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A series of novel pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine substituted at N 8 pyrazole nitrogen and at the 5-amino group was synthesized and their affinities to all four cloned human adenosine receptor subtypes were evaluated by competition binding assays using [ 3 H]-DPCPX (A 1 and A 2B ), [ 3 H]-SCH 58261 (A 2A ), and [ 3 H]-MRE3008-F20 (A 3 ) as radioligands. In particular, the structural requirements necessary for adenosine A 2B receptor recognition were investigated. These preliminary results confirm that the free amino group at the 5-position confers better A 2B affinity, while the effect of the chain at nitrogen pyrazole nucleus, the 8-position, seems to be preferred with respect to the corresponding N 7 regioisomers. The introduction of an aminoacyl chain at the 5-amino group produces better selectivity for A 2B receptors vs. A 2A , but the compounds still remain more potent for A 1 and a significant decrease (about 5-fold) of affinity at A 2B receptors was observed. Surprisingly, with these polar chains a good affinity at human A 3 adenosine receptors was also detected. The most interesting compounds in binding studies proved to be potent antagonists (nM range) in functional assays, measuring the inhibition of cAMP production induced by 100 nM NECA.

Several studies have evidenced the opposite role played by dopamine and adenosine receptors in the control of motor behavior. In line with those studies, we have previously shown that the acute administration of the A 2A receptor antagonist SCH 58261 potentiated the turning behavior induced by L-DOPA in the unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease (PD), suggesting that selective adenosine A 2A receptor antagonists might be useful as adjuncts to L-DOPA therapy. One of the most important problems in the treatment of PD is to find a drug regimen effective after chronic administration and devoid of dyskinesia-like side effects. Previous studies have shown that the sensitized turning response, developed in 6-OHDA-lesioned rats after chronic intermittent L-DOPA, is a useful model of dyskinesia. In the present study, we evaluated the effect of a chronic administration of SCH 58261 alone or in combination with L-DOPA in 6-OHDA lesioned rats in order to verify the effectiveness of SCH 58261 after repeated administration and its dyskinetic potential. Repeated administration of SCH 58261 (5 mg/kg) did not produce tolerance to its ability to potentiate L-DOPA-turning behavior. Moreover, chronic intermittent SCH 58261 (5 mg/kg) plus L-DOPA produced a stable turning behavior response during the course of the treatment, whereas L-DOPA alone produced a progressive increase in turning behavior intensity and duration (sensitization). These results suggest that SCH 58261 is effective in potentiating L-DOPA-induced turning behavior even after a chronic treatment and it does not produce, in combination with L-DOPA, motor response alterations. A 2A receptor antagonists could be a new and useful tool for the treatment of PD.

Of the four known adenosine receptors, the A 2A receptor has received much attention over the last few years. The discovery of high-affinity and selective A 2A adenosine receptor antagonists, together with the development of different genetic lines of mice lacking A 2A receptors, have greatly contributed to the new insights into the mechanisms whereby A 2A receptors modulate central nervous system functions. Efforts made using the prototypic A 2A receptor antagonists, e.g., the 8-styrylxanthine KW 6002 and the pyrazolotriazolopyrimidine SCH 58261, have shown that these drugs are effective in different models of motor impairment mimicking the main features of Parkinson's disease. Moreover, these drugs show neuroprotective properties in models of brain injury. Consistent with pharmacology, A 2A receptor knockout mice have been found to be less sensitive to both motor impairment and neurochemical changes relevant to neurodegenerative disorders. The main effect of A 2A receptor blockade or inactivation is related to selective interaction with dopamine-mediated function in the striatum. However, there are responses which appear to be independent of dopamine receptors while the mechanisms underlying neuroprotection remain to be elucidated. Overall, there are now compounds that appear to be promising for treatment of Parkinson's disease and related neurodegenerative disorders. The efforts currently ongoing to understand their efficacy in patients will make it possible to assess whether A 2A receptor blockers are a new interesting class of antiparkinsonian agents.

Caffeine is the most widely consumed of all psychoactive drugs. In doses that induce behavioral stimulation it acts primarily on adenosine A 2A and possibly A 1 receptors. Actions on the latter appear to be very much involved with regulation of the stimulatory glutamatergic input to several neuronal structures, including the striatum. Adenosine A 2A receptors are found enriched on a subpopulation of GABAergic output neurons from the striatum, where they coexist with dopamine D 2 receptors. The two receptors interact, both at the membrane level and by having opposing effects at the level of second messengers. Inhibition of adenosine A 2A receptors by the selective antagonist SCH 58261 causes motor stimulation and a decrease in the expression of immediate early genes in these striatopallidal neurons. This effect is also clearly seen when dopamine D 2 activation is blocked, either by preventing dopaminergic impulse flow or by receptor blockade. Thus, a tonic effect of endogenous adenosine is specifically antagonized by adenosine receptor antagonists such as caffeine. Despite a strictly delimited primary effect, secondary interactions between neurons lead to changes in the activity of several groups of neurons in interconnected networks. These changes likely underlie the behavioral effects of caffeine.

Recent studies have clearly shown that the adenosine A 2A receptors are present in a variety of peripheral tissues, including smooth muscle cells, heart muscle and coronary arteries, and human circulating blood elements. This paper reviews the studies performed by our research group on the A 2A receptors on human platelets, lymphocytes, and neutrophils. Affinity and potency of typical adenosine receptor ligands were compared in binding and functional studies as adenylyl cyclase, aggregation, and superoxide anion production assays. Saturation experiments, performed by using the selective A 2A adenosine receptor antagonist [ 3 H]SCH 58261 (5-amino-/-(2-phenylethyl)-2-(2-turyl)-pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5c] pyrimidine) revealed a single class of binding sites in all different preparations examined with affinity values in the nanomolar range (0.85-1.34 nM) and Bmax values ranging from 35 to 80 fmol/mg protein. Competition experiments showed that the potency order of agonists and antagonists studied was similar in all human circulating blood elements. In the functional assays, the same compounds exhibited a rank order of potency identical to that observed in binding experiments. Moreover, an excellent rank order correlation was found between cAMP accumulation, aggregation, and superoxide anion production data by adenosine receptor agonists and antagonists examined. Thermodynamic data indicated that [ 3 H]SCH 58261 binding to human lymphocytes and neutrophils is entropy and enthalpy driven, a finding in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A 2A adenosine receptors.

The recent discovery of selective antagonists for the A2A adenosine receptors has been of great help to further research in this field. One compound, SCH 58261, 5- amino-7-(β-phenylethyl)-2-(8-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, is playing a key part in a variety of studies. This review describes its pharmacological characteristics as they are emerging in various laboratories. The compound has an affinity in the low nM range (Ki value of 1–2 nM) for A2A receptors located on membranes from a variety of tissues and cell types, including rat and bovine brain striatum, human platelets, lymphocytes and neutrophils, porcine coronary arteries, and CHO cells transfected with the cloned human A2A receptors. SCH 58261 has little or no affinity up to the μM range for adenosine A2B, A3, or other G protein-coupled receptors. Selectivity for A2A vs. A1 receptors varies from 53- to 750-fold, depending on membranes or type of assay. SCH 58261 blocks A2A-receptor mediated increase of cyclic AMP formation with high potency (e.g., IC50 values between 15 and 20 nM in human white blood cells). The tritiated form of SCH 58261 specifically labels A2A receptors in discrete regions of the rat brain such as caudate-putamen, nucleus accumbens, and tuberculum olfactorium. In classic in vitro bioassays, such as A2A-receptor-mediated vasodilation in coronary arteries, SCH 58261 displays competitive antagonistic properties (e.g., pA2 value of 9.5 in porcine coronary arteries). In assays involving responses mediated by A1 or A2B receptors, SCH 58261 shows little or no activity up to concentrations about 100-fold higher than those affecting A2A receptors (higher concentrations not being testable due to its poor water solubility). In the rat, SCH 58261 enhances locomotor activity (at 3.7 mg/kg ip), increases wakefulness (10 mg/kg ip) and slightly increases both blood pressure and heart rate (10 mg/kg ip). These activities appear to be specifically mediated by A2A receptors since the drug counteracts the effect of A2A receptor agonists in some experimental paradigms. In models mimicking CNS disorders, SCH 58261 potentiates the activity of L-DOPA or dopamine receptor agonists in the 6-hydroxydopamine-lesioned rat model. Moreover, the compound reduces brain infarct size in a rat model of cerebral ischemia. Altogether, SCH 58261 and its radiolabeled form have emerged as interesting tools for better understanding the function of A2A receptors in physiological or altered conditions. Moreover, the neuroprotective properties of SCH 58261 indicate that drugs of this class have a potential for treatment of brain damage produced by Parkinson's disease or stroke. Drug Dev. Res. 42:63–70, 1997. © 1997 Wiley-Liss, Inc.

Acute stimulation of A3 adenosine receptors has been shown to intensify damage in an in vivo model of global cerebral ischemia and to induce cell death in cell cultures. To investigate the mechanism for these cytotoxic phenomena, adenosine analogs of varied receptor subtype selectivities were applied directly to rat cerebellar granule neurons in culture. Agonists of A1, A2A, and/or A2B receptors (CPA, NECA and CGS 21680) had no effect on neuronal survival during a 16 h incubation period. The highly selective adenosine A3 receptor agonist 2-chloro-N 6 ‐(3-iodobenzyl)adenosine-5´-N-methyluronamide (Cl-IB-MECA) at ≥10 μM induced cell death in a concentration-dependent fashion. A structurally related nucleoside, cladribine, which has cytotoxic properties via a non-adenosine receptor related mechanism, had no effect on cell survival. Adding dbcAMP to the culture to activate cyclic AMP-dependent protein kinases attenuated Cl-IB-MECAinduced neurotoxicity, suggesting the Cl-IB-MECA-induced neuronal cell death is mediated by an inhibition of cyclic AMP production. Glutamate (50 μM) induced cell death to a degree comparable to that induced by 10 μM Cl-IB-MECA. Furthermore, a subcytotoxic concentration of Cl-IB-MECA (1 μM) significantly augmented glutamate neurotoxicity. In contrast, the adenosine A1/A2 agonists, the adenosine A1/A2 antagonist XAC, and the A2A antagonist SCH 58261 at micromolar concentrations had no effect on neuronal viability or on glutamate neurotoxicity. These results suggest that activation of adenosine A3 receptors may play an important role in regulating neuronal survival and death in cerebellar neurons. Drug Dev. Res. 40:267‐273, 1997. © 1997 Wiley-Liss, Inc.

Progress in the identification of novel P1 and P2 receptor ligands has continued to lag behind the explosion in receptor cloning, especially in the P2 area. Nonetheless, a number of novel chemical entities and natural receptor ligands are continuing to advance in clinical trials or, alternatively have become important new tools to study receptor function. Compounds of note with activity at the P1 receptor family include NNC 21-0136 (A 1 agonist; preclinical; stroke); SCH 59761 (nonselective P1 agonist; preclinical; cardiovascular disorders); the A 1 antagonists, KFM-19 (BIIP-20; phase II) and MDL 102,503 development (status unknown) that may have therapeutic potential as cognition enhancers. KF 17837 and related A 2A -antagonists such as KW 6002 represent potential novel treatments for Parkinson's disease. SCH 58261 (A 2A receptor antagonist; preclinical) is a novel nonxanthine antagonist ligand. KW 3902 (phase II), FK- 453/FK 113453 (possibly discontinued) and CVT-124 (phase I) are A 1 receptor-selective xanthine-based antagonists that have potential in the treatment of renal diseases. NNC 53-0055 (preclinical) is the first of a new series of selective A 3 receptor agonists that modulate cytokine production. MRS 1067, MRS 1067, MRS 1097, MRS 1222, L-249, 313, and L-268, 605 (all preclinical) represent new A 3 -receptor antagonists. GP 3269 (preclinical) is an adenosine kinase inhibitor with potential efficacy in septic shock, stroke, and pain. ARL 67085 (phase II) is an ATP bioisostere that is an antagonist of the P 2T receptor that is the first of new generation of antithrombotic agents. Systemic ATP has reached phase II trials as a novel approach to metastasis regression. The pyrimidine nucleotide, UTP (phase II) is being examined as P2Y 2 receptor agonist for the treatment of cystic fibrosis.

There is evidence that adenosine may participate in the regulation of sleep and wakefulness in the mammalian central nervous system. To understand whether the adenosine receptor subtypes, A1 and A2A, are involved in the modulation of sleep and waking, we carried out electroencephalographic (EEG) studies in the rat using selective agonists and antagonists for either receptor. EEG activity was recorded for 6 h after intraperitoneal administration of drugs, and the stages of wakefulness, rapid eye movements (REM) sleep and non-REM sleep were classified thereafter. The dose-response effects of the A1 agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), and the A2A agonist, 2-hexynyl-5′-N-ethylcarboxamido-adenosine (2HE-NECA), were examined. Both drugs, CCPA (0.003–0.03 mg/kg) and 2HE-NECA (0.03–0.3 mg/kg), given at a dose range known to be effective pharmacologically did not significantly modify the sleep patterns in the whole recording period. We have also studied the effects of the selective A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthyne (DPCPX), and the A2A antagonist, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-c]-1,2,4,-triazolo-[1,5-c]-pyrimidine (5CH 58261). The results obtained with antagonists were compared with those of two non-selective adenosine antagonists, caffeine (10 mg/kg) and 9-chloro-2-(2-furyl)-5,6-dihydro-[1,2,4]-triazolo-[1,5-c]-quinazolin-5-imine (CGS 15943). Like caffeine, both CGS 15943 (0.3–10 mg/kg) and SCH 58261 (0.3–10 mg/kg), at the highest dose, increased wakefulness, while DPCPX (0.3–10 mg/kg) did not affect sleep parameters. The data indicate that potent and selective adenosine agonists have little or no effect on sleep states in the rat, while using adenosine antagonists it seems that A2A receptors are primarily involved in the modulation of wakefulness. © 1996 Wiley-Liss, Inc.