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Article

Piperazine- and Piperidine-Containing Thiazolo[5,4-d]pyrimidine Derivatives as New Potent and Selective Adenosine A2A Receptor Inverse Agonists

by
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
2
Dipartimento di Morfologia, Chirurgia e Medicina Sperimentale, Università degli Studi di Ferrara, Via Fossato di Mortara 17-19, 44121 Ferrara, Italy
*
Author to whom correspondence should be addressed.
Pharmaceuticals 2020, 13(8), 161; https://doi.org/10.3390/ph13080161
Submission received: 29 June 2020 / Revised: 20 July 2020 / Accepted: 21 July 2020 / Published: 24 July 2020
(This article belongs to the Special Issue Adenosine Receptors as Attractive Targets in Human Diseases)
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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 8, 11, and 19 possessed good drug-likeness profiles.

Graphical Abstract

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 [3,4,5]. Several A2A AR antagonists have demonstrated to improve PD motor dysfunctions in various preclinical animal models as well as in clinical studies [5]. Furthermore, neuroprotective functions were associated with the use of A2A AR antagonists thus suggesting that they may delay the onset and progression of PD [5]. The A2A AR antagonists, such as Tozadenant (SYN 115) [6], ST 1535 [7], Vipadenant [8], Preladenant (SCH 420814) [9], and Istradefylline [10], have been clinically investigated showing potential effect 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].
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 [21,22,23,24,25,26,27,28,29,30,31]. Among these, the thiazolo[5,4-d]pyrimidine one (TP series) has been deeply investigated allowing us to delineate comprehensive structure activity relationships [21,25,26,27,31]. This was possible because the central thiazolopyrimidine scaffold can 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 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 [21,25,26,27]. 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 1–8, 10–21 (Figure 2) bearing at position 5 a piperidine or a piperazine moiety directly attached to the bicyclic core (1, and 2–8, respectively) or spaced by an ethylamino chain (10 and 11–16, respectively). Moreover, a little set of compounds bearing at position 5 a methylamino (17) or a methylaminopiperidine chain (18–21) is reported.

2. Results

2.1. Chemistry

Compounds 1–8, 10–21 were prepared following a common procedure that first involved the 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 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 23–42, under microwave irradiation, delivered the target compounds 1–8, 10–21. The amines 23–24, 26, 28–30 were commercial, while 25, 27, and 38 were prepared according to the literature [33,34,35]. The ethylamine derivatives 31–37 [36,37] 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 45–51. Removal of the phthaloyl group of the latters by hydrazinolysis produced compounds 31–37.
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.

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 Table 1 and Table 2 together with those of the reference compound ZM 241,385 [41].

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 1–9 bear a piperidine (1) or a piperazine (2–9) substituted ring directly linked to the bicyclic thiazolopyrimidine core. Comparison of the hA2A AR binding activity of the piperidine substituted 1 (Ki = 594 nM) and of its corresponding piperazine analogue 3 (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 (2) or benzyl residue (3) was equally tolerated, a longer phenylethyl group (4) or a para-substituent (OCH2CH2OCH3, COOEt) on the phenyl ring of 2 (compounds 5 and 9, respectively), produced a drop in the binding activity. Introduction of a furan-2-yl methanone residue on the piperazine ring gave compound 6 which shows good hA2A AR affinity even if lower than that of 2 and 3. In contrast, the presence of an ethylamine chain yielded derivatives 7–8 endowed with higher hA2A AR affinity than that of 2 and 3. Moreover, the (pyrrolidin-1-yl)ethyl derivative 8 is also highly selective toward this receptor subtype.
With respect to derivatives 1–6 and 9, the piperidine or piperazine residue at position 5 of compounds 10–16 was shifted from the thiazolopyrimidine core by an ethylamino linker thus increasing 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 (17–21), all characterized by an aminomethyl linker between the bicyclic core and the ethylbenzoate (17) or the substituted piperidine residue (18–21), 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.

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 8, 11, 15, and 19 had no PAINS alerts, while 14 showed one alert. The topological surface area (TPSA) measures the drug ability to permeate cells. Compounds 8, 11, and 19 showed similar TPSA values less than 140 Å2 suggesting that they could 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.
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+).

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 derivative 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 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). 1H-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%.
2-(Furan-2-yl)-5-(4-phenylpiperazin-1-yl)thiazolo[5,4-d]pyrimidin-7-amine (2). Yield 30%. Mp: 219–221 °C (ethanol). 1H-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%.
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 30%. Mp: 203–204 °C (ethanol). 1H-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 (5). Yield 50%. Mp: 181–183 °C (methanol). 1H-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 7/3. Yield 25%. Mp: 227–229 °C (tetrahydrofuran/water). 1H-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, 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-Benzylpiperidin-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). Yield 60%. Mp: 218–220 °C (2-methoxyethanol). 1H-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-5,7-diamine (14). Yield 25%. Mp: 203–204 °C (ethanol). 1H-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 acetate/cyclohexane/methanol 5/4/1. Yield 17%. Mp: 242–244 °C. 1H-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 (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). Yield 17%. Mp: 188–190 °C (ethyl acetate). 1H-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 27%. Mp: 174–176 °C. 1H-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 × 30 mL) dried over Na2SO4 and concentrated under reduced pressure. The oily residue without further purification was used in the following step.
2-(4-Benzylpiperidin-1-yl)ethan-1-amine (31). Yield 85%. 1H-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.
2-(4-Benzylpiperazin-1-yl)ethan-1-amine (33) [37]. Yield 85%. 1H-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).
2-(4-Phenethylpiperazin-1-yl)ethan-1-amine (34). Yield 90%. 1H-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 used without further purification in the following step. Yield 50%. 1H-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-(2-Methoxyethyl)piperidin-4-yl)methanamine (39). Yield 60%. 1H-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-Benzylpiperidin-4-yl)methanamine (40) [39]. Yield 35%. 1H-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-(4-Methoxybenzyl)piperidin-4-yl)methanamine (41). Yield 55%. 1H-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).
(1-Phenethylpiperidin-4-yl)methanamine (42) [40]. Yield 63%. 1H-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 (acetonitrile). 1H-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 (acetonitrile). 1H-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 (acetonitrile). 1H-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%. Mp: 124–126 °C (acetonitrile). 1H-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 such in the next step. Yield 46%. 1H-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).

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).
N-((1-(4-Methoxybenzyl)piperidin-4-yl)methyl)-1-phenylmethanimine (60). Yield 90%. 1H-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× g at 4 °C and the resulting membrane pellets were used for competition binding experiments [44].

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 μg of protein/100 μL) and different 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 μM DPCPX and was always <10% of the total binding [44]. Inhibition experiments to hA2A ARs were performed incubating 1 nM of [3H]-ZM241385 with the membrane suspension (50 μg of protein/100 μL) and different concentrations of the examined compounds for 60 min at 4 °C in 50 mMTris-HCl (pH 7.4), 10 mM MgCl2. Non-specific binding was evaluated in the presence of 1 μM ZM241385 and was about 20% of the total binding [45]. Competition binding experiments to A3 ARs were carried out incubating the membrane suspension (50 μg of protein/100 μL) with 0.5 nM [125I]-N6-(4-aminobenzyl)-N-methylcarboxamidoadenosine ([125I]-ABMECA) in the presence of different concentrations of the examined compounds for an 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 μM ABMECA and was always < 10% of the total 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 https://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.

References

  1. Borea, P.A.; Gessi, S.; Merighi, S.; Vincenzi, F.; Varani, K. Pharmacology of adenosine receptors: The state of the art. Physiol. Rev. 2018, 98, 1591–1625. [Google Scholar] [CrossRef] [PubMed]
  2. Borea, P.A.; Gessi, S.; Merighi, S.; Vincenzi, F.; Varani, K. Pathological overproduction: The bad side of adenosine. Br. J. Pharmacol. 2017, 174, 1945–1960. [Google Scholar] [CrossRef] [Green Version]
  3. Al-Attraqchi, O.H.A.; Attimarad, M.; Venugopala, K.N.; Nair, A.; Al-Attraqchi, N.H.A. Adenosine A2A receptor as a potential drug target—Current status and future perspectives. Curr. Pharm. Des. 2019, 25, 2716–2740. [Google Scholar] [CrossRef]
  4. Domenici, M.R.; Ferrante, A.; Martire, A.; Chiodi, V.; Pepponi, R.; Tebano, M.T.; Popoli, P. Adenosine A(2A) receptor as potential therapeutic target in neuropsychiatric disorders. Pharmacol Res. 2019, 147, 104338. [Google Scholar] [CrossRef]
  5. Zheng, J.; Zhang, X.; Zhen, X. Development of adenosine A(2A) receptor antagonists for the treatment of Parkinson’s Disease: A recent update and challenge. ACS Chem. Neurosci. 2019, 20, 783–791. [Google Scholar] [CrossRef] [PubMed]
  6. Flor, A.M.; Moreau, J.L.; Poli, S.M.; Riemer, C.; Steward, L. 4-Hydroxy-4-methyl-piperidine-1-carboxylic acid-(4-methoxy-7-morpholin-4-yl-benzothiazol-2-yl) Amide. US Patent US20050261289, 24 November 2005. [Google Scholar]
  7. Minetti, P.; Tinti, M.O.; Carminati, P.; Castorina, M.; Di Cesare, M.A.; Di Serio, S.; Gallo, G.; Ghirardi, O.; Giorgi, F.; Giorgi, L.; et al. 2-n-Butyl-9-methyl-8-[1,2,3]-triazol-2-yl-9H-purin-6-ylamine and analogues as A2A adenosine receptor antagonists. Design, synthesis, and pharmacological characterization. J. Med. Chem. 2005, 48, 6887–6896. [Google Scholar] [CrossRef] [PubMed]
  8. Gillespie, R.J.; Bamford, S.J.; Botting, R.; Comer, M.; Denny, S.; Gaur, S.; Griffin, M.; Jordan, A.M.; Knight, A.R.; Lerpiniere, J.; et al. Antogonists of the human A(2A) adenosine receptor. Design, synthesis, and preclinical evaluation of 7-aryltriazolo[4,5-d]pyrimidines. J. Med. Chem. 2009, 52, 33–47. [Google Scholar] [CrossRef] [PubMed]
  9. Hodgson, R.A.; Bedard, P.J.; Varty, G.B.; Kazdoba, T.M.; Di Paolo, T.; Grzelak, M.E.; Pond, A.J.; Hadjtahar, A.; Belanger, N.; Gregoire, L.; et al. Preladenant, a selective A2A receptor antagonist, is active in primate models of movement disorders. Exp. Neurol. 2010, 225, 384–390. [Google Scholar] [CrossRef] [PubMed]
  10. Pinna, A. Adenosine A2A receptor antagonists in Parkinson’s disease: Progress in clinical trials from the newly approved istradefylline to drugs in early development and those already discontinued. CNS Drugs 2014, 28, 455–474. [Google Scholar] [CrossRef]
  11. Chen, J.F.; Cunha, R.A. The belated US FDA approval of the adenosine A2A receptor antagonist istradefylline for treatment of Parkinson’s disease. Purinergic Signal. 2020. [Google Scholar] [CrossRef] [PubMed]
  12. Dall’Igna, O.P.; Fett, P.; Gomes, M.G.; Souza, D.O.; Cunha, R.A.; Lara, D.L. Caffeine and adenosine A2a receptor antagonists β-amyloid (25–35)-induced cognitive deficits in mice. Exp. Neurol. 2007, 203, 241–245. [Google Scholar] [CrossRef] [PubMed]
  13. Congreve, M.; Brown, G.A.; Borodovsky, A.; Lamb, M.L. Targeting adenosine A2A receptor antagonism for treatment of cancer. Expert Opin. Drug Discov. 2018, 13, 997–1003. [Google Scholar] [CrossRef] [PubMed]
  14. Vijayan, D.; Young, A.; Teng, M.W.L.; Smyth, M.J. Targeting immunosuppressive adenosine in cancer. Nat. Rev. Cancer 2017, 17, 709–724. [Google Scholar] [CrossRef] [PubMed]
  15. Ohta, A.; Gorelik, E.; Prasad, S.J.; Ronchese, F.; Lukashev, D.; Wong, M.K.K.; Huang, X.; Caldwell, S.; Liu, K.; Smith, P.; et al. A2A adenosine receptor protects tumors from antitumor T cells. Proc. Natl. Acad. Sci. USA 2006, 103, 13132–13137. [Google Scholar] [CrossRef] [Green Version]
  16. Merighi, S.; Battistello, E.; Giacomelli, L.; Varani, K.; Vincenzi, F.; Borea, P.A.; Gessi, S. Targeting A3 and A2A adenosine receptors in the fight against cancer. Exp. Opin. Ther. Targets 2019, 23, 669–678. [Google Scholar] [CrossRef] [PubMed]
  17. Merck Sharp and Dohme Corp. A phase Ib/II Study to Evaluate the Safety and Tolerability of Preladenant as a Single Agent and in Combination with Pembrolizumab in Subjects with Advanced Malignancies. ClinicalTrials.gov NLM Identifier: NCT03099161. Available online: https://clinicaltrials.gov/ct2/show/NCT03099161 (accessed on 4 April 2017).
  18. Mediavilla-Verela, M.; Castro, J.; Chiappori, A.; Noyes, D.; Hernandez, D.C.; Allard, B.; Stagg, J.; Antonia, S.J. A novel antagonist of the immune checkpoint protein adenosine A2A receptor restores tumor infiltrating lymphocyte activity in the context of the tumor microenvironment. Neoplasia 2017, 19, 530–536. [Google Scholar] [CrossRef] [PubMed]
  19. Corvus Pharmaceuticals, Inc. A phase 1/1b, Open Label, Multicenter, Repeat-Dose, Dose-Selection Study of CPI-444 as a Single Agent and in Combination with Atezolizumab in Patients with Selected Incurable Cancers. ClinicalTrials.gov, NML Identifier NCT02655822. Available online: https://clinicaltrials.gov/ct2/show/NCT02655822 (accessed on 14 January 2016).
  20. Congreve, M.; Andrews, S.P.; Dorè, A.S.; Hollestenin, K.; Hurrell, E.; Langmead, C.J.; Mason, J.S.; Ng, I.W.; Tehan, B.; Zhukov, A.; et al. Discovery of 1,2,4-triazine derivatives as adenosine A2A antagonists using structure based drug design. J. Med. Chem. 2012, 55, 1898–1903. [Google Scholar] [CrossRef] [PubMed]
  21. Varano, F.; Catarzi, D.; Vincenzi, F.; Betti, M.; Falsini, M.; Ravani, A.; Borea, P.A.; Colotta, V.; Varani, K. Design, synthesis and pharmacological characterization of 2-(2-furanyl)thiazolo[5,4-d]pyrimidine-5,7-diamine derivatives: New potent A2A adenosine receptor inverse agonists with antinociceptive activity. J. Med. Chem. 2016, 59, 10564–10576. [Google Scholar] [CrossRef]
  22. Poli, D.; Falsini, M.; Varano, F.; Betti, M.; Varani, K.; Vincenzi, F.; Pugliese, A.M.; Pedata, F.; Dal Ben, D.; Thomas, A.; et al. Imidazo[1,2-a]pyrazin-8-amine core for the design of new adenosine receptor antagonists: Structural exploration to target the A3 and A2A subtypes. Eur. J. Med. Chem. 2017, 125, 611–628. [Google Scholar] [CrossRef]
  23. Squarcialupi, L.; Betti, M.; Catarzi, D.; Varano, F.; Falsini, M.; Ravani, A.; Pasquini, S.; Vincenzi, F.; Salmaso, V.; Sturlese, M.; et al. The role of 5-arylalkylamino- and 5-piperazino moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 A2A receptor affinity and selectivity profiles. J. Enzym. Inhib. Med. Chem. 2017, 32, 248–263. [Google Scholar] [CrossRef] [Green Version]
  24. Falsini, M.; Squarcialupi, L.; Catarzi, D.; Varano, F.; Betti, M.; Dal Ben, D.; Marucci, G.; Buccioni, M.; Volpini, R.; De Vita, T.; et al. The 1,2,4-triazolo[4,3-a]pyrazin-3-one as a versatile scaffold for the design of potent adenosine human receptor antagonists. Structural investigations to target the A2A receptor subtype. J. Med. Chem. 2017, 60, 5772–5790. [Google Scholar] [CrossRef] [PubMed]
  25. 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. Bioorg. Med. Chem. 2018, 26, 3688–3695. [Google Scholar] [CrossRef] [PubMed]
  26. Varano, F.; Catarzi, D.; Vincenzi, F.; Falsini, M.; Pasquini, S.; Borea, P.A.; Colotta, V.; Varani, K. Structure-activity relationship studies and pharmacological ch.; aracterization of N5-heteroarylalkyl-substituted-2-(2-furanyl)-thiazolo[5,4-d]pyrimidine-5,7-diamine-based derivatives as inverse agonists at human A2A adenosine receptor. Eur. J. Med. Chem. 2018, 155, 552–561. [Google Scholar] [CrossRef] [PubMed]
  27. Varano, F.; Catarzi, D.; Falsini, M.; Dal Ben, D.; Buccioni, M.; Marucci, G.; Volpini, R.; Colotta, V. Novel human adenosine receptor antagonists based on the 7-amino-thiazolo[5,4-d]pyrimidine scaffold. Structural investigations at the 2-, 5- and 7- positions to enhance affinity and tune selectivity. Bioorg. Med. Chem. Lett. 2019, 29, 563–569. [Google Scholar] [CrossRef] [PubMed]
  28. Falsini, M.; Catarzi, D.; Varano, F.; Ceni, C.; Dal Ben, D.; Marucci, G.; Buccioni, M.; Volpini, R.; Di Cesare Mannelli, L.; Lucarini, E.; et al. Antioxidant-conjugated 1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives: Highly potent and selective human A2A adenosine receptor antagonists possessing protective efficacy in neuropathic pain. J. Med. Chem. 2019, 62, 8511–8531. [Google Scholar] [CrossRef] [PubMed]
  29. Falsini, M.; Catarzi, D.; Varano, F.; Dal Ben, D.; Marucci, G.; Buccioni, M.; Volpini, R.; Di Cesare Mannelli, L.; Ghelardini, C.; Colotta, V. Novel 8-amino-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives as potent human adenosine A1 and A2Areceptorantagonists. Evaluation of their protective effect against β-amyloid induced neurotoxicity in SHSY5Y cells. Bioorg. Chem. 2019, 87, 380–394. [Google Scholar] [CrossRef]
  30. Falsini, M.; Ceni, C.; Catarzi, D.; Varano, F.; Dal Ben, D.; Marucci, G.; Buccioni, M.; Navia, A.M.; Volpini, R.; Colotta, V. New 8-amino-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives. Evaluation of different moieties on the 6-aryl ring to obtain potent and selective human A2A adenosine receptor antagonists. Bioorg. Med. Chem. Lett. 2020, 30, 127126. [Google Scholar] [CrossRef]
  31. Varano, F.; Catarzi, D.; Vincenzi, F.; Pasquini, S.; Pelletier, J.; Lopes Rangel Fietto, J.; Espindola Gelsleichter, N.; Sarlandie, M.; Guilbaud, A.; Sevigny, J.; et al. Structural investigation on thiazolo[5,4-d]pyrimidines to obtain dual-acting blockers of CD73 and adenosine A2A receptor as potential antitumor agents. Bioorg. Med. Chem. Lett. 2020, 30, 127067. [Google Scholar] [CrossRef]
  32. Shaquiquzzaman, M.; Verma, G.; Marella, A.; Akhter, M.; Akhtar, W.; Khan, M.F.; Tasneem, S.; Alam, M.M. Piperazine scaffold: A remarkable tool in generation of diverse pharmacological agents. Eur. J. Med. Chem. 2015, 102, 487–529. [Google Scholar] [CrossRef]
  33. Long, J.Z.; Jin, X.; Adibekian, A.; Li, W.; Cravatt, B.F. Characterization of tunable piperidine and piperazine carbamates as inhibitors of endocannabinoid hydrolases. J. Med. Chem. 2010, 53, 1830–1842. [Google Scholar] [CrossRef] [Green Version]
  34. Silverman, L.S.; Caldwell, J.P.; Greenlee, W.J.; Kiselgof, E.; Matasi, J.J.; Tulshian, D.B.; Arik, L.; Foster, C.; Bertorelli, R.; Monopoli, A.; et al. 3H-[1,2,4]-Triazolo[5,1-i]purin-5-amine derivatives as adenosine A2A antagonists. Bioorg. Med. Chem. Lett. 2007, 17, 1659–1662. [Google Scholar] [CrossRef] [PubMed]
  35. Chun, C.; Schmitzer, A.R. A pseudorotaxane umbrella thread with chloride transmembrane transport properties. Med. Chem. Commun. 2011, 2, 987–990. [Google Scholar] [CrossRef]
  36. Mejuch, T.; Garivet, G.; Hofer, W.; Kaiser, N.; Fansa, E.K.; Ehrt, C.; Koch, O.; Baumann, M.; Ziegler, S.; Wittinghofer, A.; et al. Small-molecule inhibition of the UNC119-cargo interaction. Angew. Chem. Int. Ed. 2017, 56, 6181–6186. [Google Scholar] [CrossRef] [PubMed]
  37. Piemontese, L.; Tomas, D.; Hiremathad, A.; Capriati, V.; Candeias, E.; Cardoso, S.M.; Chaves, S.; Santos, M.A. Donezepil structure-based hybrids as potential multifunctional anti-Alzheimer’s drug candidates. J. Enzym. Inhib. Med. Chem. 2018, 33, 1212–1224. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Kubota, D.; Ishikawa, M.; Yamamoto, M.; Murakami, S.; Hachisu, M.; Katano, K.; Ajito, K. Tricyclic pharmacophore-based molecules as novel integrin αvβ3 antagonists. Part 1: Design and synthesis of a lead compound exhibiting αvβ3/αIIbβ3 dual antagonistic activity. Bioorg. Med. Chem. 2006, 2006, 2089–2108. [Google Scholar] [CrossRef]
  39. Diouf, O.; Depreux, P.; Chavatte, P.; Paupaert, J.H. Synthesis and preliminary pharmacological results on new naphthalene derivatives as 5-HT4 receptor ligands. Eur. J. Med. Chem. 2000, 35, 699–706. [Google Scholar] [CrossRef]
  40. Furlotti, G.; Alisi, M.A.; Cazzola, N.; Ceccacci, F.; Garrone, B.; Gasperi, T.; La Bella, A.; Leonelli, F.; Loreto, M.A.; Magarò, G.; et al. Targeting serotonin 2A and adrenergic α1 receptors for ocular antihypertensive agents: Discovery of 3,4-dihydropyrazino[1,2-b]indazol-1(2H)-one derivatives. ChemMedChem 2018, 13, 1597–1607. [Google Scholar] [CrossRef] [PubMed]
  41. Caulkett, P.W.R.; Jones, G.; Collis, M.G.; Poucher, S.M. Preparation of (amino)heteroaryl[1,2,4]triazolo[1,5-a]triazines and Related Compounds as Adenosine A2 Receptor Antagonists. Eur Patent Appl EP 459702, 23 May 1991. [Google Scholar]
  42. Daina, A.; Michielin, O.; Zoete, V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717. [Google Scholar] [CrossRef] [Green Version]
  43. Daina, A.; Zoete, V. A BOILED-Egg to predict gastrointestinal absorption and brain penetration of small molecules. ChemMedChem 2016, 11, 1117–1121. [Google Scholar] [CrossRef] [Green Version]
  44. Vincenzi, F.; Targa, M.; Romagnoli, R.; Merighi, S.; Gessi, S.; Baraldi, P.G.; Borea, P.A.; Varani, K. TRR469, a potent A1 adenosine receptor allosteric modulator, exhibits anti-nociceptive properties in acute and neuropathic pain models in mice. Neuropharmacology 2014, 81, 6–14. [Google Scholar] [CrossRef] [PubMed]
  45. Varani, K.; Massara, A.; Vincenzi, F.; Tosi, A.; Padovan, M.; Trotta, F.; Borea, P.A. Normalization of A2A and A3 adenosine receptor up-regulation in rheumatoid arthritis patients by treatment with anti-tumor necrosis factor alpha but not methotrexate. Arthritis Rheum. 2009, 60, 2880–2891. [Google Scholar] [CrossRef] [PubMed]
  46. Varani, K.; Merighi, S.; Gessi, S.; Klotz, K.N.; Leung, E.; Baraldi, P.G.; Cacciari, B.; Romagnoli, R.; Spalluto, G.; Borea, P.A. [3H]MRE 3008F20: A novel antagonist radioligand for the pharmacological and biochemical characterization of human A3 adenosine receptors. Mol. Pharmacol. 2000, 57, 968–975. [Google Scholar] [PubMed]
  47. Ravani, A.; Vincenzi, F.; Bortoluzzi, A.; Padovan, M.; Pasquini, S.; Gessi, S.; Merighi, S.; Borea, P.A.; Govoni, M.; Varani, K. Role and function of A2A and A3 adenosine receptors in patients with ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. Int. J. Mol. Sci. 2017, 18, 697. [Google Scholar] [CrossRef] [PubMed]
Pharmaceuticals 13 00161 g001 550
Figure 1. A2A AR antagonists progressed into clinical testing for the treatment of Parkinson’s disease (PD) and cancer.
Figure 1. A2A AR antagonists progressed into clinical testing for the treatment of Parkinson’s disease (PD) and cancer.
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Pharmaceuticals 13 00161 g002 550
Figure 2. General structure of the designed 7-amino-2-(furan-2-yl)-thiazolo[5,4-d]pyrimidines.
Figure 2. General structure of the designed 7-amino-2-(furan-2-yl)-thiazolo[5,4-d]pyrimidines.
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Pharmaceuticals 13 00161 sch001 550
Scheme 1. Reagents and conditions. (a) n-BuOH, 200 °C MW, 20 min.
Scheme 1. Reagents and conditions. (a) n-BuOH, 200 °C MW, 20 min.
Pharmaceuticals 13 00161 sch001
Pharmaceuticals 13 00161 sch002 550
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.
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.
Pharmaceuticals 13 00161 sch002
Pharmaceuticals 13 00161 sch003 550
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.
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.
Pharmaceuticals 13 00161 sch003
Pharmaceuticals 13 00161 g003 550
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).
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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Figure 4. Predicted BOILED-Egg diagram of the analyzed compounds, from the SwissADME web tool.
Figure 4. Predicted BOILED-Egg diagram of the analyzed compounds, from the SwissADME web tool.
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Table
Table 1. Binding affinities (Ki) at hA1, hA2A, and hA3 ARs and potencies (IC50) at hA2B ARs.
Table 1. Binding affinities (Ki) at hA1, hA2A, and hA3 ARs and potencies (IC50) at hA2B ARs.
Pharmaceuticals 13 00161 i001
R5hA1AR a
Ki (nM)
(I%) e		hA2AAR b
Ki (nM)		hA2BAR c
IC50 (nM)
(I%) e		hA3AR d
Ki (nM)
(I%) e
1 Pharmaceuticals 13 00161 i002586 ± 37594 ± 48>10,000
(21%)		>10,000
(8%)
2 Pharmaceuticals 13 00161 i003296 ± 2264 ± 5>10,000
(15%)		>10,000
(18%)
3 Pharmaceuticals 13 00161 i004180 ± 1558 ± 510,000
(26%)		868 ± 82
4 Pharmaceuticals 13 00161 i0051213 ± 114237 ± 17>10,000
(11%)		197 ± 16
5 Pharmaceuticals 13 00161 i0062355 ± 213326 ± 27>10,000
(7%)		127 ± 15
6 Pharmaceuticals 13 00161 i0072766 ± 249137 ± 11>10,000
(10%)		816 ± 74
7 Pharmaceuticals 13 00161 i008345 ± 2829 ± 3>10,000
(23%)		>10,000
(34%)
8 Pharmaceuticals 13 00161 i009638 ± 5615.1 ± 1.3>10,000
(19%)		>10,000
(25%)
9f Pharmaceuticals 13 00161 i0104536 ± 312279 ± 23>10,000
(38%)		2679 ± 221
10 Pharmaceuticals 13 00161 i011725 ± 67187 ± 16>10,000
(22%)		>10,000
(22%)
11 Pharmaceuticals 13 00161 i012102 ± 98.62 ± 0.74>10,000
(13%)		>10,000
(15%)
12 Pharmaceuticals 13 00161 i013798 ± 7292 ± 8>10,000
(26%)		>10,000
(17%)
13 Pharmaceuticals 13 00161 i014522 ± 4737 ± 3>10,000
(29%)		>10,000
(33%)
14 Pharmaceuticals 13 00161 i015452 ± 3818.3 ± 1.9>10,000
(17%) 		1492 ± 126
15 Pharmaceuticals 13 00161 i016436 ± 3610.8 ± 1.0>10,000
(16%)		>10,000
(23%)
16 Pharmaceuticals 13 00161 i017>10,000
(31%)		264 ± 24>10,000 (26%)>10,000 (34%)
17 Pharmaceuticals 13 00161 i018>10,000
(29%)		>10,000
(33%)		>10,000 (17%)>10,000 (28%)
18 Pharmaceuticals 13 00161 i019524 ± 41802 ± 73>10,000
(11%)		>10,000
(28%)
19 Pharmaceuticals 13 00161 i020365 ± 2915.2 ± 1.7>10,000
(18%)		>10,000
(35%)
20 Pharmaceuticals 13 00161 i021152 ± 1188 ± 9>10,000
(24%)		>10,000
(29%)
21 Pharmaceuticals 13 00161 i022247 ± 18483 ± 34>10,000
(27%)		>10,000
(21%)
ZM 241385188 ± 160.94 ± 0.0751 ± 4672 ± 51
Data are expressed as means ± SEM. Affinity values obtained from the displacement of specific [3H]DPCPX a, [3H]ZM241383 b, or [125I]AB-MECA d binding to hA1ARs, hA2AARs, or A3ARs, respectively (n = 3–6). c Potency (IC50) in cAMP assays to hA2BARs. e Percentage of inhibition (I%) is determined at 10 μM concentration of the tested compounds. f Ref. 31.
Table
Table 2. Potency (IC50) of selected compounds on cyclic AMP assays in CHO cells expressing hA2A AR.
Table 2. Potency (IC50) of selected compounds on cyclic AMP assays in CHO cells expressing hA2A AR.
CompoundsPotency
IC50, nM		Intrinsic ActivityPharmacological Behavior
813.8 ± 1.2−44 ± 3Inverse agonist
117.42 ± 0.68−52 ± 4Inverse agonist
1415.2 ± 1.3−51 ± 5Inverse agonist
159.42 ± 0.87−67 ± 5Inverse agonist
1914.8 ± 1.4−64 ± 4Inverse agonist
ZM 2413851.42 ± 0.11−48 ± 4Inverse agonist
Data are expressed as means ± SEM.

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MDPI and ACS Style

Varano, F.; Catarzi, D.; Vigiani, E.; Vincenzi, F.; Pasquini, S.; Varani, K.; Colotta, V. Piperazine- and Piperidine-Containing Thiazolo[5,4-d]pyrimidine Derivatives as New Potent and Selective Adenosine A2A Receptor Inverse Agonists. Pharmaceuticals 2020, 13, 161. https://doi.org/10.3390/ph13080161

AMA Style

Varano F, Catarzi D, Vigiani E, Vincenzi F, Pasquini S, Varani K, Colotta V. Piperazine- and Piperidine-Containing Thiazolo[5,4-d]pyrimidine Derivatives as New Potent and Selective Adenosine A2A Receptor Inverse Agonists. Pharmaceuticals. 2020; 13(8):161. https://doi.org/10.3390/ph13080161

Chicago/Turabian Style

Varano, Flavia, Daniela Catarzi, Erica Vigiani, Fabrizio Vincenzi, Silvia Pasquini, Katia Varani, and Vittoria Colotta. 2020. "Piperazine- and Piperidine-Containing Thiazolo[5,4-d]pyrimidine Derivatives as New Potent and Selective Adenosine A2A Receptor Inverse Agonists" Pharmaceuticals 13, no. 8: 161. https://doi.org/10.3390/ph13080161

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