ABSTRACT
New amino-1,4-oxazine derived BACE-1 inhibitors were explored and various synthetic routes developed. The binding mode of the inhibitors was elucidated by co-crystallization of 4 with BACE-1 and X-ray analysis. Subsequent optimization led to inhibitors with low double digit nanomolar activity in a biochemical and single digit nanomolar potency in a cellular assays. To assess the inhibitors for their permeation properties and potential to cross the blood-brain-barrier a MDR1-MDCK cell model was successfully applied. Compound 8a confirmed the in vitro results by dose-dependently reducing Aß levels in mice in an acute treatment regimen.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Drug Discovery , Enzyme Inhibitors/pharmacology , Oxazines/pharmacology , Amyloid Precursor Protein Secretases/metabolism , Animals , Aspartic Acid Endopeptidases/metabolism , Dogs , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Madin Darby Canine Kidney Cells/drug effects , Mice , Models, Molecular , Molecular Conformation , Oxazines/chemical synthesis , Oxazines/chemistry , Structure-Activity RelationshipABSTRACT
Design and optimization of benzo- and pyrido-thiazoles/isothiazoles are reported leading to the discovery of the potent, orally bioavailable Syk inhibitor 5, which was found to be active in a rat PK/PD model. Compound 5 showed acceptable overall kinase selectivity. However, in addition to Syk it also inhibited Aurora kinase in enzymatic and cellular settings leading to findings in the micronucleus assay. As a consequence, compound 5 was not further pursued.
Subject(s)
Disease Models, Animal , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Thiazoles/pharmacology , Administration, Oral , Animals , Biological Availability , Dose-Response Relationship, Drug , Intracellular Signaling Peptides and Proteins/metabolism , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein-Tyrosine Kinases/metabolism , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Structure-Activity Relationship , Syk Kinase , Thiazoles/administration & dosage , Thiazoles/chemistryABSTRACT
Previous structure based optimization in our laboratories led to the identification of a novel, high-affinity cyclic sulfone hydroxyethylamine-derived inhibitor such as 1 that lowers CNS-derived Aß following oral administration to transgenic APP51/16 mice. Herein we report SAR development in the S3 and S2' subsites of BACE1 for cyclic sulfoxide hydroxyethyl amine inhibitors, the synthetic approaches employed in this effort, and in vivo data for optimized compound such as 11d.
Subject(s)
Amyloid Precursor Protein Secretases/drug effects , Amyloid beta-Peptides/chemistry , Aspartic Acid Endopeptidases/drug effects , Drug Discovery , Ethanolamines/pharmacology , Sulfoxides/pharmacology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/blood , Animals , Aspartic Acid Endopeptidases/antagonists & inhibitors , Brain Chemistry , Crystallography, X-Ray , Cyclization , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Ethanolamines/chemistry , Female , Inhibitory Concentration 50 , Male , Mice , Molecular Structure , Rats , Structure-Activity Relationship , Substrate Specificity , Sulfoxides/chemistryABSTRACT
Starting from lead compound 4, the 1,4-oxazine headgroup was optimized to improve potency and brain penetration. Focusing at the 6-position of the 5-amino-1,4-oxazine, the insertion of a Me and a CF3 group delivered an excellent pharmacological profile with a pKa of 7.1 and a very low P-gp efflux ratio enabling high central nervous system (CNS) penetration and exposure. Various synthetic routes to access BACE1 inhibitors bearing a 5-amino-6-methyl-6-(trifluoromethyl)-1,4-oxazine headgroup were investigated. Subsequent optimization of the P3 fragment provided the highly potent N-(3-((3R,6R)-5-amino-3,6-dimethyl-6-(trifluoromethyl)-3,6-dihydro-2H-1,4-oxazin-3-yl)-4-fluorophenyl)-5-cyano-3-methylpicolinamide 54 (NB-360), able to reduce significantly Aß levels in mice, rats, and dogs in acute and chronic treatment regimens.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Enzyme Inhibitors/chemical synthesis , Picolinic Acids/chemical synthesis , Thiazines/chemical synthesis , Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Animals , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Binding Sites , Brain/metabolism , Crystallography, X-Ray , Dogs , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/therapeutic use , Half-Life , Humans , Mice , Molecular Dynamics Simulation , Oxazines/chemistry , Picolinic Acids/pharmacokinetics , Picolinic Acids/therapeutic use , Rats , Structure-Activity Relationship , Thiazines/pharmacokinetics , Thiazines/therapeutic useABSTRACT
After identification of lead compound 6, 5-amino-1,4-oxazine BACE1 inhibitors were optimized in order to improve potency, brain penetration, and metabolic stability. Insertion of a methyl and a trifluoromethyl group at the 6-position of the 5-amino-1,4-oxazine led to 8 (NB-360), an inhibitor with a pKa of 7.1, a very low P-glycoprotein efflux ratio, and excellent pharmacological profile, enabling high central nervous system penetration and exposure. Fur color changes observed with NB-360 in efficacy studies in preclinical animal models triggered further optimization of the series. Herein, we describe the steps leading to the discovery of 3-chloro-5-trifluoromethyl-pyridine-2-carboxylic acid [6-((3R,6R)-5-amino-3,6-dimethyl-6-trifluoromethyl-3,6-dihydro-2H-[1,4]oxazin-3-yl)-5-fluoro-pyridin-2-yl]amide 15 (CNP520, umibecestat), an inhibitor with superior BACE1/BACE2 selectivity and pharmacokinetics. CNP520 reduced significantly Aß levels in mice and rats in acute and chronic treatment regimens without any side effects and thus qualified for Alzheimer's disease prevention studies in the clinic.
Subject(s)
Alzheimer Disease/prevention & control , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Drug Discovery , Enzyme Inhibitors/pharmacology , Oxazines/pharmacology , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/metabolism , Animals , Aspartic Acid Endopeptidases/metabolism , Cell Line , Dogs , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Male , Mice , Mice, Inbred C57BL , Molecular Structure , Oxazines/chemical synthesis , Oxazines/chemistry , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
A series of macrocyclic peptidic BACE-1 inhibitors was designed. While potency on BACE-1 was rather high, the first set of compounds showed poor brain permeation and high efflux in the MDRI-MDCK assay. The replacement of the secondary benzylamino group with a phenylcyclopropylamino group maintained potency on BACE-1, while P-glycoprotein-mediated efflux was significantly reduced and brain permeation improved. Several compounds from this series demonstrated acute reduction of Abeta in human APP-wildtype transgenic (APP51/16) mice after oral administration.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Aspartic Acid Endopeptidases/antagonists & inhibitors , Benzamides/chemistry , Brain/metabolism , Lactams/chemistry , Macrocyclic Compounds/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Amyloid Precursor Protein Secretases/metabolism , Animals , Aspartic Acid Endopeptidases/metabolism , Benzamides/chemical synthesis , Benzamides/pharmacology , Binding Sites , Cell Line , Crystallography, X-Ray , Humans , Lactams/chemical synthesis , Lactams/pharmacology , Macrocyclic Compounds/pharmacology , Mice , Mice, Transgenic , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The hydroxyethylene octapeptide inhibitor OM99-2 served as starting point to create the tripeptide inhibitor 1 and its analogues 2a and b. An X-ray co-crystal structure of 1 with BACE-1 allowed the design and syntheses of a series of macrocyclic analogues 3a-h covalently linking the P1 and P3 side-chains. These inhibitors show improved enzymatic potency over their open-chain analogue. Inhibitor 3h also shows activity in a cellular system.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Amyloid Precursor Protein Secretases/chemistry , Amyloid Precursor Protein Secretases/metabolism , Animals , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/metabolism , Binding Sites/physiology , Biomimetic Materials/chemistry , Biomimetic Materials/pharmacology , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Humans , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacology , Oligopeptides/chemical synthesis , Oligopeptides/pharmacology , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Protease Inhibitors/pharmacology , Protein Structure, Tertiary/physiology , Structure-Activity RelationshipABSTRACT
The macrocyclic peptidic BACE-1 inhibitors 2a-c show moderate enzymatic and cellular activity. By exchange of the hydroxyethylene- to ethanolamine-transition state mimetic the peptidic character was reduced, providing the highly potent and selective inhibitor 3. Variation of the P' moiety resulted in the macrocyclic inhibitor 14. Both macrocycles show inhibition of BACE-1 in the brain of APP51/16 transgenic mice, 3 (NB-544) after intravenous and 14 (NB-533) after oral application.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Peptide Fragments/chemistry , Protease Inhibitors/chemistry , Amyloid Precursor Protein Secretases/chemistry , Amyloid Precursor Protein Secretases/metabolism , Animals , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/metabolism , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Humans , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments/pharmacology , Protease Inhibitors/pharmacology , Protein Structure, Secondary/physiologyABSTRACT
The beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) initiates the generation of amyloid-ß (Aß), and the amyloid cascade leading to amyloid plaque deposition, neurodegeneration, and dementia in Alzheimer's disease (AD). Clinical failures of anti-Aß therapies in dementia stages suggest that treatment has to start in the early, asymptomatic disease states. The BACE-1 inhibitor CNP520 has a selectivity, pharmacodynamics, and distribution profile suitable for AD prevention studies. CNP520 reduced brain and cerebrospinal fluid (CSF) Aß in rats and dogs, and Aß plaque deposition in APP-transgenic mice. Animal toxicology studies of CNP520 demonstrated sufficient safety margins, with no signs of hair depigmentation, retina degeneration, liver toxicity, or cardiovascular effects. In healthy adults ≥ 60 years old, treatment with CNP520 was safe and well tolerated and resulted in robust and dose-dependent Aß reduction in the cerebrospinal fluid. Thus, long-term, pivotal studies with CNP520 have been initiated in the Generation Program.
Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/prevention & control , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Oxazines/therapeutic use , Alzheimer Disease/blood , Alzheimer Disease/cerebrospinal fluid , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/cerebrospinal fluid , Amyloid beta-Protein Precursor/metabolism , Animals , Aspartic Acid Endopeptidases/metabolism , Astrocytes/metabolism , Brain/pathology , Cathepsin D/antagonists & inhibitors , Cathepsin D/metabolism , Cerebral Hemorrhage/pathology , Female , Hominidae/genetics , Humans , Inflammation/pathology , Male , Mice, Inbred C57BL , Mice, Transgenic , Microglia/metabolism , Oxazines/blood , Oxazines/chemistry , Oxazines/pharmacology , Translational Research, BiomedicalABSTRACT
Molecular modeling based on the X-ray crystal structure of the Tang-Ghosh heptapeptide inhibitor 1 (OM99-2) of BACE led to the design and synthesis of a series of constrained P(1)' analogues. A cyclopentane ring was incorporated in 1 spanning the P(1)' Ala methyl group and the adjacent methylene carbon atom of the chain. Progressive truncation at the P(2)'-P(4)' sites led to a potent truncated analogue 5 with good selectivity over Cathepsin D. Using the same backbone replacement concept, a series of cyclopentane, cyclopentanone, tetrahydrofuran, pyrrolidine, and pyrrolidinone analogues were synthesized with considerable variation at the P and P' sites. The cyclopentanone and 2-pyrrolidinone analogues 45 and 57 showed low nM BACE inhibition. X-ray cocrystal structures of two analogues 5 and 45 revealed excellent convergence with the original inhibitor 1 structure while providing new insights into other interactions which could be exploited for future modifications.