Discovery, modeling, and human pharmacokinetics of N-(2-acetyl-4,6-dimethylphenyl)-3-(3,4-dimethylisoxazol-5-ylsulfamoyl)thiophene-2-carboxamide (TBC3711), a second generation, ETA selective, and orally bioavailable endothelin antagonist.

Sitaxsentan (1) (Wu et al. J. Med. Chem. 1997, 40, 1690) is our first endothelin antagonist being evaluated in clinical trials. It has demonstrated biological effects in an acute hemodynamic study in CHF (Givertz et al. Circulation 2000, 101, 2922), an open-label 20-patient pulmonary hypertension trial (Barst et al. Chest 2002, 121, 1860-1868), and a 31-patient trial in essential hypertension (Calhoun et al. AHA Scientific Sessions 2000). In a phase 2b/3 pulmonary arterial hypertension trial, once a day treatment of 100 mg of sitaxsentan statistically significantly improved 6-min walk distance and NYHA class at 12 weeks (Barst et al. Am. J. Respir. Crit. Care Med. 2004, 169, 441). We have since reported on our efforts in generating follow-up compounds (Wu et al. J. Med. Chem. 1999, 42, 4485) and recently communicated that an ortho acyl group on the anilino ring enhanced oral absorption in this category of compounds (Wu et al. J. Med. Chem. 2001, 44, 1211). Here we report an expansion of this work by substituting a variety of electron-withdrawing groups at the ortho position and evaluating their effects on oral bioavailability as well as structure-activity relationships. As a result, TBC3711 (7z) was identified as our second endothelin antagonist to enter the clinic due to its good oral bioavailability (approximately 100%) in rats, high potency (ET(A) IC(50) = 0.08 nM), and optimal ET(A)/ET(B) selectivity (441 000-fold). Compound 7z has completed phase-I clinical development and was well tolerated with desirable pharmacokinetics in humans (t(1/2) = 6-7 h, oral availability > 80%).

Recently discovered sulfonamide-, acyl sulfonamide- and carboxylic acid-based endothelin antagonists.

Endothelins (ET-1, 2 and 3) are 21-residue peptides with two disulfide bridges and a highly conserved carboxy terminal. ET-1, the most significant isoform, is a potent vasoconstrictor and mitogen that exerts its biological effects through binding to its two G protein-coupled receptors: ET(A) and ET(B). ET(A) receptors are expressed on vascular smooth muscle cells and mediate vasoconstrictive and proliferative responses to ET-1. ET(B) receptors are mainly located on endothelial cells where they clear ET-1 from circulation and mediate vasodilation via the release of nitric oxide. ET-1 has been associated with a variety of serious diseases such as pulmonary hypertension, heart failure, prostate cancer and renal dysfunction.

The critical role of the intrinsic VSMC proliferation and death programs in injury-induced neointimal hyperplasia.

Postangioplasty and in-stent restenosis remain ominous problems in percutaneous coronary intervention where good animal models of restenosis proneness and resistance are needed. We accidentally discovered that the carotid arteries (CAs) of the Harlan and Sasco substrains of Sprague-Dawley rats display drastically different restenosis phenotypes following balloon-induced endothelial denudation. When subjected to balloon injury, Sasco CAs exhibited significantly larger neointimal mass than did Harlan CAs at both days 14 and 32, as evidenced by a higher intima-to-media ratio and a greater number of intimal cells in Sasco CAs. This was due to a greater cell proliferation and to a less vigorous apoptosis of Sasco neointima, as assessed by 5-bromo-2'-deoxyuridine and terminal deoxynucleotidyl transferase-deoxyuridine nick-end labeling staining, respectively. At a cellular level, whereas vascular smooth muscle cells (VSMCs) isolated from Sasco and Harlan CAs were identical in morphology and in propensity to migrate, Sasco VSMCs proliferated more robustly and died far less, suggesting that under the exact same microenvironment, Sasco and Harlan VSMCs respond to growth and noxious stimuli in a drastically different fashion and that Sasco's significantly more robust neointimal proliferation after vascular injury in vivo can be accounted for by these intrinsic differences in VSMCs of these substrains in vitro. Sasco and Harlan Sprague-Dawley rats as well as VSMCs from these rats will prove to be powerful tools to study genes involved in the pathogenesis of restenosis.

Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2.

Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of the substrates was F-X-Q, in which F was the fluorescent group, Abz, Q was the quenching group (either Phe(NO(2)) or Tyr(NO(2))), and X was the intervening peptide. These substrates were selectively cleaved by recombinant human ACE2, as shown by MS and HPLC. Quenching efficiency increased as the peptide sequence was shortened from 8 to 3 aa, and also when Tyr(NO(2)) was used as a quenching group instead of Phe(NO(2)). Two of the optimized substrates, TBC5180 and TBC5182, produced a signal:noise ratio of better than 20 when hydrolyzed by ACE2. Kinetic measurements with ACE2 were as follows: TBC5180, K(m)=58 microM and k(cat)/K(m)=1.3x10(5)M(-1)s(-1); TBC5182, K(m)=23 microM and k(cat)/K(m)=3.5 x 10(4)M(-1)s(-1). Thus, based on hydrolysis rate, TBC5180 was a better substrate than TBC5182. However, TBC5180 was also hydrolyzed by ACE, whereas TBC5182 was not cleaved, suggesting that TBC5182 was a selective for ACE2. We conclude that these two peptides can be used as fluorescent substrates for high-throughput screening for selective inhibitors of ACE2 enzyme.

JAM2 interacts with alpha4beta1. Facilitation by JAM3.

We have previously reported that junctional adhesion molecule 2 (JAM2) adheres to T cells through heterotypic interactions with JAM3. An examination of the cation dependence of JAM2 adhesion to HSB cells revealed a Mn(2+)-enhanced binding component indicative of integrin involvement. Using neutralizing integrin antibodies, we have defined an interaction between JAM2 and alpha(4)beta(1) in T cells. The interaction is readily amenable to drug intervention as demonstrated by the ability of TBC 772, an alpha(4)-specific inhibitor, to attenuate the Mn(2+)-enhanced component. Intriguingly, the engagement of alpha(4)beta(1) by JAM2 is only enabled following prior adhesion of JAM2 with JAM3 and is not detectable in cells where JAM3 expression is absent. Supporting this observation, we show that neutralizing JAM3 serum and soluble JAM3 ectodomain inhibit not only JAM2 binding to JAM3 but also prevent JAM2/alpha(4)beta(1) interactions in T cells. We further define the first Ig-like fold of JAM2 as being competent in binding both JAM3 and alpha(4)beta(1) counter-receptors. Mutagenesis of the only acidic residue in the C-D loop of this Ig fold, namely Asp-82, has no bearing on alpha(4)beta(1) interactions, and thus JAM2 deviates somewhat from the mechanism used by other immunoglobulin superfamily cell adhesion molecules to engage integrin.

Nonpeptide endothelin antagonists in clinical development.

Endothelins (ET-1, ET-2 and ET-3) are 21-amino-acid peptides with two disulfide bonds that belong to the sarafotoxin family. ET-1, ET-2 and ET-3 are produced endogenously from preproendothelin to give big endothelins, which are cleaved by endothelin-converting enzyme (ECE) to yield the active protein. Endothelin has been shown to play important physiological and pathological roles by interacting with its G-protein-coupled receptors. There are two cloned ET receptors: the ET(A) receptor, which is selective for ET-1, and the ET(B) receptor, which binds ET-1, ET-2 and ET-3 with similar affinities. Since the discovery of endothelin, and especially since the availability of peptide ET antagonists such as BQ-123 and BQ-788, and nonpeptide compounds such as bosentan, considerable effort has been spent on better understanding the role of endothelin and its receptor antagonists. As a result, endothelin has been implicated in a variety of serious diseases, such as congestive heart failure, hypertension, pulmonary hypertension and prostate cancer. Research in pharmaceutical and biotechnology laboratories has generated many endothelin antagonists with either sulfonamide or triaryl carboxylic acid scaffolds, and a number of ET(A)-selective or nonselective ET(A)/ET(B) endothelin antagonists have entered clinical trials. This article will review the small-molecule ET(A)-selective and nonselective ET(A)/ET(B) antagonists that are under clinical evaluation, and highlight a member of this group of compounds, sitaxsentan. A summary of the medicinal chemistry that led to the identification of sitaxsentan will be presented, followed by selected animal and human clinical trial data. (c) 2001 Prous Science. All rights reserved.