Chemical Probes for the Adenosine Receptors

pharmaceuticals

Review Chemical Probes for the Adenosine Receptors

Stephanie Federico *, Lucia Lassiani and Giampiero Spalluto

Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via Licio Giorgeri 1, 34127 Trieste, Italy; [email protected] (L.L.); [email protected] (G.S.) * Correspondence: [email protected]; Tel.: +39-040-558-3671

Received: 26 September 2019; Accepted: 7 November 2019; Published: 12 November 2019

Abstract: Research on the adenosine receptors has been supported by the continuous discovery of new chemical probes characterized by more and more affinity and selectivity for the single adenosine receptor subtypes (A1,A2A,A2B and A3 adenosine receptors). Furthermore, the development of new techniques for the detection of G protein-coupled receptors (GPCR) requires new specific probes. In fact, if in the past radioligands were the most important GPCR probes for detection, compound screening and diagnostic purposes, nowadays, increasing importance is given to fluorescent and covalent ligands. In fact, advances in techniques such as fluorescence resonance energy transfer (FRET) and fluorescent polarization, as well as new applications in flow cytometry and different fluorescence-based microscopic techniques, are at the origin of the extensive research of new fluorescent ligands for these receptors. The resurgence of covalent ligands is due in part to a change in the common thinking in the medicinal chemistry community that a covalent drug is necessarily more toxic than a reversible one, and in part to the useful application of covalent ligands in GPCR structural biology. In this review, an updated collection of available chemical probes targeting adenosine receptors is reported.

Keywords: adenosine receptors; ﬂuorescent ligands; radioligands; radiotracers; covalent ligands; GPCR probes

1. Introduction Since their discovery in the mid seventies [1,2], adenosine receptors (ARs) have attracted research interest for their implication in a wide range of physiological and pathological processes (i.e., asthma, ischemia, cancer, Parkinson’s disease, etc.) [3]. As a consequence, at the same time research commenced on specific receptor probes that are essential tools for receptor characterization [4,5]. ARs exist as four different subtypes: A1,A2A,A2B and A3 ARs [6,7]. Due to the advancement in techniques for detection and characterization of receptors, and in particular of G protein-coupled receptors (GPCRs) [8–12], the availability of suitable probes is a constant need. In particular, this review covers three specific chemical probe families for ARs: radioactive, covalent and fluorescent ligands. Radioactive ligands, properly called radioligands, are the oldest class of AR probes, and still represent the principal tool in drug discovery since their use in binding assays [7]. Recently, the broad interest in radioactive ligands is due to their development as radiotracers in positron emission tomography (PET), leading to new diagnostic possibilities [13,14]. On the other hand, covalent ligands for GPCRs, which were in the past used as tools to purify, isolate or pharmacologically characterize receptors, have recently attracted the interest of the scientific community for their ability to stabilize their target protein, increasing the probability of obtaining X-ray crystal structures [10]. This strategy was successfully applied for the A1 AR subtype [15,16]. Since A2B and A3 AR crystal structures are still lacking, it is easy to imagine that several works will focus on development of covalent ligands for these receptor subtypes in the near future. Finally, the last few years have been characterized by the application of a

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Pharmaceuticals 2019, 12, x FOR PEER REVIEW 2 of 21 variety of fluorescence-based methods for GPCR structure biology and drug discovery [17]. These techniquesbiology and involve drug discovery the introduction [17]. These of atechniques fluorescent in tagvolve on athe GPCR introduction or on a GPCRof a fluorescent ligand, leading tag on to a fluorescentGPCR or on ligands, a GPCR which ligand, are leading discussed to fluorescen here [9,18–t 20ligands,]. The aimwhich of thisare discussed review is tohere give [9,18–20]. a panorama The ofaim the of available this review chemical is to give probes a panorama for the ARs of tothe researchers available chemical working probes in this fieldfor the or medicinalARs to researchers chemists workingworking onin this ARs field or other or medicinal GPCR targets. chemists working on ARs or other GPCR targets.

2. Radioligands and Radiotracers ItIt isis wellwell known,known, thatthat radioligandradioligand probes are usefuluseful forfor studyingstudying bothboth thethe distributiondistribution and functionsfunctions ofof receptors.receptors. In this class of compounds, two families of derivatives should be considered: i)i) radioligands,radioligands, generally generally tritiated tritiated or iodinated,or iodinated, for bindingfor binding studies; studies; ii) radioligands ii) radioligands used for used imaging, for 11 18 15 inimaging, general in probes general including probes including isotopes such isotopes as C,suchF as and 11C, 18O.F and 15O. InIn the first first class class of of compounds, compounds, in in the the last last deca decades,des, several several examples examples of radioligands of radioligands for forall AR all ARsubtypes, subtypes, both both agonists agonists and andantagonists, antagonists, withwith different different degrees degrees of potency of potency and selectivity and selectivity have havebeen beenreported reported and andextensively extensively reviewed reviewed [7,21–24]. [7,21– 24Our]. Our purpose purpose is to is togive give a abr briefief update update of of the the workwork developed inin thisthis fieldfield inin thisthis review.review. Considering labeled derivatives for binding studies only an agonist for A AR named Considering labeled derivatives for binding studies only an agonist for A2B2B AR named 3 [[3HH]-BAY60-6583]-BAY60-6583 (1)) waswas recentlyrecently reportedreported byby thethe groupgroup ofof Prof.Prof. C.A.C.A. MüllerMüller (Figure(Figure1 1))[ 25[25].].

Figure 1. Structure of BAY60-6583.

This partial agonist in in its its tritiated tritiated form form (the (the position position of of tritium tritium is is not not reported) reported) failed failed to to be be a agood good probe probe for for binding binding studies. studies. This This is is probably probably due due to to its moderate aaffinityffinity atat thethe humanhuman AA2B2B receptorreceptor andand highhigh levellevel ofof non-specificnon-specific binding.binding. The only results obtainedobtained usingusing thisthis radioligandradioligand indicateindicate thatthat nucleosidenucleoside and non-nucleosidenon-nucleoside agonistsagonists most probably bind the receptor in didifferentfferent conformations [[25].25]. InIn contrastcontrast toto thethe developmentdevelopment ofof tritiatedtritiated oror 125125II radioligands, radioligands, in in the last few years great eeffortsfforts have been mademade inin thethe fieldfield ofof radiotracersradiotracers forfor imagingimaging [[14].14]. InIn particular,particular, severalseveral examplesexamples ofof11 11C or 1818F derivatives for the different different AR subtypes have been reported. Regarding the 1111C derivatives, some recent examples (compounds 2–5) have been reportedreported inin Figure2 2,, in in particular, particular, regarding regarding the the A A11 andand AA2A2A ARs. One of the most studied derivatives is xanthine derivative [1-methyl-11C]8-dicyclopropylmethyl- 1-methyl-3-propylxanthine (11C-MPDX, 2). This compound has been utilized for various studies [26–28]. For example, Paul and coworkers performed in vivo binding studies in rats hypothesizing that agonists and antagonists may bind in different sites at the A1 receptor [26]. The same derivative was also utilized to evaluate A1 AR alterations in patients with chronic diffuse axonal injury [27] or to investigate the density of A1 ARs in patients with early-stage Parkinson’s disease [28]. Another interesting ligand for the A1 AR, is the pyridine derivative 3 reported by Guo et al. This compound could be considered the first brain blood barrier (BBB)-permeable A1 partial agonist with promising properties of detection of endogenous adenosine fluctuations [29]. Also in the field of A2A AR antagonists, some labeled compounds have been recently reported, in particular the styryl-xanthine derivative [7-methyl-11C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7- 11 trimethylxanthine ( C-TMSX, 4) that has been utilized for demonstrating that A2A receptors could be a future target to enhance brown adipose tissue (BAT) metabolism [30]. In addition, the non-xanthine

Pharmaceuticals 2019, 12, 168 3 of 21 labeled derivative 11C-preladenant (5) has been reported to be an interesting probe for investigating cerebral distribution of A2A ARs and other kinetic aspects such as its extensive distribution in the striatumPharmaceuticals [31,32 ].2019, 12, x FOR PEER REVIEW 3 of 21

11 O CH3 O 11 CH3 H C 3 N N NC CN O N N nC H N CH3 3 7 H2N N 11C-MPDX, 2 3

O 11CH 3 N H3C N OCH N 3 N N N N O O N N OCH3 O NN 11 N O CH3 nC3H7 OCH3 H2N

11 11C-Preladenant, 5 C-TMSX, 4 FigureFigure 2. 2.Structures Structures of of11 11CC labeled adenosine adenosine receptor receptor (AR) (AR) ligands. ligands.

Nevertheless,One of one ofthe the majormost problems studied of the usederiva of 11tivesC-labeled is compounds xanthine is the shortderivative half-life of this[1-methyl- isotope11C]8-dicyclopropylmethyl-1-methyl-3-propylxanthine (20 min), this means that the compound has to be prepared(11C-MPDX, and 2). immediately This compound utilized beforehas itsbeen inactivation. utilized for Forvarious this studies reason, [26–28]. other isotopesFor example, are preferred Paul and coworkers to 11C such performed as 18F, which in vivo has a half-lifebinding of 109 studies min. in In rats this hypothesizing ﬁeld, some promising that agonists agents and forantagonists A1,A2B mayand Abind3 receptors in different (6– 10sites) have at the been A1 receptor [26]. The same derivative was also utilized to evaluate A1 AR alterations in patients with reportedPharmaceuticals (Figure 20193)., 12, x FOR PEER REVIEW 4 of 21 chronic diffuse axonal injury [27] or to investigate the density of A1 ARs in patients with early-stage Parkinson’s disease [28]. O H O H Another interesting ligand for the A1 AR, is the pyridine derivative 3 reported by Guo et al. This nC3H7 N N R compound could beN considered the first brain blood barrier (BBB)-permeableN A1 partial agonist with promising properties of detection of endogenous adenosine fluctuations [29]. O N N O N N Also in the field of A2A AR antagonists, some labeled compounds have been recently reported, in particular the styryl-xanthine derivative 11 11 [7-methyl- C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine18F ( C-TMSX,18F 4) that has been utilized for demonstrating18F-CPFPX, that6 A2A receptors could be a futureR=H, target18F-CBCPM, to enhance7 brown adipose tissue (BAT) metabolism [30]. In addition, the non-xanthine labeled18 derivative 11C-preladenant (5) R=CH3, F-CPMMCB, 8 has been reported to be an interesting probe for investigating cerebral distribution of A2A ARs and other kinetic aspects such as its extensive distribution in the striatum [31,32]. Nevertheless, one of the major problems of the use of 11C-labeled compounds is the short 18 half-life of this isotope (20 min), this meansF that the compound has18F to be prepared and immediately utilized before its inactivation. For this reason, other isotopes are preferred to 11C such as 18F, which O N has a half-life of 109 min. In this field, some promising agents for A1, A2B and A3 receptors (6–10) O O N have been reportedS (Figure 3). O

N N N H F 18F-FE@SUPPY, 9 10

FigureFigure 3. 3.Structures Structures of of18 18F-labeled AR AR ligands. ligands.

OneOne of theof the most most used used18 F-labeled18F-labeled compounds compounds could be be consider considereded to to be be the the xanthine xanthine derivative derivative 18F-8-cyclopentyl-3-(3-ﬂuoropropyl)-1-propylxanthine18F-8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ( (1818F-CPFPX,F-CPFPX, 6).6 ).This This compound compound has has been been extensivelyextensively used used for for developing developing suitable suitable pharmacokinetic pharmacokinetic models models for for the the quantiﬁcation quantification ofof cerebralcerebral A1 ARsA [331 ARs]. [33]. It has also been utilized to visualize and quantify the in vivo occupancy of the human cerebral adenosine A1 receptor by caffeine [34], and demonstrated that its chronic use did not lead to

persistent changes in functional availability of A1 ARs [35]. Taking into account the results obtained with these derivatives, Kreft and coworkers developed other 18F-CPFPX structurally-related derivatives named CBCPM (8-cyclobutyl-1-cyclopropymethyl-3-(3-fluoropropyl)xanthine, 7) and CPMMCB (1-cyclopropylmethyl-3-(3-fluoropropyl)-8-(1-methylcyclobutyl)xanthine, 8), respectively, which showed similar behavior to the reference compound in preliminary PET studies [36]. In addition, a promising labeled derivative, named 18F-FE@SUPPY (5-(2-fluoroethyl)2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate, 9), has been developed for the A3 AR, which has been shown to be a tracer for studying pharmacokinetic aspects and mapping A3 ARs in rats [37,38]. Very recently, a 18F-labeled form of a pyrazine A2B AR antagonists (10) has been reported by Lindemann et al. [39]. Preliminary studies on this compound, which showed binding affinity in the nanomolar range towards the human A2B AR, clearly indicated the potential of this derivative as a tracer for studying this receptor subtype and provided an important basis for the development of new tracers for A2B AR [39].

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It has also been utilized to visualize and quantify the in vivo occupancy of the human cerebral adenosine A1 receptor by caffeine [34], and demonstrated that its chronic use did not lead to persistent changes in functional availability of A1 ARs [35]. Taking into account the results obtained with these derivatives, Kreft and coworkers developed other 18F-CPFPX structurally-related derivatives named CBCPM (8-cyclobutyl-1-cyclopropymethyl-3- (3-fluoropropyl)xanthine, 7) and CPMMCB (1-cyclopropylmethyl-3-(3-fluoropropyl)-8-(1-methylcyclobutyl) xanthine, 8), respectively, which showed similar behavior to the reference compound in preliminary PET studies [36]. In addition, a promising labeled derivative, named 18F-FE@SUPPY (5-(2-fluoroethyl)2,4-diethyl-3- (ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate, 9), has been developed for the A3 AR, which has been shown to be a tracer for studying pharmacokinetic aspects and mapping A3 ARs in rats [37,38]. 18 Very recently, a F-labeled form of a pyrazine A2B AR antagonists (10) has been reported by Lindemann et al. [39]. Preliminary studies on this compound, which showed binding affinity in the nanomolar range towards the human A2B AR, clearly indicated the potential of this derivative as a tracer for studying this receptor subtype and provided an important basis for the development of new tracers for A2B AR [39].

3. Covalent Ligands It is well known that covalent ligands may be considered important pharmacological tools for the study of the receptors; in fact, the irreversible blockade of the receptors could reveal details of both structural binding interactions and the patho-physiological role of the examined receptor. The strategy utilized for obtaining irreversible ligands is generally based on the introduction of chemoreactive moieties on the scaffold of well-known agonists and/or antagonists. The limiting step in preparing this kind of probe is the position on which the chemoreactive group should be introduced in order to obtain irreversible block of the receptor without losing both affinity and selectivity. For this reason, several structure–activity relationship studies (SAR) have been performed in order to optimize the structural requirements that are indispensable for obtaining irreversible probes that retain affinity and selectivity. In the field of ARs, several examples of covalent agonists and antagonists have been reported in the last decades. We wish, for this reason, to briefly summarize the results obtained in this research area. Regarding agonists for ARs, several examples of A1,A2A and A3 receptor subtypes are reported and depicted in Figures4 and5. The strategy to obtain irreversible agonists for ARs is based on the core modification of the natural nucleoside adenosine. Concerning the A1 AR subtype, examples of covalent agents are reported and summarized in Figure4[40–42]. In particular the N6 substituted derivative of adenosine, named m-DITC-ADAC (4-isothiocyanatophenylaminothiocarbonyl, DITC; adenosine amine congener or N6-[4-[[[4-[[[(2- Aminoethyl)amino]carbonyl]methyl]-anilino]carbonyl]methyl]phenyl]adenosine, ADAC) (11), proved to be a good irreversible agonist for A1 AR. It was able to mimic ischemic preconditioning in rabbits [40] and also prolong the stimulus of the His bundle (SA) interval by 2.1 fold in guinea pig isolated hearts [41]. Another interesting covalent agonist, R-AHPIA (2-azido-N-(2-(4-hydroxyphenyl)-1-methylethyl)- adenosine, 12) was reported by Klotz and coworkers [42]. This ligand was also labeled with 125I and, after UV irradiation, it irreversibly binds to the rat receptor, which was isolated for the first time as a protein of about 35 KDa [42]. A quite extensive SAR study on the adenosine nucleus for obtaining other covalent ligands for human ARs was performed by Jorg and coworkers [16], who obtained a series of compounds of general formula 13. Nevertheless no extensive pharmacological studies for their characterization were reported [16]. Pharmaceuticals 2019, 12, x FOR PEER REVIEW 5 of 21

introduced in order to obtain irreversible block of the receptor without losing both affinity and selectivity. For this reason, several structure–activity relationship studies (SAR) have been performed in order to optimize the structural requirements that are indispensable for obtaining irreversible probes that retain affinity and selectivity. In the field of ARs, several examples of covalent agonists and antagonists have been reported in the last decades. We wish, for this reason, to briefly summarize the results obtained in this research area. Regarding agonists for ARs, several examples of A1, A2A and A3 receptor subtypes are reported and depicted in Figures 4 and 5. PharmaceuticalsThe strategy2019, 12, 168to obtain irreversible agonists for ARs is based on the core modification of the5 of 21 natural nucleoside adenosine.

NCS H N O S O HN N N H H N N

N N HO O

m-DITC-ADAC, 11 HO OH

OH CH3 reactive group

HN HN N N N N Reactive group: N N N3 N N NCS PharmaceuticalsHO 2019O , 12, x FOR PEER REVIEWHO O O O 6 of 21

general formula 13. Nevertheless no extensive pharmacologicalN N studies forN their characterization H H H were reportedHO [16].OH HO OH SO2F Development of covalent A2A agonists was performed by an extensive SAR study on the R-AHPIA, 12 13 CGS21680 (2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine) scaffold, by

introduction of several chemoreactiveFigureFigure 4. 4.Structures Structures groups on ofof covalentcovalentits carboxylic A 11 ARAR acidagonists. agonists. moiety (Figure 5) [24,43,44].

Concerning the A1 AR subtype, examples of covalent agents are reported and summarized in Figure 4 [40–42]. In particular the N6 substituted derivative of adenosine, named m-DITC-ADAC (4-isothiocyanatophenylaminothiocarbonyl, DITC; adenosine amine congener or N6-[4-[[[4-[[[(2-Aminoethyl)amino]carbonyl]methyl]-anilino]carbonyl]methyl]phenyl]adenosine, ADAC) (11), proved to be a good irreversible agonist for A1 AR. It was able to mimic ischemic preconditioning in rabbits [40] and also prolong the stimulus of the His bundle (SA) interval by 2.1 fold in guinea pig isolated hearts [41]. Another interesting covalent agonist, R-AHPIA (2-azido-N-(2-(4-hydroxyphenyl)-1-methylethyl)-adenosine, 12) was reported by Klotz and coworkers [42]. This ligand was also labeled with 125I and, after UV irradiation, it irreversibly binds to the rat receptor, which was isolated for the first time as a protein of about 35 KDa [42]. A quite extensive SAR study on the adenosine nucleus for obtaining other covalent ligands for human ARs was performed by Jorg and coworkers [16], who obtained a series of compounds of

Figure 5. Structures of covalent A2A and A3 AR agonists. Figure 5. Structures of covalent A2A and A3 AR agonists.

DevelopmentThese studies of covalent led A2Ato agoniststhe discovery was performed of bya an extensivecompound SAR named study onDITC-APEC the CGS21680 (2-p(2-[(2-aminoethyl-aminocarbony-(2-carboxyethyl)phenethylamino-5lethyl)phenylethylamino]-5’-0-N-ethylcarboxamidoadenosine)N-ethyl-carboxamidoadenosine, scaﬀold, by introduction of severalAPEC) chemoreactive (14), which was groups able to on induce its carboxylic concentration acid moiety dependent (Figure coronary5)[24, 43vasodilation,44]. that persisted forThese a long studies time, even led after to the washou discoveryt. This ofsuggests a compound that compound named 14 DITC-APEC irreversibly bound (2-[(2-aminoethyl- A2A AR in aminocarbonylethyl)phenylethylamino]-5’-guinea pig coronary arteries [44]. N-ethyl-carboxamidoadenosine, APEC) (14), which was A quite similar compound (15) was obtained by introducing a reactive ester on the CGS21680 able to induce concentration dependent coronary vasodilation that persisted for a long time, even nucleus as an acylating agent. This derivative was found to be an irreversible agonist for the human A2A AR, and docking studies suggested that two lysine residues in the second extracellular loop were involved in the covalent binding. In fact, site directed mutagenesis studies confirmed that lysine 153 reacts with the active ester moiety, giving the irreversible adduct [45]. For the rat A3 AR subtype, an irreversible agonist (16) was developed by introducing a benzyl-isothiocyanate group at the N6 position of 5’-(N-methylcarboxamido)adenosine, but, except for binding experiments confirming the irreversible properties of the compound, no significative pharmacological experiments were performed [46]. In addition, several irreversible antagonists for ARs have been reported. We could classify the obtained compounds in two major classes: i) xanthine derivatives (the natural antagonists for ARs), and ii) non-xanthine derivatives. In the xanthine group, several derivatives of the well-known A1 AR antagonist DPCPX (1, 3-dipropyl-8-cyclopentyl xanthine) have been synthesized by introducing chemoreactive groups [47–49]. Among this large number of synthesized compounds, the most interesting derivative could be considered to be 8-cyclopentyl-3-N-[3-((3-(4-fluorosulphonyl)benzoyl)-oxy)-propyl]-1-N-propyl-xanthine (FSCPX, 17), which proved to be the best irreversible rat A1 AR antagonist reported [47] (Figure 6). Several pharmacological studies have been performed using FSCPX (17) as a probe, permitting better understanding of the pathophysiological role of the A1 AR [50,51]. A structurally related derivative to FSCPX (17), bearing a cyclohexyl group at the 8 position and an amido function instead of an ester at the 3 position, named DU172 (18), has been synthesized and

Pharmaceuticals 2019, 12, 168 6 of 21

after washout. This suggests that compound 14 irreversibly bound A2A AR in guinea pig coronary arteries [44]. A quite similar compound (15) was obtained by introducing a reactive ester on the CGS21680 nucleus as an acylating agent. This derivative was found to be an irreversible agonist for the human A2A AR, and docking studies suggested that two lysine residues in the second extracellular loop were involved in the covalent binding. In fact, site directed mutagenesis studies confirmed that lysine 153 reacts with the active ester moiety, giving the irreversible adduct [45]. For the rat A3 AR subtype, an irreversible agonist (16) was developed by introducing a benzyl-isothiocyanate group at the N6 position of 5’-(N-methylcarboxamido)adenosine, but, except forPharmaceuticals binding experiments 2019, 12, x FOR confirming PEER REVIEW the irreversible properties of the compound, no significative 7 of 21 pharmacological experiments were performed [46]. utilizedIn addition, to obtain several a co-crystalized irreversible structure antagonists of the forhuman ARs A have1 AR. beenWith reported.the help of Wecomputational could classify and the obtainedmutational compounds studies, inthe two structural major classes:basis for i) subtype xanthine selectivity derivatives versus (the A natural2A subtype antagonists were identified for ARs), and[15]. ii) non-xanthine derivatives. A quite similar approach for developing other A1 adenosine covalent antagonists has been In the xanthine group, several derivatives of the well-known A AR antagonist DPCPX utilized using 1 XAC, (1, 3-dipropyl-8-cyclopentyl xanthine) have been synthesized by introducing chemoreactive (N-(2-aminoethyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-acetamid groupse), as [47 a –template.49]. Among This this approach large numberled to the of discovery synthesized of several compounds, promising the most covalent interesting agents such derivative as couldm-DITC-XAC be considered (19), p-DITC-XAC to be 8-cyclopentyl-3- (20) [52], andN the-[3-((3-(4-fluorosulphonyl)benzoyl)-oxy)-propyl]-1- trifunctionalized compound 21 [53], which were N-propyl-xanthinefound to be irreversible (FSCPX, ligands17), for which rat A proved1 ARs in to binding be the studies. best irreversible In particular, rat compound A1 ARantagonist 21 was reportedfound [to47 be] (Figure 894-fold6). selective for A1 vs. A2A ARs (Figure 6) [53].

O H O H nC H nC H 3 7 N N 3 7 N N

O N N O N N

SO2F SO2F

O HN

O O

FSCPX, 17 DU172, 18

NCS

m-DITC-XAC, 19 O H R= nC H 3 7 N N O O O N N R= NCS p-DITC-XAC, 20 HN nC3H7 NH NCS NHR S R= 21

NCS

O CH3 H C 3 N N

O N N

CH3 NCS

ISC, 22 FigureFigure 6. 6.Structures Structures of of xanthine xanthine derivativesderivatives as as covalent covalent antagonists antagonists for for ARs. ARs.

By modifying the xanthine nucleus, other chemical entities have been developed by Scammells and coworkers. These studies led to some quite interesting covalent antagonists towards hamster receptors but low pharmacological characterization was reported [54]. In this field, some styrylxanthines have been investigated with the aim to obtain novel irreversible A2A AR antagonists. These studies led to the discovery of ISC (8-(3-isothiocyanatostyryl)caffeine, 22), which in binding studies was found to be a good covalent ligand at the rat receptor, with potency in the nanomolar range and good levels of selectivity (Figure 6) [55].

Pharmaceuticals 2019, 12, 168 7 of 21

Several pharmacological studies have been performed using FSCPX (17) as a probe, permitting better understanding of the pathophysiological role of the A1 AR [50,51]. A structurally related derivative to FSCPX (17), bearing a cyclohexyl group at the 8 position and an amido function instead of an ester at the 3 position, named DU172 (18), has been synthesized and utilized to obtain a co-crystalized structure of the human A1 AR. With the help of computational and mutational studies, the structural basis for subtype selectivity versus A2A subtype were identiﬁed [15]. A quite similar approach for developing other A1 adenosine covalent antagonists has been utilized using XAC, (N-(2-aminoethyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]- acetamide), as a template. This approach led to the discovery of several promising covalent agents such as m-DITC-XAC (19), p-DITC-XAC (20)[52], and the trifunctionalized compound 21 [53], which were found to be irreversible ligands for rat A1 ARs in binding studies. In particular, compound 21 was found to be 894-fold selective for A1 vs. A2A ARs (Figure6)[53]. By modifying the xanthine nucleus, other chemical entities have been developed by Scammells and coworkers. These studies led to some quite interesting covalent antagonists towards hamster receptors but low pharmacological characterization was reported [54].

PharmaceuticalsIn this field, 2019, some12, x FOR styrylxanthines PEER REVIEW have been investigated with the aim to obtain novel irreversible 8 of 21 A2A AR antagonists. These studies led to the discovery of ISC (8-(3-isothiocyanatostyryl)caffeine, 22), whichAlso in binding in the non-xanthine studies was found family, to beseveral a good examples covalent of ligand irreversible at the rat AR receptor, antagonists with potencyhave been in thereported nanomolar [56–59]. range and good levels of selectivity (Figure6)[55]. AlsoIn order in the to non-xanthineobtain covalent family, A2A severalantagonists, examples insertion of irreversible of a fluorosulfonyl AR antagonists moiety have on been the reportedwell-known [56 –59A].2A AR antagonists SCH58261 (2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo [4,3-eIn][1,2,4]triazolo[1,5- order to obtain covalentc]pyrimidin-5-amine) A2A antagonists, insertion of a fluorosulfonyland moiety on the well-knownZM241385 A(4-(2-(7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-2A AR antagonists SCH58261 (2-(2-Furanyl)-7-(2-phenylethyl)-7a][1,3,5]triazin-5-ylamino)ethyl)phenol)H-pyrazolo [4,3-e][1,2,4]triazolo[1,5- have beenc] pyrimidin-5-amine)developed. This and ZM241385approach (4-(2-(7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5- led to the discovery a][1,3,5]triazin-5-of FSPTP ylamino)ethyl)phenol)(5-amino-7-[2-(4-fluorosulfonyl)phenylethyl]-2-(2-furyl)-pryazolo[4,3- have been developed. This approach led to the discoverye]-1,2,4-triazolo[1,5- of FSPTP (5-amino-7-c]pyrimidi [2-(4-fluorosulfonyl)phenylethyl]-2-(2-furyl)-pryazolo[4,3-ne, 23) e]-1,2,4-triazolo[1,5-and c]pyrimidine,LUF744523) and LUF7445((4-((3-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5- ((4-((3-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)propyl)carbamoyl)benza][1,3,5]triazin-5-yl)amino)propyl)carbamoyl) benzeneene sulfonyl sulfonyl fluoride), fluoride), 24) as24 irreversible) as irreversible A2A AR A2A antagonistsAR antagonists (Figure (Figure 7). 7).

NH2 NH2 FO S 2 N N N O N N N

N N O N N N N O H H N FO2S FSPTP, 23 LUF7445, 24

O SO2F nC H OCH3 3 7 N N O N HN N O N H NNN O N O N N H N FO2S nC3H7 26 LUF7602, 25 Figure 7. Structures of non-xanthinic derivativesderivatives as covalent antagonists for ARs.

The use of FSPTP (23) in pharmacological studies showed that there is a large receptor reserve for the A2A AR, which mediates the increase in coronary conductance in guinea pigs [56], while LUF7445 (24), through the help of computational and mutational studies permitted identification of lysine 153 as the amino acid involved in covalent binding at the hA2A AR [58]. A quite similar approach has been utilized for developing covalent human A3 AR antagonists, leading to a derivative named LUF7602 (4-((3(8-methoxy-2,4-dioxo-3-propyl-3,4-dihydropyrido[2,1-f]purin-1(2H)yl)propyl)carbamoyl)benz enesulfonyl fluoride, 25) [57] and a pyrazolo-triazolo-pyrimidine compound (26) [59]. The first one showed that tyrosine 265 was involved in the covalent binding, while compound 26, through the help of computational studies, suggested that serine 247 or cysteine 251, both in TM6, could be responsible for covalent binding. Of course, these results clearly indicate that the two compounds have a different binding pose into the receptor binding site.

4. Fluorescent Ligands The concept of using fluorescent ligands to detect GPCRs in native conditions, including ARs, is not new [60]. However, the urgent need for new fluorescent tools for ARs is given by the possibility of applying new fluorescent techniques to deeply study the receptors in cells, in particular, receptor localization, both on the membrane surface and during their internalization. This would allow for study of desensitization, recycling and homo- or oligo-merization of the receptors [19]. A description of the techniques used to study GPCRs was recently reviewed and comprised fluorescence

Pharmaceuticals 2019, 12, 168 8 of 21

The use of FSPTP (23) in pharmacological studies showed that there is a large receptor reserve for the A2A AR, which mediates the increase in coronary conductance in guinea pigs [56], while LUF7445 (24), through the help of computational and mutational studies permitted identification of lysine 153 as the amino acid involved in covalent binding at the hA2A AR [58]. A quite similar approach has been utilized for developing covalent human A3 AR antagonists, leading to a derivative named LUF7602 (4-((3(8-methoxy-2,4-dioxo-3-propyl-3,4-dihydropyrido [2,1-f ]purin-1(2H)yl)propyl)carbamoyl)benzenesulfonyl fluoride, 25)[57] and a pyrazolo-triazolo- pyrimidine compound (26)[59]. The first one showed that tyrosine 265 was involved in the covalent binding, while compound 26, through the help of computational studies, suggested that serine 247 or cysteine 251, both in TM6, could be responsible for covalent binding. Of course, these results clearly indicate that the two compounds have a different binding pose into the receptor binding site.

4. Fluorescent Ligands The concept of using fluorescent ligands to detect GPCRs in native conditions, including ARs, is not new [60]. However, the urgent need for new fluorescent tools for ARs is given by the possibility of applying new fluorescent techniques to deeply study the receptors in cells, in particular, receptor localization, both on the membrane surface and during their internalization. This would allow for study of desensitization, recycling and homo- or oligo-merization of the receptors [19]. A description of the techniques used to study GPCRs was recently reviewed and comprised fluorescence polarization, confocal microscopy, fluorescence correlation spectroscopy (FCS), flow cytometry, FRET and bioluminescence resonance energy transfer (BRET) [18,19,61–63]. Nowadays, thanks to the progress in both synthetic and instrumental techniques, a plethora of fluorophores is available, with a diversity of applicable conjugation strategies that simplify the development of new specific fluorescent ligands [64–66]. In fact, the literature reports several examples of fluorescent ligands for all four AR subtypes. A very comprehensive review on fluorescent ligands for ARs was reported by Kozma et al. [67] and extended to some newer derivatives by another two reviews published in 2014 and 2015 [68,69]. Thus, here the most representative fluorescent ligands are reported among with the new fluorescent ligands for the ARs reported in the literature from 2015 to now. The first fluorescent ligands were developed in 1987 by Jacobson and coworkers to target the rat A1 AR. The authors conjugated the fluorophores fluorescein isothiocyanate (FITC) and nitrobenzoxadiazole (NBD) to the free amino group of the A1 agonist ADAC (FITC-ADAC, 27; NBD-ADAC, 28), maintaining affinity to the receptor (pKi@ratA1 = 8.14, 27; pKi@ratA1 = 8.36, 28). The same approach was used for conjugating FITC to the A1 antagonist XAC, FITC-XAC (29, pKi@ratA1 = 6.90) (Figure8)[60]. Therefore, fluorescent ligands for human adenosine receptors were developed both from agonists and antagonists. Concerning agonists, only few derivatizations were made from the simple adenosine scaffold. Specifically, in ABA-X-BY630 (30)[70], adenosine was conjugated by a linker at the 6 position, while in compound 31 [71], thioadenosine was used to introduce the BODIPY (boron dipyrromethene) fluorophore at the 2 position of the purine ring (Figure9). ABA-X-BY630 ( 30) was developed as an A1 ligand (pKi = 6.65), but compound 31 exhibited a preferential affinity towards the A3 AR subtype (pKi = 6.21), showing more than ten-fold selectivity against A1 and A2A ARs. More efforts were made with the more active, but still not selective, agonist NECA (5’-(N-ethylcarboxamido)adenosine). It has been studied by different groups, where all substitutions were carried out at the 6 position and linkers of different length were used, as were different fluorophores such as dansyl, NBD, BODIPY 630/650, Cy5, Texas Red, and EvoBlue30 [70,72–75]. In most cases, the introduction of a fluorophore led to A1 AR ligands. Macchia et al. developed a series of dansyl-NECA derivatives that exhibited higher affinity at the A1 AR (i.e., ABEA-dansyl, 32)[75]. Because dansyl excitation wavelength (340 nm) falls in the autofluorescence spectrum of cells and tissues, the authors substituted the dansyl group with a longer excitation wavelength fluorophore such as NBD (λex = 465 nm, λem = 535 nm). Interestingly, NBD led to a detrimental effect on A1 AR, while the affinity towards A3 AR (i.e., NBD-NECA, 33) increased [73] (Figure9). Pharmaceuticals 2019, 12, x FOR PEER REVIEW 9 of 21

polarization, confocal microscopy, fluorescence correlation spectroscopy (FCS), flow cytometry, FRET and bioluminescence resonance energy transfer (BRET) [18,19,61–63]. Nowadays, thanks to the progress in both synthetic and instrumental techniques, a plethora of fluorophores is available, with a diversity of applicable conjugation strategies that simplify the development of new specific fluorescent ligands [64–66]. In fact, the literature reports several examples of fluorescent ligands for all four AR subtypes. A very comprehensive review on fluorescent ligands for ARs was reported by Kozma et al. [67] and extended to some newer derivatives by another two reviews published in 2014 and 2015 [68,69]. Thus, here the most representative fluorescent ligands are reported among with the new fluorescent ligands for the ARs reported in the literature from 2015 to now. The first fluorescent ligands were developed in 1987 by Jacobson and coworkers to target the rat A1 AR. The authors conjugated the fluorophores fluorescein isothiocyanate (FITC) and nitrobenzoxadiazole (NBD) to the free amino group of the A1 agonist ADAC (FITC-ADAC, 27; NBD-ADAC, 28), maintaining affinity to the receptor (pKi@ratA1 = 8.14, 27; pKi@ratA1 = 8.36, 28). The Pharmaceuticalssame approach2019 ,was12, 168 used for conjugating FITC to the A1 antagonist XAC, FITC-XAC (29, pKi@ratA9 of1 = 21 6.90) (Figure 8) [60].

H N O H N O R N NH O H H nC3H7 N N N N O O N N O N N HN HO nC3H7 O NH FITC

R= FITC FITC-ADAC, 27 HO OH FITC-XAC, 29 R= NBD NBD-ADAC, 28 OH

O O N NBD = FITC = N NO S 2

N COO- O H Figure 8. Structures of the first examples of fluorescent ligands for ARs. Figure 8. Structures of the first examples of fluorescent ligands for ARs. Therefore, fluorescent ligands for human adenosine receptors were developed both from agonistsIn contrast, and antagonists. Middleton Concerning et al. developed agonists, various only NECA-BODIPY few derivatizations 630/ 650were derivatives made from as the A1 simpleligands, becauseadenosine red-emitting scaffold. Specifically, BODIPY fluorophores in ABA-X-BY630 become (30 brighter) [70], adenosine in non-aqueous was conjugated environments, by a linker therefore at specificthe 6 position, fluorescence while onin compound the plasma 31 membrane [71], thioadenosine surface can was be used more to easily introduce visualized. the BODIPY Among (boron them, ABEA-X-BY630dipyrromethene) (34 fluorophore) showed high at atheffinity 2 position for both of A the1 and purine A3 ARs ring and (Figure maintained 9). ABA-X-BY630 agonist behavior (30) was [70 ] (Figuredeveloped9). This as an allowed A1 ligand visualization (pKi = 6.65), of but the compound receptors 31 at theexhibited cell membrane a preferential by confocal affinity towards microscopy the andA3 AR quantification subtype (pK ofi its= 6.21), binding showing kinetics. more Consequently, than ten-fold it wasselectivity used as against a probe A to1 and investigate A2A ARs. allosteric More interactionsefforts were given made by small with molecules the andmore receptor active dimerization, but still [76 ,not77]. Unfortunately,selective, agonist ABEA-X-BY630 NECA (34(5’-() showedN-ethylcarboxamido)adenosine). high levels of non-specific It has cytoplasmic been studie uptake,d by different which groups, prevented where its useall substitutions in long-term studies.were carried In a work out aimedat the to6 position improve and these linkers poor imagingof different properties, length anwere additional used, as tripeptide were different linker, A-A-G,fluorophores was introduced such as dansyl, between NBD, the fluorophoreBODIPY 630/650, and the Cy5, agonist Texas to Red, obtain and ABEA-A-A-G-X-BY630. EvoBlue30 [70,72–75]. In All fourmost possible cases, the combinations introduction of of L a and fluorophore D amino led acids to wereA1 AR examined, ligands. Macchia and apart et al. from developed the (L)A-(L)A-G a series isomer,of dansyl-NECA all others showed derivatives excellent that exhibited affinity values higher at theaffinity A1 and at the A3 AR.A1 AR The (i.e., ABEA-(D)A-(D)A-G-BY630 ABEA-dansyl, 32) [75]. (35Because) agonist dansyl was selectedexcitation for wavelength further experiments (340 nm) andfalls because in the autofluorescence it showed activation spectrum of the Gsof cells protein and at thetissues, A1 AR the when authors tested substituted at high concentrations, the dansyl group this compound with a longer was ex preferentiallycitation wavelength used as a fluorophore probe for the A3 AR (Figure9). As an example, compound 35 was successfully used to investigate the internalization process of A3 ARs and their co-localization with arrestin [78]. As shown in Figure 10, among agonists, the only other compound that was conjugated to a fluorophore in order to obtain, in this case, an agonist for the A2A AR subtype was APEC (2-[2-[4-[2-(2-aminoethylcarbonyl)ethyl]phenyl]ethylamino]-50-N-ethylcarboxamidoadenosine). It was successfully functionalized with FITC, Alexa Fluor 488 (MRS5206, 36) and Alexa Fluor 532 (MRS5424, 37)[79–81]. MRS5206 (36) showed affinity towards both A2A (Ki = 149 nM) and A3 (Ki = 240 nM) ARs and was used to demonstrate that A2A agonist-induced internalization is mediated by a clathrin-dependent mechanism [80]. Instead, the APEC–Alexa Fluor 532 conjugate MRS5424 (37) was developed to be applied in FRET experiments. In fact, the A2A receptor tagged with CFP (cyan fluorescent protein) was able to emit at 480 nm when excited at about 430 nm (blue). Then, emission at 480 nm was able to excite Alexa Fluor 532 in MRS5424 (37), which consequently emitted at 554 nm (yellow). In particular, MRS5424 (37) was used for the investigation of allosterism mediated by A2A/D2 oligomerization [8,81]. Pharmaceuticals 2019, 12, x FOR PEER REVIEW 10 of 21 Pharmaceuticals 2019, 12, 168 10 of 21 su

H N R BODIPY630 NH NH2 N nNH H N N N N N N N N N N S O N N HO O HO O NH O H C CH3 3 C2H5

HO OH HO OH N+ N HO OH B- H C F F CH3 3 R= Dansyl n= 4 ABA-X-BY630, 30 31 ABEA-dansyl, 32 A1 ligand A3 ligand A1 ligand R= NBD n= 8 H NBD-NECA, 33 BODIPY630 N N NH A3 ligand H O N N

O N N structural HN O optimization C2H5

HO OH ABEA-X-BY630, 34 A1 and A3 ligand

O O H H H N N N BODIPY630 N N NH H H O O N N

O N N HN O C2H5 BY630-X-(D)-A-(D)-A-G-ABEA, 35 A3 ligand HO OH

S O O F N(CH3)2 BODIPY 630 = O Dansyl = S F B- N O N+

FigureFigure 9. 9.Structures Structures of of representative representative ﬂuorescent fluorescent agonist agonist ligands ligands for for A A1 1and and A A3 3ARs. ARs.

In contrast, Middleton et al. developed various NECA-BODIPY 630/650 derivatives as A1 ligands, because red-emitting BODIPY fluorophores become brighter in non-aqueous environments, therefore specific fluorescence on the plasma membrane surface can be more easily visualized. Among them, ABEA-X-BY630 (34) showed high affinity for both A1 and A3 ARs and maintained agonist behavior [70] (Figure 9). This allowed visualization of the receptors at the cell membrane by confocal microscopy and quantification of its binding kinetics. Consequently, it was used as a probe to investigate allosteric interactions given by small molecules and receptor dimerization [76,77]. Pharmaceuticals 2019, 12, x FOR PEER REVIEW 11 of 21

Unfortunately, ABEA-X-BY630 (34) showed high levels of non-specific cytoplasmic uptake, which prevented its use in long-term studies. In a work aimed to improve these poor imaging properties, an additional tripeptide linker, A-A-G, was introduced between the fluorophore and the agonist to obtain ABEA-A-A-G-X-BY630. All four possible combinations of L and D amino acids were examined, and apart from the (L)A-(L)A-G isomer, all others showed excellent affinity values at the A1 and A3 AR. The ABEA-(D)A-(D)A-G-BY630 (35) agonist was selected for further experiments and because it showed activation of the Gs protein at the A1 AR when tested at high concentrations, this compound was preferentially used as a probe for the A3 AR (Figure 9). As an example, compound 35 was successfully used to investigate the internalization process of A3 ARs and their co-localization with arrestin [78]. As shown in Figure 10, among agonists, the only other compound that was conjugated to a fluorophore in order to obtain, in this case, an agonist for the A2A AR subtype was APEC (2-[2-[4-[2-(2-aminoethylcarbonyl)ethyl]phenyl]ethylamino]-5′-N-ethylcarboxamidoadenosine). It was successfully functionalized with FITC, Alexa Fluor 488 (MRS5206, 36) and Alexa Fluor 532 (MRS5424, 37) [79–81]. MRS5206 (36) showed affinity towards both A2A (Ki = 149 nM) and A3 (Ki = 240 nM) ARs and was used to demonstrate that A2A agonist-induced internalization is mediated by a clathrin-dependent mechanism [80]. Instead, the APEC–Alexa Fluor 532 conjugate MRS5424 (37) was developed to be applied in FRET experiments. In fact, the A2A receptor tagged with CFP (cyan

Pharmaceuticals 2019, 12, 168 11 of 21

O NH2 H NH N N N N R N N H O N N N N N H H O N Cy5 HN O HN N H C2H5 C2H5 O

HO OH HO OH R= Alexa Fluor 488 R= Alexa Fluor 532 MRS5218, 38 MRS5206, 36 MRS5424, 37 A3 ligand A2A ligands H3C NH 2 NH - SO3 H3C - SO3 H3C a Alexa Fluor 488 = O Alexa Fluor 532 = O - SO3 - 6 SO3

5 + O - NH2 COO COO- NH+ O O H3C H3C CH3

Cy5 =

C H - - + 2 5 SO O3S N N 3

H C CH3 3 CH3 H3C

Figure 10. Structures of representative fluorescent agonist ligands for A2A and A3 ARs. Figure 10. Structures of representative fluorescent agonist ligands for A2A and A3 ARs. APEC was also used to develop quantum dot (QD) conjugates, which are well-known for their emission properties. In particular, a QD was functionalized, through a linker based on (R)-thioctic acid, with a dendron containing multiple molecules of APEC. Dendron was used to increase the solubility of the structure and to ensure a good loading of APEC on the QD. One of the prepared systems, MRS5303, showed affinity towards A2A AR, but further work is needed to obtain a good A2A probe [82]. Finally, Jacobson and coworkers reported different methanocarba derivative-based fluorescent probes for the A3 AR subtype. The functionalized scaffold is that of the A3 agonist MRS3558 ((1S,2R,3S, 4R,5S)-4-[2-chloro-6-[(3-chlorophenyl)methylamino]purin-9-yl]-2,3-dihydroxy-N-methylbicyclo[3.1.0] hexane-1-carboxamide) in which the chlorine atom at the 2 position of the purine ring was substituted with an alkyne moiety, and where different fluorophores (e.g., Cy5, squaraine-rotaxane, Alexa Fluor 488, pyrene) were attached directly or through a linker [83–85]. Compound MRS5218 (38) is a full agonist of the A3 AR, displaying an IC50 of 1.09 nM in a functional cAMP assay (Figure 10)[83]. Concerning antagonists, the most studied fluorescent probes were based on the xanthine derivative XAC. As for NECA, several linkers and different fluorophores were introduced on the free amino group in XAC in this case, leading to both A1 and A3 AR fluorescent antagonists [60,72,86–89]. Among them XAC-X-BY630 (39) and CA200645 (40) are the more representative (Figure 11)[87,90]. Pharmaceuticals 2019, 12, x FOR PEER REVIEW 12 of 21

APEC was also used to develop quantum dot (QD) conjugates, which are well-known for their emission properties. In particular, a QD was functionalized, through a linker based on (R)-thioctic acid, with a dendron containing multiple molecules of APEC. Dendron was used to increase the solubility of the structure and to ensure a good loading of APEC on the QD. One of the prepared systems, MRS5303, showed affinity towards A2A AR, but further work is needed to obtain a good A2A probe [82]. Finally, Jacobson and coworkers reported different methanocarba derivative-based fluorescent probes for the A3 AR subtype. The functionalized scaffold is that of the A3 agonist MRS3558 ((1S,2R,3S,4R,5S)-4-[2-chloro-6-[(3-chlorophenyl)methylamino]purin-9-yl]-2,3-dihydroxy-N-methylb icyclo[3.1.0]hexane-1-carboxamide) in which the chlorine atom at the 2 position of the purine ring was substituted with an alkyne moiety, and where different fluorophores (e.g., Cy5, squaraine-rotaxane, Alexa Fluor 488, pyrene) were attached directly or through a linker [83–85]. Compound MRS5218 (38) is a full agonist of the A3 AR, displaying an IC50 of 1.09 nM in a functional cAMP assay (Figure 10) [83]. Concerning antagonists, the most studied fluorescent probes were based on the xanthine derivative XAC. As for NECA, several linkers and different fluorophores were introduced on the

Pharmaceuticals 2019, 12, 168 12 of 21

HN BODIPY630 O H nC3H7 O N N O O O N N NH HN nC3H7 HN BODIPY630 XAC-X-BY630, 39 O

NH O O H nC3H7 O NH N N O O O N N NH HN nC3H7

CA200645, 40

HN BODIPY630

O HO O H NH nC3H7 O HN N N O O O O N N NH HN HO nC3H7

XAC-(S)-S-(S)-Y-BY630, 41

FigureFigure 11. 11.Structures Structures of of representative representative ﬂuorescent fluorescent xanthine xanthine antagonist antagonist ligands ligands for for A A1 and1 and A A3 3ARs. ARs.

TheThe first first aim aim waswas toto developdevelop a probe for the the A A11 AR,AR, and and from from SAR SAR analysis, analysis, it itis isknown known that that the theintroduction introduction of oflong long chains chains at atthe the 8 position 8 position of ofthe the xanthine xanthine ring ring are are well well tolerated tolerated by by the the A A1 AR.1 AR. In In fact, XAC-X-BY630 (39) showed affinity in the nanomolar range towards A1 AR and it was used to quantify ligand-receptor binding at a single cell level using FCS [87]. Instead, CA200645 (40), which bears a longer polyamido linker even if it is particularly used as an A3 florescent probe (i.e., for kinetics, allosterism and dimerization studies by FCS and nano-BRET technology [91–93]), binds potently both to A1 and A3 ARs, in fact, it was used in a high-content screening assay for both receptors [90]. Further optimization on the linker of XAC/BODIPY 630 derivatives was reported by Vernall et al., where, like NECA derivatives, the authors decided to introduce a peptide linker, instead of a naked polyamido chain like in CA200645 (40)[89]. The aim was to increase selectivity at the A3 AR by gaining specific interactions with the linker. In order to find a less expensive way to assess the SAR of this compound, the BODIPY 630 was replaced by the similarly bulky Fmoc group. The linker was made by two amino acids chosen between alanine, tyrosine, serine and asparagine. The last were chosen for their polarity and, thus, ability to make hydrogen bonds. In addition, tyrosine can also be engaged in π-π interactions. The best results were obtained with XAC-(S)-S-(S)-Y-BY630 (41), which showed a pKD of 9.12 towards the A3 AR, compared to a pKD of 7.26 towards the A1 AR [89]. Compound 41 demonstrated a long residence time at A3 AR (RT = 288 min); thus, when it was used to determine the association and dissociation constants of the ligands, it gave quite different constants with respect to those determined with classical radioligands, such as [3H]PSB-11 Pharmaceuticals 2019, 12, x FOR PEER REVIEW 13 of 21 fact, XAC-X-BY630 (39) showed affinity in the nanomolar range towards A1 AR and it was used to quantify ligand-receptor binding at a single cell level using FCS [87]. Instead, CA200645 (40), which bears a longer polyamido linker even if it is particularly used as an A3 florescent probe (i.e., for kinetics, allosterism and dimerization studies by FCS and nano-BRET technology [91–93]), binds potently both to A1 and A3 ARs, in fact, it was used in a high-content screening assay for both receptors [90]. Further optimization on the linker of XAC/BODIPY 630 derivatives was reported by Vernall et al., where, like NECA derivatives, the authors decided to introduce a peptide linker, instead of a naked polyamido chain like in CA200645 (40) [89]. The aim was to increase selectivity at the A3 AR by gaining specific interactions with the linker. In order to find a less expensive way to assess the SAR of this compound, the BODIPY 630 was replaced by the similarly bulky Fmoc group. The linker was made by two amino acids chosen between alanine, tyrosine, serine and asparagine. The last were chosen for their polarity and, thus, ability to make hydrogen bonds. In addition, tyrosine can also be engaged in π-π interactions. The best results were obtained with XAC-(S)-S-(S)-Y-BY630 (41), which showed a pKD of 9.12 towards the A3 AR, compared to a pKD of 7.26 towards the A1 AR [89].

PharmaceuticalsCompound 412019 demonstrated, 12, 168 a long residence time at A3 AR (RT = 288 min); thus, when it was13 used of 21 to determine the association and dissociation constants of the ligands, it gave quite different constants with respect to those determined with classical radioligands, such as [3H]PSB-11 ([3H]-(8R)-8-ethyl-1,4,7,8-tetrahydro-4-5HH-imidazo[2,1--imidazo[2,1-i]purin-5-one)i]purin-5-one) [ 92[92].]. This This result result suggested suggested again thatagain a that probe a probe should should be properly be properly selected selected before before its use its in use an experiment. in an experiment. XAC was also used to prepare GPCR ligand–dendrimer (GLiDe) conjugates. In particular, XAC was functionalizedfunctionalized atat the the free free amino amino group group present presen att the at 8the position, 8 position, with anwith alkyne-containing an alkyne-containing chain. Alkynechain. Alkyne was then was used then toused perform to perform a copper a copper catalyzed catalyzed click click reaction reaction with with an azido-containing an azido-containing G4 (fourth-generation)G4 (fourth-generation) polyamidoamine polyamidoamine (PAMAM) (PAMAM) dendrimer dendrimer to form to form triazoles. triazoles. Dendrimers Dendrimers were were also conjugatedalso conjugated to fluorophores to fluorophores such such as Alexa as Alexa Fluor Fl 488,uor 488, leading leading to fluorescent to fluorescent dendrimers dendrimers MRS5397 MRS5397 and MRS5399.and MRS5399. In addition,In addition, near near infrared infrared dyes dyes were were also also used used by by Jacobson Jacobson and and coworkers,coworkers, leadingleading to MRS5421 (which used NIR dye 800) andand MRS5422MRS5422 (which(which usedused NIRNIR dyedye 700).700). These compounds showed higher aaffinityffinity towards thethe AA2A2A AR,AR, followed followed by A 33 andand A A11 ARsARs [88]. [88]. A seriesseries ofof xanthine xanthine derivatives derivatives was was recently recently developed developed to obtain to obtain fluorescent fluorescent antagonists antagonists towards thetowards A2B AR the [ 94A].2B TheAR xanthine[94]. The sca xanthineffold was scaffold chosen was on the chos basisen ofon known the basis SAR of for known this receptor, SAR for which this suggestedreceptor, which that 3-unsubstituted suggested that 8-phenyl-1-propylxanthine3-unsubstituted 8-phenyl-1-propylxan derivativesthine are generally derivatives more are potent generally and selectivemore potent to A 2BandAR selective than the to corresponding A2B AR than 1,3-substitutedthe corresponding derivatives. 1,3-substituted Thus, the derivatives. BODIPY fluorophore Thus, the wasBODIPY conjugated fluorophore to the was 8-phenyl conjugated by means to the of 8-phenyl spacers of by di ffmeanserent lengths.of spacers The of bestdifferent compound lengths. of The the seriesbest compound was PSB-12105 of the (42 )series (Figure was 12), PSB-12105 which showed (42) nanomolar(Figure 12), or subnanomolarwhich showed a ffinanomolarnity towards or human,subnanomolar rat and affinity mice receptors towards and human, was consequentlyrat and mice used receptors to develop and was a binding consequently assay using used flow to cytometrydevelop a binding [94]. assay using flow cytometry [94].

Figure 12. Structures of the ﬂuorescentfluorescent xanthinexanthine AA2B2B AR antagonist PSB-12105 ( 42).

In addition,addition, scascaffoldsffoldsother other than than xanthines xanthines have have been been used used to to develop develop fluorescent fluorescent antagonists antagonists for thefor adenosinethe adenosine receptors. receptors. In particular, In particular, tricyclic tricyc antagonistslic antagonists that exhibited that exhibited high affi nityhigh and affinity selectivity and forselectivity the various for AR the subtypes various were used;AR amongsubtypes them were we can used; find [1,2,4]triazolo[1,5- among themc ]quinazolineswe can find [95], [1,2,4]-triazolo[4,3-[1,2,4]triazolo[1,5-ca]quinazolines]quinoxalin-1-ones [96[95],], pyrazolo[4,3- [1,2,4]-triazolo[4,3-e][1,2,4]triazolo[1,5-a]quinoxalin-1-onesc]pyrimidines [95,97–[96],100] andpyrazolo[4,3- benzo[4,5]imidazo[2,1-e][1,2,4]triazolo[1,5-c][1,2,4]triazinesc]pyrimidines [101 ]. [95,97–100] and benzo[4,5]imidazo[2,1-CGS15943 (9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c][1,2,4]triazines [101]. c]quinazolin-5-amine) is a well-known non-selective AR antagonist that, when conjugated to Alexa Fluor 488 by a triazole-containing linker at the 5 position, led to MRS5449 (43, Figure 13) displaying a radioligand binding Ki value of 6.4 nM at the A3 AR, and was more than 10-fold selective against A1 and A2A ARs. For these characteristics it was chosen for the establishment of an A3 AR biding assay using flow cytometry [95]. In contrast, the analog pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine derivative SCH442416 (5-Amino-7-[3-(4-methoxy)phenylpropyl]-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine), was used to design potent A2A AR fluorescent antagonists. Linkers were introduced on the hydroxy group on the phenylalkyl chain present at the 7 position of the scaffold. Three different fluorophores were introduced: Alexa Fluor 488, Tamra and BODIPY 650/665, leading to MRS5346 (44), MRS5347 (45) and MRS5418 (46), respectively (Figure 13). MRS5418 (46) showed the best affinity (Ki = 15.1 nM) and selectivity for the A2A AR [97,98], but MRS5346 (44) was used in a fluorescence polarization-based receptor binding assay at the A2A AR [99]. Ciruela and coworkers developed another member of this class of A2A antagonists, where they introduced dye 467 directly to the hydroxyl group of SCH442416 (Figure 13). SCH442416Dy647 (47) was employed to undoubtedly demonstrate the presence of A2A/D2 oligomers in native tissues of rat striatum, which is essential information for designing new therapies for Parkinson’s disease [69,100]. Pharmaceuticals 2019, 12, x FOR PEER REVIEW 14 of 21

CGS15943 (9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine) is a well- known non-sel Pharmaceuticals 2019, 12, 168 14 of 21

a Figure 13. Structures of tricyclic fluorescent A2A and A3 ARs antagonists. Alexaa Fluor 488 5 isomer. Figure 13. Structures of tricyclic ﬂuorescent A2A and A3 ARs antagonists. Alexa Fluor 488 5 isomer. Pharmaceuticals 2019, 12, x FOR PEER REVIEW 15 of 21

In contrast, the analog pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine derivative SCH442416 (5-Amino-7-[3-(4-methoxy)phenylpropyl]-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) , was used to design potent A2A AR fluorescent antagonists. Linkers were introduced on the hydroxy group on the phenylalkyl chain present at the 7 position of the scaffold. Three different fluorophores were introduced: Alexa Fluor 488, Tamra and BODIPY 650/665, leading to MRS5346 (44), MRS5347 (45) and MRS5418 (46), respectively (Figure 13). MRS5418 (46) showed the best affinity (Ki = 15.1 nM) and selectivity for the A2A AR [97,98], but MRS5346 (44) was used in a fluorescence polarization-based receptor binding assay at the A2A AR [99]. Ciruela and coworkers developed another member of this class of A2A antagonists, where they introduced dye 467 directly to the hydroxyl group of SCH442416 (Figure 13). SCH442416Dy647 (47) was employed to undoubtedly demonstrate the presence of A2A/D2 oligomers in native tissues of rat striatum, which is essential information for designing new therapies for Parkinson’s disease [69,100]. The pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold was also functionalized at the 5 position, leaving the 7 position unsubstituted and inserting a methyl group at the 8 position: substitutions that should shift selectivity towards the A3 AR [102]. FITC was used as fluorophore Pharmaceuticalsand simple 2019diamino, 12, 168 alkyl or PEG linkers were used as spacers. These derivatives exhibited equal15 of 21 low micromolar affinity for the A2A and A3 AR subtypes [99]. In an attempt to obtain more potent derivatives, an aromatic ring, which could gain some interactions with the receptor, was introduced. As expected,The pyrazolo[4,3- new compoundse][1,2,4]triazolo[1,5- (48 and 49) cdisplayed]pyrimidine nanomolar scaffold wasaffinity also at functionalized the two receptors at the (Ki 5 position, A2A leaving= 60.4 nM, the Ki 7 position A3 = 73.6 unsubstituted nM, 48; Ki A2A and = 90 inserting nM, Ki A a3 = methyl 31.8 nM, group 49) (Figure at the 813) position: [103]. substitutions that shouldVernall shift selectivityet al. towardsdeveloped the A3aAR series [102]. FITCof wasA3 usedfluorescent as fluorophore derivatives and simple of diaminothe alkyl4-amino-2-(4-methoxyphenyl)-[1,2,4]triazolo[4,3- or PEG linkers were used as spacers. Thesea]quinoxalin-1(2 derivatives exhibitedH)-one, equalby introducing low micromolar different affi nity forfluorophores the A2A and through A3 AR a subtypes polyamido [99 ].linker In an at attempt the 4 posi to obtaintion of more the scaffold potent derivatives,[96]. The presence an aromatic of the ring, whichp-methoxy could moiety gain some on the interactions phenyl group with theat the receptor, 2 position was of introduced. the triazoloquinoxaline As expected, newring compoundsand the (48introductionand 49) displayed of an acyl nanomolar on the amino affinity group at the attwo the receptors4 position, (Ki are A 2Athe= characteristics60.4 nM, Ki A 3that= 73.6 should nM, 48; Kiconfer A2A =affinity90 nM, at Ki the A 3A=3 31.8AR. nM,A mini-series49) (Figure of 13 compounds)[103]. was developed using different linkers (chains from 2 to 12 atoms containing C, N and O atoms) and fluorophores (e.g., BODIPY, Tamra Vernall et al. developed a series of A3 fluorescent derivatives of the 4-amino-2-(4-methoxyphenyl)- [1,2,4]triazolo[4,3-and Cy5). The besta]quinoxalin-1(2 compound of theH)-one, series by was introducing AV039 (50 di), ffwhicherent fluorophoresshowed a pKD through of about a 9 polyamido at the linkerhuman at A the3 AR 4 position and was of more the sca thanffold 650-fold [96]. The selective presence over of other the p receptor-methoxy subtypes moiety on(Figure the phenyl 14). This group compound was able to specifically label the A3 AR in cells containing a mixed population of ARs, at the 2 position of the triazoloquinoxaline ring and the introduction of an acyl on the amino group thus it represented an ideal tool for the study of these receptors [92,96]. at the 4 position, are the characteristics that should confer affinity at the A AR. A mini-series of Recently, Barresi et al. reported a series of benzo[4,5]imidazo[2,1-c][1,2,4]triazines3 derivatives compounds was developed using different linkers (chains from 2 to 12 atoms containing C, N and as fluorescent probes for the A1 and A2B ARs. The authors used NBD as a fluorophore, which was O atoms) and fluorophores (e.g., BODIPY, Tamra and Cy5). The best compound of the series was introduced using two linkers of different lengths at the N1 or N10 positions, in order to investigate AV039 (50), which showed a pK of about 9 at the human A AR and was more than 650-fold selective which position gives the best flexibilityD to the molecule, to best3 accommodate the orthosteric binding over other receptor subtypes (Figure 14). This compound was able to specifically label the A AR in site [101]. One compound (51) (Figure 14) showed an affinity of about 1 µM at the A1 AR and an IC3 50 cells containing a mixed population of ARs, thus it represented an ideal tool for the study of these value in the same range towards the A2B AR, and therefore was demonstrated to be a useful tool for receptorsdetecting [these92,96 ].AR subtypes in fluorescence confocal microscopy experiments on cells.

O O O H H H N O N N HN O N BODIPY630 N NBD H H O N N N N OCH3 N N N O O AV039, 50 51

A3 ligand A1 and A2B ligand Figure 14. Structures of other tricyclic fluorescent AR antagonists. Figure 14. Structures of other tricyclic ﬂuorescent AR antagonists.

5. ConclusionsRecently, Barresi et al. reported a series of benzo[4,5]imidazo[2,1-c][1,2,4]triazines derivatives as fluorescentFrom this probes review, for it is the clear A1 andthat even A2B ARs. if ARs The were authors discovered used more NBD than as a fluorophore,forty years ago, which their was introducedcomplex mechanism using two of linkers action, ofwhich different includes, lengths but is at not the limitedN1 or Nto,10 activation,positions, oligomerization in order to investigate and which position gives the best flexibility to the molecule, to best accommodate the orthosteric binding site [101]. One compound (51) (Figure 14) showed an affinity of about 1 µM at the A1 AR and an IC50 value in the same range towards the A2B AR, and therefore was demonstrated to be a useful tool for detecting these AR subtypes in fluorescence confocal microscopy experiments on cells.

5. Conclusions From this review, it is clear that even if ARs were discovered more than forty years ago, their complex mechanism of action, which includes, but is not limited to, activation, oligomerization and internalization processes, is not fully elucidated. Over the last years, great progress in the field has been obtained thanks to the advancements made in structural biology. AR probes were continuously applied to new available techniques, which allowed deep investigation of the receptors, but at the same time, obtained information that is useful for designing new, more specific, drugs and probes, which in turn has contributed to the improvement in the AR research field. In particular, a better understanding of the role and the mechanism of action of these receptors could allow design of more specific therapies for several pathological conditions in which these receptors are involved, and for that reason, ARs remain a very interesting and intriguing therapeutic target. Pharmaceuticals 2019, 12, 168 16 of 21

Author Contributions: Conceptualization, S.F. and G.S.; writing—original draft preparation, S.F., L.L., G.S.; writing—review and editing, S.F. and G.S. Funding: This research was funded by MINISTRO DELL’ISTRUZIONE, DELL’UNIVERSITÀ E DELLA RICERCA, grant number PRIN2017 2017MT3993 and by UNIVERSITÀ DEGLI STUDI DI TRIESTE grant FRA2018. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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