The A3 adenosine receptor: history and perspectives.

By general consensus, the omnipresent purine nucleoside adenosine is considered a major regulator of local tissue function, especially when energy supply fails to meet cellular energy demand. Adenosine mediation involves activation of a family of four G protein-coupled adenosine receptors (ARs): A(1), A(2)A, A(2)B, and A(3). The A(3) adenosine receptor (A(3)AR) is the only adenosine subtype to be overexpressed in inflammatory and cancer cells, thus making it a potential target for therapy. Originally isolated as an orphan receptor, A(3)AR presented a twofold nature under different pathophysiologic conditions: it appeared to be protective/harmful under ischemic conditions, pro/anti-inflammatory, and pro/antitumoral depending on the systems investigated. Until recently, the greatest and most intriguing challenge has been to understand whether, and in which cases, selective A(3) agonists or antagonists would be the best choice. Today, the choice has been made and A(3)AR agonists are now under clinical development for some disorders including rheumatoid arthritis, psoriasis, glaucoma, and hepatocellular carcinoma. More specifically, the interest and relevance of these new agents derives from clinical data demonstrating that A(3)AR agonists are both effective and safe. Thus, it will become apparent in the present review that purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health.

Antidyskinetic effect of A2A and 5HT1A/1B receptor ligands in two animal models of Parkinson's disease.

BACKGROUND: The serotonin 5-HT1A/1B receptor agonist eltoprazine suppressed dyskinetic-like behavior in animal models of Parkinson's disease (PD) but simultaneously reduced levodopa (l-dopa)-induced motility. Moreover, adenosine A2A receptor antagonists, such as preladenant, significantly increased l-dopa efficacy in PD without exacerbating dyskinetic-like behavior. OBJECTIVES: We evaluated whether a combination of eltoprazine and preladenant may prevent or suppress l-dopa-induced dyskinesia, without impairing l-dopa's efficacy in relieving motor signs, in 2 PD models: unilateral 6-hydroxydopamine-lesioned rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. METHODS: Rotational behavior and abnormal involuntary movements, or disability and l-dopa-induced dyskinesia were evaluated in 6-hydroxydopamine-lesioned rats and MPTP-treated monkeys, respectively. Moreover, in the rodent striatum, induction of immediate-early gene zif-268, an index of long-term changes, was correlated with dyskinesia. RESULTS: In 6-hydroxydopamine-lesioned rats, combined administration of l-dopa (4 mg/kg) plus eltoprazine (0.6 mg/kg) plus preladenant (0.3 mg/kg) significantly prevented or reduced dyskinetic-like behavior without impairing motor activity. Zif-268 was increased in the striatum of rats treated with l-dopa and l-dopa plus preladenant compared with vehicle. In contrast, rats treated with eltoprazine (with or without preladenant) had lower zif-268 activation after chronic treatment in both the dyskinetic and l-dopa-non-primed groups. Moreover, acute l-dopa plus eltoprazine plus preladenant prevented worsening of motor performance (adjusting step) and sensorimotor integration deficit. Similar results were obtained in MPTP-treated monkeys, where a combination of preladenant with eltoprazine was found to counteract dyskinesia and maintain the full therapeutic effects of a low dose of l-dopa. CONCLUSIONS: Our results suggest a promising nondopaminergic pharmacological strategy for the treatment of dyskinesia in PD. © 2016 International Parkinson and Movement Disorder Society.

Synthesis and biological evaluation of novel 2-amino-3-aroyl-4-neopentyl-5-substituted thiophene derivatives as allosteric enhancers of the A1 adenosine receptor.

2-Amino-3-benzoyl thiophenes have been widely reported to act as allosteric enhancers at the A1 adenosine receptor. Their activity can be increased considerably by appropriate substitutions at the 4- and 5-positions of the thiophene ring. Substituent size at the thiophene C-4 position seemed to be a factor closely related to activity, with the 4-neopentyl (2,2-dimethylpropyl) substitution showing the greatest enhanced activity. A wide series of 2-amino-3-aroyl-4-neopentylthiophene derivatives with general structure 3, characterized by the presence of different substituents (bromine, aryl and heteroaryl) at the 5-position of the thiophene ring, have been identified as potent AEs at the A1AR. With only one exception, all of the synthesized compounds proved to be superior to the reference compound PD 81,723 in a functional assay. Derivatives 3p, 3u, 3am, 3ap and 3ar were the most active compounds in binding (saturation and competition) and functional cAMP studies, being able to potentiate agonist [(3)H]CCPA binding to the A1 receptor.

Design, synthesis and biological evaluation of 3,5-disubstituted 2-amino thiophene derivatives as a novel class of antitumor agents.

In search of new compounds with strong antiproliferative activity and simple molecular structure, we designed a novel series of agents based on the 2-amino-3-alkoxycarbonyl/cyano-5-arylethylthiophene scaffold. The presence of the ethyl spacer between the 2',5'-dimethoxyphenyl and the 5-position of the thiophene ring, as well as the number and location of methoxy substitutents on the phenyl ring, played a profound role in affecting the antiproliferative activity. Among the synthesized compounds, we identified the 2-amino-3-cyano-[2-(2,5-dimethoxyphenyl)ethyl] thiophene 2c as the most promising derivative against a wide panel of cancer cell lines (IC50=17-130 nM). The antiproliferative activity of this compound appears to correlate well with its ability to inhibit tubulin assembly and the binding of colchicine to tubulin. Moreover 2c, as determined by flow cytometry, strongly induced arrest in the G2/M phase of the cell cycle, and annexin-V and propidium iodide staining indicate that cell death proceeds through an apoptotic mechanism that follows the intrinsic mitochondrial pathway.

Synthesis and biological evaluation of 2-substituted-4-(3',4',5'-trimethoxyphenyl)-5-aryl thiazoles as anticancer agents.

Antitumor agents that bind to tubulin and disrupt microtubule dynamics have attracted considerable attention in the last few years. To extend our knowledge of the thiazole ring as a suitable mimic for the cis-olefin present in combretastatin A-4, we fixed the 3,4,5-trimethoxyphenyl at the C4-position of the thiazole core. We found that the substituents at the C2- and C5-positions had a profound effect on antiproliferative activity. Comparing compounds with the same substituents at the C5-position of the thiazole ring, the moiety at the C2-position influenced antiproliferative activities, with the order of potency being NHCH(3) > Me >> N(CH(3))(2). The N-methylamino substituent significantly improved antiproliferative activity on MCF-7 cells with respect to C2-amino counterparts. Increasing steric bulk at the C2-position from N-methylamino to N,N-dimethylamino caused a 1-2 log decrease in activity. The 2-N-methylamino thiazole derivatives 3b, 3d and 3e were the most active compounds as antiproliferative agents, with IC(50) values from low micromolar to single digit nanomolar, and, in addition, they are also active on multidrug-resistant cell lines over-expressing P-glycoprotein. Antiproliferative activity was probably caused by the compounds binding to the colchicines site of tubulin polymerization and disrupting microtubule dynamics. Moreover, the most active compound 3e induced apoptosis through the activation of caspase-2, -3 and -8, but 3e did not cause mitochondrial depolarization.

Structure-activity relationships of 2-amino-3-aroyl-4-[(4-arylpiperazin-1-yl)methyl]thiophenes. Part 2: Probing the influence of diverse substituents at the phenyl of the arylpiperazine moiety on allosteric enhancer activity at the A1 adenosine receptor.

In a preliminary article, we reported the potent allosteric enhancer activity at the A(1) adenosine receptor of a small series of 2-amino-3-(4-chlorobenzoyl)-4-[4-(aryl)piperazin-1-yl)methyl]thiophene derivatives bearing electron-withdrawing or electron-releasing groups at the para-position of the phenylpiperazine moiety. In the present study, we report the development of the compounds previously studied by modifying both the number and position of substituents on the phenylpiperazine moiety, aimed at establishing a structure-activity relationship identifying additional compounds with improved activity. The nature and the position of substituents on the phenyl ring tethered to the piperazine seemed to exert a fundamental influence on the allosteric enhancer activity, with the 3,4-difluoro 4i, 3-chloro-4-fluoro 4o, and 4-trifluoromethoxy 4ak derivatives being the most active compounds in binding (saturation and competition experiments) and functional cAMP studies. This study shows that it is also possible to obtain a good separation between allosteric enhancement and antagonistic activity at the A(1) adenosine receptor.

Recent developments in A2B adenosine receptor ligands.

A selective, high-affinity A(2B) adenosine receptor (AR) antagonist will be useful as a pharmacological tool to help determine the role of the A(2B)AR in inflammatory diseases and angiogenic diseases. Based on early A(2B)AR-selective ligands with nonoptimal pharmaceutical properties, such as 15 (MRS 1754: K(i)(hA(2B)) = 2 nM; K(i)(hA(1)) = 403 nM; K(i)(hA(2A)) = 503 NM, and K(i)(hA(3)) = 570 nM), several groups have discovered second-generation A(2B)AR ligands that are suitable for development. Scientists at CV Therapeutics have discovered the selective, high-affinity A(2B)AR antagonist 22, a 8-(4-pyrazolyl)-xanthine derivative, (CVT-6883, K(i)(hA(2B)) = 22 nM; K(i)(hA(1)) = 1,940 nM; K(i)(hA(2A)) = 3,280; and K(i)(hA(3)) = 1,070 nM). Compound 22 has demonstrated favorable pharmacokinetic (PK) properties (T(1/2) = 4 h and F > 35% rat), and it is a functional antagonist at the A(2B)AR(K (B) = 6 nM). In a mouse model of asthma, compound 22 demonstrated a dose-dependent efficacy supporting the role of the A(2B)AR in asthma. In two Phase I clinical trails, 22 (CVT-6883) was found to be safe, well tolerated, and suitable for once-daily dosing. Baraldi et al. have independently discovered a selective, high-affinity A(2B)AR antagonist, 30 (MRE2029F20), 8-(5-pyrazolyl)-xanthine (K(i)(hA(2B)) = 5.5 nM; K(i)(hA(1)) = 200 nM; K(i)(hA(2A), A(3)) > 1,000, that has been selected for development in conjunction with King Pharmaceuticals. Compound 30 has been demonstrated to be a functional antagonist of the A(2B)AR, and it has been radiolabeled for use in pharmacological studies. A third compound, 58 (LAS-38096), is a 2-aminopyrimidine derivative (discovered by the Almirall group) that has high A(2B)AR affinity and selectivity (K(i)(hA(2B)) = 17 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 2,500; and K(i)(hA(3)) > 1,000 nM), and 58 has been moved into preclinical safety testing. A fourth selective, high-affinity A(2B)AR antagonist, 54 (OSIP339391 K(i))(hA(2B)) = 0.5 nM; K(i))(hA(1)) = 37 nM; K(i))(hA(2A)) = 328; and K(i))(hA(3)) = 450 nm) was discovered by the OSI group. The three highly selective, high-affinity A(2B)AR antagonists that have been selected for development should prove useful in subsequent clinical trials that will establish the role of the A(2B)ARs in various disease states.

Discovery of 8-methoxypyrazino[1,2-a]indole as a New Potent Antiproliferative Agent Against Human Leukemia K562 Cells. A Structure-Activity Relationship Study.

Identification of novel and selective anticancer agents remains an important and challenging goal in pharmacological research. The indole nucleus, frequently encountered as a molecular fragment in natural products and pharmaceutically active compounds, was employed as the initial building block for the synthesis of a series of pyrazino[1,2-a]indoles 1a-k, variably substituted at the 6, 7, 8 and 9-positions. Compound 1e, bearing the methoxy group at the 8-position of the pyrazino[1,2-a]indole nucleus was identified as a novel potent antiproliferative agent against the human chronic myelogenous leukemia K562 cell line, but it was much less active against several other cancer cell lines. Comparison of positional isomers indicated that moving the methoxy group from the 8- to the 7- or 6-position, to furnish compounds 1f and 1g, respectively, yielded inactive compounds. The analysis of structure-activity relationships observed in the series of investigated compounds may represent the basis for the design of more active molecules.

Synthesis and biological evaluation of 1-methyl-2-(3',4',5'-trimethoxybenzoyl)-3-aminoindoles as a new class of antimitotic agents and tubulin inhibitors.

The 2-(3,4,5-trimethoxybenzoyl)-2-aminoindole nucleus was used as the fundamental structure for the synthesis of compounds modified with respect to positions C-4 to C-7 with different moieties (chloro, methyl, or methoxy). Additional structural variations concerned the indole nitrogen, which was alkylated with small alkyl groups such as methyl or ethyl. We have identified 1-methyl-2-(3,4,5-trimethoxybenzoyl)-3-amino-7-methoxyindole as a new highly potent antiproliferative agent that targets tubulin at the colchicine binding site and leads to apoptotic cell death.

1,3-Dipropyl-8-(1-phenylacetamide-1H-pyrazol-3-yl)-xanthine derivatives as highly potent and selective human A(2B) adenosine receptor antagonists.

A new series of 1,3-dipropyl-8-(1-phenylacetamide-1H-pyrazol-3-yl)-xanthine derivatives has been identified as potent A(2B) adenosine receptor antagonists. The products have been evaluated for their binding affinities for the human A(2B), A(1), A(2A), and A(3) adenosine receptors. N-(4-chloro-phenyl)-2-[3-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-5-methyl-pyrazol-1-yl] (11c) showed a high affinity for the human A(2B) adenosine receptor K(i)=7nM and good selectivity (A(1), A(2A), A(3)/A(2B)>140). Synthesis and SAR of this novel class of compounds is presented herein.

Structure-activity relationship studies of a new series of imidazo[2,1-f]purinones as potent and selective A(3) adenosine receptor antagonists.

We recently described the synthesis of 1-benzyl-3-propyl-1H,8H-imidazo[2,1-f]purine-2,4-diones, new potent and selective A(3) adenosine receptor antagonists containing a xanthine core. The present work can be considered an extension of our SAR studies on related structures in which the effect of different kind of substitutions at the 1-, 3- and 8-positions has been evaluated in order to improve both the potency and the hydrophilicity of the originally synthesised molecules. The A(3) binding disposition of these compounds was also investigated through docking and 3D-QSAR studies.

Synthesis and biological evaluation of novel 1-deoxy-1-[6-[((hetero)arylcarbonyl)hydrazino]- 9H-purin-9-yl]-N-ethyl-beta-D-ribofuranuronamide derivatives as useful templates for the development of A2B adenosine receptor agonists.

The lack of molecules endowed with selective and potent agonistic activity toward the hA2B adenosine receptors has limited the studies on this pharmacological target and consequently the evaluation of its therapeutic potential. We report the design and the synthesis of the first potent (EC50 in the nanomolar range) and selective hA2B adenosine receptor agonists consisting of 1-deoxy-1-[6-[((hetero)arylcarbonyl)hydrazino]-9H-purin-9-yl]-N-ethyl-beta-D-ribofuranuronamide derivatives. The concurrent effect of 6-substitution of the purine nucleus with a ((hetero)arylcarbonyl)hydrazino function and a 2-chloro substitution has been investigated in such NECA derivatives.

From tyrosine to glycine: synthesis and biological activity of potent antagonists of the purinergic P2X7 receptor.

The characterization of the native and recombinant P2X7 receptor continues to be hindered by the lack of specific and subtype-selective antagonists with a "druglike" profile. However, a tyrosine derivative named KN-62 exhibits selective P2X7 receptor-blocking properties. As a molecular simplification of KN-62, the present study was designed to evaluate the functional antagonistic properties of a novel series of glycine derivatives characterized by the presence of different phenyl-substituted piperazine moieties. Antagonistic activity of these glycine derivatives was tested on HEK293 cells transfected with the human P2X7 receptor. The most potent P2X7 receptor antagonist identified in this study (compound 4g) contains an o-fluorine substituent on the phenylpiperazine moiety and had an IC50 of 12.1 nM. The biological responses investigated were ATP-dependent Ca2+ influx across the plasma membrane and ethidium bromide uptake.

Synthesis and biological evaluation of 2- and 3-aminobenzo[b]thiophene derivatives as antimitotic agents and inhibitors of tubulin polymerization.

Two new series of inhibitors of tubulin polymerization based on the 2-amino-3-(3,4,5-trimethoxybenzoyl)benzo[b]thiophene molecular skeleton and its 3-amino positional isomer were synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. Although many more 3-amino derivatives have been synthesized so far, the most promising compound in this series was 2-amino-6-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[b]thiophene, which inhibits cancer cell growth at subnanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.

Synthesis and biological evaluation of 2-amino-3-(3', 4', 5'-trimethoxy-phenylsulfonyl)-5-aryl thiophenes as a new class of antitubulin agents.

Bioisosterism represents one approach used by the medicinal chemist for the rational modification of lead compounds into safer and more clinically effective agents. Bioisosteres are substituents or groups that have chemical or physical similarities and that produce broadly similar biological effects. The sulfone moiety is recognized as a nonclassical bioisostere for replacement of the carbonyl group. When sulfonyl derivatives 5a-e were compared with carbonyl compounds 4a-e, the sulfone substitution dramatically decreased the antiproliferative activity of the series.

Ligands for A2B adenosine receptor subtype.

Adenosine is a naturally occurring nucleoside, which exerts its biological effects by interacting with a family of adenosine receptors known as A(1), A(2A), A(2B), and A(3). The A(2B) subtype is a low affinity receptor, which couples to stimulation of adenylyl cyclase and also leads to a rise in intracellular calcium modulating important physiological processes. Adenosine exhibiting activity at this subtype is at concentrations greater than 10 microM. The A(2B) receptors show a ubiquitous distributions, the highest levels are present in cecum, colon and bladder, followed by blood vessels, mast cells and lung. Through A(2B) receptors, adenosine also regulates the growth of smooth muscle cell populations in blood vessels, cell growth, intestinal function, inhibition of Tumor Necrosis Factor (TNF-alpha), vascular tone, and inflammatory processes such as diarrhea and asthma. Potent and selective adenosine agonists are the result of modifications of the parent ligand adenosine by substitution, namely at N(6) or C(2) position of the purine heterocycle or at the 5' position of the ribose moiety. 5'-N-ethylcarboxamidoadenosina (NECA) is one of the most potent A(2B) adenosine receptor agonist. Classical antagonists for A(2B) adenosine receptors are xanthine analogues obtained from multiple substitutions of the parent heterocycle by C(8) substitution combined with N(1) and N(3) (and sometimes N(7)) substitutions.

Synthesis and biological evaluation of 2-amino-3-(3',4',5'-trimethoxybenzoyl)-5-aryl thiophenes as a new class of potent antitubulin agents.

A new series of compounds in which the 2-amino-5-chlorophenyl ring of phenstatin analogue 7 was replaced with a 2-amino-5-aryl thiophene was synthesized and evaluated for antiproliferative activity and for inhibition of tubulin polymerization and colchicine binding to tubulin. 2-Amino-3-(3',4',5'-trimethoxybenzoyl)-5-phenyl thiophene (9f) as well as the p-fluoro-, p-methyl-, and p-methoxyphenyl substituted analogues (9i, j, and l, respectively) displayed high antiproliferative activities with IC50 values from 2.5 to 6.5 nM against the L1210 and K562 cell lines. Compounds 9i and j were more active than combretastatin A-4 as inhibitors of tubulin polymerization. Molecular docking simulations to the colchicine site of tubulin were performed to determine the possible binding mode of 9i. The results obtained demonstrated that antiproliferative activity correlated well with the inhibition of tubulin polymerization and the lengthening of the G2/M phase of the cell cycle. Moreover, a good correlation was found between these inhibitory effects and the induction of apoptosis in cells treated with the compounds.

Synthesis and biological evaluation of 2-(3',4',5'-trimethoxybenzoyl)-3-amino 5-aryl thiophenes as a new class of tubulin inhibitors.

2-(3',4',5'-Trimethoxybenzoyl)-3-amino-5-aryl/heteroaryl thiophene derivatives were synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. SARs were elucidated with various substitutions on the aryl moiety 5-position of the thienyl ring. Substituents at the para-position of the 5-phenyl group showed antiproliferative activity in the order of F=CH(3) > OCH(3)=Br=NO(2) > CF(3)=I > OEt. Several of these compounds led to arrest of HL-60 cells in the G2/M phase of the cell cycle and induction of apoptosis.

Dopamine and adenosine receptor interaction as basis for the treatment of Parkinson's disease.

Preclinical evidence strongly indicate that adenosine A(2A) receptor antagonists represent a promising class of drugs for the treatment of motor deficits associated to Parkinson's disease. The effects of adenosine A(2A) receptor antagonists were here assessed in a rat model of parkinsonian tremor induced by cholinomimetic drugs by evaluating the counteraction of tremulous jaw movements. Systemic administration of the A(2A) antagonist SCH 58261 dose-dependently reduced the magnitude of perioral tremor induced by the acetylcholinesterase inhibitor tacrine (2.5 mg/kg). Furthermore, intrastriatal infusion of SCH BT2 (5 microg/microl), a water-soluble analogue of SCH 58261, antagonized tacrine-induced jaw movements with a maximal effect in the ventrolateral striatum. On the other hand, SCH 58261 (5 mg/kg) was ineffective in blocking tremulous jaw movements stimulated by the direct muscarinic agonist pilocarpine (1 mg/kg). Taken together, these results indicate that A(2A) antagonists reduce parkinsonian tremor stimulated in rats by tacrine and that the striatum is deeply involved in the observed effect. Moreover, the ineffectiveness of SCH 58261 in blocking pilocarpine-stimulated perioral tremor suggests that the antitremorigenic effects of A(2A) antagonists described here are not related to a direct action on muscarinic receptor. The prospective of providing additional antitremor benefits considerably enhances the therapeutic potential of A(2A) antagonists.

Design and Synthesis of Potent in Vitro and in Vivo Anticancer Agents Based on 1-(3',4',5'-Trimethoxyphenyl)-2-Aryl-1H-Imidazole.

A novel series of tubulin polymerization inhibitors, based on the 1-(3',4',5'-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold and designed as cis-restricted combretastatin A-4 analogues, was synthesized with the goal of evaluating the effects of various patterns of substitution on the phenyl at the 2-position of the imidazole ring on biological activity. A chloro and ethoxy group at the meta- and para-positions, respectively, produced the most active compound in the series (4o), with IC50 values of 0.4-3.8 nM against a panel of seven cancer cell lines. Except in HL-60 cells, 4o had greater antiproliferative than CA-4, indicating that the 3'-chloro-4'-ethoxyphenyl moiety was a good surrogate for the CA-4 B-ring. Experiments carried out in a mouse syngenic model demonstrated high antitumor activity of 4o, which significantly reduced the tumor mass at a dose thirty times lower than that required for CA-4P, which was used as a reference compound. Altogether, our findings suggest that 4o is a promising anticancer drug candidate that warrants further preclinical evaluation.