Alexander L. Johnson

Identification of angiotensin II receptor subtypes

We have demonstrated the existence of two distinct subtypes of the angiotensin II receptor in the rat adrenal gland using radioligand binding and tissue section autoradiography. The identification of the subtypes was made possible by the discovery of two structurally dissimilar, nonpeptide compounds, DuP 753 and EXP655, that show reciprocal selectivity for the two subtypes. In the rat adrenal cortex, DuP 753 inhibited 80% of the total AII binding with an IC50 value on the sensitive sites of 2 x 10(-8) M, while EXP655 displaced only 20%. In the rat adrenal medulla, EXP655 gave 90% inhibition of AII binding with an IC50 value of 3.0 x 10(-8) M, while DuP 753 was essentially inactive. The combination of the two compounds completely inhibited AII binding in both tissues.

Hypotensive action of DuP 753, an angiotensin II antagonist, in spontaneously hypertensive rats. Nonpeptide angiotensin II receptor antagonists: X.

In conscious 18-21-week-old spontaneously hypertensive rats, DuP 753, a nonpeptide angiotensin II receptor antagonist, given orally at 3 and 10 mg/kg or intravenously at 3, 10, and 30 mg/kg, reduced blood pressure dose dependently. It did not alter heart rate at these doses. At 10 mg/kg i.v., DuP 753 decreased blood pressure significantly for at least 24 hours, suggesting a long duration of the antihypertensive effect. Unlike saralasin, DuP 753 did not cause a transient increase in blood pressure. The acute antihypertensive efficacy of DuP 753 was greater than that of captopril. Our data indicate that, for captopril to reduce blood pressure to a similar extent as that of DuP 753, it would need to be supplemented by a diuretic. DuP 753 did not have an acute diuretic effect. Bilateral nephrectomy, but not inhibition of prostaglandin synthesis, abolished the antihypertensive effect of DuP 753, suggesting that the antihypertensive effect of DuP 753 is dependent on an active renin-angiotensin system. Furthermore, DuP 753 inhibited the pressor response to angiotensin II but not the responses to norepinephrine, vasopressin, and Bay K 8644 (a calcium agonist). As neither DuP 753 nor captopril decreased blood pressure acutely in Wistar-Kyoto normotensive rats, our results suggest that the renin-angiotensin system plays a significant role in the control of blood pressure in spontaneously hypertensive rats.

Discrimination of angiotensin II receptor subtypes by dithiothreitol

It was of interest to evaluate whether DTT has a disciminatory effect on these two types of AII receptor in rat adrenal cortex. In this study, rat isolated adrenal cortical microsomes were treated for 20 min with either 5 mM DTT

Nonpeptide angiotensin II receptor antagonists. Studies with EXP9270 and DuP 753.

A series of nonpeptide angiotensin II (Ang II) receptor antagonists was evaluated in rat adrenal cortical microsomes for their inhibitory effects on the specific binding of [3H]Ang II, in the isolated rabbit aorta bioassay for their functional antagonism of contractile response to Ang II, and in high renin, renal-hypertensive rats for their intravenous antihypertensive effects, expressed as IC50, pA2, and intravenous ED30, respectively. Highly significant linear correlations were found between ICM and pAj (r = −0.88), between IC50, and intravenous ED30 (r = 0.79), and between pA2 and intravenous EDM (r = −0.93). In both in vitro and in vivo functional assays, none of these antagonists exhibited agonistic effects. The orally active nonpeptide Ang II receptor antagonists EXP9270 and DuP 753 (oral ED30=3.6 and 0.59 mg/kg, respectively) were selected for further characterization. These antagonists exhibited selective and competitive Ang II antagonism in rabbit aorta and guinea pig ileum. In conscious normotensive rats, DuP 753 abolished the pressor response to saralasin, suggesting that the pressor effect of saralasin is attributed to its Ang II-like activity. In addition, DuP 753 also blocked the Ang II-induced drinking response and aldosterone release in rats. These results suggest that Ang II receptor blockade is the primary mechanism of the antihypertensive effect of these nonpeptide Ang II receptor antagonists. Further, the specificity and lack of partial agonistic effects of these molecules make them potentially useful physiological probes and therapeutic agents.

Nonpeptide angiotensin II receptor antagonists.

Although the most direct way to interfere with the renin-angiotensin system (RAS) is at the level of the angiotensin II (AII) receptor, the currently available AII receptor antagonists are peptides still retaining significant agonistic properties with the obvious drawbacks of limited stability and lack of oral activity. We have characterized simple N-benzylimidazoles as weak, but selective AII receptor antagonists with a competitive mode of action. Chemical modification of these early leads led to EXP6155 and EXP6803, which show approximately 10- and 100-fold higher affinity. Oral activity was obtained for EXP7711, and in particular for EXP9654. This class of compounds displaces 3H-AII from its specific binding sites in various tissues. They competitively antagonize AII-induced responses in various in vitro and in vivo preparations, but do not influence AII-induced responses to KCl, norepinephrine, and vasopressin, nor do they affect converting enzyme or renin. In high renin models of elevated blood pressure, such as the renal hypertensive rat and sodium-depleted dog, these substances produce a sustained decrease in arterial pressure without changing heart rate after intravenous and oral (EXP7711 and EXP9654) administration. None of these compounds showed agonistic activity in any of the above test systems. In conclusion, the nonpeptide structures described herein are selective and competitive AII receptor antagonists and add another dimension to the arsenal of drugs manipulating the RAS.

Non-peptide angiotensin II receptor antagonists. II . Pharmacology of S-8308 ☆

2-Butyl-4-chloro-1-(2-nitrobenzyl)imidazole-5-acetic acid, sodium salt (S-8308), inhibited the specific binding of labeled angiotensin II (AII) to its receptor sites in rat adrenal cortical microsomes and in cultured aortic smooth muscle cells with IC50S of 15 and 4.5 microM, respectively. In the presence of S-8308 (15 microM) the dissociation constant for AII was increased 2-fold and the total number of binding sites was unaltered. In a concentration-dependent manner S-8308 blocked the 45Ca2+ influx induced by AII (3 X 10(-8) M) in rat aortic rings (IC50 7 microM) and the contractile response in rabbit aorta was competitively inhibited (pA2 = 5.74). This agent was highly specific for AII: it showed no affinity for alpha 1-adrenoceptors or Ca2+ channels and in addition, it did not alter the contractile responses to norepinephrine (10(-7) M) or KCl (55 mM). In conscious renal artery-ligated rats, S-8308 (30 mg/kg i.v.) elicited a rapid decrease of mean arterial pressure with a duration of about 30 min. The results demonstrate that S-8308 is a weak, but specific and competitive, non-peptide antagonist of AII exerting its inhibitory action at the receptor level.

Nonpeptide angiotensin II receptor antagonists. IV. EXP6155 and EXP6803.

EXP6155 (2-n-butyl-l-[4-carboxybenzyl]-4-chloroimidazole-5-acetic acid) and EXP6803 (methyl 2-n-butyl-l-[4-(2-carfooxybenzamido)ben2yl]-4-chloroimidazole-5-acetate, sodium salt) are shown to be novel, nonpeptide, antihypertensive, specific angiotensin II receptor antagonists. In rabbit aorta, they competitively inhibited the contractile response to angiotensin II with pA2 values of 6.54 and 7.20 and did not alter the response to norepinephrine or KC1. In guinea pig ileum, both agents blocked the responses to angiotensin I and II and did not alter the responses to bradykinin and acetylcholine. A similar specific angiotensin II antagonism was shown in vivo in the spinal pithed rat model. In renal artery-ligated rats, a high renin hypertensive model, EXP6155 and EXP6803 given intravenously, decreased blood pressure with ED30 of 10 and 11 mg/kg, respectively. Both compounds did not alter blood pressure when given orally at 100 mg/ kg. Unlike saralasin, EXP6155 and EXP6803 given intravenously did not cause a transient increase in blood pressure in the renal artery-ligated and nonnotensive rats. Our results indicate that EXP6155 and EXP6803 are selective angiotensin II receptor antagonists and antihypertensive agents. Since neither compound had partial agonist activities or bradykinin potentiation effects, unlike the existing peptide angiotensin II receptor antagonists and angiotensin converting enzyme inhibitors, respectively, they may represent preferred probes for studying the physiological roles of angiotensin II.

The preparation of (perfluoroalkyl)imidazoles as nonpeptide angiotensin II receptor antagonists

Abstract A series of (perfluoroalkyl)imidazoles have been prepared and are potent angiotensin II antagonists. One of these compounds, DuP 532, 4-pentafluoroethyl-2-propyl-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-5-carboxylic acid, is a selective antagonist of angiotensin II which causes a marked and long-lasting drop in blood pressure when administered orally to a renal hypertensive rat.

Nonpeptide Angiotensin II Receptor Antagonists: A Novel Class of Antihypertensive Agents

The most direct approach to block the function of the renin-angiotensin system would be to antagonize angiotensin II (AII) at the level of its receptor. However, the AII receptor antagonists currently available, such as saralasin, are peptides which still retain agonistic activity and lack oral bioavailability. We have identified the N-benzylimidazoles, S-8307 and S-8308, as weak, but selective nonpeptide AII receptor antagonists. These initial leads were subsequently converted into more potent compounds, such as EXP6155, EXP6803 and EXP7711, while maintaining the selectivity. The compounds displace 3H-AII from its specific binding sites in adrenal cortical membranes and smooth muscle cells. They competitively inhibit the vasoconstrictor response to AII in various in vivo and in vitro preparations, but do not influence those to KCl, norepinephrine, and vasopressin. Converting enzyme and renin are not affected by these agents. In renal hypertensive rats and sodium-depleted dogs our compounds cause a sustained decrease in arterial pressure following intravenous and oral (EXP7711) administration, and are devoid of agonistic properties. Taken together, these nonpeptide structures are true competitive AII receptor antagonists and represent a new class of effective antihypertensive agents.

DuP 747: sar study☆

Abstract The results of a detailed study of the structure-activity relationships in the series of compounds represented by DuP 747 are described.