Synthesis of P2Y₁₂ receptor antagonists as therapeutics and diagnostics

Abstract

The P2Y₁₂ receptor (P2Y₁₂R), a G protein-coupled receptor activated by adenosine diphosphate (ADP), has traditionally been associated with platelet aggregation and targeted for antithrombotic therapy. More recently, its expression on microglial cells in the central nervous system (CNS) has attracted considerable interest, as it plays a central role in neuroinflammatory processes and represents a promising biomarker for neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and epilepsy. Targeting microglial P2Y₁₂Rs enables modulation of its function and offers an avenue for imaging neuroinflammatory processes. In this context, the present dissertation focuses on the design, synthesis, characterization, and evaluation of novel P2Y₁₂R antagonists, with particular emphasis on radiolabeled ligands, and blood-brain barrier (BBB)-permeable compounds for both therapeutic and diagnostic applications. <br/> The first part of this work explores structural modifications of anthraquinone derivatives guided by molecular docking studies performed on sodium 1-amino-9,10-dioxo-4-((4-(phenylamino)-3-sulfonatophenyl)amino)-9,10-dihydroanthracene-2-sulfonate (<strong>PSB-0739</strong>), a potent and selective P2Y₁₂R antagonist. These studies highlighted synthetic strategies to enable substitution at the C6- and C7-positions of the anthraquinone core and to modify the aniline moiety, followed by the synthesis of the targeted final compounds via Ullmann coupling. This systematic structure-activity relationship (SAR) study provided new insights into the chemical space surrounding the anthraquinone scaffold within the binding pocket and led to the synthesis of novel P2Y₁₂R antagonist radioligand precursors as a basis for the development of radioligands. <br/> In the second part, efforts were directed towards the development of radiolabeled anthraquinone-based P2Y₁₂R antagonists. Radioligands hold promise as diagnostic tools for non-invasive imaging of microglial activation, thereby supporting early detection and monitoring of neurodegenerative disorders. However, the chemical instability of the 2-sulfo- and 2-carboxyanthraquinone derivatives, particularly the release of the C2-sulfonate or carboxylic acid groups under palladium-catalyzed hydrogenation conditions, has limited their use as indicated by three synthetic trials starting with three different precursors. To address this challenge, a model compound was developed and investigated to optimize key reaction parameters, including catalyst type, solvent system, and hydrogen pressure. These studies identified suitable conditions, thus laying the foundation for the preparation of radioligands suitable for imaging applications. <br/> The third part of this dissertation shifts focus from anthraquinones to a systematic evaluation of published P2Y₁₂R antagonists with regard to their suitability as CNS-permeable lead structures. Since microglial P2Y₁₂Rs provide an attractive marker for imaging neuroinflammation through positron emission tomography (PET), identifying scaffolds with favorable BBB-penetration is of high relevance. A comprehensive review of available antagonists was performed, combining physicochemical property analysis with <em>in silico</em> CNS penetration filters and SAR assessment. This approach identified a pyrazolyl-phenyl-carbamoyl-indole scaffold as particularly promising. Within this series, substitution patterns were defined that improve potency and hold potential of CNS bioavailability. These findings provide a roadmap for the rational design of novel P2Y₁₂R antagonists optimized for CNS applications. <br/> Building on these evaluations, the fourth part of the work describes the development and pharmacological characterization of [³H]PSB-22219, a novel non-nucleotidic P2Y₁₂R antagonist radioligand. The unlabeled compound demonstrated high metabolic stability in rat liver microsomes and selectivity over other ADP-activated receptors. [³H]PSB-22219 exhibited high-affinity binding to human P2Y₁₂Rs expressed in recombinant cell systems (<em>K</em>_D = 4.57 nM) with very low nonspecific binding. Importantly, radioligand binding assays confirmed nanomolar affinity to native receptors in human platelets (<em>K</em>_D = 2.53 nM), rat brain cortex (<em>K</em>_D = 5.35 nM), and mouse microglia (<em>K</em>_D = 269 nM), with microglia displaying exceptionally high receptor density. Autoradiography studies further demonstrated visualization of human P2Y₁₂R expression in the brain of a humanized rat model. Together, these results establish [³H]PSB-22219 as a valuable pharmacological tool for probing P2Y₁₂R biology and as a promising candidate for radiodiagnostic development. <br/> Finally, the lead structure [³H]PSB-22219 was taken as a starting point for the design, synthesis, and establishment of structure-property relationship (SPR) studies to identify novel, potent, selective, and metabolically stable P2Y₁₂R antagonists with favorable permeability properties. The identified candidates present opportunities for direct application as P2Y₁₂-targeted therapeutics as well as for chemical radiolabeling to enable CNS imaging.