Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
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  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to novel antagonists of endothelin useful as pharmaceutical agents, to methods for their production, to pharmaceutical compositions which include these compounds and a pharmaceutically acceptable carrier, and to pharmaceutical methods of treatment. More particularly, the novel compounds of the present invention are antagonists of endothelin useful in treating elevated levels of endothelin, acute and chronic renal failure, hypertension, myocardial infarction, metabolic, endocrinological and neurological disorders, congestive heart failure, endotoxic shock, subarachnoid hemorrhage, arrhythmias, asthma, preeclampsia, atherosclerotic disorders including Raynaud's disease, restenosis, angina, cancer, pulmonary hypertension, ischemic disease, gastric mucosal damage, hemorrhagic shock, ischemic bowel disease, and diabetes.
• Endothelin-1 (ET-1) , a potent vasoconstrictor, is a 21 amino acid bicyclic peptide that was first isolated from cultured porcine aortic endothelial cells.
• Endothelin-1 is one of a family of structurally similar bicyclic peptides which include; ET-2, ET-3, vasoactive intestinal contractor (VIC), and the sarafotoxins (SRTXs) .
• VOC vasoactive intestinal contractor
• SRTXs sarafotoxins
• ET-1 analogues with incorrect disulfide pairings exhibit at least 100-fold less vasoconstrictor activity.
• the flexible C-terminal hexapeptide of ET-1 has been shown to be important for binding to the ET receptor and functional activity in selected tissues. Additionally, the C-terminal amino acid (Trp-2l) has a critical role in binding and vasoconstrictor activity, since ET[l-20] exhibits approximately 1000-fold less functional activity.
• ET Endothelin is involved in many human disease states.
• Several in vivo studies with ET antibodies have been reported in disease models. Left coronary artery ligation and reperfusion to induce myocardial infarction in the rat heart, caused a four- to sevenfold increase in endogenous endothelin levels.
• Administration of ET antibody was reported to reduce the size of the infarction in a dose-dependent manner (Watanabe, T. , et al, "Endothelin in Myocardial Infarction, ⁇ Nature (Lond.) 344:114 (1990)).
• ET may be involved in the pathogenesis of congestive heart failure and myocardial ischemia
• the effectiveness and specificity of the anti-ET antibody were confirmed by its capacity to prevent renal deterioration caused by a single bolus dose (150 pmol) of synthetic ET, but not by infusion of angiotensin II, norepinephrine, or the thromboxane A 2 mimetic U-46619 in isolated kidneys (Perico, N. , et al, "Endothelin Mediates the Renal Vasoconstriction Induced by Cyclosporine in the Rat," J. Am. Soc. Nephrol. 1:76 (1990)).
• ET A and ET B The distribution of the two cloned receptor subtypes, termed ET A and ET B , have been studied extensively (Arai, H. , et al, Nature 348:730 (1990), Sakurai, T. , et al, Nature 348:732 (1990)).
• the ET A or vascular smooth muscle receptor, is widely distributed in cardiovascular tissues and in certain regions of the brain (Lin, H.Y., et- al, Proc. Natl. Acad. Sci. 88:3185 (1991)).
• the ET B receptor originally cloned from rat lung, has been found in rat cerebellum and in endothelial cells, although it is not known if the ET B receptors are the same from these sources.
• the human ET receptor subtypes have been cloned and expressed (Sakamoto, A., et al, Biochem. Biophys. Res. Chem. 178:656 (1991), Hosoda, K. , et al, FEBS Lett. 287:23 (1991)).
• the ET A receptor clearly mediates vasoconstriction and there have been a few reports implicating the ET B receptor in the initial vasodilatory response to ET (Takayanagi, R. , et al, FEBS Lett. 282:103 (1991)).
• recent data has shown that the ET B receptor can also mediate vasoconstriction in some tissue beds (Panek, R.L., et al, Biochem. Biophys. Res. Commun. 183(2):566 (1992)) .
• Plasma endothelin-1 levels were dramatically increased in a patient with malignant hemangioendothelioma (K. Nakagawa et al, Nippon Hifuka Gakkai Zasshi. 1990, 100, 1453-1456).
• the ET receptor antagonist BQ-123 has been shown to block ET-l induced bronchoconstriction and tracheal smooth muscle contraction in allergic sheep providing evidence for expected efficacy in bronchopulmonary diseases such as asthma (Noguchi, et al, Am. Rev. Respir. Pis.. 1992, 145 (4 Part 2), A858) .
• Circulating endothelin levels are elevated in women with preeclampsia and correlate closely with serum uric acid levels and measures of renal dysfunction. These observations indicate a role for ET in renal constriction in preeclampsia (Clark B.A.. , et al. Am. J. Obstet. Gvnecol., 1992, 166, 962-968). Plasma immunoreactive endothelin-1 concentrations are elevated in patients with sepsis and correlate with the degree of illness . and depression of cardiac output (Pittett J., et al, Ann Sur ⁇ .. 1991, 213(3), 262) . In addition the ET-1 antagonist BQ-123 has been evaluated in a mouse model of endotoxic shock.
• Endothelin is a potent agonist in the liver eliciting both sustained vasoconstriction of the hepatic vasculature and a significant increase in hepatic glucose output (Gandhi C.B., et al, Journal of Biological Chemistry. 1990, 265(29), 17432).
• streptozotocin-diabetic rats there is an increased sensitivity to endothelin-1 (Tammesild P.J., et al, Clin. Exp. Pharmacol. Physiol.. 1992, 19(4), 261).
• ET A antagonist receptor blockade has been found, to produce an antihypertensive effect in normal to low renin models of hypertension with a time course similar to the inhibition of ET-1 pressor responses (Basil M.K., et al, J. Hypertension. 1992, 10(Suppl 4), S49) .
• the endothelins have been shown to be arrhythmogenic, and to have positive chronotropic and inotropic effects, thus ET receptor blockade would be expected to be useful in arrhythmia and other cardiovascular disorders (Han S.-P., et al, Life Sci.. 1990, 46, 767) .
• Intracerebroventricular administration of ET-1 in rats has been shown to evoke several behavioral effects. These factors strongly suggest a role for the ETs in neurological disorders.
• the potent vasoconstrictor action of ETs on isolated cerebral arterioles suggests the importance of these peptides in the regulation of cerebrovascular tone.
• Increased ET levels have been reported in some CNS disorders, i.e., in the CSF of patients with subarachnoid hemorrhage and in the plasma of women with preeclampsia.
• Stimulation with ET-3 under conditions of hypoglycemia have been shown to accelerate the development of striatal damage as a result of an influx of extracellular calcium.
• Circulating or locally produced ET has been suggested to contribute to regulation of brain fluid balance through effects on the choroid plexus and CSF production.
• ET-1 induced lesion development in a new model of local ischemia in the brain has been described.
• Circulating and tissue endothelin immunoreactivity is increased more than twofold in patients with advanced atherosclerosis (A. Lerman, et al, New England J. Med.. 1991, 325, 997-1001). Increased endothelin immunoreactivity has also been associated with Buerger's disease (K. Kanno, et al, J. Amer. Med. Assoc. 1990, 264, 2868) and Raynaud's phenomenon (M.R. Zamora, et al, Lancet, 1990, 336, 1144-1147) . Likewise, increased endothelin concentrations were observed in hypercholesterolemic rats (T. Horio, et al, Atherosclerosis. 1991, 89, 239- 245) .
• Elevated levels of endothelin have also been measured in patients suffering from ischemic heart disease (M. Yasuda, et al, Amer. Heart J.. 1990, 119 801-806, S.G. Ray, et al, Br. Heart J.. 1992, 67, 383- 386) and either stable or unstable angina (J.T. Stewart, et al, Br. Heart J.. 1991, 66, 7-9) .
• endothelin has been suggested that the proliferative effect of endothelin on mesangial ceils may be a contributing factor in chronic renal failure (P.iT. Schultz, J. Lab. Clin. Med.. 1992, 119, 448- 449) .
• Local intra-arterial administration of endothelin has been shown to induce small intestinal mucosal damage in rats in a dose-dependent manner (S. Mirua, et al, Digestion. 1991, 48, 163-172) .
• endothelin-1 in the range of 50-500 pmol/kg into the left gastric artery increased the tissue type plasminogen activator release and platelet activating formation, and induced gastric mucosal hemorrhagic change in a dose dependent manner (I. Kurose, et al, Gut. 1992, 33, 868-871). Furthermore, it has been shown that an anti-ET-1 antibody reduced ethanol- induced vasoconstriction in a concentration-dependent manner (E. Masuda, et al, Am. J. Physiol.. 1992, 262, G785-G790) . Elevated endothelin levels have been observed in patients suffering from Crohn's disease and ulcerative colitis (S.H. Murch, et al, Lancet, 1992, 339, 381-384) .
• the present invention is a compound of Formula I
• R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, fluorenylmethyl, TS.T __) _5
• R and R are each the same or
• Rl 3 different and each is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, or fluorenylmethyl,
• R is F, Cl, Br, or I
• R a and R 3 are as defined above excluding R 3 is hydrogen, or
• R 1 is hydrogen or alkyl
• Z is
• R 2 and R 3 are each the same or different and each is as defined above,
• X and Y are the same and substituted at the same position on the aromatic ring and each may be one, two, three, or four substituents selected from the group consisting of hydrogen, halogen, alkyl, -C0 2 R 2 , wherein R 2 is as defined above,
• R, Z, X, and Y are as defined above;
• R 4 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
• aryl are each the same or different and each is hydrogen, alkyl, cycloalkyl, aryl, or hetero .
• R 2b and R 3b are each the same or different and each is as defined above for R 2b and R 3b ,
• R 1 and n are as defined above, or
• AA 2 is absent
• R 5 is hydrogen, alkyl, aryl, heteroaryl
• R 1 and n are as defined above, or AA 3 is absent;
• AA 4 and AA 5 are each independently absent or each is independently
• R 6 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, and R 1 and n are as defined above;
• R 7 is aryl or heteroaryl
• Elevated levels of endothelin have been postulated to be involved in a number of pathophysiological states including diseases associated with the cardiovascular system as well as various metabolic and endocrinological disorders.
• the compounds of Formula I are useful in the treatment of hypertension, myocardial infarction, metabolic, endocrinological and neurological disorders, congestive heart failure, endotoxic shock, subarachnoid hemorrhage, arrhythmias, asthma, and chronic and acute renal failure, preeclampsia, atherosclerotic disorders including Raynaud's disease, restenosis, angina, cancer, pulmonary hypertension, ischemic disease, gastric mucosal damage, hemorrhagic shock, ischemic bowel disease, and diabetes.
• alkyl means a straight or branched hydrocarbon radical having from 1 to 12 carbon atoms and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n- onyl, n-decyl, undecyl, dodecyl, and the like.
• alkenyl means a straight or branched unsaturated hydrocarbon radical having from 2 to 12 carbon atoms and includes, for example, ethenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-methyl-3-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 3-heptenyl, 1-octenyl, 1-nonenyl,
• alkynyl means a straight or branched triple bonded unsaturated hydrocarbon radical having from 2 to 12 carbon atoms and includes, for example, ethynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 3-heptynyl, 1-octynyl, 2-octynyl, l-nonynyl, 2-nonynyl, 3-nonynyl, 4-nonynyl, 1-decynyl, 2-decynyl, 2-undecynyl, 3-undecynyl, 3-dodecynyl, and the like.
• cycloalkyl means a saturated hydrocarbon ring which contains from 3 to 12 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, and the like.
• cycloalkylalkyl means a saturated hydrocarbon ring attached to an alkyl group wherein alkyl is as defined above.
• the saturated hydrocarbon ring contains from 3 to 12 carbon atoms. Examples of such are cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl, adamantylmethyl and the like.
• alkoxy and thioalkoxy are 0-alkyl or S-alkyl as defined above for alkyl.
• aryl means an aromatic radical which is a phenyl group, a benzyl group, a naphthyl group, a biphenyl group, a pyrenyl group, an anthracenyl group,
• alkyl is as defined above, -C-O-alkyl wherein 0 alkyl is as defined above, -C-alkyl wherein alkyl is as defined above, or aryl.
• arylalkyl means an aromatic radical attached to an alkyl radical wherein aryl and alkyl are as defined above for example benzyl, fluorenylmethyl and the like.
• heteroaryl means a heteroaromatic radical which is 2-or 3-thienyl, 2- or 3-furanyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1,2,4-triazolyl, 4- or 5-1,2,3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridinyl, 3-, 4-, or 5-pyridazinyl, 2-pyrazinyl, 2-, 4-, or 5-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolinyl, 2-, 3-, 4-, 5-, 6-, or 7-indo
• alkyl is as defined above, -C-O-alkyl wherein alkyl
• O is as defined above, -C-alkyl wherein alkyl is as defined above or phenyl.
• heterocycloalkyl means 2- or
• Halogen is fluorine, chlorine, bromine or iodine.
• the following table provides a list of abbreviations and definitions thereof used in the present invention.
• the compounds of Formula I are capable of further forming both pharmaceutically acceptable acid addition and/or base salts. All of these forms are within the scope of the present invention.
• Pharmaceutically acceptable acid addition salts of the compounds of Formula I include salts derived from nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, hydrofluoric, phosphorous, and the like, as well as the salts derived from nontoxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
• Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like.
• salts of amino acids such as arginate and the like and gluconate, galacturonate (see, for example, Berge, S. M. , et al, "Pharmaceutical Salts, " Journal of Pharmaceutical Science. 66. pp. 1-19 (1977)).
• the acid addition salts of said basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner.
• a peptide of Formula I can be converted to an acidic salt by treating with an aqueous solution of the desired acid, such that the resulting pH is less than 4.
• the solution can be passed through a C18 cartridge to absorb the peptide, washed with copious amounts of water, the peptide eluted with a polar organic solvent such as, for example, methanol, acetonitrile, aqueous mixtures thereof, and the like, and isolated by concentrating under reduced pressure followed by lyophilization.
• the free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner.
• the free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the present invention.
• Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations .are sodium, potassium, magnesium, calcium, and the like.
• Suitable amines are N,N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexyla ine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge, S. M. , et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, , pp. 1-19 (1977)).
• the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
• a peptide of Formula I can be converted to a base salt by treating with an aqueous solution of the desired base, such that the resulting pH is greater than 9.
• the solution can be passed through a C18 cartridge to absorb the peptide, washed with copious amounts of water, the peptide eluted with a polar organic solvent such as, for example, methanol, acetonitrile, aqueous mixtures thereof, and the like, and isolated by concentrating under reduced pressure followed by lyophilization.
• the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
• the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.
• Certain of the compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms, including hydrated forms, are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention.
• Certain of the compounds of the present invention possess one or more chiral centers and each center may exist in the R(D) or S(L) configuration.
• the present invention includes all enantiomeric and epimeric forms as well as the appropriate mixtures thereof.
• a preferred compound of Formula I is one wherein
• R is -N-R 2 ,
• R 3 wherein R 2 and R 3 are each the same or different and each is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, or fluorenylmethyl, -N-C-N-R 3 , wherein R 2 and R 3 are as ⁇ J j 2 defined above.
• R 9 is F, Cl, Br, or I, 0 -NH-C-R 3, wherei.n R3 is as defined above, or
• R is hydrogen
• n is zero or an integer of
• R 1 is hydrogen or alkyl
• R 2 and R 3 are each the same or different and each is as defined above and X and Y are the same and substituted at the same position on the aromatic ring and each substituent is selected from the group consisting of hydrogen, halogen, or alkyl;
• R 4 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,_ heteroaryl,
• R 2b and R 3b are each the same or different and each is hydrogen, alkyl, cycloalkyl, aryl, or heteroaryl, -0R 2b , wherein R 2b is as defined above, . 0
• R 2b wherein R 2b and R 3b are each the same or different and each is as defined above for
• R 5 is aryl, heteroaryl
• R and R are each the same or different and each is as defined above,
• R o is as defined above, or C-OR , wherein R 2b is as defined above, and n is as defined above, or AA 3 is absent; AA 4 and AA 5 are each independently absent or each is
• * g stereochemistry at CH in AA is L; or a pharmaceutically acceptable salt thereof.
• a more preferred compound of Formula I is one wherein AA 1 is
• R is -N-R 2, - k- wherein R 2 and R3 are each the same or different and each is hydrogen, alkyl, aryl, or fluorenylmethyl,
• R 2 and R 3 are '2 ⁇ j 2 as defined above.
• R 9 is F, Cl, Br, or I, or 0 -NH-C-OR
• R 10 is hydrogen, alkyl, aryl, or arylalkyl, excluding R 10 is hydrogen, Z is -0-, -S-,
• X and Y are each the same and substituted at the same position on the aromatic ring and each substituent is selected from the group consisting of hydrogen, halogen, or alkyl;
• R 4 is hydrogen, alkyl, aryl, heteroaryl
• R and R are each the same or different and each is hydrogen or alkyl
• R _,2b and-averageR3b are each the same or different and each is hydrogen or alkyl, NH
• n is an integer of 1, 2, 3, or 4 or
• R 3 is aryl, heteroaryl, 0
• R is hydrogen or alkyl
• R is hydrogen or alkyl
• -C- is hydrogen or alkyl, and n is an integer of 1, 2, 3, or 4; AA 4 and AA 5 are each independently
• R 6 is hydrogen, alkyl, cycloalkyl, or aryl, and n is an integer of 1, 2, 3, or 4;
• R 7 is aryl or heteroaryl, and n is zero or an integer of 1, 2, 3, or 4, or
• R 7 , R 1 , and n are as ( H 2 ) n R defined above,
• * g stereochemistry at CH in AA is L; or a pharmaceutically acceptable salt thereof.
• Fmoc-D-Bheg-Leu-Asp-lie-Ile-Trp Fmoc-D-Bheg-Orn-Asp-lie-Ile-Trp;
• the compounds of Formula I are valuable antagonists of endothelin.
• the tests employed indicate that compounds of Formula I possess endothelin antagonist activity.
• the compounds of Formula I were tested for their ability to inhibit [ 125 I] -ET-1( [ 125 I] -Endothelin-1) binding in a receptor assay according to the following procedures:
• the cells used were rabbit renal artery vascular smooth muscle cells grown in a 48-well dish (1 cm 2 ) (confluent cells) .
• the growth media was Dulbecco's Modified Eagles/Ham's F12 which contained 10% fetal bovine serum and antibiotics (penicillin/streptomycin/ fungizone) .
• the assay buffer was a medium 199 containing
• Amersham radioiodinated endothelin-1 [ 125 I]-ET-1 was used at final concentration of 20,000 cpm/0.25 mL (25 pM) .
• the tissue is made up of 20 M tris(hydroxy- methyljaminomethane hydrochloride (Trizma) buffer, 2 mM ethylenediaminetetra acetate, 100 ⁇ M phenylmethylsulfonyl fluoride.
• Trizma tris(hydroxy- methyljaminomethane hydrochloride
• 2.5 mL cold wash buffer 50 mM Trizma buffer
• Antagonist activity is measured by the ability of added compounds to reduce endothelin-stimulated arachidonic acid release in cultured vascular smooth muscle cells as arachidonic acid release (AAR) .
• Arachidonic Acid Loading Media LM is DME/F12 + 0.5% FCS x 0.25 mCi/mL
• arachidonic acid A ersham
• the LM was aspirated and the cells were washed once with the assay buffer (Hank's BSS + 10 mM HEPES + fatty acid-free BSA (1 mg/mL) ) , and incubated for 5 minutes with 1 mL of the prewarmed assay buffer. This solution was aspirated, followed by an additional 1 mL of prewarmed assay buffer, and further incubated for another 5 minutes. A final 5-minute incubation was carried out in a similar manner. The same procedure was repeated with the inclusion of 10 ⁇ L of the test compound (1 nM to 1 ⁇ M) and 10 ⁇ h ET-1 (0.3 nM) and the incubation was extended for 30 minutes. This solution was then collected, 10 ⁇ h of scintillation cocktail was added, and the amount of [ 3 H] arachidonic acid was determined in a liquid scintillation counter.
• Denuded rings were mounted in 20 mL organ baths containing Krebs-bicarbonate buffer (composition in mM: NaCl, 118.2; NaHC0 3 , 24.8; C1, 4.6; MgS0 4 7-H 2 0, 1.2; H 2 P0 4 , 1.2; CaCl 2 -2H 2 0; Ca-Na 2 EDTA, 0.026; dextrose, ICO), that was maintained at 37°C and gassed continuously with 5% C0 2 in oxygen (pH 7.4) . Resting tension was adjusted to 3.0 g for femoral and 4.0 g pulmonary arteries; the rings were left for 90 minutes to equilibrate.
• Krebs-bicarbonate buffer composition in mM: NaCl, 118.2; NaHC0 3 , 24.8; C1, 4.6; MgS0 4 7-H 2 0, 1.2; H 2 P0 4 , 1.2; CaCl 2 -2H 2 0; Ca-Na 2 ED
• Vascular rings were tested for lack of functional endothelium (i.e., lack of an endothelium-dependent relaxation response to carbachol (1.0 ⁇ M.) in norepinephrine (0.03 ⁇ M) contracted rings.
• Agonist peptides, ET-1 (femoral), and S6c (pulmonary) were cumulatively added at 10-minute intervals.
• the ET antagonists were added 30 minutes prior to adding the agonist and pA 2 values were calculated (Table I) .
• the compounds of Formula I may be prepared by solid phase peptide synthesis on a peptide synthesizer, for example, an Applied Biosystems 430A peptide synthesizer using activated esters or anhydrides of N-alpha-Boc protected amino acids, on PAM or MBHA resins. Additionally, the compounds of Formula I may also be prepared by conventional solution peptide synthesis. Amino acid side chains are protected as follows: BzKAsp, Glu, Ser) ,
• Each peptide resin (1.0 g) is cleaved with 9 mL of HF and 1 mL of anisole or p-cresol as a scavenger (60 minutes, 0°C) .
• the peptide resin is washed with cyclohexane, extracted with 30% aqueous HOAc, followed by glacial HOAc, concentrated under reduced pressure, and lyophilized.
• the homogeneity and composition of the resulting peptide is verified by RP-HPLC, capillary electrophoresis, thin layer chromatography (TLC) , proton nuclear magnetic resonance spectrometry (NMR) , and fast atom bombardment mass spectrometry (FAB-MS) .
• the compounds of the present invention can be prepared and administered in a wide variety of oral and parenteral dosage forms.
• the compounds of the present invention can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, s bcutaneously, mtraduodenally, or intraperitoneally.
• the compounds of the present invention can be administered by inhalation, for example, intranasally.
• the compounds of the present invention can be administered transdermally.
• the following dosage forms may comprise as the active component, either a compound of Formula I or a corresponding pharmaceutically acceptable salt of a compound of Formula I.
• pharmaceutically acceptable carriers can be either solid or liquid.
• Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
• a solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
• the carrier is a finely divided solid which is in a mixture with the finely divided active component.
• the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
• the powders and tablets preferably contain from five or ten to about seventy percent of the active compound.
• Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
• the term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included.
• Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
• a low melting wax such as a mixture of fatty acid glycerides or cocoa butter
• the active component is dispersed homogeneously therein, as by stirring.
• the molten homogenous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
• Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water propylene glycol solutions.
• liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
• Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents as desired.
• Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
• viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
• solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
• liquid forms include solutions, suspensions, and emulsions.
• These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
• the pharmaceutical preparation is preferably in unit dosage form.
• the preparation is subdivided into unit doses containing appropriate quantities of the active component.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
• the unit dosage form can be a capsules, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
• the quantity of active component in a unit dose preparation may be varied or adjusted from 0.1 mg to 100 mg preferably 0.5 mg to 100 mg according to the particular application and the potency of the active component.
• the composition can, if desired, also contain other compatible therapeutic agents.
• the compounds utilized in the pharmaceutical method of this invention are administered at the initial dosage of about 0.01 mg to about 20 mg per kilogram daily.
• a daily dose range of about 0.01 mg to about 10 mg per kilogram is preferred.
• the dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
• the linear hexapeptide is prepared by standard solid phase synthetic peptide methodology utilizing a Boc/benzyl strategy (Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis. Pierce Chemical Co., Rockford, IL, 1984) . All protected amino acids and reagents are obtained from commercial sources with the exception of N- ⁇ -Boc-DL-Bhg and are not further purified.
• the protected peptide resin is prepared on an Applied Biosystems 430A Peptide Synthesizer, utilizing protocols supplied for a dicyclohexyl- carbodiimide-mediated coupling scheme (Standard 1.0, Version 1.40) . Starting with 0.710 g of N- ⁇ -Boc-Trp-PAM resin (0.70 meq/g, 0.497 eq of
• Boc-Trp(For) total) the protected peptide is prepared by the stepwise coupling of the following amino acids (in order of addition) : N- ⁇ -Boc-Ile « 0.5H 2 O, N-Q!-Boc-Ile-0.5H 2 0, N- ⁇ -Boc-Asp(Bzl) , N- ⁇ -Boc-Leu-H 2 0, and N- ⁇ -Boc-DL-Bhg.
• a typical cycle for the coupling of an individual amino acid residue is illustrated below (reproduced from the ABI manual) :
• the peptide is liberated from the solid support, and the carboxylate of aspartic acid deprotected by treatment with anhydrous hydrogen fluoride (9.0 mL) , anisole (0.5 mL) , and dimethyl sulfide (0.5 mL) (60 minutes, 0°C) .
• anhydrous hydrogen fluoride 9.0 mL
• anisole 0.5 mL
• dimethyl sulfide 0.5 mL
• the resin is washed with diethyl ether (3 x 30 mL) and extracted with 20% HOAc in water (3 x 30 mL) and glacial HOAc (2 x 30 mL) .
• the aqueous extractions are combined, concentrated under reduced pressure, and lyophilized (360 mg) .
• the crude peptide is dissolved in 4.0 mL of 50% TFA/H 2 0, filtered through a 0.4 L syringe filter, and chromatographed on a Vydac 218TP 1022 column (2.2 x 25.0 cm, 15.0 mL/min, A: 0.1% TFA/H 2 0, B: 0.1% TFA/CH 3 CN, Gradient; 0% B for 10 minutes, 10% to 40% B over 120 minutes) .
• Two individual fractions are collected and combined based upon analysis by analytical HPLC. The combined fractions are concentrated separately under reduced pressure (10 mL) , diluted with H 2 0 (50 mL) , and lyophilized (40_0 mg/ea) .
• a saturated solution of sodium bicarbonate in water is prepared, diluted with water (1:10), chilled to 0°C, and 10 mL of the solution is added to approximately 50 mg of Ac-D-Bhg-Leu-Asp-Ile-Ile-Trp (Example 1) with stirring.
• the pH of the solution is greater than 9.
• the solution is passed through a C18 cartridge, washed with water (100 mL) , and the absorbed peptide is eluted with methanol (50 mL) , concentrated under reduced pressure, resuspended in water (50 mL) , and lyophilized (three times) to give the title compound.
• Bhg-HCl (1.70 g, 5.43 mmol) is suspended in 150 mL of p-dioxane:H 2 0 (2:1) at room temperature.
• To the stirred solution is added 1.40 g (6.42 mmol) of di-tert butyldicarbonate.
• the pH of the solution is adjusted to >9.0 with IN NaOH and maintained at between pH 9 and 10 with aliquot additions of IN NaOH, until the pH is constants
• the solution is concentrated under reduced pressure to approximately 75 mL, overlain with ethyl acetate (50 mL) , and acidified to approximately pH 2.5 with 10V aqueous HC1.
• the organic layer is separated, washed successively with 10% aqueous HC1 (2 x 50 mL) , brine (2 x 50 mL) , H 2 0 (3 x 50 mL) , and dried with MgS0 4 .
• the solution is filtered, concentrated under reduced pressure, and the oil is recrystallized from ethyl acetate:heptane (1.82 g) .

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