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.

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.

A coiled-coil-based design strategy for the thermostabilization of G-protein-coupled receptors.

Structure elucidation of inactive-state GPCRs still mostly relies on X-ray crystallography. The major goal of our work was to create a new GPCR tool that would provide receptor stability and additional soluble surface for crystallization. Towards this aim, we selected the two-stranded antiparallel coiled coil as a domain fold that satisfies both criteria. A selection of antiparallel coiled coils was used for structure-guided substitution of intracellular loop 3 of the ß3 adrenergic receptor. Unexpectedly, only the two GPCR variants containing thermostable coiled coils were expressed. We showed that one GPCR chimera is stable upon purification in detergent, retains ligand-binding properties, and can be crystallized. However, the quality of the crystals was not suitable for structure determination. By using two other examples, 5HTR2C and α2BAR, we demonstrate that our approach is generally suitable for the stabilization of GPCRs. To provide additional surface for promoting crystal contacts, we replaced in a structure-based approach the loop connecting the antiparallel coiled coil by T4L. We found that the engineered GPCR is even more stable than the coiled-coil variant. Negative-staining TEM revealed a homogeneous distribution of particles, indicating that coiled-coil-T4L receptor variants might also be promising candidate proteins for structure elucidation by cryo-EM. Our approach should be of interest for applications that benefit from stable GPCRs.

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.