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).

Development of orally active oxytocin antagonists: studies on 1-(1-[4-[1-(2-methyl-1-oxidopyridin-3-ylmethyl)piperidin-4-yloxy]-2- methoxybenzoyl]piperidin-4-yl)-1,4-dihydrobenz[d][1,3]oxazin-2-one (L-372,662) and related pyridines.

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.

Cloning and expression of the rhesus monkey oxytocin receptor.

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.

Nonpeptide oxytocin antagonists: potent, orally bioavailable analogs of L-371,257 containing a 1-R-(pyridyl)ethyl ether terminus.

Structure-activity studies on the oxytocin antagonist 1 (L-371,257) have identified a new series of high affinity, receptor-selective OT antagonists in which the N-acetyl-4-piperidinyl ether terminus in 1 has been replaced with a 1-(aryl)ethoxy group.

Progress in the development of oxytocin antagonists for use in preterm labor.

From a targeted screening effort and medicinal chemistry program, L-368,899 was selected as the first orally-active oxytocin (OT) antagonist to enter clinical trials. In animal studies, L-368,899 was shown to be a potent and selective OT antagonist and was orally bioavailable in rats, dogs and chimpanzees. L-368,899 was further shown to be a potent OT antagonist in pregnant rhesus and to inhibit spontaneous nocturnal uterine contractions. In Phase I human studies, L-368,899 was generally well-tolerated given intravenously and showed significant plasma levels after oral administration. In addition, L-368,899 blocked OT-stimulated uterine activity in postpartum women with a potency similar to that in the pregnant rhesus monkey. More recently, another structural series has been pursued, represented by L-371,257 [1-(1-(4-(N-acetyl-4-piperidinyloxy)-2-methoxybenzoyl)pip eridin-4-yl)- 1,2-dihydro-4(H)-3,1-benzoxazin-2-one]. L-371,257 exhibits high affinity (Ki, 4.6 nM) for human uterine OT receptors with high selectivity vs. human vasopressin receptors. In rat tissues in vitro, L-371,257 is a potent and competitive OT antagonist (pA2, 8.4) and, in vivo, blocks OT-stimulated uterine activity given both i.v. and intraduodenally. L-371,257 highlights the promise of this novel structural class.

Identification of an orally active, nonpeptidyl oxytocin antagonist.

L-366,509, a member of a novel class of nonpeptidyl compounds, has been characterized as an orally active oxytocin (OT) antagonist. L-366,509 exhibits a moderate binding affinity (K(i) values, 370-780 nM) for the rat, rhesus and human uterine OT receptor. L-366,509 also binds to vasopressin receptor subtypes (arginine vasopressin-V1 and V2) with measurable affinity in rat (K(i) values, 25-30 microM) and primate (K(i) values, 2-6 microM) tissues. In rat uterine slices, L-366,509 inhibits (IC50 = 1.6 microM) the stimulation of phosphatidylinositol turnover induced by OT but not bradykinin. In the rat isolated uterus, L-366,509 is a competitive and reversible OT antagonist (pA2 = 7.32). In vivo, L-366,509 given i.v. (10 mg/kg) or intraduodenally (10-50 mg/kg) to rats causes a marked and long-lasting inhibition of OT-stimulated uterine activity. OT antagonist activity in a pregnant rhesus macaque (approximately day 135 gestation) is also observed with L-366,509 after i.v. or p.o. dosing. L-366,509 represents a prototype for a new chemical class of OT antagonists with significant p.o. bioavailability.

Radioligand binding studies reveal marked species differences in the vasopressin V1 receptor of rat, rhesus and human tissues.

The [3H]arginine-vasopressin ([3H]AVP) binding site in rat, rhesus and human liver and nonpregnant human uterus was characterized and contrasted. [3H]AVP bound with high affinity (Ki values, 0.2-0.6 nM) to preparations of all tissues studied. Competition binding studies using a series of compounds from three structural classes indicate a marked species difference between the rat and primate liver AVP-V1 site. This site in rhesus and human liver however, is essentially identical, indicating that the rhesus liver is an appropriate surrogate for human tissue. These studies also indicate that the AVP-V1 site of nonpregnant human uterus and human liver is equivalent.

In vitro pharmacological profile of a novel structural class of oxytocin antagonists.

A number of structurally novel cyclic hexapeptides have been characterized as potent and selective oxytocin (OT) antagonists in vitro. As a representative of this class of compounds, L-366,948 [[cyclo(L-prolyl-D-2-naphthylalanyl-L-isoleucyl-D-pipecolyl- L-pipecolyl-D- histidyl)]] exhibited a high binding affinity (Ki, low nanomolar) for OT receptors in rat (uterus and mammary) and primate (pregnant rhesus and human myometrium) tissue with a several hundred-fold binding selectivity vs. rat arginine vasopressin (AVP)-V1 (liver) and AVP-V2 (kidney medulla) receptors. In functional assays, L-366,948 was a pure OT antagonist, blocking both OT-stimulated contraction of the isolated rat uterus (pA2, 8.5) and phosphatidylinositol turnover in uterine slices (IC50, 40 vs. 3 nM OT), with no evidence of partial agonist activity. L-366,948 was comparatively weak as an antagonist of AVP-induced contraction of the isolated rat tail artery (AVP-V1 receptor) and AVP-stimulated adenylate cyclase (AVP-V2 receptor) activity in rat kidney medulla and did not influence prostaglandin F2 alpha- or bradykinin-induced contractions of the isolated rat uterus. L-366,948 and related compounds described in this report represent new experimental tools for the study of the pharmacology and physiology of OT.

Identification of functional oxytocin receptors in lactating rat mammary gland in vitro.

The oxytocin (OT) receptor of the lactating rat mammary gland was further characterized by radioligand binding and functional assays in vitro and compared to the uterine OT receptor. In equilibrium saturation binding studies, [3H]OT bound apparently to a single site in mammary tissue with an affinity (Kd = 0.98 nM) similar to that found in the uterus (Kd = 0.68 nM). Using a variety of ligands for OT and arginine vasopressin (AVP) receptors, binding studies indicated that the recognition profile of the mammary [3H]OT binding site closely resembled that found for the uterus but was distinct from the known AVP receptor subtypes. In functional studies, OT and the highly selective OT agonist, [Thr4,Gly7]OT, were potent activators of phosphatidylinositol (PI) turnover in both mammary and uterine slices (EC50 3-5 nM). L-365,209, a novel potent and selective OT antagonist, inhibited OT-stimulated PI turnover in both tissues with similar potencies. These data provide evidence that the high-affinity [3H]OT binding site found in rat mammary tissue during lactation is a functional OT receptor coupled to PI turnover.