RESUMEN
The structure activity relationship of the prime region of conformationally restricted hydroxyethylamine (HEA) BACE inhibitors is described. Variation of the P1' region provided selectivity over Cat-D with a series of 2,2-dioxo-isothiochromanes and optimization of the P2' substituent of chromane-HEA(s) with polar substituents provided improvements in the compound's in vitro permeability. Significant potency gains were observed with small aliphatic substituents such as methyl, n-propyl, and cyclopropyl when placed at the C-2 position of the chromane.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Cromanos/química , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Sitios de Unión , Células Cultivadas , Etilaminas/síntesis química , Etilaminas/química , Etilaminas/farmacología , Concentración 50 Inhibidora , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Polo-like kinase-2 (Plk-2) has been implicated as the dominant kinase involved in the phosphorylation of α-synuclein in Lewy bodies, which are one of the hallmarks of Parkinson's disease neuropathology. Potent, selective, brain-penetrant inhibitors of Plk-2 were obtained from a structure-guided drug discovery approach driven by the first reported Plk-2-inhibitor complexes. The best of these compounds showed excellent isoform and kinome-wide selectivity, with physicochemical properties sufficient to interrogate the role of Plk-2 inhibition inâ vivo. One such compound significantly decreased phosphorylation of α-synuclein in rat brain upon oral administration and represents a useful probe for future studies of this therapeutic avenue toward the potential treatment of Parkinson's disease.
Asunto(s)
Encéfalo/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , alfa-Sinucleína/metabolismo , Animales , Sitios de Unión , Barrera Hematoencefálica/metabolismo , Femenino , Células HEK293 , Semivida , Humanos , Masculino , Ratones , Simulación de Dinámica Molecular , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-DawleyRESUMEN
Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aß generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds 30 (ELND006) and 34 (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aß generation in vivo between 30, 34, Semagacestat 41, Begacestat 42, and Avagacestat 43 in mice is made. 30 lowered Aß in the CSF of healthy human volunteers.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Pirazoles/farmacología , Quinolinas/farmacología , Receptores Notch/antagonistas & inhibidores , Sulfonamidas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/metabolismo , Animales , Área Bajo la Curva , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Perros , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Estabilidad de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos con 3 Anillos/química , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Modelos Químicos , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/farmacocinética , Quinolinas/síntesis química , Quinolinas/farmacocinética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Notch/metabolismo , Relación Estructura-Actividad , Sulfonamidas/química , Factores de Tiempo , Factor de Transcripción HES-1RESUMEN
Utilizing a structure based design approach, combined with extensive medicinal chemistry execution, highly selective, potent and novel BACE1 inhibitor 8 (BACE1 Alpha assay IC50=8nM) was made from a weak µM potency hit in an extremely efficient way. The detailed SAR and general design approaches will be discussed.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Diseño de Fármacos , Isoquinolinas/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Tiofenos/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Isoquinolinas/química , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/química , Relación Estructura-Actividad , Tiofenos/químicaRESUMEN
Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. ß-secretase-1 (BACE1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aß is a validated target for AD. Herein, the authors develop and validate a novel binding assay for BACE1 using the AlphaScreen platform that is amenable for high-throughput screening (HTS). Small-molecule BACE1 inhibitors of the hydroxyethylamine, hydantoin, and sulfamide classes were functionalized by biotin PEG linkers of varying lengths forming probes that were bound to streptavidin donor beads. BACE1 was coupled to nickel-chelate acceptor beads. Upon mixing, probes designed from all three classes registered high signal-to-background values in the AlphaScreen binding assay, where the interaction between probe and BACE1 was completely blocked by free parent compound. A probe from the hydantoin class was chosen for further optimization, where the final assay conditions of 50 nM BACE and 250 nM probe were used and Z(') values >0.75 were commonly observed. IC50 values determined by the AlphaScreen assay format exhibited ~10-fold greater sensitivity when compared with a fluorescence polarization-based activity assay. The assay was miniaturized to a 1536-well format for HTS, in which 525 000 compounds were screened.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Humanos , Hidantoínas , Concentración 50 Inhibidora , Sensibilidad y EspecificidadRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familial Parkinson's disease (PD). The kinase activity of this complex protein is increased by pathogenic mutations. Inhibition of LRRK2 kinase activity has therefore emerged as a promising approach for the treatment of PD. Herein we report our findings on a series of 4-alkylamino-7-aryl-3-cyanoquinolines that exhibit kinase inhibitory activity against both wild type and G2019S mutant LRRK2. Activity was determined in both biochemical and cellular assays. Compound 14 was further evaluated in an in vivo pharmacodynamic study and found to significantly inhibit Ser935 phosphorylation after oral dosing.
Asunto(s)
Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Quinolinas/farmacología , Animales , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Moleculares , Estructura Molecular , Mutación , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Quinolinas/síntesis química , Quinolinas/química , Relación Estructura-ActividadRESUMEN
Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of [1,2,4]triazolo[4,3-b]pyridazines that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays and show an unprecedented selectivity towards the G2019S mutant. A structural rational for the observed selectivity is proposed.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridazinas/farmacología , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Piridazinas/síntesis química , Piridazinas/química , Relación Estructura-ActividadRESUMEN
Polo-like kinase-2 (Plk-2) is a potential therapeutic target for Parkinson's disease and this Letter describes the SAR of a series of dihydropteridinone based Plk-2 inhibitors. By optimizing both the N-8 substituent and the biaryl region of the inhibitors we obtained single digit nanomolar compounds such as 37 with excellent selectivity for Plk-2 over Plk-1. When dosed orally in rats, compound 37 demonstrated a 41-45% reduction of pS129-α-synuclein levels in the cerebral cortex.
Asunto(s)
Diseño de Fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Administración Oral , Animales , Encéfalo/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Semivida , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Pteridinas/síntesis química , Pteridinas/química , Pteridinas/farmacocinética , Ratas , Relación Estructura-Actividad , Quinasa Tipo Polo 1RESUMEN
The structure-activity relationship of a series of dihydroisoquinoline BACE-1 inhibitors is described. Application of structure-based design to screening hit 1 yielded sub-micromolar inhibitors. Replacement of the carboxylic acid of 1 was guided by X-ray crystallography, which allowed the replacement of a key water-mediated hydrogen bond. This work culminated in compounds such as 31, which possess good BACE-1 potency, excellent permeability and a low P-gp efflux ratio.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico/química , Diseño de Fármacos , Isoquinolinas/farmacología , Inhibidores de Proteasas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Catálisis , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Isoquinolinas/síntesis química , Isoquinolinas/química , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/química , Relación Estructura-ActividadRESUMEN
Structure-activity relationship (SAR) of a novel, potent and metabolically stable series of benzo [3.2.1] bicyclic sulfonamide-pyrazoles as γ-secretase inhibitors are described. Compounds that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via oral dose, as well as those with high selectivity over Notch, are highlighted.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Pirazoles/farmacología , Sulfonamidas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Ratones , Pirazoles/química , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
Leucine rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of cinnoline-3-carboxamides that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays. These compounds are also shown to be potent inhibitors in a cellular assay and to have good to excellent CNS penetration.
Asunto(s)
Compuestos Heterocíclicos con 2 Anillos/química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Amidas/síntesis química , Amidas/química , Amidas/metabolismo , Animales , Sitios de Unión , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Simulación del Acoplamiento Molecular , Mutación , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , TransfecciónRESUMEN
The structure-activity relationship (SAR) of a novel, potent and metabolically stable series of sulfonamide-pyrazoles that attenuate ß-amyloid peptide synthesis via γ-secretase inhibition is detailed herein. Sulfonamide-pyrazoles that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via a single PO dose, as well as sulfonamide-pyrazoles that exhibit selectivity for inhibition of APP versus Notch processing by γ-secretase, are highlighted.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Compuestos Heterocíclicos con 3 Anillos/farmacología , Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Péptidos beta-Amiloides/metabolismo , Animales , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos con 3 Anillos/química , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
The SAR of a series of brain penetrant, trisubstituted thiophene based JNK inhibitors with improved pharmacokinetic properties is described. These compounds were designed based on information derived from metabolite identification studies which led to compounds such as 42 with lower clearance, greater brain exposure and longer half life compared to earlier analogs.
Asunto(s)
Encéfalo/metabolismo , Diseño de Fármacos , Degeneración Nerviosa/prevención & control , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Tiofenos/farmacología , Tiofenos/farmacocinética , Animales , Técnicas de Química Sintética , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Semivida , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Estereoisomerismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/químicaRESUMEN
In this Letter, we describe the evolution of selective JNK3 inhibitors from 1, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs. Strong SAR was found for substitution of the naphthalene ring, as well as for inhibitors adopting different central scaffolds. Significant potency gains were appreciated by inverting the polarity of the thione of the parent triazolothione 1, resulting in potent compounds with attractive pharmacokinetic profiles.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteína Quinasa 10 Activada por Mitógenos/antagonistas & inhibidores , Naftalenos/síntesis química , Tionas/síntesis química , Animales , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Ratones , Microsomas Hepáticos/enzimología , Estructura Molecular , Naftalenos/química , Naftalenos/farmacología , Solubilidad , Relación Estructura-Actividad , Tionas/química , Tionas/farmacologíaRESUMEN
The SAR of a series of tri-substituted thiophene JNK3 inhibitors is described. By optimizing both the N-aryl acetamide region of the inhibitor and the 4-position of the thiophene we obtained single digit nanomolar compounds, such as 47, which demonstrated an in vivo effect on JNK activity when dosed orally in our kainic acid mouse model as measured by phospho-c-jun reduction.
Asunto(s)
Encéfalo/metabolismo , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Administración Oral , Diseño de Fármacos , Enlace de Hidrógeno , Modelos Moleculares , Inhibidores de Proteínas Quinasas/síntesis química , Relación Estructura-ActividadRESUMEN
In this Letter, we describe the discovery of selective JNK2 and JNK3 inhibitors, such as 10, that routinely exhibit >10-fold selectivity over JNK1 and >1000-fold selectivity over related MAPKs, p38α and ERK2. Substitution of the naphthalene ring affords an isoform selective JNK3 inhibitor, 30, with approximately 10-fold selectivity over both JNK1 and JNK2. A naphthalene ring penetrates deep into the selectivity pocket accounting for the differentiation amongst the kinases. Interestingly, the gatekeeper Met146 sulfide interacts with the naphthalene ring in a sulfur-π stacking interaction. Compound 38 ameliorates neurotoxicity induced by amyloid-ß in human cortical neurons. Lastly, we demonstrate how to install propitious in vitro CNS-like properties into these selective inhibitors.
Asunto(s)
Aminopiridinas/química , Proteína Quinasa 10 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 9 Activada por Mitógenos/antagonistas & inhibidores , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/química , Inhibidores de Proteínas Quinasas/química , Triazinas/química , Aminopiridinas/farmacocinética , Aminopiridinas/uso terapéutico , Animales , Sitios de Unión , Sistema Nervioso Central/metabolismo , Simulación por Computador , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Proteína Quinasa 10 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Fármacos Neuroprotectores/farmacocinética , Fármacos Neuroprotectores/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Relación Estructura-Actividad , Triazinas/farmacocinética , Triazinas/uso terapéuticoRESUMEN
From high throughput screening, we discovered compound 1, the prototype for a series of disubstituted thiophene inhibitors of JNK which is selective towards closely related MAP kinases p38 and Erk2. Herein we describe the evolution of these compounds to a novel class of thiophene and thiazole JNK inhibitors that retain favorable solubility, permeability, and P-gp properties for development as CNS agents for treatment of neurodegeneration. Compound 61 demonstrated JNK3 IC(50)=77 nM and retained the excellent broad kinase selectivity observed for the series.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Quinolinas/síntesis química , Tiazoles/química , Tiofenos/química , Animales , Diseño de Fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Microsomas Hepáticos/metabolismo , Proteína Quinasa 10 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 10 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/química , Quinolinas/farmacología , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/farmacología , Tiofenos/síntesis química , Tiofenos/farmacologíaRESUMEN
In this Letter, we describe our efforts to design HEA BACE-1 inhibitors that are highly permeable coupled with negligible levels of permeability-glycoprotein activity. These efforts culminate in producing 16 which lowers Αß by 28% and 32% in the cortex and CSF, respectively, in the preclinical wild type Hartley guinea pig animal model when dosed orally at 30mpk BID for 2.5days.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Etilaminas/síntesis química , Etilaminas/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Alquilación , Enfermedad de Alzheimer , Animales , Encéfalo/metabolismo , Línea Celular , Perros , Diseño de Fármacos , Cobayas , Humanos , Indicadores y Reactivos , Inhibidores de Proteasas/farmacocinética , Unión Proteica , Relación Estructura-ActividadRESUMEN
The structure-activity relationship of the prime region of hydroxyethylamine BACE inhibitors is described. Variation in the aryl linker region with 5- and 6-membered heterocycles provided compounds such as 33 with improved permeability and reduced P-gp liability compared to benzyl amine analog 1.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Etilaminas/química , Etilaminas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/química , Ácido Aspártico Endopeptidasas/química , Cristalografía por Rayos X , Etilaminas/síntesis química , Humanos , Modelos Moleculares , Inhibidores de Proteasas/síntesis química , Unión Proteica , Relación Estructura-ActividadRESUMEN
Herein we describe further evolution of hydroxyethylamine inhibitors of BACE-1 with enhanced permeability characteristics necessary for CNS penetration. Variation at the P2' position of the inhibitor with more polar substituents led to compounds 19 and 32, which retained the potency of more lipophilic analog 1 but with much higher observed passive permeability in MDCK cellular assay.