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.

Discovery of a series of cyclohexylethylamine-containing protein farnesyltransferase inhibitors exhibiting potent cellular activity.

Synthesis of a library of secondary benzylic amines based on the Sebti-Hamilton type peptidomimetic farnesyltransferase (FTase) inhibitor FTI-276 (1) led to the identification of 6 as a potent enzyme inhibitor (IC(50) of 8 nM) which lacked the problematic thiol residue which had been a common theme in many of the more important FTase inhibitors reported to date. It has previously been disclosed that addition of o-tolyl substitution to FTase inhibitors of the general description 2 had a salutary effect on both FTase inhibition and inhibition of Ras prenylation in whole cells. Combination of these two observations led us to synthesize 7, a potent FTase inhibitor which displayed an IC(50) of 0.16 nM for in vitro inhibition of FTase and an EC(50) of 190 nM for inhibition of whole cell Ras prenylation. Modification of 7 by classical medicinal chemistry led to the discovery of a series of potent FTase inhibitors, culminating in the identification of 25 which exhibited an IC(50) of 0.20 nM and an EC(50) of 4.4 nM. In vivo tests in a nude mouse xenograft model of human pancreatic cancer (MiaPaCa cells) showed that oral dosing of 25 gave rise to impressive attenuation of the growth of this aggressive tumor cell line.

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.

Second-generation peptidomimetic inhibitors of protein farnesyltransferase demonstrating improved cellular potency and significant in vivo efficacy.

The synthesis and evaluation of analogues of previously reported farnesyltransferase inhibitors, pyridyl benzyl ether 3 and pyridylbenzylamine 4, are described. Substitution of 3 at the 5-position of the core aryl ring resulted in inhibitors of equal or less potency against the enzyme and decreased efficacy in a cellular assay against Ras processing by the enzyme. Substitution of 4 at the benzyl nitrogen yielded 26, which showed improved efficacy and potency and yet presented a poor pharmacokinetic profile. Further modification afforded 30, which demonstrated a dramatically improved pharmacokinetic profile. Compounds 26 and 29 demonstrated significant in vivo efficacy in nude mice inoculated with MiaPaCa-2, a human pancreatic tumor-derived cell line.

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.

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.

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.

[3H]A-81988, a potent, selective, competitive antagonist radioligand for angiotensin AT1 receptors.

Abbott-81988 (A-81988), 2-(N-n-Propyl-N-[(2'-[1H-tetrazol-5-yl]biphenyl-4- yl)methyl]amino)pyridine-3-carboxylic acid is a potent, competitive, non-peptidic antagonist of angiotensin AT1 receptors. A-81988 was labeled with tritium to high specific activity (16 Ci/mmol) and radioligand binding assays performed in rat liver membranes. [3H]A-81988 bound with high affinity (KD = 0.57 nM) and the KD determined from kinetics assays was similar. Non-specific binding (defined with 10(-6) M angiotensin-II) was very low (< 6% at the KD). The binding of [3H]A-81988 was competitive and exhibited appropriate pharmacological specificity for compounds acting at angiotensin AT1 receptors. These properties demonstrate that [3H]A-81988 will be a useful radioligand for studies of angiotensin AT1 receptors in various tissues.

Characterization of two endothelin converting enzymes and their preference for big endothelin-1 and -2 as substrates.

Two proteolytic activities that convert big ET to ET at neutral pH were identified in solubilized membranes prepared from rat lung. The endothelin-converting activities were partially purified by using A80227 ((2S,3R,4S)-2-([N-acetylcyclohexylalanyl-isoleucyl]amino)-1-(2-nap hthyl)-3,4-dihydroxy-6-methylheptane) coupled to an affinity-gel column (Affigel), and subsequently by concanavalin-A immobilized gel chromatography. An endothelin-converting activity was identified in the fraction containing proteins that did not bind to A80227-Affigel. This protease was sensitive to phosphoramidon, soybean trypsin inhibitor, and chymostatin, and preferred big ET-1 or big ET-2 as its substrate over bit ET-3. A second endothelin-converting activity was identified in the fraction containing proteins that bound to the A80227-coupled gel and was eluted by raising the pH. This protease exhibited activities throughout a range of pH 5.5-9.5, was inhibited by pepstatin A and A80227, and also preferred big ET-1 or big ET-2 over big ET-3 as its substrate. Both enzymes were glycoproteins based on their binding to concanavalin-A immobilized gel and were readily eluted by a buffer containing 0.5 M manopyranoside. The results from the pH and protease inhibitor profiles suggesting that these two ET-converting activities extracted from rat lung membranes are distinct and are different from the previously reported endothelin-converting enzymes.

Potent and selective inhibitors of an aspartyl protease-like endothelin converting enzyme identified in rat lung.

Two structurally distinct series of potent and selective inhibitors of an aspartyl protease-like endothelin converting enzyme (ECE) activity identified in the rat lung have been developed. Pepstatin A, which potently inhibits the rat lung ECE, served as the basis for the first series. Alternatively, selected renin inhibitors containing the dihydroxyethylene moiety were shown to be inhibitors of rat lung activity. Subsequent modifications improved inhibition of the rat lung ECE while eliminating renin activity. Both series of ECE inhibitors demonstrated a range of selectivity over Cathepsin D. Water-solubilizing moieties were appended onto selected compounds to facilitate in vivo testing. Partial reduction of the pressor response to exogenously administered Big ET-1 was observed with selected rat lung ECE inhibitors.