Subclasses of cyclic GMP-specific phosphodiesterase and their role in regulating the effects of atrial natriuretic factor.

Two subclasses of cyclic guanosine monophosphate (GMP)-specific phosphodiesterases were identified in vascular tissue from several beds. The activity of one subclass (phosphodiesterase IB) was stimulated severalfold by calmodulin and selectively inhibited by the phosphodiesterase inhibitor TCV-3B. The activity of the other subclass (phosphodiesterase IC) was not stimulated by calmodulin and was selectively inhibited by the phosphodiesterase inhibitor M&B 22,948. To assess the involvement of both subclasses in regulating cyclic GMP-dependent responses, the ability of TCV-3B and M&B 22,948 to potentiate the in vitro and in vivo responses to the endogenous guanylate cyclase stimulator atrial natriuretic factor (ANF) was evaluated. Both TCV-3B and M&B 22,948 relaxed isolated rabbit aortic and pulmonary artery rings and also potentiated the relaxant effect of ANF. In addition, both inhibitors produced small increases in urine flow and sodium excretion in anesthetized rats and potentiated the diuretic and natriuretic responses to exogenous ANF. M&B 22,948 (30 micrograms/kg/min) produced a threefold increase in the natriuretic response to simultaneously administered ANF, and TCV-3B (10 micrograms/kg/min) produced a twofold increase in the response to ANF. The results of the present experiments suggest that both the calmodulin-sensitive and calmodulin-insensitive subclasses of cyclic GMP-specific phosphodiesterase play a role in regulating the in vitro and in vivo response to ANF.

Synthesis and biological activity of 4-(diphenylmethyl)-alpha-[(4-quinolinyloxy)methyl]-1-piperazineethanol and related compounds.

A series of 4-(diphenylmethyl)-alpha-[(4-quinolinyloxy)methyl]-1-pipe razineethanol and closely related compounds was synthesized and evaluated for cardiac and vascular activity in isolated perfused rat and guinea pig hearts. Compound 1 produced greater inotropic effects in rat hearts than in guinea pig hearts, a phenomenon which was also observed with the prototype agent DPI 201-106. Compound 15 produced an inotropic effect with one-tenth the potency of compound 1. Both compounds 1 and 15 demonstrated direct inotropic and vasodilatory effects when administered iv in anesthetized dogs, although the vasodilatory activity was more pronounced with compound 15 than 1 and DPI compound. Compound 1 lacks the CN moiety which is a key structural requirement in DPI for positive inotropic activity. The synthesis, in vitro, and in vivo evaluations of these agents, and comparative data with DPI-201-106 (compound 17) are reported.

N6-(arylalkyl)adenosines. Identification of N6-(9-fluorenylmethyl)adenosine as a highly potent agonist for the adenosine A2 receptor.

Several N6-(arylalkyl)adenosines related to N6-benzyladenosine were synthesized, and their A1 and A2 adenosine receptor binding affinities were determined. The annulated derivative N6-(1-naphthylmethyl)adenosine resulted in a very potent A2 agonist (A1 Ki = 24 nM, A2 Ki = 9.1 nM), whereas N6-(9-anthracenylmethyl)adenosine was virtually inactive (A1 Ki = 9,000 nM, A2 Ki = 29,000 nM). Interestingly, the structurally similar N6-(9-fluorenylmethyl)adenosine was the most potent A2 agonist reported to date, with a Ki of 4.9 nM in A2 binding and 5.1 nM in A1 binding. The homologues N6-9-fluorenyladenosine and N6-[2-(9-fluorenyl)ethyl]adenosine showed little or no activity at either adenosine receptor. Effects of these agents on heart rate and coronary flow in the isolated rat heart paralleled their A1 and A2 binding affinities, respectively. These data suggest that for high affinity at the A2 receptor a planar hydrophobic function at a certain distance and angle from the N6 nitrogen is required.

2-(2-Aryl-2-oxoethylidene)-1,2,3,4-tetrahydropyridines. Novel isomers of 1,4-dihydropyridine calcium channel blockers.

The title compounds are novel double bond isomers of 1,4-dihydropyridine-type calcium channel blockers (CCB). These derivatives were prepared by using the Hantzsch dihydropyridine synthesis. The assignment of structure was based on spectroscopic data and a regiochemically unambiguous synthesis. Several of the analogues inhibited [3H]nitrendipine binding with IC50 values as low as 25 nM. By comparison, nifedipine, a clinically useful 1,4-dihydropyridine CCB, inhibits [3H]nitrendipine binding with an IC50 of 1.6 nM. In the Langendorff rat heart preparation, treatment with the more potent derivatives produced marked dose-related increases in coronary flow with little or no effect on heart rate or contractility, except at the highest concentrations tested. The selectivity for vascular versus cardiac effects was similar to that of nifedipine, i.e., the concentration producing vasodilation was approximately 2 orders of magnitude lower than the concentration eliciting cardiodepression. These novel isomers extend the structure-activity relationships for calcium channel blockers into a series closely related to the 1,4-dihydropyridines.

Design of a potent combined pseudopeptide endothelin-A/endothelin-B receptor antagonist, Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21 (PD 156252): examination of its pharmacokinetic and spectral properties.

The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and prolonged vasoconstrictors. It has been shown that highly potent combined ETA/ETB receptor antagonists can be developed from the C-terminal hexapeptide of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21), such as Ac-(D)Dip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) and Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21 (PD 145065). However, these compounds are relatively unstable to enzymatic proteolysis as determined in an in vitro rat intestinal perfusate assay. This instability is thought to be due to carboxypeptidase activity. In fact, incubation of PD 145065 with carboxypeptidase inhibitors greatly increased its half-life in rat intestinal perfusate. By performing a reduced amide bond and N-methyl amino acid scan, it was discovered that N-methylation of Ile-20 resulted in a compound (Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21, PD 156252) that retained full receptor affinity at both endothelin receptor subtypes along with enhanced proteolytic stability and cellular permeability. Interestingly, N-methylation of this bond allows the cis configuration to be readily accessible which greatly alters the preferred structure of the entire molecule and may be responsible for the observed enhanced metabolic stability.

Synthesis of xanthines as adenosine antagonists, a practical quantitative structure-activity relationship application.

A set of 56 8-phenylxanthines, previously tested for adenosine antagonism (adenosine A1 receptor affinity), was analyzed by quantitative structure-activity relationship (QSAR) techniques. The resulting QSAR revealed that (1) the most potent receptor binders had already been made in this series and thus suggested the termination of synthesis of compounds with additional phenyl substituents to increase potency and (2) potency was much more strongly affected by changes in ortho than para phenyl substitution. On the basis of this study, an additional 20 compounds were synthesized that contained primarily para substituents designed to increase aqueous solubility. High potency was maintained among the resulting sulfonamide derivatives (as predicted by the QSAR), and aqueous solubility was dramatically increased. Furthermore, in vitro antagonism of an adenosine receptor mediated physiological effect was demonstrated.

Calcium channel blocking and positive inotropic activities of ethyl 5-cyano-1,4-dihydro-6-methyl-2-[(phenylsulfonyl)methyl]-4-aryl-3- pyridine-carboxylate and analogues. Synthesis and structure-activity relationships.

The synthesis and pharmacological evaluation of a series of 2-[(arylsulfonyl)methyl]-4-aryl-5-cyano-1,4-dihydropyridine-3-carboxylic acid esters and analogues are described. These compounds possess a unique profile namely, calcium channel blocking and positive inotropic activities in vitro. Compound 54 was selected as the best compound in the series and was studied in detail. The synthesis and biological profiles of enantiomers of 54 are also reported. The data indicate that although the calcium channel blocking property of 54 is stereospecific the positive inotropic activity is not. Examples of 3- and 6-cyano and other closely related 1,4-dihydropyridine derivatives are described and evaluated for comparison and were found to be devoid of dual activities mentioned above.

Structure-activity relationships of the potent combined endothelin-A/endothelin-B receptor antagonist Ac-DDip16-Leu-Asp-Ile-Ile-Trp21: development of endothelin-B receptor selective antagonists.

The endothelins (ETs) are a family of bicyclic 21-amino acid-containing peptides that are highly potent and prolonged vasoconstrictors. The discovery of potent ET antagonists will facilitate the understanding of the physiological and/or pathophysiological role of ET. Structure-activity studies have revealed the importance of the C-terminal hexapeptide (residues 16-21) of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21) to the development of potent antagonists at both receptor subtypes (ETA and ETB). In particular, it has been shown that Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (Dip = 3,3-diphenylalanine) has low nanomolar affinity for the two endothelin receptor subtypes and is a functional antagonist of ET activity, both in vitro and in vivo at both receptors. Herein, we will describe the structure-activity relationships of Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) with a particular emphasis on modifications that lead to enhanced receptor affinity and/or individual receptor subtype selectivity. In particular, we will demonstrate how we utilized PD 142893 to develop ETB receptor selective ligands and the pharmacological differences that exist between species ETB receptors with respect to their affinity for C-terminal hexapeptide antagonists.

Butenolide endothelin antagonists with improved aqueous solubility.

Continued development around our ETA-selective endothelin (ET) antagonist 1 (CI-1020) has led to the synthesis of analogues with improved aqueous solubility profiles. Poor solubility characteristics displayed by 1 required a complex buffered formulation in order to conduct iv studies. To overcome the use of specific iv formulations for preclinical studies on additional drug candidates, analogues with improved aqueous solubility were desired. Several analogues were synthesized with substitution patterns that allowed for the formation of either acid or base addition salts. These derivatives had dramatically improved aqueous solubility. In addition, these analogues retained equivalent or improved ETA receptor selectivity and antagonist potency, versus 1, both in vitro and in vivo. Compound 29, which contains as a substituent the sodium salt of a sulfonic acid, has an ETA IC50 = 0.38 nM, ETA selectivity of 4200-fold, and ETA functional activity of KB = 7.8, all of which are similar or superior to those of 1. Compound 29 also has vastly superior aqueous solubility and solubility duration, compared to 1. Furthermore, 29 after iv infusion displays improved activity to 1 in preventing acute hypoxia-induced pulmonary hypertension in rats with an ED50 = 0.3 microg/kg/h.

Structure-activity relationships in a series of orally active gamma-hydroxy butenolide endothelin antagonists.

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.

Selective effects of adenosine receptor agonists upon coronary resistance and heart rate in isolated working rabbit hearts.

Dose dependent changes in heart rate (HR) and coronary resistance (CR) were determined for adenosine, 5'-N-ethylcarboxamide adenosine (NECA) and l-N6-phenylisopropyladenosine (l-PIA) over a dose range of 1 X 10(-9) to 1 x 10(-5) M. Changes in CR were determined under controlled metabolic demand conditions (constant mean aortic pressure, constant mean left atrial pressure, and constant HR). Decreases in HR were determined by allowing the paced hearts to beat spontaneously between doses for 15 seconds. Adenosine significantly decreased CR and HR at greater than or equal to 1 X 10(-5) M. NECA significantly decreased both CR and HR at greater than or equal to 3 X 10(-8) M. l-PIA significantly decreased HR at greater than or equal to 3 X 10(-8) M; however a dose at greater than or equal to 3 X 10(-6) M was required to significantly decrease CR. These results provide evidence that the coronary vasodilator action of adenosine may primarily be mediated by A2 receptors. Furthermore, the data are in support of previous observations that the bradycardic action of adenosine is principally mediated via A1 receptors.

PD 116,948, a highly selective A1 adenosine receptor antagonist.

(R)-N-(1-Methyl-2-phenylethyl) adenosine (R-PIA), an adenosine receptor agonist has both negative chronotropic activity and coronary vasodilator activity. These actions of R-PIA are proposed to be mediated by subtypes (A1 and A2) of adenosine receptors. PD 116,948 is a xanthine derivative which is a highly selective A1 adenosine receptor ligand. In this study PD 116,948 selectively antagonized the negative chronotropic activity of R-PIA in the isolated rat heart. These results are consistent with, and add further support to the hypothesis that adenosine receptor agonists mediate their negative chronotropic activity via A1 receptors and their vasodilator activity via A2 receptors.

Correlation of adenosine receptor affinities and cardiovascular activity.

Binding affinities of 28 adenosine analogs at A1 adenosine receptors (rat whole brain membranes, [3H]N6-cyclohexyladenosine, CHA), and at A2 adenosine receptors (rat striatal membranes, [3H]NECA) were compared to their EC25 values for decreasing heart rate and increasing coronary flow in the isolated rat heart. Heart rate (an A1 response) correlated with A1 binding affinity (r2 = 0.71, p less than 0.0001) but not with A2 binding affinity (r2 = 0.007, n.s.); conversely, coronary flow (an A2 response) correlated with A2 binding affinity (r2 = 0.83, p less than 0.0001) but not with A1 binding affinity (r2 = 0.05, n.s.). These results confirm that the brain A1 and A2 receptors, studied by binding methods, bear close similarities to their respective counterparts in the heart, studied by means of functional responses.

Cardiopulmonary indications for endothelin receptor antagonists: review of recent efficacy trials.

Recent clinical and experimental animal trials indicate that endogenously produced endothelin-1 (ET-1) contributes to the abnormal systemic and pulmonary vascular resistance associated with congestive heart failure (CHF) and pulmonary hypertension (PH). In experimental CHF, the chronic blockade of ET-1 actions by ET receptor antagonists clearly improves haemodynamic status, and improves cardiac structure and survival. The latter is based on limited results. In experimental PH there are consistent reports of prevention and reversal of PH, pulmonary vascular remodelling and right ventricular hypertrophy, independent of the inciting mechanisms. These results in experimental animals illustrate the potential efficacy of the ET receptor antagonists in future clinical trials. With five ET receptor antagonists in clinical development, and more on the way, their potential will soon be realised.

Species differences in the positive inotropic response to DPI 201-106, a novel cardiotonic agent.

The positive inotropic activity of the novel cardiotonic DPI 201-106 was investigated in rat and guinea pig isolated hearts. For comparative purposes, the adenylate cyclase stimulant forskolin and the sodium channel agonist veratridine were also evaluated in both species. DPI 201-106 and veratridine produced greater inotropic effects in rat hearts than in guinea pig hearts, whereas forskolin produced comparable effects. In both species the inotropic response to DPI 201-106 and veratridine, but not forskolin, was reversed by the sodium channel antagonist tetrodotoxin. These results confirm that the positive inotropic effect of DPI 201-106 is due to stimulation of the sodium channel and demonstrate for the first time that species differences exist in the inotropic response to this novel cardiotonic drug.

ETA and ETB receptors coexist on rabbit pulmonary artery vascular smooth muscle mediating contraction.

The possibility that both ETA and ETB endothelin receptor subtypes could mediate contractile activity in the same tissue was investigated in isolated, endothelium denuded rabbit pulmonary arteries. The ETB selective agonist, sarafotoxin 6c (S6c), produced potent contractile activity, equal to the non-selective ETA and ETB receptor agonist endothelin-1 (ET-1), indicating a contractile role for ETB receptors in this tissue. In addition BQ-123 (10.0 microM), the ETA selective antagonist, was only partially effective in blocking ET-1 induced contractions further indicating a contractile role for ETB receptors. However, the partial blockade by BQ-123 suggested a possible contractile role for ETA receptors. To address this possibility, ETB receptors were desensitized with a 30 minute pretreatment of S6c (0.01 microM). Under these conditions, we were able to demonstrate full ET-1 contractile activity that was now sensitive to blockade by BQ-123. The coexistence of both ETA and ETB receptors was confirmed through receptor binding experiments indicating 40/60 ratio, respectively. We conclude that 1) both ETA and ETB receptors coexist on vascular smooth muscle of rabbit pulmonary artery, 2) activation of either receptors subtype results in contraction, and 3) prolong activation of the ETB receptor subtype produces tachyphylaxis preventing further activation by S6c or ET-1.

Why big endothelin-1 lacks a vasodilator response.

The biphasic arterial blood pressure response to endothelin-1 (ET-1) results from a transient decrease, followed by a longer-lasting increase, in systemic vascular resistance. In contrast to ET-1, big endothelin-1 (bET-1) produces monophasic increases in systemic vascular resistance and arterial blood pressure. This is somewhat surprising, because bET-1 activity is reportedly due to ET-1, bET-1 being converted to ET-1 by a putative converting enzyme. In this study we tested two hypotheses that could explain the singular effect of bET-1 on the arterial vasculature: that bET-1 vasoconstriction, mediated by ETA receptors at the level of the smooth muscle, masks the release of endothelial derived vasodilators, and/or that the endothelium develops tachyphylaxis owing to prolonged activation of endothelial ETB receptors. In anesthetized rats, blockade of the vasoconstrictor activity of bET-1 with BQ-123, an ETA-receptor antagonist, did not reveal a masked bET-1 vasodilator component in the rat hindquarter. Furthermore, in the presence of bET-1 (after 3.0 nmol/kg bET-1 i.v.), low doses of ET-1 (0.03-0.3 nmol/kg) produced dose-dependent hindquarter vasodilation, indicating activation of endothelial ETB and therefore no tachyphylaxis. Collectively, these experiments suggest that i.v. administration of bET-1 results in little or no activation of endothelial ETB receptors and therefore lacks a vasodilator response.

The endothelin-A-receptor antagonist PD 180988 (CI-1034) selectively reverses the pulmonary vasoconstrictor response to hypoxia in the lamb.

Endothelin-1 (ET-1) is assigned a mediator role in the constrictor response of the pulmonary vasculature to hypoxia. Accordingly, a recently developed endothelin-A (ETA) antagonist, PD180988, was tested in the chronically instrumented newborn lamb to verify this possibility and, at the same time, to study a potential new treatment for pulmonary hypertension (PH). PD180988, given by infusion after a priming bolus, had an insignificant effect on the pulmonary circulation under normoxia, while it reversed the sustained pulmonary constriction caused by hypoxia. No appreciable change was noted under either experimental condition in the systemic circulation and cardiac contractility. We conclude that PD180988 is a selective inhibitor of hypoxic pulmonary vasoconstriction and lends itself to therapeutic use in infants.

PD 142893, SB 209670, and BQ 788 selectively antagonize vascular endothelial versus vascular smooth muscle ET(B)-receptor activity in the rat.

The purpose of this study was to determine whether vascular endothelial and vascular smooth-muscle endothelin ET(B) receptors could be quantitatively differentiated by PD 142893 (PD), SB 209670 (SB), and BQ 788 (BQ) in the same species by using closely matched experimental conditions. The isolated perfused rat kidney (vascular smooth muscle) and isolated perfused rat mesentery (vascular endothelium) were challenged with increasing bolus doses of sarafotoxin S6c in the absence and presence of antagonist. PD, SB, and BQ produced parallel concentration-dependent rightward shifts in the S6c dose-response curve in the kidney. PD and SB also produced parallel concentration-dependent rightward shifts in the S6c dose-response curve in the mesentery. In contrast, BQ produced an insurmountable antagonism. Schild-derived pA2 values for PD and SB were significantly greater for inhibiting endothelial versus smooth-muscle ET(B) receptors. Furthermore, PD and SB differed in their relative potency between the two assays. Because BQ produced an insurmountable antagonism in the mesentery, it was not possible quantitatively to compare the antagonist activity in the two assays. These results indicate that PD, SB, and BQ selectively antagonize endothelial ET(B)-receptor activity over smooth-muscle ET(B)-receptor activity.