Design and synthesis of a novel series of cyanoindole derivatives as potent γ-secretase modulators.

The discovery, design and synthesis of a new series of GSMs is described. The classical imidazole heterocycle has been replaced by a cyano group attached to an indole nucleus. The exploration of this series has led to compound 26-S which combined high in vitro and in vivo potency with an acceptable drug-like profile.

JNJ-67569762, A 2-Aminotetrahydropyridine-Based Selective BACE1 Inhibitor Targeting the S3 Pocket: From Discovery to Clinical Candidate.

The discovery of a novel 2-aminotetrahydropyridine class of BACE1 inhibitors is described. Their pKa and lipophilicity were modulated by a pending sulfonyl group, while good permeability and brain penetration were achieved via intramolecular hydrogen bonding. BACE1 selectivity over BACE2 was achieved in the S3 pocket by a novel bicyclic ring system. An optimization addressing reactive metabolite formation, cardiovascular safety, and CNS toxicity is described, leading to the clinical candidate JNJ-67569762 (12), which gave robust dose-dependent BACE1-mediated amyloid ß lowering without showing BACE2-dependent hair depigmentation in preclinical models. We show that 12 has a favorable projected human dose and PK and hence presented us with an opportunity to test a highly selective BACE1 inhibitor in humans. However, 12 was found to have a QT effect upon repeat dosing in dogs and its development was halted in favor of other selective leads, which will be reported in the future.

Direct nose to brain delivery of small molecules: critical analysis of data from a standardized in vivo screening model in rats.

The blood-brain barrier (BBB) is often a limiting factor for getting drugs in the brain. Bypassing the BBB by intranasal (IN), or also called nose to brain (NTB), route is an interesting and frequently investigated concept for brain drug delivery. However, despite the body of evidence for IN drug delivery in literature over the last decades, reproducibility and interpretation of animal data remain challenging. The objective of this project was to assess the feasibility and value of a standardized IN screening model in rats for the evaluation of direct brain delivery. A chemically diverse set of commercial and internal small molecules were tested in the in vivo model with different doses and/or formulations. Data were analyzed using different ways of ratio calculations: blood concentration at time of sacrifice, total exposure in blood (area under the curve, AUC) and the brain or olfactory bulb concentrations. The IN route was compared to another parenteral route to decide if there is potential direct brain transport. The results show that blood and tissue concentrations and ratios are highly variable and not always reproducible. Potential direct brain delivery was concluded for some compounds, however, sometimes depending on the analysis: using blood levels at sacrifice or AUC could lead to different conclusions. We conclude that a screening model for the evaluation of direct brain transport of small molecules is very difficult to achieve and a conclusion based on a limited number of animals with this variability is questionable.

Echocardiographic and hemodynamic indices of myocardial contractility simultaneously evaluated in telemetered beagle dogs: A HESI-sponsored cross-company evaluation.

INTRODUCTION: Alterations in cardiac contractility can have significant clinical implications, highlighting the need for early detection of potential liabilities. Pre-clinical methods to assess contractility are typically invasive and their translation to human measures of cardiac function are not well defined. Clinically, cardiac function is most often measured non-invasively using echocardiography. The objective of these studies was to introduce echocardiography into standard large animal cardiovascular safety pharmacology studies and determine the feasibility of this combination. METHODS: A consortia of laboratories combined their data sets for evaluation. At each site, telemetered beagle dogs, in a 4 × 4 Latin square crossover study design (n = 4), were administered either pimobendan (positive inotrope) or atenolol (negative inotrope) orally at clinically relevant dose levels. Standard telemetry parameters were collected (heart rate, mean arterial blood pressure, etc.) continuously over 24 h, as well as derived contractility endpoints: QA interval and LV +dP/dtmax. At Tmax, echocardiography was performed in conscious dogs with minimal restraint to collect contractility parameters: ejection fraction (EF) and fractional shortening (FS). RESULTS: Correlations between telemetry and echo contractility endpoints showed that, in general, a change in LV +dP/dtmax of 1000 mmHg/s translates to a 5.2% change in EF and a 4.2% change in FS. Poor correlations were shown between QA interval derived simultaneously, to both EF and FS. DISCUSSION: Comparing data from telemetry-only groups to those that included echocardiography collections showed no effect in the ability to interpret test article-related effects, providing the foundation for the inclusion of echocardiography without compromising standard telemetry data quality.

Trifluoromethyl Dihydrothiazine-Based ß-Secretase (BACE1) Inhibitors with Robust Central ß-Amyloid Reduction and Minimal Covalent Binding Burden.

The ß-site amyloid precursor protein cleaving enzyme 1 (BACE1, also known as ß-secretase) is a promising target for the treatment of Alzheimer's disease. A pKa lowering approach over the initial leads was adopted to mitigate hERG inhibition and P-gp efflux, leading to the design of 6-CF3 dihydrothiazine 8 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-cyanopicolinamide). Optimization of 8 led to the discovery of 15 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-(fluoromethoxy)pyrazine-2-carboxamide) with an excellent balance of potency, hERG inhibition, P-gp efflux, and metabolic stability. Oral administration of 8 elicited robust Aß reduction in dog even at 0.16 mg/kg. Reflecting the reduced hERG inhibitory activity, no QTc prolongation was observed at high doses. The potential for reactive metabolite formation of 15 was realized in a nucleophile trapping assay using [14 C]-KCN in human liver microsomes. Utilizing covalent binding (CVB) in human hepatocytes and the maximum projected human dosage, the daily CVB burden of 15 was calculated to be at an acceptable value of below 1 mg/day. However, hepatotoxicity was observed when 15 was subjected to a two-week tolerance study in dog, which prevented further evaluation of this compound.

Discovery of an Extremely Potent Thiazine-Based ß-Secretase Inhibitor with Reduced Cardiovascular and Liver Toxicity at a Low Projected Human Dose.

Genetic evidence points to deposition of amyloid-ß (Aß) as a causal factor for Alzheimer's disease. Aß generation is initiated when ß-secretase (BACE1) cleaves the amyloid precursor protein. Starting with an oxazine lead 1, we describe the discovery of a thiazine-based BACE1 inhibitor 5 with robust Aß reduction in vivo at low concentrations, leading to a low projected human dose of 14 mg/day where 5 achieved sustained Aß reduction of 80% at trough level.

2,4-Diaminoquinazolines as Dual Toll-like Receptor (TLR) 7/8 Modulators for the Treatment of Hepatitis B Virus.

A novel series of 2,4-diaminoquinazolines was identified as potent dual Toll-like receptor (TLR) 7 and 8 agonists with reduced off-target activity. The stereochemistry of the amino alcohol was found to influence the TLR7/8 selectivity with the ( R) isomer resulting in selective TLR8 agonism. Lead optimization toward a dual agonist afforded ( S)-3-((2-amino-8-fluoroquinazolin-4-yl)amino)hexanol 31 as a potent analog, being structurally different from previously described dual agonists ( McGowan J. Med. Chem. 2016 , 59 , 7936 ). Pharmacokinetic and pharmacodynamic (PK/PD) studies revealed the desired high first pass profile aimed at limiting systemic cytokine activation. In vivo pharmacodynamic studies with lead compound 31 demonstrated production of cytokines consistent with TLR7/8 activation in mice and cynomolgus monkeys and ex vivo inhibition of hepatitis B virus (HBV).

Optimization of 1,4-Oxazine ß-Secretase 1 (BACE1) Inhibitors Toward a Clinical Candidate.

In previous studies, the introduction of electron withdrawing groups to 1,4-oxazine BACE1 inhibitors reduced the p Ka of the amidine group, resulting in compound 2 that showed excellent in vivo efficacy, lowering Aß levels in brain and CSF. However, a suboptimal cardiovascular safety margin, based on QTc prolongation, prevented further progression. Further optimization resulted in the replacement of the 2-fluoro substituent by a CF3-group, which reduced hERG inhibition. This has led to compound 3, with an improved cardiovascular safety margin and sufficiently safe in GLP toxicity studies to progress into clinical trials.

Rational Design of Novel 1,3-Oxazine Based ß-Secretase (BACE1) Inhibitors: Incorporation of a Double Bond To Reduce P-gp Efflux Leading to Robust Aß Reduction in the Brain.

Accumulation of Aß peptides is a hallmark of Alzheimer's disease (AD) and is considered a causal factor in the pathogenesis of AD. ß-Secretase (BACE1) is a key enzyme responsible for producing Aß peptides, and thus agents that inhibit BACE1 should be beneficial for disease-modifying treatment of AD. Here we describe the discovery and optimization of novel oxazine-based BACE1 inhibitors by lowering amidine basicity with the incorporation of a double bond to improve brain penetration. Starting from a 1,3-dihydrooxazine lead 6 identified by a hit-to-lead SAR following HTS, we adopted a p Ka lowering strategy to reduce the P-gp efflux and the high hERG potential leading to the discovery of 15 that produced significant Aß reduction with long duration in pharmacodynamic models and exhibited wide safety margins in cardiovascular safety models. This compound improved the brain-to-plasma ratio relative to 6 by reducing P-gp recognition, which was demonstrated by a P-gp knockout mouse model.

Discovery of Potent and Centrally Active 6-Substituted 5-Fluoro-1,3-dihydro-oxazine ß-Secretase (BACE1) Inhibitors via Active Conformation Stabilization.

ß-Secretase (BACE1) has an essential role in the production of amyloid ß peptides that accumulate in patients with Alzheimer's disease (AD). Thus, inhibition of BACE1 is considered to be a disease-modifying approach for the treatment of AD. Our hit-to-lead efforts led to a cellular potent 1,3-dihydro-oxazine 6, which however inhibited hERG and showed high P-gp efflux. The close analogue of 5-fluoro-oxazine 8 reduced P-gp efflux; further introduction of electron withdrawing groups at the 6-position improved potency and also mitigated P-gp efflux and hERG inhibition. Changing to a pyrazine followed by optimization of substituents on both the oxazine and the pyrazine culminated in 24 with robust Aß reduction in vivo at low doses as well as reduced CYP2D6 inhibition. On the basis of the X-ray analysis and the QM calculation of given dihydro-oxazines, we reasoned that the substituents at the 6-position as well as the 5-fluorine on the oxazine would stabilize a bioactive conformation to increase potency.

Young to Middle-Aged Dogs with High Amyloid-ß Levels in Cerebrospinal Fluid are Impaired on Learning in Standard Cognition tests.

Understanding differences in Alzheimer's disease biomarkers before the pathology becomes evident can contribute to an improved understanding of disease pathogenesis and treatment. A decrease in amyloid-ß (Aß)42 in cerebrospinal fluid (CSF) is suggested to be a biomarker for Aß deposition in brain. However, the relevance of CSF Aß levels prior to deposition is not entirely known. Dogs are similar to man with respect to amyloid-ß protein precursor (AßPP)-processing, age-related amyloid plaque deposition, and cognitive dysfunction. In the current study, we evaluated the relation between CSF Aß42 levels and cognitive performance in young to middle-aged dogs (1.5-7 years old). Additionally, CSF sAßPPα and sAßPPß were measured to evaluate AßPP processing, and CSF cytokines were measured to determine the immune status of the brain. We identified two groups of dogs showing consistently low or high CSF Aß42 levels. Based on prior studies, it was assumed that at this age no cerebral amyloid plaques were likely to be present. The cognitive performance was evaluated in standard cognition tests. Low or high Aß concentrations coincided with low or high sAßPPα, sAßPPß, and CXCL-1 levels, respectively. Dogs with high Aß concentrations showed significant learning impairments on delayed non-match to position (DNMP), object discrimination, and reversal learning compared to dogs with low Aß concentrations. Our data support the hypothesis that high levels of CSF Aß in dogs coincide with lower cognitive performance prior to amyloid deposition. Further experiments are needed to investigate this link, as well as the relevance with respect to Alzheimer's disease pathology progression.

Identification and Optimization of Pyrrolo[3,2-d]pyrimidine Toll-like Receptor 7 (TLR7) Selective Agonists for the Treatment of Hepatitis B.

Pyrrolo[3,2-d]pyrimidines were identified as a new series of potent and selective TLR7 agonists. Compounds were optimized for their activity and selectivity over TLR8. This presents an advantage over recently described scaffolds that have residual TLR8 activity, which may be detrimental to the tolerability of the candidate drug. Oral administration of the lead compound 54 effectively induced a transient interferon stimulated gene (ISG) response in mice and cynomolgus monkeys. We aimed for a high first pass effect, limiting cytokine induction systemically, and demonstrated the potential for the immunotherapy of viral hepatitis.

Novel Pyrimidine Toll-like Receptor 7 and 8 Dual Agonists to Treat Hepatitis B Virus.

Toll-like receptor (TLR) 7 and 8 agonists can potentially be used in the treatment of viral infections and are particularly promising for chronic hepatitis B virus (HBV) infection. An internal screening effort identified a pyrimidine Toll-like receptor 7 and 8 dual agonist. This provided a novel alternative over the previously reported adenine and pteridone type of agonists. Structure-activity relationship, lead optimization, in silico docking, pharmacokinetics, and demonstration of ex vivo and in vivo cytokine production of the lead compound are presented.

Comparison of two different methods for measurement of amyloid-ß peptides in cerebrospinal fluid after BACE1 inhibition in a dog model.

Beta-secretase is the first cleavage enzyme of amyloid-ß protein precursor (AßPP) in the amyloidogenic pathway, leading to the formation of the plaque forming Amyloid-ß (Aß)1-42 peptide. BACE (beta-site AßPP cleaving enzyme) 1 inhibition is therefore considered to be a promising disease modifying therapy for Alzheimer's disease. An early assessment of the in vivo activity of BACE inhibitors was done in dogs since AßPP processing is the same as in humans and this species easily enables longitudinal cerebrospinal fluid (CSF) sampling. Aß changes in CSF compared to baseline are used to evaluate target engagement of the compounds. Levels of Aß1-37, Aß1-38, Aß1-40, and Aß1-42 in CSF are measured with immunoassay (Mesoscale electrochemiluminescence technology) and with an ultra high-performance liquid chromatography mass spectrometry (UPLC-MS/MS). Two experimental BACE inhibitors were evaluated. With the immunoassay, a dose dependent decrease is observed for all four Aß peptides. Measurements with the UPLC-MS/MS are in line with the immunoassay for Aß1-37, Aß1-38, and Aß1-40, however, for Aß1-42, differences are sometimes observed when comparing to changes seen in the other peptides with UPLC-MS/MS and with immunoassay results. Generally lower concentrations are measured with immunoassay. The reason for these differences is still unknown. Aß1-42 is more prone to form aggregates compared to the other peptides. One hypothesis could be that while the immunoassay only measures free Aß, bound and aggregated Aß peptides are at least partially dissolved with the UPLC-MS/MS method, since acetonitrile is added to the CSF samples. This increases variability in the concentration of Aß peptide measured with UPLC-MS/MS, especially for Aß1-42, potentially masking the compound effect on Aß1-42 levels.

A translational assessment of preclinical versus clinical tools for the measurement of cardiac contractility: comparison of LV dP/dt(max) with echocardiography in telemetry implanted beagle dogs.

INTRODUCTION: Regarding evaluation of drug-induced changes in left ventricular contractility in safety pharmacology there is still a gap in knowledge between preclinically and clinically used measurements. METHODS: As a step towards translation of preclinical to clinical outcomes, this study in telemetered dogs was initiated to compare indexes of contractility, such as LV dP/dt(max) (contractility measured as the maximum raise of pressure in the left ventricle) and LV dP/dt(max)/P (contractility measured as the maximum raise of pressure in the left ventricle, corrected for pressure) (telemetry; both commonly preclinically used) and EF (ejection fraction) and FS (fractional shortening) (echocardiography; both commonly clinically used). Different inotropic states were induced by minoxidil, milrinone, isoprenaline, clonidine, atenolol and verapamil. RESULTS: Both techniques demonstrated reproducible changes in contractility which showed a clear linear association. A change in LV dP/dt(max) of 1000 mmHg/s (in the range of 2500 to 7500 mmHg/s; in healthy dogs) corresponded with a change in ejection fraction of approximately 7% and a fractional shortening of approximately 6%. A change of 10/s LV dP/dt(max)/P (in the range of 35 to 85/s; in healthy dogs) corresponded with a change in ejection fraction of approximately 7% and a fractional shortening of 7%. DISCUSSION: The correlation found in this study could potentially enable a better--translational--assessment of the clinical relevance of changes in contractility indices measured with telemetry devices in preclinical safety studies.

BACE1 inhibition induces a specific cerebrospinal fluid ß-amyloid pattern that identifies drug effects in the central nervous system.

BACE1 is a key enzyme for amyloid-ß (Aß) production, and an attractive therapeutic target in Alzheimer's disease (AD). Here we report that BACE1 inhibitors have distinct effects on neuronal Aß metabolism, inducing a unique pattern of secreted Aß peptides, analyzed in cell media from amyloid precursor protein (APP) transfected cells and in cerebrospinal fluid (CSF) from dogs by immunoprecipitation-mass spectrometry, using several different BACE1 inhibitors. Besides the expected reductions in Aß1-40 and Aß1-42, treatment also changed the relative levels of several other Aß isoforms. In particular Aß1-34 decreased, while Aß5-40 increased, and these changes were more sensitive to BACE1 inhibition than the changes in Aß1-40 and Aß1-42. The effects on Aß5-40 indicate the presence of a BACE1 independent pathway of APP degradation. The described CSF Aß pattern may be used as a pharmacodynamic fingerprint to detect biochemical effects of BACE1-therapies in clinical trials, which might accelerate development of novel therapies.