Effect of bradykinin metabolism inhibitors on evoked hypotension in rats: rank efficacy of enzymes associated with bradykinin-mediated angioedema.

BACKGROUND AND PURPOSE: Inhibition of bradykinin metabolizing enzymes (BMEs) can cause acute angioedema, as demonstrated in a recent clinical trial in patients administered the antihypertensive, omapatrilat. However, the relative contribution of specific BMEs to this effect is unclear and confounded by the lack of a predictive pre-clinical model of angioedema. EXPERIMENTAL APPROACH: Rats were instrumented to record blood pressure and heart rate; inhibitors were infused for 35 min and bradykinin was infused during the last 5 min to elicit hypotension, as a functional marker of circulating bradykinin and relative angioedema risk. KEY RESULTS: In the presence of omapatrilat bradykinin produced dose-dependent hypotension, an effect abolished by B(2) blockade. In the presence of lisinopril (ACE inhibitor), but not candoxatril (NEP inhibitor) or apstatin (APP inhibitor), bradykinin also elicited hypotension. Lisinopril-mediated hypotension was unchanged with concomitant blockade of NEP or NEP/DPPIV (candoxatril+A-899301). However, hypotension was enhanced upon concomitant blockade of APP and further intensified in the presence of NEP inhibition to values not different from omapatrilat alone. CONCLUSIONS AND IMPLICATIONS: We demonstrated that bradykinin is degraded in vivo with an enzyme rank-efficacy of ACE>APP>>NEP or DPPIV. These results suggest the effects of omapatrilat are mediated by inhibition of three BMEs, ACE/APP/NEP. However, dual inhibition of ACE/NEP or ACE/NEP/DPPIV elicits no increased risk of angioedema compared to ACE inhibition alone. Thus, novel BME inhibitors must display no activity against APP to avoid angioedema risk due to high prevalence of ACE inhibitor therapy in patients with diabetes and cardiovascular disease.

Discovery of novel p-arylthio cinnamides as antagonists of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction. 4. Structure-activity relationship of substituents on the benzene ring of the cinnamide.

We have shown that p-arylthio cinnamides can inhibit the interaction of LFA-1 and ICAM-1, which is involved in cell adhesion and the inflammatory process. We now show that 2,3-disubstitution on the aryl portion of the cinnamide results in enhanced activity over mono substitution on the ring. The best 2,3-substituents were chlorine and trifluoromethyl groups. Compounds 39 and 40 which contain two CF3 groups have IC(50) values of 0.5 and 0.1 nM, respectively, in inhibiting JY8 cells expressing LFA-1 on their surface, from adhering to ICAM-1. The structure-activity relationship (SAR) was examined using an NMR based model of the LFA-1 I domain/compound 31 complex. One of our compounds (38) was able to reduce cell migration in two different in vivo experiments.

New antimitotic agents with activity in multi-drug-resistant cell lines and in vivo efficacy in murine tumor models.

During a screen for compounds that could inhibit cell proliferation, a series of new tubulin-binding compounds was identified with the discovery of oxadiazoline 1 (A-105972). This compound showed good cytotoxic activity against non-multi-drug-resistant and multi-drug-resistant cancer cell lines, but its utility in vivo was limited by a short half-life. Medicinal chemistry efforts led to the discovery of indolyloxazoline 22g (A-259745), which maintained all of the in vitro activity seen with oxadiazoline 1, but also demonstrated a better pharmacokinetic profile, and dose-dependent in vivo activity. Over a 28 day study, indolyloxazoline 22g increased the life span of tumor-implanted mice by up to a factor of 3 upon oral dosing. This compound, and others of its structural class, may prove to be useful in the development of new chemotherapeutic agents to treat human cancers.

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 5. Highly selective, potent, and orally active ET(A) antagonists.

The synthesis and structure-activity relationships (SAR) of a series of pyrrolidine-3-carboxylic acids as endothelin antagonists are described. The data shows an increase in selectivity when the methoxy of Atrasentan (ABT-627) is replaced with methyl, and the benzodioxole is replaced with dihydrobenzofuran. Adding a fluorine further increases the binding activity and provides a metabolically stable and orally bioavailable ET(A)-selective antagonist.

Discovery of potent antagonists of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction. 3. Amide (C-ring) structure-activity relationship and improvement of overall properties of arylthio cinnamides.

The interaction of LFA-1 and ICAM-1 plays an important role in the cell adhesion process. On the basis of previously reported SAR and structural information on the binding of our p-arylthiocinnamide series to LFA-1, we have identified the cyclic amide (C-ring) as a site for modification. Improvement in potency and, more importantly, in the physical properties and pharmacokinetic profiles of the leading compounds resulted from this modification. One of the best compounds (11f) is also shown to reduce myocardial infarct size in rat.

Novel p-arylthio cinnamides as antagonists of leukocyte function-associated antigen-1/intracellular adhesion molecule-1 interaction. 2. Mechanism of inhibition and structure-based improvement of pharmaceutical properties.

The interaction between leukocyte function-associated antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1) has been implicated in inflammatory and immune diseases. Recently, a novel series of p-arylthio cinnamides has been described as potent antagonists of the LFA-1/ICAM-1 interaction. These compounds were found to bind to the I domain of LFA-1 using two-dimensional NMR spectroscopy of 15N-labeled LFA-1 I domain. On the basis of NOE studies between compound 1 and the I domain of LFA-1, a model of the complex was constructed. This model revealed that compound 1 does not directly inhibit ICAM-1 binding by interacting with the metal ion dependent adhesion site (MIDAS). Instead, it binds to the previously proposed I domain allosteric site (IDAS) of LFA-1 and likely modulates the activation of LFA-1 through its interaction with this regulatory site. A fragment-based NMR screening strategy was applied to identify small, more water-soluble ligands that bind to a specific region of the IDAS. When incorporated into the parent cinnamide template, the resulting analogues exhibited increased aqueous solubility and improved pharmacokinetic profiles in rats, demonstrating the power of this NMR-based screening approach for rapidly modifying high-affinity ligands.

Discovery of novel p-arylthio cinnamides as antagonists of leukocyte function-associated antigen-1/intracellular adhesion molecule-1 interaction. 1. Identification of an additional binding pocket based on an anilino diaryl sulfide lead.

The interaction between leukocyte function-associated antigen-1 (LFA-1), a member of the beta(2)-integrin family of adhesion molecules, and intracellular adhesion molecule ICAM-1 (cd54) is thought to play a critical role in the inflammatory process. On the basis of an anilino diaryl sulfide screening lead 1, in combination with pharmacophore analysis of other screening hits, we have identified an adjacent binding pocket. Subsequently, a p-ethenylcarbonyl linker was discovered to be optimal for accessing this binding site. Solution-phase parallel synthesis enabled rapid optimization of the cinnamides for this pocket. In conjunction with fine-tuning of the diaryl substituents, we discovered a novel series of potent, nonpeptide inhibitors of LFA-1/ICAM-1 interaction, exemplified by A-286982 (28h), which has IC(50) values of 44 and 35 nM in an LFA-1/ICAM-1 binding assay and LFA-1-mediated cellular adhesion assay, respectively.

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 4. Side chain conformational restriction leads to ET(B) selectivity.

When the dialkylacetamide side chain of the ET(A)-selective antagonist ABT-627 is replaced with a 2,6-dialkylacetanilide, the resultant analogues show a complete reversal of receptor selectivity, preferring ET(B) over ET(A). By optimizing the aniline substitution pattern, as well as the alkoxy group on the 2-aryl substituent, it is possible to prepare antagonists with subnanomolar affinity for ET(B) and with selectivities in excess of 4000-fold. A number of these compounds also show promising pharmacokinetic profiles; a useful balance of properties is found in A-192621 (38). Pharmacology studies with A-192621 serve to reveal the role of the ET(B) receptor in modulating blood pressure; the observed hypertensive response to persistent ET(B) blockade is consistent with previous postulates and indicates that ET(B)-selective antagonists may not be suitable as agents for long-term systemic therapy.

Design, synthesis, and activity of a series of pyrrolidine-3-carboxylic acid-based, highly specific, orally active ET(B) antagonists containing a diphenylmethylamine acetamide side chain.

The endothelin (ET)-B receptor subtype is expressed on vascular endothelial and smooth muscle cells and mediates both vasodilation and vasoconstriction. On the basis of the pharmacophore of the previously reported ET(A)-specific antagonist 1, (ABT-627), we are reporting the discovery of a novel series of highly specific, orally active ET(B) receptor antagonists. Replacing the dibutylaminoacetamide group of 1 with a diphenylmethylaminoacetamide group resulted in antagonist 2 with a complete reversal of receptor specificity. Structure-activity relationship studies revealed that ortho-alkylation of the phenyl rings could further increase ET(B) affinity and also boost the ET(A)/ET(B) activity ratio of the resulting antagonists. A similar antagonism selectivity profile could also be achieved when one of the phenyl rings of the acetamide side chain was replaced with an alkyl group, preferably a tert-butyl group (10h). Combining these features with modification of the 2-aryl group of the pyrrolidine core, we have identified a potent antagonist (9k, A-308165) with over 27 000-fold selectivity favoring the ET(B) receptor and an acceptable pharmacokinetic profile (F = 24%) in rats.

Discovery of a series of pyrrolidine-based endothelin receptor antagonists with enhanced ET(A) receptor selectivity.

Endothelins, ET-1, ET-2, and ET-3 are potent vasoconstricting and mitogenic 21-amino acid bicyclic peptides, which exert their effects upon binding to the ET(A) and ET(B) receptors. The ET(A) receptor mediates vasoconstriction and smooth muscle cell proliferation, and the ET(B) receptor mediates different effects in different tissues, including nitric oxide release from endothelial cells, and vasoconstriction in certain vascular cell types. Selective antagonists of endothelin receptor subtypes may prove useful in determining the role of endothelin in various tissue types and disease states, and hence as therapeutic agents for such diseases. The pyrrolidine carboxylic acid A-127722 has been disclosed as a potent and ET(A)-selective antagonist, and is currently undergoing clinical trials. In our efforts to find antagonists with altered selectivity (ET(A)-selective, ET(B)-selective, or nonselective), we investigated the SAR of the 2-substituent on the pyrrolidine. Compounds with alkyl groups at the 2-position possessed ET(A) selectivity improved over A-127722 (1400-fold selective), with the best of these compounds showing nearly 19,000-fold selectivity.

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.

The effects of diminishing albumin binding to some Endothelin receptor antagonists.

As a pharmacological class, Endothelin-A receptor (ET(A)) antagonists are highly bound (>98%) to serum albumin. In the presence of physiological concentrations of albumin, their affinities for ET(A) decrease 10 to 100 fold. We have prepared ET(A) antagonists which exhibit lower degrees of binding to albumin, while maintaining potency and selectivity for the ET(A) receptor. The protein induced IC50 shift is reduced or eliminated in this new series of compounds. The compounds also display altered in vivo and pharmacokinetic profiles which may be consistent with their lower degree of protein binding.

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 3. Discovery of a potent, 2-nonaryl, highly selective ETA antagonist (A-216546).

Previously we have reported the discovery of ABT-627 (1, A-147627, active enantiomer of A-127722), a 2,4-diaryl substituted pyrrolidine-3-carboxylic acid based endothelin receptor-A antagonist. This compound binds to the ETA receptor with an affinity (Ki) of 0. 034 nM and with a 2000-fold selectivity for the ETA receptor versus the ETB receptor. We have expanded our structure-activity studies in this series, in an attempt to further increase the ETA selectivity. When the p-anisyl group of 1 was replaced by an n-pentyl group, the resultant antagonist 3 exhibited substantially increased ETB/ETA activity ratio, but a decreased ETA affinity. Structure-activity studies revealed that substitution and geometry of this alkyl group, and substitution on the benzodioxolyl ring, are important in optimizing this series of highly ETA selective antagonists. In particular, the combination of a (E)-2,2-dimethyl-3-pentenyl group and a 7-methoxy-1,3-benzodioxol-5-yl group provided hydrophobic compound 10b with subnanomolar affinity for human ETA receptor subtype and with an ETB/ETA activity ratio of over 130000. Meanwhile, synthetic efforts en route to olefinic compounds led to the discovery that 2-pyridylethyl (9o) and 2-(2-oxopyrrolidinyl)ethyl (9u) replacement of the p-anisyl group of 1yielded very hydrophilic ETA antagonists with potency and selectivity equal to those of 10b. On the basis of overall superior affinity, high selectivity for the ETA receptor (Ki, 0.46 nM for ETA and 13000 nM for ETB), and good oral bioavailability (48% in rats), A-216546 (10a) was selected as a potential clinical backup for 1.

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 2. Sulfonamide-based ETA/ETB mixed antagonists.

When the N,N-dialkylacetamide side chain of the highly ETA-selective endothelin antagonist ABT-627 (1; [2R,3R,4S]-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-[[N, N-dibutylamino)-carboxyl]methyl]pyrrolidine-3-carboxylic acid; A-147627) is replaced by N,S-dialkylsulfonamidoethyl, the resultant analogs retain ETA affinity, but exhibit substantial ETB affinity as well. Structure-activity studies reveal that modifications in the length of the two alkyl groups, and in the substitution on the anisyl ring, are important in optimizing this "balanced" antagonist profile. In particular the combination of an N-n-propyl group, an S-alkyl chain between four and six carbons in length, and a fluorine atom ortho to the aromatic OCH3 provides compounds with sub-nanomolar affinities for both receptor subtypes, and with ETA/ETB ratios close to 1. A number of these compounds also exhibit oral bioavailabilities (in rats) in the 30-50% range and have substantial plasma half-lives. The balanced receptor-binding profile of these potent and orally bioavailable compounds complements the ETA selectivity observed with 1.

Stimulation of atrial natriuretic peptide receptor/guanylyl cyclase- A signaling pathway antagonizes the activation of protein kinase C-alpha in murine Leydig cells.

Atrial natriuretic peptide (ANP) regulates diverse physiological responses by binding to its specific guanylyl cyclase-A receptor (Npra) which synthesizes the intracellular second messenger cGMP. To understand the molecular mechanisms of cellular signaling of ANP, we have studied its effect on the enzymatic activity of overexpressed protein kinase C (PKC) in murine Leydig tumor (MA-10) cells which were transfected with PKC-alpha cDNA. Treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II (ANG II) and endothelin-1 (ET-1) stimulated the PKC activity by 4-5-fold in PKC-alpha cDNA transfected MA-10 cells. The pretreatment of PKC-alpha transfected cells with ANP significantly inhibited the TPA-, ANG II- and ET-1-stimulated PKC activity. The agonist-stimulated PKC activity was also inhibited in the presence of 8-bromo-cGMP, however, cAMP had no effect on stimulatory PKC activity. The exposure of cells to Npra- antagonist A71915, which blocks the production of cGMP, significantly reduced the inhibitory effect of ANP on agonist-stimulated PKC activity and accumulation of intracellular cGMP in MA-10 cells. Similarly, inhibition of cGMP-dependent protein kinase by KT5823, restored the stimulatory levels of PKC activity in the presence of ANP. These results provide direct evidence that ANP antagonizes the agonist-stimulated PKC activity in MA-10 cells, involving the specific receptor Npra, its second messenger cGMP and cGMP-dependent protein kinase. Together, these findings implicate that ANP may act as a negative mediator of 'cross-talk' between PKC-alpha and Npra signaling pathway in MA-10 cells.

Potent and selective non-benzodioxole-containing endothelin-A receptor antagonists.

The benzodioxole ((methylenedioxy)benzene) group is present in a number of endothelin (ET) receptor antagonists thus far reported. As part of our own endothelin antagonist program we have developed (2R*,3R*,4S*)-1-(N,N-dibutylacetamido)-4-(1,3-benzodioxol-5- yl)-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (A-127722). This is a potent antagonist, binding to the ETA and ETB receptor subtypes with affinities (IC50) of 0.4 and 520 nM, respectively, and also contains the aforementioned benzodioxole. While this compound was seemingly optimized at its N-terminus, no effort had been directed toward understanding the contributions to binding affinity or receptor subtype selectivity conferred by the benzodioxole. Substitution by 1- or 2-naphthyl yielded weak antagonists. Oxygenated benzenes, such as p-anisyl, were potent compounds with IC50s in the low-nanomolar range. Simple deletion of either of the two oxygen atoms (dihydrobenzofurans) yielded extremely potent agents, possessing subnanomolar affinity for the ETA receptor. Additionally, the compounds showed enhanced selectivity, binding to the ETB receptor subtype in the micromolar range. This paper describes the development of this novel class of compounds.

An essential myosin light chain peptide induces supramaximal stimulation of cardiac myofibrillar ATPase activity.

The N-terminal region of skeletal myosin light chain-1 (MLC-1) binds to the C terminus of actin, yet the functional significance of this interaction is unclear. We studied a fragment (MLC-pep; residues 5-14) of the ventricular MLC-1. When added to rat cardiac myofibrils, 10 nM MLC-pep induced a supramaximal increase in the MgATPase activity at submaximal Ca2+ levels with no effect at low and maximal Ca2+ levels. A nonsense, scrambled sequence peptide had no effect at any pCa value. MLC-pep did not affect myosin KEDTA and CaATPase activities or actin-activated MgATPase activities in the absence or presence of tropomyosin. The MLC-pep did not alter the ability of troponin I to inhibit MgATPase activity. Moreover, when troponin I and troponin C were extracted from the myofibrils, the MLC-pep lost its ability to stimulate the ATPase rate. This effect was fully restored upon reconstitution of the extracted myofibrils with troponin I-troponin C complex. Thus, activation of MgATPase activity by the peptide required a full complement of thin filament regulatory proteins. Interestingly, the stimulatory effect occurred at a ratio of 4 peptides to 1 thin filament, suggesting that the peptide engages in a highly cooperative process that may involve activation of the entire thin filament.

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.

Azole endothelin antagonists. 1. A receptor model explains an unusual structure-activity profile.

The pseudotetrapeptide FR-139317 is a potent and highly selective antagonist of the endothelin-A (ET(A)) receptor; however, its peptidic nature leads to poor oral absorption characteristics which make it an unlikely drug candidate. In an attempt to improve these properties, we have replaced a portion of the amide bond framework of FR-139317 with a heterocyclic surrogate. The resultant analogs are also ET(A)-selective antagonists, but show a structure-activity profile substantially different from that of the peptidic series, particularly with regard to the requirements for the side chain group that has been incorporated into the heterocycle. The nature of the heterocycle itself also has profound effects on the activity of the compounds. Both of these surprising results can be rationalized through examination of a 3D model of ET ligand--receptor binding that has previously been developed in our laboratories.