Diaminoindanes as microsomal triglyceride transfer protein inhibitors.

The synthesis and biological activities of biarylamide-substituted diaminoindanes as microsomal triglyceride transfer protein (MTP) inhibitors are described. One of the more potent compounds, 8aR, inhibited both the secretion of apoB from Hep G2 cells and the MTP-mediated transfer of triglycerides between synthetic acceptor and donor liposomes with IC(50) values of 0.7 and 70 nM, respectively. In normolipidemic rats and dogs, oral administration of 8aR dose-dependently reduced both plasma triglycerides and total cholesterol. Moreover, in rats and dogs, 8aR also prevented the postprandial rise in plasma triglycerides following a bolus administration of a fat load. Because MTP inhibitors decrease very low density lipoprotein assembly in the liver, the potential for hepatic lipid accumulation was evaluated. In normolipidemic rats, hepatic cholesterol and triglyceride contents were dose-dependently increased by 8aR. However, hepatic lipid accumulation resulted in negligible change in total liver weight and was reversible after withdrawal of the compound.

Benzofused macrocyclic lactams as triple inhibitors of endothelin-converting enzyme, neutral endopeptidase 24.11, and angiotensin-converting enzyme.

The purpose of this study was to identify endothelin-converting enzyme (ECE) inhibitors that also possess inhibitory activity for neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE). The ortho-substituted benzofused macrocyclic lactams, such as CGS 26670, are generally potent NEP inhibitors but poor ACE inhibitors. CGS 26670 inhibited ECE activity with an IC50 of 600 nM, whereas it inhibited NEP and ACE activities with IC50 values of 0.9 and > 10,000 nM, respectively. This compound also prevented the conversion of big endothelin-1 (big ET-1) to ET-1 by denuded porcine coronary arterial smooth muscle with an IC50 of 200 nM. The ACE inhibitory activity is greatly is greatly improved in metasubstituted benzofused macrocyclic lactams. For example, CGS 26582 inhibited ECE, NEP, and ACE activities with IC50 values of 620, 4, and 175 nM, respectively. When injected at 30 mg/kg i.v. in conscious rats, followed by a challenge with big ET-1 at 1 nmol/kg i.v., this compound suppressed by 44% the increase in mean arterial blood pressure owing to the generation of ET-1 by ECE. Because ECE, NEP, and ACE play regulatory roles in cardiovascular and renal function, triple inhibitors of these enzymes may represent a novel class of agents for treatment of cardiovascular and renal diseases.

Ortho-substituted benzofused macrocyclic lactams as zinc metalloprotease inhibitors.

The design and preparation of ortho-substituted benzofused macrocyclic lactams are described. The benzofused macrocyclic lactams were designed as neutral endopeptidase 24.11 (NEP) inhibitors. Docking studies were carried out in a model of thermolysin (TLN) using the MACROMODEL and QXP modeling programs to select suitable ring sizes. These studies predicted that the 11-, 12-, and 13-membered ring macrocyclic lactams would be active in both enzymes TLN and NEP. Good predictability of experimental results, within this series, of binding to thermolysin and to a lesser extent to NEP was observed. A visual comparison, docked at the active site of TLN, is presented for thiorphan, a 10-membered ring macrocycle and an 11-membered ring benzofused macrocyclic lactam. Potent inhibition of both NEP and thermolysin was obtained. The 11-membered ring macrocycle 25a is the most potent inhibitor from this series of compounds (TLN IC50 = 68 nM; NEP IC50 = 0.9 nM). The effects of prodrug 44b administered at 10 mg/kg po on plasma atrial natriuretic peptide (ANP) levels in conscious rats was greater than 200% over a 4 h period.

Meta-substituted benzofused macrocyclic lactams as zinc metalloprotease inhibitors.

The design, synthesis, and biochemical profile of meta-substituted benzofused macrocyclic lactams are described. The meta-substituted benzofused macrocyclic lactams were designed to have a degree of flexibility allowing the amide bond to occupy two completely different conformations while maintaining sufficient rigidity to allow for strong interaction between enzyme and inhibitor. Using TFIT, a novel molecular superimposition program, it was shown that the meta analogs could be readily superimposed onto our ACE inhibitor template whereas no low-energy superimpositions of the ortho-substituted macrocycles could be found. The macrocycles were prepared by tethering aldehyde 1 derived from S-glutamic acid or S-aspartic acid to a meta-substituted phosphonium bromide 2. Homologation to a monocarboxylic acid methyl ester malonate followed by deprotection and cyclization gave the macrocyclic frame. Further manipulation gave the desired compounds. Unlike the ortho-substituted benzofused macrocyclic lactams described in the previous paper which are selective NEP inhibitors, the meta-substituted compounds are dual inhibitors of both NEP and ACE. The most potent member of this new series, compound 16a, inhibited both enzymes with an IC50 = 8 nM in NEP and 4 nM in ACE.

Dicarboxylic acid dipeptide neutral endopeptidase inhibitors.

The synthesis of three series of dicarboxylic acid dipeptide neutral endopeptidase 24.11 (NEP) inhibitors is described. In particular, the amino butyramide 21a exhibited potent NEP inhibitory activity (IC50 = 5.0 nM) in vitro and in vivo. Blood levels of 21a were determined using an ex vivo method by measuring plasma inhibitory activity in conscious rats, mongrel dogs, and cynomolgus monkeys. Free drug concentrations were 10-1500 times greater than the inhibitory constant for NEP over the course of a 6 h experiment. A good correlation of free drug concentrations was obtained when comparing values determined by the ex vivo analysis to those calculated from direct HPLC measurements. Plasma atrial natriuretic factor (exogenous) levels were elevated in rats and dogs after oral administration of 19a. Urinary volume and urinary sodium excretion were also potentiated in anesthetized dogs treated with 21a.

A rapid indirect method to determine the plasma concentrations of neutral endopeptidase inhibitors.

The selection of therapeutic agents for clinical development is principally based upon pharmacodynamic and pharmacokinetic criteria. Although many compounds are routinely tested in pharmacologic assays, direct pharmacokinetic assessment is difficult and not applicable to a large number of agents. Therefore, we have developed a rapid indirect method based on enzyme inhibition for determining the unbound concentration of NEP 24.11 inhibitors in rat plasma. In the present study, drug levels of compounds from three different classes of NEP 24.11 inhibitors: mercaptoalkyl, carboxyalkyl and aminophosphonates were compared. Studies were carried out in conscious, unrestrained rats. Arterial blood samples were obtained 10, 30, 60, 120, and 240 min after drug administration at 10 mg/kg i.v. or 30 mg/kg p.o. The blood was collected in EDTA and plasma prepared immediately. Protein bound NEP inhibitor was separated from plasma by centrifugation through an ultrafiltration membrane. Following acidification and serial dilution, the concentration of unbound inhibitor was determined in the plasma ultrafiltrate using the in vitro assay for NEP 24.11 inhibition. The results of this study indicated that the mercaptoalkyl inhibitor thiorphan was cleared rapidly from plasma, whereas, the plasma concentrations of the carboxyalkyl inhibitor CGS 23880A (UK-69,578), and the plasma concentrations of the aminophosphonate, CGS 24128, were maintained at high levels for at least 4 hours. Furthermore, the ratio of the NEP inhibitor concentration/IC50 value correlated well with the pharmacologic activity of these compounds.

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.

Effects of the ETB-selective antagonist IRL 2500 in conscious spontaneously hypertensive and Wistar-Kyoto rats.

The pharmacologic profile of a novel and selective ETB antagonist, IRL 2500 (N-(3,5-dimethylbenzoyl)-N-methyl-(D)-(4-phenylphenyl)-alany l-L-tryptophan) was examined in conscious spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. The initial vasodepressor response to endothelin-1 (ET-1) and IRL 1620 (0.5 nmol/kg, i.v.) was significantly reduced in conscious WKY rats pretreated with IRL 2500 (10 mg/kg, i.v.). The secondary and sustained pressor response to these agonists, however, was not altered by IRL 2500. The linear peptide antagonist BQ 788, although also inhibiting the initial depressor responses, attenuated the secondary pressor response to IRL 1620 and potentiated the pressor response to ET-1. IRL 2500, administered alone to naive conscious SHRs produced a -37 +/- 8 mm Hg reduction in blood pressure, followed by a secondary pressor response (+38 +/- 7 mm Hg) with a duration exceeding 90 min. Pretreatment with either the ETA-selective antagonist BQ 123 or with the nonselective ETA/ETB antagonist SB 209670 resulted in marked potentiation of the depressor response and significant attenuation of the secondary rise in pressure. These results indicate that in the conscious rat, IRL 2500 acts as an ETB1-selective antagonist. In addition, ETA receptor activation contributes to the sustained pressore response to IRL 2500 in the conscious SHR. Furthermore, IRL 2500 may also exert a non-ET receptor-mediated vasodilation in the SHR.

Dual angiotensin converting enzyme/thromboxane synthase inhibitors.

A variety of compounds were prepared to determine whether dual angiotensin converting enzyme (ACE)/thromboxane synthase (TxS) inhibition could be obtained in the same molecule. These compounds would be used to explore the concept that a dual inhibitor would have superior antihypertensive activity in the spontaneous hypertensive rat compared to an ACE inhibitor. Potent in vitro dual ACE and TxS inhibition was obtained in the same molecule with five series of compounds. Potent blood pressure lowering in the SHR was observed after oral administration of 8b and 11. However, a correlation between blood pressure lowering and the A1 pressor response inhibition was not observed. The blood pressure-lowering actions of enalapril were significantly potentiated by concurrent administration of 3, a thromboxane synthase inhibitor. Analysis of the area under the curve for 24 h showed nearly a doubling of the blood pressure-lowering effect.

Enkephalinase inhibitors. 1. 2,4-Dibenzylglutaric acid derivatives.

The synthesis of two new series of dicarboxylic acid dipeptides and two sulfhydryl-containing inhibitors are described. The in vitro enkephalinase inhibition data and some in vivo analgesic data are presented for these compounds. For the dibenzylglutaric acid series structure-activity relationships and in vivo analgesic activity are discussed. The reverse amides, i.e., 4-amino-2,4-dibenzylbutyric acid derivatives, are also discussed. Two sulfhydryl-containing inhibitors showed good in vivo potency in the mouse jump-latency hot-plate test after peripheral administration at moderate low doses.

Thromboxane synthase inhibition enhances action of converting enzyme inhibitors.

Mean arterial blood pressure was measured over a 24-hour period from the femoral artery of conscious, unrestrained spontaneously hypertensive rats. Oral administration of the angiotensin converting enzyme inhibitor CGS 16617 significantly lowered mean arterial pressure. In contrast, both the thromboxane synthase inhibitor CGS 12970 and the thromboxane receptor antagonist BM 13505 lacked an antihypertensive action in the spontaneously hypertensive rat. When administered concurrently, the thromboxane synthase inhibitor CGS 12970 potentiated the antihypertensive action of the angiotensin converting enzyme inhibitor CGS 16617. This effect was not observed with the thromboxane receptor antagonist BM 13505. In addition to CGS 16617, CGS 12970 also potentiated the hypotensive effect of two structurally dissimilar angiotensin converting enzyme inhibitors, benazapril HCL and captopril. Indomethacin blocked the thromboxane synthase inhibition-induced potentiation of the antihypertensive action of angiotensin converting enzyme inhibitors. The thromboxane synthase inhibitor CGS 12970 had no effect on the hypotension induced by hydralazine, indicating that the hypotension is not a nonspecific action related to the fall in blood pressure. These results may suggest that converting enzyme inhibition augments the levels and actions of a hormone that stimulates prostaglandin formation. It is well established that thromboxane synthase inhibitors eliminate the formation of the vasoconstrictor thromboxane A2 and allow reorientation of eicosanoid production toward the formation of vasodilating prostaglandins, which could enhance the antihypertensive action of angiotensin converting enzyme inhibitors.

Angiotensin converting enzyme inhibitors: N-substituted D-glutamic acid gamma dipeptides.

The preparation of two series of N-carbobenzoxy-gamma-D-glutamyl secondary 2S amino acids and (N-substituted gamma-D-glutamyl)indoline-2(S)-carboxylic acid dipeptides is described. In vitro inhibition of angiotensin converting enzyme (ACE) is reported for each compound, and the structure-activity relationship is discussed. Oral and iv inhibition of AI pressor response in vivo of selected compounds in Table II is also discussed. The most potent compounds in vitro, 3 and 6a, had an ACE IC50 of 7 and 2.7 X 10(-9) M, respectively.