Structural modifications requiring novel synthetic chemistry were made to the morpholine acetal human neurokinin-1 (hNK-1) receptor antagonist 4, and this resulted in the discovery of 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-ox o-1 ,2,4-triazol-5-yl)methyl morpholine (17). This modified compound is a potent, long-acting hNK-1 receptor antagonist as evidenced by its ability to displace [125I]Substance P from hNK-1 receptors stably expressed in CHO cells (IC50 = 0.09 +/- 0.06 nM) and by the measurement of the rates of association (k1 = 2.8 +/- 1.1 x 10(8) M-1 min-1) and dissociation (k-1 = 0.0054 +/- 0.003 min-1) of 17 from hNK-1 expressed in Sf9 membranes which yields Kd = 19 +/- 12 pM and a t1/2 for receptor occupancy equal to 154 +/- 75 min. Inflammation in the guinea pig induced by a resiniferatoxin challenge (with NK-1 receptor activation mediating the subsequent increase in vascular permeability) is inhibited in a dose-dependent manner by the oral preadmininstration of 17 (IC50 (1 h) = 0.008 mg/kg; IC90 (24 h) = 1.8 mg/kg), indicating that this compound has good oral bioavailbility and peripheral duration of action. Central hNK-1 receptor stimulation is also inhibited by the systemic preadministration of 17 as shown by its ability to block an NK-1 agonist-induced foot tapping response in gerbils (IC50 (4 h) = 0.04 +/- 0.006 mg/kg; IC50 (24 h) = 0.33 +/- 0.017 mg/kg) and by its antiemetic actions in the ferret against cisplatin challenge. The activity of 17 at extended time points in these preclinical animal models sets it apart from earlier morpholine antagonists (such as 4), and the piperidine antagonists 2 and 3 and could prove to be an advantage in the treatment of chronic disorders related to the actions of Substance P. In part on the basis of these data, 17 has been identified as a potential clinical candidate for the treatment of peripheral pain, migraine, chemotherapy-induced emesis, and various psychiatric disorders.

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Glucagon is a 29-amino acid peptide that is an important counter-regulatory hormone in the control of glucose homeostasis (1). Glucagon secretion from the endocrine pancreas induces an increase in hepatic glycogenolysis and gluconeogenesis, and it attenuates the ability of insulin to inhibit these processes. As such, the overall rates of hepatic glucose synthesis and glycogen metabolism are controlled by the systemic ratio of insulin and glucagon (2, 3). Therefore, glucagon antagonists have the potential to improve hepatic insulin sensitivity and to be effective hypoglycemic agents.Peptidyl glucagon antagonists and their hypoglycemic activity were first described over 15 years ago, and an extensive exploration of the structure/activity relationships of these glucagon analogs has been reported (4 -6). The hepatic receptor for glucagon was cloned recently (7,8), confirming that it is a member of the seven-transmembrane domain, G-protein-coupled receptor superfamily. This receptor superfamily has a binding pocket for small-molecule ligands within the transmembrane domain that has made it possible to identify nonpeptidyl antagonists for many receptor families in which the endogenous ligands are small peptides or proteins (9). Thus, we initiated an effort to identify non-peptidyl, orally active antagonists for the human glucagon receptor.Collins et al. (10) have described a dichloroquinoxaline glucagon antagonist with weak affinity (IC 50 ϭ 4 M) for the rat glucagon receptor. However, there have been no subsequent reports in the patent or scientific literature describing the development of potent antagonists from this series. Our initial screening efforts identified a series of triarylimidazole and triarylpyrrole compounds with significant binding affinity for the human glucagon receptor, and efforts to evaluate the structure-activity relationships of this series have lead to the identification of potent glucagon antagonists (11). In the present article, we describe the identification and characterization of a potent glucagon antagonist from this series. MATERIALS AND METHODSCharacterization of Binding Affinity and Functional Activity-Stable CHO 1 cell lines or COS cells transiently expressing the human glucagon receptor were prepared as described previously (8, 12). Antagonist binding affinity was assessed by measuring inhibition of radiolabeled glucagon binding to CHO cell membranes. Briefly, 125 I-glucagon (58 pM) binding to the membrane preparation was measured in 20 mM Tris, pH 7.4, containing 1 mM dithiothreitol, 5 g/ml leupeptin, 10 g/ml benzamidine, 40 g/ml bacitracin, 5 g/ml soybean trypsin inhibitor, and 3 M o-phenanthroline Ϯ 1 M glucagon for 1 h at room temperature. Bound cpm were recovered by filtration using a Tomtec harvester and quantified in a ␥-scintillation counter.The ability of compound to inhibit glucagon-stimulated adenylyl cyclase was assessed as described previously (12). Briefly, cells were harvested from monolayers with enzyme-free cell dissociation solution (Specialty Media, Inc.) and were...

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