In order to evaluate the in vivo pharmacology associated
In order to evaluate the in vivo pharmacology associated with EP receptor antagonism, we wished to discover antagonists presenting a higher degree of selectivity over the other prostanoid receptors. We also wanted to increase the brain–blood ratio since it is probable that centrally mediated EP agonism plays a role in certain types of pain. We now disclose the results of a SAR study directed toward the optimization of analogs of antagonist in terms of EP binding affinity, selectivity, functional activity, and pharmacokinetics. Human prostanoid receptor binding affinities were determined as described previously by Abramovitz et al. The functional antagonist properties of compounds were evaluated using a cell-based assay. An extensive SAR study was conducted on the structure of compound . Modifications made at the 3-thienyl position at best permitted preservation of the binding affinity for the EP receptor (). Surrogates for the central thiophene ring were identified since small lipophilic rings such as cyclopentene and pyrrole are tolerated (). However, no significant effects on selectivity were observed as a result of these structural modifications. In contrast, modifications at the 2-thienyl position proved more fruitful. Three classes of derivatives were identified with improved binding affinity, selectivity and/or pharmacokinetic profiles. First is a class of benzoic Syringin analogs (). The benzoic acid derivative and the nicotinic acid both show higher affinity for the EP receptor in the binding assay compared to the phenylacetic acid analog . This behavior is translated in the functional assay where antagonists and are 10- to 16-fold more potent than phenylacetic acid . Unfortunately, these two compounds are plagued with short half-lives in rats (<0.5h). The best representative of this class is picolinic acid derivative , which binds with high affinity to the human EP receptor (=6nM) and is shifted 45-fold to a of 280nM in the presence of 2% HSA. Picolinic acid is very potent in the functional assay with a of 1nM. The selectivity ratio of antagonist is at least 100-fold against the EP, EP, EP, DP, FP, and IP receptors and 25-fold versus the TP receptor. Antagonist binds with high affinity to the rat EP receptor (=12nM) and, in contrast to other benzoic acid analogs, shows good pharmacokinetics in the same species (=73%; =20μM at 4h, 10mg/kg P.O., =4h). The brain–blood ratio (0.1) is modest for this compound. A second class of analogs, the trifluoromethylketone hydrates was discovered by preparing carboxylic acid surrogates. For example, in the nicotinic acid series, the carboxylic acid moiety found in antagonist was replaced by various functional groups (). While substitution by a bis(trifluoromethyl)methanol () or a cyanohydrin () function was detrimental to binding affinity, equipotent ligands were obtained by replacing the carboxylic acid group by a 2,2,2-trifluoroethan-1-ol () or a trifluoromethylketone hydrate moiety (). This last analog binds with high affinity to the human EP receptor (=4nM), is not shifted in presence of 2% HSA (=5nM) and presents a selectivity greater than 100-fold against the EP, EP, EP, DP, FP, and IP receptors. Its selectivity versus the TP receptor is 55-fold. Compound is potent (=70nM) in the functional EP cell-based assay. Also, antagonist shows good pharmacokinetics in rats (=42%; =5.5μM at 6h, 20mg/kg P.O., =2h) and a higher brain–blood ratio of 1. Benzamide and sulfonamide analogs, such as compounds –, form the third class identified (). The benzamides and are less protein shifted and more selective than their carboxylic acid counterparts, but formulation difficulties due to insolubility and poor pharmacokinetics reduced their utility. Sulfonamide binds with high affinity to the human EP receptor (=8nM) and is shifted only 2-fold to a of 18nM in the presence of 2% HSA. The selectivity ratio of is at least 100-fold against the EP, EP, EP, DP, FP, and IP receptors and 50-fold versus the TP receptor. Compound is potent (=100nM) in the functional EP cell-based assay. Antagonist is modestly bioavailable in rats but presents an acceptable half-life (=9%; =0.4μM at 30min, 20mg/kg P.O., =2h) and a high brain–blood ratio of 7.