4-amino-6-alkyloxy-2-alkylthiopyrimidine derivatives as novel non-nucleoside agonists for the adenosine A1 receptor

Article abstract of DOI:10.1111/cbdd.12801

Three 4-amino-6-alkyloxy-2-alkylthiopyrimidine derivatives (4–6) were investigated as potential non-nucleoside agonists at human adenosine receptors (ARs). When tested in competition binding experiments, these compounds exhibited low micromolar affinity (

Full text of DOI:10.1111/cbdd.12801

Received Date : 04-Feb-2016
Revised Date : 21-Apr-2016
Accepted Date : 02-Jun-2016
Article type : Research Article
Title Page:
4-Amino-6-alkyloxy-2-alkylthiopyrimidine Derivatives as
Novel Non-nucleoside Agonists for the Adenosine A1
Receptor
Running Title: Novel non-nucleoside A1 adenosine receptor agonists
Keywords: 4-amino-6-alkyloxy-2-alkylthiopyrimidine derivatives, A1 adenosine
receptor, non-nucleoside adenosine receptor agonists, A1 AR ligands, A1 AR
efficacy, A1 AR agonists.
Barbara Cosimelli1,*, Giovanni Greco1, Sonia Laneri1, Ettore Novellino1, Antonia
Sacchi1, Maria Letizia Trincavelli2, Chiara Giacomelli2, Sabrina Taliani2, Federico
Da Settimo2, and Claudia Martini2
1 Dipartimento di Farmacia, Università di Napoli Federico II, Via Montesano 49, 80131 Napoli, Italy
2 Dipartimento di Farmacia, Università di Pisa, Via Bonanno Pisano 6, 56126 Pisa, Italy
*Corresponding Author: Barbara Cosimelli Dipartimento di Farmacia, Università di Napoli Federico
II, Via Montesano 49, 80131 Napoli, Italy Tel: +39 081678614, Fax: +39 081678107; E-mail:
barbara.cosimelli@unina.it
Abstract: Three 4-amino-6-alkyloxy-2-alkylthiopyrimidine derivatives (4-6) were investigated as
potential non-nucleoside agonists at human adenosine receptors (ARs). When tested in
competition binding experiments, these compounds exhibited low micromolar affinity (Ki values
comprised between 1.2 and 1.9 μM) for the A1 AR and no appreciable affinity for the A2A and A3
ARs. Evaluation of their efficacy profiles by measurement of intracellular cAMP levels revealed that
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4 and 5 behave as non-nucleoside agonists of the A1 AR with EC50 values of 0.47 μM and 0.87 μM,
respectively. No clear concentration-response curves could be instead obtained for 6, probably
because this compound modulates one or more additional targets, thus masking the putative
effects exerted by its activation of A1 AR The three compounds were not able to modulate A2B AR-
mediated cAMP accumulation induced by the non-selective AR agonist NECA, thus demonstrating
no affinity towards this receptor.
1. INTRODUCTION
The nucleoside adenosine plays a key role in a large variety of physiological and pathological
processes by interacting with specific receptors. Four different subclasses of adenosine receptors (ARs)
have been identified to date, A1, A2A, A2B, and A3, all belonging to the superfamily of G-protein coupled
receptors (1-3). Activation of these receptors by adenosine or a synthetic agonist determines
different intracellular events starting with inhibition (A1 and A3) or stimulation (A2A and A2B) of
adenylate cyclase. Additional molecular mechanisms coupled to occupation of ARs by agonists are
stimulation of phospholipase C (A1, A2B, and A3), activation of potassium channels and inhibition of
calcium channels (A1) (4). Being ubiquitously distributed in tissues and organs of mammalians, ARs
have been considered attractive targets for the development of agonist- and antagonist-based therapies
against a wide range of pathologies, including central nervous system (CNS) disorders, cardiac
arrhythmia, ischemic injuries, asthma, renal failure, and inflammatory diseases (5). Although several
agonists binding selectively to each of the four ARs are available for preclinical studies, the only currently
marketed AR agonists are adenosine itself, administered by intravenous infusion in the treatment of
cardiovascular acute pathologies, and a synthetic derivative of adenosine, regadenoson, administered by
intravenous injection as vasodilatator stress agent (6). Most of the currently known synthetic AR agonists,
differently from AR antagonists, are characterized by a ribose moiety or a ribose-like moiety linked to a
heterocyclic system.
The nucleoside nature of such compounds represents one of the reasons hampering their clinical use
due to a low oral bioavailability and/or a short half-life. As an example, the A1 AR agonist N6-cyclohexyl-
2'-O-methyladenosine (SDZ-WAG 994), administered to patients with heart failure, exhibits an oral
bioavailability comprised between 30% and 50% and a terminal half-life of about 30 minutes (7). AR
ligands lacking a sugar moiety have nearly always been found devoid of agonist efficacy. This has been
true until the discovery of some non-nucleoside 2-amino-4-aryl-3,5-dicyanopyridines and 4-amino-6-aryl-
5-cyano-2-thiopyrimidines. Representatives of such pyridine derivatives are capadenoson (1) and BAY
60-6583 (2) (Figure 1), reported as selective agonists at A1 AR and A2B AR, respectively (8). An exponent
of the aforementioned pyrimidine derivatives is compound 3 (Figure 1) described as a potent selective
agonist at A2A AR (9,10).
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OH
O
NC
CN
S
N
S
H2N
N
Cl
1 (capadenoson)
O
NC
CN
S
NH2
H2N
N
O
2 (BAY 60-6583)
O
HN
CH3
NC
N
O
N
H2N
N
S
N
CH2CH3
N
N
CH2CH3
3
Figure 1: Structures of compounds 1-3 chosen as representative of well-known non-nucleoside AR agonists.
Compound 4 (Figure 2) is a synthetic precursor of one of several 4-acylamino-6-alkyloxy-2-
alkylthiopyrimidines disclosed by our research group as antagonists of the human A3 AR (11).
Compounds 5 and 6 (Figure 2) were described by some of us as potential inhibitors of Wnt signaling
pathway (12). The synthesis of the above pyrimidine derivatives is summarized in Scheme 1.The
resemblance of 4-6 with 1-3 led us to evaluate whether our compounds might be endowed with AR
agonist efficacies, hopefully making them new reference structures in the perspective of lead-optimization
studies. The present paper describes the results of such investigations.
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H3C
O
CH3
CH3
O
N
O
N
N
N
N
H2N
N
S
H2N
S
H2N
S
5
6
4
Figure 2: Structures of 4-amino-6-alkyloxy-2-benzylthiopyrimidines 4-6 investigated in the present work as potential non-
nucleoside AR agonists.
R
OH
OH
N
O
N
O
N
BrCH2C6H5
R
RX
N
N
N
N
+
NaOH 1M
50-55°C, 4 h
K2CO3, DMF
reflux, 1-2 h
H2N
N
SH
H2N
SCH2C6H5
H2N
SCH2C6H5
H2N
SCH2C6H5
7
8
4 R = CH2CH2CH3
4a R = CH2CH2CH3
5 R = CH2CH(CH3)2
6 R = CH2C6H5
5a R = CH2CH(CH3)2
6a R = CH2C6H5
Scheme 1: Synthesis of compounds 4-6. X = Br for compounds 4 and 6; X = I for compound 5.
2. METHODS AND MATERIALS
The synthesis of 4 is described in ref. 11 whereas 5 and 6 are prepared as described in ref. 12
(Scheme 1). Briefly, the first step is S-alkylation of 6-hydroxy-2-mercaptopyrimidine monohydrate (7) by
reaction with benzylbromide in NaOH 1M to give 8 (80%). This latter is collected by filtration and
used, without further purification, in the next step, i.e. alkylation with an appropriate alkyl halide, in
anhydrous DMF with an excess of K2CO3, to give 4-6 and the isomers 4a-6a. The ratio 4-6:4a-6a is
about 4:1 with an overall yield comprised between 50% and 90%. Finally, 4-6 are isolated by flash-
chromatography. The affinities of 4-6 towards human A1, A2A and A3 ARs were evaluated by competition
experiments, assessing the ability of the compounds to displace specific radiolabeled ligands from
transfected CHO cells (13). The efficacy profiles of the compounds at human A1 and A2B ARs were
evaluated by assays based on intracellular cAMP measurements in transfected CHO cells (13).
3. EXPERIMENTAL
3.1. Adenosine Receptor Binding Assays. Materials.
Each compound was routinely dissolved in DMSO and diluted with assay buffer to the final concentration,
where the amount of DMSO never exceeded 2%. Percentage inhibition values of specific radiolabeled
ligand binding at 1-10 µM concentration were calculated as means
SEM of at least three
determinations. [3H]DPCPX and [3H]NECA were obtained from DuPont-NEN (Boston, MA). ADA was from
Sigma Chemical Co. (St. Louis, MO). All other reagents were from standard commercial sources and of
the highest commercially available grade. CHO cells stably expressing human A1, A2A, A2B and A3 ARs
were kindly supplied by Prof. K.-N. Klotz, Wurzburg University, Germany (13).
3.2. Human A1 Adenosine Receptors.
Aliquots of cell membranes (30 µg proteins) were incubated at 25°C for 180 min in 500 µL of binding
buffer (50 mM Tris-HCl, 2 mM MgCl2, 2 units/mL ADA, pH 7.4) containing [3H]DPCPX (3 nM) and six
different concentrations of the tested compounds. Non-specific binding was determined in the presence of
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50 µM R-PIA. The dissociation constant (Kd) of [3H]DPCPX in human A1 AR CHO cell membranes was 3
nM.
3.3. Human A2A Adenosine Receptors.
Aliquots of cell membranes (30 µg) were incubated at 25°C for 90 min in 500 µL of binding buffer (50
mM Tris-HCl, 2 mM MgCl2, 2 units/mL ADA, pH 7.4) in the presence of 30 nM of [3H]NECA and six
different concentrations of the tested compounds. Non-specific binding was determined in the presence of
100 µM R-PIA. The dissociation constant (Kd) of [3H]NECA in human A2A AR CHO cell membranes was
30 nM.
3.4. Human A3 Adenosine Receptors.
Aliquots of cell membranes (90 µg) were incubated at 25°C for 180 min in 500 µL of binding buffer (50
mM Tris-HCl, 5 mM MgCl2, 1 mM EDTA, 2 units/mL ADA, pH 7.4) in the presence of 30 nM [3H]NECA
and six different concentrations of the tested compounds. Non-specific binding was determined in the
presence of 100 µM R-PIA (13,14).
3.5. Measurement of cAMP Levels in Human A1 and A2B AR-transfected CHO cells.
Intracellular cAMP levels were measured using a competitive protein binding method (15). CHO cells,
expressing recombinant human ARs, were harvested by trypsinization. After centrifugation and
resuspension in medium, cells (~ 30,000) were plated in 24-well plates in 0.5 mL of medium. After 24 h,
the medium was removed, and the cells were incubated at 37°C for 15 min with 0.5 mL of DMEM in the
presence of ADA (1U/mL) and the phosphodiesterase inhibitor Ro20-1724 (20 μM). The antagonist
efficacy profile of the compounds at A1 AR was evaluated by assessing their ability to counteract NECA-
mediated inhibition of cAMP accumulation in the presence of 10 µM forskolin, a non-selective activator of
adenylate cyclase. The compounds (10 or 50 μM) were incubated 10 min before the addiction of the
agonist NECA (1 nM). The agonist efficacy profile of the compounds at A1 AR was evaluated by
assessing their ability to modulate intracellular cAMP levels in the presence of 10 µM forskolin and in the
absence of standard agonists. Cells were incubated in the reaction medium (15 min at 37°C) with
different concentrations of the tested compound (0.1 nM-100 µM). In the assays carried out to evaluate
the antagonist efficacy profile, cells were treated with the agonist NECA (100 nM). Following incubation,
the reaction was terminated by the removal of the medium and the addition of 0.4 N HCl. After 30 min,
lysates were neutralized with 4 N KOH, and the suspension was centrifuged at 800 rpm for 5 min. For the
determination of cAMP production, bovine adrenal cAMP binding protein was incubated with [3H]cAMP (2
nM) and 50 μL of cell lysate or cAMP standard (0-48 pmol) at 0°C for 150 min in a total volume of 300 μL.
Bound radioactivity was separated by rapid filtration through GF/C glass fiber filters and washed twice
with 4 mL 50 mM Tris/HCl, pH 7.4. The radioactivity was measured by liquid scintillation spectrometry.
3.6. Data Analysis.
All binding and functional data were analyzed using Graph-Pad Prism 5.0 (GraphPad Software Inc.,
San Diego, CA). The EC50 values were obtained using the non-linear regression curve fitting of the
concentration-response curves. The Ki values were obtained using the one-site fit curve. All values
obtained are mean values of at least three different experiments each performed in duplicate.
4. RESULTS
The binding data of the 4-amino-6-alkyloxy-2-alkylthiopyrimidine derivatives 4-6 to human A1, A2A, and
A3 ARs, expressed as Ki values, are summarized in Table 1, along with those of DPCPX, NECA, and Cl-
IBMECA, used as the reference standards. Our compounds were able to compete with the radiolabeled
antagonist [3H]DPCPX showing monophasic curves (Figure 3). The compounds displayed low micromolar
affinity towards A1 AR (Ki values comprised between 1.2 and 1.9 μM) and no appreciable affinity towards
A2A and A3 ARs (inhibition of radioligand binding less than 20% at 10 μM). In addition, when evaluated in
a functional assay, the same compounds were not able to modulate NECA-mediated cAMP accumulation
in CHO A2B AR transfected cells, thus demonstrating no affinity towards this receptor (percentage
inhibition less than 5% at 10 µM, data not shown).
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Table 1: Binding affinities of compounds 4-6 towards A1, A2A and A3 ARs in CHO transfected cells.a
hA1b Ki
(nM)
hA2Ac Ki
(nM)
hA3d Ki
(nM)
Compound
4
5
1,240 275 > 10,000
1,945 427 > 10,000
1,196 280 > 10,000
> 10,000
> 10,000
> 10,000
>10,000
6
DPCPX
NECA
Cl-IBMECA
0.5 0.03
14
890 61
337 28
16
401 25
4
3
73
5
0.22 0.02
aKi values are means SEM of four separate assays, each performed in triplicate. bDisplacement of specific [3H]DPCPX binding at
human A1 AR expressed in CHO cells. cDisplacement of specific [3H]NECA binding at human A2A R expressed in CHO cells.
dDisplacement of specific [3H]NECA binding at human A3 AR expressed in CHO cells.
As next step, we characterized the efficacy profiles (antagonism/agonism) of 4-6 towards A1 AR.
The antagonist efficacies of the compounds at this receptor were evaluated by assessing their abilities
to counteract NECA-mediated inhibition of cAMP accumulation produced by forskolin, a non-selective
activator of adenylate cyclase. As shown in Figure 4, none of the compounds exhibited significant effects
in this assay, suggesting that they do not behave as A1 AR antagonists.
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A
B
C
120
100
80
60
40
20
0
-8
-8
-8
-7
-7
-7
-6
log [4]
-5
-5
-5
120
100
80
60
40
20
0
-6
log [5]
120
100
80
60
40
20
0
-6
log [6]
Figure 3: Displacement curves of [3H]DPCPX binding by compounds 4-6 at human A1 AR expressed in CHO cells. Data are
expressed as percentage of [3H]DPCPX specific binding and represent the mean SEM of three different experiments.
The agonist profiles of the compounds towards A1 AR were evaluated by assessing their abilities to
reduce intracellular cAMP levels in the presence of forskolin and in absence of any agonist. NECA was
able to reduce the intracellular cAMP levels with a potency of 1.5 nM, in accordance with literature data
(16). In this assay, 4 and 5 exhibited a concentration-dependent inhibition of cAMP levels (Figure 5), thus
suggesting that they act as A1 AR agonists. No clear concentration-response curves could be instead
obtained for 6, probably because this compound modulates one or more additional targets, thus masking
the putative effects exerted by its activation of A1 AR.
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120
100
80
60
40
20
0
Figure 4: Effects of compounds 4-6 on NECA-mediated inhibition of cAMP levels in A1 AR CHO transfected cells. Cells were treated
with 1 nM NECA in the presence of 10 µM forskolin. When indicated, 10 or 50 µM of each compound was added. Intracellular cAMP
levels were quantified as described in the experimental section. Data are expressed as percentage of cAMP/well versus forskolin
set to 100% and represent the mean SEM of three different experiments.
5. DISCUSSION
The results of our studies indicate that the 4-amino-6-alkyloxy-2-benzylthiopyrimidines 4 and 5 can be
regarded as the first exponents of a novel class of non-nucleoside selective A1 AR agonists. Their EC50
values of 0.49 µM and 0.87 µM, respectively, are 2-2.5 fold lower than their corresponding binding
affinities towards the same receptor (Table 1).
These compounds, together with 6, were investigated by us taking into account their structural
resemblance with known non-nucleoside A1 AR agonists (Figure 2). Studies are in progress to dissect the
mechanism of action of 6 underlying its unexpected biochemical behavior, quite different from those
exhibited by 4 and 5.
The therapeutic potential of the class of non-nucleoside A1 AR agonists, now including compounds 4
and 5, stems, as mentioned, from the key role played by A1 AR in the organism (3,5) and the lack of
pharmacokinetics-related disadvantages associated with a sugar moiety.
A comparison between the structures of 4 and 5 with those reported in literature as potent AR agonists
(exemplified by 1-3) suggests that requirements for a tight ligand-receptor binding might be a cyano group
(at least one) and/or an aryl substituent directly attached to the pyridine/pyrimidine ring. With regard to the
molecular details of such interactions, J. Robert Lane et al. proposed a docking model of the A2A AR
agonist LUF5834 (17), a pyridine analog of 1 and 2, into the corresponding orthosteric binding site (18).
According to these authors, the 2-amino and 3-cyano groups of LUF5834 are both engaged in hydrogen
bonds with the Asn253 side chain of A2A AR.
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A
EC50 = 1.5 0.2 nM
120
100
80
60
40
20
0
-12
-10
-8
-6
log [NECA]
EC50 = 0.49 0.02 µM
B
120
100
80
60
40
20
0
-10
-8
log [4]
-6
-4
C
EC50 = 0.87 0.09 µM
120
100
80
60
40
20
0
-10
-8
log [5]
-6
-4
Figure 5: Effects of NECA (A), compounds 4 (B) and 5 (C) on forskolin-stimulated accumulation of cAMP in A1 AR CHO
transfected cells. Cells were treated with 10 µM forskolin in the absence or in the presence of the tested compound at different
concentrations ranging from 0.01 nM to 100 µM. Intracellular cAMP levels were quantified as described in the experimental
section. Data are expressed as percentage of cAMP/well versus forskolin set to 100% and represent the mean SEM of three
different experiments.
The preliminary study described here has opened up a whole new range of investigations on the
structure-activity relationships of this class of A1 AR agonists, which are currently in progress and will be
reported on in due course.
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LIST OF ABBREVIATIONS
ADA, adenosine deaminase;
cAMP, 3',5'-cyclic adenosine monophosphate;
CHO, chinese hamster ovary;
Cl-IBMECA, 2-Chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide;
DMEM, Dulbecco’s modified Eagle’s medium;
[3H]DPCPX, [3H]8-cyclopentyl-1,3-dipropylxanthine;
[3H]NECA, [3H]5'-N-ethylcarboxamidoadenosine;
R-PIA, (R)-N6-(2-phenylisopropyl)adenosine
SEM, standard error of mean
CONFLICT OF INTEREST
The Authors confirm that this article content has no conflict of interest.
ACKNOWLEDGEMENTS
We thank Prof. Dr. Karl-Norbert Klotz for his generous gift of transfected CHO cells expressing human
A1, A2A, A2B and A3 receptors.
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