Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists

Article abstract of DOI:10.1016/j.bmc.2018.05.048

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A₁, A_2A, A_2B and A₃ adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA₁ and hA_2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A₁/A_2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA₁ K_i = 10.2 nM; hA_2A K_i = 4.72 nM) and behaved as a potent A₁/A_2A antagonist/inverse agonist (hA₁ IC₅₀ = 13.4 nM; hA_2A IC₅₀ = 5.34 nM).

Full text of DOI:10.1016/j.bmc.2018.05.048

Accepted Manuscript
Identification of Novel Thiazolo[5,4-d]pyrimidine Derivatives as human A1 and
A2A Adenosine Receptor Antagonists/Inverse Agonists
Flavia Varano, Daniela Catarzi, Matteo Falsini, Fabrizio Vincenzi, Silvia
Pasquini, Katia Varani, Vittoria Colotta
PII:
S0968-0896(18)30541-8
https://doi.org/10.1016/j.bmc.2018.05.048
BMC 14390
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 March 2018
29 May 2018
30 May 2018
Please cite this article as: Varano, F., Catarzi, D., Falsini, M., Vincenzi, F., Pasquini, S., Varani, K., Colotta, V.,
Identification of Novel Thiazolo[5,4-d]pyrimidine Derivatives as human A1 and A2A Adenosine Receptor
Antagonists/Inverse Agonists, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.05.048
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Identification of Novel Thiazolo[5,4-d]pyrimidine Derivatives as human A1 and
A2A Adenosine Receptor Antagonists/Inverse Agonists.
Flavia Varano,a* Daniela Catarzi,a Matteo Falsini,a Fabrizio Vincenzi,b Silvia Pasquini,b
Katia Varani,b Vittoria Colottaa
a 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. b Dipartimento di Scienze Mediche, Sezione di Farmacologia, Università degli
Studi di Ferrara, via Fossato di Mortara 17-19, 44121, Ferrara, Italy.
KEYWORDS. G protein-coupled receptors; A
adenosine receptors; inverse agonists;
2A
thiazolopyrimidine derivatives, bicyclic heteroaromatic system.
Corresponding Author
*Tel: +39 055 4573732. Fax: +39 055 4573780. E-mail: flavia.varano@unifi.it.
ABBREVIATIONS
AR, adenosine receptor; cAMP, 3’,5’-cyclic adenosine monophosphate; CHO, chinese hamster
ovary;
DPCPX,
8-cyclopentyl-1,3-dipropyl-xanthine;
I-AB-MECA,
4-[((4-amino-3-
iodophenyl)methyl)amino]-5’-N-methylcarboxamidoadenosine; ZM 241385, (4-(2-[7-amino-2-(2-
furil)[1,2.4] triazolo[2,3-a][1,3,5]triazin-5-ylamino] ethyl) phenol); NECA, 5’-N-ethyl-
carboxamidoadenosine;
AB-MECA,
4-[((4-aminophenyl)methyl)amino]-5’-N-
methylcarboxamidoadenosine; TCA, trichloroacetic acid; MW, microwave.
Abstract.

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives
bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a
free amino group at position-7. The new compounds were tested for their affinity and potency at
human (h) A1, A2A, A2B and A3 adenosine receptors expressed in CHO cells. The results reveal that
the higher affinity of these new set of thiazolopyrimidines is toward the hA1 and hA2A adenosine
receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the
thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual
A1/A2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino)
group and a phenyl moiety at position 2, displayed the highest affinity (hA1 Ki = 10.2 nM; hA2A Ki
= 4.72 nM) and behaved as a potent A1/A2A antagonist/inverse agonist (hA1 IC50 = 13.4 nM; hA2A
IC50 = 5.34 nM).
1. Introduction

The nucleoside adenosine plays important roles in many pathophysiological conditions through the
modulation of cell-membrane G protein-coupled receptors known as adenosine receptors (ARs).
These receptors have been extensively characterized and subdivided in four subtypes, namely A1,
A2A, A2B and A3. The A1 and A3 ARs mediate inhibition of adenylyl cyclase, whereas adenosine
A2A and A2B ARs induce stimulation.1 ARs are considered very promising drug targets in many
pathological conditions.2-3 In particular, specific AR antagonists have gained considerable interest
for clinical development. For example, selective A1 AR antagonists have been of interest for renal
and cardiovascular disorders,4-5 while antagonism at the A2A subtype is therapeutically effective in
neurodegenerative disorders,6-7 dermal fibrosis and scarring,8-10 cancer11-12 and retinal diseases.13
The A2B antagonists are promising drug candidates for the treatment of diabetes as well as asthma
and chronic obstructive pulmonary disease (COPD),14-15 while preclinical data indicate that
selective A3 AR antagonists might be useful for glaucoma, stroke, asthma, inflammation and
cancer.16 In the last decade has emerged the idea that more effective drugs for the treatment of
neurodegenerative diseases such as Parkinson disease (PD), must influences multiples targets in a
parallel fashion. In the field of the AR ligands, dual A1 and A2A antagonists were found to be highly
active in different animal models of PD.17 Indeed, dual A1/A2A AR antagonists would not only treat
the motor symptoms of PD and potentially be neuroprotective via A2A blockade, but may also show
positive effects on the cognitive impairment associated with the disease by blocking the A1
subtypes.17
As a part of our research interest in identifying new AR ligands,18-24 we recently investigate the
thiazolo[5,4-d]pyrimidine (TP) nucleus as basis for the design of new AR antagonists (TP A, TP B,
Chart 1).25-26 Many reasons prompted us to develop this nucleus: i) the structural similarity of TP
nucleus to many bicyclic cores of known AR antagonists;6 ii) the synthetic feasibility of the TP
scaffold which has made possible to synthesize a number of products that differ in the nature of the
substituents at the C2, C5 and C7 positions of the TP core;25-26 and iii) thiazolopyrimidines have
been found to exhibit a range of biological activities particularly in the antimicrobial,27

immunosuppressive28 and pain29 areas. In addition, in a recent study, TP derivatives have been
identified as partial agonists at the A2A AR (TP C, Chart 1).30 On this basis, we explored the
structure-activity relationship (SAR) of the TP B series by introducing diverse (aryl, alkyl,
alkylaryl) amino chains at the C5 position of the TP scaffold while maintaining a 2-furanyl
Chart 1. Previously reported thiazolo[5,4-d]pyrimidine derivatives.
R6
N
O
NH2
N
N
N
N
S
N
S
HN
R5
N
N
R2
NH
O
S
N
R5
TP B
TP C
TP A
R2 = Aryl, Het, NHaryl R5 = CH3, NHaryl, NHalkylaryl, NHalkyl
R6 = H, CH2OH
NH2
7
TP455 R = 2-OCH3
R
N
S
2
KH = 3.55 fM; KL = 6.45 nM
N
5
TP400 R = 3-OCH3
KH = 5.31 fM; KL = 26 nM
O
NH
N
and a free amino group at C2 and C7 positions, respectively. This study afforded two compounds
(TP455, TP450, Chart 1) which resulted the most potent and selective A2A AR inverse agonists yet
reported.26 These compounds showed an exceptional binding behavior with two Ki affinity values
(KH and KL) the highest (KH) falling in the femtomolar range. In addition, TP455 and TP450 were
also tested in murine models of acute pain exhibiting antinociceptive activity greater than that of
morphine.26 In another study, TP455 reveals its capability to counteract the A2A AR stimulated
proliferation of different cancer cell lines.31
Due to the above encouraging results, in the present study we further explore the SARs of TP-based
AR antagonists by investigating the effect of substitutions at both the C5 and C2 positions while
maintaining a free amino group at C7 (compounds 1-18, Chart 2).

The newly synthesized derivatives can be divided into three sets according to the substituent
attached at the C2 position, as compounds bearing a phenyl (1-8), a 2-pyrazinyl (9-11) or a methyl
(12-18) group.
Chart 2. Newly synthesized thiazolo[5,4-d]pyrimidine derivatives.
R
(CH2)n
(CH2)n
1-8
NH2
7
N
S
2
N
N
R2
5
R5
S
9-11
NH
N
N
n = 0, 1, 2
R = H, 2-OCH3, 3-OCH3,
4-OCH3, 4-OH
12-18
CH3
2. Chemistry
Compounds 1-18 were prepared starting from the commercial uric acid 19, following the synthetic
procedure depicted in Scheme 1.
Thus, by reaction of the salt 2032 with benzoic or acetic anhydride the 2-phenyl or 2-methyl-
thiazolo[5,4-d]pyrimidine-5,7-diolo derivatives 2233 or 2432 were obtained, respectively. The 5,7-
diolo derivative 23, bearing at the 2-position a 2-pyrazinyl moiety, was prepared by heating in NMP
at 150 °C compound 2132 and 2-pyrazine carbonyl chloride prepared starting from the
corresponding acid.34 Treatment of the 5,7 diol derivatives 22-24 with POCl3 gave the
corresponding 5,7-dichloro compounds 25,35 26, 2733 which were reacted with 33% aqueous
solution of ammonia to afford the 7-amino-5-chloro derivatives 28-30. Reaction of these latter with
the (hetero)arylalkylamines or the arylamines of interest, under microwave irradiation at about 200
°C, provides the desired compounds 1-7, 9-18. Finally, the 5-(4-methoxyphenyl)ethylamino 7 was
hydrolyzed to its corresponding phenol 8 by treatment with BBr3 in CH2Cl2.

O
OH
N
H
N
OH
N
NH2
SH
NH2
S-
HN
a
c
N
N
NH+4
O
N
H
O
N
H
HO
HO
21
19
20
b
d
OH
N
N
N
No.
R2
No.
R5
C6H5
R2
S
HO
1
2
3
4
5
6
7
CH2C6H5
22-24
CH2(2-OCH3-C6H4)
CH2(3-OCH3-C6H4)
CH2(2-thienyl)
CH2CH2C6H5
e
22
25
28
Cl
CH2CH2(4-OCH3-
C6H4)
N
N
CH2CH2(4-OH-
C6H4)
8
R2
S
Cl
N
N
N
CH2(2-OCH3-C6H4)
CH2(3-OCH3-C6H4)
CH2(2-thienyl)
9
23
26
29
25-27
10
11
f
C6H5
12
13
14
15
16
17
18
NH2
CH2C6H5
CH2(2-OCH3-C6H4)
CH2(3-OCH3-C6H4)
CH2(2-thienyl)
CH2CH2C6H5
N
24
27
30
N
R2
CH3
S
Cl
N
28-30
CH2CH2(2-thienyl)
g
NH2
N
N
R2
S
HN
R5
N
1-6, 9-18
7
8
h
Scheme 1. Reagents and conditions: (a) [NH4]2S, KOH, H2O, 160 °C, 16h, 60% ; (b) for 22
(PhCO)2O, 170 °C, 4h, 85%, for 24 i) (CH3CO)2O, 140 °C, 1h ii) NaOH, H2O, reflux, 1h, 80%; (c)
CH3COOH, H2O, 80%; (d) 2-pyrazine carbonyl chloride, NMP, 150 °C, 18h, 75%; (e) for 25 and
27: POCl3, N, N dimethylaniline, 100 °C, 5h, 60-70%, for 26 POCl3, 170 °C MW, 40min, 70%; (f)
NH3, H2O, EtOH, reflux, 6-8h, 60-70%; (g) R5NH2, BuOH, 200 °C MW, 10-20min, 50-70%; (h)
BBr3, CH2Cl2, 40 °C, 24h, 60%.

3. Results and Discussion
All final compounds (1-18) were tested at hA1, hA2A, hA2B and hA3 ARs expressed in CHO cells.
[3H]DPCPX (A1), [3H]ZM 241385 (A2A) and [125I]AB-MECA (A3) were used in radioligand
binding assays. Because of the lack of a suitable radioligand for the A2B ARs the activity at this
receptor subtype was determined through the measurement of the inhibition of NECA-stimulated
adenylyl cyclase activity. Binding and potency data of the newly synthesized compounds (1-18) are
reported in Table 1.
Concerning the hA3 and the hA2B ARs, the greater part of the newly synthesized derivatives display
poor affinity with Ki and IC50 values above 100 nM. Compound 5, which belong to the C2 phenyl
set and bear a 5-((2-thienyl)methylamino) substituent, was the most active at both subtypes (hA3 Ki
= 112 nM, hA2B IC50 = 72 nM).
As regards the binding results at the hA1 AR, the most potent compounds (2, 3, 4, 5, 18) possess a
Ki in the range 10.2-92 nM and belong to the C2 phenyl (2, 3, 4, 5) or C2 methyl (18) set.
Analyzing the binding data at the hA2A AR it emerges that compounds 3, 4, 5 and 18 (hA2A Ki =
4.72-61 nM) are some of the most promising also at this subtype. In addition, compounds 8, 9 and
17 displayed good hA2A binding affinities with Ki values in the range 78-96 nM.
Table 1. Affinity (Ki) and potency (IC50) of the novel thiazolo[5,4-d]pyrimidines on ARs.
NH2
N
N
R2
S
N
NH
R5
1-18

Affinity values obtained from displacement of specific [3H]DPCPX [a], [3H]ZM241383 [b] or [125I]AB- MECA [c]
binding to hA1ARs, hA2AARs or A3ARs, respectively (n = 3-6). [d] Potency (IC50) in cAMP assays to hA2BARs.
Percentage of inhibition (I%) is determined at 10 µM concentration of the tested compounds. Data are expressed as
means SEM.
R2
R5
hA1AR[a]
Ki (nM)
(I%)
hA2AAR[b]
Ki (nM)
(I%)
hA3AR[c]
Ki (nM)
(I%)
hA2BAR[d]
IC50 (nM)
(I%)
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
>10000
(8%)
>10000
(1%)
>10000
(1%)
>10000
(1%)
1
CH2C6H5
37
4
116 10
275 24
358 31
2
CH2(2-OCH3-C6H4)
CH2(3-OCH3-C6H4)
CH2(2-thienyl)
CH2CH2C6H5
10.2 1.1
4.72 0.46
692 67
2622 224
2387 243
3
32
43
3
42
28
4
3
>10000
(25%)
4
4
112 10
72
6
5
121 13
485 37
242 19
112 10
406 38
403 37
2274 217
3529 315
1612 153
725 67
4380 429
6
CH2CH2(4-OCH3-
C6H4)
>10000
(33%)
7
8
CH2CH2(4-OH-C6H4)
96
79
8
7524 537
Pyrazin- CH2(2-OCH3-C6H4)
2-yl
7
4017 385
9
Pyrazin- CH2(3-OCH3-C6H4)
2-yl
10
11
12
13
14
15
16
17
18
253 23
338 31
973 82
219 18
749 58
165 13
5013 423
784 62
Pyrazin- CH2(2-thienyl))
2-yl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
547 42
333 28
281 26
195 18
531 44
140 12
608 51
532 47
135 11
212 17
313 28
446 39
1912 176
782 68
667 56
780 72
548 44
1519 146
3725 347
1810 168
5538 412
3516 287
2072 176
1105 97
134 11
CH2C6H5
CH2(2-OCH3-C6H4)
CH2(3-OCH3-C6H4)
CH2(2-thienyl))
CH2CH2C6H5
CH2CH2(2-thienyl)
78
61
7
6
92
9

In the C2 phenyl set (1-8) binding affinity at hA1 and hA2A ARs decreases in the following order 5-
phenyl-methylamino 2 > 5-phenyl-ethylamino 6 > 5-phenyl-amino 1. These results indicated that
the 5-methylamino group is the optimal linker between the TP core and the appended (hetero)aryl
ring. Moreover, the presence of the 5-((2-methoxyphenyl)methylamino) group, the same present in
TP455, afforded the most active compound (e.g compound 3: hA1 Ki = 10.2 nM, hA2A Ki = 4.72
nM) among the herein reported TP derivatives. To note that the hA2A affinity value of compound 3
(hA2A Ki = 4.72 nM) is comparable to the KL affinity value of TP455 (hA2A KL = 6.45 nM).
Furthermore, among the 5-phenyl-ethylamino derivatives 6-8, compound 8 bearing an hydroxyl
function in para position, showed the highest affinity at the A2A AR (Ki = 96 nM). In fact, the para
hydroxyl derivative 8 is about 4-fold more active than its precursor para-methoxy derivative 7
which in turn has the same affinity as the 5-phenyl-ethylamino unsubstituted 6.
The 2-(2-pyrazinyl) derivatives 9-11 were synthesized as isosters of the corresponding C2 phenyl
derivatives 3-5. Comparison of the binding data of the two subseries clearly indicated that the
substitution of the phenyl ring with a 2-pyrazinyl one is deleterious for the anchoring at the A1 ad
A2A AR subtypes. However, it is interesting to remark that compound 9, bearing a 5-((2-
methoxyphenyl)methylamino) substituent, resulted the most active also among this subseries, thus
confirming the beneficial effect of this 5-substituent for good hA1 and hA2A binding affinity.
In contrast, in the C2 methyl set the highest affinity at the hA1 and hA2A subtypes was observed in
the 5-ethylamino substituted derivatives 17 and 18 which were, respectively, about 7 and 5 fold
more active than their corresponding 5-methylamino derivatives 13 and 16. This behavior can be
explained assuming that the decreased steric hindrance at the C2 position due to the presence of a
methyl group is compensated by the increased bulk of the C5 substituent. However, also in the C2
methyl set the 5-((2-methoxyphenyl)methylamino) substituted derivative 14 exhibits the higher

affinity with respect to the other 5-methylamino substituted of the same set (compare 14 vs 13, 15,
16).
Compounds 3, 4, 5 and 18, the most interesting in terms of affinity at the hA1 and hA2A ARs, were
also evaluated for their antagonist/inverse agonist behavior toward these receptor subtypes (Table 2
and 3).
Table 2. Modulation of Forskolin-stimulated cAMP, potency (IC50) and efficacy (Emax) of selected
compounds on cyclic AMP assays in hA1 CHO cells.a
% of Forskolin-stimulated
cAMP levelsb
IC50 (nM)c
13.4 11.1
37 3
Emax (%)d
100 3
99 4
3
4
102 5
98 3
101 4
100 5
51 5
97 4
5
107 9
98 5
18
aData are expressed as means SEM. bCapability of selected compounds to modulate Forskolin-
stimulated cAMP levels (100%). Potencyc and efficacyd of the novel compounds to antagonize
CCPA (1 nM) inhibition of Forskolin-stimulated cAMP levels.
Table 3. Potency (IC50) and efficacy (Emax) of selected compounds on cyclic AMP assays in hA2A
CHO cells.a
IC50 (nM)b
5.34 0.47
48 4
Emax (%)c
55 4
IC50 (nM)d
7.63 0.61
66 6
Emax (%)e
134 13
128 11
131 11
124 12
3
4
52 4
36 4
64 5
43 4
5
83 7
58 5
102 9
18
aData are expressed as means SEM. Potency (b, d) and efficacy (c, e) of selected compounds in the
absence (b, c) or in the presence (d, e) of CGS 21680 (10 nM), respectively.
In A1 AR CHO cells, compounds 3, 4, 5 and 18 were not able to modulate Forskolin-stimulated
cAMP levels suggesting that they did not behave as agonists or inverse agonists. However, they

completely counteracted hA1 AR agonist CCPA inhibition of Forskolin-stimulated cAMP levels
behaving as antagonists (Table 2).
In A2A AR CHO cells, the selected compounds behaved as inverse agonists being able to inhibit
basal cAMP accumulation at nanomolar concentration (Table 3). Furthermore, they were able to
inhibit cAMP accumulation stimulated by the selective hA2A AR agonist CGS 21680 reaching
values lower than those of basal production as indicated by efficacy data (Table 3).
Figure 1 reports the effect of compound 3, the most interesting one, on cAMP assays in hA2A CHO
cells in the absence or in the presence of CGA 21680.
Figure 1. Inhibition of cAMP levels in hA2A CHO cells by compound 3 in the absence (A) or in the
presence (B) of CGS 21680 (10 nM). Data represent means SEM of four experiments each performed
in triplicate.
A
B
120
100
120
100
80
60
40
20
0
80
60
40
20
0
-11 -10 -9
-8
-7
-6
-5
-4
-20
-40
log [3] (M)
-12 -11 -10 -9 -8 -7 -6 -5 -4
log [3] (M)
In conclusion, this study allowed to deepen the SARs of the thiazolo[5,4-d]pyrimidine series as AR
antagonists/inverse agonists. In particular, the herein reported results reveal that the higher affinity
is toward the hA1 and hA2A AR subtypes and is tuned by the substitution pattern at both the C2 and
C5 positions. In fact, it emerges that in all subseries which differ for the substituent at C2 position,
the presence of a 5-((2-methoxyphenyl) methylamino) group affords good affinity for hA1 and A2A

ARs. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and phenyl moiety at
position 2, has shown the best affinity values for both the A1 and A2A ARs. Functional studies
evidenced that the herein reported compounds are dual A1/A2A AR antagonists/inverse agonists.
and thus could be useful for the therapeutic treatment of neurological disorders.
4. Experimental section
4.1.Chemistry
All the commercial available reagents and solvents were used as purchased from Sigma Aldrich
(Italy), without further purification. The microwave-assisted synthesis were performed using an
Initiator EXP Microwave Biotage instrument (frequency of irradiation: 2.45 GHz). Analytical silica
gel plates (0.20 mm, F254, Merck, Germany), preparative silica gel plates (2 mm, F254, Merck,
Germany) and silica gel 60 (70-230 mesh, Merck, Germany) were used for analytical and
preparative TLC, and for column chromatography, respectively. Melting points were determined in
glass capillary tubes on a Gallenkamp melting point apparatus and are uncorrected. Compounds
were named following IUPAC rules as applied by ACD/ChemSketch. Elemental analyses were
performed with a Flash E1112 Thermofinnigan elemental analyzer for C, H, N and the results are
within 0.4% of the theoretical values. The IR spectra were recorded with a Perkin-Elmer Spectrum
RX I spectrometer in Nujol mulls and data are expressed in cm-1. Proton nuclear magnetic
resonance (1H NMR) experiments were run on Bruker Avance 400 instrument at 400 MHz.Spectra
were recorded at 300 K, using DMSO-d6 as solvent. Chemical shifts were recorded in parts per
million using the residual non-deuterated solvent as the internal standard. Data are reported as
follows: chemical shift δ (ppm), multiplicity (indicated as: s, singlet; br s, broad singlet; d, doublet;
t, triplet; q, quartet; m, multiplet and combination thereof), integrated intensity, coupling costants
(J) in Hertz (Hz). Compounds 20,32 21,32 22,33 24,32 25,35 and 2733 were synthesized following the
procedures reported in the literature.

4.1.1. 2-(Pyrazin-2-yl)-thiazolo[5,4-d]pyrimidine-5,7-diol (23). To a suspension of compound 2132
(3 mmol) in anhydrous NMP (6 mL), 2-pyrazine carbonyl chloride34 (3.5 mmol) was added. The
mixture was refluxed under N2 atmosphere for 18h. After cooling the reaction mixture was diluted
with water (about 100 mL) affording a solid residue which was collected by filtration and purified
by crystallization. Yield: 70 %. Mp > 300 °C (ethylene glycol monomethyl ether). 1H NMR: δ 8.70-
8.71 (m, 1H, ar), 8.75-8.76 (m, 1H, ar), 9.27 (s, 1H, ar), 11.47 (s, 1H, OH), 12.21 (br s, 1H, OH).
Anal calcd. for (C9H5N5O2S): C, 43.72%; H, 2.04%; N, 28.33%. Anal. found: C, 44.05%; H,
2.25%; N, 28.51%.
4.1.2. 5,7-Dichloro-2-(pyrazin-2-yl)-thiazolo[5,4-d]pyrimidine (26). A suspension of the 5,7-diolo
derivative 23 (4.2 mmol) in POCl3 (10 mL) was microwave irradiated at 170°C for 40min. The
organics were evaporated under reduced pressure, then the residue was treated with ice-water (about
30g) affording a precipitate which was collected by filtration and used in the next step without
1
further purication. Yield: 70 %. H NMR: δ 8.89-8.90 (m, 1H, ar), 8.97-8.98 (m, 1H, ar), 9.54 (s,
1H, ar).
4.1.3. General procedure for the preparation of the 5-chloro-thiazolo[5,4-d]pyrimidine-7-amine
derivatives 28-30. To a suspension of the 5,7-dichloro derivatives 25-27 (4 mmol) in ethanol (10
mL), aqueous ammonia solution (33%, 15 mL) was added, and the resulting mixture was refluxed
for 6-8h. The precipitate was collected by filtration and purified by crystallization.
4.1.3.1. 5-Chloro-2-phenyl-thiazolo[5,4-d]pyrimidin-7-amine (28). Yield: 70 %. Mp 288-290 °C
1
(nitromethane). H NMR: δ 7.60 (m, 3H, ar), 8.06-8.07 (m, 2H, ar), 8.20-8.30 (br s, 2H, NH2). IR:
3124, 3278, 3467. Anal calcd. for (C11H7ClN4S): C, 50.29%; H, 2.69%; N, 21.33%. Anal. found: C,
50.44%; H, 2.72%; N, 21.47%.
4.1.3.2. 5-Chloro-2-(pyrazine-2yl)-thiazolo[5,4-d]pyrimidin-7-amine (29). Yield: 65 %. Mp > 300
°C (acetic acid). 1H NMR: δ 8.41 (br s, 2H, NH2), 8.77-8.78 (m, 1H, ar), 8.81-8.82 (m, 1H, ar), 9.43

(s, 1H, ar). Anal calcd. for (C9H5ClN6S): C, 40.84%; H, 1.90%; N, 31.75%. Anal. found: C,
41.11%; H, 2.22%; N, 31.88%.
4.1.3.3. 5-Chloro-2-methyl-thiazolo[5,4-d]pyrimidin-7-amine (30). Yield: 75 %. Mp 235-236 °C
1
dec. (ethanol). H NMR: δ 2.77 (s, 3H, CH3), 8.10 (br s, 2H, NH2). IR: 3136, 3296, 3359. Anal
calcd. for (C6H5ClN4S): C, 35.92%; H, 2.51%; N, 27.92%. Anal. found: C, 36.07%; H, 2.63%; N,
28.12%.
4.1.4. General procedure for the preparation of the thiazolo[5,4-d]pyrimidine-5,7-diamine
derivatives 1-18
The title compounds were prepared by reacting the 5-chloro-7-amino derivatives 28-30 (1 mmol)
and the suitable amines (R5NH2, 3 mmol) in n-BuOH (2 mL). The reaction mixture was microwave
irradiated at 200 °C for 10-20 min. The suspension was basified with aqueous KOH solution (50%),
and then diluted with H2O (about 100 mL) to afford a solid which was filtered and washed with
Et2O. The crude products were purified by chromatography and/or crystallization as specified
below.
4.1.4.1. N5, 2-diphenyl-thiazolo[5,4-d]pyrimidine-5,7-diamine (1). Yield: 80 %. Mp 245-248 °C
1
(ethanol). H NMR: δ 6.92 (t, 1H, ar, J = 7.2), 7.26 (t, 2H, ar, J = 7.8), 7.47-7.56 (m, 5H, 3ar +
NH2), 7.81 (d, 2H, ar, J = 7.8), 7.99 (d, 2H, ar, J = 6.6), 9.34 (s, 1H, NH). Anal calcd. for
(C17H13N5S): C, 63.93%; H, 4.10%; N, 21.93%. Anal. found: C, 64.71%; H, 4.27%; N, 22.10%.
4.1.4.2. N5-Benzyl-2-phenyl-thiazolo[5,4-d]pyrimidine-5,7-diamine (2). Yield: 85%. Mp 207-210
1
°C (ethanol). H NMR: δ 4.51 (d, 2H, J = 6.2), 7.19-7.21 (m, 2H, ar), 7.28-7.32 (m, 5H, 3 ar +
NH2), 7.50-7.52 (m, 4H, 3ar + NH), 7.92-7.94 (m, 2H, ar). IR: 3173, 3252, 3441. Anal calcd. for
(C18H15N5S): C, 64.84%; H, 4.53%; N, 21.01%. Anal. found: C, 64.63%; H, 4.39%; N, 21.15%.
4.1.4.3. N5-(2-Methoxybenzyl)-2-phenyl-thiazolo[5,4-d]pyrimidine-5,7-diamine (3). Yield: 70%.
Mp 201-204 °C (column chromatography, eluting system ethyl acetate/cyclohexane 4/6 ). 1H NMR:

δ 3.83 (s, 3H, OCH3), 4.49 (d, 2H, CH2, J = 6.0), 6.88(t, 1H, ar, J = 7.3), 6.97 (d, 1H, ar, J = 8.0),
7.18-7.22 (m, 4H, 2 ar + NH2), 7.47-7.53 (m, 4H, 3ar + NH), 7.93 (d, 2H, ar, J = 6.8). IR: 3068,
3321, 3373. Anal calcd. for (C19H17N5OS): C, 62.79%; H, 4.71%; N, 19.27%. Anal. found: C,
62.56%; H, 4.94%; N, 19.03%.
4.1.4.4. N5-(3-Methoxybenzyl)-2-phenyl-thiazolo[5,4-d]pyrimidine-5,7-diamine (4). Yield: 60%.
Mp 169-171 °C (column chromatography, eluting system ethyl acetate/cyclohexane 3/7 ). 1H NMR:
δ 3.72 (s, 3H, OCH3), 4.48 (d, 2H, CH2, J = 6.1), 6.77 (dd, 1H, ar, J = 7.4, 2.0), 6.88-6.90 (m, 2H,
ar), 7.19-7.23 (m, 3H, 1ar + NH2), 7.38 (br s, 1H, NH), 7.47-7.53 (m, 3H, ar), 7.93 (d, 2H, ar, J =
7.6). Anal calcd. for (C19H17N5OS): C, 62.79%; H, 4.71%; N, 19.27%. Anal. found: C, 62.90%; H,
4.85%; N, 19.41%.
4.1.4.5. 2-Phenyl-N5-(thiophen-2-ylmethyl)-thiazolo[5,4-d]pyrimidine-5,7-diamine (5). Yield: 72%.
Mp 186-188 °C (column chromatography, eluting system ethyl acetate/cyclohexane 3/7 ). 1H NMR:
δ 4.64 (d, 2H, CH2, J = 6.2), 6.94-6.95 (m, 1H, ar), 7.00-7.01 (m, 1H, ar), 7.25 (br s, 2H, NH2), 7.33
(d, 1H, ar, J = 4.9), 7.43 (br s, 1H, NH), 7.48-7.54 (m, 3H, ar), 7.94-7.95 (m, 2H, ar). IR: 3186.
3255. 3400. Anal calcd. for (C16H13N5S2): C, 56.61%; H, 3.86%; N, 20.63%. Anal. found: C,
56.36%; H, 4.10%; N, 20.52%.
4.1.4.6. 2-Phenyl-N5-(2-phenylethyl)-thiazolo[5,4-d]pyrimidine-5,7-diamine (6). Yield: 75%. Mp
1
180-182 °C (n-butanol). H NMR: δ 2.85 (t, 2H, CH2, J = 7.2), 3.46-3.51 (m, 2H, CH2), 6.87 (br s,
1H, NH), 7.18-7.32 (m, 7H, 5 ar + NH2), 7.46-7.54 (m, 3H, ar), 7.93-7.95 (m, 2H, ar). IR: 3147,
3266. Anal calcd. for (C19H17N5S): C, 65.68%; H, 4.93%; N, 20.16%. Anal. found: C, 65.87%; H,
5.09%; N, 20.18%.
4.1.4.7. N5-[2-(4-Methoxyphenyl)ethyl]2-phenyl-thiazolo[5,4-d]pyrimidine-5,7-diamine (7). Yield:
67%. Mp 186-187 °C (ethanol). 1H NMR: δ 2.78 (t, 2H, CH2, J = 7.1), 3.42-3.47 (m, 2H, CH2), 3.72
(s, 3H, CH3), 6.81-6.87 (m, 3H, 2ar + NH), 7.16-7.19 (m, 4H, 2 ar + NH2), 7.46-7.54 (m, 3H, ar),

7.94 (d, 2H, ar, J = 7.2). IR: 3198, 3268, 3335, 3471. Anal calcd. for (C20H19N5OS): C, 63.64%; H,
5.07%; N, 18.55%. Anal. found: C, 63.48%; H, 4.97%; N, 18.44%.
4.1.4.8. N5-(2-Methoxybenzyl)-2-(pyrazin-2-yl)-thiazolo[5,4-d]pyrimidine-5,7-diamine (9). Yield:
30%. Mp 276-278 °C (acetic acid). 1H NMR: δ 3.83 (s, 3H, CH3), 4.50 (d, 2H, CH2, J = 5.6), 6.88
(t, 1H, ar, J = 7.5), 6.97 (d, 1H, ar, J = 8.0), 7.19-7.23 (m, 2H, ar), 7.31 (br s, 1H, NH), 7.39 (br s,
2H, NH2), 8.68-8.69 (m, 2H, ar), 9.33 (s, 1H, ar). Anal calcd. for (C17H15N7OS): C, 55.88%; H,
4.14%; N, 26.83%. Anal. found: C, 56.01%; H, 4.32%; N, 26.71%.
4.1.4.9. N5-(3-Methoxybenzyl)-2-(pyrazin-2-yl)-thiazolo[5,4-d]pyrimidine-5,7-diamine (10). Yield:
40%. Mp 207-209 °C (acetic acid). 1H NMR: δ 3.73 (s, 3H, CH3), 4.50 (d, 2H, CH2, J = 6.1), 6.78
(d, 1H, ar, J = 7.4), 6.89-6.91 (m, 2H, ar), 7.22 (t, 1H, ar, J = 8.0), 7.41 (br s, 2H, NH2), 7.55 (br s,
1H, NH), 8.69-8.70 (m, 2H, ar), 9.34 (s, 1H, ar). Anal calcd. for (C17H15N7OS): C, 55.88%; H,
4.14%; N, 26.83%. Anal. found: C, 55.94%; H, 4.07%; N, 27.00%.
4.1.4.10. 2-(Pyrazin-2-yl)-N5-(thiophen-2-ylmethyl)-thiazolo[5,4-d]pyrimidine-5,7-diamine (11).
Yield: 45 %. Mp 250-252 °C (acetic acid). 1H NMR: δ 4.67 (d, 2H, CH2, J = 6.2), 6.95 (t, 1H, ar, J
= 4.1), 7.00-7.01 (m, 1H, ar), 7.33 (d, 1H, ar, J = 4.9), 7.42 (br s, 2H, NH2), 7.57 (br s, 1H, NH),
8.70-8.71 (m, 2H, ar), 9.35 (s, 1H, ar). Anal calcd. for (C14H11N7S2): C, 49.25%; H, 3.25%; N,
28.72%. Anal. found: C, 48.93%; H, 3.37%; N, 29.07%.
4.1.4.11. 2-Methyl-N5-phenyl-thiazolo[5,4-d]pyrimidine-5,7-diamine (12). Yield: 70%. Mp 214-215
°C (ethanol). 1H NMR: δ 2.68 (s, 3H, CH3), 6.89 (t 1H, ar, J = 7.3), 7.22-7.27 (m, 4H, 2 ar + NH2),
7.78 (d, 2H, ar, J = 7.7), 9.14 (s, 1H, NH). IR: 3176, 3247, 3425. Anal calcd. for (C12H11N5S): C,
56.01%; H, 4.31%; N, 27.22%. Anal. found: C, 56.35%; H, 4.50%; N, 27.31%.
4.1.4.12. N5-Benzyl-2-methyl-thiazolo[5,4-d]pyrimidine-5,7-diamine (13). Yield: 80%. Mp 170-172
°C (isopropanol). 1H NMR: δ 2.68 (s, 3H, CH3) 4.47 (d, 2H, CH2, J = 6.0), 7.00 (s, 2H, NH2), 7.13-

7.21 (m, 2H, 1 ar + NH), 7.27-7.30 (m, 4H, ar). IR: 3186, 3326, 3425. Anal calcd. for (C13H13N5S):
C, 57.54%; H, 4.83%; N, 25.81%. Anal. found: C, 57.62%; H, 4.89%; N, 25.98%.
4.1.4.13. N5-(2-Methoxybenzyl)-2-methyl-thiazolo[5,4-d]pyrimidine-5,7-diamine (14). Yield: 60%.
Mp 215-217 °C (preparative TLC eluting system ethyl acetate/cyclohexane 6/4) 1H NMR: δ 2.61 (s,
3H, CH3), 3.82 (s, 3H, OCH3), 4.44 (d, 2H, CH2, J = 6.2), 6.84-6.88 (m, 2H, ar), 6.95-7.00 (m, 3H, 1
ar + NH2), 7.16-7.22 (m, 2H, 1 ar + NH). Anal calcd. for (C14H15N5OS): C, 55.80%; H, 5.02%; N,
23.24%. Anal. found: C, 55.61%; H, 4.96%; N, 23.15%.
4.1.4.14. N5-(3-Methoxybenzyl)-2-methyl-thiazolo[5,4-d]pyrimidine-5,7-diamine (15). Yield: 65%.
Mp 168-170 °C (preparative TLC eluting system ethyl acetate/cyclohexane 6/4) 1H NMR: δ 2.61 (s,
3H, CH3), 3.71 (s, 3H, OCH3), 4.44 (d, 2H, CH2, J = 6.4), 6.75-6.78 (m, 1H, ar), 6.86-6.88 (m, 2H,
ar), 7.01 (s, 2H, NH2), 7.10 (t, 1H, ar, J = 7.0), 7.12 (t, 1H, NH, J = 6.4). Anal calcd. for
(C14H15N5OS): C, 55.80%; H, 5.02%; N, 23.24%. Anal. found: C, 55.86%; H, 5.13%; N, 23.19%.
4.1.4.15. 2-Methyl-N5-(thiophen-2ylmethyl)-thiazolo[5,4-d]pyrimidine-5,7-diamine (16). Yield:
1
50%. Mp 168-170 °C (preparative TLC eluting system ethyl acetate/cyclohexane 1/1) H NMR: δ
2.63 (s, 3H, CH3), 4.60 (d, 2H, CH2, J = 6.4), 6.91-6.93 (m, 2H, ar), 7.04 (br s, 2H, NH2), 7.18 (t,
1H, NH, J = 6.4), 7.30-7.31 (m, 1H, ar). IR: 3188, 3312. Anal calcd. for (C11H11N5S2): C, 47.63%;
H, 4.00%; N, 25.25%. Anal. found: C, 47.94%; H, 4.21%; N, 25.37%.
4.1.4.16. 2-Methyl-N5-(2-phenylethyl)-thiazolo[5,4-d]pyrimidine-5,7-diamine (17). Yield: 76%. Mp
130-133 °C (n-butanol). 1H NMR: δ 2.62 (s, 3H, CH3), 2.83 (t, 2H, CH2, J = 7.0), 3.42-3.47 (m, 2H,
CH2), 6.62 (br s, 1H, NH), 6.97 (br s, 2H, NH2), 7.19-7.21 (m, 1H, ar), 7.24-7.31 (m, 4H, ar). IR:
3187, 3251, 3315, 3360. Anal calcd. for (C14H15N5S): C, 58.92%; H, 5.30%; N, 24.54%. Anal.
found: C, 59.05%; H, 5.46%; N, 24.71%.
4.1.4.17. 2-Methyl-N5-[2-(thiophen-2-yl)ethyl]-thiazolo[5,4-d]pyrimidine-5,7-diamine (18). Yield:
1
30 %. Mp 143-145 °C (ethyl acetate/cyclohexane). H NMR: δ 2.62 (s, 3H, CH3), 3.04 (t, 2H, CH2,

J= 7.2), 3.48 (dd, 2H, CH2, J= 13.4, 7.2), 6.69 (br s, 1H, NH), 6.90-6.99 (m, 4H, 2 ar + NH2), 7.31-
7.32 (m, 1H, ar). Anal calcd. for (C12H13N5S2): C, 49.46%; H, 4.50%; N, 24.03%. Anal. found: C,
49.29%; H, 4.61%; N, 23.88%.
4.1.5. 4-[2-(7-Amino-2-phenyl-thiazolo[5,4-d]pyrimidin-5-yl)amino]ethyl]phenol (8). A solution of
BBr3 in CH2Cl2 (1 M, 6 mL) was portion-wise added to a suspension of compound 7 (2 mmol) in
anhydrous CH2Cl2 (40 mL). The suspension was stirred at 50 °C for one day and then was diluted
with ice-water (about 90 g) and vigorously stirred for 4h. After addition of a saturated solution of
NaHCO3 (10 mL), the organic phase was collected, washed with water (20 mL x 2) and dried
(Na2SO4). Evaporation at reduced pressure of the solvent afforded a solid which was purified by
1
crystallization. Yield: 60%. Mp 205-207 °C (methanol). H NMR: δ 2.74 (t, 2H, CH2, J = 7.0),
3.40-3.45 (m, 2H, CH2), 6.69 (d, 2H, ar, J = 8.0), 6.79 (br s, 1H, NH), 7.05 (d, 2H, ar, J = 8.0), 7.15
(br s, 2H, NH2), 7.46-7.54 (m, 3H, ar), 7.94 (d, 2H, ar, J = 8.2), 9.15 (s, 1H, OH). IR: 3268, 3335,
3471. Anal calcd. for (C19H17N5OS): C, 62.79%; H, 4.71%; N, 19.27%. Anal. found: C, 62.85%; H,
4.77%; N, 19.51%.
4.2. In vitro pharmacology.
4.2.1. Materials. [3H]-DPCPX ([3H]1,3-dipropyl-8-cyclopentyl-xanthine; specific activity, 120
Ci/mmol) and [125I]-ABMECA ([125I]4-aminobenzyl-5'-N-methyl-carboxamidoadenosine; specific
activity, 2200 Ci/mmol) were obtained from Perkin Elmer Research Products (Boston, MA); [3H]-
ZM 241385 ([3H](4-(2-[7-amino-2-(2-furil)[1,2.4] triazolo[2,3-a][1,3,5]triazin-5-ylamino] ethyl)
phenol); specific activity, 17 Ci/mmol) was obtained from Biotrend (Cologne, Germany). DPCPX,
NECA (N-ethylcarboxamido adenosine), AB-MECA, CGS 21680 (2-p(2-carboxyethyl)
phenethylamino-5’-N-ethylcarboxamido
adenosine),
Forskolin,
CCPA
(2-Chloro-N6-
cyclopentyladenosine) and ZM 241385 were obtained from Sigma Aldrich (St. Louis, MO).

4.2.2. Cell culture and membrane preparation. Chinese Hamster Ovary (CHO) cells transfected
with hA1, hA2A, hA2B and hA3 ARs were grown adherently and maintained in Dulbecco’s modified
Eagle’s medium with nutrient mixture F12, containing 10% fetal calf serum, penicillin (100 U/ml),
streptomycin (100 µg/ml), l-glutamine (2 mM), geneticine (G418; 0.2 mg/ml) at 37°C in 5%
CO /95% air until the use in cAMP assays.26 For membrane preparation the culture medium was
2
removed, and the cells were washed with phosphate-buffered saline and scraped off T75 flasks in
ice-cold hypotonic buffer (5 mM TrisHCl,1 mM EDTA, pH 7.4). The cell suspension was
homogenized with a Polytron, centrifuged for 30min at 40000 g at 4°C and the resulting membrane
pellet was used for competition binding experiments.26
4.2.3. Competition binding experiments. All synthesized compounds were tested for their affinity to
hA1, hA2A and hA3 ARs. Competition experiments to A1 ARs were carried out incubating 1 nM [3H]-
DPCPX with membrane suspension (50 µg of protein/100 µl) and different concentrations of the
examined compounds at 25°C for 90 min in 50 mMTrisHCl, pH 7.4. Non-specific binding was
defined as binding in the presence of 1 µM DPCPX and was always < 10% of the total binding.36
Inhibition experiments to A2A ARs were performed incubating the radioligand [3H]-ZM 241385 (1
nM) with the membrane suspension (50 µg of protein/100 µl) and different concentrations of the
examined compounds for 60 min at 4°C in 50mMTrisHCl (pH 7.4), 10 mM MgCl2. Non-specific
binding was determined in the presence of ZM 241385 (1 µM ) and was about 20% of the total
binding.26 Competition binding experiments to A ARs were carried out incubating the membrane
3
suspension (50 µg of protein/100 µl) with 0.5 nM [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
TrisHCl (pH 7.4), 10 mM MgCl2, 1 mM EDTA. Non-specific binding was defined as binding in the
presence of 1 µM ABMECA and was always < 10% of the total binding.37 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).

4.2.4. Cyclic AMP assays. The potency of the examined compounds to hA2B ARs were determined
evaluating their capability to inhibit NECA (100 nM)-stimulated cAMP levels. In hA1 ARs CHO
cells, different concentrations of the selected compounds were incubated with Forskolin (5 µM) in
the presence or in the absence of A1 agonist CCPA (1 nM) in order to evaluate their functional
behavior. Moreover, the potency of the compounds versus hA2AARs was determined studying their
capability to inhibit basal levels of cAMP. The same experiments were also performed in the
presence of CGS 21680 (10 nM). cAMP levels were then quantified by using the AlphaScreen
cAMP Detection Kit (Perkin Elmer, Boston, USA) following the manufacturer's instructions.38
Briefly, compounds were added to cell suspension in a half-area 96 well plate in the presence of
anti-cAMP acceptor beads and incubated for 30 min. Then, streptadvidin coated donor beads and
biotinylated cAMP were added and incubated for 1 hour. At the end of the experiments, plates were
read with the Perkin Elmer EnSight Multimode Plate Reader.
4.2.5. Statistical analysis. The protein concentration was determined according to a Bio-Rad
method with bovine albumin as a standard reference.26 The data are expressed as the mean SEM of
n = 4 independent experiments. Statistical analysis of the data was performed using one way
ANOVA followed by Dunnett’s post hoc test. Inhibitory binding constants, Ki, will be calculated
from the IC values according to the Cheng and Prusoff equation: K = IC /(1 + [C*]/K *), where
50
i
50
D
[C*] is the concentration of the radioligand and KD* its dissociation constant.26 IC50 values obtained
in cAMP assays were calculated by non-linear regression analysis using the equation for a sigmoid
concentration-response curve.
Author Contributions

All authors have given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
Acknowledgment
The synthetic work was financially supported by the University of Florence and the Italian Ministry
for University and Research (MIUR, PRIN 2010-2011, 20103W4779_004 project). The
pharmacological work was supported by the University of Ferrara by using internal funding.
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Graphical abstract
R
( )
n
NH2
N
N
S
N
( )
N
S
n
R2
R5
NH
N
n = 0, 1, 2
R = H, 2-OCH3,
3-OCH3, 4-OCH3,
4-OH
CH
3

Highlights
•
•
•
Synthesis of novel thiazolo[5,4-d]pyrimidine derivatives is reported
The compounds were tested for their affinity and potency at human adenosine receptors
The new thiazolo[5,4-d]pyrimidines are hA1 and A2A antagonists/inverse agonists