ABSTRACT
Antagonism of the gonadotropin releasing hormone (GnRH) receptor has resulted in positive clinical results in reproductive tissue disorders such as endometriosis and prostate cancer. Following the recent discovery of orally active GnRH antagonists based on a 4-piperazinylbenzimidazole template, we sought to investigate the properties of heterocyclic isosteres of the benzimidazole template. We report here the synthesis and biological activity of eight novel scaffolds, including imidazopyridines, benzothiazoles and benzoxazoles. The 2-(4-tert-butylphenyl)-8-(piperazin-1-yl)imidazo[1,2-a]pyridine ring system was shown to have nanomolar binding potency at the human and rat GnRH receptors as well as functional antagonism in vitro. Additional structure-activity relationships within this series are reported along with a pharmacokinetic comparison to the benzimidazole-based lead molecule.
Subject(s)
Heterocyclic Compounds/chemical synthesis , Heterocyclic Compounds/pharmacology , Piperazines/chemical synthesis , Piperazines/pharmacology , Receptors, LHRH/antagonists & inhibitors , Animals , Biological Availability , Cells, Cultured , Half-Life , Heterocyclic Compounds/pharmacokinetics , Humans , Male , Piperazines/pharmacokinetics , Rats , Rats, Sprague-DawleyABSTRACT
A series of (hetero)arylpyrimidines agonists of the Wnt-beta-catenin cellular messaging system have been prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla. Selected compounds show minimal GSK-3beta inhibition indicating that the Wnt-beta-catenin agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphorylated beta-catenin formation in bone.
Subject(s)
Imidazoles/chemistry , Pyrimidines/chemistry , Wnt Proteins/metabolism , beta Catenin/metabolism , Animals , Bone Development/drug effects , Cell Line, Tumor , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred C57BL , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Recombinant Fusion Proteins/agonists , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Skull/metabolism , Wnt Proteins/agonists , Wnt Proteins/genetics , Wnt3 Protein , Wnt3A Protein , beta Catenin/agonistsABSTRACT
A high-throughput screening campaign to discover small molecule leads for the treatment of bone disorders concluded with the discovery of a compound with a 2-aminopyrimidine template that targeted the Wnt beta-catenin cellular messaging system. Hit-to-lead in vitro optimization for target activity and molecular properties led to the discovery of (1-(4-(naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine (5, WAY-262611). Compound 5 has excellent pharmacokinetic properties and showed a dose dependent increase in the trabecular bone formation rate in ovariectomized rats following oral administration.
Subject(s)
Osteogenesis/drug effects , Piperidines/pharmacology , Pyrimidines/pharmacology , Wnt Proteins/agonists , beta Catenin/agonists , Animals , Catalytic Domain , Cell Line, Tumor , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/chemistry , Glycogen Synthase Kinase 3 beta , Humans , Mice , Models, Molecular , Piperidines/administration & dosage , Piperidines/pharmacokinetics , Pyrimidines/administration & dosage , Pyrimidines/pharmacokinetics , Rats , Signal Transduction/drug effects , Wnt Proteins/metabolism , beta Catenin/metabolismABSTRACT
A previous report described the serum LH suppression pharmacology of the 2-phenyl-4-piperazinyl-benzimidazole N-ethyluracil GnRH receptor antagonist 1 following oral administration in rats. A series of small heterocycles were appended to the 2-(4-tert-butylphenyl)-4-piperazinyl-benzimidazole template in place of the N-ethyluracil. Two imidazole analogues, 32 and 41, were shown to possess substantial in vitro potency at the target receptor (hGnRH IC(50) = 7 and 18 nM, respectively) and aqueous solubility (55 and 100 microg/mL at pH 7.4, respectively). Both compounds had high oral bioavailability in rats and 32 was further examined in an orchidectomized rat model for serum LH suppression based on increased volume of distribution over 41. Serum LH levels trended lower in orchidectomized rats following oral administration of 32.
Subject(s)
Benzimidazoles/pharmacology , Piperazines/pharmacology , Receptors, LHRH/antagonists & inhibitors , Administration, Oral , Animals , Benzimidazoles/chemistry , Benzimidazoles/pharmacokinetics , Heterocyclic Compounds/chemical synthesis , Heterocyclic Compounds/chemistry , Luteinizing Hormone/blood , Models, Animal , Piperazines/chemistry , Piperazines/pharmacokinetics , Rats , Receptors, LHRH/metabolism , Structure-Activity Relationship , Time FactorsABSTRACT
A potent, highly insoluble, GnRH antagonist with a 2-phenyl-4-piperazinylbenzimidazole template and a quinoxaline-2,3-dione pharmacophore was modified to maintain GnRH antagonist activity and improve in vitro pharmaceutical properties. Structural changes to the quinoxaline-2,3-dione portion of the molecule resulted in several structures with improved properties and culminated in the discovery of 6-([4-[2-(4-tert-butylphenyl)-1H-benzimidazol-4-yl]piperazin-1-yl] methyl)quinoxaline (WAY-207024). The compound was shown to have excellent pharmacokinetic parameters and lowered rat plasma LH levels after oral administration.
Subject(s)
Benzimidazoles/chemical synthesis , Quinoxalines/chemical synthesis , Receptors, LHRH/antagonists & inhibitors , Administration, Oral , Animals , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Binding, Competitive , Biological Availability , Half-Life , Humans , In Vitro Techniques , Luteinizing Hormone/blood , Male , Microsomes, Liver/metabolism , Orchiectomy , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Quinoxalines/chemistry , Quinoxalines/pharmacology , Radioligand Assay , Rats , Structure-Activity RelationshipABSTRACT
Using a cell-based assay, we have identified a new series of Notch-sparing gamma-secretase inhibitors from HTS screening leads 2a and 2e. Lead optimization studies led to the discovery of analog 8e with improved gamma-secretase inhibitory potency and Notch-sparing selectivity.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Chemistry, Pharmaceutical/methods , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Receptors, Notch/metabolism , Alzheimer Disease/drug therapy , Amyloid/chemistry , Amyloid Precursor Protein Secretases/chemistry , Carbon/chemistry , Drug Design , Drug Evaluation, Preclinical , Humans , Models, Chemical , Receptors, Notch/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
Antagonism of the gonadotropin releasing hormone (GnRH) receptor has shown positive clinical results in numerous reproductive tissue disorders such as endometriosis, prostate cancer and others. Traditional therapy has been limited to peptide agonists and antagonists. Recently, small molecule GnRH antagonists have emerged as potentially new treatments. This article describes the discovery of 2-phenyl-4-piperazinylbenzimidazoles as small molecule GnRH antagonists with nanomolar potency in in vitro binding and functional assays, excellent bioavailability (rat %F>70) and demonstrated oral activity in a rat model having shown significant serum leuteinizing hormone (LH) suppression.
Subject(s)
Benzimidazoles/administration & dosage , Benzimidazoles/chemistry , Piperazines/chemistry , Receptors, LHRH/antagonists & inhibitors , Administration, Oral , Animals , Benzimidazoles/chemical synthesis , Cross-Linking Reagents/chemistry , Glycolates/chemistry , Humans , Luteinizing Hormone/blood , Male , Methylation , Molecular Structure , Piperazine , Rats , Rats, Sprague-Dawley , Receptors, LHRH/metabolism , Structure-Activity RelationshipABSTRACT
Estrogens play a critical role in the regulation of cellular proliferation, differentiation, and apoptosis. Evidence indicates that this regulation is mediated by a complex interface of direct control of gene expression (so-called "genomic action") and by regulation of cell-signaling/phosphorylation cascades (referred to as the "non-genomic", or "extranuclear" action). However, the mechanisms of the non-genomic action of estrogens are not well defined. We have recently described the identification of a novel scaffold protein termed MNAR (modulator of non-genomic action of estrogen receptor), that couples conventional steroid receptors with extranuclear signal transduction pathways, thus potentially providing additional and tissue- or cell-specific level of steroid hormone regulation of cell functions. We have demonstrated that the MNAR is required for ER alpha (ERa) interaction with p60(src) (Src), which leads to activation of Src/MAPK pathway. Our new data also suggest that activation of cSrc in response to E2 leads to MNAR phosphorylation, interaction with p85, and activation of the PI3 and Akt kinases. These data therefore suggest that MNAR acts as an important scaffold that integrates ERa action in regulation of important signaling pathways. ERa non-genomic action has been suggested to play a key role in estrogen-induced cardio-, neuro-, and osteo-protection. Therefore, evaluation of the molecular crosstalk between MNAR and ERa may lead to development of functionally selective ER modulators that can separate between beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS and the "detrimental", proliferative effects in reproductive tissues and organs.
Subject(s)
Estrogen Receptor alpha/metabolism , Estrogens/metabolism , Gene Expression Regulation, Neoplastic , Phosphatidylinositol 3-Kinases/metabolism , Trans-Activators/physiology , src-Family Kinases/metabolism , Catalytic Domain , Cell Line, Tumor , Cell Proliferation , Co-Repressor Proteins , Enzyme Activation , Humans , Phosphorylation , Proto-Oncogene Proteins pp60(c-src)/metabolism , Signal Transduction , Structure-Activity Relationship , Trans-Activators/metabolism , Transcription FactorsABSTRACT
We have identified novel estrogen receptor alpha (ERalpha) antagonists using both cell-based and computer-based virtual screening strategies. A mammalian two-hybrid screen was used to select compounds that disrupt the interaction between the ERalpha ligand binding domain (LBD) and the coactivator SRC-3. A virtual screen was designed to select compounds that fit onto the LxxLL peptide-binding surface of the receptor, based on the X-ray crystal structure of the ERalpha LBD complexed with a LxxLL peptide. All selected compounds effectively inhibited 17-beta-estradiol induced coactivator recruitment with potency ranging from nano-molar to micromolar. However, in contrast to classical ER antagonists, these novel inhibitors poorly displace estradiol in the ER-ligand competition assay. Nuclear magnetic resonance (NMR) suggested direct binding of these compounds to the receptors pre-complexed with estradiol and further demonstrated that no estradiol displacement occurred. Partial proteolytic enzyme digestion revealed that, when compared with 17-beta-estradiol- and 4 hydroxy-tamoxifen (4-OHT) bound receptors, at least one of these compounds might induce a unique receptor conformation. These small molecules may represent new classes of ER antagonists, and may have the potential to provide an alternative for the current anti-estrogen therapy.