Practical synthesis of capromorelin, a growth hormone secretagogue, via a crystallization-induced dynamic resolution.

A practical synthesis of capromorelin (1), a growth hormone secretagogue, is described that utilizes as a key step a crystallization-induced dynamic resolution (CIDR) of (±)-3a-benzyl-2-methyl-4,5,6,7-tetrahydro-2H-pyrazolo[4,3-c]pyridin-3(3aH)-one [(±)-2] by L-tartaric acid salt formation, yielding (R)-2.L-tartaric acid in high chemical yield (>85%) and with diastereomeric excess (de) of ∼98%. Treatment of (R)-2.L-tartaric acid with ammonium hydroxide provided (R)-2 without loss of chiral purity. In situ generated (R)-2 was coupled with (R)-3-(benzyloxy)-2-(2-(tert-butoxycarbonyl)-2-methylpropanamido)propanoic acid [(R)-3] to give predominantly a single diastereomer of N-Boc-protected capromorelin [(1R,3aR)-4]. This process was used to prepare bulk quantities of capromorelin from (±)-2 to support preclinical toxicology studies.

A potentiator of orthosteric ligand activity at GLP-1R acts via covalent modification.

We report that 4-(3-(benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), which behaves as a positive allosteric modulator at the glucagon-like peptide-1 receptor (GLP-1R), covalently modifies cysteines 347 and 438 in GLP-1R. C347, located in intracellular loop 3 of GLP-1R, is critical to the activity of BETP and a structurally distinct GLP-1R ago-allosteric modulator, N-(tert-butyl)-6,7-dichloro-3-(methylsulfonyl)quinoxalin-2-amine. We further show that substitution of cysteine for phenylalanine 345 in the glucagon receptor is sufficient to confer sensitivity to BETP.

Development of a selective activity-based probe for glycosylated LIPA.

Loss of LIPA activity leads to diseases such as Wolman's Disease and Cholesterol Ester Storage Disease. While it is possible to measure defects in LIPA protein levels, it is difficult to directly measure LIPA activity in cells. In order to measure LIPA activity directly we developed a LIPA specific activity based probe. LIPA is heavily glycosylated although it is unclear how glycosylation affects LIPA activity or function. Our probe is specific for a glycosylated form of LIPA in cells, although it labels purified LIPA regardless of glycosylation.

Mechanistic characterization of a 2-thioxanthine myeloperoxidase inhibitor and selectivity assessment utilizing click chemistry--activity-based protein profiling.

Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Despite a high level of interest in MPO as a therapeutic target, there have been limited reports about MPO inhibitors that are suitable for evaluating MPO in pharmacological studies. 2-Thioxanthine, 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (A), has recently been reported to inhibit MPO by covalently modifying the heme prosthetic group. Here we report a detailed mechanistic characterization demonstrating that A possesses all the distinguishing features of a mechanism-based inactivator. A is a time-dependent MPO inhibitor and displays saturable inactivation kinetics consistent with a two-step mechanism of inactivation and a potency (k(inact)/K(I) ratio) of 8450 ± 780 M⁻¹ s⁻¹. MPO inactivation by A is dependent on MPO catalysis and is protected by substrate. A reduces MPO compound I to compound II with a second-order rate constant of (0.801 ± 0.056) × 106 M⁻¹ s⁻¹, and its irreversible inactivation of MPO occurs prior to release of the activated inhibitory species. Despite its relatively high selectivity against a broad panel of more than 100 individual targets, including enzymes, receptors, transporters, and ion channels, we demonstrate that A labels multiple other protein targets in the presence of MPO. By synthesizing an alkyne analogue of A and utilizing click chemistry-activity-based protein profiling, we present that the MPO-activated inhibitory species can diffuse away to covalently modify other proteins, as reflected by the relatively high partition ratio of A, which we determined to be 15.6. This study highlights critical methods that can guide the discovery and development of next-generation MPO inhibitors.

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.

Deconstruction of activity-dependent covalent modification of heme in human neutrophil myeloperoxidase by multistage mass spectrometry (MS(4)).

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.

Discovery and evaluation of spirocyclic derivatives as antagonists of the neuropeptide Y5 receptor.

A novel series of spirocyclic derivatives was synthesized and evaluated as NPY Y5R antagonists for the treatment of obesity. Cis and trans analogs 7a and 8a were equipotent in a Y5R binding assay (K(i)'s ≤ 1 nM) and displayed good stability in human and rat liver microsome preparations. Compound 7a failed to demonstrate weight loss activity in a diet-induced obese (DIO) rat model at unbound drug levels in the brain that exceeded the Y5R K(i) value by 25-fold over a 24-h time-period.

Identification of spirocyclic piperidine-azetidine inverse agonists of the ghrelin receptor.

The discovery of spirocyclic piperidine-azetidine inverse agonists of the ghrelin receptor is described. The characterization and redressing of the issues associated with these compounds is detailed. An efficient three-step synthesis and a binding assay were relied upon as the primary means of rapidly improving potency and ADMET properties for this class of inverse agonist compounds. Compound 10 n bearing distributed polarity in the form of an imidazo-thiazole acetamide and a phenyl triazole is a unit lower in logP and has significantly improved binding affinity compared to the hit molecule 10a, providing support for further optimization of this series of compounds.

Discovery and evaluation of pyrazolo[1,5-a]pyrimidines as neuropeptide Y1 receptor antagonists.

A novel series of pyrazolo[1,5-a]pyrimidine derivatives was synthesized and evaluated as NPY Y1R antagonists. High binding affinity and selectivity were achieved with C3 trisubstituted aryl groups and C7 substituted 2-(tetrahydro-2H-pyran-4-ylamino)ethylamine moieties. Efforts to find close analogs with low plasma clearance in the rat and minimal p-glycoprotein efflux in the mouse were unsuccessful. Compound 2f (CP-671906) inhibited NPY-induced increases in blood pressure and food intake after iv and icv administration, respectively, in Sprague-Dawley (SD) rat models. Oral administration of compound 2f resulted in a modest, but statistically significant, reduction in food intake in a Wistar rat model of feeding behavior. Small inhibitions of food intake were also observed in an overnight fasting/refeeding model in SD rats. These data suggest a potential role for Y1R in the regulation of food intake in rodents.

Quantitative in vitro and in vivo pharmacological profile of CE-178253, a potent and selective cannabinoid type 1 (CB1) receptor antagonist.

BACKGROUND: Cannabinoid 1 (CB1) receptor antagonists exhibit pharmacological properties favorable for the treatment of obesity and other related metabolic disorders. CE-178253 (1-[7-(2-Chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[1,5-a]-[1,3,5]triazin-4-yl]-3-ethylaminoazetidine-3-carboxylic acid hydrochloride) is a recently discovered selective centrally-acting CB1 receptor antagonist. Despite a large body of knowledge on cannabinoid receptor antagonists little data exist on the quantitative pharmacology of this therapeutic class of drugs. The purpose of the current studies was to evaluate the quantitative pharmacology and concentration/effect relationships of CE-178253 based on unbound plasma concentration and in vitro pharmacology data in different in vivo preclinical models of FI and energy expenditure. RESULTS: In vitro, CE-178253 exhibits sub-nanomolar potency at human CB1 receptors in both binding (Ki = 0.33 nM) and functional assays (Ki = 0.07 nM). CE-178253 has low affinity (Ki > 10,000 nM) for human CB2 receptors. In vivo, CE-178253 exhibits concentration-dependent anorectic activity in both fast-induced re-feeding and spontaneous nocturnal feeding FI models. As measured by indirect calorimetry, CE-178253 acutely stimulates energy expenditure by greater than 30% in rats and shifts substrate oxidation from carbohydrate to fat as indicated by a decrease the respiratory quotient from 0.85 to 0.75. Determination of the concentration-effect relationships and ex vivo receptor occupancy in efficacy models of energy intake and expenditure suggest that a greater than a 2-fold coverage of the Ki (50-75% receptor occupancy) is required for maximum efficacy. Finally, in two preclinical models of obesity, CE-178253 dose-dependently promotes weight loss in diet-induced obese rats and mice. CONCLUSIONS: We have combined quantitative pharmacology and ex vivo CB1 receptor occupancy data to assess concentration/effect relationships in food intake, energy expenditure and weight loss studies. Quantitative pharmacology studies provide a strong a foundation for establishing and improving confidence in mechanism as well as aiding in the progression of compounds from preclinical pharmacology to clinical development.

Bioisosteric replacement of the hydrazide pharmacophore of the cannabinoid-1 receptor antagonist SR141716A. Part I: potent, orally-active 1,4-disubstituted imidazoles.

A new series of CB(1) receptor antagonists incorporating an imidazole-based isosteric replacement for the hydrazide moiety of rimonabant (SR141716) is disclosed. Members of this imidazole series possess potent/selective binding to the rCB(1) receptor and exhibit potent hCB(1) functional activity. Isopropyl analog 9a demonstrated activity in the tetrad assay and was orally-active in a food intake model.

Diabetes area patent participation analysis - part II: years 2011-2016.

INTRODUCTION: Diabetes is a metabolic disease characterized by elevated levels of plasma glucose. When untreated, diabetes increases the risk of developing co-morbidities such as cardiovascular disease. Several drugs, often used as part of combination therapies, have been approved to treat the disease, but these drugs will eventually fail to effectively control blood glucose levels, at which point insulin replacement therapy is required. A medical need exists for new antidiabetic drugs that exhibit good efficacy with improved safety/toleration profiles and can be added on top of existing therapies, or that can provide additional benefits beyond glucose lowering such as pancreatic beta (ß)-cell protection. AREAS COVERED: This review analyzes drug targets and applicants of patents that published between 2011-2016 claiming novel small or large molecules for the treatment of diabetes, and compares the results to the 2008-2010 time period. EXPERT OPINION: A majority of patent activity around the discovery of new antidiabetic drugs in 2011-2016 was directed against 15 targets, most of which were also the focus of drug discovery efforts in the 2008-2010 time period. The top targets by total patent counts were DPP4, GLP1R, INSR, GPR119, and SGLT2 (SLC5A2). With the exception of GPR119, these are the pharmacological targets of some of the best-selling antidiabetic drugs currently on the market. The top targets of patent families with the largest size counts, a metric useful in assessing patent value and applicant interest, were AMPK, CALCR, DPP4, and GLP1R. The patent analysis identified several emerging targets with greater patent activity in 2011-2016 compared to 2008-2010, including FFAR1, FFAR4, and FGFR1. Most of the patent activity in 2011-2016 was directed at established and precedented diabetes targets, the modulation of which may lead to improvements in glucose control and a delay in the progression of the disease. Few targets were identified that promote pancreatic ß-cell regeneration and ß-cell health, areas where future opportunities may exist for developing transformative drug therapies that may potentially lead to cures for diabetes.

Discovery and in Vitro Optimization of 3-Sulfamoylbenzamides as ROMK Inhibitors.

Inhibitors of the renal outer medullary potassium channel (ROMK) show promise as novel mechanism diuretics, with potentially lower risk of diuretic-induced hypokalemia relative to current thiazide and loop diuretics. Here, we report the identification of a novel series of 3-sulfamoylbenzamide ROMK inhibitors. Starting from HTS hit 4, this series was optimized to provide ROMK inhibitors with good in vitro potencies and well-balanced ADME profiles. In contrast to previously reported small-molecule ROMK inhibitors, members of this series were demonstrated to be highly selective for inhibition of human over rat ROMK and to be insensitive to the N171D pore mutation that abolishes inhibitory activity of previously reported ROMK inhibitors.

A Small-Molecule Anti-secretagogue of PCSK9 Targets the 80S Ribosome to Inhibit PCSK9 Protein Translation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that downregulates low-density lipoprotein (LDL) receptor (LDL-R) levels on the surface of hepatocytes, resulting in decreased clearance of LDL-cholesterol (LDL-C). Phenotypic screening of a small-molecule compound collection was used to identify an inhibitor of PCSK9 secretion, (R)-N-(isoquinolin-1-yl)-3-(4-methoxyphenyl)-N-(piperidin-3-yl)propanamide (R-IMPP), which was shown to stimulate uptake of LDL-C in hepatoma cells by increasing LDL-R levels, without altering levels of secreted transferrin. Systematic investigation of the mode of action revealed that R-IMPP did not decrease PCSK9 transcription or increase PCSK9 degradation, but instead caused transcript-dependent inhibition of PCSK9 translation. In support of this surprising mechanism of action, we found that R-IMPP was able to selectively bind to human, but not E. coli, ribosomes. This study opens a new avenue for the development of drugs that modulate the activity of target proteins by mechanisms involving inhibition of eukaryotic translation.

Melanocortin-4 receptor modulators for the treatment of obesity: a patent analysis (2008-2014).

The central melanocortin system and particularly the melanocortin-4 receptor (MC4R) subtype, plays an important role in the regulation of body weight. The discovery of orally active MC4R agonists suitable for evaluation in human clinical trials as weight loss agents has attracted considerable interest over the past decade, but has proved challenging, in part because of cardiovascular and behavioral side effects. Currently, the only MC4R agonist in clinical trials is a peptide identified as RM-493. To avoid some of the undesirable side effects associated with MC4R activation, new pharmacological approaches for modulating the MC system have been investigated. In this article, we provide a review of the MC4R patent landscape from 2008 to 2014 and analyze the physicochemical properties of compounds described herein.

Latest approaches for the treatment of obesity.

INTRODUCTION: Obesity is a body weight disorder characterized by excess adiposity that increases the risk for developing co-morbidities such as type 2 diabetes. A large medical need exists for new anti-obesity treatments capable of promoting 10% or greater weight loss, with minimal side effects. AREAS COVERED: The authors describe the application of monogenic forms of rare obesity and genome-wide association studies in selecting critical pathways for drug discovery. Furthermore, they review in detail several pathways and pharmacological targets in the central nervous system (e.g., the leptin-melanocortin axis, the opioid system, GLP-1/GLP-1 system, and FGF21/FGFR1c/ß-Klotho axis) that play an important role in the regulation of feeding behavior and energy homeostasis. Special focus is given to new strategies that engage well-known targets via novel mechanisms in order to circumvent issues seen with previous drug candidates that failed in the clinic. Finally, the authors discuss the recent developments around fixed-dose combinations, targeted polypharmacology, and non-traditional combinations of drugs and devices. EXPERT OPINION: The future for new weight-loss approaches to treat obesity looks promising. Current therapies have shown modest effects on weight loss in the general obese population but will have greater impact in smaller homogeneous sub-populations of obese subjects using personalized medicine. Drug combinations that target multiple, complementary pathways have the potential to promote double-digit weight loss in a broader, heterogeneous patient population. Furthermore, the development of advanced subcutaneous delivery technologies has opened up opportunities to develop breakthrough peptide and biologic agents for the treatment of obesity.

MAP4K4 Is a Threonine Kinase That Phosphorylates FARP1.

Mitogen-activated protein kinase 4 (MAP4K4) regulates the MEK kinase cascade and is implicated in cytoskeletal rearrangement and migration; however, identifying MAP4K4 substrates has remained a challenge. To ascertain MAP4K4-dependent phosphorylation events, we combined phosphoproteomic studies of MAP4K4 inhibition with in vitro assessment of its kinase specificity. We identified 235 phosphosites affected by MAP4K4 inhibition in cells and found that pTP and pSP motifs were predominant among them. In contrast, in vitro assessment of kinase specificity showed that MAP4K4 favors a pTL motif. We showed that MAP4K4 directly phosphorylates and coimmunoprecipitates with FERM, RhoGEF, and pleckstrin domain-containing protein 1 (FARP1). MAP4K4 inhibition in SH-SY5Y cells increases neurite outgrowth, a process known to involve FARP1. As FARP1 and MAP4K4 both contribute to cytoskeletal rearrangement, the results suggest that MAP4K4 exerts some of its effects on the cytoskeleton via phosphorylation of FARP1.

Discovery of Selective Small Molecule Inhibitors of Monoacylglycerol Acyltransferase 3.

Inhibition of triacylglycerol (TAG) biosynthetic enzymes has been suggested as a promising strategy to treat insulin resistance, diabetes, dyslipidemia, and hepatic steatosis. Monoacylglycerol acyltransferase 3 (MGAT3) is an integral membrane enzyme that catalyzes the acylation of both monoacylglycerol (MAG) and diacylglycerol (DAG) to generate DAG and TAG, respectively. Herein, we report the discovery and characterization of the first selective small molecule inhibitors of MGAT3. Isoindoline-5-sulfonamide (6f, PF-06471553) selectively inhibits MGAT3 with high in vitro potency and cell efficacy. Because the gene encoding MGAT3 (MOGAT3) is found only in higher mammals and humans, but not in rodents, a transgenic mouse model expressing the complete human MOGAT3 was used to characterize the effects of 6f in vivo. In the presence of a combination of diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2) inhibitors, an oral administration of 6f exhibited inhibition of the incorporation of deuterium-labeled glycerol into TAG in this mouse model. The availability of a potent and selective chemical tool and a humanized mouse model described in this report should facilitate further dissection of the physiological function of MGAT3 and its role in lipid homeostasis.

Discovery of 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999): A Highly Selective Mechanism-Based Myeloperoxidase Inhibitor for the Treatment of Cardiovascular Diseases.

Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Clinical evidence suggests a causal role for MPO in various autoimmune and inflammatory disorders including vasculitis and cardiovascular and Parkinson's diseases, implying that MPO inhibitors may represent a therapeutic treatment option. Herein, we present the design, synthesis, and preclinical evaluation of N1-substituted-6-arylthiouracils as potent and selective inhibitors of MPO. Inhibition proceeded in a time-dependent manner by a covalent, irreversible mechanism, which was dependent upon MPO catalysis, consistent with mechanism-based inactivation. N1-Substituted-6-arylthiouracils exhibited low partition ratios and high selectivity for MPO over thyroid peroxidase and cytochrome P450 isoforms. N1-Substituted-6-arylthiouracils also demonstrated inhibition of MPO activity in lipopolysaccharide-stimulated human whole blood. Robust inhibition of plasma MPO activity was demonstrated with the lead compound 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999, 8) upon oral administration to lipopolysaccharide-treated cynomolgus monkeys. On the basis of its pharmacological and pharmacokinetic profile, PF-06282999 has been advanced to first-in-human pharmacokinetic and safety studies.

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.