L-368,899 [1S-((-7,7-dimethyl-2-endo-(2S-amino-4-(methylsulfonyl)bu~ramido)-bicyclo(2.2.1)-heptan-l-yl)methanesulfonyl)-4-(2-methylphenyl)piper~inel was characterized in vitro and in vivo as a potent and selective, orally bioavailable oxytocin (OT) antagonist. L-368,899 exhibits high affinity for rat (Ki, 3.6 nM) and human (Ki, 13 nM) uterine OT receptors with selectivity versus liver arginine-vasopressin (AVP)-VI and kidney AVP-V2 receptors in both species. In vitro, L-368,899 is a potent and competitive OT antagonist in the rat isolated uterus (pA2, 8.9) and mouse anococcygeus muscle (pA2, 8.3) and is inactive against a number of different contractile agents in these preparations. L-368,899 also inhibits OT-stimulated phosphatidylinositol turnover in rat uterine slices and exhibits no agonist-like activity in any of the in vitro assays. In vivo, L-368,899 selectively antagonizes the uterine contractile effects of OT in the anesthetized rat given both i.v. and intraduodenally (i.d.) (AD50; 350 Fg/kg i.v., 7 mg/kg i.d.) with a moderate duration of action. In near-term pregnant rhesus macaques, L-368,899 is a potent inhibitor of OT-stimulated uterine activity (AD50, 27 pg/kg i.v.) and OT-mediated spontaneous, nocturnal uterine contractions. L-368,899 is orally bioavailable in several species and, combined with adequate aqueous solubility, represents a potential new tocolytic agent for both oral and i.v. use. o 1993 WiIey-Liss, inc.

The previously reported oxytocin antagonist L-371,257 (2) has been modified at its acetylpiperidine terminus to incorporate various pyridine N-oxide groups. This modification has led to the identification of compounds with improved pharmacokinetics and excellent oral bioavailability. The pyridine N-oxide series is exemplified by L-372,662 (30), which possessed good potency in vitro (Ki = 4.1 nM, cloned human oxytocin receptor) and in vivo (intravenous AD50 = 0.71 mg/kg in the rat), excellent oral bioavailability (90% in the rat, 96% in the dog), good aqueous solubility (>8.5 mg/mL at pH 5.2) which should facilitate formulation for iv administration, and excellent selectivity against the human arginine vasopressin receptors. Incorporation of a 5-fluoro substituent on the central benzoyl ring of this class of oxytocin antagonists enhanced in vitro and in vivo potency but was detrimental to the pharmacokinetic profiles of these compounds. Although lipophilic substitution around the pyridine ring of compound 30 gave higher affinity in vitro, such substituents were a metabolic liability and caused shortfalls in vivo. Two approaches to prevent this metabolism, addition of a cyclic constraint and incorporation of trifluoromethyl groups, were examined. The former approach was ineffective because of metabolic hydroxylation on the constrained ring system, whereas the latter showed improvement in plasma pharmacokinetics in some cases.

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The oxytocin receptor belongs to the family of G-protein-coupled receptors (GPCRs) characterized by seven transmembrane spanning domains and mediates numerous neurotransmitter and hormonal functions. The cloning of this receptor was initiated to validate the use of the rhesus monkey (Macaca mulatta) as a viable animal model for therapeutic development of oxytocin receptor antagonists by ruling out potential species variations that are sometimes present among GPCRs. The rhesus monkey oxytocin receptor was cloned by the polymerase chain reaction (PCR) and expressed transiently in 293/EBNA cells. The cDNA encodes a protein of 389 amino acids and is highly homologous to that from other species, especially the human receptor which exhibits 97% identity to the rhesus protein. The cloned receptor shows a very similar pharmacological profile to the human oxytocin receptor for a variety of agonists and antagonists from various structural classes. These results substantiate the validity of the rhesus monkey as a useful model for the evaluation of human therapeutics.