Mitigating a Bioactivation Liability with an Azetidine-Based Inhibitor of Diacylglycerol Acyltransferase 2 (DGAT2) En Route to the Discovery of the Clinical Candidate Ervogastat.

We recently disclosed SAR studies on systemically acting, amide-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) that addressed metabolic liabilities with the liver-targeted DGAT2 inhibitor PF-06427878. Despite strategic placement of a nitrogen atom in the dialkoxyaromatic ring in PF-06427878 to evade oxidative O-dearylation, metabolic intrinsic clearance remained high due to extensive piperidine ring oxidation as exemplified with compound 1. Piperidine ring modifications through alternate N-linked heterocyclic ring/spacer combination led to azetidine 2 that demonstrated lower intrinsic clearance. However, 2 underwent a facile cytochrome P450 (CYP)-mediated α-carbon oxidation followed by azetidine ring scission, resulting in the formation of ketone (M2) and aldehyde (M6) as stable metabolites in NADPH-supplemented human liver microsomes. Inclusion of GSH or semicarbazide in microsomal incubations led to the formation of Cys-Gly-thiazolidine (M3), Cys-thiazolidine (M5), and semicarbazone (M7) conjugates, which were derived from reaction of the nucleophilic trapping agents with aldehyde M6. Metabolites M2 and M5 were biosynthesized from NADPH- and l-cysteine-fortified human liver microsomal incubations with 2, and proposed metabolite structures were verified using one- and two-dimensional NMR spectroscopy. Replacement of the azetidine substituent with a pyridine ring furnished 8, which mitigated the formation of the electrophilic aldehyde metabolite, and was a more potent DGAT2 inhibitor than 2. Further structural refinements in 8, specifically introducing amide bond substituents with greater metabolic stability, led to the discovery of PF-06865571 (ervogastat) that is currently in phase 2 clinical trials for the treatment of nonalcoholic steatohepatitis.

Mechanistic Characterization of Long Residence Time Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2).

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triacylglycerol (TAG) synthesis. Genetic knockdown or pharmacological inhibition of DGAT2 leads to a decrease in very-low-density lipoprotein TAG secretion and hepatic lipid levels in rodents, indicating DGAT2 may represent an attractive therapeutic target for treatment of hyperlipidemia and hepatic steatosis. We have previously described potent and selective imidazopyridine DGAT2 inhibitors with high oral bioavailability. However, the detailed mechanism of DGAT2 inhibition has not been reported. Herein, we describe imidazopyridines represented by PF-06424439 (1) and 2 as long residence time inhibitors of DGAT2. We demonstrate that 1 and 2 are slowly reversible, time-dependent inhibitors, which inhibit DGAT2 in a noncompetitive mode with respect to the acyl-CoA substrate. Detailed kinetic analysis demonstrated that 1 and 2 inhibit DGAT2 in a two-step binding mechanism, in which the initial enzyme-inhibitor complex (EI) undergoes an isomerization step resulting in a much higher affinity complex (EI*) with overall apparent inhibition constants ( Ki*app values) of 16.7 and 16.0 nM for 1 and 2, respectively. The EI* complex dissociates with dissociation half-lives of 1.2 and 1.0 h for 1 and 2, respectively. A binding assay utilizing 125I-labeled imidazopyridine demonstrated that the level of imidazopyridine binding to DGAT2 mutant enzymes, H161A and H163A, dramatically decreased to 11-17% of that of the wild-type enzyme, indicating that these residues are critical for imidazopyridines to bind to DGAT2. Taken together, imidazopyridines may thus represent a promising lead series for the development of DGAT2 inhibitors that display an unprecedented combination of potency, selectivity, and in vivo efficacy.

Optimization of amide-based EP3 receptor antagonists.

Prostaglandin E receptor subtype 3 (EP3) antagonism may treat a variety of symptoms from inflammation to cardiovascular and metabolic diseases. Previously, most EP3 antagonists were large acidic ligands that mimic the substrate, prostaglandin E2 (PGE2). This manuscript describes the optimization of a neutral small molecule amide series with improved lipophilic efficiency (LipE) also known as lipophilic ligand efficiency (LLE) ((a) Nat. Rev. Drug Disc.2007, 6, 881; (b) Annu. Rep. Med. Chem.2010, 45, 380).

Design and synthesis of aryl sulfonamide-based nonsteroidal mineralocorticoid receptor antagonists.

Hit-to-lead medicinal chemistry efforts are described starting from a screening hit 1, leading to a new class of aryl sulfonamide-based MR antagonist, exemplified by 17, that possesses favourable MR binding affinity, selectivity profile against closely related NHRs, physicochemical properties and metabolic stability.

Discovery of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as potent agonists of TGR5 via sequential combinatorial libraries.

Optimization of a high-throughput screening hit led to the discovery of a new series of 5-phenoxy-1,3-dimethyl-1H-pyrazole-4-carboxamides as highly potent agonists of TGR5. This novel chemotype was rapidly developed through iterative combinatorial library synthesis. It was determined that in vitro agonist potency correlated with functional activity data from human peripheral blood monocytes.

From partial to full agonism: identification of a novel 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole as a full agonist of the human GPR119 receptor.

A novel GPR119 agonist based on the 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole scaffold was designed through lead optimization starting from pyrazole-based GPR119 agonist 1. The design is centered on the conformational restriction of the core scaffold, while minimizing the change in spatial relationships of two key pharmacophoric elements (piperidine-carbamate and aryl sulfone).

Design of Next-Generation DGAT2 Inhibitor PF-07202954 with Longer Predicted Half-Life.

Diacylglycerol O-acyltransferase 2 (DGAT2) inhibitors have been shown to lower liver triglyceride content and are being explored clinically as a treatment for non-alcoholic steatohepatitis (NASH). This work details efforts to find an extended-half-life DGAT2 inhibitor. A basic moiety was added to a known inhibitor template, and the basicity and lipophilicity were fine-tuned by the addition of electrophilic fluorines. A weakly basic profile was required to find an appropriate balance of potency, clearance, and permeability. This work culminated in the discovery of PF-07202954 (12), a weakly basic DGAT2 inhibitor that has advanced to clinical studies. This molecule displays a higher volume of distribution and longer half-life in preclinical species, in keeping with its physicochemical profile, and lowers liver triglyceride content in a Western-diet-fed rat model.

Discovery of Ervogastat (PF-06865571): A Potent and Selective Inhibitor of Diacylglycerol Acyltransferase 2 for the Treatment of Non-alcoholic Steatohepatitis.

Discovery efforts leading to the identification of ervogastat (PF-06865571), a systemically acting diacylglycerol acyltransferase (DGAT2) inhibitor that has advanced into clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) with liver fibrosis, are described herein. Ervogastat is a first-in-class DGAT2 inhibitor that addressed potential development risks of the prototype liver-targeted DGAT2 inhibitor PF-06427878. Key design elements that culminated in the discovery of ervogastat are (1) replacement of the metabolically labile motif with a 3,5-disubstituted pyridine system, which addressed potential safety risks arising from a cytochrome P450-mediated O-dearylation of PF-06427878 to a reactive quinone metabolite precursor, and (2) modifications of the amide group to a 3-THF group, guided by metabolite identification studies coupled with property-based drug design.

Design and evaluation of a 2-(2,3,6-trifluorophenyl)acetamide derivative as an agonist of the GPR119 receptor.

The design and synthesis of a GPR119 agonist bearing a 2-(2,3,6-trifluorophenyl)acetamide group is described. The design capitalized on the conformational restriction found in N-ß-fluoroethylamide derivatives to help maintain good levels of potency while driving down both lipophilicity and oxidative metabolism in human liver microsomes. The chemical stability and bioactivation potential are discussed.

Stereoselective construction of halogenated quaternary stereogenic centers via catalytic asymmetric Diels-Alder reaction.

Highly enantioselective Diels-Alder reactions of alpha-halo-alpha,beta-unsaturated ketones with Lewis acid-activated chiral oxazaborolidine 1 are described. The reaction with alpha-fluoroenones provided the corresponding cyclohexane derivatives having a fluorinated quaternary stereogenic center with up to 99% de and 94% ee. The reaction with alpha-bromo cyclic enones provided the corresponding bromo bicyclic adducts with up to 99% de and 95% ee. A brominated cis-fused bicyclic adduct derived from 2-bromocyclopenten-1-one and Dane's diene was converted to the trans-fused bicyclic system via reductive alkylation with the bulky aluminum reagent aluminum tris(2,6-diphenylphenoxide) (ATPH). With this process, formal syntheses of (+)-estrone and norgestrel have been demonstrated.

Discovery of PF-06835919: A Potent Inhibitor of Ketohexokinase (KHK) for the Treatment of Metabolic Disorders Driven by the Overconsumption of Fructose.

Increased fructose consumption and its subsequent metabolism have been implicated in metabolic disorders such as nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH) and insulin resistance. Ketohexokinase (KHK) converts fructose to fructose-1-phosphate (F1P) in the first step of the metabolic cascade. Herein we report the discovery of a first-in-class KHK inhibitor, PF-06835919 (8), currently in phase 2 clinical trials. The discovery of 8 was built upon our originally reported, fragment-derived lead 1 and the recognition of an alternative, rotated binding mode upon changing the ribose-pocket binding moiety from a pyrrolidinyl to an azetidinyl ring system. This new binding mode enabled efficient exploration of the vector directed at the Arg-108 residue, leading to the identification of highly potent 3-azabicyclo[3.1.0]hexane acetic acid-based KHK inhibitors by combined use of parallel medicinal chemistry and structure-based drug design.

Deuterium isotope effects in drug pharmacokinetics II: Substrate-dependence of the reaction mechanism influences outcome for cytochrome P450 cleared drugs.

Two chemotypes were examined in vitro with CYPs 3A4 and 2C19 by molecular docking, metabolic profiles, and intrinsic clearance deuterium isotope effects with specifically deuterated form to assess the potential for enhancement of pharmacokinetic parameters. The results show the complexity of deuteration as an approach for pharmacokinetic enhancement when CYP enzymes are involved in metabolic clearance. With CYP3A4 the rate limiting step was chemotype-dependent. With one chemotype no intrinsic clearance deuterium isotope effect was observed with any deuterated form, whereas with the other chemotype the rate limiting step was isotopically sensitive, and the magnitude of the intrinsic clearance isotope effect was dependent on the position(s) and extent of deuteration. Molecular docking and metabolic profiles aided in identifying sites for deuteration and predicted the possibility for metabolic switching. However, the potential for an isotope effect on the intrinsic clearance cannot be predicted and must be established by examining select deuterated versions of the chemotypes. The results show how in a deuteration strategy molecular docking, in-vitro metabolic profiles, and intrinsic clearance assessments with select deuterated versions of new chemical entities can be applied to determine the potential for pharmacokinetic enhancement in a discovery setting. They also help explain the substantial failures reported in the literature of deuterated versions of drugs to elicit a systemic enhancement on pharmacokinetic parameters.

Identification of Morpholino-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-ones as Nonsteroidal Mineralocorticoid Antagonists.

A novel series of morpholine-based nonsteroidal mineralocorticoid receptor antagonists is reported. Starting from a pyrrolidine HTS hit 9 that possessed modest potency but excellect selectivity versus related nuclear hormone receptors, a series of libraries led to identification of morpholine lead 10. After further optimization, cis disubstituted morpholine 22 was discovered, which showed a 45-fold boost in binding affinity and corresponding functional potency compared to 13. While 22 had high clearance in rat, it provided sufficient exposure at high doses to favorably assess in vivo efficacy (increased urinary Na+/K+ ratio) and safety. In contrast to rat, the dog and human MetID and PK profiles of 22 were adequate, suggesting that it could be suitable as a potential clinical asset.

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.

"Designer acids": combined acid catalysis for asymmetric synthesis.

Lewis and Brønsted acids can be utilized as more-effective tools for chemical reactions by sophisticated engineering ("designer acids"). The ultimate goal of such "designer acids" is to form a combination of acids with higher reactivity, selectivity, and versatility than the individual acid catalysts. One possible way to take advantage of such abilities may be to apply a "combined acids system" to the catalyst design. The concept of combined acids, which can be classified into Brønsted acid assisted Lewis acid (BLA), Lewis acid assisted Lewis acid (LLA), Lewis acid assisted Brønsted acid (LBA), and Brønsted acid assisted Brønsted acid (BBA), can be a particularly useful tool for the design of asymmetric catalysis, because combining such acids will bring out their inherent reactivity by associative interaction, and also provide more-organized structures that allow an effective asymmetric environment.

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.

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 1 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 sp(3)-hybridized carbon center in the core of the molecule for simultaneous improvement of N-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate 9 (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models.

Asymmetric protonation of lithium enolates of alpha-amino acid derivatives with alpha-amino acid-based chiral Brønsted acids.

The reaction of lithium enolates of alpha-amino acid derivatives with chiral amides, easily synthesized from L-tert-leucine, gives corresponding optically active unnatural alpha-amino acid derivatives with up to 87% ee.

Identification of Tetrahydropyrido[4,3-d]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-d]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 16, 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 16 could be used to drive plasma exposures sufficient to test the effects of systemic activation of TGR5.

Glucokinase activators.

In this review we highlight recently disclosed progress in the field of small-molecule activators of the human glucokinase enzyme. Several of the reported chemotypes possess structural features that diverge from known leads; some of these modifications appear to be specifically designed to modulate tissue selectivity or discrete parameters of enzyme function (e.g., S0.5 v Vmax). This review will inform the reader of the extent of continued effort being directed toward discovery of a first-in-class drug for Type II diabetes mellitus that functions through this target. Patents were selected from those published in December 2009 up to November 2011; foreign filings were translated where possible to understand the claims and biological techniques utilized to characterize the reported glucokinase activators. Overall, there appears to be a recent trend leading to reduced patent filings for small-molecule glucokinase activators. There are many possible explanations for this trend; however, it is likely that the field has reached maturity and that the downturn of new disclosures represents the transition of many of these programs to the clinic.