WO1997035864A1 - Phosphoramidates and phosphinic amides, their use to modulate the activity of endothelin - Google Patents

Abstract

Phosphoramidates and phosphinic amides and related compounds are provided. Also provided are methods that use the compounds for modulating the activity of the endothelin family of peptides are provided. In particular, compounds having formula (IIa) or (II), in which A, B, R?1 and R2¿ are as described are provided. Also provided are methods for treating endothelin-mediated disorders by administering effective amounts of one or more of these compounds or prodrugs thereof. Methods for elucidating the physiological and pathophysiological roles of endothelin, as well as isolating endothelin receptors are also provided.

Description

PHOSPHORAMIDATES AND PHOSPHINIC AMIDES, THEIR USE TO MODULATE THE ACTIVITY OF ENDOTHELIN

For any U.S. national purposes, this application is a continuation-in-part 5 of U.S. application Serial No. 08/624,706, filed March 26, 1 996, entitled "PHOSPHORAMIDATES, PHOSPHINIC AMIDES AND RELATED COMPOUNDS AND THE USE THEREOF TO MODULATE THE ACTIVITY OF ENDOTHELIN" to Ming Fai Chan and Erik Verner. The subject matter of this application is herein incorporated by reference.

10 For international purposes the benefit of prioriy to U.S. application

Serial No. 08/624,706, filed March 26, 1 996, is claimed. FIELD OF THE INVENTION

The present invention relates to the compounds that modulate the activity of the endothelin family of peptides. In particular, phosphoramidate compounds, phosphinic amide compounds and related compounds and use of these compounds as endothelin antagonists are 5 provided.

BACKGROUND OF THE INVENTION

The vascular endothelium releases a variety of vasoactive sub¬ stances, including the endothelium-derived vasoconstrictor peptide, endothelin (ET) (see, e.g. , Vanhoutte et al. (1 986) Annual Rev. Physiol.

1 0 48: 307-320; Furchgott and Zawadski ( 1 980) Nature 288: 373-376) . Endothelin, which was originally identified in the culture supernatant of porcine aortic endothelial cells (see, Yanagisawa et al. ( 1 988) Nature 332: 41 1 -41 5), is a potent twenty-one amino acid peptide vasoconstric¬ tor. It is the most potent vasopressor known and is produced by

1 5 numerous cell types, including the cells of the endothelium, trachea, kidney and brain. Endothelin is synthesized as a two hundred and three amino acid precursor preproendothelin that contains a signal sequence which is cleaved by an endogenous protease to produce a thirty-eight (human) or thirty-nine (porcine) ammo acid peptide. This intermediate, referred to as big endothelin, is processed in vivo to the mature biologically active form by a putative endothelin-convertmg enzyme (ECE) that appears to be a metal-dependent neutral protease (see, e.g. , Kashiwabara et al. ( 1 989) FEBS Lttrs. 247: 337-340). Cleavage is required for induction of physiological responses (see, e.g. , von Geldern et al. ( 1 991 ) Peptide Res. 4: 32-35). In porcine aortic endothelial cells, the thirty-nine ammo acid intermediate, big endothelin, is hydrolyzed at the Trp ¹-Val²² bond to generate endothelin- 1 and a C-terminal fragment. A similar cleavage occurs in human cells from a thirty-eight ammo acid intermediate. Three distinct endothelin isopeptides that exhibit potent vasoconstrictor activity have been identified. They are endothelιn-1 , endothelιn-2 and endothelιn-3.

The isopeptides are encoded by a family of three genes (see, e.g. , Inoue et al. ( 1 989) Proc. Natl. Acad. Sci. USA 86: 2863-2867; see, also Saida et al. ( 1 989)J. Biol. Chem. 264: 1 461 3-1461 6) . The nucleotide sequences of the three human genes are highly conserved within the region encoding the mature 21 amino acid peptides and the C-termmal portions of the peptides are identical. Endothelιn-2 is (Trp⁶, Leu⁷) endothelin- 1 and endothelin-3 is (Thr²,Phe⁴,Thr⁵,Tyr⁶,Lys⁷,Tyr¹⁴) endothelιn-1 . The endothelin peptides exhibit numerous biological activities in vitro and in vivo. Endothelin provokes a strong and sustained vasoconstriction in vivo in rats and in isolated vascular smooth muscle preparations; it also provokes the release of eicosanoids and endothelium-derived relaxing factor (EDRF) from perfused vascular beds Intravenous administration of endothelin- 1 and in vitro addition to vascular and other smooth muscle tissues produce long-lasting pressor effects and contraction, respectively (see, e.g. , Bolger et al. ( 1 991 ) Can. J. Phvsiol. Pharmacol. 69: 406-41 3) . In isolated vascular strips, for example, endothelin- 1 is a potent (EC₅₀ = 4 x 1 0 ¹⁰ M), slow acting, but persistent, contractile agent. A single dose in vivo elevates blood pressure in about twenty to thirty minutes. Endothelm-induced vasoconstriction is not affected by antagonists to known neurotransmitters or hormonal factors, but is abolished by calcium channel antagonists. The effect of calcium channel antagonists, however, is most likely the result of inhibition of calcium influx, since calcium influx appears to be required for the long-lasting contractile response to endothelin. Endothelin also mediates renin release, stimulates ANP release and induces a positive inotropic action in guinea pig atria. In the lung, endothelin- 1 acts as a potent bronchoconstrictor (Maggi et al. ( 1 989) Eur. J. Pharmacol. 1 60: 1 79-1 82) . Endothelin increases renal vascular resistance, decreases renal blood flow, and decreases glomerular filtrate rate. It is a potent mitogen for glomerular mesangial cells and invokes the phosphoinoside cascade in such cells (Simonson et al. (1 990) J. Clin. Invest. 85: 790-797). Release of endothelins from cultured endothelial cells is modulated by a variety of chemical and physical stimuli and appears to be regulated at the level of transcription and/or translation. Expression of the gene encoding endothelin-1 is increased by chemical stimuli, including adrenaline, thrombin and Ca^{2 +} ionophore. The production and release of endothelin from the endothelium is stimulated by angiotensin II, vasopressin, endotoxin, cyclosporine and other factors (see, Brooks et al. ( 1 991 ) Eur. J. Pharm. 1 94: 1 1 5-1 1 7), and is inhibited by nitric oxide. Endothelial cells appear to secrete short-lived endothelium-derived relaxing factors (EDRF) , including nitric oxide or a related substance (Palmer et al. ( 1 987) Nature 327: 524-526), when stimulated by vasoactive agents, such as acetylcholine and bradykinin. Endothelin-induced vasoconstriction is also attenuated by atrial natriuretic peptide (ANP) .

There are specific high affinity binding sites (dissociation constants in the range of 2-6 x 10^'10 M) for the endothelins in the vascular system and in other tissues, including the intestine, heart, lungs, kidneys, spleen, adrenal glands and brain. Binding is not inhibited by catecholamines, vasoactive peptides, neurotoxins or calcium channel antagonists. Endothelin binds and interacts with receptor sites that are distinct from other autonomic receptors and voltage dependent calcium channels. Competitive binding studies indicate that there are multiple classes of receptors with different affinities for the endothelin isopeptides. The sarafotoxins, a group of peptide toxins from the venom of the snake Atractaspis einqadensis that cause severe coronary vasospasm in snake bite victims, have structural and functional homology to endothelin- 1 and bind competitively to the same cardiac membrane receptors (Kloog et al. ( 1 989) Trends Pharmacol. Sci. 10: 21 2-214).

Two distinct endothelin receptors, designated ET_A and ET_B, have been identified and DNA clones encoding each receptor have been isolated (Arai et al. (1990) Nature 348: 730-732; Sakυrai et al. (1 990) Nature 348: 732-735) . Based on the amino acid sequences of the proteins encoded by the cloned DNA, it appears that each receptor contains seven membrane spanning domains and exhibits structural similarity to G-protein-coupled membrane proteins. Messenger RNA encoding both receptors has been detected in a variety of tissues, including heart, lung, kidney and brain. The distribution of receptor subtypes is tissue specific (Martin et al. ( 1 989) Biochem. Biophys. Res. Commun. 1 62: 1 30-1 37). ET_A receptors appear to be selective for endothelin-1 and are predominant in cardiovascular tissues. ET_B receptors are predominant in noncardiovascular tissues, including the central nervous system and kidney, and interact with the three endothelin isopeptides (Sakurai et al. ( 1 990) Nature 348: 732-734) . In addition, ET_A receptors occur on vascular smooth muscle, are linked to vasoconstriction and have been associated with cardiovascular, renal and central nervous system diseases; whereas ET_B receptors are located on the vascular endothelium, linked to vasodilation (Takayanagi et al. ( 1 991 ) FEBS Lttrs. 282: 103- 106) and have been associated with broncho- constrictive disorders.

By virtue of the distribution of receptor types and the differential affinity of each isopeptide for each receptor type, the activity of the endo¬ thelin isopeptides varies in different tissues. For example, endothelin-1 inhibits ¹²⁵l-labelled endothelin-1 binding in cardiovascular tissues forty to seven hundred times more potently than endothelin-3. ¹²⁵l-labelled endo- thelin-1 binding in non-cardiovascular tissues, such as kidney, adrenal gland, and cerebellum, is inhibited to the same extent by endothelin-1 and endothelin-3, which indicates that ET_A receptors predominate in cardiovascular tissues and ET_B receptors predominate in non- cardiovascular tissues.

Endothelin plasma levels are elevated in certain disease states (see, e.g. , International PCT Application WO 94/27979, and U.S. Patent No. 5,382,569, which disclosures are herein incorporated in their entirety by reference) . Endothelin-1 plasma levels in healthy individuals, as measured by radioimmunoassay (RIA), are about 0.26-5 pg/ml. Blood levels of endothelin- 1 and its precursor, big endothelin, are elevated in shock, myocardial infarction, vasospastic angina, kidney failure and a variety of connective tissue disorders. In patients undergoing hemodialysis or kidney transplantation or suffering from cardiogenic shock, myocardial infarction or pulmonary hypertension levels as high as 35 pg/ml have been observed (see, Stewart et al. ( 1 991 ) Annals Internal Med. 1 1 4: 464-469) . Because endothelin is likely to be a local, rather than a systemic, regulating factor, it is probable that the levels of endothelin at the endothelium/smooth muscle interface are much higher than circulating levels. Elevated levels of endothelin have also been measured in patients suffering from ischemic heart disease (Yasuda et al. ( 1 990) Amer. Heart _sL 119:801 -806, Ray et al. ( 1 992) Br. Heart J. 67:383-386). Circulating and tissue endothelin immunoreactivity is increased more than twofold in patients with advanced atherosclerosis (Lerman et al. ( 1 991 ) New Engl. J. Med. 325:997- 1001 ). Increased endothelin immunoreactivity has also been associated with Buerger's disease (Kanno er a/. ( 1 990) J. Amer. Med. Assoc. 264:2868) and Raynaud's phenomenon (Za ora et al. ( 1 990) Lancet 336 1 144-1 147) . Increased circulating endothelin levels were observed in patients who underwent percutaneous transluminal coronary angioplasty (PTCA) (Tahara et al. ( 1 991 ) Metab. Clin. Exp.

40: 1 235- 1 237; Sanjay et al. ( 1991 ) Circulation 84(SUPPI. 4):726), and in individuals with pulmonary hypertension (Miyauchi et al. ( 1 992) Jpn. J . Pharmacol.58:279P; Stewart et al. ( 1 991 ) Ann.lnternal Medicine 1 14:464-469) . Thus, there is clinical human data supporting the correlation between increased endothelin levels and numerous disease states.

Endothelin agonists and antagonists

Because endothelin is associated with certain disease states and is implicated in numerous physiological effects, compounds that can interfere with endothelin-associated activities, such as endothelin- receptor interaction and vasoconstrictor activity, are of interest. Compounds that exhibit endothelin antagonistic activity have been identified. For example, a fermentation product of Streptomyces misakiensis, designated BE-1 8257B, has been identified as an ET_A receptor antagonist. BE-18257B is a cyclic pentapeptide, cyclo(D-Glu-L- Ala-allo-D-lle-L-Leu-D-Trp), which inhibits ¹²⁵l-labelled endothelin-1 binding in cardiovascular tissues in a concentration-dependent manner (IC₅₀1.4 μM in aortic smooth muscle, 0.8 μM in ventricle membranes and 0.5 μM in cultured aortic smooth muscle cells), but fails to inhibit binding to receptors in tissues in which ET_B receptors predominate at concentrations up to 100 μM. Cyclic pentapeptides related to BE- 18257B, such as cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123), have been synthesized and shown to exhibit activity as ET_A receptor antagonists (see, U.S. Patent No.5,114,918 to Ishikawa et al; see, also, EP A1 0 436189 to BANYU PHARMACEUTICAL CO., LTD (October 7, 1991)). Studies that measure the inhibition by these cyclic peptides of endothelin-1 binding to endothelin-specific receptors indicate that these cyclic peptides bind preferentially to ET_A receptors. Other peptidic and non-peptidic ET_A antagonists have been identified (see, e.g., 5,352,800, 5,334,598, 5,352,659, 5,248,807, 5,240,910, 5,198,548, 5,187,195, 5,082,838). These include other cyclic peptides, acyltripeptides, hexapeptide analogs, certain antraquinone derivatives, indanecarboxylic acids, certain N-pyrimidylbenzenesulfonamides, certain benzenesulfon- amides, and certain naphthalenesulfonamides (see, e.g., Nakajima et al. (1991) J. Antibiot.44:1348-1356: Miyata era/. (1992) J. Antibiot. 45:74-8; Ishikawa etal. (1992) J.Med. Chem.35:2139-2142: U.S. Patent No.5, 114,918 to Ishikawa et al ; EP A1 0569193; EP A1 0558 258; EP A1 0436189 to BANYU PHARMACEUTICAL CO., LTD (October 7, 1991); Canadian Patent Application 2,067,288; Canadian Patent Application 2,071,193; U.S. Patent No. 5,208,243; U.S. Patent No.5,270,313; Cody et al (1993) Med. Chem. Res.3:154-162; Miyata etal (1992) J. Antibiot 45:1041-1046: Miyata et al (1992) J. Antibiot 45:1029-1040, Fujimoto et al (1992) FEBS Lett.305:41-44; Oshashi et al ( 1002) J. Antibiot 45: 1 684-1 685; EP A1 0 496 452, Clozel et al ( 1 993) Nature 365:759-761 : Internationai Patent Application W093/08799; Nishikibe et al ( 1 993) Life Sci. 52:71 7-724; and Benigni et al ( 1 993) Kidney Int. 44:440-444. U.S. Patent No. 5,464,853 and others) . In general, the identified compounds have activities in in vitro assays as ET_A antagonists at concentrations on the order of about 50 μM 100 μM or less. A number of such compounds have also been shown to possess activity in in vivo animal models. Very few, if any, selective ET_B antagonists have been described Certain phosphonic acid derivatives and related compounds have been identified that have activity as ECE inhibitors (see, e.g. , U.S. Patent Nos. 5,481 ,030, 5,476,847, 5,438,046, 5,380,921 and 5,330,978); there does not appear to be any evidence that any of these compounds inhibit the interaction of an endothelin peptide with an endothelin receptor.

Endothelin agonists and antagonists as therapeutic agents It has been recognized that compounds that exhibit activity at IC₅₀ or EC₅₀ concentrations on the order of 10^"4M or lower in standard in vitro assays that assess endothelin antagonist or agonist activity have pharmacological utility (see, e.g. , U.S. Patent Nos. 5,352,800,

5,334,598, 5,352,659, 5,248,807, 5,240,910, 5, 1 98,548, 5, 1 87, 1 95, 5,082,838 and 5,464,853). By virtue of this activity, such compounds are considered to be useful for the treatment of hypertension such as peripheral circulatory failure, heart disease such as angina pectoπs, cardiomyopathy, arteriosclerosis, myocardial infarction, pulmonary hyper¬ tension, vasospasm, vascular restenosis, Raynaud's disease, cerebral stroke such as cerebral arterial spasm, cerebral ischemia, late phase cerebral spasm after subarachnoid hemorrhage, asthma, bronchocon¬ striction, renal failure, particularly post-ischemic renal failure, cyclosporine nephrotoxicity such as acute renal failure, colitis, as well as other inflammatory diseases, endotoxic shock caused by or associated with endothelin, and other diseases in which endothelin has been implicated. Thus, in view of the numerous physiological effects of endothelin and its association with certain diseases, endothelin is believed to play a critical role in these pathophysiological conditions (see, e.g. , Saito et al ( 1 990) Hypertension 1 5: 734-738; Tomita et al. ( 1 989) N. Engl. J. Med. 321 : 1 1 27; Kurihara et al ( 1 989) J. Cardiovasc. Pharmacol. 1 3(Suppl. 5) : S1 3-S1 7: Doherty ( 1 992) J. Med. Chem. 35: 1 493-1 508; Morel et al ( 1 989) Eur. J. Pharmacol. 1 67: 427-428) . More detailed knowledge of the function and structure of the endothelin peptide family should provide insight into the progression and treatment of such conditions.

To aid in gaining further understanding of and to develop treat- ments for endothelin-mediated or related disorders, there is a need to identify compounds that modulate or alter endothelin activity. Identifi¬ cation of compounds that modulate endothelin activity, such as com¬ pounds that act as specific antagonists or agonists, may not only aid in elucidating the function of endothelin, but may yield in therapeutically useful compounds. In particular, compounds that specifically interfere with the interaction of endothelin peptides with ET_A, ET_B or other recep¬ tors should be useful in identifying essential characteristics of endothelin peptides, should aid in the design of therapeutic agents, and may be useful as disease specific therapeutic agents. Therefore, it is an object herein to provide compounds that have the ability to modulate the biological activity of one or more of the endothelin isopeptides. It is another object to provide compounds that have use as endothelin antagonists. It is also an object to use compounds that specifically interact with or inhibit the interaction of endothelin peptides with endothelin receptors as therapeutic agents for the treatment of endothelin-mediated diseases and disorders and/or as reagents for the identification of endothelin receptor subtypes and/or for the elucidation of the physiological and pathophysiological roles of endothelin.

SUMMARY OF THE INVENTION

Phosphoramidates, phosphinic amides and related compounds and methods using the compounds for modulating the interaction of an endothelin peptide with endothelin receptors are provided. In particular, phosphoramidate compounds, phosphinic amide compounds and related compounds, compositions containing these compounds and methods using the compounds for inhibiting the binding of an endothelin peptide to ET_A and/or ET_B receptors are provided.

The methods are effected by contacting endothelin receptors with one or more of the compounds prior to, simultaneously with, or subsequent to contacting the receptors with an endothelin peptide.

The compounds provided herein have formula I:

O A - P - N - Ar¹ _{( ! )}

B H

in which Ar¹ is selected from among substituted and unsubstituted alkyl groups or aryl and heteroaryl groups containing one or more, preferably one ring or two to three fused rings and from 3 up to about 21 members in the ring(s), in which the heteroaryl groups contain one to three heteroatoms selected from among O, S and N. In particular Ar¹ is selected from substituted or unsubstituted groups that include, but are not limited to, the following: naphthyl, phenyl, biphenyl, quinolyl, thienyl, furyl, isoquinolyl, pyrrolyl, pyridyl, indolyl, oxadiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyπmidyl, benzo[b]furyl, benzo[b]thιenyl and other such aryl and heteroaryl groups. Preferred among these groups are 5 to 6 membered aryl groups and heteroaryl groups that contain one or two heteroatom(s) . Ar¹ is unsubstituted or is substituted with one or more substituents, preferably selected from among NH₂, NO₂, halide, pseudohahde, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsufinyl, arylsulfonyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms and are either straight or branched chains or cyclic, and the aryl and heteroaryl portions contain from about 4 to about 1 6 members in the ring.

Ar¹ is preferably a five or six membered aryl or heteroaryl ring having at least two substituents selected independently from the group consisting of halide, pseudohahde, alkyl, alkoxy, alkenyl or alkynyl and is more preferably selected from among thienyl, furyl, isoquinolyl, pyrrolyl, pyridyl, indolyl, oxadiazolyl, pyrazolyl, isoxazolyl, isothiazolyl and pyπmidyl.

A and B are any groups such that the resulting compound inhibits binding by 50% of an endothelin peptide to an endothelin receptor, compared to binding in the absence of the compound or in the absence of the receptor, at a concentration of the compound of less than about 100 //M In particular, A and B are independently selected from among halo, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, thioalkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsufinyl, arylsulfonyl, heteroaryloxy, heteroarylamino, heteroarylthio, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido and ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms, preferably about 1 to 6 carbon atoms, and are either straight or branched chains or cyclic, and in which the aryl and heteroaryl portions are substituted or unsubstituted and contain from about 4 to about 1 6 members in the ring, preferably about 4 to 8 members. Preferably A and B are independently selected from among halo, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, haloalkyl, aryloxy, arylamino, arylthio, heteroaryloxy, heteroarylamino, heteroarylthio and haloaryl. A and B, in certain embodiments, are each independently selected from alkyl, alkenyl, alkynyl, alkoxy, or A and B are respectively Ar -X_p and Ar³-Y₀ in which o and p are 0 or 1 and Ar² and Ar³ are each independently selected from aryl and heteroaryl, and particularly aryl and heteroaryl groups that have 4 to 7 members, preferably 5 to 7 members in the ring. In particular, Ar² and Ar³ are each independently selected from among phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrrolyl, pyrazinyl, thienyl and furyl.

Ar¹ is, in certain embodiments, selected from groups such as:

where each R, which is the substituent or represents the substituent(s), which may be the same or different, on each group, is independently selected from NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsufinyl, arylsulfonyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 1 4 carbon atoms and are either straight or branched chains or cyclic, and the aryl and heteroaryl portions contain from about 4 to about 1 6 members in the ring. R is preferably H, halide, pseudohalide, alkyl, alkoxy, alkenyl or alkynyl, and is most preferably H, lower alkyl, lower alkoxy or halide.

In preferred embodiments, Ar¹ is an N-(5-isoxazotyl), N-(3- isoxazolyl) or N-pyridazinyl, preferably N-3-pyridazinyl, and A and B, independently, are aryl or aryloxy groups, alkyl, alkoxy, alkenyl, and are preferably phenyl, phenoxy, lower alkyl, lower alkenyl or lower alkoxy. In the embodiments described in detail herein, Ar¹ is generally an isoxazolyl group and the compounds are represented by the formula II:

in which R¹ and R² are independently selected from H, NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylaryl, aryloxy, arylamino, arylthio, haloaryl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms and are either straight or branched chains or cyclic, and the aryl and heteroaryl portions contain from about 4 to about 16 members, with the proviso that R² is not halide, pseudohalide, alkylsufinyl, alkylsulfonyl, arylsufinyl, arylsulfonyl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl or arylcarbonyl. In the preferred compounds herein, R¹ and R² are independently selected from among lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl, halide, pseudohalide or H; R² is not halide or pseudohalide. In certain preferred embodiments described herein, A and B are aryl, aryloxy, arylthioxy, heteroaryl, heteroaryloxy or heteroarylthioxy that preferably contain 4 to 7 member, more preferably 5 or 6 members in each ring. Of interest herein are compounds in which A and B are 5 to 6-membered aryloxy, aryl, heteroaryloxy, and heteroaryl groups. Of particular interest are compounds in which A and/or B are phenyl and phenoxy. In particular, in certain embodiments, A and B are each independently selected from alkyl, alkenyl, alkynyl, alkoxy, or A and B are respectively Ar²-X_p and Ar³-Y₀ in which o and p are 0 or 1 , and Ar² and Ar³ are each independently selected from aryl and heteroaryl, and particularly aryl and heteroaryl groups that have 4 to 7 members, preferably 5 to 7 members in the ring. Ar² and Ar³ are preferably each independently selected from among phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrrolyl, pyrazinyl, thienyl and furyl, and more preferably phenyl, pyrimidyl, pyrrolyl, thienyl and furyl, and most preferably phenyl and pyrimidyl.

Thus, among the compounds provided herein are compounds that have formula III:

in which R¹ and R² are each independently selected from H, N0₂ 2,' N *3' halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkylaryl, alkoxy, alkylamino, alkylthio, alkylsufinyl, alkylsulfonyl, aryloxy, arylalkyl, arylamino, arylthio, aryisufinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido, ureido, in which the alkyl, alkenyl, alkynyl portions are straight or branched chains of from about 1 up to about 10 carbons, preferably 1 to about 5 or 6 carbons, and in which the aryl portions contain from 3 up to about 1 0 carbons, preferably 6 carbons, with the proviso that R² is not halide, pseudohalide, alkylsufinyl, alkylsulfonyl, aryisufinyl, arylsulfonyl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl or arylcarbonyl;

X and Y are each independently selected from among 0, N, NR¹³, S or (CH₂)_n in which n is 0 to 10, preferably 0 to 3, more preferably 0 or 1 , most preferably 0; o and p are independently 0 or 1 ;

R¹³ contains up to about 30 carbon atoms, preferably 1 to 10, more preferably 1 to 6 and is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heterocycle, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl, C(O)R¹⁴ and S(0)_nR¹⁴ in which n is 0-2; R¹⁴ is selected from among hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heterocycle, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl; R¹³ and R¹⁴ are unsubstituted or are substituted with one or more substituents each selected independently from Z, which is hydrogen, halide, pseudohalide, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocycle, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl, and preferably selected from among H, a straight or branched carbon chain, preferably containing 1 to 6, more preferably 1 to 3, carbons, halide, alkoxyalkyl, or haloalkyl; and R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are each independently selected from among any of (i), (ii) , (iii), (iv) or (v):

(i) R³, R\ R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are each independently selected from among H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkoxyalkyl, alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylalkyl, alkylaryl, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido, ureido, or at least two of R³, R⁴, R⁵, R⁶ and R⁷ or R⁸, R⁹, R¹⁰, R^{1 1} and R¹² form alkylenedioxy, in which the alkyl, alkenyl, alkynyl portions are straight or branched chains of from about 1 up to about 1 0 carbons, preferably 1 to about 5 or 6 carbons, and in which the aryl portions contain from 3 up to about 1 0 carbons, preferably 6 carbons; or (ii) R⁴ and R⁷ and/or R¹⁰ and R^{1 1} together are substituted or unsubstituted 1 , 3-butadienyl, 4-dimethylamino-1 ,3 butadienyl, 1 -chloro- 1 ,3-butadienyl, 1 -aza- 1 ,3-butadienyl or 2-aza-1 ,3-butadienyl groups; and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are as defined in (i) above; or alternatively,

(iii) R⁷ and R³ and/or R ¹ and R¹² together are substituted or unsubstituted 1 , 3-butadienyl, 4-dimethylamino- 1 ,3 butadienyl, 1 -chloro- 1 ,3-butadienyl, 1 -aza- 1 ,3-butadienyl or 2-aza-1 ,3-butadienyl groups; and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are as defined in (i) above; or

(iv) R³, R⁵, and R⁷ and R¹⁰, R^{1 1} and R⁸ are H or are as defined in (i); and R⁴ and R⁶ and/or R⁹ and R¹² are each independently selected from alkyl, alkoxy, halide, aminoalkyl, dialkylaminoalkyl, in which the alkyl and alkoxy groups contain from 1 to 10, preferably 1 to 6 carbons, and are straight or branched chains, and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are as defined in (i) above; or (v) any two of R³, R⁴, R⁵, R⁶, and R⁷, and/or any two of R⁸, R⁹, R¹⁰,

R^{1 1} and R¹², which are each selected as in (i) form fused carbocyclic or heterocyclic rings.

In particularly preferred embodiments described herein, the compounds have formula III, in which R¹ and R² are independently selected from among lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl, halide, pseudohalide or H, with the proviso that R² is not halide or pseudohalide.

In the preferred compounds, X and Y are independently selected from among (CH₂)_n in which n is 1 to 3, and O; p and o are 0 or 1 ; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are independently selected from among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl and lower haloalkyl and at least three of R³, R⁴, R⁵, R⁶ and R⁷ are hydrogen and at least three of R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are hydrogen. In more preferred compounds, R² is selected from among lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl; and R¹ is lower alkyl or halide, particularly bromide or chloride.

Of the compounds described herein, those that inhibit or increase an endothelin-mediated activity by about 50% at concentrations of less than about 10 μM are preferred. More preferred are those that inhibit or increase an endothelin-mediated activity by about 50% at concentrations of less than about 5 μM and most preferably less than about 1 μM. The preferred IC₅₀ concentrations are set forth with reference to the in vitro assays exemplified herein.

Also among the most preferred compounds, for use in the methods provided herein, are those that are ET_A or ET_B selective. A compound is considered selective for a particular endothelin receptor subtype if it inhibits endothelin binding to the receptor at an IC₅₀ concentration that is at least 10-fold lower than its IC₅₀ concentration for other endothelin receptor subtypes. In particular, compounds that interact with ET_A receptors with an IC₅₀ of less than about 10 μM, preferably less than 1 μM, but with ET_B receptors with an IC₅₀ of greater than about 10 μM or compounds that interact with ET_B receptors with an IC₅₀ of less than about 10 μM, preferably less than 1 μM, but with ET_A receptors with an IC₅₀ of greater than about 10 μM are preferred.

Of particular interest herein are compounds that inhibit binding of endothelin to ET_A receptors at lower concentrations, preferably at least 5- to 1 0-fold, more preferably at least 50-fold and most preferably 1 00-fold, lower than they inhibit binding to ET_B receptors.

Preferred compounds are ET_A receptor selective or bind to ET_A receptors with an IC₅₀ of less than about 20 μM, more preferably less than about 1 0 μM and most preferably less than about 5μM.

Preferred compounds also include compounds that are ET_B receptor selective or that competitively inhibit binding of endothelin-1 to ET_B receptors at IC₅₀ concentrations of less than about 20 μM.

Pharmaceutical compositions formulated for administration by an appropriate route and by appropriate means, containing effective concentrations of one or more of the compounds provided herein or pharmaceutically acceptable salts or esters thereof, that deliver amounts effective for the treatment of hypertension, stroke, asthma, shock, ocular hypertension, glaucoma, renal failure, inadequate retinal perfusion or other conditions that are in some manner mediated by an endothelin peptide or that involve vasoconstriction or whose symptoms can be ameliorated by administration of an endothelin antagonist or agonist, are also provided. Particularly preferred compositions are those that deliver amounts effective for the treatment of hypertension or renal failure. The effective amounts and concentrations are effective for ameliorating any of the symptoms of any of the disorders.

Methods for treatment of endothelin-mediated disorders, including but not limited to, hypertension, asthma, shock, ocular hypertension, glaucoma, inadequate retinal perfusion and other conditions that are in some manner mediated by an endothelin peptide, or for treatment of disorders that involve vasoconstriction or that are ameliorated by administration of an endothelin antagonist or agonist are provided.

In particular, methods of treating endothelin-mediated disorders by administering effective amounts of the compounds, prodrugs or other suitable derivatives of the compounds are provided. Such disorders include but are not limited to hypertension, cardiovascular diseases, cardiac diseases including myocardial infarction, pulmonary hypertension, erythropoietin-mediated hypertension, respiratory diseases and inflammatory diseases, including asthma, bronchoconstriction, ophthalmologic diseases, gastroenteric diseases, renal failure, endotoxin shock, menstrual disorders, obstetric conditions, wounds, anaphylactic shock, hemorrhagic shock, and other diseases in which endothelin mediated physiological responses are implicated. Treatment involves, for instance, administration of effective amounts of one or more of the compounds provided herein in pharmaceutically acceptable carriers as provided herein. Preferred methods of treatment are methods for treatment of hypertension and renal failure.

More preferred methods of treatment are those in which the compositions contain at least one compound that inhibits the interaction of endothelin- 1 with ET_A or ET_B receptors at an IC₅₀ of less than about 10 M, preferably less than about 5 μM and more preferably less than about 1 μM. Other preferred methods are those in which the compositions contain one or more compounds that is (are) ET_A selective or one or more compounds that is (are) ET_B selective. Methods in which the compounds are ET_A selective are for treatment of disorders, such as hypertension; and methods in which the compounds are ET_B selective are for treatment of disorders, such as asthma, that require bronchodilation. In practicing the methods, effective amounts of compositions containing therapeutically effective concentrations of the compounds formulated for oral, intravenous, local and topical application for the treatment of hypertension, cardiovascular diseases, cardiac diseases, including myocardial infarction, respiratory diseases, including asthma, inflammatory diseases, ophthalmologic diseases, gastroenteric diseases, renal failure, immunosuppressant-mediated renal vasoconstriction, erythropoietin-mediated vasoconstriction, endotoxin shock, anaphylactic shock, hemorrhagic shock, pulmonary hypertension, and other diseases in which endothelin mediated physiological responses are implicated are administered to an individual exhibiting the symptoms of one or more of these disorders. The amounts are effective to ameliorate or eliminate one or more symptoms of the disorders.

Methods for the identification and isolation of endothelin receptor subtypes are also provided. In particular, methods for detecting, distinguishing and isolating endothelin receptors using the disclosed compounds are provided.

In addition, methods for identifying compounds that are suitable for use in treating particular diseases based on their preferential affinity for a particular endothelin receptor subtype are provided. Also provided are methods for elucidating the physiological and/or pathophysiological roles of endothelin using the compounds disclosed herein.

Articles of manufacture containing packaging material, a compound provided herein, which is effective for ameliorating the symptoms of an endothelin-mediated disorder, antagonizing the effects of endothelin or inhibiting binding of an endothelin peptide to an ET receptor with an IC₅₀ of less than about 50 μM, preferably 1 0 μM, within the packaging material, and a label that indicates that the compound or salt thereof is used for antagonizing the effects of endothelin, treating an endothelin-mediated disorder, or inhibiting the binding of an endothelin peptide to an ET receptor are provided. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference.

As used herein, endothelin (ET) peptides include peptides that have substantially the amino acid sequence of endothelin-1 , endothelin-2 or endothelin-3 and that act as potent endogenous vasoconstrictor peptides.

As used herein, an endothelin-mediated condition is a condition that is caused by abnormal endothelin activity or one in which compounds that inhibit endothelin activity have therapeutic use. Such diseases include, but are not limited to hypertension, cardiovascular disease, asthma, inflammatory diseases, ophthalmologic disease, menstrual disorders, obstetric conditions, gastroenteric disease, renal failure, pulmonary hypertension, endotoxin shock, anaphylactic shock, or hemorrhagic shock. Endothelin-mediated conditions also include conditions that result from therapy with agents, such as erythropoietin and immunosuppressants, that elevate endothelin levels.

As used herein an effective amount of a compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Typically, repeated administration is required to achieve the desired amelioration of symptoms. As used herein, an endothelin agonist is a compound that potentiates or exhibits a biological activity associated with or possessed by an endothelin peptide.

As used herein, an endothelin antagonist is a compound, such as a drug or an antibody, that inhibits endotheiin-stimulated vasoconstriction and contraction and other endothelin-mediated physiological responses. The antagonist may act by interfering with the interaction of the endothelin with an endothelin-specific receptor or by interfering with the physiological response to or bioactivity of an endothelin isopeptide, such as vasoconstriction. Thus, as used herein, an endothelin antagonist interferes with endotheiin-stimulated vasoconstriction or other response or interferes with the interaction of an endothelin with an endothelin- specific receptor, such as ET_A receptors, as assessed by assays known to those of skill in the art. The effectiveness of potential agonists and antagonists can be assessed using methods known to those of skill in the art. For example, endothelin agonist activity can be identified by its ability to stimulate vasoconstriction of isolated rat thoracic aorta or portal vein ring segments (Borges et al. ( 1 989) "Tissue selectivity of endothelin" Eur. J. Pharmacol. 1 65: 223-230) . Exemplary assays are set forth in the

EXAMPLES. As noted above, the preferred IC₅₀ concentration ranges are set forth with reference to assays in which the test compound is incubated with the ET receptor-bearing cells at 4°C. Data presented for assays in which the incubation step is performed at the less preferred 24°C are identified. It is understood that for purposes of comparison, these concentrations are somewhat higher than the concentrations determined at 4°C.

As used herein, the biological activity or bioactivity of endothelin includes any activity induced, potentiated or influenced by endothelin in vivo. It also includes the ability to bind to particular receptors and to induce a functional response, such as vasoconstriction. It may be assessed by in vivo assays or by in vitro assays, such as those exemplified herein. The relevant activities include, but are not limited to, vasoconstriction, vasorelaxation and bronchodilation. For example, ET_B receptors appear to be expressed in vascular endothelial cells and may mediate vasodilation and other such responses; whereas ET_A receptors, which are endothelin- 1 -specific, occur on smooth muscle and are linked to vasoconstriction Any assay known to those of skill in the art to measure or detect such activity may be used to assess such activity (see, e.g. , Spokes et al. ( 1 989) J. Cardiovasc. Pharmacol. 1 3(Suppl. 5_1:S1 91 -S1 92; Spinella et al ( 1 991 ) Proc. Natl. Acad. Sci. USA 88: 7443-7446; Cardell et al. (1 991 ) Neurochem. Int. 1 8:571 -574); and the Examples herein). As used herein, the IC₅₀ refers to an amount, concentration or dosage of a particular test compound that achieves a 50% inhibition of a maximal response, such as binding of endothelin to tissue receptors, in an assay that measures such response.

As used herein, EC₅₀ refers to a dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.

As used herein a phosphoramidate and/or phosphinic amide or related compounds that is ET_A selective refers to one that exhibits an IC₅₀ that is at least about 10-fold lower with respect to ET_A receptors than ET_B receptors.

As used herein, a phosphoramidate and/or phosphinic amide or related compound that is ET_B selective refers to one that exhibits an IC₅₀ that is at least about 10-fold lower with respect to ET_B receptors than ET_A receptors.

As used herein, a phosphoramidate refers to compounds, such as those of compound of formula III in which o and p are 1 ; a phosphinic amide refers to compounds in which o and p are 0, and related compounds refer to compounds in which one of o and p is 0 and the other is 1 .

As used herein, pharmaceutically acceptable salts, esters or other derivatives of the compounds include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs. For example, hydroxy groups can be esterified or etherified. As used herein, treatment means any manner in which the symptoms of a conditions, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use as contraceptive agents. As used herein, amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition. As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin Iayer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.

As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.

As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or pro¬ perties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, e.g. , Nogrady ( 1 985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392). For example, succinyl-sulfathiazole is a prodrug of 4-amino-N-(2-thiazoyl)ben- zenesulfonamide (sulfathiazole) that exhibits altered transport characteristics. As used herein, "halogen" or "halide" refers to F, Cl, Br or I.

As used herein, pseudohalides are compounds that behave substantially similar to halides. Such compounds can be used in the same manner and treated in the same manner as halides (X , in which X is a halogen, such as Cl or Br). Pseudohalides include, but are not limited to cyanide, cyanate, thiocyanate, selenocyanate and azide.

As used herein, lower alkyl, lower alkenyl, and lower alkynyl refer to carbon chains having less than about 6 carbons. In preferred embodiments of the compounds provided herein that include alkyl, alkenyl, or alkynyl portions include lower alkyl, lower alkenyl, and lower alkynyl portions.

As used herein aryl refers to cyclic groups containing from 3 to 1 5 or 1 6 carbon atoms, preferably from 5 to 10. Aryl groups include, but are not limited to groups, such as phenyl, substituted phenyl, napthyl, substituted naphthyl, in which the substituent is lower alkyl, halide, O, or lower alkoxy. Preferred aryl groups are lower aryl groups that contain less than 7 carbons in the ring structure.

As used herein, the nomenclature alkyl, alkoxy, carbonyl, etc. are used as is generally understood by those of skill in this art. For example, as used herein alkyl refers to saturated carbon chains that contain one or more carbons; the chains may be straight or branched or include cyclic portions or may be cyclic.

As used herein, "haloalkyl" refers to a lower alkyl radical in which one or more of the hydrogen atoms are replaced by halogen including, but not limited to, chloromethyl, trifluoromethyl, 1 -chloro-2-fluoroethyl and the like.

As used herein, "haloalkoxy" refers to RO- in which R is a haloalkyl group. As used herein, "aminocarbonyl" refers to -C(0)NH₂.

As used herein, "alkoxycarbonyl" refers to -C(0)OR in which R is alkyl, preferably lower alkyl or aryl, preferably lower aryl.

As used herein, "alkoxy" and "thioalkoxy" refer to RO- and RS-, in which R is alkyl, preferably lower alkyl or aryl, preferably lower aryl.

As used herein, "haloalkoxy" refers to RO- in which R is a haloalkyl group.

As used herein, heteroaryl refers to ring structures that include at least one carbon atom and at least one non-carbon atom, such as N, S, P and O. The rings may be single rings or two or more fused rings.

As used herein, any corresponding N-(4-halo-3-methyl-5-isox- azolyl), N-(4-halo-5-methγl-3-isoxazolyl), N-(3,4-dimethyl-5-isoxazolyl) , N- (4-halo-5-methyl-3-isoxazolyl), N-(4-halo-3-methyl-5-isoxazolyl), N-{4,5- dimethyl-3-isoxazolyl) derivative thereof refers to compounds in which A, B, X and Y are the same as the compound specifically set forth, but Ar¹ is N-(4-halo-3-methyl-5-isoxazolyl), N-(4-halo-5-methyl-3-isoxazolyl), N- (3,4-dimethyl-5-isoxazolyl), N-(4-halo-5-methyl-3-isoxazolyl), N-(4-halo-3- methyl-5-isoxazolyl), or N-(4,5-dimethyl-3-isoxazolyl) in which halo is any halide, preferably Cl or Br. As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, ( 1972) Biochem. 11:942-944). A. Compounds for use in treating endothelin-mediated diseases

Compounds and methods for treating endothelin-mediated diseases using the compounds of formula I are provided . In particular, among the compounds provided herein are those in which Ar¹ is an isoxazolyl group and the compounds are represented by the formula 11:

in which R¹ and R² are independently selected from H, NH₂, N0₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, aryisufinyl, arylsulfonyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms and.are either straight or branched chains or cyclic, and the aryl and heteroaryl portions contain from about 4 to about 1 6 members, with the proviso that R² is not halide, pseudohalide, alkylsufinyl, alkylsulfonyl, aryisufinyl, arylsulfonyl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl or arylcarbonyl. In the preferred compounds herein, R¹ and R² are independently selected from among lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl, halide, pseudohalide or H; R² is not halide or pseudohalide. In certain preferred embodiments described herein, A and B are aryl, aryloxy, arylthioxy, heteroaryl, heteroaryloxy or heteroarylthioxy that preferably contain 4 to 7 members, more preferably 5 or 6 members in each ring. Of interest herein are compounds in which A and B are 5 to 6-membered aryloxy, aryl, heteroaryloxy, and heteroaryl groups. Of particular interest are compounds in which A and/or B are phenyl and phenoxy. In particular, in certain embodiments, A and B are each independently selected from alkyl, alkenyl, alkynyl, alkoxy, or A and B are respectively Ar²-X_p and Ar³-Y₀ in which o and p are 0 or 1 , and Ar² and Ar³ are each independently selected from aryl and heteroaryl, and particularly aryl and heteroaryl groups that have 4 to 7 members, preferably 5 to 7 members in the ring. Ar² and Ar³ are preferably each independently selected from among phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrrolyl, pyrazinyl, thienyl and furyl, and more preferably phenyl, pyrimidyl, pyrrolyl, thienyl and furyl, and most preferably phenyl and pyrimidyl.

Of interest herein are compounds in which Ar² and Ar³ are 5- to 6- membered aryl and heteroaryl groups. Of particular interest are compounds in which A and/or B are phenyl and phenoxy.

Thus, among the compounds provided herein are compounds that have formula III:

in which R¹ and R² are each independently selected from H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, aryisufinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido, ureido, in which the alkyl, alkenyl, alkynyl portions are straight or branched chains of from about 1 up to about 10 carbons, preferably 1 to about 5 or 6 carbons, and in which the aryl portions contain from 3 up to about 1 0 carbons, preferably 6 carbons;

X and Y are each independently selected from among O, N, NR¹³, S or (CH₂)_n in which n is 1 to 10, preferably 1 to 3, more preferably 1 ; o and p are independently 0 or 1 ;

R¹³ contains up to about 30 carbon atoms, preferably 1 to 1 0, more preferably 1 to 6 and is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heterocycle, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl, C(O)R¹⁴ and S(0)_nR¹⁴ in which n is 0-2; R¹⁴ is selected from among hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heterocycle, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl; R¹³ and R¹⁴ are unsubstituted or are substituted with one or more substituents each selected independently from Z, which is hydrogen, halide, pseudohalide, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocycle, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl, and preferably selected from among H, a straight or branched carbon chain, preferably containing 1 to 6, more preferably 1 to 3, carbons, halide, alkoxyalkyl, or haloalkyl; and

R³, R\ R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independently selected from among any of (i), (ii), (iii), (iv) or (v):

(i) R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are each independently selected from among H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkoxyalkyl, alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido, ureido, or at least two of R³, R⁴, R⁵, R⁶ and R⁷ or R⁸, R⁹, R¹⁰, R^{1 1} and R¹² form alkylenedioxy where the alkyl, alkenyl, alkynyl portions are straight or branched chains of from about 1 up to about 1 0 carbons, preferably 1 to about 5 or 6 carbons, and in which the aryl portions contain from 3 up to about 1 0 carbons, preferably 6 carbons;

(ii) R⁴ and R⁷ and/or R¹⁰ and R¹¹ together are substituted or unsubstituted 1 , 3-butadienyl, 4-dimethylamino-1 ,3 butadienyl, 1 -chloro- 1 ,3-butadienyl, 1 -aza-1 , 3-butadienyl or 2-aza-1 ,3-butadienyl groups; and the others of R³, R\ R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are as defined in (i) above; or alternatively,

(iii) R⁷ and R³ and/or R¹¹ and R¹² together are substituted or unsubstituted 1 , 3-butadienyl, 4-dimethylamino-1 , 3 butadienyl, 1 -chloro- 1 ,3-butadienyl, 1 -aza-1 ,3-butadienyl or 2-aza-1 ,3-butadienyl groups; and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are as defined in (i) above; or

(iv) R³, R⁵, and R⁷ and'R¹⁰, R^{1 1} and R⁸ are H are as defined in (i); and R⁴ and R⁶ and/or R⁹ and R¹² are each independently selected from alkyl, alkoxy, halide, aminoalkyl, dialkylaminoalkyi, in which the alkyl and alkoxy groups contain from 1 to 10, preferably 1 to 6 carbons, and are straight or branched chains, and the others of R³, R\ R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are as defined in (i) above; or

(v) any two of R³, R⁴, R⁵, R⁶, and R⁷, and/or any two of R⁸, R⁹, R¹⁰, R¹¹ and R¹², which are each selected as in (i) form fused carbocyclic or heterocyclic rings.

In particularly preferred embodiments described herein, the compounds have formula III, in which R¹ and R² are independently selected from among lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl, halide, pseudohalide or H, with the proviso that R² is not halide or pseudohalide. ln the preferred compounds, X and Y are independently selected from among (CH₂)_n where n is 0 or 1 , and O; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are independently selected from among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl and lower haloalkyl and at least three of R³, R⁴, R⁵, R⁶ and R⁷ are hydrogen and at least three of R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are hydrogen.

In more preferred compounds, R² is selected from among lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl; and R¹ is lower alkyl or halide, particularly bromide or chloride. In the presently preferred compounds, the compounds have formula III in which X and Y are independently O or S, preferably O, and o and p are both either 0 or 1 . Compounds in which o and p are 1 are phosphoramidates, and compounds in which o and p are 0 are phosphinic amides. 1 . Phosphoramidates for use in treating endothelin-mediated diseases

Compounds and methods for treating endothelin-mediated diseases using the compounds of formula III are provided. In particular, the compounds of formula III in which X and Y are O have formula IV:

in which R¹ and R² are each independently selected from H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, aryisufinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido, ureido, in which the alkyl, alkenyl, alkynyl portions are straight or branched chains of from about 1 up to about 10 carbons, preferably 1 to about 5 or 6 carbons, and in which the aryl portions contain from 3 up to about 10 carbons, preferably 6 carbons; and R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² preferably are each independently selected from among H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkoxyalkyl, alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido, ureido, or at least two of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R 1 2 form alkylenedioxy, in which the alkyl, alkenyl, alkynyl portions are straight or branched chains of from about 1 up to about 1 0 carbons, preferably 1 to about 5 or 6 carbons, and in which the aryl portions contain from 3 up to about 1 0 carbons, preferably 6 carbons are provided. In more preferred of these embodiments, R is H, halide, pseudohalide, lower alkyl, lower alkenyl or lower haloalkyl, and R² is H, lower alkyl, lower alkenyl and lower haloalkyl; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are independently selected from among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl and lower haloalkyl, and preferably, at least three of R³, R⁴, R⁵, R⁶ and R⁷ are hydrogen and at least three of R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are hydrogen.

In the most preferred of these embodiments in which the compounds have formula IV, R¹ is halide, pseudohalide or lower alkyl, preferably lower alkyl or Br or Cl; R² is lower alkyl, preferably methyl or ethyl; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are independently selected from among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl and lower haloalkyl, preferably lower alkyl or hydrogen, and most preferably hydrogen.

2. Phosphinic amides for use in treating endothelin-mediated diseases

Compounds and methods for treating endothelin-mediated diseases using the compounds of formula I are provided. In particular, compounds in which Ar¹ is 5-isoxazole or 3-isoxazole and A and B are each phenyl are provided. In particular, compounds having formula V:

in which R¹ and R² are each independently selected from H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, aryisufinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido, ureido, or at least two of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² form alkylenedioxy, in which the alkyl, alkenyl, alkynyl portions are straight or branched chains of from about 1 up to about 10 carbons, preferably 1 to about 5 or 6 carbons, and in which the aryl portions contain from 3 up to about 10 carbons, preferably 6 carbons; and

R³, R\ R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independently selected from among H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkoxyalkyl, alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido, ureido, in which the alkyl, alkenyl, alkynyl portions are straight or branched chains of from about 1 up to about 10 carbons, preferably 1 to about 5 or 6 carbons, and in which the aryl portions contain from 3 up to about 10 carbons, preferably 6 carbons are provided. ln the more preferred of the embodiments in which the compounds have formula V, R¹ is halide, pseudohalide or lower alkyl, preferably lower alkyl or Br or CI; R² is lower alkyl, preferably methyl or ethyl; and R³, R\ R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are independently selected from among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl and lower haloalkyl, preferably lower alkyl or hydrogen, and most preferably hydrogen.

In more preferred of these embodiments, R¹ is halide, pseudohalide or lower alkyl, preferably lower alkyl or bromine and R² is lower alkyl, preferably methyl or ethyl; and

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are each independently selected from among H, NHf, halide, pseudohalide, lower alkyl, lower alkenyl and lower haloalkyl, preferably lower alkyl or hydrogen, and most preferably hydrogen.

B. Activities of the compounds

Exemplary compounds were synthesized and tested using the exemplified assays (see, EXAMPLES) and selected results are set forth in Table 1 :

TABLE 1

COMPOUND ET_A (//MΓ ET_B ( Mf ^τ

N-(3,4-dimethyl-5-isoxazolyl)- 2.5 > 100 diphenylphosphoramidate

N-(3,4-dimethyl-5-isoxazolyl)diphenylphosphinic 7.1 > 100 amide

N-(4-bromo-3-met yl-5-isoxa- 0.58 77 zolyDdiphenylphosphoramidate COMPOUND ET_A (μM) ET_B (μM)^M

N-(4-bromo-3-methyl-5-ιsoxa- 0.84 85 zolyOdiphenylphosp inic amide

N-(4-bromo-3-methyl-5-ιsoxa- 20 > 100 zolyDdiethylphosphoramidate

* results generally from 1 , 2 or 3 experiments with the same preparation

¹ assay performed with incubation at 4° C. As described in the

Examples, incubation at the higher temperature reduces the activity by a factor of 2- to about 10-compared to the activity at 4° C

- data not available or measured as % inhibition @ 1 00 M

% = % inhibition @ 100 μM

D. Preparation of the compounds

Preparation of exemplary compounds is described in detail in the examples. The compounds provided herein may be synthesized according to a method discussed in general below and set forth in the Examples by selecting appropriate starting materials.

In general, the syntheses involve the condensation of a chlorophosphate or phosphinic chloride with an aminoisoxazole in dry pyridine or in tetrahydrofuran (THF) and sodium hydride. The chlorophosphates or phosphinic chlorides and aminoisoxazoles either can be obtained commercially or synthesized according to methods available to those of skill in this art.

In particular, the compounds may be prepared by reacting an appropriate phosphinic chloride or chlorophosphate with 5-amino- isoxazoles substituted at the 3 and 4 positions, such as 4-bromo-3- methyl-5-aminoisoxazole, in tetrahydrofuran (THF) solution containing a base, such as sodium hydride. Following the reaction, the mixture is diluted with ethyl acetate and washed with HCI. The organic Iayer is dried over anhydrous magnesium sulfate, filtered and concentrated. The residue is purified by flash chromatography using ethyl acetate/hexanes followed by recrystallization using chloroform and hexanes. The N-(4-haloisoxazolyl)phosphoramidates and N-(4- haloisoxazolyDphosphinic amides can be prepared by condensation of an amino-4-haloisoxazole with a chlorophosphate or phosphinic chloride, respectively, in THF with sodium hydride as a base. For example, N-(4- bromo-3-methyl-5-isoxazolyl)diphenylphosphoramidate was prepared from 4-bromo-3-methyl-5-aminoisoxazole and diphenyl chlorophosphate in THF and sodium hydride. N-(4-bromo-3-methyl-5-isoxazolyl)diethyl- phosphoramidate was prepared from 4-bromo-3-methyl-5-aminoisoxazo!e and diethyl chlorophosphate.

Alternatively, the phosphoramidates and/or phosphinic amides can be prepared from the corresponding phosphinic chloride or chlorophosphate and the appropriately substituted aminoisoxazole in pyridine. Following the reaction, the mixture is diluted with ethyl acetate, washed with HCI and the organic Iayer dried, filtered and concentrated. The residue is purified by flash chromatography using methanol/chloroform, followed by recrystallization from ethyl acetate or chloroform and hexanes. The N-(alkylisoxazolyl)phosphoramidates and N-(alkylisoxazolyl)- phosphinic amides can be prepared by condensing an alkylaminoisoxazole with a chlorophosphate or phosphinic chloride, respectively, in dry pyridine. For example, the N-(3,4-dimethyl-5-isoxazolyl)phosphor- amidates and N-(3,4-dimethyl-5-isoxazolyl)phosphinic amides can be prepared from 3,4-dimethyl-5-aminoisoxazole and the appropriately substituted chlorophosphate or phosphinic chloride in pyridine. Prodrugs and other derivatives of the compounds suitable for administration to humans may also be designed and prepared by methods known to those of skill in the art (see, e.g. , Nogrady ( 1 985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392) .

Compounds listed and described have been synthesized and tested for activity in in vitro assays and, in some cases, in in vivo animal models. Nuclear magnetic resonance spectroscopic (NMR), mass spectrometric, infrared spectroscopic and high performance liquid chromatographic analyses indicated that the synthesized compounds have structures consistent with those expected for such compounds and are generally at least about 98% pure. All of the compounds exemplified or described herein exhibited" activity as endothelin antagonists. D. Evaluation of the bioactivity of the compounds Standard physiological, pharmacological and biochemical procedures are available for testing the compounds to identify those that possess any biological activities of an endothelin peptide or the ability to interfere with or inhibit endothelin peptides. Compounds that exhibit in vitro activities, such as the ability to bind to endothelin receptors or to compete with one or more of the endothelin peptides for binding to endothelin receptors can be used in the methods for isolation of endothelin receptors and the methods for distinguishing the specificities of endothelin receptors, and are candidates for use in the methods of treating endothelin-mediated disorders. Thus, compounds of formulae l-V that are endothelin antagonists or agonists may be identified using such screening assays. 1. Identifying compounds that modulate the activity of an endothelin peptide

The compounds are tested for the ability to modulate the activity of endothelin-1. Numerous assays are known to those of skill in the art for evaluating the ability of compounds to modulate the activity of endothelin (see, e.g., U.S. Patent No.5,114,918 to Ishikawa et al; EP

A1 0436189 to BANYU PHARMACEUTICAL CO., LTD. (October 7,

1991 ); Borges et al. ( 1989) Eur. J. Pharm. 165: 223-230; Filep et al.

(1991) Biochem. Biophvs. Res. Commun. 177: 171-176). In vitro studies may be corroborated with in vivo studies (see, e.g., U.S. Patent No.5,114,918 to Ishikawa etal; EP A1 0436189 to BANYU PHARMACEUTICAL CO., LTD. (October 7, 1991)) and pharmaceutical activity thereby evaluated..Such assays are described in the Examples herein and include the ability to compete for binding to ET_A and ET_B receptors present on membranes isolated from cell lines that have been genetically engineered to express either ET_A or ET_B receptors on their cell surfaces.

The properties of a potential antagonist may be assessed as a function of its ability to inhibit an endothelin induced activity in vitro using a particular tissue, such as rat portal vein and aorta as well as rat uterus, trachea and vas deferens (see e.g. , Borges, R., Von Grafenstein, H. and Knight, D.E., Tissue selectivity of endothelin, Eur. J. Pharmacol 165:223-230. (1989)). The ability to act as an endothelin antagonist in vivo can be tested in hypertensive rats, ddy mice or other recognized animal models (see, Kaltenbronn et al. (1990) J. Med. Chem.33:838- 845, see, also, U.S. Patent No.5,114,918 to Ishikawa et al; and EP A1 0436189 to BANYU PHARMACEUTICAL CO., LTD (October 7, 1991); see, also Bolger et al. (1983) J. Pharmacol. Exp. Ther.225291-309). Using the results of such animal studies, pharmaceutical effectiveness may be evaluated and pharmaceutically effective dosages determined. A potential agonist may also be evaluated using in vitro and in vivo assays known to those of skill in the art.

Endothelin activity can be identified by the ability of a test compound to stimulate constriction of isolated rat thoracic aorta (Borges et al. (1 989) "Tissue selectivity of endothelin" Eur. J. Pharmacol. 1 65: 223-230) . To perform the assay, the endothelium is abraded and ring segments mounted under tension in a tissue bath and treated with endothelin in the presence of the test compound. Changes in endothelin induced tension are recorded. Dose response curves may be generated and used to provide information regarding the relative inhibitory potency of the test compound. Other tissues, including heart, skeletal muscle, kidney, uterus, trachea and vas deferens, may be used for evaluating the effects of a particular test compound on tissue contraction. Endothelin isotype specific antagonists may be identified by the ability of a test compound to interfere with endothelin binding to different tissues or cells expressing different endothelin-receptor subtypes, or to interfere with the biological effects of endothelin or an endothelin isotype (Takayanagi et al. ( 1 991 ) Reg. Pep. 32: 23-37, Panek et al. ( 1992) Biochem. Biophys. Res. Commun. 1 83: 566-571 ) . For example, ET_B receptors are expressed in vascular endothelial cells, possibly mediating the release of prostacyclin and endothelium-derived relaxing factor (De Nucci et al. ( 1 988) Proc. Natl. Acad. Sci. USA 85:9797) . ET_A receptors are not detected in cultured endothelial cells, which express ET_B receptors.

The binding of compounds or inhibition of binding of endothelin to ET_B receptors can be assessed by measuring the inhibition of endothelin- 1 -mediated release of prostacyclin, as measured by its major stable metabolite, 6-keto PGF_{l σ}, from cultured bovine aortic endothelial cells (see, e.g. , Filep et al. ( 1 991 ) Biochem. and Biophys Res. Commun. 1 77: 1 71 - 1 76) . Thus, the relative affinity of the compounds for different endothelin receptors may be evaluated by determining the inhibitory dose response curves using tissues that differ in receptor subtype. Using such assays, the relative affinities of the compounds for ET_A receptors and ET_B receptors have been and can be assessed. Those that possess the desired properties, such as specific inhibition of binding of endothelin-1 , are selected. The selected compounds that exhibit desirable activities may be therapeutically useful and are tested for such uses using the above-described assays from which in vivo effectiveness may be evaluated (see, e.g. , U.S. Patent No. 5,248,807; U.S. Patent No. 5,240,910; U.S. Patent No. 5, 1 98,548; U.S. Patent No. 5, 1 87, 1 95; U.S. Patent No. 5,082,838; U.S. Patent No. 5,230,999; published Canadian Application Nos. 2,067,288 and 2071 1 93; published Great Britain Application No. 2,259,450; Published International PCT Application No. WO 93/08799; Benigi et al. ( 1 993) Kidney International 44:440-444; and Nirei et al. ( 1 993) Life Sciences 52: 1 869-1 874) . Compounds that exhibit in vitro activities that correlate with in vivo effectiveness will then be formulated in suitable pharmaceutical compositions and used as therapeutics.

The compounds also may be used in methods for identifying and isolating endothelin-specific receptors and aiding in the design of compounds that are more potent endothelin antagonists or agonists or that are more specific for a particular endothelin receptor. 2. Isolation of endothelin receptors

A method for identifying endothelin receptors is provided. In practicing this method, one or more of the compounds is linked to a support and used in methods of affinity purification of receptors. By seiecting compounds with particular specificities, distinct subclasses of ET receptors may be identified .

One or more of the compounds may be linked to an appropriate resin, such as Affi-gel, covalently or by other linkage, by methods known to those of skill in the art for linking endothelin to such resins (see, Schvartz et al. ( 1 990) Endocrinology 1 26: 321 8-3222). The linked compounds can be those that are specific for ET_A or ET_B receptors or other subclass of receptors.

The resin is pre-equilibrated with a suitable buffer generally at a physiological pH (7 to 8). A composition containing solubilized receptors from a selected tissue are mixed with the resin to which the compound is linked and the receptors are selectively eluted. The receptors can be identified by testing them for binding to an endothelin isopeptide or analog or by other methods by which proteins are identified and characterized. Preparation of the receptors, the resin and the elution method may be performed by modification of standard protocols known to those of skill in the art (see, e.g. , Schvartz et al. (1990) Endocrinology 1 26: 321 8-3222).

Other methods for distinguishing receptor type based on differential affinity to any of the compounds herein are provided. Any of the assays described herein for measuring the affinity of selected compounds for endothelin receptors may also be used to distinguish receptors subtypes based on affinity for particular compounds provided herein. In particular, an unknown receptor may be identified as an ET_A or ET_B receptor by measuring the binding affinity of the unknown receptor for a compound provided herein that has a known affinity for one receptor over the other. Such preferential interaction is useful for determining the particular disease that may be treated with a compound prepared as described herein. For example, compounds with high affinity for ET_A receptors and little or no affinity for ET_B receptors are candidates for use as hypertensive agents; whereas, compounds that preferentially interact with ET_B receptors are candidates for use as anti-asthma agents. E. Formulation and administration of the compositions Effective concentrations of one or more of the phosphoramidate or phosphinic amide compounds of formulae l-V or pharmaceutically acceptable salts, esters or other derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle.

In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as tween, or dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts of the compounds or prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.

The concentrations of the compounds are effective for delivery of an amount, upon administration, that ameliorates the symptoms of the endothelin-mediated disease. Typically, the compositions are formulated for single dosage administration.

Upon mixing or addition of the phosphoramidate or phosphinic amide compound(s), the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined. Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.

The active compounds can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration.

Preferred modes of administration include oral and parenteral modes of administration.

The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems (see, e.g. , U.S. Patent No. 5, 1 14,91 8 to Ishikawa et al ; EP A1 0 436 1 89 to BANYU PHARMACEUTICAL CO. , LTD (October 7, 1 991 ); Borges et al. ( 1 989) Eur. J. Pharm. 1 65: 223-230; : Filep et al. ( 1 991 ) Biochem. Biophys. Res. Commun. 177: 171 -176) and then extrapolated therefrom for dosages for humans.

The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, the amount that is delivered is sufficient to treat the symptoms of hypertension. Typically a therapeutically effective dosage should produce a serum concentration of active ingredient of from about 0.1 ng/ml to about 50-100 μg/ml. The pharmaceutical compositions typically should provide a dosage of from about 0.01 mg to about 2000 mg of compound per kilogram of body weight per day. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

If oral administration is desired, the compound should be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth and gelatin; an excipient such as starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, and fruit flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics. For example, if the compound is used for treating asthma or hypertension, it may be used with other bronchodilators and antihypertensive agents, respectively. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA) ; buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Parental preparations can be enclosed in ampules, disposable syringes or multiple dose vials made of glass, plastic or other suitable material.

If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Patent No. 4,522,81 1 .

The active compounds may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for preparation of such formulations are known to those skilled in the art.

The compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Such solutions, particularly those intended for ophthalmic use, may be formulated as 0.01 % - 1 0% isotonic solutions, pH about 5-7, with appropriate salts. The compounds may be formulated as aerosols for topical application, such as by inhalation (see, e.g. , U.S. Patent Nos. 4,044, 1 26, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment inflammatory diseases, particularly asthma). Finally, the compounds may be packaged as articles of manufacture containing packaging material, a compound provided herein, which is effective for antagonizing the effects of endothelin, ameliorating the symptoms of an endothelin-mediated disorder, or inhibiting binding of an endothelin peptide to an ET receptor with an IC₅₀ of less than about 50 μM, preferably about 20 μM or less, more preferably less than 10 μM, within the packaging material, and a label that indicates that the compound or salt thereof is used for antagonizing the effects of endothelin, treating endothelin-mediated disorders or inhibiting the binding of an endothelin peptide to an ET receptor. The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLE 1 N-(3,4-dimethyl-5-isoxazolyl)diphenylphosphoramidate 3,4-Dimethyl-5-aminoisoxazole (0.34 g, 3.0 mmol) and diphenyl chlorophosphate (0.62 ml 3.3mmol) were stirred in pyridine (7ml) at ambient temperature for 1 h, then diluted with ethyl acetate ( 1 00 ml) and washed with 2% HCI (3 x 100 ml). The organic Iayer was dried (MgSO₄), filtered and concentrated to collect 0.73 g of a light orange oil. Flash chromatography (2% methanol/chloroform) followed by recrystallization from chloroform and hexanes provided 0.31 g (30%) of white crystals, m.p. 97-99° C. EXAMPLE 2

N-(3,4-dimethyl-5-isoxazolyl)diphenylphosphinic amide

N-(3,4-dimethyl-5-isoxazolyl)diphenyl phosphinic amide was prepared by the method of Example 1 with 3,4-dimethyl-5-amino isoxazole (0.22 g, 2.0 mmol), diphenyl phosphinic chloride (0.6 ml, 3.0 mmol) and pyridine (5 ml). Flash chromatography (5% methanol/chloro¬ form) followed by recrystallization from ethyl acetate and hexanes provided 0.38g (61 %) of white crystals, m.p. 1 67-1 70°C.

EXAMPLE 3

N-(4-bromo-3-methyl-5-isoxazolyl)diphenylphosphoramidate

NaH (60% oil dispersion, 84 mg, 2.1 mmol) was added to 4- bromo-3-methyl-5-amino isoxazole (0.1 8 g, 1 .0 mmol) in THF (5ml) at ambient temperature. After 1 0 min. of stirring diphenyl chlorophosphate (0.23 ml, 1 .1 mmol) was added. The reaction solution was stirred 1 hr at ambient temperature, then diluted with ethyl acetate d OOmls) and washed with 2% HCI (2 x 100 ml) . The organic was dried (MgSO₄), filtered and concentrated. Flash chromatography (50% ethyl acetate/hexane) followed by recrystallization from chloroform and hexanes provided 0.26 g (64%) of tan crystals, m.p. 1 1 6.5-1 1 8.5° C.

EXAMPLE 4

N-(4-bromo-3-methyl-5-isoxazolyl)diphenylphosphinic amide

N-(4-bromo-3-methyl-5-isoxazolyl)diphenyl phosphinic amide was prepared by the method of Example 3 with 4-bromo-3-methyl-5-amino isoxazole (0.1 8 g, 1 .0 mmol), THF (5 ml), NaH (60% oil dispersion, 88 mg, 2.2 mmol), and diphenyl phosphinic chloride (0.23 ml, 1 .2 mmol). Recrystallization three times from chloroform and hexanes provided 0.1 7 g (45%) of faint pink crystals, m.p. 1 78.5- 1 81 ° C. EXAMPLE 5

N-(4-bromo-3-methyl-5-isoxazolyl)diethylphosphoramidate

N-(4-bromo-3-methyl-5-isoxazolγl)diethyl phosphoramidate was prepared by the method of Example 3 with 4-bromo-3-methyl-5-amino isoxazole (0.27 g, 1 .5 mmol), THF (8 ml), NaH (60% oil dispersion,

0. 1 5 g, 3.8 mmol) and diethyl chlorophosphate (0.25 ml, 1 .7 mmol) .

Recrystallization two times from chloroform and hexanes provided 0.24 g (51 %) of light yellow crystals, m.p. 78-80° C.

EXAMPLE 6

Assays for identifying compounds that exhibit endothelin antagonistic and/or agonist activity

Compounds that are potential endothelin antagonists are identified by testing their ability to compete with ¹²⁵l-labeled ET-1 for binding to human ET_A receptors or ET_B receptors present on isolated cell membranes. The effectiveness of the test compound as an antagonist or agonist of the biological tissue response of endothelin can also be assessed by measuring the effect on endothelin induced contraction of isolated rat thoracic aortic rings. The ability of the compounds to act as antagonists or agonists for ET_B receptors can be assessed by testing the ability of the compounds 'to inhibit endothelin-1 induced prostacyclin release from cultured bovine aortic endothelial cells.

A. Endothelin binding inhibition - Binding Test #1 : Inhibition of binding to ET_A receptors

TE 671 cells (ATCC Accession No. HTB 1 39) express ET_A receptors. These cells were grown to confluence in T-1 75 flasks. Cells from multiple flasks were collected by scraping, pooled and centrifuged for 1 0 min at 1 90 X g. The cells were resuspended in phosphate buffered saline (PBS) containing 10 mM EDTA using a Tenbroeck homogenizer. The suspension was centrifuged at 4°C at 57,800 X g for 1 5 min, the pellet was resuspended in 5 ml of buffer A (5mM HEPES buffer, pH 7.4 containing aprotinin ( 100 KlU/ml)) and then frozen and thawed once. 5 ml of Buffer B (5 mM HEPES Buffer, pH 7.4 containing 1 0 mM MnCI₂ and 0.001 % deoxyribonuclease Type 1 ) was added, the suspension mixed by inversion and then incubated at 37 °C for 30 minutes. The mixture was centrifuged at 57,800 X g as described above, the pellet washed twice with buffer A and then resuspended in buffer C (30 mM HEPES buffer, pH 7.4 containing aprotinin ( 1 00 KlU/ml) to give a final protein concentration of 2 mg/ml and stored at -70°C until use.

The membrane suspension was diluted with binding buffer (30 mM HEPES buffer, pH 7.4 containing 1 50 mM NaCl, 5mM MgCI₂, 0.5% Bacitracin) to a concentration of 8 μg/50 μl. ¹²⁵l-endothelin-1 (3,000 cpm, 50 mL) was added to 50 μL of either: (A) endothelin- 1 (for non specific binding) to give a final concentration 80 nM); (B) binding buffer (for total binding); or (C) a test compound (final concentration 1 nM to 100 μM) . The membrane suspension (50 μL), containing up to 8 μg of membrane protein, was added to each of (A), (B), or (C) . Mixtures were shaken, and incubated at 4°C for 1 6-1 8 hours, and then centrifuged at 4°C for 25 min at 2,500 X g. Alternatively, the incubation can be conducted at other temperatures, such as 24°C. When incubated at 24°C, the IC₅₀ concentrations are 2- to 1 0-fold higher than when the incubation is conducted at 4°C.

The supernatant, containing unbound radioactivity, was decanted and the pellet counted on a Genesys multiwell gamma counter. The degree of inhibition of binding (D) was calculated according to the following equation: (C) - (A)

% D = 1 00 - X 100

(B) - (A)

Each test was generally performed in triplicate. B. Endothelin binding inhibition - Binding Test #2: Inhibition of binding to ET_B receptors

COS7 cells were transfected with DNA encoding the ET_B receptor,

The resulting cells, which express the human ET_B receptor, were grown to confluence in T-1 50 flasks. Membrane was prepared as described above. The binding assay was performed as described above using the membrane preparation diluted with binding buffer to a concentration of 1 μg/50 μl.

Briefly, the COS7 cells, described above, that had been transfected with DNA encoding the ET_B receptor and express the human ET_B receptor on their surfaces were grown to confluence in T-1 75 flasks. Cells from multiple flasks were collected by scraping, pooled and centrifuged for 10 min at 190 X g. The cells were resuspended in phosphate buffered saline (PBS) containing 10 mM EDTA using a Tenbroeck homogenizer. The suspension was centrifuged at 4°C 57,800 X g for 1 5 min, the pellet was resuspended in 5 ml of buffer A (5mM

HEPES buffer, pH 7.4 containing aprotinin ( 100 KlU/ml)) and then frozen and thawed once. Five ml of Buffer B (5 M HEPES Buffer, pH 7.4 containing 10 mM MnCI₂ and 0.001 % deoxyribonuclease Type 1 ) was added, the suspension mixed by inversion and then incubated at 37°C for 30 minutes. The mixture was centrifuged at 57,800 X g as described above, the pellet washed twice with buffer A and then resuspended in buffer C (30 mM HEPES buffer, pH 7.4 containing aprotinin ( 100 KlU/ml) to give a final protein concentration of 2 mg/ml.

The binding assay was performed as described above using the membrane preparation diluted to give 1 μg/50 μl of binding buffer. C. Test for activity against endothelin-induced contraction of isolated rat thoracic aortic rings

The effectiveness of the test compound as an antagonist or agonist of the biological tissue response of endothelin also is assessed by measuring the effect on endothelin induced contraction of isolated rat thoracic aortic rings (see, e.g. , Borges et al ( 1 989) Eur. J. Pharmacol. 1 65:223-230) or by measuring the ability to contract the tissue when added alone.

Compounds to be tested are prepared as 1 00 μM stocks. If necessary to effect dissolution, the compounds are first dissolved in a minimum amount of DMSO and diluted with 1 50 mM NaCl. Because DMSO can cause relaxation of the aortic ring, control solutions containing varying concentrations of DMSO were tested.

The thoracic portion of the adult rat aorta is excised, the endothelium abraded by gentle rubbing and then cut into 3 mm ring segments. Segments are suspended under a 2 g preload in a 10 ml organ bath filled with Krebs'- Henseleit solution saturated with a gas mixture of 95% O₂ and 5% CO₂ ( 1 1 8 mM NaCl, 4,7 mM KCl, 1 .2 mM MgS0₄, 1 .2 mM KH₂PO₄, 25 mM NaHC0₃, 2.5 mM CaCI₂, 10 mM D- glucose).

There is a correlation between activity as an antagonist of endothelin-induced thoracic aortic ring contraction and activity as an inhibitor of binding of endothelin to endothelin receptors. The pA₂ is a linear function of the log of the IC₅₀. D. Assay for identifying compounds that have agonist and/or antagonistic activity against ET_B receptors

1. Stimulation of prostacyclin release

Since endothelin-1 stimulates the release of prostacyclin from cultured bovine aortic endothelial cells, the compounds that have agonist or antagonist activity are identified by their ability to inhibit endothelin- 1 induced prostacyclin release from such endothelial cells by measuring 6- keto PGF_{l σ} substantially as described by (Filep et al. ( 1991 ) Biochem. Biophys. Res. Commun. 1 77 1 71 -1 76. Bovine aortic cells are obtained from collagenase-treated bovine aorta, seeded into culture plates, grown in Medium 1 99 supplemented with heat inactivated 1 5% fetal calf serum, and L-glutamine (2 mM), penicillin, streptomycin and fungizone, and subcultured at least four times. The cells are then seeded in six-well plates in the same medium. Eight hours before the assay, after the cells reach confluence, the medium is replaced. The cells are then incubated with a) medium alone, b) medium containing endothelin-1 ( 1 0 nM), c) test compound alone, and d) test compound + endothelin-1 (10 nM).

After a 1 5 min incubation, the medium is removed from each well and the concentrations of 6-keto PGF_{1 σ} are measured by a direct immunoassay. Prostacyclin production is calculated as the difference between the amount of 6-keto PGF_1σ released by the cells challenged with the endothelin- 1 minus the amount released by identically treated unchallenged cells. Compounds that stimulate 6-keto PGF_lσ release possess agonist activity and those which inhibit endothelin- 1 6-keto PGF_{l σ} release possess antagonist activity. 2. Inhibition of sarafotoxin 6c induced contraction

Sarafotoxin 6c is a specific ET_B antagonist that contracts rat fundal stomach strips. The effectiveness of tests compounds to inhibit this sarafotoxin 6c-induced contraction of rat fundal stomach strips is used as a measure ET_B antagonist activity. Two isolated rat fundal stomach strips are suspended under a 1 g load in a 10 ml organ bath filled with Krebs'-Henseleit solution containing 10 M cyclo(D-Asp-Pro-D-Val-Leu-D- Trp) (BQ-1 23; see, U.S. Patent No. 5, 1 14,91 8 to Ishikawa et al ), 5 μM indomethacin, and saturated with a gas mixture of 95% O₂/5% C0₂. Changes in tension are measured isometrically and recorded using a Grass Polygraph coupled to a force transducer. Sarafotoxin 6c is added cumulatively to one strip while the second strip is preincubated for 1 5 min with a test compound prior to addition of cumulative doses of sarafotoxin 6c. The effects of the test compounds on the concentration- response curve for sarafotoxin 6c are examined.

E. Deoxycorticosterone acetate (DOCA)-salt hypertensive rat model for assessing in vivo activity of compounds

Another model useful for assessing the in vivo activity of compounds is the deoxycorticosterone acetate (DOCA)-salt hypertensive rat model. To perform these tests, silastic MDX4-4210 elastomer implants containing 47 mg (DOCA) are prepared according to the method of Ornmsbee et al. (( 1 973) the J. Pharm. Sci. 62:255-257). Briefly, DOCA is incorporated into silicon rubber implants for sustained release. To prepare the implants the DOCA is incorporated into unpolymerized silicone rubber, catalyst is added and the mixture is cast in a hemicylindrical shape.

Sprague Dawley rats (7-8 weeks old) are unilaterally nephrectomized under ketamine anesthesia and a DOCA-implant is placed on the left lateral dorsal abdomen of the animal. The rats are allowed to recover for three weeks. During recovery they are permitted free access to normal rat chow and 0.9% NaCl drinking solution in place of drinking water. The rats develop hypertension within 3 weeks.

All animals are used in the tests between 21 and 30 days post surgery. The mean arterial blood pressure in these animals ranges from 165-200 mm Hg.

On the day of experimentation, catheters are inserted under brevital anesthesia into the right femoral artery for measurement of blood pressure, and into the right femoral vein for administration of a selected compound. The animals are placed in a restrainer and allowed to recover for a minimum of 60 min or until a steady mean arterial blood pressure is recorded . At that time, the selected compound or control vehicle is administered either intravenously, as a 60 minute infusion, or orally by oral gavage. Blood pressure is recorded continuously for a further 10 hrs.

F. Effect of Intravenous administration on ET-1 -induced pressor responses in conscious, autonomically blocked rats; a model for assessing [n vivo activity of selected compounds Male Sprague Dawley rats (250-450 g) are anesthetized (Brevital

50 mg/kg, IP) and cannulae were placed in the femoral artery to measure mean arterial pressure (MAP) and in the femoral vein for intravenous drug administration. Animals are placed in a restrainer and allowed to regain consciousness. Thirty minutes later autonomic blockade is administered (atropine methyl nitrate, 3 mg/kg, IV, followed by propranalol, 2 mg/kg, IV). An hour later animals receive a bolus injection of vehicle (0.5 ml) followed thirty minutes later by intravenous bolus administration of ET-1 (Control, 1 μg/kg). Following recovery from this challenge, test - compounds are administered by intravenous bolus administration (0.5 ml) and then re-challenged with ET-1 thirty minutes later. Results are expressed as the percent inhibition of the ET- 1 -induced pressor response after administration of the test compound compared to the pressor response induced by the control ET-1 challenge. In some cases a third ET-1 challenge is administered ninety minutes after administration of the test compound.

G. Results

The IC₅₀ for each of the compounds of the preceding Examples for ET_A and ET_B receptors has been measured. Most of the compounds have an IC₅₀ of less than 1 0 μM (measured at 4° C) for either or both of the ET_A and ET_B receptors. The tested compounds have an IC₅₀ less than about 10-20 μM, some have an IC₅₀ less than about 5 μM and some of the compounds have an IC₅₀ less than about 1 μM. A number of the compounds, such as N-(4-bromo-3-methyl-5-isoxazolyl)diphenyl- phosphoramidate and N-(4-bromo-3-methyl-5-isoxazolyl)diphenyl- phosphoramidate have an IC₅₀ for ET_A receptors that is substantially less ( 10 to 100-fold or more) than for ET_B receptors, and, thus are selective for ET_A receptors.

Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims.

Claims

WE CLAIM:

1 . A compound of formula I: o

A - P - N -Ar¹ B H

or a pharmaceutically acceptable salt or ester thereof,

wherein:

Ar¹ is alkyl or an aryl or heteroaryl group containing one ring or two or three fused or unfused rings and from 3 up to about 21 members in the ring(s), in which the heteroaryl groups contain one to three heteroatoms selected from among O, S and N; and A and B are independently selected from among halide, pseudo¬ halide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, thioalkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, aryisufinyl, arylsulfonyl, heteroaryloxy, heteroarylamino, heteroarylthio, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido and ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 1 4 carbon atoms, and are either straight or branched chains or cyclic, and in which the aryl and heteroaryl portions contain from 4 to 1 6 members in the ring.

2. A compound of claim 1 , wherein Ar¹ is selected from naphthyl, phenyl, biphenyl, quinolyl, thienyl, furyl, isoquinolyl, pyrrolyl, pyridyl, indolyl, oxadiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyrimidyl, benzo[b]furyl, benzo[b]thienyl.

3. A compound of claim 1 , wherein: Ar¹ is a 5 to 6 membered aryl group or heteroaryl group that contains one or two heteroatom(s).

4. A compound of any of claims 1 -3, wherein Ar¹ is unsubstituted or is substituted with one or more substituents selected from among NH₂, N0₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, aryisufinyl, arylsulfonyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 1 4 carbon atoms and are either straight or branched chains or cyclic, and the aryl and heteroaryl portions contain from about 4 to about 1 6 members in the ring.

5. A compound of claim 1 or claim 4, wherein Ar¹ is selected from among alkyl, styryl, thienyl, furyl, isoquinolyl, pyrrolyl, pyridyl, indolyl, oxadiazolyl, pyrazolyl, isoxazolyl, isothiazolyl and pyrimidyl.

6. A compound of claim 1 or claim 4, wherein:

Ar¹ is alkyl or is selected from:

each group is unsubstituted or substituted with one or more substituents R; and each R, which may be the same or different, is independently selected from H, NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsufinyl, arylsulfonyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms and are either straight or branched chains or cyclic, and the aryl and heteroaryl portions contain from about 4 to about 1 6 members.

7. A compound of claim 6, wherein each R is selected from among H, halide, pseudohalide, alkyl, alkoxy, alkenyl or alkynyl.

8. A compound of claim 1 , wherein: A has the formula Ar²-X_p— and and B has the formula Ar³-Y₀— in which o and p are 0 or 1 ;

Ar² and Ar³ are each independently selected from aryl and heteroaryl groups; and

X and Y are each independently selected from among O, N, NR¹³, S or (CH₂)_n in which n is 0 to 10, preferably 0 to 3, more preferably 0 or 1 , most preferably 0.

9. A compound of claim 8, wherein Ar² and Ar³ are each independently selected from among phenyl, pyridyl, pyrimidyl, pyridazinyl, pyrrolyl, pyrazinyl, thienyl and furyl.

1 0. A compound of any of claims 1 , and 4-9, wherein Ar¹ is isoxazolyl or N-pyridazinyl.

1 1 . A compound of any of claims 1 and 4-1 0, wherein Ar¹ is N- pyridazinyl.

1 2. A compound of any of claims 1 , 4, 6-8 and 10, wherein Ar¹ is isoxazolyl.

1 3. A compound of any of claims 1 and 4-1 0, wherein Ar¹ is an isoxazolyl group of formula:

R¹ and R² are independently selected from H, NH₂, NO₂, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkyloxy, haloalkyl, alkylsufinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, aryisufinyl, arylsulfonyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, substituted or unsubstituted amido, substituted or unsubstituted ureido, in which the alkyl, alkenyl and alkynyl portions contain from 1 up to about 14 carbon atoms and are either straight or branched chains or cyclic, and the aryl portions contain from about 4 to about 1 6 carbons, with the proviso that R² is not halide, pseudohalide, alkylsufinyl, alkylsulfonyl, aryisufinyl, arylsulfonyl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl or arylcarbonyl.

14. A compound of claim 1 3, wherein R¹ and R² are independently selected from among lower alkyl, lower alkenyl, lower alkynyl, lower haloalkyl, halide, pseudohalide or H; R² is not halide or pseudohalide.

1 5. A compound of claim 1 3 that has formula III:

wherein:

X and Y are each independently selected from among O, N, NR¹³, S or (CH₂)_n in which n is 0 to 10; o and p are independently 0 or 1 ;

R¹³ contains up to about 30 carbon atoms and is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heterocycle, aralkyl, aralkoxy, cycloalkyl, halide, cycloalkenyl, cycloalkynyl, haloalkyl, alkoxyalkyl, alkC(O)R¹⁴ and S(O)_nR¹⁴ in which n is 0-2; R¹⁴ is selected from among hydrogen, alkyl, alkenyl, alkynyl, aryl, alkylaryl, heterocycle, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl; R¹³ and R¹⁴ are unsubstituted or are substituted with one or more substituents each selected independently from Z, which is hydrogen, halide, pseudohalide, alkyl, alkoxy, alkenyl, alkynyl, aryl, heterocycle, aralkyl, aralkoxy, cycloalkyl, cycloalkenyl, cycloalkynyl;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are each independently selected from among any of (i), (ii) , (iii) , (iv) or (v):

(i) R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each independently selected from among H, NHOH, NH₂, NO₂, N₃, halide, pseudohalide, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, alkoxy, alkylamino, alkylthio, alkoxyalkyl, alkylsulfinyl, alkylsulfonyl, aryloxy, arylamino, arylthio, arylsulfinyl, arylsulfonyl, haloalkyl, haloaryl, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, arylcarbonyl, formyl, amido, ureido, or at least two of R³, R⁴, R⁵, R⁶ and R⁷ or R⁸, R⁹, R¹⁰, R^{1 1} and R¹² form alkylenedioxy, in which the alkyl, alkenyl, alkynyl portions are straight or branched chains of from about 1 up to about 10 carbons, and in which the aryl portions contain from 3 up to about 10 carbons;

(ii) R⁴ and R⁷ and/or R¹⁰ and R^{1 1} together are substituted or unsubstituted 1 , 3-butadienyl, 4-dimethylamino- 1 ,3 butadienyl, 1 -chloro- 1 ,3-butadienyl, 1 -aza-1 ,3-butadienyl or 2-aza-1 ,3-butadienyl groups; and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are as defined in (i) above; or alternatively,

(iii) R⁷ and R³ and/or R¹¹ and R¹² together are substituted or unsubstituted 1 , 3-butadienyl, 4-dimethylamino- 1 ,3 butadienyl, 1 -chloro- 1 ,3-butadienyl, 1 -aza-1 ,3-butadienyl or 2-aza-1 ,3-butadienyl groups; and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are as defined in (i) above; or

(iv) R³, R⁵, and R⁷ and R¹⁰, R^{1 1} and R⁸ are H are as defined in (i); and R⁴ and R⁶ and/or R⁹ and R¹² are each independently selected from alkyl, alkoxy, halide, aminoalkyl, dialkylaminoalkyi, in which the alkyl and alkoxy groups contain from 1 to 10, preferably 1 to 6 carbons, and are straight or branched chains, and the others of R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are as defined in (i) above; or

(v) any two of R³, R⁴, R⁵, R⁶, and R⁷, and/or any two of R⁸, R⁹, R¹⁰, R¹¹ and R¹², which are each selected as in (i) form fused carbocyclic or heterocyclic rings.

1 6. A compound of claim 1 5, wherein R¹³ is alkyl, alkoxyalkyl, or haloalkyl in which the alkyl group is a straight or branched carbon chain.

1 7. A compound of claim 1 6, wherein R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are independently selected from among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl and lower haloalkyl and at least three of R³, R⁴, R⁵, R⁶ and R⁷ are hydrogen and at least three of R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are hydrogen.

1 8. A compound of claim 1 5 that has formula IV:

1 9. A compound of claim 1 5 or claim 1 8, wherein R¹³ is a straight or branched carbon chain, halide, alkoxyalkyl, or haloalkyl.

20. A compound of claim 1 9, wherein R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹ and R¹² are independently selected from among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl and lower haloalkyl and at least three of R³, R⁴, R⁵, R⁶ and R⁷ are hydrogen and at least three of R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are hydrogen.

21 . A compound of claim 1 5 that has formula V:

22. A compound of claim 21 , wherein R³, R\ R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selected from among H, NH₂, halide, pseudohalide, lower alkyl, lower alkenyl and lower haloalkyl and at least three of R³, R\ R⁵, R⁶ and R⁷ are hydrogen and at least three of R⁸, R⁹, R¹⁰, R^{1 1} and R¹² are hydrogen.

23. A compound of claim 1 that is N-(4-bromo-3-methyl-5-isoxa- zolyDdiethylphosphoramidate.

24. A compound of claim 1 that is N-(3,4-dimethyl-5-isoxa- zolyDdiphenylphosphoramidate.

25. A compound of claim 1 that is N-(3,4-dimethyl-5-isoxa- zolyDdiphenylphosphinic amide.

26. A compound of claim 1 that is N-(4-bromo-3-methyl-5-isoxa- zolyDdiphenylphosphoramidate.

27. A compound of claim 1 that is N-(4-bromo-3-methyl-5-isoxa- zolyDdiphenylphosphinic amide.

28. A pharmaceutical composition, comprising a compound of any of claims 1 -27 or a pharmaceutically acceptable salt or acid of a compound of any of claims 1 -27 in a pharmaceutically acceptable carrier.

29. A method for the treatment of endothelin-mediated diseases, comprising administering an effective amount of one or more compounds of any of claims 1 -27 or pharmaceutically acceptable salts or acids of the compounds of any of claims 1 -27, wherein the effective amount is sufficient to ameliorate one or more of the symptoms of the disease.

30. The method of claim 29, wherein the disease is selected from the group consisting of hypertension, cardiovascular disease, asthma, pulmonary hypertension, inflammatory diseases, ophthalmologic disease, menstrual disorders, obstetric conditions, wounds, gastroenteric disease, renal failure, immunosuppressant-mediated renal vasoconstriction, erythropoietin-mediated vasoconstriction, endotoxin shock, pulmonary hypertension, anaphylactic shock and hemorrhagic shock.

31 . The method of claim 29, wherein the disease is selected from the group consisting of asthma and inflammatory diseases.

32. A method for inhibiting the binding of an endothelin peptide to endothelin_A (ET_A) or endothelin_B (ET_B) receptors, comprising contacting the receptors with an endothelin peptide and with one or more compounds of any of claims 1 -27, wherein: the contacting is effected prior to, simultaneously with or subsequent to contacting the receptors with the endothelin peptide.

33. A method for altering endothelin receptor-mediated activity, comprising contacting endothelin receptors with one or more compounds of any of claims 1 -27.

34. A pharmaceutical composition formulated for single dosage administration, comprising an effective amount of one or more compounds of any of claims 1 -27, wherein the amount is effective for ameliorating the symptoms of an endothelin-mediated disease.

35. An article of manufacture, comprising packaging material and a compound of any of claims 1 -27 or pharmaceutically acceptable salt or acid of a compound of any of claims 1 -27 contained within the packaging material, wherein the compound or salt or acid thereof is effective for antagonizing the effects of endothelin, ameliorating the symptoms of an endothelin-mediated disorder, or inhibiting the binding of an endothelin peptide to an ET receptor with an IC₅₀ of less than about 10 μM; and the packaging material includes a label that indicates that the compound or salt thereof is used for antagonizing the effects of endothelin, inhibiting the binding of endothelin to an endothelin receptor or treating an endothelin-mediated disorder.

36. Use of a compound of any of claims 1 -27 for the treatment of endothelin-mediated disorders.

37. Use of a composition of claim 28 for the treatment of endothelin-mediated disorders.

38. Use of the compound of any of claims 1 -27 for formulation of a medicament for treatment of an endothelin-mediated disorder.