ABSTRACT
Development of a series of highly kinome-selective spleen tyrosine kinase (Syk) inhibitors with favorable druglike properties is described. Early leads were discovered through X-ray crystallographic analysis, and a systematic survey of cores within a selected chemical space focused on ligand binding efficiency. Attenuation of hERG ion channel activity inherent within the initial chemotype was guided through modulation of physicochemical properties including log D, PSA, and pKa. PSA proved most effective for prospective compound design. Further profiling of an advanced compound revealed bacterial mutagenicity in the Ames test using TA97a Salmonella strain, and subsequent study demonstrated that this mutagenicity was pervasive throughout the series. Identification of intercalation as a likely mechanism for the mutagenicity-enabled modification of the core scaffold. Implementation of a DNA binding assay as a prescreen and models in DNA allowed resolution of the mutagenicity risk, affording molecules with favorable potency, selectivity, pharmacokinetic, and off-target profiles.
Subject(s)
Amides/pharmacology , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Spleen/enzymology , Amides/chemical synthesis , Amides/chemistry , Crystallography, X-Ray , Dose-Response Relationship, Drug , Ether-A-Go-Go Potassium Channels/genetics , Ether-A-Go-Go Potassium Channels/metabolism , Humans , Models, Molecular , Molecular Structure , Mutagenicity Tests , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein-Tyrosine Kinases/metabolism , Spleen/drug effects , Structure-Activity RelationshipABSTRACT
Intramolecular inverse electron demand cycloadditions of isatin-derived 1,2,4-triazines with acetylenic dienophiles tethered by amidations or transesterifications proceed in excellent yields to produce lactam- or lactone-fused α-carbolines. Beginning with various isatins and alkynyl dienophiles, a pilot-scale library of eighty-eight α-carbolines was prepared by using this robust methodology for biological evaluation.
ABSTRACT
The chemistry of 1,2,3,4-tetrahydro-1,5-naphthyridines and 2,3,4,5-tetrahydro-1H-pyrido[3,2-b]azepines has been explored with the goal of discovering reactions at N1 suitable for library development. Epoxide openings, palladium-catalyzed N-arylations, DEPBT-promoted acylations, and urea formation through the reaction with isocyanates were all successful. The epoxide opening chemistry using homochiral epichlorohydrin, with epoxide reclosure and a second nucleophilic opening led to the preparation of a small 24-membered library.
ABSTRACT
A primary limitation in the development and use of screens to identify factors that regulate mammalian pre-mRNA splicing has been the development of sensitive reporter assays. Alternative splicing typically involves relatively small (< 10-fold) changes in isoform ratios. Therefore, reporter constructs designed to allow direct analysis of isoform expression historically have at most a 10-fold window of discrimination between a positive signal and background. Here we describe the design and application of a reporter cell line that makes use of the phenomenon of transcriptional synergy to amplify the detection of changes in splicing, such that a three- to five-fold change in splicing pattern is observed as a 30- to 50-fold change in GFP expression. Using this cell line we have identified two small molecules, from a library of approximately 300 synthetic compounds, that can induce partial repression of a variable exon from the CD45 gene. We propose that the concept of transcription-based amplification of signal will allow the development of true high-throughput screening approaches to identify effectors of mammalian alternative splicing.
Subject(s)
Alternative Splicing , Genes, Reporter , Green Fluorescent Proteins/metabolism , RNA Splicing , Transcription, Genetic , Cell Line , Exons , Gene Library , Leukocyte Common Antigens/drug effects , Leukocyte Common Antigens/genetics , Luminescent Proteins/drug effects , Luminescent Proteins/genetics , Lymphocyte Activation/drug effects , RNA Precursors/metabolism , Sensitivity and Specificity , Signal Transduction , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Tetradecanoylphorbol Acetate/pharmacology , TransfectionABSTRACT
The intramolecular inverse-electron-demand Diels-Alder reaction between imidazoles and 1,2,4-triazines linked by a trimethylene tether from the imidazole N1 position to the triazine C3 proceed in excellent yields to produce 1,2,3,4-tetrahydro-1,5-naphthyridines. The reaction proceeds by a cycloaddition with subsequent loss of nitrogen, followed by a presumed stepwise loss of a nitrile. The analogous intramolecular cycloadditions employing a tetramethylene tether also proceeded to give 2,3,4,5-tetrahydro-1H-pyrido[3,2-b]azepines in acceptable yields. The reaction to produce the tetrahydro-1,5-naphthyridines can also be promoted with microwave irradiation.
