ABSTRACT
The SAR development is described for a series of N-acyl pyrrolidine inhibitors of the Hepatitis C virus RNA-dependent RNA polymerase, NS5B, from tractable Delta21 enzyme inhibitors to an example with antiviral activity in a cellular assay (HCV replicon).
Subject(s)
Antiviral Agents/pharmacology , Chemistry, Pharmaceutical/methods , Hepacivirus/chemistry , Hepacivirus/genetics , Pyrrolidines/antagonists & inhibitors , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Replicon/genetics , Viral Nonstructural Proteins/pharmacology , Antiviral Agents/chemistry , Drug Design , Enzyme Inhibitors/pharmacology , Inhibitory Concentration 50 , Models, Chemical , Molecular Conformation , RNA, Viral/chemistry , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effectsABSTRACT
Optimization of a pyrrolidine-based template using structure-based design and physicochemical considerations has provided a development candidate 20b (3082) with submicromolar potency in the HCV replicon and good pharmacokinetic properties.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/drug effects , Pyrrolidines/chemical synthesis , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , Biological Availability , Chlorocebus aethiops , Hepacivirus/enzymology , Models, Molecular , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , RNA-Dependent RNA Polymerase/chemistry , Rats , Stereoisomerism , Structure-Activity Relationship , Vero CellsABSTRACT
A recombinant vaccinia virus, expressing the NS3-to-NS5 region of the N clone of hepatitis C virus (HCV), was generated and utilized both in a gel-based assay and in an enzyme-linked immunosorbent assay (ELISA) to evaluate the pyrrolidine-5,5-trans-lactams, a series of inhibitors of the HCV NS3/4A protease. The absolute levels of processed, mature HCV nonstructural proteins in this system were found to decrease in the presence of the trans-lactams. Monitoring of this reduction enabled end points and 50% inhibitory concentrations to be calculated in order to rank the active compounds according to potency. These compounds had no effect on the transcription or translation of the NS3-5 polyprotein at concentrations shown to inhibit NS3/4A protease, and they were shown to be specific inhibitors of this protease. The ELISA, originally developed using the vaccinia virus expression system, was modified to utilize Huh-7 cells containing an HCV replicon. Results with this assay correlated well with those obtained with the recombinant vaccinia virus assays. These results demonstrate the utility of these assays for the characterization of NS3/4A protease inhibitors. In addition, inhibitors of other viral targets, such as polymerase and helicase, can be evaluated in the context of the replicon ELISA.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Lactams/pharmacology , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Cell Line , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Hepacivirus/enzymology , Humans , Lactams/chemistry , Microbial Sensitivity Tests/methods , Replicon , Vaccinia virus/enzymology , Vaccinia virus/genetics , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
[reaction: see text] In this, the first of two Letters, we describe how a P3/P4 urea linking unit was used to greatly enhance the biochemical and replicon potency of inhibitors based upon the pyrrolidine-5,5-trans-lactam template. Compound 7b demonstrated a 100 nM IC(50) in the replicon cell-based surrogate HCV assay.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/enzymology , Lactams/chemistry , Protease Inhibitors/chemical synthesis , Pyrrolidines/chemistry , Urea/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Crystallography, X-Ray , Hepacivirus/drug effects , Hepacivirus/physiology , Inhibitory Concentration 50 , Molecular Conformation , Molecular Structure , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effectsABSTRACT
A series of chiral, (S)-proline-alpha-methylpyrrolidine-5,5-trans-lactam serine protease inhibitors has been developed as antivirals of human cytomegalovirus (HCMV). The SAR of the functionality on the proline nitrogen has shown that derivatives of para-substituted phenyl ureas > para-substituted phenyl sulfonamides > para-substituted phenyl carboxamide for activity against HCMV deltaAla protease, producing para-substituted phenyl ureas with single figure nM potency (K(i)) against the viral enzyme. The SAR of the functionality on the lactam nitrogen has defined the steric and electronic requirements for high human plasma stability while retaining good activity against HCMV protease. The combination of high potency against HCMV deltaAla protease and high human plasma stability has produced compounds with significant in vitro antiviral activity against human cytomegalovirus with the 6-hydroxymethyl benzothiazole derivative 72 being equivalent in potency to ganciclovir. The parent benzothiazole 56 had good pharmacokinetics in dogs with 29% bioavailability and good brain and ocular penetration in guinea pigs.
