2). ESI-MS: positive mode m/z 276.0 [M+H]+, 297.9
[M+Na]+, 315.9 [M+K]+.
Synthesis of 9-ethyl-8-(furan-2-yl)-N6-phenethyl-9H-purine-2,6-diamine (13): Compound 12 (75 mg,
0.29 mmol) was added to phenethylamine (3 mL) and left to react under stirring at r.t. until completion after
4 h. The phenethylamine was removed under vacuum and after preparation of a dry silica gel slurry, the
crude mixture was chromatographed with a flash column, eluted with CHCl3. Compound 13 was obtained as
a pure vitreous solid in 90% yield. 1H-NMR (DMSO-d6, 400 MHz) δ = 1.27 (t, J = 7.2 Hz, 3H, CH3); 2.89 (t,
J = 7.6 Hz, 2H, CH2-Ph); 3.61 (bs, 2H, NH-CH2); 4.23 (q, J = 7.0 Hz, 2H, N-CH2); 6.00 (bs, 2H, NH2); 6.69
(m, 1H, H-Furyl); 6.95 (d, J = 3.6 Hz, 1H, H-Furyl); 7.18 (m, 1H, H-Ph); 7.28 (m, 4H, H-Ph); 7.45 (br s, 1H,
16

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NH); 7.87 (s, 1H, H-Furyl). ESI-MS: positive mode m/z 349.1 [M+H]+, 371.1 [M+Na]+. Elemental analysis
calcd for C19H20N6O: C, 65.50; H, 5.79; N, 24.12; found: C, 65.77; H, 5.93; N, 23.85.
Synthesis of 9-ethyl-N2,N6-diphenethyl-9H-purine-2,6-diamine (15): To 2-phenylethylamine (2 mL) the
2,6-dichloro-9-ethylpurine (14 [38]; 200 mg; 0.92 mmol) was added in a steel vial and left to react at 120 °C
for 16 h. After concentration under vacuum, a dry silica slurry was prepared and purified over a flash column
chromatography eluting with CH2Cl2 – cC6H12 (75:25 to 100% CH2Cl2) to afford 15 as a light yellowish solid
after crystallization with CH3CN. Compound 15 was obtained with 90% yield. M.p. = 102-104 °C; 1H-NMR
(DMSO-d6, 400 MHz) δ = 1.34 (t, J = 7.6 Hz, 3H, NCH2CH3), 2.85 (t, J = 7.2 Hz, 2H, CH2Ph), 2.88 (t, J =
8.0 Hz, 2H, CH2Ph), 3.47 (q, J = 6.4 Hz, 2H, N2CH2), 3.63 (br s, 2H, N6CH2), 3.99 (q, J = 7.6 Hz, 2H,
NCH2CH3), 6.45 (br s, 1H, N2H), 7.15-7.27 (m, 11H, 2 x Ph and N6H), 7.70 ppm (s, 1H, H-8). ESI-MS:
positive mode m/z 387.1 [M+H]+, 409.0 [M+Na]+. Elemental analysis calcd for C23H26N6: C, 71.47; H, 6.78;
N, 21.74; found: C, 71.82; H, 6.91; N, 21.55.
Synthesis of 8-bromo-9-ethyl-N2,N6-diphenethyl-9H-purine-2,6-diamine (16): To a mixture of 15 (50 mg;
0.13 mmol), sodium acetate (16 mg; 0.19 mmol), and acetic acid (0.56 mL) bromine (0.4 mL; 0.78 mmol)
were added. The mixture was then stirred at r.t. for 12 h, diluted with H2O (10 mL) and extracted with
CH2Cl2 (3x10 mL). The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to
dryness. The crude was purified by flash column chromatography eluting with cC6H12-CH2Cl2 (10:90) to
CH2Cl2/CH3OH (99.75:0:25) to afford 16 as a yellow powder after crystallization from cC6H12 and Et2O. 16
Was obtained with 85% yield. M.p. = 250 °C (dec); 1H-NMR (DMSO-d6, 400 MHz) δ = 1.25 (t, J = 6.4 Hz,
3H, NCH2CH3), 2.85 (m, 3H, CH2Ph), 2.99 (m, 2H, CH2Ph), 3.44 (m, 2H, N2CH2), 3.60 (br s, 2H, N6CH2),
3.98 (q, J = 6.8 Hz, 2H, NCH2CH3), 6.66 (m, 2H, N2H & N6H), 7.13-7.56 (m, 10H, H-Ph). ESI-MS: positive
mode m/z 465.2 [M+H]+, 467.3 [M+Na]+. Elemental analysis calcd for C23H25BrN6: C, 59.36; H, 5.41; N,
18.06; found: C, 59.61; H, 5.79; N, 17.83.
General procedure for the preparation of the 8-alkoxypurine derivatives 18 and 19: Sodium hydroxide
(200 mg; 5 mmol) was in turn added to methanol or ethanol (33 mL) at 70 °C and left until dissolution. To
the obtained solutions, 16 (70 mg, 0.15 mmol) was added together with another portion of sodium hydroxide
(200 mg; 5 mmol) and the reaction mixture was left under stirring, at the same temperature, for the time
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necessary to the reaction completion. The volatiles were evaporated and a dry silica slurry was prepared with
the crude residue and used for the flash chromatography, to be eluted with the suitable solvent.
9-Ethyl-8-methoxy-N2,N6-diphenethyl-9H-purine-2,6-diamine (18): The title compound was obtained by
reaction of 16 with methanol for 17 h. Purification by column chromatography eluted with nC6H14 – AcOEt
(90:10 to 70:30) gave 18 as vitreous solid with 80% yield. 1H-NMR (DMSO-d6) δ = 1.21 (t, J = 7.2 Hz, 3H,
NCH2CH3), 2.85 (m, 4H, 2 x CH2Ph), 3.44 (q, J = 6.4 Hz, 2H, N2CH2), 3.62 (br s, 2H, N6CH2), 3.81 (q, J =
6.8 Hz, 2H, NCH2CH3), 4.00 (s, 3H, OCH3), 6.29 (br s, 1H, N2H), 6.87 (br s, 1H, N6H), 7.17-7.26 ppm (m,
10H, H-Ph). ESI-MS: positive mode m/z 417.0 [M+H]+, 438.9 [M+Na]+. Elemental analysis calcd for
C24H28N6O: C, 69.21; H, 6.78; N, 20.18; found: C, 69.51; H, 6.92; N, 19.99.
8-Ethoxy-9-ethyl-N2,N6-diphenethyl-9H-purine-2,6-diamine (19): The title compound was obtained by
reaction of 16 with ethanol for 19 h. Purification by column chromatography eluted with nC6H14 – AcOEt
(90:10 to 80:20) gave 19 as vitreous solid with 67% yield. 1H-NMR (DMSO-d6) δ = 1.22 (t, J = 6.8 Hz, 3H,
NCH2CH3), 1.35 (t, J = 6.8 Hz, 3H, OCH2CH3), 2.85 (m, 4H, 2 x CH2Ph), 3.44 (q, J = 6.0 Hz, 2H, N2CH2),
3.62 (br s, 2H, N6CH2), 3.80 (q, J = 7.2 Hz, 2H, NCH2CH3), 4.41 (q, J = 7.2 Hz, 2H, OCH2CH3), 6.27 (br s,
1H, N2H), 6.82 (br s, 1H, N6H), 7.15-7.26 ppm (m, 10H, H-Ph). ESI-MS: positive mode m/z 431.1 [M+H]+.
Elemental analysis calcd for C25H30N6O: C, 69.74; H, 7.02; N, 19.52; found: C, 69.95; H, 7.39; N, 19.31.
Synthesis of 9-ethyl-8-phenethoxy-N2,N6-diphenethyl-9H-purine-2,6-diamine (20): To a solution of 16
(70 mg; 0.15 mmol) in dry tetrahydrofuran (2 mL) sodium hydroxide (30 mg; 0.75 mmol) and 2-
phenylethanol (120 µL; 1.00 mmol) were added. The reaction was poured at reflux for 72 h. The solvent was
removed under vacuum and the crude residue purified by silica gel column, dry silica slurry, eluting with
nC6H14 – AcOEt (90:10 to 85:15). The obtained solid was crystallized from nC6H14-AcOEt and 20 was
obtained with 37% yield as off-white powder. M.p. = 250 °C (dec.); 1H-NMR (DMSO-d6) δ = 1.12 (t, J = 6.8
Hz, 3H, NCH2CH3), 2.84 (m, 4H, 2 x CH2Ph), 3.08 (t, J = 6.4 Hz, 2H, OCH2CH2Ph), 3.43 (q, J = 6.8 Hz, 2H,
N2CH2), 3.61 (br s, 2H, N6CH2), 3.74 (q, J = 8.0 Hz, 2H, NCH2CH3), 4.56 (q, J = 7.2 Hz, 2H, OCH2), 6.28
(br s, 1H, N2H), 6.83 (br s, 1H, N6H), 7.16-7.30 (m, 15H, H-Ph). ESI-MS: positive mode m/z 507.1 [M+H]+,
529.0 [M+Na]+. Elemental analysis calcd for C31H34N6O: C, 73.49; H, 6.76; N, 16.59; found: C, 73.68; H,
6.97; N, 16.35.
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4.2 Biological evaluation
Cell culture. CHO cells stable transfected with hARs were grown in Dulbecco’s modified Eagle’s medium
(DMEM) with nutrient mixture F12 supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin,
100 µg/ml streptomycin, 2.5 µg/ml amphotericin B, 0.1 mg/ml geneticine and 1 mM sodium pyruvate. They
were cultured at 37 °C in a humidified atmosphere of 5% CO2/95% air [38, 48].
Membrane preparation. Crude membranes for radioligand binding experiments were prepared by collecting
cells (CHO stably transfected with hA1, hA2A and hA3 ARs) in ice-cold hypotonic buffer (5 mM Tris/HCl, 2
mM EDTA, pH 7.4). The cell suspension was homogenized on ice (Ultra-Turrax, 2x20 sec at full speed) and
the homogenate was spun for 10 min (4 °C) at 3200 rpm. The supernatant was centrifuged for 50 min at
37000 rpm at 4 °C. The membrane pellet was resuspended in the specific binding buffer (hA1AR: 50 mM
Tris/HCl buffer, pH 7.4; hA2AAR: 50 mM Tris/HCl, 50 mM MgCl2, pH 7.4; hA3AR: 50 mM Tris/HCl, 10
mM MgCl2, 1 mM EDTA, pH 8.25), frozen in liquid nitrogen at a protein concentration of 2–4 mg/mL and
stored at -80 °C.
Radioligand binding. Dissociation constants of unlabelled compounds (Ki values) were determined in
radioligand competition experiments, where a fixed concentration of radioligand (1 nM [3H]CCPA, KD = 1.1
nM; 10 nM [3H]NECA, KD = 20 nM; [3H] HEMADO, KD = 1.5 nM) was incubated in a 96-well plate with
10 µg of specific receptor (hA1, hA2A and hA3 ARs, respectively) cell membrane preparations and increasing
concentrations of the compound understudy. Non-specific binding was determined in the presence of 1 mM
theophylline for hA1AR and 100 µM (R)-N6-phenyliso-propyladenosine (R-PIA) for both hA2AAR and
hA3AR. All binding data were calculated by non-linear curve fitting with Prism 5.0 programme (GraphPAD
Software, San Diego, CA, USA). Each concentration was tested three-five times in triplicate and the values
are given as the mean ± standard errors (S.E.). The activity of the compounds at the hA2BAR was evaluated
as inhibition of NECA-stimulated adenylyl cyclase activity.
GloSensor cAMP Assay. Cells, stably expressing the hA2A or hA2B AR and transiently the biosensor, were
harvested in CO2-independent medium and were counted in a Neubauer chamber. The desired number of
cells was incubated in equilibration medium containing a 3% v/v GloSensor cAMP reagent stock solution,
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10% FBS, and 87% CO2 independent medium. After 2 h of incubation at rt, the cells were dispensed in the
wells of a 384-well plate and, when a steady-state basal signal was obtained, the NECA reference agonist or
the understudy compounds, at different concentrations, were added. Initially, the ability of the selected
compounds to stimulate the cAMP production was evaluated, but any results were obtained. Subsequently,
their antagonist profile was evaluated by assessing their ability to counteract NECA-induced increase of
cAMP accumulation. The cells were incubated in the reaction medium (10 min at rt) with different
understudy molecule concentrations and then treated with NECA. After 10 min, various luminescence
measurements were performed at different incubation times [38, 48].
Functional antagonism in isolated rat aorta. All animal testing was carried out according to European
Communities Council Directive of 24 November 1986 (86/609/EEC).
Male Wistar rats (275-300 g) were killed by cervical dislocation and the aorta was isolated, freed from
adhering connective tissue, and set up rapidly under 1 g resting tension in 10 mL organ baths containing in
Krebs solution of the following composition (mM): NaCl, 118.4; KCl, 4.7; CaCl2, 1.9; MgSO4, 1.2;
NaH2PO4, 1.2; NaHCO3, 25; glucose, 11.7. The preparation was kept at 37° C and aerated with 5% CO2: 95
% O2 at pH 7.4 [49]. After at least a 2-hs equilibration period a sub-maximal contractions of aorta rings were
obtained by PGF2α (3 µM).
Concentration-response curves were constructed by cumulative addition of the A2A selective agonist CGS
21680 which gives relaxation of the tissue [47]. The concentration of agonist in the organ bath was increased
approximately 3-fold at each step, with each addition being made only after the response to the previous
addition had attained a minimal level and remained steady. Relaxations were recorded by means of an
isometric transducer connected to the MacLab system PowerLab/800. After 30 minutes the dose-response
curve was repeated in the presence of compounds 1, 13 (used at 1000 nM) or the reference antagonist ZM
241385 (used at 300 nM). In addition, parallel experiments in which tissues did not receive any antagonist
were run in order to check any variation in sensitivity.
The IC50 values were obtained according to the following equation:
Kb= IC50/[1+(A/EC50)K]
in which A = [Ago] = 300 nM, and the K = slope = 0.99.
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The Kb values were obtained as follow:
Kb= [B]/(DR-1)
where DR is the ratio of EC50 values of agonist after and before treatment with antagonist concentration [B].
Statistical analysis. Responses were expressed as percentage of the maximal relative luminescence units
(RLU). Concentration–response curves were fitted by a nonlinear regression with the Prism 5.0 programme
(GraphPAD Software, San Diego, CA, USA). The antagonist profile of the two compounds was expressed as
IC50. The IC50 value is the concentration of antagonists that produces 50% inhibition of the agonist effect.
Each concentration was tested three-five times in triplicate and the values are given as geometric means with
95% confidence intervals in parentheses [50].
4.3 Molecular Modeling
A high-resolution crystal structures of the hA2AAR in complex with ZM241385 was retrieved from the
Protein Data Bank (http://www.rcsb.org; pdb code: 5NM4; 1.7-Å resolution [41]). The structure was
modified by rebuilding the missing IL3 segment and by mutating some residues to restore the wild type
sequence. The Homology Modelling tool of MOE [51] was used for this task. Hydrogen atoms were added
and energetically minimized. A homology model of the human A3AR was built following a procedure
previously reported [43]. The Homology Modelling tool of MOE was employed even for this task. All
compound structures were docked into the binding site of the hA2A and hA3 ARs structure using the genetic
algorithm docking tool of CCDC Gold [42] with default efficiency settings and by selecting GoldScore as
scoring function [42].
ACKNOWLEDGEMENTS
This work was supported by the University of Camerino (Progetto FAR FPI000042) and by a grant from the
Ministry of Research (PRIN N° 2015E8EMCM_008, 2015).
CONFLICTS OF INTEREST
The authors declare no conflict of interest.
21

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Tables
Table 1. Biological activity data of the synthesized compounds at the human ARs.
NH2
N
X
N
NH
N
N
N
N
N
N
N
N
N
N
R
R
R
X
R1
N
H
V, VI 1, 2
VII, VIII, 7-10, 13
15-20
,
hA1AR
hA2AAR
hA2BAR
hA3AR
CPD
X
R
R1
(Ki nM)a
(Ki nM)b
(IC50 nM)c
(Ki nM)d
690
V
VI
1
NH
O
Br
Br
150
23
19
3,100
1,090
(250-1,900)
569
(440–734)
1.7
9.4
(7.0-13)
3.8
(3.4-4.2)
2,340
(1,340 - 4,110)
18
(17-18)
NH
O
2-furyl
5.8
(4.0 - 8.4)
2.2
(2.1 - 2.3)
521
(467 – 580)
16
(11 – 22)
2
2-furyl
2,210
VII
VIII
7
NH
O
Br
H
1,540
280
2,440
60,900
>40,000
(1,460-3,350)
Br
H
11,700
19,300
>100,000
6,404
(6,321-6.482)
4,635
(4,581-4,720)
NH
O
H
NH2
NH2
NH2
NH2
NH2
nd
nd
5,959
(3,570 – 8,348)
5,044
(2,861 – 7,227)
3,404
(3,032 – 3,775)
8
H
Br
1,931
(1,855-2,008)
266
(251-296)
5,895
(5,735-6,108)
1,192
(1,104-1,265)
9
NH
O
2,671
(2,535-2,730)
1,080
(998-1,160)
1,115
(1,035-1,198)
10
13
15
16
17
18
19
20
Br
>30,000
96
(70-121)
12
(7.3-16)
4,000
(3,816-4,230)
20
(14-27)
NH
2-furyl
H
440
(401-486)
593
(545-630)
13
(9.3-18)
>30,000
nd
602
(463 - 740)
251
(188 - 314)
481
(376 - 586)
Br
293
(226 - 360)
104
(78 - 130)
45
(35 - 55)
2-furyl
OCH3
OCH2CH3
O(CH2)2C6H5
>30,000
nd
798
(595 - 941)
217
(171 - 263)
585
(452 - 717)
546
(423 - 669)
250
(197 - 303)
183
(145 - 220)
>30,000
1,062
(823 – 1,301)
> 30,000
>30,000
nd
aDisplacement of specific [3H]-CCPA binding at human A1AR expressed in CHO cells, (n=3-6).
bDisplacement of specific [3H]-NECA binding at human A2AAR expressed in CHO cells. cIC50 values of the
inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells expressing human A2BAR (nd = not
d
determined). Displacement of specific [3H]-HEMADO binding at human A3AR expressed in CHO cells.
Data are expressed as geometric means, with 95% confidence limits.
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Table 2. Inhibitory effect of a set of selected compounds on the NECA-induced cAMP production in CHO
cells. Data are expressed as IC50 values (the respective Ki values at the A2AAR are indicated).
hA2AAR
(Ki nM)
hA2AR
(IC50 nM)
411
(398-424)
CPD
V
19
1.7
108
(50-166)
VI
1
171
(135-207)
3.8
7
4,635
5,044
266
>50,000
>50,000
8
5,753
(4,776-6,730)
9
10
13
1,080
12
>50,000
962
(696-1,228)
Table 3. Functional activity of compounds 1 and 13 in rat aorta expressed as IC50 nM in comparison with the
reference agonist CGS 21680 and antagonist ZM 241385. In the table are reported also the Ki data obtained
from binding studies at A2AAR subtype.
a
b
EC50
IC50
Ki
CPD
(nM)
(nM)
(nM)
280
(228-332)
23
(18-27)
CGS 21680
64
(50-77)
1.2
(0.9-1.4)
ZM 241385
136
(93-179)
3.8
(3.4-4.2)
1
589
(487-690)
12
(7.3-16)
13
b
aEC50 is the concentration of agonists that gives half-maximal response. IC50 is the concentration of
antagonists required to produce 50% inhibition of the CGS 21680 relaxation effect. Data are means of 2-4
separated experiments. Values are given as geometric means with 95% confidence intervals in parentheses.
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Figure legends
Fig. 1. Known and designed compounds of general formula A.
Fig. 2. Design of N6,8-disubstituted 2-amino-9-ethyladenines. A. Previously reported N6,8-disubstituted 9-
ethyladenines [31]. B. Comparison of compounds in panel A (compound VII as example) with 2,8-
disubstituted 9-ethyladenines (compound V as example) and definition of respective pharmacophore
features, where Ha, Hd, and Hy are H-bond acceptor, H-bond donor and Hydrophobic pharmacophoric
features, respectively. C. Superimposition of compounds VII and V with matching of pharmacophore
features; compound V presents and additional H-bond donor group indicated by the arrow. D. Design of N6-
phenylalkyl substituted 2-amino-9-ethyladenine derivatives; the 2-amino group is inserted to restore the H-
bond donor feature suggested in panel; example of designed compound; E. The same example compound,
rotated view. F. Designed N6,8-disubstituted 2-amino-9-ethyladenines to be synthesized and tested in this
work.
Fig. 3. Designed compounds of general formula C.
Fig. 4. Assignment of the alkylation site on compounds 5 and 6 by [1H]-NMR NOE experiment. In the figure
are illustrated the irradiated protons in blue and the protons giving an increased signal in red. Only in the
case of compound 6, an increased signal of the CH3 and also at the H-N(6) was obtained upon irradiation of
the CH2 protons. This demonstrated that the CH2 is close to the N6-position, hence the alkylation site is the 7-
position.
29