RESUMEN
Lufotrelvir was designed as a first in class 3CL protease inhibitor to treat COVID-19. Development of lufotrelvir was challenged by its relatively poor stability due to its propensity to epimerize and degrade. Key elements of process development included improvement of the supply routes to the indole and lactam fragments, a Claisen addition to homologate the lactam, and a subsequent phosphorylation reaction to prepare the prodrug as well as identification of a DMSO solvated form of lufotrelvir to enable long-term storage. As a new approach to preparing the indole fragment, a Cu-catalyzed C-O coupling using oxalamide ligands was demonstrated. The control of process-related impurities was essential to accommodate the parenteral formulation. Isolation of an MEK solvate followed by the DMSO solvate ensured that all impurities were controlled appropriately.
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We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI)-MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision, and accuracy. Subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro absorption, distribution, metabolism, elimination, pharmacokinetic and biomarker analysis, and HT parallel medicinal chemistry.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masa por Ionización de Electrospray , AcústicaRESUMEN
High-throughput experimentation (HTE) has emerged as an important tool in drug discovery, providing a platform for preparing large compound libraries and enabling swift reaction screening over wide-ranging conditions. Recent advances in automated high-density, material-sparing HTE have necessitated the development of rapid analytics with sensitivity and resolution sufficient to identify products and/or assess reaction performance in a timely and data-rich manner. Combination of an ultrathroughput (UT) reader platform with Acoustic Droplet Ejection-Open Port Interface-Mass Spectrometry (ADE-OPI-MS) provides the requisite speed and sensitivity. Herein, we report the application of ADE-OPI-MS to HTE in the areas of parallel medicinal chemistry and reaction screening.
RESUMEN
PDE8B is a cAMP-specific isoform of the broader class of phosphodiesterases (PDEs). As no selective PDE8B inhibitors had been reported, a high throughput screen was run with the goal of identifying selective tools for exploring the potential therapeutic utility of PDE8B inhibition. Of the numerous hits, one was particularly attractive since it was amenable to rapid deconstruction leading to inhibitors with very high ligand efficiency (LE) and lipophilic ligand efficiency (LLE). These triazolopyrimidines were optimized for potency, selectivity and ADME properties ultimately leading to compound 42. This compound was highly potent and selective with good bioavailability and advanced into pre-clinical development.
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3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Diabetes Mellitus/enzimología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Pirimidinas/química , Pirimidinas/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus/tratamiento farmacológico , Descubrimiento de Drogas , Inhibidores Enzimáticos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ligandos , Microsomas Hepáticos/metabolismo , Unión Proteica , Pirimidinas/metabolismo , Ratas , Relación Estructura-Actividad , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
Replacement of the central, para-substituted fluorophenyl ring in the γ-secretase inhibitor 1 (BMS-708,163) with the bicyclo[1.1.1]pentane motif led to the discovery of compound 3, an equipotent enzyme inhibitor with significant improvements in passive permeability and aqueous solubility. The modified biopharmaceutical properties of 3 translated into excellent oral absorption characteristics (~4-fold ↑ C(max) and AUC values relative to 1) in a mouse model of γ-secretase inhibition. In addition, SAR studies into other fluorophenyl replacements indicate the intrinsic advantages of the bicyclo[1.1.1]pentane moiety over conventional phenyl ring replacements with respect to achieving an optimal balance of properties (e.g., γ-secretase inhibition, aqueous solubility/permeability, in vitro metabolic stability). Overall, this work enhances the scope of the [1.1.1]-bicycle beyond that of a mere "spacer" unit and presents a compelling case for its broader application as a phenyl group replacement in scenarios where the aromatic ring count impacts physicochemical parameters and overall drug-likeness.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Compuestos Bicíclicos con Puentes/síntesis química , Oxadiazoles/síntesis química , Pentanos/síntesis química , Sulfonamidas/síntesis química , Administración Oral , Animales , Disponibilidad Biológica , Encéfalo/metabolismo , Compuestos Bicíclicos con Puentes/farmacocinética , Compuestos Bicíclicos con Puentes/farmacología , Línea Celular , Perros , Femenino , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Oxadiazoles/farmacocinética , Oxadiazoles/farmacología , Pentanos/farmacocinética , Pentanos/farmacología , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Distribución TisularRESUMEN
This paper describes the design and synthesis of a novel series of dual inhibitors of acetyl-CoA carboxylase 1 and 2 (ACC1 and ACC2). Key findings include the discovery of an initial lead that was modestly potent and subsequent medicinal chemistry optimization with a focus on lipophilic efficiency (LipE) to balance overall druglike properties. Free-Wilson methodology provided a clear breakdown of the contributions of specific structural elements to the overall LipE, a rationale for prioritization of virtual compounds for synthesis, and a highly successful prediction of the LipE of the resulting analogues. Further preclinical assays, including in vivo malonyl-CoA reduction in both rat liver (ACC1) and rat muscle (ACC2), identified an advanced analogue that progressed to regulatory toxicity studies.
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Acetil-CoA Carboxilasa/antagonistas & inhibidores , Bencimidazoles/síntesis química , Hipoglucemiantes/síntesis química , Indazoles/síntesis química , Indoles/síntesis química , Pirazoles/síntesis química , Compuestos de Espiro/síntesis química , Animales , Bencimidazoles/química , Diseño de Fármacos , Humanos , Hipoglucemiantes/química , Indazoles/química , Indoles/química , Isoenzimas/antagonistas & inhibidores , Hígado/enzimología , Músculo Esquelético/enzimología , Pirazoles/química , Relación Estructura-Actividad Cuantitativa , Ratas , Compuestos de Espiro/químicaRESUMEN
The Diels-Alder reaction is not limited to 1,3-dienes. Many cycloadditions of enynes and a smaller number of examples with 1,3-diynes have been reported. These "dehydro"-Diels-Alder cycloadditions are one class of dehydropericyclic reactions which have long been used to generate strained cyclic allenes and other novel structures. CCSD(T)//M05-2X computational results are reported for the cycloadditions of vinylacetylene and butadiyne with ethylene and acetylene. Both concerted and stepwise diradical routes have been explored for each reaction, with location of relevant stationary points. Relative to 1,3-dienes, replacement of one double bond by a triple bond adds 6-6.5 kcal/mol to the activation barrier; a second triple bond adds 4.3-4.5 kcal/mol to the barrier. Product strain decreases the predicted exothermicity. In every case, a concerted reaction is favored energetically. The difference between concerted and stepwise reactions is 5.2-6.6 kcal/mol for enynes but diminishes to 0.5-2 kcal/mol for diynes. Experimental studies on intramolecular diyne + ene cycloadditions show two distinct reaction pathways, providing evidence for competing concerted and stepwise mechanisms. Diyne + yne cycloadditions connect with arynes and ethynyl-1,3-cyclobutadiene. This potential energy surface appears to be flat, with only a minute advantage for a concerted process; many diyne cycloadditions or aryne cycloreversions will proceed by a stepwise mechanism.
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A metabolism-based approach toward the optimization of a series of N-arylsulfonamide-based γ-secretase inhibitors is reported. The lead cyclohexyl analogue 6 suffered from extensive oxidation on the cycloalkyl motif by cytochrome P450 3A4, translating into poor human liver microsomal stability. Knowledge of the metabolic pathways of 6 triggered a structure-activity relationship study aimed at lowering lipophilicity through the introduction of polarity. This effort led to several tetrahydropyran and tetrahydrofuran analogues, wherein the 3- and 4-substituted variants exhibited greater microsomal stability relative to their 2-substituted counterparts. Further reduction in lipophilicity led to the potent γ-secretase inhibitor and 3-substituted oxetane 1 with a reduced propensity toward oxidative metabolism, relative to its 2-substituted isomer. The slower rates of metabolism with 3-substituted cyclic ethers most likely originate from reductions in lipophilicity and/or unfavorable CYP active site interactions with the heteroatom. Preliminary animal pharmacology studies with a representative oxetane indicate that the series is generally capable of lowering Aß in vivo. As such, the study also illustrates the improvement in druglikeness of molecules through the use of the oxetane motif.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Éteres Cíclicos/síntesis química , Sulfonamidas/síntesis química , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Éteres Cíclicos/metabolismo , Éteres Cíclicos/farmacología , Humanos , Técnicas In Vitro , Ratones , Microsomas Hepáticos/metabolismo , Oxidación-Reducción , Receptores Notch/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Distribución TisularRESUMEN
Inhibition of acetyl-CoA carboxylases (ACCs), a crucial enzyme for fatty acid metabolism, has been shown to promote fatty acid oxidation and reduce body fat in animal models. Therefore, ACCs are attractive targets for structure-based inhibitor design, particularly the carboxyltransferase (CT) domain, which is the primary site for inhibitor interaction. We have cloned, expressed, and purified the CT domain of human ACC2 using baculovirus-mediated insect cell expression system. However, attempts to crystallize the human ACC2 CT domain have not been successful in our hands. Hence, we have been using the available crystal structure of yeast CT domain to design human ACC inhibitors. Unfortunately, as the selectivity of the lead series has increased against the full-length human enzyme, the potency against the yeast enzyme has decreased significantly. This loss of potency against the yeast enzyme correlated with a complete lack of binding of the human-specific compounds to crystals of the yeast CT domain. Here, we address this problem by converting nine key active site residues of the yeast CT domain to the corresponding human residues. The resulting humanized yeast ACC-CT (yCT-H9) protein exhibits biochemical and biophysical properties closer to the human CT domain and binding to human specific compounds. We report high resolution crystal structures of yCT-H9 complexed with inhibitors that show a preference for the human CT domain. These structures offer insights that explain the species selectivity of ACC inhibitors and may guide future drug design programs.
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Acetil-CoA Carboxilasa/antagonistas & inhibidores , Acetil-CoA Carboxilasa/química , Dominio Catalítico , Inhibidores Enzimáticos/química , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/química , Acetil-CoA Carboxilasa/genética , Animales , Línea Celular , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Especificidad de la Especie , Spodoptera , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
Screening Pfizer's compound library resulted in the identification of weak acetyl-CoA carboxylase inhibitors, from which were obtained rACC1 CT-domain co-crystal structures. Utilizing HTS hits and structure-based drug discovery, a more rigid inhibitor was designed and led to the discovery of sub-micromolar, spirochromanone non-specific ACC inhibitors. Low nanomolar, non-specific ACC-isozyme inhibitors that exhibited good rat pharmacokinetics were obtained from this chemotype.
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Acetil-CoA Carboxilasa/antagonistas & inhibidores , Acetil-CoA Carboxilasa/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Inhibidores Enzimáticos/farmacocinética , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Modelos Moleculares , Ratas , Bibliotecas de Moléculas Pequeñas/farmacocinética , Relación Estructura-ActividadRESUMEN
The development of a series of novel 1,2,3,4-tetrahydroisoquinolin-1-ones as antagonists of G protein-coupled receptor 40 (GPR40) is described. The synthesis, in vitro inhibitory values for GPR40, in vitro microsomal clearance and rat in vivo clearance data are discussed. Initial hits displayed high rat in vivo clearances that were higher than liver blood flow. Optimization of rat in vivo clearance was achieved and led to the identification of 15i, whose rat oral pharmacokinetic data is reported.
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Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Tetrahidroisoquinolinas/síntesis química , Tetrahidroisoquinolinas/farmacocinética , Administración Oral , Animales , Química Farmacéutica/métodos , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Concentración 50 Inhibidora , Ligandos , Tasa de Depuración Metabólica , Modelos Químicos , Ratas , Relación Estructura-Actividad , Tetrahidroisoquinolinas/químicaRESUMEN
Using a combination of parallel and directed synthesis, the discovery of a highly potent and selective series of adenosine A3 agonists was achieved. High aqueous solubility, required for the intended parenteral route of administration, was achieved by the presence of one or two basic amine functional groups.
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Agonistas del Receptor de Adenosina A3 , Adenosina/síntesis química , Adenosina/farmacología , Adenosina/análogos & derivados , Humanos , Conformación Molecular , Solubilidad , Estereoisomerismo , Relación Estructura-Actividad , Agua/químicaRESUMEN
Selective adenosine A(3) agonists have potential utility for the prevention of perioperative myocardial ischemic injury. Herein, we report on the discovery and synthesis of compound 7. This amino nucleoside agonist possesses unprecedented levels of selectivity for the human adenosine A(3) receptor.