ABSTRACT
Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aß42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aß42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed robust lowering of CSF Aß42 levels.
Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/metabolism , Animals , Drug Discovery , Macaca mulatta , Mice , Mice, Transgenic , Rats , Rats, Sprague-DawleyABSTRACT
Alzheimer's disease is a major unmet medical need with pathology characterized by extracellular proteinaceous plaques comprised primarily of ß-amyloid. γ-Secretase is a critical enzyme in the cellular pathway responsible for the formation of a range of ß-amyloid peptides; one of which, Aß42, is believed to be responsible for the neuropathological features of the disease. Herein, we report 4,4 disubstituted piperidine γ-secretase inhibitors that were optimized for in vitro cellular potency and pharmacokinetic properties in vivo. Key agents were further characterized for their ability to lower cerebral Aß42 production in an APP-YAC mouse model. This structural series generally suffered from sub-optimal pharmacokinetics but hypothesis driven lead optimization enabled the discovery of γ-secretase inhibitors capable of lowering cerebral Aß42 production in mice.
Subject(s)
Amides/chemical synthesis , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Piperidines/chemistry , Alzheimer Disease/drug therapy , Amides/pharmacology , Amyloid beta-Peptides/biosynthesis , Animals , Brain/drug effects , Brain/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Mice , Peptide Fragments/biosynthesisABSTRACT
Synthesis, SAR, and evaluation of aryl triazoles as novel gamma secretase modulators (GSMs) are presented in this communication. Starting from the literature and in-house leads, we evaluated a range of five-membered heterocycles as replacements for olefins commonly found in non-acid GSMs. 1,2,3-C-aryl-triazoles were identified as suitable replacements which exhibited good modulation of γ-secretase activity, excellent pharmacokinetics and good central lowering of Aß42 in Sprague-Dawley rats.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Triazoles/chemical synthesis , Triazoles/pharmacology , Amyloid beta-Peptides/metabolism , Animals , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Inhibitory Concentration 50 , Molecular Structure , Protein Binding , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Triazoles/metabolismABSTRACT
Synthesis, SAR and evaluation of styrenyl quinazolinones as novel gamma secretase modulators are presented in this communication. Starting from literature and in-house leads we evaluated a range of quinazolinones which showed good modulation of γ-secretase activity.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Quinazolinones/chemistry , Quinazolinones/pharmacology , Alzheimer Disease/drug therapy , Alzheimer Disease/enzymology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Cell Line , Humans , Inhibitory Concentration 50 , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/metabolism , Protein Binding , Quinazolinones/pharmacokinetics , Rats , Rats, Sprague-DawleyABSTRACT
The development of a novel series of purines as gamma-secretase modulators for potential use in the treatment of Alzheimer's disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based gamma-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Alphabeta42 in an APP-YAC transgenic mouse model.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Peptide Fragments/antagonists & inhibitors , Purines/chemistry , Purines/therapeutic use , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Peptides/metabolism , Animals , Humans , Mice , Mice, Transgenic , Peptide Fragments/metabolism , Purines/pharmacology , Receptors, Notch/metabolism , Structure-Activity RelationshipABSTRACT
We report herein a novel series of difluoropiperidine acetic acids as modulators of gamma-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective beta-difluorination with Selectfluor. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Abeta42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250mg/kg per day, AUC(0-24)=2100microMh) did not exhibit Notch-related effects.
Subject(s)
Acetates/chemistry , Amyloid Precursor Protein Secretases/metabolism , Fluorine/chemistry , Piperidines/chemistry , Acetates/chemical synthesis , Acetates/pharmacokinetics , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Animals , Diazonium Compounds/chemistry , Disease Models, Animal , Mice , Mice, Transgenic , Peptide Fragments/genetics , Peptide Fragments/metabolism , Piperidines/chemical synthesis , Piperidines/pharmacokinetics , Rats , Receptors, Notch/metabolismABSTRACT
Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with gamma-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-end-stage Tal1/Ink4a/Arf+/- leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSI-treated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T-ALL and suggests that inhibition of the mTOR and NOTCH1 pathways may have added efficacy.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Carrier Proteins/metabolism , Cyclic S-Oxides/pharmacology , Disease Models, Animal , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Receptor, Notch1/metabolism , Thiadiazoles/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Animals , Apoptosis , Basic Helix-Loop-Helix Transcription Factors/physiology , Blotting, Western , Carrier Proteins/genetics , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p16/physiology , Flow Cytometry , Humans , Immunoenzyme Techniques , Mice , Mice, Transgenic , Phosphotransferases (Alcohol Group Acceptor)/genetics , Proto-Oncogene Proteins/physiology , Receptor, Notch1/genetics , Signal Transduction , T-Cell Acute Lymphocytic Leukemia Protein 1 , TOR Serine-Threonine Kinases , Tumor Cells, CulturedABSTRACT
Tyrosine nitration is a well-established protein modification that occurs in disease states associated with oxidative stress and increased nitric oxide synthase activity. Nitration of specific tyrosine residues has been reported to affect protein structure and function, suggesting that 3-nitrotyrosine formation may not only be a disease marker but may also be involved in the pathogenesis of some diseases and in normal regulatory processes. It has been, however, difficult to identify sites of nitration. We describe a method that combines specific isolation of nitrated proteins with mass spectrometric determination of the amino acid sequence and the site of nitration of individual proteins. A complex protein mixture, e.g., serum or cell lysate, was enriched for nitrotyrosine-containing proteins by immunoprecipitation with antinitrotyrosine antibodies. The nitrotyrosines were then reduced to aminotyrosines with a strong reducing agent in parallel in-gel and in-solution procedures. Using nitrated human serum albumin as a model, we reduced the disulfide bonds with dithiothreitol and alkylated the free sulfhydryl groups with iodoacetamide. The nitrotyrosines were next reduced to aminotyrosines with sodium dithionite, and-at pH 5.0-cleavable biotin tags were selectively attached to the aminotyrosines and the albumin was then digested with trypsin. The biotinylated tryptic peptides were purified on a streptavidin affinity column and identified by mass spectrometry. We have also purified nitrated human serum albumin from an enriched sample of SJL mouse plasma and confirmed its identity by peptide mass fingerprinting and MASCOT.
