CA2072390A1 - Endothelin analogues with alpha-amine substitution at residue 20 - Google Patents

Abstract

Abstract ENDOTHELIN ANALOGUES WITH ALPHA-Endothelin receptor antagonism is exhibited by novel compounds of the formula wherein:
;
R9 is alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, or aralkyl;
R10 is alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, cycloalkylalkyl, cycloalkenyl, or cycloalkenylalkyl;
or R9 and R10 together are an alkylene chain of three to five carbons (e.g., such that A20 is prolyl), optionally substituted with one or two alkyl, alkoxy, aryl, aralkyl, cycloalkyl, or hydroxy groups;
and the remaining symbols are as defined in the specification.

Description

3 ~ ~
~ ~575 ENDOTHELIN ANALOGUES WITH ALPHA-This invention relates to polypeptide endothelin (ET) analogues useful as ET receptor antagonists, and in particular to ET analogues having a-amine substitutions at residue 20.

Compounds of the formula I

E-Al-A2-A3-A~-~5-A6-A7-As-As Al 3 -Al ~ _Al S _Al 6 -Al7-Al8-Al9-A2o-A2l-O~I
are endothelin receptor antagonists that are useful, inter alia, as antihypertensive agents.
Throughout this specification, the symbols in formula I are defined as follows:
A1 and A15 are each independently polar -NH-ICH-C(O)-amino acid residues, or Al is R1-C-S--NH-CH-C(O)- R2 and A1 5 iS -S-¢-R7wherein A1 and A1 5 are S,S-crosslinked;
A2, A~, A5, A6, A7, A9, A12, and A1 6 are each independently amino acid residues;

~3~2~

A3 and ~11 are each independently polar -NH-CH-C(O)-amino acid residues, or A3 is R3-~-S--NH-~H-C(O~- R4 and Al1 is ~ -R5 wherein A3 and A1l are S,S-crosslinked;
A8 iS an aliphatic, aromatic, acidic, or polar ami~o acid residue;
A10 is an aliphatic, aromatic, D- or L-acidic, or polar amino acid residue;
Al 3 iS an aliphatic, aromatic or polar amino acid residue;
Al 4 iS an aromatic or aliphatic amino acid residue;
Al 7 iS an aliphatic or polar amino acid residue;
A18 is an acidic, polar, aliphatic or aromatic amino acid residue;
A19 is an aliphatic amino acid residue A20 is -N-CH-C(O)- ;
R9Rl0 A21 is an aromatic amino acid residue;
R1 and R2 are each independently hydrogen or lower alkyl, or Rl and R2 together with the carbon atom to which they are attached are cycloalkyl;
R3 and R4 are each independently hydrogen or lower alkyl, or R3 and R4 together with the carbon atom to which they are attached are cycloalkyl;

~72~a H~575 ~3--Rs and R6 are each independently hydrogen or lower alkyl, or R5 and R6 together with the carbon atom to which they are attached are cycloalkyl;
R7 and R8 are each independently hydrogen or lower alkyl, or R7 and R8 together with the carbon atom to which they are attached are cycloalkyl;
R9 is alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, or aralkyl; and R10 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, or cycloalkenylalkyl;
or R9 and R10 together are an alkylene chain of three to five carbons (e.g., such that A20 is prolyl), optionally substituted with one or two alkyl, alkoxy, aryl, aralkyl, cycloalkyl, or hydroxy groups.
The following moieties are preferred for the foregoing symbols:
R9 is lower alkyl;
R10 is lower alkyl;
-NH-~H-C(O)- , A1 is Rl-¢-S--NH-~H-C(O)-A3 is R3-C-S--NH-CH-C(O)- , A11 is -S-C-R5 2~ 3~
~575 -N~-CH-C(O)- , and A1s is -S-~-R7 wherein Al and ~15 are S,S-crosslinked and A3 and A11 are S,S-crossli~ked;
A7 is norleucyl, methionyl, leucyl, lysyl, seryl, or threonyl;
A8 is aspartyl;
A1~ is L-glutamyl, D-glutamyl, glutaminyl, or alanyl;
Al 3 iS tyrosyl, phenylalanyl, or asparaginyl;
Al4 is tyrosyl or phenylalanyl;
A1 7 iS leucyl or glutaminyl;
Al8 is aspartyl, asparaginyl, alanyl, or leucyl;
A19 is isoleucyl or valyl;
A21 is tryptophyl or napthylalanyl; and A2 A4 A5, A6, A9, Al 2 and A1 6 are each independently amino acid residues.
Most preferred are the compounds wherein:
at least one of R1 and R2 is lower alkyl or R1 and R2 together with the carbon atom to which they are attached are cycloalkyl, R3 and R4 are hydrogen, at least one of Rs and R6 is lower alkyl or R5 and R6 together with the carbon atom to which they are attached are cycloalkyl, and R7 and R3 are hydrogen; or Rl and R2 are hydrogen, at least one of R3 and R4 is lower alkyl or R3 and R4 together with the carbon atom to which they are attached are cycloalkyl, R5 and R6 are hydrogen, and at least ~7~3~
~A575 one of R7 and R8 is lower alkyl or R7 and R8 together with the carbon atom to which they are attached are cycloalkyl;
R9 is methyl;
R10 is 2-butyl;
A2 is seryl or threonyl;
A4 is seryl, phenylalanyl, asparaginyl, or lysyl;
A5 is seryl, threonyl, or aspartyl;
A6 is leucyl, methionyl, lysyl, seryl or threonyl;
A9 is D- or L-lysyl, alanyl or glutamyl;
A1~ is valyl or leucyl;
A1 6 iS alanyl, histidyl or phenylalanyl; and A20 is N-methylisoleucyl or prolyl.

Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.
The term "amino acid residue" refers to moieties of the formula R'~
" ~ fi including natural, modified and synthetic moieties in L-form unless otherwise specified, wherein:
R is hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, heteroaryl, heteroarylalkyl, mercaptoalkyl, mercaptoalkenyl, mercaptoaryl, alkylthioalkyl, hydroxyalkyl, hydroxyaryl, hydroxyaralkyl, dihydroxyaryl, dihydroxyaralkyl, aryloxyalkyl, aminoalkyl, aminoguanidinoalkyl, guanidinoalkyl, carboxyalkyl, carboalkoxyalkyl, carboxamidoalkyl, dicarboxyalkyl, alkylcarbonyl, heterocyclo, heterocycloalkyl, -PO~, (P04 ~- alkyl, ( P04 )- aryl, ( P04 )- aralkyl, ( P04 )-heterocyclo, or (PO~)-heterocycloalkyl; and R' is hydrogen; or R and R' together with the carbon and nitrogen atoms to which they are attached complete a 3- to 7-membered heterocyclo (e.g., pyrrolidinyl for proline residues) group.
The terms "alkyl" and "alkoxy" refer to both straight and branched chain groups having 1 to 10 carbon atoms. Those groups having one to four carbon atoms are preferred. The terms "lower alkyl" and "lower alkoxy" refer to groups of 1 to 4 carbon atoms.
The term "aryl" or "ar" refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 10 carbon atoms in the ring portion, such as phenyl and naphthyl.
The terms "cycloalkyl" and "cycloalkenyl"
refer to cyclic hydrocarbon groups having 3 to 7 carbon atoms in the ring.
The term "heterocyclo" refers to fully saturated, partially saturated, and unsaturated mono- or bicyclic groups having 5 to 6 atoms in each ring and one to four heteroatoms in at least one ring, wherein the heteroatoms can comprise one or two oxygen atoms, one or two sulfur atoms J

and/or one to four nitrogen atoms, and wherein an available carbon or nitrogen atom can be substituted with hydroxyl, lower alkyl, lower alkoxy, halo, mercapto r amino or carboxyl.
Exemplary heterocyclo groups are pyrrolidinyl, pyrazolidinyl, imidazolyl, imidazolidinyl, thiazolidinyl, oxa201idinyl, indolyl, pyridyl and the like.
The term "heteroaryl" refers to aromatic heterocyclo groups, such as imidazolyl, indolyl, oxazolyl, pyridyl, pyrrolyl and the like.
The term "acidic" as used with respect to amino acid residues refers to residues that would be negatively charged at about pH 6 to 7, such as residues derived from aspartic acid, glutamic acid, and the like.
The term "polar" as used with respect to amino acid residues refers to residues derived from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, lysine, serine, threonine, tyrosine, ornithine and the like.
The term "aromatic" as used with respect to amino acid residues refers to amino acid residues wherein R is aryl, aralkyl, heteroaryl, hetero-arylalkyl, mercaptoaryl, hydroxyaryl, dihydroxy-aryl, hydroxyaralkyl, dihydroxyaralkyl, aryloxyalkyl, (P04 )-aryl, (P04 )-aralkyl, (P04 )-heteroaryl, or (P04 )-heteroarylalkyli for example, moieties derived from histidine, phenylalanine, tryptophan, tyrosine and the like.

H~575 The term "aliphatic" as used with respect to amino acid residues refers to amino acid residues having hydrocarbon sidechains that are not aromatic, such as residues derived from alanine, isoleucine, leucine, valine and the like (but not proline). The term "aliphatic" thus includes residues wherein R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, and cycloalkenylalkyl.
The term "natural amino acid residue" refers to alanyl, argininyl, asparaginyl, aspartyl, cysteinyl, glutamyl, glutaminyl, glycyl, histidyl, isoleucyl, leucyl, lysyl, methionyl, phenylalanyl, prolyl, seryl, threonyl, tryptophyl, tyrosyl, or valyl.
The compounds of formula I are antagonists of ET-l, ET-2, and/or ET-3 and are useful in treatment of all endothelin-dependent disorders.
They are especiaily useful as antihypertensive agents. By the administration of a composition having one (or a combination) of the compounds of this invention, the blood pressure of a hypertensive mammalian (e.g., human) host is reduced.
The compounds of the present invention are also useful in the treatment of disorders related to renal, glomerular, and mesangial cell function, including chronic renal failure, glomerular injury, renal damage secondary to old age, nephrosclerosis (especially hypertensive nephrosclerosis), nephrotoxicity (including nephrotoxicity related to imaging and contrast agents), and the like. The ~2~

_g_ compounds of this invention may also be useful in ~he treatment of disorders related to paracrine and endocrine function.
The compounds of the present invention are also useful as anti-ischemic agents for the treatment of, for example, heart, renal and cerebral ischemia and the like.
In addition, the compounds of this invention are also believed to be useful as:
anti-arrhythmic agents;
anti-anginal agents;
anti-fibrillatory agents;
anti-asthmatic agents;
agents to increase the ratio of HDL-cholesterol to total serum cholesterol in the blood;
therapy for myocardial infarction;
therapy for peripheral vascular disease (e.g., Raynaud's disease);
anti-thrombotic agentsi anti-atherosclerotic agents;
treatment of cardiac hypertrophy (e.g., hypertrophic cardiomyopathy);
treatment of pulmonary hypertensioni additives to cardioplegic solutions for 5 cardiopulmonary bypasses;
adjuncts to thrombolytic therapy;
treatment of central nervous system vascular disorders; for example, as anti-stroke agents, anti-migraine agents, and therapy for subarachnoid hemorrhage;

treatment of central nervous system behavorial disorders, including psychiatric conditions such as depression, mania, anxiety and schizophrenia;
anti-diarrheal agents;
therapy for dysmenorrhea;
therapy for tinnitus and other auditory and vestibulatory disorders;
alleviation of the various forms of oedema;
reversal of adriamycin resistance;
regulation of cell growth;
treatment of glaucoma, hepatoxicity, sudden death, drug-induced tardive dyskinesia, allergies, electrolyte imbalance, muscular dystrophy and 5 cancer.
The compounds of this invention can also be formulated in combination with endothelin converting enzyme (ECE) inhibitors, such as phosphoramidon and thiorphan; platelet activating factor tPAF) antagonists; angiotensin II (AII) receptor antagonists; renin inhibitors; angiotensin converting enzyme (ACE) inhibitors such as captopril, zofenopril, fosinopril, ceranapril, alacepril, enalapril, delapril, pentopril, quinapril, ramipril, lisinopril, and salts of such compounds; neutral endopeptidase (NEP) inhibitors;
calcium channel blockers; potassium channel activators; beta-adrenergic agents; antiarrhythmic agents; diuretics, such as chlorothiazide, hydro-chlorothiazide, flumethiazide, hydroflumethiazide,bendroflumethiazide, methylchlorothiazide, trichloro-methiazide, polythiazide or benzothiazide as well as ethacrynic acid, tricrynafen, chlorthalidone, 2 ~

furosemide, musolimine, bl~etanide, triamterene, amiloride and spironolactone and salts of such compounds; thrombolytic agents such as tissue plasminogen activator (tPA), recombinant tPA, streptokinase, urokinase, prourokinase, and anisoylated plasminogen streptokinase activator complex (APSAC, Eminase, Beecham Laboratories). If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described below and the other pharmaceutically active agent within its approved dosage range. The compounds of this invention may also be formulated with or useful in conjunction with antifungal and immunosuppressive agents such as amphotericin B, cyclosporins and the like to counteract the glomerular contraction and nephrotoxicity secondary to such compounds. The compounds of this invention may also be used in conjunction with hemodialysis.
The compounds of formula I can be formulated in compositions such as sterile solutions of suspensions for parenteral administration. About 0.1 to 500 milligrams of a compound of formula I is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, etc., in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in these compositions or preparations is such that a suitable dosage in the range indicated is obtained.
The compounds of the present invention may be prepared as follows. This preparation may be carried out on an automated peptide synthesizer (e.g., Biosearch 9600) using standard software protocols.
A compound of the formula II
Prot-A21 -O-PAM ~ESIN
wherein Prot is an amino-protecting group (e.g., t-butoxycarbonyl) attached at the amino terminus and A21 is A2l or a residue derived therefrom having a sidechain-protecting group if A2l is an amino acid residue having a reactive sidechain functional group. Exemplary reactive sidechain functional groups are hydroxyl, carboxyl, mercapto, amino, guanidino, imidazolyl, indolyl and the like.
Compound II is treated with, in sequence:
(a) a deprotecting agent (e.g., trifluoro-acetic acid) in an inert solvent (e.g., methylene chloride) in the presence of one or more cation scavengers (e.g., dimethylphosphite, anisole);
(b) a tertiary base (e.g., diisopropyl-ethylamine); and (c) an amino acid of the formula III
Prot-A20 -OH
(wherein A20 is A20 or a residue derived therefrom having a sidechain-protecting group if A20 has a reactive sidechain functional group) in an inert solvent (e.g., dimethylformamide) in the presence of a coupling reagent (e.g., diisopropylcarbodiimide~
in an inert solvent (e.g., methylene chloride);
to form an amino acid of the formula ~ '3 -13~

IV
Prot-A20'-A2l~-O-PAM RESIN
Presence of an agent which suppresses racemization or dehydration (e.g., hydroxybenzotriazole) is optional. Step (c~ above may be followed by treatment with an amino acid acetylating agent (e.g., acetylimidazole) which acetylates or "caps"
unreacted amino acids. When Prot is a base-labile protecting group (e.g., fluorenylmethoxycarbonyl), step (a) above is carried out with a base (e.g., piperidine, morpholine) in an inert solvent (e.g., dimethylformamide) and step (b) is unnecessary.
The foregoing process is repeated with the resin-linked amino acid chain until the N-terminal amino acid residue (A1) has been coupled to the polypeptide. One may also use multiple peptide synthesis techniques, which are generally known in the art. See, e.g., Tjoeng et al., "Multiple Peptide Synthesis Using A Single Support (MPS3)", Int. J. Protein Peptide Res., 35 (1990), 141-146.
After the polypeptide chain is complete, it may be cleaved from the resin with an acid, such as hydrofluoric acid, trifluoromethanesulfonic acid and the like. When Prot is a base-labile protecting group, a mild acid such as trifluoro-acetic acid and the like may be used.
As noted above, sidechain protecting groups may be used in this process for sidechains having reactive functionalities, such as hydroxyl, carboxyl, amino, mercapto, guanidino, imidazolyl, indolyl and the like. The particular protecting groups used for any amino acid residues depend upon the sidechains to be protected and are generally known in the art. Exemplary sidechain protecting groups are benzyl, halocarbobenzoxy, and the like for hydroxyl; cyclohexyl, benzyl and the like for carboxyl; 4-methylbenzyl, 4-methoxybenzyl, ~7~3~

acetamidomethyl and the like for mercapto;
carbobenzoxy, halocarbobenzoxy and the like for amino; 2,4-dinitrophenyl, benzyloxymethyl and the like for imidazolyl; formyl and the like for indolyl; and tosyl, nitro and the like for guanidino.
Sidechain protecting groups may be removed, if desired, by treatment with one or more deprotecting agents in an inert solvent or solvent mixture (e.g., dimethylformamide, methylene chloride). Suitable deprotecting agents are generally known in the art. Exemplary deprotecting agents are thiophenol, mercaptoethanol and the like for removing 2,4-dinitrophenyl; trifluoroacetic acid and the like for butoxycarbonyl; hydrofluoric acid, trifluoromethanesulfonic acid and the like for several different protecting groups.
The disulfide bonds in the compound of formula I are formed by oxidation with, for example, air/ammonium hydroxide, potassium ferricyanide, air/urea/ammonium hydroxide, water/methanol/iodine, and the like. The preferxed Rl to R8 moieties cause higher yields of the desired 1-15 and 3-11 disulfide bonds upon oxidation.
The invention will now be further described by the following working examples, which are preferred embodiments of the invention. These examples are meant to be illustrative rather than limiting. Unless otherwise indicated, all reactions are conducted at about room temperature using a Biosearch 9600 automated peptide synthesizer.

L-Penicillaminyl-L-seryl-L-cysteinyl-L-seryl-L-seryl-L-leucyl-L-norleucyl-L-aspartyl-L-lysyl-L-glutamyl-L~penicillaminyl-L-valyl-L-tyrosyl-L-phenylalanyl~L~cysteinyl-L-histidyl-L-leucyl-L-aspartyl-L-isoleucyl-N-methyl-L-isoleucyl-L-tryptophan ! S,S-crosslinked (1,15:3,11); and L-Cysteinyl-L-seryl-L-penicillaminyl-L-seryl-L-seryl-L-leucyl-L-norleucyl-L-aspartyl-L-lysyl-L-glutamyl-L-cysteinyl-L-valyl-L-tyrosyl-L-phenyl-alanyl-L-penicillaminyl-L-histidyl-L-leucyl-L-aspartyl-L-isoleucyl-N-methyl-L-isoleucyl-L-tryptophan, S,S-crosslinked (1,15:3,11) The compounds were synthesized by solid phase techniques performed manually or on a Biosearch 9600 Automated Peptide Synthesizer using t-Boc amino acids in accordance with the instructions of the manufacturer.
In accordance with the above description, the following procedures were used for the chemical synthesis of novel endothelin derivatives.
Preparation began with a resin of the nitrogen-protected carboxyl terminal amino acid, which is sidechain protected if reactive. The successive amino acids were added according to Procedure A
(single-coupled) or Procedure B (double-coupled).

Procedure A
1) Wash once with dichloromethane (CH2Cl2);
2) Treat for one minute with CH2Cl2:TFA:anisole:
dimethylphosphite (50.5:45:2.5:2 by volume);

3) Treat for 20 minutes with CH2Cl2:TFA:Anisole:
Dimethyl phosphite (50.5:45:2.5:2.0 by volume);

4) Wash once with CH2 C12;

5) Wash once with dimethylformamide (DMF);

~-16~

6) Wash three times with CH2Cl2;

7) Treat three times for 50 seconds with 10%
(v/v) diisopropylethylamine (DIPEA) in CH2C12;

8) Wash three times with CH2Cl2;

9) Couple the suitably protected Boc-amino acid to the ~-~mine of the resin-bound peptide using one of the following protocols:
a) i) combine the suitably protected Boc-amino acid (7.1 equivalents) dissolved in dimethylformamide and diisopropyl-carbodiimide dissolved in CH2Cl2 to afford a 0.4 M solution of the amino acid O-acylisourea in (1:1) DMF/CH2Cl2, ii) add this mixture to the resin, and iii) mix the suspension for 2 hours;
b) i) dissolve the suitably protected Boc-amino acid (10 eguivalents), HOBT
(10 equivalents) and DCC (10 eguivalents) in 1:1 DMF:CH2Cl2 (0.1 M), ii) add the amino acid HOBT ester solution to the resin, and iii) mix the suspension for 2 hours; or c) i) dissolve the suitably protected Boc-amino acid (10 equivalents) and DCC (5 equivalents) in CH2Cl2, ii) stir the solution for 15 minutes, iii) filter off dicyclohexylurea, iv) add DMF to the filtrate, v) remove CH2C12 by rotary evaporation to afford a 0.1 M solution of the symmetrical anhydride in DMF, vi) add this solution to the resin, and vii~ mix the suspension for 2 hours;

10) Wash three times with CH2Cl2;
ll) Wash once with lQ% DIPEA for 40 seconds;

12) Wash three times with CH~ Cl2;
13) Treat for 30 minutes with acetylimidazole in DMF (0.3 M);
14) Wash three times with DMF.
s Procedure B
For amino acids which reguired double coupling, after step 12 of Schedule A, steps 9-12 are repeated.
Example_l 5.0 g of Boc-W(formyl)-Pam-resin (0.48 meg/g; Bachem Inc., Torrance, CA) was subjected to Procedure B (first coupling using 9b, second coupling using 9c) to introduce Boc-N-methyl-I-OH followed by Boc-I-OH-~ H2O. The resulting BOC-I-(N-Me-I)-W(formyl)-Pam-resin (0.6 g) was subjected to either Procedure A
(single c~uple using Step 9a) or B (double couple using Step 9a), as indicated, with the required seguence of amino acids introduced in order, as Boc-D(Chx)-OH (B), Boc-L-OH-H20 (B), Boc-H(DNP)-OH isopropanol (B), Boc-C(MeBn)-OH (A), Boc-F-OH
(A), Boc-Y(2BrZ)-OH (A), Boc-V-OH (B), Boc-Pen-(MeOBn)-OH (B), Boc-E(chx)-OH (B), Boc-K(2ClZ)-OH
(A), Boc-D(chx)-OH (B), Boc-Nle-OH (A), Boc-L-OH-H2O (B), Boc-S(Bn)-OH (A), Boc-S(Bn)-OH (A), Boc-C(MeBn)-OH (A), Boc-S(Bn)-OH (A), Boc-Pen (MeOBn)-OH (B). The protected peptidyl resin was treated with TFA:CH2Cl2:anisole (45:52.5:2.5 v/v/v) for 1 minute, then 20 minutes, washed three times with CH2Cl2, txeated with a mixture of 8 mL of DMF

3 9 ~

and 2 mL of thiophenol for 30 minutes, washed twice with DMF, treated with a mixture of 8 mL of DMF and 2 mL of thiophenol for 1 hour, washed twice with DMF, twice with water, twice with ethanol and three times with CH2Cl2 and dried under nitrogen to give the TFA salt of the protected peptidyl resin containing unprotected His.
The peptidyl resin was suspended in HF
containing 10% anisole for 1 hour at 0C. The HF
was removed by evaporation under vacuum. The peptide/resin mixture was washed three times with 1:1 cyclohexane:ether. The peptide was extracted with 50% aqueous acetic acid (3 x 25 mL) and with neat TFA (3 x 25 mL). The combined filtrates were evaporated to a gum, which was suspended in water, and the solution was lyophilized to afford the unoxidized sulfhydryl peptide as a yellow solid.
The crude peptide was dissolved in 1.2 L of degassed 0.2% ammonium hydroxide and air was bubbled through the solution for 4 hours. The solution was stirred overnight and eluted through a 5 g Sep-pak cartridge (trifunctional octadecylsilane). After washing the cartridge with water, the peptide was eluted with aqueous acetonitrile and the solution was membrane-filtered and subjected to preparative HPLC on an octadecylsilane column using water:CH3CN gradients containing 0.1% TFA. Fractions were analyzed by analytical reversed phase HPLC and those containing product with a minimum purity of 95%
were pooled and lyophilized to afford 9.3 mg of ~723~

the trifluoroacetate salt of Example l (1.6%) as a white solid. MS (M+~) 2545.4.

Example 2 0.58 g of Boc-I-(N-Me~ W(formyl)-Pam-resin (see Example 1) was subjected to either Procedure A
(single couple using Step 9a~ or B (double couple using Step 9a), as indicated, with the required sequence of amino acids introduced in order, as Boc-D(chx)-OH (B), Boc-L-OH H20 (B), Boc-H(DNP) OH-isopropanol (B), Boc-Pen~MeOBn)-OH (B), Boc-F-OH
(A), Boc-Y(2BrZ)-OH (A), Boc-V-OH (B), Boc-C(MeBn)-OH (A), Boc-E(chx)-OH (B), Boc-K(2ClZ)-OH (A), Boc-D(chx)-OH (B), Boc-Nle-OH (A), Boc-L-OH H2O
(B), Boc--S(Bn)-O~ (A), Boc-S(Bn)-O~ (A), Boc-Pen (MeOBn)-OH (B), Boc-S(Bn)-OH (A), Boc-C(MeBn)-OH
(A). The protected peptidyl resin was treated with TFA:CH2Cl2:anisole (45:52.5:2.5 v/v/v) for 1 minute, then 20 minutes, washed three times with CH2Cl2, treated with a mixture of 8 mL of DMF and 2 mL of thiophenol for 30 mi.nutes, washed twice with DMF, treated with a mixture of 8 mL of DMF and 2 mL
of thiophenol for 1 hour, washed twice with DMF, twice with water, twice with ethanol and three times with CH2Cl2 and dried under nitrogen to give the TFA salt of the protected peptidyl resin containing unprotected His.
The peptidyl resin was suspended in HF
containing 10% anisole for l hour at 0C. The HF
was removed by evaporation under vacuum. The peptide/resin mixture was washed three times with 1:1 cyclohexane:ether. The peptide was extracted with 50% aqueous acetic acid (3 x 25 mL) and with neat TFA (3 x 25 mL). The combined filtrates were evaporated to a gum, which was suspended in water, and the solution was lyophilized to afford the unoxidized sulfhydryl peptide as a yellow solid.

2~7~

The crude peptide was dissolved in 1.2 L of degassed 0.2~ ammonium hydroxide and air was bubbled through the solution for 4 hours. The solution was stirred overnight and eluted through a 5 g Sep-pak cartridge (trifunctional octadecylsilane). After washing the cartridge with water, the peptide was eluted with aqueous acetonitrile and the solution was membrane-filtered and subjected to preparative HPLC on an octadecylsilane column using water:CH3CN gradients containing 0.1% TFA. Fractions were analyzed by analytical reversed phase HPLC and those containing product with a minimum purity of 95%
were pooled and lyophilized to afford 14.5 mg o~
the trifluoroacetate salt of Example 2 (2.4%) as a white solid. MS (M+H) 2545.4.

ExamPle 3 L-Cysteinyl-L-seryl-L-penicillaminyl-L-seryl-L-seryl-L-leucyl-L-norleucyl-L-aspartyl-L-lysyl-L-glutamyl-L-cysteinyl-L-valyl-L-tyrosyl-L-phenyl-alanyl-L-penicillaminyl-L-hist~dyl-L-leucyl-L-aspartyl-L-isoleucyl-L-prolyl-L-tryptophan, S,S-crosslinked (1,15:3,11) 0.5 g of Boc-W-Pam-resin (O.56 meq/g;
Bachem Inc., Torrance, CA) was subjected to either Procedure A (single couple using step 9a) or B
(double couple using step 9a), as indicated, with the required sequence of amino acids introduced in order, as Boc-P-OH (2), Boc~I-OH ~ H2O (B), Boc-D(chx)-OH (B), Boc-L-OH-H20 (A), Boc-H(DNP)-OH-isopropanol (A), Boc-Pen(MeOBn)-OH
(A), Boc-F-OH (A), Boc-Y(2Br2)-OH (A), Boc-V-OH

HA5 ~ 7 2 3 (B), Boc-C(MeBn)-OH (B), Boc-E(chx) O~ (A), Boc-K(2ClZ)-OH (A), Boc-D(chx)-O~ (A), Boc-Nle~O~
(A), Boc-L-OH ~2O (A), Boc-S~Bn)-OH (A), Boc-S(Bn)-OH (A), Boc-Pen(MeOBn)-OH (A), Boc-S(Bn)-O~ (A), Boc-C(MeBn)-O~ (A). The protected peptidyl resin was treated with TFA:CH~ Cl2: anisole (45:52.5:2.5 v/v/v) for 1 minute, then 20 minutes, washed 3 times with CH2Cl2, treated with a mixture of 8 mL of DMF and 2 mL of thiophenol for 30 minutes, washed twice with DMF, treated with a mixture of 8 mL of DMF
and 2 mL of thiophenol for 1 hour, washed twice with DMF, twice with water, twice with ethanol and three times with CH2Cl2 and dried under nitrogen to give the TFA salt of the protected peptidyl resin containing unprotected His.
The peptidyl resin was suspended in HF
containing 10% anisole for 1 hour at 0C. The HF
was removed by evaporation under vacuum. The peptide/resin mixture was washed three times with 1:1 cyclohexane:ether. The peptide was extracted with 50% a~ueous acetic acid (3 x 25 mL), with 50%
aqueous TFA (3 x 25 mL) and with neat TFA
(3 x 25 mL). The combined filtrates were evaporated to a gum, which was suspended in water, and the solution was lyophilized to afford the unoxidized sulfhydryl peptide as a yellow solid.
The crude peptide was dissolved in 1.4 L
of degassed 0.2% ammonium hydroxide and air was bubbled through the solution for 4 hours. The solution was stirred overnight and eluted through a 5 g Sep-pak cartridge (trifunctional octadecylsilane). After washing the cartridge ~7~3~
~575 with water, the peptide was eluted with aqueous acetonitrile and ~he solution was membrane-filtered and subjected to preparative HPLC on an octadecylsilane column using water:C~3CN gradients containing 0.1% TFA.
Fractions were analyzed by analytical reversed phase HPLC and those containing product with a minimum purity of 95% were pooled and lyophilized to afford 22 mg of the trifluoroacetate salt of Example 3 (3.1%~ as a white solid.
MS (M+~) 2513.1.
The abbreviations used throughout this specification have the following meanings:
Bn Benzyl BOC tert-butoxycarbonyl 2-BrZ 2-bromo-benzyloxycarbonyl Chx cyclohexyl 2-ClZ 2-chlorobenzyloxycarbonyl DCC dicyclohexylcarbodiimide DIPEA diisopropylethylamine DMF dimethylformamide DNP 2,4-dinitrophenyl HOBT hydroxybenzotriazole MeBn 4-methylbenæyl MeOBn 4-methoxybenzyl Nle norleucyl PAM phenylacetamidomethyl Pen penicillaminyl TFA trifluoroacetic acid

Claims (16)

1. A compound of the formula wherein:
A1 and A15 are each independently polar amino acid residues, or A1 is and A15 is wherein A1 and A15 are S,S-crosslinked;
A2, A4, A5, A6, A7, A9, A12, and A16 are each independently amino acid residues;
A3 and A11 are each independently polar amino acid residues, or A3 is and A11 is wherein A3 and A11 are S,S-crosslinked;
A8 is an aliphatic, aromatic, acidic, or polar amino acid residue;
A10 is an aliphatic, aromatic, D- or L-acidic, or polar amino acid residue;
A13 is an aliphatic, aromatic or polar amino acid residue;
A14 is an aromatic or aliphatic amino acid residue;
A17 is an aliphatic or polar amino acid residue;
A18 is an acidic, polar, aliphatic or aromatic amino acid residue;

A19 is an aliphatic amino acid residue;
A20 is ;
A21 is an aromatic amino acid residue;
R1 and R2 are each independently hydrogen or lower alkyl, or R1 and R2 together with the carbon atom to which they are attached are cycloalkyl;
R3 and R4 are each independently hydrogen or lower alkyl, or R3 and R4 together with the carbon atom to which they are attached are cycloalkyl;
R5 and R6 are each independently hydrogen or lower alkyl, or R5 and R6 together with the carbon atom to which they are attached are cycloalkyl;
R7 and R8 are each independently hydrogen or lower alkyl, or R7 and R8 together with the carbon atom to which they are attached are cycloalkyl;
R9 is alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, or aralkyl; and R10 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, or cycloalkenylalkyl;
or R9 and R10 together are an alkylene chain of three to five carbons, optionally substituted with one or two alkyl, alkoxy, aryl, aralkyl, cycloalkyl, or hydroxy groups.

2. The compound of Claim 1, wherein R9 is lower alkyl.

3. The compound of Claim 1, wherein R9 is methyl.

4. The compound of Claim 1, wherein R10 is lower alkyl.

5. The compound of Claim 1, wherein R10 is 2-butyl.

6. The compound of Claim 1, wherein:
A1 is , A3 is , A11 is , and A15 is , wherein A1 and A15 are S,S-crosslinked and A3 and A11 are S,S-crosslinked;
at least one of R1 and R2 is lower alkyl or R1 and R2 together with the carbon atom to which they are attached are cycloalkyl; R3 and R4 are hydrogen, at least one of R5 and R6 is lower alkyl, or R5 and R6 together with the carbon atom to which they are attached are cycloalkyl; and R7 and R8 are hydrogen; or R1 and R2 are hydrogen; at least one of R3 and R4 is lower alkyl or R3 and R4 together with the carbon atom to which they are attached are cycloalkyl; R5 and R6 are hydrogen; and at least one of R7 and R8 is lower alkyl or R7 and R8 together with the carbon atom to which they are attached are cycloalkyl.

7. The compound of Claim 6, wherein lower alkyl is methyl and cycloalkyl is cyclopropyl.

8. The compound of Claim 1, wherein:
A7 is norleucyl or methionyl;
A8 is aspartyl;
A10 is L-glutamyl, D-glutamyl, glutaminyl, or alanyl;
A13 is tyrosyl, phenylalanyl, or asparaginyl;
A14 is tyrosyl or phenylalanyl;
A17 is leucyl or glutaminyl;
A18 is aspartyl, asparaginyl, alanyl, or leucyl;
A19 is isoleucyl or valyl; and A21 is tryptophyl or napthylalanyl.

9. The compound of Claim 6, wherein:
A7 is norleucyl or methionyl;
A8 is aspartyl;
A10 is L-glutamyl, D-glutamyl, glutaminyl, or alanyl;
A13 is tyrosyl, phenylalanyl, or asparaginyl;
A14 is tyrosyl or phenylalanyl;
A17 is leucyl or glutaminyl;
A18 is aspartyl, asparaginyl, alanyl, or leucyl;
A19 is isoleucyl or valyl; and A21 is tryptophyl or napthylalanyl.

10. The compound of Claim 8, wherein:
A2 is seryl or threonyl;
A4 is seryl, phenylalanyl, asparaginyl, or lysyl;
A5 is seryl, threonyl, or aspartyl;
A6 is leucyl, methionyl, lysyl, seryl or threonyl;
A9 is D- or L-lysyl, alanyl or glutamyl;
A12 is valyl or leucyl; and A16 is alanyl, histidyl or phenylalanyl.

11. The compound of Claim 9, wherein:
A2 is seryl or threonyl;
A4 is seryl, phenylalanyl, asparaginyl, or lysyl;
A5 is seryl, threonyl, or aspartyl;
A6 is leucyl, methionyl, lysyl, seryl or threonyl;
A9 is D- or L-lysyl, alanyl or glutamyl;
A12 is valyl or leucyl; and A16 is alanyl, histidyl or phenylalanyl.

12. The compound of Claim 1 wherein A20 is N-methylisoleucyl.

13. The compound of Claim 1 wherein A20 is prolyl.

14. The compound of Claim 1, selected from the group consisting of:
L-Penicillaminyl-L-seryl-L-cysteinyl-L-seryl-L-seryl-L-leucyl-L-norleucyl-L-aspartyl-L-lysyl-L-glutamyl-L-penicillaminyl-L-valyl-L-tyrosyl-L-phenylalanyl-L-cysteinyl-L-histidyl-L-leucyl-L-aspartyl-L-isoleucyl-N-methyl-L-isoleucyl-L-tryptophan, S,S-crosslinked (1,15:3,11);
L-Cysteinyl-L-seryl-L-penicillaminyl-L-seryl-L-seryl-L-leucyl-L-norleucyl-L-aspartyl-L-lysyl-L-glutamyl-L-cysteinyl-L-valyl-L-tyrosyl-L-phenylalanyl-L-penicillaminyl-L-histidyl-L-leucyl-L-aspartyl-L-isoleucyl-N-methyl-L-isoleucyl-L-tryptophan, S,S-crosslinked (1,15:3,11); and L-Cysteinyl-L-seryl-L-penicillaminyl-L-seryl-L-seryl-L-leucyl-L-norleucyl-L-aspartyl-L-lysyl-L-glutamyl-L-cysteinyl-L-valyl-L-tyrosyl-L-phenylalanyl-L-penicillaminyl-L-histidyl-L-leucyl-L-aspartyl-L-isoleucyl-L-prolyl-L-trypto-phan, S,S-crosslinked (1,15:3,11).

15. A method of treating endothelin-related disorders in a mammal, which comprises administering an effective amount of a compound of Claim 1.

16. A method of treating renal, glomerular, or mesangial cell disorders, which comprises administering an effective amount of a compound of Claim 1.