Structural studies identify angiotensin II receptor blocker-like compounds as branched-chain ketoacid dehydrogenase kinase inhibitors.

The mammalian mitochondrial branched-chain ketoacid dehydrogenase (BCKD) complex is a multienzyme complex involved in the catabolism of branched-chain amino acids. BCKD is regulated by the BCKD kinase, or BCKDK, which binds to the E2 subunit of BCKD, phosphorylates its E1 subunit, and inhibits enzymatic activity. Inhibition of the BCKD complex results in increased levels of branched-chain amino acids and branched-chain ketoacids, and this buildup has been associated with heart failure, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. To find BCKDK inhibitors for potential treatment of these diseases, we performed both NMR and virtual fragment screening and identified tetrazole-bearing fragments that bind BCKDK at multiple sites. Through structure-based virtual screening expanding from these fragments, the angiotensin receptor blocker class antihypertension drugs and angiotensin receptor blocker-like compounds were discovered to be potent BCKDK inhibitors, suggesting potential new avenues for heart failure treatment combining BCKDK inhibition and antihypertension.

5-Aryltetrazoles from Direct C-H Arylation with Aryl Bromides.

A mild, direct C-H arylation of 1-substituted tetrazoles to 5-aryltetrazoles is developed using a Pd/Cu cocatalytic system with readily available aryl bromides. The methodology avoids late-stage usage of azides and tolerates a wide range of functionalities.

High-Throughput Ligand Screening Enables the Enantioselective Conjugate Borylation of Cyclobutenones to Access Synthetically Versatile Tertiary Cyclobutylboronates.

Cyclobutane rings are important in medicinal chemistry, yet few enantioselective methods exist to access this scaffold. In particular, cyclobutylboronates are receiving increasing attention in the literature due to the synthetic versatility of alkylboronic esters and the increasing role of boronic acids in drug discovery. Herein, a conjugate borylation of α-alkyl,ß-aryl/alkyl cyclobutenones is reported leading to the first synthesis of enantioenriched tertiary cyclobutylboronates. Cyclobutanones with two stereogenic centers are obtained in good to high yield, with high enantioselectivity and diastereoselectivity. Vital to this advance are the development of a novel approach to α,ß unsymmetrically disubstituted cyclobutenone substrates and the use of a high-throughput chiral ligand screening platform. The synthetic utility of both the boronic ester and ketone functionalities is displayed, with remarkable chemoselectivity for either group being possible in this small ring scaffold.

Utilizing on- and off-line monitoring tools to follow a kinetic resolution step during flow synthesis.

In situ reaction monitoring tools offer the ability to track the progress of a synthetic reaction in real time to facilitate reaction optimization and provide kinetic/mechanistic insight. Herein, we report the utilization of flow NMR, flow IR, and other off-line spectroscopy tools to monitor the progress of a flow chemistry reaction. The on-line and off-line tools were selected to facilitate the stereoselective kinetic resolution of a key racemic monomer, which lacked a chromophore, making conventional reaction monitoring difficult. Copyright © 2016 John Wiley & Sons, Ltd.

Discovery of a novel Kv7 channel opener as a treatment for epilepsy.

Facilitating activation, or delaying inactivation, of the native Kv7 channel reduces neuronal excitability, which may be beneficial in controlling spontaneous electrical activity during epileptic seizures. In an effort to identify a compound with such properties, the structure-activity relationship (SAR) and in vitro ADME for a series of heterocyclic Kv7.2-7.5 channel openers was explored. PF-05020182 (2) demonstrated suitable properties for further testing in vivo where it dose-dependently decreased the number of animals exhibiting full tonic extension convulsions in response to corneal stimulation in the maximal electroshock (MES) assay. In addition, PF-05020182 (2) significantly inhibited convulsions in the MES assay at doses tested, consistent with in vitro activity measure. The physiochemical properties, in vitro and in vivo activities of PF-05020182 (2) support further development as an adjunctive treatment of refractory epilepsy.

A Second-Generation Oral SARS-CoV-2 Main Protease Inhibitor Clinical Candidate for the Treatment of COVID-19.

Despite the record-breaking discovery, development and approval of vaccines and antiviral therapeutics such as Paxlovid, coronavirus disease 2019 (COVID-19) remained the fourth leading cause of death in the world and third highest in the United States in 2022. Here, we report the discovery and characterization of PF-07817883, a second-generation, orally bioavailable, SARS-CoV-2 main protease inhibitor with improved metabolic stability versus nirmatrelvir, the antiviral component of the ritonavir-boosted therapy Paxlovid. We demonstrate the in vitro pan-human coronavirus antiviral activity and off-target selectivity profile of PF-07817883. PF-07817883 also demonstrated oral efficacy in a mouse-adapted SARS-CoV-2 model at plasma concentrations equivalent to nirmatrelvir. The preclinical in vivo pharmacokinetics and metabolism studies in human matrices are suggestive of improved oral pharmacokinetics for PF-07817883 in humans, relative to nirmatrelvir. In vitro inhibition/induction studies against major human drug metabolizing enzymes/transporters suggest a low potential for perpetrator drug-drug interactions upon single-agent use of PF-07817883.

Discovery of the Potent and Selective MC4R Antagonist PF-07258669 for the Potential Treatment of Appetite Loss.

The melanocortin-4 receptor (MC4R) is a centrally expressed, class A GPCR that plays a key role in the regulation of appetite and food intake. Deficiencies in MC4R signaling result in hyperphagia and increased body mass in humans. Antagonism of MC4R signaling has the potential to mitigate decreased appetite and body weight loss in the setting of anorexia or cachexia due to underlying disease. Herein, we report on the identification of a series of orally bioavailable, small-molecule MC4R antagonists using a focused hit identification effort and the optimization of these antagonists to provide clinical candidate 23. Introduction of a spirocyclic conformational constraint allowed for simultaneous optimization of MC4R potency and ADME attributes while avoiding the production of hERG active metabolites observed in early series leads. Compound 23 is a potent and selective MC4R antagonist with robust efficacy in an aged rat model of cachexia and has progressed into clinical trials.

Small molecule branched-chain ketoacid dehydrogenase kinase (BDK) inhibitors with opposing effects on BDK protein levels.

Branched chain amino acid (BCAA) catabolic impairments have been implicated in several diseases. Branched chain ketoacid dehydrogenase (BCKDH) controls the rate limiting step in BCAA degradation, the activity of which is inhibited by BCKDH kinase (BDK)-mediated phosphorylation. Screening efforts to discover BDK inhibitors led to identification of thiophene PF-07208254, which improved cardiometabolic endpoints in mice. Structure-activity relationship studies led to identification of a thiazole series of BDK inhibitors; however, these inhibitors did not improve metabolism in mice upon chronic administration. While the thiophenes demonstrated sustained branched chain ketoacid (BCKA) lowering and reduced BDK protein levels, the thiazoles increased BCKAs and BDK protein levels. Thiazoles increased BDK proximity to BCKDH-E2, whereas thiophenes reduced BDK proximity to BCKDH-E2, which may promote BDK degradation. Thus, we describe two BDK inhibitor series that possess differing attributes regarding BDK degradation or stabilization and provide a mechanistic understanding of the desirable features of an effective BDK inhibitor.

Discovery of microsomal triglyceride transfer protein (MTP) inhibitors with potential for decreased active metabolite load compared to dirlotapide.

Analogues related to dirlotapide (1), a gut-selective inhibitor of microsomal triglyceride transfer protein (MTP) were prepared with the goal of further reducing the potential for unwanted liver MTP inhibition and associated side-effects. Compounds were designed to decrease active metabolite load: reducing MTP activity of likely human metabolites and increasing metabolite clearance to reduce exposure. Introduction of 4'-alkyl and 4'-alkoxy substituents afforded compounds exhibiting improved therapeutic index in rats with respect to liver triglyceride accumulation and enzyme elevation. Likely human metabolites of select compounds were prepared and characterized for their potential to inhibit MTP in vivo. Based on preclinical efficacy and safety data and its potential for producing short-lived, weakly active metabolites, compound 13 (PF-02575799) advanced into phase 1 clinical studies.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro , and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.

C-Aryl glycoside inhibitors of SGLT2: Exploration of sugar modifications including C-5 spirocyclization.

Modifications to the sugar portion of C-aryl glycoside sodium glucose transporter 2 (SGLT2) inhibitors were explored, including systematic deletion and modification of each of the glycoside hydroxyl groups. Based on results showing activity to be quite tolerant of structural change at the C-5 position, a series of novel C-5 spiro analogues was prepared. Some of these analogues exhibit low nanomolar potency versus SGLT2 and promote urinary glucose excretion (UGE) in rats. However, due to sub-optimal pharmacokinetic parameters (in particular half-life), predicted human doses did not meet criteria for further advancement.

Sulfonamide Synthesis via Calcium Triflimide Activation of Sulfonyl Fluorides.

A method using calcium triflimide [Ca(NTf2)2] as a Lewis acid to activate sulfonyl fluorides toward nucleophilic addition with amines is described. The reaction converts a wide array of sterically and electronically diverse sulfonyl fluorides and amines into the corresponding sulfonamides in good yield.

Discovery and Characterization of (R)-6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates.

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.

2-Aryl-3-methyloctahydrophenanthrene-2,3,7-triols as potent dissociated glucocorticoid receptor agonists.

A significant improvement in agonist activity of the previously described 2-aryloctahydrophenanthrene-2,3,7-triol series of dissociated glucocorticoid receptor agonists (DAGRs) was achieved by modifying the substitution at C3 from (S)-3-hydroxy to (R)-3-hydroxy-3-methyl. The IC50 of the prototype 13 in the efficacy assay measuring repression of IL-1 induced MMP-13 expression was 3.5 nM, exhibiting 87% of the maximal effect of dexamethasone (DEX). It displayed a dissociated profile by exhibiting 42% of the maximal effect of DEX in a mouse mammary tumor virus (MMTV) luciferase reporter transactivation assay. Compound 13 and analogues containing heterocyclic replacements for the C2 phenyl and modified B rings showed high repression of TNFα production in human whole blood, with IC50 values (43-167 nM) approaching the level of DEX (21 nM). On the basis of X-ray structures and force field calculations, the overall potency of this series was attributed to a favorable conformation of the C2α phenyl, induced by the neighboring C3α methyl.

Discovery and preclinical characterization of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-[3,4-b]pyrazine (PF470): a highly potent, selective, and efficacious metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulator.

A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (1), a systematic structure-activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of 14 to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-mediated type IV hypersensitivity in a 90-day NHP regulatory toxicology study.

Synthesis of α,α-difluoroethyl aryl and heteroaryl ethers.

Fluorine plays a critical role in modern medicinal chemistry due to its unique properties, and new methods for its incorporation into target molecules are of high interest. An efficient new method for the preparation of aryl-α,α-difluoroethyl ethers (4) via addition of aryl and heteroaryl alcohols (1) to commercially available 2-bromo-1,1-difluoroethene (2) and subsequent hydrogenolysis is presented. This procedure is an attractive alternative to existing methods that employ harshly reactive fluorinating systems such as xenon difluoride and hydrogen fluoride.

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.

Octahydrophenanthrene-2,7-diol analogues as dissociated glucocorticoid receptor agonists: discovery and lead exploration.

As exemplified by the lead compound 2, octahydrophenanthrene-2,7-diol analogues exhibit the profile of a pathway-selective or "dissociated" agonist of the glucocorticoid receptor (GR), retaining the potent activity that glucocorticoids have for transrepression (as measured by inhibition of IL-1 induced MMP-13 expression) but showing an attenuated capacity for transactivation (as measured in an MMTV luciferase reporter assay). With the guidance of a homology model of the GR ligand binding domain, structural modifications to 2 were carried out that were successful in replacing the allyl and propynyl side chains with groups likely to be more chemically stable and less likely to produce toxic metabolites. Key to success was the introduction of an additional hydroxyl group onto the tricyclic carbon framework of the series.