Pharmacology of A-216546: a highly selective antagonist for endothelin ET(A) receptor.

Endothelins, 21-amino acid peptides involved in the pathogenesis of various diseases, bind to endothelin ET(A) and ET(B) receptors to initiate their effects. Here, we characterize the pharmacology of A-216546 ([2S-(2,2-dimethylpentyl)-4S-(7-methoxy-1,3-benzodioxol-5-yl )-1-(N,N-di(n-butyl) aminocarbonylmethyl)-pyrrolidine-3R-carboxylic acid), a potent antagonist with > 25,000-fold selectivity for the endothelin ET(A) receptor. A-216546 inhibited [125I]endothelin-1 binding to cloned human endothelin ET(A) and ET(B) receptors competitively with Ki of 0.46 and 13,000 nM, and blocked endothelin-1-induced arachidonic acid release and phosphatidylinositol hydrolysis with IC50 of 0.59 and 3 nM, respectively. In isolated vessels, A-216546 inhibited endothelin ET(A) receptor-mediated endothelin-1-induced vasoconstriction, and endothelin ET(B) receptor-mediated sarafotoxin 6c-induced vasoconstriction with pA2 of 8.29 and 4.57, respectively. A-216546 was orally available in rat, dog and monkey. In vivo, A-216546 dose-dependently blocked endothelin-1-induced pressor response in conscious rats. Maximal inhibition remained constant for at least 8 h after dosing. In conclusion, A-216546 is a potent, highly endothelin ET(A) receptor-selective and orally available antagonist, and will be useful for treating endothelin-1-mediated diseases.

2,4-Diarylpyrrolidine-3-carboxylic acids--potent ETA selective endothelin receptor antagonists. 1. Discovery of A-127722.

We have discovered a novel class of endothelin (ET) receptor antagonists through pharmacophore analysis of the existing non-peptide ET antagonists. On the basis of this analysis, we determined that a pyrrolidine ring might replace the indian ring in SB 209670. The resultant compounds were readily prepared and amenable to extensive SAR studies. Thus a series of N-substituted trans,trans-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)pyrroli din e-3- carboxylic acids (8) have been synthesized and evaluated for binding at ET(A) and ET(B) receptors. Compounds with N-acyl and simple N-alkyl substituents had weak activity. Compounds with N-alkyl substituents containing ethers, sulfoxides, or sulfones showed increased activity. Much improved activity resulted from compounds where the N-substituents were acetamides. Compound 17u (A-127722) with the N,N-dibutylacetamide substituent is the best of the series. It has an IC(50)=0.36 nM for inhibition of ET-1 radioligand binding at the ET(A) receptor, with a 1000-fold selectivity for the ET(A) vs the ET(B) receptor. It is also a potent inhibitor (IC(50)=0.16 nM) of phosphoinositol hydrolysis stimulated by ET-1, and it antagonized the ET-1-induced contraction of the rabbit aorta with a pA(2)=9.20. The compound has 70% oral bioavailability in rats.

Pharmacological characterization of A-127722: an orally active and highly potent ETA-selective receptor antagonist.

Endothelins (ET) are potent vasoactive peptides implicated in the pathogenesis of a number of vascular diseases. The effects of ET on mammalian organs and cells are initiated by binding to ETA or ETB receptors. In this report, we document the pharmacology of A-127722, a novel ETA-selective receptor antagonist. A-127722 inhibits [125I]ET-1 binding to cloned human ETA and ETB receptors competitively with Ki values of 69 pM and 139 nM, respectively. A-127722 exhibits a dose-dependent inhibition of ET-1-induced arachidonic acid release in human pericardium smooth muscle cells with a pA2 value of 10.5 and inhibits ET-1-induced vasoconstriction in isolated rat aorta with a pA2 value of 9.2. In vivo, A-127722 dose-dependently blocks the pressor response to ET-1 (0.3 nmol/kg i.v.) in conscious rats. Statistically significant (P < .05) antagonism is seen at doses greater than 0.1 mg/kg p.o. Maximal inhibition, at 10 mg/kg, remains constant for at least 8 hr after dosing. No effect is seen on the ETB-mediated transient vasodepressor effect of exogenous ET-1. In conclusion, A-127722 is ETA-selective, orally bioavailable and efficacious for inhibiting the effects of ET in the rat, and A-127722 is the most potent ET receptor antagonist yet reported.

In vivo characterization of a phosphoramidon-sensitive endothelin-converting enzyme in the rat.

Experiments were conducted to characterize the nature of a phosphoramidon-sensitive endothelin-converting enzyme in vivo by evaluating the pressor response to a bolus intravenous (i.v.) injection of endothelin family peptides following administration of phosphoramidon in anesthetized Sprague-Dawley rats. Phosphoramidon given i.v. at 10 mg/kg completely prevented the pressor response to human big endothelin-1-(1-38) (big ET-1). The EC50 for phosphoramidon was determined to be in the range of 1 to 3 mg/kg. The pressor response to big ET-1 60 min after phosphoramidon injection was attenuated by roughly 60% indicating a long inhibitory half-life. Very high doses of big ET-1 (> 20 mg/kg) were capable of over-riding the effect of phosphoramidon and produced characteristic pressor responses suggesting that the inhibition by phosphoramidon can be considered competitive in nature. Human big endothelin-3-(1-41) (big ET-3) produced significant increases in arterial pressure although with less potency and efficacy compared to big ET-1. The pressor response to big ET-3 was also inhibited by phosphoramidon. Phosphoramidon does not act indirectly by interfering with ET-1 receptor-mediated actions since the inhibitor has no effect on the in vivo pressor response to ET-1 and does not antagonize [125I]ET-1 receptor binding or constrictor responses in vitro. These results are consistent with the idea that a phosphoramidon-sensitive endothelin-converting enzyme is capable of cleaving both big ET-1 and big ET-3 to the active peptides in the rat.

Atrial natriuretic factor and endothelin interactions in control of vascular tone.

Endothelin-1 (ET-1) is reported to be the most powerful constrictor of blood vessels known. Atrial natriuretic factor is a potent relaxor of contracted vessels. This study examined the potential interaction between these vasoactive peptides on rabbit aortic rings. ET-1 contracted the rings in a dose-dependent manner with an EC50 (-log M) of 9.78 +/- 0.16. Atriopeptin III (APIII) completely relaxed ET-1 (10(-9) M)-contracted rings with an EC50 of 9.63 +/- 0.07 and methoxamine contracted rings with an EC50 of 9.18 +/- 0.09. Pretreatment of aortic rings with APIII (up to 3 x 10(-6) M) did not alter the normal ET-1 dose-response curve with respect to potency but did diminish the maximal contraction achieved. An interesting additional finding was that the temporal nature of ET-1-induced contraction is remarkably dose-variable. In conclusion, the potent contractile effects of ET-1 on vascular smooth muscle can be effectively reversed but not prevented by APIII.

Atrial natriuretic peptide antagonists: biological evaluation and structural correlations.

A collection of analogues of atrial natriuretic peptide (ANP) were screened for their ability to inhibit ANP-induced cGMP stimulation. The antagonists revealed through this screen are structurally related; almost all are substituted at either aspartate-13 or phenylalanine-26. This tendency is consistent throughout several families of small ANP analogues, suggesting that these two amino acid residues are involved in the process of ANP/cGMP signal transduction. One compound, A74186, was studied in some detail. A74186 is a potent inhibitor of the activation of guanylate cyclase by ANP; it acts with a pA2 of 7.12 in rat vascular smooth muscle cells and shifts the ANP/cGMP dose-response curve by 3 orders of magnitude at a 10 microM concentration. It also inhibits cGMP release in vivo, and at an infusion rate of 10 micrograms/kg-min it completely abolishes ANP-induced natriuresis and diuresis. A74186 does not, however, antagonize the hypotensive or vasorelaxant effects of ANP; in fact it acts as an agonist in these assays. It thus appears that cGMP, although it may mediate the renal responses to ANP, is not responsible for the vascular and hemodynamic effects that result from the action of the hormone.

Atrial natriuretic peptide (ANP) does not inhibit the basal vascular tone present in the in situ blood-perfused dog gracilis muscle.

This study evaluated the effects of rat ANP(5-28) infusion into the blood-perfused dog gracilis muscle at concentrations ranging from 30 to 10,000 pg/ml. The vasculature of gracilis muscles from anesthetized beagle dogs was isolated and pump-perfused at constant flow with blood utilizing an extracorporeal circuit. Maximal vasodilatory capacity was determined by adenosine injection. ANP was infused into the arterial circuit to produce increasing arterial blood concentrations. Each infusion lasted 10 min. Systemic arterial pressure, central venous pressure, cardiac output and heart rate did not change during ANP infusion into the gracilis vasculature. ANP at arterial blood concentrations up to 10,000 pg/ml did not produce significant vasodilation although the vasculature showed pronounced vasodilation in response to adenosine. In vitro experiments showed that ANP had much less vasorelaxant activity in dog femoral artery and saphenous vein than in rabbit aorta. Therefore, rat ANP(5-28) at concentrations within and well above physiological and pharmacological ranges does not inhibit the basal vascular tone present in the innervated, blood-perfused dog gracilis muscle in situ.