Piperazine- and Piperidine-Containing Thiazolo[5,4-d]pyrimidine Derivatives as New Potent and Selective Adenosine A_2A Receptor Inverse Agonists

Abstract

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.

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, A_2A, A_2B, and A₃ adenosine receptors (ARs). The A₁ and A₃ receptors are principally coupled to G_i/o proteins thus inducing an inhibitory effect on adenylyl cyclase and reducing cAMP production, while the A_2A and A_2B receptors stimulate the production of cAMP via G_s 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 A_2A AR is located both peripherally and centrally, with the highest expression levels in the striatum, olfactory tubercle, and the immune system. The A_2A 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 A_2A 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 A_2A AR antagonists thus suggesting that they may delay the onset and progression of PD [5]. The A_2A 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 A_2A AR antagonists prevent amyloid beta toxicity accompanied by improvement of spatial memory [12].

Figure 1.

A_2A 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 A_2A AR as a new target for cancer immunotherapy [13,14]. In fact, the A_2A AR represents an important immune checkpoint for T cells and NK cells and its activation induces suppression of immune cells response. Considering the increased A_2A AR expression in activated tumor infiltrating T cells, it is thus clear that this mechanism is important to favor tumor escape [15]. Moreover, A_2A 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 A_2A AR antagonists to avoid immunoescaping of tumor cells and tumor development is evident. Indeed, four A_2A 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) A_2A 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 hA_2A 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 A_2A 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 A_2A 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.

Figure 2.

General structure of the designed 7-amino-2-(furan-2-yl)-thiazolo[5,4-d]pyrimidines.

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).

Scheme 1.

Reagents and conditions. (a) n-BuOH, 200 °C MW, 20 min.

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.

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.

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.

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, hA_2A, 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, hA_2A, and hA₃ ARs. Activities of compounds 1–8, 10–21 at the hA_2B AR subtype was determined by measuring the inhibition of NECA stimulated adenylyl cyclase activity in CHO cells expressing the hA_2B receptor. Compounds 8, 11, 14–15, and 19, the best in terms of hA_2A AR affinity and selectivity, were also evaluated for their functional behavior. Hence, compounds were tested to assess their ability to modulate cAMP production in hA_2A CHO cells. All pharmacological data are reported in Table 1 and Table 2 together with those of the reference compound ZM 241,385 [41].

Table 1.

Binding affinities (K_i) at hA₁, hA_2A, and hA₃ ARs and potencies (IC₅₀) at hA_2B ARs.

	R5	hA1AR a Ki (nM) (I%) e	hA2AAR b Ki (nM)	hA2BAR c IC50 (nM) (I%) e	hA3AR d Ki (nM) (I%) e
1		586 ± 37	594 ± 48	>10,000 (21%)	>10,000 (8%)
2		296 ± 22	64 ± 5	>10,000 (15%)	>10,000 (18%)
3		180 ± 15	58 ± 5	10,000 (26%)	868 ± 82
4		1213 ± 114	237 ± 17	>10,000 (11%)	197 ± 16
5		2355 ± 213	326 ± 27	>10,000 (7%)	127 ± 15
6		2766 ± 249	137 ± 11	>10,000 (10%)	816 ± 74
7		345 ± 28	29 ± 3	>10,000 (23%)	>10,000 (34%)
8		638 ± 56	15.1 ± 1.3	>10,000 (19%)	>10,000 (25%)
9 f		4536 ± 312	279 ± 23	>10,000 (38%)	2679 ± 221
10		725 ± 67	187 ± 16	>10,000 (22%)	>10,000 (22%)
11		102 ± 9	8.62 ± 0.74	>10,000 (13%)	>10,000 (15%)
12		798 ± 72	92 ± 8	>10,000 (26%)	>10,000 (17%)
13		522 ± 47	37 ± 3	>10,000 (29%)	>10,000 (33%)
14		452 ± 38	18.3 ± 1.9	>10,000 (17%)	1492 ± 126
15		436 ± 36	10.8 ± 1.0	>10,000 (16%)	>10,000 (23%)
16		>10,000 (31%)	264 ± 24	>10,000 (26%)	>10,000 (34%)
17		>10,000 (29%)	>10,000 (33%)	>10,000 (17%)	>10,000 (28%)
18		524 ± 41	802 ± 73	>10,000 (11%)	>10,000 (28%)
19		365 ± 29	15.2 ± 1.7	>10,000 (18%)	>10,000 (35%)
20		152 ± 11	88 ± 9	>10,000 (24%)	>10,000 (29%)
21		247 ± 18	483 ± 34	>10,000 (27%)	>10,000 (21%)
ZM 241385		188 ± 16	0.94 ± 0.07	51 ± 4	672 ± 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, hA_2AARs, or A₃ARs, respectively (n = 3–6). ^c Potency (IC₅₀) in cAMP assays to hA_2BARs. ^e Percentage of inhibition (I%) is determined at 10 μM concentration of the tested compounds. ^f Ref. 31.

Table 2.

Potency (IC₅₀) of selected compounds on cyclic AMP assays in CHO cells expressing hA_2A AR.

Compounds	Potency IC50, nM	Intrinsic Activity	Pharmacological Behavior
8	13.8 ± 1.2	−44 ± 3	Inverse agonist
11	7.42 ± 0.68	−52 ± 4	Inverse agonist
14	15.2 ± 1.3	−51 ± 5	Inverse agonist
15	9.42 ± 0.87	−67 ± 5	Inverse agonist
19	14.8 ± 1.4	−64 ± 4	Inverse agonist
ZM 241385	1.42 ± 0.11	−48 ± 4	Inverse agonist

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 hA_2A AR (8.62 nM < K_i < 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 hA_2A AR binding activity of the piperidine substituted 1 (K_i = 594 nM) and of its corresponding piperazine analogue 3 (K_i = 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 hA_2A 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 hA_2A 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 hA_2A AR affinity displaying a K_i value of 8.6 nM. Compared to the latter, the (furan-2-yl)methanonepiperazine derivative 15 shows a similar binding activity (K_i = 10.8 nM) but is more selective toward the hA_2A AR. Compound 14, characterized by the same side chain of Preladenant, possesses high hA_2A AR affinity (K_i = 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 (K_i = 15.2 nM) and a good selectivity toward the A_2A subtype is reached.

Selected compounds 8, 11, 14–15, and 19, the best in terms of hA_2A AR affinity and selectivity, were also evaluated in functional assays to assess their ability to modulate cAMP production in hA_2A 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 hA_2A 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 Ų suggesting that they could permeate cell membranes. The Consesus log P_o/w (octanol/water partition coefficient) values indicated rather a reasonable absorption (1.71 < Consensus log P_o/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 Ų); 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).

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 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, hA_2A, hA_2B, 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, hA_2A, 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 hA_2A 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*]/K_D*), where [C*] is the concentration of the radioligand and K_D* is its dissociation constant [46]. K_i 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 hA_2A AR inverse agonist activities. Among them, compound 11 bearing a phenylpiperazine-ethylamino chain at position 5, showed the highest hA_2A 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 hA_2A 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.