M. A. Flynn

Summary: The effects of different amino acids incorporated into the 16 and 17 positions of the C-terminal hexapeptide of ET-1 were examined. Structure-activity relationships (SAR) of the ET receptor antagonists PD 142893 [Ac-(D-Dip16-L-Leu17-L-Asp-L-Ile-L-Ile-L-Trp) (D-Dip = 3,3-D-diphenylalanine)] and PD 145065 [Ac-(D-Bhg16-L-Leu17-L-Asp-L-Ile-L-Ile-L-Trp) (D-Bhg = 5H-dibenzyl[a,d]cycloheptene-10,11-dihydro-glycine)] uncovered certain requirements for high potency. The disodium salt of PD 145065 has 4.0 and 15 nM binding affinity (IC50 values) for the ETA (rabbit renal artery vascular smoothmuscle cells) and ETB receptor (rat cerebellum), respectively. The compound is also an antagonist of ET-1- and SRTX-6c-stimulated vasoconstrictor activity, with pA2 values of 6.9 (rabbit femoral artery, ETA assay) and 7.1 (rabbit pulmonary artery, ETB assay). The tripeptidic ETA antagonist FR 139317 was found to be less active in the rabbit femoral artery, with a pA2 value of 6.0, and inactive in the rabbit pulmonary artery. Substitution of acidic and basic residues at position 17 in PD 142893 and PD 145065 indicates differences in selectivity. Incorporation of bulky non-natural amino acids at position 16 has led to potent nonselective analogues, including Ac-D-Bheg16-L-Leu-L-Asp-L-Ile-L-Ile-L-Trp [D-Bheg (5H-dibenzo[a,d]cycloheptene glycine)]. The in vivo effects of single-bolus doses of selected ET antagonists on depressor and pressor responses to ET-1 in anesthetized ganglion-blocked rats were evaluated. The disodium salt of PD 145065 dose dependently inhibited the depressor and pressor responses induced by ET-1 infusion, whereas PD 142893 inhibited only the depressor component up to 10 μmol/kg. Duration of action studies were carried out with PD 142893, PD 145065, and FR 139317 in conscious, chronically prepared, normotensive rats over 5 days. Five groups of animals were dosed with ET-1 (1.0 nmol/kg) at 5, 20, 60, and 120 min after administration of the antagonist (10 μmol/kg i.v. bolus). The results indicate that FR 139317 has a very short duration of action on the pressor component, whereas PD 142893 and PD 145065 both showed some blockade of the depressor component of the ET challenge at 2 h postdose.

The design of potent and selective non-peptide antagonists of endothelin-1 (ET-1) and its related isopeptides are important tools defining the role of ET in human diseases. In this report we will describe the detailed structure-activity relationship (SAR) studies that led to the discovery of a potent series of butenolide ETA selective antagonists. Starting from a micromolar screening hit, PD012527, use of Topliss decision tree analysis led to the discovery of the nanomolar ET(A) selective antagonist PD155080. Further structural modifications around the butenolide ring led directly to the subnanomolar ETA selective antagonist PD156707, IC50's = 0.3 (ET(A)) and 780 nM (ET(B)). This series of compounds exhibited functional activity exemplified by PD156707. This derivative inhibited the ETA receptor mediated release of arachidonic acid from rabbit renal artery vascular smooth muscle cells with an IC50 = 1.1 nM and also inhibited the ET-1 induced contraction of rabbit femoral artery rings (ETA mediated) with a pA2 = 7.6. PD156707 also displayed in vivo functional activity inhibiting the hemodynamic responses due to exogenous administration of ET-1 in rats in a dose dependent fashion. Evidence for the pH dependence of the open and closed tautomerization forms of PD156707 was demonstrated by an NMR study. X-ray crystallographic analysis of the closed butenolide form of PD156707 shows the benzylic group located on the same side of the butenolide ring as the gamma-hydroxyl and the remaining two phenyl groups on the butenolide ring essentially orthogonal to the butenolide ring. Pharmacokinetic parameters for PD156707 in dogs are also presented.