Design and Synthesis of Clinical Candidate PF-06852231 (CVL-231): A Brain Penetrant, Selective, Positive Allosteric Modulator of the M4 Muscarinic Acetylcholine Receptor.

Selective activation of the M4 muscarinic acetylcholine receptor subtype offers a novel strategy for the treatment of psychosis in multiple neurological disorders. Although the development of traditional muscarinic activators has been stymied due to pan-receptor activation, muscarinic receptor subtype selectivity can be achieved through the utilization of a subtype of a unique allosteric site. A major challenge in capitalizing on this allosteric site to date has been achieving a balance of suitable potency and brain penetration. Herein, we describe the design of a brain penetrant series of M4 selective positive allosteric modulators (PAMs), ultimately culminating in the identification of 21 (PF-06852231, now CVL-231/emraclidine), which is under active clinical development as a novel mechanism and approach for the treatment of schizophrenia.

PF-04859989 as a template for structure-based drug design: identification of new pyrazole series of irreversible KAT II inhibitors with improved lipophilic efficiency.

The structure-based design, synthesis, and biological evaluation of a new pyrazole series of irreversible KAT II inhibitors are described herein. The modification of the inhibitor scaffold of 1 and 2 from a dihydroquinolinone core to a tetrahydropyrazolopyridinone core led to discovery of a new series of potent KAT II inhibitors with excellent physicochemical properties. Compound 20 is the most potent and lipophilically efficient of these new pyrazole analogs, with a k(inact)/K(i) value of 112,000 M(-1)s(-1) and lipophilic efficiency (LipE) of 8.53. The X-ray crystal structure of 20 with KAT II demonstrates key features that contribute to this remarkable potency and binding efficiency.

Heterocyclic methylsulfone hydroxamic acid LpxC inhibitors as Gram-negative antibacterial agents.

The synthesis and antibacterial activity of heterocyclic methylsulfone hydroxamates is presented. Compounds in this series are potent inhibitors of the LpxC enzyme, a key enzyme involved in the production of lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria. SAR evaluation of compounds in this series revealed analogs with potent antibacterial activity against challenging Gram-negative species such as Pseudomonas aeruginosa and Klebsiella pneumoniae.

Influence of activated charcoal on desorption kinetics and biodegradation of phenanthrene in soil.

The observed strong sorption of polycyclic aromatic hydrocarbons (PAHs) to black carbon (BC) presents potential implications for PAH bioaccessibility in soils. The effects of BC on the desorption kinetics and mineralization of phenanthrene in four soils was investigated after 1, 25, 50, and 100 d soil-PAH contact time, using sequential hydroxypropyl-ß-cyclodextrin (HPCD) extractions in soils amended with 0, 0.1, 1, and 5% (dry wt. soil) activated charcoal (AC, a form of BC). The rapidly (%F(rap)) and slowly (%F(slow)) desorbing phenanthrene fractions and their rate constants were determined using a first-order two-compartment (biphasic) desorption model. A minimum 7.8-fold decrease in %F(rap) occurred when AC was increased from 0 to 5%, with a corresponding increase in %F(slow). Desorption rate constants followed the progression k(rap) (% h(-1)) > k(slow) (% h(-1)) and were in the order of 10(-1) to 10(-2) and 10(-3) to 10(-4), respectively. Linear regressions between %F(rap) and the fractions degraded by a phenanthrene-degrading inoculum (%F(min)) indicated that slopes did not approximate 1 at concentrations greater than 0% AC; %F(min) often exceeded %F(rap), indicating a fraction of sorbed phenanthrene (%F(slow)) remained microbially accessible. Therefore, HPCD-desorption kinetics alone may not be an adequate basis for the prediction of the bioaccessibility of PAHs to microorganisms or bioremediation potential in AC-amended soils.

Idiopathic Intracranial Hypertension: A Case Study of Patient Engagement in the Treatment of a Chronic Disease.

Idiopathic intracranial hypertension is a rare neurological disorder characterized by increased intracranial pressure, which can lead to visual loss and headaches. While medical therapy exists, weight loss is the only disease-modifying treatment. Weight loss is the only therapy that leads to sustained resolution of papilledema. Involving the patient in their disease management through patient engagement is a way to improve disease outcomes, and strengthen the therapeutic relationship. This feature discusses an overview of the disease, a patient's experience, and a physician's perspective.

Tuberculosis among persons born in the Philippines and living in the United States, 2000-2007.

OBJECTIVES: We examined demographic, clinical, and treatment outcome characteristics of Filipinos with tuberculosis (TB) in the United States. METHODS: We calculated TB case rates from US Census Bureau population estimates and National Tuberculosis Surveillance System data for US-born non-Hispanic Whites and for US residents born in the Philippines, India, China, Cambodia, Vietnam, Pakistan, and Korea--countries that are major contributors to the TB burden in the United States. We compared Filipinos with the other groups through univariate and multivariate analyses. RESULTS: Of 45,504 TB patients, 15.5% were Filipinos; 43.0% were other Asian/Pacific Islander groups; and 41.6% were Whites. Per 100 000 persons in 2007, the TB rate was 73.5 among Cambodians, 54.0 among Vietnamese, 52.1 among Filipinos, and 0.9 among Whites. Filipinos were more likely than other groups to be employed as health care workers and to have used private health care providers but less likely to be HIV positive and to be offered HIV testing. CONCLUSIONS: The relatively high TB rate among Filipinos indicates that TB control strategies should target this population. Providers should be encouraged to offer HIV testing to all TB patients.

A general strategy for the synthesis of cyclic N-aryl hydroxamic acids via partial nitro group reduction.

We describe a generalized approach to stereocontrolled synthesis of substituted cyclic hydroxamic acids (3-amino-1-hydroxy-3,4-dihydroquinolinones) by selective reduction of substituted 2-nitrophenylalanine substrates. Compounds in this series have antibacterial properties and have also recently been reported as KAT II inhibitors. The key nitrophenyl alanine intermediates are prepared enantioselectively in excellent yield by phase transfer catalyzed alkylation of the corresponding nitrobenzyl bromides. The scope and limitations of the reductive cyclization transformation have been explored with attention to the effects of substitution pattern and electronics on reaction efficiency and byproduct formation. In addition, a novel activated trifluoroethyl ester cyclization strategy has been developed as an alternate approach to the most sterically demanding systems in this series.

Intramolecular Ring-Opening Decomposition of Aryl Azetidines.

The ring strain present in azetidines can lead to undesired stability issues. Herein, we described a series of N-substituted azetidines which undergo an acid-mediated intramolecular ring-opening decomposition via nucleophilic attack of a pendant amide group. Studies were conducted to understand the decomposition mechanism enabling the design of stable analogues.

Identification and Development of an Irreversible Monoacylglycerol Lipase (MAGL) Positron Emission Tomography (PET) Radioligand with High Specificity.

Monoacylglycerol lipase (MAGL), a serine hydrolase extensively expressed throughout the brain, serves as a key gatekeeper regulating the tone of endocannabinoid signaling. Preclinically, inhibition of MAGL is known to provide therapeutic benefits for a number of neurological disorders. The availability of a MAGL-specific positron emission tomography (PET) ligand would considerably facilitate the development and clinical characterization of MAGL inhibitors via noninvasive and quantitative PET imaging. Herein, we report the identification of the potent and selective irreversible MAGL inhibitor 7 (PF-06809247) as a suitable radioligand lead, which upon radiolabeling was found to exhibit a high level of MAGL specificity; this enabled cross-species measurement of MAGL brain expression (Bmax), assessment of in vivo binding in the rat, and nonhuman primate PET imaging.

Toward the discovery of potent inhibitors of botulinum neurotoxin A: development of a robust LC MS based assay operational from low to subnanomolar enzyme concentrations.

The development of a sensitive, yet reliable assay for the analysis of botulinum neurotoxin A (BoNT/A) inhibitors is described; using this assay a new protease inhibitor was characterized and found to be one of the most potent inhibitors reported to date.

Discovery of Trifluoromethyl Glycol Carbamates as Potent and Selective Covalent Monoacylglycerol Lipase (MAGL) Inhibitors for Treatment of Neuroinflammation.

Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log  D, improving solubility, and minimizing chemical lability. Compound 15 achieves excellent CNS exposure, extended 2-AG elevation effect in vivo, and decreased brain inflammatory markers in response to an inflammatory challenge.

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.

Discovery and characterization of a novel class of pyrazolopyrimidinedione tRNA synthesis inhibitors.

A high-throughput phenotypic screen for novel antibacterial agents led to the discovery of a novel pyrazolopyrimidinedione, PPD-1, with preferential activity against methicillin-resistant Staphylococcus aureus (MRSA). Resistance mapping revealed the likely target of inhibition to be lysyl tRNA synthetase (LysRS). Preliminary structure-activity relationship (SAR) studies led to an analog, PPD-2, which gained Gram-negative antibacterial activity at the expense of MRSA activity and resistance to this compound mapped to prolyl tRNA synthetase (ProRS). These targets of inhibition were confirmed in vitro, with PPD-1 showing IC50s of 21.7 and 35 µM in purified LysRS and ProRS enzyme assays, and PPD-2, 151 and 0.04 µM, respectively. The highly attractive chemical properties of these compounds combined with intriguing preliminary SAR suggest that further exploration of this compelling novel series is warranted.

The first Pummerer cyclisations on solid phase. Convenient construction of oxindoles enabled by a sulfur-link to resin.

Alpha-sulfanyl N-aryl acetamides, attached to resin via the sulfur atom, undergo efficient Pummerer cyclisation upon activation of the sulfur link, to give oxindoles; heterocyclic products can be cleaved from the resin in a traceless manner using samarium(II) iodide.

Chemoproteomics demonstrates target engagement and exquisite selectivity of the clinical phosphodiesterase 10A inhibitor MP-10 in its native environment.

Phosphodiesterases (PDEs) regulate the levels of the second messengers cAMP and cGMP and are important drug targets. PDE10A is highly enriched in medium spiny neurons of the striatum and is an attractive drug target for the treatment of basal ganglia diseases like schizophrenia, Parkinson's disease, or Huntington's disease. Here we describe the design, synthesis, and application of a variety of chemical biology probes, based on the first clinically tested PDE10A inhibitor MP-10, which were used to characterize the chemoproteomic profile of the clinical candidate in its native environment. A clickable photoaffinity probe was used to measure target engagement of MP-10 and revealed differences between whole cell and membrane preparations. Moreover, our results illustrate the importance of the linker design in the creation of functional probes. Biotinylated affinity probes allowed identification of drug-interaction partners in rodent and human tissue and quantitative mass spectrometry analysis revealed highly specific binding of MP-10 to PDE10A with virtually no off-target binding. The profiling of PDE10A chemical biology probes described herein illustrates a strategy by which high affinity inhibitors can be converted into probes for determining selectivity and target engagement of drug candidates in complex biological matrices from native sources.

Impact of black carbon on the bioaccessibility of organic contaminants in soil.

The ability of carbonaceous geosorbents (CGs) such as black carbon (BC) to extensively sorb many common environmental contaminants suggests that they potentially possesses qualities useful to the sequestration of harmful xenobiotics within contaminated land. Presently, however, there is limited understanding of the implications for the bioaccessibility, mobility and environmental risk of organic contaminants while sorbed to BC in soil and sediment, in addition to the inherent toxicity of BC itself to terrestrial flora and fauna. We review both the processes involved in and factors influencing BC sorption characteristics, and ultimately consider the impacts BC will have for bioavailability/bioaccessibility, toxicity and risk assessment/remediation of contaminated land. We conclude that while the application of BC is promising, additional work on both their toxicity effects and long-term stability is required before their full potential as a remediation agent can be safely exploited.

Structure-Based Design of Irreversible Human KAT II Inhibitors: Discovery of New Potency-Enhancing Interactions.

A series of aryl hydroxamates recently have been disclosed as irreversible inhibitors of kynurenine amino transferase II (KAT II), an enzyme that may play a role in schizophrenia and other psychiatric and neurological disorders. The utilization of structure-activity relationships (SAR) in conjunction with X-ray crystallography led to the discovery of hydroxamate 4, a disubstituted analogue that has a significant potency enhancement due to a novel interaction with KAT II. The use of k inact/K i to assess potency was critical for understanding the SAR in this series and for identifying compounds with improved pharmacodynamic profiles.

Discovery of Brain-Penetrant, Irreversible Kynurenine Aminotransferase II Inhibitors for Schizophrenia.

Kynurenine aminotransferase (KAT) II has been identified as a potential new target for the treatment of cognitive impairment associated with schizophrenia and other psychiatric disorders. Following a high-throughput screen, cyclic hydroxamic acid PF-04859989 was identified as a potent and selective inhibitor of human and rat KAT II. An X-ray crystal structure and (13)C NMR studies of PF-04859989 bound to KAT II have demonstrated that this compound forms a covalent adduct with the enzyme cofactor, pyridoxal phosphate (PLP), in the active site. In vivo pharmacokinetic and efficacy studies in rat show that PF-04859989 is a brain-penetrant, irreversible inhibitor and is capable of reducing brain kynurenic acid by 50% at a dose of 10 mg/kg (sc). Preliminary structure-activity relationship investigations have been completed and have identified the positions on this scaffold best suited to modification for further optimization of this novel series of KAT II inhibitors.

Pyridone methylsulfone hydroxamate LpxC inhibitors for the treatment of serious gram-negative infections.

The synthesis and biological activity of a new series of LpxC inhibitors represented by pyridone methylsulfone hydroxamate 2a is presented. Members of this series have improved solubility and free fraction when compared to compounds in the previously described biphenyl methylsulfone hydroxamate series, and they maintain superior Gram-negative antibacterial activity to comparator agents.

One-pot (1-ethoxycarbonylcyclopropyl)triphenylphosphonium tetrafluoroborate ring-opening and Wittig reaction.

An efficient method was developed for the synthesis of 2-methylene-4-substituted ethyl butyrates via cyclopropyl opening followed by a Wittig reaction. The desired products were formed in a two-step, one-pot reaction sequence. Alternatively, the key intermediate ylide 2 was isolable and could be stored under oxygen-free conditions and subsequently utilized. A variety of nucleophiles were found to open the commercially available cyclopropane 1. The resulting ylide reacted with aldehydes to provide E-olefinic products.