[1,2,4]Triazolo[1,5-c]pyrimidines as Tools to Investigate A3 Adenosine Receptors in Cancer Cell Lines.

The A3 adenosine receptor is an interesting target whose role in cancer is controversial. In this work, a structural investigation at the 2-position of the [1,2,4]triazolo[1,5-c]pyrimidine nucleus was performed, finding new potent and selective A3 adenosine receptor antagonists such as the ethyl 2-(4-methoxyphenyl)-5-(methylamino)-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxylate (20, DZ123) that showed a Ki value of 0.47 nM and an exceptional selectivity profile over the other adenosine receptor subtypes. Computational studies were performed to rationalize the affinity and the selectivity profile of the tested compounds at the A3 adenosine receptor and the A1 and A2A adenosine receptors. Compound 20 was tested on both A3 adenosine receptor positive cell lines (CHO-A3 AR transfected, THP1 and HCT16) and on A3 negative cancer cell lines, showing no effect in the latter and a pro-proliferative effect at a low concentration in the former. These interesting results pave the way to further investigation on both the mechanism involved and potential therapeutic applications.

Structure-based virtual screening discovers potent and selective adenosine A1 receptor antagonists.

Development of subtype-selective leads is essential in drug discovery campaigns targeting G protein-coupled receptors (GPCRs). Herein, a structure-based virtual screening approach to rationally design subtype-selective ligands was applied to the A1 and A2A adenosine receptors (A1R and A2AR). Crystal structures of these closely related subtypes revealed a non-conserved subpocket in the binding sites that could be exploited to identify A1R selective ligands. A library of 4.6 million compounds was screened computationally against both receptors using molecular docking and 20 A1R selective ligands were predicted. Of these, seven antagonized the A1R with micromolar activities and several compounds displayed slight selectivity for this subtype. Twenty-seven analogs of two discovered scaffolds were designed, resulting in antagonists with nanomolar potency and up to 76-fold A1R-selectivity. Our results show the potential of structure-based virtual screening to guide discovery and optimization of subtype-selective ligands, which could facilitate the development of safer drugs.

Dual A1/A3 Adenosine Receptor Antagonists: Binding Kinetics and Structure-Activity Relationship Studies Using Mutagenesis and Alchemical Binding Free Energy Calculations.

Drugs targeting adenosine receptors (AR) can provide treatment for diseases. We report the identification of 7-(phenylamino)-pyrazolo[3,4-c]pyridines L2-L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo[3,4-c]pyridine (A17) displaying the highest affinity at both receptors with a long residence time of binding, as determined using a NanoBRET-based assay. Two binding orientations of A17 produce stable complexes inside the orthosteric binding area of A1R in molecular dynamics (MD) simulations, and we selected the most plausible orientation based on the agreement with alanine mutagenesis supported by affinity experiments. Interestingly, for drug design purposes, the mutation of L2506.51 to alanine increased the binding affinity of A17 at A1R. We explored the structure-activity relationships against A1R using alchemical binding free energy calculations with the thermodynamic integration coupled with the MD simulation (TI/MD) method, applied on the whole G-protein-coupled receptor-membrane system, which showed a good agreement (r = 0.73) between calculated and experimental relative binding free energies.

Adenosine receptor antagonists: Recent advances and therapeutic perspective.

Adenosine is an endogenous purine-based nucleoside expressed nearly in all body tissues. It regulates various body functions by activating four G-protein coupled receptors, A1, A2A, A2B, and A3. These receptors are widely acknowledged as drug targets for treating different neurological, metabolic, and inflammatory diseases. Although numerous adenosine receptor inhibitors have been developed worldwide, achieving target selectivity is still a big hurdle in drug development. However, the identification of specific radioligands-based affinity assay, fluorescent ligands, and MS-based ligand assay have contributed to the development of selective and potent adenosine ligands. In recent years various small heterocyclic-based molecules have shown some promising results. Istradefylline has been approved for treating Parkinson's in Japan, while preladenant, tozadenant, CVT-6883, MRS-1523, and many more are under different phases of clinical development. The present review is focused on the quest to develop potent and selective adenosine inhibitors from 2013 to early 2021 by various research groups. The review also highlights their biological activity, selectivity, structure-activity relationship, molecular docking, and mechanistic studies. A special emphsesis on drug designing strategies has been also given the manuscript. The comprehensive compilation of research work carried out in the field will provide inevitable scope for designing and developing novel adenosine inhibitors with improved selectivity and efficacy.

The Medicinal Chemistry of Caffeine.

The purine alkaloid caffeine is the most widely consumed psychostimulant drug in the world and has multiple beneficial pharmacological activities, for example, in neurodegenerative diseases. However, despite being an extensively studied bioactive natural product, the mechanistic understanding of caffeine's pharmacological effects is incomplete. While several molecular targets of caffeine such as adenosine receptors and phosphodiesterases have been known for decades and inspired numerous medicinal chemistry programs, new protein interactions of the xanthine are continuously discovered providing potentially improved pharmacological understanding and a molecular basis for future medicinal chemistry. In this Perspective, we gather knowledge on the confirmed protein interactions, structure activity relationship, and chemical biology of caffeine on well-known and upcoming targets. The diversity of caffeine's molecular activities on receptors and enzymes, many of which are abundant in the CNS, indicates a complex interplay of several mechanisms contributing to neuroprotective effects and highlights new targets as attractive subjects for drug discovery.

X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists.

We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2A AR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2A AR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A2A AR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2A AR, an emerging target in immuno-oncology.

Assessment of Pharmacokinetics and Metabolism Profiles of SCH 58261 in Rats Using Liquid Chromatography-Mass Spectrometric Method.

5-Amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c) pyrimidine (SCH 58261) is one of the new chemical entities that has been developed as an adenosine A2A receptor antagonist. Although SCH 58261 has been reported to be beneficial, there is little information about SCH 58261 from a drug metabolism or pharmacokinetics perspective. This study describes the metabolism and pharmacokinetic properties of SCH 58261 in order to understand its behaviors in vivo. Rats were used as the in vivo model species. First, an LC-MS/MS method was developed for the determination of SCH 58261 in rat plasma. A GastroPlus™ simulation, in vitro microsomal metabolic stability, and bile duct-cannulated studies were also performed to understand its pharmacokinetic profile. The parameter sensitivity analysis of GastroPlus™ was used to examine the factors that influence exposure when the drug is orally administered. The factors are as follows: permeability, systemic clearance, renal clearance, and liver first-pass effect. In vitro microsomal metabolic stability indicates how much the drug is metabolized. The extrapolated hepatic clearance value of SCH 58261 was 39.97 mL/min/kg, indicating that the drug is greatly affected by hepatic metabolism. In vitro microsomal metabolite identification studies revealed that metabolites produce oxidized and ketone-formed metabolites via metabolic enzymes in the liver. The bile duct-cannulated rat study, after oral administration of SCH 58261, showed that a significant amount of the drug was excreted in feces. These results imply that the drug is not absorbed well in the body after oral administration. Taken together, SCH 58261 showed quite a low bioavailability when administered orally and this was likely due to significantly limited absorption, as well as high metabolism in vivo.

Huntington's Disease: A Review of the Known PET Imaging Biomarkers and Targeting Radiotracers.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a CAG expansion mutation in the huntingtin gene. As a result, intranuclear inclusions of mutant huntingtin protein are formed, which damage striatal medium spiny neurons (MSNs). A review of Positron Emission Tomography (PET) studies relating to HD was performed, including clinical and preclinical data. PET is a powerful tool for visualisation of the HD pathology by non-invasive imaging of specific radiopharmaceuticals, which provide a detailed molecular snapshot of complex mechanistic pathways within the brain. Nowadays, radiochemists are equipped with an impressive arsenal of radioligands to accurately recognise particular receptors of interest. These include key biomarkers of HD: adenosine, cannabinoid, dopaminergic and glutamateric receptors, microglial activation, phosphodiesterase 10 A and synaptic vesicle proteins. This review aims to provide a radiochemical picture of the recent developments in the field of HD PET, with significant attention devoted to radiosynthetic routes towards the tracers relevant to this disease.

Conjugable A3 adenosine receptor antagonists for the development of functionalized ligands and their use in fluorescent probes.

Compounds able to simultaneously bind a biological target and be conjugated to a second specific moiety are attractive tools for the development of multi-purpose ligands useful as multi-target ligands, receptor probes or drug delivery systems, with both therapeutic and diagnostic applications. The human A3 adenosine receptor is a G protein-coupled receptor involved in many physio-pathological conditions, e.g. cancer and inflammation, thus representing a promising research target. In this work, two series of conjugable hA3AR antagonists, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine nucleus, were developed. The introduction of an aromatic ring at the 5 position of the scaffold, before (phenylacetamido moiety) or after (1,2,3-triazole obtained by click chemistry) the conjugation is aimed to increase affinity and selectivity towards the hA3AR receptor. As expected, conjugable compounds showed good affinity towards the hA3AR. In order to prove their potential in the development of hA3AR ligands for different purposes, compounds were also functionalized with fluorescent probes. Unfortunately, conjugation decreased affinity and selectivity for the target as compared to the hA2AAR. Computational studies identified specific non-conserved residues of the extracellular loops which constitute a structural barrier able to discriminate between ligands, giving insights into the rational development of new highly selective ligands.

Identification of new potent A1 adenosine receptor antagonists using a multistage virtual screening approach.

The use of antagonists for each adenosine receptor (AR) subtype as potent clinical candidates is of growing interest due to their involvement in the treatment of various diseases. The recent resolution of several A1 and A2A ARs X-ray structures provides opportunities for structure-based drug design. In this study, we describe the discovery of novel A1AR antagonists by applying a multistage virtual screening approach, which is based on random forest (RF), e-pharmacophore modeling and docking methods. A multistage virtual screening approach was applied to screen the ChemDiv library (1,492,362 compounds). Among the final hits, 22 compounds were selected for further radioligand binding assay analysis against human A1AR, and 18 compounds (81.82% success) exhibited nanomolar or low micromolar binding potency (Ki). Then, we selected six compounds (pKi > 6) to further evaluate their antagonist profile in a cAMP functional assay, and we found that they had low micromolar antagonistic activity (pIC50 = 5.51-6.38) for the A1AR. Particularly, four of six compounds (pKi > 6) showed very good affinity (pKi = 6.11-7.13) and selectively (>100-fold) for A1AR over A2AAR. Moreover, the novelty analysis suggested that four of six compounds (pKi > 6) were dissimilar to existing A1AR antagonists and hence represented novel A1AR antagonists. Further molecular docking and molecular dynamics (MD) studies showed that the three selective compounds 15, 20 and 22 were stabilized (RMSlig value ≤ 2 Å) inside the binding pocket of A1AR with similar orientations to the docking pose in 100-ns MD simulations, whereas they escaped from the binding area of A2AAR with larger values of RMSlig (RMSlig ≥ 2 Å). We hope that these findings provide new insights into the discovery of drugs targeting A1AR and facilitate research on new drugs and treatments for A1AR-related human pathologies.

Substituted 4-phenylthiazoles: Development of potent and selective A1, A3 and dual A1/A3 adenosine receptor antagonists.

Adenosine acts as a powerful signaling molecule via four distinct G protein-coupled receptors, designated A1, A2A, A2B and A3 adenosine receptors (ARs). A2A and A2B ARs are Gs-coupled, while A1 and A3 ARs inhibit cAMP production via Gi proteins. Antagonists for A1 and A3 ARs may be useful for the treatment of (neuro)inflammatory diseases including acute kidney injury and kidney failure, pulmonary diseases, and Alzheimer's disease. In the present study, we optimized the versatile 2-amino-4-phenylthiazole scaffold by introducing substituents at N2 and C5 to obtain A1 and A3 AR antagonists including dual-target compounds. Selective A1 antagonists with (sub)nanomolar potency were produced, e.g. 11 and 13. These compounds showed species differences being significantly more potent at the rat as compared to the human A1 AR, and were characterized as inverse agonists. Several potent and selective A3 AR antagonists, e.g. 7, 8, 17 and 22 (Ki values of 5-9 nM at the human A3 AR) were prepared, which were much less potent at the rat orthologue. Moreover, dual A1/A3 antagonists (10, 18) were developed showing Ki values between 8 and 42 nM. Docking and molecule dynamic simulation studies using the crystal structure of the A1 AR and a homology model of the A3 AR were performed to rationalize the observed structure-activity relationships.

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.

2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual A1/A3 adenosine receptor antagonists: Synthesis and structure-activity relationships.

From a collection containing more than 1500 academic compounds, in silico screening identified a hit for the human A1 adenosine receptor containing a new purine scaffold. To study the structure activity relationships of this new chemical series for adenosine receptors, a library of 24 purines was synthesized and tested in radioligand binding assays at human A1, A2A, A2B and A3 adenosine receptor subtypes. Fourteen molecules showed potent antagonism at A1, A3 or dual A1/A3 adenosine receptors. This purine scaffold is an important source for novel biochemical tools and/or therapeutic drugs.

Modulation of Adenosine Receptors and Antioxidative Effect of Beer Extracts in in Vitro Models.

The fight against neurodegenerative diseases is promoting the searching of nutrients, preferably of wide consumption, with proven effects on health. Beer is widely consumed and has potential benefits on health. In this work, three different extracts from dark beer (DB), non-alcoholic beer (NAB), and lager beer (LB) were assayed at 30 min and 24 h in rat C6 glioma and human SH-SY5Y neuroblastoma cells in order to study their possible protective effects. Cell viability and adenosine A1, A2A, A2B, and A3 receptor gene expression and protein levels were measured in control cells and in cells challenged with hydrogen peroxide as an oxidant stressor. Among the three extracts analyzed, DB showed a greater protective effect against H2O2-induced oxidative stress and cell death. Moreover, a higher A1 receptor level was also induced by this extract. Interestingly, A1 receptor level was also increased by NAB and LB extracts, but to a lower extent, and the protective effect of these extracts against H2O2 was lower. This possible correlation between protection and A1 receptor level was observed at 24 h in both C6 and SH-SY5Y cells. In summary, different beer extracts modulate, to a different degree, adenosine receptors expression and protect both glioma and neuroblastoma cells from oxidative stress.

Recent Advances of In-Silico Modeling of Potent Antagonists for the Adenosine Receptors.

The rapid advancement of computer architectures and development of mathematical algorithms offer a unique opportunity to leverage the simulation of macromolecular systems at physiologically relevant timescales. Herein, we discuss the impact of diverse structure-based and ligand-based molecular modeling techniques in designing potent and selective antagonists against each adenosine receptor (AR) subtype that constitutes multitude of drug targets. The efficiency and robustness of high-throughput empirical scoring function-based approaches for hit discovery and lead optimization in the AR family are assessed with the help of illustrative examples that have led to nanomolar to sub-micromolar inhibition activities. Recent progress in computer-aided drug discovery through homology modeling, quantitative structure-activity relation, pharmacophore models, and molecular docking coupled with more accurate free energy calculation methods are reported and critically analyzed within the framework of structure-based virtual screening of AR antagonists. Later, the potency and applicability of integrated molecular dynamics (MD) methods are addressed in the context of diligent inspection of intricated AR-antagonist binding processes. MD simulations are exposed to be competent for studying the role of the membrane as well as the receptor flexibility toward the precise evaluation of the biological activities of antagonistbound AR complexes such as ligand binding modes, inhibition affinity, and associated thermodynamic and kinetic parameters.

Novel 8-amino-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives as potent human adenosine A1 and A2A receptor antagonists. Evaluation of their protective effect against ß-amyloid-induced neurotoxicity in SH-SY5Y cells.

In this work, an enlarged series of 1,2,4-triazolo[4,3-a]pyrazin-3-ones was designed to target the human (h) A2A adenosine receptor (AR) or both hA1 and hA2A ARs. The novel 8-amino-1,2,4-triazolopyrazin-3-one derivatives 1-25 featured a phenyl or a benzyl pendant at position 2 while different aryl/heteroaryl substituents were placed at position 6. Two compounds (8 and 10) endowed with high affinity (Ki = 7.2 and 10.6 nM) and a complete selectivity for the hA2A AR were identified. Moreover, several derivatives possessed nanomolar affinity for both hA1 and hA2A ARs (both Ki < 20 nM) and different degrees of selectivity versus the hA3 AR. Two selected compounds (10 and 25) demonstrated ability in preventing ß-amyloid peptide (25-35)-induced neurotoxicity in SH-SY5Y cells. Results of docking studies at the hA2A and hA1 AR crystal structures helped us to rationalize the observed affinity data and to highlight that the steric hindrance of the substituents at the 2- and 6-position of the bicyclic core affects the binding mode in the receptor cavity.

Novel human adenosine receptor antagonists based on the 7-amino-thiazolo[5,4-d]pyrimidine scaffold. Structural investigations at the 2-, 5- and 7-positions to enhance affinity and tune selectivity.

This paper describes the synthesis of novel 7-amino-thiazolo[5,4-d]pyrimidines bearing different substituents at positions 2, 5 and 7 of the thiazolopyrimidine scaffold. The synthesized compounds 2-27 were evaluated in radioligand binding (A1, A2A and A3) and adenylyl cyclase activity (A2B and A2A) assays, in order to evaluate their affinity and potency at human adenosine receptor subtypes. The current study allowed us to support that affinity and selectivity of 7-amino-thiazolo[5,4-d]pyrimidine derivatives towards the adenosine receptor subtypes can be modulated by the nature of the groups attached at positions 2, 5 and 7 of the bicyclic scaffold. To rationalize the hypothetical binding mode of the newly synthesized compounds, we also performed docking calculations in human A2A, A1 and A3 structures.

[1,2,4]Triazolo[1,5-c]pyrimidines as adenosine receptor antagonists: Modifications at the 8 position to reach selectivity towards A3 adenosine receptor subtype.

[1,2,4]Triazolo[1,5-c]pyrimidine is a promising platform to develop adenosine receptor antagonists. Here, we tried to investigate the effect of the substituent at the 8 position of [1,2,4]triazolo[1,5-c]pyrimidine derivatives on affinity and selectivity at the human A3 adenosine receptor subtype. In particular, we have introduced both esters and amides, principally with a benzylic nature. In addition, a small series of 5-substituted [1,2,4]triazolo[1,5-c]pyrimidines was designed in order to complete the structure-activity relationship analysis. Several of these new compounds showed affinity towards human A3 adenosine receptor in the low nanomolar range, with the most potent derivative of the series bringing a 4-ethylbenzylester at the 8 position (compound 18, hA3AR Ki = 1.21 nM). Docking studies performed on the synthesized compounds inside models of human A1, A2A and A3 adenosine receptors showed similar binding modes, comparable with the typical crystallographic binding mode of the inverse agonist ZM-241,385.

Discovery of Novel Adenosine Receptor Antagonists through a Combined Structure- and Ligand-Based Approach Followed by Molecular Dynamics Investigation of Ligand Binding Mode.

An intense effort is made by pharmaceutical and academic research laboratories to identify and develop selective antagonists for each adenosine receptor (AR) subtype as potential clinical candidates for "soft" treatment of various diseases. Crystal structures of subtypes A2A and A1ARs offer exciting opportunities for structure-based drug design. In the first part of the present work, Maybridge HitFinder library of 14400 compounds was utilized to apply a combination of structure-based against the crystal structure of A2AAR and ligand-based methodologies. The docking poses were rescored by CHARMM energy minimization and calculation of the desolvation energy using Poisson-Boltzmann equation electrostatics. Out of the eight selected and tested compounds, five were found positive hits (63% success). Although the project was initially focused on targeting A2AAR, the identified antagonists exhibited low micromolar or micromolar affinity against A2A/A3, ARs, or A3AR, respectively. Based on these results, 19 compounds characterized by novel chemotypes were purchased and tested. Sixteen of them were identified as AR antagonists with affinity toward combinations of the AR family isoforms (A2A/A3, A1/A3, A1/A2A/A3, and A3). The second part of this work involves the performance of hundreds of molecular dynamics (MD) simulations of complexes between the ARs and a total of 27 ligands to resolve the binding interactions of the active compounds, which were not achieved by docking calculations alone. This computational work allowed the prediction of stable and unstable complexes which agree with the experimental results of potent and inactive compounds, respectively. Of particular interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides, and carbonyloxycarboximidamide derivatives were found to be selective and possess a micromolar to low micromolar affinity for the A3 receptor.