Amino-3, 5-dicyanopyridines targeting the adenosine receptors ranging from pan ligands to combined A1/A2B partial agonists

Article abstract of DOI:10.3390/ph12040159

The amino-3,5-dicyanopyridine derivatives belong to an intriguing series of adenosine receptor (AR) ligands that has been developed by both academic researchers and industry. Indeed, the studies carried out to date underline the versatility of the dicyanopyridine scaffold to obtain AR ligands with not only a wide range of affinities but also with diverse degrees of efficacies at the different ARs. These observations prompted us to investigate on the structure–activity relationships (SARs) of this series leading to important previously reported results. The present SAR study has helped to confirm the 1H-imidazol-2-yl group at R² position as an important feature for producing potent AR agonists. Moreover, the nature of the R¹ substituent highly affects not only affinity/activity at the hA₁ and hA_2B ARs but also selectivity versus the other subtypes. Potent hA₁ and hA_2B AR ligands were developed, and among them, the 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(prop-2-en-1-yloxy)phenyl]pyridine-3,5-dicarbonitrile (3) is active in the low nanomolar range at these subtypes and shows a good trend of selectivity versus both the hA_2A and hA₃ ARs. This combined hA_1/hA_2B partial agonist activity leads to a synergistic effect on glucose homeostasis and could potentially be beneficial in treating diabetes and related complications.

Full text of DOI:10.3390/ph12040159

pharmaceuticals
Article
Amino-3,5-Dicyanopyridines Targeting the
Adenosine Receptors. Ranging from Pan Ligands to
Combined A1/A2B Partial Agonists
Daniela Catarzi 1,*, Flavia Varano 1, Katia Varani 2, Fabrizio Vincenzi 2 , Silvia Pasquini 2,
Diego Dal Ben 3 , Rosaria Volpini 3 and Vittoria Colotta 1
1
Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica
e Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy;
flavia.varano@unifi.it (F.V.); vittoria.colotta@unifi.it (V.C.)
Dipartimento di Scienze Mediche, Sezione di Farmacologia, Università degli Studi di Ferrara,
2
Via Fossato di Mortara 17-19, 44121 Ferrara, Italy; vrk@unife.it (K.V.); fabrizio.vincenzi@unife.it (F.V.);
silvia.pasquini@unife.it (S.P.)
Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università degli Studi di Camerino,
3
via S.Agostino 1, 62032 Camerino (MC); Italy; diego.dalben@unicam.it (D.D.B.);
rosaria.volpini@unicam.it (R.V.)
*
Correspondence: daniela.catarzi@unifi.it
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 September 2019; Accepted: 18 October 2019; Published: 22 October 2019
Abstract: The amino-3,5-dicyanopyridine derivatives belong to an intriguing series of adenosine receptor
(AR) ligands that has been developed by both academic researchers and industry. Indeed, the studies carried
out to date underline the versatility of the dicyanopyridine scaffold to obtain AR ligands with not only
a wide range of affinities but also with diverse degrees of efficacies at the different ARs. These observations
prompted us to investigate on the structure–activity relationships (SARs) of this series leading to
important previously reported results. The present SAR study has helped to confirm the 1H-imidazol-2-yl
group at R2 position as an important feature for producing potent AR agonists. Moreover, the nature
of the R1 substituent highly affects not only affinity/activity at the hA1 and hA2B ARs but also
selectivity versus the other subtypes. Potent hA1 and hA2B AR ligands were developed, and among them,
the2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(prop-2-en-1-yloxy)phenyl]pyridine-3,5-dicarbonitrile
(3) is active in the low nanomolar range at these subtypes and shows a good trend of selectivity versus both
the hA2A and hA3 ARs. This combined hA1/hA2B partial agonist activity leads to a synergistic effect on
glucose homeostasis and could potentially be beneficial in treating diabetes and related complications.
Keywords: G protein-coupled receptors; adenosine A2B receptor ligands; adenosine A1 receptor
ligands; aminopyridine-3,5-dicarbonitriles; ligand-adenosine receptor modeling studies
1. Introduction
Adenosine is a ubiquitous autacoid that has been reported to play important roles in various
tissues by acting through four different adenosine receptors (ARs) classified as A1, A2A, A2B, and A3
receptors [1]. These ARs belong to the G protein–coupled receptors superfamily, and each of them has
different functions although with some overlap. Depending on their coupling to G-protein, they can
either increase or decrease intracellular cAMP levels by affecting adenylate cyclase (AC) activity.
However, other effector systems have been proposed [
and A2A ARs, the lowest for A2BAR, and intermediate affinity for the A3 subtype. Selective agonists
and antagonists have been developed for each of the ARs [ ], and their discovery opened up new
avenues for potential drugs treatment of a variety of conditions such as neurodegenerative disorders,
2]. Adenosine has the highest affinity for the A1
3,4
Pharmaceuticals 2019, 12, 159; doi:10.3390/ph12040159
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Pharmaceuticals 2019, 12, 159
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chronic inflammatory diseases, asthma, and many other pathophysiological states that are known to
be associated with changes in adenosine levels [3].
Dysregulated intracellular nucleotide concentrations can be produced by altered and defective
cellular metabolism leading to important metabolic pathologies such as diabetes mellitus [3,5,6].
This disease is increasing worldwide due to aging, incorrect lifestyle, and eating habits, and its
complications are of great clinical and socioeconomic impact. All four ARs are involved in the
regulation of islet hormone secretion and have the potential of being drug targets for the treatment of
diabetes [
for diabetes treatment, even if there is discrepancy in the studies reported in literature, which may be
ascribed to the different experimental conditions employed [ ]. However, it is reported that agonists
of this AR subtype may be useful for the therapy of this pathology [3].
The role of the A1AR in diabetes seems to be clearer [ ]. In fact, improvement of insulin
sensitivity and glucose homeostasis by A1 activation has been well recognized for many years,
thus the promotion of A1AR agonists as drug candidates [ ]. Since full A1AR agonists are
generally characterized by cardiovascular side effects, partial A1AR agonists could represent the
best choice to avoid cardiovascular complications [ ]. For this reason, a synergistic effect on
3]. Currently, much attention has been devoted to A2BAR agonists as potential new drugs
3,7
3,8
5,6
9
glucose homeostasis targeting both A1 and A2B ARs could be of interest for treating diabetes and its
complications. It is also worth noting that A1AR is involved in lipid metabolism, so the development
of A1AR agonists as lipolytic agents is reported to be useful in cardiovascular disease–associated
pathologies [10]. While the pathophysiological roles of the A1AR are well known, the A2B subtype
is the least characterized because of the paucity of potent and selective agonists. BAY-606583
(2-{[6-Amino-3,5-dicyano-4-(4-(cyclopropylmethoxy)phenyl)pyridin-2-yl]thio}acetamide) is one
of the most potent and selective A2BAR agonists known to date [11]. However, we recently
produced
a new series of amino-3,5-dicyanopyridines as BAY-606583 analogues [12];
most of the compounds are potent human (h) A2B AR partial agonists. Among them,
the2-{[(1H-imidazol-2-yl)methyl]thio}-6-amino-4-[4-(cyclopropylmethoxy)phenyl]pyridine-3,5-dicarbonitrile
(P453, Figure 1) turns out to be three-fold more active than BAY-606583, although it is less selective.
Compound P453 was used for experiments in in vitro models of short-term synaptic plasticity and
ischemic-like condition [13]. In particular, compound P453 was shown to be a functional A2BAR agonist
whose activation decreases PPF (paired pulse facilitation) by increasing glutamate release at presynaptic
terminals and delays A1AR-mediated fEPSP inhibition during a two-minute oxygen and glucose deprivation
(OGD) insult in the rat CA1 hippocampus.

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NH
CN
2
CN
N
1
2
R
R
Reference compounds
Currently reported 1-20
H
N
R2 =
R1 = aryl, benzyl, methyl
S
N
H
N
S
R3
1-13
LUF series R1 =
CH2OH
N
R4 =
4
N
N
14-16, 19
NH
R
R3 = H, OCH3, OH
R2 =
N
N
OCH3
17
P453
R1 =
O
18
20
H
COOCH3
S
Figure 1. Lead structures and currently reported amino-3,5-dicyanopyridine-based adenosine
receptor ligands.
From a structure–activity point of view, the new findings confirm the 1H-imidazol-2-yl
group in the R2 pendant as an important feature for producing potent AR agonists.
Certain 2-amino-4-aryl-6-(1H-imidazol-2-yl-methylsulfanyl)-pyridine-3,5-dicarbonitrile compounds,
belonging to the LUF series (Figure 1), were already reported to display nanomolar affinity for all
the ARs, including the A2B subtype for which they showed, in general, considerable affinity and
efficacy [14]. The growing interest in this class is also dictated by the fact that non-nucleoside AR
agonists seem to be more versatile for pharmacological studies, showing fewer species differences than
the adenosine-like ones [
versatility of the amino-3,5-dicyanopyridine scaffold for producing AR ligands with not only a wide
range of affinities but, interestingly, with different degrees of efficacy at the different ARs [11 12 14 19].
4]. Moreover, the studies carried out to date are sufficient to underline the
,
,
–
On this basis, the 1H-imidazol-2-yl group was used to design other amino-3,5-dicyanopyridine
derivatives in order to deeply investigate the influence of the R1 substituent at the 4-position on AR
affinity and selectivity. Moreover, other modifications were performed at R2 to enrich what is known
about how the nature of the substituent at this position modulates AR affinity and selectivity.
2. Results
2.1. Chemistry
Compounds
according to the procedure reported in Schemes 1–3.
the 2-(((1H-Imidazol-2-yl)methyl)thio)-6-amino-4-(4-ethoxyphenyl)pyridine-3,5-dicarbonitrile
previously reported in reference [12], was prepared. For the synthesis of derivatives 13, and 20, the
strategic intermediates are represented by the 6-sulfanyl-substituted compounds 28 12 14 18 20 23],
1–
4,
6
–
20,
and their corresponding intermediates were synthesized
Following the same procedures,
5,
1–4, 6–
–40
[
,
,
,
–
which were prepared as depicted in Scheme 1. The suitable aldehyde was reacted with malononitrile
and thiophenol, in boiling water, in the presence of a catalytic amount of basic alumina to yield
the 4-aryl- and 4-benzyl-6-phenylsulfanyl-derivatives 21
yield. When these latter were treated with anhydrous sodium sulfide in anhydrous DMF at 80 ◦C,
followed by acidification with HCl, the free thiol 28 38 12 18 20 21], 40 14] were obtained in
–27 [18,24–26], with moderate to good
–
[
,
,
,
[

Pharmaceuticals 2019, 12, 159
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high yields. The preparation of the 4-methyl-6-sulfanyl compound 39
[
22
,23] was performed by
reacting the (1-ethoxyethylidene)propanedinitrile 41
containing EtONa. The target dicyanopyridines
6-sulfanyl intermediates 28–40 with 2-(bromomethyl)-1H-imidazole hydrobromide (
methyl chloroacetate (20) in the presence of sodium hydrogen carbonate at room temperature (rt).
[
22] with 2-cyanothioacetamide in boiling ethanol
13, and 20 were obtained by reaction of the
13) or
1–4, 6–
1–4, 6–
NH2
NH2
CN
R1
CN
R1
CN
CN
EtO
a
N
N
b
OHC
R1
c
HS
S
41
CN
CN
28-40
21-27
d
NH2
CN
R1
N
S
R
CN
N
1-4, 6-13 R =
20
N
H
R = COOCH3
R1
compd
1, 28
R1
compd
R1
compd
O
7, 33, 21
12, 38, 26
Cl
F
O
2, 29
3, 30
8, 34, 22
9, 35, 23
CH3
13, 39
O
O
20, 40, 27
CF3
OCH3
4, 31
6, 32
10, 36, 24
11, 37, 25
CH3
NH
SCH3
O
◦
Scheme 1. Reagents and conditions. (a) malononitrile, thiophenol, basic Al2O3, H2O, 100 C; (b)
Na2S, anhydrous DMF, 80 ◦C; 1N HCl, room temperature (rt); (c) 2-cyanothioacetamide, EtONa,
absolute EtOH, reflux; d) RCH2Cl, NaHCO3, anhydrous DMF, rt.

Pharmaceuticals 2019, 12, 159
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NH2
CN
NH2
CN
CN
a
N
N
R2
Cl
42
CN
b
H
N
14 R2 =
15 R2 =
16 R2 =
17 R2 =
NHCH
2
N
NH2
NHCH2CH2OH
NHCH CH
CN
N
OCH
3
2
2
N
N
CN
18
Scheme 2. Reagents and conditions. (a) 1-(1H-imidazol-2-yl)methanamine for 14, ethanolamine for
15, 2-(4-methoxyphenyl)ethanamine for 16, or imidazole (for 17), triethylamine, THF/EtOH 2:1, reflux;
(b) H2, 10% Pd/C, absolute EtOH, 30 Psi.
NH2
NH2
CN
CN
a
N
N
HO
S
NH
CN
CN
OCH3
OCH3
19
27
Scheme 3. Reagents and conditions. (a) 2-aminoethanol, DMF, 100 ◦C.
To replace the thiomethyl linker at the 6-position with an aminomethyl bridge, compounds
14
–16 were synthesized (Scheme 2) starting from the 6-chloro derivative 42, obtained with a reported
procedure [27], and the suitable commercially available amine. The reaction was carried out in
the presence of triethylamine and in a refluxing mixture of THF/EtOH. The same procedure was
applied for the synthesis of the 6-(1H-imidazol-1-yl)-derivative 17, which was obtained in high yield.
Elimination of the 6-substituent was realized in compound 18 which was synthesized by catalytic
reduction (10% Pd/C) of 42 with hydrogen at 30 Psi in absolute EtOH.
Finally, the 6-[(2-hydroxyethyl)amino]-derivative 19 was synthesized (Scheme 3) starting from the
intermediate 27 [24] by exploiting the good property of the 6-phenylthio function as the leaving group
in nucleophilic substitution [15]. The reaction was performed at 100 ◦C in DMF using an excess of
2-aminoethanol as nucleophile.
2.2. Pharmacological Assays
The amino-3,5-dicyanopyridines 1–20 were tested for their affinity at hA1, hA2A, and hA3 ARs,
stably transfected in Chinese Hamster Ovary (CHO) cells, and were also studied as hA2B agonists by
evaluating their stimulatory effect on cAMP production in CHO cells, stably expressing the hA2B AR.
Compounds 3, 8, and 11, the most interesting in terms of combined affinity/activity at hA1 and hA2B
ARs, were evaluated for their A1 pharmacological profile in the cAMP assay, where each compound
was tested to assess its capability to modulate Forskolin-stimulated cAMP levels in the absence or
presence of the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA) (1 nM). All pharmacological
data are reported in Tables 1 and 2.

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Table 1. Binding affinities (Ki) at hA1, hA2A, and hA3 ARs and potencies (EC50) at hA2B ARs.
NH2
N
N
R1
R2
1-20
cAMP Assays
N
Binding Experiments a
EC50 (nM) b
Ki (nM) or I%
Efficacy, c
%
e
d
f
hA2B
hA2A
hA1
hA3
R1
R2
N
S
>1000
(12%)
O
1
25%
4%
574 ± 46
N
H
N
S
227 ± 21
O
O
O
2
3
26%
1%
17%
1%
1%
8%
N
H
(36%)
N
S
27 ± 3
16 ± 2
77 ± 6
N
H
(56%)
N
N
N
S
S
S
>1000
(14%)
4
5g
6
8%
N
H
11.7 ± 1.2
8.21 ± 0.73
83 ± 7
221 ± 19
38 ± 3
85 ± 6
48 ± 4
O
N
H
(62%)
12 ± 1
NH
N
H
(100%)
O
N
S
S
S
109 ± 12
7
8
30 ± 4
0.78 ± 0.08
419 ± 32
5.32 ± 0.42
8.32 ± 0.78
31%
865 ± 68
139 ± 12
32%
850 ± 62
410 ± 38
21%
N
H
Cl
F
(32%)
N
18 ± 2
N
H
(76%)
N
>1000
(4%)
9
N
H
CF3
CH3
SCH3
N
N
N
N
N
S
78 ± 6
10
11
12
13
47 ± 4
227 ± 21
8%
212 ± 19
388 ± 29
1%
N
H
(78%)
S
36 ± 3
N
H
(30%)
S
260 ± 24
N
H
(44%)
S
>1000
(1%)
CH3
74 ± 8
331 ± 32
510 ± 53
N
H
NH
>1000
(1%)
14
15
140 ± 12
943 ± 87
115 ± 10
19%
1%
N
H
>1000
(1%)
NHCH2CH2OH
1%

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Table 1. Cont.
cAMP Assays
EC50 (nM) b
Binding Experiments a
Ki (nM) or I%
Efficacy, c
%
e
d
f
hA2B
hA2A
hA1
hA3
R1
R2
NH
>1000
16
1%
1%
1%
OCH 3
(1%)
>1000
(1%)
N
N
17
18
27%
15%
1%
25%
5%
>1000
(1%)
H
16%
>1000
(1%)
19
20
NHCH2CH2OH
SCH2COOCH3
14%
2%
1%
15%
5%
OCH3
OCH3
O
>1000
(1%)
987 ± 81
N
S
N
H
9.52 ± 0.91
P453 g
235 ± 24
2.4 ± 1
764 ± 72
28 ± 1
474 ± 45
(70%)
N
S
N
H
LUF5833
19 ± 7
171 ± 109
h
(81%)
a
Ki values are means
±
SEM of four separate assays each performed in triplicate. Percentage of inhibition (I%)
b
is determined at 1
µ
M concentration of the tested compounds. EC50 values are means
±
SEM of four separate
M concentration, in comparison with
c
assays each performed in triplicate. Efficacy of the tested compound at 1
µ
NECA (1µ
M = 100%). d Displacement of specific [3H]DPCPX competition binding to hA1CHO cells. e Displacement
of specific [3H]ZM241385 competition binding to hA2AhCHO cells. Displacement of specific [125I]AB-MECA
f
g
competition binding to hA3CHO cells. Reference [12]. Reference [14].
Table 2. Modulation of Forskolin-stimulated cAMP levels of selected amino-3,5-dicyanopyridine
derivatives on cyclic cAMP assay in hA1 CHO cells.a.
Efficacy, b
%
compd
EC50 (nM) c
3
8
11
39 ± 4
34 ± 3
52 ± 4
100 ± 9
19.6 ± 1.7
1.34 ± 0.11
8.87 ± 0.64
1.52 ± 0.12
DPCPX
a
Potency values (EC50) are expressed as means
triplicate. The compounds have been tested on Forskolin at 5
±
SEM of four independent cAMP experiments, each performed in
b
µ
M concentration. Efficacy of the novel compounds
were normalized by using the efficacy value of the reference compound DPCPX (set at 100%) that showed the
c
higher capability to increase cAMP production. Potency of the novel compounds to increase Forskolin-stimulated
cAMP levels.
2.3. Molecular Docking Studies
To simulate the binding mode of the herein reported dicyanopyridines at hA1 and hA2B ARs,
molecular docking studies were performed on the cryo-EM structure of the agonist-bound hA1AR
(pdb code: 6D9H; 3.6-Å resolution [28]) and on a homology model of the hA2BAR developed by using
the crystal structure of agonist-bound hA2AAR as a template (pdb code: 2YDO; 3.0-Å resolution [29]).
The obtained hA2BAR homology model was checked using the Protein Geometry Monitor application
within MOE (Molecular Operating Environment 2019.0101) [30] by inspecting the structural quality of
the protein model (backbone bond lengths, angles, and dihedrals, Ramachandran
ϕ-ψ dihedral plots,
and quality of side chain rotamer and non-bonded contact). The two AR structures were then used as
target for the docking analysis of the synthesized derivatives. The docking studies were performed
using the MOE docking tool (induced fit docking and optimization protocol) and Gold software [31].
For each compound, the top-score docking pose at each AR model, according to at least two out of
three scoring functions, was selected for final ligand-target interaction analysis.

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Docking conformations at hA1 and hA2A ARs of selected dicyanopyridine compounds are reported
in Figures 2 and 3, respectively (see the Discussion Section).
Figure 2. Docking conformations at hA1AR. The simulated binding mode of compound
8
is shown (
A)
with detail of key receptor residues involved in ligand-target interaction or (
the binding cavity as molecular surface.
B) with representation of

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Figure 3. Docking conformations at hA2BAR. The simulated binding mode of compound 6 is shown,
with detail of key receptor residues involved in ligand–target interaction.
3. Discussion
3.1. Structure–Activity Relationships
The biological and pharmacological data for the newly synthesized amino-3,5-dicyanopyridine
derivatives 20 are reported in Tables 1 and 2 together with those of the reference compounds LUF5833
(2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-phenylpyridine-3,5-dicarbonitrile) [14] and P453 [12].
All the derivatives that interact with the hA2BAR (2, 3, 5 8, 10 12) display EC50 values from 12 to
260 nM behaving as partial agonists, with the only exception being compound , previously reported as
trifluoroacetate salt [32], which shows a full agonist profile. Some compounds (17 20), selected among
1
–
–
–
6
–
those that had no activity at the hA2BAR were also evaluated to investigate their antagonistic effect on
cAMP production stimulated by 50-(N-ethylcarboxamido)adenosine (NECA) 100 nM in hA2BCHO
cells. These functional studies reveal their inability to inhibit NECA-stimulated cAMP levels (I%
(data not shown). Most of the compounds have generally modest to null hA2A and hA3 AR affinity
with the only exception of the two pairs of compounds and 10 and and that bind, respectively,
the hA2A and hA3 ARs with nanomolar affinity. Best results have been obtained in the hA1AR binding
experiments. In particular, all the 6-(imidazole-2-ylmethyl)sulfanyl derivatives reported herein ( 13),
with the only exception being compounds , and 12, have from high to good hA1AR affinity.
≤
3)
6
5
6
1–
1, 2, 9
In particular, some compounds showed Ki values in the low nanomolar range and in general below
10 nM. Functional studies at the hA1 subtype (data are reported in Table 2) indicated that the tested
compounds 3, 8, and 11 behave as partial agonists and that their relative potencies correlate well
with the Ki values obtained in the hA1 binding experiments. All the newly synthesized compounds
showed different and not easily predictable behavior depending on both R1 and R2 substituents.
However, the data shown in Table 1 indicate that the binding at the hA2BAR subtype depends strictly
on the substitution at the R1 position [12].

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Modifications of the para-substituent at the R1 pendant of the lead compound P453 were performed
to evaluate the influence on hA2BAR activity and selectivity. 6-(Imidazole-2-ylmethyl)sulfanyl
derivatives
were synthesized. Only compounds
while derivative and derivatives and
the different steric hindrance of the para-substituent with respect to P453. In particular, derivative
1–
5, bearing different cycloalkyl- or cycloalkenyl-methyloxy substituents at R1 position,
3
and
are, respectively, scarcely active or inactive, probably due to
is
5 maintain the hA2BAR activity in the nanomolar range,
2
1
4
5
endowed with hA2BAR activity comparable to that of the lead P453, despite a low or null selectivity
versus the other AR subtypes. Introduction of a para-acetamido substituent on the 4-phenyl moiety led
to compound 6, which is the only amino-3,5-dicyanopyridine in the whole series having a full agonist
profile at the hA2BAR. The para-acetamido substituent, in addition to possessing an oxygen atom as
H-bond acceptor, also contains the NH donor group that could have some influence in determining
the pharmacological profile of this derivative as observed for the dicyanopyridine of the LUF series
(Figure 1) [33,34].
Activity at the hA2BAR is also affected by the electronic effects of the para-substituent on the
4-phenyl ring. In fact, the corresponding EC50 values fall in the low nanomolar range for compounds
8
(para-fluoro) and 11 (para-thiomethyl) and move to higher values by passing to derivatives 10
(para-methyl) and (para-chloro), up to the total inactivity of , bearing a para-trifuoromethyl group.
Homologation of the 4-phenyl moiety of LUF5833 led to the 4-benzyl-susbstituted derivative 12
7
9
,
which maintains modest hA2B activity. In contrast, its replacement with a methyl group was highly
detrimental leading to compound 13, which is completely avoid of any activity at the hA2BAR
subtype. In this first set of compounds (1–13), bearing a 6-(imidazole-2-ylmethyl)sulfanyl moiety
on the dicyanopyridine scaffold, the hA1AR affinity depends on the para-phenyl substitution as
observed for hA2B receptor activity trend. In fact, many compounds show mixed hA1/hA2B combined
affinity/activity. Looking at Table 1, we can observe different activity profiles—for example, compound
whose biological data fall in the same range of values (EC50 at hA2BAR and Ki at the other subtypes)
can be considered in effect as a pan ligand. Compound is interesting in terms of its combined
hA1/hA2B AR partial agonist activity, exhibiting a good trend of selectivity versus both the hA2A
and hA3 subtypes. The data of derivatives and 11, which lose a little in selectivity but behave as
6,
3
8
hA1/hA2B partial agonists, are also noteworthy. Moreover, compound 8, binding the hA1AR in the
sub-nanomolar range, is the most potent hA1AR ligand among the herein reported dicyanopyridine
series. When the 6-sulfanyl linker of LUF5833 was replaced by a 6-amino bridge, compound 14 was
obtained. A dramatic decrease of hA2BAR activity was observed with respect to the LUF derivative but
the effect was detrimental also for the other ARs. Replacement of the imidazolyl moiety at R2 position
with different substituents in terms of both steric hindrance and capability to engage hydrogen-bonding
led to derivatives 15–20, which show null affinity/activity for each of the AR subtypes. The only
exception are compounds 15 and 20 endowed with hA1AR binding affinity in the micromolar range.
Compounds 17–20, besides being inactive as hA2B agonists, do not even work as antagonists.
3.2. Molecular Docking Investigation and In Silico ADMET Prediction.
The simulated binding mode at both the hA1 and hA2B AR structures, generally associated to
the best score, presents the herein reported compounds oriented similarly to analogous derivatives
previously reported as hAR ligands (Figure 2A) [12,35,36]. In detail, the pyridine scaffold is located
in the AR cavity in correspondence to the purine moiety of Ado observed at the hA1AR cryo-EM
structure and at the hA2AAR template, and interacts with the side chains of hA1AR residues Phe171
extracellular loop (EL) 2 and Ile2747.39 (Phe173 and Ile2767.39 in hA2BAR). For clarity, in this section,
derivative
8 has been considered the reference compound to define the position of the substituents
on the pyridine nucleus. Thus, starting from the N1 position, the amino and the sulfanyl functions
occupy positions 2 and 6, respectively. The 3-cyano and 2-amino groups make H-bonds with the amide
function of Asn2546.55. An additional H-bond is observed between the 2-amino group and the hA1AR
residue Glu172 (EL2, Glu174 in hA2BAR), while the 5-cyano group points toward transmembrane

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(TM) 2 (i.e., Ala662.61 in hA1AR, Ala642.61 in hA2BAR) and TM7 (i.e., His2787.43 in hA1AR, His2807.43
in hA2BAR) residues. The 2-sulfanyl substituent is located at the entrance of the binding cavity,
providing interaction with residues of TM1, TM2, and TM7. Binding data show that in general an aryl
moiety at R1 is the type of substituent providing highest affinity at ARs. Its replacement with a smaller
methyl group (compound 13) or a larger (and differently oriented) benzyl substituent (12) leads to
a significant decrease (if not loss) of affinity. In this sense, the 4-aryl moiety well fits the depth of the
binding cavity at both receptors, by occupying the available space in a more complete and efficient
way with respect to the methyl (13) or benzyl group (12) (Figure 2B). Looking at the para-position
of the 4-aryl moiety, biological data show that the size of the substituent at this level is critical for
compound affinity, with the highest obtained with a small group. At the hA1AR receptor, the space
available around the para-position is limited, allowing the insertion of small residues and not well
tolerating ramified substituents. Accordingly, compounds bearing a bulky, non-linear para-substituent
(compounds
4 and 6) are endowed with lower hA1AR affinity with respect to other compounds of the
series active in the low nanomolar range.
At the hA2BAR, the impact of the size of the para-substituent on compound affinity
appears analogous to that observed at the hA1AR, with the exception of compound
a para-acetamido group. This compound is among the most potent derivatives of the series at
the hA2BAR. Figure 3 shows the binding mode of compound at the hA2BAR, with indication of
the key residues involved in ligand interaction. The para-acetamido function engages a double
6 bearing
6
H-bond interaction with Thr893.36 and His2516.52
.
This double polar interaction with the
receptor is also given by the 50-amide groups of the hA2AAR agonists NECA [29], UK-432097
(6-[2,2-di(phenyl)ethylamino]-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxyoxolan-2-yl]-N-[2-[(1-
pyridin-2-ylpiperidin-4-yl)carbamoylamino]ethyl]purine-2-carboxamide)
[
37
]
and CGS-21680
(4-[2-[[6-amino-9-(N-ethyl- -D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic
β
acid) [38], as observed from X-ray structures of the hA2AAR in complex with these two compounds.
The insertion of an amide group at the 50-position of nucleosides is a typical modification of the
Ado scaffold to improve its affinity and potency at the hA2A and hA2B ARs. This observation helps to
explain the high activity of compound 6 at the hA2BAR.
The potential use of derivatives belonging to this series as therapeutic agents depends not only
on their pharmacodynamics but also pharmacokinetics, the latter including Adsorption, Distribution,
Metabolism, Excretion, and Toxicity (ADME/T). A preliminary study of the theoretical ADMET profiles
of compound
3 was performed using the methodology developed by pKCSM, a novel approach to the
prediction of pharmacokinetic and toxicity properties [39]. A complete summary of all the parameters
evaluated for the selected compound is reported in the Supplementary Materials. It is worth noting
that no violation of the Lipinski’s rule of five (RO5) (molecular weight (MW) < 500; log P < 5; number of
H.-bond donors (HBD) < 5, and acceptors (HBA) < 10) was evidenced, making derivative
drug-like candidate [40]. Nevertheless, while some bioavailability and toxicity parameters fall in the
safety range, others indicate some points of concerns for compound . In interpreting the contrasting
3 a promising
3
results, it is important to consider that in silico evaluation of the ADMET parameters for a group of
approved antidiabetic drugs [41] evidenced not only violation of some of the RO5 (25%) but also the
toxicity parameters as the major failures (2–33%).
4. Conclusions
This study, which has led to some amino-3,5-dicyanopyridines differently substituted at R1 and
R2 positions, provides greater insight into the SARs of this series as AR ligands. In particular, it has
emerged that the affinity/activity at the hA1 and hA2B ARs depends strictly on the R1 substitution
which also affects the selectivity versus the other subtypes. Moreover, the 1H-imidazol-2-yl
group at R2 position was confirmed to be an essential feature for potent AR agonists. In fact,
looking at the biological and pharmacological data of this series, it is usual to find a combined
AR affinity/activity, even if most of the compounds have generally modest to null hA2A and hA3

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AR affinity. Many of the dicyanopyridines herein reported interact with the hA2B subtype with
IC50 values from 12 to 260 nM behaving as partial agonists. The best results were obtained in the
hA1AR binding assays where most of the compounds show from high to good affinity at this receptor,
the
2-amino-4-(4-fluorophenyl)-6-[(1H-imidazol-2-ylmethyl)sulfanyl]pyridine-3,5-dicarbonitrile
emerging as the most active compound with partial agonist profile.
The compounds showing combined hA1/hA2B AR partial agonist activity,
as derivatives and 11 deserve the greatest attention. In particular,
the2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(prop-2-en-1-yloxy)phenyl]pyridine-3,5-dicarbonitrile
) is active in the low nanomolar range at both these subtypes and shows a good trend of selectivity
(8
)
a
a
3,
8
,
,
(3
versus both the hA2A and hA3 ARs. This combined hA1/hA2B partial agonist activity, leading to
a synergistic effect on glucose homeostasis, could be of interest in the development of potentially
useful agents for treating diabetes and its complications. The in silico calculated ADMET profiles
indicated that some bioavailability and toxicity parameters fall in the safety range, while others
suggest some points of concerns for compound
3. Thus, further studies will be devoted in the future to
thoroughly investigate on the toxicity of compound
3
. We are however confident in considering as
3
a promising lead for the development of new antidiabetics with improved pharmacokinetics and safer
therapeutic potential.
5. Materials and Methods
5.1. Chemistry
5.1.1. General Methods
Analytical silica gel plates (Merck F254, Sigma-Aldrich, Milan, Italy), preparative silica gel plates
(Merck F254, 2 mm), and silica gel 60 (Merck, 70-230 mesh) were used for analytical and preparative TLC,
and for column chromatography, respectively. All melting points were determined on a Gallenkamp
(U.K) melting point apparatus and are uncorrected. Elemental analyses were performed with a Flash
E1112 Thermo Finnigan elemental analyzer for C, H, abd N (Thermo Fisher Scientific, Milan, Italy),
and the results were within 0.4% of the theoretical values. All final compounds revealed purity
±
not less than 95%. The IR spectra were recorded with a Perkin-Elmer Spectrum RX I spectrometer
(Perkin-Elmer, Milan, Italy) in Nujol mulls and are expressed in cm-1. NMR spectra were recorded on
a Bruker Avance 400 spectrometer (Bruker, Milan, Italy) (400 MHz for 1H NMR and 100 MHz for 13
C
NMR). The chemical shifts are reported in δ (ppm) and are relative to the central peak of the residual
non-deuterated solvent, which was DMSO-d6. The following abbreviations are used: s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, Ar = aromatic protons. Compounds
28–
32 were synthesized as reported in reference [12]. When available, melting point and/or 1H NMR
values were in accordance to literature data.
5.1.2. Synthesis of the target compounds 1–4, 6–13, and 20
Sodium hydrogen carbonate (2 mmol) and the suitable halomethyl-derivative (1 mmol) were
added to a solution of the suitable mercapto-compound (28-40 [12,14,18,20–23], 1 mmol) in anhydrous
DMF (1 mL). The reaction mixture was stirred at room temperature (rt) until disappearance of the
starting material (TLC monitoring). Then, water was added (25 mL) to precipitate a solid that was
collected by filtration and washed with water. The crude product was triturated with Et2O (5 mL),
collected by filtration and purified.
2-Amino-4-[4-(cyclobutylmethoxy)phenyl]-6-[(1H-imidazol-2-ylmethyl)sulfanyl]pyridine-3,5-
dicarbonitrile (1)
The crude product was purified by preparative TLC using EtOAc/cyclohexane/MeOH 6:4:1 as
eluting system. Yield 20%; mp 214-216 ◦C. 1H NMR (DMSO-d6) 11.87 (s, 1H, NH); 8.10 (s, 2H, NH2);
7.46 (d, 2H, Ar, J = 7.8 Hz); 7.09 (d, 2H, Ar, J = 7.8 Hz); 7.08 (s, 1H, Ar); 6.96 (s, 1H, Ar); 4.49 (s, 2H,

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CH2); 4.03 (d, 2H, OCH2, J = 6.0 Hz); 2.70-2.60 (m, 1H, CH); 2.08-2.07 (m, 2H, CH2); 1.91-1.88 (m, 4H,
2CH2). IR 3385; 3320; 3217; 2210. Anal. Calc. for C22H20N6OS
2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(2-methylpropoxy)phenyl]pyridine-3,5-
dicarbonitrile (2)
The crude product was purified by preparative TLC using EtOAc/cyclohexane /MeOH 5:5:1 as
eluting system. Yield 31%; mp 230-232 ◦C. 1H NMR (DMSO-d6) 11.84 (s, 1H, NH); 8.14 (s, 2H, NH2),
7.47 (d, 2H Ar, J = 8.7 Hz); 7.10 (d, 2H Ar, J = 8.6 Hz); 7.08 (s, 1H, Ar); 6.85 (s, 1H, Ar); 4.50 (s, 2H, CH2);
3.84 (d, 2H, OCH2, J = 6.3 Hz); 2.09-2.02 (m, 1H, CH); 1.00 (d, 6H, 2 CH3, J = 6.7 Hz); IR 3387; 3315;
3215; 2208. Anal. Calc. for C21H20N6OS
2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(prop-2-en-1-yloxy)phenyl]pyridine-3,5-
dicarbonitrile (3)
The crude product was purified by preparative TLC using DCM/MeOH 9:1 as eluting system.
Yield 21%; mp 216-218 ◦C. 1H NMR (DMSO-d6) 11.86 (s, 1H, NH); 8.09 (s, 2H, NH2); 7.48 (d, 2H Ar,
J = 8.5 Hz); 7.12 (d, 2H, Ar, J = 8.6 Hz); 7.07 (s, 1H, Ar); 6.85 (s, 1H, Ar); 6.11-6.04 (m, 1H, CH); 5.40
(d, 1H, CH); 5.30 (d, 1H, CH); 4.62 (d, 2H, OCH2, J = 5.2 Hz); 4.48 (s, 2H, CH2). Anal. Calc. for
C20H16N6OS
2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-{4-[(2-methylprop-2-en-1-yl)oxy]phenyl}
pyridine-3,5- dicarbonitrile (4)
The crude product was purified by column chromatography using EtOAc/cyclohexane /MeOH
6:3:1 as eluting system. Yield 36%; mp 224-226 ◦C. 1H NMR (DMSO-d6) 11.85 (s, 1H, NH); 8.08 (s, 2H,
NH2); 7.48 (d, 2H, Ar, J = 8.4 Hz); 7.12 (d, 2H Ar, J = 8.4 Hz); 7.07 (s, 1H, Ar); 6.85 (s, 1H, Ar); 5.09 (s, 1H,
CH); 4.99 (d, 1H, CH); 4.56 (s, 2H, CH2); 4.49 (s, 2H, OCH2); 1.79 (s, 3H, CH3); 13C NMR (DMSO-d6)
167.19; 161.03; 160.95; 159.11; 143.71; 141.67; 131.75; 131.28; 126.93; 116.58; 115.93; 115.80; 113.79; 94.22;
87.00; 72.14; 27.75; 20.32; IR 3404; 3377; 3336; 2210. Anal. Calc. for C21H18N6OS
N-(4-{2-Amino-3,5-dicyano-6-[(1H-imidazol-2-ylmethyl)sulfanyl]pyridin-4-yl}phenyl) acetamide (6)
The crude product was purified by column chromatography using EtOAc/cyclohexane /MeOH
8.2:1.5:0.9 as eluting system. Yield 61%; mp 275-278 ◦C. 1H NMR (DMSO-d6) 11.84 (s, 1H, NH); 10.22 (s,
1H, NH); 8.11 (s, 2H, NH2); 7.73 (d, 2H, Ar, J = 8.3 Hz); 7.46 (d, 2H, ar, J = 8.4 Hz); 7.05 (s, 1H, Ar); 6.86
(s, 1H, Ar); 4.49 (s, 2H, CH2); 2.09 (s, 3H, CH3); IR 3387; 3211; 2214; 1683. Anal. Calc. for C19H15N7OS
2-Amino-4-(4-chlorophenyl)-6-[(1H-imidazol-2-ylmethyl)sulfanyl]pyridine-3,5-dicarbonitrile (7)
1
Yield 42%; mp 229-231 ◦C dec (EtOAc). H NMR (DMSO-d6) 11.87 (br s, 1H, NH); 8.2 (br s, 2H,
NH2); 7.65 (d, 2H, Ar, J = 8.0 Hz); 7.59 (d, 2H, Ar, J = 8.0 Hz); 7.08 (s, 1H, Ar); 6.85 (s, 1H, Ar); 4.50 (s,
2H, CH2); IR 3336; 3244; 2220; 1683. Anal. Calc. for C17H11ClN6S
2-Amino-4-(4-fluorophenyl)-6-[(1H-imidazol-2-ylmethyl)sulfanyl]pyridine-3,5-dicarbonitrile (8)
1
Yield 80%; mp 223-225 ◦C dec (EtOAc). H NMR (DMSO-d6) 11.9 (br s, 1H, NH); 8.2 (br s, 2H,
NH2); 7.60 (m, 2H, Ar); 7.45 (t, 2H, Ar, J = 8.8 Hz); 7.0 (br s, 2H, Ar); 4.50 (s, 2H, CH2); IR 3314; 3145;
2229. Anal. Calc. for C17H11FN6S
2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(trifluoromethyl)phenyl]pyridine-3,5-
dicarbonitrile (9)
Yield 80%; mp 233-235 ◦C dec (EtOH). 1H NMR (DMSO-d6) 11.85 (br s, 1H, NH); 8.22 (br s, 2H,
NH2); 7.97 (d, 2H, Ar, J = 7.8 Hz); 7.79 (d, 2H, Ar, J = 7.8 Hz); 7.08 (s, 1H, Ar); 6.86 (s, 1H, Ar);4.55 (s,
2H, CH2); IR 3527; 3185; 2437. Anal. Calc. for C18H11F3N6S
2-[(1H-Imidazol-2-il)metiltio]-6-ammino-4(4-metilfenil)piridina-3,5-dicarbonitrile (10)

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Yield 67%; mp 259-261 ◦C dec (EtOH). 1H NMR (DMSO-d6) 11.85 (br s, 1H, NH); 8.14 (br s, 2H,
NH2); 7.40 (d, 2H, Ar, J = 8.0 Hz); 7.38 (d, 2H, Ar, J = 8.0 Hz); 7.07 (br s, 2H, Ar); 4.50 (s, 2H, CH2); 2.39
(s, 3H, CH3). IR 3518; 3129; 2213. Anal. Calc. for C18H14N6S
2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(methylsulfanyl)phenyl]pyridine-3,5-
dicarbonitrile (11)
Yield 95%; mp 257-259 ◦C dec (MeOH). 1H NMR (DMSO-d6) 11.87 (br s, 1H, NH); 8.12 (br s, 2H,
NH2); 7.47 (d, 2H, Ar, J = 8.0 Hz); 7.42 (d, 2H, Ar, J = 8.0 Hz); 7.08 (br s, 1H, Ar); 6.85 (br s, 1H, Ar);
4.50 (s, 2H, CH2); 2.51 (s, 3H, CH3). 13C NMR (DMSO-d6) 166.65; 160.26; 158.33; 143.00; 142.31; 130.19;
129.43; 125.69; 115.81; 93.54; 86.35; 27.23; 14.47. IR 3420; 3390; 2223. Anal. Calc. for C18H14N6S2
2-Amino-4-benzyl-6-[(1H-imidazol-2-ylmethyl)sulfanyl]pyridine-3,5-dicarbonitrile (12)
Yield 70%; mp 246-248 ◦C dec (EtOH). 1H NMR (DMSO-d6) 11.82 (br s, 1H, NH); 8.14 (br s, 2H,
NH2); 7.34 (d, 2H, Ar, J = 8.0 Hz); 7.24 (m, 3H, Ar); 6.95 (br s, 2H, Ar); 4.47 (s, 2H, CH2); 4.08 (s, 2H,
CH2). 13C NMR (DMSO-d6) 166.73; 160.33; 159.10; 142.89; 136.61; 129.38; 128.78; 127.64; 115.61; 93.99;
86.95; 38.84; 27.15; 14.47. IR 3431; 3357; 2377. Anal. Calc. for C18H14N6S
2-Amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-methylpyridine-3,5-dicarbonitrile (13)
Yield 66%; mp 267-269 ◦C dec (EtOH). 1H NMR (DMSO-d6) 11.87 (br s, 1H, NH); 8.12 (br s, 2H,
NH2); 7.02 (br s, 2H, Ar); 4.51 (s, 2H, CH2), 2.43 (s, 3H, CH3). IR 3455; 3363; 2473. Anal. Calc. for
C12H10N6S
Methyl{[6-amino-3,5-dicyano-4-(4-methoxyphenyl)pyridin-2-yl]sulfanyl}acetate (20)
Yield 80%; mp 218-220 ◦C (EtOH). 1H NMR (DMSO-d6) 7.96 (br s, 2H, NH2); 7.51 (d, 2H, Ar,
J = 8.8 Hz); 7.12 (d, 2H, Ar; J = 8.8 Hz)); 4.20 (s, 2H, CH2), 3.84 (s, 3H, CH3), 3.70 (s, 3H, CH3). IR 3426;
3397; 3325; 3224; 2209. Anal. Calc. for C17H14N4O3S
5.1.3. Synthesis of the Target Compounds 14–17
The suitable commercially available amino-derivative (1-(1H-imidazol-2-yl)methanamine, for
compound 14; ethanolamine, for 15; 2-(4-methoxyphenyl)ethanamine, for 16; or imidazole (for
compound 17 (1.18 mmol), and triethylamine (1.18 mmol) were sequentially added to a solution of the
2-chloro-derivative 42 [27] (0.59 mmol) in a mixture THF/EtOH 2:1 (7.5 mL). The reaction mixture was
heated at reflux until the disappearance of the starting material (TLC monitoring) and then cooled at rt.
After dilution with water (20 mL), a solid precipitated was collected and washed with water and Et2O.
2-Amino-6-[(1H-imidazol-2-ylmethyl)amino]-4-phenylpyridine-3,5-dicarbonitrile (14)
Yield 57%; mp 208-210 ◦C (EtOH). 1H NMR (DMSO-d6) 11.64 (br s, 1H, NH); 8.39 (br s, 2H, NH2);
7.88 (t, 1H, NH, J = 5.5 Hz); 7.61-7.45 (m, 5H, Ar); 7.04 (br s, 1H, Ar); 6.83 (br s, 1H, Ar);4.56 (d, 2H,
CH2, J = 5.6 Hz). IR 3360; 3322; 3186; 2207. Anal. Calc. for C17H13N7
2-Amino-6-[(2-hydroxyethyl)amino]-4-phenylpyridine-3,5-dicarbonitrile (15)
Yield 64%; mp 230-231 ◦C (EtOH). 1H NMR (DMSO-d6) 7.55-7.53 (m, 3H, Ar); 7.47-7.45 (m, 2H,
ar); 7.37 (br s, 2H, NH2); 7.20 (t, 1H, NH, J = 5.1 Hz); 4.74 (t, 1H, OH, J = 5.2 Hz); 3.54 (m, 2H, CH2);
3.47 (m, 2H, CH2). IR 3472; 3345; 3223; 2204. Anal. Calc. for C15H13N5O
2-Amino-6-{[2-(4-methoxyphenyl)ethyl]amino}-4-phenylpyridine-3,5-dicarbonitrile (16)
Yield 80%; mp >300 ◦C (EtOH/DMF). 1H NMR (DMSO-d6) 7.54-7.53 (m, 3H, Ar); 7.51-7.45 (m, 3H,
2 Ar + NH); 7.39 (br s, 2H, NH2); 7.20 (d, 2H, Ar, J = 8.4 Hz); 6.87 (d, 2H, Ar, J = 8.4 Hz); 3.73 (s, 3H,
CH3); 3.57-3.51 (m, 2H, CH2); 2.80 (t, 2H, CH2, J = 8.0 Hz). IR 3451; 3342; 3244; 2201. Anal. Calc. for
C22H19N5O

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2-Amino-6-(1H-imidazol-1-yl)-4-phenylpyridine-3,5-dicarbonitrile (17)
Yield 77%; mp 253-255 ◦C (EtOH). 1H NMR (DMSO-d6) 8.40 (br s, 2H, NH2), 8.36 (s, 1H, Ar), 7.81
(s, 1H, Ar), 7.63 (m, 5H, Ar), 7.18 (s, 1H, Ar). IR 3399; 2220; 1681;1466. Anal. Calc. for C16H10N6
5.1.4. Synthesis of 2-amino-4-phenylpyridine-3,5-dicarbonitrile (18)
Ten per cent Pd/C (0.1 g) was added to a warm solution of the 2-chloro-derivative 42 [27] (0.98
mmol, 0.25 g) in absolute EtOH (50 mL). The mixture was hydrogenated in a Parr apparatus, at 30 Psi
for 6 h. The catalyst was filtered off and the solvent was distilled to a small volume under reduced
pressure to yield a yellow solid, which was collected and washed with Et2O. Yield 33%; mp 244-246 ◦C
1
(2- Propanol). H NMR (DMSO-d6) 8.66 (s, 1H, ar), 8.10 (br s, 2H, NH2), 7.52-7.60 (m, 5H, ar). IR 3328;
3140; 2224; 1660. Anal. Calc. for C13H8N4
5.1.5. 2-Amino-6-[(2-hydroxyethyl)amino]-4-(4-methoxyphenyl)pyridine-3,5-dicarbonitrile (19)
A suspension of the 6-phenylsulfanyl derivative 27 [24] (1.81 mmol) and an excess of ethanolamine
(7.25 mmol) in anhydrous DMF (7.8 mL) was heated at 100 ◦C for 5h. After cooling at rt, water (30
mL) was added. A solid precipitated, which was collected by filtration and washed with water and
Et2O. Yield 90%; mp 248-250 ◦C (EtOH). 1H NMR (DMSO-d6) 7.43 (d, 2H, Ar, J = 8.8 Hz); 7.32 (br s, 2H,
NH2); 7.14 (t, 1H, NH), 7.09 (d, 2H, Ar, J = 8.8 Hz)); 4.74 (t, 1H, OH), 3.84 (s, 3H, CH3), 3.56-3.53 (m, 2H,
CH2), 3.50-3.47 (m, 2H, CH2). IR 3331; 3226; 2204. Anal. Calc. for C16H15N5O2
5.1.6. Synthesis of 4-substituted-2-amino-6-(phenylsulfanyl)pyridine-3,5-dicarbonitriles 21–27
[18,24–26]
A suspension of the suitable aldehyde (8.9 mmol), malononitrile (20.8 mmol), thiophenol (10.4
mmol), and basic alumina (0.019 mol) in water (20 mL) was stirred at 100 ◦C until the disappearance
of the starting material (TLC monitoring). After cooling at rt, the aqueous layer was separated by
decantation from the yellow sticky mass. The residue was treated with a mixture EtOH/Et2O 1:2 (3
mL) yielding a solid which was collected by filtration and extracted with boiling EtOAc (100 mLx2).
After distillation of the combined and dried (Na2SO4) organic layers, the crude product was triturated
with a little of Et2O/EtOH 1:1 and collected by filtration. The compounds were used without further
purification unless otherwise stated.
2-Amino-4-(4-chlorophenyl)-6-(phenylsulfanyl)pyridine-3,5-dicarbonitrile (21) [24]
Yield 75%; mp 236-237 ◦C (lit mp 236-237 ◦C)
2-Amino-4-(4-fluorophenyl)-6-(phenylsulfanyl)pyridine-3,5-dicarbonitrile (22) [25]
Yield 78%; mp 220-222 ◦C (lit mp 221-223 ◦C)
2-Amino-6-(phenylsulfanyl)-4-[4-(trifluoromethyl)phenyl]pyridine-3,5-dicarbonitrile (23) [18]
Yield 64%; mp 199-201 ◦C (EtOH). 1HNMR (DMSO-d6) 7.98 (d, 2H, J = 7.8 Hz); 7.92 (br s, 2H,
NH2); 7.82 (d, 2H, Ar, J = 7.8 Hz); 7.63-7.61 (m, 2H, Ar); 7.52-7.51 (m, 3H, Ar). IR 3320; 3211; 2243. Anal.
Calc. for C20H11F3N4S
2-Amino-4-(4-methylphenyl)-6-(phenylsulfanyl)pyridine-3,5-dicarbonitrile (24) [24]
Yield 74%; mp 220-221 ◦C (lit mp 215-216 ◦C).
2-Amino-4-[4-(methylsulfanyl)phenyl]-6-(phenylsulfanyl)pyridine-3,5-dicarbonitrile (25) [26]
Yield 75%; mp 244-246 ◦C (lit mp 254-256 ◦C).
2-Amino-4-benzyl-6-(phenylsulfanyl)pyridine-3,5-dicarbonitrile (26)

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Yield 70%; mp 247-249 ◦C (MeOH). 1HNMR (DMSO-d6) 7.81 (br s, 2H, NH2); 7.60-7.57 (m, 2H,
Ar); 7.47-7.50 (m, 3H, Ar); 7.39-7.35 (m, 2H, Ar); 7.29-7.25 (m, 3H, Ar); 4.12 (s, 2H, CH2). IR 3329; 3225;
2198. Anal. Calc. for C20H14N4S
2-Amino-4-(4-methoxyphenyl)-6-(phenylsulfanyl)pyridine-3,5-dicarbonitrile (27) [24]
Yield 66%; mp 252-254 ◦C (lit mp 254-255 ◦C).
5.1.7. Synthesis of 2-amino-4-substituted-6-sulfanylpyridine-3,5-dicarbonitriles 33–40 [14,18,20,21]
An excess of anhydrous sodium sulfide (33 mmol) was added to a stirred solution of the suitable
sulfanyl derivative 21-27 [18,24–26] (10 mmol) in anhydrous DMF (1 mL), maintained at rt and under
nitrogen atmosphere. The reaction mixture was heated at 80 ◦C until the disappearance of the starting
material (TLC monitoring). Then, 6N HCl (25 mL) was drop by drop added to obtain a precipitate,
which was collected by filtration and washed with water (20 mL) and Et2O (5 mL). Compounds were
used without further purification unless otherwise stated.
2-Amino-4-(4-chlorophenyl)-6-sulfanylpyridine-3,5-dicarbonitrile (33) [20,21]
Yield 80%; mp 202-204 ◦C (lit mp 110◦C). 1HNMR (DMSO-d6) 13.12 (br s, 1H, SH); 8.01 (sl, 2H,
NH2); 7.65 (d, 2H, Ar, J = 8.0 Hz); 7.58 (d, 2H, Ar, J = 8.0 Hz). IR 3320; 3192; 2220. Anal. Calc. for
C13H7ClN4S
2-Amino-4-(4-fluorophenyl)-6-sulfanylpyridine-3,5-dicarbonitrile (34) [18]
Yield 86%; mp 234-236 ◦C. 1HNMR (DMSO-d6) 13.03 (br s, 1H, SH); 8.01(br s, 2H, NH2); 7.58-7.52
(m, 2H, Ar); 7.38 (m, 2H, Ar). IR 3319; 3182; 2224.
2-Amino-6-sulfanyl-4-[4-(trifluoromethyl)phenyl]pyridine-3,5-dicarbonitrile (35) [18]
◦
1
Yield 90%; mp 212-214 C (MeOH). HNMR (DMSO-d6) 13.15 (br s, 1H, SH); 8.07 (d, 2H, Ar,
J = 8.0 Hz); 7.8 (d, 2H, Ar, J = 8.0 Hz). IR 3310; 3193; 2225.
2-Amino-4-(4-methylphenyl)-6-sulfanylpyridine-3,5-dicarbonitrile (36) [18]
Yield 83%; mp 228-230 ◦C. 1HNMR (DMSO-d6) 13.03 (br s, 1H, SH); 8.0 (br s, 2H, NH2); 7.41-7.37
(m, 4H, Ar). IR 3322; 3190; 2215.
2-Amino-4-[4-(methylsulfanyl)phenyl]-6-sulfanylpyridine-3,5-dicarbonitrile (37)
Yield 82%; mp 188-190 ◦C (EtOH). 1HNMR (DMSO-d6) 13.1 (br s, 1H, SH); 8.03 (br s, 2H, NH2),
7.46 (d, 2H, Ar, J = 8.0 Hz); 7.41 (d, 2H, Ar J = 8.0 Hz); 2.51 (s, 3H, CH3). IR 3311; 3195; 2222. Anal.
Calc. for C14H10N4S2
2-Amino-4-benzyl-6-sulfanylpyridine-3,5-dicarbonitrile (38)
◦
1
Yield 80%; mp 231-233 C (EtOH). HNMR (DMSO-d6) 12.96 (br s, 1H, SH); 7.82 (br s, 2H);
7.37-7.35 (m, 2H, Ar); 7.30-7.28 (m, 3H, Ar); 4.01 (s, 2H, CH2). IR 3319; 3280; 2229. Anal. Calc. for
C14H10N4S
2-Amino-4-(4-methoxyphenyl)-6-sulfanylpyridine-3,5-dicarbonitrile (40) [14]
Yield 80%; mp 261-262 ◦C (EtOH). 1H NMR (DMSO-d6) 7.96 (br s, 2H, NH2); 7.52 (d, 2H, Ar,
J = 8.8 Hz); 7.13 (d, 2H, Ar; J = 8.8 Hz)); 3.85 (s, 3H, CH3). IR 3289; 3182; 2213.
5.1.8. Synthesis of 2-amino-4-methyl-6-sulfanylpyridine-3,5-dicarbonitrile (39) [22,23]
Compound 41 [22] (23 mmol) and 2-cyanothioacetamide (22.3 mmol) were consequentially added
to an ethanolic solution of EtONa (570 mg of Na in 50 mL absolute EtOH). The reaction mixture was
heated at reflux for 4 h. After evaporation of the solvent at small volume, a yellow solid precipitated
which was collected under suction, washed with water and recrystallized. Yield 90%; mp 226-228 ◦C

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1
(EtOH) (lit mp 225 ◦C [23]; lit mp 120-123 ◦C [22]). H NMR (DMSO-d6) 12.94 (br s, 1H, SH); 7.1 (br s,
2H, NH2); 2.25 (s, 3H, CH3). IR 3338; 3253; 2248.
5.1.9. Synthesis of 2-amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (42) [27]
Malononitrile (15.2 mmol) was portion-wise added to a solution of triethyl orthobenzoate (8mmol)
and pyridine (0.6 mL) at 100 ◦C. Heating was continued for 6 h, and after cooling at 0 ◦C, 12 M HCl
(2.7 mL) was drop by drop added to the mixture. The suspension was heated at 100 ◦C for 2 h, and the
resulting precipitate was then collected by filtration and suspended in NaHCO3 saturated solution (5
mL). After stirring at room temperature for 30 min, the solid was filtered by suction and washed with
water. Yield 61%; mp >300 ◦C (MeOH) (lit mp >250 ◦C). 1H NMR (DMSO-d6) 8.41 (br s, 2H, NH2),
7.61-7.57 (m, 5H, Ar); IR 3356; 3179; 2225;1660;1564.
5.2. Pharmacological Assays
5.2.1. Cell Culture and Membrane Preparation.
CHO cells transfected with hA1, hA2A, hA2B, and hA3 ARs were grown adherently and maintained
in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, Milan, Italy) with nutrient mixture F12,
containing 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 µg/mL), l-glutamine (2
mM) and geneticin (G418; 0.2 mg/mL) at 37◦C in 5% CO2/95% air until the use in cAMP assays [42].
For membrane preparation, the culture medium was removed, and the cells were washed with
phosphate-buffered saline and scraped off T75 flasks in ice-cold hypotonic buffer (5 mM Tris HCl, 1
mM EDTA, pH 7.4). The cell suspension was homogenized with a Polytron, centrifuged for 30 min at
40000 g at 4◦C, and the resulting membrane pellet was used for competition binding experiments [42].
5.2.2. Competition Binding Experiments
All synthesized compounds have been tested for their affinity to hA1, hA2A
and hA3 ARs. Competition experiments to hA1 ARs were carried out incubating 1
nM [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) with membrane suspension (50
of protein/100
L) and different concentrations of the examined compounds at 25◦C for
90 min in 50 mM Tris HCl, pH 7.4. Non-specific binding was defined as binding
in the presence of 1
,
µg
µ
µ
M DPCPX and was always < 10% of the total binding [42].
Inhibition experiments to hA2A ARs were performed incubating the radioligand [3H]-ZM241385
(4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol) (1 nM) with the
membrane suspension (50
compounds for 60 min at 4◦C in 50 mM Tris HCl (pH 7.4), 10 mM MgCl2. Non-specific binding
was determined in the presence of ZM241385 (1 M) and was about 20% of the total binding [43].
Competition binding experiments to A3 ARs were carried out incubating the membrane suspension
µg of protein/100 µL) and different concentrations of the examined
µ
(50 µg of protein/100 µ
L) with 0.5 nM [125I]-N6-(4-aminobenzyl)-N-methylcarboxamidoadenosine
([125I]-ABMECA) in the presence of different concentration of the examined compounds for an
incubation time of 120 min at 4◦C in 50 mM Tris HCl (pH 7.4), 10 mM MgCl2, and 1 mM EDTA.
Non-specific binding was defined as binding in the presence of 1µM ABMECA and was always < 10% of
the total binding [44]. Bound and free radioactivity were separated by filtering the assay mixture through
Whatman GF/B glass fiber filters using a Brandel cell harvester (Brandel Instruments, Unterföhring,
Germany). The filter bound radioactivity was counted by Packard Tri Carb 2810 TR scintillation
counter (Perkin Elmer, Boston, MA, USA).
5.2.3. Cyclic AMP Assays.
CHO cells transfected with hAR subtypes were washed with phosphate-buffered saline,
detached with trypsin and centrifuged for 10 min at 200 g. Cells were seeded in 96-well white
half-area microplate (Perkin Elmer, Boston, USA) in a stimulation buffer composed of Hank Balanced

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Salt Solution (Sigma-Aldrich, Milan, Italy), 5 mM HEPES, 0.5 mM Ro 20-1724, and 0.1% BSA. cAMP
levels were then quantified by using the AlphaScreen cAMP Detection Kit (Perkin Elmer) following
the manufacturer’s instructions [45]. At the end of the experiments, plates were read with the Perkin
Elmer EnSight Multimode Plate Reader.
5.2.4. Data Analysis
The protein concentration was determined according to a Bio-Rad method with bovine albumin
as a standard reference. Inhibitory binding constant (Ki) values were calculated from those of IC50
according to Cheng & Prusoff equation Ki = IC50/(1+[C*]/KD*), where [C*] is the concentration of the
radioligand and KD* its dissociation constant [44]. Ki and IC50 values were calculated by non-linear
regression analysis using the equation for a sigmoid concentration-response curve (Graph-PAD Prism,
San Diego, CA, USA).
5.3. Molecular Modelling
Homology modelling and energy minimization studies were carried out using MOE (Molecular
Operating Environment, C.C.G., Inc., 1255 University St., Suite 1600, Montreal, Quebec, Canada, H3B
3X3, version 2019.0101) suite [30]. A homology model of the hA2B AR was built using the X-ray
structure of the hA2A AR in complex with Ado as template (pdb code: 2YDO; 3.0-Å resolution [29]).
A multiple alignment of the hAR primary sequences was built within MOE as preliminary step.
The boundaries identified from the used X-ray crystal structure of hA2A AR were then applied for the
corresponding sequences of the TM helices of the hA2B AR. The missing loop domains were built by the
loop search method implemented in MOE. Once the heavy atoms were modelled, all hydrogen atoms
were added, and the protein coordinates were then minimized with MOE using the AMBER10 force
field until the Root Mean Square (RMS) gradient of the potential energy was less than 0.05 kJ mol−1 Å−1
.
Reliability and quality of the model were checked using the Protein Geometry Monitor application
within MOE, which provides a variety of stereochemical measurements for inspection of the structural
quality in a given protein, like backbone bond lengths, angles and dihedrals, Ramachandran
ϕ-ψ
dihedral plots, and quality of side chain rotamer and non-bonded contact. The cryo-EM structure of
the agonist-bound hA1 AR (pdb code: 6D9H; 3.6-Å resolution [28]) was added of hydrogen atoms and
energetically minimized following the same protocol above described; the coordinates of the heavy
atoms were kept fixed in this task.
All compound structures were docked into the binding site of the hA2B and hA1 ARs structures
using as docking tools the Induced Fit docking protocol of MOE and the genetic algorithm docking
tool of CCDC Gold [31]. The Induced Fit docking protocol of MOE is divided into the following
several stages:
•
Conformational Analysis of ligands. The algorithm generated conformations from a single 3D
conformation by conducting a systematic search. In this way, all combinations of angles were
created for each ligand.
•
Placement. A collection of poses was generated from the pool of ligand conformations using Alpha
Triangle placement method. Poses were generated by superposition of ligand atom triplets and
triplet points in the receptor binding site. The receptor site points are alpha sphere centres which
represent locations of tight packing. At each iteration, a random conformation was selected,
a random triplet of ligand atoms and a random triplet of alpha sphere centres were used to
determine the pose.
•
•
Scoring. Poses generated by the placement methodology were scored using the Alpha HB scoring
function, which combines a term measuring the geometric fit of the ligand to the binding site and
a term measuring hydrogen bonding effects.
Induced Fit. The generated docking conformations were subjected to energy minimization within
the binding site and the protein sidechains are included in the refinement stage. In detail,

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the protein backbone is set as rigid while the side chains are not set to “free to move” but are set to
“tethered”, where an atom tether is a distance restraint that restrains the distance not between two
atoms but between an atom and a fixed point in space.
•
Rescoring. Complexes generated by the Induced Fit methodology stage were scored using the
Alpha HB scoring function. Gold tool was used with default efficiency settings through MOE
interface, by selecting GoldScore as scoring function [31].
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/12/4/159/s1:
Table S1. Combustion analysis data of the newly synthesized compounds 20 26 37 38, and derivative 33
Table S2. Calculated molecular properties and ADMET parameters for compound 3.
1–4
,
6–
,
,
,
.
Author Contributions: D.C. conceived and supervised the research work, and wrote the manuscript; F.V. (Flavia
Varano) designed and performed the synthetic and analytical experiments; V.C. supervised the synthetic and
analytical experiments, and analyze the data; K.V. and F.V. (Fabrizio Vincenzi) designed the pharmacological
experiments, and process the data; S.P. performed the pharmacological experiments; D.D.B and R.V. conceived
and performed the docking studies, and analyzed the results.
Funding: The work was financially supported by the Italian Ministry for University and Research (MIUR, PRIN
2010-2011, 20103W4779_004 project) and an intramural grant from the University of Florence (ex 60%).
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ABMECA
AC
N6-(4-Aminobenzyl)-N-methylcarboxamidoadenosine
Adenylate Cyclase
ADMET
Absorption, Distribution, Metabolism, Excretion,
Toxicity
Ado
Adenosine
AR
Adenosine Receptor
BSA
Bovine Serum Albumin
2-Chloro-N6-cyclopentyladenosine
Chinese Hamster Ovary
N6-Cyclopentyladenosine
8-Cyclopentyl-1,3-dipropylxanthine
Extracellular loop
CCPA
CHO
CPA
DPCPX
EL
EtOAc
DMF
DMSO
EDTA
HEPES
Ethyl Acetate
Dimethylformamide
Dimethylsulfoxide
Ethylendiaminotetracetic Acid
4-(2-Hydroxyethyl)-1-piperazine-1-ethane sulfonic
Acid
MOE
NECA
rt
Molecular operating environment;
50-(N-Ethylcarboxamido)adenosine
room temperature
SARs
THF
TM
Structure-Activity Relationships
Tetrahydrofuran
Transmembrane
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