Insights into the Novel Hydrolytic Mechanism of a Diethyl 2‑Phenyl-2-(2-arylacetoxy)methyl Malonate Ester-Based Microsomal Triglyceride Transfer Protein (MTP) Inhibitor

Inhibition of intestinal and hepatic microsomal triglyceride transfer protein (MTP) is a potential strategy for the treatment of dyslipidemia and related metabolic disorders. Inhibition of hepatic MTP, however, results in elevated liver transaminases and increased hepatic fat deposition consistent with hepatic steatosis. Diethyl 2-((2-(3-(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-2-ylcarboxamido)­phenyl)­acetoxy)­methyl)-2-phenylmalonate (JTT-130) is an intestine-specific inhibitor of MTP and does not cause increases in transaminases in short-term clinical trials in patients with dyslipidemia. Selective inhibition of intestinal MTP is achieved via rapid hydrolysis of its ester linkage by liver-specific carboxylesterase(s), resulting in the formation of an inactive carboxylic acid metabolite <b>1</b>. In the course of discovery efforts around tissue-specific inhibitors of MTP, the mechanism of JTT-130 hydrolysis was examined in detail. Lack of ¹⁸O incorporation in <b>1</b> following the incubation of JTT-130 in human liver microsomes in the presence of H₂¹⁸O suggested that hydrolysis did not occur via a simple cleavage of the ester linkage. The characterization of atropic acid (2-phenylacrylic acid) as a metabolite was consistent with a hydrolytic pathway involving initial hydrolysis of one of the pendant malonate ethyl ester groups followed by decarboxylative fragmentation to <b>1</b> and the concomitant liberation of the potentially electrophilic acrylate species. Glutathione conjugates of atropic acid and its ethyl ester were also observed in microsomal incubations of JTT-130 that were supplemented with the thiol nucleophile. Additional support for the hydrolysis mechanism was obtained from analogous studies on diethyl 2-(2-(2-(3-(dimethylcarbamoyl)-4-(4′-trifluoromethyl)-[1,1′-biphenyl]-2-ylcarboxamido)­phenyl)­acetoxy)­ethyl)-2-phenylmalonate (<b>3</b>), which cannot participate in hydrolysis via the fragmentation pathway because of the additional methylene group. Unlike the case with JTT-130, ¹⁸O was readily incorporated into <b>1</b> during the enzymatic hydrolysis of <b>3</b>, suggestive of a mechanism involving direct hydrolytic cleavage of the ester group in <b>3</b>. Finally, 3-(ethylamino)-2-(ethylcarbamoyl)-3-oxo-2-phenylpropyl 2-(3-(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-2-ylcarboxamido)­phenyl)­acetate (<b>4</b>), which possessed an <i>N</i>,<i>N</i>-diethyl-2-phenylmalonamide substituent (in lieu of the diethyl-2-phenylmalonate motif in JTT-130) proved to be resistant to the hydrolytic cleavage/decarboxylative fragmentation pathway that yielded <b>1</b>, a phenomenon that further confirmed our hypothesis. From a toxicological standpoint, it is noteworthy to point out that the liberation of the electrophilic acrylic acid species as a byproduct of JTT-130 hydrolysis is similar to the bioactivation mechanism established for felbamate, an anticonvulsant agent associated with idiosyncratic aplastic anemia and hepatotoxicity

Chemical Probe Identification Platform for Orphan GPCRs Using Focused Compound Screening: GPR39 as a Case Example

Orphan G protein-coupled receptors (oGPCRs) are a class of integral membrane proteins for which endogenous ligands or transmitters have not yet been discovered. Transgenic animal technologies have uncovered potential roles for many of these oGPCRs, providing new targets for the treatment of various diseases. Understanding signaling pathways of oGPCRs and validating these receptors as potential drug targets requires the identification of chemical probe compounds to be used in place of endogenous ligands to interrogate these receptors. A novel chemical probe identification platform was created in which GPCR-focused libraries were screened against sets of oGPCR targets, with a goal of discovering fit-for-purpose chemical probes for the more druggable members of the set. Application of the platform to a set of oGPCRs resulted in the discovery of the first reported small molecule agonists for GPR39, a receptor implicated in the regulation of insulin secretion and preservation of beta cells in the pancreas. Compound <b>1</b> stimulated intracellular calcium mobilization in recombinant and native cells in a GPR39-specific manner but did not potentiate glucose-stimulated insulin secretion in human islet preparations

Evaluation and Synthesis of Polar Aryl- and Heteroaryl Spiroazetidine-Piperidine Acetamides as Ghrelin Inverse Agonists

Several polar heteroaromatic acetic acids and their piperidine amides were synthesized and evaluated as ghrelin or type 1a growth hormone secretagogue receptor (GHS-R1a) inverse agonists. Efforts to improve pharmacokinetic and safety profile was achieved by modulating physicochemical properties and, more specifically, emphasizing increased polarity of our chemical series. <i>ortho</i>-Carboxamide containing compounds provided optimal physicochemical, pharmacologic, and safety profile. pH-dependent chemical stability was also assessed with our series

Discovery of an <i>in Vivo</i> Tool to Establish Proof-of-Concept for MAP4K4-Based Antidiabetic Treatment

Recent studies in adipose tissue, pancreas, muscle, and macrophages suggest that MAP4K4, a serine/threonine protein kinase may be a viable target for antidiabetic drugs. As part of the evaluation of MAP4K4 as a novel antidiabetic target, a tool compound, <b>16</b> (PF-6260933) and a lead <b>17</b> possessing excellent kinome selectivity and suitable properties were delivered to establish proof of concept <i>in vivo</i>. The medicinal chemistry effort that led to the discovery of these lead compounds is described herein together with <i>in vivo</i> pharmacokinetic properties and activity in a model of insulin resistance

Identification of Tetrahydropyrido[4,3‑<i>d</i>]pyrimidine Amides as a New Class of Orally Bioavailable TGR5 Agonists

Takeda G-protein-coupled receptor 5 (TGR5) represents an exciting biological target for the potential treatment of diabetes and metabolic syndrome. A new class of high-throughput screening (HTS)-derived tetrahydropyrido­[4,3-<i>d</i>]­pyrimidine amide TGR5 agonists is disclosed. We describe our effort to identify an orally available agonist suitable for assessment of systemic TGR5 agonism. This effort resulted in identification of <b>16</b>, which had acceptable potency and pharmacokinetic properties to allow for in vivo assessment in dog. A key aspect of this work was the calibration of human and dog in vitro assay systems that could be linked with data from a human ex vivo peripheral blood monocyte assay that expresses receptor at endogenous levels. Potency from the human in vitro assay was also found to correlate with data from an ex vivo human whole blood assay. This calibration exercise provided confidence that <b>16</b> could be used to drive plasma exposures sufficient to test the effects of systemic activation of TGR5

Discovery of PF-5190457, a Potent, Selective, and Orally Bioavailable Ghrelin Receptor Inverse Agonist Clinical Candidate

The identification of potent, highly selective orally bioavailable ghrelin receptor inverse agonists from a spiro-azetidino-piperidine series is described. Examples from this series have promising in vivo pharmacokinetics and increase glucose-stimulated insulin secretion in human whole and dispersed islets. A physicochemistry-based strategy to increase lipophilic efficiency for ghrelin receptor potency and retain low clearance and satisfactory permeability while reducing off-target pharmacology led to the discovery of <b>16h</b>. Compound <b>16h</b> has a superior balance of ghrelin receptor pharmacology and off-target selectivity. On the basis of its promising pharmacological and safety profile, <b>16h</b> was advanced to human clinical trials

Discovery and Preclinical Characterization of 6‑Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]‑1<i>H</i>‑indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy

Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (<b>7</b>). Compound <b>7</b> was advanced to first-in-human trials for the treatment of diabetic nephropathy

Discovery and Optimization of Imidazopyridine-Based Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2)

The medicinal chemistry and preclinical biology of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) is described. A screening hit <b>1</b> with low lipophilic efficiency (LipE) was optimized through two key structural modifications: (1) identification of the pyrrolidine amide group for a significant LipE improvement, and (2) insertion of a sp³-hybridized carbon center in the core of the molecule for simultaneous improvement of <i>N</i>-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate <b>9</b> (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models

Characterization of cGAS enzyme activity.

<p>Measurement of cGAMP production was conducted by LC-MS as described in Methods. (A) Time course of cGAS (15 nM) activity; (B) titration of dsDNA activation of cGAS (1nM) activity; (C) cGAS enzyme titration; (D) inhibition of cGAS (1 nM) activity by CuBr.</p

Binding affinities and <i>in vitro</i> activities of cGAS inhibitors.

<p>Binding affinities and <i>in vitro</i> activities of cGAS inhibitors.</p