Piperazine-and piperidine-containing thiazolo[5,4-d]pyrimidine derivatives as new potent and selective adenosine a2a receptor inverse agonists

Article abstract of DOI:10.3390/ph13080161

The therapeutic use of A_2A adenosine receptor (AR) antagonists for the treatment of neurodegenerative disorders, such as Parkinson and Alzheimer diseases, is a very promising approach. Moreover, the potential therapeutic role of A_2A AR antagonists to avoid both immunoescaping of tumor cells and tumor development is well documented. Herein, we report on the synthesis and biological evaluation of a new set of piperazine-and piperidine-containing 7-amino-2-(furan-2-yl)thiazolo[5,4-d]pyrimidine derivatives designed as human A_2A AR antagonists/inverse agonists. Binding and potency data indicated that a good number of potent and selective hA_2A AR inverse agonists were found. Amongst them, the 2-(furan-2-yl)-N⁵-(2-(4-phenylpiperazin-1-yl)ethyl)thiazolo[5,4-d]pyrimidine-5,7-diamine 11 exhibited the highest A_2A AR binding affinity (K_i = 8.62 nM) as well as inverse agonist potency (IC₅₀ = 7.42 nM). In addition, bioinformatics prediction using the web tool SwissADME revealed that 8, 11, and 19 possessed good drug-likeness profiles.

Full text of DOI:10.3390/ph13080161

pharmaceuticals
Article
Piperazine- and Piperidine-Containing
Thiazolo[5,4-d]pyrimidine Derivatives as New Potent
and Selective Adenosine A2A Receptor Inverse Agonists
Flavia Varano 1,*, Daniela Catarzi 1, Erica Vigiani 1, Fabrizio Vincenzi 2, Silvia Pasquini 2,
Katia Varani 2 and Vittoria Colotta 1
1
Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino,
Sezione di Farmaceutica e Nutraceutica, Universita’degli Studi di Firenze, Via Ugo Schiff 6,
50019 Sesto Fiorentino (FI), Italy; daniela.catarzi@unifi.it (D.C.); erica.vigiani@unifi.it (E.V.);
vittoria.colotta@unifi.it (V.C.)
2
Dipartimento di Morfologia, Chirurgia e Medicina Sperimentale, Università degli Studi di Ferrara,
Via Fossato di Mortara 17-19, 44121 Ferrara, Italy; fabrizio.vincenzi@unife.it (F.V.);
silvia.pasquini@unife.it (S.P.); katia.varani@unife.it (K.V.)
*
Correspondence: flavia.varano@unifi.it
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 29 June 2020; Accepted: 21 July 2020; Published: 24 July 2020
Abstract: The therapeutic use of A2A adenosine receptor (AR) antagonists for the treatment of
neurodegenerative disorders, such as Parkinson and Alzheimer diseases, is a very promising
approach. Moreover, the potential therapeutic role of A2A AR antagonists to avoid both
immunoescaping of tumor cells and tumor development is well documented. Herein, we
report on the synthesis and biological evaluation of a new set of piperazine- and piperidine-
containing 7-amino-2-(furan-2-yl)thiazolo[5,4-d]pyrimidine derivatives designed as human A2A
AR antagonists/inverse agonists. Binding and potency data indicated that a good number
of potent and selective hA2A AR inverse agonists were found.
Amongst them, the
2-(furan-2-yl)-N5-(2-(4-phenylpiperazin-1-yl)ethyl)thiazolo[5,4-d]pyrimidine-5,7-diamine 11 exhibited
the highest A2A AR binding affinity (Ki = 8.62 nM) as well as inverse agonist potency (IC50 = 7.42 nM).
In addition, bioinformatics prediction using the web tool SwissADME revealed that
possessed good drug-likeness profiles.
8, 11, and 19
Keywords: G protein-coupled receptors; adenosine receptors; adenosine A2A receptor ligands;
thiazolo[5,4-d]pyrimidines
1. Introduction
Adenosine is an endogenous purinergic nucleoside which interferes in many physiological states
related to cardiovascular, immune, and neurological functions. Extracellular adenosine acts via four
distinct G protein-coupled membrane receptors, namely A1, A2A, A2B, and A3 adenosine receptors
(ARs). The A1 and A3 receptors are principally coupled to Gi/o proteins thus inducing an inhibitory
effect on adenylyl cyclase and reducing cAMP production, while the A2A and A2B receptors stimulate
the production of cAMP via Gs proteins [1]. ARs are distributed all over in the body and elevated
adenosine levels and/or upregulation of ARs have been detected in many pathological conditions [
2].
The A2A AR is located both peripherally and centrally, with the highest expression levels in the striatum,
olfactory tubercle, and the immune system. The A2A AR is a very promising target in the field of
neurodegenerative pathologies, mainly Parkinson’s (PD) and Alzheimer’s (AD) diseases [
A2A AR antagonists have demonstrated to improve PD motor dysfunctions in various preclinical animal
models as well as in clinical studies [ ]. Furthermore, neuroprotective functions were associated with
3–5]. Several
5
Pharmaceuticals 2020, 13, 161; doi:10.3390/ph13080161
www.mdpi.com/journal/pharmaceuticals

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the use of A2A AR antagonists thus suggesting that they may delay the onset and progression of PD [
The A2A AR antagonists, such as Tozadenant (SYN 115) [ ], ST 1535 [ ], Vipadenant [ ], Preladenant
(SCH 420814) [ ], and Istradefylline [10], have been clinically investigated showing potential effect
5].
6
7
8
9
in the treatment of PD. In particular, Istradefylline received marketing approval in Japan in 2013 as
NOURIAST® (Figure 1) and in 2019 was approved by the US Food and Drug Administration (FDA)
for PD [11]. In the case of AD, it is well established that A2A AR antagonists prevent amyloid beta
toxicity accompanied by improvement of spatial memory [12].
Figure 1. A2A AR antagonists progressed into clinical testing for the treatment of Parkinson’s disease
(PD) and cancer.
Recently a large amount of research focused on the A2A AR as a new target for cancer
immunotherapy [13,14]. In fact, the A2A AR represents an important immune checkpoint for T
cells and NK cells and its activation induces suppression of immune cells response. Considering
the increased A2A AR expression in activated tumor infiltrating T cells, it is thus clear that this
mechanism is important to favor tumor escape [15]. Moreover, A2A AR is expressed also in tumor
cells and its stimulation induces and increases cell proliferation, chemotaxis and migration, thus
favoring tumor growth and metastasis [16]. The potential therapeutic role of A2A AR antagonists
to avoid immunoescaping of tumor cells and tumor development is evident. Indeed, four A2A AR
antagonists, including Preladenant [17], PBF-509 [18], CPI-444 [19], and AZD4635 [20] have entered
clinical development as anticancer drugs alone and in combination with other agents (Figure 1).
Our group previously synthesized some potent human (h) A2A AR antagonists/inverse agonists
belonging to different chemical classes [21
(TP series) has been deeply investigated allowing us to delineate comprehensive structure activity
relationships [21 25 27 31]. This was possible because the central thiazolopyrimidine scaffold can
–31]. Among these, the thiazolo[5,4-d]pyrimidine one
,
– ,
be easily decorated by at least three different substituents at positions 2, 5, and 7, to explore diverse
sites of interaction. To obtain potent and selective hA2A AR antagonists/inverse agonists, the

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thiazolopyrimidine core must exhibit an exocyclic amine group at position 7 and a furan-2-yl moiety at
position 2. In contrast, substituents endowed with variable properties, such as the steric hindrance,
seems to be tolerated at position 5. In fact, good to high A2A AR affinity was observed when an
(hetero)aryl or alkyl residue was attached by diverse linkers at position 5 of the thiazolopyrimidine
scaffold [21,25–27]. In particular, in a recent paper by us some interesting results were obtained when
the linker was a piperazine moiety directly attached to the bicyclic core or spaced by an ethylamino
chain [31]. It has to be noted that piperazine derivatives are reported to elicit a broad spectrum of
pharmacological activities. In fact, this heterocycle is present in many well-known drugs belonging to
diverse pharmacological classes [32].
Thus, to further investigate the structure-activity relationships of the 7-amino-2-(furan-2-yl)-
thiazolo[5,4-d]pyrimidines as A2A AR antagonists/inverse agonists, in the present paper we describe
the synthesis of the new derivatives
piperazine moiety directly attached to the bicyclic core (
ethylamino chain (10 and 11 16, respectively). Moreover, a little set of compounds bearing at position
1
–
8,
10–
21 (Figure 2) bearing at position 5 a piperidine or a
1
, and , respectively) or spaced by an
2–8
–
5 a methylamino (17) or a methylaminopiperidine chain (18–21) is reported.
Figure 2. General structure of the designed 7-amino-2-(furan-2-yl)-thiazolo[5,4-d]pyrimidines.
2. Results
2.1. Chemistry
Compounds
obtainment of the 7-amino-5-chloro-2-(furan-2-yl)-thiazolo[5,4-d]pyrimidine 22 and of the appropriate
amine tails 23 42. Then, the two building blocks were reacted together to provide the desired
1–8, 10–21 were prepared following a common procedure that first involved the
–
compounds (Scheme 1).
The 7-amino-5-chloro-thiazolo[5,4-d]pyrimidine 22 was prepared as previously described [21].
The reaction of the latter with an excess of the proper amine 23
delivered the target compounds 10 21. The amines 23 24 26
35]. The ethylamine derivatives 31–
–
,
42, under microwave irradiation,
28 30 were commercial, while 25
37 [36,37]
1
–
8
,
–
–
,
–
,
27, and 38 were prepared according to the literature [33
–
were synthesized as outlined in Scheme 2.
Briefly, 4-benzylpiperidine 23 and the N1-substituted piperazines 24
–
28,
43 [38]
were alkylated in standard conditions with N-(2-bromoethyl)phalimide 44 to achieve the
N-(2-ethylsubstituted)phtalimide derivatives 45
hydrazinolysis produced compounds 31–37.
–51. Removal of the phthaloyl group of the latters by

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X
R
CH
23, 1, 31, 10
24, 2, 32, 11
25, 3, 33, 12
26, 4, 34, 13
27, 5, 35, 14
28, 6, 36, 15
N
N
N
N
N
N
N
29, 7
30, 8
37, 16
R
38, 17
39, 18
40, 19
41, 20
42, 21
Scheme 1. Reagents and conditions. (a) n-BuOH, 200 ◦C MW, 20 min.
X
R
CH
23, 45, 31
24, 46, 32
25, 47, 33
26, 48, 34
27, 49, 35
N
N
N
N
N
N
28, 50, 36
43, 51, 37
Scheme 2. Reagents and conditions. (a) For 45–50: CH3CN, K2CO3, reflux, 14 h; for 51: CH3CN, Et3N,
reflux, 24 h; (b) MeOH, NH2NH2·H2O, reflux, 2 h.

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Finally, the 4-aminomethyl-piperidine derivatives 39
–42 [39,40] were prepared starting from
the commercial 4-aminomethylpiperidine 52, following the reported procedure (Scheme 3) [31].
The reaction of the latter with benzaldehyde in absolute ethanol gave the imino derivative 53 [39]
which was then reacted with the proper alkyl(aryl)halide 54–57 to furnish, in satisfactory yields,
the corresponding N-substituted piperidine derivatives 58
–
61 [39,40]. Acidic hydrolysis of the
protecting imino group of the latter gave the desired 39–42.
R
54, 58, 39
55, 59, 40
56, 60, 41
57, 61, 42
Scheme 3. Reagents and conditions. (a) EtOH, benzaldehyde, reflux, 24 h; (b) CH3COCH3, K2CO3, rt,
12 h; (c) CH2Cl2/H2O, oxalic acid, reflux, 3 h.
2.2. Pharmacological Assays
Binding affinities of compounds 1–8, 10–21 for the hA1, hA2A, and hA3 AR subtypes, expressed in
Chinese Hamster Ovary (CHO) cells, were determined in radioligand competition experiments. In the
binding affinity assays, the competition of ligands for specific binding of [3H]DPCPX, [3H]ZM241385,
and [125I]AB-MECA, respectively was measured to hA1, hA2A, and hA3 ARs. Activities of compounds
1–8, 10–21 at the hA2B AR subtype was determined by measuring the inhibition of NECA stimulated
adenylyl cyclase activity in CHO cells expressing the hA2B receptor. Compounds 8, 11, 14–15, and 19,
the best in terms of hA2A AR affinity and selectivity, were also evaluated for their functional behavior.
Hence, compounds were tested to assess their ability to modulate cAMP production in hA2A CHO
cells. All pharmacological data are reported in Tables 1 and 2 together with those of the reference
compound ZM 241,385 [41].

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Table 1. Binding affinities (Ki) at hA1, hA2A, and hA3 ARs and potencies (IC50) at hA2B ARs.
NH2
NH2
HO
N
N
N
S
N
N
N
O
R5
N
N
N
O
H
1-21
ZM 241385
hA1AR a
Ki (nM)
(I%) e
hA2BAR c
hA3AR d
Ki (nM)
(I%) e
hA2AAR b
Ki (nM)
R5
IC50 (nM)
(I%) e
>10,000
(21%)
>10,000
(8%)
1
586 ± 37
594 ± 48
>10,000
(15%)
>10,000
(18%)
2
3
4
5
296 ± 22
180 ± 15
64 ± 5
58 ± 5
10,000
(26%)
868 ± 82
197 ± 16
127 ± 15
>10,000
(11%)
1213 ± 114
2355 ± 213
237 ± 17
326 ± 27
>10,000
(7%)
>10,000
(10%)
6
7
2766 ± 249
345 ± 28
137 ± 11
29 ± 3
816 ± 74
>10,000
(23%)
>10,000
(34%)
>10,000
(19%)
>10,000
(25%)
8
638 ± 56
15.1 ± 1.3
279 ± 23
>10,000
(38%)
9 f
4536 ± 312
2679 ± 221
>10,000
(22%)
>10,000
(22%)
10
11
12
725 ± 67
102 ± 9
187 ± 16
8.62 ± 0.74
92 ± 8
>10,000
(13%)
>10,000
(15%)
>10,000
(26%)
>10,000
(17%)
798 ± 72
>10,000
(29%)
>10,000
(33%)
13
14
522 ± 47
452 ± 38
37 ± 3
>10,000
(17%)
18.3 ± 1.9
1492 ± 126
>10,000
(16%)
>10,000
(23%)
15
436 ± 36
10.8 ± 1.0
264 ± 24
>10,000
(31%)
16
17
18
>10,000 (26%)
>10,000 (17%)
>10,000 (34%)
>10,000 (28%)
>10,000
(29%)
>10,000
(33%)
>10,000
(11%)
>10,000
(28%)
524 ± 41
802 ± 73
>10,000
(18%)
>10,000
(35%)
19
20
365 ± 29
152 ± 11
15.2 ± 1.7
88 ± 9
OCH3
N
>10,000
(24%)
>10,000
(29%)
H
N
>10,000
(27%)
>10,000
(21%)
21
247 ± 18
188 ± 16
483 ± 34
ZM 241385
0.94 ± 0.07
51 ± 4
672 ± 51
a
Data are expressed as means
±
SEM. Affinity values obtained from the displacement of specific [3H]DPCPX
,
[3H]ZM241383 b, or [125I]AB-MECA d binding to hA1ARs, hA2AARs, or A3ARs, respectively (n = 3–6). Potency
c
e
(IC50) in cAMP assays to hA2BARs. Percentage of inhibition (I%) is determined at 10
µ
M concentration of the
f
tested compounds. Ref. 31.

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Table 2. Potency (IC50) of selected compounds on cyclic AMP assays in CHO cells expressing hA2A AR.
Potency
IC50, nM
Intrinsic
Activity
Pharmacological
Behavior
Compounds
8
11
14
15
19
13.8 ± 1.2
7.42 ± 0.68
15.2 ± 1.3
9.42 ± 0.87
14.8 ± 1.4
1.42 ± 0.11
−44 ± 3
−52 ± 4
−51 ± 5
−67 ± 5
−64 ± 4
−48 ± 4
Inverse agonist
Inverse agonist
Inverse agonist
Inverse agonist
Inverse agonist
Inverse agonist
ZM 241385
Data are expressed as means ± SEM.
3. Discussion
3.1. Structure-Activity Relationships
Binding and potency data of the newly synthesized compounds 1–8, 10–21, and of the previously
reported derivative 9 [31] are summarized in Table 1.
Most of the tested compounds (
2–3, 6–8, 10–15, 19–20) displayed high to good affinity for the hA2A
AR (8.62 nM < Ki < 187 nM). Instead, no significant affinity was detected for the off-target hARs with
the exception of that of compounds
3–
5
,
11
,
20 which bind the hA1 (
3, 11, 20) and the hA3 subtypes
(4–5) with good affinities.
Compounds
the bicyclic thiazolopyrimidine core. Comparison of the hA2A AR binding activity of the piperidine
substituted (Ki = 594 nM) and of its corresponding piperazine analogue (Ki = 58 nM), both bearing an
appended benzyl group, indicates that the piperazine linker is preferred. Analyzing the effect of different
substituents on the piperazine ring, the data indicate that while an appended phenyl ( ) or benzyl residue
) was equally tolerated, a longer phenylethyl group ( ) or a para-substituent (OCH2CH2OCH3, COOEt)
on the phenyl ring of (compounds and 9, respectively), produced a drop in the binding activity.
Introduction of a furan-2-yl methanone residue on the piperazine ring gave compound which shows
good hA2A AR affinity even if lower than that of and . In contrast, the presence of an ethylamine
chain yielded derivatives endowed with higher hA2A AR affinity than that of and . Moreover,
the (pyrrolidin-1-yl)ethyl derivative 8 is also highly selective toward this receptor subtype.
With respect to derivatives and , the piperidine or piperazine residue at position 5 of
compounds 10 16 was shifted from the thiazolopyrimidine core by an ethylamino linker thus increasing
1–9
bear a piperidine (1) or a piperazine (2–9) substituted ring directly linked to
1
3
2
(3
4
2
5
6
2
3
7–8
2
3
1–6
9
–
chain flexibility. In general this structural change leads to an improved binding affinity with only two
exceptions. In fact, while derivative 12 is slightly less active than its homologue 3, the ethylbenzoate
derivative 16 is equiactive to 9. Among the herein reported compounds, the phenylpiperazine derivative
11 possesses the highest hA2A AR affinity displaying a Ki value of 8.6 nM. Compared to the latter, the
(furan-2-yl)methanonepiperazine derivative 15 shows a similar binding activity (Ki = 10.8 nM) but is
more selective toward the hA2A AR. Compound 14, characterized by the same side chain of Preladenant
,
possesses high hA2A AR affinity (Ki = 18.3 nM) similar to that of 11 and 15, and is also highly selective.
Finally, the binding results of the last set of compounds (17
linker between the bicyclic core and the ethylbenzoate (17) or the substituted piperidine residue (18
–
21), all characterized by an aminomethyl
21),
–
indicate that only in one case, i.e., the benzyl piperidine derivative 19, a high affinity (Ki = 15.2 nM)
and a good selectivity toward the A2A subtype is reached.
Selected compounds 8, 11, 14–15, and 19, the best in terms of hA2A AR affinity and selectivity,
were also evaluated in functional assays to assess their ability to modulate cAMP production in hA2A
CHO cells (Table 2, Figure S1). All the tested compounds behaved as inverse agonists since they
were able to inhibit basal cAMP accumulation. In particular, according to their nanomolar hA2A AR
affinities, compounds
8, 11, 14–15, and 19 show IC50 values spanning from 15.2 to 7.42 nM and also in
this assay derivative 11 is the most active.

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3.2. In Silico ADME Prediction
Compounds 8, 11, 14–15, and 19 were also evaluated in silico to test their “drug-likeness” profiles
on the basis of the absorption, distribution, metabolism, and excretion (ADME) properties. Calculations
were performed by the SwissADME web service (http://www.swissadme.ch developed by the Molecular
Modeling Group of the Swiss Institute of Bioinformatics) that gives free access to a pool of fast yet
robust predictive models for small molecules pharmacokinetic properties [42]. The data evaluated for
the selected compounds are summarized in the Supplementary Materials (Table S1).
Investigated molecules possessed several favorable ADME properties. All compounds obeyed the
Lipinsky’s rule of five indicating drug-likeness. Moreover, they possessed good probability to have at
least 10% oral bioavailability in rat or measurable Caco-2 permeability. SwissADME returns warnings
if the molecule under evaluation contains fragments that could yield a false positive biological output
(PAINS Pan Assay Interference Structures). Compounds
14 showed one alert. The topological surface area (TPSA) measures the drug ability to permeate cells.
Compounds
11, and 19 showed similar TPSA values less than 140 Å2 suggesting that they could
8, 11, 15, and 19 had no PAINS alerts, while
8
,
permeate cell membranes. The Consesus log Po/w (octanol/water partition coefficient) values indicated
rather a reasonable absorption (1.71 < Consensus log Po/w < 3.34), while the log S values defined
moderate solubility in the body.
The bioavailability radars (Figure 3) are the drug-likeness graphs of analyzed compounds
presented in the form of a hexagon with each of the vertices representing a parameter (lipophilicity,
size, polarity, solubility, flexibility, and saturation) that define a bioavailable drug. The pink region is
the suitable physicochemical space for oral bioavailability. The radar plot of the molecule, represented
by the red distorted hexagon, has to fall entirely in the pink area to be considered drug-like. From
the graphs in Figure 3, it was found that while compounds 8, 11, and 19 were orally bioavailable,
compounds 14 and 15 were not, because of being too polar and 14 also too flexible.
Figure 3. Bioavailability radars for the analyzed compounds, from the SwissADME web tool.
LIPO = lipophilicity (XLOGP3 between
−
0.7 and 5.0); SIZE (molecular weight between 150 and
500 g/mol); POLAR = polarity (TPSA between 20 and 130 Å2); INSOLU = solubility (log S not
higher than 6); INSATU = saturation (fraction of carbons in the sp3 hybridization not less than 0.25);
FLEX = flexibility (no more than nine rotatable bonds).

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Finally, the BOILED-egg (Brain Or IntestinaL EstimateD) method (Figure 4) allows predicting
simultaneously two keys in vivo ADME parameters, i.e., the passive gastrointestinal absorption (HIA)
and brain access (BBB) [43]. While all studied compounds had no BBB permeability (none in the yellow
region), compounds 8, 11, and 19 exert high HIA (in the white region) and compounds 14 and 15 were
not permeable (in the grey region). Moreover, they all were predicted as actively effluxed by Pgp
(blue dots = PGP+).
Figure 4. Predicted BOILED-Egg diagram of the analyzed compounds, from the SwissADME web tool.
4. Materials and Methods
4.1. Chemistry
4.1.1. General Methods
The microwave-assisted syntheses were performed using an Initiator EXP Microwave Biotage
instrument (frequency of irradiation: 2.45 GHz). Analytical silica gel plates (Merck F254, Kenilworth,
NJ, USA), preparative silica gel plates (Merck F254, 2 mm), and silica gel 60 (Merck, 70–230 mesh) were
used for analytical and preparative TLC, and for column chromatography, respectively. All melting
points were determined on a Gallenkamp melting point apparatus and are uncorrected. Elemental
analyses were performed with a FlashE1112 Thermofinnigan elemental analyzer for C, H, N and
the results were within 0.4% of the theoretical values. All final compounds revealed a purity not
±
less than 95%. Compounds were named following IUPAC rules as applied by ChemDrawUltra 9.0.
The IR spectra were recorded with a Perkin-Elmer Spectrum RX I spectrometer in Nujol mulls and are
expressed in cm−1. NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz for
1H-NMR and 100 MHz for 13C-NMR). The chemical shifts are reported in δ (ppm) and are relative to
the central peak of the solvent which was CDCl3 or DMSOd6. The following abbreviations are used:
s: Singlet, d: Doublet, t: Triplet, m: Multiplet, br: Broad, and ar: Aromatic protons.
4.1.2. General Procedure for the Synthesis of 1–8, 10–21
The proper amine 23–42 (3 mmol) was added to a solution of the 5-chloro-2-(furan-
2-yl)thiazolo[5,4-d]pyrimidin-7-amine deriv◦ative 22
[21] (1 mmol) in n-BuOH (2 mL). The reaction
mixture was microwave irradiated at 200 C for 20 min, then cooled at room temperature and
basified with an aqueous KOH solution (50%). Addition of water afforded a solid which was

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collected by filtration and washed with Et2O. The crude material was purified by crystallization or
by chromatography.
5-(4-Benzylpiperidin-1-yl)-2-(furan-2yl)thiazolo[5,4-d]pyrimidin-7-amine (
1
). Yield 51%. Mp:
197–199 ◦C (acetonitrile). H-NMR (DMSO-d6): 1.06–1.15 (m, 2H), 1.60 (d, 2H, J = 12 Hz), 1.77 (br s,
1H), 2.77 (t, 2H, J = 13 Hz), 4.64 (d, 2H, J = 12 Hz), 6.72–6.73 (m, 1H, ar), 7.04–7.05 (m, 1H, ar), 7.17–7.30
(m, 7H, 5ar + NH2), 7.90 (s, 1H, ar). Anal. calcd. for (C21H21N5OS): C, 64.43%; H, 5.41%; N, 17.89%.
Anal. found: C, 64.55%; H 5.77%; N 18.13%.
1
2-(Furan-2-yl)-5-(4-phenylpiperazin-1-yl)thiazolo[5,4-d]pyrimidin-7-amine (
2
). Yield 30%. Mp:
219–221 ◦C (ethanol). H-NMR (CDCl3): 3.27 (t, 4H, J = 5.1 Hz), 4.02 (t, 4H, J = 5.1 Hz), 5.49 (br s, 2H,
NH2), 6.57–6.58 (m, 1H, ar), 6.91 (t, 1H, ar, J = 7.3 Hz), 6.99–7.02 (m, 3H, ar), 7.29–7.33 (m, 2H, ar),
7.57–7.58 (m, 1H, ar). 13C-NMR (DMSO-d6): 164.99, 159.26, 157.29, 151.56, 148.58, 146.30, 129.42, 125.09,
119.63, 116.32, 113.20, 110.26, 48.87, 44.20. IR: 3172, 3213, 3300, 3392. Anal. calcd. for (C19H18N6OS): C,
60.30%; H, 4.79%; N, 22.21%. Anal. found: C, 60.43%; H, 5.08%; N, 22.49%.
1
5-(4-Benzylpiperazin-1-yl)-2-(furan-2-yl)thiazolo[5,4-d]pyrimidin-7-amine (3). The crude product
was purified by column chromatography, eluting system ethyl acetate/cyclohexane 7/3. Yield 40%. Mp:
181–183 ◦C. 1H-NMR (DMSO-d6): 2.34–2.44 (m, 4H), 3.51 (s, 2H), 3.73–3.75 (m, 4H), 6.72–6.73 (m, 1H,
ar), 7.06–7.08 (m, 1H, ar), 7.23–7.34 (m, 7H, 5 ar + NH2), 7.90–7.91 (m, 1H, ar).13C-NMR (DMSO-d6):
164.97, 159.24, 157.21, 148.58, 146.12, 138.54, 124.44, 129.24, 128.67, 128.58, 127.43, 127.31, 113.19, 110.16,
62.59, 53.01, 44.27. IR: 3149, 3172, 3211, 3304. Anal. calcd. for (C20H20N6OS): C, 61.21%; H, 5.14%; N,
21.41%. Anal. found: C, 61.48%; H, 5.51%; N, 21.74%.
2-(Furan-2-yl)-5-(4-phenethylpiperazin-1-yl)thiazolo[5,4-d]pyrimidin-7-amine (4). The crude
product was purified by column chromatography, eluting system ethyl acetate/cyclohexane 7/3. Yield
1
30%. Mp: 203–204 ◦C (ethanol). H-NMR (DMSO-d6): 2.52–2.57 (m, 6H), 2.75–2.79 (m, 2H), 3.72–3.77
(s, 4H), 6.72–6.73 (m, 1H, ar), 7.06–7.07 (m, 1H, ar), 7.17–7.31 (m, 7H, 5 ar + NH2), 7.91–7.92 (s, 1H,
ar).13C-NMR (DMSO-d6): 164.97, 159.26, 157.20, 148.56, 146.09, 140.86, 129.11, 128.70, 126.30, 124.93,
113.22, 110.18, 60.25, 53.06, 44.30, 33.17. IR: 3280, 3421. Anal. calcd. for (C21H22N6OS): C, 62.05%; H,
5.46%; N, 20.67%. Anal. found: C, 61.98%; H, 5.54%; N, 21.03%.
2-(Furan-2-yl)-5-(4-(4-(2-methoxyethoxy)phenyl)piperazin-1-yl)thiazolo[5,4-d]pyrimidin-7-amine
1
(5
). Yield 50%. Mp: 181–183 ◦C (methanol). H-NMR (DMSO-d6): 3.05–3.08 (m, 4H). 3.29 (s, 3H), 3.62
(t, 2H, J = 5.0 Hz), 3.86–3.89 (m, 4H), 4.01 (t, 2H, J = 5 Hz), 6.72–6.73 (m, 1H, ar), 6.84 (d, 2H, ar, J = 9.0
Hz), 6.94 (d, 2H, ar, J = 9.0 Hz), 7.07–7.08 (m, 1H, ar), 7.32 (br s, 2H, NH2), 7.91 (s, 1H, ar). Anal. calcd.
for (C22H24N6O3S): C, 58.39%; H, 5.35%; N, 18.57%. Anal. found: C, 58.68%; H, 5.58%; N, 18.79%.
(4-(7-Amino-2-(furan-2-yl)thiazolo[5,4-d]pyrimidin-5-yl)piperazin-1-yl)(furan-2-yl)methanone (6).
The crude product was purified by column chromatography, eluting system ethyl acetate/cyclohexane
1
7/3. Yield 25%. Mp: 227–229 ◦C (tetrahydrofuran/water). H-NMR (DMSO-d6): 3.74–3.81 (m, 8H),
6.65–6.66 (m, 1H, ar), 6.74–6.75 (m, 1H, ar), 7.04–7.05 (m, 1H, ar), 7.08–7.09 (m, 1H, ar), 7.39 (s, 2H,
NH2), 7.87 (s, 1H, ar), 7.92 (s, 1H, ar). 13C-NMR (CDCl3): 165.47, 159.31, 158.89, 156.28, 148.70, 147.91,
144.07, 143.82, 125.30, 116.65, 112.33, 111.38, 110.07, 60.41, 44.38, 26.91, 21.07, 14.21. IR: 3429, 3307, 3209,
1620. Anal. calcd. for (C18H16N6O3S): C, 54.54%; H, 4.07%; N, 21.20%. Anal. found: C, 54.00%; H,
4.29%; N, 21.39%.
5-(4-(2-(Dimethylamino)ethyl)piperazin-1-yl)-2-(furan-2-yl)thiazolo[5,4-d]pyrimidin-7-amine (7).
The product was purified by column chromatography, eluting system chloroform/methanol/ammonium
hydroxide 8.5/1.5/0.15. Yield 38%. Mp: 183–186 ◦C. 1H-NMR (DMSO-d6): 2.15 (s, 6H), 2.36–2.44 (m,
8H), 3.69–3.71 (m, 4H), 6.72–6.73 (m, 1H, ar), 7.06–7.07 (m, 1H, ar), 7.26 (s, 2H, NH2), 7.90–7.91 (m, 1H,
ar). Anal. calcd. for (C17H23N7OS): C, 54.67%; H, 6.21%; N, 26.25%. Anal. found: C, 55.01%; H, 6.54%;
N, 26.39%.
2-(Furan-2-yl)-5-(4-(2-(pyrrolidin-1-yl)ethyl)piperazin-1-yl)thiazolo[5,4-d]pyrimidin-7-amine (8).
The product was purified by column chromatography, eluting system chloroform/methanol/ammonium
hydroxide 8.5/1.5/0.15. Yield 25%. Mp: 181–183 ◦C. 1H-NMR (DMSO-d6): 1.59–1.65 (m, 4H), 2.40–2.49
(m, 12H), 3.65–3.69 (m, 4H), 6.72–6.73 (m, 1H, ar), 7.06–7.07 (m, 1H, ar), 7.29 (s, 2H, NH2), 7.90–7.91 (m,

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1H, ar). Anal. calcd. for (C19H25N7OS): C, 57.12%; H, 6.31%; N, 24.54%. Anal. found: C, 56.89%; H,
6.45%; N, 24.77%.
N5-(2-(4-Benzylpiperi◦din-1-yl)ethyl)-2-(furan-2-yl)thiazolo[5,4-d]pyrimidine-5,7-diamine (10).
Yield 22%. Mp: 155-157 C (ethyl acetate). 1H-NMR (CDCl3): 1.28–1.31 (m, 2H), 1.34–1.37 (m,
1H), 1.56–1.57 (m, 2H), 1.95 (t, 2H, J = 11.2 Hz), 2.55–2.57 (m, 4H), 2.91 (d, 2H, J = 11.2 Hz), 3.48–3.52
(m, 2H), 5.48 (br s, 2H, NH2), 5.58 (br s, 1H, NH), 6.57–6.58 (m, 1H, ar), 6.97–6.98 (m, 1H, ar), 7.15–7.32
(m, 5H, ar), 7.56 (s, 1H, ar). 13C-NMR (DMSO-d6): 165.01, 160.30, 157.45, 148.63, 140.86, 129.43,
128.56, 126.15, 113.15, 109.98, 57.77, 53.86, 42.87, 37.89, 32.26. IR: 3323, 3169, 3116. Anal. calcd. for
(C23H26N6OS): C, 63.57%; H, 6.03%; N, 19.34%. Anal. found: C, 63.88%; H, 6.36%; N, 19.21%.
2-(Furan-2-yl)-N5-(2-(4-phenylpiperazin-1-yl)ethyl)thiazolo[5,4-d]pyrimidine-5,7-diamine (11).
1
Yield 60%. Mp: 218–220 ◦C (2-methoxyethanol). H-NMR (CDCl3): 2.67–2.72 (m, 6H), 3.23–3.25 (m,
4H), 3.56–3.60 (m, 2H), 5.49 (br s, 2H, NH2), 5.58 (br s, 1H, NH), 6.57–6.58 (m, 1H, ar), 6.88 (t, 1H, ar,
J = 7.2 Hz), 6.95–6.99 (m, 3H, ar), 7.27–7.31 (m, 2H, ar), 7.57 (s, 1H, ar). 13C-NMR (DMSO-d6): 160.36,
157.49, 151.54, 148.64, 129.36, 119.17, 115.77, 113.14, 110.01, 57.55, 53.25, 48.70, 38.83. IR: 3334, 3263,
3165, 3115. Anal. calcd. for (C21H23N7OS): C, 59.84%; H, 5.50%; N, 23.26%. Anal. found: C, 60.19%; H,
5.55%; N, 23.55%.
N5-(2-(4-Benzylpiperazin-1-yl)ethyl)-2-(furan-2-yl)thiazolo[5,4-d]pyrimidine-5,7-diamine (12).
The product was purified by column chromatography, eluting system ethyl acetate/cyclohexane/
methanol 6.5/2/1.5. Yield 45%. Mp: 133–135 ◦C. 1H-NMR (CDCl3): 2.54–2.60 (m, 10H), 3.50–3.54 (m,
4H), 5.53 (br s, 2H, NH2), 5.63 (br s, 1H, NH), 6.56–6.58 (m, 1H, ar), 6.97–6.98 (m, 1H, ar), 7.28–7.34 (m,
5H, ar), 7.56 (s, 1H, ar).13C-NMR (DMSO-d6): 160.29, 157.44, 148.61, 138.68, 133.04, 129.26, 127.31, 125.56,
113.12, 110.00, 62.54, 57.47, 53.24, 53.08. IR: 3331, 3265, 3174, 3082. Anal. calcd. for (C22H25N7OS): C,
60.67%; H, 5.79%; N, 22.51%. Anal. found: C, 60.49%; H, 6.09%; N, 22.43%.
2-(Furan-2-yl)-N5-(2-(4-phenethylpiperazin-1-yl)ethyl)thiazolo[5,4-d]pyrimidine-5,7-diamine (13).
The product was purified by column chromatography, eluting system ethyl acetate/cyclohexane/
methanol 6.5/2/1.5. Yield 37%. Mp: 151–153 ◦C. 1H-NMR (CDCl3): 2.61–2.66 (m, 12H), 2.82–2.86 (m,
2H), 3.48–3.57 (m, 2H), 5.51 (br s, 2H, NH2), 5.60 (br s, 1H, NH), 6.56–6.58 (m, 1H, ar), 6.98–6.99 (m, 1H,
ar), 7.20–7.33 (m, 5H, ar), 7.56 (s, 1H, ar). 13C-NMR (DMSO-d6): 160.30, 157.46, 148.63, 140.95, 133.00,
129.07, 128.24, 125.60, 113.15, 109.99, 60.24, 57.50, 53.25, 53.21, 33.22. IR: 3325, 3259, 3184, 3105. Anal.
calcd. for (C23H27N7OS): C, 61.45%; H, 6.05%; N, 21.81%. Anal. found: C, 61.63%; H, 6.34%; N, 22.11%.
2-(Furan-2-yl)-N5-(2-(4-(4-(2-methoxyethoxy)phenyl)piperazin-1-yl)ethyl)thiazolo[5,4-d]pyrimidine-
1
5,7-diamine (14). Yield 25%. Mp: 203–204 ◦C (ethanol). H-NMR (CDCl3): 2.67–2.68 (m, 6H), 3.13–3.14
(m, 4H), 3.47 (s, 3H), 3.55–3.59 (m, 2H), 3.74–3.76 (m, 2H), 4.09–4.11 (m, 2H), 5.50 (br s, 2H, NH2), 5.57 (br
s, 1H, NH), 6.57–6.58 (m, 1H, ar), 6.90 (br s, 4H, ar), 6.98–6.99 (m, 1H, ar), 7.56 (s, 1H, ar). 13C-NMR
(CDCl3): 159.78, 156.65, 152.94, 148.76, 145.92, 143.96, 118.05, 115.41, 112.26, 109.88, 71.20, 67.75, 59.19,
56.85, 53.05, 50.51, 38.31. IR: 3325, 3263, 3167. Anal. calcd. for (C24H29N7O3S): C, 58.16%; H, 5.90%; N,
19.78%. Anal. found: C, 58.29%; H, 5.63%; N, 20.05%.
(4-(2-((7-Amino-2-(furan-2-yl)thiazolo[5,4-d]pyrimidin-5-yl)amino)ethyl)piperazin-1-yl)(furan-
2-yl)methanone (15). The product was purified by column chromatography, eluting system ethyl
acetate/cyclohexane/methanol 6/4/0.5. Yield 33%. Mp: 163–165 ◦C. 1H-NMR (CDCl3): 2.57–2.59 (m,
4H), 2.65 (t, 2H, J = 5.9 Hz), 3.54–3.58 (m, 2H), 3.85 (br s, 4H), 5.53–5.55 (m, 3H, NH + NH2), 6.49–6.50
(m, 1H, ar), 6.57–6.58 (m, 1H, ar), 6.98–6.99 (m, 1H, ar), 7.01–7.02 (m, 1H, ar), 7.50 (s, 1H, ar), 7.57 (s, 1H,
ar). 13C-NMR (DMSO-d6): 164.97, 160.31, 158.70, 157.45, 148.61, 147.51, 145.07, 115.91, 113.15, 111.71,
110.00, 57.33, 53.27, 38.65. Anal. calcd. for (C20H21N7O3S): C, 54.66%; H, 4.82%; N, 22.31%. Anal.
found: C, 54.94%; H, 5.18%; N, 22.67%.
Ethyl 4-(4-(2-((7-amino-2-(furan-2-yl)thiazolo[5,4-d]pyrimidin-5-yl)amino)ethyl)piperazin-1-yl)
benzoate (16). The product was purified by column chromatography, eluting system ethyl
◦
1
acetate/cyclohexane/methanol 5/4/1. Yield 17%. Mp: 242–244 C. H-NMR (CDCl3): 1.27 (t, 3H,
J = 7.0 Hz), 2.59–2.72 (m, 6H), 3.33–3.38 (m, 4H), 3.57–3.58 (m, 2H), 4.35 (q, 2H, J = 7.0 Hz), 4.49 (br s,
1H, NH), 5.52 (br s, 2H, NH2), 6.57 (s, 1H, ar), 6.89 (d, 2H, ar, J = 8.7 Hz), 6.98–6.99 (m, 1H, ar), 7.57–7.59

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(m, 1H, ar), 7.95 (d, 2H, ar, J = 8.7 Hz). Anal. calcd. for (C24H27N7O3S): C, 58.40%; H, 5.51%; N, 19.86%.
Anal. found: C, 58.67%; H, 5.82%; N, 20.10%.
Ethyl 4-(((7-amino-2-(furan-2-yl)thiazolo[5,4-d]pyrimidin-5-yl)amino)methyl)benzoate
(17).
The product was purified by column chromatography, eluting system ethyl acetate/cyclohexane
4/6. Yield 36%. Mp: 218–220 ◦C. 1H-NMR (DMSO-d6): 1.31 (t, 3H, J = 7.3 Hz), 4.29 (q, 2H, J = 7.3 Hz),
4.53–4.61 (m, 2H), 6.70–6.71 (m, 1H, ar), 7.03–7.07 (m, 1H, ar), 7.20 (s, 2H, NH2), 7.41–7.50 (m, 3H, 2ar +
NH), 7.86–7.94 (m, 3H, ar). 13C-NMR (DMSO-d6): 164.95, 159.22, 157.22, 148.55, 146.15, 143.28, 142.87,
124.95, 113.23, 110.21, 65.38, 61.86, 53.02, 44.45. Anal. calcd. for (C19H17N5O3S): C, 57.71%; H, 4.33%;
N, 17.71%. Anal. found: C, 57.58%; H, 4.65%; N, 17.88%.
2-(Furan-2-yl)-N5-((1-(2-methoxyethyl)piperidin-4-yl)methyl)thiazolo[5,4-d]pyrimidine-5,7-diamine
(18). The product was purified by column chromatography, eluting system chloroform/methanol 8/2.
Yield 20%. Mp: 143–146 ◦C. 1H-NMR (CDCl3): 1.45–1.48 (m, 2H), 1.79–1.82 (m, 4H), 2.07–2.11 (m, 2H),
2.59–2.63 (m, 2H), 3.04–3.06 (m, 2H), 3.33–3.37 (m, 4H), 3.57 (t, 2H, J = 5.5 Hz), 5.03 (t, 1H, NH, J = 5.8Hz),
5.49 (br s, 2H, NH2), 6.56–6.58 (m, 1H, ar), 6.97–6.98 (m, 1H, ar), 7.56 (s, 1H, ar). 13C-NMR (DMSO-d6):
165.03, 160.63, 157.41, 148.67, 113.17, 109.96, 70.36, 58.46, 57.77, 53.95, 47.12, 35.85, 30.21. IR: 3315, 3261,
3178. Anal. calcd. for (C18H24N6O2S): C, 55.65%; H, 6.23%; N, 21.63%. Anal. found: C, 55.98%; H, 5.98%;
N, 21.77%.
N5-((1-Benzylpiperidin-4-yl)methyl)- 2-(furan-2-yl)thiazolo[5,4-d]pyrimidine-5,7-diamine (19).
1
Yield 17%. Mp: 188–190 ◦C (ethyl acetate). H-NMR (DMSO-d6): 1.12–1.20 (m, 2H), 1.50–1.55 (m, 1H),
1.64–1.67 (m, 2H), 1.87 (t, 2H, J = 10.7 Hz), 2.78 (d, 2H, J = 11.3 Hz), 3.15 (t, 2H, J = 6.2 Hz), 3.40 (s, 2H),
6.71–6.72 (m, 1H, ar), 6.87 (br s, 1H, NH), 7.03–7.04 (m, 1H, ar), 7.11 (br s, 2H, NH2), 7.21–7.33 (m, 5H,
ar), 7.89 (s, 1H, ar). 13C-NMR (DMSO-d6): 160.62, 157.41, 148.67, 139.24, 129.15, 128.54, 127.19, 113.13,
109.92, 62.94, 53.51, 36.07, 30.37. IR: 3311, 3263, 3201. Anal. calcd. for (C22H24N6OS): C, 62.83%; H,
5.75%; N, 19.98%. Anal. found: C, 63.15%; H, 5.59%; N, 20.21%.
2-(Furan-2-yl)-N5-((1-(4-methoxybenzyl)piperidin-4-yl)methyl)thiazolo[5,4-d]pyrimidine-5,7-
diamine (20). The product was purified by column chromatography, eluting system chloroform/methanol
8/2. Yield 15%. Mp: 182–183 ◦C. 1H-NMR (CDCl3): 1.34–1.40 (m, 2H), 1.61–1.78 (m, 3H), 1.96 (t, 2H,
J = 11.0 Hz), 2.92 (d, 2H, J = 10.7 Hz), 3.34 (t, 2H, J = 6.1 Hz), 3.46 (s, 2H), 3.82 (s, 3H), 5.01 (br s, 1H, NH),
5.47 (br s, 2H, NH2), 6.57 (m, 1H, ar), 6.86 (d, 2H, J = 8.3 Hz), 6.96–6.97 (m, 1H, ar), 7.24 (d, 2H, ar, J = 8.3
Hz), 7.56 (s, 1H, ar). 13C-NMR (DMSO-d6): 160.62, 158.72, 157.51, 148.62, 130.94, 130.41, 113.94, 113.18,
109.99, 62.29, 55.49, 53.37, 47.14, 36.09, 30.30. IR: 3313, 3255, 3197. Anal. calcd. for (C23H26N6O2S): C,
61.31%; H, 5.82%; N, 18.65%. Anal. found: C, 61.49%; H, 5.75%; N, 18.78%.
2-(Furan-2-yl)-N5-((1-phenethylpiperidin-4-yl)methyl)thiazolo[5,4-d]pyrimidine-5,7-diamine (21).
The product was purified by column chromatography, eluting system chloroform/methanol 8/2. Yield
◦
1
27%. Mp: 174–176 C. H-NMR (CDCl3): 1.40–1.43 (m, 2H), 1.82–1.85 (m, 3H), 2.06–2.11 (m, 2H),
2.59–2.62 (m, 2H), 2.83–2.84 (m, 2H), 3.06–3.11 (m, 2H), 3.36–3.39 (m, 2H), 5.03 (br s, 1H, NH), 5.47 (br s,
2H, NH2), 6.57–6.58 (m, 1H, ar), 6.98–6.99 (m, 1H, ar), 7.22–7.30 (m, 5H, ar), 7.56 (s, 1H, ar). 13C-NMR
(DMSO-d6): 160.62, 157.41, 148.66, 141.05, 129.11, 128.67, 126.21, 113.13, 109.87, 60.59, 53.52, 47.18, 36.09,
33.35, 30.35. IR: 3311, 3267, 3197. Anal. calcd. for (C23H26N6OS): C, 63.57%; H, 6.03%; N, 19.34%. Anal.
found: C, 63.72%; H, 6.39%; N, 19.51%.
4.1.3. General Procedure for the Synthesis of 31–32
In a 50 mL flask, equipped with a magnetic stirrer and reflux condenser, the proper phthalimide
derivatives 45–46 (7 mmol), hydrazine hydrate (10 mmol), and methanol (50 mL) were added.
The resulting mixture was refluxed for 2 h, cooled down to room temprature and concentrated under
reduced pressure. The remaining residue was dissolved in a NaOH aqueous solution (1 M, 30 mL),
washed with ethyl acetate (3
The oily residue without further purification was used in the following step.
×
30 mL) dried over Na2SO4 and concentrated under reduced pressure.

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1
2-(4-Benzylpiperidin-1-yl)ethan-1-amine (31). Yield 85%. H-NMR (CDCl3): 1.26–1.36 (m, 2H),
1.50–1.66 (m, 3H), 1.90 (t, 2H, J = 10.7 Hz), 2.39 (t, 2H, J = 6.3 Hz), 2.54–2.56 (m, 2H), 2.79 (t, 2H, J = 6.3
Hz), 2.88 (d, 2H, J = 11.6 Hz), 7.15–7.31 (m, 5H, ar).
2-(4-Phenylpiperazin-1-yl)ethan-1-amine (32) [36]. Yield 63%. 1H-NMR (CDCl3): 2.51 (t, 2H,
J = 5.8 Hz), 2.63–2.66 (m, 4H), 2.86 (t, 2H, J = 5.8 Hz), 3.22–3.24 (m, 4H), 6.87 (t, 1H, ar, J = 7.2 Hz), 6.95
(d, 2H, ar, J = 8.1 Hz), 7.28 (t, 2H, ar, J = 7.9 Hz).
4.1.4. General Procedure for the Synthesis of 33–36
In a 50 mL flask, equipped with a magnetic stirrer and reflux condenser, the proper phthalimide
derivatives 47–50 (7 mmol), hydrazine hydrate (10 mmol), and methanol (50 mL) were added.
The resulting mixture was refluxed for 2 h, cooled down to room temprature, and concentrated under
reduced pressure. The remaining residue was treated with diethyl ether and the solid was filtered and
used without further purification in the following step.
1
2-(4-Benzylpiperazin-1-yl)ethan-1-amine (33) [37]. Yield 85%. H-NMR (CDCl3): 2.10 (br s, 4H),
2.34–2.56 (m, 6H), 2.82 (t, 2H, J = 6.0 Hz), 3.52 (s, 2H), 7.26–7.33 (m, 5H, ar).
1
2-(4-Phenethylpiperazin-1-yl)ethan-1-amine (34). Yield 90%. H-NMR (CDCl3): 1.73 (br s, 4H),
2.46 (t, 2H, J = 6.2 Hz), 2.56–2.65 (m, 8H), 2.81–2.85 (m, 4H), 7.20–7.32 (m, 5H, ar).
2-(4-(4-(2-Methoxyethoxy)phenyl)piperazin-1-yl)ethan-1-amine (35). Yield 30%. 1H-NMR
(CDCl3): 2.51 (t, 2H, J = 6.1 Hz), 2.63–2.65 (m, 4H), 2.85 (t, 2H, J = 6.1 Hz), 3.11–3.13 (m, 4H),
3.46 (s, 3H), 3.74 (t, 2H, J = 4.7 Hz), 4.10 (t, 2H, J = 4.7 Hz), 6.87–6.92 (m, 4H, ar).
(4-(2-Aminoethyl)piperazin-1-yl)(furan-2-yl)methanone (36). Yield 60%. 1H-NMR (CDCl3):
2.47–2.54 (m, 6H), 2.83 (t, 2H, J = 6.1 Hz), 3.83 (br s, 4H), 6.49–6.50 (m, 1H, ar), 6.99–7.00 (m, 1H, ar),
7.49 (s, 1H, ar).
4.1.5. Ethyl 4-(4-(2-aminoethyl)piperazin-1-yl)benzoate (37)
In a 50 mL flask, equipped with a magnetic stirrer and reflux condenser, the proper phthalimide
derivatives 51 (7 mmol), hydrazine hydrate (8.4 mmol), and methanol (30 mL) were added. The resulting
mixture was refluxed for 2 h, cooled down to room temprature and concentrated under reduced
pressure. The remaining residue was treated with a solution of HCl 1 M and the solid residue was
filtered. The acidic solution was alkalinized with Et3N and the obtained precipitate was filtered and
1
used without further purification in the following step. Yield 50%. H-NMR (DMSO-d6): 1.29 (t, 3H,
J = 7.1 Hz), 2.34 (t, 2H, J = 6.5 Hz), 2.48–2.60 (m, 4H), 2.65 (t, 2H, J = 6.5 Hz), 3.29–3.31 (m, 4H), 4.24 (q,
2H, J = 7.1 Hz), 6.97 (d, 2H, ar), 7.78 (d, 2H, ar).
4.1.6. General Procedure for the Synthesis of 39–42
To a solution of 58–61 (5.6 mmol) in dichloromethane (60 mL), oxalic acid (6.3 mmol) was added.
The solution was diluted with water (30 mL) and refluxed under vigorous stirring for 3 h. After cooling,
the aqueous layer was isolated, washed twice with dichloromethane (30 mL), added with a NaOH
aqueous solution (1 M, pH 9–10), and extracted with chloroform (50 mL
×
3). The organic layer was
dried over Na2SO4, evaporated under reduced pressure, and the obtained oily residue was used as
such in the next step.
1
(1-(2-Methoxyethyl)piperidin-4-yl)methanamine (39). Yield 60%. H-NMR (DMSO-d6): 1.01–1.11
(m, 3H), 1.60-1.63 (m, 2H), 1.86 (t, 2H, J = 10.7 Hz), 2.35–2.43 (m, 4H), 2.81–2.84 (m, 2H), 3.22 (s, 3H)
3.39 (t, 2H, J = 6.0 Hz).
1
(1-Benzylpiperidin-4-yl)methanamine (40) [39]. Yield 35%. H-NMR (DMSO-d6): 1.15–1.17 (m,
3H), 1.62–1.65 (m, 2H), 1.86 (t, 2H, J = 11.5 Hz), 2.38–2.40 (m, 2H), 2.78 (d, 2H, J = 11.5 Hz), 3.41 (s, 2H),
7.23–7.33 (m, 5H, ar).
1
(1-(4-Methoxybenzyl)piperidin-4-yl)methanamine (41). Yield 55%. H-NMR (CDCl3): 1.27–1.29
(m, 3H), 1.70–1.72 (m, 2H), 1.94 (t, 2H, J = 11.2 Hz), 2.58–2.59 (m, 2H), 2.92 (d, 2H, J = 11.6Hz), 3.46 (s,
2H), 3.82 (s, 3H), 6.87 (d, 2H, ar, J = 8.5 Hz), 7.24 (d, 2H, ar, J = 8.5 Hz).

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1
(1-Phenethylpiperidin-4-yl)methanamine (42) [40]. Yield 63%. H-NMR (CDCl3): 1.27–1.31 (m,
3H), 1.75–1.78 (m, 2H), 2.03 (t, 2H, J = 10.7 Hz), 2.58–2.62 (m, 4H), 2.81–2.86 (m, 2H), 3.05 (d, 2H, J = 10.8
Hz), 7.21–7.32 (m, 5H, ar).
4.1.7. General Procedure for the Synthesis of 45–50
In a 100 mL flask, equipped with a reflux condenser and a magnetic stirrer, benzyl piperidine 23
or the proper piperazine 24–28 (5 mmol), alkyl bromide 44 (5 mmol), K2CO3 (10 mmol), and MeCN
(30 mL) were added. The resulting mixture was refluxed for 14 h. The warm suspension was filtered
and the resulting filtrate was concentrated under reduced pressure. The crude material was purified
by crystallization.
2-(2-(4-Benzylpiperidin-1-yl)ethyl)isoindoline-1,3-dione (45). Yield 54%. Mp: 100–102 ◦C
(acetonitrile). 1H-NMR (CDCl3): 1.18–1.29 (m, 2H), 1.48–1.54 (m, 1H), 1.60–1.63 (m, 2H), 1.97 (t,
2H, J = 10.8 Hz), 2.51–2.52 (m, 2H), 2.61 (t, 2H, J = 6.9 Hz), 2.97 (d, 2H, J = 11.3 Hz), 3.84 (t, 2H, J = 6.9
Hz), 7.13–7.15 (m, 2H, ar), 7.17–7.21 (m, 1H, ar), 7.26–7.30 (m, 2H, ar), 7.72–7.75 (m, 2H, ar), 7.85–7.87
(m, 2H, ar). IR: 1770, 1708, 1705. Anal. calcd. for (C22H24N2O2): C, 75.85%; H, 6.94%; N, 8.04%. Anal.
found: C, 76.17%; H, 7.23%; N, 8.33%.
2-(2-(4-Phenylpiperazin-1-yl)ethyl)isoindoline-1,3-dione (46) [36]. Yield 45%. Mp: 152–154 ◦C
1
(acetonitrile). H-NMR (CDCl3): 2.69–2.74 (m, 6H), 3.14–3.17 (m, 4H), 3.89 (t, 2H, J = 6.5 Hz), 6.85 (t,
1H,ar, J = 7.3 Hz), 6.91–6.93 (m, 2H, ar), 7.24–7.26 (m, 2H, ar), 7.72–7.75 (m, 2H, ar), 7.85–7.88 (m, 2H,
ar). IR: 1712. Anal. calcd. for (C20H21N3O2): C, 71.62%; H, 6.31%; N, 12.53%. Anal. found: C, 71.95%;
H, 6.52%; N, 12.88%.
◦
2-(2-(4-Benzylpiperazin-1-yl)ethyl)isoindoline-1,3-dione (47) [37]. Yield 43%. Mp: 88–90 C
1
(acetonitrile). H-NMR (CDCl3): 2.44 (br s, 4H), 2.57 (br s, 4H), 2.66 (t, 2H, J = 6.7 Hz), 3.49 (s, 2H), 3.83
(t, 2H, J = 6.7 Hz), 7.24–7.33 (m, 5H, ar), 7.72–7.75 (m, 2H, ar), 7.85–7.87 (m, 2H, ar). Anal. calcd. for
(C21H23N3O2): C, 72.18%; H, 6.63%; N, 12.03%. Anal. found: C, 72.39%; H, 6.50%; N, 12.29%.
◦
2-(2-(4-Phenethylpiperazin-1-yl)ethyl)isoindoline-1,3-dione (48). Yield 42%. Mp: 131–133 C
1
(acetonitrile). H-NMR (CDCl3): 2.52–2.69 (m, 12H), 2.74–2.82 (m, 2H), 3.85 (t, 2H, J = 6.7 Hz), 7.19–7.22
(m, 3H, ar), 7.27–7.31 (m, 2H, ar), 7.71–7.75 (m, 2H, ar), 7.84–7.88 (m, 2H, ar). Anal. calcd. for
(C22H25N3O2): C, 72.70%; H, 6.93%; N, 11.56%. Anal. found: C, 72.81%; H, 6.81%; N, 11.43%.
2-(2-(4-(4-(2-Methoxyethoxy)phenyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (49). Yield 36%.
1
Mp: 124–126 ◦C (acetonitrile). H-NMR (CDCl3): 2.69–2.74 (m, 6H), 3.04–3.06 (m, 4H), 3.46 (s, 3H),
3.74 (t, 2H, J = 4.8 Hz), 3.88 (t, 2H, J = 6.6 Hz), 4.08 (t, 2H, J = 4.8 Hz), 6.87 (s, 4H, ar), 7.72–7.74 (m, 2H,
ar), 7.84–7.87 (m, 2H, ar). IR: 1697. Anal. calcd. for (C23H27N3O4): C, 67.46%; H, 6.65%; N, 10.26%.
Anal. found: C, 67.55%; H, 7.01%; N, 10.33%.
2-(2-(4-(Furan-2-carbonyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (50). Yield 61%. Mp:
◦
146–148 C (acetonitrile). 1H-NMR (CDCl3): 2.57–2.60 (m, 4H), 2.69 (t, 2H, J = 6.4 Hz), 3.74 (br
s, 4H), 3.85 (t, 2H, J = 6.4 Hz), 6.47–6.48 (m, 1H, ar), 6.97–6.98 (m, 1H, ar), 7.48–7.49 (m, 1H, ar), 7.72–7.76
(m, 2H, ar), 7.85–7.89 (m, 2H, ar). Anal. calcd. for (C19H19N3O4): C, 64.58%; H, 5.42%; N, 11.89%. Anal.
found: C, 64.67%; H, 5.69%; N, 12.17%.
4.1.8. Ethyl 4-(4-(2-(1,3-dioxoisoindolin-2-yl)ethyl)piperazin-1-yl)benzoate (51)
In a 100 mL flask, equipped with a reflux condenser and a magnetic stirrer, the aryl piperazine 43
(5 mmol), alkyl bromide 44 (5 mmol), Et3N (6 mmol), and MeCN (60 mL) were added. The resulting
mixture was refluxed for 24 h. The solution was concentrated under reduced pressure and the oily
residue was treated with water (20 mL). The solid was filtered, washed with diethyl ether, and used as
1
such in the next step. Yield 46%. H-NMR (DMSO-d6):
δ 1.28 (t, 3H, J = 7.5 Hz), 2.53–2.65 (m, 6H),
3.22–3.27 (m, 4H), 3.70–3.75 (m, 2H), 4.23 (q, 2H, J = 7.5 Hz), 6.96 (d, 2H, ar, J = 7.9 Hz), 7.76 (d, 2H, ar,
J = 7.9 Hz), 7.81–7.92 (m, 4H, ar).

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4.1.9. General Procedure for the Synthesis of 58–61
Benzaldehyde (5 mmol) was added to a solution of 4-aminomethylpiperidine 52 (5 mmol) in
absolute ethanol (10 mL), and the mixture was heated under reflux for 24 h. After cooling, the solvent
was removed by evaporation at reduced pressure. Oily N-(piperidin-4-ylmethyl)-1-phenylmethanimine
53 [39] was thus obtained, and used in a subsequent reaction without further purification. The imine
derivative 53 (2.6 mmol) was dissolved in acetone (15 mL) containing potassium carbonate (5.1 mmol)
and the proper bromide derivative 54–57 (3.1 mmol). The mixture thus obtained was stirred at room
temperature for 12 h, then suspension was filtered and the solvent was evaporated at reduced pressure.
The remaining oily residue without further purification was used in the next step.
N-((1-(2-Methoxyethyl)piperidin-4-yl)-1-phenylmethanimine (58). Yield 67%. 1H-NMR
(DMSO-d6): 1.04–1.14 (m, 1H), 1.17–1.26 (m, 1H), 1.57–1.67 (m, 4H), 1.89–1.94 (m, 1H), 2.41–2.44
(m, 2H), 2.84–2.93 (m, 2H), 3.23 (s, 3H), 3.38–3.45 (m, 4H), 7.42–7.45 (m, 3H, ar), 7.72–7.74 (m, 2H, ar),
8.30 (s, 1H, CH).
N-((1-Benzylpiperidin-4-yl)methyl)-1-phenylmethanimine (59) [39]. Yield 90%. 1H-NMR
(DMSO-d6): 1.22–1.25 (m, 2H), 1.62–1.65 (m, 3H), 1.91 (t, 2H, J = 10.9 Hz), 2.78–2.81 (m, 2H),
3.42–3.45 (m, 4H), 7.18–7.28 (m, 5H, ar), 7.40–7.44 (m, 3H, ar), 7.70–7.72 (m, 2H, ar), 8.30 (s, 1H, CH).
1
N-((1-(4-Methoxybenzyl)piperidin-4-yl)methyl)-1-phenylmethanimine (60). Yield 90%. H-NMR
(DMSO-d6): 1.15–1.27 (m, 2H), 1.59–1.65 (m, 3H), 1.83–1.93 (m, 2H), 2.79–2.81 (m, 2H), 3.38 (s, 2H),
3.45–3.46 (m, 2H), 3.73 (s, 3H), 6.86 (d, 2H, ar, J = 8.5 Hz), 7.19 (d, 2H, ar, J = 8.5 Hz), 7.41–7.45 (m, 3H,
ar), 7.72–7.74 (m, 2H, ar), 8.30 (s, 1H, CH).
N-((1-Phenethylpiperidin-4-yl)methyl)-1-phenylmethanimine (61) [40]. Yield 90%. 1H-NMR
(DMSO-d6): 1.11–1.27 (m, 2H), 1.59–1.73 (m, 3H), 1.94 (t, 2H, J = 10.8 Hz), 2.69–2.73 (m, 2H), 3.11–3.14
(m, 2H), 3.45–3.47 (m, 2H), 3.73 (t, 2H, J = 7.2 Hz), 7.15–7.33 (m, 5H, ar), 7.44–7.45 (m, 3H, ar), 7.73–7.74
(m, 2H), 8.31 (s, 1H, CH).
4.2. Pharmacological Assays
4.2.1. Cell Culture and Membrane Preparation
CHO cells transfected with hA1, hA2A, hA2B, and hA3 ARs were grown adherently and
maintained in Dulbecco’s modified Eagle’s medium with nutrient mixture F12, containing 10%
fetal calf serum, penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2 mM), geneticin (G418;
0.2 mg/mL) at 37 ◦C in 5% CO2/95% air [44]. For membrane preparation, the cells were washed with
phosphate-buffered saline and scraped off T75 flasks in an ice-cold hypotonic buffer (5 mMTris-HCl,
1 mM EDTA, pH 7.4). The cell suspensions were homogenized with a Polytron, centrifuged for
30 min at 40,000
experiments [44].
×
g at 4 ◦C and the resulting membrane pellets were used for competition binding
4.2.2. Competition Binding Experiments
All synthesized compounds have been tested for their affinity to hA1, hA2A, and hA3
ARs. Competition experiments to hA1 ARs were performed incubating 1 nM [3H]-8-cyclopentyl-
1,3-dipropylxanthine ([3H]-DPCPX) with membrane suspension (50
µ
g of protein/100
concentrations of the examined compounds at 25 ◦C for 90 min in 50 mM TrisHCl, pH 7.4. Non-specific
binding was defined as binding in the presence of 1 M DPCPX and was always <10% of the total
binding [44]. Inhibition experiments to hA2A ARs were performed incubating 1 nM of [3H]-ZM241385
µL) and different
µ
with the membrane suspension (50
compounds for 60 min at 4 C in 50 mMTris-HCl (pH 7.4), 10 mM MgCl2. Non-specific binding
µ
g of protein/100
µ
L) and different concentrations of the examined
◦
was evaluated in the presence of 1
Competition binding experiments to A3 ARs were carried out incubating the membrane suspension
(50 g of protein/100
L) with 0.5 nM [125I]-N6-(4-aminobenzyl)-N-methylcarboxamidoadenosine
125I]-ABMECA) in the presence of different concentrations of the examined compounds for an
µM ZM241385 and was about 20% of the total binding [45].
µ
µ
([

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incubation time of 120 min at 4 ◦C in 50 mMTris-HCl (pH 7.4), 10 mM MgCl2, 1 mM EDTA. Non-specific
binding was defined as binding in the presence of 1 M ABMECA and was always < 10% of the total
µ
binding [46]. 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 in a Packard Tri Carb 2810 TR scintillation
counter (Perkin Elmer, Waltham, MA, USA).
4.2.3. Cyclic AMP Assays
CHO cells transfected with hAR subtypes were washed with phosphate-buffered saline, detached
with trypsin, and centrifuged for 10 min at 200 g. Cells were seeded in a 96-well white half-area
×
microplate (Perkin Elmer, Boston, USA) in a stimulation buffer composed of Hank Balanced Salt
solution, 5 mM HEPES, 0.5 mM Ro 20-1724, 0.1% BSA, 1 IU/mL adenosine deaminase. cAMP levels
were then quantified by using the AlphaScreencAMP detection kit (Perkin Elmer, Waltham, MA, USA)
following the manufacturer’s instructions [47]. At the end of the experiments, plates were read with
the Perkin Elmer EnSight Multimode Plate Reader.
4.2.4. Data Analysis
The protein concentration was determined according to a Bio-Rad method with bovine albumin
as a standard reference. Inhibitory binding constant (Ki) values were calculated from those of IC50
according to the Cheng and Prusoff equation Ki = IC50/(1 + [C*]/KD*), where [C*] is the concentration
of the radioligand and KD* is its dissociation constant [46]. Ki and IC50 values were calculated by
the non-linear regression analysis using the equation for a sigmoid concentration-response curve
(Graph-PAD Prism, San Diego, CA, USA).
5. Conclusions
In conclusion, the herein reported structural investigation has led to a good number of new
7-amino-2-(furan-2-yl)-thiazolo[5,4-d]pyrimidines, featuring piperidine or piperazine substituents
at position 5, endowed with potent and selective hA2A AR inverse agonist activities. Among them,
compound 11 bearing a phenylpiperazine-ethylamino chain at position 5, showed the highest hA2A
AR binding affinity and potency. Furthermore, the SwissADME prediction indicated that compounds
8, 11, and 19 exhibited good drug-likeness properties.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/13/8/161/s1,
Figure S1: Inhibition curves of cAMP levels in hA2A CHO cells by selected compounds in comparison with
the reference compound ZM 241385. Table S1: Selected physicochemical and pharmacokinetic properties and
drug-likeness predictions of analyzed compounds 8, 11, 14, 15, 19.
Author Contributions: F.V. (Flavia Varano) conceived and supervised the research work, and wrote the manuscript;
D.C. designed and performed the synthetic and analytical experiments; E.V. performed the synthetic experiments;
V.C. supervised the synthetic and analytical experiments, and analyzed the data; K.V. and F.V. (Fabrizio Vincenzi)
designed the pharmacological experiments, and processed the data; S.P. performed the pharmacological
experiments. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by an intramural grant from the University of Florence (Fondi Ateneo
Ricerca 2019).
Conflicts of Interest: The authors declare no conflict of interest.
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