Structure-Based Optimization of Selective and Brain Penetrant CK1δ Inhibitors for the Treatment of Circadian Disruptions.

Neuropsychiatric disorders such as major depressive disorders and schizophrenia are often associated with disruptions to the normal 24 h sleep wake cycle. Casein kinase 1 (CK1δ) is an integral part of the molecular machinery that regulates circadian rhythms. Starting from a cluster of bicyclic pyrazoles identified from a virtual screening effort, we utilized structure-based drug design to identify and reinforce a unique "hinge-flip" binding mode that provides a high degree of selectivity for CK1δ versus the kinome. Pharmacokinetics, brain exposure, and target engagement as measured by ex vivo autoradiography are described for advanced analogs.

Discovery of a Series of Substituted 1H-((1,2,3-Triazol-4-yl)methoxy)pyrimidines as Brain Penetrants and Potent GluN2B-Selective Negative Allosteric Modulators.

Herein, we describe a series of substituted 1H-((1,2,3-triazol-4-yl)methoxy)pyrimidines as potent GluN2B negative allosteric modulators. Exploration of several five- and six-membered heterocycles led to the identification of O-linked pyrimidine analogues that possessed a balance of potency and desirable ADME profiles. Due to initial observations of metabolic saturation, early metabolite identification studies were conducted on compound 18, and the results drove further iterative optimization efforts to avoid the formation of undesired saturating metabolites. The comprehensive investigation of substitution on the pyrimidine moiety of the 1H-1,2,3-triazol-4-yl)methoxy)pyrimidines allowed for the identification of compound 31, which demonstrated high GluN2B receptor affinity, improved solubility, and a clean cardiovascular profile. Compound 31 was profiled in an ex vivo target engagement study in rats at a 10 mg/kg oral dose and achieved an ED50 of 1.7 mg/kg.

Evaluating EEG complexity metrics as biomarkers for depression.

Nonlinear EEG analysis offers the potential for both increased diagnostic accuracy and deeper mechanistic understanding of psychopathology. EEG complexity measures have previously been shown to positively correlate with clinical depression. In this study, resting state EEG recordings were taken across multiple sessions and days with both eyes open and eyes closed conditions from a total of 306 subjects, 62 of which were in a current depressive episode, and 81 of which had a history of diagnosed depression but were not currently depressed. Three different EEG montages (mastoids, average, and Laplacian) were also computed. Higuchi fractal dimension (HFD) and sample entropy (SampEn) were calculated for each unique condition. The complexity metrics showed high internal consistency within session and high stability across days. Higher complexity was found in open-eye recordings compared to closed eyes. The predicted correlation between complexity and depression was not found. However, an unexpected sex effect was observed, in which males and females exhibited different topographic patterns of complexity.

Discovery and SAR studies of 2-alkyl-3-phenyl-2,4,5,6,7,8-hexahydropyrazolo[3,4-d]azepines as 5-HT7/2 inhibitors leading to the identification of a clinical candidate.

We report here the synthesis and characterization of a dual 5-HT7 / 5-HT2 receptor antagonist 3-(4-Fluoro-phenyl)-2-isopropyl-2,4,5,6,7,8-hexahydro-1,2,6-triaza-azulene (4j). 4j is a high affinity 5-HT7 and 5-HT2A receptor ligand having a pKi = 8.1 at both receptors. It behaves as an antagonist in an in vitro functional assay for 5-HT2A and as an inverse agonist in an in vitro functional assay for 5-HT7. In a validated in vivo model for central 5-HT7 activity in rats, blockade of 5-carboxamidotryptamine (5-CT) induced hypothermia, 4j shows efficacy at low doses (ED50 = 0.05 mg/kg, p.o., 1 h) and maximal efficacy was observed at 0.3 mg/kg p.o. with a corresponding plasma concentration of ~27 ng/ml. In a validated in vivo model for central 5-HT2A activity, blockade of 2,5-dimethoxy-4-iodoamphetamine (DOI) induced head-twitches in mice, 4j shows efficacy at low doses with an ED50 = 0.3 mg/kg p.o. Ex vivo receptor binding studies demonstrate that 4j occupied 5-HT2A receptor binding sites in the frontal cortex of the rat brain with an ED50 in good agreement with the ED50 value for central functional effect mediated by 5-HT2A receptor (ED50 = 0.8 mg/kg, p.o., 1 h).

Substituted Azabicyclo[2.2.1]heptanes as Selective Orexin-1 Antagonists: Discovery of JNJ-54717793.

The orexin system consists of two neuropeptides (orexin-A and orexin-B) that exert their mode of action on two receptors (orexin-1 and orexin-2). While the role of the orexin-2 receptor is established as an important modulator of sleep wake states, the role of the orexin-1 receptor is believed to play a role in addiction, panic, or anxiety. In this manuscript, we describe the optimization of a nonselective substituted azabicyclo[2.2.1]heptane dual orexin receptor antagonist (DORA) into orally bioavailable, brain penetrating, selective orexin-1 receptor (OX1R) antagonists. This resulted in the discovery of our first candidate for clinical development, JNJ-54717793.

Translational evaluation of novel selective orexin-1 receptor antagonist JNJ-61393215 in an experimental model for panic in rodents and humans.

Orexin neurons originating in the perifornical and lateral hypothalamic area project to anxiety- and panic-associated neural circuitry, and are highly reactive to anxiogenic stimuli. Preclinical evidence suggests that the orexin system, and particularly the orexin-1 receptor (OX1R), may be involved in the pathophysiology of panic and anxiety. Selective OX1R antagonists thus may constitute a potential new treatment strategy for panic- and anxiety-related disorders. Here, we characterized a novel selective OX1R antagonist, JNJ-61393215, and determined its affinity and potency for human and rat OX1R in vitro. We also evaluated the safety, pharmacokinetic, and pharmacodynamic properties of JNJ-61393215 in first-in-human single- and multiple-ascending dose studies conducted. Finally, the potential anxiolytic effects of JNJ-61393215 were evaluated both in rats and in healthy men using 35% CO2 inhalation challenge to induce panic symptoms. In the rat CO2 model of panic anxiety, JNJ-61393215 demonstrated dose-dependent attenuation of CO2-induced panic-like behavior without altering baseline locomotor or autonomic activity, and had minimal effect on spontaneous sleep. In phase-1 human studies, JNJ-61393215 at 90 mg demonstrated significant reduction (P < 0.02) in CO2-induced fear and anxiety symptoms that were comparable to those obtained using alprazolam. The most frequently reported adverse events were somnolence and headache, and all events were mild in severity. These results support the safety, tolerability, and anxiolytic effects of JNJ-61393215, and validate CO2 exposure as a translational cross-species experimental model to evaluate the therapeutic potential of novel anxiolytic drugs.

Design, Synthesis, and Preclinical Evaluation of 3-Methyl-6-(5-thiophenyl)-1,3-dihydro-imidazo[4,5-b]pyridin-2-ones as Selective GluN2B Negative Allosteric Modulators for the Treatment of Mood Disorders.

Selective inhibitors of the GluN2B subunit of N-methyl-d-aspartate receptors in the ionotropic glutamate receptor superfamily have been targeted for the treatment of mood disorders. We sought to identify structurally novel, brain penetrant, GluN2B-selective inhibitors suitable for evaluation in a clinical setting in patients with major depressive disorder. We identified a new class of negative allosteric modulators of GluN2B that contain a 1,3-dihydro-imidazo[4,5-b]pyridin-2-one core. This series of compounds had poor solubility properties and poor permeability, which was addressed utilizing two approaches. First, a series of structural modifications was conducted which included replacing hydrogen bond donor groups. Second, enabling formulation development was undertaken in which a stable nanosuspension was identified for lead compound 12. Compound 12 was found to have robust target engagement in rat with an ED70 of 1.4 mg/kg. The nanosuspension enabled sufficient margins in preclinical toleration studies to nominate 12 for progression into advanced good laboratory practice studies.

Putative role of GPR139 on sleep modulation using pharmacological and genetic rodent models.

GPR139 is a G-protein coupled receptor expressed in circumventricular regions of the habenula and septum. Amino acids L-tryptophan and L-phenylalanine have been shown to activate GPR139 at physiologically relevant concentrations. The aim of the present study was to investigate the role of GPR139 on sleep modulation using pharmacological and genetic (GPR139 knockout mice, KO) rodent models. To evaluate the effects of GPR139 pharmacological activation on sleep, rats were orally dosed with the selective GPR139 agonist JNJ-63533054 (3-30 mg/kg). When acutely administered at the beginning of the light phase, the GPR139 agonist dose-dependently reduced non-rapid eye movement (NREM) latency and increased NREM sleep duration without altering rapid eye movement (REM) sleep. This effect progressively dissipated upon 7-day repeated dosing, suggesting functional desensitization. Under baseline conditions, GPR139 KO mice spent less time in REM sleep compared to their wild type littermates during the dark phase, whereas NREM sleep was not altered. Under conditions of pharmacologically enhanced monoamine endogenous tone, GPR139 KO mice showed a blunted response to citalopram or fluoxetine induced REM sleep suppression and an attenuated response to the wake promoting effect of amphetamine. These findings indicate an emerging role of GPR139 in the modulation of sleep states.

Pharmacologic Characterization of JNJ-42226314, [1-(4-Fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone, a Reversible, Selective, and Potent Monoacylglycerol Lipase Inhibitor.

The serine hydrolase monoacylglycerol lipase (MAGL) is the rate-limiting enzyme responsible for the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) into arachidonic acid and glycerol. Inhibition of 2-AG degradation leads to elevation of 2-AG, the most abundant endogenous agonist of the cannabinoid receptors (CBs) CB1 and CB2. Activation of these receptors has demonstrated beneficial effects on mood, appetite, pain, and inflammation. Therefore, MAGL inhibitors have the potential to produce therapeutic effects in a vast array of complex human diseases. The present report describes the pharmacologic characterization of [1-(4-fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone (JNJ-42226314), a reversible and highly selective MAGL inhibitor. JNJ-42226314 inhibits MAGL in a competitive mode with respect to the 2-AG substrate. In rodent brain, the compound time- and dose-dependently bound to MAGL, indirectly led to CB1 occupancy by raising 2-AG levels, and raised norepinephrine levels in cortex. In vivo, the compound exhibited antinociceptive efficacy in both the rat complete Freund's adjuvant-induced radiant heat hypersensitivity and chronic constriction injury-induced cold hypersensitivity models of inflammatory and neuropathic pain, respectively. Though 30 mg/kg induced hippocampal synaptic depression, altered sleep onset, and decreased electroencephalogram gamma power, 3 mg/kg still provided approximately 80% enzyme occupancy, significantly increased 2-AG and norepinephrine levels, and produced neuropathic antinociception without synaptic depression or decreased gamma power. Thus, it is anticipated that the profile exhibited by this compound will allow for precise modulation of 2-AG levels in vivo, supporting potential therapeutic application in several central nervous system disorders. SIGNIFICANCE STATEMENT: Potentiation of endocannabinoid signaling activity via inhibition of the serine hydrolase monoacylglycerol lipase (MAGL) is an appealing strategy in the development of treatments for several disorders, including ones related to mood, pain, and inflammation. [1-(4-Fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone is presented in this report to be a novel, potent, selective, and reversible noncovalent MAGL inhibitor that demonstrates dose-dependent enhancement of the major endocannabinoid 2-arachidonoylglycerol as well as efficacy in models of neuropathic and inflammatory pain.

Pharmacology of JNJ-28583113: A novel TRPM2 antagonist.

Transient receptor potential melastatin type 2 (TRPM2) is a cation channel activated by free intracellular ADP-ribose and reactive oxygen species. TRPM2 signaling has been linked to the pathophysiology of CNS disorders such as neuropathic pain, bipolar disorder and Alzheimer's disease. In this manuscript, we describe the discovery of JNJ-28583113, a potent brain penetrant TRPM2 antagonist. Ca2+ flux assays in cells overexpressing TRPM2 and electrophysiological recordings were used to test the pharmacology of JNJ-28583113. JNJ-28583113 was assayed in vitro on GSK-3 phosphorylation levels, cell death, cytokine release in microglia and unbiased morphological phenotypic analysis. Finally, we dosed animals to evaluate its pharmacokinetic properties. Our results showed that JNJ-28583113 is a potent (126 ±â€¯0.5 nM) TRPM2 antagonist. Blocking TRPM2 caused phosphorylation of GSK3α and ß subunits. JNJ-28583113 also protected cells from oxidative stress induced cell death as well as morphological changes induced by non-cytotoxic concentrations of H2O2. In addition, inhibiting TRPM2 blunted cytokine release in response to pro-inflammatory stimuli in microglia. Lastly, we showed that JNJ-28583113 was brain penetrant but not suitable for systemic dosing as it was rapidly metabolized in vivo. While the in-vitro pharmacology of JNJ-28583113 is the best in class, its in-vivo properties would need optimization to assist in further probing key roles of TRPM2 in CNS pathophysiology.

In vivo Characterization of a Selective, Orally Available, and Brain Penetrant Small Molecule GPR139 Agonist.

Recently, our group along with another demonstrated that GPR139 can be activated by L-phenylalanine (L-Phe) and L-tryptophan (L-Trp) at physiologically relevant concentrations. GPR139 is discretely expressed in brain, with highest expression in medial habenula. Not only are the endogenous ligands catecholamine/serotonin precursors, but GPR139 expressing areas can directly/indirectly regulate the activity of catecholamine/serotonin neurons. Thus, GPR139 appears expressed in an interconnected circuit involved in mood, motivation, and anxiety. The aim of this study was to characterize a selective and brain penetrant GPR139 agonist (JNJ-63533054) in relevant in vivo models. JNJ-63533054 was tested for its effect on c-fos activation in the habenula and dorsal striatum. In vivo microdialysis experiments were performed in freely moving rats to measure basal levels of serotonin or dopamine (DA) in prefrontal cortex (mPFC) and nucleus accumbens (NAc). Finally, the compound was profiled in behavioral models of anxiety, despair, and anhedonia. The agonist (10-30 mg/kg, p.o.) did not alter c-fos expression in medial habenula or dorsal striatum nor neurotransmitter levels in mPFC or NAc. JNJ-63533054 (10 mg/kg p.o.) produced an anhedonic-like effect on urine sniffing, but had no significant effect in tail suspension, with no interaction with imipramine, no effect on naloxone place aversion, and no effect on learned helplessness. In the marble burying test, the agonist (10 mg/kg p.o.) produced a small anxiolytic-like effect, with no interaction with fluoxetine, and no effect in elevated plus maze (EPM). Despite GPR139 high expression in medial habenula, an area with connections to limbic and catecholaminergic/serotoninergic areas, the GPR139 agonist had no effect on c-fos in medial habenula. It did not alter catecholamine/serotonin levels and had a mostly silent signal in in vivo models commonly associated with these pathways. The physiological function of GPR139 remains elusive.

1H-Pyrrolo[3,2-b]pyridine GluN2B-Selective Negative Allosteric Modulators.

Herein, we disclose a series of selective GluN2B negative allosteric modulators containing a 1H-pyrrolo[3,2-b]pyridine core. Lead optimization efforts included increasing brain penetration as well as decreasing cytochrome P450 inhibition and hERG channel binding. The series was also optimized to reduce metabolic turnover in human and rat. Compounds 9, 25, 30, and 34 have good in vitro GluN2B potency and good predicted absorption, but moderate to high projected clearance. They were assessed in vivo to determine their target engagement. All four compounds achieved >75% receptor occupancy after an oral dose of 10 mg/kg in rat. Compound 9 receptor occupancy was measured in a dose-response experiment, and its ED50 was found to be 2.0 mg/kg.

Discovery of Imidazo[1,2-a]pyrazines and Pyrazolo[1,5-c]pyrimidines as TARP γ-8 Selective AMPAR Negative Modulators.

This report discloses the discovery and characterization of imidazo[1,2-a]pyrazines and pyrazolo[1,5-c]pyrimidines as selective negative modulators of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) associated with transmembrane AMPAR regulatory protein γ-8. Imidazopyrazine 5 was initially identified as a promising γ-8 selective high-throughput screening hit, and subsequent structure-activity relationship optimization yielded subnanomolar, brain penetrant leads. Replacement of the imidazopyrazine core with an isosteric pyrazolopyrimidine scaffold improved microsomal stability and efflux liabilities to provide 26, JNJ-61432059. Following oral administration, 26 exhibited time- and dose-dependent AMPAR/γ-8 receptor occupancy in mouse hippocampus, which resulted in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.

α6-Containing Nicotinic Acetylcholine Receptor Reconstitution Involves Mechanistically Distinct Accessory Components.

Acetylcholine gates a large family of nicotinic receptor cation channels that control neuronal excitation and neurotransmitter release. These receptors are key targets for neuropsychiatric disorders; however, difficulties in expressing nicotinic acetylcholine (nACh) receptors hamper elaboration of their pharmacology and obscure elucidation of their biological functions. Particularly intriguing are α6-containing nACh receptors, which mediate nicotine-induced dopamine release in striatum-nucleus accumbens. Using genome-wide cDNA screening, we identify three accessory proteins, ß-anchoring and -regulatory protein (BARP), lysosomal-associated membrane protein 5 (LAMP5), and SULT2B1, that complement the nACh receptor chaperone NACHO to reconstitute α6ß2ß3 channel function. Whereas NACHO mediates α6ß2ß3 assembly, BARP primarily enhances channel gating and LAMP5 and SULT2B1 promote receptor surface trafficking. BARP knockout mice show perturbations in presynaptic striatal nACh receptors that are consistent with BARP modulation of receptor desensitization. These studies unravel the molecular complexity of α6ß2ß3 biogenesis and enable physiological studies of this crucial neuropharmacological target.

Lead Optimization of 5-Aryl Benzimidazolone- and Oxindole-Based AMPA Receptor Modulators Selective for TARP γ-8.

Glutamate mediates fast excitatory neurotransmission via ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The trafficking and gating properties of AMPA receptors (AMPARs) can be amplified by transmembrane AMPAR regulatory proteins (TARPs), which are often expressed in localized brain regions. Herein, we describe the discovery, lead optimization, and preclinical characterization of 5-arylbenzimidazolone and oxindole-based negative modulators of AMPARs associated with TARP γ-8, the primary TARP found in hippocampus. High-throughput screen lead 4 was optimized for potency and brain penetration to provide benzimidazolone 3, JNJ-55511118.1 Replacement of the benzimidazolone core in 3 with an oxindole mitigated reactive metabolite formation and led to the identification of 18 (GluA1/γ-8 pIC50 = 9.7). Following oral dosing in rats, 18 demonstrated robust target engagement in hippocampus as assessed by ex vivo autoradiography (ED50 = 0.6 mg/kg, plasma EC50 = 9 ng/mL).

Neuropsychopharmacology of JNJ-55308942: evaluation of a clinical candidate targeting P2X7 ion channels in animal models of neuroinflammation and anhedonia.

Emerging data continues to point towards a relationship between neuroinflammation and neuropsychiatric disorders. ATP-induced activation of P2X7 results in IL-1ß release causing neuroinflammation and microglial activation. This study describes the in-vitro and in-vivo neuropharmacology of a novel brain-penetrant P2X7 antagonist, JNJ-55308942, currently in clinical development. JNJ-55308942 is a high-affinity, selective, brain-penetrant (brain/plasma of 1) P2X7 functional antagonist. In human blood and in mouse blood and microglia, JNJ-55308942 attenuated IL-1ß release in a potent and concentration-dependent manner. After oral dosing, the compound exhibited both dose and concentration-dependent occupancy of rat brain P2X7 with an ED50 of 0.07 mg/kg. The P2X7 antagonist (3 mg/kg, oral) blocked Bz-ATP-induced brain IL-1ß release in conscious rats, demonstrating functional effects of target engagement in the brain. JNJ-55308942 (30 mg/kg, oral) attenuated LPS-induced microglial activation in mice, assessed at day 2 after a single systemic LPS injection (0.8 mg/kg, i.p.), suggesting a role for P2X7 in microglial activation. In a model of BCG-induced depression, JNJ-55308942 dosed orally (30 mg/kg), reversed the BCG-induced deficits of sucrose preference and social interaction, indicating for the first time a role of P2X7 in the BCG model of depression, probably due to the neuroinflammatory component induced by BCG inoculation. Finally, in a rat model of chronic stress induced sucrose intake deficit, JNJ-55308942 reversed the deficit with concurrent high P2X7 brain occupancy as measured by autoradiography. This body of data demonstrates that JNJ-55308942 is a potent P2X7 antagonist, engages the target in brain, modulates IL-1ß release and microglial activation leading to efficacy in two models of anhedonia in rodents.

A Dipolar Cycloaddition Reaction To Access 6-Methyl-4,5,6,7-tetrahydro-1H-[1,2,3]triazolo[4,5-c]pyridines Enables the Discovery Synthesis and Preclinical Profiling of a P2X7 Antagonist Clinical Candidate.

A single pot dipolar cycloaddition reaction/Cope elimination sequence was developed to access novel 1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridine P2X7 antagonists that contain a synthetically challenging chiral center. The structure-activity relationships of the new compounds are described. Two of these compounds, (S)-(2-fluoro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyrimidin-2-yl)-6-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridin-5-yl)methanone (compound 29) and (S)-(3-fluoro-2-(trifluoromethyl)pyridin-4-yl)(1-(5-fluoropyrimidin-2-yl)-6-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridin-5-yl)methanone (compound 35), were found to have robust P2X7 receptor occupancy at low doses in rat with ED50 values of 0.06 and 0.07 mg/kg, respectively. Compound 35 had notable solubility compared to 29 and showed good tolerability in preclinical species. Compound 35 was chosen as a clinical candidate for advancement into phase I clinical trials to assess safety and tolerability in healthy human subjects prior to the initiation of proof of concept studies for the treatment of mood disorders.

Evaluation of JNJ-54717793 a Novel Brain Penetrant Selective Orexin 1 Receptor Antagonist in Two Rat Models of Panic Attack Provocation.

Orexin neurons originating in the perifornical and lateral hypothalamic area are highly reactive to anxiogenic stimuli and have strong projections to anxiety and panic-associated circuitry. Recent studies support a role for the orexin system and in particular the orexin 1 receptor (OX1R) in coordinating an integrative stress response. However, no selective OX1R antagonist has been systematically tested in two preclinical models of using panicogenic stimuli that induce panic attack in the majority of people with panic disorder, namely an acute hypercapnia-panic provocation model and a model involving chronic inhibition of GABA synthesis in the perifornical hypothalamic area followed by intravenous sodium lactate infusion. Here we report on a novel brain penetrant, selective and high affinity OX1R antagonist JNJ-54717793 (1S,2R,4R)-7-([(3-fluoro-2-pyrimidin-2-ylphenyl)carbonyl]-N-[5-(trifluoromethyl)pyrazin-2-yl]-7-azabicyclo[2.2.1]heptan-2-amine). JNJ-54717793 is a high affinity/potent OX1R antagonist and has an excellent selectivity profile including 50 fold versus the OX2R. Ex vivo receptor binding studies demonstrated that after oral administration JNJ-54717793 crossed the blood brain barrier and occupied OX1Rs in the rat brain. While JNJ-54717793 had minimal effect on spontaneous sleep in rats and in wild-type mice, its administration in OX2R knockout mice, selectively promoted rapid eye movement sleep, demonstrating target engagement and specific OX1R blockade. JNJ-54717793 attenuated CO2 and sodium lactate induced panic-like behaviors and cardiovascular responses without altering baseline locomotor or autonomic activity. These data confirm that selective OX1R antagonism may represent a novel approach of treating anxiety disorders, with no apparent sedative effects.

4-Methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridine-Based P2X7 Receptor Antagonists: Optimization of Pharmacokinetic Properties Leading to the Identification of a Clinical Candidate.

The synthesis and preclinical characterization of novel 4-(R)-methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridines that are potent and selective brain penetrant P2X7 antagonists are described. Optimization efforts based on previously disclosed unsubstituted 6,7-dihydro-4H-triazolo[4,5-c]pyridines, methyl substituted 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazines, and several other series lead to the identification of a series of 4-(R)-methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridines that are selective P2X7 antagonists with potency at the rodent and human P2X7 ion channels. These novel P2X7 antagonists have suitable physicochemical properties, and several analogs have an excellent pharmacokinetic profile, good partitioning into the CNS and show robust in vivo target engagement after oral dosing. Improvements in metabolic stability led to the identification of JNJ-54175446 (14) as a candidate for clinical development. The drug discovery efforts and strategies that resulted in the identification of the clinical candidate are described herein.

NACHO Mediates Nicotinic Acetylcholine Receptor Function throughout the Brain.

Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of α and ß subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4ß2-, and α6-containing nAChRs, respectively. By contrast, GABAA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs.