Identification of potent and selective oxytocin antagonists. Part 2: further investigation of benzofuran derivatives

The paper covers continuing efforts to discover novel, potent and selective oxytocin antagonists. Further benzofuran derivatives with potent oxytocin antagonist activity and good pharmacokinetic parameters are reported. Efforts to improve the in vivo activity of the series are described.     

Nonpeptide oxytocin antagonists: analogs of L-371,257 with improved potency.

Structure-activity studies on the oxytocin antagonist 1 (L-371,257; Ki = 9.3 nM) have led to the identification of a related series of compounds containing an ortho-trifluoroethoxyphenylacetyl core which are orally bioavailable and have significantly improved potency in vitro and in vivo, e.g., compound 8 (L-374,943; Ki = 1.4 nM).     

A Gly/Ala switch contributes to high affinity binding of benzoxazinone-based non-peptide oxytocin receptor antagonists

Non-peptide antagonists of the oxytocin receptor (OTR) have been developed to prevent pre-term labour. The benzoxazinone-based antagonists L-371,257 and L-372,662 display pronounced species-dependent pharmacology with respect to selectivity for the OTR over the V(1a) vasopressin receptor. Examination of receptor sequences from different species identified Ala(318) in helix 7 of the human OTR as a candidate discriminator required for high affinity binding. The mutant receptor [A318G]OTR was engineered and characterised using ligands representing many different chemical classes. Of all the ligands investigated, only the benzoxazinone-based antagonists had decreased affinity for [A318G]OTR. Molecular modelling revealed that Ala(318) provides a direct hydrophobic contact with a methoxy group of L-371,257 and L-372,662.     

Pharmacological chaperones for the oxytocin receptor increase oxytocin responsiveness in myometrial cells

Oxytocin is a potent uterotonic agent administered to nearly all patients during childbirth in the United States. Inadequate oxytocin response can necessitate Cesarean delivery or lead to uterine atony and postpartum hemorrhage. Thus, it may be clinically useful to identify patients at risk for poor oxytocin response and develop strategies to sensitize the uterus to oxytocin. Previously, we showed that the V281M variant in the oxytocin receptor (OXTR) gene impairs OXTR trafficking to the cell surface, leading to a decreased oxytocin response in cells. Here, we sought to identify pharmacological chaperones that increased oxytocin response in cells expressing WT or V281M OXTR. We screened nine small-molecule agonists and antagonists of the oxytocin/vasopressin receptor family and identified two, SR49059 and L371,257, that restored both OXTR trafficking and oxytocin response in HEK293T cells transfected with V281M OXTR. In hTERT-immortalized human myometrial cells, which endogenously express WT OXTR, treatment with SR49059 and L371,257 increased the amount of OXTR on the cell surface by two- to fourfold. Furthermore, SR49059 and L371,257 increased the endogenous oxytocin response in hTERT-immortalized human myometrial cells by 35% and induced robust oxytocin responses in primary myometrial cells obtained from patients at the time of Cesarean section. If future studies demonstrate that these pharmacological chaperones or related compounds function similarly in vivo, we propose that they could potentially be used to enhance clinical response to oxytocin.     

2,5-Diketopiperazines as potent and selective oxytocin antagonists 1: Identification, stereochemistry and initial SAR

This paper covers efforts to discover orally active potent and selective oxytocin antagonists. Screening pooled libraries identified a novel series of 2,5-diketopiperazine derivatives with antagonist activity at the human oxytocin receptor. We report the initial structure-activity relationship investigations and the determination of the stereochemistry of the most potent compounds.     

Discovery and optimization of highly ligand-efficient oxytocin receptor antagonists using structure-based drug design

A novel oxytocin antagonist was identified by ‘scaffold-hopping’ using Cresset FieldScreen molecular field similarity searching. A single cycle of optimization driven by an understanding of the key pharmacophoric elements required for activity led to the discovery of a potent, selective and highly ligand-efficient oxytocin receptor antagonist. Selectivity over vasopressin receptors was rationalized based on differences in the structure of the natural ligands.     

Downregulation of Oxytocin Receptor Decreases the Length of Projections Stimulated by Retinoic Acid in the U-87MG Cells

Oxytocin is a neuropeptide widely expressed in the brain. Oxytocin plays a role in both proliferation and differentiation of various cells. Previous studies have suggested that oxytocin could affect the morphology of neuronal cells, therefore the objective of the present study was to test whether (1) oxytocin receptor stimulation/inhibition by specific ligands may change cell morphology and gene expression of selected cytoskeletal proteins (2) oxytocin receptor silencing/knockdown may decrease the length of cell projections (3) oxytocin receptor knockdown may affect human glioblastoma U-87MG cell survival. We confirmed the stimulatory effect of retinoic acid (10 µM) and oxytocin (1 µM) on projection growth. The combination of retinoic acid (10 µM) and oxytocin receptor antagonist (L-371,257, 1 µM) decreased projections length. Contrary to our assumptions, oxytocin receptor silencing did not prevent stimulation of length of projection by retinoic acid. Retinoic acid’s and oxytocin’s stimulation of projections length was significantly blunted in U-87MG cells with oxytocin receptor knockdown. Cell viability was significantly decreased in U-87MG cells with oxytocin receptor knockdown. Significantly higher levels of mRNA for cytoskeletal proteins drebrin and vimentin were observed in response to oxytocin incubation for 48 h. The data obtained in the present study clearly show that oxytocin induces formation and elongation of cell projections in astrocyte-like U-87MG cells. The effect is mediated by oxytocin receptors and it is accompanied by an increase in gene expression of drebrin and vimentin. Thus, oxytocin receptor signaling, particularly in the glial cells, may play an important role in native cell life, differentiation processes, and tumor progression, as well.     

Oxytocin induces intracellular Ca2+ release in cardiac fibroblasts from neonatal rats

Pituitary neuropeptide oxytocin is increasingly recognised as a cardiovascular hormone, in addition to its many regulatory roles in other organ systems. Studies in atrial and ventricular myocytes from the neonatal and adult rats have identified synthesis of oxytocin and the expression of oxytocin receptors in these cells. In cardiac fibroblasts, the most populous non-myocyte cell type in mammalian heart, the oxytocin receptors have not been described before. In the present study, we have investigated the direct effects of oxytocin on intracellular Ca²⁺ dynamics in ventricular myocytes and fibroblasts from new born rats. In myocytes, oxytocin increased the frequency of spontaneous Ca²⁺ transients and decreased their amplitude. Our data suggest that oxytocin receptors are also present and functional in the majority of cardiac fibroblasts. We used selective oxytocin receptor inhibitor L-371,257 and a number of intracellular Ca ²⁺ release blockers to investigate the mechanism of oxytocin induced Ca²⁺ signalling in cardiac fibroblasts. Our findings suggest that oxytocin induces Ca²⁺ signals in cardiac fibroblasts by triggering endoplasmic reticulum Ca²⁺ release via inositol trisphosphate activated receptors. The functional significance of the oxytocin induced Ca²⁺ signalling in cardiac fibroblasts, especially for their activation into secretory active myofibroblasts, remains to be investigated.     

Oxytocin receptor blockade reduces acquisition but not retrieval of taste aversion and blunts responsiveness of amygdala neurons to an aversive stimulus

When gastrointestinal sickness induced by toxin injection is associated with exposure to novel food, the animal acquires a conditioned taste aversion (CTA). Malaise is accompanied by a surge in oxytocin release and in oxytocin neuronal activity; however, it is unclear whether oxytocin is a key facilitator of aversion or merely its marker. Herein we investigated whether blockade of the oxytocin receptor with the blood–brain barrier penetrant oxytocin receptor antagonist L-368,899 is detrimental for the acquisition and/or retrieval of lithium chloride (LiCl)-dependent CTA to a saccharin solution in mice. We also examined whether L-368,899 given prior to LiCl affects neuronal activity defined through c-Fos immunohistochemistry in select brain sites facilitating CTA acquisition. L-368,899 given prior to LiCl caused a 30% increase in saccharin solution intake in a two-bottle test, but when the antagonist was administered before the two-bottle test, it failed to diminish the retrieval of an existing CTA. LiCl administration increased c-Fos expression in the hypothalamic paraventricular and supraoptic nuclei, area postrema, nucleus of the solitary tract and basolateral and central (CNA) nuclei of the amygdala. L-368,899 injected before LiCl reduced the number of c-Fos positive CNA neurons and brought it down to levels similar to those observed in mice treated only with L-368,899. We conclude that oxytocin is one of the key components in acquisition of LiCl-induced CTA and the aversive response can be alleviated by the oxytocin receptor blockade. Oxytocin receptor antagonism blunts responsiveness of CNA to peripherally injected LiCl.     

Development of L-689,065 - the prototype of a new class of potent 5-lipoxygenase inhibitors

The combination of a 5-phenylpyridine substituent and a novel, substituted thiopyrano[2,3,4-c,d]indole ring system yields a potent and selective 5-lipoxygenase inhibitor, L-689,065.     

1,2-Disubstituted indole, azaindole and benzimidazole derivatives possessing amine moiety: a novel series of thrombin inhibitors

A novel series of 1,2-disubstituted indole, azaindole and benzimidazole derivatives possessing an amine moiety was identified as thrombin inhibitors. An indole with basic diamine moieties (12a) was the most potent thrombin inhibitor in the series with Kass= 197 x 10(6) L/mol.     

Benzoyl and cinnamoyl nitrogen mustard derivatives of benzoheterocyclic analogues of the tallimustine: synthesis and antitumour activity

A series of benzoyl and cinnamoyl nitrogen mustards tethered to different benzoheterocycles and to oligopyrroles structurally related to netropsin consisting of two pyrrole-amide units and terminating with an amidine moiety have been synthesised and a structure--activity relationship determined. Derivatives 3--10 have been evaluated for their sequence selective alkylating properties and cytotoxicity against human K562 leukaemia cells. They are 2- to 50-fold less cytotoxic than tallimustine, with compound 8 being the most potent member of this series. Among tallimustine isosters, the compounds with an indole 3 or benzothiophene 6 are 4-fold less cytotoxic than tallimustine, while the compounds with an N-methyl indole or benzofuran showed a 7- and 14-fold reduced cytotoxic potency, respectively. Our preliminary results indicate that these derivatives preferentially bind to AT-rich sequence with a sequence selectivity similar to tallimustine.     

Estrogen receptor ligands. Part 16: 2-Aryl indoles as highly subtype selective ligands for ERalpha

A novel class of indole ligands for estrogen receptor alpha have been discovered which exhibit potent affinity and high selectivity. Substitution of the bazedoxifene skeleton to the linker present in the HTS lead 1a provided 22b which was found to be 130-fold alpha-selective and acted as an antagonist of estradiol activity in uterine tissue and MCF-7 cancer cells.     

Structure-activity relationship investigations of a potent and selective benzodiazepine oxytocin antagonist

We have investigated the structure-activity relationships of the 1- and 3-substituents and replacements of the 5-phenyl group of GW405212X 1, a potent selective oxytocin antagonist. The effect of these modifications on oxytocin binding antagonism and on pharmacokinetic parameters is reported.     

Discovery of novel 3,5-disubstituted indole derivatives as potent inhibitors of Pim-1, Pim-2, and Pim-3 protein kinases

A series of novel 3,5-disubstituted indole derivatives as potent and selective inhibitors of all three members of the Pim kinase family is described. High throughput screen identified a pan-Pim kinase inhibitor with a promiscuous scaffold. Guided by structure-based drug design, SAR of the series afforded a highly selective indole chemotype that was further developed into a potent set of compounds against Pim-1, 2, and 3 (Pim-1 and Pim-3: IC(50)≤2nM and Pim-2: IC(50)≤100nM).     

The discovery and synthesis of novel adenosine receptor (A(2A)) antagonists

In high throughput screening of our file compounds, a novel structure 1 was identified as a potent A(2A) receptor antagonist with no selectivity over the A1 adenosine receptor. The structure-activity relationship investigation using 1 as a template lead to identification of a novel class of compounds as potent and selective antagonists of A(2A) adenosine receptor. Compound 26 was identified to be the most potent A(2A) receptor antagonist (Ki = 0.8 nM) with 100-fold selectivity over the A1 adenosine receptor.     

Synthesis and SAR of selective benzothiophene, benzofuran, and indole-based peroxisome proliferator-activated receptor delta agonists

Recent literature has suggested the benefit of selective PPARdelta agonists for the treatment of atherosclerosis and other disease states associated with the metabolic syndrome. Herein we report the synthesis and structure-activity relationships of a series of novel and selective PPARdelta agonists. Our search began with identification of a novel benzothiophene template which was modified by the addition of various thiazolyl, isoxazolyl, and benzyloxy-benzyl moieties. Further elucidation of the SAR led to the identification of benzofuran and indole based templates. During the course of our research, we discovered three new chemical templates with varying degrees of affinity and potency for PPARdelta versus the PPARalpha and PPARgamma subtypes.     

Inhibition of Excitatory Amino Acid-Stimulated Phosphoinositide Hydrolysis in the Neonatal Rat Hippocampus by 2-Amino-3-Phosphonopropionate

The effects of excitatory amino acid agonists and alpha-amino-omega-phosphonocarboxylic acid antagonists on phosphoinositide hydrolysis in hippocampal slices of the 7-day neonatal rat were examined. Significant stimulation of [3H]inositol monophosphate formation was observed with ibotenate, quisqualate, L-glutamate, L-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, L-homocysteate, and kainate. N-Methyl-D-aspartate had no effect. Of these agonists, ibotenate and quisqualate were the most potent and efficacious. Stimulations by ibotenate and quisqualate were partially inhibited by L-2-amino-4-phosphonobutyrate (10(-3) M), but this antagonist had no effect on L-glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, or kainate. At 10(-3) M, D,L-2-amino-3-phosphonopropionate completely inhibited ibotenate and quisqualate stimulations, partially inhibited L-glutamate stimulation, and had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-, kainate-, or carbachol-induced [3H]inositol monophosphate formation. Concentration-effect experiments showed D,L-2-amino-3-phosphonopropionate to be five times more potent as an antagonist of ibotenate-stimulated phosphoinositide hydrolysis than L-2-amino-4-phosphonobutyrate. Thus in the neonatal rat hippocampus, like in the adult rat brain, D,L-2-amino-3-phosphonopropionate is a selective and relatively potent inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis. Because this glutamate receptor is uniquely sensitive to D,L-2-amino-3-phosphonopropionate, these studies provide further pharmacological evidence for the existence of a novel excitatory amino acid receptor subtype that is coupled to phosphoinositide hydrolysis in brain.     

Identification of an indole series of prostaglandin D2 receptor antagonists

A novel indole series of PGD2 receptor (DP receptor) antagonists is presented. Optimization of this series led to the identification of potent and selective DP receptor antagonists. In particular, antagonists 35 and 36 were identified with Ki values of 2.6 and 1.8 nM, respectively. These two antagonists are also potent in a DP functional assay where they inhibit the PGD2 induced cAMP production in platelet rich plasma with IC50 values of 7.9 and 8.6 nM, respectively. The structure-activity relationships of this indole series of DP receptor antagonists will also be discussed.     

The discovery of GSK221149A: a potent and selective oxytocin antagonist

Optimisation of a series of oxazole diketopiperazines has led to the discovery of a very potent and selective oxytocin antagonist GSK221149A. GSK221149A has been shown to inhibit oxytocin-induced uterine contractions in the anaesthetised rat.