TAK-994, a Novel Orally Available Brain-Penetrant Orexin 2 Receptor-Selective Agonist, Suppresses Fragmentation of Wakefulness and Cataplexy-Like Episodes in Mouse Models of Narcolepsy.

Loss of orexin neurons is associated with narcolepsy type 1 (NT1), which is characterized by multiple symptoms including excessive daytime sleepiness and cataplexy. Orexin 2 receptor (OX2R) knockout (KO) mice, but not orexin 1 receptor (OX1R) KO mice, show narcolepsy-like phenotypes, thus OX2R agonists are potentially promising for treating NT1. In fact, in early proof-of-concept studies, intravenous infusion of danavorexton, an OX2R-selective agonist, significantly increased wakefulness in individuals with NT1. However, danavorexton has limited oral availability. Here, we report pharmacological characteristics of a novel OX2R agonist, TAK-994 [N-{(2S,3S)-1-(2-hydroxy-2-methylpropanoyl)-2-[(2,3',5'-trifluorobiphenyl-3-yl)methyl]pyrrolidin-3-yl}methanesulfonamide sesquihydrate]. TAK-994 activated recombinant human OX2R (EC50 value of 19 nM) with > 700-fold selectivity against OX1R and activated OX2R-downstream signaling similar to those by orexin peptides in vitro. Oral administration of TAK-994 promoted wakefulness in normal mice but not in OX2R KO mice. TAK-994 also ameliorated narcolepsy-like symptoms in two mouse models of narcolepsy: orexin/ataxin-3 mice and orexin-tTA;TetO diphtheria toxin A mice. The wake-promoting effects of TAK-994 in orexin/ataxin-3 mice were maintained after chronic dosing for 14 days. These data suggest that overall in vitro and in vivo properties, except oral availability, are very similar between TAK-994 and danavorexton. Preclinical characteristics of TAK-994 shown here, together with upcoming clinical study results, can improve our understanding for orally available OX2R agonists as new therapeutic drugs for NT1 and other hypersomnia disorders. SIGNIFICANCE STATEMENT: Narcolepsy type 1 (NT1) is caused by a loss of orexin neurons, and thus an orexin 2 receptor (OX2R) agonist is considered to address the underlying pathophysiology of NT1. Oral administration of TAK-994, a novel OX2R agonist, promoted wakefulness in normal mice, but not in OX2R knockout mice, and ameliorated fragmentation of wakefulness and cataplexy-like episodes in mouse models of narcolepsy. These findings indicate that TAK-994 is an orally available brain-penetrant OX2R-selective agonist with potential to improve narcolepsy-like symptoms.

Deep Learning-Based Seminal Vesicle and Vas Deferens Recognition in the Posterior Approach of Robot-Assisted Radical Prostatectomy.

OBJECTIVE: To develop a convolutional neural network to recognize the seminal vesicle and vas deferens (SV-VD) in the posterior approach of robot-assisted radical prostatectomy (RARP) and assess the performance of the convolutional neural network model under clinically relevant conditions. METHODS: Intraoperative videos of robot-assisted radical prostatectomy performed by the posterior approach from 3 institutions were obtained between 2019 and 2020. Using SV-VD dissection videos, semantic segmentation of the seminal vesicle-vas deferens area was performed using a convolutional neural network-based approach. The dataset was split into training and test data in a 10:3 ratio. The average time required by 6 novice urologists to correctly recognize the SV-VD was compared using intraoperative videos with and without segmentation masks generated by the convolutional neural network model, which was evaluated with the test data using the Dice similarity coefficient. Training and test datasets were compared using the Mann-Whitney U-test and chi-square test. Time required to recognize the SV-VD was evaluated using the Mann-Whitney U-test. RESULTS: From 26 patient videos, 1 040 images were created (520 SV-VD annotated images and 520 SV-VD non-displayed images). The convolutional neural network model had a Dice similarity coefficient value of 0.73 in the test data. Compared with original videos, videos with the generated segmentation mask promoted significantly faster seminal vesicle and vas deferens recognition (P < .001). CONCLUSION: The convolutional neural network model provides accurate recognition of the SV-VD in the posterior approach RARP, which may be helpful, especially for novice urologists.

Deep learning for hetero-homo conversion in channel-domain for phase aberration correction in ultrasound imaging.

Echo imaging in ultrasound computed tomography (USCT) using the synthetic aperture technique is performed with the assumption that the speed of sound is constant in the system. However, tissue heterogeneity causes a mismatch between the predicted arrival time and the actual arrival time of the echo signal, which will result in phase aberration, leading to the quality degradation of the reconstructed B-mode image. The conventional correction methods that use the correlation of each different channel require the presence of strong point scatterers and involve the problem of local solutions due to excessive correction. In this study, we propose a novel approach to correcting the signal distortion due to sound speed heterogeneity using a deep neural network (DNN). The DNN was trained to convert the distorted radio frequency (RF) inputs for the heterogeneous medium to the distortion-free RF outputs for the homogeneous medium. The network with U-net architecture using ResNet-34 as a backbone was trained using the hetero-homo corresponding channel-domain RF data generated via numerical simulations. The trained network performed phase aberration correction in the channel-domain RF, with the B-mode images reconstructed with the corrected RF demonstrating a higher contrast and an improved resolution compared with uncorrected cases. It was also demonstrated that the DNN model is robust to both varied reflection intensities and varied sound speed heterogeneities. The successful results demonstrated that the proposed DNN-based method is effective for phase aberration correction in US imaging.

Design and synthesis of aryl-piperidine derivatives as potent and selective PET tracers for cholesterol 24-hydroxylase (CH24H).

Cholesterol 24-hydroxylase (CH24H, CYP46A1) is a cytochrome P450 family enzyme that maintains the homeostasis of brain cholesterol. Soticlestat, a potent and selective CH24H inhibitor, is in development as a therapeutic agent for Dravet syndrome and Lennox-Gastaut syndrome. Herein, we report the discovery of aryl-piperidine derivatives as potent and selective CH24H positron emission tomography (PET) tracers which can be used for dose guidance of a clinical CH24H inhibitor and as a diagnostic tool for CH24H-related pathology. Starting from compound 1 (IC50 = 16 nM, logD = 1.7), which was reported as a CH24H inhibitor with lower lipophilicity, a18F-labeling site (3-fluoroazetidine) was incorporated by structure-based drug design (SBDD) utilizing the co-crystal structure of a compound 1 analog. Subsequent optimization to adjust key parameters for PET tracers, such as potency, lipophilicity, brain penetration, and unbound plasma protein binding, enabled compounds 3f (IC50 = 8.8 nM) and 3g (IC50 = 8.7 nM) as PET imaging candidates. Selectivity of these compounds for CH24H was validated by a brain distribution study using CH24H-WT and KO mice. In non-human primate PET imaging, [18F]3f and [18F]3g showed similar regional uptake in the brain, indicating that these tracers were specific to the CH24H-expressed regions and validated the expression of CH24H in the living brain by different tracers.

Molecular mechanism of the wake-promoting agent TAK-925.

The OX2 orexin receptor (OX2R) is a highly expressed G protein-coupled receptor (GPCR) in the brain that regulates wakefulness and circadian rhythms in humans. Antagonism of OX2R is a proven therapeutic strategy for insomnia drugs, and agonism of OX2R is a potentially powerful approach for narcolepsy type 1, which is characterized by the death of orexinergic neurons. Until recently, agonism of OX2R had been considered 'undruggable.' We harness cryo-electron microscopy of OX2R-G protein complexes to determine how the first clinically tested OX2R agonist TAK-925 can activate OX2R in a highly selective manner. Two structures of TAK-925-bound OX2R with either a Gq mimetic or Gi reveal that TAK-925 binds at the same site occupied by antagonists, yet interacts with the transmembrane helices to trigger activating microswitches. Our structural and mutagenesis data show that TAK-925's selectivity is mediated by subtle differences between OX1 and OX2 receptor subtypes at the orthosteric pocket. Finally, differences in the polarity of interactions at the G protein binding interfaces help to rationalize OX2R's coupling selectivity for Gq signaling. The mechanisms of TAK-925's binding, activation, and selectivity presented herein will aid in understanding the efficacy of small molecule OX2R agonists for narcolepsy and other circadian disorders.

Discovery of Novel 3-Piperidinyl Pyridine Derivatives as Highly Potent and Selective Cholesterol 24-Hydroxylase (CH24H) Inhibitors.

Cholesterol 24-hydroxylase (CH24H or CYP46A1) is a brain-specific cytochrome P450 enzyme that metabolizes cholesterol into 24S-hydroxycholesterol (24HC) for regulating brain cholesterol homeostasis. For the development of a novel and potent CH24H inhibitor, we designed and synthesized 3,4-disubstituted pyridine derivatives using a structure-based drug design approach starting from compounds 1 (soticlestat) and 2 (thioperamide). Optimization of this series by focusing on ligand-lipophilicity efficiency value resulted in the discovery of 4-(4-methyl-1-pyrazolyl)pyridine derivative 17 (IC50 = 8.5 nM) as a potent and highly selective CH24H inhibitor. The X-ray crystal structure of CH24H in complex with compound 17 revealed a unique binding mode. Both blood-brain barrier penetration and reduction of 24HC levels (26% reduction) in the mouse brain were confirmed by oral administration of 17 at 30 mg/kg, indicating that 17 is a promising tool for the novel and selective inhibition of CH24H.

Quantification of monoacylglycerol lipase and its occupancy by an exogenous ligand in rhesus monkey brains using [18F]T-401 and PET.

Monoacylglycerol lipase (MAGL) is a cytosolic serine hydrolase that cleaves monoacylglycerols into fatty acids and is a potential target for the novel treatment of CNS disorders related to the endocannabinoid system and neuroinflammation. We have developed [18F]T-401 as a selective Positron emission tomography (PET) imaging agent for MAGL. In this study, we determined an analytical method to quantify MAGL availability and its occupancy by an exogenous inhibitor in rhesus monkey brains using [18F]T-401-PET. In rhesus monkeys, regional time-activity curves were described well when using an extended 2-tissue compartment model that accommodated the formation of a radiometabolite in the brain. This model yielded reliable estimates of the total distribution volume (VT), and the rank order of VT was consistent with known regional activity of MAGL enzyme in primates. The pretreatment of monkeys with JW642 resulted in a dose-dependent reduction of [18F]T-401 retentions in the brain, and VT. Lassen's graphical analysis indicated a VND of 0.69 mL/cm3 and a plasma JW642 concentration of 126 ng/mL for inhibiting the specific binding by 50%. [18F]T-401 and the method established can be used for quantification of MAGL in healthy brain and in disease conditions, and is suitable for evaluations of target engagement at cerebral MAGL.

Preclinical characterization of [18F]T-008, a novel PET imaging radioligand for cholesterol 24-hydroxylase.

PURPOSE: Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that plays a major role in brain cholesterol homeostasis by converting cholesterol into 24S-hydroxycholesterol. The selective CH24H inhibitor soticlestat (TAK-935) is being pursued as a drug for treatment of seizures in developmental and epileptic encephalopathies. Herein, we describe the successful discovery and the preclinical validation of the novel radiolabeled CH24H ligand (3-[18F]fluoroazetidin-1-yl){1-[4-(4-fluorophenyl)pyrimidin-5-yl]piperidin-4-yl}methanone ([18F]T-008) and its tritiated analog, [3H]T-008. METHODS: In vitro autoradiography (ARG) studies in the CH24H wild-type (WT) and knockout (KO) mouse brain sections were conducted using [3H]T-008. PET imaging was conducted in two adult rhesus macaques using [18F]T-008. Each macaque received two test-retest baseline scans and a series of two blocking doses of soticlestat administered prior to [18F]T-008 to determine the CH24H enzyme occupancy. PET data were analyzed with Logan graphical analysis using plasma input. A Lassen plot was applied to estimate CH24H enzyme occupancy by soticlestat. RESULTS: In ARG studies, binding of [3H]T-008 was specific to CH24H in the mouse brain sections, which was not observed in CH24H KO or in wild-type mice after pretreatment with soticlestat. In rhesus PET studies, the rank order of [18F]T-008 uptake was striatum > cortical regions > cerebellum, which was consistent with CH24H distribution in the brain. Pre-blocking with soticlestat reduced the maximum uptake and increased the washout in all brain regions in a dose-dependent manner. Calculated global occupancy values for soticlestat at a dose of 0.89 mg/kg were 97-98%, indicating maximum occupancy. CONCLUSION: The preclinical in vitro and in vivo evaluation of labeled T-008 demonstrates that [18F]T-008 is suitable for imaging CH24H in the brain and warrants further studies in humans.

Discovery of Soticlestat, a Potent and Selective Inhibitor for Cholesterol 24-Hydroxylase (CH24H).

Cholesterol 24-hydroxylase (CH24H, CYP46A1), a brain-specific cytochrome P450 (CYP) family enzyme, plays a role in the homeostasis of brain cholesterol by converting cholesterol to 24S-hydroxycholesterol (24HC). Despite a wide range of potential of CH24H as a drug target, no potent and selective inhibitors have been identified. Here, we report on the structure-based drug design (SBDD) of novel 4-arylpyridine derivatives based on the X-ray co-crystal structure of hit derivative 1b. Optimization of 4-arylpyridine derivatives led us to identify 3v ((4-benzyl-4-hydroxypiperidin-1-yl)(2,4'-bipyridin-3-yl)methanone, IC50 = 7.4 nM) as a highly potent, selective, and brain-penetrant CH24H inhibitor. Following oral administration to mice, 3v resulted in a dose-dependent reduction of 24HC levels in the brain (1, 3, and 10 mg/kg). Compound 3v (soticlestat, also known as TAK-935) is currently under clinical investigation for the treatment of Dravet syndrome and Lennox-Gastaut syndrome as a novel drug class for epilepsies.

Design, Synthesis, and Evaluation of 11C-Labeled 3-Acetyl-Indole Derivatives as a Novel Positron Emission Tomography Imaging Agent for Diacylglycerol Kinase Gamma (DGKγ) in Brain.

Diacylglycerol kinase gamma (DGKγ) is a subtype of DGK enzyme, which catalyzes ATP-dependent conversion of diacylglycerol to phosphatidic acid. DGKγ, localized in the brain, plays an important role in the central nervous system. However, its function has not been widely investigated. Positron emission tomography (PET) imaging of DGKγ validates target engagement of therapeutic DGKγ inhibitors and investigates DGKγ levels under normal and disease conditions. In this study, we designed and synthesized a series of 3-acetyl indole derivatives as candidates for PET imaging agents for DGKγ. Among the synthesized compounds, 2-((3-acetyl-1-(6-methoxypyridin-3-yl)-2-methyl-1H-indol-5-yl)oxy)-N-methylacetamide (9) exhibited potent inhibitory activity (IC50 = 30 nM) against DGKγ and desirable physicochemical properties allowing efficient blood-brain barrier penetration and low levels of undesirable nonspecific binding. The radiolabeling of 9 followed by PET imaging of wild-type and DGKγ-deficient mice and rats indicated that [11C]9 ([11C]T-278) specifically binds to DGKγ and yields a high signal-to-noise ratio for DGKγ in rodent brains.

Design, Synthesis, and Evaluation of [18F]T-914 as a Novel Positron-Emission Tomography Tracer for Lysine-Specific Demethylase 1.

Histone methylation is associated with the pathophysiology of neurodevelopmental disorders. Lysine-specific demethylase 1 (LSD1) catalyzes histone demethylation in a flavin adenine dinucleotide (FAD)-dependent manner. Thus, inhibiting LSD1 enzyme activity could offer a novel way to treat neurodevelopmental disorders. Assessing LSD1 target engagement using positron-emission tomography (PET) imaging could aid in developing therapeutic LSD1 inhibitors. In this study, PET probes based on 4-(2-aminocyclopropyl)benzamide derivatives that bind irreversibly to FAD found in LSD1 were examined. By optimizing the profiles of brain penetrance and brain-penetrant metabolites, T-914 (1g) was identified as a suitable PET tracer candidate. PET studies in nonhuman primates demonstrated that [18F]1g had heterogeneous brain uptake, which corresponded to known LSD1 expression levels. Moreover, brain uptake of [18F]1g was reduced by coadministration of unlabeled 1g, demonstrating blockable binding. These data suggest that [18F]1g warrants further investigation as a potential PET tracer candidate for assessing target engagement of LSD1.

Discovery of a Potent and Orally Bioavailable Melatonin Receptor Agonist.

To develop potent and orally bioavailable melatonin receptor (MT1 and MT2) agonists, a novel series of 5-6-5 tricyclic derivatives was designed, synthesized, and evaluated. The synthesized indeno[5,4-d][1,3]oxazole, cyclopenta[c]pyrazolo[1,5-a]pyridine, indeno[5,4-d][1,3]thiazole, and cyclopenta[e]indazole derivatives showed potent binding affinities for MT1/MT2 receptors. Further optimization of these derivatives based on their metabolic stability in human hepatic microsomes revealed that (S)-3b ((S)-N-[2-(2-methyl-7,8-dihydro-6H-indeno[5,4-d][1,3]oxazol-8-yl)ethyl]acetamide) was a potent MT1 and MT2 ligand (MT1, Ki = 0.031 nM; MT2, Ki = 0.070 nM) with good metabolic stability in human hepatic microsomes. Moreover, compound (S)-3b showed good BBB permeability in rats, and its in vivo pharmacological effects were confirmed by its sleep-promotion ability in cats.

Design, Synthesis, and Evaluation of (2-Aminocyclopropyl)phenyl Derivatives as Novel Positron Emission Tomography Imaging Agents for Lysine-Specific Demethylase 1 in the Brain.

Dysregulation of histone H3 lysine 4 (H3K4) methylation is implicated in the pathogenesis of neurodevelopmental disorders. Lysine-specific demethylase 1 (LSD1) determines the methylation status of H3K4 through flavin adenine dinucleotide (FAD)-mediated histone demethylation. Therefore, LSD1 inhibition in the brain can be a novel therapeutic option for treating these disorders. Positron emission tomography (PET) imaging of LSD1 allows for investigating LSD1 expression levels under normal and disease conditions and validating target engagement of therapeutic LSD1 inhibitors. This study designed and synthesized (2-aminocyclopropyl)phenyl derivatives with irreversible binding to LSD1 as PET imaging agents for LSD1 in the brain. We optimized lipophilicity of the lead compound to minimize the risk of nonspecific binding and identified 1e with high selectivity over monoamine oxidase A and B, which are a family of FAD-dependent enzymes homologous to LSD1. PET imaging in a monkey showed a high uptake of [18F]1e to regions enriched with LSD1, indicating its specific binding to LSD1.

Discovery of a Novel and Brain-Penetrant O-GlcNAcase Inhibitor via Virtual Screening, Structure-Based Analysis, and Rational Lead Optimization.

O-GlcNAcase (OGA) has received increasing attention as an attractive therapeutic target for tau-mediated neurodegenerative disorders; however, its role in these pathologies remains unclear. Therefore, potent chemical tools with favorable pharmacokinetic profiles are desirable to characterize this enzyme. Herein, we report the discovery of a potent and novel OGA inhibitor, compound 5i, comprising an aminopyrimidine scaffold, identified by virtual screening based on multiple methodologies combining structure-based and ligand-based approaches, followed by sequential optimization with a focus on ligand lipophilicity efficiency. This compound was observed to increase the level of O-GlcNAcylated protein in cells and display suitable pharmacokinetic properties and brain permeability. Crystallographic analysis revealed that the chemical series bind to OGA via characteristic hydrophobic interactions, which resulted in a high affinity for OGA with moderate lipophilicity. Compound 5i could serve as a useful chemical probe to help establish a proof-of-concept of OGA inhibition as a therapeutic target for the treatment of tauopathies.

Synthesis of low-molecular weight fucoidan derivatives and their binding abilities to SARS-CoV-2 spike proteins.

Fucoidan derivatives 10-13, whose basic sugar chains are composed of repeating α(1,4)-linked l-fucopyranosyl residues with different sulfation patterns, were designed and systematically synthesized. A structure-activity relationship (SAR) study examined competitive inhibition by thirteen fucoidan derivatives against heparin binding to the SARS-CoV-2 spike (S) protein. The results showed for the first time that 10 exhibited the highest inhibitory activity of the fucoidan derivatives used. The inhibitory activity of 10 was much higher than that of fondaparinux, the reported ligand of SARS-CoV-2 S protein. Furthermore, 10 exhibited inhibitory activities against the binding of heparin with several mutant SARS-CoV-2 S proteins, but was found to not inhibit factor Xa (FXa) activity that could otherwise lead to undesirable anticoagulant activity.

Soticlestat, a novel cholesterol 24-hydroxylase inhibitor shows a therapeutic potential for neural hyperexcitation in mice.

Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that converts cholesterol into 24S-hydroxycholesterol, the primary mechanism of cholesterol catabolism in the brain. The therapeutic potential of CH24H activation has been extensively investigated, whereas the effects of CH24H inhibition remain poorly characterized. In this study, the therapeutic potential of CH24H inhibition was investigated using a newly identified small molecule, soticlestat (TAK-935/OV935). The biodistribution and target engagement of soticlestat was assessed in mice. CH24H-knockout mice showed a substantially lower level of soticlestat distribution in the brain than wild-type controls. Furthermore, brain-slice autoradiography studies demonstrated the absence of [3H]soticlestat staining in CH24H-knockout mice compared with wild-type mice, indicating a specificity of soticlestat binding to CH24H. The pharmacodynamic effects of soticlestat were characterized in a transgenic mouse model carrying mutated human amyloid precursor protein and presenilin 1 (APP/PS1-Tg). These mice, with excitatory/inhibitory imbalance and short life-span, yielded a remarkable survival benefit when bred with CH24H-knockout animals. Soticlestat lowered brain 24S-hydroxycholesterol in a dose-dependent manner and substantially reduced premature deaths of APP/PS1-Tg mice at a dose lowering brain 24S-hydroxycholesterol by approximately 50%. Furthermore, microdialysis experiments showed that soticlestat can suppress potassium-evoked extracellular glutamate elevations in the hippocampus. Taken together, these data suggest that soticlestat-mediated inhibition of CH24H may have therapeutic potential for diseases associated with neural hyperexcitation.

Discovery of novel 4-phenyl-2-(pyrrolidinyl)nicotinamide derivatives as potent Nav1.1 activators.

The voltage-gated sodium channel, Nav1.1, is predominantly expressed in parvalbumin-positive fast spiking interneurons and has been genetically linked to Dravet syndrome. Starting from a high throughput screening hit isoxazole derivative 5, modifications of 5 via combinations of IonWorks and Q-patch assays successfully identified the nicotinamide derivative 4. Its increasing decay time constant (tau) of Nav1.1 currents at 0.03 µM along with significant selectivity against Nav1.2, Nav1.5, and Nav1.6 and acceptable brain exposure in mice was observed. Compound 4 is a promising Nav1.1 activator that can be used to analyze pathophysiological functions of the Nav1.1 channel towards treating various central nervous system diseases.

Design, Synthesis, and Evaluation of (4 R)-1-{3-[2-(18F)Fluoro-4-methylpyridin-3-yl]phenyl}-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one ([18F]T-401) as a Novel Positron-Emission Tomography Imaging Agent for Monoacylglycerol Lipase.

Monoacylglycerol lipase (MAGL) is a cytosolic serine hydrolase involved in endocannabinoid and inflammatory signaling. Positron-emission tomography (PET) imaging of MAGL serves to validate target engagement of therapeutic MAGL inhibitors as well as to investigate MAGL levels under normal and disease conditions. However, PET radioligands with reversible binding kinetics for MAGL, which allow quantitative assessment of MAGL, are hitherto unavailable. In this study, we designed and synthesized fluoro-containing PET probes starting from a recently identified piperazinyl pyrrolidine-2-one derivative with reversible binding to MAGL. By tailoring the lipophilicity of the molecule to optimize nonspecific binding and blood-brain barrier permeability, we successfully identified two compounds that show high uptake to regions enriched with MAGL. PET imaging of wild-type and MAGL-deficient mice as well as a macaque monkey indicated that [18F]5 ((4 R)-1-{3-[2-(18F)fluoro-4-methylpyridin-3-yl]phenyl}-4-[4-(1,3-thiazol-2-ylcarbonyl)piperazin-1-yl]pyrrolidin-2-one, [18F]T-401) specifically binds to MAGL with adequate reversibility, yielding a high contrast for MAGL within an appropriate imaging time.

Design, Synthesis, and Evaluation of Piperazinyl Pyrrolidin-2-ones as a Novel Series of Reversible Monoacylglycerol Lipase Inhibitors.

Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyzes 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, inhibition of MAGL is an attractive therapeutic target for CNS disorders, particularly neurodegenerative diseases. In this study, we report the structure-based drug design of novel piperazinyl pyrrolidin-2-ones starting from our hit compounds 2a and 2b. By enhancing the interaction of the piperazinyl pyrrolidin-2-one core and its substituents with the MAGL enzyme via design modifications, we identified a potent and reversible MAGL inhibitor, compound ( R)-3t. Oral administration of compound ( R)-3t to mice decreased AA levels and elevated 2-AG levels in the brain.