CA2033198A1 - Atrial peptide derivatives - Google Patents

Atrial peptide derivatives

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Publication number
CA2033198A1
CA2033198A1 CA002033198A CA2033198A CA2033198A1 CA 2033198 A1 CA2033198 A1 CA 2033198A1 CA 002033198 A CA002033198 A CA 002033198A CA 2033198 A CA2033198 A CA 2033198A CA 2033198 A1 CA2033198 A1 CA 2033198A1
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Prior art keywords
arg
boc
cys
ile
phe
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Abandoned
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CA002033198A
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French (fr)
Inventor
Scott D. Lucas
Todd W. Rockway
Steven K. Davidsen
A. Mitchell Thomas
Thomas W. Von Geldern
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Abbott Laboratories
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Individual
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cardiology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Endocrinology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

ABSTRACT

Novel atrial peptides of the formula are disclosed which have useful hypotensive, natriuretic and diuretic activity and in which R1 is selected from hydrogen, AcetylArg, Aha, Arg, Cit, His, Lys, Orn and Ser-Ser; R2 is a sulfur-containing group; R3 is a hydrophobic amino acid or dipeptide; R4 is a dipeptide spacer, or a tripeptide containing a dipeptide spacer plus a basic amino acid residue;
R5 is a hydrophobic amino acid; R6 is a peptide of up to three amino acids; R7 is selected from Cha, Phe, Cha-Arg, Phe-Arg, (D)Phe-Arg, Phe-(D)Arg, Phe?Arg, Leu-Arg, Ala-Arg, Arg and Gly-Ala; and R8 is a sulfur-containing group, as well as pharmaceutically acceptable salts, esters and amides thereof.

Description

~3~3 ~ ~

ATRIAL PEPTIDE D~:RIVATIVES -~echnical Field The present invention relates to derivatives of atrial natriuretic polypeptides (ANP) which exhibit useful hypotensive, natriuretic, diuretic, renovasodilating, renoprotective, vasorelaxant and smooth muscle relaxant activity.

B~ckg~_und of the Invent1on Heretofore, hypertension has been classified as either hypertension having clear factor disease or hypertension having obscure factor disease. Hypertension having clear factor disease is also known as secondary hypertension and involves mainly hypertension caused by kidney disease, endocrine disease, toxemia of pregnancy, arterial embolism, atherosclerosis, central nervous system disease and the like.
The conditions of secondary hypertension can be improved by treatment of the factor disease.
However, eighty to ninety per cent of all hypertensive patients suffer from hypertension having obscure factor disease, commonly known as essential hypertension. The treatment of patients having essential hypertention has been affected by agents such as hypotensive diuretics or vasodilators which provide symptomatic relief. With these antihypertensive agents side effects, including lightheadedness, fatique, nausea, respiratory distress, headache, palpitations, depression and impotence are common.
Recently, alpha-human atrial polypeptide (alpha-hANP) has been isolated from human atrium and identified as a 3 ~ r~ ~

polypeptide consisting of twenty eight amino acids having the following formula:

Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-I

S -S
I

Ile-Gly-Ala-Gln-Ser-Gly-Leu-Giy-Cys-Asn-Ser-Phe-Arg-Tyr Alpha-hANP i.s a peptide hormone which reacts with specific receptors on the plasma membrane of cells in target tissues and effects a number of hemodynamic and metabolic events in humans. It has been suggested that the above alpha-hANP is one of the substances involved in the control of hypertension, congestive heart failure, cirrhosis, renal failure, cerebral edema and the maintenance of normal extracellular fluid volume. It has also been reported that the diuretic action of alpha-hANP is about 1000 times as strong as that of furosemide which is used as a hypotensive diuretic.
Others have found that alpha-hANP increases urinary sodium concentration and urinary output.
Known analo~s of alpha-hANP have been reported to show biological potency that correlates well with their affinity for the alpha-hANP receptors (Jacobs, J.W.; Vlasuk, G.P.; Rosenblatt, M.
Endocrinoloay and Metabolism Clinics of North Am~rl~, 16, 63-77 (1987)). However, alpha-hANP or derivatives thereof which are chemically and enzymatically stable and have therapeutically useful potency and duration of action have not been commercially available. For this reason, the search for alpha-hANP derivatives with these favorable properties has been actively pursued. In addition, it is considered desirable to prepare analogs of ANP in order to isolate the regions of ANP responsible for binding to the cellular plasma membrane receptor and also for eliciting subsequent 2~'3~ ~

C. C

biological activities mediated via receptor occupation. U.S. Patent No. 4,757,048 discloses alpha-hANP analogs which vary in size from 8 to ~5 amino acid residues. The compounds of the present invention are distinctly different from the~e prior art compounds in several important aspects, specifically in their amino acid sequences and in their ability to elicit receptor-mediated synthesis of cGMP.
Additionally, there are no known analogs of ANP which are characterized by incorporation within the disulfide ring of a truncated C-terminal amino acid sequence as described in the present invention.

Summary of the Invention In accordance with the present invention, novel peptides are provided which exhibit useful hypotensive, natriuretic, diuretic, renovasodilating, reno-protective, smooth muscle relaxant and vasorelaxant activities. The compounds of the invention include biologically active peptides having the following amino acid sequence:
I' s s R1-R2-R3-R4-R5-R6-R7 R8 (Formula I) or a pharmaceutically acceptable salt, ester or amide thereof.

In the above formula, R is selected from hydrogen, AcetylArg, Aha, Arg, Cit, His, Lys, Orn and Ser-Ser;

R2 is a sulfur-containing group;

R3 is a hydrophobic amino acid or dipeptide;

c R4 is a dipeptide spacer, or a tripeptide containing a dipeptide spacer plus a basic amino acid residue;

R5 is a hydrophobic amino acid;

R6 is a peptide of up to three amino acids;

R is selected from Cha, Phe, Cha-Arg, Phe-Arg, (D)Phe-Arg, Phe-(D)Arg, Phe~Arg, Leu-Arg, Ala-Arg, Arg and Sly-Ala; and R8 is a sulfur-containing group.

The present invention also relates to the use of the atrial peptide derivatives of formula ~I) in the treatment of ANP-related diseases.
The invention further relates to compositions comprising a therapeutically effective amount of the polypeptide of formula (I) and a pharmaceutically acceptable carrier or diluent.

Detailed Descriptlon of the Tnvention The present invention provides novel peptides which exhibit useful hypotensive, natriuretic, diuretic, renovasodilating, reno-protective, smooth muscle relaxant and vasorelaxant activities. The compounds of the invention comprise peptides having the following amino acid sequence:

S--S - I

Rl-R2-R3_R4_R5_R6_R7 R8 (Formula I) or a pharmaceutically acceptable salt, ester or amide thereo~, 2~33 C

wherein Rl is selected from hydrogen, AcetylArg, Aha, Arg, Cit, His, Lys, Orn and Ser-Ser, and is preferably hydrogen (when R2 is Mpa) or a basic amino acid;

R is a sulfur-containing group;

R3 is a hydrophobic amino acid or dipeptide, examples of which include Phe, ~D)Phe, Phe-Gly, Phg, (D)Phg, Cha, (D)Cha, Chg, Cha-(D)Ala, (D)Ala-Cha, Arg-Cha, Pic, Tic and 3,5-diiodoTyr (3,5-I2Tyr), and is preferably a hydrophobic amino acid containing a cycloalkyl group;

R4 is a dipeptide spacer such as Gly-Gly or (D)Ala-Ala, or a tripeptide of the formula X-Y where X is a dipeptide spacer and Y is a basic amino acid, as for example Gly-Gly-Arg, Gly-Gly-(D)Arg, Gly-Gly-Lys, Gly-Arq-Arg, (D)Ala-Arg-Arg, (D)Ala-Ala-Arg and (D)Ala-(D)Ala-Arg;

R is a hydrophobic amino acid;

R6 is a peptide of up to three amino acids, such as Asp-Arg-Ile, (D)Asp-Arg-Ile, Asp-Arg-(D)Ile, ~Asp-Arg-Ile, Asp-Arg-Ala, Asp-Arg-~va, Asp-Ala-Ile, Glu-Arg-Ile, His-Arg-Ile or Ala-Arg-Ile;

R is selected from Cha, Phe, Cha-Arg, Phe-Arg, (D)Phe-Arg, Phe-(D)Arg, Phe~Arg, Leu-Arg, Ala-Arg, Arg and Gly-Ala; and R8 is a sulfur-containing group.

The methods of the preSent invention comprise the use of atrial peptide derivatives of formula (I) in the treatment of r~

ANP-related diseases i~cluding, for example, congestive heart failure, renal failure and hypertension.

The pharmaceutical compositions of the invention include compositions comprislng a therapeutically effecti~e amount of a polypeptide of formula (I) and a pharmaceutically acceptable carrier or diluent.

Representative of the preferred compounds of this invention include the following polypeptides, as well as their pharmaceutically acceptable salts, esters and amides:

Arg-Cys-Cha-(D)Ala-Gly-Gly-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Ser-Ser-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Ser-Ser-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Cha-Arg-Cys;

Arg-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-~D~Ala-Cha-Gly-Gly-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Ser-Ser-Cys-Cha-Gly-Gly-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Cys-Cha-Gly-Gly-Arg--Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Cys-Arg-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-(D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

~ 3 Arg-Cys-Tic-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Phe-Gly-Gly-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

His-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

. .
Orn-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

AcetylArg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Cit-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Cys-NH2;

Arg-Cys-3,5-I2Tyr-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-ASp-Arg-Ile-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Lys-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-(D)Asp-Arg-Ile-Phe-Arg-Cys-NH2;

(D)Arg-cys-cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-phe-Arg-cys-NH

Arg-Cys-Chg-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

2~3~ 9 C C

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ala-Phe-Arg-Cys-NH2;

Arg-Cys-(D)Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Ala-Arg-Ile-Ala-Arg-Cys;

Mpa-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Arg-Cys-Pic-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Cys-Cha-Gly-Gly-Arg-Ile-(D)Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ala-Phe-Arg-Cys-NH2;

Cys-Cha-Gly-Gly-Arg-Ile~Asp-Ala-Ile-Phe-Arg-Cys-NH2;

Mpa-Cha-Gly-Gly-Arg-Ile-beta-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-(D)Ile-Phe-Arg-Cys-NH2;

Cys-Cha-Gly-Gly-Arg-Nva-Asp-Arg-Nva-Phe-Arg-Cys-NH2;

Cys-(D)Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Mpa-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-(D)Arg-Cys-NH2;

Mpa-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-(D)Cys-NH2;

Mpa-Cha-~D)Ala-Arg-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys~NH2;

~ t,~ ?f ~

Cys-Phe-Gly-Gly-Arg-Leu-Asp-Arg-Ile-Leu Arg-Cys-NH2;

Mpa-Cha-Gly-Gly-Arg-Ile-Glu-Arg-Ile-Phe-Arg-Cys-NH2;

Mpa-Cha Gly-Gly-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Mpa-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-(D)Phe-Arg-Cys-NH2;

Aha-Cys-Cha-(D)Ala-(D)Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Mpa-Cha-Gly-Gly-Arg-Ile-His-Arg-Ile-Phe-Arg-Cys-NH2;

Mpa-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Mpa-Cha-(D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-(D)Cys-NH2; and Mpa-Cha-(D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-~Phe~Arg]-(D)Cys-NH2.

Preferred compounds of this invention include the following polypeptides, as well as their pharmaceutically acceptable salts, esters and amides:

Ser-Ser-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Ser-Ser-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Cha-Arg-Cys;

Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Cys-Arg-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

2 ~i ~J 3 {

Arg-Cys-Cha-~D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

His-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Lys-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

(D)Arg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ala-Phe-Arg-Cys-NH2;

Arg-Cys-Cha-Gly-Gly-Arg-Ile-Ala-Arg-Ile-Ala-Arg-Cys;

Mpa-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ala-Phe--Arg-Cys-NH2;

Cys-Cha-Gly-Gly-Arg-Ile-Asp-Arg-(D)Ile-Phe-Arg-Cys-NH2;

Cys-(D)Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Mpa-Cha-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-(D)Cys-NH2;

Mpa-Cha-(D)Ala-Arg-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

Aha-Cys-Cha-(D)Ala-(D)Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys-NH2;

2~3~8 C

Mpa-Cha-Gly-Gly-Arg-Ile-Glu-Arg-Ile-Phe-Arg-Cys-NH2; and Mpa-Cha-(D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-[Phe~Arg]-(D)Cys-NH2.

The amino acid components of the above compounds are designated by conventional abbreviations as follows:

Amino Acid ~b~revia~e~ Designation One Letter Code Alanine Ala A
6-Aminohexanoic acid Aha Aminoisobutyric acid Aib Arginine . Arg R
Aspartic acid Asp D
Citrulline Cit Cyclohexylalanine Cha Cyclohexylglycine Chg Cysteine Cys C
Glutamic acid Glu E
Glycine Gly G
Histidine His H
a-Homocysteine hCys Isoleucine Ile Leucine Leu L
Lysine Lys K
Mercaptopropionic acid Mpa Methionine Met M
Norvaline Nva Ornithine Orn Penicillamine Pen Phenylalanine Phe F

~3 C

~minL~ A~breviated_~esiqn~tion One Letter Co~e Phenylalanyl-Arginine reduced amide (Na[1-(2-amino-3~phenyl-propyl)]arginine) [Phe~Arg]
Phenylglycine Phg Per-Hydroindane carboxylic acid Pic Serine Ser S
Tetrahydroisoquinoline-3-carboxylic acid Tic Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V

All of the above amino acids can be either in the ~ or 2 configuration as well as mixtures thereof including a racemic mixture of the two isomers. Unless expressly stated otherwise, all of the amino acids are optically active and have the L-configuration.
The term "AcetylArg" as used herein refers to a group wherein an acetyl group is attached through the carbonyl carbon of the acetyl group to the alpha amino group of the amino acid arginine.
The term "acidic amino acid" as used herein refers to amino acids which have a group, in addition to the alpha-carboxylic acid group, which exists in ionized form in neutral aqueous solution, as for example Asp, Glu and the like.
The term "basic amino acid" as used herein refers to amino acids which have a group, in addition to the alpha-amino group, which exists in protonated form in neutral aqueous solution, as for example Lys, Orn, Arg and the like.

2 ~

The term "cycloalkyl" as used herein refers to a three to seven carbon cyclic radical. These rad~cals can be unsubstituted, or they can be substituted provided that any such substituents not interfere with the affinity of the peptide for atrial natriuretic polypeptide receptors.
The term "hydrophobic amino acid" as used herein refers to amino acids which lack an affinity for water, such as Ala, Ile, Leu, Val, Met, Nva, Phe, Phg, Cha, Chg, Tic, and the like.
The term "sulfur-containing group" as used herein refers to a group having a sulfur substituent which is available to participate in bonding to form a disulfide bridge, as for example Cys, Mpa, Pen and hCys.
By "pharmaceutically acceptable salts, esters, or amides"
it is meant those salts, esters and amides which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. Examples of pharmaceutically acceptable, nontoxic addition salts are salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid.
Other pharmaceutically acceptable salts include nitrate, bisulfate, formate, valerate, lactate, fumarate, oleate, palmitate, stearate, laurate, borate, benzoate, mesylate, ascorbate, p-toluene-sulfonate, glucoheptonate, lactobioate, lauryl sulfate and the like or metal salts such as sodium, potassium, calcium or magnesium salts or amino salts such as ammonium, triethylamine salts, and the like and they may be prepared according to conventional methods.

~33~ ~8 C

Examples of pharmaceutically acceptable, non-toxic esters of the compounds of Formula (I) include Cl to C6 alkyl esters wherein the alkyl group is straight or branched chain.
Acceptable esters also include C5 to C7 cycloalkyl esters. C1 to C4 alkyl esters are preferred. Esters of the compounds of Formula ~I) may be prepared according to conventional methods.
Examples of pharmaceutically acceptable, nontoxic amides of the compounds of Formula (I) include amides derived from ammonia, primary Cl to C6 alkyl amines and secondary C1 to C6 dialkyl amines wherein the alkyl groups are straight or branched chain. In the case of secondary amines the amine may also be in the form of a five or six membered heterocycle containing one nitrogen atom. Amides derived from ammonia, C1 to C3 alkyl primary amines and Cl to C2 dialkyl secondary amines are preferred. Amides of the compounds of Formula (I) are prepared according to the conventional methods.
The daily dose of the compounds of the present invention may be suitably defined, according to the condition of the patient, by those skilled in the art, but generally may be administered in an amount of 0.2 - 250 mg/kg body weight. Unit dosage compositions may contain such amounts of submultiples thereof to make up the daily dose.
The amount of active ingredient that may may be combined with carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factoxs including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the patient; the route of adrninistration and the rate of excretion of the specific compoundi drug combination used and the severity of the particular disease being treated.

2~33 C

The compounds of the present invention may be administered orally, parenterally, by inhalation spray, ~ectally or topically in unit dosage formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants and vehicles as desired. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
Injectable preparations, as for example sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, as for example a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycol which are solid at ordinary temperature but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
Solid dosage forms for oral administration may include capsules, tablets, pills, powders and ~ranules. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, as for example lubricating agents such as magnesium stearate. In the 2~31.

C

case o~ capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art such as water. Such compositions may also comprise adjuvants, such as wetting agents; emulsifying and suspending agents; and sweetening, flavoring and perfuming agents.
The novel polypeptides and salts thereof of the invention can be utilized effectively as vasorelaxant agents, diuretics, natriuretic agents, renovasodilators or renoprotective agents.
~ccordingly the present invention further relates to vasorelaxant, diuretic, natriuretic, renovasodilating or renoprotective compositions comprising a novel polypeptide having the general formula (I) or salts thereof as an active component.
The polypeptides of the invention may be prepared by the synthetic method of elongation of a peptide chain through condensation of one amino acid by one, or several amino acids, or by a combination of these methods in accordance with conventional peptide synthesis methods.
The condensation of two amino acids, the condensation of an amino acid with a peptide or the condensation of one peptide with another peptide may be effected in acccrdance with conventional condensation methods such as the azide method, the mixed acid anhydride method, the DCC
(dicyclohexylcarbodiimide) method, the active ester method (p-nitrophenyl ester method, N-hydroxysuccinic acid imide ester method, cyanomethyl ester method and the like), the Woodward reagent K method, the DCC-HOBT (1-hydroxy-benzotriazole) method and the like. These condensation reactions may be done by either solution methods or solid phase synthetic methods.

~3 C

When the peptide chain is elongated by solid phase methods, the C-terminal amino acid is linked to an insoluble carrier.
Any insoluble carrier may be used which can produce a detachable bond by reacting with a carboxyl group in a C-terminal amino acid, as for example halomethyl resins such as chloromethyl or bromomethyl resins; hydroxymethyl resin;
aminomethyl resin; benzhydrylamine resin and t-alkyloxycarbonyl hydrazide resin.
As in conventional polypeptide synthesis, branched chain amino and carboxyl groups at alpha and omega positions in amino acids may be protected and deprotected if necessary. The protecting groups for amino groups which can be used involve, for example, benzyloxycarbonyl ~Z), o-chloro-benzyloxycarbonyl((2-Cl)Z), p nitrobenzyloxycarbonyl (Z(NO2)), p-methoxybenzyloxycarbonyl (Z(OMe)), t-butoxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobornealoxycarbonyl, adamantyloxycarbonyl (Adoc), 2-(4-biphenyl)-2-propyloxy carbonyl (Bpoc), 9-fluorenyl-methoxycarbonyl (Fmoc), methylsulfonylethoxy carbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl (Nps), diphenylphosphinothioyl (Ppt) and dimethylphosphino-thioyl (Mpt).
The examples of protecting groups for carboxyl groups involve, for example, benzyl ester (OBzl), cyclohexyl ester, 4-nitrobenzyl ester (OBzlNO2), t-butyl ester (OtBu), 4-pyridylmethyl ester (OPic) and the like.
In the course of the synthesis of the following peptides, specific amino acids having functional groups other than amino and carboxyl groups in the branched chain such as arginine, -cysteine, serine and the like may be protected, if necessary, with suitable protecting groups. It is preferable that, for example, the guanidino group (N ) in arginine may be protected with nitro, p-toluenesulfonyl (Tos), benzyloxycarbonyl (z), 2~31~8 C C

adamantyloxycarbonyl (Adoc), p-metho~ybenzenesulfonyl, 4-methoxy-2,6-dimethyl-benzenesulfonyl (Mts) and the like, and the thiol group in cysteine may be protected with benzyl, p-methoxybenzyl, triphenylmethyl, acetamidomethyl, ethylcarbamyl, 4-methylbenzyl (4-MeBæl), 2,4,6,-trimethylbenzyl (Trnb) and the llke, and the hydroxy group in serine may be protected with benzyl (Bzl), t-butyl, acetyl, tetrahydropyranyl (THP) and the like.
The compounds of the invention are prepared by standard solid phase peptide synthesis conditions as described in "Solid Phase Peptide Synthesis" by John M Stewart and Janis D.
Young~ Second Edition (1984) and illustrated in Examples 1 and 2 in the experimental section.
The compounds of the invention may also be prepared by partial solid phase synthesis, fragment condensation methods and classical solution methods as exemplified by the methods described in "Peptide Synthesis", Second Edition, M. Bodansky, Y.S. Klausner and M.A. Ondetti (1976).
The ~oregoing may be better understood by reference to the following examples which are provided for illustration and not limitation of the practice of the invention.

Example 1 ArcTinyl~N~-Tos)-Cysteinyl(4-~eBzl)-CYCloh~x~lalanyl-(~L~
Alanyl-Glycyl-Glycyl-Arginyl(N~-Tos)-Teucyl-Aspa~tyl(beta-cyclohexyl)-ArginyLl~-Tos)-Isoleucyl-phenylalanyl--Argin Tos)-Cysteinyl-p-Methylbenzhydrylamine prQ-~ected Pepti~e ResLn~ ~-Resin (p-methylbenzhydrylamine functionalized styrene-divinylbenzene copolymer, 0.5 g, commercially available from Bachem, Inc.) was placed in a solid phase peptide synthesis ~3~3~ ~

vessel and amino acids were attached to the peptide resin sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg~NG-Tos), Boc-Asp~beta-cyclohexyl), Boc-Leu, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-(D)Ala, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), according to the protocol outlined in Agenda A to yield the protected peptide resin: Arginyl-Cysteinyl(4-MeBzl)-Cyclohexylalanyl-(D)Alanyl-Glycyl-Glycyl-Arginyl(NG-Tos)-Leucyl-Aspartyl(beta-cyclohexyl)-Arginyl(NG-Tos)-Isoleucyl-Phenylalanyl-Arginyl(NG-Tos)-Cysteinyl(4-MeBzl)-p-Methylbenzhydrylamine resin.
Following the synthesis, the protected peptide was removed from the reaction vessel by washing the resin three times with 20 mL N,N-dimethyl formamide (DMF) into a 60 mL sintered glass funnel, followed by washing the resin three times with 30 mL
methylene chloride (CH2Cl2). The resin was dried n vacuo for three hours, then weighed.

~enda A

1. Deblock: 50% Trifluoroacetic acid ~TFA) in CH2Cl2 containing 2% Anisole, 0.5% Ethanedithiol tv/v/v).
2. Neutralization: 10% Diisopropylethylamine: ~DIEA) in CH2C12 ~v/v) .
3. Single Coupling: 0.2 ~ Boc-amino acid derivative in DMF, 0.2 M diisopropylcarbodiimide in CH2Cl2, reaction time 60 minutes.
4. Resin Clean Up: 10% DIEA in CH2Cl2.
5. Single Coupling: 0.2 M Boc-amino acid derivative ~same as Step 3) in DMF; 0.2 ~ diisopropylcarbodiimide in CH2Cl2, reaction time 60 min.
6. Cap: 0.3 _ Acetylimidazole in DMF.
7. Go to next amino acid residue.

~3~9 C
8. Upon attachment of the final amino acid to the growing peptide chain, the protecting group (t-Boc) is removed as in Step 1.

~m~LlQ ~

Arginyl-Cysteinyl-Cyclohexylalanyl-(D)Alanyl-Glycyl-Glycyl-Arginyl-LeuCyl ~ art.yl.-Arginyl-Isole-l;Lçyl-ph~ Lan Arginyl-Cysteinyl Carboxamide Cyclic ~isulfide.

Protected resin (0.50 g) of Example 1 was treated with 1.3 mL anisole and 13 mL liquid hydrogen fluoride (HF) for 60 minutes at 0 C. The HF and anisole were removed from the resin in vacuo and the resin was washed, under a nitrogen atmosphere, with 2 X 25 mL portions of diethyl ether or ethyl acetate. The crude peptide was extracted from the resin by treatment with 6 X 50 mL portions of deoxygenated 20% aqueous acetic acid. The acetic acid solution was diluted to 1.5 L
with distilled water and the pH of the solution was adjusted to about 7.2 using concentrated ammonium hydroxide. The solution was oxidized with 17 mL of O.OlN iodine solution (in ethanol). The resultant yellow solution was stirred for 60 minutes at room temperature while the pH was maintained at about 7.2. The excess iodine was removed from the solution by first adjusting the pH to about S.S with S0 mL glacial acetic acid and then adding 3-6 mL of O.OlN sodium thiosulfate solution. The combined aqueous solution was applied to a column containing 300 g XAD-16 molecular adsorbent resin (commercially available from Rohm and Haas Company). The ~-sample was desalted by first washing the column with 3 L
distilled water. The sample was eluted from the column with l.S L 50% aqueous ethanol. The combined ethanol fractions were concentrated to approximately 100 mL ln vacuo, taken up in an 2~3~9 {

additional 100 mL distilled water and lyophilized to a dry amorphous powder. The dry powder was purified by High Performance Liquid Chromatography (HPLC) using 0.1% aqueous trifluoroacetic acid tTFA) and acetonitrile as organic modifiers. The preparative HPLC was performed using a Dynamax ~RAININ) Clg reverse-phase column ~2.1 cm i.d. X 25 cm) at a flow rate of 12-15 mL/min. The sample was purified by linear solvent-gradient elution from 10% acetonitrile/90% aq. solvent (0.1% TFA in H2O) to 3S% acetonitrile/65% aqueous solvent.
Analytical HPLC was performed using a VYDAC 218 TP lO micron Clg reverse-phase column (0.46 cm i.d. X 20 cm). There was obtained 2.5 mg of the title compound as a white powder.

Amino acid Analysis: Ala (1.1), Arg (4.0), Asp (1.1), Cha tO.9), Cys (1.4), Gly ~2.2), Ile (1.2) Leu ~1.2), Phe (1.2).
FAB ~M+H) at M/Z: 1672.
The 300 MHz lH NM~ spectrum was found to be consistent with the proposed structure.

E~m~le 3 Seryl-Seryl-Cysteinyl-Cyclohexyl~lanyl-Glycyl-Glycyl-Arqinyl-Isoleucyl-As~artyl-Arginyl-Ts~le-~cyl-Phenyla~nyl-Arginyl-Cysteinyl Cyclic Disulfide Protected peptide resin ~0.50 q), prepared (as described in Example 1) from Boc-Cys~4-MeBzl)-O- Merrifield resin (commercially available from Bachem, Inc.) and the amino acids ~-added sequentially in the following order: Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(N -Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Ser~Bzl), Boc-Ser(Bzl), was treated with HF

2~33 C. {

(liquid) and purified as described in Example 2. The title compound was obtained as a white powder/ 17 mg.

Amino Acid Analysis: Arg (3.0), Asp ~1.0), Cys (0.8), Gly (2.1), Ile (2.0), Phe tl.0), Ser ~2.0).
FAB (M+H) at M/Z: 1621.
The 300 MHæ H NMR spectrum was found to be consistent with the proposed structure.

~xample 4 Seryl-Seryl-Cysteinyl-Cyclohexylalanvl-Glycyl-Glvcvl-Arainvl-I~oleucyl-As~artvl-Arginyl-Isoleucvl-cvclQh~ylalanyl-Arain Cysteinyl Cyclic n;sulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from Boc-Cys(4-MeBzl)-0-Merrifield resin and the amino acids added sequentially in the following order: Boc-Arg(N -Tos), Boc-Cha, Boc~Ile, ~oc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Ser(Bzl), 80c-Ser(Bzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 18.1 mg.

Amino Acid Analysis: Arg (3.0), Asp (1.0), Cha (2.0), Cys (1.5), Gly (2.0), Ile (2.0), Ser (1.5).
FAB (M+H) at M/Z: 1628.
The 300 MHz lH spectrum was found to be consistent with the proposed structure. -2 ~

Ex~m~le S

~L~inyl-cy~teinyl-phenylala~yl-Glycyl-Glycyl-~rainyl-Isol.eucyl-As~rtvl-Arainyl-Isoleucyl-Phenvlalanvl-Arainvl-Cysteinyl Carbo~amide Cvclic Disulfide Protected peptide resin (0.50 g), prepared ~as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos~, Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Phe, Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 15 mg.

Amino Acid Analysis: Arg (3.9), Asp (0.9), Cys (1.8), Ile ~2.1), Gly (1.9), Phe (2.0).
FAB (M+H) at M~Z: 1597.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

~E.xam~le 6 Arainyl-Cvsteinvl-~D)Alanvl-Cvclohexylalanyl-Glvcyl-Glvcvl-~lainYl=Ls~l-Aspartvl-Arainvl-Isoleucyl-phenylalan ~rginyl-Cysteinvl Carboxamid~ Cvc].ic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Leu, Boc-Arg(N -Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-(D)Ala, Boc-Cys(4-MeBzl), Boc-Arg(N5-3 ~L ~ ~

Tos), was ~reated with HF (liquid) and purified as describedin Example 2. The title compound was obtained as a white powder, 15 mg.

Amino Acid Analysis: Ala (0.9), Arg (4.1), Asp (0.9), Cha (1.0), Cys (1.7), Gly (2.0), Ile (1.1), Leu (1.1), Phe (1.0).
FAB (M+H) at M/Z: 1672.
The 300 MHz lH NMR spectrum was found to be consistent with the proposed structure.

Example 7 Arginyl-Cvsteinyl-Cyclohexylalanyl-Glycyl.-Glycyl-Arqinyl-soleucyl-Aspartyl-Arqi~Lvl-Isoleucvl-Phenvlalanyl-Arqinyl-Cysteinyl Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from Boc-Cys~4-MeBzl)-O-Merrifield resin and the amino acids added sequentially in the following order: Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(N -Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), was treated with HF
(liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 13 mg.

Amino Acid Analysis: Arg (4.1), Asp (l.0), Cha (1.0), Cys (1.9), Gly (l.9), Ile (2.0), Phe (l.0).
FAB (M+H) at M/Z: 1604. --The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

2~3~

Exa~gle 8 ~er~l-~eryl=Cysteinyl-Cyclohexylalanyl-Glvcvl-Gl~cvl-t~)-~rginyl-Isoleucyl-~s~artyl-Arainyl-Isoleucyl-Phenylalanyl-~rginyl-Cysteinyl Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from Boc-Cys(4-MeBzl)-O-Merrifield resin and the amino acids added sequentially in the following order: Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-(D)Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Ser(Bzl), Boc-Ser(Bzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 17.5 mg.

Amino Acid Analysis: Arg (3.2), Asp (1.0), Cha (0.9), Cys (1.8), Gly (1.9), Ile (2.0), Phe (0.9), Ser (1.4).
FAB (M+H) at M/Z: 1620.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

Example 9 Cysteinyl-Cyclohexylalanyl-Glycyl-Glycyl-Arginyl-Isoleucyl-Cyclic Disulfide Protected peptide resin (0.50 g), prepared ~as described in Example 1) from Boc-Cys(4-MeBzl)-O-Merrifield resin and the --amino acids added sequentially in the following order: Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg~N -Tos), Boc-Gly, Boc-Glyl Boc-Cha, Boc-Cys(4-MeBzl)~ was treated with HF (liquid) and 2~3~ 9~

purified as described in Example 2. The title compound was obtained as a white powder, 25.1 mq.

Amino Acid Analysîs: Arg t3.0), Asp (1.0), Cha (1.0), Cys (1.7), Gly ~2.1), Ile (2.0), Phe (1.0).
FAB (M+H) at MJZ: 1446.
The 300 MHz 1H NMR spectrum was found to be consistent with the proposed structure.

Example 10 ysteinyl-Arg;nvl-Cvclohexylalanvl-Glycyl-Glycyl-Ar~in~l~
Isoleucyl-As~artyl-Arginyl-Isoleucyl-Phenylalanyl-Arginyl-Cysteinyl Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from Boc-Cys(4-MeBzl)--O-Merrifield resin and the amino acids added sequentially in the following order: Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Arg(NG-Tos), Boc-Cys(4-MeBzl), was treated with HF
(liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 18 mg.

Amino Acid Analysis: Arg (3.9), Asp (0.9), Cha (1.0), Cys (0.6), Gly (2.1), Ile ~2.0), Phe (1.0).
FAB (M+H) at M/Z: 1602.
The 300 MHz lH NMR spectrum was found to be consistent with the proposed structure.

2~3~3 98 ~am~le 11 Arginyl-Cysteinyl-Cyclohexylalanyl-Glycyl-Glycyl-Arginyl-.Isoleucyl-Aspartyl-Arg;nyl-Isoleucyl-Phenylalanyl-Arainyl-Cysteinyl Carboxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-Me8zl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp~beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Arg(N -Tos)~ was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 12.4 mg.

Amino Acid Analysis: Arg (3.9), Asp (1.0), Cha ~1.0), Cys ~1.2), Gly (2.1), Ile (2.0), Phe (l.0).
FAB (M+H) at M/Z: 1602.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

Example 12 ~L5inyl-cysteinyl-cyclohexvlalanvl-~D)A]anvl-Alanvl-Arq;n Isoleucyl-Aspartyl-A~ainyl-Isoleucvl-Phenylalanyl-Arginyl-Cysteinyl Carboxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos~, Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(N -Tos), Boc-Ala, Boc-(D)Ala, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), was 2~ g~

-2~-treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 40 mg.

Amino Acid Analysis: Ala-~1.9), Arg (4.0), Asp (0.9), Cha ~1.0), Cys (1.7), Ile (2.0), Phe (1.0).
FAB (M+H) at M/Z: 1629.
The 300 MHz lH NMR spectrum was found to be consistent with the proposed structure.

Exam~le 13 A~ginyl Cysteinyl-Tetrahydroisoquinolin-3-ovl-Glvcyl-Glycyl-a~gin~ soleucyl-Aspartyl-Arginyl-Isoleucyl-phenylalan a~iny1-Cysteinyl Carboxam;de Cvcli5-~L~LL~id~

Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Tic, Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), was treated with HF (liquid) and purified as described in Example 2. The title co~pound was obtained as a white powder, 14.6 mg.

Amino Acid Analysis: Arg (4.0), Asp (1.1), Cys (1.7), Gly (1.9), Ile (2.1), Phe (1.0).
FAB (M+H) at M/Z: 1607.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

~331 C C

~xample 14 A~ai~yl-Cysteinyl-Phenylalanyl-Glycyl-Glycyl-Isoleucyl-arty]-Arginyl-Isoleucyl-phenyL~la-nyl-Arqinyl-cystein Carboxamide Cyclic Disulflde Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylben~hydrylamine resin and ~he amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Gly, Boc-Gly, Boc-Phe, Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), was treated with HF
(liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 31 mg.

Amino Acid Analysis: Arg (3.0), Asp (1.0), Cys (1.8), Gly (1.9), Ile (2.1), Phe (2.0).
FAB (M+H) at M/Z: 1439.
The 300 MHz lH NMR spectrum was found to be consistent with the proposed structure.

~x~mple 15 ~listidyl-Cysteinyl-Cyclohexylalanyl-Glycyl-Glv~vl-~rqinyl-,Isoleucyl-Aspa~;yl-ALg~nyl-Isoleucyl-phe~lalany-L-Ar~in Cyst~inyl Carboxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydryamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-His(N-Im-Tos), was 2 ~

treated with HF ~liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 13 mg.

Amino Acid Analysis: Arg (2.9), Asp (0.9), Cha (1.03, Cys (1.5), Gly (1.9), His ~0.8), Ile (2.0), Phe (1.0).
FAB (M+H) at M/Z: 1583.
The 300 MHz H NMR was found to be consistent with the proposed structure.

Example 16 arg;nyl-Cy~teinyl-Cyclohexylalanyl-Glycvl-Glvcyl-Arainyl-Phenylalanyl-As artvl-Arainyl-Isoleucyl-Phenylalanyl-Arginyl-Cysteinyl Carhoxamide Cyclic Disulfide Protected peptide resin (O.S0 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Phe, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 25.1 mg.

Amino Acid Analysis: Arg (4.0), Asp (0.9), Cha (1.0), Cys (1.8), Gly (1.9), Ile (1.1), Phe (2.1).
FAB (M+H) at M/Z: 1635.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

C

E,xam~le 17 Cysteinyl-Cyclohexylalanyl-Glvcvl-Glyçyl-Arginyl-Isoleucyl-Aspartyl- ~qin~l-~ oleucyl-E~henylalanvl-Arginyl-Cysteinyl Carboxamlde Cyclic Visulfi~

Protected peptide resin (0.31 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg~N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), was treated with HF
(liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 2.3 mg.

Amino Acid Analysis: Arg (2.7), Asp (0.8), Cha (0.9), Cys (1.6), Gly (1.8), Ile (1.9), Phe (1.1).
FAB tM+H) at M/Z: 1445.
The 300 MHz 1H NMR spectrum was consistent with the proposed structure.

~ample 18 Ornithinvl-Cvsteinyl-Cyclohexylalanvl-Glycyl-Glycyl-Arglnyl-Isoleucyl-As~artyl-~r~inyl-Isoleucyl-Phenylalanyl-Arainyl-Cysteinvl Car~mide Cy~lis~L~LLEi~Q

Protected peptide resin (0.37 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(N -Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys, Boc~Orn(Cbæ), was treated with H~

2~33198 C

~liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 20.0 mg.

Amino Acid Analysis: Arg (3.0), Asp t0.9), Cha (1.0), Cys (1.7), Gly (2.0), Ile (2.1), Orn (1.0), Phe (1.0).
FAB (M+H) at M/Z: 1559.
The 300 MHz 1H NMR spectrum was found to be consistent with the proposed structure.

Example 19 . .
AcetylArqinyl-Cy~teinyl-Cyclohe~ylsl~n~l-Glycyl-Glycyl-AraiQvl-I~oleucyl-Aspartyl-Arginyl-Tsol.eucyl-Phenylalanyl-Ar~in~ yste.~nyl Carboxamide Cycl~ Dl~lf;de Protected peptide resin (0.30 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(~ -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(N -Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys, Boc-Arg(N -Tos), was treated with HF and purified as described in Example 2. The N-terminal Arginyl residue was acetylated prior to purification with 0.3 acetylimidazole in DMF (according to Step 6, Agenda A of Example 1). The title compound was obtained as a white powder, 2.3 mg.

Amino Acid Analysis: Arg (4.1), Asp (1.0), Cha (1.0), Cys (1.6), Gly (1.9), Ile (2.0), Phe (1.1).
FAB (M+H) at M/Z: 1645.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

~33~

~Q

Citrullinyl=Cysteinyl-Cyclohexylalanyl-Glycyl-Glycyl-Ar~inYl=
eu~y~ sRa~-~y-l~ rginyl-Isoleucyl-~h ~ anyl-Arain Cysteinyl Car~oxamide Cycl;c Disulfide Protected peptide resin (0.42 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys~4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(~-Meszl), Boc-Cit, was treated with HF (liquid) and purlfied as described in Example 2. ~he title compound was obtained as a white powder, 24.9 mg.

Amino Acid Analysis: Arg (3.0), Asp (l.0), Cys (1.2), Gly (1.6), Ile (1.6), Phe (1.2).
FAB (M+H) at M/Z: 1602.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.
~m~

~inyl-Cysteinyl-Cyclohexylalanyl-Glycyl-Glycy1-Ar inyl-T~sQ~-e-uc-y~ spartyl-Ar~invl-Iso]eucyl-phenylalanyl-cyste - S~lh5~5~L~LCyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(N -Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), was treated with HF

2g33~

(liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 20.6 mg.

Amino Acid Analysis: Arg ~3.0), Asp (1.0), Cha (1.0), Cys (1.7), Gly (1.9), Ile (2.0), Phe ~1.1).
FAB (M+H) at M~Z: 1447.
The 300 MH lH NMR spectrum was found to be consisten~ with the proposed structure.

Exam~le 22 Arainyl-Cysteinyl-3,5-~iiodotyrosinyl-C,lycyl-Glycyl-Arginyl-Isoleucyl-Aspartyl-Arainyl-Isol.eucyl-Phenylalanyl-Arginyl-Cysteinyl Carboxamide Cy~lic D;sulfide Protected peptide (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg~N -Tos), Boc-Phe, Boc-Ile, Boc-Arq(NG-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Tyr(2-BrCbz), Boc-Cys(4-MeBl), Boc-Arg(N -Tos), was treated with HF (liquid) and purified as described in Example 2. Iodination of the tyrosine occurred during the oxidation (i.e. cyclization) of the peptide with 0.01 N iodine solution as described in Example 2. The title compound was obtained as a white powder, 25 mg.

Amino Acid Analysis: Arg (4.0), Asp (1.1), Cys ~1.4), Gly (1.9), Ile (2.0), Phe (1.0), Tyr (1.0).
FAB (M+H) at M/Z: 1865.
The 300 MHz 1H NMR spectrum was found to be consistent with the proposed structure.

2~ 3~9~

Exam~le 23 Arginyl-Cysteinyl-CyclQhexylalanyl-Glycyl-Glycyl-Arginyl-Isol ellcyl-Aspartyl-Arginyl -Isol eucyL-Arginyl -C~ste; n~l Carboxamide Cycli~ D;sulfide Protected peptide resin ~0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(~-MeBzl), Boc-Arg(NG-Tos), Boc-Ile, Boc-Arg~NG-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), was treated with HF (liquid) as described in Example 2. The title compound was obtained as a white powder, 28 mg.

Amino Acid Analysis: Arg (9.2), Asp (1.1), Cha (1.0), Cys (1.6), Gly ~2.0), Ile ~1.8).
FAB ~M+H) at M/Z: 1455.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

~xample 24 Arqinyl-Cy~teinyl-Cyclohexylalanyl-Glycyl-Glycyl-[cyclo~Lysiny~-Isoleucyl-Aspartyl)l-Arg;nyl-Isole ~h~nyl~. ~anyl=A~qiny-L=
Cysteinyl CarbQxamide Cyclic Disulfide Protected peptide resin (0.43 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Lys-Ile-Asp, Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), ~3~

Boc-Arg(N -Tos), was treated with HF ~liquid) and purified as described in Example 2. The side chain amino group of lysine in the tripeptide Lys-Ile-Asp was cyclized with the side chain carboxylic acid group of aspartic acid by standard DpeA
coupling techniques. The tripeptide was incorporated during the solid phase synthesis as the Boc-protected cyclized tripeptide acid dissolved in N-methyl pyrrolidone. The title compound was obtained as a white powder, 2.8 mg.

Amino Acid Analysis: Arg (3.2), Asp (0.8), Cha (1.0), Cys (0.9), Gly (2.0), Ile (2.0), Lys ~0.9), Phe (1.1).
FAB (M+H) at M/Z: 1556.
The 300 MHz lH NMR spectrum was found to be consistent with the proposed structure.

Exam~le 25 ,a;~.ginyl-Cysteinyl-CYclohexvlalanyl-Glvcyl-Glycvl-Arc~invl-Isoleucyl-Aspartyl-Arainvl-Isoleucvl-Glvc~l-Alan~l-Cysteinyl .S~r~mide Cyclic Disulf-de Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Ala, Boc-Gly, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(N -Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 15 mg.

Amino Acid Analysis: Ala (1.0), Arg (3.0), Asp (1.0), Cha (l.0), Cys (1.8), Gly (3.0), Ile (2.2).
FAB (M+H) at M/Z: 1426.

2~3~9 C C

The 300 MHz lH NMR spectrum was found to be consistent with the proposed structure.

x~mple 26 ~inyl-Cysteinyl-Cyclohexylalanyl-Clyc~L-Glycyl-Arginyl-Isoleu~yl-(D~AspaL~yl-Arainyl-Iso~eucyl-Phenylalanyl-Arginyl-Cysteinyl Carboxamide Cyclic Disulfide Protected peptide resin (O.S0 g), prepared ~as described in Example l) from p-methylbenzhydrylamine resin and the amino acids added in the following order: Boc-Cys(q-MeBzl), Boc-ArgtNG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-(D)-Asp(beta-Bzl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBæl), Boc-Arg(NG-Tos), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 15.3 mg.

Amino Acid Analysis: Arg ~3.7), Asp (1 1), Cha (0.9), Cys (1.4), Gly (2.2), Ile (2.2), Phe (0.9) FAB (M+H) at M/Z: 1602.
The 300 MHz 1H NMR spectrum was found to be consistent with the proposed structure.

~mPle 27 (D)Arainyl-Cvsteinyl-CycloheXylal~nyl-Glycyl-C.lycyl-~rainyl-Cystein~l Carboxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cyst4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-ArgtN -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg~NG-Tos), Boc-Gly, Boc Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-(D)Arg(NG-Tos), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 18.7 mg.

Amino Acid Analysis: Arg (4.0), Asp (0.9), Cha (1.0), Cys (1.3), Gly (2.1), Ile (2.2), Phe (0.9) FAB (M+H) at M/Z: 1601.
The 300 MHz 1H NMR spectrum was found to be consistent with the proposed structure.

~xample 28 Arginyl-Cysteinyl-Cyclohexyl~lycine-Glvcyl-G1ycyl-Arinyl-Isoleucyl-Aspartyl-Ar~inyl-Isoleucyl-Phenylalanyl-Arainyl-~yg~einyl Carboxamide Cyclic D;sulfide Protected peptide resin (0.50 g), prepared (as described in Example l) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-~sp(beta-cyclohexyl), Boc-Ile, Boc-Arg~N -Tos), Boc-Gly, Boc-Gly, Boc-Chg, Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 30 mg.

Amino Acid Analysis: Arg (4.2), Asp (0.8), Cys (1.9), Gly (1.8), Ile (2.1), Phe (l.0).
FAB (M+H) at M/z: 1588.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

2~319~

Example 29 ~rginyl-Cysteiny ~Cyclohexylalanyl-Glycyl-Glycvl-Arainyl-Isc-leu ~L-Aspartyl=arginyl-Alanyl~henylalanvl-Arainyl-Cysteinyl Car~oxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ala, Boc-Arg(NG Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), was treated with HF
tliquid) and purified as described in Example 20 The title compound was obtained as a white powder, 30 mg.

Amino Acid Analysis: Ala (1.0), Arg (4.2), Asp (0.9), Cha (1.0), Cys (1.3), Gly ~1.8), Ile (1.2), Phe (1.0).
FAB (M~H) at M/Z: 1560.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

Exam~le 3Q

Arginyl-Cysteinyl-(D)Cyclohexylalanyl-Glycyl-Glycyl-Arqinyl-Isoleucyl-Asnartvl-~rginyl-Isoleucyl-Phenylalanyl-A~inyl-~ Lo~ L~ c~-lic Disulfide Protected peptide resin (O.S0 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-(D~Cha, Boc-Cys(4-MeBzl), Boc-Arg~NG-Tos), was 3 ~ r~3 (5~

treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 8 mg.

Amino Acid ~nalysis: Arg (3.7), Asp ~1.1), Cha (1.1), Cys (0.7), Gly ~2.1), Ile (2.0), Phe (1.0) FAB (M+H) at M/Z: 1602.
The 300 1H NMR spectrum was found to be consistent with the proposed structure.

Example 31 Arainyl-Cysteinyl-Cyclohexylalanyl-Gl~y1-Glycyl-Rrqinyl-I~oleucyl-Alanyl-Arainvl-Isoleucvl-Alan~l-Arainvl-Cvsteinyl Cyc1ic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from Boc-Cys(4-MeBzl)-0-Merrifield resin and the amino acids added sequentially in the following order: Boc-Arg(N -Tos), Boc-Ala, Boc-Ile, Boc-Arg(N -Tos), Boc-Ala, Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 22.4 mg.

Amino Acid Analysis: Ala (1.7), Arg (4.3), Cha (1.0), Cys (1.7), Gly (1.9), Ile (2.0).
FAB (M+H) at M/Z: 1482.
The 300 MHz H NMR was found to be consistent with the proposed structure.

2~3~

~xamplç~

MercaDto~ropionyl-Cyclohexylalanyl-Glycyl-Glycyl-Arginyl-lsoleu~yl-As~artyl-Arginyl-Isoleucyl-Phenylalanvl-Arainyl-Cysteinyl Carboxamide Cyclic Disulfide Protected peptide resin (0.30 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Mpa(4-MeBzl), was treated with HF (liquid) and purified as des~ribed in Example 2. The title compound was obtained as a white powder, 27.0 mg.

Amino Acid Analysis: Arg (2.9), Asp (1.0), Cha (1.0), Cys (1.2), Gly (2.1), Ile (2.0), Phe (1.0).
FAB (M+H) at M~Z:.1430.
The 300 MHz 1H NMR spectrum was found to be consistent with the proposed structure.

Exa~ple l~

A~ginyl-Cysteinyl-Cyclohexylalanyl-Glycyl-Glycyl-(D)Argin Isoleu~yl-Aspartyl-Arginyl-Isoleucyl-phenylalanyl-~rqin Cysteinyl Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1~ from Boc-Cys(4-MeBzl)-O-Merrifield resin and the amino acids added sequentially in the following order: Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc--(D)Arg(N -Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(9-MeBzl), Boc-Arg(N -Tos), was treated with 2~3319 {

HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 40 mg.

Amino Acid Analysis: Arg (4.0), Asp ~1.0), Cys ~1.8), Gly (2.0), Ile (2.1), Phe (1.0).
FAB (M+H) at M/Z: 1602.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

~am~le 34 Arainyl-Cysteinyl-Per-~ydroin~n~c~onyl-Glycyl-Glycyl-Arginyl-Isoleucyl-Aspartyl-Arginyl-Isoleucyl-Phenylalanyl-~rginyl=~ysteinyl Cyclic Disulfide Protected peptide resin (0.97 g), prepared (as described in Example 1) from Boc-Cys(4-MeBzl)-O-Merrifield resin and the amino acids added sequentially in the following order: Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Pic, Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), was treated with HF
(liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 81 mg.

Amino Acid Analysis: Arg (3.6), Asp (1.0), Cys (1.5), Gly (1.9), Ile (2.3), Phe (1.2).
FAB (M+H) at M/Z: 1614.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

C

-~3-Example 35 Seryl-Seryl-Cysteinyl-Phenylalanyl-Gly~yl-Glycyl-Arginyl-lsoleucyl-Aspar~yl-Ar.ginyl=I~ol.eucyl-Phenylalanyl-Ar~inyl-Cysteinyl ~ lii9-Protected peptide resin (0.79 g), prepared ~as described in Example 1) from Boc-Cys(4-MeBzl)-O-Merrifield resin and the amino acids added sequentially in the following order: Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(N -Tos), Boc-Gly, Boc-Gly, Boc-Phe, Boc-Cys(4-MeBzl), Boc-Ser(Bzl), Boc-Ser(Bzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 70 mg.

Amino Acid Analysis: Arg (2.7), Asp (1.0), Cys (1.8), Gly (2.0), Ile (2.0), Phe (1.9), Ser (1.8).
FAB (M+H) at M~Z: 1615.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

~xam~le 36 Cysteinyl-Cyclohexylalan~l-Glycyl-Glycyl-Arginyl-Isoleucyl-(D)Aspartyl-Arainyl-ISoleuCyl-Phenylalanyl-Arginyl-C~çinyl Car~oxamide Cyclic Di.sulfide Protected peptide resin ~0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg~N -Tos), Boc-(D)Asp(beta-Bzl), Boc-Ile, Boc-Arg(N -Tos), Boc-Gly, Boc-2Q3~

Gly, Boc-Cha, Boc-Cys(4-MeBzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 37.6 mg.

Amino Acid Analysis: Arg (3.1), Asp (1.0), Cha (0.9), Cys (1.7), Gly (1.8), Ile (2.1), Phe (0.9).
FAB (M+H) at M/Z: 1445.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

Exam~18_37 Cy~invl-Cvclohexvlalanvl-Glvcyl-Glycyl-~rainvl-Isoleucvl-~s~artyl-Arainvl-Alanvl-Phenvlalanvl-i~Lsinyl-Cyste;nyl Carboxamid~ Cvcl;c n i su 1 f ide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ala, Boc-Arg~N5-Tos), Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(9-MeBzl), was treated with HF
(liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 30.7 mg.

Amino Acid Analysis: Ala (l.0), Arg (3.1), Asp (1.0), Cha (0.9), Cys (l.S), Gly (1.8), Ile (1.1), Phe (1.0).
FAB (M+H) at M/Z: 1903.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

~ysteinyl-Cyclohexylalanyl=G~ =Glycyl-Ara;nyl-I~oleucyl-Aspartyl-Alanyl-IsoleuGyl-Pheny ~lanyl-Arqinyl-Cysteinyl Carboxamide Cyclic ~isulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Ala, Boc-Asp(beta-cyclohexyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 16.0 mg.

Amino Acid Analysis: Ala (1.0), Arg (2.0), Asp (1.1), Cys (1.4), Gly (1.9), Ile (2.0), Phe (1.0).
FAB (M+H) at M/Z: 1360.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

Example 39 Mercaptopropionyl-Cyclohexylalanvl-Glycyl-Glycyl-~rainyl-leucyl-beta-~spartyl-Arainyl-Isoleucyl-Phenylalanyl-Arqinyl-Cysteinyl Carboxamide Cyclic D.isulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(N -Tos), Boc-Phe, Boc-Ile, Boc-Arg(N -Tos), Boc-Asp(alpha-Bzl), Boc-Ile, Boc-Arg(N -Tos), Boc-Gly, Boc-Gly, Boc-Cha, Mpa(4-MeBzl~, was treated with HF (liquid) and 9 ~

purified as described in Exa~ple 2. The title compound was obtained as a white powder, S0.0 mg.

Amino Acid Analysis: Arg (3.1), Asp ~1.0), Cha (0.8), Cys (0.5), Gly (1.9), Ile (1.9), Phe (1.0).
FAB (M+H) at M/Z: 1430.
The 300 MHz 1H NMR spectrum was found to be consistent with the proposed structure.

Example 40 Cysteinyl-CyclQh~xylalanyl-Glycyl-Glycyl-Ara;nyl-Isoleuoy~-~pa~L-Arginyl-(~)Isoleucyl-Phenylalanyl-Arginyl-cystein Carboxamide Cyclic Disulfide Protected peptide resin (0.45 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg~NG-Tos), Boc-Phe, Boc-(D)Ile, Boc-Arg(N -Tos), Boc-Asp~beta-cyclohexyl), Boc-Ile, Boc-Arg~N -Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys~9-MeBzl), was treated with HF
(liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 28.0 mg.

Amino Acid Analysis: Arg ~3.2), Asp ~1.0), Cha ~1.0), Cys ~1.8), Gly ~2.1), Ile ~1.8), Phe (1.1).
FAB ~M+H) at M/Z: 1445.
The 300 MHz H NMR spectrum was found to be consistent with the proposed structure.

2 ~ 9 C

~m~le 4l Cysteinyl-Cyclohexylalanyl-Glycyl-Glycyl-A~sinyl-Norvalyl-~sp~;~l=arginyl-Norvalyl-Phenylalanyl-Arainvl-Cys~eir~l Carboxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared ~as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Nva, Boc-Arg(NG-Tos), Boc-Asp(~-Bzl), Boc-Nva, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Boc-Cys(4-MeBzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 23 mg.

Amino Acid Analysis: Arg (3.1), Asp (0.9), Cys (1.7~, Gly (2.0), Nva (2.0), Phe (1.0) plus Cha.
FAB (M-~H)+ at M/Z: 1417.
The 500 MHz 1H NMR spectrum was found to be consistent with the proposed structure.

E~mple 42 Cysteinyl-(D)Phenvlqlvcyl-Glycyl-Glycyl-Ara;nvl-Isoleucvl-Aspar~yl-Arainvl-Isoleucyl-Phenylalanyl-Arginyl-Cysteinyl Carhoxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(~-Bzl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-(D)Phg, Boc-Cys(4-MeBzl), was treated with HF (liquid) and 319~

purified as described in ~xample 2. The title compound was obtained as a white powder, 25 mg.

Amino Acid Analysis: Arg (3.0~, Asp (1.0), Cys ~1.8), Gly ~2.0), Ile (2.0), Phe (1.0).
FAB (M+H)+ at M/Z: 1425.
The 500 MHz lH NMR spectrum was found to be consistent for the proposed structure.

~xample 43 3-Mercapto~ropionyl-Cyclohexylalanyl-Glycyl-GlycyL-Arainyl-lsoleucyl-AsDartyl-Arginyl-Isoleucyl-phenylaL~nyl-(D)Arain Cysteinyl Carboxa~lde Cyc]ic D;sulf;de Protected peptide resln ~0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-(D)Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(~-tert-butyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Mpa(4-MeBzl), was treated with ~F (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 19.1 mg.

Amino Acid Analysis: Arg (2.9), Asp (1.0), Cys (0.5), Gly (2.0), Ile (2.1), Phe (1.0) plus Cha, Mpa.
FAB (M+H)+ at M/Z: 1431.
The 500 MHz lH NMR spectrum was found to be consistent for the proposed structure.

FlxamPl~ 44.

3-Merca~topLopLs~l-cyclohexylalanyl-Glycyl-Glycyl-p~rgin Tsoleucyl-Aspartyl-Arginyl-Isoleucyl-phenylalanyl-Ar~in tD)~ einyl Carboxamide Cyclic nisulfide Protected peptide resin ~0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-(D)Cys~4-MeBzl), Boc-Arg(NG-Tos~, Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(~-benzyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Mpa(4-MeBzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 12.0 mg.

Amino Acid Analysis: Arg (3.0), Asp (1.0), Cys (0.5), Gly (1.7), Ile (2.0), Phe (1.0) plus Cha, Mpa.
FAB (M+H)+ at M/Z: 1430.
The 500 MHz 1H NMR spectrum was found to be consistent for the proposed structure.

~x~ e ~ ~

3-Mercaptopropionyl-Cyclohexylalanyl-(D)Alanyl-Arg;nyl-Ar~;nyl-Isoleucyl-Aspartyl-Arainyl-Isoleucyl-Phenylalanyl-Ar~inyl-Cysteinyl Carboxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(~-tert-butyl), Boc-Ile, Boc Arg(NG-Tos), Boc-Arg(NG-Tos), Boc-(D)Ala, Boc-Cha, Mpa~4-MeBzl), was treated with HF

~, ~

(liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 51 mg.

Amino Acid Analysis: Ala (0.9), Arg (4.2), Asp (0.9), Cys (0.4), Ile (2.0), Phe (1.0) plus Cha, Mpa.
FAB (M+H)+ at M/Z: 1545.
The 500 MHz lH NMR spectrum was found to be consistent for the proposed structure.

Exam~le 46 Cyst~inyL-~henylalanyl-Glycyl-Glycyl-Arainyl-~rg;nyl-I.eucyl-As~artyl-Arainyl-Isoleucyl-Leucyl-Arqinyl-Cysteinyl Carhoxam;de Cvclic Disulfi~e Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Leu, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(~-Bzl), Boc-Leu, Boc-Arg(NG-Tos), Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Phe, Boc-Cys(4-MeBzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 54 mg.

Amino Acid Analysis: Arg (4.2), Asp (0.9), Cys (1.8), Gly (0.9), Ile (1.0), Leu (2.1), Phe (1.0).
FAB at M/Z: 1505.
The 500 MHz lH NMR spectrum was found to be consistent for the proposed structure. --2 ~ ~ 3 C

.F.xam~lg 47 3-Mercapto~ro~lonyl-Cyclohexylalanyl-Glycyl-Glycyl-ArainyL-Isoleucyl-Glutamyl-~r iny]-Isoleucyl-phenyl~lanyl-~rsin~l=
Cysteinyl Carboxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc Cys(9-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Glu(Bzl), Boc-Ile, Boc-Arg(NG-Tos?, Boc-Gly, Boc-Gly, Boc-Cha, Mpa(4-MeBzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 140 mg.

Amino Acid Analysis: Arg (3.1), Cys (0.4), Glu (1.0), Gly (2.1), Ile (2.0), Phe (1.0) plus Cha, Mpa.
FAB (M+H)+ at M/Z: 1445.
The 500 MHz lH NMR spectrum was found to be consistent for the proposed structure ~m~ 1 ~ q 8 3-Mercaptopro~ionyl-Cyclohexylalanyl-Glycyl-Glycyl-(D)Arqinyl-Isoleucyl-As~artyl-Arginyl-ISOleucyl-Phenylalanyl-Arginyl-Cysteinyl Carb~amide Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino -acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc~Asp(~-Bzl), Boc-Ile, Boc-(D)Arg(NG Tos), Boc-Gly, Boc-Gly, Boc-Cha, Mpa(4-MeBzl), was treated with HF (liquid) and ~3 C

purified as descri~ed in Example 2. The title compound was obtained as a white powder, 47 mg.

Amino Acid Analysis: Arg (3.0), Asp (1.0), Cys (0.5), Gly (1.5), Ile (1.6), Phe (1.0) plus Cha, Mpa.
FAB (M+H)+ at M/Z: 1430.
The 500 MHz 1H NMR spectrum was found to be consistent for the proposed structure.

E~A~mple ~9 . .
3-Merc~ptopr~ionyl-Cyclohexylalanyl-Glvcvl-Glvcvl-Ar~invl-Isoleucyl-As~artyl-Arainyl-lsoleucyl-(D) Phenylalanyl-Arginyl-~ysteinyl Carboxamide Cvclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-(D)Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(~-tert-butyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Mpa(4-MeBzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 12.0 mg.

Amino Acid Analysis: Arg (3.1), Asp (1.0), Cys (0.6), Gly (2.0), Ile ~2.0), Phe (1.1) plus Cha, Mpa.
FAB (M+H)+ at M/Z: 1431.
The 500 MHz lH NMR spectrum was found to be consistent for the proposed structure. --2 ~ 9 8 C C

~xample 50 6-~minLohexanoyl-Cysteinyl-Cy~lohexylalanvl-(D)Alanyl-[D)Ala~yl-~ i ~ l ~ ucyl-~spartvl-Arqinvl-Isoleu ~l-Phenylalanyl-A~ainvl-Cvsteinyl Carbnxamide Cvcl;c ~isulfide Protected peptide resin (O.S0 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino ac~ds added sequentially in the following order: Boc-Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(~-Bzl), Boc-Ile, Boc-Arg(NG-Tos), Boc-(D)Ala, Boc-~D)Ala, Boc-Cha, Boc-Cys(4-MeBzl), Boc-Aha, was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 39 mg.

Amino Acid Analysis: Ala (l.9), Arg (3.2), Asp (l.0), Cys (1.6), Ile (2.0), Phe (l.0) plus Cha.
FAB (M+H)+ at M/Z: 1587.
The 500 MHz lH NMR spectrum was found to be consistent for the proposed structure ~c~m 3-Merca~topropionyl-Cyclohexvlalanyl-Glvcvl-C,lvcYl-arqinvl-Isoleucyl-Histidinvl-Ar~inyl-Tsoleucvl-Phenvlalarlvl-Ar~inyl-Cysteinyl Carboxamide Cyclic Disu!fide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino - ~
acids added sequentially in the following order: Boc-Cys~4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-His(Nim-Tos), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Cha, Mpa(4-MeBzl), was treated with HF (liquid) and 2q~3~

-5~-purified as described in Example 2. The title compound was obtained as a white powder, 10.0 mg.

Amino Acid Analysis: Arg (3.2), Cys ~0.5), Gly (2.5), His (0.9), Ile (1.9), Phe (1.0) plus Cha, Mpa.
FAB (M+H)+ at M/Z: 1453.
The 500 MHz lH NMR spectrum was found to be consistent for the proposed structure.

~ample 52 ;~=,M,e ~ DtoprQplo~yl-Phenylalanyl-Glycyl-Glycyl-Arainvl-I~oleucyl-Aspar~yl-Arginyl-Isoleucyl-Phenylalanyl-Arainyl-Cysteinyl Carboxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-Cys(4-MeBzl) r Boc-Arg~NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(~-Bzl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Gly, Boc-Gly, Boc-Phe, Mpa(4-MeBzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 25 mg.

Amino Acid Analysis: Arg (3.0), Asp (0.9) Cys (0.5), Gly (2.0), Ile (2.0), Phe (2.0) plus Mpa.
FAB (M+H)+ at M/Z: 1424.
The 500 MHz 1H NM~ spectrum was found to be consistent for the proposed structure. ~

. ~

~ample 53 3-Mercaptopropionyl-cyclQhe~~ nyl-~D~lany~ anyL--~rginyl=
Isoleucyl-~spar~yl-Arainyl-I~nl~y~--phenyl2l~nyl-~rain (D)Cyst~inyl Carboxamide Cyclic Disulfide Protected peptide resin (0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-(D)Cys(4-MeBzl), Boc-Arg(NG-Tos), Boc-Phe, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(b-tert-butyl), Boc-Ile, Boc-Arg(NG-Tos), Boc-Ala, Boc-(D)Ala, Boc-Cha, Mpa(4-MeBzl), was treated with HF (liquid) and purified as described in Example 2. The title compound was obtained as a white powder, 33.1 mg.

Amino Acid Analysis: Ala (1.6), Arg (3.2), Asp (1.0), Cys (0.5), Ile (2.0), Phe (1.0) plus Cha, Mpa.
FAB (M+H)+ at M/Z: 1459.
The 500 MHz lH NMR spectrum was found to be consistent for the proposed structure.

~xample 54 3-Mercapto~ropionyl-CyclohexylalanYI-(D)AlanYl-Alanyl-A~qinyl=
IsoleucyL-Aspartyl-Arqinyl-Isoleucyl-NQ r3- (1-Phenyl-2-aminopro~ionyl)lArginyl-(D)Cysteinyl Carboxamide Cycl;c ~is~lfide Protected peptide resin ~0.50 g), prepared (as described in Example 1) from p-methylbenzhydrylamine resin and the amino acids added sequentially in the following order: Boc-(D)Cys(4-MeBzl), Boc-Phe-~rg(NG-Tos) reduced amide, Boc-Ile, Boc-Arg(NG-Tos), Boc-Asp(~-tert-butyl), Boc-Ile, Boc-Arg(NG-Tos~, 2~31 C

Boc-Ala, Boc-(D)Ala, Boc-Cha, Mpa(4-MeBzl), was treated with HF tliquid) and purified as described in Example 2. The title compound was obtained as a white powder, 7.4 mg.

Amino Acid Analysis: Ala ~2.0), Arg(2.2), Asp (0.8), Cys (0.5), Ile (1.6) plus Cha, Mpa.
FAB ~M+H)+ at M/Z: 1445.
The 500 MHz lH NMR spectrum was found to be consistent for the proposed structure.

E~am~le 55 The representative compounds of the invention described in the above examples are summarized in Table 1, below:

Table 1 ANP Aqon;st Sequences E~ample :~Q......... ~L~n~

2 R-C-Cha-(D)A-G-G-R-L-D-R-I-F-R-C-Amide 3 S-S-C-Cha-G-G-R-I-D-R-I-F-R-C
4 S-S-C-Cha-G G-R-I-D-R-I-Cha-R-C
R-C-F-G-G-R-I-D-R-I-F-R-C-Amide 6 R-C-(D)A-Cha-G-G-R-L-D-R-I-F-R-C-Amide 7 R-C-Cha-G-G-R-I-D-R-I-F-R-C
8 S-S-C-Cha-G-G-(D)R-I-D-R-I-F-R-C
9 C-Cha-G-G-R-I-D-R-I-F-R-C ~-C-R-Cha-G-G-R-I-D-R-I-F-R-C
11 R-C-Cha-G-G-R-I-D-R-I-F-R-C-Amide 12 R-C-Cha-(D)A-A-R-I-D-R-I-F-R-C-Amide 13 R-C-Tic-G-G-R-I-D-R-I-F-R-C-Amide ~3~8 ~ r 9~ ~

14 R-C-F-G-G-I-D-R-I-F-R-C-Amide H-C-Cha-G-G-R-I-D-R-I-F-R-C-Amide 16 R-C-Cha-G-G-R-F-D-R-I-F-R-C-Amide 17 C-Cha-G-G-R-I-D-R-I-F-R-C-Amide 18 Orn-C-Cha-G-G-R-I-D-R-I-F-R-C-Amide 19 AcetylR-C-Cha-G-G-R-I-D-R-I-F-R-C-Amide Cit-C-Cha-G-G-R-I-D-R-I-F-R-C-Amide 21 R-C-Cha-G-G-R-I-D-R-I-F-C-Amide 22 R-C-3,5-I2Y-G-G-R-I-D-R-I-F-R-C-Amide 23 R-C-Cha-G-G-R-I-D-R-I-R-C-Amide 24 R-C-Cha-G-G-cyclo(~-I-D)-R-I-F-R-C-Amide R-C-Cha-G-G-R-I-D-R-I-G-A-C-Amide 26 R-C-Cha-G-G-R-I-(D)D-R-I-F-R-C-Amide 27 (D)R-C-Cha-G-G-R-I-D-R-I-F-R-C-Amide 28 R-C-Chg-G-G-R-I-D-R-I-F-R-C-Amide 29 R-C-Cha-G-G-R-I-D-R-A-F-R-C-Amide R-C-~D)Cha-G-G-R-I-D-R-I-F-R-C-Amide 31 R-C-Cha-G-G-R-I-A-R-I-A-R-C
32 Mpa-Cha-G-G-R-I-D-R-I-F-R-C-Amide 33 R-C-Cha-G-G-(D)R-I-D-R-I-F-R-C
34 R-C-Pic-G-G-R-I-D-R-I-F-R-C
S-S-C-F-G-G-R-I-D-R-I-F-R-C
36 C-Cha-G-G-R-I-(D)D-R-I-F-R-C-Amide 37 C-Cha-G-G-R-I-D-R-A-F-R-C-Amide 38 C-Cha-G-G-R-I-D-A-I-F-R-C-Amide 39 Mpa-Cha-G-G-R-I-betaD-R-I-F-R-C-Amide C-Cha-G-G-R-I-D-R-(D)I-F-R-C-Amide 41 C-Cha-G-G-R-Nva-D-R-Nva-F-R-C-Amide 42 C-(D)F-G-G-R-I-D-R-I-F-R-C-Amide --43 Mpa-Cha-G-G-R-I-D-R-I-F-(D)R-C-Amide 44 Mpa-Cha-G-G-R-I-D-R-I-F-R-(D)C-Amide Mpa-Cha-(D)A-R-R-I-D-R-I-F-R C-Amide 46 C-F-G-G-R-R-L-D-R-I-L-R-C-Amide ~31 C

47 Mpa-Cha-G-G-R-I-E-R-I-F R-C-Amide 48 Mpa-Cha-G-G-(D)~-I-D-R-I-F-R-C-Amide 49 Mpa-Cha-G-G-R-I-D-R-I-~D)F-R-C-Amide Aha-C-Cha-(D)A-(D)A-R-I-D-R-I-F-R-C-Amide 51 Mpa-Cha-G-G-R-I-H-R-I-F-R-C-Amide 52 Mpa-F-G-G-R-I-D-R-I-F-R-C-Amide 53 Mpa-Cha-(D)A-A-R-I-D-R-I-F-R-(D)C-Amide 54 Mpa-Cha-(D)A-A-R-I-D-R-I-[F~R]-(D)C-Amide Other peptides of the invention which can be synthesized according to the methods of Examples 1 and 2 include the following:

Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Cys-Phe-Gly-Gly-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Cys-Phe-Gly-Gly-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
Cys-Phe-Gly-Gly-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-Gly-Gly-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-Gly-Gly-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-Gly--Gly-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-Gly-Gly-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-Gly-Gly-(D)Arq-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-Gly-Gly-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-Gly-Gly-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-Gly-Gly-Ile-Leu-Arg-Ile-Phe--Arg-Cys;
His-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-Gly-Gly-~D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-Gly-Gly-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-Cys-Gly-Gly-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-Gly-Gly-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
hCys-Phe-(D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

hCys-Phe-(D)Ala-Ala-~D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
hCys-Phe-(D)Ala-Ala-Arg-Leu-Asp-Arg-Ile~Phe-Arg-Cys;
hCys-Phe-(D)Ala-Ala-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
hCys-Phe-(D)Ala-Ala-Ile-Asp-Arg-Ile-Phe-Arg-Cysi Ser-Ser-Cys-Phe-(D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-~D)Ala-Ala-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-~D)Ala-Ala-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-(D)Ala-Ala-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Arg-Cys-Phe-(D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-(D)Ala-Ala-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-(D)Ala-Ala-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-(D)Ala-Ala-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-(D)Ala-Ala-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Cys-Phe-Gly-Gly-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-Gly-Gly-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-Gly-Gly-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-Gly-Gly-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys Arg-Cys-Phe-Gly-Gly-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-Gly-Gly-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-Gly-Gly-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-Gly-Gly-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-Cys-Gly-Gly-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-Gly-Gly-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
hCys-Phe-(D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
hCys-Phe-(D)Ala-Ala-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
hCys-Phe-(D)Ala-Ala-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
hCys-Phe-(D)Ala-Ala-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
hCys-Phe-(D)Ala-Ala-Ile-Asp-Arg-Ile-Phe-Arg-Cys; ~-Ser-Ser-Cys-Phe-(D~Ala-Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-(D)Ala-Ala-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-(D)Ala-Ala-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
Ser-Ser-Cys-Phe-(D)Ala-Ala-Ile-Asp-Arg-Ile-Phe-Arg-Cys;

2~3~98 C C, Arg-Cys-Phe-(D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-(D)Ala-Ala-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-(D)Ala-Ala-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys;
Arg-Cys-Phe-~D)~la-Ala-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-(D)Ala-Ala-Ile-~sp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-(D)Ala-Ala-Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-(D)Ala-Ala-(D)Arg-Ile-Asp-Arg-Ile-Phe-Arg-Cys;
His-Cys-Phe-(D)Ala-Ala-Arg-Leu-Asp-Arg-Ile-Phe-Arg-Cys; and His-Cys-Phe-(D)Ala-Ala-Arg-Phe-Asp-Arg-Ile-Phe-Arg-Cys.

~xam~le 56 p~e~aration of Plasma Membranes `- Adrenal cell membranes were prepared for use in binding assays as follows: Weighed frozen mature rabbit adrenals (Pel-Freez) were pulverized in a liquid-nitrogen-cooled mortar. The plasma membrane powder was transferred to assay buffer (50mM HEPES (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid), 100 mM NaCl, 5 mM EGTA (ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N',N'-tetraacetic acid), 1 mM
PMSF (phenyl methylsulfonyl fluoride), 0.01% Brij-35 detergent, 0.01~ Bovine Serum Albumin, 0.1% Bacitracin, 5 mM
MnCl2, 1 micromolar leupeptin, 1 micromolar phosphoramidon, pH
7.4) and homogenized in a Polytron three times at 10 seconds each time. The homogenate was centrifuged at 20,000 g for 15 min at 4C. The supernatant was decanted. The pellet was resuspended in assay buffer and homogenized with the Polytron (2 X 10 sec) and again centrifuged at 20,000 g at 4C. The --supernatant decantation and pellet resuspension were repeated two additional times. The final homogenate was diluted to a concentration of 0.16 mg/mL (wet tissue weight) in assay buffer and stored at -80C until used.

2Q33~98 C

Exa~El~_57 ANP Receptor Bindina Assa~

The in vitro receptor binding of the compounds of the present invention was studied as follows: 0.53 mg rabbit adrenal plasma membranes were incubated with 26.1 pM 125I-rat ANP and various concentrations of competing analogs for 90 min at 4C in a total volume of 0.2 mL of assay buffer. Bound 5I-rat ANP was separated from free ligand by rapid filtration through 0.1% polyethylenimine-treated 240-1 glass-fiber filter strips ~Cambridge Technology), followed by rapid washing with ice cold saline. The filters were counted for radioactivity in a LKB 1277 Gammamaster counter with 76%
counting efficiency. Triplicate determinations of bound 125I-rat ANP were used for data analysis. Binding data was analyzed by computer assisted non-linear regression analysis using the LIGAND program (Munson, P.J. and M. Rodbard, Analyti~al Biochemistry, 107, 220-239 (1980)) for determination of density of binding sites on the membrane and for determination of the competing analog's affinity for the binding sites. In the case of selective analogs only the low affinity binding values (pKL) are reported. Table 2, below, which contains the ANP receptor binding data, demonstrates the affinity of the atrial peptide derivatives of the present invention for ANP
binding receptor sites.

2~3~

~ re~eptor billin~ data EX. NQ ~i Ex- NQ ~i ~, No. ~i 2 6.3q 20 5.70 38 5.44 3 6.94 21 6.97 39 6.11 4 6.93 22 S .88 40 6.84 6.06 23 6.71 41 5.94 6 6.22 24 7.24 42 6.90 7 6.34 25 5.91 43 6.42 8 5.66 26 6.22 A 44 6.02 9 6.14 27 6.75 45 6.68 6.60 28 6.23 46 6.01 11 6.63 29 6.63 47 6.37 12 6.69 30 6.45 48 6.45 13 5.61 31 6.61 49 5.19 14 5.05 32 6.74 50 6.75 6.73 33 6.14 51 6.36 16 6.17 34 6.32 52 6.05 17 6.56 35 6.10 54 7.20 18 6.20 36 5.45 19 6.43 37 6.73 = Negative logarithm of ligand binding constant 2~33~98 {, Exam~le 5~

In Vi~o Testina The in ~lYQ effect of the compounds of the present invention on blood pressure and sodium output were studied as follows: Male Sprague-Dawley rats (4-12 rats weighing 200-250 mg each) were anesthetized with inactin administered intraperotoneally at a dose of 100 mg/kg. The test compounds were infused intravenously in 0.1% bovine serum albumin (BSA) in saline solution at a rate of l mL per hour for 15 minutes at each dose level. For routine screening the doses administered were 0.1, 0.3, 1.0, 3.0, 10.0 and 30.0 ~g/kgJmin.
Blood pressure, urine volume output, sodium and potassium secretion were measured over a 15 minute period at each dose.
Test compounds were regarded as active if they produced either a three-fold increase in sodium excretion or a 5% decrease in blood pressure at the doses tested. The results for change in blood pressure and sodium output are shown in Table 3 and demonstrate the ability of the compounds of the present invention to modify vascular and renal function.

Table 3 ~fect on Blo~d Pressure an~_Sodium Output Example oSe (~a/ka) ~ B.P.~) (N) ~ Sodium* ~N) 4 0.100-0.20 8 0.22 5 0.3000.11 8 0.24 5 --l.0003.11 8 0.44 5 3.000~3.14 8 0.52 5 lO.000-7.25 8 0.82 5 30.00G-8.45 8 1.07 5 ~Q~3~ ~38 C

Pose illq/kq) a B.P. (%) ~L ~ Sodium*

0 . 1000 . 35 8 0 . 11 6 _ _ 0.3000.86 8 0.18 6 1.0~0-0.76 8 0.29 6 3 . 000-3 . 30 8 0 . 43 6 10.000-4.84 8 0.49 6 30 . 000 -6 . 79 8 0 . 59 6 6 0.100-0. 95 4 -0.07 4 0 . 300-1 . 25 4 -0 . 02 4 1 . 000-3 . 65 4 -0 . 13 4 3 . 000-5 . 30 4 0 . 04 4 10 . 000 -7 . 93 4 0 . 04 4 30 . 000 -9 . 30 4 0 . 02 4 7 0 . 100-0 . 47 6 0 . 10 6 0 . 300-1 . 20 6 0 . 14 6 1 . 000-2 . 90 6 0 . 3 6 6 3, 000-7 . 32 6 0 . 57 6 10 . 000 -8 . 88 6 0 . 75 6 30 . 000 -7 . 08 6 1 . 12 6 8 0 . 1002 . 50 8 0 . 21 6 0.3000.86 8 0.22 6 1.000-0.89 8 0.42 6 3 . ooo-2 . 05 8 0 . 60 6 lO . 000 -4 . 55 8 0 . 64 6 30 . 000 -7 . 7 9 8 0 . 84 6 ~33~ ~8 ~-` C

~xample Dose (~q~Xq) ~ ~.P.(~) (N) Sodiu~* ~L

0.100 -2.60 6 0.05 5 0.300 -3.47 6 0.05 5 l.000 -4.68 6 0.17 5 3.000 -6.90 6 0.34 5 10.000 -12.00 6 0.62 5 30.000 -16.88 6 0.82 5 0.100 -1.98 12 -0.05 8 0.300 -3.51 12 0.00 8 1.000 -4.60 12 0.06 8 3.000 -5.25 12 0.22 8 10.000 -7.42 12 0.38 8 30.000 -7.92 12 0.77 8 11 0.100 -1.49 12 -0.01 9 0.300 -2.27 12 0.06 9 l.000 -4.64 12 0.07 9 3.000 -7.64 12 0.28 9 10.000 -10.93 12 0.46 9 30.000 -12.42 12 0.85 8 12 0.100 -3.08 5 0.25 9 0.300 -4.22 5 0.55 4 1.000 -8.70 5 0.66 4 3.000 -12.46 5 0.91 4 10.000 -17.54 5 1.14 4 30.000 -24.98 5 1.09 4 ~3 ~~ C, Table 3 (continued) Example Dose (~q/kq) ~ B.P.(~) (N) ~ Sodiu~*

19 Q.100 -0.60 4 -0.03 4 0.300 -8.10 4 -0.10 4 1.000 -7.07 4 -0.13 4 3.000 -9.13 4 0.01 4 10.000 -9.20 4 0.14 30.000 -9.65 4 0.18 4 0.100 -0.25 6 -0.23 5 0.300 -3.72 6 -0.20 5 1.000 -8.60 6 0.15 5 3.000 -14.17 6 0.78 5 10.000 -17.95 6 0.97 5 30.000 -18.90 6 1.38 5 17 0.100 -0.42 11 0.1~ 4 0.300 -1.22 11 0.25 4 1.000 -2.49 11 0.56 4 3.000 -4.70 11 0.66 4 10.000 -8.63 11 0.67 4 30.000 -15.83 11 0.93 4 18 0.100 1.21 7 0.17 5 0.300 -0.63 7 0.17 5 1.000 -2.7~ 7 0.18 5 3.000 -~.57 7 0.25 5 10.000 -6.46 7 0.40 5 30.000 -11.19 7 0.90 5 2~33~9~

~able 3 (CQn~ilU~L
Exampl~ Dose (Ua/kqL a B . ~ ) tN) ~ Sodium* (N) 0.100 -1.35 6 -0.28 5 0.300 -4.87 6 0.01 5 1.000 -8.03 6 -0.01 5 3.000 -10.20 6 0.29 5 10.000 -15.67 6 0.62 5 30.000 -22.23 ~ 0.74 5 26 0.100 -1.20 13 0.09 9 0.300 -0.79 13 0.41 9 1.000 -0.63 13 0.52 9 3.000 -1.55 13 0.60 9 10.000 -3.58 13 0.58 9 30.000 -6.83 13 0.58 9 27 0.100 -2. q8 10 -0.03 9 0.300 -4.85 10 -0.07 9 1.000 -9.20 10 0.03 9 3.000 -11.32 10 0.06 9 10.000 -15.77 10 0.24 9 30.000 -18.54 10 0.49 9 31 0.100 -0.28 6 0.07 5 0.300 -1.93 6 0.32 5 1.000 -3.55 6 0.37 5 3.000 -3.43 6 0.38 5 10.000 -4.03 6 0.46 5 30.000 -7.18 6 0.49 5 ~ ~'33~

C

.

Table 3 (continued) Example Dose (~/kq) ~ ~.P.(~) (Nl ~ Sodium* (N) 32 0.100 1.52 8 -0.03 6 0.300 0.7~ 8 -0.11 6 1.000 -0.90 8 -0.12 6 3.000 -6.06 8 0.37 6 10.000 -15.14 8 0.78 6 30.000 -18.61 8 0.89 6 36 0.100 0.22 8 0.07 4 0.300 -0.82 8 0.01 4 1.000 -1.51 8 0.02 4 3.000 -3.01 8 0.15 4 10.000 -4.25 8 0.19 4 30.000 -6.50 8 0.27 4 37 0.100 1.66 8 0.06 6 0.300 2.66 8 0.14 6 1.000 1.70 8 0.12 6 3.000 0.65 8 0.08 6 10.000 -0.04 7 0.33 6 30.000 -1.0q 8 0.22 6 38 0.100 -2.01 8 -0.03 6 0.300 -2.13 8 -0.10 6 1.000 -2.17 8 -0.04 6 3.000 -4.82 8 0.21 6 10.000 -6.01 8 0.35 6 30.000 -5.28 8 0.48 6 2 ~3 J e~ ~ t) ~3 Table 3 -(~Qn-~-inued) l~ Dose (~q/kg) A B.P~ L Sodi~m* l~L

0.100 -1.11 8 -0.01 4 0.300 -2.03 8 0.11 4 1.000 -1.67 8 -0.28 4 3.000 -2.09 8 -0.18 4 10.000 -2.24 8 -0.14 4 30.000 -2.89 8 0.01 4 42 0.100 0.30 11 -0.04 4 0.300 0.78 11 0.28 4 1.000 0.86 11 0.40 4 3.000 0.51 11 0.26 4 10.000 -3.47 11 0.58 4 30.000 -8.52 11 0.81 4 43 0.100 1.70 8 -0.12 5 0.300 1.67 8 -0.15 5 1.000 1.34 8 -0.19 5 3.000 1.79 8 0.18 5 10.000 -2.85 8 0.04 5 30.000 -3.50 8 0.30 5 44 0.100 -0.35 8 -0.01 4 0.300 -2.50 8 -0.36 4 1.000 -6.24 8 -0.51 4 3.000 -8.59 8 0.83 10.000 -9.45 8 0.72 4 30.000 -14.10 8 0.68 4 C, ~able 3 (continued) Example Dose ~ka) ~ s P.(%) (~) ~ Sodium*

0.100 -0.89 8 0.07 6 0.300 -1.80 8 0.00 6 1.000 -3.26 8 0.17 6 3.000 -4.70 8 0.20 6 10.000 -4.31 8 0.13 6 30.000 -12.11 8 0.18 6 46 0.100 2.38 8 -0.04 5 0.300 -0.86 8 -0.10 5 l.OQ0 -2.85 8 -0.06 5 3.000 -4.39 8 -0.02 5 10.000 -6.89 8 0.17 5 30.000 -8.61 8 0.36 5 47 0~100 -0.35 8 0.03 6 0.300 -1.60 8 -0.11 6 l.000 -3.90 8 0.05 6 3.000 -6.66 8 0.38 6 10.000 -15.09 8 0.58 6 30.000 -21.08 8 0.75 6 0.100 -1.99 8 0.14 5 0.300 -2.64 8 0.07 5 l.000 -5.38 8 -0.03 5 3.000 -9.01 8 0.31 5 10.000 -13.91 8 0.44 5 30.000 -3~.71 7 0.14 5 x = sodium excretion = log(ttc) where t = treated, c = control 2~33~9 C

Fx~mple 5 Treatment of Post-Ischemi~ Acute Ren~l Failu~

The ability of the compound of Example 11 to improve renal function after ischemically-induced renal failure was studied as follows: Male Sprague-Dawley rats were anesthetized with Inactin and surgically prepared for typical renal clearance measurements. Surgical preparation was followed by a 1 hour equilibration period. Bilateral ischemia was induced by complete occlusion of both renal arteries.
After 30 minutes of ischemia, the test compound was infused intravenously at a dose of lO ~g/kg/rnin for two hoursO
Separate groups of animals were also treated with vehicle alone or ANF(1-28) (human ANF).
The results of testing are shown in Tables 4-6, below.
Both the compound of Example 11 and ANF(1-28) produced significant short- and long-term improvement in post-ischemic renal function as measured in increased glomerular filtration rate, reduced plasma creatinine concentration, and reduced gross anatomical damage. These results demonstrate the renoprotective effect of the compounds of the invention and their usefulness in restoring kidney function in acute renal failure.

Table 4 Effect on Glomerular Filtration Rate Glomerular Filtration Rate Group(ml~min/lOOq body wt~

Vehicle0.19 + 0.04 8 Compound ll 0.39 ~ 0.07 7 ANF(1-28)0.40 ~ 0.04 6 2 ~

Tahle 5 Ef~e~t 9n Plasma Creatinine Concent~ation (mg%) nays Post-Ischemia.
1 _2_ _~_ 4 Vehicle (N=8) Mean 0.47 5.05 5.49 3.72 2.22 S.E. 0.11 0.41 1.19 0.99 0.62 Compound 11 ~N=7) Mean 0.52 2.34 1.24 0.99 0.84 S.E. 0.07 0.58 0.71 0.60 0.40 ANF(1-28) (N=5) Mean 0.33 3.29 2.72 1.51 1.32 S.E. 0.08 0.61 0.77 0.71 0.51 Table 6 Gross Anatomical Evalua~i~n Grou~ ~~aluation Score* (Day 4) Vehicle 1.8 i 0.1 Compound 11 1.0 + 0.1 ANF(1-28) 1.7 + 0.4 * Ratings are from 0 to 4 indicating least to greatest damage.

3 ~ ~ 8 The present invention has been described in terms of specific embodiments set forth in detail. It should be understood, however, that these embodiments are presented by way of illustration only, and that the invention is not necessarily limited thereto. For example, although synthetic methods are presented for the preparation of the peptides of the present invention, it is to be understood that these peptides can be prepared by enzymatic and recombinant DNA
methodology. Other modifications and variations suggested by this disclosure and apparent to those skilled in the art are intended to be within the spirit and scope of the claims that follow.

Claims (5)

WHAT IS CLAIMED IS:
1. A compound of the formula:

or a pharmaceutically acceptable salt, ester or amide thereof, wherein R1 is selected from hydrogen, AcetylArg, Aha, Arg, Cit, His, Lys, Orn and Ser-Ser;

R2 is a sulfur-containing group;

R3 is a hydrophobic amino acid or dipeptide;

R4 is a dipeptide spacer or a tripeptide of the formula X-Y, where X is a dipeptide spacer and Y is a basic amino acid;

R5 is a hydrophobic amino acid;

R6 is a peptide of up to three amino acids;

R7 is selected from Cha, Phe, Cha-Arg, Phe-Arg, (D)Phe-Arg, Phe-(D)Arg, Phe?Arg, Leu-Arg, Ala-Arg, Arg and Gly-Ala; and R8 is a sulfur-containing group.
2. The compound of Claim 1 wherein R1 is a basic amino acid; R3 is a hydrophobic amino acid containing a cycloalkyl;
and R6 is a peptide of up to three amino acids containing an acidic amino acid.
3. The compound of Claim 1 wherein R1 is hydrogen; R2 is mercaptopropionic acid; R3 is a hydrophobic amino acid containing a cycloalkyl; and R6 is a peptide of up to three amino acids containing an acidic amino acid.
4. A compound selected from the group consisting of:

;

;

;

;

;

or a pharmaceutically acceptable salt, ester, or amide thereof.
5. A pharmaceutical composition comprising a therapeutically effective amount of the compound of Claim 1 and a pharmaceutically acceptable carrier or a diluent.
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US5208218A (en) * 1988-09-19 1993-05-04 Ludwig Institute For Cancer Research T cell growth factor glycoproteins
US5846932A (en) * 1993-11-12 1998-12-08 Genentech, Inc. Receptor specific atrial natriuretic peptides
US5665704A (en) * 1993-11-12 1997-09-09 Genentech, Inc. Receptor specific atrial natriuretic peptides
US6525022B1 (en) 1993-11-12 2003-02-25 Genentech, Inc. Receptor specific atrial natriuretic peptides
WO2004011498A2 (en) * 2002-07-31 2004-02-05 Conjuchem Inc. Long lasting natriuretic peptide derivatives
WO2007115175A2 (en) 2006-03-30 2007-10-11 Palatin Technologies, Inc. Cyclic natriuretic peptide constructs
US8580746B2 (en) 2006-03-30 2013-11-12 Palatin Technologies, Inc. Amide linkage cyclic natriuretic peptide constructs
JP2009532385A (en) 2006-03-30 2009-09-10 パラティン・テクノロジーズ・インコーポレーテッド Linear natriuretic peptide construct
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US4757048A (en) * 1985-11-05 1988-07-12 Biotechnology Research Associates J.V. Synthetic analogs of atrial natriuretic peptides
US4721704A (en) * 1986-05-09 1988-01-26 Peninsula Laboratories, Inc. Potent synthetic atrial peptide analogs
US4804650A (en) * 1986-10-28 1989-02-14 Biotechnology Research Associates, J.V. Analogs of atrial natriuretic peptides
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