Exploring the 7-oxo-thiazolo[5,4-d]pyrimidine core for the design of new human adenosine A3 receptor antagonists. Synthesis, molecular modeling studies and pharmacological evaluation

Article abstract of DOI:10.1016/j.ejmech.2015.04.010

A new series of 5-methyl-thiazolo[5,4-d]pyrimidine-7-ones bearing different substituents at position 2 (aryl, heteroaryl and arylamino groups) was synthesized and evaluated in radioligand binding assays to determine their affinities at the human (h) A₁, A_2A, and A₃ adenosine receptors (ARs). Efficacy at the hA_2B and antagonism of selected ligands at the hA₃ were also assessed through cAMP experiments. Some of the new derivatives exhibited good to high hA₃AR affinity and selectivity versus all the other AR subtypes. Compound 2-(4-chlorophenyl)-5-methyl-thiazolo[5,4-d]pyrimidine-7-one 4 was found to be the most potent and selective ligand of the series (K_i hA₃ Combining double low line 18 nM). Molecular docking studies of the reported derivatives were carried out to depict their hypothetical binding mode in our hA₃ receptor model.

Full text of DOI:10.1016/j.ejmech.2015.04.010

European Journal of Medicinal Chemistry 96 (2015) 105e121
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Exploring the 7-oxo-thiazolo[5,4-d]pyrimidine core for the design of
new human adenosine A3 receptor antagonists. Synthesis, molecular
modeling studies and pharmacological evaluation
,
Flavia Varano a *, Daniela Catarzi a, Lucia Squarcialupi a, Marco Betti a, Fabrizio Vincenzi b,
Annalisa Ravani b, Katia Varani b, Diego Dal Ben c, Ajiroghene Thomas c, Rosaria Volpini c,
Vittoria Colotta a
a Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica e Nutraceutica, Universita' di Firenze,
Polo Scientifico, Via Ugo Schiff 6, 50019 Sesto Fiorentino, FI, Italy
b Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Universita' di Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy
c School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Via S. Agostino 1, 62032 Camerino, MC, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 5-methyl-thiazolo[5,4-d]pyrimidine-7-ones bearing different substituents at position 2
(aryl, heteroaryl and arylamino groups) was synthesized and evaluated in radioligand binding assays to
determine their affinities at the human (h) A1, A2A, and A3 adenosine receptors (ARs). Efficacy at the hA2B
and antagonism of selected ligands at the hA3 were also assessed through cAMP experiments. Some of
the new derivatives exhibited good to high hA3AR affinity and selectivity versus all the other AR sub-
types. Compound 2-(4-chlorophenyl)-5-methyl-thiazolo[5,4-d]pyrimidine-7-one 4 was found to be the
most potent and selective ligand of the series (Ki hA3 ¼ 18 nM). Molecular docking studies of the reported
derivatives were carried out to depict their hypothetical binding mode in our hA3 receptor model.
© 2015 Elsevier Masson SAS. All rights reserved.
Received 1 December 2014
Received in revised form
31 March 2015
Accepted 3 April 2015
Available online 4 April 2015
Keywords:
G protein-coupled receptors
Adenosine receptor antagonists
Thiazolopyrimidine
Bicyclic heteroaromatic systems
Ligand-receptor modeling studies
1. Introduction
in several pathological conditions. Selective modulation of each
receptor subtype has been recognized as therapeutically useful in
Purine nucleoside adenosine regulates a large number of
pathophysiological processes through activation of four known
receptor subtypes (A1, A2A, A2B and A3) which are cell surface G
protein-coupled receptors [1,2]. Activation of adenosine re-
ceptors (ARs) typically inhibits (A1 and A3) or stimulates (A2A and
many disorders such as cerebral ischemia, asthma, renal failure,
CNS disorders, inflammatory and neurodegenerative diseases
[1e6].
Among the ARs, the A3AR is the latest to be cloned and phar-
macologically characterized, consequently its role in several ther-
apeutic fields, including ischemia, inflammation and cancer, is still
controversial. In particular, antagonists of the A3AR have been
shown to be attractive as novel potential anti-inflammatory drugs
as well as for the treatment of glaucoma and in cancer chemo-
therapy [7e11]. Moreover, in the CNS, the A3AR is expressed in
neurons and glial cells which are recognized as structural supports
for neurons and as active participants in various neurodegenerative
diseases [12e14].
The putative therapeutic applications of human (h) A3AR an-
tagonists as well as the growing need of pharmacological tools to
clarify the A3 AR roles has made the search for new potent and
selective hA3AR antagonists an emerging and stimulating medici-
nal chemistry topic [7].
A
2B) adenylyl cyclase, but other second messengers can be
modulates the most important being inositol trisphosphate (IP3)
and diacylglycerol (DAG). Therefore, adenosine receptors (ARs)
are valid targets for the development of new therapeutic agents
Abbreviations: MRS 1220, 9-Chloro-2- (2-furanyl)-5-((phenylacetyl)amino)-
[1,2,4]triazolo[1,5-c]quinazoline; NECA, 50 -N-ethyl-carboxamidoadenosine; cAMP,
cyclic adenosine monophosphate; DPCPX, 8-cyclopentyl-1,3-dipropyl-xanthine;
ZM-241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamin
o]ethyl)phenol; I-AB-MECA, N6-(4-amino-3- iodobenzyl)-50 -N-methylcarboxamido
adenosine.
* Corresponding author.
E-mail address: flavia.varano@unifi.it (F. Varano).
http://dx.doi.org/10.1016/j.ejmech.2015.04.010
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

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F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
In our laboratory we have also devoted much effort to the study
of AR antagonists belonging to different heterocyclic classes, and
have identified many potent hA3AR antagonists [15e30]. Recently,
we disclosed
a class of bicyclic hA3AR antagonists, the 2-
arylpyrazolo[4,3-d]pyrimidin-7-one (PP) derivatives endowed
with high hA3AR affinity (Ki ¼ 1.2e72 nM) and selectivity (Fig. 1)
[23]. Molecular modeling studies indicated that PP derivatives are
anchored at the binding site through two stabilizing hydrogen-
bonding interactions that involve, as hydrogen accepting groups,
the 7-oxo function and the nitrogen atom at position-1 [23].
Therefore, to develop a new class of compounds targeting the
ARs, we design the thiazolo[5,4-d]pyrimidine-7-one (TP) de-
rivatives 1e33 (Fig. 2). The thiazolo pyrimidine core maintains a
size and shape similar to the pyrazolo pyrimidine nucleus, as well
as the structural features (the 7-oxo function and the nitrogen atom
at position-1) able to engage the above mentioned crucial in-
teractions with the receptor site.
Fig. 2. Herein reported thiazolo[5,4-d]pyrimidine (Series TP).
Structureeaffinity relationship (SAR) analysis of PP derivatives
allowed us to deduce other structural features that could be
important for the hA3AR receptoreligand interaction. In particular,
the presence of a small lipophilic substituent, such as a methyl or an
ethyl group, at the 5-position of the PP scaffold, significantly en-
hances the hA3AR binding affinity of the ligands, probably because
these groups engage hydrophobic bonds with a lipophilic receptor
pocket of limited size [23]. Hence, the 5-position of all the newly
synthesized TP derivatives (compounds 1e33) was functionalized
with a methyl group.
In addition, the TP derivatives were obtained following chemical
synthetic procedures much more easier than those used for the
synthesis of compounds PP. This feature has made it possible to
synthesize several compounds that differ in the nature of the
substituent at position-2 of the TP core and, thus, has made it
possible to deeply investigate the interaction between the sub-
stituent at that position and the receptor cleft that receives it.
In the PP lead series, the presence of a 4-methoxy or a 4-methyl
group on the 2-phenyl ring was found to increase hA3AR affinity
[23]. Therefore, in the new series, these substituents and others
with different electronic and steric properties (Cl, F, CF3, COOH, OH)
were probed at para- and meta-positions of the 2-phenyl ring
(compounds 1e13).
analyzed to assess their hA3 antagonistic activity.
Along with the pharmacological evaluation, we carried out
molecular modeling studies of the new derivatives to better
rationalize the available SARs.
2. Chemistry
Compounds 1e33 were synthesized as depicted in Schemes
1e5. Briefly, the 2-aryl derivatives 1e13, the 2-benzyl substituted
14 and the 2-heteroaryl thiazolopyrimidines 15e18, were obtained
starting from the commercially available 2-amino malonate hy-
drochloride which was reacted with suitable acyl chlorides to yield
the amides 34e49 (Scheme 1) [31e37]. Most of the acyl chlorides
Moreover, further groups were introduced at position-2 of the
TP nucleus: i.e. heterocyclic substituents (compounds 15e20), and
a benzyl (compound 14) or arylamino chain (compounds 21e28), to
space the phenyl moiety from the bicyclic thiazolo pyrimidine core.
Finally, in some selected 2-aryl- and 2-arylamino-substituted
compounds, the hydrogen accepting 7-oxo group was replaced by a
7-ethoxy function (compounds 29e31 and 32e33, respectively)
which shows the same capability of engaging a hydrogen bond but
is more cluttered than the corresponding 7-oxo moiety.
The new derivatives (1e33) were tested to evaluate their affin-
ities at hA1, hA2A and hA3ARs and their efficacy at hA2BAR. Two
selected compounds, i.e. the 2-phenyl-thiazolopyrimidine-7-one 1
and the 2-(4-chlorophenyl)-thiazolopyrimidine-7-one 4, were also
Scheme 1. Reagents and conditions: a) for 34, 35, 46, 47: R2COCl, Py, room tempera-
ture, for 36e45, 48e49: R2COCl, NaHCO3, Et2O/H2O, 0 ꢀC; b) acetamidine hydrochlo-
ride, NaOEt, EtOH, reflux; c) i) P2S5, Py, reflux; ii) conc HCl, NMP, reflux; d) 1.5 M NaOH,
H2O2, H2O, room temperature; e) BBr3, CH2Cl2 reflux; f) CrO3, conc H2SO4, H2O, room
temperature.
Fig. 1. Previously reported 2-arylpyrazolo[4,3-d]pyrimidin-7-one derivatives (Series
PP).

F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
107
Scheme 2. Reagents and conditions: a) benzoylisothiocyanate, acetone, reflux; b) 6 M HCl, reflux; c) 2,5-diethoxytetrahydrofuran, acetic acid, 90 ꢀC; d) 37% aqueous formaldehyde,
40% aqueous glyoxal, ammonium acetate, acetic acid, 70 ꢀC.
were commercial except the nicotinoyl and isonicotinoyl chlorides
which were synthesized following literature procedures [38,39].
Allowing the amides 34e49 to react with an excess of acetamidine
hydrochloride in the presence of sodium ethoxide, the corre-
sponding 4,6-dihydroxy-2-methyl-pyrimidine-5-amido derivatives
50e65 were isolated [40]. By refluxing the latter in pyridine with
P2S5 and, successively, by treating the mixture with concentrated
HCl in N-methyl-2-pyrrolidone and successively with hydrogen
peroxide at room temperature, the corresponding 7-oxo-thiazolo
[5,4-d]pyrimidine derivatives 1e6, 9e18 were obtained [40].
Demethylation with BBr3 of the 2-(4-methoxy-phenyl) derivative 2
afforded the 2-(4-hydroxyphenyl) substituted 7. Otherwise,
oxidation with CrO3 of 2-(4-methyl-phenyl) derivative 3 yielded
the corresponding 2-(4-carboxyphenyl) compound 8.
[5,4-d]pyrimidine-2-amino derivative 67 as key intermediate was
followed (Scheme 2). 67 was synthesized by reaction of the com-
mercial 5-amino-4,6-dichloro-2-methylpyrimidine with benzoyli-
sothiocyanate and subsequent acid-catalyzed hydrolysis of the
obtained 7-chloro-2-benzoylamino-thiazolo[5,4-d]pyrimidine 66.
By reacting the intermediate 2-amino-thiazolopyrimidine 67 either
with 2,5-diethoxytetrahydrofuran, or formaldehyde glyoxale and
ammonium acetate, in acetic acid, the 2-(pyrrol-1-yl)- and the 2-
(imidazol-1-yl)- 19 and 20, respectively, were obtained.
Scheme 3 shows the synthesis of the thiazolopyrimidine-2-
arylamino derivatives 21e28. Allowing the commercially available
5-amino-4,6-dichloro-2-methylpyrimidine to react with suitable
arylisothiocyanate in the presence of cesium carbonate, the corre-
sponding 7-chloro-5-methyl-N-(aryl)-thiazolo[5,4-d]pyrimidin-2-
amines 68e75 were obtained, which were treated with sodium
acetate and acetic acid to afford the corresponding 7-oxo-
For the synthesis of the 2-heteroaryl-thiazolopyrimidines
19e20 another procedure involving the 5-methyl-7-oxo-thiazolo
Scheme 3. Reagents and conditions: a) arylisothiocyanate, Cs2CO3, acetonitrile, 50 ꢀC, b) CH3COONa, acetic acid, mw or conventional heating 140 ꢀC.

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F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
Scheme 4. Reagents and conditions: a) POCl3, N,N-dimethylaniline, reflux; b) DBU, abs EtOH, mw 160 ꢀC.
thiazolopyrimidine-2-arylamino derivatives 21e28.
reported PP-1A as reference, are listed in Table 1.
The 7-ethoxy-thiazolopyrimidine derivatives 29e31 and 32e33
were obtained as reported in Schemes 4 and 5, respectively. By
reaction with POCl3, the 7-oxo-2-aryl-thiazolopyrimidines 1, 3, 4
were transformed into the corresponding 7-chloro-2-aryl-thiazo-
lopyrimidine derivatives 76e78 [41] which were microwave irra-
diated at 160 ꢀC in the presence of ethanol and 1,8-diazabicyclo
[5.4.0]undec-7-ene (DBU) to give the 7-ethoxy derivatives 29e31
(Scheme 4). Finally, the 7-chloro-2-arylamino derivatives 68 and 72
were treated respectively with an aqueous solution of NaOH or
EtONa in absolute ethanol to afford the corresponding 7 ethoxy-2-
arylamino 32 and 33 (Scheme 5).
4. Results and discussion
4.1. Structureeaffinity relationship studies
As clearly shown in Table 1, some of the synthesized compounds
(1e33) possess good hA3AR affinity and high selectivity being
almost inactive at all the other ARs. These data are very encour-
aging and suggest that the thiazolo[5,4-d]pyrimidine ring system
can be considered as a new scaffold for the development of potent
and selective hA3AR antagonists. In fact, the 2-phenyl parent 1
(Ki ¼ 85 nM) displays an hA3AR binding affinity value only 5-fold
lower than the corresponding PP-1A derivative.
3. Pharmacology
It is quite evident (Table 1) that the highest hA3AR binding af-
finity is attributable to the thiazolo pyrimidine derivatives bearing
a 7-oxo moiety and an aryl group at position-2 (compounds 1e13).
In particular, the 7-oxo-2-(4-chlorophenyl)- derivative 4 emerges
as the most interesting compound, showing a nanomolar hA3AR
affinity (Ki ¼ 18 nM) which is 5-fold higher than that of the parent 1.
The introduction of groups with different electronic and lipophilic
properties in para- or meta-position on the appended 2-phenyl ring
of 1, differently affects the affinity at the hA3AR. In particular, as
already observed for the 4-chloro substituent (see compound 4),
the presence of a lipophilic 4-methoxy or 4-methyl group, seems to
positively affect hA3AR affinity. Indeed, the 2-(4-methoxyphenyl)-
2 and the 2-(4-methylphenyl)- 3 display comparable binding af-
finity that is higher than that of the 2-phenyl unsubstituted 1. In
contrast, the presence of the hydrophobic 4-trifluoromethyl group
exerts a deleterious effect on hA3AR binding affinity (compare 6
All the newly synthesized compounds (1e33) were evaluated in
radioligand binding assays to determine their affinities for hA1,
hA2A and hA3ARs expressed in CHO cells. In particular [3H]
DPCPX [3H]ZM-241385 and [125I]AB-MECA were used as radio-
ligands to hA1, hA2A and hA3ARs, respectively. The efficacy of the
compounds to hA2BAR was investigated by evaluating their capa-
bility to inhibit (100 nM) NECA-stimulated adenylyl cyclase activity.
Antagonism of selected ligands, i.e. the 2-phenyl-thiazolo[5,4-d]
pyrimidin-7-one 1 and the 2-(4-chlorophenyl)-thiazolo[5,4-d]pyr-
imidin-7-one 4, at hA3AR was also assessed through cAMP exper-
iments. by evaluating their capability to block the inhibition of
cAMP production mediated by Cl-IB-MECA (100 nM).
Affinity data for hA1, hA2A and hA3ARs (expressed as Ki values or
I%), and IC50 values or I% for hA2B and hA3AR, derived from the
cAMP assays, of compounds 1e33 along with the previously
Scheme 5. Reagents and conditions: a) 20% aqueous NaOH, EtOH, reflux; b) EtONa, EtOH, reflux.

F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
109
Table 1
Binding Affinity (Ki) at hA1, hA2A, and hA3ARs and Potencies (IC50) at hA2B and hA3ARs.
Comp
R2
Binding experimentsa
cAMP assays
Ki (nM) or I %
IC50 (nM) or I%
b
c
d
e
f
hA1
hA2A
hA3
hA2B
hA3
221 24
1
C6H5
11%
10%
17%
1%
1%
1%
1%
1%
1%
1%
24%
1%
1%
11%
1%
1%
37%
4%
1%
1%
24%
1%
20%
33%
8%
12%
44%
30%
11%
1%
6%
2%
3%
1%
1%
1%
2%
85
38
33
18
9
4
4
2
1%
3%
3%
1%
17%
1%
1%
1%
4%
1%
1%
1%
17%
1%
1%
1%
2%
1%
3%
1%
1%
1%
1%
1%
1%
1%
1%
1%
1%
1%
1%
1%
1%
2%
2
3
4
4-OCH3-C6H4
4-CH3-C6H4
4-Cl-C6H4
1%
42
4
5
4-F-C6H4
15%
37%
1%
107
32%
15%
1%
9
6
7
8
9
4-CF3-C6H4
4-OH-C6H4
4-COOH-C6H4
3-OCH3-C6H4
3-CH3-C6H4
3-Cl-C6H4
3-F-C6H4
3-CF3-C6H4
CH2eC6H5
3-pyridyl
4-pyridyl
2-furyl
2-thienyl
1%
12%
31%
10%
1%
15%
1%
1%
1%
1%
1%
28%
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
PP-1Ag
384 36
826 79
109 11
15%
427 46
33%
15%
20%
248 21
227 19
8%
45%
36%
47%
49%
113 10
450 42
48%
pyrrol-1-yl
imidazol-1-yl
NHe C6H5
9%
1%
14%
1%
18%
18%
1%
NH-(4-OCH3-C6H4)
NH-(4-CH3-C6H4)
NH-(4-Cl-C6H4)
NH-(3-OCH3-C6H4)
NH-(3-CH3-C6H4)
NH-(3-Cl-C6H4)
NH-(3,4-Cl2-C6H3)
C6H5
4-CH3-C6H4
4-Cl-C6H4
NHeC6H5
NH-(3-OCH3-C6H4)
e
8%
21%
16%
1%
1%
9%
3%
25%
1%
25%
6%
5%
10%
45%
110 12
16
2
a
Ki values are means SEM of four separate assays each performed in duplicate. Percentage of inhibition (I %) is determined at 1
Displacement of specific [3H]DPCPX competition binding assays to hA1CHO cells.
mM concentration of the tested compounds.
b
c
d
e
f
Displacement of specific [3H]ZM241385 competition binding to hA2ACHO cells.
Displacement of specific [125I]AB-MECA competition binding to hA3CHO cells.
cAMP experiments in hA2BCHO cells, stimulated by 200 nM NECA. Percentage of inhibition (I%) is determined at 1 mM concentration of the tested compounds.
IC50 values are expressed as means SEM of four separate cAMP experiments in hA3CHO cells, in the presence of 100 nM Cl-IB-MECA.
Ref. [23].
g
with 1). The 2-(4-fluorophenyl) substituted compound 5 does not
show any appreciable difference in the hA3 AR affinity with respect
to the 2-phenyl derivative 1. Finally, introduction of a hydrophilic 4-
hydroxyl or 4-carboxy substituent turns out to be detrimental for
hA3AR affinity (compare 7, 8 with 1). It is interesting to point out
that the movement of the methoxy, methyl or chloro group, from
the para-to the meta-position, results in an important drop of the
hA3 affinity (compare 9, 10, 11, to 2, 3, 4 respectively). On the
contrary, the same shift of the fluorine substituent does not affect
the hA3AR binding affinity (compare 12 to 5, respectively).
phenyl derivative 1 with those of the 2-(2-thienyl)- 18 and the 2-
(pyrrol-1-yl)- substituted 19, indicates a substantial bioisosterism
of these substituents as regards the interaction with the hA3 re-
ceptor site, even if the 2-heterocyclic substituted compounds 18
and 19 are about 3-fold less active than the 2-phenyl derivative 1.
On the contrary, replacement of the phenyl ring with a 2-(3-
pyridyl)- (15), a 2-(4-pyridyl)- (16), a 2-(2-furyl)- (17) or with a 2-
(imidazol-1yl)- (20) leads to a substantial loss of hA3AR affinity.
Continuing to investigate the SARs of these new hA3AR ligands,
we decided to distance the 2-phenyl moiety of compound 1 from
the bicyclic core via a methylene (compound 14) or an amino
(compound 21) linker. In both cases, the hA3 receptor binding is
maintained but the affinity decreases. Thus, with the aim of
Replacement of the 2-phenyl ring of 1 with different heterocy-
clic residues (compounds 15e20) leads to results which are difficult
to explain. In fact, a comparison of the hA3 affinity value of the 2-

110
F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
improving hA3AR binding activity of the 2-phenylamino
substituted compound 21, we inserted on the appended 2-phenyl
ring those substituents that in the 2-aryl series afforded the high-
est hA3AR binding affinity. Thus, we introduced a methoxy, a
methyl group or a chlorine atom either in para- (22e24) or meta-
position (25e27) on the appended phenylamino chain. Overall,
these substituents do not exert the desired effect observed in the 2-
aryl substituted series (1e13). The only exceptions are the 3-
methoxy and 3-methyl groups that make compounds 25 and 26,
respectively, more active than the unsubstituted parent compound
21, while a chlorine atom in the same position does not affect hA3
binding.
Differences between the SARs of the 2-aryl and the 2-arylamino
substituted compounds are also evidenced by the different binding
behavior of the 7-ethoxy derivatives of the two series 29e31 and
32e33. In fact, in the 2-aryl-7-ethoxy derivatives 29e31 we
observe a dramatic drop of the hA3AR binding activity compared to
their corresponding 7-oxo compounds 1, 3e4. In contrast, the 2-
arylamino-7-ethoxy 32 and 33 and their corresponding 7-oxo- 21
and 25 bind to the hA3AR with similar affinities.
The selected compounds 1 and 4 were tested to evaluate their
antagonistic potencies to modulate Cl-IB-MECA inhibited cAMP
accumulation in CHO cells expressing the hA3 receptor. In accor-
dance with the hA3AR affinity values, the IC50 results (Table 1)
showed that the tested compounds are hA3AR antagonists
endowed with significant potencies.
4.2. Molecular modeling studies
A molecular docking analysis was performed on a homology
model of hA3AR developed by using the X-ray structure of the
hA2AAR as template (pdb code: 3EML; 2.6 Å resolution [42]). This
crystal structure has been solved in complex with a high affinity
antagonist (ZM241385), hence presenting an inactive conformation
and a cavity suitable as a binding site for docking analysis [43]. The
sequence alignment of the four human ARs is shown within
Supplementary data. The obtained hA3AR homology model was
checked by using the Protein Geometry Monitor application within
MOE [44], which provides a variety of stereochemical measure-
ments for inspection of the structural quality in a given protein,
such as backbone bond lengths, angles and dihedrals, Ramachan-
dran 4-
j dihedral plots, and sidechain rotamer and nonbonded
contact quality.
A preliminary docking analysis was performed by manually
docking the high affinity antagonist MRS 1220 (Ki hA3AR ¼ 0.65 nM
[45]) structure within the hA3AR model binding site. The obtained
hA3AR-MRS 1220 complex was then subjected to energy minimi-
zation refinement and to Monte Carlo analysis (with Schrodinger
Macromodel [46] software) to explore the favorable binding con-
formations, with the input structure consisting of the ligand and a
shell of receptor amino acids within 6 Å distance from it. The co-
ordinates of the remaining receptor residues were kept fixed.
During the Monte Carlo conformational search (10,000 steps), the
input structure was modified by random changes in torsion angles
(for all input structure residues), and molecular position (for the
ligand). Hence, the ligand was left free to be continuously re-
oriented and re-positioned within the binding site and the
conformation of both ligand and internal shell residues could be
explored and reciprocally relaxed. This stage allowed us to provide
an hA3AR binding site conformation able to accommodate the
analyzed antagonists. The best receptor-MRS 1220 complex was
subjected to energy minimization. The RMSD value (calculated
within MOE; only backbone atoms were considered) from the
comparison of the initial and the final model was 0.32 Å. This value
raises to 0.53 Å considering only the receptor residues subjected to
Fig. 3. A. Side view of the hA3AR model with indication of the compounds binding
mode (compound 4 is shown). B. Detailed view of the binding mode of the synthesised
compounds. The compound 4 is shown. The picture displays, in particular, the inter-
action of the scaffold with the receptor residues. The double H-bond interaction of the
compound scaffold with the sidechain of Asn250 is clearly visible. C. Detailed view of
the binding mode of the compound PP-1A.
Monte Carlo conformational search.
Once the MRS 1220 compound was removed, the hA3AR model
was then used as a target for the docking analysis of the synthe-
sized derivatives. All ligand structures were optimized using RHF/
AM1 semi-empirical calculations (with the software package
MOPAC [47] implemented in MOE) and then docked into the
binding site of the hA3 AR model by using the MOE Dock tool. Top-

F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
111
Fig. 5. A. Detail of the interaction of the 2-substituent with the TM2-TM3 residues
(compound 4 is shown). The TM2-TM3 subcavity (see text for details) is represented as
molecular surface. B. Superimposition of the compounds 2 and 25 binding mode. The
methoxy group in position meta of the appended phenyl ring of compound 25 results
superimposed to the same substituent in position para of the 2 phenyl ring of com-
pound 2.
The compounds are relatively small compared to the cavity
volume, hence they may present different binding modes as evi-
denced by the docking results. The most populated (and lowest
scoring) family of docking conformations presents the ligand
almost orthogonally oriented with respect to the receptor axis
(Fig. 3), with the bicyclic scaffold located in proximity of TM
(transmembrane) domains 6 and 7 and EL (extracellular loop) 2
domain and the 2-substituent positioned between TM2 and TM3
segments. Thus, docking results suggest for the newly synthesized
compounds a different binding mode than the above cited pyr-
azolopyrimidine derivatives (Series PP) from which these com-
pounds were designed. In fact, the S-3 and N-4 atoms of the
thiazolopyrimidine scaffold are oriented toward the extracellular
environment, apparently not involved in any direct interaction with
receptor residues, while the N-1 atom and the carbonyl group at 7-
position point toward the core of the protein. The 7-oxo function
and the NH group in 6-position make a double H-bond with the
Asn2506.55 residue (as superscript it is indicated the residue ID
according to the numbering system suggested by Ballesteros and
Weinstein [48]). This interaction is the main polar interaction be-
tween the compounds and the receptor and hence seems the key
stabilizing factor for this binding mode of the ligands.
Fig. 4. A. Detail of the interaction of the 2-substituent with the TM2-TM3 residues
(compounds 1, 14, and 21 are shown). The TM2-TM3 subcavity (see text for details) is
represented as molecular surface.
score docking poses of each compound were subjected to energy
minimization and then rescored using three available methods
implemented in MOE: the London dG and the Affinity dG scoring
tools and the dock-pKi predictor (see experimental section for de-
tails). For each compound, the top-score docking poses, according
to at least two out of three scoring functions, were selected for final
ligandetarget interaction analysis.
The 2-substituent differently affects the affinity of the com-
pounds on the basis of its volume and chemical-physical profile.
When this substituent is an aromatic group directly bound to the
scaffold (compounds 1e13 and 15e20), the best affinity values are
observed when this group presents a marked hydrophobic profile.

112
F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
Hence, insertion in position 2 of a phenyl ring (compound 1) or a
para-substituted phenyl moiety presenting methoxy or methyl
groups, or a chlorine atom (compounds 2, 3, 4, respectively) leads to
good affinity for the hA3AR, while a fluorine atom or a trifuor-
omethyl, hydroxyl, or carboxy group at the same position leaves
unchanged or decreases affinity at the same receptor. Docking re-
sults show that the 2-substituent is inserted into a narrow sub-
pocket (Figs. 4 and 5A) given by residues belonging to TM2
(Val652.57, Leu682.60, Ala692.61, and Val722.64) and TM3 (Met863.28
and Leu903.32). The chemical-physical profile of these residues
provides a marked hydrophobic environment for this subpocket
and explains why the insertion of too polar or hydrophilic groups is
detrimental for the compound affinity. In this sense, the insertion of
heterocycles presenting polar atoms (i.e. pyridyl or furyl groups as
in compounds 15e17) seems detrimental, while introduction of
heterocycles with a more pronounced hydrophobic profile (i.e.
thienyl or pyrrol-1-yl groups as in compounds 18e19) maintains
affinity for the receptor. Given the above cited interaction with the
Asn2506.55 residue, which provide a first general stabilizing factor
of this binding mode of the ligands, the geometric and chemical-
physical matching of the 2-substituent and the TM2-TM3 sub-
cavity provides the second stabilizing feature and also a factor
providing high or low affinity of the analyzed compounds for the
A3AR. As indicated above, this subcavity is quite narrow and this
feature explains why the presence of a fluorine atom at the meta-
position of the 2-phenyl ring is accepted (compound 12), while
insertion, in the same position, of bigger functions like a methyl
group or a chlorine atom (compounds 10e11), leads to a drop of
hA3AR affinity, contrary to what is observed for the same kind of
substitutions at the para-position.
When the aromatic group at position 2 is spaced from the
thiazolopyrimidine scaffold by a methylene or amine linker as in
compounds 14 and 21e28, respectively, a general decrease in af-
finity is observed. Docking results suggest that introduction of the
linker makes the phenyl ring being differently oriented than the
analogue ring directly bound to the scaffold. Fig. 4 shows a com-
parison between the docking pose of compound 1 (whose phenyl
ring is directly bound to the scaffold; Fig. 4A) and the binding mode
of compounds 14 and 21 (whose phenyl ring is bound to the scaf-
fold trough a methyl or an amine as spacers; Fig. 4B and C,
respectively). As a consequence, these compounds generally fail to
match both the above cited stabilizing factors (the double H-bond
with Asn2506.55 and the fitting into the TM2-TM3 subcavity). An
exception is given when a small hydrophobic group is inserted at
the meta-position of the appended phenyl ring (i.e. a methoxy or a
methyl group as in compounds 25 and 26, respectively). In this case,
the small hydrophobic group gets similarly oriented to the
analogue group inserted in para-position on the 2-phenyl ring
directly bound to the bicyclic scaffold (Fig. 5, panel B). We must
underline that this phenomenon is not observed for compound 27
which presents a chlorine atom at the meta-position of the 2-
phenylamine substituent, but is not endowed with affinity for the
receptor as the 2-(4-chlorophenyl) derivative 4.
Fig. 6. Detail of the interaction of the 7-position of compounds 25 (A) and 33 (B) with
the A3AR residues. The A3AR subcavity (see text for details) is represented as molecular
surface. C. Interaction between the same two compounds and the A3AR residues
estimated by the IF-E 6.0 tool; only residues in proximity of the compound 7-position
are here considered.
Finally, considering the derivatives presenting
a 7-ethoxy
function (compounds 29e33), affinity for the A3AR is observed
only in the case of compound 33 presenting a methoxy group in
the meta-position of the 2-phenylamine substituent. Respect to
the 7-oxo analogue 25, compound 33 lacks the H-bond donor
function at position 6. The comparison of the affinity data of these
two compounds suggests that the conversion of the 7-oxo func-
tion in a 7-ethoxy group and the consequent loss of an H-bond
donor function in 6-position are not detrimental for the binding
with the receptor. On the other hand, we cannot exclude that the
two H-bond acceptor functions at position 6 and 7 of this ligand
may interact with the amine function of Asn2506.55 sidechain. We
compared the binding mode of these two compounds by consid-
ering the residues in proximity of the 6- and 7-position of the
ligands. As described in Fig. 6(AeB), the introduction of an ethoxy
group in 7-position (compound 33, Fig. 6B) makes the ethyl chain
being inserted within a set of hydrophobic aminoacids (Leu903.32
,
F168, M1775.38, W2436.48, L2466.51, and Ile2496.54). The interaction
of compounds 25 and 33 with the surrounding residues was
estimated with the aid of the IF-E 6.0 tool [49] retrievable at the
SVL exchange service (Chemical Computing Group, Inc. SVL

F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
113
exchange: http://svl.chemcomp.com). The script calculates and
5.1.1. General procedure for the synthesis of 2-(aroylamino)
displays atomic and residue interaction forces as 3D vectors. It also
calculates the per-residue interaction energies (values in kcal
molꢁ1), where negative and positive energy values are associated
to favorable and unfavorable interactions, respectively. This
method was already used by us for the analysis of ligandetarget
interaction for a series of potent A3AR and A2BAR agonists [50,51].
Focusing in particular on the interaction between the 7-position
and the surrounding A3AR residues, results (Fig. 6C) show that
as expected there is a quite stronger interaction between com-
pound 25 and Asn2506.55 respect to compound 33. At the same
time it is also observable that for some residues the interaction is
better with the latter compound (Phe168, W2436.48, L2466.51, and
Ile2496.54). This data could suggest on the one hand that the
presence of a double H-bond interaction with Asn2506.55 (i.e.
compound 25) may be important in terms of stabilization of the
binding mode and gain of high affinity, on the other hand that the
conversion of the 7-oxo group in an ethoxy function (compound
33) provides a more marked hydrophobic character that increase
the interaction with non-polar residues of the cavity and possibly
makes easier its desolvation during the process of interaction with
the receptor. These considerations could be applied in general to
all analogues presenting a different 7-group. Nevertheless, we
must also notate that docking results do not provide clear expla-
nations regarding the loss of affinity of compounds 29e31, while
for compound 32 the same considerations as for compound 21 can
be made.
propanedioic acid diethyl ester derivatives (34e35, 46e47)
The title compounds were prepared by reacting equimolar
amounts of diethyl 2-aminomalonate hydrochloride (14.2 mmol)
and the suitable aroyl chloride in anhydrous pyridine (30 mL). The
mixture was stirred at room temperature for 2 h (compounds 34
and 35) or 10 h (compounds 46 and 47). Evaporation at reduced
pressure of the solvent afforded a residue which was treated with
H2O (120 mL for compounds 34 and 35, 40 mL for compounds 46
and 47) and EtOH (5 mL). The resulting solid was collected by
filtration, washed with water and recrystallised.
5.1.1.1. Diethyl ((phenylcarbonyl)amino)propanedioate 34 [31].
Yield 90%; m.p. 60e62 ꢀC (Et2O) (litt m.p. 63 ꢀC ethyl acetate/
heptanes 1/10); 1H NMR: 1.23 (t, 6H, 2 CH3, J ¼ 7.1 Hz), 4.15 (m, 4H, 2
CH2), 5.31 (d, 1H, CH, J ¼ 7.6 Hz), 7.50 (t, 2H, ar, J ¼ 7.7 Hz), 7.59 (t,
1H, ar, J ¼ 7.2 Hz), 7.91 (d, 2H, ar, J ¼ 7.2 Hz), 9.31 (d, 1H, NH,
J ¼ 7.6 Hz). IR: 3312, 1747, 1639. Anal. Calc. for C14H17NO5.
5.1.1.2. Diethyl (((4-methoxyphenyl)carbonyl)amino)propanedioate
35 [31]. Yield 89%; m.p. 103e107 ꢀC (Et2O) (litt. m.p. 106 ꢀC); 1H
NMR: 1.23 (t, 6H, 2 CH3, J ¼ 7.1 Hz), 3.83 (s, 3H, OCH3), 4.16e4.26 (m,
4H, 2 CH2), 5.29 (d, 1H, CH, J ¼ 7.5 Hz), 7.03 (d, 2H, ar, J ¼ 8.9 Hz),
7.90 (d, 2H, ar, J ¼ 8.9 Hz), 9.15 (d, 1H, NH, J ¼ 7.5 Hz). IR: 3300, 1753,
1631. Anal. Calc. for C15H19NO6.
5.1.1.3. Diethyl ((pyridin-3-ylcarbonyl)amino)propanedioate 46.
Yield 78%; m.p. 76e78 ꢀC (Et2O); 1H NMR: 1.23 (t, 6H, 2 CH3,
J ¼ 7.1 Hz), 4.17e4.26 (m, 4H, 2 CH2), 5.34 (d, 1H, CH, J ¼ 7.5 Hz), 7.57
(dd, 1H, ar, J ¼ 7.7, 4.9 Hz), 8.27 (d, 1H, ar, J ¼ 7.7 Hz), 8.77 (d, 1H, ar,
J ¼ 4.9 Hz), 9.05 (s, 1H, ar), 9.60 (d, 1H, NH, J ¼ 7.5 Hz). IR: 3304,
1753, 1643. Anal. Calc. for C13H16N2O5.
4.3. Conclusion
In the present study we have identified the thiazolo[5,4-d]pyr-
imidin-7-one bicyclic system as a new scaffold for obtaining potent
and selective hA3 AR antagonists. In fact, some of the newly syn-
thesized compounds showed high hA3AR affinity and selectivity
versus all the other AR subtypes, the best being the 2-(4-
chlorophenyl)-5-methyl-thiazolo[5,4-d]pyrimidin-7-one 4 which
possesses the highest hA3AR affinity value (Ki ¼ 18 nM). Molecular
docking of the newly synthesized compounds allows us to repre-
sent their hypothetical binding mode to our hA3 receptor model as
well as to rationalize the observed SARs.
5.1.1.4. Diethyl ((pyridin-4-ylcarbonyl)amino)propanedioate 47.
Yield 79%; m.p. 60e62 ꢀC (Et2O); 1H NMR: 1.23 (t, 6H, 2 CH3,
J ¼ 7.1 Hz), 4.12e4.26 (m, 4H, 2 CH2), 5.33 (d,1H, CH, J ¼ 7.6 Hz), 7.81
(d, 2H, ar, J ¼ 5.6 Hz), 8.77 (d, 2H, ar, J ¼ 5.6 Hz), 9.68 (d, 1H, NH,
J ¼ 7.6 Hz). IR: 3313, 1743, 1645. Anal. Calc. for C13H16N2O5.
5.1.2. General procedure for the synthesis of 2-(aroylamino)
propanedioic acid diethyl ester derivatives (36e45, 48e49)
Suitable aroyl chloride (13.6 mmol) was added dropwise to a
solution of diethyl aminomalonate hydrochloride (14.2 mmol) and
sodium hydrogen carbonate (35.6 mmol) in Et2O (40 mL) and H2O
(30 mL) cooled at 0 ꢀC. The solution was warmed to room tem-
perature and stirred for 2 h. The organic layer was washed with 1 M
HCl (20 mL) then water (30 mL), dried (Na2SO4) and concentrated
to give the desired amide which was collected by filtration and
recrystallised.
5. Experimental section
5.1. Chemistry
The microwave-assisted synthesis were performed using an
Initiator EXP Microwave Biotage instrument (frequency of irradia-
tion: 2.45 GHz). Analytical silica gel plates (Merck F254), prepara-
tive silica gel plates (Merck F254, 2 mm) and silica gel 60 (Merck,
70e230 mesh) were used for analytical and preparative TLC, and for
column chromatography, respectively. All melting points were
determined on a Gallenkamp melting point apparatus and are
uncorrected. Elemental analyses were performed with a Flash
E1112 Thermofinnigan elemental analyzer for C, H, N and the re-
sults were within 0.4% of the theoretical values. The IR spectra
were recorded with a PerkineElmer Spectrum RX I spectrometer in
Nujol mulls and are expressed in cmꢁ1. The NMR spectra were
obtained with a Bruker Avance 400 instrument (400 MHz for 1H
NMR and 100 MHz for 13C NMR). The chemical shifts are reported in
5.1.2.1. Diethyl (((4-methylphenyl)carbonyl)amino)propanedioate 36
[32]. Yield 92%; m.p. 102e103 ꢀC (Et2O); 1H NMR: 1.22 (t, 6H, 2 CH3,
J ¼ 7.1 Hz), 3.37 (s, 3H, CH3), 4.16e4.25 (m, 4H, 2 CH2), 5.29 (d, 1H,
CH, J ¼ 7.5 Hz), 7.31 (d, 2H, ar, J ¼ 8.0 Hz), 7.82 (d, 2H, ar, J ¼ 8.0 Hz),
9.23 (d, 1H, NH, J ¼ 7.5 Hz). IR: 3292, 1754, 1638. Anal. Calc. for
C15H19NO5
5.1.2.2. Diethyl (((4-chlorophenyl)carbonyl)amino)propanedioate 37
[33]. Yield 93%; m.p. 91e94 ꢀC (Et2O); 1H NMR: 1.23 (t, 6H, 2 CH3,
J ¼ 7.1 Hz), 4.12e4.27 (m, 4H, 2 CH2), 5.30 (d, 1H, CH, J ¼ 7.5 Hz), 7.58
(d, 2H, ar, J ¼ 8.5 Hz), 7.93 (d, 2H, ar, J ¼ 8.5 Hz), 9.44 (d, 1H, NH,
J ¼ 7.5 Hz). IR: 3312, 1748, 1643. Anal. Calc. for C14H16ClNO5.
d
(ppm) and are relative to the central peak of the solvent which
was always DMSO-d6. The following abbreviations are used:
s ¼ singlet, d ¼ doublet, t ¼ triplet, m ¼ multiplet, br ¼ broad, and
ar ¼ aromatic protons. Scanned 1H and 13C NMR spectra of some
selected compounds (1, 3, 4, 11, 18, 25, 26, 33) are reported in the
Supplementary data.
5.1.2.3. Diethyl (((4-fluorophenyl)carbonyl)amino)propanedioate 38
[34]. Yield 90%; m.p. 79e81 ꢀC (Et2O) (litt. m.p. 108e109 ꢀC); 1H

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F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
NMR: 1.23 (t, 6H, 2 CH3, J ¼ 7.1 Hz), 4.14e4.27 (m, 4H, 2 CH2), 5.30
(d, 1H, CH, J ¼ 7.6 Hz), 7.34 (t, 2H, ar, J ¼ 8.9 Hz), 7.99 (dd, 2H, ar,
J ¼ 5.4, 2.1 Hz), 9.39 (d,1H, NH, J ¼ 7.6 Hz). IR: 3358,1751,1658. Anal.
Calc. for C14H16FNO5.
5.1.3. General procedure for the synthesis of N-(4,6-dihydroxy-2-
methylpyrimidin-5-yl)carboxyamide derivatives (50e65)
A suspension of acetamidine hydrochloride (15.6 mmol) and
sodium (31.2 mmol) in ethanol (44 mL) was stirred at 30 ꢀC for
10 min and then added with the suitable amide 34e49 (5.2 mmol).
The reaction mixture was refluxed for 4 h then concentrated in
vacuo. The solid product was filtered off, washed with ethanol,
dissolved in the minimum amount of water (about 20 mL, 10 mL for
compounds 62 and 63) and acidified to pH 2e3 with 6 M HCl (to pH
4e5 with acetic acid for compounds 62 and 63). The precipitate was
collected, washed with acetone and recrystallised.
5.1.2.4. Diethyl
(((4-(trifluoromethyl)phenyl)carbonyl)amino)pro-
panedioate 39. Yield 92%; m.p. 103e105 ꢀC (Et2O); 1H NMR: 1.23 (t,
6H, 2 CH3, J ¼ 7.1 Hz), 4.17e4.26 (m, 4H, 2 CH2), 5.33 (d, 1H, CH,
J ¼ 7.5 Hz), 7.90 (d, 2H, ar, J ¼ 8.0 Hz), 8.10 (d, 2H, ar, J ¼ 8.0 Hz), 9.62
(d, 1H, NH, J ¼ 7.5 Hz). IR: 3294, 1754, 1646. Anal. Calc. for
C
15H16F3NO5.
5.1.2.5. Diethyl (((3-methoxyphenyl)carbonyl)amino)propanedioate
40. Yield 92%; m.p. 54e56 ꢀC (Et2O); 1H NMR: 1.23 (t, 6H, 2 CH3,
J ¼ 7.1 Hz), 3.82 (s, 3H, OCH3), 4.15e4.27 (m, 4H, 2 CH2), 5.31 (d, 1H,
CH, J ¼ 7.6 Hz), 7.15 (dd, 1H, ar, J ¼ 5.7, 1.8 Hz), 7.39e7.51 (m, 3H, ar),
9.34 (d, 1H, NH, J ¼ 7.6 Hz). IR: 3298, 1747, 1640 Anal. Calc. for
5.1.3.1. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)benzamide
50.
Yield 49%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.29 (s, 3H, CH3),
7.48 (t, 2H, ar, J ¼ 6.9 Hz), 7.55 (t, 1H, ar, J ¼ 6.9 Hz), 7.94 (d, 2H, ar,
J ¼ 6.9 Hz), 9.07 (s, 1H, NH), 12.05 (br s, 2H, 2 OH). IR: 3258, 2670,
1641. Anal. Calc. for C12H11N3O3.
C
15H19FNO6.
5.1.3.2. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)-4-
methoxybenzamide 51. Yield 52%; m.p. >300 ꢀC (acetic acid); 1H
NMR: 2.29 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 7.01 (d, 2H, ar, J ¼ 8.4 Hz),
7.92 (d, 2H, ar, J ¼ 8.4 Hz), 8.89 (s, 1H, NH), 12.01 (br s, 2H, 2 OH). IR:
3155, 2598, 1639: Anal. Calc. for C13H13N3O4.
5.1.2.6. Diethyl (((3-methylphenyl)carbonyl)amino)propanedioate 41
[35]. Yield 91%; m.p. 53e56 ꢀC (Et2O); 1H NMR: 1.23 (t, 6H, 2 CH3,
J ¼ 7.1 Hz), 2.37 (s, 3H, CH3), 4.16e4.27 (m, 4H, 2 CH2), 5.31 (d, 1H,
CH, J ¼ 7.6 Hz), 7.36e7.39 (m, 2H, ar), 7.69e7.74 (m, 2H, ar), 9.24 (d,
1H, NH, J ¼ 7.6 Hz). IR: 3281, 1753, 1642. Anal. Calc. for C15H19NO5.
5.1.3.3. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)-4-
methylbenzamide 52. Yield 53%; m.p. >300 ꢀC (acetic acid); 1H
NMR: 2.28 (s, 3H, CH3), 2.37 (s, 3H, CH3), 7.28 (d, 2H, ar, J ¼ 7.8 Hz),
7.85 (d, 2H, ar, J ¼ 7.8 Hz), 8.98 (s, 1H, NH), 12.02 (br s, 2H, 2 OH). IR:
3230, 2617, 1644. Anal. Calc. for C13H13N3O3.
5.1.2.7. Diethyl (((3-chlorophenyl)carbonyl)amino)propanedioate 42.
Yield 94%; m.p. 84e86 ꢀC (Et2O); 1H NMR: 1.23 (t, 6H, 2 CH3,
J ¼ 7.1 Hz), 4.17e4.27 (m, 4H, 2 CH2), 5.31 (d, 1H, CH, J ¼ 7.5 Hz), 7.55
(t, 1H, ar, J ¼ 7.9 Hz), 7.67 (dd, 1H, ar, J ¼ 7.9, 1.7 Hz), 7.87 (d, 1H, ar,
J ¼ 7.9), 7.97 (d, 1H, ar, J ¼ 1.7 Hz), 9.51 (d, 1H, NH, J ¼ 7.5 Hz). IR:
3281, 1751, 1643. Anal. Calc. for C14H16ClNO5.
5.1.3.4. 4-Chloro-N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)benza-
mide 53. Yield 55%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.28 (s, 3H,
CH3), 7.56 (d, 2H, ar, J ¼ 8.0 Hz), 7.96 (d, 2H, ar, J ¼ 8.0 Hz), 9.16 (s,
1H, NH), 12.14 (br s, 2H, 2 OH). IR: 3236, 2603, 1643. Anal. Calc. for
5.1.2.8. Diethyl (((3-fluorophenyl)carbonyl)amino)propanedioate 43
[36]. Yield 89%; m.p. 72e74 ꢀC (Et2O); 1H NMR: 1.23 (t, 6H, 2 CH3,
J ¼ 7.1 Hz), 4.14e4.25 (m, 4H, 2 CH2), 5.31 (d,1H, CH, J ¼ 7.6 Hz), 7.45
(t, 1H, ar, J ¼ 8.6 Hz), 7.57 (dd, 1H, ar, J ¼ 7.5, 6.2 Hz), 7.72 (d, 1H, ar,
J ¼ 10.0 Hz), 7.77 (d,1H, ar, J ¼ 7.0 Hz), 9.45 (d,1H, NH, J ¼ 7.6 Hz). IR:
3295, 1755, 1642. Anal. Calc. for C14H16FNO5.
C12H10ClN3O3.
5.1.3.5. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)-4-
fluorobenzamide 54 [40]. Yield 53%; m.p. >300 ꢀC (acetic acid); 1H
NMR: 2.28 (s, 3H, CH3), 7.31 (t, 2H, ar, J ¼ 8.7 Hz), 8.01 (t, 2H, ar,
J ¼ 5.9 Hz), 9.12 (s, 1H, NH), 12.06 (br s, 2H, 2 OH). IR: 3240, 2610,
1645. Anal. Calc. for C12H10FN3O3.
5.1.2.9. Diethyl
(((3-(trifluoromethyl)phenyl)carbonyl)amino)pro-
panedioate 44. Yield 91%; m.p. 89e92 ꢀC (Et2O); 1H NMR: 1.23 (t,
6H, 2 CH3, J ¼ 7.1 Hz), 4.16e4.28 (m, 4H, 2 CH2), 5.35 (d, 1H, CH,
J ¼ 7.6 Hz), 7.77 (t, 1H, ar, J ¼ 7.7 Hz), 7.98 (d, 1H, ar, J ¼ 7.7 Hz), 8.21
(d, 1H, ar, J ¼ 7.8 Hz), 8.27 (s, 1H, ar), 9.60 (d, 1H, NH, J ¼ 7.6 Hz). IR:
3303, 1749, 1640. Anal. Calc. for C15H16F3NO5.
5.1.3.6. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)-4-(tri-
fluoromethyl)benzamide 55. Yield 54%; m.p. >300 ꢀC (acetic acid);
1H NMR: 2.29 (s, 3H, CH3), 7.87 (d, 2H, ar, J ¼ 8.0 Hz), 8.13 (d, 2H, ar,
J ¼ 8.0 Hz), 9.33 (s, 1H, NH), 12.32 (br s, 2H, 2 OH). Anal. 3243, 2618,
1634. Calc. for C13H10F3N3O3.
5.1.2.10. Diethyl ((phenylacetyl)amino)propanedioate 45 [37].
Yield 90%; m.p. 66e68 ꢀC (Et2O) (litt. m.p. 68 ꢀC); 1H NMR: 1.19 (t,
6H, 2 CH3, J ¼ 7.1 Hz), 3.58 (s, 2H, CH2), 4.11e4.23 (m, 4H, 2 CH2),
5.22 (d, 1H, CH, J ¼ 7.5 Hz), 7.22e7.32 (m, 5H, ar), 9.02 (d, 1H, NH,
J ¼ 7.5 Hz) IR: 3316, 1746, 1648. Anal. Calc. for C15H19NO5.
5.1.3.7. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)-3-
methoxybenzamide 56. Yield 46%; m.p. 288e290 ꢀC (acetic acid);
1H NMR: 2.28 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 7.10 (d, 1H, ar,
J ¼ 8.2 Hz), 7.38 (t, 1H, ar, J ¼ 7.8 Hz), 7.49e7.53 (m, 2H, ar), 9.05 (s,
1H, NH), 12.01 (br s, 2H, 2 OH). IR: 3152, 2630, 1639. Anal. Calc. for
5.1.2.11. Diethyl
((furan-2-ylcarbonyl)amino)propanedioate
48.
Yield 88%; m.p. 60e63 ꢀC (Et2O); 1H NMR: 1.22 (t, 6H, 2 CH3,
J ¼ 7.1 Hz), 4.14e4.26 (m, 4H, 2 CH2), 5.22 (d, 1H, CH, J ¼ 7.5 Hz),
6.66e6.68 (m, 1H, ar), 7.29 (d, 1H, ar, J ¼ 3.5 Hz), 7.91 (d, 1H, ar,
J ¼ 0.9 Hz), 9.05 (d, 1H, NH, J ¼ 7.5 Hz). IR: 3335, 1749, 1651. Anal.
Calc. for C12H15NO6.
C13H13N3O4.
5.1.3.8. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)-3-
methylbenzamide 57. Yield 55%; m.p. >300 ꢀC (acetic acid); 1H
NMR: 2.28 (s, 3H, CH3), 2.37 (s, 3H, CH3), 7.35e7.36 (m, 2H, ar),
7.73e7.77 (m, 2H, ar), 9.00 (s, 1H, NH), 12.01 (br s, 2H, 2 OH). IR:
3251, 2611, 1624. Anal. Calc. for C13H13N3O3.
5.1.2.12. Diethyl ((thienyl-2-ylcarbonyl)amino)propanedioate 49.
Yield 91%; m.p. 74e77 ꢀC (Et2O); 1H NMR: 1.23 (t, 6H, 2 CH3,
J ¼ 7.1 Hz), 4.15e4.27 (m, 4H, 2 CH2), 5.29 (d, 1H, CH, J ¼ 7.8 Hz),
7.18e7.20 (m, 1H, ar), 7.83 (d, 1H, ar, J ¼ 4.9 Hz), 7.97 (d, 1H, ar,
J ¼ 3.0 Hz), 9.36 (d, 1H, NH, J ¼ 7.8 Hz). IR: 3281, 1734, 1632 Anal.
Calc. for C12H15NO5S.
5.1.3.9. 3-Chloro-N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)benza-
mide 58. Yield 54%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.28 (s, 3H,
CH3), 7.52 (t,1H, ar, J ¼ 7.9 Hz), 7.62 (d,1H, ar, J ¼ 8.0 Hz), 7.90 (d, 2H,
ar, J ¼ 7.8 Hz), 7.98 (s, 1H, ar), 9.20 (s, 1H, NH), 12.07 (br s, 2H, 2 OH).

F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
115
IR: 3255, 2621, 1644. Anal. Calc. for C12H10ClN3O3.
5.1.4.1. 5-Methyl-2-phenyl-thiazolo[5,4-d]pyrimidin-7-one
1.
Yield 72%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.42 (s, 3H, CH3),
7.56e7.57 (m, 3H, ar), 7.99e8.01 (m, 2H, ar), 12.77 (br s, 1H, NH). 13
NMR: 162.53, 162.29, 157.28, 156.80, 137.28, 133.04, 131.62, 129.84
(2C), 127.07 (2C), 21.58. IR: 1662, 1592. Anal. Calc. for C12H9N3OS.
C
5.1.3.10. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)-3-
fluorobenzamide 59. Yield 52%; m.p. >300 ꢀC (acetic acid); 1H NMR:
2.29 (s, 3H, CH3), 7.40 (t, 1H, ar, J ¼ 7.9 Hz), 7.54 (dd, 1H, ar, J ¼ 14.0,
7.5 Hz), 7.73 (d, 2H, ar, J ¼ 9.3 Hz), 7.80 (d, 1H, ar, J ¼ 7.5 Hz), 9.15 (s,
1H, NH), 12.08 (br s, 2H, 2 OH). IR: 3242, 2614, 1643. Anal. Calc. for
5.1.4.2. 2-(4-Methoxyphenyl)-5-methyl-thiazolo[5,4-d]pyrimidin-7-
one 2. Yield 80%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.40 (s, 3H,
CH3), 3.85 (s, 3H, OCH3), 7.11 (d, 2H, ar, J ¼ 8.8 Hz), 7,94 (d, 2H, ar,
J ¼ 8.8 Hz), 12.72 (br s, 1H, NH). IR: 1692, 1604. Anal. Calc. for
C
12H10FN3O3.
5.1.3.11. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)-3-(tri-
fluoromethyl)benzamide 60. Yield 51%; m.p. >300 ꢀC (acetic acid);
1H NMR: 2.28 (s, 3H, CH3), 7.74 (t, 1H, ar, J ¼ 7.7 Hz), 7.93 (t, 1H, ar,
J ¼ 7.5 Hz), 8.24 (d, 2H, ar, J ¼ 8.00 Hz), 8.29 (s, 1H, ar), 9.37 (s, 1H,
NH), 12.16 (br s, 2H, 2 OH). IR: 3371, 2680, 1625. Anal. Calc. for
C13H11N3O2S.
5.1.4.3. 5-Methyl-2-(4-methylphenyl)-thiazolo[5,4-d]pyrimidin-7-
one 3. Yield 69%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.39 (s, 3H,
CH3), 2.41 (s, 3H, CH3), 7.37 (d, 2H, ar, J ¼ 8.0 Hz), 7.89 (d, 2H, ar,
J ¼ 8.0 Hz), 12.75 (br s, 1H, NH). 13C NMR:162.68, 162.08, 157.14,
156.78, 141.72, 137.22, 130.48, 130.40 (2C), 127.04 (2C), 21.58, 21.47.
IR: 1689, 1601. Anal. Calc. for C13H11N3OS.
C
13H10F3N3O3.
5.1.3.12. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)-2-
phenylacetamide 61. Yield 48%; m.p. >300 ꢀC (acetic acid); 1H NMR:
2.24 (s, 3H, CH3), 3.58 (s, 2H, CH2), 7.22e7.32 (m, 5H, ar), 8.91 (s, 1H,
NH), 11.96 (br s, 2H, 2 OH). IR: 3256, 2610, 1630. Anal. Calc. for
5.1.4.4. 2-(4-Chlorophenyl)-5-methyl-thiazolo[5,4-d]pirimidin-7-one
4. Yield 82%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.42 (s, 3H, CH3),
7.63 (d, 2H, ar, J ¼ 8.6 Hz), 8.02 (d, 2H, ar, J ¼ 8.6 Hz), 12.78 (br s, 1H,
NH). 13C NMR: 162.53, 161.18, 157.53, 156.76, 137.32, 136.22, 131.89,
129.92 (2C), 128.77 (2C), 21.61. IR: 1690, 1595. Anal. Calc. for
C
13H13N3O3.
5.1.3.13. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)pyridine-3-
carboxamide 62. Yield 47%; m.p. >300 ꢀC (acetic acid); 1H NMR:
2.29 (s, 3H, CH3), 7.53 (s, 1H, ar), 8.27 (d, 1H, ar, J ¼ 7.4 Hz), 8.73 (s,
2H, ar), 9.09 (s, 1H, ar), 9.29 (s, 1H, NH), 12.08 (br s, 2H, 2 OH). IR:
3349, 2678, 1640. Anal. Calc. for C11H10N4O3.
C12H8ClN3OS.
5.1.4.5. 2-(4-Fluorophenyl)-5-methyl-thiazolo[5,4-d]pyrimidin-7-
one 5. Yield 76%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.41 (s, 3H,
CH3), 7.39e7.43 (m, 2H, ar), 8.05e8.07 (m, 2H, ar), 12.75 (br s, 1H,
NH). IR: 1693, 1591. Anal. Calc. for C12H8FN3OS.
5.1.3.14. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)pyridine-4-
carboxamide 63. Yield 49%; m.p. >300 ꢀC (acetic acid); 1H NMR:
2.25 (s, 3H, CH3), 7.84 (d, 2H, ar, J ¼ 5.1 Hz), 8.73 (d, 2H, ar,
J ¼ 5.1 Hz), 9.31 (s, 1H, NH), 11.98 (br s, 2H, 2 OH). IR: 3422, 3242,
2722, 1658. Anal. Calc. for C11H10N4O3.
5.1.4.6. 5-Methyl-2-(4-trifluoromethyl-phenyl)-thiazolo[5,4-d]pyr-
imidin-7-one 6. Yield 74%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.43
(s, 3H, CH3), 7.92 (d, 2H, ar, J ¼ 7.9 Hz), 8.21 (d, 2H, ar, J ¼ 7.9 Hz),
12.83 (br s, 1H, NH). IR: 1682, 1594. Anal. Calc. for C13H8F3N3OS.
5.1.3.15. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)furan-2-
carboxamide 64. Yield 48%; m.p. >300 ꢀC (acetic acid); 1H NMR:
2.27 (s, 3H, CH3), 6.64 (s, 1H, ar), 7.21 (d, 1H, ar, J ¼ 2.2 Hz), 7.85 (s,
1H, ar), 8.88 (s, 1H, NH), 12.06 (br s, 2H, 2 OH). IR: 3259, 2640, 1642.
Anal. Calc. for C10H9N3O4.
5.1.4.7. 2-(3-Methoxyphenyl)-5-methyl-thiazolo[5,4-d]pyrimidin-7-
one 9. Yield 71%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.41 (s, 3H,
CH3), 3.87 (s, 3H, OCH3), 7.14 (dd, 1H, ar, J ¼ 5.7, 2.5 Hz), 7.45e7.56
(m, 3H, ar), 12.75 (br s, 1H, NH). IR: 1688, 1596. Anal. Calc. for
C
13H11N3O2S.
5.1.3.16. N-(4,6-dihydroxy-2-methylpyrimidin-5-yl)thiophene-2-
carboxamide 65. Yield 49%; m.p. >300 ꢀC (acetic acid); 1H NMR:
2.28 (s, 3H, CH3), 7.17 (t, 1H, ar, J ¼ 7.0 Hz), 7.77 (d, 1H, ar, J ¼ 4.9 Hz),
7.91 (d, 1H, ar, J ¼ 2.9 Hz), 9.09 (s, 1H, NH), 12.17 (br s, 2H, 2 OH). IR:
3218, 2679, 1628. Anal. Calc. for C10H9N3O3S.
5.1.4.8. 5-Methyl-2-(3-methylphenyl)-thiazolo[5,4-d]pyrimidin-7-
one 10. Yield 74%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.41 (s, 6H,
CH3), 7.37e7.39 (m, 1H, ar), 7.50 (t, 1H, ar, J ¼ 7.6 Hz), 7.68e7.83 (m,
2H, ar), 12.76 (br s, 1H, NH). IR: 1669, 1595. Anal. Calc. for
C13H11N3OS.
5.1.4. General procedure for the synthesis of 2-(alkyl)aryl-5-methyl-
thiazolo[5,4-d]pyrimidin-7-one derivatives (1e6, 9e18)
5.1.4.9. 2-(3-Chlorophenyl)-5-methyl-thiazolo[5,4-d]pirimidin-7-one
11. Yield 76%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.42 (s, 3H, CH3),
7.57e7.64 (m, 2H, ar), 7.96 (d, 1H, ar, J ¼ 7.3 Hz), 8.02 (s, 1H, ar),
12.80 (br s, 1H, NH). 13C NMR: 160.70, 157.65, 156.76, 134.90, 134.56,
131.76, 131.26, 126.30 (2C), 125.84 (2C), 21.62. IR: 1681, 1590. Anal.
Calc. for C12H8ClN3OS.
A suspension of the suitable N-(4,6-dihydroxy-2-methylpyri-
midin-5-yl)carboxyamide 50e65 (3.7 mmol) and phosphorus
pentasulfide (8.5 mmol) in anhydrous pyridine (20 mL) was
refluxed for 4e6 h. The solvent was evaporated in vacuo and the
residue was treated with water (100 mL, 25 mL for compounds 15
and 16). The resulting solid was filtered off and then refluxed for 6 h
with conc HCl (0.3 mL) in NMP (8 mL). The mixture was cooled to
room temperature and added with brine (100 mL) to afford a solid
which was collected by filtration. The product was dissolved in
1.5 M NaOH (30 mL) and to this solution, mantained at room
temperature, a water solution of H2O2 (35% H2O2 0.93 mL, H2O
5 mL) was added dropwise. The mixture was stirred at room tem-
perature for 1e2 h and then acidified to pH 2e3 with 6 M HCl (to
pH 4e5 with acetic acid, for compounds 15 and 16). The resulting
solid was collected by filtration, washed with water, and
recrystallised.
5.1.4.10. 2-(3-Fluorophenyl)-5-methyl-thiazolo[5,4-d]pyrimidin-7-
one 12. Yield 78%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.42 (s, 3H,
CH3), 7.40e7.44 (m, 1H, ar), 7.59e7.64 (m, 1H, ar), 7.80e7.86 (m, 2H,
ar), 12.81 (br s, 1H, NH). IR: 1670, 1587. Anal. Calc. for C12H8FN3OS.
5.1.4.11. 5-Methyl-2-(3-trifluoromethyl-phenyl)-thiazolo[5,4-d]pyr-
imidin-7-one 13. Yield 75%; m.p. >300 ꢀC (acetic acid); 1H NMR:
2.43 (s, 3H, CH3), 7.81e7.83 (m,1H, ar), 7.93e7.95 (m,1H, ar), 8.28 (s,
2H, ar), 12.84 (br s, 1H, NH). IR: 1694, 1592. Anal. Calc. for
C13H8F3N3OS.

116
F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
5.1.4.12. 2-Benzyl-5-methyl-thiazolo[5,4-d]pyrimidin-7-one
14.
5.1.8. 2-Amino-5-Methyl-thiazolo[5,4-d]pyrimidin-7-one 67
A suspension of compound 66 (1.6 mmol) in 6 M HCl (20 mL)
was refluxed for 48 h. The solution was concentrated to half of its
original volume, and then neutralized with NaHCO3 saturated so-
lution to give a solid which was collected, washed with water, and
recrystallised. Yield 65%; m.p. >300 ꢀC (ethanol); 1H NMR: 2.28 (s,
3H, CH3), 7.38 (s, 2H, NH2), 12.27 (s, 1H, NH). IR: 3346, 3253, 3136,
1651. Anal. Calc. for C6H6N4OS.
Yield 80%; m.p. 239e241 ꢀC (methanol); 1H NMR: 2.36 (s, 3H, CH3),
4.38 (s, 2H, CH2), 7.27e7.32 (m, 1H, ar), 7.36e7.37 (m, 4H, ar), 12.64
(br s, 1H, NH). IR: 1676, 1587. Anal. Calc. for C13H11N3OS.
5.1.4.13. 5-Methyl-2-(pyridin-3-yl)-thiazolo[5,4-d]pyrimidin-7-one
15. Yield 58%; m.p. >300 ꢀC (2-methoxyethanol); 1H NMR: 2.43 (s,
3H, CH3), 7.59e7.62 (m, 1H, ar), 8.37 (d, 1H, ar, J ¼ 8.0 Hz), 8.73 (d,
1H, ar, J ¼ 4.6 Hz), 9.17 (s, 1H, ar), 12.83 (br s, 1H, NH). IR: 1701, 1586.
Anal. Calc. for C11H8N4OS.
5.1.9. 5-Methyl-2-(1H-pyrrol-1-yl)-thiazolo[5,4-d]pyrimidin-7-one
19
To a hot (90 ꢀC) solution of compound 67 (0.8 mmol) in acetic
acid (10 mL), a solution of 2,5-diethoxytetrahydrofuran (2.5 mmol)
in acetic acid (5 mL) was added dropwise. The reaction mixture was
heated at 90 ꢀC for 40 min and then cooled. The precipitate was
collected, washed with diethyl ether, and recrystallised. Yield 53%;
m.p. >300 ꢀC (acetic acid); 1H NMR: 2.17 (s, 3H, CH3), 6.41 (t, 2H, ar,
J ¼ 1.8 Hz), 7.54 (t, 2H, ar, J ¼ 1.9 Hz), 12.76 (br s, 1H, NH). IR: 1665.
Anal. Calc. for C10H8N4OS.
5.1.4.14. 5-Methyl-2-(pyridin-4-yl)-thiazolo[5,4-d]pyrimidin-7-one
16. Yield 55%; m.p. >300 ꢀC (2-methoxyethanol); 1H NMR: 2.43 (s,
3H, CH3), 7.95 (d, 2H, ar), 8.77 (d, 2H, ar), 12.87 (br s, 1H, NH). IR:
1705, 1592. Anal. Calc. for C11H8N4OS.
5.1.4.15. 2-(Furan-2-yl)-5-methyl-thiazolo[5,4-d]pyrimidin-7-one 17.
Yield 71%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.40 (s, 3H, CH3),
6.78 (s, 1H, ar), 7.28e7.29 (m, 1H, ar), 7.98 (s, 1H, ar), 12.76 (br s, 1H,
NH). IR: 1662, 1592. Anal. Calc. for C10H7N3O2S.
5.1.10. 2-(1H-Imidazol-1-yl)-5-methyl-thiazolo[5,4-d]pyrimidin-7-
one 20
5.1.4.16. 5-Methyl-2-(thiophen-2-yl)-thiazolo[5,4-d]pyrimidin-7-one
18. Yield 73%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.40 (s, 3H, CH3),
7.22 (t, 1H, ar, J ¼ 3.8 Hz), 7.77 (d, 1H, ar, J ¼ 3.8 Hz), 7.85 (d, 1H, ar,
J ¼ 4.2 Hz), 12.77 (br s, 1H, NH). 13C NMR: 162.02, 157.30, 156.53,
156.42, 136.68, 136.62, 130.96, 129.50, 129.08, 21.56. IR: 1672, 1592.
Anal. Calc. for C10H7N3OS2.
Aqueous formaldehyde (37%, 1.2 mmol), aqueous glyoxal
(40%, 1.2 mmol), and ammonium acetate (1.2 mmol) were
added to a suspension of compound 67 (1.2 mmol) in acetic
acid (7 mL). The mixture was heated at 70 ꢀC for 2 h and
then cooled to room temperature and neutralized with NaHCO3
saturated solution. The solid was collected, washed with
water, and recrystallised. Yield 72%; m.p. >300 ꢀC (2-
methoxyethanol); 1H NMR: 2.41 (s, 3H, CH3), 7.21 (s, 1H, ar),
7.92 (s, 1H, ar), 8.50 (s, 1H, ar), 12.85 (s, 1H, NH). IR: 1689, 1581.
Anal. Calc. for C9H7N5OS.
5.1.5. 2-(4-Hydroxyphenyl)-5-methyl-thiazolo[5,4-d]pyrimidin-7-
one 7
A 1 M solution of BBr3 in dichloromethane (2.9 mL) was added
to a suspension of compound 2 (0.48 mmol) in anhydrous
dichloromethane (70 mL). The resulting mixture was refluxed,
under nitrogen atmosphere, for 24 h. The suspension was added
with brine (100 mL) and stirred at room temperature for 30 min.
The organic solvent was removed under reduced pressure and the
precipitate was collected, washed with diethyl ether and recrys-
tallised. Yield 48%; m.p. >300 ꢀC (acetic acid); 1H NMR: 2.39 (s, 3H,
CH3), 6.91 (d, 2H, ar, J ¼ 8.8 Hz), 7.82 (d, 2H, ar, J ¼ 8.8 Hz), 10.21 (br
s, 1H, OH), 12.70 (br s, 1H, NH). IR: 1669, 1599. Anal. Calc. for
5.1.11. General procedure for the synthesis of N-aryl-7-chloro-5-
methyl-thiazolo[5,4-d]pyrimidin-2-amines 68e75
To a solution of 5-amino-4,6-dichloro-2-methyl-pyrimidine
(5.6 mmol) in acetonitrile (20 mL), Cs2CO3 (11.2 mmol) and the
suitable arylisothiocyanate (5.6 mmol) were added. The suspension
was stirred at 50 ꢀC until disappearance (5e30 h) of the starting
materials (TLC monitoring: eluting system ciclohexane/ethyl ace-
tate 7:3). Then, another 50 mL of acetonitrile was added and the
suspension was heated at 50 ꢀC and filtered while hot. The filtrate,
after the addition of 5 g of silica gel (70e230 mesh), was stirred at
room temperature for 20 min and filtered. The solvent was
removed in vacuum, and the residue was collected and
recrystallized.
C
12H9N3O2S.
5.1.6. 4-(5-Methyl-7-oxo-6,7-dihydro-thiazolo[5,4-d]pyrimidin-2-
yl)-benzoic acid 8
To a suspension of compound 3 (1.6 mmol) in conc H2SO4
(3.8 mL), maintained at room temperature, a solution of CrO3
(14.8 mmol) in H2O (4.2 mL) was slowly added. The reaction
mixture was stirred at room temperature for 2 h, and then was
added with H2O (100 mL). The precipitate was collected by filtra-
5.1.11.1. 7-Chloro-5-methyl-N-phenyl-thiazolo[5,4-d]pyrimidin-2-
amine 68. Reaction time 20 h. Yield 65%; m.p. 217e218 ꢀC
(ethanol); 1H NMR: 2.63 (s, 3H, CH3), 7.13 (t, 1H, ar, J ¼ 7.4 Hz), 7.43
(t, 2H, ar, J ¼ 8.2 Hz), 7.77 (d, 2H, ar, J ¼ 8.2 Hz), 11.08 (s, 1H, NH). IR:
3255, 3197, 1584, 1446. Anal. calc. for C12H9ClN4S
tion, washed with H2O and recrystallised. Yield 65%; m.p. >300 ꢀ
C
(acetic acid); 1H NMR: 2.43 (s, 3H, CH3), 8.04e8.20 (m, 4H, ar),12.82
(br s, 1H, NH), 13.25 (br s, 1H, OH). IR: 1685, 1596. Anal. Calc. for
C
13H9N3O3S.
5.1.11.2. 7-Chloro-N-(4-methoxyphenyl)-5-methyl-thiazolo[5,4-d]
pyrimidin-2-amine 69. Reaction time 30 h. Yield 75%; m.p.
208e209 dec ꢀC (ethanol); 1H NMR: 2.61 (s, 3H, CH3), 3.77 (s, 3H,
OCH3), 7.01 (d, 2H, ar, J ¼ 8.0 Hz), 7.66 (d, 2H, ar, J ¼ 8.0 Hz), 10.91 (s,
1H, NH). IR: 3256, 1511, 1463. Anal. calc. for C13H11ClN4OS.
5.1.7. N-(7-Chloro-5-methyl-thiazolo[5,4-d]pyrimidin-2-yl)
benzamide 66
The title compound was obtained by refluxing for 3 h a sus-
pension of equimolar amounts of 5-amino-4,6-dichloro-2-
methylpyrimidine (5.6 mmol) and benzoylisothiocyanate in
acetone (10 mL). The mixture was cooled to room temperature and
the solid was filtered off and recrystallised. Yield 87%; m.p.
258e260 ꢀC (ethanol); 1H NMR: 2.72 (s, 3H, CH3), 7.60 (t, 2H, ar,
J ¼ 7.4 Hz), 7.71 (t, 1H, ar, J ¼ 7.4 Hz), 8.18 (d, 2H, ar, J ¼ 7.4 Hz), 13.50
(s, 1H, NH). IR: 3182, 1666. Anal. Calc. for C13H9ClN4OS.
5.1.11.3. 7-Chloro-5-methyl-N-(4-methylphenyl)-thiazolo[5,4-d]pyr-
imidin-2-amine 70. Reaction time 30 h. Yield 48%; m.p. 212e213 ꢀC
(ethanol); 1H NMR: 2.30 (s, 3H, CH3), 2.62 (s, 3H, CH3), 7.22 (d, 2H,
ar, J ¼ 8.3 Hz), 7.63 (d, 2H, ar, J ¼ 8.3 Hz), 10.98 (s, 1H, NH). IR: 3235,
1621, 1458. Anal. calc. for C13H11ClN4S.

F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
117
5.1.11.4. 7-Chloro N-(4-chlorophenyl)-5-methyl-thiazolo[5,4-d]pyr-
imidin-2-amine 71. Reaction time 6 h. Yield 51%; m.p. 216e217 ꢀC
(ethanol); 1H NMR: 2.63 (s, 3H, CH3), 7.48 (d, 2H, ar, J ¼ 8.8 Hz), 7.80
(d, 2H, ar, J ¼ 8.8 Hz), 11.20 (s, 1H, NH). IR: 3264, 1625, 1462. Anal.
calc. for C12H8Cl2N4S.
7.75 (d, 2H, ar, J ¼ 8.9 Hz), 10.60 (s, 1H, NH), 12.53 (br s, 1H, NH). IR:
3291, 3198, 1652. Anal. calc. for C12H9ClN4OS.
5.1.12.5. 2-((3-Methoxyphenyl)amino)-5-methyl-thiazolo[5,4-d]pyr-
imidin-7-one 25. Reaction time 23 h. Yield 85%; m.p. 286e287 ꢀC
(ethanol); 1H NMR: 2.34 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 6.61 (d, 1H,
ar, J ¼ 7.9 Hz), 7.19e7.27 (m, 2H, ar), 7.42 (s,1H, ar), 10.45 (s, 1H, NH),
12.51 (br s, 1H, NH). 13C NMR: 160.32, 158.13, 155.88, 155.69, 154.36,
141.89, 134.06, 130.24, 110.54, 107.51, 104.33, 55.49, 21.25. IR: 3246,
3082, 1688. Anal. calc. for C13H12N4O2S.
5.1.11.5. 7-Chloro-N-(3-methoxyphenyl)-5-methyl-thiazolo[5,4-d]
pyrimidin-2-amine 72. Reaction time 20 h. Yield 73%; m.p.
206e207 ꢀC (ethanol); 1H NMR: 2.63 (s, 3H, CH3), 3.79 (s, 3H,
OCH3), 6.70 (dd,1H, ar, J ¼ 8.1,1.9 Hz), 7.20 (dd,1H, ar, J ¼ 8.1,1.9 Hz),
7.32 (t, 1H, ar, J ¼ 8.1 Hz), 7.59 (t, 1H, ar, J ¼ 2.0 Hz), 11.08 (s, 1H, NH).
IR: 3198, 1577, 1438. Anal. calc. for C13H11ClN4OS.
5.1.12.6. 5-Methyl-2-((3-methylphenyl)amino)-thiazolo[5,4-d]pyr-
imidin-7-one 26. Reaction time 28 h. Yield 91%; m.p. 290e291 ꢀC
(methanol); 1H NMR: 2.31 (s, 3H, CH3), 2.33 (s, 3H, CH3), 6.84 (d, 1H,
ar, J ¼ 7.5 Hz), 7.23 (t,1H, ar, J ¼ 7.8 Hz), 7.46 (s,1H, ar), 7.55 (d,1H, ar,
J ¼ 7.8 Hz), 10.37 (s, 1H, NH), 12.51 (br s, 1H, NH). 13C NMR: 158.28,
155.88, 155.63, 154.26, 140.78, 138.69, 134.10, 129.33, 123.37, 118.45,
115.29, 21.78, 21.25. IR: 3196, 3143, 1686, 1463. Anal. calc. for
5.1.11.6. 7-Chloro-5-methyl-N-(3-methylphenyl)-thiazolo[5,4-d]pyr-
imidin-2-amine 73. Reaction time 21 h. Yield 76%; m.p. 240e241
dec ꢀC (ethanol); 1H NMR: 2.34 (s, 3H, CH3), 2.62 (s, 3H, CH3), 6.95
(d, 1H, ar, J ¼ 7.2 Hz), 7.31 (t, 1H, ar, J ¼ 7.6 Hz), 7.52 (s, 1H, ar), 7.63
(d, 1H, ar, J ¼ 7.6 Hz), 11.01 (s, 1H, NH). IR: 3246, 1560, 1462. Anal.
calc. for C13H11ClN4S.
C13H12N4OS.
5.1.11.7. 7-Chloro-N-(3-chlorophenyl)-5-methyl-thiazolo[5,4-d]pyr-
imidin-2-amine 74. Reaction time 24 h. Yield 47%; m.p. 244e245
dec ꢀC (ethanol); 1H NMR: 2.64 (s, 3H, CH3), 7.17 (dd, 1H, ar, J ¼ 8.0,
1.0 Hz), 7.44 (t, 1H, ar, J ¼ 8.0 Hz), 7.60 (dd, 1H, ar, J ¼ 8.0, 1.0 Hz),
8.04 (s, 1H, ar), 11.26 (s, 1H, NH). IR: 3246, 1429, 1577. Anal. calc. for
5.1.12.7. 2-((3-Chlorophenyl)amino)-5-methyl-thiazolo[5,4-d]pyr-
imidin-7-one 27. Reaction time 30 min. Yield 93%; m.p. >300 ꢀC
(ethyl acetate); 1H NMR: 2.34 (s, 3H, CH3), 7.06 (d, 1H, ar, J ¼ 7.9 Hz),
7.37 (t, 1H, ar, J ¼ 8.0 Hz), 7.59 (d, 1H, ar, J ¼ 8.0 Hz), 8.01 (s, 1H, ar),
10.67 (br s, 1H, NH), 12.57 (br s, 1H, NH). IR: 3271, 3187, 1673. Anal.
calc. for C12H9ClN4OS.
C
12H8Cl2N4S.
5.1.11.8. 7-Chloro-N-(3,4-dichlorophenyl)-5-methyl-thiazolo[5,4-d]
5.1.12.8. 2-((3,4-Dichlorophenyl)amino)-5-methyl-thiazolo[5,4-d]
pyrimidin-7-one 28. Reaction time 30 min. Yield 81%; m.p. >300 ꢀC
(ethanol); 1H NMR: 2.34 (s, 3H, CH3), 7.53e7.61 (m, 2H, ar), 8.18 (d,
1H, ar, J ¼ 2.3 Hz), 10.79 (br s, 1H, NH), 12.60 (br s, 1H, NH). IR: 3203,
3148, 1664. Anal. calc. for C12H8Cl2N4OS.
pyrimidin-2-amine 75. Reaction time
5 h. Yield 52%; m.p.
243e244 ꢀC (ethanol); 1H NMR (DMSO-d6): 2.64 (s, 3H, CH3),
7.61e7.68 (m, 2H, ar), 8.25 (d, 1H, ar, J ¼ 2.4 Hz),11.36 (s, 1H, NH). IR:
3266, 1624, 1578, 1460. Anal. calc. for C12H7Cl3N4S.
5.1.12. General procedure for the synthesis of 2-arylamino-5-
methyl-thiazolo[5,4-d]pyrimidin-7-ones 21e28
5.1.13. General procedure for the synthesis of 2-aryl-7-chloro-5-
methyl-thiazolo[5,4-d]pyrimidine derivatives (76e78)
Sodium acetate (2.9 mmol) was added to a suspension of the
suitable N-aryl-7-chloro-5-methyl-[1,3]thiazolo[5,4-d]pyrimidin-
2-amine 68e75 (1.4 mmol) in acetic acid (10 mL). The suspension
was refluxed 18e28 h (compounds 21e22, 25e26) or microwave
irradiated at 140 ꢀC for 30e60 min (compounds 23e24, 27e28).
The mixture was cooled at room temperature, the crude product
was collected by filtration, washed with water and recrystallized.
A suspension of compounds 1, 3, 4 (5 mmol), N,N-
dimethylaniline (1.15 mL) and POCl3 (20 mL) was refluxed for 3 h.
The solvent was evaporated under pressure, and the residue was
added with brine (100 mL). The precipitate was collected and
washed with water. The crude products were unstable upon
recrystallization; however, they were pure enough to be used
without further purification.
5.1.12.1. 5-Methyl-2-(phenylamino)-thiazolo[5,4-d]pyrimidin-7-one
21. Reaction time 18 h. Yield 82%; m.p. >300 ꢀC (2-
methoxyethanol); 1H NMR: 2.34 (s, 3H, CH3), 7.02 (t, 1H, ar,
J ¼ 7.7 Hz), 7.35 (t, 2H, ar, J ¼ 8.0 Hz), 7.71 (d, 2H, ar, J ¼ 8.0), 10.45 (s,
1H, NH), 12.50 (broad s, 1H, NH). IR: 3489, 3245, 1669. Anal. calc. for
5.1.13.1. 7-Chloro-5-methyl-2-phenyl-thiazolo[5,4-d]pyrimidine 76.
Yield 92%; 1H NMR: 2.75 (s, 3H, CH3), 7.62e7.69 (m, 3H, ar),
8.15e8.16 (m, 2H, ar).
5.1.13.2. 7-Chloro-5-methyl-2-(4-methylphenyl)-thiazolo[5,4-d]py-
rimidine 77. Yield 94%; 1H NMR: 2.40 (s, 3H, CH3), 2.76 (s, 3H, CH3),
7.45 (d, 2H, ar, J ¼ 8.0 Hz), 8.04 (d, 2H, ar, J ¼ 8.0 Hz).
C
12H10N4OS.
5.1.12.2. 2-((4-Methoxyphenyl)amino)-5-methyl-thiazolo[5,4-d]pyr-
imidin-7-one 22. Reaction time 19 h. Yield 61%; m.p. >300 ꢀC
(ethanol); 1H NMR: 2.32 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 6.94 (d, 2H,
ar, J ¼ 9.0 Hz), 7.60 (d, 2H, ar, J ¼ 9.0), 10.25 (s, 1H, NH), 12.46 (br s,
1H, NH). IR: 3252, 1690. Anal. calc. for C13H12N4O2S.
5.1.13.3. 7-Chloro-2-(4-chlorophenyl)-5-methyl-thiazolo[5,4-d]py-
rimidine 78. Yield 94%; 1H NMR: 2.76 (s, 3H, CH3), 7.70 (d, 2H, ar,
J ¼ 8.6 Hz), 8.17 (d, 2H, ar, J ¼ 8.6 Hz).
5.1.14. General procedure for the synthesis of 2-aryl-7-ethoxy-5-
methyl-thiazolo[5,4-d]pyrimidine derivatives (29e31)
A suspension of compounds 76e78 (0.8 mmol), DBU (1.2 mmol)
in absolute ethanol (8 mL) was microwave irradiated at 160 ꢀC for
90 min. Evaporation of the solvent gives a solid which was
collected, washed with diethyl ether and recrystallised.
5.1.12.3. 5-Methyl-2-((4-methylphenyl)amino)-thiazolo[5,4-d]pyr-
imidin-7-one 23. Reaction time 60 min. Yield 88%; m.p. >300 ꢀC
(methanol); 1H NMR: 2.27 (s, 3H, CH3), 2.33 (s, 3H, CH3), 7.16 (d, 2H,
ar, J ¼ 8.4 Hz), 7.59 (d, 2H, ar, J ¼ 8.4 Hz), 10.35 (s, 1H, NH), 12.49 (br
s, 1H, NH). IR: 3205, 1685, 1462. Anal. calc. for C13H12N4OS.
5.1.12.4. 2-((4-Chlorophenyl)amino)-5-methyl-thiazolo[5,4-d]pyr-
imidin-7-one 24. Reaction time 30 min. Yield 80%; m.p. >300 ꢀC
(methanol); 1H NMR: 2.34 (s, 3H, CH3), 7.41 (d, 2H, ar, J ¼ 8.9 Hz),
5.1.14.1. 7-Ethoxy-5-methyl-2-phenyl-thiazolo[5,4-d]pyrimidine 29.
Yield 45%; m.p. 134e136 ꢀC (ethanol); 1H NMR: 1.46 (t, 3H, CH3,
J ¼ 7.0 Hz), 2.65 (s, 3H, CH3), 4.63 (q, 2H, CH2, J ¼ 7.0 Hz), 7.61 (m,

118
F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
3H, ar), 8.08 (m, 2H, ar). Anal. calc. for C14H13N3OS.
corresponding sequences of the TM helices of the hA3AR. The
missing loop domains were built by the loop search method
implemented in MOE. Once the heavy atoms were modeled, all
hydrogen atoms were added, and the protein coordinates were
then minimized with MOE using the AMBER99 [52] force field. The
minimizations were performed by steepest descent steps followed
by conjugate gradient minimization until the RMS gradient of the
5.1.14.2. 7-Ethoxy-5-methyl-2-(4-methylphenyl)-thiazolo[5,4-d]py-
rimidine 30. Yield 68%; m.p. 146e149 ꢀC (methanol); 1H NMR: 1.45
(t, 3H, CH3, J ¼ 7.0 Hz), 2.40 (s, 3H, CH3), 2.64 (s, 3H, CH3), 4.62 (q,
2H, CH2, J ¼ 7.0 Hz), 7.40 (d, 2H, ar, J ¼ 8.0 Hz), 7.97 (d, 2H, ar,
J ¼ 8.0 Hz). Anal. calc. for C15H15N3OS.
potential energy was less than 0.05 kJ molꢁ1
Å
ꢁ1. Reliability and
5.1.14.3. 2-(4-Chlorophenyl)-7-ethoxy-5-methyl-thiazolo[5,4-d]py-
rimidine 31. Yield 56%; m.p. 148e150 ꢀC (ethanol); 1H NMR: 1.46 (t,
3H, CH3, J ¼ 7.0 Hz), 2.64 (s, 3H, CH3), 4.61 (q, 2H, CH2, J ¼ 7.0 Hz),
7.65 (d, 2H, ar, J ¼ 8.5 Hz), 8.10 (d, 2H, ar, J ¼ 8.5 Hz). Anal. calc. for
quality of the model was checked using the Protein Geometry
Monitor application within MOE, which provides a variety of ste-
reochemical measurements for inspection of the structural quality
in a given protein, like backbone bond lengths, angles and di-
C
14H12ClN3OS.
hedrals, Ramachandran 4-j dihedral plots, and sidechain rotamer
and nonbonded contact quality.
5.1.15. 7-Ethoxy-5-methyl-N-phenyl-thiazolo[5,4-d]pyrimidin-2-
amine 32
5.2.3. Preliminary analysis with MRS 1220
A solution of the 7-chloro derivative 68 (0.6 mmol) in ethanol
(10 mL) was treated with an aqueous solution of NaOH (20%,
20 mL). The mixture was refluxed for 5 h, then cooled to room
temperature. Addition of H2O afforded a precipitate which was
collected, washed with water and recrystallised. Yield 43%; m.p.
105e106 ꢀC (ethanol); H1 NMR: 1.41 (t, 3H, CH3, J ¼ 7.0 Hz), 2.54 (s,
3H, CH3), 4.55 (q, 2H, CH2, J ¼ 7.0 Hz), 7.06 (t, 1H, ar, J ¼ 7.1 Hz), 7.39
(t, 2H, ar, J ¼ 7.4 Hz), 7.72 (d, 2H, ar, J ¼ 7.8 Hz), 10.67 (br s, 1H, NH).
Anal. calc. for C14H14N4OS.
A preliminary docking analysis was performed by manually
docking MRS 1220 structure within the hA3AR model binding site.
The obtained hA3AR-MRS 1220 complex was then subjected to
energy minimization refinement and to Monte Carlo analysis to
explore the favorable binding conformations. This analysis was
conducted by Monte Carlo Conformational Search protocol imple-
mented in Schrodinger Macromodel. The input structure consisted
of the ligand and a shell of receptor amino acids within the speci-
fied distance (6 Å) from the ligand. The coordinates of the
remaining receptor residues were kept fixed. During the Monte
Carlo conformational searching, the input structure was modified
by random changes in user-specified torsion angles (for all input
structure residues), and molecular position (for the ligand). Hence,
the ligand was left free to be continuously re-oriented within the
binding site and the conformation of both ligand and internal shell
residues could be explored and reciprocally relaxed. The method
consisted of 10,000 Conformational Search steps with MMFF94s
[53e59] force field. The best hA3AR-MRS 1220 complex was saved
and subjected to energy minimization with the same protocol as
above. This protocol was recently used to prepare hA3AR models for
docking and dynamics studies of nucleoside agonists at the same
receptor [50,60].
5.1.16. 7-Ethoxy-N-(3-methoxyphenyl)-5-methyl-thiazolo[5,4-d]
pyrimidin-2-amine 33
Sodium ethoxide (2.4 mmol) was added to a solution of the 7-
chloro derivative 72 (1.2 mmol) in absolute ethanol (5 mL). The
reaction mixture was refluxed, under nitrogen atmosphere, for 7 h
and then cooled to room temperature. The precipitate was collected
by filtration, resuspended in H2O (5 mL), and acidified to pH 4e5
with acetic acid. The solid was collected, washed with water and
recrystallised. Yield 63%; m.p. 150e151 ꢀC (ethanol); 1H NMR: 1.40
(t, 3H, CH3, J ¼ 7.1 Hz), 2.54 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 4.54 (q,
2H, CH2, J ¼ 7.1 Hz), 6.65 (dd, 1H, ar, J ¼ 8.1, 1.8 Hz), 7.21 (dd, 1H, ar,
J ¼ 8.1, 1.8 Hz), 7.29 (t, 1H, ar, J ¼ 8.1 Hz), 7.47 (s, 1H, ar), 10.67 (s, 1H,
NH). 13C NMR: 161.47, 160.36, 160.30, 158.84, 158.10, 141.63, 130.33,
129.18, 110.88, 108.18, 104.63, 62.62, 55.43, 25.67, 14.86. Anal. calc.
for C15H16N4O2S.
5.2.4. Molecular docking analysis
All compound structures were docked into the binding site of
the hA3AR model using the MOE Dock tool. This method is divided
into a number of 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 Trian-
gle Matcher placement method. Poses were generated by super-
position of ligand atom triplets and triplet points in the receptor
binding site. The receptor site points are alpha sphere centers
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 centers were used to
determine the pose. Scoring. Poses generated by the placement
methodology were scored using two available methods imple-
mented in MOE, the London dG scoring function which estimates
the free energy of binding of the ligand from a given pose, and
Affinity dG scoring which estimates the enthalpic contribution to
the free energy of binding. The top 30 poses for each ligand were
output in a MOE database.
5.2. Molecular modeling studies
5.2.1. Computational methodologies
All molecular modeling studies were performed on a 2 CPU (PIV
2.0e3.0 GHZ) Linux PC. Homology modeling, energy minimization,
and docking studies were carried out using Molecular Operating
Environment (MOE, version 2010.10) suite [44]. Manual docking
and Monte Carlo studies of the MRS 1220 binding mode were done
using MOE and Schrodinger Macromodel (ver. 8.0) [46] with
Schrodinger Maestro interface. Compounds docking analyses were
then performed with MOE. All ligand structures were optimized
using RHF/AM1 semiempirical calculations and the software
package MOPAC [47] implemented in MOE was utilized for these
calculations.
5.2.2. Homology modeling of the human A3AR
The homology model of the hA3AR was built using the X-ray
structure of the antagonist-bound hA2AAR as template (pdb code:
3EML; 2.6 Å resolution [42]). A multiple alignment of the AR pri-
mary sequences was built within MOE as preliminary step. The
sequence alignment of the four human ARs is shown within
Supplementary data. The boundaries identified from the used X-ray
crystal structure of hA2AAR were then applied for the
5.2.5. Post docking analysis
The five top-score docking poses of each compound were then
subjected to AMBER99 force field energy minimization until the
RMS gradient of the potential energy was less than

F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
119
0.05 kJ molꢁ1
Å
ꢁ1. Receptor residues within 6 Å distance from the
examined ligands for 120 min at 4 ꢀC [61]. Non-specific binding
was defined as binding in the presence of 1 M AB-MECA and was
ligand were left free to move, while the remaining receptor co-
ordinates were kept fixed. AMBER99 partial charges of receptor and
MOPAC output partial charges of ligands were utilized. Once the
compound-binding site energy minimization was completed, re-
ceptor coordinates were fixed and a second energy minimization
stage was performed leaving free to move only compound atoms.
MMFF94 force field was applied. For each compound, the mini-
mized docking poses were then rescored using London dG and Af-
finity dG scoring functions and the dock-pKi predictor. The latter tool
allows estimating the pKi for each ligand using the “scoring.svl”
script retrievable at the SVL exchange service (Chemical Computing
Group, Inc. SVL exchange: http://svl.chemcomp.com). The algo-
rithm is based on an empirical scoring function consisting of a
directional hydrogen-bonding term, a directional hydrophobic
interaction term, and an entropic term (ligand rotatable bonds
immobilized in binding). For each compound, the top-score dock-
ing poses according to at least two out of three scoring functions
were selected for final ligandetarget interaction analysis. The in-
teractions between the ligands 25 and 33 and the receptors binding
site were analyzed by using the IF-E 6.0 tool [49] retrievable at the
SVL exchange service. The program calculates and displays the
atomic and residue interaction forces as 3D vectors. It also calcu-
lates the per-residue interaction energies, where negative and
positive energy values (expressed as kcal molꢁ1) are associated to
favorable and unfavorable interactions, respectively. A shell of
residues contained within a 10 Å distance from ligand were
considered for this analysis.
m
about 20% of total binding. Bound and free radioactivity were
separated by filtering the assay mixture through Whatman GF/B
glass fiber filters using a Brandel cell harvester. The filter bound
radioactivity was counted by Scintillation Counter Packard Tri
Carb 2810 TR with an efficiency of 62%.
5.3.2. Measurement of cyclic AMP levels in CHO cells transfected
with hA2B or hA3ARs
CHO cells transfected with hAR subtypes were washed with
phosphate-buffered saline, diluted tripsine and centrifuged for
10 min at 200g. The cells (1 ꢂ 106 cells/assay) were suspended in
0.5 mL of incubation mixture (mM): NaCl 15, KCl 0.27, NaH2PO4
0.037, MgSO4 0.1, CaCl2 0.1, Hepes 0.01, MgCl2 1, glucose 0.5, pH
7.4 at 37 ꢀC, 2 IU/mL adenosine deaminase, and 4-(3-butoxy-4-
methoxybenzyl)-2-imidazolidinone (Ro 20e1724) as phosphodi-
esterase inhibitor and preincubated for 10 min in a shaking bath
at 37 ꢀC. The potency of antagonists to hA2BAR was determined
by the inhibition of NECA (200 nM)-induced cyclic AMP pro-
duction [62]. In addition, the potency of antagonists to hA3ARs
was determined in the presence of forskolin 1
mM and Cl-IB-
MECA (100 nM) that mediated inhibition of cyclic AMP levels
[61]. The reaction was terminated by the addition of cold 6%
trichloroacetic acid (TCA). The TCA suspension was centrifuged at
2000g for 10 min at 4 ꢀC, and the supernatant was extracted four
times with water saturated diethyl ether. The final aqueous so-
lution was tested for cyclic AMP levels by a competition protein
binding assay. Samples of cyclic AMP standard (0ꢁ10 pmol) were
added to each test tube containing [3H] cAMP and incubation
buffer (trizma base 0.1 M, aminophylline 8.0 mM, 2-
mercaptoethanol 6.0 mM, pH 7.4). The binding protein pre-
pared from beef adrenals was added to the samples previously
incubated at 4 ꢀC for 150 min, and, after the addition of charcoal,
was centrifuged at 2000g for 10 min. The clear supernatant was
counted in a Scintillation Counter Packard Tri Carb 2810 TR with
an efficiency of 58%.
5.3. Pharmacology
5.3.1. Human cloned A1, A2A, and A3AR receptor binding assay
All synthesized compounds were tested to evaluate their af-
finity to hA1, hA2A and hA3ARs stably expressed in CHO cells. The
cells were grown adherently and maintained in Dulbecco's
modified Eagles medium with nutrient mixture F12 (DMEM/F12)
without nucleosides, containing 10% fetal calf serum, penicillin
(100 U/mL), streptomycin (100 mg/mL), L-glutamine (2 mM) and
Geneticin (G418, 0.2 mg/mL) at 37 ꢀC in 5% CO2, 95% air. For
membrane preparation the culture medium was removed and the
cells were washed with PBS and scraped off T75 flasks in ice-cold
hypotonic buffer (5 mM Tris HCl, 2 mM EDTA, pH 7.4). The cell
suspension was homogenized with Polytron and the homogenate
was spun for 10 min at 1000ꢂg. The supernatant was then
centrifuged for 30 min at 100,000ꢂg [28]. The membrane pellet
was suspended in: a) 50 mM Tris HCl buffer pH 7.4 for A1ARs; b)
50 mM Tris HCl, 10 mM MgCl2 buffer pH 7.4 for A2AARs; c) 50 mM
Tris HCl, 10 mM MgCl2, 1 mM EDTA buffer pH 7.4 for A3ARs. The
cell suspension was incubated with 2 IU/mL of adenosine deam-
inase for 30 min at 37 ꢀC. The membrane preparation was used to
perform binding experiments. Displacement experiments of [3H]
5.3.3. Data analysis
The protein concentration was determined according to a Bio-
Rad method with bovine albumin as a standard reference [61].
Inhibitory binding constant (Ki) values were calculated from those
of IC50 according to Cheng & Prusoff equation Ki ¼ IC50/(1 þ [C*]/
KD*), where [C*] is the concentration of the radioligand and KD* its
dissociation constant [61]. A weighted non linear leastesquares
curve fitting program LIGAND was used for computer analysis of
inhibition experiments [61]. IC50 values obtained in cyclic AMP
assay were calculated by nonlinear regression analysis using the
equation for a sigmoid concentrationeresponse curve (Graph-PAD
Prism, San Diego, CA, U.S.A) [61,62].
DPCPX (1 nM) to hA1 CHO membranes (50
m
g of protein/assay)
Acknowledgments
and at least six to eight different concentrations of the compounds
for 120 min at 25 ꢀC in 50 mM Tris HCl buffer pH 7.4 were per-
formed [30]. Non-specific binding was determined in the pres-
The synthetic work was financially supported by the University
of Florence, the Italian Ministry for University and Research (MIUR,
PRIN 2010-2011, 20103779_04 project) and the Ente Cassa di Ris-
parmio di Firenze (2013.0664A2202.3926 project). The molecular
modeling work has been carried out with financial support from
the University of Camerino, Italy, and the Italian Ministry for Uni-
versity and Research, Rome, Italy.
ence of 1
m
M of DPCPX (ꢃ10% of the total binding). Binding of [3H]
ZM-241385 (1 nM) to hA2ACHO membranes (50
mg of protein/
assay) was performed using 50 mM Tris HCl buffer, 10 mM MgCl2
pH 7.4 and at least six to eight different concentrations of antag-
onists studied for an incubation time of 60 min at 4 ꢀC [30]. Non-
specific binding was determined in the presence of 1
mM ZM
241385 and was about 20% of total binding. Competition binding
Appendix A. Supplementary data
experiments to hA3CHO membranes (50 mg of protein/assay) and
0.5 nM [125I]AB-MECA, 50 mM Tris HCl buffer, 10 mM MgCl2, 1 mM
EDTA, pH 7.4 and at least six to eight different concentrations of
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.04.010.

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F. Varano et al. / European Journal of Medicinal Chemistry 96 (2015) 105e121
of novel 4-modified-2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives
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