"Dual Anta-Inhibitors" of the A2A Adenosine Receptor and Casein Kinase CK1delta: Synthesis, Biological Evaluation, and Molecular Modeling Studies.

Based on a screening of a chemical library of A2A adenosine receptor (AR) antagonists, a series of di- and tri-substituted adenine derivatives were synthesized and tested for their ability to inhibit the activity of the enzyme casein kinase 1 delta (CK1δ) and to bind adenosine receptors (ARs). Some derivatives, here called "dual anta-inhibitors", demonstrated good CK1δ inhibitory activity combined with a high binding affinity, especially for the A2AAR. The N6-methyl-(2-benzimidazolyl)-2-dimethyamino-9-cyclopentyladenine (17, IC50 = 0.59 µM and KiA2A = 0.076 µM) showed the best balance of A2AAR affinity and CK1δ inhibitory activity. Computational studies were performed to simulate, at the molecular level, the protein-ligand interactions involving the compounds of our series. Hence, the dual anta-inhibitor 17 could be considered the lead compound of new therapeutic agents endowed with synergistic effects for the treatment of chronic neurodegenerative and cancer diseases.

Dual Anta-Inhibitors Targeting Protein Kinase CK1δ and A2A Adenosine Receptor Useful in Neurodegenerative Disorders.

Currently, the number of patients with neurodegenerative pathologies is estimated at over one million, with consequences also on the economic level. Several factors contribute to their development, including overexpression of A2A adenosine receptors (A2AAR) in microglial cells and up-regulation and post-translational alterations of some casein kinases (CK), among them, CK-1δ. The aim of the work was to study the activity of A2AAR and CK1δ in neurodegeneration using in-house synthesized A2A/CK1δ dual anta-inhibitors and to evaluate their intestinal absorption. Experiments were performed on N13 microglial cells, which were treated with a proinflammatory CK cocktail to simulate an inflammatory state typical of neurodegenerative diseases. Results showed that the dual anta-inhibitors have the ability to counteract the inflammatory state, even if compound 2 is more active than compound 1. In addition, compound 2 displayed an important antioxidant effect similar to the reference compound ZM241385. Since many known kinase inhibitors are very often unable to cross lipid bilayer membranes, the ability of A2A/CK1δ double anta-inhibitors to cross the intestinal barrier was investigated by an everted gut sac assay. HPLC analysis revealed that both compounds are able to cross the intestinal barrier, making them promising candidates for oral therapy.

Machine Learning Scoring Functions for Drug Discovery from Experimental and Computer-Generated Protein-Ligand Structures: Towards Per-Target Scoring Functions.

In recent years, machine learning has been proposed as a promising strategy to build accurate scoring functions for computational docking finalized to numerically empowered drug discovery. However, the latest studies have suggested that over-optimistic results had been reported due to the correlations present in the experimental databases used for training and testing. Here, we investigate the performance of an artificial neural network in binding affinity predictions, comparing results obtained using both experimental protein-ligand structures as well as larger sets of computer-generated structures created using commercial software. Interestingly, similar performances are obtained on both databases. We find a noticeable performance suppression when moving from random horizontal tests to vertical tests performed on target proteins not included in the training data. The possibility to train the network on relatively easily created computer-generated databases leads us to explore per-target scoring functions, trained and tested ad-hoc on complexes including only one target protein. Encouraging results are obtained, depending on the type of protein being addressed.

4-Heteroaryl Substituted Amino-3,5-Dicyanopyridines as New Adenosine Receptor Ligands: Novel Insights on Structure-Activity Relationships and Perspectives.

A new set of amino-3,5-dicyanopyridines was synthesized and biologically evaluated at the adenosine receptors (ARs). This chemical class is particularly versatile, as small structural modifications can influence not only affinity and selectivity, but also the pharmacological profile. Thus, in order to deepen the structure-activity relationships (SARs) of this series, different substituents were evaluated at the diverse positions on the dicyanopyridine scaffold. In general, the herein reported compounds show nanomolar binding affinity and interact better with both the human (h) A1 and A2A ARs than with the other subtypes. Docking studies at hAR structure were performed to rationalize the observed affinity data. Of interest are compounds 1 and 5, which can be considered as pan ligands as binding all the ARs with comparable nanomolar binding affinity (A1AR: 1, Ki = 9.63 nM; 5, Ki = 2.50 nM; A2AAR: 1, Ki = 21 nM; 5, Ki = 24 nM; A3AR: 1, Ki = 52 nM; 5, Ki = 25 nM; A2BAR: 1, EC50 = 1.4 nM; 5, EC50 = 1.12 nM). Moreover, these compounds showed a partial agonist profile at all the ARs. This combined AR partial agonist activity could lead us to hypothesize a potential effect in the repair process of damaged tissue that would be beneficial in both wound healing and remodeling.

A2A Adenosine Receptor Antagonists: Are Triazolotriazine and Purine Scaffolds Interchangeable?

The A2A adenosine receptor (A2AAR) is one of the four subtypes activated by nucleoside adenosine, and the molecules able to selectively counteract its action are attractive tools for neurodegenerative disorders. In order to find novel A2AAR ligands, two series of compounds based on purine and triazolotriazine scaffolds were synthesized and tested at ARs. Compound 13 was also tested in an in vitro model of neuroinflammation. Some compounds were found to possess high affinity for A2AAR, and it was observed that compound 13 exerted anti-inflammatory properties in microglial cells. Molecular modeling studies results were in good agreement with the binding affinity data and underlined that triazolotriazine and purine scaffolds are interchangeable only when 5- and 2-positions of the triazolotriazine moiety (corresponding to the purine 2- and 8-positions) are substituted.

A patent review of adenosine A2B receptor antagonists (2016-present).

INTRODUCTION: A2B adenosine receptor (A2BAR) plays a crucial role in pathophysiologic conditions associated with high adenosine release, typical of airway inflammatory pathologies, gastrointestinal disorders, cancer, asthma, type 2 diabetes, and atherosclerosis. In some pathologies, simultaneous inactivation of A2A and A2BARs is desirable to have a synergism of action that leads to a greater efficacy of the pharmacological treatment and less side effects due to the dose of drug administered. In this context, it is strongly required to identify molecules capable of selectively antagonizing A2BAR or A2A/A2BARs. AREAS COVERED: The review provides a summary of patents, published from 2016 to present, on chemicals and their clinical use. In this paper, information on the biological activity of representative structures of recently developed A2B or A2A/A2B receptor ligands is reported. EXPERT OPINION: Among the four P1 receptors, A2BAR is the most inscrutable and the least studied until a few years ago, but its involvement in various inflammatory pathologies has recently made it a pharmacological target of high interest. Many efforts by the academy and pharmaceutical companies have been made to discover potential A2BAR and A2A/A2BARs drugs. Although several compounds have been synthesized only a few molecules have entered clinical trials.

A3 Adenosine Receptor Antagonists with Nucleoside Structures and Their Anticancer Activity.

The overexpression of the A3 adenosine receptor (AR) in a number of cancer cell types makes it an attractive target for tumor diagnosis and therapy. Hence, in the search for new A3AR ligands, a series of novel 2,N6-disubstituted adenosines (Ados) was synthesized and tested in radioligand binding and functional assays at ARs. Derivatives bearing a 2-phenethylamino group in the N6-position were found to exert higher A3AR affinity and selectivity than the corresponding N6-(2,2-diphenylethyl) analogues. 2-Chloro-N6-phenylethylAdo (15) was found to be a potent full A3AR agonist with a Ki of 0.024 nM and an EC50 of 14 nM, in a cAMP accumulation assay. Unlike 15, the other ligands behaved as A3AR antagonists, which concentration-dependently reduced cell growth and exerted cytostatic activity on the prostate cancer cell line PC3, showing comparable and even more pronounced effects with respect to the ones elicited by the reference full agonist Cl-IB-MECA. In particular, the N6-(2,2-diphenylethyl)-2-phenylethynylAdo (12: GI50 = 14 µM, TGI = 29 µM, and LC50 = 59 µM) showed the highest activity proving to be a potential antitumor agent. The cytostatic effect of both A3AR agonist (Cl-IB-MECA) and antagonists (12 and other newly synthesized compounds) confirm previous observations according to which, in addition to the involvement of A3ARs, other cellular mechanisms are responsible for the anticancer effects of these ligands.

Design and Synthesis of Novel Thiazolo[5,4-d]pyrimidine Derivatives with High Affinity for Both the Adenosine A1 and A2A Receptors, and Efficacy in Animal Models of Depression.

New compounds with a 7-amino-2-arylmethyl-thiazolo[5,4-d]pyrimidine structure were synthesized and evaluated in vitro for their affinity and/or potency at the human (h) A1, hA2A, hA2B, and hA3 adenosine receptors (ARs). Several compounds (5, 8-10, 13, 18, 19) were characterized by nanomolar and subnanomolar binding affinities for the hA1 and the hA2A AR, respectively. Results of molecular docking studies supported the in vitro results. The 2-(2-fluorobenzyl)-5-(furan-2yl)-thiazolo[5,4-d]pyrimidin-7-amine derivative 18 (hA1 Ki = 1.9 nM; hA2A Ki = 0.06 nM) was evaluated for its antidepressant-like activity in in vivo studies, the forced swimming test (FST), the tail suspension test (TST), and the sucrose preference test (SPT) in mice, showing an effect comparable to that of the reference amitriptyline.

Adenosine Receptors as Neuroinflammation Modulators: Role of A1 Agonists and A2A 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 A2AAR. Evidence suggests that A1AR activation produces a neuroprotective effect and A2AARs 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 KiA1AR = 550 nM, KiA2AAR = 24,800 nM, and KiA3AR = 5560 nM, α = 0.70, EC50A1AR = 832 nM) and the newly synthesized in house compound 8-chloro-9-ethyl-2-phenethoxyadenine (C2 KiA2AAR = 0.75 nM; KiA1AR = 17 nM and KiA3AR = 227 nM, IC50A2AAR = 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-1ß, 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.

The Anti-Inflammatory and Pain-Relieving Effects of AR170, an Adenosine A3 Receptor Agonist, in a Rat Model of Colitis.

The pharmacological activation of A3 receptors has shown potential usefulness in the management of bowel inflammation. However, the role of these receptors in the control of visceral hypersensitivity in the presence of intestinal inflammation has not been investigated. The effects of AR170, a potent and selective A3 receptor agonist, and dexamethasone (DEX) were tested in rats with 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis to assess their tissue inflammatory parameters. The animals received AR170, DEX, or a vehicle intraperitoneally for 6 days, starting 1 day before the induction of colitis. Visceral pain was assessed by recording the abdominal responses to colorectal distension in animals with colitis. Colitis was associated with a decrease in body weight and an increase in spleen weight. The macroscopic damage score and tissue tumor necrosis factor (TNF), interleukin 1ß (IL-1ß), and myeloperoxidase (MPO) levels were also enhanced. AR170, but not DEX, improved body weight. Both drugs counteracted the increase in spleen weight, ameliorated macroscopic colonic damage, and decreased TNF, IL-1ß, and MPO tissue levels. The enhanced visceromotor response (VMR) in rats with colitis was decreased via AR170 administration. In rats with colitis, AR170 counteracted colonic inflammatory cell infiltration and decreased pro-inflammatory cytokine levels, thereby relieving visceral hypersensitivity.

Approaches for designing and discovering purinergic drugs for gastrointestinal diseases.

INTRODUCTION: Purines finely modulate physiological motor, secretory, and sensory functions in the gastrointestinal tract. Their activity is mediated by the purinergic signaling machinery, including receptors and enzymes regulating their synthesis, release, and degradation. Several gastrointestinal dysfunctions are characterized by alterations affecting the purinergic system. AREAS COVERED: The authors provide an overview on the purinergic receptor signaling machinery, the molecules and proteins involved, and a summary of medicinal chemistry efforts aimed at developing novel compounds able to modulate the activity of each player involved in this machinery. The involvement of purinergic signaling in gastrointestinal motor, secretory, and sensory functions and dysfunctions, and the potential therapeutic applications of purinergic signaling modulators, are then described. EXPERT OPINION: A number of preclinical and clinical studies demonstrate that the pharmacological manipulation of purinergic signaling represents a viable way to counteract several gastrointestinal diseases. At present, the paucity of purinergic therapies is related to the lack of receptor-subtype-specific agonists and antagonists that are effective in vivo. In this regard, the development of novel therapeutic strategies should be focused to include tools able to control the P1 and P2 receptor expression as well as modulators of the breakdown or transport of purines.

New 8-amino-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives. Evaluation of different moieties on the 6-aryl ring to obtain potent and selective human A2A adenosine receptor antagonists.

In this work, further structural investigations on the 8-amino-2-phenyl-6-aryl-1,2,4-triazolo[4,3-a]pyrazin-3-one series were carried out to achieve potent and selective human A2A adenosine receptor (AR) antagonists. Different ether and amide moieties were attached at the para-position of the 6-phenyl ring, thus leading to compounds 1-9 and 10-18, respectively. Most of these moieties contained terminal basic rings (pyrrolidine, morpholine, piperidine and substituted piperazines) which were thought to confer good physicochemical and drug-like properties. Compounds 11-16, bearing the amide linker, possessed high affinity and selectivity for the hA2A AR (Ki = 3.6-11.8 nM). Also derivatives 1-9, featuring an ether linker, preferentially targeted the hA2A AR but with lower affinity, compared to those of the relative amide compounds. Docking studies, carried out at the hA2A AR binding site, highlighted some crucial ligand-receptor interactions, particularly those provided by the appended substituent whose nature deeply affected hA2A AR affinity.

Update on novel purinergic P2X3 and P2X2/3 receptor antagonists and their potential 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.

Non-Nucleoside Agonists of the Adenosine Receptors: An Overview.

Potent and selective adenosine receptor (AR) agonists are of pharmacological interest for the treatment of a wide range of diseases and conditions. Among these derivatives, nucleoside-based agonists represent the great majority of molecules developed and reported to date. However, the limited availability of compounds selective for a specific AR subtype (i.e., A2BAR) and a generally long and complex synthetic route for largely substituted nucleosides are the main drawbacks of this category of molecules. Non-nucleoside agonists represent an alternative set of compounds able to stimulate the AR function and based on simplified structures. This review provides an updated overview on the structural classes of non-nucleoside AR agonists and their biological activities, with emphasis on the main derivatives reported in the literature. A focus is also given to the synthetic routes employed to develop these derivatives and on molecular modeling studies simulating their interaction with ARs.

Amino-3,5-Dicyanopyridines Targeting the Adenosine Receptors Ranging from Pan Ligands to Combined A1/A2B Partial Agonists.

The amino-3,5-dicyanopyridine derivatives belong to an intriguing series of adenosine receptor (AR) ligands that has been developed by both academic researchers and industry. Indeed, the studies carried out to date underline the versatility of the dicyanopyridine scaffold to obtain AR ligands with not only a wide range of affinities but also with diverse degrees of efficacies at the different ARs. These observations prompted us to investigate on the structure-activity relationships (SARs) of this series leading to important previously reported results. The present SAR study has helped to confirm the 1H-imidazol-2-yl group at R2 position as an important feature for producing potent AR agonists. Moreover, the nature of the R1 substituent highly affects not only affinity/activity at the hA1 and hA2B ARs but also selectivity versus the other subtypes. Potent hA1 and hA2B AR ligands were developed, and among them, the 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(prop-2-en-1-yloxy)phenyl]pyridine-3,5-dicarbonitrile (3) is active in the low nanomolar range at these subtypes and shows a good trend of selectivity versus both the hA2A and hA3 ARs. This combined hA1/hA2B partial agonist activity leads to a synergistic effect on glucose homeostasis and could potentially be beneficial in treating diabetes and related complications.

Antioxidant-Conjugated 1,2,4-Triazolo[4,3-a]pyrazin-3-one Derivatives: Highly Potent and Selective Human A2A Adenosine Receptor Antagonists Possessing Protective Efficacy in Neuropathic Pain.

New 8-amino-6-aryl-1,2,4-triazolo[4,3-a]pyrazin-3-ones were designed to obtain dual antioxidant-human A2A adenosine receptor (hA2A AR) antagonists. Two sets of compounds were synthesized, the first featuring phenol rings at the 6-position, the second bearing the lipoyl and 4-hydroxy-3,5-di-tertbut-benzoyl residues appended by different linkers on the 6-phenyl ring. Several new triazolopyrazines (1-21) were potent and selective hA2A AR antagonists (Ki = 0.17-54.5 nM). Compounds 11, 15, and 21, featuring antioxidant moieties, and compound 12, lacking the antioxidant functionality, reduced oxaliplatin-induced toxicity in microglia cells, the most active being the lipoyl-derivative 15 and the (4-hydroxy-3,5-di-tert-butyl)benzoyl-analogue 21 which were effective in reducing the oxygen free radical level. The lipoyl-derivative 15 was also able to revert oxaliplatin-induced neuropathy in the mouse. In vivo efficacy of 15 makes it a promising neuroprotective agent in oxidative stress-related diseases.

New A2A adenosine receptor antagonists: a structure-based upside-down interaction in the receptor cavity.

Adenosine receptor antagonists are generally based on heterocyclic core structures presenting substituents of various volumes and chemical-physical profiles. Adenine and purine-based adenosine receptor antagonists have been reported in literature. In this work we combined various substituents in the 2, 6, and 8-positions of 9-ethylpurine to depict a structure-affinity relationship analysis at the human adenosine receptors. Compounds were rationally designed trough molecular modeling analysis and then synthesized and evaluated at radioligand binding studies at human adenosine receptors. The new compounds showed affinity for the human adenosine receptors, with some derivatives endowed with low nanomolar Ki data, in particular at the A2AAR subtype. The purine core proves to be a versatile core structure for the development of novel adenosine receptor antagonists with nanomolar affinity for these membrane proteins.

Modifications on the Amino-3,5-dicyanopyridine Core To Obtain Multifaceted Adenosine Receptor Ligands with Antineuropathic Activity.

A new series of amino-3,5-dicyanopyridines (1-31) was synthesized and biologically evaluated in order to further investigate the potential of this scaffold to obtain adenosine receptor (AR) ligands. In general, the modifications performed have led to compounds having high to good human (h) A1AR affinity and an inverse agonist profile. While most of the compounds are hA1AR-selective, some derivatives behave as mixed hA1AR inverse agonists/A2A and A2B AR antagonists. The latter compounds (9-12) showed that they reduce oxaliplatin-induced neuropathic pain by a mechanism involving the alpha7 subtype of nAchRs, similar to the nonselective AR antagonist caffeine, taken as the reference compound. Along with the pharmacological evaluation, chemical stability of methyl 3-(((6-amino-3,5-dicyano-4-(furan-2-yl)pyridin-2-yl)sulfanyl)methyl)benzoate 10 was assessed in plasma matrices (rat and human), and molecular modeling studies were carried out to better rationalize the available structure-activity relationships.

New sensible method to quantize the intestinal absorption of receptor ligands.

In recent years, special attention has been paid to the A3 adenosine receptor (A3AR) as a possible pharmacological target to treat intestinal inflammation. In this work, it was set up a novel method to quantify the concentration of a promising anti-inflammatory agent inside and outside of intestinal barrier using the everted gut sac technique. The compound chosen for the present study is one of the most potent and selective A3AR agonist reported so far, named AR 170 (N6-methyl-2-phenylethynyl-5'-N-methylcarboxamidoadenosine). In order to evaluate the intestinal absorption of AR 170 the radioligand binding assay in comparison with HPLC-DAD was used. Results showed that the compound is absorbed via passive diffusion by paracellular pathway. The concentrations determined in the serosal (inside the sac) fluid by radioligand binding assay are in good agreement with those obtained through the widely used HPLC/MS protocol, demonstrating the reliability of the method. It is worthwhile to note that the radioligand binding assay allows detecting very low concentrations of analyte, thus offering an excellent tool to measure the intestinal absorption of receptor ligands. Moreover, the AR 170 quantity outside the gut sac and the interaction with A3AR could presuppose good topical anti-inflammatory effects of this compound.