Activation of the glucagon-like peptide-1 (GLP-1) receptor stimulates insulin release, lowers plasma glucose levels, delays gastric emptying, increases satiety, suppresses food intake, and affords weight loss in humans. These beneficial attributes have made peptide-based agonists valuable tools for the treatment of type 2 diabetes mellitus and obesity. However, efficient, and consistent delivery of peptide agents generally requires subcutaneous injection, which can reduce patient utilization. Traditional orally absorbed small molecules for this target may offer improved patient compliance as well as the opportunity for co-formulation with other oral therapeutics. Herein, we describe an SAR investigation leading to small-molecule GLP-1 receptor agonists that represent a series that parallels the recently reported clinical candidate danuglipron. In the event, identification of a benzyloxypyrimidine lead, using a sensitized high-throughput GLP-1 agonist assay, was followed by optimization of the SAR using substituent modifications analogous to those discovered in the danuglipron series. A new series of 6-azaspiro[2.5]octane molecules was optimized into potent GLP-1 agonists. Information gleaned from cryogenic electron microscope structures was used to rationalize the SAR of the optimized compounds.

Our previous work on the optimization of a new class of small molecule PCSK9 mRNA translation inhibitors focused on empirical optimization of the amide tail region of the lead PF-06446846 (1). This work resulted in compound 3 that showed an improved safety profile. We hypothesized that this improvement was related to diminished binding of 3 to non-translating ribosomes and an apparent improvement in transcript selectivity. Herein, we describe our efforts to further optimize this series of inhibitors through modulation of the heterocyclic head group and the amine fragment. Some of the effort was guided by an emerging cryo electron microscopy structure of the binding mode of 1 in the ribosome. These efforts led to the identification of 15 that was deemed suitable for evaluation in a humanized PCSK9 mouse model and a rat toxicology study. Compound 15 demonstrated a dose dependent reduction of plasma PCSK9 levels. The rat toxicological profile was not improved over that of 1, which precluded 15 from further consideration as a clinical candidate.

The scalable route to PF-07059013 (3), a non-covalent modulator of hemoglobin for the treatment of sickle cell disease, is discussed. Optimization of the discovery route is presented, examining bond connections, late-stage Buchwald-Hartwig C-O coupling, and palladium content reduction strategies. The first process chemistry route to deliver 11 kg of the final API is also discussed.

The α7 nicotinic acetylcholine receptor is a calcium permeable, ligand-gated ion channel that modulates synaptic transmission in the hippocampus, thalamus, and cerebral cortex. Previously disclosed work described PNU-120596 that acts as a powerful positive allosteric modulator of the α7 nicotinic acetylcholine receptor. The initial structure–activity relationships around PNU-120596 were gleaned from screening a large thiazole library. Independent systematic examination of the aryl and heteroaryl groups resulted in compounds with enhanced potency and improved physico-chemical properties culminating in the identification of 16 (PHA-758454). In the presence of acetylcholine, 16 enhanced evoked currents in rat hippocampal neurons. In a rat model of impaired sensory gating, treatment with 16 led to a reversal of the gating deficit in a dose-dependent manner. These results demonstrate that aryl heteroaryl ureas, like compound 16, may be useful tools for continued exploration of the unique biology of the α7 nicotinic acetylcholine receptor.

A series of tertiary amides was identified as lead matter for a new medicinal chemistry program. Analysis of analogs made for structure activity studies and physicochemical property measurements revealed that some of these tertiary amides were prone to acyl migration. A series of compounds designed to assess the propensity for acyl migration was synthesized. Qualitative time and temperature kinetics were followed up with quantitative kinetic measurements using 1H NMR techniques. Many of the examined tertiary amides from this series were shown to migrate under range of pHs and temperatures. Our finding serves as a cautionary note for others working with amides of a similar nature.

Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited because they require injection. Herein, we describe the discovery of the orally bioavailable, small-molecule, GLP-1R agonist PF-06882961 (danuglipron). A sensitized high-throughput screen was used to identify 5-fluoropyrimidine-based GLP-1R agonists that were optimized to promote endogenous GLP-1R signaling with nanomolar potency. Incorporation of a carboxylic acid moiety provided considerable GLP-1R potency gains with improved off-target pharmacology and reduced metabolic clearance, ultimately resulting in the identification of danuglipron. Danuglipron increased insulin levels in primates but not rodents, which was explained by receptor mutagensis studies and a cryogenic electron microscope structure that revealed a binding pocket requiring a primate-specific tryptophan 33 residue. Oral administration of danuglipron to healthy humans produced dose-proportional increases in systemic exposure (NCT03309241). This opens an opportunity for oral small-molecule therapies that target the well-validated GLP-1R for metabolic health.

Non-enzymatic dynamic kinetic resolution (DKR) of secondary alcohols by enantioselective acylation using an isothiourea-derived HyperBTM catalyst and racemization of slowly reacting alcohol by Bäckvall's ruthenium complex is reported. The DKR approach features high enantioselectivities (up to 99:1), employs easy-to-handle crystalline 4-nitrophenyl isobutyrate as the acylating reagent, and proceeds at room temperature and under an ambient atmosphere. The stereoinduction model featuring cation-π system interactions between the acylated HyperBTM catalyst and π electrons of an alcohol aryl subunit has been elaborated by DFT calculations.

Sickle cell disease (SCD) is a severe genetic disorder that impacts approximately 20 million people worldwide.1 The causative β6 Glu-Val substitution is a gain of function mutation; in the deoxygenated state, the mutant protein, Hb S, can form polymers, leading to red blood cell sickling and precipitating downstream consequences, including vaso-occlusion (pain crisis), hemolytic anemia, stroke and related pathophysiology.2, 3 Polymerization is exponentially dependent on deoxy Hb S concentration.4 Thus, relatively small changes in deoxyHb S concentration will significantly impact polymerization, red blood cell sickling, and ultimately the clinical course of the disease. Pharmacologic evidence for the benefit of reducing the concentration of deoxyHb S arises from studies of covalent modification of Hb S, where stabilization of the oxygenated conformation increases Hb O2 affinity, reduces RBC sickling, extends RBC half-life, and ultimately reduces the frequency of vaso-occlusive crisis (VOC).5-8 In clinical trials, ex vivo carbamylation of patient blood led to improvements in hemolytic anemia; treated patients exhibited a 2.7 g/dl increase in hemoglobin, a 58% decrease in reticulocytes, a 65% decrease in irreversibly sickled cells, and an 80% decrease in frequency of painful crises.8 Subsequent oxyHb S stabilizing molecules were developed based on the observation that benzaldehyde derivatives formed stable, covalent Schiff bases with hemoglobin. The most advanced of this class of molecules is the covalent compound Voxelotor (GBT 440, Oxbryta), which was approved by the FDA in 2019 for the treatment of SCD. In the pivotal study, 59% of patients in the higher dose group (1500 mg/day) had increases of 1 g/dl or greater in hemoglobin, with a mean hemoglobin modification of 26.5%.9 While the impact of covalent Hb S modifiers on hemolytic anemia is well established, the rise in hemoglobin observed for voxelotor falls short of the effects observed by ex vivo carbamylation, and was not accompanied by a significant effect on the frequency of VOC. This suggests that the therapeutic potential of hemoglobin modification has not been fully realized. PF-7059013 is a non-covalent modifier of hemoglobin that stabilizes the oxygenated state (see Gopalsamy et al.). Here we present the impact this molecule has on a well-established mouse model of sickle cell disease. Treatment with PF-7059013 demonstrated robust changes in key markers of hemolytic anemia in the Townes mouse model,10 suggesting it has the potential to be a potent and efficacious therapy for SCD. PF-07059013 was orally administered to Townes SCD model mice twice daily at a dose of 200 mg/kg for 15 days. This dose was selected as it was expected to result in approximately 25% hemoglobin coverage.11 At 30 minutes post the initial dose, total blood concentrations for individual animals were 2–4 mM, consistent with the low total blood clearance observed in the single dose administration studies. These values translate to approximately 40%–60% hemoglobin coverage, based on measured hemoglobin concentrations. The high total blood concentrations observed following the initial dose were maintained for the duration the 15-day dose period (Supporting Information S1). Animals treated with PF-07059013 show a significant stabilization of the oxygenated state. The average p50 decreased by 53.7% (±21.2%) in the treated group, relative to vehicle, and the average p20, a more sensitive marker of compound occupancy, decreased by 84.4% (±2.6%) in the treated group relative to vehicle (Supporting Information S1). As expected from the large shifts in oxygen affinity, blood from animals in the treated group showed significant reductions in RBC sickling. Under stringent hypoxic conditions, treatment with PF-07059013 resulted in a 37.8% (±9%) decrease in RBC sickling (Figure 1(A)). Consistent with reduction of RBC sickling, following 15 days of dosing, mice treated with PF-07059013 showed significant improvement in markers of hemolytic anemia. PF-07059013 treated animals showed a 42.4% (±4.2%) increase in hemoglobin, with a mean increase in hemoglobin of 5 g/dl, as well as a 30.9% (±0.7%) increase in hematocrit, and a 39.2 (±9.3%) increase in red blood cells relative to vehicle. All of the changes were statistically significant, and the increases restored the hemoglobin, hematocrit, and RBC counts of the treated group to values similar to wild type (C7BL/6) mice (Figure 1(B)). In addition, treatment with PF-07059013 resulted in a 54.7% (±2.4%) decrease in reticulocytes (Figure 1(B)). Note, PF-07059013 achieves consistently high levels of hemoglobin occupancy in in vivo studies using the Townes SCD murine model, which were sustained for the duration of dosing. Taken together, these results indicate that a non-covalent molecule has the potential to be efficacious for the treatment of sickle cell disease, and that the presence of a reactive aldehyde is not a requirement for potency. Comparing the activity of early covalent modifiers in the Townes model with PF-07059013 is not possible, as the development of many of those molecules predated the development of the transgenic mouse models. However, it is possible to compare the performance of PF-07059013 in the Townes SCD mouse with previously published Voxelotor pre-clinical data in the Townes model. Oksenberg et al. report that twice-daily oral dosing of Townes SCD mice with Voxelotor/GBT 440 at 100 or 150 mg/kg for 9–12 days resulted in hemoglobin occupancies ranging from 11%–39.7%.12 The pharmacodynamic effects of Voxelotor were strongly correlated with the degree of hemoglobin occupancy, as only the animals attaining >30% occupancy (4/14) showed changes in reticulocyte count or red blood cell half-life relative to vehicle. Thus, PF-07059013 achieved high degrees of hemoglobin coverage upon twice-daily oral dosing at 200 mg/kg for 15 days. As PF-07059013 binds ditopically to Hb (two compound: one tetramer), the dose is comparable to the doses used in the Voxelotor animal studies,12 as PF-07059013 requires twice as much compound to achieve the same hemoglobin occupancy percentages. In contrast with Voxelotor preclinical studies12 all of the animals dosed with PF-07059013 (n = 7) achieved >40% hemoglobin occupancy. The consistently high occupancy level across all animals leads to a uniform improvement in markers of hemolytic anemia. Similarly, all PF-07059013 treated animals showed decreases in RBC sickling, ranging from 35.3%–45.5%. The observed decrease in RBC sickling with PF-07059013 treatment is consistent with the decrease observed for Voxelotor in Townes SCD mice that had high Hb occupancy.12 The role of increases in hemoglobin in the overall pathology of sickle cell disease, particularly as it relates to VOC, is not completely understood. In the Voxelotor pivotal trial, 59% of patients treated with 1500 mg/day experienced hemoglobin increases of 1 g/dl or greater (average = 1.1 g/dl), and showed reductions in reticulocytes and bilirubin, consistent with improvements in hemolytic anemia, following 24 weeks of dosing.9 Post-hoc analysis of the pivotal trial results indicated that patients who achieved a hemoglobin level of 10 g/dl or greater had reduced incidence of VOC (50/179), with the greatest benefit observed in the small group of patients that had hemoglobin levels of 12 g/dl or greater (10/179).13 These data are consistent with the observation that increased hemoglobin can lead to reductions in VOC, provided RBC sickling is sufficiently impeded; Diedrich et al. demonstrated a substantial reduction in VOC frequency following weekly extracorporeal carbamylation.8 After 3 months of treatment, hemoglobin had increased by an average of 2.7 g/dl to an average of 8.8 g/dl and occurrences of VOC decreased by 80%.8 An increase in hemoglobin alone is likely not sufficient, as SCD patients undergoing exchange transfusions still experience VOC.14 Ex vivo carbamylation was most efficacious when hemoglobin occupancy was above 35%, suggesting achieving and maintaining high levels of hemoglobin occupancy may be crucial for making the maximum reduction in RBC sickling, reducing hemolysis, and increasing hemoglobin. These data indicate that it is possible to correlate the hemoglobin increase mediated by stabilization of the oxygenated state to resolution of VOC, and further suggest that the size of the increase in hemoglobin may be an important influencer of other clinical outcomes. Based in part on the magnitude and consistency of the response in the Townes SCD mouse model presented here, clinical studies of PF-07059013 are currently underway. The authors wish to thank Dr. Carlo Brugnara (Boston Children's Hospital) for assistance in obtaining SCD patient blood, and for valuable project discussions. We thank Dr. John Kelly (Northeastern University Co-op program), Joseph Nneji (Northeastern University Co-op program), and Victoria Ball (Northeastern University Co-op Program) for assistance in hemoglobin purification. We thank Jazmyne Lopez (Pfizer Occupational Health and Wellness) for coordinating healthy human blood sample collection, and Dr. David Karanian and Dr. Jamie DaSilva (Pfizer Drug Safety Research and Development) for providing whole blood from relevant toxicology species. All authors listed were employees of Pfizer Inc and declare no conflict of interest. Kelly M. Knee: designed research, performed research, analyzed data, wrote the paper. Reema Jasuja: designed research, performed research, analyzed data. Amey Barakat: performed research, analyzed data. Dharani Rao: performed research, analyzed data. Zane Wenzel: performed research, analyzed data. Jayasankar Jasti: performed research, analyzed data. Jonathan Novak: performed research, analyzed data. Kevin Beaumont: designed research, analyzed data. David W. Piotrowski: designed research, analyzed data. Phil Jeffery: designed research, analyzed data. Christine Bulawa: designed research, analyzed data. John E. Murphy: analyzed data, designed research. Jay M. Janz: performed research, designed research, analyzed data, wrote the paper. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1. Supporting information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

DNA Encoded Libraries have shown promise as a valuable technology for democratizing the hit discovery process. Although DEL provides relatively inexpensive access to libraries of unprecedented size, their production has been hampered by the idiosyncratic needs of the encoding DNA tag relegating DEL compatible chemistry to dilute aqueous environments. Recently Reversible Adsorption to Solid Support (RASS) has been demonstrated as a promising method to expand DEL reactivity using standard organic synthesis protocols. Here we demonstrate a suite of on-DNA chemistries to incorporate medicinally relevant and C–S, C–P and N–S linkages into DELs, which are underrepresented in the canonical methods.

DNA-encoded library (DEL) technology has the potential to dramatically expedite hit identification in drug discovery owing to its ability to perform protein affinity selection with millions or billions of molecules in a few experiments. To expand the molecular diversity of DEL, it is critical to develop different types of DNA-encoded transformations that produce billions of molecules with distinct molecular scaffolds. Sequential functionalization of multiple C-H bonds provides a unique avenue for creating diversity and complexity from simple starting materials. However, the use of water as solvent, the presence of DNA, and the extremely low concentration of DNA-encoded coupling partners (0.001 M) have hampered the development of DNA-encoded C(sp(3))-H activation reactions. Herein, we report the realization of palladium-catalyzed C(sp(3))-H arylation of aliphatic carboxylic acids, amides and ketones with DNA-encoded aryl iodides in water. Notably, the present method enables the use of alternative sets of monofunctional building blocks, providing a linchpin to facilitate further setup for DELs. Furthermore, the C-H arylation chemistry enabled the on-DNA synthesis of structurally-diverse scaffolds containing enriched C(sp(3)) character, chiral centers, cyclopropane, cyclobutane, and heterocycles.

DNA-encoded library (DEL) technology has the potential to dramatically expedite hit identification in drug discovery owing to its ability to perform protein affinity selection with millions or billions of molecules in a single experiment. To expand the molecular diversity of DEL, it is critical to develop different types of DNA-encoded transformations that produces billions of molecules with distinct molecular scaffolds. Sequential functionalization of multiple C–H bonds provides a unique avenue for creating diversity and complexity from simple starting materials. However, the use of water as solvent, the presence of DNA, and the extremely low concentration of DNA-encoded coupling partners (0.001 M) have hampered the development DNA-encoded C(sp3)–H activation reactions. Herein, we report the realization of palladium-catalyzed C(sp3)–H arylation of aliphatic carboxylic acids, amides and ketones with DNA-encoded aryl iodides in water. Notably, the present method enables the use of alternative sets of bifunctional building blocks, and facilitates access to certain setups for DELs. Furthermore, sequential C–H arylation chemistry enabled the on-DNA synthesis of structurally-diverse scaffolds containing enriched C(sp3) character, chiral centers, cyclopropane, cyclobutane, and heterocycles.

Preparative chemical reactions that occur efficiently under dilute, buffered, aqueous conditions in the presence of biomolecules find application in ligation, peptide synthesis, polynucleotide synthesis and sequencing. However, the identification of functional groups or reagents that are mutually reactive with one another, but unreactive with biopolymers and water, is challenging. Here we show that cobalt catalysts will react with the alkenes of unsaturated tertiary amines under dilute, aqueous, buffered conditions and promote efficient cycloisomerization, in many cases mediating a formal Friedel-Crafts reaction. We find the constraining conditions of biorthogonal chemistry to be beneficial for reaction efficiency as we obtain superior conversion at low catalyst concentration and maintain competent rates in dilute conditions. The efficiency at high dilution in the presence of buffer and nucleobases suggests that these conditions may find use on or in the presence of biomolecules.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (β6 Glu → Val) on the β-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.

The synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor 3 is described. This complex structure contains a tetrazole modified by a chiral hemiaminal carbonate prodrug. A regioselective tin-mediated alkylation was utilized to access the N-1 alkylated tetrazole isomer, and a highly selective enzymatic hydrolysis efficiently provided the desired prodrug enantiomer. A Suzuki–Miyaura coupling was employed for the final fragment union, which was challenging due to base sensitivity of the prodrug. This route was enabled and used to manufacture multikilogram quantities of API 3 in an efficient manner.

Sickle cell disease (SCD) is a severe genetic disorder caused by a single point mutation on the β-chain of adult hemoglobin (Hb A), β6 Glu→Val (Hb S). In the deoxygenated state Hb S polymerizes, leading to RBC sickling and precipitating all downstream consequences, including vaso-occlusion (pain crisis), hemolytic anemia, and stroke. Over time, these features cause significant organ damage and eventual organ failure, dramatically impacting both quality of life and expected lifespan. Numerous small molecules which covalently bind to Hb S have been evaluated clinically, however, the molecules that have demonstrated clinical efficacy all carry a reactive aldehyde group. The reactive aldehyde, a moiety that has the potential to react with any free amine, forms a covalent Schiff base with the N-terminal amine of the α1-Val. At least one member of this class of molecules, Tucaresol, showed a significant safety signal attributed to off-target Schiff base formation. An early investigation of covalent hemoglobin modification, extracorporeal carbamylation, both improved anemia and decreased the frequency of vaso-occlusive events by 80%, when there was a sufficiently high level of modification (30-50%). These results suggest that a molecule that binds Hb S and stabilizes the oxygenated state can impact both hemolytic anemia and vaso-occlusive crisis, if the molecule can achieve the necessary degree of hemoglobin modification. PFE-001 is a non-covalent molecule which binds selectively to Hb S and stabilizes the oxygenated state. Biochemical and biophysical studies show that PFE-001 binds specifically to Hb with double digit nanomolar potency and exhibits strong in vivo partitioning into RBCs. In a two-week multiple dose study using Townes SCD model animals (200 mg/kg, twice daily), PFE-001 significantly improved markers of hemolytic anemia, increased oxygen affinity, and reduced RBC sickling. Following 15 days of treatment blood drawn from PFE-001 treated animals and exposed to intense hypoxic conditions (4% O2, 4 hr) showed a 37.8% reduction in sickling compared to vehicle treated mice. Oxygen affinity was increased, demonstrated by a 53.7% reduction in p50 and an 84.4% reduction in p20 in the PFE-001 treated group. Hemoglobin levels in mice treated with PFE-001 increased by 42%, a mean increase of 5 g/dL. Hematocrit in the PFE-001 treated group increased to 42%, in contrast to 29% in the vehicle group. Reticulocyte percentages were reduced from 53% in vehicle treated animals to 24% in PFE-001 treated animals. In addition to the significant impact PFE-001 had on hemolytic anemia, a 10% reduction in sVCAM-1 levels in the PFE-001 treated group indicates a small but statistically significant improvement in vasculopathy following 15 days of treatment. This improvement in vasculopathy suggests that PFE-001 has the potential to address vaso-occlusive crisis in addition to anemia. In total, the in vitro and in vivo data suggest that PFE-001 is a potent, selective, and effective inhibitor of Hb S polymerization and RBC sickling. PFE-001 can reduce hemolytic anemia, improve vasculopathy, increase oxygen affinity, and reduce RBC sickling under hypoxic conditions. Plans for advancement of PFE-001 to clinical trials are in progress. Disclosures Knee: Pfizer Inc: Employment. Jasuja:Pfizer Inc.: Employment. Barakat:Pfizer Inc.: Employment. Rao:Pfizer Inc.: Employment. Wenzel:Pfizer Inc.: Employment. Sahasrabudhe:Pfizer Inc.: Employment. Narula:Pfizer Inc.: Employment. Jasti:Pfizer Inc.: Employment. Chang:Pfizer Inc.: Employment. Beaumont:Pfizer Inc.: Employment. Piotrowski:Pfizer Inc.: Employment. Janz:Pfizer Inc.: Employment.

Herein, we present the adaptation of reversible adsorption to solid support (RASS) for a DEL setting, which allows reactions to be performed in organic solvents at near anhydrous conditions opening previously inaccessible chemical reactivities to DEL. The RASS approach enabled the rapid development of C(sp2)-C(sp3) decarboxylative cross-couplings with broad substrate scope, an electrochemical amination (the first electrochemical synthetic transformation performed in a DEL context), and improved reductive amination conditions. We believe that RASS will offer expedient access to new DEL reactivities, expanded chemical space, and ultimately more drug-like libraries.

[This corrects the article DOI: 10.1371/journal.pbio.2001882.].