Antiplatelet Effects of Selected Xanthine-Based Adenosine A2A and A2B Receptor Antagonists Determined in Rat Blood.

The platelet aggregation inhibitory activity of selected xanthine-based adenosine A2A and A2B receptor antagonists was investigated, and attempts were made to explain the observed effects. The selective A2B receptor antagonist PSB-603 and the A2A receptor antagonist TB-42 inhibited platelet aggregation induced by collagen or ADP. In addition to adenosine receptor blockade, the compounds were found to act as moderately potent non-selective inhibitors of phosphodiesterases (PDEs). TB-42 showed the highest inhibitory activity against PDE3A along with moderate activity against PDE2A and PDE5A. The antiplatelet activity of PSB-603 and TB-42 may be due to inhibition of PDEs, which induces an increase in cAMP and/or cGMP concentrations in platelets. The xanthine-based adenosine receptor antagonists were found to be non-cytotoxic for platelets. Some of the compounds showed anti-oxidative properties reducing lipid peroxidation. These results may provide a basis for the future development of multi-target xanthine derivatives for the treatment of inflammation and atherosclerosis and the prevention of heart infarction and stroke.

Anti-Inflammatory Activities of 8-Benzylaminoxanthines Showing High Adenosine A2A and Dual A1/A2A Receptor Affinity.

Chronic inflammation plays an important role in the development of neurodegenerative diseases, such as Parkinson's disease (PD). In the present study, we synthesized 25 novel xanthine derivatives with variable substituents at the N1-, N3- and C8-position as adenosine receptor antagonists with potential anti-inflammatory activity. The compounds were investigated in radioligand binding studies at all four human adenosine receptor subtypes, A1, A2A, A2B and A3. Compounds showing nanomolar A2A and dual A1/A2A affinities were obtained. Three compounds, 19, 22 and 24, were selected for further studies. Docking and molecular dynamics simulation studies indicated binding poses and interactions within the orthosteric site of adenosine A1 and A2A receptors. In vitro studies confirmed the high metabolic stability of the compounds, and the absence of toxicity at concentrations of up to 12.5 µM in various cell lines (SH-SY5Y, HepG2 and BV2). Compounds 19 and 22 showed anti-inflammatory activity in vitro. In vivo studies in mice investigating carrageenan- and formalin-induced inflammation identified compound 24 as the most potent anti-inflammatory derivative. Future studies are warranted to further optimize the compounds and to explore their therapeutic potential in neurodegenerative diseases.

Xanthine-Dopamine Hybrid Molecules as Multitarget Drugs with Potential for the Treatment of Neurodegenerative Diseases.

Multitarget drugs based on a hybrid dopamine-xanthine core were designed as potential drug candidates for the treatment of neurodegenerative diseases. Monoamine oxidase B (MAO-B) inhibitors with significant ancillary A2A adenosine receptor (A2AAR) antagonistic properties were further developed to exhibit additional phosphodiesterase-4 and -10 (PDE4/10) inhibition and/or dopamine D2 receptor (D2R) agonistic activity. While all of the designed compounds showed MAO-B inhibition in the nanomolar range mostly combined with submicromolar A2AAR affinity, significant enhancement of PDE-inhibitory and D2R-agonistic activity was additionally reached for some compounds through various structural modifications. The final multitarget drugs also showed promising antioxidant properties in vitro. In order to evaluate their potential neuroprotective effect, representative ligands were tested in a cellular model of toxin-induced neurotoxicity. As a result, protective effects against oxidative stress in neuroblastoma cells were observed, confirming the utility of the applied strategy. Further evaluation of the newly developed multitarget ligands in preclinical models of Alzheimer's and Parkinson's diseases is warranted.

Cyclin-Dependent Kinases (CDK) and Their Role in Diseases Development-Review.

Cyclin-dependent kinases (CDKs) are involved in many crucial processes, such as cell cycle and transcription, as well as communication, metabolism, and apoptosis. The kinases are organized in a pathway to ensure that, during cell division, each cell accurately replicates its DNA, and ensure its segregation equally between the two daughter cells. Deregulation of any of the stages of the cell cycle or transcription leads to apoptosis but, if uncorrected, can result in a series of diseases, such as cancer, neurodegenerative diseases (Alzheimer's or Parkinson's disease), and stroke. This review presents the current state of knowledge about the characteristics of cyclin-dependent kinases as potential pharmacological targets.

8-Benzylaminoxanthine scaffold variations for selective ligands acting on adenosine A2A receptors. Design, synthesis and biological evaluation.

A library of 34 novel compounds based on a xanthine scaffold was explored in biological studies for interaction with adenosine receptors (ARs). Structural modifications of the xanthine core were introduced in the 8-position (benzylamino and benzyloxy substitution) as well as at N1, N3, and N7 (small alkyl residues), thereby improving affinity and selectivity for the A2A AR. The compounds were characterized by radioligand binding assays, and our study resulted in the development of the potent A2A AR ligands including 8-((6-chloro-2-fluoro-3-methoxybenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12d; Ki human A2AAR: 68.5 nM) and 8-((2-chlorobenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12h; Ki human A2AAR: 71.1 nM). Moreover, dual A1/A2AAR ligands were identified in the group of 1,3-diethyl-7-methylxanthine derivatives. Compound 14b displayed Ki values of 52.2 nM for the A1AR and 167 nM for the A2AAR. Selected A2AAR ligands were further evaluated as inactive for inhibition of monoamine oxidase A, B and isoforms of phosphodiesterase-4B1, -10A, which represent classical targets for xanthine derivatives. Therefore, the developed 8-benzylaminoxanthine scaffold seems to be highly selective for AR activity and relevant for potent and selective A2A ligands. Compound 12d with high selectivity for ARs, especially for the A2AAR subtype, evaluated in animal models of inflammation has shown anti-inflammatory activity. Investigated compounds were found to display high selectivity and may therefore be of high interest for further development as drugs for treating cancer or neurodegenerative diseases.

Novel, Dual Target-Directed Annelated Xanthine Derivatives Acting on Adenosine Receptors and Monoamine Oxidase B.

Annelated purinedione derivatives have been shown to act as possible multiple-target ligands, addressing adenosine receptors and monoaminooxidases. In this study, based on our previous results, novel annelated pyrimido- and diazepino[2,1-f]purinedione derivatives were designed as dual-target-directed ligands combining A2A adenosine receptor (AR) antagonistic activity with blocking monoamine oxidase B. A library of 19 novel compounds was synthesized and biologically evaluated in radioligand binding studies at AR subtypes and for their ability to inhibit MAO-B. This allowed 9-(2-chloro-6-fluorobenzyl)-3-ethyl-1-methyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-dione (13 e; Ki human A2A AR: 264 nM and IC50 human MAO-B: 243 nM) to be identified as the most potent dual-acting ligand from this series. ADMET parameters were estimated in vitro, and analysis of the structure-activity relationships was complemented by molecular-docking studies based on previously published X-ray structures of the protein targets. Such dual-acting ligands, by selectively blocking A2A AR, accompanied by the inhibition of dopamine metabolizing enzyme MAO-B, might provide symptomatic and neuroprotective effects in, among others, the treatment of Parkinson disease.

Novel multi-target directed ligands based on annelated xanthine scaffold with aromatic substituents acting on adenosine receptor and monoamine oxidase B. Synthesis, in vitro and in silico studies.

N9-Benzyl-substituted imidazo-, pyrimido- and 1,3-diazepino[2,1-f]purinediones were designed as dual-target-directed ligands combining A2A adenosine receptor (AR) antagonistic activity with blockade of monoamine oxidase B (MAO-B). A library of 37 novel compounds was synthesized and biologically evaluated in radioligand binding studies at AR subtypes and for their ability to inhibit MAO-B. A systematic modification of the tricyclic structures based on a xanthine core by enlargement of the third heterocyclic ring or attachment of various substituted benzyl moieties resulted in the development of 9-(2-chloro-6-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-dione (9u; Ki human A2AAR: 189 nM and IC50 human MAO-B: 570 nM) as the most potent dual acting ligand of the series displaying high selectivity versus related targets. Moreover, some potent, selective MAO-B inhibitors were identified in the group of pyrimido- and 1,3-diazepino[2,1-f]purinediones. Compound 10d (10-(3,4-dichlorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-f]purine-2,4(3H,6H)-dione) displayed an IC50 value at human MAO-B of 83 nM. Analysis of structure-activity relationships was complemented by molecular docking studies based on previously published X-ray structures of the protein targets. An extended biological profile was determined for selected compounds including in vitro evaluation of potential hepatotoxicity calculated in silico and antioxidant properties as an additional desirable activity. The new molecules acting as dual target drugs may provide symptomatic relief as well as disease-modifying effects for neurodegenerative diseases, in particular Parkinson's disease.

Tricyclic xanthine derivatives containing a basic substituent: adenosine receptor affinity and drug-related properties.

A library of 27 novel amide derivatives of annelated xanthines was designed and synthesized. The new compounds represent 1,3-dipropyl- and 1,3-dibutyl-pyrimido[2,1-f]purinedione-9-ethylphenoxy derivatives including a CH2CONH linker between the (CH2)2-amino group and the phenoxy moiety. A synthetic strategy to obtain the final products was developed involving solvent-free microwave irradiation. The new compounds were evaluated for their adenosine receptor (AR) affinities. The most potent derivatives contained a terminal tertiary amino function. Compounds with nanomolar AR affinities and at the same time high water-solubility were obtained (A1 (Ki = 24-605 nM), A2A (Ki = 242-1250 nM), A2B (Ki = 66-911 nM) and A3 (Ki = 155-1000 nM)). 2-(4-(2-(1,3-Dibutyl-2,4-dioxo-1,2,3,4,7,8-hexahydropyrimido[2,1-f]purin-9(6H)-yl)ethyl)phenoxy)-N-(3-(diethylamino)propyl)acetamide (27) and the corresponding N-(2-(pyrrolidin-1-yl)ethyl)acetamide (36) were found to be the most potent antagonists of the present series. While 27 showed CYP inhibition and moderate metabolic stability, 36 was found to possess suitable properties for in vivo applications. In an attempt to explain the affinity data for the synthesized compounds, molecular modeling and docking studies were performed using homology models of A1 and A2A adenosine receptors. The potent compound 36 was used as an example for discussion of the possible ligand-protein interactions. Moreover, the compounds showed high water-solubility indicating that the approach of introducing a basic side chain was successful for the class of generally poorly soluble AR antagonists.

Molecular and functional interaction between GPR18 and cannabinoid CB2 G-protein-coupled receptors. Relevance in neurodegenerative diseases.

GPR18, still considered an orphan receptor, may respond to endocannabinoids, whose canonical receptors are CB1 and CB2. GPR18 and CB2 receptors share a role in peripheral immune response regulation and are co-expressed in microglia, which are immunocompetent cells in the central nervous system (CNS). We aimed at identifying heteroreceptor complexes formed by GPR18 and CB1R or CB2R in resting and activated microglia. Receptor-receptor interaction was assessed using energy-transfer approaches, and receptor function by determining cAMP levels and ERK1/2 phosphorylation in heterologous cells and primary cultures of microglia. Heteroreceptor identification in primary cultures of microglia was achieved by in situ proximity ligation assays. Energy transfer results showed interaction of GPR18 with CB2R but not with CB1R. CB2-GPR18 heteroreceptor complexes displayed particular functional properties (heteromer prints) often consisting of negative cross-talk (activation of one receptor reduces signaling arising from the partner receptor) and cross-antagonism (the response of one of the receptors is blocked by a selective antagonist of the partner receptor). Activated microglia showed the heteromer print (negative cross-talk and bidirectional cross-antagonism) and increased expression of CB2R and GPR18. Due to the important role of CB2R in neuroprotection, we further investigated heteroreceptor occurrence in primary cultures of microglia from transgenic mice overexpressing human APPSw,Ind, an Alzheimer's disease model. Microglial cells from transgenic mice showed the heteromer print and functional interactions that were similar to those found in cells from wild-type animals that were activated by treatment with lipopolysaccharide and interferon-γ. Our results suggest that GPR18 and its heteromers may play important roles in neurodegenerative processes.

Evaluation of antidepressant-like and anxiolytic-like activity of purinedione-derivatives with affinity for adenosine A2A receptors in mice.

BACKGROUND: It has recently been suggested that the adenosine A2A receptor plays a role in several animal models of depression. Additionally, A2A antagonists have reversed behavioral deficits and exhibited a profile similar to classical antidepressants. METHODS: In the present study, imidazo- and pyrimido[2,1-f]purinedione derivatives (KD 66, KD 167, KD 206) with affinity to A2A receptors but poor A1 affinity were evaluated for their antidepressant- and anxiolytic-like activity. The activity of these derivatives was tested using a tail suspension and forced swim test, two widely-used behavioral paradigms for the evaluation of antidepressant-like activity. In turn, the anxiolytic activity was evaluated using the four-plate test. RESULTS: The results showed the antidepressant-like activity of pyrimido- and imidazopurinedione derivatives (i.e. KD 66, KD 167 and KD 206) in acute and chronic behavioral tests in mice. KD 66 revealed an anxiolytic-like effect, while KD 167 increased anxiety behaviors. KD 206 had no effect on anxiety. Furthermore, none of the tested compounds increased locomotor activity. CONCLUSION: Available data support the proposition that the examined compounds with adenosine A2A receptor affinity may be an interesting target for the development of antidepressant and/or anxiolytic agents.

Tuberculosis in Antelopes in a Zoo in Poland--Problem of Public Health.

Bovine tuberculosis is an infectious disease that occurs in many species of both domestic and wild animals, as well as those held in captivity. The etiological factor is the acid resistant bacillus (Mycobacterium bovis or Mycobacterium caprae), which is characterized by the major pathogenicity among mycobacteria belonging to the Mycobacterium tuberculosis complex. The material from 8 antelopes from the zoo, suspected for tuberculosis were examined, and M. bovis strains were isolated from 6 of them. The spoligotyping method showing spoligo pattern 676763777777600. In Poland, this spoligotype has not been observed so far.