CA2133090A1 - Endothelin antagonists ii - Google Patents

Abstract

Novel antagonists of endothelin are described, as well as methods for the preparation and pharmaceutical compositions of the same, which are useful in treating elevated levels of endothelin, acute and chronic renal failure, hypertension, myocardial infarction, metabolic, endocrinological, 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. In the claimed new compounds, the N-terminal 16-His is replaced by a building-block of formula (.alpha.).

Description

~V093/212192 ~ 3 3 ~9 0 PCT/US93/036$8 D. ~
1' `

ENDOTHELIN ANTAGO~ISTS II

BACKGROUND OF THE INVENTION
-;
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, ~i:
and to pharmaceutical methods of treatment. More particularly, the novel compounds of the present invention are an~agonists of endothelin useful in :
treating elevated leveis of endothelin, acute and .
chronic renal failure, hypertension, myocardial infarction, metabolic, endocrinological and neurological disorders, congestive heart failure, ~ ~endotoxic shock, subarachnoid hemorrhage, arrhythmias, ; : :asthm~ preeclamp~ia, atherosclerotic disorders including Raynaud's disease, re~tenosis, angina, cancer,~ pul nary hypertension, ischemic disease, gastri:c mucosal damage, hemorrhagic shock, ischemic ; bowel disease, and diabetes.
: .
Endothelin-l (ET-i), a potent vasoconstrictor, is ;2~ ~ a ~1 amino acid bicyclic peptide that was first isolat@d from cultured porcine aortic endothelial : cells. Endothelin-l, i8 one of a family of structurally similar bicyclic peptide~ which include;
ET-2, ET-3, vasoactive intestinal contractor (VIC), :
~ and ~the sarafotoxinsl~SRTXs). T~q unique bicyclic structure and corresponding arrangement of the disulfide bridges of ET-l, which are the same for the -.
endothelins, VIC, and the sarafotoxins, ha~ led to :~
significant speculation as to the importance of the resulting induced 3econdary structure to receptor : binding and functional activity. ET-l analogues with -~ incorrect disulfide pairings exhibit at least l00-fold less vasoconstrictor activity. The flexible : ' ' W093/21219 PCT/USg3/0365~
21~309(~

-2- ~;
C-terminal hexapeptide of ET-1 has been ~hown to be important for binding to the ET receptor and functional acti~ity in selected tissues.
Additionally, the C-terminal ~mino acid ~Trp-21) ~as a ``~
S critical role in binding and vasoconætrictor acti~ity, since ET[1-20] exhibits approximately 1000-fold less functional activity. -Endothelin is involved in many human disease states.
Several in vivo studies with ET antibodies ha~e been reported in disea~e models. Left coronary artery ligation and reperfusion to induce myocardial infarction in th2 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,~ ond.) 344:114 (~990)). Thus, ET
may be in~olved in the pathogenesis of congestive -~
heart failure and myocardial ischemia ~;Margulies, K.B., et al, "Increased Endothelin in Experimental Heart FaiIure," Circulation 82:2226 ~: : ( l 9 g O ) ) ~
Studies by Kon and colleagues using anti-ET
antibodies in an ischemic kidney model, to deactivate endogenous ET, indicated the peptide's invol~ement in acute renal ischemic injury ~Kon, V., et al, "Glomerular Actions of Endothelin In Vivo, n J. Clin. ~"
Invest. 83:1762 tl989)). In isolated kidneys, ~ preexposed to specifiic a~tiendot;helin antibody and then challenged with cyclosporine, the renal perfusate flow and glomerular filtration rate increased, while . ~;~
renal resistance decreased as compared with isolated ~ `
kidneys preexpo~ed to a nonimmunized rabbit serum. -The effecti~eness and pecificity of the anti-ET
antibody were confirmed by its capacity to pre~ent renal deterioration caused by a single bolus dose w093/21219 2 1 3 3 0 9 ~ PCT/~S93/0365~ ~

(150 pmol) of synthetic ET, but not by infuision of angiotensin II, norepinephrine, or the thromboxane A2 ~:.
mimetic U-46619 in isolated kidneys ~Perico, N., et al, "Endothelin Mediate~ the Renal Vasoconstriction Induced by Cyclosporine in the Rat,~ J. Am. Soc. .
Nephrol. l:76 ~l990)).
Others have reported inhibition of ET-l or ET-2-induced va~oconstriction in rat isolated thoracic aorta using a monoclonal antibody to ET-l (Koshi, T., et al, ~Inhibition of E~dothelin (ET)-l and ET-2- :
Induced Yasoconstriction by Anti-ET-l Monoclonal Antibody, n Chem. Pharm. Bull., 39:1295 (l99l)).
Combined adr~inistration of ET-l and ET-l antibody to rabbits showed significant inhibition of the blood `:
pre~sure (BP) and renal blood flow responses (Miyamori, I., et al, Systemic and Regional Effects of Endothelin in Rabbits: Effects of Endothelin Antibody,~ Clin. Exp. Pharmacol. Physiol., 17:691 (1990)):-: ~20~ Other investigators have reported that infusion : of ET-specific antibodies into spontaneously :~
hypertensive rats ~SHR) decreased mean arterial pres3ure (MAP), and increased glomerular filtration rate and renal blood flow. In the control study with .
~ normotensive Wistar-Ryoto rats (WRY) there were no significant changes in these parameters (Ohno, A.
Effects of Endothelin-Specific Antibodies and Endothelin in Spontaneously Hypertensive Rats,~
J. Tok~o Women~s Med. Coll., 6l:95l (l99l)).
~n additi~n,~elevated levels of endothelin have j -been reported in several disease states (see Table I
below).
Burnett and co-workers recently demonstrated that exogenous infusion of ET (2.5 ng/kg/mL) to : 35 anestheti~ed dogs, producing a doubling of the :
circulating concentration, did have biological actions : (Lerman, A., et al, ~Endothelin has Biological Actions :

W093t2121s PCT~US93/03658_ ~
21330!~ j at Pathophysiological Concentrations,~ Cirsulation 83:1808 (l99l)). Thus heart rate and cardiac output decreased in association with increased renal and ~ystemic vascular resistances and antinatriuresis~ ~
These studie~ support a role for endothelin in the ;
regulation of cardiovascular, renal, and endocrine function.
In the anesthetized dog with congestive heart failure, a significant two- to threefold elevation of circulating ET levels has been reported (Cavero, P.G., et al, n Endothelin in Experimental Congestive Heart Failure in the Anesthetized Dog, n Am. J. PhYsiol.
259:F312 ~l990)), and studies in humans have shown similar increases (Rodeheffer, R.J., et al, n Circulating Plasma Endothelin Correlates With the Severity of Congestive Heart Failure in Humans,~ ;
Am. J. Hypertension 4:9A (l99l)j. When ET was chronically infused into male rats, to determine ; wheeher a ~long-term increase in circulating ET levels would cause a sustained elevation in mean arterial blood pressure, significant, su~tained, and dose-dependent increases in mean arterial BP were observed.
Similar results were obserYed with ET-3 although larger~doses were required (~ortenson, L.H., et al, 25~ ~ nChroni~: Hypertension Produced by Infu~ion of Endothelin in Rats, n Hypertension, 15:729 (l990)3. ``
~; ~The distribution of the two cloned receptor ~`
- ~ subtypes, termed ETA and ETB, have been studied extensively (Arai, H., et al, Nature 348:730 tl990), Sakura~, T.~, et~ al, ature 348:732 (l990)!). The ET~
or Yascular smooth muscle receptor, i~ widely distributed in cardiovascular tissues and in certain regions of the brain (~in, H.Y., et al, Proc. Natl.
Acad. Sci. 88:3185 (l99l)). The ETB receptor, originally cloned from rat lung, has been found in rat cerebellum and in endothelial cells, although it is not known if the ETB receptors are the same from these ~;

W093/21219 2 1 3 3 0 ~ O PCT/US93~036~8 .... ~ ' ' .

sources. The human ET receptor subtypes ha~e been cloned and expre~sed (Sakamoto, A., et al, Biochem.
Biophys._Res. Chem. ~78:656 (1991), Hosoda, K., et al, FE~S Lett. 2~7:23 (l991)). The ETA receptor clearly mediates vasoconstriction and there have been a few reports implicating the ETB receptor in the initial vasodilatory response to ET (Takayanagi, R., et al, FEBS Lett. 282:103 (1991)). However, recent data has shown that the ETB receptor can also mediate vasoconstriction in some tissue beds (Panek, R.L., et al, Biochem. Biophys. Res._Commun. 1~3(2):566 (1992)).
Comparison of the receptor affinities of the ETs and SRTXs in rats and atri~ (ETA) or cerebellum and hippocampus (ET9), indicate that SRTX-c is a selective ETB ligand ~Williams, D.L., et al, Biochem. Biophys. -~
~es. Commun., 175:556 (1991)). A recent ~tudy showed that selective ET8 agonists caused only vasodilation in the rat aortic ring, possibly through the release of EDRF from the endothelium (ibid~. Thus, reported ~-~
selective ETB agonists, for example, the linear analog ;~
ET[1,3,11,15-Ala] and truncated analogs ET16-21, 1,3,11,15-Ala], ETl8-21,11,15-Ala], and N-Acetyl-ET[10-21,11,15-Ala~ caused vasorelaxation in 2S isoIated, endothelium-intact porcine pulmonary arteries (Saeki, T., et al, Biochem. Bio~hys. Res. ~;~
Commun. 179:286 (1991)). However, some ET analogs are poten~ vasocon~trictors in the rabbit pulmonary artery, a tissue that appears to possess an ETB
y, nonselective type ~ofl receptor (ibid).
Plasma endothelin-l levels were dramatically increa~ed in a patient with malignant hemangioendothelioma (K. Nakagawa et al, Nip~on Hifuka ~`
Gakkai Zasshi, l990, 100, 1453-14S6).
The ~T receptor antagonist BQ-123 has been shown to block ET-1 induced bronchoconstriction and tracheal smooth muscle contraction in allergic sheep providing WO93/2121~ 21~3og o PCT/US93/03658 evidence for expected efficacy in bronchopulmonary diseases such as asthma (Noguchi, et al, Am Rev.
Respir. Dis., 1992, 145 (4 Part 2), A~58).
Circulating endothelin levels are elevated in s 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. Gynecol., 1992, 166, 962-968).
Plasma immunoreactive endothelin-l concentrations are elevated in patients with sepsiis and correlate ~ith the degree of illness and depression of cardiac output (Pittett J., et al, Ann Surq., l99l, 213(3), -262).
In addition the ET-l antagonist ~Q-123 has been evaluated in a mouse model of endotoxic shock. This ETA antagonist significantly increased the survival rat~e~in~this model (Toshiaki M., et al, 20.12.90.
EP~0 4~36 l~as Al).
~ ~End~thelin is a potent agonist in the li~er -`-eliciting both sustained vasoconstriction of the hepatic vasculature and a significant increase in `~
hepatic glucose output (Gandhi C.B., et al, Journal of iolo~ical~Chemistry, l990, 265~29), 17432). In ~,`
streptozotocin-diabetic rats there i~ an increased sensiti~ity to endothelin-l (Tammesild P.J., et al, Clin.~Ex~. PharmacoI. PhY~iiol , 1992, l9(4), 261). In ; addition increased levels of plasma ET-l have been obser~ed in microalbuminuric insulin-dependent ii diabete!s mellltuslpatients indicating a role for ET in endocrine disorders such as diabetes (Collier A., et al, Diabetes Care, 992, 15(8), 1038). . `
` ETA antagonist re¢eptor blockade ha~i been found to produce an antihypertensi~e effect in normal to low renin modeiis of hypertenision with a time courise similar to the inhibition of ET-l pre~sor responses ~-(3asil M.~., et al, J. Hvpertension, 199~, ~' . .
.
~`

W O 93/21219 2 1 3 3 0 9 0 PC~r/US93/03658 ~

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).
The widespread localization of the endothelins and their receptors in the central nervous system and -cerebrovascular circulation has been described -(Nikolov R.K., et al, Druqs of Today, 1992, 28(5), 303-310). Intracerebroventricular administration of ET-1 in rats has bee~ shown to evoke several behavioral effects. The~e factor~ strongly suggest a role for the ETs in neurological disorders. The potent vasoconstrictor action of ETs on isolated cerebral arteriolec suggests the importance of these -~ peptides in the regu~lation of cerebrovascular tone. ~~
Increased~ET~levels have been reported in some CNS
disorders,~i.e., in the CSF of patients with 20 ~ ~subarachnoid hemorrhage and in the plasma of women ~-w~ith~preecliampsia. 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 ~uggested to~contribu~te to regulation of brain fluid balance ~through effects on the choroid plexus and CSF
production. ET-l induced lesion development in a new model of local ischemia in the brain has been ;~
described.~ l !1 ' ii i` ". , ~:
Circulating and tissue endothelin immunoreacti~ity is increased more than twofold in patient~ with advanced atherosclerosi~ (A. ~erman, et al, New Enqland J. Med., 1991, 325, 997-1001).

3~ Increased endothelin immunoreactivity has also been associ~ted with Buerger'9 di~ea9e (~. Ranno, et al, J.
` Amer. Med. Assoc., 1990, 264, 2868) and Raynaud's -~' 2 i 33090 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, ~9,~239-245).
An increase of circulating endothelin le~els was observed in patients that underwent percutaneous transluminal coronary angioplasty (PTCA) (A. Tahara, et al, Metab. Clin. Exp., 1991, 40, 1235-1237, K.
Sanjay, et al, Circulation, 1991, 84(Suppl. 4), 726).
Increased plasma levels of endothelin have been measured in rats (T.J. Stelzner, et al, Am. J.
Physiol., 1992, ~62, L614-~620) and individuals (T. Miyauchi, et al, Jpn. J._Pharmacol., 1992, 58, 279P, D.J. Stewart, et al, Ann. Internal Medicine, 1991, 114 464-469) with pulmonary hypertension. --~ Elevated levels of endothelin have also been measured in patients suffering from ischemic heart disease~(M. Yasuda, et al, Amer. Heart J., 1990, 119 20 ~ ~801-806, ~S.G. Ray, et al, Br. Heart J., 1992, 67, 3~3- -`
386)~and either stable or unstable angina /J.T.
Stewart, et al, Br. Heart J., 1991, 66, 7-9).
Infusion of an endothelin antibody lh prior to and lh after a 60 minute period of renal ischaemia 25~ resulted in changes in renal function versus control.
In addition, an increase in glomerular platelet-activating factor was attributed to endothelin (A. ;~
~opez-Farre, et al, J. Physioloqy, l991, 444, 513-522). In patients with chronic renal failure as well ; as in patientsion,regular hemodialysis tr,eatment mean plasma endothelin levels were significantly increased (F. Stockenhuber, et al, Clin. Sci. l~ond.), 1992, 82, 255-258). In addition it has been suggested that the prolifarative effect of endothelin on mesangial cells may bé a contrlbuting factor in chronic renal failure (P.J. Schultz, J. Lab. Clin. Med., 1992, 119, 448-449).

, WO93/21219 2 1 3 3 o (3 o PCTIUS93/03658 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, Diqestion, 1991, 48, 163-172). Administration S of endothelin-1 in the range o~ 50-500 pmol/kg into the left gastric artery increa3ed the tissue type ;~
plasminogen activator release and platelet activating formation, and induced gastric mucosal hemorrhagic change in a dose dependent manner (I. Xurose, 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. Masud2, et al, Am. J. Physiol., 1992, 262, G785-G790). Elevated endothelin levels have been observed in patient~ suffering from Crohn's disease :~
and ulcerative colitis (S.H. Murch, et al, Lance.t, 1992, 339, 381-384)~
Recently the nonpeptide endothelin antagonist RO 46-2005 has been reported to be effective in models .
of~acute renal ischemia and subarachnoid hemorrhage in ~-~ rats~(3rd International Conference on Endothelin, `~:
- :~ Houston, Texas, February 1993). In addition, the ETA -antagonist BQ^123 has been shown to prevent early cerebral vasospasm following subarachnoid hemorrhage (M. Clozel and H. Watanabe, Life Sci., 52:825-834 (1993)).

: .

~: ' ; ;.
,' ~

' WO 93~21219 213 3 ~) 9 0 PCI/US93/03658 TABLE I . Plasma Concentrations of ET- 1 in Humans ET Plasma C ndition NormalLevel~ Reported Control( /mL) Atherosclerosis 1.4 3.2 pmo~/L
Surgical operation 1.5 7.3 Buerger's disea~e 1.6 4.8 Takayasu's arteritis 1.6 5.3 Cardiogenic shock 0.3 3.7 :-Congestive heart failure ~CHF) 9.7 20.4 Mild CHF 7.1 11.1 ~.
Se~ere CHF 7.1 13.8 Dilated cardio~yopathy 1.6 7.1 Preeclampsia 10.4 pmol/L 22.6 pmol/L
Pulmonary hypertension 1.45 3.S
Acute myocardial infarction 1.5 3.3 (~everal reports) 6.0 11.0 0.76 ~.9S :
0.50 3.8 Subarachnoid hemorrhage 0.4 2.2 Crohn's Disease 0-24 fmol/mg 4-6~ fmol/mg -~
Ulcerati~e coliti6 0-24 fmol/mg 20-50 fmol/mg --Cold pres~or test 1.2 8.4 Raynaud'6 phenomenon 1.7 5.3 ~:
Raynaud'6/hand cooling 2.8 5.0 Hemodialysis c7 10.9 (several reports) 1.88 4.59 .~:
Chronic renal failure 1.88 10.1 :
Acute renal failure 1.5 10.4 ~:
~remia before he:modialy~i6 0.96 1.49 Uremia after hemodialysi6 0.96 2.19 Es~ential hypertension 18.5 33.9 Sep~i6 6yndrome 6.1 19.9 Postoperative cardiac 6.1 11.9 I~flammatory arthritide~ l.S 4.2 ~:
Malignant hemangioendothelioma 4.3 16.2 : 35 (after removal) ,~ -Rovero, P., et al, British Journal of PharmacolQ~y 01, pages 232-236 (1990) disclosed various analogs of the C-terminal hexapeptide of ET-l, none of which wére reported;to bé antagonists of ET-1.
Doherty, A. M., et al, Ab~tract, Second International Conference on Endothelin, Tsukuba, Japan, December 9, 1990, and the published manuscript ~45 (J. Cardiovasc. Pharm. 17 (Suppl. 7), 1991, pp. 559-561) disclosed various analog~ of the C-terminal hexapeptide of ET-1, none of which ex~libited any functional activity.
~.

~ 93/21219 2 1 3 ~ O 9 ~ PCT/US93/03658 - 1 1 - .
Copending United States Patent Appli~ation Serial Number 07/995,480 discloses a ~eries of novel antagonist~ of endothelin.
However, we have surprisingly and unexpected~y found that a series of C-terminal hexapep~ide and related analogs of ET-l are receptor antagoni~ts of -;~
endothelin. Additional data for the activity of this ;
series of peptides iY found in the followins -~
referenceQ (W.L. Cody, et al, J. Med. Chem., 1992, 35, ~;;
3301-3303., D.M. LaDouceur, et al, FASEB, lg92).
~ ~.
SUMMARY OF TEE INVENTION

Accordingly, the present irl~ention is a compound of Formula I
AAl - AA2 - AA3 - AA4 - AA5 AA6 ~;

I

wherein AAl is ~-' :'.

R-C-C -`~ ZS X~ Y

wh~rein R is!,hydrogen~
alkyl, alkenyl, alkynyl, , ; cycloalkyl, ~35 cycloalkylalkyl, aryl, heteroaryl, fluorenylmethyl, - ,- .

WO93/21219 ` 2 1 3 3 0 9 0 PCT/US93/0365~

, :., N R2, wherein R2 and R3 are each the same or R3 different and each is hydrogen, alkyl, ~
alkenyl, .
alkynyl, .:
cycloalkyl, ~:
cycloalkylalkyl, ~-aryl, :-arylalkyl, heteroaryl, or ~-~
fluorenylmethyl, O ,~
-C-OR2, wherein R2 is as defined above, -oR2, wherein R2 is as defined above, O
-N-C-N-R3, wherein R2 and R3 are as defined 2 1 2 above, O .
-C-C~R9)3, wherein R9 is F, Cl, ~r, or~
-CH2-OR2, wherein R2 is as defined above f '"
o ~.~
: 3 ~-:~ -N-C-R , `.
~: 30 ¦Za wherein R2a is hydrogen or alkyl and R3 i9 ag defined above, ;

: -N-C-OR , 2a wherein R2a and R3 are as defined above excluding R3 i8 hydrogen, or -O
-C-R2, wherein R2 is as defined above, Rl is hydrogen or alkyl, . ~'' ~"~93/21219 2 `1 3 3~0 9 ~ PCT/VS93/03658 i Z is :;' -O-, -S ()m~
wherein m is zero or an integer of~ -1 or 2, -~
-N-, wherein R2 is as defined above, 12 :~
-(CH2)n-, wherein n is zero or an integer :~
of 1, 2, 3, or 4, - ( CH2 ) n- CH=CH- ( CX2 ) n~
wherein n is as defined abo~e, O ,~, Il , -C-, -CRl-, wherein R1 and R2 are as defined IR2 above, or ~ ~:
~ ~ : R2 . ':~

: : 13 wherein R2 and R3 are each the same or 25 ~ 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 sub6tituents selected from the group consisting of :30 : ~ hydrogen, ,, -: ~ :
: halogen, :alkyl, -CO2R2, wherein R2 is as defined above, ::
li ~ -CONR2,! whierein:R2 an`d R3 are'as defined ;
: ~ 35 13 ~-- R abo~e, NR2, wherein R2 and R3 are as defined above, or ~ ~;

WO93/21219 213 3 O 9 ~ PCTIUS93/03658~;

-14- ;-::
nitro or ~:

O ~';, R--C--C-- ~
S X--~--Y

wherein R, Z, X, a~d Y are as defined above; ~:
~
AA2 is Rl O ,.
~ * 11 ~
--N--C~C---Rl ( CH2 ) n `:
wherein R4 is , ~
~ hydrogen, .1-:~-: 20 ~ alkenyl, : `~;
alky~yl, cycloalkyl, aryl, heteroaryl, ;25~ ~ ~ N R3b, 2b wherein R2b and ~3b are each the same or different and each i8 ~ hydrogen, ~ . ....
lkyl, cycloalkyl, aryl, or ~ , heteroaryl, -OR2b, wherein R2b i8 as defined above, -;`

- C - N- R3b, ~ 40 R2b :, : ~
:, ;~.

93/~1219 213309~0 PCr/U593/03658 wherein R2b and R3b are each the same or different and .:
each i~ as def ined abotre f or R2b 3.nd R3b, O
-C-R2b, wherei~ R2~ is as defined abo~e, .
NH , NH c N~ R2b wherein R2b is as defined -~
above, or ~.
O
- 15 -C-OR~, wherein R2b is as de~ined abovei, and :;~
Rl and n axe as def ined above, or - AA2 iS absent;
: ~ is : ~ ; 20 ~ : Rl O
--N--C--C--Rl ( CIH2 ) n Rs ~i 25 i wherein R5 i~
: : hydrogen, alkyl, aryl, heteroaryl, , ,:
~-~`: 30~ : o ~ 2~
~ :35 wherein R2b and R3b are : ; each the same or different and each i9 as ~, - -, ,, def ined above, ". ~ :

..

WO93~21219 PCT/US93/03658~ ^~
2133~9D

O '~
2b, wherein R2~ is as defined above, or -C-OR2b, wherein R2b is as defined above, and Rl and n are as defined above, or AA3 is absent; ~::
AA4 and AA5 are each independently absent or each is independently Rl O ,~
C--C-- .
Rl ~ IH2) n R6 `

wherein R6 is hydrogen, alkyl, 20 ~ alkenyl, alkynyl, cycloalkyl, ;
aryl, or heteroaryl, and i2~5~ :: Rl and n are as defined above;
~iS
R ~,!' N--C*-R

- ~ : :Rl: ( CH2 ) n r R7 ;
wherein R7 is ~.
aryl or ~.
~;~- . heteroaryl, .
:
~: ', R8 i~3 ' , O :
-C-OR1, wherein Rl i~ as defined `-.
above, ~ :

"~O 93~21219213 3 0 9 0 PC~r/US93/03658 ,, '' ;

-OR1, wherein Rl is as defined :.~
above, -O
5-C-~-R1, wherein R1 is as defined Il abo~e, or .
-CH2-ORl, wherein Rl is as defined above, and 10Rl and n are as defined above;

stereochemi~try at C in A~ 2, AA3, A~4, or AA5 iS
D, L, or ~L and 3tereochemistry at C in AA6 is L; or a pharmaceutically acceptable salt thereof.
. Ele~ated levels of endothelin ha~e been postulated to be involved in a nu~ber of pathophysiological states including diseases 20~ ~associated with:the cardiovascular system~às well as ~ari~ous metabolic and endoc~inological disorders. As antagonists of endothelin, the compounds~of Formula I
are:~useful in~the treatment of hYpertension, :`.
myQcardial infarction, metabolic, endocrinological and :neurolog:ical disorders, congestive heart~failure, endot~oxic shock, subarachnoid hemorrhage, arrhythmias, asthma, and chronic~and acute renal faiIure, presclampsia, atherasclerotic di~orders including -Raynaud'3~ diseas~e, re~tenosis, angina, cancer, ~ pulmonary~hypertension, ischemic disPase, ~astric mucosal damage, hemorrhagic ~hock, i~chemic bowel `~
~; disease,-and d-~abetes,.~
A still further embodiment of the prqsent nvention is a pharmaceutical composition for 35 ~. admini3tering an effe¢tive amount o a compound of `~
Formula I in unit dosage form in the treatment method~ :
me~tioned above.
FinaIly, the present in~ention i~ directed to method~ for production of a compound of Formula I. ~.

' WO93/21219 PCT/US93/03658,.~
2 :1 3 3 '0 ,~ O
-18- :~
DETAILED DESCRIPTION OF THE INVENTION :

In the compounds of Formula I, the term "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-nonyl, n-decyl, undecyl, dodecyl, and the like.
The term "alkenyl" means a straigh~ or branched unsaturated hydrocarbon radical having from 2 to 12 carbon atoms alld 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, 1-decenyl, 1-undecenyl, l-dodecenyl, and the like.
The term "alkynyl" means a straight or branched triple~bonded unsaturated hydrocarbon radical having rom~2 to 12 carbon atoms and includes, for example, e~hynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, .
1-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 3-heptynyl, 1-octynyl, 2-octynyl, '~
1-nonynyl, 2-nonynyl, 3-nonynyl, 4-nonynyl, 1-decynyl, 2-decynyl, 2-undecynyl, 3-undecynyl, 3-dodecynyl, and ~ the like.
The term "cycloalkyl" means a saturated hydrocarbon ring which contains from 3 to 12 carbon atoms, for example,~ cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, and the like.
30 ~i The term~ cyçloalkylalkyl n 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. ~xamples of -~ such are cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl, adamantylmethyl and the like.
The terms "alkoxy n and "thioalkoxy n are O-alkyl or S-alkyl as defined above for alkyl.

~: ' ' ~ .

"'~93/21219 ~1 3 3 0 9 0 PCT/US93/03658 .

-l9- :
The term "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, 3,3-diphenylalanyl, lO,ll-dihydro-5H-dibenzo~a,d]~ ~
(cyclohepten-5-yl)glycyl, or a fluorenyl group and the :
like, unsubstituted or substituted by l to 4 substituents selected from alkyl as defined above, alkoxy as defined above, thioalkoxy as defined above, ~.

hydroxy, thiol, nitro, halogen, amino, -NH-C-alkyl .
O
Il -wherein alkyl is as defined above, -C-O-alkyl wherein O ::
a}kyI is as defined above, -C-alkyl wherein alkyl is : as defined above, or aryl.
The: term~-ar~yla1kyl"~means an aromatic radical 20 ~ a:ttac ~ d~to~an:~alkyl~radical~wherein aryl and alkyl ~-aré~as~def;ined~above~for example benzyl,:
fluorenylmethyl~and~the like. . -`
The term~"heteroaryl" means a:heteroaromatic ~-radical~which is 2-or 3-thienyl, 2- or 3-furanyl, 2-25~ or 3-~pyrr~l l, 2-, 4-,:or 5-imidazolyI, 3-,~ 4-, or `5~pyrazo}yl,:~2-~, 4-,::or 5-thiazolyl, 3-, 4-, or `~

5~-i ~ iazolyl,-~2-, 4-,~ or~5-~oxàzolyl, 3-, 4-, or '.`
isoxazolyl,;~:~3;-~or 5-1,2,4~-triazolyl, 4- or ~r~ 5~1~ 2~,~`3-eriazolyl,~ tetrazolyl, 2-, 3-,::or 4-pyridinyl, 30~ 3~ 4~ or~:~5~-:pyridazinyl, 2-pyrazinyl, 2-, 4-, or ~.
5-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or ;`
8-~quinolinyl,,/~ 3~ 4i- ,! 5-, 6-l, 7-, or 8-iqoquinolinyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-,~4~ 5-, 6-, or 7-benzo[b]thienyl, or 2-, 4-, 5-, .~6-,~or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, a-, 4-, 5-, 6-, or 7~benzothiazolyl, uniubstituted~or substituted by 1 to 2 substituents elected from alkyl as defined above, aryl as defined ;~.

~ .

WO93/21219 2 1 ~ 3 0 9 0 PCT/US93/036~_ above, alkoxy as defined abo~e, thioalkoxy as defined above, hydroxy, thiol, ~

Il ~ , .
nitro, halogen, formyl, amino, -NH-C-alkyl wherein O ~, alkyl is as defined above, -C-O-alkyl wherein alkyl O
' 11 ,''.'`
is as defined above, -C-alkyl wherein alkyl i~i as ~i:
de~ined above or phenyl.
The term "heterocycloalkyl" means 2- or ~`
3-tetrahydrothieno, 2- or 3-tetrahydrofurano, 2- or .-3-pyrrolidino, 2-, 4-, or 5-thiazolidino, 2-, 4-, or -~
5-oxazolidino, 2-, 3-, or 4-piperidino, N-morpholinyl ~`
or N-thiamorpholinyl. :.
"Halogen" is fluorine, chlorine, bromine or ~-iodine.
: The f~ollowing table:provides a lis~ of a`bbreviations and definitions thereof used in the pre~ént invention.

: : .
~.

~. .
, . . ..
.,::

,':

' ~93/21219 2 1 ~ ~ O 9 0 PCT/US93/03658 TA~LE
Abbreviation~ Amino Acid Ala Alanine Arg Arginine Asn Asparaglne Asp Aspartic acid -;~
Cy5 Cysteine ;
Glu Glutamic acid Gln Glutamine ~, Gly G3.ycine .
His Histidine -", Ile Isoleucine Leu Leucine ~`
. ~ .
Lys~ ysi~e - Met ;Methionlne~ ~' 'Phe~ Phenylalanine Pro~ Prollne S~er:~ Serine ,,, 2Q Thr Threonine ~.
Trp Tryptophan~
Tyr Tyrosine Val ~ ~ Valine ``i 25' : ~,Abbre~i~tion* Modified and Unusual Amino Acid Bhg 10,11-Dihydro-5H-dibenzo[a,d]- ,~
~ " ,'" !~ cycljQhepten-5-yl,)glycine, or ;~
- ~-Amino-10,11-dihydro-5H-dibenzo-[a,d]cycloheptene-5-acetic acid , . Bip (Paraphenyl)phenylalanine :~

: ~ If the optical actiYity of the amino acid is other -:
'~ than ~S),~ the amino acid or abbreviation is ~: preceded by the appropriate configuration D(R) or ~, ' D~(RS).

WO93~21219 2 1 3 3 ~ 9 o PCT/US93/0365~

-22- ~;
Abbre~iation* Modified and Unusual Amino Acid (cont) `~
Dip 3,3-Diphenylalanine 3Hyp 3-Hydroxyproline ;
4Hyp 4-Hydroxyproline ~
N-MePhe N-Methylphenylalanine '~;
N-MeAsp N-Methylaspartic acid Nva Norvaline Nle Norleucine .;
Orn ~ Ornithine ~
Abu 2-.Aminobutyric acid ,~.
Alg 2-Amino-4-pentenoic acid '~'!'~'~'`
(Allylglycine) Arg(N02) NG-nitroarginine Atm~ 2-Amino-3-(2-amino-5-thiazole)propanoic acid ,~
Cpn~ : ~ 2-Amino-3-cyclopropanepropanoic acid Cy~clopropylalanine) 15 ~ Chx ~ Cyolohexylalanine (Hexahydrophenyl-;Emg ~ ~ 2-Amino-4, 5 ~RS) -epoxy-4-pentenoic acid ;::
His~(Dnp) ;Nim- 2,4-Dinitrophenylhistidine HomoGlu : 2-Aminoadipic acid : HomoPhe : ~ 2-Amino-5-phenylpentanoic acid Homophenylalanine) Met(O) Methionine sulfoxide :
: Met (2) / ilMelthionine sulfone / l~ -Nal 3-(1'-Naphthyl)alanine : 2-Nal 3-(2'-Naphthyl)alanine ;,. .: ~, :Nia 2-Amino-3-cyanopropanoic acid . (Cyanoalanine) : ~25 ~: ~ Pgl Phenylglycine ; P~y 2-Aminopentanoic acid (Propylglycine) ~
, ~ . ...
. .

- ' .

~093/21219 ~1 3 3 0 g o P~T/US93/03658 Abbrevi~tion* Modified and_Unusual Amino_Acid (cont) .
Pha 2-Amino-6-(1-pyrrolo)-hexanoic acid Pyr 2-Amino-3-(3-pyridyl)-propanoic acid `
(3-Pyridylalanine) Tic 1,2,3,4-Tetrahydro-3- .
i~oquinolinecarboxylic acid ~-~
Tza 2~Amino-3-(4-thiazolyl)-propanoic acid :.
:: Tyr(Ot-Bu) O-tertiary butyl-tyrosine !'i~
Tyr(OMe) O-Methyl-tyrosine Tyr(OEt). O-Ethyl-tyrosine Trp(For) Nin-Formyl-tryptophan ~- 10 Bheg 5H~-Di~enzo~a,d]cycloheptene glycine Txg 9U-Thioxanthene glycine ~;

viation: ~Protectin~ Group Acetyl~
15~ Ada~ l-Adamantyl~acetic acid Adoc ~: Adamant~loxycarbonyl ~:
Bzl Benzyl ;~
MeBzl :4-Methylbenzyl Z~ Benzylo~cycarbonyl 0.~ 2~-Br~-Z~ ortho-Bromobenzy}oxycarbonyl 2-Cl-Z :~ ortho~Chlorobenzyloxycarbo~yl 30m: ~ Benzyloxymethyl t ' Boc i ~ , tertia ~ ~Bueyl~oxycarbonyl TBS tertiary Butyldimethylsilyl 25~ ;~Dnp ~ 2,4-Dinltrophenyl . For ; Formyl Fmoc 9-Fluorenylmethyloxycarbonyl ~ N02- ~ Nitro ; : :: Tos 4-Toluenesulfonyl (to~yl) .. . ~ , , ,: :

WO93/21219 ~1 3 3 0 .S O pcT/us93/036sa~ ~:

Abbreviation Protectina Group (cont) Trt Triphenylmethyl (trityl) Ada l-Adamantyl acetic acid ~z Benzylcarbonyl ~`:
tBu t-Butylcarbonyl ~
CF3CO Trifluoroacetyl ~-Cxl Cyclohexylacetyl Cxl(U) Cyclohexylurea Et Propionyl Pya 3-Pyridylacetyl Me(U) Methylurea Abbre~iat1on Solvents and Reagents HO~c Acetic acid ~
CH3CN Acetonitrile ::
: DCM Dichloromethane . .;
DCC N,N'-Dicyclohexylcarbodii~ide DIEA N,N-Diisopropylethylamine DMF Dimethylformamide ~-`
HCl Hydrochloric acid -;: ~ HF Hydrofluoric acid , ~
HOBt l-Hydroxybenzotriazole KOH Potasqium hydroxide TFA Trifluoroacetic acid : 25 MBEA Resin Methylbenzhydrylamine resin PAM Resin 4-(Oxymethyl)-phenylacetamidomethyl .~ ~ resin ~

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.

WO93/2121g n go PCT/US93/03658 -25- :~
Pharmaceutically acceptable acid addition salts of the compounds of Formula I include salts derived from nonto~ic inorganic acid~ 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 l~
include sulfate, pyrosulfate, bisulfate, sulfite, ~`
bisulfite, nitrate, phosphate, monohydrogenphosphate, .
dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, su:ccinate, suberate, sebacate, fumarate, maleate, màndelate,:~benzoate, chlorobenzoate, methylbenzoate, ~:
dini~robenzoate, phthalate, benzenesulfonate, :~ eoluenesulf~onate, phenylacetate, citrate, lactate, : mal~eate, tartrate,:methanesulfonate, and the like.
Also~contemplated:are salts of amino acids such as arginate and the like and gluconate, galacturonate : (s~ee,~for~e ampl:e, Berge, S. M., et al, Phàrmaceutical Salt:s, n Journal of Pharmaceutical ~; : 25 ~ :Science, 66, pp. 1-19 (1977)). -The:acid addition salts of said basic compounds ~ are~prepared by coneacting the free base form with a ;~: : :: suffic~ient amount of:the desired acid to produce the ~ ~ saIt in the conventional manner. Preferably a peptide :~ 30 of Fonmula` I can be!con~erted to an acidi,c salt by I .treating with an aqueous solution of the desired acid, -~- sùch that the re ulting pH is les~ than 4. The ;
; solution can be~::pa~sed through.a C18 cartridge to ~ absorb the peptide, washed with copious amounts of ~ 35 water, the peptide eluted with a polar organic solvent ~ such as, for example, methanol, acetonitrile, aqueous , .. . . .
~ mixtures thereof, and the like, and isolated by `.
~ :, , W093/21219 PCT/US93/03658 ~'~
i~

concentrating under reduced pressure followed by lyophilization. The free base form ~ay be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner. The free ba~e 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. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, - and procaine (see, for example, Berge, S. M., et al., "Pharmaceutical Salts, n Journal of Pharmaceutical Science;, 66, pp. l-l9 (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. Preferably, a peptide of Formula I can be converted to a ba~e 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 Cl8 cartridge to absorb the peptide, washed with copious amounts of wate~r, the pept~ide eluted with a polar organic solvent such as, for example, methanol, acetonitrile, aqueous mixtures thereof, and the li~e, and isolated by ; concqntrating 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 re~pective qalt forms ~40 93/21219 ~l33~9 PC~r/US93/03658 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 mo-e 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 iS: O ~ ~
~: : R - CH - C - ```

X - ~ - y wherein R i9 -N-R2, wherein R2 and R3 are each the same or different and each is hydrogen, alkyl, alkenyl, alkynyl, cy~loalkyl, cycloalkylalkyl, ~- - aryl, - ~ arylalkyl, ~; 35 heteroaryl, or fluorenylmethyl, ,:
~,:

WO93/21219 213 3 D 9 0 PCT/US93/0365~

O
-N-C-N-R3, wherein R2 and R3 are as ~:
1 2 1 2 defined above, -C-C(R9)3, wherein R9 is F, Cl, Br, or I, 1 0 l ~ ,' -NH-C-R3, wherein R3 is as defined above, or O

-~H-C-OR , wherein R3 is as defined above excluding R3 is hydrogen, :~
Z is - O-, -20 -S ()m~ ':
wherein m is zero or an integer of ::~
l or 2, `:~-N-, wherein R2 i9 as defined above, - ~ 25 : 1 ~2 :
- ~CH2)n, wherein n is zero or an integer of 1, 2, 3, or 4, ~;
-(CH2)n-CHSCH-~CH2)n-, wherein n is as defined above, 3Q ~ ~
~:.: : - C- :.
CH-, wherein Rl is hydrogen or alkyl, O

--C I : ' ..
'3 -~ 40 ~ wherein R2 and R3 are each the same or .
different and each is as defined above and `
, ::

w093/21219 2133~) PCT/US93/036a8 -29- ::~
X and Y are the same and ~ubstituted at the same ;
position on the aromatic ring and each substituent is selected from the group consisting of .
hydrogen, halogen, or -alkyl;
AA2 is O
- NH - CH - C -( IH2)n R4 `

wherein R4 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, ~- ~ 20 heteroaryl, - N - R3b, . , . : ' R2b wherein R2b and R3b are each . .25 the same or different and each i9 :: hydroge~, :
alkyl, :1 cycloalkyl, ~:
aryl, or ~-~
` I ! ; i ` ` hèteroaryl~
-OR2b, wherein R2b i9 as defined above, ;:

3 -C-N-R3b, ;

::

.~

W O 93/~1219 2 1 3 3 PC~r/US93/03658 -30- ~:
wherein R2b and R3b are each the same or different and each i9 as defined above for R2b and R3b O
-C-R2b, wherein R2~ is as defined above, 1~
-NH-C-NH-R2b, wherein R2b i~ as defined above, or O , -c-oR2b, wherein R2b is a~
defined above, and :~`
n is a9 defined a~ove or AA2 is absent;
AA3 is ~20 : ~ - NH - CH - C -t CIH2 ) n RS `:

: wherein R5 is aryl, ``;heteroaryl, ~;
~ ~ o , ~.:
3 0 ~ - C - N- R3b, ``
2b : wherein R2b and R3b are each the same or different and teach i-q asl ' ~`
: 35 defined above, ~T-R2b, wherein R2b i8 as defined .
above, or . ,.

' WO93/21219 ~33~9~ PCT/US93/03658 -31- ~
o -C-OR2b, wherein R2b is as defined above, and n is as defined above, or AA3 is absent;
AA4 and AA5 are each independently absent or each is O . .~
* 11 - NH - CH - C -( IH2) n : ~
R6 ~;:

wherein R6 is hydrogen :
alkyl, .~.
alkenyl, -.
alkynyl, cycloalkyl, ` aryl, or ~ ~ heteroaryl, and n-is as defined above;
AA6 is ~.

25 ~ ~ tfH2)n ` wherein R7 is aryl or ; 30 : heteroaryl, and n is as defined above, or -~-O
-NH-~CH-C--N-R1 ; 35 ¦ ¦ wherein R7, Rl, and n are as ` (CH2)n Rl defined above, : ~ 17 ~ ` , W093/21219 2~ a9u PCT/US93/03658 stereochemistry at CH in AA1, AA2, AA3, AA4, or AA5 is D~L, or D~, and -* ~.
5stereochemistry at CH in AA6 is L; or a pharmaceutically acceptable salt thereof.
A more preferred compound of Formula I is one wherein AAl is .
* 11 , ~:
R-CH - C -X~ Y '.'~

15wherein R is -N-R , :

wherein R2 and R3 are each the same or different and each:is 20~ hydrogen,~ :
alkyl, ~ :
aryl, or : fluorenylmethyl, N-C-N-:R3, wherein R2 and R3 are R2 R2 as~defined above, C-C(R9)l,wherein R9 i9 F, Cl, Br, or I, 1l ~ ~
: ~ 3~ -NH-C-OR10, wherein R10 i9 hydrogen, -~ ~ : : alkyl, aryl, or ~;;
. arylalkyl, excluding RlO
is hydrogen, Z is O-, : 40 -S~

WO g3/21219 2 l 3 3 0 ~ U PCr/US93io3658 -~CH2)n, wherein n is zero or an --integer of 1, 2, 3, or 4, or - ~CH2)n-CH=CH- ~CH2)n' wherein n is zero or an integer of 1 or 2 and - 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 con~isting of hydrogen, halogen, or alkyl;
AA2 iC
l~i O
NH-CH-C- , ..

~ 1 2)n ,~
;~ : wherein R4 i~ hydrogen, ;:
: alkyl, aryl, heteroaryl, .
N-R3b , :
2b wherein R2b and R3b are : ~ ~ each the same or different and each is hydrogen or alkyl, ~-;

- ~ 35 -C-N-R3b ' R2b .~ ~ - wherein R2b and R3b are ~:
each the same or ~ different and each is hydrogen or alkyl, ~;

, ~ ;
:: , .
~ ' ~

h 1 ~ ~ U
W093/21219 PCT/US93fO3658 , -34- : .
NH . -~, -NH-C-NH-R2b wherein R2b i5 as ~;
defined above, ;~-~
or O ~- ~
OR2b wherein R2b is as defined above, and n is an integer of l, 2, 3, or 4 or AA2 is absent; ~.
~;A3 is o ~`

-NH-fH-C~
(CIH2)n . R5 wherein R5 is `,~,!'.
20~ ~ : aryl, heteroaryl, N~R3b wherèin R3b i9 25 : ~ : hydrogen or :~
alkyl, 0 " -:
2b ~wherein R2 i9 ~a ~ hydrogen or ~ `.
; alkyl, or ; Il oR2b~ wherein R2b is -: ~ hydrogen or ::~

: n is an integer of l, 2, 3, or 4;
A~4 and AAs are each independently O
40~ NH CH

(CH2)n ~ ;
~: 45 R :~
~: ~

: :
~, :
.
, ~ ..

WO93/21219 2l.3 ~ n g~ PCT/US93/036~8 ``':

wherein R6 is hydrogen, ;.
alkyl, cycloalkyl, or .
aryl, and S n is an integer of l, 2, 3, or 4; , *
- NH - fH - C02H
(fH2) n `- .
R7 -: .
wherein R7 is aryl or heteroaryl, and ~',.
n i8 zero or an integer of l, 2, 3, : 15 or 4, or ...
O ~, - NH - CH - C - - N- Rl ~ -¦ wherein R7, Rl, and n are as 20 ~ ~2:'n~ defined above,~

R~
stereochemistry;at CH in:AA1, ~ , A~3, AA4, or AA5 is 2s~ ",D'~ t;

st:e:reochemistry:at CH in A~6~is L; or a pharmaceutically:accepeable sale thereof.
pà~ticularly~ valuable~ are~
-30~ L-~B'hg-~eu-Asp:;Ile-Il,e-Trp; ~, :D^~Bhg-Leu-Asp-I}e'-~Ile-Trp; ~
Ac-'L-Bhg~-~eu-Asp-Ile-Ile-Trp;
Ac-D~-Bhg-~eu-Asp-Ilè-Ile-Trp;
Ac-D-Bhg-Orn-Asp-Ile-Ile-Trp;
~ : 35 ~ ~c-D-~hg-~ys-Asp-Ile-Ile-Trp; i "
"~ : : Ac-D-Bhg-Asp-Asp-Ile-Ile-Trp;
; Ac-D-Bhg-Glu-Asp-Ile-Ile-Trp; ,:
' 'Ac-D-Bhg-Phe-Asp-Ile-Ile-Trp; - ~.'' Ac-D-Bhg-Arg-Asp-Ile-Ile-Trp;
~ ;4Q -~ Ac-D-Bhg-Asp-Ile-Ile-Trp; -~-"~ Fmoc-D-Bhg-Leu-Asp-Ile-Ile~Trp;
~ : Fmoc-D-Bhg-Orn-Asp-Ile-Ile-Trp;

., ,~ . ~ . ;, .

WO93/21219 2 ~ ~ ~ O 9 ~ PCT~US93/~36~8 Fmoc-D-Bhg-Lys-Asp-Ile-Ile-Trp;
Fmoc-D-Bhg-Asp-Asp-Ile-Ile-Trp;
Fmoc-D-~hg-Glu-Asp-Ile-Ile-Trp; ~;
Fmoc-D-Bhg-Phe-Asp-Ile-Ile-Trp;
Fmoc-D-Bhg-Arg-Asp-Ile-Ile-Trp;
Fmoc-D-Bhg-Asp-Ile-Ile-Trp;
Ac-D-Bhg-Leu-Phe-Ile-Ile-Trp; ~'-Ac-D-Bhg-Leu-Asn-Ile-Ile-Trp; -.
. Ac-D-Bhg-Leu-Glu-Ile-Ile-Trp;
Ac-D-Bhg-Leu-Gln-Ile-Ile-Trp;
Ac-D-Bhg-Leu-Tyr-Ile-Ile-Trp; ~.
Ac-D-Bhg-Leu-l-Nal-Ile-Ile-Trp;
Ac-D-Bhg-Leu-2-Nal-Ile-Ile-Trp;
Ac-D-Bhg-Leu-Trp-Ile-IIe-Trp;
Ac-D-Bhg-Leu-Asp-Val-Ile-Trp;
Ac-D-Bhg-Leu-Asp-Ile-Val-Trp; ~-~
Ac-D-Bhg-Leu-Asp-Chx-Ile-Trp;
Ac-D-Bhg-Leu-Asp-Ile-Chx-Trp;
Ac-D-Bhg-:Arg-Asp-Ile-Chx-Trp; . ~;
.
Ac-D-~hg-Lys-Asp-IIe-Chx-Trp;
Ac-D-Bhg-Orn-Asp-Ile-Chx-Trp;
Ac-D-Bhg-Asp-Asp-Ile-Chx-Trp;
Ac-D-Bhg-Glu-Asp-Ile-Chx-Trp; ~`
. Fmoc-D-Bhg-~eu-:Phe-Ile-Ile-Trp;
25 . Fmoc-D-Bhg-Leu-Asn-Ile-Ile-Trp;
Fmoc-D-Bhg-Leu-Glu-Ile-Ile-Trp;
Fmoc:-D~-Bhg-Leu-Gln-Ile-Ile-Trp;
- : Fmoc-D-Bhg-Leu- Tyr -Ile-Ile-Trp;
Fmoc-D-Bhg-Leu-Asp-Val-Ile-Trp;
30 . ,Fm",oc~-~-Bhg,~eu-~sp~,Ile-Val-Trp;
Fmoc-~D-Bhg-Leu-Asp-Chx-Ile-Trp;
Fmoc-~D-Bhg-Arg-ABp-chx-Ile-Trp;
' Fmoc-D-Bhg-Ly~-Asp-Chx-Ile-Trp;
Fmoc-D-Bhg-Orn-Asp-Chx-Ile-Trp; , 35 .~ Fmoc-D-Bhg-Asp-Asp-Chx-Ile-Trp;
Fmoc-D-Bhg-Glu-Asp-Chx-Ile-Trp;
~: Fmoc-D-Bhg-Leu-Asp-Ile-Chx-Trp;
~:, : ':

WO93/21219 2 1 3 3 0 9 0 PCT/US93/036~8 ~
. ;

Fmoc-D-~hg-Arg-Asp-Ile-Chx-Trp;
Fmoc-D-Bhg-Lys-Asp-Ile-Chx-Trp; :~
Fmoc-D-Bhg-Orn-Asp-Ile-Chx-Trp;
Fmoc-D-Bhg-Asp-Asp-Ile-Chx-Trp;
Fmoc-D-~hg-Glu-Asp-Ile-Chx-Trp;
Ac-D-Bheg-Leu-Asp-Ile-Ile-Trp;
Ac-D-Bheg-Orn-Asp-Ile-Ile-Trp;
Ac-D-Bheg-Lys-Asp-Ile-Ile-Trp;
Ac-D-Bheg-Asp-Asp-Ile-Ile-Trp;
Ac-D-Bheg-Glu-Asp-Ile-Ile-Trp;
Ac-D-Bheg-Phe-Asp-Ile-Ile-Trp; :
Ac-D-Bheg-Arg-Asp-Ile-Ile-Trp;
Ac-D-Bheg-Asp-Ile-Ile-Trp; ;~
Fmoc-D-Bheg-Leu-Asp-Ile-Ile-Trp; :~
.
Fmoc-D-Bheg-Orn-Asp-Ile-Ile-Trp; `~-; Fmoc-D-Bheg-Lys-Asp-Ile-Ile-Trp; ` ::~
: Fmoc-D-Bheg-Asp-Asp-Ile-Ile~-Trp;
: Fmoc-D-Bheg-Glu-Asp-Ile-Ile-Trp; ;~
Fmoc-D-Bheg-Phe-Asp-Ile-Ile-Trp;
20 ~ Fmoc-D-~heg-Arg-Asp-Ile-Ile-Trp;
: Fmoc-~-Bheg-Asp-Ile-Ile-Trp; -;
Ac-D-Bheg-~eu-Phe~-Ile-Ile-Trp;
Ac-D-Bheg-Leu-Asn-Ile-Ile-Trp;
Ac-D-Bheg-Beu-Glu-Ile-Ile-Trp;
25`~ Ac-D-~Bheg-~eu-Gln-I}e-Ile-Trp;
Ac~-D~-~Bheg-Leu-Tyr-Ile-Ile-Trp - Ac-D-Bheg-Leu-l-Nal-Ile-Ile-Trp;
Ac-D-Bheg-~eu-2-Nal-Ile-Ile-Trp;
Ac-D-Bheg-Leu-Trp-Ile-Ile-Trp;
30 . ~ Ac-D-Bheg-Leu-As~-Va1-Ile-Trp;

~ : Ac-D-Bheg-Leu-Asp-Ile-Val-Trp;
s~ Ac-~-Bheg-~eu-A~p-Chx-Ile-Trp;
. Ac-D-Bhe~-~eu-Asp-Ile-Chx-Trp;
Ac-D-Bheg-Arg-A p-Ile-Chx-Trp;
35~ Ac-D-Bheg-Bys-Asp-Ile-Chx-Trp; ..
Ac-D-Bheg-Orn-Asp-Ile-Chx-Trp;
Ac-D-Bheg-Asp-Asp~Ile-Chx-Trp;

- - . ;, .
.

. ~
'.,:,`

WO93~21219 2 1 ~ 3 0 Q~ PCT/US93/03658 Ac-D-Bheg-Glu- Asp - I le-Chx-Trp;
Fmoc-D-Bheg-Leu-Phe-Ile-Ile-Trp;
Fmoc-D-Bheg-Leu-Asn-Ile-Ile-Trp;
Fmoc - D -Bheg-Leu-Glu-Ile-Ile-Trp;
Fmoc-D-Bheg-Leu-Gln-Ile-Ile-Trp;
Fmoc-D-Bheg-Leu-Tyr-Ile-Ile-Trp;
Fmoc-D-Bheg-Leu-Asp-Val-Ile-Trp;
Fmoc-D-Bheg-Leu-Asp-Ile-Val-Trp;
Fmoc-D-Bheg-Leu-Asp-Chx-Ile-Trp;
Fmoc-D-Bheg-Arg-Asp-Chx-Ile-Trp;
E~noc-D-Bheg-~ys-Asp-Chx-Ile-Trp;
hmoc-D-Bheg-Orn-Asp-Chx-Ile-Trp;
- Fmoc-D-Bheg-Asp-Asp-Chx-Ile-Trp;
Fmoc-D-Bheg-Glu-Asp-Chx-Ile-Trp;
Fmoc-D-Bheg-Leu-;Asp-Ile-Chx-Trp;
Fmoc-D-Bheg-Arg-Asp-Ile-Chx-Trp;
Fmoc-D-Bheg-Lys-Asp-I1e-Chx-Trp;
Fmoc-D-Bheg-Orn-Asp-Ile-Chx-Trp;
Fmoc-D-Bheg-Asp-Asp-Ile-Chx-Trp;
20~ Fmo~-D-Bheg-Glu-Asp-Ile-Chx-Trp;
Ac-D-Txg-Beu-Asp-Ile-Ile-Trp;
Ac-D-Txg-Orn-Asp-Ile-Ile-Trp;
` Ac-D-Txg-~ys-Asp-Ile-Ile-Trp;
~:~ Ac-~-Txg-Asp-Asp-Ile-Ile-Trp;
Ac-D-Txg-GIu-Asp:-Ile-Ile-Trp;
Ac-D-Txg-Phe-Asp-Ile-Ile-Trp;
Ac-D-~xg-~Arg-Asp-Ile-Ile-Trp;
,: , Ac-D--Txg-Asp-Ile-Ile-Trp; -~
Fmoc-D-Txg-Leu-Asp-Ile-Ile-Trp;
. ~ . Fmoc-D-Tx~-O~nT~sp,Ile-Ile-~Trp;
Fmoc-D-Txg- ~y9 -Asp-Ile-Ile-Trp;
Fmoc-D-Txg-Asp-Asp-Ile-Ile-Trp; ;:-Fmoc-D-Txg-Glu-Asp-Ile-Ile-Trp;
- ~ Fmoc-D-Txg-Phe-Asp-Ile-Ile-Trp;
35~ Fmoc-D-Txg-Arg-Asp-Ile-Ile-Trp;
: Fmoc-D-Txg-Asp-Ile-Ile-Trp;
Ac-D-Txg-~eu-Phe-Ile-Ile-Trp;
~, ~, W O 93/~1219 .P ~ /US93/03658 2,1 339 `
-39- ~:
Ac-D-Txg-Leu-Asn-Ile-Ile-Trp;
Ac-D-Txg-Leu-Glu-Ile-Ile-Trp;
Ac-D-Txg-Leu-Gln-Ile-Ile-Trp;
Ac-D-Txg-Leu-Tyr-Ile-Ile-Trp; ~ :
Ac-D-Txg-Leu-1-.Nal-Ile-Ile-Trp;
Ac-D-Txg-Leu-2-Nal-Ile-Ile-Trp; :
Ac D-Txg-Leu-Trp-Ile-Ile-Trp;
Ac-D-Txg-Leu-Asp-Val-Ile-Trp;
Ac-D-Txg-Leu-Asp-Ile-Val-Trp;
~c-D-Txg-Leu-Asp-Chx-Ile-Trp; ~;~
Ac-D-Txg-Leu-Asp-Ile-Chx-Trp;
Ac-D-Txg-Arg-Asp-Ile-Chx-Trp;
Ac-D-Txg-Lys~Asp-Ile-Chx-Trp; :~
Ac-D-Txg-Orn-Asp-Ile-Chx-Trp;
1~ Ac-D-Txg-Asp-Asp-Ile-Chx-Trp;
Ac-D-Txg-Glu-Asp-Ile-Chx-Trp;
Fmoc-D-Txg-Leu-Phe-Ile-Ile-Trp;
Fmoc-D-Txg-Leu-Asn~-Ile-Ile-Trp;
Fmoc-D-Txg-Leu-Glu-Ile-Ile-Trp;
Fmoc-D-Txg-Leu-Gln-Ile-Ile-Trp;
Fmoc-D-Txg-Leu-Tyr-Ile-Ile-Trp; ~:
Fmoc-D-Txg-Leu-Asp-Val-Ile-Trp;
; : Fmoc-D-Txg-Leu-Asp-Ile-Val-Trp;
- .
Fmoc-D-Txg-Leu-Asp-Chx-Ile-Trp;
Fmoc-D-Txg-Arg-~sp-Chx-Ile-Trp;
Fmoc-D-Txg-Lys-Asp-Chx-Ile-Trp;
Fmoc-D-Txg-Orn-Asp-Chx-Ile-Trp;
Fmoc-D-Txg-Asp-A p-Chx-Ile-Trp;
Fmoc-D-Txg-Glu-Asp-Chx-Ile-Trp;
Fmoc-D-Txg-Leu-Asp-Ile-Chx-Trp;
: Fmoc-D-Txg-Arg-Asp-Ile-Chx-Trp;
: Fmoc-D-Txg-~ys-Asp-Ile-Chx-Trp;
Fmoc-D-Txg-Orn-Asp-Ile-Chx-Trp;
:~ Fmoc-D-Txg-Asp-Asp-Ile-Chx-Trp;
Fmoc-D-Txg-Glu-Asp-Ile-Chx-Trp;
Et-D-Bhg-Leu-Asp-Ile-Ile-Trp;
Bz-D-Bhg-Leu-Asp-Ile-Ile-Trp;

W093/21219 2 1 3 3 0 9 0 PCT/US93/036~8 ,~

Pya-D-Bhg-Leu-Asp-Ile-Ile-Trp;
Cxl-D-Bhg-Leu-Asp-Ile-Ile-Trp;
Ada-D-Bhg-~eu-Asp-Ile-Ile-Trp;
Cxl(U)-D-Bhg-Leu-Asp-Ile-Ile-Trp;
Me(U)-D-Bhg-Leu-A~p-Ile-Ile-Trp;
tBu-D-Bhg-Leu-A~p-Ile-Ile~Trp;
CF3CO-D-Bhg-Leu-Asp-Ile-Ile-Trp; ~
Et-D-Bheg-Leu-Asp-Ile-Ile-Trp; ~, Bz-D-Bheg-Leu-Asp-Ile-Ile-Trp;
Pya-D-Bheg-Leu-Asp-Ile-Ile-Trp; -~
Cxl-D-Bheg-Leu-Asp-Ile-Ile-Trp; -~
Ada-D-Bheg-Leu-Asp-Ile-Ile-Trp;
Cxl(U)-D-Bheg-Leu-Asp-Ile-Ile-Trp; -~
Me(U)-D-Bheg-Leu-Asp-Ile-Ile-Trp;
tBu-D-~heg-~eu-Asp-Ile-Ile-Trp;
- ~ CF3CO-D-Bheg-Leu-Asp-Ile-Ile-Trp;
Ac-D-Bhg-~eu-Asp;-Phe-Ile-Trp;
Ac-D-Bhg-Orn-Asp-Phe-Ile-Trp;
Ac-D~-Bhg-~ys-Asp-Phe-Ile-Trp; ;~-~
~ AG~D~-B ~-Asp-Asp-Phe-Ile-Trp;
Ac~-D-~hg-G}u- ~ -Phe-Ile-Trp;
Ac~-D~-Bhg-~Phe-Asp-Phe~-Ile-Trp;
Ac-~D-~hg-Arg-Asp-Phe-Ile-Trp;
Ac-D-~heg-heu-Asp-Phe-Ile-T~p;
~-; 25 ~ ;Ac-~D-Bheg-Orn-Asp-~Phe-I1e-T~; ;` Ac-~D-Bheg-Lys-A~p-Phe-Ile-Trp;
Ac-D-Bheg-Asp-Asp-Phe-Ile-Trp;
Ac-~D-~heg-Glu-Asp~-Phe-Ile-Trp;
Ac-D-Bheg-Phe-Asp-Phe-Ile-Trp; and 30 ~ Aci-D-~Bheg-~Argl-A~p,-PIhe-Ile-Trp;
or a pharmaceutically acceptable acid or base addition ~-salt~thereof. ~ ~
The compounds~of Formula I are ~aluable antagonists of endothelin. The tests employed ~"
35 ~ indi~cate that compounds of Fonmula I po~-Ress endothel1n antagonist acti~ity. Thus, the compounds of Pormula I were tested for their ability to inhibit ::

W O 93/212~9 ~ ~ 3 3 o ~ ~ PC~r/US93/03658 [125I] -ET-1([125I]-Endothelin-~) binding in a receptor assay according to the following procedures~

E~nDOl~EhIN ~UECEPTOR BI~nDING ~k5SAY-A (ERBA-A)~ .:
I~rrACT CELL BI~nDING OF [125I]-ET-l `~`~

Materials and Terms Used:
Cell The cells used were rabbit renal artery vascular smooth muscle cells grown in a 48-well dish (1 cm2) (confluent cells).

Growth Medla The growth media was Dulbecco' 9 Modified Eagles/Ham's F12 which contained 10~ fetal bovine serum and antibiotics (penicillin/streptomycin/
fungizone).

As~ay Buffer -~
The~assay buffer was a medium 199 containing ' Hanks salts and 25 mM Hepes buffer (Gibco 380-23SOAJ~
supplemented~ with penicillin/streptomycin/fungizone 0.~5~ and~bovine ~erum albumin (1 mg/mL~

t125~ ET 1 Amersham radioiodinated endothelin~ 25I]-ET-1 was`used~at final concentration of 20,000 cpm/0.25 mL
(25 pM).: -~

~:: 30 . Protocol `~
First, add 0.5 mL warm assay buffer '(described ! '~
above) to the aspirated growth media and preincubate for 2 to.3 hours in a 37C water bath (do not put back ;~
in the 5% carbon dioxide). Second, remove the assay i:
-~ ~:35:~ buffers, place the dish on ice, and add 150 ~L of cold :.
assay buffer described above to each well. Third, add 50~mL each of cold l125I]-ET-1 and competing ligand to -~

., '.
`, , W093/21219 2 I ~ 3 o ,q o PCT/US93/03658 j,._ the solution ~at the same time if possible). Next, place dish in a 37~C water bath for about 2 hours and gently agitate the dish every 15 minutes. Discard the radioactive incubation mixture in the sink and wash wells 3 times with 1 mL of cold phosphate buffered saline. Last, add 250 mL of 0.25 molar sodium hydroxide, agitate for l hour on a rotator, and then transfer the sodium hydroxide extract to gamma counting tubes and count the radioactivity.
ENDOT~ELIN RECEPTOR BINDING ASSAY-B (ERBA-B) [125I]-ET-l B~NDING IN RAT CEREBELLAR M}~BRANES

Materials and Terms Used:
Ti~Que Buffer The tissue is made up o~ 20 mM tris(hydroxy- -~
methyl)aminome~thane hydrochloride ~Trizma) buffer, 2 mM ethylenediaminetetra acetate, l00 ~M --phenylmethylsulf~onyl fluoride.
Tlssu-~Preparat~o~
First, thaw one aliquot of frozen rat cerebellar membranes (2 mg protein in 0.5 mL). Next, add 0.5 mL
membrane aliquot to 4.5 mL cold tis~ue buffer, ; 2S polytron at 7,500 revolutions per mi~ute for `
l0 seconds. Fina1ly, dilute tissue suspension l/100 (0.l m~ suspension + 9.9 mL tissue buffer), polytron again,~and pla~e ice. ~i D~lutlq~ BUff~r Medium 199 with Hank's salts plus 25 mM Hepes +
1 mg/mL bovine serum albumin.

-~ - - [l25I]-~T_l -~ 35 - Amersham [l25I]-ET-l (aliquots of 2 x l06 cpm per 100 m~ aliquot of ~l25I]-ET-l with 5.2 mL dilution W O 93/2121~ 21 3 3 n ~ ~ i PC~r/US93~03658 buffer, place on ice until use (final concentration will be 20,000 cpm per tube, or 25 pM). `

Protocol ~ '~
Add 50 ~L each of cold ~125]-ET-1 and competing '' ligand to tubes on ice. Mix in 150 ~L of tissue to ;
each tube, vortex briefly, then tap to force a}l liquids to bottom (total assay volume - 250 ~L). Then' place the tubes in a 37C water bath for 2 hours. ~
Add 2.5 mL cold wash buffer (50 mM Trizma buffer) ~' to each tube, filter, and then wash tube with additional 2.5 mL wash buffer and add to filter.
,i .
Finally, wash filters~ wlth an additional 2.5 mL of '~
cold wash buffer. ~'^
Count filters for radioactivity in gamma counter.
~.

IN VIIqUD~ nII8ITION Q~ ET-l STIMInL~TD lURAC~aIDONIC
ACID ~;iU~LE/USE~ IN ClnLTonUED RL~8BIT V~USC5ILiUR SMOOql~
20 ~ JnJSC~E~CEI~LS 8Y CCnDPqnn~DS OF FORDfCnL~ I

Antagonist activity is measured by the ability of added~;co~ ounds to reduce endothelin-stimulated ara ~ i~donic acid release~in cultured vascular smooth''`' -25~ muscie~cel1s as arachidonic acid re}ease (AAR).
3H~ Arachidonic~Acid~Loading Media (LM) is DME~F12~+~0.5~FCS x ~0.25~mCi/mL ~3H] arachidonic acid ( ~ rsham)~ Confluent monolayers of cultured rabbit , , - ~ ;
renal artery vascular smooth muscle cells were '~ 30~ ~ incuba~ed in ~Ot,5 ~ of the ~M ov~r 18 hou!rs, at 37C, in 5% C02. The ~M was aspirated and the cells were washed once with the assay buffer (Hank's BSS + lO mM
E~S + fa~tty acid-free BSA (1 mg/m~)), and incubated for 5~mlnutes with 1 m~ of the prewarmed assay buffer.
35~ This~soiution was aspirated, followed by an additional 1 m~ of prewarmed a9say buffer, and further incubated '~
for another 5 minutes. A final 5-minute incubation , - :

WO93/21219 2 1 3 3 0 9 ~ PCT/US93/03658_ was carried out in a similar manner. The same procedure was repeated with the inclusion of lO ~L of the te~t compound (l nM to l ~M) and lO ~L ET-l (0.3 nM) and the incubation was extended for 30 minutes. This solution was then collected, lO ~L
of scintillation cocktail was added, and the amount of [3H] arachidonic acid was determined in a li~uid scintillation counter.

I~ VITRO ANTAGONISM OF ET-l STIMnLATED .-VASOCONSTRICTION IN 'l~: RABBTT FEMORAL ARTERY (ETA) ~-AND SARAFOTOXIN 6c `~
STIMnLATED VASO~ONSTRICTION IN T~E RABBIT P~LMONARY
ARTERY (ETB) -~ -Male New Zealand rabbits were killed by cervical dislocation and exsanguination. Femoral and pulmonary arteries~were~ isolated, cleaned of connecti~e tissue, and cut into 4-mm rings. The endothelium was denuded by placing the rings over hypodermic tubing (32 guage ~~
for femoral rings and 28 guage for pulmonary rings, Small Parts, Inc, Miami, Florida) and gently rolling .
them.~ Denuded rings were mounted in 20 mL organ baths ~-containing Krebs-bicarbonate buffer tcomposition in ~"
25~ mM: NaCl, 1}8.2; NaHC03, 24.8; KCl, 4.6; MgS04 7-H2O, 1.2; KH2P04, 1.2; Ca~l2-2H20; Ca-Na2 EDTA, ~-0.026; dextrase, lO.0), that was maintained at 37C
and gassed continuously with 5% C2 in oxygen IpH 7.4). Resting ten~ion was adju~ted to 3.0 g for 30 - femoralland 4.0 g pulm~nary arteries; the rings were -~
left for 90 minutes to equilibrate. Vascular rings ~ were tested for lack of functional endothelium (i.e., ,~
- lack of an endothelium-dependent relaxation response , to carbachol (l.0 ~M) in norepinephrine (0.03 ~M) contracted rings. Agonist peptide~, ET-l (femoral), and S6c (pulmonary), were cumulati~ely added at lO-minute intervals. The ET antagoniQts were added `

W093/21~19 2l.3 3 n ~ 0 PCT/US93/~3658 30 minutes prior to adding the agonist ancl pA 2 values were calculated (Table I).
The data in Table I below show the endothelin receptor binding and antagonist acti~ity of representative compounds of Formula I.

: ':
'' ,;

, .:

'' `
, ~ ~ .

W O 93/212t9 ~ P ~ /US93/036~8 . ,,-`~ O '.

H E~ ~ 0 (~ . :.

X O O ~D ~0 O ~ O O ., O ~_ O ~ O O O O '~
~U '¢ ~0 O O N O O O O O .
O H
'~' _ ~1 ,~ n N Ul ~
o ~¢ ~ o ,~ o r ~1 o ~n o o - ~:
gJ ~ ~ o ~ ~ o o o ~ o o . .~
O H
~1 ,s,~
O _ ~ CD 11~ N If- ~
~ ~,~ ~ O U~ m o ~ o o ~
W~ O O ~ O O O O O O r ' :~
V~ ~ ~ ~ ~ ~ '~
~ : ' ~
~' ~ ' ,U ~ ''.. ', - O ~
O I ~ t~ ' :-~ U ID ..
m ~ ,, ,, ~H - ~ 1~ H H ~ l . .
~:! ~
~ ~ U ~ ~ t) ID .-.
O ~1 ~ ~ ~ :'' H O. ~ , , ~~ . ~ H ~ .`
- ~ i ~ : .
o o. ,~ ~ a. o. mD. a. D. ~:
H H ~ m ,~ ~:
~ 3 j j E~ ~ o ~ ~
tn ! S~ C
tn cn I
c~ ~c s ~ a a a a n . . ~
'~.
- ~D ~ Dr~a~ ~-~ , ~

U~

W O 93/21219 2 1 3 3 ~ (3 ~ PC~r/US93/03658 .. . i`

General Method for Preparinq Compounds of Formula 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 u~ing 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 ~ynthesis. Amino acid side chains are protected as follows: Bzl(A~p, Glu, Ser), 2-Cl-Z~Lys), 2-Br-Z(Tyr), Bom(His), For(Trp), and MeBzl(Cys). Each peptide resin (1.0 g) is cleaved -with 9 mL of HF and 1 mL of ani~ole or p-cre~ol as a scavenger (60 minutes, 0C). The peptide resin is washed with cyclohexane, extracted with 30~ aqueous -`
HOAc, followed by glacial HOAc, concentrated under redùced pressure, and lyophilized. (A peptide conta~lning For(Trp) is dissolved in 0C, the pH is ;adjusted~to~12~.5 with lN ROH (2 minutes), neutralized 20 ~ with~glacial HOAc, desalted on Cl8 (as described below)~, and lyophilized. The crude peptide is puri~fied by preparative reversed phase high performance liquid chromatography IRP-HPLC) on a C18 column ~(~.2 x 25.0 cm, 15.0 mL/min) with a linear-25~ gradient of O.l~ TFA in water to O.l~ TFA in ; acetonitrile and~lyophilized. The homogeneity and h composition of the resulting peptide i8 verified by RP-HP~C, capillary~electrophoresis, thin layer chromatography (T~C), proton nuclear magnetic resonance spect,rom,etry ,(NMR~, an~ fast atom bombardment mass spectrometry (FA~-MS).
~ ; The compounds of the present invention can be - ~ ~ prepared and administered in a wide variety of oral and parenteral dosage forms. Thus, the compounds of the~pre~ent invention can be administered by injection, that is, intravenously, intramuscularly, ~ ~ intracutaneously, subcutaneously, intraduodenally, or :, W093/21219 2 1 3 3 1~ 9 0 PCT/US93/03658 ~

-48- ;~
intraperitoneally. Also, the compounds of the present invention can be administered by inhalation, for example, intranasally. Additiona1ly, the compounds of the present invention can be administered transdermally. It will be obvious to tho~e skilled in the art that 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. ;;
For preparing pharmaceutical compositions from the compounds of the present invention, 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, fla~oring agents, binders, preservatives, tablet-disintegrating agents, or an encapsulating material. ` ~--In powders, the carrier is a finely divided solid which is in a mixture with the finely divided acti~e componene. `~
- In tablets, the active component is mixed with ~;
the carrier ha~ing the necessary binding properties in ~`~
suitable proportions and compacted in the shape and - size desired.
The powders and tablets preferably contain f rom fi~e or ten to about seventy percent of the acti~e compound. Suitable carriers are magnesium carbonate, magnesium s!teaF~te? ta,lc, ,sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, ~ sodium carboxymethylcellulose, a low melting wax, `~- cocoa butter, and the like. The tenm "preparation" is intended to include the f ormulation 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, `:`
.;

W093/2l2l9 2 ~ 3 3 0 ~ ~ PCT/US93/03658 l~

which is thu~ 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 administratio~.
For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and 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. For parenteral injection liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
Aqueous solutions suitable for oral use can be prepared by dissol~ing 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 agentq.
Also included are ~olid form preparations which -~ are intended to be converted, shortly before use, to ~~
liquid form preparations for oral administration.
~i Such li~uid for~s incl~de~solutions, suspensions, and emulsions. These preparations may contain, in -~
addition to the activa 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. In such form the preparation is W O 93/21219 2 l ~ 3 0 Q O PC~r/US93tO3658 `~

-50~
subdivided into unit do~es 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. Also, 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 O.S mg to 100 mg according to the ;~
particular application and the potency of the active 'i~
component. The composition can, if desired, also `
lS contain other compatible therapeutic agents.
In therapeutic use as antagonist of endothelin, the compounds utilized in the pharmaceutical method of ''~
this invention are a'dministered at the initial dosage 'of~ bout O.D1 mg~to~about 20 mg per kilogram daily. A '~
20 ~ da~ily~dose~range of about O.Ql mg to about 10 mg per '-kilogram~is~preferred. The dosages, however, may be varied depending upon the~ requirements of the patient, '~
the severity of the condition being treated, and the '~'compour,d being employed.~ Determination of the proper ~, 25~ 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 co~mpound. m ereafter, the dosage is increa~ed by small increments until the optimum effect under the ~ circumstànces is reached. For convenience, the total '~
daily do~age may be divided and administered in portions during the day, if desired. ''"
'The following nonlimiting examples illustrate the inventors' preferred methods for preparing the ~ 35~ compounds of the invention. -~

,: ~ `.. '.' ; ~'.
`, ;;

WO93/21219 2 1 3 3 ~ 9 0 PCT/US93/03658 -51- `~
ExAMoeBE 1 Ac-D-Bhg-Leu-Asp-Ile-Ile-Trp 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., ~:
Rock~ord, IL, l984). All protected amino acids and ~.:
reagents are obtained from commercial ~ources 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- ;
carbod~imide-mediated coupling scheme (Standard l.0, Version 1.40). Starting with 0.710 g of N~ oc-Trp-PAM resin (0.70 meq/g, 0.497 meq 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 0.5H20, : -N-~-Boc-Ile 0.5H2O, N-~-Boc-Asp~BzI), N-a-Boc-Leu H2O, : 20 and N-a-Boc-DL-Bhg. A typical cycle for the coupling :~
of an individual amino acid residue is illustrated below (reproduced from the ABI manual):
All the single couple RV cycles conform to the : following pattern:
25 : l) 33% TFA in DCM for 80 seconds - 2) 50~ TFA in DCM for 18.5 minutes 3) Three DCM washes ~- 4) lO~ DIEA in DMF for 1 minute 5) lO~ DIEA in DMF for 1 minute ~;::-.

6) Five DMF washe~ . :-7) Coupling period ! i .:
: 8) Fi~e DCM washes After the coupling of N-~-Boc-DL-Bhg, the Boc . group is removed with the end-NH2 cycle ~l.0l2 g).
The peptide is liberated from the solid support, ~:
and ~he carboxylate of aspartic acid deprotected by W093/21219 213~ PCT/US93/03658 -52~
treatment with anhydrous hydrogen fluoride (9.0 mL), anisole (O.5 mL), and dimethyl sulfide (O.5 mL) (60 minutes, 0C). After removing the hydrogen fluoride under a strei~m of nitrogen, the re~in 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 dissol~ed in 4.0 mL of 50~ TFA/H2O, filtered through a 0.4 L ~yringe filter, and chromatographed on a Vydac 218TP l022 column (2.2 x 25.0 cm, l~.0 m~/min, A: 0.1% TFA/H2O, ;~`
B: 0.l~ TFA/CH3CN, Gradient; 0% B for l0 minut~s, l0 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 ~l0 mL), diluted with~H2O (50 mL), and lyophilized (40.0 mg/ea). Separation into the tWQ diastereomers ~20 (Isomers A and B) is effected under these conditions tR~ Isomer A 15.63 min., Isomer B 16.79 min.). The late~running peak fractions (Isomer B) are repurified -~
under the same experimental conditions with a gradient of 30% to 50~ B over 120 minutes at lS mL/min to afford purified product. ~Acetylation is carried out with 20 mg of Isomer B in 90% acetic acid foIlowed by addition of acetic anhydride (5 m~) and stirring overnight. ~After evaporat~on and drying the product Ac-D-Bhg-Leu-Asp-Ile-Ile-Trp is 99~ pure by HP~C.
~ ~Vydac 218 TP 101,22Icolymn (2.2 x 25.0 cm, 15.0 mL/min.
A: 0.1% TFA/CH3CN, Gradient 20% to 86% B over ;
22 min.)] tR - 18.66 minutes. The homogeneity and structure of the resulting peptide is confinmed by analytical HP~C. Proton Nuclear Magnetic Re~onance Spectroscopy (Hl-NMR) and Fast Atom Bombardment Mass Spectroscopy (FAB-MS), M~Na 972.0, M+2Na+ 995.9.

W093~21219 21 3 ~ ~ ~ O PCT/US93/03658 .~

-53- .
In a process analogous to Example l using the appropriate amino acids, the corresponding compounds :
of Formula I are prepared as follows: ~:

. EXAMPhE 2 D-Bhg-Leu Asp-Ile-Ile-Trp; FAB-MS, M+l 907.4.

EXAMPLE 3 ~
L-Bhq-Leu-As~-Ile-Ile-Trp; FAB-MS, M+l 907.4. -~ EXAMPLE 4 ~
Ac h-Bhq-Leu-Asp- le-Ile-Trp; FAB-MS, M+l 950Ø ~ .

EXAMPhE 5 Ac-D-Tx~-~eu-Asp-Ile-I1e-Trp; FA3-MS, M+Na 977Ø

EXAMPhE 6 Ac-D-Bheq-Leu-As~-Ile-Ile-Trp, FAB-MS, M+l 970.3. .~

20~ EXAMPhE 7 Ac-D-~hq-Orn-Asp-Ile-Ile-Trp; FAB-MS, M+l 951.2. .;
':,: .., EXAMPhE 8 Ac-D-3hq-Glu-Asp-Ile-Ile-T~p; FAB-MS, M+Na 988.8. ..

EXAMPLE 9 :~
Disodium~L~ of:Ac-D-3hg-~eu-Asp-Ile-Ile-Trp ~ ~
:A:~s~aturated~solution of sodium bicarbonate in ~.;.
~ water is prepared, diluted with water (l:lO), chilled ".
:~ 30 - l~to 0lC,, and lQ lm,L jofjt,he,j~o1ution! is added to :`.
approximately 50 mg of Ac-D-Bhg-~eu-Asp-Ile-Ile-Trp ~.
(Example l) wi-th stirring. The pH of the solution is : greater than 9. After lO minutes, the solution is ;.
pa~sed:through a Cl8 cartridge, washed with water 35: ~ (100 mL), and the absorbed peptide is eluted with methanol ~50 mL), concentrated under reduced pressure, ~:;
- . . .

~ ~ , :;

WO9~/21219 1 3 3 ~ ~ o PCT/US93/036~8 i~$r .:

resuspended ln water (50 mL), and lyophilized (three times) to give the title co~pound.
Ac-D-Bhg-Leu-Asp-Ile-Ile-Trp. disodium sal~; FAB-MS, M+l 950.4, M+Na 972.l, M+2Na 994.3.
~
EXAMPLE lO ~-Boc-Bhg .~ ~
Bhg HCl (1.70 g, 5.43 mmol) is suspended in ;~
150 mL of p-dioxane:H20 (2:1) at room temperature. To the stirred solution is added l.40 g (6.42 mmol) of di-tert butyldicarbonate. The pH of the solution i5 :
adjusted to ~9.0 with lN NaOH and maintained at between pH 9 and lO with aliquot additions of lN NaOH, until the pH is constant~ The solution is concentrated under reduced pressure to approximately ~
75 mL, overlain with ethyl acetate (50 imh), and -acidified to approximately~pH 2.5~ with lO~ aqueous HCl. The organic layer is~Yeparated, washed successively with 10~ aqueous HCl (2 x 50 mL~, brine ~2 x 50 mL), H~20 (3 x 50 mL), and driled with MgS04.
The solution i9 ~iltered, concentrated under reduced pressure, and the oil is recrystallized from ethyl acetate:heptane (1.82 g). The white~solid is ,;
characterized by proton NMR, fast atom bombardment mass spectrometry (M+1=368), and elemental analysis. i`-: ~

: - `

'~
' '

Claims (45)

1. A compound of Formula I

AA1,AA2-AA3-AA4-AA5-AA6 I
wherein AA1 is wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, fluorenylmethyl, , wherein R2 and R3 are each the same or different and each is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, or fluorenylmethyl, -?-OR2, wherein R2 is as defined above, -OR2, wherein R2 is as defined above, , wherein R2 and R3 are as defined above, -?-C(R9)3, wherein R9 is F, Cl, Br, or I, -CH2-OR2, wherein R2 is as defined above, , wherein R2a is hydrogen or alkyl and R3 is as defined above, , wherein R2a and R3 are as defined above excluding R3 is hydrogen, or -?R3, wherein R2 is as defined above, R1 is hydrogen or alkyl, Z is -O-, -S(O)?, wherein m is zero or an integer of 1 or 2, , wherein R2 is as defined above, -(CH2)n-, wherein n is zero or an integer of 1, 2, 3, or 4, -(CH2)n-CH=CH-(CH2)n-, wherein n is as defined above, -?- , , wherein R1 and R2 are as defined above, or , wherein R2 and R3 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, -CO2R2, wherein R2 is as defined above, , wherein R2 and R3 are as defined above, , wherein R2 and R3 are as defined above, or nitro or wherein R, Z, X, and Y are as defined above;

AA2 is wherein R4 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, , wherein R2b and R3b are each the same or different and each is hydrogen, alkyl, cycloalkyl, aryl, or heteroaryl, -OR2b, wherein R2b is as defined above, , wherein R2b and R3b are each the same or different and each is as defined above for R2b and R3b, -?-R2b, wherein R2b is as defined above, wherein R2b is as defined above, or -?-OR2b, wherein R2b is as defined above, and R1 and n are as defined above, AA2 is absent;
AA3 is wherein R5 is hydrogen, alkyl, aryl, heteroaryl, , wherein R2b and R3b are each the same or different and each is as defined above, -?-R2b, wherein R2b is as defined above, or -OR2b, wherein R2b is as defined above, and R1 and n are as defined above, or AA3 is absent;

AA4 and AA5 are each independently absent or each is independently wherein R6 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, and R1 and n are as defined above;
wherein R7 is aryl or heteroaryl, R8 is -?-OR1, wherein R1 is as defined above, -OR1, wherein R1 is as defined above, , wherein R1 is as defined above, or -CH2-OR1, wherein R1 is as defined above, and R1 and n are as defined above;

stereochemistry at ? in AA1, AA2, AA3, AA4, or AA5 is D, L, or DL and stereochemistry at ? in AA6 is L; or a pharmaceutically acceptable salt thereof.

2. A compound according to Claim 1, in which AA1 is wherein R is , wherein R2 and R3 are each the same or different and each is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, or fluorenylmethyl, , wherein R2 and R3 are as defined above, -?-C(R9)3, wherein R9 is F, Cl, Br, or I, -NH-?-R3, wherein R3 is as defined above, or -NH-?-OR3, wherein R3 is as defined above excluding R3 is hydrogen, Z is -O-, -S(O)?, wherein m is zero or an integer of 1 or 2, , wherein R2 is as defined above, -(CH2)n, wherein n is zero or an integer of 1, 2, 3, or 4, -(CH2)n-CH=CH-(CH2)n-, wherein n is as defined above, -?-, , wherein R1 is hydrogen or alkyl, or , wherein R2 and R3 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;
AA2 is wherein R4 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, , wherein R2b and R3b are each the same or different and each is hydrogen, alkyl, cycloalkyl, aryl, or heteroaryl, -OR2b, wherein R2b is as defined above, , wherein R2b and R3b are each the same or different and each is as defined above for R2b and R3b, -?-R2b, wherein R2b is as defined above, wherein R2b is as defined above, or -?-OR2b, wherein R2b is as defined above, and n is as defined above or AA2 is absent;
AA3 is wherein R5 is aryl, heteroaryl, , wherein R2b and R3b are each the same or different and each is as defined above, -?-R2b, wherein R2b is as defined above, or -?-OR2b, wherein R2b is as defined above, and n is as defined above, or AA3 is absent;

AA4 and AA5 are each independently absent or each is wherein R6 is hydrogen alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, and n is as defined above;
AA6 is wherein R7 is aryl or heteroaryl, and n is as defined above, or wherein R7, R1, and n are as defined above, stereochemistry, at ?H in AA1, AA2, AA3, AA4, or AA5 is D,L, or DL, and stereochemistry at ?H in AA6 is L; or a pharmaceutically acceptable salt thereof.

3. A compound according to Claim 2, in which AA1 is wherein R is , wherein R2 and R3 are each the same or different and each is hydrogen, alkyl, aryl, or fluorenylmethyl, , wherein R2 and R3 are as defined above, -?-C(R9)3, wherein R9 is F, Cl, Br, or I, or -NH-?-OR10, wherein R10 is hydrogen, alkyl, aryl, or arylalkyl, excluding R10 is hydrogen, Z is -O-, -S-, -NH-, -(CH2)n, wherein n is zero or an integer of 1, 2, 3, or 4, or -(CH2)n-CH=CH-(CH2)n, wherein n is zero or an integer of 1 or 2 and 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;
AA2 is , wherein R4 is hydrogen, alkyl, aryl, heteroaryl, , wherein R2b and R3b are each the same or different and each is hydrogen or alkyl, , wherein R2b and R3b are each the same or different and each is hydrogen or alkyl, wherein R2b is as defined above, or -?-OR2b, wherein R2b is as defined above, and n is an integer of 1, 2, 3, or 4 or AA2 is absent;
AA3 is , wherein R5 is aryl, heteroaryl, , wherein R3b is hydrogen or alkyl, -?-R2b, wherein R2b is hydrogen or alkyl, or -?-OR2b, wherein R2b is hydrogen or alkyl, and n is an integer of 1, 2, 3, or 4;
AA4 and AA5 are each independently wherein R6 is hydrogen, alkyl, cycloalkyl, or aryl, and n is an integer of 1, 2, 3, or 4;
AA6 is wherein R7 is aryl or heteroaryl, and n is zero or an integer of 1, 2, 3, or 4, or wherein R7, R1, and n are as defined above, stereochemistry at ?H in AA1, AA2, AA3, AA4, or AA5 is D, L, or DL and stereochemistry at ?H in AA6 is L; or a pharmaceutically acceptable salt thereof.

4. A compound according to Claim 3 selected from the group consisting of:

;

;

;

;

;

;
; and .

5. A method of inhibiting elevated levels of endothelin comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

6. A pharmaceutical composition adapted for administration as an antagonist of endothelin comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent, or carrier.

7. A method of treating hypertension comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

8. A pharmaceutical composition adapted for administration as an antihypertensive agent comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

9. A method of treating metabolic and endocrine disorders comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

10. A pharmaceutical composition adapted for administration as an agent for treating metabolic and endocrine disorders comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

11. A method of treating congestive heart failure and myocardial infarction comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

12. A pharmaceutical composition adapted for administration as an agent for treating congestive heart failure and myocardial infarction comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

13. A method of treating endotoxic shock comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

14. A pharmaceutical composition adapted for administration as an agent for treating endotoxic shock comprising a therapeutically effective amount of a compound according to Claim 1 in a mixture with a pharmaceutically acceptable excipient, diluent or carrier.

15. A method of treating subarachnoid hemorrhage comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

16. A pharmaceutical composition adapted for administration as an agent for treating subarachnoid hemorrhage comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

17. A method of treating arrhythmias comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

18. A pharmaceutical composition adapted for administration as an agent for treating arrhythmias comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

19. A method of treating asthma comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

20. A pharmaceutical composition adapted for administration as an agent for treating asthma comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

21. A method of treating acute and chronic renal failure comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

22. A pharmaceutical composition adapted for administration as an agent for treating acute and chronic renal failure comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

23. A method of treating preeclampsia comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

24. A pharmaceutical composition adapted for administration as an agent for treating preeclampsia comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

25. A method of treating diabetes comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

26. A pharmaceutical composition adapted for administration as an agent for treating diabetes comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent or carrier.

27. A method of treating neurological disorders comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

28. A pharmaceutical composition adapted for administration as an agent for treating neurological disorders comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent, or carrier.

29. A method of treating pulmonary hypertension comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

30. A pharmaceutical composition adapted for administration as an agent for treating pulmonary hypertension comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent, or carrier.

31. A method of treating ischemic disease comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

32. A pharmaceutical composition adapted for administration as an agent for treating ischemic disease comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent, or carrier.

33. A method of protecting against gastric mucosal damage or treating ischemic bowel disease comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

34. A pharmaceutical composition adapted for administration as an agent for protecting against gastric mucosal damage or treating ischemic bowel disease comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable-excipient, diluent, or carrier.

35. A method of treating atherosclerotic disorders including Raynaud's disease comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

36. A pharmaceutical composition adapted for administration as an agent for treating atherosclerotic disorders including Raynaud's disease comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent, or carrier.

37. A method of treating restenosis comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

38. A pharmaceutical composition adapted for administration as an agent for treating restenosis comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent, or carrier.

39. A method of treating angina comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

40. A pharmaceutical composition adapted for administration as an agent for treating angina comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent, or carrier.

41. A method of treating cancer comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

42. A pharmaceutical composition adapted for administration as an agent for treating cancer comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient, diluent, or carrier.

43. A method of treating hemorrhagic shock comprising administering to a host suffering therefrom a therapeutically effective amount of a compound according to Claim 1 in unit dosage form.

44. A pharmaceutical composition adapted for administration as an agent for treating hemorrhagic shock comprising a therapeutically effective amount of a compound according to Claim 1 in admixture with a pharmaceutically acceptable excipient diluent, or carrier.

45. A method of preparing a compound of Formula I

I

wherein AA1 is wherein R is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, heteroaryl, fluorenylmethyl, , wherein R2 and R3 are each the same or different and each is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, or fluorenylmethyl, -?-OR2, wherein R2 is as defined above, -OR2, wherein R2 is as defined above, , wherein R2 and R3 are as defined above, -?-C(R9)3, wherein R9 is F, Cl, Br, or I, -CH2-OR2, wherein R2 is as defined above, , wherein R2a is hydrogen or alkyl and R3 is as defined above, , wherein R2a and R3 are as defined above excluding R3 is hydrogen, or -?-R2, wherein R2 is as defined above, R1 is hydrogen or alkyl, Z is -O-, -S(O)?, wherein m is zero or an integer of 1 or 2, , wherein R2 is as defined above, -(CH2)n-, wherein n is zero or an integer of 1, 2, 3, or 4, -(CH2)n-CH=CH-(CH2)n-, wherein n is as defined above, -?-, , wherein R1 and R2 are as defined above, or , wherein R2 and R3 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, -CO2R2, wherein R2 is as defined above, , wherein R2 and R3 are as defined above, , wherein R2 and R3 are as defined above, or nitro or wherein R, Z, X, and Y are as defined above;
AA2 is wherein R4 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, , wherein R2b and R3b are each the same or different and each is hydrogen, alkyl, cycloalkyl, aryl, or heteroaryl, -OR2b, wherein R2b is as defined above, , wherein R2b and R3b are each he same or different and each is as defined above for R2b and R3b, -?-R2b, wherein R2b is as defined above, , wherein R2b is as defined above, or , wherein R2b is as defined above, and R1 and n are as defined above, or AA2 is absent;

AA3 is wherein R5 is hydrogen, alkyl, aryl, heteroaryl, wherein R2b and R3b are each the same or different and each is as defined above, -?-R2b, wherein R2b is as defined above, or -?R2b, wherein R2b is as defined above, and R1 and n are as defined above, or AA3 is absent;
AA4 and AA5 are each independently absent or each is independently wherein R6 is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, and R1 and n are as defined above;
AA6 is wherein R7 is aryl or heteroaryl, R8 is -?-OR1, wherein R1 is as defined above, -OR1, wherein R1 is as defined above, , wherein R1 is as defined above, or -CH2-OR1, wherein R1 is as defined above, and R1 and n are as defined above stereochemistry at ? in AA1, AA2, AA3, AA4, or AA5 is D, L, or DL and stereochemistry at ? in AA6 is L; or a pharmaceutically acceptable salt thereof comprising sequential stepwise coupling of the amino acids selected from AA1, AA2, AA3, AA4, AA5, or AA6 to the preceding amino acid using conventional peptide synthesis methodology and after conventional deprotection to afford a compound of Formula I and, if desired, converting a compound of Formula I to a pharmaceutically acceptable salt of a compound of Formula I by conventional methodology and, if further desired, converting the obtained pharmaceutically acceptable salt of a compound of Formula I to a compound of Formula I by conventional methodology.