In medicinal chemistry, purification and characterization of organic compounds is an ever-growing challenge, with an increasing number of compounds being synthesized at a decreased scale of preparation. In response to this trend, we developed a parallel medicinal chemistry (PMC)-tailored platform, coupling automated purification to mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) on a range of synthetic scales (similar to 3.0-75.0 mu mol). Here, the generation and acquisition of 1.7 mm NMR samples is fully integrated into a high-throughput automated workflow, processing 36 000 compounds yearly. Utilizing dead volume, which is inaccessible in conventional liquid handling, NMR samples are generated on as little as 10 mu g without consuming material prioritized for biological assays. As miniaturized PMC synthesis becomes the industry standard, we can now obtain quality NMR spectra from limited material. Paired with automated structure verification, this platform has the potential to allow NMR to become as important for high-throughput analysis as ultrahigh performance liquid chromatography (UPLC)-MS.

Low-molecular weight chiral amines are valuable components in medicinal chemistry as they serve as core templates, linking units, and substituent appendages. The piperidine scaffold is particularly useful among privileged small amines, with substituted variants having a great number of potential regio- and diastereoisomers, which allow for high stereochemical definition to enable a variety of productive protein interactions. Herein, we describe the successful enablement, scale-up, and delivery of >400 g of a single isomer, (3S,5S)-1-((benzyloxy)carbonyl)-5-fluoropiperidine-3-carboxylic acid (>98% de and >96% ee), via 450 g-scale biocatalytic desymmetrization and 335 g-scale flow photochemical decarboxylative fluorination.

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.

The disposition of the hepatoselective ACC inhibitor PF-05221304 (Clesacostat) was studied after a single 50-mg oral dose of [14C]-PF-05221304 to healthy human subjects.Mass balance was achieved with 89.9% of the administered dose recovered in urine and faeces, over the 11-day study period. The total administered radioactivity excreted in faeces and urine was 81.7 and 8.2%, respectively. Unchanged PF-05221304 accounted for 35.6% of the radioactive dose in faeces, suggesting ∼64% of the administered dose was absorbed.PF-05221304 was principally metabolised via oxidative and reductive pathways involving: (a) N-dealkylation, (b) isopropyl group monohydroxylation to yield enantiomeric metabolites (M2a and M2b), (c) hydroxylation on the 3-azaspiro[5.5]undecan-8-one moiety to metabolites M5 and 519c, and (d) carbonyl group reduction to enantiomeric alcohol metabolites M3, and M4. Secondary metabolites (521a, 521b, and 533), derived from a combination of oxidation and reduction of the primary metabolites accounted for ∼14.8% of the dose. In plasma, unchanged PF-05221304 represented 96.1% circulating radioactivity. Metabolites M1, M2b, and M2a represented 1.94, 1.76, and 0.18% of circulating radioactivity, respectively.Overall, these data suggest that PF-05221304 is well absorbed in humans and eliminated largely via phase I metabolism.

The ability to predict chemical structure from DNA sequence has to date been a necessary cornerstone of DNAencoded library technology. DNA-encoded libraries (DELs) are typically screened by immobilized affinity selection and enriched library members are identified by counting the number of times an individual compound's sequence is observed in the resultant dataset. Those with high signal reads (DEL hits) are subsequently followed up through off-DNA synthesis of the predicted small molecule structures. However, hits followed-up in this manner often fail to translate to confirmed ligands. To address this low conversion rate of DEL hits to off-DNA ligands, we have developed an approach that eliminates the reliance on chemical structure prediction from DNA sequence. Here we describe our method of combining non-combinatorial resynthesis on-DNA following library procedures as a rapid means to assess the probable molecules attached to the DNA barcode. Furthermore, we apply our Bead-Assisted Ligand Isolation Mass Spectrometry (BALI-MS) technique to identify the true binders found within the mixtures of on-DNA synthesis products. Finally, we describe a Normalized Enrichment (NE) metric that allows for the quantitative assessment of affinity selection in these studies. We exemplify how this combined approach enables the identification of putative hit matter against a clinically relevant therapeutic target bisphosphoglycerate mutase, BPGM.

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.

(R)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)-1-(2-(5-(6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)ethan-1-one (PF-5190457) was identified as a potent and selective inverse agonist of the ghrelin receptor [growth hormone secretagogue receptor 1a (GHS-R1a)]. The present translational bed-to-bench work characterizes the biotransformation of this compound in vivo and then further explores in vitro metabolism in fractions of human liver and primary hepatocytes. Following oral administration of PF-5190457 in a phase 1b clinical study, hydroxyl metabolites of the compound were observed, including one that had not been observed in previously performed human liver microsomal incubations. PF-6870961 or (R)-1-(2-(5-(2-hydroxy-6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)ethan-1-one was biosynthesized using liver cytosol, and the site of hydroxylation was shown to be on the pyrimidine using nuclear magnetic resonance spectroscopy. The aldehyde oxidase (AO) inhibitor raloxifene and the xanthine oxidase inhibitor febuxostat inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. However, greater inhibition was observed with raloxifene, indicating AO is a dominant enzyme in the biotransformation. The intrinsic clearance of the drug in human liver cytosol was estimated to be 0.002 ml/min per milligram protein. This study provides important novel information at three levels: 1) it provides additional new information on the recently developed novel compound PF-5190457, the first GHS-R1a blocker that has moved to development in humans; 2) it provides an example of a reverse translational approach where a discovery in humans was brought back, validated, and further investigated at the bench level; and 3) it demonstrates the importance of considering the molybdenum-containing oxidases during the development of new drug entities. SIGNIFICANCE STATEMENT PF-5190457 is a novel ghrelin receptor inverse agonist that is currently undergoing clinical development for treatment of alcohol use disorder. PF-6870961, a major hydroxyl metabolite of the compound, was observed in human plasma, but was absent in human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation on the pyrimidine ring was characterized. Inhibitors of aldehyde oxidase and xanthine oxidase inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. This information is important for patient selection in subsequent clinical studies.

Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) alpha 1 beta 1 gamma 1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of beta 1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK alpha 1 beta 1 gamma 1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.

In this open-label study (NCT02142920), we investigated the distribution, pharmacokinetics, and metabolism of the pan-class-I isoform phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor gedatolisib (PF-05212384), following a single intravenous administration in healthy male subjects. A single, 89-mg, intravenous dose of gedatolisib was associated with a favorable safety profile in the 6 healthy subjects evaluated. Peak plasma concentrations for unchanged gedatolisib and total radioactivity were observed at the end of the 30-minute infusion. The only observed drug-related material in plasma was the parent drug, gedatolisib. Terminal half-life for plasma gedatolisib was similar to 37 hours. Following the dose, 66%-73% of drug-related material was recovered in the feces. Metabolism of gedatolisib was trace; only 1 oxidative metabolite, M5, was identified in feces (<1% of total dose). Identification of gedatolisib in feces suggests that biliary and/or intestinal secretion of unchanged parent drug significantly contributes to gedatolisib clearance.

C-X-C chemokine receptor type 7 (CXCR7) is involved in cardiac and immune pathophysiology. We report the discovery of a novel 1,4-diazepine CXCR7 modulator, demonstrating for the first time the role of pharmacological CXCR7 intervention in cardiac repair. Structure-activity-relationship (SAR) studies demonstrated that a net reduction in lipophilicity (log D) and an incorporation of saturated ring systems yielded compounds with good CXCR7 potencies and improvements in oxidative metabolic stability in human-liver microsomes (HLM). Tethering an ethylene amide further improved the selectivity profile (e.g., for compound 18, CXCR7 Ki = 13 nM, adrenergic α 1a Kb > 10 000 nM, and adrenergic β 2 Kb > 10 000 nM). The subcutaneous administration of 18 in mice led to a statistically significant increase in circulating concentrations of plasma stromal-cell-derived factor 1α (SDF-1α) of approximately 2-fold. Chronic dosing of compound 18 in a mouse model of isoproterenol-induced cardiac injury further resulted in a statistically significant reduction of cardiac fibrosis.

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 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

The thiouracil derivative PF-06282999 [2-(6-(5-chloro-2-methoxyphenyl)4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide] is an irreversible inactivator of myeloperoxidase and is currently in clinical trials for the potential treatment of cardiovascular diseases. Concerns over idiosyncratic toxicity arising from bioactivation of the thiouracil motif to reactive species in the liver have been largely mitigated through the physicochemical (molecular weight, lipophilicity, and topological polar surface area) characteristics of PF-06282999, which generally favor elimination via nonmetabolic routes. To test this hypothesis, pharmacokinetics and disposition studies were initiated with PF-06282999 using animals and in vitro assays, with the ultimate goal of predicting human pharmacokinetics and elimination mechanisms. Consistent with its physicochemical properties, PF-06282999 was resistant to metabolic turnover from liver microsomes and hepatocytes from animals and humans and was devoid of cytochrome P450 inhibition. In vitro transport studies suggested moderate intestinal permeability and minimal transporter-mediated hepatobiliary disposition. PF-06282999 demonstratedmoderate plasmaprotein binding across all of the species. Pharmacokinetics in preclinical species characterized by low to moderate plasma clearances, good oral bioavailability at 3to 5-mg/kg doses, and renal clearance as the projected major clearance mechanism in humans. Human pharmacokinetic predictions using single-species scaling of dog and/or monkey pharmacokinetics were consistent with the parameters observed in the first-inhuman study, conducted in healthy volunteers at a dose range of 20–200 mg PF-06282999. In summary, disposition characteristics of PF-06282999 were relatively similar across preclinical species and humans, with renal excretion of the unchanged parent emerging as the principal clearance mechanism in humans, which was anticipated based on its physicochemical properties and supported by preclinical studies.

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.

The thiouracil derivative PF-06282999 [2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide] is an irreversible inactivator of myeloperoxidase and is currently in clinical trials for the potential treatment of cardiovascular diseases. Concerns over idiosyncratic toxicity arising from bioactivation of the thiouracil motif to reactive species in the liver have been largely mitigated through the physicochemical (molecular weight, lipophilicity, and topological polar surface area) characteristics of PF-06282999, which generally favor elimination via nonmetabolic routes. To test this hypothesis, pharmacokinetics and disposition studies were initiated with PF-06282999 using animals and in vitro assays, with the ultimate goal of predicting human pharmacokinetics and elimination mechanisms. Consistent with its physicochemical properties, PF-06282999 was resistant to metabolic turnover from liver microsomes and hepatocytes from animals and humans and was devoid of cytochrome P450 inhibition. In vitro transport studies suggested moderate intestinal permeability and minimal transporter-mediated hepatobiliary disposition. PF-06282999 demonstrated moderate plasma protein binding across all of the species. Pharmacokinetics in preclinical species characterized by low to moderate plasma clearances, good oral bioavailability at 3- to 5-mg/kg doses, and renal clearance as the projected major clearance mechanism in humans. Human pharmacokinetic predictions using single-species scaling of dog and/or monkey pharmacokinetics were consistent with the parameters observed in the first-in-human study, conducted in healthy volunteers at a dose range of 20-200 mg PF-06282999. In summary, disposition characteristics of PF-06282999 were relatively similar across preclinical species and humans, with renal excretion of the unchanged parent emerging as the principal clearance mechanism in humans, which was anticipated based on its physicochemical properties and supported by preclinical studies.

The contribution of drug metabolites to the pharmacologic and toxicologic activity of a drug can be important; however, for a variety of reasons metabolites can frequently be difficult to synthesize. To meet the need of having samples of drug metabolites for further study, we have developed biosynthetic methods coupled with quantitative NMR spectroscopy (qNMR) to generate solutions of metabolites of known structure and concentration. These quantitative samples can be used in a variety of ways when a synthetic sample is unavailable, including pharmacologic assays, standards for in vitro work to help establish clearance pathways, and/or as analytical standards for bioanalytical work to ascertain exposure, among others. We illustrate five examples of metabolite biosynthesis and qNMR. The types of metabolites include one glucuronide and four oxidative products. Concentrations of the isolated metabolite stock solutions ranged from 0.048 to 8.3 mM, with volumes from approximately 0.04 to 0.150 ml in hexadeutarated dimethylsulfoxide. These specific quantified isolates were used as standards in the drug discovery setting as substrates in pharmacology assays, for bioanalytical assays to establish exposure, and in variety of routine absorption, distribution, metabolism, and excretion assays, such as protein binding and determining blood-toplasma ratios. The methods used to generate these materials are described in detail with the objective that these methods can be generally used for metabolite biosynthesis and isolation.

The contribution of drug metabolites to the pharmacologic and toxicologic activity of a drug can be important; however, for a variety of reasons metabolites can frequently be difficult to synthesize. To meet the need of having samples of drug metabolites for further study, we have developed biosynthetic methods coupled with quantitative NMR spectroscopy (qNMR) to generate solutions of metabolites of known structure and concentration. These quantitative samples can be used in a variety of ways when a synthetic sample is unavailable, including pharmacologic assays, standards for in vitro work to help establish clearance pathways, and/or as analytical standards for bioanalytical work to ascertain exposure, among others. We illustrate five examples of metabolite biosynthesis and qNMR. The types of metabolites include one glucuronide and four oxidative products. Concentrations of the isolated metabolite stock solutions ranged from 0.048 to 8.3 mM, with volumes from approximately 0.04 to 0.150 ml in hexadeutarated dimethylsulfoxide. These specific quantified isolates were used as standards in the drug discovery setting as substrates in pharmacology assays, for bioanalytical assays to establish exposure, and in variety of routine absorption, distribution, metabolism, and excretion assays, such as protein binding and determining blood-to-plasma ratios. The methods used to generate these materials are described in detail with the objective that these methods can be generally used for metabolite biosynthesis and isolation.

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.

Tofacitinib is a novel, oral Janus kinase inhibitor. The objectives of this study were to summarize the pharmacokinetics and metabolism of tofacitinib in humans, including clearance mechanisms. Following administration of a single 50-mg C-labeled tofacitinib dose to healthy male subjects, the mean (standard deviation) total percentage of administered radioactive dose recovered was 93.9% (63.6), with 80.1% (63.6) in the urine (28.8% parent), and 13.8% (61.9) in feces (0.9% parent). Tofacitinib was rapidly absorbed, with plasma concentrations and total radioactivity peaking at around 1 hour after oral administration. The mean terminal phase half-life was approximately 3.2 hours for both parent drug and total radioactivity. Most (69.4%) circulating radioactivity in plasma was parent drug, with all metabolites representing less than 10% each of total circulating radioactivity. Hepatic clearance made up around 70% of total clearance, while renal clearance made up the remaining 30%. The predominant metabolic pathways of tofacitinib included oxidation of the pyrrolopyrimidine and piperidine rings, oxidation of the piperidine ring side-chain, N-demethylation and glucuronidation. Cytochrome P450 (P450) profiling indicated that tofacitinib was mainly metabolized by CYP3A4, with a smaller contribution from CYP2C19. This pharmacokinetic characterization of tofacitinib has been consistent with its clinical experience in drug-drug interaction

Tofacitinib is a novel, oral Janus kinase inhibitor. The objectives of this study were to summarize the pharmacokinetics and metabolism of tofacitinib in humans, including clearance mechanisms. Following administration of a single 50-mg (14)C-labeled tofacitinib dose to healthy male subjects, the mean (standard deviation) total percentage of administered radioactive dose recovered was 93.9% (±3.6), with 80.1% (±3.6) in the urine (28.8% parent), and 13.8% (±1.9) in feces (0.9% parent). Tofacitinib was rapidly absorbed, with plasma concentrations and total radioactivity peaking at around 1 hour after oral administration. The mean terminal phase half-life was approximately 3.2 hours for both parent drug and total radioactivity. Most (69.4%) circulating radioactivity in plasma was parent drug, with all metabolites representing less than 10% each of total circulating radioactivity. Hepatic clearance made up around 70% of total clearance, while renal clearance made up the remaining 30%. The predominant metabolic pathways of tofacitinib included oxidation of the pyrrolopyrimidine and piperidine rings, oxidation of the piperidine ring side-chain, N-demethylation and glucuronidation. Cytochrome P450 (P450) profiling indicated that tofacitinib was mainly metabolized by CYP3A4, with a smaller contribution from CYP2C19. This pharmacokinetic characterization of tofacitinib has been consistent with its clinical experience in drug-drug interaction studies.