The principal mechanism by which caffeine and other alkylxanthines act as physiological stimulants is by blocking the effects of the ubiquitous neuromodulator adenosine [1,2]. Adenosine has a depressant action in the brain, heart, kidneys and other organs and is believed to mediate its effects via four adenosine receptor subtypes, termed A1, A2A, A2B and A3 (Table 1). Extracellular adenosine, produced locally in response to increased activity or stress, activates adenosine receptors of the A1 and A2A subtypes. The A2B subtype has a considerably higher threshold for activation by endogenous adenosine [3]. Such feedback mechanisms allow the organ to compensate for the increased activity or stress by decreasing energy demand, a feedback generally associated with A1 receptor activation, and by increasing oxygen supply (e.g. by vasodilation and inhibition of platelet and neutrophil function), a feedback generally associated with A2 receptor activation.
Recently, the A3 adenosine receptor subtype has been cloned [4], and its pharmacological [5-7] and regulatory [8] characteristics studied. This exciting development has opened new therapeutic vistas in the adenosine field [9]. The A3 receptor has a unique SAR (structure activity relationship) profile [10], tissue distribution [6,11] and effector coupling [4,8,12]. Activation of A3 receptors requires relatively high, i.e. pathological, concentrations of adenosine; the Ki value of adenosine at the rat A3 receptor is approximately 1 µM versus 10 and 30 nM at rat A1 and A2A receptors, respectively [9]. Thus, the physiological role of A3 receptors may be very different from those of A1 and A2A subtypes.
All of the adenosine receptors are members of the G protein-coupled receptor family and possess seven transmembrane helical regions [2,45]. Furthermore, it is clear that these receptors are structurally distinct from P2 purinoceptors. Site-directed mutagenesis of the A1 and A2A receptors [13,14,46] has yielded much insight into structure function relationships and the emerging receptor models are approaching a degree of resolution that promises to assist in the design of improved ligands. The aim of this article is to review the ever-increasing array of ligands that have been shown to interact with adenosine receptors. Further information on adenosine receptor classification is available in The RBI Handbook of Receptor Classification and Signal Transduction (Cat. No. T-172).
Adenosine Derivatives. There is a tremendous impetus for the development of therapeutic agents based on selective interactions with one of the four subtypes of adenosine receptors. In brain, exogenously administered adenosine receptor agonists have proven to be exceptionally efficient in producing neuroprotection [15-17]. Furthermore, adenosine has been shown to be involved in pain, cognition, movement and sleep [2,47]. The neuroprotective effect of A1 receptor activation is, in part, due to counteracting the damaging effects of excessive glutamate release [18]. Adenosine receptor agonists, which are almost exclusively derivatives of adenosine, have been sought as potential anti-arrhythmic, anti-lipolytic (thus anti-diabetic) and neuroprotective agents (A1), and hypotensive and anti-psychotic agents (A2A) [2]. A3 receptor agonists have potential as prophylactic neuroprotective agents [16,17]. The release of inflammatory mediators from mast cells in response to A3 receptor activation has been proposed to be responsible for the resultant hypotensive effects [44].
Highly selective ligands for adenosine A1 and A2A receptors [2] have been designed using both classical medicinal chemical approaches and a functionalized congener approach. By the latter approach, a chemically functionalized chain is incorporated at a specific site on a pharmacophore (e.g. adenosine amine congener, ADAC, Figure 1) leading to increased flexibility of substitution and enhancement of potency/selectivity via distal interactions at the receptor. In general, for adenosine receptor agonists, optimal modification of the N6-position with hydrophobic moieties, such as ADAC, R(-)-N6-(2-Phenylisopropyl)adenosine (R-PIA), N6-Cyclohexyladenosine (CHA) and N6-Cyclopentyladenosine (CPA) (Figure 1) has provided nanomolar potency and selectivity for A1 receptors. The affinities of many N6-substituted adenosine derivatives at A3 receptors are intermediate between their respective A1 and A2A affinities [10]. SPA (Figure 1) has selectivity for peripheral A1 receptors, due to its diminshed ability to cross biological membranes. This compound is available through the National Institute of Mental Health (NIMH) Chemical Synthesis Program.
Although most N6-substituted adenosine derivatives are A1 selective, the agonist DPMA (Figure 3) is 30-fold selective for the A2A receptor [19]. Evaluation of 2-position modifications of the highly potent, non-selective agonist 5'-N-Ethylcarboxamidoadenosine (NECA) (Figure 2) led to the identification of CGS 21680 (Figure 3), which is 140-fold selective for the A2A versus A1 receptor, with a Ki value of 21 nM [20]. Its ethylenediamine conjugate, APEC (Figure 3), a functionalized congener, can be radioiodinated as the p-aminophenylacetyl conjugate iodo-PAPA-APEC [34]. Its non-iodinated derivative, PAPA-APEC and its precursor APEC, are also available through the NIMH Chemical Synthesis Program. APEC is highly potent as a locomotor depressant when administered peripherally [21], unlike CGS 21680 which apparently does not cross the blood brain barrier. Both CGS 21680 and APEC are inactive at A2B receptors [3]. The 2-alkynyl-5'-uronamide derivative HE-NECA (Figure 3) is a potent A2A agonist (Ki 2.2 nM) [22], and also binds to A3 receptors (Ki 26 nM).
We recently reported the first A3 selective adenosine receptor agonists [7,10,23,24]. The non-selective adenosine receptor agonist N6-2-(4-Aminophenyl)ethyladenosine (APNEA) (Figure 2) had been used previously to activate A3 receptors, in combination with a xanthine such as 8-(p-Sulfophenyl)theophylline (SPT) (Figure 2) to eliminate non A3 receptor-mediated effects [44]. One principle of achieving true A3 receptor selectivity among adenosine derivatives is the combination of optimal substitutions at the N6- and 5'-positions of adenosine [10]. Specifically, among alkyl, cycloalkyl and aralkyl N6-substituents, a benzyl group is favored, due to its diminished potency at A1 and A2A receptors. The A3 selectivity enhancing effects of N6-benzyl modification are additive with the A3 affinity enhancing effects of the 5'-uronamido function, as in NECA. The first such hybrid molecule to show A3 receptor selectivity was N6-Benzyl-5'-N-ethylcarboxamidoadenosine (N6-benzyl-NECA ) (Figure 4) [10]. In a comparison of various 5'-uronamido groups in mono-substituted adenosine derivatives, the 5'-N-methyluronamide group [23] had a particularly favorable A3 versus A1/A2A receptor affinity. Empirical and SAR studies [24,25] of substituent effects on the N6-benzyl group have shown that substitution at the 3-position with sterically bulky groups, such as the iodo group, is optimal, leading to the development of the highly potent A3 receptor agonist IB-MECA (Figure 4). IB-MECA is 50-fold selective for A3 versus either A1 or A2 receptors in vitro [23] and appears to be highly selective for A3 receptors in vivo [7]. Although not selective, radioiodinated I-AB-MECA (Figure 2) is widely used as a high affinity radioligand for A3 receptors [26], with Kd values at cloned rat and human A3 receptors of 1.48 and 0.59 nM, respectively. AB-MECA (Figure 4) serves as the precursor to this radioligand. Urea substitution at the N6-position led to unexpected agonist potency and provided slight A3 receptor selectivity [27].
2-Substitution in combination with modifications at N6- and 5'-positions was found to further enhance A3 receptor selectivity [24]. Cl-IB-MECA (Figure 4), which displayed a Ki value of 0.33 nM, is selective for A3 versus A1 and A2A receptors by 2500- and 1400-fold, respectively. Cl-IB-MECA will be available from RBI through the NIMH synthesis program later this year. Certain N6-benzyl derivatives of adenosine also inhibit the Na+-independent adenosine transporter, yet IB-MECA and Cl-IB-MECA have been shown not to interact appreciably with this site [24].
In in vivo studies, chronic IB-MECA dramatically improved the histopathological and neurological outcome and preserved short term memory following cerebral ischemia in gerbils [16]. As with A1 receptor selective agents, a regimen-dependent inversion of the therapeutic result was also seen after administration of IB-MECA in the stroke model, with the acute administration resulting in an extensive deterioration. Chronic IB-MECA was protective in chemically-induced (N-methyl-D-aspartate or penta-methylenetetrazole ) seizures [17]. Significant improvement in seizure latency, neurological impairment and survival was observed. In electrically-induced seizures, chronic but not acute IB-MECA reduced postepileptic mortality. It is unknown whether the protective effect of chronically administered IB-MECA is related to effect on blood flow, neuronal mechanism, or both.
A3 Adenosine receptors have been proposed to play a role in the pathophysiology of cerebral ischemia, a condition where phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PIPLC) activation is known to occur. In both striatal and hippocampal slices, selective A3 adenosine receptor agonists, such as IB-MECA, stimulated PIPLC in a concentration-dependent manner [12]. In striatum, the potency order for adenosine receptor agonists was identical to their potency order for binding to cloned rat A3 receptors. Stimulation of PIPLC was abolished by guanosine-5'-O-(2-thiodiphosphate) , confirming the involvement of a G protein-coupled receptor. In agreement with the insensitivity of the cloned rat A3 receptor to xanthines, stimulation of PIPLC by adenosine analogues was only modestly antagonized by xanthine derivatives and at much higher concentration than needed for blocking adenosine A1, A2A and A2B receptors. Thus, stimulation of PIPLC represents a transduction mechanism for A3 receptors in mammalian brain and perhaps A3 receptor-mediated increases of inositol phosphates in the ischemic brain may contribute to neurodegeneration by raising intracellular calcium levels [12,16]. In a separate study, A3 adenosine receptor agonists induced intracellular Ca2+ release and apoptosis in human leukemia cells [48].
While an interaction with Gq-like proteins results in stimulation of PIPLC, A3 receptors may also interact with Gialpha-2 and Gialpha-3, leading to inhibition of adenylyl cyclase [8]. In a functional assay of rat A3 receptors expressed in Chinese Hamster Ovary (CHO) cells, Cl-IB-MECA inhibited adenylyl cyclase with an IC50 of 67 nM [24].
Xanthine Derivatives. Adenosine receptor antagonists, of which xanthines and a number of fused heterocyclic compounds are representative [2], have been under development as anti-asthmatic, anti-depressant, anti-arrhythmic, renal protective, anti-Parkinsonian and cognition enhancing drugs. Theophylline and caffeine have only moderate affinity (Ki > 10 µM) and are essentially non-selective for A1/A2 receptors [2] (Figure 2). Increasing chain-length at positions 1 and 3 increases affinity. Selective antagonists displaying nanomolar potency for A1 receptors include many 8-aryl and 8-cycloalkyl xanthine derivatives, such as xanthine amine congener (XAC ) and 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) (Figure 1). DPCPX is approximately 500-fold selective for A1 receptors. The acidic xanthine, BWA 522 (Figure 1) is 19-fold and 32-fold A1 selective versus A2A and A3 receptors, respectively (all in the rat) [6,28]. SPT (Figure 2) is useful as a peripheral acting antagonist. Some interesting newer 8-substituted xanthines include: KW-3902, which has potent diuretic properties [29]; KF15372, which is even more potent and A1 selective than DPCPX in guinea-pig forebrain [30]; and KFM-19, a potent A1 selective compound with sufficient aqueous solubility to display good bioavailability [31] (Figure 1). KF15372 and KFM-19 are currently under development as cognition enhancers. Many 8-substituted xanthines are A1 receptor selective; however, very few such as the 8-styrylxanthines KF17837 [32] and 8-(3-Chlorostyryl)caffeine (CSC; Figure 3) are A2A selective [33].
The A3 adenosine receptor cloned from rat [4] was shown to be unique among the subtypes in that agonist action is not antagonized by xanthines, such as theophylline. To date, A3 selective antagonists have not yet been reported [28]. Typical Ki values at rat A3 receptors of roughly 100 µM have been obtained for many xanthines that have nearly nanomolar potency at the A1 or A2A subtypes, and these xanthines do not effectively antagonize agonist-elicited inhibition of adenylyl cyclase [28]. In an effort to synthesize A3 receptor antagonists, we have attempted to maximize the affinity of xanthine derivatives at this binding site. The presence of an anionic group on the xanthine tended to diminish the affinity at A1 and A2A receptors. One such xanthine is MRS512 (Figure 4), which has a Ki value at rat A3 receptors of 9.4 µM and is 7-fold selective for rat A3 versus A2A receptors. The affinity of certain xanthines is highly species dependent, such as BWA 522 (Figure 1), which is more potent in binding in the human (Ki 18 nM) and sheep (Ki 3 nM) homologues of the A3 receptor [6,11,28] than in the rat (Ki 1.17 µM). The differences in antagonist affinity and the low degree of homology (~70%) among A3 receptors of different species raises the question whether these clones represent a single subtype. In rodents, 1 µM XAC has been used in pharmacological experiments in vitro and in vivo [16] for distinguishing A3 receptors (Ki 29 µM, rat) from A1 and A2A receptors, at which it is much more potent.
It has been proposed [10,13] that the ribose moiety of adenosine, which is relatively more important for high affinity binding to A3 receptors than at other subtypes, is coordinated to a histidine residue that is conserved among all the adenosine receptors in the seventh transmembrane helical domain. This is one of two conserved histidine residues essential for ligand recognition in A1 and A2 receptors [13,14,46]; the other histidine, which occurs in the sixth transmembrane helical domain, is absent in A3 receptors. Consequently, we tested the hypothesis that a means of anchoring xanthines in the A3 binding site is by adding a sugar moiety at the 7-position to form xanthine-7-ribosides. At rat brain A3 receptors, 1,3-dibutylxanthine-7-riboside was found to bind with a Ki value of 6 µM [10], whereas the parent xanthine, 1,3-dibutylxanthine, displayed a Ki value of 143 µM. Thus, the presence of the ribose moiety enhances affinity of xanthines at rat A3 receptors, while at A1 receptors the xanthine-7-riboside derivatives are, as a rule, less potent than the parent xanthines [34]. Functionally, 1,3-dibutylxanthine-7-riboside, as a structural hybrid of classical A1/A2A agonist and antagonist molecules, appeared to act as a partial agonist at rat A3 receptors [10], providing hope that this was a means of designing antagonists. However, upon structural modification that increased the potency and selectivity of the xanthine ribosides at A3 receptors, full agonism was observed [35]. Specifically, the structural parallel between adenosine derivatives and the xanthine-7-ribosides was maintained with respect to A3 receptor affinity. This parallel lead to the design of DBXRM (Figure 4), having a Ki value of 229 nM at A3 receptors with 160-fold, and greater than 400-fold selectivity, for A3 versus A1 and A2A receptors, respectively [35]. The selectivity of this compound is a result of incorporation of the 5'-methyluronamide group, found to enhance A3 selectivity in IB-MECA, and optimization of the alkyl chain length at positions 1 and 3. Unlike 1,3-dibutylxanthine-7-riboside, DBXRM acted as a full agonist in the rat A3 receptor-mediated inhibition of adenylyl cyclase. Thus, there was a tendency towards greater efficacy as the affinity increased within the same series of compounds [34].
Non-Xanthine Antagonist Ligands. Numerous structurally diverse, non-xanthine, adenosine receptor antagonists (Figure 3) have also been identified, many of which are not well defined in terms of SAR. Among the first classes of heterocycles found to antagonize the effects of adenosine receptor agonists were the tricyclic non-xanthine antagonists, including the triazoloquinazolines [36] and the triazoloquinoxalines, including the A2 selective antagonist CP 66,713 [21] (Figure 3). CGS 15943 (Figure 2), is a potent adenosine receptor antagonist with slight selectivity for the A2A receptor (IC50 3 nM) [36]. Other potent A1 selective antagonists have been derived from adenine, including N-0861 (Figure 1) [37,38]. An attempt to design A3 selective adenines by applying the structural principles of recognition derived from adenosine agonists resulted in loss of potency and selectivity. The recent reports [38,39] of the highly potent and selective non-xanthine A2A receptor antagonists SCH-58261 and ZM241385 (Figure 3) have removed a major obstacle in the characterization of the function of adenosine A2 receptors. The non-xanthine ZM241385 is the most selective A2A antagonist (6800-fold) yet reported [38]. Radioiodination of ZM241385 has provided a highly potent and selective A2A antagonist radioligand [40]. Alloxazine is approximately 10-fold selective for A2B versus either A1 or A2A adenosine receptors [41] (Figure 3).
Recently, we identified novel adenosine receptor ligands as a result of screening diverse chemical libraries for lead structures for developing A3 adenosine antagonists [42,43]. A number of classes of non-xanthine adenosine antagonists have already been reported, including various nitrogen heterocycles [2] and several classes of non-nitrogen heterocycles [43]. For example we recently reported that tetrahydrobenzothiophenones, e.g. BTH4 (Figure 2), bind to adenosine receptors in the micromolar range [43]. Folic acid, pyridopyrimidinone, cytochalasin H, 11-hydroxy-tetrahydrocarbazolenine, the adenosine uptake inhibitor dipyridamole, certain sulfonylpiperazines (e.g. HA-100), and a number of other heterocyclic substances displaced specific [125I]-AB-MECA binding to rat A3 adenosine receptors selectively, although weakly [42].
Summary. Highly selective agonists and antagonists have been designed for A1 and A2A receptors. Selective agents are potentially useful in the treatment of cardiovascular, renal and central nervous system disorders. The A3 receptor has been characterized and shown to be a distinct receptor subtype, through cloning and the synthesis of selective agonists. A3 receptor selective antagonists are currently sought. The effects of A3 receptor activation on the inflammatory system and in the brain suggests the use of A3 selective agents for asthma, inflammatory diseases and cerebroprotection.
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