Design, synthesis and evaluation of reversible and irreversible monoacylglycerol lipase PET tracers using a ‘tail-switching’ strategy on a piperazinyl azetidine skeleton

Objectives: Selective inhibition of monoacylglycerol lipase (MAGL) represents a novel therapeutic approach for CNS disorders. Reported MAGL PET tracers suffered from relative high lipophilicity, which is often linked with fast metabolic clearance, poor in vivo stability, and high propensity for off-target promiscuity. Furthermore, the binding mechanism was confined to be irreversible. The objective of this work was to develop both irreversible and reversible MAGL PET tracers with favorable lipophilicity and pharmacokinetics using a ‘tail switching’ strategy on a piperazinyl azetidine skeleton, which demonstrated high binding specificity in vitro and in vivo.

Methods: A focused library of piperazinyl azetidine derived carbamates/ureas 1-6 as irreversible MAGL inhibitor candidates and piperazinyl azetidine derived amides 7-16, as reversible MAGL inhibitor candidates were synthesized (Figs. 1A&1B) and subjected to pharmacology evaluation, docking studies and physiochemical evaluation. The most promising irreversible inhibitor 1 and reversible inhibitors 8 & 15 were identified by [³H]2-OG binding and activity-based protein profiling (ABPP) assays, and further radiolabeled with diverse methods, including [¹¹C]COCl_2, [¹¹C]CO₂ fixation, ¹¹C-methylation and ¹⁸F-fluorination. Dynamic PET studies were carried out in Sprague-Dawley rats for 90 min in a Siemens Inveon scanner. The uptake/washout, biodistribution and binding specificity of these radioligands were investigated.

Results: Irreversible MAGL inhibitor 1 (IC₅₀ = 0.88 nM) and reversible inhibitors 8 (IC₅₀ = 2.7 nM) & 15 (IC₅₀ = 11.7 nM) were selected for further radiotracer development based on [³H]2-OG and ABPP binding assays (Figs. 1C-1F). Possible molecular interactions (H-bonding and π-π stacking interactions) between these lead compounds and MAGL, and the corresponding binding domain were also identified through molecular docking studies (Fig. 1G). Irreversible lead compound [¹¹C]1 were radio-synthesized in average 18% and 2.5% RCYs (decay-corrected) utilizing [¹¹C]COCl₂ and [¹¹C]CO₂ fixation, respectively. Reversible lead compounds [¹¹C]8 and [¹⁸F]15 were isolated in average 25% and 39% RCYs (decay-corrected) utilizing ¹¹C-methylation and ¹⁸F-fluorination, respectively (Fig. 1H). For all radioligands, high radiochemical purity (>99%) and molar activity (>30 GBq/μmol) were achieved, and no signs of radiolysis were detected up to 90 min. [¹¹C]1 readily penetrated the blood-brain-barrier and exhibited heterogeneous distribution (Fig. 1I). Pretreatment of KML29 (3 mg/kg) significantly decreased the uptake of [¹¹C]1 in the whole brain as well as various brain regions, suggesting excellent in vivo binding specificity (Fig. 1J). Despite limited brain uptake (Figs. 1K-1L), the reversible radioligands [¹¹C]8 and [¹⁸F]15 also exhibited excellent binding specificity towards MAGL in the periphery as depicted by ex vivo blocking studies (Fig. 1M). PET imaging studies of [¹¹C]8 and [¹⁸F]15 in Pgp/Bcrp knockout mice indicated that these two radioligands have intensive interactions with ABC efflux transporters, which to some extent explains the reason of their limited brain accumulation (Figs. 1N-1O). Whole body biodistribution studies suggested urinary and hepatobiliary elimination for these three radioligands. Radiometabolite analysis demonstrated excellent stability of [¹¹C]1 in rat brain and reasonable stability of [¹¹C]8 & [¹⁸F]15 in rat blood samples.

Conclusions: We have developed both irreversible and reversible PET tracers utilizing a ‘tail switching’ strategy on a piperazinyl azetidine skeleton. [¹¹C]1 was identified as a potent and promising irreversible PET probe with favorable lipophilicity and pharmacokinetics for neuroimaging of MAGL. The studies of ligands [¹¹C]8 & [¹⁸F]15 may pave the way for reversible MAGL PET probe development.

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