The low affinity A2B adenosine receptor, like any other adenosine receptor subtype, belongs to the super-family of seven transmembrane domain G protein-coupled receptors (7TMs GPCR) and is classified by the GPCR database in the family of rhodopsin like receptors (Class A of GPCR). It has been cloned from various species, including rat and human, and its sequences are highly similar across species, ranging from 85% identity between human and mouse and 95% identity between rat and mouse. The A2B receptors show a ubiquitous distribution, the highest levels are present in cecum, colon and bladder, followed by blood vessels, lung, eye and mast cells. Through A2B receptors adenosine seems to cause mast cells degranulation, vasodilation, cardiac fibroblast proliferation, inhibition of Tumor Necrosis Factor (TNF-α), increased synthesis of interleukin-6 (IL-6), stimulation of Cl- secretion in intestinal epithelia and hepatic glucose production. Hence, A2B adenosine receptor agonists could be useful in the treatment of cardiac diseases like hypertension or myocardial infarction and in the management of septic shock, while antagonists may serve as novel drugs for asthma, Alzheimers disease, cystic fibrosis and type-II diabetes. No potent and selective A2B agonists have been reported so far, 5-N-ethylcarboxamidoadenosine (NECA) is one of the most active. The monosubstitution on N6-position of adenosine is well tolerated and that position appears to be a useful site for increasing A2B potency. Among substituents in 2-position of adenosine only 1- alkynyl chains are effective for A2B potency. In particular, the (S)-2-hydroxypropynyl substituents brought about the highest activity demonstrating that the A2B receptors discriminate between (R) and (S) diastereomers. Hence, (S)-2-phenylhydroxypropynylNECA (PHPNECA), with an EC50 = 0.22 μM, proved to be the most potent A2B agonist reported so far. Classical antagonists for adenosine receptors are alkylxanthines which show considerable potency at A2B receptors. Para substituted 1,3-dialkyl-8-phenylxanthines possess high affinity in binding assays, the 3- unsubstituted 1-alkyl analogues resulted more A2 B selective with the 8-[4-[(N-(2- hydroxyethyl)carboxamidomethyl)oxy]phenyl]-1-propylxanthine (60) showing the highest affinity (Ki = 1.2 nM) and selectivity (A1 / A2B = 60, A2A / A2B = 1,790, A3 / A2B = 360) . Among non-xanthine derivatives very promising are substituted purines, in which combination of appropriate substituents in 2-, 8-, and 9-position could lead to very potent and selective A2B antagonists.

The pathological condition of neuroinflammation is caused by the activation of the neuroimmune cells astrocytes and microglia. The autacoid adenosine seems to be an important neuromodulator in this condition. Its main receptors involved in the neuroinflammation modulation are A₁AR and A_2AAR. Evidence suggests that A₁AR activation produces a neuroprotective effect and A_2AARs block prevents neuroinflammation. The aim of this work is to elucidate the effects of these receptors in neuroinflammation using the partial agonist 2′-dCCPA (2-chloro-N⁶-cyclopentyl-2′-deoxyadenosine) (C1 K_iA₁AR = 550 nM, K_iA_2AAR = 24,800 nM, and K_iA₃AR = 5560 nM, α = 0.70, EC₅₀A₁AR = 832 nM) and the newly synthesized in house compound 8-chloro-9-ethyl-2-phenethoxyadenine (C2 K_iA_2AAR = 0.75 nM; K_iA₁AR = 17 nM and K_iA₃AR = 227 nM, IC₅₀A_2AAR = 251 nM unpublished results). The experiments were performed in in vitro and in in vivo models of neuroinflammation. Results showed that C1 was able to prevent the inflammatory effect induced by cytokine cocktail (TNF-α, IL-1β, and IFN-γ) while C2 possess both anti-inflammatory and antioxidant properties, counteracting both neuroinflammation in mixed glial cells and in an animal model of neuroinflammation. In conclusion, C2 is a potential candidate for neuroinflammation therapy.

In this work, we have highlighted data reported in the literature trying to draw a complete picture of the structures and biological activity of agonists and orthosteric antagonists of P2X receptors. Actually, only few P2X receptor agonists have been found and most of them are derived from modification of the natural ligand ATP and they are P2X receptor subtype unselective. In particular, BzATP (9) is one of the most potent P2X receptor agonists with EC₅₀ value in the nanomolar range at some subtypes. Differently from agonists, P2X receptor antagonists belong to different chemical classes such as high molecular weight aryl polysulfonate molecules like suramin and its simplified derivatives and anthraquinone compounds. All these molecules proved to be non selective at P2X receptors, and they are endowed with micromolar activity and not favourable pharmacokinetic properties due to the presence of several charged groups. Also modification of the natural ligand ATP led to the discovery of P2X receptor antagonists like TNP-ATP (29), which, although not selective, showed high potency at P2X1, P2X3 (IC₅₀ of 0.006 µM and 0.001 µM, respectively), and heteromeric P2X2/3 receptors. Also the dinucleotide inosine polyphosphate Ip₅I (33) was found to be a potent and selective antagonist at P2X1 vs P2X3 receptors with IC₅₀ = 0.003 µM. A significant improvement has been gained from the interest of pharmaceutical companies that in the last years discovered, through the use of high-throughput screening, potent and selective antagonists endowed with novel structures, some of which are currently in clinical trials for several therapeutic applications.

Introduction: Purinergic P2X3-P2X2/3 receptors are placed in nociceptive neurons’ strategic location and show unique desensitization properties, hence they represent an attractive target for many pain related diseases. Therefore a broad interest from academic and pharmaceutical scientists has focused on the search for P2X3 and P2X2/3 receptor ligands and has led to the discovery of numerous new selective antagonists. Some of them have been studied in clinical trials for the treatment of pathological conditions such as bladder disorders, gastrointestinal and chronic obstructive pulmonary diseases. Areas covered: This review provides a summary of the patents concerning the discovery of P2X3 and/or P2X2/3 receptor antagonists published between 2015 and 2019 and their potential clinical use. Thus, the structures and biological data of the most representative molecules are reported. Expert opinion: The 2016 publication of the crystallographic structure of the human P2X3 receptor subtype gave an improvement of published patents in 2017. Hence, a great number of small molecules with dual antagonist activity on P2X3-P2X2/3 receptors, a favorable pharmacokinetic profile, and reasonable oral bioavailability was discovered. The most promising compounds are the phenoxy-diaminopyrimidines including gefapixant (AF-219), and the imidazo-pyridines like BLU-5937, which are in phase III and phase II clinical trials, respectively, for refractory chronic cough.

P2X3 Receptor Ligands: Structural Features and Potential Therapeutic Applications