Synthesis and Structure-Activity Relationships of N-(4-Benzamidino)-Oxazolidinones: Potent and Selective Inhibitors of Kallikrein-Related Peptidase 6.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins. Aberrant expression of KLK6 has been found in different cancers and neurodegenerative diseases, and KLK6 is currently studied as a potential target in these pathologies. We report a novel series of KLK6 inhibitors discovered in a high-throughput screen within the European Lead Factory program. Structure-guided design based on docking studies enabled rapid progression of a hit cluster to inhibitors with improved potency, selectivity and pharmacokinetic properties. In particular, inhibitors 32 ((5R)-3-(4-carbamimidoylphenyl)-N-((S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) and 34 ((5R)-3-(6-carbamimidoylpyridin-3-yl)-N-((1S)-1-(naphthalen-1-yl)propyl)-2-oxooxazolidine-5-carboxamide) have single-digit nanomolar potency against KLK6, with over 25-fold and 100-fold selectivities against the closely related enzyme trypsin, respectively. The most potent compound, 32, effectively reduces KLK6-dependent invasion of HCT116 cells. The high potency in combination with good solubility and low clearance of 32 make it a good chemical probe for KLK6 target validation in vitro and potentially in vivo.

Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1.

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

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.

Identification of a Novel Positron Emission Tomography (PET) Ligand for Imaging ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE-1) in Brain.

Alzheimer's disease (AD) is characterized by accumulation of ß-amyloid (Aß) plaques and neurofibrillary tau tangles in the brain. ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a key role in the generation of Aß fragments via extracellular cleavage of the amyloid precursor protein (APP). We became interested in developing a BACE1 PET ligand to facilitate clinical assessment of BACE1 inhibitors and explore its potential in the profiling and selection of patients for AD trials. Using a set of PET ligand design parameters, compound 3 (PF-06684511) was rapidly identified as a lead with favorable in vitro attributes and structural handles for PET radiolabeling. Further evaluation in an LC-MS/MS "cold tracer" study in rodents revealed high specific binding to BACE1 in brain. Upon radiolabeling, [18F]3 demonstrated favorable brain uptake and high in vivo specificity in nonhuman primate (NHP), suggesting its potential for imaging BACE1 in humans.

Azetidine and Piperidine Carbamates as Efficient, Covalent Inhibitors of Monoacylglycerol Lipase.

Monoacylglycerol lipase (MAGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the CNS. MAGL catalyzes the conversion of 2-AG to arachidonic acid (AA), a precursor to the proinflammatory eicosannoids such as prostaglandins. Herein we describe highly efficient MAGL inhibitors, identified through a parallel medicinal chemistry approach that highlighted the improved efficiency of azetidine and piperidine-derived carbamates. The discovery and optimization of 3-substituted azetidine carbamate irreversible inhibitors of MAGL were aided by the generation of inhibitor-bound MAGL crystal structures. Compound 6, a highly efficient and selective MAGL inhibitor against recombinant enzyme and in a cellular context, was tested in vivo and shown to elevate central 2-AG levels at a 10 mg/kg dose.

The Discovery of a Novel Phosphodiesterase (PDE) 4B-Preferring Radioligand for Positron Emission Tomography (PET) Imaging.

As part of our effort in identifying phosphodiesterase (PDE) 4B-preferring inhibitors for the treatment of central nervous system (CNS) disorders, we sought to identify a positron emission tomography (PET) ligand to enable target occupancy measurement in vivo. Through a systematic and cost-effective PET discovery process, involving expression level (Bmax) and biodistribution determination, a PET-specific structure-activity relationship (SAR) effort, and specific binding assessment using a LC-MS/MS "cold tracer" method, we have identified 8 (PF-06445974) as a promising PET lead. Compound 8 has exquisite potency at PDE4B, good selectivity over PDE4D, excellent brain permeability, and a high level of specific binding in the "cold tracer" study. In subsequent non-human primate (NHP) PET imaging studies, [18F]8 showed rapid brain uptake and high target specificity, indicating that [18F]8 is a promising PDE4B-preferring radioligand for clinical PET imaging.

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.

Chemoproteomic profiling reveals that cathepsin D off-target activity drives ocular toxicity of ß-secretase inhibitors.

Inhibition of ß-secretase BACE1 is considered one of the most promising approaches for treating Alzheimer's disease. Several structurally distinct BACE1 inhibitors have been withdrawn from development after inducing ocular toxicity in animal models, but the target mediating this toxicity has not been identified. Here we use a clickable photoaffinity probe to identify cathepsin D (CatD) as a principal off-target of BACE1 inhibitors in human cells. We find that several BACE1 inhibitors blocked CatD activity in cells with much greater potency than that displayed in cell-free assays with purified protein. Through a series of exploratory toxicology studies, we show that quantifying CatD target engagement in cells with the probe is predictive of ocular toxicity in vivo. Taken together, our findings designate off-target inhibition of CatD as a principal driver of ocular toxicity for BACE1 inhibitors and more generally underscore the power of chemical proteomics for discerning mechanisms of drug action.

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.

Evidence of low toxicity of oil sands process-affected water to birds invites re-evaluation of avian protection strategies.

Exposure to water containing petroleum waste products can generate both overt and subtle toxicological responses in wildlife, including birds. Such exposure can occur in the tailings ponds of the mineable oil sands, which are located in Alberta, Canada, under a major continental flyway for waterfowl. Over the 40 year history of the industry, a few thousand bird deaths have been reported following contact with bitumen on the ponds, but a new monitoring programme demonstrated that many thousands of birds land annually without apparent harm. This new insight creates an urgent need for more information on the sublethal effects on birds from non-bitumen toxicants that occur in the water, including naphthenic acids, polycyclic aromatic hydrocarbons, heavy metals and salts. Ten studies have addressed the effects of oil sands process-affected water (OSPW), and none reported acute or substantial adverse health effects. Interpretive caution is warranted, however, because nine of the studies addressed reclaimed wetlands that received OSPW, not OSPW ponds per se, and differences between experimental and reference sites may have been reduced by shared sources of pollution in the surrounding air and water. Two studies examined eggs of birds nesting >100 km from the mine sites. Only one study exposed birds directly and repeatedly to OSPW and found no consistent differences between treated and control birds in blood-based health metrics. If it is true that aged forms of OSPW do not markedly affect the health of birds that land briefly on the ponds, then the extensiveness of current bird-deterrent programmes is unwarranted and could exert negative net environmental effects. More directed research on bird health is urgently needed, partly because birds that land on these ponds subsequently migrate to destinations throughout North America where they are consumed by both humans and wildlife predators.

Health of domestic mallards (Anas platyrhynchos domestica) following exposure to oil sands process-affected water.

Bitumen extraction from the oil sands of northern Alberta produces large volumes of process-affected water that contains substances toxic to wildlife. Recent monitoring has shown that tens of thousands of birds land on ponds containing this water annually, creating an urgent need to understand its effects on bird health. We emulated the repeated, short-term exposures that migrating water birds are thought to experience by exposing pekin ducks (Anas platyrhynchos domestica) to recycled oil sands process-affected water (OSPW). As indicators of health, we measured a series of physiological (electrolytes, metabolites, enzymes, hormones, and blood cells) and toxicological (metals and minerals) variables. Relative to controls, juvenile birds exposed to OSPW had higher potassium following the final exposure, and males had a higher thyroid hormone ratio (T3/T4). In adults, exposed birds had higher vanadium, and, following the final exposure, higher bicarbonate. Exposed females had higher bile acid, globulin, and molybdenum levels, and males, higher corticosterone. However, with the exception of the metals, none of these measures varied from available reference ranges for ducks, suggesting OSPW is not toxic to juvenile or adult birds after three and six weekly, 1 h exposures, but more studies are needed to know the generality of this result.

Design and selection parameters to accelerate the discovery of novel central nervous system positron emission tomography (PET) ligands and their application in the development of a novel phosphodiesterase 2A PET ligand.

To accelerate the discovery of novel small molecule central nervous system (CNS) positron emission tomography (PET) ligands, we aimed to define a property space that would facilitate ligand design and prioritization, thereby providing a higher probability of success for novel PET ligand development. Toward this end, we built a database consisting of 62 PET ligands that have successfully reached the clinic and 15 radioligands that failed in late-stage development as negative controls. A systematic analysis of these ligands identified a set of preferred parameters for physicochemical properties, brain permeability, and nonspecific binding (NSB). These preferred parameters have subsequently been applied to several programs and have led to the successful development of novel PET ligands with reduced resources and timelines. This strategy is illustrated here by the discovery of the novel phosphodiesterase 2A (PDE2A) PET ligand 4-(3-[(18)F]fluoroazetidin-1-yl)-7-methyl-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazine, [(18)F]PF-05270430 (5).

The synthesis and in vivo evaluation of [18F]PF-9811: a novel PET ligand for imaging brain fatty acid amide hydrolase (FAAH).

INTRODUCTION: Fatty acid amide hydrolase (FAAH) is responsible for the enzymatic degradation of the fatty acid amide family of signaling lipids, including the endogenous cannabinoid (endocannabinoid) anandamide. The involvement of the endocannabinoid system in pain and other nervous system disorders has made FAAH an attractive target for drug development. Companion molecular imaging probes are needed, however, to assess FAAH inhibition in the nervous system in vivo. We report here the synthesis and in vivo evaluation of [(18)F]PF-9811, a novel PET ligand for non-invasive imaging of FAAH in the brain. METHODS: The potency and selectivity of unlabeled PF-9811 were determined by activity-based protein profiling (ABPP) both in vitro and in vivo. [(18)F]PF-9811 was synthesized in a 3-step, one-pot reaction sequence, followed by HPLC purification. Biological evaluation was performed by biodistribution and dynamic PET imaging studies in male rats. The specificity of [(18)F]PF-9811 uptake was evaluated by pre-administration of PF-04457845, a potent and selective FAAH inhibitor, 1h prior to radiotracer injection. RESULTS: Biodistribution studies show good uptake (SUV~0.8 at 90 min) of [(18)F]PF-9811 in rat brain, with significant reduction of the radiotracer in all brain regions (37%-73% at 90 min) in blocking experiments. Dynamic PET imaging experiments in rat confirmed the heterogeneous uptake of [(18)F]PF-9811 in brain regions with high FAAH enzymatic activity, as well as statistically significant reductions in signal following pre-administration of the blocking compound PF-04457845. CONCLUSIONS: [(18)F]PF-9811 is a promising PET imaging agent for FAAH. Biodistribution and PET imaging experiments show that the tracer has good uptake in brain, regional heterogeneity, and specific binding as determined by blocking experiments with the highly potent and selective FAAH inhibitor, PF-04457845.

Chiral sulfinamide/achiral sulfonic acid cocatalyzed enantioselective protonation of enol silanes.

The application of chiral sulfinamides and achiral sulfonic acids as a cocatalyst system for enantioselective protonation reactions is described. Structurally simple, easily accessible sulfinamides were found to induce moderate-to-high ee's in the formation of 2-aryl-substituted cycloalkanones from the corresponding trimethylsilyl enol ethers.

Pd-catalyzed C-H bond functionalization on the indole and pyrrole nucleus.

This review details recent developments in the Pd-catalyzed C-H bond arylation and alkenylation of indoles and pyrroles, aromatic heterocycles that are frequently displayed in natural products and medicinal agents.

Oxidative Pd(II)-catalyzed C-H bond amination to carbazole at ambient temperature.

We report a new Pd(II)-catalyzed C-H bond amination reaction to form carbazoles, an important motif that is prevalent in a range of systems. The catalytic amination process operates under extremely mild conditions and produces carbazole products in good to excellent yields. Carbazoles possessing complex molecular architecture can also be formed using this reaction, highlighting its potential in natural product synthesis applications. Preliminary mechanistic investigations reveal the reaction proceeds through a Pd(II)/Pd(IV) manifold and that reductive elimination from a high oxidation state Pd(IV) complex facilitates the mild conditions of this transformation.

Bend-ribbon forming gamma-peptides.

Homo- and heterochiral tetrameric gamma-peptide derivatives in which the backbone is constrained by a five-membered ring populate a bend-ribbon conformation in solution stabilized by intramolecular hydrogen bonds.