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.

A covalent BTK ternary complex compatible with targeted protein degradation.

Targeted protein degradation using heterobifunctional chimeras holds the potential to expand target space and grow the druggable proteome. Most acutely, this provides an opportunity to target proteins that lack enzymatic activity or have otherwise proven intractable to small molecule inhibition. Limiting this potential, however, is the remaining need to develop a ligand for the target of interest. While a number of challenging proteins have been successfully targeted by covalent ligands, unless this modification affects form or function, it may lack the ability to drive a biological response. Bridging covalent ligand discovery with chimeric degrader design has emerged as a potential mechanism to advance both fields. In this work, we employ a set of biochemical and cellular tools to deconvolute the role of covalent modification in targeted protein degradation using Bruton's tyrosine kinase. Our results reveal that covalent target modification is fundamentally compatible with the protein degrader mechanism of action.

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.

Snapshots and ensembles of BTK and cIAP1 protein degrader ternary complexes.

Heterobifunctional chimeric degraders are a class of ligands that recruit target proteins to E3 ubiquitin ligases to drive compound-dependent protein degradation. Advancing from initial chemical tools, protein degraders represent a mechanism of growing interest in drug discovery. Critical to the mechanism of action is the formation of a ternary complex between the target, degrader and E3 ligase to promote ubiquitination and subsequent degradation. However, limited insights into ternary complex structures exist, including a near absence of studies on one of the most widely co-opted E3s, cellular inhibitor of apoptosis 1 (cIAP1). In this work, we use a combination of biochemical, biophysical and structural studies to characterize degrader-mediated ternary complexes of Bruton's tyrosine kinase and cIAP1. Our results reveal new insights from unique ternary complex structures and show that increased ternary complex stability or rigidity need not always correlate with increased degradation efficiency.

Synthesis of PEG-Functionalized Amines Using Ruthenium-Catalyzed Hydrogen Borrowing.

The polyethylene glycol (PEG) moiety has become increasingly important in medicinal chemistry. Herein, we describe the PEG functionalization of amines via hydrogen borrowing reductive amination. This was accomplished using the [Ru(p-cymene)Cl2]2 catalyst and phosphorus-containing ligand dppf or DPE to yield a variety of PEGylated primary and secondary amine products. Furthermore, we illustrate the utility of this method with the synthesis of quetiapine (Seroquel) in 62% isolated yield.

Delineating the role of cooperativity in the design of potent PROTACs for BTK.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

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.

Novel 3-fluoro-6-methoxyquinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV.

Novel (non-fluoroquinolone) inhibitors of bacterial type II topoisomerases (NBTIs) are an emerging class of antibacterial agents. We report an optimized series of cyclobutylaryl-substituted NBTIs. Compound 14 demonstrated excellent activity both in vitro (S. aureus MIC90=0.125µg/mL) and in vivo (systemic and tissue infections). Enhanced inhibition of Topoisomerase IV correlated with improved activity in S. aureus strains with mutations conferring resistance to NBTIs. Compound 14 also displayed an improved hERG IC50 of 85.9µM and a favorable profile in the anesthetized guinea pig model.

Optimization of a Dicarboxylic Series for in Vivo Inhibition of Citrate Transport by the Solute Carrier 13 (SLC13) Family.

Inhibition of the sodium-coupled citrate transporter (NaCT or SLC13A5) has been proposed as a new therapeutic approach for prevention and treatment of metabolic diseases. In a previous report, we discovered dicarboxylate 1a (PF-06649298) which inhibits the transport of citrate in in vitro and in vivo settings via a specific interaction with NaCT. Herein, we report the optimization of this series leading to 4a (PF-06761281), a more potent inhibitor with suitable in vivo pharmacokinetic profile for assessment of in vivo pharmacodynamics. Compound 4a was used to demonstrate dose-dependent inhibition of radioactive [(14)C]citrate uptake in liver and kidney in vivo, resulting in modest reductions in plasma glucose concentrations.

Discovery and characterization of novel inhibitors of the sodium-coupled citrate transporter (NaCT or SLC13A5).

Citrate is a key regulatory metabolic intermediate as it facilitates the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake, by blocking the sodium-coupled citrate transporter (NaCT or SLC13A5), has been suggested as a potential therapeutic approach to treat metabolic disorders. NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions. The studies herein report the identification and characterization of a novel small dicarboxylate molecule (compound 2) capable of selectively and potently inhibiting citrate transport through NaCT, both in vitro and in vivo. Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT. The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints.

Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors.

Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymatic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry additional risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biologically relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.

One-pot oxidation and rearrangement of propargylamines and in situ pyrazole synthesis.

Reported here are procedures for a one-pot oxidation and rearrangement of propargylamines to synthesize enaminones, with supporting mechanistic studies. Also reported are the extended one-pot syntheses of pyrazoles, including celecoxib and various heterocyclic compounds.

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.

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.

Potent inhibitors of LpxC for the treatment of Gram-negative infections.

In this paper, we present the synthesis and SAR as well as selectivity, pharmacokinetic, and infection model data for representative analogues of a novel series of potent antibacterial LpxC inhibitors represented by hydroxamic acid.

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.

Effect of solvent and temperature on the lithium-bromine exchange of vinyl bromides: reactions of n-butyllithium and t-butyllithium with (E)-5-bromo-5-decene.

The outcome of reactions of (E)-5-bromo-5-decene (1), a representative vinyl bromide, with t-BuLi or n-BuLi at 0 degrees C and room temperature, respectively, in a variety of solvent systems has been investigated. Vinyl bromide 1 does not react with t-BuLi in pure heptane; however, the presence of even small quantities of an ether in a predominantly heptane medium resulted in virtually complete consumption of 1 at 0 degrees C, resulting in nearly the same distribution of products, including 60-80% of (Z)-5-decenyllithium, regardless of the solvent composition. Vinyl bromide 1 reacts slowly with n-BuLi at room temperature in a variety of ether and heptane-ether mixtures to afford a mixture of products including significant quantities of recovered starting material. The results of these experiments demonstrate that lithium-bromine exchange between a vinyl bromide and either t-BuLi or n-BuLi at temperatures significantly above -78 degrees C is not an efficient method for the generation of a vinyllithium.