ABSTRACT
The M2 isoform of pyruvate kinase is an emerging target for antitumor therapy. In this letter, we describe the discovery of 2-((1H-benzo[d]imidazol-1-yl)methyl)-4H-pyrido[1,2-a]pyrimidin-4-ones as potent and selective PKM2 activators which were found to have a novel binding mode. The original lead identified from high throughput screening was optimized into an efficient series via computer-aided structure-based drug design. Both a representative compound from this series and an activator described in the literature were used as molecular tools to probe the biological effects of PKM2 activation on cancer cells. Our results suggested that PKM2 activation alone is not sufficient to alter cancer cell metabolism.
Subject(s)
Benzimidazoles/chemistry , Carrier Proteins/agonists , Membrane Proteins/agonists , Pyrimidinones/chemistry , Thyroid Hormones/agonists , Binding Sites , Carrier Proteins/metabolism , Cell Line , Computer-Aided Design , Drug Evaluation, Preclinical , High-Throughput Screening Assays , Humans , Kinetics , Membrane Proteins/metabolism , Molecular Docking Simulation , Protein Binding , Protein Structure, Tertiary , Pyrimidinones/chemical synthesis , Pyrimidinones/metabolism , Structure-Activity Relationship , Thyroid Hormones/metabolism , Thyroid Hormone-Binding ProteinsABSTRACT
(S)-1-((4-(3-(6-Amino-5-methoxypyridin-3-yl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)propan-2-ol, 1, was recently identified as a potent inhibitor of the oncogenic kinase bRAF. Compounds containing 3-methoxy-2-aminopyridine, as in 1, comprised a promising lead series because of their high ligand efficiency and excellent ADME profile. However, following metabolic oxidation, compounds in this series also demonstrated two significant safety risks: mutagenic potential and time-dependent drug-drug interaction (TDI). Metabolite identification studies revealed formation of a reactive metabolite. We hypothesized that minimizing or blocking the formation of such a metabolite would mitigate the safety liabilities. Our investigation demonstrated that structural modifications which either reduced the electron density of the 3-methoxy-2-aminopyridine ring or blocked the reactive site following metabolic oxidation were successful in reducing TDI and AMES mutagenicity.
Subject(s)
Aminopyridines/chemistry , Aminopyridines/metabolism , Electrons , Humans , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Molecular Structure , Mutagenicity Tests , Oxidation-Reduction , Time FactorsABSTRACT
Oxobenzimidazoles (e.g., 1), a novel series of androgen receptor (AR) antagonists, were discovered through de novo design guided by structure-based drug design. The compounds in this series were reasonably permeable and metabolically stable, but suffered from poor solubility. The incorporation of three dimensional structural features led to improved solubility. In addition, the observation of a 'flipped' binding mode of an oxobenzimidazole analog in an AR ligand binding domain (LBD) model, led to the design and discovery of the novel oxindole series (e.g., 2) that is a potent full antagonist of AR.
Subject(s)
Androgen Receptor Antagonists/chemical synthesis , Antineoplastic Agents/chemical synthesis , Benzimidazoles/chemical synthesis , Indoles/chemical synthesis , Receptors, Androgen/chemistry , Androgen Receptor Antagonists/pharmacology , Antineoplastic Agents/pharmacology , Benzimidazoles/pharmacology , Cell Line, Tumor , Drug Design , Drug Discovery , Humans , Indoles/pharmacology , Ligands , Male , Models, Molecular , Prostatic Neoplasms , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Androgen/metabolism , Solubility , Structure-Activity RelationshipABSTRACT
High throughput cell-based screening led to the identification of 3-aryloxy lactams as potent androgen receptor (AR) antagonists. Refinement of these leads to improve the ADME profile and remove residual agonism led to the discovery of 12, a potent full antagonist with greater oral bioavailability. Improvements in the ADME profile were realized by designing more ligand-efficient molecules with reduced molecular weights and lower lipophilicities.
Subject(s)
Drug Discovery , Lactams/pharmacology , Prostatic Neoplasms/drug therapy , Receptors, Androgen/chemistry , Dose-Response Relationship, Drug , High-Throughput Screening Assays , Humans , Lactams/chemical synthesis , Lactams/chemistry , Male , Models, Molecular , Molecular Structure , Prostatic Neoplasms/surgery , Receptors, Androgen/metabolism , Stereoisomerism , Structure-Activity RelationshipABSTRACT
An aryloxy tetramethylcyclobutane was identified as a novel template for androgen receptor (AR) antagonists via cell-based high-throughput screening. Follow-up to the initial "hit" established 5 as a viable lead. Further optimization to achieve full AR antagonism led to the discovery of 26 and 30, both of which demonstrated excellent in vivo tumor growth inhibition upon oral administration in a castration-resistant prostate cancer (CRPC) animal model.
Subject(s)
Androgen Antagonists/chemical synthesis , Antineoplastic Agents/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Cyclobutanes/chemical synthesis , Pyrazoles/chemical synthesis , Administration, Oral , Androgen Antagonists/pharmacokinetics , Androgen Antagonists/pharmacology , Androgens/chemical synthesis , Androgens/pharmacokinetics , Androgens/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line , Cyclobutanes/pharmacokinetics , Cyclobutanes/pharmacology , Drug Resistance, Neoplasm , High-Throughput Screening Assays , Humans , Ligands , Male , Mice , Mice, Nude , Models, Molecular , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-2,2,4,4-tetramethylcyclobutyl}imidazo[1,2-a]pyrimidine-3-carboxamide (1) was recently identified as a full antagonist of the androgen receptor, demonstrating excellent in vivo tumor growth inhibition in castration-resistant prostate cancer (CRPC). However, the imidazo[1,2-a]pyrimidine moiety is rapidly metabolized by aldehyde oxidase (AO). The present paper describes a number of medicinal chemistry strategies taken to avoid the AO-mediated oxidation of this particular system. Guided by an AO protein structure-based model, our investigation revealed the most probable site of AO oxidation and the observation that altering the heterocycle or blocking the reactive site are two of the more effective strategies for reducing AO metabolism. These strategies may be useful for other drug discovery programs.
Subject(s)
Aldehyde Oxidase/chemistry , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Cyclobutanes/chemistry , Imidazoles/chemistry , Pyrimidines/chemistry , Aldehyde Oxidase/metabolism , Catalytic Domain , Humans , Imidazoles/chemical synthesis , Imidazoles/metabolism , Models, Molecular , Oxidation-Reduction , Protein Binding , Pyrimidines/chemical synthesis , Pyrimidines/metabolism , Stereoisomerism , Structure-Activity RelationshipABSTRACT
PF-00868554 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, which exerts its inhibitory effect by binding to the thumb base domain of the protein. It is a potent and selective inhibitor, with a mean 50% inhibitory concentration of 0.019 microM against genotype 1 polymerases and a mean 50% effective concentration (EC(50)) of 0.075 microM against the genotype 1b-Con1 replicon. To determine the in vitro antiviral activity of PF-00868554 against various HCV strains, a panel of chimeric replicons was generated, in which polymerase sequences derived from genotype 1a and 1b clinical isolates were cloned into the 1b-Con1 subgenomic reporter replicon. Our results indicate that PF-00868554 has potent in vitro antiviral activity against a majority (95.8%) of genotype 1a and 1b replicons, with an overall mean EC(50) of 0.059 microM. PF-00868554 showed no cytotoxic effect in several human cell lines, up to the highest concentration evaluated (320 microM). Furthermore, the antiviral activity of PF-00868554 was retained in the presence of human serum proteins. An in vitro resistance study of PF-00868554 identified M423T as the predominant resistance mutation, resulting in a 761-fold reduction in susceptibility to PF-00868554 but no change in susceptibility to alpha interferon and a polymerase inhibitor that binds to a different region. PF-00868554 also showed good pharmacokinetic properties in preclinical animal species. Our results demonstrate that PF-00868554 has potent and broad-spectrum antiviral activity against genotype 1 HCV strains, supporting its use as an oral antiviral agent in HCV-infected patients.
Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Animals , Antiviral Agents/pharmacokinetics , Cell Line , Dogs , Drug Resistance, Viral , Genotype , Hepacivirus/enzymology , Humans , Macaca fascicularis , Male , Phenotype , Protein Binding , Rats , Rats, Sprague-Dawley , Replicon/drug effectsABSTRACT
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 novel class of non-nucleoside HCV NS5B polymerase inhibitors has been identified from screening. A co-crystal structure revealed an allosteric binding site in the protein that required a unique conformational change to accommodate inhibitor binding. Herein we report the structure-activity relationships (SARs) of this novel class of dihydropyrone-containing compounds that show potent inhibitory activities against the HCV RNA polymerase in biochemical assays.