ABSTRACT
Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A K(d) = 7.5 nM, Aurora-B K(d) = 48 nM), FLT3 kinase (K(d) = 6.2 nM), and FLT3 mutants including FLT3-ITD (K(d) = 38 nM) and FLT3(D835Y) (K(d) = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20-35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4-11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children.
Subject(s)
Antineoplastic Agents/chemical synthesis , Imidazoles/chemical synthesis , Leukemia, Myeloid, Acute/drug therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Purines/chemical synthesis , Pyrazoles/chemical synthesis , Pyridines/chemical synthesis , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Administration, Oral , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Aurora Kinase A , Aurora Kinase B , Aurora Kinases , Biological Availability , Cell Line, Tumor , Drug Screening Assays, Antitumor , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Female , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Male , Mice , Mice, Inbred BALB C , Microsomes, Liver/metabolism , Models, Molecular , Mutation , Neoplasm Transplantation , Purines/chemistry , Purines/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Transplantation, Heterologous , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in the regulation of Ca(2+) release from inositol 1,4,5-triphosphate-sensitive stores. U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) has been extensively used as a pharmacological inhibitor of PLC to elucidate the importance of this enzyme family in signal transduction pathways. U73122 has an electrophilic maleimide group, which readily reacts with nucleophiles such as thiols and amines. In the current study the conjugation of U73122 to common components of cell culture medium, namely l-glutamine, glutathione, and bovine serum albumin (BSA), was demonstrated. The half-life of U73122 on incubation with phosphate-buffered saline (PBS), Hanks' buffered saline solution (with 2 mM glutamine), optimized basal nutrient medium (MCDB131, without BSA), complete medium, Dulbecco's modified Eagle's medium (with 2 mM l-glutamine) was approximately 150, 60, 32, 30, and 18 min, respectively. However, U73122 was not recoverable from medium supplemented with 0.5% BSA. U73122 underwent hydrolysis of the maleimide group when incubated with PBS. Glutamine conjugates of U73122 were identified in cell culture medium. Furthermore, the inhibition of epidermal growth factor-stimulated Ca(2+) release in a human epidermoid carcinoma cell line (A431) by U73122 was substantially reduced by the presence of BSA in a time-dependent manner. In complex cellular assays, the availability of U73122 to inhibit PLC may be limited by its chemical reactivity and lead to the misinterpretation of results in pharmacological assays.
Subject(s)
Artifacts , Biological Assay , Calcium Signaling/drug effects , Culture Media/chemistry , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Pyrrolidinones/pharmacology , Type C Phospholipases/antagonists & inhibitors , Cell Line, Tumor , Culture Media/metabolism , Drug Stability , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Epidermal Growth Factor/pharmacology , Estrenes/chemistry , Estrenes/metabolism , Glutamine/chemistry , Glutathione/chemistry , Half-Life , Humans , Protein Binding , Pyrrolidinones/chemistry , Pyrrolidinones/metabolism , Serum Albumin, Bovine/chemistry , Time FactorsABSTRACT
Five 1-benzazepine heterocycles were synthesized by utilizing transition-metal-catalyzed processes in key bond-forming steps. exo-Methylene and methyl substituents were introduced at position 5, as well as a unit of unsaturation between positions 3 and 4, with benzoyl or benzyl N-substituents. Solution- and solid-state structures were examined, using dynamic NMR spectroscopy and X-ray crystallography, corroborated by molecular mechanics calculations. Greater amide distortion is associated with a more stable ground-state structure, which is in turn more reluctant to undergo conformational changes.
Subject(s)
Amides/chemistry , Benzazepines/chemistry , Heterocyclic Compounds, 2-Ring/chemistry , Benzazepines/chemical synthesis , Crystallography, X-Ray , Cyclization , Heterocyclic Compounds, 2-Ring/chemical synthesis , Models, Chemical , Molecular Conformation , Structure-Activity RelationshipABSTRACT
Partially and fully reduced forms of benzo-fused eight- to ten-membered nitrogen heterocycles (1-benzazecines, 1-benzazonines and 1-benzazecines) have been prepared. Conformational features, transannular distances and dynamic behavior were studied using X-ray crystallography and VT NMR spectroscopy. The amide moiety in the nine-membered benzazonine ring 5b favors N-pyramidization, whereas the ten-membered benzazecine 5c adopts an amide twist. Molecular mechanics calculations reveals a correlation between the amide twist (tau) and ring stability. The dynamic behavior of the heterocycles in solution were also found to be dependent on the extent and nature of the amide distortion. We thus conclude that ring strain of these medium-sized heterocyclic rings is relieved through amide distortion, which leads to a more stable structure.
