Design and discovery of C2-fluoroalkyl iminothiazine dioxides as BACE inhibitors.

This paper describes the structure-activity-relationships of novel fluoroalkyl substituents at the C2 position of iminothiazine dioxide beta secretase inhibitors. Key discoveries include reduced amidine basicity and its effect on Pgp, cell potency, and efficacy in various preclinical in vivo efficacy animal models. Findings from these structure-activity-relationships are discussed.

Bioprinted 3D Primary Human Intestinal Tissues Model Aspects of Native Physiology and ADME/Tox Functions.

The human intestinal mucosa is a critical site for absorption, distribution, metabolism, and excretion (ADME)/Tox studies in drug development and is difficult to recapitulate in vitro. Using bioprinting, we generated three-dimensional (3D) intestinal tissue composed of human primary intestinal epithelial cells and myofibroblasts with architecture and function to model the native intestine. The 3D intestinal tissue demonstrates a polarized epithelium with tight junctions and specialized epithelial cell types and expresses functional and inducible CYP450 enzymes. The 3D intestinal tissues develop physiological barrier function, distinguish between high- and low-permeability compounds, and have functional P-gp and BCRP transporters. Biochemical and histological characterization demonstrate that 3D intestinal tissues can generate an injury response to compound-induced toxicity and inflammation. This model is compatible with existing preclinical assays and may be implemented as an additional bridge to clinical trials by enhancing safety and efficacy prediction in drug development.

Accuracy of data buoys for measurement of cyanobacteria, chlorophyll, and turbidity in a large lake (Lake Erie, North America): implications for estimation of cyanobacterial bloom parameters from water quality sonde measurements.

Microcystin (MCY)-producing harmful cyanobacterial blooms (cHABs) are an annual occurrence in Lake Erie, and buoys equipped with water quality sondes have been deployed to help researchers and resource managers track cHABs. The objective of this study was to determine how well water quality sondes attached to buoys measure total algae and cyanobacterial biomass and water turbidity. Water samples were collected next to two data buoys in western Lake Erie (near Gibraltar Island and in the Sandusky subbasin) throughout summers 2015, 2016, and 2017 to determine correlations between buoy sonde data and water sample data. MCY and nutrient concentrations were also measured. Significant (P < 0.001) linear relationships (R2 > 0.75) occurred between cyanobacteria buoy and water sample data at the Gibraltar buoy, but not at the Sandusky buoy; however, the coefficients at the Gibraltar buoy differed significantly across years. There was a significant correlation between buoy and water sample total chlorophyll data at both buoys, but the coefficient varied considerably between buoys and among years. Total MCY concentrations at the Gibraltar buoy followed similar temporal patterns as buoy and water sample cyanobacterial biomass data, and the ratio of MCY to cyanobacteria-chlorophyll decreased with decreased ambient nitrate concentrations. These results suggest that buoy data are difficult to compare across time and space. Additionally, the inclusion of nitrate concentration data can lead to more robust predictions on the relative toxicity of blooms. Overall, deployed buoys with sondes that are routinely cleaned and calibrated can track relative cyanobacteria abundance and be used as an early warning system for potentially toxic blooms.

Discovery of a Tetrahydrobenzisoxazole Series of γ-Secretase Modulators.

The design and synthesis of a new series of tetrahydrobenzisoxazoles as modulators of γ-secretase activity and their structure-activity relationship (SAR) will be detailed. Several compounds are active γ-secretase modulators (GSMs) with good to excellent selectivity for the reduction of Aß42 in the cellular assay. Compound 14a was tested in vivo in a nontransgenic rat model and was found to significantly reduce Aß42 in the CNS compartment compared to vehicle-treated animals (up to 58% reduction of cerebrospinal fluid Aß42 as measured 3 h after an acute oral dosing at 30 mg/kg).

Chronic Verubecestat Treatment Suppresses Amyloid Accumulation in Advanced Aged Tg2576-AßPPswe Mice Without Inducing Microhemorrhage.

Verubecestat is a potent BACE1 enzyme inhibitor currently being investigated in Phase III trials for the treatment of mild-to-moderate and prodromal Alzheimer's disease. Multiple anti-amyloid immunotherapies have been dose-limited by adverse amyloid related imaging abnormalities such as vasogenic edema (ARIA-E) and microhemorrhage (ARIA-H) observed in human trials and mice. Verubecestat was tested in a 12-week nonclinical study for the potential to exacerbate microhemorrhage (ARIA-H) profiles in 18-22-month-old post-plaque Tg2576-AßPPswe mice. Animals were treated with verubecestat or controls including the anti-Aß antibody analog of bapineuzumab (3D6) as a positive control for ARIA induction. ARIA-H was measured using in-life longitudinal T2*-MRI and Prussian blue histochemistry at study end. Verubecestat reduced plasma and cerebrospinal fluid Aß40 and Aß42 by >90% and 62% to 68%, respectively. The ARIA-H profile of verubecestat-treated mice was not significantly different than controls. Anti-Aß treatment significantly increased ARIA-H detected by Prussian blue staining; however, anti-Aß antibody treatment did not impact plaque status. Verubecestat treatment significantly suppressed the accumulation of total levels of brain Aß40 and Aß42 and Thioflavin S positive plaque load. Stereological analysis of cortex and hippocampus plaque load similarly revealed significantly reduced area of Aß immunoreactivity and reduced plaque number in verubecestat-treated animals compared to controls. The absence of elevated ARIA events in verubecestat-treated mice was associated with a significant reduction in the level of accumulated CNS amyloid pathology and brain Aß peptides; effects consistent with the desired therapeutic mechanism of verubecestat in AD patients. These data will be compared with longitudinal MRI profiles from ongoing clinical trials.

Inhibition of O-GlcNAcase leads to elevation of O-GlcNAc tau and reduction of tauopathy and cerebrospinal fluid tau in rTg4510 mice.

BACKGROUND: Hyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies. METHODS: Genetic and pharmacological approaches are used to evaluate the pharmacodynamic response of OGA inhibition. A panel of quantitative biochemical assays is established to assess the effect of OGA inhibition on pathological tau reduction. A "click" chemistry labeling method is developed for the detection of O-GlcNAcylated tau. RESULTS: Substantial (>80%) OGA inhibition is required to observe a measurable increase in O-GlcNAcylated proteins in the brain. Sustained and substantial OGA inhibition via chronic treatment with Thiamet G leads to a significant reduction of aggregated tau and several phosphorylated tau species in the insoluble fraction of rTg4510 mouse brain and total tau in cerebrospinal fluid (CSF). O-GlcNAcylated tau is elevated by Thiamet G treatment and is found primarily in the soluble 55 kD tau species, but not in the insoluble 64 kD tau species thought as the pathological entity. CONCLUSION: The present study demonstrates that chronic inhibition of OGA reduces pathological tau in the brain and total tau in the CSF of rTg4510 mice, most likely by directly increasing O-GlcNAcylation of tau and thereby maintaining tau in the soluble, non-toxic form by reducing tau aggregation and the accompanying panoply of deleterious post-translational modifications. These results clarify some conflicting observations regarding the effects and mechanism of OGA inhibition on tau pathology, provide pharmacodynamic tools to guide human dosing and identify CSF total tau as a potential translational biomarker. Therefore, this study provides additional support to develop OGA inhibitors as a treatment for Alzheimer's disease and other neurodegenerative tauopathies.

Discovery of a 3-(4-Pyrimidinyl) Indazole (MLi-2), an Orally Available and Selective Leucine-Rich Repeat Kinase 2 (LRRK2) Inhibitor that Reduces Brain Kinase Activity.

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein which contains a kinase domain and GTPase domain among other regions. Individuals possessing gain of function mutations in the kinase domain such as the most prevalent G2019S mutation have been associated with an increased risk for the development of Parkinson's disease (PD). Given this genetic validation for inhibition of LRRK2 kinase activity as a potential means of affecting disease progression, our team set out to develop LRRK2 inhibitors to test this hypothesis. A high throughput screen of our compound collection afforded a number of promising indazole leads which were truncated in order to identify a minimum pharmacophore. Further optimization of these indazoles led to the development of MLi-2 (1): a potent, highly selective, orally available, brain-penetrant inhibitor of LRRK2.

Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer's Disease.

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aß levels in rats and nonhuman primates and CSF Aß levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

The BACE1 inhibitor verubecestat (MK-8931) reduces CNS ß-amyloid in animal models and in Alzheimer's disease patients.

ß-Amyloid (Aß) peptides are thought to be critically involved in the etiology of Alzheimer's disease (AD). The aspartyl protease ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of Aß, and BACE1 inhibition is thus an attractive target for the treatment of AD. We show that verubecestat (MK-8931) is a potent, selective, structurally unique BACE1 inhibitor that reduced plasma, cerebrospinal fluid (CSF), and brain concentrations of Aß40, Aß42, and sAPPß (a direct product of BACE1 enzymatic activity) after acute and chronic administration to rats and monkeys. Chronic treatment of rats and monkeys with verubecestat achieved exposures >40-fold higher than those being tested in clinical trials in AD patients yet did not elicit many of the adverse effects previously attributed to BACE inhibition, such as reduced nerve myelination, neurodegeneration, altered glucose homeostasis, or hepatotoxicity. Fur hypopigmentation was observed in rabbits and mice but not in monkeys. Single and multiple doses were generally well tolerated and produced reductions in Aß40, Aß42, and sAPPß in the CSF of both healthy human subjects and AD patients. The human data were fit to an amyloid pathway model that provided insight into the Aß pools affected by BACE1 inhibition and guided the choice of doses for subsequent clinical trials.

Design and synthesis of water soluble ß-aminosulfone analogues of SCH 900229 as γ-secretase inhibitors.

In this paper we describe our strategy to improve the aqueous solubility of SCH 900229, a potent PS1-selective γ-secretase inhibitor for the treatment of Alzheimer's disease. Incorporation of ionizable amino groups into the side chain terminal generates water soluble ß-aminosulfone analogues of SCH 900229 that maintain robust in vitro potency and in vivo efficacy.

Substituted 4-morpholine N-arylsulfonamides as γ-secretase inhibitors.

The design, synthesis, SAR, and biological profile of a substituted 4-morpholine sulfonamide series of γ-secretase inhibitors (GSIs) were described. In several cases, the resulting series of GSIs reduced CYP liabilities and improved γ-secretase inhibition activity compared to our previous research series. Selected compounds demonstrated significant reduction of amyloid-ß (Aß) after acute oral dosing in a transgenic animal model of Alzheimer's disease (AD).

Structure-Based Design of an Iminoheterocyclic ß-Site Amyloid Precursor Protein Cleaving Enzyme (BACE) Inhibitor that Lowers Central Aß in Nonhuman Primates.

We describe successful efforts to optimize the in vivo profile and address off-target liabilities of a series of BACE1 inhibitors represented by 6 that embodies the recently validated fused pyrrolidine iminopyrimidinone scaffold. Employing structure-based design, truncation of the cyanophenyl group of 6 that binds in the S3 pocket of BACE1 followed by modification of the thienyl group in S1 was pursued. Optimization of the pyrimidine substituent that binds in the S2'-S2″ pocket of BACE1 remediated time-dependent CYP3A4 inhibition of earlier analogues in this series and imparted high BACE1 affinity. These efforts resulted in the discovery of difluorophenyl analogue 9 (MBi-4), which robustly lowered CSF and cortex Aß40 in both rats and cynomolgus monkeys following a single oral dose. Compound 9 represents a unique molecular shape among BACE inhibitors reported to potently lower central Aß in nonrodent preclinical species.

Discovery of a Novel, Potent Spirocyclic Series of γ-Secretase Inhibitors.

In the present paper, we described the design, synthesis, SAR, and biological profile of a novel spirocyclic sulfone series of γ-secretase inhibitors (GSIs) related to MRK-560. We utilized an additional spirocyclic ring system to stabilize the active chair conformation of the parent γ-secretase inhibitors. The resulting series is devoid of the CYP2C9 inhibition liability of MRK-560. A few representative analogs were assessed in a nontransgenic animal model of Alzheimer's disease (AD), demonstrating reduction of amyloid-ß (Aß) in the CNS after acute oral dosing. A spirocyclic phosphonate was identified as the optimal ring system for both potency and pharmacokinetics. Compared to GSIs studied in the clinic, representative spirocyclic phosphonate 18a(-) features improved selectivity for the inhibition of the PS-1 isoform of γ-secretase (33-fold vs PS-2), which may alleviate the adverse effect profile of the clinical GSIs.

MLi-2, a Potent, Selective, and Centrally Active Compound for Exploring the Therapeutic Potential and Safety of LRRK2 Kinase Inhibition.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of familial and sporadic Parkinson's disease (PD). That the most prevalent mutation, G2019S, leads to increased kinase activity has led to a concerted effort to identify LRRK2 kinase inhibitors as a potential disease-modifying therapy for PD. An internal medicinal chemistry effort identified several potent and highly selective compounds with favorable drug-like properties. Here, we characterize the pharmacological properties of cis-2,6-dimethyl-4-(6-(5-(1-methylcyclopropoxy)-1H-indazol-3-yl)pyrimidin-4-yl)morpholine (MLi-2), a structurally novel, highly potent, and selective LRRK2 kinase inhibitor with central nervous system activity. MLi-2 exhibits exceptional potency in a purified LRRK2 kinase assay in vitro (IC50 = 0.76 nM), a cellular assay monitoring dephosphorylation of LRRK2 pSer935 LRRK2 (IC50 = 1.4 nM), and a radioligand competition binding assay (IC50 = 3.4 nM). MLi-2 has greater than 295-fold selectivity for over 300 kinases in addition to a diverse panel of receptors and ion channels. Acute oral and subchronic dosing in MLi-2 mice resulted in dose-dependent central and peripheral target inhibition over a 24-hour period as measured by dephosphorylation of pSer935 LRRK2. Treatment of MitoPark mice with MLi-2 was well tolerated over a 15-week period at brain and plasma exposures >100× the in vivo plasma IC50 for LRRK2 kinase inhibition as measured by pSer935 dephosphorylation. Morphologic changes in the lung, consistent with enlarged type II pneumocytes, were observed in MLi-2-treated MitoPark mice. These data demonstrate the suitability of MLi-2 as a compound to explore LRRK2 biology in cellular and animal models.

Use of molecular modeling aided design to dial out hERG liability in adenosine A(2A) receptor antagonists.

Molecular modeling was performed on a triazolo quinazoline lead compound to help develop a series of adenosine A2A receptor antagonists with improved hERG profile. Superposition of the lead compound onto MK-499, a benchmark hERG inhibitor, combined with pKa calculations and measurement, identified terminal fluorobenzene to be responsible for hERG activity. Docking of the lead compound into an A2A crystal structure suggested that this group is located at a flexible, spacious, and solvent-exposed opening of the binding pocket, making it possible to tolerate various functional groups. Transformation analysis (MMP, matched molecular pair) of in-house available experimental data on hERG provided suggestions for modifications in order to mitigate this liability. This led to the synthesis of a series of compounds with significantly reduced hERG activity. The strategy used in the modeling work can be applied to other medicinal chemistry programs to help improve hERG profile.

Analysis of tau post-translational modifications in rTg4510 mice, a model of tau pathology.

BACKGROUND: Microtubule associated protein tau is the major component of the neurofibrillary tangles (NFTs) found in the brains of patients with Alzheimer's disease and several other neurodegenerative diseases. Tau mutations are associated with frontotemperal dementia with parkinsonism on chromosome 17 (FTDP-17). rTg4510 mice overexpress human tau carrying the P301L FTDP-17 mutation and develop robust NFT-like pathology at 4-5 months of age. The current study is aimed at characterizing the rTg4510 mice to better understand the genesis of tau pathology and to better enable the use of this model in drug discovery efforts targeting tau pathology. RESULTS: Using a panel of immunoassays, we analyzed the age-dependent formation of pathological tau in rTg4510 mice and our data revealed a steady age-dependent accumulation of pathological tau in the insoluble fraction of brain homogenates. The pathological tau was associated with multiple post-translational modifications including aggregation, phosphorylation at a wide variety of sites, acetylation, ubiquitination and nitration. The change of most tau species reached statistical significance at the age of 16 weeks. There was a strong correlation between the different post-translationally modified tau species in this heterogeneous pool of pathological tau. Total tau in the cerebrospinal fluid (CSF) displayed a multiphasic temporal profile distinct from the steady accumulation of pathological tau in the brain. Female rTg4510 mice displayed significantly more aggressive accumulation of pathological tau in the brain and elevation of total tau in CSF than their male littermates. CONCLUSION: The immunoassays described here were used to generate the most comprehensive description of the changes in various tau species across the lifespan of the rTg4510 mouse model. The data indicate that development of tauopathy in rTg4510 mice involves the accumulation of a pool of pathological tau that carries multiple post-translational modifications, a process that can be detected well before the histological detection of NFTs. Therapeutic treatment targeting tau should therefore aim to reduce all tau species associated with the pathological tau pool rather than reduce specific post-translational modifications. There is still much to learn about CSF tau in physiological and pathological processes in order to use it as a translational biomarker in drug discovery.

Characterization of a novel vasopressin V1b receptor antagonist, V1B-30N, in animal models of anxiety-like and depression-like behavior.

Overactivity of the hypothalamic-pituitary-adrenal (HPA) axis has been linked to affective disorders such as anxiety and depression. Dampening HPA activity has, therefore, been considered as a possible means of treating affective disorders. Given the important role of vasopressin in modulating the HPA axis, one strategy has focused on inhibiting activity of the vasopressin 1b (V1b) receptor. In animals, V1b receptor antagonists reduce plasma stress hormone levels and have been shown to have an anxiolytic-like effect. Recently, V1B-30N was identified as a highly potent V1b receptor antagonist with selectivity over other vasopressin receptors, which is evaluated here in rodent models of anxiety-like and depression-like behaviors. V1B-30N (1-30mg/kg, IP) dose-dependently reduced separation-induced vocalizations in rat pups without producing any sedative effects in the animals. Similarly, V1B-30N (3-30mg/kg, IP) dose-dependently reduced separation-induced vocalizations in guinea pig pups. In a conflict assay, conditioned lick suppression, V1B-30N (3-30mg/kg, IP) increased punished licking. To assess antidepressive-like properties, V1B-30N (1-30mg/kg) was tested in the mouse and rat forced-swim tests but was found to be inactive. These results are consistent with previous findings with other V1b antagonists, which suggest that acute pharmacological antagonism of the V1b receptor has anxiolytic-like but not antidepressant-like properties.

Effects of the selective adenosine A2A receptor antagonist, SCH 412348, on the parkinsonian phenotype of MitoPark mice.

Adenosine A2A receptors are predominantly localized on striatopallidal gamma-aminobutyric acid (GABA) neurons, where they are colocalized with dopamine D2 receptors and are involved in the regulation of movement. Adenosine A2A receptor antagonists have been evaluated as a novel treatment for Parkinson's disease and have demonstrated efficacy in a broad spectrum of pharmacological and toxicological rodent and primate models. Fewer studies have been performed to evaluate the efficacy of adenosine A2A receptor antagonists in genetic models of hypodopaminergic states. SCH 412348 is a potent and selective adenosine A2A receptor antagonist that shows efficacy in rodent and primate models of movement disorders. Here we evaluated the effects of SCH 412348 in the MitoPark mouse, a genetic model that displays a progressive loss of dopamine neurons. The dopamine cell loss is associated with a profound akinetic phenotype that is sensitive to levodopa (l-dopa). SCH 412348 (0.3-10mg/kg administered orally) dose dependently increased locomotor activity in the mice. Moreover, SCH 412348 retained its efficacy in the mice as motor impairment progressed (12-22 weeks of age), demonstrating that the compound was efficacious in mild to severe Parkinson's disease-like impairment in the mice. Additionally, SCH 412348 fully restored lost functionality in a measure of hind limb bradykinesia and partially restored functionality in a rotarod test. These findings provide further evidence of the anti-Parkinsonian effects of selective adenosine A2A receptor antagonists and predict that they will retain their efficacy in both mild and severe forms of motor impairment.

In Vivo Characterization of a Novel γ-Secretase Inhibitor SCH 697466 in Rodents and Investigation of Strategies for Managing Notch-Related Side Effects.

Substantial evidence implicates ß-amyloid (Aß) peptides in the etiology of Alzheimer's disease (AD). Aß is produced by the proteolytic cleavage of the amyloid precursor protein by ß- and γ-secretase suggesting that γ-secretase inhibition may provide therapeutic benefit for AD. Although many γ-secretase inhibitors have been shown to be potent at lowering Aß, some have also been shown to have side effects following repeated administration. All of these side effects can be attributed to altered Notch signaling, another γ-secretase substrate. Here we describe the in vivo characterization of the novel γ-secretase inhibitor SCH 697466 in rodents. Although SCH 697466 was effective at lowering Aß, Notch-related side effects in the intestine and thymus were observed following subchronic administration at doses that provided sustained and complete lowering of Aß. However, additional studies revealed that both partial but sustained lowering of Aßand complete but less sustained lowering of Aß were successful approaches for managing Notch-related side effects. Further, changes in several Notch-related biomarkers paralleled the side effect observations. Taken together, these studies demonstrated that, by carefully varying the extent and duration of Aß lowering by γ-secretase inhibitors, it is possible to obtain robust and sustained lowering of Aß without evidence of Notch-related side effects.

Structure activity relationship studies of tricyclic bispyran sulfone γ-secretase inhibitors.

An investigation is detailed of the structure activity relationships (SAR) of two sulfone side chains of compound (-)-1a (SCH 900229), a potent, PS1-selective γ-secretase inhibitor and clinical candidate for the treatment of Alzheimer's disease. Specifically, 4-CF(3) and 4-Br substituted arylsulfone analogs, (-)-1b and (-)-1c, are equipotent to compound (-)-1a. On the right hand side chain, linker size and terminal substituents of the pendant sulfone group are also investigated.