ABSTRACT
Presented here are initial structure-activity relationship (SAR) studies on a series of novel heteroaryl fused tetracyclic indole-based inhibitors of the hepatitis C viral polymerase, NS5B. The introduction of alternative heterocyclic moieties into the indolo-fused inhibitor class significantly expands the reported SAR and resulted in the identification of pyridino analogs, typified by compounds 44 and 45 that displayed excellent potency against the NS5B polymerase of both HCV 1a and HCV 1b genotypes.
Subject(s)
Amides/chemistry , Hepacivirus/drug effects , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/chemical synthesis , Viral Nonstructural Proteins/antagonists & inhibitors , Allosteric Regulation , Amides/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds/pharmacology , Humans , Indoles/chemistry , Indoles/pharmacology , Inhibitory Concentration 50 , Structure-Activity RelationshipABSTRACT
Herein, we present initial SAR studies on a series of bridged 2-arylindole-based NS5B inhibitors. The introduction of bridging elements between the indole N1 and the ortho-position of the 2-aryl moiety resulted in conformationally constrained heterocycles that possess multiple additional vectors for further exploration. The binding mode and pharmacokinetic (PK) properties of select examples, including: 13-cyclohexyl-6-oxo-6,7-dihydro-5H-indolo[2,1-d][1,4]benzodiazepine-10-carboxylic acid (7) (IC(50)=0.07 µM, %F=18), are reported.
Subject(s)
Enzyme Activation/drug effects , Hepacivirus/enzymology , Heterocyclic Compounds/chemical synthesis , Heterocyclic Compounds/pharmacology , Indoles/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Drug Design , Heterocyclic Compounds/chemistry , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
Hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B) is required for viral replication. Crystal structures of the NS5B apoprotein show that the finger and thumb domains interact to encircle the active site, and that inhibitors defined by P495 resistance that bind to the thumb-finger interface displace the Δ1 finger loop and disrupt this structure. Since crystal structures may not reveal all of the conformations of a protein in solution we have developed an alternative method, using limited trypsin protease digestion, to investigate the impact of inhibitors as well as substrates on the movement of the Δ1 loop. This assay can be used to study NS5B under conditions that support enzymatic activity. In the absence of inhibitors, no specific region of NS5B was hypersensitive to trypsin, and no specific intermediate cleavage products were formed. Binding of P495-site inhibitors to NS5B induced specific trypsin hypersensitivity at lysine residues 50 and 51. Previously characterized inhibitors and mutant polymerases were used to link this specific trypsin hypersensitivity to movement of the Δ1 loop. Trypsin hypersensitivity identical to the inhibitor pattern was also induced by the binding of the RNA template. The addition of primer to the NS5B-template complex eliminated the hypersensitivity. The data are consistent with displacement of the Δ1 finger loop from the thumb by the binding of template, and reversal by the addition of primer or NTP. Our results complement inhibitor-enzyme co-crystal studies, and the assay provides a rapid and sensitive method to study dynamic changes in HCV NS5B polymerase conformation under conditions that support functional activity.
Subject(s)
Antiviral Agents/pharmacology , Gene Expression/drug effects , Hepacivirus/enzymology , Protein Conformation , RNA-Dependent RNA Polymerase/chemistry , Viral Nonstructural Proteins/chemistry , Antiviral Agents/metabolism , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , Hepacivirus/genetics , Hepacivirus/physiology , Ligands , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Protein Structure, Tertiary , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/antagonists & inhibitors , RNA-Dependent RNA Polymerase/metabolism , Structure-Activity Relationship , Trypsin/metabolism , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects , Virus Replication/geneticsABSTRACT
Several semi-synthetic bis- and mono-O-alkyl nocathiacin derivatives were synthesized and evaluated for antibacterial activity. Mono-O-alkyl N-hydroxyindole analogues 3a-l were prepared by regioselective alkylation. Bis-O-alkyl nocathiacins 4a-f were obtained by treatment with base and excess electrophile. A one-pot protection-alkylation-deprotection strategy was developed for the preparation of mono-O-alkyl hydroxypyridine analogues 5a,b. Most of the bis- and mono-O-alkyl nocathiacins maintained good in vitro activity but showed reduced in vivo efficacy when compared with the natural product. The excellent in vivo activity and improved water solubility of phosphate analogues 3m and 4g suggest their use as potential pro-drugs.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Peptides/chemical synthesis , Peptides/pharmacology , Alkylation/drug effects , Enterococcus faecalis/drug effects , Enterococcus faecalis/growth & development , Intercellular Signaling Peptides and Proteins , Microbial Sensitivity Tests/statistics & numerical data , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Streptococcus pneumoniae/drug effects , Streptococcus pneumoniae/growth & developmentABSTRACT
Synthesis of phosphonooxymethyl derivatives of ravuconazole, 2 (BMS-379224) and 3 (BMS-315801) and their biological evaluation as potential water-soluble prodrugs of ravuconazole are described. The phosphonooxymethyl ether analogue 2 (BMS-379224) and N-phosphonooxymethyl triazolium salt 3 (BMS-315801) were both prepared from ravuconazole (1) and bis-tert-butyl chloromethylphosphate, but under two different conditions. Both derivatives were highly soluble in water and converted to the parent in alkaline phosphatase, and also in vivo (rat). However, BMS-315801 was found to be less stable than BMS-379224 in water at neutral pH. BMS-379224 (2) has proved to be one of the most promising prodrugs of ravuconazole that we tested, and it is currently in clinical evaluation as an intravenous formulation of the broad spectrum antifungal azole, ravuconazole.
