Design and Synthesis of Clinical Candidate PF-06751979: A Potent, Brain Penetrant, ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Lacking Hypopigmentation.

A major challenge in the development of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors for the treatment of Alzheimer's disease is the alignment of potency, drug-like properties, and selectivity over related aspartyl proteases such as Cathepsin D (CatD) and BACE2. The potential liabilities of inhibiting BACE2 chronically have only recently begun to emerge as BACE2 impacts the processing of the premelanosome protein (PMEL17) and disrupts melanosome morphology resulting in a depigmentation phenotype. Herein, we describe the identification of clinical candidate PF-06751979 (64), which displays excellent brain penetration, potent in vivo efficacy, and broad selectivity over related aspartyl proteases including BACE2. Chronic dosing of 64 for up to 9 months in dog did not reveal any observation of hair coat color (pigmentation) changes and suggests a key differentiator over current BACE1 inhibitors that are nonselective against BACE2 in later stage clinical development.

Aminomethyl-Derived Beta Secretase (BACE1) Inhibitors: Engaging Gly230 without an Anilide Functionality.

A growing subset of ß-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogues with an excellent balance of ADME properties and potency; however, potential drug-drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound 16, which exhibits robust in vivo efficacy as a BACE1 inhibitor.

Utilizing structures of CYP2D6 and BACE1 complexes to reduce risk of drug-drug interactions with a novel series of centrally efficacious BACE1 inhibitors.

In recent years, the first generation of ß-secretase (BACE1) inhibitors advanced into clinical development for the treatment of Alzheimer's disease (AD). However, the alignment of drug-like properties and selectivity remains a major challenge. Herein, we describe the discovery of a novel class of potent, low clearance, CNS penetrant BACE1 inhibitors represented by thioamidine 5. Further profiling suggested that a high fraction of the metabolism (>95%) was due to CYP2D6, increasing the potential risk for victim-based drug-drug interactions (DDI) and variable exposure in the clinic due to the polymorphic nature of this enzyme. To guide future design, we solved crystal structures of CYP2D6 complexes with substrate 5 and its corresponding metabolic product pyrazole 6, which provided insight into the binding mode and movements between substrate/inhibitor complexes. Guided by the BACE1 and CYP2D6 crystal structures, we designed and synthesized analogues with reduced risk for DDI, central efficacy, and improved hERG therapeutic margins.

Discovery of a series of efficient, centrally efficacious BACE1 inhibitors through structure-based drug design.

The identification of centrally efficacious ß-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease (AD) has historically been thwarted by an inability to maintain alignment of potency, brain availability, and desired absorption, distribution, metabolism, and excretion (ADME) properties. In this paper, we describe a series of truncated, fused thioamidines that are efficiently selective in garnering BACE1 activity without simultaneously inhibiting the closely related cathepsin D or negatively impacting brain penetration and ADME alignment, as exemplified by 36. Upon oral administration, these inhibitors exhibit robust brain availability and are efficacious in lowering central Amyloid ß (Aß) levels in mouse and dog. In addition, chronic treatment in aged PS1/APP mice effects a decrease in the number and size of Aß-derived plaques. Most importantly, evaluation of 36 in a 2-week exploratory toxicology study revealed no accumulation of autofluorescent material in retinal pigment epithelium or histology findings in the eye, issues observed with earlier BACE1 inhibitors.

Cerebrospinal fluid ß-Amyloid turnover in the mouse, dog, monkey and human evaluated by systematic quantitative analyses.

BACKGROUND: Reducing brain ß-amyloid (Aß) via inhibition of ß-secretase, or inhibition/modulation of γ-secretase, has been widely pursued as a potential disease-modifying treatment for Alzheimer's disease. Compounds that act through these mechanisms have been screened and characterized with Aß lowering in the brain and/or cerebrospinal fluid (CSF) as the primary pharmacological end point. Interpretation and translation of the pharmacokinetic (PK)/pharmacodynamic (PD) relationship for these compounds is complicated by the relatively slow Aß turnover process in these compartments. OBJECTIVE: To understand Aß turnover kinetics in preclinical species and humans. METHODS: We collected CSF Aß dynamic data after ß- or γ-secretase inhibitor treatment from in-house experiments and the public domain, and analyzed the data using PK/PD modeling to obtain CSF Aß turnover rates (kout) in the mouse, dog, monkey and human. RESULTS: The kout for CSF Aß40 follows allometry (kout = 0.395 × body weight(-0.351)). The kout for CSF Aß40 is approximately 2-fold higher than the turnover of CSF in rodents, but in higher species, the two are comparable. CONCLUSION: The turnover of CSF Aß40 was systematically examined, for the first time, in multiple species through quantitative modeling of multiple data sets. Our result suggests that the clearance mechanisms for CSF Aß in rodents may be different from those in the higher species. The understanding of Aß turnover has considerable implications for the discovery and development of Aß-lowering therapeutics, as illustrated from the perspectives of preclinical PK/PD characterization and preclinical-to-clinical translation.

Cerebrospinal fluid amyloid-ß (Aß) as an effect biomarker for brain Aß lowering verified by quantitative preclinical analyses.

Reducing the generation of amyloid-ß (Aß) in the brain via inhibition of ß-secretase or inhibition/modulation of γ-secretase has been pursued as a potential disease-modifying treatment for Alzheimer's disease. For the discovery and development of ß-secretase inhibitors (BACEi), γ-secretase inhibitors (GSI), and γ-secretase modulators (GSM), Aß in cerebrospinal fluid (CSF) has been presumed to be an effect biomarker for Aß lowering in the brain. However, this presumption is challenged by the lack of quantitative understanding of the relationship between brain and CSF Aß lowering. In this study, we strived to elucidate how the intrinsic pharmacokinetic (PK)/pharmacodynamic (PD) relationship for CSF Aß lowering is related to that for brain Aß through quantitative modeling of preclinical data for numerous BACEi, GSI, and GSM across multiple species. Our results indicate that the intrinsic PK/PD relationship in CSF is predictive of that in brain, at least in the postulated pharmacologically relevant range, with excellent consistency across mechanisms and species. As such, the validity of CSF Aß as an effect biomarker for brain Aß lowering is confirmed preclinically. Meanwhile, we have been able to reproduce the dose-dependent separation between brain and CSF effect profiles using simulations. We further discuss the implications of our findings to drug discovery and development with regard to preclinical PK/PD characterization and clinical prediction of Aß lowering in the brain.

Application of the bicyclo[1.1.1]pentane motif as a nonclassical phenyl ring bioisostere in the design of a potent and orally active γ-secretase inhibitor.

Replacement of the central, para-substituted fluorophenyl ring in the γ-secretase inhibitor 1 (BMS-708,163) with the bicyclo[1.1.1]pentane motif led to the discovery of compound 3, an equipotent enzyme inhibitor with significant improvements in passive permeability and aqueous solubility. The modified biopharmaceutical properties of 3 translated into excellent oral absorption characteristics (~4-fold ↑ C(max) and AUC values relative to 1) in a mouse model of γ-secretase inhibition. In addition, SAR studies into other fluorophenyl replacements indicate the intrinsic advantages of the bicyclo[1.1.1]pentane moiety over conventional phenyl ring replacements with respect to achieving an optimal balance of properties (e.g., γ-secretase inhibition, aqueous solubility/permeability, in vitro metabolic stability). Overall, this work enhances the scope of the [1.1.1]-bicycle beyond that of a mere "spacer" unit and presents a compelling case for its broader application as a phenyl group replacement in scenarios where the aromatic ring count impacts physicochemical parameters and overall drug-likeness.

Metabolism-directed design of oxetane-containing arylsulfonamide derivatives as γ-secretase inhibitors.

A metabolism-based approach toward the optimization of a series of N-arylsulfonamide-based γ-secretase inhibitors is reported. The lead cyclohexyl analogue 6 suffered from extensive oxidation on the cycloalkyl motif by cytochrome P450 3A4, translating into poor human liver microsomal stability. Knowledge of the metabolic pathways of 6 triggered a structure-activity relationship study aimed at lowering lipophilicity through the introduction of polarity. This effort led to several tetrahydropyran and tetrahydrofuran analogues, wherein the 3- and 4-substituted variants exhibited greater microsomal stability relative to their 2-substituted counterparts. Further reduction in lipophilicity led to the potent γ-secretase inhibitor and 3-substituted oxetane 1 with a reduced propensity toward oxidative metabolism, relative to its 2-substituted isomer. The slower rates of metabolism with 3-substituted cyclic ethers most likely originate from reductions in lipophilicity and/or unfavorable CYP active site interactions with the heteroatom. Preliminary animal pharmacology studies with a representative oxetane indicate that the series is generally capable of lowering Aß in vivo. As such, the study also illustrates the improvement in druglikeness of molecules through the use of the oxetane motif.

Quantitative pharmacokinetic/pharmacodynamic analyses suggest that the 129/SVE mouse is a suitable preclinical pharmacology model for identifying small-molecule γ-secretase inhibitors.

Alzheimer's disease (AD) poses a serious public health threat to the United States. Disease-modifying drugs slowing AD progression are in urgent need, but they are still unavailable. According to the amyloid cascade hypothesis, inhibition of ß- or γ-secretase, key enzymes for the production of amyloid ß (Aß), may be viable mechanisms for the treatment of AD. For the discovery of γ-secretase inhibitors (GSIs), the APP-overexpressing Tg2576 mouse has been the preclinical model of choice, in part because of the ease of detection of Aß species in its brain, plasma, and cerebrospinal fluid (CSF). Some biological observations and practical considerations, however, argue against the use of the Tg2576 mouse. We reasoned that an animal model would be suitable for GSI discovery if the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of a compound for Aß lowering in this model is predictive of that in human. In this study, we assessed whether the background 129/SVE strain is a suitable preclinical pharmacology model for identifying new GSIs by evaluating the translatability of the intrinsic PK/PD relationships for brain and CSF Aß across the Tg2576 and 129/SVE mouse and human. Using semimechanistically based PK/PD modeling, our analyses indicated that the intrinsic PK/PD relationship for brain Aßx-42 and CSF Aßx-40 in the 129/SVE mouse is indicative of that for human CSF Aß. This result, in conjunction with practical considerations, strongly suggests that the 129/SVE mouse is a suitable model for GSI discovery. Concurrently, the necessity and utilities of PK/PD modeling for rational interpretation of Aß data are established.