Spin dynamics phenomena of a cerium(III) double-decker complex induced by intramolecular electron transfer.

Switchable spin dynamic properties in single-molecule magnets (SMMs) via an applied stimulus have applications in single-molecule devices. Many SMMs containing heavy lanthanoid ions with strong uniaxial magnetic anisotropy have been reported to exhibit SMM characteristics in the absence of an external magnetic field. On the other hand, SMMs containing light lanthanoid cerium(III) (Ce3+) ions exhibit field-induced slow magnetic relaxation. We investigated the chemical conversion of a diamagnetic Ce4+ ion (4f0) to a paramagnetic Ce3+ ion (4f1) in Ce-phthalocyaninato double-decker complexes (TBA+[Ce(obPc)2]- (1) and TBA+[Ce(Pc)2]- (2)) which exhibit field-induced SMM behaviour due to a 4f1 system. The phthalocyaninato ligands with electron-donating substituents (obPc2- = 2,3,9,10,16,17,23,24-octabutoxyphthalocyaninato) in 1 have a significant effect on the valence state of the Ce ion, which is reflected in its magnetic properties due to the mixed valence state of the Ce ion. Given that Ce double-decker complexes with π-conjugated ligands undergo intramolecular electron transfer (IET) to the Ce ion mixed valence state, characterised by a mixture of 4f0 and 4f1 configurations, we examined the dynamic disorder inherent in IET influencing magnetic relaxation.

Cationic Oligopeptides with Amino Groups as Synthetic Nucleolar Localization Signals for the Rational Design of Nucleolus-Staining Probes.

Cationic oligopeptides with amino groups were found to function as synthetic nucleolar localization signals for directing various fluorophores to the nucleolus with high selectivity in the cells with a view toward the development of nucleolus-staining probes.

Bright and Light-Up Sensing of Benzo[c,d]indole-oxazolopyridine Cyanine Dye for RNA and Its Application to Highly Sensitive Imaging of Nucleolar RNA in Living Cells.

Small molecular weight probes that can show a fluorescence signaling response upon binding to RNAs are promising for RNA imaging in living cells. Live-cell RNA imaging probes that can achieve a large light-up ability (>100-fold) and high Φbound value for RNA (>0.50) have been rarely reported to date. Here, benzo[c,d]indole-oxazolopyridine (BIOP), an unsymmetrical monomethine cyanine analogue, was newly developed as a bright and large light-up probe for imaging of nucleolar RNA in living cells. BIOP served as a yellow-emissive probe (λem = 570 nm) and exhibited a significant light-up response upon RNA binding (770-fold) with a high Φbound value (0.52). We demonstrated the advantages of BIOP over a commercially available RNA-staining probe, SYTO RNA select, for robust and sensitive RNA sensing by a systematic comparison of fluorescent properties for RNA. In addition, BIOP was found to possess high membrane permeability and low cytotoxicity in living cells. The examination of live-cell imaging revealed that BIOP exhibited emission in the nucleolus upon binding to nucleolar RNA much stronger than that of SYTO RNA select. Furthermore, BIOP facilitated the highly sensitive imaging of nucleolar RNA, in which 50 nM BIOP can stain nucleolar RNA in living cells with a 20 min incubation.

Deep-red fluorogenic cyanine dyes carrying an amino group-terminated side chain for improved RNA detection and nucleolar RNA imaging.

The introduction of an amino-group-terminated side chain into deep-red emissive benzo[c,d]indole-quinoline monomethine cyanine dye has led to the improved detection of RNAs as well as the imaging of nucleolar RNAs in cells.

Disposition and Metabolism of [14C]Lemborexant in Healthy Human Subjects and Characterization of Its Circulating Metabolites.

Lemborexant is a novel dual orexin receptor antagonist recently approved for the treatment of insomnia in the United States and Japan. Here, disposition and metabolic profiles were investigated in healthy human subjects. After single oral administration of 10 mg [14C]lemborexant (100 µCi), plasma concentrations of lemborexant and radioactivity peaked at 1 hour postdose and decreased biphasically. Cumulative recovery of the administered radioactivity within 480 hours was 86.5% of the dose, with 29.1% in urine and 57.4% in feces. Unchanged lemborexant was not detected in urine but accounted for 13.0% of the dose in feces, suggesting that the main elimination pathway of lemborexant was metabolism. Metabolite analyses revealed that the major metabolic pathways of lemborexant are oxidation of the dimethylpyrimidine moiety and subsequent further oxidation and/or glucuronidation. In plasma, lemborexant was the dominant component, accounting for 26.5% of total drug-related exposure. M4, M9, M10, and M18 were detected as the major radioactive components; M10 was the only metabolite exceeding 10% of total drug-related exposure. Although M4, M9, and M10 showed binding affinity for orexin receptors comparable to that of lemborexant, their contributions to the sleep-promoting effects of lemborexant are likely low because of the limited brain penetration by P-glycoprotein. Exposure comparison between humans and nonclinical toxicology species confirmed that plasma exposure of M10 was higher in at least one animal species compared with that in humans, indicating that there is no disproportionate metabolite in humans, as defined by International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use M3(R2) and U.S. Food and Drug Administration Metabolite in Safety Testing guidance; therefore, no additional toxicology studies are needed. SIGNIFICANCE STATEMENT: This study provides detailed data of the disposition and metabolism of lemborexant, a novel therapeutic drug for insomnia, in humans, as well as a characterization of the circulating metabolites and assessment of their contributions to efficacy and safety. The information presented herein furthers our understanding of the pharmacokinetic profiles of lemborexant and its metabolites and will promote the safe and effective use of lemborexant in the clinic.

Preclinical in vivo characterization of lemborexant (E2006), a novel dual orexin receptor antagonist for sleep/wake regulation.

STUDY OBJECTIVES: To present results from in vivo studies underlying the preclinical development of lemborexant (E2006), a novel dual orexin (hypocretin) receptor antagonist for sleep/wake regulation. METHODS: Rodent (wild-type rats and wild-type and orexin neuron-deficient [orexin/ataxin-3 Tg/+] mice) studies were performed to evaluate the effects of single-dose oral lemborexant (1-300 mg/kg) on orexin-induced increases in plasma adrenocorticotropic hormone (ACTH), locomotor activity, vigilance state measures (wakefulness, nonrapid eye movement [non-REM] sleep, rapid eye movement [REM] sleep), ethanol-induced anesthesia, and motor coordination, and the effects of multiple-dose oral lemborexant (30 mg/kg) on vigilance state measures. Active comparators were almorexant and zolpidem. Pharmacokinetics were assessed after single-dose lemborexant in mice and rats. RESULTS: Lemborexant prevented the orexin-promoted increase in ACTH in rats, therefore demonstrating inhibition of the orexin signaling pathway. Furthermore, lemborexant promoted sleep in wild-type mice and rats. Lemborexant promoted REM and non-REM sleep at an equal rate (there was no change in the REM sleep ratio). In contrast, zolpidem reduced REM sleep. The sleep-promoting effect of lemborexant was mediated via the orexin-peptide signaling pathway as demonstrated by a lack of sleep promotion in orexin neuron-deficient mice. Chronic dosing was not associated with a change in effect size or sleep architecture immediately postdosing. Lemborexant did not increase the sedative effects of ethanol or impair motor coordination, showing good safety margin in animals. Pharmacokinetic/pharmacodynamic data for mice and rats were well aligned. CONCLUSIONS: These findings supported further clinical evaluation (ongoing at this time) of lemborexant as a potential candidate for treating insomnia and other sleep disorders.

In Vitro and In Silico Characterization of Lemborexant (E2006), a Novel Dual Orexin Receptor Antagonist.

Orexin (hypocretin) neuropeptides have, among others, been implicated in arousal/sleep control, and antagonizing the orexin signaling pathway has been previously demonstrated to promote sleep in animals and humans. This mechanism opens up a new therapeutic approach to curb excessive wakefulness in insomnia disorder rather than to promote sleep-related signaling. Here we describe the preclinical pharmacological in vitro and in silico characterization of lemborexant ((1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide)), a dual orexin receptor antagonist (DORA), as a novel experimental therapeutic agent for the symptomatic treatment of insomnia disorder and compare its properties to two other DORAs, almorexant and suvorexant. Lemborexant binds to both orexin receptors and functionally inhibits them in a competitive manner with low nanomolar potency, without any species difference apparent among human, rat, and mouse receptors. Binding and dissociation kinetics on both orexin receptors are rapid. Lemborexant is selective for both orexin receptors over 88 other receptors, transporters, and ion channels of important physiologic function. In silico modeling of lemborexant into the orexin receptors showed that it assumes the same type of conformation within the receptor-binding pocket as suvorexant, the π-stacked horseshoe-like conformation.

Development of Novel PET Probes for Central 2-Amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic Acid Receptors.

We document the development of PET probes for central AMPA receptors and their application to in vivo animal imaging. An initial screening of perampanel derivatives was performed to identify probe candidates. Despite the high autoradiographic contrast yielded by several radioligands, rat PET scans did not support their in vivo suitability. Further focused derivatization and a second screening by ex vivo LC-MS measurements led to the selection of 2-[1-(3-methylaminophenyl)-2-oxo-5-(pyrimidin-2-yl)-1,2-dihydropyridin-3-yl]benzonitrile, 21a, and its analogues as candidates. [(11)C]21a was shown by autoradiography to specifically bind to the neocortex and hippocampus, consistent with AMPA receptor localization. PET imaging with [(11)C]21a demonstrated moderate uptake of radioactivity in rat and monkey brains, with the retention of radiosignals being consistent with that from the autoradiogram data, and the uptake was blocked by pretreatment with unlabeled 21a in a dose-dependent manner. The current approach has facilitated the discovery of a PET probe potentially suitable for translational research and development focused on AMPA receptors.

Discovery of (1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006): A Potent and Efficacious Oral Orexin Receptor Antagonist.

The orexin/hypocretin receptors are a family of G protein-coupled receptors and consist of orexin-1 (OX1) and orexin-2 (OX2) receptor subtypes. Orexin receptors are expressed throughout the central nervous system and are involved in the regulation of the sleep/wake cycle. Because modulation of these receptors constitutes a promising target for novel treatments of disorders associated with the control of sleep and wakefulness, such as insomnia, the development of orexin receptor antagonists has emerged as an important focus in drug discovery research. Here, we report the design, synthesis, characterization, and structure-activity relationships (SARs) of novel orexin receptor antagonists. Various modifications made to the core structure of a previously developed compound (-)-5, the lead molecule, resulted in compounds with improved chemical and pharmacological profiles. The investigation afforded a potential therapeutic agent, (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006), an orally active, potent orexin antagonist. The efficacy was demonstrated in mice in an in vivo study by using sleep parameter measurements.

Design, synthesis, and structure-activity relationships of a series of novel N-aryl-2-phenylcyclopropanecarboxamide that are potent and orally active orexin receptor antagonists.

Herein we describe the design, synthesis, and structure-activity relationships (SARs) of a novel phenylcyclopropane series represented by 7 and 33 b as antagonists of orexin 1 and orexin 2 receptors. With 4 serving as the initial lead for the development of orexin antagonists, exploration of SAR resulted in improved binding affinity for orexin 1 and orexin 2 receptors. Among the synthesized compounds, 33 b ((-)-N-(5-cyanopyridin-2-yl)-2-[(3,4-dimethoxyphenyl)oxymethyl]-2-phenylcyclopropanecarboxamide) exhibited potent in vitro activity and oral efficacy in animal sleep measurement experiments. The results of our study suggest that compound 33 b may serve as a valuable template for the development of new orexin receptor antagonists.

A small-animal pharmacokinetic/pharmacodynamic PET study of central serotonin 1A receptor occupancy by a potential therapeutic agent for overactive bladder.

Serotonin 1A (5-HT1A) receptors have been mechanistically implicated in micturition control, and there has been a need for an appropriate biomarker surrogating the potency of a provisional drug acting on this receptor system for developing a new therapeutic approach to overactive bladder (OAB). Here, we analyzed the occupancy of 5-HT1A receptors in living Sprague-Dawley rat brains by a novel candidate drug for OAB, E2110, using positron emission tomography (PET) imaging, and assessed the utility of a receptor occupancy (RO) assay to establish a pharmacodynamic index translatable between animals and humans. The plasma concentrations inducing 50% RO (EC50) estimated by both direct and effect compartment models were in good agreement. Dose-dependent therapeutic effects of E2110 on dysregulated micturition in different rat models of pollakiuria were also consistently explained by achievement of 5-HT1A RO by E2110 in a certain range (≥ 60%). Plasma drug concentrations inducing this RO range and EC50 would accordingly be objective indices in comparing pharmacokinetics-RO relationships between rats and humans. These findings support the utility of PET RO and plasma pharmacokinetic assays with the aid of adequate mathematical models in determining the in vivo characteristics of a drug acting on 5-HT1A receptors and thereby counteracting OAB.

Synthesis and evaluation of novel radioligands for positron emission tomography imaging of the orexin-2 receptor.

Orexin receptors (OXRs) in the brain have been implicated in diverse physiological and neuropsychiatric conditions. Here we describe the design, synthesis, and evaluation of OXR ligands related to (1R,2S)-2-(((2-methyl-4-methoxymethylpyrimidin-5-yl)oxy)methyl)-N-(5-fluoropyridin-2-yl)-2-(3-fluorophenyl)cyclopropanecarboxamide (9a) applicable to positron emission tomography (PET) imaging. Structural features were incorporated to increase binding affinity for OXRs, to enable carbon-11 radiolabeling, and to adjust lipophilicity considered optimal for brain penetration and low nonspecific binding. 9a displayed nanomolar affinity for OXRs, and autoradiography using rat brain sections showed that specific binding of [(11)C]9a was distributed primarily to neocortical layer VI and hypothalamus, consistent with reported localizations of orexin-2 receptors (OX2Rs). In vivo PET study of [(11)C]9a demonstrated moderate uptake of radioactivity into rat and monkey brains under deficiency or blockade of P-glycoprotein, and distribution of PET signals in the brain was in agreement with autoradiographic data. Our approach and findings have provided significant information for development of OX2R PET tracers.

Dual involvement of G-substrate in motor learning revealed by gene deletion.

In this study, we generated mice lacking the gene for G-substrate, a specific substrate for cGMP-dependent protein kinase uniquely located in cerebellar Purkinje cells, and explored their specific functional deficits. G-substrate-deficient Purkinje cells in slices obtained at postnatal weeks (PWs) 10-15 maintained electrophysiological properties essentially similar to those from WT littermates. Conjunction of parallel fiber stimulation and depolarizing pulses induced long-term depression (LTD) normally. At younger ages, however, LTD attenuated temporarily at PW6 and recovered thereafter. In parallel with LTD, short-term (1 h) adaptation of optokinetic eye movement response (OKR) temporarily diminished at PW6. Young adult G-substrate knockout mice tested at PW12 exhibited no significant differences from their WT littermates in terms of brain structure, general behavior, locomotor behavior on a rotor rod or treadmill, eyeblink conditioning, dynamic characteristics of OKR, or short-term OKR adaptation. One unique change detected was a modest but significant attenuation in the long-term (5 days) adaptation of OKR. The present results support the concept that LTD is causal to short-term adaptation and reveal the dual functional involvement of G-substrate in neuronal mechanisms of the cerebellum for both short-term and long-term adaptation.

Orexin-A (hypocretin-1) is possibly involved in generation of anxiety-like behavior.

Orexins (hypocretins) are neuropeptides expressed specifically in neurons in the lateral hypothalamic area and are known to be involved in the regulation of vigilance and feeding behavior. However, the relationship between orexin and emotional behaviors like anxiety is still poorly understood. Therefore, in this report we evaluated the effect of intracerebroventricular injection of orexin-A in two major anxiety tests, the light-dark exploration test (mouse) and the elevated plus-maze test (mouse, rat). Orexin increased time spent in the dark compartment in the light-dark test and time spent on the closed arms in the elevated plus-maze test. These results were not caused by a hypothetical sedative or activity-inducing effect of orexin-A because spontaneous locomotor activity did not alter upon orexin-A application under novel conditions. We therefore suggest an anxiogenic effect of orexin-A. To our knowledge, this is the first report about a relationship between orexin-A and anxiety.