Complex peptide macrocycle optimization: combining NMR restraints with conformational analysis to guide structure-based and ligand-based design.

Systematic optimization of large macrocyclic peptide ligands is a serious challenge. Here, we describe an approach for lead-optimization using the PD-1/PD-L1 system as a retrospective example of moving from initial lead compound to clinical candidate. We show how conformational restraints can be derived by exploiting NMR data to identify low-energy solution ensembles of a lead compound. Such restraints can be used to focus conformational search for analogs in order to accurately predict bound ligand poses through molecular docking and thereby estimate ligand strain and protein-ligand intermolecular binding energy. We also describe an analogous ligand-based approach that employs molecular similarity optimization to predict bound poses. Both approaches are shown to be effective for prioritizing lead-compound analogs. Surprisingly, relatively small ligand modifications, which may have minimal effects on predicted bound pose or intermolecular interactions, often lead to large changes in estimated strain that have dominating effects on overall binding energy estimates. Effective macrocyclic conformational search is crucial, whether in the context of NMR-based restraints, X-ray ligand refinement, partial torsional restraint for docking/ligand-similarity calculations or agnostic search for nominal global minima. Lead optimization for peptidic macrocycles can be made more productive using a multi-disciplinary approach that combines biophysical data with practical and efficient computational methods.

Discovery of novel pyridinones as MGAT2 inhibitors for the treatment of metabolic disorders.

Inhibition of monoacylglycerol transferase 2 (MGAT2) has recently emerged as a potential therapeutic strategy for the treatment of metabolic diseases such as obesity, diabetes and non-alcoholic steatohepatitis (NASH). Metabolism studies with our clinical lead (1) suggested variability in in vitro glucuronidation rates in liver microsomes across species, which made projection of human doses challenging. In addition, the observation of deconjugation of the C3-C4 double bond in the dihydropyridinone ring of 1 in solution had the potential to complicate its clinical development. This report describes our lead optimization efforts in a novel pyridinone series, exemplified by compound 33, which successfully addressed both of these potential issues.

Structure-activity relationship study of central pyridine-derived TYK2 JH2 inhibitors: Optimization of the PK profile through C4' and C6 variations.

Efforts directed at improving potency and preparing structurally different TYK2 JH2 inhibitors from the first generation of compounds such as 1a led to the SAR study of new central pyridyl based analogs 2-4. The current SAR study resulted in the identification of 4h as a potent and selective TYK2 JH2 inhibitor with distinct structural differences from 1a. In this manuscript, the in vitro and in vivo profiles of 4h are described. The hWB IC50 of 4h was shown as 41 nM with 94% bioavailability in the mouse PK study.

Optimization of physicochemical properties of pyrrolidine GPR40 AgoPAMs results in a differentiated profile with improved pharmacokinetics and reduced off-target activities.

GPR40 AgoPAMs are highly effective antidiabetic agents that have a dual mechanism of action, stimulating both glucose-dependent insulin and GLP-1 secretion. The early lipophilic, aromatic pyrrolidine and dihydropyrazole GPR40 AgoPAMs from our laboratory were highly efficacious in lowering plasma glucose levels in rodents but possessed off-target activities and triggered rebound hyperglycemia in rats at high doses. A focus on increasing molecular complexity through saturation and chirality in combination with reducing polarity for the pyrrolidine AgoPAM chemotype resulted in the discovery of compound 46, which shows significantly reduced off-target activities as well as improved aqueous solubility, rapid absorption, and linear PK. In vivo, compound 46 significantly lowers plasma glucose levels in rats during an oral glucose challenge yet does not demonstrate the reactive hyperglycemia effect at high doses that was observed with earlier GPR40 AgoPAMs.

Ni/Photoredox-Catalyzed C(sp2)-C(sp3) Cross-Coupling of Alkyl Pinacolboronates and (Hetero)Aryl Bromides.

Utilizing quinoline as a mild, catalytic additive, broadly applicable conditions for the Ni/photoredox-catalyzed C(sp2)-C(sp3) cross-coupling of (hetero)aryl bromides and alkyl pinacolboronate esters were developed, which can be applied to both batch and flow reactions. In addition to primary benzylic nucleophiles, both stabilized and nonstabilized secondary alkyl boronic esters are effective coupling partners. Density functional theory calculations suggest that alkyl radical generation occurs from an alkyl-B(pin)-quinoline complex, which may proceed via an energy transfer process.

Pharmacokinetics of 40 kDa Polyethylene glycol (PEG) in mice, rats, cynomolgus monkeys and predicted pharmacokinetics in humans.

Conjugation with polyethylene glycol (PEG), PEGylation, has been considered a useful tool to improve drug-like properties of novel small molecules and biologics in drug discovery. PEG40 or 40 kDa PEG is a double-branched PEG, routinely employed to improve the pharmacokinetics (PK) of therapeutics, including successful marketed products such as Pegasys® and Omontys®. However, less is known about the extent of contribution of PEG40 to the overall PK of the PEGylated product. Considering the half-life of PEG40 conjugated PEGylated products ranges from 1 to 14 days in human, this information is immensely valuable. After successfully developing a high sensitivity NMR based analytical method to quantitate PEG40 in mice serum after intravenous (IV) administration (Khandelwal et al., 2019), here, we extend its application to measure PEG40 in serum after IV administration and subcutaneous (SC) absorption in routinely employed non-clinical species in drug discovery, namely, mice, rats and cynomolgus monkeys. We utilized non-compartmental analysis and compartmental modeling to characterize the PK of PEG40 in these non-clinical species. Finally, we employed allometric scaling and Wajima (MRT-Css) method to predict the PK of PEG40 in human after IV administration and SC absorption. In general, our data shows that intrinsic PK parameters of PEG40 in mice, rats and cynomolgus monkeys are in the range of published literature values for PEG40-conjugated products, unless saturable clearance mechanisms are involved. We observed a bioavailability (F) of ~68% in CD-1 mice after SC administration of PEG40. In rats, the clearance (CL) and volume of distribution at steady state (Vss) after IV infusion of PEG40 were 0.079 mL/min/kg and 0.19 L/kg, respectively; and SC bioavailability was ~20%. In cynomolgus monkeys, after IV infusion, CL and Vss of PEG40 were 0.037 mL/min/kg and 0.20 L/kg, respectively; and SC bioavailability was ~69%. In addition, our findings indicate flip-flop kinetics of PEG40 in rodents, but not in cynomolgus monkeys. Finally, in human, intrinsic CL and Vss of PEG40 were projected to be 0.02 mL/min/kg (0.084 L/h) and 0.22 L/kg, respectively. This comprehensive report of PK of PEG40 in non-clinical species and its subsequent prediction in humans is expected to be useful to drug discovery and development scientists for efficient decision-making and optimal resource utilization.

Discovery of BMS-753426: A Potent Orally Bioavailable Antagonist of CC Chemokine Receptor 2.

To improve the metabolic stability profile of BMS-741672 (1a), we undertook a structure-activity relationship study in our trisubstituted cyclohexylamine series. This ultimately led to the identification of 2d (BMS-753426) as a potent and orally bioavailable antagonist of CCR2. Compared to previous clinical candidate 1a, the tert-butyl amine 2d showed significant improvements in pharmacokinetic properties, with lower clearance and higher oral bioavailability. Furthermore, compound 2d exhibited improved affinity for CCR5 and good activity in models of both monocyte migration and multiple sclerosis in the hCCR2 knock-in mouse. The synthesis of 2d was facilitated by the development of a simplified approach to key intermediate (4R)-9b that deployed a stereoselective reductive amination which may prove to be of general interest.

Azatricyclic Inverse Agonists of RORγt That Demonstrate Efficacy in Models of Rheumatoid Arthritis and Psoriasis.

Structure-activity relationship studies directed toward the replacement of the fused phenyl ring of the lead hexahydrobenzoindole RORγt inverse agonist series represented by 1 with heterocyclic moieties led to the identification of three novel aza analogs 5-7. The hexahydropyrrolo[3,2-f]quinoline series 5 (X = N, Y = Z=CH) showed potency and metabolic stability comparable to series 1 but with improved in vitro membrane permeability and serum free fraction. This structural modification was applied to the hexahydrocyclopentanaphthalene series 3, culminating in the discovery of 8e as a potent and selective RORγt inverse agonist with an excellent in vitro profile, good pharmacokinetic properties, and biologic-like in vivo efficacy in preclinical models of rheumatoid arthritis and psoriasis.

Tricyclic-Carbocyclic RORγt Inverse Agonists-Discovery of BMS-986313.

SAR efforts directed at identifying RORγt inverse agonists structurally different from our clinical compound 1 (BMS-986251) led to tricyclic-carbocyclic analogues represented by 3-7 and culminated in the identification of 3d (BMS-986313), with structural differences distinct from 1. The X-ray co-crystal structure of 3d with the ligand binding domain of RORγt revealed several key interactions, which are different from 1. The in vitro and in vivo PK profiles of 3d are described. In addition, we demonstrate robust efficacy of 3d in two preclinical models of psoriasis-the IMQ-induced skin lesion model and the IL-23-induced acanthosis model. The efficacy seen with 3d in these models is comparable to the results observed with 1.

Novel Tricyclic Pyroglutamide Derivatives as Potent RORγt Inverse Agonists Identified using a Virtual Screening Approach.

Employing a virtual screening approach, we identified the pyroglutamide moiety as a nonacid replacement for the cyclohexanecarboxylic acid group which, when coupled to our previously reported conformationally locked tricyclic core, provided potent and selective RORγt inverse agonists. Structure-activity relationship optimization of the pyroglutamide moiety led to the identification of compound 18 as a potent and selective RORγt inverse agonist, albeit with poor aqueous solubility. We took advantage of the tertiary carbinol group in 18 to synthesize a phosphate prodrug, which provided good solubility, excellent exposures in mouse PK studies, and significant efficacy in a mouse model of psoriasis.

Tricyclic sulfones as potent, selective and efficacious RORγt inverse agonists - Exploring C6 and C8 SAR using late-stage functionalization.

In order to rapidly develop C6 and C8 SAR of our reported tricyclic sulfone series of RORγt inverse agonists, a late-stage bromination was employed. Although not regioselective, the bromination protocol allowed us to explore new substitution patterns/vectors that otherwise would have to be incorporated at the very beginning of the synthesis. Based on the SAR obtained from this exercise, compound 15 bearing a C8 fluorine was developed as a very potent and selective RORγt inverse agonist. This analog's in vitro profile, pharmacokinetic (PK) data and efficacy in an IL-23 induced mouse acanthosis model will be discussed.

Discovery of BMS-986251: A Clinically Viable, Potent, and Selective RORγt Inverse Agonist.

Novel tricyclic analogues were designed, synthesized, and evaluated as RORγt inverse agonists. Several of these compounds were potent in an IL-17 human whole blood assay and exhibited excellent oral bioavailability in mouse pharmacokinetic studies. This led to the identification of compound 5, which displayed dose-dependent inhibition of IL-17F production in a mouse IL-2/IL-23 stimulated pharmacodynamic model. In addition, compound 5 was studied in mouse acanthosis and imiquimod-induced models of skin inflammation, where it demonstrated robust efficacy comparable to a positive control. As a result of this excellent overall profile, compound 5 (BMS-986251) was selected as a clinically viable developmental candidate.

Rationally Designed, Conformationally Constrained Inverse Agonists of RORγt-Identification of a Potent, Selective Series with Biologic-Like in Vivo Efficacy.

RORγt is an important nuclear receptor that regulates the production of several pro-inflammatory cytokines such as IL-17 and IL-22. As a result, RORγt has been identified as a potential target for the treatment of various immunological disorders such as psoriasis, psoriatic arthritis, and inflammatory bowel diseases. Structure and computer-assisted drug design led to the identification of a novel series of tricyclic RORγt inverse agonists with significantly improved in vitro activity in the reporter (Gal4) and human whole blood assays compared to our previous chemotype. Through careful structure activity relationship, several potent and selective RORγt inverse agonists have been identified. Pharmacokinetic studies allowed the identification of the lead molecule 32 with a low peak-to-trough ratio. This molecule showed excellent activity in an IL-2/IL-23-induced mouse pharmacodynamic study and demonstrated biologic-like efficacy in an IL-23-induced preclinical model of psoriasis.

One-Step Diastereoselective Pyrrolidine Synthesis Using a Sulfinamide Annulating Reagent.

This communication highlights the use of chiral sulfinamides as nitrogen nucleophiles in intermolecular aza-Michael reactions. When chiral sulfinamides are coupled to a chloroethyl group, the corresponding novel annulating reagents can be used to streamline the stereoselective synthesis of complex pyrrolidine-containing molecules. As a result, it has enabled a medicinal chemistry campaign for the synthesis of biologically active RORγt inverse agonists.

Pharmacokinetics of 40 kDa PEG in rodents using high-field NMR spectroscopy.

Conjugation of macromolecular drugs to polyethylene glycol (PEG) improves their therapeutic potential by reducing their rate of degradation, thereby extending the drugs half life. As a substantial component of the drug, it is necessary to measure the pharmacokinetic (PK) characteristics of PEG in vivo. A quantitative NMR-based method was developed and successfully applied to measuring double-branched polyethylene glycol 40 kDa (PEG40) in serum samples, enabling determination of PK parameters of PEG40 in preclinical species. NMR is ideal for measuring such polymers because a single, sharp peak is obtained for all the equivalent methylene protons; this amplifies the signal and makes the method insensitive to polymeric heterogeneity. High field NMR (600 MHz) with proton-observe cryoprobe technology allowed for analysis of samples in 300 nM range. Mice received 50 mg/kg of PEG40 intravenously (IV) and serum samples were collected at regular intervals for up to 72 h after dosing. The serum samples were analyzed for PEG40 using the NMR method and PK parameters were calculated using non-compartmental analysis. The volume of distribution was determined to be 0.17 L/kg for IV dosing, indicating limited distribution to interstitial space. A low clearance and observed half life of 18 h is consistent with previous reports on the PK properties of a variety of different PEG molecules ranging from 3 kDa to 190 kDa using 125I-labeled PEG in mice. The current NMR technique is easy to implement and does not require labeling of the PEG. Additionally, this is the first report, to our knowledge, of NMR spectroscopy application to PK profiling in serum.

Use of a Conformational-Switching Mechanism to Modulate Exposed Polarity: Discovery of CCR2 Antagonist BMS-741672.

We encountered a dilemma in the course of studying a series of antagonists of the G-protein coupled receptor CC chemokine receptor-2 (CCR2): compounds with polar C3 side chains exhibited good ion channel selectivity but poor oral bioavailability, whereas compounds with lipophilic C3 side chains exhibited good oral bioavailability in preclinical species but poor ion channel selectivity. Attempts to solve this through the direct modulation of physicochemical properties failed. However, the installation of a protonation-dependent conformational switching mechanism resolved the problem because it enabled a highly selective and relatively polar molecule to access a small population of a conformer with lower polar surface area and higher membrane permeability. Optimization of the overall properties in this series yielded the CCR2 antagonist BMS-741672 (7), which embodied properties suitable for study in human clinical trials.

Protected Chloroethyl and Chloropropyl Amines as Conformationally Unrestricted Annulating Reagents.

The purpose of this letter is to document the use of protected chloroethyl and chloropropyl amines as conformationally unrestricted ambiphilic reagents that undergo annulation reactions with Michael acceptors. This reaction is wide in scope and utilizes reagents that are commercially available, inexpensive, and stable. Furthermore, this reaction is easy to execute and proceeds rapidly.

1H NMR-based lipidomics of rodent fur: species-specific lipid profiles and SCD1 inhibitor-related dermal toxicity.

A method is described that allows noninvasive identification and quantitative assessment of lipid classes present in sebaceous excretions in rodents. The method relies on direct high-field proton NMR analysis of common group lipid protons in deuterated organic solvent extracts of fur. Extracts from as little as 15 mg of fur from rat, mouse, and hamster provided acceptable results on a 600 MHz NMR equipped with a cryogenically cooled proton-observe probe. In rats, sex- and age-related differences in lipid composition are larger than differences in fur collected from various body regions within an individual and much larger than interanimal differences in age- and sex-matched specimens. The utility of this method to noninvasively monitor drug-induced sebaceous gland atrophy in rodents is demonstrated in rats dosed with a stearoyl-CoA desaturase 1 (SCD1) inhibitor. In this model, a 35% reduction in sebum lipids, extracted from fur, was observed. Finally, structural elucidation of cholesta-7,24-dien-3ß-ol ester as the most prominent, previously unidentified sebum sterol ester in male Syrian hamsters is described. The utility of this method for drug and cosmetic safety and efficacy assessment is discussed.

Characterization of organic anion-transporting polypeptide (Oatp) 1a1 and 1a4 null mice reveals altered transport function and urinary metabolomic profiles.

Organic anion-transporting polypeptides (Oatp) 1a1 and 1a4 were deleted by homologous recombination, and mice were characterized for Oatp expression in liver and kidney, transport in isolated hepatocytes, in vivo disposition of substrates, and urinary metabolomic profiles. Oatp1a1 and Oatp1a4 proteins were undetected in liver, and both lines were viable and fertile. Hepatic constitutive messenger RNAs (mRNAs) for Oatp1a4, 1b2, or 2b1 were unchanged in Oatp1a1⁻/⁻ mice, whereas renal Oatp1a4 mRNA decreased approximately 50% (both sexes). In Oatp1a4⁻/⁻ mice, no changes in constitutive mRNAs for other Oatps were observed. Uptake of estradiol-17ß-D-glucuronide and estrone-3-sulfate in primary hepatocytes decreased 95 and 75%, respectively, in Oatp1a1⁻/⁻ mice and by 60 and 30%, respectively, in Oatp1a4⁻/⁻ mice. Taurocholate uptake decreased by 20 and 50% in Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, respectively, whereas digoxin was unaffected. Plasma area under the curve (AUC) for estradiol-17ß-D-glucuronide increased 35 and 55% in male and female Oatp1a1⁻/⁻ mice, respectively, with a concurrent 50% reduction in liver-to-plasma ratios. In contrast, plasma AUC or tissue concentrations of estradiol-17ß-D-glucuronide were unchanged in Oatp1a4⁻/⁻ mice. Plasma AUCs for dibromosulfophthalein increased nearly threefold in male Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, increased by 40% in female Oatp1a4⁻/⁻ mice, and were unchanged in female Oatp1a1⁻/⁻ mice. In both lines, no changes in serum ALT, bilirubin, and cholesterol were noted. NMR analyses showed no generalized increase in urinary excretion of organic anions. However, urinary excretion of taurine decreased by 30-40% and was accompanied by increased excretion of isethionic acid, a taurine metabolite generated by intestinal bacteria, suggesting some perturbations in intestinal bacteria distribution.