Recent developments in adenosine receptor ligands and their potential as novel drugs

Abstract

Medicinal chemical approaches have been applied to all four of the adenosine receptor (AR) subtypes (A₁, A_2A, A_2B, and A₃) to create selective agonists and antagonists for each. The most recent class of selective AR ligands to be reported is the class of A_2BAR agonists. The availability of these selective ligands has facilitated research on therapeutic applications of modulating the ARs and in some cases has provided clinical candidates. Prodrug approaches have been developed which improve the bioavailability of the drugs, reduce side-effects, and/or may lead to site-selective effects. The A_2A agonist regadenoson (Lexiscan®), a diagnostic drug for myocardial perfusion imaging, is the first selective AR agonist to be approved. Other selective agonists and antagonists are or were undergoing clinical trials for a broad range of indications, including capadenoson and tecadenoson (A₁ agonists) for atrial fibrillation, or paroxysmal supraventricular tachycardia, respectively, apadenoson and binodenoson (A_2A agonists) for myocardial perfusion imaging, preladenant (A_2A antagonist) for the treatment of Parkinson’s disease, and CF101 and CF102 (A₃ agonists) for inflammatory diseases and cancer, respectively. This article is part of a Special Issue entitled: “Adenosine Receptors”.

1. Introduction

Extracellular adenosine acts on a family of four cell surface receptors termed adenosine receptors (ARs) of which there exist four subtypes: A₁, A_2A, A_2B, and A₃ [1,2]. The ARs are G protein-coupled receptors (GPCRs) and consist of a single polypeptide chain that transverses the membrane from the extracellular side beginning at the N terminus to form seven transmembrane helices (TMs). The A₁ and A₃ receptors preferentially couple to G_i protein to inhibit adenylate cyclase and consequently the production of cyclic AMP (cAMP), and the A_2A and A_2B subtypes stimulate the production of cAMP by coupling to G_s or G_o. These two subtype pairs also share higher sequence identity: the human A₁ and A₃ARs are 49% identical, and the human A_2A and A_2BARs are 59% identical.

The human A_2AAR became the first non-rhodopsin, non-adrenergic GPCR for which an X-ray crystallographic structure was reported [3]. The availability of this physically determined structure has aided in recent drug discovery efforts [4,5], as did theoretical homology models previously. The initial structure of the A_2AAR contained ZM241385 (53), only one of the many known potent antagonists of nanomolar affinity. There is now an effort to crystallize the receptor with other ligands and to crystallize other AR subtypes. Another structural issue to be considered in drug discovery is the phenomenon of GPCR dimerization, which can have a major effect on the pharmacological behavior. The ARs have been proposed to participate in both homo- and heterodimerization or even oligomerization [6,7]. For example, a well-established A_2AAR/D₂ dopamine receptor heterodimer occurs in the striatum and is the target of drug discovery for antagonists to treat Parkinson’s disease [8–10].

The effects of activation of ARs tend to be cytoprotective in many organs and tissues under a wide variety of physiological conditions. The levels of extracellular adenosine can rise substantially in response to stress, such as hypoxic stress, and the resultant activation of ARs acts to adapt to the stress [1,2]. Extracellular adenosine concentrations may rise as a result of the release of the breakdown of extracellular ATP or from intracellular sources, which leads to the activation of ARs in the vicinity. These protective responses may take the form of decreased energy demand (e.g. bradycardia), increased energy supply (e.g. vasodilation or angiogenesis), ischemic preconditioning (e.g. in the heart or brain), inhibition of the release of excitotoxic neurotransmitters, suppression of cytokine-induced apoptosis, or reduced inflammatory response [1,2].

Medicinal chemical approaches have been applied to all four of the AR subtypes to create selective agonists and antagonists for each (see Figs. 1–9 and Tables 1–3). The most recent class of selective AR ligands to be reported is the class of A_2BAR agonists [11–13]. The availability of these selective ligands has facilitated research on therapeutic applications of modulating the ARs and in some cases has provided clinical candidates. It must be kept in mind that there is a marked species dependency of ligand affinity at the ARs, and that the same ligand (especially antagonists of the A₃AR) could be selective for a given subtype in one species (e.g. human) and lose or reverse that selectivity in another species (e.g. rat) [14,15,17–20]. Therefore, caution must be used when characterizing new ligands and when using them in pharmacological experiments. In addition to receptor subtype selectivity, major considerations in the design of new ligands have been bioavailability and metabolic stability.

Fig. 1.

A₁ adenosine receptor agonists.

Fig. 9.

Radioligands for positron emission tomography (PET) studies.

Table 1.

Adenosine receptor affinities of agonists.

		Ki (nM)a
		A1	A2A	A2Bb	A3
1	Adenosinec [35]	ca. 100 (h)	310 (h)	15,000 (h)	290 (h)
		73 (r)	150 (r)	5100 (r)	6500 (r)
A1-selective agonists
2	R-PIA	2.04 (h) [128]	220 (r) [129]	150,000 (h) [130]	33 (h) [132]
		1.2 (r) [129]			158 (r) [133]
				19,000 (m) [131]
3	CPA [109]	2.3 (h)	794 (h)	18,600 (h)	72 (h)
4	CCPA	0.83 (h) [109]	2270 (h) [109]	18,800 (h) [109]	38 (h) [109]
		1.3 (r) [134]	950 (r) [134]		237 (r) [134]
		0.1 (rb) [134]			37.7 (rb) [134]
5	(S)-ENBA [19]	0.34 (r)	477 (r)	ndd	282 (h)
					915 (r)
6	Capadenoson (BAY68-4986)	nd	nd	nd	nd
7	GW493838	nd	nd	nd	nd
8	Selodenoson (DTI-0009)	nd	nd	nd	nd
9	GR79236 [109]	3.1 (r)	1300 (h)	nd	nd
10	Tecadenoson [109]	6.5 (p)	2315 (h)	nd	nd
11	CVT-3619 (GS 9667) [135]	55 (h)	> 10,000 (h)	>50,000 (h)	> 1000 (h)
12	AMP579 [136]	5.0 (r)	56 (r)	nd	nd
13	SDZ WAG 994 [137]	23 (p)	25,000 (p)	Inactive (p)	nd
14	NNC 21-0136 [93]	10 (r)	630 (r)	nd	nd
A2A-selective agonists
15	CGS21680	289 (h) [109]	27 (h) [109]	>10,000 (h) [109]	67 (h) [109]
		1800 (r) [134]	19 (r) [134]
		120 (rb) [134]			584 (r) [134]
				>10,000 (r) [134]	673 (rb) [134]
16	Apadenoson (ATL-146e) [109]	77 (h)	0.5 (h)	nd	45 (h)
17	ATL-313	nd	nd	nd	nd
18	UK-432097 [44]	nd	4 (h)	nd	nd
19	Regadenoson (CV-3146) [109]	>10,000 (h)	290 (h)	>10,000 (h)	> 10,000 (h)
20	[138]	>10,000 (h)	5.4 (h)	9866 (h)	1640 (h)
21	GW328267X [138]	882 (h)	2.3 (h)	51 (h)	4.2 (h) (antagonist)
22	Sonedenoson (MRE-0094) [139]	>10,000 (h)	490 (h)	>10,000 (h)	nd
23	Binodenoson (WRC-0470) [45]	48,000 (h)	270 (h)	430,000 (h)	903 (h)
25	[45]	400 (r)	372 (r)	nd	3640 (h)
			50 (m)
A2B-selective agonists
26	[46]	1050 (h)	1550 (h)	82 (h)	> 5000 (h)
27	MRS3997 [47]	253 (h)	150 (h)	128 (h)	90 (h)
28	BAY 60-6583	>10,000 (h)c, [140]	> 10,000 (h)c, [140]	3–10 (h) [140]	> 10,000 (h)c, [140]
				330 (m)e, [141]
				750 (d)e, [141]
				340 (rb)e, [141]
A3-selective agonist
29	IB-MECA (CF101) [109]	51 (h)	2900 (h)	11,000 (h)	1.8 (h)
30	Cl-IB-MECA (CF102)	220 (h) [109]	5360 (h) [109]	>10,000 (h) [134]	1.4 (h) [109]
		280 (r) [134]	470 (r) [134]		0.33 (r) [134]
		35 (m) [134]	~10,000 (m) [134]	>10,000 (m) [134]	0.18 (m) [134]
31	[51]	245 (h)	> 10,000 (h)	nd	2.25 (h)
32	[52]	32,800 (h)	41,700 (h)	>30,000 (h)	0.44 (h)
33	LJ529 [142]	193 (h)	223 (h)	nd	0.38 (h)
34	MRS3558 (CF502)	260 (h) [109]	2330 (h) [109]	>10,000 (h) [109]	0.29 (h) [109]
		105 (r) [134]	1080 (r) [134]		1.0 (r) [134]
		15.8 (m) [134]	10,400 (m) [134]		1.49 (m) [134]
35	MRS5151 [143]	14,900 (h)	~10,000 (h)	nd	2.38 (h)
		10,500 (m)	> 10,000 (m)		24.4 (m)
36	CP608,039 [144]	7300 (h)	nd	nd	5.8 (h)
		1750 (rb)			83 (rb)
37	CP532,903 [103]	898 (m)	> 10,000 (m)	>10,000 (m)	9.0 (m)

^ah = human; d = dog; m = mouse; p = pig; r = rat; rb = rabbit.

^bMost data are from functional studies.

^cData from functional studies.

^dnd = no data available.

^eData from radioligand binding studies versus the antagonist radioligand [³H]MRS1754.

Table 3.

Adenosine receptor affinities of ligands used for positron emission tomography.

		Ki (nM)a
		A1	A2A	A2B	A3
88	CPFPX	1.26 (h) [29]	940 (h) [29]	ndb	nd
		0.63 (r) [29]	812 (r) [29]
		1.37(p) [29]
		0.18 (c) [29]
89	FR194921	2.91 (h) [185]	> 100 (h,r,m) [185]	nd	> 100 (h) [185]
		4.96 (r) [185]
		6.49 (m) [185]
90	(E)-KF17837	390 (r) [186]	7.9 (r) [186] (E/Z)	1500 (h) [186]	nd
			1.0 (r) [186] (E)
91	IS-DMPX	8.9 (r) [123]	> 8000 (r) [123]	nd	nd
92	BS-DMPX	1200 (r) [187]	8.2 (r) [187]	>10,000 (h) [188]	> 10,000 (h) [188]
93	KF18446	nd	5.9 (r) [124]	nd	nd
94	FE@SUPPY	6050 (r) [189]	9670 (r) [189]	nd	9.67 (h) [189]

^ah = human; pig; m = mouse; p = pig; r = rat; a few A_2B data are from functional (cAMP) studies.

^bnd = no data available.

Synthetic adenosine agonists are under development as therapeutic agents. The half-life of adenosine in circulation is very short (~1 s), due to the action of enzymes that convert it to inosine (adenosine deaminase) or phosporylate it to 5′-AMP (adenosine kinase), or due to its uptake through nucleoside transporters (such as the equilibrative transporter ENT1) [1,2,21]. Therefore, analogues of adenosine for selective activation of ARs tend to prevent these processes and thereby lengthen the half-life. For example, the A₃-selective agonist IB-MECA (29) has a half-life of 8–9 h in man [22]. Adenosine itself is in use as an AR agonist for the treatment of paroxysmal supraventricular tachycardia (through the A₁ receptor) and in radionuclide myocardial perfusion imaging (through the A_2A receptor). For those applications, the short half-life of adenosine is advantageous. There are many selective and potent synthetic AR agonists that have been introduced as research tools and for consideration to be used in humans. So far, only one synthetic adenosine agonist (A_2AAR agonist regadenoson, 19, Lexiscan^™) is in clinical use, and that, so far, is for a diagnostic purpose rather than therapeutic use. One major consideration in the development of AR agonists is that desensitization of the receptor can occur after agonist binding, resulting in downregulation of the receptor. Thus, AR responses can desensitize rapidly, typically on the scale of less than one hour [23].

Synthetic adenosine antagonists have also been explored for potential therapeutic applications. Various early analogues of the xanthines, which greatly increased the AR subtype-selectivity over the naturally occurring alkylxanthines, tended to be hydrophobic and poorly water-soluble and consequently of low bioavailability [24–26]. More recently introduced AR antagonists and prodrug approaches have overcome some of these issues [e.g. 25–28].

The introduction of numerous radioligands for the ARs has aided in the drug discovery process. Thus, the primary screen of newly synthesized compounds in many AR drug discovery efforts has consisted of convenient radioligand binding assays [24]. Furthermore, both agonist and antagonist ligands containing positron-emitting radioisotopes have been introduced for 3-dimensional in vivo imaging of the receptors [29]. Such ligands for positron emission tomography (PET) might prove useful for diagnostic as well as research purposes. Now fluorescent ligands have been introduced for characterization of the ARs [30–32]. Some of these spectroscopic probes are suitable for compound screening and avoid the use of radioisotopes.

2. Adenosine receptor agonists

The structure–activity relationship (SAR) of adenosine derivatives as AR agonists has been exhaustively probed. Nearly all of the known AR agonists are derivatives of purine nucleosides, either adenosine (1) or xanthosine (Figs. 1–4 and Table 1). Therefore, in screens of structurally diverse chemical libraries, most of the hits will typically provide antagonists, rather than agonists. One exception to that generalization is the class of 2-aminopyridine-3,5-dicarbonitrile derivatives that act as agonists at ARs with varied degrees of subtype selectivity [33–35].

Fig. 4.

A₃ adenosine receptor agonists.

2.1. A₁-selective agonists

The SAR of adenosine ligands at the A₁AR was recently reviewed [36]. The earliest synthetic analogues of adenosine, such as N⁶-[(R)-phenylisopropyl]adenosine (2, R-PIA), to be characterized at the ARs tended to be selective for the A₁AR (Fig. 1). In general, substitution of adenosine at the N⁶-position with a wide range of alkyl, cycloalkyl, and arylalkyl groups increases selectivity for the A₁AR. In addition, any modification at the N⁶-position precludes the action of adenosine deaminase, which rapidly degrades adenosine itself, in vivo.

N⁶-Cycloalkyl substitution has been the most successful and general means of achieving selectivity for the A₁AR. N⁶-Cyclopentyladenosine (3, CPA) and its 2-chloro analogue (4, CCPA) are among the most potent and selective A₁AR in wide use as pharmacological agents. As with many other N⁶-substituted adenosine analogues, these two derivatives display considerable affinity at the A₃AR. In fact, CCPA was shown to act as an antagonist of the human A₃AR with a K_i value of 35 nM [37]. It was noted that the bicyclic analogue S-ENBA (5) has subnanomolar affinity at the A₁AR and has less residual affinity than CPA (3) or CCPA (4) for other AR subtypes [19]. Bayer Co. (Germany) discovered 2-amino-3,5-dicyanopyridine derivatives, e.g. capadenoson (6), as non-nucleoside-derived adenosine receptor agonists [33,36]. Besides 6 several A₁-selective adenosine derivatives, including GW493838 (7), selodenoson (8), GR79236 (9), tecadenoson (10), and CVT-3619 (GS9667, 11) have been evaluated in clinical trials for various indications (see below).

2.2. A_2A-selective agonists

The SARs of ligands at the A_2AAR have been reviewed recently [38,39]. Substitution of adenosine at the 2-position, especially with (thio)ethers, secondary amines, and alkynes, has resulted in many synthetic analogues selective for the A_2AAR. The presence of a 5′-N-alkyluronamide modification, as found in the potent nonselective agonist NECA, a 5′-N-ethyluronamide, tends to maintain or enhance the selectivity for the A_2AAR. These two modifications are present in the widely used A_2AAR agonist CGS21680 (15) (Fig. 2). The 2-(2-phenylethyl)amino modification of adenosine was particularly conducive to enhanced affinity at the A_2AAR and is present in an extended chain in CGS21680 (15). The carboxylate group at the terminal position of CGS21680 was found to act as a general site for chain extension and derivatization with bulky groups, including fluorescent groups and dendrimeric polymers [40,41], without losing high affinity of binding to the receptor. In receptor docking of agonist structures [42,43], this chain is pointing toward the extracellular face of the receptor, which has relaxed structural constraints relative to the main TM binding site. The 2-(2-cyclohexylethyl)amino modification of adenosine also favored high affinity at the A_2AAR. Extended substituents are also present at the 2-position of the more recently introduced A_2AAR, such as apadenoson (ALT-146e, 16) and ATL-313 (17), which are 5′-uronamide modified analogues.

Fig. 2.

A_2A adenosine receptor agonists.

Certain N⁶-position substitutions have also been found to increase the affinity at the A_2AAR. An example of this is the class of N⁶-(2,2-diphenylethyl)adenosine analogues, such as UK-432097 (18). Regadenoson (Lexiscan^™, 19) [191] has been introduced as a diagnostic for stress testing due to its vasodilatatory effects, and apadenoson (16) is developed for the same application.

An inverse amide structure in the 4′-position as in the C2,N⁶-substituted adenosine analogue 20, which is additionally lacking the oxygen atom in the ribose-analogous cyclopentane ring, is also well tolerated by the A_2AAR. Furthermore, the 4′-hydroxymethylene group in adenosine derivatives can be exchanged for a tetrazolyl residue as in GW328267X (21) (Fig. 2).

Several A_2A-selective agonists including UK-432097 (18), sonedenoson (22), and binodenoson (23) have been clinically evaluated (see below). A major problem with the systemic application of A_2A agonists as anti-inflammatory therapeutics has been their potent hypotensive effects. Recently, efforts have been undertaken to obtain A_2A agonists which show site-specific action. A_2A agonists, such as 18 have been developed for the treatment of bronchial inflammation (constructive pulmonary disease, COPD) by inhalation with limited systemic exposure [44]. In another approach 5′-phosphate prodrugs of A_2A agonists have been prepared (e.g. 24) which are to be preferably cleaved releasing the A_2A agonist 25 at sites of inflammation where ecto-5′-nucleotidase (CD73) is highly expressed [45].

2.3. A_2B-selective agonists

The SAR of adenosine agonists at the A_2BAR was recently reviewed [11,13]. Substitution of adenosine at the N⁶-position with a narrow range of aryl groups increases affinity at the A_2BAR. Also, very specific modification of the 5′- and C2-positions complements this increased affinity at the A_2BAR (e.g. compound 26) (Fig. 3). Thus, combinations of narrowly defined modifications have resulted in compounds that interact selectively with A_2BAR [11,46] or that activate the A_2BAR along with the A_2AAR (MRS3997, 27) [47].

Fig. 3.

A_2B adenosine receptor agonists.

BAY 60-6583 (28) is one of the 2-aminopyridine-3,5-dicarbonitrile derivatives found to activate the ARs [33]. This compound appears to be an A_2BAR-selective agonist [48,49].

2.4. A₃-selective agonists

The SAR of ligands at the A₃AR was recently reviewed [50]. Substitution with an N⁶-benzyl group or substituted benzyl group increases selectivity for the A₃AR in both human and rat (e.g. 29, 33) (Fig. 4). Even bulky substituents as in compound 31 are well tolerated [51]. The N⁶-methyl (e.g. compound 32) and ethyl groups also favor A₃AR in human [52]. As with A_2AAR agonists, the NECA-like 5′-uronamide modification has also been found to be conducive to selectivity in A₃AR agonists. IB-MECA (29) and its 2-chloro analogue Cl-IB-MECA (30) are prototypical and widely used agonists of the A₃AR. Cl-IB-MECA (30) is more A₃AR selective (~2000-fold compared to the A₁AR) than IB-MECA (~50-fold compared to the A₁AR). The 4′-thioadenosine derivative LJ-529 (33), which is otherwise equivalent to Cl-IB-MECA, acts as a highly potent and selective A₃AR agonist with a subnanomolar affinity [53].

The ribose ring is normally freely twisting in solution and can adopt a range of conformations. The North conformation of the ribose ring was found to be the preferred conformation for binding to the A₃AR. It is possible to chemically freeze this preferred conformation in analogues containing a [3.1.0]bicyclohexane ring system in place of the ribose 5-membered ring. This observation was utilized in the design of more potent and selective analogues such as MRS3558 (34), which displays nanomolar affinity at the A₃AR [54]. The selectivity of MRS3558 for the A₃AR is evident in a comparison of human ARs and rat ARs, but at the mouse ARs only 10-fold selectivity for the A₃AR vs. the A₁AR was observed. A third (alkynyl) substituent at the 2 position in MRS5151 (35) remedied this issue of species-dependent selectivity [55].

Recently, a PAMAM dendrimer conjugate of a chemically functionalized AR agonist was reported to bind to and activate the A₃AR selectively with nanomolar affinity [56]. Such macromolecular receptor ligands can display pharmacological properties that are qualitatively different in comparison to the monomeric agonists.

3. Adenosine receptor antagonists

The prototypical AR antagonists were alkylxanthine derivatives. The stimulants caffeine (38) and theophylline (39) are natural products that behave as weak and nonselective AR antagonists (Fig. 5). The structure–activity relationship (SAR) of xanthine derivatives as AR antagonists has been exhaustively probed. The effects of receptor subtype selectivity of substitution at the 1-, 3-, 7-, and 8-positions have been explored in detail [15]. However, many newer, highly selective AR antagonists are more chemically diverse than the xanthines and contain nonpurine heterocyclic core structures (Figs. 5–8 and Table 2). Various classes of AR antagonists and their synthetic methods have been reviewed [21,24,57,58].

Fig. 5.

A₁ adenosine receptor antagonists.

Fig. 8.

A₃ adenosine receptor antagonists.

Table 2.

Adenosine receptor affinities of antagonists.

		Ki (nM)a
		A1	A2A	A2B	A3
Non-selective antagonists
38	Caffeine	10,700 (h) [145]	23,400 (h) [67]	33,800 (h) [14]	13,300 (h) [145]
		44,900 (h) [67]	9560 (h) [145]	10,400 (h) [149]	>100,000 (r) [133]
		41,000 (r) [146]	45,000 (r) [147]	20,500 (h) [150]
		44,000 (r) [147]	32,500 (r) [148]	30,000 (r) [131]
		47,000 (gp) [20]	48,000 (r) [145]	13,000 (m) [131]
		44,000 (c) [20]
39	Theophylline	6770 (h) [127]	1710 (h) [127]	9070 (h) [149]	22,300 (h) [145]
		14,000 (r) [151]	6700 (h) [145]	74,000 (h) [150]	86,400 (h) [127]
		8740 (r) [145]	22,000 (r) [151]	15,100 (r) [149]	>100,000 (r) [133]
		7060 (gp) [152]	25,300 (r) [145]	5630 (m) [141]	85,000 (r) [154]
		4710 (rb) [152]		11,000 (gp) [153]	>100,000 (d) [155]
		9050 (s) [152]		17,700 (rb) [141]
		6330 (c) [152]		38,700 (d) [141]
A1-selective antagonists
40	DPCPX (CPX)	3.0 (h) [25]	129 (h) [127]	51 (h) [25]	795 (h) [156]
		0.50 (r) [25]	60 (h) [25]	63.8 (h) [149]	243 (h) [25]
		1.0 (r) [149]	157 (r) [148]	186 (r) [149]	509 (h) [155]
		0.18 (r) [152]	500 (r) [149]	200 (r) [153]	3960 (h) [127]
		1.06 (gp) [152]		86.2 (m) [141]	>10,000 (r) [25]
		3.9 (gp) [20]		145 (gp) [153]	43,000 (r) [155]
		0.21 (rb) [152]		96.0 (rb) [141]	708 (rb) [155]
		0.10 (s) [152]		147 (d) [141]	115 (d) [155]
		0.05 (c) [152]		132 (d) [153]
		0.29 (c) [20]
		11.4 (d) [155]
41	Rolofylline (KW3902, NAX)	0.72 (h) [61]	108 (h) [61]	296 (h) [157]	4390 (h) [157]
		8.0 (h) [157]	673 (h) [157]
		0.19 (r) [158]	380 (r) [158]
		12.6 (r) [61]	510 (r) [61]
42	Toponafylline (BG-9928)	7.4 (h) [157]	6410 (h) [157]	90 (h) [157]	>10,000 (h) [157]
		3.9 (mk) [159]	943 (mk) [159]
		1.3 (r) [157]	2440 (r) [157]
		29 (d) [159]	4307 (d) [159]
43	Naxifylline (BG9719, CVT-124)	0.45 (h) [61]	1100 (h) [61]	611 (h) [158]	4810 (h) [158]
		12 (h) [158]	1660 (h) [158]	1010 (m) [141]
		0.67 (r) [61]	1250 (r) [61]	470 (rb) [141]
				742 (d) [141]
44	PSB-36	0.7 (h) [25]	980 (h) [25]	187 (h) [25]	2300 (h) [25]
		0.124 (r) [25]	552 (r) [25]		6500 (r) [25]
45	FK-453	18 (h) [109]	1300 (h) [109]	980 (h) [109]	>10,000 (h) [109]
46	SLV320	1.00 (h) [160]	398 (h) [160]	3981 (h) [160]	200 (h) [160]
		2.51 (r) [160]		501 (r) [160]
47	Thiazole derivative	57.4 (h) [63]	6250 (h) [63]	>1000 (r) [63]	2160 (h) [63]
		4.83 (r) [63]	> 1000 (r) [63]
48	L-97-1	580 (h) [161]	> 100,000 (h) [161]	>100,000 (h) [161]	ndb
A2A-selective antagonists
49	Istradefylline (KW6002)	841 (h)c	12 (h) [162]	>10,000 (h)c	4470 (h)c
		230 (r)c	91.2 (h)c
			2.2 (r) [163]
			4.46 (r) [164]
50	CSC (Ki MAO-B=80.6 nM) [164]	28,000 (r) [165]	54 (r) [165]	8200 [165]	>10,000 (r) [133]
51	MSX-2	900 (r) [26]	8.04 (r) [26,148]	>10,000 (h) [26]	>10,000 (h) [26]
		2500 (h) [26]	5.38 (h)d, [26]
			14.5 (h)e, [26]
52	CGS15943	3.5 (h) [18]	1.2 (h) [18]	32.4 (h) [141]	35 (h) [18]
		6.4 (r) [18]		9.07 (m) [141]
53	ZM-241385	774 (h) [109]	1.6 (h) [109]	75 (h) [109]	743 (h) [109]
54	Vipadenant (BIIB014, V2006)	68 (h) [166]	1.3 (h) [166]	63 (h) [166]	1005 (h) [166]
55	SCH-442416	1110 (h) [109]	4.1 (h) [32]	>10,000 (h) [109]	>10,000 (h) [109]
56		2720 (r) [72]	18.3 (r) [72]	3420 (h) [72]	489 (h) [72]
57	SCH-58261	725 (h) [109]	5.0 (h) [109]	1110 (h) [109]	1200 (h) [109]
58	Preladenant (SCH-420814)	>1000 (h) [65]	0.9 (h) [65]0	>1000 (h) [65]	>1000 (h) [65]
59	ST-1535	71.8 (h) [167]	6.6 (h) [167]9	352.3 (h) [167]	>1000 (h) [167]
60	SYN-115	nd	nd	nd	nd
61		2.8 (h) [75]	0.0038 (h) [75]	nd	nd
			0.14 (h) cAMP [75]
62		228.4 (h) [76]	0.38 (h) [76]	nd	nd
A2B-selective antagonists
63	MRS1754	403 (h) [168]	503 (h) [168]	1.97 (h) [168]	570 (h) [168]
		16.8 (r) [168]	612 (r) [168]	12.8 (r) [168]
				16.6 (r) [153]
				3.39 (m) [141]
				9.12 (gp) [153]
				1.79 (rb) [141]
				12.8 (d) [141]
				12.3 (d) [153]
66	MRE-2029-F20	200 (h) [169]	> 1000 (h) [169]	5.5 (h) [169]	>1000 (h) [169]
65	PSB-603	>10,000 (h) [14]	> 10,000 (h) [14]	0.553 (h) [14]	>10,000 (h) [14]
		>10,000 (r) [14]	> 10,000 (r) [14]	KD 0.403 (h) [14]
				KD 0.351 (m) [14]
67	GS 6201 (CVT-6883)	1940 (h) [170]	3280 (h) [170]	22 (h) [170]	1070 (h) [170]
64	PSB-1115	>10,000 (h) [156]	24,000 (r) [151]	53.4 (h) [156]	>10,000 (h) [156]
		2200 (r) [151]
68	ATL 802	369 (h) [168]	654 (h) [168]	2.36 (h) [168]	>1000 (h) [168]
		9583 (m) [168]1	8393 (m) [168]	8.58 (m) [168]	>10,000 (m) [168]
69	LAS38096	2821 (h) [171,172]	> 1000 (h) [171,172]	17 (h) [171,172]	1043 (h) [171,172]
70		nd	965 (h) [173]	3.5 (h) [173]	nd
71		100 (h) [174]	51 (h) [174]	8 (h) [174]	nd
				21 (h) cAMP [174]
72		931 (h) [174]	239 (h) [174]	4 (h) [174]	3754 (h) [174]
73		2444 (h) [175]	2126 (h) [175]	11 (h) [175]	>1000 (h) [175]
74	OSIP	37 (h) [176]	328 (h) [176]	0.41 (h) [176]	450 (h) [176]
75	QAF805	186 (h) [177]	1775 (h) [177]	3.4 (h) [177]	10.2 (h) [177]
A3-selective antagonists
76	MRS1523	>10,000 (h) [134]	3660 (h) [134]	>10,000 (h) [134]	18.9 (h) [109]
		15,600 (r) [134]	2050 (r) [134]		113 (r) [134]
				>10,000 (m) [134]	731 (m) [134]
77	MRE3008-F20	1200 (h) [109]	141 (h) [109]	2100 (h) [109]	0.82 (h) [109]
78		562 (h) [178]	778 (h) [178]	>10,000 (h) [178]	0.108 (h) [178]
79	VUF-5574	≥10,000 (r) [179]	≥10,000 (r) [179]	nd	4.03 (h) [179]
80	KF26777	1800 (h) [180]	470 (h) [180]	620 (h) [180]	0.20 (h) [180]
81	PSB-10	1700 (h) [181]	2700 (h) [181]	nd	0.441 (h) [28]
		805 (r) [28]	6040 (r) [28]
82	PSB-11	1640 (h) [181]	1280 (h) [181]	2100 (m) [28]	2.34 (h) [181]
		440 (r) [181]	2100 (r) [181]		KD 4.9 (h) [182]
83		>1000 (h) [183]	> 1000 (h) [183]	>1000 (h) [183]	1.2 (h) [183]
84	MRS5147	1760 (h) [55]	1600 (h) [55]	nd	0.73 (h) [55]
85	MRS5127	3040 (h) [55]	1080 (h) [55]	nd	1.44 (h) [55]
86	LJ1251	2490 (h) [184]	341 (h) [184]	nd	4.16 (h) [184]
87	OT7999	>10,000 (h) [50]	> 10,000 (h) [50]	>10,000 (h) [50]	0.95 (h) [50]

^ah = human; c = cow; d = dog; gp = guinea pig; m = mouse; r = rat; rb = rabbit; s = sheep; a few A_2B data are from functional (cAMP) studies.

^bnd = no data available.

^cUnpublished data (Müller et al.).

^dRecombinant receptors (expressed in CHO cells).

^eNative receptors (post-mortem brain).

3.1. A₁-selective antagonists

A₁-selective AR antagonists have recently been reviewed [36,59]. In general, modifications of the xanthine core structure at the 8-position with aryl or cycloalkyl groups have led to high affinity and selectivity for the A₁AR. Highly selective xanthine antagonists of the A₁AR have been reported. Many contain a cycloalkyl substitution at the 8-position. For example, the 8-cyclopentyl derivative DPCPX or alternately abbreviated CPX (40, 8-cyclopentyl-1,3-dipropylxanthine) (Fig. 5) is highly selective and of nanomolar affinity at the rat A₁AR and is still selective, to a lesser degree, at the human A₁AR. A bicycloalkyl group is present in the 8-(3-noradamantyl) group of rolofylline (41, KW-3902, MK-7418) [60]. Another 8-bicycloalkyl xanthine analogue naxifylline (BG9719, 42) was even more selective for the A₁AR, with a K_i ratio human A_2A/A₁ of 2400 compared with a ratio of 150 for KW-3902 (41). While BG9719 is highly selective for A₁AR compared to the human A_2BAR, the selectivity of the related A₁AR antagonist BG 9928 (42) is only ~10-fold. The 3-(3-hydroxypropyl)-substituted 1-butyl-8-noradamantylxanthine (44, PSB-36) shows a particularly high affinity and A₁-selectivity [25]. Phosphate prodrugs of 3-(3-hydroxypropyl)xanthine derivatives show greatly improved water-solubility [25,62].

A variety of A₁-selective antagonists with a non-xanthine structure has been developed [18,36,59], including the pyrazolopyridine derivative FK-453 (45) and the 7-deazaadenine derivative SLV320 (46), both of which have been evaluated in clinical trials. Recently, a 2-aminothiazole derivative (47) showing high A₁ affinity and selectivity has been developed [63].

3.2. A_2A-selective antagonists

Recent developments in the field of A_2A antagonists have been described [64–66]. Modification of xanthines at the 8-position with alkenes (notably styryl groups) has led to selectivity for the A_2AAR. The 8-styrylxanthine istradefylline (49, KW6002) was among the first A_2AAR antagonists reported (Fig. 6). Some 8-styrylxanthine derivatives, such as CSC (50, 8-(3-chlorostyryl)caffeine), were later found to inhibit monoamine oxidase-B in addition to the A_2AAR [67]. The phosphate prodrug MSX-3 (51a) and the _L-valine ester prodrug MSX-4 (51b) have been prepared as water-soluble prodrugs of the potent and selective A_2A antagonist MSX-2 (51) [26,27]. Both are now broadly used as pharmacological tools in particular for in vivo studies [e.g. 67–70].

Fig. 6.

A_2A adenosine receptor antagonists.

Substituting various heterocyclic ring systems in place of the xanthine core has led to exceptionally high affinity and selectivity at the A_2AAR. An early example of a heterocyclic structure proposed as an A_2AAR antagonist was the triazoloquinazoline CGS15943 (52), which was later demonstrated to be only slightly selective. Later refinement of the triazoloquinazoline by addition of a third ring or alteration of the pattern of N inclusion in the heterocyclic system greatly improved the A_2AAR selectivity. The triazolotriazine ZM241385 (53), the triazolopyrimidine vipadenant (54, BII014, V2006), and the pyrazolotriazolopyrimidine SCH442416 (55) are examples of highly potent A_2AAR antagonists of later generation. ZM241385 (53) also binds to the human A_2BAR with moderate affinity, and in both tritiated and iodinated form has been used as a radioligand at that receptor [71]. A recently described analogue (56) shows somewhat higher selectivity [72].

The affinity of SCH 442416 (55) at the human A_2AAR was originally reported as K_i 0.048 nM [73], however later reports of binding assays have placed it in the low nanomolar range (4.1 nM in [32]). Related compounds include SCH 58261 (57) and preladenant (SCH 420814, 58). The latter is undergoing clinical trials for the treatment of Parkinson’s disease (see below).

Examples for further non-xanthine A_2A antagonists are the adenine derivative ST-1535 (59) and the benzothiazole derivative SYN-115 (60), both of which are being clinically evaluated. Very recently, benzofurans [74], 7-imino-2-thioxo-thiazolo[4,5-d]pyrimidines [75] (e.g. 61) and the related thiazolotriazolopyrimidinethiones [76] (e.g. 62) have been described as new potent A_2A-selective AR antagonists.

3.3. A_2B-selective antagonists

A_2B AR antagonists have recently been reviewed [12,13]. Certain modifications of the xanthine core structure at the 8-position with aryl groups have been found to result in selectivity for the A_2BAR [15]. The first antagonist to be reported was MRS1754 (63) (Fig. 7). Later, groups at Univ. of Bonn, Germany (PSB-1115 (64), PSB-603 (65)), Univ. of Ferrara, Italy (MRE-2029-F20, 66), at CV Therapeutics (now Gilead Sciences, CVT-6883, GS6201, 67) and at Adenosine Therapeutics (now Clinical Data Inc., ATL802, 68) improved on the degree of selectivity and/or the water solubility of the xanthines as A_2BAR antagonists. For example, GS-6201 (CVT-6883, 67) has a selectivity of 88-fold vs. A₁, 149-fold vs. A_2A, and 49-fold vs. A₃ARs (human species).

Fig. 7.

A_2B adenosine receptor antagonists.

PSB-603 (65) shows a particularly high affinity and selectivity, not only in humans, but also in rodents. PSB-1115 (64) exhibits high water-solubility and is therefore useful for in vivo studies, however its A_2B affinity and selectivity is lower than that for other A_2B antagonists. Besides xanthines, 2-aminopyrimidine derivatives, such as LAS38096 (69), 2-aminothiazolopyrimidines (e.g. 70), benzothiazoles (e.g. 71) [174], pyridine derivatives (e.g. 72, 73) [171,175], have been developed as A_2B antagonists. Compound 72 was shown to be metabolically unstable.

3.4. A₃-selective antagonists

Review articles on A₃AR antagonists have been previously published [16,17,77]. After it was recognized that the xanthines tended to be less potent as antagonists at the A₃AR in comparison to the other AR subtypes, chemically diverse sources were examined for possible interactions with the A₃AR. In an initial broad screen, several classes of nonxanthine antagonists were identified for the A₃AR: 1,4-dihydropyridines, pyridines, and flavones. The pyridine derivative MRS1523 (76) (Fig. 8) has become a useful tool since it shows relatively high affinity not only for the human but also for the rat A₃ receptor. Later it was noted that the potent nonselective AR antagonist CGS15943 (52) could be modified to produce A₃AR selectivity, two of the most potent and selective compounds at human A₃ receptors being MRE3008-F20 (77) and 78. The urea-substituted quinazoline derivative VUF5574 (79) also possesses high A_2B affinity and selectivity. Some tricyclic xanthines (80–82) have been found to be very potent and selective antagonists at human A₃ receptors showing increased water-solubility due to a basic nitrogen atom in the additional imidazole ring. Recently described A₃-selective antagonists include pyrazolopyrimidinones (e.g. 83). Many A₃ antagonists are much more potent at human as compared to rat A₃ ARs.

The principles for converting selective A₃AR agonists into selective A₃AR antagonists are based on either a conformationally constrained ribose-like ring or one that is truncated at the 4′-position (i.e., missing the ribose CH₂OH group entirely). Thus, the nucleoside derivatives MRS5147 (84) and its 3-iodo analogue MRS5127 (85) are highly selective A₃AR ligands generally across species. MRS5127 (84) was recently reported as a radioligand selective for the A₃AR [78]. The truncated 4′-thioadenosine derivative LJ-1251 (86), which acts as a A₃AR antagonist across species, was shown to lower intraocular pressure when applied topically [79,80].

4. Adenosine receptor ligands for diagnostic and therapeutic use

A selection of clinically used or evaluated AR ligands is collected in Table 4.

Table 4.

Therapeutic drugs, imaging agents, and clinical candidates that act through adenosine receptors.

Compound	Selectivity	Company	Indication or use (phase)a
Agonists
Adenosine (1) (Adenocard, Adenoscan)	A1, A2A	Astellas	Paroxysmal supraventricular tachycardia (approved), myocardial perfusion imaging (approved), other uses in testing
AMP579 (12)	A1, A2	Aventis	Myocardial infarction (discontinued)
Apadenoson (16, Stedivaze, BMS068645, ATL146e)	A2A	Clinical Data	Myocardial perfusion imaging (III)
ATL-1222	A2A	Clinical Data	Acute inflammatory conditions (preclinical)
ATL-313 (17)	A2A	Clinical Data	Ophthalmic disease (preclinical)
BAY 60-6583 (28)	A2B	Bayer	Atherosclerosis (preclinical)
Binodenoson (23, WRC-0470, MRE-0470)	A2A	Aderis, King	Myocardial perfusion imaging (III)
BVT.115959	A2A	Biovitrum	Diabetic neuropathic pain (II)
Capadenoson (6, BAY68-4986, nonnucleoside)	A1	Bayer Schering	Atrial fibrillation, chronic treatment (II)
Cl-IB-MECA (30, CF102)	A3	Can-Fite	Liver cancer (I–II)
CP608,039 (36)	A3	Pfizer	Cardiac ischemia (discontinued)
GS 9667 (11, CVT-3619)	A1	Gilead	Hypertriglyceridemia associated with diabetes (I)
GR79236 (9)	A1	GlaxoSmithKline	Pain, hyperlipidemia (I, discontinued)
GW493838 (7)	A1	GlaxoSmithKline	Peripheral neuropathic pain (II, discontinued)
IB-MECA (29, CF101)	A1	Can-Fite	Rheumatoid arthritis, psoriasis, dry eye, and other autoimmune inflammatory diseases (II), glaucoma (II)
MRS3558 (34, CF502)	A3	Can-Fite	Autoimmune inflammatory diseases (preclinical)
NNC-21-0136 (14)	A1	Novo Nordisk	Stroke, neurodegeneration (discontinued)
INO 8875 (PJ-875)	A1	Inotek Pharmaceuticals	Glaucoma (II); atrial fibrillation (discontinued)
Regadenoson (19, Lexiscan, CV-3146)	A2A	Gilead and Astellas	Myocardial perfusion imaging (approved)
RPR749	A1	Aventis	Hyperlipidemia (I)
SDZ WAG 94 (13)	A1	Sandoz/Novartis	Diabetes
Selodenoson (8)	A1	Aderis	Atrial fibrillation (II)
Sonedenoson (22, MRE-0094)	A2A	King	Diabetic foot ulcers, wound healing (II)
Tecadenoson (10, CVT-510)	A1	Gilead	Paroxysmal supraventricular tachycardia (III)
UK 432097 (18)	A2A	Pfizer	COPD (II)
Antagonists
ATL 844	A2B	Clinical Data and Novartis	Asthma and/or diabetes
Naxifylline (43, BG-9719)	A1	Biogen-Idec	Heart failure (renal function) (discontinued)
Caffeine (38)		McMaster University	Apnea
CPFPX (88)	A1	Research Center Jülich, Germany	PET imaging (18F)
FK-453 (45)	A1	Astellas	Acute renal failure
GS 6201 (67, CVT-6883)	A2B	Gilead	Asthma (I)
Istradefylline (49, KW6002)	A2A	Kyowa-Hakko Kogyo	Parkinson’s disease (III, discontinued)
KF26777 (80)	A3	Kyowa-Hakko Kogyo	Asthma (preclinical)
LAS38096 (69)	A2B	Almirall	Anti-inflammatory (preclinical)
LAS101057 [192]	A2B	Almirall	Antiasthmatic (I)
L-97-1 (48)	A1	Endacea	Sepsis (preclinical)
MRE2029-F20 (66)	A2B	King	Anti-inflammatory (preclinical)
OSIP339391 (74)	A2B	OSI	Asthma (preclinical)
OT-7999 (87)	A3	Otsuka	Glaucoma (preclinical)
Preladenant (58, SCH-420814)	A2A	Schering-Plough	Parkinson’s disease (III)
SCH-442416 (55)	A2A	Schering-Plough	PET imaging (11C)
QAF805 (75)	A2B, A3	Novartis	Asthma (I)
Rolofylline (41, KW3902, NAX)	A1	NovaCardia and Merck	Heart failure (renal function) (discontinued)
SLV320 (46)	A1	Solvay	Heart failure (renal function)
ST-1535 (59)	A2A	Sigma-Tau	Parkinson’s disease (I)
SYN-115 (60)	A2A	Synosia Therapeutics/UCB (after Synosia Therapeutics)	Parkinson’s disease (II), addiction
Theophylline/aminophylline (39)	A1	King Faisal University	Recovery after anaesthesia
Toponafylline (42, BG-9928)	A1	Biogen-Idec	Heart failure (renal function) (IIb)
Vipadenant (54, BIIB014, V2006)	A2A	Vernalis and Biogen-Idec	Parkinson’s disease (II)

^aMany of the clinical trials indicated are no longer current.

4.1. Agonists

Adenosine (1) itself for a long time was the only adenosine agonist to be used in humans. It is in widespread use in the treatment of paroxysmal supraventricular tachycardia (Adenocard®) due to its activation of A₁ receptors, and as a diagnostic for myocardial perfusion imaging (Adenoscan®, Astellas Pharma, Inc.) utilizing its A_2A-activating effects resulting in vasodilation. In addition, adenosine is being evaluated in several clinical trials for the treatment of inflammation, neuropathic and perioperative pain, and cardioprotection.

AMP579 (12) is a mixed agonist at A₁ and A_2AARs. It was in clinical trials for myocardial ischemic preconditioning and reperfusion injury. It was tolerated in patients with end-stage renal disease, but in a placebo-controlled trial of patients undergoing primary percutaneous transluminal coronary angioplasty it failed to reduce infarct size [81,82]. Recently it was also shown to activate the A_2BAR, which may account for its cardioprotective properties in the rabbit heart [83].

A₁AR agonists are useful in preclinical models of cardiac arrythmia and ischemia and in pain. Adenosine agonists are also of interest for the treatment of sleep disorders [84,85]. A_2A agonists exhibit anti-inflammatory and immunosuppressive effects [86]. Activation of the A_2B AR protects against vascular injury [87]. A₃AR agonists have been proposed for the treatment of a wide range of autoimmune inflammatory conditions, such as rheumatoid arthritis, inflammatory bowel diseases, psoriasis, etc. [88–90], and also for cardiac and brain ischemia.

4.1.1. A₁-selective agonists

A₁-selective (partial) agonists have been clinically evaluated for the treatment of paroxysmal supraventricular tachycardia, atrial fibrillation, or angina pectoris (capadenoson (6), selodenoson (8), tecadenoson (10), and PJ-875), hypertriglyceridemia and type II diabetes (GR79236 (9), RPR-749, and GS9667/CVT-3619 (11)) and neuropathic pain (GW493838 (7), GR79236 (9)). Partial agonists are usually preferred to avoid receptor desensitization and to possibly achieve a certain tissue selectivity of the effects. The A₁AR agonist SDZ WAG94 (13) was one of the first agonists of this subtype to progress to clinical trials, i.e. for consideration for treatment of diabetes [91].

A₁AR agonists have antiischemic effects in the heart and brain. Recently, A₁AR activation was shown to mediate neuroprotective effects through microglial cells [92]. Various A₁AR agonists have been shown to be neuroprotective in ischemic and seizure models. However, the peripheral side effects of A₁AR agonists could be severe. The A₁AR agonist NNC-21-0136 (14) was previously in clinical development for the treatment of stroke and other neurodegenerative conditions [93]. It was found empirically to provide some degree of in vivo selectivity for the CNS in comparison to peripheral cardiovascular actions of adenosine that was not based on subtype selectivity.

Other A₁AR-selective agonists are intended for activation of the receptor at peripheral locations. The A₁AR-selective adenosine derivative GR79236 (9) has analgesic and anti-inflammatory actions in humans and animals [94]. The A₁AR-selective agonist GW493838 (7) was also under evaluation for pain management. RPR749 (Aventis) and its methylated metabolite are orally active and selective adenosine A₁AR agonists that inhibit lipolysis in adipocytes and lower plasma triglyceride levels [95]. GS-9667 (11, CVT-3619), a partial agonist of the A₁AR, is in development as an antilipolytic agent. It acts as a full agonist of the A₁AR in the inhibition of adenylate cyclase in adipocytes, which have a large receptor reserve and/or higher efficacy of coupling of the receptor to G_i. However, it is a partial agonist in the cardiovascular system and therefore lacks cardiovascular side effects.

A₁AR agonists are of interest for use in treating cardiac arrhythmias, and it recently was suggested that a partial agonist of this subtype would have advantages over a full agonist for this use [96]. The A₁AR-selective agonist selodenoson (formerly DTI-0009, 8) has been in clinic trials for treatment of acute and chronic control of tachycardia and topical treatment of diabetic foot ulcers (Aderis Pharmaceuticals). It was formulated for intravenous administration to control heart rate during acute attacks and for oral administration in the chronic management of atrial fibrillation. The nonnucleoside AR agonist BAY 68-4986 (capadenoson, 6) is under investigation for atrial fibrillation and for the treatment of angina.

4.1.2. A_2A-selective agonists

The 2-substituted A_2AAR agonists apadenoson (16, ATL-146e), binodenoson (23, MRE-0470 or WRC-0470), and sonedenoson (22, MRE0094) have been cardiovascular clinical candidates [97–99]. Such agonists are of interest for use as vasodilatory agents in cardiac imaging (like adenosine itself, marketed as Adenoscan®) and in suppressing inflammation [100]. Regadenoson (19, CVT-3146, Lexiscan®) is already approved for diagnostic imaging [101]. The A_2A-agonist BVT.115959 (Biovitrum; structure not disclosed) has been claimed to show higher A_2A affinity at pH 7.0 as compared to pH 7.4 and is currently in phase II for the treatment of diabetic neuropathic pain. Since decreased pH values are found in pathological, e.g. inflamed tissues, this would result in tissue-selective effects and reduced side-effects. Two selective A_2A agonists developed by Adenosine Therapeutics (now Clinical Data) are in preclinical development for acute inflammatory conditions (ATL-1222, structure not disclosed) and ophthalmic disease (ATL-313, 17).

4.1.3. A₃-selective agonists

The two A₃AR agonists that are currently in clinical trials contain the 5′-N-methyluronamide modification and have nanomolar affinity at the receptor. Thus CF101 (29, Can-Fite Biopharma) and Cl-IB-MECA (30, CF102) are in trials for autoimmune inflammatory disorders and for liver cancer, respectively. CF101 (29) was recently demonstrated to be efficacious in clinical trials of rheumatoid arthritis, psoriasis, and dry eye disease [102]. Further clinical trials are planned for glaucoma and osteoarthritis. Two other A₃AR agonists CP-608,039 (36) and its N⁶-(2,5-dichlorobenzyl) analogue CP-532,903 (37) [103] were previously under development for cardioprotection. MRS3558 (34, CF502) is in preclinical development for the treatment of autoimmune diseases.

4.2. Antagonists

The non-selective AR antagonists caffeine (38) and theophylline (39) have been used as drugs for various indications. Caffeine (38) is mainly applied for CNS stimulation to restore alertness and to counteract fatigue, for the treatment of pain (e.g. headache, migraine) typically in combination with analgesics such as acetylsalicylic acid and/or paracetamol/acetaminophen, and for the treatment of apnoea in premature babies [104]. Theophylline or its salt aminophylline (39) are mainly applied for the treatment of bronchial asthma and COPD as a second-line treatment [105], although their use is now limited as a result of side effects on the central nervous system and the renal system. Theophylline may also be used for the prevention of sleep apnea in adults and for apnea of prematurity as a substitute for caffeine. A clinical study is currently performed using aminophylline for recovery after sevoflurane anaesthesia, since sevoflurane indirectly leads to an activation of A₁ARs.

A large number of synthetic AR antagonists that are much more potent and selective than the prototypical alkylxanthines have been introduced, although none have yet been approved for clinical use. Potent and selective AR antagonists display therapeutic potential as kidney protective (A₁), antifibrotic (A_2A), neuroprotective, antiasthmatic (A_2B), and antiglaucoma (A₃) agents [105–108].

4.2.1. A₁ receptor antagonists

Various A₁AR antagonists, xanthines and non-xanthines, have been or are currently being explored for clinical applications [109] for heart failure, and for improving renal function and treatment of acute renal failure. The xanthine derivative BG9719 (43), containing an epoxide ring, is highly selective, while the selectivity of the more water-soluble, metabolically more stable toponafylline (42) for the human A₁AR compared to the human A_2BAR is roughly 10. The 8-cyclopentyl derivative DPCPX (40), also known as CPX, which is selective for the A₁AR in the rat with nanomolar affinity but less selective at the human AR subtypes, has been in clinical trials for cystic fibrosis through a non-AR related mechanism [110]. The highly selective A₁AR antagonist L-97-1 (48, Endacea Inc.) is relatively well water-soluble and in late preclinical development for the treatment of asthma and sepsis [111]. As in the cases of DPCPX (40), rolofylline (41), naxifylline (BG 9719, 42), and others a persistent problem in the development of A₁AR antagonists has been low water-solubility and low bioavailability [18,112]; thus, A₁AR antagonists, e.g. toponafylline (43) and L-97-1 (48), with good water solubility are preferable clinical candidates.

Nonxanthine antagonists of the A₁AR have also been shown to have high receptor subtype selectivity, e.g. FK453 (45) [113] and SLV 320 (46, Solvay Pharmaceuticals) [114]. For example, SLV 320 is in clinical trials as an intravenous treatment for acute decompensated heart failure with renal impairment.

4.2.2. A_2A receptor antagonists

Several selective A_2A antagonists have been evaluated in clinical trials for the treatment of Parkinson’s disease. The first one has been istradefylline (49, KW6002), which did not reach the endpoint of phase III clinical trials, but additional trials are planned [115]. The non-xanthine derivatives preladenant (58, SCH420814; phase III), vipadenant (54, BII014, V2006; phase II), ST-1535 (59; phase I), as well as SYN-115 (60, phase II). Further potential indications include other neurodegenerative diseases, such as Alzheimer’s disease, restless legs syndrome, depression, and addiction.

4.2.3. A_2B receptor antagonists

Modification of xanthines at the 8 position with certain aryl groups has given rise to preclinical candidates that are selective for the A_2BAR (e.g. 67, GS-6201, CVT-6883, Gilead Sciences). GS-6201 is the first selective A_2B antagonist to be clinically evaluated for the treatment of asthma. Other A_2B-selective xanthine and nonxanthine derivatives include ATL844 (structure not disclosed), MRE2029-F20 (66), LAS38096 (69), LAS101057, and OSIP339391 (74), which are intended for treatment of asthma and/or inflammatory diseases. The aminothiazole derivative QAF 805 (75), a mixed A_2B/A₃-antagonist, has failed to attenuate bronchial hyperresponsiveness to inhaled AMP in a phase 1b clinical trial in asthmatics [116], but has also been investigated for other indications.

4.2.4. A₃ adenosine receptor antagonists

Cyclized derivatives of xanthines, such as PSB-11 (82), are A₃AR-selective, and similar compounds have been explored by Kyowa Hakko (e.g. 80). Selective A₃AR antagonists, such as the heterocyclic derivatives OT-7999 (87), are being studied for the treatment of glaucoma [117], and other such antagonists are under consideration for treatment of cancer, stroke, and inflammation [10,118]. No A₃AR antagonists have yet reached human trials.

5. Radioligands for in vivo PET imaging of adenosine receptors

With the established relevance of ARs to human disease states, it has been deemed useful to develop high affinity imaging ligands for these receptors, for eventual diagnostic use in the CNS and in the periphery. Ligands for in vivo positron emission tomographic (PET) imaging of A_1, A_2A, and A₃ARs have been developed (Fig. 9 and Table 3). For example, the xanthine [¹⁸F]CPFPX (88), similar in structure to DPCPX and the nonxanthine [¹¹C]FR194921 (89) have been developed as centrally-active PET tracers for imaging of the A₁AR in the brain [119].

¹¹C-labeled (E)-KF17837 (90) was proposed as a potential PET radioligand for mapping the adenosine A_2A receptors in the heart and brain [120,121]. ¹¹C labeled (E)-8-(3-chlorostyryl)-1,3-dimethyl-7-[¹¹C]methylxanthine ([¹¹C]CSC, 50) proved to accumulate in the striatum, and PET studies on rabbits showed a fast brain uptake of [¹¹C]CSC, reaching a maximum in less than 2 min [122]. Further styrylxanthine derivatives labeled with ¹¹C were tested as in vivo probes [123]. [7-Methyl-¹¹C]-(E)-3,7-dimethyl-8-(3-iodostyryl)-1-pro-pargylxanthine ([¹¹C]IS-DMPX, 91) and [7-methyl-¹¹C]-(E)-8-(3-bromostyryl)-3,7-dimethyl-1-propargylxanthine ([¹¹C]BS-DMPX, 92) showed K_i affinities of 8.9 and 7.7 nM respectively, and high A_2A/A₁ selectivity values. Unfortunately, biological studies proved that the two ligands were only slightly concentrated in the striatum, and that the two compounds were not suitable as in vivo ligands because of low selectivity for the striatal A_2A receptors and a high degree of nonspecific binding [123]. A useful A_2A PET ligand for in vivo imaging proved to be [¹¹C]KF18446 (93), also named (¹¹C)TMSX [124]. Ex vivo autoradiography for this molecule showed a high striatal uptake and a high uptake ratio of the striatum to the other brain regions. In 2001 the synthesis and evaluation of [¹¹C]KW-6002 (49) was reported. This molecule showed high retention in the striatum but it bound also to extra-striatal regions [190]. [¹¹C]SCH442416 (55) has recently been explored as a PET agent in the non-invasive in vivo imaging of the human A_2AAR [73,125].

Recently, an A₃AR PET ligand, [18F]FE@SUPPY (94), based on a series of pyridine A₃AR antagonists, was introduced [126]. Several nucleoside derivatives that bind with nanomolar affinity at the A₃AR and that contain ⁷⁶Br for PET imaging were recently reported, including the antagonist MRS5147 (84) [127].

6. Concluding remarks

In conclusion, selective agonists and antagonists for all four adenosine receptor subtypes have been developed and diagnostic and therapeutic applications are being explored. The first selective AR agonist, the A_2A agonist regadenoson (Lexiscan®), has been approved as a diagnostic drug for myocardial perfusion imaging. Many other selective agonists and antagonists for the various receptor subtypes are undergoing clinical trials for a broad range of indications. Although some trials of the selective ligands have been discontinued, the most advanced drugs so far have been capadenoson and tecadenoson (A₁ agonists for atrial fibrillation, or paroxysmal supraventricular tachycardia, respectively), apadenoson and binode-noson (A_2A agonists for myocardial perfusion imaging), preladenant (A_2A antagonist) for the treatment of Parkinson’s disease, and CF101 and CF102 (A₃ agonists for inflammatory diseases and cancer, respectively).