Structural Investigations on 2-Amidobenzimidazole Derivatives as New Inhibitors of Protein Kinase CK1 Delta.

Protein kinase CK1δ (CK1δ) is a serine-threonine/kinase that modulates different physiological processes, including the cell cycle, DNA repair, and apoptosis. CK1δ overexpression, and the consequent hyperphosphorylation of specific proteins, can lead to sleep disorders, cancer, and neurodegenerative diseases. CK1δ inhibitors showed anticancer properties as well as neuroprotective effects in cellular and animal models of Parkinson's and Alzheimer's diseases and amyotrophic lateral sclerosis. To obtain new ATP-competitive CK1δ inhibitors, three sets of benzimidazole-2-amino derivatives were synthesized (1-32), bearing different substituents on the fused benzo ring (R) and diverse pyrazole-containing acyl moieties on the 2-amino group. The best-performing derivatives were those featuring the (1H-pyrazol-3-yl)-acetyl moiety on the benzimidazol-2-amino scaffold (13-32), which showed CK1δ inhibitor activity in the low micromolar range. Among the R substituents, 5-cyano was the most advantageous, leading to a compound endowed with nanomolar potency (23, IC50 = 98.6 nM). Molecular docking and dynamics studies were performed to point out the inhibitor-kinase interactions.

"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.

The possible role of the nucleoside adenosine in countering skin aging: A review.

Skin aging is a complex biological process. Skin aspect is considered as a sign of well-being and of beauty. In view of this, noninvasive and/or minimally invasive anti-aging strategies were developed. Adenosine, a well-known nucleoside, may play a role in skin rejuvenation. Adenosine receptors belong to the G protein-coupled receptors superfamily and are divided into four subtypes: A1 , A2A , A2B , and A3 . The adenosine receptors expressed by skin are mainly the A1 and A2A subtypes. In the hypodermis, adenosine through the A1 receptor stimulates lipogenesis and adipogenesis. In the dermis, adenosine through the A2A receptor subtype stimulates collagen production. Moreover, the nucleoside increases new DNA synthesis and subsequently protein synthesis in dermal cells. Activation of adenosine receptors by interacting with various skin layers may induce a decrease in the amount of wrinkles, roughness, dryness, and laxity. This article has reviewed the mechanisms through which adenosine modulates biological mechanisms in the skin tissues and the effect of preparations containing adenosine or its derivatives on the skin.

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.

Casein Kinase 1δ Inhibitors as Promising Therapeutic Agents for Neurodegenerative Disorders.

Casein kinase 1 (CK1) belongs to the serine-threonine kinase family and is expressed in all eukaryotic organisms. At least six human isoforms of CK1 (termed α, γ1-3, δ and ε) have been cloned and characterized. CK1δ isoform modulates several physiological processes, including DNA damage repair, circadian rhythm, cellular proliferation and apoptosis. Therefore, CK1δ dysfunction may trigger diverse pathologies, such as cancer, inflammation and central nervous system disorders. Overexpression and aberrant activity of CK1δ have been connected to hyperphosphorylation of key proteins implicated in the development of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases and Amyotrophic Lateral Sclerosis. Thus, CK1δ inhibitors have attracted attention as potential drugs for these pathologies and several compounds have been synthesized or isolated from natural sources to be evaluated for their CK1δ inhibitory activity. Here we report a comprehensive review on the development of CK1δ inhibitors, with a particular emphasis on structure-activity relationships and computational studies, which provide useful insight for the design of novel inhibitors.

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.

A2A Adenosine Receptor Antagonists and their Potential in Neurological Disorders.

Endogenous nucleoside adenosine modulates a number of physiological effects through interaction with P1 purinergic receptors. All of them are G protein-coupled receptors, and, to date, four subtypes have been characterized and named A1, A2A, A2B, and A3. In recent years, adenosine receptors, particularly the A2A subtype, have become attractive targets for the treatment of several neurodegenerative disorders, known to involve neuroinflammation, like Parkinson's and Alzheimer's diseases, multiple sclerosis, and neuropsychiatric conditions. In fact, it has been demonstrated that inhibition of A2A adenosine receptors exerts neuroprotective effects counteracting neuroinflammatory processes and astroglial and microglial activation. The A2A adenosine receptor antagonist istradefylline, developed by Kyowa Hakko Kirin Inc., was approved in Japan as adjunctive therapy for the treatment of Parkinson's disease, and very recently, it was also approved by the US Food and Drug Administration. These findings pave the way for new therapeutic opportunities, so, in this review, a summary of the most relevant and promising A2A adenosine receptor antagonists will be presented along with their preclinical and clinical studies in neuroinflammation related diseases.

Multipotent Stromal Cells from Subcutaneous Adipose Tissue of Normal Weight and Obese Subjects: Modulation of Their Adipogenic Differentiation by Adenosine A1 Receptor Ligands.

Adenosine A1 receptor (A1R) activation, stimulating lipogenesis and decreasing insulin resistance, could be useful for metabolic syndrome management in obese subjects. Since full A1R agonists induce harmful side-effects, while partial agonists show a better pharmacological profile, we investigated the influence of two derivatives of the full A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA), C1 and C2 behaving as A1R partial agonists in animal models, on the adipogenic differentiation of stromal/stem cells (ASCs) from human subcutaneous adipose tissue, which mainly contribute to increase fat mass in obesity. The ASCs from normal-weight subjects showed increased proliferation and A1R expression but reduced adipogenic differentiation compared to obese individual-derived ASCs. Cell exposure to CCPA, C1, C2 or DPCPX, an A1R antagonist, did not affect ASC proliferation, while mainly C2 and DPCPX significantly decreased adipogenic differentiation of both ASC types, reducing the activity of glycerol-3-phosphate dehydrogenase and the expression of PPARγ and FABP-4, all adipogenic markers, and phosphorylation of Akt in the phosphatidylinositol-3-kinase pathway, which plays a key-role in adipogenesis. While requiring confirmation in in vivo models, our results suggest that A1R partial agonists or antagonists, by limiting ASC differentiation into adipocytes and, thereby, fat mass expansion, could favor development/worsening of metabolic syndrome in obese subjects without a dietary control.

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.

Combined Therapy of A1AR Agonists and A2AAR Antagonists in Neuroinflammation.

Alzheimer's, Parkinson's, and multiple sclerosis are neurodegenerative diseases related by neuronal degeneration and death in specific areas of the central nervous system. These pathologies are associated with neuroinflammation, which is involved in disease progression, and halting this process represents a potential therapeutic strategy. Evidence suggests that microglia function is regulated by A1 and A2A adenosine receptors (AR), which are considered as neuroprotective and neurodegenerative receptors, respectively. The manuscript's aim is to elucidate the role of these receptors in neuroinflammation modulation through potent and selective A1AR agonists (N6-cyclopentyl-2'- or 3'-deoxyadenosine substituted or unsubstituted in 2 position) and A2AAR antagonists (9-ethyl-adenine substituted in 8 and/or in 2 position), synthesized in house, using N13 microglial cells. In addition, the combined therapy of A1AR agonists and A2AAR antagonists to modulate neuroinflammation was evaluated. Results showed that A1AR agonists were able, to varying degrees, to prevent the inflammatory effect induced by cytokine cocktail (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and interferon (IFN)-γ), while A2AAR antagonists showed a good ability to counteract neuroinflammation. Moreover, the effect achieved by combining the two most effective compounds (1 and 6) in doses previously found to be non-effective was greater than the treatment effect of each of the two compounds used separately at maximal dose.

Efficacy of acetylcholinesterase inhibitors in Alzheimer's disease.

Alzheimer's disease (AD), the most common cause of adult-onset dementia is characterized by a progressive decline of cognitive functions accompanied by behavioral manifestations. The main class of drugs currently used for the treatment of AD are acetylcholinesterase/cholinesterase inhibitors (ChE-Is). The first ChE-I licensed for symptomatic treatment of AD was tacrine. The ChE-Is currently available in the market are donepezil, rivastigmine and galantamine as tacrine is no longer in use, due to its hepatotoxicity. According to mechanism of action the ChE-Is are classified as short-acting or reversible agents such as tacrine, donepezil, and galantamine, as intermediate-acting or pseudo-irreversible agent such as rivastigmine. Overall, the efficacy of the three ChE-Is available in the market is similar and the benefit of administration of these compounds is mild and may not be clinically significant. Due to gastrointestinal side effects of these drugs, medicinal chemistry and pharmaceutical delivery studies have investigated solutions to improve the pharmacological activity of these compounds. In spite of the limited activity of ChE-Is, waiting for more effective approaches, these drugs still represent a pharmacotherapeutic resource for the treatment of AD. Other approaches in which ChE-Is were investigated is in their use in combination with other classes of drugs such as cholinergic precursors, N-methyl-d-aspartate (NMDA) receptor antagonists and antioxidant agents. After many years from the introduction in therapy of ChE-Is, the combination with other classes of drugs may represent the chance for a renewed interest of ChE-Is in the treatment of adult-onset dementia disorders.

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.

Discovery of first-in-class multi-target adenosine A2A receptor antagonists-carbonic anhydrase IX and XII inhibitors. 8-Amino-6-aryl-2-phenyl-1,2,4-triazolo [4,3-a]pyrazin-3-one derivatives as new potential antitumor agents.

This paper describes identification of the first-in-class multi-target adenosine A2A receptor antagonists-carbonic anhydrase (CA) IX and XII inhibitors, as new potential antitumor agents. To obtain the multi-acting ligands, the 8-amino-2,6-diphenyltriazolo[4,3-a]pyrazin-3-one, a potent adenosine hA2A receptor (AR) antagonist, was taken as lead compound. To address activity against the tumor-associated CA isoforms, it was modified by introduction of different substituents (OH, COOH, CONHOH, SO2NH2) on the 6-phenyl ring or on a phenyl pendant connected to the former through different spacers. Among the new triazolopyrazines 1-23, the most active were those featuring the sulfonamide residue. Derivative 20, featuring a 4-sulfonamidophenyl residue attached through a CONH(CH2)2CONH spacer at the para-position of the 6-phenyl ring, showed the best combination of activity at the three targets. In fact, it inhibited both the tumor-associated hCA IX and XII isozymes at nanomolar concentration (Ki = 5.0 and 27.0 nM), and also displayed a quite good affinity for the hA2A AR (Ki = 108 nM). Compound 14, bearing the 4-sulfonamidophenyl residue linked at the para-position of the 6-phenyl ring by a CONH spacer, was remarkable because both its hA2A AR affinity and hCA XII inhibitory potency were in the low nanomolar range (Ki = 6.4 and 6.2 nM, respectively). Molecular docking studies highlighted the interaction mode of selected triazolopyrazines in the hA2A AR recognition pocket and in the active site of hCA II, IX and XII isoforms.

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.