Isoindolines: a new series of potent and selective endothelin-A receptor antagonists.

1,3-Disubstituted isoindolines have been discovered as a new class of potent functional ET(A) selective receptor antagonists through pharmacophore analysis of existing nonpeptide endothelin antagonists. The structure-activity relationships for both the trans and the cis series of isoindolines are discussed.

Anilides of (R)-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

The optimization of a series of anilide derivatives of (R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase (PDHK) is described that started from N-phenyl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide 1 (IC(50) = 35 +/- 1.4 microM). It was found that small electron-withdrawing groups on the ortho position of the anilide, i.e., chloro, acetyl, or bromo, increased potency 20-40-fold. The oral bioavailability of the compounds in this series is optimal (as measured by AUC) when the anilide is substituted at the 4-position with an electron-withdrawing group (i.e., carboxyl, carboxyamide, and sulfoxyamide). N-(2-Chloro-4-isobutylsulfamoylphenyl)-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropionamide (10a) inhibits PDHK in the primary enzymatic assay with an IC(50) of 13 +/- 1.5 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts, lowers blood lactate levels significantly 2.5 and 5 h after oral doses as low as 30 micromol/kg, and increases the ex vivo activity of PDH in muscle, kidney, liver, and heart tissues. However, in contrast to sodium dichloroacetate (DCA), these PDHK inhibitors did not lower blood glucose levels. Nevertheless, they are effective at increasing the utilization and disposal of lactate and could be of utility to ameliorate conditions of inappropriate blood lactate elevation.

Differential inhibition of the prejunctional actions of angiotensin II in rat atria by valsartan, irbesartan, eprosartan, and losartan.

The effects of valsartan and other nonpeptide angiotensin II type 1 (AT(1)) receptor blockers on the prejunctional actions of angiotensin II were investigated in the isolated left atria of rat. Norepinephrine stores in rat atria were loaded with [(3)H]norepinephrine, and neuronal norepinephrine release was deduced from the radioactivity efflux. Angiotensin II (10(-9) to 10(-6) M) produced concentration-dependent enhancement of the electrical stimulation-induced efflux of [(3)H]norepinephrine from the preparation. Pretreatment of tissues with valsartan, irbesartan, eprosartan, or losartan (10(-8) to 10(-6) M) produced concentration-dependent inhibitions of the stimulation-induced efflux of radioactivity observed in the presence of angiotensin II (10(-7) M). The AT(1) receptor blockers did not decrease the "basal stimulation-induced overflow of radioactivity but rather selectively inhibited the angiotensin II-mediated augmentation of the response. Regression analyses of the inhibition of the angiotensin II-mediated response by valsartan, irbesartan, eprosartan, and losartan revealed corresponding log IC(50) values (log M, with 95% confidence intervals) of -7.78 (-8.19, -7.51), -7.65 (-8.02, -7.40), -7.12 (-7. 37, -6.86), and -6.75 (-7.00, -6.40), indicating that the IC(50) values for valsartan and irbesartan are significantly lower than those for eprosartan and losartan. Thus, valsartan is a potent inhibitor of the prejunctional facilitatory effect of angiotensin II on the release of norepinephrine from peripheral sympathetic nerves. This implies that the therapeutic domain of valsartan may be extended to include pathophysiological conditions such as congestive heart failure wherein prejunctional angiotensin II receptors apparently play a significant role. Whether the high potency of valsartan translates into a significant clinical advantage relative to the other agents tested remains to be ascertained.

Secondary amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

N'-methyl-N-(4-tert-butyl-1,2,5,6-tetrahydropyridine)thiourea, SDZ048-619 (1), is a modest inhibitor (IC(50) = 180 microM) of pyruvate dehydrogenase kinase (PDHK). In an optimization of the N-methylcarbothioamide moiety of 1, it was discovered that amides with a small acyl group, in particular appropriately substituted amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, are inhibitors of PDHK. Utilizing this acyl moiety, herein is reported the rationale leading to the optimization of a series of acylated piperazine derivatives. Methyl substitution of the piperazine at the 2- and 5-positions (with S and R absolute stereochemistry) markedly increased the potency of the lead compound (>1,000-fold). Oral bioavailability of the compounds in this series is good and is optimal (as measured by AUC) when the 4-position of the piperazine is substituted with an electron-poor benzoyl moiety. (+)-1-N-[2,5-(S, R)-Dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropanamide (14e) inhibits PDHK in the primary enzymatic assay with an IC(50) of 16 +/- 2 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts with an EC(50) of 57 +/- 13 nM, diminishes lactate significantly 2.5 h post-oral-dose at doses as low as 1 micromol/kg, and increases the ex vivo activity of PDH in muscle, liver, and fat tissues in normal Sprague-Dawley rats. These PDHK inhibitors, however, do not lower glucose in diabetic animal models.

Triterpene and diterpene inhibitors of pyruvate dehydrogenase kinase (PDK).

Several oximes of triterpenes with a 17-beta hydroxyl and abietane derivatives are inhibitors of pyruvate dehydrogenase kinase (PDK) activity. The oxime 12 and dehydroabietyl amine 2 exhibit a blood glucose lowering effect in the diabetic ob/ob mouse after a single oral dose of 100 micromol/kg. However, the mechanism of the blood glucose lowering effect is likely unrelated to PDK inhibition.

Characterization of CGS 31447, a potent and nonpeptidic endothelin-converting enzyme inhibitor.

Optimization of an aminophosphonic acid series of compounds for inhibition of endothelin-converting enzyme (ECE) has led to the discovery of CGS 31447. This compound reversibly inhibited the activity of recombinant human ECE-1 with an IC50 value of 21 nM. The effect of CGS 31447 was not due to nonspecific chelation of the zinc ion at the catalytic center of ECE-1 by the phosphonic acid of the inhibitor. Determination of kinetic parameters of ECE-1 in the presence of 5-15 nM CGS 31447 revealed the competitive nature of the compound; a K1 of 7 nM was obtained. CGS 31447 infused at concentrations of 0.01, 0.1, and 1.0 microM inhibited the mean increase in big ET-1-induced pressor responses in isolated and perfused rat kidneys by 7, 39, and 68%, respectively, compared with the controls. These results demonstrate that CGS 31447 is a potent, reversible, and competitive inhibitor of ECE-1.

A novel model of conduit coronary constriction reveals local actions of endothelin-1 and prostaglandin F2alpha.

We evaluated the effects of endothelin-1 (ET-1) and prostaglandin F2alpha (PGF2alpha) selectively on conduit coronary artery diameter using a novel approach in which the local concentration of vasoactive agent was controlled and maintained in vivo. ET-1 and PGF2alpha were applied topically (100 microl every 3 min) to the external surface of the left circumflex coronary artery (LCx) in anesthetized dogs or to the bathing medium of isolated canine LCx rings in parallel in vitro experiments. The dose-dependent constrictions obtained in vivo and in vitro were similar with each agent. Single, approximately maximally effective concentrations of PGF2alpha evoked an initial rapid contraction followed by a slow and sustained larger contraction in both preparations. In contrast, single concentrations of ET-1 elicited a rapid constriction that partially recovered (50-80%) in the ensuing 1.5-2 h despite continuous exposure to ET-1. After the ET-1 constriction reversed, PGF2alpha could still elicit a contraction, indicating a homologous endothelin receptor desensitization. Both agents maximally decreased conduit artery cross-sectional area in vivo by approximately 40% without significantly changing LCx resistance. Thus this in situ technique revealed effects of ET-1 and PGF2alpha on a localized segment of coronary artery that were not discernible with either intravenous or intracoronary administration.

In vitro and in vivo evaluation of an endothelin inhibitor reveals novel K+ channel opening activity.

A low molecular weight endothelin (ET-1) inhibitor (Ex. 127, European Patent Application 404 525 A2, Takeda Chemical Ind., 1991), CGS 26061, was synthesized and evaluated to determine its mechanism of action. CGS 26061 (10 microM) failed to inhibit binding of [125I]ET-1 in porcine thoracic aorta and was without effect on ET-1-induced [3H]inositol phosphate accumulation in A7r5 cells. However, CGS 26061 relaxed porcine coronary arterial rings precontracted with ET-1. In addition, contractions to PGF2 alpha and low K+ (20 mM) but not high K+ were attenuated, suggesting that CGS 26061 (1, 10 microM) is a potassium channel opener. Patch-clamp experiments confirmed the K+ channel activity (0.1-10 microM). The originally re ported inhibition of ET-1-induced pressor responses by Ex. 127 (CGS 26061) was not replicated in the anesthetized dog or conscious rat nor was it shown to be antihypertensive in SHR. These data have identified CGS 26061 as a novel K+ channel opener with a unique cardiovascular profile.

The unusual binding properties of the endothelin receptor antagonist CGS 27830 distinguishes receptor/agonist interactions.

CGS 27830 [meso-1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-4-(3- nitrophenyl)-3-pyridine carboxylic acid anhydride] is a nonpeptidic, insurmountable, endothelin (ET) receptor antagonist with approximately 10- to 20-fold selectivity for ETA receptors. CGS 27830 exhibits unusual binding properties which depend on the receptor and ligand: standard saturation binding experiments (coincubation of membranes with ligand in the absence or presence of antagonist) suggest that CGS 27830 is a competitive inhibitor of [125I]IRL 1620 binding to ETB receptors in rat cerebellar membranes (i.e., there was a change of apparent Kd with no change of maximum binding), but a noncompetitive inhibitor of [125I]IRL 1620 binding to ETB receptors in rat lung membranes (i.e., significant loss of total binding was observed). Although the antagonist appears to be a noncompetitive inhibitor of [125I]IRL 1620 binding to ETB receptors in rat lung membranes, CGS 27830 appears to be a competitive inhibitor of [125I]ET-1 binding to the same receptors as well as to ETA receptors in A7r5 cell membranes. Thus, CGS 27830 can distinguish [125I]IRL 1620 binding to ETB receptors in rat cerebellar and lung membranes, but not ET-1 binding to ETB receptors in these tissues. These unusual binding properties demonstrate that rat lung and cerebellum ETB receptors interact differently with IRL 1620 or ET-1.

Delineation of endothelin receptor subtypes in rat and rabbit aortas.

The receptor subtypes mediating tension responses to endothelin-1 (ET-1) in isolated rat and rabbit aortic preparations were examined with various ET receptor antagonists. In the presence of 3 microM IRL 2500 (an ETB-selective antagonist), SB 209670 (an ETA/ETB antagonist) induced monophasic inhibitions of the ET-1 dose-response curves of both rat (pKB 9.1) and rabbit aortas (pKB 8.9). In the presence of IRL 2500, PD 156707 (an ETA antagonist) also caused a similar monophasic inhibition of the agonist-induced dose-response curve of the rat aorta (pKB 7.7) but produced a biphasic inhibition of the curve obtained with the rabbit aorta. PD 156707, however, produced monophasic inhibitions of the ET-1-induced dose-response curve of rabbit aortas pretreated either with BQ 788 (an ETB-selective antagonist) or desensitized with sarafotoxin S6c (an ETB-selective agonist). The receptor subtypes mediating the contractile responses to ET-1 were further characterized by examining the effects of the antagonists on the binding of [125I]ET-1 to these two tissues. In the presence of 1 nM IRL 2500, SB 209670 displaced [125I]ET-1 binding in both rat and rabbit aortic membrane preparations in a monophasic manner, with similar potencies (IC50 1.2-1.6 nM). Similar results were also ontained with PD 156707 in rat aortic membrane (IC50 0.27 nM). In contrast, the data obtained with PD 156707 using rabbit aortic membrane were best-fitted with a two-site model (site 1, IC50 0.21 nM, 67% of Bmax; site 2, 100 nM and 33%, respectively). Therefore, the constrictor response to ET-1 in the rat aorta is mediated exclusively by the ETA receptor, whereas that in the rabbit aorta is additionally mediated by an ETB receptor subtype that is sensitive to SB 209670 and BQ 788 but insensitive to IRL 2500.

Characterization of a potent and selective endothelin-B receptor antagonist, IRL 2500.

IRL 2500 [N-(3,5-dimethylbenzoyl)-N-methyl-(D)-(4-phenylphenyl)-alany l-L- tryptophan] inhibited the binding of [125I]-endothelin-1 (ET-1) to human ETB (IC50 1.3 +/- 0.2 nM) and ETA (IC50 94 +/- 3 nM) receptors expressed in transfected Chinese hamster ovary (CHO) cells. In in vitro studies, IRL 2500 inhibited the sarafotoxin S6c (STX6c)-mediated contraction of the dog saphenous vein (pKb 7.77) and the STX6c-induced relaxation of the preconstricted rabbit mesenteric artery (pKb 6.92). In the anesthetized rat, IRL 2500 (10 mg/kg, i.v.) inhibited the initial transient decrease in mean arterial pressure (MAP) induced by the ETB-selective agonist IRL 1620 (0.5 nmol/kg, i.v.). IRL 2500 also attenuated the IRL 1620-mediated increase in renal vascular resistance (RVR) in the anesthetized rat. Therefore, IRL 2500 is a potent and selective ETB receptor antagonist that can be used to delineate ET responses mediated by the ETB receptor.

Inhibition by nickel of endothelin-1-induced tension and associated 45Ca movements in rabbit aorta.

Contractions induced by 10 nM endothelin-1 (ET) in the rabbit aortic media intimal layer were inhibited by prior exposure to 100 microM Ni++ (33.1%) or to a Ca(++)-free buffer (80.2%) but were unaffected by pretreatment with 0.1 microM nifedipine. Contractions elicited by phenylephrine (1 nM-100 microM) or K+ (10-50 mM) were not inhibited by 100 microM Ni++ but those induced by ET in tissues submaximally precontracted with 20 mM K+ were selectively antagonized by the divalent cation. The mechanism for the inhibitory action of Ni++ was ascertained by an examination of the effects of the cation on ET-induced alterations in the cellular distribution and mobilization of Ca++. Efflux of 45Ca from the muscle into a solution without added Ca++ was not altered by ET. Total or cellular 45Ca uptake (uptake after exposure to La and low temperature), at either low- or high-affinity sites in resting muscles was also not affected by the peptide. However, low-affinity cellular 45Ca retention in muscles depolarized with high K+ levels (160 mM) was significantly enhanced (45.1%) by ET. Ni++ did not alter 45Ca retention in control and K(+)-treated muscles but it blocked the additional incremental 45Ca uptake associated with ET (in the presence of high K+). Thus, Ni++ produced a selective blockade of an ET-activated Ca++ influx pathway, distinct from the dihydropyridine-sensitive L-type Ca++ channels, in rabbit aortic smooth muscle. This action by Ni++ apparently inhibits subsequent contractile responses of the muscle to ET.

Conversion of proendothelin-1 into endothelin-1 by aspartylproteases.

The ability of cathepsin D, chymosin, pepsin and renin to produce endothelin-1 (ET-1) from proendothelin-1 (proET-1) was compared. No significant conversion was observed when proET-1 was incubated with up to 1 U of renin for 15 min at 37 degrees C. Cathepsin D generated, as well as degraded, ET-1 rapidly. Net production of ET-1 reached a maximum when 0.003 U of cathepsin D was used, and about 16% of the initial proET-1 was detected as ET-1 by HPLC. Pepsin up to 1 U converted proET-1 into ET-1 dose-dependently with a maximum of 71% conversion. A further increase of the amount of pepsin in the reaction mixture produced nonspecific cleavage of ET-1. Less than 10% of ET-1 remained in the presence of 15 U of pepsin. Chymosin also generated ET-1 dose-dependently, and a complete conversion was obtained at 1 U of enzyme. Greater than 1 U of chymosin only slightly degraded ET-1; at least 80% of ET-1 was still present when 15 U of chymosin was included in the assay. Other properties associated with the conversion of proET-1 into ET-1 by chymosin were investigated. Similar to authentic ET-1, the product of chymosin treatment caused contraction of isolated rabbit aortic rings, and pre-incubation of chymosin with pepstatin A abolished this contractile response.(ABSTRACT TRUNCATED AT 250 WORDS)