ABSTRACT
The human immunodeficiency virus type-1 (HIV-1) encodes three enzymes essential for viral replication: a reverse transcriptase, a protease, and an integrase. The latter is responsible for the integration of the viral genome into the human genome and, therefore, represents an attractive target for chemotherapeutic intervention against AIDS. A drug based on this mechanism has not yet been approved. Benzyl-dihydroxypyrimidine-carboxamides were discovered in our laboratories as a novel and metabolically stable class of agents that exhibits potent inhibition of the HIV integrase strand transfer step. Further efforts led to very potent compounds based on the structurally related N-Me pyrimidone scaffold. One of the more interesting compounds in this series is the 2-N-Me-morpholino derivative 27a, which shows a CIC95 of 65 nM in the cell in the presence of serum. The compound has favorable pharmacokinetic properties in three preclinical species and shows no liabilities in several counterscreening assays.
Subject(s)
HIV Integrase Inhibitors/chemical synthesis , HIV Integrase/chemistry , HIV-1/drug effects , Morpholines/chemical synthesis , Pyrimidinones/chemical synthesis , Administration, Oral , Animals , Biological Availability , Blood Proteins/metabolism , Cell Line, Tumor , Dogs , HIV Integrase Inhibitors/pharmacokinetics , HIV Integrase Inhibitors/pharmacology , HIV-1/enzymology , HIV-1/physiology , Humans , Macaca mulatta , Morpholines/pharmacokinetics , Morpholines/pharmacology , Protein Binding , Pyrimidinones/pharmacokinetics , Pyrimidinones/pharmacology , Rats , Stereoisomerism , Structure-Activity Relationship , Virus Replication/drug effectsABSTRACT
A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C9 in human liver microsomes is described. In contrast to the conventional diclofenac 4'-hydroxylation assay, the new method does not require high performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP2C9-mediated 4'-hydroxylation of diclofenac labeled with tritium in the 4' position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent, and sensitive to CYP2C9 inhibitors and inhibitory monoclonal antibodies, but not to inhibitors of or antibodies against other P450 enzymes. Competition experiments using tritiated and unlabeled diclofenac indicated that CYP2C9-mediated diclofenac 4'-hydroxylation exhibits positive cooperativity and no significant kinetic isotope effect or NIH shift. IC(50) values for 18 structurally diverse chemical inhibitors were not significantly different from those determined in the diclofenac 4'-hydroxylation assay, using HPLC-tandem mass spectrometry. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classic diclofenac 4'-hydroxylation assay, which is one of the most widely used methods to assess the potential for CYP2C9 inhibition of new chemical entities.
Subject(s)
Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Enzyme Inhibitors/analysis , Radiometry/methods , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/pharmacology , Aryl Hydrocarbon Hydroxylases/metabolism , Automation , Cytochrome P-450 CYP2C9 , Diclofenac/analogs & derivatives , Diclofenac/analysis , Diclofenac/metabolism , Enzyme Inhibitors/pharmacology , Humans , Hydroxylation , Microsomes, Liver/metabolism , Reproducibility of Results , Theophylline/analogs & derivatives , Theophylline/pharmacology , Tritium/analysis , WaterABSTRACT
A new class of the HCV NS5b RNA-dependent RNA polymerase inhibitors, the dihyroxypyrimidinecarboxylic acid derivative, was designed from a diketoacid and meconic acid derivative discovered by screening. Mechanism of action and essential moieties required for activity were identified. The corresponding N-methylpyrimidinone was also prepared; both classes are novel, reversible, and selective inhibitors of the HCV NS5b polymerase with improved druglike characteristics.
Subject(s)
Keto Acids/chemical synthesis , Pyrimidines/chemical synthesis , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism , Animals , Binding Sites , Drug Design , Hepacivirus/enzymology , In Vitro Techniques , Isomerism , Keto Acids/chemistry , Keto Acids/pharmacology , Microsomes, Liver/metabolism , Models, Molecular , Pyrimidines/chemistry , Pyrimidines/pharmacology , RNA-Dependent RNA Polymerase/chemistry , Rats , Structure-Activity RelationshipABSTRACT
SAR on the phenethylamide 1 (Ki 1.2 microM) in the P2- and the P'-position led to potent inhibitors, one of which showed good exposure and low clearance when administered intramuscularly to rat.
