ABSTRACT
The HCV RNA-dependent RNA polymerase has emerged as one of the key targets for novel anti-HCV therapy development. Herein, we report the optimization of the dihydropyrone series inhibitors to improve compound aqueous solubility and reduce CYP2D6 inhibition, which led to the discovery of compound 24 (PF-00868554). Compound 24 is a potent and selective HCV polymerase inhibitor with a favorable pharmacokinetic profile and has recently entered a phase II clinical evaluation in patients with genotype 1 HCV.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/enzymology , Pyrones/chemical synthesis , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Triazoles/chemical synthesis , Administration, Oral , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Crystallography, X-Ray , Cytochrome P-450 CYP2D6 Inhibitors , Dogs , Macaca fascicularis , Microsomes, Liver/metabolism , Models, Molecular , Pyrones/pharmacokinetics , Pyrones/pharmacology , Rats , Rats, Sprague-Dawley , Solubility , Stereoisomerism , Structure-Activity Relationship , Triazoles/pharmacokinetics , Triazoles/pharmacologyABSTRACT
The discovery and optimization of a novel class of carbon-linked dihydropyrones as allosteric HCV NS5B polymerase inhibitors are presented. Replacement of the sulfur linker atom with carbon reduced compound acidity and greatly increased cell permeation. Further structure-activity relationship (SAR) studies led to the identification of compounds, exemplified by 23 and 24, with significantly improved antiviral activities in the cell-based replicon assay and favorable pharmacokinetic profiles.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/enzymology , Pyrones/chemical synthesis , Viral Nonstructural Proteins/antagonists & inhibitors , Administration, Oral , Allosteric Regulation , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Biological Availability , Caco-2 Cells , Cell Line, Tumor , Half-Life , Humans , Permeability , Pyrones/chemistry , Pyrones/pharmacology , Rats , Stereoisomerism , Structure-Activity Relationship , Viral Nonstructural Proteins/geneticsABSTRACT
A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude.
