RESUMEN
The KEAP1-NRF2-mediated cytoprotective response plays a key role in cellular homoeostasis. Insufficient NRF2 signaling during chronic oxidative stress may be associated with the pathophysiology of several diseases with an inflammatory component, and pathway activation through direct modulation of the KEAP1-NRF2 protein-protein interaction is being increasingly explored as a potential therapeutic strategy. Nevertheless, the physicochemical nature of the KEAP1-NRF2 interface suggests that achieving high affinity for a cell-penetrant druglike inhibitor might be challenging. We recently reported the discovery of a highly potent tool compound which was used to probe the biology associated with directly disrupting the interaction of NRF2 with the KEAP1 Kelch domain. We now present a detailed account of the medicinal chemistry campaign leading to this molecule, which included exploration and optimization of protein-ligand interactions in three energetic "hot spots" identified by fragment screening. In particular, we also discuss how consideration of ligand conformational stabilization was important to its development and present evidence for preorganization of the lead compound which may contribute to its high affinity and cellular activity.
Asunto(s)
Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Propionatos/metabolismo , Unión Proteica/efectos de los fármacos , Sitios de Unión , Línea Celular , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/química , Conformación Molecular , Factor 2 Relacionado con NF-E2/química , Propionatos/síntesis química , Propionatos/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The antioxidant natural product sulforaphane (SFN) is an oil with poor aqueous and thermal stability. Recent work with SFN has sought to optimize methods of formulation for oral and topical administration. Herein we report the design of new analogs of SFN with the goal of improving stability and drug-like properties. Lead compounds were selected based on potency in a cellular screen and physicochemical properties. Among these, 12 had good aqueous solubility, permeability and long-term solid-state stability at 23⯰C. Compound 12 also displayed comparable or better efficacy in cellular assays relative to SFN and had in vivo activity in a mouse cigarette smoke challenge model of acute oxidative stress.
Asunto(s)
Antioxidantes/farmacología , Ciclobutanos/farmacología , Descubrimiento de Drogas , Isotiocianatos/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antioxidantes/síntesis química , Antioxidantes/farmacocinética , Línea Celular , Ciclobutanos/síntesis química , Ciclobutanos/farmacocinética , Expresión Génica , Hemo-Oxigenasa 1/genética , Humanos , Isotiocianatos/síntesis química , Isotiocianatos/farmacocinética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones Endogámicos C57BL , Estructura Molecular , Estrés Oxidativo/efectos de los fármacos , Ratas , Solubilidad , Relación Estructura-Actividad , Sulfóxidos , Tiocarbamatos/síntesis química , Tiocarbamatos/farmacocinética , Tiocarbamatos/farmacologíaRESUMEN
KEAP1 is the key regulator of the NRF2-mediated cytoprotective response, and increasingly recognized as a target for diseases involving oxidative stress. Pharmacological intervention has focused on molecules that decrease NRF2-ubiquitination through covalent modification of KEAP1 cysteine residues, but such electrophilic compounds lack selectivity and may be associated with off-target toxicity. We report here the first use of a fragment-based approach to directly target the KEAP1 Kelch-NRF2 interaction. X-ray crystallographic screening identified three distinct "hot-spots" for fragment binding within the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment hit to molecules with nanomolar affinity for KEAP1 while maintaining drug-like properties. This work resulted in a promising lead compound which exhibits tight and selective binding to KEAP1, and activates the NRF2 antioxidant response in cellular and in vivo models, thereby providing a high quality chemical probe to explore the therapeutic potential of disrupting the KEAP1-NRF2 interaction.
Asunto(s)
Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Células Cultivadas , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/química , Ratones , Factor 2 Relacionado con NF-E2/química , Unión ProteicaRESUMEN
Activation of the Nrf2 stress pathway is known to play an important role in the defense mechanism against electrophilic and oxidative damage to biological macromolecules (DNA, lipids, and proteins). Chemical inducers of Nrf2 such as sulforaphane, dimethyl fumarate (Tecfidera®), CDDO-Me (bardoxolone-methyl), and 3-(dimethylamino)-4-((3-isothiocyanatopropyl)(methyl)amino)cyclobut-3-ene-1,2-dione (a synthetic sulforaphane analogue; will be referred to as ) have the ability to react with Keap1 cysteine residues, leading to activation of the Antioxidant Response Element (ARE). Due to their electrophilic nature and poor matrix stability, these compounds represent great challenges when developing bioanalytical methods to evaluate in vivo exposure. like SFN reacts rapidly with glutathione (GSH) and nucleophilic groups in proteins to form covalent adducts. In this work, three procedures were developed to estimate the exposure of in a non-GLP 7 day safety study in rats: (1) protein precipitation of blood samples with methanol containing the free thiol trapping reagent 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) to measure GSH- and N-acetylcysteine conjugated metabolites of ; (2) an Edman degradation procedure to cleave and analyze N-terminal adducts of at the valine moiety; and (3) treatment with ammonium hydroxide to measure circulating free- and all sulfhydryl bound .
Asunto(s)
Factor 2 Relacionado con NF-E2/metabolismo , Pruebas de Toxicidad , Animales , Área Bajo la Curva , Cromatografía Liquida , Masculino , Ratas , Estándares de Referencia , Espectrometría de Masas en TándemRESUMEN
A novel series of N-substituted tropane derivatives was characterized as potent muscarinic acetylcholine receptor antagonists (mAChRs). Kinetic washout studies showed that the N-endosubstituted analog 24 displayed much slower reversibility at mAChRs than the methyl-substituted parent molecule darotropium. In addition, it was shown that this characteristic appeared to translate into enhanced which duration of action in a mouse model of bronchonstriction.
Asunto(s)
Antagonistas Muscarínicos/síntesis química , Tropanos/síntesis química , Animales , Enfermedades Bronquiales/tratamiento farmacológico , Diseño de Fármacos , Ratones , Antagonistas Muscarínicos/farmacología , Receptores Muscarínicos/efectos de los fármacos , Relación Estructura-Actividad , Tropanos/farmacologíaRESUMEN
Design and syntheses of a novel series of muscarinic antagonists are reported. These efforts have culminated in the discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide (4a) as a potent and pan-active muscarinic antagonist as well as a functionally active compound in a murine model of bronchoconstriction. The compound has also displayed pharmacokinetic characteristics suitable for inhaled delivery.
Asunto(s)
Compuestos de Bifenilo/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Antagonistas Muscarínicos/química , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Receptores Muscarínicos/química , Administración por Inhalación , Animales , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/farmacocinética , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Descubrimiento de Drogas , Humanos , Ratones , Antagonistas Muscarínicos/síntesis química , Antagonistas Muscarínicos/farmacocinética , Ratas , Receptores Muscarínicos/metabolismo , Relación Estructura-ActividadRESUMEN
Novel tropane derivatives were characterized as muscarinic acetylcholine receptor antagonists (mAChRs). Through optimization of the structure-activity relationship around the tropane scaffold, the quaternary ammonium salt 34 was identified as a very potent M(3) mAChR antagonist. The compound was functionally active and displayed greater than 24 h duration of action in a mouse model of bronchoconstriction.
Asunto(s)
Compuestos de Bifenilo/síntesis química , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Antagonistas Muscarínicos/síntesis química , Tropanos/síntesis química , Animales , Disponibilidad Biológica , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Bronquios/efectos de los fármacos , Bronquios/fisiología , Broncoconstricción/efectos de los fármacos , Células CHO , Calcio/metabolismo , Cricetinae , Cricetulus , Diseño de Fármacos , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Antagonistas Muscarínicos/química , Antagonistas Muscarínicos/farmacología , Contracción Muscular , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Ensayo de Unión Radioligante , Ratas , Receptor Muscarínico M1/fisiología , Receptor Muscarínico M2/fisiología , Receptor Muscarínico M3/fisiología , Estereoisomerismo , Relación Estructura-Actividad , Tropanos/química , Tropanos/farmacologíaRESUMEN
An aza-Darzens reaction, involving the addition of chloromethylphosphonate anions to enantiopure sulfinimines, has been developed for the asymmetric synthesis of aziridine 2-phosphonates. Best results involve cyclization of the syn and anti diastereomerically pure alpha-chloro-beta-amino phosphonates to cis- and trans-N-sulfinyl aziridine 2-phosphonates, respectively, with n-BuLi. A transition-state hypothesis is proposed wherein the chloromethylphosphonate anion adds to the C-N bond on the side that is opposite the bulky p-tolyl sulfinyl group. The N-sulfinyl group is easily removed by treatment with MeMgBr or TFA/MeOH, which affords the NH-aziridines in good yield. Using transfer hydrogenation conditions, the NH-aziridines were regioselectively opened to the corresponding enantiopure alpha-amino phosphonates without N-activation and in excellent yield.
Asunto(s)
Aminas/química , Aziridinas/síntesis química , Organofosfonatos/síntesis química , Compuestos de Azufre/química , Catálisis , Cromatografía en Capa Delgada , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Estructura Molecular , EstereoisomerismoRESUMEN
[reaction: see text] Diels-Alder reactions of enantiomerically enriched 2H-azirine 3-phosphonates and dienes stereoselectively furnish optically pure, bicyclic aziridine adducts that on hydrogenation afford the first examples of enantiopure quaternary piperidine phosphonates.