Optimization of 2-Amino-4,6-diarylpyrimidine-5-carbonitriles as Potent and Selective A1 Antagonists.

We herein document a large collection of 108 2-amino-4,6-disubstituted-pyrimidine derivatives as potent, structurally simple, and highly selective A1AR ligands. The most attractive ligands were confirmed as antagonists of the canonical cyclic adenosine monophosphate pathway, and some pharmacokinetic parameters were preliminarilly evaluated. The library, built through a reliable and efficient three-component reaction, comprehensively explored the chemical space allowing the identification of the most prominent features of the structure-activity and structure-selectivity relationships around this scaffold. These included the influence on the selectivity profile of the aromatic residues at positions R4 and R6 of the pyrimidine core but most importantly the prominent role to the unprecedented A1AR selectivity profile exerted by the methyl group introduced at the exocyclic amino group. The structure-activity relationship trends on both A1 and A2AARs were conveniently interpreted with rigorous free energy perturbation simulations, which started from the receptor-driven docking model that guided the design of these series.

Adenosine A2AR/A1R Antagonists Enabling Additional H3R Antagonism for the Treatment of Parkinson's Disease.

Adenosine A1/A2A receptors (A1R/A2AR) represent targets in nondopaminergic treatment of motor disorders such as Parkinson's disease (PD). As an innovative strategy, multitargeting ligands (MTLs) were developed to achieve comprehensive PD therapies simultaneously addressing comorbid symptoms such as sleep disruption. Recognizing the wake-promoting capacity of histamine H3 receptor (H3R) antagonists in combination with the "caffeine-like effects" of A1R/A2AR antagonists, we designed A1R/A2AR/H3R MTLs, where a piperidino-/pyrrolidino(propyloxy)phenyl H3R pharmacophore was introduced with overlap into an adenosine antagonist arylindenopyrimidine core. These MTLs showed distinct receptor binding profiles with overall nanomolar H3R affinities (Ki < 55 nM). Compound 4 (ST-2001, Ki (A1R) = 11.5 nM, Ki (A2AR) = 7.25 nM) and 12 (ST-1992, Ki (A1R) = 11.2 nM, Ki (A2AR) = 4.01 nM) were evaluated in vivo. l-DOPA-induced dyskinesia was improved after administration of compound 4 (1 mg kg-1, i.p. rats). Compound 12 (2 mg kg-1, p.o. mice) increased wakefulness representing novel pharmacological tools for PD therapy.

Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A1 Receptor in Living Cells.

The adenosine A1 receptor (A1AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A1AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A1AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A1AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A1AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A1AR molecular pharmacology and signaling in living cells.

The influence of selective A1 and A2A receptor antagonists on the antidepressant-like activity of moclobemide, venlafaxine and bupropion in mice.

OBJECTIVE: The main goal of our study was to investigate whether a selective antagonism of the adenosine A1 or A2A receptors is able to enhance the antidepressant activity of commonly prescribed drugs. MATERIALS AND METHODS: All experiments were carried out on male Albino Swiss mice. The forced swim test and the tail suspension test were used to evaluate the antidepressant-like potential. Drug concentrations in animals' serum and brains were measured by high-performance liquid chromatography. KEY FINDINGS: The antidepressant potential of moclobemide (1.5 mg/kg), venlafaxine (1 mg/kg) and bupropion (10 mg/kg) was enhanced by a co-administration with 3,7-dimethyl-1-propargylxanthine (DMPX; an antagonist of adenosine A2A receptors; 3 mg/kg) or 8-cyclopentyl-1,3-dipropylxanthine (an antagonist of adenosine A1 receptors; 1 mg/kg). However, significant interactions between the tested substances were detected only in the experiments with DMPX. The nature of the observed interplays is rather pharmacodynamic than pharmacokinetic, because neither serum nor brain concentrations of the used drugs were significantly increased. CONCLUSIONS: Blockage of the adenosine receptors (particularly the A2A subtypes) could be considered in future as a novel, promising part of the combined antidepressant therapy. However, further studies on this subject are needed.

Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists.

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A1, A2A, A2B and A3 adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA1 and hA2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A1/A2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA1 Ki = 10.2 nM; hA2A Ki = 4.72 nM) and behaved as a potent A1/A2A antagonist/inverse agonist (hA1 IC50 = 13.4 nM; hA2A IC50 = 5.34 nM).

Evaluation of 2-benzylidene-1-tetralone derivatives as antagonists of A1 and A2A adenosine receptors.

Antagonists of the adenosine receptors (A1 and A2A ) are thought to be beneficial in neurological disorders, such as Alzheimer's and Parkinson's disease. The aim of this study was to explore 2-benzylidene-1-tetralone derivatives as antagonists of A1 and/or A2A adenosine receptors. In general, the test compounds were found to be selective for the A1 adenosine receptor, with only three test compounds possessing affinity for both the A1 and A2A adenosine receptor. The 2-benzylidene-1-tetralones bearing a hydroxyl substituent at either position C5, C6 or C7 of ring A displayed favourable adenosine A1 receptor binding, while C5 hydroxy substitution led to favourable A2A adenosine receptor affinity. Interestingly, para-hydroxy substitution on ring B in combination with ring A bearing a hydroxy at position C6 or C7 provided the 2-benzylidene-1-tetralones with both A1 and A2A adenosine receptor affinity. Compounds 4 and 8 displayed the highest A1 and A2A adenosine receptor affinity with values below 7 µm. Both these compounds behaved as A1 adenosine receptor antagonists in the performed GTP shift assays. In conclusion, the 2-benzylidene-1-tetralone derivatives can be considered as lead compounds to design a new class of dual acting adenosine A1 /A2A receptor antagonists that may have potential in treating both dementia and locomotor deficits in Parkinson's disease.

Synthesis and Pharmacological Evaluation of Identified and Putative Metabolites of the A1 Adenosine Receptor Antagonist 8-Cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX).

The A1 adenosine receptor (A1 AR) antagonist [18 F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([18 F]CPFPX), used in imaging human brain A1 ARs by positron emission tomography (PET), is stable in the brain, but rapidly undergoes transformation into one major (3-(3-fluoropropyl)-8-(3-oxocyclopenten-1-yl)-1-propylxanthine, M1) and several minor metabolites in blood. This report describes the synthesis of putative metabolites of CPFPX as standards for the identification of those metabolites. Analysis by (radio)HPLC revealed that extracts of human liver microsomes incubated with no-carrier-added (n.c.a.)[18 F]CPFPX contain the major metabolite, M1, as well as radioactive metabolites corresponding to derivatives functionalized at the cyclopentyl moiety, but no N1-despropyl species or metabolites resulting from functionalization of the N3-fluoropropyl chain. The putative metabolites were found to displace the binding of [3 H]CPFPX to the A1 AR in pig brain cortex at Ki values between 1.9 and 380 nm and the binding of [3 H]ZM241385 to the A2A AR in pig striatum at Ki values >180 nm. One metabolite, a derivative functionalized at the ω-position of the N1-propyl chain, showed high affinity (Ki 2 nm) to and very good selectivity (>9000) for the A1 AR.

Adenosine A1( )receptors are selectively coupled to Gα(i-3) in postmortem human brain cortex: Guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding/immunoprecipitation study.

By means of guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding assay combined with immunoprecipitation using anti-Gα subunit antibody, we recently reported 5-HT2A receptor- and M1 muscarinic acetylcholine receptor-mediated Gαq activation in rat cerebral cortical membranes (Odagaki et al., 2014). In the present study, this method has been applied to postmortem human brains, with focusing on adenosine receptor-mediated G-protein activation. In the exploratory experiments using a series of agonists and the antibodies specific to each Gα subtypes in the presence of low (10 nM) or high (50 µM) concentration of GDP, the most prominent increases in specific [(35)S]GTPγS binding in the membranes prepared from human prefrontal cortex were obtained for the combinations of adenosine (1mM)/anti-Gαi-3 in the presence of 50 µM GDP as well as 5-HT (100 µM)/anti-Gαq and carbachol (1mM)/anti-Gαq in the presence of 10nM GDP. Adenosine-induced activation of Gαi-3 emerged only when GDP concentrations were increased higher than 10 µM, and the following experiments were performed in the presence of 300 µM GDP. Adenosine increased specific [(35)S]GTPγS binding to Gαi-3 in a concentration-dependent manner to 251.4% of the basal unstimulated binding, with an EC50 of 1.77 µM. The involvement of adenosine A1 receptor was verified by the experiments using selective agonists and antagonists at adenosine A1 or A3 receptor. Among the α subunits of Gi/o class (Gαi-1, Gαi-2, Gαi-3, and Gαo.), only Gαi-3 was activated by 1mM adenosine, indicating that human brain adenosine A1 receptor is coupled preferentially, if not exclusively, to Gαi-3.

Selectivity is species-dependent: Characterization of standard agonists and antagonists at human, rat, and mouse adenosine receptors.

Adenosine receptors (ARs) have emerged as new drug targets. The majority of data on affinity/potency and selectivity of AR ligands described in the literature has been obtained for the human species. However, preclinical studies are mostly performed in mouse or rat, and standard AR agonists and antagonists are frequently used for studies in rodents without knowing their selectivity in the investigated species. In the present study, we selected a set of frequently used standard AR ligands, 8 agonists and 16 antagonists, and investigated them in radioligand binding studies at all four AR subtypes, A1, A2A, A2B, and A3, of three species, human, rat, and mouse. Recommended, selective agonists include CCPA (for A1AR of rat and mouse), CGS-21680 (for A2A AR of rat), and Cl-IB-MECA (for A3AR of all three species). The functionally selective partial A2B agonist BAY60-6583 was found to additionally bind to A1 and A3AR and act as an antagonist at both receptor subtypes. The antagonists PSB-36 (A1), preladenant (A2A), and PSB-603 (A2B) displayed high selectivity in all three investigated species. MRS-1523 acts as a selective A3AR antagonist in human and rat, but is only moderately selective in mouse. The comprehensive data presented herein provide a solid basis for selecting suitable AR ligands for biological studies.

Chaperoning of the A1-adenosine receptor by endogenous adenosine - an extension of the retaliatory metabolite concept.

Cell-permeable orthosteric ligands can assist folding of G protein-coupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A1-adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A1-receptor-Y(288)A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A1 antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase, and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound to an A1-selective antagonist with an affinity indistinguishable from the wild-type A1 receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V2-vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O2) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A1-receptor-Y(288)A within 1 hour. These findings were recapitulated for the wild-type A1 receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A1 receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A1 receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress.

Homodimerization of adenosine A1 receptors in brain cortex explains the biphasic effects of caffeine.

Using bioluminescence resonance energy transfer and proximity ligation assays, we obtained the first direct evidence that adenosine A1 receptors (A1Rs) form homomers not only in cell cultures but also in brain cortex. By radioligand binding experiments in the absence or in the presence of the A1Rs allosteric modulator, adenosine deaminase, and by using the two-state dimer receptor model to fit binding data, we demonstrated that the protomer-protomer interactions in the A1R homomers account for some of the pharmacological characteristics of agonist and antagonist binding to A1Rs. These pharmacological properties include the appearance of cooperativity in agonist binding, the change from a biphasic saturation curve to a monophasic curve in self-competition experiments and the molecular cross-talk detected when two different specific molecules bind to the receptor. In this last case, we discovered that caffeine binding to one protomer increases the agonist affinity for the other protomer in the A1R homomer, a pharmacological characteristic that correlates with the low caffeine concentrations-induced activation of agonist-promoted A1R signaling. This pharmacological property can explain the biphasic effects reported at low and high concentration of caffeine on locomotor activity.

Targeting adenosine receptors with coumarins: synthesis and binding activities of amide and carbamate derivatives.

OBJECTIVES: With the aim of finding the structural features governing binding activity and selectivity against adenosine receptors (ARs), several 3-subtituted coumarins with amide (compounds 3-6) and carbamate (7-9) functions were synthesized. To study its possible influence on the binding activity and selectivity, a hydroxyl substituent was also introduced at position 4 of the coumarin moiety. METHODS: A new series of coumarins (3-9) were synthesized and evaluated by radioligand binding studies towards ARs. KEY FINDINGS: None of the 4-hydroxy derivatives (4, 8 and 9) showed binding affinity for any of the ARs. None of the compounds interacted with the hA(2B) AR (K(i) > 100,000 nM). Compounds 3, 5, 6 and 7 had different activity profiles with dissimilar binding affinity and selectivity towards human A1, A(2A) and A3 ARs. CONCLUSIONS: The most remarkable derivative is compound 7, which presents the best affinity and selectivity for the A3 adenosine receptor (K(i) = 5500 nM).

Overcoming the genotoxicity of a pyrrolidine substituted arylindenopyrimidine as a potent dual adenosine A(2A)/A(1) antagonist by minimizing bioactivation to an iminium ion reactive intermediate.

2-Amino-4-phenyl-8-pyrrolidin-1-ylmethyl-indeno[1,2-d]pyrimidin-5-one (1) is a novel and potent selective dual A(2A)/A(1) adenosine receptor antagonist from the arylindenopyrimidine series that was determined to be genotoxic in both the Ames and Mouse Lymphoma L5178Y assays only following metabolic activation. Compound 1 was identified as a frame-shift mutagen in Salmonella typhimurium tester strain TA1537 as indicated by a significant dose-dependent increase in revertant colonies as compared to the vehicle control. The metabolic activation-dependent irreversible covalent binding of radioactivity to DNA, recovery of 1 and its enamine metabolite from acid hydrolysis of covalently modified DNA, and protection of covalent binding to DNA by both cyanide ion and methoxylamine suggest that the frame-shift mutation in TA1537 strain involved covalent binding instead of simple intercalation to DNA. Compound 1 was bioactivated to endocyclic iminium ion, aldehyde, epoxide, and α,ß-unsaturated keto reactive intermediates from the detection of cyano, oxime, and glutathione conjugates by data-dependent high resolution accurate mass measurements. Collision-induced dissociation of these conjugates provided evidence for bioactivation of the pyrrolidine ring of 1. The epoxide and α,ß-unsaturated keto reactive intermediates were unlikely to cause the genotoxicity of 1 because the formation of their glutathione adducts did not ameliorate the binding of compound related material to DNA. Instead, the endocyclic iminium ions and amino aldehydes were likely candidates responsible for genotoxicity based on, first, the protection afforded by both cyanide ion and methoxylamine, which reduced the potential to form covalent adducts with DNA, and, second, analogues of 1 designed with low probability to form these reactive intermediates were not genotoxic. It was concluded that 1 also had the potential to be mutagenic in humans based on observing the endocyclic iminium ion following incubation with a human liver S9 preparation and the commensurate detection of DNA adducts. An understanding of this genotoxicity mechanism supported an evidence-based approach to selectively modify the structure of 1 which resulted in analogues being synthesized that were devoid of a genotoxic liability. In addition, potency and selectivity against both adenosine A(2A) and A(1) receptors were maintained.

Differential contributions of adenosine to hypoxia-evoked depressions of three neuronal pathways in isolated spinal cord of neonatal rats.

BACKGROUND AND PURPOSE: Hypoxic effects on neuronal functions vary significantly with experimental conditions, but the mechanism for this is unclear. Adenosine has been reported to play a key role in depression of neuronal activities in the CNS during acute hypoxia. Hence, we examined the effect of acute hypoxia on different spinal reflex potentials and the contribution of adenosine to them. EXPERIMENTAL APPROACH: Spinal reflex potentials, monosynaptic reflex potential (MSR), slow ventral root potential (sVRP) and dorsal root potential (DRP), were measured in the isolated spinal cord of the neonatal rat. Adenosine release was measured by using enzymatic biosensors. KEY RESULTS: In the spinal cord preparation isolated from postnatal day 5-8 rats at 27°C, acute hypoxia induced adenosine release and depressed three reflex potentials. However, in postnatal day 0-3 rats at 27°C, the hypoxic-induced adenosine release and depression of MSR were negligible, while the depression of sVRP and DRP were perceptible responses. In postnatal day 0-3 rats at 33°C, hypoxia evoked adenosine release and depression of MSR. An adenosine A(1) receptor selective antagonist and a high [Ca(2+)](o), which suppressed adenosine release, abolished the hypoxic-induced depression of MSR but not those of sVRP and DRP. CONCLUSIONS AND IMPLICATIONS: Hypoxic-induced depression of MSR depends on adenosine release, which is highly susceptible to age, temperature and [Ca(2+)](o). However, a large part of the depressions of DRP and sVRP are mediated via adenosine-independent mechanisms. This differential contribution of adenosine to depression is suggested to be an important factor for the variable effects of hypoxia on neuronal functions.