ABSTRACT
Polycomb Repressive Complex 2 (PRC2) plays an important role in transcriptional regulation during animal development and in cell differentiation, and alteration of PRC2 activity has been associated with cancer. On a molecular level, PRC2 catalyzes methylation of histone H3 lysine 27 (H3K27), resulting in mono-, di-, or trimethylated forms of H3K27, of which the trimethylated form H3K27me3 leads to transcriptional repression of polycomb target genes. Previously, we have shown that binding of the low-molecular-weight compound EED226 to the H3K27me3 binding pocket of the regulatory subunit EED can effectively inhibit PRC2 activity in cells and reduce tumor growth in mouse xenograft models. Here, we report the stepwise optimization of the tool compound EED226 toward the potent and selective EED inhibitor MAK683 (compound 22) and its subsequent preclinical characterization. Based on a balanced PK/PD profile, efficacy, and mitigated risk of forming reactive metabolites, MAK683 has been selected for clinical development.
Subject(s)
Histones , Neoplasms , Animals , Enzyme Inhibitors , Histones/metabolism , Humans , Methylation , Mice , Neoplasms/drug therapy , Polycomb Repressive Complex 2ABSTRACT
MAK683 (N-((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)-8-(2-methylpyridin-3-yl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine) is a potent and orally bioavailable EED inhibitor for the potential treatment in oncology. Pharmacokinetics (PK) in preclinical species are characterised by low to moderate plasma clearances, high oral exposure, and moderate to high oral bioavailability at the dose of 1-2 mg/kg.A species comparison of the metabolic pathways of MAK683 has been made using [14C]MAK683 incubations with liver microsomes and hepatocytes from rat, dog, cynomolgus monkey, and human. Overall, the in vitro hepatic metabolism pathway of MAK683 in all five species was very complex. A total of 60 metabolites with 19 metabolites >1.5% of the total integrated area in the radiochromatogram of at least one species were identified in five species (rat, mouse, dog, monkey, and human).The primary in vitro hepatic oxidative metabolism pathway identified in humans involved 2-hydroxylation of the dihydrofuran ring to form alcohol (M28), which was in a chemical equilibrium favouring the formation of its aldehyde form. The aldehyde was then oxidised to the carboxylic acid metabolite (M26) or reduced to the O-hydroxyethylphenol (M29). N-dealkylation (M1), 3-hydroxylation of the dihydrofuran ring (M27), N-oxidation of the pyridine moiety (M53), and sulphate conjugation of M28 to form M19 were also important biotransformation pathways in human hepatocytes. The above major human hepatic metabolic pathways were also observed across the animal species (rat, mouse, dog, and monkey) mostly providing precursors for the formation of other metabolites via further oxygenation, glucuronidation, and sulphation pathways.No human-specific metabolites were observed. In addition, in vivo biotransformation was also conducted in bile-duct cannulated (BDC) rat. The metabolism in BDC rat was similar to those observed the in vitro hepatocytes.
Subject(s)
Ectoderm , Neoplasms , Animals , Dogs , Hepatocytes/metabolism , Macaca fascicularis , Mice , Microsomes, Liver/metabolism , Polycomb Repressive Complex 2/metabolism , RatsABSTRACT
The predominant expression of phosphoinositide 3-kinase δ (PI3Kδ) in leukocytes and its critical role in B and T cell functions led to the hypothesis that selective inhibitors of this isoform would have potential as therapeutics for the treatment of allergic and inflammatory disease. Targeting specifically PI3Kδ should avoid potential side effects associated with the ubiquitously expressed PI3Kα and ß isoforms. We disclose how morphing the heterocyclic core of previously discovered 4,6-diaryl quinazolines to a significantly less lipophilic 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine, followed by replacement of one of the phenyl groups with a pyrrolidine-3-amine, led to a compound series with an optimal on-target profile and good ADME properties. A final lipophilicity adjustment led to the discovery of CDZ173 (leniolisib), a potent PI3Kδ selective inhibitor with suitable properties and efficacy for clinical development as an anti-inflammatory therapeutic. In vitro, CDZ173 inhibits a large spectrum of immune cell functions, as demonstrated in B and T cells, neutrophils, monocytes, basophils, plasmocytoid dendritic cells, and mast cells. In vivo, CDZ173 inhibits B cell activation in rats and monkeys in a concentration- and time-dependent manner. After prophylactic or therapeutic dosing, CDZ173 potently inhibited antigen-specific antibody production and reduced disease symptoms in a rat collagen-induced arthritis model. Structurally, CDZ173 differs significantly from the first generation of PI3Kδ and PI3Kγδ-selective clinical compounds. Therefore, CDZ173 could differentiate by a more favorable safety profile. CDZ173 is currently in clinical studies in patients suffering from primary Sjögren's syndrome and in APDS/PASLI, a disease caused by gain-of-function mutations of PI3Kδ.
ABSTRACT
Drug metabolism studies are performed in drug discovery to identify metabolic soft spots, detect potentially toxic or reactive metabolites and provide an early insight into potential species differences. The relative peak area approach is often used to semi-quantitatively estimate the abundance of metabolites. Differences in the liquid chromatography-mass spectrometry responses result in an underestimation or overestimation of the metabolite and misinterpretation of results. The relative MS response factors (RF) of 132 structurally diverse drug candidates and their 233 corresponding metabolites were evaluated using a capillary-liquid chromatography/high-resolution mass spectrometry system. All of the synthesized metabolites discussed here were previously identified as key biotransformation products in discovery investigations or predicted to be formed. The most commonly occurring biotransformation mechanisms such as oxygenation, dealkylation and amide cleavage are represented within this dataset. However, relatively few phase II metabolites were evaluated because of the limited availability of authentic standards. Approximately 85% of these metabolites had a relative RF in the range between 0.2 (fivefold under-prediction) and 2.0 (twofold over-prediction), and the median MS RF was 0.6. Exceptions to this included very small metabolites that were hardly detectable. Additional experiments performed to understand the impact of the MS platform, flow rate and concentration suggested that these parameters do not have a significant impact on the RF of the compounds tested. This indicates that the use of relative peak areas to semi-quantitatively estimate the abundance of metabolites is justified in the drug discovery setting in order to guide medicinal chemistry efforts. Copyright © 2017 John Wiley & Sons, Ltd.
Subject(s)
Drug Discovery/methods , Pharmaceutical Preparations/metabolism , Biotransformation , Chromatography, High Pressure Liquid/methods , Databases, Pharmaceutical , Humans , Mass Spectrometry/methods , Metabolome , Pharmaceutical Preparations/chemistryABSTRACT
Inhibition of the lipid kinase PI3Kδ is a promising principle to treat B and T cell driven inflammatory diseases. Using a scaffold deconstruction-reconstruction strategy, we identified 4-aryl quinazolines that were optimized into potent PI3Kδ isoform selective analogues with good pharmacokinetic properties. With compound 11, we illustrate that biochemical PI3Kδ inhibition translates into modulation of isoform-dependent immune cell function (human, rat, and mouse). After oral administration of compound 11 to rats, proximal PD markers are inhibited, and dose-dependent efficacy in a mechanistic plaque forming cell assay could be demonstrated.
ABSTRACT
In vitro metabolic identification studies with a PI3K-α inhibitor lead molecule 1 identified a single predominant site of oxidative metabolism to be occurring within a tert.butyl moiety. Modification of the tert.butyl group within the lead molecule 1, to the corresponding d9-tert.butyl analogue 2, led to an increase in both the in vitro and in vivo metabolic stability. This increase in metabolic stability resulted in a 2-fold increase in the oral bioavailability measured in the rat, and a 3-fold increase in potency in a chronic in vivo study in the mouse, for 2 when compared to 1.
Subject(s)
Deuterium/metabolism , Enzyme Inhibitors/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Amides/chemistry , Animals , Biological Availability , Class I Phosphatidylinositol 3-Kinases , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacokinetics , Kinetics , Phosphoinositide-3 Kinase Inhibitors , Proline/chemistry , Rats , Thiazoles/chemistry , Urea/chemistryABSTRACT
Cancer Osaka thyroid (COT) kinase is an important regulator of pro-inflammatory cytokines in macrophages. Thus, pharmacologic inhibition of COT should be a valid approach to therapeutically intervene in the pathogenesis of macrophage-driven inflammatory diseases such as rheumatoid arthritis. We report the discovery and chemical optimization of a novel series of COT kinase inhibitors, with unprecedented nanomolar potency for the inhibition of TNFα. Pharmacological profiling in vivo revealed a high metabolism of these compounds in rats which was demonstrated to be predominantly attributed to aldehyde oxidase. Due to the very low activity of hepatic AO in the dog, the selected candidate 32 displayed significant blood exposure in dogs which resulted in a clear prevention of inflammation-driven lameness. Taken together, the described compounds both potently and selectively inhibit COT kinase in primary human cells and ameliorate inflammatory pathologies in vivo, supporting the notion that COT is an appropriate therapeutic target for inflammatory diseases.
Subject(s)
Drug Discovery , Imidazoles/pharmacology , MAP Kinase Kinase Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Quinolines/pharmacology , Animals , Crystallography, X-Ray , Dogs , Dose-Response Relationship, Drug , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , MAP Kinase Kinase Kinases/metabolism , Male , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins/metabolism , Quinolines/chemical synthesis , Quinolines/chemistry , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
Design and optimization of benzo- and pyrido-thiazoles/isothiazoles are reported leading to the discovery of the potent, orally bioavailable Syk inhibitor 5, which was found to be active in a rat PK/PD model. Compound 5 showed acceptable overall kinase selectivity. However, in addition to Syk it also inhibited Aurora kinase in enzymatic and cellular settings leading to findings in the micronucleus assay. As a consequence, compound 5 was not further pursued.
Subject(s)
Disease Models, Animal , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Thiazoles/pharmacology , Administration, Oral , Animals , Biological Availability , Dose-Response Relationship, Drug , Intracellular Signaling Peptides and Proteins/metabolism , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein-Tyrosine Kinases/metabolism , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Structure-Activity Relationship , Syk Kinase , Thiazoles/administration & dosage , Thiazoles/chemistryABSTRACT
A cyclisation within a 4',5-bisthiazole (S)-proline-amide-urea series of selective PI3Kα inhibitors led to a novel 4,5-dihydrobenzo[1,2-d:3,4-d]bisthiazole tricyclic sub-series. The synthesis and optimisation of this 4,5-dihydrobenzo[1,2-d:3,4-d]bisthiazole sub-series and the expansion to a related tricyclic 4,5-dihydrothiazolo[4,5-h]quinazoline sub-series are described. From this work analogues including 11, 12, 19 and 23 were identified as potent and selective PI3Kα inhibitor in vivo tool compounds.
Subject(s)
Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Quinazolines/chemistry , Quinazolines/pharmacology , Thiazoles/chemistry , Thiazoles/pharmacology , Animals , Caco-2 Cells , Class I Phosphatidylinositol 3-Kinases , Female , Humans , Mice, Nude , Models, Molecular , Neoplasms/drug therapy , Neoplasms/enzymology , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Quinazolines/pharmacokinetics , Quinazolines/therapeutic use , Structure-Activity Relationship , Thiazoles/pharmacokinetics , Thiazoles/therapeutic useABSTRACT
LC-MS based drug metabolism studies are effective in the optimization stage of drug discovery for rapid partial structure identification of metabolites. However, these studies usually do not provide unambiguous structural characterization of all metabolites, due to the limitations of MS-based structure identification. LC-MS-SPE-NMR is a technique that allows complete structure identification, but is difficult to apply to complex in vivo samples (such as bile collected during in vivo drug metabolism studies) due to the presence, at high concentrations, of interfering endogenous components, and potentially also dosage excipient components (e.g. polyethylene glycols). Here, we describe the isolation and structure characterization of seven metabolites of the drug development candidate 1-isopropyl-4-(4-isopropylphenyl)-6-(prop-2-yn-1-yloxy) quinazolin-2(1H)-one from a routine metabolism study in a bile-duct cannulated rat by LC-MS-SPE. The metabolites were isolated from bile and urine by repeated automatic trapping of the chromatographic peak of each metabolite on separate Oasis HLB SPE columns. The micropreparative HPLC/MS was performed on an XBridge BEH130 C18 HPLC column using aqueous formic acid/acetonitrile/methanol as mobile phase for the gradient elution. Mass spectrometric detection was performed on a LTQ XL linear ion trap mass spectrometer using electrospray ionization. Desorption of each metabolite was performed after the separation sequence. NMR spectra ((1)H, (13)C, 2D ROESY, HSQC and HMBC were measured on a Bruker AVANCE III spectrometer (600 MHz proton frequency) equipped with a 1.7 mm (1)H{(13)C,(15)N} Bruker Biospin's TCI MicroCryoProbe™.
Subject(s)
Bile/metabolism , Quinazolines/chemistry , Quinazolines/metabolism , Animals , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Male , Mass Spectrometry , Molecular Structure , Quinazolines/isolation & purification , Quinazolines/urine , Rats , Rats, Sprague-Dawley , Solid Phase ExtractionABSTRACT
We describe the discovery of selective and potent Syk inhibitor 11, which exhibited favorable PK profiles in rat and dog and was found to be active in a collagen-induced arthritis model in rats. Compound 11 was selected for further profiling, but, unfortunately, in GLP toxicological studies it showed liver findings in rat and dog. Nevertheless, 11 could become a valuable tool compound to investigate the rich biology of Syk in vitro and in vivo.
Subject(s)
Arthritis, Experimental/drug therapy , Drug Discovery , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Arthritis, Experimental/chemically induced , Collagen , Disease Models, Animal , Dogs , Dose-Response Relationship, Drug , Female , Humans , Intracellular Signaling Peptides and Proteins/blood , Intracellular Signaling Peptides and Proteins/metabolism , Liver/drug effects , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Conformation , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/chemistry , Protein-Tyrosine Kinases/blood , Protein-Tyrosine Kinases/metabolism , Rats , Rats, Inbred Lew , Structure-Activity Relationship , Syk KinaseABSTRACT
We describe two series of Syk inhibitors which potently abrogate Syk kinase function in enzymatic assays, cellular assays and in primary cells in the presence of blood. Introduction of a 7-aminoindole substituent led to derivatives with good kinase selectivity and little or no hERG channel inhibition (3b, 10c).
Subject(s)
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/blood , Protein Kinase Inhibitors/blood , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/blood , Humans , Indoles/blood , Indoles/chemistry , Indoles/pharmacology , Protein Kinase Inhibitors/chemistry , Syk KinaseABSTRACT
Phosphatidylinositol-3-kinase α (PI3Kα) is a therapeutic target of high interest in anticancer drug research. On the basis of a binding model rationalizing the high selectivity and potency of a particular series of 2-aminothiazole compounds in inhibiting PI3Kα, a medicinal chemistry program has led to the discovery of the clinical candidate NVP-BYL719.
Subject(s)
Drug Discovery , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Thiazoles/pharmacology , Animals , Biological Availability , Cell Line , Dogs , Dose-Response Relationship, Drug , Female , Humans , Mice , Models, Molecular , Molecular Structure , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/chemistryABSTRACT
A novel series of agonists at the benzodiazepine binding site of the GABA(A) receptor was prepared by functionalizing a known template. Adding substituents to the pyrazolone-oxygen of CGS-9896 led to a number of compounds with selectivities for either α2- or α1-containing GABA(A) receptor subtypes offering an entry into indications such as anxiety and insomnia. In this communication, structure-activity relationship and efforts to increase in vitro stabilities are discussed.
Subject(s)
Benzodiazepines/chemistry , GABA-A Receptor Agonists/chemical synthesis , Pyrazoles/chemistry , Receptors, GABA-A , Benzodiazepines/metabolism , Binding Sites , Drug Stability , GABA-A Receptor Agonists/chemistry , GABA-A Receptor Agonists/pharmacology , Inhibitory Concentration 50 , Ligands , Molecular Structure , Pyrazoles/metabolism , Receptors, GABA-A/metabolism , Structure-Activity RelationshipABSTRACT
A focused chemical optimization effort of compound 1 based on metabolite elucidation is described, resulting in 15i, a highly potent and selective mGlu5 receptor antagonist with an improved pharmacokinetic profile compared to 1. Characterization of 15i in vivo in the fear-potentiated startle (FPS) paradigm revealed a robust reduction of conditioned fear behavior. This effect nicely correlates with the rat brain pharmacokinetics.