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Monoacylglycerol lipase (MAGL) is a key enzyme involved in the metabolism of the endogenous signaling ligand 2-arachidonoylglycerol, a neuroprotective endocannabinoid intimately linked to central nervous system (CNS) disorders associated with neuroinflammation. In the quest for novel MAGL inhibitors, a focused screening approach on a Roche library subset provided a reversible benzoxazinone hit exhibiting high ligand efficiency. The subsequent design of the three-dimensional cis -hexahydro-pyrido-oxazinone ( cis -HHPO) moiety as benzoxazinone replacement enabled the combination of high MAGL potency with favorable ADME properties. Through enzymatic resolution an efficient synthetic route of the privileged cis -(4 R ,8 S ) HHPO headgroup was established, providing access to the highly potent and selective MAGL inhibitor 7o . Candidate molecule 7o matches the target compound profile of CNS drugs as it achieves high CSF exposures after systemic administration in rodents. It engages with the target in the brain and modulates neuroinflammatory processes, thus holding great promise for the treatment of CNS disorders.

The cannabinoid receptor type 1 (CB 1 R) is pivotal within the endocannabinoid system regulating various signaling cascades with effects in appetite regulation, pain perception, memory formation, and thermoregulation. Still, understanding of CB 1 R's cellular signaling, distribution, and expression dynamics is very fragmentary. Real-time visualization of CB 1 R is crucial for addressing these questions. Selective drug-like CB 1 R ligands with a defined pharmacological profile were investigated for the construction of CB 1 R fluorescent probes using a reverse design-approach. A modular design concept with a diethyl glycine-based building block as the centerpiece allowed for the straightforward synthesis of novel probe candidates. Validated by computational docking studies, radioligand binding, and cAMP assay, this systematic approach allowed for the identification of novel pyrrole-based CB 1 R fluorescent probes. Application in fluorescence-based target-engagement studies and live cell imaging exemplify the great versatility of the tailored CB 1 R probes for investigating CB 1 R localization, trafficking, pharmacology, and its pathological implications.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo , optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency ( K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P -glycoprotein-mediated efflux from the brain. 3 H and 14 C labeled RNB-61 showed apparent K d values of <4 nM toward human CB2R in both cell and tissue experiments. The 6,800-fold selectivity over CB1 receptors and negligible off-targets in vitro , combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands., Competing Interests: The authors declare the following competing financial interest(s): Christoph Ullmer, Antonello Caruso, Thomas Hartung, Roland Degen, Matthias Mller, Sabine Grner and Uwe Grether are employees and shareholders of F. Hoffmann-La Roche AG. The remaining authors declare no potential competing interests., (© 2024 The Authors. Published by American Chemical Society.)

New potent, selective monoacylglycerol lipase (MAGL) inhibitors based on the azetidin-2-one scaffold ((±)- 5a - v , (±)- 6a - j , and (±)- 7a - d ) were developed as irreversible ligands, as demonstrated by enzymatic and crystallographic studies for (±)- 5d , (±)- 5l , and (±)- 5r . X-ray analyses combined with extensive computational studies allowed us to clarify the binding mode of the compounds. 5v was identified as selective for MAGL when compared with other serine hydrolases. Solubility, in vitro metabolic stability, cytotoxicity, and absence of mutagenicity were determined for selected analogues. The most promising compounds ((±)- 5c , (±)- 5d , and (±)- 5v ) were used for in vivo studies in mice, showing a decrease in MAGL activity and increased 2-arachidonoyl- sn -glycerol levels in forebrain tissue. In particular, 5v is characterized by a high eudysmic ratio and (3 R ,4 S )- 5v is one of the most potent irreversible inhibitors of h / m MAGL identified thus far. These results suggest that the new MAGL inhibitors have therapeutic potential for different central and peripheral pathologies.

The cannabinoid type 2 receptor (CB2) has been implicated in a variety of central and peripheral inflammatory diseases, prompting significant interest in the development of CB2-targeted diagnostic and therapeutic agents. A validated positron emission tomography (PET) radioligand for imaging CB2 in the living human brain as well as in peripheral tissues is currently lacking. As part of our research program, we have recently identified the trisubstituted pyridine, [ 18 F]RoSMA-18-d 6 , which proved to be highly suitable for in vitro and in vivo mapping of CB2 in rodents. The aim of this study was to assess the performance characteristics of [ 18 F]RoSMA-18-d 6 in nonhuman primates (NHPs) to pave the way for clinical translation. [ 18 F]RoSMA-18-d 6 was synthesized from the respective tosylate precursor according to previously reported procedures. In vitro autoradiograms with NHP spleen tissue sections revealed a high binding of [ 18 F]RoSMA-18-d 6 to the CB2-rich NHP spleen, which was significantly blocked by coincubation with the commercially available CB2 ligand, GW405833 (10 μM). In contrast, no specific binding was observed by in vitro autoradiography with NHP brain sections, which was in agreement with the notion of a CB2-deficient healthy mammalian brain. In vitro findings were corroborated by PET imaging experiments in NHPs, where [ 18 F]RoSMA-18-d 6 uptake in the spleen was dose-dependently attenuated with 1 and 5 mg/kg GW405833, while no specific brain signal was observed. Remarkably, we observed tracer uptake and retention in the NHP spinal cord, which was reduced by GW405833 blockade, pointing toward a potential utility of [ 18 F]RoSMA-18-d 6 in probing CB2-expressing cells in the bone marrow. If these observations are substantiated in NHP models of enhanced leukocyte proliferation in the bone marrow, [ 18 F]RoSMA-18-d 6 may serve as a valuable marker for hematopoietic activity in various pathologies. In conclusion, [ 18 F]RoSMA-18-d 6 proved to be a suitable PET radioligand for imaging CB2 in NHPs, supporting its translation to humans.

Pharmacological modulation of cannabinoid receptor type 2 (CB 2 R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB 2 R, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CB 2 R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB 2 R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB 2 R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB 2 R by exploiting fluorogenic O -nitrobenzoxadiazole ( O- NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O- NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N -sulfonyl pyridone ( N -SP) and N -acyl- N -alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB 2 R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.

Monoacylglycerol lipase (MAGL) is one of the key enzymes in the endocannabinoid system. Inhibition of MAGL has been proposed as an attractive approach for the treatment of various diseases. In this study, we designed and successfully synthesized two series of piperazinyl pyrrolidin-2-one derivatives as novel reversible MAGL inhibitors. ( R )-[ 18 F] 13 was identified through the preliminary evaluation of two carbon-11-labeled racemic structures [ 11 C] 11 and [ 11 C] 16 . In dynamic positron-emission tomography (PET) scans, ( R )-[ 18 F] 13 showed a heterogeneous distribution and matched the MAGL expression pattern in the mouse brain. High brain uptake and brain-to-blood ratio were achieved by ( R )-[ 18 F] 13 in comparison with previously reported reversible MAGL PET radiotracers. Target occupancy studies with a therapeutic MAGL inhibitor revealed a dose-dependent reduction of ( R )-[ 18 F] 13 accumulation in the mouse brain. These findings indicate that ( R )-[ 18 F] 13 ([ 18 F]YH149) is a highly promising PET probe for visualizing MAGL non-invasively in vivo and holds great potential to support drug development.

Neuroinflammation is an adaptive response of the central nervous system to diverse potentially injurious stimuli, which is closely associated with neurodegeneration and typically characterized by activation of microglia and astrocytes. As a noninvasive and translational molecular imaging tool, positron emission tomography (PET) could provide a better understanding of neuroinflammation and its role in neurodegenerative diseases. Ligands to translator protein (TSPO), a putative marker of neuroinflammation, have been the most commonly studied in this context, but they suffer from serious limitations. Herein we present a repertoire of different structural chemotypes and novel PET ligand design for classical and emerging neuroinflammatory targets beyond TSPO. We believe that this Perspective will support multidisciplinary collaborations in academic and industrial institutions working on neuroinflammation and facilitate the progress of neuroinflammation PET probe development for clinical use.

The utilization of an activatable, substrate-based probe design in combination with a cellular targeting approach has been rarely explored for cancer imaging on a small-molecule basis, although such probes could benefit from advantages of both concepts. Cysteine proteases like cathepsin S are known to be involved in fundamental processes associated with tumor development and progression and thus are valuable cancer markers. We report the development of a combined dual functional DOTAM-based, RGD-targeted internally quenched fluorescent probe that is activated by cathepsin S. The probe exhibits excellent in vitro activation kinetics which can be fully translated to human cancer cell lines. We demonstrate that the targeted, activatable probe is superior to its nontargeted analog, exhibiting improved uptake into α ν β 3 -integrin expressing human sarcoma cells (HT1080) and significantly higher resultant fluorescence staining. However, profound activation was also found in cancer cells with a lower integrin expression level, whereas in healthy cells almost no probe activation could be observed, highlighting the high selectivity of our probe toward cancer cells. These auspicious results show the outstanding potential of the dual functionality concept combining a substrate-based probe design with a targeting approach, which could form the basis for highly sensitive and selective in vivo imaging probes.

Pharmacological modulation of cannabinoid type 2 receptor (CB 2 R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB 2 R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB 2 R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB 2 R specificity was demonstrated by competition experiments in living cells expressing CB 2 R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.

Despite the broad implications of the cannabinoid type 2 receptor (CB2) in neuroinflammatory processes, a suitable CB2-targeted probe is currently lacking in clinical routine. In this work, we synthesized 15 fluorinated pyridine derivatives and tested their binding affinities toward CB2 and CB1. With a sub-nanomolar affinity ( K i for CB2) of 0.8 nM and a remarkable selectivity factor of >12,000 over CB1, RoSMA-18- d 6 exhibited outstanding in vitro performance characteristics and was radiofluorinated with an average radiochemical yield of 10.6 ± 3.8% ( n = 16) and molar activities ranging from 52 to 65 GBq/μmol (radiochemical purity > 99%). [ 18 F]RoSMA-18- d 6 showed exceptional CB2 attributes as demonstrated by in vitro autoradiography, ex vivo biodistribution, and positron emission tomography (PET). Further, [ 18 F]RoSMA-18- d 6 was used to detect CB2 upregulation on postmortem human ALS spinal cord tissues. Overall, these results suggest that [ 18 F]RoSMA-18- d 6 is a promising CB2 PET radioligand for clinical translation.

The cannabinoid type 2 (CB2) receptor has emerged as a valuable target for therapy and imaging of immune-mediated pathologies. With the aim to find a suitable radiofluorinated analogue of the previously reported CB2 positron emission tomography (PET) radioligand [ 11 C]RSR-056, 38 fluorinated derivatives were synthesized and tested by in vitro binding assays. With a K i (hCB2) of 6 nM and a selectivity factor of nearly 700 over cannabinoid type 1 receptors, target compound 3 exhibited optimal in vitro properties and was selected for evaluation as a PET radioligand. [ 18 F] 3 was obtained in an average radiochemical yield of 11 ± 4% and molar activities between 33 and 114 GBq/μmol. Specific binding of [ 18 F] 3 to CB2 was demonstrated by in vitro autoradiography and in vivo PET experiments using the CB2 ligand GW-405 833. Metabolite analysis revealed only intact [ 18 F] 3 in the rat brain. [ 18 F] 3 detected CB2 upregulation in human amyotrophic lateral sclerosis spinal cord tissue and may thus become a candidate for diagnostic use in humans.

Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB 2 R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB 2 R-selective bifunctional probe that covalently captures CB 2 R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB 2 R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.

Upregulation of the cannabinoid type 2 receptors (CB 2 R) unveils inflammation processes of pathological disorders, such as cancer, pain, or neurodegenerative diseases. Among others, CB 2 R agonist A-836339 has been labeled with carbon-11 for PET imaging of the CB 2 R and displayed promising results in a mouse model of Alzheimer's disease. The aim of the present work was to develop fluorinated analogs of A-836339 for labeling with fluorine-18 to design a new PET tracer for CB 2 R imaging. Seven fluorinated analogs of A-836339 were synthesized in two to three steps and their binding affinities and selectivities for both the human and the mouse CB 2 R were measured as well as their early ADME profiles. Among them, compound 2f (K ihCB2R = 0.1 nM, K ihCB1R /K ihCB2R = 300) displayed high affinity and selectivity for CB 2 R but also promising lipophilicity, kinetic solubility, and membrane permeation properties and was further selected for in vitro metabolism studies. Incubation of 2f with human or rat liver microsomes followed by LC/MS analysis revealed the presence of six different metabolites mainly resulting from oxidation reactions. A tosylated precursor of 2f was synthesized in two steps and radiolabeled with fluorine-18 to afford [ 18 F]2f in 15 ± 5% radiochemical yield and a molar activity of 110 ± 30 GBq/μmol. Autoradiographies of rat spleen and biodistribution studies in healthy rats including pretreatments with either CB 2 R or CB 1 R-specific compounds suggested that [ 18 F]2f is a specific tracer for the CB 2 R in vivo. We have therefore demonstrated here that [ 18 F]2f is a promising novel tracer for imaging CB 2 R in vivo using PET. Further investigation in animal models of inflammation will follow.

As part of our efforts to develop CB2 PET imaging agents, we investigated 2,5,6-substituted pyridines as a novel class of potential CB2 PET ligands. A total of 21 novel compounds were designed, synthesized, and evaluated for their potency and binding properties toward human and rodent CB1 and CB2. The most promising ligand 6a was radiolabeled with carbon-11 to yield 16 ([(11)C]RSR-056). Specific binding of 16 to CB2-positive spleen tissue of rats and mice was demonstrated by in vitro autogadiography and verified in vivo in PET and biodistribution experiments. Furthermore, 16 was evaluated in a lipopolysaccharid (LPS) induced murine model of neuroinflammation. Brain radioactivity was strikingly higher in the LPS-treated mice than the control mice. Compound 16 is a promising radiotracer for imaging CB2 in rodents. It might serve as a tool for the investigation of CB2 receptor expression levels in healthy tissues and different neuroinflammatory disorders in humans.